Journal of Controlled Release 209 (2015) 229–237

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Journal of Controlled Release

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

a r t i c l e i n f o a b s t r a c t

Article history: Capreomycin sulfate (CS) is a second-line drug used for the treatment of multidrug-resistant tuberculosis (MDR-
Received 28 July 2014 TB). The adverse effects profile and uncomfortable administration scheme of CS has led to the development of
Received in revised form 24 April 2015 formulations based on liposomes and polymeric microparticles. However, as CS is a water-soluble peptide that
Accepted 3 May 2015
does not encapsulate properly into hydrophobic particulate matrices, it was necessary to reduce its aqueous sol-
Available online 5 May 2015
ubility by forming the pharmacologically active capreomycin oleate (CO) ion pair. The aim of this research was to
Keywords:
develop a new formulation of CO for intramuscular injection, based on biodegradable microparticles that encap-
Capreomycin oleate sulate CO in order to provide a controlled release of the drug with reduced local and systemic adverse effects.
PLGA The CO-loaded microparticles prepared by spray drying or solvent emulsion-evaporation were characterized in
DPPC their morphology, encapsulation efficiency, in vitro/in vivo kinetics and tissue tolerance. Through scanning elec-
Microparticles tron microscopy it was confirmed that the microparticles were monodisperse and spherical, with an optimal size
Spray drying for intramuscular administration. The interaction between CO and the components of the microparticle matrix
Intramuscular was confirmed on both formulations by X-ray powder diffraction and differential scanning calorimetry analyses.
Tuberculosis
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 sol-
vent 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 tubercu-
losis [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: pabltorr@udec.cl (P. Torres-Vergara), cargomez@udec.cl
(C. Gómez-Gaete).
1
Carolina Gómez-Gaete and Pablo Torres-Vergara are both senior authors.
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 Strepto-
myces 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

http://dx.doi.org/10.1016/j.jconrel.2015.05.001
0168-3659/© 2015 Elsevier B.V. All rights reserved.
230 A. Cambronero-Rojas et al. / Journal of Controlled Release 209 (2015) 229–237
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 consid-
erably 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 ad-
ministration of inhalable powders could be a significant step forward in
terms of efficacy, there is still a problem that needs to be solved for pa-
tients 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, reduc-
tion 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 1,2-
dipalmitoyl-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 partic-
ulate system that encapsulates CO, aiming for a potential application in
patients who suffer from MDR-TB.
2. Materials and methods
2.1. Materials
Capreomycin sulfate, sodium oleate and hyaluronic acid sodium salt
95% (HA) were purchased from Sigma-Aldrich (USA). Poly (lactic-co-
glycolic acid) (PLGA 50:50) Resomer® RG502 was provided from
Boehringer Ingelheim (Germany) and 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine (DPPC) by Genzyme Pharmaceuticals (Switzerland).
All other chemicals and reagents were of the highest purity grade
commercially available.
2.2. Capreomycin oleate preparation
Capreomycin oleate was prepared by the method described by
Schoubben et al. [18] with modifications. Briefly, a 3.5 M sodium oleate
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)):
mass of the powder collected
% yield ¼  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 sol-
vent 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
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 buff-
er 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.
2.5. Capreomycin oleate loading within microparticles
The extraction of CO from the microparticle matrix was performed
by adding 1 mL of methanol or acetonitrile on a weighted amount of
 A. Cambronero-Rojas et al. / Journal of Controlled Release 209 (2015) 229–237
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.
actual amount of capreomycin
Encapsulation efficiency ¼  100:
theoretical amount
2.6. Morphological analysis and particle size
Samples were analyzed in a JMS-6380 LV scanning electronic micro-
scope (JEOL, Japan). The microparticles obtained by solvent emulsion-
evaporation 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 measur-
ing 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).
D90‐D10
Span ¼
D50
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 alumi-
num 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°.
2.9. In vitro release kinetics of capreomycin oleate from microparticles
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 pre-
hydrated 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
231
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 sam-
ples 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
ð2Þ
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 CO-
loaded 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 sin-
gle 60 mg/kg dose. Samples of blood were withdrawn at predetermined
time intervals by cardiac puncture and after obtaining the required vol-
ume 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 analy-
sis. The concentration of capreomycin in serum was determined with
the HPLC method described above. From the serum concentrations ob-
tained after each treatment, a time-concentration curve was construct-
ed and certain pharmacokinetic parameters were determined including
area under the curve (AUC) calculated by the trapezoidal rule method,
ð3Þ 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 in-
tegrity 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) paraformalde-
hyde dissolved in phosphate buffer saline (pH 7.4), then impregnated
and embedded in paraffin. Each sample was cut with a RM 2145 micro-
tome (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
3.1. Optimization of microparticle formulation and morphological analysis
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 polysaccha-
ride 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
232 A. Cambronero-Rojas et al. / Journal of Controlled Release 209 (2015) 229–237
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 aggre-
gation (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 emulsion-
evaporation 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 be-
cause 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 experi-
ments. The evidence from reports that tested microparticle systems de-
signed for pulmonary [30,31] and intramuscular [32] delivery of drugs
has proved that despite the smaller particle size and subsequent phago-
cytosis, 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.
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).
3.2. Drug loading
The encapsulation efficiency value obtained for the formulation pre-
pared 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 drop-
lets 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 degrada-
tion 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 crystal-
line 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 degrada-
tion 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 emulsion-
evaporation, 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 ob-
served 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.
 A. Cambronero-Rojas et al. / Journal of Controlled Release 209 (2015) 229–237
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 organi-
zation 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 micro-
particles prepared by solvent emulsion-evaporation (Fig. 4, bottom)
showed a similar outcome, suggesting that CO is dispersed at a molecu-
lar level in the microparticles obtained by both methods.
Table 1
Microparticle size of the selected formulations shown as number distribution.
Method Diameters (μm) ± DS
D10 D50 D90
Spray drying 2.06 ± 0.71 7.85 ± 4,27 9.14 ± 2.03
Solvent emulsion-evaporation 1.01 ± 0.08 2.94 ± 0,25 3.12 ± 0.96
233
3.4. In vitro release studies
The results of release kinetics performed on free CS and CO, CO-
loaded 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 dis-
solution 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 formu-
lations 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 re-
leased 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 mi-
croparticles 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 hy-
drated. Moreover, the mechanisms involved on the release of drug
from PLGA microparticles are well known and the profiles obtained in
Span 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
0.87 drug is slowly released from the polymeric matrix that undergoes
0.72
through a hydration and erosion process. Finally, the third phase
234 A. Cambronero-Rojas et al. / Journal of Controlled Release 209 (2015) 229–237
Fig 3. DSC curves. Top: HA (A), DPPC (B), CO (C), physical mixture (D), unloaded micro-
particles (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 reduc-
tion 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 rate-
limiting 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].
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).
The encapsulation of CO into lipophilic microparticules had an even
more marked effect on the disposition of the drug. From the time con-
centration 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, re-
spectively. The fast rise in serum concentrations of capreomycin can
be explained by the transfer of drug that is bound or trapped in the sur-
face of the microparticles [22,38], which may be favored by the abun-
dant 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 for-
mulations becomes another rate-limiting factor that will modify the in-
tramuscular absorption of capreomycin from the site of injection, thus
reducing the peak concentrations in serum and consequently the elim-
ination rate, resulting finally in an increased residence time. Due to the
exploratory nature of this study it was not possible to characterize in de-
tail 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.
 A. Cambronero-Rojas et al. / Journal of Controlled Release 209 (2015) 229–237
Fig 5. Top: dissolution kinetics of CS and CO as free drug in HEPES buffer under sink con-
ditions. 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 high-
light. A single 20 mg/kg dose of CO-loaded microparticles provides
sustained concentrations of the drug for at least 8 days, which are con-
siderably over the minimum inhibitory concentration (MIC) value
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: CO-
loaded microparticles. Results are expressed as mean ± SD (n = 3).
235
(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 García-
Contreras et al. [12,39] have the advantage of a reduced systemic expo-
sure 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 com-
pliance, 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
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 stud-
ied 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 in-
flammation 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 tol-
erability, 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 dif-
ferences 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
This work has successfully attempted to develop two
microparticulate formulations that provide a controlled release of
capreomycin after their intramuscular administration. The pharmacoki-
netic 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. Howev-
er, the main difference between the studied formulations is that the
spray-dried microparticles can be prepared at a larger scale and encap-
sulate 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
236 A. Cambronero-Rojas et al. / Journal of Controlled Release 209 (2015) 229–237
Table 2
Pharmacokinetic parameters of the studied formulations. Results are expressed as mean ± SD (n = 3).
Formulation Time
(h)
CS 0–8
CO 0–96
Spray-dried microparticles 0–240
Solvent emulsion-evaporation microparticles
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).
be focused on testing the formulation in higher mammalian models in
order to assess its safety and efficacy compared to the current treatments.
Disclosure
The authors report no conflicts of interest in this work.
Acknowledgments
This work was funded by The University of Concepción, through the
DIUC 212.074.048-1.0 grant. The authors would like to acknowledge the
help of Dr. Francisco Mucientes and Dr. Miguel Zárraga for their advice
with histological analysis and preparation of capreomycin oleate,
respectively. Mr. Cambronero-Rojas is supported by the International
Cooperation Agency of Chile (AGCI) and INNOVA BIOBIO 12.211-
EM.TES and he would like to thank Laboratory Pasteur SA.
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