Establishment of a reference formulation for bioequivalence assessment of rifampicin-containing FDCs: an essential step towards improving tuberculosis treatment.

SETTING: Selection of a reference product for bioequivalence studies of rifampicin (RMP) in prequalifying fixed-dose combinations (FDC) for worldwide distribution through the WHO is critical. OBJECTIVE: To investigate the feasibility of establishing FDC formulations as reference products for bioequivalence studies of RMP in prequalification programmes. DESIGN: A biostudy was conducted as an open, two-period randomised cross-over trial. Two three-drug FDCs containing RMP, isoniazid and ethambutol hydrochloride were administered to a group of 22 volunteers with a wash-out period of 1 week. Plasma samples were collected and analysed for the concentration of RMP and desacetyl-RMP, a major active metabolite of RMP, up to 24 h. Pharmacokinetic parameters of RMP were calculated: Cmax, AUC0-24, Tmax, kel and absorption efficiencies. RESULTS: No significant difference was observed between the administered formulations with respect to the major pharmacokinetic parameters Cmax, Tmax and AUC0-24 when evaluated by parametric (two-way ANOVA) and non-parametric (Hauschke's analysis) statistical analysis. The concentration of RMP falls within the reported acceptable therapeutic range. CONCLUSION: FDCs can be developed as a reference product for bioequivalence studies.     

Determination of rifampicin bioequivalence in a three-drug FDC by WHO and indian protocols: effect of sampling schedule and size.

SETTING: To promote the quality assurance of fixed-dose combination (FDC) formulations, the World Health Organization (WHO) has prepared a convenient simplified protocol for the determination of rifampicin (RMP) bioequivalence. During the development of this protocol, it was proved that sampling time up to 8 h can determine the rate and extent of RMP absorption. However, this protocol utilises 20 volunteers in contrast to other local regulatory requirements of a minimum of 12 volunteers. The different sample sizes utilised in these protocols may affect the sensitivity of the bioequivalence outcome. OBJECTIVE: To determine the effect of sampling size and schedule on RMP bioequivalence when two different protocols are used. DESIGN: A bioequivalence trial was conducted with a study design of 20 volunteers and 24 h sampling time, which fulfils the requirements of both the WHO and Indian regulatory protocols. Pharmacokinetic and statistical analysis was done by stepwise reduction in sample size and schedule. RESULT: Bioequivalence limits of RMP were unaffected by a reduced sample size of 12 volunteers and 8 h sampling time. CONCLUSION: Minimising sample size after validation for borderline and poor quality FDC formulations can further reduce the cost of conducting bioequivalence trials.     

Minimum sample size and sampling time requirements for assessment of rifampicin bioequivalence from FDC formulations.

SETTING: The WHO- and IUATLD-recommended protocol for rifampicin (RMP) bioequivalence utilises 20-22 volunteers and 8 h, whereas the requirement of other regulatory authorities is 12 volunteers with a 24 h sampling schedule. Differing sampling size and time requirements may change the outcome of RMP bioequivalence. OBJECTIVE: To determine the minimal sample size and time required to assess RMP bioequivalence from FDC formulations. DESIGN: Bioequivalence studies were conducted that fulfilled the criteria of the WHO and Indian regulatory protocols. From earlier studies, retrospective pharmacokinetic evaluation, power of the test and bioequivalence limits were also calculated using 8-22 volunteers and sampling points of 8-24 h. Pharmacokinetic and statistical evaluations from three representative studies showing low, moderate and high intra-subject variability are given to determine minimum requirements for RMP bioequivalence. RESULT: It was found that a sampling schedule up to 8 h was sufficient to compare the absorption process of RMP. There was no influence of reduced sample size on bioequivalence estimates of RMP that showed low or moderate variability. However, in a study showing higher variation, a sample size of 14-16 subjects was found to be optimal. CONCLUSION: It is possible to reduce the sample size requirement for determination of RMP bioequivalence using the WHO protocol.     

Quality control of anti-tuberculosis FDC formulations in the global market: part II-accelerated stability studies.

OBJECTIVE: To determine the quality and performance of rifampicin (RMP) containing fixed-dose combination (FDC) formulations of anti-tuberculosis drugs sourced from the international market with respect to physical, chemical and dissolution properties after storage at accelerated stability conditions (40 degrees C/75% relative humidity) and to identify appropriate storage specifications. METHODS: Formulations across different companies and combinations were subjected to 6-month accelerated stability testing in packaging conditions recommended by the manufacturer. Various pharmacopeial and nonpharmacopeial tests for tablets were performed for 3- and 6-month samples. RESULTS: All the formulations were found to be stable, where extent of dissolution was within +/- 10% of that of the initial value, and all formulations passed the pharmacopeial limits for assay and content uniformity of 90-110% and +/- 15% of average drug content, respectively. CONCLUSIONS: Good quality RMP-containing FDCs that remain stable after 6-month accelerated stability testing are available in the marketplace.     

Substandard tuberculosis drugs on the global market and their simple detection.

SETTING: The prevalence of substandard anti-tuberculosis drugs is unknown. To maximize the effectiveness of tuberculosis (TB) control efforts, simple, inexpensive drug quality screening methods are needed. DESIGN: Isoniazid (INH) and rifampin (RMP) single- and fixed-dose combination (FDC) formulations were collected from selected TB programs and pharmacies in Colombia, Estonia, India, Latvia, Russia and Vietnam. Samples were screened using a recently developed thin-layer chromatography (TLC) kit. All abnormal samples and a 40% random sample of normal formulations were further analyzed using confirmatory techniques. Samples outside of 85% to 115% of stated content, and/or containing compounds other than the stated drug, were defined as being substandard. RESULTS: Overall, 10% (4/40) of all samples, including 13% (4/30) RMP samples, contained <85% of stated content. More FDCs (5/24, 21%) than single-drug samples (2/16, 13%) were substandard. A comparison of TLC with the confirmatory analysis for RMP analysis showed a sensitivity of 100% (4/4), a specificity of 92% (24/26), a positive predictive value (PPV) of 67% (4/6), and a negative predictive value (NPV) of 100% (24/24). An analysis of INH showed a specificity of 90% (9/10). However, sensitivity, PPV, and NVP could not be determined. CONCLUSION: A substantial number of anti-tuberculosis drugs from several countries, in particular FDCs, were found to be substandard. Such drugs may contribute to the creation of drug-resistant TB. TLC is an effective, convenient, and inexpensive method for the detection of substandard drugs.     

Plasma pooling: utility in expediting bioequivalence assessment of rifampicin-containing fixed-dose combinations.

To improve the rapidity of the registration process of rifampicin (RMP) containing fixed-dose combination (FDC) formulations, a plasma pooling methodology was used to increase the throughput of bioanalysis of plasma samples from bioequivalence trials of FDCs. Plasma samples of a biostudy were analysed for RMP using traditional analysis methods as well as a plasma pooling method (volunteer and time pooling). Both methods produced similar results, with less than 15% variability in both volunteer and time pooling. The plasma pooling method for bioanalysis was validated. Further studies are required to identify and reduce the percentage variability.     

Regional gastrointestinal permeability of rifampicin and isoniazid (alone and their combination) in the rat.

OBJECTIVE: To determine if rifampicin (RMP) and isoniazid (INH) are absorbed from different gastrointestinal tract (GIT) sites, and to ascertain the feasibility of producing new fixed-dose combination (FDC) products containing RMP and INH by segregating drug delivery at different sites along the GIT. DESIGN: Permeability of RMP and INH (alone and in combination) was determined in various segments of rat GIT (stomach, duodenum, jejunum and ileum) at concentrations of respectively 2.4 and 1.2 mg/ml, using a ligated loop technique. Drug analysis was performed by HPLC. Extent of absorption was considered as the total drug disappearing from the loop. Permeability was correlated with solubility and decomposition data at pH corresponding to different GIT sites. RESULTS: RMP was well absorbed from the stomach due to its solubility, which was maximum between pH 1-2. INH was poorly absorbed from the stomach, but was well absorbed from all three segments of the intestine. In combination, RMP disappearance was enhanced in the presence of INH in the stomach and jejunum, but INH disappearance was not influenced by RMP. CONCLUSION: The study shows higher in situ RMP disappearance in the presence of INH, attributable to drug degradation due to catalysis by INH. As the two drugs show regional specific permeability, FDCs without reduced RMP bioavailability resulting from its decomposition in the presence of INH can be designed by segregating delivery of the two drugs by around 3-4 h. RMP should be released in the stomach and INH in the intestine.     

Rifampicin mono-resistant Mycobacterium tuberculosis in Bujumbura, Burundi: results of a drug resistance survey.

SETTING: Bujumbura, Burundi. OBJECTIVES: To determine resistance levels of Mycobacterium tuberculosis (TB) to the main anti-tuberculosis drugs after 11 years of a DOTS programme using a WHO-recommended partially intermittent 6-month rifampicin (RMP) first-line regimen and fixed-dose drug combinations (FDCs). DESIGN: Drug susceptibility testing of systematic samples of M. tuberculosis isolated from newly registered sputum smear-positive cases in the capital during a 15-month period (2002-2003). RESULTS: Of 496 strains from new cases, 16.1% showed resistance to any drug, 6.3% to isoniazid (INH), 2.0% to RMP (1.4% multidrug-resistant TB [MDR-TB]), 13.3% to streptomycin and 1.6% to ethambutol. Among 69 strains from previously treated cases, the prevalence of resistance was 30%, 19%, 15% (12% MDR-TB strains), 25% and 6%, respectively. CONCLUSION: Levels of drug resistance in Bujumbura are higher than average for Africa, despite long-term use of the DOTS strategy with FDCs and a ban on sales of TB drugs. Most worrying is the appearance of MDR-TB and RMP-resistant, INH-susceptible strains in new cases. Although a survey cannot prove that high HIV prevalence, elevated levels of resistance to some other drugs and irregular intake allowed acquisition of drug resistance, the effectiveness and safety of 6-month regimens with (partially) intermittent RMP throughout under such conditions should be investigated.     

The physical and chemical stability of anti-tuberculosis fixed-dose combination products under accelerated climatic conditions.

OBJECTIVE: To determine the physical and chemical stability of anti-tuberculosis fixed-dose combinations (FDC) of rifampicin (RMP), isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) sold on the Indian market. METHODS: The products were stored for 3 months under ICH/WHO accelerated conditions (40 degrees C / 75% RH), with and without the original packaging in the presence and absence of light. RESULTS: The initial RMP, INH and PZA content was found to be within the range of 90-110% of the label claim. However, the products were found to have some chemical instability even initially; one of the tablets also showed physical instability. Under accelerated conditions, the unpackaged products underwent severe changes, whereas both physical and chemical changes were also observed in the packaged formulations. The physical changes were stronger under lighted conditions. A significant finding is that PZA and perhaps EMB may play a catalytic role in the interaction between INH and RMP. CONCLUSION: This study suggests that, unless they are packed in barrier packaging, anti-tuberculosis FDC formulations should be considered unstable, and due consideration should be given to their development pharmaceutics, packaging and stability testing.     

Evaluation of rifampicin bioequivalence in fixed-dose combinations using the WHO/IUATLD recommended protocol.

For an accurate assessment of rifampicin bioequivalence from fixed-dose combinations (FDCs), and to reduce the time and cost constraints associated with bioequivalence studies, the World Health Organization and the International Union Against Tuberculosis and Lung Disease have developed a simplified screening protocol. This study was undertaken with the objective of testing the applicability of this protocol for all types of FDCs. Data were obtained for volunteers common to three studies, and pharmacokinetic parameters were evaluated by different statistical tests. From the results, it has been demonstrated that the simplified screening protocol is suitable for evaluating the bioequivalence of rifampicin in all the types of FDCs available on the market.     

Pattern of mycobacterial resistance to four anti-tuberculosis drugs in pulmonary tuberculosis patients in the state of Qatar after the implementation of DOTS and a limited expatriate screening programme.

OBJECTIVE: To determine the resistance pattern of Mycobacterium tuberculosis to four anti-tuberculosis drugs in pulmonary tuberculosis patients in the State of Qatar after the implementation of DOTS and an expatriate screening programme on arrival. METHOD: A state-wide, population-based, retrospective analysis of all cases of pulmonary tuberculosis with positive M. tuberculosis culture reported to the Division of Public Health TB Unit from January 1996 to December 1998. M. tuberculosis sensitivity testing was done by the Bactec method for isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB). The results were interpreted as a daily change of the growth index of test vials (with drug) compared with controls. RESULTS: There were 406 isolates with positive M. tuberculosis culture. Sixty-one (15%) were resistant to one or more of the four anti-tuberculosis drugs, of which 58 (95%) were from newly diagnosed cases (primary) and three (5%) were from previously treated cases (acquired). Primary resistance was as follows: any resistance 15%, INH 12.4%, RMP 2%, SM 5.2%, EMB 0.8% and multidrug resistance (MDR, resistance to INH and RMP at least) was found in 0.8%. Acquired resistance was as follows: any resistance 15%, INH 15%, RMP 5%, SM 5% and MDR 5%. CONCLUSION: The prevalence of resistance to four anti-tuberculosis drugs is strikingly low due to the limited expatriate screening programme (chest radiography) and implementation of DOTS. The four-drug regimen is recommended for the initial phase of therapy until the results of sensitivity testing are known.     

Low serum concentrations of anti-tuberculosis drugs and determinants of their serum levels.

SETTING: Low serum concentrations of anti-tuberculosis drugs have occasionally been associated with treatment failure. OBJECTIVE: To determine the prevalence of low serum concentrations of anti-tuberculosis drugs and to identify the determinants of drug concentrations. DESIGN: Venous blood was obtained 2 h after drug ingestion, and serum levels of isoniazid (INH), rifampicin (RMP), ethambutol (EMB), pyrazinamide (PZA), acetyl INH and 25-desacetyl RMP were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Patients with human immunodeficiency virus co-infection and gastrointestinal disease or diarrhoea were excluded. RESULTS: Among 69 enrolled TB patients, the prevalence of a low 2 h serum concentration of at least one anti-tuberculosis drug was 46.4%. Prevalences of a low concentration of INH, RMP, EMB or PZA were 15.2%, 23.5%, 22.4% and 4.5%, respectively. By multivariate linear regression analysis, the serum concentrations of INH, RMP and PZA were positively associated with dose per kg of body weight (P < 0.05). Moreover, INH concentration was associated with acetyl INH/INH ratio (beta = -8.588, P < 0.001) and EMB concentration was associated with calculated creatinine clearance (beta = -0.025, P < 0.001). CONCLUSION: Low concentrations of anti-tuberculosis drugs are common, and although the clinical significance of low concentrations remains uncertain, it may be necessary to optimise drug doses by therapeutic drug monitoring, especially in patients with an inadequate clinical response to chemotherapy.     

Fixed-dose combination chemotherapy (Rifater/Rifinah) for active pulmonary tuberculosis in Taiwan: a two-year follow-up.

SETTING: Veterans General Hospital-Taipei, Taiwan. OBJECTIVE: To assess the efficacy and safety of a fixed-dose combination (FDC) of Rifater (RFT)/Rifinah (RFN) in the treatment of newly diagnosed smear-positive pulmonary tuberculosis. DESIGN: Patients were randomly assigned to two 6-month short-course chemotherapy regimens. One group of patients was treated with FDCs and another was given the four component drugs (INH, RMP, EMB and PZA) as separate formulations. RESULTS: The 105 patients enrolled in the study were divided into two treatment groups. Fifty-one patients who had completed treatment without interruption, 26 in the FDC group and 25 in the separate regimen, were eligible for analysis at the end of 2 years. Among the patients with a drug susceptibility test result available, four in the FDC group had bacilli resistant to pyrazinamide. In the separate regimen group, two patients had bacilli resistant to ethambutol and six had bacilli resistant to pyrazinamide. The two regimens were of similar effectiveness with regard to sputum conversion, compliance and radiological improvement. No patient with FDC treatment developed gastointestinal symptoms, visual disturbance or peripheral neuropathy (P < 0.05). However, FDC treatment resulted in drug-induced fever in one patient. One patient (3.8%) in the FDC group relapsed 5 months after completing treatment. CONCLUSION: This study suggests that the two regimens had similar effectiveness in the treatment of smear-positive pulmonary tuberculosis. However, the fewer adverse drug events among those patients treated with the FDC regimen suggests that it has a better safety profile.     

Widespread distribution of a single drug rifampicin formulation of inferior bioavailability in South Africa.

A pharmacokinetic study of rifampicin, isoniazid, pyrazinamide and ethambutol in 118 tuberculosis patients revealed low and variable concentrations of rifampicin after 2 months of treatment on standard daily doses. A group of 53 patients exposed to specific batches of formulations containing rifampicin alone showed particularly low and variable levels of the drug. The national drug regulatory authority subsequently withdrew the batches in question, as sufficient bioavailability data had not been submitted after what the manufacturer had considered to be a minor formulation change. The evidence supports initiatives to implement bioavailability testing of new formulations (and of established formulations subsequent to changes in the manufacturing process) prior to distribution. Concerns about the bioavailability of rifampicin-containing products, including those with adequate dissolution profiles, should not be confined to fixed-dose combination anti-tuberculosis drugs, but should also be applied to single drug formulations.     

Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs.

OBJECTIVE: A multicentre evaluation was performed to assess two rapid low-cost methods, MTT (3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) and resazurin assays, for testing the susceptibility of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and streptomycin (SM). METHODS: Thirty coded M. tuberculosis strains were sent to seven laboratories located in Latin America, representing six countries. Each site performed the colorimetric assays, MTT and resazurin, blind for the first-line drugs RMP, INH, EMB and SM. The minimum inhibitory concentration results obtained were compared to the conventional proportion method on Lowenstein-Jensen medium. RESULTS: After establishing the breakpoint concentrations, excellent results were obtained for RMP, INH and EMB, with levels of specificity and sensitivity of between 96% and 99%. CONCLUSION: MTT and resazurin assays are promising, accessible new alternative methods for middle- and low-resource countries that need low-cost methods to perform rapid susceptibility testing of M. tuberculosis to key anti-tuberculosis drugs.     

Rifampicin-resistant Mycobacterium tuberculosis: susceptibility to isoniazid and other anti-tuberculosis drugs

Based on data from 14 Supranational Tuberculosis (TB) Reference Laboratories worldwide, the proportion of rifampicin (RMP) resistant isolates that were isoniazid (INH) susceptible by phenotypic drug susceptibility testing varied widely (0.5-11.6%). RMP-resistant isolates that were INH-susceptible had significantly lower rates of resistance to other first- and second-line anti-tuberculosis drugs (except rifabutin) compared to multidrug-resistant isolates. RMP resistance is not always a good proxy for a presumptive diagnosis of multidrug-resistant TB, which has implications for use of molecular assays that identify only RMP resistance-associated DNA mutations.     

Anti-tuberculosis drug susceptibility testing of Mycobacterium bovis BCG Tokyo strain.

SETTING: The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine is the only vaccine against tuberculosis (TB), owing to its valuable protective effects and low virulence. However, it can occasionally cause systemic infection in immunocompromised hosts. Isoniazid (INH), rifampicin (RMP), streptomycin (SM) and ethambutol (EMB) are known to be effective anti-tuberculosis drugs and are used for the treatment of BCG infections. Unfortunately, there are few studies of the susceptibility of BCG vaccine strains to these drugs. OBJECTIVE: To measure the minimum inhibitory concentrations (MICs) of BCG Tokyo vaccine products for anti-tuberculosis drugs and assess vaccine safety in terms of drug susceptibility. DESIGN: We measured the MIC for one seed and five product lots of BCG Tokyo strain for INH, RMP, SM and EMB using Middlebrook 7H11 agar plates. RESULTS: The MIC results for INH were 0.06 and 0.125 mg/ml for the product and seed lots, respectively. The MIC results for RMP, SM and EMB were 0.25-0.5, 0.25 and 2-4 microg/ml, respectively. CONCLUSION: Our results indicate that the BCG Tokyo strain was susceptible to the major anti-tuberculosis drugs and treatable even in cases of severe adverse events, including systemic infection.     

The potential use of urinary excretion data for assessing the relative bioavailability of rifampicin in fixed dose combination anti-tuberculosis formulations.

SETTING: The perceived need for simple, non-invasive methods of assessing the relative bioavailability of rifampicin in fixed-dose combination (FDC) anti-tuberculosis formulations. OBJECTIVE: To compare the performance of methods based on urinary excretion data with those utilising plasma concentration-time profiles to assess the relative bioavailability of rifampicin in combined and single-drug formulations. DESIGN: A two-period randomised crossover bioequivalence study in healthy male volunteers with a 1 week washout period between treatments. Plasma rifampicin concentrations were measured at 0, 1, 2, 4, 6 and 8 hours after each drug administration using a high performance liquid chromatography (HPLC) method. The rifampicin and desacetylrifampicin content of complete urinary collections made from 0-4 and 4-8 hours after dosage were determined using both the HPLC and a much simpler colorimetric procedure. RESULTS: There was good agreement between the relative bioavailability of the formulations using plasma and urinary excretion data, although the precision of the urinary-based estimates was slightly less than those derived from the plasma findings. There was also good agreement between the HPLC and colorimetric estimates of the combined urinary excretion of rifampicin plus desacetylrifampicin. CONCLUSIONS: Urinary excretion data may be used for ongoing quality control to confirm that commercial combined rifampicin-containing formulations that were initially shown to be satisfactory continue to be so.     

A step in the global effort to control hypertension: Fixed dose combination antihypertensive drugs

Several fixed dose combinations (FDCs) of antihypertensive drugs have recently been added to the World Health Organization model list of essential medications. FDCs have advantages in the management of hypertension compared to single drug tablets including improved adherence, greater blood pressure lowering and are associated with reduced cardiovascular complications. FDCs can also reduce ethnic, and age‐related variation in blood pressure lowering and have similar or reduced adverse effects relative to single‐drug therapy. Best hypertension control practices from the World Health Organization HEARTS program advocates the use of FDC in simple directive treatment protocols. FDC in simple directive protocols was viewed as a key success factor in the control of chronic infections (eg, tuberculosis, HIV). Unfortunately, implementing simple directive protocols with FDC has had substantial opposition from hypertension experts. Hypertension organizations and experts need to familiarize themselves with best practices in hypertension control, their supporting evidence and to become advocates.     

Adverse drug reactions associated with first-line anti-tuberculosis drug regimens.

BACKGROUND: Standard treatment of active tuberculosis (TB) consists of isoniazid (INH), rifampin (RMP), pyrazinamide (PZA) and ethambutol (EMB). Although this regimen is effective in treating active TB, it is associated with many adverse drug reactions (ADRs) and poses a significant challenge to completion of treatment. OBJECTIVES: To examine the incidence of major ADRs and risk factors associated with first-line anti-tuberculosis medications. METHODS: This study evaluated patients receiving treatment for active TB from a population-based database (2000-2005). The nature of the ADRs, likelihood of association with the study medications and severity were evaluated. RESULTS: A total of 1061 patients received treatment, of whom 318 (30%) had at least one major ADR. The overall incidence of all major ADRs was 7.3 events per 100 person-months (95%CI 7.2-7.5): 23.3 (95%CI 23.0-23.7) when on all four first-line drugs, 13.6 (95%CI 13.3-14.0) when on RMP, INH and PZA, and 2.4 (95%CI 2.3-2.6) when on INH and RMP. Adjusted hazard ratio (HR) revealed that combination regimens containing PZA, females, subjects aged 35-59 and >or=60 years, baseline aspartate aminotransferase >or=80 U/l and drug resistance were associated with any major event. CONCLUSIONS: First-line anti-tuberculosis drugs are associated with significant ADRs. There are several risk factors associated with the development of ADRs, including exposure to regimens containing PZA.