N-nitrosoephedrine and N-nitrosopseudoephedrine: their dissolution, absorption, blood clearance and elimination studies.

Dissolution and absorption rates (in vitro); clearance from blood and elimination rates of N-nitrosoephedrine (NEP) and N-nitrosopseudophedrine (NPEP) in mice were determined. The two isomers obeyed first-order kinetics and from the slope of the regression line, the rate constant for each study was obtained. These constants were 0.023, 0.038 min-1 (dissolution); 0.192, 0.225 h-1 (stomach absorption); 2.898, 1.980 h-1 (intestinal absorption); 0.33, 0.55 h-1 (blood clearance) and 0.373, 0.393 h-1 (elimination from whole animal) of NEP and NPEP, respectively, and were tested by Student's t-test. Significant differences in the dissolution, absorption and blood clearance rates of NPEP from those of NEP have been observed (P less than 0.05-0.01). These differences are expected to exert their influence on the metabolic rates and the carcinogenic and/or spectral properties of NEP and NPEP, in a related pattern.

Production of nitrosamines from ephedrine, pseudoephedrine and extracts of Ephedra foliata under physiological conditions.

N-Nitrosoephedrine (NEP) and N-nitrosopseudoephedrine (NPEP) were synthesised at 5 degrees C using different concentrations of various acids. The reaction with acetic acid gave the highest yield (85%) of N-nitrosamine. Ephedrine and pseudoephedrine were reacted with nitrite under physiological conditions (37 degrees C, pH 1-3) to form NEP and NPEP. The yield of NEP, which is a known carcinogen, and NPEP were the highest (18.5%) at pH 2. Aqueous and alcoholic extracts of Ephedra foliata (100 g dry wt), nitrosated under physiological conditions, produced 0.77 mg and 8.3 mg, respectively, as total nitrosamines. This indicated the potential of the nitrosamine formation from the plant extracts specified.

The potential of carboxypeptidase G2-antibody conjugates as anti-tumour agents. I. Preparation of antihuman chorionic gonadotrophin-carboxypeptidase G2 and cytotoxicity of the conjugate against JAR choriocarcinoma cells in vitro.

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been linked to a monoclonal antibody (W14A) raised to human chorionic gonadotrophin. The coupling efficiency and retention of antibody and enzymatic activities are compared for three separate methods of preparing 1:1 conjugates. Preliminary in vitro studies on the cytotoxicity of the free enzyme and the conjugated enzyme towards JAR choriocarcinoma cells are reported. Despite the limitations of the in vitro model, it could be demonstrated that a significant proportion of 10(6) choriocarcinoma cells lost viability when exposed to either free or conjugated enzyme for 72 hours at concentrations of carboxypeptidase G2 of 1-3 units ml-1 of medium.

Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method.

A folate-degrading enzyme, carboxypeptidase G2, has been purified on a large scale from Pseudomonas sp. strain RS-16. Homogeneous enzyme was obtained by a three-step procedure involving ion-exchange chromatography and a novel triazine dye (affinity) chromatography step which utilizes Zn2+ to promote adsorption of the enzyme. Enzyme was selectively eluted by the use of a chelating agent (EDTA) and a step change in pH. The enzyme is a dimeric protein (Mr 83000) with two identical subunits of 41800 and contains four atoms of zinc per enzyme molecule, which are required for full activity. The enzyme follows Michaelis-Menten kinetics with Km values of 4.0 microM for folate, 8.0 microM for methotrexate and 34.0 microM for 5-methyltetrahydrofolate, the predominant form of reduced folate found in plasma.