Chemotherapy of Mycobacterium tuberculosis infections in mice with a combination of isoniazid and rifampicin entrapped in Poly (DL-lactide-co-glycolide) microparticles

Manisha Dutt and G. K. Khuller,*

Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strategies to improve patient compliance in tuberculosis chemotherapy include the use of sustained release drug delivery systems. In this study, Poly (DL-lactide-co-glycolide) (PLG) microparticles containing a combination of isoniazid and rifampicin were developed as sustained release carrier systems. A single dose of PLG microparticles exhibited a sustained release of isoniazid and rifampicin in vivo up to 7 and 6 weeks, respectively. Free drugs (in combination) injected in the same doses were detectable in vivo up to 24 h only. One dose of PLG microparticles cleared bacteria more effectively from lungs and liver in an experimental murine model of tuberculosis after low-dose PLG combination drug therapy and in liver after high-dose PLG combination drug therapy as compared with a daily administration of the free drugs. These results suggest that PLG microparticles offer an improvement for tuberculosis chemotherapy over the conventional treatment.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The incidence of mycobacterial infections has increased rapidly in recent years; one-third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB).1 Chemotherapy of tuberculosis is complicated by the need for multi-drug regimens given over long periods. Current short-course chemotherapy (SCC) involves daily administration of isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA) for a period of 6–9 months. Therapy for TB may be further complicated by patient non-compliance and the development of multi-drug resistant (MDR) strains. Directly observed therapy short course (DOTS) is the strategy advocated by the WHO for the global control of TB.2 Although DOTS is generally effective, it has several operational difficulties, particularly in developing countries. Therefore, if all the component drugs could be administered once a month, rather than daily, treatment and control of TB would be improved.

Various drug carrier systems have been investigated in recent years, including liposomes,3 polymeric implants4 and microspheres,5 notably polymer-based microsphere delivery systems. The polymer used most frequently is Poly (DL-lactide-co-glycolide) or PLG, a copolymer of lactic acid and glycolic acid. PLG polymers are completely biodegradable, biocompatible and non-immunogenic.6 PLG polymers have recently been used as carriers for INH,7 RIF8 and clofazimine9 for treatment of mycobacterial infections. PLG polymers are versatile both in their formulation, which includes films, fibres, rods and implants,7 and their mode of delivery, which may be parenteral,10 oral11 or aerosol.12 They are capable of sustained drug release over days to several weeks depending on the polymer used.13

The present study was planned to develop a PLG-based microparticle carrier system containing entrapped INH and RIF in combination, to be administered subcutaneously. The disposition of the drugs was evaluated in plasma and various organs of mice. Chemotherapeutic efficacy and toxicity were also investigated against experimental murine TB.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and drugs

Poly (DL-lactide-co-glycolide) (50:50) resomer RG 506, mol. wt 2500, was purchased from Boehringer Ingleheim (Germany), polyvinyl alcohol, 87–89% hydrolysed (mol. wt 13–23000), INH and RIF were obtained from Sigma Chemical Co. (St Louis, MO, USA). Kits for alkaline phosphatase (ALP), serum glutamate pyruvate transaminase (SGPT) and total bilirubin estimation were purchased from Boehringer Mannheim (Germany). All other reagents were of analytical grade and obtained from standard companies.

Animals

Mice (laca strain) of both sexes (4–5 weeks old and weighing 18–20 g) obtained from Central Animal House, PGIMER, Chandigarh, India, were used in this study and were housed in an animal house facility under natural light conditions. The animals were fed a standard pellet diet and water as required. The project was approved by the Institute Animal Ethics Committee.

Culture

The culture of M. tuberculosis H37Rv was originally obtained from the National Collection of Type Cultures (NCTC), London, UK, and was maintained on Youman's modified medium.

Preparation of PLG microparticles

‘Hardened’ PLG microparticles were prepared by the double emulsion solvent evaporation procedure as described by Edwards et al.12 with slight modifications. The drug: polymer ratio was kept at 60:40 (w/w) for INH and 1:20 (w/w) for RIF.

Briefly, 135 mg of INH in 2 mL of phosphate-buffered saline (PBS) was emulsified on ice into 90 mg of PLG in 2 mL of dichloromethane (DCM) by probe sonication. RIF, 8.5 mg, in 1 mL of PBS was emulsified on ice into 170 mg PLG in 1 mL DCM by probe sonication in darkness under slow N2 pressure. The primary emulsion was stabilized in 1 mL of 20% (w/v) aqueous polyvinyl alcohol solution. The microparticles were stirred continuously overnight and collected the next day by centrifugation. They were washed several times with PBS and finally suspended in PBS. Empty PLG microparticles were prepared by substituting normal saline/PBS for the antibiotic.

Determination of drug content of microparticles

The entrapment of drugs in hardened PLG microparticles was determined by lysing them with 5% SDS (w/v) in 0.1 M NaOH. The drugs released in the supernatant were assayed by standard protocols. INH was estimated by the spectrofluorimetric method of Scott & Wright.14 RIF was evaluated spectrophotometrically as described by Deol & Khuller.3

Size of PLG microparticles

The size of hardened PLG microparticles was determined on a particle size analyser, CIS-1, Galai, Israel (based on laser light scattering) at the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi. A volume probability density graph was generated from which the mean volume diameter was calculated.

In vivo combination drug disposition studies from PLG microparticles

Hardened PLG microparticles containing drugs (INH/RIF) were prepared separately. They were mixed together in a 1:1 ratio (v/v) for combination drug disposition studies. Mice were divided into four groups, each of seven or eight animals. The doses of INH and RIF used in treated mice were 75 and 85 mg/kg, respectively. Group 1 was injected with free drugs in combination; group 2 were given hardened PLG microparticles; group 3 received PBS/normal saline; group 4 received empty PLG microparticles.

Mice were bled via the retro-orbital plexus at 12, 24, 48 and 72 h and then weekly for a period of 6–8 weeks. In addition, animals were killed at various intervals and drug concentrations were determined in 10% organ homogenates (100 mg of organs homogenized in 1 mL of PBS). Plasma INH was estimated by the spectrofluorimetric method of Scott & Wright14 after precipitation of plasma proteins. The RIF concentrations in plasma and tissue homogenates were determined by a microbiological assay15 with a sensitivity of 0.01 µg. Results were expressed as concentration of drugs (mg/L) obtained in tissue homogenates at various intervals.

Experimental infection and chemotherapy studies

Mice were inoculated via the lateral tail vein with 1.5 x 105 viable bacilli of M. tuberculosis H37Rv in a volume of 0.1 mL of 0.9% sterile NaCl solution. The animals were divided into six groups each containing seven to nine animals. Fifteen days after inoculation, infection was confirmed by Ziehl–Neelsen staining of tissue smears of lungs, liver and spleen after killing two animals.

Two different doses of the drug combination were used in this study. INH and RIF were given in combination at doses of 75 and 85 mg/kg, estimated as equivalent to a human dose for a 70 kg human16 (‘high dose’ group), or at half these concentrations, i.e. 37.5 and 42.5 mg/kg (‘low dose’ group). Mice are fast drug metabolizers and therefore it would be appropriate to give high doses. All treatments were administered via the subcutaneous route in mice. Groups 1 and 2 were injected with PBS and empty hardened PLG microparticles (without drugs), respectively, and served as controls. Groups 3 and 4 received the free drugs in combination at high and low doses. Groups 5 and 6 received the same doses of combination drugs (high and low) encapsulated in hardened PLG microparticles.

Drug therapy using PLG microparticles was administered as a single subcutaneous dose while free drugs in combination were administered daily subcutaneously for a period of 6 weeks. Seven days after the last therapeutic dose, the animals were killed.

Quantification of mycobacteria

Lungs, liver and spleen were homogenized and cultured on plates containing Youman's modified medium supplemented with 1% bovine serum albumin in order to count colony forming units (cfu).17 The numbers of cfu were determined 21 days after inoculation.

Toxicity studies

The levels of ALP, SGPT and total bilirubin were determined in plasma samples of mice on days 3 and 6 after last injections.

Statistical analysis

The data were analysed by one-way analysis of variance (ANOVA) to compare the control and treatment groups. Free drugs and PLG treatment groups were also compared by Student's two-tailed t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Encapsulation efficiency of the microparticles

The percentage entrapment of INH and RIF in hardened PLG microparticles was found to be 10–11 and 12–14%, respectively.

Particle sizing studies

The particle size analysis for the hardened PLG microparticles exhibited a volume mean diameter of 11.75 and 11.64 µm for INH- and RIF-containing microparticles, respectively.

In vivo combination drug disposition studies from PLG microparticles

In vivo drug disposition studies in mice were carried out by subcutaneous administration of a single dose of microparticles containing a combination of drugs. Subsequently, the plasma and organ disposition of both INH and RIF were monitored at different time intervals. Figure 1Go depicts the mean plasma concentrations of INH obtained after the administration of hardened PLG microparticles containing a combination of drugs. INH release from hardened PLG microparticles occurred from the third day with a peak plasma concentration of 7.49 ± 0.40 mg/L. An almost constant release phase of INH was observed from day 7 to day 21 with concentrations ranging between 2.09 ± 0.10 mg/L and 2.89 ± 0.10 mg/L. The release of INH then increased to a concentration of 4.02 ± 0.25 mg/L on day 27. A sustained release of INH was observed up to 49 days in plasma with concentrations of INH ranging between 2.85 ± 0.5 mg/L on day 27 and 5.55 ± 0.18 mg/L on day 49. Free INH at the same dose showed release in plasma only at 12 h, with a concentration of 1.66 ± 0.21 mg/L.



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Figure 1. Mean plasma levels of INH obtained after the administration of hardened PLG microparticles containing a combination of drugs. All values are mean ± S.D. for three or four animals.

 
Table IGo represents the mean plasma concentration of RIF obtained after the administration of PLG microparticles. A biphasic release of RIF was observed in plasma with an initial release at 12 h, with a concentration of <0.5 mg/L. However, between 24 and 72 h, RIF was not detectable in plasma (i.e. no zones of inhibition in the micobiological assay). A sustained release of RIF was again observed from day 7 to day 42, with a concentration of <0.5 mg/L of plasma (i.e. zones of inhibition too small to permit accurate measurement of antibiotic concentration). When free RIF was administered in mice, release was detected only up to 24 h, with a concentration of <0.5 mg/L of plasma.


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Table I. Plasma concentrations of RIF obtained after administration of free and PLG-encapsulated drug Concentration of RIF (mg/L)a
 
When the tissue disposition of drugs was examined, sustained release of INH was detected in all the organs examined between days 3 and 49 (Figure 2Go). The concentrations of INH obtained at all time points were found to be higher than the MIC of INH. When the same dose of free INH was administered to mice, INH release was detected only in the liver at 24 h with a concentration of 0.65 ± 0.2 mg/L of the tissue homogenates.



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Figure 2. Tissue disposition of INH following administration of hardened PLG microparticles containing a combination of drugs. All values are mean ± S.D. for three or four animals. Symbols: {blacksquare}, lungs; , liver; {square}, spleen.

 
Table IIGo illustrates the tissue disposition of RIF in mice at different time intervals. A sustained release of RIF from hardened PLG microparticles was observed in all the organs (lungs, liver and spleen) of mice between days 3 and 21, with a concentration of <0.5 mg/L of the tissue homogenates. Sustained release of RIF was, however, observed only in spleen between days 27 and 49, with a concentration of <0.5 mg/L of the tissue homogenates. Free RIF administered at the same dose showed release in lungs, liver and spleen only at 24 h, with the maximal concentration of RIF in the liver (3.33 ± 0.3 mg/L of tissue homogenates).


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Table II. Disposition of RIF in various organs of mice Concentration of RIF (mg/L)a free RIF RIF in PLG microparticles
 
Chemotherapeutic efficacy of a combination of free and PLG-encapsulated anti-tubercular drugs

Figure 3Go shows the log cfu in mice after treatment with a low dose (37.5 mg/kg of INH + 42.5 mg/kg of RIF) of the drugs. Treatment in all the groups resulted in a significant clearance of bacilli compared with the controls (P < 0.001). One injection of low-dose PLG combination drug treatment showed a significant reduction in the number of cfu in lungs, liver and spleen by 1.47, 1.87 and 0.84 log units, respectively, as compared with the controls (P < 0.001). These results indicate a significantly better clearance of bacilli in lungs and liver by 4.75- and 5.09-fold, respectively, in PLG combination drug treatment compared with daily free combination drug treatment (P < 0.001).



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Figure 3. Cfu of M. tuberculosis in different organs of mice after chemotherapy with free drugs in combination () and hardened PLG microparticles containing a combination of drugs at low doses ({square}) ({blacksquare}, control). All values are mean ± S.D. of three or four animals. ***P < 0.001 (level of significance for free drugs and PLG microparticles with respect to controls).

 
Figure 4Go shows the log cfu of M. tuberculosis in different organs of mice after high-dose treatment with free combination drugs and PLG combination drugs. Treatment in all the groups resulted in a significant (P < 0.001) clearance of bacilli compared with controls. One injection of high-dose PLG combination drug treatment showed a significant (P < 0.001) reduction in number of cfu in lungs, liver and spleen by 1.69, 2.05 and 1.37 log units, respectively, compared with controls. These results indicate that PLG combination therapy showed a significantly better clearance of bacilli in liver by 4.05-fold and an equivalent clearance of bacilli in spleen as compared with free combination drug therapy (P < 0.001). Free combination therapy in high dosage showed better lung clearance of bacilli compared with PLG combination drug treatment (P < 0.01).



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Figure 4. M. tuberculosis cfu in different organs of mice after chemotherapy with free drugs in combination () and hardened PLG microparticles containing a combination of drugs at high doses ({square}) ({blacksquare}, control). All values are mean ± S.D. of three or four animals. ***P < 0.001 (level of significance for free drugs and PLG microparticles with respect to controls).

 
Evaluation of the in vivo toxicity of the drugs

In vivo toxicity of the PLG combination drugs and free combination drugs were evaluated on days 3 and 6 after the last therapeutic dose. Table IIIGo shows the levels of ALP, SGPT and total bilirubin in plasma after combination drug therapy. As is evident from this table, there was no apparent change in these parameters compared with the controls in any of the treatment groups.


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Table III. Concentrations of ALP, SGPT and total bilirubin in plasma of mice after combination drug therapy
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study describes the use of polymer-based delivery systems as carriers for two of the front-line antitubercular drugs, INH and RIF, which form the mainstay of the chemotherapy of tuberculosis. However, these drugs exhibit toxic side-effects such as hepatotoxicity over long-term administration. Also, there is a need for daily administration of the drugs because of their short half-lives.

In the present study, we have observed that a combination of anti-tubercular drugs (INH and RIF) in hardened PLG microparticles exhibits a sustained release of the drugs up to 7 weeks in mice, while free drugs at the same doses showed release only up to 24 h. It has been observed that particles with a size range >10 µm remain at the site of injection forming a depot from where the entrapped contents of the polymer are released slowly.18 Such depots can release drugs up to several months until the entire polymer is biodegraded. In this study the particles investigated were >10 µm, so that they could exhibit sustained release over prolonged periods.

The results show that a single injection of drugs in PLG microparticles significantly reduces the number of bacilli compared with controls. PLG microparticles containing a combination of drugs in lungs, liver and spleen after low-dose therapy and liver after high-dose therapy show a significantly better clearance of bacilli than a daily administration of free combination of drugs.

Previous studies using PLG-based delivery systems have shown the effectiveness of these polymeric delivery systems as sustained release carriers in both mice19 and rabbits.20 However, most of these systems involved implants, which had inevitable practical limitations.

Reports have also shown this technology to be effective both ex vivo in macrophages8 and in in vivo studies in mice5 but reduction in cfu was observed only up to 26 days after infection in the latter study. In this communication, we have shown for the first time that a combination of antitubercular drugs entrapped in PLG microparticles can be used therapeutically, when injected once in 7 weeks. This system could effectively reduce cfu in tissues of mice compared with daily free drug administered groups, without causing toxicity.

These observations are encouraging and indicate that microsphere-based systems are a potential alternative to conventional chemotherapy of tuberculosis.


    Notes
 
* *Corresponding author. Tel: +91-172-747585, ext. 282, 274; Fax: +91-172-744401/745078; E-mail: medinst{at}pgi.chd.nic.in Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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16 . Paget, G. E. & Barnes, J. M. (1964). In Evaluation of Drug Activities: Pharmacometrics, Vol. 1, (Lawrence, D. R. & Bacharach, A. L., Eds). Academic Press, New York.

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Received 30 October 2000; returned 22 December 2000; revised 18 January 2001; accepted 23 February 2001