New channel blocker BIIA388CL blocks delayed rectifier, but not A-type potassium current in central neurons.

A new substance (R,S)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-yl)-2-cyclohexyl-N-(3,3-diphenylpropyl)-acetamide hydrochloride (BIIA388Cl), which demonstrates neuroprotective properties in animal models, was examined for its action on K(+) currents in acutely isolated rat hippocampal neurons using the patch-clamp/concentration clamp techniques in the whole-cell configuration. The delayed rectifier K(+)-current (I(DR)) was strongly inhibited by externally applied BIIA388Cl, while the transient A-current (I(A)) remained virtually unaffected. Block of I(DR) by the pre-applied BIIA388Cl was revealed as a rapid decay of the current indicating direct interaction of the drug with the open state of the channel. The removal of the block upon repolarization was also rapid (tau=22 ms). The dose-response relationship for the blocking action of BIIA388Cl revealed an IC(50) value of 300 nM for the peak I(DR), whereas the IC(50) value for I(DR) measured 300 ms after the onset of depolarization was 120 nM. The blocking action of BIIA388Cl on I(A) was at least 200 times less potent. These data allow us to conclude that BIIA388Cl is an effective and selective blocker of I(DR). This current is the main pathway for the loss of intracellular potassium by depolarized neurons. Selective obstruction of this pathway could be useful for neuroprotection.

New aspects of the clinical pharmacology of clonidine.

Using a newly developed radioimmunoassay, we observed the pharmacokinetics and the pharmacologic effects of clonidine simultaneously (mean arterial pressure, plasma catecholamines) in normotensive subjects following single and multiple administration of infusions, tablets, and Perlongets given in varying doses. The following findings were established: (1) The terminal elimination half-life of clonidine was 20 to 25 hours. (2) The pharmacokinetics were modified by an enterohepatic circulation. (3) The pharmacokinetics of clonidine were linear. (4) Clonidine was 100 percent bioavailable in tablets and Perlongets. (5) The pharmacokinetic and pharmacodynamic properties of the drug remained stable during multiple dosing. (6) Following cessation of clonidine medication, no overshooting was observed.

Broad-spectrum cation channel inhibition by LOE 908 MS reduces infarct volume in vivo and postmortem in focal cerebral ischemia in the rat.

Cation channels conduct calcium, sodium and potassium, cations that are likely deleterious in the evolution of focal ischemic injury. We studied the effects of a novel, broad-spectrum inhibitor of several cation channels, LOE 908 MS, on acute ischemic lesion development with diffusion-weighted magnetic resonance imaging (DWI) and on cerebral perfusion with perfusion imaging (PI) in vivo and on cerebral infarct size using 2,3,5-triphenyltetrazolium chloride (TTC) staining postmortem. A total of 18 male Sprague-Dawley rats underwent 90 min of middle cerebral artery occlusion (MCAO) and were randomly and blindly assigned to either LOE 908 MS or vehicle starting 30 min after inducing focal ischemia and continuing for 4 h. Whole-brain DWI and multislice PI were done before initiation of treatment and repeated frequently for the next 3.5 h. DWI-derived lesion volume at 4 h showed a significant difference in favor of the drug treated group (P=0.03), whereas PI-derived perfusion deficit volumes did not significantly differ between the groups. The postmortem infarct volume at 24 h was significantly attenuated in the treated group in comparison to controls (P=0.0001) and neurological score was significantly better in the treated group (P<0.02). Blocking several distinct cation channels with LOE 908 MS significantly reduced infarct size and improved neurological outcome without observable adverse effects in this focal ischemia model.

A solid phase radioimmunoassay for digoxin and its acylated derivatives.

A solid phase radioimmunoassay (RIA) is presented for the determination of digoxin and its acylated derivatives. In the procedure the antiserum is covalently bound to bromacetylcellulose. Therefore the free and the immunologically bound digoxin fractions can easily be separated by centrifuging. The radioactivity in the supernatant representing the unbound digoxin is proportional to the digoxin concentration in the sample. This modification of the RIA for digoxin is far more time saving than the separation procedure using dextran-coated charcoal as an absorbent; nevertheless both methods are equally sensitive, specific and reliable. The use of the solid phase assay is demonstrated comparing the bioavailability of various digoxin derivatives.

Omega-conotoxin GVIA potently inhibits the currents mediated by P2X receptors in rat DRG neurons.

We examined effects of omega-conotoxin previously known as a selective blocker of N-type calcium channels, on the adenosine triphosphate (ATP)-induced currents in the rat dorsal root ganglion neurons. These neurons express at least two types of ionotropic purinoreceptors: P2X3 receptors that have very rapid desensitization kinetics and P2X2/X3 heterooligomeric receptor, which exhibits slow desensitization. We have found that omega-conotoxin GVIA potently inhibits the inward currents mediated by both receptor types. This effect was specific for the receptor subtypes: the IC(50) value for responses evoked by 10 microM ATP was 21.2 +/- 1.7 nM for the P2X3 receptor-mediated responses and 3.84 +/- 0.43 microM for slower responses mediated by P2X2/X3 heteropolymers. The efficacy of another type of omega-conotoxin, MVIIC, is much lower: at 10 microM the latter toxin inhibited the rapidly desensitizing response by 65% and the slowly desensitizing response by 18%. The effects of both toxins were reversible and independent on the membrane potential. Omega-Conotoxin GVIA shifted the dose dependence for the agonistic action of ATP on P2X3 receptors to higher concentrations without producing any effect on the kinetics of the response. It is suggested that omega-conotoxin allosterically modulates the receptor properties, rather than competes for the agonist binding site.

Mass spectral analysis of glucuronides from sympathomimetic hydroxyphenylalkylaminoethanols.

A mass spectral method is described for the structure determination of glucuronic acid conjugates of hydroxyphenylalkylaminoethanol-type drugs. Trimethylsilylation and application of the GLC-mass spectral technique yield mass spectra with sufficient information for the identification of all structural subunits.

Pharmacokinetics and metabolism of 14C-labelled alinidine in man and dogs.

Radioactively labelled alinidine was administered intravenously (10 mg) and orally (40 mg) to 5 healthy volunteers and beagle dogs (3 animals for each administration route: 0.1 mg/kg body weight i.v. and 1 mg/kg body weight p.o.). Alinidine was totally absorbed in both species. Regardless of the route of administration man excreted the drug via the kidneys within 12 hours, almost entirely in the unchanged form. The blood plasma curves in man followed a multiexponential decline (t 1/2 alpha : 35 sec, t 1/2 beta : 44 min, t 1/2 gamma : 210 min). The maximum plasma levels of the drug were recorded in man, 45 min after oral administration. However, the rather slow decline of plasma radioactivity observed in dogs, corresponded well with the delayed urinary excretion of alinidine (50% of the administered dose) in this species. Dogs metabolized the drug extensively; seven different metabolites including the parent compound were isolated from canine urine. considerable interindividual differneces were found concerning the quantitive but not the qualitative metabolic pattern of alinidine in dogs. Structural analysis by mass spectrometry revealed oxidation, hydroxylation, and cleavage products of alinidine, altered in its imidazolin and/or allylic moiety. In both species no traces of clonidine were found, which was a predicted metabolite formed by the removal of the allylic sidechain of alinidine.

[Drug research and insurance medicine]. / Pharma-Forschung und Versicherungsmedizin.

The inflation in hospital financing has fallen off. The new regulations brought in with the health reform law (Gesundheitsreformgesetz) could help to cut hospital running costs and lead to a more economical form of medical treatment. At present a prognosis of these cost reductions is not possible. The new list of charges for medical treatment (Bundespflegesatzverordnung) brought in by the government in 1986 also has helped in reducing expenditure. Especially the requirement for self-budgeting and performance-costing has lead to a reduced inflation rate in medical treatment costs. An effective control of medical treatment costs demands more management not only in administrative but also in medical areas as well. Improving hospital economy must not automatically lead to a reduction in the quality of the service provided.

Drug effects on myocardial 45Ca uptake in conscious rats.

The myocardial content of 45Ca++ in conscious rats is increased by single s.c. injections of sympathomimetics. A dose dependent inhibition of this effect is achieved by simultaneous administration of calcium antagonists or beta-receptor blocking agents. The myocardial 45Ca++ content of conscious rats is increased by i.v. administration of dibutyrylcycloadenosinemonophosphate (DBcAMP). The effect of the cyclic nucleotide is suppressed only by a calcium antagonist but not by a beta-receptor blocker. The following conclusions may be drawn: 1. Calcium antagonists and beta-sympatholytics have different sites of action in the heart. 2. The lack of DBcAMP-antagonism of the beta-sympatholytics permits a simple discrimination between both types of substances. After pretreatment with sympathomimetics for 7 days (0.3 mg/kg isoprenaline s.c.), neither high doses of isoprenaline nor doses of DBcAMP and aminophylline increased the myocardial 45Ca content in rats. This effect only lasted briefly (for about two weeks); the site of its action is so far unknown.

Pharmacokinetics and pharmacodynamics of transdermally administered clonidine.

Clonidine was applied to the skin of healthy volunteers once weekly by means of a Transdermal Therapeutic System (TTS). The plasma concentration and renal excretion of clonidine, and its effects on mean arterial blood pressure (MAP) and heart rate (HR) were recorded for 7 days, followed by a three-day observation period when a second TTS was applied. Subjective side effects were semiquantitatively recorded. Four different TTS formulations were tested; of which TTS-RP 600679 was the most effective. Following application of this formulation, the plasma level of the drug built-up up during the first 2 days and then remained stable for 120 h at therapeutic concentrations between 0.5 and 0.7 ng/ml; MAP was consistently reduced. During the steady state period the daily urinary clonidine excretion was in the same range as during chronic administration of Catapres tablets 0.15 mg every 12 h, or Catapres Perlongets 0.25 mg every 24 h. Transdermal clonidine applications renewed weekly provide the following therapeutic advantages: 1. patients are protected continuously throughout the entire steady state period; 2. daily fluctuations in plasma clonidine concentration are minimized, which may result in a marked reduction in side effects; and, 3. drug compliance should be improved.

Development and quality control of a highly sensitive radioimmunoassay for alinidine.

A new precise and sensitive radioimmunoassay (RIA) for alinidine (N-allyl-clonidine) has been developed. Synthesis and analysis of the hapten (4-carboxy-alinidine = STH 2329), as well as the production of the antibody in rabbits, are described in detail. At a final dilution of 1 : 1000, the resulting immune serum binds 50% of a tritiated alinidine standard (50 pg). The detection limit of the present RIA for alinidine is 50 pg/ml plasma. The intra-assay coefficient of variance (VC) is lower than 4% (N = 10) for any standard concentration; the inter-assay VC does not exceed 8.7%. There is no cross reactivity of any alinidine metabolite or congener with the antibody. The low detection limit of the assay, 10(-3) of therapeutically relevant alinidine blood levels, brings about several analytical advantages, which are discussed in detail. Quality control tests were performed in comparison with two reference methods (HPLC). Concerning assay sensitivity, specificity, reliability, and expenditure in costs or sample volumes, the RIA turned out to be the optimal method for routine analysis of alinidine in biological fluids. An example for practical use of the assay is given, evaluating the pharmacokinetics of alinidine in beagle dogs. From the accumulated renally-excreted total radioactivities, the enteral absorption of the drug was calculated (91%); the bioavailability of orally administered alinidine was derived from the corresponding areas under the blood plasma concentration curves of the radioimmunologically-evaluated parent compound (72%).

[Metabolite isolation for mass spectrometrical determination of new drugs (author's transl)]. / Isolierung von Metaboliten neuer Arzneimittel zur massenspektrometrischen Strukturaufklärung.

Only very minute concentrations of metabolites of highly effective and consequently low dosed drugs can be detected in urine. In order to identify the structure of these compounds they must be concentrated by a factor up to 1:10(6). Isolation of metabolites, especially when present as conjugates, becomes more complicated the greater the polarity. In this paper some methods for isolation are presented using column- and thin-layer chromatography as well as extraction and dialysis, finally their practical application and their most effective combination are described. Priority must be given to solving two problems: firstly, a rough pre-purification is necessary to eliminate physiological components from the urine such as salts, urea, etc., and secondly, the mixture of metabolites has to be separated into single metabolites. Examples are given to demonstrate the practicability of these methods for the isolation and structural identification of metabolites belonging to varying structural classes.

New aspects of the pharmacokinetics and pharmacodynamics of clonidine in man.

Using considerably improved analytical methods, the kinetics and effects of clonidine were observed in healthy volunteers over periods of time more than 3 times longer than those previously reported. The high sensitivity and small work load of the newly developed method permitted the performance of low-dose and multipledose trials. 1. The complete bioavailability of clonidine and its elimination half-life (20 to 25.5 h) remained constant after single and multiple doses. 2. Approximately 62% of a given dose was excreted unchanged in the urine, independent of the quantity administered (0.075, 0.15, 0.2, 0.25 or 0.3 mg), the drug formulation (solution, tablet, Perlonget) or of the mode of administration (i.v., p.o.; single or multiple doses). 3. As the pharmacokinetics of the drug were affected by entero-hepatic circulation, it cannot be described by a conventional, open one or two compartment model. 4. The time courses of the plasma clonidine concentration and its drug effects ran asynchronously. 5. On cessation of chronic clonidine administration, blood pressure and plasma catecholamine levels increased to pretreatment levels without exhibiting any "overshoot" reaction.

A newly developed precise and sensitive radioimmunoassay for clonidine.

A new precise and sensitive radioimmunoassay for clonidine has been developed. Synthesis and analysis of the hapten (4-carboxy-clonidine; St 1984) as well as antibody production in rabbits are described in detail. At a final dilution of 1:1000 the resulting immune serum binds 50% of a tritiated clonidine standard containing 1 ng of clonidine. The detection limit of the presented radioimmunoassay for clonidine is 0.1 ng/ml. The coefficient of variation did not exceed 4.3% for any of 7 standard determinations with 5 replicates. There was no relevant cross-reactivity of inactive clonidine metabolites apart from 4-OH-clonidine. To avoid any errors from cross-reaction clonidine was selectively and quantitatively extracted into diethylether from unknown plasma samples. Following concentration of the extracts even such low concentrations as 20 pg of clonidine/ml plasma were detectable. With the radioimmunoassay applied in pharmacokinetic studies a maximal clonidine concentration in blood plasma of healthy human volunteers was determined to 0.6 ng/ml 1.5 h after oral administration of 150 micrograms.

Alinidine pharmacokinetics following acute and chronic dosing.

1 Alinidine (N-allyl clonidine) pharmacokinetics were investigated in healthy volunteers following acute administration of 40 mg orally and intravenously (i.v.) and chronic administration of 40 mg daily and twice daily for 8 days. 2 After acute oral administration the following values were obtained; Cmax -- 166.5 +/- 18.5 ng/ml at 1.8 +/- 0.7 h (mean +/- s.d., n = 5); AUC -- 1122.9 ng ml-1 h; VdSS -- 190.71 and T1/2 -- 4.2 h, and after i.v. administration: AUC -- 1046.7 ng ml-1 h; VdSS -- 190.71 and T1/2 4.2 h. 3 Clonidine was identified in plasma and urine samples following oral and i.v. administration; clonidine Cmax was 0.26 +/- 0.06 ng/ml at 8.4 +/- 2.2 h and 0.5 +/- 0.2 ng/ml at 4.8 +/- 2.5 following oral and i.v. alinidine respectively. Urinary excretion of clonidine represented 0.1% of the administered dose of alinidine. 4 During administration of alinidine 40 mg daily for 8 days, peak and trough plasma levels reached steady state after day 2 (223.1 +/- 123.9 and 9.03 +/- 6.7 ng/ml respectively). During alinidine 40 mg twice daily for 8 days peak and trough plasma levels on day 2 were 356.2 +/- 92.0 and 80.0 +/- 35.8 ng/ml respectively, these levels did not change (P greater than 0.05) between days 2 and 8. Urine elimination of alinidine did not change (P greater than 0.05) between days 5, 6, 7 and 8. 5 Clonidine plasma concentration following alinidine 40 mg daily and twice daily were 0.47 +/- 0.18 and 0.84 +/- 0.21 ng/ml respectively 2 h after administration on day 2 and did not change (P less than 0.05) between days 2-8. 6 It is unlikely that clonidine formed from alinidine contributes to the pharmacological action of alinidine.

Proof of the linearity of the pharmacokinetics of alinidine in man.

The pharmacokinetics of alinidine was investigated in two groups of volunteers: Group I (N=5) received on two occasions single doses of 14C-labelled drug given orally (40 mg) or intravenously (10 mg); Group II (N=6) received single oral doses 10, 30, or 90 mg dissolved in 20 ml water. The samples from Group I were analysed by two different and independent methods (RIA and counting total radioactivity). The results obtained by the two methods were identical, since the compound was not metabolized. The plasma concentrations and renal excretion data obtained from both groups were individually fitted to an open three compartment model. Independent of the route of administration and of the doses given, similar pharmacokinetic parameters were calculated for each group and each trial. The half lives of the distribution and elimination phases were t1/2 alpha: 36-41s, t1/2 beta: 9.9-11.1 min and t 1/2 gamma: 2.7-3.8h. There was a linear relationship between the dose administered and the resulting areas under the plasma concentration curves (AUC). Following a lag period (tau =0.19-0.22h), the peak plasma concentration was reached 0.6-1.2h after oral administration. Oral alinidine was 100% bioavailable.