Subcutaneous nanoparticle-based antitubercular chemotherapy in an experimental model

JAC Journal of Antimicrobial Chemotherapy (2004) 54, 266–268 DOI: 10.1093/jac/dkh260 Advance Access publication 5 May 2004 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 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 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-coglycolide) 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 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 508C 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)100. 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 drugloaded PLG-NP (5 mg of drug-loaded PLG-NP comprised a therapeutic dose, which was suspended in 100 mL 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 – 1), the mean residence time (MRT) and the absolute .......................................................................................................................................................................................................................................................................................................................................................................................................................... *Corresponding author. Tel: +91-172-2747585, ext. 5174-75; Fax: +91-172-2744401, 2745078; E-mail: .......................................................................................................................................................................................................................................................................................................................................................................................................................... 266 JAC vol.54 no.1 q The British Society for Antimicrobial Chemotherapy 2004; all rights reserved. Nanoparticle-based antitubercular chemotherapy 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 mL of isotonic saline. Experimental infection and chemotherapy For the chemotherapeutic studies, the mice were inoculated via the tail vein with 1105 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 mL 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 untreate (...truncated)