crystals
Article
A New Hemihydrate of Valacyclovir Hydrochloride
Shuai Zhang 1 , Meiqi Zheng 1 , Mengqing Zhou 1 , Tian Chen 2 , Naixing Wang 2 , Zhaoxia Zhang 1
and Guoqing Zhang 1, *
1
2
*
Engineering Research Center for Eco-Dyeing and Finishing of Textiles, Ministry of Education,
Zhejiang Sci-Tech University, Hangzhou 310018, China; [email protected] (S.Z.);
[email protected] (Mei.Z.); [email protected] (Men.Z.); [email protected] (Z.Z.)
Zhejiang Charioteer Pharmaceutical Co., Ltd., Taizhou 317321, China; [email protected] (T.C.);
[email protected] (N.W.)
Correspondence: [email protected]; Tel.: +86-571-8684-3588
Academic Editors: Helmut Cölfen and Mei Pan
Received: 28 March 2017; Accepted: 11 May 2017; Published: 16 May 2017
Abstract: Several crystal forms of valacyclovir hydrochloride, including two anhydrous and three
hydrates, were investigated in this study. At the same time, a new hemihydrate of valacyclovir
hydrochloride was first discovered and its properties were characterized by PXRD, TGA, DSC,
and Raman in this study. The hemihydrate shows a distinctive PXRD pattern and a melting point of
209 ◦ C with a water weight loss of 2.42% from the thermal analysis. The Raman spectra show a few
distinctive peaks in the region of 1250–1400 cm−1 due to different crystal forms. The thermostability
testing suggests it is a stable crystal form and remain the same for several months under high
temperature and humidity. All these crystal forms show good dissolubility in the water at room
temperature with excess 100 mg/mL.
Keywords: valacyclovir hydrochloride; polymorphs; hemihydrate; stability
1. Introduction
Polymorphism of a drug compound usually results from the possibility of at least two different
spatial arrangements of the molecules in the crystal lattice, or in some cases, from variations in
molecular conformation, including crystalline and amorphous forms as well as solvate and hydrate
forms [1–4].
Due to difference in the solid–state structure, polymorphs can be imparted with different chemical
and physical properties, including melting point, chemical reactivity, apparent solubility, dissolution
rate, and optical and mechanical properties of solid dosage forms [5–7], which may affect subsequent
process and/or manufacture of drug products.
Valacyclovir hydrochloride (VCV), [L-Valine, 2-[(2-amino-1, 6-dihydro-6-oxo-9H-purin-9-yl)
methoxy] ethyl ester, monohydrochloride], as shown in Figure 1, is the L-valyl ester prodrug of
acyclovir and has demonstrated antiviral activity against herpes simplex virus types, 1 (HSV-1) and 2
(HSV-2) and varicella-zoster virus (VZV) both in vitro and in vivo [8–13]. Although VCV has been used
as one of the most important antiviral drugs, study on its polymorphism and pseudopolymorphism is
still in negligible quantity. So far, two anhydrous and several hydrates crystalline forms of valacyclovir
hydrochloride were reported in the US patent 6107302, US 6849736 B2 and US 0021444 as well as their
preparation processes [14–16]. Based on the complexity and influence on the subsequent drug dosage,
it is necessary to further investigate the polymorphism of VCV.
Crystals 2017, 7, 140; doi:10.3390/cryst7050140
www.mdpi.com/journal/crystals
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NH2
O
H
N
HN
·
O
H2N
CH3
N
N
HCl
CH3
O
O
Figure1.1.Molecular
Molecular structure
structure scheme
Figure
schemeof
ofVCV.
VCV.
In this work, a new crystal form—hemihydrate—was obtained by recrystallization technology,
In thiswith
work,
new crystal
form—hemihydrate—was
obtainedusing
by recrystallization
technology,
together
thea other
four crystal
forms of VCV were investigated
powder X-ray diffraction
together
with
the
other
four
crystal
forms
of
VCV
were
investigated
using
powder
X-ray
diffraction
(PXRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). In addition,
(PXRD),
differential
scanning
calorimetry
(DSC), and thermogravimetric
analysis
(TGA).
In addition,
the testing
for apparent
solubility
and thermostability
was also carried out
for deep
understanding
thetheir
testing
for
apparent
solubility
and
thermostability
was
also
carried
out
for
deep
understanding
pharmaceutical potential.
their pharmaceutical potential.
2. Results and Discussion
2. Results and Discussion
According to above experiments, the five solid forms of VCV were successfully prepared and
According
to aboveproperties
experiments,
solid forms
VCVDSC,
wereand
successfully
prepared and
their
physicochemical
were the
alsofive
characterized
byof
PXRD,
TGA.
their physicochemical properties were also characterized by PXRD, DSC, and TGA.
2.1. X-ray Diffraction
2.1. X-ray Diffraction
Although crystal structure determination from an analysis of single-crystal diffraction can
Although
crystal structure
determination
from an
of single-crystal
diffraction
can provide
provide
the fundamental
structural
understanding
of analysis
polymorphic
solids, the difficulty
of preparing
theafundamental
structural
understanding
of
polymorphic
solids,
the
difficulty
of
preparing
a suitable
suitable single-crystal precludes this technique from being used on a routine basis
for
characterization
of most
crystalfrom
forms.
Since
most
substances
were
obtained as microsingle-crystal
precludes
thisdrug
technique
being
used
on drug
a routine
basis for
characterization
of most
crystalline
powders,
technologywere
becomes
theaspredominant
tool powders,
for the the
routine
drug
crystal forms.
Sincethe
mostPXRD
drug substances
obtained
micro-crystalline
PXRD
characterization
of
polymorphs
[4].
In
this
study,
no
any
suitable
single-crystal
of
the
five
VCV
crystal
technology becomes the predominant tool for the routine characterization of polymorphs [4]. In this
forms
prepared under
experiment
conditions.
The X-ray diffraction
study,
nowas
anysuccessfully
suitable single-crystal
of thecurrent
five VCV
crystal forms
was successfully
prepared data
under
relevant
to the five
powder crystalline
are summarized
in Table
current
experiment
conditions.
The X-rayforms
diffraction
data relevant
to the1.five powder crystalline forms
are summarized in Table 1.
Table 1. Reflections of the five crystal forms of VCV.
Form I
2θ(°) Intensity (%)
3.66Form
100
I
8.58
2.6
◦
2θ(9.48
) Intensity
6.0 (%)
3.66 9.8
100
10.60
8.58 24.4
2.6
10.86
9.48 7.8
6.0
13.36
10.60 3.9
9.8
16.44
10.86 2.0
24.4
20.20
13.36 2.9
7.8
21.42
16.44 3.9
21.80 7.2
20.20 2.0
24.00 3.2
21.42 2.9
26.76 2.1
21.80 7.2
27.28 5.2
24.00
26.76
27.28
3.2
2.1
5.2
Table
1.IIReflections of the
of VCV.
Form
Formfive
III crystal formsForm
IV
2θ(°) Intensity (%)
3.56
Form100
II
8.58
5.2
◦
2θ( 9.38
) Intensity
5.2 (%)
3.56 100
10.52
8.6
8.58 15.8
5.2
10.78
9.38 5.2
12.08
0.9
10.52 6.1
8.6
13.26
10.78 15.8
14.42
2.5
12.08 5.3
0.9
16.36
13.26 6.1
20.04 2.5
14.42 2.5
21.34 2.2
16.36 5.3
23.92 9.3
20.04 2.5
25.94 1.8
21.34 2.2
26.82 2.4
23.92 9.3
27.22 4.1
25.94 1.8
26.82 2.4
27.22 4.1
2θ(°) Intensity (%)
3.46
100
Form III
6.94
93.1
◦
2θ( 7.88
) Intensity
13.2(%)
3.46 100
8.46
10.9
6.94 93.1
9.30
13.3
7.88
10.46 13.2
9.5
8.46
11.64 10.9
8.4
9.30 13.3
13.02
11.5
10.46
9.5
14.44 12.9
11.64
8.4
15.34 9.2
13.02
11.5
16.25 11
14.44 12.9
20.78 11.4
15.34 9.2
23.47 6.6
16.25 11
23.90 9.9
20.78 11.4
24.54 19.5
23.47 6.6
26.18 11.0
23.90 9.9
27.16 19.6
24.54 19.5
27.80 11.6
26.18
27.16
27.80
11.0
19.6
11.6
2θ(°) Intensity (%)
3.66
63.1
Form IV
8.66
41.1
◦
2θ( )10.62
Intensity
13.8(%)
3.66
10.92 63.1
48.5
8.66
12.18 41.1
18.3
10.62
13.32 13.8
12.1
10.92
14.56 48.5
51.1
12.18
15.20 18.3
14.3
13.32
12.1
16.48 52.2
14.56
51.1
16.96 22.5
15.20
14.3
18.16 33.8
16.48 52.2
20.10 34.1
16.96 22.5
20.62 7.1
18.16 33.8
21.28 15.3
20.10 34.1
21.88 25.7
20.62 7.1
22.72 8.8
21.28 15.3
24.02 100
21.88 25.7
24.46 15.9
22.72 8.8
25.13 6.8
24.02 100
25.94 21.1
24.46 15.9
26.26 60.8
25.13 6.8
26.92 10.6
25.94
21.1
27.26 60.8
8.4
26.26
28.10 10.6
7.9
26.92
28.38 8.4
6.5
27.26
29.00 7.9
16.2
28.10
29.70 6.5
8.8
28.38
30.64 16.2
18.1
29.00
29.70 8.8
30.64 18.1
Form V
2θ(°) Intensity (%)
3.50 V33.6
Form
6.58
58.7
◦
2θ( ) Intensity
(%)
9.18
100
3.50
11.3233.6
18.8
6.58
15.3458.7
13.3
9.18
15.62 10016.9
11.32
16.16 18.8
10.4
15.34
17.02 13.3
16.1
15.62
16.9
19.14 18.8
16.16
10.4
19.66 9.0
17.02
16.1
21.26 19.6
19.14 18.8
22.86 15.0
19.66 9.0
24.16 10.8
21.26 19.6
26.30 8.4
22.86 15.0
27.34 17.8
24.16 10.8
27.86 15.3
26.30 8.4
28.76 3.4
27.34 17.8
27.86 15.3
28.76 3.4
Crystals 2017, 7, 140
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From the diffraction patterns, one can see that Form I, II, IV and V are consistent as previously
Crystals 2017, 7, 140
3 of 9
reported crystal forms. However, the PXRD pattern of the Form III—with characteristic diffraction
◦ , 7.9◦ , 8.5◦ , 9.3◦ , 13◦ , 14.4◦ , 16.3◦ , 20.8◦ , 24.5◦ , and 27.2◦ —is completely distinctive
peaks at 2θ
3.5◦the
, 6.9diffraction
From
patterns, one can see that Form I, II, IV and V are consistent as previously
from reported
those ofcrystal
the other
forms.theIn
particular,
Form
III exhibited
verydiffraction
characteristic
forms.four
However,
PXRD
pattern of
the Form
III—with several
characteristic
◦ , 13◦ , 20.8◦ and 23.5◦ , where no any peak can be detected in the patterns of other
peakspeaks
at 6.9at◦ ,2θ
7.9
3.5°, 6.9°, 7.9°, 8.5°, 9.3°, 13°, 14.4°, 16.3°, 20.8°, 24.5°, and 27.2°—is completely distinctive
from
those of the
other
fourIII
forms.
In particular,
III exhibited
several
veryIn
characteristic
peaks this
forms.
It indicates
that
Form
should
be a new Form
polymorphic
form
of VCV.
order to confirm
6.9°, 7.9°,analysis
13°, 20.8°including
and 23.5°, where
no any
peak
can be detected
the patterns
of other
forms.
It new
point,atthermal
DSC and
TGA
technology
wereinused
to further
analyze
the
indicates
that
Form
III
should
be
a
new
polymorphic
form
of
VCV.
In
order
to
confirm
this
point,
polymorphic form.
thermal analysis including DSC and TGA technology were used to further analyze the new
polymorphic
form. Calorimetry
2.2. Differential
Scanning
The
DSC thermograms
of the five crystal forms of VCV are presented in Figure 2. For Form I, II,
2.2. Differential
Scanning Calorimetry
and III—different crystal forms—as hydrates containing a certain of crystal water, there are significant
The DSC thermograms of the five crystal forms of VCV are presented in Figure 2. For Form I, II,
◦C
endothermic
peaks on crystal
their DSC
scans because
water lossa in
the temperature
rangethere
of 60–140
and III—different
forms—as
hydratesofcontaining
certain
of crystal water,
are
(Figure
2a). DSC
curves ofpeaks
FormonI and
showed
two partially
endothermic
peaks
at
significant
endothermic
their II
DSC
scans because
of wateroverlapping
loss in the temperature
range
of
◦
70 and
130 °C
C respectively,
which
means
there
areIItwo
different
types ofoverlapping
crystal water
in the crystal
60–140
(Figure 2a). DSC
curves
of Form
I and
showed
two partially
endothermic
◦ Cdifferent
peakswhereas
at 70 andonly
130 °C
respectively,
which
means
there are
types
of crystal
in thethere
structure,
one
endothermic
event
at about
141two
is seen for
Form
III. Inwater
addition,
◦
crystal
structure,
whereas
only
one
endothermic
event
at
about
141
°C
is
seen
for
Form
III.
In
addition,
are not any endothermic peaks in 60–140 C on the runs of Form IV and Form V, which indicates that
are not
endothermic
peaks in 60–140 °C on the runs of Form IV and Form V, which indicates
both there
of them
are any
anhydrous
compounds.
that
both
of
them
are
anhydrous
compounds.
The melt/decomposition events
of the five forms were shown in Figure 2b. For Form I, the melting
The melt/decomposition events of the five forms were◦ shown in Figure 2b. For Form I, the melting
and decomposition simultaneously occurs at about 205 C because there is only a single exothermic
and decomposition simultaneously occurs at about 205 °C because there is only a single exothermic peak
peak on
onthe
theDSC
DSCcurve,
curve,
while Forms II, IV, and V have two significant melting and decomposition
while Forms II, IV, and V have two significant melting and decomposition stages,
stages,
respectively.
Melting
is an important
parameter
for a polymorphic
drug,
to some
respectively. Melting point point
is an important
parameter
for a polymorphic
drug, which to
somewhich
extent can
extentbecan
betoused
to identify
different
crystal
form. temperatures
The meltingoftemperatures
theestimated
four forms
used
identify
different crystal
form.
The melting
the four forms of
were
and were
estimated
listed
in Table
2. Form
III has transformation
a solid-solid transformation
about
197 ◦itC,
means
listed and
in Table
2. Form
III has
a solid-solid
at about 197 °C, at
which
means
is which
probably
already
turned
into
another
crystalline
form
after
the
dehydration
process.
it is probably already turned into another crystalline form after the dehydration process.
Heat Flow Endo Up(mW)
(a)
(b)
( Ⅰ)
( Ⅱ)
(Ⅲ)
(Ⅳ)
(Ⅴ)
60 80 100 120 140 160
180
200
220
240
Temperature(℃)
Figure 2. DSC thermograms of all the polymorphic forms of VCV, (a) water lease stage and (b)
Figure 2. DSC thermograms of all the polymorphic forms of VCV, (a) water lease stage and
melting/decomposition stage.
(b) melting/decomposition stage.
Table 2. Differential scanning calorimetry data for all the forms of VCV.
Table 2. Differential scanning calorimetry data for all the forms of VCV.
Items
Form I
Melting point (°C)
205
Items
Form I
Transition 1 (°C)
53–139
Melting Transition
point (◦ C)2 (°C)
205 /
Transition 1 (◦ C)
Transition 2 (◦ C)
53–139
/
Form II
206
Form II
60–140
183–196
206
Form III
209
Form III
124–141
186–206
209
60–140
183–196
124–141
186–206
Form IV
227
Form IV
/
/ 227
/
/
Form V
223
Form V
/
/ 223
/
/
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2017, 7,
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140
44 of
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2.3. Thermogravimetric Analysis
2.3. Thermogravimetric Analysis
The water contents of all the forms were determined by TGA and the curves are illustrated in
The water contents of all the forms were determined by TGA and the curves are illustrated in
Figure 3. The three hydrates showed significant water release steps from Figure 3a, and Form I and
Figure 3. The three hydrates showed significant water release steps from Figure 3a, and Form I and
II were respectively calculated to be 6.93 wt% and 4.78 wt% in the temperature range of 50–140 °C,
II were respectively calculated to be 6.93 wt% and 4.78 wt% in the temperature range of 50–140 ◦ C,
but it is 2.42 wt% for Form III in a higher temperature range of 123–145 °C. The above results are
but it is 2.42 wt% for Form III in a higher temperature range of 123–145 ◦ C. The above results are
respectively consistent with water content theoretically calculated from sesquihydrate, monohydrate,
respectively consistent with water content theoretically calculated from sesquihydrate, monohydrate,
and hemihydrate. Whereas it is clear that Form IV and V had no any weight loss in the same
and hemihydrate. Whereas it is clear that Form IV and V had no any weight loss in the same
temperature zone from Figure 3b, this suggest they are anhydrous crystal forms.
temperature zone from Figure 3b, this suggest they are anhydrous crystal forms.
105
100
Form Ⅰ
FormⅡ
(a)
95
Weight(%)
70
80
50
100
150
75
Ⅱ
70
65
60
FormⅣ
85
Ⅰ
80
FormⅤ
90
FormⅢ
85
(b)
100
95
90
75
105
80
65
120
Ⅲ
60
55
50
130
55
140
100
200
300
100
200
300
Temperature(℃)
Figure
VCV polymorphs:
polymorphs: (a)
(a) Form
Form I,
I, Form
Form II
II and
and Form
Form III;
III; (b)
(b) Form
Form IV
IV and
and Form
Form V,
V,
Figure 3.
3. TGA
TGA curves
curves of
of VCV
the
the insert
insert is
is the
the profiles
profiles of
of the
the derivative
derivative curves.
curves.
In addition,
addition,the
theprofiles
profilesofofthe
thederivative
derivative
curves
insert
in Figure
3a show
there
are two
weight
In
curves
insert
in Figure
3a show
there
are two
weight
loss
loss stages
for Forma
II Form
while III
Form
has
only
one.results
Theseare
results
arecorresponding
totally corresponding
stages
for Forma
I and IIIand
while
has III
only
one.
These
totally
with the
with profiles
the DSCshown
profiles
shown2.inInFigure
2. Inwater
fact, each
lease stage
usually
stands for
a specific
DSC
in Figure
fact, each
lease water
stage usually
stands
for a specific
combination
combination
mode
between
water
and VCV
molecules.
suggests
that bonding
there aremodes
two bonding
mode
between
water
and VCV
molecules.
This
suggests This
that there
are two
among
modes
among
water
with
VCV
molecular
in
VCV
hydrates
for
Form
I
and
II
but
only
one
for
water with VCV molecular in VCV hydrates for Form I and II but only one for Form III. FormForm
DSC
III. Form
and
Formwater
III, theloss
single
water
loss process
actually
confirms
it is a
and
TGA DSC
results
ofTGA
Formresults
III, theofsingle
process
actually
confirms
that it
is a purethat
hydrate
pure hydrate
crystalline
with
half crystal water.
crystalline
form
with halfform
crystal
water.
2.4. Raman Spectroscopy
As well
well known,
known,Raman
Ramanspectroscopy
spectroscopy
a reliable
technology
to differentiate
crystal
is aisreliable
technology
to differentiate
crystal
forms.forms.
From
From
the Raman
spectra
theVCV
five VCV
crystal
in Figure
the most
distinctive
region
can
be
the Raman
spectra
of theof
five
crystal
formsforms
in Figure
4, the4,most
distinctive
region
can be
seen
−
1
−1
seen
between
and 1400
. Inregion,
this region,
V show
peaks,
and
IV have
between
12501250
and 1400
cm cm
. In this
FormForm
I and IVand
show
threethree
peaks,
FormForm
II andII IV
have
four
four
peaks,
but Form
IIIonly
has only
two peaks.
peaks,
but Form
III has
two peaks.
From Table 3, Form I has two peaks at 1308.5 and 1351.3 cm−1 with a small peak at 1392.3 cm−1 ,
whereas Form II has three peaks in the region at 1299.4, 1331.7, and 1352.3 cm−1 with a small peak at
1387.3 cm−1 . The new crystal Form III shows two peaks at 1307.1 and 1353.3 cm−1 . The anhydrate
Form IV has four peaks at 1298.4, 1331.7, 1352.8, and 1386.8 cm−1 , whereas another anhydrate form V
shows three peaks at 1306.1, 1348.8, and 1393.36 cm−1 . It is clear that different crystal forms exhibit
distinctive Raman spectra.
Crystals 2017, 7, 140
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5 of 9
5 of 9
Form I
Form II
Form III
Form IV
Form V
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
Wavenumber (cm-1)
Figure
4. The
TheRaman
Ramanspectra
spectra
forms
of valacyclovir
(all spectra
collected
at
Figure 4.
of of
thethe
fivefive
forms
of valacyclovir
(all spectra
were were
collected
at room
room
temperature).
temperature).
−1 withofaValacyclovir.
Table
3. Summary
of Raman
Positions
the Five
From Table
3, Form
I has two
peaksPeak
at 1308.5
andfor
1351.3
cmForms
small peak at 1392.3 cm−1,
whereas Form
II has three peaks in the region at 1299.4, 1331.7, and 1352.3 cm−1 with a small peak at
Form I
Form II
Form III
Form IV
Form V
Raman Shift (cm−1 )
−1
1387.3 cm−1. The new
crystal
IIIpeaks
shows
two peaks
at 1307.1
1353.3
anhydrate
Form
Three peaks
at 409.4,FormFour
at 409.9,
Three peaks
at 471.5, and
Four
peaks atcm
409.4, . TheFive
peaks at 409.4, 438.1,
400–600
472.6 and 501.8
470.4, 493.1 and 536.4
504.0 and 527.7
439.9, 490.3 and 538.1
470.9, 503.5 and 578.6
−1
IV has four peaks at 1298.4, 1331.7, 1352.8, and 1386.8 cm , whereas another anhydrate form V shows
Five peaks at 630.2, 642.1, Five peaks at 629.6, 642.6, Five peaks at 634.5, 643.2, Five peaks at 626.9, 940.9,
Four peaks at 630.7,
600–800
−1. It is clear that different crystal forms exhibit distinctive
three
peaks at 1306.1,
1348.8,
cm785.2
687.9, 751.2 and
691.1, 752.7 and 782.5
685.7, 754.8 and 783.1
692.2, 751.7 and 780.4
644.8, 754.3
and 777.3and 1393.36
Five peaks at 1023.0,
Three peaks at 1019.4,
Four peaks at 1021.9,
Four peaks at 1010.1,
Four peaks at 1019.9,
Raman
spectra.
1000–1200
1067.8, 1097.1 and 1171.3
Three peaks at 1308.5,
1351.3 3.
andSummary
1392.3
Table
1200–1400
Five peaks at 1416.7,
1450.0, 1479.7, 1571.0
andForm
1629.3 I
1400–1700
Raman
Shift (cm−1)
1065.3 and 1177.8
1067.8, 1096.6 and 1176.3
Four peaks at 1299.4,
Two peaks at 1307.1 and
1352.3Peak
and 1387.3
1353.3 for the Five
of1331.7,
Raman
Positions
Six peaks at 1411.3,
1454.5, 1473.8, 1569.5,
1606.6
and II
1666.5
Form
Five peaks at 1420.7,
1452.5, 1480.7, 1570.6
and Form
1629.3 III
1064.2, 1089.4 and 1176.3
1067.3, 1091.5 and 1172.3
Four peaks at 1298.4,
Three peaks at 1306.1,
1331.7, 1352.8
and 1386.8 1348.8 and 1393.3
Forms
of Valacyclovir.
Six peaks at 1457.4,
1473.8, 1565.1, 1607.0,
1633.6Form
and 1667.0
IV
Five peaks at 1416.2,
1451.0, 1477.7, 1568.5
and 1625.9
Form V
Three peaks at
Four peaks at
Three peaks at
Four peaks at
Five peaks at
409.9, 470.4, 493.1
471.5, 504.0 and
409.4, 439.9, 490.3
409.4, 438.1, 470.9,
501.8
and 536.4
527.7
and 538.1
503.5 and 578.6
As well known,
theat solid-state
structure
has
significant
influence
solubility
Four peaks
Five peaks
at
Fivea peaks
at
Five peakson
at the apparent
Five peaks
at
of600–800
the drug substance.
In
this
study,
the
apparent
solubility
of
polymorphic
solids
of
VCV
were
630.7, 644.8,
630.2, 642.1, 687.9,
629.6, 642.6, 691.1,
634.5, 643.2, 685.7,
626.9, 940.9, 692.2,
754.3
and
777.3
751.2
and
785.2
752.7
and
782.5
754.8
and
783.1
751.7
and
780.4
measured and listed in Table 4. It is obvious that all the crystal forms have excellent dissolubility with
Five peaks at
excess 100 mg/mL
in the ultrapure
water.
theattwo anhydrous
V—show
Three peaks
at However,
Four peaks
Four peaksForms—IV
at
Fourand
peaks
at
1023.0, 1067.8,
1000–1200
1019.4,
1065.3
and
1021.9,
1067.8,
1010.1,
1064.2,
1019.9,
1067.3,
best apparent solubility
1097.1 and with about 100 mg/mL compared with the other hydrates. The results of the
1177.8
1096.6 and 1176.3
1089.4 and 1176.3
1091.5 and 1172.3
present study are
consistent with
the commonly observed
trend that
the aqueous solubility
of hydrate
1171.3
Three
peaks at
Four
Four peaks at
Three peaks at
compounds can
be significantly
lesspeaks
thanat their anhydrous
forms
[17].
Two peaks at
1200–1400
1308.5, 1351.3
1299.4, 1331.7,
1298.4, 1331.7,
1306.1, 1348.8 and
1307.1 and 1353.3
and 1392.3
1352.3 and 1387.3
1352.8 and 1386.8
◦
Table 4. Apparent Solubility of all the Forms of VCV in Water (25 C). 1393.3
Five peaks at
Six peaks at
Five peaks at
Six peaks at
Five peaks at
1416.7, 1450.0,
1411.3, 1454.5,
1420.7, 1452.5,
1457.4, 1473.8,
1416.2, 1451.0,
1400–1700
Crystals
Apparent
Solubility
Crystal
after 1607.0,
Solubility Determination
1479.7, 1571.0
1473.8,
1569.5, (mg/mL)
1480.7, 1570.6
andform
1565.1,
1477.7, 1568.5 and
1606.6 145
and 1666.5
1629.3
1633.6 and
1667.0
1625.9
Form Iand 1629.3
Form
I
2.5.
Apparent Solubility
400–600
409.4, 472.6 and
Form II
143
Form II
118
Form III
Form IV
147
Form I
As well
the solid-state 161
structure has a significant influence Form
on theVapparent solubility of
Formknown,
V
Form Solubility
III
2.5. Apparent
the drug substance. In this study, the apparent solubility of polymorphic solids of VCV were
measured
listed in Table
4. It is obvious
allcompleted,
the crystal forms
have excellent
dissolubility
with
Once and
the solubility
measurements
hadthat
been
the remaining
crystals
were collected
excess
100 mg/mL
in the
ultrapure
water. suggest
However,
the
two anhydrous
and V—show
and
subjected
to PXRD
testing.
The results
that
Forms
I, II, III, andForms—IV
V did not transform
into
best
apparent
solubility
with
about
100
mg/mL
compared
with
the
other
hydrates.
The
of the
other crystals forms, but Form IV spontaneously converted to Form I during the time results
of solubility
present studyItare
consistent
with the commonly
observed
trend
the aqueous
equilibrium.
is easily
to understand
why solubility
of Form
IVthat
is close
to thosesolubility
hydrates.of hydrate
compounds can be significantly less than their anhydrous forms [17].
Form V
161
Form V
Once the solubility measurements had been completed, the remaining crystals were collected
and subjected to PXRD testing. The results suggest that Forms I, II, III, and V did not transform into
other crystals forms, but Form IV spontaneously converted to Form I during the time of solubility
Crystals 2017, 7, 140
6 of 9
equilibrium. It is easily to understand why solubility of Form IV is close to those hydrates.
2.6. Stability
Stability Studies
Studies
2.6.
Stability of
of aa drug
drug compound
compound is
is also
also an
an important
important property
Stability
property during
during pharmaceutical
pharmaceutical production.
production.
A
stability
study
for
Form
III
was
performed
by
storing
some
sample
under
a condition
of 40of°C,
◦ C,
A stability study for Form III was performed by storing some sample under
a condition
4075%
RH
for
16
weeks.
During
the
testing,
several
batches
of
specimens
were
in
turn
taken
out
at
intervals
75% RH for 16 weeks. During the testing, several batches of specimens were in turn taken out at
for PXRDforand
DSCand
measurements
to monitor
their structure
changes.
As shown
in Figure
5a, 5a,
no
intervals
PXRD
DSC measurements
to monitor
their structure
changes.
As shown
in Figure
impurity
diffraction
peaks
were
detected
in
the
PXRD
patterns
of
all
the
specimens,
which
means
the
no impurity diffraction peaks were detected in the PXRD patterns of all the specimens, which means
crystal
structure
stillstill
retained
its original
form.
the
crystal
structure
retained
its original
form.
Intensity(counts)
(a)
(E)
(D)
(C)
(B)
(A)
5
10
15
20
25
30
35
40
Heat Flow Endo Up(mW)
2-Theta(°)
(b)
(E)
(D)
(C)
(B)
(A)
100
200
Temperature(℃)
Figure
5. The
The PXRD
PXRD patterns
(a) and
and DSC
Figure 5.
patterns (a)
DSC curves
curves (b)
(b) for
for Form
Form III
III stored
stored for
for 16
16 weeks,
weeks, several
several specimens
specimens
were
taken
out
and
tested
at
(A)
0
week,
(B)
4
weeks,
(C)
8
weeks,
(D)
12
weeks,
(E)
16
weeks.
were taken out and tested at (A) 0 week, (B) 4 weeks, (C) 8 weeks, (D) 12 weeks, (E) 16 weeks.
◦ C the
inin
the
temperature
range
of 50–110
°C on
From Figure 5b,
5b, broad
broadendothermic
endothermicpeaks
peakscan
canbebeseen
seen
the
temperature
range
of 50–110
on
DSCDSC
curves
of specimens
after
stored
forfor
four
the
curves
of specimens
after
stored
fourweeks,
weeks,which
whichmeans
meansaawater
water lease
lease process.
process. However,
change the
the crystal
crystal
considering relevant unchanged
unchanged PXRD patterns,
patterns, the presence of the water does not change
structure
of
the
hemihydrate,
so
it
should
be
not
crystal
water
but
absorption
water.
In
addition,
it is
structure of the hemihydrate, so it should be not crystal water but absorption water. In addition,
it is noticed that no recognizable distinction was viewed from the melting points of all the specimens,
which further suggests that their crystal form is not changed after a long storage time. Above results
indicate that hemihydrate of VCV has good stability and suitable for use in solid drug dosage.
3. Materials and Methods
3.1. Materials
VCV raw material was obtained from Zhejiang Charioteer Medical Technology Co., Ltd.
(Zhejiang, China). Solvents—such as ethanediol, trichloromethane, acetone, ethyl alcohol,
and isopropanol—were purchased from Tianjing YongDa Chemical Reagent Co., Ltd. (Tianjing, China)
Crystals 2017, 7, 140
7 of 9
with a purity exceeding 99.5%. All aqueous solutions were prepared using Milli-Q deionized (DI)
Water with a resistivity of 10.2 MΩ·cm and total organic content <5 ppb.
3.2. Preparation of Polymorphs and Hydrates
The five crystal forms—including two anhydrous and three hydrates—were prepared and named
as Forms I, II, III, IV, and V respectively (as shown in Table 5).
Table 5. The Five Crystal Forms of VCV, Including Two Anhydrous and Three Hydrates.
Number
Form I
Form II
Form III
Form IV
Form V
Crystal form
Sesquihydrate
Monohydrate
Hemihydrate
Anhydrous one
Anhydrous two
Form I, II, IV, and V of VCV could be obtained by recrystallizing from the mixed solvent of water
and isopropanol. The VCV solution with 20 wt% raw VCV was first heated up to 80 ◦ C and refluxed
for one hour then cooled down to room temperature until crystals precipitation in full. During this
process, Forms I, II, IV, and V can be prepared respectively by adjusting the percentage of water in
mixed solvent. In fact, Form I can be obtained from mixed solvent with 10 wt% water, Form II in a
water content range of 6–8 wt%, and Form IV when the water content is lower than 5%. In particular,
Form V was obtained in the mixture with 8% water content by quenching to zero.
For the preparation of Form III, 1 g VCV raw material was dissolved in 10 mL ethanediol at
room temperature, the solution was then heated up to 40 ◦ C under stirring. A mixture of 20 mL
trichloromethane and 20 mL isopropanol was prepared and cooled to zero. The VCV solution was then
dropped into the mixed solvent with a rate of 1 mL/min, the crystals would settle out soon during this
process. At last, the precipitation was filtered and dried in an oven at 50 ◦ C.
3.3. Powder X-ray Diffraction Measurements
Powder X-ray diffraction (PXRD) patterns of all the forms of VCV were collected on a powder
X-ray diffractometer (Thermal ARL X’TRA, Thermal Technology Co., Santa Rosa, CA, USA) equipped
with Cu Kα radiation (λ = 1.54178 Å), operating with a 40 mA current and 40 kV voltage. The data
was recorded at a continuous scanning rate of 1.2◦ /min over a 2θ angular range of 3–40◦ with a 0.02◦
step size.
3.4. Thermal Analysis
Thermal analysis was carried out using a differential scanning calorimetry (PYRIS Diamond
DSC, PerkinElmer Co., Waltham, MA, USA) calibrated using the indium and zinc standards. These
VCV samples were respectively weighted and encapsulated in aluminum pans and then heated from
40–250 ◦ C at a heating rate of 5◦ /min under nitrogen gas flow of 20 mL/min.
Thermogravimetric analysis (TGA) was performed on a thermogravimetric analyzer (PYRIS 1
TGA, PerkinElmer Co., Waltham, MA, USA) which is calibrated using standard weight and the Curie
temperature of magnetic metal. About 4 mg of samples were placed in a platinum crucible and heated
from ambient temperature to 300 ◦ C at a rate of 10 ◦ C/min with a 20 mL/min dry nitrogen purge.
3.5. Raman Measurement
Raman spectra were recorded on a */PI Action Tri Vista Raman microscope (Princeton Instruments,
Trenton, NJ, USA) equipped with a 20× objective and utilizing a 633 nm laser. The scan range was 100
to 3600 cm−1 , using three scans of 30 s length each per spectrum.
3.6. Apparent Solubility Measurement
The chromatography analysis was performed on an Agilent 1100 (Agilent Co., Santa Clara, CA,
USA) HPLC equipped with an Agilent G1316B UV Detector and an octadecylsilyl silica gel C18 column
Crystals 2017, 7, 140
8 of 9
with a pore size of 10 nm. The mobile phase consisted of 0.02 mol/L monopotassium phosphate and
methyl alcohol (4:1, v/v) at a flow rate of 1.0 mL/min. The column eluent was monitored at 280 nm for
10 min and the injection volume was 20 µL at a column temperature of 25 (±2) ◦ C.
In a typical testing, a sample was respectively weighted 40, 80, 120, 160, 200, 240, and 280 mg and
added carefully into glass vials with 1.0 mL ultrapure water, then oscillated for 15 hours at 25 ◦ C until
the solution reached apparent equilibrium. Afterwards, 20 µL solution was collected through 0.22 µm
syringe filters and diluted into 10.0 mL mobile phase for HPLC testing.
3.7. Stability Testing
In order to evaluate the stability of the new crystal form product, a long-term stability testing
was carried out under certain temperature and humility. During this process, Form III sample was
stored under an accelerated condition of 40 ◦ C and 75% RH for 16 weeks. The crystal transformation
of the sample, including crystal structure and thermal behavior, was monitored periodically by PXRD
and DSC.
4. Conclusions
In this study, a few of VCV crystal forms were obtained by recrystallization in different mixed
solvents. At the same time, a new polymorphic form—hemihydrate of VCV—was first discovered and
its physicochemical properties were investigated by PXRD, DSC, TGA, and Raman.
The PXRD pattern for the new crystal form is completely different from the other polymorphs
reported previously. It has melting point measured to be 209 ◦ C from the DSC curve, and a weight loss
of 2.42 wt% calculated from TGA profile in the temperature range of 123–145 ◦ C, which confirms that
it is a hemihydrate of VCV.
The stability testing suggested the new polymorphic form is a stable crystal form and will remain
the same for several months under high temperature and humidity. The results suggest that it has
potential to be an active pharmaceutical ingredient (API) using in the drug dosage.
Acknowledgments: The authors would like to express the gratitude to the financial support from the Young
Researchers Foundation of Zhejiang Provincial Top Key Academic Discipline of Chemical Engineering and
Technology, Zhejiang Sci-Tech University (ZYG2015007), Zhejiang Provincial Natural Science Foundation under
Grant No. LY16B010002.
Author Contributions: Guoqing Zhang, Shuai Zhang, and Mengqing Zhou conceived and designed
the experiments; Shuai Zhang, Mengqing Zhou, Meiqi Zheng, and Zhaoxia Zhang performed the
experiments; Shuai Zhang and Mengqing Zhou analyzed the data; Tian Chen and Naixing Wang contributed
reagents/materials/analysis tools; Shuai Zhang, Mengqing Zhou, and Guoqing Zhang wrote the paper.
Conflicts of Interest: The authors report no conflicts of interest and are responsible for the content and writing of
the paper.
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