Formation of the ternary inclusion complex of limaprost with α- And β-cyclodextrins in aqueous solution

Article abstract of DOI:10.1248/cpb.c14-00733

The inclusion mode of Limaprost in the presence of α- and β-cyclodextrins (CDs) was investigated to gain insight into the stabilization mechanism of Limaprost-alfadex upon the addition of β-CD in the solid state. The inclusion sites of α- and β-CDs were s

Full text of DOI:10.1248/cpb.c14-00733

318
Chem. Pharm. Bull. 63, 318–325 (2015)
Vol. 63, No. 5
Regular Article
Formation of the Ternary Inclusion Complex of Limaprost with
α- and β-Cyclodextrins in Aqueous Solution
,a
Yasuo Inoue,* Noboru Sekiya,b Masanobu Yamamoto,b Daisuke Iohara,c
Fumitoshi Hirayama,c and Kaneto Uekamad
a CMC Regulatory and Analytical R&D, Ono Pharmaceutical Co., Ltd.; 3–1–1 Sakurai Shimamoto-cho, Mishima-
b
gun, Osaka 618–8585, Japan: Pharmaceutical R&D, Ono Pharmaceutical Co., Ltd.; 3–1–1 Sakurai Shimamoto-cho,
c
Mishima-gun, Osaka 618–8585, Japan: Faculty of Pharmaceutical Sciences, Sojo University; 4–22–1 Ikeda, Nishi-
d
ku, Kumamoto 860–0082, Japan: and DDS Research Institute, Sojo University; 4–22–1 Ikeda, Nishi-ku, Kumamoto
860–0082, Japan.
Received October 21, 2014; accepted February 25, 2015
The inclusion mode of Limaprost in the presence of α- and β-cyclodextrins (CDs) was investigated to
gain insight into the stabilization mechanism of Limaprost-alfadex upon the addition of β-CD in the solid
state. The inclusion sites of α- and β-CDs were studied by NMR spectroscopic and kinetic methods. With the
addition of α- and β-CDs, displacements in 13C chemical shifts of prostaglandin F2α (PGF2α) were observed
in the ω-chain and the five-membered ring, respectively, of the drug. Similar shift changes were observed
1
with the addition of both α- and β-CDs. In two-dimensional (2D) H-NMR spectra, intermolecular correla-
tion peaks were observed between protons of PGF2α and protons of both α- and β-CDs, suggesting that PGF2α
interacts with α- and β-CDs to form a ternary complex by including the ω-chain with the former CD and
the five-membered ring with the latter. In kinetic studies in aqueous solution, Limaprost was degraded to
17S,20-dimethyl-trans-Δ2-PGA1 (11-deoxy-Δ10) and 17S,20-dimethyl-trans-Δ2-8-iso-PGE1 (8-iso). The addition
of α-CD promoted the dehydration to 11-deoxy-Δ10, while β-CD promoted the isomerization to 8-iso, under
these conditions. In the presence of both α- and β-CDs, dehydration and isomerization were also accelerated,
supporting the formation of the ternary Limaprost/α-CD/β-CD complex.
Key words Limaprost alfadex; α-cyclodextrin; β-cyclodextrin; ternary inclusion complex; NMR spectros-
copy; kinetics
α-, β- and γ-Cyclodextrins (CDs) are cyclic oligosaccharides formulated as the α-CD complex, Limaprost-alfadex, in com-
in which six, seven and eight D-glucose units, respectively, are mercially available tablets, Opalmon® tablets, to ensure its
linked by α-1,4 glycosidic bonds.1–6) The outside of the ring is dose uniformity and to improve its aqueous solubility, because
hydrophilic, while the center cavity is hydrophobic. Because of the very low content, 5µg, in each tablet.24–26)
CDs form inclusion complexes by encapsulating other com-
Limaprost in Opalmon® tablets is chemically stable in
pounds into their hydrophobic cavity, natural and chemically Press-through-Package, but once the package is opened and
modified CDs are widely used as pharmaceutical and food placed in humid conditions, Limaprost degrades to 17S,20-
additives. Many studies of CD inclusion complex formations dimethyl-trans-Δ2-PGA1 (referred to as 11-deoxy-Δ10) and
have been conducted to improve physicochemical proper- 17S,20-dimethyl-trans-Δ2-8-iso-PGE1 (referred to as 8-iso), as
ties of drugs, such as poor water solubility,7–10) degradation shown in Fig. 1. We have reported previously that the addi-
upon heating11–15) or by light irradiation,16–19) and oxidation.20) tion of dextran improved the stability of Limaprost-alfadex in
However, most of these studies have examined the effect of a humid conditions,27–29) probably due to the increased interac-
single type of CD. Only a few reports focus on the effect of tion of the drug with α-CD in the presence of dextran.30,31)
two types of CDs. For example, Nikouei et al. reported that Furthermore, we have found that the addition of β-CD to the
the simultaneous addition of both α-CD and 2-hydoxypropyl- Limaprost/α-CD (Limaprost-alfadex) improved the stability of
β-CD (HP-β-CD) improved the solubility of Cyclosporine A the drug in humid conditions.32) The stabilization of Limaprost
(CyA), a poorly water-soluble drug, compared to the addition by employing two CDs may be due to the formation of a ter-
of one CD.21) A similar synergistic effect on the solubility has nary complex with the two CDs, but the inclusion mode of the
been reported for dexamethasone by the addition of γ-CD and prostaglandin derivative in the presence of two CDs does not
HP-γ-CD.22,23) Although these reports focused on the synergis- yet confirmed.
tic effect of two CDs on drug solubility, the inclusion mode
between the two CDs and the drug has not been studied in of Limaprost with two CDs, α-CD and β-CD, in solution. The
detail. interaction between the prostaglandin derivative and the two
In this study, we focus on the inclusion complex formation
1
Limaprost is a prostaglandin E1 (PGE1) derivative that CDs was investigated by H- and 13C-NMR spectroscopy. In
is approved for “treatment of various ischemic symptoms addition, the effects of α-CD and β-CD on the stability of
such as ulcers, pain, and sensation of coldness of the hands Limaprost in aqueous solution were evaluated to estimate the
and feet associated with thromboangiitis obliterans” and for inclusion mode of the drug.
“improvement in subjective symptoms and walking ability,
which accompany lumbar spinal canal stenosis.” Limaprost is
*To whom correspondence should be addressed. e-mail: ya.inoue@ono.co.jp
© 2015 The Pharmaceutical Society of Japan

Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
319
Fig. 1. Limaprost Decomposition Pathway
Fig. 2. 13C-NMR Spectrum of PGF2α (50 mM) in a 0.1M Sodium Borate/D2O Buffer (pH 9.3) at 25°C
a JEOL JNM-A500 NMR system (external magnetic field of
Materials Limaprost-alfadex is specified in the Japanese 500MHz) at 25°C and accumulated 10000 times.
Experimental
1
Pharmacopoeia (JP) XVI. All excipients used in this study
Two-Dimensional (2D) H-NMR Studies PGF2α (10 mM)
were purchased in accordance with the specifications of JP was dissolved in a 0.1M sodium borate/D2O solution (pH
XVI or Japanese Pharmaceutical Excipients. Deionized dou- meter reading of 9.3) and α- or β-CDs (10mM) was added to
ble-distilled water was used. All other chemicals and solvents the solution. 2D-NMR measurements (rotating frame nuclear
were of analytical reagent grade.
Overhauser enhancement spectroscopy (ROESY)) were con-
13C-NMR Studies: Interaction of Prostaglandin F2α ducted for the sample solution on a JEOL JNM-ECA500
(PGF2α) with α- and β-CDs PGF2α (50 mM) was dissolved in NMR system (external magnetic field of 500MHz) at 30°C
a 0.1M sodium borate/D2O solution (pH meter reading of 9.3). and accumulated 32 times.
A predetermined concentration of α- or β-CD (0–50mM) was
Degradation of Limaprost in the CD Solutions α-CD
added to the solution. 13C-NMR measurements of the solution (10mg), β-CD (10mg) or the mixture of α- and β-CDs (10mg
were acquired on a JEOL JNM-A500 NMR system (external each) was dissolved in 5.0mL of phosphate buffer (pH 9.0).
magnetic field of 500MHz) at 25°C and accumulated 10000 Then, 25µL of an ethanoic solution of Limaprost (10.0mg/
times. Chemical shifts were given as parts per million (ppm) mL) was added. The final concentrations of Limaprost and
down field from that of trimethylsilyl propionic acid sodium CDs were 0.13mM, 2.0 (α-CD) and 1.8 (β-CD) mM, respec-
salt as an external reference with an accuracy of 0.005ppm.
tively, where CDs were more than 10-folds higher than that
13C-NMR Studies: Effect of β-CD on PGF2α/α-CD System of Limaprost (0.13mM) and then most of the guest was in the
and That of α-CD on PGF2α/β-CD System PGF2α (10 mM) complex form. The solution was kept at 25°C and subjected
and α-CD (10mM) were dissolved in a 0.1M sodium borate/ to HPLC analysis at predetermined times. Limaprost and its
D2O solution (pH 9.3). β-CD (0–20mM) was subsequently degradation products, 11-deoxy-Δ10 and 8-iso, were analyzed
added to the solution, and then the 13C-NMR spectra were using a Prominence UFLC system (Shimadzu, Kyoto, Japan),
acquired. Similarly, α-CD (0–20mM) was added to a solution which consisted of a pump, auto-injector, UV-Vis detector,
of PGF2α (10 mM) and β-CD (10mM), and then 13C-NMR mea- and a ODS C18 column (5µm, 4.6mm×15cm; YMC, Tokyo,
surements were performed. All measurements were taken on Japan). The mobile phase was a mixture of 0.02M KH2PO4

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(pH 4.2), acetonitrile and propan-2-ol at 9:5:2 ratio and a particular, C2 and C3 in the α-chain and C18, C19, and C20
flow rate of 1.0mL/min, and the detection was at 215nm and in the ω-chain exhibited significant downfield shifts, but the
35°C.
shift changes of the five-membered ring were minimal. These
results indicate that α-CD preferentially interacts with the
long alkyl chain, α- or ω-chain of PGF2α. On the other hand,
Results and Discussion
Interaction of PGF2α with One Type of CD in Solution the signals from the five-membered ring of PGF2α were sig-
Limaprost is poorly water-soluble and chemically labile in nificantly shifted as the concentration of β-CD increased (Fig.
water.27) Therefore, PGF2α was used as a model compound in 3b), whereas the minor changes were observed for the carbons
the NMR experiments, because it has a similar structure to of the ω-chain. These shift changes indicate that β-CD with a
Limaprost and is relatively stable in aqueous media. Figure 2 larger cavity interacts preferentially with the five-membered
shows the 13C-NMR spectrum of PGF2α and Table 1 summa- ring of PGF2α. The small shift changes in the α-chain may be
rizes its chemical shifts. The displacements of the 13C chemi- due to intermolecular hydrogen bonding between the hydroxyl
cal shifts of PGF2α by the addition of α- or β-CD are shown group of the β-CD and the carboxylate ion of included PGF2α,
in Fig. 3. The addition of α-CD altered the chemical shifts as reported previously.33,34)
mainly for the α- and ω-chains of PGF2α and the displacement
Interaction of PGF2α with Two Types of CDs (α- and
increased as the α-CD concentration increased (Fig. 3a). In β-CD) in Solution To reveal the interaction of PGF2α with
two types of CDs, NMR measurements were conducted in
the presence of α-CD and β-CD. PGF2α (10 mM) and α-CD
(10 mM) were dissolved in a 0.1M sodium borate/D2O solution
(pH 9.3), and then β-CD was added at various concentrations
Table 1. 13C-NMR Chemical Shift of PGF2α (50mM) in a 0.1M Sodium
Borate/D2O Buffer (pH 9.3) at 25°C
(0–20mM). Similarly, α-CD (0–20mM) was added to a solution
α-Chain
Five-membered ring
ω-Chain
of PGF2α (10 mM) and β-CD (10mM). Figures 4a and b show
the changes in the 13C chemical shifts of PGF2α upon the ad-
dition of β- and α-CDs to the PGF2α/α-CD and PGF2α/β-CD
systems, respectively. In Fig. 4a, the shift changes of PGF2α
at the initial point, i.e., in the absence of β-CD, were those
induced by the addition of only α-CD. It was apparent from
Fig. 4a that the shift displacements of the α- and ω-chains at
the initial point were not significantly changed by the addition
of β-CD, whereas that of C12 carbon of the five-membered
ring was significantly changed and the change was more
pronounced as the β-CD concentration increased. The results
Attribution
Attribution
PGF2α
Attribution
PGF2α
PGF2α
(13C)
(13C)
(13C)
1
2
3
4
5
6
7
183.54
36.21
24.91
26.80
130.66
128.75
26.01
8
9
48.48
71.09
42.09
75.96
54.42
13
14
15
16
17
18
19
20
133.72
134.84
73.11
37.33
24.69
31.11
22.19
13.56
10
11
12
Fig. 3a. Effects of α-CD on the 13C-NMR Chemical Shifts of PGF2α (50 mM) in a 0.1M Sodium Borate/D2O Buffer (pH 9.3) at 25°C
Fig. 3b. Effects of β-CD on the 13C-NMR Chemical Shifts of PGF2α (50 mM) in a 0.1M Sodium Borate/D2O Buffer (pH 9.3) at 25°C

Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
321
Fig. 4a. Effects of β-CD on the 13C-NMR Chemical Shifts of PGF2α (10 mM) in the Presence of α-CD (10mM) in a 0.1M Sodium Borate/D2O Buffer
(pH 9.3) at 25°C
Fig. 4b. Effects of α-CD on the 13C-NMR Chemical Shifts of PGF2α (10 mM) in the Presence of β-CD (10mM) in a 0.1M Sodium Borate/D2O Buffer
(pH 9.3) at 25°C
Table 2. 13C-NMR Chemical Shifts of PGF2α (10mM) in the Presence
of α-CD (10mM) and β-CD (10mM) in a 0.1M Sodium Borate/D2O Buffer
(pH 9.3) at 25°C
PGF2α/β-CD binary system (Fig. 3b(A)), due to the intermo-
lecular hydrogen bonding between the guest and host mol-
ecules, as described above. However, the shift changes of the
five-membered ring in PGF2α/α-CD/β-CD system tended to
be smaller than those of the PGF2α/β-CD system, as shown
in Table 2(A). The 13C chemical shifts of PGF2α of the five-
membered ring and the ω-chain in the presence of α-CD and
β-CD are summarized in Table 2. The shift changes of C8
(−0.38ppm), C10 (−0.36ppm), and C12 (0.78ppm) observed in
the PGF2α/β-CD system decreased to −0.23ppm, −0.17ppm
and 0.53ppm, respectively, in the co-presence of both α- and
β-CD, while the shift of C11 was small. These results suggest
that the preexisting α-CD may slightly weaken the interaction
between the five-membered ring and β-CD.
Figure 4b shows the effect of α-CD on the chemical shifts
of the PGF2α/β-CD system. The significant downfield shifts
were observed for 17–20 carbons of the ω-chain as the α-CD
concentration increased. In addition, as shown in Table 2(B),
the shift changes of the ω-chain (C18, 19, 20) in the bi-
nary PGF2α/α-CD system were similar to those in the ternary
(A) Five-membered ring
β-CD with
Attribution (13C)
β-CDa)
preexisting α-CDb)
8
9e)
10
11f)
12
−0.38
—
−0.23
—
−0.36
0.13
−0.17
0.21
0.78
0.53
(B) ω-Chain
α-CDc)
α-CD with
Attribution (13C)
preexisting β-CDd)
17
18
19
20
0.55
0.86
0.84
0.74
0.92
0.84
0.84
0.61
a) (PGF2α+β-CD (10mM))−(PGF2α) (ppm): Chemical shifts of PGF2α (10mM) in PGF2α/α-CD/β-CD system. This suggests that α-CD interacts
the presence of β-CD (10mM). b) (PGF2α+β-CD (10mM)+α-CD (10mM))−(PGF2α
)
preferentially with the ω-chain and the pre-existing β-CD
does not significantly interfere with the interaction of α-CD
with the ω-chain. The downfield shift of the C17 carbon in
the ω-chain was larger in the ternary system than in the α-CD
binary system. The ω-chain was included more deeply in the
α-CD cavity or some conformational changes occurred at this
carbon in the ternary system, compared with the binary sys-
tem, because of the flexible property of the ω-chain. However,
the reason for this large shift is not obvious at the present
(ppm): Chemical shifts of PGF2α (10mM) in the presence of β-CD (10mM) with pre-
existing α-CD (10mM). c) (PGF2α+α-CD (10mM))−(PGF2α) (ppm): Chemical shifts
of PGF2α (10mM) in the presence of α-CD (10mM). d) (PGF2α+α-CD (10mM)+β-CD
(10mM))−(PGF2α) (ppm): Chemical shifts of PGF2α (10mM) in the presence of α-CD
(10mM) with preexisting β-CD (10mM). e) Values not shown (“—”) for C9 because
its spectrum overlaps with that of CD. f) Shift did not change for C11 most likely
because the chemical shift of C11 is unaffected by the addition of α- or β-CD (shift
change of 0.08 with the addition of 10mM α-CD, and 0.13 with the addition of 10mM
β-CD).
suggested that β-CD interacted with the five-membered ring time. On the other hand, the β-CD-induced shift change of
of PGF2α even in the presence of α-CD. The C3 carbon in C12 of the five-membered ring slightly decreased upon the ad-
the α-chain shifted downfield, as observed similarly in the dition of α-CD (Fig. 4b). The addition of high concentrations

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Chem. Pharm. Bull.
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of α-CD may weaken the interaction between β-CD and the to investigate the inclusion sites of PGF2α by α- and β-CDs.
five-membered ring. The collective 13C-NMR results showed PGF2α, α-CD, and β-CD were dissolved in a 0.1M sodium
that when adding α- and β-CDs to PGF2α solutions, α-CD borate/D2O solution (pH 9.3) at a molar ratio of 1:1:1, and
preferentially interacts with the ω-chain, while β-CD interacts the 2D-NMR (ROESY) spectra were measured. This molar
with the five-membered ring, although the interaction strength ratio was selected because it is reported that PGF2α interacts
of α- and β-CDs with PGF2α in the ternary complex is differ- with each CD at 1:1 molar ratio.35) The ROESY spectrum of
ent from those in the binary complex.
2D H-NMR Spectroscopic Studies of PGF2α in the Pres- H13 and H14 protons and the H8 and H12 protons (Fig. 5A).
ence of α- and β-CDs 2D-NMR studies were conducted When α-CD was added, the additional correlation peaks ap-
PGF2α gave the intramolecular correlation peaks between the
1
Fig. 5. 2D-ROESY Spectrum in a 0.1M Sodium Borate/D2O Buffer (pH Meter Reading 9.3) at 25°C
(A) PGF2a, (B) PGF2α in the presence of α-CD, (C) PGF2α in the presence of β-CD, (D) PGF2α in the presence of α- and β-CDs.

Vol. 63, No. 5 (2015)
Chem. Pharm. Bull.
323
Fig. 6. Changes in (A) Limaprost, (B) 11-Deoxy-Δ10, and (C) 8-Iso in the Presence of CDs in a Phosphate Buffer (pH 9.0)
◇: Limaprost, □: Limaprost+α-CD, △: Limaprost+β-CD, ○: Limaprost+α-CD+β-CD.
peared between the ω-chain protons (H17, 18, 19, 20) of PGF2α degradation, i.e., it accelerated the degradation to 8-iso,
and the H3′ and H5′ protons of α-CD (Fig. 5B). Since the H3′ whereas negligibly to 11-deoxy, as shown in Figs. 6B and C.
and H5′ protons of α-CD are located inside of the cavity, these When both α- and β-CDs were added, the degradations to
correlation peaks indicated that α-CD included the ω-chain of 11-deoxy-Δ10 and 8-iso were both accelerated and the profiles
PGF2α. Additionally, the correlation peaks were observed be- of the degradation rates were almost the same as those for
tween the H2 proton of PGF2α and the H2′ and H4′ protons of adding a single CD.
α-CD which are located outside of the cavity. This is probably
The degradation to 11-deoxy-Δ10 is thought to proceed via
due to the interaction of the α-chain of PGF2α with the out- the E2 elimination mechanism (Chart 1A); the deprotonation
side of α-CD cavity, because the α-chain is not significantly occurs at the C10 position on Limaprost, and the hydroxyl
involved in the inclusion complexation. Figure 5C shows the group at the C11 position is subsequently eliminated. We
ROESY measurements after adding β-CD to PGF2α. The pro- confirmed that α-CD preferentially includes the ω-chain of Li-
tons on the five-membered ring of PGF2α (H7, H8, H10, H12) maprost. The secondary hydroxyl group of α-CD located near
showed the correlation peaks with the internal protons of the the five membered ring may act as a general base catalyst in
β-CD cavity (H3′, H5′), indicating that β-CD included the aqueous solutions to promote the dehydration to 11-deoxy-Δ10.
five-membered ring of PGF2α. When both α- and β-CDs are On the other hand, the degradation to 8-iso likely proceeds via
added, the correlation peaks appeared between the H3′ and the enol intermediate (8-ene-9-ol) or its conjugate base (Chart
H5′ protons of the CDs and the protons of the ω-chain, as 1B). The epimerizing C8 carbon in the intermediate has a
well as the five-membered ring of PGF2α, as shown in Fig. 5D. planar conformation, i.e., sp2 hybrid orbital (Chart 1B). There-
These results clearly demonstrate that both the ω-chain and fore, when the C8 carbon is forced to adapt this conformation,
five-membered ring of PGF2α participate in the ternary com- the epimerization is accelerated. Monkhouse et al. reported39)
plexation with α- and β-CDs.
that the isomerization of PGA2 was faster than that of PGA1.
Effect of CDs on the Stability of Limaprost in Solu- This is due to that two double bonds in a PGA2 molecule
tion It is reported that prostaglandins are chemically labile force the α- and ω-chains to align in a parallel conformation
in aqueous media and their rates of degradation are affected through a nonbonding interaction, while PGA1 with one dou-
by CDs.36,37) For example, sulfobutylether–β-CD (SBE–β-CD) ble bond has no interaction. In this conformation, the reactive
suppresses the degradation of PGE1 derivatives in acid or C12 carbon is apt to take the planar conformation, accelerat-
basic conditions, while other CDs accelerate the dehydra- ing the isomerization. Furthermore, NMR spectroscopic and
tion of the five-membered ring of the derivatives.38) To gain potentiometric studies suggested that the terminal carboxylic
insight into the inclusion mode for the Limaprost/α-CD/β-CD acid of PGF2α interacts with the hydroxyl groups of β-CD by
ternary complex, the effects of α- and β-CDs on the stability hydrogen bonding,33,34) as described above. Therefore, when
of Limaprost in alkaline solutions (pH 9.0 phosphate buffer) β-CD preferentially encapsulates the five-membered ring of
were investigated. As shown in Fig. 6, Limaprost degraded Limaprost, it may fix the α-side chain of Limaprost in a con-
in the alkaline solution, producing the two main products, formation favorable for enol formation, which promoting the
11-deoxy-Δ10 and 8-iso, where the rate to 8-iso was slightly isomerization. As shown in Fig. 6B, β-CD had the negligible
faster than that of 11-deoxy. The degradation of Limaprost effect on the dehydration into 11-deoxy-Δ10, which is probably
was accelerated by the addition of CDs in the condition. due to its larger cavity diameter and to free access of catalytic
The degradation rates (k) determined from Fig. 6A were water molecules to Limaprost. These results suggest that the
1.19×10−2 µg·mL−1·h−1 without CD, 1.58×10−2 µg·mL−1·h−1 respective inclusion sites of α- and β-CDs on Limaprost do not
with 2.0mg/mL α-CD, 1.88×10−2 µg·mL−1·h−1 with 2.0mg/ change in the co-presence of the two CDs, i.e., α- and β-CDs
mL β-CD, and 2.04×10−2 µg·mL−1·h−1 with both 2.0mg/ still encapsulate the ω-chain and the five-membered ring of
mL α-CD and 2.0mg/mL β-CD. The degradation rate of the Limaprost, respectively. Figure 7 schematically depicts the in-
ternary system was faster than those of the binary system. clusion mode estimated from the above results. α- and β-CDs
It was noteworthy that α-CD promoted the degradation to preferentially encapsulates the ω-chain and the five-membered
11-deoxy-Δ10, but negligibly affected the degradation to 8-iso. ring of Limaprost, respectively, to form the ternary complex,
On the other hand, β-CD showed the opposite effect on the although the fraction of two binding sites changes depending

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Chem. Pharm. Bull.
Vol. 63, No. 5 (2015)
Fig. 7. Proposed Inclusion Modes of the Limaprost/α-CD/β-CD Ternary
Complex in Water
Chart 1A. Reaction Mechanism for Dehydration of Limaprost
solid state, although the degradation mechanism of the drug in
the solid state is different from that in aqueous solution.
Conflict of Interest Yasuo Inoue, Noboru Sekiya and
Masanobu Yamamoto are employees of Ono Pharmaceutical
Co., Ltd.
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Chart 1B. Reaction Mechanism for Isomerization of Limaprost
on concentrations of CDs.
Conclusion
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On the basis of NMR spectroscopic and kinetic studies, we
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and the five-membered ring of Limaprost to form the ternary
complex. In a previous study, we reported that the chemi-
cal stability of Limaprost in the solid state was significantly
improved by the co-addition of both α- and β-CDs.32) The
ternary complex formation of Limaprost with α- and β-CDs
probably contributes to the stabilization of Limaprost in the

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