Effect of Absorption Promoters on Subcutaneous Absorption of Human Epidermal Growth Factor in Rats

Effect of Absorption Promoters on Subcutaneous Absorption of Human Epidermal Growth Factor in Rats TERUOMURAMMI*, MASAFUMIMISAKI*,YOSHlE KOJIMA",MIE YAMADA*, MANAMIYUKI*, AMAGASE**, TOHRUFUWA*, AND NOBORU YATA*' YUTAKA HIGASHI*,HARUNOBU Received April 17, 1992, from the *Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, 1-2-3Kasumi, Minami-ku, Hiroshima 734, Japan, and the *Laboratory of Pharmacology, Central Research Laboratories, Wakunaga Pharmaceutical Company Ltd., Accepted for publication August 12, 1992. 1624 Shimo-kohtachi, Koda-cho, Takada-gun, Hiroshima 729-64, Japan. -

___

Abstract 0 Subcutaneous administration of human epidermal growth factor (hEGF) to rats gave a significantlysmaller value of area under the curve (AUC) of concentration in plasmaof immunoreactive hEGF versus time than intravenous administration, probably because the slow entry rate into the blood circulation and consequently the enzymic degradation of hEGF at the injection site. In the present study, absorption promoters such as sodium caprate, Nacylamino acids, disodium ethylenediaminetetraacetate (EDTA), and sodium glycocholatewere used because they were expected to inhibitthe enzymic degradationof hEGF at the injection site and to facilitate the entry of hEGF into the blood circulation. Coadministration of an absorption promoter with hEGF significantly increased the entry rate and AUC value of immunoreactive hEGF compared with the case without the absorption promoter. The enzymic degradation of hEGF in the supernatant of the rat subcutaneous tissue homogenates and in the buffer solution containing leucine aminopeptidase or protease was markedly inhibited by the presence of the absorption promoters except EDTA. On the other hand, only EDTA increased the initial entry rate of FITC-dextran (M, 4000), which is not metabolized at the injection site, although all absorption promoters including EDTA markedly increased the extravasation of Evans blue. Thus, the increased subcutaneous bioavailability of hEGF in the presence of absorption promoters (except EDTA) was mainly attributedto the inhibitoryeffect of absorptionpromoters against the enzymic degradation of hEGF at the subcutaneous tissues.

Bergar e t al.1 reported that degradation of polypeptides at t h e injection site i s involved int h e i r subcutaneous absorption. The e n t r y r a t e o f polypeptides i n t o the blood circulation after subcutaneous administration w i l l be restricted by t h e i r large molecular weights, l o w membrane permeabilities, and possib l e enzymic degradation at the injection site.2 To increase t h e subcutaneous absorption of porcine i n s u l i n by inhibiting enzymic degradation at t h e injection site, t h e use o f enzyme inhibitors such as bacitracin ( i n s u l i n protease and glutathion-insulin transdehydrogenase inhibitor), leupepcine (cathepsine-B inhibitor), and phosphoramide (thermolysin and collagenase i n h i b i t o r ) have been reported.3.4 In t h e present study, absorption promoters such as sodium caprate, N-acylamino acids, disodium EDTA, or sodium glycocholate were examined €or t h e i r abilities t o increase t h e subcutaneous absorption of human epidermal g r o w t h factor (hEGF), an endogeneous polypeptide composed o f 53 amino acids. These adjuvants have been reported t o increase t h e rectal and nasal membrane permeabilities t o sodium ampicillin, insulin, or hEGF, w h i c h are naturally unabsorbable w i t h o u t any aids in rats.5-'3 Additionally, some o f these absorption promoters, such as sodium caprate and sodium glycocholate, have been k n o w n t o inhibit leucine aminopeptidase activity, w h i c h acts as an enzymatic barrier t o i n s u l i n absorption through t h e mucosal membrane.6s7J2

236 IJournal of PharmaceuticalSciences Vol. 82, No. 3, March 1993

Experimental Section Materials-hEGF (purity, 199.9%) was produced by Wakunaga Pharmaceutical Company Ltd. (Hiroshima, Japan) with genetic technology. Reagents used for the determination of hEGF were the same as described previously.5 N-Lauroylalanine (C12-A) and N-decanoylphenylalanine ((210-PA),used as absorption promoters, were synthesized in the same manner as described p r e v i o u ~ l yLeucine .~ protease (Type VII), L-leucine pnaphaminopeptidase (Type IV-S), tylamide hydrochloride, L-alanine P-naphtylamide hydrobromide, L-arginine j3-naphtylamide hydrochloride, and fluorescein isothiocyanate-dextran (Mr,4000; FD-4) were purchased from Sigma Chemical Company (St. Louis, MO). Sodium caprate, disodium EDTA, sodium glycocholate, and Evans blue were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). All other reagents were of analytical reagents grade and were used without further purification. Effect of Absorption Promoters on Subcutaneous Absorption of hEGF o r FD-4-Male Sprague-Dawley rats weighing 200-230 g were anesthetized by an intraperitoneal injection of sodium pentobarbital (Nembutal solution, Abbott laboratories) at a dose of 30 mg/kg and were kept supine on a surface controlled at 37 "C. hEGF was dissolved in pH 7.4, 0.05 M Tris-HC1 buffer solution containing polysorbate 80 (Tween 80, 0.01%)at a concentration of 100 pg/mL. Polysorbate 80 was used to minimize the adsorption of hEGF to experimental glass vessels. An absorption promoter was also mixed in the hEGF solution at different concentrations. After mixing all ingredients, pH and osmolarity of the solution were again adjusted to pH 7.4 with 1 N NaOH and 280 mOsm/kg with NaC1, respectively. The dosing solution of FD-4 was prepared in the same manner as hEGF. The final concentration of FD-4 in the dosing solution was 10 pmol/mL. Osmolarity of the solution was determined with a n osmometer (Osmostat, OM-6020, Kyoto Daiichi Kagaku Company, Kyoto, Japan). Subcutaneous administration of the drug solution was performed a t the ventral side at a dosing volume of 0.5 mL/'kg. Blood was collected from a jugular vein at appropriate time intervals and immediately centrifuged to obtain plasma samples. Plasma samples were stored at -30 "C until analysis. Effect of Absorption Promoters on the Extravasation of Evans Blue-Evans blue is known to be bound completely by plasma protein and its extravasation is not normally observed. Fifteen min after the intravenous administration of Evans blue (20 mg/kgl into the tail vein of a n anesthetized rat, 50 FL of the buffer solution without absorption promoter or the solution containing an absorption promoter (25 or 50 mM EDTA, 50 or 100 mM sodium caprate; 15 mM sodium glycocholate; 15 mM N-lauroylalanine, 15 mM N-decanoylphenylalanine) was intracutaneously injected at the shaved ventral side of the rat. The extravasation of intravenously administered Evans blue at the injection site of a n absorption promoter and at the underlying tissue (dermis, subcutaneous tissue) was visually examined 10 min after the injection of a n absorption promoter solution. Effect of Absorption Promoters on t h e Enzymic Degradation of hEGF In Vitro-The degradation of hEGF in the absence or presence of absorption promoter was determined with the supernatant of subcutaneous tissue homogenates and a 0.1 M phosphate buffer solution (pH 7.3) containing a protease or a leucine arninopeptidase. Freshly isolated parietal peritoneal tissue was homogenized with a ninefold volume of ice-cold 0.9% NaCl solution. The homogenates

0022-3549/93/0300-0236$02.50/0 0 1993, American PharmaceuticalAssociation

were centrifuged at 9000 x g for 20 min, and the supernatant (-10 mg protein/mL) was separated. Protease or leucine aminopeptidase was dissolved in pH 7.3 phosphate buffer at 1 or 0.1 unit/mL. One milliliter of hEGF solution (1 pg hEGF/mL in pH 7.3 phosphate buffer) and 0.5 mL of buffer solution with or without an absorption promoter were mixed and the resultant mixture was incubated at 37 "C for 3 min. The supernatant of the tissue homogenate or the enzyme solution (0.5 mL), prewarmed at 37"C, was added to the mixture to initiate the enzymic reaction. Aliquots (1 mL) of the solution were periodically taken, put into a test tube containing 0.1 mL of 0.2 N HCl to terminate enzymic reaction, and stored at -30 "C until analysis. Effect of absorption promoters on leucine, alanine, or arginine aminopeptidase activity in the supernatant of subcutaneous tissue homogenates was carried out in the same manner as described by Hirai et al.6 Leucine, alanine, or arginine naphtylamide was dissolved in 0.05 M, pH 7.0 phosphatebuffer solution at a concentration of 0.4 mg/mL. The amino acid naphtylamide solution (0.25 mL) was mixed with an equal volume of the same buffer solution with or without an absorption promoter at different concentrations. After incubation of the mixture at 37 "C for 3 min, 30 pL of the supernatant of subcutaneous tissue homogenates were added. Incubation was made at 37 "C for 15 min. The enzymic reaction was terminated by adding 1 mL of 0.2 N HC1, followed by addition of 1 mL of 4% p-dimethylaminobenzaldehydeethanol solution. After standing for 20 min at room temperature, the absorbance of the mixture was measured at 450 nm. The residual aminopeptidase activity in the supernatant of subcutaneoustissue homogenates was calculatedfrom the absorbance difference at 450 nm in the absence and the presence of an absorption promoter. Analytical Method-The concentration of hEGF in the plasma and the supernatant of tissue homogenates was determined by enzymeimmunoassay.5 The specificity of this assay has been tested with different pleiotypic polypeptides such as insulin, somatostatin, or mouse EGF. However, no cross-reaction was found. The practical detection limit of hEGF in this assay was 0.1 ng hEGFlmL. The concentration of FD-4 in the plasma was determinedfluorometricdly. The plasma sample (50 pL) was diluted with 2 mL of pH 7.4,0.05 M Tris-HC1 buffer solution and the concentration of FD-4 was determined with a fluorescence spectrophotometer (F-3000, Hitachi Ltd., Japan) at emission and excitation wavelengths of 494 and 516 nm, respectively.

Results Effect of Absorption Promoters on t h e Subcutaneous Absorption of hEGF-hEGF solution (50 pgkg) was subcutaneously administered with or without an absorption promoter. Profiles of concentration of immunoreactive hEGF in plasma versus time (concentration-time profiles) are shown in Figure 1.By coadministration of an absorption promoter,

;i 0.2

2

0

30

60

90

120

150

2

160

Time, min

Figure 1-Enhanced concentrations of hEGF in plasma after subcutaneous administration with an absorption promoter in rats. Dose of hEGF was 50 pglO.5 mUkg. Key: (0) no adjuvant; (0)50 mM EDTA; (A)15 mM sodium glycocholate, (A) 100 mM sodium caprate; (0) 15 mM Klauroylalanine; (m) 15 mM Wdecanoylphenylalanine. Each point represents the mean of four trials.

the peak plasma level (C,,) of hEGF was significantly increased compared with that in the absence of the absorption ,,, the time required to reach the promoter. The value of C C ,, (tmm), and the value of area under the plasma concentration-time curve from time 0 to infinity (AUC) are summarized in Table I, with the AUC value after the intravenous administration of hEGF at a dose of 50 pg/kg. The C,, and AUC values of hEGF in the presence of a n absorption promoter were significantly greater than those in the absence of an absorption promoter. In particular, when hEGF was subcutaneously administered with 50 mM EDTA, 100 mM sodium caprate, 15 mM N-lauroylalanine, or 15 mM N-decanoylalanine, the AUC value of hEGF was almost the same as that of the intravenous administration at the same dose. Effect of Absorption Promoter on t h e Subcutaneous Absorption of FD-4-The effect of absorption promoters on the subcutaneous absorption of FD-4, a nondegradable model compound, at the subcutaneous injection site, was examined. Figure 2 shows that only EDTA markedly increases the initial entry rate of FD-4 into the blood circulation. On the other hand, other absorption promoters, such as sodium caprate and N-lauroylalanine, suppressed to some extent the initial entry rate of FD-4, probably because of the increased viscosity of the dosing solution caused by the presence of an absorption promoter. Effect of Absorption Promoters on t h e Extravasation of Evans Blue-When buffer solution alone was intracutaneously injected, no extravasation of intravenously administered Evans blue at the injection site and the subcutaneous tissue under the injection site was observed. Severe extravasations of Evans blue at the injection site, but not at the underlying tissues, was observed by the injection of EDTA. On the other hand, the extent of the extravasation of Evans blue at the injection site of other absorption promoters such as sodium caprate, sodium glycocholate, N-lauroylalanine, and N-decanoylphenylalanine was much less than that by EDTA. However, extravasation of Evans blue by such absorption promoters was severe at the subcutaneous tissue under the injection site. Thus, a marked difference was observed in the action of EDTA and other lipophilic absorption promoters, although all absorption promoters used in the present study were shown to have a n increasing effect on the permeability of the blood capillary vessels. Effect of Absorption Promoters on t h e Enzymic Degradation of hEGF-hEGF is degraded in the solution containing the supernatant of subcutaneous tissue homogenates, protease, or leucine aminopeptidase (Figures 3 and 4). However, i n the presence of a n absorption promoter such as sodium caprate or N-acylamino acid in the enzymic solution, the degradation of hEGF was significantly suppressed (Figure 3 and 4). The inhibitory effect of absorption promoters against the aminopeptidase activity in the supernatant of subcutaneous tissue homogenates was investigated with a synthetic substrate. As summarized in Table 11, the leucine, alanine, and arginine aminopeptidase activities were markedly inhibited in the presence of a n absorption promoter such as sodium caprate, N-acylamino acids, and sodium glycocholate, depending on the concentration of the absorption promoter. On the other hand, the inhibitory effect of EDTA was relatively small.

Discussion The AUC values after subcutaneous administration of hEGF at various doses were always smaller than those after the intravenous administration of the corresponding dose as reported previously.5 The values of AUC of hEGF in plasma after subcutaneous and intravenous administrations at a dose of 50 pgkg were 0.47 2 0.02 and 2.24 ? 0.45 pg min/mL,

-

Journal of Pharmaceutical Sciences I 237 Vol. 82, No. 3, March 1993

Table I-Enhanced

Subcutaneous Absorption of hEGF by Absorptlon Promoters In Rats'

Concentration,

Promoter

,, C

mM

EDTA

Sodium glycocholate Sodium caplate NLauroylalanine NDecanoylphenylalanine

min 10

5.18 f 0.04 31.65-C 1.70 22.61 1.09 20.27 f 1.09 10.702 1.12 13.34f 0.77 7.73 i 0.30 11.51 f 1.19 8.91 f 0.85 12.02f 1.02 7.76 f 0.68

-e 50 12.5 15 3.75 100 25 15 3.75 15 3.75

None (iv)d None (sc)'

tllaxl

ng/mLb

5

5 5 6.31.3 16.76.7 10 23.36.7 10 16.76.7 30

AUC, yg . min/mLb

Percent Increased'

1.20 2 0.04 0.44f 0.02 1.24 f 0.04 1.09 f 0.07 0.90f 0.10 0.63 f 0.09 1.84 2 0.08 0.81 -t 0.07 1.37f 0.13 0.68? 0.05 1.33 f 0.09 1.482 0.12

100 282 248 205 1 43 418 184 31 1 155 302 336

-~ ~

Dose of hEGF was 50 pg/0.5 mUkg; each value represents the mean f standard error of the mean of 3-4 trials. The values of,,C and AUC in the presence of absorption promoter are significantly higher than that in the absence of absorption promoter [None (sc),p < 0.051. Increase (%) was calculated by dividing the AUC value in the presence with that in the absence of absorption promoter. iv, Intravenous. '-, Not applicable. 'SC, Subcutaneous. a

1

L

c

30

0

60

90

120

Time, m i n

Figure 2-Effect of absorption promoters on subcutaneousabsorption of FITC-dextran (M,, 4000) in rats. Dose of FITC-dextran was 5 ymol/0.5 mUkg. Key: (0)no adjuvant; (0)50 mM EDTA; (0)50 mM sodium caprate; (A)15 mM Klauroylalanine. Each point represents the mean of

three to five trials.

lZot 110

I

5 10

20

40

Time, m i n

0 5 10

20 Time, m i n

40

Figure 4-Effect of absorption promoters on the enzymic degradation of hEGF by (A) protease and (6)leucine aminopeptidase at 37 "C. Initial concentration in the reaction mixture: hEGF, 500 ng/mL (A, B); protease, 0.25 uniffml (A); leucine aminopeptidase,0.025unit/mL (6).Key: (0)no 25 mM sodium caprate; (0)50 mM sodium caprate; (A) adjuvant; (0) 3.75 mM Klauroylalanine; (A)7.5 mM ff-lauroylalanine. Each point

T

L

0

T

represents the mean of four trials.

A

1

0

s

1

10

I

20 Time, m i n

I

30

4b

Flgure &Effect of absorption promoters on the enzymic degradation of

hEGF in the supernatant of rat subcutaneous tissue homogenates at 37 "C. Key: (0)no adjuvant; (0) 25 mM sodium caprate; (A) 3.75 mM

Nlauroylalanine.The error bars represent the standard error of the mean (n

=

4).

respectively. On the other hand, the difference of the AUC value of hEGF between subcutaneous and intravenous administrations became smaller at a higher dose (500 kg/kg). Above results suggest that the enzymic loss of hEGF at the subcutaneous injection site before transferring to the systemic blood circulation will be involved in the subcutaneous absorption of hEGF. In the present study, a dose of 50 p g k g was used for examining the effect of a n absorption promoter on the subcutaneous absorption of hEGF because the lower 238 'I Journal of Pharmaceutical Sciences Vol. 82,No. 3, March 1993

dose of hEGF was expected to be more susceptible to enzymic degradation. Also, the inhibitory effect of an absorption promoter against the enzymic degradation of hEGF must be effectively reflected on the AUC value of hEGF. As shown in Figure 1and Table I, subcutaneous absorption of hEGF was significantly increased in the presence of an absorption promoter. In particular, the increased AUC in the presence of 50 mM EDTA, 15 mM N-lauroylalanine, and 15 mM N-decanoylalanine were almost comparable with that after intravenous administration. However, in the presence of 100 mM sodium caprate, the AUC value was greater than that after intravenous administration. The reason for this is not clear at present. Previously, sodium glycocholate and sodium caprate were reported to inhibit the activity of leucine aminopeptidase at the rectal or nasal membranes and to increase the hypoglycemic activity of insulin administered in the rectal or nasal cavity.6.7Aminopeptidases such as leucine, alanine, glutamine, and arginine aminopeptidases exist in various mucosal membranes and act as an enzymic barrier for peptide absorption.12Aminopeptidases were also found in the supernatant of subcutaneous tissue homogenates (Table 11) and degraded hEGF (Figures 3 and 4). Activities of aminopeptidases in the supernatant of the subcutaneous tissue homogenates were markedly inhibited by various absorption promot-

Table Il-Effect of Absorption Promoters on the Activities of Leuclne, Alanine, and Arginine Aminopeptidases in Rat Subcutaneous Tlssue Homogenates'

Promoter

Concentration, mM

Leucine Aminopeptidase,%

Alanine Aminopeptidase,%

Arginine Aminopeptidase, %

50 25 12.5 15 7.5 3.8 100 50 25 12.5 15 7.5 3.8 15 7.5 3.8

100 66.4 f 3.5 78.4 f 2.8 87.1 2.7 13.1 f 0.3 15.7 2 0.7 20.0 c 0.6 7.3 f 0.1 7.5 ? 0.3 7.2 c 0.1 11.6 2 0.9 7.6 f 0.2 8.4 f 1.1 11.7 f 1.3 9.1 f 0.5 8.1 f 0.1 11.3 t 0.9

100 49.0 ? 0.1 72.4 f 2.8 91.4 2 1.9 14.1 -C 0.4 23.3 2 1.7 23.3 f 2.0 1.8 0.1 2.3 t 0.1 6.1 f 1.0 10.4 f 1.3 1.9 f 0.1 2.8 & 0.2 6.0 f 0.5 1.7 ? 0.2 3.3 f 1.0 6.1 t 0.7

100 44.8 % 4.0 65.2 f 2.4 83.3 5 5.1 21 .o f 0.9 20.0 f 2.7 34.4 f 1.8 2.6 f 0.4 3.0 f 0.7 2.9 5 0.2 5.4 5 0.6 3.2 4 0.7 2.4 f 0.4 2.5 a 0.2 2.1 a 0.3 2.0 5 0.1 4.7 5 0.4

None EDTA 2Na

Sodium glycocholate Sodium caprate

NLauroylalanine NDecanoylphenylalanine

*

a The enzymic activity of each aminopeptidase in the supernatant of peritoneal tissue homogenates was determined with correspondingamino acids naphtylamide at 37 "Cfor 15 min; each value (mean ? standard error of the mean of four trials) represents the percentage of residual activity of arninopeptidase after 15 min.

ers, except EDTA. Thus, the increasing effect of absorption promoters, except EDTA, on the rate and extent of subcutaneous absorption of hEGF may be mainly attributed to their inhibitory effectsagainst the enzymic degradation of hEGF in subcutaneous tissues, like some enzymic inhibitors for insulin.3.4Additionally,Okumura et al.14J5 recently reported that the healing effect of hEGF topically applied on open wounds or second-degree burns was markedly improved by using ointment containing hEGF and a protease inhibitor such as nafamostat mesilate or gabexate mesilate, possibly due to the stabilization of hEGF at the subcutaneous tissues. Besides the inhibitory effect of absorption promoters against the enzymic degradation of hEGF, other factors, such as increase in the diffusion rate of a drug to the blood vessel from the injection site1.2 andor increase in the blood capillary vessel permeability to the drug,2 should also be taken into consideration for the mechanism of the increased absorption effects of absorption promoters. In fact, all absorption promoters examined in the present study caused severe extravasation of Evans blue, although the appearance of the extravasation of Evans blue was different between EDTA and other absorption promoters. This difference may be because of the difference in the lipophilicities of the promoters. For example, absorption promoters except EDTA (i.e., lipophilic absorption promoters) were easily absorbed from the rectal membrane, probably by transcellular passive difision.gJ3 On the other hand, [14C]EDTA,a hydrophilic compound, was not absorbed (data not shown), indicating that the transcellular diffusion of EDTA is doubtful. In the present study, EDTA may remain at the injection site for a long time and not penetrate into subcutaneous tissues because of its low lipophilicity, whereas other lipophilic absorption promoters can diffuse easily into the surrounding tissue by transcellular passive diffusion. EDTA caused a rapid subcutaneous absorption of hEGF (Figure 1) and increased the plasma AUC value of hEGF (Table I), although its inhibitory effect against the aminopeptidase activity was small (Table 11). This fact may indicate that the effect of EDTA of increasing subcutaneous absorption of hEGF was mainly attributed to the increase in the blood capillary vessel permeability to hEGF and/or the diffusion rate of hEGF through the subcutaneous tissues. This possible mechanism for EDTA was confirmed with a nondegradable model compound, FD-4 (Figure 2). The initial entry rate of FD-4 into the blood circulation was increased only in the

presence of EDTA. The reason absorption promoters other than EDTA failed to increase the blood capillary vessel permeability to FD-4 is not clear. However, it may be considered that lipophilic absorption promoters demonstrate the inhibitory effect against the aminopeptidase activities only at the injection site and underlying tissues because of their rapid disappearance from the injection site. In conclusion, we demonstrated that the rate and the extent of subcutaneous absorption of hEGF were enhanced by absorption promoters such as sodium caprate, N-acylamino acids, EDTA, and sodium glycocholate. The increasing effect of these absorption promoters, with the exception of EDTA, was mainly attributed to the inhibitory effect against the enzymic degradation of hEGF at the subcutaneous injection site. On the other hand, the increasing effect of EDTA was mainly attributed to the increase of the blood capillary vessel permeability to hEGF.

References and Notes 1. Bergar, M.; Halban, P. A.; Girardier, L.; Seydoux, J.; Offord, R. E.; Renold, A. E. J. Cell. Biol. 1967, 71, 159. 2. Ballard, B. E. J. Pharm. Sci. 1968,57, 357. 3. Hori, R.; Komada, F.; Okumura, K. J. Pharm. Sci. 1983,72,435. 4. Komda, F.; Okumura, K.; Hori, R. J. Pharmacobio-mn. 1983, 8, 33.

5. Murakami, T.; Kawakita, H.; Kishimoto, M.; Higashi, Y.; Amagase, H.; Hayashi, T.; Nojima, N.; Fuwa, T.; Yata, N. Znt. J. Pharm. 1988,46,9. 6. Hirai. S.: Yashiki, T.: Mima. H. Int. J. Pharm. 1981, 9. 173. 7. Mishlrnal M.; Wakita, Y.; Nakano, M. J. Pharmucobio-Dyn.

1987. 10. 624. 8. Yatai N.'; Higashi, Y.; Murakami, T.; Yamajo, R.; Wu, W. M.; Taku,Y.; Sasaki, Y.; Hideshima, Y. J. Pharrnucobio-Dyn. 1983, 6, S-78. 9. Wu, W. M.; Murakami, T.; Higashi, Y.; Yata, N. J. Pharm. Sci. 1987, 76, 508. 10. Murakami, T.; Sasaki, Y.; Yamajo, R.; Yata, N. Chem. Pharm. Bull. 1985,32, 1948. 11. Murakami, T.; Kishimoto, M.; Kawakita, H.; Misaki, M.; Kojima, I.; Higashi, Y.; Amagase, H.; Fuwa, T.; Yata, N. Eur. J. Drug Metub. Pharmacokinet. 1991, Special issue RO. IIZ, 125. 12. Stratford. R. E. Jr.: Lee. N V . H. L. Int. J. Pharm. 1986, 30. 7 3 . 13. Wu, W. M.; Murakami, T.; Yamajo, R.; Higashi, Y.; Yata, N. J. Pharm. Set. 1989, 78,499. 14. Okumura, K.; Kiyohka, Y.; Komada, F.; Iwakawa, S.; Hirai, M.; Fuwa, T. Pharm. Res. 1990,12, 1289. 15. Kiyohara, Y.; Komada, F.; Iwakawa, S.; Hirai, M.; Fuwa, T.; Okumura, K. J . Pharmaeobio-Dyn. 1991,14, 47.

Journal of PharmaceuticalSciences / 239 Vol. 82,No. 3, March 1993