A Study on Solubility Enhancement of Etravirine by Crystal Engineering Method

*Corresponding Author:

Sayani Bhattacharyya

Department of Pharmaceutics, Krupanidhi College of Pharmacy, Chikka Bellandur, Varthur, Bangalore 560035, India

E-mail: [email protected]

Date of Received 22 October 2021
Date of Revision 12 January 2022
Date of Acceptance 11 May 2022
Indian J Pharm Sci 2022;84(3):575-585  

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Etravirine, an antiretroviral agent, used in the treatment of human immunodeficiency virus belongs to biopharmaceutical classification system classification IV. The reported solubility of the drug is 0.0169 mg/ ml. In the present study, an attempt was made to enhance the solubility of etravirine by crystal engineering technique. The cocrystallization method was carried out using 12 different coformers and each coformer was studied in two different stoichiometric ratios. A preliminary screening of all the cocrystals was done by determination of melting point and solubility. A statistical evaluation of all the cocrystals on solubility was carried out at a significance level of p<0.05. The best cocrystals were subjected to drug content, in vitro drug release, solid-state study (fourier transform infrared spectroscopy, differential scanning calorimetry, powder x-ray diffraction) and stability study for 3 mo. Coformer benzoic acid showed a significant improvement in etravirine solubility in the drug:coformer ratio of 1:1 and 1:2. The drug:benzoic acid ratio of 1:2 was found to have more solubility and showed enhanced dissolution compared to pure drug. The in vitro dissolution rate of the drug:benzoic acid ratio of 1:2 was found to be more than 90 % in 60 min. Therefore, it can be concluded that the cocrystallization method with benzoic acid as coformer can be a promising approach for solubility improvement of etravirine.


Etravirine, cocrystal, crystal engineering, solubility enhancement, dissolution

The crucial factors that determine the oral
bioavailability of drugs are solubility and permeability.
Among the biopharmaceutical factors, solubility of
the drug is the key factor for the successful delivery
of drugs, as it determines the systemic exposure in
terms of dissolution particularly when administered
orally. At the time of development and formulation,
the pharmaceutical industries face major challenges
in improving the bioavailability of the drugs having
poor solubility. There are various methods available
to improve the bioavailability of the drugs including
micronization, nanonization, salt formation, micellar
solubilizations and complexation, etc[1]. The modification
of crystal habit of the Active Pharmaceutical Ingredient
(API) either in amorphous form, anhydrous form
or polymorphism suffers from many drawbacks in
preserving the stability of the component, balancing
hygroscopicity and removal of toxic solvents used in the
crystallization process. Therefore, to fine-tune the API
properties like solubility, stability and micromeritics,
the co-crystallization process is found to be beneficial
for the pharmaceutical industry[2]. Cocrystals are
homogeneous crystalline materials comprised of at least two different components incorporated in the
same crystal lattice with a defined stoichiometry[3].
Cocrystal structures are made up of noncovalent
interactions between the API and the coformers which
lead to the formation of supramolecular synthons[4].
The supramolecular assembly not only fine-tunes the
physical properties like solubility, hygroscopicity and
stability of the API in its crystal structure but also
helps to address the flow property, compressibility and

Etravirine is an antiretroviral agent used in the
treatment of Human Immunodeficiency Virus type 1
(HIV-1) infection. It is classified as a Non-Nucleoside
Reverse Transcriptase Inhibitor (NNRTI). It belongs
to Biopharmaceutical Classification System (BCS)
class IV molecule. The problems associated with class IV include low aqueous solubility, poor permeability,
significant food effect which lead to low and variable
bioavailability. The reported solubility of etravirine is
0.0169 mg/ml[5]. Reported literature showed that the
improvement of solubility of etravirine was studied
by solid dispersion and spray drying methods[6,7]. The
present study focuses on the development of cocrystals
of etravirine using different coformers in different
molar ratios and evaluating their effects on melting
point, solubility, and percentage (%) drug release.

Materials and Methods

Etravirine was obtained as a gift sample from Apotex
Pharmachem India Pvt. Ltd. Bangalore Karnataka.
All other conformers and chemicals were of analytical
grade, obtained from SD fine chemicals, Bangalore.

Method of preparation of etravirine cocrystal:

Etravirine cocrystals were prepared using twelve
different coformers. The twelve different coformers
used are mannitol, sodium saccharin, salicylic acid,
tartaric acid, benzoic acid, urea, succinic acid, citric
acid, ascorbic acid, caffeine, oxalic acid and magnesium
stearate. The drug was mixed stoichiometrically with
the coformers in a ratio of 1:1 and 1:2 in a mortar and
pestle for 30 min. Few drops of 20 % w/v ethanol-water
mixture were added during grinding[8]. The mixture was
dried overnight at ambient temperature and stored in
a desiccator[9]. The mixture was passed through sieve
number 60 and stored in a glass vial for further studies.

Determination of melting point:

The melting point of the pure drug and its co crystals
was determined by the capillary method. In this method,
the powder sample was introduced into the capillary
tube by gently tapping. The capillary tube was tied with
a thermometer and was placed in the heated thiele tube
containing liquid paraffin. The temperature was raised
slowly until the transition happened to the liquid state.
The temperature at which the sample started melting
was recorded.

Determination of drug content:

The sample equivalent to 5 mg was dissolved in 25 ml
of methanol. A quantity of 5 mg of drug was dissolved
in 25 ml of methanol in a separate volumetric flask. The
solutions were diluted suitably with methanol and the
absorbance was estimated spectrophotometrically using
methanol as blank at 311 nm. The % drug content was
determined using the following formula. All findings were carried out in triplicates.

% Drug content=Absorbance×100/Standard absorbance

Determination of solubility:

The solubility of etravirine was determined by
dissolving 25 mg equivalent of etravirine cocrystal in a
flask containing 50 ml of distilled water. The solutions
were vortexed for 2 min and placed afterward to a rotary
shaker at 100 rpm for 48 h at room temperature. After
that 0.5 ml slurry was withdrawn from each sample
and filtered through 0.22 μm AST works disposable
syringe filter. After suitable dilution with methanol, the
concentration was measured spectrophotometrically
at 311 nm. The solubility study was performed in
triplicates for each sample to minimize the error[10].

Selection of dissolution media by estimation of
Gibbs free energy:

The best cocrystals of etravirine were taken for
solubility study in different dissolution media like 0.01
M hydrochloric acid solution, 1 % w/v Sodium Lauryl
Sulfate (SLS) solution and phosphate buffer pH 7.4.
Cocrystal equivalent to 100 mg was dispersed in 250 ml
of the selected medium and placed in a rotary shaker for
48 h at room temperature. The solubility of the samples
in different media was determined after suitable
dilution. From the solubility data of the cocrystals in
different media, Gibbs free energy was calculated using
the following equation where RT=Ideal gas constant,
S0=Solubility of coformers in the selected media and
SS=Solubility of etravirine in the selected media[11].

ΔG=-2.303 RT log S0/SS

Drug release study:

The dissolution study was carried out for the best
cocrystals of etravirine in 900 ml of 1 % SLS in United
States Pharmacopeia (USP) paddle-type II apparatus.
An equivalent of 100 mg etravirine was used to study
the release from the cocrystals. The stirring speed
was maintained at 50 rpm and the bath temperature
was kept at 37°±1°. The dissolution was carried out
for 60 min. The sample was withdrawn from time to
time and replaced with a fresh solution of media. The
samples after dilution with methanol were analyzed
spectrophotometrically at 311 nm. The analysis for
each study point was performed in triplicates[7].

Particle size determination:

The particle size determination of the best cocrystals was carried out in Malvern zeta sizer at a detection
angle of 173° at 25°. A specific amount of sample was
dispersed in millipore water (2 mg/ml) and sonicated
for 10 min. The sample was further diluted with water
and scanned to determine the particle size.

Fourier Transform Infrared (FTIR) spectroscopy

The cocrystals of etravirine were taken for interaction
study. The sample was analyzed using FTIR 8400S
device Shimadzu, Japan using the Potassium Bromide
(KBr) pellet technique from a range of 4000 cm-1 to 400
cm-1. The spectra were generated for the pure drug with
the best coformer in both the ratio of 1:1 and 1:2[12].

Differential Scanning Calorimetry (DSC) study:

The thermal analysis of the samples was carried out
using DSC 60 (Shimadzu) for the pure drug and the
best cocrystals in both ratios. Around 5 mg sample was
placed in the aluminum pan under a nitrogen flow rate
of 20 ml/min, with a gradual increase in temperature by
10o per min from 30° to 300°. An empty aluminum pan
was used as a reference standard. The thermograms and
the phase transition behavior were recorded for further

Powder X-Ray Diffraction (PXRD):

PXRD diffraction pattern of the pure drug and the
different ratios of the best cocrystal were studied in
the Bruker D8 diffractometer instrument. A Copper K-alpha 1 (Cu K-α 1) tube was used as the source and
the instrument was set at 40 kV and 30 mA. A scan was
carried out from 5º to 59.98º 2θ at 2θ scan step of 0.03º
at a scan step time of 0.8 s. The diffraction patterns were
recorded for further analysis of the reflection angle and
peak intensity[8].

Stability studies:

The best cocrystals were stored in sealed glass vials and
kept at 40°±2° at a relative humidity of 75 %±5 % for
3 mo. The samples were analyzed for solubility, drug
content and in vitro drug release every 30 d[9].

Results and Discussion

Crystal engineering of poorly soluble drug etravirine
was carried out by screening of different coformers.
The solvent drop grinding method was used to prepare
different cocrystals of etravirine. The melting point of
each formulation was determined to estimate the entropy
of the crystal lattice of the cocrystals. The melting point
of the cocrystals of etravirine with various coformers
in 1:1 and 1:2 ratios is listed in Table 1. The pure drug
showed a melting point of 265°. It was found that all the
coformers in the stoichiometric ratio could bring down
the melting point of the cocrystals. Among them, the
coformer benzoic acid showed the least melting point
of the cocrystal of etravirine in both the stoichiometric
ratios. This lowering of melting point is an indication
of the asymmetric molecular structure of the cocrystals.
The drug content for all the cocrystals was found to be
within the range of 88 % to 91 % as shown in Table 1.

Coformer Code for ratio 1:1 Melting point of cocrystal (1:1) (°) Solubility of cocrystal (1:1) (mg/ml) Drug content (%) Code for ratio 1:2 Melting point of cocrystal (1:2) (°) Solubility of cocrystal (1:2) (mg/ml) Drug content (%)
D-Mannitol ETM1 249±0.05 0.221±0.004 90.01±4.50 ETM2 251±0.02 0.031±0.001 88.23±1.76
Sodium saccharin ETSS1 257±0.03 0.23±0.015 89.21±1.09 ETSS2 283±0.06 0.076±0.001 89.27±2.08
Salicylic acid ETSA1 212±0.05 0.231±0.016 89.53±2.13 ETSA2 187±0.03 0.181±0.011 88.52±3.60
Tartaric acid ETTA1 209±0.02 0.398±0.011 90.45±5.01 ETTA2 191±0.03 0.255±0.020 90.97±2.34
Benzoic acid ETBA1 169±0.03 0.920±0.017 90.61±3.52 ETBA2 176±0.06 1.011±0.013 89.93±2.06
Citric acid ETCA1 177±0.07 0.066±0.005 89.45±2.66 ETCA2 187±0.06 0.034±0.001 88.66±4.27
Urea ETU1 183±0.04 0.022±0.001 89.88±2.90 ETU2 167±0.08 0.018±0.001 89.10±2.54
Succinic acid ETSU1 176±0.05 0.078±0.001 91.03±5.12 ETSU2 184±0.06 0.075±0.004 88.54±2.98
Ascorbic acid ETAA1 197±0.02 0.117±0.016 90.78±1.45 ETAA2 196±0.02 0.123±0.007 88.44±1.38
Caffeine ETC1 182±0.06 0.029±0.015 90.09±3.06 ETC2 213±0.04 0.083±0.002 90.67±2.71
Oxalic acid ETOA1 196±0.06 0.046±0.001 89.11±3.22 ETOA2 190±0.06 0.084±0.002 90.40±2.13
Magnesium stearate ETMS1 249±0.04 0.070±0.008 89.31±4.12 ETMS2 269±0.06 0.019±0.001 89.54±2.04

Table 1: Melting Point and Solubility of Etravirine With Different Coformers

The solubility study of the cocrystals revealed that
the solubility of etravirine was increased with all the
coformers compared to the pure drug. The solubility
data of the different cocrystals were compared using
Dunnett’s test at a significant level p<0.05 using
GraphPad Prism 5 software. It was found that the effect
of different coformers on etravirine solubility was
statistically significant in 1:1 ratio of drug and coformer
except for urea and caffeine as mentioned in Table 2.
The solubility was not found statistically significant for
mannitol, citric acid and urea at a drug and coformer
ratio 1:2. These differences in observation might be
attributed to the mismatch of structural fit between the
drug and coformers[14]. The solubility study revealed
that among the 12 different coformers, benzoic acid
was found to increase the solubility of the drug in
water significantly as represented in fig. 1. The drug
and benzoic acid at a stoichiometric ratio of 1:1 and 1:2
showed a drastic increase in the solubility of the drug.
An approximately 54-folds and 60-folds increase in
aqueous solubility was observed in cocrystals (ETBA1
and ETBA2) respectively. Therefore, the cocrystals
ETBA1 and ETBA2 were taken for further evaluation.

Dunnett’s multiple comparison test Drug:Coformer=1:1 Drug:Coformer=1:2
Mean difference q Significant, p<0.05 Mean difference q Significant, p<0.05
Pure drug vs. mannitol -0.2044 25.1 Yes -0.01528 2.384 No
Pure drug vs. sodium saccharin -0.2119 26.03 Yes -0.05899 9.207 Yes
Pure drug vs. salicylic acid -0.2163 26.56 Yes -0.1651 25.76 Yes
Pure drug vs. tartaric acid -0.382 46.91 Yes -0.2384 37.2 Yes
Pure drug vs. benzoic acid -0.9031 110.9 Yes -0.9873 154.1 Yes
Pure drug vs. citric acid -0.04996 6.136 Yes -0.01689 2.635 No
Pure drug vs. urea -0.005013 0.6157 No -0.00157 0.2455 No
Pure drug vs. succinic acid -0.0621 7.627 Yes -0.05855 9.138 Yes
Pure drug vs. ascorbic acid -0.1001 12.3 Yes -0.1061 16.56 Yes
Pure drug vs. caffeine -0.01223 1.502 No -0.0666 10.39 Yes
Pure drug vs. oxalic acid -0.02904 3.566 Yes -0.06673 10.41 Yes

Table 2: Dunnett’s Test


Fig. 1: Solubility of cocrystals of etravirine with different coformers in different stoichiometric ratios, () 01:01 and () 01:02

To establish the solubility in different dissolution media, a 100 mg dose equivalent of ETBA1 and ETBA2 were
tested for solubility in 0.01 M hydrochloric acid, 1 %
w/v SLS solution and phosphate buffer 7.4. The study
revealed that the solubility of both the cocrystals was
high in 1 % w/v SLS solution and resulted in minimum
Gibbs free energy, presented in Table 3. Therefore the
dissolution study of the cocrystals was carried out in 1
% w/v SLS solution.

Medium Solubility (mg/ml) ETBA1

DG (kJ/mol)
Solubility (mg/ml) ETBA2

DG (kJ/mol)
0.01 M hydrochloric acid 0.396±0.12 -7.81±0.01 0.413±0.14 -7.92±0.04
1 % w/v SLS solution 1.98±0.25 -11.80±0.05 2.478±0.18 -12.35±0.03
Phosphate buffer pH 7.4 0.594±0.11 -8.82±0.02 0.619±0.43 -8.92±0.04

Table 3: Selection Of Dissolution Medium

The release study of etravirine from the selected
cocrystals of benzoic acid was compared with the
equivalent amount of pure drug of etravirine in the
same dissolution condition. The % cumulative drug
release vs. time graph is presented in fig. 2. The pure
drug showed a release of 10 % in 10 min and 41 % over
60 min. But both the cocrystals ETBA1 and ETBA2
showed an improved drug release in 10 min, and finally
and approximately 86 % and 93 % of drug release
for 60 min was observed for ETBA1 and ETBA2
respectively. The cocrystal ETBA2 showed higher
release compared to ETBA1. The high release may be
due to the formation of a weaker crystalline structure
with the stoichiometric proportion of the coformer. The
greater dissolution of the cocrystals over pure etravirine
proved the enhancement of solubility of etravirine in
presence of coformer.


Fig. 2: Comparative release study of pure drug with cocrystals of benzoic acid, ( ) Pure drug; ( ) ETBA1 and ( )

The particle size of the formulation ETBA1 and ETBA2
was found to be 571.2 and 550.6 nm respectively as
shown in fig. 3. The nano-size range of the cocrystal
also confirms the high rate of dissolution.


Fig. 3: Particle size of (A) ETBA1 and (B) ETBA2

The FTIR spectra of pure drug and cocrystals of
benzoic acid in different ratios are shown in fig. 4.
The pure drug showed the characteristic peaks at 2220
cm-1 for the presence of aromatic -C≡N group, at 1305
cm-1 for the presence of C-O stretching, at 1238 cm-1 for the C-N stretching and a C-Br stretching at 684
cm-1[15]. All the characteristic peaks are well preserved
in the formulations ETBA1 and ETBA2. The spectra
revealed neither appearance nor disappearance of any
characteristic peaks of etravirine in the cocrystals.
Therefore, it indicates that there is no incompatibility or interaction between the drug and benzoic acid.
The stretching of -C≡N group in ETBA1 and ETBA2
indicated the formation of H bonding between etravirine
and benzoic acid and the formation of an amorphous


Fig. 4: FTIR spectra of (A) Pure drug and its cocrystals (B) ETBA1 and (C) ETBA2

The DSC thermograms are presented in fig. 5.
Thermograms revealed that the phase transition of the
pure drug occurred in the range of 263.75°. The DSC
thermograms showed a shift of peak to 174° and 178°
for the formulations ETBA1 and ETBA2 respectively.
This shift might be due to the strong non-covalent
interaction between etravirine and benzoic acid.
Therefore, a new crystalline arrangement might have
formed which had altered the physiochemical property
of the drug inside the cocrystal that resulted in reduced
melting point and improved solubility.


Fig. 5: DSC thermograms of (A) Pure drug etravirine and its cocrystals (B) ETBA1 and (C) ETBA2

The peak intensities of pure drug and the cocrystals
of benzoic acid at various diffraction angles are
represented in Table 4 and fig. 6. The diffraction pattern
of the pure drug exhibited peaks of high intensities
and indicated the crystalline form of the drug which
was in confirmation with the DSC results. The PXRD
pattern of the cocrystals also revealed that all the major
peaks of the pure drug were preserved and confirmed
the observations of compatibility study with FTIR[15].
The PXRD pattern of the physical mixture of the
cocrystals showed the appearance of broader peaks
with low intensities compared to the pure drug. The low
intensities of the peaks were attributable to the effect
of coformer in altering the physicochemical properties
of etravirine. The lowest intensities of the peaks in the
formulation ETBA2 indicated a strong transformation
of the crystallinity of the pure drug and that might
have led to the increase in solubility and improved


Fig. 6: PXRD of (A) Pure drug etravirine and its cocrystals (B) ETBA1 and (C) ETBA2

Etravirine (2θ) % Intensities ETBA1 (2θ) % Intensities ETBA2 (2θ) % Intensities
9.35 100 9.3 16.46 9.32 7.99
19.59 51.6 19.65 16.65 19.58 10.05
22.1 9.14 22.07 5.3 22.07 2.52
23.7 42.68 23.89 3.03 23.57 1.43
26.06 24.29 26.61 16.66 26.62 7.06
28.66 19.28 28.62 12.2 28.65 4.64
29.53 12.04 29.54 6.06 29.59 2.55
32.11 8.59 32.96 6.47 32.17 1.82
36.49 17.13 37.4 3.09 36.1 0.83
45.19 4.71 45.09 2.07 45.14 1.15
47.27 3.51 47.58 1.92 48.63 1.09
9.35 100 9.3 16.46 9.32 7.99
19.59 51.6 19.65 16.65 19.58 10.05

Table 4: Peak Intensities of Pure Drug and The Cocrystals of Benzoic Acid

The above characterizations of the cocrystals of
etravirine and its improved solubility can be further
supported by the acid dissociation constant (ΔpKa)
of the drug and the coformer. The Food and Drug
Administration (FDA) guidance for the industry also
says that the difference in pKa between drug and
coformer should be <1, to be classified as cocrystal[17].
In the present study, the ΔpKa of the drug and coformer benzoic acid was found to be less than 1.

The 3 mo stability study of the cocrystals indicated
that there were no major changes in the solubility, drug
content and release of drug as indicated in Table 5. Both
the cocrystals were found to be stable at the storage
condition of 40°±2° at a relative humidity of 75 %.

Solubility (mg/ml) % Drug content % Drug release Solubility (mg/ml) % Drug content % Drug release
30 0.91±0.012 88.0±2.13 85.87±1.10 0.989±0.015 90.02±1.05 92.89±2.11
60 0.90±0.02l 87.67±1.16 85.6±1.15 0.975±0.01 89.95±0.90 92.65±1.25
90 0.89±0.011 87.60±1.01 85.34±1.21 0.97±0.016 89.9±1.12 92.43±1.16

Table 5: Stability Study of ETBA1 and ETBA2

In conclusion, the crystal engineering of etravirine with
coformers can be regarded as an effective method in
improving solubility and dissolution of etravirine. In
the present research, a screening of twelve different
coformers was carried out. Among them, benzoic
acid was found to be the most promising coformer
based on the solubility study. The amount of benzoic
acid in the cocrystals was also kept within the
Generally Recognized As Safe (GRAS) limit[18].
The observation from the compatibility, thermal and
surface characteristics study supported the formation
of cocrystal of etravirine with benzoic acid. The
improvement in solubility and dissolution of etravirine
was attributed to the altered solid-state characteristics of
the drug in the coformer. The significant improvement
in the water solubility resulted in high drug release in
the dissolution medium of 1 % w/v SLS solution. The
results from the stability study indicated the firmness
of the products. Therefore, it can be concluded that the
co-crystallization approach and use of benzoic acid as
a coformer can be suitably employed to improve the solubility of etravirine.


Authors are highly obliged to the management
and principal of Krupanidhi College of Pharmacy,
Bangalore for providing the necessary infrastructure to
conduct the research work.

Conflict of interests:

The authors declared no conflict of interest.


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