Enantioselective CE analysis of hepatic ketamine metabolism in different species in vitro

Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently in most animal species. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations is described. Racemic ketamine was incubated with pooled microsomes from humans, horses and dogs over a 3 h time interval with frequent sample collection. CE data revealed that ketamine is metabolized enantioselectively to norketamine (NK), dehydronorketamine and three hydroxylated NK metabolites in all three species. The metabolic patterns formed differ in production rates of the metabolites and in stereoselectivity of the hydroxylated NK metabolites. In vitro pharmacokinetics of ketamine N-demethylation were established by incubating ten different concentrations of racemic ketamine and the single enantiomers of ketamine for 8 min and data modeling was based on Michaelis-Menten kinetics. These data revealed a reduced intrinsic clearance of the S-enantiomer in the racemic mixture compared with the single S-enantiomer in human microsomes, no difference in equine microsomes and the opposite effect in canine microsomes. The findings indicate species differences with possible relevance for the use of single S-ketamine versus racemic ketamine in the clinic.

Robust expression of CCR3 as a single basophil selection marker in flow cytometry

Basophil activation tests (BAT) rely on different combinations of basophil selection and activation markers. Whereas activation markers, especially CD63, are widely validated, the most suitable and robust marker for basophil selection is still a matter of debate.

Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals

Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values used in previous modeling efforts. Bioconcentration factors (BCF) predicted using the experimental binding data were substantially higher than the predicted values obtained in earlier studies and correlated poorly with measured BCF values in fish. One possible explanation for this finding is that chemicals bound to proteins can desorb rapidly and thus contribute to metabolic turnover of the chemicals. This hypothesis remains to be investigated in future studies, ideally with chemicals of higher hydrophobicity.

In vitro and in vivo efficacies of mefloquine-based treatment against alveolar echinococcosis

Alveolar echinococcosis (AE) is caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis and causes severe disease in the human liver, and occasionally in other organs, that is fatal when treatment is unsuccessful. The present chemotherapy against AE is based on mebendazole and albendazole. Albendazole treatment has been found to be ineffective in some instances, is parasitostatic rather than parasiticidal, and usually involves the lifelong uptake of large doses of drugs. Thus, new treatment options are urgently needed. In this study we investigated the in vitro and in vivo efficacy of mefloquine against E. multilocularis metacestodes. Treatment using mefloquine (20 muM) against in vitro cultures of metacestodes resulted in rapid and complete detachment of large parts of the germinal layer from the inner surface of the laminated layer within a few hours. The in vitro activity of mefloquine was dependent on the dosage. In vitro culture of metacestodes in the presence of 24 muM mefloquine for a period of 10 days was parasiticidal, as determined by murine bioassays, while treatment with 12 muM was not. Oral application of mefloquine (25 mg/kg of body weight administered twice a week for a period of 8 weeks) in E. multilocularis-infected mice was ineffective in achieving any reduction of parasite weight, whereas treatment with albendazole (200 mg/kg/day) was highly effective. However, when the same mefloquine dosage was applied intraperitoneally, the reduction in parasite weight was similar to the reduction seen with oral albendazole application. Combined application of both drugs did not increase the treatment efficacy. In conclusion, mefloquine represents an interesting drug candidate for the treatment of AE, and these results should be followed up in appropriate in vivo studies.

In vitro efficacy of dicationic compounds and mefloquine enantiomers against Echinococcus multilocularis metacestodes

The current chemotherapy of alveolar echinococcosis (AE) is based on benzimidazoles such as albendazole and has been shown to be parasitostatic rather than parasiticidal, requiring lifelong duration. Thus, new and more efficient treatment options are urgently needed. By employing a recently validated assay based on the release of functional phosphoglucose isomerase (PGI) from dying parasites, the activities of 26 dicationic compounds and of the (+)- and (-)-erythro-enantiomers of mefloquine were investigated. Initial screening of compounds was performed at 40 muM, and those compounds exhibiting considerable antiparasitic activities were also assessed at lower concentrations. Of the dicationic drugs, DB1127 (a diguanidino compound) with activities comparable to nitazoxanide was further studied. The activity of DB1127 was dose dependent and led to severe structural alterations, as visualized by electron microscopy. The (+)- and (-)-erythro-enantiomers of mefloquine showed similar dose-dependent effects, although higher concentrations of these compounds than of DB1127 were required for metacestode damage. In conclusion, of the drugs investigated here, the diguanidino compound DB1127 represents the most promising compound for further study in appropriate in vivo models for Echinococcus multilocularis infection.

Lung on Chip: In vitro HGF effects on injured alveolar A549 epithelial cells in microfluidic system

Microfluidic systems have become competitive tools in the invitro modelling of diseases and promising alternatives to animal studies. They allow obtaining more invivo like conditions for cellular assays. Research in idiopathic pulmonary fibrosis could benefit from this novel methodological approach to understand the pathophysiology of the disease & develop efficient therapies. The use of hepatocyte growth factor (HGF) for alveolar reepithelisation is a promising approach. In this study, we show a new microfluidic system to analyse the effects of HGF on injured alveolar epithelial cells. Microfluidic systems in polydimethylsiloxane were fabricated by soft lithography. The alveolar A549 epithelial cells (10,000 cells) were seeded and studied in these microfluidic systems with media perfusion (1μl/30min). Injury tests were made on the cells by the perfusion with media containing H2O2 or bleomycin. The degree of injury was then assessed by a metabolic and an apoptotic assays. Wound assays were also performed with a central laminar flow of trypsin. Monitoring of wound closure with HGF vs control media was assessed. The alveolar A549 epithelial cells grew and proliferated in the microfluidic system. In the wound closure assay, the degree of wound closure after 5 hours was (53.3±1.3%) with HGF compared to (9.8±2.4%) without HGF (P <0.001). We present a novel microfluidic model that allows culture, injury and wounding of A549 epithelial cells and represents the first step towards the development of an invitro reconstitution of the alveolar-capillary interface. We were also able to confirm that HGF increased alveolar epithelial repair in this system.

In vitro hemodynamic evaluation of ventricular suction conditions of the EVAHEART ventricular assist pump

Purpose: Mismatches between pump output and venous return in a continuous-flow ventricular assist device may elicit episodes of ventricular suction. This research describes a series of in vitro experiments to characterize the operating conditions under which the EVAHEART centrifugal blood pump (Sun Medical Technology Research Corp., Nagano, Japan) can be operated with minimal concern regarding left ventricular (LV) suction. Methods: The pump was interposed into a pneumatically driven pulsatile mock circulatory system (MCS) in the ventricular apex to aorta configuration. Under varying conditions of preload, afterload, and systolic pressure, the speed of the pump was increased step-wise until suction was observed. Identification of suction was based on pump inlet pressure. Results: In the case of reduced LV systolic pressure, reduced preload (=10 mmHg), and afterload (=60 mmHg), suction was observed for speeds =2,200 rpm. However, suction did not occur at any speed (up to a maximum speed of 2,400 rpm) when preload was kept within 10-14 mmHg and afterload =80 mmHg. Although in vitro experiments cannot replace in vivo models, the results indicated that ventricular suction can be avoided if sufficient preload and afterload are maintained. Conclusion: Conditions of hypovolemia and/or hypotension may increase the risk of suction at the highest speeds, irrespective of the native ventricular systolic pressure. However, in vitro guidelines are not directly transferrable to the clinical situation; therefore, patient-specific evaluation is recommended, which can be aided by ultrasonography at various points in the course of support.

Improved serodiagnosis of alveolar echinococcosis of humans using an in vitro-produced Echinococcus multilocularis antigen

Serology is an important tool for the diagnosis of alveolar echinococcosis (AE) in humans. In order to improve serodiagnostic performance, we have developed an in vitro-produced Echinococcus mulilocularis metacestode vesicle fluid (EmVF) antigen for application in an immunoblot assay. Immunoblot analysis of EmVF revealed an abundant immunoreactive band triplet of 20-22 kDa, achieving a sensitivity of 100% based on the testing of sera from 62 pre-operative and pre-treatment cases of active and inactive AE. Thus, the EmVF-immunoblotting allowed the specific detection of cases seronegative by the Em2- and/or EmII/3-10-ELISA, usually attributable to abortive, inactive cases of AE. The specificity of the EmVF-immunoblotting did not allow discrimination between AE and cystic echinococcosis (CE) but was 100% with respect to non-Echinococcus parasitic infections or cancer malignancies. Based on the findings of this study, it is recommended that the current ELISA test combination (Em2- and II/3-10-ELISA) be complemented with EmVF-immunoblotting, allowing an improved diagnosis of both clinical and subclinical forms of AE, including those associated with E. multilocularis-specific antibody reactivities not detectable by ELISA.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

In vitro resistance of articular chondrocytes to 5-Aminolevulinic acid based photodynamic therapy

OBJECTIVE: 5-Aminolevulinic acid based photodynamic therapy (5-ALA-PDT) has revealed promising results in the treatment of inflammatory joint diseases due to the sensitivity of inflamed synovial tissue. For 5-ALA-PDT to be safe and beneficial for intra-articular applications, resistance of chondrocytes is essential to prevent cartilage damage. As no data yet exist, the aim of the present study was to assess in vitro the response of the chondrocytes to 5-ALA-PDT and to compare with osteoblasts and synovial tissue derived cells. METHODS: Bovine articular chondrocytes, osteoblasts, and synovial cells were subjected to 5-ALA-PDT in cell culture. The PpIX accumulation and the function of the cells were assessed for up to 12 days. RESULTS: Bovine chondrocytes showed lower PpIX fluorescence upon incubation with 5-ALA (0.0-2.0 mM) for 4 hours as compared to osteoblasts and synovial cells suggesting a low PpIX accumulation. After incubation with 0.5 mM 5-ALA and application of light at a dose of 20 J/cm2, chondrocytes were functionally not affected (collagen type II and aggrecan mRNA, glycosaminoglycan synthesis) whereas a decrease in the proportion of viable cells was observed in osteoblasts and synovial cells (2+/-2% and 14+/-8%, respectively; chondrocytes 91+/-13%). Chondrocytes showed a 58% reduction of 5-ALA uptake using [3H]5-ALA as compared to osteoblasts and a lower mitochondrial content as assessed by the activity of the mitochondrial marker enzyme citrate synthase (9.2+/- 3.6 mU/mg protein) than osteoblasts (32.6+/-10.5 mU/mg) and synovial cells (60.0+/-10.8 mU/mg). The reduced uptake of 5-ALA and/or the low mitochondrial content, an adaptation to their in vivo environment and the site of PpIX synthesis, presumably explains the lower PpIX content in chondrocytes and their resistance against 5-ALA-PDT. CONCLUSION: 5-ALA-PDT might represent a treatment strategy in inflammatory joint diseases without endangering the cartilage function. However, further in vitro and in vivo experiments are required to confirm this data in the authentic environment of chondrocytes, the articular cartilage.

Design and in vitro characterization of highly sst2-selective somatostatin antagonists suitable for radiotargeting

Radiolabeled sst 2 and sst 3 antagonists are better candidates for tumor targeting than agonists with comparable binding characteristics (Ginj, M.; Zhang, H.; Waser, B.; Cescato, R.; Wild, D.; Erchegyi, J.; Rivier, J.; Mäcke, H. R.; Reubi, J. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16436-16441.). Because most of the neuroendocrine tumors express sst 2, we used the known antagonists acetyl- pNO 2Phe (2)- c[ dCys (3)-Tyr (7)- dTrp (8)-Lys (9)-Thr (10)-Cys (14)]- dTyr (15)-NH 2 ( 1) (Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharmacol. 1996, 50, 709-715. Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharmacol. 1997, 51, 170; Erratum.) and H-Cpa (2)- c[ dCys (3)-Tyr (7)- dTrp (8)-Lys (9)-Thr (10)-Cys (14)]-2Nal (15)-NH 2 ( 7) (Hocart, S. J.; Jain, R.; Murphy, W. A.; Taylor, J. E.; Coy, D. H. J. Med. Chem. 1999, 42, 1863-1871.) as leads for analogues with increased sst 2 binding affinity and selectivity. Among the 32 analogues reported here, DOTA- pNO 2Phe (2)- c[ dCys (3)-Tyr (7)- dAph (8)(Cbm)-Lys (9)-Thr (10)-Cys (14)- dTyr (15)-NH 2 ( 3) and DOTA-Cpa (2)- c[ dCys (3)-Aph (7)(Hor)- dAph (8)(Cbm)-Lys (9)-Thr (10)-Cys (14)]- dTyr (15)-NH 2 ( 31) had the highest sst 2 binding affinity and selectivity. All of the analogues tested kept their sst 2 antagonistic properties (i.e., did not affect calcium release in vitro and competitively antagonized the agonistic effect of [Tyr (3)]octreotide). Moreover, in an immunofluorescence-based internalization assay, the new analogues prevented sst 2 internalization induced by the sst 2 agonist [Tyr (3)]octreotide without being active by themselves. In conclusion, several analogues (in particular 3, 31, and 32) have outstanding sst 2 binding and functional antagonistic properties and, because of their DOTA moiety, are excellent candidates for in vivo targeting of sst 2-expressing cancers.

CD69 upregulation on T cells as an in vitro marker for delayed-type drug hypersensitivity

BACKGROUND: T cells play a key role in delayed-type drug hypersensitivity reactions. Their reactivity can be assessed by their proliferation in response to the drug in the lymphocyte transformation test (LTT). However, the LTT imposes limitations in terms of practicability, and an alternative method that is easier to implement than the LTT would be desirable. METHODS: Four months to 12 years after acute drug hypersensitivity reactions, CD69 upregulation on T cells of 15 patients and five healthy controls was analyzed by flow cytometry. RESULTS: All 15 LTT-positive patients showed a significant increase of CD69 expression on T cells after 48 h of drug-stimulation exclusively with the drugs incriminated in drug-hypersensitivities. A stimulation index of 2 as cut-off value allowed discrimination between nonreactive and reactive T cells in LTT and CD69 upregulation. T cells (0.5-3%) showed CD69 up-regulation. The reactive cell population consisted of a minority of truly drug reactive T cells secreting cytokines and a higher number of bystander T cells activated by IL-2 and possibly other cytokines. CONCLUSIONS: CD69 upregulation was observed after 2 days in all patients with a positive LTT after 6 days, thus appearing to be a promising tool to identify drug-reactive T cells in the peripheral blood of patients with drug-hypersensitivity reactions.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.