ANTITUBERCULOUS ACTIVITY OF ISONIAZID IN COMBINATIONWITH SULFISOXAZOLE (GANTRISIN) 1.
THE EFFECT OF ISONIAZID-SULFISOXAZOLE IN VITRO/\

Masukazu NAITO, Rinzo WATANABE, Shunji TSUKUMA
and Kazuo IBA
The Tuberculosis Research Institute, Kyoto University, JapanThe Division of Chemotherapy (Director: Prof. M. Naito)(Received for publication August 10, 1960)

INTRODUCTION

The antituberculous activity of the sulfanilamides has been investigated since
the drugs were first found. The discovery of Tibione was probably related tothose investigations.
At present, however, sulfanilamides are rarely used in the treatment oftuberculosis.
In 1952 Isoniazid (INH) became available in this laboratory.
Considering the difference in antibacterial spectrum between INH and streptomycin (SM), the present authors administered Sulfisoxazole (SI) in combination
with INH to some pulmonary tuberculosis patients who were believed to havemixed infections, and it became apparent that SI was effective not only against
the bacteria which caused the mixed infection, but also against Mycobacterium

tuberculosis when it was used with INH.
Investigations on the antituberculous activity of INH and SI were therefore
begun in this laboratory, and the first data obtained were reported at the 29th
annual meeting of the Japanese Association for Tuberculosis l ) held in 1954, andthe results of the clinical studiesZ-7) of the INH -51 regimen for pulmonary tuberculosis in this institute have been successively reported since 1955.
In 1956 Ushiba, Sunahara, Shimamura, Koyama, Takahashi, Tomita and Gomi 8 )undertook similar research and reported similar results with satisfaction.
Since then many investigations of this regimen have been carried out in thiscountry.
The present report concerns the in vitro effect of this combined use of thedrugs which was elucidated in this laboratory.

48

Masukazu NAITO, Rinzo WATANABE, Shunji TSUKUMA and Kazuo IBA

EXPERIMENTSI.

In Vitro Studies on the Tuberculostatic Effects of INH, SM or P AS in
Combination with SI and Other Sulfanilamide Derivatives
Methods:

The drugs were evaluated for in vitro activity against M. tuberculosis var.
hominis (H37Rv) in 2 ml. of Kirchner's liquid medium9 ) containing bovine serumin the concentration of 10 per cent.
The solutions of drugs used were those marketed for injection. Tubes ofeach drug-combination and appropriate controls were inoculated with approximately 0.04 mg. wet weight of M. tuberculosis in fine suspension in the firstexperiment, 0.8 mg. in the second experiment and 0.0008 to 0.8 mg. in the third
experiment.
The suspension was prepared by the following method. A portion of a surface pellicle culture which had grown on Kirchner's medium for ten days washalf dried on aseptic filter paper for 30 minutes and weighed. A definite amountof Mycobacterium was put into a flask containing glass beads. Then the flask
was shaken, while physiological saline solution was added drop by drop. Thissuspension was diluted to a definite concentration of Mycobacterium with physiological saline solution.
Incubation was carried out at 37°C in an incubator. At the end of four weeks
the tubes were examined and the bacteriostatic activity of the drug or combinationof drugs was recorded as the least amount of the drug or combination which
completely prevented the growth of the Mycobacterium.
Results:

The results are illustrated in tables 1 to 3.
In the first experiment (table 1) the combined effects of INH, SM or PAS
with various concentrations of sulfanilamide derivatives were studied. The minimum inhibitory concentration (MIC) of INH used in combination with various
concentrations of sulfanilamide derivatives was half to one-sixth of that of INH
used alone. When SM or PAS was combined with sulfanilamide derivatives noadditive effect was found. In every experiment it was shown that SI increased
the bacteriostatic effect of INH when they were used together. It is well known
that SI is one of the sulfanilamide derivatives, which rarely cause gastric trouble,
the most frequent side effect of many sulfanilamide derivatives. In the subsequent
experiments, therefore, only SI was employed as the sulfanilamide derivative to
be used in combination with INH.
In the second experiment (table 2) 2 mI. of the medium was inoculated with
0.8 mg. wet weight of Mycobacterium. The MIC of INH used in combination withten times as much SI was 0.032 r per mI., while the MIC of INH used alone was
0.125 r per mI.

49

Antituberculous activity of isoniazid in combination with sulfisoxazole 1.
Table 1.

Minimum Inhibitory Concentrations of Antituberculous Agents(INH, SM and PAS) in Combination with Various Concentrations of Sulfanilamide Derivatives (SA) (in vitro)
Ratio of antituberculous agent and sulfanilamide

Drug used in

INH:SA

SM:SA

PAS: SA

combination
1: 25

1:3

1: 10 I

1:3 1 : 1/211 : 1/3

1:5
I

-

None

0.08

0.06

0.06

0.06

0.06

0.12

1.56

1.25

3.13

1.56

Sulfisoxazole

0.03

0.02
0.02

0.03

0.03

0.03
0.03

0.02

1.0

4.0

1.56

0.04

2.0

4.0

2.5
2.5

0.08
0.16

2.0

2.5
2.5

1.56
1.56

Sulfapyridine
Homosulfamine

0.03
0.06

1.56

Sulfamerazine

0.03
0.12

0.03

0.03
0.06

0.5

4.0
2.0

Sulfathiazole

0.02

0.03

0.06

0.04

2.0

2.0

2.5

1.56

I Sulfadiazine

0.12

0.06

0.12

0.12

1.0

2.0

1.56

1.56

0.03

0.12

0.12

I SUlfapyrimi~_in_e---,_ _-,2.0

1.56 II* Figures indicate the minimum inhibitory concentration of INH, SM or PAS
in 'Y per mI.
Table 2.

MIC of INH in Combination with SI in vitro

Drug

MIC of INH ('Y/mI.)

INH

0.125

INH+SI (1: 10)

0.032

In the third experiment (table 3) the combined effect was studied withvarious amounts of inoculum, and found to be present when the concentrationof inoculum was higher than 0.04 mg. per m!. of medium. But a combined effectwas not apparent when the concentration of inoculum was less than 0.004 mg.
per mL of medium.
Table 3.

Effect of Inoculum Size on Tuberculostatic Activity of INH and SI-------

Concentration of Mycobacteria in the medium
Drug0.04 mg./m!.

0.4 mg./m!.
I

INH

*1.0

INH+SI (1: 10)

0.1

I

I

0.1

0.004 mg./m!.

I

0.004 mg./m!.

0.005

0.005

0.005

0.005

I

I
0.02

I

I

* Figures indicate the MIC of INH ('Y per mI.).
II.

In Vitro Studies on the Tuberculostatic Effect of SI

Methods:(1)

Three different experiments were carried out as follows:
An inoculum of 0.01 mg. of H37Rv per m!. of Kirchner's liquid medium9 )50Masukazu NAITO, Rinzo WATANABE, Shunji TSUKUMA and Kazuo IBA

with 10 per cent serum was used. The suspension of the inoculum was prepared
as previously described.
(2) Dubos liquid medium with the same inoculum.
(3) Inoculum of 0.02 mg. in each tube of 5 mI. of Oka-Katakura's egg medium10 ).
Cultures employing Kirchner's and Oka-Katakura's medium were macroscopically read after a four-week incubation at 37°C and culture employing Dubos
medium after a two-week incubation.
Results:

As stated in table 4, in every medium the MIC of SI after a two-week-incubation was one-half or one-fourth of the MIC after a four-week-incubation.
As stated in table 5, after a four-week-incubation the MIC of 51 in acidmedia was less than in neutral or alkaline (Kirchner's Medium). In summary,
51 has a weak but definite tuberculostatic action in vitro.
Table 4.

MIC of 51 in Different Media and Different Periods of Incubation

Media

I

Period (days)I

MIC of 51 (ylm!.)

14

31.3

Kirchner's M.

28

125.0
I

--~_.-

14

7.8

28

15.6

14

15.6

28

31.3

Dubos M.

Oka-Katakura's egg M.

Table 5.
pH

MIC of 51 ('lIm!.)

5.5

31.3

6.0
6.5
7.0

250.0

7.5
8.0

III.

Effect of pH of Media on MIC of 51

62.5
500.0
500.0
500.0

Effect of pH on Tuberculostatic Activity of INH alone and in Combination
with SI

Methods:

The pH of Kirchner's medium containing 10 per cent serum was adjusted
to 5.5, 6.5 and 7.5 with NaOH or RCI. The concentration of the inoculum was
0.04 mg. per mI. of the medium.

Antituberculous activity of isoniazid in combination with sulfisoxazole 1.

51

Results:

The MIC of INH was 0.125 r per ml. at pH 6.0 to 7.0 and 0.0625 r per mI. at
pH 5.5. The combined effect of the two drugs was more marked in an acid thanin a neutral or alkaline medium (table 6).
Table 6.

Effect of pH on the Combined Use of 1NH and S1-~-~---------

pH-------- ----

1NH alone

1NH+S1 (1: 10)

5.5*0.0625

0.015

6.0
6.5

0.125
0.125

0.031
0.062

7.0

0.125

0.125

7.5

0.06250.125-~~------,---* Figures indicate the M1C of 1NH (7 per m!.).
IV.

The Bacteriostatic Effect of INH and SI In Vitro on M. Tuberculosis
Resistant to SM or PAS

Methods:

The methods were those of the previous experiment (D, but M. tuberculosis,
strain H37Rv resistant to 100 r of SM per ml. and that resistant to 50 r of PAS
per m!. were employed as inocula. The concentration of the inoculum was 0.02
mg. per ml. of the medium.
Results:

As may be seen in table 7, the combined effect of INH and SI was recognizedon both SM and PAS resistant strains.
Table 7.

The Combined Effect of 1NH and S1 on M. Tuberculosis
Resistant to SM or to PAS

Drug
1NH alone*0.025-----~----

1NH +51 (1: 10)

M. Resistant to PAS

M. Resistant to SM

0.025
--

0.0062~---------

0.0062~---------* Figures indicate the M1C of 1NH (7/m!.).
V.

The Bactericidal Effect of INH and SI In Vitro

Methods:

1) Physiological saline solution was placed in 2 m!. amounts in centrifuge
tubes, the drugs were added to the first tube and serial dilutions were madethrough the tubes. Each tube was then inoculated with 0.05 mg. of M. tuberculosis
and incubated for 24 hours at 37°C.

52

Masukazu NAITO, Rinzo WATANABE, Shunji TsUKUMA and Kazuo IBA

2) Kirchner's medium containing 10 per cent serum was placed in 2 mI.
amounts in the centrifuge tubes, the drugs were added to the first tube andserial dilutions were made through the tubes.
and incubation was for 4 weeks at 37°C.

Inoculation was the same as above

After incubation each tube was centrifuged for 15 minutes at 3,000 r.p.m.,the supernatant was discarded, physiological saline solution was added to theprecipitate and the tubes were centrifuged as above. The washing process was
repeated three times, and after the final washing the precipitate was dispersed
in approximately 0.1 mI. of physiological saline solution.
One loop-full of the suspension was then smeared on 2 egg media (OkaKatakura's), and incubated for 4 to 6 weeks.

Results:
The bactericidal effects of INH and 51 for M. tuberculosis in physiologicalsaline solution at pH 5.5, 6.5 and 7.5 with a 24-hour-incubation are shown intable 8. The minimum bactericidal concentration of INH alone was 12.5 r per ml.
at pH 5.5, 25.0 r per m!. at pH 6.5, and 25.0 r per m!. at pH 7.5. The minimum
bactericidal concentration of INH in combination with three or ten times as much
51 was approximately 1.56 r per m!.
Table 8.

Bactericidal Effects of INH Combined with SI in Physiological
Saline Solution with a 24-Hour Incubation

pH of medium

I

5.5

I

6.5
-~

I

7.5

*12.5( ~)25.0( -)

25.0( -)

6.25( +)

12.5( +)

12.5( + )1.56( -)

1.56( -)

0.78( +)

0.78( +)

INH alone

INH +SI (1: 3)I

3.13( -)
1.56( + )I

1.56( - )1.56( - )0.78( +)

0.78( +)

INH+SI (1: 10)I

I

O.39( -)
O.19( +)* Figures indicate the concentration of INH ('Y / ml.).
( - ) : Bactericidal( + ): Not bactericidal

The bactericidal effects of INH and 51 for M. tuberculosis in Kirchner's
medium at various pH values with a four-week incubation are shown in table 9.
The minimum bactericidal concentration of INH alone-was 6.25 r per ml. at pH 5.5,
12.5 r per m!. at pH 6.5 and 7.5, and that of INH in combination with three or
ten times as much 51 was approximately 1.56 r per m!.

Antituberculous activity 0/ isoniazid in combination with sulfisoxazole 1.
Table 9.

53

Bactericidal Effects of INH Combined with SI in Kirchner's
Media with a Four-Week Incubation

pH of medium

5.5

6.5

*6.25( INH alone

7.5

I

I

-

12.5( -))

12.5( -)
I

3.13( + )!

6.25( +)

6.25( +)
I

1.56( -)

1.56( - )0.78( +)

0.78( + )0.78( -.)

3.13 ( - )1.56 ( - )0.39( +)

1.56( + )0.78( +)

1.56( -:)

INH -+ SI (1: 3)

0.78(

I

+:

)

I

INH+SI (1: 10)* Figures indicate the concentration of INH (/, 1m!.).
( - ) : Bactericidal

VI.

( + ): Not bactericidal

Development of Resistance of M. Tuberculosis to INH through Successive
Transfers in Media Containing Increasing Concentrations of INH alone
and of INH pIus SI

Methods:

Tubes with 2 ml. of Kirchner's media with 10 per cent serum containing
various dilutions of INH alone or INH plus SI were inoculated with 0.04 mg. of
M. tuberculosis, prepared as described above. Monthly, after the MIC of INH
was estimated, the Mycobacterium growing in the test medium containing thehighest concentration of drugs was transferred into the second, newly prepared
test media containing the drugs in higher concentrations. The test media were
adjusted at pH between 5.4 and 5.6 (acid), 6.6 and 6.8 (neutral) and 7.2 and 7.4(alkaline) .
Table 10.

Development of Resistance of M. tuberculosis to INH on Successive Transfers in the Media Containing Increasing Concentration of the Drugs
pH

Acid

Neutral-------------- ----c---

GenerationI

I----------c--------+---

Not inhibited
INH alone
Inhibited1--------;--

Not inhibited*0.005I

INH+SI (1: 25) I

1

0.1

I III
I

I

1.0 I 0.1

I

II
10.0

I III
I

25.0

I
0.25

I II
0.02

I III
I

10.0

~-1-1~IIMI----u;-J:(;OlOO:O~~1100.00.005 [ 0.01 I

INH+SI (1: 3)
Inhibitedi II

Alkaline--- ---

0.01

0.1

0.02

0.02

1.0 I 0.02

0.02

1.0

1---;:-02I----u;-~·~lMl~~U;:O

N~~h;b;ted -~~Ol-r;;~-I O~lJ~o;_l_~or~J{l~I-(W21-1~~__-.!nhib!~~~~~~_ 1.0 1.0J~ i_~·1_L_1_~:~J_ 0.1_:_0=_1~0~ii* Figures indicate the concentration of INH ("11m!.).

54

Masukazu NAITO, Rinzo WATANABE, Shunji TSUKUMA and Kazuo IBA

Results:

Development of resistance of M. tuberculosis to INH on successive transfers
in media containing increasing concentrations of INH and of INH plus SI atvarious pH values is illustrated in table 10. It may be seen that emergence ofresistance of Mycobacterium tuberculosis to INH was delayed by adding three or
ten times as much SI, especially in neutral media.
VII. Development of Resistance of M. Tuberculosis to INH in Media Containing
Fixed Concentrations of INH Alone and of INH Plus SI
Methods:

Tubes with 2 ml. of Kirchner's serum medium containing various concentrations of INH or INH plus SI were inoculated with 0.04 mg. of M. tuberculosis.
At intervals of one month the supernatants were discarded aseptically, andthe precipitates were re-suspended in newly prepared media containing the same
concentrations of the drugs, and each time the resistance of Mycobacterium to
INH was estimated in egg media.
Results:

The results are shown in table 11, in which it may be seen again that the
emergence of resistance of Mycobacterium to INH was delayed in media containing INH plus SI.
Table 11.

Development of Resistance of M. tuberculosis to INH by Culturingin Media Containing a Fixed Concentration of the Drugs

pH

Acid

Generation

I

I

II

Neutral

I III
i

I

II
I

I

Alkaline
III

I

I

II

I III

Not inhibited

0.0125 100.0 100.0 0.0125 4.0

0.25

4.0

4.0

Inhibited

0.025 200.0 200.0 0.025 8.0

0.5

8.0

8.0

INH alone
Not inhibited

0.00311.0

4.0

0.0062 0.0062

0.1

10.0031 0.0062[ 0.0062

Inhibited

0.00622.0

8.0 0.0125 0.0125 0.2

10.0062 0.0125 0.0125

INH+SI (1: 3)

1

Not inhibited

0.00310.05 0.25 0.0031 0.0062

Inhibited

0.0062

0.1

0.5

l

0.25 0. 0031 0.0062 0.0062

INH+SI (1: 10)
0.0062 0.0125 0.5

1

I 0.0062 0.0125 0.0125
1

DISCUSSION

The combined effect of INH, SM or PAS and various sulfanilamide derivatives,
especially SI, on M. tuberculosis var. hominis, strain H37Rv in vitro was studied.
In Kirchner's liquid medium containing 10 per cent serum, the sulfanilamide
derivatives, especially sr, increased the bacteriostatic effect of INH. Although
the combined effect of INH and SI was clearly noted when the concentration of

Antituberculous activity of isoniazid in combination with sulfisoxazole I.

55

the inoculum was higher than 0.04 mg. wet weight per mI., it was not obvious
when the concentration was lower than 0.004 mg. wet weight per ml.
Sulfanilamide derivatives did not increase the bacteriostatic effect of SM or
PAS.
The MIC of SI was 125.0 r per ml. in Kirchner's medium, 31.3 r per ml. in
egg medium and 15.6 r per ml. in Dubos medium. The MIC was lower in acidthan in neutral or alkaline media. Thus, SI has a weak but definite tuberculostatic
effect in vitro.
The combined effect of INH and SI was also more marked in acid than inneutral or alkaline media.
It is worth emphasizing that this combination is effective even on Mycobacteria
resistant to SM or to PAS.
The tuberculocidal effect of INH has been well known to be relatively higher
than that of other tuberculostatic drugs. In the present studies it was foundthat SI increased the tuberculocidal effect of INH markedly. The minimum
bactericidal concentration of INH used with SI was approximately 1.56 r per ml.
This concentration can probably be achieved in human body fluids.
The aim of combined use of antituberculous agents is, of course, not only
the increase of bacteriostatic effect, but also the delay of the development ofdrug resistance. Therefore the in vitro effect of SI on the development of resistance of Mycobacterium to INH was studied. Both in the experiment withsuccessive transfers in media containing increasing concentrations of INH and inthat with media containing a fixed concentration of INH, SI was noted to delay
the emergence of resistance of Mycobacterium to INH.
Thus the combined effect of INH and SI in vitro was demonstrated clearly.
The mode of this synergic action, results of animal experiments and clinicalstudies will be successively reported.
SUMMARY

1)
2)
3)
4)
5)

A synergic bacteriostatic effect was demonstrated in the combined use of INH
and Sulfisoxazole (SI) in vitro.
SI was found to have a weak but definite tuberculostatic effect in vitro.
This combined effect was recognized even with SM and PAS resistantstrains.
SI also increased the tuberculocidal effect of INH.
The emergence of resistance of Mycobacterium to INH in vitro was markedly
delayed in the presence of SI.

56

Masukazu NAITO, Rinzo W ATANABE, Shunji TSUKUMA and Kazuo IBA

REFERENCES
1)

Naito, M., Watanabe, R., Maekawa, N., Rineno, Y., Nakamura, A., and Iba, K.: Kekkaku,
29: 248, 1954.
2) Naito, M.: Kekkaku no Rinsho, 3: 458, 1955.
3) Naito, M.: Jap. Arch. Internal Med. 2: 639, 1955.
4) Naito, M.: Japan.]. Clin. Tuberc. 15: 674, 1956.
5) Naito, M.: Naika, 2: 540, 1958.
6) Yoshida, T.: Rep. Tuberc. Res. lnst., Kyoto University, 6: 259, 1958.
7) Naito, M.: V International Congress on Diseases of the Chest, 315, 1958.
8) Ushiba, D., Sunahara, S., Shimamura, K., Koyama, Y., Takahashi, K., Tomita, Y. and Gomi, J.:
Japan. J. Clin. Tuberc. 15: 664, 1956.
9) Kirchner, 0.: Zbl. Bakt. I, Org., 124: 403, 1932.
10) Oka, S., Katakura, T. and Ishikawa, Y.: Tohoku 19aku Zasshi, 21: 692, 1937.