Presence Of Mouse Plasma Research Articles

Impact of plasma protein binding on cargo release by thermosensitive liposomes probed by fluorescence correlation spectroscopy.

Thermosensitive liposomes (TSLs) whose phase-transition temperature (Tm) lies slightly above body temperature are ideal candidates for controlled drug release via local hyperthermia. Recent studies, however, have revealed disruptive shifts in the release temperature Tr in mouse plasma, which are attributed to undefined interactions with blood proteins. Here, we study the effects of four major plasma proteins - serum albumin (SA), transferrin (Tf), apolipoprotein A1 (ApoA1) and fibrinogen (Fib) - on the temperature-dependent release of fluorescein di-β-D-galactopyranoside (FDG) from TSLs. The amount of fluorescein released was quantified by fluorescence correlation spectroscopy (FCS) after hydrolysis of FDG with β-galactosidase (β-Gal). This approach is more sensitive and thus superior to previous release assays, as it is impervious to the confounding effects of Triton on conventional fluorescence measurements. The assay determines the molar release ratio, i.e. the number of molecules released per liposome. We show that shifts in the Tr of release do not reflect protein affinities for the liposomes derived from adsorption isotherms. We confirm a remarkable shift in induced release towards lower temperatures in the presence of mouse plasma. In contrast, exposure to rat or human plasma, or fetal bovine serum (FBS), has no effect on the release profile.

Abstract A239: Preclinical characterization of ABT-348, a kinase inhibitor targeting the Aurora, VEGFR/PDGFR, and SRC kinase families.

Abstract ABT-348 is a novel ATP-competitive inhibitor of Aurora kinases (Aurora B, C and A, IC50 values of 7, 1 and 120 nM, respectively). The activity against Aurora B is reflected in cellular assays of inhibition of histone H3 phosphorylation (IC50 value of 21 nM), induction of polyploidy (IC15 value of 12 nM) and inhibition of colony formation of human pancreatic carcinoma cells (MiaPaCa, IC50 value of 4 nM). Consistent with enzyme inhibition, ABT-348 inhibited the autophosphorylation of the respective enzyme in nocodazole-arrested cells (IC50 values of 13, 13 and 189 nM for Aurora B, C and A). Potency (IC50 values) in the cellular assays also correlated with inhibition of proliferation of cell lines derived from leukemia (0.3 nM, MV-4–11), lymphoma (4 nM, DOHH2), and solid tumors (MiaPaCa, 4 nM; SW620, 6 nM; OVCAR5, 7 nM; and HCT-15, 6 nM). Inhibition of Aurora B activity in vivo by ABT-348 was confirmed by measuring phosphorylation of histone H3 in circulating tumor cells obtained from an engrafted leukemia model (RS4;11). Inhibition of histone H3 phosphorylation was observed 4 hours after dosing that persisted in a dose-dependent manner for at least 8 hours. The extent of inhibition at 4 hours was related to the plasma concentration of ABT-348 (IC50: 3.6 μM), which was in close agreement with the value determined for inhibition of histone H3 phosphorylation in cells in the presence of mouse plasma (3.3 μM). In addition to its Aurora enzyme activity, evaluation of ABT-348 for inhibitory activity across 128 kinases revealed a unique kinome profile “signature” by virtue of its potent binding activity (Ki values <30 nM) against the VEGFR/PDGFR families and the SRC family of cytoplasmic tyrosine kinases (LYN, BLK, LCK, ABL, FYN, and SRC). The potent VEGFR/PDGFR binding activity was reflected in enzyme assays (IC50 values <10 nM) performed at 1 mM ATP and also correlated with inhibition of RTK auto-phosphorylation in cells for KDR, FLT3, CSF-1R, KIT, PDGFR and. These activities translated into potent inhibition of VEGF-stimulated endothelial cell proliferation (IC50 value <0.3 nM). Evidence that ABT-348 blocked VEGF-mediated responses in vivo was provided by the molecule's potent activity (ED50 value of 0.2 mg/kg, IV) in blocking VEGF-mediated vascular permeability in the uterus and its ability to induce dose-dependent increases in plasma PIGF in mice. Activity against the Src kinase family was evident in the anti-proliferative activity of ABT-348 against BCR ABL expressing tumor cells and cells expressing the gleevec-resistant BCR-ABL T315I mutation (IC50 values of 47 and 260 nM). Based upon these favorable in vitro and in vivo activities, ABT-348 was evaluated and found to be effective in leukemia (RS4;11, regression) xenografts. These results provide the rationale for clinical assessment of ABT-348 as a therapeutic agent in the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A239.

Isosorbide-based cholinesterase inhibitors; replacement of 5-ester groups leading to increased stability

Isosorbide-2-carbamate-5-esters are highly potent and selective butyrylcholinesterase inhibitors with potential utility in the treatment of Alzheimer’s Disease (AD). They are stable in human plasma but in mouse plasma they undergo hydrolysis at the 5-ester group potentially attenuating in vivo potency. In this paper we explore the role of the 5-position in modulating potency. The focus of the study was to increase metabolic stability while preserving potency and selectivity. Dicarbamates and 5-keto derivatives were markedly less potent than the ester class. The 2-benzylcarbamate-5-benzyl ether was found to be potent (IC 50 52 nM) and stable in the presence of mouse plasma and liver homogenate. The compound produces sustained moderate inhibition of mouse butyrylcholinesterase at 1 mg/kg, IP.

Inhibition of fibrinolysis by a synthetic urokinase inhibitor enhances lung colonization of metastatic murine mammary tumor cells

We have investigated the role of coagulation and fibrinolysis during the metastatic lung colonization of F3II mouse mammary carcinoma cells. The selective synthetic urokinase inhibitor B623 significantly enhanced lung colonization and blocked the antimetastatic effect of heparin when administered i.p. during the first stages of metastasis formation. In B623-treated mice the overall activity of the fibrinolytic system was reduced and circulating urokinase was specifically inhibited by this agent. In vitro studies demonstrated that B623 induces the aggregation of F3II cells in the presence of mouse plasma and facilitates the entrapment of tumor cells in a fibrin gel matrix. Our data suggest that imbalances of fibrin deposition and removal may dramatically influence metastatic lung colonization.

Liposomes as vaccine carriers: Incorporation of soluble and particulate antigens in giant vesicles

Giant liposomes (mean diameter 5.5 μm) composed of egg phosphatidylcholine or distearoyl phosphatidylcholine, phosphatidyl glycerol, cholesterol and triolein were prepared by a double emulsion technique. They were then mixed with model particulate (killed Bacillus subtilis, and killed Bacille Calmette-Guérin) and soluble (tetanus toxoid) vaccines and freeze-dried. Rehydration of the powder resulted in the generation of vesicles of similar diameter and diameter range, containing up to 27% (mean value) of the materials used for entrapment. Separation of entrapped from non-entrapped materials was carried out by sucrose gradient centrifugation ( B. subtilis and BCG) or centrifugation at 600 × g (toxoid). Light microscopy of liposomes containing B. subtilis labelled with fluorescein isothiocyanate revealed the presence of bacteria in individual vesicles which, in separate studies, were also found to entrap latex particles (0.5 and 1.0 μm diameter). Bacteria-containing liposomes could be freeze-dried in the presence of trehalose with most (83–87%) of the entrapped material recovered within the vesicles on reconstitution with saline. Liposomes were also shown to retain quantitatively their content of B. subtilis and, to a lesser extent, toxoid in the presence of mouse plasma at 37°C and in situ after intramuscular injection into mice, for up to 24 h. Since liposomes are known (Gregoriadis, G. (1990) Immunol. Today 11, 89) to act as immunological adjuvants and vaccine carriers, giant vesicles containing microbes (live or attenuated if needed since the conditions of entrapment are mild) and, when appropriate, soluble antigens, could be used as multiple vaccines to ensure simultaneous presentation of antigens to immunocompetent cells.

Interaction of Staphylococcus aureus cells and silk threads in vitro and in mouse skin

Staphylococcus aureus cell suspension was epicutaneously inoculated on the back skin of cyclophosphamide-treated mice with silk stitches and these sites were occluded. Biopsy specimens were taken from three mice at 1, 3, 6, 12, 24, 48, and 72 h after inoculation and were examined by electron microscopy. Fibril-like structures (glycocalyx) were seen around the S. aureus cells at 1 h. At 3 h, they had extended towards the silk threads. There were microcolonies on the surfaces of the silk threads and at 12 h the S. aureus cells were enclosed in membrane-like structures. The electron density of the membrane-like structures increased over time. After ruthenium red staining, the membrane-like structures and the fibril-like structures were stained positive, suggesting that these structures contain polysaccharide components. With a combination chemotherapy using clarithromycin and ofloxacin, S. aureus cells in the membrane-like structures were degenerated, whereas the use of clarithromycin or ofloxacin alone had little effect. Chlorhexidin gluconate and povidone iodine were effective if they were able to reach the biofilm. The fibril-like structures appeared in vitro only in the presence of silk threads, and were enhanced by the presence of mouse plasma. These structures did not form with formaldehyde-killed S. aureus cells. Thus, S. aureus cells may interact with foreign bodies to form biofilms, thereby evading the effect of antibacterial agents.

Emergence of hamster fibroblast tumors in nude mice--evidence for in vivo selection leading to loss of growth factor requirement.

The Chinese hamster lung fibroblast cell line (CC139) has high anchorage dependence for growth and has retained the high serum dependence of secondary cultures of adult fibroblasts. This cell line is tumorigenic in nude mice; however, the resulting tumor cells have different properties than those of the cell line injected. The tumor-derived cells had strongly reduced or even lost both the high anchorage and the high serum dependence of CC139 cells. This finding suggests that an in vivo selection is necessary for CC139 cells to acquire the malignant phenotype. After mutagenesis, which increases the frequency of CC139 colony formation in agarose up to 8-fold, we selected and analyzed 15 anchorage-independent colonies. No correlation between the colony-forming ability in agarose and serum-growth factor requirement for DNA synthesis was observed. Each of these clones were injected into nude mice and the growth factor dependence of the ensuing tumor cells was compared to that of corresponding injected cells. All of the anchorage-independent colonies with the exception of one (A71), had acquired in vivo a stable phenotype allowing for partial or total escape of growth factor requirement. A71, the only clone which maintained the same growth factor requirement after two passages in vivo (A71 T1 and A71 T2) had already gained, in vitro, the minimal growth factor "relaxation" compatible with in vivo growth. A71 and A71 T1 tumor cells arrested in G0/G1 can reinitiate DNA synthesis in the presence of mouse plasma, low concentrations of serum, or thrombin. The fact that none of the tumors analyzed (more than 20) were found to have retained the high serum dependence of CC139 cells strongly suggests that the partial loss of serum growth factor requirement acquired in vivo is an essential malignant character for bypassing the hormonal growth restraints imposed by the host upon CC139 cells.

Lysophosphatidylserine-induced release of intra-cellular amines in mice.

In the presence of mouse plasma, lysophosphatidylserine stimulates histamine secretion from isolated mast cells. The extensive modification of carbohydrate metabolism produced by lysophosphatidylserine in mice was largely prevented by the antihistaminic drug, pyrilamine. However, to prevent completely the change in carbohydrate metabolism induced by lysophosphatidylserine the administration of an antihistamine and an adrenoceptor antagonist was required. It is concluded that the effect of lysophosphatidylserine in mice is due to release of intracellular amines. Histamine and catecholamines are involved.

An explanation of the biological action of toxotoxin based on some in vitro experiments.

The present investigation has failed to show any correlation between the lethal activity of Toxotoxin and the viscosity of its solutions. In vitro experiments in the presence of mouse plasma revealed a dual character of Toxotoxin, this being partly thromboplastin and partly coprecipitant to fibrin. In vivo, the thromboplastic activity, though in itself checked by fibrinolysis, was thought to initiate the coprecipitation of Toxotoxin, thus causing the formation of a massive clot which fibrinolysis would be unable to resolve.