International Journal of Pharmaceutics 608 (2021) 121119

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International Journal of Pharmaceutics

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

Implications of phase solubility/miscibility and drug-rich phase formation

on the performance of co-amorphous materials: The case of Darunavir
co-amorphous materials with Ritonavir and Indomethacin as co-formers
Sushant Shete a, 1, Sai Charan Reddy a, 1, Yarlagadda Dani Lakshman a, Sai Krishna
Anand Vullendula a, Chetan Hasmukh Mehta b, Usha Yogendra Nayak b, Swapnil Dengale a, c, *
a
Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal 576104, India
b
Department of Pharmaceutics, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal 576104, India
c
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, Changsari 781101, India

A R T I C L E I N F O A B S T R A C T

Keywords: The present study was designed to investigate the contribution of solid-state and the impact of composite drug-
Co-amorphous rich phase generated as a consequence of pH shift on the maximum achievable supersaturation of co-amorphous
Solubility formulations. The co-amorphous phases of weak base-weak base-pair i.e. Ritonavir and Darunavir were prepared
Dissolution
in anticipation of studying the effect of drug-rich phase consequent to pH shift. While the co-amorphous phases
Supersaturation
of weak base-Weak acid pair i.e. Darunavir and Indomethacin were studied to understand the manifestation of
Drug rich phase
LLPS the solid-state drug: co-former miscibility in the absence of drug rich phase. Thermodynamically, the lowering of
the supersaturation was found commensurate with the mole fraction of the respective component (Drug/Co-
former) within the co-amorphous materials for both Darunavir: Ritonavir and Darunavir: Indomethacin pair.
Kinetically, for Darunavir: Ritonavir co-amorphous materials, the shift in the pH from acidic to the neutral side
led to the generation of drug-rich phase and subsequent LLPS. The free drug concentration achieved in the bulk
of the solution was found dependent upon the mole fraction of the respective component within the drug-rich
phase. The relative mole fraction of each component within the composite drug-rich phase is dictated by pH-
dependent solubility and molecular weight of the individual components.

1. Introduction components within each other is a prerequisite for a successful forma­

The co-amorphous formulations are becoming a credible alternative
to the Solid Dispersion (SD) technology for the stabilization of high-
energy, efficient amorphous solids (Chavan et al., 2016; Dengale
et al., 2016; Laitinen et al., 2013). From a wider perspective, co-
amorphous formulations are considered a type of SD (Nair et al.,
2020). However, instead of polymers, the co-amorphous formulations
employ small molecular weight co-formers to form a homogeneous
single-phase amorphous system. The employment of small molecular
weight co-formers offers certain advantages over polymers which
include high drug loading, less hygroscopicity, longer physical stability
(Korhonen et al., 2017; Laitinen et al., 2017). As co-amorphous formu­
lations are single-phase systems of two or more small molecular weight
components, the ideal mixing and miscibility/solubility of all
tion of co-amorphous systems. In that way, they are often sub-classified
as glass solutions (Dengale et al., 2016; Yarlagadda et al., 2021). How­
ever, there are thermodynamic consequences for multicomponent
single-phase glass solutions. If the mixing of the solutes/Drug-coformer
is ideal and entropically driven, which is the case for co-amorphous
formulations, then there is a change in the chemical potential, and
subsequently the free drug concentration of the solutes. For a two-
component single-phase co-amorphous system, the chemical potential
μ1 of component 1 is given by
μ1 = μ01 + RTInx (1)
where μ1 and μ01 are the chemical potentials of component 1 in the
amorphous mixture and pure supercool liquid state, respectively. x is the

* Corresponding author at: National Institute of Pharmaceutical Education and Research (NIPER)-Guwahati, Changsari 781101, India.
E-mail addresses: swapnil.jd@manipal.edu, swapnil@niperguwahati.in (S. Dengale).
1
Both authors have contributed equally.

https://doi.org/10.1016/j.ijpharm.2021.121119
Received 17 May 2021; Received in revised form 16 September 2021; Accepted 17 September 2021
Available online 21 September 2021
0378-5173/© 2021 Elsevier B.V. All rights reserved.
S. Shete et al. International Journal of Pharmaceutics 608 (2021) 121119

mole fraction of component 1 in the amorphous mixture, R is the gas 2015b).

constant, and T is the absolute temperature. In the above context, it is evident that the single-phase glassy co-
amorphous phases are amenable for compromised supersaturation.
μ2 = μ02 + RTIn(1 − x) (2)
The causality of this compromised supersaturation lies with the solid-
state and solution phase behavior alike. The presence of a co-former
where μ2 and μ02 are the chemical potentials of component 2 in the
in the single glassy solid state offsets the drug release, while the gen­
amorphous mixture and pure supercool liquid state, respectively.
eration of drug-rich phase during dissolution preferentially sequester
Therefore, it is evident from the above equations that the chemical
free drug from the bulk of the solution. Therefore, the present study was
potential of the components is reduced by an amount that is directly
designed to investigate the contribution of solid-state and the impact of
proportional to the mole fraction of each component present in the bi­
composite drug rich phase generated as a consequence of pH shift on the
nary mixture (Taylor and Zhang, 2016; Trasi and Taylor, 2015a, 2015b).
maximum achievable supersaturation of co-amorphous formulations.
The manifestation of this thermodynamic phenomenon is demonstrated
The co-amorphous phases of Ritonavir (RTV) and Darunavir (DRV) were
for multicomponent solid dispersion in the literature. Trasi and Taylor
prepared in anticipation of studying the effect of drug-rich phase
(2015a, 2015b), studied the dissolution and flux behavior of multi­
consequent to pH shift. Both Ritonavir and Darunavir are weak bases
component solid dispersion of Ritonavir-Lopinavir. The group observed
with pKa 2.7 and 2.4 respectively. They are also phase-soluble within
that the maximum achievable supersaturation and flux were depressed
each other, as evident by melting point depression, and therefore ideal
in the multi-component SD than the SD of the respective individual
for the preparation of single-phase co-amorphous systems (Dengale
component. The offset in the free drug concentration and flux was found
et al., 2015; Nguyen and Van den Mooter, 2014). The second co-
consistent with the mole fraction of each component within SD (Trasi
amorphous formulation was prepared as a comparator where a weak
and Taylor, 2015a, 2015b). Similar observations were reported for the
acid Indomethacin (IND) with pKa 4.0 was employed as a co-former. As
multicomponent SD of Ritonavir and Atazanavir. Furthermore, the
Indomethacin is a strong glass former (Dengale et al., 2014; Moura
dissolution and flux of ternary SD of Ritonavir, Atazanavir, and Lopi­
Ramos et al., 2004), the weak acid: weak base pair would be devoid of
navir were found decreased by a factor of 1/3rd compared with their
the drug-rich phase of Indomethacin, which would enable the investi­
individual amorphous solid dispersion counterpart (Alhalaweh et al.,
gation of the phase behavior of Darunavir in the absence of drug rich
2016). The co-amorphous formulations also demonstrated an analogous
phase of the co-former. Notwithstanding the pharmacodynamics or
trend. Anand et al., (2017), found the lowering of maximum achievable
pharmacokinetic rationale, the drugs were selected as model compounds
supersaturation consistent with the mole fraction of individual compo­
based on their thermodynamic and physicochemical properties to
nents within the co-amorphous system (Sai Krishna Anand et al., 2018).
demonstrate the solution phase behavior of co-amorphous formulations.
The amorphous phase dissolves in an aqueous solvent till its amor­
phous solubility is reached, concurrently, water penetrates the amor­
2. Materials and methods
phous phase till it saturates with water leading to the formation of a
saturated supercooled-liquid phase. Once the concentration of solute
2.1. Materials
breaches the amorphous solubility in the given solvent, in the absence of
recrystallization, phase separation occurs. Consequently, two immis­
Darunavir (Fig. 1A) and Indomethacin (Fig. 1B) were received as a
cible phases will separate due to the solubility gap between them. The
kind gift from Hetero Drugs Limited, Hyderabad, India. Ritonavir
phase with metastable glass or colloidal aggregates is termed as Drug-
(Fig. 1C) was obtained from Mylan pharmaceuticals limited, Hyderabad,
Rich Phase (DRP), and the maximum solute concentration present in
India. Analytical grade chemicals like Potassium Dihydrogen Ortho­
the bulk of the solution is regarded as Drug-Lean-Phase (DLP). This
phosphate, Sodium Hydroxide, Hydrochloric Acid, and Phosphoric Acid
whole phenomenon is called either Liquid-Liquid Phase Separation
were procured from Loba chemicals, Mumbai, India. HPLC grade sol­
(LLPS) if the glass transition temperature of the precipitated supercooled
vents like Acetonitrile, Methanol, and Isopropyl Alcohol were purchased
liquid phase is less than the room/experimental temperature or Glass-
from Finar Limited, Ahmedabad, India. Ultrapure water was obtained
Liquid Phase Separation (GLPS) if the glass transition temperature of
from SIEMEN ultra-clear purification system (Munich, Germany)
the precipitated liquid phase is more than the room/experimental
installed in the laboratory.
temperature (Li and Taylor, 2018; Taylor and Zhang, 2016; Trasi and
Taylor, 2015a, 2015b). For co-amorphous formulations of weakly basic
2.2. Methods
compounds, the drug-rich phase is likely to be formed in response to the
ascending gradient pH, wherein the unionized form of the weak base
2.2.1. Preparation of co-amorphous phases of Darunavir-Ritonavir (DR)
will predominate in the neutral pH of the small intestine, eventually
A total 1000 mg of Darunavir and Ritonavir mixture in the molar
precipitating as a supercooled liquid phase (Hsieh et al., 2015, 2012;
ratio 1:1 (D1R1, 548.39 mg R and 451.61 mg Darunavir), 1:2 (D2R1,
Sakhare et al., 2019). Provided that both the drug and the co-former are
377.79 mg Ritonavir and 622.21 mg Darunavir), and 2:1 (D1R2, 708.34
weakly basic, the drug-rich phase for co-amorphous formulations is
mg Ritonavir and 291.66 mg Darunavir) were taken into a separate
expected to be the composite of the drug and the co-former. Recent
porcelain dish. The mixture was melted at a heating rate of 10 ◦ C/min in
studies have demonstrated that the environment-sensitive fluorescence
a heating mantle. The porcelain dish with the melted mixture was
probe pyrene registers a more hydrophobic environment in the presence
immediately transferred into a previously cooled glass desiccator
of the drug-rich phase as compared to a single-phase aqueous solution
(-80 ◦ C). The desiccator along with the porcelain dish was sealed using
(Raina et al., 2015; Trasi and Taylor, 2015a, 2015b). Due to this, any
paraffin wax and the whole assembly was stored in an ultra-low freezer
additional solute present concomitantly in the solution may partition
at − 80 ◦ C for 4 h. Afterward, the samples were retrieved from the freezer
into the drug-rich phase lowering the free drug concentration of either
and pulverized using ceramic mortar and pestle to get a fine and uniform
or both the species. It implies that the drug-rich phase has the potential
co-amorphous material. The samples were then stored in a vacuum
to interact physico-chemically with additional species that are present in
desiccator until further use.
the solution. Trasi and Taylor (2015a, 2015b), demonstrated that the
activity and free drug concentration achieved after the dissolution of the
2.2.2. Preparation of co-amorphous phases of Darunavir-Indomethacin
multicomponent amorphous system are not only dependent on the mole
(DI)
fraction of individual components within the dispersion (As explained in
A 1000 mg mixture of Darunavir and Indomethacin in the molar ratio
the earlier paragraph) but also found to be dictated by the mole fraction
of 1:1 (D1I1, 604.85 mg Darunavir, and 395.140 mg Indomethacin), 1:2
of each component in the drug rich phase (Trasi and Taylor, 2015a,
(D1I2, 433.54 mg Darunavir, and 566.452 mg Indomethacin), and 2:1

2
S. Shete et al. International Journal of Pharmaceutics 608 (2021) 121119

Fig. 1. Chemical structures of Darunavir (A), Indomethacin (B), and Ritonavir (C).

(D2I1, 762.60 mg Darunavir, and 237.39 mg Indomethacin) were taken 2.3. Van Krevelen group contribution approach
in a separate round-bottomed flask. Methanol (ICH class-II solvent) was
added to dissolve the mixture of drugs. The solvent was evaporated in Solubility parameters (δ) for given drugs were calculated by the Van
rotary evaporation apparatus at 45 ◦ C. The round-bottomed flask was Krevelen group contribution approach, where cohesive energy is
then stored in a vacuum desiccator for 2 days for drying. Later the regarded as the indicator of attractive forces between constituent mol­
samples were scraped and stored in a vacuum desiccator for further ecules in a material. The total cohesive energy is the sum of three types
experiments. of forces, namely, dispersion forces (Ed), polar forces (Ep), and
Note: The quench cool method could not be implemented due to hydrogen bonding (Eh).
excessive degradation of the components involved in the preparation of
Ecoh = Ed + Ep + Eh (3)
co-amorphous phases of Darunavir and Indomethacin.
The equation for the solubility parameter is as follows,
2.2.3. Solid state characterization
X-ray powder diffractograms were recorded using Rigaku miniflex- δ = δ2d + δ2p + δ2h
2
(4)
600 X-ray diffractometer, which was operated at 600 W (X-ray tube), Where,
with a voltage of 40 Kv, and a fixed tube current of 15 mA. The in­ √̅̅̅̅̅̅̅̅̅̅̅̅
∑ 2
strument employed a graphite monochromator and a standard scintil­ ∑ Fpi
√̅̅̅̅̅̅̅̅̅̅̅̅
∑
Fdi Ehi
lation counter as a detector. Diffraction intensities were measured by the δd = δp = δh =
Vm Vm Vm
fixed time step scanning method in the range of 5-40◦ (2θ).
FTIR spectra of the prepared samples were obtained by the Shimadzu Where Fdi is the molar attraction constant due to dispersive com­
FTIR-8300 system over a range of 4000–500 cm− 1 (25 scans, resolution ponents, Fpi is the dipole–dipole interactions, Ehi is the hydrogen
4 cm− 1). Further, AT-FTIR spectra were measured by Bruker Alfa-II bonding contribution in the chemical structure, and Vm is the molar
spectrophotometer. volume (Fedors, 1974; Lakshman et al., 2020; Van Krevelen and Te
DSC measurements were recorded using the Shimadzu DT-60 appa­ Nijenhuis, 2009).
ratus. Approximately 5 mg of sample was placed in a flat bottomed
aluminum pan of 0.1 mm thickness and crimped with an aluminum lid. 3. Computational approach
The crimped sample was placed in a DSC furnace and heated from 25 to
250 ◦ C at a heating rate of 10 ◦ C/min under a stream of nitrogen gas Computational studies were performed to find the gas-phase free
(Flow 10 cc/min). energy of Indomethacin and Ritonavir with Darunavir. Schrodinger
Solid-state phase Solubility/Miscibility of Drug-Coformer software of version 3.0 (Schrödinger, LLC, New York) was used for

3
S. Shete et al.
performing the gas-phase free energy calculations. MS Maestro interface
was employed to prepare 3D chemical structures from the 2D structures
using the 2D Sketcher tool (Rocha-Rinza et al., 2011).
3.1. Solubility determination
3.1.1. Solubility by shake flask method
The solubility of pure drugs and co-amorphous materials was esti­
mated by the shake flask method. Excess material was added to the vials
containing 2 mL of USP phosphate buffer pH 6.8, and the resultant
suspension was shaken at 120 rpm in an orbital shaker at 37 ◦ C for 24 h.
Samples were collected and centrifuged at 10000 rpm for 10 min at
37 ◦ C using Remi C24 centrifuge. The supernatant collected was trans­
ferred to the HPLC vial and diluted as appropriate with the mobile
phase. The samples were analyzed using the HPLC method mentioned in
the supplementary material (Supplementary material, Tables S1 and
S2).
3.1.2. Amorphous solubility determination
The amorphous solubility of individual drugs was determined by the
solvent quench method in USP phosphate buffer (pH 6.8). A 100 μL of
10 mg/mL methanolic solution of the respective drug was spiked into 10
mL of USP phosphate buffer (pH 6.8) containing 10 μg/mL HPMC as a
crystallization inhibitor. The resultant suspension was stirred for 10 min
at 37 ◦ C and then centrifuged at 10000 rpm for 10 min using Microcon®
centrifugal filters (MWCO 10Kda, Pore size 3 nm, Merck, Germany). The
filtered samples were diluted appropriately with the mobile phase. The
samples were analyzed by the HPLC method as described in the sup­
plementary material (Supplementary material, Tables S1 and S2).
3.2. Dissolution by pH shift method
An amount of physical mixture (PM) or co-amorphous materials
equivalent to 135 mg of Darunavir was weighed and equilibrated with
100 mL, 100 mM HCl solution (pH 1.5) using a magnetic stirrer. After
10 min of equilibration, a sample was withdrawn. Further, the whole
solution (Suspension) was transferred into 200 mL potassium dihy­
drogen orthophosphate buffer (75 mM, pH 6.8) which was maintained
at 37 ◦ C. The stirring was continued till 120 min and samples were
withdrawn at predetermined time intervals. The collected samples were
centrifuged for 10 min at 10000 rpm. The supernatant was suitable
diluted with the mobile phase. The diluted samples were analyzed using
the HPLC method described in the supplementary material (Supple­
mentary material, Tables S1 and S2).
3.3. Determination of Liquid-Liquid Phase Separation (LLPS)
After the transfer of the acidic milieu into the phosphate buffer pH
6.8, there was evident precipitation of the dissolved material. Therefore,
the following experiments were done to confirm the occurrence of
Liquid-Liquid Phase Separation (LLPS)
3.4. DLS method
The samples were monitored for Dynamic Light Scattering employ­
ing Nano Zeta Sizer (Nano ZS, Malvern Instruments, Westborough, MA,
USA). The samples were introduced in the zeta sizer using a clear zeta
cell and, analyzed for particle size and dispersity.
3.5. UV extinction method
The second aliquot of the samples was analyzed at the non-absorbing
wavelength i.e., 400 nm for the given set of compounds using a UV
Spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan).
Generation of colloidal drug-rich phase scatters the UV light, and the
scattering can be detected at a non-absorbing wavelength which
4
International Journal of Pharmaceutics 608 (2021) 121119
confirms LLPS.
3.6. Investigation of appearance of drug rich phase by fluorescence probe
method
Pyrene is an environment-sensitive fluorescence probe, which pref­
erentially mixes with the lipophilic drug-rich phase. Therefore, the
relative lipophilicity of the drug-rich phase formed after the dissolution
of co-amorphous materials and in response to the change in the pH of the
media was determined by the fluorescence probe method. A pyrene
concentration of 1 μm was prepared in 100 mM phosphate buffer, pH
2.0. The co-amorphous materials were equilibrated with this solution
and the pH was shifted towards the higher side till 7.0. At every unit
increment in pH, the aliquot from the dissolution vessel was retrieved
and analyzed at an excitation wavelength of 332 nm, and the emission
spectrum was monitored between 350 nm and 450 nm by employing a
spectrofluorimeter (Model: RF5301 pc, Shimadzu Scientific In­
struments, Kyoto, Japan). The local polarity was monitored by using the
ratio of the emission peak at 383 nm (I3) to that of the peak at 373 nm
(I1).
4. Permeability by everted gut sac method
Ex-vivo studies were conducted on the ileum obtained from Wistar
rats in accordance with CPCSEA guidelines and were approved by the
Institutional Animal Ethics Committee (IAEC/KMC/121/2019), Man­
ipal Academy of Higher Education (MAHE). The Wistar rats weighing
220 ± 10 gm fasted for 12 h before they were sacrificed by cervical
dislocation and a midline incision was made in the abdomen to obtain
the small intestine. The recovered intestine was washed thoroughly with
a saline solution. The intestine was cut 5 cm above the ileocecal junction
to recover the ileum part of the small intestine.
In order to estimate the permeation from the mucosal to the serosal
side, the ileal segment was everted using a capillary tube and washed
with saline solution. The proximal end of the segment was ligated to a
receptor silicone tube and, the distal end was ligated tightly to create a
closed compartment. The segment was then suspended in a glass beaker
consisting of 50 mL equilibrated test solution in phosphate buffer. The
test solution was prepared by equilibrating 135 mg equivalent co-
amorphous material in phosphate buffer (pH 2.0) and then shifting
the pH to 6.8 to generate a drug-rich phase. The bulk of the test solution
in the glass beaker which represents the donor compartment is main­
tained at 37 ◦ C and continuously bubbled with oxygen to maintain the
tissue viability. The enclosed everted sac, which represents the acceptor
compartment was filled with 1.5 mL phosphate buffer pH 6.8. After
suspending the everted sac into the assembly, the samples were with­
drawn at predetermined time intervals using long-necked oral gavage
attached to a syringe, and the equivalent volume of phosphate buffer
was immediately replenished. The flux was calculated from the
following formula once the asymptote reached,
dc 1
Flux(J) = * (5)
dt A
Where dc/dt is a change in the concentration per unit time at the
serosal side and, A is the area of ileum sac available for permeation.
5. Results and discussion
5.1. Solid-state characterization
The conversion of crystalline drugs into the amorphous state was
confirmed by the powder X-ray diffraction technique. Pure Ritonavir
showed numerous diffraction peaks confirming the crystalline nature.
However, pure Darunavir diffractogram was devoid of any Bragg’s
diffraction suggesting the disordered nature thereof. All the diffracto­
grams of the prepared co-amorphous materials of Ritonavir: Darunavir
S. Shete et al.
exhibited diffuse halo confirming the amorphous nature of the samples
(Supplementary Material, Fig. S1). Co-amorphous materials are single-
phase systems of multiple components wherein all involved compo­
nents are miscible with each other leading to the formation of a uniform
molecularly dissolved phase. One of the methods to confirm the for­
mation of a single-phase system is DSC, where a single glass transition
event confirms the phase solubility of the components. However,
disappointingly a single glass transition temperature could not be
spotted for the prepared co-amorphous materials even at the heating
rates as high as 30 ◦ C (Supplementary material, Fig. S2). The failure to
detect the glass transition temperature can be attributed to the usage of
conventional DSC instruments which could not detect the nuanced
transitions. However, the phase solubility of the drugs in the co-
amorphous phases was established by alternative methods (solubility
parameter, Gibbs free energy, and melting point depression) discussed
in the following sections of the manuscript. Furthermore, FTIR experi­
ments were conducted to probe the specific intermolecular interaction
between Ritonavir and Darunavir (Supplementary material, Fig. S3).
The FTIR spectrum of the physical mixture of Ritonavir and Darunavir
showed a sharp peak at 3300 cm− 1. The peak can be attributed to O-H
stretching from hydroxyl groups of both drugs. However, the sharpness
of the peak indicates the possibility of the presence of free hydroxyl
groups. The respective hydroxyl group from Ritonavir and Darunavir are
hindered (Fig. 1A and C), which may prevent their participation in
hydrogen bonding in the ordered state. Therefore, such hydroxyl groups
can give rise to the sharp peaks at 3300 cm− 1 consistent with the alco­
holic vapor phase. In the spectrum of the physical mixture, the medium
intensity peak at 1630 cm− 1 and relatively sharp peak at 1510 cm− 1 can
be attributed respectively to C = O stretching and –N-H bending of
amide linkage present in both the compounds. The medium intensity
peak at 1730 cm− 1 can be assigned the C = O group bonded to Oxygen
from Ritonavir and Darunavir. The negative inductive effect of Oxygen
reduces the length of the carbonyl bond and thus increases its force
constant and the frequency of the absorption (Silverstein et al., 2014).
All the above-discussed peaks were significantly attenuated in the
spectra of co-amorphous Ritonavir: Darunavir materials (Supplemen­
tary material, Fig. S3). Nevertheless, there were no significant shifts in
terms of frequency/wavenumber for the peaks of co-amorphous mate­
rials as compared to the physical mixture. The only evident change in
the spectra of co-amorphous materials was the significant broadening of
the O-H stretching peak at 3300 cm− 1. Further, the FT-IR data was
corroborated by measuring the IR spectra by employing the FT-NIR in­
strument. The AT-FTIR spectra were shown in figure (Supplementary
material, Fig. S4), where no wavenumber shift in the AT-FTIR spectra
was observed for both the systems, albeit the transmittance intensity of
the peaks was found reduced significantly which points towards the
change in the chemical environment but not the possibility of
interactions.
The diffractogram of Indomethacin exhibited strong diffraction
peaks confirming crystalline nature. While diffuse halo was observed for
Darunavir indicative of disordered phase. All the diffractograms of the
prepared co-amorphous materials of Darunavir: Indomethacin demon­
strated diffuse halo confirming the amorphous nature of prepared ma­
terials (Supplementary material, Fig. S5). Like Ritonavir: Darunavir co-
amorphous materials, the thermograms of Darunavir: Indomethacin co-
amorphous materials did not show a single glass transition temperature
(Supplementary material, Fig. S6). However, in the thermograms, the
weak endothermic event was observed at 140 ◦ C which corresponds to
the melting point of α-Indomethacin and indicates the possibility of
trace polymorphic conversion. Further, the FTIR spectrum (Supple­
mentary material, Fig. S7) of Darunavir and Indomethacin physical
mixture showed relatively strong O-H stretching vibrations of hindered
hydroxyl groups from Darunavir at 3300 cm− 1. The peak at 2900 cm− 1
can be assigned to the C-H stretching of alkyl groups from both Dar­
unavir and Indomethacin. In the carbonyl region, the strong peak at
1720 cm− 1 was believed to be an overlap of carbonyl stretching of the
5
International Journal of Pharmaceutics 608 (2021) 121119
carboxylic moiety of dimerized Indomethacin and amide linkage with
Oxygen heteroatom in the vicinity of Darunavir (Silverstein et al., 2014).
The IR spectra of prepared co-amorphous materials resemble the spec­
trum of physical mixture of Darunavir and Indomethacin, except the
spectra of co-amorphous materials exhibited peak broadening (Supple­
mentary material, Fig. S7), which indicates the change in the chemical
environment due to the formation of an amorphous phase. The same
results were corroborated by AT-FTIR spectra (Fig. S8).
5.2. Solid-state phase solubility/miscibility of drug-coformer
A drug: co-former miscibility is a prerequisite to forming a single-
phase glassy amorphous system. For such systems, above the glass
transition temperature, a drug and co-former dissolve in each other
leading to the formation of a single-phase supercooled liquid phase,
while the annealing of the mixture below the glass transition tempera­
ture yields glassy undercooled melt. Though the phase solubility/
miscibility is responsible to form a stable system, it also manifests into
certain performance limitations for co-amorphous materials which are
discussed in this article. Therefore, it is imperative to establish the phase
solubility between a drug and co-former pair, which was established by
theoretical, computational, and experimental approaches. Theoreti­
cally, the drug: co-former miscibility was calculated by Hilderband and
Hansen approach using the Van Krevelen group contribution values
(Table 1). For both the pairs i.e., Darunavir: Ritonavir and Darunavir:
Indomethacin, the respective Hilderband solubility parameter differ­
ence is less than 7 Mpa1/2 which indicates completely miscible systems.
Further, the Hansen solubility parameter revealed the relative contri­
bution of polarity, dispersibility, and hydrogen bonding in forming
single-phase miscible systems. For Darunavir: Ritonavir, all three pa­
rameters have comparable values. However, for Darunavir: Indometh­
acin, the values for δd and δh have a significant difference, which
indicates a larger contribution of polarity in the phase solubility.
Further, gas-phase free energies were computed for the given drugs to
find the probable phase solubility between Darunavir: Ritonavir and
Darunavir: Indomethacin pairs. For both the pairs, the energy of in­
teractions was found negative, which indicated the high probability of
ideal mixing and single-phase formation (Table 2). Though the Dar­
unavir: Ritonavir phase is expected to have high stability as compared to
Darunavir: Indomethacin due to the least energy of mixing. Further­
more, the drug-drug miscibility of the co-amorphous pairs was experi­
mentally established by employing the melting point depression
approach. The Flory-Huggins interaction parameters (□) were calcu­
lated by using melting point depression data for the given pair which
was found to be 0.3731 and − 0.1752 for Ritonavir: Darunavir and
Darunavir: Indomethacin pairs respectively (Supplementary material
S9, S10, and S11). The interaction parameter (□) values close to zero
indicate the ideal mixing behavior between the two components of the
system.
5.3. Solubility
To establish the net solubility advantage/compromise of drugs from
co-amorphous materials over their respective crystalline and amorphous
counterparts, the equilibrium solubilities of neat amorphous and crys­
talline drugs along with the co-amorphous materials were determined
(Table 3) (El-Badry et al., 2009; Ilevbare and Taylor, 2013; Kasten et al.,
2017; US FDA, 2008). The amorphous solubility of Ritonavir and
Indomethacin was estimated by the solvent quench method. While the
solubility of pure Darunavir that is present in the disordered form at
room temperature was taken as an amorphous solubility. The solubility
results of Darunavir from Ritonavir: Darunavir co-amorphous materials
are represented in Fig. 2(B). It was evident that the amorphous Dar­
unavir exhibited the highest solubility, while the solubility of Darunavir
from co-amorphous material was found depressed more than that of the
pure amorphous Darunavir. In the literature, it has been established that
S. Shete et al. International Journal of Pharmaceutics 608 (2021) 121119

Table 1
Drug:Co-former miscibility calculated by Van-Krevlen group contribution method.
Substance Molecular Weight (g/ Density (g/ Molecular Volume (cm3/ Melting Point ΔH fusion (kJ/ Solubility Parameter (MPa1/2)
mol) cm3) mol) (K) mol)
Hilderbrand Hansen

δd δp δh

Ritonavir 720.94 1.2 ± 0.1 600.78 399.15 57.9 27.18 13.62 5.16 9.83
Indomethacin 357.8 1.3 ± 0.1 275.23 425.15 43.5 22.52 21.70 5.77 0.32
Darunavir 547.66 1.34 ± 0.1 408.70 347.15 106.0 28.60 13.24 5.45 8.72

Table 2
Gas phase free energy calculations for co-amorphous materials.
SN Co-amorphous system Energy (Hartree)

Drug Co-former Drug-coformer complex Interaction

1 Darunavir-Ritonavir − 2140.1541 − 2939.948 − 5080.1794 − 0.077398

2 Darunavir-Indomethacin − 2140.1436 − 1549.5364 − 3689.7299 − 0.049846

Table 3
Experimental and reported solubility of pure Ritonavir, Indomethacin, and Darunavir (n = 3).
SN. Drug Amorphous solubility (μg/mL) Crystalline solubility (μg/mL) Degree of supersaturation

Reported Obtained Reported Obtained

1. Ritonavir 18.8 ± 0.07a 38.61 ± 0.75 1.3 ± 0.20a 4.36 ± 0.58 8.85
36.93 ± 1.521 4.45 ± 0.331
2. Indomethacin 26.7 ± 1.0b1 630.06 ± 17.50 2232 217 ± 8.3 2.90
~6503
3. Darunavir 150b4 227.16 ± 7.29 NA NA NA
a b
All solubility values were determined in phosphate buffer pH 6.8 (50 mM) except, Phosphate buffer 6.8 (100 mM), water.
1
Ilevbare and Taylor, 2013.
2
El-Badry et al., 2009.
3
Kasten et al., 2017.
4
US FDA, 2008.

Fig. 2. Predicted and experimental equilibrium solubility of Ritonavir (A), and Darunavir from co-amorphous materials (B). Horizontal lines represent the predicted
concentration limits after applying Co-former Reduction Factor (CFRF) in USP Phosphate buffer (pH 6.8) at 37 ◦ C (n = 3, Error bars represent Standard Deviation).

the maximum achievable supersaturation advantage of a solute from
ideally phase soluble multicomponent amorphous systems decreases as
a function of mole fraction of the solute in that multi-component system
(Alhalaweh et al., 2016; Li and Taylor, 2018). Ritonavir: Darunavir
forms a single phase co-amorphous system as obvious from the solid-
state characterization studies (Section 3.2). Therefore, to confirm that
the offset in the solubility of Darunavir was consistent with its mole
fraction within the co-amorphous material, the theoretical solubility of
drugs from multicomponent co-amorphous systems was calculated using
the following formula,
Sa = S0 .X (6)
Where Sa is the solubility of drug from multicomponent (Co-amor­
phous) system, S0 is the solubility of neat amorphous form, and X is the
mole fraction of the solute in the multicomponent system. The extent of
suppression of maximum achievable supersaturation is dependent on

6
S. Shete et al.
the mixing state, composition, and strength of drug-drug interactions.
Further, it has been demonstrated in the literature that the amorphous
solubility also decreases after significant water/moisture absorption. For
the given pairs, the ideal mixing behavior was assumed based on the
spectroscopic and thermodynamic data which showed a lack of in­
teractions and interaction parameter (□) values close to zero respec­
tively. The calculated solubility values were displayed as horizontal
lines in Fig. 2B, where the decrease in the solubility of Darunavir from
the co-amorphous materials was found consistent with its mole fraction
in the respective co-amorphous materials. The same pattern was
observed for Ritonavir solubility (Fig. 2A). Though the solubility of Ri­
tonavir from co-amorphous materials was found less than its amorphous
counterpart, these solubility values were still well in the supersaturation
region yielding 2–3-fold solubility improvement over crystalline Rito­
navir. With regards to the solubility of Darunavir: Indomethacin co-
amorphous materials, the solubility of both Darunavir and Indometh­
acin were decreased as a function of mole fraction thereof in the
respective co-amorphous materials (Fig. 3A, B). However, the solubility
of Darunavir was found marginally higher than the calculated solubility
for all co-amorphous materials. The increase in the ionic strength of the
solution due to ionized Indomethacin may be attributed to the higher
than the calculated values as we found that the solubility of Darunavir
was increased as a function of ionic strength. Though the change in the
ionic strength due to the ionization of Indomethacin was found to be
marginal (̃ 2 mM), the increase in the solubility of Darunavir in the
presence of Indomethacin was also found to be marginal commensurate
with the small increase in the ionic strength.
There is strong precedence in the literature regarding the compro­
mised maximum achievable supersaturation of drugs in multicompo­
nent amorphous systems. According to equations (1) and (2), the
chemical potential of a drug is reduced by an amount that is directly
related to the mole fraction of each component present in the binary
amorphous materials (Alhalaweh et al., 2016; Li and Taylor, 2018;
Purohit and Taylor, 2015). The co-amorphous materials are considered
glass solutions, where the components are miscible with each other to
form a single-phase system. It was evident from the solid-state charac­
terization that all the co-amorphous materials formed molecularly dis­
solved systems. Therefore, it is expected for the participating
components of the co-amorphous materials that owing to their respec­
tive miscibility in the solid-state, the chemical potential thereof will be
reduced commensurate with the mole fraction of the individual
component within the co-amorphous materials. When such co-
Fig. 3. Predicted and experimental equilibrium solubility of Darunavir (A), and Indomethacin from co-amorphous materials (B). Horizontal lines represent the
predicted concentration limits after applying Co-former Reduction Factor (CFRF) in USP Phosphate buffer (pH 6.8) at 37 ◦ C (n = 3, Error bars represent Stan­
dard Deviation).
7
International Journal of Pharmaceutics 608 (2021) 121119
amorphous materials dissolve in a solvent, the solubility of an individ­
ual component after dissolving in the solvent gets offset in proportion
with the reduced chemical potential in the solid-state. Trasi and Taylor
(2015a, 2015b), investigated the dissolution of multicomponent SDs of
Ritonavir and Lopinavir. The solution concentration in the dissolution
media for both Ritonavir and Lopinavir decreased when compared with
the individual amorphous drugs (Trasi and Taylor, 2015a, 2015b).
Similar observations were recorded for multicomponent SDs of Ritona­
vir, Lopinavir, and Atazanavir, wherein the dissolution advantage of
drugs from multicomponent SD was depressed to 1/3rd of the individual
amorphous drugs (Alhalaweh et al., 2016). Anand et al., (2018), studied
the dissolution and solubility of co-amorphous Ritonavir: Lopinavir and
the maximum achievable supersaturation of drugs from the co-
amorphous materials were found decreased than their pure amor­
phous counterparts (Sai Krishna Anand et al., 2018).
Overall, the solubility compromise of Darunavir in the presence of
Indomethacin was less pronounced than that in the presence of Rito­
navir (Fig. 4). Though both the solutes (i.e., Indomethacin and Ritona­
vir) in their respective co-amorphous materials formed single-phase
amorphous systems, the lesser molecular weight of Indomethacin
translated into the relatively less compromise in the solubility of the
second solute i.e., Darunavir in the co-amorphous materials. To
comprehend the decrease in the supersaturation potential of a drug from
multicomponent systems as compared to their respective single
component amorphous counterpart, we intend to define a factor called
as Co-Former Reduction Factor (CFRF). To put simply, CFRF is a ratio of
Sa (equation (6)) which is the solubility of an amorphous drug in the
multicomponent system to the maximum attainable supersaturation i.e.,
amorphous solubility of a pure amorphous form (S0),
Sa
CFRF = (7)
S0
It is evident from the equation that CFRF will always be in a fraction
for ideally soluble multicomponent systems and dependent upon the
molecular fraction of a drug in the multicomponent amorphous system.
As the molecular weight of the co-former/solute shifts towards the lower
side, the value of CFRF for the other component in the multicomponent
system approaches unity. To get the comparative perspective of the
studied co-amorphous systems, the CFRF for Darunavir: Ritonavir and
Darunavir: Indomethacin systems were calculated and summarized in
Table 4. The CFRF for Darunavir from Darunavir: Indomethacin system
exhibited a higher value than that from Darunavir: Ritonavir co-
S. Shete et al.
Fig. 4. Equilibrium solubility of Darunavir from Darunavir: Ritonavir Vis a Vis Darunavir: Indomethacin co-amorphous materials in USP Phosphate buffer (pH 6.8) at
37 ◦ C (n = 3, Error bars represent Standard Deviation).
Table 4
Co-former reduction factor (CFRF) for co-amorphous materials.
System/Molar ratio 1:1 M 1:2 M 2:1 M
Darunavir: Ritonavir
Darunavir 0.44 0.28 0.63
Ritonavir 0.44 0.56 0.31
Darunavir: Indomethacin
Darunavir 0.61 0.44 0.76
Indomethacin 0.39 0.57 0.25
amorphous materials. This is attributed to the lower Indomethacin
molecular weight. Therefore, to minimize the compromise in the su­
persaturation due to the presence of co-former/or additional solute in
the co-amorphous systems, it would be a wise strategy to implement a
low molecular weight co-former. However, the majority of low molec­
ular weight co-formers like citric acid possess the weak glass-forming
ability (Wu et al., 2018), albeit the compounds like Indomethacin
with relatively low molecular weight and good glass-forming ability
must be screened and kept in the toolbox of formulators (Dengale et al.,
2014; Moura Ramos et al., 2004). The other strategy to avoid the
compromise in the supersaturation potential or even exceed the
maximum achievable solubility is to pair weak acids like amino acids as
a co-former with a weak base or another way round to form co-
amorphous salts. In the co-amorphous salts, the solubility is not
dictated by the mole fraction of individual drugs but instead is a result of
the solubility product of the co-amorphous salt. Oftentimes, the solu­
bility of drugs from the co-amorphous salts is higher than their respec­
tive amorphous solubility. Kasten et al., (2017) demonstrated that the
weak acid Indomethacin forms a co-amorphous salt with Lysine which
demonstrated 38 fold solubility improvement over amorphous Indo­
methacin (Kasten et al., 2017). A similar pattern was demonstrated for
the co-amorphous salts of Naproxen: Arginine (Kasten et al., 2018).
5.4. Dissolution
The dissolution of prepared co-amorphous materials was performed
by the pH shift method. It has been established that SDs and co-
amorphous materials generate supersaturation as a result of the
gradient pH of GIT. This is relevant, for supersaturation drug delivery
systems of weakly basic drugs. Where the drug precipitates in the
supercooled liquid form in response to a change in the pH (Chegireddy
et al., 2020; Hsieh et al., 2012; Indulkar et al., 2015; Sakhare et al.,
2019). Therefore, the pH shift dissolution method was employed to
8
International Journal of Pharmaceutics 608 (2021) 121119
mimic the physiological conditions. The dissolution profiles of Dar­
unavir from corresponding Darunavir: Indomethacin and Darunavir:
Ritonavir co-amorphous materials are represented in Fig. 5. The uniform
trend of higher Darunavir release from Darunavir: Indomethacin than
that from Darunavir: Ritonavir materials was observed through all
molar ratios. The dissolution of Ritonavir and Indomethacin from their
respective co-amorphous materials was also found compromised than
their pure amorphous counterparts. Surprisingly, the extent of decrease
in Darunavir and Ritonavir dissolution from Darunavir: Ritonavir co-
amorphous materials are not consistent with the solubility results
(Figs. 2, 5, and 6). However, the divergence of the extent of Darunavir
and Indomethacin dissolution from their respective solubility trends
from Darunavir: Indomethacin co-amorphous materials is significantly
minimal.
Therefore, these results prompt us to consider the hypothesis that,
unlike solubility, the dissolution trends are not dictated by the mole
fraction of individual drugs within co-amorphous materials. From the
thermodynamic perspective, the drug-rich phase is a metastable state
characterized by the formation of particles of colloidal dimensions.
Whilst the bulk of the solution is regarded as a drug lean phase. This
phenomenon of phase separation is termed as Liquid-Liquid Phase
Separation (LLPS) (Taylor and Zhang, 2016). Trasi and Taylor (2015a,
2015b), demonstrated that the reduction in the amorphous solubility of
a given compound is dependent on the mole fraction of that drug in the
composite drug-rich phase. These observations were derived from
generating the drug-rich phase of model compounds by employing the
solvent quench approach, where a concentrated methanolic solution of
the drug was spiked in a solution containing HPMC as a crystallization
inhibitor (Trasi and Taylor, 2015a, 2015b). However, for Darunavir:
Ritonavir co-amorphous materials, the mere dissolution of the co-
amorphous materials does not lead to the generation of drug-rich
phase and LLPS, though both the compounds are expected to be in
supercooled liquid form due to their high glass-forming ability. Never­
theless, the pH shift provides the kinetic impetus required for the for­
mation of drug-rich phase and LLPS, for weak bases. As both Darunavir
and Ritonavir are weak bases and strong glass formers, they tend to
precipitate in the composite supercooled liquid form after shifting the
pH from acidic to the neutral side. This is thermodynamically possible
due to the phase solubility and ideal mixing of Ritonavir and Darunavir.
To confirm that Ritonavir and Darunavir co-amorphous materials form a
drug-rich phase after pH shift and undergo LLPS, the fluorescence of
environment-sensitive probe Pyrene was monitored at 373 (I1) and 383
nm (I3). An increase in the ratio I3/I1 of pyrene indicates the decrease in
the polarity environment of the probe (Supplementary materials,
S. Shete et al. International Journal of Pharmaceutics 608 (2021) 121119

Fig. 5. Dissolution profiles of Darunavir from Darunavir: Ritonavir and Darunavir: Indomethacin co-amorphous materials. D1R1 and D1I1 (A), D1R2 and D1I2 (B),
D2R1 and D2I1 (C). (Horizontal dotted lines represent the concentration limits of amorphous Darunavir and Darunavir concentration after applying Colloidal
Reduction Factor-CLRF) (n = 3, Error bars represent Standard Deviation).

Fig. S12) as a function of pH. The inflection point corresponds to the

formation of drug rich phase and subsequent LLPS for the respective co-
amorphous material. The asymptote was reached beyond pH 3.5, the
point which is approximately 1 unit past the respective pKa of Darunavir
(2.39) and Ritonavir (2.7), indicating the completion of drug-rich phase
formation. Further to corroborate the drug-rich formation during pH
shift dissolution, we subjected the samples from dissolution experiments
after shifting the pH from acidic to neutral side to UV-extinction and DLS
studies. In UV-extinction studies, the increase in the light scattering at
non-absorbing wavelength as a function of increase in pH, which indi­
cated the formation of drug rich phase and subsequent LLPS in response
to change in pH (Data not shown). Further, DLS studies substantiated the
particle size and count for the resulting suspensions of Darunavir: Ri­
tonavir co-amorphous materials. The results of DLS studies are

Table 5
Fig. 6. Dissolution profiles of Ritonavir from Darunavir: Ritonavir co- Dynamic Light Scattering (DLS) analysis showing the particle size, and particle
amorphous materials. (Horizontal dotted lines represent the concentration count.
limits of Ritonavir concentration after applyingColloidal Reduction Factor- Sample Particle size Count Rate Poly Dispersity Zeta Potential
CLRF) (n = 3, Error bars represent Standard Deviation). (nm) (KCPS) Index (PDI) (mV)

D1R1 122.4 140.0 1.00 − 12.2

D2R1 1314 289.6 0.368 − 13.6
D1R2 575.9 193.3 0.655 − 7.29

9
S. Shete et al. International Journal of Pharmaceutics 608 (2021) 121119

summarized in Table 5. The results confirmed the particle formation of
colloidal dimensions except for Ritonavir: Darunavir 1:2 M ratio which
exhibited particle size 1314 nm. The significantly higher particle count
confirmed the presence of drug-rich phase with moderate stability (zeta
potential between − 7.29 and − 13.60).
Therefore, it is logical to assume that the concentration achieved
during the pH shift dissolution of Darunavir: Ritonavir co-amorphous
phases is dependent upon the mole fraction of the respective drug pre­
sent in the precipitated composite drug-rich phase. The relative amount
of individual drugs within the precipitated composite drug-rich phase
will be dictated by the differential solubilities of the weak bases across
the pH range. The mole fraction of individual components within the
composite drug-rich phase can be calculated by using differential solu­
bility values of the components at various pH. For a multicomponent co-
amorphous system, the compromised supersaturation can be calculated
by the following equation,
S1 = S01 *X1
S2 = S02 *X2
where S01 and S02 are the maximum achievable supersaturation of
components 1 and 2 respectively. X1 and X2 are mole fractions of the
respective component in the composite drug-rich phase. These fractions
represent the compromise in the maximum achievable supersaturation;
therefore they are also regarded as Colloidal Reduction Factor (CLRF).
The CLRF can be calculated by the following formula,
X1 (CLRF) =
(x1 μ1 )pH1.5 − (x1 μ1 )pH6.8 1
X (10)
MW 1 F desirable physicochemical properties. The desirability of a co-former
(x2 μ2 )pH1.5 − (x2 μ2 )pH6.8 1
X2 (CLRF) = X (11)
MW 2 F logically relevant pH range i.e., 2 to 7 then the possibility of a composite
where x1 and μ1 are the concentration and activity coefficient of
component − 1, and x2 and μ2 are the concentration and activity coef­
ficient of component-2 in the multicomponent amorphous system. MW1
and MW2 are molecular weights of respective components. Factor F can
be calculated by the following formula,
[
(x1 μ1 )pH1.5 − (x1 μ1 )pH6.8
] [
(x2 μ2 )pH1.5 − (x2 μ2 )pH6.8
] the solubility gradient for weakly basic compounds, the higher the mole
F= + (12)
MW 1 MW 2
It is evident from Eqs. (10) and (11) that a steeper gradient in the pH-
dependent solubility combined with the lower intrinsic solubility of the
drug at intestinal pH 6.8 will result in a relatively higher amount of drug
rich phase than a drug with less steep pH-dependent solubility and
comparatively higher solubility at pH 6.8. To check this hypothesis, we
calculated the mole fraction of Darunavir and Ritonavir in the composite
drug-rich phase by using their respective solubility values at pH 2.0 and
6.8. The calculations were based on several assumptions. First, it was
assumed that the mixing between two components is ideal. Second, the
drug-rich phase is in the supercooled liquid form. After considering the
mole fractions/CLRF of Darunavir and Ritonavir within the precipitate
drug rich phase, the corresponding theoretical dissolution limits were
predicted by Eqs. (8) and (9), which are represented by horizontal lines
in Figs. 5 and 6. The Root Mean Square Error (RMSE) was calculated
between the experimental dissolution profile and predicted values after
applying CLRF (Table 6). The practical dissolution profile was found
analogous with the predicted values for Darunavir and Ritonavir from
their respective co-amorphous materials, while the RMSE values are
larger for Darunavir and Indomethacin dissolution from co-amorphous
materials. Furthermore, to corroborate the results, we prepared the
composite drug rich phase of Darunavir and Ritonavir by solvent quench
method by adding 1% methanolic solutions exceeding their respective
amorphous solubilities into 50 mM phosphate buffer pH 2.0, then the pH
of this solution was increased up to 6.8, which consequently lead to the
formation of composite supercooled drug rich phase. The solution con­
centrations were determined from sampling the supernatant, whilst the
precipitate was dried and analyzed for establishing the relative amount
of each drug in the drug-rich phase. The results are represented in Fig. 7,
where the free drug concentration of Darunavir and Ritonavir is plotted
as a function of the mole fraction of Darunavir in the drug-rich phase.
The free drug concentration of both the drugs in the solution is dictated
by their respective mole fraction within the drug-rich phase.
(8)
For Darunavir: Indomethacin co-amorphous materials, owing to the
employment of a weak acid i.e. Indomethacin as a co-former, the solu­
(9)
tion phase of dissolved co-amorphous materials will be devoid of any
composite drug-rich phase even after shifting the pH towards the neutral
side. Hence, the offset in the dissolution of Darunavir and Indomethacin
from the co-amorphous materials was found consistent with the
respective mole fractions of solutes within the co-amorphous materials
(Fig. 8).
To achieve the maximum achievable supersaturation and keep the
co-former mediated compromised dissolution at the minimum level, it is
advisable from the formulation perspective to employ a co-former with
must be adjudged on a case-to-case basis. If the drug and co-former,
both possess weak basicity and comparable pKa values in the physio­
drug-rich phase formation and subsequently compromised supersatu­
ration are highly likely after pH shift. Contrary, if the drug: co-former
pair is acid-base then the solution after pH shift will be devoid of com­
posite drug-rich phase, enabling the achievement of maximum possible
supersaturation. Furthermore, the solubility gradient across the physi­
ologically relevant pH range is also an important determinant. Steeper
fraction of drug in the resultant composite drug rich phase leading to
comparatively less compromised dissolution. Therefore, to keep the
lowering of the principal drug’s supersaturation at a minimum level for
weakly basic drug-co-former co-amorphous materials, it is preferable to
use a co-former with a less steep pH-dependent solubility than the
principal drug.

Table 6
Root mean squared error (RMSE) values for dissolution profile.
Materials/Molar RMSE (μg/mL)
ratio
Darunavir Darunavir Ritonavir Indomethacin
(DR) (DI)
Fig. 7. Concentration of Darunavir and Ritonavir in the supernatant as a
1:1M 10.19 51.81 2.00 39.46 function of mole fraction of Darunavir in the drug rich phase. Dotted and solid
1:2 M 12.94 12.62 4.23 127.37
lines represent predicted concentration of respective drug by employing Eqs.
2:1 M 9.72 34.27 4.73 13.69
(8) and (9).

10
S. Shete et al.
Fig. 8. Dissolution profiles of Indomethacin from Darunavir: Indomethacin co-
amorphous materials. (Horizontal dotted lines represent the concentration
limits of Indomethacin concentration after applying Co-former Reduction
Factor-CFRF). (n = 3, Error bars represent Standard Deviation).
Fig. 9. Ex-vivo Permeability/Flux of Darunavir (A), Ritonavir (B), and Indomethacin (C). (n = 3, Error bars represent Standard Deviation).
11
International Journal of Pharmaceutics 608 (2021) 121119
5.5. Ex-vivo permeability studies
Permeability is an important determining factor of bioavailability.
The permeability of co-amorphous materials was determined by the ex-
vivo everted gut sack method (Mariappan and Singh, 2003). The
permeability of Darunavir from co-amorphous materials was found
reduced as compared to individual amorphous Darunavir. The perme­
ability of Darunavir was lowered to a greater extent from Darunavir:
Ritonavir materials than Darunavir: Indomethacin counterparts
(Fig. 9A). Similarly, the permeability of Ritonavir from their respective
co-amorphous materials was also found compromised (Fig. 9B). This
compromise in the permeability was found consistent with the free drug
concentration achieved during dissolution experiments, which in turn is
dependent on the mole fraction of the respective drug achieved within
the drug-rich phase. However, the permeability of Ritonavir from D1R1
co-amorphous material is an exception to this trend, which can be
attributed to the permeability driven by nanoparticles than the free drug
concentration. The permeability of Indomethacin from co-amorphous
phases was not found depressed significantly from amorphous Indo­
methacin (Fig. 9C). This can be attributed to the limited permeability of
Indomethacin due to the predominance of ionized species thereof in the
ileum milieu (pH 6.8).
It has long been established that permeability is dictated by the de­
gree of supersaturation, and maximum flux is directly proportional to
S. Shete et al.
the amount of free drug available for absorption (Indulkar et al., 2016;
Miller et al., 2012; Raina et al., 2014). Trasi and Taylor (2015a, 2015b),
reported that the thermodynamic activity of a solute in the multi-
component system is reduced as compared to the activity of individual
amorphous solutes leading to reduced permeability. The study reported
that the flux of Ritonavir and Lopinavir in the multi-component solu­
tion-phase decreased when compared with the flux of individual
amorphous drugs (Trasi and Taylor, 2015a, 2015b). Alhalaweh et al,
(2016), devised an equation to predict the permeability of multicom­
ponent systems, where the reduced maximum permeability for multi­
drug formulation can be predicted from the following equation,
J1 = J01 *X (13)
where J1 is the permeability at the amorphous solubility of component 1
in the bulk aqueous solution in the presence of drug rich phase con­
taining a second solute. J01 is the permeability at the amorphous solu­
bility of component 1 alone and X is the mole fraction of component 1 in
the drug-rich phase. By using the Eq. (13), the authors predicted the
compromised flux of Ritonavir: Atazanavir solid dispersion with respect
to their respective single drug amorphous dispersion and found the
predictions consistent with the experimental results obtained by Caco-2
cell permeability studies (Alhalaweh et al., 2016). However, there are
certain exceptions in the literature where the permeability was found
dependent on the particle size achieved during LLPS than the free drug
concentration. Anand et al., (2018), reported that the permeability of
Ritonavir from Ritonavir: Lopinavir co-amorphous materials exceeded
that of the single component amorphous Ritonavir. The anomalous
finding was attributed to the generation of particles of nano-dimension
during dissolution (Sai Krishna Anand et al., 2018). Therefore, it is
adventitious to choose a co-former that can have a limited effect on the
supersaturation of the principal drug, which could translate into
permeability.
6. Conclusion
Co-amorphous formulations are becoming credible alternatives to
solid dispersion technology. Unlike polymeric solid dispersions, co-
amorphous formulations employ small molecular weight compounds
as co-formers which result in better stability, high drug loading, and
improved performance. Drug-Co-former miscibility/solubility is a pre­
requisite for a stable co-amorphous system formation. However, such
miscibility/solubility has thermodynamic and kinetic consequences on
the performance of the co-amorphous systems. Thermodynamically, the
presence of co-former and miscibility thereof with the principal drug
leads to the lowering of the maximum achievable supersaturation. Such
lowering of the supersaturation was found commensurate with the mole
fraction of the respective component (Drug/Co-former) within the co-
amorphous materials. Kinetically, for co-amorphous phases of weakly
basic drugs, the shift in the pH from acidic to the neutral side leads to the
generation of drug-rich phase and subsequent LLPS. Herein, the free
drug concentration achieved in the bulk of the solution is dependent
upon the mole fraction of the respective component within the drug-rich
phase. The relative mole fraction of each component within the com­
posite drug-rich phase is dictated by pH-dependent solubility and mo­
lecular weight of the individual components. Hence, it is advisable to use
a co-former with lower molecular weight and less steep pH-dependent
solubility to keep the compromise in the supersaturation at the mini­
mum level. Further, employing weakly basic drug and weakly acidic co-
former helps in generating the drug-rich phase, which is devoid of co-
former fraction, consecutively leading to the relatively higher degree
of supersaturation and permeability than weak base-weak base
combination.
12
International Journal of Pharmaceutics 608 (2021) 121119
CRediT authorship contribution statement
Sushant Shete: Formal analysis, Funding acquisition, Investigation,
Methodology. Sai Charan Reddy: Formal analysis, Funding acquisition,
Investigation, Methodology. Yarlagadda Dani Lakshman: Resources,
Data curation, Software, Supervision, Validation, Writing-Review and
editing. Sai Krishna Anand Vullendula: Resources, Data curation,
Supervision, validation, Writing-Review and editing. Chetan Hasmukh
Mehta: Software. Usha Yogendra Nayak: Software, Supervision.
Swapnil Dengale: Conceptualization, Supervision, Project administra­
tion, Writing-original draft, Writing-Review and editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors would want to thank Manipal College of Pharmaceutical
Sciences (MCOPS), Manipal Academy of Higher Education (MAHE) for
providing the research infrastructure to execute the project. Further, the
kind help of Dr. Lalit Kumar, Assistant Professor-selection grade, MCOPS
in recording AT-FTIR spectra is also acknowledged.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2021.121119.
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