Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology: Update 2017.

Therapeutic drug monitoring (TDM) is the quantification and interpretation of drug concentrations in blood to optimize pharmacotherapy. It considers the interindividual variability of pharmacokinetics and thus enables personalized pharmacotherapy. In psychiatry and neurology, patient populations that may particularly benefit from TDM are children and adolescents, pregnant women, elderly patients, individuals with intellectual disabilities, patients with substance abuse disorders, forensic psychiatric patients or patients with known or suspected pharmacokinetic abnormalities. Non-response at therapeutic doses, uncertain drug adherence, suboptimal tolerability, or pharmacokinetic drug-drug interactions are typical indications for TDM. However, the potential benefits of TDM to optimize pharmacotherapy can only be obtained if the method is adequately integrated in the clinical treatment process. To supply treating physicians and laboratories with valid information on TDM, the TDM task force of the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) issued their first guidelines for TDM in psychiatry in 2004. After an update in 2011, it was time for the next update. Following the new guidelines holds the potential to improve neuropsychopharmacotherapy, accelerate the recovery of many patients, and reduce health care costs.

[Therapeutic drug monitoring in psychiatry. A brief summary of the new consensus paper by the task force on TDM of the AGNP]. / Therapeutisches Drug-Monitoring in der Psychiatrie. Kurze Zusammenfassung des neuen Konsensuspapiers der Arbeitsgruppe TDM der AGNP.

In October 2011 the Task Force Therapeutic Drug Monitoring of the Association for Neuropsychopharmacology and Pharmacopsychiatry (AGNP) published an update (Pharmacopsychiatry 2011, 44: 195-235) of the first version of the consensus paper on therapeutic drug monitoring (TDM) published in 2004. This article summarizes the essential statements to make them accessible to a wider readership in German speaking countries.

AGNP consensus guidelines for therapeutic drug monitoring in psychiatry: update 2011.

Therapeutic drug monitoring (TDM), i. e., the quantification of serum or plasma concentrations of medications for dose optimization, has proven a valuable tool for the patient-matched psychopharmacotherapy. Uncertain drug adherence, suboptimal tolerability, non-response at therapeutic doses, or pharmacokinetic drug-drug interactions are typical situations when measurement of medication concentrations is helpful. Patient populations that may predominantly benefit from TDM in psychiatry are children, pregnant women, elderly patients, individuals with intelligence disabilities, forensic patients, patients with known or suspected genetically determined pharmacokinetic abnormalities or individuals with pharmacokinetically relevant comorbidities. However, the potential benefits of TDM for optimization of pharmacotherapy can only be obtained if the method is adequately integrated into the clinical treatment process. To promote an appropriate use of TDM, the TDM expert group of the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) issued guidelines for TDM in psychiatry in 2004. Since then, knowledge has advanced significantly, and new psychopharmacologic agents have been introduced that are also candidates for TDM. Therefore the TDM consensus guidelines were updated and extended to 128 neuropsychiatric drugs. 4 levels of recommendation for using TDM were defined ranging from "strongly recommended" to "potentially useful". Evidence-based "therapeutic reference ranges" and "dose related reference ranges" were elaborated after an extensive literature search and a structured internal review process. A "laboratory alert level" was introduced, i. e., a plasma level at or above which the laboratory should immediately inform the treating physician. Supportive information such as cytochrome P450 substrate and inhibitor properties of medications, normal ranges of ratios of concentrations of drug metabolite to parent drug and recommendations for the interpretative services are given. Recommendations when to combine TDM with pharmacogenetic tests are also provided. Following the guidelines will help to improve the outcomes of psychopharmacotherapy of many patients especially in case of pharmacokinetic problems. Thereby, one should never forget that TDM is an interdisciplinary task that sometimes requires the respectful discussion of apparently discrepant data so that, ultimately, the patient can profit from such a joint eff ort.

AGNP Consensus Guidelines for Therapeutic Drug Monitoring in Psychiatry: Update 2011.

Therapeutic drug monitoring (TDM), i. e., the quantification of serum or plasma concentrations of medications for dose optimization, has proven a valuable tool for the patient-matched psychopharmacotherapy. Uncertain drug adherence, suboptimal tolerability, non-response at therapeutic doses, or pharmacokinetic drug-drug interactions are typical situations when measurement of medication concentrations is helpful. Patient populations that may predominantly benefit from TDM in psychiatry are children, pregnant women, elderly patients, individuals with intelligence disabilities, forensic patients, patients with known or suspected genetically determined pharmacokinetic abnormalities or individuals with pharmacokinetically relevant comorbidities. However, the potential benefits of TDM for optimization of pharmacotherapy can only be obtained if the method is adequately integrated into the clinical treatment process. To promote an appropriate use of TDM, the TDM expert group of the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) issued guidelines for TDM in psychiatry in 2004. Since then, knowledge has advanced significantly, and new psychopharmacologic agents have been introduced that are also candidates for TDM. Therefore the TDM consensus guidelines were updated and extended to 128 neuropsychiatric drugs. 4 levels of recommendation for using TDM were defined ranging from "strongly recommended" to "potentially useful". Evidence-based "therapeutic reference ranges" and "dose related reference ranges" were elaborated after an extensive literature search and a structured internal review process. A "laboratory alert level" was introduced, i. e., a plasma level at or above which the laboratory should immediately inform the treating physician. Supportive information such as cytochrome P450 substrate- and inhibitor properties of medications, normal ranges of ratios of concentrations of drug metabolite to parent drug and recommendations for the interpretative services are given. Recommendations when to combine TDM with pharmacogenetic tests are also provided. Following the guidelines will help to improve the outcomes of psychopharmacotherapy of many patients especially in case of pharmacokinetic problems. Thereby, one should never forget that TDM is an interdisciplinary task that sometimes requires the respectful discussion of apparently discrepant data so that, ultimately, the patient can profit from such a joint effort.

[Therapeutic drug monitoring (TDM) of psychotropic drugs: a consensus guideline of the AGNP-TDM group]. / Le dosage plasmatique des médicaments psychotropes a des fins thérapeutiques: recommandations du groupe d'experts AGNP-TDM.

In psychiatry, therapeutic drug monitoring (TDM) is an established procedure for most psychotropic drugs. However, as its use in everyday clinical practice is far from optimal, the AGNP-TDM group has worked out consensus guidelines to assist psychiatrists and laboratories involved in drug analysis. Based on a thorough analysis of available literature, 5 levels of recommendation were defined with regard to TDM of psychoactive drugs, from 1) (strongly recommended) to 5) (not recommended). A list of indications for TDM, alone or in combination with pharmacogenetic tests is presented. Instructions are given with regard to preparation of TDM, analytical procedures, reporting and interpretation of results and the use of information for patient treatment. Using the consensus guideline will help to ensure optimal clinical benefit of TDM.

Widening potential for Ca2+ antagonists: non-L-type Ca2+ channel interaction.

In addition to their well characterized interaction with the alpha 1 subunit of the voltage-dependent L-type Ca2+ channel, certain Ca2+ antagonists have been reported to modulate an increasing number of cellular functions as diverse as extrusion of cytotoxic substances, or cleavage of cAMP by phosphodiesterase. Some of these interactions (such as the reversal of multidrug resistance by Ca2+ antagonists for the treatment of lymphoma patients) have already been exploited clinically; some (such as protection of ischemic tissue by Ca2+ antagonists interacting with mitochondrial sites) open new therapeutic issues. In this survey of the non-L-type channel Ca2+ antagonist target structures known to date, Gerald Zernig evaluates the available data and emphasizes common characteristics shared by the seemingly diverse target structures. Research on these sites might help to understand yet unexplained effects of Ca2+ antagonists and possibly lead to the development of novel drugs with higher selectivity for non-L-type Ca2+ channel structures.

Pharmacotherapy of alcohol dependence.

The current pharmacotherapeutic approaches to alcohol dependence, together with the results of a number of clinical trials, are reviewed in this article. Despite the somewhat disappointing clinical results, pharmacotherapeutic interventions did lead to some small, but significant, improvements in alcohol abstinence rates.

Morphological integrity of single adult cardiac myocytes isolated by collagenase treatment: immunolocalization of tubulin, microtubule-associated proteins 1 and 2, plectin, vimentin, and vinculin.

Single cardiac myocytes were isolated from hearts of 9 to 12-week-old rats by means of collagenase (100 U/ml). After assessment of their functional integrity they were processed for immunofluorescence microscopy of the cytoskeletal proteins tubulin, microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), plectin, vimentin, and vinculin. Antibodies to tubulin decorated a delicate filamentous network that apparently was unrelated to any sarcomeric organization. The distribution of MAP-1 and MAP-2 was strikingly different from that of tubulin, as both antigens were confined to Z-line structures. These structures were also prominently stained by affinity-purified antibodies to plectin and a monoclonal antibody to vimentin. Co-distribution of plectin and vimentin was also observed at the former intercalated disk region of the heart cell. Anti-vinculin antibodies decorated an intricate meshwork consisting of delicate filaments with predominantly irregular orientation and occasional assembly into whorls. These immunolocalization data indicate that the cell shape and cytoskeletal architecture characteristic of cardiac myocytes in tissues is maintained in single isolated cells. Furthermore, intermediate filaments rather than microtubules seem to be instrumental in the preservation of cell morphology.

Different stereoselective effects of (R)- and (S)-propafenone: clinical pharmacologic, electrophysiologic, and radioligand binding studies.

Propafenone is a class 1c antiarrhythmic agent with moderate beta-blocking activity as a result of a structural similarity to beta-adrenoceptor antagonists. In a randomized, double-blind crossover exercise study, eight healthy volunteers were examined before and 2 1/2 hours after oral administration of 300 mg (R,S)-, 150 mg (R)-, and 150 mg (S)-propafenone hydrochloride. The mean rate pressure product was significantly reduced by (R,S)-propafenone hydrochloride (-5.2%; p = 0.045) and half-dosed (S)-propafenone hydrochloride (-5.9%; p = 0.013), whereas the (R)-enantiomer caused no significant changes. There was a significant difference between the effects of (R)- and (S)-propafenone (p = 0.033). In beta-adrenoceptor-binding inhibition experiments with (S)-(125I)iodocyanopindolol in a sarcolemma-enriched cardiac membrane preparation, the eudismic ratio of (S)- over (R)-propafenone was 54. On the spontaneously beating Langendorff-perfused guinea pig heart, 3 x 10(-6) mol/L of both (R)- and (S)-propafenone resulted in significant changes (p less than 0.01) on His bundle conduction (+79% +/- 27% and +69% +/- 9%), as well as comparable decreases in the maximal rate of pacing with 1:1 conduction of the atrial (-54% +/- 10% and -57% +/- 8%) and ventricular myocardium (-42% +/- 6% and -43% +/- 6%), indicating equal effects in sodium channel-dependent antiarrhythmic class 1 activity. Thus (R)- and (S)-propafenone exert different beta-blocking actions but equal effects on the sodium channel-dependent antiarrhythmic class 1 activity. More specific antiarrhythmic class 1 therapy with reduction of beta-blocking side effects may be attained with optically pure (R)-propafenone hydrochloride instead of the currently used racemic mixture.

Hydrophobic calcium channel ligands: methodical problems and their solution.

Niguldipine is a 1,4-dihydropyridine derivative that combines L-type Ca2+ channel-blocking effects and alpha 1-adrenolytic activity within a single molecule, exemplifying a novel approach in the treatment of hypertension. As niguldipine is a very hydrophobic compound, it (1) readily adsorbs to surfaces of the plastic-ware often used in radioligand binding assays and (2) partitions into the hydrophobic membrane compartments. Both phenomena decrease the actual free drug concentration in radioligand-binding assays and lead to gross underestimation of the affinity of niguldipine (and other hydrophobic ligands) for the 1,4-dihydropyridine binding domain of the L-type Ca2+ channel or for alpha 1A adrenoceptors, respectively. Partitioning of the hydrophobic molecules into the membrane phase leads to a dependence of the Ki value on "total receptor" concentration despite mathematic corrections of the experimentally determined IC50 values. The Ki dependence was mimicked by adding denatured membranes (devoid of high-affinity receptor-binding activity) to native membrane preparations. Loss to pipet tips and tubes was avoided by a special dilution protocol. Partitioning into the hydrophobic membrane compartments needed more elaborate correction procedures.

Acute and prolonged effects of clocinnamox and methoclocinnamox on nucleus accumbens dopamine overflow.

The mu opioid antagonist clocinnamox (CCAM) insurmountably inhibits opioid self-administration. In contrast, CCAM's prodrug, methoclocinnamox (MCCAM), acts as a weak partial agonist in this paradigm when given acutely and inhibits opioid self-administration for up to 5 days. In vivo microdialysis was employed to determine if these effects are paralleled in basal and opioid-stimulated dopamine (DA) overflow in the rat nucleus accumbens (NAC). When given acutely, CCAM (10 mg/kg s.c.) was essentially without effect. CCAM also markedly attenuated the overflow of DA induced by heroin (0.5 mg/kg s.c.; 200% of DA baseline) 24 h later. In contrast, MCCAM (10 mg/kg s.c.) acutely increased NAC DA overflow to 200-245% baseline within 30 min. NAC DA remained at this elevated level for the whole 3-h period of the experiment. Even after 24 h, NAC DA overflow of MCCAM-pretreated animals remained elevated at 165% of VEH-treated animals. Administration of heroin did not result in any further elevation of NAC DA release under these conditions. Thus, the suggested therapeutic profile of MCCAM, i.e., an acute partial agonistic reinforcing effect followed by antagonism of the reinforcing effects of subsequently abused opioids, was confirmed in NAC DA overflow, a neurochemical correlate of the reinforcing effects of drugs of abuse. The most parsimonious explanation for MCCAM's effect on NAC DA overflow is that it acted as an essentially irreversible partial agonist.

In vivo apparent affinity and efficacy estimates for mu opiates in a rat tail-withdrawal assay.

Experiments in a rat tail-withdrawal assay tested the hypothesis that the magnitude and pattern of antagonism of mu opiate agonists by the insurmountable mu opioid antagonist clocinnamox are inversely related to agonist efficacy. In addition, these experiments examined whether this antagonism could be quantified to yield apparent affinity and efficacy estimates for the pharmacological characterization of five opiate agonists. Etonitazene, etorphine, morphine, buprenorphine, and GPA 1657 produced dose-dependent increases in tail-withdrawal latency until 100% maximum possible effect (%MPE) was obtained. Morphine required a higher dose of clocinnamox for a 50% reduction in maximal antinociceptive effect than did buprenorphine or GPA 1657. In contrast, no dose of clocinnamox tested decreased the %MPE for etonitazene or etorphine. These data suggest a rank order of relative efficacy of etonitazene > or = etorphine > morphine > or = GPA 1657 > or = buprenorphine. Similarly, numerical analysis of these data yielded the following apparent affinity and efficacy estimates: etonitazene (0.38 mg/kg, 128); etorphine (0.68 mg/kg, 125); morphine (50 mg/kg, 38), GPA 1657 (6.6, 39); and buprenorphine (0.042 mg/kg, 2.2). These data illustrate that in vivo affinity and efficacy estimates for a number of agonists are remarkably similar across different methods of analysis and are useful for drug classification.

Clocinnamox inhibits the intravenous self-administration of opioid agonists in rhesus monkeys: comparison with effects on opioid agonist-mediated antinociception.

The effects of CCAM, an insurmountable mu opioid receptor antagonist, were studied on the intravenous self-administration and thermoantinociception of alfentanil and nalbuphine, high- and low-efficacy opioid agonists, respectively, in rhesus monkeys. A single dose of 0.1 mg/kg CCAM IV reduced alfentanil's reinforcing potency in an FR30 TO 45s schedule 10-fold within a 24-h period. The maximum response rates remained essentially unchanged. At 1 mg/kg, CCAM caused a 300-fold shift of the alfentanil dose-response curve and also depressed the maximum response rates. CCAM also blocked insurmountably responding for nalbuphine, which was essentially abolished in two of three animals after a dose of 0.1 mg/kg CCAM and in all animals after 1 mg/kg. The acute insurmountable antagonism of alfentanil and nalbuphine self-administration by CCAM was used to determine the (relative initial) efficacy values of both agonists. Efficacy values, tau, were 391 for alfentanil and 196 for nalbuphine; the apparent in vivo dissociation constants, KA, were 0.16 mg/kg per injection (i.e., 350 nmol/kg per injection) for alfentanil and 0.14 mg/kg (370 nmol/kg per injection) for nalbuphine. In comparison, in a rhesus monkey 50 degrees C warm-water tail withdrawal assay, the tau values were 11 for alfentanil and 0.92 for nalbuphine, and the KA values were 0.2 mg/kg (440 nmol/kg) for alfentanil and 0.15 mg/kg (400 nmol/kg) for nalbuphine. Therefore, it seems that the higher potency of alfentanil and nalbuphine in self-administration as compared to thermal antinocieption in rhesus monkeys is predominantly due to a larger efficacy of the same agonist in self-administration (i.e., a larger receptor pool) rather than differences in apparent in vivo affinity.

In vivo estimates of efficacy at 5-HT1A receptors: effects of EEDQ on the ability of agonists to produce lower-lip retraction in rats.

RATIONALE: Maximal responses are often used as a measure of intrinsic activity or efficacy, but cannot be directly equated to efficacy. Using irreversible antagonists, estimates of efficacy can be obtained that may be less dependent on specific conditions. OBJECTIVES: To characterize the intrinsic activity of serotonin (5-HT)1A agonists by examining the effects of an irreversible antagonist on their ability to produce 5-HT1A receptor-mediated responses. METHODS: The effects of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on the ability of 5-HT1A agonists to produce lower-lip retraction (LLR) in rats were studied. RESULTS: In the absence of EEDQ, each 5-HT1A agonist produced full effects, the rank order of potency being: S 14506 > 8-OH-DPAT > buspirone > ipsapirone. EEDQ decreased the number of 5-HT1A binding sites and shifted the dose-response curves (DRCs) of each agonist either to the right or, at higher EEDQ doses, to the right and downward. The manner in which these shifts occurred, however, differed among the compounds. For each agonist, all DRCs obtained after different doses of EEDQ were fitted to models proposed by Furchgott and Black and Leff, and the results indicated the following rank order of efficacy: ipsapirone < buspirone approximately 8-OH-DPAT < S 14506. 5-HT1A agonist-induced LLR appears to be mediated by 5-HT1A receptors, because the 5-HT1A antagonist, WAY 100635, shifted the agonist DRCs to the right in a parallel and dose-related manner, with pA2 values ranging from 7.8 to 8.1. Moreover, pretreatment with WAY 100635 protected against the antagonist activity of EEDQ. CONCLUSIONS: The results suggest that the effects of EEDQ on the ability of 5-HT1A agonists to produce LLR in rats may be useful to obtain estimates of their apparent efficacy at 5-HT1A receptors.

Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase ('enkephalinase') gene.

The biological inactivation of enkephalins by neutral endopeptidase (enkephalinase, NEP, EC3.4.24.11) represents a major mechanism for the termination of enkephalinergic signalling in brain. A pharmacological blockade of NEP-activity enhances extracellular enkephalin concentrations and induces opioid-dependent analgesia. Recently, knockout mice lacking the enzyme NEP have been developed [Lu et al., J. Exp. Med. 1995;181:2271-2275]. The present study investigates the functional consequences and biochemical compensatory strategies of a systemic elimination of NEP activity in these knockout mice. Using biochemical and behavioural tests we found that the lack of NEP activity in brain is not compensated by enhanced activities of alternative enkephalin-degrading enzymes. Also no change in enkephalin biosynthesis was detectable by in situ methods quantifying striatal proenkephalin-mRNA levels in NEP-deficient mice compared with wildtype. Only a 21% reduction of mu receptor density in crude brain homogenates of NEP knockout mice was observed, while delta- and kappa-opioid receptor densities were unchanged. This receptor downregulation was also confirmed functionally in the hot-plate paradigm. NEP knockouts developed normally, but showed enhanced aggressive behaviour in the resident-intruder paradigm, and altered locomotor activity as assessed in the photobeam system. Thus, although NEP plays a substantial role in enkephalinergic neurotransmission, the biochemical adaptations within the opioid system of NEP-deficient mice are of only modest nature.

Opioid receptor modulation of feeding-evoked dopamine release in the rat nucleus accumbens.

Feeding is associated with increases in the activity of the mesolimbic dopamine (DA) system which originates in the ventral tegmental area (VTA) and projects heavily to the nucleus accumbens. The present study used in vivo brain microdialysis to assess the contribution of opioid receptors in feeding-evoked DA release in the nucleus accumbens. Feeding in 18 h food-deprived rats increased DA release by about 50% above baseline. Systemic injection of the opioid receptor antagonist naltrexone (1 mg/kg, s.c.) blocked the effect of feeding on DA release and reduced the amount of food consumed. Unilateral application of naltrexone (100 microM) in the VTA via a microdialysis probe failed to affect the DA response to feeding, the amount of food consumed, or the latency to eat. In contrast, intra-VTA naltrexone significantly reduced the effect of systemic heroin (0.5 mg/kg, s.c.) on accumbal DA release. These results indicate that: (1) opioid receptor activation is a component of the neural substrates of deprivation-induced feeding: (2) opioid receptors in the VTA do not contribute significantly to feeding-associated increases in DA release in the nucleus accumbens; and (3) heroin-induced increases in accumbal DA release are mediated, at least in part, by opioid receptors in the VTA.

Stereospecific regulation of [3H]inositol monophosphate accumulation by calcium channel drugs from all three main chemical classes.

Depolarization of [3H]inositol-prelabelled rat cortical slices through the elevation of extracellular K+ levels leads to increased accumulation of [3H]inositol phosphates. In the presence of 18 mM K+, Ca2+ channel activators selectively stimulated the formation of [3H]inositol monophosphate ([3H]IP1) whereas Ca2+ channel blockers were inhibitory. Blockade of the Na+ channel by 1 microM tetrodotoxin had no effect but chelation of extracellular Ca2+ abolished the response. The enantiomers of the benzoxadiazol 1,4-dihydropyridine 202-791 showed opposite stereospecific regulation of [3H]IP1 formation: (+)-(S)-202-791 stimulated (252%; ED50: 88 nM), whereas (-)-(R)-202-791 inhibited (65% inhibition, ED50: 602 nM). The (-) enantiomer of Bay K 8644 was a potent [3H]IP1 stimulator (258%; ED50: 82 nM). While (+)-Bay K 8644 was inactive in the presence of 18 mM K+, it completely inhibited the (-)-Bay K 8644-induced stimulation with a Ki of 103 nM. Representatives of the other two main classes of Ca2+ channel blockers (phenylalkylamines and benzothiazepines) inhibited K+ depolarization-induced and (-)-Bay K 8644 enhanced [3H]IP1 formation in a dose-dependent, stereospecific manner. The results show that Ca2+ channel blockers are efficient modulators of depolarization-induced and Ca2+ channel activator-induced [3H]inositol monophosphate formation in brain, and demonstrate the functional coupling of three distinct drug receptor sites on neuronal Ca2+ channels.

The mitochondrial high-capacity low-affinity (+/-)-[3H]nitrendipine binding site is regulated by nucleotides.

The high-capacity, low-affinity (+/-)-[3H]nitrendipine binding site in the inner mitochondrial membrane from guinea-pig heart is regulated by purine and pyrimidine nucleotides. The rank order in (+/-)-[3H]nitrendipine binding inhibition assays (with decreasing potency) was: ATP (IC50 11.8 microM) = adenosine 5'-O-(2-thiotriphosphate (ATP gamma S) greater than 5'-adenylylimidodiphosphate (AppNHp) greater than ADP much greater than GTP = ITP = CTP greater than UTP greater than guanosine 5'-tetraphosphate (GT4P) greater than guanosine 5'-O-(2-thiotriphosphate) (GTP gamma S) greater than 5'-guanylylimidodiphosphate (GppNHp) greater than IDP greater than CDP greater than GDP. There was no effect of AMP, adenosine 3':5'-cyclic monophosphate (cAMP), adenosine, UDP, NAD, and NADP. The ATP effect was fully reversible upon wash-out. Adenine nucleotides and analogs had a (+/-)-[3H]nitrendipine binding inhibition profile in mitochondrial membranes from guinea-pig liver or kidney similar to that obtained in heart mitochondrial membranes. In heart mitochondria, 0.3 mM ATP decreased the Bmax from 1.69 +/- 0.04 nmol/mg protein to 0.73 +/- 0.24 nmol/mg protein whilst it decreased the KD only moderately, from 521 +/- 50 to 352 +/- 43 nM, in equilibrium saturation studies. In kinetic studies, ATP slowed down the dissociation rate of the (+/-)-[3H]nitrendipine binding site complex from 0.016 +/- 0.004 to 0.0042 +/- 0.0002 min-1 but it also decreased the association rate constant from 1.52 +/- 0.15 to 0.41 +/- 0.28 10(4).M-1.min-1, yielding a kinetically determined KD (1024 nM) identical to the control KD (1053 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoselective binding of niguldipine enantiomers to alpha 1A-adrenoceptors labeled with [3H]5-methyl-urapidil.

[3H]5-Methyl-urapidil, a potent antihypertensive derivative of urapidil, binds to alpha 1A-adrenoceptors in rat brain cortex membranes with a dissociation constant (KD) of 0.89 nM and a Bmax of 116 fmol/mg protein. The ligand does not bind to purified liver cell membranes (alpha 1B-adrenoceptors). [3H]5-Methyl-urapidil also labels 5-HT1A receptors in brain membranes (KD: 0.84 nM and Bmax: 235 fmol/mg protein). (+/-)-Niguldipine, a novel 1,4-dihydropyridine with Ca2+-antagonistic as well as alpha 1A-adrenoceptor blocking properties, is a competitive inhibitor of [3H]5-methyl-urapidil binding to alpha 1A-adrenoceptors. In contrast to those for prazosin, the Ki values for niguldipine were highly dependent on the membrane protein concentration, indicating partitioning of niguldipine into hydrophobic compartments unavailable for alpha-adrenoceptor interaction. The extrapolated, 'true' Ki values were as follows: (+/-)-niguldipine: 0.298 nM, (-)-niguldipine: 3.12 nM, (+)-niguldipine: 0.145 nM.