Buclizine crystal forms: First Structural Determinations, counter-ion stoichiometry, hydration, and physicochemical properties of pharmaceutical relevance

Article abstract of DOI:10.1016/j.ijpharm.2020.119840

Buclizine (BCZ) is a chiral synthetic piperazine derivative which has antihistaminic, anti-muscarinic and antiemetic properties, and has been reintroduced as an appetite stimulant, especially for pediatric patients. Structural information about this drug, as well as other buclizine crystalline forms (solvates, salts and co-crystals) including the BCZ free-base (BCZ-FB), is non-existent. Here, we present for the first time the crystal structure of the monohydrochloride monohydrate salt of BCZ (BCZHCl·H₂O), and of its anhydrous form, BCZHCl. Interestingly, BCZHCl·H₂O was obtained by recrystallization from the raw material (BCZH₂Cl₂) in ethanol:water solution showing that BCZ anhydrous dihydrochloride salt changes easily to a monohydrochloride monohydrate salt modification, which raise concerns about formulation quality control. BCZHCl·H₂O and BCZHCl crystallize in the orthorhombic space groups (Pna2₁ and Pca2₁) belonging to the mm2 point group and are thus classified as non-centrosymmetric achiral structures (NA). Intuitively, we expect these salts to crystallize in a space group with a center of symmetry, since less than 5% of the known racemic compounds crystallize in the NA type. The crystal structures of BCZH₂Cl₂ and BCZ-FB were not determined, but their existence was verified by other techniques (chloride ion analysis, PXRD, HPLC, FT-IR, DSC, TGA) and by comparison of the obtained results with those found for BCZHCl. Additionally, we have also performed an evaluation of the equilibrium solubility (at six different aqueous media) and the dissolution profile of the BCZHCl salt compared to the raw material and BCZ-FB. Different equilibrium solubility values were found comparing the three forms in acidic and neutral pH ranges and all of them were insoluble at pH > 7.0. Moreover, tablets prepared with BCZH₂Cl₂, BCZHCl or BCZ-FB show significant differences in terms of dissolution profile.

Full text of DOI:10.1016/j.ijpharm.2020.119840

International Journal of Pharmaceutics 589 (2020) 119840
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Buclizine crystal forms: First Structural Determinations, counter-ion
stoichiometry, hydration, and physicochemical properties of pharmaceutical
relevance
a,c
a
a
Monalisa Bitencourt , Olimpia Maria Martins Santos Viana , Andre Luiz Machado Viana ,
a
a,b,⁎
a,b,⁎
Jennifer Tavares Jacon Freitas , Cristiane Cabral de Melo
, Antonio Carlos Doriguetto
a
b
c
Faculty of Pharmaceutical Sciences, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 701 Alfenas, Minas Gerais 37130-001, Brazil
Institute of Chemistry, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 701, Alfenas, Minas Gerais 37130-001, Brazil
Novartis Pharmanalytica AS, Via Serafino Balestra, 31, 6600 Locarno, Switzerland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Buclizine (BCZ) is a chiral synthetic piperazine derivative which has antihistaminic, anti-muscarinic and an-
tiemetic properties, and has been reintroduced as an appetite stimulant, especially for pediatric patients.
Structural information about this drug, as well as other buclizine crystalline forms (solvates, salts and co-crys-
tals) including the BCZ free-base (BCZ-FB), is non-existent. Here, we present for the first time the crystal
Buclizine
Counter-ion stoichiometry
Hydration
Equilibrium solubility
Dissolution profile
Structure determination
Crystal engineering
structure of the monohydrochloride monohydrate salt of BCZ (BCZHCl·H
2
O), and of its anhydrous form, BCZHCl.
Cl ) in ethanol:water
solution showing that BCZ anhydrous dihydrochloride salt changes easily to a monohydrochloride monohydrate
salt modification, which raise concerns about formulation quality control. BCZHCl·H O and BCZHCl crystallize
Interestingly, BCZHCl·H
2
O was obtained by recrystallization from the raw material (BCZH
2
2
2
in the orthorhombic space groups (Pna2
1
and Pca2 ) belonging to the mm2 point group and are thus classified as
1
non-centrosymmetric achiral structures (NA). Intuitively, we expect these salts to crystallize in a space group
with a center of symmetry, since less than 5% of the known racemic compounds crystallize in the NA type. The
crystal structures of BCZH
2
Cl and BCZ-FB were not determined, but their existence was verified by other
2
techniques (chloride ion analysis, PXRD, HPLC, FT-IR, DSC, TGA) and by comparison of the obtained results with
those found for BCZHCl. Additionally, we have also performed an evaluation of the equilibrium solubility (at six
different aqueous media) and the dissolution profile of the BCZHCl salt compared to the raw material and BCZ-
FB. Different equilibrium solubility values were found comparing the three forms in acidic and neutral pH ranges
and all of them were insoluble at pH > 7.0. Moreover, tablets prepared with BCZH
2
2
Cl , BCZHCl or BCZ-FB
show significant differences in terms of dissolution profile.
1
. Introduction
drugs: meclizine, cetirizine, cyclizine and hydroxyzine (Peraman et al.,
2
015). Due its antiemetic action, BCZ is particularly applied for the
Buclizine (BCZ), (R,S)-1-(4-tert-butybenzyl)-4-(4-chlorobenzhydryl)
treatment of migraine (in combination with other analgesics) and in the
prevention of motion sickness (nausea, vomiting), although it has been
used in some countries for the man.agement of allergic disorders
(Mostafa and Al-Badr, 2011; Conklin and Nesbitt, 1958). Nausea and
emesis (or vomiting) are triggered by stimulation of either the che-
moreceptor trigger zone (CTZ) or the emesis center in the central ner-
vous system. BCZ acts by binding and blocking the histamine and
piperazine, is a sedating antihistaminic drug (Kuminek et al., 2012;
Mostafa and Al-Badr, 2011) administered as a syrup and tablets in its
dihydrochloride salt modification (Scheme1) under several brand
names: Aphilan, Bucladin-S, Buclina, Longifene, Posdel, Postadoxine,
Postafen, Postafeno, Softran, Vibazine (Mostafa and Al-Badr, 2011).
Structurally, this compound is very similar to other antihistaminic
Abbreviations: CTZ, Chemoreceptor Trigger Zone; BCS, Biopharmaceutical Classification System; BCZ, Buclizine; BCZ-FB, Buclizine free-base; CSD, Cambridge
Structural Database; FDA, Food and Drug Administration; PXRD, Powder X-ray Diffraction; FT-IR, Fourier Transform Infrared; HPLC, High Performance Liquid
Chromatography, SCXRD, Single-Crystal X-ray Diffraction; DSC, Differential Scanning Calorimetry; TGA, Thermogravimetric Analysis; BCZH
2
Cl , Buclizine
2
Dihydrochloride; BCZHCl, Buclizine Monohydrochloride
⁎
Corresponding authors at: Institute of Chemistry, Federal University of Alfenas, Rua Gabriel Monteiro da Silva, 701 Alfenas, Minas Gerais 37130-001, Brazil.
E-mail addresses: crisbrizoti@gmail.com (C.C. de Melo), doriguetto@unifal-mg.edu.br (A.C. Doriguetto).
https://doi.org/10.1016/j.ijpharm.2020.119840
Received 11 June 2020; Received in revised form 13 August 2020; Accepted 30 August 2020
Available online 02 September 2020
0378-5173/ © 2020 Elsevier B.V. All rights reserved.

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
crystals); a racemic compound (the two enantiomers coexisting in equal
quantities in the same crystal); or a pseudoracemate/solid solution (the
two enantiomers coexisting in an unordered manner in the same crystal
(
Mitchell, 1998). Indeed, before this work, such information was un-
known due to the lack of X-ray structures available on the Cambridge
Structural Database (Allen, 2002). A CSD search revealed no entry for
BCZ dihydrochloride salt and other BCZ crystalline forms (solvates,
salts and co-crystals), including the BCZ free-base.
In this study, we report the X-ray structures of the mono-
hydrochloride monohydrate salt of BCZ (hereafter BCZHCl·H O) and of
2
its anhydrous form (hereafter BCZHCl) for the first time. In contrast to
the BCZHCl, which was obtained through the deprotonation of
BCZH
2
Cl
2
, BCZHCl·H O resulted from the recrystallization of the com-
2
mercial raw material (BCZH
2
Cl ). The mechanism by which this anionic
2
change occurs is unknown, but a chloride ion-selective analysis was
employed to determine the authenticity of the powder raw material and
the corresponding monohydrochloride BCZ salts (hydrate and anhy-
drous forms).
Scheme 1. Molecular Structure of Buclizine Dihydrochloride.
Although it has not been possible to determine the X-ray structures
of BCZH
2
Cl and BCZ free-base (hereafter BCZ-FB), their existence was
2
muscarinic receptors in the emesis center, thereby preventing the ac-
tivation of CTZ and reducing nausea and vomiting (Mostafa and Al-
Badr, 2011; Conklin and Nesbitt, 1958; Watcha, 2002). More recently,
BCZ has been reintroduced as an appetite stimulant for weight gain in
children and adults (Mostafa and Al-Badr, 2011; Babu, 2011). The or-
exigenic effect is not well understood, but it is thought to be due to its
hypoglycemic action.
confirmed by Powder X-ray diffraction (PXRD), Fourier Transform
Infrared
spectroscopy
(FT-IR),
High
Performance
Liquid
Chromatography (HPLC) analysis, Differential Scanning Calorimetry
(DSC) and Thermogravimetric Analysis (TGA) techniques. Additionally,
this study also presents an evaluation of the equilibrium solubility (at
six different aqueous media) and the dissolution profile of BCZHCl
compared to the raw material (BCZH Cl ) and BCZ-FB.
2
2
Even in its saline form (dihydrochloride), as expected in medicine,
BCZ is known as a low solubility drug (Mostafa and Al-Badr, 2011), but
is not specified as belonging to class II (low solubility and high per-
meability) or IV (low solubility and low permeability) in the Bio-
pharmaceutical Classification System (BCS). Independently, BCZ is ex-
pected to show low dissolution rates due to its low solubility, which
often cause limited bioavailability (Yu et al., 2002; Amidon et al, 1995).
Especially if the drug is BCS class II, where the limiting step is the drug
release from the dosage form and the solubility in the gastrointestinal
fluid and not the absorption, its bioavailability would be modulated
only by increasing the aqueous solubility (Yu et al., 2002; Amidon et al,
2. Experimental section
2.1. Materials and reagents
Anhydrous sodium acetate, acetic acid, phosphoric acid, hydro-
chloric acid, trihydrated sodium acetate, methanol, potassium hy-
drogen phosphate, acetonitrile and sodium lauryl sulfate (LSS) from
TM
TM
TM
Merck ; sodium hydroxide from Fluka ; chloroform from Synth
;
TM
TM
ethanol from Dinâmica ; acetone from Sigma-Aldrich . Ultrapure
TM
water from Milli-Q- Merck-Millipore . A commercial raw material
1
995).
The definition of drug solubility is based on the highest dose
sample of BCZH Cl was obtained from D.K. Pharmachem Pvt. Ltd,
2
2
Mumbai, India. Raw material and all solid forms obtained during the
crystal engineering procedures (see section 2.2) were stored at room
temperature (25 °C) protected from light and moisture.
strength of an immediate release product, i.e. with no less than 85% of
the dose dissolved within 30 min, according to the United States Food
and Drug Administration (US-FDA) BCS criteria (FDA, 2017). There-
fore, a drug can be considered highly soluble (BCS class I or III) when
the highest dose strength is soluble in 250 mL or less of aqueous media
over the pH range from 1.0 to 7.5. The volume of 250 mL comes from
typical bioequivalence study protocols that prescribe the administration
of a drug product to fasting human volunteers with a glass of water (Yu
et al., 2002; Amidon et al, 1995).
2.2. Samples
2.2.1. Buclizine dihydrochloride (BCZH Cl )
2
2
The raw material (99.2% +/- 0.8 of BCZH Cl by HPLC analysis)
2
2
-
was used as received in the characterization part (API:Cl ratio analysis,
PXRD, FT-IR TGA and DSC analyses), which confirmed its solid phase
identity (See Discussion), and also in the comparative physicochemical
studies involving the other two BCZ forms (BCZHCl and BCZ-FB).
Interestingly, recrystallization procedures (Beckmann, 2013; Gao et al.,
2017) using raw material as a solute resulted in single crystals of
BCZHCl·H O instead of BCZH Cl (see 2.2.2). Indeed, attempts either to
Since water is the preferential solvent for liquid pharmaceutical
formulations, several methods have been developed to improve the
solubility of poorly water-soluble drugs including the reduction of
particle size, the use of solid dispersions, microemulsions, inclusion
complexes (drug-cyclodextrin complexes), co-solvents and salt formers,
besides others (Savjani et al., 2012). For acid and basic drugs, salt
formation is by far the most appropriate method for increasing solu-
bility and the dissolution rate (Serajuddin, 2007; De Melo et al, 2016;
Cruz-Cabeza, 2012).
2
2
2
obtain BCZH Cl single crystals good enough for a single-crystal X-ray
2
2
diffraction experiment or to determine the crystal structure of
BCZH Cl from powder X-ray diffraction data were unsuccessful.
2
2
BCZ free-base is a dibasic molecule with pKa values of 2.12 and 6.55
so can therefore form both mono- and di-salts by protonation of the
piperazine nitrogens (ACD/ChemSketch, 2018). Furthermore, it con-
tains a chiral center (C7 atom, Scheme 1), which is being marketed as
an equimolar mixture of two different optical isomers or enantiomers.
This, in turn, implies that the BCZ raw material as well as the other
buclizine crystalline forms recrystallized from it may belong to one of
three classes: a conglomerate (a physical mixture of pure enantiomers
2.2.2. Buclizine monohydrochloride monohydrate (BCZHCl·H O).
2
To obtain single crystals of BCZHCl·H O, 3.0 mg of the raw material
2
were dissolved in 50 mL of an ethanol:water (1:1, v:v) solution at 75 °C.
The heated solution was filtered and allowed to reach 25 °C. Then, this
solution was transferred to a flask capped with a parafilm layer punc-
tured with small holes. During the evaporation process, the flask was
maintained at 8 °C without the direct incidence of light. Needle-shaped
crystals of BCZHCl·H O were grown after 21 days by slow evaporation
2
2

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
of the solvent (Beckmann, 2013; Gao et al., 2017). The most suitable
single crystals were removed from the crystallization solution and sto-
rage in coin capsules. To avoid evaporation of the solvent and conse-
quently amorphization of the crystals, they are covered by a thin layer
of vegetable oil. The coin capsules were kept at room temperature
protected from the light. Attempts to obtain sufficient pure poly-
data reduction and multi-scan method absorption correction. The
structures were solved by direct methods and refined by full-matrix
2
least squares on F using the SHELXL-2018/3 software (Sheldrick,
2015). All non-hydrogen atoms were found from the electronic density
map constructed by Fourier synthesis and refined with anisotropic
thermal parameters. All hydrogen atoms (with exception of the water
hydrogen atoms) were placed in idealized positions in a riding model
with fixed C—H (or N—H) bond lengths of 0.98, 0.93, 0.96, 0.97 and
crystalline BCZHCl·H
2
O for the characterization part and the com-
parative physicochemical studies were unsuccessful.
0
.98 Å for the amino, aromatic, methyl, methylene and methine groups,
2
.2.3. Buclizine monohydrochloride (BCZHCl)
respectively. The isotropic thermal parameters of all hydrogens (except
the water hydrogens) depended on the equivalent isotropic thermal
displacements of the atoms bonded to them [Uiso(H) = 1.2Ueq(N-
amino, C-aromatic, C-methylene, C-methine) or 1.5Ueq(C-methyl)]. In
To obtain BCZHCl polycrystalline material, 1.0 g of the raw material
(
BCZH
2
Cl ) was solubilized at room temperature (25 °C) in 500 mL of a
2
methanol:water mixture (1:1, v:v). The solution was sonicated for
-
1
1
0 min followed by neutralization with drops of 1.0 mol L NaOH
the low temperature (150 K) structure of BCZHCl·H O, electron density
2
solution until it became cloudy, which took place at approximately pH
peaks corresponding to positions of the water hydrogen atoms were
identified and assigned at approximately 1 Å from the oxygen atom
(O1). Their positions (distances) nonetheless were restrained using
DFIX (O1-H1/O1-H2 = 0.84(2) Å) and DANG (H1-H2 = 1.34(4) Å)
instructions (Sheldrick, 2015), with Uiso(H1/H2) values = 1.5Ueq
(O1). For the room temperature (293 K) dataset, one of the water hy-
drogens is shared between the water oxygen and the amino nitrogen
(assigned in accordance with residual peaks without distance re-
straints), giving a final model after occupancy refinement of 23%
water/77% buclizine monohydrochloride and 77% hydroxide ion/23%
buclizine dihydrochloride. As previously observed for BCZ analogous
structures (Deb et al, 2015; Mohanty et al., 2015; Song et al, 2012), the
chlorine atom (Cl1) of 1-[(4-chlorophenyl)(phenyl)methyl]piper-
azinediium moiety was disordered over the two-symmetric benzene
carbon atoms (C1 and C11, Scheme 1). In all structures, the chlorine
atom was disordered over the Cl1-C1/Cl1-C11′ positions, with refined
site-occupation factors of 0.835(4)/0.165(4) (BCZHCl), 0.889(6)/
4
.8. As expected, this pH is at the middle of the pH range between the
two calculated BCZ pKa values (pKa1 = 2.12 and pKa2 = 6.55, See
section 3.3). The solution flask was capped with a parafilm layer
(
punctured with small holes) and stored without the direct incidence of
light. It is important to emphasize that the precipitate formed soon after
the BCZH Cl neutralization was amorphous (Figure S1, Supplementary
2
2
material). Crystalline BCZHCl without the presence of an amorphous
halo in the PXRD experiment was obtained by leaving the precipitate to
stand in contact with the stock solution for at least 10 days. In this way,
after 13 days, the precipitate on the flask bottom was separated from
the solution and dried in a desiccator containing silica for 24 h. The
generation of the BCZHCl form was confirmed by PXRD data (See
Discussion) and its chemical purity (99.6% +/- 0.3) attested by HPLC
analysis. The BCZHCl polycrystalline material was stored without the
direct incidence of light for the further analysis. The freshly prepared
BCZHCl polycrystalline material was also used in recrystallization
procedures in order to grow BCZHCl single-crystals that were good
enough for the single crystal X-ray experiment. After several attempts,
single-crystals were successfully obtained by vapor diffusion crystal-
lization method (Beckmann, 2013; Gao et al., 2017) as follow: ap-
proximately 2.0 mg of the BCZHCl form was dissolved in 0.5 mL of
acetone within a small open container (beaker of 10 mL), which was
placed in a larger container (vial of 50 mL) containing 2.5 mL of
chloroform. The outer container was sealed and the vapor chloroform
solution diffusion process into the acetone container was performed at
0
.111(6) (BCZHCl·H
2
O at 150 K) and 0.940(4)/0.060(4) (BCZHCl·H O
2
at 293 K). The occupancy factors were assigned using the free variables
and restraining the sum of them equal to 1.000 (FVAR SHELXL-2018/3
instruction) (Sheldrick, 2015). The Flack parameter for BCZHCl dataset
(
Cu radiation dataset) was reliably refined (Flack, 2003, 1983; Cianci
et al, 2005; Parsons and Flack, 2004) to 0.04(3) ((R)-isomer) using the
BASF/TWIN SHELXL-2018/3 instruction (Sheldrick, 2015). Refinement
of the Flack parameter for BCZHCl·H O datasets (both Mo radiation
2
datasets) at 150 K and 293 K was 0.08(16) and −0.03(10) respectively,
giving an inconclusive indication of the absolute structure, since the
standard uncertainty values are above 0.10. A favorable outcome was
obtained from the Bijvoet analysis reported by Hooft et al. (Hooft et al.,
2
5 °C. Plate-shaped colorless crystals were obtained after 15 days.
2
.2.4. Buclizine free-base (BCZ-FB)
2
008, 2010) and implemented in the PLATON program (Spek, 2009).
To obtain BCZ-FB polycrystalline material, 1.0 g of the raw material
was solubilized at 25 °C in 500 mL of ethanol:water (1:1, v:v) mixture.
However, due to the low Friedel coverage, this analysis was incon-
clusive for the low temperature dataset. The probability that the (R)-
isomer had been correctly assigned was 1.00 for either P2 (probability
assuming two possibilities, i.e. one enantiomer or the other) or P3
(probability assuming three possibilities, including the two possible
enantiomers plus the third option of a racemic twin). The resulting
value of the Hooft parameter y was 0.03(3). Therefore, since BCZHCl
-
1
After sonication for 10 min, 1.0 mol L NaOH solution was trickled into
the vial until pH 5.8 was reached (optimized value to produce BCZ-FB
absent of BCZHCl), which produced a significant amount of a white
colloidal precipitate. The remaining procedures were the same as those
applied to obtain the polycrystalline BCZH
2
2
Cl sample. The generation
of BCZ-FB was confirmed by HPLC (chemical purity of 99.5% +/-
and BCZHCl·H
2
O were solved respectively in the Pna2
1
and Pca2 polar
1
0
.01), PXRD, IR, DSC, and TGA data (See Discussion). Unfortunately,
several attempts using different crystal growth protocols to obtain BCZ-
FB single crystals that were good enough for a single-crystal X-ray
diffraction experiment were unsuccessful. Attempts to determine the
crystal structure of BCZ-FB from powder X-ray diffraction data were
also unsuccessful.
space groups (non-centrosymmetric space group containing mirror
planes), both are racemic crystals, so it obviously makes no sense to
establish the BCZ absolute configuration from the absolute structure.
The MERCURY (version 3.9) software program was used for crystal-
lographic analysis and artwork representations, in which the disorder
was omitted for the sake of clarity, i.e. only the chlorine atom with
highest site-occupation factors -linked to C1- was included. The crystal
data and details of data collection and refinement are shown in Table 1.
2.3. Single crystal X-ray diffraction analysis (SCXRD)
CCDC 1,912,183 (BCZHCl), CCDC 1,912,184 (BCZHCl·H O at 293 K)
2
Suitable BCZHCl·H O and BCZHCl single-crystals were previously
2
selected for the X-ray measurements. The data collections were per-
and CCDC 2,008,948 (BCZHCl·H O at 150 K) contain the supplemen-
2
TM
tary crystallographic data for this paper. The data can be obtained free
of charge from The Cambridge Crystallographic Data Centre (CCDC) via
www.ccdc.cam.ac.uk/getstructures. (Allen, 2002).
formed on an Agilent SuperNova diffractometer, equipped with dual
source of radiation (Cu and Mo) and an Atlas S2 CCD detector at 293
and 150 K (only for BCZHCl·H O crystal). The CrysalisPro software
2
(
Agilent, 2010) was used for data collection, unit cell determination,
3

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Table 1
Crystal data and structure refinement parameters for BCZ salts.
BCZHCl·H
2
O
BCZHCl·H
2
O
BCZHCl
Formula
C
28
H
36Cl
2
N
2
O
C
28
H
36Cl
2
N
2
O
C
28
H
2 2
34Cl N
Formula weight (g mol−1)
Temperature (K)
Wavelength (Å)
Crystal System
487.49
150(2)
0.71073
487.49
293(2)
0.71073
469.47
293(2)
1.54184
Orthorhombic
Orthorhombic
Orthorhombic
Space group
Pna2
1
Pna2
1
Pca2
1
Unit cell dimensions (Å)
a = 25.018(2)
b = 15.6230(11)
c = 6.9000(6)
2697.0(4)
a = 25.156(2)
b = 15.7503(15)
c = 6.9637(6)
2759.2(4)
a = 11.6629(3)
b = 10.5092(3)
c = 21.0777(7)
2583.45(13)
4, 1
3
Volume (Å )
Z, Ź
4, 1
4, 1
−
3
Calc. Density (g cm
)
1.201
1.174
1.207
Absorp. coefficient (mm−1)
F(000)
0.263
0.257
2.379
1040
1040
1000
Data collect. θ range (°)
Index ranges
2.732 to 29.729
−34 ≤ h ≤ 33
3.199 to 29.712
−31 ≤ h ≤ 29
−21 ≤ k ≤ 13
−9 ≤ l ≤ 6
12,637
4.195 to 73.451
14 ≤ h ≤ 14
−13 ≤ k ≤ 8
−25 ≤ l ≤ 26
22,462
−
−
16 ≤ k ≤ 19
7 ≤ l ≤ 9
Reflections collected
20,478
Independent reflections
Completeness to θmax. (%)
Data/restraints/parameters
5957 [R(int) = 0.0714]
89.0
5510 [R(int) = 0.0408]
97.3
5030 [R(int) = 0.0589]
100.0
5957/4/318
5510/1/313
1.068
5030/1/295
1.079
2
Goodness-of-fit on F
1.119
Final R indices [I > 2σ(I)]
R indices (all data)
R1 = 0.0898, wR2 = 0.1997
R1 = 0.1220, wR2 = 0.2199
0.08(16)
R1 = 0.0603, wR2 = 0.1192
R1 = 0.1010, wR2 = 0.1366
−0.02(10)
R1 = 0.0649, wR2 = 0.1830
R1 = 0.0685, wR2 = 0.1871
0.04(3)
Absolute structure parameter
Largest diff. peak and hole (e.Å−3)
0.83 and −0.33
0.22 and −0.15
0.47 and −0.40
2.4. Powder X-ray diffraction analysis (PXRD)
2.8. Residual chloride quantification
The PXRD analyses were performed at room temperature (293 K) on
A mass of 100 mg of BCZ-FB, BCZHCl, BCZHCl·H2O and BCZH
2
Cl
2
TM
a Rigaku
(Japan) Ultima IV X-ray diffractometer using the CuKα
(raw material) was separately weighed, transferred to a 5 mL volu-
metric flask and solubilized in chloroform p.a. (Anidrol, Brazil). Liquid-
liquid extraction was performed by adding 5 mL of water to a 25 mL
falcon centrifuge tube and centrifuging this for 10 min at 3000 g. The
aqueous part was separated and quantified in a spectrophotometer
radiation (λ = 1.5418 Å) generated in a sealed tube energized at a
voltage of 40 kV and current of 30 mA. For the experimental analysis,
samples were placed on the standard sample holder and then scanned
continuously. The data were collected with a 2θ step size of 0.02°, scan
−
1
TM
speed of 1° 2θ min
and angular range from 3° to 35° in 2θ.
(Shimadzu , Japan, model UV-1800), by colorimetric reaction using a
commercial kit (mercury and iron thiocyanate) (Labtest Diagnóstica,
Brazil), according to the manufacturer's instructions. As expected, the
free base did not quantify residual chloride, the BCZHCl and
2
.5. Thermal analysis.
BCZHCl·H
2
O forms showed similar residual chloride values and the
showed twice the residual chloride value compared with
Differential scanning calorimetry (DSC) experiments were obtained
−
1
BCZH Cl
2
2
under a dynamic atmosphere of nitrogen (50 mL min ) with a heat
−
1
TM
these forms, confirming the nominal API:counter-ion ratios (1:0, 1:1 or
flow of 10 °C min , between 30 and 260 °C, on a NEZSTCH DSC,
model DSC Syrius 3500 (Germain). Approximately 3 mg of sample was
placed in an open aluminum crucible. The instrument was calibrated
with an indium standard. Thermogravimetric analysis (TGA) and
Differential thermal analysis (DTA) curves were performed using about
1
:2).
2
.9. Equilibrium solubility of BCZ forms.
Six aqueous media were used to determine the equilibrium solubi-
3
mg of sample in open aluminum crucibles, on Thermobalance SII
−
1
TM
lity of BCZ-FB, BCZHCl and BCZH Cl : ultrapure water, 0.01 mol L
Nano Technology , model TGA/DTA-7300 (Japan). The curves were
2
2
−1 -1
−1
HCl, 0.1 mol L
HCl, pH 4.5 acetate buffer (0.050 mol L ), pH 6.8
obtained under a dynamic atmosphere of nitrogen flow (50 mL min ),
-
1
-1
−
1
(0.072 mol L ) and pH 7.5 phosphate buffers (0.085 mol L ), according
to Resolution Number 031/2010 and Technical Note Number 003/2013
with a heat flow of 10 °C min , between 30 and 500 °C.
(
ANVISA, 2013). Previously sieved amounts (particle size from 200
2
.6. Fourier transform infrared (FT-IR) spectroscopy
TM
mesh (75 μm) to 270 mesh (53 μm) using a BerTel
granulometric
analysis sieves, Brazil) of the three BCZ forms were individually added
in flasks (n = 3) containing 4 mL of each medium and vortexed for
TM
The spectra were recorded on a Shimadzu
spectrophotometer,
model Prestige-21 (Japan), using KBr pellets. The transmittance mode
1
min until the saturation of these solutions. Subsequently, the flasks,
−
1
data were collected at 25 °C: resolution 4 cm , 64 scans, range of
protected from light, were agitated at 150 rpm and at 25 °C, using an
−
1
4
000–400 cm
.
TM
IKA
orbital shaker table, model Ks 501 digital (Switzerland). After
7
2 h of stirring, the samples were filtered through a nylon syringe filter
2
.7. Optical rotation measurements
(0.45 μm, ValuPrep 25 mm, Pall) and diluted properly in a mobile
phase (methanol:water 4:1 v:v, pH 2.6). The amount of the three forms
in each media was determined using a validated stability-indicating
HPLC method (Kuminek et al., 2012), which was partially validated
(selectivity, linearity, accuracy and precision) in this work (Figures S2
and S3 and Tables S1-S4, Supplementary material) according to the
0
.2 g of BCZH
2
Cl (raw material) was solubilized at 25 °C in 500 mL
2
of methanol:water (1:1, v:v) mixture. Optical rotation measurements
TM
were performed on a Quimis automatic polarimeter, model Q-760 M
(
Brazil), confirming that the raw material bulk is a racemic mixture.
4

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Resolution RDC No. 166 of 2017 (ANVISA, 2017). The HPLC mea-
reference product, and Tt is the cumulative percentage dissolved at
each of the selected n time points of the test product. The factor f2
measures the closeness between the two dissolution profiles, and values
between 50 and 100 indicate similarity between two profiles (FDA,
1997).
TM
surements were performed on an Agilent model HP1200 HPLC (USA)
TM
using a Waters
X bridge C18 (150 × 4.6 mm-5 µm) column. The
solubility of BCZ forms was statistically compared, using the ANOVA
test with 95% of confidence. When the p-value is less than 0.05, the
samples are considered different. The pH of the saturated solutions was
measured before and after the stirring period. The remaining solid was
separated from the supernatant and dried in a desiccator containing
silica for 7 days. Thereafter, it was evaluated by PXRD to verify whether
the resulting material was the same as the starting material.
3. Results and Discussion
3.1. Single-Crystal X-ray diffraction Analysis
3
.1.1. Chiral molecules crystal packing types
Optical rotation measurements performed here confirm that raw
2.10. Production of BCZ tablets with different BCZ forms
material is a racemic mixture. Therefore, BCZ can adopt crystal packing
types as follows: a racemic compound with a 1:1 stoichiometry (oc-
currence, 90–95%); a mechanical mixture (conglomerate) of single
crystals containing only homochiral molecules (5–10%); and a solid
solution crystal containing an equal number of enantiomers in a
random distribution (less than 1%) (Deb et al, 2015). Since chiral
molecules cannot be superimposed on an image of themselves created
by the action of a mirror, center of inversion or an improper rotation
axis, crystals containing only one enantiomer must adopt a non-cen-
trosymmetric chiral structure (type NC) associated with point groups 1,
Three batches containing 60 tablets each were prepared to evaluate
whether there is difference in solubility and dissolution between the
three formulations: batch 1 (containing BCZ-FB), batch 2 (containing
BCZHCl) and batch 3 (containing BCZH
2
Cl ). First, the placebos (in-
2
active ingredients) were prepared using the following composition:
microcrystalline cellulose (33.9%), lactose monohydrate (33.9%),
starch (29.8%), povidone K30 (1.4%) and magnesium stearate (1.0%).
Then, the amount of each BCZ form (particle size from 200 mesh to 270
mesh) was based on the proportion of BCZ in commercial oral solid
dosages which contain 25 mg of its dihydrochloride solid modification:
2
, 222, 4, 422, 3, 32, 6, 622, 23 and 432. This rule also applies to
conglomerates. On the other hand, achiral molecules can crystallize as
an NC structure as well as a centrosymmetric achiral structure (type
CA) associated with point group containing inversion center or non-
centrosymmetric achiral structure (type NA) associated with a point
group without an inversion center but containing a mirror and/or im-
proper rotation axis. Finally, for racemic compounds and solid solution
crystals, beyond the NC, there are also CA and NA types (Deb et al,
BCZ-FB (21.5 mg), BCZHCl (23.5 mg) and BCZH
2
Cl (25.0 mg). These
2
ingredients were slowly mixed in an aluminum container for five
minutes. The equivalent of an average weight (320 mg) of each for-
mulation was weighed into a single rotary tablet compression machine
TM
Lemaq model LM08B (Brazil), which was used to produce the tablets
(
n = 60) by direct compression.
All of the prepared formulations contain the same average weight
2
015). Considering only racemic compounds, the frequency of CA, NA
and excipients as Buclina®, the reference medicine of Sanofi
Pharmaceutical industry. Additionally, the proportion of excipients was
based according to the percentage used in the production of solid do-
sage forms (Aulton, 2016).
and NC are ~ 95%, 4.5–5% and ~ 0.02%, respectively (Deb et al,
015).
Here, the BCZHCl·H
solved in the space groups Pca2
are the most predominant space groups of NA structures. The R-isomer,
which is related to the S-isomer by the glide planes of Pca2 and Pna2
represents both BCZHCl and BCZHCl·H O asymmetric units (Fig. 1).
The main crystallographic data for these are summarized in Table 1.
2
2
O and BCZHCl structures were respectively
1
and Pna2 , where Cc and Pc together
1
The compression strength used to produce all tablets was the same
(
4.2 Kgf). The reference value adapted to the strength was 4.0 to 4.5
1
1
,
Kgf. Determination of average weight, friability, assay and uniformity
of the tablets were also determined according to USP 40 (USP, 2017).
2
(
Tables S5 –S9, Supplementary material).
3
.1.2. BCZHCl structure description
2
.11. Dissolution profile of BCZ tablets with different BCZ forms
+
The BCZHCl structure presents one buclizine monocation (BCLH
)
and one chloride anion in the asymmetric unit. The protonation of the
piperazine ring occurs at atom N2, which is the N atom that is most
distant from the electronegative chlorophenyl moiety (Fig. 1a). The
piperazine group (N1-C14-C15-N2-C16-C17) adopts a slightly distorted
The solubility analysis was the starting point to choose the ideal
dissolution medium (pH 4.5 sodium acetate buffer) for the dissolution
profile analysis. The dissolution profile of each formulation was per-
TM
formed in an Dissolutor Sotax model AT 7smart, multi-bath (n = 8)
chair conformation with the puckering parameters Q, θ and φ of
(
apparatus II) (Switzerland), using a previously developed and vali-
ο
0
.598(6) Å, 175.7(5)° and 152(8) . For an ideal chair conformation, the
dated method (Kuminek et al., 2012), with the following conditions:
ο
value of θ must be 0 or 180 . The substituents in BCZHCl (4-chloro-
diphenylmethane and 1-methyl-4-tert-butylbenzene) occupy the equa-
torial positions of the piperazine ring. Selected torsion angles involving
the piperazine ring and the substituents for BCZHCl compared to
9
00 mL of pH 4.5 sodium acetate buffer + 1.0% sodium lauryl sulfate
at 37.0 ± 1.0 °C, apparatus II (paddle) and rotation of 75 rpm. Ali-
quots of 5 mL were collected from each vessel (n = 6), at sampling
times of 5, 10, 15, 20, 30 and 60 min, followed by the immediate re-
placement of identical volumes of dissolution medium. The aliquots
were filtered directly into vials using a 0.45 μm nylon syringe filter
BCZHCl·H O are given in Table S12 (Supplementary material).
2
In the BCZHCl crystal packing (Figure S5, Supplementary material),
the ion-pair is stabilized by a medium charge-assisted H-bond (Fig. 1a)
(
0.45 μm, ValuPrep 25 mm, Pall) . The amount of dissolved buclizine at
-
involving the protonated N2 atom of the drug and the Cl anion
each sampling time was determined by HPLC method chromatography
at a wavelength of 230 nm according to a validated method (Figures S2
and S4 and Tables 10 and 11, Supplementary material).
The similarity of the dissolution profiles was determined by the si-
milarity factor (f2) calculated as follows:
(
N2 − H2a•••Cl2). The ion-pairs are also connected to each other via
two additional non-classical H-bonds (C13
− H13•••Cl2 and
C17 − H17b•••Ct3) that contribute by linking the symmetry-related
ion-pairs (where A and B are related by a glide plane perpendicular to
the b-axis) into 1D chains (…A-B-A-B…) along the [100] direction
0
.5
1
n
n
(Fig. 2). These chains are themselves connected along the [010] di-
f 2 = 50xlog 1 +
(Rt Tt)2
x100
t=1
rection through C15 − H15a•••Ct1 and C16 − H16b•••Ct2 H-bonds
(
Fig. 3a). In fact, the C − H•••π interactions (Fig. 3b) seem to fix the
Where, n is the number of selected time points, Rt is the cumulative
percentage dissolved at each of the selected n time points of the
orientation adopted by the phenyl moieties, by reducing the rotation
freedom around the C4-C7, C8-C7 and C18-C19 σ bonds. The overall 3D
5

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Fig. 1. ORTEP view of the asymmetric unit of
BCZHCl (a) and BCZHCl·H O (b) structures at 293
2
and 150 K, respectively. Thermal ellipsoids for non-
hydrogen atoms are drawn at a 50% probability level
with the numbering scheme. Hydrogen bonds be-
tween the piperazine nitrogen atoms and chlorine
counter-ion or water molecule are given in dashed
cyan lines.
packing is also stabilized by non-classical H-bonds (C10 − H10•••Cl1)
as well as other types of interactions (C − H•••H − C van der Waals
contacts) (Figure S6, Supplementary material). The hydrogen bond
geometries for BCZHCl are listed in Table 2.
raw material has twice the residual chloride value compared with
BCZHCl and BCZHCl·H
ion ratios (1:1 or 1:2).
2
O forms, confirming the nominal API:counter-
In order to obtain a less disordered structure, the BCZHCl·H O da-
2
taset collected at 150 K was chosen for the structure description. The
+
asymmetric unit comprises one buclizine monocation (BCZH ), with
3
.1.3. The BCZHCl·H
Contrary to our expectation of recrystallizing the raw material
BCZH Cl ), the obtained single crystals were confirmed to be the
monohydrochloride monohydrate salt (BCZHCl·H O). It is important to
emphasize that the residual chloride quantification analysis show that
2
O (150 K) structure description
the nitrogen N2 atom protonated, and one chloride anion as a counter-
ion besides a water molecule (Fig. 1b). Also here, the piperazine ring
adopts a distorted chair conformation, with the puckering parameters
Q, θ, and φ of 0.573(6) Å, 177.0(6), and 220(14)°, respectively.
(
2
2
2
Fig. 2. View of the 1D chain along the [100] direction of the BCZHCl structure. The symmetry-related drug molecules are represented in white and magenta colors.
The red spheres are centroids of the drug phenyl rings. The hydrogen bonds are represented by dashed cyan lines. The H atoms non-participating in the interactions
have been omitted for clarity.
6

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Fig. 3. View of the 2D hydrogen-bonded pattern depicting the non-classical C-H•••π interactions (dashed cyan lines) along the [100] direction (b). Detailed view of
the inter-chain interactions (C-H•••π) in BCZHCl packing (b). Ct is the centroid of the drug phenyl rings. For clarity, only the H atoms involving in interactions are
shown.
Table 2
Although the substituents in the BCZHCl·H O occupy the equatorial
2
Hydrogen bond geometry for BCZHCl, where D = Donor and A = Acceptor.
position of the piperazine ring, they show a conformational geometry
that was slightly different from the corresponding isomer in the BCZHCl
structure (Fig. 4 and Table S12, Supplementary material).
ο
Distance (Å)
Angle ( )
Interaction
D…
H
D…A
H…A D-H…A
Symmetry code
In the crystal, the protonated N2 atom acts as H-bond donor to the
chloride anion, resulting in
a medium charge-assisted H-bond
N2 − H2a•••Cl2
C13 − H13•••Cl2
C10 − H10•••Cl1
0.98 3.012(4) 2.08
0.93 3.735(8) 2.82
0.93 3.637(8) 2.87
158
166
141
143
173
128
x, y, z
(
N2 − H2•••Cl2, Fig. 5). Since the chloride and water molecules are
+
−1/2 + x, −1−y, z
+1/2−x, y, −1/2 + z
1/2 + x, −y, z
both intercalated between the drug molecules (BCZH ), the ion-pair
propagates by translation along the [001] direction through H-bonds
involving the O1 atom as a H-bond donor and the chloride and piper-
azine N1 atoms as H-bond acceptors (Fig. 5).
C15 − H15a•••Ct1 0.97 3.548(7) 2.72
C16 − H16a•••Ct2 0.97 3.942(6) 2.98
C17 − H17b•••Ct3 0.97 3.514(6) 2.83
1/2 + x, −y, z
−1/2 + x, −1−y, z
The ion-pair is also stabilized by non-classical H-bonds between the
3
chloride anion and the sp carbon atoms, C16 and C18 of the drug
(
Fig. 5, Table 3). The drug molecules in these columnar stacks (Fig. 6a)
interact with the drug molecules of the neighboring columns along the
[
010] direction by C-H•••π interactions (C27 − H27b•••Ct1 and
C27 − H27c•••Ct2) (Fig. 6b). As in the anhydrous structure (BCZHCl),
Fig. 4. Overlay of the backbones of BCZHCl and BCZHCl·H O isomers ((R)-
2
isomers). The structures were matched considering all piperazine homologous
non-hydrogen atoms using the Mercury structure overlay calculation (RMS
Cartesian displacement = 0.032). For clarity, the hydrogen atoms were
omitted.
Fig. 5. Ionic chain along [001] direction additionally stabilized by water H-
bonds and non-classical C-H…Cl interactions (depicted in dashed cyan lines).
7

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Table 3
characterization by other techniques.
The infrared spectra of the BCZH Cl
shown in Fig. 9. The BCZH Cl spectrum is similar to previously pub-
Geometric parameters of the BCZHCl·H
and A = Acceptor.
2
O hydrogen bonds, where D = Donor
, BCZHCl and BCZ-FB forms are
2
2
2
2
Angle (ο)
lished (Mostafa and Al-Badr, 2011) spectra presenting characteristic
Distance (Å)
−1 −1
peaks in the 3600–1500 cm
range at 3051 cm
(C–H stretch aro-
D…
−1
−1
+
Interaction
H
D…A
H…A
D-H…A
Symmetry code
matic), 2961 cm
(C–H stretch aliphatic) and 2347 cm
(R
3
–N –H
−
1
stretch). Since tertiary amines do not absorb in the 3700–3100 cm
N2 − H2•••Cl2
0.98
3.067(6)
2.904(8)
3.205(5)
3.596(7)
3.607(8)
3.689(8)
3.712(9)
2.09
173
x, y, z
−1
region, the broad peak at 3420 cm viewed in the BCZH
2
2
Cl spectrum
O1 − H1b•••N1
O1 − H1a•••Cl2
C16 − H16b•••Cl2
C18 − H18b•••Cl2
C27 − H27b•••Ct1
C27 − H27c•••Ct2
0.85(5)
0.85(6)
0.97
2.10(4)
2.41(5)
2.72
159(7)
157(7)
151
x, y, z
x, y, 1 + z
x, y, 1 + z
x, y, 1 + z
x, −1 + y, z
x, −1 + y, z
is probably due to sample moisture (Larkin, 2017; Silverstein and
Webster, 2005). Indeed, tertiary amine salt groups are expected to show
0.97
2.76
146
-l
a broad and intense peak located in the range from 2770 to 2380 cm
0.96
2.73
173
+
assigned to an N –H stretching vibration (Thompson et al., 1965).
0.97
2.91
136
Therefore, the broad band observed to either BCZH
2
Cl
2
or BCZHCl with
−1
−1
respective maximum intensity at 2347 cm
and 2326 cm
is the
these interactions seem to fix the orientation adopted by phenyl moi-
eties, by reducing the rotation freedom around the C4-C7, C8-C7 and
C18-C19 σ bonds. The resulting 2D columnar layers pack along the
distinguishing feature of the salt form of BCZ relative to its free base.
+
Additionally, it is observed that the intensity of the N –H stretch band
is higher for BCZH
2
Cl
2
than BCZHCl, taking the sharp peak at
−
1
[
100] direction through van der Waals contacts (Fig. 7).
2961 cm
lated to the number of protonated piperazine nitrogen atoms (two in
BCZH Cl and one in BCZHCl) and could therefore be used to distin-
as a reference. The difference in intensity could be corre-
2
2
3.2. Characterization of the BCZ-FB, BCZHCl and BCZH
2
Cl bulk material
2
guish the two salt forms. Another notable difference between the salt
−1
forms is the band at 2806 cm observed for BCZHCl only. Methylene
groups next to the nitrogen atom in tertiary amines result in bands
The experimental PXRD pattern of the powder raw material (Figure
S7, Supplementary material) is equivalent to the experimental PXRD
pattern of BCZH Cl reported in the literature (Mostafa and Al-Badr,
−1
(
doublets at slightly different frequencies) at ~ 2800 cm
which are
2
2
absent from the BCZH
2
Cl spectrum due to the protonation of both
2
2
011). The experimental and calculated PXRD patterns of BCZHCl
piperazine nitrogen atoms (Auton, 2016). The protonation increases the
C–H binding constant displacing these bands to a region of higher fre-
quency, causing them to overlap with other bands expected for this
match well with each other, indicating that the raw material was
completely converted to the BCZ monohydrochloride (Fig. 8). The ex-
perimental PXRD pattern of the BCZ-FB form differs from the PXRD
patterns of the salts indicating that the molecular crystal was obtained.
It was not possible to obtain a significant sampling of the powder
−1
spectral range (e.g., those ones at 3051 and 2961 cm ). However,
when one of the piperazine nitrogen atoms became deprotonated in the
BCZHCl form, and the characteristic shift in the C–H stretching of
corresponding to the BCZHCl·H O crystalline form, which restrained its
2
Fig. 6. View of the crystal packing onto ac (right) and ab (left) planes showing the columnar ionic chains (a) and columnar layers (b) along the [001] direction. The
drug molecules related by symmetry operations are represented at the same color. Hydrogen atoms not involved in H-bonds were omitted for clarity.
8

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Fig. 7. View of the crystal packing onto the ab plane. The C-H…π H-bonds (dashed cyan lines) are responsible for linking the drugs along the [010] direction. The
water molecules and also the hydrogen atoms non-participating of H-bonds were omitted for clarity.
Fig. 9. IR spectra (in KBr pellet) of BCZH
2
Cl , BCZHCl and BCZ-FB.
2
Fig. 8. Experimental PXRD patterns of BCZH
2
Cl (raw material), BCZ-FB and
2
BCZHCl forms. The calculated PXRD pattern of BCZHCl was also included to
ensure the purity of the BCZHCl powder sample.
BCZH Cl DSC curve shows two endothermic peaks at 206 and 243 °C
2
2
attributed to the beginning of its decomposition, since it is accompanied
by a weight loss starting in the respective TGA curve (Fig. 10a). The
weight loss of approximately 14.0% in the interval of 200–250 °C was
attributed to the release occurring in two steps (highlighted by the DTG
curve) of two HCl molecules (14.0%). The complete decomposition of
methylene groups next to tertiary amines was observed. As expected,
the spectral changes were even more remarkable for BCZ-FB. The band
+
attributed to the N –H stretch is completely absent, confirming that the
two piperazine nitrogen atoms are deprotonated. Additionally, the
−
1
BCZH
2
Cl
2
is reached before 350 °C. The thermal behavior of the
double bands at 2800 and 2761 cm
(from C–H methylene groups
BCZHCl form is remarkably similar to that of BCZH
2
Cl (Fig. 10b). In
2
next to tertiary amines) which are only observed in the BCZ-FB spec-
trum also confirms the deprotonation of both nitrogen atoms. In sum-
mary, the infrared spectroscopy analysis confirms the raw material as
spite of its 1:1 salt feature, the chloride amount (weight loss 7.0%
corresponding to one HCl molecule, calc. = 7.0%) from the BCZHCl
form is also released in two steps (DSC peaks at 227 and 240 °C). In-
deed, the thermal decomposition similarity of the two salt forms is more
clearly seen by the DTA curves (Figure S8, Supplementary material).
The DSC curve of the BCZ-FB form shows a single endothermic peak at
BCZH
2
Cl and the BCZ-FB crystallization for which the structural de-
2
termination was not possible; this also enables us to differentiate the
three BCZ solid modifications.
The DSC and TGA curves of BCZH
2
Cl , BCZHCl and BCZ-FB are
2
1
05 °C corresponding to its melting (Fig. 10c). The TGA results show
shown in Fig. 10. The complete DTA curves collected simultaneously to
that the molten BCZ start its decomposition at ~ 250 °C which is
the TGA curves are shown in Figure S8 (Supplementary material). The
9

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Fig. 11. The calculated pH-dependent acid
−
base BCZ species
2
+
+
+ 2
1
distribution.
2 = [BCZH2 ]/C = (1 + (Ka1/[H ]) + (Ka1 × Ka2/[H ] ))
;
+
+
0
+ 2
1
= [BCZH ]/C = 2 (Ka1/[H ]); 0 = [BCZ ]/C = 2 (Ka1 × Ka1/[H ] ); where
2
+
+
0
0
+
1
+
2
= 1 and C = [BCZH2 ] + [BCZH ] + [BCZ ]. Curves show pH in
steps of 0.1.
3.3. Equilibrium solubility
Since BCZ is a weakly basic drug (weak electrolyte), its aqueous
solubility is controlled by both the solution pH and the dissociation
constant (pKa). However, to the best of our knowledge, neither ex-
perimental nor calculated dissociation constants associated with the
protonation/deprotonation of the two BCZ piperazine nitrogen atoms
Ka1
Ka2
2
+
+
+
0
+
(
BCZH
2
BCZH + H
BCZ + 2H ) have been published to
2.12 and
date. Therefore, we used here the values (pKa1
=
pKa2 = 6.55) calculated using the ChemSketch software from ACDLabs
to sequentially calculate the pH-dependent acid-base BCZ species dis-
tribution (Fig. 11). It is important to mention that pKa1 refers to the N1
deprotonation, which is the piperazine nitrogen atom linked to the [(4-
chlorophenyl) phenylmethyl] moiety (Scheme 2) as confirmed by the
BCZHCl structure (item 3.1.2).
No significant variation was observed when comparing the equili-
brium pH values (pH ) with either the nominal (pH of the pure media)
f
or starting (pH , measured immediately after placing the solid forms in
i
contact with the media) pH values, except for the saturated solutions
used to determine the equilibrium solubility of the two BCZ salt forms
-
1
in ultrapure water pH = 6.8 and in 0.01 mol L HCl medium (Table 4).
The pH reduction for the BCZHCl (pH
f
=
3.3) and BCZH
2
Cl
2
(
pH = 2.8) solutions using ultrapure water as medium was expected
f
due to the hydrolysis of the cationic BCZ species released into the so-
lution from these solid acidic salt forms.
-
1
Considering the 0.01 mol L HCl media (pH
f
from 1.9 to 2.9), the
Cl (Fig. 12 and
order of solubility was BCZ-FB > BCZHCl > BCZH
2
2
Table 4). This behavior was expected due to the probable formation of
2
+
+
BCZH
2
and/or BCZH species in solution at pH less than 4.5
(
Fig. 11). The lower solubility of BCZHCl and BCZH
2
2
Cl compared to
BCZ-FB in HCl media could be attributed to the solubility suppression of
the hydrochloride salt forms due to common ion effects. This hypothesis
is reinforced by a reduction in the respective solubility values when the
-
1
concentration of the HCl medium is increased to 0.1 mol L (Fig. 12
Fig. 10. DSC (in red) and TGA (in blue) curves for the (a) BCZH
2
Cl , (b)
2
and Table 4). It is also noted that the BCZ-FB > BCZHCl > BCZH
2
Cl
2
BCZHCl and (c) BCZ-FB. The inset in the top right position is the TGA 1st de-
-1
trend is kept in the 0.1 mol L HCl media (pH
f
= 1.2). Therefore, the
rivative curve (DTG).
BCZH
2
Cl form (raw material) is the one that had the lowest solubility
2
in the HCl media.
completed in a single step until 350 °C. In summary, the thermal ana-
lyses show signatures of the three forms and suggest a single-phase
characteristic.
In ultrapure water medium, the BCZ-FB was insoluble (at least un-
detectable) and BCZH Cl (final pH = 2.8) was more soluble than that
2
2
of BCZHCl (final pH = 3.3) reversing the trend observed in the HCl
media (Fig. 12 and Table 4). This result suggests that without the
common ion effect, the balance of the exothermic process involving the
10

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Scheme 2. pH-dependent BCZ species.
solvation of BCZ ionic/neutral species versus the endothermic process
involving their release from the crystalline lattices, favors the BCZH Cl
in water
the equilibrium establishment. The PXRD patterns show that either
2
2
BCZH
2
Cl or BCZHCl form remain as unique solid phase irrespective the
2
solubility. In contrast to BCZHCl, the solubility of BCZH
2
Cl
2
media (Fig. 13 and Figures S9-S13, Supplementary material). On the
one hand, our PXRD patterns also show that the BCZ-FB form is con-
verted to the BCZHCl form during the establishment of equilibrium in
medium is higher than in both HCl media (Fig. 12 and Table 4).
The solubility trend observed in ultrapure water media
-
1
(
BCZH
2
Cl
2
> BCZHCl > BCZ-FB(insoluble)) is kept in acetate buffer pH
the two media (0.1 and 0.01 mol L HCl media) in which it was soluble
(Fig. 13 and Figure S9, Supplementary material). The conversion occurs
4
.5 with a significant decrease in the solubility of the respective salt
forms. However, since the solubility reduction of the BCZHCl form is
more pronounced, the BCZH Cl form became approximately 5-fold
more soluble than the BCZHCl one (Fig. 12 and Table 4). It is also
0
+
+
via protonation (BCZ + H → BCZH ) of the released neutral species
from BCZ-FB, which is completely dissolved, followed by precipitation
with the chloride ion from the HCl media. Because of this conversion,
the BCZ-FB solubility in the HCl media cannot be considered actual
values. As expected, in all media where the BCZ forms were insoluble
(at least not detectable) no phase transition was observed (Figures S10-
S13, Supplementary material).
2
2
observed that the solubility of BCZH
2
Cl in acetate buffer pH 4.5 is
2
-1
comparable to those reached in the 0.1 and 0.01 mol L HCl media.
Finally, the three BCZ forms are insoluble in the pH 6.8 and pH 7.5
buffers, which is an expected behavior considering that the pH of these
media are greater than the BCZ pKa2 (Figs. 11 and 12). The p-value of
ANOVA evaluation was 0.0 (less than 0.05) for all forms, in the media
-
1
-1
HCl 0.01 mol L , HCl 0.1 mol L , sodium acetate pH 4.5 and water in a
significance level of 5% showing that the three solid forms have distinct
solubilities.
3.4. Dissolution profile
Despite the higher and discriminative solubility values found for the
BCZ forms in water and HCl media, the acetate buffer pH 4.5 medium
was chosen for the dissolution profile assay performed here. The HCl
To be considered a class I or II in the BCS (Yu et al., 2002), the
highest dosage of BCL (25 mg) must be soluble in 250 mL of aqueous
−1
media, which means reaching solubility values of at least 100 µg mL
.
media were discarded due to the BCZ-FB → BCZH
2
Cl phase transition
2
Since the solubility values found here are below this limit, the three
BCZ forms should be classified as a class II (low solubility and high
permeability) or IV (low solubility and low permeability) API.
The remaining solid material in equilibrium with the solutions used
to determine the solubility of the crystal forms was analyzed by PXRD
to determine whether the starting crystal forms are maintained during
observed during the establishment of equilibrium (Fig. 13 and Figure
S6, Supplementary material), whereas the water medium was discarded
due to the pH variation. The solubility results also indicated the need
for surfactant addition to the chosen dissolution medium (ANVISA,
2013; Aulton, 2016) in order to reach the sink condition criteria (the
dissolution profile assay considering the highest commercial dosage
with 25 mg of BCZH
2
Cl ) (Table S13, Supplementary material). In order
2
Table 4
Equilibrium solubility (μg mL−1) data for BCZ-FB, BCZHCl and BCZH
BCZ-FB
. The initial pH (pH
) and final pH at the equilibrium (pH
BCZHCl
)* of each solution are also given.
BCZH Cl
12.2 ± 0.8
2
Cl
2
i
f
2
2
0
0
.1 mol L-1 HCl (pH = 1.0)
.01 mol L-1 HCl (pH = 2.0)
51.1 ± 1.9
pH = pH = 1.2
75.7 ± 1.9
pH = 2.1 pH
30.3 ± 1.1
i
f
pH
i
= 1.1 pH
f
= 1.2
pH = 1.1 pH
19.9 ± 1.2
pH = 2.4 pH
16.3 ± 0.9
pH = pH = 4.5
34.8 ± 1.6
pH = 3.4 pH
i
f
= 1.2
36.0 ± 3.1
i
f
= 2.9
= 4.5
= 5.1 pH = 4.6
pH
3.1 ± 0.1
pH = pH
i
= pH
f
= 2.1
= 4.5
i
f
= 1.9
a
Acetate buffer pH 4.5
ND
pH
ND
i
= pH
f
i
f
i
f
Ultrapure water pH = 6.8
Phosphate buffer pH 6.8
Phosphate buffer pH 7.5
18.4 ± 0.7
pH = 3.9 pH
pH
i
f
i
f
= 3.3
i
f
= 2.8
ND
ND
ND
pH
i
= pH
= pH
f
f
= 6.8
= 7.5
pH
i
= pH
= pH
f
f
= 6.8
= 7.5
pH
i
= pH
f
f
= 6.8
= 7.5
ND
ND
ND
pH
i
pH
i
pH
i
= pH
*
After 72 h-stirring;
a
ND = not detectable.
11

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
Fig. 12. pH-dependent equilibrium solubility values of BCZ-FB, BCZHCl, and BCZH
2
Cl
2
(values and standard deviation in Table 4). ND = not detectable.
Fig. 13. Experimental PXRD patterns of BCZH
2
Cl , BCZHCl, and BCZ-FB before
2
Fig. 14. Comparative dissolution profile between BCZ-FB, BCZHCl and
BCZH Cl in buffer pH 4.5 + 1.0% sodium lauryl sulfate dissolution medium.
-1
and after the equilibrium solubility test in the 0.01 mol L HCl medium.
2
2
to establish the smallest necessary amount of surfactant (FDA, 1997) to
the dissolution profile assay, profiles containing 0.5 and 1.0% of so-
a better interaction of neutral molecules with the non-polar chains of
SLS.
dium lauryl sulfate (SLS) were carried out for the BCZH
2
Cl form. The
2
results (Table S14 and Figure S14, Supplementary material) led us to
select the medium containing 1.0% of SLS due to its higher release rate
4
. Conclusions
(
(
11.0% great than in the 0.5% SLS medium) and FDA guidance criteria
variation coefficient below 20% and 10% for the initial and final times,
Buclizine monohydrochloride monohydrate (BCZHCl·H O) and its
2
anhydrous form, BCZHCl, were solved and refined in the non-cen-
respectively) (FDA, 1997).
trosymmetric space groups Pna2
1
and Pca2
Cl
1
2
2
, respectively. Since re-
) resulted in BCZHCl·H
Cl was not determined.
The comparative dissolution profile study using the optimized
conditions was carried for the three BCZ forms (Fig. 14) reaching a
dissolution percentage at 60 min of 91, 88, and 55% for BCZ-FB,
crystallization of the raw material (BCZH
2
2
O
single crystals, the X-ray structure of BCZH
2
Efforts to solve the buclizine free-base (BCZ-FB) were hindered by
weakly diffracting crystals, however, its existence was successfully
confirmed by a series of techniques and by comparison of the obtained
results with those found for the raw material and BCZHCl form. In the
BCZH
2
Cl , and BCZHCl, respectively. The similarity factors (F2) cal-
2
culated from the dissolution profiles (see item 2.9) are 44, 26, and 19
comparing BCZH
2
Cl
2
vs. BCZ-FB, BCZH
2
2
Cl vs. BCZHCl, and BCZ-FB vs.
BCZHCl, respectively. Therefore, all forms are different from each other
in terms of performance because the three combinations have an F2 less
than 50. The larger percentage of release for BCZ-FB could be attributed
to a better interaction of the neutral species with the non-polar chains
of SLS compared to the saline forms. In contrast to the salt forms, the
BCZ-FB tablets have a high percentage of release, which could be due to
+
FT-IR spectrum, the vibrational mode relative to the piperazine N –H
stretching was used to differentiate the buclizine salts from the free-
base; the broad and intense peak seen in the salt’s spectra at
−
1
2
770–2380 cm was absent in the BCZ-FB one. Also, for the salts, the
DSC curves showed two endothermic peaks in the range of 200–250 °C
corresponding to the decomposition process, whereas for the free-base,
12

M. Bitencourt, et al.
International Journal of Pharmaceutics 589 (2020) 119840
there was only one peak at 105 °C due to melting. Moreover, the TGA
profile of BCZ-FB was significantly different from the salts. In contrast
Authorship
to a single step process, the thermal degradation of BCZH
2
Cl
2
and
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript. All
authors contributed equally.
BCZHCl salts was characterized by two step events associated with the
loss of two and one HCl molecules besides the drug itself, respectively.
2
+
As a result of protonation and the formation of BCZH
2
and/or
+
BCZH species, BCZ-FB showed the highest solubility values in HCl
Appendix A. Supplementary data
media. On the other hand, due to the common ion effect, BCZH
BCZHCl salts showed the lowest ones. In ultrapure water medium, BCZ-
FB was insoluble (at least undetectable) and BCZH Cl was more so-
luble than BCZHCl, thus reversing the trend observed in HCl media.
2
Cl
2
and
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijpharm.2020.119840.
2
2
This trend (BCZH
2
Cl
2
> BCZHCl > BCZ-FB (insoluble)) was also
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