Pharmaceutical cocrystals of niclosamide

Article abstract of DOI:10.1021/cg300784v

Niclosamide (NCL) is an anthelmintic BCS class II drug of low solubility and high permeability. Pharmaceutical cocrystals of NCL were prepared with GRAS molecules, such as caffeine (CAF), urea (URE), p-aminobenzoic acid (PABA), theophylline (THPH), nicoti

Full text of DOI:10.1021/cg300784v

Article
pubs.acs.org/crystal
Pharmaceutical Cocrystals of Niclosamide
Palash Sanphui, S. Sudalai Kumar, and Ashwini Nangia*
School of Chemistry, University of Hyderabad, Prof. C. R. Rao Road, Central University P.O., Gachibowli, Hyderabad 500 046, India
S
* Supporting Information
ABSTRACT: Niclosamide (NCL) is an anthelmintic BCS class II drug of low
solubility and high permeability. Pharmaceutical cocrystals of NCL were prepared
with GRAS molecules, such as caffeine (CAF), urea (URE), p-aminobenzoic acid
(PABA), theophylline (THPH), nicotinamide (NCT), and isonicotinamide
(INA), to improve drug solubility. Neat grinding, wet granulation, and slow
evaporation methods were successful to make niclosamide cocrystals. All new
crystalline forms were characterized by X-ray diffraction, differential scanning
calorimetry, and IR-Raman spectroscopy to confirm their purity and
homogeneity. X-ray crystal structures provided details of hydrogen bonding,
molecular packing, and drug···coformer interactions. The intermolecular O−
H···O hydrogen bond from the hydroxyl donor to the carbonyl acceptor in the
niclosamide crystal structure was replaced by an acceptor atom of the coformer in
cocrystal structures. Cocrystals with nicotinamide and isonicotinamide were
characterized by 13C ss-NMR spectroscopy because their single crystals could not be obtained. All cocrystals, except NCL−
PABA, showed a faster powder dissolution rate than the reference active pharmaceutical ingredient (API). Niclosamide−
theophylline acetonitrile solvate showed the highest solubility (6 times compared to the API) among all the crystalline forms.
NCL−THPH cocrystals showed comparably good dissolution (5 times faster than the drug) up to 90 min. The solubility
advantage of the cocrystal was diminished by transformation to insoluble niclosamide monohydrate within 1 h of the dissolution
experiment in 40% i-PrOH−water. Equilibrium solubility experiments showed that all cocrystals as well as the pure API
transformed to NCL monohydrate within 24 h in 40% i-PrOH−water slurry medium. Among the cocrystals studied, NCL−NCT
and NCL−INA exhibited better stability under accelerated humidity conditions (75% RH, 40 °C), but they did not have the
same solubility advantage as the fast dissolving species NCL−THPH.
drug cocrystal will often dissolve faster than the API. Cocrystals
can offer the dual advantage of high solubility and good stability
of the API in the same solid phase.3a,c,i,j The analysis of
dissolution kinetics for a series of cocrystals with the reference
API will provide a better understanding on the role of hydrogen
bonding and coformer nature on drug dissolution and
solubility.3a−d,k
INTRODUCTION
■
Approximately 40% of drugs in the marketplace and 70% of
new chemical entities in the discovery pipeline are poorly
water-soluble. An improvement in the solubility of oral drugs
can enhance their bioavailability and therapeutic potential.
Identifying the optimum solid form of an active pharmaceutical
ingredient (API) is a challenge in clinical development, a stage
in the drug discovery chain that has assumed an even greater
significance in the current decade of fewer new chemical
entities being launched.1 Most APIs are crystalline solids at
room temperature and are commonly delivered as a solid oral
dosage form (such tablet, capsule, etc.). It is well established
that different solid forms (e.g., polymorphs, hydrates, solvates,
amorphous, salts, and cocrystals) of the same compound have
different physicochemical properties such as solubility,
bioavailability, stability, tableting, etc.2 Cocrystals consist of
two or more solid components (at ambient conditions) in a
definite stoichiometric ratio held together by noncovalent
interactions, for example, hydrogen bonds. A pharmaceutical
cocrystal3 is a multicomponent solid form of an API with a
GRAS (Generally Regarded As Safe)4 partner molecule. Since
pharmaceutical cocrystals are molecular complexes held
together by hydrogen bonds, the API is not modified covalently
and hence the active drug species at the site of biological action
is the same. Second, if the coformer has high solubility then the
Niclosamide5 (chemical name 2,5-dichloro-4-nitrosalicylani-
lide, Figure 1, NCL hereafter) is an anthelmintic drug used for
the treatment of worm infestations in humans and animals. It
inhibits the replication of several acute respiratory syndrome
coronavirus.6 NCL is available in two dosage forms, tablets and
suspensions. It is a BCS Class II drug marketed as Niclocide
tablet (500 mg dose, dose number Do = 200). Niclosamide is
among the essential orally administered drugs with inclusive
data.7 It is practically insoluble in water, sparingly soluble in
organic solvents such as diethyl ether, THF, ethyl acetate,
dioxane, etc. and is usually formulated as a suspension. The
liquid dosage form contains an appropriate quantity of the drug
mixed in a small volume. Both niclosamide anhydrate and
niclosamide monohydrate are available for drug formulation.8
Received: June 9, 2012
Revised: July 18, 2012
Published: July 24, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structure of niclosamide (NCL) and coformers caffeine (CAF), urea (URE), p-aminobenzoic acid (PABA), theophylline
(THPH), nicotinamide (NCT), and isonicotinamide (INA). All niclosamide cocrystals are of 1:1 stoichiometry.
However, given the high affinity of niclosamide anhydrate for
water, various suspension formulations transform to cement-
like sediments of the hydrate during storage.9 Two mono-
hydrates, an anhydrate, and a few solvates of niclosamide are
reported in the literature.5b Photolysis of niclosamide in pH 5
buffer is 4.3 times faster than pH 9 buffer and 1.5 times faster
than in pH 7 buffer medium.10 Niclosamide has a high
tendency to make solvates, similar to the drug axitinib.11 The
aqueous solubility of niclosamide is 13−15 mg/L and the
solubility order is anhydrous ≫ solvates > hydrates.5c
of the API is reported. Solvent-free crystallization by melting
and sublimation15 afforded a guest-free structure of niclosa-
mide. Niclosamide cocrystals (1:1) were obtained from dry
EtOAc and CH3CN solvents after grinding the components in
mortar-pestle. Dry solvents were necessary in cocrystallization;
otherwise hydrate formation was a side product. Wet
granulation16 enhances reaction kinetics to give improved
yields of cocrystals. The formation of new solid-state forms was
established by FT-IR, melting point, and PXRD. Crystallo-
graphic parameters for niclosamide and its novel cocrystals are
summarized in Table 1, and ORTEP diagrams are displayed in
Figure S1, Supporting Information.
The pharmaceuticals cocrystals approach was attempted to
modify the solubility of Niclosamide and prevent trans-
formation to hydrate, because the latter product has even
lower solubility (0.45−0.55 mg/L). Niclosamide hydration is
similar to caffeine and nitrofurantoin hydrate transformation.12
Anhydrous white niclosamide transforms to greenish mono-
hydrate within one month of storage at ambient conditions.
Hydrates are generally not preferred for clinical purpose
because of lower solubility and variable drug activity with water
composition in the hydrate. NCL−phosphate and NCL−
ethanolamine salts of higher solubility are reported in recent
patents.13 However, hydration is a common byproduct of salts
and in this respect cocrystals are preferred even though they
provide only modest solubility advantage (4−20 times)
compared to salts (500−1000 times).14 Moreover, the neutral
functional groups in niclosamide molecule (amide, phenol)
preclude salt formation and pharmaceutical cocrystallization is a
promising alternative to improve solubility and dissolution rate.
There are no niclosamide cocrystals reported in the literature to
improve API pharmacokinetics. Pharmaceutical cocrystals of
niclosamide with GRAS molecules (caffeine, urea, p-amino-
benzoic acid, theophylline, nicotinamide, and isonicotinamide;
see Figure 1) were prepared by solvent drop grinding and
characterized using IR-Raman spectroscopy, thermal differential
scanning calorimetry (DSC) and thermogravimetric analysis
(TGA), single crystal and powder X-ray diffraction (PXRD),
and solid-state NMR spectroscopy.
CRYSTAL STRUCTURE ANALYSIS
■
Niclosamide (NCL). Niclosamide crystallized in the
monoclinic space group P21/c with one molecule in the
asymmetric unit. The molecule is locked in a near planar
conformation through intramolecular N−H···O hydrogen bond
(1.76 Å, 142°). Intermolecular O−H···O hydrogen bond (1.72
Å, 173°) from hydroxyl to carbonyl group extends in a chain
along the c-axis (Figure 2a). Hydrogen bonds were normalized
using Platon (Table 2). Niclosamide molecules are connected
by Cl···NO2 interaction (3.22 Å) in a dimer motif of graph set
notation17 R2 (12) ring. π−π stacking of layers (at 3.34 and
2
3.39 Å separation) along the b-axis (Figure 2b) completes the
structure description. The crystal structure of guest-free
niclosamide is different from its monohydrates5a where water
molecules are present in the cavity formed by niclosamide
molecules, which stabilize the hydrate structure through two
water to niclosamide O−H···O hydrogen bonds.
Niclosamide−Caffeine (NCL−CAF, 1:1). The crystal
structure of NCL−CAF (1:1) cocrystal was solved in
monoclinic space group P21/c. The intramolecular N−H···O
hydrogen bond (1.76 Å, 143°) of niclosamide persists in the
cocrystal structure. Two niclosamide molecules form C−H···O
2
dimer (2.26 Å, 141°) of R2 (12) ring motif (Figure 3a). There
is a strong O−H···O bond between the components (1.66 Å,
173°) from O−H of niclosamide to the carbonyl acceptor of
caffeine. The molecules are arranged in ABBA fashion along the
a-axis through π−π stacking (3.32 Å) (Figure 3b).
RESULTS AND DISCUSSION
■
Niclosamide has a strong tendency to form solvates when
crystallized from solution. Anhydrous niclosamide, monohy-
drates HA and HB, and THF, tetraethylene glycol, DMF,
DMSO, MeOH solvates of niclosamide were characterized by
PXRD, DSC, and FT-IR. Crystal structures of the mono-
hydrate, tetrahydrofuran and tetraethylene glycol solvates are
known.5a However, no guest-free X-ray single crystal structure
Niclosamide−Urea (NCL−URE, 1:1). The asymmetric unit
in space group P21/c contains one molecule each of
niclosamide and urea. Intramolecular N−H···O hydrogen
bond (1.79 Å, 139°) in niclosamide makes available the OH
donor for the O−H···O hydrogen bond (1.62 Å, 176°) with
urea in an orthogonal orientation (angle between molecular
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Powder X-ray Diffraction. Niclosamide was purchased as
an anhydrous powder from Sigma-Aldrich (Hyderabad, India)
and used without further purification. Storage at ambient
conditions of 30−40 °C and 50−75% relative humidity
(Hyderabad climate) gave a greenish material of niclosamide
monohydrate within one month. The transformation was
monitored by PXRD and color change. Niclosamide hydrate
reverted back to the anhydrous form by heating at 100 °C for
5−6 h. PXRD19 is a diagnostic tool to differentiate between
cocrystals, anhydrate, hydrate, and solvate forms of drugs at a
Figure 2. (a) O−H···O interaction between niclosamide molecules
results in a layered structure along the c-axis in niclosamide. (b)
Niclosamide molecules form π−π stacking interaction between the
layers parallel to the b-axis.
precision of typically Δ2θ >
0.2°. The PXRD pattern of
niclosamide cocrystals (discussed earlier) matched with the
calculated lines from the crystal structure, confirming the purity
and homogeneity of the bulk phases (Figure S2). Niclosamide−
nicotinamide (NCL−NCT, 1:1) and niclosamide−isonicotina-
mide (NCL−INA, 1:1) cocrystals were obtained from dry
CH3CN by solvent-assisted grinding for 15 min. However,
diffraction quality single crystals could not be obtained by
crystallization from different solvents. The cocrystal stoichiom-
etry was ascertained as 1:1 API/coformer based on mixing ratio
planes 83.8°). The urea α-tape motif18 along the c-axis (N−
H···O: 09 Å, 145°; 1.98 Å, 152°) is bonded to amide CO
through the N−H···O hydrogen bond (1.99 Å, 152°). Two
4
niclosamide and two urea molecules form a cyclic R6 (20) ring
motif (Figure 4a). Urea and niclosamide molecules are
arranged in an ABAB fashion such that π−π stacking of
niclosamide (3.34 Å) and the α-network translation of urea
(N···N 4.38 Å) are about roughly matched in distance
separation (Figure 4b), due to slight up−down tilt of urea
molecules in the linear array.
1
and H NMR of the crystal in DMSO-d6. The product solids
were ascertained as new phases by PXRD. There are noticeable
differences in the peak positions of NCL−NCT and NCL−
INA cocrystals compared to the pure API and coformer
diffraction lines (Figure 8). Two types of niclosamide
monohydrates (HA and HB) are known in the literature.5b
PXRD of niclosamide monohydrate HA (no 3D coordinates
reported in the CSD) exhibited characteristic reflections at 2θ
9.47, 11.40, 16.83, 22.43, and 25.54 0.2°. Monohydrate HB
(CSD refcode OBEQER)5a exhibited characteristic lines at 2θ
10.37, 13.07, 17.63, 19.02, and 21.0 0.2°.
Thermal Stability of Niclosamide Cocrystals. The
thermal stability and phase transition of drugs are important
to study physicochemical and pharmacokinetic properties. DSC
indicated that NCL−THPH and NCL−NCT showed a second
endotherm before melting, perhaps due to phase transition or
decomposition. Evolution of CH3CN from NCL−THPHS
solvate occurred at 82 °C and the second endotherm for
melting point of the desolvated cocrystal was observed at 209
°C. DSC of niclosamide and its remaining cocrystals exhibited
single endotherm peaks confirming thermal stability of the new
solid phases (Figure 9). Melting points are listed in Table 3.
TGA supports the exact equimolar stoichiometry of niclosa-
mide−theophylline CH3CN solvate (calc. 7.32%, obsd. 7.48%,
Figure S3). The single endotherm in NCL−CAF, NCL−URE,
and NCL−INA cocrystals suggested no decomposition prior to
melting. The melting point of cocrystals is in between that of
the API and the coformer.
IR and Raman Spectroscopy. Infrared and Raman
spectroscopy20 are quantitative tools for the characterization
and identification of different solid-state forms. These spectra
are based on the vibrational modes of a compound and are
extremely sensitive to the structure, hydrogen bonding,
molecular conformations, and environment of the API. In
general phenolic OH absorbs strongly in the stretching region
of 3700−3584 cm−1. According to Moffat et al.,20d principal
peaks of niclosamide analyzed in KBr disk appear at 1572 (N−
H bend), 1515 (NO2 asymmetric), 1613 (CC), 1650 (C
O), and 1218 (C−O) cm−1. In the IR spectra (KBr disk) of
niclosamide anhydrate and cocrystals, some of the bands were
shifted relative to that in the pure API and coformer (Table 4).
A change in both carbonyl and amide IR stretching frequencies
indicated the formation of a new cocrystal phase. OH stretching
Niclosamide−PABA (NCL−PABA, 1:1). NCL−PABA
(1:1) cocrystal (P1) contains a COOH dimer of PABA in
̅
2
R2 (8) ring motif (H···O: 1.63 Å, 179°) hydrogen bonded to
niclosamide through NH···O2N and CH···O2N hydrogen
bonds (2.19 Å, 154°; 2.58 Å, 154°) along the c-axis (Figure
5a). Two niclosamide and two PABA molecules form a
4
tetramer of R6 (16) ring motif through both N−H···O and O−
H···N hydrogen bonds (2.02 Å, 157°; 1.82 Å, 173°) parallel to
the (200) plane (Figure 5b). Niclosamide molecules connect to
the next layer by π−π stacking (3.38 Å).
Niclosamide−Theophylline (NCL−THPH, 1:1). NCL−
THPH (1:1) was crystallized from i-PrOAc and the crystal
structure was solved in the monoclinic space group P21/c. The
API and theophylline form intermolecular hydrogen bond
(H···O: 1.69 Å, 179°) from the hydroxyl donor of the drug to
the carbonyl acceptor of theophylline. Theophylline molecules
2
form a centrosymmetric carboxamide dimer of R2 (10) ring
through the N−H···O bond (1.84 Å, 168°) (Figure 6a). There
is a weak C−H···O interaction (2.24 Å, 142°) between
inversion-related niclosamide molecules. These tetramer units
are connected to the next layer by an auxiliary C−H···O
interaction (2.56 Å, 144°) from theophylline aromatic ring C−
H to O2N acceptor of niclosamide along the c-axis in a sheet
structure (Figure 6b). Two niclosamide and two theophylline
molecules are arranged in an ABBA fashion.
Niclosamide−Theophylline Acetonitrile Solvate
(NCL−THPHS−CH3CN, 1:1:1). Niclosamide−theophylline
CH3CN solvate (abbreviated NCL−THPHS) was crystallized
from EtOAc−CH3CN mixture (1:1) and its crystal structures
solved in triclinic space group P1. An intermolecular O−H···O
̅
hydrogen bond (1.66 Å, 174°) between the API and
theophylline connects the molecules. Two theophylline
molecules form centrosymmetric carboxamide dimer through
2
the N−H···O hydrogen bond (1.75 Å, 175°) of R2 (10) ring
motif (Figure 7a). Acetonitrile solvent molecules are present in
channels down the c-axis (Figure 7b), similar to niclosamide
THF solvate.5a A characteristic feature of the crystal is
desolvation of acetonitrile from the crystal lattice within 1 h
and loss of crystallinity at ∼80 °C (discussed later).
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Table 2. Hydrogen Bonds in Crystal Structures (Neutron-Normalized Distance)
interaction
d(H···A) (Å)
d(D···A) (Å)
∠D−H···A (deg)
symmetry code
intramolecular
intramolecular
NCL
N1−H1···Cl1
N1−H1···O4
O4−H2···O3
C3−H3···O2
C5−H5···Cl2
C6−H6···O3
N1−H1···Cl1
N1−H1···O4
O4−H2···O5
C5−H5···O3
2.36
1.76
1.72
2.50
2.70
2.23
2.42
1.76
1.66
2.27
2.21
2.26
2.24
2.32
2.79
2.39
2.45
1.80
1.62
3.07
2.09
2.58
2.56
1.99
1.98
2.69
2.12
2.24
2.63
2.42
1.82
1.82
2.03
2.19
2.14
2.39
2.35
1.85
1.69
1.84
2.24
2.50
2.11
2.51
2.27
2.46
1.65
1.74
2.47
1.79
2.24
2.35
2.14
2.37
2.71
2.47
2.902(2)
2.630(2)
2.693(2)
3.363(2)
3.777(2)
2.896(2)
2.918(2)
2.640(3)
2.640(3)
3.183(3)
2.876(3)
3.304(3)
2.732(4)
2.779(4)
3.275(2)
3.387(4)
2.959(1)
2.644(1)
2.600(1)
3.630(1)
2.974(1)
3.530(1)
3.279(1)
2.921(1)
2.918(1)
3.751(1)
2.813(1)
3.238(1)
3.365(1)
2.936(2)
2.668(2)
2.803(3)
2.981(3)
3.132(3)
2.827(3)
3.401(3)
2.927(3)
2.654(4)
2.676(4)
2.835(5)
3.165(5)
3.436(5)
2.807(4)
3.246(4)
2.740(4)
2.942(5)
2.629(3)
2.749(3)
2.927(3)
2.650(2)
3.149(3)
3.150(3)
2.811(3)
3.429(3)
3.628(3)
3.542(3)
113
142
172
136
176
117
109
142
176
141
117
161
105
103
107
152
111
139
176
116
145
157
128
152
152
168
119
153
124
111
139
173
157
155
119
156
115
135
179
168
142
144
119
124
104
105
174
174
107
141
140
129
117
163
142
170
x, 1/2 − y, −1/2 + z
1 − x, 1 − y, −z
−x, 1/2 + y, 1/2− z
intramolecular
NCL−CAF (1:1)
intramolecular
intramolecular
1 − x, 1 − y, 1 − z
−x, 2 − y, −z
intramolecular
C6−H6···O3
C11−H11··O9
C14−H14B··O9
C16−H16A··O5
C16−H16A··Cl1
C20−H20B··O1
N1−H1···Cl1
N1−H1···O4
O4−H2···O5
N3−H3A···Cl2
N3−H3A···O5
N3−H3B···Cl2
N3−H3B···O2
N4−H4A···O3
N4−H4B···O5
C3−H3···Cl2
C6−H6···O3
C11−H11···O1
C12−H12···O5
N1−H1···Cl1
N1−H1···O4
O4−H2···N3
N3−H3A···O3
N3−H3B···O1
C6−H6···O3
1 − x, −y, −z
intramolecular
intramolecular
1 − x, 1 − y, 1 − z
−x, −3/2 + y, 1/2 − z
intramolecular
NCL−URE (1:1)
intramolecular
1 − x, −y, −z
−1 + x, 1/2 − y, 1/2+z
x, 1/2 − y, 1/2 + z
−1 + x, y, z
−x, 1/2 + y, 1/2 − z
x, y, z
x, 1/2 − y, 1/2 + z
1 − x, −1/2+ y, 1/2 − z
intramolecular
1 + x, y, −1 + z
1 − x, −y, −z
intramolecular
NCL−PABA (1:1)
intramolecular
x, y, z
1 − x, −y, 1 − z
x, −1 + y, 1 + z
intramolecular
C11−H11···O6
N1−H1···Cl1
N1−H1···O4
O4−H2···O6
N5−H5A···O5
C5−H5···O3
x, −1 + y, z
intramolecular
NCL−THPH (1:1)
intramolecular
2 − x, 1 − y, −z
−x, 2 − y, −z
1 − x, 1 − y, −z
x, 3/2 − y, 1/2 + z
intramolecular
C16−H16···O1
C6−H6···O3
C19−H19A···O2
C19−H19B···O5
C20−H20B···N6
O4−H2···O5
N5−H5A···O6
N1−H1···Cl1
N1−H1···O4
C2−H2···O4
C10−H10···O2
C11−H11···O4
C12−H12···N3
C21−H21A···Cl2
C21−H21B···O6
1 − x, 1/2 + y, −1/2 − z
intramolecular
intramolecular
NCL−THPHS (1:1:1)
1 + x, y, z
1 − x, 1 − y, 1 − z
intramolecular
intramolecular
1 − x, 1 − y, 2 − z
2 − x, 1 − y, 1 − z
intramolecular
1 + x, y, z
1 − x, −y, 1 − z
1 − x, 1 − y, 2 − z
frequencies at 3490 and 3577 cm−1 are characteristic of
niclosamide hydrate. Similarly Raman spectra differences in
carbonyl, amide as well as nitro stretching frequencies were
noted (Table 5). Though single crystals were not possible for
NCL−NCT and NCL−INA, changes in IR (Figure S4) and
Raman frequencies clearly suggested new solid phases.
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Figure 3. (a) NCL−CAF (1:1) cocrystal forms O−H···O and C−H···O hydrogen bond in a 1D chain. (b) π −π stacking interaction (3.37 Å)
between the aromatic rings of niclosamide and also with caffeine in two successive layers along the a-axis.
4
Figure 4. (a) Niclosamide and urea molecules form cyclic R6 (20) ring motif between carbonyl oxygen of niclosamide molecule and urea N−H. (b)
Two antiparallel urea tape form a column in which niclosamide molecules are stacked through π−π interactions at 3.34 Å.
Figure 5. (a) NCL−PABA cocrystals interact through NH2···NO2 hydrogen bond along the c-axis. (b) Two niclosamide and two PABA molecules
4
form R6 (16) ring through N−H···O and O−H···N hydrogen bond.
Figure 6. (a) Niclosamide and theophylline molecules arrange as ABBA fashion interacting through O−H···O and N−H···O hydrogen bonds in
NCL−THPH cocrystals. (b) Tetramer motif consists of two niclosamide and two theophylline molecules further interacted through C−H···O
interaction between niclosamide and theophylline along the c-axis completing the overall packing.
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Figure 7. (a) Theophylline carboxamide dimer followed by intermolecular hydrogen bond between niclosamide and theophylline as NCL−THPH
cocrystal. (b) Acetonitrile resides in the channel created by niclosamide and theophylline molecules down the a-axis.
Figure 8. PXRD peaks of NCL−NCT and NCL−INA cocrystal with
that of niclosamide and the coformer confirm a new solid phase.
Solid-State NMR Spectroscopy. Solid-state 13C NMR
spectroscopy21 provides structural information on differences in
hydrogen bonding, molecular conformations, molecular mobi-
lity, and short-range interactions. To confirm the purity as well
1
as stoichiometry of cocrystals, solution H NMR spectra were
recorded in DMSO-d6 for NCL−NCT and NCL−INA and
further characterized by 13C ss-NMR spectroscopy (Figure 10).
Analysis of chemical shifts (in ppm) for NCL−NCT and
NCL−INA cocrystals along with that for the API and coformer
(Table S1) showed that the carbonyl peak of niclosamide is
deshielded from 161.7 to 163.1 ppm and that for nicotinamide
moves upfield from 168.9 to 167.7 ppm (corresponding shifts
in NCL−INA are 161.8, 164.9, and 168.3 ppm), clear
indication for new cocrystals. The absence of an abrupt change
in chemical shift (5−10 ppm) for carbonyl peak and also large
pKa differences rule out salt formation. Both solution and ss-
NMR spectra indicate 1:1 stoichiometry for NCL−NCT and
NCL−INA.
Figure 9. (a, b) DSC endotherms of niclosamide cocrystals. The single
Physical Form Stability. One of the most important
endotherm at a temperature different from the melting points of the
pure components is indicative of a homogeneous cocrystal phase.
characteristics to be determined for any solid drug form is how
stable it is to changes in relative humidity and temperature so
Caffeine (mp 227−228 °C), urea (mp 132−135 °C), PABA (mp
that it can be stored without affecting drug activity.
187−189 °C), theophylline (mp 270−274 °C), nicotinamide (mp
Pharmaceutical cocrystals, for example, indomethacin−saccha-
rin, ibuprofen−nicotinamide, flurbiprofen−nicotinamide,3i,22
are shown to be less hygroscopic than the pure APIs.
Anhydrous niclosamide at 37 °C and 75% RH converted to
monohydrate within 2 weeks.5b In contrast niclosamide
cocrystals were stable at accelerated ICH conditions23 of 40
°C and 75% RH for up to 7 weeks as judged by PXRD. NCL−
128−131 °C), isonicotinamide (mp 157−158 °C).
PABA, NCL−URE, and NCL−CAF cocrystals were stable for
2, 4, and 6 weeks respectively (Figure 11). The reference drug
NCL and NCL−THPHS converted to niclosamide mono-
hydrate within 1 week (Figure S5, Supporting Information).
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dissolution rate to NCL−THPHS for 90 min and the second
highest solubility (181.2 mg/L) in powder dissolution experi-
ment at 2 h. NCL−CAF had intermediate solubility (108.4 mg/
L) between NCL and NCL−THPHS. The remaining
cocrystals, such as NCL−URE, NCL−NCT, and NCL−INA,
showed comparatively lower solubility of 67.3, 55.9, and 62.3
mg/L. Surprisingly the solubility of NCL−PABA (28.2 mg/L)
was lower than that of the API. The intermolecular N−H···O
hydrogen bond distance between the API and PABA is long
(1.82 Å), and we surmise that the weak link may result in faster
dissociation of cocrystal and hydration of the free API thereby
resulting in the observed low solubility of the drug. To
summarize, NCL−THPHS and NCL−THPH are 6.3 and 5.1
times more soluble than niclosamide at 2 h. Recently Smith et
al.24 reported quercetin−caffeine (1:1) and quercetin−caffeine
MeOH solvate (1:1:1) with 14- and 8-fold increase in solubility
of quercetin and 10-fold bioavailability compared to quercetin
dihydrate. Generally solvates are not preferred in API
formulation because of their instability, phase transformation,
and volatile byproduct. Here acetonitrile solvate crystals were
not stable at ambient conditions and moreover CH3CN is a
class II solvent25 with permissible concentration limit of 410
ppm. In comparison, NCL−THPH cocrystal appears to be the
best choice in terms of good stability and improved solubility.
Most drugs exert their therapeutic effect within 4−6−8 h of
oral administration. High coformer solubility confers improved
solubility to the drug cocrystal.3a,c,26 NCT−URE seems to be
an exception since the high solubility of urea (1.1 g/mL) did
not reflect in the cocrystal (only 2-fold higher). Again
theophylline (solubility 8 mg/mL) is the least soluble cofomer
but NCL−THPH cocrystal exhibited the second highest
solubility. The melting points of cocrystals are lower than
that of niclosamide, and there was no simple relation between
melting point and solubility. NCL−PABA cocrystal of lowest
melting point (186 °C) is the least soluble and NCL−INA has
highest melting point (228 °C) and two times more soluble.
Solubility is a thermodynamic quantity and usually taken as
the concentration of the solute at 24 or 48 h after mixing in a
solvent. The miniaturized shake-flask method27 was used to
determine equilibrium solubility of niclosamide cocrystals. The
amount of drug required in this method is more to ensure that
undissolved solid phase is in equilibrium with the saturated
solution. This method is therefore unsuited for those drug
forms that are metastable and undergo phase transformation
during the slurry conditions of dissolution. Equilibrium
solubility was measured by dissolving the crystalline form
(100 mg) in a minimum amount of 40% i-PrOH−water solvent
Table 3. Melting Point Comparison of Niclosamide
Cocrystals
mp of
mp of API
(°C)
coformer
mp of cocrystals (°C),
(°C)
(DSC, TPeak)
NCL
229−230
NCL−CAF
NCL−URE
NCL−PABA
NCL−THPH
NCL−THPHS
NCL−NCT
NCL−INA
227−228
132−135
187−189
270−274
270−274
128−131
155−158
204−207
209−210
186−188
209−210
83−87, 209−210
215−216
227−229
NCL−THPH was stable for 2 weeks but after that started
transforming to the hydrate. There is possibility of NCL−
THPH hydrate forms after 2−3 weeks in humidity chamber as
judged by DSC and TGA (Figure S6). No hydrate was
observed for NCL−NCT and NCL−INA cocrystals at 7 weeks
in the same conditions (Figure S5). Stability profile of
niclosamide cocrystals are summarized in Table 6. NCL−
NCT and NCL−INA cocrystals (even though no single crystal
XRD is available) are the most stable and did not transform to
monohydrate after 7 weeks.
Solubility of Niclosamide Cocrystals. Fast dissolution
and high solubility, referred to as bioavailability, are necessary
for a solid dosage form to exert its therapeutic effect. Since
different crystal forms have different lattice energies and
enthalpies, it follows that their solubility will be different.
Niclosamide is sparingly soluble in water (13
3 mg/L).
Vantonder et al.5c carried out dissolution experiments of
niclosamide and its monohydrates (HA and HB) in 40% i-
PrOH−water medium. Solubility of niclosamide increased to
43 mg/L in alcohol−water mixture. Powder dissolution
experiments of niclosamide and its cocrystals were carried out
on a U.S. Pharmacopeia (USP) approved dissolution tester in
the same medium (40% i-PrOH−water) by the rotating paddle
method (100 rpm) on powder sieved to 200 μm particle size.
Powder dissolution curves for niclosamide cocrystals are shown
in Figure 12. An increase in solubility can increase the tendency
for phase transformation to hydrate. The cocrystals transformed
to hydrate within 1 h of dissolution experiment as indicated by
the UV−vis absorption peak at 221 nm5d for niclosamide
monohydrate. Among the crystalline forms studied, niclosa-
mide−theophylline acetonitrile solvate (NCL−THPHS) ex-
hibited the highest dissolution rate and solubility (223.1 mg/L
at 2 h). NCL−THPH cocrystal exhibited comparable
Table 4. FT-IR Stretching Modes (νs, cm−1) of Niclosamide Cocrystals
N−H
C−O
NO2 asymm
stretch
NO2 sym
stretch
O−H stretch
N−H stretch
bend
CC stretch
CO stretch
stretch
NCL
3242.7, 3199.5
1570.4
1569.7
1556.5
1545.0
1547.8
1555.9
1552.3
1613.5, 1588.9 1652.7
1217.9
1218.1
1216.4
1225.3
1217.7
1218.7
1219.0
1520.9
1517.3
1508.1
1512.0
1504.5
1509.0
1508.6
1348.4, 1329.7
1348.2, 1328.3
1340.0, 1323.3
1342.9, 1321.1
1344.1, 1320.5
1343.8, 1323.5
1340.3, 1321.4
NCL HYD
NCL−CAF
NCL−URE
NCL−PABA
NCL−THPH
NCL−THPHS
3577.3, 3490.4 3241.5, 3197.9
3256.2
1613.2, 1604.8 1680.1, 1652.9
1605.1, 1581.5 1705.3, 1674.9
3491.6, 3438.5 3352.9, 3249.5
1605.3
1606.6
1601.4
1604.0
1673.9
3461.9
3364.1, 3253.9
3260.4, 3160.6
3262.3, 3126.1
1672.0
1697.5, 1674.7
1698.0, 1673.0,
1639.5
NCL−NCT
NCL−INA
3409.0
3445.2
3305.4, 3235.8,
3168.8
1557.5
1560.7
1607.4, 1582.6 1737.2, 1712.7,
1665.0
1225.0
1220.1
1518.4
1523.0
1347.5, 1322.7
1344.5, 1323.8
3237.3, 3159.6
1609.2, 1583.2 1707.4, 1665.0
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Table 5. FT-Raman Stretching Modes (νs, cm−1) of Niclosamide Cocrystals
C−H stretch
N−H bend
CC stretch
CO stretch
C−O stretch
NO2 asymm stretch
NO2 sym stretch
NCL
3069.5, 3103.8
3070.1, 3102.2
3081.7
1560.9
1562.1
1548.0
1543.3
1547.9
1535.2
1542.2
1587.3
1649.7
1216.5
1509.5
1509.2
1348.1, 1325.8
1348.6, 1327.3
1340.0, 1318.8
1345.2, 1317.7
1340.4, 1322.4
1343.9, 1317.8
1342.5, 1316.4
1321.7, 1347.4
1343.7, 1323.1
NCL HYD
NCL−CAF
NCL−URE
NCL−PABA
NCL−THPH
NCL−THPHS
NCL−NCT
NCL−INA
1588.3
1650.5
1217.7
1600.3, 1578.9
1582.2
1707.8, 1676.9
1675.3
1215.4
3098.2, 3073.0
3075.0
1222.9
1599.0, 1580.8
1583.3
1663.9
1216.7
1508.0
1506.5
1506.2
1519.8
1524.3
3066.8
1726.3, 1674.5
1675.1
1215.9
3126.4, 3067.4
3072.7
1583.4
1218.1
1585.1
1661.2
1219.1
3084.6
1583.2
1665.2
1233.2, 1223.0
toward hydration. All cocrystals exhibited faster dissolution rate
and higher stability toward hydration than the reference drug
except NCL−PABA. The search for new GRAS coformers with
better improvement in solubility and stability continues to be
explored.
EXPERIMENTAL SECTION
■
Niclosamide and other GRAS coformers were purchased from Sigma-
Aldrich (Hyderabad, India) and used directly for experiments. All
other chemicals were of analytical or chromatographic grade. Melting
points were measured on a Fisher-Johns melting point apparatus.
Water filtered through a double deionized purification system
(AquaDM, Bhanu, Hyderabad, India) was used in all experiments.
Single crystals were obtained via slow evaporation of stoichiometric
amounts of starting materials in an appropriate solvent after grinding
in a mortar-pestle. Cocrystals and hydrate/solvate were characterized
by IR, PXRD, DSC, TGA, and single crystal X-ray diffraction (SC-
XRD).
Figure 10. Solid-state 13C NMR spectra of NCL−NCT and NCL−
INA cocrystal. The change in carbonyl peak shift indicates a new solid
phase.
Niclosamide, NCL. Niclosamide crystals were obtained from
sublimation at 190−200 °C. Good quality rectangular plate crystal
appeared after 4−5 h. Single crystals were also obtained by
crystallization from AcOH. mp 229−230 °C.
NCL−CAF (1:1) Cocrystal. 100 mg (0.31 mmol) of niclosamide
and 59.4 mg (0.31 mmol) of caffeine were ground in mortar-pestle for
15 min after adding 5 drops of dry EtOAc, and then kept for
crystallization in 10 mL of EtOAc. Suitable square block crystals
appeared at ambient conditions after 3−4 days. mp 204−206 °C.
NCL−URE (1:1) Cocrystal. 100 mg (0.31 mmol) of niclosamide
and 18.4 mg (0.31 mmol) of urea were ground in mortar-pestle for 15
min after adding 5 drops of dry EtOAc, and then kept for
crystallization in 10 mL of EtOAc. Suitable thick plate crystals were
harvested at ambient conditions after 3−4 days. mp 209−210 °C.
NCL−PABA (1:1) Cocrystal. 100 mg (0.31 mmol) of niclosamide
and 41.9 mg (0.31 mmol) of PABA were ground in mortar-pestle for
15 min after adding 5 drops of dry EtOAc, and then kept for
crystallization in 10 mL of EtOAc. Suitable block crystals were
harvested at ambient conditions after 3−4 days. mp 186−188 °C.
NCL−THPH (1:1) Cocrystal. 100 mg (0.31 mmol) of niclosamide
and 55.1 mg (0.31 mmol) of theophylline were ground in mortar-
pestle for 15 min after adding 5 drops of dry isopropyl acetate, and
then kept for crystallization in 10 mL of isopropyl acetate. Suitable
block crystals were harvested at ambient conditions after 3−4 days. mp
209−210 °C.
NCL−THPHS (1:1:1) Complex. 100 mg (0.31 mmol) of
niclosamide and 55.1 mg (0.31 mmol) of theophylline were ground
in mortar-pestle for 15 min after adding 5 drops of dry acetonitrile,
and then kept for crystallization in 10 mL of EtOAc−acetonitrile (1:1)
mixture. Suitable block crystals were harvested at ambient conditions
after 3−4 days. mp 209−210 °C. Desolvation temperature for
acetonitrile solvate 83−87 °C.
NCL−NCT (1:1) and NCL−INA (1:1) Cocrystal. 100 mg (0.31
mmol) of niclosamide and 37.4 mg (0.31 mmol) of nicotinamide (or
isonicotinamide) were ground in a mortar-pestle for 15 min after
adding 5 drops of dry EtOAc. No single crystals were obtained in
several solvents attempted. Solution 1H NMR (DMSO-d6 solvent) and
(5 mL) and stirred for 24 h at 37 °C. Niclosamide showed
higher solubility (43 mg/L) in alcohol−water mixture, which is
in reality the solubility of niclosamide monohydrate after 24 h
of slurry. The dissolved drug concentration at 2 and 24 h of
dissolution experiment are listed in Table 7. All crystalline
forms converted to niclosamide monohydrate (HA) after 24 h
(as confirmed by IR spectroscopy) and for this reason solubility
of all cocrystals is very close.
CONCLUSION
■
Modification of the physicochemical properties of a crystalline
API by introducing a pharmaceutically acceptable compound is
a current strategy in drug formulation and dosage optimization.
Niclosamide is almost insoluble in water. Pharmaceutical
cocrystals of niclosamide are reported with GRAS molecules
for the first time. Niclosamide is prone to solvate and hydrate
formation and hence there are limitations to the wet-
granulation method for cocrystal preparation. Dry solvents
are required to prepare niclosamide cocrystals by solvent-
assisted grinding. All new crystalline forms were characterized
by spectroscopy, thermal analysis, and X-ray diffraction. The
hydrogen bonds in niclosamide structure are changed by the
presence of coformers containing COOH, CONH, OH, NH
functional groups. NCL−THPHS solvate had the fastest
dissolution rate (6 times), but it was prone to hydrate
formation within 2−3 days under ICH conditions. The
permissible concentration limit of class II solvent acetonitrile
is 410 ppm. In comparison, NCL−THPH showed the second
best dissolution rate (5 times) and moderate stability (2 weeks)
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Figure 11. PXRD stack plots of (a) NCL−CAF, (b) NCL−URE, (c) NCL−PABA, and (d) NCL−THPH kept in accelerated ICH conditions of 40
°C and 75% RH. Peaks at 2θ 7.86, 9.47, 10.37, 11.40, 16.83, 19.02° suggest formation of niclosamide hydrate after 1 week.
Table 6. Stability of Niclosamide Cocrystals under ICH
a
Conditions of 40 °C and 75% RH
1W
2W
3W
4W
5W
6W
7W
NCL
X
X
X
X
X
X
X
NCL−CAF
NCL−URE
NCL−PABA
NCL−THPH
NCL−THPHS
NCL−NCT
NCL−INA
√
√
√
√
X
√
√
X
√
√
X
√
X
√
X
X
X
X
X
X
X
X
X
√
X
X
X
X
X
X
X
X
X
X
X
√
√
√
√
√
√
√
√
√
√
√
√
√
√
a
W = week, X = hydrate formation started, √ = no hydrate formation.
solid-state 13C NMR suggested that cocrystals stoichiometry is 1:1 of
niclosamide and nicotinamide/isonicotinamide. mp NCL−NCT (1:1)
215−216 °C and NCL−INA (1:1) 227−229 °C.
X-ray Crystallography. Single crystal obtained from the
crystallization solvent(s) was mounted on the goniometer of Oxford
Gemini (Oxford Diffraction, Yarnton, Oxford, UK) or Bruker Smart
(Bruker-AXS, Karlsruhe, Germany) equipped with Mo−Kα radiation
(λ = 0.71073 Å) source. Data reduction was performed using
CrysAlisPro 171.33.55 software.28 Crystal structures were solved and
refined using Olex2−1.029 with anisotropic displacement parameters
for non-H atoms. Hydrogen atoms were experimentally located
through the Fourier difference electron density maps in all crystal
structures. All aromatic C−H atoms were geometrically fixed using
HFIX command in SHELX-TL program of Bruker-AXS.30 A check of
Figure 12. Powder dissolution curve for niclosamide cocrystals in 40%
i-PrOH−water. NCL (black), NCL−CAF (green), NCL−URE (red),
NCL−PABA (blue), NCL−THPH (dark yellow), NCL−THPHS
(navy blue), NCL−NCT (magenta), and NCL−INA (yellow).
the final .cif file with PLATON31 did not show any missed symmetry.
X-Seed32 was used to prepare the figures and packing diagrams.
Crystallographic parameters of crystal structures are summarized in
Table 1. Hydrogen bond distances in Table 2 are neutron-normalized
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USA) for known concentration solutions in 40% i-PrOH−water
medium. The mixed solvent system was selected because niclosamide
has higher solubility in this medium. The slope of the plot from the
standard curve gave the molar extinction coefficient (ε) by applying
Beer−Lambert’s law. Equilibrium solubility was determined in 40% i-
PrOH−water medium using the shake-flask method.27 To determine
the equilibrium solubility, 100 mg of each solid material was stirred for
24 h in 5 mL of 40% i-PrOH−water at 37 °C, and the absorbance was
measured at 337 nm. The concentration of the saturated solution was
calculated at 24 h, which is referred to as the equilibrium solubility of
the stable solid form.
For powder dissolution studies of niclosamide and its cocrystals, the
starting solids were sieved in ASTM standard mesh sieves to provide
samples with particle size of approximately 200 μm and then directly
poured into 900 mL of dissolution medium, and the paddle rotation
was fixed at 100 rpm. Dissolution experiments were continued up to 2
h at 37 °C. At regular intervals of 5−10 min, 5 mL of the dissolution
medium was drawn and replaced by an equal volume of fresh medium
to maintain a constant volume. The amount of niclosamide dissolved
was calculated from the plot of dissolved niclosamide (mg/L) vs time
(min).
Table 7. Solubility Profile of Niclosamide Cocrystals
solubility at 37 °C
absorption
(24 h) in 40% i-
solubility at 37 °C in 40%
coefficient (ε,
PrOH−water
i-PrOH−water (mg L−1
)
a
mM−1 cm−1
)
(mg L−1
)
at 2 h of dissolution
NCL
16.95
19.18
42.8
56.7
35.53
NCL−
108.45 (x3.0)
CAF
NCL−
13.35
19.89
13.01
16.02
14.72
15.87
84.3
32.8
60.4
56.2
53.1
71.9
67.34 (x1.9)
28.22 (x0.8)
181.19 (x5.1)
223.09 (x6.3)
55.97 (x1.6)
62.33 (x1.7)
URE
NCL−
PABA
NCL−
THPH
NCL−
THPHS
NCL−
NCT
NCL−
INA
a
Number in parentheses indicates number of times higher solubility of
cocrystal compared to reference niclosamide.
ASSOCIATED CONTENT
* Supporting Information
Additional NMR, IR, PXRD and DSC plots, and crystallo-
graphic .cif files are available free of charge via the Internet at
http://pubs.acs.org.
■
S
to fix the D−H distance to its accurate neutron value in the X-ray
crystal structures (O−H 0.983 Å, N−H 0.82 Å, C−H 1.083 Å).
Crystallographic .cif files (CCDC Nos. 892330−\892335) are available
at www.ccdc.cam.ac.uk/data_request/cif or as part of Supporting
Information.
Powder X-ray Diffraction. Bulk samples were analyzed by PXRD
with a Bruker AXS D8 powder diffractometer (Bruker-AXS, Karlsruhe,
Germany). Experimental conditions: Cu−Kα radiation (λ = 1.54056
Å); 40 kV; 30 mA; scanning interval 5−50° 2θ at a scan rate of 1°/
min; time per step 0.5 s. The experimental PXRD patterns and
calculated X-ray lines from crystal structures were compared to
confirm purity of the bulk phase using Powder Cell.33
Thermal Analysis. DSC and TGA were performed on a Mettler
Toledo DSC 822e module and a Mettler Toledo TGA/SDTA
851emodule, respectively. Samples were placed in open alumina pans
for TGA and in crimped but vented aluminum sample pans for DSC.
A typical sample size is 4−6 mg for DSC and 9−12 mg for TGA. The
temperature range was 30−250 at 2 °C/min for DSC and at 10 °C/
min for TGA. Samples were purged with a stream of dry N2 flow at
150 mL/min for DSC and 50 mL/min for TGA.
Vibrational Spectroscopy. A Thermo-Nicolet 6700 FT-IR
spectrometer (Waltham, MA, USA) with NXR FT-Raman Module
(Nd:YAG laser source, 1064 nm wavelength) was used to record IR
and Raman spectra. IR spectra were recorded on samples dispersed in
KBr pellets. Raman spectra were recorded on samples contained in
standard NMR diameter tubes or on compressed samples contained in
a gold-coated sample holder.
AUTHOR INFORMATION
Corresponding Author
*E-mail: ashwini.nangia@gmail.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank DST (JC Bose fellowship SR/S2/JCB-06/2009) and
CSIR (Pharmaceutical cocrystals 01(2410)/10/EMR-II) for
research funding, and DST (IRPHA) and UGC (PURSE grant)
for providing instrumentation and infrastructure facilities. P.S.
and S.S.K. thank UGC and CSIR for a fellowship.
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