J. Pharm. Pharmacol. 1999, 51: 159–164
`Received September 14, 1998
`Accepted September 15, 1998
`
`# 1999 J. Pharm. Pharmacol.
`
`Improving the Oral Bioavailability of Albendazole in Rabbits by
`the Solid Dispersion Technique
`
`N A O N O R I K O H R I , Y A S U K O Y A M A Y O S H I * , H E X I N * , K E N I S E K I * , N A O K I S A T O { ,
`S A T O R U T O D O { A N D K A T S U M I M I Y A Z A K I *
`
`Department of Pharmacy, Hokkaido College of Pharmacy, *Department of Pharmacy and {First
`Department of Surgery, Hokkaido University Hospital, School of Medicine, Hokkaido University,
`Kita-14-jo, Nishi-5-chome, Kita-ku, Sapporo 060-8648, Japan
`
`Abstract
`We have investigated the oral bioavailability of granules of albendazole, a drug used for
`treating echinococcosis in man, prepared by the solid dispersion technique.
`Rapid dissolution and supersaturation were observed when hydroxypropylmethyl-
`cellulose and hydroxypropylmethylcellulose phthalate were used as carriers in the solid
`dispersion. They inhibited the crystallization of albendazole from the supersaturated
`solution and maintained an amorphous state for 8 h. Gastric acidity-controlled rabbits
`were used to evaluate the variation in absorption after oral administration of the
`albendazole solid dispersion. For rabbits with low gastric acidity the bioavailability of
`orally administered albendazole in the granular form prepared by solid dispersion was
`more than three times that of albendazole in physical mixtures.
`These results suggest that the bioavailability of albendazole in solid dispersions might be
`high even if there is a great variation in the gastric pH of patients.
`
`Alveolar echinococcosis is considered to be the
`most lethal type of helminthiasis in man, and the
`number of such cases is increasing, especially in
`northern hemisphere countries (Kumar & Chatto-
`padhyay 1992). Albendazole has a wide-spectrum
`anthelminthic effect and has been used clinically in
`inoperable or disseminated hydatidosis (Uchino et
`al 1993; Ishizu et al 1997; Luchi et al 1997).
`Clinical studies, including our previous study (Sato
`et al 1994), have shown a great
`inter-subject
`variability in the bioavailability of albendazole
`(Marriner et al 1986; Jung et al 1992), possibly
`because of the poor water-solubility of the drug.
`When taken with a fatty meal, absorption of
`albendazole was
`improved fivefold (approx.)
`(Lange et al 1998). In a pharmaceutical investiga-
`tion, the addition of surfactants, co-solvents or a
`solid dispersion mixed with polyvinylpyrrolidone
`improved the rate of dissolution of albendazole (del
`Estal et al 1994; Torrado et al 1996a, b). However,
`few such preparations have been used clinically.
`
`Correspondence: K. Miyazaki, Department of Pharmacy,
`Hokkaido University Hospital, School of Medicine, Hokkaido
`University, Kita-14-jo, Nishi-5-chome, Kita-ku, Sapporo 060-
`8648, Japan.
`
`Our previous study showed that intestinal absorp-
`tion of albendazole was significantly lower for
`rabbits with low gastric acidity than for those with
`high gastric acidity, because of the poor solubility
`of albendazole in weakly acidic and neutral solu-
`tions (Kohri et al 1998). In the current study we
`have attempted to improve the rate of dissolution of
`albendazole, by using a solid dispersion technique,
`and to evaluate oral bioavailability after adminis-
`tration of the solid dispersion to gastric acidity-
`controlled rabbits.
`
`Materials and Methods
`
`Chemicals
`Albendazole sulphoxide was provided by Smith-
`Kline Beecham (Madrid, Spain). Albendazole and
`phenacetin were purchased from Sigma (St Louis,
`MO). Mebendazole was provided by Janssen Kyowa
`(Tokyo,
`Japan). Lansoprazole (Takepron) was
`obtained from Takeda (Tokyo, Japan). Hydroxy-
`propylmethylcellulose
`(TC-5)
`and
`hydroxy-
`propylmethylcellulose phthalate
`(HP-55) were
`obtained from Shin-Etsu (Tokyo, Japan). All other
`
`1017-0001
`
`Petition for Inter Partes Review of
`U.S. Patent No. 6,881,745
`
`PETITIONERS' EXHIBIT 1017
`
`

`
`160
`
`NAONORI KOHRI ET AL
`
`Table 1. Composition (g) of albendazole preparations.
`
`Preparation
`
`Albendazole TC-5 HP-55 Lactose
`0(cid:1)1
`1(cid:1)5
`0(cid:1)1
`0(cid:1)5
`0(cid:1)1
`0(cid:1)1
`
`–
`–
`1(cid:1)0
`0(cid:1)5
`
`–
`0(cid:1)5
`
`ately replaced with an equal volume of 2(cid:1)5 M
`KH2PO4 containing 16(cid:1)72% (w/v) NaOH to adjust
`the pH of the medium to 6(cid:1)5. The test solution was
`analysed by high-performance liquid chromato-
`graphy (HPLC) with an Hitachi L-6000 constant
`flow pump and a Hitachi L-4000 UV detector
`operating at 310 nm. Compounds were separated on
`a 10 cm 6 6 mm ERC-ODS 1161 reversed-phase
`column, particle size 3 mm (Erma Optical Works).
`The mobile phase was 0(cid:1)05 M phosphate buffer (pH
`7(cid:1)0)–acetonitrile, 55 : 45 with the pH was adjusted
`to 6(cid:1)5 with phosphoric acid; the flow rate was
`1(cid:1)0 mL min71. Mebendazole was used as internal
`standard.
`
`In-vivo absorption study
`Experiments were performed on white male rabbits,
`2(cid:1)5–3(cid:1)5 kg, with a 14-day wash-out period between
`doses. Low gastric-acidity rabbits were obtained by
`a method reported elsewhere (Kohri et al 1998).
`Briefly, rabbits were fasted for one day before the
`absorption study, but water was freely available.
`On the day of the experiment, hard gelatin capsules
`(JP XIII, No.3) containing lansoprazole (6 mg), an
`H(cid:135)-pump inhibitor, were administered orally at
`0900 and 2100 h. At 0900 h on the day of the study
`water (10 mL) was given orally through a plastic
`catheter, and gastric juice was withdrawn by suc-
`tion. The pH of the gastric juice was determined
`with pH paper (Toyo Roshi, Tokyo) for each rabbit.
`For
`intravenous
`administration
`a
`solution
`(5 mg mL71) of albendazole sulphoxide, an active
`metabolite of albendazole, in dimethylsulphoxide
`was
`injected through the ear marginal vein
`(1 mg kg71). Plasma samples were collected from
`the marginal ear vein at predetermined intervals by
`means of a heparinized syringe. The assay for
`albendazole sulphoxide in plasma was performed
`according to a method reported elsewhere (Kohri et
`al 1998). Plasma (600 mL) was mixed with
`Na2B4O7 solution (0(cid:1)1 M, 3 mL) containing phena-
`cetin as internal standard for HPLC and extracted
`with chloroform (6 mL). The organic
`layer
`(500 mL) was evaporated, the residue was recon-
`stituted with mobile phase (0(cid:1)1 mL), and the
`resulting solution was analysed by the HPLC
`method used for in-vitro solubility studies. The
`mobile phase was 0(cid:1)05 M phosphate buffer–
`acetonitrile, 75 : 25.
`
`Analysis of plasma data
`The parameters of the appropriate pharmacokinetic
`model were estimated using the MULTI program
`(Yamaoka et al 1981). The area under the plasma
`
`Physical mixture
`Solid dispersion with
`TC-5
`Solid dispersion with
`HP-55
`Solid dispersion with
`TC-5 and HP-55
`TC-5(cid:136) hydroxypropylmethylcellulose, HP-55(cid:136) hydroxy-
`propylmethylcellulose phthalate.
`
`–
`1(cid:1)0
`
`–
`0(cid:1)5
`
`chemicals were of the highest grade available and
`used without further purification.
`
`Preparation of dosage forms
`A physical mixture was prepared by mixing
`albendazole and lactose with a pestle and mortar;
`the mixture obtained was passed through a 100-
`mesh sieve.
`Solid dispersions were prepared by use of a sol-
`vent method. Albendazole and the polymers were
`dissolved
`in
`ethanol–dichloromethane
`(1 : 1,
`75 mL) at room temperature and mixed with lac-
`tose. For hydroxypropylmethylcellulose phthalate
`preparations, albendazole and the polymer were
`dissolved in 75 mL acetone (50–60(cid:14)C), and lactose
`was not included. The solution was then evaporated
`immediately at 45(cid:14)C on a water bath. The residue
`was dried for 12 h under vacuum and passed
`through a 100-mesh sieve. Powder X-ray dif-
`fractometry was performed with an RU-300
`(Rigaku Denki, Japan; CuKa; 50 kV; 100 mA;
`3(cid:14) min71). The chemical compositions of prepara-
`tions are shown in Table 1.
`
`In-vitro dissolution study
`Dissolution experiments were performed by the JP
`XIII paddle method at an agitation speed of
`100 rev min71 at 37(cid:14)C. Each preparation contain-
`ing 10 mg albendazole was added to 900 mL JP 2nd
`fluid. Test medium (400 mL) was removed at
`appropriate intervals and filtered through a 0(cid:1)45-mm
`membrane filter (Toyo Roshi, Tokyo).
`A dissolution test by the pH-shift method was
`conducted to simulate drug transition from the
`stomach to the small intestine (Kondo et al 1994).
`A sample equivalent
`to 10 mg albendazole was
`added to the test medium (pH 1(cid:1)2; 500 mL). After
`1 h, medium (10 mL) was removed and immedi-
`
`1017-0002
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`

`
`IMPROVING THE ORAL BIOAVAILABILITY OF ALBENDAZOLE
`
`161
`
`concentration–time curve from 0 to 24 h (AUC0 – 24 h)
`was calculated by the linear trapezoidal rule. Because
`albendazole was completely metabolized to alben-
`dazole sulphoxide in rabbits, the bioavailability was
`calculated according to the equation:
`Bioavailability (cid:136)
`(cid:133)AUCalbendazole sulphoxide oral=doseoral(cid:134)=
`(cid:133)AUCalbendazole sulphoxide intravenous=
`(cid:133)1(cid:134)
`doseintravenous(cid:134)
`where AUCalbendazole sulphoxide oral and AUCalbenda-
`represent
`the AUC0 – 24 h
`zole
`sulphoxide
`intravenous
`sulphoxide
`after oral
`values of
`albendazole
`administration of albendazole and intravenous
`administration of albendazole sulphoxide, respec-
`tively.
`Statistical differences between high and low
`gastric-acidity groups were assessed by use of
`Student’s t-test.
`
`Results and Discussion
`
`Dissolution study
`hydroxypropylmethyl-
`Low molecular-weight
`cellulose (TC-5) has been used as a carrier for solid
`dispersion formulations to enhance the dissolution
`rate and saturated solubility of several water-
`insoluble drugs (Sugimoto et al 1982; Honbo et al
`1987; Kohri et al 1992; Kagayama et al 1993). In
`this study, therefore, solid dispersions were pre-
`pared by use of TC-5 and by a solvent method. The
`chemical compositions of preparations are sum-
`marized in Table 1. The addition of lactose was
`necessary because a solid dispersion of TC-5 only
`was too hard to mill. Figure 1 shows the dissolution
`profiles of solid dispersions in JP III 2nd fluid (pH
`6(cid:1)8). The rate of dissolution was rapid for all solid
`dispersions, and for both formulations containing
`TC-5 the highest supersaturated concentration was
`reached after 0(cid:1)5 h. Although the supersaturated
`concentration of the formulation prepared with HP-
`55 only subsequently decreased, both formulations
`with TC-5 maintained their maximum concentra-
`tions for 8 h. Figure 2 shows the dissolution profiles
`of solid dispersions in media of pH 1(cid:1)2–6(cid:1)5, used to
`simulate dissolution in the gastrointestinal tract. At
`pH 1(cid:1)2 albendazole dissolved completely from all
`the preparations except that containing HP-55 only;
`it crystallized rapidly when the pH was changed to
`6(cid:1)5. However,
`formulations
`containing TC-5
`maintained their supersaturated concentrations for
`long periods, suggesting that the crystallization of
`
`Figure 1. Dissolution behaviour of albendazole from solid
`dispersions in 900 mL JP XIII 2nd test fluids at 37(cid:14)C: j,
`physical mixture; d, solid dispersion with hydroxypropyl-
`methylcellulose and hydroxypropylmethylcellulose phthalate;
`s, solid dispersion with hydroxypropylmethylcellulose; u,
`solid dispersion with hydroxypropylmethylcellulose phthalate.
`Each point represents the mean(cid:6) standard deviation of results
`from three measurements.
`
`Figure 2. Dissolution behaviour of albendazole from solid
`dispersions in media of pH 1(cid:1)2 – 6(cid:1)5: j, physical mixture; d,
`solid dispersion with hydroxypropylmethylcellulose
`and
`hydroxypropylmethylcellulose phthalate; s, solid dispersion
`with hydroxypropylmethylcellulose; u, solid dispersion with
`hydroxypropylmethylcellulose phthalate.
`
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`

`
`162
`
`NAONORI KOHRI ET AL
`
`prevents albendazole crystallization in a neutral
`medium.
`
`Physical characterization of the solid dispersion
`Figure 3 shows the powder X-ray diffraction pat-
`terns of the physical mixture and of the solid dis-
`persion containing both TC-5 and HP-55. For the
`physical mixture, the peaks located at 7(cid:1)3(cid:14) and
`24(cid:1)7(cid:14) (2y) correspond to albendazole crystals; the
`others correspond to lactose crystals. Peaks from
`albendazole crystals are absent from the patterns
`obtained from solid dispersions with a drug-to-
`polymer ratio of 1 : 10, indicating that albendazole
`is present as the amorphous state in these for-
`mulations. Moreover, this amorphous state did not
`change over a period of 15 months at 4(cid:14)C in a
`screw-cap vial.
`
`Absorption study
`The plasma concentrations of albendazole sulph-
`oxide after oral administration of the preparations
`to gastric acidity-controlled rabbits are shown in
`Figure 4. The pharmacokinetic parameters are
`summarized in Table 2. For both groups the Cmax
`(maximum plasma concentration) and AUC of the
`solid dispersion (which contained both TC-5 and
`HP-55) were higher than those of the physical
`mixture. Bioavailability from the solid dispersion
`was almost 100% in the normal gastric acidity
`group (gastric pH values were 1 (approx.)) and
`for the low gastric-acidity group was 3(cid:1)2 times
`that from the physical mixture (gastric pH values
`were >5).
`On the basis of these results we suggest that when
`the solid dispersion was administered to rabbits
`with normal gastric acidity both albendazole and
`TC-5 dissolved completely in the stomach. As the
`solid dispersion shifted to the small intestine, HP-
`55 began to dissolve. Thus, crystallization of
`albendazole was prevented by both polymers. This
`speculation is supported by the prolonged MRT
`(mean residence time) and the almost 100% bio-
`availability observed when the preparation was
`administered to rabbits with normal gastric acidity.
`When the solid dispersion was administered to
`rabbits with low gastric acidity, the dissolution of
`albendazole in the stomach was
`four
`times
`(approx.) that from the physical mixture, and the
`supersaturated
`concentration
`of
`albendazole
`remained in the small intestine for a long time,
`because of the presence of the polymers. Moreover,
`polymers remaining undissolved in the stomach
`dissolved in the small intestine. We conclude that
`
`Figure 3. Powder X-ray diffraction patterns of albendazole
`physical mixture and solid dispersions. A. Albendazole crys-
`tals, B. 1 : 1 TC-5-HP-55, C. 1 : 1 : 1 TC-5-HP-55-lactose, D.
`physical mixture 1 : 5 : 5 : 5 albendazole-TC-5-HP-55-lactose,
`E. solid dispersion 1 : 5 : 5 : 5 albendazole-TC-5-HP-55-lactose,
`F. solid dispersion 1 : 5 : 5 : 5 albendazole-TC-5-HP-55-lactose
`after storage in a screw-cap vial at 4(cid:14)C for 15 months.
`TC-5 (cid:136) hydroxypropylmethylcellulose,
`HP-55 (cid:136) hydroxy-
`propylmethylcellulose phthalate.)
`
`albendazole in these two solid dispersions is
`retarded by the presence of polymers. These results
`suggest that the formulation containing both TC-5
`and HP-55 not only has excellent solubility but also
`
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`
`

`
`IMPROVING THE ORAL BIOAVAILABILITY OF ALBENDAZOLE
`
`163
`
`Table 2. Pharmacokinetic parameters after administration of albendazole (5 mg kg71) to rabbits in a cross-over study.
`
`Parameter
`
`Normal gastric acidity
`
`Low gastric acidity
`
`Solid dispersion
`Physical mixture
`Solid dispersion
`Physical mixture
`1(cid:1)4(cid:6) 0(cid:1)2*
`0(cid:1)5(cid:6) 0(cid:1)2
`2(cid:1)4(cid:6) 0(cid:1)4
`2(cid:1)1(cid:6) 0(cid:1)2
`Cmax (mg mL71)
`6(cid:1)7(cid:6) 1(cid:1)1
`6(cid:1)3(cid:6) 1(cid:1)6
`3(cid:1)7(cid:6) 0(cid:1)7
`4(cid:1)0(cid:6) 0(cid:1)8
`Tmax (h)
`24(cid:1)8(cid:6) 5(cid:1)8*
`6(cid:1)4(cid:6) 2(cid:1)6
`31(cid:1)8(cid:6) 4(cid:1)7*
`22(cid:1)4(cid:6) 3(cid:1)5
`AUC0 – 24 h (mg h mL71)
`4(cid:1)6(cid:6) 0(cid:1)8
`4(cid:1)8(cid:6) 1(cid:1)6
`5(cid:1)8(cid:6) 1(cid:1)7
`2(cid:1)8(cid:6) 0(cid:1)4
`t1=2 (h)
`13(cid:1)3(cid:6) 2(cid:1)3
`12(cid:1)0(cid:6) 3(cid:1)2
`10(cid:1)6(cid:6) 2(cid:1)0*
`7(cid:1)7(cid:6) 1(cid:1)1
`MRT (h)
`68(cid:1)8(cid:6) 13(cid:1)2*
`21(cid:1)3(cid:6) 9(cid:1)9
`94(cid:1)9(cid:6) 12(cid:1)9*
`67(cid:1)6(cid:6) 17(cid:1)9
`Bioavailability (%)
`Values are means(cid:6) standard error (n(cid:136) 5). *P < 0(cid:1)05 compared with corresponding values using the physical mixture.
`Cmax(cid:136) maximum concentration, Tmax(cid:136) time of maximum concentration, AUC0 – 24 h(cid:136) area under the plasma concentration –
`time curve from 0 to 24 h, t1=2 (cid:136) plasma half-life, MRT(cid:136) mean residence time.
`
`Figure 4. Mean plasma concentrations of albendazole sulphoxide after oral administration of physical mixture (n, s) and solid
`dispersion (m, d) to normal acidity rabbits (A) and to low acidity rabbits (B) at a dose of 5 mg kg71. Each point represents the
`mean(cid:6) standard error of results from five rabbits.
`
`the bioavailability of albendazole in solid disper-
`sions might be improved even if there is a great
`variation in the gastric pH of patients.
`
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`
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