VOL. 12

NO. 2

CHEMOTHERAPY

Assay

methodfluidbyforbiologicallyagar

diffusion

mycobacterium

117

activeethambutol

methodusingas test

organismin bodysaprophytic

YUTAKA KANAZAWA & TOSHIO KURAMATA
Niigata Railway Hospital(Received August 21, 1963)
Agar diffusion method e.g. cylinder-plate
has been widely used as the standard
the assay in the antibiotic

the thin-agar

cylin-

method as the assay procedure of antibio-

tics in body fluid, whichusual cylinder-platetechniques

was more sensitive

thanmethod.

Authors3-7) reported

the thin-agarcylinder-plate

for the assay of biologically active levelsof synthetic-nitrofurans ,chemotherapeutics,
isoniazidandi. e. sulfonamides,ethionamide,

inbodyfluids.
THOMAS at al. 8) reported
for the assay

an agar diffusion methodof ethambutol*using

M. smegmatis

607 as test organism,

which was useful to determine

the peak concentrationin serum after drug admini-stration.
In the present
the application
to theassay

ethambutol

paper authorsof thin-agar

intendto describe

cylinder-plateof ethambutol

in bodymethodfluids using

high sensitive

mycobacterium.

EXPERIMENTAL

MATERIALS

AND METHODS
Testorganism : A saprophytic,

mycobacterium

non-chromogenic

11-7, which had been isolated

soil as the test organism

fromfor isoniazid.

Culture media : KIRCHNER's agar medium containing 10% serum or albumin and 1.5% agas was used
for the assay plate.
with purified
useful.
* Ethambutol

agar

Usual nutrient
eliminated

fatty

agar

mediumacid was also

is the generic name reserved for thedextrorotaryform of 2, 2-(ethylendiimino)-di1-butanol by Lederle Laboratories Division ofAmerican Cyanamid Company8-10).
** Ethambutol was supplied by Kaken ChemicalCompany.

Usualnutrient

broth

DUBOS' albumin liquidfield, because of its sim-plicity and accuracy.
MIYAMURA et al. 1,2) reported
der-platemethodprocedure forwithmedium0.05%

Tween 80 or

was used

for theinoculum of the test organism.
Preparation of samples : Normal plasma containing-

ethambutol** in the following concentration, 8, 4,2, 1, O.5, 0 pg/mI were prepared as standard fortesting of plasma taken after drug administration_
Preparation of the inoculum of the test organism :Usual nutrient broth with Tween 80 or DUBOS'medium was inoculated with M. H-7 and incubatedfor approximately 24 hours at 37°C.
Preparation of the assay plate : KIRCHNER'sagar
medium melted and kept at 50°C was inoculatedwith 0.2% broth culture of M. H-7. Petri-dishes(inside diameter 90 mm) with plain bottoms wereplaced on a level surface and 5 ml of the inoculated
medium was distributed evenly and allowed to harden. If dishes of other sizes were used, amount ofmedium in one plate was altered to provide thinlayer of the inoculated agar medium.
Assay procedure : Cylinders were placed on theinoculated agar and filled with standard samples andtest ones. The plates were incubated at 37°C for
approximately 48 hours. Then diameter of each
inhibition zone was measured and averaged.
Estimation of concentration : On semi-log paper,the diameter of the inhibition zone was plotted against the log of ethambutol concentration and thestandard curve representing the relation between thedrug concentration and the diameter of inhibitionzone was drawn. Then the concentration of the testsample was estimated by comparison with standardcurve.
EXPERIMENTAL RESULTS
Comparison between different kinds of mycobacteria as test organism : Effect of different kinds of
mycobacteria i. a. M. 607, M. grass, [M. phlei, and118

CHEMOTHERAPY

MAR. 1964

fi9.2 Relationbetween the concentrationof ethambutoland theinhibition zonesby the thin agar cup method using M./1-7as
the test organism

Fig.1 Ef!fct different kinds of
mgcobacteria as test organisms
ondiameter ofinhibition zonemm
40Diameter ofinhibition zonemm

Concentration.777177

40z0in Plasma in gutter" 30o30-8441.2±2J/7:\14\i ..//// 0.5

32.0±1.8 33.1±0322.8 2-±0.9 24.411.9/4.3 ± 24 16.3±2.S

120L

•-•
0-Buffer(pH70)
PlasmaAverageof 6 plates

10i

1
24 /P/m/Concentration of elhambaidin plasma

0.5

124Concentrationof ethambutol

Fig.3 Bloodlevel after oral administration of
500my ethambutol in 4 adults

M. H-7, on the diameter of inhibition zone weretested. M. H-7, growing clear zone, was most sensitive to the drug, M. 607, however, was lesssensitive, as shown in Fig. 1.
Relation between the diameter of inhibition zone
and the concentration of ethambutol : The relation
between the diameter of inhibition zone and thelogarithmic concentration was linear in the range
from 1.0 to 8. 0 Pg/ml, as shown in Fig. 2.

11/m1
10Average

L

1,3Li

0.50:20.

••

•

•

2/44

•

6•

•
•

6

F2'

Hours after administration
Fis.4 Urinarylevel and urinary recoveryafter oral
adminlistration of 500myethambutolin 4 adults

log0=-EU—EShSh —ES1 logA

1000•500-•••(L)10050

plasma and urine levels were determined in 4 adults.
As shown in Fig. 3 and Fig. 4 the drug showed
active level in blood for approximately 8 hours pe-Average

24-----

68
10
12
Hours after administration
Urinaryrecoveryin 24 hours

Case no1
2
3
4

riod and was excreted in urine, in highly activelevel.
Influence of organ homogenates on the activity ofethambutol : Inactivation rate of the agent in organ
homogenates i.e. lung, liver, and kidney of mousewere tested by using above mentioned assay method.
No deterioration was observed in its activity in 2-----

5

Consequently, the concentration under test wascalculated from the formula :where, 0 was the ratio of highandand test dose, A
was the ratio of high and low dose, Sh, Si and U
were the diameters of standard high, standard lowand test samples, respectively.
And the lower
limit of the concentration measure was approximately 0. 6 pg/ml.
Absorption and excretion of ethambutol . After
single oral administration of 500 mg of ethambutol,

8 Pk'hours

at 37°C,

Distribution
tion

datain Fig.

18mean

360
266
299
370

72
5360
74

as shown

in organsby intubation
6 provided

65%

in Fig.

of ethambutolof ethambutol

administration

14

that

5.

in organs: Concentra-of micefollowing

wasdetermined

thedrug.
developedoral
The
ac-VOL. 12 NO. 2

CHEMOTHERAPY

Fii.5 Inactivation rate of .ethambutolin organ homogenate of mouse

119

Table 11'0010--Case
No.

Kidney1Lung 2gLiver10'1342

Microbiologically active levels in termsof ethambutol
concentration(,ugiml)in the resected lung tissues following
over 3 days oral administrationofethambutol 1, 000 mg per day in 4 adults.
CavityCaseousI Normalwall
mass
1lung
4. 0, 2.4
1. 1, 1.8
2.0
3. 3, 1.9

<0. 6

5.2
1.2
2.5
2.3

Hours at 37°C
Fig6 Biologically
activeconcentration
inbodyfluidsandorgansofmiceafteroraladministration
ofethambutol(100.1140

721f:

ethambutol was absorbed orally and distributed inorgans and excreted in urine in active form. And
the agent was assumed to be of effective in thetreatment of lung, kidney and urinary tract tuberculosis, because it developed significant microbiologically active levels, especially in lung, kidney andurine.
SUMMARY
1) Microbiologically active ethambutol was assayed to the lower limit of 0.6 ,ug/m1 in plasmausing mycobacteria M. H-7 as the test organism.
2) After oral administration, ethambutol developed active blood level for several hours period anddistributed in organ tissues including lung andkidney then excreted mainly in urine in biologically
active form.

101
10,
Lik

17 \lb16'8/Heart
Spleentive levels in kidney, liver and lung and then wasexcreted mainly in urine in active form.
Active level of ethambutolsue : Microbiologicallylung tissues resectednistration

activelungconcentrationof ethambutol

tis-in thejust after over 3 days'

admi-

1,000 mg per day elimina-ting other chemotherapy,
dulous patients.

in resected

was measured

in 4 tuber-

Active levels were observed in thetissue of normal lung and cavity
in a caesous mass,wall, but no trace

growinghighly sensitive

REFERENCES
1)

acid-fast

to ethambutol

bacillus

M. H-7,and having some pro-

perties in common with human tubercle bacillus:appeared to be the suitable test organism for assaying ethambutol,

a new antituberculous

peutic.
Fromthe results

mentioned

assay

their appreciation to Dr. T. NAKAJIMA,Director of
Surgical Department of Niigata Health Insurance
Hospital, for his supplying resected lung tissues.
These results were reported at the April 1963
Meeting of the Japanese Society for Tuberculosis
and the June 1963 Meeting of the Japan Society ofChemotherapy.

as shown in Table 1.
DISCUSSION

The rapidly

Acknowledgement : The authors wish to express

obtainedmethod,itchemothera-by using
wastheabove

indicated

thatS. MIYAMURAand Y. KANAZAWA: Assay forpenicillin and streptomycin in body fluid using
thin-agar cylinder-plate method. J. Antibiotics, Ser. B, 13(5) : 295, 1950.
2) S. MIYAMURAand Y. KANAZAWA. Assay ofantibiotics in body fluid by cup method. Rinsho (Clinic) 4: 678, 1951.
3) Y. KANAZAWA
and T. KURAMATA. Assay of
biologically active sulfonamide in body fluid.
Chemotherapy 8(5) : 478, 1960.
4) Y. KANAZAWA
and T. KURAMATA: Assay of

120

CHEMOTHERAPY

biologically active nitrofurans in body fluidby thin-agar cylinder-plate method. Chemotherapy 9(6) 481, 1961.
5) Y. KANAZAWA
and T. KURAMATA: Assay of
biologically active INH in body fluid by agar
diffusion method. Chemotherapy 8(5) : 451,
1960.
6) Y. KANAZAWA
and T. KURAMATA Assay of
biologically active INH in body fluid by agar
diffusion method using non-pathogenic acidfast bacillus. Clinics of Respiratory Organs
16(11) : 681,1960.
7) Y. KANAZAWA: Assay of ethionamide in bodyfluid by agar diffusion method, Kekkaku (Tuberculosis) 37 : 530, 1962.

MAR. 1964

8) J. P. THOMAS,C. 0. BAUGHN,R. G. WILKINSON'and R. G. SHEPHERD: A new synthetic compound with antituberculous activity in mice :ethambutol (dextro-2, 2'-(ethylendiimino)-di1-butanol). Am. Rev. Res. Dis. 83 (6) : 891,1961.
9) A. G. KARLSON: Therapeutic effect of ethambutol (dextro-2, 2'-(ethylenediimino)-di-l-butanol) on experimental tuberculosis in guinea
pigs. Am. Rev. Res. Dis. 84(6) : 902, 1961.
10) A.G.KARLSON: The in vitro activity of ethambutol against tubercle bacilli and other microorganisms. Am. Rev. Res. Dis. 84(6) : 905,1961.