For the standardization of lepromin, it is necessary to know the available period of potency, and also to run a trial on prolonged preservation by the freeze-dry procedure. A batch of lepromin was divided in two parts; one part was lyophilized without addition of phenol and preserved in a refrigerator for 3 and 5 years, while the other part was kept, as a suspension containing phenol, in a refrigerator for the same period. After the storage, the lyophilized lepromins returned immediately to stable suspensions with the addition of 0.5% phenol solution. The bacterial counts of these lepromins, using the pinhead method described by Hanks et al., were not different from the standard value, i.e. 150-160 million per ml, while that of the lepromins kept in a refrigerator were reduced to about 74% and 70% of the original count after 3 and 5 years, respectively.
Comparative tests with these lepromins were carried out by examining the size of the Fernandez and Mitsuda reactions in leprosy patients, the results being shown in Tables 2 and 3. It was found that the Mitsuda reactions caused by lepromin preserved as a suspension for 5 years were clearly weaker than that of the lepromin lyophilized during the same period, and that the Fernandez reactions were not different for the two lepromins. In the case of the lepromins preserved for 3 years, there was no significant difference in the potency causing the Fernandez as well as the Mitsuda reactions, whether the lepromin had been lyophilized or not.
Accordingly, it is concluded that a lepromin can be preserved for 3 years in a refrigerator without appreciable loss of potency. Lyophilization of lepromin was found to be suited for prolonged preservation, at least for 5 years, without change in the bacterial count and potency of the lepromin.

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This study was carried out to examine the minimum inoculum, the differences in the strain, sex and age of mice, and the multiplication of M. lepraemurium in the hind footpads of mice and hamsters which had been treated with thymectomy, thymectomy plus the administration of immunosuppressive agents, injection of Freund's imcomplete adjuvant, and gamma-ray irradiation (approximately 1200 rad) in thymectomized mice.
The strain of mice used were ICR, SW/A, CF#I, ddY-F and dd, and the hamsters used were the golden hamster.
The results obtained are summarized as follows:
1) The minimum inoculum was approximately 5.0×10² bacilli per each hind footpad.
2) Nodular swelling in the hind footpad of the mice was occasionally greater in the male than in the female, but that of the hamsters was less in the male than in the female, as was true in thymectomized hamster. Also, the differences for the age of 4 weeks (adults) and that of 6-8 months (old) in the female mouse were less in the old than in the adult. Furthermore, no significant difference was observed between the CF#I strain of mice and the others.
3) In the thymectomized mice and hamsters, leproma-formation in the hind footpad was inhibited at an early stage after the inoculation, but accelerated at a later stage after the inoculation. This difference was apparently observed in the neonatally thymectomized mice. In addition, leproma-formation was strongly inhibited by the administration of antilymphocytic serum (ALS).
4) The effect of immunosuppressive agents on the multiplication of M. lepraemurium was apparent in the testosterone propionate or progesterone-administration, but such other agents as Cortisone, Thyroxine, Pituitary hormone, Reserpine, Hydroxyurea, Mitomycin C, 5-Fluorouracil and 6-Mercaptopurine, did not show good results in our investigation. However, in the case of the hamsters leproma-formation was inhibited by the administration of progesterone.
5) The count of M. lepraemurium and leproma-formation in the footpads was strongly inhibited by an injection of an adjuvant and by gamma-irradiation.
It may be concluded from these results that the multiplication of M. lepraemurium in the footpad of mice and hamsters was mainly influenced by the presence or absence of immunity, i.e., the depression of circulating lymphocyte levels in the granuloma formation.

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To find out a favorable method for the analysis of various sulfone drugs and their metabolites, the separation of 55 compounds relating to the present drugs was studied by thin-layer chromatography (TLC).
The separation of DDS, DDSO, 4, 4'-Diaminodiphenyl sulfide (DDSD) and their mono-N-acetylates could easily be achieved on a thin-layer plate. When benzene/ethylacetate (2/3) was used as a developing system, the order of their Rf values was S>SO₂>SO and NH₂>NHCOCH₃. Also, the order was found to be R, R': NO₂>H>OCH₂CH₂OC₂ H₅>NH2NH>OH, NH₂NHCOCH3, NHCH₂CH₂OH>COOH, NHCH₂COOH in the case of 4-R-C₆H_4/SO₂C₆H₄-R'-4'. When the same solvent system was used, no clear difference was found between the Rf value of a diphenyl sulfone compound (DS) and that of the corresponding diphenyl sulfonamide (DSA) or the diphenyl ethyl sulfonamide (DESA), though the Rf value of the former was somewhat higher than that of the latter two.
Applying this method, a sample of human urine was examined after the oral intake of DDSD. The metabolites identified were DDSD, the mono-N-acetylate (MAcDDSD) and DDS (all of them faintly detected), and MAcDDS, unchanged DDSO, and MAcDDSO (all of them markedly detected).
It was also coincident with the result of the experiment, that the MAcDDSO was more labile under auto-oxidation than DDSO when they were allowed to stand in ethylacetate. Therefore, it can be said that this experiment suggests the reason why a considerable portion of DDSO is found to be existent as MAcDDS in the urine sample, even though this oxidation is presumed to be accelerated by TPN-linked liver microsomal function. However, in the urine sample of a rabbit, DDS, MAcDDS, MAcDDSO, and unchanged DDSO could merely be detected.
Several authors had already reported the reduction of some dialkyl sulfoxides to the corresponding sulfides by an enterobacteria^{5a, b)} or their oxidation by liver microsomes^5c) However, no report hitherto mentioned the reduction of diaryl sulfoxdes in vivo.
In the second step, the separation of some water-soluble labile derivatives or conjugates of DDS was carried out to certify the main metabolites of Promin by TLC. A compound named Promin A (PrA) was prepared for this purpose, together with ¹⁴C-PrA, and intraveneously injected into several rabbits, which were synthesized by sodium glucuronate (GNa), glucuronic amide (GNH₂) or GK-6-¹⁴C respectively, instead of glucose in the case of Promin. All of them were more labile than Promin in aqueous solution.
As the result, it was found that the main metabolites in the rabbit urine were DDS and mono-N-glucosiduronate (DDSG). This result suggested that a main metabolite of Promin might be DDS mono-N-glucoside, especially if the Rf values of DDS mono-and di-N-glucoside were compared with those of the corresponding mono- and di-N-glucosidu-ronate on a TLC plate, even though DDS N-glucosides were more labile than the N-glucosiduronates, and also the mono-N-glucoside as a standard material could not be isolated yet.
In spite of the report by Dr. Sweet^6a) and Dr. Bushby^6b) who had respectively presumed and maintained the existence of DDS mono-N-glucose sulfonite (mono-Promin) as a main metabolite of Promin, this predominant desulfinition was again certified in the case of PrA.
In addition, because the measured radioactivity in the rabbit urine and the plasma was calculated to be too low to explain the total activity in ¹⁴C-PrA injected into the rabbit, the GK-6-¹⁴C freed from ¹⁴C-PrA was supposed to be separately catabolized in vivo, though the relation between the xylulose cycle could not be explained.

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The authors synthesized certain pyrazine derivatives. Each compound was designated as follows and employed in the experiments. (Fig. 1).
For an experiment on their antibacterial actions, the compounds were examined for their effects on Mycobacterium tuberculosis, H37Rv, as compared with the first. The results showed that Pycazid 200 r<, PBS-110 r<, PBS-2 5 r<, diluted soultions, PBS-1 and PBS-2 were effective in checking the growth of H37Rv but the effect of Pycazid was weak.
On acute toxicity to mice, the results of the experiment were as follows.
Pycazid 150.5mg/kg
LD 50 PBS-1 1510.0mg/kg
PBS-2 1540.0mg/kg
The effects of the three compounds on the respiration and blood pressure of rabbits did not show any action to 100ml of 0.02% solution.
No effects was found on the peripheral blood vessels of rabbits to 15ml of each solution (0.02%).
Actions on an excised toad heart; 5-10mg/20ml of PBS-1 and PBS-2 showed no action. But, 1mg/20ml of Pycazid caused an increase of amplitude lasting for minutes. The heart beat become irregular and came to a stillstand at the systolic position with a dose of 20mg/20ml.
Actions on rabbit intestines: The actions of the compounds on the motility and tone of an excised small intestine were tested by the Magnus's method. Neither PBS-1 nor PBS-2 showed any action. The intestinal tone was barely strained with 10mg/20ml of Pycazid, but a gradual decrease caused by application of Pycazid in the tone of the intestine could be antagonized by an after-treatment with BaCl₂.
The spasmogenic activity of acetylcholine was reduced remarkably with the application of Pycazid.
The pharmacological actions and toxicities of PBS-1 and PBS-2 were very weak, PBS-1 and PBS-2 were designated to be used in clinical treatment tests on leprosy.

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Reliability of the screening method employing the footpads of mice in the search for antileprous drugs had mainly been evaluated in the use of drugs which had already been established to be effective in the treatment of leprosy, tuberculosis or malarial disease, or otherwise, in the use of some masked compounds of DDS, which were expected to be longer-active and also lower-toxic than DDS.
In the present study, however, the authors employed, in addition, some compounds which had not been clinically tested on mycobacterial diseases, together with some of 60 compounds which have been newly synthesized and consisted of three series.
Each was given orally to the mice, mixed in the diet at concentrations of 0.1 mg or 0.5mg/g, or injected intramuscularly in a corresponding dosage into the dorsal muscle once a week.
The strains of leprosy bacilli employed were N Aneta P2 or B 2409 P12, and the transmission was carried out to inject one of the strain into both of the hind footpads of the experimental mice. The bacterial growth was examined by counting bacilli in each slice of the foot pad covering a period of approximately eight months. A serious deviation in the bacterial counts was observed within a group. However, in most cases, the bacterial counts in the right and the left hind foot pads of each mouse were comparable.
As exceptional cases, the results of INAH, 1314 TH, and Ethambutol were contradictory to their respective clinical findings, and s-DDS, the chemical structure of which is.similar to DDS, and especially to Promacetin, was inff ective, though the results stated in this report are merely based on the continuous method.
To elucidate the origin of such a contradication, the concentrations of these exceptional case drugs in the footpads or in the blood of mice were measured by a radio-tracer techique. When based on the results of this experiment, the relative difficulty of these drugs to arrive in the footpads of mice was supposed to not play an important partt in the effectiveness of these drugs in the footpad method. Therefore, this contradiction may be caused by a difference between the mode of action of a drug against the parasitic leprosy bacilli in the macrophages of the footpads of mice and that in the human lepra cells. The Aryl Propiolthieanilides are considered to be promising.
Subsequent to this study, various kinds of compounds untested on any infections or newly synthesized, together with numerous antibiotics, will be tested by the mouse footpad method in the next stage.

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With the progress of metabolic and especially of histopathological investigations, a different view or diduction upon the mode of action of sulfones has been reported, in which it was advocated that the inhibitory mechanism of sulfones against leprosy bacilli differed from such a simple in vitro competition between sulfonamide and the folic acid biosynthesis by usual micro-organisms. Because the studies on the sulfonamide-type inhibition were still in limited status in the field of mycobacteria, a series of studies were performed step by step to elucidate these problems.
In this report, the inhibitory effect of DDS against the intake of some radioactive nutrients by numerous strains of mycobacteria was examined in comparison with that of s-DDS (DDS 2-sulfonamide) and DDSA (4, 4'-diaminodiphenyl sulfonamide), together with that of some PAS derivatives, two of them were newly synthesized.
As the result, the intake inhibition of PABA was generally found in the phase of partialy inhibited growth of mycobacteria. Whole the drugs employed except hydrazino-PAS derivatives showed this inhibition against all the strains employed, while only DDS was effective on leprosy patients or on the growth of leprosy bacilli in the footpads of mice. Also, this inhibition was limitedly seen in the case of Promin, even after 96 hours' incubation.
Although an inhibition of DDS against the intake of glutamate was slightly detected, the intake inhibition of tested nutrients other than PABA could not be clearly detected.
As for the PAS derivatives, PAS methylester (PASMe), whose carboxylic radical was masked, showed an intake inhibition of PABA, though it was somewhat inferior to that of PAS. Therefore, the relationship between the chemical structures of the PAS derivatives and their in vitro growth-inhibitory action on five strains of mycobacteria was examined, in which three strains of human tubercle bacilli showing resistance to some antituberculous drugs were included. As the result, it was found that PAS hydrazide (PASH) had the highest action even on PAS-resistant strains and that PASMe showed an effect similar to PAS. However, the derivation of the NH₂-radical of the PAS derviative into the corresponding hydrazine compound brought about a lowering of the action. while, in the case of sulfones and sulfonamides, it was well-known that this derivation brings about a favorable result. The result of IR spectrophotometric analysis showed that only PAS formed an intramolecular hydrogen bond among these PAS derivatives. Thus, the mechanism of the in vitro action by PASH was supposed to be different from that by PAS. Both the hydrolysis of PASMe into PAS and the decarboxylation of PAS by M. phlei growing in Dubos' broth nutrient fluid could not be detected.
To find out any relation between these in vitro results and the mode of action of sulfones in vivo, at the first step, the distribution of 3H-labled Promin in leprous nodules of 17 leprosy patients was examined, and it was found that the effective concentrations of DDS in Promin were calculated to be only 3-4μg at 1 hour, 1.5-0.5μg at 6 hours, and below 0.5μg at 12 hours per 1g (wet weight) of each nodule after an injection.
Based on these results, a bacteriological re-consideration was mentioned, especially concerning the relation between the mode of action of sulfones in vitro and in vivo.

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