- Email: [email protected]
Inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis
International Journal of Antimicrobial Agents 26 (2005) 298–303
Inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis A. Zahoor, Sadhna Sharma, G.K. Khuller ∗ Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India Received 7 April 2005; accepted 19 July 2005
Abstract Pharmacokinetic and chemotherapeutic studies have been carried out with aerosolised alginate nanoparticles encapsulating isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA). The nanoparticles were prepared by cation-induced gelification of alginate and were 235.5 ± 0 nm in size, with drug encapsulation efficiencies of 70–90% for INH and PZA and 80–90% for RIF. The majority of particles (80.5%) were in the respirable range, with mass median aerodynamic diameter of 1.1 ± 0.4 m and geometric standard deviation of 1.71 ± 0.1 m. The relative bioavailabilities of all drugs encapsulated in alginate nanoparticles were significantly higher compared with oral free drugs. All drugs were detected in organs (lungs, liver and spleen) above the minimum inhibitory concentration until 15 days post nebulisation, whilst free drugs stayed up to day 1. The chemotherapeutic efficacy of three doses of drug-loaded alginate nanoparticles nebulised 15 days apart was comparable with 45 daily doses of oral free drugs. Thus, inhalable alginate nanoparticles can serve as an ideal carrier for the controlled release of antitubercular drugs. © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Alginate; Antitubercular drugs; Tuberculosis; Nebulisation
1. Introduction The failure of antitubercular chemotherapy is mainly due to patient non compliance [1], which is attributed to the requirement of multidrug administration daily or several times a week for at least 6 months. Although attempts to improve patient non compliance by applying modified drug delivery systems for antimycobacterial agents are encouraging [2], higher polymer consumption [3], lower drug entrapment [4], higher cost, use of toxic substances and organic solvents during preparation [5,6], and surgical requirements [7] are all drawbacks associated with these drug delivery systems. Moreover, these particulate systems (liposomes, microparticles and nanoparticles) used as drug carriers are rapidly taken up from the blood by mononuclear phagocytes, especially by the Kupffer cells in the liver, which is a drawback to gaining access to other target sites in ∗ Corresponding author. Tel.: +91 172 275 5174/75; fax: +91 172 274 4401/5078. E-mail address: [email protected] (G.K. Khuller).
the body, e.g. the lungs [8]. In addition, the high frequency of pulmonary tuberculosis demands the development of novel drug delivery approaches that enhance the bioavailability of drugs at the level of lungs. In recent years, one of the best ways to achieve higher drug levels in the lungs has been the development of new formulations (nanoparticle-based) that are directly delivered to the lungs via the aerosol route [9]. The present study was planned with an aim of developing a natural polymer-based inhalable drug delivery system to overcome the limitations associated with various drug delivery systems. Sodium alginate, a natural polymer (d-mannuronic acid and ␣-l-guluronic acid) with properties such as an aqueous matrix environment, high gel porosity and biocompatibility, and approved by the US Food and Drug Administration (FDA) for oral use [10,11], was used to prepare nanoparticles encapsulating three antitubercular drugs (ATDs). Alginate nanoparticles containing isoniazid (INH), pyrazinamide (PZA) and rifampicin (RIF) were developed and characterised, and pharmacokinetic and pharmacodynamic evaluation was carried out via the aerosol route in guinea pigs.
0924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2005.07.012
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
2. Materials and methods 2.1. Chemicals Sodium alginate (medium viscosity, 3500 cps for a 2% w/v solution), chitosan (minimum 85% deacetylated), INH, RIF and PZA were obtained from Sigma Chemical Co. (St Louis, MO). Middlebrook 7H10 agar was obtained from Difco (Detroit, MI). All other reagents were of analytical grade obtained from standard companies. 2.2. Animals Guinea pigs of either sex, weighing 250–350 g, were obtained from Haryana Agricultural University, Hisar, India. Animals were housed in animal isolators (NU 605-600E, Series 6; NuAire Instruments, Plymouth, MN) and provided with pellet diet/water ad libitum. The study was approved by the Institute’s Animal Ethics Committee.
299
where final drug concentration includes both forms (encapsulated + free) and alginate final concentration is the concentration of alginate in the final solution, i.e. after the addition of CaCl2 and chitosan. RIF was analysed by microbiological assay that was specific for the drug, using Bacillus subtilis (MTCC 441) as the indicator strain (sensitivity 0.25 g/mL, linearity 0.25–10 g/mL) [12]. INH was estimated by a spectrofluorimetric method based on the principle that in the presence of salicylaldehyde, the drug forms a hydrazone that is soluble in isobutanol and a highly fluorescent compound that can be measured at an excitation wavelength of 392 nm and emission of 478 nm (sensitivity 0.1 g/mL, linearity 0.1–10 g/mL) [13]. PZA was analysed spectrophotometrically based on the formation of a coloured complex between the drug and sodium nitroprusside at alkaline pH which was measured at 495 nm (sensitivity 5.0 g/mL, linearity 5–10 g/mL) [14]. 2.5. Nebulisation of alginate nanoparticles
2.3. Culture Mycobacterium tuberculosis H37 Rv, originally obtained from National Collection of Type Cultures (NCTC), was maintained on modified Youman’s media. 2.4. Development and characterisation of drug-loaded alginate nanoparticles Alginate nanoparticles were prepared by the method of Rajaonarivony et al. [8] with slight modifications. The principle involves cation-induced controlled gelification of alginate. Briefly, calcium chloride (0.5 mL, 18 mM) was added to 9.5 mL of sodium alginate solution (0.06%) containing different amounts of INH, PZA and RIF. The initial ratio of drug:polymer was 7.5:1 (% w/w). Two millilitres of chitosan solution (0.05%) was added, followed by stirring for 30 min and storage at room temperature overnight. Drugloaded nanoparticles were recovered by centrifugation at 19 000 rpm for 35 min and washed thrice with distilled water. Drug-free nanoparticles were also prepared in the same manner. Alginate nanoparticles were characterised for their size and polydispersity index on a Zetasizer 1000 HS (Malvern Instruments, Malvern, UK). The amount of drug entrapped in alginate nanoparticles was calculated by estimating the amount of unentrapped drug recovered in the supernatant and pellet washings after centrifugation. The drug encapsulation efficiency was calculated as the percentage of drug entrapped in alginate nanoparticles compared with the initial amount of drug. The drug loading capacity (L) of alginate nanoparticles was determined as described by Rajaonarivony et al. [8] using the following equation: L(% w/w) =
A compressor nebuliser system (Medel Aerofamily, Italy) was used in the study. Each animal received drugs suspended in 4 mL of 0.9% NaCl via an appropriately modified paediatric facemask connected to the nebuliser, with an exposure time of 4 min, to receive the dose as described in our previous studies [2]. Nebulised alginate nanoparticles were sized on a 7stage Andersen Cascade Impactor (Andersen Samplers, Inc., Atlanta, GA) and evaluated for mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). An adjustable bleed valve on the pump that required periodic calibration controlled the flow rate through the impactor. The pump required 12 V automotive type battery (DC unit) drawing a current of 11 amps. 2.6. In vivo drug disposition studies Throughout the study, the drugs were used at therapeutic dosage (INH, 10 mg/kg; RIF, 12 mg/kg and PZA 15 mg/kg body weight). Since the dose was different for the three drugs, the initial amount of each drug required for nanoparticle 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, 7.5fold lesser alginate was used in the preparation process. The basic procedure for nanoparticle preparation remained the same as discussed above. For a 400 g guinea pig, 21.3 mg of dried nanoparticles constituted a therapeutic dose containing 4.8 mg RIF + 4 mg INH + 10 mg PZA.
Final drug concentration − drug concentration in supernatant Alginate final concentration
300
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
For single-dose drug disposition studies, guinea pigs were grouped as follows, with six animals in each group: Group 1, oral free drugs; Group 2, free drugs, aerosol; Group 3, nanoparticles loaded with ATDs, aerosol; and Group 4, nebulised drug-free nanoparticles (a positive control to explore the influence of alginate nanoparticles on drug estimations). The animals were bled at several time points and plasma was analysed for ATDs as described earlier. The area under the plasma drug concentration over time curve (AUC) was calculated using data analysis tools in SigmaPlot software (version 8.0) and further used to compute the relative bioavailability of each drug. Relative bioavailability = (AUC0–∞ of oral encapsulated drugs/AUC0–∞ of oral free drugs) × (dose of oral free drugs/dose of oral encapsulated drugs). Animals were sacrificed at different time points and drug levels were determined in 20% w/v tissue homogenates (lungs, liver and spleen) as described for plasma.
analysed by analysis of variance (ANOVA) to compare the control and treated groups.
4. Results 4.1. Physicochemical characterisation of alginate nanoparticles Alginate nanoparticles had an average size of 235.5 ± 0 nm, with a polydispersity index of 0.439. Drug encapsulation efficiency was observed to be 80–90% for RIF and 70–90% for INH and PZA. The drug loading capacity of alginate nanoparticles was 586–654 mg, 480–600 mg and 468–576 mg for RIF, INH and PZA, respectively, per 100 mg of alginate. 4.2. Aerodynamic characterisation of nebulised alginate nanoparticles
2.7. Experimental infection and chemotherapy Animals were infected intramuscularly with 1.5 × 105 viable bacilli of M. tuberculosis H37 Rv in 0.1 mL of 0.9% NaCl. Infection was confirmed by the presence of mycobacteria (by acid-fast staining) in tissue smears of spleens and lungs of two to three animals after 20 days. Animals were then divided into the following groups, with six animals in each group: Group I, untreated control; Group II, oral free drugs; Group III, drug-loaded nanoparticles, aerosol; and Group IV, empty nanoparticles, aerosol. Control animals received phosphate-buffered saline only, whilst other groups received the same drug doses as described under ‘in vivo studies’. Free drugs were administered daily, whilst the schedule of nanoparticle-based therapy was 15 days apart (based on the in vivo drug release studies in plasma and organs). Animals were sacrificed after 45 days of chemotherapy. Lungs and spleen were removed aseptically and homogenised in 3 mL of sterile normal saline. Then, 50 L of 1:100 dilution (prepared in normal saline) of tissue homogenates was plated on Middlebrook 7H10 agar plates. Colony-forming units (CFUs) were enumerated 30 days post inoculation. 2.8. Biochemical hepatotoxicity To evaluate hepatotoxic effects of ATDs, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin levels were evaluated on day 46 of chemotherapy using standard kits obtained from Transasia Bio-medicals Limited (Daman, India).
3. Statistical analysis The pharmacokinetic data, including bioavailability, were analysed by Student’s unpaired t-test, and CFU data were
Aerodynamic characterisation revealed that nearly 80.5% of nebulised alginate nanoparticle droplets were in the size range 0.4–2.1 m (respirable range), with a MMAD of 1.1 ± 0.4 m and a GSD of 1.71 ± 0.1 m. 4.3. In vivo drug disposition studies When the alginate nanoparticles were administered through nebulisation, the drugs were detected in plasma from 3 h onwards. INH, RIF and PZA were observed up to 14, 10 and 14 days, respectively. In contrast, free drugs were cleared from the circulation within 12–24 h (Fig. 1). All the pharmacokinetic parameters, which include maximum plasma concentration (Cmax ), time to reach maximum plasma concentration (Tmax ) and AUC0–∞ , were found to be higher for alginate nanoparticle-encapsulated drugs upon administration via the aerosol route, resulting in a significant (P < 0.001) increase in relative bioavailability of encapsulated drugs compared with free drugs (Table 1). All drugs were detected in tissues, i.e. lungs, liver and spleen, above the minimum inhibitory concentration (MIC) up to 15 days (Fig. 2) [15], thus prompting us to design the fortnightly schedule of nebulised drug-loaded alginate nanoparticles for chemotherapy. 4.4. Chemotherapeutic efficacy Administration of nebulised alginate nanoparticles fortnightly (three doses spaced 15 days apart) for 45 days and of oral free drugs administered daily for 45 days to M. tuberculosis-infected guinea pigs resulted in undetectable mycobacterial CFUs in lung and spleen homogenates (1:100 dilution) (Table 2). However, guinea pigs administered empty alginate nanoparticles and untreated controls exhibited comparable CFU counts.
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
301
Fig. 1. Plasma drug profile following a single nebulisation of antitubercular drug-loaded alginate nanoparticles and free drugs to guinea pigs. Values are mean ± S.D. of six animals. RIF, rifampicin; INH, isoniazid; PZA, pyrazinamide.
4.5. Hepatotoxicity studies On the basis of serum bilirubin, ALT and ALP, the formulation did not produce any hepatotoxicity (Table 3). The normal range of these analytes has been previously established in healthy guinea pigs [10]. Thus, there was no biochemical evidence of hepatotoxicity in either group.
5. Discussion To overcome the daily dosing problem of tuberculous therapy, the application of drug delivery systems is a novel
strategy [16]. Alginate nanoparticles are a drug delivery system bearing the benefits of sustained release properties of nanoparticles, but also carry additional advantages, including the least use of organic solvents, no involvement of toxic molecules, least use of equipment, and reduced reticuloendothelial system uptake due to the stealth nature of alginate [8]. The present study explores the therapeutic potential of alginate nanoparticles encapsulating ATDs against experimental tuberculosis through the aerosol route in guinea pigs. We have modified the original protocol for nanoparticle production at two steps, namely replacement of poly-l-lysine with chitosan and reduction in the polymer:drug ratio. These modifications have great implications, as chitosan is a com-
Table 1 Pharmacokinetics of nebulised antitubercular drug-loaded alginate nanoparticles compared with oral free drugs in guinea pigs Group Oral free drugs Rifampicin Isoniazid Pyrazinamide
Cmax (g/mL)
Tmax (h)
Kel
T1/2 (h)
AUC0–∞ (g · h/mL)
Relative bioavailability
0.8 ± 0.18 2.5 ± 0.13 22.0 ± 1.63
1 1 0.5
−0.15 ± 0.0 −0.21 ± 0.01 −0.1 ± 0.01
4.5 ± 0.53 3.1 ± 0.53 6.5 ± 1.43
8.37 ± 1.10 10.93 ± 1.90 185.00 ± 10.00
1.00 1.00 1.00
Aerosol alginate nanoparticles Rifampicin 1.12 ± 0.02 Isoniazid 4.90 ± 0.3 Pyrazinamide 35.00 ± 1.1
96 168 144
−0.158 ± 0.01 −0.59 ± 0.01 −4.41 ± 0.09
4.39 ± 0.06 1.17 ± 0.3 0.16 ± 0.02
138.46 ± 5.32 521.45 ± 32.4 7776.00 ± 50.34
16.54 47.71 42.02
Cmax , maximum plasma concentration; Tmax , time to reach maximum plasma concentration; Kel , elimination half-life; T1/2 , absorption half-life; AUC, area under the plasma drug concentration over time curve.
302
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
Fig. 2. Tissue levels of antitubercular drugs on day 15 following aerosol administration of alginate nanoparticles to guinea pigs. Values are mean ± S.D. of six animals. RIF, rifampicin; INH, isoniazid; PZA, pyrazinamide.
pletely non-toxic molecule [17] whilst poly-l-lysine has been reported to have some toxicity [18]. In most drug delivery systems, the higher polymer (encapsulating material) consumption becomes a limiting factor because of cost and the final
dose size of the formulation (which is one of the critical factors for administration); thus, reduction in the polymer:drug ratio is a major concern in drug delivery technology. The higher drug loading observed for alginate nanoparticles in
Table 2 Chemotherapeutic efficacy of aerosolised alginate nanoparticle encapsulating antitubercular drugs against experimental tuberculosis in guinea pigs Group
Untreated controls Empty alginate nanoparticles every 15 days, aerosol (three doses) Drug-loaded alginate nanoparticles every 15 days, aerosol (three doses) Free drugs daily, orally (45 doses)
log10 CFUsa Lung (right caudal lobe)
Spleen (whole organ)
5.8 ± 0.1 5.8 ± 0.3* <1.0b <1.0b
5.9 ± 0.1 5.9 ± 0.1 <1.0b <1.0b
CFUs, colony-forming units. a Results are based on visible growth of Mycobacterium tuberculosis on Middlebrook 7H10 agar on day 21 post inoculation. Results are mean ± S.D. (N = 5–6). b Value <1.0 indicates no detectable CFUs following the inoculation of 50 L of neat and 1:10 diluted tissue homogenates. * P > 0.05 according to ANOVA. Table 3 Evaluation of aerosolised antitubercular drug-loaded alginate nanoparticles for biochemical hepatotoxicity in Mycobacterium tuberculosis H37 Rv-infected guinea pigs Group
Serum bilirubin (mg/100 mL)
Serum ALT (U/L)
Serum ALP (U/L)
Untreated controls (N = 5) Nebulised drug-loaded alginate formulation every 15 days (three doses) (N = 5) Oral free drugs daily (45 doses) (N = 5)
0.10–1.00 0.28–0.37 0.26–0.42
19–70 17–30 20–42
10–70 9–74 43–55
ALT, alanine aminotransferase; ALP, alkaline phosphatase.
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
this study can be attributed to the higher drug:polymer ratio as well as the high gel porosity of alginate [8]. The prolonged plasma retention of drugs encapsulated in alginate nanoparticles compared with free drugs might be attributed to their longer half-life coupled with the sustained release potential and the affinity of alginate for amino groups in the case of INH and PZA [8]. In the encapsulated form, RIF stayed in the plasma for up to day 10, whilst INH and PZA were found to reside for up to 14 days. This variation in residence time of RIF in plasma compared with INH and PZA could be due to the hydrophobic nature of this drug and hence its predilection towards tissues rather than plasma. The bioavailabilities of drugs encapsulated in alginate nanoparticles were significantly increased (P < 0.001), as evident from the plasma profile (increased Cmax and Tmax ) and pharmacokinetic evaluation (increased Cmax and AUC0–∞ ) compared with free drugs. The detection of INH, RIF and PZA in tissues demonstrated that alginate nanoparticles encapsulating ATDs, when administered through the aerosol route, not only increased local (lung) drug bioavailability but also the bioavailability of each drug at other sites of the body. The retention of all the drugs in organs, i.e. lungs, liver and spleen, above the MIC for 15 days following nebulisation of alginate nanoparticles favours their application against tuberculosis where infection is largely localised in tissues. Accordingly, a fortnightly schedule of chemotherapy was designed for alginate nanoparticles (Table 2). It was observed that only three nebulised doses of this formulation, administered every 15 days, were equiefficient to 45 doses of oral free drugs administered daily in achieving undetectable CFUs. The formulation was found to elicit no signs of hepatotoxicity, supporting their safety. The chemotherapeutic benefits of liposomes [4], poly-(dl-lactide-co-glycolide) (PLG) microparticles, PLG nanoparticles [2] and alginate beads [3] have been demonstrated previously. The main advantages of the abovementioned drug delivery systems include the sustained release of drugs over longer durations, prevention of premature drug degradation and reduction in drug toxicity. The various disadvantages include higher polymer consumption, lower drug entrapment, higher cost and risk of organic residuals. Alginate nanoparticles bear the advantages of all these systems whilst simultaneously overcoming all of their limitations, thus representing the most suitable carrier for the delivery of ATDs. This study reports for the first time the application of alginate nanoparticles as carriers of ATDs to improve their therapeutic benefit and in turn patient compliance for effective tuberculosis control. As tuberculosis therapy requires multiple drugs in different combinations depending on the patient category, future studies will be required to encapsulate other ATDs and to evaluate them in the same way. Later,
303
clinical trials will be needed to evaluate this system before use in humans.
References [1] Burman WJ, Cohn DL, Rietmeijer CA, Judson FN, Sbarbaro JA, Reves RR. Noncompliance with directly observed therapy for tuberculosis. Epidemiology and effect on the outcome of treatment. Chest 1997;111:1168–73. [2] Pandey R, Zahoor A, Sharma S, Khuller GK. Nanoparticle encapsulated antitubercular drugs as a potential oral drug delivery system against murine tuberculosis. Tuberculosis (Edinb) 2003;83:373–8. [3] Ain QU, Sharma S, Khuller GK, Garg SK. Alginate-based oral drug delivery system for tuberculosis: pharmacokinetic and therapeutic effects. J Antimicrob Chemother 2003;51:931–8. [4] Labana S, Pandey R, Sharma S, Khuller GK. Chemotherapeutic activity against murine tuberculosis of once weekly administered drugs (INH and RIF) encapsulated in liposomes. Int J Antimicrob Agents 2002;20:301–4. [5] Bala I, Hariharan S, Ravi Kumar MNV. PLGA nanoparticles in drug delivery: the state of the art. Crit Rev Ther Drug Carrier Syst 2004;21:387–422. [6] Shah SP, Misra A. Development of liposomal amphotericin B dry powder inhaler formulation. Drug Deliv 2004;11:247–53. [7] Hurley L, Andersen BR. Biodegradable implants from poly-(alphahydroxy acid) polymers for isoniazid delivery. Int J Tuberc Lung Dis 1999;3:1015–24. [8] Rajaonarivony M, Vauthier C, Couarraze G, Puisieux F, Couvreur P. Development of a new drug carrier made from alginate. J Pharm Sci 1993;82:912–7. [9] Jacobs C, Muller RH. Production and characterization of a budesonide nanosuspension for pulmonary administration. Pharm Res 2002;19:189–94. [10] Pandey R, Khuller GK. Chemotherapeutic potential of alginate– chitosan microspheres as antitubercular drug carriers. J Antimicrob Chemother 2004;53:635–40. [11] Tonnesen HH, Karlsen J. Alginate in drug delivery systems. Drug Dev Ind Pharm 2002;28:621–30. [12] Saito H, Tomioka H. Therapeutic efficacy of liposome-entrapped rifampin against M. avium complex infection induced in mice. Antimicrob Agents Chemother 1989;4:429–33. [13] Scott EM, Wright RC. Fluorimetric determination of isonicotinic acid hydrazide in serum. J Lab Clin Med 1967;70:355–60. [14] Gurumurthy P, Nair NGK, Sarma GR. Methods for the estimation of pyrazinamide and pyrazinoic acid in body fluids. Indian J Med Res 1980;71:129–34. [15] Rattan A. Development of new drugs for tuberculosis. In: Gupta, S, Sood, OP, editors. Tuberculosis. Proceedings of the 7th Round Table Conference. New Delhi, India: Ranbaxy Research Foundations; 2000. p. 113–22. [16] Pandey R, Sharma A, Zahoor A, Sharma S, Khuller GK, Prasad B. Poly(dl-lactide-co-glycolide) nanoparticle-based inhalable sustained drug delivery system for experimental tuberculosis. J Antimicrob Chemother 2003;52:981–6. [17] Berrada M, Serreqi A, Dabbarh F, Owusu A, Gupta A, Lehnert S. A novel non-toxic camptothecin formulation for cancer chemotherapy. Biomaterials 2005;26:2115–20. [18] Wen J, Mao HQ, Li W, Lin KY, Leong KW. Biodegradable polyphosphoester micelles for gene delivery. J Pharm Sci 2004;93:2142–57.
Inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis A. Zahoor, Sadhna Sharma, G.K. Khuller ∗ Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India Received 7 April 2005; accepted 19 July 2005
Abstract Pharmacokinetic and chemotherapeutic studies have been carried out with aerosolised alginate nanoparticles encapsulating isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA). The nanoparticles were prepared by cation-induced gelification of alginate and were 235.5 ± 0 nm in size, with drug encapsulation efficiencies of 70–90% for INH and PZA and 80–90% for RIF. The majority of particles (80.5%) were in the respirable range, with mass median aerodynamic diameter of 1.1 ± 0.4 m and geometric standard deviation of 1.71 ± 0.1 m. The relative bioavailabilities of all drugs encapsulated in alginate nanoparticles were significantly higher compared with oral free drugs. All drugs were detected in organs (lungs, liver and spleen) above the minimum inhibitory concentration until 15 days post nebulisation, whilst free drugs stayed up to day 1. The chemotherapeutic efficacy of three doses of drug-loaded alginate nanoparticles nebulised 15 days apart was comparable with 45 daily doses of oral free drugs. Thus, inhalable alginate nanoparticles can serve as an ideal carrier for the controlled release of antitubercular drugs. © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Alginate; Antitubercular drugs; Tuberculosis; Nebulisation
1. Introduction The failure of antitubercular chemotherapy is mainly due to patient non compliance [1], which is attributed to the requirement of multidrug administration daily or several times a week for at least 6 months. Although attempts to improve patient non compliance by applying modified drug delivery systems for antimycobacterial agents are encouraging [2], higher polymer consumption [3], lower drug entrapment [4], higher cost, use of toxic substances and organic solvents during preparation [5,6], and surgical requirements [7] are all drawbacks associated with these drug delivery systems. Moreover, these particulate systems (liposomes, microparticles and nanoparticles) used as drug carriers are rapidly taken up from the blood by mononuclear phagocytes, especially by the Kupffer cells in the liver, which is a drawback to gaining access to other target sites in ∗ Corresponding author. Tel.: +91 172 275 5174/75; fax: +91 172 274 4401/5078. E-mail address: [email protected] (G.K. Khuller).
the body, e.g. the lungs [8]. In addition, the high frequency of pulmonary tuberculosis demands the development of novel drug delivery approaches that enhance the bioavailability of drugs at the level of lungs. In recent years, one of the best ways to achieve higher drug levels in the lungs has been the development of new formulations (nanoparticle-based) that are directly delivered to the lungs via the aerosol route [9]. The present study was planned with an aim of developing a natural polymer-based inhalable drug delivery system to overcome the limitations associated with various drug delivery systems. Sodium alginate, a natural polymer (d-mannuronic acid and ␣-l-guluronic acid) with properties such as an aqueous matrix environment, high gel porosity and biocompatibility, and approved by the US Food and Drug Administration (FDA) for oral use [10,11], was used to prepare nanoparticles encapsulating three antitubercular drugs (ATDs). Alginate nanoparticles containing isoniazid (INH), pyrazinamide (PZA) and rifampicin (RIF) were developed and characterised, and pharmacokinetic and pharmacodynamic evaluation was carried out via the aerosol route in guinea pigs.
0924-8579/$ – see front matter © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2005.07.012
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
2. Materials and methods 2.1. Chemicals Sodium alginate (medium viscosity, 3500 cps for a 2% w/v solution), chitosan (minimum 85% deacetylated), INH, RIF and PZA were obtained from Sigma Chemical Co. (St Louis, MO). Middlebrook 7H10 agar was obtained from Difco (Detroit, MI). All other reagents were of analytical grade obtained from standard companies. 2.2. Animals Guinea pigs of either sex, weighing 250–350 g, were obtained from Haryana Agricultural University, Hisar, India. Animals were housed in animal isolators (NU 605-600E, Series 6; NuAire Instruments, Plymouth, MN) and provided with pellet diet/water ad libitum. The study was approved by the Institute’s Animal Ethics Committee.
299
where final drug concentration includes both forms (encapsulated + free) and alginate final concentration is the concentration of alginate in the final solution, i.e. after the addition of CaCl2 and chitosan. RIF was analysed by microbiological assay that was specific for the drug, using Bacillus subtilis (MTCC 441) as the indicator strain (sensitivity 0.25 g/mL, linearity 0.25–10 g/mL) [12]. INH was estimated by a spectrofluorimetric method based on the principle that in the presence of salicylaldehyde, the drug forms a hydrazone that is soluble in isobutanol and a highly fluorescent compound that can be measured at an excitation wavelength of 392 nm and emission of 478 nm (sensitivity 0.1 g/mL, linearity 0.1–10 g/mL) [13]. PZA was analysed spectrophotometrically based on the formation of a coloured complex between the drug and sodium nitroprusside at alkaline pH which was measured at 495 nm (sensitivity 5.0 g/mL, linearity 5–10 g/mL) [14]. 2.5. Nebulisation of alginate nanoparticles
2.3. Culture Mycobacterium tuberculosis H37 Rv, originally obtained from National Collection of Type Cultures (NCTC), was maintained on modified Youman’s media. 2.4. Development and characterisation of drug-loaded alginate nanoparticles Alginate nanoparticles were prepared by the method of Rajaonarivony et al. [8] with slight modifications. The principle involves cation-induced controlled gelification of alginate. Briefly, calcium chloride (0.5 mL, 18 mM) was added to 9.5 mL of sodium alginate solution (0.06%) containing different amounts of INH, PZA and RIF. The initial ratio of drug:polymer was 7.5:1 (% w/w). Two millilitres of chitosan solution (0.05%) was added, followed by stirring for 30 min and storage at room temperature overnight. Drugloaded nanoparticles were recovered by centrifugation at 19 000 rpm for 35 min and washed thrice with distilled water. Drug-free nanoparticles were also prepared in the same manner. Alginate nanoparticles were characterised for their size and polydispersity index on a Zetasizer 1000 HS (Malvern Instruments, Malvern, UK). The amount of drug entrapped in alginate nanoparticles was calculated by estimating the amount of unentrapped drug recovered in the supernatant and pellet washings after centrifugation. The drug encapsulation efficiency was calculated as the percentage of drug entrapped in alginate nanoparticles compared with the initial amount of drug. The drug loading capacity (L) of alginate nanoparticles was determined as described by Rajaonarivony et al. [8] using the following equation: L(% w/w) =
A compressor nebuliser system (Medel Aerofamily, Italy) was used in the study. Each animal received drugs suspended in 4 mL of 0.9% NaCl via an appropriately modified paediatric facemask connected to the nebuliser, with an exposure time of 4 min, to receive the dose as described in our previous studies [2]. Nebulised alginate nanoparticles were sized on a 7stage Andersen Cascade Impactor (Andersen Samplers, Inc., Atlanta, GA) and evaluated for mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). An adjustable bleed valve on the pump that required periodic calibration controlled the flow rate through the impactor. The pump required 12 V automotive type battery (DC unit) drawing a current of 11 amps. 2.6. In vivo drug disposition studies Throughout the study, the drugs were used at therapeutic dosage (INH, 10 mg/kg; RIF, 12 mg/kg and PZA 15 mg/kg body weight). Since the dose was different for the three drugs, the initial amount of each drug required for nanoparticle 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, 7.5fold lesser alginate was used in the preparation process. The basic procedure for nanoparticle preparation remained the same as discussed above. For a 400 g guinea pig, 21.3 mg of dried nanoparticles constituted a therapeutic dose containing 4.8 mg RIF + 4 mg INH + 10 mg PZA.
Final drug concentration − drug concentration in supernatant Alginate final concentration
300
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
For single-dose drug disposition studies, guinea pigs were grouped as follows, with six animals in each group: Group 1, oral free drugs; Group 2, free drugs, aerosol; Group 3, nanoparticles loaded with ATDs, aerosol; and Group 4, nebulised drug-free nanoparticles (a positive control to explore the influence of alginate nanoparticles on drug estimations). The animals were bled at several time points and plasma was analysed for ATDs as described earlier. The area under the plasma drug concentration over time curve (AUC) was calculated using data analysis tools in SigmaPlot software (version 8.0) and further used to compute the relative bioavailability of each drug. Relative bioavailability = (AUC0–∞ of oral encapsulated drugs/AUC0–∞ of oral free drugs) × (dose of oral free drugs/dose of oral encapsulated drugs). Animals were sacrificed at different time points and drug levels were determined in 20% w/v tissue homogenates (lungs, liver and spleen) as described for plasma.
analysed by analysis of variance (ANOVA) to compare the control and treated groups.
4. Results 4.1. Physicochemical characterisation of alginate nanoparticles Alginate nanoparticles had an average size of 235.5 ± 0 nm, with a polydispersity index of 0.439. Drug encapsulation efficiency was observed to be 80–90% for RIF and 70–90% for INH and PZA. The drug loading capacity of alginate nanoparticles was 586–654 mg, 480–600 mg and 468–576 mg for RIF, INH and PZA, respectively, per 100 mg of alginate. 4.2. Aerodynamic characterisation of nebulised alginate nanoparticles
2.7. Experimental infection and chemotherapy Animals were infected intramuscularly with 1.5 × 105 viable bacilli of M. tuberculosis H37 Rv in 0.1 mL of 0.9% NaCl. Infection was confirmed by the presence of mycobacteria (by acid-fast staining) in tissue smears of spleens and lungs of two to three animals after 20 days. Animals were then divided into the following groups, with six animals in each group: Group I, untreated control; Group II, oral free drugs; Group III, drug-loaded nanoparticles, aerosol; and Group IV, empty nanoparticles, aerosol. Control animals received phosphate-buffered saline only, whilst other groups received the same drug doses as described under ‘in vivo studies’. Free drugs were administered daily, whilst the schedule of nanoparticle-based therapy was 15 days apart (based on the in vivo drug release studies in plasma and organs). Animals were sacrificed after 45 days of chemotherapy. Lungs and spleen were removed aseptically and homogenised in 3 mL of sterile normal saline. Then, 50 L of 1:100 dilution (prepared in normal saline) of tissue homogenates was plated on Middlebrook 7H10 agar plates. Colony-forming units (CFUs) were enumerated 30 days post inoculation. 2.8. Biochemical hepatotoxicity To evaluate hepatotoxic effects of ATDs, serum alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin levels were evaluated on day 46 of chemotherapy using standard kits obtained from Transasia Bio-medicals Limited (Daman, India).
3. Statistical analysis The pharmacokinetic data, including bioavailability, were analysed by Student’s unpaired t-test, and CFU data were
Aerodynamic characterisation revealed that nearly 80.5% of nebulised alginate nanoparticle droplets were in the size range 0.4–2.1 m (respirable range), with a MMAD of 1.1 ± 0.4 m and a GSD of 1.71 ± 0.1 m. 4.3. In vivo drug disposition studies When the alginate nanoparticles were administered through nebulisation, the drugs were detected in plasma from 3 h onwards. INH, RIF and PZA were observed up to 14, 10 and 14 days, respectively. In contrast, free drugs were cleared from the circulation within 12–24 h (Fig. 1). All the pharmacokinetic parameters, which include maximum plasma concentration (Cmax ), time to reach maximum plasma concentration (Tmax ) and AUC0–∞ , were found to be higher for alginate nanoparticle-encapsulated drugs upon administration via the aerosol route, resulting in a significant (P < 0.001) increase in relative bioavailability of encapsulated drugs compared with free drugs (Table 1). All drugs were detected in tissues, i.e. lungs, liver and spleen, above the minimum inhibitory concentration (MIC) up to 15 days (Fig. 2) [15], thus prompting us to design the fortnightly schedule of nebulised drug-loaded alginate nanoparticles for chemotherapy. 4.4. Chemotherapeutic efficacy Administration of nebulised alginate nanoparticles fortnightly (three doses spaced 15 days apart) for 45 days and of oral free drugs administered daily for 45 days to M. tuberculosis-infected guinea pigs resulted in undetectable mycobacterial CFUs in lung and spleen homogenates (1:100 dilution) (Table 2). However, guinea pigs administered empty alginate nanoparticles and untreated controls exhibited comparable CFU counts.
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
301
Fig. 1. Plasma drug profile following a single nebulisation of antitubercular drug-loaded alginate nanoparticles and free drugs to guinea pigs. Values are mean ± S.D. of six animals. RIF, rifampicin; INH, isoniazid; PZA, pyrazinamide.
4.5. Hepatotoxicity studies On the basis of serum bilirubin, ALT and ALP, the formulation did not produce any hepatotoxicity (Table 3). The normal range of these analytes has been previously established in healthy guinea pigs [10]. Thus, there was no biochemical evidence of hepatotoxicity in either group.
5. Discussion To overcome the daily dosing problem of tuberculous therapy, the application of drug delivery systems is a novel
strategy [16]. Alginate nanoparticles are a drug delivery system bearing the benefits of sustained release properties of nanoparticles, but also carry additional advantages, including the least use of organic solvents, no involvement of toxic molecules, least use of equipment, and reduced reticuloendothelial system uptake due to the stealth nature of alginate [8]. The present study explores the therapeutic potential of alginate nanoparticles encapsulating ATDs against experimental tuberculosis through the aerosol route in guinea pigs. We have modified the original protocol for nanoparticle production at two steps, namely replacement of poly-l-lysine with chitosan and reduction in the polymer:drug ratio. These modifications have great implications, as chitosan is a com-
Table 1 Pharmacokinetics of nebulised antitubercular drug-loaded alginate nanoparticles compared with oral free drugs in guinea pigs Group Oral free drugs Rifampicin Isoniazid Pyrazinamide
Cmax (g/mL)
Tmax (h)
Kel
T1/2 (h)
AUC0–∞ (g · h/mL)
Relative bioavailability
0.8 ± 0.18 2.5 ± 0.13 22.0 ± 1.63
1 1 0.5
−0.15 ± 0.0 −0.21 ± 0.01 −0.1 ± 0.01
4.5 ± 0.53 3.1 ± 0.53 6.5 ± 1.43
8.37 ± 1.10 10.93 ± 1.90 185.00 ± 10.00
1.00 1.00 1.00
Aerosol alginate nanoparticles Rifampicin 1.12 ± 0.02 Isoniazid 4.90 ± 0.3 Pyrazinamide 35.00 ± 1.1
96 168 144
−0.158 ± 0.01 −0.59 ± 0.01 −4.41 ± 0.09
4.39 ± 0.06 1.17 ± 0.3 0.16 ± 0.02
138.46 ± 5.32 521.45 ± 32.4 7776.00 ± 50.34
16.54 47.71 42.02
Cmax , maximum plasma concentration; Tmax , time to reach maximum plasma concentration; Kel , elimination half-life; T1/2 , absorption half-life; AUC, area under the plasma drug concentration over time curve.
302
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
Fig. 2. Tissue levels of antitubercular drugs on day 15 following aerosol administration of alginate nanoparticles to guinea pigs. Values are mean ± S.D. of six animals. RIF, rifampicin; INH, isoniazid; PZA, pyrazinamide.
pletely non-toxic molecule [17] whilst poly-l-lysine has been reported to have some toxicity [18]. In most drug delivery systems, the higher polymer (encapsulating material) consumption becomes a limiting factor because of cost and the final
dose size of the formulation (which is one of the critical factors for administration); thus, reduction in the polymer:drug ratio is a major concern in drug delivery technology. The higher drug loading observed for alginate nanoparticles in
Table 2 Chemotherapeutic efficacy of aerosolised alginate nanoparticle encapsulating antitubercular drugs against experimental tuberculosis in guinea pigs Group
Untreated controls Empty alginate nanoparticles every 15 days, aerosol (three doses) Drug-loaded alginate nanoparticles every 15 days, aerosol (three doses) Free drugs daily, orally (45 doses)
log10 CFUsa Lung (right caudal lobe)
Spleen (whole organ)
5.8 ± 0.1 5.8 ± 0.3* <1.0b <1.0b
5.9 ± 0.1 5.9 ± 0.1 <1.0b <1.0b
CFUs, colony-forming units. a Results are based on visible growth of Mycobacterium tuberculosis on Middlebrook 7H10 agar on day 21 post inoculation. Results are mean ± S.D. (N = 5–6). b Value <1.0 indicates no detectable CFUs following the inoculation of 50 L of neat and 1:10 diluted tissue homogenates. * P > 0.05 according to ANOVA. Table 3 Evaluation of aerosolised antitubercular drug-loaded alginate nanoparticles for biochemical hepatotoxicity in Mycobacterium tuberculosis H37 Rv-infected guinea pigs Group
Serum bilirubin (mg/100 mL)
Serum ALT (U/L)
Serum ALP (U/L)
Untreated controls (N = 5) Nebulised drug-loaded alginate formulation every 15 days (three doses) (N = 5) Oral free drugs daily (45 doses) (N = 5)
0.10–1.00 0.28–0.37 0.26–0.42
19–70 17–30 20–42
10–70 9–74 43–55
ALT, alanine aminotransferase; ALP, alkaline phosphatase.
A. Zahoor et al. / International Journal of Antimicrobial Agents 26 (2005) 298–303
this study can be attributed to the higher drug:polymer ratio as well as the high gel porosity of alginate [8]. The prolonged plasma retention of drugs encapsulated in alginate nanoparticles compared with free drugs might be attributed to their longer half-life coupled with the sustained release potential and the affinity of alginate for amino groups in the case of INH and PZA [8]. In the encapsulated form, RIF stayed in the plasma for up to day 10, whilst INH and PZA were found to reside for up to 14 days. This variation in residence time of RIF in plasma compared with INH and PZA could be due to the hydrophobic nature of this drug and hence its predilection towards tissues rather than plasma. The bioavailabilities of drugs encapsulated in alginate nanoparticles were significantly increased (P < 0.001), as evident from the plasma profile (increased Cmax and Tmax ) and pharmacokinetic evaluation (increased Cmax and AUC0–∞ ) compared with free drugs. The detection of INH, RIF and PZA in tissues demonstrated that alginate nanoparticles encapsulating ATDs, when administered through the aerosol route, not only increased local (lung) drug bioavailability but also the bioavailability of each drug at other sites of the body. The retention of all the drugs in organs, i.e. lungs, liver and spleen, above the MIC for 15 days following nebulisation of alginate nanoparticles favours their application against tuberculosis where infection is largely localised in tissues. Accordingly, a fortnightly schedule of chemotherapy was designed for alginate nanoparticles (Table 2). It was observed that only three nebulised doses of this formulation, administered every 15 days, were equiefficient to 45 doses of oral free drugs administered daily in achieving undetectable CFUs. The formulation was found to elicit no signs of hepatotoxicity, supporting their safety. The chemotherapeutic benefits of liposomes [4], poly-(dl-lactide-co-glycolide) (PLG) microparticles, PLG nanoparticles [2] and alginate beads [3] have been demonstrated previously. The main advantages of the abovementioned drug delivery systems include the sustained release of drugs over longer durations, prevention of premature drug degradation and reduction in drug toxicity. The various disadvantages include higher polymer consumption, lower drug entrapment, higher cost and risk of organic residuals. Alginate nanoparticles bear the advantages of all these systems whilst simultaneously overcoming all of their limitations, thus representing the most suitable carrier for the delivery of ATDs. This study reports for the first time the application of alginate nanoparticles as carriers of ATDs to improve their therapeutic benefit and in turn patient compliance for effective tuberculosis control. As tuberculosis therapy requires multiple drugs in different combinations depending on the patient category, future studies will be required to encapsulate other ATDs and to evaluate them in the same way. Later,
303
clinical trials will be needed to evaluate this system before use in humans.
References [1] Burman WJ, Cohn DL, Rietmeijer CA, Judson FN, Sbarbaro JA, Reves RR. Noncompliance with directly observed therapy for tuberculosis. Epidemiology and effect on the outcome of treatment. Chest 1997;111:1168–73. [2] Pandey R, Zahoor A, Sharma S, Khuller GK. Nanoparticle encapsulated antitubercular drugs as a potential oral drug delivery system against murine tuberculosis. Tuberculosis (Edinb) 2003;83:373–8. [3] Ain QU, Sharma S, Khuller GK, Garg SK. Alginate-based oral drug delivery system for tuberculosis: pharmacokinetic and therapeutic effects. J Antimicrob Chemother 2003;51:931–8. [4] Labana S, Pandey R, Sharma S, Khuller GK. Chemotherapeutic activity against murine tuberculosis of once weekly administered drugs (INH and RIF) encapsulated in liposomes. Int J Antimicrob Agents 2002;20:301–4. [5] Bala I, Hariharan S, Ravi Kumar MNV. PLGA nanoparticles in drug delivery: the state of the art. Crit Rev Ther Drug Carrier Syst 2004;21:387–422. [6] Shah SP, Misra A. Development of liposomal amphotericin B dry powder inhaler formulation. Drug Deliv 2004;11:247–53. [7] Hurley L, Andersen BR. Biodegradable implants from poly-(alphahydroxy acid) polymers for isoniazid delivery. Int J Tuberc Lung Dis 1999;3:1015–24. [8] Rajaonarivony M, Vauthier C, Couarraze G, Puisieux F, Couvreur P. Development of a new drug carrier made from alginate. J Pharm Sci 1993;82:912–7. [9] Jacobs C, Muller RH. Production and characterization of a budesonide nanosuspension for pulmonary administration. Pharm Res 2002;19:189–94. [10] Pandey R, Khuller GK. Chemotherapeutic potential of alginate– chitosan microspheres as antitubercular drug carriers. J Antimicrob Chemother 2004;53:635–40. [11] Tonnesen HH, Karlsen J. Alginate in drug delivery systems. Drug Dev Ind Pharm 2002;28:621–30. [12] Saito H, Tomioka H. Therapeutic efficacy of liposome-entrapped rifampin against M. avium complex infection induced in mice. Antimicrob Agents Chemother 1989;4:429–33. [13] Scott EM, Wright RC. Fluorimetric determination of isonicotinic acid hydrazide in serum. J Lab Clin Med 1967;70:355–60. [14] Gurumurthy P, Nair NGK, Sarma GR. Methods for the estimation of pyrazinamide and pyrazinoic acid in body fluids. Indian J Med Res 1980;71:129–34. [15] Rattan A. Development of new drugs for tuberculosis. In: Gupta, S, Sood, OP, editors. Tuberculosis. Proceedings of the 7th Round Table Conference. New Delhi, India: Ranbaxy Research Foundations; 2000. p. 113–22. [16] Pandey R, Sharma A, Zahoor A, Sharma S, Khuller GK, Prasad B. Poly(dl-lactide-co-glycolide) nanoparticle-based inhalable sustained drug delivery system for experimental tuberculosis. J Antimicrob Chemother 2003;52:981–6. [17] Berrada M, Serreqi A, Dabbarh F, Owusu A, Gupta A, Lehnert S. A novel non-toxic camptothecin formulation for cancer chemotherapy. Biomaterials 2005;26:2115–20. [18] Wen J, Mao HQ, Li W, Lin KY, Leong KW. Biodegradable polyphosphoester micelles for gene delivery. J Pharm Sci 2004;93:2142–57.