The antimicrobial activity of M₁ and M₂, two metabolites of SF-837, are studied in vitro and in vivo. The results obtained are as follows :
1) M₁ and M₂ showed antimicrobial activity against Gram-positive bacteria, but it was somewhat inferior to that of SF-837.
2) M₁ was active against clinical isolates of Staphylococcus aureus, but it was less active as compared with those of SF-837, kitasamycin and josamycin. M₁ and SF-837 were inactive against clinical isolates of Escherichia coli.
3) Rate of binding to serum protein of M₁ and SF-837, at the concentrations of 100-1, 000 mcg potency per 1 ml of calf serum was 14.1-7.5% and 28.4-13.6% respectively, apparently smaller in M₁.
4) In experimental mice infections with Staphylococcus aureus and Streptococcus pyogenes no significant differences were noted in ED₅₀ as compared with the differences in in vitro activities. Against infections with Salmonella enteritidis both M₁ and SF-837 were confirmed to be inactive.

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The influence of inoculum size on the determination of MIC of sulfonamides has been investigated. by agar or broth dilution method using MUELLER-HINTON medium mainly as an assay medium.
It was found that, when the overnight broth cultures were used as inoculum cell suspension, the majority of the test strains used was found highly resistant, while if the inocula sizes were decreased to less than one-hundredth of the original overnight broth cultures, many of them showed sensitive, although some strains remained resistant, and in this condition, relatively constant and reproducible. values of MIC were obtained. Thus, if such small inoculum size was used, sensitive and resistant cultures were distinguished clearly with each other. The inoculum size, at which the fluctuation. of MIC takes place, ranged from 10⁶ to 10⁵ cells per ml according to the kind of test medium and test. microbe.
Through the analysis of bacterial growth curve in the liquid medium containing sulfonamide, it was. proved that the sensitive strains were affected at same degree by the drug regardless to whether the inoculum size was large or small.
According to the results described above, in the determination of MIC of sulfonamide it should be recommended that one loop or drop of more than 100 times dilution of overnight broth culture is used. as inoculum size.

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Several strains of S. pyogenes, E. coli K. pneumoniae, Shigella and Proteus which were determined. for both the degree of virulence to the mouse and sensitivity in vitro to the sulfonamides were used for the present studies. Using these pathogens, therapeutic activities of the sulfonamides were investigated in mice infected by the intraperitoneal route with a variety of challenge doses.
In sensitive strains, it was found that levels of ED₅₀ had changed remarkably by challenge dose of the infected strains and a therapeutic effect of the drug was obtained clearly when below 10⁶ viable cells per mouse had been challenged.
Such results as above mentioned were also observed, to a certain degree, on strains sensitive to the drug in the level of 100-800 mcg/ml, but for high resistant strains, scarcely any therapeutic effects were observed, even when the challenge doses were very small.
When a certain level of challenge dose less than 10⁶ viable cells per mouse was used in all test cultures and the therapeutic effects were compared with each other definite correlation was observed between MIC and ED₅₀ with only a few exceptions. That is to say, degree of the therapeutic effect was dependent mainly on levels of the MIC and challenge dose of the test strain, and then it would seem that there is no remarkable influence of virence of the strain to the effect of the drugs.

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2-Methoxy-4-sulfanilamidopyrimidine (OS-3376), a new sulfanilamide synthesized in our laboratory, has an in vitro antibacterial spectrum similar to sulfadimethoxine, sulfisomezole and sulfamethizole which were used as control drugs, and little difference was observed among them in the antibacterial potency.
The concentrations of OS-3376 in blood and tissues or urine after the oral administration in mouse and rabbit were compared with the concentrations of sulfamonomethoxine and sulfamethizole in the experiments under the same conditions. The blood peak level of OS-3376 obtained at 30 minutes (mouse) or 1 hour (rabbit) after administration and its disappearance was later than sulfamethizole but sooner than sulfamonomethoxine in mouse, while in rabbit, it was rather later and comparatively high concentrations were observed. Mouse tissue assays showed kidney had the highest concentration of OS-3376 and other organs exhibited the concentrations as follows in order of blood, lung, liver and spleen. The drug was excreted in urine with high concentration and the urinary recovery rate was higher than sulfamonomethoxine.
The therapeutic effect of OS-3376 has been shown distinctly in mice infected with Staphylococcus aureus and Streptococcus pyogenes, though this effect was rather slight as comparing with sulfamonomethoxine and sulfadimethoxine.

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Distribution of penicillin G and other 7 antibiotics to the erythrocytes of male mice was investigated 1 and 4 hours after subcutaneous injections.
Their transfer into the erythrocytes was almost proportional to their concentrations and the drugs used were arranged in order of decreasing transfer : chlorabulin, chloramphenicol, tetracycline, streptomycin, penicillin G, erythromycin and aminobenzylpenicillin.
Concentrations of penicillin G and aminobenzylpenicillin which are water-soluble and weak acids in plasma, red cell membranes and intracellular materials of red cells were decreased in order of that in plasma, membranes and intracellular materials.
Concentrations of streptomycin and kanamycin which are water-soluble and weakly alkaline were decreased in order of that in plasma, membranes and intracellular materials. However, concentration in the membranes was almost equal to that in intracellular materials in the case of application of a small dose.Concentrations of tetracycline which is fat-soluble and a weakly acidic were decreased in order of that in plasma, intracellular materials and membranes.
Concentrations of erythromycin which is fat-soluble and a weakly alkaline were almost equal in plasma, membranes and intracellular materials.
Concentratoins of chloramphenicol and chlorabulin which are fat-soluble and neutral were decreased. in order of that in intracellular materials, plasma and membranes or in order of that in plasma, intracellular materials and membranes.
Although blood levels after 4 hours were lower than those after 1 hour, concentrations in red blood cells after 4 hours fell only slightly.

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Transfer of penicillin G and other 26 antibiotics into erythrocytes was investigated.
Transfer rates of penicillin antibiotics into red blood cells were at the range 3.02 to 11.41% and were small in order of phenoxypropylpenicillin, phenoxyethylpenicillin, cephaloridine, carbenicillin, penicillin G and aminobenzylpenicillin.
Transfer rates of glycoside antibiotics were at the range 1.73 to 5.05% and were small in order of kanendomycin, streptomycin, aminosidine and gentamicin.
Transfer rates of tetracycline antibiotics were at the range 5.61 to 9.58% and were small in order of tetracycline, demethylchlortetracycline, minocycline, doxycycline, methacycline and oxytetracycline.
Transfer rates of chloramphenicol antibiotics were at the range 4. 73 to 17. 83% and were small in order of thiophenicol, thiophenicol glycinate, chlorabulin and chloramphenicol.
Transfer rates of macrolide antibiotics were at the range 4. 0 to 13. 04% and were small in order of rifampicin, josamycin, lincomycin, 7-chlorolincomycin, josamycin aspartate, acetylspiramycin and spiramycin.
Transfer rates of antibiotics into ghost cells were lower than those into red blood cells. However, their orders of antibiotics as mentioned above were almost identical.
Transfer of antibiotics into red blood cells were lowered by addition of plasma or its fraction. However, concentrations of antibiotics in intracellular materials and membranes of red blood cells were increased by addition of adenine or adenosine triphosphate.

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