ABSTRACT: PURPOSE: The aim of this study was to elucidate the effects of sex hormones that are secreted during gestation from the placenta on ABCG2 mRNA and protein expression levels by using the placental cell line BeWo. METHODS: We investigated the effects of estrogens (estrone, 17-β-estradiol and estriol) on the expression level of ABCG2 mRNA by RT-PCR. The expression level of ABCG2 protein was analyzed by Western blot analysis. We also investigated the localization of ABCG2 in BeWo cells by Western blot analysis of the plasma membrane fraction and by immunohistochemistry. RESULTS: It was found that all estrogens induce the expression of ABCG2 mRNA in a concentration-dependent manner. Furthermore, Western blot analysis showed that 17-β-estradiol induces the expression of ABCG2 protein.
Abstract PURPOSE: Overactivation of nitric oxide and protein kinase C (PKC) pathway has been reported to play a role in the pathogenesis of vascular hyporesponsiveness of endotoxic shock. In this study we investigated the role of nitric oxide and PKC in lipopolysaccharide (LPS) mediated vascular hyporeactivity. METHODS: Contraction to phenylephrine and endothelium-dependent and independent vasodilation in the presence and absence of a nonspecific NO inhibitor (L-NAME) and potent PKC inhibitor (chelerythrine) were examined.
Abstract: PURPOSE: To evaluate the permeability enhancing effects of octylglucoside (OG) for molecules with poor absorption such as insulin by in vitro cell models. METHODS: Transepithelial electrical resistance (TEER) was monitored to ensure monolayer integrity. Permeability was ascertained using paracellular markers. Markers and insulin were dissolved in Hanks balanced salt solution and placed on the apical side of the cells in Transwell® plates and allowed to diffuse under sink conditions. RESULTS: The effect of OG on the permeability of molecules across both monolayers was concentration and time dependent. Enhanced transport of the three molecules was observed across both monolayers treated with OG as compared to untreated monolayers.
ABSTRACT Purpose: The aim of this work was to evaluate the pulmonary antimetastatic activity and the systemic toxicity of camptothecin-loaded microspheres. Methods: PCL microspheres containing camptothecin (CPT) were prepared by the emulsion solvent/evaporation method and characterized according to their encapsulation efficiency, particle size, morphology, and drug release. The ability of CPT to inhibit the lung metastasis was verified using an experimental mouse model intravenously injected with metastatic B16-F10 melanoma cells. The microspheres and the free drug were given intraperitoneally at a dose of 7 mg/kg at intervals of three or five days for 24 days.
ABSTRACT. Purpose: To test the effect of 72 h water deprivation on the non-renal clearance (CL) of DA-8159 in a rat model of dehydration. DA-8159 is mainly metabolized via CYP3A1/2 and the expression and mRNA level of CYP3A1/2 are not affected by dehydration. Methods: DA-8159 (30 mg/kg) was administered intravenously or orally to male control Sprague–Dawley rats and rat model of dehydration. Results: As expected, after intravenous administration, the CLNR values of DA-8159 were comparable between two groups of rats.
Abstract Increased oxidative stress has been implicated in the mechanisms of delayed neuronal cell death following cerebral ischemic insult. In this study, we investigated whether safranal, an active constituent of Crocus sativus L. stigmas, may ameliorate ischemia-reperfusion injury (IRI)-induced oxidative damage in rat hippocampus. Male NMRI rats were divided into six groups, namely, sham, control, ischemia and ischemia treated with safranal (four groups). The transient global cerebral ischemia was induced using four-vessel-occlusion method for 20 min. Safranal was injected intraperitoneally (727.5 mg/kg, 363.75 mg/kg, 145.5 mg/kg, and 72.75 mg/kg body weight) 5 min. prior to reperfusion and the administration was continued every 24 hours for 72 hours after induction of ischemia. The markers of oxidative stress including thiobarbituric acid reactive substances (TBARS), total sulfhydryl (SH) groups and antioxidant capacity of hippocampus (using FRAP assay) were measured.
Abstract PURPOSE. The generation of reactive oxygen species and lipid peroxidation are associated with tissue injury following ischemic insult; therefore, the use of antioxidants appears rational in the improvement of kidney diseases therapy. The aim of the present study was to assess the effect of aqueous saffron extract (Crocus sativus L.) and its active constituent, crocin, on oxidative stress following renal ischemia-reperfusion injury (IRI) in rats. METHODS. The cellular redox status (thiobarbituric acid reactive species (TBARS) and total thiol levels) and antioxidant power (using ferric reducing/antioxidant power test) were assessed in control and ischemic groups. The left kidney was exposed to warm ischemia for 60 min followed by reperfusion for 90 min.
Purpose: A facile method was established to enzymatically synthesize rhapontigenin from the glycosylated parent compound rhaponticin. A novel and simple high-performance liquid chromatographic method was developed for the determination of rhapontigenin. The assay was successfully applied to both the in vitro and in vivo metabolic kinetic study of rhapontigenin. Methods: Serum, or microsomes (0.1 mL) was precipitated with acetonitrile after addition of the internal standard, daidzein. Separation was achieved on an amylose tris 3,5 dimethylphenylcarbamate column (150 x 4.6mm, ID, 5μm) with UV detection at 324nm. Hep G2 hepatoma cells were treated with rhapontigenin or rhaponticin (0-250 μg/mL) and cell viability was measured. Results: The calibration curves were linear ranging from 0.5 to 100 mg/mL. The mean extraction efficiency was > 99%.
ABSTRACT Expression of both pro- and anti-inflammatory mediators are influenced by various factors such as rheumatic diseases, myocardial infarction, angina, aging, obesity and pharmacotherapy. This has therapeutic consequences. Clearance of highly bound and efficiently metabolized drugs may be reduced in the presence of inflammation amounting to increased circulating drug concentration. In the meantime, various cardiovascular receptors are down-regulated in the presence of pro-inflammatory mediators. Consequently, conditions such as rheumatoid arthritis, aging and obesity results in reduced response to drugs such as verapamil despite increased drug concentration.
Abstract PURPOSE. The present study was carried out to evaluated acute and subacute toxicity of a hydroalcoholic extract from aerial parts of Wedelia paludosa (Asteraceae). METHODS. Toxicity of W. paludosa was evaluated in Swiss mice after ingestions of the extract during one day (acute model) and during 15 days (subacute model). RESULTS. The results showed that the LD50 of the extract is higher than 4000 mg/kg and the subacute treatment did not shows any change in corporal weight and hematological parameters. However, a change in liver weight but not in hepatic enzymes was observed. This suggests that the liver function is not altered by Wedelia paludosa in this study. Some changes in the creatinine content were observed, but could not be related with the extract dose. CONCLUSIONS. The results suggest that the plant seems to be destituted of toxic effects in mice.
FDA utilizes a risk assessment strategy to evaluate the potential immunogenicity of novel protein therapeutics and licensed products following manufacturing changes.
The first two parts of this article discussed FDA's assessment of the critical factors influencing the immunogenicity of such products and the consequences of immune responses to treated patients. The first part focused on the clinical consequences of immune responses to protein therapeutics, while the second part examined product- and patient-related factors that influence immunogenicity. This third and final part focuses on the effects of changes in manufacturing and the utility of animal models in assessing a product's immunogenicity.
EFFECTS OF MANUFACTURING CHANGES ON IMMUNOGENICITY
Abstract :Dynorphins, such as dynorphin A(1–13) (Dyn A(1–13)), have been shown to enhance analgesia in morphine-tolerant animals, despite their very short half-life after intravenous administration. The potential use of dynorphins in humans is therefore of interest. This laboratory has recently evaluated the metabolic fate of stabilized dynorphin derivatives. This study was conducted to evaluate whether such stabilized derivatives, ie, [N-Met-Tyr1]-Dynorphin A(1–13) (N-MT Dyn A, stabilized at the N-terminal end) and [N-Met-Tyr1]-Dynorphin A(1–13) amide (N-MT Dyn A amide, stabilized at the C- and N-terminal ends), would enhance the antinociceptive activity of morphine not only after intravenous administration but also after subcutaneous and pulmonary delivery. Intravenous administration of N-MT Dyn A (5 µmol/kg) and N-MT Dyn A amide (5 µmol/kg) to morphine-tolerant rats resulted in significantly higher tail-flick latencies than those observed for the saline group.
This work was presented in part at the American Society of Clinical Pharmacology and Therapeutics Annual Meeting, March 24-27, 2004, Miami Beach, FL.
Abstract: Biliary excretion is an important route of elimination and the biliary tract is a potential site of toxicity for many drugs and xenobiotics. Quantification of biliary excretion in healthy human volunteers is logistically challenging and is rarely defined during drug development. The current study uses a novel oroenteric tube coupled with a specialized clinical protocol to examine the pharmacokinetics of 99mTechnetium (Tc-99m) mebrofenin, a compound that undergoes rapid hepatic uptake and extensive biliary excretion. A custom-made multilumen oroenteric tube was positioned in the duodenum of healthy human volunteers. Subjects were positioned under a gamma camera and 2.5 mCi of Tc-99m mebrofenin was administered intravenously.
The objective of the study was to determine the region of maximum permeation of salmon calcitonin (sCT) through the gastrointestinal tract and to investigate the mechanism of permeation. For regional permeability determination, male Sprague-Dawley rats (250-300 g) were anesthetized and the gastrointestinal tissues were isolated. Stomach, duodenum, jejunum, ileum, or colon tissues were mounted on Navicyte side-by-side diffusion apparatus. Salmon calcitonin solutions (50 µM in phosphate-buffered saline, pH 7.4, 37°C) were added to the donor side, and the samples were removed from the receiver compartment and analyzed by competitive radioimmunoassay (RIA). For mechanistic studies, Caco-2 cells were grown on Transwell inserts (0.4-µm pore size, 0.33 cm2 area) in a humidified 37°C incubator (with 5% CO2).
The presence of halogens within the classical cannabinoid structure leads to large variations in the compounds’ potencies and affinities for the CB1 receptors. To explore the structure activity relationships within this class of analogs we have used a series of halogen-substituted (-)-Δ8-tetrahydrocannabinol analogs and compared their affinities for the CB1 cannabinoid receptor.Our results indicate that halogen substitution at the end-carbon of the side chain leads to an enhancement in affinity with the bulkier halogens (Br, I) producing the largest effects. Conversely, 2-iodo substitution on the phenolic ring leads to a 2-fold reduction in affinity while iodo-substitution in the C1′-position of the side chain lowers the compound’s affinity for CB1 by more than 8-fold. The pharmacophoric requirements resulting from halogen-substitution are explored using computer modeling methods.
The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes.
We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry.
mRNA expression profiles had previously been measured in Caco-2 cells (human colonic carcinoma cells) using either custom-designed spotted oligonucleotide arrays or Affymetrix GeneChip oligonucleotide arrays. The Caco-2 cells used were from different clones and were examined under slightly different culture conditions commonly encountered when Caco-2 cells are used as a model tissue for studying intestinal transport and metabolism in different laboratories. In this study, we compared gene expression profiles of Caco-2 cells generated with different arrays to assess the validity of conclusions derived from the 2 independent studies, with a focus on changes in transporter and ion channel mRNA expression levels on Caco-2 cell differentiation. Significant changes in expression levels upon differentiation were observed with 78 genes, with probes common to both arrays. Of these, 18 genes were upregulated and 36 genes were downregulated.
The toxicity of 2 new synthetic lipids, 1,2-dioleoyl-rac-glycerol-3-dodecaethylene glycol, GDO-12 (lipid 1) and 1,2-distearoyl-rac-glycerol-3-dodecaethylene glycol, GDS-12 (lipid 2) has been evaluated in acute and subchronic toxicity studies. Acute oral toxicity studies in male and female rats documented no deaths or treatment-related signs at high doses.
The lipids were individually administered (by gavage) to male and female Sprague-Dawley rats at concentrations of 250, 500, and 1000 mg/Kg bodyweight for 28 days. All animals survived the duration of the study, with no significant changes in clinical signs, hematological parameters, organ weights, ophthalmology evaluations, or histopathological findings. These studies establish that both GDO-12 (lipid 1) and GDS-12 (lipid 2) are nontoxic in rats following oral administration. The no-observed-adverse-effect level ranged between 250 mg/Kg and 1000 mg/Kg following oral administration.
The purpose of this study was to assess whether male rats whose testosterone levels were suppressed to castration levels (<0.5 ng/mL) for a 1-year period by the sustained delivery of orntide acetate, a GnRH antagonist, would return to fertility (ie, produce offspring) after serum testosterone returned to control levels. Male rats comprising a treatment group (orntide microspheres, dose = 27 mg/kg/y), a vehicle control group, and a control group of proven male breeders were used. For the treatment and vehicle control groups, serum orntide and testosterone levels were monitored at periodic intervals for 14 months from the initiation of treatment. After serum testosterone levels returned to vehicle control levels and orntide serum levels were no longer discernible for the treated group, each of the animals was housed with 2 drug-naive, female, proven breeders.
Thrombopoietin, TPO, a 353 amino acid cytokine, is a primary regulator of platelet production that was cloned recently. A target-mediated (platelet receptors) pharmacokinetic model was developed to characterize the disposition of TPO. Receptor-mediated endocytosis was assigned as the major elimination pathway in the model. A nonspecific binding compartment was also incorporated into the model. TPO concentration vs time profiles from a published phase 1 and 2 clinical trial were used to apply this model. Noncompartmental analysis demonstrated that TPO exhibits nonlinear kinetics. The proposed model captured the concentration-time profiles relatively well. The first-order internalization rate constant was estimated as 0.1 h-1. The endogenous binding capacity was estimated as 164.0 pM.
Multidrug resistance-associated protein 1 (MRP1) is one of the major proteins shown to mediate efflux transport of a broad range of antitumor drugs, glucuronide conjugates, and glutathione, in addition to endogenous substrates. Significant differences in substrate selectivity were reported for murine and human MRP1. As preclinical drug disposition and pharmacokinetics studies are often conducted in rats, we have recently cloned the rat MRP1 (rMRP1) and demonstrated that rMRP1 expressed in transfected cells effluxes calcein, a commonly used fluorescence substrate for human MRP1. To further characterize the rat ortholog of MRP1, we isolated a cell line stably expressing recombinant rMRP1. These cells were tested for their ability to transport calcein and a range of chemotherapeutic drugs. Our results showed that cells expressing rMRP1 consistently efflux calcein at a rate 5-fold greater than control cells.