Spray Dried Aerosol Particles of Pyrazinoic Acid Salts for Tuberculosis Therapy. [Corrected].

Tuberculosis is the most serious infectious disease caused by a single organism, Mycobacterium tuberculosis (Mtb). The standard of care is a protracted and complex drug treatment regimen made more complicated and of longer duration by the incidence of multiple and extensively drug resistant disease. Pulmonary delivery of aerosols as a supplement to the existing regimen offers the advantage of delivering high local drug doses to the initial site of infection and most prominent organ system involved in disease. Pyrazinamide is used in combination with other drugs to treat tuberculosis. It is postulated that the action of pyrazinoic acid (POA), the active moiety of pyrazinamide, may be enhanced by local pH adjustment, when presented as a salt form. POA was prepared as leucine (POA-leu) and ammonium salts (POA-NH4), spray dried, and characterized in terms of physicochemical properties (melting point, crystallinity, moisture content), aerodynamic performance (aerodynamic particle size distribution, emitted dose), and in vitro inhibitory effect on two mycobacteria (Mtb and Mycobacterium bovis). Particles were prepared in sizes suitable for inhalation (3.3 and 5.4 µm mass median aerodynamic diameter and 61 and 40% of the aerodynamic particle size distribution less than 4.46 µm, as measured by inertial impaction, for POA-leu and POA-NH4, respectively) and with properties (stoichiometric 1:1 ratio of salt to drug, melting points at ∼180 °C, with water content of <1%) that would support further development as an inhaled dosage form. In addition, POA salts demonstrated greater potency in inhibiting mycobacterial growth compared with POA alone, which is promising for therapy.

Pharmacokinetic/pharmacodynamic parameters and the choice of high-dosage rifamycins.

SETTING: Clinical trials and the behaviour of bacterial persisters. OBJECTIVE: To explain why the efficacies of isoniazid (INH) and rifamycins during the treatment of tuberculosis (TB) are related not to the area under the curve (AUC)/minimum inhibitory concentration (MIC), but to peak drug concentrations. DESIGN: We examined the response in clinical trials with patients treated with INH alone and divided into slow and rapid acetylators of INH. RESULTS: The efficacy of INH is best related to peak concentrations, as repeated peaks can kill low-degree resistant mutants. A similar process might result in repeated peak concentrations of rifamycins killing low-tolerance persisters. CONCLUSIONS: If the efficacy of rifamycins is best related to peak concentrations, we can explain the discrepancy between mouse studies on daily rifapentine (RPT) and the failure to accelerate elimination of TB from sputum in the TBTC Study 29A, as daily RPT greatly increases the AUC but not the peak concentrations. High dosage rifampicin may be better able than RPT to cause high peaks.

Moxifloxacin and pyrazinamide susceptibility testing in a complex case of multidrug-resistant tuberculosis.

Multidrug-resistant tuberculosis (MDR-TB) is a public health problem of global concern. It is critical that drug susceptibility testing (DST) methods accurately predict clinical response. We present a patient with a challenging case of MDR-TB with additional resistance to quinolones and pyrazinamide. Treatment with a regimen including high-dosage moxifloxacin, based on additional genotypic and phenotypic DST, produced excellent results. This case highlights the possibility of treatment with high-dose fluoroquinolones despite apparent bacterial resistance to these agents. Improved DST methods are necessary for both agents. Development of genotypic approaches may offer a susceptibility profile rapidly, enabling early introduction of individualised treatments.

The near future: improving the activity of rifamycins and pyrazinamide.

While we wait for improved new anti-tuberculosis drugs, the main aim for improving current treatment should be to optimize the use of the two current drugs, rifampicin and the pro-drug pyrazinamide, which are responsible to a similar extent for the entire sterilizing activity of current therapy. The rifamycin activity could be improved by increasing the dose size of rifampicin or by daily dosing with long acting rifapentine. Increasing the dose size of pyrazinamide is limited by toxicity but an alternative approach is to use inhalation with pyrazinoic acid, as an adjunct to standard oral therapy. This would acidify pulmonary lesions, thus increasing the bactericidal activity of the orally administered pyrazinamide. Because pyrazinoic acid is the active moiety, it should also increase overall pyrazinamide activity and, because most resistance arises in the pncA gene that converts pyrazinamide to pyrazinoic acid, it should act on most pyrazinamide resistant strains. Inhalation technology allows delivery of drug to lesions rapidly and without first pass toxicity. The properties of drug containing microparticles and nanoparticles during inhalation and storage are reviewed. Spray-dried larger Trojan particles in which the smaller encapsulated particles can reside should be able to improve localisation within alveoli and avoid some storage problems.

Timing of relapse in short-course chemotherapy trials for tuberculosis.

We have reviewed the results from 15 tuberculosis treatment trials initiated by the British Medical Research Council between 1970 and 1983 in Africa and East Asia. Of 574 relapses, 447 (78%) occurred within 6 months of stopping treatment and 525 (91%) within 12 months. We suggest that investigators should consider terminating follow-up after the last enrolled patient completes 6 months following treatment, while continuing to follow patients enrolled earlier until that time. Compared to following all patients to 24 months, the total trial duration could be reduced by 18 months, with no increase in patient numbers in a non-inferiority design.

Bactericidal activity of moxifloxacin on exponential and stationary phase cultures of Mycobacterium tuberculosis.

The bactericidal activity of moxifloxacin, alone and in combination with isoniazid and rifampin, was studied on exponential and stationary phase cultures of Mycobacterium tuberculosis H37 Rv strain, the standard strain which is a wild type of M. tuberculosis strain, not exposed to any environment, susceptible to all anti-tuberculosis drugs. Moxifloxacin alone was highly bactericidal, being intermediate in activity between isoniazid and rifampin on both types of culture. The speed of activity was slow with the stationary phase culture, causing a reduction from 6.41 log(10)cfu/ml to 2.70 log(10)cfu/ml on day 6 with the higher moxifloxacin concentration of 4 microg/ml and to 4.08 log(10)cfu/ml with the lower concentration of 0.25 microg/ml. When added to isoniazid, its activity against both exponential and stationary phase cultures was increased. However, when it was added to rifampin, no increase in activity was found with either type of culture. Addition of moxifloxacin to isoniazid and rifampin resulted in a slight increase in activity against the exponential culture but a considerable increase against the stationary culture with counts below the limit of detection at 4 and 6 days with both moxifloxacin concentrations. The synergism found with isoniazid, but not with rifampin, supports the view that isoniazid should be included in combinations with moxifloxacin during the therapy of pulmonary tuberculosis.

A Phase II study of the sterilising activities of ofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis.

SETTING: Current treatment for pulmonary tuberculosis (TB) might be shortened by the incorporation of fluoroquinolones (FQs). OBJECTIVES: A Phase II study aimed to assess the sterilising activities of three novel regimens containing FQs before a Phase III trial of a 4-month regimen containing gatifloxacin (GFX). DESIGN: A total of 217 newly diagnosed smear-positive patients were randomly allocated to one of four regimens: isoniazid (INH), pyrazinamide and rifampicin (RMP) with either ethambutol, GFX, moxifloxacin (MFX) or ofloxacin (OFX) for 2 months. At the end of the study, RMP and INH were given for 4 months. The rates of elimination of Mycobacterium tuberculosis were compared in the regimens using non-linear mixed effects modelling of the serial sputum colony counts (SSCC) during the first 8 weeks. RESULTS: After adjustment for covariates, MFX substitution appeared superior during the early phase of a bi-exponential fall in colony counts, but significant and similar acceleration of bacillary elimination during the late phase occurred with both GFX and MFX (P = 0.002). Substitution of OFX had no effect. These findings were supported by estimates of time to conversion, using Cox regression, but there were no significant differences in proportions culture-negative at 8 weeks. CONCLUSIONS: GFX and MFX improve the sterilising activity of regimens and might shorten treatment; their progression into Phase III trials therefore seems warranted.

Comparison of the sterilising activities of the nitroimidazopyran PA-824 and moxifloxacin against persisting Mycobacterium tuberculosis.

OBJECTIVES: To measure the bactericidal activity of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis, strain H37Rv, in the three Hu/Coates models of bacterial persistence, in comparison with the activity of moxifloxacin (MXF), a drug shown to be likely to shorten treatment in current clinical trials. METHODS: The bactericidal activity of a wide range of PA-824 and MXF concentrations was tested against a 100-day static, starved, anaerobically-adapted culture in Model 1. In Models 2 and 3, rifampicin (RMP) 100 mg/ml was added to the 100-day culture for 5-7 days and bactericidal activities against surviving tolerant bacilli were tested by addition of the test drugs after removal of RMP in Model 2, and during exposure to RMP in Model 3. RESULTS AND DISCUSSION: PA-824 exhibited little bactericidal activity at low concentrations up to 1.25 microg/ml in each of these models, but high concentrations of >or=10 microg/ml showed considerable bactericidal activity, sufficient to kill all bacilli in Model 3, and appreciably greater than with MXF. However, as PA-824 is 94% plasma bound, concentrations of free drug sufficient to reach the zone of high bactericidal activity may not be obtained in cavities of pulmonary tuberculosis.

Clinical development of anti-tuberculosis drugs.

In the clinical development of new anti-tuberculosis drugs, the most important step is efficient Phase II studies to show whether the drug is likely to be able to shorten treatment and with what other drugs it has the greatest sterilizing activity. The use of non-linear mixed effects modelling applied to serial sputum cfu counts appears to be the most effective technique, but we know little about the optimal design of such novel studies. A paper in the current journal reports on the relative efficiencies of various timing patterns in sampling sputum.

Sterilising action of pyrazinamide in models of dormant and rifampicin-tolerant Mycobacterium tuberculosis.

SETTING: Pyrazinamide (PZA) is an effective sterilising drug in tuberculosis, but its mode of action is controversial. OBJECTIVE: To test the bactericidal activity of 1.56-100 microg/ml PZA in Hu/Coates models of dormant and rifampicin (RMP) tolerant Mycobacterium tuberculosis. METHODS: In model 1, bactericidal activity was tested in pH 5.5 medium against 4-day, 30-day or 100-day static, hypoxic cultures. In models 2 and 3, 100 microg/ml RMP was added to a 100-day culture and PZA was added either during incubation with RMP in model 3, or after resuspension in RMP-free medium in model 2. RESULTS: Model 1: cfu counts on the 100-day and 30-day cultures fell by a maximum of about 1.6 log cfu/ml with increasing culture age, PZA concentration and incubation period, while counts on the 4-day culture showed little change. Model 2: cfu counts at the end of 7 days of recovery showed little bactericidal activity. Model 3: viable bacilli were almost completely eliminated. Bactericidal activity in these models increased with decreasing metabolic bactericidal activity, as measured by the uptake of [3H] uridine into bacterial RNA. CONCLUSION: PZA differs from other anti-tuberculosis drugs in showing greater bactericidal activity the slower the bacillary metabolic activity, hence its great value as a sterilising drug, likely to remain as an effective companion drug with newer sterilising drugs.

Moxifloxacin and gatifloxacin in an acid model of persistent Mycobacterium tuberculosis.

Studies in the mouse and in humans suggest that use of moxifloxacin and gatifloxacin may shorten the duration of treatment of pulmonary tuberculosis. We describe here the in vitro findings with gatifloxacin and moxifloxacin in regimens similar to those that might be used in the treatment of tuberculosis. The bactericidal activities of moxifloxacin and gatifloxacin were measured alone and in different combinations with isoniazid, rifampicin and pyrazinamide against a 30-day, stationary phase culture, at a pH of 5.9. There was a rapid, irregular fall in colony counts during the first 4 days followed by a slower consistent kill during days 4-21 with a mean kill of -0.36 (SD=2.74) and -0.106 (SD=0.011) log(10)CFU/ml/day, respectively. The 4-21-day kill is considered the best assessment of bactericidal activity against persisting bacilli that prolong treatment. The substitution of either of the quinolones for isoniazid in the control regimen of rifampicin, pyrazinamide and isoniazid did not increase bactericidal activity with log CFU of 5.00 and 4.88, but did result in increased bactericidal action with the log CFU of 4.11 and 4.10 for moxifloxacin and gatifloxacin respectively. Moxifloxacin and gatifloxacin had closely similar activities in all drug combinations. Adding moxifloxacin or gatifloxacin to the control regimen resulted in a significant increase in bactericidal action, considered sufficient to reduce the treatment duration.

Drug resistance in tuberculosis.

A drug-resistant strain of Mycobacterium tuberculosis is defined as one differing from the tight distribution of wild strains that have not come into contact with the drug concerned. Sensitivity tests are performed by the absolute concentration method, the resistance ratio method or the proportion method. The hypothesis underlying the proportion method is that there are appreciable differences in inoculum size so that there should be an association between the proportion on drug-free medium and the proportion on drug-containing medium. This hypothesis was not supported by a study on ethionamide-resistant strains. It indicated that variation in the proportion on drug-free medium was due to clumping of the bacilli in the inoculum rather than to differences in the number of bacilli. Hence, the use of the proportion method introduces errors in susceptibility testing. While the method can produce reliable results, it is more time consuming than a minimal inhibitory concentration determination, and should not be adopted as a standard method.

Bactericidal action of gatifloxacin, rifampin, and isoniazid on logarithmic- and stationary-phase cultures of Mycobacterium tuberculosis.

The bactericidal activity of gatifloxacin, alone and in combination with isoniazid and rifampin, was studied on both exponential- and stationary-phase cultures of Mycobacterium tuberculosis strain H37Rv. On log-phase cultures, the bactericidal activity of gatifloxacin at 4 microg/ml was rapid and was very similar to that of isoniazid. At concentrations of 0.25 and 4 microg/ml, gatifloxacin enhanced the activity of isoniazid. Killing of the stationary-phase culture was biphasic. During the first 2 days, gatifloxacin at 4 microg/ml slightly increased the limited bactericidal activities of isoniazid and rifampin. However, no further additional bactericidal activity was found during further incubation with isoniazid alone or when gatifloxacin was added to either isoniazid or rifampin. This suggested that the stationary-phase culture contained a mixture of occasionally dividing bacilli that were killed during the first 2 days and true static persisters in the residual population that mimicked those in human lesions. In view of the failure of gatifloxacin to add to the sterilizing activity of isoniazid or rifampin during days 2 to 6 of exposure in the stationary-phase culture, it is unlikely to be a sterilizing drug that can be used to shorten the duration of treatment appreciably when it is added to present treatment regimens.

Split-drug regimens for the treatment of patients with sputum smear-positive pulmonary tuberculosis--a unique approach.

OBJECTIVE: To evaluate the efficacy of split-drug regimens for treatment of patients with sputum smear-positive pulmonary tuberculosis in south India. DESIGN: Randomized controlled clinical trial where eligible patients were randomly allocated to: (i) 2RE(3)HZ(3)(alt)/4RH(2) (split I): rifampicin plus ethambutol given on one day and isoniazid plus pyrazinamide the next day for first 2 months followed by rifampicin plus isoniazid twice weekly for 4 months, or (ii) 3RE(3)HZ(3)(alt)/3RH(2) (split II): similar to regimen 1, except duration was 3 months in each phase, or (iii) 2REHZ(3)/4RH(2) (control): rifampicin, isoniazid, ethambutol and pyrazinamide, given thrice weekly for 2 months followed by isoniazid and rifampicin twice weekly for 4 months. All patients were followed up clinically and bacteriologically every month up to 2 years and every 6 months for up to 5 years. RESULTS: A favourable response (cultures negative for Mycobacterium tuberculosis during the last 2 months of treatment) was observed in 91% of 407 patients in split I, 94% of 415 in split II and 89% of 418 in the control regimen. Ninety-one per cent of 370 patients in split I, 93% of 389 in split II and 90% of 370 in control regimens had quiescent disease at the end of 60 months. Gastrointestinal symptoms were more frequent under the control regimen (P = 0.01). CONCLUSION: Split-drug regimens were as effective as the control regimen in terms of favourable response at the end of treatment and quiescent disease at 5 years, and caused fewer gastrointestinal side-effects.

The early bactericidal activity of streptomycin.

SETTING: Patients with sputum smear-positive, newly diagnosed pulmonary tuberculosis studied at Tygerburg Hospital, Cape Town, for their early response to streptomycin (SM). OBJECTIVE: To determine the standard early bactericidal activity (EBA), namely the fall in viable counts of tubercle bacilli in 16-hour sputum collections during the first 2 days of treatment with SM. DESIGN: Patients were randomised to logarithmically spaced daily doses of 7.5, 15 or 30 mg/kg SM. A comparison by standard biological assay methods was then made with previous estimations of the EBA of paromomycin in doses of 7.5 and 15 mg/kg. RESULTS: An EBA of 0.133 obtained with 30 mg/kg SM differed significantly from zero (P = 0.0009), while the EBAs of 0.043 with 15 mg/kg and -0.025 with 7.5 mg/kg did not so differ. A linear regression equation of EBA = -0.2587 + 0.2627 log10 dose was obtained with significant slope (P = 0.007). Paromomycin was estimated to be 1.745 more potent than SM with wide 95% confidence limits (0.6-28.6), indicating that it cannot be considered more potent than SM. CONCLUSIONS: The low EBAs show that SM has low, dose-related, bactericidal activity in cavities, consistent with results from clinical trials. If streptomycin-resistant bacilli are present, paromomycin is probably the aminoglycoside of choice.