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The lungs are a well-recognized site for drug administration. Properly tailored drug delivery systems can improve the treatment of lung infections by achieving higher local drug concentrations and reducing systemic exposure.[2, 3] Among other treatments, tuberculosis (TB) therapy may benefit from this strategy.
The efficient clearance of exogenous substances from the lungs complicates the successful development of suitable pulmonary anti-TB drug delivery systems. Various approaches to this problem have been reported, such as the use of polymeric microparticles,[4-6] large porous particles, liposomes[8-12] and polymeric nanoparticles.[13, 14]
These approaches are all aimed at enhancing infected alveolar macrophage targeting. In fact, the Mycobacterium tuberculosis can survive within cells inhibiting lysosome fusion, and this peculiarity makes the alveolar macrophages therapeutic targets. However, the conventional TB therapy is not able to ensure adequate intracellular delivery.[16, 17] In this regard, inhalation may help overcome some of the well-known drawbacks of TB therapy, such as high dose and toxicity, by enhancing local drug accumulation. To do so, rapid drug absorption into the blood stream should be avoided.
Beyond the aforementioned microencapsulation approaches, indeed modulation of drug water solubility is commonly used. Sparingly or highly water-soluble active molecules show often a high burst of drug release from pharmaceutical formulations as they solubilize fast in body fluids. This is the case of injectable second-line antitubercular drugs (sl-ATDs), such as capreomycin (C) and kanamycin (K), and fluoroquinolones like ofloxacin (Ofx).
As a result, they are promptly available for systemic absorption, and in addition, unfavourable physicochemical properties can prevent them from being extensively internalized into cells and from diffusing into tissues.
This is an important aspect as to treat complex forms of TB, like latent and multidrug-resistant TB (MDR-TB), drugs are required to access highly hydrophobic environments where dormant and persistent bacilli find shelter. Moreover, hypoxia, low-nutrient exchange and acidic pH conditions, characterizing the persistent bacilli environment, together with the lack of relevant models of human MDR and latent TB infection limit the successful development of adequate therapies and prevention.[19, 20] These issues represent a tremendous obstacle to the complete eradication of TB infection and explain, together with socio-economic aspects, the widespread latent TB incidence.
Attempts have been made by formulating C as inhalable dry powder;[21-23] nevertheless, such approach is not likely to solve the problem of low tissue penetration and high absorption in the blood stream. Other likely more effective approaches include ionic coupling of C with long-chain fatty acids, such as oleic acid.
Complexation with metals, such as palladium (Pd), may be an alternative. This approach was previously reported for fluoroquinolones, such as Ofx, that in addition has been used to formulate an insoluble dry powder for lung delivery. In spite of the concerns regarding the use of heavy metals in pharmaceutical formulations,[27-32] Pd has been employed to produce a number of active compounds.[33-37] Of course, such concerns limit the development of Pd-bearing medicines; however, data on Pd toxicity are controversial, and as yet fate, adverse reactions as well as accumulation of Pd in the body are insufficiently described in literature. In particular, the effect of the metal when inhaled is unclear, and formulation, therapeutic regime and administration strategy are likely to strongly influence Pd fate and toxicity profile.
The pulmonary administration of the complexes in the form of dry powders may provide sl-ATD with an improved capacity of tissue penetration and an enhanced localized action. According to that, lower dosages and shorter treatments may be conceivable. In this case, the potential benefit of TB therapy improvement may counterbalance the risk of the administration of Pd compounds, especially considering the high sl-ATD systemic toxicity.
In light of these considerations, the present work focused on the synthesis and characterization of new sl-ATD-Pd complexes potentially useful for pulmonary administration in MDR-TB. Based on the previously published complexation strategy for OfxPd, novel sl-ATD-Pd complexes of C (CPd) and K (KPd) with Pd were synthesized and structurally characterized to determine the reaction kinetics and the properties of the complexes. Being OfxPd already reported in literature, the data obtained in this work were compared with those published. The products were then compared with the parent drugs for activity against an M. tuberculosis strain, and in particular, an infected macrophage model was used to test the intracellular activity of such compounds.
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While OfxPd formation and structure basically matched the work of Vieira et al. with a monodentate binding of PdCl2 on the carboxyl and carbonyl groups of the fluoroquinolone, the kinetics of formation measured, in the case of KPd and perhaps CPd, suggests the possibility of multiple co-existing species.
The difficulty in resolving CPd and KPd structure was due to the molecular complexity of the parent drugs, which are characterized by different forms occurring in different ratios and all participated to the binding with the metal. Therefore, different forms of the complexes can be inferred and the stoichiometry ratios of 1 : 3 for CPd and 2 : 3 for KPd may just be the overall combination of several possible species.
In the case of CPd, the molecule is characterized by four different isoforms: IA, IB, IIA and IIB.[43, 44] As observed from NMR signals, the form I is predominant over the form II that lacks of the β-lysine moiety, while forms A and B occur in a nearly 55-45 ratio, which is consistent with the work of Nomoto et al. From our data, it seems that Pd could give rise not only to intramolecular but also intermolecular binding even in light of the involvement of the lateral β-lysine residue. However, the formation of intermolecular complexes is a remote possibility because of the size of C that produces a considerable hindrance that opposes the vicinal binding of a second C molecule. It is thereby conceivable that the 1 : 3 ratio in CPd could be the result of the intracellular binding of Pd, which can however give rise to multiple species.
This situation may also fit the KPd case, which is almost totally in the form A, where the ratio of 2 : 3 may depict alternation of 1 : 1 and 1 : 2 species. This may exclude the unlikely possibility that Pd could bind two K molecules, which is not consistent with the presence of Cl detected by EDX. Such a hypothesis can be ruled out even considering the high repulsion that would be generated by the proximity of two large K molecules linked to the same centre. This high thermodynamic energy could not conceive the high log Kf value for KPd formation.
Even though we could not determine the nature of binding of Pd in CPd and KPd, the evidence previously obtained indicating the consistent presence of Cl suggests the same situation observed for OfxPd, where the metal binds as PdCl2 in a monodentate way. Of course, a deeper investigation is required to unravel the possible different structures produced by these complicated interactions, but this study is beyond the aims of the present work.
The structural complexity of the complexes may explain the activity reduction observed for CPd compared with the free drug with a significant increase of the MIC value. It must be underlined that all MIC values here reported have been only estimated, as this study was aimed at investigating a comparative activity rather than an absolute efficacy of these compounds. Therefore, all MIC should not be considered as absolute values.
In the case of C-Pd, the presence of Pd may affect the uptake through the bacterial wall. Residues as the β-lysine side chain provide peptide antibiotics with the capacity to interact with bacterial cell walls. The β-lysine residue in C is one of the sites of binding of Pd, and this may partially impair the peptide ability to penetrate bacterial membrane. Another factor affecting uptake may be the insolubility of CPd that, on the one side, may enhance macrophage intake but, on the other side, may reduce the amount of the compound available for bacteria cell uptake over time.
In the case of K, activity was not affected by Pd binding as the amine groups that allow aminoglycoside interaction with bacterial walls are only partially occupied by the metal.
The hydrophobicity provided by Pd binding seemed to partially enhance the intracellular efficacy of the complexes compared with the drugs. In the case of Cpd, such enhanced penetration may have balanced the negative effect previously discussed.
Another important factor to be considered is the stability of Pd binding in the intracellular environment. Even though the Kf values suggest a high binding capacity of the metal, the activity results seem to support a possible liberation of the drug from the complex in the cell milieu. In fact, although in OfxPd the metal binds the quinolone centres recognized as responsible for the killing activity, the complex showed even better performance compared with the free drug. This may infer that the active moiety is freed from the metal inside cells, hypothesis that requires further investigation.