Konjac glucomannan microcarriers: an application in the treatment of pulmonary tuberculosis

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Respirable konjac glucomannan microparticles as antitubercular drug carriers: Effects of in vitro and in vivo interactions

Publication . Guerreiro, Filipa; Pontes, Jorge Filipe; Gaspar, Maria Manuela; Rosa Da Costa, Ana; Faleiro, Maria Leonor; Grenha, Ana

Pulmonary delivery of drugs is potentially beneficial in the context of lung disease, maximising drug concentrations in the site of action. A recent work proposed spray-dried konjac glucomannan (KGM) microparticles as antitubercular drug (isoniazid and rifabutin) carriers to treat pulmonary tuberculosis. The present work explores in vitro and in vivo effects of these microparticles, focusing on the ability for macrophage uptake, the exhibited antibacterial activity and safety issues. Efficient uptake of KGM microparticles by macrophages was demonstrated in vitro, while the antitubercular activity of the model drugs against Mycobacterium bovis was not affected by microencapsulation in KGM microparticles. Despite the good indications provided by the developed system, KGM is not yet approved for pulmonary applications, which is a limiting characteristic. To reinforce the available data on the performance of the material, safety parameters were evaluated both in vitro and in vivo, showing promising results. No significant cell toxicity was observed at concentrations considered realistic for lung delivery approaches (up to 125 & mu;g/mL) when lung epithelial cells and macrophages were exposed to KGM microparticles (both drug-loaded and unloaded). Finally, no signs of systemic or lung inflammatory response were detected in mice after receiving 10 administrations of unloaded KGM microparticles.

2023-09Journal article

Restricted access

Konjac glucomannan microcarriers: an application in the treatment of pulmonary tuberculosis

Publication . Guerreiro, Filipa Raquel Horta; Grenha, Ana; Faleiro, Leonor

Tuberculosis remains one of the leading causes of death worldwide.1 The conventional therapy of this infection is based on the intake of a combination of several antitubercular drugs for a period that could reach 2 years.2 The prolonged treatment of tuberculosis has limited the therapeutic success, as it fosters the non-compliance of the patient and, consequently, the emergence of drug resistance.3 Thus, the development of new approaches to treat tuberculosis is demanded to address the limitations of the current treatment. Considering that 80% of tuberculosis cases are pulmonary, the direct delivery of antitubercular drugs to the lungs has been explored as therapeutic alternative in recent years. This approach is thought to potentially allow to decrease the dose and frequency of drug administration, while reducing the treatment duration and the systemic side effects associated to the conventional therapy of tuberculosis.4 Its success requires the engineering of suitable carriers, which must reach the alveolar region, where the macrophages infected with Mycobacterium tuberculosis are located, and undergo phagocytosis by these cells.5 On the other side, specific receptors existing on macrophage surface may be used as drug targets in a strategy where drug carriers may, again, play a relevant role. At this moment, there are few excipients approved for pulmonary delivery applications, which hinders the development of drug carriers.6 The work entailing this PhD thesis proposes the development of inhalable microparticles based on konjac glucomannan (KGM), which are targeted to the alveolar macrophages. KGM is a polysaccharide composed by mannose and glucose units.7 The rational beyond the proposed strategy relies on the fact that the presence of mannose on KGM microparticles may mediate a preferential recognition by the mannose receptors present on the macrophage surface and potentiate their phagocytosis via these receptors.8 Inhalable microparticles were produced in the form of dry powders by a technique of spray-drying. In order to have KGM with the suitable properties for microparticle production using this technique, the polymer was initially submitted to an acid hydrolysis, which was demonstrated to not affect its physicochemical characteristics (composition and glucose/mannose ratio) and permitted a successful processing by spray-drying to produce microparticles. Isoniazid (INH) and rifabutin (RFB) are first-line antitubercular drugs and were associated to KGM microparticles as model drugs. Various formulations of drug-loaded KGM microparticles were produced (KGM/INH/RFB = 10/1/0.5, 10/1/1, 10/2/0.5, w/w) and subsequently characterised. Drug association efficiency varied within 66% and 91%, and the release of the drugs in conditions resembling the alveolar environment showed slower release of RFB compared with INH, but complete release of both drugs within 24 h. Regarding to the aerodynamic characteristics, which are of utmost importance in lung delivery strategies, KGM microparticles exhibited aerodynamic diameters around 3 μm, regardless of the drug amounts, which evidences suitability to reach the alveolar zone. Additionally, the spherical shape and the geometric size of approximately 2 μm displayed by KGM microparticles has proven adequate for macrophage internalisation, as shown in an in vitro assay. In fact, approximately 100% of macrophage-like THP-1 cells in culture demonstrated to phagocytose the microparticles. Despite the uptake was demonstrated, macrophage activation upon exposure to microparticles was not observed. As mentioned before, a relevant limitation of the pulmonary drug delivery field relies on the shortness of excipients approved for inhalation. One of the main reasons for that is the unknown fate of materials after deposition in the lungs. In this work, the swelling of (unloaded) KGM microparticles was observed to occur (40% - 50%) upon liquid contact, but size reduction (> 62% in 90 min) in presence of β-mannosidase, an enzyme present in the lung, was further demonstrated, indicating potential biodegradability upon inhalation. The preservation of antibacterial effect of the used drugs after spray-drying was demonstrated using Mycobacterium bovis, evidencing an absence of effect of the process of microencapsulation. In fact, the minimum inhibitory concentration (MIC) remained similar to that determined for the free drugs. However, a preliminary study indicated that the amount of drug corresponding to MIC was not enough to kill the bacteria after infection of the macrophages (macrophage-like THP-1 cells) with M. bovis. The number of bacteria surviving in macrophages only decreased on the ninth day of infection. Moreover, the continued exposure of bacteria to KGM microparticles for 7 days suggested the development of a certain degree of drug resistance by M. bovis. Finally, to better support the proposal of using KGM in lung delivery applications, the safety of KGM microparticles was evaluated in vitro and in vivo. With regards to the in vitro tests, these focused on the evaluation of toxicological profile in respiratory cells (alveolar epithelium and macrophages). Despite a wide range of concentrations was assessed to have a more comprehensive knowledge on the effect of the material and the developed drug carrier, at the concentrations expected to be realistic in approaches of lung delivery (up to 125 μg/mL) drug-loaded KGM microparticles did not show overt cell toxicity. An in vivo assay was also performed which focused on evaluating the toxicity of the material itself, as it is not approved for lung delivery applications. Mice receiving daily administration of unloaded KGM microparticles by inhalation for a period of two weeks have shown no signs of systemic or lung inflammatory response, which is in line with the results of macrophage activation obtained in vitro. Moreover, histological examination of the lung revealed no differences upon inhalation, comparing with control naïve mice, although some unexpected observations transversal to all groups require clarification. The results further demonstrated the development of eosinophilia, which is typically associated to allergic reactions. Nevertheless, no alterations were observed in serum IgE upon inhalation, which opposes to the eosinophilic effect. Overall, despite some positive indications, regulations on safety evaluation include a vast number of parameters to assess and further studies are required to deepen the assessment and support the safety of KGM for lung delivery applications. Considering the whole set of data generated throughout the work, encouraging indications were given on the potential of KGM to be used as a pharmaceutical excipient in the formulation of inhalable antitubercular drug carriers for pulmonary tuberculosis treatment and, potentially, in other applications benefiting from macrophage targeting.

2021-10-13Doctoral thesis

Open access

Engineering of konjac glucomannan into respirable microparticles for delivery of antitubercular drugs

Publication . Guerreiro, Filipa; Swedrowska, Magda; Patel, Roshnee; Floréz- Fernández, Noelia; Torres, María Dolores; Rosa Da Costa, Ana M.; Forbes, Ben; Grenha, Ana

Few medically-approved excipients are available for formulation strategies to endow microcarriers with improved performance in lung drug targeting. Konjac glucomannan (KGM) is a novel, biocompatible material, comprising mannose units potentially inducing macrophage uptake for the treatment of macrophage-mediated diseases. This work investigated spray-dried KGM microparticles as inhalable carriers of model antitubercular drugs, isoniazid (INH) and rifabutin (RFB). The polymer was characterised and different polymer/drug ratios tested in the production of microparticles for which respirability was assessed in vitro. The swelling of KGM microparticles and release of drugs in simulated lung fluid were characterised and the biodegradability in presence of beta-mannosidase, a lung hydrolase, determined. KGM microparticles were drug loaded with 66-91% association efficiency and had aerodynamic diameter around 3 mu m, which enables deep lung penetration. The microparticles swelled upon liquid contact by 40-50% but underwent size reduction (>62% in 90 min) in presence of beta-mannosidase, indicating biodegradability. Finally, drug release was tested showing slower release of RFB compared with INH but complete release of both within 24 h. This work identifies KGM as a biodegradable polymer of natural origin that can be engineered to encapsulate and release drugs in respirable microparticles with physical and chemical macrophage-targeting properties.

2021Journal article

Open access

Inhalable locust bean gum microparticles co-associating isoniazid and rifabutin: therapeutic assessment in a murine model of tuberculosis infection

Publication . Grenha, Ana; Alves, Ana D.; Guerreiro, Filipa; Pinho, Jacinta; Simões, Sandra; Almeida, António José; Gaspar, Maria Manuela

Tuberculosis is a leading cause of death worldwide. Although the development of new antimycobacterial drugs is an obvious and necessary strategy to address the disease, improving the therapeutic performance of drugs already approved constitutes a valuable alternative approach. As the lung is the most affected organ, where M. tuberculosis is able to survive and proliferate, the direct pulmonary delivery of antitubercular drugs comprises a highly promising therapeutic strategy. In this work, spray-dried locust bean gum (LBG) microparticles were used to deliver a combination of two first line antitubercular drugs, isoniazid (INH) and rifabutin (RFB), to the alveolar zone, where macrophages hosting the bacteria reside. LBG is expected to mediate favoured macrophage uptake of microparticles, leading to enhanced therapeutic effect. The therapeutic effect of LBG/INH/RFB microparticles was evaluated in a murine model infected with M. tuberculosis, strain H37Rv and compared with oral co-therapy of INH and RFB in the free form. The pulmonary administration of LBG/INH/RFB microparticles 5 times per week was the only treatment schedule that provided negative growth index values in lung (-0.22), spleen (-0.14) and liver (-0.26) even using a lower therapeutic dose for both antibiotics. For the control group, the respective values were +1.95, +0.75 and +0.96.

2020-02Journal article

Restricted access