Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh160 012, India
Received 3 November 2003; returned 8 December 2003; revised 17 December 2003; accepted 6 January 2004
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Abstract |
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Methods: Alginatechitosan microspheres encapsulating three frontline anti-tuberculous drugs (ATDs), rifampicin, isoniazid and pyrazinamide, were formulated. A therapeutic dose and a half-therapeutic dose of the microsphere-encapsulated ATDs were orally administered to guinea pigs for pharmacokinetic/chemotherapeutic evaluations, respectively.
Results: The drug encapsulation efficiency ranged from 65% to 85% with a loading of 220280 mg of drug per gram microspheres. Administration of a single oral dose of the microspheres to guinea pigs resulted in sustained drug levels in the plasma for 7 days and in the organs for 9 days. The half-life and mean residence time of the drugs were increased 13- to 15-fold by microsphere encapsulation, along with an enhanced relative/absolute bioavailability. The sustained release and increase in bioavailability were also observed with a sub-therapeutic dose of the microspheres. In Mycobacterium tuberculosis H37Rv-infected guinea pigs, administration of a therapeutic dose of microspheres spaced 10 days apart produced a clearance of bacilli equivalent to conventional treatment for 6 weeks. The most important observation, however, was the documentation of therapeutic benefit with a half-therapeutic dose of the microspheres administered weekly.
Conclusion: Alginatechitosan microspheres hold promise as a potential natural polymer-based oral ATD carrier for better management of TB.
Keywords: tuberculosis, polymers, bioavailability, chemotherapy
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Introduction |
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Materials and methods |
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Sodium alginate (medium viscosity, 3500 cps for a 2% w/v solution), chitosan (minimum 85% deacetylated), rifampicin, isoniazid and pyrazinamide were obtained from Sigma Chemical Company (St Louis, MO, USA). All other reagents were of analytical grade obtained from standard companies.
Animals
Dunkin Hartley guinea pigs of either sex (300400 g), obtained from Hisar Agricultural University, Hisar (India) were used in the study. The animals were fed standard pellet diet and water ad libitum. The study was approved by the Institutes Ethics Committee.
Culture
The culture of Mycobacterium tuberculosis H37Rv, originally obtained from the National Collection of Type Cultures (NCTC), London, UK, was maintained on Youmans modified medium.
Determination of MICs
The MIC90 of each drug was determined by the broth dilution method. A 500 µL inoculum of M. tuberculosis H37Rv (3 x 108 cells/mL) was added to specially designed flat-bottomed tubes containing a single ATD (rifampicin/isoniazid 0.020.3 mg/L or pyrazinamide 4.020.0 mg/L) in 5 mL Youmans media at 37°C under shaking conditions. The relative percentage growth was plotted against the concentration of each ATD.
Preparation of alginatechitosan microspheres
The principle involved was the cation-induced gelation of alginate,7 for the simultaneous encapsulation of rifampicin, isoniazid and pyrazinamide. Briefly, 10 mg of each drug was dissolved in 2 mL distilled water containing methanol (methanol/water 1:4 v/v) for the complete solubilization of rifampicin. To it was added 1.2 mL of sodium alginate solution (25 g/L). After thorough mixing, 2030 min were allowed to elapse in order to make the solution bubble-free. The mixture was passed through a peristaltic pump and allowed to fall dropwise at 60 drops/min into 50 mL of 0.1 M calcium chloride solution containing 30 mg chitosan (i.e. drug/alginate/chitosan 1:1:1) at pH 4.5. The beads formed instantaneously and were left as such for 810 h at room temperature. Subsequently, the beads were recovered by filtration, washed twice with distilled water and dried at room temperature. The yield was approximately 50 mg.
Characterization of alginatechitosan microspheres
The microspheres were weighed just after filtration/washing and then after drying in order to determine the water content. Eighty dried beads obtained from four separate experiments (20 beads were selected from each experiment) were measured with an oculomicrometer to obtain the mean particle diameter. Twenty micrograms of dried microspheres were put in 3 mL of 0.1 M PBS (pH 7.5) at 37°C under shaking conditions for 2448 h for lysis and drug release. Rifampicin was assayed by microbiological method (sensitivity 0.25 µg/mL),8 which was specific for the drug, using Bacillus subtilis (MTCC 441) as the indicator strain. Isoniazid was estimated by spectrofluorimetry (sensitivity 0.1 µg/mL)9 and pyrazinamide by spectrophotometry (sensitivity 5.0 µg/mL).10 The percentage encapsulation efficiency for each drug was calculated by the formula: (amount of drug released from the lysed microspheres/amount of drug initially taken to prepare the microspheres) x 100.
The drug loading capacity for each drug (expressed as mg drug/g microspheres) was calculated by the formula: amount of drug (mg) released from the lysed microspheres/amount of microspheres (g) put for lysis.
The residual methanol was determined by headspace gas chromatography and expressed as parts per million (ppm).
In vitro dissolution studies
Twenty micrograms of drug-loaded microspheres was added to 10 mL of simulated gastric fluid (SGF, 0.1 M HCl, pH 1.2) and simulated intestinal fluid (SIF, phosphate buffer, pH 7.5), both without enzymes, prepared according to the US Pharmacopeia.11 The in vitro release of drugs was assessed at room temperature up to 72 h by drawing 1 mL aliquots at various time points and replacing an equal amount of dissolution media. The methods of drug estimation were as described above. The results were expressed as the ratio of drug released relative to the amount of encapsulated drug, expressed as a percentage.
Preparation of microspheres for in vivo studies
The drug doses used throughout the study were either therapeutic dose (rifampicin 12 mg/kg + isoniazid 10 mg/kg + pyrazinamide 25 mg/kg body weight) or half-therapeutic dose. Since the doses were different for the three drugs, the initial amount of each drug required for microsphere preparation was calculated by the formula: (amount of drug required per animal/mean drug encapsulation efficiency) x 100.
Once the total drug quantities required were known, equivalent amounts of alginate and chitosan were used in the preparation process (to maintain drug/alginate/chitosan ratios at unity). The basic procedure for microsphere preparation remained the same as discussed above. For a 400 g guinea pig, 50 mg of dried microspheres constituted a therapeutic dose (containing 4.8 mg rifampicin + 4 mg isoniazid + 10 mg pyrazinamide) whereas 25 mg comprised a half-therapeutic dose.
In vivo drug disposition studies
The guinea pigs were divided into the following groups for single dose studies: Group 1, oral ATD-loaded microspheres at therapeutic dose (n = 8); Group 2, oral ATD-loaded microspheres at half-therapeutic dose (n = 8); Group 3, oral free (non-encapsulated) drugs in combination at therapeutic dose (n = 6); and Group 4, intravenous (iv, through the lateral leg vein) free (non-encapsulated) drugs in combination at therapeutic dose (n = 6). The microspheres were fed orally with a spatula whereas the free drugs were suspended in isotonic saline and administered orally/iv. The drugs were assayed in plasma collected at various time points. In addition, the animals were killed at different intervals to determine the drug content in 20% organ homogenates of lungs, liver and spleen. The drug concentrations were expressed as mg/L plasma or organ homogenates.
Pharmacokinetic analysis
The plasma drug concentration versus time data were used to determine various pharmacokinetic parameters. Peak plasma concentration (Cmax) and time taken to reach Cmax (Tmax) were obtained by visual data inspection. Elimination rate constant (kel) was calculated by regression analysis whereas elimination half-life (t1/2) was calculated from the equation 0.693/kel. The area under the concentrationtime curve (AUC0t) was determined by the trapezoidal rule. The terminal AUC0 was obtained by dividing the last measurable plasma drug concentration by kel. AUMC/AUC, i.e. area under moment curve (AUMC)/area under curve (AUC), gave the mean residence time (MRT). Relative bioavailability of encapsulated drugs was computed by the formula:
whereas
yielded the absolute bioavailability.
Biochemical hepatotoxicity studies
Guinea pigs were divided into the following groups: Group 1, ATD-loaded microspheres (therapeutic dose) every 10 days (three oral doses) (n = 6); Group 2, drug-free/empty microspheres every 10 days (three oral doses) (n = 5); and Group 3, free (non-encapsulated) drugs daily orally for 25 days (n = 5). On day 26, blood was collected from the animals in each group and the sera were analysed immediately for total bilirubin, alanine aminotransferase (ALT) and alkaline phosphatase (ALP) using standard kits.
Chemotherapeutic efficacy
Guinea pigs were infected intramuscularly with 1 x 105 viable bacilli of M. tuberculosis H37Rv in 0.1 mL sterile isotonic saline. Twenty days post-infection, the animals were divided into the following groups for oral chemotherapy: Group 1, ATD-loaded microspheres at therapeutic dose, every 10 days (five doses) (n = 6); Group 2, ATD-loaded microspheres at half-therapeutic dose, every 7 days (seven doses) (n = 6); Group 3, empty microspheres every 7 days (seven doses) (n = 5); Group 4, free (non-encapsulated) drugs (freshly prepared by suspending in isotonic saline) daily at therapeutic dose for 46 days (n = 6); and Group 5, untreated controls (n = 5). The animals were killed on day 46. The right caudal lung lobe and spleen (whole organ) were homogenized in 3 mL sterile saline. Fifty microlitres of 1:10 and 1:100 diluted homogenates were inoculated on Middlebrook 7H10 agar base. Colony forming units (cfu) were enumerated on day 25 post-inoculation. The results were expressed as log10 cfu per right caudal lung lobe or spleen.
Statistical analysis
The colony data were analysed by Students unpaired t-test.
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Results |
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A concentration-dependent decrease in the percentage growth was observed with increasing concentrations of each ATD against the bacterial strain. The MIC90 was 0.2 mg/L for rifampicin, 0.3 mg/L for isoniazid and 8.0 mg/L for pyrazinamide.
Characterization of microspheres
One hundred milligrams of the wet formulation produced 1520 mg of dry beads indicating a water content of 8085%. The microspheres were almost spherical with a mean (±S.D.) size of 70 ± 4 µm. The mean drug encapsulation efficiency (±S.D.) was found to be 83 ± 2% for rifampicin, 65 ± 6% for isoniazid and 69 ± 6% for pyrazinamide. The mean drug loading (±S.D.) was 270 ± 8 mg for rifampicin, 230 ± 8 mg for isoniazid and 235 ± 5 mg for pyrazinamide per gram of microspheres. Approximately 330 ppm of methanol was present in the finished product.
In vitro drug release profile
There was nominal release (less than 7% of the encapsulated drug) in the SGF throughout the 72 h study period. In the SIF, the release of rifampicin was less (16%) compared with isoniazid (20.6%) or pyrazinamide (22.1%) in the initial 6 h. Subsequently, there was a slow but sustained release of each drug, limited to less than 3% of the encapsulated drug.
In vivo drug disposition studies
A single oral administration of ATD-loaded microspheres resulted in sustained plasma drug levels for 7 days with the therapeutic dose and 5 days with the half-therapeutic dose (Figure 1). However, drugs were not detected beyond 12 h of oral dosing with free, non-encapsulated drugs. Drugs administered by the iv route attained instantaneous peak plasma levels and were subsequently cleared by 12 h. Drug concentrations were maintained in the organs until day 9 in case of the therapeutic dose of microspheres and day 7 in the case of the half-therapeutic dose (Table 1). Unencapsulated drugs were cleared from the organs by 24 h.
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Drugs encapsulated in alginatechitosan microspheres attained Cmax at 24 h as against 1 h in the case of orally administered parent drugs. In case of iv free drugs, the Cmax was achieved instantaneously. Because of a slower rate of elimination (kel), the encapsulated drugs exhibited a substantial increase in t1/2 (8- to 15-fold) and MRT (8.8- to 13-fold) and consequently, the AUC0. There was a striking improvement in the bioavailability of all three drugs (Table 2).
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As is evident from Table 3, the administration of drug-loaded or drug-free microspheres did not produce an increase in serum bilirubin, ALT or ALP. There was no evidence of any biochemical hepatotoxicity with respect to the control animals.
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Treatment with either a therapeutic dose of ATD-loaded microspheres (five doses), a half-therapeutic dose of microspheres (seven doses) or parent drugs (46 doses) all resulted in undetectable (<1.0 based on the lowest dilution tested) cfu in lungs/spleen. The results clearly show that the therapeutic potential of alginatechitosan microspheres lies not merely in reducing the dosing frequency, but also the dose itself as exemplified by the fact that the half-therapeutic dose of the formulation also resulted in bacterial clearance. Untreated controls exhibited comparable bacterial load (P > 0.05) to animals receiving empty microspheres (Table 4).
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Discussion |
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The formulation process resulted in small microspheres (6575 µm) with a high drug-encapsulation efficiency (6585%) and drug loading. The simple alginate microspheres reported previously6 were larger (90100 µM) and exhibited much lower drug encapsulation, especially for isoniazid and pyrazinamide (2543%). The polyionic complexation between chitosan and alginate depends on the pH of the dissolution medium. A decrease in the pH leads to shrinkage in the alginate gel and a reduced permeability of the alginatechitosan microspheres.13 In a neutral/alkaline medium, the interpolymeric complex swells and disintegrates to release the drugs, assisted by the sequestration of calcium ions by the phosphate present in the SIF. Hence, in vitro drug release was higher in the SIF compared with the SGF. Chitosan acts as a reinforcing polymer to retard the erosion of alginate microspheres, which explains the slow, but sustained in vitro drug release.14
A single oral dose of alginatechitosan microspheres encapsulating ATDs at therapeutic dosages maintained sustained drug levels in the plasma for 7 days. By comparison, the non-encapsulated parent drugs were cleared by 12 h (Figure 1). In case of simple alginate microspheres, the sustained drug release is restricted to no more than 96108 h.6 Furthermore, in this study, the encapsulated drugs exhibited a 15-fold increase in t1/2 as well as a 13-fold increase in MRT. These factors resulted in a striking improvement in the bioavailability of encapsulated drugs (Table 2). In particular, the relative bioavailability was enhanced by 17- to 19-fold as against 1.5- to 9-fold in the case of simple alginate microspheres.
Therapeutic concentrations of the ATDs were maintained in the tissues for as long as 9 days (Table 1) compared to just 2 days for the non-encapsulated parent drugs. Furthermore, a half-therapeutic dose of ATD-loaded microspheres was also able to produce a satisfactory drug release profile: it was observed that sustained drug levels could still be maintained for 5 days in plasma (Figure 1) and 7 days in organs (Table 1). Although the enhancement in t1/2 and MRT were lower with the half-therapeutic compared with the therapeutic dosage of microspheres, the values were nevertheless 8- to 9-fold higher than those obtained with free drugs. It should also be emphasized that despite the dose reduction, the bioavailability remained uncompromised (Table 3). The mechanism of sustained drug release is attributable to the fact that alginate is a mucoadhesive polymer; the enhanced gastrointestinal residence time is likely to be responsible for the improvement in drug bioavailability.4 In addition, polycationic macromolecules such as chitosan not only stabilize the alginate microspheres but also control the porosity of alginate to enhance the sustained release effect. The ability of chitosan to modulate the intestinal tight junctions is an added virtue, which helps the encapsulated drugs in crossing the permeability barriers.5 However, despite the increase in bioavailability, drug-related toxicity did not occur in our study. Preliminary toxicological evaluation showed that repeated administration of the microspheres every 10 days to guinea pigs was safe in terms of biochemical hepatotoxicity (Table 3).
Once the feasibility of reduction in dose/dosing frequency as well as formulation safety were established, the chemotherapeutic efficacy was evaluated in M. tuberculosis-infected guinea pigs. With the knowledge that the drug levels were maintained in the organs for 9 days (with a single therapeutic dose) or 7 days (with half-therapeutic dose) (Table 1), the ATD-loaded microspheres were administered every 10 days for the therapeutic dose and every 7 days for the half-therapeutic dose. Both of these treatment schedules resulted in undetectable cfu in the organs, as did conventional therapy (Table 4). Thus, 46 conventional doses could be brought down to five therapeutic doses. Bacterial clearance still occurred when the drug dosages were halved, thereby confirming the systems potential in terms of therapeutic benefit and potential cost savings. Other authors have reported on the encapsulation of isoniazid in alginatechitosan microspheres; however, no in vivo studies were carried out.15
Our previous work with an alginate-based ATD delivery system,6 and the alginatechitosan system being discussed here, bear distinct formulation characteristics. Adjustments to drug/polymer ratio and the use of chitosan are some of the critical factors which enable the current system to outrank the simple alginate system,6 in terms of drug-encapsulation efficiency, biodistribution profile and pharmacokinetic variables. The most encouraging observation, however, is the ability of the formulation to show a therapeutic benefit with just half the therapeutic dose.
In summary, alginatechitosan-based ATD delivery systems provide an economical means for a much needed reduction in dose/dosing frequency and offer hope for a better tomorrow in the management of TB.
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Footnotes |
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References |
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