New dipyridamole salt with improved dissolution and oral bioavailability under hypochlorhydric conditions

Article abstract of DOI:10.2133/dmpk.DMPK-12-RG-139

The aim of this study was to develop new dipyridamole (DP) salts with pH-independent solubility for improving oral bioavailability under hypochlorhydria. Salt screening was carried out using nine counterions by the temperature gradient method. Six DP salts were obtained, and there was marked improvement in dissolution behavior for all DP salts in a neutral medium. Most DP salts were stable under accelerated conditions. On the basis of the dissolution and stability data, DP tosylate (DP/TS) was selected as a promising DP salt. The pharmacokinetics of DP and the promising DP salt were assessed in normal rats and omeprazole-treated rats as a hypochlorhydric model. After oral administration of DP/TS (10mg-DP/ kg) in normal rats, enhanced DP exposures with increased C_max and AUC_0-3 were observed compared with those with DP by ca. 2.8- and 1.7-fold, respectively. There was ca. 1 h delay of T_max and ca. 62% reduction of AUC_0-3 for DP in omeprazole-treated rats compared with those for DP in normal rats; however, oral absorption for DP/TS under hypochlorhydria was almost identical to that in normal rats. The newly developed DP/TS might provide better therapeutic efficacy in clinical use for hypochlorhydric patients.

Full text of DOI:10.2133/dmpk.DMPK-12-RG-139

Drug Metab. Pharmacokinet. 28 (5): 383–390 (2013).
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)
Regular Article
New Dipyridamole Salt with Improved Dissolution and
Oral Bioavailability under Hypochlorhydric Conditions
1
1
2
Chika TANIGUCHI1,2, Ryo INOUE , Masashi KATO , Kazuhiro YAMASHITA ,
2
2
1
1,
*
Yohei KAWABATA , Koichi WADA , Shizuo YAMADA and Satomi ONOUE
1
Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences,
University of Shizuoka, Shizuoka, Japan
2
Department of Chemistry, Manufacturing and Control, Kobe Pharma Research Institute,
Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan
Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: The aim of this study was to develop new dipyridamole (DP) salts with pH-independent solu-
bility for improving oral bioavailability under hypochlorhydria. Salt screening was carried out using nine
counterions by the temperature gradient method. Six DP salts were obtained, and there was marked
improvement in dissolution behavior for all DP salts in a neutral medium. Most DP salts were stable under
accelerated conditions. On the basis of the dissolution and stability data, DP tosylate (DP/TS) was selected
as a promising DP salt. The pharmacokinetics of DP and the promising DP salt were assessed in normal rats
and omeprazole-treated rats as a hypochlorhydric model. After oral administration of DP/TS (10 mg-DP/
kg) in normal rats, enhanced DP exposures with increased Cmax and AUC0-3 were observed compared with
those with DP by ca. 2.8- and 1.7-fold, respectively. There was ca. 1 h delay of Tmax and ca. 62% reduction
of AUC0-3 for DP in omeprazole-treated rats compared with those for DP in normal rats; however, oral
absorption for DP/TS under hypochlorhydria was almost identical to that in normal rats. The newly
developed DP/TS might provide better therapeutic efficacy in clinical use for hypochlorhydric patients.
Keywords: dipyridamole; salt screening; dissolution; bioavailability; hypochlorhydria
with hypochlorhydric subjects compared with that in control
Introduction
subjects.9) In addition to low secretion of hydrochloric acid, gastric
Dipyridamole [2,6-bis(diethanolamino)-4,8-dipiperidinopyrimi-
do[5,4-d]pyrimidine] (DP) is a thromboxane synthase inhibitor
that is clinically used as a vasodilatory agent and an antithrombotic
agent.1,2) DP is well known to be a weakly basic drug with a pKa
value of 6.43) and to exhibit pH-dependent solubility, with high
solubility at acidic pH and low solubility above pH 4.4) DP is
categorized as a Biopharmaceutical Classification System (BCS)
class II compound.4) For BCS class II compounds with high
permeability and low solubility, drug absorption is often limited
by drug solubility and dissolution.5) In clinical practice, a hypo-
chlorhydric situation often occurs in young infants, ca. 30% of
elderly,6) subjects infected with Helicobactor pylori7) or human
immunodeficiency virus (HIV),8) and patients treated with anti-
ulcer drugs. In previous clinical research, oral absorption of DP
was decreased in famotidine-treated patients with increased gastric
pH, and the oral bioavailability of DP was reduced by ca. 60%
pH could elevate after a meal,9) and elevated gastric pH leads to
incomplete oral absorption of DP with pH-dependent solubility,
possibly leading to inconsistent clinical outcomes. Therefore,
enhancement of DP solubility at neutral pH could improve oral
bioavailability in hypochlorhydric patients.
Various techniques have already been developed to enhance
solubility and dissolution behavior. In formulation development,
solid dispersion,10,11) micronization,12,13) cyclodextrin complexa-
tion14) and a microenvironmental pH-modifier approach15–17) have
already been performed to enhance the dissolution behavior of DP.
In particular, the microenvironmental pH-modifier approach might
be promising because of the simplified manufacturing process
and cost performance. In our previous study, new DP immediate-
release formulations were developed using the microenvironmental
pH-modifier approach, and these DP formulations successfully
improved dissolution behavior and oral absorption under hypo-
Received December 5, 2012; Accepted January 22, 2013
J-STAGE Advance Published Date: February 19, 2013, doi:10.2133/dmpk.DMPK-12-RG-139
*
To whom correspondence should be addressed: Satomi ONOUE, Ph.D., Department of Pharmacokinetics and Pharmacodynamics, School of
Pharmaceutical Sciences, University of Shizuoka, 52-1, Yada, Suruga-ku, Shizuoka 422-8526, Japan. Tel. ©81-54-264-5633, Fax. ©81-54-264-
5635, E-mail: onoue@u-shizuoka-ken.ac.jp
This work was supported in part by a Grant-in-Aid for Young Scientists (B) (No. 24590200; S. Onoue) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan.
383

384
Chika TANIGUCHI, et al.
chlorhydric conditions.18,19) In the pharmaceutical industry, salt
formation is a crucial step in drug development since it has a huge
impact on physicochemical properties of drug substances.20–22) In
particular, salt formation has been identified as the most common
and effective method of increasing solubility and dissolution rates
of both acidic and basic drugs without modifying their chemical
structures, and salt formation also provides a means of altering the
hygroscopicity, stability, impurity profile and particle character-
istics of a drug at reasonable cost. For DP, salt formation could also
be a beneficial approach to improve dissolution and oral absorp-
tion. However, there are a limited number of reports and patents on
salt formation of DP,23) and their biopharmaceutical properties are
far less well known.
Scanning electron microscopy (SEM)
Representative SEM images of DP samples were taken using a
scanning electron microscope, VE-7800 (Keyence Corporation,
Osaka, Japan), without Au or Pt coating. For the SEM observa-
tions, each sample was fixed on an aluminum sample holder using
double-sided carbon tape.
Powder X-ray diffraction (PXRD) analysis: The PXRD
pattern was collected with a D8 Advance (Bruker AXS GmbH,
Karlsruhe, Germany) with Cu K¡ radiation generated at 40 mA
and 35 kV. Data were obtained from 3° to 35° (2ª) at a step size of
0.2° and scanning speed of 6°/min.
Thermal analysis: TG/DTA was performed using a Simulta-
neous DTA-TG Apparatus DTG-60 (Shimadzu Co., Ltd., Kyoto,
Japan). TG/DTA thermograms were collected in an aluminum
open-pan system using a sample weight of ca. 3 mg and a heating
rate of 10°C/min with nitrogen purge at 20 mL/min.
The present study aimed to develop new DP salts with
pH-independent solubility to improve oral bioavailability under
hypochlorhydric conditions. DP salts were prepared using nine
counterions by the temperature gradient method. The physico-
chemical properties of the obtained DP salts were characterized by
polarized light microscopy (PLM), scanning electron microscopy
(SEM), powder X-ray diffraction (PXRD) and thermogravimetric
and differential thermal analyses (TG/DTA). Spectroscopic char-
acterization of DP and DP salts was carried out using ultraviolet
(UV) spectral analysis to anticipate a possible change of photo-
sensitivity by salt formation. A promising salt was selected on
the basis of the results on the physicochemical properties. A
pharmacokinetic study on the promising DP salt was also per-
formed to clarify the possible improvement in oral bioavailability
using omeprazole-treated rats as a hypochlorhydric model.
Determination of water content in DP salts: The water
content in DP salts was determined using a Karl Fischer Moisture
Titrator, MKA-520 (Kyoto Electronics Manufacturing Co., Ltd.,
Kyoto, Japan).
Ion chromatography: Ion chromatography was performed
using an HIC-20A with a suppressor type of conductivity detector
(Shimadzu Co., Ltd.), and Shim-pack IC-SA2 (Shimadzu Co.,
Ltd.) was used as a column for anion analysis. The solution of
0.6 mM sodium carbonate–12 mM sodium hydrogen carbonate was
used as a mobile phase at a constant flow rate of 1.0 mL/min.
Inductively coupled plasma atomic emission spectrometry
(ICP-AES): ICP-AES was performed using an ICP emission
spectrometer ICPS-8100 (Shimadzu Co., Ltd.) at 180.731 nm for
sulfur and 178.278 nm for phosphorus with argon gas as the carrier
gas.
Materials and Methods
Chemicals: Dipyridamole (DP) was produced by Boehringer
Ingelheim GmbH (Ingelheim, Germany), and the specification tests
were carried out according to the Japanese pharmacopeia (15th
edition). L-Tartaric acid (TA) was purchased from Tartarica Treviso
S.r.l. (Villorba, Italy). Citric acid monohydrate (CA) was purchased
from Jungbunzlauer Ladenburg GmbH (Ladenburg, Germany).
Fumaric acid (FA) was purchased from Bartek Ingredients Inc.
(Ontario, Canada). Ethyl acetate, tetrahydrofuran (THF), toluene,
2-propanol, hydrochloric acid (HCl), phosphoric acid (PA), sulfuric
acid (SA), p-toluenesulfonic acid monohydrate (TS), benzenesul-
fonic acid monohydrate (BS) and maleic acid (MA) were pur-
chased from Wako Pure Chemical Industries (Osaka, Japan).
Methanol and acetonitrile were purchased from Thermo Fisher
Scientific Inc. (Waltham, MA). All other chemicals were purchased
from commercial sources.
Moisture sorption analysis: Dynamic vapor sorption (DVS)
was recorded using an Automated Water Sorption Analyser (DVS-
1, Surface Measurement Systems Ltd., London, UK). For mois-
ture sorption analysis, ca. 5 mg of each sample was stored under
humidity levels that varied between 0 and 95% RH at 25°C.
Dipyridamole determination in salts: The amount of DP
in the DP salt was determined using an HPLC system with UV
detection at 410 nm, Waters Alliance 2695 with Dual- absorbance
detector 2487 (Waters Corporation, Milford, MA). An ODS column
(particle size: 5 µm, column size: 3.0 mm © 60 mm; Inertsil ODS-2,
GL Sciences, Inc., Torrance, CA) was used and column temperature
was maintained at 40°C. Samples were separated using an isocratic
mobile phase consisting of 0.48 M ammonium formate buffer
(pH 6.5), methanol and acetonitrile (580:240:180) with a flow rate
of 1.0 mL/min, and the retention time of DP was ca. 15 min. Purity
was calculated against standard solution.
Preparation of dipyridamole salts: Crystalline salts of
dipyridamole were prepared using a 500-mg scale. Salt screening
for DP was carried out in methanol, acetonitrile, 2-propanol, ethyl
acetate, THF and toluene. The resultant products were filtrated
and dried under a nitrogen atmosphere. The obtained solids
were characterized by ion chromatography, inductively coupled
plasma atomic emission spectrometry (ICP-AES), PXRD and
TG/DTA.
Stability testing:
Stress stability testing
For stress stability study, ca. 30 mg of each DP salt was poured
into a 25 mL brown glass bottle. The samples were stored at 40 «
2°C/75 « 5% relative humidity (RH) in a stability chamber, SRH-
15VEVJ2 (Nagano Science Co. Ltd., Osaka, Japan), and 60 « 2°C
in a stability chamber, LH21-15M (Nagano Science Co. Ltd.),
for 12 weeks. After storage, the samples were evaluated to purity
according to the section on Dipyridamole determination in salts.
Photostability testing
Micrographic images:
Polarized light microscopy (PLM)
Representative PLM images of DP samples were taken using a
BX53-P microscope (Olympus Co. Ltd., Tokyo, Japan). DP sam-
ples were examined under various conditions, including differential
interference contrast, using slightly uncrossed polars and a red
wave compensator.
For photostability testing, each DP salt containing ca. 5 mg of
DP was weighed exactly and spread in a 25 mL clear glass bottle.
The samples were stored in a SUNTEST XLS+ (Atlas Material
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)

Dipyridamole Salts with Improved Absorption under Hypochlorhydria
385
Technology LLC, Chicago, IL) and the amount of DP remaining
in the DP salt was determined by HPLC as described in the section
on Dipyridamole determination in salts. The UVA/B and visible
light irradiation (300–800 nm) was carried out at 25°C with an
irradiance of 250 W/m2 for 1 h and 24 h.
Each blood sample (200 µL) was centrifuged at 10,000 © g for
5 min at 4°C to prepare plasma samples. To 50 µL of plasma
sample, 150 µL of methanol was added, and the solution was
centrifuged at 3,000 © g for 10 min. The supernatant was filtrated
through a 0.2-µm filter, and then the filtrate was analyzed by an
internal standard method using a Waters Acquity UPLC system
(Waters), which included a binary solvent manager, sample man-
ager, column compartment and SQD connected with MassLynx
software. An Acquity UPLC BEH C 18 column (particle size:
1.7 µm, column size: 2.1 mm © 50 mm; Waters) was used, and the
column temperature was maintained at 40°C. The samples were
separated using a gradient mobile phase consisting of acetonitrile
(A) and 5 mM ammonium acetate (B) with a flow rate of 0.25
mL/min. The gradient conditions of the mobile phase were 0–0.5
min, 40% A; 0.5–2.5 min, 40–95% A; 2.5–3.0 min, 95% A; and
3.0–3.5 min, 40% A. Analysis was carried out using selected ion
recording (SIR) for specific m/z 429 and 505 for DP, and DP was
detected at a retention time of 1.53 min.
Dissolution properties:
Preliminary dissolution testing
Dissolution screening was carried out for 60 min at 200 rpm in
20 mL of 0.05 M monobasic potassium phosphate buffer solu-
tion (pH 6.8) using a small volume dissolution tester, the µ-DISS
ProfilerTM (Pion Inc., Woburn, MA) at 37°C. The DP salt was
weighed to keep the total amount of DP in the dissolution vessel
constant at ca. 3 mg. A sample volume of 5 mL was withdrawn
at 60 min, filtrated through a 0.45-µm membrane filter and
then measured with a UV-visible spectrophotometer UV-2550
(Shimadzu Co., Ltd.) at 298 nm.
Dissolution testing
Dissolution tests were carried out for 2 h by the paddle method
at 50 rpm in 900 mL of 0.05 M monobasic potassium phosphate
buffer solution (pH 6.8) and 0.1 M HCl solution (pH 1.0) using
the dissolution tester system with a UV automatic flow system,
NTR-6100 (Toyama Sangyo Co., Ltd., Osaka, Japan), at 37°C.
The sample was weighed to keep the total amount of DP in the
dissolution vessel constant at 50 mg. Samples were measured at
the indicated times with an automatic UV flow cell at 298 nm for
pH 6.8 buffer solution and at 283 nm for 0.1 M HCl solution. The
supersaturation level was calculated from the actual DP concen-
tration in the dissolution medium (C) and equilibrium DP solubility
(Ceq) as the C/Ceq value.
Statistical analysis: For statistical comparisons, one-way ana-
lysis of variance (ANOVA) with pairwise comparison by Fisher’s
least significant difference procedure was used. A p value of less
than 0.05 was considered significant for all analyses.
Results and Discussion
Preparation and physicochemical properties of dipyrida-
mole salts: In the present study, salt screening of dipyridamole
(DP) salts was conducted with nine counterions by the temperature
gradient method, and these counterions were selected on the basis
of oral toxicity and their pKa values, as described in Table 125)
;
UV spectral analysis: DP samples were dissolved in 50%
methanol solution at a final concentration of 0.02 mg/mL. UV-
visible absorption spectra were recorded with a UV-visible spectro-
photometer, the UV-2550 (Shimadzu Co., Ltd.).
this was because a pKa difference between acid and base (¦pKa)
of at least 2 or 3 is generally required to form a salt.26,27) Salt
screening was firstly carried out in methanol, acetonitrile, 2-
propanol, ethyl acetate, THF or toluene, and, on the basis of crys-
tallization kinetics and yields, 2-propanol and THF were found to
be suitable crystallization solvents (data not shown). The DP salts
with six counterions were successfully obtained, and the yield
of these salts was over 50% (Table 1); however, the other three
acids, namely L-tartaric acid (TA), fumaric acid (FA) and citric acid
(CA), did not form any DP salts. The differences in pKa values
between DP and each of these three counterions were below 3.5,
and those for the other six counterions were above 4. This finding
suggested that ¦pKa values greater than 4 might be required for
DP salt formation. Morphology of the DP and DP salts prepared in
this study was assessed by SEM and PLM. Intense birefringence
was observed in crystalline DP and the six DP salts in PLM
appearance (Fig. 1A), and morphological difference was observed
between DP and the DP salts in SEM images (Fig. 1B). SEM
micrographs revealed a tablet-shaped morphology of DP, plate-like
morphology of DP tosylate (DP/TS), irregular needle-like mor-
phology of DP besylate (DP/BS) and rock-like morphology of
DP hydrochloride (DP/HCl), DP phosphate (DP/PA), DP sulfate
(DP/SA) and DP maleate (DP/MA). The physical state of the six
DP salts obtained was evaluated by PXRD (Fig. 2A) and TG/DTA
(Fig. 2B). DP and the six DP salts exhibited typical diffraction
patterns, and significant differences in PXRD patterns among the
samples were observed, suggesting that the six DP salts could be
different crystal forms and could not remain as a free base of DP.
On the basis of the results from TG/DTA, DP and all six DP salts
showed thermal events, and the extrapolated onset temperature of
the endothermic peak (Ton) for each sample was different. DP/HCl
Diffuse reflectance UV-visible spectral analysis: The diffuse
reflectance UV-visible spectra were measured using a UV-visible
spectrophotometer, the UV-2550, equipped with an ISR-2200
integrating sphere attachment (Shimadzu Co., Ltd.). Barium sulfate
was used as the standard, and the Kubelka-Monk function was
applied to convert diffuse reflectance data to the absorbance
spectra.
Pharmacokinetics:
Animals
Male Sprague–Dawley rats, weighing ca. 354 « 11.6 g (10–11
weeks of age; Japan SLC, Shizuoka, Japan), were housed two per
cage in the laboratory with free access to food and water, and
maintained on a 12-h dark/light cycle in a room with controlled
temperature (24 « 1°C) and RH (55 « 5%). Animals were fasted
for 18 h before oral administration of DP samples. In this study,
rats were pretreated with 1 mL of omeprazole suspension (p.o.,
30 mg/kg) to develop an experimental hypochlorhydric model for
pharmacokinetic study as reported previously.24) All procedures
used in the present study were conducted in accordance with the
guidelines approved by the Institutional Animal Care and Ethical
Committee of the University of Shizuoka.
UPLC/ESI-MS analysis for plasma concentration of dipyridamole
Blood samples (200 µL) were collected from the tail vein at the
indicated periods (5, 15 and 30 min, and 1, 1.5, 2, 3, 4, 8 and 12 h)
after oral administration of 0.5 mL of DP suspension or DP/TS
suspension (10 mg DP/kg). A suspension of DP and DP/TS was
immediately administered just after preparation of the suspension.
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)

386
Chika TANIGUCHI, et al.
Table 1. The results for preparation of dipyridamole salts
¦pKa
(DP–counterion)
Yield
(%)
Counterion
MWa
pKaa
Crystallization solvent
Stoichiometryb
Hydrochloric acid (HCl)
Phosphoric acid (PA)
Sulfuric acid (SA)
36.46
98.00
98.08
¹6
1.96, 7.12, 12.32
¹3, 1.92
10.4
4.44
10.4
7.74
5.70
3.38
3.37
3.27
4.48
2-propanol
2-propanol
2-propanol
THF
1:1 (H2O)
1:2
1:2
62.7
47.0
58.6
93.7
p-Toluenesulfonic acid (TS)
Benzenesulfonic acid (BS)
L-Tartaric acid (TA)
Fumaric acid (FA)
172.20
158.17
150.09
116.07
192.12
116.07
¹1.34
0.7
1:2
1:2
2-propanol
2-propanol
2-propanol
2-propanol
2-propanol
48.6
3.02, 4.36
3.03, 4.38
3.13, 4.76, 6.40
1.92, 6.23
Not gained
Not gained
Not gained
1:1
Not gained
Not gained
Not gained
91.3
Citric acid (CA)
Maleic acid (MA)
aData from Handbook of Pharmaceutical Salt (2002).25)
bStoichiometry was confirmed using ion chromatography, ICP-AES and KF.
Fig. 1. Micrographic images of dipyridamole and dipyridamole salts
(A) Polarized light microscopy and (B) scanning electron microscopy
observations. (i) DP, (ii) DP/HCl, (iii) DP/PA, (iv) DP/SA, (v) DP/TS,
(vi) DP/BS and (vii) DP/MA. Each bar represents 20 µm.
Fig. 2. Characterizations of dipyridamole and dipyridamole salts
(A) Powder X-ray diffraction patterns, (B-1) thermograms and (B-2) differential
thermal analysis. (i) DP, (ii) DP/HCl, (iii) DP/PA, (iv) DP/SA, (v) DP/TS,
(vi) DP/BS and (vii) DP/MA.
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)

Dipyridamole Salts with Improved Absorption under Hypochlorhydria
387
Table 2. The physicochemical and dissolution properties of dipyridamole salts
Stability, 12 weeks
% of DP remaininga
Photostabilityb
% of DP remaininga
DVS isothermc
change in mass (%)
Dissolution at pH 6.8
DP salt
pH value of
test medium
after testing
DP dissolved
at 60 min (%)
40°C/75% RH
60°C closed
1 h
24 h
25°C/95% RH
DP
98.8
101.3
101.9
98.3
98.9
102.4
100.5
98.4
99.6
102.4
100.7
100.0
98.8
98.5
98.9
99.4
100.4
96.7
93.7
98.1
10
21
33
46
43
33
28
6.78
6.78
6.78
6.78
6.80
6.78
6.77
0.051
3.842
0.218
5.552
1.907
0.682
0.135
DP/HCl
DP/PA
DP/SA
DP/TS
DP/BS
DP/MA
100.2
98.5
98.3
98.6
98.5
97.1
100.3
95.7
aRemaining to initial sample.
bUV and visible light irradiation (250 W/m2).
cSample was stored under humidity between 0 and 95% RH at 25°C.
and DP/SA exhibited endothermic weight loss, and the Ton of DP/
HCl and DP/SA were at 62°C and 173°C, respectively. The results
of PXRD and TG/DTA suggested that the obtained DP salts were
different salt forms. The stoichiometry of the DP salts was assessed
using ion chromatography and ICP-AES (data not shown). The
molar ratio of counterion to DP in DP/PA, DP/SA, DP/TS and DP/
BS was calculated to be approximately 2:1, and that in DP/MA and
DP/HCl was approximately 1:1 (Table 1). The endothermic weight
loss of DP/HCl in TG was ca. 2.8% from 60 to 80°C (Fig. 2B-1),
and this finding, taken together with Karl-Fischer titration, was
indicative of monohydrate.
TS and DP/BS were selected as representative samples for this
analysis because SA, TS and BS are strong acids and could interact
with DP strongly compared with the other weak acids. UV spectral
patterns in 50% methanol solution for all three DP salts were
almost the same; however, their spectral patterns were different
from that of DP. All samples showed strong absorption in visible
light (400–700 nm), and the maximum absorption of DP was
shifted from 412 nm to 405–406 nm by salt formation (Fig. 3A).
Thus, the UV spectral patterns of DP salts were found to be
different from that of DP, and this result was in agreement with the
UV-visible diffuse reflectance spectra in a solid state (Fig. 3B).
This finding suggested that the light sensitivity of DP salts could
be different from that of DP; therefore, a photostability study was
performed for the DP salts. DP and the DP salts were exposed
to simulated sunlight consisting of UVA/B and visible light (250
W/m2) for the indicated periods (Table 2). Decreases of the
remaining DP were observed in DP/TS and DP/BS by ca. 3% and
6%, respectively; however, most DP salts showed no significant
changes in the amount of remaining DP after light exposure
compared with DP. Although the UV spectral pattern of DP
salts shifted to that of DP, the DP salts exhibited no significant
difference in photostability compared with DP. On the basis of the
stability data, some DP salts were a little less stable under accel-
erated conditions and light exposure; however, most DP salts were
almost as stable as DP.
Chemical and photochemical stability studies on dipyrida-
mole salts: Stability testing of DP and the six DP salts was
performed under accelerated conditions (40°C/75% RH and 60°C)
for 12 weeks to clarify the influence of salt formation on the
chemical stability (Table 2). The appearance of DP and DP salts
did not change after storage. According to the HPLC measurement
of aged samples, very small unidentified peaks were observed in
DP salts (data not shown); however, most samples maintained the
high potency (>98%) even after storage at accelerated conditions.
After storage at 60°C for 12 weeks, only DP/MA exhibited slight
degradation (ca. 4%), suggesting the potential influence of co-
existing MA on the chemical stability of DP. In general, the
hygroscopic property of a drug substance tends to impact upon its
chemical and physicochemical stability; therefore, water sorption
of DP and DP salts was evaluated. DP salts exhibited higher water
sorption than DP. In particular, DP/SA and DP/HCl showed
remarkably higher water sorption, suggesting that the stability of
DP salts might be influenced by humidity conditions (Table 2).
However, after storage at 40°C/75% RH for 12 weeks, the amount
of remaining DP in all DP salts including DP/HCl and DP/SA did
not change, showing that the stability of DP salts might not be
influenced by moisture. The physical states of DP and DP/TS as
representative samples were evaluated by PXRD and TG/DTA, and
neither DP nor DP/TS showed any transition after storage under
accelerated conditions for 12 weeks (data not shown). In a previous
study, the photo-oxidation reaction of DP coexisting with hydrate
silica was found to become higher than that of DP by itself.28)
Interaction between DP and counterions in salt formation has the
potential to change the UV-absorbing property of DP, and a change
in the UV-absorbing property might also influence photostability.
Therefore, spectroscopic analyses of DP and DP salts were carried
out using UV spectral analysis in solution and diffuse reflectance
UV-visible solid-state absorption spectral analysis. DP/SA, DP/
Dissolution behavior of dipyridamole salts: The preliminary
dissolution testing of DP and six DP salts was carried out in 20
mL of 0.05 M phosphate buffer (pH 6.8) at 37°C using a small
volume dissolution tester to confirm possible improvement of drug
dissolution. The drug dissolution level of DP reached 10% of
the total drug amount at 60 min because of poor DP solubility at
pH 6.8 (ca. 6 µg/mL at 37°C) (Table 2).29) In contrast, the six DP
salts exhibited a higher level of dissolved drug at pH 6.8 by at least
2-fold compared with DP. In particular, the DP dissolved levels
of DP/SA, DP/TS and DP/BS improved by 4.6-, 4.3- and 3.3-fold
compared with DP, respectively. After dissolving DP salts, pH
values of the test medium did not change from 6.8 (Table 2). As a
result, counterions containing DP salts could modulate pH values
in the diffusion area during the dissolution of DP salts, resulting in
improved solubility of DP in medium at neutral pH. According to
the results from stability testing, preliminary dissolution testing,
DP/SA and DP/TS might be promising salt forms, although DP/SA
was found to be hygroscopic, possibly leading to high variability
in its potency. In this context, DP/TS was selected as a promising
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)

388
Chika TANIGUCHI, et al.
Fig. 4. Dissolution profiles of dipyridamole and dipyridamole salt at (A)
pH 1.0 and (B) pH 6.8
DP; DP/TS. Each bar represents mean « SD of
experiments.
Fig. 3. UV spectral patterns of dipyridamole and dipyridamole salts
(A) UV absorption spectra and (B) UV-visible diffuse reflectance spectra. Black
dotted line, DP; black solid line, DP/SA; red solid line, DP/TS; and blue solid
line, DP/BS.
,
,
3 independent
salt for further study. Dissolution tests of DP and DP/TS were
performed in 0.1 M HCl solution (pH 1.0) and 0.05 M phosphate
buffer solution (pH 6.8) using a regular pharmacopeial dissolution
tester to simulate normal and hypochlorhydric conditions (Fig. 4).
The dissolution profiles at pH 1.0 of DP and DP/TS were almost
identical and reached 50 µg/mL DP concentration as almost 100%
of the total drug amount within 15 min because of high DP solu-
bility at pH 1.0 (53.0 mg/mL at 37°C).29) At pH 6.8, DP exhibited
poor dissolution behavior and a maximum dissolution level of
ca. 5 µg/mL. In contrast, DP/TS showed rapid dissolution, and the
maximum dissolved level of DP/TS was much higher than that of
DP, by ca. 5-fold (Fig. 4B). The supersaturation level (C/Ceq) of
DP/TS was found to be ca. 4 at 60 min, and the level seemed to
gradually decrease to ca. 2.2 at 6 h. Thus, the supersaturation state
was still maintained for at least 6 h, and no significant increase of
turbidity was observed nor any precipitation. Considering these
results, the newly developed DP/TS could be a promising salt and
have the potential to improve oral absorption under hypochlo-
rhydric conditions.
hypochlorhydria with increased gastric pH because DP had pH-
dependent solubility.9) In our previous studies, DP formulations
with enhanced dissolution behavior at neutral pH were developed
using a microenvironmental pH-modifier approach, and this
formulation exhibited improved oral bioavailability in hypochlo-
rhydric model rats.18) In this study, some DP salts were devel-
oped, and these salts showed pH-independent dissolution behavior.
This prompted us to clarify the potential improvement of oral
absorption; therefore, the pharmacokinetic behaviors of DP and
DP/TS were evaluated in normal rats and omeprazole-treated rats.
The pharmacokinetic behavior of DP in normal rats was assessed
after oral administration of DP and DP/TS (10 mg DP/kg) in order
to compare it with that in hypochlorhydric model rats. The plasma
concentration-time profiles of DP in rats are shown in Figure 5,
and the related pharmacokinetic parameters are described in
Table 3. In normal rats, the maximum concentration (Cmax) and
area under the curve of plasma concentration versus time from 0 h
to 3 h (AUC0–3) values of DP/TS were higher than those of DP by
2.8- and 1.7-fold, respectively; however, the AUC0–3 values for DP
and DP/TS were not statistically different, possibly due to high
variability. On the basis of the AUC value of intravenously admin-
Pharmacokinetic profiling of dipyridamole salt: In previ-
ous clinical studies, DP showed low oral bioavailability under
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)

Dipyridamole Salts with Improved Absorption under Hypochlorhydria
389
istered DP (3.0 mg/kg),19) absolute bioavailabilities of DP and DP/
TS were calculated to be ca. 19% and 24%, respectively. Thus,
oral bioavailabilities of DP and DP/TS were statistically identical,
whereas there was significant difference in their Cmax values. In
addition to the normal rats, pharmacokinetic behaviors of DP and
DP/TS were also characterized in hypochlorhydric model rats. The
rats were treated with 30 mg/kg omeprazole by oral administration
to prepare them as hypochlorhydric model rats. After treatment
with omeprazole, the gastric pH of rats rose to about 6 within
1 h, and high gastric pH was maintained for about 5 h.24) The
pharmacokinetic behavior after oral administration of DP in
omeprazole-treated rats was found to be lower than that in normal
rats, and the AUC0–3 values in omeprazole-treated rats and normal
rats were 48.4 « 10.3 ng0h/mL and 127.7 « 32.9 ng0h/mL, respec-
tively. In addition, delayed oral absorption of DP was observed in
omeprazole-treated rats compared with normal rats, and Tmax values
of DP in omeprazole-treated rats and normal rats were ca. 1.3 h
and ca. 0.4 h, respectively. On the other hand, the pharmaco-
kinetic behavior after oral administration of DP/TS in omeprazole-
treated rats was almost identical to that in normal rats. The values
of AUC0–3 and Tmax in omeprazole-treated rats were 198.5 « 21.8
ng h/mL and ca. 0.3 h, respectively, and did not differ significantly
from those in normal rats. From the pharmacokinetic behavior of
DP/TS taken together with the pH-independent dissolution behav-
ior, salt formation could be a promising approach for improving the
oral bioavailability of DP.
In our previous studies, DP granules with 30% loading of
TS (DPG/TS30) were developed using the microenvironmental
pH-modifier approach, and the DPG/TS30 showed significant
improvement in dissolution behavior at pH 6.8 and oral absorption
in hypochlorhydric rats.18) In both salt formation and micro-
environmental pH-modifier approaches, modulation of pH value in
the diffusion area led to pH-independent dissolution behavior,
and these approaches could be effective to improve oral absorption
for drug substances with pH-dependent solubility. In particular,
salt formation could be a promising and simplified approach in
pharmaceutical pre-formulation; however, sometimes it is chal-
lenging to obtain appropriate salts with expected molecular prop-
erties. For some drugs, preparation of stable salts may not be
feasible, and free form may be preferred.30) In such a case, the
microenvironmental pH-modifier approach might be alternatively
used in the formulation development. In addition to these solu-
bilization strategies, a number of efficacious approaches have been
developed, and, to enhance the therapeutic potential of drugs with
pH-dependent solubility, careful selection of a strategic approach
would be necessary on the basis of cost of goods, molecular
properties and clinical needs.
In conclusion, salt screening of DP was conducted using nine
counterions by the temperature gradient method, and six DP salts
were obtained in the present study. All six DP salts showed
improved dissolution behavior at neutral pH compared with DP.
On the basis of the physicochemical properties, including dissolu-
tion behavior and stability, DP/TS was selected as a promising salt
to achieve pH-independent dissolution behavior. In the pharmaco-
kinetic study using hypochlorhydric model rats, DP/TS showed
ca. 4-fold higher oral absorption in comparison with DP, and the
pH-independent dissolution behavior of DP might explain the
Fig. 5. Plasma dipyridamole concentrations in normal and hypochlorhy-
dric rats after oral administration (10 mg DP/kg) of (A) DP and (B) DP/TS
, normal rats;
4 experiments.
, hypochlorhydric rats. Data represent mean « SE of
Table 3. Pharmacokinetic parameters of dipyridamole and dipyridamole salt following oral administration in normal and omeprazole-treated rats
DP (10 mg DP/kg; p.o.)
Omeprazole-treated rats
DP/TS (10 mg DP/kg; p.o.)
Normal rats
Normal rats
Omeprazole-treated rats
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
87.1 « 23.6
0.38 « 0.07
2.23 « 0.23
269.2 « 43.1
127.7 « 32.9
18.6 « 3.0
31.1 « 11.3
1.33 « 0.57
2.42 « 0.67
96.8 « 25.8
48.4 « 10.3
6.7 « 1.8
239.8 « 19.3#
0.31 « 0.06
1.55 « 0.64
341.2 « 75.2
213.6 « 26.7
23.5 « 5.2
211.1 « 74.7*
0.31 « 0.06*
1.22 « 0.25
AUC (ng0h/mL)
AUC0–3 (ng0h/mL)
Oral BA (%)
Oral BA0–3 (%)
390.9 « 20.9*
198.5 « 21.8*
27.0 « 1.4*
10.0 « 2.6
3.8 « 0.8
16.7 « 2.1
15.5 « 1.7*
Cmax, maximum concentration; T1/2, half-life; AUC, area under the curve of plasma concentration; BA, bioavailability. Values are expressed as mean « SE from
#
4 experiments. p < 0.05 between DP and DP/TS in normal rats. * p < 0.05 between DP and DP/TS in omeprazole-treated rats.
Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX)

390
Chika TANIGUCHI, et al.
dextrin complex in healthy volunteers after single and multiple doses.
Eur. J. Drug Metab. Pharmacokinet., 16: 197–201 (1991).
15) Siepe, S., Herrmann, W., Borchert, H. H., Lueckel, B., Kramer, A., Ries,
A. and Gurny, R.: Microenvironmental pH and microviscosity inside pH-
controlled matrix tablets: an EPR imaging study. J. Control. Release, 112:
72–78 (2006).
16) Siepe, S., Lueckel, B., Kramer, A., Ries, A. and Gurny, R.: Assessment of
tailor-made HPMC-based matrix minitablets comprising a weakly basic
drug compound. Drug Dev. Ind. Pharm., 34: 46–52 (2008).
17) Siepe, S., Lueckel, B., Kramer, A., Ries, A. and Gurny, R.: Strategies for
the design of hydrophilic matrix tablets with controlled microenviron-
mental pH. Int. J. Pharm., 316: 14–20 (2006).
enhanced oral absorption under hypochlorhydria. Salt formation is
a valuable method for enhancement of dissolution behavior and
oral absorption, and the newly developed DP salt might provide
better therapeutic efficacy in clinical use for hypochlorhydric
patients.
Acknowledgments: The authors are grateful to Dr. Yukinori
Yamauchi for his excellent support on physicochemical character-
ization.
18) Taniguchi, C., Inoue, R., Kawabata, Y., Yamashita, K., Wada, K.,
Yamauchi, Y., Yamada, S. and Onoue, S.: Novel formulations of
dipyridamole with microenvironmental pH-modifiers for improved
dissolution and bioavailability under hypochlorhydria. Int. J. Pharm.,
434: 148–154 (2012).
19) Onoue, S., Inoue, R., Taniguchi, C., Kawabata, Y., Yamashita, K., Wada,
K., Yamauchi, Y. and Yamada, S.: Improved dissolution and pharmaco-
kinetic behavior of dipyridamole formulation with microenvironmental
pH-modifier under hypochlorhydria. Int. J. Pharm., 426: 61–66 (2012).
20) Tarsa, P. B., Towler, C. S., Woollam, G. and Berghausen, J.: The
influence of aqueous content in small scale salt screening—improving hit
rate for weakly basic, low solubility drugs. Eur. J. Pharm. Sci., 41: 23–30
(2010).
References
1) Zhuplatov, S. B., Masaki, T., Blumenthal, D. K. and Cheung, A. K.:
Mechanism of dipyridamole’s action in inhibition of venous and arterial
smooth muscle cell proliferation. Basic Clin. Pharmacol. Toxicol., 99:
431–439 (2006).
2) Chakrabarti, S. and Freedman, J. E.: Dipyridamole, cerebrovascular
disease, and the vasculature. Vascul. Pharmacol., 48: 143–149 (2008).
3) Kostewicz, E. S., Brauns, U., Becker, R. and Dressman, J. B.: Forecasting
the oral absorption behavior of poorly soluble weak bases using solubility
and dissolution studies in biorelevant media. Pharm. Res., 19: 345–349
(2002).
4) Zhou, R., Moench, P., Heran, C., Lu, X., Mathias, N., Faria, T. N., Wall,
D. A., Hussain, M. A., Smith, R. L. and Sun, D.: pH-dependent
dissolution in vitro and absorption in vivo of weakly basic drugs:
development of a canine model. Pharm. Res., 22: 188–192 (2005).
5) Kawabata, Y., Wada, K., Nakatani, M., Yamada, S. and Onoue, S.:
Formulation design for poorly water-soluble drugs based on biopharma-
ceutics classification system: basic approaches and practical applications.
Int. J. Pharm., 420: 1–10 (2011).
6) Champagne, E. T.: Low gastric hydrochloric acid secretion and mineral
bioavailability. Adv. Exp. Med. Biol., 249: 173–184 (1989).
7) Lahner, E., Annibale, B. and Delle Fave, G.: Systematic review:
Helicobacter pylori infection and impaired drug absorption. Aliment.
Pharmacol. Ther., 29: 379–386 (2009).
8) Shelton, M. J., Adams, J. M., Hewitt, R. G., Steinwandel, C., DeRemer,
M., Cousins, S. and Morse, G. D.: Effects of spontaneous gastric
hypoacidity on the pharmacokinetics of zidovudine and didanosine.
Pharmacotherapy, 17: 438–444 (1997).
9) Russell, T. L., Berardi, R. R., Barnett, J. L., O’Sullivan, T. L., Wagner,
J. G. and Dressman, J. B.: pH-related changes in the absorption of
dipyridamole in the elderly. Pharm. Res., 11: 136–143 (1994).
10) Chen, S., Zhu, J., Ma, F., Fang, Q. and Li, Y.: Preparation and
characterization of solid dispersions of dipyridamole with a carrier
“copolyvidonum Plasdone S-630.” Drug Dev. Ind. Pharm., 33: 888–899
(2007).
11) Onoue, S., Kojo, Y., Aoki, Y., Kawabata, Y., Yamauchi, Y. and Yamada,
S.: Physicochemical and pharmacokinetic characterization of amorphous
solid dispersion of tranilast with enhanced solubility in gastric fluid and
improved oral bioavailability. Drug Metab. Pharmacokinet., 27: 379–387
(2012).
12) Sanganwar, G. P. and Gupta, R. B.: Nano-mixing of dipyridamole drug
and excipient nanoparticles by sonication in liquid CO2. Powder
Technol., 196: 36–49 (2009).
13) Onoue, S., Sato, H., Kawabata, Y., Mizumoto, T., Hashimoto, N. and
Yamada, S.: In vitro and in vivo characterization on amorphous solid
dispersion of cyclosporine A for inhalation therapy. J. Control. Release,
138: 16–23 (2009).
14) Ricevuti, G., Mazzone, A., Pasotti, D., Uccelli, E., Pasquali, F., Gazzani,
G. and Fregnan, G. B.: Pharmacokinetics of dipyridamole-beta-cyclo-
21) Serajuddin, A. T.: Salt formation to improve drug solubility. Adv. Drug
Deliv. Rev., 59: 603–616 (2007).
22) Tsutsumi, S., Iida, M., Tada, N., Kojima, T., Ikeda, Y., Moriwaki, T.,
Higashi, K., Moribe, K. and Yamamoto, K.: Characterization and
evaluation of miconazole salts and cocrystals for improved physico-
chemical properties. Int. J. Pharm., 421: 230–236 (2011).
23) López-Solera, I., Perez, J. M., Monge, M. A., Alvarez-Valdes, A.,
Masaguer, J. R., Alonso, C. and Navarro-Ranninger, C.: Synthesis,
crystal structure, and biological activity of a Pt-dipyridamole salt.
J. Inorg. Biochem., 56: 233–242 (1994).
24) Wada, I., Otaka, M., Jin, M., Odashima, M., Komatsu, K., Konishi, N.,
Matsuhashi, T., Horikawa, Y., Ohba, R., Itoh, H. and Watanabe, S.:
Expression of HSP72 in the gastric mucosa is regulated by gastric acid in
rats-correlation of HSP72 expression with mucosal protection. Biochem.
Biophys. Res. Commun., 349: 611–618 (2006).
25) Stahl, P. H. and Wermuth, C. G.: Monographs on acids and bases.
In Stahl, P. H. (ed.): Handbook of Pharmaceutical Salts: Properties,
Selection, and Use, Zurich, Verlag Helvetica Chimica Acta, 2002,
pp. 266–327.
26) Childs, S. L., Stahly, G. P. and Park, A.: The salt-cocrystal continuum: the
influence of crystal structure on ionization state. Mol. Pharm., 4: 323–338
(2007).
27) Kim, H., Gao, J. and Burgess, D. J.: Evaluation of solvent effects on
protonation using NMR spectroscopy: implication in salt formation. Int.
J. Pharm., 377: 105–111 (2009).
28) Kigasawa, K., Shimizu, H., Hayashida, S. and Ohkubo, K.: Decom-
position and stabilization of drugs. XX. Photodecomposition and
stabilization of dipyridamol. Yakugaku Zasshi, 104: 1191–1197 (1984).
29) Heigoldt, U., Sommer, F., Daniels, R. and Wagner, K. G.: Predicting
in vivo absorption behavior of oral modified release dosage forms
containing pH-dependent poorly soluble drugs using a novel pH-adjusted
biphasic in vitro dissolution test. Eur. J. Pharm. Biopharm., 76: 105–111
(2010).
30) Morris, K. R., Fakes, M. G., Thakur, A. B., Newman, A. W., Singh,
A. K., Venit, J. J., Spagnuolo, G. J. and Serajuddin, A. T.: An integrated
approach to the selection of optimal salt form for a new drug candidate.
Int. J. Pharm., 105: 209–217 (1994).
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