Novel Ethanol-in-Fluorocarbon Microemulsions for Topical Genetic Immunization

Purpose. Traditionally, vaccines have been administered by needle injection. Topical immunization through the intact skin with either protein- or DNA-based vaccines has attracted much attention recently. We sought to enhance the immune responses induced by DNA-based vaccines after topical application by developing novel ethanol-in-fluorocarbon (E/F) microemulsion systems to aid in the delivery of plasmid DNA (pDNA). Methods. Ten different fluorosurfactants were selected or synthesized and screened by pseudo-phase-diagram construction for their ability to form E/F microemulsions. Plasmid DNA was successfully incorporated into E/F microemulsions using several different fluorosurfactants and perfluorooctyl bromide as the continuous fluorocarbon phase. For several reasons, Zonyl® FSN-100 (an ethoxylated nonionic fluorosurfactant) was selected for further studies. In vivo studies were performed in mice to assess pDNA expression in skin and immunologic responses after topical application of this system using a luciferase-encoding plasmid (CMV-luciferase) and a CMV--galactosidase-encoding plasmid, respectively. Results. Plasmid DNA incorporated into E/F microemulsion using FSN-100 as the surfactant was found to be stable. After topical application of this E/F microemulsion system, significant enhancements in luciferase expression and antibody and T-helper type-1 biased immune responses were observed relative to those of naked pDNA in saline or ethanol. For example, with the E/F microemulsion system, the specific serum IgG and IgA titers were increased by 45-fold and over 1000-fold, respectively. Conclusion. A novel fluorocarbon-based microemulsion system for potential DNA vaccine delivery was developed.     

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.     

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.     

Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.     

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.     

A New Specific Enzyme Immunoassay Allowing an Efficient Pharmacokinetic Evaluation of γ-Cyclodextrin After Intravenous Administration to Rats

Purpose. Because of its ability to form complexes with drugs, -cyclodextrin is of great potential value in pharmaceutical formulations. The biological fate of -cyclodextrin must therefore be considered in safety evaluation, using sensitive and specific methods applicable to biological fluids. Methods. Antibodies were raised against -cyclodextrin, allowing the development of a new enzyme immunoassay. The analytical characteristics of this assay were evaluated. Rats were given a single intravenous 25 mg/kg dose of -cyclodextrin. Plasma and urine samples were collected and assayed. Results. This new enzyme immunoassay was sensitive (limit of detection close to 94 pg/mL) and suitable for quantification of -cyclodextrin in urine and plasma after methanol extraction. The use of different linear and cyclic compounds demonstrated the high specificity of the assay. After i.v. administration, the concentration of -cyclodextrin rapidly decreased in the plasma while the molecule was probably distributed into the tissues. Although urinary elimination predominates, only 50% of the injected -cyclodextrin was recovered in urine, suggesting enzymatic degradation and/or tissular storage. Conclusions. This assay may provide important information on the fate of -cyclodextrin inclusion complexes dedicated to drug-delivery using various modes of administration (oral, parenteral, transmucosal or dermal).     

Thermodynamics of Binding of Neutral Molecules to Sulfobutyl Ether β-Cyclodextrins (SBE-β-CDs): The Effect of Total Degree of Substitution

Purpose. To understand the role of degree of substitutionon binding of molecules to -Cyclodextrins (-CDs) with varyingdegrees of sulfobutyl ether (SBE) substitution. Methods. Using UV spectroscopy, complexation constants ofmolecules to SBE--CDs were estimated as a function of temperature,allowing for calculation of thermodynamic parameters, including the enthalpyand entropy of binding. Results. Binding constants of various molecules toSBE--CDs did not show a uniform trend to total degree of SBEsubstitution. However, a distinct pattern was observed with the enthalpy andentropy of complexation. The results showed the complexation of substratesto SBE--CDs to be more entropy-favored as the number of SBE groupsincreased. This favorable entropy of interaction was compensated by a lessfavorable enthalpy of interaction. Conclusions. Enthalpy and entropy of complexation providedadditional insight into the role that the alkylsulfonate groups may play inthe complexation of molecules with SBE--CDs.     

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.     

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.     

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.     

Pulmonary Absorption of Insulin Mediated by Tetradecyl-β-Maltoside and Dimethyl-β-Cyclodextrin

Purpose. To determine if tetradecyl--maltoside (TDM) and dimethyl--cyclodextrin (DMCD) enhance pulmonary absorption of insulin and to investigate if they do so by a reversible action on respiratory epithelium. Methods. Insulin formulated with saline, TDM, or DMCD was administered intratracheally, after laryngoscopic visualization, as a spray to anesthetized rats. Reversibility studies were conducted in intact rats by administering insulin at different time points after administration of TDM or DMCD. The pharmacodynamics and pharmacokinetics of insulin formulations were assessed by measuring plasma glucose and plasma insulin concentrations. Results. When insulin formulated with increasing concentrations (0.06-0.25%) of TDM or DMCD were administered to anesthetized rats, there was a concentration-dependent decrease in plasma glucose and increase in plasma insulin concentrations. The relative bioavailability of insulin formulations containing TDM was higher (0.34-0.84%) than that of formulations containing DMCD (0.19-0.48%). When insulin was administered 120 min after an agent was administered, in the reversibility study, no significant change in plasma glucose and insulin levels occurred compared to control. Conclusions. Both TDM and DMBCD enhance pulmonary absorption of insulin, with TDM being more efficacious than DMCD in enhancing insulin absorption via pulmonary administration. The effects of TDM and DMCD on respiratory epithelium are reversible, and the epithelium reestablishes its normal physiologic barrier 120 min after exposure to these agents.     

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.     

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

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

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.     

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.     

Synthesis and β-Adrenergic Activities of R-Fluoronaphthyloxypropanolamine

Purpose. Many biogenic amines where an aromatic proton is substituted with fluorine have exhibited pharmacological properties that are dependent on the position of fluorine on the aromatic ring. For example, 6-fluoroepinephrine is selective for -adrenergic receptors whereas the 2-fluoroisomer is selective for -receptors. Aryloxypropanolamines are -receptor agonists or antagonists, depending on the aryl group and its substituents. We therefore hypothesized that fluorine substitution on the aromatic ring could lead to significant biological effects in this class. A target with fluorine on naphthyl group of a known -antagonist was chosen for investigation. Methods. Synthesis of the target compound began with fluoronaphthalene and involved introduction of 4-hydroxy group by Friedel-Crafts acylation followed by Baeyer Villiger oxidation. The side chain was introduced stereoselectively using the chiral synthon (2R)-glycidyl 3-nitrobenzenesulfonate, a Sharpless epoxidation technique. The epoxide was opened with t-butyl amine. HPLC methods were used to characterize %ee of the enantiomer. Results. The target compound was synthesized in several hundred milligram quantity, and in good yield and high enantiomeric excess, showing practicality of the synthetic scheme. It exhibited potent binding activities on -adrenergic receptors, and was found to be two times selective for ₂-receptors over ₁. Conclusions. The current report demonstrates that aromatic fluorine substitution on -adrenergic ligands can be achieved, and that such can be used to obtain binding selectivity between receptors.     

Transport of Biologically Active Interferon-gamma Across Human Skin in Vitro

Purpose. Several studies have suggested epidermal uptake of cytokines, such as interferons, can be facilitated using topical liposomal formulations. We have evaluated the in vitro transport of biologically active recombinant human interferon- (rhIFN-) into and through split-thickness human skin to assess this possibility. Methods. Skin samples were exposed to rhIFN- under various conditions involving hydrated and dry surface conditions in the presence and absence of liposomes. A new low-level ELISA and an anti-viral bioassay were used to quantitate transported rhIFN-. Immunohistochemical staining for ICAM-1 expression by keratinocytes was used to visualize the extent and distribution of rhIFN- transport. Results. Apparent steady-state transport of rhIFN- occurred within the first 5 hours of exposure with approximately 10% of transported rhIFN- demonstrating bioactivity. While the permeability of rhIFN- across human skin under drying conditions was enhanced by the presence of liposomes, no augmentation of permeability was observed when the skin was kept hydrated. Liposomal formulations of rhIFN-; had greater transport rates than aqueous formulations when the applied formulations were allowed to dry after dosing. Conclusions. Our results demonstrate the transport of biologically active rhIFN- across human skin in vitro and suggest a role for stratum corneum hydration as one possibility for the augmented cytokine transport.     

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.     

Synthesis and Cytotoxic Evaluation of a Series of γ-Substituted γ-Aryloxymethyl-α-methylene-γ-butyrolactones Against Cancer Cells

Purpose. The main objective of this investigation was to explore thecytotoxic structure-activity relationships of -substituted -aryloxymethyl--methylene--butyrolactones against cancer cells. Methods. The target compounds were synthesized in two stepscommencing with aryl-OH which was treated with a bromomethyl ketonefollowed by the Reformatsky-type condensation. Results. Seven types of -methylene--butyrolactones were evaluatedin vitro against 60 human cancer cell lines derived from nine cancercell types. The average values of log G₅₀ indicated that for thearylportion, potencies of these -methylene--butyrolactones are in adecreasing order of quinolin-2(1H)-one (or 2-hydroxyquinoline, 21,–5.89) > quinoline (19, –5.79) > 2-methylquinoline (20, –5.69)> 8-hydroxyquinoline (17, –5.64) > 2-naphthalene (16, –5.59)> benzene (15, –4.90). The same order was obtained for both log TGIand log LC₅₀. However, for the -substituent, the potencies are in adecreasing order of biphenyl (16f–21f) > phenyl and4-substituted phenyl (16b-e–21b-e) > methyl (16a–21a). Conclusions. Unlike cardiovascular activities of -methylene--butyrolactones in which a -methyl substituent is necessary for vasorelaxingeffect while a phenyl or a halogen-substituted phenyl is prefer for theantiplatelet activities, a -biphenyl substituent proved to be the bestfor their cytotoxicities against various cancer cell lines tested.     

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.