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.

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.

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.

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.

The use of murine monoclonal antibodies without purification of antigen in the serodiagnosis of tuberculosis.

A serum diagnostic test for tuberculosis has been devised on the basis of competitive inhibition by human sera of the binding of 125I-labelled murine monoclonal antibodies (Mabs) to a solid-phase bound pressate of M. tuberculosis. Five monoclonal antibodies binding to distinct antigenic determinants of the organism were used as structural probes which conferred their stringent combining site specificities to the polyclonal mixture of human antibodies. Sera from patients but not from healthy controls competed effectively with the binding of 125I-labelled Mabs to M. tuberculosis-coated polyvinyl plates. This inhibition technique eliminated the need for elaborate purification of antigen used in previous serological methods. Some Mabs gave considerably more positive results than others. The best combination of tests used 2 Mabs and yielded a positive result in 71% of 41 patients with smear-positive pulmonary tuberculosis. This approach is applicable in principle to the serodiagnosis of other human bacterial diseases.

A computer-assisted bacteriology reporting and information system.

A computer system for reporting and recording all specimens processed by the routine bacteriology laboratory at the Royal Postgraduate Medical School is described. Features of interest are the method of input using a mixture of 3-character alphanumeric codes and numbers, cumulative reporting to the wards, and selective listing of relevant previous results for the patient, which is available to technical and supervisory staff during processing of the specimen. The relative value to the wards and the laboratory of each type of information transfer has been assessed. Overall the use of a computer has resulted in higher quality bench work and more accurate reporting. It seems little more expensive than a previous manual system, although it has transferred work from the technical to the clerical staff.

Use of the Phoenix system for bacteriology.

A dedicated microbiology data processing system with remote batched job entry to an obsolete computer, has been superseded by the inclusion of bacteriology in an on-line interactive clinical pathology system which had previously incorporated chemical pathology and haematology. The original Phoenix system has been adapted to allow for the entry of bacteriology data using mnemonic codes and to deal with the problems caused by the longer processing time of bacteriology specimens. Particular advantages of the new system include the immediate linkage of all specimens for each patient and an easy recall and display of results in the laboratories and on the wards.

Selective Kirchner medium in the culture of specimens other than sputum for mycobacteria.

Over a two-year period, 2949 non-sputum specimens were cultured on two slopes of Löwenstein-Jensen (LJ) medium, two bottles of Kirchner liquid medium, made selective by adding polymyxin B, carbenicillin, trimethoprim and amphotericin B, and a selective 7H11 agar slope. Pus and CSF were inoculated into this set without prior treatment, but other specimens were decontaminated with sulphuric acid. Tissues and fluids were also inoculated without decontamination into additional selective media. The use of the selective media as well as the LJ slopes increased the yield of specimens with cultures of tubercle bacilli from 34 to 53 and decreased specimens with all media contaminated from 34 to 2. Results almost as good, 52 positive specimens and five totally contaminated, would have been obtained by the addition of a single selective Kirchner bottle to the two LJ slopes, and this is recommended for routine use.

Selective media in the isolation of tubercle bacilli from tissues.

Direct culture on Lowenstein-Jensen slopes and on three media made selective for tubercle bacilli by the addition of four antibacterial agents was compared with guinea-pig inoculation on 490 tissue specimens. Tubercle bacilli were obtained from 15 specimens by culture and 14 by guinea-pig inoculation; only one specimen was positive by guinea-pig and not by culture. The most efficient culture medium was a selective 7H11 slope. Routine guinea-pig inoculation has been replaced by a wider range of culture procedures.

Assay of rifampicin in serum.

Two methods for the assay of rifampicin in serum are described. The first is a conventional plate diffusion method, measuring concentrations down to 0.02 mug/ml, and the second a chemical extraction followed by measurement of the inhibition of uptake of (14)C-uridine by Staphylococcus aureus, which estimates in the range of 0.02 to 0.001 mug/ml. The methods were used to measure serum concentrations in man following doses of about 1050 mg and 75 mg rifampicin.

Examination of operation specimens from patients with spinal tuberculosis for tubercle bacilli.

Operation specimens from 52 Hong Kong patients considered to have tuberculosis of the spine were sent at -78 degrees C to London where they were cultured on two Löwenstein-Jensen medium slopes, a slope of 7H11 medium made selective with antibiotics and in two bottles of selective liquid Kirchner medium. Cultures of M tuberculosis, usually with only scanty colonies, were obtained from 37 of the patients. Pus and caseous matter yielded more positive cultures and more numerous colonies than other specimens, but eight of the 37 patients yielded positive cultures only from tissues lining abscess walls or from bone or intervertebral disc specimens. Cultures in Kirchner medium were never contaminated and twice as many were positive as cultures on the slopes; nine patients had cultures positive only on Kirchner medium. It is recommended that (i) a variety of operation specimens, always including pus or caseous material in volumes of at least 1 ml, should be sent for bacteriology; (ii) specimens should be cultured in selective Kirchner medium, with or without Löwenstein-Jensen slopes; (iii) cultures should be incubated for a minimum of 8-9 wk.

Comparison of machine and manual staining of direct smears for acid-fast bacilli by fluorescence microscopy.

Comparisons were made in Lusaka and in London between manual staining and staining in an automatic machine with auramine-phenol of direct smears of sputum and other types of specimen for acid-fast bacilli. No evidence was obtained of carry-over of acid-fast bacilli from positive to negative smears during machine staining. There was improved contrast between bacilli and the background in smears prepared with the machine.

Protein synthesis is shutdown in dormant Mycobacterium tuberculosis and is reversed by oxygen or heat shock.

Oxygen-limiting conditions are critical to the survival of the bacteria in tuberculosis. Mycobacterium tuberculosis can survive anaerobiosis in vitro for long periods of time only after a gradual transition to a microaerophilic stationary phase. The underlying mechanism behind stationary phase adaption needs to be elucidated. The protein profiles of Mycobacterium tuberculosis during long-term stationary phase growth and under strict anaerobic incubation were monitored by [35S]methionine labelling, SDS-PAGE and fluorography. These experiments have established that protein synthesis gradually decreased over 50 days in the long-term stationary phase cultures which were considered to be microaerophilic. There was an 80% linear decrease in the level of total protein synthesis during the first 40 days of microaerophilic growth and then the rate of protein synthesis faded quickly. For the first time we have shown that total protein synthesis shutdown occurred when bacilli were incubated under further anaerobic conditions. Viability, estimated by cfu counts, remained constant during stationary phase growth and under anaerobic incubation. Furthermore, when oxygen was introduced into the anaerobic culture, protein synthesis restarted. Also heat shock at 45 degrees C, 48 degrees C and 50 degrees C rapidly induced protein synthesis in stationary and anaerobic cultures. These data indicate that dormant bacteria shut down protein synthesis but remain responsive to specific stimuli which restore protein synthesis. In addition the dormant bacilli induced by anaerobiosis developed more heat resistance than nondormant organisms.

Role of individual drugs in the chemotherapy of tuberculosis.

During the course of chemotherapy, certain drugs are predominant in their bactericidal activities. Isoniazid is responsible for an initial kill of about 95% of organisms during the first 2 days of treatment. Its bactericidal role is then replaced by rifampicin and pyrazinamide during the intensive phase. In the continuation phase with an isoniazid/rifampicin regimen, rifampicin is the only effective drug against persisters, as shown by the similarity of response by patients with initially isoniazid-resistant or sensitive strains. If the continuation phase regimen does not contain rifampicin but does contain isoniazid, the dominant bactericidal drug is isoniazid. In this case, the response of patients with initial isoniazid resistance is appreciably less good than in those with sensitive organisms. The review suggests exploration in randomised control trials of a continuation phase of rifampicin (or rifapentine) alone. It also suggests the importance of the dose size of rifampicin and the need for exploring a higher dose. Finally, it emphasises the importance of finding drugs that act on persisting organisms that are phenotypically but not genetically resistant to rifampicin.

How drug resistance emerges as a result of poor compliance during short course chemotherapy for tuberculosis.

OBJECTIVE: To explore mechanisms by which drug resistance might arise as a result of poor compliance during short course chemotherapy. DESIGN: Four theoretical mechanisms are first described. RESULTS: Examples of the way the mechanisms probably operate are taken from: 1) a study of once-weekly chemotherapy with streptomycin and isoniazid, and 2) the pattern of drug susceptibility in cultures from patients who relapsed after the end of treatment. CONCLUSION: Good compliance is vitally important. The value of a fourth drug in the initial phase of chemotherapy in preventing resistance is questioned. An explanation for mono-resistance to rifampicin in patients with the acquired immune deficiency syndrome (AIDS) is suggested.

Bioavailability of Chinese rifapentine during a clinical trial in Hong Kong.

SETTING: A clinical trial of rifapentine in Hong Kong. OBJECTIVE: Assessment of the bioavailability of the Chinese rifapentine used in the trial. DESIGN: The content of rifapentine in serum samples taken from 287 patients during the administration of four batches of the drug was measured by microbiological assay. RESULTS: An initial comparison of areas under curve obtained in a random allocation to 40 patients of rifapentine either of Western or Chinese origin indicated that the bioavailability of the Chinese drug was 74% of the Western drug. The bioavailability of the second batch was found to be about 66% of the Western drug. The dose of the last two batches of rifapentine was therefore increased from the planned 600 mg to 750 mg, or briefly to 900 mg; serum concentrations were then similar to those obtained with the Western drug. Bioavailability did not change during the use of each drug batch. CONCLUSION: A comparison of the results obtained in the trial with the initial two batches and the final batches will estimate the effects of rifapentine dose size on its efficacy and toxicity.