Capreomycin oleate microparticles for intramuscular administration: Preparation, in vitro release and preliminary in vivo evaluation

Journal of Controlled Release 209 (2015) 229–237

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Capreomycin oleate microparticles for intramuscular administration: Preparation, in vitro release and preliminary in vivo evaluation Adrián Cambronero-Rojas a,b, Pablo Torres-Vergara a,⁎,1, Ricardo Godoy a, Carlos von Plessing a, Jacqueline Sepúlveda c, Carolina Gómez-Gaete a,⁎,1 a b c

Faculty of Pharmacy, University of Concepción, Concepción, Chile Hospital Dr. Fernando Escalante Pradilla, CCSS, San José, Costa Rica Department of Pharmacology, Faculty of Biological Sciences, University of Concepción, Chile

a r t i c l e

i n f o

Article history: Received 28 July 2014 Received in revised form 24 April 2015 Accepted 3 May 2015 Available online 5 May 2015 Keywords: Capreomycin oleate PLGA DPPC Microparticles Spray drying Intramuscular Tuberculosis

a b s t r a c t Capreomycin sulfate (CS) is a second-line drug used for the treatment of multidrug-resistant tuberculosis (MDRTB). The adverse effects profile and uncomfortable administration scheme of CS has led to the development of formulations based on liposomes and polymeric microparticles. However, as CS is a water-soluble peptide that does not encapsulate properly into hydrophobic particulate matrices, it was necessary to reduce its aqueous solubility by forming the pharmacologically active capreomycin oleate (CO) ion pair. The aim of this research was to develop a new formulation of CO for intramuscular injection, based on biodegradable microparticles that encapsulate CO in order to provide a controlled release of the drug with reduced local and systemic adverse effects. The CO-loaded microparticles prepared by spray drying or solvent emulsion-evaporation were characterized in their morphology, encapsulation efficiency, in vitro/in vivo kinetics and tissue tolerance. Through scanning electron microscopy it was confirmed that the microparticles were monodisperse and spherical, with an optimal size for intramuscular administration. The interaction between CO and the components of the microparticle matrix was confirmed on both formulations by X-ray powder diffraction and differential scanning calorimetry analyses. The encapsulation efficiencies for the spray-dried and emulsion-evaporation microparticles were 92% and 56%, respectively. The in vitro kinetics performed on both formulations demonstrated a controlled and continuous release of CO from the microparticles, which was successfully reproduced on an in vivo rodent model. The results of the histological analysis demonstrated that none of the formulations produced significant tissue damage on the site of injection. Therefore, the results suggest that injectable CO microparticles obtained by spray drying and solvent emulsion-evaporation could represent an interesting therapeutic alternative for the treatment of MDR-TB. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Tuberculosis (TB) is an infectious bacterial disease considered as a major cause of illness and death in many countries [1,2]. In humans, this disease is mainly caused by contagion with Mycobacterium tuberculosis [3] and pulmonary TB is the commonest clinical presentation [4,5]. Pharmacological treatment of TB is basically a combination of drugs that must be given (orally or via injection) under a strict scheme, which varies according to the degree of resistance held by the bacterial strain that infects the patient [6]. Total compliance to TB treatments is difficult to achieve because of their length, the amount of drugs administered, the severity of certain adverse effects and the route of administration

⁎ Corresponding authors at: Facultad de Farmacia, Universidad de Concepción, Barrio Universitario s/n, Concepción, Chile. E-mail addresses: [email protected] (P. Torres-Vergara), [email protected] (C. Gómez-Gaete). 1 Carolina Gómez-Gaete and Pablo Torres-Vergara are both senior authors.

http://dx.doi.org/10.1016/j.jconrel.2015.05.001 0168-3659/© 2015 Elsevier B.V. All rights reserved.

of some drugs, which can be very uncomfortable for most of patients [7]. Failure to comply can lead to the apparition of M. tuberculosis strains that are resistant to first-line drugs. Multidrug-resistant tuberculosis (MDR-TB) is often the result of a failed treatment with first line drugs due to patient non-compliance, prescription of a wrong/incomplete drug scheme, or an ineffective directly observed treatment short course (DOTS) therapy. Treatment of MDR-TB involves the use of second-line drugs, which are more expensive and have more adverse effects than first-line drugs [8]. Capreomycin sulfate (CS), a peptide obtained from strains of Streptomyces capreolum, is a second-line drug used to treat patients infected with isoniazid and rifampicin-resistant M. tuberculosis strains [9,10]. Despite its efficacy, the adverse effects profile of CS and administration route, based on repeated intramuscular injections for several days, reduce the probabilities of achieving a full recovery from the disease [11]. As CS is an effective drug when tolerated by the patient, this subject has been a matter of interest for many research groups that have addressed the issue with the current advances in microparticle

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technology. Nowadays, pulmonary delivery of inhalable powders [12–14], liposomes [15,16] and polymeric microparticles [17,18] has been extensively studied as potential approaches because, presumably, the drug could reach the lungs with a reduced systemic distribution. To date, there is a work that attempted to test an inhalable CS formulation in healthy volunteers with interesting results [14]. However, in the case of liposomes and microparticles the works of Giovagnoli et al. [15,17] and Ricci et al. [16] showed that the chemical nature of CS limits considerably its encapsulation into hydrophobic matrices because of its high aqueous solubility. In order to overcome this limitation, Schoubben et al. [18] proposed that reducing the aqueous solubility of capreomycin through a simple ion pair exchanging reaction with a hydrophobic counterion could increase the drug loading within the microparticle and therefore allowing its use as a inhalable dosage form. The results showed that the ion pairing of capreomycin with sodium oleate formed capreomycin oleate, a product with reduced solubility and capable of being loaded at larger amounts into poly(D,L-lactide-co-glycolide) (PLGA) microparticles. Furthermore, recently it has been demonstrated that CO pharmacological activity is comparable to the showed by CS [19]. Although pulmonary TB constitutes the majority of cases and the administration of inhalable powders could be a significant step forward in terms of efficacy, there is still a problem that needs to be solved for patients that suffer extrapulmonary TB. Therefore, a valid alternative of taking advantage of the known features present in PLGA-based microparticulate systems, including controlled release of drug, reduction of adverse effects and increased stability of the active drug among others, is developing a depot formulation that can be administered through intramuscular (IM) injection. On recent years, the increasing number of depot formulations based in microparticles demonstrates the IM route could be an improvement in terms of compliance [20]. The preparation of PLGA microparticles through the solvent emulsion-evaporation method is a well-documented procedure and easy to implement, but there are other alternatives of excipients and preparation methods that could be more efficient when considering a large-scale production. The spray drying method is one of the most reliable ways to achieve the latter purpose because the machinery is designed to allow escalation of the process and also, the resulting particulate systems can exhibit some useful features [21]. The excipients used for preparation of spray-dried microparticles, including 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and hyaluronic acid (HA) have demonstrated to be highly stable, biocompatible and especially, capable to provide a controlled release of drug [21,22]. In the present work, the preparation of capreomycin oleate microparticles for intramuscular administration by spray drying and solvent emulsion-evaporation is described. Also, this report provides the first evidence of the in vitro/in vivo performance showed by a particulate system that encapsulates CO, aiming for a potential application in patients who suffer from MDR-TB.

solution was dropwise added to a 1 M aqueous solution of CS under magnetic stirring (700 rpm) in order to obtain the CO ion pair. The obtained suspension was centrifuged at 12,000 rpm for 10 min at room temperature and then the supernatant was discarded, repeating the procedure once. The washed product was reconstituted in 40 mL of nanopure water and then freeze dried for 15 h. The resulting solid (CO) was collected in an amber bottle and stored at 8 °C. 2.3. Microparticle preparation 2.3.1. Spray drying method DPPC and HA microparticles were prepared by the spray drying method, according to the procedure described by Gómez et al., with modifications [21]. DPPC and CO were dissolved in ethanol and HA in ultra-pure water. Then, a 75/25 (v/v) mixture of both ethanolic and aqueous solutions was prepared, keeping a solid concentration in the final solution at 2 g/L. The mixture was fed into a B-290 mini spray dryer (Büchi, Switzerland), which was set up with the following conditions: inlet temperature: 110 °C, outlet temperature: 60–65 °C, air flow rate: 500 L/h, feed-flow rate: 17 mL/min, aspiration: 100%, nozzle diameter: 0.7 mm. Powder samples were stored at room temperature under vacuum in a dessicator immediately after their spray-drying to limit the uptake of moisture. It has been shown in a previous work that environmental moisture leads to a modification of the supramolecular organization of chemicals within particles [21]. The yield was calculated using the following equation (Eq. (1)): % yield ¼

mass of the powder collected  100: initial mass of solids in the solution prior to spray drying

ð1Þ

2.3.2. Solvent emulsion-evaporation method PLGA microparticles loaded with CO were prepared through the solvent emulsion-evaporation method [23]. Briefly, CO was dissolved in 0.3 mL of ethanol and 2.2 mL of acetone. The resulting solution was mixed with 300 mg of PLGA dissolved in 2.5 mL dichloromethane. The organic solution was then pre-emulsified with 20 mL of a 0.25% w/v PVA aqueous solution by shaking it at 3200 rpm for 1 min with a Vortex Genie 2 (Scientific Industries Inc. USA). In order to obtain a microemulsion, the pre-emulsion was homogenized for 30 s at 8000 rpm with a mixer system (Heidolph, Germany). The organic phase was evaporated at room temperature under gentle agitation (700 rpm) and the microparticle suspension was then completed to 20 g by weight with nanopure water. Amber vials were used throughout the process to provide protection against UV light, which degrades capreomycin [24]. 2.4. Quantification of capreomycin in samples

2. Materials and methods

Capreomycin sulfate, sodium oleate and hyaluronic acid sodium salt 95% (HA) were purchased from Sigma-Aldrich (USA). Poly (lactic-coglycolic acid) (PLGA 50:50) Resomer® RG502 was provided from Boehringer Ingelheim (Germany) and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) by Genzyme Pharmaceuticals (Switzerland). All other chemicals and reagents were of the highest purity grade commercially available.

The amount of capreomycin within the microparticles and its concentration in liquid samples (e.g. medium for in vitro release and rat serum) was determined by HPLC using a validated method for each matrix, based on the conditions described by Rossi et al. [25] with modifications. Analyses were performed in a LaChrom Elite system (Merck-Hitachi, Japan), using a Kromasil Reverse Phase C-18 column (5 μm, 250 mm), a mobile phase composed of acetonitrile-KH2PO4 buffer solution (pH 2.2; 0.2 M) with 0.2% of heptafluorobutyric acid (7.5:92.5 v/v) at isocratic flow rate of 1 ml min−1. The UV detection of capreomycin was set up at 268 nm.

2.2. Capreomycin oleate preparation

2.5. Capreomycin oleate loading within microparticles

Capreomycin oleate was prepared by the method described by Schoubben et al. [18] with modifications. Briefly, a 3.5 M sodium oleate

The extraction of CO from the microparticle matrix was performed by adding 1 mL of methanol or acetonitrile on a weighted amount of

2.1. Materials

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spray-dried and emulsion-evaporation microparticles, respectively. For the latter, a previous extra step had to be included, as the particles were obtained by this last method. Briefly, a weighted amount of suspension was centrifuged at 14,000 rpm for 15 min at 25 °C in a Z320 centrifuge (Hermle Scientific, Germany) and the supernatant was discarded, leaving the particles ready for the extraction procedure. The encapsulation efficiency was assessed as the ratio between the determined and theoretical amounts of CO loaded (Eq. (2)) and all the measurements were carried out in triplicate, expressing the variation as S.D. Encapsulation efficiency ¼

actual amount of capreomycin  100: theoretical amount

ð2Þ

2.6. Morphological analysis and particle size Samples were analyzed in a JMS-6380 LV scanning electronic microscope (JEOL, Japan). The microparticles obtained by solvent emulsionevaporation were isolated by centrifugation to remove any excess of PVA and then were reconstituted in ultrapure water. Samples were coated with a gold layer of 150 Å of thickness, using a S 150 metallizer (Edwards, England). The particle size of the microparticles was described by the volume-number mean diameter, calculated from the number distribution estimated (assuming spherical shape) by measuring approximately 200 particles located in an arbitrarily chosen area, with the TRO111-020 software (JEOL, Japan). Span, a mathematical value related to the granulometric dispersion, was calculated according to Eq. (3). Span ¼

D90‐D10 D50

ð3Þ

where D10, D50 and D50 are the particles diameters at 10, 50 and 90%, respectively, of the accumulated number-distribution calculated from the sample. 2.7. Differential scanning calorimetry DSC analyses were carried out in a DSC-822e system (Mettler Toledo, Germany) coupled to a Haake EK90/MT Intra-Cooler (Haake, Germany). Approximately 1 mg of sample was weighted into an aluminum pan and the analysis parameters were set up in the range of 20–300 °C, with a scanning rate 10 °C/min under a dynamic nitrogen atmosphere (N2 flow rate: 20 mL/min). 2.8. Powder X-ray diffraction (XRD) X-ray diffraction patterns of drugs, excipients, blank an CO-loaded microparticles were obtained from an EndeavorD4 X-ray diffractometer (Brüker, Germany), built with a fine-focus, Ni filtered Cu Kα1 (λ = 1.54 A) beam, operated at 40 kV and 20 mA. Samples were mounted on a plate volume of about 1 cm3 and the analyses were performed at room temperature in the 2θ range between 5° and 45°.

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Medcenter Einrichtungen, Germany) set up at 37 °C under magnetic stirring at 100 rpm. At predetermined time intervals; 1 mL of medium was withdrawn, being replaced with an equal volume of fresh and warm medium. After filtration (0.22 μm PVDF filter), samples were stored at 4 °C until their analysis by HPLC as described above. The samples proved to be stable in medium for at least 30 days at 4 °C (data not shown) and all the experiments were performed at least in triplicate. 2.10. In vivo studies 2.10.1. Pharmacokinetic study The in vivo experiments were conducted according to the University of Concepcion Ethical Committee Regulations for Animal Handling. Male and female Sprague Dawley rats (250–300 g) obtained from the University animal unit were used and kept in groups of 5 per cage, at room temperature with free access to food and water. CS, CO and COloaded microparticles were administered intramuscularly (n = 3 for each treatment) into semi-membranosus or semi-tendinosus muscles, at a dose equivalent to 20 mg/kg of capreomycin [26]. The specimens were anesthetized with an intraperitoneal solution of ketamine in a single 60 mg/kg dose. Samples of blood were withdrawn at predetermined time intervals by cardiac puncture and after obtaining the required volume of blood the specimen was sacrificed by cervical dislocation. Serum samples were obtained by centrifugation of the collected blood at 5000 rpm for 5 min and were immediately stored at −20 °C until analysis. The concentration of capreomycin in serum was determined with the HPLC method described above. From the serum concentrations obtained after each treatment, a time-concentration curve was constructed and certain pharmacokinetic parameters were determined including area under the curve (AUC) calculated by the trapezoidal rule method, maximum concentration reached (Cmax) and time necessary to reach the maximum serum concentration (tmax). 2.10.2. Histological analysis The effect of CS, CO, blank and CO-loaded microparticles on tissue integrity at the site of injection was assessed by histological analysis of muscle samples exposed to the compounds and formulations. Rats were sacrificed by cervical dislocation at predetermined time intervals post-injection in order to obtain samples from semi-membranous and semi-tendinous muscles. Samples were fixed in 4% (w/v) paraformaldehyde dissolved in phosphate buffer saline (pH 7.4), then impregnated and embedded in paraffin. Each sample was cut with a RM 2145 microtome (Leica Microsystems, Switzerland) in order to obtain a 10 μm in thickness slice that was treated in xylol to eliminate the paraffin and finally stained with Mayer's hematoxylin and eosin. After staining, samples were mounted in a glass slide prior to optical microscope observation with 10× and 40× zoom objectives. The analysis of samples included the study of the following markers of tissue injury: presence of inflammatory cells, damage of muscle fibers, hyperemia in epimysium, hyperemia in perimysium, hyperemia in endomysium, hemorrhage in perimysium, hemorrhage in epimysium and presence of fibroblasts. 3. Results and discussion

2.9. In vitro release kinetics of capreomycin oleate from microparticles

3.1. Optimization of microparticle formulation and morphological analysis

The in vitro release analysis of CO from the microparticles was performed under sink conditions (CO aqueous solubility 40 μg/mL), using 10 mM HEPES buffer saline (150 mM NaCl, pH 7.4) as medium. An accurately weighted amount of microparticles, equivalent to 2 mg of CO, was resuspended in 10 mL of medium and added into a prehydrated dialysis membrane pore size of 12,000 kDa. In the case of microparticles obtained by solvent emulsion-evaporation, they were centrifuged at 6000 rpm for 10 min before being resuspended. The filled membranes were placed into beakers containing 240 mL of medium. Experiments were carried out within a Venticell oven (MMM

For preparation of microparticles by the spray drying method, DPPC and HA were used as matrix components. DPPC has a high entrapment capacity of lipophilic compounds [22,27,28], while HA is a polysaccharide that has been used to improve the physical properties of the powders obtained by spray drying and also to modulate the release of active compounds [22,29]. In order to optimize the physical and morphological properties of the final formulation, the excipient ratio was modified but maintaining the amount of CO (5% w/w) constant in the mixture. Microparticles with increasing concentrations of HA (5, 15 and 25% w/w) were elaborated and the amount of DPPC was

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adjusted to keep the total solids concentration at 2 g/L. Independent of the ratio of the excipients the yield of the process remained at 45 ± 6%. The micrographs of the prepared formulations are shown in Fig. 1. At the lowest HA amount used (Fig. 1A), the particles formed aggregates and this behavior can be attributed to the tendency of DPPC to form highly cohesive powders. Increasing the HA concentration to 15% w/w did not revert the aggregation (Fig. 1B) but at 25% w/w, the resulting particles showed a spheric shape with a smooth surface and less aggregation (Fig. 1 C). The formulation composed of DPPC 70%, HA 25% and 5% CO gave microparticles with a volume-number mean diameter of 8.86 μm and was selected for further studies. In the case of microparticles prepared by solvent emulsionevaporation method (Fig. 2), the formulation parameters were adjusted in function of the amount of CO used, as the amount of PLGA 50:50 were kept constant. The microparticles prepared with 1 and 5 mg of CO (Fig. 2A and B, respectively) were spherical, monodisperse and without presence of drug crystals in their surface. When the amount of CO was increased to 10 mg (Fig. 2C) the particles had crystals adsorbed in its surface, whereas particles containing 15 mg (Fig. 2D) resulted to be extremely agglomerated and with a great number of pores, probably because PLGA was unable to encapsulate such amount of drug. As the presence of crystals in the surface is not desired when it comes to achieving a controlled release of drug from the polymeric matrix, the formulation loaded with 5 mg provided the best results in this regard, with a volume-number mean diameter of 3.60 μm. The particle size distribution and Span value of the selected formulations prepared by both methods are summarized in Table 1. Regarding the size of the chosen microparticulate formulations, the selection was done to overcome certain shortcomings related to the spray-drying technique, including condensation in the drying chamber, increase of the output temperature and clogging of the spraying nozzle due to the increased viscosity of solutions with a higher concentration of solids (larger particle size), among others. Therefore, the emulsion/ evaporation microparticles were prepared with a particle size as similar as possible in order to make a better comparison of the results obtained in further studies. Although it has been clearly demonstrated that formulations with a particle size b 10 μm can suffer phagocytosis from macrophages surrounding the site of injection, this issue was assumed to not be a major inconvenience at the moment of outlining the in vivo experiments. The evidence from reports that tested microparticle systems designed for pulmonary [30,31] and intramuscular [32] delivery of drugs has proved that despite the smaller particle size and subsequent phagocytosis, it is possible to reach therapeutic plasma concentrations or at least modify the pharmacokinetics to the point of achieving measurable levels in the target tissue when compared to the non-encapsulated drug. Furthermore, the concerns about the safety of polymer-based pulmonary delivery systems in chronic treatments helped to make the decision of using the intramuscular route in this study.

3.2. Drug loading The encapsulation efficiency value obtained for the formulation prepared by spray drying was 92 ± 8% (n = 5). This result is consistent with those described by several reports that regarded this method as being capable of providing high encapsulation values, because the droplets formed by atomizing the solution contain exactly equal amounts of the components present in the mixture, so each formed droplet becomes a microparticle when dried [33]. On the other hand, the emulsion-evaporation method did not provide the same outcome, despite the change in lipophilicity done to capreomycin in order to increase its encapsulation into hydrophobic matrices. The amount of CO incorporated into the particles was 56 ± 7% (n = 5), value that could be explained by the diffusion of drug from the microdroplets during the evaporation phase [34], or a reduced interaction between CO and the polymeric matrix. 3.3. Structural analysis In order to evaluate any possible interactions between CO and the formulation components, DSC and XRD analyses were carried out on both microparticulate formulations. The DSC curves obtained from the formulation components used in the method of spray drying (Fig. 3, top) show for HA a wide endothermic band in the temperature range of 30 to 150 °C, which may reflect loss of the volatile components, while at 230 °C there is an exothermic peak attributed to the degradation of the polysaccharide (Fig. 3 top, curve A) [35]. In the case of DPPC, two characteristic endothermic peaks were observed including a pretransition peak corresponding to a gel-to-ripple (Tp) phase at ~67 °C and the bilayer main phase transition (Tm) to the liquid crystalline phase at ~72 °C (Fig. 3 top, curve B) [36,37]. The CO curve (Fig. 3 top, curve C) shows a typical endothermic band corresponding to degradation of the peptidic fraction that conforms the hydrophobic ion pair [18]. The DSC curve of the physical mixture shows again the previously mentioned peaks of DPPC and HA but not the peak corresponding to CO, probably due to the low amount of drug present in the mixture (Fig. 3. top, curve D). The thermograms of both unloaded and CO-loaded microparticle formulations are similar as they only show the main phase transition temperature of DPPC (Fig. 3 top, curves E and F, respectively). In the case of microparticles prepared by solvent emulsionevaporation, DSC curves (Fig. 3, bottom) for CO, PLGA, physical mixture and unloaded or CO-loaded microparticles were generated as well. Thermograms for PLGA and unloaded microparticles exhibited a glass temperature transition around 40 °C (Fig. 3 bottom, curves B and D). The endothermic band corresponding to denaturation of CO was not observed for both the physical mixture and CO-loaded microparticles (Fig. 3 bottom, curves C and E), which only have the endothermic peak associated with the glass transition temperature of the polymer.

Fig. 1. SEM micrographs of microparticles prepared by the spray-drying method. Percentage of HA employed in the formulation (% w/w): 5 (A), 15 (B), 25 (C).

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Fig. 2. SEM micrographs of microparticles prepared by the solvent emulsion-evaporation method. Amount of CO employed in the formulation (mg): 1 (A), 5 (B), 10 (C), 15 (D).

The absence of a CO-related peak can be attributed again to the low amount present in the formulation, since the physical mixture did not show the band associated to denaturation of the compound. Confirmation of the physical state of CO within the microparticles was done by powder X-ray diffraction. The diffractogram of pure DPPC showed peaks in the range of 21–23° 2θ indicating the presence of a multi-lamellar bilayer structure (Fig. 4 top, curve A) [36], while HA shows the expected lack of peaks characteristic of amorphous structure (Fig. 4 top, curve B). The diffraction pattern of CO confirms its crystalline nature (Fig. 4 top, curve C) and the physical mixture of the components used for preparation of the microparticles revealed the presence of peaks that belong CO (Fig. 4 top, curve D), but those were not observed in the drug-loaded microparticle diffractogram (Fig. 4 top, curve F). The X-ray pattern of the DPPC/HA microparticles greatly differs from the patterns obtained from the raw materials, suggesting a different organization of both components within the microparticles and an interaction between them. These interactions together with the dispersion of CO at the molecular level within the particulate matrix might impact the release of drug from the formulation. The results obtained for the microparticles prepared by solvent emulsion-evaporation (Fig. 4, bottom) showed a similar outcome, suggesting that CO is dispersed at a molecular level in the microparticles obtained by both methods.

Table 1 Microparticle size of the selected formulations shown as number distribution. Method

Spray drying Solvent emulsion-evaporation

Diameters (μm) ± DS

Span

D10

D50

D90

2.06 ± 0.71 1.01 ± 0.08

7.85 ± 4,27 2.94 ± 0,25

9.14 ± 2.03 3.12 ± 0.96

0.87 0.72

3.4. In vitro release studies The results of release kinetics performed on free CS and CO, COloaded spray-dried and solvent emulsion-evaporation microparticles release profiles are displayed in Fig. 5. As it was expected, the dissolution and diffusion of free CS from the dialysis bag was fast and complete after 8 h (Fig. 5, top). For CO the dissolution and diffusion rate was slower, demonstrating that the chemical modification can change the rate of dissolution of capreomycin under the described experimental conditions. The release profiles of CO from microparticles show that both formulations exhibit a similar behavior (Fig. 5, bottom). In the first 8 h there is a burst release that can be attributed to the adsorbed drug on the surface or encapsulated in the outermost layers of the particles. From then, the release of CO became continuous, reaching approximately a 50% of released drug after 16 days. This behavior remained fairly constant but the spray-dried particles released the totality of the drug after 26 days, while solvent emulsion-evaporation particles continued releasing drug until day 28. XRD analyses from previous reports have demonstrated that the microparticles prepared with DPPC and HA have a particular structure that allows a controlled release of drug as HA is “sandwiched” between DPPC polar head groups [21]. The interaction between CO and the matrix may be determined by the oleate group, which could be embedded within the hydrophobic fractions delaying the release of drug until HA is hydrated. Moreover, the mechanisms involved on the release of drug from PLGA microparticles are well known and the profiles obtained in this study suggest that the release of CO follows a triphasic pattern. The first phase corresponds to a fast release of CO that is adsorbed on the surface of the particle, followed by a second phase in which the drug is slowly released from the polymeric matrix that undergoes through a hydration and erosion process. Finally, the third phase

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Fig 3. DSC curves. Top: HA (A), DPPC (B), CO (C), physical mixture (D), unloaded microparticles (E), CO-loaded microparticles (F). Bottom: CO (A), PLGA (B), physical mixture (C), unloaded microparticles (D), CO-loaded microparticles (E).

Fig 4. XRD diffraction patterns. Top: DPPC (A), HA (B), CO (C), physical mixture (D), unloaded microparticles (E), CO-loaded microparticles (F). Bottom: CO (A), PLGA (B), physical mixture (C), unloaded microparticles (D), CO-loaded microparticles (E).

of faster release is often attributed to the onset of the erosion process [38].

3.5. In vivo pharmacokinetic study The time-concentration profiles of the studied formulations and their pharmacokinetic parameters are presented in Fig. 6 and Table 2, respectively. The profile obtained from an intramuscular administration of CS (Fig. 6, top) is consistent with the available data, which describes capreomycin as a drug that is quickly excreted after the intramuscular administration of an aqueous solution [11]. The influence of the reduction in CS aqueous solubility is showed in the CO time-concentration profile, as there is a significant modification in the pharmacokinetic behavior of capreomycin, expressed through an increase in tmax and a reduction of Cmax. In terms of pharmacokinetic parameters, CO reaches its peak concentration at 24 h post-application (20.52 μg/mL), whereas for CS, Cmax (29.32 μg/mL) is reached 1 h after its administration. Despite the observed changes, the outcome is not so surprising because as capreomycin is a drug whose elimination goes under first-order kinetics [39,40], its rate is a function of the concentration present in serum, so the administration of a salt with reduced solubility will influence the absorption from the site of injection, becoming the ratelimiting step that alters the elimination phase of the drug. The use of insoluble salts is a well-documented method employed to increase the residence time of drugs that are quickly excreted when administered intramuscularly [9,10].

The encapsulation of CO into lipophilic microparticules had an even more marked effect on the disposition of the drug. From the time concentration profiles (Fig. 6, bottom), it can be said that both spray-dried and solvent emulsion-evaporation microparticles are capable to keep sustained serum concentrations of capreomycin for at least 6 days, with a tmax of 8 h and an observed Cmax of 13.75 and 15.29 μg/mL, respectively. The fast rise in serum concentrations of capreomycin can be explained by the transfer of drug that is bound or trapped in the surface of the microparticles [22,38], which may be favored by the abundant and stable blood muscular irrigation. The oscillant pattern after reaching Cmax is probably a product of the controlled release of CO from the microparticle, whose structure suffers a gradual erosion that provides free drug and leads to a complete disintegration of the matrix [41]. The obtained AUC values for the microparticulate formulations could be justified by employing the same approach utilized to describe the differences between the pharmacokinetics of CS and CO. In this case, the controlled and sustained release of drug provided by both formulations becomes another rate-limiting factor that will modify the intramuscular absorption of capreomycin from the site of injection, thus reducing the peak concentrations in serum and consequently the elimination rate, resulting finally in an increased residence time. Due to the exploratory nature of this study it was not possible to characterize in detail the kinetics involved on each phase, but from what is observed in the calculated pharmacokinetic parameters and time-concentration profiles the outcome supports the proposed hypothesis.

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(2 μg/mL) reported for capreomycin sulfate on virulent strains of M. tuberculosis [11] and a significantly larger exposure to CO, expressed as AUC. Capreomycin oleate has demonstrated to show activity over susceptible M. tuberculosis strains that is comparable to the obtained with CS [19], so in this regard it can be assumed that a CO formulation should be capable to perform well in a infection in vivo challenge study. If the results of this study are compared to previous publications that tested their formulations on animal and human models, it must be noted that the inhalable powder developed by Fiegel et al. and GarcíaContreras et al. [12,39] have the advantage of a reduced systemic exposure to capreomycin, but still require the administration of multiple doses in order to maintain concentrations within the therapeutic range. The phase I trial of an inhalable capreomycin formulation in healthy volunteers performed by Dharmadhikari et al. [14] confirms this statement. From a practical perspective, depot IM formulations are generally well tolerated and can solve many issues related to compliance, as patients do not need to be reminded about taking their drugs and is possible to sort out cases in which the patient is not collaborative. 3.6. Histological analysis

Fig 5. Top: dissolution kinetics of CS and CO as free drug in HEPES buffer under sink conditions. Bottom: Release kinetics of CO from microparticles in HEPES buffer under sink conditions. Results are expressed as mean ± SD (n = 3).

Summarizing the obtained results, there are some points to highlight. A single 20 mg/kg dose of CO-loaded microparticles provides sustained concentrations of the drug for at least 8 days, which are considerably over the minimum inhibitory concentration (MIC) value

The results from the histological analysis performed on biopsies of semi-membranous and semi-tendinous muscle exposed to a single dose of CO-loaded microparticles prepared by both methods are shown on Fig. 7. From the micrographs, it can be observed that after 8 h of injection there was presence of inflammatory cells with hyperemia in endomysium and perimysium (Fig. 7A and D). Hemorrhage in the studied areas was mild to moderate without presence of eosinophils and scarce fibroblast growth. In biopsies obtained at 48 h (Fig. 7B and E) there is a clear decrease of inflammatory cells and an improvement in the lesions. At 192 h (Fig. 7C and F), the tissue showed no signs of inflammation or hyperemia. The population of fibroblasts and satellite cells was low at 48 and 192 h of sampling, without signs of necrosis. As can be seen, the findings are consistent with the events that arise after an intramuscular injection [42,43]. It must be noted that the obtained results must be considered as a preliminary evaluation and should be reproduced with multiple doses of the selected formulations in order to conclude if the encapsulation of capreomycin into microparticles has a beneficial effect in terms of tolerability, as a single dose not necessarily reflects the changes in tissue integrity that would appear under a chronic treatment. Nevertheless, as the injection of CO-loaded microparticles did not show significant differences regarding tissue damage and wound recovery when compared to the exposure to free CS or CO and blank microparticles (data not shown), the outcome could set a trend for further studies. Furthermore, the amount of evidence that supports the biocompatibility and biodegradability of PLGA and DPPC would lead to think that a chronic intramuscular treatment with microparticulate formulations is feasible [44]. 4. Conclusion

Fig 6. Time–concentration profiles obtained from intramuscular administration of the studied formulations to Sprague-Dawley rats. Top: CS and CO as free drug. Bottom: COloaded microparticles. Results are expressed as mean ± SD (n = 3).

This work has successfully attempted to develop two microparticulate formulations that provide a controlled release of capreomycin after their intramuscular administration. The pharmacokinetic analysis of the in vivo study performed in rats showed that both formulations kept sustained concentrations of the drug for several days without significant tissue damage on the site of injection. However, the main difference between the studied formulations is that the spray-dried microparticles can be prepared at a larger scale and encapsulate more efficiently CO in this particular case, so these advantages constitute a selling point for pharmaceutical companies and health organizations that are interested in counting with a new formulation capable to give an effective treatment of MDR-TB. Further work will

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Table 2 Pharmacokinetic parameters of the studied formulations. Results are expressed as mean ± SD (n = 3). Formulation

Time (h)

AUC 0–t (μg × h × mL−1)

Observed tmax (h)

Observed Cmax (μg/mL)

CS CO Spray-dried microparticles Solvent emulsion-evaporation microparticles

0–8 0–96 0–240

60.13 ± 1.13 747.61 ± 62.07 2210.01 ± 73.11 2127.49 ± 202.64

1 24 8 8

29.32 ± 1.27 20.52 ± 0.74 13.75 ± 2.15 15.29 ± 1.84

Fig. 7. Micrographies (10×) of rat muscle biopsies inoculated with CO-loaded microparticles prepared by the spray drying method (a–c), group inoculated with CO-loaded microparticles prepared by solvent emulsion-evaporation method (d–f), at different time points: 8 h (left column), 48 h (central column) and 192 h (right column).

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