J.

of Supercritical Fluids 87 (2014) 921

Contents lists available at ScienceDirect

The Journal of Supercritical Fluids

journal homepage: www.elsevier.com/locate/supflu

A comparative evaluation between utilizing SAS supercritical uid

technique and solvent evaporation method in preparation of
Azithromycin solid dispersions for dissolution rate enhancement
Ehsan Adeli
The International Branch, Shahid Beheshti University of Medical Sciences, Tehran, Iran

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

Article history: Azithromycin is a poorly water-soluble drug with a lower dissolution rate which resulted in poor bioavail-
Received 13 July 2013 ability after oral administration. The aim of this study was to enhance Azithromycin dissolution by a
Received in revised form solid dispersion (SD) using solvent evaporation and supercritical uid based on solvent-anti-solvent
17 December 2013
technique. Solid dispersions of Azithromycin were prepared with various concentrations of PEG 6000,
Accepted 19 December 2013
Sorbitol and Poloxamer 188, SLS (in ternary systems). All samples were studied for the drug solubility.
The formulations were also characterized by IR, DSC, XRD and SEM. The solubility and dissolution rate
Keywords:
were remarkably improved in case of most SDs prepared with of PEG 6000 (in binary systems, 1:6 ratio)
Azithromycin
Solubility
and both surfactants (ternary systems) compared to the related PMs and pure Azithromycin. But the
Dissolution rate best result was obtained in the dispersion (Azithromycin:PEG 6000:SLS) with a weight ratio of (1:4:2).
Solid dispersion SASSCF processes were signs of less crystallinity of the drug due to the transformation of its crystalline
Solvent evaporation stat into amorphous state. The analysis of dissolution data indicated that enhanced drug dissolution can
Supercritical uid be achieved where the SDs obtained in the supercritical uid process was consisted of PEG 6000 and
SLS. The dissolution rate and solubility of Azithromycin improved signicantly with PEG 6000 and SLS
utilizing SAS-supercritical uid.
2014 Elsevier B.V. All rights reserved.

1. Introduction as Azithromycin are bacteriostatic agents that inhibit protein

1.1. Azithromycin
Azithromycin with chemical IUPAC name [1]:
[2R-(2R, 3S, 4R, 5R, 8R, 10R, 11R, 12S, 13S, 14R)]-13-
[2,6-Dideoxy-3-C-methyl-3O-methyl--l-ribo-hexopyranosyl)oxy]-
2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-
trideoxy-3-(dimethylamino)--d-xylo-hexopyranosyl]oxy]-1-oxa-6-
azacyclopentadecan-15-one and the following chemical structure
(Fig. 1) is an Azalide (nitrogen-containing macrolide) and a subclass
of Macrolide antibiotics. Azithromycin is a white crystalline powder
with a molecular formula of (C38 H72 N2 O12 2H2 O) and a molecular
weight of 748.984 g mol1 [1]. This drug belongs to class II of BCS1
[2,3]. Azithromycin is derived from Erythromycin, with a methyl-
substituted nitrogen atom incorporated into the lactone ring, thus
making the lactone ring 15-membered [1]. Azithromycin is one of
the best-selling antibiotics in the world [4]. Macrolide antibiotics
synthesis by reversible binding to the 50S ribosomal subunits of
sensitive organisms. Azithromycin is very active against Moraxella
catarrhalis, Pasteurella multocida, Chlamydia spp., Mycoplasma
pneumoniae, Legionella pneumophila, Borrelia burgdorferi, Fusobac-
terium spp., and Neisseria gonorrhoeae. Azithromycin has enhanced
activity against Mycobacterium avium-intracellulare, as well as
against some protozoa (e.g., Toxoplasma gondii, Cryptosporidium,
and Plasmodium spp. [5,6]. Azithromycin administered orally is
absorbed rapidly and distributed widely throughout the body,
except to the brain and CSF. Azithromycin with actions and uses
similar to those of Erythromycin [6] is given in the treatment of
respiratory-tract infections (including otitis media), and in skin
and soft-tissue infections. Azithromycin may also be used for the
prophylaxis, and as a component of regimens in the treatment of
M. avium complex (MAC) infections. It is used in some countries
for the prophylaxis of endocarditis in at-risk patients unable to
take penicillin. It is also used in the management of trachoma and
typhoid [6].

Correspondence to: The International Branch, Shahid Beheshti University of

1.2. Solid dispersion
Medical Sciences, No. 19, Shahid Abbasspour Street, Vali-Asr Avenue, Tehran, Iran.
Tel.: +98 2188209623; fax: +98 2188209623.
E-mail address: ehs.adeli@gmail.com As early as in 1961, Sekiguchi [7,8] developed the concept of
1
Biopharmaceutics classication system. solid dispersion to enhance absorption of poorly water-soluble

0896-8446/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.supu.2013.12.020
10 E. Adeli / J. of Supercritical Fluids 87 (2014) 921
Fig. 1. Chemical structure of anhydrous Azithromycin.
drugs. Later, Goldberg et al. [912] demonstrated that a certain
fraction of the drug may also be molecularly dispersed in the
matrix, forming solid solutions, while other investigators [13,14]
reported that the drug may be embedded in the matrix as amor-
phous materials [15,16]. On the basis of these considerations, Chiou
and Riegelman [17] dened solid dispersion (SD) as The dispersion
of one or more active ingredients in an inert excipient or a matrix,
where the active ingredients could exist in nely crystalline, solu-
bilized, or amorphous states. Particle size reduction often leads to
improvement in dissolution rate of poorly soluble drugs through an
increase in effective surface area. However, for practical purposes,
it is difcult to reduce particle sizes of drug in capsules or Tablets
to below the 25 m range, while signicantly higher particle size
is generally preferred during drug product development for ease of
handling, formulating and manufacturing. Solid dispersion, in con-
trast, could dissolve a portion of the drug immediately in contact
with the gastrointestinal (GI) uid [18], resulting in a saturated or
supersaturated solution for rapid absorption, and the excess drug
could precipitate in the GI uid in a very nely divided state. The
literatures on the application of solid dispersion in improving the
dissolution rate and oral bioavailability of poorly water-soluble
drugs have been reviewed by Lunar and Dressman [19]. In addi-
tion to the aforementioned formulation options, dissolution rate of
poorly water-soluble drug can be enhanced by converting the drug
into its amorphous form [20]. Solid dispersion formulations, by sta-
bilizing amorphous drugs, can provide signicant advantages. The
core steps involved in the formation of solid dispersion between a
drug and polymer are:
Fig. 2. Schematic diagram of SASSCF apparatus.
(1) Transforming drug and polymer from their solid state to uid
or uid-like state through processes such as melting, dissolving
in solvent or co-solvent, or subliming a process that is not so
commonly used.
(2) Mixing the components in their uid state.
(3) Transforming the uid mixture into solid phase.
1.3. Supercritical uid (SCF)
A supercritical uid (SCF) is a substance whose temperature
and pressure are simultaneously above its critical point. CO2 -
Supercritical is a good solvent for water-insoluble as well as
water-soluble compounds (drugs) under suitable conditions of
temperature and pressure. Therefore CO2 -supercritical has poten-
tial as an alternative to conventional organic solvents used in
solvent-based processes for forming solid dispersions due to its
favorable properties of being non-toxic and inexpensive [2124].
The process consists of the following steps:
(1) Charging the bioactive material and suitable polymer into the
autoclave;
(2) The addition of supercritical CO2 under precise conditions of
temperature and pressure, that causes the polymer to swell;
(3) Mechanical stirring in the autoclave; and
(4) Rapid depressurization of the autoclave vessel through a
computer-controlled orice to obtain desired particle size;
The temperature conditions used in this process are fairly
mild (3575 C), which allows the handling of heat sensitive
biomolecules, such as enzymes and proteins [2429]. Fig. 2 shows
representative schematic of the solvent-anti-solvent (SAS) super-
critical uid process. Firstly, the solute dissolves in a liquid organic
solvent mixture, and a gas is employed to precipitate the solute. Gas
is injected as anti-solvent, into the solution in an adiabatic cham-
ber; in addition, we will obtain a uniform mixture, when it becomes
supersaturated with the gas during which the drug precipitates
in the form of the ne particles. The samples are washed with
additional anti-solvent to eliminate the remainder of the solvent
[26,2933].
2. Materials and methods
2.1. Materials
Azithromycin dihydrate (Pharmacopeia grade) and polyethyl-
ene glycol 6000 (PEG 6000, Analytical grade) were purchased from
 E. Adeli / J. of Supercritical Fluids 87 (2014) 921 11

Table 1
Composition of binary systems.

Carrier Azithromycin:Carrier Formulation code Preparation method

PEG 1:1 SD1 Solvent evaporation

6000 1:2 SD2 Solvent evaporation
1:4 SD3 Solvent evaporation
1:6 SD4 Solvent evaporation
1:8 SD5 Solvent evaporation
1:6 PM4 Physical mixing
Sorbitol 1:1 SD6 Solvent evaporation
1:2 SD7 Solvent evaporation
1:4 SD8 Solvent evaporation
1:6 SD9 Solvent evaporation
1:8 SD10 Solvent evaporation
1:4 PM8 Physical mixing

Fluka (Fluka, USA). Poloxamer 188 (Pluronic F-68) was purchased
from BASF (BASF, Germany, Analytical grade), SLS, Sorbitol, Potas-
sium dihydrogen orthophosphate, Sodium hydroxide and Ethanol
96% (Analytical grade) were purchased from Merck, USA. Ethanol
99.8% (HPLC grade) was purchased from Fluka, USA. All other chem-
icals and solvents used were of analytical grades.
2.2. Methods
2.2.1. Solid dispersions preparation
2.2.1.1. Binary systems preparation. Solid dispersion was prepared
using Azithromycin and different carrier, such as PEG 6000 and
Sorbitol [34,35] in various ratios of 1:1, 1:2, 1:4, 1:6 and 1:8. In
order to prepare solid dispersions by solvent evaporation method,
the sufcient amount (100 mg) of Azithromycin and carriers were
dissolved in Ethanol (96%) with constant stirring at 200 RPM for
45 min. The mixture was kept in the oven for 24 h at 40 C and then
at room temperature for 4872 h until a dry cake was obtained.
Dry batches were sieved with 100 mesh. Samples were stored away
from light and heat for the next experiments (Table 1).
2.2.1.2. Ternary systems preparation. After reviewing the results of
the dissolution rate of the drug, the ternary solid dispersion sys-
tems by Poloxamer 188 and SLS were prepared (Table 2) using the
method described in the preceding paragraph (previous section)
with various ratios of 1:5:1, 1:4:3, and 1:3:3.
2.2.1.3. Ternary systems using SASSCF. Apparatus of SASSCF (Thar
SAS 50, Technologies Co., USA) was used for preparation of
Azithromycin solid dispersions. The SAS 50 system is made up of
the following components:
(1) CO2 tank
(2) Low pressure heat exchanger (HE1)
(3) High pressure pumps
(4) High pressure heat exchanger (HE2)
(5) Solvent pump
(6) Particle collection vessel consisting of the main body (218 mm
internal height, 54 mm internal diameter and 0.5 L volume)
Fig. 2 shows the schematic diagram of the Thar SAS system using
supercritical carbon dioxide (SC CO2 ) as an anti-solvent. Firstly,
Azithromycin and PEG 6000 and surfactant (Poloxamer 188 or SLS)
were dissolved in a minimum volume of Ethanol as an organic
solvent. Then the CO2 ows from the tank and passes through
the low-pressure heat exchanger and using a cooling bath oper-
ating was cooled down at 4 C to assure liquid state in the pump.
The liqueed CO2 was then pumped into particle collection ves-
sel using a high-pressure pump through the spray nozzle. CO2
was heated using another heat exchanger (HE2) before enter-
ing the precipitation vessel. The drug solution and SC CO2 were
pumped into the particle formation vessel at speeds of 0.2 and
2 ml/min, respectively. The drug solution of carriers and CO2 ow-
rate were adjusted via using computer software. At the beginning
of the experiment, Ethanol was sprayed for 10 min to establish the
steady state condition. After that, the solvent pump was used to
spray the solution of the drug and polymer and surfactant into
the precipitation vessel through the spray nozzle. The pressure in
particle collection vessel was controlled using an automatic back-
pressure regulator (ABPR) and a temperature controller regulates
the amount of heat being applied to the heating jacket. Rapidly mix-
ing between the organic solvent and SC CO2 and the fast diffusion
of supercritical CO2 into the organic solvent produces a supersat-
urated solution and causes the formulation to precipitate as ne
particles, which were collected on a 0.22 m Nylon lter, placed
on top of 5 m metal frit. Approximately 150200 ml of solution of
the drug and polymer and surfactant was sprayed into the precip-
itation vessel. Once sufcient powder was collected, the solution
pump was switched off while supercritical CO2 was continuously
pumped into the precipitation vessel to wash-up the remaining sol-
vent residual for approximately 6080 min. The precipitation vessel
was then de-pressurized gradually to atmospheric pressure using
the vent valves. Finally, the powder was collected from the inside
of the precipitator for further characterization. In order to nd out

Table 2
Composition of ternary systems.

Surfactant Azithromycin:PEG 6000:Surfactant Formulation code Preparation method

Poloxamer 188 1:5:1 SD11 Solvent evaporation

1:4:2 SD12 Solvent evaporation
1:3:3 SD13 Solvent evaporation
1:4:2 SF-SD14 SAS-Supercritical uid
1:4:2 PM12 Physical mixing
SLS 1:5:1 SD15 Solvent evaporation
1:4:2 SD16 Solvent evaporation
1:3:3 SD17 Solvent evaporation
1:4:2 SF-SD18 SAS-Supercritical uid
1:4:2 PM16 Physical mixing
12 E. Adeli / J. of Supercritical Fluids 87 (2014) 921
the variability of the processing method, two sets of experiments
were repeated three times each [24,31,36].
2.2.2. Physical mixtures preparation
Physical mixtures were prepared by manually mixing with vari-
ous weight ratios in a porcelain mortar for 5 min. Physical mixtures
remained in a closed container away from light and kept at ambient
temperature to next tests.
3. Characterization studies
3.1. Assay of Azithromycin dihydrate
Outset the Azithromycin solution was prepared (5 mg/ml)
in phosphate buffer with pH = 6.0 using a UV spectropho-
tometer (SHIMADZU UV mini 1240 Spectrophotometer, Japan).
Using the Standard calibration curve determined the amount of
Azithromycin. Concentration range of required linearity observed
in 230 g/ml Azithromycin solution in phosphate buffer pH = 6.0
with the correlation coefcient (R2 = 0.9998) [37].
3.2. Drug content
Accurately 100 mg of solid dispersions to the equivalent of
Azithromycin were weighed and dissolved in quantity of phosphate
buffer pH = 6.0. The solutions were ltered then for evaluation of
drugs content, samples after suitable dilution was determined at
215 nm [37] by a UV spectrophotometer (SHIMADZU UV mini 1240
Spectrophotometer, Japan). The percentage yield of each formula-
tion was also calculated.
3.3. Saturation solubility measurements
In order to measurement of saturation solubility, it should
be added the excess amount of the samples to the phosphate
buffer pH = 6.0 under certain conditions. Hence, the excess of
Azithromycin, solid dispersions and the physical mixture (approx-
imately 100 mg) were added to glass ask (100 ml) containing of
phosphate buffer pH = 6.0, then samples were stirred by a magnetic
stirrer at 200 RPM at 25 C for 24 h. Next, samples were ltered, suit-
ably diluted and analyzed by a UV spectrophotometer at 215 nm.
3.4. In vitro dissolution measurements
The solubility and dissolution rate of SDs as well as related phys-
ical mixtures (PMs) and the pure drug were determined in pH = 6.0.
To evaluate the in vitro dissolution, studies of pure Azithromycin
and samples were performed using a US Pharmacopeia type II dis-
solution test apparatus. The samples equivalent to 100 mg plain
Azithromycin were added to 900 ml of phosphate buffer pH = 6.0
at 37 0.5 C and stirred at 100 RPM (Erweka DT6R, Germany)
[38]. Aliquots of 5 mL were collected at specied time intervals
and replaced with a fresh dissolution medium. Samples ltered and
were analyzed spectrophotometrically at 215 nm. The dissolution
studies were carried out in triplicate times at the sample.
3.5. Dissolution efciency (DE)
Dissolution efciency is a parameter for evaluation of in vitro
dissolution data. This parameter is equal to the total area under
the curve of the dissolution curve at time t. Also DE expressed
as percentage of the area of the rectangle described by 100%
dissolution at the same time. Dissolution efciency (DE%) was
calculated to compare the dissolution data [39]:
 t 
0
y.dt
Dissolution efciency (DE%) = 100
yt100
3.6. Statistical analysis
All data obtained from dissolution and solubility studies were
compared using analysis of variance (ANOVA) with the Tukeys post
test [40,41].
3.7. Infrared (IR) spectroscopy
The samples were studied by IR spectroscopy. IR studies deter-
mined drug carrier interactions. IR spectra were recorded on
samples prepared in potassium bromide (KBr) disks using an IR
spectrophotometer (PerkinElmer 843 System, USA). Samples were
powdered and prepared in KBr disks by means of a hydrostatic
press under high pressure using special dies. The scanning range
was 2004000 cm1 at a resolution of 1 cm1 . An average of scans
was taken 20 cm1 .
3.8. Differential scanning calorimetric (DSC)
Differential scanning calorimetry (DSC) measurements were
recorded using an instrument (SHIMADZU DSC-60, Japan). The
calorimeter was calibrated using Indium as standard at 156.6 1 C.
Accurately 710 mg of samples were weighed and placed in sealed
Aluminum pans (Al-pan) and heated from 10.0 C to 170.0 C at a
rate of 10 C/min under atmosphere of Nitrogen gas (30 ml/min).
An empty pan was used as reference.
3.9. X-ray diffraction (XRD)
To evaluate the crystalline properties of prepared samples,
XRD pattern was measured by PHILIPS PW 1800 X-Ray Diffrac-
tion Machine (The Netherlands) using Ni-ltered, Cu-K radiation,
a current of 30 mA, and a voltage of 40 kV. The continuous scanning
of the instrument was operated at the speed of 1 /min over a range
2 of 5 90 2 1 . Intervals to record 2: 0.02 was considered.
3.10. Scanning electron microscopy (SEM)
The surface morphology of samples was determined using an
analytical scanning electron microscope (VEGA\\\\TESCAN-LMU,
Czech Republic, equipped with thermo-emission cathode [Balzers
Union Ltd., Balzers, Lichtenstein]). The samples were placed on
sample disk carrier carbon stub (10 mm diameter, 3 mm height)
and coated with gold under vacuum (0.25 Torr). The samples were
monitored then an image was generated using a 30 kV electron
beam.
4. Results and discussion
Dissolution and solubility studies were carried out at four levels.
The rst level: Includes the study of binary solid dispersion sys-
tems and preparation of the binary solid dispersion systems using
the solvent evaporation.
The second level: Includes the study of ternary solid dispersion
systems and preparation of the ternary solid dispersion using the
solvent evaporation.
The third level: At this level, the best formulation of the rst and
second levels was used for preparation of SAS-SCF. Then a compar-
ison was performed between two methods.
 E. Adeli / J. of Supercritical Fluids 87 (2014) 921 13

Table 3
Drug content and saturation solubility of pure Azithromycin, solid dispersions and the physical mixture containing Azithromycin and PEG 6000 (n = 3, mean SD).

Formulation code Preparation type Drug:PEG 6000 Theoretical drug content Assayed drug content Saturation
solubility
(g/mL)

Amount in mg Expressed in % Amount in mg Expressed in %

Azithromycin Pure drug 1:0 100 100 99.92 1.27 99.92 85 1.08
SD1 Solid dispersion 1:1 50 100 49.82 0.33 99.64 185 1.04
SD2 Solid dispersion 1:2 33.3 100 33.08 0.21 99.33 220 1.93
SD3 Solid dispersion 1:4 20 100 19.77 0.11 98.85 231 1.27
SD4 Solid dispersion 1:6 14.3 100 14.06 0.05 98.32 237 1.78
SD5 Solid dispersion 1:8 11.1 100 10.86 0.04 97.84 240 1.51
PM4 Physical mixture 1:6 14.3 100 14.18 0.12 99.16 119 1.38

Table 4
Drug content and saturation solubility of pure Azithromycin, solid dispersions and the physical mixture containing Azithromycin and Sorbitol (n = 3, mean SD).

Formulation code Preparation type Drug:Sorbitol Theoretical drug content Assayed drug content Saturation
solubility
(g/mL)

Amount in mg Expressed in % Amount in mg Expressed in %

Azithromycin Pure drug 1:0 100 100 99.92 1.27 99.92 85 1.08
SD6 Solid dispersion 1:1 50 100 49.83 0.53 99.66 175 1.35
SD7 Solid dispersion 1:2 33.3 100 33.11 0.17 99.43 190 1.44
SD8 Solid dispersion 1:4 20 100 19.80 0.15 98.99 203 1.11
SD9 Solid dispersion 1:6 14.3 100 14.10 0.05 98.63 196 2.02
SD10 Solid dispersion 1:8 11.1 100 10.91 0.03 98.29 191 1.14
PM8 Physical mixture 1:4 20 100 19.89 0.18 99.44 112 1.28

The fourth level: Finally, was performed further testing also on
selected samples; in order to conrm the amorphous state and no particle size.
interaction between drug and carriers.
4.1. Solubility studies
4.1.1. Saturation solubility
4.1.1.1. Binary systems. As shown in Tables 3 and 4, SD3, SD4 and
SD5 having 231 g/ml, 237 g/ml and 240 g/ml, respectively, are
the highest solubility among all other samples. SD4 (Drug: PEG
Increase of the solubility of the drug is evident. It is due to the Nano
4.1.2. In vitro dissolution rate
4.1.2.1. Binary systems. As shown in Table 7 and in Figs. 3 and 4,
SD4 and SD5 at 60 min have 42.90% and 40.99% of the drug release,
respectively. Therefore SD4 and SD5 are the highest dissolution,
50
45
Cumulative Drug Released (%)

6000, ratio 1:6 by the weight) is the highest solubility among the 40
Pure Drug
binary systems (pure drug solubility only is 85 g/ml). Perhaps 35
PM4
hydrophilicity of PEG 6000 has led to increase of solubility SD4 30
SD1
(discussed further will be explained in the in vitro dissolution stud- 25
SD2
ies section). Although, the SD5 has further saturation solubility but 20
SD3
the percentage polymer of SD4 is less than SD5, therefore SD4 is a 15
SD4
suitable candidate for the ternary systems. 10
SD5
5
0
4.1.1.2. Ternary systems. The results of the ternary systems solubil- 0 10 20 30 40 50 60 70
ity are shown in Tables 5 and 6. All samples of ternary systems were Time (Min)
prepared with considering the ratio of SD4 as ternary systems. With
development of ternary solid dispersion systems using two types Fig. 3. In vitro dissolution release prole of pure Azithromycin, solid dispersions,
and the physical mixture containing Azithromycin and PEG 6000 (n = 3, mean SD).
of surfactants, the amount of surfactant should be less than the
CMC (critical micelle concentration). Thus, three formulations were
prepared for each surfactant using solvent evaporation. Saturation 45

solubility studies show satisfactory results for most formulations 40

Cumulative Drug Released (%)

ternary systems. According to solvent evaporation as preparation 35 Pure Drug

method and citations to Tables 5 and 6, saturation solubility of SD12 30 PM8
is 414 g/ml and SD16 is 436 g/ml. SD16 was selected as desirable 25 SD6
sample. Saturation solubility of SD16 is 5.13 times more than other 20 SD7
formulations of the ternary systems. 15 SD8

10 SD9
SD10
4.1.1.3. Ternary systems using SASSCF. At this stage, SF-SD14 and 5
SF-SD18 were prepared using SASSCF and the ratios of the SD4 for- 0
0 10 20 30 40 50 60 70
mulation. Saturation solubility was measured in phosphate buffer
Time (Min)
at pH = 6.0. Saturation solubility of SF-SD14 and SF-SD18 has grown
tremendously (Tables 5 and 6). For example, saturation solubility of Fig. 4. In vitro dissolution release prole of pure Azithromycin, solid dispersions,
SF-SD18 is 871 g/ml (10.25 fold more than pure drug solubility). and the physical mixture containing Azithromycin and Sorbitol (n = 3, mean SD).
14 E. Adeli / J. of Supercritical Fluids 87 (2014) 921

Table 5
Drug content and saturation solubility of pure Azithromycin, solid dispersions and the physical mixture containing Azithromycin, PEG6000 and Poloxamer 188 (n = 3,
mean SD).

Formulation code Preparation type Drug:PEG 6000:SLS Theoretical drug content Assayed drug content Saturation
solubility
(g/mL)

Amount in mg Expressed in % Amount in mg Expressed in %

Azithromycin Pure drug 1:0:0 100 100 99.92 1.27 99.92 85 1.08
SD11 Solid dispersion 1:5:1 14.3 100 13.92 0.17 97.34 378 1.76
SD12 Solid dispersion 1:4:2 14.3 100 13.89 0.38 97.10 414 0.86
SD13 Solid dispersion 1:3:3 14.3 100 13.90 1.11 97.19 402 1.25
SF-SD14 Solid dispersion 1:4:2 14.3 100 13.48 0.34 94.29 817 1.33
PM12 Physical mixture 1:4:2 14.3 100 13.99 1.09 97.84 309 2.22

Table 6
Drug content and saturation solubility of pure Azithromycin, solid dispersions and the physical mixture containing Azithromycin, PEG 6000 and SLS (n = 3, mean SD).

Formulation code Preparation type Drug:PEG 6000:SLS Theoretical drug content Assayed drug content Saturation
solubility
(g/mL)

Amount in mg Expressed in % Amount in mg Expressed in %

Azithromycin Pure drug 1:0:0 100 100 99.92 1.27 99.92 85 1.08
SD15 Solid dispersion 1:5:1 14.3 100 13.90 0.03 97.17 405 1.12
SD16 Solid dispersion 1:4:2 14.3 100 13.86 0.02 96.95 436 1.09
SD17 Solid dispersion 1:3:3 14.3 100 13.89 1.05 97.13 410 1.01
SF-SD18 Solid dispersion 1:4:2 14.3 100 13.43 0.14 93.91 871 1.55
PM16 Physical mixture 1:4:2 14.3 100 13.98 0.06 97.78 317 1.22

among all other samples and hence SD4 or SD5 are a good candi-
date for the preparation of ternary systems. There are many reasons
for this increase [42]. Perhaps at this point, increase of satura-
tion solubility can be explained by four reasons. These reasons are
respectively [4345]:
(1) High hydrophilicity of the polymer
(2) Suitable concentration of polymer around the molecule of drug
(3) The balance between the above items (1 and 2)
(4) The type of drug (regarding the effect of the functional groups)
SD4 (Azithromycin:PEG 6000 with 1:6 ratio) with
DE30,SD4 = 30.09 and 34.33% cumulative drug released at 30 min
and according to the less used polymer has the highest percentage
of drug release among binary systems. This sample is the top
choice of binary systems (The DE parameter conrms the same
argument also). SD4 used the basic formulation for the preparation
of ternary systems.
4.1.2.2. Ternary systems. The results of ternary of ternary systems
are shown in Table 7 and Figs. 5 and 6. Ternary systems were
prepared using two types of surfactant (surfactant concentration
should be less than of the critical micelle concentration). Hence
for each surfactant, it was made new formulation using solvent
evaporation method (Table 2). The dissolution rate of most ternary
systems shows satisfactory results. According to the results (Table 7
and Figs. 5 and 6), SD12 release 76.20% drug during at 60 min
and SD16 at the same time (60 min) released 86.12% drug (3.91
times more than of intact drug). While at the same time, pure
Azithromycin only released 22.00% drug. In addition, the reasons

Table 7
Dissolution parameters for pure Azithromycin, various solid dispersion systems and physical mixtures.

Formulation code Preparation type Preparation method Drug:Carrier:Surfactant DE10 (%) DE30 (%)

Azithromycin Pure drug 1:0:0 4.50 1.12 14.32 0.99

SD1 Solid dispersion Solvent evaporation 1:1:0 14.28 1.37 23.22 1.08
SD2 Solid dispersion Solvent evaporation 1:2:0 15.49 0.38 25.11 0.87
SD3 Solid dispersion Solvent evaporation 1:4:0 15.65 2.16 27.43 1.05
SD4 Solid dispersion Solvent evaporation 1:6:0 18.11 1.39 30.09 1.34
SD5 Solid dispersion Solvent evaporation 1:8:0 16.90 1.35 28.56 0.53
SD6 Solid dispersion Solvent evaporation 1:1:0 11.37 0.29 20.61 0.39
SD7 Solid dispersion Solvent evaporation 1:2:0 13.98 0.45 25.83 0.86
SD8 Solid dispersion Solvent evaporation 1:4:0 14.30 0.10 26.49 0.79
SD9 Solid dispersion Solvent evaporation 1:6:0 15.55 0.30 27.01 1.55
SD10 Solid dispersion Solvent evaporation 1:8:0 14.76 0.33 26.30 0.66
SD11 Solid dispersion Solvent evaporation 1:5:1 25.56 1.31 47.18 1.82
SD12 Solid dispersion Solvent evaporation 1:4:2 30.40 1.99 56.83 0.42
SD13 Solid dispersion Solvent evaporation 1:3:3 26.08 1.02 48.64 1.81
SF-SD14 Solid dispersion SAS-Supercritical uid 1:4:2 69.25 1.44 90.39 1.75
SD15 Solid dispersion Solvent evaporation 1:5:1 28.99 0.86 49.58 1.11
SD16 Solid dispersion Solvent evaporation 1:4:2 36.12 1.09 61.12 1.59
SD17 Solid dispersion Solvent evaporation 1:3:3 30.05 0.87 52.58 1.49
SF-SD18 Solid dispersion SAS-Supercritical uid 1:4:2 81.37 1.55 95.24 1.09
PM4 Physical mixture Physical mixing 1:6:0 5.31 2.01 15.15 2.09
PM8 Physical mixture Physical mixing 1:4:0 8.10 1.11 15.92 1.52
PM12 Physical mixture Physical mixing 1:4:2 18.56 0.67 29.76 1.40
PM16 Physical mixture Physical mixing 1:4:2 20.88 1.09 36.14 1.73
 E. Adeli / J. of Supercritical Fluids 87 (2014) 921 15

120
Cumulative Drug Released (%)

100
Pure Drug
80 PM12
SD11
60
SD12
40 SD13
SF-SD14
20

0
0 10 20 30 40 50 60 70
Time (Min)

Fig. 5. In vitro dissolution release prole of pure Azithromycin, solid dispersions,

and the physical mixture containing Azithromycin, PEG 6000 and Poloxamer 188
(n = 3, mean SD)

expressed in saturation solubility of the binary systems section.

That includes particle size reduction, increased wettability, reduced
drug accumulation (aggregation) in the dissolution media, the
increase of surface area of drug particles and also very ne particles
are more reasons for drug release. Hence SD16 was selected among
all of the ternary samples (DE30,SD12 = 56.83 and DE30,SD16 = 61.12,
respectively). Further experiments and SCF technique were per-
formed based on SD16 formulation. Perhaps, because the high
solubility of SD17 (compared to SD16) is less than the concen-
tration of the polymer around the drug, it avoids more release of
the drug that DE% determines it. The added surfactant increased
more dissolution rate. This is due to the fact that the compound
(as Azithromycin) was insoluble and as the surface-active agent
was added in the system, it increased the wetting ability of the
compound and thereby increased the solubility of the drug. Also
this might be due to the fact and studies that the viscosity of the
dissolution media was decreased by the surfactant, therefore, the Fig. 7. IR spectra of: (A) Azithromycin, (B) PEG 6000, (C) SLS, (D) PM4, (E) SD4, (F)
dissolution rate of the drug was increased. PM16, (G) SD16 and (H) SF-SD18.
Here it also has ultrane nanometer particles in size; increase
the surface area of the drug particles and spherical particles fol-
lowed by increased wettability led to better results compared to method and particle size, was obtained much desired results, so
other formulations [46]. that;
Firstly, was released a large amount of the drug that in the early
minutes;
4.1.2.3. Ternary systems using SASSCF. At this stage, SF-SD14 and
Second, about 98% of drug was released.
SF-SD18 were prepared using SD16 formulation, and SCF technique.
Supercritical uid processing is more powerful in controlling the
In addition, in vitro dissolution rates of them were measured in
particle size and the physical transformation nature of this tech-
phosphate buffer pH = 6.0 and conditions previously is mentioned.
nique will not interfere with the interactions between drug and
Accordingly, the dissolution rate of SF-SD14 and SF-SD18 has grown
carrier, it is possible to utilize this method to control the in vitro per-
signicantly (Table 7 and Figs. 5 and 6). So that SF-SD14 at 30 min
formance stability issue. As a result, due to the drug wettability due
was drug released 92.12% and SF-SD18, 97.14% was drug released
to the formation of ne and uniform particles in the SASSCF, the
(at the same time). Also at 60 min, SF-SD18, 99.56% drug is released,
sample could be improved more efciently. It can be expected that
that is all the more. This is due to the mechanism of derived the
the improvement in the Azithromycin dissolution rate will increase
its bioavailability, but further efforts must be made to verify disso-
120 lution mechanism of the Azithromycin:PEG 6000:SLS formulations
[4749].
Cumulative Drug Released (%)

100
Pure Drug Comparison of the effect of increasing the solubility and disso-
80 PM16 lution rate of excipients in this study:
SD15
60
SD16 PEG6000 > Sorbitol
40 SD17
SF-SD18
20 SLS + PEG6000 > Polxamer407 + PEG6000
0
0 10 20 30 40 50 60 70 A comparison between inuence of methods for increasing the
Time (Min) solubility and dissolution rate in this study:

Fig. 6. In vitro dissolution release prole of pure Azithromycin, solid disper-

SASSCF(SLS + PEG6000) > SolventEvaporation(SLS + PEG6000)
sions, and the physical mixture containing Azithromycin, PEG 6000 and SLS (n = 3,
mean SD)
16 E. Adeli / J. of Supercritical Fluids 87 (2014) 921
Table 8
DSC multi thermogram data from a representative sample of Azithromycin, PEG 6000, SLS, PM4 and SD16.
Sample Azithromycin PEG 6000
TOnset ( C) 22.34 21.46
109.45 58.38
PEG 6000 is better than Sorbitol because:
(1) In present study, solid dispersion was prepared with PEG 6000
that shows the presence of amorphous form conrmed by the
characterization study.
(2) Hydrophilicity of soluble carrier (PEG 6000) > Sorbitol.
(3) The viscosity of the medium around the particles for PEG
6000 < Sorbitol.
(4) In order to obtain desirable results, a balance must be achieved
between those two factors.
Further experiments were carried out on selected samples of
the SD4 and SD15. That it will continue.
4.2. IR spectroscopy studies
The characteristic peaks of Azithromycin (Fig. 7A), PEG 6000
(Fig. 7B), SLS (Fig. 7C), physical mixtures (Fig. 7D and F) and solid
dispersions are presented in Fig. 7E, G and H. All basic peaks of
Azithromycin observed at wavenumbers 1724 cm1 (C O Car-
bonyl ester stretch) 1190 cm1 (C O C asymmetrical stretching)
1049 cm1 (C O C symmetrical stretching) were retained in phys-
ical mixtures and solid dispersions, which clearly indicate that no
interaction exists between pure drug and carriers. The main peaks
of PEG 6000 are at 1108 cm1 for C O C stretch, 2907 cm1 for C H
Fig. 8. DSC multi thermograms of: (A) Azithromycin, (B) PEG 6000, (C) SLS, (D) PM4, (E) SD4, (F) PM16, (G) SD16 and (H) SF-SD18.
SLS PM4 PM16 SD4 SD16
12.70 31.17 11.80 56.82 53.10
105.26 57.14 57.63 149.27
99.49
141.86
stretch and 3244 cm1 O H stretch. Also IR spectra of SLS show
these peaks: CH2 asymmetric stretching appears at 2917 cm1
and 2951 cm1 for C H stretch and 1655 cm1 for S O. IR spec-
troscopic studies ruled out any potential interactions between the
drug, carriers and surfactants (Fig. 7). Since the main signals of the
drug and the carrier appeared in SD and PM samples, no interac-
tion seems to be occurred in both sample preparations. It can be the
main reason for the observed major peaks of Azithromycin, carri-
ers and surfactants in the physical mixture. Comparing the SD and
PM spectra and the untreated drug, a broad peak of O H stretch-
ing vibration with a slight shift to lower wavenumbers could be
observed in SD12, SF-SD14, SD16 and SF-SD18 that indicates the
possibility of intermolecular hydrogen bonding between the drug
and the carrier. On the other hand, CH3 bending vibration peak
shifted to the lower wavenumber in both solid dispersion and phys-
ical mixture that might be attributed to the van der Walls forces
between Azithromycin and PEG. In addition, it can be integrated
with other peaks [50].
4.3. DSC studies
DSC studies revealed a possible physical interaction between
the drugand the carriers. As can be seen in the Table 8 and Fig. 8,
 E. Adeli / J. of Supercritical Fluids 87 (2014) 921 17

Fig. 9. XRD diffractograms of: (A) Azithromycin, (B) PEG 6000, (C) SLS, (D) PM4, (E) SD4, (F) PM16, (G) SD16 and (H) SF-SD18.

Azithromycin exhibited sharp and completely clear main endother-
mic peak at 120.87 C (109.45127.77 C). That peak is an identied
indicator of the drug. The main endothermic peaks of the PEG 6000
and SLS, SD4, SD16, PM4 and PM16 are shown in Fig. 8. Decompo-
sition endothermic peak of plain drug is depicted at 144.28 C in
illustrator Fig. 8A. Elimination, shift or relocation of some peaks can
be resolved as drug in carriers. The height of the melting peak solid
dispersions of Azithromycin is slightly decreased; and also from the
polymer content in the physical mixture with a higher percentage
of carriers is decreased is more signicant. Determination of the
peak on thermograms may prove the presence of Azithromycin in
crystalline form. Thermograms for Azithromycin solid dispersions
with PEG 6000 demonstrate a distinct shift of endothermic peak
observed for SF-SD18 was diffuse and broad compared to the
sharp intense peak of the conforming the formation of SF-SD18
carrier alone (Fig. 8). In general, disappearance of the drug melting
endothermic peak could be attributed to the lack of crystalline
structure of Azithromycin in the sample or the solubility of drug
in melted carrier during the test. Since in the next section, the
XRD spectra proved that the drug was in crystalline and not
amorphous state in SD sample, so the second condition might have
been occurred during the DSC analysis. The same thing could be
18 E. Adeli / J. of Supercritical Fluids 87 (2014) 921

Fig. 10. SEM microphotograph of: (A) Azithromycin; 500, (B) SD4; 500, (C) SD4; 5000, (D) SD16; 500 (E) SD16; 2500 and (F) SF-SD18; 500.
 E. Adeli / J. of Supercritical Fluids 87 (2014) 921
Table 9
Drug content of selected formulations after stability studies according to ICH guidelines (n = 3, mean SD).
Storage conditions Azithromycin SD4
40 2 C
75 5%RH
Time
0 days 99.92 1.27 98.32 0.44
60 days 99.90 0.14 98.25 2.09
120 days 99.88 0.78 98.18 1.07
happened for PM sample in the DSC pan (Endothermic peaks at
58.38 C and 105.26 C in the thermograms of PEG 6000 and SLS
[Table 8]).
4.4. XRD studies
XRD patterns are one of the most important methods to identify
and study the crystalline structure of materials and assessment of
solid dispersion systems. The diffractogram of pure Azithromycin,
PEG 6000, SLS, solid dispersion samples and physical mixture sam-
ples were depicted in Fig. 9. The XRD diffractogram of Azithromycin
(Fig. 9A) shows sharp peaks at diffraction angles (2) of 9.56 ,
7.72 , 20.67 , 19.59 , 23.80 , 17.31 , 28.68 and 26.55 . These peaks
clearly are evident in Fig. 9. The main peaks of PEG 6000 (Fig. 9B)
and SLS (Fig. 9C); at (2 value) 19.09 and 20.70 , respectively, are
shown in Fig. 9. In optimized solid dispersion, less intense and
wide diffraction peaks of Azithromycin are observed, which may be
attributed to the adsorption process in which some of amorphous
drug may be crystallized due to higher temperature (Fig. 9C and D).
The XRD peaks of untreated Azithromycin could be detected in SDs
and PMs at the same 2 values which conrmed that drug remains
crystalline in solid dispersion and physical mixture and no crys-
tallinity major changes (formation of amorphous state) occurred
during the process of preparation. However, the intensity of the
peaks of Azithromycin in both samples was signicantly less than
that of untreated drug, which could be attributed to the lower per-
centage of drug incorporated in samples preparation compared to
the carrier. The signals were generally stronger in physical mix-
tures than in solid dispersions. These results suggest that there
was no chemical interaction between Azithromycin and carriers.
Approximately Azithromycin pattern crystalline is maintained in
solid dispersions and physical mixtures. In general, we can say that
there is no interaction between drug and polymer or surfactant.
Thus, a decrease in the drug crystallinity might be responsible for
the improvement in dissolution of Azithromycin. This reduction of
crystallinity of SCF samples is much higher than other samples.
4.5. SEM studies
The microphotographs of Azithromycin and solid dispersions
are shown at different magnications in Fig. 10. Azithromycin
consisted of discrete, large cubic-like shape crystal structures
(Fig. 10A) with rough edges. Characteristic cubic shape crystals of
Azithromycin were observed in the photomicrograph of pure drug.
SEM of the solid dispersions reveals irregular particles with several
microscopic cracks and crevices, which provide additional surface
for deposition of the drug particles. These structures were also cov-
ered on their surfaces by the ne particles. Microphotograph of
Fig. 10BD belongs to binary solid dispersion systems and Fig. 10E
illustrates of ternary solid dispersion. In all of the SEMs, ne parti-
cles of drug disperse in context of PEG 6000. This illustration looked
like a matrix with spherical micro particles, at lower magnication.
However, at greater magnication, the spherical micro particles
revealed an aggregate, dense packing of the large crystal struc-
tures. The enhancement in dissolution rates is due to reduction in
drug crystallinity and size. This decreased particle size or increased
19
SD12 SD16 SF-SD14 SF-SD18
97.10 1.33 96.95 1.51 94.29 0.80 93.91 1.77
97.00 1.87 96.81 0.06 94.28 1.10 93.88 1.91
96.89 1.99 96.80 1.00 94.19 2.52 93.79 2.28
surface area was responsible for an increase in dissolution rate and
saturation solubility of SLS over PEG 6000 (Fig. 10D and E). The SEM
of SASSCF (Fig. 10F) for samples shows ne spherical particles led
to increase of contact surface and consequently emerge the amor-
phous properties of the samples. SEM studies further suggested that
the surface properties of Azithromycin and carriers were lost dur-
ing the formation of solid dispersion system by SAS-supercritical
uid or solvent evaporation and solidication resulting in the dis-
persion of the drug molecules within the carrier matrix. Molecules
of the drug must be dispersed or soluble or particle size is reduced
in the solid dispersions without crystal formation in order to it
enhances the dissolution prole. These observations also exhibit
that the results from DSC study are tenable. In short, we can say
that the percentage of crystalline drug is reduced but the drug has
not became amorphous. Perhaps this is due to increased dissolution
prole. There is no evidence of the prior crystallinity percentage of
the drug crystals, which it conrms the previous ndings based on
XRD patterns.
4.6. Physical stability studies
In order to the optimized formulations were charged for accel-
erated stability studies as per ICH2 guidelines. For a period of
120 days studies at 40 2 C and 75 5% RH in environmental
test chamber (Electrolux, Germany), the samples (each 100 mg,
n = 3) were kept for stability. The samples were kept in glass vials
sealed with rubber plugs. 10 mg of stored samples was taken out
in 15, 30, 60, 90 and 120 days, they were analyzed for drug con-
tent and physical change. The results did not show a signicant
change. So we can conclude that in this period samples are stable
(Table 9).
5. Conclusion
The general conclusions that the hydrophilic polymer as PEG
6000 increased solubility and dissolution rate of Azithromycin.
This happened in binary solid dispersion systems using solvent
evaporation method (SD4, 1:6 ratios). In other hand, the prepara-
tion of the ternary solid dispersion by solvent evaporation using
surfactants increased further solubility and dissolution rate of
Azithromycin (SD16, 1:4:2 ratios). For the solid dispersions; the
decrease of Azithromycin crystallinity and dispersed state of the
drug resulting in its higher wettability are mainly reason for
dissolution rate enhancement. These results are in agreement with
those of differential scanning calorimetry and X-ray assays. The
increase in dissolution from the physical mixtures was probably
due to the wetting and solubilizing effect of the carrier, which
could reduce the interfacial tension between the drug and the
dissolution medium, thus leading to a higher dissolution rate. This
phenomenon can be ascribed in the physical mixtures to a higher
wettability of Azithromycin in presence of carriers. The physi-
cal mixtures have a water-soluble carrier (PEG 6000, Sorbitol),
2
International conference on harmonization of technical requirements for regis-
tration of pharmaceuticals for human use.
20 E. Adeli / J. of Supercritical Fluids 87 (2014) 921
solubility enhancer (PEG 6000), lm-forming polymers (PEG 6000)
and emulsifying agents or active surface agents (SLS, Poloxamer).
Therefore this agent can enhance the solubility of drugs (any
compounds) alone without any processing.
Finally, the utilization of SASSCF technique for preparation of
ternary solid dispersion systems (SF-SD18, 1:4:2 ratio) increased
extraordinary solubility and dissolution rate of Azithromycin. This
is due to the formation of very ne and spherical particles as
nanometer size. Results of IR, DSC, XRD and SEM showed lit-
tle change in the crystalline form of the drug. These changes
were not statistically signicant (P < 0.05) and it is not notice-
able. The current study indicated the potential of supercritical
uid-based technologies in enhancing of the dissolution rate of
poorly soluble drugs from the solid dispersion of drugs and
polymers and ingredients commonly used in pharmaceutical
industry.
Acknowledgment
The paper is taken from a part of Pharm.D. Thesis of Ehsan Adeli,
The International Branch, Shahid Beheshti University of Medical
Sciences, Tehran, Iran.
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