Subcutaneous nanoparticle-based antitubercular chemotherapy in an experimental model

Rajesh Pandey and G. K. Khuller*

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

Received 10 February 2004; returned 22 March 2004; revised 26 March 2004; accepted 6 April 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Poly (DL-lactide-co-glycolide) (PLG) nanoparticles encapsulating three front-line antitubercular drugs, i.e. rifampicin, isoniazid and pyrazinamide, were prepared by the multiple emulsion technique and administered subcutaneously to mice for pharmacokinetic/chemotherapeutic study. A single subcutaneous dose of drug-loaded PLG nanoparticles resulted in sustained therapeutic drug levels in the plasma for 32 days and in the lungs/spleen for 36 days. The mean residence time and absolute bioavailability were increased several-fold as compared with unencapsulated drugs. Further, drug-loaded PLG nanoparticles resulted in undetectable bacterial counts in the lungs and spleen of Mycobacterium tuberculosis-infected mice, thereby demonstrating a better chemotherapeutic efficacy, as compared with daily free drug treatment. Hence, injectable PLG nanoparticles hold promise for increasing drug bioavailability and reducing dosing frequency for better management of tuberculosis.

Keywords: poly (DL-lactide-co-glycolide) , tuberculosis , bioavailability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Tuberculosis (TB) continues to be a leading cause of mortality in spite of the availability of an effective chemotherapeutic regimen.1 The fact that a TB patient needs to take multiple antitubercular drugs (ATDs) daily for at least 6 months is largely responsible for patient non-compliance and therapeutic failure. The development of a controlled-release ATD formulation is a possible solution to this problem. Recently, poly (DL-lactide-co-glycolide) nanoparticles (PLG-NP) were explored to ascertain their suitability as antitubercular drug carriers for oral2/aerosol3 administration. In addition, drug dosing frequency could be reduced to once every 10 days, instead of daily with conventional therapy. Here, we report on the single subcutaneous administration of PLG-NP and its chemotherapeutic potential in a murine TB model.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
PLG-NP co-encapsulating rifampicin, isoniazid and pyrazinamide were prepared by the multiple emulsion technique4 with slight modifications,2 and vacuum dried. The particle size, determined by photon correlation spectroscopy, ranged from 186–290 nm (polydispersity index: 0.38±0.04). The PLG-NP were lysed in 0.1 M sodium hydroxide at 50°C for 10 min to release the drug contents. The percentage drug encapsulation efficiency, with respect to initial amount of drug taken, was calculated by the formula: (amount of drug released from the lysed PLG-NP/amount of drug initially taken to prepare the nanoparticles)x100. The drug-loading capacity for each drug was calculated by the formula: [amount of drug (mg) released from the lysed PLG-NP/amount of PLG-NP (g) put for lysis], expressed as mg drug/g PLG-NP. The drug assay sensitivities were 0.25 mg/L for rifampicin (microbiological assay), 0.1 mg/L for isoniazid (spectrofluorimetric assay) and 5.0 mg/L for pyrazinamide (spectrophotometric assay).3

In vivo drug disposition studies

The study was approved by the Institute's Animal Ethics Committee. Different groups of laca mice were administered subcutaneous drug-loaded PLG-NP (5 mg of drug-loaded PLG-NP comprised a therapeutic dose, which was suspended in 100 µL of isotonic saline just before injection) and subcutaneous/oral/intravenous (iv) free drugs (n=16 in each case). The drug doses were rifampicin 12 mg/kg + isoniazid 10 mg/kg + pyrazinamide 25 mg/kg body weight. The control mice were administered with subcutaneous empty PLG-NP or isotonic saline (n=15). At different time intervals (6 and 12 h, and days 1, 2, 3, 7, 11, 15, 21, 28 and 32–36), the mice were bled (six mice per time point in each group) and the drug levels assayed in the plasma. The plasma drug concentration over time data were used to calculate the area under the concentration–time curve (AUC0–{infty}), the mean residence time (MRT) and the absolute bioavailability of each drug. In addition, the animals were sacrificed at various time points, and drug levels were determined in 20% lung/spleen homogenates prepared by homogenizing 50 mg of the tissue in 250 µL of isotonic saline.

Experimental infection and chemotherapy

For the chemotherapeutic studies, the mice were inoculated via the tail vein with 1x105 bacilli of Mycobacterium tuberculosis H37Rv in 0.1 mL of sterile isotonic saline. The infected animals were maintained in biological safety cabinets (Nuaire Cabinets, Model NU-605-600E, Series 6). Fifteen days later, the establishment of infection was confirmed by Ziehl–Neelsen staining of lung/spleen homogenates of two or three animals. Mice were then divided into various groups: groups 1 (n=6) and 2 (n=6) were administered drug-loaded PLG-NP and free drugs, respectively, once subcutaneously; group 3 (n=6) was administered free drugs (prepared freshly by suspending the drugs in 50 µL of isotonic saline) orally daily (conventional chemotherapy) for 5 weeks; and groups 4 (n=5) and 5 (n=5) received a single dose of subcutaneous empty PLG-NP and isotonic saline, respectively. The animals were sacrificed on day 36 following the initiation of chemotherapy. Fifty microlitres of undiluted, 1:100 diluted and 1:1000 diluted aliquots of lung/spleen homogenates were inoculated on Middlebrook 7H10 agar base supplemented with OADC. Colony forming units (cfu) were enumerated on day 21 post-inoculation and the data were analysed by one way analysis of variance (ANOVA) followed by unpaired Student's t-test to compare the untreated and treated groups.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The drug encapsulation efficiency was 56.9±2.7% for rifampicin, 66.3±5.8% for isoniazid and 68.0±5.6% for pyrazinamide. The drug-loading capacity varied from 570–680 mg/g polymer for each drug. A sustained drug release in the plasma for up to 32 days was observed for each drug, following a single subcutaneous dose of PLG-NP. Therapeutic drug concentrations were maintained for up to 36 days in the lungs (rifampicin 0.84±0.1 mg/L, isoniazid 0.99±0.1 mg/L and pyrazinamide 8.1±1.2 mg/L homogenate) and spleen (rifampicin 0.8±0.1 mg/L, isoniazid 1.33±0.3 mg/L and pyrazinamide 8.33±2.0 mg/L homogenate). After the administration of free drugs, the latter were undetectable in the plasma beyond 10–12 h and in the organs beyond 24–48 h of subcutaneous/oral/iv administration. The PLG-NP possibly form a depot at the injection site from where the drugs may be slowly released into the circulation. The coating of nanoparticles with polyvinyl alcohol imparts considerable stability to the particles.5 These factors explain the sustained drug release which resulted in a higher MRT and absolute bioavailability observed with PLG-NP (Table 1). Further, a single subcutaneous shot of drug-loaded PLG-NP resulted in undetectable cfu, whereas a single subcutaneous injection of free drugs resulted in a high bacterial load in the organs of M. tuberculosis-infected mice (Table 2). Although colony counting was performed on day 21 post-inoculation, the cfu plates were observed for 60 days and no visible growth appeared in the case of subcutaneous drug-loaded PLG-NP. However, the daily administration of oral free drugs for 5 weeks resulted in a 2.2 log10 cfu reduction (P<0.001 as compared with the untreated controls). Splenic enlargement and tubercles were observed in untreated controls and animals receiving empty PLG-NP or subcutaneous free drugs. It is clear that a single subcutaneous injection of drug-loaded PLG-NP demonstrated a better chemotherapeutic efficacy as compared with 35 doses of oral free drugs. Although from the patient's point of view, subcutaneous dosing is not a favourite route of drug administration, it is expected that a reduction in dosing frequency from 35 conventional doses to one subcutaneous dose would significantly improve patient compliance and help in the better management of TB.


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Table 1. Pharmacokinetic studies following a single subcutaneous dose of drug-loaded PLG-NP as compared with free drugs in mice

 

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Table 2. Chemotherapeutic efficacy of a single subcutaneous injection of drug-loaded PLG-NP against murine tuberculosis

 
In our previous reports,6,7 the microsphere technology suffered from several demerits, such as a low drug encapsulation efficiency, high polymer consumption and difficulties in the inclusion of pyrazinamide for injectable preparations; these were overcome by the nanosphere technology.1,2 The present communication, employing subcutaneous PLG-NP, suggests a reduction in dosing frequency to one, and merits further evaluation in a higher animal model.


    Footnotes
 
* Corresponding author. Tel: +91-172-2747585, ext. 5174-75; Fax: +91-172-2744401, 2745078; Email: gkkhuller{at}yahoo.co.in


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Fauci, A. S. (2001). Infectious diseases: considerations for the 21st century. Clinical Infectious Diseases 32, 675–85.[CrossRef][ISI][Medline]

2 . Pandey, R., Zahoor, A., Sharma, S. et al. (2003). Nanoparticle encapsulated antitubercular drugs as a potential oral drug delivery system against experimental murine tuberculosis. Tuberculosis (Edinburgh, Scotland) 83, 373–8.

3 . Pandey, R., Sharma, A., Zahoor, A. et al. (2003). Poly (dl-lactide-co-glycolide) nanoparticle-based inhalable sustained drug delivery system for experimental tuberculosis. Journal of Antimicrobial Chemotherapy 52, 981–6.[Abstract/Free Full Text]

4 . Lamprecht, A., Ubrich, N., Hombreiro, P. M. et al. (1999). Biodegradable monodispersed nanoparticles prepared by pressure homogenization-emulsification. International Journal of Pharmaceutics 184, 97–105.[CrossRef][ISI][Medline]

5 . Jain, R., Shah, N. H., Malick, A. W. et al. (1998). Controlled drug delivery by biodegradable poly(ester) devices: different preparative approaches. Drug Development and Industrial Pharmacy 24, 703–27.[ISI][Medline]

6 . Dutt, M. & Khuller, G. K. (2001). Chemotherapy of Mycobacterium tuberculosis infections in mice with a combination of isoniazid and rifampicin entrapped in poly (dl-lactide-co-glycolide) microparticles. Journal of Antimicrobial Chemotherapy 47, 829–35.[Abstract/Free Full Text]

7 . Dutt, M. & Khuller, G. K. (2001). Therapeutic efficacy of poly (dl-lactide-co-glycolide)-encapsulated antitubercular drugs against Mycobacterium tuberculosis infection induced in mice. Antimicrobial Agents and Chemotherapy 45, 363–6.[Abstract/Free Full Text]