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The effect of cupric ion on the stability and antibacterial activity of penicillins, benzylpenicillin and 2, 6-dimethoxyphenylpenicillin, in phosphate buffer solution and phosphate broth kept at pH 5.0 was investigated. The degradation was found to obey first-order kinetics to produce the corresponding penicillenic acids, though the formation of penicilloic acid analogs have been postulated in other studies. Catalytic and complexing effect of cupric ion on the stability of penicillins was revealed in relatively lower and higher concentrations of the metal ion respectively, where an appreciable difference in the effect was noted between buffer solution and broth. Apparent stability constants for the proposed complex between penicillins and cupric ion were estimated to be log Kapp=2.65 and 2.91 with benzylpenicillin and 2, 6-dimethoxyphenylpenicillin, respectively. The antibacterial activity against Staphylococcus aureus was not apparently affected by the addition of cupric ion, which indicates an apparent lack of correlation between the stability and the activity of the drug in broth. This is presumably because the probable enhancing effect of the metal ion on the cell wall permeability of the penicillins outweighs the other effects investigated.

Disappearance of nitrofuran derivatives in pH 7.0 culture media and in the media inoculated with Staphylococcus aureus was investigated at 37°. From the media inoculated with the bacteria, rapid disappearance which followed a biexponential profile was observed in contrast to the monoexponential one observed with the media excluding the bacteria. Apparent rate of disappearance was dependent on the bacterial contents, concentration of the drugs. and the compositions of the media. Generally, vinyl derivatives were more susceptible to the process than azomethine ones. Equilibrium dialysis behavior, extent of the binding of the drugs to the crude membrane fraction of the bacteria, and the effect of medium compositions on the disappearance as well as the binding tendency of the drugs together with the inhibitory effect of p-chloromercurybenzoate suggest the possible involvement of a specialized transport process in the disappearance of nitrofurans from the culture media inoculated with the bacteria. The implications of these results from in vitro biological activity standpoint are discussed.

The relationships between stability in culture media and in vitro anti-bacterial activity against Bacillus Calmette-Guerin (BCG) were investigated with furyl methyl ketone isonicotinoylhydrazone (FKI), glucose isonicotinoylhydrazone (G-INAH), and sodium pyruvate isonicotinoylhydrazone (P-INAH). FKI was found to be essentially equal to isoniazid in its activity under the equimolar basis. Thus its stability which was dominantly affected by several amino acids resulted to have no influence on the evaluation of the activity. On the other hand, a significant relationship was found among G-INAH and P-INAH, which can not inhibit growth of BCG until they release sufficiently large amount of isoniazid by hydrolysis. P-INAH was found to be hydrolyzed with such an extraordinary rapidity as 6 and 23 times greater than G-INAH in Kirchner and Long medium, respectively. This instability inversely related to the enhancement of its activity which consequently resulted in the same one as isoniazid. While G-INAH was observed to be hydrolyzed in Kirchner medium about 4 times as rapidly as in Long medium and it showed to have greater activity in the former than in the latter. The release rate of isoniazid might be inversely proportional to the stability and directly to the activity of these drugs. Another in vitro experiment with drug exposure times suggested that the bactericidal effect of drugs was probably displayed in the relatively early stage of test incubation period.

With furylmethylketone isonicotinoylhydrazone (FKI) used as an example, factors affecting drug stability in various culture media and its implications on the in vitro activity tests have been studied. In buffer solution (pH 2-10), FKI was stable in the neutral pH and unstable in acidic and alkaline pH regions. It became evident that in culture media the degradative reaction was greatly influenced by medium components such as serum and amino acids. The rate of degradation was four to ten times more rapid than in buffer solutions and considerable amounts of isoniazid were released long before one could expect growth of test organisms in culture. Among the medium components tested, amino acids such as asparagine and glutamic acid exhibited a dominant effect and the data seemed to indicate non-protonated amino group as catalytic species. Suggestions are made relative to the significance of these observations for the in vitro evaluation of anti-bacterial agents.