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.. -1.

New Results in the Chemotherapy of Mycobacterioses J M. Rosenfeld, J. P. Freerksen, E. Retzlaff and S. Hokama'

Since Rifampicin (RAMP) has become available for tbl' chemotherapy of myco­bacterioses', new schemes for successful first treatment of tuberculosis have become pos­sible. Now the same good results can be ob­tained in the chemotherapy of chronic tuberculosis, resistant to many antitubercu­lollS drugs. This is why we should also anew investigate the practicability of RAMP in the tr~atment of so-called atypical myco­bacterioses. As the evaluation of such data on a clinical basis is not possible because of the rarity of cases, and the bacteriologic determination of Tesistance does not give a satisfying answeT as to the therapeutic ef­fect of drugs used alone or in combination, we wish to offer. in the following pages, ways for the assessment of therapeutic ef­fects in animals OT in man. One of the ex­perimental methods is screening the theTapy in infected animals.

Mice infected with M. kansasii will show readily reproducible courses of infection, which, as worked out by Yoshida (3) (Fig. 1), give a good therapeutic response to com­bination therapy including Rifampicin. These strains are often as sensitive as M. tuberculosis H.·7Rv in vitro to Rifampicin. The large number of mycobacteria in these slowly developing infections makes neces­sary somewhat higher dosages in com­parison with those used for M. tuberculosis. Rifampicin in triple or quadruple combina­tion with streptomycin (SM), Isoxyl , cer­tain sulfonamides, ethionamide, isoniazid (INH) or ethambutol (EMB), offers some promising aspects for therapy. Generally speaking, it is best to recommend a quad­ruple combination.

Beside the therapy of swimmingpool­infections, the determinRtion of an effec­tive treatment of infections with !If. mnrinum is verv important because of its :,malogy to M. leprae. Infections with M. marinum in mice are virulent enough to serve also as an experimental model for the evaluation of

1 Forschnngsinstilut Borstel, Institute for Ex­perimental Biology and Medicine, BOTstel, 'Vest Germany.

358

antimycoba<.:terial drugs. Intmvenous injec_ tion at first causes skin lesions in the tail feet . mouth and ears (in male animals, U; addition. the testicles) with large numbeTs of mycobacteria (Fig. 2) . LateT on We find pTogTessive mycobacteTioses of the lung (Fig. 3). In survival-time tests (under ex­tTemcly hard conditions), as well as in the tTeatment of these serious peTipheral proc­esses, Rifampicin at a dosage of 20 mgm. and 50 mgm ~ Ikgm. was again very effective (Fig. 4). Similar Tesults (as with the effec­tive dose of 10 mgm./kgm. of Rifampicin ) were obtained in spite of the clinical Course of the infection with 50 mgm./kgm. of B.663, 100 mgm. /kgm. of sulfamethoxypyra_ zine, 50 mgm. /kgm. of ethionamide and 50 mgm. / kgm. of DDS accoTding to the sur­vival time of the animals (Fig. 5). Ten mgm./ kgm. of INH and 100 mgm./ kgm. of LedeTmycin were shown to be less effective, and 100 mgm. / kgm. of Isoxyl caused the lowest effect. In these expeTiments only Rifampicin could significantly control the peTipheTal processes. A combination of Rifampicin with one of the above men­tioned tuberculostatics showed some thera­pe.utic improvement compaTed with ' the activity of the partner, but the effectivity of this combination was not better than that of Rifampicin alone. The combination with Ledermycin Tesulted in a decrease of activity. In most experiments, however. the course of generalizing infections was effec­tively influenced by treatment with twin combinations (Fig. 6); the most active are Rifampicin and B.663 (Fig. 7), and Rifampi­cin and ethionamide (Fig. 8). Not so good Tesults weTe obtained by combinations of Rifampicin and Kelfizine, DDS (Fig. 9) or Isoxyl. The combination of Rifampicin and INH showed lower activity than Rifampicin alone. The individual effects of Rifampicin and Ledermycin are nearly nullified when these two drugs are combined-similaTly to theiT activity against the peripheral lesions (Fig. 10). No decrease of activity was to be obseTved by adding 10 mgm.lkgm. of l!\TJI to 20 mgm./ kgm. of Rifampicin and 100

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360 International Journal of L eprosy 1971

FIG. 2. Infection with M. marinum in mice. Skin lesions on tail. Ziehl-Neelsen staining of my­cobacteria.

FIG. 3. Final lung processes after infection with M. marinum. Laboratory animal, mouse.

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39,2 Rosenfeld et al.: Cherrwtherapy of Mycobacterioses

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50 mg/J:g Rifampicin

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10 mg/J:g INH 100 mg/J:g Sullamefhoxypyrazin

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FIG. 12. Example showing effectivity of therapy on serious processes eight days after state­ment of therapy.

mgm./kgm. of sulfamethoxypyrazine; in this case the effectivity of this drug was again to be seen (Fig. 11). The combination of these three drugs is effective in both cases, i.e., the generalizing and the peripheral in­fection. Similar results were obtained by 50 mgm./kgm. of Rifampicin as mono therapy. The treatment of the serious peripheral processes by 20 .mgm. /kgm. of Rifampicin alone showed some failures, whereas the triple combination of Rifampicin, sulfa­methoxypyrazine ~nd INH, as well as the mOno therapy by 50 mgm. /kgm. of Rifam­picin, produced a complete healing of the ulcerations in all animals (Fig. 12). The results gathered from monotherapy and twin combinations offer the possibility of several other and perhaps better triple com­binations.

The fact that the drugs used for the treatment of leprosy, viz., DDS, B.663 and su}fonamides, are also effective against M.

marinum infections and show promJSmg effects in combination with Rifampicin, may be an indication for the use of such infec­tions as analog models for leprosy itself.

As "atypical" mycobacteria are often less virulent, the building of experimental mod­els with laboratory animals encounters many difficulties. Large numbers of mycobacteria are necessary as infection doses. This ac­celerates the course of the infections on the one hand and on the other brings about obstacles in analyzing the data. For this reason we developed a special kind of serum activity determination which enables us to compare the activity of several chemo­therapeutic agents against diHerent myco­bacterial strains and species. This method has the advantage of being independent of the virulence of the mycobacteria for labora­tory animals.

For this kind of experiment we show the serum activity (Fig. 13) after oral applica-

370

Strain

ifvt·phlei fsN 109

ifvt·kansasii RS 8316

ifvt.ulcerans SN 1.21

InterMtioMl ]ouTMl of Leprosy 1971

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o ++++++#-#-*-++-++*i+it+iHiH1~H-I 1 - - (+) I f+)

1 - - - - - - - - - - - - - - - - -r::' 3 -----------------_ 3 -----------------5 - - - - - f+) - - - - - + - - - - f+) -++ 5 ------.----~-----+ 7 -----------------~ 7 -----------+-----+ 9 f-H ----+------* 9 ---~-+-+--~+-++-+++*.ffmffi~ 2~ -+- + + _+_ + _+_ 41- -++ #- #- _++- # 41- #- * ++- I+f . Hi 2~ - - fH f+) + + f.H f+) + + -++ #- #- * -++ #- I+f I+i WK+++_+_ .ff##.ff +H+H+H+H

o f+) + + + + + * ++- -# #- -# ++ I-H +H H-l 1+1 f+t I f+I 1 - - - - (+) _+_ H7 + -+- f-H 1 ----++----++----~~ 3 - - - - - 1:+) - - - - - + - - - - - * 3 -----~-----+-----* 5 - - - (_+_) + + - - - + + #- - - - + -1+ ffj.

5 ----~-+-----+*----*~ 7 - - - - - (+) - - - - f-H + - - - - f+)J -H 7 ----~+----~-+-----+-I+ 9 - - - - E:I-) + - - - - + + - - - - _1+ -# 9 + + + + + + +H ++ -t+- ++ ++ ++ +ti !-H i+1 i+1 ffl +ti

21. f+7 + _+_ _+_ -+- _+_ 1+ _++ _1+ _++ -1+ 4+ -++ tH i-H i+i I-H I-H 21. (+1 (+) + + + -+- + _++ -1+ -1+ -l+ -1+ _f+. #I #I 1+1 H+ ++i WK + + + _+_ -If- _# --f+. -1+ +H fH +tI tH

o ~+++++_#H_++_++_++Ht+ifflfflffl+Hffl 1 --~~++-+-++-f+.**+#*i-H++if-H 1 - - - - - (-+1 - - - (+) --it -» - - + -+l +fi 3 -----~---~+-++---+~f-H 3 ----~~----+-*-++--~#~~ 5 ----~~--~+*--it+**mm~ 5 ----~~--~+-++_++f++_++~#I~ 7 -----E-H- +4+*--tf--+t++-li_H 7 - - - - - H-) - - (+l + _1+ +t - - ++ -HI f.H .fH 9 ----~~--~+-I+#---I+i-Hi-Hf.H 9 --~~~~+#++++-I+++m+-Hffifflffl~ 2~ - (+) (+) f+-' (+1 f+) + ++- -++ ++ 4+ _++ _1+ -++I -1+-1 +H H-I t+I 2~ - f+) f+l f+) f+) f+) -+- -++ -++ -# -++- +t -+t +_H H1 I+l /+j ++I

WK + + + + -+t -++- -+t -+t +H +H +H +H

FIG. 13. Determination of serum activity in rabbits after oral application of 50 mgm./kgm. Rifampicin against mycobacterial

39, 2

Strain

Mmarinum SN 125~

"M.smegma tis SN 7

iM.avium 13839

lM.fortuitum SN 203

Rosenfeld et al.: Chemotherapy of Mycobacterioses

Serumdilution

o ++++++*~~*#*+#~~fflm~ 1 -8-)++++~~ ~-t+++-..J+ ~~~~W#l

l' H- + + + + + -f+ -++- f+ ~ ++- 4t ~ #f t+-l ~ +# f+j

3 -~++++-****~~ffim~ffl~ 3 --++++H+*f+-t+*~~~ffiffl~ 5 ---~++*-++4t**-t+~~~#f~~ 5 --~~~~+***-t+*~~ffi~ffl~ 7 --~H-+++****-t+~~~ffl~ffi 7 --~~++~*-t+-++-**~~~ffl~~ 9 -++++++*-++***ffiffi~~~~ 9 -~++++H-++4t***fflfflfflfflfflffl 2~ + + + + + + -++ -++ -++ -l+ * -t+ t+f ffl ~ ++t ~ ffl 2~ f+-) + + + + + * * -++ -++ -++ -ti- ffi H-l +H I+i I+f ffl

WK + + + -tt- * * f# H-l +H o ++++++-tt--++*-++***ffl~ffl~ffl 1 ~+++..J+*-++-t+*-++~~~t+f~ffl 1 --~+++#-t+**..J+*t+f~~fflfflffl 3 --~+++*-++**-t+*ffl~~l+it+f~ 3 --~+++#4+#-t+**~~~t+ft+fffl 5 --~+++**-t+-t+-t+-t+t+ft+fmt+fl+it+f 5 --~~++++-*****ffl~t+fffi~ffl 7 -- I ~+++-t+-l+++-***~~t+f~~1+f 7 --~+++-++-t+++-++-**t+ft+fffit+fl+i~ 9 --~+++**++-***ffl~~ffl~* 9 --~++****-++*ffl~~ _ m~1+f

2 ~ - 1:+-) H--) + + + ++- ++- -l+ * ++- -+I- t+i ffl ~ ffl Itt #l 2~ - t+-l C+) + + + ++- ++- ..J+ -t+ * --+t H1 +tt ffl +# t+f I+f

WK + + + + . ---H- -++- ++- -++ m I+i +tt t+f 0 ++++++++-++-++-*-++-++ 1 E+) E+- f+-) 1:+-) H--) H--) -4 -++ --# ++- -++ -+I-

1 -+++++****-++-+1-

3 1:+-) (j- f+--l f+-) -#-- -++ +t +t -++ -++ 5 - E+-l E+) E+) + + -++- -++ -++ +t -++- ++-

7 - &-1 E-rl + + + 4+ -+f- ++- ++- * -++-7 E+--l++++****** 9 - ~) + + + + -++- -++- ..J+ -++ * * 9 (+) + + + + + +I -++- -+t #- ++- *

2~ 4- + _-+- + + + -++- -tt- ++ ++- 4+ -f+ 2~ + + + + + + it -I+- #- ++- -++ -++-

WK + + + + -++ -I+- -++ +I-

o ++++++ff#-++--++*-+l-ffit+ffflffiffim 1 ++++++**-t+*-++--+tffiffl~ffl+tt* 1 ++++++#-*#-~*-++ffll+iffif#ffll+i 3 ++++++*#-t+***~~I+ft+fffll+f 3 ++++++***-H---+t-+l-t+fffifflt+ft+fl+f 5 ++++++*-++**++-++-ffi+ttffifflt+f~ 5+ +++++-++-*---H-*-t+-++-ffi~t+ft+fl+iffl 7 ++++++**+t*-++-*~~fflt+ffflffl 7 + + + + + + +f- -++ -t+ ++- +I- -t+ +H Ht ffl #I 1+1 ffl

9 ++++++***++-+I-~t+fffl+tt#lffl~ 9 ++++++**-f+*-+I--+f-~ffi#lt+f~Ht ~ ++++++*~-++-tt--++*+tt+ttt+ft+fffll+i ~++ ++++*-++**-+I-*~fflfflt+ft+ft+f

WK + + + + -++ -++ .f+ -++ +H -1+1 ffl t++

strains. Medium: Lockemann. Serum dilution 2-6 and 2-1 'liith an addition of 4% serum. Inoculation : 6 x 10- 4 mgm.

371

372 International Journal of Leprosy 197]

FIG. 14. Example of the growth control +, ++, +++.

tion of 50 mgm./kgm. of Rifampicin against inoculations with diHerent mycobacteria. Blood samples were taken I, 3, 5, 7, 9 and 24 hours after application of Rifampicin at every given point of time from two rab­bits. A mixture of 1 ml. of the serum with 3 ml. / culture-broth (Lockemann) was pre­pared, and by 1:2 steps diluted up to 1:128, i.e., 36 readings over one day. The test tubes were inoeulated with 6 X 10- 4 mgm. mycobacteria. The resulting growth of the mycobacteria was labelled as (+), +, ++ and +++; C_' means that no bac­terial growth could be observed. These results were compared with the zero values of bacterial growth and control runs with­out drugs. The results of total inhibition are pOinted out by a dark }jne. The inhibi­tion may be unchanged after three read­ings, e.g., with M. phlei, or may be de­creased when mycobacteria grow in num­bers in the control (+, ++ and +++) tubes (Fig. 14), e.g., with M. ulcerans. The differences in the inhibition of several my­cobacterial strains with varying sensitivity at the same serum concentrations are sig­ni£cant.

In order to check the usefulness of this method and to demonstrate the inhibition

effect of diHerent schemes Fig. 15 has been prepared. It' shows the serum activity destination with rabbit serum according to the model in Fig. 13. Fifteen diHerent strains of 10 species were tested under 10 thera­peutic regimens. Laek of bacterial growth (' - ') was analyzed and illustrated only by the height of the column, which is a direct representation of the inhibition grade. The time and the grade of serum activity are determined in the same manner. An arrow­shaped column represents activity going be­yond the measured concentrations. The horizontal readings show the therapeutic effect of a regimen; vertical readings show the inhibitory effectivity of one medication against the strains tested. These data give us the following important results:

Rifampicin is effective at a dosage of 50 mgm./ kgm. according to our test conditions (1:2 dilution, inoculation with 6 X 10- · mgm. mycobacteria) against M. tuberculo­sis, M. bovis, M. kansasii, M. ulcerans, M. intracellulare, M. phlei SN 109, M. marinum and one strain of M smegmatis There are some extremely sensitive mycobacteria, as M. bovis Vallee , M. phlei SN 109 and, as earlier described (2), but not shown in this figure, some strains of M. tuberculosis and

39, 2

Strain

M. bov.

Vall ••

M. kansasii

8316

M. avium

13839

f.1. avium

M in/racel/.

Ov.

M ulcuans

M. in/race/l.

SN ~03

M marinum

M. marinum

SN 1301

M. phlei

SN 109

M. scrof.

10022

M. (orlUilum

SN 203

SN 7

/rf. sm~gmatls

SN ' 6

Rosenfeld et al.: Chemotherapy of Mycobacterioses 373

INH RAMP Ifamelll - RAMP DDS B 663 10 mg/kg 50 mg/kg oxypyrazin 50 mg/kg 50 mg/kg 100 mg/kg

IJ 2J 00 mglkg • INH 20 2J 2J 2J Sulfamel

oxypyraztn /00 mg/ kg

2J

EMB Isoxyl SM ETH OOmg/ kg 200mg/kg 20 mg/kg 50mg/kg

IJ 2J IJ IJ

findin S oflolal inhibilion f rOINI (max. 36J

FIG. 15. Survey of serum activity determination in rabbits. Height and duration of the total inhibition labelled as column. 1) After single application. 2) After eight days' pre treatment.

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-39, 2 Rosenfeld et al.: Chemotherapy of Mycobacterioses 375

RAMP 10 RAMP 10 Sulf. 20 DDS 20 8663 ETH + +

SuIt 20 Su/f 20 mg/kg +

mg/kg 2 x 300 10 mg/kg 10mg/kg

INH 5 mg/kg (- 7mg/kg

mg/kg

Mkansas;;

8316

M ulcerans

SN 421

Mmar;num

SN 1254

M rub.

H37 Rv

WK. + .". .,. + -No .,. + -No +H- + ++ ~ + -No ~ + * ~ + -II- -FIG. 17. Survey of serum activity detennination in healthy test persons. Assessment as in Fig. 15.

M. intrpcellulare. These data are always in agreement with the sensitivity tests and are extremely valuable as a basis for the evaluation of therapeutic dosages. M. tu­berculosis H37Rv and M. kansasii 8316 are very similar in their sensitivity. M. marinum is less sensitive than M. tuberculosis H37Rv.

Sulfonamides might be a very important factor in the therapy of "atypical" myco­bacterioses; e.g., sulfamethoxypyrazine had the broadest spectrum of activity among the here tested substances and even inhibits rapidly growing mycobacteria which up to now have been a serious problem for chemotherapy. Sulfamethoxypyrazine is more effective against several strains and remarkably better tolerated than DDS. Ex­periments for comparison of several sul­fonamides will be continued.

\1ost significant is the increasing in­hibitory effect by combining Rifampicin, sulfamethoxypyrazine and INH in a triple combination, the activity of which exceeds most frequently the mentioned measure range. Even if there are small or no inhibi­tion effects after monotherapy we can ob­serve an increase in therapeutic effectivity alter combination treatment, e.g., against ll. avium infections.

Streptomycin also has an inhibitory ef­fect on most of the mycobacteria tested. It has important therapeutic value even in relatively low concentrations against infec­tions with M. kansasii and M. avium, as shown earlier (I).

Isoniazid and ethionamide show only a small spectrum of activity. The effectivity of ethionamide is already evident in mono­therapy against M. kansasii and M. marinum infections in mice. Our experimental data show that it is a good drug for combination therapy, too. INH can be used as third or fourth drug in several combinations.

Another drug, B.663, causes extremely high serum levels after application of a single dose of 100 mgm./ kgm., and shows a very broad antibiotic spectrum. The high dose is not indicated for long term therapy in rabbits.

Isoxyl in a dose of 200 mgm./ kgm. is active against strains of M. tuberculosis, M. hovis, M. kansasii and M. ulceran.~, and, according to a control growth '+', even against M. intracellulare, M. marinum, M. scrofuLaceum and some M. smegmatis strains. It is suitable, too, for combination therapy against infections with the above mentioned mycobacteria.

376 J IIt c nWtlOlW/ J (mrllll/ OJ L c proSIj

The test conditiuns of ethambutol show (,xtraun.linarily unsatisfactory results in rab­bits,

The usefulness of these data, found in rabbits, for chemotherapy in man is de­pendent on whether the tolerated doses will cause a corresponding antibacterial activity. These problems can easily be solved by means of the above described method as being worked out for M. marinum and other mycobacteria ( Fig. 16). From Fig. 17 we can gather that twin or triple combinations administered in toler­able doses reveal excellent results. It is also shown that the tested sulfonamide has a better or equal activity, as compared with DDS at the same dosage, against M. mar­inurn and M. ulcerans. A well tolerated dose of su]famethoxypyrazine is more valu­able against M. kansasii than DDS, which on the one hand is very active, but in the concentrations used is already too toxic for therapy in man. Already a single dose of ' 10 mgm./ kgm. of B.663 inhibits growth of M. marinurn and M. tuberculosis H37Rv according to control growth '+'. This dmg shows significant accumulation in the body so that a blood level control after long-tenn treatment is necessary; nevertheless, its scrum activitv in man is irrefutable , too.

As the se'nsitivity data are extremely variable, even for one 1.ind of "atypical" my­cobacteria-more variable than in the spe­cies M. tuberculosis-it is necessary to work out an individual therapy in man. The

, dosages and combinations can be easily evaluated by means of the above mentioned method, res'ulting in a specific treatment of "atypical" mycobacterioses. In the S:lme way it is possible to cure infections caused bv M. ovium, only if a plan of therapy is f~rmulated by the b est combination of

tolerait'd drugs und er p erma.nent eontrol of th t' seTllm level ( the same IS d~e. to poly_ resistant tu bereuloses ). The prehmmary ad­vice of the regimen can easily be screened in rabbit experiments.

SUMMARY

1. By means of the method briefly de­scribed it is possible to evaluate drug com. binations against almost any kind of myco­bacteriosis.

2. By including serum of patients or healthy test persons we can adapt the ex. perimentally gained results to conditions in man.

3. Although a large group of substances when used alone are effective against all mycobacterioses, it is, however, to be rec. ommended that for the purpose of therapy they be employed in combinations of sev. eral drugs.

4. The combinations of chemotherapeutic agents found by this experimental screen. ing allow successful therapy at dosages

. which are well tolerated by men. 5. The combination of Rifampicin, suI.

famethoxypyrazine and INH proved to be therapeutically effective against most my­cobacterioses. Screening fqr other combina­tions of therap eutic value will b e continued.

REFERENCES 1. ROSENFELD, M. Chemotherapie sogenannter

atypischer Mycobaeteriosen. Beitr. Khn. Tuberk. 137 (1968) 115-129.

2. ROSENFELD, M. Rifampicin, MyambutoL Isoxyl, and Capreomycin as combination partners in animal experiments. Antibiotica et Chemotherapia 16 (1970) 501-515.

3. YOSIDDA, F. Experimental therapy with Rifampicin. Antibiotica et Chemotherapil 16 (1970) 416-423.