Enhancement of Solubility and Bioavailability of β-Lapachone Using Cyclodextrin Inclusion Complexes

Purpose. To explore the use of cyclodextrins (CD) to form inclusion complexes with -lapachone (-lap) to overcome solubility and bioavailability problems previously noted with this drug. Methods. Inclusion complexes between -lap and four cyclodextrins (-, -, -, and HP-CD) in aqueous solution were investigated by phase solubility studies, fluorescence, and ¹H-NMR spectroscopy. Biologic activity and bioavailability of -lap inclusion complexes were investigated by in vitro cytotoxicity studies with MCF-7 cells and by in vivo lethality studies with C57Blk/6 mice (18-20 g). Results. Phase solubility studies showed that -lap solubility increased in a linear fashion as a function of -, -, or HP-CD concentrations but not -CD. Maximum solubility of -lap was achieved at 16.0 mg/ml or 66.0 mM with HP-CD. Fluorescence and ¹H-NMR spectroscopy proved the formation of 1:1 inclusion complexes between -CD and HP-CD with -lap. Cytotoxicity assays with MCF-7 cells showed similar biologic activities of -lap in -CD or HP-CD inclusion complexes (TD₅₀ = 2.1 M). Animal studies in mice showed that the LD₅₀ value of -lap in an HP-CD inclusion complex is between 50 and 60 mg/kg. Conclusions. Complexation of -lap with HP-CD offers a major improvement in drug solubility and bioavailability.     

Compressed Donut-Shaped Tablets with Zero-Order Release Kinetics

Purpose. Simple uncoated compressed tablets with a central hole (donut-shape) are proposed to provide a constant drug release over a long period of time (>20 hrs). The effect of hole size and drug solubility on the release kinetics is investigated. Methods. The donut-shaped polyethylene oxide (PEO, Mw = 4 × l0⁶) tablets (600 mg and 12 mm diameter) are bored with a drill bit (3/32, 7/64, 1/8, and 5/32). Results. The release of theophylline from the donut-shaped tablets is zero order (80 – 90% release) before rapidly decreasing. As the hole size is increased from 7/64 to 5/32, the release rate increases and the release time is shortened. However, the release of theophylline from the donut-shaped tablet with a hole size of 3/32 follows the same anomalous release profile from a tablet without a hole. As drug solubility increases, the duration of linear drug release is shortened to 65 – 70% release followed by a severe tailing at the later stage of the release. Conclusions. Donut-shaped PEO tablets with a hole provide zero-order release kinetics because the effect of the releasing surface area on the release kinetics is reduced.     

Understanding and Predicting Drug Delivery from Hydrophilic Matrix Tablets Using the “Sequential Layer” Model

Purpose. The objectives of this work were (i) to study and understand the physicochemical phenomena which are involved in the swelling and drug release from hydrophilic matrix tablets using the sequential layer model; and (ii) to predict the effect of the initial radius, height and size of the tablets on the resulting drug release profiles. Methods. Tablets were prepared by direct compression, using hydroxypropyl methylcellulose (HPMC) grades with different average molecular weights as matrix-forming polymers. The in vitro release of chlorpheniramine maleate, propranolol HCl, acetaminophen, theophylline and diclofenac sodium was studied in phosphate buffer (pH 7.4) and 0.1 M HCl, respectively. The initial drug loading varied from 1 to 70%, while the radius and height of the tablets varied from 1 to 8 mm. Results. The sequential layer model considers water and drug diffusion with non-constant diffusivities and moving boundary conditions, non-homogeneous polymer swelling, drug dissolution, and polymer dissolution. We showed that this model was able to predict the resulting drug release kinetics accurately in all cases. Conclusions. The sequential layer model can be used to elucidate the swelling and drug release behavior from hydrophilic matrix tablets and to simulate the effect of the device geometry on the drug release patterns. Hence, it can facilitate the development of new pharmaceutical products.     

Biodegradable PLGA Microspheres Loaded with Ganciclovir for Intraocular Administration. Encapsulation Technique,in Vitro Release Profiles,and Sterilization Process

Purpose. The purpose of this work was to obtain a sterilized formulation consisting of biodegradable microspheres of poly (DL-lactide-co-glycolide) (PLGA) for intraocular sustained release of ganciclovir. Methods. Microspheres were prepared using a dispersion of ganciclovir in fluorosilicone oil (FSiO) that was further dispersed in an acetone solution of PLGA [50/50 and inherent viscosity 0.41 dl/g], and emulsified in silicone oil with a surfactant. Once prepared, the formulation was exposed with an effective radiation dose of 2.5 megarads. The release rate data of ganciclovir from the sterilized and nonsterilized batches were compared using the similarity factor (f₂). Results. The dispersion of the drug in FSiO contributed to achieving a drug payload of up to 95% of the theoretical in the 300-500 m microspheres. Ten mg released ganciclovir in vitroat 1.3 g/h for the first 21 days, but decreased to 0.2 g/h from day 25 until the end of the release study (42 days). No significant differences in the amounts of encapsulated drug (=0.05) were observed between the sterilized and nonsterilized microspheres. Furthermore, dissolution profiles of formulations behaved similarly before and after gamma radiation exposure. Conclusions. The technique of microsphere preparation described resulted in high ganciclovir loading (95%) and prolonged drug release. The ganciclovir formulation behaved similarly before and after the sterilization process.     

Stabilizing and Solubilizing Effects of Sulfobutyl Ether β-Cyclodextrin on Prostaglandin E1 Analogue

Purpose. Parent cyclodextrins are known to accelerate the degradations such as dehydration and isomerization of E-type prostaglandins in neutral and alkaline solutions. The objective of this study was to attempt the stabilization and solubilization of E₁-type prostaglandin analogue in aqueous solution by biocompatible cyclodextrin derivatives. Methods. The interaction of an E₁-type prostaglandin, methyl 7-[(1R,2R,3R)-3-hydroxy-2-[(E)-(3S)-3-hydroxy-4-(m-methoxymethylphenyl)1-butenyl]-5-oxocyclopentyl]-5-thiaheptanoate (MEester) with cyclodextrins (CyDs) was studied by spectroscopies and the solubility method. The degradation of MEester was monitored by high-performance liquid chromatography. Results. ¹H-nuclear magnetic resonance spectroscopic studies indicated that MEester forms 1:1 inclusion complexes with -, -, and -CyDs in solutions, where -CyD interacts with the -side chain containing methyl ester moiety of the drug, whereas - and -CyDs preferentially include around the five-membered ring and both side chains of the drug. Parent -CyD and hydrophilic derivatives, such as 2-hydoxypropyl-- and --CyDs, sulfobutyl ether -CyD (SBE--CyD) and maltosyl -CyD showed higher solubilizing abilities against MEester over parent - and -CyDs. SBE--CyD and 2,6-dimethyl--CyD (DM--CyD) significantly decelerated the degradation of MEester, particularly the base-catalyzed dehydration, in neutral and alkaline solutions, whereas other CyDs accelerated the degradation. The acid-catalyzed degradation of MEester (pH < 3) was decelerated by the addition of CyDs, especially -CyD. Conclusions. SBE--CyD with low hemolytic activity and low toxicity is useful as a pharmaceutical carrier for the preparation of injectable MEester, because of its higher stabilizing and solubilizing effects on MEester. Furthermore, SBE--CyD can be useful as a stabilizing agent for drugs, that are subject to base-catalyzed degradations, probably because of the electric repulsion between anionic charges of the sulfobutyl moiety and catalytic anionic species such as hydroxide ion.     

Inhibition by the putative potassium channel opener pinacidil of the electrically-evoked release of endogenous dopamine and noradrenaline in the rat vas deferens

Summary The effect of pinacidil on the release of endogenous noradrenaline and dopamine from the sympathetic innervation of the rat vas deferens was examined. Amine release was evoked by electrical stimulation (1, 2, 5 and 10 Hz) or by depolarization with high potassium (75 mmol/l) in the medium. Dopamine and noradrenaline were measured by means of high pressure liquid chromatography with electrochemical detection.Pinacidil (1, 5, 10 and 50 mol/l) produced a concentration-dependent inhibition of the electrically stimulated (2 Hz) overflow of noradrenaline and dopamine. Only pinacidil 50 mol/l increased the spontaneous loss of dopamine and noradrenaline. The inhibitory effects of pinacidil (5 mol/l) on amine overflow were also observed at other frequencies of stimulation (1, 5 and 10 Hz). The magnitude of the inhibitory effect on noradrenaline release was approximately the same at all frequencies (63% to 56% reduction); for dopamine, the higher the frequency of stimulation, the greater the inhibitory effect of pinacidil (up to 73% reduction). When the preparations were continuously stimulated for 70 min at 2 Hz, pinacidil (5 mol/l) reduced the overflow of dopamine and noradrenaline during the first 40 or 30 min of stimulation only. The addition of phentolamine (1 mol/l) to the perifusion medium slightly reduced the inhibitory effect of pinacidil on amine overflow, but the inhibition by pinacidil remained statistically significant. Tetraethylammonium (10 mmol/l) completely abolished the inhibitory effect of pinacidil (10 mol/l). Pinacidil (5 mol/l) did not reduce the potassium-evoked release of the amines.The results demonstrate that pinacidil impairs transmitter release from the sympathetic innervation of the rat vas deferens, probably as a consequence of the opening of potassium channels. Send offprint request to P. Soares-da-Silva at the above adress     

Inactivation and Aggregation of β-Galactosidase in Lyophilized Formulation Described by Kohlrausch-Williams-Watts Stretched Exponential Function

Purpose. To examine whether the empirical Kohlrausch-Williams-Watts (KWW) equation is applicable not only to protein aggregation but also to protein denaturation in lyophilized formulations. Lyophilized -galactosidase (-GA) formulations containing polyvinylalcohol and methylcellulose were used as model formulations. The possibility of predicting storage stability based on the temperature dependence of the estimated parameters of inactivation/aggregation—time constant () and its distribution () is discussed. Methods. Protein aggregation in lyophilized -GA formulations at 10-70°C and 6-43% relative humidity was determined as a function of time by size exclusion chromatography. Enzyme activity was also determined using 2-nitrophenyl--D-galactopyranoside as a substrate. Results. Inactivation and aggregation of -GA were describable with the empirical KWW equation, regardless of whether the temperature was above or below the NMR relaxation-based critical mobility temperature (T_mc) or whether protein molecules with different degrees of deformation resulting from stresses during lyophilization exist in the formulation. The estimated parameter for protein aggregation decreased rapidly as temperature increased beyond T_mc because the mobility of polymer molecules increased in the initial stages of glass transition. The time required for 10% enzyme to aggregate (t₉₀) calculated from the and parameters exhibited a change in temperature dependence gradient near T_mc. In contrast, t₉₀ for protein inactivation exhibited temperature dependence patterns varying with the excipients. Conclusions. The t₉₀ calculated from the estimated and parameters was found to be a useful parameter for evaluating the stability of lyophilized -GA formulations. The prediction of t₉₀ by extrapolation was possible in the temperature range in which did not rapidly vary with temperature.     

A New Mathematical Model Quantifying Drug Release from Bioerodible Microparticles Using Monte Carlo Simulations

Purpose. The major objectives of this study were to 1) develop a new mathematical model describing all phases of drug release from bioerodible microparticles; 2) evaluate the validity of the theory with experimental data; and 3) use the model to elucidate the release mechanisms in poly(lactide-co-glycolide acid)-based microspheres. Methods. 5-Fluorouracil-loaded microparticles were prepared with an oil-in-water solvent extraction technique and characterized in vitro. Monte Carlo simulations and sets of partial differential equations were used to describe the occurring chemical reactions and physical mass transport phenomena during drug release. Results. The new mathematical model considers drug dissolution, diffusion with nonconstant diffusivities and moving boundary conditions, polymer degradation/erosion, time-dependent system porosities, and the three-dimensional geometry of the devices. In contrast with previous theories, this model is able to describe the observed drug release kinetics accurately over the entire period of time, including 1) initial burst effects; 2) subsequent, approximately zero-order drug release phases; and 3) second rapid drug release phases. Important information, such as the evolution of the drug concentration profiles within the microparticles, can be calculated. Conclusions. A new, mechanistic mathematical model was developed that allows further insight into the release mechanisms in bioerodible microparticles.     

β2-Adrenergic Receptor Genotype-Related Changes in cAMP Levels in Peripheral Blood Mononuclear Cells After Multiple-Dose Oral Procaterol

Purpose. To evaluate the ₂-adrenergic receptor (₂AR) genotype frequency in the Japanese population and the relationship between ₂AR genotype at amino acid position 16 (₂AR-16) and desensitization to ₂-agonist ex vivo. Methods. The ₂AR genotypes at amino acid positions 16, 27, and 164 of 92 healthy Japanese subjects were determined by polymerase chain reaction-restriction fragment-length polymorphism. The relationship between the ₂AR-16 genotype and the desensitization to ₂-agonist was examined in 10 male subjects ex vivo. Procaterol tablet (HCl salt, 50g, Meptin®) was given orally for 5 days, and peripheral blood was obtained before and after 5 days of consecutive medications followed by the assessment of the intracellular cAMP levels in peripheral blood mononuclear cells after incubation with or without procaterol hydrochloride (0-1000 ng/mL). Results. Allele frequency was Arg16:Gly16 = 46%:54%, Gln27:Glu27 = 92%:8%, and Thr164:Ile164 = 100%:0%, respectively. The cAMP levels were increased by incubation with procaterol hydrochloride, and the increase was suppressed after 5 days of consecutive medications. The suppression was more significant in the homozygote for Gly16 than the homozygote for Arg16. Conclusions. The desensitization to ₂-agonist was associated more frequently with the mutation at ₂AR-16 (Gly16).     

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P_2Y-purinoceptor, which is activated by adenosine 5-O-(2-thiodiphosphate) (ADPßS), and a separate receptor for ,-methylene ATP (,-MeATP). Effects of several as yet incompletely characterized P₂-purinoceptor antagonists at these receptors were examined.The concentration-relaxation curve of ADPßS was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonic acid (XAMR0721; at 1000 M only), pyridoxalphosphate-6-azophenyl-2,5-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 M only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADPßS had slopes <1. The concentration-relaxation curve of ,-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 M). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against ,-MeATP had slopes >1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against ,-MeATP than against ADPßS. 2-Methylthio ATP (MeSATP; 1 M) and ATP (100 M) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides.The selective effect of XAMR0721 against ,-MeATP confirms the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P_2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P_2Y-purinoceptor in turkey erythrocytes, was very weak at the P_2Y-receptor of the taenia, thus supporting the existence of pharmacologic P_2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas deferens, is the most selective P_2X-(versus P_2Y-) purinoceptor antagonist presently known, although its effect on the degradation of nucleotides must be kept in mind.     

Effect of ω-conotoxin GVIA on release of 3H-γ-aminobutyric acid from slices of rat neostriatum

Summary -Conotoxin GVIA (-CT) diminished the potassium-induced in vitro release of ³H--aminobutyric acid (³H-GABA) from slices of rat neostriatum in a manner which depended on the concentration of potassium. -CT (0.1 nmol/l) decreased the release of ³H-GABA induced by 25 mmol/l K⁺ from 11.6% to 6.1% of tissue content, ie. by 48%, while it did not affect the release of ³H-GABA caused by 20 mmol/l K⁺, which was 4.8% of tissue content. However, in the presence of a polyclonal antiserum or cysteamine (600 mol/l), both of which diminish the effects of endogenous somatostatin, 0.1–10 nmol/l -CT decreased the release of ³H-GABA induced by 20 mmoles/l K⁺ by 40%. It is concluded that -CT did not only inhibit GABA-neurones, but had an additional inhibitory effect on somatostatin neurones which are known to depress the release of ³H-GABA. It is further concluded that neuronal interactions, which are possible in brain slice preparations, may impede the interpretation of effects of drugs, especially if agents are used which affect basic mechanisms of transmitter release and thus the release of various transmitters from neurones. Send offprint requests to D. K. Meyer at the above address     

Intradermal Injection of Transforming Growth Factor-β1 Gene Enhances Wound Healing in Genetically Diabetic Mice

Purpose. To evaluate the biologic effect of direct cutaneous TGF-1 gene delivery on impaired wound healing models using genetically diabetic mice. Methods. Diabetic mice (C57BKS.Cg-m +/+ Leprdb female mice) with 1 cm × 1 cm excisional wounds were intradermally injected with 60 g of plasmid DNA encoding TGF-1 gene. The wound closure was measured up to 14 days postwounding. At days 7 and 14 postwounding, sections of skin were taken for hematoxylin and eosin and Masson's trichome staining to examine the morphology and collagen deposition. The cell proliferation and TGF-1 gene expression were studied using immunohistochemical stainings for 5-bromo-2-deoxy-uridine and for TGF-1. Results. A higher cell proliferation rate and a denser and more organized new extracellular matrix were observed in the treated wound site. Complete wound closure was detected as early as 7 days for TGF-1-treated group in comparison with 11-14 days for the untreated, control plasmid DNA- and PBS-treated groups. Conclusion. A single intradermal injection of TGF-1 plasmid DNA was sufficient to enhance wound healing. This approach represents a new strategy that may be applied to the treatment of excisional wounds in human diabetic patients.     

Plasma insulin levels and β-adrenoceptor antagonists

Summary The effect of -adrenoceptor antagonists on the intravenous glucose tolerance test was investigated in conscious dogs. dl-Celiprolol (cardioselective with ISA=intrinsic sympathomimetic activity) 200 and 1000 g/kg i.v., dl-metoprolol (cardio-selective without ISA) 200 and 1000 g/kg i.v., dl-pindolol (non-selective with ISA) 5 and 25 g i.v. and l-bupranolol (non-selective without ISA) 10 and 50 g/kg i.v. were used in the study. The influence of -adrenoceptor antagonists on the plasma glucose and immunoreactive insulin following the intravenous glucose tolerance test were evaluated by calculating the respective areas under the plasma curve.The present investigtion clearly demonstrates the marked difference between the various -adrenoceptor antagonists on heart rate and, especially on metabolic parameters. dl-Metoprolol, a -adrenoceptor antagonist with cardioselectivity and without ISA can be assumed not to alter plasma insulin level and glucose assimilation. l-Bupranolol, a non-selective -adrenoceptor antagonist without ISA reduces plasma insulin level and probably enhances peripheral glucose uptake, resulting in an unchanged glucose tolerance. dl-Celiprolol or dl-pindolol, -adrenoceptor antagonists with ISA, but cardioselective or non-selective enhance both, basal insulin level and insulin level after glucose stimulation but must be assumed to decrease peripheral glucose uptake since here too glucose tolerance was unchanged.     

Effects of GABA,dopamine, and substance P on the release of newly synthesized 3H-5-hydroxytryptamine from rat substantia nigra in vitro

Summary The effects of GABA, substance P and dopamine on the release of newly synthesized ³H-5-HT were investigated, using slices of rat substantia nigra superfused with l-³H-tryptophan in vitro. GABA (50 M) had no inhibitory effect on the potassium-evoked-release of ³H-5-HT. Substance P (50 M) and eledoisin (50 M) stimulated the spontaneous release of ³H-5-HT. This effect seems to be indirect and is possibly mediated by dopaminergic neurones, since the dopamine antagonist drug -flupenthixol (1 M) abolished the substance P-evoked release of 5-HT. Furthermore, it was found that substance P (10 M) stimulated ³H-dopamine release from nigral slices in vitro and the dopaminergic agonist apomorphine (50 M) also stimulated ³H-5-HT release. Substance P may, therefore, activate nigral dopaminergic neurones which then release dopamine from their dendrites. The release of dopamine may in turn stimulate 5-HT release from terminals of the raphe-nigral pathway.     

The selective P2X purinoceptor agonist, β,γ-methylene-L-adenosine 5′-triphosphate,discriminates between smooth muscle and neuronal P2X purinoceptors

The effects of the putative selective P_2X purinoceptor agonist, ,-methylene-l-adenosine 5-triphosphate (me-l-ATP), were determined at rat neuronal and smooth muscle P_2X purinoceptors.Me-l-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 M. In contrast, the archetypal P_2X purinoceptor agonist, , methylene ATP (meATP;1–100 M), produced concentration-related depolarisation responses with a mean EC₅₀ value of 10.8 M. The depolarising effects of meATP were not attenuated by me-l-ATP (100 M). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 M), but not me-l.-ATP (1–300 M), evoked rapid ( < 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, me-d-ATP and meATP competed with high affinity for [³H]Lx meATP binding sites, with mean pIC₅₀ values of 7.7 and 8.3, respectively. However, me-l-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 M (mean pIC₅₀ value 4.1).In prostatic segments of the rat vas deferens, me-l-ATP (1–100 M) and meATP (0.3–100 M) each produced concentration-related contractile responses with mean EC₅₀ values of 17.1 and 3.6 M, respectively. Me-l-ATP (1–10 M) evoked fast inward currents in freshly dispersed vas deferens smooth muscle cells, indicative of an action at ligand-gated ion channels. Binding sites in vas deferens membranes labelled using 1 nM [³H]meATP exhibited high affinity for me-l-ATP, meATP and me-d-ATP with mean PIC₅₀ values of 7.7, 8.4 and 7.3, respectively.These results indicate that me-l-ATP exhibits neither agonist nor antagonist properties at P_2X purinoceptors on rat vagal neurones and possesses only very low affinity for [³H]meATP binding sites in rat brain. In contrast, me-l-ATP is a potent, high affinity agonist at smooth muscle P_2X purinoceptors of the rat vas deferens. This selective agonist action of me-l-ATP suggests that P_2X purinoceptors in smooth muscle and neurones are different and represent distinct P_2X purinoceptor subtypes.     

Prodrugs for Improved Oral β-Estradiol Bioavailability

Prodrugs of -estradiol (1) were prepared with the objective of improving its oral bioavailability. -Estradiol-3-acetylsalicylate (2), -estradiol-3-salicylate (3), and -estradiol-3-anthranilate (4) were synthesized. With these prodrugs the 3-phenolic hydroxy group of estradiol was protected, so that first-pass conjugative metabolism could be reduced. Prodrug hydrolysis rates in dog and human plasma in vitro were determined. Deacetylation of estradiol-3-acetylsalicylate was much more rapid than its hydrolysis to estradiol. In dogs, oral estradiol bioavailability after administration of 2 and 4 was 17-fold and 5-fold higher, respectively, than after oral 1.     

Influence of N-allyl-normetazocine on acetylcholine release from brain slices: involvement of muscarinic receptors

Summary The effects of (±)N-allyl-normetazocine on the release of acetylcholine from different areas of guinea-pig and rat brain were investigated. 1. The drug did not modify the electrically (2 Hz) evoked tritium efflux from guinea-pig cerebral cortex, thalamus and caudate nucleus slices, preloaded with ³H-choline 0.1 mol/l and superfused with Krebs solution containing hemicholinium-3 10 mol/l. 2. (±)N-allyl-normetazocine 10 mol/l. enhanced the evoked ³H efflux from guinea-pig brain slices superfused with Krebs solution containing physostigmine 30 mol/l or oxotremorine 0.3 -1 gmol/l; the effect was naloxone-insensitive and was abolished by atropine 0.15 mol/l, but not by pirenzepine 1 mol/l. 3. (±)N-allyl-normetazocine 5 mol/l enhanced the electrically evoked release of endogenous acetylcholine as well, in a naloxone-insensitive way. 4. Both (±) and (+)N-allyl-normetazocine were without effect on ³H efflux from rat caudate nucleus slices electrically stimulated at 0.2 Hz frequency, after preloading with ³H-choline and during superfusion with hemicholinium-3. 5. The results are discussed in view of the antimuscarinic properties of the drug. Send offprint requests to A. Siniscalchi     

β 1- andβ 2-adrenoceptor binding and functional response in right and left atria of rat heart

Summary The properties of ₁- and ₂-adrenoceptors in right and left atria of rat heart, and their roles in mediating chronotropic and inotropic responses to-adrenoceptor agonists were examined. [¹²⁵I](-)-pindolol (¹²⁵IPIN) bound saturably and specifically to a single class of high affinity sites in homogenates of both right and left atria. Thek ₁'s for association in right and left atria were 6.5×10⁹ l/mol-min and 2.3×10⁹ l/mol-min respectively, while thek _–1's for dissociation were 0.20 min^–1 and 0.17 min^–1. The kinetically determinedK _D's were 75 pmol/l in right and 30 pmol/l in left atria and were similar to the equilibriumK _D's determined from Scatchard analysis of saturation isotherms of specific¹²⁵IPIN binding. Inhibition of¹²⁵IPIN binding by-adrenoceptor antagonists was stereoselective and the order of potency was timolol > 1-propranolol > d-propranolol > sotalol. Inhibition by ₁- and ₂-adrenoceptor subtype selective antagonists yielded flat displacement curves with low Hill coefficients. Nonlinear regression analysis of displacement by ₁-selective (practolol, atenolol and metoprolol) and ₂-selective (ICI 118,551) antagonists gave estimates of the proportion of ₁- and ₂-adrenoceptors present in rat atria. Right atria contained 67±4.2% ₂-adrenoceptors and 33±4.2% ₂-adrenoceptor, while left atria contained 67±2.8% ₁- and 33±2.8% ₂-adrenoceptors. Increases in the rate of spontaneously beating right atria and the force of electrically driven left atria caused by-adrenoceptor agonists were also measured. pA₂ values for non-subtype selective-adrenoceptor antagonists in inhibiting isoprenaline-induced increases in rate and force were highly correlated withK _D values determined for specific¹²⁵IPIN binding. pA₂ values for ₁- and ₂-selective antagonists in inhibiting isoprenaline-induced increases in rate and force correlated well with the pK _D values of these drugs in binding to ₁-adrenoceptors, but not with the pK _D values in binding to ₂-adrenoceptors. Dose-response curves for stimulation of both rate and force by the ₂-selective agonists procaterol and zinterol were shifted to a much greater extent by selective blockade of ₁-adrenoceptors with metoprolol than by selective blockade of ₂-adrenoceptors with ICI 118,551, suggesting that these compounds caused their effects by activating ₁-adrenoceptors. These results suggest that ₁- and ₂-adrenoceptors coexist in both left and right atria of rat heart in approximately a 21 ratio, however only ₁-adrenoceptors mediate the chronotropic and inotropic effects of-adrenoceptor agonists.Supported by a grant from the American Heart Association — Georgia Affiliate     

The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties

Summary Four groups of narcotic analgesic drugs have been assessed for their opiate activities by using three binding assays and three pharmacological bioassays. In the binding assays, their inhibition constants (K _I, nM) were determined against the binding of the -ligand, [³H]-[d-Ala ² ,MePhe ⁴ , Gly-ol⁵]enkephalin, of the -ligand, [³H]-[d-Ala ² ,d-Leu ⁵]enkephalin and of the -ligand, [³H]-(±)-ethylketazocine after suppression of - and -binding by 100 nM of the unlabelled -ligand and 100 nM of the unlabelled -ligand. The pharmacological agonist or antagonist activities were assayed on the guinea-pig ileum, mouse vas deferens and rat vas deferens.The first group of compounds were pure agonists in all three pharmacological bioassays. The majority of the compounds showed preference to -binding but phenazocine and particularly etorphine had also high affinities to the - and -binding sites.The second group consisted of N-allyl and N-cyclopropylmethyl homologues of the morphine, 3-hydroxymorphinan and normetazocine series which had agonist and antagonist activities in the guinea-pig ileum and mouse vas deferens but were pure antagonists in the rat vas deferens. In the binding assays, -binding and -binding were prominent.The third group was made up by the ketazocine-like compounds which in the guinea-pig ileum and mouse vas deferens were pure agonists and in the rat vas deferens pure antagonists. The binding spectrum showed particularly high binding to the -binding site.The fourth group was the antagonists which were devoid of agonist activity with the exception of diprenorphine and Mr 2266 which had retained some agonism. The binding spectrum showed considerable variation, naloxone in low concentration being a selective -antagonist, Mr 2266 having high affinities to the - and -binding sites and diprenorphine having considerable affinities to the -, - and -binding sites.Since each of the four groups of compounds, whether pure agonists, agonist-antagonists, ketazocine-like drugs or pure antagonists, shows independent varittions in the affinities to the - and -binding sites, their different pharmacological behaviour cannot be solely due to difference in the binding spectra.     

Influence of Hydrodynamics and Particle Size on the Absorption of Felodipine in Labradors

Purpose. To study the influence of GI hydrodynamics and drug particle size on felodipine absorption in the dog. Methods. Labradors fistulated at midjejunum were used to selectively study the influence of hydrodynamics and particle size on the in vivodissolution and absorption of the poorly soluble, lipophilic drug felodipine. A combination of infusion and oral administration of either normal saline or a 5% glucose solution was used to maintain fasted and establish fed state motility patterns, respectively. The absorption characteristics of both a micronized (8 m) and a coarse fraction (125 m) of felodipine were subsequently studied under these two motility patterns. Results. A reduction in particle size led up to an approximate 22-fold increase in maximum plasma concentration and up to an approximate 14-fold increase in area under the curve, with a commensurate decrease in the time at which the maximum plasma concentration occurred. Although the absorption of felodipine from the solution and micronized suspension was not influenced by a change in the hydrodynamics, felodipine was absorbed from the coarse suspension almost twice as well in the fed state as under fasted conditions. Conclusions. Absorption from coarse suspensions of felodipine was sensitive to luminal hydrodynamics, whereas micronized suspensions were not. However, the particle size seems to have a much more important influence on the bioavailability of felodipine than the hydrodynamics per se.