IN VIVO ASSESSMENT OF ORAL ADMINISTRATION OF PROBUCOL

NANOPARTICLES IN RATS （ラット経口投与におけるナノ微粒子化プロブコールの評価）

２００８

JYUTARO SHUDO

首藤 十太郎

CONTENTS Page#

ABSTRACT 1

INTRODUCTION 2

EXPERIMENTAL

CHAPTER 1: Stability and in vivo absorption of probucol nanoparticles

obtained from probucol/PVP/SDS ground mixtures 5

CHAPTER 2: In vivo comparison in the case that the particle size

obtained from ternary GM with PVP K12 is larger

than that from ternary GM with PVP K17 17

RESULTS AND DISCUSSION

CHAPTER 1: Stability and in vivo absorption of probucol nanoparticles

obtained from probucol/PVP/SDS ground mixtures

1. Stability study of probucol/PVP/SDS

ternary GM suspension 21

2. Drug absorption from the ternary GM 26

3. Investigation of drug absorption from

binary GM with SDS 30

CHAPTER 2: In vivo comparison in the case that the particle size

obtained from ternary GM with PVP K12 is larger

than that from ternary GM with PVP K17

1. Verification of the effect of nanoparticle surface

condition with PVP and SDS on the absorption 35

CONCLUSION 38

REFERENCES 39

ACKNOWLEDGEMENTS 48

LIST OF PUBLICATION 49

THESIS COMMITTEE 50

MAIN TEXT

- 1 -

ABSTRACT

Pharmacokinetic profiles of probucol were evaluated after oral

administration of the various nano-suspensions in rats. Probucol nanoparticles

were prepared by co-grinding with various molecular weights of

polyvinylpyrrolidone (PVP K12, PVP K17 and PVP K30) and sodium dodecyl

sulfate (SDS). The average particle sizes of probucol after dispersing the

ternary ground mixtures (GMs), probucol/PVP K12/SDS, probucol/PVP

K17/SDS and probucol/PVP K30/SDS into distilled water were 28, 75 and 89

nm, respectively, compared to the unprocessed probucol was 27 µm. The

ternary GM suspensions with PVP K17/SDS and PVP K30/SDS were stable at

25 °C. However the particle size of probucol from the ternary GM with PVP

K12/SDS gradually increased was attributable to insufficient surface coverage

of the nanoparticles with PVP K12-SDS micelle complex. Pharmacokinetic

profiles of probucol indicated that variation in particle surface condition

covered with PVP and SDS in addition to the particle size affected the

improvement of in vivo absorption of probucol. The ternary GM with PVP

K12/SDS exhibited a superior improvement of probucol absorption compared to

the GMs with PVP K17/SDS and PVP K30/SDS. The binary GM with PVP or

SDS and physical mixtures with PVP and/or SDS did not show significant

differences in the area under the plasma concentration-time curve compared to

the unprocessed probucol. In conclusion, preparation of probucol

nanoparticles by co-grinding with PVP K12 and SDS could be a promising

method for bioavailability enhancement.

- 2 -

INTRODUCTION

Probucol, 4,4’-{(l-methylethylidene)bis(thio)}-bis{2,6-bis(l,l-dimethylethyl)}phenol, is

mainly used as a cholesterol-lowering agent.1, 2) Various studies suggest that

probucol lowers serum cholesterol by increasing the fractional rate of low

density lipoprotein (LDL) catabolism in the final metabolic pathway for

cholesterol elimination from the body.3-5) Probucol also inhibits the oxidation

and tissue deposition of LDL cholesterol, thereby inhibiting atherogenesis.3, 6-8)

Probucol can not be injected intravenously due to the poor water solubility (5

ng/mL at 25 °C).3, 9, 10) Since The bioavailability of probucol is very poor ( up

to 6%), LD50 of probucol in rat is 5 g/kg through oral administration.11) The

tablet form of probucol is absorbed at different rates and in different amounts

depending on the individual patients.3) When probucol was co-administered

with arachis oil, the absorption enhancement of probucol in rats was

observed.12) Thus, the formulation seems to be important to improve the

bioavailability.

Reducing the particle size is one of the ideas to improve the

bioavailability of drugs with poor absorption.13, 14) Several cutting-edge

technologies such as high pressure homogenization, spray dry, supercritical

fluid processing, precipitation techniques and grinding are currently utilized to

generate drug nanoparticles.15-24) Co-grinding, where a drug is ground together

with excipients, is a promising method for an effective reduction of particle size.

A certain number of studies revealed that the co-ground mixture

enhanced dissolution, resulting in the improvement of oral absorption and

- 3 -

bioavailability.25-28) Wet co-grinding method has been known as an effective

method to produce stable nanosuspensions.29-33) Liversidge and Cundy

proposed a preparation method for crystalline nanoparticle by wet co-grinding

of danazol with polyvinylpyrrolidone (PVP).29) Oral absorption of poorly

water-soluble drugs in nanocrystal form was remarkably improved.29, 30)

However, the wet process may cause drug degradation by hydrolysis during the

preparation. In addition, a drying process should be required to prepare the

solid dosage form. Compared with the wet co-grinding method, the dry

co-grinding process has some advantages for solid pharmaceutical applications

due to its simple preparation with free solvents. However, in vivo absorption

experiments have been rarely reported.

Our previous studies demonstrated that drug nanoparticle was

successfully produced by the dry co-grinding of a poorly water-soluble drug

with polymer and surfactant.34-36) The combined use of polymer and a

surfactant contributed to the effective stabilization of solid particles by the

adsorption of a polymer-surfactant complex on the particle.37-39) PVP and

sodium dodecyl sulfate (SDS) have been used in a variety of pharmaceutical

formulations.40, 41) The drug/PVP K17/SDS was profitably applied for the

preparation of hydrophobic drug nanoparticles.34, 36) The nanosuspension

prepared from the ground mixture (GM) dispersed into distilled water exhibited

good stability as the particle agglomeration was effectively inhibited by the

adsorption of both PVP and SDS on the surface of crystalline particles.36) 13C

NMR studies revealed that the nanoparticle formation and stabilization were

attributable to grinding-induced solid-state interactions among components of

- 4 -

the drug/PVP/SDS ternary system.42-44) The present study was designed to

investigate the effects of particle size and PVP molecular weight on the oral

absorption of probucol. Pharmacokinetic profiles of probucol nanoparticles

were evaluated after oral administration in rats. Different sizes of probucol

nanoparticles were produced by co-grinding with various molecular weights of

PVP (i.e. PVP K12, PVP K17 or PVP K30) and SDS. The Probucol/PVP/SDS

at the weight ratio of 1:3:0.5 was employed because it was one of the most

appropriate ratios to reduce particle size with the least ratio of SDS.42) Effects

of the ternary composition of the GM on the improvement of absorption were

also discussed.

- 5 -

EXPERIMENTAL

CHAPTER 1

Stability and in vivo absorption of probucol nanoparticles

obtained from probucol/PVP/SDS ground mixtures

Materials

Probucol, 4,4’-{(l-methylethylidene)bis(thio)}-bis{2,6-bis(l,l-dimethylethyl)} phenol,

was supplied by Daiichi Sankyo Co., Ltd. (Tokyo, Japan).

Polyvinylpyrrolidone, PVP K12 (MW ∼2,500), was obtained from BASF Japan

Ltd. (Tokyo, Japan). PVP K17 (MW ∼10,000) and PVP K30 (MW ∼50,000)

were obtained from ISP Technologies, Inc. (Texas City, TX, USA). Sodium

dodecyl sulfate (SDS) was purchased from Wako Pure Chemical Industries Ltd.

(Osaka, Japan). These chemical structures are shown in Fig. 1.

Hydroxypropylcellulose was purchased from Shin-Etsu Chemical Co., Ltd.

(Tokyo, Japan). α-Tocopherol acetate, ethyl acetate, methanol, trifluoroacetic

acid and acetonitrile were purchased from Sigma-Aldrich Co. (St. Louis, MO,

USA). All other chemicals used were of reagent grades.

Probucol

M.W.: 516.84

Melting point: 160 ºC

pKa: 10.24

Sodium dodecyl sulfate (SDS)

Anionic surfactant

M.W.: 228.38

Melting point: 204-207 ºC

CMC at 25 ºC=8.15 mM＝2.0 mg/mL

Polyvinylpyrrolidone (PVP)

Nonionic water soluble polymer

PVP K12: M.W.: ∼2,500

PVP K17: M.W.: ∼10,000

PVP K30: M.W.: ∼50,000

Fig. 1 Chemical structures of probucol, polyvinylpyrrolidone and sodium

dodecyl sulfate.

- 6 -

- 7 -

Preparation of physical mixture (PM)

Probucol (0.67 g), PVP (2.00 g) and SDS (0.33 g) (weight ratio of

1:3:0.5) were physically mixed in a glass vial using a vortex mixer for 5 min

(Fig. 2). For the binary physical mixture, probucol (0.75 g) and PVP (2.25 g)

or probucol (2.00 g) and SDS (1.00 g) were physically mixed following the

same method as described above.

Preparation of ground mixture (GM)

Probucol (0.67 g), PVP (2.00 g) and SDS (0.33 g) (weight ratio of

1:3:0.5) were physically mixed and then ground in a vibrational rod mill

(TI-500ET, CMT Co., Ltd., Fukushima, Japan) for 30 min (Fig. 2). The

grinding was performed under temperature control and monitoring at 10 ± 5 °C.

The grinding cell and rod were made of stainless steel. For the binary ground

mixture, probucol (0.75 g) and PVP (2.25 g) or probucol (2.00 g) and SDS (1.00

g) were ground respectively by the same method as described above.

Probucol SDSPVP

Grinding by vibrational rod mill

Ground mixture (GM)

Mixing at a weight ratio of 1:3:0.5 by vortex

mixer

Physical mixture (PM)

Probucol SDSPVP

Grinding by vibrational rod mill

Ground mixture (GM)

Mixing at a weight ratio of 1:3:0.5 by vortex

mixer

Physical mixture (PM)

Fig. 2 Preparation process of probucol/PVP/SDS physical mixture and ground

mixture.

- 8 -

- 9 -

Particle size analysis

The GM was dispersed into distilled water, then sonicated for 2 min to

make the suspension. The drug concentration in the suspension was 0.50

mg/mL. The Japanese Pharmacopoeia (JP XIV) first fluid (pH 1.2) and second

fluid (pH 6.8) were also used as liquid media instead of distilled water. The

particle size was determined by the dynamic light scattering method using

Microtrac UPA, with a measurement range of 0.003-6 µm (Nikkiso Co., Ltd.,

Tokyo, Japan) and by the laser diffractive scattering method using Microtrac

FRA, with a measurement range of 0.1-700 µm (Nikkiso Co., Ltd., Tokyo,

Japan).

Stability study

Stability studies of the GM suspensions were investigated by particle

size analysis after storage at 25°C for 0 (initial), 1, 2, 3, 7, 10, 12, 24 and 48 h.

Quantitative determination of nanoparticles

The GM suspensions were filtered through a 0.8 µm membrane filter

(Millipore Corp., Tokyo, Japan) and then diluted with acetonitrile. The

concentration in nanoparticle fractions was determined by HPLC (LC-6A,

Shimadzu Corp., Kyoto, Japan). The mobile phase (acetonitrile/water =

185/15, v/v) was delivered at a flow rate of 1.0 mL/min through a C-18 column

(Inertsil ODS-2, 5 µm, 4.5 x 150 mm, GL Sciences Inc., Tokyo, Japan) at 40 °C

and the detection wavelength was 220 nm. The concentrations of probucol in

- 10 -

the samples were determined by the linear equation obtained from the standard

curve(y =26088x-2223, correlation coefficient =0.9999).

In vivo absorption study

Probucol formulations were administered by oral gavages to male

Sprague Dawley rats. Twenty-seven adult rats with a weight of 270-330 g were

obtained from Charles River Laboratories Inc. (Wilmington, MA, USA). All

rats were made to fast overnight prior to the dose administration and remained

fasting until 6 h after the dose administration. The sample suspensions were

prepared with sonication by dispersing the probucol formulations into distilled

water to obtain a probucol concentration of 25 mg/mL, and then administered to

the rats at 8 mL/Kg by oral gavage. In the case of unprocessed probucol, due

to its low wettability, 0.5% hydroxypropylcellulose solution was used instead of

distilled water to facilitate the dispersion. Blood samples (∼0.3 mL) were

collected into vacutainer tubes containing EDTA, 0, 1, 2, 3, 4, 7, 10, 24 and 48 h

after drug administration. After this collection, the blood samples were

centrifuged at approximately 2800 rpm (∼1000 x g) at 2-8 °C for about 15 min.

Each plasma specimen was collected, and stored at ∼ -80 °C until analysis. No

adverse reaction was observed following dose administration during the study.

Determination of probucol in rat plasma by HPLC

- 11 -

Preparation of plasma samples

Probucols in the plasma samples were determined by HPLC.45-47)

α-Tocopherol acetate was used as internal standard.48) A solution of internal

standard was prepared with ethyl acetate at 10 µg/mL. Samples were prepared

as follows: 50 µL of plasma were extracted with 1 mL of internal standard

solution in polypropylene tubes. Samples were then sonicated for 5 min,

vortexed for 5 min and centrifuged at 12,000 rpm for 10 min. Supernatants

were transferred into glass test tubes. A blank (50 µL of blank plasma

extracted with internal standard) and double blank (50 µL of blank plasma

extracted with blank ethyl acetate) were also prepared. Samples were dried

under nitrogen, reconstituted with 100 µL of methanol, and transferred into a

glass insert in an autosampler vial. The plasma sample preparation scheme is

shown in Fig. 3.

50μL of plasma

(Plastic tube)

1mL of Internal Standard solution

Sonication for 5 min

Vortex for 5 min

Centrifuge at 12,000 rpm for 10 min

Dried with N2

HPLC assay

Supernatants

(Glass tube)

Add 100μL of Me-OH and transfer solution to HPLC Vial

50μL of plasma

(Plastic tube)

1mL of Internal Standard solution

Sonication for 5 min

Vortex for 5 min

Centrifuge at 12,000 rpm for 10 min

Dried with N2

HPLC assay

Supernatants

(Glass tube)

Add 100μL of Me-OH and transfer solution to HPLC Vial

Fig. 3 The plasma sample preparation scheme.

- 12 -

Preparation of standard samples

Standards were prepared in the same manner as samples (using blank

plasma) and reconstituted with methanol containing probucol at known

concentrations. Eight calibration standards were prepared for probucol

concentration in plasma, between 0.02 and 8.75 µg/mL probucol in plasma.

The limit of quantitation was described by the lowest concentration of the

standard (0.02 µg/mL).

0.000.020.040.060.080.100.12

0.140.16

0.00 2.00 4.00 6.00 8.00 10.00

Plasma concentration (μg/mL)

Prob

ucol

area

/ IS

area

Series1Linear (Series1)

y=0.0157x+0.0007R2=0.9987

0.000.020.040.060.080.100.12

0.140.16

0.00 2.00 4.00 6.00 8.00 10.00

Plasma concentration (μg/mL)

Prob

ucol

area

/ IS

area

Series1Linear (Series1)

y=0.0157x+0.0007R2=0.9987

(The graph of the linear standard curve)

HPLC conditions

The probucol concentration in plasma samples was determined by HPLC

(HP 1100, Agilent Technologies Inc., Santa Clara, CA, USA). 45-47) The mobile

phase was delivered at a flow rate of 1.5 mL/min through a reverse-phase

column (Zorbax 300SB-C8, 5 µm, 4.6 x 150 mm, Agilent Technologies Inc.,

Santa Clara, CA, USA) at 40 °C and the detection wavelength was 220 nm.

- 13 -

- 14 -

The injection volume was 20 µL. The gradient was the change in the

proportion of phase A solvent (0.1% trifluoroacetic acid in water) and phase B

solvent (0.1% trifluoroacetic acid in acetonitrile) that make up the mobile phase.

The steps of gradient shown were: phase B solvent concentration in the mobile

phase was 75% (at 0 min) and changed from 75% to 90% (after 10 min) and

returned from 90% to 75% (after 15 min). A representative chart sample is

shown in Fig. 4.

Fig. 4 A representative HPLC chart, probucol retention time is around 3.4

min.

- 15 -

- 16 -

HPLC data analysis

Analysis of the plasma samples was carried out using Agilent Chem

Station software (V. A. 09. 01, Agilent Technologies Inc., Santa Clara, CA,

USA). The concentrations of probucol in the samples were determined by the

linear equation obtained from the standard curve(y=0.0157x+0.0007, correlation

coefficient =0.9987).

Statistic analysis

Data are expressed by the mean ± S.E.. The area under the plasma

concentration-time curve (AUC) value (0-48 h) of the plasma profiles was

calculated using the logarithmic and linear trapezoidal rules. P-values were

calculated using F-test and T-test in Microsoft Excel 2003 and less than 0.05

considered significant.

- 17 -

CHAPTER 2

In vivo comparison in the case that the particle size

obtained from ternary GM with PVP K12 is larger than

that from ternary GM with PVP K17

Materials

Ingredients used in chapter 2 were the same as those described in

materials in chapter 1.

Preparation of ground mixture (GM) with PVP K12 (40nm)

Probucol (0.67 g), PVP K12 (2.00 g) and SDS (0.33 g) (weight ratio of

1:3:0.5) were physically mixed and then ground in a vibrational rod mill

(TI-500ET, CMT Co., Ltd., Fukushima, Japan) for the certain time to adjust the

particle size in suspension to 40nm. The grinding was performed under

temperature control and monitoring at 10 ± 5 °C. The grinding cell and rod

were made of stainless steel.

Preparation of ground mixture (GM) with PVP K17 (36nm)

Probucol (0.67 g), PVP K17 (2.00 g) and SDS (0.33 g) (weight ratio of

1:3:0.5) were physically mixed and then ground in a vibrational rod mill

(TI-500ET, CMT Co., Ltd., Fukushima, Japan) for 30 min. The grinding was

performed under temperature control and monitoring at -180 ± 5 °C. The

- 18 -

grinding cell and rod were made of stainless steel.

In vivo absorption study

In vivo absorption study was conducted in a manner similar to the study

method described in in vivo absorption study in chapter 1, but six adult rats with

a weight of 350-400 g were obtained from Charles River Laboratories Inc.

(Wilmington, MA, USA). No adverse reaction was observed following dose

administration during the study.

Determination of probucol in rat plasma by HPLC

Preparation of plasma samples

Plasma samples were prepared in a manner similar to the method

described in preparation of plasma samples in chapter 1.

Preparation of standard samples

Standards were prepared in the same manner as samples (using blank

plasma) and reconstituted with methanol containing probucol at known

concentrations. Five calibration standards were prepared for probucol

concentration in plasma, between 0.05 and 4.00 µg/mL probucol in plasma.

The limit of quantitation was described by the lowest concentration of the

standard (0.05 µg/mL).

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.00 1.00 2.00 3.00 4.00 5.00

Plasma concentration (μg/mL)

Prob

ucol

area

/ IS

area

Series1Linear (Series1)

y=0.2807x-0.005R2=0.9997

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.00 1.00 2.00 3.00 4.00 5.00

Plasma concentration (μg/mL)

Prob

ucol

area

/ IS

area

Series1Linear (Series1)

y=0.2807x-0.005R2=0.9997

(The graph of the linear standard curve)

HPLC conditions

The probucol concentration in plasma samples was determined by HPLC

(HP 1200, Agilent Technologies Inc., Santa Clara, CA, USA). 45-47) The mobile

phase was delivered at a flow rate of 1.5 mL/min through a reverse-phase

column (Zorbax 300SB-C8, 5 µm, 4.6 x 150 mm, Agilent Technologies Inc.,

Santa Clara, CA, USA) at 40 °C and the detection wavelength was 220 nm.

The injection volume was 20 µL. The gradient was the change in the

proportion of phase A solvent (0.1% trifluoroacetic acid in water) and phase B

solvent (0.1% trifluoroacetic acid in acetonitrile) that make up the mobile phase.

The steps of gradient shown were: phase B solvent concentration in the mobile

phase was 75% (at 0 min) and changed from 75% to 90% (after 10 min) and

returned from 90% to 75% (after 15 min).

HPLC analysis

- 19 -

- 20 -

Analysis of the plasma samples was carried out using Agilent Chem

Station for LC 3D systems software (Rev. 13. 02. 01, Agilent Technologies Inc.,

Santa Clara, CA, USA). The concentrations of probucol in the samples were

determined by the linear equation obtained from the standard

curve(y=0.2807x+0.005, correlation coefficient =0.9997).

Data analysis

Data are expressed by the mean ± S.E.. The area under the plasma

concentration-time curve (AUC) value (0-48 h) of the plasma profiles was

calculated using the logarithmic and linear trapezoidal rules.

- 21 -

RESULTS AND DISCUSSION

CHAPTER 1

Stability and in vivo absorption of probucol nanoparticles

obtained from probucol/PVP/SDS ground mixtures

1. Stability study of probucol/PVP/SDS ternary GM suspension

The mixing ratio of probucol/PVP/SDS at the weight ratio of 1:3:0.5

was employed for ternary ground mixtures (GMs) and ternary physical mixtures

(PMs) in this study, in order to reduce the amount of excipients and the

administration volume though our previous experiments confirmed that the best

suitable ratio was 1:3:1.42)

Variations in particle size of probucol obtained from probucol/PVP/SDS

ternary GMs dispersed into distilled water and physiological fluids, JP XIV first

fluid (pH 1.2) and JP XIV second fluid (pH 6.8), are shown in Fig. 5. After

dispersion into distilled water, the initial particle sizes of probucol nanoparticles

obtained from probucol/PVP K12/SDS, probucol/PVP K17/SDS and

probucol/PVP K30/SDS ternary GMs were 28, 75 and 89 nm, respectively.

During the storage period, the size of probucol nanoparticles obtained from the

ternary GM prepared with PVP K12 increased steadily, while those of the

ternary GMs prepared with PVP K17 and PVP K30 showed slight increases

only in the initial stage then became constant. Finally, the particle size of the

ternary GM with PVP K12 was higher than that of the ternary GMs prepared

with PVP K17 or PVP K30.

0

30

60

90

120

150

0 10 20 30 40 50

Mea

npa

rticl

e si

ze (n

m)

(A)

(B)

0

30

60

90

120

150

0 10 20 30 40 50

Mea

n pa

rticl

e si

ze (n

m)

Storage time (h)

(C)

0

30

60

90

120

150

0 10 20 30 40 50

Mea

n pa

rticl

e si

ze (n

m)

PVP K12

PVP K17

PVP K30

0

30

60

90

120

150

0 10 20 30 40 50

Mea

npa

rticl

e si

ze (n

m)

(A)

(B)

0

30

60

90

120

150

0 10 20 30 40 50

Mea

n pa

rticl

e si

ze (n

m)

Storage time (h)

(C)

0

30

60

90

120

150

0 10 20 30 40 50

Mea

n pa

rticl

e si

ze (n

m)

PVP K12

PVP K17

PVP K30

Fig. 5 Variation in the particle size of probucol nanoparticles obtained from

ternary ground mixtures of probucol/PVP/SDS at the weight ratio of 1:3:0.5

after dispersion in (A) distilled water, (B) JP first fluid and (C) JP second fluid,

at various time intervals and at a temperature of 25 °C. - 22 -

- 23 -

The initial probucol particle size of the ternary GM with PVP K12

increased from 28 nm in distilled water to 48 nm in JP first fluid, and 56 nm in

JP second fluid. In contrast, the particle size obtained from the ternary GMs

with PVP K17 and PVP K30 slightly decreased from 75 and 89 nm in water to

69 and 81 nm in JP first fluid, and 75 and 85 nm in JP second fluid, respectively.

Changes in the particle size with time, for all ternary GMs, exhibited a

comparable trend regardless of the dispersion media. The results demonstrated

that the probucol particle size was effectively reduced at the initial stage when

the low molecular weight PVP was employed. In pH-adjusted conditions, the

particle size of probucol prepared with PVP K12 became large, while the other

two PVPs showed little influence on the particle size. It was assumed that the

surface conditions of nanoparticles obtained from PVP K12/SDS might be

different from those of PVP K17/SDS and PVP K30/SDS. It was clarified that

SDS formed micelles together with the PVP polymer as a micelle-like aggregate

in PVP/SDS solution.49-57) For the ternary GMs with PVP K17 and PVP K30,

the surfaces of probucol nanoparticles were probably covered by PVP-SDS

micelle complex as a layered structure, so that ternary GMs with PVP K17 and

PVP K30 were able to become stable in the suspensions. For the ternary GM

with PVP K12, PVP K12 and SDS might not make a PVP-SDS complex with

micelle-like structure, due to the shortest length of the chain (the molecular

weight was 1/4 of PVP K17, 1/20 of PVP K30). The PVP K12 and SDS might

cover insufficiently the surface of probucol nanoparticles in the suspension.

Therefore ternary GM with PVP K12 induced a relatively rapid agglomeration

of probucol nanoparticles. Possible models of the probucol nanoparticle

- 24 -

formation from the probucol/PVP/SDS GMs52) are shown in Fig. 6.

Insufficient surface coverage with PVP K12 and SDS of probucol nanoparticles

SDS moleculeProbucol nanoparticle PVP polymer

Ternary GMs with PVP K17 and with PVP K30 are dispersed into water.

∼90 nm

(Probucol/PVP K17/SDS GM and Probucol/PVP K30/SDS GM)

Ternary GM with PVP K12 is dispersed into water.

(Probucol/PVP K12/SDS GM)

∼30 nm

PVP-SDS complex with micelle-like structure.

Insufficient surface coverage with PVP K12 and SDS of probucol nanoparticles

SDS moleculeProbucol nanoparticle PVP polymer SDS moleculeProbucol nanoparticle PVP polymer

Ternary GMs with PVP K17 and with PVP K30 are dispersed into water.

∼90 nm

(Probucol/PVP K17/SDS GM and Probucol/PVP K30/SDS GM)

Ternary GM with PVP K12 is dispersed into water.

(Probucol/PVP K12/SDS GM)

∼30 nm

PVP-SDS complex with micelle-like structure.

Reference; Pongpeerapat et al., Int. J. Pharm., in press (2008)

Fig. 6 Possible models of the formation of probucol nanoparticles from the

ternary GMs with PVP and SDS.

- 25 -

- 26 -

2. Drug absorption from the ternary GM

Pharmacokinetics of different probucol formulations administered by

oral gavages to male Sprague Dawley rats was evaluated. The plasma

concentration-time profiles of probucol after oral administration in rats are

shown in Fig. 7. The in vivo absorptions were remarkably improved by

probucol/PVP/SDS ternary GMs compared to those of the ternary PM and

unprocessed probucol. After the administration of ternary GMs, the plasma

concentrations increased rapidly and peaks were observed after 3-4 h.

Pharmacokinetic parameters following oral administration of the

probucol/PVP/SDS ternary GM suspensions are presented in Table 1.

Interestingly, the suspension obtained from the ternary GM prepared with PVP

K12 remarkably increased AUC and the maximum drug concentration (Cmax)

values of probucol, 41.3-fold (P < 0.005) and 17.0-fold (P < 0.005) compared to

the respective values of unprocessed probucol. For ternary GMs with PVP

K17 and PVP K30 cases, the AUC and Cmax values increased 10.7-fold (P <

0.05) and 7.7-fold (P < 0.02), 5.6-fold (P < 0.05) and 3.3-fold (P < 0.02),

respectively. There were no significant differences in AUC and Cmax values

between the ternary PM and the unprocessed probucol. These results

suggested that additives used have no effect on in vivo absorption of the

unprocessed probucol.

Plas

ma

conc

entra

tion

(µg/

mL)

0

0.3

0.6

0.9

1.2

0 10 20 30 40 5

Probucol/PVP K12/SDS GM

Probucol/PVP K17/SDS GMProbucol/PVP K30/SDS GM

Probucol/PVP K12/SDS PM

0

Unprocessed probucol

Time (h)

Plas

ma

conc

entra

tion

(µg/

mL)

0

0.3

0.6

0.9

1.2

0 10 20 30 40 5

Probucol/PVP K12/SDS GM

Probucol/PVP K17/SDS GMProbucol/PVP K30/SDS GM

Probucol/PVP K12/SDS PMUnprocessed probucol

Time (h)0

Fig. 7 Effects of molecular weight of PVP on plasma concentration of

probucol following oral administration of the probucol/PVP/SDS ternary

ground mixture suspension. Results are expressed as mean ± S.E. (n = 3).

- 27 -

- 28 -

Table 1 Pharmacokinetic parameters of probucol following oral administration

of probucol/PVP/SDS ternary suspensions. Suspension Cmax (µg/mL) Tmax (h) AUC (µg h/mL) Enhancement

ratioa

Unprocessed probucol 0.07 ± 0.01 3.7 ± 0.3 0.31 ± 0.02 1.0

Probucol/PVP K12/SDS GM 1.19 ± 0.06*** 4.0 ± 0.0 12.81 ± 0.77*** 41.3

Probucol/PVP K17/SDS GM 0.54 ± 0.06** 3.0 ± 0.6 3.33 ± 0.49* 10.7

Probucol/PVP K30/SDS GM 0.23 ± 0.03** 3.3 ± 0.3 1.74 ± 0.22* 5.6

Probucol/PVP K12/SDS PM 0.05 ± 0.00 3.7 ± 0.3 0.54 ± 0.15 1.7

Cmax = maximum drug concentration, Tmax = time of maximum concentration,

AUC= area under the plasma concentration-time curve.

Results are expressed as mean ± S.E. (n = 3).

a Enhancement ratio was calculated according to the following equation:

The ratio = The corresponding AUC/AUC of unprocessed probucol.

***P<0.005; **P<0.02; *P<0.05, compared to the corresponding parameters of

the unprocessed probucol suspension.

- 29 -

The particle size reduction could lead to the enhancement of the in vivo

absorption of probucol. But stability results shown in Fig. 5 demonstrated that

the probucol particle obtained from ternary GM with PVP K12 has different

characteristics from those of ternary GMs with PVP K17 and PVP K30. Not

only the size of probucol nanoparticles but also the different surface states

covered by PVP and SDS seemed to influence the in vivo absorption of

probucol. Despite the fact that the particle size obtained from the ternary GM

with PVP K17 was somewhat similar to that from the ternary GM with PVP

K30, ternary GM with PVP K17 enhanced the absorption better than the ternary

GM with PVP K30. It was considered that the long chain length of high

molecular weight PVP (PVP K30) contributed to the thick layer structure of the

PVP/SDS complex on the surface of nanoparticles. The thick layer structure

might be attributed to the difficulty of probucol molecules to be dissolved and

absorbed. On the contrary, the smallest nanoparticles without the layer

structure, which were generated by ternary GM with PVP K12, might facilitate

dissolution and absorption, though the stability in water is not so good as shown

in Fig. 5.

Palin and Wilson reported co-administration with arachis oil enhanced

the in vivo absorption of probucol in rats which AUC0-16 value was 11.74±1.55

µg h/mL.12) Generally, oil dissolution system has an advantage for lipophilic

drug absorption,58) however this study clarified that water dispersion with

ternary GM with PVP K12 even improved the in vivo absorption of probucol as

the comparison in AUC between ternary GM with PVP K12 and arachis oil.

Furthermore, it is understood that ternary GM with PVP K12 can also enhance

- 30 -

the absorption speed of probocol because more than 0.4 µg/mL plasma

concentration was observed at 1 hour after the administration despite the plasma

concentration from arachis oil was below the detection limit.

3. Investigation of drug absorption from binary GM with SDS

The effects of probucol/PVP K12 and probucol/SDS binary GMs on

plasma concentration of probucol through oral administration of the suspension

are shown in Fig. 8. The probucol/PVP K12 binary GM demonstrated a

similar pharmacokinetics profile to that of the binary PMs and unprocessed

probucol. On the other hand, the probucol/SDS binary GM showed small

absorption improvement compared to all other binary mixtures and the

unprocessed probucol. Table 2 also presents pharmacokinetic parameters of

binary systems. The AUC and Cmax values of the probucol/SDS GM increased

3.5-fold (not statistically significant) and 2.4-fold (P < 0.005) compared to the

unprocessed probucol, respectively.

Mean particle sizes of probucol obtained from the binary systems and

ternary PM are shown in Table 3. The size of the unprocessed probucol was

27.7 µm. Probucol/PVP K12 and probucol/SDS GMs showed no significant

particle size reduction compared to the unprocessed probucol even though

absorption improvement in probucol/SDS GM was observed. However, there

still remains the possibility that the particle size in the range of the submicron

region might not be detected due to the detection limit of the FRA particle size

analyzer. A filtrate sample of the probucol/SDS GM suspension was prepared

with a 0.8 µm membrane filter to investigate whether the micronized particles

- 31 -

exist or not. HPLC quantitative analysis revealed that approximately 17% of

the probucol was recovered as nanometer-sized particles in the suspension of

probucol/SDS binary GM. The average particle size was around 287 nm.

These results supported the reason that there was insignificant absorption

improvement derived from the probucol/SDS binary GM. Because probucol

nanoparticles from the probucol/SDS binary GM were unstable in JP first

fluid,36) their structures were different from those of ternary GMs, which were

covered with PVP and SDS.

Time (h)

0

0.05

0.10

0.15

0.20

0 10 20 30 40 5

Plas

ma

conc

entra

tion

(µg/

mL)

0

Probucol/SDS GM

Probucol/SDS PM

Probucol/PVP K12 GM

Probucol/PVP K12 PM

Time (h)

0

0.05

0.10

0.15

0.20

0 10 20 30 40 5

Plas

ma

conc

entra

tion

(µg/

mL)

Probucol/SDS GM

Probucol/SDS PM

Probucol/PVP K12 GM

Probucol/PVP K12 PM

0

Fig. 8 Effects of grinding with PVP K12 or SDS on plasma concentration of

probucol following oral administration of the suspension. Results are

expressed as mean ± S.E. (n = 3).

- 32 -

- 33 -

Table 2 Pharmacokinetic parameters of probucol following oral administration

of probucol/PVP and probucol/SDS binary suspensions. Suspension Cmax (µg/mL) Tmax (h) AUC (µg h/mL) Enhancement

ratioa

Unprocessed probucol 0.07 ± 0.01 3.7 ± 0.3 0.31 ± 0.02 1.0

Probucol/PVP K12 PM 0.06 ± 0.00 3.3 ± 0.7 0.36 ± 0.06 1.2

Probucol/PVP K12 GM 0.05 ± 0.00 2.7 ± 0.3 0.53 ± 0.16 1.7

Probucol/SDS PM 0.05 ± 0.00 3.7 ± 0.3 0.49 ± 0.05 1.6

Probucol/SDS GM 0.17 ± 0.01* 3.0 ± 0.0 1.09 ± 0.21 3.5

Cmax = maximum drug concentration, Tmax = time of maximum concentration,

AUC= area under the plasma concentration-time curve.

Results are expressed as mean ± S.E. (n = 3).

a Enhancement ratios were calculated according to the following equation:

The ratio = The corresponding AUC/AUC of unprocessed probucol.

*P<0.05 compared to the corresponding parameters of the unprocessed

probucol suspension.

- 34 -

Table 3 Mean particle size of probucol obtained from the binary and ternary

physical mixtures and ground mixtures in distilled water, measured by FRA

(0.1-700 µm).

Suspension Mean particle size (µm)

Unprocessed probucol 27.7 ± 0.6

Probucol/PVP K12 PM 42.3 ± 0.4

Probucol/PVP K12 GM 17.3 ± 0.4

Probucol/SDS PM 46.0 ± 0.3

Probucol/SDS GM 58.4 ± 2.4

Probucol/PVP K12/SDS PM 47.1 ± 0.1

Probucol/PVP K12/SDS GM 0.028 ± 0.001a

Results are expressed as mean ± S.D. (n = 3).

a Particle size was measured by UPA (0.003-6 µm).

- 35 -

CHAPTER 2

In vivo comparison in the case that the particle size

obtained from ternary GM with PVP K12 is larger than

that from ternary GM with PVP K17

1. Verification of the effect of nanoparticle surface condition with PVP and

SDS on the absorption

Probucol in vivo absorption rate was remarkably improved by ternary

GM with PVP K12 compared to that of ternary GM with PVP K17 due to its

smallest particle size in chapter 1. But, insufficient surface coverage with PVP

K12 and SDS of the particles in the suspension also seemed to enhance the in

vivo absorption of probucol. To evaluate if both of smallest particle size and

insufficient surface coverage influence the absorption improvement, ternary

GMs with PVP K12 and with PVP K17, which can generate smaller particle

size than PVP K12 in suspension, were prepared. Specially, ternary GM with

PVP K17 was ground under the temperature of -180 °C to reduce the size under

50 nm. In chapter 2, the nanoparticles obtained from ternary GMs with PVP

K12 and with PVP K17 in suspensions were 40 nm and 36 nm, respectively.

Pharmacokinetic parameters following oral administration of the

probucol/PVP/SDS ternary GM suspensions are presented in Table 4A. The

AUC and Cmax values of probucol from ternary GM with PVP K12 were 23.64

µg h/mL and 1.61 µg/mL, respectively. For the ternary GM with PVP K17

case, the AUC and Cmax values were 13.42 µg h/mL and 1.29 µg/mL,

respectively.

- 36 -

Table 4 The percentage of pharmacokinetic parameters of probucol/PVP

K17/SDS GM in probucol/PVP K12/SDS GM on (A) in vivo absorption study

in chapter 2 and (B) in vivo absorption study in chapter 1. (A)

Probucol/PVP K12/SDS GM (40nm)

Probucol/PVP K17/SDS GM (36nm) Percentage (%)a

AUC (µg h/mL) 23.64 ± 7.19 13.42 ± 1.11 56.77

Cmax (µg/mL) 1.61 ± 0.48 1.29 ± 0.07 80.12

(B) Probucol/PVP K12/SDS GM (28nm)

Probucol/PVP K17/SDS GM (75nm) Percentage (%)a

AUC (µg h/mL) 12.81 ± 0.77 3.33 ± 0.49 26.00

Cmax (µg/mL) 1.19 ± 0.06 0.54 ± 0.06 45.38

AUC= area under the plasma concentration-time curve, Cmax = maximum drug

concentration.

Results are expressed as mean ± S.E. (n = 3).

a The percentage was calculated according to the following equation:

The value = AUC or Cmax of Probucol/PVP K17/SDS GM divided by

AUC or Cmax of Probucol/PVP K12/SDS GM multiplied by 100.

- 37 -

However, the PK results in chapter 2 (Table 4A) were unexpectedly

higher than those in chapter 1 (Table 4B). It might be the reason that the

stronger feeding impact begun 6 h after the dose administration influenced the

increasing of in vivo probucol absorption in chapter 2.12) Therefore the PK

values were converted into percentage values in order to compare the results

between chapter 2 and chapter 1, because the results in chapter 2 could not be

compared directly with those in chapter 1. Higher percentage value means the

absorption improvement obtained from ternary GM with PVP K17 approaches

or overtakes that from ternary GM with PVP K12.

In chapter 2, the percentages of AUC and Cmax values obtained from

ternary GM with PVP K17 (36nm) in those of ternary GM with PVP K12

(40nm) were 56.77 % and 80.12 %, respectively. On the contrary, in chapter 1,

the percentages of AUC and Cmax values obtained from ternary GM with PVP

K17 (75nm) in those of ternary GM with PVP K12 (28nm) were 26.00 % and

45.38 %, respectively, as shown in Table 4. These results suggested that

smaller particle size with PVP K17 (75nm→36nm) may influence the higher

absorption improvement (AUC: 26.00→56.77 %, Cmax: 45.38→80.12 %).

Moreover, it might be clarified that insufficient surface coverage of the probucol

nanoparticles with PVP K12 and SDS in the suspension plays an important role

in enhancing in vivo drug absorption because the AUC value (23.64 µg h/mL)

derived from ternary GM with PVP K12 can still overtake the value (13.42 µg

h/mL) derived from ternary GM with PVP K17 even the particle size (40nm) is

larger than PVP K17 (36nm).

- 38 -

CONCLUSION

It was demonstrated that the ternary ground mixtures of

probucol/PVP/SDS significantly enhanced the bioavailability of probucol. The

ternary GM with PVP K12 showed a superior absorption improvement

compared to the GMs with PVP K17 and PVP K30. The results suggested that

not only reduced particle size of probucol but also different particle surface

conditions covered with PVP and SDS could influence the in vivo absorption of

probucol. On the contrary, the ternary PM and all binary GMs did not show

significant differences in the AUC value compared to the unprocessed probucol.

Preparation of probucol nanoparticles by co-grinding with PVP K12 and SDS

was confirmed as the promising method for the bioavailability enhancement.

- 39 -

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- 48 -

ACKNOWLEDGMENTS

To my supervisor, Professor Dr. Keiji Yamamoto, I would like to express

all my gratitude and sincere appreciation for his encouragement and wise

guidance from the beginning to the end of this research work. I am deeply

indebted to him for his assistance. He gave me warm hospitality, kindness and

understanding my study.

I would like to express all my sincere gratitude to Associate Professor Dr.

Kunikazu Moribe, for the continuous support, discussion and technical expertise

throughout the dissertation process. He always helped me with his competent

advice for my study.

I would like to express all my sincere gratitude to Assistance Professor

Mr. Kenjiro Higashi, for the continuous support and technical expertise

throughout the dissertation process.

I am grateful to Dr. Adchara Pongpeerapat and Mr. Chalermphon

Wanawongthai for their helpful assistances and supports during the all stage of

my research. I couldn’t obtain the fruitful data without your helps.

I also wish to express my hearty thanks to all members of the Laboratory

of Pharmaceutical Technology for their supports all along my study.

I would like to thank Daiichi Sankyo Co., Ltd. (Tokyo, Japan) for their

kind offer of probucol.

Finally, I would like to express tremendous gratitude to my wife (Yoko),

my family (Haruka, Natsumi), my parents, my brother, my sister and my best

friends for their emotional supports and encouragements.

- 49 -

LIST OF PUBLICATION

This thesis is based on the following publication:

Shudo J., Pongpeerapat A., Wanawongthai C., Moribe K., Yamamoto K.: In

vivo assessment of oral administration of probucol nanoparticles in rats. Biol.

Pharm. Bull., 31, in press (2008).

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THESIS COMMITTEE

This thesis, conducted for the Degree of Doctor of Philosophy

(Pharmaceutical Sciences) was examined by the following committee,

authorized by the Graduate School of Medical and Pharmaceutical Sciences,

Chiba University, Japan.

Professor Toshiharu Horie, Ph.D., Chairman

(Graduate School of Medical and Pharmaceutical Sciences, Chiba University)

Professor Yasushi Arano, Ph.D.

(Graduate School of Medical and Pharmaceutical Sciences, Chiba University)

Professor Nobunori Satoh, Ph.D.

(Graduate School of Medical and Pharmaceutical Sciences, Chiba University)