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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 Engineering Research Center for Eco-Dyeing and Finishing of Textiles, Ministry of Education,
Zhejiang Sci-Tech University, Hangzhou 310018, China; yyz291059810@163.com (S.Z.);
meiqizheng1994@163.com (Mei.Z.); zhoumq34@163.com (Men.Z.); zhangzx@zstu.edu.cn (Z.Z.)
2 Zhejiang Charioteer Pharmaceutical Co., Ltd., Taizhou 317321, China; Chentian@charioteer.cn (T.C.);
wangnaixing@charioteer.cn (N.W.)
* Correspondence: zgq@zstu.edu.cn; 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


Crystals 2017, 7, 140 2 of 9
Crystals 2017, 7, 140 2 of 9

O NH2
H CH3

N · HCl
HN
O CH3
N N
H2N O
O

Figure1.1.Molecular
Figure Molecular structure
structure scheme
schemeof
ofVCV.
VCV.

In this work, a new crystal form—hemihydrate—was obtained by recrystallization technology,


In thiswith
together work,thea other
new crystal form—hemihydrate—was
four crystal obtainedusing
forms of VCV were investigated by recrystallization technology,
powder X-ray diffraction
together with the other four crystal forms of VCV were investigated using powder
(PXRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). In addition, X-ray diffraction
(PXRD), differential
the testing scanning
for apparent calorimetry
solubility (DSC), and thermogravimetric
and thermostability was also carried out analysis
for deep(TGA). In addition,
understanding
thetheir
testing for apparent solubility
pharmaceutical potential. and thermostability was also carried out for deep understanding
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
their to aboveproperties
physicochemical experiments,
were thealsofive solid forms
characterized byof VCVDSC,
PXRD, wereand successfully
TGA. prepared and
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
provide crystal structure
the fundamental determination
structural from an
understanding of analysis of single-crystal
polymorphic diffraction
solids, the difficulty can provide
of preparing
theafundamental structural understanding of polymorphic solids, the difficulty
suitable single-crystal precludes this technique from being used on a routine basis of preparing a suitable
for
characterization
single-crystal of most
precludes thisdrug crystalfrom
technique forms. Since
being most
used on drug substances
a routine basis forwere obtained as micro-
characterization of most
crystalline
drug powders,
crystal forms. SincethemostPXRD technologywere
drug substances becomes theaspredominant
obtained micro-crystallinetool powders,
for the the
routine
PXRD
characterization of polymorphs [4]. In this study, no any suitable single-crystal of
technology becomes the predominant tool for the routine characterization of polymorphs [4]. In this the five VCV crystal
forms
study, nowas
anysuccessfully prepared under
suitable single-crystal of thecurrent
five VCVexperiment conditions.
crystal forms The X-ray diffraction
was successfully prepared data
under
relevant
current to the five
experiment powder crystalline
conditions. The X-rayforms are summarized
diffraction in Table
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 Table 1.IIReflections of the
Form Formfive
III crystal formsForm
of VCV.
IV Form V
2θ(°) Intensity (%) 2θ(°) Intensity (%) 2θ(°) Intensity (%) 2θ(°) Intensity (%) 2θ(°) Intensity (%)
3.66Form 100
I 3.56Form100
II 3.46 100
Form III 3.66
Form IV 63.1 3.50 V33.6
Form
8.58 2.6 8.58 5.2 6.94 93.1 8.66 41.1 6.58 58.7

2θ(9.48
) Intensity ◦ ◦ ◦ ◦
6.0 (%) 2θ( 9.38
) Intensity
5.2 (%) 2θ( 7.88
) Intensity
13.2(%) 2θ( )10.62
Intensity
13.8(%) 2θ( ) Intensity
9.18 (%)
100
3.66 9.8
10.60 100 3.56 100
10.52 8.6 3.46 100
8.46 10.9 3.66
10.92 63.1
48.5 3.50
11.3233.618.8
10.868.58 24.4
2.6 8.58 15.8
10.78 5.2 6.94 93.1
9.30 13.3 8.66
12.18 41.1
18.3 6.58
15.3458.713.3
9.48 7.8
13.36 6.0 9.38 5.2
12.08 0.9 7.88
10.46 13.2
9.5 10.62
13.32 13.8
12.1 9.18
15.62 10016.9
10.60 3.9
16.44 9.8 10.52 6.1
13.26 8.6 8.46
11.64 10.9
8.4 10.92
14.56 48.5
51.1 11.32
16.16 18.8
10.4
10.86 2.0
20.20 24.4 10.78 15.8
14.42 2.5 9.30 13.3
13.02 11.5 12.18
15.20 18.3
14.3 15.34
17.02 13.3
16.1
13.36 2.9
21.42 7.8 12.08 5.3
16.36 0.9 10.46 9.5
14.44 12.9 13.32 12.1
16.48 52.2 15.62 16.9
19.14 18.8
16.44 3.9
21.80 7.2
13.26 6.1
20.04 2.5
11.64 8.4
15.34 9.2
14.56 51.1
16.96 22.5
16.16 10.4
19.66 9.0
20.20 2.0
24.00 3.2
14.42 2.5
21.34 2.2
13.02 11.5
16.25 11
15.20 14.3
18.16 33.8
17.02 16.1
21.26 19.6
21.42 2.9 16.36 5.3 14.44 12.9 16.48 52.2 19.14 18.8
26.76 2.1 23.92 9.3 20.78 11.4 20.10 34.1 22.86 15.0
21.80 7.2 20.04 2.5 15.34 9.2 16.96 22.5 19.66 9.0
27.28 5.2 25.94 1.8 23.47 6.6 20.62 7.1 24.16 10.8
24.00 3.2 21.34 2.2 16.25 11 18.16 33.8 21.26 19.6
26.82 2.4 23.90 9.9 21.28 15.3 26.30 8.4
26.76 2.1 23.92 9.3 20.78 11.4 20.10 34.1 22.86 15.0
27.22 4.1 24.54 19.5 21.88 25.7 27.34 17.8
27.28 5.2 25.94 1.8 23.47 6.6 20.62 7.1 24.16 10.8
26.18 11.0 22.72 8.8 27.86 15.3
26.82 2.4 23.90 9.9 21.28 15.3 26.30 8.4
27.16 19.6 24.02 100 28.76 3.4
27.22 4.1 24.54 19.5 21.88 25.7 27.34 17.8
27.80 11.6 24.46 15.9
26.18 11.0 22.72 8.8 27.86 15.3
25.13 6.8
27.16 19.6 24.02 100 28.76 3.4
25.94 21.1
27.80 11.6 24.46 15.9
26.26 60.8
25.13 6.8
26.92 10.6
25.94 21.1
27.26 60.8
26.26 8.4
28.10 10.6
26.92 7.9
28.38 8.4
27.26 6.5
29.00 7.9
28.10 16.2
29.70 6.5
28.38 8.8
30.64 16.2
29.00 18.1
29.70 8.8
30.64 18.1
Crystals 2017, 7, 140 3 of 9

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
peaks at 2θ 3.5◦the
From
◦ , 7.9◦ , 8.5◦ , 9.3◦ , 13◦ , 14.4◦ , 16.3◦ , 20.8◦ , 24.5◦ , and 27.2◦ —is completely distinctive
, 6.9diffraction patterns, one can see that Form I, II, IV and V are consistent as previously
from reported
those ofcrystalthe other
forms.four forms.theIn
However, particular,
PXRD pattern ofForm III exhibited
the Form III—with several verydiffraction
characteristic characteristic
at 6.9at◦ ,2θ
peakspeaks 7.9 ◦ , 13◦ , 20.8◦ and 23.5◦ , where no any peak can be detected in the patterns of other
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
forms. those of the
It indicates thatother
Form fourIII
forms.
should In particular,
be a new Form III exhibited
polymorphic form several veryIn
of VCV. characteristic peaks this
order to confirm
point,atthermal
6.9°, 7.9°,analysis
13°, 20.8°including
and 23.5°, where
DSC and no any
TGA peak can be detected
technology wereinused
the patterns of other
to further forms.
analyze theIt new
indicates
polymorphic form. that Form III should be a new polymorphic form of VCV. In order to confirm this point,
thermal analysis including DSC and TGA technology were used to further analyze the new
polymorphic
2.2. Differential form. Calorimetry
Scanning
The DSC thermograms
2.2. Differential of the five crystal forms of VCV are presented in Figure 2. For Form I, II,
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,
endothermic peaks on crystal
their DSC scans because ◦C
and III—different forms—as hydratesofcontaining
water lossa in the temperature
certain rangethere
of crystal water, of 60–140
are
(Figure 2a). DSC
significant curves ofpeaks
endothermic FormonI andtheir II
DSCshowed two partially
scans because of wateroverlapping endothermic
loss in the temperature rangepeaks
of at
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
structure,
peakswhereas
at 70 andonly one
130 °C endothermic
respectively, which event
means at about
there are ◦ Cdifferent
141two is seen for Form
types III. Inwater
of crystal addition,
in thethere
crystal structure, whereas only one ◦
endothermic event at about 141 °C is seen
are not any endothermic peaks in 60–140 C on the runs of Form IV and Form V, which indicates that for Form III. In addition,
both there
of them are not
are any endothermic
anhydrous peaks in 60–140 °C on the runs of Form IV and Form V, which indicates
compounds.
that both of them are anhydrous
The melt/decomposition events compounds.
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 onthetheDSCDSCcurve,
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 point point
respectively. Melting is an important
is an important parameter parameter for a polymorphic
for a polymorphic drug, which to drug,
somewhich to some
extent can
extentbecanused betoused to identify
identify different
different crystal form.crystal form. temperatures
The melting The meltingoftemperatures
the four forms of theestimated
were four forms and were
estimated
listed and listed
in Table in Table
2. Form 2. Form
III has III has transformation
a solid-solid a solid-solid transformation
at about 197 °C, at about
which 197 ◦itC,
means is which
probably means
already turned into another crystalline form after the dehydration process.
it is probably already turned into another crystalline form after the dehydration process.

(a) (b)
Heat Flow Endo Up(mW)

( 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 Form II Form III Form IV Form V
Melting point (°C) 205 206 209 227 223
Items Form I Form II Form III Form IV Form V
Transition 1 (°C) 53–139 60–140 124–141 / /
point (◦ C)2 (°C)
Melting Transition 205 / 206
183–196 209
186–206 / 227 / 223
Transition 1 (◦ C) 53–139 60–140 124–141 / /
Transition 2 (◦ C) / 183–196 186–206 / /
Crystals 2017, 7,
Crystals 2017, 7, 140
140 44 of
of 99

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.

105 105

100
Form Ⅰ (a) (b)
FormⅡ 100
95 95
FormⅤ
90
90
85 FormⅢ FormⅣ
85
Weight(%)


80
80
75
50 100 150
75
70 Ⅱ

70
65

60 80 120 65

55 60

50 55
130 140

100 200 300 100 200 300

Temperature(℃)
Figure 3. TGA
Figure 3. TGA curves
curves of VCV polymorphs:
of VCV 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,
the
the insert
insert is
is the
the profiles
profiles of
of the
the derivative
derivative curves.
curves.

In addition,
In addition,thetheprofiles
profilesofofthe
thederivative
derivative curves
curves insert
insert in Figure
in Figure 3a show
3a show there
there are two
are two weightweight
loss
loss stages
stages for Forma
for Forma I and IIIand
whileII Form
while III
Form
has III
onlyhas only
one. one.results
These Theseare
results arecorresponding
totally totally corresponding
with the
with profiles
DSC the DSCshown
profiles shown2.inInFigure
in Figure 2. Inwater
fact, each fact, each
lease water lease stage
stage usually usually
stands stands for
for a specific a specific
combination
combination
mode between mode
water between
and VCV water and VCV
molecules. Thismolecules.
suggests This suggests
that there that bonding
are two there aremodes
two bonding
among
water with VCV molecular in VCV hydrates for Form I and II but only one for Form III. FormForm
modes among water with VCV molecular in VCV hydrates for Form I and II but only one for DSC
III. Form
and TGA DSC and
results ofTGA
Formresults
III, theofsingle
Formwater
III, theloss
single water
process loss process
actually actually
confirms confirms
that it is a purethat it is a
hydrate
pure hydrate
crystalline crystalline
form with halfform with
crystal half crystal water.
water.

2.4. Raman Spectroscopy


As well
well known,
known,Raman
Ramanspectroscopy
spectroscopy is aisreliable
a reliable technology
technology to differentiate
to differentiate crystalcrystal
forms.forms.
From
From the Raman
the Raman spectra
spectra of theof theVCV
five five VCVcrystalcrystal
formsforms in Figure
in Figure 4, the4,most
the most distinctive
distinctive region region can
can be be
seen
between
seen 12501250
between and 1400 cm cm
and 1400 −1 − 1
. In this. Inregion, FormForm
this region, I and IVand
show threethree
V show peaks, FormForm
peaks, II andII IV
andhave four
IV have
peaks,
four but Form
peaks, III has
but Form IIIonly two peaks.
has only 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 5 of 9
Crystals 2017, 7, 140 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 TheRaman
4. The
Figure 4. Ramanspectra
spectra
of of
thethe
fivefive forms
forms of valacyclovir
of valacyclovir (all spectra
(all spectra were were collected
collected at
at room
room temperature).
temperature).

From Table Table 3. Summary


3, Form I has twoof Raman
peaksPeak Positions
at 1308.5 andfor the Five
1351.3 cmForms
−1 withofaValacyclovir.
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
Raman Shift (cm−1 ) Form I Form II Form III Form IV Form V
1387.3 cm−1. The new crystal
Three peaks at 409.4,FormFour
IIIpeaks
shows two peaks
at 409.9, at 1307.1
Three peaks at 471.5, and 1353.3
Four peaks atcm409.4, . TheFive
−1 anhydrate Form
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
IV has four peaks at 1298.4, 1331.7, 1352.8, and 1386.8 cm , whereas another anhydrate form V shows
−1
Four peaks at 630.7, 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,
three peaks at 1306.1,
600–800 1348.8,
644.8, 754.3 and 777.3and 1393.36 cm785.2
687.9, 751.2 and
−1. It is clear that different crystal forms exhibit distinctive
691.1, 752.7 and 782.5 685.7, 754.8 and 783.1 692.2, 751.7 and 780.4

Raman spectra.
1000–1200
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,
1067.8, 1097.1 and 1171.3 1065.3 and 1177.8 1067.8, 1096.6 and 1176.3 1064.2, 1089.4 and 1176.3 1067.3, 1091.5 and 1172.3
Three peaks at 1308.5, Four peaks at 1299.4, Two peaks at 1307.1 and Four peaks at 1298.4, Three peaks at 1306.1,
1200–1400
Table
1351.3 3.
andSummary
1392.3 of1331.7,
Raman 1352.3Peak Positions
and 1387.3 1353.3 for the Five Forms of Valacyclovir.
1331.7, 1352.8 and 1386.8 1348.8 and 1393.3
Five peaks at 1416.7, Six peaks at 1411.3, Five peaks at 1420.7, Six peaks at 1457.4, Five peaks at 1416.2,
1400–1700
Raman 1450.0, 1479.7, 1571.0 1454.5, 1473.8, 1569.5, 1452.5, 1480.7, 1570.6 1473.8, 1565.1, 1607.0, 1451.0, 1477.7, 1568.5
andForm
1629.3 I Form
1606.6 and II
1666.5 and Form
1629.3 III 1633.6Form IV
and 1667.0 Form V
and 1625.9
Shift (cm−1)
Three peaks atFour peaks at Three peaks at Four peaks at Five peaks at
2.5. Apparent Solubility
400–600 409.4, 472.6 and 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,
Four peakstheat solid-state structure
Five peaks at has
Fivea peaks
significant
at influence
Five peaksonat the apparent
Five peakssolubility
at
of600–800
the drug substance.
630.7, 644.8, In this study, the apparent
630.2, 642.1, 687.9, solubility
629.6, 642.6, 691.1, of polymorphic
634.5, 643.2, 685.7,solids of VCV were
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
measured and listed in Table 4. It is obvious that all the crystal forms have excellent dissolubility with 751.7 and 780.4
Five peaks at
excess 100 mg/mL in the ultrapure water.
Three peaks at However, theattwo anhydrous
Four peaks Four peaksForms—IV
at Fourand V—show
peaks at
1023.0, 1067.8,
1000–1200
best apparent solubility 1019.4, 1065.3 and 1021.9, 1067.8, 1010.1, 1064.2, 1019.9, 1067.3,
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
1171.3 the commonly observed trend that the aqueous solubility of hydrate
compounds can be significantly
Three peaks at lesspeaks
Four thanat their anhydrous
Two peaks at
forms [17].
Four peaks at Three 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
Table 4. Apparent Solubility of all the Forms of VCV in Water (25 C). 1393.3
1352.3 and 1387.3 1352.8 and 1386.8

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
1479.7, 1571.0 1473.8, 1569.5, (mg/mL) Crystal
1480.7, 1570.6 andform after 1607.0,
1565.1, Solubility Determination
1477.7, 1568.5 and
Form Iand 1629.3 1606.6 145
and 1666.5 1629.3 1633.6 and 1667.0
Form I 1625.9
Form II 143 Form II
Form Solubility
2.5. Apparent III 118 Form III
Form IV 147 Form I
As well
Formknown,
V the solid-state 161
structure has a significant influence Form
on theVapparent solubility of
the drug substance. In this study, the apparent solubility of polymorphic solids of VCV were
measured
Once and listed in Table
the solubility 4. It is obvious
measurements hadthat
beenallcompleted,
the crystal forms have excellent
the remaining dissolubility
crystals with
were collected
excess
and 100 mg/mL
subjected to PXRDin the ultrapure
testing. water. suggest
The results However, the
that two anhydrous
Forms I, II, III, andForms—IV and V—show
V did not transform into
other crystals forms, but Form IV spontaneously converted to Form I during the time results
best apparent solubility with about 100 mg/mL compared with the other hydrates. The of the
of solubility
present studyItare
equilibrium. consistent
is easily with the commonly
to understand observed
why solubility trend
of Form IVthat the aqueous
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
2.6. Stability Studies
Studies
Stability of
Stability of aa drug
drug compound
compound is is also
also an
an important
important property
property during
during pharmaceutical
pharmaceutical production.
production.
A stability study for Form III was performed by storing some
A stability study for Form III was performed by storing some sample under sample under a condition of 40of°C,
a condition 4075%
◦ C,
RH for 16 weeks. During the testing, several batches of specimens were in turn
75% RH for 16 weeks. During the testing, several batches of specimens were in turn taken out at taken out at intervals
for PXRDforand
intervals PXRDDSCand measurements
DSC measurements to monitor their structure
to monitor changes.
their structure As shown
changes. in Figure
As shown 5a, 5a,
in Figure no
impurity diffraction peaks were detected in the PXRD patterns of all the specimens,
no impurity diffraction peaks were detected in the PXRD patterns of all the specimens, which means which means the
crystal
the structure
crystal structurestillstill
retained its original
retained form.
its original form.

Intensity(counts) (a)

(E)

(D)

(C)

(B)
(A)
5 10 15 20 25 30 35 40

2-Theta(°)

(b)
Heat Flow Endo Up(mW)

(E)

(D)

(C)

(B)

(A)

100 200

Temperature(℃)

Figure The PXRD


5. The
Figure 5. PXRD patterns (a) and
patterns (a) and DSC
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.

From Figure 5b,


5b, broad
broadendothermic
endothermicpeaks peakscan
canbebeseen inin
seen the temperature
the range
temperature of 50–110
range ◦ C the
°C on
of 50–110 on
DSCDSC
the curves of specimens
curves after
of specimens stored
after forfor
stored four
fourweeks,
weeks,which
whichmeans
meansaawater
water lease
lease process.
process. However,
considering relevant unchanged
unchanged PXRD patterns,
patterns, the presence of the water does not change
change the
the crystal
crystal
structure of the hemihydrate, so it should be not crystal water but absorption water. In
structure of the hemihydrate, so it should be not crystal water but absorption water. In addition, addition, it is
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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