Journal of Antimicrobial Chemotherapy (2004) 53, 635640 DOI: 10.

1093/jac/dkh139 Advance Access publication 3 March 2004

Chemotherapeutic potential of alginatechitosan microspheres as anti-tubercular drug carriers

Rajesh Pandey and G. K. Khuller*
Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh160 012, India
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Received 3 November 2003; returned 8 December 2003; revised 17 December 2003; accepted 6 January 2004

Objectives: This study was designed to develop alginatechitosan microspheres as drug carriers to reduce dose/dosing frequency in the management of tuberculosis (TB), which otherwise demands prolonged chemotherapy. 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

Introduction
The controlled delivery of antimycobacterial agents may be accomplished by employing various polymeric drug carriers. Although experience with synthetic polymers is extensive and encouraging,13 the recent trend has been to shift towards natural polymers.4 The major advantage of natural polymers (e.g. alginate and chitosan) includes their low cost and compatibility with the encapsulation of a wide range of drugs, with minimal use of organic solvents. Furthermore, bio-adhesion, stability, safety and their approval for human use by the US FDA are additional advantages.4,5 The encapsulation of three frontline anti-tuberculous drugs (ATDs), rifampicin, isoniazid and pyrazinamide, in alginate microspheres demonstrated promising chemotherapeutic potential.6 Subsequent work showed that a few critical adjustments in the formulation process, especially the incor-

poration of chitosan, were not only capable of improving the drug(s) encapsulation efficiency and bioavailability, but also of reducing the dose and dosing frequency. The development and pharmacokinetic/ chemotherapeutic evaluation of the new formulation of alginate chitosan microspheres in a guinea pig model is reported here.

Materials and methods

Chemicals
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.

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*Corresponding author. Tel: +91-172-747-585, ext. 5174/75; Fax: +91-172-744-401, 745-078; E-mail: gkkhuller@yahoo.co.in
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R. Pandey and G. K. Khuller

5 mL Youmans media at 37C 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.

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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 37C under shaking conditions for 2448 h for lysis and drug release. Rifampicin was assayed by microbiological method (sensitivity 0.25 g/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 g/mL)9 and pyrazinamide by spectrophotometry (sensitivity 5.0 g/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) 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).

Figure 1. The profile of anti-tuberculous drugs in plasma following the single oral administration of ATD-loaded alginatechitosan microspheres and free drugs (in combination) to guinea pigs. (a) Plasma rifampicin; (b) plasma isoniazid; and (c) plasma pyrazinamide. Values are means S.D. of eight animals for alginatechitosan microspheres and six animals for free drugs.

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.

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.

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) 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

Determination of MICs
The MIC90 of each drug was determined by the broth dilution method. A 500 L inoculum of M. tuberculosis H37Rv (3 108 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

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Alginatechitosan microspheres as anti-tubercular drug carriers

Table 1. Tissue drug levels following the oral administration of drug-loaded alginatechitosan microspheres to guinea pigsa Rifampicin (mg/L homogenate) therapeutic dose (n = 8) Day 7 lung liver spleen Day 9 lung liver spleen 0.80 0.10 1.00 0.20 0.80 0.10 0.33 0.04 0.75 0.10 0.50 0.08 half-therapeutic dose (n = 8) 0.25 0.00 0.53 0.10 0.52 0.07 0.00 0.00 0.00 0.00 0.00 0.00 Isoniazid (mg/L homogenate) therapeutic dose (n = 8) 1.20 0.20 1.50 0.20 1.50 0.30 0.21 0.03 0.37 0.05 0.22 0.03 half-therapeutic dose (n = 8) 0.80 0.10 0.93 0.10 0.85 0.20 0.00 0.00 0.00 0.00 0.00 0.00 Pyrazinamide (mg/L homogenate) therapeutic dose (n = 8) 14.00 3.20 18.00 4.00 17.00 3.00 8.30 1.00 8.80 1.10 7.30 1.00 half-therapeutic dose (n = 8) 13.00 3.00 15.00 3.00 15.00 2.00 0.00 0.00 0.00 0.00 0.00 0.00
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Values are mean S.D. aIn case of oral/iv free drugs, the drugs were not detectable beyond day 2.

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.

Biochemical hepatotoxicity studies

Guinea pigs were divided into the following groups: Group 1, ATDloaded 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.

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.

Chemotherapeutic efficacy
Guinea pigs were infected intramuscularly with 1 105 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.

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:

Statistical analysis
The colony data were analysed by Students unpaired t-test.

AUCalginate drugs Doseoral free drugs ------------------------------------- ------------------------------------, AUCoral free drugs Dosealginate drugs
whereas

Results
MIC of anti-tuberculous drugs against M. tuberculosis H37Rv
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.

AUCalginate drugs Doseiv free drugs ------------------------------------ -----------------------------------AUCiv free drugs Dosealginate drugs
yielded the absolute bioavailability.

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alginatechitosan encapsulated drugs (half-therapeutic dose) (n = 8)

Characterization of microspheres
Table 2. Pharmacokinetic parameters of antitubercular drugs following the oral administration of alginatechitosan encapsulated drugs compared to oral/iv free drugs in guinea pigs

23.8 4.00 71.10 4.00 34.40 5.00 2.00 0.00 0.17 0.00 24.00 0.00 0.13 0.03 0.14 0.02 0.01 0.00 5.30 0.80 4.95 0.70 69.30 6.40 6.60 1.00 3.86 0.73 75.50 6.00 185.00 10.00 202.00 8.10 3538.00 88.00

Pyrazinamide

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 m. 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.

24.80 4.00 24.00 0.00 0.01 0.00 53.30 4.90 58.40 4.50 1940.00 60.00

alginatechitosan encapsulated drugs (therapeutic dose) (n = 8)

21.00 19.10

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.

iv free drugs (therapeutic dose) (n = 6)

oral free drugs (therapeutic dose) (n = 6)

1.00

17.50

19.20

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alginatechitosan encapsulated drugs (half-therapeutic dose) (n = 8)

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 halftherapeutic dose (Table 1). Unencapsulated drugs were cleared from the organs by 24 h.

2.34 0.40 24.00 0.00 0.02 0.00 34.70 4.10 58.40 5.00 162.70 10.10

1.00 29.90 17.60 1.00 20.30 18.40

alginatechitosan encapsulated drugs (therapeutic dose) (n = 8)

2.14 0.40 24.00 0.00 0.01 0.00 53.30 4.10 70.80 4.60 191.50 13.00

Isoniazid

alginatechitosan encapsulated drugs oral free drugs iv free drugs (half-therapeutic (therapeutic (therapeutic dose) (n = 8) dose) (n = 6) dose) (n = 6)

28.50 3.10 0.02 0.00 0.41 0.06 1.69 0.30 2.41 0.70 18.80 2.90

Pharmacokinetic analysis
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).

1.71 0.30 2.00 0.00 0.20 0.02 3.47 0.70 5.50 0.70 10.93 1.90

1.08 0.20 24.00 0.00 0.02 0.00 34.70 3.90 56.90 5.00 85.00 7.70

alginatechitosan oral free drugs iv free drugs encapsulated (therapeutic (therapeutic drugs (therapeutic dose) (n = 6) dose) (n = 6) dose) (n = 8)

Biochemical hepatotoxicity
As is evident from Table 3, the administration of drug-loaded or drugfree 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.

Rifampicin

1.59 0.30 24.00 0.00 0.01 0.00 57.80 4.10 73.00 6.00 154.20 12.00

25.40 4.10 0.02 0.00 0.39 0.05 1.80 0.30 2.51 0.60 16.50 3.00

Chemotherapeutic efficacy
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

Cmax, mg/L 1.22 0.20 Tmax, h 2.00 0.00 kel 0.16 0.03 t1/2, h 4.30 0.70 MRT, h 6.20 1.00 AUC0, 8.37 1.10 mg.h/L Relative 1.00 bio-availability Absolute 0.51 bio-availability

1.00

9.35

10.30

0.58

1.00

10.20

17.30

0.92

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Values are mean S.D.

Alginatechitosan microspheres as anti-tubercular drug carriers

Table 3. Evaluation of oral ATD-loaded alginatechitosan microspheres for biochemical hepatotoxicity Group ATD-loaded microspheres every 10 days (three doses) (n = 6) Empty microspheres every 10 days (three doses) (n = 5) Free drugs daily (26 doses) (n = 5) Serum bilirubin (mg/100 mL) 0.310.58 0.280.51 0.340.46 Serum ALT (U/L) 2847 2135 3350 Serum ALP (U/L) 4052 3657 2542

The normal range was ascertained in the laboratory by analysing the sera of 10 healthy guinea pigs: Total bilirubin = 0.11.0 mg/100 mL; ALT = 1570 U/L; ALP = 1570 U/L.

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Table 4. Chemotherapeutic efficacy of alginatechitosan encapsulated anti-tubercular drugs against experimental tuberculosis in guinea pigs Log10 cfu Groups Untreated controls (n = 5) Empty alginatechitosan microspheres every 7 days, orally (seven doses) (n = 5) Drug loaded alginatechitosan microspheres (half-therapeutic dose) every 7 days, orally (seven doses) (n = 6) Drug loaded alginatechitosan microspheres (therapeutic dose) every 10 days, orally (five doses) (n = 6) Free drugs daily, orally (46 doses) (n = 6) lung (right caudal lobe) 5.8 0.1 5.8 0.3* <1.0a <1.0a <1.0a spleen (whole organ) 5.9 0.1 5.8 0.3* <1.0a <1.0a <1.0a

Results are mean S.D. aNo detectable cfu following the inoculation of 50 L of 1:10 and 1:100 diluted homogenates. *P > 0.05 (Students t-test), with respect to the untreated controls.

controls exhibited comparable bacterial load (P > 0.05) to animals receiving empty microspheres (Table 4).

Discussion
The oral delivery of ATDs using synthetic polymers, though capable of providing a sustained drug release2 and therapeutic benefit,3 suffers from two major drawbacks: the high cost of polymers and the need to use organic solvents in the formulation development process. Natural polymers are endowed with properties which make them ideal drug delivery carriers. Alginate is a favourite vehicle for the delivery of a wide range of therapeutic agents. Alginate-based systems are known to work better when used in conjunction with polycationic stabilizers such as chitosan.12 This study describes the formulation of alginatechitosan microspheres for the controlled release of ATDs with the aim of reducing the dosing frequency as well as the dose in TB chemotherapy. The formulation process resulted in small microspheres (6575 m) with a high drug-encapsulation efficiency (6585%) and drug loading. The simple alginate microspheres reported previously6 were larger (90100 M) 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

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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 halftherapeutic 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.

References
1. Quenelle, D. C., Winchester, G. A., Staas, J. K. et al. (2001). Treatment of tuberculosis using a combination of sustained-release rifampin-loaded microspheres and oral dosing with isoniazid. Antimicrobial Agents and Chemotherapy 45, 163744. 2. Ain, Q., Sharma, S. & Khuller, G. K. (2002). Role of poly (DL-lactideco-glycolide) in development of sustained oral delivery systems for antitubercular drug(s). International Journal of Pharmaceutics 239, 3746. 3. Ain, Q., Sharma, S. & Khuller, G. K. (2003). Chemotherapeutic potential of orally administered poly (lactide-co-glycolide) microparticles containing isoniazid, rifampicin and pyrazinamide against experimental tuberculosis. Antimicrobial Agents and Chemotherapy 47, 30057. 4. Tonnesen, H. H. & Karlsen, J. (2002). Alginate in drug delivery systems. Drug Development and Industrial Pharmacy 28, 62130. 5. Hejazi, R. & Amiji, M. (2003). Chitosan-based gastrointestinal delivery systems. Journal of Controlled Release 89, 15165. 6. Ain, Q., Sharma, S., Khuller, G. K. et al. (2003). Alginate based oral drug delivery system for tuberculosis: pharmacokinetics and therapeutic effects. Journal of Antimicrobial Chemotherapy 51, 9318. 7. Gonzalez-Rodriguez, M. L., Holgado, M. A., Sanchez-Lafuente, C. et al. (2002). Alginate/chitosan particulate systems for sodium diclofenac release. International Journal of Pharmaceutics 232, 22534. 8. Saito, H. & Tomioka, H. (1989). Therapeutic efficacy of liposome entrapped rifampicin against Mycobacterium avium complex infections induced in mice. Antimicrobial Agents and Chemotherapy 33, 42931. 9. Scott, E. H. & Wright, R. C. (1967). Fluorimetric determination of isonicotinic acid hydrazide in plasma. Journal of Laboratory and Clinical Medicine 70, 35560. 10. Gurumurthy, P., Nair, N. G. K. & Sarma, G. R. (1980). Methods for the estimation of pyrazinamide and pyrazinoic acid in body fluids. Indian Journal of Medical Research 71, 12934. 11. Anonymous. (1995). US Pharmacopoeia USP 23/NF18. United States Pharmacopoeial Convention, Rockville, MD, USA. 12. Ravi Kumar, M. N. V. (2000). Nano and microparticles as controlled drug delivery devices. Journal of Pharmacy and Pharmaceutical Sciences 3, 23458. 13. You, J.-O., Park, S.-B., Park, H.-Y. et al. (2001). Preparation of regular sized Ca-alginate microspheres using membrane emulsification method. Journal of Microencapsulation 18, 52132. 14. Takka, S. & Acarturk, F. (1999). Calcium alginate microparticles for oral administration: 1: effect of sodium alginate type on drug release and drug entrapment efficiency. Journal of Microencapsulation 16, 27590. 15. Lucinda-Silva, R. M. & Evangelista, R. C. (2003). Microspheres of alginatechitosan containing isoniazid. Journal of Microencapsulation 20, 14552.

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