In vitro effects of cadmium on two different animal cell models.

The aim of this study was to assess comparatively the effects of cadmium on two different in vitro cell models, a cell line derived from proximal tubule renal cells (LLC-PK1) and haemocytes or blood cells of mussels (Mytilus galloprovincialis). Cells were seeded in 96-well microplates and exposed in vitro to different concentrations of cadmium (CdCl(2)) ranging from 10 to 2000 microM for haemocytes and from 1 to 100 microM for LLC-PK1 cells, added to the culture medium. After 24 h of exposure, different assays were performed on haemocytes: neutral red uptake, phagocytosis of neutral red-stained zymosan, XTT assay, activity of lysosomal acid phosphatase and demonstration of the actin cytoskeleton using TRITC-labeled phalloidin. Cell viability expressed as LC50 was 750 microM when using the neutral red assay and 400 microM with the XTT assay. The phagocytic ability and the activity of acid phosphatase increased significantly in cells treated with Cd in a non dose-dependent manner. Doses of Cd above 100 microM caused disruption of the actin cytoskeleton. In LLC-PK1 cells, cell viability expressed as LC50 was found to be around 40 microM when using the neutral red assay and 50-60 microM with MTT and LDH assays, respectively. These results show that mussel haemocytes are in general more resistant to Cd exposure than LLC-PK1 cells. Furthermore, Cd appears to stimulate phagocytic and lysosomal activities in haemocytes in vitro.     

In vivo MRI in different models of experimental epilepsy

In order to investigate epilepsy, that is one of the most common neurological disorders, in the last decades different animal models have been proposed. Prevention, diagnosis, treatment and basic knowledge have been improved by the mean of these models. Numerous animal models have been developed in epilepsy research, both for generalized and for simple/complex partial seizures. Animal models for generalized seizures include sensory (light, noise, movement, etc) or electrical stimulations and genetic models. Models for focal seizures include topical or systemic application of pro-convulsive compounds or electrical stimulation. Baboons, mice, rats, rabbits, and Fayoumi chicken have been extensively used in this regard. Since 1983, when magnetic resonance spectroscopy was used to evaluate for the first time in vivo alterations induced by status epilepticus in rabbit, an increasing interest for the neuroimaging perspective has led to new insights in the study of epileptic disorders. In the early 1990s experimental studies provided evidence for the feasibility of magnetic resonance imaging analysis and detection of tissue damage in kainic acid-induced epilepsy in rat. In the following years a wealth of data has been obtained by the mean of functional MRI and/or by diffusion-weighted images. The studies reported in the literature of the last decades indicate in vivo magnetic resonance of epilepsy model as valuable and extremely informative tool.     

In vitro metabolism of hyperforin in rat liver microsomal systems.

Hyperforin is an important active component of St. John's wort (Hypericum perforatum) that has been suggested to be responsible for the St. John's wort antidepressive effects and herbal-drug interactions. In this study, the in vitro metabolism profile of hyperforin was investigated using liver microsomes from male and female Sprague-Dawley rats, with or without induction by phenobarbital or dexamethasone. Four major Phase I metabolites, named 19-hydroxyhyperforin, 24-hydroxyhyperforin, 29-hydroxyhyperforin, and 34-hydroxyhyperforin, were isolated by high performance liquid chromatography and identified by mass spectrometry and NMR. Results suggest that hydroxylation is a major biotransformation of the hyperforin pathway in rat liver and that inducible cytochrome P450 3A (CYP450 3A) and/or CYP2B may be the major cytochrome P450 isoforms catalyzing these hydroxylation reactions.     

In vitro release of chloramphenicol from different suppository bases.

The release of chloramphenicol from different suppository bases has been investigated. It is proved that the release was dependent on drug solubility in the base, solubility of the base in test medium, and the melting point of the base and its chemical composition.     

In vitro effects of the antiallergy compound, CI-949, on interleukin-1 and 2 release, and on mitogen and alloantigen responsiveness

CI-949 (5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H -indole- 2-carboxamide, L-arginine salt), an antiallergy compound, was found to be a weak inhibitor of IL-1 release from LPS-stimulated murine peritoneal exudate cells and human peripheral blood leukocytes, with IC50S of 186.2 and 267.9 microM, respectively. CI-949 was also a poor inhibitor of release of IL-2 from Con A-stimulated rat splenocytes (37% inhibition at 100 microM). CI-949 did produce concentration-related inhibition of the response of human lymphocytes to PHA and Con A (IC50S = 44.7 and 21.5 microM, respectively) as well as in the mixed lymphocyte reaction (MLR) (IC50 = 16.8 microM). The clinical significance of these latter findings is unknown at present.     

Vehicle effects on in vitro release of tiaprofenic acid from different topical formulations

The aim of the present study was to investigate the in vitro release properties of tiaprofenic acid (TA) from different topical vehicles. Carbopol 940 gel, chitosan gel, two types of emulsion-based ointment formulations (o/w and w/o) and hydrophilic petrolatum USP were prepared with 2% drug content. Drug release from all vehicles through a standard cellophane membrane was evaluated by using Franz-type diffusion cells. In vitro release study results showed that the diffusion coefficients of the drug from vehicles rank according to the following order: Carbopol 940 gel (D = 3.11 x 10(-7) +/- 0.54 cm(2)/s) > chitosan gel (D = 0.27 x 10(-7) +/- 0.08 cm(2)/s) > emulsion-based ointment (o/w) (D = 0.18 x 10(-7) +/- 0.05 cm(2)/s) > emulsion-based ointment (w/o) (D = 0.13 x 10(-7) +/- 0.02 cm(2)/s) > hydrophilic petrolatum USP (D = 0.02 x 10(-7) +/- 0.01 cm(2)/s). Carbopol 940 gel base showed significantly higher drug release than other vehicles (P < 0.001). These results indicated that Carbopol 940 gel base is a good candidate for the topical delivery of TA, giving significantly higher drug release than the other vehicles.     

Functional and antiischaemic effects of monoacetyl-vitexinrhamnoside in different in vitro models

• 1.1. Functional and antiischaemic effects of monoacetyl-vitexinrhamnoside (AVR), a flavonoid with phosphodiesterase (PDE)-inhibitory properties contained in Crataegus species (Hawthorn, Rosaceae) were studied in several in-vitro models.
• 2.2. In rabbit isolated femoral artery rings, AVR concentration-dependently reduced developed tension. Vasodilation by AVR was reduced after inhibiting EDRF formation by L-N^G-nitro arginine.
• 3.3. In spontaneously-beating Langendorff-guinea pig hearts, AVR concentration-dependently enhanced heart-rate, contractility, lusitropy and coronary flow.
• 4.4. In isolated electrically-driven Langendorff-rabbit hearts, acute regional ischemia (MI) was induced by coronary artery occlusion and quantified from epicardial NADH-fluorescence photography. AVR (5 × 10⁻⁵ mol/l) induced a slight numerical increase of left ventricular pressure and coronary flow (p > 0.05). MI was reduced (p < 0.05).
• 5.5. Monoacetyl-vitexinrhamnoside is an inodilator whose vasodilatory action may be mediated in part by EDRF in addition to PDE-inhibition. Monoacetyl-vitexinrhamnoside does possess marked antiischemic properties even in isolated hearts, suggesting an improvement of myocardial perfusion.

     

In vitro investigation of lipid implants as a controlled release system for interleukin-18

Operating on the inductive and effective phases of an anti-tumor immune response and uncovering pivotal functions that may reduce cancer cell growth, interleukin-18 (IL-18) appears to be an attractive candidate for the sustained local adjuvant immunotherapeutic treatment of brain gliomas. The objective of this work was to develop IL-18 loaded lipid implants as a controlled delivery system. For the preparation of protein loaded triglyceride matrix material, a solid-in-oil (s/o) dispersion technique was chosen for which protein particles in the micrometer range were first prepared by co-lyophilization with polyethylene glycol (PEG). Implants of 1 mm diameter, 1.8 mm height and 1.8 mg weight were manufactured by compression of the powder mixture in a specially designed powder compacting tool. The in vitro release behavior of 125I-Bolton-Hunter-radiolabeled IL-18 was assessed in a continuous-flow system. A cell culture assay was established for the determination of bioactivity of released IL-18. Implants showed a continuous release of 10-100 ng IL-18 per day for 12 days. A progressive integrity loss was observed with ongoing release, which would be related to protein degradation during incubation. The initially released fraction proved complete retention of bioactivity, indicating that the manufacturing procedure had no detrimental effects on protein stability.     

淫羊藿苷对骨质疏松模型小鼠骨组织中IL-6表达的影响

目的观察淫羊藿苷对骨质疏松模型小鼠骨组织中白介素-6（IL-6）表达的影响,探讨淫羊藿苷的抗骨质疏松作用机制。方法取雌性小鼠于无菌条件下切除双侧卵巢,复制骨质疏松小鼠模型。造模小鼠随机分为：模型,己稀雌酚、淫羊藿苷高、中、低剂量组,以假手术小鼠作为对照组。各组动物按各自药物和剂量灌胃给药,每日1次,连续2个月。停药次日,脱颈椎处死动物,取右侧股骨常规脱钙、石蜡包埋、切片,用免疫组化方法观察骨组织白介素-6（IL-6）的表达。结果与对照组比较,模型组小鼠骨组织中IL-6阳性破骨细胞灰度值明显降低;与模型组比较,淫羊藿苷高、中剂量组小鼠骨组织中IL-6阳性破骨细胞灰度值明显增加。结论淫羊藿苷通过降低骨质疏松模型小鼠骨组织中IL-6的表达,从而减少骨吸收。     

Nicotinic acid induces antinociceptive and anti-inflammatory effects in different experimental models

Although in vitro studies have shown that nicotinic acid inhibits some aspects of the inflammatory response, a reduced number of in vivo studies have investigated this activity. To the best of our knowledge, the effects induced by nicotinic acid in models of nociceptive and inflammatory pain are not known. Per os (p.o.) administration of nicotinic acid (250, 500 or 1000 mg/kg, − 1 h) inhibited the first and the second phases of the nociceptive response induced by formalin in mice. Nicotinic acid (250 or 500 mg/kg, − 1 and 3 h) also inhibited the mechanical allodynia induced by carrageenan in rats, a model of inflammatory pain. However, in a model of nociceptive pain, exposure of mice to a hot-plate, nicotinic acid was devoid of activity. In addition to inhibiting the nociceptive response in models of inflammatory pain, nicotinic acid (250 or 500 mg/kg, p.o., − 1 and 3 h) inhibited paw edema induced by carrageenan in mice and rats. Picolinic acid (62.5 or 125 mg/kg, p.o., − 1 h), a nicotinic acid isomer, inhibited both phases of the nociceptive response induced by formalin, but not paw edema induced by carrageenan in mice. The other nicotinic acid isomer, isonicotinic acid, was devoid of activity in these two models. In conclusion, our results represent the first demonstration of the activity of nicotinic acid in experimental models of nociceptive and inflammatory pain and also provide further support to its anti-inflammatory activity. It is unlikely that conversion to nicotinamide represents an important mechanism to explain the antinociceptive and anti-inflammatory activities of nicotinic acid. The demonstration of new activities of nicotinic acid, a drug that has already been approved for clinical use and presents a positive safety record, may contribute to raise the interest in conducting clinical trials to investigate its usefulness in the treatment of painful and inflammatory diseases.     

In vitro evaluation of intestinal fluoride absorption using different cell models

The main routes of fluoride (F) exposure are drinking water and certain foods; consequently, intestinal absorption is an important stage in the study of F exposure. In the present study, different cell models [Caco-2, HT29-MTX and various proportions of Caco-2/HT29-MTX)] were used to evaluate intestinal transport of F. The influence of cell type, pH, mucus layer, bile salts and food matrices on the apparent permeability coefficient (P(app)) was evaluated. The results show that a higher proportion of HT29-MTX in the monolayer produces an increase in F permeability, although the mucus layer secreted by HT29-MTX decreases F transport. The results also show that taurocholic acid, a component of bile salts, and acid pH increase F permeability, whereas the presence of a food matrix (rice) decreases intestinal transport of F. In all cases, alterations in F permeability were closely related with modulation of cell junctions.     

In vivo and in vitro effects of endotoxin on prostaglandin release from rat lung.

1 The release of prostaglandins (PGE2, PGF2 alpha, PGI2) and thromboxane A2 (TxA2) from rat lung exposed to E. coli endotoxin, in vivo or in vitro has been studied. 2 Lung strips of endotoxin-treated rats demonstrated a preferential increase of TxA2 and PGF2 alpha and a decrease in the ratio PGI2/TxA2. 3 These data suggest that changes in the proportions of arachidonic acid metabolites might play a role in the pulmonary pathophysiology during endotoxin shock. 4 Incubation of lung strips with endotoxin in vitro failed to stimulate prostaglandin release; paradoxically, it suppressed both the spontaneous and the ionophore-induced prostaglandin release. 5 These findings suggest that the increase in prostaglandin release by the lung following endotoxin administration in vivo is probably mediated by factor(s) generated in endotoxaemia and is not due to a direct action of endotoxin on the lung tissue.     

黄藤素缓释片释放度的测定及其释药特性

目的　进行黄藤素缓释片释放度的测定及释药规律的研究。方法　采用紫外分光光度法测定黄藤素缓释片的释放度,并进行释药规律的研究。结果　测定波长为342nm ,黄藤素在3. 0～1 0 . 0 μg·ml-1 范围内线性关系良好(r =0 . 9999)。其体外释放特性符合Higuchi模式。结论　黄藤素缓释片释药机理是扩散和骨架溶蚀协同作用,而非单纯Fick扩散。     

In vitro models of proarrhythmia

Proarrhythmia models use electrophysiological markers to predict the risk of torsade de pointes (TdP) in patients. The set of variables used by each model to predict the torsadogenic propensity of a drug has been reported to correlate with clinical outcome; however, these reports should be interpreted cautiously as no model has been independently assessed. Each model is discussed along with its merits and shortcomings; none, as yet, having shown a predictive value that makes it clearly superior to the others. As predictive as these models may become, extrapolation of results directly to the clinic must be exercised with caution. The use of in silico models, from subcellular to whole system, is rapidly beginning to form the first line of screening activity in many drug discovery programmes, indicating that biological experimentation may become secondary to analysis by simulation. In vitro proarrhythmia models challenge current perceptions of appropriate surrogates for TdP in man and question existing non-clinical strategies for assessing proarrhythmic risk. The rapid emergence of such models, compounded by the lack of a clear understanding of the key proarrhythmic mechanisms has resulted in a regulatory reluctance to embrace such models. The wider acceptance of proarrhythmia models is likely to occur when there is a clear understanding and agreement on the key proarrhythmia mechanisms. With greater acceptance and ongoing improvements, these models have the potential to unravel the complex mechanisms underlying TdP.     

In vitro effects of organophosphorus anticholinesterases on muscarinic receptor-mediated inhibition of acetylcholine release in rat striatum

The aim of the present study was to evaluate the in vitro modulation of muscarinic autoreceptor function by the organophosphorus (OP) anticholinesterases chlorpyrifos oxon, paraoxon, and methyl paraoxon. Acetylcholine (ACh) release was studied by preloading slices from rat striatum with [3H]choline and depolarizing with potassium (20 mM) in perfusion buffer containing hemicholinium-3 (to prevent reuptake of radiolabeled choline). Under these conditions, chlorpyrifos oxon, paraoxon, and methyl paraoxon (0.1-10 microM) all reduced ACh release in a concentration-dependent manner. Addition of the carbamate acetylcholinesterase (AChE) inhibitor physostigmine (20 microM) to the perfusion buffer also decreased ACh release. When physostigmine was present, the three oxons had no additional effect on ACh release. Concentration-dependent inhibition of AChE activity in striatal slices perfused with chlorpyrifos oxon (0.1, 1, and 10 microM) suggested AChE inhibition was responsible for oxon-mediated alterations in ACh release. To differentiate between direct and indirect actions of the OP toxicants on muscarinic autoreceptors, we compared the effects of the oxons on ACh release under two conditions, i.e., tissues were perfused with buffer containing only hemicholinium-3 or with buffer containing hemicholinium-3, physostigmine, and the nonselective muscarinic receptor blocker atropine (100 nM). In the presence of only hemicholinium-3, concentration-dependent inhibition of ACh release was again noted for all oxons, similar to the effects of the muscarinic agonists carbachol and cis-dioxolane. In the presence of physostigmine and atropine, the relative potencies of all agents were markedly reduced. Interestingly, carbachol, cis-dioxolane, paraoxon, and methyl paraoxon all decreased ACh release as before, but chlorpyrifos oxon (100-300 microM) actually increased ACh release. Together, the results suggest that chlorpyrifos oxon, paraoxon, and methyl paraoxon can activate muscarinic autoreceptors indirectly through inhibition of AChE. Both paraoxon and methyl paraoxon also directly activate whereas chlorpyrifos oxon blocks muscarinic autoreceptor function. Qualitative differences in the direct actions of these oxons at this presynaptic regulatory site could contribute to differential toxicity with high-dose exposures.