pH-Dependent passive and active transport of acidic drugs across Caco-2 cell monolayers.

The aim of this study was to investigate pH-dependent passive and active transport of acidic drugs across Caco-2 cells. Therefore, the bidirectional pH-dependent transport of two acidic drugs, indomethacin and salicylic acid, across Caco-2 cells was studied in the physiological pH range of the gastrointestinal tract. The transport of both drugs decreased with increased pH, as expected from the pH-partition hypothesis. Net absorption occurred when the basolateral pH exceeded the apical pH. Concentration dependence and transporter inhibition studies indicated passive transport for indomethacin and a mixture of pH-dependent passive and active influx for salicylic acid. Unexpectedly, active and passive drug transport results were indistinguishable in temperature dependency studies. The transport of salicylic acid (apical pH 5.0; basolateral pH 7.4) was partly blocked by inhibitors of the proton-dependent transporters MCT1 (SLC16A1) and OATP-B (SLC21A9, SLCO2B1). This study shows that the asymmetry in bidirectional transport of acidic drugs is affected by both passive and active components in the presence of pH gradients across Caco-2 cells. Thus, combined studies of concentration-dependency and transport-inhibition are preferred when acidic drug transport is studied in a pH gradient. The findings of this in vitro study can be extrapolated to in vivo situations involving an acidic microclimate.     

Evaluation of permeability enhancement of hydrophilic compounds and macromolecular compounds by bile salts through rabbit corneas in-vitro

The effect of bile salts, sodium taurocholate (TC-Na) and sodium taurodeoxycholate (TDC-Na), on the permeability of hydrophilic compounds and macromolecular compounds through the rabbit cornea in-vitro was examined. 6-Carboxyfluorescein and glutathione were used as low molecular weight hydrophilic model compounds and FITC-dextran (mol. wt 4000) and insulin were used as relatively macromolecular model compounds. TC-Na (2 and 10 mM) marginally increased the corneal permeabilities to hydrophilic compounds and macromolecular compounds. TDC-Na (2 and 10 mM) markedly increased the corneal permeabilities of these compounds.     

Distribution of liposome-incorporated carboxyfluorescein in rabbit eyes

The penetration of liposomal drugs into rabbit eyes was investigated using 6-carboxyfluorescein (6-CF) as a water-soluble model substance. It was possible to incorporate 4.1 mg 6-CF into the aqueous space of 1 ml of liposomes produced with reverse phase technique. In comparison to aqueous 6-CF, liposomal 6-CF reached extremely high concentrations in all parts of the eye, and was released slowly over many days. The highest concentration of liposomal 6-CF (55 micrograms g-1) was found in the sclera 30 minutes after injection. The corresponding value of aqueous 6-CF was 5 micrograms g-1. After 7 days, liposomal 6-CF was still detectable at the subconjunctival injection location as well as in the sclera, retina, chorioid and cornea. In clinical examination and fluorescence photography no side effects were observed except for moderate and transitory conjunctivitis. In conclusion, liposomes are appropriate drug carriers for delivery of drugs to the posterior eye segment. This method is, however, limited to drugs with chemical properties which permit incorporation in pharmacologically relevant amounts.     

Distribution of liposome-incorporated carboxyfluorescein in rabbit eyes

Abstract

The penetration of liposomal drugs into rabbit eyes was investigated using 6-carboxyfluorescein (6-CF) as a water-soluble model substance. It was possible to incorporate 4.1 mg 6-CF into the aqueous space of 1 ml of liposomes produced with reverse phase technique. In comparison to aqueous 6-CF, liposomal 6-CF reached extremely high concentrations in all parts of the eye, and was released slowly over many days. The highest concentration of liposomal 6-CF (55 μgg^?1) was found in the sclera 30 minutes after injection. The corresponding value of aqueous 6-CF was 5μ^?1.

After 7 days, liposomal 6-CF was still detectable at the subconjunctival injection location as well as in the sclera, retina, chorioid and cornea. In clinical examination and fluorescence photography no side effects were observed except for moderate and transitory conjunctivitis.

In conclusion, liposomes are appropriate drug carriers for delivery of drugs to the posterior eye segment. This method is, however, limited to drugs with chemical properties which permit incorporation in pharmacologically relevant amounts.     

Encapsulation of biological active phenolic compounds extracted from wine wastes in alginate-chitosan microbeads

Grapes (Vitis vinifera) are produced in large amounts worldwide and mostly are used for winemaking. Their untreated wastes are rich in valuable secondary metabolites, such as phenolics. Thus, in this study, white and red wine wastes (Malagouzia and Syrah variety) were investigated for their added value phenolics, which were analysed by high performance liquid chromatography (HPLC) and electrospray ionisation-mass spectrometry (ESI/MS) and subsequently encapsulated in several polymers. Extracts from all wastes gave high amounts of total phenolics (13?±?2.72–22?±?2.69?mg?g^?1) and possessed high antioxidant activity (67–97%). In addition to their significant antibacterial activity against gram-negative and gram-positive bacteria, interesting results were also obtained from their anti-inflammatory and antiplatelet activity, in vitro. Encapsulation of the extracts was selective, leaving out most of sugars and other organic compounds when alginate-chitosan was used. Encapsulation efficiency recorded for all extracts ranged from 55% to 79%. Release studies were also performed in several solutions aiming in their commercial use in food and pharmaceutical industries.     

Inhibitory action of furosemide on the active chloride transport of papillary heart muscle in rabbit

The action of furosemide on the intracellular chloride activity, ai(Cl), of rabbit papillary muscle was investigated with Cl ion selective microelectrodes. With furosemide concentrations greater than 1 microM, ai(Cl) decreased. Furthermore, the decrease in ai(Cl) was larger as the furosemide concentration increased. Lowering the extracellular sodium concentration, [Na]o, by replacing Na with choline, significantly reduced the effect of furosemide on ai(Cl). The results provide evidence that the membrane of rabbit heart cells possess a furosemide sensitive chloride transport mechanism related to [Na]o.     

Contractile effects of jellyfish toxin extracted from Carybdea rastonii on isolated rabbit aorta

Effects of the toxic component of jellyfish (Carybdea rastonii) (pCrTX) on the smooth muscle tension of isolated rabbit thoracic aorta were examined. pCrTX, at concentrations higher than 10(-7) g/ml, caused slowly developing tension that reached its maximum after about 1 hr. This contraction was partially inhibited by pretreatment of the tissue with phentolamine (5 X 10(-6) M) or indomethacin (10(-5) M). The contraction induced by pCrTX was partially inhibited by nicardipine (10(-7) M) and markedly augmented by Bay k8644 (10(-6) M). In low-Na+ solution, the rate of rise of the pCrTX-induced contraction was significantly reduced. Removal of external Ca2+ inhibited the pCrTX-induced contraction by about 30%, while chlorpromazine, trifluoperazine, prenylamine and papaverine (10(-4) M) completely inhibited the contraction. pCrTX itself did not cause any contraction in saponin-skinned smooth muscle and had no effect on the Ca2+-induced contractile tension. It has been reported that pCrTX-induced contraction is attributable to the release of endogenous catecholamines and also to the increase in Ca2+ influx in smooth muscle (Azuma et al., 1986). The present results confirmed the previous suggestion and further suggested that a portion of the contraction is due to release of prostaglandin(s) and also to the direct effect on smooth muscle which is not dependent on Ca2+ influx.     

Retinal toxicity of intraocular vancomycin and ceftazidime in vitrectomized rabbit eyes

The purpose of this study was to evaluate the retinal toxicity of vancomycin and ceftazidime combined into an infusion solution that was intraoculary given after or during vitrectomy. Forty albino rabbits were divided into 4 groups of 10 each. Vitrectomized right eyes of groups 1, 2, and 3 were given recommended doses of vancomycin and ceftazidime alone or combined, while right eyes in the fourth group were vitrectomized using an infusion solution to which was added ceftazidime and vancomycin combination. Toxicity was tested with electroretinography (ERG) and light microscopy. ERG and light microscopy did not show any toxicity signs associated with vancomycin or ceftazidime alone or with combined therapy. Vancomycin and/or ceftazidime can reliably and effectively be used combined in an infusion solution at recommended doses after and during vitrectomy. This treatment modality does not have any toxic effects to retinal structures and is an alternative method to separate injections of the two antimicrobial agents.     

Ocular toxicity of ethylene chlorohydrin and ethylene glycol in rabbit eyes

Toxicity and irritation of ethylene chlorohydrin and ethylene glycol were assessed in rabbit eyes following multiple topical or multiple intraocular (anterior chamber) administrations. Ethylene glycol was nontoxic and nonirritating at 0.4% concentration following topical and intraocular administration. Ocular toxicity at higher test concentrations consisted of conjunctival redness, chemosis, flare, and iritis. Ethylene chlorohydrin was nontoxic and nonirritating at 1.0% and 0.5% concentrations following topical and intraocular administration, respectively. Ocular toxicity at higher test concentrations consisted of conjunctival redness, chemosis, discharge, flare, iritis, pannus, transient corneal opacity (topical route), nontransient corneal opacity (intraocular route), lens capsule rupture, and opaque lens.     

改性壳聚糖膜在兔眼中的降解及组织病理学观察

目的了解兔眼中壳聚糖膜的降解与生物相容性 ,观察眼组织的病理学变化。方法对兔眼实施小梁切除术 ,将不同相对分子质量改性壳聚糖膜 ,植入板层巩膜瓣下 ,分别于术后 1、4、1 2周处死 ,摘除眼球 ,病理切片 ,观察组织反应。结果在植入早期 1、4周时 ,局部组织出现明显的炎症反应 ,随时间的推移 ,改性壳聚糖膜的降解速度加快 ,1 2周时 ,已降解为碎屑状 ,炎症反应基本消失。结论改性壳聚糖膜植入兔眼内可降解吸收 ,对兔眼组织不产生病理性损害     

Electrical effects of okadaic acid extracted from black sponge on rabbit sinus node.

1. Palmitoyl carnitine (10-1000 microM) resembled Bay K 8644 (10-1000 nM) in that it directly contracted rat aortic rings which were partially depolarized with K+ (12 mM). However, the effects of Bay K 8644 were reduced in the presence of endothelium whereas the presence of the endothelium hardly affected the palmitoyl carnitine-induced contractions, which occurred at high concentrations (greater than 10 microM). 2. Lower concentrations of palmitoyl carnitine (0.3-30 microM; EC50 1.1 microM), but not Bay K 8644, carnitine or palmitic acid, antagonized the relaxant effects of acetylcholine in rat aorta. The antagonism was specific for endothelium-dependent relaxations, in that the relaxations to ATP and the calcium ionophore A23187 were also non-competitively antagonized, albeit at slightly higher concentrations, whereas the direct relaxant effects of sodium nitroprusside were unaffected. Palmitoyl carnitine therefore antagonizes the effects or the release of endothelial-derived relaxant factor (EDRF). The inhibitory effects were reversed on prolonged washout, indicating that the effects were not due to destruction of the endothelial cells. 3. In superfusion experiments, palmitoyl carnitine inhibited the release of EDRF from rat aorta but did not affect the responsiveness to exogenous EDRF, indicating a site of action at the endothelial cell. In superfusion experiments, palmitoyl carnitine, and lysophosphatidyl choline, caused direct relaxations of the aorta, indicating EDRF release, prior to inhibition of release evoked by receptor stimulation. These substances may modulate vascular responsiveness under certain conditions.     

纤溶酶和分散酶诱发兔眼玻璃体后脱离

目的：评价纤溶酶和分散酶诱发玻璃体后脱离(PVD)的安全性和有效性。方法：48只兔均分6组。组1,5眼内注射分散酶0.025 U,组2为0.1 U；组3,6注射纤溶酶1 U,组4为4 U。组5,6术后15 min取眼球行组织化学检测,其余组术后7 d内行临床检查、视网膜电流图(ERG)及扫描、透射电镜检测。结果：扫描电镜证实组1,2,4均能诱发出部分或完全性PVD(8/8,8/8,8/8),除组3(6/8)。组5,6组织化学显示均发生眼内炎(8/8,8/8)。ERG显示组1,2暗适应眼最大电反应的a,b波振幅均显著下降(均P＜0.01),组3,4无显著改变(均P＞0.05)。组1,2眼出现视网膜结构损伤、视网膜出血和白内障发生。结论：眼内注射纤溶酶和分散酶均能诱发出PVD,前者眼内毒性反应小,后者大。     

Determination of the toxicity of intravitreal minocycline in rabbit eyes

Purpose: To evaluate the retinal toxicity of intravitreal minocycline in rabbit eyes.

Methods: Intravitreal injection of minocycline with concentrations of 1000, 500, 250, 125 and 62.5?μg in 0.1?ml was performed in 10 New Zealand albino rabbits. Each concentration was injected into two rabbit eyes. For each dose, normal saline was injected in one contralateral eye and the other fellow eye remained non-injected. Electrophysiologic testing was performed before and 4 weeks after injections. The eyes were enucleated 4 weeks after injections and examined using light microscopy.

Results: The clinical examination was unremarkable after injections. Electroretinography recordings were significantly affected at all doses in at least one of the a- or b-waves of photopic or scotopic responses. Histopathologic examination revealed marked atrophy and loss of integrity in all retinal layers in all minocycline injected eyes. Contralateral eyes were normal.

Conclusion: In our study, intravitreal minocycline was toxic to the retina in albino rabbits even at a concentration of 62.5?µg/0.1?ml.     

In vitro permeation characteristics of moxifloxacin from oil drops through excised goat, sheep, buffalo and rabbit corneas

The objective of present investigation was to study the in vitro permeation characteristics of moxifloxacin from oil drops through freshly excised goat, sheep, buffalo and rabbit corneas. Moxifloxacin, 0.043 to 0.048% (w/v) ophthalmic solutions with or without (0.5% v/v) benzyl alcohol were made in arachis, castor, cottonseed, olive, soybean, sunflower and sesame oils. Permeation studies were conducted by putting 1 ml oil formulation on cornea (0.50 cm2) fixed between donor and receptor compartments of an all glass modified Franz diffusion cell and measuring the drug permeated in receptor (containing 10 ml bicarbonate ringer, pH 7.4 at 37 degrees C under stirring) by spectrophotometry at 291 nm, after 120 min. Post permeation corneal hydration was measured to assess corneal damage. The study was designed with paired corneas i.e. one cornea of an animal received formulation without benzyl alcohol while the contralateral cornea received formulation with benzyl alcohol. Moxifloxacin ophthalmic solution in castor oil showed maximum permeation with all the corneas. Addition of benzyl alcohol, a preservative, to oil drops reduced permeation of moxifloxacin from each oil drop, with corneas of all the species. Partition experiments with moxifloxacin oil drops and phosphate buffer (pH 7.4) indicated higher partitioning of drug in the oil phase, in presence of benzyl alcohol. Thus results of permeation are consistent with the partition characteristics of drug between oil and aqueous phase. Corneal hydration obtained with all the formulations was between 75 to 80% indicating no corneal damage.     

Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM

The purpose was to develop a general mathematical model for estimating passive permeability and efflux transport parameters from in vitro cell culture experiments. The procedure is applicable for linear and non-linear transport of drug with time, <10 or >10% of drug transport, negligible or relevant back flow, and would allow the adequate correction in the case of relevant mass balance problems. A compartmental kinetic approach was used and the transport barriers were described quantitatively in terms of apical and basolateral clearances. The method can be applied when sink conditions are not achieved and it allows the evaluation of the location of the transporter and its binding site. In this work it was possible to demonstrate, from a functional point of view, the higher efflux capacity of the TC7 clone and to identify the apical membrane as the main resistance for the xenobiotic transport. This methodology can be extremely useful as a complementary tool for molecular biology approaches in order to establish meaningful hypotheses about transport mechanisms.     

Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM

The purpose was to develop a general mathematical model for estimating passive permeability and efflux transport parameters from in vitro cell culture experiments. The procedure is applicable for linear and non-linear transport of drug with time,?<10 or?>10% of drug transport, negligible or relevant back flow, and would allow the adequate correction in the case of relevant mass balance problems. A compartmental kinetic approach was used and the transport barriers were described quantitatively in terms of apical and basolateral clearances. The method can be applied when sink conditions are not achieved and it allows the evaluation of the location of the transporter and its binding site. In this work it was possible to demonstrate, from a functional point of view, the higher efflux capacity of the TC7 clone and to identify the apical membrane as the main resistance for the xenobiotic transport. This methodology can be extremely useful as a complementary tool for molecular biology approaches in order to establish meaningful hypotheses about transport mechanisms.     

Extraction of active clonidine-displacing substance from bovine lung and comparison with clonidine-displacing substance extracted from other tissues.

Crude methanolic clonidine-displacing substance (CDS) extracted from bovine lung competed for radioligand binding from alpha2-adrenoceptors and I2-sites present in rat brain membranes, and from I1-sites present in rat brain and kidney membranes. There was no difference in the competition of [3H]clonidine binding to alpha2-adrenoceptors present in either rat or rabbit brain membranes by the crude CDS extract and therefore either tissue could be used to estimate the number of units of CDS present in extracts. Further purification by reverse phase high performance liquid chromatography (RP-HPLC), with UV detection, of extracts obtained from bovine lung, brain and rat brain exhibited similar three-peak profiles, previously reported. Corresponding fractions competed for radioligand binding to alpha2-adrenoceptors present in rat brain membranes, eluting between 19 and 23 min, which corresponded with the middle peak of the three-peaks. Therefore, we propose the CDS-like material eluting from all these tissues to be similar. Interestingly, CDS extracted from bovine adrenal glands under the same conditions showed a similar three-peak profile, but did not repeat the displacement of binding just at 19-23 min, but at every time point after 4 min. This suggests this tissue could represent a source of CDS in this species.     

从白术总多糖中抗肿瘤有效成分的变化试论GAP的重要性

目的:研究白术药材中总多糖(AMP-O1)的质量,探讨其影响因素及控制方法.方法:采用水提、醇沉、透析和冷冻干燥等方法从白术药材中提AMP-O1.以AMP-O1中抗肿瘤活性成分AMP-Ⅰ的含量以及中性糖、糖醛酸、蛋白质含量为指标对药材质量进行评价.结合文献报道论述在白术药材生产中影响药材质量的各种因素和施行GAP管理的重要性.结果:发现从同一产地所购6批白术药材质量存在极大差异.结论:白术药材中AMP-O1质量受生产中多种因素综合影响,为保证其产量和质量,在生产过程中施行GAP管理是非常重要的.     

Comparison between active and passive drug transport in human intestinal epithelial (caco-2) cells in vitro and human jejunum in vivo

Drug transport rates in Caco-2 monolayers were compared with those obtained in the human jejunum in vivo. Permeability coefficients unbiased by the hydrodynamic conditions were calculated in order to allow direct comparison of the two models. The rapidly (passively) transported drugs naproxen, antipyrine and metoprolol had comparable permeability coefficients in Caco-2 cells and in human jejunum. The permeability coefficients of the slowly (passively) transported, hydrophilic drugs, terbutaline and atenolol, 79- and 27-fold lower, respectively, in Caco-2 cells than in jejunum. The carrier-mediated transport rates of L-dopa, L-leucine and D-glucose were also much slower in Caco-2 cells than in human jejunum. The lower permeability of the actively transported compounds and of atenolol and terbutaline is consistent with the colonic origin of the Caco-2 cells. The results indicate that Caco-2 monolayers can be used to predict passive drug transport in humans, while prediction of transport by carrier-mediated systems may require a scaling factor, due to a low expression of carriers in this cell line.