Pharm Res (2019) 36:9

https://doi.org/10.1007/s11095-018-2538-7

RESEARCH PAPER

Physicochemical Characterization and Pharmacokinetics

of Agomelatine-Loaded PLGA Microspheres
for Intramuscular Injection
Hongjuan Zhang 1 & Chenguang Pu 1 & Qiao Wang 1 & Xinyi Tan 1 & Jingxin Gou 1 & Haibing He 1 &
Yu Zhang 1 & Tian Yin 1 & Yanjiao Wang 1 & Xing Tang 1

Received: 27 March 2018 / Accepted: 29 October 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

ABSTRACT ABBREVIATIONS
Purpose The aim of this study was to design agomelatine AGM Agomelatine
loaded long acting injectable microspheres, with an eventual AUC Area under the curve
goal of reducing the frequency of administration and improv- Cmax Maximum concentration
ing patient compliance in treatment of depression. DALYs Disability adjusted life years
Methods AGM-loaded microspheres were prepared by an DAS Drug and statistics software
O/W emulsion solvent evaporation method. The physico- DSC Differential scanning calorimetry
chemical properties and in vitro performance of the micro- EMA European Medicines Agency
spheres were characterized. The pharmacokinetics of different FDA Food and drug administration
formulations with various particle sizes and drug loadings MDD Major depressive disorder
were evaluated. PLGA Poly (D,L-lactide- co-glycolide)
Results AGM-loaded microspheres with drug loading of PXRD Powder X-ray diffraction
23.7% and particle size of 60.2 μm were obtained. The SD Standard deviation
in vitro release profiles showed a small initial burst release SDS Sodium dodecyl sulfate
(7.36%) followed by a fast release, a period of lag time SEM Scanning electron microscopy
and a second accelerated release. Pore formation and T1/2 Plasma half-life
pore closure were observed in vitro, indicating that the Tmax Peak time
release of drug from microspheres is dominated by UPLC-MS/MS Ultra-performance liquid
water-filled pores. Pharmacokinetic studies showed that chromatography-tandem mass spectrometry
AGM microspheres could release up to 30 days in vivo at YLDs Years lived with disability
a steady plasma concentration. As well, particle size and
drug loading could significantly influence the in vivo re-
lease of AGM microspheres. INTRODUCTION
Conclusions AGM-loaded microspheres are a promising car-
rier for the treatment of major depressant disorder. Depression is a common heterogeneous disorder charac-
terized clinically by low spirit, mental retardation, and
exercise suppression. It has been estimated that depression
KEY WORDS agomelatine . PLGA microspheres . pore is expected to be the second highest cause of morbidity by
formation and pore closure . release in vitro . release in vivo 2020 (1). As well, patients with major depressive disorder
(MDD) in the risk of death from suicide, accident, coro-
nary heart disease, stroke and respiratory disease is in-
creasing (2–4). The estimated global calculated years lived
* Yanjiao Wang with disability (YLDs) and disability adjusted life years
TangLab@126.com (DALYs) of MDD are 8.2% (5.9-10.8%) and 2.5% (1.9-
3.2%) respectively (5). Furthermore, not only do patients
1
School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua with MDD suffer from increased serious physical illness,
Road, Shenyang 110016, China but they are also affected by decreased social function.
 9 Page 2 of 11
MDD has a high rate of relapse and high levels of dys-
function, and so has serious consequences for both the
depressed individuals and their families (6,7). At present,
the clinical treatment of depression is mainly drug based
treatments, and the efficiency of the antidepressant drugs
is only fully achieved when taken regularly and for a long
period. It is recommended that the treatment cycle for
depression is at least one year to minimize the risk of
recurrence (8,9). However, most studies have shown that
more than 40% of patients discontinue the treatment
within 1 month, and approximately 56% of patients dis-
continue the treatment within 4 months (9). It is known
that tolerability and psychological problems weaken the
capacity of antidepressants, and that a steady and
sustained plasma concentration is critical for the treat-
ment of depression (10,11). Hence, in order to achieve
good clinical efficiency, it is of importance to develop a
long-acting injection of antidepressants.
Agomelatine (AGM, Fig. 1) is a novel small molecule
antidepressant with melatonergic MT1/MT2 receptor ag-
onist and 5-HT2C receptor antagonist activity. It was ap-
proved by the EMA in 2009, and was thought to be able
to improve the sleep of people with MDD by synchroniz-
ing the circadian rhythms. It also showed improved toler-
ability and a low propensity for drug withdrawl, sexual
dysfunction and weight gain compared to other antide-
pressants (3,6). According to its chemical structure,
agomelatine is a water-insoluble small molecule drug. Its
solubility increase with temperature and the effective con-
centration is very low due to its new mechanism of action
(12,13). At present, agomelatine is only commercially
available in oral tablets. However, various shortcomings
severely restrict clinical use of agomelatine tablets. First,
due to its short half-life (1–2 h) and severe first-pass effect
in the liver, orally administered agomelatine often shows a
low absolute bioavailability (5% of oral dose), severe he-
patic metabolism and further elevated aminotransferases
Fig. 1 Chemical structure of agomelatine.
Pharm Res (2019) 36:9
(2,7). Clinically, the use of agomelatine tablets requires
liver function testing, which severely limits the use of the
drug (9). Also, daily medication is particularly inconve-
nient for people with MDD, as they often refuse to take
medicine because of psychological problems. Therefore, it
is ideal to develop a depot-like system which can retain
the therapeutic concentration of agomelatine in plasma
for a long time by releasing the drug in a controlled man-
ner for patients with MDD to ensure stable recovery and
to prevent relapse. Thus, promising polymeric micro-
spheres can be applied to many hydrophobic drugs.
With the development of drug delivery systems, depot-
like sustained-release drug delivery systems including in-
jectable drug suspensions, oil-based injectable solutions,
polymer-based in-situ formings and polymeric micro-
spheres that can release drugs longer than one week have
attracted much attention in the pharmaceutical field (14).
These systems offer numerous advantages over traditional
drug delivery systems, including predictable drug release
period, improved drug stability, reduced dosing frequen-
cy, increased patient compliance, decreased side effects
and an overall cost reduction of medical care (14).
Among various sustained-released drug delivery sys-
tems, polymeric microspheres, particularly PLGA–based
microspheres, are the most widely used and commercially
successful due to their ability to encapsulate a variety of
drugs, high bioavailability, biocompatibility and sustained
release (15). PLGA, certified by FDA to be used for drug
delivery systems, not only has good biodegradability and
biocompatibility, but can also tailor the release rate by
changing the molecular weight or the ratio of the two
monomers. Today, several commercial products are based
on PLGA microspheres, including Risperdal Consta® (ris-
peridone, Janssen, Inc.), Vivitrol® (naltrexone, Alkermes,
Inc.) and Leupron Depot® (leuprolide acetate, Abbot,
Inc.) and more (14,16). Many different types of processes
are commonly employed for the preparation of polymer
microspheres, including emulsion-solvent extraction/evap-
oration, spray drying, interfacial polymerization and coac-
ervation (16,17). Until now, the preparation process and
many properties of PLGA microspheres including encap-
sulation efficiency, release and degradation rates have been
studied in detail (18–21). However, many obstacles hinder
the development of insoluble drug microsphere products.
On one hand, traditional emulsion solvent evaporation
techniques and spray drying techniques may result in pores
in the microspheres during the solvent removal process.
Too many drugs can then diffuse through the holes, lead-
ing to an unwanted burst release in the initial stage of
release. On the other hand, drug loading limitation is a
significant problem for insoluble drugs (22). Particularly
for hydrophobic drugs with relatively big water solubility,
it is difficult to achieve a good encapsulation. In addition,
Pharm Res (2019) 36:9
potential toxicity caused by residual solvent inside the mi-
crospheres can be a concern (22).
In this work, in order to improve the bioavailability, reduce
the side effects caused by the first pass effect of the oral tablets
and achieve sustained release of AGM for the treatment of
MDD, AGM-loaded PLGA microspheres were first devel-
oped using the traditional emulsion solvent evaporation meth-
od. Preparation, characterization, in vitro and in vivo release
properties of AGM-loaded PLGA microspheres were subse-
quently evaluated in detail.
MATERIALS AND METHODS
Materials and Animals
Poly (D, L-lactide-co-glycolide) (PLGA) (LA/GA: 75:25, Mw:
70 kDa, carboxylic acid end group) was purchased from
Jinandaigang Biomaterials (Jinan, China). Poly vinyl alcohol
(KURARAY POVAL 22–88, 87.0-89.0% hydrolyzed) was
purchased from Kuraray Co., Ltd. (Osaka, Japan).
Agomelatine (AGM) was provided by Benyuan Pharmacy
Co., Ltd. (Benxi, China). Dichloromethane (DCM), acetoni-
trile and all the other reagents were of chromatographic
grade.
Male Sprague-Dawley rats, with the average weight of
180–220 g, were obtained from the Experiment Animal
Center of Shenyang Pharmaceutical University (Shenyang,
China). All procedures were performed according to the
guidelines issued by the Ethical Committee of Shenyang
Pharmaceutical University (SYPU-IACUC-C2017–0512-
001). All efforts were made to minimize animal suffering and
to limit the number of animals used with the approval of the
Animal Ethics Committee of Shenyang Pharmaceutical
University.
Preparation of AGM-Loaded PLGA Microspheres
An O/W emulsion solvent evaporation technique was used to
prepare AGM-loaded PLGA microspheres (23,24). Briefly,
300 mg PLGA and 129 mg AGM were dissolved in 1.5 mL
DCM to obtain an organic phase. The organic phase was
cooled to 4°C, slowly added to 30 mL pre-cooled 1% (w/v)
PVA aqueous solution and then stirred (Ultra-Turrax
TP18/10, IKA, Germany) at a set speed for 2 min to form
the O/W emulsion. The obtained emulsion was added to
120 mL distilled water to dilute the emulsion, and stirred at
300 rpm for 4 h to evaporate the solvent. The resulting mi-
crospheres were collected by centrifugation at 3000 rpm for
5 min, washed three times with distilled water to remove re-
sidual organic solvent and PVA, lyophilized (VirTis
AdVantage Plus Bench Top Freeze Dryer, SP industries,
Inc., USA) and finally stored at 4°C.
Page 3 of 11 9
Characterization of AGM-Loaded PLGA Microspheres
Particle Size and Morphology
Particle size and distribution of AGM-loaded PLGA micro-
spheres were investigated by a laser diffraction particle size
analyzer (BT-9300S, Bettersize Co., Ltd., Dandong, China).
The microspheres were re-dispersed in distilled water prior to
analysis. The mean size and size distribution were expressed
by D50 (volume weighted mean diameter) and Span (Span
value = (D90-D10)/D50) respectively.
The surface morphology of the microspheres was exam-
ined using scanning electron microcopy (SEM) (SU8010,
Hitachi., Ltd., Japan). Freeze-dried microspheres were spread
on double-sided conductive adhesive tape, which was previ-
ously attached to a copper stub, and then the sample was
coated with a thin layer of gold under an argon atmosphere
and observed by SEM. SEM photos of the in vitro release study
were also obtained.
X-Ray Diffraction Analysis
To investigate the physical state of AGM within the micro-
spheres, AGM, blank microspheres, physical mixture of AGM
and blank microspheres and AGM-loaded microspheres were
studied by powder X-ray diffraction (PXRD) (PANanalyical,
Almelo, The Netherlands) using Nifiltered Cu Kα radiation.
The experiment was conducted at a voltage of 40 KV and
30 mA. Samples were analyzed in a 2θ range of 5–60°.
Differential Scanning Calorimetry
AGM, blank microspheres, physical mixture of AGM and
blank microspheres and microsphere products were studied
by differential scanning calorimetry (DSC) technique using a
METTLER TOLEDO DSC1 (METTLER TOLEDO,
Switzerland). 5 mg samples were placed in an aluminum
pan and heated at a rate of 10°C/min, using dry atmosphere
of nitrogen as carrier gas, across a temperature range of 30 to
150°C.
HPLC-UV Analysis and Drug Loading
Quantitative analysis of agomelatine in various samples was
performed using a Hitachi high pressure liquid chromatogra-
phy (HPLC) system consisting of a Hitachi Chromaster 5110
HPLC pump, a Hitachi Chromaster 5210 autosampler, a
Hitachi Chromaster 5310 column oven and a Hitachi
Chromaster 5410 UV detector. For analysis, a C18
reversed-phase column (Haito Pack ODS, 250 × 4.6 mm,
5 μm) was used, with the mobile phase of acetonitrile:water
50:50 (v/v) and the flow rate was of 1 mL/min. UV detection
was performed at 230 nm.
 9 Page 4 of 11
A direct extraction method was used to determine the
loading content and encapsulation efficiency of AGM in
microspheres accurately. In brief, approximately 10 mg
microspheres were dissolved in acetonitrile and sonicated
for 1 min. Then the samples were filtered through a
0.22 μm membrane and determined by HPLC. Drug
loading and encapsulation efficiency were calculated using
the following equations:
weight of AGM in microspheres
Drug loadingð%Þ ¼
weight of microspheres
 100 ð1Þ
Encapsulation efficiencyð%Þ
Actual AGM loading
¼  100 ð2Þ
Theoretical AGM loading
In Vitro Release and Degradation Evaluation
The optimal formulation performance in vitro was investi-
gated as follows. Briefly, 10 mg AGM-loaded micro-
spheres were suspended in 10 mL release medium (0.5%
SDS, 0.02% NaN3) (25,26). All the samples were incubat-
ed in a shaking water bath (Zhicheng Inc., China) at 37 ±
1°C and 100 rpm. At predetermined sampling points,
samples were centrifuged at 3000 rpm for 10 min
(USTC chuangxing Co. Ltd. Zonkia Branch, China).
9 mL supernatant were separated for the HPLC assay,
and fresh medium with equal volume was added in the
meantime. All release tests were conducted in triplicate.
The amount of released drug during the first 24 h was
designated as Bburst release^. In order to evaluate its re-
lease mechanism and degradation, SEM was used to ob-
serve the microspheres at different time points during the
release test.
In Vivo Pharmacokinetic Study
The in vivo performance of AGM-loaded microspheres
was investigated, to further examine the influence of par-
ticle size and drug loading on the in vivo release of AGM-
loaded microspheres. The pharmacokinetics of three
AGM-loaded microsphere formulations and AGM solu-
tion were investigated. The dosing of rats in the study
was calculated based on the oral dose of humans.
After 7 days acclimatization, rats were randomly di-
vided into four groups (n = 6). All animals were fasted
for 12 h with access to water prior to dosing. Group 1
received AGM solution (AGM carboxymethylcellulose
Pharm Res (2019) 36:9
suspension) as a single dose of 2.6 mg/kg by mouth to
investigate the oral bioavailability and the minimum ef-
fective blood concentration of AGM in rats. Group 2,
Group 3 and Group 4 received different AGM micro-
spheres formulations (formulation MS1, MS2 and MS3
respectively) by intramuscular injection at the right
hind-leg muscle as a single dose of 10 mg/kg. At
predetermined intervals, approximately 0.3 mL blood
samples were collected via the orbital vein with hepa-
rinized tubes. The sampling time points for AGM solu-
tion was 10, 20, 30, 45, 60, 90, 120, 240, 360, 480,
720, 1440 and 2160 min after administration by mouth
and 0.5, 1, 2, 4, 8, 12 h and 1, 2, 3, 5,7, 9, 12, 15, 18,
21, 24, 27, 30 and 35 days for the three AGM micro-
sphere groups. The heparinized blood samples were
separated immediately by centrifugation at 4000 rpm
for 10 min and stored at −80°C until subsequent ex-
traction and analysis.
Plasma samples were processed as follows. First,
100 μL of plasma samples, 20 μL of indapamide
(500 ng/mL, internal standard) and 20 μL of acetonitrile
were mixed by vortex for 3 min. Next, 2 mL of ethyl
acetate were added to the mixture and vortexed for
10 min to extract the mixture. After centrifugation at
10,000 rpm for 10 min, 1.5 mL of the organic layer were
removed and dried with nitrogen. Finally, after removal
of the solvent, the residue was reconstituted with 100 μL
of mobile phase, and the plasma concentration of AGM
was d etermined u sing ultra p erformance liqu id
chromatography-tandem mass spectrometry (UPLC-MS/
MS, Water Corp., Milford, MA).
The obtained data were analyzed using a non-
compartmental method with drug and statistics (DAS) soft-
ware (version 2.0, Mathematical Pharmacology Professional
Committee of China, Shanghai, China).
Storage Stability
A preliminary stability study of the optimized formulation
was carried out. Freeze-dried microspheres were kept at
4°C and room temperature, respectively. Samples were
taken on 0 d, 7 d and 28 d to observe its fluidity, and
their in vitro release were determined.
Statistical Analysis
A paired student’s t test was used for statistical analysis,
with p < 0.05 considered the minimum level of signifi-
cance. Pharmacokinetic parameters were obtained using
a non-compartmental method with drug and statistics
(DAS) software.
Pharm Res (2019) 36:9
RESULTS AND DISCUSSION
Optimizing the Formulation of Microspheres
The traditional emulsification solvent evaporation method
was employed to prepare AGM-loaded microspheres. The
parameters of the preparation process and formulation
were optimized by assessing major factors including drug
loading, encapsulation efficiency, particle size distribution
and burst release (in terms of the cumulative drug release
after 1 day). In the preliminary study, it was found that a
high encapsulation efficiency could be achieved by in-
creasing the concentration of PLGA, but the burst release
of AGM-loaded PLGA microspheres was mainly related
to the particle size. In order to obtain microspheres with
low burst release, the formulation was optimized by pre-
paring microspheres of different particle sizes through
varying the stirring speed. As shown in Table I, the par-
ticle size of the microspheres could be adjusted by chang-
ing the stirring speed of emulsification, with a higher stir-
ring speed resulting in smaller microspheres. It was also
found that as the particle size increased, burst release
from the microspheres decreased significantly (27).
Finally, considering that a very large particle size could
restrict the syringeability of the formulation, formulation
E was chosen as the optimal formulation.
A simple direct extract method was used to determine
the loading content and encapsulation efficiency of AGM
in the microspheres accurately. The determined drug
loading of the optimized formulation was 23.7% and the
average encapsulation efficiency was 78.5%. It is well
known that the solubility of the drug in water is one of
the major factors determining the encapsulation efficiency
of drugs in an emulsion solvent evaporation process
(23,27). Drugs with high water solubility always tend to
leak into the outer water phase, causing a low encapsula-
tion efficiency (27). However, AGM is an insoluble drug
with a slightly higher solubility, and the solubility of AGM
in water increases with temperature, which is not condu-
cive to encapsulation of the drug. In order to improve the
encapsulation efficiency, the organic phase and the water
phase was precooled to a low temperature, which could
Table I Relationship of Particle Size and Burst Release of AGM-Loaded
PLGA Microspheres
Formulation A B C D
Stirring speed (rpm) 12,000 10,000 8000 6000
Mean particle size (μm) 23.0 31.0 36.5 50.4
Drug loading (%) 21.8 22.9 22.5 23.4
Encapsulation efficiency (%) 72.5 75.9 74.5 77.8
Burst release (%) 25.6 22.6 16.4 11.4
Page 5 of 11 9
prevent the drug from migrating to the water phase by
decreasing the solubility of AGM in water and increasing
the viscosity of the organic phase. In addition, it was re-
ported that increasing the viscosity of the organic phase
by increasing the polymer concentration or molecular
weight could increase the drug encapsulation (28). Thus,
PLGA with a relatively high molecular weight and a high
concentration of 200 mg/mL was used to achieve high
drug encapsulation and long duration of drug release.
Characterization of AGM Microspheres
The optimized AGM microspheres (Formulation E) were
characterized in terms of particle size, size distribution, mor-
phology, drug loading and encapsulation efficiency.
As shown in Fig. 2, AGM microspheres have a mean
diameter of 60.2 μm and a narrow particle size distribution
(Span = 0.2). It has been reported that larger particle size of
microspheres could lead to a higher the encapsulation effi-
ciency. (28). As well, the particle size and its distribution can
significantly affect the in vitro release in the microspheres
system. Therefore, microspheres with a relatively big parti-
cle size are beneficial for obtaining a high drug encapsula-
tion and slow drug release.
The SEM images (Fig. 3a and b) revealed smooth surfaces
structure with no pores and drug crystals. They confirmed
that the particle size of AGM microspheres was evenly distrib-
uted, which is consistent with that obtained from the laser
diffraction particle size analyzer.
The physical state of AGM within the microspheres
was investigated using the PXRD technique. As the phys-
ical properties of high molecular weight PLGA are not
suitable for PXRD testing, blank microspheres instead of
PLGA was used in this study. Figure 4 showed the PXRD
patterns of AGM, blank microspheres, microspheres with
20% AGM loading, the physical mixture of AGM and
blank microspheres (AGM: blank microspheres = 20: 80).
Several distinct peaks in the PXRD of AGM were consis-
tent with reports indicating that AGM is present in a
crystalline form II (29). However, they showed that only
a faint crystal diffraction peak appeared in microspheres
when the drug loading was 20%, and thus it can be con-
cluded that the drug in microspheres with 20% AGM
loading mainly existed in the amorphous state, and a
small part of AGM existed in a crystalline state.
Further investigation of the physical state of AGM within
E F
the microspheres was performed using DSC. Figure 5 showed
4000 2000 the DSC pattern of AGM, blank microspheres, physical mix-
60.2 100.9 ture of AGM and blank microspheres and AGM-loaded mi-
23.7 22.5 crospheres (20% drug loading). The AGM powder and phys-
78.5 77.4 ical mixtures showed obvious endothermic peaks at approxi-
7.3 7.1 mately 109°C. By contrast, there were two small endothermic
peaks at near 78°C and 97°Cin the microspheres with drug
 9 Page 6 of 11
Fig. 2 Particle size distribution of
AGM microspheres (75/25 PLGA
70 kDa; stirring speed: 4000 rpm/
min; drug loading = 20.31%;
encapsulation efficiency =
70.23%).
loading of 20%. This confirms that a small portion of AGM
exists in a crystalline state, but the majority of drug was dis-
persed in the polymer matrix in an amorphous state, which is
consistent with the PXRD data. The endothermic peaks near
78°C and 97°C were below the melting point of the drug,
which inferred that a small part of AGM and PLGA form
cocrystals, as AGM contains a secondary amide group which
can be involved in supramolecular heterosynthons with the
carboxyl end of PLGA (30).
It is well known that hydrophobic drugs tend to crystallize
inside the microspheres, and phase separation occurs within
microspheres at high drug loadings, whereas the drug was
dispersed in the polymer in an amorphous state at lower drug
loading (17,31). In our study, both PXRD and DSC results
confirmed that there was a very small amount of AGM dis-
persed in a crystal state in the microspheres, and thus it could
be concluded that the maximal drug embedding capacity in
the PLGA was less than 20%.
In Vitro Release Behavior of AGM Microspheres
Drug release from PLGA-based drug delivery systems can
follow a combination of several mechanisms. In general,
diffusion and degradation/erosion are two main release
mechanisms of microspheres, It is commonly believed that
the release rate of microspheres is diffusion-controlled
Fig. 3 SEM images of AGM
microspheres (75/25 PLGA
70 kDa; stirring speed: 4000 rpm/
min; D50 = 61.33; drug loading =
20.07%; encapsulation
efficiency = 71.20%).
Pharm Res (2019) 36:9
initially, and degradation/erosion controlled during the
final stages of the release period (32) .
Figure 6 showed the in vitro release profile of AGM-loaded
microspheres. Only about 7.36% AGM was released from
PLGA microspheres in the first day in vitro, indicating a small
burst release of AGM-loaded PLGA microspheres. From day
2 to day 14, drug was released quickly from the microspheres
at a nearly constant rate of about 1.45% per day. However,
from day 14 to day 70, a lag time occurred and the drug was
released at a rate of approximately 0.45% per day. In the next
35 days, the second accelerated release occurred and drug was
released quickly at a nearly constant rate of 1.06% per day in
this stage. The above results indicated that the pattern of drug
release in vitro was different from the typical triphasic release
profile, which can be divided into three stages: an initial burst
release, lag time depending on the Mw and end-capping of
the polymer with a slow or absent diffusion-controlled release,
and finally a second erosion-accelerated release (17).
However, in this study it showed that a rapid burst release
phase was followed by a period of near zero-order quick re-
lease, a lag phase and a final second accelerated release.
Except for the first day’s burst release, the drug release
profiles of different release durations of AGM-loaded micro-
spheres could all be fitted to a zero order model, and accept-
able regression coefficients were achieved for all release phases
(Table II). These results indicated that the drug release from
Pharm Res (2019) 36:9 Page 7 of 11 9

Fig. 4 The PXRD curve of AGM

(a), blank microspheres (b), AGM-
loaded microspheres (c), physical
mixture of AGM and blank
microspheres (d).

PLGA microspheres was predominately governed by diffusion
of drug through connected channels in the microspheres,
which are formed by the presence of drug.
To further investigate the release mechanism of AGM-
loaded microspheres in vitro, SEM images of AGM-loaded
microspheres in different release stages were obtained
(Fig. 7). It can be observed that the microspheres kept their
shape and integrity over 10 weeks, until they disintegrated
completely, suggesting that the slow-degradation region was
at the surface, that is, that the internal degradation of the
microspheres was faster than at the surface. At the same time,
the surface of the microspheres began to show a small quantity
of big hydrophilic pores after 1 day, which was formed due to
the diffusion of the drug. In other words, the initial burst
release was attributed to the diffusion of non-encapsulated
drug particles on the surface or drug molecules close to the
surface. As the release continued, the surface of the micro-
spheres became uneven and the hydrophilic pores became
larger and more numerous. In this phase, a fast release oc-
curred at a nearly constant rate of 1.45% per day and this is
consistent with SEM results. However, after 2 weeks, the SEM
images clearly showed the closure of the big pores at the sur-
face, and the pores seemed to be still closed after 10 weeks,
which caused the lag time of drug from day 14 to day 70.
Next, many detectable small pores began to form after
10 weeks which can be seen in Fig. 7. This suggested that

Fig. 5 The DSC curves of AGM

(a), blank microspheres (b), AGM-
loaded microspheres (c), physical
mixture of AGM and blank
microspheres (d).
 9 Page 8 of 11 Pharm Res (2019) 36:9

Fig. 6 In vitro release profiles of AGM-loaded microspheres (the optimal

formulation: drug loading = 20%, D50 = 60 μm, containing 2.0 mg AGM)
in release medium (0.5% SDS solution, containing 0.02% NaN3). Each point
represents the mean ± SD; n = 3.

the second accelerated release was attributed to a large num-

ber of newly formed small pores.
The above in vitro results indicated that the release of
drug from the microspheres was mainly through water-
filled pores. The initial burst release was attributed to
the diffusion of the drug in the surface of the micro-
spheres. However, the microsphere release in the later
phase was mostly the result of diffusion through water-
filled pores. Figure 8 showed the in vitro release process
of AGM-loaded microspheres. In general, the overall re-
lease mechanism of microspheres can be explained as fol-
lows. First, when the microspheres are immersed in an
aqueous release medium, the drug from the surface
spreads to the medium and hydration occurs, which is
faster than erosion (33,34). PLGA absorbs a large amount
of water and temporarily increases the pressure inside the
microspheres, which leads to the formation of big pores in
the low density area on the surface of the microspheres.
As the amount of the water increases inside the micro- Fig. 7 SEM images of AGM microspheres in the different release stages.
spheres, and the auto-catalytic phenomenon within the Each arrowhead represents a pore.
microspheres means the internal degradation of the mi-
crospheres is faster than at the surface, the pores become rearrangement of the PLGA chains. This means that
bigger and more numerous (32,34). Drug is mainly re- any significant increase in pressure can be compensated
leased from the big pores in this phase. Then, as the by swelling and arrangement of the polymer chains. The
release progresses, the volume of the water inside in- big pores in the surface of the microspheres later started
creases, as well as the mobility and the ability for to close in the release medium (35–37), but with further
hydrolysis of the PLGA and internal self-catalysis, a lot of
Table II Evaluation of Drug Release Kinetics of AGM-Loaded new pores formed and the drug tended to release from the
Microspheres newly formed pores. As well, a small amount of crystal-
lized or aggregated hydrophobic drug may reduce the
Phase Burst release(%) 2–14 day 14–70 day 70–105 day
release rate from another aspect. In general, the release
Slope 7.36 1.45 0.448 1.056 rate of microspheres is governed by the pore formation
R2 0.9806 0.9878 0.9989 and pore closure which occur simultaneously. The release
rate of microspheres in different release phases are kept
Pharm Res (2019) 36:9 Page 9 of 11 9

Fig. 8 The in vitro drug release

process of AGM-loaded
microspheres.

constant, which is common for drugs encapsulated in 30 days with almost no burst release in vivo, and the bioavail-
PLGA drug delivery systems. Studies showed that small ability of AGM was dramatically increased when it was pre-
porous microspheres prepared by high-Mw, hydrophobic pared into sustained release microspheres. Three different
PLGA with low polymer chain mobility is more likely to AGM microspheres were released in vitro for 3 months, with
release drugs in a constant rate (34). a burst release of 25%, 7.0% and 7.0% respectively, but when
released in vivo for 30 days demonstrated a burst release of
In Vivo Pharmacokinetics of AGM Microspheres 10.0%, 9.0% and 6.0% (calculated by the AUC (0-1 day)
versus AUC (0-∞)), respectively. This indicated that in vitro re-
The in vitro release studies showed that the AGM-loaded mi- lease test could predict the burst release of AGM microspheres
crospheres exhibited a promising drug release behavior, as
reflected by a small burst release followed by a slow release.
To further evaluate the in vivo release behavior of different
AGM microspheres, the pharmacokinetics of oral AGM solu-
tion, which could evaluate the oral bioavailability and the
minimum effective blood concentration of AGM in rats, and
three different formulations (MS1, MS2 and MS3) of AGM
microspheres were investigated.
The mean plasma concentration-time profiles of AGM in
rats after oral administration of AGM solution and intramus-
cular administration of AGM microspheres are shown in
Fig. 9. The main calculated pharmacokinetic parameters were
listed in Table III.
As can be seen in Fig. 9a and Table III, the AGM solution
was absorbed by the mouth quickly and the plasma concen-
tration reached the peak concentration (Cmax) 32.6 ng/mL in
approximately 1 h. The plasma concentration decreased be-
low 5 ng/mL after a single oral dose of 2.6 mg/mL AGM
solution to the rats after 24 h, suggesting that the minimun
effective plasma concentration was about 5 ng/mL.
As seen in Fig. 9b, after intramuscular injection of three
different AGM microspheres, the plasma concentration of
AGM reached a peak concentration (109.5, 37.4 and
20.3 ng/mL for MS1, MS2 and MS3 respectively), and then
the concentration declined gradually. The blood concentra-
tion dropped below 5 ng/mL in 30 days. Compared to AGM
solution administrated intragastrically, the T1/2 value of
AGM microspheres was prolonged to 96.5 h, 99.6 h and
178.4 h, respectively, which was approximately 6.3, 6.6 and
11.8-fold longer than that of oral AGM solution.
Furthermore, the AUC of three different AGM microspheres Fig. 9 Pharmacokinetic results of AGM. (a) mean concentration-time profile
was approximately 4.8, 3.3 and 2.5-fold more than the calcu- of AGM after a single oral dose of 2.6 mg/mL AGM solution to the rats, (b)
plasma concentration- time profile of AGM after intramuscular injection of
lated AUC of AGM oral solution of the equivalent dose, re- AGM-loaded microspheres (10 mg/kg). MS1: drug loading = 20%, D50 =
spectively. These results further confirmed that AGM micro- 30 μm, MS2: drug loading = 7%, D50 = 30 μm, MS3: drug loading = 20%,
spheres could provide a sustained release of approximately D50 = 60 μm.
 9 Page 10 of 11 Pharm Res (2019) 36:9

Table III The Non-

Compartmental Model Parameters Formulations AGM solution MS1 MS2 MS3
of AGM after i.g. Administration of
2.6 mg/mL AGM Solution and AUC(0-t) (mg/L·h) 330.9 ± 52.5 6779.0 ± 1106.3 5025.2 ± 841.6 3709.2 ± 778.7
Intramuscular Administration of AUC(0-∞) (mg/L·h) 461.3 ± 106.5 7285.6 ± 1149.6 7564.1 ± 3940.2 5560.0 ± 996.3
AGM Microspheres (10 mg/mL) AUC(0-1 day) (mg/L·h) – 680.6 ± 111.1 451.6 ± 75.6 224.4 ± 47.1
(Mean ± SD; n = 6)
T1/2 (h) 15.2 ± 7.3 96.5 ± 16.6 99.6 ± 33.3 178.4 ± 27.0
Cmax(mg/L) 32.6 ± 12.5 109.5 ± 17.6 37.4 ± 1.4 20.3 ± 4.7
Tmax (h) 1.0 ± 0.0 1.0 ± 0.9 3.3 ± 4.0 –
Burst release (%) – 10.0 9.0 6.0

in vivo. It is significant to develop an in vitro release assay with
good in vitro and in vivo correlation (IVIVC), which can be used
not only to predict the in vivo behavior of the formulation and
optimize the formation during formulation development, but
also be used to control the quality of the product in the prep-
aration of the product process, and so further work will focus
on establishing a new in vitro release assay with a good in vitro
and in vivo relationship.
Comparing MS1 with MS2, it can be inferred that drug
loading could significantly influence the burst release of the
microsphere in vivo. The lower the drug loading could result in
the smaller the burst release in vivo. However, the AUC of
MS1 was bigger than that of MS2, indicating that AGM
was eliminated quickly in the blood and the rate of elimination
of AGM is faster than absorption. Comparing MS1 with
MS3, it was apparent that particle size had a significant effect
on the release of microspheres in vivo. It can be seen that the
bigger the particle size, the smaller the burst release in vivo,
suggesting that a relatively big particle is beneficial for achiev-
ing a sustained release in vivo with a small burst release. As is
shown in Table III, T1/2: MS1 < MS2 < MS3, Cmax: MS1 >
MS2 > MS3, burst release in vivo: MS1 > MS2 > MS3. The
results concluded that MS3 showed the best sustained release
characteristics and minimal burst release, which was very im-
portant for reducing the side effects caused by a too high
concentration in the blood in vivo.
Storage Stability
The stability of the optimized formulation was measured at
4°C and room temperature for 30 days, respectively. As
shown in Table IV, microspheres at room temperature tended
to agglomerate and its burst release became slowly. However,
there was no significant change in fluidity and the burst release
when the microspheres were stored at 4°C.
CONCLUSION
AGM-loaded PLGA microspheres with smooth surface
and narrow particle size distribution were prepared by
the emulsion solvent evaporation method. The drug load-
ing content and encapsulation efficiency of AGM micro-
spheres were 23.7% and 78.5% respectively. DSC and
PXRD analysis demonstrated that the majority of drug
was dispersed in the polymer matrix in an amorphous
state, and only a small part of AGM existed in a crystal-
line state.
In vitro release studies showed that AGM microspheres
released slowly with a small burst release of 7.0% followed
by a fast release, a lag time and a second accelerated
release in vitro. SEM images showed pore formation and
pore closure in different release stages, indicating that the
main release mechanism of the microspheres was diffusion
through water filled pores in vitro.
Over 30 days, a sustained and steady plasma concen-
tration of AGM could be achieved after a single intramus-
cular injection of AGM microspheres. In vivo release stud-
ies also showed that particle size and drug loading had
significant effects on the release of AGM-loaded micro-
spheres. These results suggested the potential use of
AGM-loaded PLGA microspheres for treatment of de-
pression over a long time period.

Table IV The Stability Results of

the Optimized Formulation Condition 0 day 7 days 28 days

4 °C 25 °C 4 °C 25 °C 4 °C 25 °C

Fluidity good good good Slightly agglomeration good agglomeration

Burst release in vitro 8.96 8.49 8.18 6.99 8.88 6.94
Pharm Res (2019) 36:9
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