Which form of dopamine is the substrate for the human dopamine transporter: the cationic or the uncharged species?

The question of which is the active form of dopamine for the neuronal dopamine transporter is addressed in HEK-293 cells expressing the human dopamine transporter. The Km value for [3H]dopamine uptake fell sharply when the pH was increased from 6.0 to 7.4 and then changed less between pH 7.4 and 8.2. The KI for dopamine in inhibiting the cocaine analog [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane binding displayed an identical pH dependence, suggesting that changes in uptake result from changes in dopamine recognition. Dopamine can exist in the anionic, neutral, cationic, or zwitterionic form, and the contribution of each form was calculated. The contribution of the anion is extremely low (

Molecular basis for the inhibition of 1,4-dihydropyridine calcium channel drugs binding to their receptors by a nonspecific site interaction mechanism.

The "membrane bilayer" pathway (Rhodes, D. G., J. G. Sarmiento, and L. G. Herbette. 1985. Mol. Pharmacol. 27:612-623.) for 1,4-dihydropyridine calcium channel drug (DHP) binding to receptor sites in cardiac sarcolemmal membranes has been extended to include the interaction of amphiphiles within the lipid bilayer. These studies focused on the ability of the Class III antiarrhythmic agents bretylium and clofilium to nonspecifically inhibit DHP-receptor binding in canine cardiac sarcolemma. Clofilium was found to inhibit nimodipine binding with an inhibition constant of approximately 5 microM, whereas bretylium had no effect on nimodipine binding. Small angle x-ray diffraction was then used to examine the differential ability of these two Class III agents to inhibit DHP-receptor binding. The time-averaged locations of bretylium, clofilium, and nimodipine in bovine cardiac phosphatidylcholine (BCPC) bilayers (supplemented with 13 mol% cholesterol) were determined to a resolution of 9 A. The location of bretylium as dominated by its phenyl ring in BCPC bilayers was found to be at the hydrocarbon core/water interface, similar to that of the dihydropyridine ring of nimodipine. The location of clofilium as dominated by its phenyl ring was found to be below the hydrocarbon/core water interface within the hydrocarbon chain region of the bilayer, similar to that of the phenyl ring of nimodipine. The location of the dihydropyridine ring portion of nimodipine has previously been shown by neutron diffraction to be located at the hydrocarbon core/water interface of native sarcoplasmic reticulum, consistent with the small angle x-ray data from model membranes in this paper. Therefore, we speculate that the nonspecific inhibition arises from the interaction of clofilium's phenyl ring with the site on the calcium channel receptor where the phenyl ring portion of nimodipine must interact. The DHP-receptor binding pathway would then involve both nonspecific (membrane) and specific (protein) binding components, both of which are necessary for receptor binding.

Bretylium tosylate binds preferentially to muscarinic receptors labelled with [3H]oxotremorine M (SH or 'high affinity' receptors) in rat heart and brain cortex.

Bretylium tosylate is an antiarrhythmic agent. In guinea pig atria it showed the properties of a competitive muscarinic (cholinergic) antagonist and could distinguish between two muscarinic receptor classes or states in cardiac membranes. We decided to further investigate its binding properties at muscarinic cholinergic receptors of the rat heart and brain (cortex), keeping in mind the recently discovered heterogeneity of muscarinic receptor protein. Bretylium tosylate recognized two receptor classes or states in the heart with Ki values of 0.9 and 11 microM. All cardiac membrane receptors showed a homogeneous (11 microM) Ki value for the drug in the presence of GTP in the incubation medium, or after in vivo pretreatment with islet activating protein (IAP). Bretylium tosylate was able (but only at a high concentration, 1 mM) to slow the dissociation kinetics of the tracer, which suggests that it also bound to an allosteric site on the muscarinic receptor, or that it affected the receptor environment. In the brain cortex, as in the heart, bretylium tosylate displayed a high affinity for receptors labelled with the agonist [3H]oxotremorine M (Ki value: 0.8 microM for the SH-or cardiac-type high-affinity receptors), and a 8- to 10-fold lower affinity for cortex M and L receptors. These data suggest that the antagonist bretylium tosylate had binding properties in rat cardiac membranes analogous to those of the partial agonist pilocarpine and that it interacted with a single type of receptor.

Inactivation of acetylcholinesterase with a bretylium tosylate photoaffinity probe.

Azidobretylium tosylate (ABT), the p-azido analogue of bretylium tosylate, has been synthesized to serve as a photoaffinity probe for bretylium binding sites. Bretylium tosylate has antiarrhythmic action and also interacts with amiloride-sensitive sodium ion transport sites. Acetylcholinesterase was used as a model protein, and both bretylium and ABT are reversible inhibitors of this enzyme. The kinetic inhibition constants (Ki) were determined to be 40 microM for bretylium tosylate and 6 microM for ABT. The azido compound is photochemically labile and apparently irreversibly inactivates the enzyme. The rate was retarded by the addition of bretylium tosylate or 4-oxo-N,N,N-trimethylpentanaminium iodide (OTI). Sephadex G-25 chromatography further demonstrated the irreversible nature of the photoinactivation. Since ABT binds at or near the acetylcholinesterase active site, it may be a useful probe for the characterization of the enzyme active site.

Clinical pharmacokinetics of bretylium.

Bretylium is a class III antiarrhythmic agent which is used for the management of serious and refractory ventricular tachyarrhythmias. It exhibits a complex pharmacokinetic profile which is poorly understood. The drug is poorly absorbed following oral administration, and its oral bioavailability is in the region of 18 to 23%. Peak plasma concentrations occur at 1 to 9 hours after oral ingestion, and following oral doses of 5 mg/kg average 76 ng/ml, which is 28-fold lower than those achieved after equivalent intravenous doses. Approximately 75% of a bretylium dose is absorbed within 24 hours of intramuscular administration. Peak plasma concentrations occur at 30 to 90 minutes after intramuscular administration and range from 670 to 1500 ng/ml in subjects receiving 4 mg/kg. Bretylium is negligibly bound to plasma proteins (1-6%). Although drug tissue concentrations have not been reported in humans, high values for the apparent volume of distribution suggest extensive tissue binding. In animals, bretylium is progressively taken up by the myocardium over a period of 12 hours, and at 12 hours after bolus administration, myocardial concentrations exceed plasma concentrations 6 to 12 times. It is also avidly taken up by adrenergic nerves in animals. Bretylium is almost entirely cleared by the renal route and its total body clearance is closely correlated with renal clearance. Available data suggest that bretylium exhibits a complex pharmacokinetic profile which has been described by a 3-compartment model in subjects receiving intravenous dosing. The terminal elimination half-life ranges from 7 to 11 hours following oral, intramuscular and intravenous administration, and renal clearance is about 600 ml/min after intravenous administration.(ABSTRACT TRUNCATED AT 250 WORDS)

[Elimination of organic cations from the eye]. / Elimination organischer Kationen aus dem Auge.

Substances injected into the vitreous cavity leave the eye partly by diffusion into the flowing aqueous, but sometimes also by other mechanisms. To look for such other mechanisms removing organic cations from the rabbit eye, 5 labeled quaternary ammonium compounds were injected intravitreally, mixed with labeled sucrose. The latter leaves essentially by way of the aqueous only. After predetermined times had elapsed the rabbits were killed, and the radioactivity remaining in each eye was measured after combustion of the dried eye bulb. The anticholinergic drug Emepronium disappeared faster than sucrose but only after a delay of many hours. The main experiments were therefore concerned with the time interval 24-48 h after injection. The N- hexylhomologue of Emepronium ' Cetihex ' and the non-sedative antihistamine Aprobit were not eliminated faster than sucrose during this time interval. Since the molecular weights of these quaternaries are similar to that of sucrose and their free diffusion rate should be similar, the present experiments over a limited time interval have proved the existence of an elimination mechanism (besides aqueous flow) only for Emepronium. Tetramethylammonium and tetraethylammonium disappeared markedly faster than sucrose. Since there is no satisfactory way to take their much lower molecular weight into account, the question of a special system for these cations remains open.

Tissue distribution of [14C]bretylium tosylate in rats.

The distribution of [14C]bretylium tosylate in the body and the relationship between tissue and plasma concentrations was determined following intravenous administration of the drug to Charles River rats. The renal excretion of bretylium was rapid in rats and follows an active process. On the average, 50% of the administered dose was excreted in the urine within 1 hr. In the postequilibrium phase, the plasma concentration declined with a half-life of 5 hr. Bretylium concentrations in all tissues, except the heart, declined rapidly according to a triexponential equation. The liver and kidney bretylium concentrations declined in parallel to the plasma concentration with mean tissue-plasma concentration ratios of 6.04 and 12.3, respectively, in the beta phase. However, the concentration of bretylium in the heart increased gradually and peaked at 2 hr, with a tissue-plasma concentration ratio of 121, which, in turn, declined to a value of greater than 60 after 8 hr. The data indicated that (a) bretylium is rapidly distributed into the liver and kidney immediately after reaching the systemic circulation; (b) the distribution into the heart occurs at a slower rate compared with the other organs, and the drug has a high affinity to the myocardium; and (c) since the heart is the site of action and there is no direct correlation between the concentrations in myocardium and plasma, the antiarrhythmic effect of bretylium may not be related to the plasma concentration.

Bretylium kinetics in renal insufficiency.

Bretylium kinetics were examined in patients with varying degrees of renal impairment after a single intravenous dose of bretylium tosylate. Maximum plasma concentrations achieved at the end of the infusion, when normalized to the dose, correlated strongly with creatinine clearance. Drug disposition from plasma was biexponential, with a short distributive phase, but drug elimination was reduced, especially in patients with creatinine clearance below 30 ml/min X 1.73 m2. There was reduction in renal and total clearance and prolongation of t 1/2, with deteriorating renal function. In one patient who was reevaluated after a year, there was 76% reduction in the total clearance, corresponding to 43% deterioration of renal function. The difference of 33% between these values is due to a reduction of nearly 36% in volume of distribution, caused by the further deterioration of the renal function. Six-hour hemodialysis procedure on two anephric patients, resulted in an apparent one- to threefold increase in the computed bretylium clearance during dialysis, but the fraction of the total body load eliminated during the same period was not proportionally significant. The strong linear relationships between renal and total clearance, beta, and the creatinine clearance, may be helpful in adjusting dosage regimens for bretylium in patients with renal dysfunction.

Pharmacokinetics of bretylium in dogs and the effect of hemoperfusion on elimination.

The pharmacokinetics of bretylium in dogs and the efficacy of hemoperfusion with a resin column in its removal from the body following intravenous administration of bretylium tosylate were investigated. Five mongrel dogs weighing 18-26 kg were given a bolus dose of 15 mg/kg. Serial blood samples were taken for 24 hr. Hemoperfusion, through a resin column, was then initiated and continued for 4 hr under pentobarbital anesthesia. During hemoperfusion, arterial and venous blood samples were collected several times; venous blood samples were then withdrawn for an additional 8 hr. Urine was collected from each dog in three portions for up to 48-54 hr. Pharmacokinetics of bretylium in dogs could be characterized by a two-compartment open model with a distribution half-life of 7 min and biological half-life of 15.9 +/- 1.9 hr. Plasma levels declined rapidly from approximately 20 microgram/ml at 6 min to less than 2 microgram/ml within 1 hr. The ratio of intercompartmental rate constants, k12/k21, was 16.7, and the volume of the central compartment and apparent volume of distribution were 0.245 and 5.22 liter/kg, respectively, indicating a wide distribution of bretylium into the tissues. Plasma dialysis clearance averaged 29.7 ml/min, which is 30% of the total body clearance (98.8 ml/ min). These data suggest that resin hemoperfusion may not be useful in the treatment of bretylium intoxication.

Bretylium pharmacokinetics and bioavailabilities in man with various doses and modes of administration.

The pharmacokinetics and bioavailabilities of bretylium tosylate were studied in 9 male normal volunteers by 60 min constant rate intravenous infusions of 200, 300, and 400 mg, by intramuscular injection of 300 and 400 mg, by oral administration of 100, 200, and 400 mg in solution, and by oral administration of 200 mg tablets. The latter studies were repeated in the same 5 volunteers which were also studied by all modes of administration and at several doses. Intravenous studies showed a sum of 3 exponentials to characterize plasma level-time studies with a terminal half-life of 535 +/- 32 (S.E.M.) min (n = 12). The urinary recovery of unchanged drug was 77 +/- 4(S.E.M.)(n = 14). Half-lives within a subject were correlated and independent of dose. Intramuscular administration showed an apparent half-life of first-order invasion of 79 +/- 13 (S.E.M.) min (n = 6) with no apparent dose dependency and a urinary recovery of unchanged drug of 95.4 +/- 3.2 (S.E.M.) per cent with a terminal half-life similar to the intravenous studies. Oral solutions had smaller lag times of absorption [17 +/- 4(S.E.M.) min] than tablets [56 +/- 9 (S.E.M.) min] and longer apparent half-lives of first-order absorption [231 +/- 23 (S.E.M.) min] than tablets [87 +/- 15 (S.E.M.) min]. The tablets had slightly greater bioavailabilities [27 +/- 2.3 (S.E.M.) per cent] than the oral solutions [22.1 +/- 2.2 (S.E.M.) per cent] but with no apparent dose dependencies. Renal clearances were the same for all modes of administration. Means +/- S.E.M. were 735 +/- 32, i.v., 686 +/- 38, i.m., and 623 +/- 57 ml min-1, p.o. Apparent overall volumes of distribution were 589 +/- 401, i.v. and 450 +/- 671 (S.E.M.), i.m. The i.v. studies in three dogs confirmed the three-compartment body model and the high overall volumes of distribution, had terminal half-lives similar to humans and had renal clearances of 84, 164, and 207 ml min-1 that were in excess of glomerular filtration. There were no significant changes of cardiovascular parameters with the time course of the drug in the body and no significant drug-affected clinical parameters. The only consistent side effect was a generally transient nasal congestion at plasma peak time on intravenous administration.

Cardiac drug overdose.

Toxicity from cardiac drugs is a particular management challenge since the manifestations of an acute overdose and the initial indications for the drug are often similar. Plasma drug levels are essential but must be interpreted in light of the clinical picture. Hypotension, due to either vasodilatation or decreased myocardial contractility, and arrhythmias are the principal cardiac manifestations, but noncardiac effects are sometimes more troublesome. An antiarrhythmic agent of the same class should not be used in treating an arrhythmia resulting from an overdose.

Pharmacokinetics of [14C]bretylium tosylate in rats.

The pharmacokinetics of bretylium tosylate were investigated in eight male Charles River rats. Each animal received an intravenous dose (10 mg/kg) of [14C)bretylium tosylate. Serial blood samples, urine, and feces were collected for up to 72 hr. Bretylium concentrations in plasma and amounts excreted in urine and feces were determined by scintillation counting. On the average, 88 and 95% of the dose were recovered in urine and feces in 24 and 72 hr, respectively. Urinary recovery accounted for 65.6 of the dose while 29.7% was excreted in the feces. Bretylium concentrations in plasma declined triexponentially and were fitted to a three-compartment open model. Bretylium has a very high apparent volume of distribution (15 liters/kg), and its beta half-life averaged 5.5 hr. Mean values of the apparent volume of the central compartment, plasma clearance, renal clearance, and excretion rate constants of bretylium in rats were 1 liter/kg, 1.93 liters/hr/kg, 1.27 liters/hr/kg, and 1.24 hr-1, respectively. The results indicate that: (a) bretylium is strongly bound to the tissues and is eliminated by active urinary secretion and by biliary excretion in rats, and (b) there are strong similarities between the pharmacokinetics of bretylium in humans and rats and that this animal model might be suitable for interaction studies with other drugs.

Clinical pharmacokinetics of intravenous and oral bretylium tosylate in survivors of ventricular tachycardia or fibrillation: clinical application of a new assay for bretylium.

We studied 12 patients receiving either chronic oral (p.o.) maintenance bretylium and/or acute intravenous (i.v.) bretylium to evaluate drug efficacy and pharmacokinetics. All patients were survivors of ventricular tachycardia or fibrillation. A new assay for bretylium was applied, and it proved sensitive and reliable. After single intravenous dosing, bretylium was eliminated from serum with a mean rate constant of lambda iv1 = 0.0515 hr-1 and a corresponding elimination half-life of tiv1/2 = 13.5 hr (7 studies), similar to previous results in normals. Total body clearance averaged 428 ml/min, of which virtually all was accounted for by renal clearance. Seven patients responding to intravenous bretylium were transferred to oral drug. During chronic therapy (mean dose, 41 mg/kg/day bretylium tosylate), mean minimum steady-state concentration of bretylium was 186 ng/ml (range, 72-461) and was accurately predicted, within experimental error, by using the elimination rate constant determined for oral, but not intravenous, drug in normal subjects (lambda po1 = 0.115 hr-1). Determination of average steady-state concentration (Css) yielded similar conclusions. Mean 24 hr urinary excretion of bretylium during oral therapy was 18.3% (range, 9-13%). These results lend validity to earlier observations in normals and suggest route and concentration dependence of disposition. Transfer to oral bretylium allowed continued control of sustained ventricular tachycardia in all 7 patients and of unsustained ventricular tachycardia in 5. Orthostatic hypotension in 4 responded to protriptyline. Six were discharged on bretylium, with a mean follow-up of 12.2 months (range, 1-25.5). Four maintained a favorable response, and 2 died suddenly at 1 and 3 months. We conclude that further evaluation or oral bretylium is justified; attempts should be made to increase steady-state concentrations during oral therapy.

Oral and intravenous bretylium disposition.

To compare the oral and intravenous disposition of bretylium tosylate in man, 10 normal male subjects were randomly assigned single doses of 5 mg/kg bretylium tosylate either orally or intravenously and crossed over 2 wk later to the opposite route (20 studies). Each experiment included sampling for drug in serum and urine over 48 hr. Bretylium tosylate was assayed by gas chromatography. Kinetic analysis provided the following mean [coefficient of variation] results: 100FPo, 22.6% [40.2%]; ClrIV, 300 ml/min [27.8%]; ClrPo, 1.268 mg/min [54.8%]; ClBIV, 299 ml/min [31.9%]; f, 101% [8.7%]; Vdss, 3.37 l/kg [30.5%]; lambda lIV 0.0510 [12.8%]; lambda lPG, 0.115 [52.7%]hr-1; elimination half-life (t 1/2) after intravenous bretylium tosylate, 13.6 hr, and after oral bretylium tosylate, 6.0 hr (harmonic means). Bretylium tosylate binding to plasma proteins in normal volunteer samples was found to be negligible. The results indicate extensive tissue binding of bretylium tosylate. Oral doses of bretylium tosylate are only partially absorbed. Bretylium tosylate is eliminated entirely by the kidneys as unchanged drug. The greater renal clearance after oral than intravenous bretylium tosylate, and the greater elimination rate constant and shorter oral bretylium tosyulate t 1/2 are of interest but no explanation is available.

Pharmacokinetics of bretylium in man after intravenous administration.

The pharmacokinetic profile of bretylium was studied in four normal male volunteers using a new sensitive EC-GC procedure for its quantitative in biological fluids. The plasma concentrations and urinary excretion rates following the constant i.v. infusion of a single 4 mg/kg dose of bretylium tosylate declined biexponentially and the data were fitted to a two-compartment model with a renal and a nonrenal route of elimination. The drug had a mean half-life (t1/2 beta) of 7.8 hr and apparent volume of distribution (Vd, beta) of 8.18 liters/kg. The renal clearance, which was 6 times that of the glomerular filtration rate, accounted for almost 84% of the total body clearance and correlated linearly with the subjects' creatinine clearance. The observed side effects of bretylium were mild and similar to those of other adrenergic blocking agents.

Studies of uptake of the bretylium analogue, iodobenzyltrimethylammonium iodide, by non-primate, monkey and human hearts.

Uptake of (+/-)-[3H]-noradrenaline, [14C]-bretylium and [125I]-o-iodobenzyltrimethylammonium iodide (RIBA) by rat heart was studied by the Langendorff technique. All three compounds showed significant uptake. 2 Corticosterone and 17-beta-oestradiol inhibited the uptake of all three compounds by rat heart, a finding consistent with extraneuronal uptake (uptake2). 3 [131I]-RIBA was injected intravenously into pigs and monkeys (M. speciosus). Myocardial samples taken from pigs killed 1 and 2 h after injection showed significant uptake. No significant uptake was found in myocardial samples of monkeys killed 10 min, 2 h and 24 h, respectively, after injection. 4 Four normal human volunteers received [125I]-RIBA intravenously and the image of the precordial area was followed by means of scintillation camera for the first 4 h after injection. In two of the subjects, the scintigrams were repeated at 22 and 23 h after injection, respectively. No evidence of myocardial uptake was observed. 5 These results suggest the possibility that man and at least one other primate species may differ from lower species with regard to uptake.

Series on pharmacology in practice. 2. Antiarrhythmic drug therapy.

The selection of appropriate antiarrhythmic drug therapy depends on a knowledge of the drugs available, their spectrum of action, their pharmacokinetics, and their major side effects. It is important to know how the pharmacokinetics of a drug vary with different disease states so that appropriate adjustments to dosage can be made. Drugs with similar actions can be assigned into groups, and five different groups can be identified. The commonly used antiarrhythmic drugs are reviewed, and some of the newer drugs are discussed.

Imaging the adrenal medulla with an I-131-labeled antiadrenergic agent.

Tissue distributions of four antiadrenergic agents labeled with iodine-125 have been determined in dogs. [125I] ortho-iodobenzldimethyl-2-hydroxyethyl ammonium and [125I] ortho-iodobenzyldimethylethyl ammonium show highly selective uptake in the adrenal medulla. Studies of molecular structure-distribution indicate that both the nature of the cationic head and the ring position of the iodine atom greatly influence adrenal specificity. Distinct images of dogs' adrenal medulla have been obtained 4 days after i.v. injection of 1.5 mCi of [131I] ortho-iodobenzyldimethyl-2-hydroxyethyl ammonium.