International Journal of Antimicrobial Agents 24 (2004) 93–94

Letter

Nebulization of liposome encapsulatedantitubercular drugs in guinea pigs

Rajesh Pandey, Sadhna Sharma, G.K. Khuller∗

Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India

Sir,The chemotherapeutic efficacy of liposome encapsulated

antitubercular drugs (ATDs) by intravenous (i.v.) routeagainst experimental tuberculosis (TB) has recently beendemonstrated in our laboratory[1,2]. However, because ofthe pain/discomfort associated with parenteral therapy andkeeping in mind that pulmonary TB is the commonest form,the present study was planned to evaluate the biodistributionof inhalable liposome encapsulated rifampicin (RIF) andisoniazid (INH) in guinea pigs. Multilamellar liposomeswere prepared as described before[1], with the exceptionthat only phosphatidylcholine and cholesterol (molar ratio:2:1.5) were used as the lipid constituents. After lysing theliposomes with 1% (v/v) Triton X-100, the released drugswere assayed[1]. The drug encapsulation efficiency was40–45% for RIF and 8–12% for INH. The final drug: lipidratios were 0.22 for RIF and 0.14 for INH. Separate prepa-rations were made for RIF/INH and mixed 1:1 (v/v) beforeuse.

A compressor-nebulizer system (Medel Aerofamily, Italy;pressure: 250 kPa, nebulizer air flow: 5.5 l/min) was usedto nebulize the liposomal suspension. Aerodynamic charac-terisation on a 7 stage Andersen Cascade Impactor showedthat 94% of the generated aerosols were in the respirablerange (≤6�M) with a mass median aerodynamic diameterof 0.96 ± 0.06�M and geometric standard deviation of2.3 ± 0.4�M, suitable for bronchoalveolar drug delivery.Each animal received the drugs (free or liposomal) sus-pended in 4 ml of isotonic saline via a mask connected tothe nebulizer, with an exposure time of 4 min[3].

Dunkin Hartley guinea pigs were administered free drugsonce i.v. and free drugs/liposome encapsulated drugs oncevia nebulizer (n = 8 in each group). The drug doses wereselected according to the surface area of the animals, i.e.RIF 46.5 and INH 23.25 mg/kg body weight. The animals

∗ Tel.: +91-172-747-585x5174-75; fax:+91-172-744-401/745-078.E-mail address: gkkhuller@yahoo.co.in (G.K. Khuller).

were bled and the drugs levels were assayed in plasma atdifferent time points. A single nebulization of liposomaldrugs could maintain therapeutic drug levels in the plasmafrom 45 min onwards up to 48 h, whereas no drugs couldbe detected beyond 24 h post-nebulization when free drugswere used. Aerosolised liposomal drugs exhibited compara-ble (RIF) or lower (INH) peak plasma concentration (Cmax)compared with aerosolised free drugs. Further, aerosolisedliposomal drugs showed a significantly enhanced (P <

0.001, Student’st-test) half-life (t 1/2) and mean residencetime (MRT) which might be due to their slower rate of elim-ination (Kel). With respect to i.v. free drugs (which werecleared by 10 h), the absolute bioavailability was better inthe case of RIF compared with INH (Table 1).

The animals were sacrificed at various time points toanalyse the drug content in 20% lung homogenates. Be-fore preparing the homogenates, the alveolar macrophageswere isolated by bronchoalveolar lavage[4], lysed with5% (w/v) SDS and the released drugs were assayed. It wasobserved that aerosolised liposomal drugs were present inthe lungs (RIF: 0.25 ± 0�g/ml, INH: 0.29 ± 0.03�g/mlhomogenate) as well as in the alveolar macrophages (RIF:0.15 ± 0.02�g/105 cells, INH: 0.1 ± 0.03�g/105 cells;approximately 2× 105 viable cells were isolated from eachanimal) until day 5 post-nebulization. Aerosolised/i.v. freedrugs, however, were undetectable in the lungs/macrophagesbeyond 48 h. The data clearly shows the ability of nebulizedliposomes to target the alveolar macrophages, which are theabode of tubercle bacilli. It is known that the nebulizationof liposomal drug leads to sustained pulmonary drug levels[5] as observed in our study, and the safety of liposomaldrugs is also well established[6].

From the patient’s point of view, nebulization of lipo-somes would be perhaps more acceptable compared withi.v. liposomes. Our studies suggest that the controlled drugrelease as well as macrophage targeting may obviate theneed for daily ATD dosing and consequently, liposomebased inhalotherapy would be a novel approach towards themanagement of pulmonary TB.

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94 R. Pandey et al. / International Journal of Antimicrobial Agents 24 (2004) 93–94

Table 1Biodistribution and pharmacokinetics of nebulized liposome encapsulated antitubercular drugs compared with free drugs in guinea pigs

Rifampicin Isoniazid

Free, i.v. Free, aerosol Liposomal, aerosol Free, i.v. Free, aerosol Liposomal, aerosol

Duration of drug stayin plasma (h)

10 24 48 10 24 48

Cmax (�g/ml)a 40 ± 3.9 1.48± 0.09 1.48± 0.09 32± 3.1 1.98± 0.09 0.61± 0.1Kel

a −0.39 ± 0.5 −0.065± 0.004 −0.03 ± 0.003 −0.38 ± 0.04 −0.137± 0.02 −0.049± 0.005t 1/2 (h)a 1.78 ± 0.3 10.66± 2.1 16.12± 3.2 1.82± 0.3 5.06± 0.9 14.14± 2MRT (h)a 2.06 ± 0.3 10.85± 2.4 19.98± 3.5 2.46± 0.4 7.75± 1 17.75± 3.1Absolute bioavailability 1 0.82 0.93 1 0.43 0.38Duration of drug stay in

lungs including alveolarmacrophages (h)

48 48 120 48 48 120

a Values are mean± S.D., n = 8.

References

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[2] Pandey R, Sadhna S, Khuller GK. Liposome based antituberculardrug therapy in a guinea pig model of tuberculosis. Int J AntimicrobAgents 2004;23:414–5.

[3] Pandey R, Sharma A, Zahoor A, Sadhna S, Khuller GK, Prasad B.Poly (dl-lactide-co-glycolide) nanoparticle based inhalable sustained

drug delivery system for experimental tuberculosis. J AntimicrobChemother 2003;52:981–6.

[4] Edelstein PH, Beer KB, DeBoynton ED. Influence of growth tem-perature on virulence ofLegionella pneumophila. Infect Immun1987;55:2701–5.

[5] Webb MS, Boman NL, Wiseman DJ, et al. Antibacterial efficacyagainst an in vivoSalmonella typhimurium infection model and phar-macokinetics of a liposomal ciprofloxation formulation. AntimicrobAgents Chemother 1998;42:45–52.

[6] Thomas DA, Myers MA, Wichert B, Schreier H, Gonzalez-Rothi RJ.Acute effects of liposome aerosol inhalation on pulmonary functionin healthy human volunteers. Chest 1991;99:1268–70.