Research paper
Capreomycin supergenerics for pulmonary tuberculosis treatment: Preparation, in vitro, and in vivo characterization

https://doi.org/10.1016/j.ejpb.2012.11.005Get rights and content

Abstract

The pulmonary route is one of the main strategies investigated to improve tuberculosis therapy. The aim of this study was to develop a simple and scalable method to produce capreomycin inhalable powders to use as supergeneric. In vitro antimycobacterial activity and in vivo acute toxicity were assessed using agar proportion susceptibility test on Mycobacterium tuberculosis and chicken chorioallantoic membrane assay, respectively. Capreomycin and three different hydrophobic counterions, namely oleate, linoleate, and linolenate, were combined in solution to obtain hydrophobic ion-pairs that were successively spray-dried. Ion-pairing efficiency was influenced by the spray-dryer employed to produce the powder. In the case of capreomycin oleate, both instruments, mini and nano spray-dryer, were suitable to maintain a high ion-paired content, while for capreomycin linoleate and linolenate, mini spray-dryer was the most appropriate instrument. The three formulations showed morphology and particle sizes potentially suitable for inhalation. Capreomycin oleate and linoleate showed the same efficacy of capreomycin sulfate against M. tuberculosis, while capreomycin linolenate showed a reduced efficacy, even though strain growth was inhibited at 10−4 mycobacterial inoculum. In vivo acute toxicity studies evidenced the lowest toxic potential for capreomycin oleate when compared to the single components or the other two salts. Overall, capreomycin oleate seems to possess the most promising characteristics to be used as supergenerics in pulmonary tuberculosis treatment.

Introduction

Tuberculosis (TB) is an infectious bacterial disease of which Mycobacterium tuberculosis is recognized as the etiological agent. Even though the etiology was elucidated more than a century ago, TB is still one of the most serious diseases caused by a single pathogen [1], [2]. In fact, the World Health Organization (WHO) reported, in 2010, 1.4 million deaths, 8.8 million new and relapse cases, and 5.7 million new and recurrent TB patients treated [3].

The failure of the disease control is surely due to TB epidemiology. In fact, even though all countries are affected by TB, most cases (∼85%) are registered in Africa (∼30%) and in Asia (∼55%), where social and health conditions are not optimal, and the access to medicines is limited or completely lacking. In addition, TB chemotherapy features can be also considered responsible for the lack of disease control. At the moment, the recommended therapies, known as DOTS (directly observed treatment, short-course) and DOTS-Plus, consist of four co-administered drugs (6-month therapy), with the addition of second line antitubercular drugs (therapy duration up to 24 months) in the case of multi-drug-resistant (MDR) strains [3]. These long-term multi-drug therapies are characterized by low patient compliance and high rates of discontinuation responsible for the insurgence of resistant strains, known as MDR and extensive drug-resistant (XDR) strains [4].

In this scenario, the scientific community is attempting to solve this complex problem adopting different strategies, such as the development of new and more effective antitubercular drugs [5], [6] and vaccines [7], [8], the use of new drug cocktails [9], and the reformulation of currently used drugs in more effective and less toxic forms [10], [11].

The latter strategy is focused mainly on the production of drug inhalable powders and/or on antitubercular embedding in micrometric and/or nanometric particulate systems, able to target the site of infection. Considering that the site of primary infection is the lungs, and about 75% of the TB cases are referred to pulmonary tuberculosis; a reliable improvement of the therapy could be achieved by using inhalation as route of administration [12]. The combination of inhalation therapy, with the use of adequate (insoluble) particulate systems, provides the possibility to target alveolar macrophages [13], the M. tuberculosis host cells [14], [15].

Second line antitubercular drugs are the most interesting compounds to formulate in novel drug delivery systems due to their high toxicity and low incidence of resistance. In this respect, capreomycin sulfate, a nonapeptide included in the injectable second line antitubercular drugs [3], has been extensively evaluated for inhalation therapy both in the form of respirable powder [16], [17], [18] or encapsulated in particulate carriers [19], [20]. Capreomycin sulfate respirable powder has been evaluated in a phase I clinical trial with promising results [21].

The aim of this study was to develop a simple and scalable production methodology for capreomycin hydrophobic ion-pairs (HIP), in order to obtain low-soluble inhalable powders to use as supergenerics. To reduce capreomycin systemic absorption and maximize intracellular drug concentration, efforts were made to decrease as much as possible the non-ion-paired capreomycin content in the formulations. The rational of using fatty acid capreomycin salts as supergenerics is twofolds. Fatty acid counterions, by conferring lipophilicity to the salts, allow the production of insoluble particles able to be taken up by infected macrophages. Once released in the lysosomal acidic environment, fatty acids can reactivate or sensibilize dormant bacteria. In fact, different lipids have been found involved in the formation and reactivation of Mycobacterium smegmatis “nonculturable” cells and oleic acid, in concentration between 0.05 and 3 μg/mL, showed the most efficient reactivating effect [22]. It is speculated that capreomycin HIPs, in particular CO, could be used to improve TB therapy outcomes using a “shock and kill” strategy. This novel approach has been recently proposed and successfully tested in vitro for HIV-1 eradication [23], [24]. In the specific case of TB, dormant bacteria are considered responsible for TB reactivation, and this strategy could turn out to be very interesting by considering the difficulties encountered in eradicating dormant mycobacteria [9]. The antimicrobial activity of the different HIPs was assessed in vitro on M. tuberculosis, while in vivo acute toxicity has been evaluated using chick embryo chorioallantoic membrane (CAM) assay.

Section snippets

Materials

Capreomycin sulfate (CS), sodium oleate (SO), and sodium linoleate (SL) were purchased from Sigma-Aldrich Chemical (Milan, Italy), while sodium linolenate (SLn) was obtained from Nu-Chek, Inc. (Elysian, MN, USA). All other chemicals and reagents were of the highest purity grade commercially available.

Hydrophobic ion-pair preparation

Capreomycin oleate (CO) and linoleate (CL) were prepared using ethanol–water mixtures as solvent, while capreomycin linolenate (CLn) was prepared in a mixture of acetone and water. Briefly, CS and

HIP powder preparation and characterization

In a previous paper, CO was prepared from aqueous solutions and successively processed by high pressure homogenization to reduce particle dimensions [34]. In this work, the preparation method was improved, and two additional HIP powders were prepared. Instead of water, a mixture of ethanol and water was used to prepare CO and CL, while acetone and water were used to obtain CLn. The ratio between the two solvents has been adjusted to allow single component complete dissolution and guarantying

Conclusion

Three different hydrophobic capreomycin salts were successfully produced using mini spray-dryer, while CO was also efficiently obtained using nano spray-dryer. CO, characterized by particle dimensions potentially suitable for inhalation, high ion-paired capreomycin content, and antimycobacterial activity comparable to CS, was the most promising to produce a supergeneric. Comparing the toxicity profile of capreomycin oleate and sulfate salts on chicken embryo chorioallantoic membrane, oleate

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