In vitro biocompatibility studies with the experimental anticancer agent BIBX1382BS.

The novel anticancer agent BIBX1382BS is a representative of the human epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. BIBX1382BS, for parenteral use, is formulated pharmaceutically as a lyophilized product containing 100 mg BIBX1382BS per dosage unit. This in vitro study was performed to establish the optimal intravenous administration conditions (infusion concentration and infusion rate) for the forthcoming clinical absolute oral bioavailability study of BIBX1382BS. BIBX1382BS infusion solutions have a low pH in order to keep the substance in solution. We therefore decided to investigate the hemolytic and precipitation potential of the drug in vitro. Also, the ratio of formulation (F) solution volume and a blood simulans (B) volume necessary to reach the physiological pH, expressed as the FB-ratio, was determined in vitro. On the basis of the results obtained, it is advised to administer BIBX1382BS infusion at a concentration of 1 mg/ml and a maximum infusion rate of 10 ml/min. This article describes the in vitro biocompatibility screening program.

Immortalization of rat hepatocytes by fusion with hepatoma cells. I. Cloning of a hepatocytoma cell line with bile canaliculi.

Hepatocytoma (HPCT) hybrid cells were obtained by fusion of cultured rat hepatocytes with Fao Reuber hepatoma cells H35 by polyethylene glycol treatment. Surviving cells were cloned in HAT (hypoxanthine-aminopterine-thymidine)/ouabain medium and propagated in cell lines over 80 passages. Morphological criteria were chosen to allow differentiation of the clones into two types of cells: 1) Type I cells which formed irregular cell layers, lacked contact inhibition and resembled the parental Fao hepatoma cells and 2) type II cells, which proliferated in monolayer cultures, exhibited contact inhibition during growth in culture plates and formed bile canaliculi thereby resembling cultured hepatocytes by phenotype. Bile canaliculi were absent in type I clones and Fao cells. One particular type II clone 1E3 was studied in detail. These cells formed bile canaliculi sealed by tight junctions and were comparably polarized as cultured hepatocytes. They expressed canalicular antigen B10, canalicular aminopeptidase N, and even secreted the fluorescent bile acid derivative NBD-cholate into the canalicular lumen. This type of HPCT cells lacked malignancy by tests in vivo and in vitro, and contained 110 +/- 5 chromosomes. The cells were considered to represent an immortalized hepatocyte-like cell line.

A comparative biochemical, pharmacological and immunological study of Clostridium novyi alpha-toxin, C. difficile toxin B and C. sordellii lethal toxin.

The three clostridial cytotoxins, i.e. alpha-toxin of C. novyi (Tox alpha-nov), toxin B of C. difficile (ToxB-dif) and lethal toxin of C. sordellii (LT-sor) consist of single peptide chains of about 200,000 (Tox alpha-nov), 250,000 (LT-sor) and 275,000 (ToxB-dif) mol. wts. ToxB-dif and LT-sor but not Tox alpha-nov cross-reacted with rabbit polyclonal antibodies. Toxicity upon i.v. injection in mice was similar (LD50, 100 hr, 50-200 ng/kg) and was characterized by a slowly developing fluid loss into the interstitial space. When injected into the rat paw the toxins caused a delayed local edema lasting for days. In vitro the three toxins provoked a persistent retraction of endothelial cells cultured from pig pulmonary artery. ToxB-dif and Tox alpha-nov triggered the accumulation of F-actin in the perinuclear region at the expense of the tight peripheral bands whereas LT-sor led to a random loss of microfilament structure. The toxins inhibited uridine incorporation into endothelial or chicken embryonic cells whereas T 84 cells responded by an about 10-fold increase of uridine incorporation. Neither toxin ADP-ribosylated actin. The similarities between the three cytotoxins warrant their arrangement into a common group which perturbs the microfilament system.

Microinjection of alpha-toxin from Clostridium novyi type A promotes meiotic maturation in Xenopus laevis oocytes.

Microinjection of purified alpha-toxin into Xenopus laevis oocytes induced meiotic maturation provided insulin was present in the medium. Induction of maturation as indicated by breakdown of the germinal vesicle depended on the amount of intracellular alpha-toxin (with a detection limit of 2 ng/oocyte) and on the concentration of insulin. The hormone concentrations used were inactive when given alone and so was alpha-toxin (1 micrograms/ml) applied from the outside. The results demonstrate an intracellular target for alpha-toxin whereas an extracellular target is apparently lacking in oocytes.

Pharmacological and biochemical studies of cytotoxicity of Clostridium novyi type A alpha-toxin.

The actions of apparently homogeneous alpha-toxin from Clostridium novyi type A were studied in order to develop an in vitro system which closely mimics its in vivo effects and to search for the mode of poisoning. Time to death (by intravenous injection of mice) was inversely related to dose, with a detection limit of about 200 ng/kg of body weight at 100 h. Injections of 2.5 ng or more into the rat paw led to a slowly (maximum after about 30 h) developing, dose-dependent edema which was useful as a quantitative in vivo assay based on volumetry. Vascular leakage was due to gap formation between endothelial cells. Similarly, endothelial cells cultured from pig pulmonary artery lost their "cobblestone" arrangement after a dose-dependent lag period of some hours after poisoning. The morphological changes were accompanied by depression of uptake or incorporation of [3H]uridine. A quantitative in vitro assay was established on the inhibition of [3H]uridine incorporation. As in animals, the action of alpha-toxin started with a few nanograms per milliliter and proceeded slowly for at least 1 day but became resistant to antitoxin within 2 h of exposure. The toxin action is not limited to endothelial cells, since chicken embryonic cells, a mouse fibroblast line (L-929), and a rat phaeochromocytoma line (PC-12) behaved similarly. Alpha-toxin was found to differ from other bacterial toxins investigated whose modes of action are already known.

High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets.

A protein-tyrosine kinase (PTK, EC 2.7.1.112) from human platelets was purified with high yield. Purification of the enzyme involved sequential chromatography on casein-agarose, tyrosine-agarose, heparin-Sepharose and hydroxylapatite. The procedure resulted in substantially enriched 54/52 kDa polypeptides on SDS-polyacrylamide gel electrophoresis and a yield of about 25% in PTK activity. About 250 micrograms of purified protein could be obtained from 1 g of cell protein. The purification factor varied between 1000 and 1500. Determination of the molecular mass of the purified PTK under nondenaturating conditions by molecular sieve chromatography revealed that the enzyme is a monomer of about 50 kDa. Among various protein substrates tested, casein was most prominently phosphorylated. All substrates were exclusively phosphorylated at tyrosine residues. Autophosphorylation at tyrosine residues of the 54/52 kDa proteins was observed in the presence of Mg2+ or Mn2+. At each purification step, the 54/52 kDa proteins were precipitated by sera from tumor-bearing rabbits immunoprecipitating pp60src, but not by control sera. The amount of the immunoprecipitated purified 54/52 kDa phosphoproteins was directly proportional to the amount of antiserum used. Partial peptide mapping by V8 proteinase showed a 26 kDa tyrosine-phosphorylated fragment for the 54 and the 52 kDa proteins as well as for the pp60c-src molecules of intact platelets. All these data indicated that purified PTK is closely related to pp60c-src of human platelets. Using casein as a substrate for the purified enzyme, the Km for ATP was 4 microM and the Vmax for the reaction was 2.0 nmol/min per mg.