ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2001, p. 363–366 0066-4804/01/$04.00⫹0 DOI: 10.1128/AAC.45.1.363–366.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 1
Therapeutic Efficacy of Poly(DL-Lactide-Co-Glycolide)-Encapsulated Antitubercular Drugs against Mycobacterium tuberculosis Infection Induced in Mice MANISHA DUTT
AND
G. K. KHULLER*
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India Received 28 June 2000/Returned for modification 26 August 2000/Accepted 25 October 2000
Poly(DL-lactide-co-glycolide) (PLG) microparticles were developed as carriers for isoniazid and rifampin in order to improve compliance of tuberculous chemotherapy. Antitubercular drugs encapsulated in PLG polymers and injected in a single dose subcutaneously resulted in a sustained release (up to 6 weeks) of drugs in various organs of mice. Further, Mycobacterium tuberculosis H37Rv-infected animals given a single shot of chemotherapy in PLG microparticles exhibited a better or equivalent clearance of CFU in various organs compared to those given a daily administration of free drugs. phosphate-buffered saline (PBS) or normal saline. At different time intervals, mice were sacrificed, and their organs, such as lungs, liver and spleen, were removed. Ten-percent tissue homogenates of the organs were prepared in 0.05 M PBS (pH 7.2), and levels of INH and RIF in the organs were determined by spectrofluorimetric assay (14) and by microbiological assay, respectively (13). Results were expressed as the concentrations of drugs obtained in micrograms per milliliter of tissue homogenates. To investigate the chemotherapeutic potential of drug-containing PLG-mps against tuberculous infections, mice were inoculated intravenously via the lateral tail vein with 1.5 ⫻ 105 viable bacilli of M. tuberculosis H37Rv in a volume of 0.1 ml of 0.9% sterile NaCl solution. Fifteen days postinoculation, establishment of infection was confirmed by Ziehl-Neelsen staining of whole-tissue homogenates of lungs, liver, and spleen from three to four animals. Mice were then divided into various groups, each containing seven to nine animals. Two different doses of the drugs for chemotherapy were used in the study. INH and RIF doses of 75 and 85 mg/kg of body weight, respectively, were referred to as the “high dose” in the study, and the drugs at half the concentrations, i.e., 37.5 and 42.5 mg/kg of body weight, were referred to as the “low dose.” The selected doses of the drugs were based on calculations involv-
In the last decade, tuberculosis (TB) has reemerged as one of the leading causes of death (15). Short-course chemotherapy forms the backbone of antitubercular chemotherapy. Firstline drugs for therapy for TB are generally effective when used properly. However, it has been suggested that one of the major reasons for the increased numbers of multidrug-resistant strains of Mycobacterium tuberculosis is inefficient therapy, probably due to lack of compliance by patients (7). Tuberculocidal therapies that reduce the dosing schedule of the drugs should greatly increase patient compliance. In this regard, microencapsulation technology using various types of polymers could be used to deliver the required doses of the drugs for prolonged time periods by a single shot without causing any toxicity. For this purpose, various types of polymers, notably those of lactic and glycolic acids and their copolymers, such as poly(DL-lactide-co-glycolide) (PLG), have been employed as antitubercular drug delivery vehicles (6, 8). PLG polymers, which are completely biodegradable and biocompatible (1), can be easily formulated into various types of delivery vehicles (5) and administered by various routes (3, 4). In the present communication, we report on the use of PLG microparticles (PLG-mps) as carriers for two of the major front-line antitubercular drugs, i.e., isoniazid (INH) and rifampin (RIF), and their chemotherapeutic potential against experimental tuberculosis in a murine model. PLG-mps containing entrapped antitubercular drugs (INH and RIF) were prepared by the double-emulsification solvent evaporation procedure as described by Edwards et al. (3) with slight modifications. Different groups of 6- to 8-week-old laca mice (weight, 18 to 20 g) of either sex were injected subcutaneously with drugs (INH and RIF) encapsulated in PLG-mps and with free drugs (INH and RIF). The doses of INH and RIF were 75 and 85 mg/kg of body weight of mice, respectively. Controls consisted of mice administered empty PLG-mps and
TABLE 1. Drug disposition studies of different organs of mice after administration of PLG-mps containing antitubercular drugs Treatment groupa
PLG-INH Free INH PLG-RIF Free RIF
* Corresponding author. Mailing address: Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160 012, India. Phone: 747 585, ext. 282, 274. Fax: 744 401, 745 078. E-mail:
[email protected].
a
Time period
Day Day Day Day Day Day
3 42 1 3 42 1
Concn of drugs in organs (g/ml)b Lungs
Liver
Spleen
3.91 ⫾ 0.43 1.66 ⫾ 0.41 NDc 0.5 ⫾ 0 ⬍0.5 ⫾ 0 0.5 ⫾ 0
4.2 ⫾ 0.25 2.49 ⫾ 1.8 1.26 ⫾ 0.11 0.5 ⫾ 0 ⬍0.5 ⫾ 0 3.60 ⫾ 0.4
4.35 ⫾ 0.43 3.33 ⫾ 0 0.4 ⫾ 0.2 ⬍0.5 ⫾ 0 ⬍0.5 ⫾ 0 0.5 ⫾ 0
All groups were administered high dose of drugs. All values are means ⫾ standard deviations of results for four to five animals at each time point. c ND, not detected. b
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FIG. 1. Log CFU in mouse lungs (a), liver (b), and spleen (c) after high- and low-dose chemotherapy of infected animals with INH and RIF. Triple asterisks indicate a P value of ⬍0.001 level of significance for free drugs and drugs given in PLG-mps with respect to controls, determined by one-way analysis of variance. Values are means ⫾ standard deviations for three to four animals.
ing body weight of mice. A dose equivalent to that for a 70-kg adult human was determined for mice (12). Mice, being fast metabolizers of drugs, would naturally be required to be administered the high doses of the drug(s). All the drug treatments were administered via the subcutaneous route. Groups 1 and 2 were administered PLG-mps containing INH and RIF at high doses. Groups 3 and 4 were administered PLG-mps con-
taining INH and RIF at low doses. Groups 5 and 6 were administered free INH and RIF at high doses. Groups 7 and 8 were administered free INH and RIF at low doses. Groups 9 and 10 consisted of controls administered blank PLG-mps and PBS or normal saline. Drug therapy using PLG-mps was administered as a single subcutaneous dose, while free drug treatment was given daily subcutaneously for a period of 6 weeks.
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FIG. 1—Continued.
Statistical analysis was done using one-way analysis of variance and further by using the Student’s t test to compare the free drug and PLG treatment groups. Mice were bled on days 3 and 6 post-completion of chemotherapy. The levels of alkaline phosphatase, serum glutamate pyruvate transaminase, and total bilirubin in the plasma samples were determined by using Boehringer Mannheim (Mannheim, Germany) kits for evaluation of hepatotoxicity, if induced. Animals were sacrificed on day 7 post-completion of therapy. The organs (lungs, liver, and spleen) were homogenized and cultured on plates containing modified Youman’s medium supplemented with 1% bovine serum albumin for enumeration of CFU. After 3 to 4 weeks, colonies were counted and CFU were determined as described earlier (11). The results of this study are presented in Table 1, which shows the concentrations of drugs (INH and RIF) obtained in various organs of mice after administration of free drugs and drugs encapsulated in PLG-mps. A sustained release of both INH and RIF was obtained in the lungs, liver, and spleen at different time intervals ranging from day 3 post-administration of high-dose PLG-mps to day 42. Administration of free drugs showed drug concentrations in various organs up to the first day only, and later release was not seen in any tissues. The data presented here are for only two time points. Drug concentrations obtained were much higher than the MICs of the drugs. Figure 1a shows the log CFU obtained in the lungs of mice after high- and low-dose chemotherapy of infected animals with INH and RIF. Treatment in all the groups resulted in a significant (P ⬍ 0.001) clearance of bacilli compared to the controls. Daily high-dose treatment with free drugs resulted in a significantly better clearance of bacilli in INH (P ⬍ 0.01) and RIF (P ⬍ 0.001) groups compared to single-dose PLG-INH and PLG-RIF treatment, respectively. Low-dose treatment
with PLG resulted in a significantly better clearance of PLGINH (P ⬍ 0.001), by 2.45-fold, and PLG-RIF (P ⬍ 0.001), by 3.32-fold, compared to daily free INH and RIF treatment, respectively. Figure 1b shows the log CFU obtained in the livers of mice after high-dose and low-dose chemotherapy of infected animals with INH and RIF. Treatment in all the groups resulted in a significant (P ⬍ 0.001) clearance of bacilli compared to the controls. High-dose PLG-INH treatment showed a significant (P ⬍ 0.001) reduction in the number of CFU by 3.01 log units compared to the controls and showed a significantly (P ⬍ 0.001) better clearance of bacilli, by 5.02-fold, compared to daily free INH treatment. The clearance of bacilli observed for high-dose PLG-RIF treatment was comparable to that for free RIF treatment. One dose of PLG-INH and PLG-RIF treatment in the low-dose group in the liver showed a clearance of bacilli equivalent to that of the free drugs. Figure 1c shows the log CFU obtained in the spleens of mice after high-dose and low-dose chemotherapy of infected animals with INH and RIF. Treatment in all the groups resulted in a significant (P ⬍ 0.001) clearance of bacilli compared to the controls. High-dose INH treatment showed equivalent clearances of bacilli in PLG-INH and free INH treatment groups. However, high-dose PLG-RIF treatment resulted in a significantly (P ⬍ 0.01) better clearance of bacilli, by 1.97-fold, than free RIF treatment. In the low-dose group, PLG-INH treatment showed a significantly (P ⬍ 0.001) better clearance of CFU, by 9.4-fold, than free INH treatment. The clearance obtained with low-dose RIF treatment was equivalent in the PLG-RIF and free RIF groups. In addition, hepatotoxicity studies revealed that there was no change in the levels of alkaline phosphatase, serum glutamate pyruvate transaminase, and total bilirubin compared to those for the controls after the completion of chemotherapy.
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These results suggest that these formulations do not induce any hepatotoxicity on a biochemical basis. The results of this study reveal that by using microsphere technology, it is possible to effectively treat an M. tuberculosis infection in a murine model. It was demonstrated that a single injection of drugs encapsulated in PLG-mps could significantly reduce the number of CFU in various organs up to 6 weeks of treatment in mice compared to results with a daily subcutaneous dose of free drugs. Daily subcutaneous doses of the free drugs were used in order to accomplish significant reductions in CFU similar to those seen with PLG treatment. Previous studies using implants in both mice (6) and rabbits (9) have shown the effectiveness of microsphere technology as sustained-release carriers of anti-TB drugs, but they had limitations in terms of surgical insertions of implants and tedious preparation methods. Encouraging reports have also shown this technology to be effective both ex vivo in macrophages (2) and in vivo studies with mice (12), but a reduction in CFU was observed only up to 26 days postinfection. In this report, we have shown for the first time that PLG can function as an effective sustained release carrier of antitubercular drug(s) up to 6 weeks in all target organs of mice. In addition, the CFU data correlate with results of the drug release studies, indicating that the drug was effective in significant reduction of CFU from the organs. In brief, this study suggests that a daily drug dose could be effectively replaced with one-shot chemotherapy using PLG polymers without inducing any hepatotoxicity. This technology would improve patients’ compliance, the lack of which is the major reason for the development of multidrug-resistant strains of mycobacteria. REFERENCES 1. Anderson, J. M., and M. S. Shive. 1997. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv. Drug Deliv. Rev. 28:5–24.
2. Barrow, E. L. W., G. A. Winchester, J. K. Staas, D. C. Quenelle, and W. W. Barrow. 1998. Use of microsphere technology for the targeted delivery of rifampicin to Mycobacterium tuberculosis-infected macrophages. Antimicrob. Agents Chemother. 42:2682–2689. 3. Edwards, D. A., J. Hanes, G. Caponetti, J. Hrkach, A. B. Jebria, M. L. Eskew, J. Mintzes, D. Deaver, N. Lotan, and R. Langer. 1997. Large porous particles for pulmonary drug delivery. Science 276:1868–1871. 4. Gangadharam, P. R. J., D. R. Ashtekar, D. C. Farhi, and D. L. Wise. 1991. Sustained release of isoniazid in vivo from a single implant of a biodegradable polymer. Tubercle 72:115–122. 5. Gangadharam, P. R. J., M. Hill, L. Kesavalu, D. R. Ashtekar, K. Parikh, and D. L. Wise. 1989. Sustained release of isoniazid in vivo from a biodegradable polymer implant. Proceedings of the 24th Joint Conference on Tuberculosis. U.S.-Japan Cooperative Medical Science Programme. Tubercle 1989:54–58. 6. Gangadharam, P. R. J., S. Kailasam, S. Srinivasan, and D. L. Wise. 1994. Experimental chemotherapy of tuberculosis using single dose treatment with isoniazid in biodegradable polymers. J. Antimicrob. Chemother. 33: 265–271. 7. Goble, M. 1994. Drug resistance, p. 259–284. In L. N. Friedman (ed.), Tuberculosis. Current concepts and treatment. CRC Press, Boca Raton, Fla. 8. Hsu, Y. Y., J. D. Gresser, D. J. Trantolo, C. M. Lyons, P. R. J. Gangadharam, and D. L. Wise. 1994. In vitro controlled release of isoniazid from poly (lactide-co-glycolide) matrices. J. Control. Release 31:223–228. 9. Kailasam, S., D. Daneluzzi, and P. R. J. Gangadharam. 1994. Maintenance of therapeutically active levels of isoniazid for prolonged periods in rabbits after a single implant of biodegradable polymer. Tuber. Lung Dis. 75:361– 365. 10. Paget, G. E., and J. M. Barnes. 1964. In D. R. Lawrence and A. L. Bacharach (ed.), Evaluation of drug activities: pharmacometrics, vol. 1. Academic Press, New York, N.Y. 11. Pancholi, P., V. K. Vinayak, and G. K. Khuller. 1989. Immunogenicity of ribonucleic acid protein fraction of Mycobacterium tuberculosis encapsulated in liposomes. J. Med. Microbiol. 29:131–139. 12. Quenelle, D. C., J. K. Staas, G. A. Winchester, E. L. W. Barrow, and W. W. Barrow. 1999. Efficacy of microencapsulated rifampicin in Mycobacterium tuberculosis-infected mice. Antimicrob. Agents Chemother. 43:1144–1151. 13. Saito, H., and H. Tomoika. 1989. Therapeutic efficacy of liposome-entrapped rifampicin against Mycobacterium avium complex infections induced in mice. Antimicrob. Agents Chemother. 33:429–433. 14. Scott, E. H., and R. C. Wright. 1967. Fluorimetric determination of isonicotinic acid hydrazide in serum. J. Lab. Clin. Med. 70:355–360. 15. World Health Organization. 1997. Treatment of tuberculosis: guidelines for national programmes, 2nd ed. World Health Organization publication no. TB/97.220. World Health Organization, Geneva, Switzerland.
