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Ixeris sonchifolia Hance is an herb distributed in northeastern part of China and has been used by natives to invigorate circulation. In the present study, bioactivity-guided fractionation of I. sonchifolia Hance extract was performed with the aim to isolate and identify the compounds underlying the potential protective effects against ischemia brain injury. Among the four fractions isolated from the herb extract, the ethyl acetate fraction was found to scavenge DPPH radicals, induce ARE-dependent transcriptional activity and upregulate Nrf2 protein levels. The isolation work focused on this fraction revealed the presence of two categories of compounds: flavonoids and sesquiterpene lactones. Among the five isolated flavonoids, luteolin was evaluated to possess direct and indirect antioxidant activities by scavenging free radicals and inducing the upregulation of ARE-dependent phase II enzymes. Concomitant with the findings from the cell-based assays, in the middle cerebral artery occlusion-induced ischemia rat model, administration of luteolin at 4 mg/kg displayed neuroprotective effects by reducing infarct area and inhibiting neuronal cell death. In summary, the obtained results suggest that flavonoids in I. sonchifolia Hance, in particular luteolin, contribute at least partly to the neuroprotective effects against ischemia-induced cellular injury and can be potentially developed for treatment of ischemia-reperfusion induced diseases.

The intention of this study was to develop and validate a simple, sensitive, and accurate high performance liquid chromatography (HPLC) method for quantification of trans-resveratrol in rat plasma. Trans-resveratrol was recovered from plasma samples by liquid-liquid extraction. A reverse phase HPLC column was used and the assay was performed at 35°C through isocratic delivery (1 mL · min−1) of acetonitrile and phosphate buffer solution, pH 7.0 (30:70 v/v). UV absorbance was monitored at 320 nm. Plasma samples were stable (>99%) at −80°C for 3 months. The recovery of trans-resveratrol from plasma was >95%. The HPLC method was validated. The retention times of trans-resveratrol and carbamazepine were 5.5 and 10.4 min, respectively. The calibration curve was linear (R2 = 0.9999) within the range of 5–1,000 ng · mL−1. The lower limits of detection and quantification of trans-resveratrol were 2.5 and 5 ng · mL−1, respectively. The intra-day and inter-day variations were 95%. This simple H...

A new lanostane-type triterpene, 29-hydroxypolyporenic acid C (8), was isolated from the dried sclerotia of Poria cocos together with eight other known compounds pachymic acid (1), dehydropachymic acid (2), 3-acetyloxy-16alpha-hydroxytrametenolic acid (3), polyporenic acid C (4), 3-epi-dehydropachymic acid (5), 3-epi-dehydrotumulosic acid (6), tumulosic acid (7), and dehydrotumulosic acid (9). The compounds were identified by spectral analysis and comparison with spectroscopic data reported in the literatures. Although none of the nine (1 to 9) compounds showed promising antioxidant activity, 1 through 6 and 8 showed good cytotoxicity against human lung cancer cell line A549 and human prostate cancer cell line DU145. Interestingly, all these compounds exhibited better cytotoxicity towards A549 than DU145 cells.

Purpose. To establish whether anionic liposomes interact with the low-density lipoprotein (LDL) receptor, to determine the role of lipoproteins in this interaction, and whether the association causes functional delivery of encapsulated drugs.

An in vitro cell culture model based on a rat alveolar macrophage (AM) cell line, NR8383, was used to determine if poly( l -lactide) (PLA) microspheres prepared by the precipitation with a compressed antisolvent (PCA) method can be taken up by AMs and activate AMs. To examine cellular uptake of microspheres, microspheres were labeled with rhodamine 6G. Using fluorescence microscopy, the uptake of microspheres by NR8383 cells was followed as a function of time, microsphere concentration, and susceptibility to lysosomotropic agents. To determine if microspheres can activate NR8383 cells, the oxidative burst and production of TNF-α by NR8383 cells following microsphere treatment was measured. Uptake of microspheres by NR8383 cells was dependent on microsphere concentration and appeared to occur via endocytosis, as uptake was significantly inhibited by the putative lysosomotropic agents, ammonium chloride and chloroquine. Furthermore, the microspheres do not appear to activate NR8383 cells, since microsphere exposure results in negligible oxidative burst and TNF-α production in NR8383. Microspheres prepared by the PCA method hold great potential in targeting drugs to AMs and, therefore, may be of utility for the treatment of diseases in which AMs play an important role, such as tuberculosis (TB).

Utilization of primary cultured brain capillary endothelial cells (BCECs) as an in vitro model of the blood-brain barrier (BBB) depends on the extent to which cultured BCECs retain the in vivo characteristics. Recently, we have reported that consistent isolation of BCECs that mimic the in vivo BBB depends on whether a specific ratio between the weight of the isolation enzyme (collagenase/dispase) and the weight of the capillaries present during the isolation is used. Since it is possible for the same weight of an enzyme to possess different activity levels, it is felt that activity rather than weight of an enzyme should be used in arriving at the above ratio. Therefore, using bovine brain as the source of BCECs, we have quantified the amount of collagenase/dispase needed for optimal isolation of BCECs and retention of their phenotypic properties in terms of collagenase/dispase activity per g of capillaries. Monolayers of bovine BCECs isolated at 0.15 or 0.30 units of collagenase and 2.06 or 4.12 units dispase per g of capillaries gave the best overall quality as judged by their permeability characteristics and the activities of angiotensin converting enzyme, alkaline phosphatase and gamma-glutamyl transpeptidase.

Multidrug resistance-associated protein 4 (MRP4) is an organic anion efflux pump capable of transporting nucleoside, nucleotide analogs, and cyclic nucleotide. MRP4 could have an influence on the resistance and transport of the two oxazaphosphorines, cyclophosphamide (CP) and ifosfamide (IF). V/HepG2 (HepG2, hepatoma cells stably transfected with an empty vehicle plasmid) and MRP4/HepG2 (HepG2 cells stably expressing MRP4) were exposed to CP and IF in the absence or presence of various MRP4 inhibitors. HepG2 and HEK293 human kidney cells were also used to investigate the inducing potency of oxazaphosphorines on the MRP4 expression. In this study, insertion of MRP4 gene in HepG2 cells was found to confer significant resistance to CP and IF in the 48-h drug-exposure assays. In the presence of various MRP4 inhibitors, the resistance to CP and IF was then partially reversed. These indicate that CP and IF are highly possible substrates of MRP4. In addition, CP and clofibrate (CFB), a reported MRP4 inducer, in vivo significantly increased the MRP4 expression at both protein level and mRNA level in HEK293 cells at higher concentrations, while IF significantly decreased the MRP4 expression at mRNA level at lower concentration and had no effect at higher concentrations. However, all tested compounds (CP, IF, and CFB) did not change the MRP4 protein expression in HepG2 cells. CP and CFB are cell-specific and concentration-dependent MRP4 inducers. The finding may have implications in the CP- or IF-based chemotherapy.

When two or more tight-binding inhibitors are present in an enzyme assay, the equation that relates the initial velocity v to the concentration of reactants cannot be written in an algebraically explicit form. Rather, for n inhibitors it is an implicit polynomial equation of degree n + 1 with respect to v. The complexity of the polynomial coefficients dramatically increases with each added inhibitor. Solving the transcendental rate equation by traditional methods of numerical mathematics has proven tedious because of the sensitivity of these methods to initial estimates and because of the existence of multiple roots. However, the equation can be rearranged into a convenient recursive form, one in which the velocity appears on both sides and the solution is found iteratively. The algebraic form of the recursive rate equation is remarkably simple and differs from the rate equation for classical rather than tight-binding inhibition only by an added term. The numerical stability and the speed of convergence were tested on the case of two competitive inhibitors. Initial estimates of velocity that spanned 12 orders of magnitude converged within five iterations. The velocities computed with the recursive method for a single tight-binding inhibitor were identical with the values predicted by the Morrison equation. The method is used to analyze experimental data for the inhibition of rat liver dihydrofolate reductase by mixtures of the anticancer drug methotrexate and its metabolic precursor form, methotrexate-α-aspartate (a prodrug)

An in vitro liposome-cell association system has been developed that will allow the study of uptake and metabolism of liposomes by cultured cells at nanomolar lipid concentrations. The fate of cell associated liposomes is followed through the liposome encapsulated marker, methotrexate. Detection is based on the inhibition of dihydrofolate reductase by methotrexate, after its release from cells through boiling. Methotrexate in phospha-tidylglycerol (PG) liposomes is taken up by cells and then subsequently lost from the cells. Uptake is partially blocked by monensin. Loss from the cells is blocked by metabolic inhibitors, monensin, ammonium chloride, and chloroquine. Methotrexate in distearoylphosphatidylglycerol (DSPG) liposomes is taken up by cells slowly, and there is minimal lost of methotrexate after uptake. Pulse studies show that metabolism of PG liposomes after endocytosis is occurring at a much higher rate than that of DSPG liposomes, and substantial retention of encapsulated methotrexate o...

Until recently it was believed that opioid uptake was mediated by passive diffusion; however, we recently demonstrated that brain: plasma partitioning of fentanyl reduced by verapamil, likely by in...

Pim-1 kinase phosphorylates substrates whose activities are linked to proliferation, survival, differentiation, and apoptosis. Although pim-1 is induced by hormones and cytokines, the hormonal control and contribution of Pim-1 to mammary gland development have not been evaluated. We examined Pim-1 expression in mammary cell lines, investigated whether Pim-1 levels could be altered in breast epithelia by mammogenic hormones, and evaluated Pim-1 expression during mammary development. We found that Pim-1 was elevated in most mammary carcinoma cell lines and progesterone increased Pim-1 protein to some extent in non-tumorigenic mammary epithelia. Pim-1 expression in situ was consistent with the documented profile of progesterone activity in mouse mammary glands. Pim-1 nuclear localization correlated with cytoplasmic distribution for its substrate, p21(CIP/Waf1), and we found that Pim-1 and p21 associate in vitro. Our results suggest that Pim-1 expression may be regulated by progesterone during mammary development and Pim-1 associates with p21 in mammary epithelial cells.

To evaluate if pulmonary delivery of microparticles loaded with a prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), can target alveolar macrophages (AM) and reduce metabolism of INH, an HPLC-MS/MS assay with automated online extraction for quantification of INH and its metabolite acetylisoniazid (AcINH) in plasma and AMs was developed and validated. Reproducibility in rat plasma and homogenate of a rat AM cell line, NR8383, for INH and AcINH showed excellent precision and accuracy with calibration curves exhibiting linearity within a range of 1-250ng/ml of INH and 0.05-50ng/ml of AcINH (r(2)>0.99). The validated methods were successfully applied to pharmacokinetic study of INHMS-loaded microparticles in rats, demonstrating efficient targeting of AMs and reduction of INH metabolism.

Pachymic acid (PA) is a natural triterpenoid known to inhibit the phospholipase A2 (PLA(2)) family of arachidonic acid (AA)-producing enzymes. PLA(2) is elevated in prostatic adenocarcinoma and conversion of AA to prostaglandins leads to AKT pro-survival activity. In this study, we investigated the effect of PA on the growth of human prostate cancer cells. PA significantly reduced cell proliferation and induced apoptosis in a dose- and time-dependent fashion, with androgen-insensitive DU145 prostate cancer cells showing greater growth inhibition relative to androgen-responsive LNCaP. Despite elevated protein expression of the cell cycle inhibitor, p21, apoptosis occurred in the absence of cell cycle arrest. PA-treatment decreased Bad phosphorylation, increased Bcl-2 phosphorylation, and activated caspases-9 and -3, suggesting that PA initiated apoptosis through mitochondria dysfunction. PA-treatment also decreased the expression and activation of proteins within the AKT signal pathway. We speculate that PA influenced apoptosis by reducing prostaglandin synthesis and AKT activity.

The purpose of this study was to assess the effect of mild heat and heat preconditioning on the uptake and permeability of a P‐glycoprotein (P‐gp) substrate, rhodamine 123 (R123), in a cell culture model of the blood–brain barrier (BBB). An immediate goal was to determine whether prior mild heat treatment could render brain microvessel endothelial cells more resistant to future heat stress and affect BBB drug permeation by future ultrasound‐induced mild heat (USMH) treatment. To address this issue, the expression level of two proteins, P‐gp and heat shock protein 70 (Hsp70), and their effects on uptake of R123 and permeability of R123 and [ 14 C]‐sucrose in combination with mild heat and P‐gp modulator PSC833 during and after mild heat treatment in heat‐preconditioned and heat‐unconditioned bovine brain microvessel endothelial cell (BBMEC) monolayers were studied. Mild heat caused a significant increase in BBB permeability of R123 and [ 14 C]‐sucrose when compared with control and PSC833. Exposure of BBMECs to heat preconditioning caused a slight but insignificant decrease in cellular uptake of R123 both during and immediately after mild heat treatment. Heat preconditioning also caused a slight but insignificant decrease in permeability of R123 and [ 14 C]‐sucrose in BBMEC monolayers during mild heat treatment. Because exposure of BBMEC monolayers to mild heat did not affect P‐gp expression but slightly affected Hsp70 expression, a heat preconditioning that results in a reinforcement of the BBB other than increased expression of P‐gp is suggested. However, heat preconditioning is not sufficient to override the permeation‐enhancing effects of mild heat because mild heat caused a significant increase in R123 uptake and permeability of R123 and [ 14 C]‐sucrose in both heat‐preconditioned and heat‐unconditioned cells. Because Hsp70 is known to play a major role in cellular repair and protective mechanisms, our results would imply a relative benign nature of mild heat treatment. Because heating produced by ultrasonic waves can be controlled and localized to a small volume within the tissue, the present results also suggest that USMH could play a pivotal role in the treatment of brain tumors and other brain‐related diseases.

We have examined the effects of verapamil and PSC 833 on cellular uptake and release of rhodamine 123 (R123) in two human cancer cell lines. Both verapamil and PSC 833 were able to increase R123 accumulation in the multidrug resistant (MDR) MV522/Q6 and KB-8-5 lines in the release study. However, the effects of these drugs on R123 accumulation during accumulation study were quite different. Incubation with PSC 833 increased R123 accumulation in both MDR lines. By contrast, incubation with verapamil only increased R123 accumulation in the KB-8-5 line. The failure of verapamil to increase R123 accumulation in the MV522/Q6 cells can be attributed to the presence of a carrier system in the parent MV522 cells that recognizes both R123 and verapamil, but not PSC 833, as substrates. These results imply that performing R123 accumulation study without first ascertaining possible role of a carrier system for cellular uptake of R123 and putative P-gp modulators might inadvertently lead one to draw improper conclusions on P-gp activity.

The apical (AP)-to-basolateral (BL) transport of [3H]biotin across Caco-2 cell monolayers grown on a Transwell polycarbonate membrane was shown to be dependent on days in culture, reaching a maximum at 10 days. The AP-to-BL transport of [3H]biotin was always much greater than the BL-to-AP transport. AP-to-BL transport of [3H]biotin was concentration-(Km = 1.49 microM and Vmax = 1.86 pmol.mg protein-1.minute-1), temperature-, Na(+)- and energy-dependent, and proceeded against a concentration gradient in the BL side. [3H]Biotin transport was inhibited by unlabeled biotin and several structural analogues (e.g., desthiobiotin and biotin methyl ester) but not by taurocholic acid. No metabolism of [3H]biotin was detected in the AP-to-BL transport across Caco-2 cell monolayers. This study demonstrates that [3H]biotin transport in Caco-2 cells occurs by a carrier-mediated process.

We have developed a two-compartment growth inhibition assay that can provide information about leakage, metabolism and delivery of liposome-dependent drugs under cell culture conditions, and at drug concentrations that are relevant to drug delivery. Two cell lines are grown in separate compartments separated from each other by a 0.1 micron polycarbonate membrane. The membrane allows free drugs to diffuse rapidly from one compartment to another, and does not allow liposomes to diffuse through. Liposomes are added to the first compartment, which contains target cells. The extent of leakage caused by these cells is determined by the growth inhibition of non-target cells in the second compartment. We show that methotrexate and methotrexate-gamma-aspartate leak rapidly and almost completely when encapsulated in phosphatidylglycerol/cholesterol (67:33) liposomes. In contrast, there is only 42% leakage when the drugs are encapsulated in distearoylphosphatidylglycerol/cholesterol (67:33) liposomes. We also demonstrate that the target cells (CV1-P) may partially degrade encapsulated methotrexate-gamma-aspartate to methotrexate. Therefore, methotrexate-gamma-aspartate may be a lysosomally cleaved pro-drug of methotrexate.