Preparation of a clofazimine nanosuspension for intravenous use and evaluation of its therapeutic efficacy in murine Mycobacterium avium infection

K. Petersa, S. Leitzkeb, J. E. Diederichsc, K. Bornerd, H. Hahnb, R. H. Müllera and S. Ehlerse,*

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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clofazimine nanosuspensions were produced by high pressure homogenization and the formulation was optimized for lyophilization. Characterization of the product by photon correlation spectroscopy, laser diffraction and Coulter counter analysis showed that the clofazimine nanosuspensions were suitable for iv injection with a particle size permitting passive targeting to the reticuloendothelial system. Following iv administration to mice of either the nanocrystalline or a control liposomal formulation at a dose of 20 mg clofazimine/kg bodyweight, drug concentrations in livers, spleens and lungs reached comparably high concentrations, well in excess of the MIC for most Mycobacterium avium strains. When C57BL/6 mice were experimentally infected with M. avium strain TMC 724, nanocrystalline clofazimine was as effective as liposomal clofazimine in reducing bacterial loads in the liver, spleen and lungs of infected mice. Nanocrystalline suspensions of poorly soluble drugs such as riminophenazines are easy to prepare and to lyophilize for extended storage and represent a promising new drug formulation for intravenous therapy of mycobacterial infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite recent advances in antimycobacterial chemotherapy, disseminated Mycobacterium avium infection continues to be a clinically important problem in AIDS patients.1–4 The rapid emergence of bacterial resistance even to novel effective drugs such as clarithromycin has intensified the search for suitable alternatives in combination chemotherapy.5–8 Clofazimine, a riminophenazine drug, was found to have good activity against M. avium both in vitro and in vivo.9–11 Use of this drug is potentially restricted because of its toxic side-effects and its poor solubility, making it unsuitable for intravenous use in patients with drug malabsorption. Recently, liposomal encapsulation of clofazimine was shown to overcome both of these problems, resulting in good therapeutic activity in a murine model of M. avium infection.12

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.13–15 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 (100–1000 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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

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 0.3–0.5 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 30–40 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 14–21 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 Mann–Whitney U-test. Haematoxylin–eosin stains were performed on 4% formalin/PBS-fixed and paraffin-embedded cranial liver lobes to assess the extent of inflammation and toxicity.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Physical characterization of clofazimine nanosuspensions

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 IGo).


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Table I. Size characteristics (PCS mean diameter, PI and LD(99)) of clofazimine nanosuspensions and clofazimine liposomes
 
For comparison, one batch was also produced by con-tinuous homogenization but applying identical parameters (1500 bar, 10 cycles). The result was a PCS diameter reduced by 220 nm and an LD(99) value reduced by 3.30 µm, giving mean values of 381 nm and 2.14 µm, respectively (Table IGo).

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 IGo).

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 IIGo 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).


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Table II. PCS mean diameters and LD(99) values of the clofazimine nanosuspension before drying and the reconstituted clofazimine nanosuspension after lyophilization with and without additives
 
Assessment of suitability of the clofazimine nanosuspensions for intravenous administration

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 IIIGo), 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 IGo shows the PCS diameter and the PI of this preparation. Table IIIGo 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


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Table III. Number of particles of various sizes in clofazimine preparations, Intralipid 10% and Lipofundin 20%
 
Repeated iv injections of clofazimine nanosuspensions for iv use and liposomes at a dose of 500 µg clofazimine (representing 20 mg/kg bodyweight) were tolerated by female C57BL/6 mice without any discernible untoward side-effects. Macroscopic and microscopic evaluations of the livers of mice yielded inconspicuous results immediately after injection and 1 week after treatment. In contrast, iv injection with 500 µg clofazimine solubilized in 10% DMSO/PBS was highly toxic to mice, resulting in rapid death of all mice treated.

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 IVGo). 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.


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Table IV. Clofazimine concentrations (mg/kg tissue) in spleen, liver and lungs of mice 2 h after iv administration of 20 mg/kg bodyweight of either clofazimine nanosuspension or clofazimine liposomes
 
Therapeutic efficacy of nanocrystalline and liposomal clofazimine in murine M. avium infection

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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We prepared successfully a formulation of a clofazimine nanosuspension that can be lyophilized easily for extended storage and that compares favourably with a liposomal form of clofazimine in terms of its therapeutic efficacy in a murine model of disseminated M. avium infection. While liposomal preparations of clofazimine and amikacin were demonstrated previously to be highly effective in experimental M. avium infections,12,20 there are problems concerning the long-term stability of liposomes during storage as aqueous dispersions. Difficulties also often arise when lyophilized liposomes are reconstituted. Since drug inclusion rates into liposomes are frequently low, a therapeutically satisfactory formulation may not always be achieved. In contrast, nanosuspensions can be shown to be physically stable as aqueous dispersions for up to 2 years (K. Peters, unpublished work). Using optimized compositions, as shown here for clofazimine, nanosuspensions may be easily redispersed after storage. As nanosuspensions are composed of pure drug particles, high doses may be administered in relatively small injection volumes.

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. 60–90% within 5–10 min). Within 5–10 min, 2–20% of the drug can be found in the spleen, a varying fraction in the lungs and 0.005–1% 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.23–26

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.



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Figure. Bacterial loads in (a) liver, (b) spleen and (c) lungs of mice following infection with 1 x 105 cfu of M. avium TMC724 and treatment with 20 mg/kg bodyweight nanocrystalline clofazimine (•), liposome-encapsulated clofazimine ({blacktriangleup}) or no treatment ({square}). Data represent the means ± S.D. of four or five mice per group.

 

    Acknowledgments
 
We thank H. Hartwig and A. Graebert for expert technical assistance. This work was supported by grant MU 708/9-2 from the Deutsche Forschungsgemeinschaft.


    Notes
 
* Corresponding author. Tel: +49-4537-188481; Fax: +49-4537-188686; E-mail: sehlers{at}fz-borstel.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 4 January 1999; returned 4 August 1999; revised 1 September 1999; accepted 9 September 1999