WO2016174573A1

WO2016174573A1 - Process for producing polysaccharide microparticles for alveolar macrophage targeting, microparticles obtained therein and use thereof

Info

Publication number: WO2016174573A1
Application number: PCT/IB2016/052353
Authority: WO
Inventors: Ana Margarida MOUTINHO GRENHA; Ana Bernardina COTRIM DIAS ALVES; Susana Isabel GUERREIRO RODRIGUES; Filipa Raquel DA SILVA MATOS PEREIRA; Ludmylla CUNHA; Filipa GUERREIRO
Original assignee: Universidade Do Algarve
Priority date: 2015-04-29
Filing date: 2016-04-26
Publication date: 2016-11-03
Also published as: PT116887B; PT116887A; EP3288537A1

Abstract

The present invention relates to a process for producing polysaccharide microparticles for alveolar macrophage targeting, using single step spray-drying. The presente invention also relates to the polysaccharide microparticles, obtained by the process of the invention, and to such polysaccharide microparticles for use in the treatment of tuberculosis, as carriers for drugs used for treating such disease.

Description

DESCRIPTION

"PROCESS FOR PRODUCING POLYSACCHARIDE MICROPARTICLES FOR ALVEOLAR MACROPHAGE TARGETING, MICROPARTICLES OBTAINED

THEREIN AND USE THEREOF"

FIELD OF THE INVENTION

The present invention relates to microencapsulation techniques of drugs, useful in the production of microparticles for the treatment of tuberculosis, using polysaccharide microparticles obtained by single step spray-drying.

BACKGROUND OF THE INVENTION

Alveolar macrophages are resident cells of the lung, particularly of the alveolar zone, being directly involved in the progress of some diseases, such as tuberculosis. In that case, macrophages host the bacteria responsible for the disease {Mycobacterium tuberculosis) and are therefore a privileged therapeutic target. As alveolar macrophages reside in the alveoli, any drug intended at macrophage targeting is required to reach this zone of the respiratory tract. Inhalation of drugs aimed at reaching the alveolar zone requires the design of specific carriers that are endowed with the adequate aerodynamic characteristics to enable the desired distribution within the lung. To permit so, an aerodynamic diameter of 0.5 - 3 μπι is usually the referred range. Polymeric microparticles are the carriers proposed more frequently for this objective of drug inhalation, because their aerodynamic properties can be tailored to fit specific requirements. Spray-drying, in turn, is the most frequently used method to obtain microparticles. This is a microencapsulation technique known to produce microparticles with tunable properties depending on the optimisation of the parameters involved in the proper spray-drying procedure, thus fitting the need of permitting tuning the microparticles to exhibit specific characteristics. Macrophages exhibit several surface receptors that might be used to mediate targeting to these cells. Actually, these receptors are those involved in the recognition of pathogenic agents. Among the described receptors, the C-type lectin receptors and the scavenger receptors are the two families reported to be involved in most cases of polysaccharide recognition. The first family includes the mannose receptor, which has reported ability to recognise mannose, fucose, iV-acetylglucosamine and sulphated sugars, which are present in many polysaccharide substances. In turn, the second family has demonstrated affinity for several polysaccharides such as chondroitin sulphate, dextran sulphate and fucoidan, among others. This enhanced ability for recognition of certain units, makes the macrophages highly targetable with materials comprising these residues, namely polysaccharides. Currently, carriers proposed for inhalable therapy of tuberculosis are usually devoid of specific targeting strategies and their application is only based on adequate aerodynamic properties and on the natural ability of macrophages for the unspecific phagocytosis of particulate matter. Approaches including active targeting abilities are based on the surface modification of preformed antitubercular drug-loaded carriers with mannose residues. Mannosylated carriers are, thus, the unique described as having specific targeting ability towards alveolar macrophages. The preformed carriers are frequently composed by synthetic polymers, namely polyesters, of which the most often used are polylactic (PLA) and poly (lactic- co-glycolic) acid (PLGA) .

GRENHA A. et al , Natural carriers for application in tuberculosis treatment in Journal of Microencapsulation: Micro and Nano Carriers, Volume 30, Issue 3, 2013, describe the usefulness of using alveolar macrophages as therapeutic target in antitubercular therapy. It is mentioned that nano and microparticles composed for instance of natural polymers, such as the polysaccharides chitosan or hyaluronic acid, could be used for this end.

LAURIENZO P., Marine polysaccharides in pharmaceutical applications: an overview in Marine Drugs, 2010; 8 (9) : 2435-2465, describes that alginate microparticles permitted higher bioavailability and reduction of adverse effects when compared with the administration of antitubercular drugs in the free form.

KUNDAWALA A. et al , Preparation of Microparticles containing rifampicin as dry powder formulation: in vitro studies on aerossol performance in American Journal of Pharmtech Research 2(4), 2012, describe the production, by spray-drying, of a dry powder formulation of rifampicin consisting of chitosan microparticles, for an application in tuberculosis therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the microparticle morphology, observed by scanning electron microscopy (SEM) , wherein CRG denotes carrageenan, FUC denotes fucoidan, KGM denotes Konjac glucomannan, LBG denotes locust bean gum and GG denotes guar gum (partially hydrolysed) . Figure 2 shows comparative uptake of microparticles composed of different polysaccharides and polyvinyl alcohol (PVA, control), by NR8383 cells (rat alveolar macrophages), upon 2 hours exposure to 50 - 60 g/cm² of fluorescently-labelled microparticles (mean ± SEM, n > 3), wherein CRG denotes carrageenan, FUC denotes fucoidan, denotes LBG denotes locust bean gum and GG denotes guar gum (partially hydrolysed) . SUMMARY OF THE INVENTION

The invention refers to microparticles produced by a single-step spray-drying process, which are based on selected polysaccharides regarding the objective of alveolar macrophage targeting. The parameters of the spray- drying process are optimised to permit the production of microparticles that evidence aerodynamic properties suitable to reach the alveolar zone, where alveolar macrophages reside. The microparticles are aimed at providing an inhalable strategy for tuberculosis therapy, thus requiring macrophage targeting. For the purpose of demonstrating the ability of the microparticles to target macrophages, unloaded microparticles were produced and used.

DE TAILED DESCRIPTION OF THE INVENTION

The present invention uses natural materials and, namely polysaccharides, which are absolutely advantageous, because if polysaccharides composed of the mentioned recognisable residues are used, it becomes possible producing targeted drug carriers in a single step, without the need to include targeting ligands at a posterior phase. Additionally, polysaccharides are obtained at a low cost, have high probability of evidencing biocompatibility and are very flexible structures. The strategy of designing polysaccharide carriers for inhalable tuberculosis therapy encompasses the single-step production of a carrier that naturally targets the macrophages.

In the present invention, polysaccharides composing the microparticles were selected for their chemical structure, as this characteristic is the one specifically mediating the active targeting of macrophages. Selected polysaccharides include locust bean gum, guar gum, fucoidan, glucomannan, carrageenan, xanthan gum, glucan, dextran sulfate, chondroitin sulfate. All these polysaccharides bear in their structure, as natural components, chemical groups (namely sulphate) or residues (i.e. mannose, fucose, sulfate, iV-acetylglucosamine, galactose, etc) that are described to potentially undergo a direct recognition by alveolar macrophage surface receptors. The idea relying on this strategy of direct macrophage targeting was proven with some of the mentioned materials . Locust bean gum is a galactomannan, having mannose and galactose residues in an average molar ratio of 4/1.

Guar gum is a galactomannan, having mannose and galactose residues in an average molar ratio of 2/1.

Fucoidan has fucose units in its structure and also sulfate groups. Glucomannan has glucose and mannose units in its structure, in a molar ratio of 1/1.6.

To produce the microparticles , the first step consists on the solubilisation of the polysaccharides. Each polysaccharide was used individually and the concentration of polymer varied within 1.5 and 2% (w/v) . The lower concentration mentioned (1.5%) was used when 2% was too viscous to enable spray-drying. These concentrations were found adequate to obtain microparticles with Feret ' s diameter around 1-2 μπι, which is known to favour macrophage capture. Several aspects required optimisation prior to spray-drying :

Locust bean gum required grinding before solubilisation.

Locust bean gum required heating at 85 °C for 30 minutes to solubilise.

Carrageenan required heating at 50 °C for 30 minutes to solubilise.

The spray-dryer was used in open mode configuration and compressed air was the applied gas. The spray-drying conditions were variable, depending on the material being used. For the set of specified polysaccharides the conditions were: inlet temperature between 140 °C and 180 °C, aspirator between 80% and 90%, solutions feed rate between 0.7 and 2 mL/min.

Depending on the selected matrix material (polysaccharide) , the resulting microparticles either evidenced a spherical shape or a convoluted shape (Figure 1) . Of the set of polysaccharides mentioned above as possible materials, microparticles of locust bean gum, guar gum, fucoidan, carrageenan and glucomannan were produced to demonstrate the concept. The characteristics of microparticles are summarised in Table 1. Feret diameters were determined to be between 1.4 and 1.8 μπι, while real densities ranged between 1.0 and 1.7 g/cm³, which resulted in theoretical aerodynamic diameters varying within 1.7 μπι and 2.3 μπι. Overall, the characteristics indicate the theoretical suitability for deep lung inhalation.

Although not directly affecting the character of the invention, these microparticles were tested for their ability to encapsulate two different first-line antitubercular drugs in association, isoniazid and rifabutin, as required in tuberculosis therapy (single therapy is not recommended) . The encapsulation efficiencies varied between 74 and 89% for isoniazid, and 57 and 92% for rifabutin .

Objects of the Invention The first object of the invention is a process for producing polysaccharide microparticles for alveolar macrophage targeting, using single step spray-drying, comprising : a) dissolving the polysaccharide in order to obtain a concentration of polymer between 1.5 and 2% (w/v) ;

b) spray-drying the obtained solution, in a single step, using the following spray-drying conditions:

i) inlet temperature between 140 °C and 180 °C,

ii) aspirator between 80% and 90%,

iii) solutions feed rate between 0.8 and 2 mL/min.

Preferably, the polysaccharide is selected from the group consisting of locust bean gum, fucoidan, guar gum, carrageenan, glucomannan, xanthan gum, glucan, dextran sulfate and chondroitin sulfate.

Particularly preferred polysaccharides are locust bean gum, fucoidan, guar gum and carrageenan.

The spray-dryer is normally used in open mode configuration and the the gas applied by the spray-dryer is compressed air.

The second object of the invention consists of polysaccharide microparticles for alveolar macrophage targeting, obtained by the process described above, having Feret diameters between 1.0 and 2.0 μπι, real densities between 1.0 and 1.7 g/cm³ and theoretical aerodynamic diameters between 1.7 μπι and 2.3 μπι.

Usually, the polysaccharide microparticles have Feret diameters between 1.4 and 1.8 μπι. The third object of the invention consists of such polysaccharide microparticles for use in the treatment of tuberculosis, as carriers for drugs used for treating such disease.

One preferred drug combination is an association of isoniazid and rifabutin.

Preparation Examples

The following Examples are intended to illustrate the invention, but are not limitative of the scope of the invention, which is only determined by the contents of the claims .

Example 1

Locust bean gum (LBG) microparticles: 1 g of LBG is grinded in a mortar for 10 min, after which 5 mL HC1 0.1 M are added, while grinding continues until complete mixture of LBG and HC1. Purified water previously heated to 85 °C is then added, up to a final volume of 50 mL (LBG concentration is 2%, w/v) . The dispersion is maintained under magnetic stirring for 30 min and subsequently placed on a water bath at 85 °C under slow stirring for additional 30 min. At the end, the dispersion is kept under stirring at room temperature overnight, until the moment of spray- drying. Spray-drying operating parameters are: inlet temperature: 160 °C; aspirator: 85%; feed rate: 0.8 mL/min; and spray flow rate: 473 L/h. Microparticle production yield is 70%.

Example 2

Guar gum (GG) microparticles: 1 g of GG (partially hydrolysed) is dissolved in 50 mL of purified water under stirring for 20 min (GG concentration is 2%, w/v) . After dissolution, mannitol is added and dissolved in the GG solution to a final concentration of 0.5% (m/v) , in order to improve the final aerodynamic characteristics of the microparticles. The final solution is maintained under stirring for 20 min before spray-drying. Spray-drying operating parameters are: inlet temperature: 160 ± 2 °C; aspirator: 80%; feed rate: 1.0 mL/min; and spray flow rate: 473 L/h. Microparticle production yield is 73%.

Example 3 Konjac glucomannan (KGM) microparticles: 1 g of

KGM (partially hydrolysed) is dissolved in 50 mL of purified water, at 70 °C, under stirring for 45 min (KGM concentration is 2%, w/v) . Spray-drying operating parameters are: inlet temperature: 170 ± 2 °C; aspirator: 90%; feed rate: 0.7 mL/min; and spray flow rate: 473 L/h. Microparticle production yield is 69%.

Characteristics of the Microparticles characteristics of polysaccharide-based microparticles , in according to the invention, proposed to target alveolar macrophages in the ambit of tuberculosis therapy, are shown in Table 1.

Table 1

*Theoretical estimation (Aerodynamic diameter = Feret ' s diameter) x real density .

In vitro Assays

The proof-of-concept regarding the alveolar macrophage targeting ability was performed in vitro, using two cell culture models. Human THP-1 cells might be differentiated (using phorbolmyristate acetate, PMA) to exhibit macrophage phenotype and develop characteristics of alveolar macrophages. NR8383 cells are rat alveolar macrophages. Alveolar macrophages have scavenger receptors on the surface, including the mannose receptor (CD206) , reported as capable of recognising mannose, fucose units and sulphated sugars. The differentiation of THP-1 cells by ΡΜΆ is reported to not induce these receptors, while they are described to be present in NR8383 cells. While all the selected polysaccharides display structural units capable of undergoing specific recognition by macrophages, polyvinyl alcohol (PVA) is devoid of such moieties and was used as control.

For the effects of this assay, which involves macrophage capture of the microparticles and a subsequent determination of the number of macrophages from a population that phagocytosed the microparticles, each polysaccharide was previously labelled with a fluorescent marker before the spray-drying process. Microparticles composing each formulation were insufflated over cell layers in 6-well plates and incubated for 2 hours. After that period, the cells were recovered and analysed by flow cytometry, to examine the compositions favouring macrophage capture . Locust bean gum, fucoidan, guar gum and carrageenan were the polysaccharides undergoing the experimental test of uptake, apart from the control PVA. Flow cytometry determines the number of macrophages in the sample and quantifies the amount of macrophages that exhibit fluorescence, which is a consequence of having phagocytosed microparticles that are fluorescently labelled. A control test was performed using non- fluorescent microparticles. In that case, no macrophages are detected as emitting a fluorescent signal or the detected signal is not significant (maximum obtained was 1%) . NR8383 cells give the most meaningful results, as this is the cell line exhibiting the receptor of interest. As seen in Figure 2, upon the contact with a dose of 50-60 g/cm² of microparticles of each polymer, locust bean gum is the polymer showing higher affinity for macrophages, inducing 95% capture, followed by guar gum and carrageenan, which have a similar result (around 70% capture) . Curiously the uptake observed for fucoidan microparticles did not overpass 30%, inclusive remaining at a lower level comparing with the control PVA. The increase of the dose of contact to 220-250 g/cm² resulted in a general increase to values around 100% for all polysaccharides, indicating a dose-dependent effect (data not shown) . Locust bean gum, guar gum and the control PVA were also tested in macrophage-differentiated THP-1 cells at the concentration of 50-60 g/cm², which resulted in an increase of capture to values around 90-100% in all cases, possibly as a consequence of the absence of the specific mannose receptor that could differentiate the uptake. This indicates that the presence of such receptor indeed enables a specific targeting .

The other polysaccharides that were indicated also hold the ability to potentiate phagocytosis, as they bear units potentially recognised by macrophage surface receptors .

Claims

1. A process for producing polysaccharide microparticles for alveolar macrophage targeting, using single step spray-drying, comprising:

a) dissolving the polysaccharide in order to obtain a concentration of polymer between 1.5 and 2% (w/v) ;

i) inlet temperature between 140 °C and 180 °C,

ii) aspirator between 80% and 90%,

iii) solutions feed rate between 0.8 and 2 mL/min.

2. The process according to claim 1, wherein the polysaccharide is selected from the group consisting of locust bean gum, fucoidan, guar gum, carrageenan, glucomannan, xanthan gum, glucan, dextran sulfate and chondroitin sulfate.

3. The process according to claim 2, wherein the polysaccharide is selected from the group consisting of locust bean gum, fucoidan, guar gum and carrageenan.

4. The process according to any one of claims 1 to 3, wherein the spray-dryer is used in open mode configuration .

5. The process according to any one of claims 1 to 4, wherein the gas applied by the spray-dryer is compressed air.

6. Polysaccharide microparticles for alveolar macrophage targeting, obtained by process of any one of claims 1 to 5, having Feret diameters between 1.0 and 2.0 μπι, real densities between 1.0 and 1.7 g/cm³ and theoretical aerodynamic diameters between 1.7 μπι and 2.3 μπι .

7. The polysaccharide microparticles according to claim 6, having Feret diameters between 1.4 and 1.8 μπι.

8. The polysaccharide microparticles according to claim 6 or 7, for use in the treatment of tuberculosis, as carriers for drugs used for treating such disease.

9. The polysaccharide microparticles for use according to claim 8, wherein the the drug is an association of isoniazid and rifabutin.