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.     

Cardiac ventricular β2-adrenoceptors in guinea-pigs and rats are localized on the coronary endothelium

Summary In mammalian heart tissue ₂ are known to coexist with ₁. In the present study, evidence that ₂ in guinea-pig and rat ventricles are primarily localized on the coronary endothelium is provided by competition binding studies with the subtype-selective -adrenoceptor antagonists ICI 89.406 (₁) and ICI 118.551 (₂) on four different plasma membrane preparations. (1) Following density gradient centrifugation of cardiac ventricular microsomes from rats or guinea-pigs, endothelial plasma membranes migrated at slightly higher density than the sarcolemmal membranes, as verified by endothelial (angiotensin converting enzyme) and sarcolemmal markers (adenylate cyclase, [³H] ouabain binding). At the activity peak of angiotensin converting enzyme, the relative amount of ₂ in guinea-pigs and rats was 25% and 65%, respectively. (2) On sarcolemmal membranes corresponding to the activity peak of adenylate, cyclase, -adrenoceptors consisted of the ₁ exclusively (guinea-pig), or to at least 90% (rat). (3) Cultures of coronary endothelial cells derived from guinea-pigs revealed only ₂. (4) Isolated guinea-pig cardiomyocytes contained only ₁, a finding recently established in rat myocytes as well.     

Differential effects of α-β-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries

Summary The effects of ,-methylene-adenosine triphosphate, (,-methylene ATP, a P₂-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with ³H-noradrenaline.Exposure to ,-methylene ATP (0.1 mol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of ,-methylene ATP (1 mol/l). In WKY tail arteries, ,-methylene ATP (1 mol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.In SHR tail arteries prelabelled with ³H-noradrenaline, ,-methyleneATP (1 mol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, ,-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mol/l), ,-methylene ATP (30 mol/l), a stable agonist at P₂-receptors, or 60 mmol/l KCl. These effects of ,-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mol/l), but were practically abolished by the addition of ,-methylene ATP (1 mol/l).In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of ,-methylene ATP (1 mol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of ,-methylene ATP not involving P₂ receptors cannot be entirely excluded.     

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.     

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.     

Transforming growth factor β1 is not involved in 2,3,7,8-tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin-dependent release from contact-inhibition in WB-F344 cells

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is the most potent tumor promoter ever tested in rodents. Although it is known that most of the effects of TCDD are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact-inhibition is one characteristic hallmark in tumorigenesis. In WB-F344 cells, TCDD induces a release from contact-inhibition, which is manifested by a two- to three-fold increase in DNA-synthesis when TCDD (1 nM) is given to confluent cells. Since proliferation of epithelial cells is known to be inhibited by transforming growth factor (TGF-) we investigated whether decreased TGF- expression mediates TCDD-dependent release from contact-inhibition in WB-F344 cells. Expression of TGF- (type II) receptor in WB-F344 cells was shown by Western blot analysis. Exposure of exponentially growing WB-F344 cells to 0.1 ng/ml TGF-1 resulted in a 40% decrease in DNA synthesis, which could be blocked by pre-incubation with a neutralizing anti-TGF-1 antibody indicating that the TGF- receptor in WB-F344 cells is functionally active. Pre-incubation of confluent, G1-arrested cultures with the neutralizing anti-TGF-1 antibody did not lead to an increase in DNA synthesis, ruling out an involvement of TGF-1 in mediating contact-inhibition in WB-F344 cells. In accordance with this, Western blot analysis revealed that protein expression of TGF-1 was neither upregulated in confluent cultures nor decreased after TCDD treatment. We therefore conclude that TGF-1 is not involved in contact-inhibition nor in TCDD-dependent release from contact-inhibition in WB-F344 cells.     

Both β1- and β2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

Summary The involvement of ₁- and ₂-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with blockers (usually ₁-selective) and 9 patients not treated with blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. ₁-adrenoceptors were activated by (–)-noradrenaline during ₂-adrenoceptor blockade with 50 nmol/l ICI 118551. ₂-adrenoceptors were activated by (–)-adrenaline during ₁-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (–)noradrenaline and (–)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (–)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a blocker and 17/17 atrial strips from 15/15 patients chronically treated with blockers. ICI 118551 (50 nmol/l) blocked the (–)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with blockers and 17/20 atrial strips from 15/18 patients chronically treated with blockers. The incidence of arrhythmic contractions evoked by both (–)-noradrenaline and (–)-adrenaline was higher in chronically blocked patients than in non blocked patients. We conclude that both ₁- and ₂-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic ₁-adrenoceptor blockade. Correspondence to: A. J. Kaumann at the Clinical Pharmacology Unit, University of Cambridge, as above     

The effect of isoprenaline on plasma concentrations of immunoreactive β-endorphin and β-lipotropin in the conscious rat

Summary The effect of the -adrenoceptor agonist isoprenaline on the plasma concentrations of -endorphin (-E) and -lipotropin (-LPH) was investigated in conscious rats. Isoprenaline (i.m.) elevated plasma -endorphin-like immunoreactivity (-EI) as measured by radioimmunoassay of unextracted plasma, with peak values 24 min after drug administration. This effect was dose-dependent. The lowest effective dose of isoprenaline was 15 g kg^–1; 240 g kg^–1 exerted a maximum effect, raising plasma -EI about ten-fold above control values. Plasma vasopressin concentrations also increased in response to isoprenaline following a timecourse identical to that of plasma -EI. (±)-Propranolol (1 mg kg^–1) but not phentolamine (10 mg kg^–1) rendered isoprenaline (240 g kg^–1) injections almost ineffective. Because of the cross-reactivity of -LPH in the radioimmunoassay used, plasma was extracted by means of a cation exchange resin and subjected to gel chromatography on a Sephadex G-50 column, avoiding artefactual degradation of the peptides. In isoprenaline-treated rats about 50% of the -EI behaved similar to human -LPH, whereas 45% co-migrated with human -E; immunoreactivity corresponding to -LPH or -E comprised about 70% or 30%, respectively, in the plasma extract of vehicle-treated rats. Dexamethasone pretreatment reduced the isoprenaline-induced increase in plasma -EI by 87%, but left the simultaneous elevation of plasma vasopressin concentrations unchanged.These data demonstrate that isoprenaline stimulates -LPH and -E release in vivo. The possibility of an interrelationship between vasopressin and -E release is discussed.     

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.     

Proteolytic maturation of transforming growth factor-α

Summary Transforming growth factor- (TGF-) is a mitogenic peptide hormone produced extracellularly, by tumor cells, and by virally and chemically transformed cells in culture. TGF- is almost certainly derived from its precursor protein (pro-TGF-) by limited endoproteolysis, but physiologically relevant processing enzyme(s) of the pro-TGF- protein and the cellular or subcellular compartment in which processing takes place are not known with certainty. We previously detailed [Cappelluti, E. and Harris, R.B., Biochemistry, 32 (1993) 551] the discovery, characterization and purification of novel, elastase-like enzymes (molecular weight 38 000) from oncogenically transformed rat liver epithelial cells or cultural Schwann cells transfected with SV40-large T antigen. The elastase-like enzyme appeared to be specifically induced in the transformed epithelial cells compared with the level of enzyme in the nontransformed parental cells. In the intervening time, other elastase-like serine proteinases have been implicated in processing pro-TGF- in other human carcinoma cell lines. We now report that the elastase-like enzymes, purified from transformed Schwann or liver epithelial cells, are inhibited in a time- and concentration-dependent fashion with three differently substituted monocyclic -lactam-based compounds originally developed as specific inhibitors of polymorphonuclear leukocyte elastase, thus further supporting the elastase-like character of the putative pro-TGF- processing enzymes. We also report the presence of the elastase-like enzyme in two different human malignant mammary cell lines, but even though MCF-7 cells receiving high doses of radiation in vitro show an increased level of expression of TGF-, the elastase-like enzyme does not appear to be induced in these cells following irradiation.     

Transforming growth factor-β1 is not involved in TCDD-dependent release from contact inhibition in WB-F344 cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent tumor promoter ever tested in rodents. Although it is known that most of the effects of TCDD are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact inhibition is one characteristic hallmark in tumorigenesis. In WB-F344 cells TCDD induces a release from contact inhibition which is manifested by a two- to threefold increase in DNA synthesis when TCDD (1 nM) is applied to confluent cells. Since proliferation of epithelial cells is known to be inhibited by TGF-, we investigated whether decreased TGF-₁ mediates TCDD-dependent release from contact inhibition in WB-F344 cells. Expression of TGF- (type II) receptor in WB-F344 cells was analyzed by Western blot analysis. Exposure of 0.1 ng/ml TGF-₁ to exponentially growing WB-F344 cells resulted in a 40% decrease in DNA synthesis, which was blocked by preincubation with a neutralizing anti-TGF-₁ antibody, indicating that the TGF- receptor in WB-F344 cells is functionally active. Preincubation of confluent, G₁-arrested cultures with the neutralizing anti-TGF-₁-antibody did not lead to an increase in DNA synthesis, ruling out an involvement of TGF-₁ in mediating contact inhibition in WB-F344 cells. In accord with this, Western blot analysis revealed that protein expression of TGF-₁ is neither upregulated in confluent cultures nor decreased after TCDD treatment. We conclude that TGF-₁ is not involved in contact inhibition or in TCDD-dependent release from contact inhibition in WB-F344 cells.     

β-Adrenergic Receptor Polymorphisms: Cardiovascular Disease Associations and Pharmacogenetics

The -adrenergic receptors (AR) play important roles in cardiovascular function and disease, and both agonists and antagonists are widely used in various settings for treatment of cardiovascular disease. Both the ₁AR and ₂AR genes have several polymorphisms that are common in the population and result in encoding of different amino acids. More importantly, in vitro functional studies suggest that these polymorphisms have functional significance. In this review we summarize the literature on the relationship between the AR polymorphisms and cardiovascular disease as well as the literature on the impact of these polymorphisms on drug response. Additionally, the polymorphisms in both the ₁AR and ₂AR genes are in linkage disequilibrium; thus, the relevance of single polymorphism vs. haplotype analysis is discussed. Further study of the AR genetic polymorphisms is likely to enhance our understanding of cardiovascular disease and improve our use of -agonists and -antagonists in treatment of cardiovascular disease.     

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.     

In vivo pharmacological effects of dihydro-β-erythroidine,a nicotinic antagonist,in mice

The comparative in vivo pharmacology of mecamylamine and dihydro--erythroidine (DHE) in mice was studied. Modulation of the behavioral effects (antinociception, hypomotility, motor impairment and hypothermia) of nicotine in mice by DHE and mecamylamine were carried out. After SC administration, DHE and mecamylamine were nearly equipotent in blocking nicotine's effects except for antinociception, in which mecamylamine was clearly more potent. Intrathecal injection of DHE was also effective in blocking the antinociceptive effect of nicotine. In vivo interaction of DHE with calcium and calcium channels, involved in the central actions of nicotine, showed that intrathecal administration of DHE failed to reduce the antinociception induced by diverse drugs which increase intracellular calcium such as thapsigargin, (±)-BAYK 8644 and calcium, indicating that this antagonist does not affect calcium-dependent mechanisms involved in antinociception. On the other hand, mecamylamine blocked the antinociceptive effect of the calcium modulatory drugs, suggesting that it may be acting on calcium-dependent mechanisms involved in the intracellular signaling process. We conclude that DHE, a nicotinic neuromuscular antagonist, is able to block some of the central actions of nicotine after systemic and intrathecal administration. The mechanism of blockade is different from that of mecamylamine, a classical ganglionic antagonist, and may involve a direct action of DHE on nicotine receptor.     

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.     

Local modulation of adrenal catecholamines release by beta-2 adrenoceptors in the anaesthetized dog

Summary The release of adrenal catecholamines into the adrenal vein elicited by splanchnic nerve stimulation, was evaluated in the presence of a -adrenoceptor agonist and both -1 and -2 adrenoceptor antagonists in anaesthetized and vagotomized dogs. Stimulations (0.5 V pulses of 2 ms duration for 3 min at 1 Hz) were applied before and after the i.v. infusion of the -adrenoceptor agonist, isoproterenol (0.1 /kg/min). While maintaining the infusion of isoproterenol, either ICI 118551 (0.3 mg/kg), a selective -2 adrenoceptor antagonist, or 204-155 (0.2 mg/kg), a selective -1 adrenoceptor antagonist (Sandoz Co., Dorval, PQ, Canada), were injected intravenously and the stimulation was repeated. The results show that isoproterenol increased significantly both pre-stimulation basal levels and the stimulated release of catecholamines. These potentiated responses were significantly reversed by ICI 118551, but not by 204155. These results suggest that the release of adrenal catecholamines is locally modulated by a positive feedback mechanism through activation of -2 adrenoceptors. Send offprint requests to S. Foucart     

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.     

Effects of intrathecal or intracerebroventricular pretreatment with pertussis toxin on antinociception induced by β-endorphin or morphine administered intracerebroventricularly in mice

We have previously demonstrated that -endorphin and morphine, when administered supraspinally, produce antinociception by activating different descending pain inhibitory systems in both rats and mice. However, the signal transduction mechanisms involved in the descending pain-inhibitory systems that are activated by -endorphin and morphine administered intracerebroventricularly (i.c.v.) have not been characterized. Therefore, in the present study, the effects of intrathecal (i.t.) and i.c.v. pretreatments with pertussis toxin (PTX) on antinociception induced by -endorphin or by morphine administered i.c.v. were studied in ICR mice. Antinociception was assessed by the tail-flick assay and by the hotplate assay. Intrathecal pretreatment with PTX (0.5 g) for 6 days effectively reduced the inhibition of the tail-flick response induced by -endorphin (1 g) or by morphine (1 g) administered i.c.v. However, i.t. pretreatment with PTX was not effective in reducing the inhibition of the hot-plate response induced by -endorphin or by morphine administered i.c.v. Intracerebroventricular pretreatment with PTX (0.5 g) for 6 days effectively reduced the inhibition of the tail-flick and hot-plate responses induced by morphine (1 g), but not that induced by -endorphin (1 g), administered i.cv. Our results suggest that there are PTX-sensitive G proteins coupled to the spinal descending pain inhibitory systems that are activated by -endorphin and morphine administered i.c.v. At a supraspinal level, i.cv. morphine- but not -endorphin-induced antinociception is mediated by PTX-sensitive G proteins. Correspondence to: Hong W. Suh at the above address     

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.     

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.