bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the transport of thyrotropin-releasing hormone (TRH) analogues in Caco-2 cell monolayers
Authors: Urayama Akihiko , Yamada Shizuo , Deguchi Yoshiharu , Ohmori Yuki , Kimura Ryohei
Published: June 2003

The transport mechanisms of thyrotropin-releasing hormone (TRH) and its pharmacologically active analogues ((3-methyl-His2)TRH (MeTRH), taltirelin, montirelin, azetirelin) across Caco-2 cell monolayers were characterized. The results of kinetic analysis showed a linear relationship between the concentration (over the range 0.5–10 mm) and apical-to-basolateral transport rate of these agents. The permeability coefficients (Papp) of these agents were not substantially different from each other, and their (Papp) ratios of the basolateral-to-apical over the apical-to-basolateral transport were close to one (0.73–1.23). The cellular transport of [3H]MeTRH at low concentrations (3–15 nm) showed a linear relationship between the concentration and transport rate. The transport of [3H]MeTRH in Caco-2 cell monolayers was neither affected by TRH nor TRH analogues, and there was little difference in (Papp) values between [3H]MeTRH and [14C]mannitol. The cell-per-medium ratio of [3H]MeTRH in the cellular uptake experiment was similar to the value of [14C]mannitol. A large excess of TRH and MeTRH did not significantly influence cell-per-medium ratios of [3H]MeTRH in Caco-2 cell monolayers. The k'IAM value, which represents lipophilicity, was decreased in the following order: montirelin > taltirelin > TRH > azetirelin, and the values varied from 0.234 to 1.028. These results indicate that a paracellular passive diffusion may be the major route for the transport of TRH and its analogues in Caco-2 cell monolayers.
Tolerance to the hypnotic and electroencephalographic effect of gamma-hydroxybutyrate in the rat: pharmacokinetic and pharmacodynamic aspects
Authors: Van Sassenbroeck Diederik K , de Paepe Peter , Belpaire Frans M , Boon Paul A , Buylaert Walter A
Published: June 2003

Tolerance to gamma-hydroxybutyrate (GHB) has been suggested in illicit users and has been described for the hypnotic effect in the rat. The aim of this study was to investigate whether tolerance is also observed for the EEG effect, and whether the EEG can give insight into the pharmacodynamic aspects of GHB tolerance. In three series of experiments, rats were pre-treated with either the GHB precursor gamma-butyrolactone (GBL) or saline intraperitoneally twice daily. In the first series, a reduction in sleeping time was observed in the GBL pre-treated rats compared with controls. In the second series, a fast infusion of GHB (300 mg kg−1 over 5 min) was given after 10 days pre-treatment. The GHB plasma concentration-time curves showed a slightly faster decrease in GHB concentration in the GBL pre-treated rats, suggesting a small induction of the GHB metabolism (Vmax = 2882 ± 457 μg min−1 kg−1 vs 2205 ± 315 μg min−1 kg−1, P<0.01). In contrast to controls, GBL pre-treated rats did not lose righting reflex. In the third series, a slow infusion of 480 mg kg−1 h−1 was given after 7 days pre-treatment, which allowed fitting a sigmoid Emax model to the EEG amplitude versus GHB plasma concentration curve. This showed reduced end-organ sensitivity to GHB in the GBL pre-treated rats (EC50 (concentration required to obtain 50% depression of the baseline effect) = 653 ± 183 μg mL−1 vs 323 ± 68 μg mL−1, P < 0.001). In conclusion, chronic pre-treatment with gamma-butyrolactone in the rat results in a reduced sleeping time and this tolerance is reflected by the EEG. This can mainly be explained by reduced end-organ sensitivity.
Kinetics of metabolism and degradation of mometasone furoate in rat biological fluids and tissues
Authors: Teng X W , Cutler D J , Davies N M
Published: June 2003

Mometasone furoate (MF) is a potent glucocorticoid developed for the treatment of glucocorticoid-responsive inflammatory disorders. The in-vitro and ex-vivo kinetics of the degradation and metabolism of MF were studied in selected biological fluids of rat and subcellular fractions of different rat tissues. In-vitro, MF was found to degrade slowly into four products in serum and urine, and metabolized rapidly and extensively in rat liver, minimally in extrahepatic tissues, including intestine, stomach, lung and kidney. Further investigation found that the microsomal fraction was the major intracellular site of MF 6β-hydroxylation in rat liver. Using chemical inhibitors, CYP3A was found to be the major enzyme involved in the in-vitro MF 6β-hydroxylation in rat liver microsomes. Enzyme kinetic studies in rat liver microsomes showed that the overall metabolic process of MF followed biphasic Michaelis-Menten kinetics, while 6β-hydroxylation obeyed monophasic Michaelis-Menten kinetics. The kinetic parameters derived from the kinetic models along with the enzyme inhibition studies suggest that MF is mainly metabolized via 6β-hydroxylation mediated by CYP3A primarily, and also biotransformed via other pathway(s) catalysed by other enzymes in rat liver in-vitro.
Hepatic biotransformation of the new calcium-mimetic agent, RWJ-68025, in the rat and in man — API-MS/MS identification of metabolites
Authors: Wu Wu-Nan , McKown Linda A , Rybczynski Philip J , Demarest Keith
Published: June 2003

The in-vitro biotransformation of a new calcium-mimetic agent and benzenemethanamine analogue, RWJ-68025, was studied after incubation with rat and human hepatic S9 fractions in the presence of an NADPH-generating system. Unchanged RWJ-68025 (44–48% of the sample) plus 12 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS and MS/MS data, and ethyl derivatization for phenolic and carboxylic metabolites. Four metabolic pathways for RWJ-68025 were proposed: pathway 1, O-demethylation; pathway 2, phenyl oxidation; pathway 3, methyl oxidation; and pathway 4, N-dealkylation/acetylation. Pathway 1 formed a major metabolite, O-desmethyl-RWJ-68025 (M1; RWJ-68311; 26% in rat; 16% in human fraction). Pathway 2 produced one major (M2; 12–17% in rat and human fraction) and two minor phenolic metabolites (M4 and M5; all <1% in both species), and in conjunction with step 1, formed hydroxy-M1 (M3; 4–5% in both species). Pathways 3 and 4 formed seven minor oxidized metabolites (M6–M12). RWJ-68025 was extensively metabolized in the rat and human hepatic S9 fractions.
The effects of phytoestrogenic isoflavones on the formation and disposition of paracetamol sulfate in the isolated perfused rat liver
Authors: Lucas Anthony N , Nation Roger L , Milne Robert W , Reynolds Geoffrey D , Evans Allan M
Published: June 2003

This study examines the potential for the phytoestrogenic isoflavones, a type of complementary medicine, to be involved in pharmacokinetic interactions in the liver. Rat livers were isolated and perfused to steady state, in single-pass mode, with either 5 μm paracetamol (n=6), or 5 μm paracetamol with a 50:50 molar mixture of genistein and biochanin A or daidzein and formononetin, at a total isoflavone concentration of 1 and 10 μm (n = 6 for each mixture at each concentration). At 1 μm, neither isoflavone mixture had any effect, while at 10 μm both mixtures decreased the clearance of paracetamol and the formation clearance to paracetamol sulfate. Genistein and biochanin A (10 μm) also increased the biliary extraction of hepatically-generated paracetamol sulfate. Additional livers were perfused with an infusion of 5 μm  14C-paracetamol in the absence (n = 4), or presence, of a 10 μm genistein and biochanin A mixture (n = 4). Analysis of washout perfusate and bile samples (up to 30min after stopping the infusion) revealed that the isoflavones reduced the first-order rate constant for paracetamol sulfate transport into perfusate, but not for transport into bile. The results indicate that isoflavones can reduce the formation of paracetamol sulfate and that its enhanced excretion into bile arises from the inhibition of sinusoidal efflux transport.
In-vitro antileishmanial and trypanocidal activities of arsonoliposomes and preliminary in-vivo distribution in BALB/c mice
Authors: Antimisiaris Sophia G , Ioannou Panayiotis V , Loiseau Philippe M
Published: June 2003

We have studied the antiprotozoal activity of some recently prepared and characterized arsonoliposome formulations. Plain arsonoliposomes and phosphatidylcholine arsonoliposomes prepared with palmitoyl- (C16) or lauroyl-(C12) acyl side chain arsonolipids showed in-vitro antileishmanial activity after a 72-h incubation period against wild-type promastigote forms of Leishmania donovani. The IC50 values ranged from 0.40 to 11.6 μm arsonolipid. Interestingly, all preparations tested were found to be significantly more potent against amphotericin B- or miltefosine-resistant promastigote forms of L. donovani, with IC50 values ranging between 0.21- and 2.33-μm arsonolipid. When tested in-vitro against Trypanosoma brucei brucei, all arsonoliposome formulations were found to have anti-trypanosomal activity after a 24-h incubation period. The fact that the corresponding arsonolipids (dissolved in dimethyl sulfoxide) were found not to be potent against the Leishmania promastigotes or the trypanosomes tested suggested that the formation of liposomes possibly influenced the mode of interaction between the active lipid and the parasites modulating their potency. In addition, a preliminary in-vivo study in BALB/c mice was performed for the initial evaluation of the biodistribution of arsonoliposomes. The accumulation of arsenic in the BALB/c mouse liver in relatively high amounts was an additional advantage of this approach for anti-protozoal therapy, especially for visceral leishmaniasis where parasites are located mainly in the liver.
Two lead drug designs based on chloramphenicol as the parent structure, which express alkylation activity with potential for clinical applications
Authors: Bartzatt Ronald
Published: June 2003

Chloramphenicol is a bacteriostatic antibiotic which acts primarily as an inhibitor of bacterial protein synthesis. Modification of chloramphenicol structure was accomplished by replacing the two hydroxyl groups (—OH) with a chlorine atom for LEAD-1 or a chloroethyl ether group (—OCH2CH2Cl) for LEAD-2. The resulting daughter compounds expressed significant alkylating activity at physiological temperature of 37°C and at pH 7.4. Alkylation activity was evaluated after reaction with guanosine 5′-diphosphate (GDP), l-serine, l-glutamic acid, and p-chloroaniline. The partition coefficient (log P) was determined for chloramphenicol, LEAD-1 and LEAD-2 to be 0.854, 3.409 and 3.10, respectively. The molecular dipole of chloramphenicol, LEAD-1 and LEAD-2 was calculated to be 5.804, 3.961 and 4.097 Debye, respectively. All three structures showed zero violations of the Rule of 5, which indicates good bioavailability. Values for polar surface area (TPSA) indicate an intestinal absorption of 51% and 35% for LEAD-1 and LEAD-2, respectively. The reduction to only one proton donor in LEAD-1 and LEAD-2 structures (chloramphenicol having three proton donors) indicates an improvement of membrane penetration compared to chloramphenicol. 13C NMR analysis of molecular structures was accomplished and fast atom bombardment mass spectrometry analysis of a reaction mixture showed LEAD-1 alkylation of guanosine 5′-diphosphate.
Impairment of the renal p-aminohippurate transport induced by 6-hydroxydopamine
Authors: Cojocel C , Thomson M S
Published: June 2003

In this study, the effects of 6-hydroxydopamine (6-OHDA) on renal p-aminohippurate transport were investigated in-vitro using rat renal cortical slices. Cisplatin, a known nephrotoxin, was used as positive control. Renal cortical slices were incubated for 60 min in a cisplatin-containing medium (0.83–5.0 μm) at 37°C under a 100% O2 atmosphere. In another series of experiments, renal cortical slices were incubated in a 3.33 μm cisplatin-containing medium for 15–120min or in a cisplatin-free medium. Subsequently, for each series of experiments, kidney slices were incubated at 25°C for 90 min in a media containing p-aminohippurate. In a further series of experiments, renal cortical slices were incubated for 60 min in a 6-OHDA containing medium (3.125–100 μm) at 37°C under a 100% O2 atmosphere. In another series of experiments, renal cortical slices were incubated in a 50 μm 6-OHDA-containing medium for 15–120min or in 6-OHDA-free medium. Subsequently, for each series of experiments, kidney slices were incubated at 25°C for 90 min in a media containing p-aminohippurate. The results of this study where slices were incubated in 6-OHDA- or cisplatin-containing media indicate that both 6-OHDA and cisplatin induced a time- and concentration-dependent decrease in p-aminohippurate accumulation by renal cortical slices. Therefore, similarly to cisplatin, 6-OHDA causes functional injury of renal proximal tubule cells, leading to impairment of transport processes across the cell membrane.
Effects of continuous exposure to digoxin on MDR1 function and expression in Caco-2 cells
Authors: Takara Kohji , Tsujimoto Masayuki , Ohnishi Noriaki , Yokoyama Teruyoshi
Published: June 2003

The Caco-2 cell line has been used widely for studying intestinal permeability and several transport functions, and express the multidrug resistance transporter MDR1/P-glycoprotein. Previously, the transient exposure to digoxin for 24h was found to induce MDR1 mRNA in Caco-2 cells. Here, a digoxin-tolerant Caco-2 subline (Caco/DX) was newly established by the continuous exposure of Caco-2 cells to digoxin, and the effects of continuous exposure to digoxin on MDR1 were examined. The 50% growth inhibitory concentration (IC50) values for digoxin in Caco-2 and Caco/DX cells were 17.2 and 81.4 nm, respectively. The IC50 values for paclitaxel, an MDR1 substrate, were 1.0 and 547 nm, respectively, whereas the cytotoxicity of 5-fluorouracil was comparable in both cells. The uptake and efflux of Rhodamine123, an MDR1 substrate, in Caco/DX cells were significantly less and greater, respectively, than those in Caco-2 cells, and these transports were affected by the addition of ciclosporin. The expression of MDR1 mRNA in Caco/DX cells was approximately 2- and 1.7-fold compared with Caco-2 cells and Caco-2 cells treated with 100 nm digoxin for 24 h, respectively. On the other hand, MRP1 mRNA in Caco/DX cells was unchanged. These observations confirmed that the continuous exposure to digoxin, as well as the transient exposure, induced MDR1 in Caco-2 cells.
Extracts of various species of Epilobium inhibit proliferation of human prostate cells
Authors: Vitalone Annabella , Guizzetti Marina , Costa Lucio G , Tita Beatrice
Published: June 2003

This study examined whether various species of Epilobium, a phytotherapeutic agent used in folk medicine as a treatment for benign prostatic hyperplasia, may have an antiproliferative effect in PZ-HPV-7 human prostatic epithelial cells in-vitro. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) test, [methyl-3H]thymidine incorporation into DNA and flow cytometry analysis were used to evaluate cell proliferation. Ethanolic extracts of E. spicatum, E. rosmarinifolium and E. tetragonum inhibited DNA synthesis in PZ-HPV-7 cells. While at high concentrations all extracts were cytotoxic, DNA synthesis was also decreased at levels that caused no or little cytotoxicity. Treatment of cells with Epilobium extracts did not result in a formation of DNA fragments (evaluated by the TUNEL assay) or chromatin condensation (assessed by Hoechst staining). Flow cytometry analysis indicated that Epilobium extracts inhibit the progression of the cell cycle from the G0/G1 phase. These results suggest that extracts of Epilobium inhibit proliferation of human PZ-HPV-7 cells in-vitro by affecting progression of the cell cycle. This study provides some initial biological plausibility for the use of Epilobium extracts in benign prostatic hyperplasia.
Astilbin suppresses delayed-type hypersensitivity by inhibiting lymphocyte migration
Authors: Cai Yu , Chen Ting , Xu Qiang
Published: June 2003

This study examined the effects of astilbin, a flavanone, on delayed-type hypersensitivity reactions and its mechanisms of action on cell migration. Astilbin significantly inhibited the sheep-red-blood-cell-induced footpad reaction and picryl-chloride-induced ear dermatitis without affecting the organ weights, when administered during the effector phase but not the induction phase. The flavanone also significantly inhibited the migration to gelatin of spleen cells isolated from mice with ear dermatitis in a transwell system. Furthermore, the isolated spleen cells from normal mice were incubated with astilbin in the presence of concanavalin A, or those from mice with ear dermatitis were cultured with astilbin. In the supernatants collected, the activity of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 was remarkably inhibited by astilbin. These results suggest that astilbin may inhibit delayed-type hypersensitivity reactions through selectively suppressing the lymphocyte functions, including cell migration, via down-regulating MMP activity.
Selectin-blocking semisynthetic sulfated polysaccharides as promising anti-inflammatory agents
Authors: Höpfner M , Alban S , Schumacher G , Rothe U , Bendas G
Published: June 2003

Selectin-induced leucocytes rolling along the endothelial surface of blood vessels initiate a complex adhesion cascade, which is an essential step in the cellular immune response. Consequently, blocking the binding between the selectins and their ligands represents a promising strategy for suppressing pathological inflammatory reactions. This study describes the effects of an unfractionated heparin and a low-molecular-weight heparin and a series of structurally well-defined semisynthetic glucan sulfates on selectin-mediated cell-rolling with respect to inhibition. To simulate the blood flow characteristics of postcapillary venules, the rolling experiments were performed in a dynamic-flow-chamber system with immobilized selectins and selectin ligand-carrying U937 cells. The influence of the test compounds on cell rolling was measured by the percentage of adherent cells after a certain flow time and the velocity of the rolling cells. Whereas the test compounds displayed no inhibitory effect on E-selectin-mediated cell rolling, they efficiently blocked the rolling induced by P-selectin. The glucan sulfates were much more active than either unfractionated heparin or low-molecular-weight heparin, or the standard inhibitor Sialyl LewisX. Their inhibitory potency turned out to be strongly dependent on various structural parameters, such as sulfation pattern and molecular weight. In conclusion, the semisysnthetic glucan sulfates represent promising candidates in the development of selectin blocking agents.
In-vitro crystallization of indinavir in the presence of ritonavir and as a function of pH
Authors: Li Lilian Y , Rodríguez-Hornedo Naír , Heimbach Tycho , Fleisher David
Published: June 2003

The aim of this study was to investigate the in-vitro crystallization of indinavir as a function of pH alteration and in the presence of another protease inhibitor, ritonavir. Crystallization processes were studied for indinavir sulfate, indinavir free base and a commercial indinavir capsule dosage form, respectively. Crystallization induction times were determined with varying initial concentration of supersaturated solution, and in the presence or absence of seed material. In-vitro induction times were found to be significantly shorter for the indinavir capsule dosage form compared with that of indinavir sulfate and indinavir free base. Induction times were inversely proportional to the final concentration in pH 7 buffer for all materials, and were significantly shortened in the presence of seeds. The crystal morphology of indinavir varied under different crystallization conditions. This study demonstrated the potential for precipitation of indinavir upon pH elevation, while also suggesting that the presence of impurities or seeding material significantly shortens the induction time for indinavir crystal formation. This induction time period falls well within the gastric emptying time following the intake of a high-caloric meal, and within small intestinal transit time. The results of this study are in agreement with the clinical observation that a high-calorie protein meal significantly reduces the oral bioavailability of indinavir in man, accompanying a pH elevation in the stomach.
Protective effect of N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine (PF 9601N), a novel MAO-B inhibitor, on dopamine-lesioned PC12 cultured cells
Authors: Perez V , Romera M , Lizcano J M , Marco J L , Unzeta M
Published: June 2003

Oxidative stress may play a role in the pathogenesis of Parkinson's disease. We have standardised a new model of dopaminergic-cell toxicity that uses dopamine, which is able to generate free radicals, as a toxin. The effect of this catecholamine on cell viability (MTT staining) was dose-dependent, reaching 65% of cell loss at a dopamine concentration of 300 μm. In this model, the protective effect of a novel MAO-B inhibitor, N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine (PF 9601N), was studied and compared with the effect of l-deprenyl assayed under the same experimental conditions. Whereas PF 9601N (50 μm and 100 μm) showed a significant protective effect, this was not the case with l-deprenyl. This different behaviour could be explained in terms of difference in antioxidant capacity. The toxicity induced in PC12 cells by 300 μm dopamine was partially reversed by incubating it in the presence of GBR-12909, a dopamine-transporter blocker. The results indicated that, besides the intracellular toxicity effect of dopamine, another non-specific extracellular mechanism could be involved.
Polycation gene delivery systems: escape from endosomes to cytosol
Authors: Cho Yong Woo , Kim Jong-Duk , Park Kinam
Published: June 2003

Clinical success of gene therapy based on oligonucleotides (ODNs), ribozymes, RNA and DNA will be greatly dependent on the availability of effective delivery systems. Polycations have gained increasing attention as a non-viral gene delivery vector in the past decades. Significant progress has been made in understanding complex formation between polycations and nucleic acids, entry of the complex into the cells and subsequent entry into the nucleus. Sophisticated molecular architectures of cationic polymers have made the vectors more stable and less susceptible to binding by enzymes or proteins. Incorporation of specific ligands to polycations has resulted in more cell-specific uptake by receptor-mediated mechanisms. However, there are still other barriers limiting the transfection efficiency of polycation gene delivery systems. There is a consensus that polycation-DNA complexes (polyplexes) enter cells via the endocytotic pathway. It is not clearly understood, however, how the polyplexes escape (if they do) from endosomes, how DNA is released from the polyplexes or how the released DNA is expressed. The primary focus of this article is to review various polycation gene delivery systems, which are designed to translocate DNA from endosomes into cytosol. Many polycation gene delivery systems have tried to mimic the mechanisms that viruses use for the endosomal escape. Polycation gene delivery systems are usually coupled with synthetic amphipathic peptides mimicking viral fusogenic peptides, histidine-based gene delivery systems for pH-responsive endosomal escape, polycations with intrinsic endosomolytic activity by the proton sponge mechanism and polyanions to mimic the anionic amphiphilic peptides.
Restoration of antibacterial activity of β-lactams by epigallocatechin gallate against β-lactamase-producing species depending on location of β-lactamase
Authors: Zhao Wei-Hua , Asano Nozomi , Hu Zhi-Qing , Shimamura Tadakatsu
Published: June 2003

The combined effects of (–)-epigallocatechin gallate (EGCg) and β-lactams were investigated against various β-lactamase-producing clinical isolates, including 21 Staphylococcus aureus, 6 Escherichia coli, 3 Klebsiella pneumoniae and 8 Serratia marcescens strains. Penicillin in combination with EGCg at 12.5μg mL−1 showed the most potent synergy against 100% penicillinase-producing S. aureus. However, cefotaxime or imipenem in combination with higher concentration of EGCg (100 μg mL−1) only showed slight synergy against 2 of 17 Gram-negative rods. Similar to the effect on the penicillinase from S. aureus, however, EGCg also directly inhibited the extracted β-lactamases from the Gram-negative rods, thereby protecting β-lactams from inactivation. The different effects of the combinations on different β-lactamase-producing species were confirmed to be related to the cellular locations of β-lactamases. In contrast to a 32.7% extracellular fraction of total β-lactamase activity in a penicillinase-producing S. aureus, the fractions were 0.6%, 0.6% and 1.2% in a TEM-derived extended-spectrum β-lactamase-producing E. coli, an inhibitor-resistant β-lactamase-producing K. pneumoniae and an IMP-producing S. marcescens, respectively. In conclusion, the combination of penicillin with EGCg showed potent synergy against penicillinase-producing S. aureus in-vitro. The combinations of β-lactams and EGCg against β-lactamase-producing Gram-negative rods do indicate a limitation owing to the cellular location of β-lactamases.
Microemulsions as colloidal vehicle systems for dermal drug delivery. Part IV: investigation of microemulsion systems based on a eutectic mixture of lidocaine and prilocaine as the colloidal phase by dynamic light scattering
Authors: Shukla Anuj , Krause Annett , Neubert Reinhard H H
Published: June 2003

Stable oil-in-water (o/w) microemulsions used as vehicles for dermal drug delivery have been developed using lidocaine (lignocaine) and prilocaine in oil form (eutectic mixture), a blend of a high (Tween 80, hydrophilic-lipophilic balance (HLB) = 15.0) and a low (Poloxamer 331, HLB = 1.0) HLB surfactant and propylene glycol-water as hydrophilic phase. These microemulsions were able to solubilize up to 20% eutectic mixture of lidocaine and prilocaine without phase separation. The dispersity of the oil phase was investigated by dynamic light scattering. Small colloidal droplets for stable microemulsions of 5∼10 nm were observed. At constant surfactant and hydrophilic phase concentration, increasing the total drug concentration in the microemulsion resulted in an increase in the droplet size of the dispersed, colloidal phase. It was observed that a monolayer of surfactant surrounds the oil (eutectic mixture) core. Colloidal droplets of the microemulsion interact via hard sphere with supplementary attractive interaction. This observed interparticle attractive interaction could explain the observed phase behaviour with respect to change in the basicity of the hydrophilic phase as well as the increase in volume fraction of the dispersed, colloidal phase. It was also observed that the stability and size of this dispersed phase depends on the pH of the composition. Because these microemulsions formed stable, isotropic systems in the range of pH 9.5 to 10.4 with alkali buffer or NaOH solution instead of water as hydrophilic phase, so one can produce microemulsions in this pH area.
De-agglomeration of micronized benzodiazepines in dissolution media measured by laser diffraction particle sizing
Authors: Zhao Feng-Ying , Stewart Peter J
Published: June 2003

The objective of this research was to develop a method to characterize the degree of particle agglomeration using laser diffraction particle sizing, following the addition of benzodiazepine interactive mixtures to water. Interactive mixtures of diazepam, nitrazepam and oxazepam (up to 20% w/w) were prepared by mixing micronized benzodiazepines with lactose granules (250–355μm). Micronized sodium lauryl sulfate and cetrimide (up to 5% w/w) were added to the benzodiazepine-lactose interactive mixes to produce ternary mixtures. Particle size distributions of benzodiazepines, after addition of the interactive mixtures to water, were determined using laser diffraction particle sizing. Bimodal distributions representing dispersed particles and agglomerates were observed initially after lactose carrier dissolution. Partial agglomerate to dispersed particle transition occurred during a 60-min observation period for all mixtures, reaching a constant level of agglomeration after this time. Interactive mixtures with higher benzodiazepine concentrations displayed transition profiles with higher levels of agglomeration. The presence of surfactant in interactive mixtures dramatically decreased agglomeration. Sodium lauryl sulfate was more effective than cetrimide in dispersing agglomerates. The shape of the transition curves during de-agglomeration demonstrated the presence of stable agglomerates that remained after the initial transition; these may be important in explaining dissolution and absorption rates.
Effect of clotrimazole on microsomal metabolism and pharmacokinetics of albendazole
Authors: Merino G , Molina A J , García J L , Pulido M M , Prieto J G , Alvarez A I
Published: June 2003

Albendazole is a broad spectrum anthelmintic drug widely used in human and veterinary medicine. Intestinal and hepatic albendazole metabolism leads to albendazole sulfoxide (active metabolite) and albendazole sulfone (inactive metabolite) formation. Microsomal sulfonase activity can be abolished by in-vitro interaction with clotrimazole and pharmacokinetic studies confirm this interaction. After albendazole incubation, albendazole sulfone formation was completely inhibited by 50 μm clotrimazole in intestinal incubations and a 50% inhibition was observed in hepatic incubations. The lower inhibition constant (Ki) value observed in the intestinal incubations (9.4 ± 1.0 μm) compared with the hepatic counterparts (23.3 ± 15.8 μm) pointed to a greater affinity of the enzymatic systems in the intestine. Regarding the formation of albendazole sulfoxide, an inhibition close to 50% was observed in liver and intestine at 10 μm clotrimazole. The pharmacokinetic parameters obtained following the oral co-administration of albendazole sulfoxide and clotrimazole corroborated the in-vitro inhibition of albendazole sulfone formation, since the ratio of the area under the plasma concentration-time curves for the sulfoxide/sulfone (AUCABZSO/AUCABZSO2) was significantly higher (38.1%). In addition, the AUC and Cmax for albendazole sulfone were significantly lower. The effect of clotrimazole was also studied after prolonged treatment. Hepatic microsomal metabolism of albendazole was induced after 10 days of clotrimazole administration, with significant increases in formation of albendazole sulfoxide (40%) and sulfone (27%). These results offer further insight into the metabolism of benzimidazole drugs and highlight the difficulty involved in human therapy with these anthelmintics, since after prolonged treatment the drug interactions are affected differentially.
Evaluation of the estimation of midazolam concentrations and pharmacokinetic parameters in intensive care patients using a bayesian pharmacokinetic software (PKS) according to sparse sampling approach
Authors: Bolon M , Bastien O , Flamens C , Paulus S , Salord F , Boulieu R
Published: June 2003

The aim of the study was to assess the performance of a bayesian program (PKS System, Abbott) for predicting midazolam concentrations and pharmacokinetic parameters in intensive care patients by comparing the pharmacokinetic parameters estimated by PKS to those calculated according to rich data. The study involved 42 patients receiving midazolam infusion for two hours or for several days. The program was used to predict plasma midazolam concentrations after feedback of 1, 2 or 3 concentrations. High correlation between observed and estimated concentrations was shown (r2 > 0.992). Mean prediction error, mean absolute prediction error and root mean squared error were low for the patients of the reference and validation groups. From two or three feedback concentrations, midazolam pharmacokinetic parameters estimated by PKS were statistically comparable with those obtained using a rich pharmacokinetic analysis (P > 0.05 paired Wilcoxon test). Thus, PKS is useful for predicting midazolam concentrations and pharmacokinetic parameters when at least two feedback concentrations are known. This software seems to be appropriate for providing significant help to the clinician for midazolam dosage adjustment, according to midazolam concentrations and clinical sedation.
The effects of mebendazole on P4501A activity in rat hepatocytes and HepG2 cells. Comparison with tiabendazole and omeprazole
Authors: Baliharová V , Skálová L , Maas R F M , de Vrieze G , Bull S , Fink-Gremmels J
Published: June 2003

Mebendazole is a benzimidazole anthelmintic widely used in veterinary and human therapy. Among benzimidazole derivatives, several drugs with inducing effect on cytochromes P450 can be found. However, the induction capacity of mebendazole on P450s has not been explored yet. In this study, the effects of mebendazole on P4501A activity was tested in primary cultures of rat hepatocytes and in human hepatoma HepG2 cell line. Two known P4501A inducers with benzimidazole structure, tiabendazole and omeprazole, were also included in the experiments with the aim of studying structure-induction relationships. After 24-, 48- and 72-h incubation of rat hepatocytes and HepG2 cells with drugs in various concentrations (0.1–100μm), enzyme activity associated with P4501A1/2 (EROD, MROD) was measured. In addition, the P4501A1/2 protein levels in both in-vitro systems were determined by Western-blotting. Mebendazole provoked a significant increase in P4501A1/2 protein expression and P4501A activity in both in-vitro systems. Omeprazole caused a significant dose-dependent increase of P4501A activity only in HepG2 cells. Although tiabendazole treatment led to significant increase of P4501A protein level, no effect on P4501A activity was observed in either system. The results demonstrate that mebendazole possesses the ability to significantly induce P4501A. Thus, pharmacological and toxicological consequences of P4501A induction should be taken into account in human therapy. The structure-induction relationships and differences between in-vitro systems used are discussed.
Relaxation of rat arteries by urocortin: effects of gender and diabetes
Authors: Sanz Elena , Fernández Nuria , Monge Luis , Climent Belén , Diéguez Godofredo , Garcia-Villaló Angel Luis
Published: June 2003

Urocortin is a peptide recently identified, structurally related to corticotropin releasing factor (CRF). We have compared the effects of urocortin in different vascular beds, and have investigated whether there are gender differences in these effects or whether they are altered by diabetes. We have studied the response of isolated segments (2-mm long) from basilar, coronary and tail arteries to urocortin. The segments were obtained from male and female, normoglycaemic and strepto-zotocin-induced diabetic rats. In the arterial segments precontracted with endothelin-1, urocortin produced concentration-dependent relaxation, and the order of sensitivity was: tail > basilar > coronary. This relaxation was similar in arteries from male and female, diabetic and normoglycaemic rats. In tail arteries from normoglycaemic male rats, the cyclooxygenase inhibitor meclofenamate (10−5M) increased the relaxation to urocortin, and the inhibitor of nitric oxide synthesis Nω-nitro-L-arginine methyl ester (L-NAME, 10−4M) or the potassium-channel-blocker charybdotoxin (10−7M) did not modify it. In tail arteries from normoglycaemic female rats meclofenamate, charybdotoxin or L-NAME did not modify the relaxation to urocortin. These results suggested that urocortin produced vasodilation which showed regional differences between basilar, coronary and tail arteries, but was not affected by diabetes. The mechanisms underlying this relaxation in tail arteries might differ between males and females.
A novel D2-dopaminergic and α2-adrenoceptor receptor agonist induces substantial and prolonged IOP decrease in normotensive rabbits
Authors: Savolainen Jouko , Rautio Jarkko , Razzetti Roberta , Järvinen Tomi
Published: June 2003

The effects of a novel and selective D2-dopaminergic/α2-adrenoceptor agonist, CHF1035, and its metabolite CHF1024 on intraocular pressure (IOP) were determined in rabbits. Because CHF1035 is a mixture of two enantiomers, CHF1800 (+) and CHF1810 (-), pure enantiomers were also studied to determine possible differences in IOP-decreasing ability depending on the stereochemistry of the molecule. CHF1035, CHF1800 (+), CHF1810 (-), CHF1024, brimonidine and 0.9% NaCl were administered topically to rabbits and IOP was then measured at fixed time intervals. The dose-response profile (0.01–1.0% w/v) was determined for CHF1035. CHF1035 and its metabolite CHF1024 significantly lowered IOP in the treated eyes. CHF1035 showed a maximum IOP decrease (7.6 ± 1.5 mmHg) 5 h post-dosing, whereas the metabolite CHF1024 showed a maximum decrease in IOP (7.0 ± 0.8 mmHg) 3 h post-dosing. The maximum IOP decrease produced by CHF1035 in the treated eye was comparable with that produced by brimonidine (7.8 ± 0.9 mmHg), but CHF1035 had a significantly longer duration of action. Unlike brimonidine, CHF1035 and CHF1024 did not decrease IOP in the untreated eye. CHF1810 (-) lowered the IOP more than CHF1800 (+). No irritation, evaluated as eyelid closure, was observed after topical administration of any of the compounds. Only in the case of CHF1035 1% solution, two rabbits out of six closed the eye for 30–45s. In conclusion, CHF1035 and its metabolite CHF1024 significantly decreased the IOP in rabbits, and are potential novel IOP lowering agents. Especially, CHF1035 produced a substantial decrease in IOP for a prolonged period of time, and thus may prove useful in glaucoma therapy.
Evaluation of the synergistic adverse effects of concomitant therapy with statins and fibrates on rhabdomyolysis
Authors: Matzno Sumio , Tazuya-Murayama Keiko , Tanaka Hiromi , Yasuda Shinya , Mishima Motohiro , Uchida Takahiro , Nakabayashi Toshikatsu , Matsuyama Kenji
Published: June 2003

Rhabdomyolysis is a severe adverse effect of hypolipidaemic agents such as statins and fibrates. We evaluated this muscular cytotoxicity with an in-vitro culture system. Cellular apoptosis was determined using phase-contrast and fluorescein microscopic observation with Hoechst 33342 staining. L6 rat myoblasts were treated with various statins and bezafibrate under various conditions. With statins only, skeletal cytotoxicity was ranked as cerivastatin > fluvastatin > simvastatin > atorvastatin > pravastatin in order of decreasing potency. Combined application of fibrates enhanced ator-vastatin-induced myopathy, which causes little apoptosis alone. These results suggest that statins and fibrates synergistically aggravate rhabdomyolysis.
Dosing time dependency of doxorubicin-induced cardiotoxicity and bone marrow toxicity in rats
Authors: To Hideto , Ohdo Shigehiro , Shin Mikiko , Uchimaru Hiroki , Yukawa Eiji , Higuchi Shun , Fujimura Akio , Kobayashi Eiji
Published: June 2003

Cardiac toxicity caused by doxorubicin (adriamycin) is a serious dose-limiting factor in the clinical situation. However, the influence of doxorubicin dosing time has not been clarified from the viewpoints of cardiotoxic development and its mechanism. In this study, we have investigated the dosing time dependency of doxorubicin-induced cardiotoxicity and bone marrow toxicity after repeated administration of doxorubicin in rats. When doxorubicin (5 mg kg−1, i.p.) was administered every seven days (total of 30 mg kg−1) at 3, 9, 15 or 21h after the light was turned on (HALO), toxic death was significantly higher in the 9 HALO treated group than the other groups. When doxorubicin was injected every seven days for 28 days at 9 or 21 HALO, we measured the levels of creatine kinase, malondialdehyde (MDA; an index of lipid peroxide), and glutathione peroxidase (GPx) as markers of cardiotoxicity. On days 14 and 28, creatine kinase levels were significantly higher in the 9-HALO group compared with the 21-HALO group (P < 0.01, respectively). On day 14, MDA levels increased significantly in the 9 HALO group compared with the 21 HALO group (P < 0.01). A single dose of doxorubicin was administered at 9-h or 21-h after the light was turned on to investigate the dosing-time-dependent difference of the pharmacokinetics. The area under the plasma time-concentration curve showed a significant increase at 9 HALO compared with 21 HALO (P < 0.05). These results suggested that the dosing-time-dependent difference of cardiotoxicity induced by doxorubicin was closely related to the daily variation of doxorubicin pharmacokinetics. In conclusion, the choice of optimal dosing time based on the chronopharmacokinetics of doxorubicin may decrease the cardio-toxicity and enable the practice of effective and safe chemotherapy of doxorubicin.
Effects of glycyrrhetinic acid derivatives on hepatic and renal 11β-hydroxysteroid dehydrogenase activities in rats
Authors: Shimoyama Yoshihito , Hirabayashi Kazuhiro , Matsumoto Hiroatsu , Sato Toshitsugu , Shibata Shoji , Inoue Hideo
Published: June 2003

The purpose of this study was to examine the structure and activity relationships of glycyrrhetinic acid derivatives on the inhibition of hepatic and renal 11β-hydroxysteroid dehydrogenases (HSDs) in rats. Furthermore, we explored whether inflammatory effect of the derivatives is involved in the inhibition of 11β-HSD activity. 18β-Glycyrrhetinic acid (Ia) potently inhibited 11β-HSD activity of hepatic (IC50 (concentration giving 50% inhibition of cortisone production) = 0.09 μm) and renal (IC50 = 0.36 μm) homogenate. The inhibitory effect of 18β-glycyrrhetol (Id) modified at the 30-position of glycyrrhetinic acid was weaker than that of glycyrrhetinic acid itself. 18β-24-Hydroxyglycyrrhetinic acid (Ie), oxidized at the 24-position, remarkably reduced the inhibitory activity for both enzymes. 18β-11-Deoxoglycyrrhetinic acid (IIc) showed the same inhibitory effect as glycyrrhetinic acid on hepatic 11β-HSD activity, but less effect on renal 11β-HSD activity. Furthermore, the inhibitory activity of 18β-deoxoglycyrrhetol (IIa), modified at the 11- and 30-position, was markedly decreased. Dihemiphthalate derivatives (IIb, IIIb and IVb) of deoxoglycyrrhetol (IIa), 18β-olean-9(11), 12-diene-3β, 30-diol (IIIa) and olean-11, 13(18)-diene-3β, 30-diol (IVa), which are anti-inflammatory agents, also showed weak inhibition against both hepatic and renal 11β-HSDs. While glycyrrhetinic acid (200 mg kg−1, p.o.) significantly inhibited 11β-HSD activity in rat liver and kidney at 3 h after administration, compound IVb (100 mg kg−1, p.o.) had no effect on either enzyme activity. In addition, the circulating corticosterone level was slightly increased by glycyrrhetinic acid but not by compound IVb. These results suggest that the anti-inflammatory effects of compound IVb, derived from glycyrrhetinic acid, are not due to accumulation of steroids induced by the inhibition of 11β-HSD activity. Our data also showed that the 11-, 24- and 30-positions of glycyrrhetinic acid may play important roles in the differential inhibitory effects on 11β-HSD isozyme activity.
Effect of monensin liposomes on the cytotoxicity of anti-My9-bR immunotoxin
Authors: Shaik Madhu Sudhan , Jackson Tanise L , Singh Mandip
Published: June 2003

The purpose of the study was to evaluate the utility of monensin liposomes in the enhancement of in-vitro cytotoxicity, apoptosis and in-vivo antitumour activity of anti-My9-bR immunotoxin. Monensin liposomes were prepared and studied for the enhancement of in-vitro cytotoxicity and apoptotic response of anti-My9-bR immunotoxin against both sensitive and resistant human promyelocytic leukemia HL-60 cells by MTS/PES method and acridine orange staining, respectively. Further, the in-vivo cytotoxicity enhancement of anti-My9-bR immunotoxin by monensin liposomes was studied in a survival model of severe combined immunodeficient (SCID) mice bearing intraperitoneal HL-60 tumours. The in-vitro cytotoxicity of anti-My9-bR immunotoxin was enhanced 580 fold and 4.7 fold against sensitive and resistant HL-60 cells, respectively, by monensin liposomes (5 times 10−8m). The combination of anti-My9-bR immunotoxin (50 ng mL−1) with monensin liposomes (5 times 10−8m) produced apoptosis in 40% of cells, whereas the apoptotic response was minimal (< 10%) in anti-My9-bR immunotoxin- or monensin liposome (alone)-treated HL-60 (resistant) cells. In SCID mice bearing HL-60 tumours, anti-My9-bR immunotoxin (75 μg kg−1 administered intravenously every other day for a total of five courses) showed a median survival time of 20 days, which was no different than that of vehicle control- or monensin liposome-treated mice. However, anti-My9-bR immunotoxin (75 μg kg−1) in combination with monensin liposomes (4 μg kg−1 monensin), administered every other day for a total of five courses, was found to prolong the survival of 20% of mice for more than 46 days. Our results indicate that, despite anti-My9-bR immunotoxin being ineffective in the HL-60 tumour model, its combination with monensin liposomes could improve the antitumour response.
Insurmountable antagonism of AT-1015, a 5-HT2 antagonist, on serotonin-induced endothelium-dependent relaxation in porcine coronary artery
Authors: Rashid Mamunur , Nakazawa Mikio , Nagatomo Takafumi
Published: June 2003

The purpose of this study was to examine the inhibitory effects of AT-1015, a newly synthesized 5-HT2 receptor antagonist, on serotonin-induced endothelium-dependent relaxation in U 46619 (5 times 10−9m)-precontracted porcine coronary artery pre-incubated with ketanserin (3 times 10−6m), and then compare its effects with another potent 5-HT2 antagonist, ritanserin. The investigation showed that AT-1015 (10−8−10−6m) caused rightward shift with significant inhibition of maximum relaxation response induced by serotonin in porcine coronary artery with endothelium. Ritanserin caused a rightward shift of serotonin-induced relaxation without decreasing maximum response at 10−9 and 10−8m, but it inhibited the maximum relaxation response at 10−7m. The study showed that AT-1015 and ritanserin had no inhibitory effect on bradykinin-induced relaxation in porcine coronary artery with endothelium. Thus, these findings suggested that AT-1015 at concentrations of 10−8−10−6m caused noncompetitive blockade of serotonin-induced endothelium-dependent relaxation in porcine coronary artery. The antagonistic effects of AT-1015 on serotonin-induced relaxation were different from that of ritanserin, except at 10−7m ritanserin. The variation of inhibitory effects between these two 5-HT2 antagonists may be due to the different chemical structure and/or interaction sites at the receptor.