Oral Administration Of Compound Research Articles

Two xanthones from Swertia punicea with hepatoprotective activities in vitro and in vivo

Ethnopharmacological relevanceSwertia punicea Hemsl. (Gentianaceae) is more commonly known as “Ganyan-cao” and used mainly as a traditional Chinese folk medicine for the treatment of acute bilious hepatitis, cholecystitis, fever, intoxification and jaundice. Materials and methodsThe active hepatoprotective constituents of Swertia punicea were purified using various column chromatography techniques. The structures of two isolated compounds were determined on the basis of spectroscopic data interpretation such as NMR analysis. The hepatoprotective activities of isolated compounds were evaluated by using hepatotoxicity in vitro and dimethylnitrosamine-induced rat hepatic fibrosis in vivo, respectively. ResultsTwo xanthones, 1, 7-dihydroxy-3, 4, 8-trimethoxyxanthone (1) and bellidifolin (2) were isolated from the stems of Swertia punicea. The compounds 1 and 2 exhibited notable hepatoprotective activities against carbon tetrachloride (CCl4) -induced HepG2 cell damage, and effectively alleviated the levels of aspartate transaminase (AST), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malonic dialdehyde (MDA) induced by CCl4 in a concentration-dependent manner. Co-treatment with compound 2 significantly increased the cell viability compared with N-acetyl-p-aminophenol (APAP) treatment. Compound 2 also alleviated APAP-induced hepatotoxicity by increasing glutathione (GSH) content and decreasing hydroxyl free radical (·OH) levels and reactive oxygen specises (ROS) production. In addition, the protective effect of compound 1 significantly alleviated DMN-induced liver inflammation and fibrosis. Oral administration of compound 1 recovered the reduction of albumin (ALB) and reversed the elevation of serum alanine transaminase (ALT), AST and total bilirubin (TBIL) in dimethylnitrosamine (DMN)-induced fibrotic rats. Severe oxidative stress induced in fibrotic rats was evidenced by a 1.5-fold elevation in MDA and a fall in the SOD activity, and treatment with compound 1 protected against these adverse effects. Recovery of rat liver tissue against DMN-induced hepatocellular necrosis, inflammatory changes and hepatic fibrosis by compound 1 is also confirmed by H&E and Masson stained histopathological evaluation of liver tissue. ConclusionTwo xanthones from Swertia punicea exhibited hepatoprotective activities in vitro (compounds 1 and 2) and in vivo (compound 1), respectively.

Pharmacological Evaluation of Selectiveα2c-Adrenergic Agonists in Experimental Animal Models of Nasal Congestion

Nasal congestion is one of the most troublesome symptoms of many upper airways diseases. We characterized the effect of selective α2c-adrenergic agonists in animal models of nasal congestion. In porcine mucosa tissue, compound A and compound B contracted nasal veins with only modest effects on arteries. In in vivo experiments, we examined the nasal decongestant dose-response characteristics, pharmacokinetic/pharmacodynamic relationship, duration of action, potential development of tolerance, and topical efficacy of α2c-adrenergic agonists. Acoustic rhinometry was used to determine nasal cavity dimensions following intranasal compound 48/80 (1%, 75 µl). In feline experiments, compound 48/80 decreased nasal cavity volume and minimum cross-sectional areas by 77% and 40%, respectively. Oral administration of compound A (0.1-3.0 mg/kg), compound B (0.3-5.0 mg/kg), and d-pseudoephedrine (0.3 and 1.0 mg/kg) produced dose-dependent decongestion. Unlike d-pseudoephedrine, compounds A and B did not alter systolic blood pressure. The plasma exposure of compound A to produce a robust decongestion (EC(80)) was 500 nM, which related well to the duration of action of approximately 4.0 hours. No tolerance to the decongestant effect of compound A (1.0 mg/kg p.o.) was observed. To study the topical efficacies of compounds A and B, the drugs were given topically 30 minutes after compound 48/80 (a therapeutic paradigm) where both agents reversed nasal congestion. Finally, nasal-decongestive activity was confirmed in the dog. We demonstrate that α2c-adrenergic agonists behave as nasal decongestants without cardiovascular actions in animal models of upper airway congestion.

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M1 and M4 muscarinic acetylcholine receptors agonists

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M1 and M4 muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M1 and M4 agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.

Improvement of Pulmonary Function by Oral Treatment with a VLA-4 Antagonist in a Mouse Asthmatic Model

We investigated in vivo efficacies of the newly synthesized VLA-4 antagonist Compound A {trans-4-[1-[[2,5-Dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid} on Ascaris antigen–induced airway inflammation and hyperresponsiveness in a murine asthmatic model. Oral administration of Compound A significantly inhibited eosinophil infiltration into BALF and airway hyperresponsiveness 48 h after the antigen challenge. Histologic analysis of the lung sections confirmed the BALF result and revealed suppression of edema and mucus hyperplasia at 8 and 48 h after the challenge, respectively. These findings clearly show that orally active Compound A has therapeutic potential for treatment of asthma.

Simultaneous determination of a p38 MAP kinase inhibitor and its amide hydrolyzed metabolite in Cynomolgus monkey plasma by LC–MS/MS, and its application to a toxicokinetic study

A LC–MS/MS method was developed for the determination of a p38 MAP kinase inhibitor (Compound I) and its amide hydrolyzed metabolite (M7) in Cynomolgus monkey plasma over the concentration range of 1.00–1000 ng/mL. Stable isotope labeled compounds (d 3-Compound I and d 3-M7) were used as internal standards (IS). Samples were prepared using protein precipitation in the 96-well format with a 30 μL plasma sample volume. Chromatographic separation was performed with reversed-phase liquid chromatography on a Varian Monochrom C 18 (100 mm × 2.00 mm, 5 μm) analytical column. The mobile phases were 5 mM ammonium formate in acetonitrile/water (95/5, v/v) pH 7.0 and 5 mM ammonium formate in acetonitrile/water (5/95, v/v) pH 7.0. Gradient elution, at a flow rate of 550 μL/min, was used to separate Compound I and M7. Positive atmospheric pressure chemical ionization was utilized with detection by multiple reaction monitoring (MRM). Total run time was about 3.2 min. This method was validated following the current Food and Drug Administration (FDA) guidance for bioanalytical method validation. The intra- and inter-day precision (% CV) and accuracy (% bias) at all concentrations tested were below 15% for both analytes. The mean recoveries for Compound I, M7, d 3-Compound I, and d 3-M7 were 106%, 107%, 108% and 105%, respectively. The method was successfully applied to support a GLP toxicokinetic study in Cynomolgus monkeys after oral administration of Compound I. A total of 48 samples (∼12.5% of the total number of samples) were selected for incurred sample reanalysis (ISR). The % difference between the reassay concentrations and the original concentrations were all less than 20% of their mean values and met the acceptance criteria for ISR.

A Nonsecosteroidal Vitamin D Receptor Modulator Ameliorates Experimental Autoimmune Encephalomyelitis without Causing Hypercalcemia

Vitamin D receptor (VDR) agonists are currently the agents of choice for the treatment of psoriasis, a skin inflammatory indication that is believed to involve an autoimmune component. 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active metabolite of vitamin D, has shown efficacy in animal autoimmune disease models of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and type I diabetes. However, the side effect of 1,25-(OH)2D3 and its synthetic secosteroidal analogs is hypercalcemia, which is a major impediment in their clinical development for autoimmune diseases. Hypercalcemia develops as a result of the action of VDR agonists on the intestine. Here, we describe the identification of a VDR modulator (VDRM) compound A that was transcriptionally less active in intestinal cells and as a result exhibited less calcemic activity in vivo than 1,25-(OH)2D3. Cytokine analysis indicated that the VDRM not only modulated the T-helper cell balance from Th1 to Th2 effector function but also inhibited Th17 differentiation. Finally, we demonstrate that the oral administration of compound A inhibited the induction and progress of experimental autoimmune encephalomyelitis in mice without causing hypercalcemia.

Preparation of blood-brain barrier-permeable paclitaxel-carrier conjugate and its chemotherapeutic activity in the mouse glioblastoma model

The per oral administration of compound 1, prepared by covalently attaching paclitaxel to a molecular carrier, shows good anti-tumor activity in the orthotopic mouse model of glioblastoma. The anti-tumor effects may be attributable to the cytotoxicity as well as the anti-angiogenic activity of the paclitaxel conjugate or paclitaxel itself in the brain parenchyma.

Characterization of α 4β 1 (CD49d/CD29) on equine leukocytes: Potential utility of a potent α 4β 1 (CD49d/CD29) receptor antagonist in the treatment of equine heaves (recurrent airway obstruction)

The purpose of this study was to characterize the α 4β 1 receptor (CD49d/CD29, very late antigen-4, VLA-4) on circulating equine leukocytes and to evaluate the intrinsic potency of an α 4β 1 receptor antagonist (Compound B) in the horse. Ultimately, these studies would allow us to determine the suitability of treating recurrent airway obstruction (RAO; heaves) affected horses by blocking the cellular recruitment of lymphocytes and neutrophils into the lung. The data demonstrates the α 4β 1 integrin is present on horse lymphocytes and neutrophils (fluorescence-assisted cell sorter, FACS) and can bind low molecular weight α 4β 1 antagonists (Compounds A and B) with high affinity. K D values for the binding of Compound A to non-activated α 4β 1 on isolated horse PBMCs (peripheral blood mononuclear cells) and activated neutrophils were 17 pM and 27 pM, respectively. Compound B was identified as a suitable antagonist for performing a series of in vivo experiments. Compound B was found to possess excellent potency in horse whole blood, possessing IC 50 and IC 90 values of 39 pM and 172 pM, respectively. This represents a 3.9-fold molar excess of drug over the α 4β 1 concentration in blood. Following oral administration of Compound B (5 mg/kg) to beagle dogs and rhesus monkeys, rapid and sustained α 4β 1 receptor occupancy (>80%) was achieved and maintained for a period of 24 h. When Compound B was administered intravenously to the horse, by either a slow or rapid infusion at a dose of 0.3 mg/kg, receptor blockade of >80% was observed out to 24 h with a concomitant leukocytosis. We believe that Compound B possesses suitable intrinsic and pharmacological properties to be evaluated clinically in horses affected by RAO.

Inhibition of dipeptidyl peptidase IV by novel inhibitors with pyrazolidine scaffold

Inhibition of dipeptidyl peptidase IV (DPP-IV) activity has been reported to improve nutrient-stimulated insulin secretion through the stabilization of glucagon-like peptide (GLP-1). In the present study, we identified novel DPP-IV inhibitors of pyrazolidine derivatives (Compounds 1 and 2) and characterized their biological effects in vitro and in vivo. Compound 1, an isoleucine pyrazolidide with a phenyl urea group, inhibited rat plasma DPP-IV, porcine kidney DPP-IV, as well as human Caco-2 DPP-IV with IC 50 values of 1.70, 2.26, and 2.02 μM, respectively. Because of the poor pharmacokinetic properties of Compound 1, further optimization was carried out, leading to the discovery of Compound 2, which had similar in vitro activities. Compound 2 acted as a selective and competitive inhibitor of DPP-IV. MALDI-TOF mass spectrometric analysis proved that the compound (20 μM) effectively blocked the degradation of active GLP-1 peptide by 61%. Although similar in in vitro potency, marked improvement of in vivo efficacy and pharmacokinetic properties was seen with Compound 2. Oral administration of Compound 2 resulted in potent and rapid inhibition of circulating DPP-IV in C57BL/6J mice, with ED 50 values of 26 mg/kg (s.c.) and 42 mg/kg (p.o.). In addition, this compound improved glucose tolerance in ob/ob mice, as determined by an oral glucose tolerance test (OGTT). These results indicate that Compound 2 is a potent and selective DPP-IV inhibitor with oral anti-hyperglycemic activity in vivo.

Simultaneous determination of a novel KDR kinase inhibitor and its N-oxide metabolite in human plasma using 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry

To support pharmacokinetic studies, a selective and sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the simultaneous determination of a novel KDR kinase inhibitor ( 1) and its active metabolite ( 2) in human plasma. The method is fully automated using a Packard MultiPROBE II system and a TomTec Quadra 96 liquid handling workstation to perform sample preparation and solid-phase extraction (SPE). Following the extraction on a mixed-mode SPE using Oasis MCX 96-well plate, the analytes were separated on a Aquasil C18 column (50 mm × 2.1 mm, i.d., 3 μm) with a mobile phase consisting of acetonitrile/ammonium acetate buffer (5 mM, pH 5.0) (60/40, v/v). The run time for each injection was 4.5 min with the retention times of approximately 2.0 and 2.7 min for 1 and 2 respectively, at a flow rate of 0.25 mL/min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) under the positive ion mode with a turbo ion-spray interface. The linear ranges of the calibration curves were 0.05–400 ng/mL for 1 and 0.1–400 ng/mL for 2 on a PE Sciex API 4000 LC–MS/MS system. The lower limits of quantitation (LLOQ) of the assay were 0.05 and 0.1 ng/mL for 1 and 2 respectively, when 0.4 mL of plasma was processed. Intra-day assay precision (using five standard curves prepared by spiking compounds to five lots of plasma) was less than 4.9% for 1 and less than 9.6% for 2 on each concentration. Assay accuracy was found to be 95.1–104.6% of nominal for 1 standards and 93.5–105.6% for 2 standards. QC samples were stable when kept at room temperature for 4 h, at −70 °C for 10 days, and after three freeze–thaw cycles. The extraction recoveries were 80%, 83% and 84% for 1 and 2 and I.S. respectively, and no significant matrix effects were observed. The method was successfully applied to plasma samples from clinical studies after oral administration of compound 1.

Activation of Insulin Signal Transduction Pathway and Anti-diabetic Activity of Small Molecule Insulin Receptor Activators

We recently described the identification of a non-peptidyl fungal metabolite (l-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T. , Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974-977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.

Stereoselective determination of a new antidepressant, E2011, and its diastereoisomer as a metabolite by high-performance liquid chromatography

A stereoselective HPLC method has been developed for the determination of E2011 (compound I) and one of its metabolites and diastereoisomers, ER-20593 (compound II), in plasma. The two substances and the internal standard were extracted from plasma, followed by two purification steps. In the first step, a minicolumn, Bond Elut C 1 8, was used and in the second step, another minicolumn, Bond Elut Si, was used for purification of the compounds. After the purification, the compounds were analyzed by HPLC with two Chiralpak AD columns. In this procedure, compounds I and II were stable and there was no chiral inversion. The within-day and the between-day assays were performed in rat plasma, where compounds I and II existed simultaneously. The within-day and the between-day precisions of compound I were 2.0∼10.1% and 1.3∼7.1%, and the within-day and the between-day accuracies were −8.2∼+3.0% and −6.6∼+4.0% in the concentration range 0.003–10 μg ml −1. The within-day and the between-day precisions of compound II were 1.7∼16.9% and 0.9∼4.5% and the within-day and the between-day accuracies were −9.0∼+2.4% and −5.6∼+3.8% in the concentration range of 0.005-0.5 μg ml −1. QC samples for compound I and II were stable for at least 3 months. The method was applied to measure the levels of compound I and II in the rat plasma after oral administration of compound I.

Simultaneous Determination of a New Dihydropyridine Calcium Blocker and Its Pyridine Metabolite in Dog Plasma by Column Switching High-Performance Liquid Chromatography with Electrochemical and Ultraviolet Detection

A highly sensitive, selective and rapid reversed-phase liquid chromatographic method using a column-switching technique has been developed for the simultaneous determination of a new dihydropyridine calcium blocker, methyl (±)-4, 7-dihydro-3- isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2, 3-b]pyridine-5-carboxylate (I) and its pyridine metabolite, methyl 3-isobutyl-6-methyl-4-(3-nitrophenyl)thieno[2, 3-b]pyridine-5-carboxylate (II), in dog plasma. Compounds I and II were determined with electrochemical detection at +0.50V and by ultraviolet detection at 250nm, respectively. The limits of the determination for compounds I and II were 0.2 and 2ng ml-1, respectively, using 1ml of dog plasma. Good liner relationships were found between the peak areas and the plasma concentrations of compounds I and II in the ranges from the lower limit of determination to 200ng ml-1. The method was applied to preliminary pharmacokinetic studies after oral administration of compound I to beagle dogs.

Cholinergic vasodilatory effect of an imidazoindole on the nephrosclerosis of hypertensive rats

Pyridino [1,2-a] imidazo [5,4-b] indole (Compound 1) has been reported previously as an antihypertensive agent. Parenteral introductions of Compound 1 in spontaneously hypertensive rats (SHR) and in normotensive dogs have been found to reduce systolic blood pressure (SBP) in those animal models. Later studies have shown cholinomimetic effects of Compound 1 in animal tissue preparations and in live animals. Our experiments showed that daily oral administration of Compound 1 minimized the development of hypertensive nephrosclerosis and prevented premature death in male SHR. Compound 1 also potentiated cholinergic activity in isolated rabbit hearts. It is suggested that cholinergic vasodilation by Compound 1 is responsible for the hypotensive effect and prevention of nephrosclerosis in SHR.

Effects of an α2-adrenoceptor antagonist on glucose tolerance in the genetically obese mouse ( [formula omitted][formula omitted

This study examines the effects of a relatively selective α 2-adrenoceptor antagonist, 8-( l-piperazinyl)imado-[1,2-α] pyrazine (compound A), and the preferential α 2-agonist clonidine on blood glucose, glucose tolerance, and plasma insulin levels in the C57BL 6J ob ob mouse and its lean littermate. While clonidine raised blood glucose levels and impaired glucose tolerance, oral administration of compound A resulted in decreased blood glucose levels, as well as improved glucose tolerance in ob ob mice. Insulin levels in ob ob mice treated with clonidine were significantly reduced, while compound A raised insulin levels threefold and blocked the effects of clonidine when co-administered to the same animals. Clonidine-induced hyperglycemia in lean littermates was not accompanied by a decrease in insulin levels, while a small but significant increase in insulin levels was observed by compound A administration. Glycogen synthesis in diaphragm of ob ob mice was enhanced after oral administration of compound A and was accompanied by an increase in plasma insulin levels. Concomitant treatment with a potent somatostatin analog to inhibit insulin release blocked the effects of the α 2-adrenoceptor antagonist, compound A. These observations suggest that the α 2-antagonist studied, increased plasma insulin levels with an accompanying reduction in blood glucose and an improvement in glucose tolerance in a genetic model of insulin resistance. Differential sensitivity to α 2-agonist in these genetically obese mice, ob ob , was demonstrated by decreased insulin levels due to clonidine administration.