Polyvinyl Derivatives as Novel Interactive Polymers for Controlled Gene Delivery to Muscle

Purpose. DNA plasmids (pDNA) can be taken up by and expressed in striated muscle after direct intramuscular injection. We have developed interactive polymeric gene delivery systems that increase pDNA bioavailability to muscle cells by both protecting pDNA from nucleases and controlling the dispersion and retention of pDNA in muscle tissue. Methods. A DNA plasmid, containing a CMV promoter and a -galactosidase reporter gene (CMV--gal), was injected either in saline or formulated in polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) solutions. Interactions between PVP and pDNA were assessed by dynamic dialysis, Isothermal Titration Calorimetry (ITC), and Fourier-Transformed Infra Red (FT-IR) spectroscopy. Formulations (50 µl) were injected into rat tibialis muscles after surgical exposure. Immuno-histochemistry for -gal was used to visualize the sites of expression in muscle. Results. -gal expression using pDNA in saline reached a plateau while -gal expression using PVP formulations increased linearly in the dose range studied (12.5–150 µg pDNA injected) and resulted in an increase in the number and distribution of cells expressing -gal. The interaction between PVP and pDNA was found to be an endothermic process governed largely by hydrogen-bonding and results in protection of pDNA from extracellular nucleases. Conclusions. Significant enhancement of gene expression using interactive polyvinyl-based delivery systems has been observed. The improved tissue dispersion and cellular uptake of pDNA using polyvinyl-based systems after direct injection into muscle is possibly due to osmotic effects.     

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.     

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.     

Distribution of Gacyclidine Enantiomers in Spinal Cord Extracellular Fluid

Purpose. Determination of the pharmacokinetics of gacyclidineenantiomers, a non-competitive NMDA antagonist, in plasma and spinal cordextracellular fluid (ECF) of rats. Methods. Implantation of microdialysis probes in spinal cord (T9).Serial collection of plasma samples and ECF dialysates over 5 hoursafter IV bolus administration of (±)-gacyclidine (2.5 mg/kg). Plasmaprotein binding determined in vivo by equilibrium dialysis. ChiralGC/MS assay. Results. Plasma concentrations of (+)-gacyclidine were 25% higherthan those of (–)-gacyclidine over the duration of the experiment inall animals. Plasma concentrations decayed in parallel in a biphasicmanner (t_1/2 9 min; t_1/2 90 min) with no significant differencebetween enantiomers. Clearance and volume of distribution of(–)-gacyclidine were approximately 20% higher than those of its opticalantipode (CL: 248 vs 197 ml.kg^–1.min^–1;Vd: 31.6 vs 23.5 l/kg).Protein binding (90%) was not stereoselective. Both gacyclidineenantiomers were quantifiable in spinal cord ECF 10 min after drugadministration and remained stable over the duration of the experimentin spite of changing blood concentrations. Penetration of(–)-gacyclidine was significantly higher (40%) than that of (+)-gacyclidine inall animals. Yet, exposure of spinal cord ECF was similar for bothenantiomers, and not correlated with plasma AUCs. Conclusions. The disposition of gacyclidine enantiomers isstereoselective. Both enantiomers exhibit a high affinity for spinal cord tissueand their distribution may involve a stereoselective and active transportsystem. This hypothesis could also explain the discrepancy betweendrug concentrations in plasma and spinal cord ECF.     

Differences in ligand binding profiles between cloned rabbit and human 5-HT1Dα and 5-HT1Dβ receptors: ketanserin and methiothepin distinguish rabbit 5-HT1D receptor subtypes

The study of serotonin receptor function has been complicated by the extreme molecular diversity of serotonin receptor subtypes, the lack of selective agonists and antagonists for many of the subtypes, and divergence in the pharmacological properties of a single receptor subtype across different animal species. An example of this pharmacological diversity between species homologues is provided by the 5-HT_1D receptor subfamily. To further advance the ability to characterize and pharmacologically compare functional responses mediated by native 5-HT_1D receptors, we have cloned the 5-HT_1D and 5-HT_1D receptor subtypes from the rabbit and evaluated their pharmacological profiles using radioligand binding assays. The deduced amino acid sequences of the rabbit 5-HT_1D and 5-HT_1D receptor genes displayed 60% overall identity [75% transmembrane (TM) identity] to each other and > 90% overall identity (95% TM identity) to their corresponding human homologues. Two compounds were identified in binding assays which discriminated between the closely-related 5-HT_1D receptors. Ketanserin exhibited high affinity (pK_i = 7.66) and selectivity ( > 20-fold) for the 5-HT_1D receptor while methiothepin displayed high affinity (pK_i = 7.86) and selectivity (16-fold) for the 5-HT_1D receptor subtype. The rabbit and human recombinant 5-HT_1D receptors showed significant intraspecies (rabbit 5-HT_1D vs. 5HT_1D) and interspecies (i.e. rabbit vs. human 5-HT_1D) similarities in their ligand binding profiles. These data suggest that 5-HT_1D-mediated responses in rabbit preparations may provide information relevant to the pharmacology of the 5-HT_1D receptor subtypes in humans.     

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.     

Involvement of Multidrug Resistance Associated Protein 1 (Mrp1) in the Efflux Transport of 17β Estradiol-D-17β-Glucuronide (E217βG) Across the Blood-Brain Barrier

Purpose.The purpose of present study is to investigate the involvement of multidrug resistance-associated protein 1 (Mrp1), Mrp2, and P-glycoprotein (Mdr1a) in the efflux transport of 17—estradiol-D-17-glucuronide (E₂17G) across the blood—brain barrier (BBB). Methods. The expression of Mrp1 and Mrp2 at the BBB was investigated by RT-PCR and Western blot analyses. The time profiles of the remaining radioactivity of [³H]E₂17G in the brain were compared in wild-type, Mdr1a/Mdr1b and Mrp1 knockout mice and normal and Mrp2-deficient mutant rats [Sprague-Dawley and Eisai hyperbilirubinemic rats (EHBR), respectively] after intracerebral microinjection. Results. RT-PCR and Western blot analyses revealed the expression of Mrp1 in isolated rat brain capillary; however, RT-PCR was unable to detect any expression of Mrp2. Significant elimination of E₂17G was observed in wild-type mice at a rate constant of 0.007 min^–1, which was significantly decreased (0.004 min^–1) in Mrp1 knockout mice. In contrast, there was no difference in the efflux of E₂17G from the brain in wild-type and Mdr1a/Mdr1b knockout mice and in normal and EHBR. No significant difference was observed in the accumulation of E₂17G by brain slices prepared from wild-type and Mrp1 knockout mice. Conclusion. Mrp1, but not Mrp2, is involved in the excretion of E₂17G at the BBB and provides a barrier function by extruding conjugated metabolites into the blood.     

Alinidine reverses the descending staircase of isolated rat atria by an antimuscarinic action

Summary Contractile force of isolated atria from most mammalian species increases with the rate of electrical stimulation, resulting in an ascending staircase. In contrast, in the rat, contractile force decreases with increasing rate of stimulation (descending staircase). The bradycardic and antianginal drug alinidine (5.7–91.2 mol/l) reversed the descending staircase to ascending by a positive inotropic effect at higher stimulation rates. Maximal positive inotropy was obtained with 45.6 mol/l, a concentration which also caused maximal bradycardia in spontaneously beating atria. Concentrations of 1 mol/l of the antimuscarinic compounds atropine as well as the quarternary salt ipratropium bromide also reversed the descending staircase of rat atria. Addition of alinidine did not cause any further increase in force of contraction under these conditions. Addition of 1 mol/l physostigmine to isolated left atria from guinea pigs for blockade of acetylcholinesterase decreased contractility at all stimulation rates, but did not change the ascending character of the staircase. Alinidine antagonized the negative inotropic effect of physostigmine. The known antimuscarinic action of alinidine was quantified in electrically driven (0.25 Hz) left rat atria by antagonism of the negative inotropic effect of oxotremorine (0.01–10 mol/l). Alinidine acted as a strictly competitive antagonist with a pA₂ of 5.82. In isolated papillary muscle from guinea pigs, pretreated with reserpine for depletion of catecholamines, carbachol (0.1–3000 mol/l) exerted positive inotropic effects. Alinidine antagonized also this effect in a competitive fashion with a pA₂ value of 5.58. Investigations of the specific antimuscarinic compounds pirenzepine, methoctramine and AF-DX 116 in these models indicate that the negative inotropic effect in atria is mediated by M_2a-receptors while the positive inotropic effect in papillary muscle is mediated by either the M_2e-receptor or a yet unidentified muscarinic subreceptor.It is concluded that the descending staircase of electrically stimulated rat atria is due to the release of acetylcholine as well as to the short duration of its action potential which decreases further upon muscarinic stimulation, thus leading to a compromise of excitation-coupled calcium influx and negative inotropy, particularly at higher stimulation rates. Therefore, antimuscarinic drugs including alinidine reverse the descending staircase. Under physiological conditions of impulse generation and in the absence of vagal activity an ascending staircase is to be expected in rat atria as well.     

Novel Direct Curve Comparison Metrics for Bioequivalence

Purpose. The object of this work was to devise four new direct curve comparison (DCC) metrics and examine each metric's distribution properties and performance characteristics. Methods. DCC metrics, Cmax, and AUCi were calculated from two bioequivalence studies of three sustained release carbamazepine formulations, where a range of profile similarity was observed. DCC metric values and their confidence intervals were compared to Cmax and AUCi. Results. The DCC metrics , _m, _a, and _s exhibited more favorable distributions than Cmax and AUCi ratios, which were frequently skewed. The DCC metrics performed differently than Cmax and AUCi ratios in profile comparisons due to the nature of the DCC metrics. Unlike Cmax and AUCi, the DCC metrics utilize all data points to directly compare entire profiles. Each DCC metric appears to measure exposure in a single assessment. Possible bioequivalence acceptance criteria are: 1.40, _m 0.35, _a 0.27, and _s 0.102. Conclusions. These DCC metrics, particularly p_m, are promising bioequivalence metrics for exposure.     

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.     

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.     

Pharmacokinetics and safety of ILX23-7553, a non-calcemic-vitamin D3 analogue, in a phase I study of patients with advanced malignancies

Purpose: Differentiation therapy is an alternative to chemotherapy with potentially less toxicity, improved quality of life, and survival. We conducted a phase I trial of ILX23-7553, a formulation of 1,25-dihydroxy-16-ene-23-yne-vitamin D₃, a 1,25-dihydroxyvitamin D₃ analog with preclinically demonstrated antitumor and differentiating effects and diminished hypercalcemic effects. Patients and methods: The protocol consisted of five daily oral treatments during 14-day cycles at 15 dose levels from 1.3 to 45.0g/m²/day. We treated 42 heavily pretreated patients who had a variety of malignancies with 162 treatment cycles, and obtained pharmacokinetics from three patients at the two highest dose levels. Results: There were no grade 3 or 4 toxicities. Grade 1–2 toxicities included diarrhea, nausea, fatigue, constipation, and one grade 1 hypercalcemia. Average day 6 calcium was 9.26 ± 0.55mg/dl in cycle 1 and 9.30 ± 0.67mg/dl in cycle 2. Pharmacokinetics at dose levels 14 (40g/m²/day) (1 patient) and 15 (45g/m²/day) (2 patients) demonstrated an average C _max of 30.4 ± 7.8pg/ml (0.07nM) and 104 ± 38.2pg/ml (0.25nM), and AUCs of 222.5 ± 225.2pg·h/ml and 855 ± 536pgh/ml, respectively. Eight patients (19%) had stable disease. While in vitro effects have been reported at these concentrations, they were at least 10-fold lower than ED₅₀s, and the study was terminated before an MTD was reached. Conclusion: The drug is safe and has potential benefits at serum concentrations where effects begin to be noted in vitro. Further study is needed with a reformulated higher unit dose compound to determine the safety and efficacy of higher serum concentrations.     

Co-Administration of a Water-Soluble Polymer Increases the Usefulness of Cyclodextrins in Solid Oral Dosage Forms

Purpose. The aim of this study was to investigate the effect of cyclodextrins (-CD, HP--CD and (SBE)_7m--CD), and co-administration of a water-soluble polymer (HPMC) and cyclodextrins, on the oral bioavailability of glibenclamide in dogs. Methods. Effects of cyclodextrins on the aqueous solubility of glibenclamide, with and without hydroxypropylmethylcellulose (HPMC), were determined by a phase-solubility method. Solid inclusion complexes were prepared by freeze-drying. Glibenclamide was administered orally and intravenously to beagle dogs. Results. Aqueous solubility of glibenclamide increased as a function of cyclodextrin concentration, showing an A_L-type diagram for -CD and an A_p-type diagrams for both of the -CD derivatives studied. HPMC enhanced the solubilising effect of cyclodextrins, but did not affect the type of phase-solubility diagram. Orally administered glibenclamide and its physical mixture with HP--CD showed poor absolute bioavailability, while orally administered glibenclamide/cyclodextrin-complexes significantly enhanced the absolute bioavailability of glibenclamide. Orally administered glibenclamide/-CD/HPMC and glibenclamide/(SBE)_7m--CD/HPMC complexes showed similar absolute bioavailability compared to formulations not containing HPMC, even though 80% (in the case of (SBE)_7m--CD) or 40% (in the case of -CD) less cyclodextrin was used. Conclusions. The oral bioavailability of glibenclamide was significantly increased by cyclodextrin complexation. HPMC increased the solubilising effect of cyclodextrins and, therefore, the amount of cyclodextrin needed in the solid dosage form was significantly reduced by their co-administration. In conclusion, the pharmaceutical usefulness of cyclodextrins in oral administration may be substantially improved by co-administration of a water-soluble polymer.     

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.     

Studies on Agents with Mixed NO-Dependent Vasodilating and β-Blocking Activities

Purpose. A series of derivatives having a propranolol-like moiety linked to NO-donor furoxan substructures were synthesized. The main objective of this investigation was to obtain agents with mixed NO-dependent vasodilating and -blocking activities. Methods. Most of the target compounds were synthesized from the appropriate furoxans bearing XCH₂CH₂NH₂ (X = O, S, SO₂) chains at the 4 position of the ring, using A1(C₂H₅)₃ in methylene chloride solution and (±)2,3-epoxypropyl 1-naphtyl ether. Two of the final products (X = CONH) were obtained by coupling the appropriate furoxancarboxylic acids with N-[2-hydroxy-3-(l-naphthoxy)propyl]-ethylenediamine. ₁- and ₂-blocking activities were examined on isolated guinea pig right atria and on guinea pig trachea respectively. Vasodilating properties were assessed on endothelium denuded strips of rat aorta. Results. Some derivatives behave as well balanced 'hybrids' displaying NO-dependent vasodilating and -blocking properties in the same concentration range. Some others display either prevalent -blocking or vasodilating activity. Generally speaking hybrid formation lowers the affinity for -receptors, in particular for ₂-type, to give an increase in ₁/₂ selectivity. Conclusions. The furoxan system is a flexible tool in designing analogues of propranolol whose NO-donating and -blocking properties are modulated over a wide range.     

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.     

Effect of Testosterone Suppression on the Pharmacokinetics of a Potent GnRH Receptor Antagonist

Purpose. The expression of cytochrome P450 enzymes (CYPs) in animals and humans is under complex hormonal regulation. Chronic treatment with drugs that alter sex hormone levels such as GnRH receptor agonists or antagonists may affect the expression of hormone-dependent CYPs, and as a result the pharmacokinetics of drugs metabolized by them. Methods. Enzyme kinetic parameters were obtained by incubating AG-045572 (0.1-30 M) with human or rat liver microsomes, or expressed CYP3A4 and CYP3A5. The pharmacokinetics of AG-045572 (10 mg/kg i.v. or 20 mg/kg p.o.) were studied in intact male, female, castrated male and male rats pretreated with AG-045572 for 4 days. Results. AG-045572 is metabolized by CYP3A in both rats and humans. The K_m values were similar in male and female human, female rat liver microsomes, and expressed CYP3A4 and CYP3A5 (0.39, 0.27, 0.28, 0.25, and 0.26 M, respectively). The K_m in male rat liver microsomes was 1.5 M, suggesting that in male and female rats AG-045572 is metabolized by different CYP3A isozymes. The oral bioavailability of AG-045572 in intact male rats was 8%, while in female or castrated male rats it was 24%. Pretreatment of intact male rats with AG-045572 i.m. for 4 days resulted in suppression of testosterone to castrate levels, accompanied by an increase in oral bioavailability of AG-045572 to 27%. In the same experiment, the male-specific pulsatile pattern of growth hormone remained unchanged with slightly elevated baseline levels. Conclusions. The potent GnRH receptor antagonist AG-045572 is metabolized by hormone-dependent CYP3A. As a result, suppression of testosterone by pretreatment with AG-045572 feminized its own pharmacokinetics.     

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.     

Sodium Dependence of Nitrofurantoin Active Transport Across Mammary Epithelia and Effects of Dipyridamole,Nucleosides, and Nucleobases

Purpose. The sodium dependence and effects of nucleoside and nucleobase transport inhibitors were determined to ascertain the role of sodium dependent nucleoside or nucleobase transporters in nitrofurantoin active transport across mammary epithelia. Methods. Five lactating female rats received steady-state intravenous infusions of nitrofurantoin with and without the broad-based inhibitor dipyridamole. In the CIT3 murine model of lactation, ¹⁴C-nitrofurantoin basolateral to apical permeability was examined in the presence of varying sodium concentrations, purine and pyrimidine nucleosides and nucleobases, and dipyridamole. Results. Dipyridamole effectively inhibited ¹⁴C-nitrofurantoin flux across CIT3 cells, with K_i = 0.78 M (95% C.I. = 0.11 to 5.3 M) and significantly decreased the milk-to-serum ratio of nitrofurantoin from 29.2 5.0 to 11.0 6.3 without changing systemic clearance. Nitrofurantoin active transport was significantly inhibited by complete sodium replacement. Adenosine and guanosine significantly inhibited nitrofurantoin permeability (54.5 2.6 (l/hr)/cm² and 50.7 0.6 (l/hr)/cm², respectively, vs. control 90.5 4.6 (l/hr)/cm²) but uridine, thymidine, and the nucleobases had no effect. Conclusions. Nitrofurantoin active transport was sodium dependent and inhibited by dipyridamole, adenosine, and guanosine, but known sodium dependent nucleoside or nucleobase transporters were not involved.     

Metabolism,Distribution, and Transdermal Permeation of a Soft Corticosteroid,Loteprednol Etabonate

The soft corticosteroid, loteprednol etabonate (chloromethyl 17-ethoxycarbonyloxy-11-hydroxy-3-oxoandrosta-1,4-diene-17-carboxylate), I, was designed based on the inactive metabolite approach. Accordingly, I should be metabolized by hydrolysis to the corresponding inactive cortienic acid derivative, II. The in vitro and in vivo metabolism of I indeed yielded mainly this inactive metabolite, which is more hydrophilic and thus readily eliminated from the body. Relatively high levels of I were found in tissues after intravenous administration of the drug in rats. The permeability of I through hairless mouse skin was comparable to what has been found for related hard steroids, without significant metabolism taking place in the skin.