Bactericidal Activity In Vitro of Various Rifamycins against Mycobacterium avium and Mycobacterium tuberculosis 1- 3

LEONID B. HEIFETS, PAMELA J. LINDHOLM-LEVY, and MARCELLA A. FLORY Introduction

There have been a number of reports indicating the bactericidal activity of rifampin against Mycobacterium tuberculosis (1-5), but the bactericidal activity of other rifamycins against these organisms is lesswellknown (6, 7). Because Mycobacterium avium disease is becoming the second most widely spread mycobacterial infection, especially in connection with the growing pandemic of AIDS, the use of rifamycins in the treatment of M. avium infection requires certain justifications. The first step would be an appropriate comparison of the in vitro activity of different rifamycins against these organisms, particularly of the bactericidal potency of these drugs, but to date there have been only a few such reports (8-11). The mode of action ofrifamycins against M. avium complex is basically the same as against M. tuberculosis and is related to inhibition of the DNA-dependent RNA-polymerase. Nevertheless, these organisms show higher drug resistance than do M. tuberculosis organisms, reported to be related to differences in the permeability of the cell walls of these two species (12, 13). Any agent that can change the permeability barrier may enhance the inhibition of M. avium by rifamycins, and probably the bactericidal effect as well (12). We reported previously (14) that combinations of rifampin plus ethambutol or rifabutin plus ethambutol have a synergistic bactericidal effect on M. avium, creating a substantial decrease in the minimal bactericidal concentrations (MBCs) of each drug in these combinations. The same synergisticeffect can be expectedbetween ethambutol and newer rifamycins, especially taking into account a suggestion that the effect of ethambutol may be to overcome the cell-wall permeability barrier and potentiate synergisticeffects with other drugs (15). Besidesthe perspectives of such combinations, quantitative evaluations of the degree of susceptibility of M. avium to rifabutin (16)and rifampin (17) singly indicated that a substantial 626

SUMMARY Minimal Inhibitory and bactericidal concentrations (MICs and MBCs) of rlfampln (RMP), rlfabutln (RBT), rltapentlne (RPT), CGP·7040,and P·DEA, were determined for 50 M. ar/um strains In 7H12liquid medium radiometrically under various pH conditions. Half were Isolated from patients with AIDS and the other half from patients without AIDS but with pulmonary disease. The MICs and MBCs were also determined In 7H12broth for M. tuberculosis strains. The MIC results obtained with M. tuberculos;s strains, and the serum peak levels In humans, were used as standards for Interpretation of the MICs and MBCs of the rlfamyclns tor M. ar;um. The bactericidal activity of all rlfamyclns tor M. arium was substantially lower than tor M. tuberculosis. The majority of M. ar/um strains was within the "susceptible" category, e.g., comparable to susceptible M. tuberculosis strains, when tested with CGP-7040 and RPT, and all of them were "moderately susceptible" when tested with P-DEA.On the basis of In rltro bacteriostatic and bactericidal activity, It seems that three agents, RPT, P-DEA, and CGP-7040 have more potential than do RMP and RBT against M. ar;um disease. AM REV RESPIR DIS 1990; 141:626-630

percentage of the strains were susceptible to the concentrations of these drugs equivalent to the highest MICs found for susceptible M. tuberculosis strains and to the peak serum concentrations. A quantitative comparison of in vitro activity of different rifamycins, based on their MICs and minimal bactericidal concentrations (MBCs), is the best available means for selecting the most promising drug of this class for chemotherapy of M. avium infection. The aims of this study were: (1)to compare the bactericidal potency of various conventional and experimental rifamycins against M. avium by determining the MBCs, (2)to compare the MICs of these drugs, and (3) to evaluate the MICs and MBCs for M. avium on the basis of comparison with the same values found for M. tuberculosis. Methods Antimicrobial Agents Eleven drugs were initially included in this study. Eight drugs were obtained from Merrell Dow Research Institute (Cincinnati, OH): rifapentine (RPT, also designated cyclopentylrifampin and MDL473); 25-desacetylmetabolite of RPT (25-D); rifampin analogs coded PR-3, PR-4, M-77, AF-DMI, P-DEA, and AF08. Rifabutin (RBT, ansamycin LM427) was obtained from Farmitalia Carlo Erba (Milan, Italy). CGP-7040 was obtained from CibaGeigy (Basel, Switzerland). Rifampin (RMP) was purchased from Sigma Chemical (St.

Louis, MO). A high concentration of each drug was made in DMSO, and working solutions weremade in bottled distilled water (Abbott Laboratories, North Chicago, IL). Aliquots of working solutions were stored at - 70 0 C until needed.

Test Strains Fifty M. avium strains, identified by the GenProbe method, were included in this study. Half of these strains were isolated from the blood of patients with acquired immune deficiency syndrome (AIDS), and half from the sputum of patients without AIDS but with pulmonary disease. Smooth transparent colonies were picked from 7HIO agar plates and subcultured in 7H9 broth. The choice of transparent type colonies was based on the wellknown fact that this colonial variant most closely resemblesthe organism in vivo, where-

(Received in original form May 8, 1989 and in revised form August 15, 1989) 1 From the National Jewish Center for Immunology and Respiratory Medicine, and the Departments of Microbiology and Immunology and of Medicine, University of Colorado Health Sciences Center, Denver, Colorado. 2 Supported by Public Health Service Contract l-AI-72636 with the National Institute of Allergy and Infectious Diseases and by the Kathryn and Gilbert Miller Fund for Research in Mycobacterial Disease. 3 Correspondence and requests for reprints should be addressed to Leonid Heifets, M.D., National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206.

627

RlFAMYClNS' MINIMAL INIHIBI10AY CONCENTRATIONS AGAINST II. AVIUII

as the opaque type colonies are generally con sidered to be a result of an in-laboratory transformation. To ensure that only transparent colonies were used in our experiments, a fresh- . frozen aliquot of the original 7H9 broth subculture, preserved at - 70 0 C, was used for each experiment. Drug-susceptible M. tuberculoslSstrains, H37Rv and 15strains isolated from patients prior to treatment, were also preserved in frozen samples.

MIC Determination The MIC was defined as the lowest concentration ofa drug that inhibited more than 990/0 of the bacterial population within an S-day period of cultivation in 7H12 broth inoculated with 10' to 10' cfu/ml, The MIC was determined radiometrically in the BACTEC 460TB system (Johnston Laboratories, Towson, MD) on the basis of comparison of growth in drug-containing vials with the growth in the drug-free vial that represents 1% of the bacterial population (1:100 control). The concentration of drug in the presence of which the daily increase in the radiometric Growth Index (GI) and final GI readings were lower than those in the 1:100 control was considered to have inhibited more than 99% of the bacterial population and was therefore defined as the MIC. Our previous reports (7, 16, IS) and the current study have justified this approach for both M. tuberculosis and M. avium, showing that the MICs determined by sampling and plating from the same vials were the same as those determined radiometrically. More technical details are given in these reports (7, 16, IS).

MBC Determination The MBC was defined as the lowest concentration of a drug that killed more than 99% ofthe bacterial population, 10' to 106 cfu/ml in the exponential phase, within a period of 15 days. We suggested previously (1S) that in the field of mycobacteriology this 99% killing criterion is more accurate and reproducible than the 99.9% most often used in other fields of clinical microbiology (19, 20). Duplicate 7Hl2 broth vials were inoculated in the same way as for a MIC determination, to produce an initial concentration of viable bacteria of 10' to 10' cfu/ml, and were allowed to incubate drug-free until growth wasin the exponential phase and the number of cfu/ml was 10' to 106 • Our previous studies (16, IS) showed that for M. avium this bacterial concentration was reflected in radiometric GI 20-S0, and for M. tuberculosiswhen the GI was approximately 500 (1S). This approach has also been validated in the current study with four M. tuberculosis and four M. avium strains listed in tables 3 and 4. The drugs were added to achieve 1, 2, 4, S, 16,32, and 64 times the previously determined MIC. Samples for determining the number of cfu/ml were taken after initial inoculation, when drugs were added, and from alternate vials at various other time points and diluted based on the GI reading, and then 0.5 ml of the dilution was

TABLE 1 MICs OF FIVE RIFAMYCINS FOR 16 M. TUBERCULOSIS STRAINS TESTED AT pH VALUES OF 6.8 AND 6.0 Number of Strains Inhibited at: pH 6.8

Concentration

(pglml) 0.015 0.03 0.06 0.12 0.25 0.5

pH 6.0

CGP

RBT

P-DEA

RPT

RMP

CGP

RBT

P·DEA

RPT

RMP

1 14 1 0 0 0

1 10 5 0 0 0

0 0 1

5

0 0 2

6

0 8 8 0 0 0

0 0 0 6

3

0 0 4 4

7 8 0

7 4 0 0 0

9 1 0 0 0

7 7 0

11 2 0 0 0

9 1

7 1

ate pH conditions as close as possible to those at the inflammation site. The MICs of these drugs for M. tuberculosis were in a narrow range, and there was little or no difference in MICs found at pH 6.8 and 6.0 (table 1). The highest MICs for M. tuberculosis, found in these observations with 16susceptible strains, were subsequently used as breakpoints for considering as tentatively "susceptible" the strains of M. avium for which the MICs were at this breakpoint or lower. The MICs for M. avium were tested with the 50 strains described above. The cumulative percentage distribution of Results these strains, by the degree of their susceptibility to four rifamycins, is shown Preliminary Screening The MICs of the 11 drugs were deter- in figure 1. Data on RBf are not included mined against 12 M. avium strains in in this figure because this drug produces 7H12 broth at pH 6.8 by employing sev- a much lower peak serum concentration en concentrations of each drug between than do the other drugs, and therefore 0.25 and 16.0 ug/rnl, On the basis of this a comparison by the MIC values in mipreliminary screening the following five crograms per milliliter would not be apdrugs were dropped from further studies propriate. Assuming that the other drugs because they were less active than the others: 25-D, PR-4, M-77, AF-DMI, and AF-08. Onemore drug, PR-3, which had lDO pH 5.0 been found to be very active in these 90 80 studies (see table 4 below), was also exo WI 70 cluded because ofits high toxicity, reportl: 60 e 50 ed to us later by the manufacturer. iii 4ll inoculated onto each of duplicate 7H11 agar plates. The inoculum was distributed by tilting the plates (not by use of a spreader); the plate surface was sufficiently dry to effect absorption in about 10min. A 10-3 dilution was used to reduce the concentration of drugs below the previously determined MIC, obviating the possibility of drug carry-over effect. Plates seeded with such samples, taken from vials containing concentrations subsequently found to be MBCs, showed no growth; therefore, the cfu/ml was calculated from the colony counts on plates inoculated with 10-1 dilutions. After 12 to 14 days of incubation at 37 0 C in a 5% CO 2 atmosphere, all plates were counted, and cfu/ml were calculated.

Minimal Inhibitory Concentrations MICs of the remaining five drugs, rifampin, rifabutin, rifapentine, CGP-7040, and P-DEA, were tested under various pH conditions: at pH 6.8 and 6.0 for M. tuberculosis strains, and at pH 6.8 and 5.0 for M. avium strains. A test at pH 5.0 with M. avium was designed to simulate the pH within macrophages, where a significant part of the in vivo bacterial population of these organisms may accumulate. M. tuberculosis does not grow sufficiently at pH lower than 6.0, and testing at pH 6.0 was conducted to ere-

~

30

~

'0

a

L-+---'f'::::..-F.:..;:---.--,----.---.-----,

c7l

..-n o---r' "

0100

;!. lID ~ 80

'ii

70

:>

60 50

~

U

I/ , ' I

II

30

0

"

(."

0

/ !' ,/ " .,1 ,l ... o ................

20 '0 ~O.03

0.01

0.12

0.25

0.5

1.0

2.0

• .0

~ 8.0

~g/ml

Fig. 1. Distribution of 50 M. avium strains (cumulative %) by the MICs of four rifamycins tested at pH values of 6.8 and of 5.0.

628

HEIFElS, LINDHOLM-LEVY, AND FLORY

TABLE 2 MICs (J1g1ml> OF FIVE RIFAMYCINS FOR 50 M. AVIUM STRAINS TESTED AT pH VALUES OF 6.8 AND OF 5.0 pH 6.8

Drug

MIC50 MICIO

CGP P-DEA RPT RMP RBT

0.06 0.11 0.58 1.0 0.32 0.82 1.15 4.75 0.17 -0.5

pH 5.0

Moderately Moderately Susceptible Susceptible Resistant Susceptible Susceptible Resistant MICso MICeo 98.0 6.0 82.0 14.0 38.0

2.0 94.0 18.0 74.0 32.0

0.05 0.45 0.42 1.3 1.13

0 0 0 12.0 30.0

can produce the same peak serum concentration (Cmax) in humans, a comparison by the MIC values presented in figure 1 indicates that at both the pH values the lowest MICs were found for CGP7040, the next most active were P-DEA and RPT, followed by RMP. The analysis of these data, including that on RBT, is presented in table 2. Because M. aviurn isolates usually have a wide range of susceptibility to most of the known antimycobacterial agents, we present in table 2 not only the calculated MICso and MIC 90 but also the percentage distribution of the tested strains, divided into three categories: "susceptible," "moderately susceptible," and "resistant." The assignment of a strain to the "susceptible" category is based on the breakpoint that is double the highest MIC found for drug-susceptible M. tuberculosis strains under the same experimental conditions (table 1):0.5 ug/ml for RMP and P-DEA, and 0.12 ug/ml for the three other drugs. The second breakpoint, designating the "moderately susceptible" category, is a concentration that is about half the lowest known Cmax achievable with the usual therapeutic doses of 600 mg for RMP and 300 mg for RBT. This second breakpoint was 4.0 ug/ml for RMP, RPT, and P-DEA, 2 ug/ml for CGP-7040, and 0.25 ug/ml for RBT. M. avium strains were designated "resistant" when the MIC was at or above Cmax. The results obtained

0.11 0.94 1.4 7.25 3.8

96.0 0 62.0 22.0 5.0

4.0 100.0 36.0 62.0 8.0

with the M. avium strains at pH 6.8 (table 2) indicate that almost all these strains were "susceptible" or "moderately susceptible" to three drugs: RPT, CGP-7040, and P-DEA. The highest percentage of "resistant" strains was found with RBT and RMP. The activity was not greatly affected in tests performed at pH 5.0, especially with RPT and CGP-7040, and the conclusion about the relative activity in these conditions remained the same: most active were RPT, CGP-7040, and P-DEA, followed by RBT and RMP (table 2 and figure 1).

Minimal Bactericidal Concentrations MBCs of the five most active rifamycins against drug-susceptible M. tuberculosis strains were substantially lower than the Cmax for these drugs, and the MBC/ MIC ratios were very low (table 3). This bactericidal activity of rifamycins, found in our experimental model using four M. tuberculosis strains, is in agreement with the numerous reports in which the bactericidal activity was tested in other media, mostly against only one strain (2-6). This fact provided a background for evaluation of the bactericidal activity of these drugs against M. avium. The MBCs of only two rifamycins, P-DEA and CGP-7040, were close to the peak serum level (table 4). The bactericidal activity of RMP, RBT, and RPT was somewhat poorer, showing higher MBCs.

TABLE 3 MBC AND MBC/MIC RATIOS OF RIFAMYCINS FOR M. TUBERCULOSIS MBCs (}lglml) and the MBC/MIC Ratios Rifampin

Rifabutin

0 0 2.0 16.0 87.0

Rifapentine

P-DEA

CGP-7040

Strain

MBC

Ratio

MBC

Ratio

MBC

Ratio

MBC

Ratio

MBC

Ratio

H37Rv 1620-4 131-4 3181-4

0.5 0.25 0.12 0.25

2 2 1 2

0.125 0.125 0.125 0.125

4 4 4 4

0.50 0.12 0.06 0.12

8 2 1 2

2.0 0.5 0.5 0.5

8 4 2 2

0.5 0.12 0.06 0.06

8 4 2 2

Discussion On the basis of comparison ofthe in vitro bactericidal activity of various rifamycins, and taking into account the MIC ranges found at different pH values, the drugs tested in this study can be placed in the following order by their activity against M. avium: CGP-7040, P-DEA, RPT, RBT, and RMP. This conclusion has been made by correlation of the results obtained with M. avium strains to the MICs found for susceptible M. tuberculosis strains, and by comparison of the MIC and MBC values with the Cmax. Our data on the activity of these drugs against M. tuberculosis, used in this study as one of the standards for evaluation of their activity against M. avium, correspond well with previous findings by other investigators. Rifapentine was reported to have inhibitory activity that was the same as or higher than that of RMP(21-23), and although it was more effective in mouse models (24, 25), it was reported to be less bactericidal than RMP (26). We found in experiments with M. tuberculosis that RPT had an even greater inhibitory activity and had bactericidal activity that was equal to or greater than that of RMP. A major advantage of RPT over RMP is its 4- to 5-fold longer half-life (24, 27, 28), which makes it more suitable for use in intermittent chemotherapy of tuberculosis (6, 25, 26). Another advantage of RPT over RMP, especially important for M. avium, is its accumulation within macrophages, reported to be about 60 times higher than in the extracellular fluid (29), whereas RMP accumulated only 5-fold (30). CGP-7040, a long-lasting rifamycin (31, 32) that has a half-life (30 to 40 h) almost 10times that of RMP and has almost the same Cmax in humans (5.6 versus 11.5 ug/ml), was reported to have the same inhibitory activity as RMP against M. tuberculosis H37Rv (31). The MICs of CGP-7040, determined in agar plates for 23 M. tuberculosis strains, were lower than the RMP MICs for the same strains (22). Our observations with 16 susceptible M. tuberculosis strains confirmed that CGP-7040 has greater in vitroinhibitory activity when the MICs are determined in 7H12 broth as well. In addition, we found that the MBCs of CGP-7040 were the same as or lower than the MBCs of RMP in experiments with four M. tuberculosis strains. Rifabutin (RBT) has been reported to be more active than RMP against M.

629

RIFAMYCINS' MINIMAL INIHIBITORY CONCENTRATIONS AGAINST II. AVIUII

TABLE 4 MBC AND MBC/MIC RATIO OF SIX RIFAMYCINS FOR M. AVIUM MBCs (pglml) and MBC/MIC Ratios Rifampin Strain At pH 6.8 211-5 3337-4 3350-4 9141 At pH 5.0 211-5 3337-4 3350-4 9141

MBC

Ratio

64.0 128.0 2.0 128.0

32 64 4 16

16.0 64.0 16.0 128.0

32 16 16 16

Rifabutin MBC

Ratio

Rifapentine MBC

16.0 16.0 256 >32.0 > 128 > 32.0 1.0 32.0 16 16.0 16.0 16 4.0 32.0 8.0 128.0

16.0 32 16 >32.0 16.0 8 128 32.0

tuberculosis (33, 34). Although MICs of RBT were indeed lower than MICs of RMP (22), the Cmax achievable in humans (lessthan 0.4IJ,g/ml) (16)is at least 10 times lower than that of RMP, although the ratio of Cmax to the MIC is about the same for both drugs. The MBC/MIC ratios, as we reported previously (7, 8), are also within the same range for both drugs. In conclusion, our data about bactericidal and inhibitory activity of RPT and CGP-7040 confirmed the superiority of these two drugs over RMP against M. tuberculosis. Moreover, we found that RBT is not more activethan RMP against these organisms. P-DEA was found to havethe same MICs as rifampin but higher MBCs in experiments with four M. tuberculosis strains. With regard to the activity of the rifamycins against M. avium, we found that only two drugs, P-DEA and CGP-7040, were bactericidal in concentrations close to Cmax, when compared with the other tested rifamycins. For M. avium the MBC/MIC ratios of these drugs were much higher than they werefor M. tuberculosis, which is in agreement with previous findings with regard to RBT (7-9) and CGP-7040 (35). It was also reported previously that the inhibitory activity of RPT and CGP-7040was greater than that of RMP for M. avium (22, 36). These findings were confirmed in our studies determining MICs in 7H12 broth. The MICs of RBT for M. avium were found by us and other investigators (16, 36, 37) to be much lowerthan the MICs of RMP, but taking into account the at least 10fold difference in the achievable serum concentrations, these data should not be considered evidencethat RBf is more active than RMP. In fact, the percentage of strains "susceptible" and "moderately

P-DEA

CGP-7040

Ratio

MBC

Ratio

128 > 128 128 64

8.0 8.0 4.0 8.0

16 8 8 16

> 16.0 >256 8.0 64 8.0 64 4.0 64

32 >64 32 64

16.0 8.0 8.0 4.0

32 8 16 8

> 16.0 > > 16.0 > 16.0 4.0

MBC

Ratio

256 128 256 64

PR-3 MBC

Ratio

16.0 32.0 8.0 32.0

16 16 4 16

8.0 > 16.0 4.0 8.0

256 >64 8 16

susceptible," as defined in this report, was about the same for both drugs. The percentage of "susceptible" and "moderately susceptible" M. avium strains was greatest for CGP-7040, followed by P-DEA and RPT, and then by RBT and RMP (figure 1), placing the tested drugs in about the same order as by their bactericidal potency. The fact that neither MICs nor MBCs have been adversely affected by lowering the pH to 5.0 might be a way to predict the activity of these drugs against mycobacteria multiplying within macrophages, where the intracellular pH is at least that low.The fact that high concentrations are achievable within macrophages (29, 30) suggests that some of the rifamycins will be even more active against the intracellular bacterial population. Such an assumption has to be confirmed in a macrophage model. Our statement that RPT, P-DEA, and CGP-7040are more promising against M. avium than are RBT and RMP will require appropriate clinical observations before the best choice among these drugs can be made. The MICs and MBCs of three drugs, RPT, P-DEA, and CGP7040, found in this study, suggest that at least one of them may be more effective than RMP and RBT for the treatment of M. avium disease. Acknowledgment The writers thank the companies that provided them with the experimental rifamycins, L. Landskroner for artwork, and C. J. Queen for the preparation of the manuscript. References 1. Pallanza R, Arioli v, Furesz S, Bolzoni G. Rifampicin: a new rifamycin. Fortschr Arzneimittelforsch 1967; 17:529-33. 2. Dickinson JM, Mitchison DA. Bactericidal activity in vitro and in the guinea pig of isoniazid, rifampicin and ethambutol. Thbercle 1976;57:251-8.

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