Physicochemical properties and inclusion complex formation of δ-cyclodextrin

|UMOP|AN

European Journal of Pharmaceutical Sciences 3 (1995) 153-162

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PIIRM,CEITICIL SCIENCES 11

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Physicochemical properties and inclusion complex formation of -cyclodextrin Izuru Miyazawaa, Haruhisa Ueda a'*, Hiromasa Nagase a, Tomohiro Endo a, Shoichi Kobayashi b, Tsuneji Nagai c aDepartment of Physical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, 4,41 Ebara 2-chome Shinagawa-ku, Tokyo 142, Japan bNational Food and Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba. Ibaraki 305, Japan CFaculty of Pharmaceutical Sciences, Hoshi University, Tokyo, Japan Received 19 September 1994; revised 30 January 1995

Abstract

•-Cyclodextrin (CD) is a cyclic oligosaccharide composed of nine a-l,4-1inked D-glucose units. Its physicochemical properties have been characterized only in terms of its X-ray crystal structure (Fujiwara et al., 1990). A method for the purification of ~-CD was examined, and its physicochemical properties and complexation abilities were elucidated and compared with those of a-, /3-, and 7-CD (Kobayashi et al., 1986; Miyazawa et al., 1993). Purification of 8-CD from the commercially available CD powder was mainly carried out with the combined treatment of HPLC and column chromatographies. The molecular weight of tS-CD was determined by FAB-MS, and the cyclic structure of ~-CD was identified by tH-NMR and ~3C-NMR. The aqueous solubility of tS-CD was greater than that of/3-CD but less than that of a - C D or 3,-CD. No surface activity of ~-CD was observed. 8-CD did not exhibit any hemolytic activity at 4.0 × 10 -2 M 8-CD, which was close to its saturated solution. The acid-catalyzed hydrolysis increased in the following order: a - C D
1. Introduction Cyclodextrin (CDi is a common name for cyclic oligosaccharides composed of a number of a-l,4-1inked D-glucoses, in which numbers 6, 7, and 8 are well known as a-, /3-, and y-CD, respectively. Owing to their annular cavity of 5-8 A, they are able to form an inclusion complex with a variety of guest molecules. They and their

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derivatives have been well studied and used in many fields. The earlier papers by French et al. (1965) and French (1957) provided the first definitive evidence for the existence of large ringed cyclodextrins (CDs) composed of a number of a-l,4-1inked D-glucose units, in which numbers 9, 10, 11, 12, and 13 are named 8-, e-, ~'-, r/-, 0-CD, respectively. However, they could not be studied in detail because of the difficulties in their purification and preparation in reasonable yields. Recently, the X-ray crystal structure of 8-CD, a cyclic oligosaccharide composed of nine a-l,4-1inked D-glucoses, has been character-

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I. Miyazawa et al. / European Journal of Pharmaceutical Sciences 3 (1995) 153-162

ized (Fujiwara et al., 1990), but its physicochemical properties are not at all clear. Based on the above considerations, the present study was planned to obtain a large amount of purified 6-CD. Furthermore, its physicochemical properties and complexation abilities were elucidated and compared with those of t~-, /3-, and y-CD.

2. Experimental section

held constant with a water bath and a column oven SSC-2100 (Senshu Kagaku). For preparative chromatography, the columns used for HPLC were a Senshu Pak. ODS-5251-SS (250 m i n x 2 0 4') (Senshu Kagaku) and an Asahipak NH2P-50 (300 mm x 21.5 d~) (Showa Denko). A column (30 x 2.8 cm i.d.) packed with Silica C~8 FS-1820 (Organo Co., Ltd, Tokyo, Japan) was also used for pre-fractionation of CDs. The columns used for analytical HPLC were Senshu Pak. ODS-1251-SS (250 mm x4.6 4~) (Senshu Kagaku), Hibar LiChro CART LiChrospher 100 NH 2 (250 m m x 4.0 4~) (Cica-MERCK, Darmstdt, Germany), and Asahipak NH2P-50 (250 mm x 4.5 40 (Showa Denko).

Materials. a- and /3-CD were supplied from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). y-CD was supplied from Wacker Chemie GmbH (Munich, Germany) and Nihon Shokuhin Kako Co., Ltd.. They were used after recrystalization from water. CD powder (Dexypearl K-50) was purchased from Ensuiko Sugar Refining Co., Ltd. (Yokohama, Japan). fl-amylase (1,4-aGlucan maltohydrolase) was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), and glucoamylase (1,4-a-Glucan glucohydrolase) was purchased from Seikagaku Kogyo Co., Ltd. (Tokyo, Japan). Pullulanase (a-l,6-glucosidase, Promozym 200L ® (Novo Industri A/S, Denmark)) was kindly donated by Novo Nordisk Bioindustry Co., Ltd. (Chiba, Japan). All other chemical drugs and solvents were from commercial sources and were used without further purification. The water used in all experiments was Milli-Q Water.

Apparatus for measurement of FAB-MS spectra and NMR spectra. The FAB-MS was performed with a JEOL SX-102A mass spectrometer (Jeol, Tokyo, Japan) with a Magic Bullet (MB) and/or glycerin as matrix; the acceleration was - 1 0 kV. ~H-NMR spectra and 13C-NMR spectra at 400 MHz were observed with a JEOL-GX-400 spectrometer (Jeol, Tokyo, Japan). Furthermore, 1HNMR, 13C-NMR, ~H-aH COSY two dimensional NMR and 13C-1HCOSY two-dimensional NMR spectra at 500 MHz were determined on a JEOLGX-500 spectrometer (Jeol). The samples were dissolved in 99.8% D20. Chemical shifts are expressed in ppm downfield from the signal of external tetramethylsilane.

Apparatus and columns for preparative and analytical methods. Preparative HPLC was performed with a SSC Flow System 3100J pump (Senshu Kagaku, Tokyo, Japan), with a Shodex DEGAS KT-16 degaser (Showa Denko, Tokyo, Japan), and an ERC-7530 refractive index (R.I.) monitor (Erma Optical Works, Tokyo, Japan) or an ERC-7521 R.I. monitor (Erma Optical Works). HPLC analyses of CDs were performed with a SSC Flow System 3100J pump (Senshu Kagaku), with a Shodex DEGAS KT-16 degaser (Showa Denko), and an ERC-7521 R.I. monitor (Erma Optical Works). For the performance of HPLC experiments, the column temperature was

Preparation of 6-CD mixture. Crude 6-CD was prepared as shown in Fig. 1. CD powder (Dexypearl K-50, 640 g) was dissolved in acetate buffer (pH 5.2) and allowed to react with flamylase, pullulanase, and yeast at 30°C, 72 h, using their enzyme catalyzed reaction. The supernatant was centrifuged by the addition of some organic solvents such as tetrachloroethane, bromobenzene, and ethanol, then a-, /3-, and y-CD, their derivatives and dextrin were removed. The supernatant containing the large ringed CDs was attained on treatments of deionization and decolorization and then was treated with pure glucoamylase to remove the contami-

I. Miyazawa et al. / European Journal of Pharmaceutical Sciences 3 (1995) 153-162 CD powder 640 g 1) dissolution in acetate buffer (pH 5.2) 800 ml 2) add. B-amylase 1.6 g pullulanase 8 ml yeast 20 g 3) shake 100 s.p.m, at 30°C for 72 h 4) centrifuge 14000 g at 10°C for 15 min [ residue

1 supernatant 5) add. bromobenzene 20 ml tetrachloroethane 20 ml ethanol 400 ml 6) shake at 4°C for 12 h 7) centrifuge 14000 g at 4°C for 15 min I residue

I supernatant 8) deionization ( Amberllite MB-3 ) 9) decolorization ( active carbon ) 10) filler through successive layers of filter paper and Celite t 1) add. glucoamylase 500 Units 12) add. acetone 150 ml 13) centrifuge 14000 g at 4°C for 10 min

I precipitate ( 6-CD mixture : 14.64 g )

supernatant

14) purification by column chromatography 6-CD (235.3mg)

]

Fig. 1. Purfication method of crude 6-CD (1).

nant dextrin. Finally, the supernatant was allowed to react with acetone, and the precipitate was obtained as a 8-CD mixture (14.64 g).

Purification of 3-CD. 3-CD was purified as shown in Fig. 2. The 8-CD mixture (14.64 g) was fractionated by H P L C using a column of Senshu Pak. ODS-5251-SS with 5% methanol-water at a flow-rate of 6.0 m l / m i n . Fig. 3 shows the chrom a t o g r a m of H P L C . The fraction of HO-3 in Fig. 2 and Fig. 3 was collected and evaporated; the yield of dry powder fr6m fraction HO-3 (Product A, n a m e d for convenience) was circa 1.52 g. Product A was mainly composed of 8-CD, with /3-CD and noncyclic oligosacharides as contaminants. This product A was fractionated by conventional chromatography on a column packed with Silica Cla FS-1820 with gradient elution on a 0 - 6 0 % methanol-water at a flow rate of about 3 m l / m i n . Approximately 100 ml of eluate was

155

collected in 30 separate flasks. Each fraction was examined by HPLC. Five h u n d r e d ml of eluent (fr. OP-7, fraction numbers 21-25 in Fig. 2) contained a mixture enriched in 6-CD. The yield of dry powder from fr. OP-7 (Product B, named for convenience) was circa 311.8 mg. Product B was further purified by semipreparative H P L C on a column of Asahipak with 40% acetonitrile-water at a flow rate of 4.5 ml/ rain. Fig. 4 shows the c h r o m a t o g r a m of HPLC. Three peaks were obtained, and they were n a m e d ft. NH-1, fr. NH-2 and fr. NH-3. The main fraction NH-2 (Product C, n a m e d for convenience) was obtained in a yield of circa 235.3 mg as a dry powder.

Pysicochemical properties of CDs. The solubility of 8-CD was determined using two different methods. Milli-Q water (25 + 0.1°C) was carefully added to a glass vessel containing 200 mg of 6-CD. The quantity of Milli-Q water varied progressively from 0.01 to 0.1 ml. The samples were vigorously shaken for 1 min periods at 10-min intervals at 2 5 - 0 . 1 ° C . The cycle was continued until 6-CD dissolved completely. The total volume of added Milli-Q water was then measured, and the saturated solubility was calculated. In another m e t h o d , a saturated solution of 8-CD was prepared according to the usual way. Its supernatant was subjected to H P L C on an analytical column of Asahipak NH2P-50 with 55% acetonitrile-water at a flow rate of 0.7 ml/ min. For reference, the solubilities of a-, /3-, and 3,-CD were determined in the same way. Optical rotation measurements were taken on a SEPA-200 digital polarimeter (Horiba, Kyoto, Japan) with an accuracy of +--0.002. Surface tension measurements were taken on a Wilhelmy surface tensiometer (Kyowa Kaimenkagaku Co., Ltd., Tokyo, Japan) with an accuracy o f ± 0.2 m N / m . Milli-Q water was used, and the glass vessel was treated with 20% sulfuric acid before each measurement. Acid-catalyzed hydrolysis of 8-CD.

Two hundred mg of 8-CD was dissolved in 10 ml of 1 N HC1, and the reaction solution was heated in a

156

1. Miyazawa et al. / European Journal of Pharmaceutical Sciences 3 (1995) 153-162 Crude 6-CD (14.64 g) purification by HPLC (OOS column) I fr. HO-1

I HO-2

I HO-3 (1.52 g)

I HO-4

purification by column chromatography (ODS column)

I

I

fr. OP-1 OP-2 MeOH (0%) (1%) Volume (200 ml) (200 ml)

I I I t J i OP-3 OP-4 OP-5 OP-6 OP-7 ( 311.8 mg ) OP-8 (3%) (5%) (7%) (10%) (30%) (60 %) (500 ml) (200 rnl) (200 ml) (600 ml) (600 ml) (500 ml)

purification by HPLC (NH2 column)

[ fr.NH-1

NH-2

NH-3

6-CD ( 235.3mg )

Fig. 2. Purfication method of 6-CD (2). fr.NH-2

fr HO-1 fr.HO-2 f--- .---]

fr.HO-3 i

fr.NH-1 r-q

fr.HO-4 • i

0

20

40

_t rnin

Fig. 3. Purification of 8-CD mixture by HPLC (ODS Column) column; Senshu Pak. ODS-5251-SS; eluent, CH3OH-H20 (6:100); flow rate, 6.0 ml/min.

heating bath at 60°C. The pH of the sample solution was ascertaine.d to be the same before and after the reaction. Samples of the reaction

0

10

20 min

Fig. 4. Purification of 6-CD mixture by HPLC (NH 2 Column) column; Asahipak NH2P-50; eluent, CH3CN-H20 (40:60); flow rate, 4.5 ml/min.

solution (0.3 ml portions) were taken at appropriate intervals and neutralized by the addition of 0.3 ml of 1 N NaOH containing /3-CD (1.8 w/

l. Miyazawa et al. / European Journal of Pharmaceutical Sciences _3 (1995) 153-162

v%) as an internal standard for HPLC. From 10 to 15 txl samples were injected into the H P L C on a column of Senshu Pak. ODS-1251-SS with 10% methanol-water at a flow rate of 1.0 ml/min. The hydrolyzed products of g-CD such as heptaose of linear saccharides had no influence on the HPLC chromatogram of 6-CD under these conditions. The kinetic data conformed to the first-order rate law.

157

pore membrane filter and analyzed by spectrophotometry at 238 nm of SP. An apparent stability constant,K, was calculated from the initial straight line portion of the phase solubility diagrams according to the following equation: slope K = intercept (1-slope)

(1)

Hemolysis studies of 6-CD.

Human erythrocytes were separated from human blood by centrifugation (3000 rpm for 15 min), washed 3 times with isotonic phosphate buffer (pH 7.4) and resuspended to produce a hematocrit of 5%. Aliquots (0.1 ml) of the erythrocyte suspension were added to isotonic phosphate buffer (4 ml) containing 6-CD, and the mixture was gently agitated for 30 min at 37°C. After centrifugation (3000 rpm for 15 min), the optical density of the supernatant was measured for hemoglobin at 541 nm using a Ubest-30 Double Beam spectrophotometer (JASCO Co., Ltd., Tokyo, Japan). The results were expressed as % total hemolysis by comparison with a sample of complete hemolysis in water.

Solubility studied for poorly water soluble drugs. An excess amount of drug was added to the aqueous solutions containing 6-CD (15 mg/ ml). The solutions were sonicated three times for 10 minutes at 30-minute intervals and then shaken at 2 5 - 0.1°C. After an equilibrium was attained (approximately 3 days), the solution was filtered through a 0.45 izm Millipore membrane filter. A portion of each sample was diluted and analyzed by spectrophotometry at suitable wavelengths, and then the solubility was calculated. For reference, the sOlubilization ability of a-, /3-, T-CD was determined in the same way.

Solubility studies of spironolactone (SP). Solubility measurements were carried out according to Higuchi and Connors (1965). Excess amounts of SP were added to aqueous solutions containing various concentrations of CDs, and the mixtures were shaken at 25-+ 0.5°C. A f t e r an equilibrium was attained (approximately 3 days), an aliquot of solution was filtered through a 0.45 tzm Milli-

3. Results and discussion

3.1. Identification of 8-CD The chromatographic behavior of the product C (fr. NH-2 in Fig. 4) was compared with that of the well-recognized standard of a-, 13-, Y-, and 8-CD. The 8-CD used as a standard was the same sample as employed in X-ray crystal structure analysis. Product C was subjected to analytical HPLC columns in three different modes. Fig. 5 shows the HPLC elution profiles of a standard mixture of CDs on the Hibar kiChrospher 100 NH 2 column (Fig. 5(a)), on an Asahipak NH2P-50 column (Fig. 5(c)), and on a Senshu Pak. ODS-1251-SS column (Fig. 5(e)). The elution sequence with the NHz-bonded silica and acetonitrile-water system was followed by the order of molecular size. The elution time provides qualitative information about the molecular size of the sugar. By contrast, the Cls-bonded silica column showed the difficulty of highly efficient separation of a-, Y-, and 6-CD. The elution time of product C on three different columns was in agreement with that of the standard 8-CD. The purity of product C was 98% as a 8-CD by HPLC determinations. Fig. 6 shows the FAB-MS of product C. In the mass spectra of product C, high parent-ion peaks appeared at 1459 and 1481, corresponding to the molecular weights plus proton (M + H) + and sodium ion (M + Na) +, respectively. The molecular weight of product C was determined to be 1459, and this value corresponded to that of 6-CD ( C 6 H l 0 0 5 ) 9. Consequently, product C was identical to 6-CD Fig. 7 shows the 13C-1H COSY two dimensional N M R spectrum of 6-CD at 500 MHz. The

1. Miyazawaet al. /

158

European Journal of

(a) c~-, 13-, y-, and 6 - C O

PharmaceuticalSciences3 (1995) 153-162

( c ) ct-, 13-, y - , a n d

6-CD

( e ) oL-, 13-, y - , a r i d 6 - C D

ct '7

-f

aB r ~

/L_

Z

7

0

10

20

0

min

(b) P r o d u c t C

10

20

min

0

(d) P r o d u c t C

20

(f) Product

40

60

min

40

60

min

C

\ 0

10

20

min

cotumn : Hibar LiChro C A R T L i C h r o s p h e r 100 NH2 eluent : C H 3 C N - H 2 0 ( 70 : 30) flow rate : 1.0 m l / m i n

0

10

20

min

0

20

column : Senshu Pak O D S - 5 2 5 1 - S S eluent : C H 3 O H - H 2 0 ( 8 ; 95 ) flow rate : 1.0 ml ~rain

column : Asahipak N H 2 P - 5 0 eluent : C H 3 C N - H 2 0 ( 55 : 45 ) flow rate : 0.7 m l / m i n

Fig. 5. HPLC chromatograms of a-, fl-, y- and 8-CD. [M+Na] + 1481.4 ( M a t r i x : MB) 428889

4

1300

1400

1500

1600

m/z

[M+H] 1459.4

j

i 800

~.~..~.,t ,t. ~L.,. 1

900

1o0o

1100

1200

1300

......... i 1480

Jt

+

(Matrix : glycerin) ..

~. • i

l 5oe m/z

Fig. 6. FAB-mass spectra of 8-CD.

peaks of H1, H3, H2, and H4 of 8-CD were recognized, but the peaks of H5, and H6 were 1 not completely assigned by the H - N M R spectrum. The 13C-NMR chemical shifts of 8-CD are also summarized with those of other CDs in

Table 1 (Gelb et al., 1982). There was no large difference in the chemical shifts of the ~3C-NMR between 8-CD and the other CDs. This suggested that 8-CD had a typical cyclic structure in analogy with the other CDs.

I. Miyazawa et al.

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European Journal of Pharmaceutical Sciences 3 (1995) 153-162

C~

C2

C4

C3.1 05

I

I

C6

t~l

~

ir

H4

H5 H5 H3

Fig. 7. t3C- 1H COSY NMR spectrum of 6-CD.

3.2. Physicochemical properties of 6-CD

Table 1 ~3C-NMR chemical shifts of CDs Carbon

o~-CDa't'

/3-CD a'b

y-CD a'b

6-CD b

1 2 3 4 5 6

102.5 72.8 74.4 82.3 73.1 61.5

102.87 72.10 74.11 82.16 72.87 61.35

102.68 73.35 73.96 81.50 72.84 61.30

102.72 74.86 75.53 80.98 74.10 63.05

Table 2 lists some physicochemical properties of a-, /3-, y-, and 6-CD such as aqueous solubility, surface activity, optical properties, acidcatalyzed hydrolysis rate and internal cavity diameter (Uekama, 1989; Uekama et al., 1988). The aqueous solubility of 6-CD was greater than that of/3-CD but less than that of o~-, or y-CD. No large difference in the aqueous solubility of 6-CD could be detected in two different methods. The low solubility of /3-CD may be a

" G e l b et al. (1982). b ppm downfield from external Me4Si at 30°C in D 2 0 soln.

Table 2 Physicochemical properties of CDs CD

Glucose unit n

Molecular weight

Aqueous b solubility (g per 100 ml)

Surface b'c tension (nM/m)

[a] 25

Acid-catalyzed hydrolysis rate (h - t )

Internal cavity diameter (/~)

a-CD /3-CD y-CD 6-CD

6 7 8 9

973 1135 1297 1459 a

14.5 d 1.85 d 23.2 d 8.19

73 73 73 72

+150.5 +162.5 +177.4 +187.5

0.11 0.13 0.23 0.63

4.7-5.2 a 6.0-6.4 d 7.5-8.3 ~ 10.3-11.2 e

"Determined by FAB-MS. ~ Observed at 25°. ¢ Concentration of CD was 0.1% in water, d K. Uekama (1981). "T. Fujiwara et al. (1990).

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I. Miyazawa et al. / European Journal of Pharmaceutical Sciences 3 (1995) 153-162

consequence of intramolecular hydrogen bonding between hydroxyl groups along the edges of the ring, which prevents adequate hydration by water molecules (Fr6mming et al., 1994). In addition, it would seem possible that solubility differences of CDs could be due to different interactions in the crystal states. The low solubility of 6-CD in comparison with that of a-, and y-CD seems to be the same mechanism as described above. /3-, and 6-CD have an odd number of glucose units, so they may have a relatively similar distorted conformation of the macrocyclic ring. If more detailed solubility data of e-, ~'-, "q-, and 0-CD could be obtained, it might become apparent that there is a relationship between the numbers of glucose units and the relative solubilities of CDs. No surface activity of 6-CD was observed; it was about the same as that of water (71.92 mN/m). The values of optical rotation increased with an increasing number of glucose units in the order of: a - C D y-CD >/3-CD > a-CD. The present finding suggested that the stability of the macrocyclic ring of CDs decreased with an increase in the number of glucose units.

3.3. Hemolytic effects Fig. 8 shows the hemolytic effects of a-,/3-, y-, and 6-CD on human erythrocytes in an isotonic phosphate buffer (pH 7.4). The CD-induced hemolysis was reported to be due to membrane destruction elicited by the dissolution and removal of membrane components (Irie et al., 1982). The hemolytic activity of 6-CD was not exhibited at 4.0 × 10 - 2 M 8-CD which was close to a

y

,L

E -7-

0

I

J

J

O A

1

2

3

4

Conc. of CDs ( × 1 0 "2M)

Fig. 8. Hemolytic effects of CDs on human erythrocytes in isotonic phosphate buffer (pH 7.4) at 37°C. Key: (O), a-CD; (F]), fl-CD; (A), y-CD; (©), 6-CD. saturated solution. This suggested that 6-CD had less affinity for membrane components of erythrocytes than did other CDs (Irie et al., 1982). This striking characteristic of 6-CD seems to be useful for the application of preparing parenteral dosage forms.

3.4. Inclusion ability of 6-CD The inclusion abilities of 6-CD for some poorly water-soluble drugs were studied by measuring their solubilization abilities. Table 3 shows the effects of 6-CD on the solubility of slightly soluble or insoluble drugs in water. 6-CD did not show any significant solubilization effect on these drugs. However, in the case of a guest molecule such as spironolactone (SP) and digitoxin, which has a steroidal framework, the enhancement of solubility of the guest molecule by 6-CD was greater than that by a-CD. In order to discuss the mechanism of inclusion complex formation of SP with 6-CD, the behavior of inclusion complex formations for SP with /3% y-, 6-CD in an aqueous solution was studied using the solubility method. Fig. 9 shows the phase solubility diagrams obtained for SP with CDs in water. The/3-CD and y-CD systems showed typical Bs type solubility curves, where the initial rising portions are followed by plateau regions, and then the total SP concentration decreased with the precipitation of micro-crys-

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I. Miyazawa et al. / European Journal of Pharmaceutical Sciences 3 (1995) 153-162

Table 3 Effects of CDs on solubility of slightly soluble or insoluble drugs in water at 25°C Drug

Anthracene Amphotericin B Ajmalicine Aimaline Carbamazepine Digitoxin Spironolactone 9,10-Dibromoanthracene 3,4,9,10-Perylenetetra caroxylic Dianhydride

Solubility in water (mg per 100 ml)

Solubility in 15 mg/ml CDs solution (mg per 100 ml)

0.0068 1.006 5.570 47.80 17.08 0.92 4.62 0.0039 0.522

0.0063 0.895 7.730 138.88 19.24 3.66 14.95 0.0049 0.904

a-CD

/3-CD (0.9) (0.9) (1.4) (2.9) (1.1) (4.0) (3.2) (1.2) (1.7)

0.0594 1.062 9.935 132.88 126.35 17.16 29.89 0.0057 0.667

(8.7) (1.1) (1.8) (2.8) (7.4) (18.6) (6.5) (1.5) (1.3)

7-CD

8-CD

0.0073 (1.1) 1.354 (1.4) 10.324 (1.9) 72.68 (1.5) 79.61 (4.7) 20.06 (21.8) 86.80 (18.8) 0.0448 (11.3) 0.757 (1.5)

0.0044 0.989 6.916 58.01 31.99 13.91 24.86 0.0070 0.670

(0.7) (1.0) (1.2) (1.2) (1.9) (15.1) (5.4) (1.8) (1.3)

The solubilization rate is summarized in parentheses. 3 o o

2 o "6 d O

16

26

36

,;

s;

s;

Conc. of CDs (xl0ai) Fig. 9. Phase solubility diagrams of spironolactone-CDs systems in water at 25°C. Key: ([~), fl-CD; (&), y-CD; (©), 8-CD.

talline complexes (Seo et al., 1983; Yusuff et al., 1991). On the other hand, the solubility of SP increased linearly as a function of 6-CD concentration, and the solubility curve can be generally classified as being of type AL. In sharp contrast, no precipitation was observed for the 6-CD system. The solubility of SP increased about 30-fold in the presence of 3-CD (4.5 x 10 -z

M). The apparent stability constant (K), as a tentative measure of inclusion complexation, was estimated from Eq. (1) based on the assumption that a 1:1 complex initially formed. The magnitude of K values calculated from the initial rising portion of solubility diagrams was found to

decrease in the order:/3-CD (1.3 x 104 M -1) > 7CD (3.9 x 103 M -1) > 6-CD (8.2 x 10 2 M-l). This suggested that 8-CD had a weak complex forming ability with SP in comparison with/3-CD and 7-CD. This phenomenon was presumed to be due to the high flexibility of 6-CD ring and behaviour of complexed water molecules in the 6-CD cavity (Fr6mming et al., 1994). In conclusion, a method for the purification of ~-CD was established, and its physicochemical properties and complexation abilities were elucidated and compared with those of a-, /3-, and 7-CD. 8-CD did not show any significant solubilization effect on several kinds of molecules except for SP and digitoxin in this experiment. The details of the interaction between 6-CD and many other chemical molecules will be published elsewhere in the near future.

Acknowledgements

We would like to thank Dr. K. Koizumi of Faculty of Pharmaceutical Sciences, Mukogawa Women's University, for helpful discussion and measurement of the X3C-1H COSY two-dimensional NMR spectrum of 6-CD. We gratefully acknowledge the generous supply of Silica C18 FS-1820 from Organo Co. Ltd.. Thanks are due also to Miss T. Tomaru, Miss K. Meguro, Miss A. Itoh, Mr. M. Kashiwazaki, Mr. Y. Suzuki, Miss K. Itayama, Miss M. Iwasaki, Miss J.

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Utsuno, Mr. T. Nakao, and Mr. N. Futatsugi, for their assistance.

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