Anooshirvan Shayeganpour

Activation of antigen-specific T cell-responses by mannan-decorated PLGA nanoparticles.

ABSTRACTPurposeMannosylation of vaccines is a promising strategy to selectively target vaccine antigens to the mannose receptor expressed on dendritic cells (DCs). The purpose of this study was to investigate the effect of mannan (MN) chemically conjugated to poly(D, L-lactide-co-glycolic acid) (PLGA) nanoparticles (NPs) on antigen-specific T-cell responses elicited by a model antigen (ovalbumin, OVA) loaded in PLGA-NPs.MethodsIn vitro T-cell proliferation assay was done to assess the ability of DCs treated with OVA-NPs (±MN decoration) to induce antigen-specific T-cell activation. The efficacy of this vaccination strategy was further evaluated in vivo, where T-cell proliferation was performed to evaluate activation of T-cell responses in lymph nodes and spleens isolated from the vaccinated mice.ResultsOur results demonstrate that MN-decorated antigen-loaded PLGA-NPs simultaneously enhanced antigen-specific CD4+ and CD8+ T-cell responses compared to non-decorated NPs.ConclusionsMN decoration of PLGA-NPs is a promising strategy for enhancing antigen-specific T-cell responses.

The impact of experimental hyperlipidemia on the distribution and metabolism of amiodarone in rat

The tissue distribution and hepatic microsomal metabolism of amiodarone were studied in a hyperlipidemic rat model. Rats were rendered hyperlipidemic by the intraperitoneal injection of poloxamer 407. Other normolipidemic animals given saline in place of poloxamer 407 were used as control animals. After single intravenous injection of amiodarone HCl (25mg/kg) rats were anesthetized and plasma and tissue specimens were obtained. Liver microsomal protein was harvested and used to measure velocity of desethylamiodarone formation from amiodarone and cytochrome P450 (CYP) protein expression. Hyperlipidemia caused large increases in plasma concentrations of amiodarone. In tissues, however, concentrations of drug selectively increased, decreased or did not change. In heart, the site of action of the drug, as well as liver and spleen, amiodarone concentrations increased. In other tissues such as kidney, lung and brain, concentrations decreased. No changes were seen in fat or thyroid. Decreases were observed in liver metabolic efficiency, and expression of CYP3A1/2 and 2C11. No changes were seen in CYP2B1/2, 2C6, 2D1 or 1A2. This experimental hyperlipidemia caused a complex pattern of changes in tissue distribution of AM. In addition, there are decreases in the expression of some important rat CYP isoenzymes.

Determination of the enzyme(s) involved in the metabolism of amiodarone in liver and intestine of rat: the contribution of cytochrome P450 3A isoforms.

In humans, cytochrome P450 3A (CYP3A4) is a major enzyme involved in the metabolism of amiodarone (AM) to its major metabolite, desethylamiodarone (DEA). In rat, a commonly used animal model, metabolism of AM has not been well studied. To determine whether DEA is formed by CYP3A isoenzymes in the rat, microsomal protein was harvested from liver and intestine of male Sprague-Dawley rats. The metabolism of AM in each tissue was assessed utilizing chemical and immunological inhibitors. Ketoconazole, a presumed inhibitor of CYP3A1/2, significantly inhibited formation of DEA by hepatic and intestinal microsomes. However, based on the DEA formation kinetics in both microsomal preparations, it appeared that more than one cytochrome P450 enzyme was involved in the process. Coincubation of AM with microsomes and anti-CYP3A2 confirmed the role of CYP3A2 in the metabolism of AM in liver. DEA was also formed by rat recombinant CYP1A1 and CYP3A1, and was inhibited by ketoconazole; hence the participation of these enzymes in the intestinal DEA formation is likely. However, anti-CYP2B1/2 or -CYP1A2 antibodies had no effect on DEA formation. In rats given oral or intravenous AM, oral ketoconazole caused significant increases in area under the concentration versus time curve (AUC) of oral and i.v. treated rats and greater than 50% decreases in the total body clearance and Vdss of i.v. treated rats. Although low to undetectable concentrations of DEA were a limitation for determination of AUC of DEA in vivo, it was confirmed that ketoconazole could cause a significant increase in AM concentrations in rat.

Encapsulation of P-glycoprotein inhibitors by polymeric micelles can reduce their pharmacokinetic interactions with doxorubicin.

Co-administration of P-glycoprotein (P-gp) inhibitors such as cyclosporine A (CyA) and its analogue valspodar with doxorubicin (DOX) can result in diminished clearance of DOX, leading to accentuated toxicity. The purpose of this study was to evaluate whether the effect of these P-gp inhibitors on the pharmacokinetics of DOX can be avoided through their encapsulation in polymeric micelles. Cyclosporine A or valspodar was physically encapsulated in methoxypoly(ethylene oxide)-b-poly(ε-caprolactone) (PEO-b-PCL) micelles using co-solvent evaporation method. The commercially available DOX was administered as a single dose of 5mg/kg intravenously to Sprague-Dawley rats either alone or 30min following a single intravenous dose (10mg/kg) of either CyA or valspodar as part of conventional or polymeric micellar formulation. Co-administration of DOX with either Sandimmune® or valspodar in the conventional Cremophor EL-based formulation was associated with greater than 50% reduction in DOX clearance (CL). Although there was nearly 40% reduction in the CL of DOX with the polymeric micellar formulation of CyA, there was only 6% reduction in CL of DOX upon co-administration with the polymeric micellar formulation of valspodar. In conclusion, encapsulation of cyclosporines, particularly valspodar, in polymeric micelles was shown to reduce their effects on the pharmacokinetics of DOX in rat.

The influence of hyperlipoproteinemia on in vitro distribution of amiodarone and desethylamiodarone in human and rat plasma.

PurposeTo study the effect of hyperlipoproteinemia on in vitro distribution of amiodarone (AM) and its prevalent metabolite desethylamiodarone (DEA) in human and rat plasma.Materials and MethodsHuman and rat normolipidemic (NL) and hyperlipidemic (HL) plasma were spiked with AM and DEA. The fractions (high and low density lipoproteins, triglyceride rich lipoproteins and lipoprotein deficient plasma) were separated using ultracentrifugation.ResultsHuman and rat displayed similar patterns in terms of association of AM and DEA in NL plasma, in which the highest and lowest associations were observed in lipoprotein deficient (LPDP) and triglyceride (TRL) rich plasma fractions, respectively. In HL a substantial shift was observed in partitioning of AM and DEA mostly to TRL. The shift of AM and DEA into TRL fraction of HL plasma was more drastic for rat than human. In HL, association of AM with rat LPDP and HDL fractions were 10 and 26-fold lower than in the corresponding human fractions, respectively. The DEA:AM ratio in rat, but not human, was significantly affected by HL.ConclusionHL caused a major shift of AM and DEA to TRL fraction in both species. The findings were consistent with the higher AM concentrations previously noted in HL rats given the drug.

An essential role for Rab27a GTPase in eosinophil exocytosis

Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab‐related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a‐deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a+ structures, some of which colocalized with CD63+ crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a‐dependent eosinophil degranulation in airway inflammation.

The Immunosuppressive Activity of Polymeric Micellar Formulation of Cyclosporine A: In Vitro and In Vivo Studies

We have previously developed micelles of methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) as vehicles for the solubilization and delivery of cyclosporine A (CsA). These micelles were able to reduce the renal uptake and nephrotoxicity of CsA. The purpose of the current study was to test the efficacy of polymeric micellar formulation of CsA (PM-CsA) in suppressing immune responses by either T cells or dendritic cells (DCs). The performance of PM-CsA was compared to that of the commercially available formulation of CsA (Sandimmune®). Our results demonstrate that PM-CsA could exert a potent immunosuppressive effect similar to that of Sandimmune® both in vitro and in vivo. Both formulations inhibited phenotypic maturation of DCs and impaired their allostimulatory capacity. Furthermore, both PM-CsA and Sandimmune® have shown similar dose-dependent inhibition of in vitro T cell proliferative responses. A similar pattern was observed in the in vivo study, where T cells isolated from both PM-CsA-treated and Sandimmune®-treated mice have shown impairment in their proliferative response and IFN-γ production at similar levels. These results highlight the potential of polymeric micelles to serve as efficient vehicles for the delivery of CsA.

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin‐dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT‐PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet‐activating factor, and secretory IgA‐Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock‐down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin‐4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist‐induced degranulation.

Effects of intestinal constituents and lipids on intestinal formation and pharmacokinetics of desethylamiodarone formed from amiodarone.

To model the impact of intestinal components associated with a high fat meal on metabolism of amiodarone, rat everted intestinal sacs were evaluated for their ability to metabolize the drug to its active metabolite (desethylamiodarone) under a variety of conditions. The preparations were obtained from fasted rats or rats pretreated with 1% cholesterol in peanut oil. After isolation of the tissues, the intestinal segments were immersed in oxygenated Krebs Henseleit buffer containing varying concentrations of bile salts, cholesterol, lecithin and lipase with or without soybean oil emulsion as a source of triglycerides. Amiodarone uptake was similar between the five 10‐cm segments isolated distally from the stomach. Desethylamiodarone was measurable in all segments. Based on the metabolite‐to‐drug concentration ratio within the tissues, there was little difference in metabolic efficiency between segments for any of the treatments. Between treatments, however, it appeared that the lowest level of metabolism was noted in rats pretreated with 1% cholesterol in peanut oil. This reduction in metabolic efficiency was not observed in gut sacs from the fasted rats to which soybean oil emulsion was directly added to the incubation media. Despite the apparent reduction in intestinal metabolism, there was no apparent change in the ratio of metabolite‐to‐drug area under the plasma concentration versus time ratios of fasted rats and those given 1% cholesterol in peanut oil, suggesting that the intestinal presystemic formation of desethylamiodarone is not substantial.