a Department of Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin, Kelchstrasse 31, D-12169 Berlin; b Department of Medical Microbiology, Free University of Berlin, Hindenburgdamm 27, D-12203 Berlin; c Max-Delbrück-Centre for Molecular Medicine, Robert-Rössle-Strasse 10, D-13122 Berlin; d Department of Clinical Chemistry and Pathobiochemistry, Benjamin-Franklin Hospital, Free University of Berlin, Hindenburgdamm 30, D-12203 Berlin; e Molecular Infection Biology, Research Centre Borstel, Parkallee 22, D-23845 Borstel, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of drugs in the form of nanosuspensions was shown to be a more cost-effective and technically simpler alternative, particularly for poorly soluble drugs, and to yield a physically more stable product than liposome dispersions.1315 With this technique, the drug, dispersed in water, is ground by shear forces, i.e. high pressure homo-genization, to particles with a mean diameter in the nanometre range (1001000 nm). The fineness of the dispersed particles causes them to dissolve more quickly owing to their higher dissolution pressure and leads to an increased saturation solubility. This may enhance the bioavailability of drugs compared with other microparticular systems. If the dissolution velocity of the drug particles is low enough in vivo, the nanosuspensions will have the passive targeting advantages of colloidal drug carriers.16
Using the experimental model of murine M. avium infection, we performed studies with a novel nanocrystalline suspension of clofazimine in order to test its suitability for intravenous chemotherapy and compared its therapeutic efficacy with that of clofazimine liposomes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
M. avium TMC724 (originally obtained from Dr F. Collins, Trudeau Institute, Saranac Lake, NY, USA) was passaged in C57BL/6 mice twice and cultured in Middlebrook 7H9 medium (Difco, Detroit, MI, USA) supplemented with oleic acid, albumin, dextrose and catalase (OADC; Becton Dickinson, Heidelberg, Germany) to mid-logarithmic phase. Aliquots were frozen at 70°C until needed. An inoculum of bacteria was prepared by thawing an aliquot and diluting it in phosphate-buffered saline (PBS). Mice were infected iv into a lateral tail vein with 1 x 105 cfu bacteria in 0.2 mL PBS. The natural course of infection with this strain in mice has been described previously in detail.17 The MIC of clofazimine for this strain of M. avium was determined to be 0.5 mg/L (courtesy of Dr S. Rüsch-Gerdes, National Reference Center for Mycobacteria, Borstel, Germany).
Chemical reagents
Clofazimine (3-(4-chloroanilino)-10-(4-chlorophenyl)-2,10-dihydro-2-isopropyliminophenazine) was supplied by Ciba Geigy (Basel, Switzerland). Phospholipon 90 was a gift from Nattermann Phospholipid (Köln, Germany) and Lipoid E80 from the Lipoid KG (Ludwigshaven, Germany). Lipofundin 10% was obtained from Braun-Melsungen (Melsungen, Germany) and Intralipid 20% from Kabi Pharmacia (Erlangen, Germany). Mannitol and all other chemicals were purchased from Sigma (Deisenhofen, Germany).
Preparation of clofazimine formulations
In order to produce the nanosuspensions, the clofazimine powder (2%) was dispersed in an aqueous solution, containing 0.5% Pluronic F68, 0.6% Phospholipon 90, 0.25% sodium cholic acid and 5.6% mannitol, with an UltraTurrax stirrer T 25 (Janke und Kunkel, Staufen i. Br., Germany). The resulting coarse pre-dispersion was homogenized at a pressure of 1500 bar and 10 cycles using an APV Gaulin Micron LAB 40 homogenizer (APV Homogenizer, Lübeck, Germany). The size reduction process resulted in a suspension in the nanometre range, i.e. a nanosuspension. To further reduce the number of particles larger than 5 µm, the clofazimine nanosuspension was processed by a separation step before administration. For this purpose, the nanosuspensions were centrifuged at 1000g for 10 min and the pellet discarded. All data presented are the mean values of three different batches produced under identical conditions.
For lyophilization, the nanosuspensions were dispensed into 10 mL vials (2 mL each vial) and transferred to a Gamma 2-20 freeze-dryer (Christ, Osterode i. H., Germany). Drying lasted for 48 h at 20°C, with a secondary cycle of 3 h with the temperature adjusted to 20°C. The pressure during drying was 1030 mbar.
Liposomes were prepared by a modified ethanol injection method18 followed by high pressure homogenization. Lipoid E80 and cholesterol at a ratio of 9:1 (w/w) were used as lipid phase (10%). The lipid compounds were dissolved in ethanol and pumped slowly (30 mL/h) into the aqueous phase which was constantly stirred with an Ultra-Turrax T25 equipped with a N18G dispersing tool at 8000 rpm. Pumping was performed through a fine needle with a speed-controlled Braun Perfuser (Braun-Melsungen). For maximum homogenization, a specially manufactured dispersing beaker with armoured walls was used. After removal of ethanol and excess water in a rotary evaporator, the liposome batches were adjusted to pH 5.5 and high-pressure homogenized at 1500 bar, two cycles with a APV Gaulin Micron LAB 40. The final formulation was sterilized by filtration under laminar air flow into sterile brown-glass vials.
Physical characterization of clofazimine formulations
Particle size analysis was performed by photon correlation spectroscopy (PCS) (Malvern ZetaSizer IV, Malvern Instruments, Malvern, UK), with a Coulter counter Multisizer II (Coulter Electronics, Krefeld, Germany) equipped with a 30 µm capillary, and by laser diffraction (LD) (Mastersizer E, Malvern Instr., Malvern, UK) particle size analysis. PCS measurements yield the hydrodynamic diameter and a polydispersity index (PI) as a measure of the width of the size distribution. The PI is zero for an ideal monodisperse sample and 0.30.5 for broad distributions. Coulter counter calculations were performed using particle counts without further correction. From the laser diffractometry data the diameter 99%, the LD(99), was used to characterize the nanosuspension. The LD(99) signifies that 99% of the particles are below the indicated size. The diameters were calculated on the basis of the volume distribution.
Determination of clofazimine concentrations in mouse tissues
Experiments involving mice were approved by the local Ethics Committee and the Berlin Senate. Female C57BL/6 mice (8 weeks old, three mice per group) were given a single iv injection of a nanocrystalline or liposomal formulation containing 500 µg clofazimine (20 mg/kg bodyweight) and were killed 2 h later. Lungs, livers and spleens of treated mice were homogenized (one part tissue and nine parts methanol/glacial acetic acid 9:1 (v/v)) and stored at 20°C. After thawing, samples were centrifuged and the pellet extracted twice with a 10-fold (w/v) volume of an extraction solution consisting of methanol/glacial acetic acid 9:1 (v/v). Clofazimine concentrations in the pooled extractions were determined by HPLC,19 with a cation exchange column (Nuclesil SA, 125 x 4 mm, particle size 5 µm, Macherey & Nagel, Düren, Germany) with a guard column of Perisorb RP18 (30 x 4 mm, particle size 3040 µm, E. Merck, Darmstadt, Germany). The mobile phase consisted of 750 mL acetonitrile and 250 mL 0.1 M aqueous phosphoric acid. The mixture was adjusted to pH 3.82 with 2 M NaOH, the final concentration of sodium was 25 mmol/L. The absorption of the column eluant was recorded at 495 nm. The detection limit was 8 mg/kg organ weight for each tissue.
Therapeutic efficacy of clofazimine formulations in M. avium-infected mice
Female C57BL/6 mice (8 weeks old) were infected intravenously with a bacterial suspension containing 1 x 105 cfu of M. avium strain TMC724, as previously described.17,20 Treatment with nanocrystalline or liposomal clofazimine was started on day 7 post-infection and was continued twice-weekly for 3 weeks, to give a total of six injections per mouse. Clofazimine formulations were administered at 500 µg per injection, representing 20 mg clofazimine/kg bodyweight, in a volume of 200 µL. Clofazimine has very low solubility in aqueous media and can only be solubilized by the addition of a suitable solvent, e.g. dimethylsulphoxide (DMSO). The use of soluble clofazimine at this concentration was prohibitive owing to the high concentration (10%) and toxicity of the solvent DMSO. Two days after either the first or the third week of treatment, groups of four or five mice were killed and bacterial loads determined in liver, spleen and lungs by plating 10-fold serial dilutions of homogenized tissues on to 7H10 agar plates supplemented with OADC. Colonies were counted following incubation for 1421 days at 37°C in a humidified atmosphere. No effect owing to drug carryover was observed, in that early and late serial dilutions gave rise to cfus proportional to the dilution factor. The data are presented as log10 cfu/organ ± S.D. (four or five mice per group). Statistical analysis was by the MannWhitney U-test. Haematoxylineosin stains were performed on 4% formalin/PBS-fixed and paraffin-embedded cranial liver lobes to assess the extent of inflammation and toxicity.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The PCS diameter represents the mean diameter of the bulk population of particles and the LD(99) value is a sensitive parameter of the microparticle content. The powder pre-dispersion had an LD(99) value of 53.63 µm and a PCS diameter greater than the upper limit of measurement (3000 nm). Both parameters decreased continuously with the number of cycles in the discontinuous process, reaching 5.44 µm and 601 nm, respectively, after the 10th cycle (Table I).
|
The batch used for in vivo testing (referred to below as the clofazimine nanosuspension for iv use) was produced with the discontinuous process because production was possible under aseptic conditions. The discontinuous process was followed by a separation/centrifugation step resulting in almost the same particle size distribution as that obtained with the continuous process (Table I).
Optimization of parameters for lyophilizing clofazimine nanosuspensions
Hydrophobic interaction may cause the clofazimine par-ticles to aggregate during the freeze-drying process. In order to prevent this aggregation, the addition of di- and monosaccharides at various concentrations were tested. Table II shows the mean PCS diameter and the LD(99) value for the nanosuspension without additives, with 2% and 8% trehalose and with 8% mannitol. In some cases, after the freeze-drying process, the mean PCS diameter and the LD(99) value of the reconstituted powder were smaller than those of the suspension before drying. This effect of particle size reduction was also observed during storage and attributed to the presence of loose aggregates in freshly prepared nanosuspensions. Similarly, the nanosuspensions stabilized with 2% and 8% trehalose had a smaller LD(99) value than the suspension before drying. The PCS diameter remained unchanged (2% trehalose) or increased by about 20 nm (8% trehalose) after reconstitution. In the case of the formulation with 8% mannitol the LD(99) value and PCS diameter were almost identical in comparison with the suspension before drying. The powder lyophilized without further additives was slightly viscous and sticky, although this is not reflected in the particle size distribution. Therefore, stabilization with trehalose yielded optimal results. Lyophilization proved to be a suitable method for stabilizing clofazimine nanosuspensions and may also be useful for producing a powder for further pharmaceutical processing (e.g. granulation, tableting).
|
Capillary blockade after iv administration can be caused by an excessive number of particles larger than 5 µm. Although the number of such particles in the homogenized clofazimine nanosuspensions was far smaller than in fat emulsions used for parenteral nutrition (Table III), the nanosuspension was further processed by a separation centrifugation step. The resultant clofazimine nanosuspension for iv use was adjusted to a drug content of 0.18%. For purposes of comparison, liposomes containing 0.16% clofazimine, 9% Lipoid E80 and 1% cholesterol were also prepared and analysed. Table I
shows the PCS diameter and the PI of this preparation. Table III
depicts the number of particles >1 µm, >2 µm and >5 µm in the clofazimine nanosuspension before undergoing the separation step, in the clofazimine nanosuspension for iv use and in the clofazimine liposomes. In the clofazimine nanosuspension there was a drop in the number of particles in all three size groups after the separation step. The clofazimine nanosuspension for iv use had a considerably larger number of particles >1 µm than the liposomal clofazimine preparation, but fewer particles >2 µm and almost the same number of particles >5 µm. The formulations were therefore regarded as suitable for iv injection because the number of particles >5 µm was far below that of standard fat emulsions for parenteral nutrition.14
|
Clofazimine concentrations in vivo after intravenous administration of nanocrystalline and liposomal preparations
Two hours after a single injection of either 500 µg liposomal or nanocrystalline clofazimine into mice, the liver, spleen and lungs were homogenized and clofazimine concentrations determined by HPLC. Highest clofazimine concentrations were measured in spleens and livers whereas drug concentrations in the lungs were significantly lower (Table IV). Clofazimine concentrations were always higher than the MIC for M. avium TMC 724 (0.5 mg/L) and, indeed, the MIC described for most strains of M. avium.10 There were no significant differences between the two formulations with respect to organ distribution or tissue concentration of clofazimine, but there was a tendency for the nanoparticle form to accumulate to higher concentrations in the liver than the liposomal formulation.
|
One week after infection, livers of mice showed few signs of inflammation upon histological examination, while livers of untreated mice examined 4 weeks post-infection were characterized by numerous granulomatous infiltrations with well-defined boundaries, as described previously.17 In contrast, animals treated with either liposomal or nanocrystalline clofazimine showed only a few granulomas in the liver at this time point. Histologically, no signs of hepatotoxicity were apparent in either treatment group.
After the first week of treatment with either clofazimine formulation, bacterial loads in livers, spleens and lungs did not differ from those of untreated animals (Figure 1). Continued therapy, however, resulted in reduced bacterial counts in the livers, and bacterial replication in spleens and lungs was suppressed (Figure 1). By the end of therapy, bacterial loads in all three organs examined were significantly lower in treated animals than in untreated controls (P < 0.05).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fate of intravenously injected colloids is a function of particle size. After iv injection, particles >7 µm and large agglomerates of smaller particles are filtered out by the capillary bed in the lung. Smaller particles are deposited in liver, spleen and bone marrow by the cells of the reticuloendothelial system (RES).21 In the case of particles >150 nm rapid hepatic clearance by Kupffer cells is predominant (c. 6090% within 510 min). Within 510 min, 220% of the drug can be found in the spleen, a varying fraction in the lungs and 0.0051% in the bone marrow.22 If this correlation is applied to the clofazimine nanosuspension for iv use with a mean diameter (PCS) of 385 nm and an LD(99) value of 2.28 µm, the fate of the intravenously administered nanoparticles should be RES sequestration, effectively targeting the drug to the principal host cells in mycobacterial infections, i.e. tissue macrophages. This assumes that the clofazimine nanoparticles do not dissolve within the first 10 min after administration. This is likely as clofazimine has low solubility in water (0.3 g/L at pH 7.8). Our finding that iv injection of clofazimine nanosuspensions results in tissue concentrations comparable to those achieved after application of a liposomal formulation supports the notion that nanoparticles are equally well suited for targeting drugs to the RES.
However, comparable relative organ distribution patterns of clofazimine in rats were described by Mamidi et al.23 after oral administration of clofazimine 20 mg/kg bodyweight, and by Kailasam et al.9 after implantation of a biodegradable clofazimine-containing polymer. Although the bioavailability of a clofazimine preparation suitable for intravenous use is certainly superior to oral application, the organ distribution of clofazimine is apparently influenced more profoundly by the characteristics of the drug itself than by the delivery system used.2326
Some clinical trials have demonstrated that clofazimine-containing regimens may be less effective against M. avium infection than other combination chemotherapies.7,8 Along the same lines, the addition of clofazimine to an effective regimen of clarithromycin and ethambutol for M. avium complex bacteraemia in AIDS patients did not contribute to the clinical response,5 while a recent study showed clofazimine to be equally effective as rifabutin in combination with clarithromycin and ethambutol.27 It is, however, clear that there continues to be a need for second-line drugs to combat this potentially life-threatening opportunistic infection in AIDS patients. In this respect, clofa-zimine was shown recently to be particularly useful when resistance to clarithromycin emerges.28 More significantly, the use of clofazimine and its derivatives may have to be reconsidered for the treatment of multidrug-resistant strains of Mycobacterium tuberculosis, as several riminophenazines have been found to be effective against these strains.11
The purpose of this study was to demonstrate that nanocrystalline preparations of poorly soluble drugs such as clofazimine are feasible and useful adjuncts to current chemotherapeutic strategies. In view of the similarly high degree of drug accumulation in target organs and the excellent therapeutic efficacy that can be achieved with clofazimine nanosuspensions when directly compared with conventional colloidal drug carriers, nanocrystalline riminophenazines warrant further investigation as part of a back-up combination chemotherapy for mycobacterial infections.
|
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Benson, C. A. (1997). Disseminated Mycobacterium avium complex infection: implications of recent clinical trials on prophylaxis and treatment. AIDS Clinical Reviews, 27187.
3 . Sesin, G. P., Manzi, S. F. & Pacheco, R. (1996). New trends in the drug therapy of localized and disseminated Mycobacterium avium complex infection. American Journal of Health and Systematic Pharmacotherapy 53, 258590.
4 . Tartaglione, T. A. (1996). Therapeutic options for the management and prevention of Mycobacterium avium complex infection in patients with the acquired immunodeficiency syndrome. Pharmacotherapy 16, 17182.[ISI][Medline]
5 . Chaisson, R. E., Keiser, P., Pierce, M., Fessel, W. J., Ruskin, J., Lahart, C. et al. (1997). Clarithromycin and ethambutol with or without clofazimine for the treatment of bacteremic Mycobacterium avium complex disease in patients with HIV infection. AIDS 11, 3117.[ISI][Medline]
6 . Dube, M. P., Sattler, F. R., Torriani, F. J., See, D., Havlir, D. V., Kemper, C. et al. (1997). A randomized evaluation of ethambutol for prevention of relapse and drug resistance during treatment of Mycobacterium avium complex bacteremia with clarithromycin-based combination therapy. California Collaborative Treatment Group. Journal of Infectious Diseases 176, 122532.[ISI][Medline]
7 . May, T., Brel, F., Beuscart, C., Vincent, V., Perronne, C., Doco-Lecompte, T. et al. (1997). Comparison of combination therapy regimens for treatment of human immunodeficiency virus-infected patients with disseminated bacteremia due to Mycobacterium avium. Clinical Infectious Diseases 25, 6219.[ISI][Medline]
8
.
Shafran, S. D., Singer, J., Zarowny, D. P., Phillips, P., Salit, I., Walmsley, S. L. et al. (1996). A comparison of two regimens for the treatment of Mycobacterium avium complex bacteremia in AIDS: rifabutin, ethambutol, and clarithromycin versus rifampin, ethambutol, clofazimine and ciprofloxacin. Canadian HIV Trials Network Protocol 010 Study Group. New England Journal of Medicine 335, 37783.
9 . Kailasam, S., Wise, D. L. & Gangadharam, P. R. (1994). Bioavailability and chemotherapeutic activity of clofazimine against Mycobacterium avium complex infections in beige mice following a single implant of a biodegradable polymer. Journal of Antimicrobial Chemotherapy 33, 2739.[Abstract]
10 . Lindholm-Levy, P. J. & Heifets, L. B. (1988). Clofazimine and other rimino-compounds: minimal inhibitory and minimal bactericidal concentrations at different pHs for Mycobacterium avium complex. Tubercle 69, 17986.[ISI][Medline]
11 . Mehta, R. T., Keyhani, A., McQueen, T., Rosenbaum, B., Rolston, K. & Tarrand, J. J. (1993). In vitro activities of free and liposomal drugs against Mycobacterium aviumM. intracellulare complex and M. tuberculosis. Antimicrobial Agents and Chemotherapy 37, 25847.[Abstract]
12 . Mehta, R. T. (1996). Liposome encapsulation of clofazimine reduces toxicity in vitro and in vivo and improves therapeutic efficacy in the beige mouse model of disseminated Mycobacterium avium M. intracellulare complex infection. Antimicrobial Agents and Chemotherapy 40, 1893902.[Abstract]
13 . Merisko-Liversidge, E., Sarpotdar, P., Bruno, J., Hajj, S., Wei, L. et al. (1996). Formulation and antitumor evaluation of nanocrystalline suspensions of poorly soluble anticancer drugs. Pharmaceutical Research 13, 2728.[ISI][Medline]
14 . Müller, R. H. & Peters, K. (1997). Nanosuspensions for the formulation of poorly soluble drugs. I. Preparation by a sizereduction technique. International Journal of Pharmaceutics 160, 22937.[ISI]
15 . Westesen, K. & Siekmann, B. (1995). Preparation and physicochemical characterization of aqueous dispersions of coenzyme Q10 nanoparticles. Pharmaceutical Research 12, 2018.[ISI][Medline]
16 . Müller, R. H. (1991). In vivo distribution of carriers. In Colloidal Carriers for Controlled Drug Delivery and Targeting, pp. 21174. CRC Press, Inc., Boca Raton, FL.
17 . Hänsch, H. C. R., Smith, D. A., Mielke, M. E. A., Hahn, H., Bancroft, G. J. & Ehlers, S. (1996). Mechanisms of granuloma formation in murine Mycobacterium avium infection: the contribution of CD4+ T cells. International Immunology 8, 1299310.[Abstract]
18 . Batzri, S. & Korn, E. D. (1973). Single bilayer liposomes prepared without sonication. Biochimica et Biophysica Acta 298, 10159.[ISI][Medline]
19 . Borner, K., Hartwig, H., Leitzke, S., Hahn, H., Müller, R. H. & Ehlers, S. (1998). HPLC determination of clofazimine in tissue and serum of mice after intravenous administration of nanocrystalline or liposomal formulations. International Journal of Antimicrobial Agents 11, 759.[ISI]
20
.
Leitzke, S., Bucke, W., Borner, K., Müller, R. H., Hahn, H. & Ehlers, S. (1998). Rationale for and efficacy of prolonged-interval treatment using liposome-encapsulated amikacin in experimental Mycobacterium avium infection. Antimicrobial Agents and Chemotherapy 42, 45961.
21 . Illum, L. & Davis, S. S. (1982). The targeting of drugs parenterally by use of microspheres. Journal of Parenteral Science and Technology 36, 24251.[Medline]
22 . Kreuter, J. (1994). Drug targeting with nanoparticles. European Journal of Drug Metabolism and Pharmacokinetics 3, 2536.
23 . Mamidi, N. V., Rajasekhar, A., Prabhakar, M. C. & Krishna, D. R. (1995). Tissue distribution and deposition of clofazimine in rats following subchronic treatment with or without rifampicin. Arzneimittelforschung 45, 102931.[Medline]
24 . Kansal, R. G., Gomez-Flores, R., Sinha, I. & Mehta, R. T. (1997). Therapeutic efficacy of liposomal clofazimine against Mycobacterium avium complex in mice depends on size of initial inoculum and duration of infection. Antimicrobial Agents and Chemotherapy 41, 1723.[Abstract]
25 . Jagannath, C., Reddy, M. V., Kailasam, S., O'Sullivan, J. F. & Gangadharam, P. R. (1995). Chemotherapeutic activity of clofa-zimine and its analogues against Mycobacterium tuberculosis. In vitro, intracellular, and in vivo studies. American Journal of Respiratory and Critical Care Medicine 151, 10836.[Abstract]
26 . O'Connor, R., O'Sullivan, J. F. & O'Kennedy, R. (1995). The pharmacology, metabolism, and chemistry of clofazimine. Drug Metabolism Reviews 27, 591614.[ISI][Medline]
27 . Cohn, D. L., Fisher, E. J., Peng, G. T., Hodges, J. S., Chesnut, J., Child, C. C. et al. (1999). A prospective randomized trial of four three-drug regimens in the treatment of disseminated Mycobacterium avium complex disease in AIDS patients: excess mortality associated with high-dose clarithromycin. Terry Birn Community Programs for Clinical Research on AIDS. Clinical Infectious Diseases 29, 12533.[ISI][Medline]
28 . Dube, M. P., Torriani, F. J., See, D., Havlir, D. V., Kemper, C. A., Leedom, J. M. et al. (1999). Successful short-term suppression of clarithromycin-resistant Mycobacterium avium complex bacteremia in AIDS. California Collaborative Treatment Group. Clinical Infectious Diseases 28, 1368.[ISI][Medline]
Received 4 January 1999; returned 4 August 1999; revised 1 September 1999; accepted 9 September 1999