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.     

Synergistic Effect of Formulated Plasmid and Needle-Free Injection for Genetic Vaccines

Purpose. A plasmid-based gene expression system was complexed with protective, interactive, and non-condensing (PINC) polymer system and administered with Medi-Jector, a needle-free injection device (NFID), to achieve high and sustained levels of antigen-specific antibodies in blood circulation. Methods. Human growth hormone (hGH) or bacterial -galactosidase gene expression plasmids driven by a cytomegalovirus (CMV) promoter were formulated in saline or complexed with a PINC polymer, polyvinylpyrrolidone (PVP), and intramuscularly or subcutaneously administered into dogs and pigs using a 22-gauge needle or a NFID. The hGH-specific IgG titers in serum were measured by an ELISA. -galactosidase expression was measured in injected muscles by an enzymatic assay or immunohistochemistry. The effect of NFID on DNA stability and topology was assessed by gel electrophoresis. Results. Intramuscular (i.m.) or subcutaneous (s.c.) injection of a hGH expression plasmid pCMV-hGH (0.05-0.5 mg/kg) in dogs and pigs elicited antigen-specific IgG antibody titers to expressed hGH. With both routes of injection, pDNA delivery by a NFID was superior to pDNA injection by needle. The magnitude of hGH-specific IgG titers with NFID was 15–20-fold higher than needle injection when pDNA was complexed with PVP, and only 3–4-fold higher with pDNA in saline. The transfection efficiency in the injected muscle, as measured by -galaclosidase expression, following i.m. injection of pCMV--galaclosidase/PVP, was not significantly different between needle and NFID-injected groups. Conclusions. These data demonstrate that the combination of pDNA/ PVP complexes and a NFID act synergistically to achieve high and sustained levels of antigen-specific IgG response to expressed antigen. This gene delivery approach may offer advantage over needle injection of naked DNA for the development of genetic vaccines.     

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.     

Bilayer Films for Mucosal (Genetic) Immunization via the Buccal Route in Rabbits

Purpose. The oral buccal mucosa may be an ideal site for mucosal immunization, allowing for the needle-free administration of cost-effective vaccines. A novel mucoadhesive bilayer film was developed to test the feasibility of this route of immunization in rabbits. Methods. Bilayer films were developed using different ratios of Noveon and Eudragit S-100 as the mucoadhesive layer and a pharmaceutical wax as the impermeable backing layer. Optimal 3/8-inch films were post-loaded with 100 g of plasmid DNA (CMV--gal) or -galactosidase protein. The in vitro release rates and stability of the postloaded antigens were determined. The films were applied to the buccal pouch of rabbits on days 0, 7, and 14, and the humoral and splenocyte proliferative immune responses to -gal were determined through day 28 and compared to those responses after conventional subcutaneous injection of adjuvanted protein. Results. The weight ratio of Noveon and Eudragit S-100 had a significant effect on adhesion time of bilayer films. Postloaded plasmid DNA and -gal remained stable after being released from bilayer films (release of 60-80% in 2 h for both). Buccal immunization using novel bilayer films (109 ± 6-m thickness) containing plasmid DNA led to comparable antigen-specific IgG titer to that of subcutaneous protein injection. All rabbits immunized with plasmid DNA via the buccal route but none by the subcutaneous route with protein antigen demonstrated splenocyte proliferative immune responses. Conclusion. The feasibility of buccal (genetic) immunization with these novel bilayer films was demonstrated.     

Tumor-Targeted Gene Delivery Using Poly(Ethylene Glycol)-Modified Gelatin Nanoparticles: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">in Vivo</Emphasis> Studies

Purpose To develop safe and effective systemically administered nonviral gene therapy vectors for solid tumors, DNA-containing poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticles were fabricated and evaluated in vitro and in vivo.Methods Reporter plasmid DNA encoding for -galactosidase (pCMV-) was encapsulated in gelatin and PEGylated gelatin nanoparticles using a water-ethanol solvent displacement method under controlled pH and temperature. Lewis lung carcinoma (LLC) cells in culture were transfected with the pCMV- in the control and nanoparticle formulations. Periodically, the expression of -galactosidase in the cells was measured quantitatively using an enzymatic assay for the conversion of o-nitrophenyl--d-galactopyranoside (ONPG) to o-nitrophenol (ONP). Qualitative expression of -galactosidase in LLC cells was observed by staining with 5-bromo-4-chloro-3-indolyl--d-galactopyranoside (X-gal). Additionally, the plasmid DNA-encapsulated gelatin and PEGylated gelatin nanoparticles were administered intravenously (i.v.) and intratumorally (i.t.) to LLC-bearing female C57BL/6J mice. At various time points postadministration, the animals were sacrificed and transgene expression in the tumor and liver was determined quantitatively by the ONPG to ONP enzymatic conversion assay and qualitatively by X-gal staining.Results Almost 100% of the pCMV- was encapsulated in gelatin and PEGylated gelatin nanoparticles (mean diameter 200 nm) at 0.5% (w/w) concentration. PEGylated gelatin nanoparticles efficiently transfected the LLC cells and the -galactosidase expression, as measured by the ONPG to ONP enzymatic conversion assay at 420 nm absorbance, increased starting from 12 h until 96 h post-transfection. The efficient expression of LLC cells was also evident by the X-gal staining method that shows blue color formation. The in vivo studies showed significant expression of -galactosidase in the tumor following administration of DNA-containing PEGylated gelatin nanoparticles to LLC-bearing mice by both i.v. and i.t. routes. Following i.v. administration of pCMV- in PEGylated gelatin nanoparticles, for instance, the absorbance at 420 nm per gram of tumor increased from 0.60 after 12 h to 0.85 after 96 h of transfection. After i.t. administration, the absorbance values increased from 0.90 after 12 h to almost 1.4 after 96 h.Conclusions The in vitro and in vivo results of this study clearly show that a long-circulating, biocompatible and biodegradable, DNA-encapsulating nanoparticulate system would be highly desirable for systemic delivery of genetic constructs to solid tumors.     

Structural and Functional Differences Between Glycosylated and Non-glycosylated Forms of Human Interferon-β (IFN-β)

Purpose. Two recombinant IFN- products have been approved for the treatment of multiple sclerosis, a glycosylated form with the predicted natural amino acid sequence (IFN--la) and a non-glycosylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN--lb). The structural basis for activity differences between IFN--la and IFN--lb, is determined. Methods. In vitro antiviral, antiproliferative and immunomodulatory assays were used to directly compare the two IFN- products. Size exclusion chromatography (SEC), SDS-PAGE, thermal denaturation, and X-ray crystallography were used to examine structural differences. Results. IFN-- la was 10 times more active than IFN-- Ib with specific activities in a standard antiviral assay of 20 × 10⁷ lU/mg for IFN--la and 2 × 10⁷ lU/mg for IFN--lb. Of the known structural differences between IFN--la and IFN--lb, only glycosylation affected in vitro activity. Deglycosylation of IFN--la produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate. Deglycosylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN--lb revealed large, soluble aggregates that had reduced antiviral activity (approximated at 0.7 × 10⁷ lU/mg). Crystallographic data for IFN--la revealed that the glycan formed H-bonds with the peptide backbone and shielded an uncharged surface from solvent exposure. Conclusions. Together these results suggest that the greater biological activity of IFN--la is due to a stabilizing effect of the carbohydrate on structure.     

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.     

Studies on the chemical constituents from the roots of <Emphasis Type="Italic">Platycodon grandiflorum</Emphasis>

Three known monodesmosidic saponins: 3-O--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid, 3-O--d-glucopyranosyl polygalacic acid, and 3-O--d-glucopyranosyl-(13)--d-glucopyranosyl polygalacic acid; and two known nonsaponin compounds: a mixed compound of n-tetracosanoic acid (lignoceric acid), n-hexacosanoic acid (cerotic acid), and n-octacosanoic acid, and -monopalmitin; were isolated for the first time from the root of Platycodon grandiflorum A. DC. together with another seven known compounds: platycoside G₁ (3-O--d-glucopyranosyl-(16)--d-glucopyranosyl-(16)--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid 28-O--d-xylopyranosyl-(14)--l-rhamnopyranosyl-(12)--l-arabinopyranoside), deapio-platycodin D, Polygalacin D, deapio-platycodin D₃, platycoside A, -spinasterol, and -spinasteryl-3-O--d-glucopyranoside. Alkaline hydrolysis of platycoside G₁ afforded a new monodesmosidic prosaponin: 3-O--d-glucopyranosyl-(16)--d-glucopyranosyl-(16)--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid. Their chemical structures were elucidated on the basis of their spectral data and chemical evidence.     

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     

Intestinal Absorption of Acyclovir β-Glucoside: Comparative Study with Acyclovir,Guanosine, and Kinetin β-Glucoside

Purpose. To characterize the intestinal absorption of a -glucose conjugate of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV) and compare it to ACV and its analogues in terms of stability and transport by Na⁺/glucose cotransporter (SGLT1). Methods. ACVglc was enzymatically synthesized using cellulase. Intestinal absorption experiments were performed with rat everted small intestine. Conformation of the glucose moiety was analyzed by NMR spectroscopy. Results. The ACVglc was stable on the mucosal side, and was transported to the serosal side in all regions of the small intestine. However, significant contribution of SGLT1 to the transport of ACVglc was not observed. NMR spectroscopic analysis indicated that the glucose conformation of ACVglc was the ⁴C₁ chair form, identical to (-glucose or SGLT1-transportable -glucosides reported previously. Therefore, other factors such as molecular size and charge due to aglycone may cause no transport of ACVglc by SGLT1. On the other hand, the hydrophilicity of ACVglc was much higher than of ACV, suggesting water solubility-derived improvement of intestinal absorption of ACV. Conclusions. ACVglc is stable and absorbable, but it is not transported by SGLT1. No involvement of SGLT1 in the ACVglc transport is not due to glucose conformation.     

Reversal of tumor-induced immunosuppression by TGF-beta inhibitors

The immune system is responsible for the early detection and destruction of newly transformed malignant cells. Some transformed cells become immunologically invisible by passive avoidance of immune surveillance (i.e., when tumor cells are immunologically indistinguishable from normal cells). Other transformed cells actively secrete cytokines that effectively blind the immune system to the presence of abnormal antigens on the tumor cell surface. Transforming growth factor- (TGF-), which is expressed by a majority of malignant tumors, is the most potent immunosuppressor and therefore, the most likely cytokine to be responsible for the latter phenomenon. In addition to playing a key role in tumor-induced immunosuppression, TGF- stimulates angiogenesis. Interestingly, tumor cells eventually become refractory to TGF--mediated growth arrest, either due to loss of TGF- receptors or due to dysregulation in TGF- signaling pathways. Neutralization of TGF- or inhibition of its production is an effective method of cancer treatment in variety of animal models. Several agents targeting TGF- are in the early stages of development and include anti-TGF- antibodies, small molecule inhibitors of TGF-, Smad inhibitors and antisense gene therapy. Since tumors may express more than one isoform of TGF-, these new drugs should target all three TGF- isoforms produced by human tumors. The effects of therapies targeting TGF- are likely to be synergistic with cytotoxic chemotherapy and immunotherapy. Reversal of TGF--induced immunosuppression is a new and promising approach to cancer therapy, with potential applications in other diseases such as AIDS.     

NMR Studies of Retinoid-Protein Interactions: The Conformation of [13C]-β-Ionones Bound to β-Lactoglobulin B

Purpose. Vitamin A (retinol) and its metabolites comprise the natural retinoids. While the biological action of these molecules are thought to be primarily mediated by ca. 55 kDa nuclear retinoic acid receptors, a number of structurally similar 15-20 kDa proteins are involved in the transport, and possibly metabolism, of these compounds. The milk protein -lactoglobulin B (-LG) is an 18 kDa protein which binds retinol and may be involved in oral delivery of retinol to neonates. -LG also binds drugs and other natural products and is of potential interest as a protective delivery vehicle. Methods. To examine the conformation of the model retinoid -ionone both in solution and when bound to -LG, NMR and computational methods have been employed. Results. Taken together, NMR studies of -ionone in solution measuring scalar and dipolar coupling, as well as CHARMm calculations, suggest -ionone prefers a slightly twisted 6-s-cis conformation. Isotope-edited NMR studies of ^l3C-labeled -ionones bound to -LG, primarily employing the HMQC-NOE experiment, suggest -ionone also binds to -LG in its 6-s-cis conformation. Conclusions. The methods employed here allow estimates of protein-bound ligand conformation. However, additional sites of ligand labeling will be necessary to aid in binding site localization.     

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.     

Deformation Behaviors of Tolbutamide, Hydroxypropyl-β-Cyclodextrin, and Their Dispersions

Purpose. The deformation behaviors of compressedfreeze-dried and spray-dried tolbutamide/hydroxypropyl--cyclodextrinmolecular dispersions were evaluated and compared with similarly preparedtolbutamides (TBM), hydroxypropyl--cyclodextrins (HP--CD) and astheir physical dispersions. Methods. TBM, HP--CD, and their 1:1 moleculardispersions were prepared by freeze-drying and spray-drying, and physicaldispersions of TBM and HP--CD were blended. Deformation properties ofthe prepared materials were evaluated by using a compaction simulator andconstants derived from Heckel plots. Molecular dynamics (MD) simulationswere performed in order to gain a molecular-level view on the deformationbehavior of TBM-HP--CD inclusion complex. Results. The freeze-dried TBM polymorphic form II was lessprone to overall particle deformation than the spray-dried stable form I.Formation of molecular dispersions decreased the plastic and elasticbehaviors of these materials. Also, the MD simulations showed a reducedmolecular flexibility of the TBM-HP--CD inclusion complex, as comparedto HP--CD. Conclusions. The formation of TBM and HP--CDmolecular dispersion resulted in more rigid molecular arrangements, whichwere less prone to deformation than either HP--CDs or physicaldispersions. The results showed how differing molecular, solid, particle,and powder state properties affect the deformation properties of thematerials studied.     

Controlled Systemic Absorption and Increased Anesthetic Effect of Bupivacaine Following Epidural Administration of Bupivacaine-Hydroxypropyl-β-Cyclodextrin Complex

Purpose. To investigate the influence of complexation between bupivacaine and hydroxypropyl--cyclodextrin (HP--CD) on the systemic absorption and on the pharmacodynamic effect of bupivacaine following epidural administration in a rabbit model. Methods. Bupivacaine and bupivacaine-HP--CD complex were administered according to a randomized and cross-over design in six rabbits chronically instrumented with an epidural catheter. The plasma concentrations of bupivacaine and the duration and intensity of the motor blockade were evaluated. Results. Complexation with HP--CD led to a decrease in the maximum plasma concentration of bupivacaine. Individual absorption kinetics evaluated by Loo-Riegelman absorption analysis indicated that systemic absorption resulted from two parallel first-order processes. Only the faster absorption phase was slowed by complexation with HP--CD. The duration of the motor blockade was increased almost twice but the intensity was not modified. Conclusions. Complexation with HP--CD could be a promising drug delivery system to improve the therapeutic index of bupivacaine.     

The Interaction of Charged and Uncharged Drugs with Neutral (HP-β-CD) and Anionically Charged (SBE7-β-CD) β-Cyclodextrins

Purpose. The objective of this work was to determine the role that charge might play in the interaction of charged and uncharged drugs with neutral (2-hydroxypropyl--cyclodextrin, HP--CD) and anionically charged (SBE7--CD) modified -cyclodextrins. SBE7--CD is a sulfobutyl ether, sodium salt, derivative variably substituted on the 2-, 3- and the 6-positions of -cyclodextrin. The number seven refers to the average degree of substitution. Methods. The binding of the acidic drugs, indomethacin, naproxen and warfarin and the basic drugs, papaverine, thiabendazole, miconazole and cinnarizine with the two cyclodextrins was determined at 25°C as a function of pH and cyclodextrin concentration by the phase-solubility method. Results. Except for miconazole and cinnarizine (A_P-type diagrams), all other materials studied displayed A_L-type diagrams. By comparing the binding constants of both the charged and uncharged forms of the same drugs to both HP--CD and SBE7--CD, the following conclusions could be drawn. The binding constants for the neutral forms of the drugs were always greater with SBE7--CD than with HP--CD. For the anionic agents, the binding constants between SBE7--CD and HP--CD were similar while the binding constants for the cationic agents with SBE7--CD were superior to those of HP--CD, especially when compared with the neutral form of the same drug. Conclusions. A clear charge effect on complexation, attraction in the case of cationic drugs and perhaps inhibition in the case of anionic drugs, was seen with the SBE7--CD.     

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 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.     

Different effects of vinblastine on the polymerization of isotypically purified tubulins from bovine brain

Vinblastine, a highly successful antitumor drug, targets the tubulin molecule. Tubulin, the subunit protein of microtubules, consists of an - and a -subunit, both of which consist of isotypes encoded by different genes. We have purified three isotypes of bovine brain tubulin, namely, _II, _III and _IV. Microtubule associated protein-2 (MAP2) and Tau-induced assembly of these isotypes were compared in the presence and absence of vinblastine. MAP2-induced assembly of unfractionated tubulin and all the isotypes except _II tubulin was resistant to 1M vinblastine. Vinblastine at low concentrations (<10M) progressively inhibited the assembly of all of the isotypes but the vinblastine concentration required for inhibition of MAP2-induced microtubule assembly was minimal for _II. The tau-induced assembly of unfractionated tubulin and _III were equally sensitive to 1M vinblastine whereas _II and _IV were much more sensitive to vinblastine. The microtubules obtained in the presence of tau from unfractionated tubulin, _II and _IV could be easily aggregated by 20M vinblastine whereas such as aggregation of microtubules obtained from _III and tau required approximatedly 40M vinblastine. Our results suggest that among the tubulin isotypes, _II is the most sensitive to vinblastine in the presence of MAPs while _III is the most resistant and this intrinsic resistance of _III dimers persists in the polymeric form of _III tubulin as well. These results may be relevant to the therapeutic and toxic actions of vinblastine.     

Die Beziehungen von Lernvermögen und Spontanmotilität alternder Ratten unter Behandlung mit Natrium-Pentamethylenhydroxybutyrat

Summary The response rate in conditioned reflexes in aged rate is augmented by sodium -pentamethylene--hydroxybutyrate. Doses of sodium -pentamethylene--hydroxybutyrates stimulating the central nervous system, neither enhance spontaneous motor activity nor depress appetite.