Kinetics and Mechanism of Degradation of Zileuton,a Potent 5-Lipoxygenase Inhibitor

Zileuton (N-(1-benzo[b]thien-2-ylethyl)N-hydroxyurea) is a powerful 5-lipoxygenase inhibitor. The chemical degradation of Zileuton and related hydroxyurea derivatives was studied in aqueous solutions as a function of pH and temperature. The pH profile for the degradation of Zileuton shows an acid-catalyzed region at pH values below 2, water hydrolysis of the protonated form at pH values from 3 to 8, and water hydrolysis of the unprotonated form at pH values greater than 9. Hydrolysis of the hydroxyurea moiety to give the hydroxylamine derivative represents the main degradation pathway for Zileuton. This product, however, is not stable and is present at low concentrations at pH values below 6 and not observed at pH values greater than 7. Further decomposition of the hydroxylamine derivative leads to the observed degradation products. Air oxidation to the isomeric oximes accounts for the observed products at pH values greater than 7. Hydrolysis of the oximes to the ketone derivative accounts for the observed products at pH values 2 to 6. Parallel decomposition pathways to the alcohol derivative were noted under strongly acidic conditions, pH 0 to 2.     

铂类抗癌新药双环铂的体内外抗肿瘤活性

目的观察铂类抗癌新药双环铂的体内外抗肿瘤活性及其对细胞周期的影响。方法MTT法观察双环铂对人肿瘤细胞增殖的抑制作用;小鼠动物肿瘤模型观察双环铂对肿瘤的体内抑制作用;碘化丙锭PI染色流式细胞术分析药物所致人卵巢癌细胞A2780细胞的周期变化。结果双环铂于体外抑制多种人肿瘤细胞增殖,体内抑制小鼠肉瘤S180、肝癌Heps及黑色素瘤B16的生长,并导致A2780细胞S期细胞明显增加及G2/M期细胞少许增加。结论双环铂具有明显的体内外抗肿瘤活性,并影响肿瘤细胞的细胞周期分布。     

Food Effect in Humans: Predicting the Risk Through In Vitro Dissolution and In Vivo Pharmacokinetic Models

In vitro and in vivo experimental models are frequently used to assess a new chemical entity’s (NCE) biopharmaceutical performance risk for food effect (FE) in humans. Their ability to predict human FE hinges on replicating key features of clinical FE studies and building an in vitro-in vivo relationship (IVIVR). In this study, 22 compounds that span a wide range of physicochemical properties, Biopharmaceutics Classification System (BCS) classes, and food sensitivity were evaluated for biorelevant dissolution in fasted- and fed-state intestinal media and the dog fed/fasted-state pharmacokinetic model. Using the area under the curve (AUC) as a performance measure, the ratio of the fed-to-fasted AUC (FE ratio) was used to correlate each experimental model to FE ratio in humans. A linear correlation was observed for the in vitro dissolution-human IVIVR (R² = 0.66, % mean square error 20.7%). Similarly, the dog FE ratio correlated linearly with the FE ratio in humans (R² = 0.74, % mean square error 16.25%) for 15 compounds. Data points near the correlation line indicate dissolution-driven mechanism for food effect, while deviations from the correlation line shed light on unique mechanisms that can come into play such as GI physiology or unusual physicochemical properties. In summary, fed/fasted dissolution studies and dog PK studies show a reasonable correlation to human FE, hence are useful tools to flag high-risk NCEs entering clinical development. Combining kinetic dissolution, dog FE model and in silico modeling one can study FE mechanism and formulation strategies to mitigate the FE risk.KEY WORDS: biorelevant dissolution, dog food effect model, food effect, in vitro-in vivo relationship     

Effect of Probenecid on Fluorescein Transport in the Central Nervous System Using In Vitro and In Vivo Models

Purpose. The purpose of this study was to characterize the function of multidrug resistance-associated proteins (MRPs) (or MRP-like organic anion transport systems) in the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) using both an in vitro BBB model and an in vivo microdialysis model.Methods. In vitro functional studies were performed using bovine brain microvessel endothelial cells (BBMEC). The accumulation of fluorescein, an anionic fluorescent dye, in BBMEC was determined with and without the presence of inhibitors of various efflux transport proteins. In vivo microdialysis simultaneously monitored fluorescein concentrations in cortical extracellular fluid and cerebrospinal fluid. The effect of probenecid on the in vivo distribution of fluorescein was studied using a balanced crossover design in the rat.Results. In vitro experiments showed that probenecid, indomethacin, LY-329146, and all MRP inhibitors significantly increased (two- to threefold) the accumulation of fluorescein in BBMEC, whereas LY-335979, a P-gp inhibitor, had no effect on the accumulation of fluorescein. Probenecid significantly increased fluorescein plasma concentration and the plasma free fraction in vivo. The distribution of fluorescein across the BBB and BCSFB was enhanced by 2.2- and 1.9-fold, respectively, when probenecid was coadministered, even after correction for increased fluorescein plasma concentrations and free fraction. Conclusions. These results demonstrate that MRPs or MRP-like transport system(s) may play an important role in fluorescein distribution across both BBB and BCSFB. This study showed that microdialysis proved to be a powerful in vivo technique for the study of transport systems in the central nervous system, and in vitro/in vivo correlations are possible using these model systems.     

In Vitro and In Vivo Antibacterial Activity of Gliotoxin Alone and in Combination with Antibiotics against Staphylococcus aureus

Multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital-acquired and community infections and pose a challenge to the human health care system. Therefore, it is important to find new drugs that show activity against these bacteria, both in monotherapy and in combination with other antimicrobial drugs. Gliotoxin (GT) is a mycotoxin produced by Aspergillus fumigatus and other fungi of the Aspergillus genus. Some evidence suggests that GT shows antimicrobial activity against S. aureus in vitro, albeit its efficacy against multidrug-resistant strains such as MRSA or vancomycin-intermediate S. aureus (VISA) strainsis not known. This work aimed to evaluate the antibiotic efficacy of GT as monotherapy or in combination with other therapeutics against MRSA in vitro and in vivo using a Caenorhabditis elegans infection model.     

重组人骨形态发生蛋白-7体内外生物学活性研究

采用体外、体内两种方法同时测定原核表达重组人BMP 7(rhBMP 7)的生物学活性 ,建立rhBMP 7活性测定的标准并为推广其临床应用提供依据。在体外 ,PNPP底物比色法和MTT比色法结果表明 ,10倍、10 0倍稀释上清组与细胞共同培养 72h后碱性磷酸酶 (ALP)水平和活细胞增殖数明显高于阴性对照组 (P <0 .0 5 ) ,而与标准品无显著性差异 (P >0 .0 5 )。在体内 ,将rhBMP 7植入小鼠肌间隙后 3周 ,见软骨岛和编织骨生成 ;而将rhBMP 7与胶原载体复合后植入 ,可见大量骨小梁、骨髓腔和板层骨 ,血管和骨髓丰富 ;成骨率均为8/ 8。ALP水平和钙含量 ,rhBMP 7组与标准品组相比 ,无显著性差异 (P >0 .0 5 ) ;复合组则明显高于单独植入rhBMP 7和胶原两组 (P <0 .0 5 )。     

In Vitro and In Vivo Activity of 16,17-dehydro-epipregnanolones: 17,20-Bond Torsional Energy Analysis and D-ring Conformation

Purpose. Certain neuroactive pregnane steroids (also known as “epalons”) are allosteric modulators of the GABA_A receptor and have been shown to be potent anticonvulsants, anxiolytics, sedative/hypnotics, and anesthetic agents. The purpose of this study was to calculate the structural consequences of introduction of a double bond in the 16,17-position and to determine if this modification would selectively reduce sedative activity, but maintain the potent anticonvulsant activity of neuroactive steroids. Methods. We have studied the biochemical and behavioral effects of introducing a 16,17 double bond into the naturally occurring neuroactive steroids, 3α-hydroxy-5α-pregnan-20-one (3α,5α-P) and 3α-hydroxy-5β-pregnan-20-one (3α,5β-P) and three synthetic neuroactive steroid derivatives, 3α-hydroxy-3β-methyl-5α-pregnan-20-one (3α,3βMe,5α-P), 3α-hydroxy-5α-androstane (3α,5α-A), and alphaxalone (3α,5α-11-one-P). Results. The 16-ene analogs of most of these neuroactive steroids were found to be 7- and 16-fold less potent in inhibiting [³⁵S]TBPS binding to GABA_A receptors and in a similar fashion, had reduced anticonvulsant and sedative potency in proportional amounts. The exception was the androstane (3α,5α-A) without a 17-acetyl group, that had virtually identical IC₅₀ and ED₅₀ values for the saturated and unsaturated derivatives. Calculation of the torsional energy profile for each of the 17-acetyl side chain conformations showed that the conformational energy minima found in the α,β-unsaturated keto systems, produce an orientation of the 20-keto group that is rotated by 165 degrees when compared to the non-conjugated acetyl group (determined by X-ray crystallography and its minimum energy conformation). Conclusions. The modified orientation of the 20-keto group of neuroactive steroids containing a 16-ene, provides an explanation for their decreased biological activity overall, but did not lead to an enhanced protective index.     

In Vitro and In Vivo Evaluation of the Enhancing Activity of Glycyrrhizin on the Intestinal Absorption of Drugs

Purpose. The enhancing activity of dipotassium glycyrrhizinate (Grz) on the intestinal absorption of drugs has been demonstrated in an in vitro study using Caco-2 cell monolayers and in an in vivo absorption study in rats. Methods. The hydrolysis of Grz by luminal content and mucosa of the rat colon was investigated. The absorption-enhancing activity of Grz and its hydrolysates was estimated by changes in transepithelial electrical resistance (TEER) and the permeation of sodium fluorescein (Flu-Na) in Caco-2 cell monolayers. It was further evaluated through the absorption of salmon calcitonin (sCT) in the rat colon. Results. Grz was not hydrolyzed to glycyrrhetinylmonoglucuronide (GrMG) and glycyrrhetinic acid (GA) by colonic mucosa, but, rather by the -glucuronidase in colonic flora. The hydrolysis of Grz to GrMG was extremely slow and the GrMG produced was rapidly regenerated to GA. Grz and GrMG had no effect on TEER nor on the permeability of Flu-Na across Caco-2 cell monolayers. On the other hand, GA decreased TEER and increased the permeability of Flu-Na in a dose-dependent manner. However, Grz and GrMG enhanced the plasma calcium-lowering effect of sCT after administration in the rat colon. The coadministration of sCT and GA in the rat colon induced the strongest plasma calcium-lowering effect and the highest plasma concentration of sCT. Conclusions. The in vivo enhancing-activity of Grz in the absorption of drugs is dependent on GA, a hydrolysis product of Grz resulting from the action of -glucuronidase in intestinal flora.     

LYG-202, a Newly Synthesized Flavonoid,Exhibits Potent Anti-angiogenic Activity In Vitro and In Vivo

LYG-202 (C₂₅H₃₀N₂O₅) is a newly synthesized flavonoid that has been confirmed to possess an antitumor effect, but the mechanism is unclear. Our present study was performed to identify the anti-angiogenic activity of this novel compound in vitro and in vivo. LYG-202 inhibited vascular endothelial growth factor (VEGF) stimulated migration and tube formation of human umbilical vein endothelial cells and arrested microvessel outgrowth from rat aortic rings in vitro. Meanwhile, LYG-202 suppressed the neovascularization of Chicken Chorioallantoic Membrane in vivo. Mechanistic studies revealed that LYG-202 suppressed the VEGF-induced tyrosine phosphorylation of KDR/Flk-1 (VEGFR-2) as well as its downstream protein kinases activation, by decreasing phosphorylated forms of serine/threonine kinase Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase. LYG-202 exerts anti-angiogenic activity both in vitro and in vivo, and these results suggest that it deserves further investigation as a promising anti–tumor angiogenesis compound.     

Polyamine Aza-Cyclic Compounds Demonstrate Anti-Proliferative Activity In Vitro But Fail to Control Tumour Growth In Vivo

Cationic polyamines such as the poly(propylenimine) dendrimers (DAB16) are anti-tumour agents (Dufes et al., 2005, Cancer Res 65:8079–8084). Their mechanism of action is poorly understood, but the lack of in vivo toxicity suggests cancer specificity. To explore this polyamine pharmacophore we cross-linked the aza-cyclic compound, hexacyclen, with 1,4-dibromobutane or 1,8-dibromooctane to yield the polyamines [poly(butylhexacyclen)—CL4] or [poly(octylhexacyclen)—CL8] respectively, both free of primary amines. We characterised the compounds and their respective nanoparticles and examined their interaction with the putative targets of the cationic polyamines: the cell membrane and DNA. Like DAB 16, CL4 and CL8 bind plasmid DNA and all three compounds interrupted the cell cycle of A431 epidermoid carcinoma cells in the S-phase. Additionally all three compounds disrupted erythrocyte membranes, with CL8 and DAB 16 being more active, in this respect, than CL4. CL4 (IC₅₀ = 775.1 μg mL^? 1) and CL8 (IC₅₀ = 8.4 μg mL^? 1), in a similar manner to DAB 16, were anti-proliferative against A431 cells; although unlike DAB 16, CL4 and CL8 were not tumouricidal against A431 xenografts in mice, indicating that primary amines may play an important role in the in vivo activity of DAB 16.     

In Vitro/in Vivo Models for Peptide Oral Absorption: Comparison of Caco-2 Cell Permeability with Rat Intestinal Absorption of Renin Inhibitory Peptides

Pharmaceutical Research -     

Cellular and Molecular Mechanisms of Action of Transcranial Direct Current Stimulation: Evidence from In Vitro and In Vivo Models

Transcranial direct current stimulation is a noninvasive technique that has been experimentally tested for a number of psychiatric and neurological conditions. Preliminary observations suggest that this approach can indeed influence a number of cellular and molecular pathways that may be disease relevant. However, the mechanisms of action underlying its beneficial effects are largely unknown and need to be better understood to allow this therapy to be used optimally. In this review, we summarize the physiological responses observed in vitro and in vivo, with a particular emphasis on cellular and molecular cascades associated with inflammation, angiogenesis, neurogenesis, and neuroplasticity recruited by direct current stimulation, a topic that has been largely neglected in the literature. A better understanding of the neural responses to transcranial direct current stimulation is critical if this therapy is to be used in large-scale clinical trials with a view of being routinely offered to patients suffering from various conditions affecting the central nervous system.     

卡莫司汀聚乳酸微球的体外释放及其在大鼠脑组织中的分布研究

目的:探讨卡莫司汀聚乳酸微球(BCNU-PLA-MS)的体外释药过程及其在大鼠脑组织中的分布。方法:应用高效液相色谱法检测BCNU在磷酸盐缓冲溶液(PBS)和大鼠脑组织内自PLA-MS中释放的药物含量;应用3H标记BCNU,检测3H-BCNU-PLA-MS在正常大鼠脑组织及血清中的分布。结果:BCNU-PLA-MS在PBS和大鼠脑组织中均可持续释药2wk以上。在PBS中,其1、3、15d时药物释放率分别约为15%、50%、90%;在大鼠脑组织内,其4h、1d、3d时药物释放率分别约为8%、16%、60%,并可持续释药15d。大鼠药物植入处药物浓度是其它检测点的6～70倍。结论:BCNU-PLA-MS具有良好的缓释功能,而且安全性、生物相容性较好。     

Biomarkers of Exposure to Zearalenone in In Vivo and In Vitro Studies

The measurement of human exposure to mycotoxins is necessary for its association with adverse health effects. This exposure is usually estimated from contamination levels of foodstuffs, which are the primary source of toxin exposure, and data on food consumption patterns. However, variations in contamination level, intestinal absorption, toxin distribution, and excretion lead to individual variations in toxin exposure that can be more readily measured with a biomarker. This review deals with the latest literature information about ZEN biomarkers in humans, animals, and cell line cultures. Their presence in urine, biomarkers that have effects in the kidney, liver, reproductive system and blood and biomarkers of cell response have been reported. It has highlighted the importance of determining α-zearalenol and β-zearalenol biomarkers to estimate the probable dietary intake (PDI) of a specific population or to characterize the severity of exposure to ZEN in animals or cell lines. α-ZEL and β-ZEL are cytotoxic by inhibiting cell proliferation, total protein and DNA syntheses, in this sense, an induction of expression proteins Hsp27 and Hsp70 was observed, and an increase in gene expression (TLR4, NF-kBp65, TNF-α, IL-1β, IL-6, IL-8, MGMT, α-GST, Hsp70, Nrf2, L-Fabp, HO-1, MAPK8), the determination of which indicates an oxidative stress effect. The integrity of the cell or tissue membrane is assessed by lactate dehydrogenase (LDH), which increase at exposure of ZEN (84.2 µM), and the proportions of some fatty acids of the renal tissue membrane were increased at treatments with ZEN. This review allows starting future studies of animal and population exposure in parallel with those of health effects works.