Nasal Insulin Delivery with Dimethyl-β-Cyclodextrin as an Absorption Enhancer in Rabbits: Powder More Effective than Liquid Formulations

The nasal absorption of insulin using dimethyl--cyclodextrin (DMCD) as an absorption enhancer in rabbits was studied. The nasal administration of insulin/DMCD liquid formulations did not result in significant changes in serum insulin and blood glucose concentrations. In contrast, previous experiments in rats showed that the addition of DMCD to the liquid nasal formulation resulted in an almost-complete insulin absorption, with a concomitant strong hy-poglycaemic response. Apparently, the effect of the cyclodextrin derivative on insulin absorption differs between animal species following nasal delivery of insulin/DMCD solutions. On the other hand, nasal administration of the lyophilized insulin/DMCD powder dosage form in rabbits resulted in increased serum insulin concentrations, and a maximum decrease in blood glucose of about 50%. The absolute bioavailability of the nasally administered insulin/DMCD powder was 13 ± 4%, compared to 1 ± 1% for both an insulin/ DMCD liquid and an insulin/lactose powder formulation. It is concluded that insulin powder formulations with DMCD as an absorption enhancer are much more effective than liquid formulations.     

Differential Effects of Sulfate and Sulfobutyl Ether of β-Cyclodextrin on Erythrocyte Membranes in Vitro

The hemolytic activity of -cyclodextrin (-CyD) on rabbit erythrocytes was reduced by the introduction of negatively-charged groups onto the hydroxyls of -CyD; the membrane disrupting abilities decreased in the order of -CyD > 2-hydroxypropyl--CyD (HP--CyD) > sulfobutyl--CyD (SB--CyD) >> -CyD sulfate (S--CyD). Under pre-hemolytic concentrations, both -CyD and SB--CyD induced shape changes of membrane invagination on the erythrocytes. In sharp contrast, S--CyD showed biphasic effect on the shape of the erythrocytes; i.e. the crenation at relatively low concentrations and the invagination at higher concentrations. The S--CyD-induced membrane crenation arose from a direct action on the membranes rather than cell metabolism-mediated effects. Unlike -CyD, S--CyD was found to bind to the erythrocytes and may be confined to the outer surface of the membrane bilayer, which may expand the exterior layer relative to the cytoplasmic half, thereby inducing the cells to crenate. On the other hand, the membrane invagination mediated by the three - CyDs was initiated by extracting specific membrane lipids from the cells, depending upon their inclusion abilities, subsequently leading to the lysis of the cells. These results indicate that SB--CyD and S--CyD interact with the erythrocyte membranes in a differential manner and possess lower membrane disrupting abilities than the parent -CyD and HP--CyD.     

The metabolism of the amyloid precursor protein and its relevance to Alzheimer's disease

Summary The pathology of Alzheimer's disease is primarily characterized by the deposition of -amyloid/A peptide as the major component of senile or neuritic plaques. The A peptide is produced as a result of proteolytic cleavage of the transmembrane protein precursor, APP, during its normal cellular metabolism. The free amino terminus of the A peptide is generated by an endopeptidic cleavage between Met⁶⁷¹-Asp⁶⁷² by a protease termed -secretase. Increased cleavage at this site takes place in a rare, inherited double mutation (Lys⁶⁷⁰-Met⁶⁷¹ to Asn⁶⁷⁰-Leu⁶⁷¹), leading to increased A production and consequent development of Alzheimer's disease on an accelerated time scale in the affected individuals, underscoring the pathological importance of -secretase activity. Cellular studies provide direct evidence that inhibition of -secretase activity would appear to be effective in inhibiting A production as a rational approach to developing therapeutics for the disease.     

The Pharmacokinetics of β-Cyclodextrin and Hydroxypropyl-β-cyclodextrin in the Rat

Hydroxypropyl--cyclodextrin was analyzed by HPLC using postcolumn complexation with phenolphthalein and negative colorimetric detection, with a detection limit of 20 µg/ml. The pharmacokinetics of -cyclodextrin and of hydroxypropyl--cyclodextrin were studied after intravenous administration to permanently cannulated rats. The pharmacokinetic behavior of both cyclodextrins was similar to that of inulin, showing rapid distribution over extracellular fluids. Elimination occurred through glomerular filtration. When a dose of 200 mg/kg -cyclodextrin was administered the elimination rate was decreased, probably as a result of nephrotoxicity of -cyclodextrin. Within 24 hr after administration most of the cyclodextrin dose was recovered unchanged in urine. After oral administration, only insignificant amounts of intact -cyclodextrin were absorbed from the gastrointestinal tract.     

Enhancement of the Antiinflammatory Effect of Ethyl 4-Biphenylyl Acetate in Ointment by β-Cyclodextrin Derivatives: Increased Absorption and Localized Activation of the Prodrug in Rats

Ethyl 4-biphenylyl acetate (EBA) is a prodrug of the antiinflammatory 4-biphenylyl acetic acid (BPAA). The inclusion complexes of EBA with -cyclodextrin (-CyD), heptakis(2,6-di-O-methyl)--cyclodextrin (DM--CyD), and 2-hydroxypropyl--cyclodextrin (HP--CyD) at a molar ratio of 1:2 (EBA:cyclodextrin) were prepared and used to make hydrophilic antiinflammatory ointments. The in vitro release of EBA from the ointments was enhanced by complexation in the order of -CyD < DM--CyD HP--CyD. The improvement correlated with the improved solubility and not with the decreased diffusibility observed to occur upon the complexation of EBA. In vivo the complexation with cyclodextrin derivatives increased both the release of EBA from the vehicle and its conversion in the underlying tissue to BPAA, but the total of EBA and BPAA in the tissue was decreased. In vitro studies confirmed that the effects of cyclodextrin derivatives on the conversion were exerted indirectly. The combination of the enhanced release and of the enhanced prodrug hydrolysis by esterases in the site where the antiinflammatory action is required resulted in increased therapeutic effects. In the model of carrageenan-induced acute edema in rat paw, the complexation improved the therapeutic effects over those of EBA alone in the order of -CyD < DM--CyD < HP--CyD. HP--CyD may be a particularly useful cyclodextrin derivative since it improves the topical availability and does not irritate tissues.     

Oral bioavailability of CHF1194, an inclusion complex of piroxicam and β-cyclodextrin,in healthy subjects under single dose and steady-state conditions

CHF1194 is an inclusion complex of -cyclodextrin with the nonsteroidal anti-inflammatory drug piroxicam. In man, -cyclodextrin acts as a carrier of piroxicam. As the inclusion complex of piroxicam--cyclodextrin is wettable and more water soluble, the absorption rate of the drug is increased whilst its other pharmacokinetic characteristics remain unchanged.The aim of the present study in 12 healthy subjects was to compare the oral bioavailability of 20 mg piroxicam in a CHF1194 tablet and a plain piroxicam capsule after a single dose and after two weeks of once daily administration, and also to assess the plasma levels and urinary excretion of -cyclodextrin after CHF1194 administration. The two treatments were administered in cross-over fashion, separated by a wash-out period of three weeks. Piroxicam, 5-hydroxypiroxicam and -cyclodextrin were monitored in plasma and urine for 120 h after the first and last doses.Clinical tolerance was excellent and no adverse event occurred during either phase of the study. The extent of absorption of piroxicam from the CHF1194 tablet after the single dose was equivalent to that after the plain piroxicam capsule, within confidence limits of less than 80–125%. After repeated dosing, CHF1194 yielded the same steady-state systemic concentrations of piroxicam and 5-hydroxypiroxicam as the reference capsule, and similar excretion pattern of the metabolite. After both single and multiple dosing, piroxicam was absorbed more rapidly after CHF1194, an expected consequence of the complexation of piroxicam with -cyclodextrin. This may be of therapeutic interest as it might accelerate the onset of pain relief.The pharmacokinetics of piroxicam was linear after the doses used here, suggesting that long term treatment with CHF1194 should not require any change in dosing regimen. Even after 14 days of repeated administration of CHF1194, -cyclodextrin could not be detected in plasma or urine, suggesting that in man the unchanged oligosaccharide was absorbed to a very small extent.     

Effect of Cyclodextrins on Protein Binding of Drugs: The Diflunisal/Hydroxypropyl-β-Cyclodextrin Model Case

The binding of diflunisal to hydroxypropyl--cyclodextrin (HPCD), bovine serum albumin (BSA), human serum albumin (HSA), normal human plasma, and mixed solutions of HPCD/ protein was studied at 25°C, pH 7.4, by potentiometry using an electrode selective to diflunisal. The experimental data for diflunisal/ HPCD fit well to the 1:1 binding model. The binding of diflunisal with each of the studied proteins was compatible with a model having two independent classes of binding sites. The binding of diflunisal in mixed solutions HPCD/BSA, HPCD/HSA, and HPCD/plasma increased considerably when the HPCD concentration was increased. The binding behavior of the two biomolecules in the mixed solutions of HPCD/BSA or HPCD/ HSA was described with an additive model formulated on the basis of the estimates of the binding parameters of diflunisal derived from the separate experiments with each one of the binders tested. The lower than theoretical binding observed in HPCD/plasma solutions was ascribed to the competitive displacement of diflunisal from the HPCD cavity by plasma cholesterol.     

Cyclodextrins as Mucosal Absorption Promoters of Insulin. II. Effects of β-Cyclodextrin Derivatives on α-Chymotryptic Degradation and Enteral Absorption of Insulin in Rats

The relative effectiveness of two -cyclodextrin derivatives, i.e., dimethyl--cyclodextrin (DMCD) and hydroxypropyl--cyclodextrin (HPCD), in enhancing enteral absorption of insulin was evaluated in the lower jejunal/upper ileal segments of the rat by means of an in situ closed loop method. The incorporation of 10% (w/v) DMCD to a 0.5 mg/ml porcine-zinc insulin solution dramatically increased insulin bioavailability from a negligible value (~0.06%) to 5.63%, when administered enterally at a dose of 20 U/kg. However, addition of 10% (w/v) HPCD did not improve enteral insulin uptake significantly with a bioavailability of only 0.07%. Similarly, the pharmacodynamic relative efficacy values obtained after the enteral administration of 20 U/kg insulin, 20 U/kg insulin with 10% HPCD, and 20 U/kg insulin with 10% DMCD were 0.24%, 0.26%, and 1.75%, respectively. Biodegradation studies of 0.5 mg/ml insulin hexamers by 0.5 µM -chymotrypsin revealed no inhibitory effect on the enzymatic activity by the two cyclodextrins. On the contrary, the apparent first-order rate constant increased significantly in the presence of 10% DMCD, suggesting insulin oligomer dissociation by DMCD. Histopathological examination of the rat intestine was performed to detect tissue damage following enteral administration of the -cyclodextrin derivatives. Light microscopic inspection indicated no observable tissue damage, thereby arguing direct membrane fluidization as the primary mechanism for enhanced insulin uptake. This study indicates the feasibility of using cyclodextrins as mucosal absorption promoters of proteins and peptide drugs.     

Comparative behavioral characterization of the neuroactive steroids 3α-OH,5α-pregnan-20-one and 3α-OH,5β-pregnan-20-one in rodents

Pregnan steroids have been shown to possess anesthetic, hypnotic, anticonvulsant and anxiolytic properties. In this study, two endogenous neuroactive steroid isomers, 3-hydroxy-5-pregnan-20-one (3,5-P) and 3-hydroxy-5-pregnan-20-one 3,5-P), were studied for differences in their pharmacological properties using behavioral assays. 3,5-P and 3,5-P were similar in their potencies and efficacies in blocking pentylenetetrazol-induced seizures in mice (ED₅₀: 3,5-P=2.8 mg/kg and 3,5-P=3.0 mg/kg). Similarly, both neuroactive steroids produced roto-rod deficits within the same range of potency (TD₅₀:3,5-P=18.8 mg/kg and 3,5-P=21.2 mg/kg). However, in animal models of anxiety, subtle differences were observed between the two isomers. In both the light/dark transition test and elevated plus-maze, 3,5-P was more efficacious than 3,5-P, though both compounds had similar potencies. In the Geller-Seifter test, 3,5-P was more potent and efficacious than 3,5-P. Neither compound had significant effects on unpunished responding within the dose range tested. Both compounds produced similar biphasic curves in the locomotor test. All together, the data indicate that 3,5-P and 3,5-P have similar anticonvulsant activity, but the 5-isomer possesses more potent and efficacious anxiolytic properties than the 5-isomer.     

Nasal Absorption Enhancement of 17β-Estradiol by Dimethyl-β-Cyclodextrin in Rabbits and Rats

A new formulation for nasal administration containing 17-estradiol (E₂) with dimethyl--cyclodextrin (DMC) as a solubilizer and absorption enhancer is described. Nasal administration of this E₂-DMC formulation gave a significantly higher E₂ absorption than an E₂ suspension in both rabbits and rats. Relative to an intravenous injection of the E₂-DMC formulation, absolute bioavailabilities of 94.6 and 67.2% were calculated for the nasal E₂-DMC formulation in rabbits and rats, respectively. Differences in bioavailability may have resulted from differences in experimental animal conditions. The effects on human nasal ciliary activity of the E₂-DMC formulation were studied with an in vitro method. The formulation was found to exert only a minor effect on ciliary beat frequency. Thus, nasal delivery of E₂, using a cyclodextrin inclusion formulation, may have potential for clinical application, e.g., in the therapy of postmenopausal disorders.     

The influence of hyperthyroidism on β-adrenoceptor-mediated relaxation of isolated small mesenteric arteries

We investigated the influence of hyperthyroidism on relaxant responses of small mesenteric resistance arteries to -adrenoceptor agonists and to compounds stimulating the corresponding second-messenger system. Hyperthyroidism was induced by feeding rats for 28 days with 5 mg/kg L-thyroxine (T4)-containing rat chow. This treatment produced a stable hyperthyroid state, as indicated by several biochemical/metabolic and haemodynamic parameters. Preparations of small mesenteric arteries were mounted in an isometric wire myograph. Subsequently, concentration-effect curves were determined for isoproterenol, noradrenaline and salbutamol as well as for forskolin, dibutyryl-cAMP and theophylline. We also determined concentration-effect curves to the -adrenoceptor agonists in the presence of ICI 118,551 and CGP 20712A (i.e., in the presence of a selective ₂- and ₁-adrenoceptor antagonist, respectively). Apparent pA₂-values were calculated to determine which -adrenoceptor subtype causes vasodilation. These experiments indicate that -adrenoceptor-mediated vasodilation involves both ₁- and ₂-adrenoceptors in mesenteric resistance vessels of both hyperthyroid and control rats. In our experiments hyperthyroidism has a sensitizing influence on vascular responses induced by the -adrenoceptor agonist isoproterenol and the selective ₂-adrenoceptor agonist salbutamol. Sensitization to isoproterenol was abolished in the presence of ICI 118,551, whereas it was emphasized in the presence of CGP 20712A. Although this was not fully supported by the results obtained with noradrenaline, these results indicate that the sensitization to -adrenoceptor agonists is probably limited to the ₂-adrenoceptor/G-protein complex and not associated with alterations of the corresponding second messenger system.     

Effects of metal cations on [3H]α,β-methylene ATP binding in rat vas deferens

In this study we have examined the effect of metal cations (as their chloride salts) on the binding of [³H],-methylene ATP ([³H]meATP) to rat vas deferens membranes using a vacuum filtration receptor binding assay. Whereas NaCl and KCl (0.01 and 30 mM) did not affect total binding of 1 nM [³H]meATP, several divalent and trivalent cation salts markedly increased binding. The trivalent cation salts, FeCl₃ and AlCl₃ (0.1 to 100 M), produced the greatest increases in total binding of [³H]meATP, however, their effects were most probably due to precipitation of the radioligand. In contrast, several divalent cations, at concentrations between 1 M and 1–10 mM, increased total binding of [³H]meATP to rat vas deferens by between 87% and 215% while having no effect on either filter binding or non specific binding. The following pEC₅₀ values for potentiating binding of the radioligand were obtained: ZnCl₂ (5.44), MnCl₂ (4.52), CaCl₂ (4.17), CoCl₂ (4.06), MgCl₂ (3.67) and BaCl₂ (3.10). Both EDTA and EGTA (0.01–1 mM) inhibited the binding of the radioligand.The effects of ZnCl₂, CaCl₂ and MgCl₂ were examined in saturation studies. In the absence of added divalent cations, [³H]meATP labelled both high (pK_d = 9.15) and low (pK_d = 7.06) affinity binding sites. The affinity of the radioligand for its high affinity sites was increased by 3 mM CaCl₂ (pK_d = 9.56) and by 30 M ZnCl₂ (pK_d = 9.46) but not by 3 mM MgCl₂. The B_max of the low affinity site for [³H]meATP was increased (approximately 4 fold) by both 3 mM MgCl₂ and 30 M ZnCl₂ but not by 3 mM CaCl₂. The selective effect of CaCl₂ on the high affinity binding sites enabled these sites to be labelled in the presence of 3 mM CaCl₂ using a low concentration of [³H]meATP (1 nM); the sites exhibited the binding characteristics expected of the P_2x purinoceptor. The selective effect of MgCl₂ on the low affinity binding sites enabled these sites to be labelled in the presence of 3 mM MgCl₂ and using a high concentration of [³H]meATP (100 nM). A comparison of the binding characteristics of the high and low affinity sites for [³H]meATP revealed several other differences, in addition to their cation selectivity. First, the adenine analogues ADP, meATP and adenosine tetraphosphate possessed between 13 and 62 fold higher affinity for the high affinity [³H]meATP binding sites than for the low affinity binding sites. Secondly, GTP--S and pyrophosphate were selective ligands for the low affinity [³H]meATP binding sites possessing approximately 43 and 1995 fold, respectively, higher pIC₅₀ values at the low affinity sites than at the high affinity sites. Finally, treatment of the membranes with 0.01–1 mM N-ethyl maleimide increased low affinity binding of the radioligand while not affecting binding to the high affinity sites. The binding characteristics of the low affinity sites suggest that they do not equate with functional P_2x purinoceptors; their identity remains to be determined. There was evidence for heterogeneity of both the high and low affinity sites for [³H]meATP since competition curves to several nucleotide and polyphosphate compounds displayed Hill slopes less than unity.In conclusion the present study has demonstrated that cations have a marked effect on the binding of [³H]meATP in rat vas deferens. Of particular interest was the ability of CaCl₂ to increase the affinity of the radioligand for its high affinity sites enabling these sites to be selectively labelled, while the ability of MgCl₂ to increase the B_max of the low affinity binding sites enabled these sites to be selectively labelled. Correspondence to: A. D. Michel at the above address     

Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.     

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.     

The Effect of Parenterally Administered Cyclodextrins on Cholesterol Levels in the Rat

The inclusion complex formation of intravenously administered hydroxypropyl--cyclodextrin and -cyclodextrin with endogenous lipids was studied. We tested the hypothesis that complex formation of endogenous cholesterol with cyclodextrins in the bloodstream leads to extraction of cholesterol from the large lipoprotein particles. The relatively small cholesterol–cyclodextrin complexes then leave the bloodstream via capillary pores, and dissociation of the complex in the extravascular compartment finally causes redistribution of cholesterol from blood to tissue. This hypothesis is supported by the following experimental findings. Intravenous administration of cyclodextrins led to a transient decrease in plasma cholesterol levels in a dose-dependent manner, and in vitro cholesterol-cyclodextrin complexes passed dialysis membranes with a molecular weight cutoff of 6000–8000. Further, cyclodextrins increased protein binding of the steroidal drug spironolactone, probably through removal of cholesterol from plasma protein binding sites. Finally, extravascular redistribution was directly demonstrated in histological studies of the kidneys. Glomerular filtration of the cholesterol–cyclodextrin complex is followed by dissociation of the complex in the ultrafiltrate, resulting in cholesterol accumulation in the proximal tubule cells. The cholesterol--cyclodextrin complex has a limited aqueous solubility. Crystallization of this complex in renal tissue might explain the nephrotoxicity of parenterally administered -cyclodextrin. The absence of such crystallization might explain the lower nephrotoxicity of hydroxypropyl--cyclodextrin after intravenous administration.     

Protection Afforded by Maltosyl-β-cyclodextrin Against α-Chymotrypsin-Catalyzed Hydrolysis of a Luteinizing Hormone-Releasing Hormone Agonist, Buserelin Acetate

Purpose. The present study addresses how maltosyl--cyclodextrin (G₂--CyD) impacts upon the -chymotrypsin-catalyzed hydrolysis of buserelin acetate, an agonist of luteinizing hormone-releasing hormone with emphasis upon the direct effect of G₂--CyD on the activity of the protease. Methods. Kinetic and solubility studies were performed in isotonic phosphate buffer (pH 7.4) at 25°C and 37°C. The interaction of -chymotrypsin with G₂--CyD in the buffer solution was examined by differential scanning calorimetry. Results. G₂--CyD decelerated the -chymotrypsin-catalyzed hydrolysis of buserelin acetate to give the 1–3 tripeptide and the 4–9 hexapeptide fragments. This deceleration can be explained solely by a nonproductive encounter between a complex of the substrate with G₂--CyD and the protease at relatively low CyD concentrations, while the direct inhibitory effect of G₂--CyD on the proteolytic activity made a considerable contribution to the overall deceleration of the hydrolysis at higher CyD concentrations. Calorimetric studies indicate the presence of intermediate states in the thermal unfolding of -chymotrypsin, simultaneously accompanied by the autolysis. By contrast, a two-state thermal unfolding of -chymotrypsin was observed in the presence of G₂--CyD, suggesting reduced proteolytic activity upon binding to G₂--CyD. Conclusions. These results suggest that G₂--CyD at higher concentrations inhibits the proteolytic action of -chymotrypsin through direct interaction with the protease, as well as through the formation of a non-productive complex with the substrate.     

Interleukin-1β and tumor necrosis factor-α inhibit the release of [3H]-noradrenaline from mice isolated atria

In the present study, we have investigated the ability of four cytokines, interleukin-1, interleukin-2, interleukin-6 and tumor necrosis factor-, to modulate the stimulation-induced outflow of radioactivity from isolated superfused mouse atria which where pre-incubated with [³H]-noradrenaline. The tissues were subjected twice to field stimulation (5 Hz frequency, 50 mA intensity, 2 ms pulses for 60 s) and the drugs were added prior to the second stimulation in order to assess their modulatory effects.The results show that mouse recombinant interleukin-1 and tumor necrosis factor- inhibited the stimulation-induced release of radioactivity from the isolated mouse atria. The effect of interleukin-1 was blocked by a human recombinant interleukin-1 receptor antagonist. The inhibitory effect of interleukin-1 was also abolished by the cyclooxygenase inhibitor, diclofenac (1 mol/1) suggesting that the action of interleukin-1 might be mediated through the formation of prostaglandins. The effect of interleukin-1 appears to be time-dependent, since a stronger inhibition of radioactivity release was observed when the incubation time was increased from 20 to 65 minutes before the second stimulation. Interleukin-2 and interleukin-6 were ineffective in modulating release under these experimental conditions.The ability of interleukin-1 and tumor necrosis factor- to inhibit noradrenaline release suggests that mediators of the immune system produced locally may modulate the activity of the sympathetic nervous system.     

Differentiation of cardiac chronotropic and inotropic effects of β-adrenoceptor agonists

Summary The relative inotropic and chronotropic activity of -adrenoceptor agonists was studied in the noradrenaline-depleted, anaesthetized cat. Terbutaline, a selective ₂-adrenoceptor agonist, gave at a certain dose a more pronounced chronotropic than inotropic response, while a new ₁-selective adrenoceptor agonist (–)-H 80/62 produced the same degree of chronotropic and inotropic stimulation. The results indicate that there is some difference between the -adrenoceptors in the sinus node mediating chronotropic stimulation and -adrenoceptors in the ventricular myocardium mediating stimulation of the contractile force. It has been shown that there are both ₁- and ₂-adrenoceptors in the heart (Carlsson et al., 1972). In the light of this finding it is hypothetized that there are differences in the relative distribution of ₁- and ₂-adrenoceptors in the sinus node and in the myocardium. Although ₁ is the predominant type of -adrenoceptor in both regions, the ₁: ₂ concentration ratio seems to be higher in the myo-cardium, than in the sinus node.     

Design and Evaluation of an Osmotic Pump Tablet (OPT) for Prednisolone,a Poorly Water Soluble Drug,Using (SBE)7m-β-CD

Purpose. The purpose of this study was to develop a controlled-porosity osmotic pump tablet (OPT) for poorly water soluble drugs using a sulfobutyl ether--cyclodextrin, (SBE)_7m--CD or Captisol, which acted as both a solubilizer and as an osmotic agent. Methods. Prednisolone (PDL) was chosen as a model drug for this study. The release of PDL from osmotic pump devices and tablets was studied. In vivo absorption of PDL from OPT was evaluated in male beagle dogs. Results. PDL release from the osmotic pump tablet with (SBE)_7m--CD was complete. Another cyclodextrin, hydroxypropyl--cyclodextrin (HP--CD), and a sugar mixture of lactose and fructose resulted in incomplete release. Although PDL release from the OPT with (SBE)_7m--CD and the sugar formulation displayed mainly zero-order release characteristics, the tablet utilizing HP--CD showed apparent first-order release characteristics. An in vivo absorption study in dogs correlated very well with the in vitro release profiles using the Japanese Pharmacopoeia dissolution method. Conclusions. The present results confirm that (SBE)_7m--CD can serve as both the solubilizer and the osmotic agent for OPT of PDL, and modify the input rate of PDL without compromising oral bioavailability.     

β2-Adrenoceptor-mediated positive inotropic effect of adrenaline in human ventricular myocardium

Summary Experiments were designed to unravel the relative contribution of ₁- and ₂-adrenoceptors to the positive inotropic effects of adrenaline and noradrenaline in isolated tissues of left ventricular myocardium of man. We also analyzed relationships between the fractions of human left ventricular ₁- and ₂-adrenoceptors, estimated from binding assays, and stimulation of adenylate cyclase and contractile force by adrenaline and noradrenaline. 1) Selective blockade of ₂-adrenoceptors by erythro(±)-(-methyl-indan-4-yloxy)-3-isopropylaminobutan-2-ol (ICI 118,551) attenuated the increase of contractile force caused by adrenaline but not by noradrenaline, suggesting some involvement of ₂-adrenoceptors. Selective blockade of ₂-adrenoceptors without affecting ₁-adrenoceptors still enabled both adrenaline and noradrenaline to cause maximum possible increases of contractile force through ₁-adrenoceptors. 2) A direct involvement of ₂-adrenoceptors became manifest by selectively antagonizing ₁-adrenoceptors by 1-[2((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]3-[4(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712 A) without affecting ₂-adrenoceptor. ₂-adrenoceptors can mediate half of the maximum increase of contractile force elicited by low concentrations of adrenaline and also contribute to the increase of contractile force caused by high concentrations of noradrenaline. 3) -adrenoceptors were labelled in membrane particles with ³H-(–)-bupranolol in the absence (₁ & ₂) and presence of 500 nmol/l CGP 20712 A (₂). 71 % of the -adrenoceptors Were ₁ and 29% ₂. Binding inhibition experiments with CGP 20712 A and ICI 118,551 yielded 74% ₁ and 26% ₂-4) With the help of ICI 118,551 and CGP 20712 A it was found that, in membrane particles, 33–36% and 64–67% of maximum stimulation of the adenylate cyclase by noradrenaline was mediated through ₁- and ₂-adrenoceptors, respectively. Adrenaline caused 11–25% and 75–89% of maximum cyclase stimulation through ₁- and ₂-adrenoceptors, respectively. 5) The contribution of ₁- and ₂-adrenoceptors to the positive inotropic effects of adrenaline and noradrenaline cannot be inferred straightforwardly from biochemical estimates of receptor fractions and fractional adenylate cyclase stimulation. Send offprint requests to A. J. Kaumann, ICI Pharmaceutical Division, Mereside Alderley Park, Macclesfield, Cheshire SK10 4TG, UK