Osthole inhibited the activity of CYP2C9 in human liver microsomes and influenced indomethacin pharmacokinetics in rats.

Osthol, a pharmacologically active ingredient in various traditional Chinese medicines, is predominantly metabolized by CYP2C9. It may be co-administered with other drugs which are metabolized by CYP2C9 in clinical medicine. However, CYP2C9*1/*2/*3 genotype on the pharmacokinetics of osthole and its metabolic diversity between rat and human are unclear.In this study, we investigated the effects of osthole on enzyme activity of CYP2C11/CYP2C9 in rat liver microsomes (RLMs) and human liver microsomes (HLMs), to distinguish metabolic manner of osthole in different species. Interestingly, we found that osthole inhibits the activity of CYP2C11 in a non-competitive manner in RLMs, while inhibits CYP2C9 activity in a competitive manner in pooled HLMs. Then, the effects of CYP2C9*1/*2/*3 allele on the pharmacokinetics of osthole were identified. In human CYP2C9 isoform, the Ki value of 21.93 µM (CYP2C9*1), 18.10 µM (CYP2C9*2), 13.12 µM (CYP2C9*3) indicate that there are individual differences in the inhibition of osthole on CYP2C9 activity.We investigated how the indomethacin pharmacokinetics was affected by osthole in SD rat. To estimate the area under the curve (AUC), maximum plasma concentration (Cmax) and apparent clearance (CL/F), indomethacin (10 mg/kg) was given orally combined with osthole (20 mg/kg) in adult SD rat. We found the value of PK on indomethacin, such as the AUC0-∞, was from 176.40 ± 17.29 to 173.74 ± 27.69 µg/ml h-1, Cmax from 9.02 ± 1.24 to 9.89 ± 0.82 µg/ml and CL/F from 0.11 ± 0.01 to 0.12 ± 0.04 mg/kg/h which were unsignificantly changed compared with the control groups. However, the Tmax was prolonged from 2.00 ± 0.00 h to 7.33 ± 1.15 h, and T1/2 increased from 8.38 ± 2.30 h to 11.37 ± 2.11 h. These results indicate that osthole could potentially affect the metabolism of indomethacin in vivo.

Attenuating Effect of Telfairia occidentalis on Oxidative Stress in Indomethacin-induced Gastric Ulcer in Male Rats.

Peptic ulcer is a major health challenge with high morbidity and mortality all over the world. This study investigated the involvement of oxidative stress in the healing and protective potentials of aqueous leave extract of Telfairia occidentalis (TO) on indomethacin induced gastric ulcers in adult Sprague Dawley male rats. The rats were divided into 6 groups (A-F) of 5 rats each, with A as normal control, B received single oral administration of 40mg/kg indomethacin without treatment for 4 hours; C received 40mg/kg indomethacin without treatment for 4 hours and scarified after 72 hours; D received 100mg/kg aqueous leave extract of TO for 7 days without ulcer induction; E (pre-treated test group) received 40mg/kg indomethacin after being pre-treated with 100mg/kg aqueous leave extract of TO daily for 7 days. Group F (Posttreated test) received 40mg/kg of indomethacin and treated four hours later with 100mg/kg aqueous leave extract of TO daily for 7 days. The results revealed changes in gastric macroscopic architecture of the mucosa, and changes in ulcer indices and oxidative stress markers levels in group B-F. These changes comparatively suggested that the leave-extract of Telfairia occidentalis has gastro-protective with minimal healing potentials mediated through reduced oxidative stress.

Ex vivo evaluation of a microneedle array device for transdermal application.

A new approach of transdermal drug delivery is the use of microneedles. This promising technique offers the potential to be broadly used for drug administration as it enables the dramatic increase in permeation of medicaments across the stratum corneum. The potential of microneedles has evolved to spawn a plethora of potential transdermal applications. In order to advance the microneedle capabilities and possibly revolutionize advanced drug delivery, this study introduces a novel transdermal electro-modulated hydrogel-microneedle array (EMH-MNA) device composed of a nano-porous, embeddable ceramic microneedle array as well as an optimized EMH for the electro-responsive delivery of indomethacin through the skin. The ex vivo permeation as well as drug release experiments were performed on porcine skin tissue to ascertain the electro-responsive capabilities of the device. In addition, the microbial permeation ability of the microneedles across the viable epidermis in both microneedle-punctured skin as well as hypodermic needle-punctured skin was determined. Ex vivo evaluation of the EMH-MNA device across porcine skin demonstrated that without electro-stimulation, significantly less drug release was obtained (±0.4540mg) as compared to electro-stimulation (±2.93mg).

Gastro-protective effects of isobrucein B, a quassinoid isolated from Picrolemma sprucei.

Infusions of Picrolemma sprucei roots, stems and leaves are used in traditional medicine throughout the Amazon region from the Guianas to Brazil and Peru in the treatment of gastritis, intestinal helminths and malaria. As there are no studies describing its mode of action in providing a gastroprotective effect, we determined herein that one of the main constituents found in P. sprucei infusions, the quassinoid isobrucein B (IsoB), reduces some of the pathophysiological effects in a mouse model of non-steroidal anti-inflammatory drug (NSAID)-induced gastritis and provides mechanisms of action. Then, IsoB (1.17 g) was isolated from the roots and stems (6.5 kg) of P. sprucei. Its structure was confirmed by 1D and 2D (1)H and (13)C NMR, ESI-tof-MS, IR and UV. C57BL/6 strain mice were subcutaneously injected with IsoB (0.5-5 mg kg(-1)) or vehicle before oral administration of indomethacin and sacrificed later at different time points. Gastric damage was assessed by measuring lesion length. Leukocyte migration was evaluated based on leukocyte rolling and adhesion using intravital microscopy in the mesenteric microcirculation and tissue MPO activity. Stomach extract cytokine (TNFα, IL-1ß and KC/CXCL1) and prostaglandin E2 (PGE2) levels were measured by ELISA and RIA, respectively. IsoB pre-treatment (0.5-5.0 mg kg(-1)) significantly reduced the formation of indomethacin-induced stomach lesions in a dose-dependent manner. The decrease in stomach lesions was associated with less observed leukocyte rolling, decreased leukocyte adhesion and less neutrophil infiltration (MPO activity). IsoB (1 mg kg(-1)) pre-treatment did not prevent indomethacin-induced decreases in stomach PGE2 levels. However, IL-1ß and KC/CXCL1 levels were inhibited by this same IsoB dosage, whereas TNF-α was unchanged. IsoB may be a prototypic compound to provide protective effects against NSAID-induced gastritis and possibly other gastropathies. Moreover, IsoB gastroprotective action may be due to a reduction in IL-1ß and KC/CXCL1 production/release and leukocyte rolling, adhesion and migration.

Microemulsion for simultaneous transdermal delivery of benzocaine and indomethacin: in vitro and in vivo evaluation.

This study investigated simultaneous transdermal delivery of indomethacin and benzocaine from microemulsion. Eucalyptus oil based microemulsion was used with Tween 80 and ethanol being employed as surfactant and cosurfactant, respectively. A microemulsion formulation comprising eucalyptus oil, polyoxyethylene sorbitan momooleate (Tween 80), ethanol and water (20:30:30:20) was selected. Indomethacin (1% w/w) and benzocaine (20% w/w) were incorporated separately or combined into this formulation before in vitro and in vivo evaluation. Application of indomethacin microemulsion enhanced the transdermal flux and reduced the lag time compared to saturated aqueous control. The same trend was evident for benzocaine microemulsion. Simultaneous application of the two drugs in microemulsion provided similar enhancement pattern. The in vivo evaluation employed the pinprick method and revealed rapid anesthesia after application of benzocaine microemulsion with the onset being 10 min and the action lasting for 50 min. For indomethacin microemulsion, the analgesic effect was recorded after 34.5 min and lasted for 70.5 min. Simultaneous application of benzocaine and indomethacin provided synergistic effect. The onset of action was achieved after 10 min and lasted for 95 min. The study highlighted the potential of microemulsion formulation in simultaneous transdermal delivery of two drugs.

Anti-ulcerogenic mechanisms of the sesquiterpene lactone onopordopicrin-enriched fraction from Arctium lappa L. (Asteraceae): role of somatostatin, gastrin, and endogenous sulfhydryls and nitric oxide.

Arctium lappa L. has been used in folk medicine as a diuretic, depurative, and digestive stimulant and in dermatological conditions. The mechanisms involved in the anti-ulcerogenic activity of the sesquiterpene onopordopicrin (ONP)-enriched fraction (termed the ONP fraction), obtained from A. lappa leaves, were studied. The gastroprotective mechanism of the ONP fraction was evaluated in experimental in vivo models in rodents, mimicking this disease in humans. ONP fraction (50 mg/kg, p.o.) significantly inhibited the mucosal injury induced by ethanol/HCl solution (75%), indomethacin/bethanecol (68.9%), and stress (58.3%). When the ONP fraction was investigated in pylorus ligature, it did not induce alteration in the gastric volume but did modify the pH and total acid concentration of gastric juice. ONP fraction significantly increased serum somatostatin levels (82.1±4.1 vs. control group 12.7±4 pmol/L) and decreased serum gastrin levels (62.6±6.04 vs. control group 361.5±8.2 µU/mL). Mucus production was not significantly altered by the ONP fraction. Gastroprotection by the ONP fraction was completely inhibited by N-ethylmaleimide treatment and did not modify the effect in the animals pretreated with l-N(G)-nitroarginine methyl ester. These results suggest an antisecretory mechanism involved with the antiulcerogenic effect of the ONP fraction. However, only endogenous sulfhydryls play an important role in gastroprotection of the ONP fraction.

Paracoccidioides brasiliensis uses endogenous and exogenous arachidonic acid for PGE x production.

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids during fungal infections plays a critical role on fungal biology as well as on host immune response modulation. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, Bt79, Pb192) produce prostaglandin E(x) (PGE(x)). Moreover, we asked if P. brasiliensis could use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) enzyme, as reported for mammalian cells. A possible association between this prostanoid and fungus viability was also assessed. Our results showed that all strains, independently of their virulence, produce high PGE(x) levels on 4 h culture that were reduced after 8 h. However, in both culture times, higher prostanoid levels were detected after supplementation of medium with exogenous AA. Treatment with indomethacin, a COX inhibitor, induced a reduction on PGEx, as well as in fungus viability. The data provide evidence that P. brasiliensis produces prostaglandin-like molecules by metabolizing either endogenous or exogenous AA. Moreover, the results suggest the involvement of these mediators on fungal viability.

Design and in vitro evaluation of formulations with pH and transit time controlled sigmoidal release profile for colon-specific delivery.

The primary objective of the study was to develop a pH and transit time controlled sigmoidal release polymeric matrix for colon-specific delivery of indomethacin. Tablet matrices were prepared using a combination of hydrophilic polymers (polycarbophil or carbopol) having pH-sensitive swelling properties with hydrophobic polymer ethyl cellulose. The prepared matrices were characterized for physical properties and in vitro release kinetics. The presence of ethyl cellulose in a hydrophilic polymer matrix resulted in a sigmoidal in vitro drug release pattern with negligible-to-very low drug release in the initial phase (0-6 h) followed by controlled release for 14-16 h. The retardation in initial release can be attributed to the presence of ethyl cellulose that reduced swelling of hydrophilic polymer(s), while in the later portion, polymer relaxation at alkaline pH due to the ionization of acrylic acid units on carbopol and polycarbophil resulted in enhanced drug release. Thus, a sigmoidal release pattern was obtained that could be ideal for colonic delivery of indomethacin in the potential treatment of colon cancer.

Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from nonhepatotoxic drugs? An analysis of 18 drugs with consideration of intrinsic clearance and daily dose.

In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.

Permeation studies of indomethacin from different emulsions for nasal delivery and their possible anti-inflammatory effects.

The purpose of this research was to develop an emulsion formulation of indomethacin (IND) suitable for nasal delivery. IND was incorporated into the oil phases of oil in water (O/W) and water in oil (W/O) emulsions. For this purpose, different emulsifying agents (Tween 80, Span 80 and Brij 58) were used in two emulsion formulations. When the effects of several synthetic membranes (nylon, cellulose, cellulose nitrate) were compared with the sheep nasal mucosa, the cellulose membrane and sheep nasal mucosa showed similar permeation properties for O/W emulsion (P > 0.05). To examine the absorption characteristics of IND, the anti-inflammatory properties of intravenous solution of IND, intranasal O/W emulsions of IND (with or without enhancers) and intranasal solution of IND (IND-Sol) were investigated in rats with carrageenan-induced paw edema. When citric acid was added to the nasal emulsion, the anti-inflammatory activity was similar to that of intravenous solution (P > 0.05). Finally, it was concluded that, intranasal administration of IND emulsion with citric acid may be considered as an alternative to intravenous and per oral administrations of IND to overcome their adverse effects.

Prostaglandin involvement in follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles.

The aim of the present study was to evaluate the role of prostaglandin (PG) on proliferation of granulosa cells from prehierarchical small yellow follicles (SYF) of buff laying hens. The granulosa layers were separated by mechanic method and dispersed into single cells. After 16 h pre-incubation in 0.5% FCS medium, the medium was replaced with serum-free medium, which was supplemented with 10 microg/ml insulin, 5 microg/ml transferrin and 3 x 10(-8)M selenite. Cells were challenged with PGE1 and FSH for 24 h and then assessed for proliferation. The results showed that PGE(1) (0.1-10 ng/ml) had a similar proliferating effect as FSH on granulosa cells, and these stimulating effects were restrained by the PGE receptor antagonist SC19220 at 10(-7) to 10(-5)M. Prostaglandin synthase antagonist indomethacin (10(-7) to 10(-5)M) suppressed FSH-induced increase in the number of granulosa cells in a dose-dependent manner. Downstream activation of protein kinase A by forskolin-activated adenylate cyclase resulted in elevated proliferation of granulosa cells, an effect unobserved by phorbol-12-myristrate-13-acetate-activated protein kinase C. In addition, PGE1-stimulated proliferation of granulosa cells was hindered by H89 (PKA inhibitor) but not by H7 (PKC inhibitor). Furthermore, the proliferating cell nuclear antigen labeling index (PCNA-LI) of granulosa cells displayed similar changes with the number of cells. These results indicated that PGE1 promoted the proliferation of granulosa cells from SYF and was also involved in mediating FSH-stimulated intracellular PKA signal transduction.

Metabolic O-demethylation does not alter the influence of isoflavones on the biophysical properties of membranes and MRP1-like protein transport activity.

Multidrug resistance transporter MRP1 could be effectively inhibited by some flavonoids. The influence of the two pairs of isoflavones: formononetin/daidzein and biochanin A/genistein on the efflux of fluorescent substrate of MRP1-like protein from erythrocytes and biophysical properties of lipid membranes has been compared. Compounds in each pair differ by the substituent in position 4' of B ring of isoflavone molecule. In the process of O-demethylation, CH(3) group (present in formonetin and biochanin A) is replaced by hydrogen (daidzein, genistein). Inhibition of MRP1-like protein transport activity by methylated and demethylated isoflavones was very similar. Their influence on lipid thermotropic properties and fluidity of lipid bilayer was not also significantly different.

Alginate-pectin-poly-L-lysine particulate as a potential controlled release formulation.

Drug delivery particulates were prepared using alginate, polylysine and pectin. Theophylline, chlorothiazide and indomethacin were used as the model drugs for in-vitro assessments, and mannitol was the model for assessing paracellular drug absorption across Caco-2 cell monolayers. Alginate and pectin served as the core polymers and polylysine helped to strengthen the particulates. Use of pectin specially helped in forming a more robust particulate that was more resistant in acidic pH and modulated the release profiles of the encapsulated model drugs in the alkaline pH. Alginate and pectin were also found to enhance the paracellular absorption of mannitol across Caco-2 cell monolayers by about three times. The release rate could be described as a first-order or square-root time process depending on the drug load. Use of alginate-polylysine-pectin particulates is expected to combine the advantages of bioadhesion, absorption enhancement, and sustained release. This particulate system may have potential use as a carrier for drugs that are poorly absorbed after oral administration.

Characteristics of shed snake skin permeability to indomethacin and fatty alcohols.

To investigate the utilities of a shed snake skin as a model membrane for preclinical studies of transdermal drug delivery, the flux of indomethacin was determined under various conditions by using a diffusion cell. The flux of fatty alcohols was determined and compared with that in human skin reported in references. The esterase activity of shed snake skin was also determined. It was found that the flux of indomethacin decreased with an increase of pH and the amount of ethanol in a vehicle. The flux of indomethacin increased by the addition of Azone, N-methyl-2-pyrrolidone and N,N-dimethyl-m-toluamide in the cream. The flux of fatty alcohols in shed snake skin was greater than that reported in human skin, and shed snake skin had similar esterase activity to human skin.

Distribution of indomethacin in human milk and estimation of its milk to plasma ratio in vitro.

1. The distribution of indomethacin in fat and protein fractions of colostrum and mature milk as well as its milk to plasma drug concentration ratio (M/P ratio) were determined in vitro. 2. The extent of plasma protein binding of indomethacin (5-20 microg ml(-1)) was > or = 99.6%. The protein binding of indomethacin in colostrum was 46.0% at pH 7.4. The lower protein content of mature milk compared with colostrum was associated with a significant decrease in the extent of drug protein binding (46 +/- 1.93 to 35 +/- 1.0 s.e. mean). Protein binding was also decreased significantly in 8% fat mature milk (20.3 +/- 2.4 s.e mean) but was constant over the pH range 7.4 to 6.8. 3. About 40% of indomethacin added to milk was associated with the fatty layer. The indomethacin M/P ratio determined by equilibrium dialysis was less than 0.01. Hence the maximum infant daily dose was estimated to be 0.006 mg kg(-1). 4. Our results indicate that indomethacin transfers to milk by simple diffusion according to its physicochemical properties, and that treatment with indomethacin is not a contraindication to breast feeding.

Xenobiotic triacylglycerol formation in isolated hepatocytes.

The formation of neutral lipophilic metabolites from five xenobiotic carboxylic acids was studied in isolated rat hepatocytes. Oleic acid was used as a positive control. Rates of formation of lipids lay in the order: oleic acid greater than phytanic acid greater than ibuprofen greater than 3-phenoxybenzoic acid greater than indomethacin and 3-phenylbutanoic acid (rates were undetectable with the last two substrates). The process was saturable with the maximum rates at about 0.5 mM substrate concentration. Supplementation of the hepatocyte system with glycerol enhanced the yields of lipid products. The hepatocytes also effectively modelled the in vivo metabolism of ibuprofen, 3-phenoxybenzoic acid and 3-phenylbutanoic acid with oxidations and classical conjugation reactions predominating over xenobiotic lipid formation.

Cod liver oil inhibits indomethacin induced gastropathy without affecting its bioavailability and pharmacological activity.

The upper gastrointestinal toxicity is one of the most common side effects associated with the use of nonsteroidal anti-inflammatory drugs (NSAIDs). Many attempts to prepare potent NSAIDs free from gastrotoxicity have failed. Hence, development of formulations to mask the gastropathy of NSAIDs are warranted. The present study was undertaken to investigate the effect of concomitant use of cod liver oil (CLO) on pharmacological activity and gastropathy of indomethacin in rats. The animals were treated with CLO (5 and 10 ml/kg body weight) along with indomethacin (30 mg/kg, body weight). Blood samples were collected for analysis of indomethacin at 0.25, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0 and 24 hours. The anti-inflammatory activity of indomethacin alone and in combination with CLO was studied using carrageenan-induced paw oedema. Our studies related to the effect of these drugs on gastrointestinal tract showed that concurrent use of CLO protects gastric mucosa against indomethacin induced depletion of gastric wall mucus, non protein sulfhydryl (NP-SH) levels and gastric lesions. The result of this study also showed that the concurrent use of the CLO does not affect the bioavailability and anti-inflammatory activity of indomethacin while it inhibits the ulcerogenic effect of indomethacin in a dose dependent manner. These findings suggest that NSAIDs formulations containing CLO may reduce gastrotoxicity without affecting their therapeutic efficacy.

A new anti-inflammatory derivative of imidazole which is less ulcerogenic than indomethacin in rats.

The mechanism of the anti-inflammatory activity and ulcerogenicity of (2-dimethylamino-1(2)-methyl) ethyl ester of the 1-(2-carboxyethyl)-2-(p-chlorophenyl)-4,5-bis-(p-methoxyphenyl)-imidazole (A-162-ester) was compared with that of indomethacin in rats and enzymatic preparations derived from other species. A-162-ester was found to be deesterified in plasma to A-162. A-162-ester was about 3 times less anti-inflammatory and about 17 times less ulcerogenic than indomethacin. A-162-ester, when given orally, decreased prostaglandin I2 (PGI2) biosynthesis by gastric mucosa. The IC50 was close to the ulcerogenic ED50. Indomethacin--in the same test--was 37 times more potent than A-162-ester and the PGI2 inhibition and ulcerogenic dose-response curves for indomethacin were parallel. In other in vitro systems of prostaglandin (PG) biosynthesis, the inhibitory activity of A-162 was comparable to that of indomethacin. It is concluded that the ulcerogenicity of indomethacin results from a high affinity of this drug to gastric mucosal wall PG-synthetase which leads to decreased PGI3 formation at this site. The relatively low ulcerogenicity of A-162-ester most probable results from a lower affinity of this drug to the same site.