Activation of methotrexate-phenylalanine by monoclonal antibody--carboxypeptidase A conjugate for the specific treatment of ovarian cancer in vitro.

Monoclonal antibody 4E3 directed against a glycosylated surface protein on human ovarian teratocarcinoma cells (CRL-1572 cell line) was conjugated to bovine carboxypeptidase A (CPA) using a 3400 Da polyethylene glycol chain bearing an N-hydroxysuccinimide group at both ends. The conjugate preparation was purified by fast protein liquid chromatography on a Superose 12/30 HR column. The 4E3-CPA conjugate was recovered in the third fraction by SDS-PAGE analysis. The specific binding of the 4E3-CPA conjugate to CRL-1572 cells was confirmed by a FACS analysis and the enzymatic activity of the conjugate remained while tested with hippuryl-L-phenylalanine. In vitro cytotoxic assays on CRL-1572 cells showed that the prodrug methotrexate-phenylalanine (MTX-Phe) alone was non-toxic (ID50 > 1000 ng ml-1) but was selectively converted to MTX when the cells were pretreated with 50 micrograms ml-1 4E3-CPA conjugate, which enhanced considerably the pharmacological activity of the prodrug with an ID50 of 70 ng ml-1. The co-culture assays with CRL-1572 and MRC-5 cells (human normal lung diploid fibroblast cell lines) demonstrated the specificity of the 4E3-CPA conjugate for the CRL-1572 cells since no cytotoxicity was observed on the MRC-5 cells. When both cell lines were mixed in ratios ranging from 1:10,000 to 1:5 (CRL-1572:MRC-5), the significant increase in the ID25 was correlated with the proportion of tumoral cells present in the cell inoculum. These results suggest that MTX-Phe combined with 4E3-CPA conjugate is a promising model for a more selective and localised anti-cancer chemotherapy based on the ADEPT concept.

Effects of articaine on intrapulpal, mandibular, and femoral pressures in dogs.

Articaine, a new local anesthetic and the first substance of the amide type with a thiophene ring, has been studied to evaluate its effects on intrapulpal blood pressure (IPP) and mandibular and femoral pressures (MAP, FAP) after injections in the posterior mental foramen (PMF). Eight mongrel dogs of either sex, 9-12 months of age weighing from 15-25 kg were anesthetized. The PMF and the middle foramen were uncovered to expose the vascular-nerve bundle. The mandibular artery was dissected, cannulated, and filled with a heparinized normal saline solution. A 27-gauge needle was placed into the PMF for the injections of the local anesthetic. Into the ipsilateral canine, a cannula hermetically sealed and filled with heparinized saline solution was inserted. All hemodynamic measurements (IPP, MAP, FAP) were recorded with a precalibrated polygraph. The results obtained allow us to conclude that articaine 4% with epinephrine 1:100,000 injected in the PMF (0.3 ml), produces a drop of the intrapulpal blood pressure due to a strong vasoconstriction, whereas this effect is less pronounced at the MAP level and almost inexistent in the FAP.