QUANTITATIVE EVALUATION OF THE ACTIVITY AND EXPRESSION OF ESTERASE IN THE RAT SMALL INTESTINE

小腸におけるエステラーゼ発現レベルと代謝能の 定量的解析に関する研究

2006 眞﨑 賢治

QUANTITATIVE EVALUATION OF THE ACTIVITY AND EXPRESSION OF ESTERASE IN THE RAT SMALL INTESTINE

Kenji Masaki

QUANTITATIVE EVALUATION OF THE ACTIVITY AND EXPRESSION OF ESTERASE IN THE RAT SMALL INTESTINE

Kenji Masaki

Intestinal metabolism plays an important role in the bioavailability of oral drugs and activation of prodrugs. Most drug-metabolizing enzymes in the liver are also found in the small intestine but the enzyme expression level is generally lower in the small intestine than the liver.

Carboxylesterase (CES) effectively catalyzes not only the hydrolysis of xenobiotics and numerous endogenous compounds but also that of ester-type prodrugs in the small intestine and liver. For ester-containing drugs, such as prodrugs, intestinal hydrolysis is one of the major determinants of their pharmacokinetics and pharmacodynamics.

In this study, the in situ single-pass perfusion method was preferentially used for pharmacokinetic analysis of the intestinal first-pass hydrolysis, because this method could maintain the intestinal architecture with respect to metabolism, absorption, and exsorption. The major enzyme involved in intestinal hydrolysis was identified in the in situ single-pass perfusion experiment. In addition, the effect of aging on the hydrolyzing capacity and expression level of rat intestinal CES isozymes in the jejunum and the ileum was investigated.

1) Rat jejunum and intestinal blood vessels were perfused simultaneously in order to evaluate the first-pass hydrolysis of O-Isovaleryl-propranolol (Isovaleryl-PL), a model ester-compound.

When Isovaleryl-PL was absorbed from the jejunal lumen, more than 90% of compound permeated into the blood vessel as PL. In addition, the estimated hydrolysis clearance from the in vitro hydrolysis study was 3.43 mL/min/10 cm jejunum, which was 25-fold greater than the observed CL

(0.14 ± 0.02 mL/min/10 cm jejunum). Isovaleryl-PL was almost completely hydrolyzed to PL and Isovaleric acid (IVA) in the epithelial cells at a rate limited by its uptake.

Subsequently, PL and IVA were transported into both vascular (pH 7.4) and luminal sides (pH 6.5) of the cells according to a mechanism based on pH-partitioning. Therefore, intestinal hydrolysis led to an increase of the permeability coefficient (P

) of Isovaleryl-PL due to preferential efflux of PL from epithelial cells into the vascular and luminal sides rather than Isovaleryl-PL.

2) An enzyme involved in the first-pass hydrolysis of Isovaleryl-PL was determined under the

single-pass perfusion experimental condition that in the presence of Bis-p-nitrophenyl

phosphate (BNPP), a specific CES inhibitor. Pre-treatment with BNPP (400 μM, 40 min) could

specifically inhibit CESs without any effect on the Aminopeptidase activity and the membrane

transport activity. When Isovaleryl-PL was perfused into the jejunal lumen under BNPP treatment condition, its absorption clearance was increased and hydrolysis clearance was decreased in comparison with control. CES was responsible for about 85% of the intestinal first-pass hydrolysis of Isovaleryl-PL which was calculated by percent of decrease of hydrolysis clearance. P

of Isovaleryl-PL was lower than that of the control, but still higher than that of PL due to the 23% remaining hydrolase activity.

3) The extent of contribution of CES in the in situ hydrolysis (85%) is consistent with the results from the studies of in vitro BNPP inhibition in mucosal S9 fraction (90% and 84% for Valeryl- and Isovaleryl-PL, respectively). Hydrolysis of 11 PL ester derivatives showed slightly higher activity in the jejunal S9 than in the ileal S9, with the same substrate specificity between the jejunal and ileal S9. V

values for Valeryl-PL and p-Nitrophenyl acetate (PNPA) in the jejunal S9 fraction were 1.7–1.8 fold higher than that in the ileal S9, which agreed with the jejunum/ileum ratio (about 1.5-fold) of mRNA expression levels for the CES2 isozymes, AB010635 and AY034877. The proximal-to-distal gradient of decreasing CES activity was less steep than that of CYP2B activity for 16β-hydroxylation of Testosterone, which was three-fold higher in rat jejunum than ileum.

4) In the jejunal S9 fraction prepared from each group of rats of different ages (8, 46 and 90 weeks), V

values for PNPA in 90 weeks were slightly higher than that in 8 and 46 weeks with similar K

values. In the ileal S9, V

values gradually increased with aging. Hydrolysis of 11 PL derivatives showed same substrate specificity in different aged rat intestine. Moreover, mRNA expression level of both major CES2 isozymes increased with aging in rat small intestine, and the similar elevation of mRNA of CES2 isozyme was observed in rat liver. In contrast, mRNA level of a major hepatic CES1 iszymes (Hydrolase A and Hydrolase B/C) decreased with aging. However, no correlation could be observed between mRNA expression of CES2 isozymes and kdel receptor isozymes, which are important proteins for retaining CES isozymes at their C-terminal tetra-peptide (HXEL), in the intestine and liver.

These findings indicate that the intestine markedly contributes to first-pass hydrolysis of prodrug and CESs mainly involves in the hydrolysis in the jejunum and ileum as well as liver.

The expression level of CES isozymes is slightly varied according to age and intestinal region.

Design of an orally administrated prodrug will be successful by controlling the CES mediated

intestinal hydrolysis.

小腸におけるエステラーゼ発現レベルと代謝能の定量的解析に関する研究

生命薬科学専攻 病態薬効解析学分野 眞﨑 賢治

生理活性既知の薬物に, 薬理不活性な置換基を導入して, 親薬物の持つ問題点を克服す る分子修飾法としてプロドラッグ化がある. 経口投与されたプロドラッグは, 小腸や肝臓の初 回代謝によって完全に復元されることが望ましく

,

Carboxylesterase

(CES)

加水分解における

CES

.

in situ

single-pass

解酵素を同定し, 吸収動態について速度論的に解析した. また, ラット空腸および回腸での 水解活性の差異を明らかにし, 加齢による基質特異性および

mRNA

1)

Isovaleryl-PL

90%以上が代謝物の PL

IVA)

PL

pH

IVA

)

in vitro

K

, V

mL/min/10 cm

1/25

)

PL

7

Isovaleryl-PL

Isovaleryl-PL

Isovaleryl-PL

PL

Isovaleryl-PL

2)

CES

CES

Bis(p-nitrophenyl) phosphate (BNPP)

400

μM

40 min

Aminopeptidase

を及ぼさずに

CES

Isovaleryl-PL

single-pass

85%が CES

PL

成が減少するため, P_effは低下した. しかしながら, BNPP処理においても加水分解活性が

23%

残存するため, 細胞内取り込み→加水分解→細胞外排出の一連の流れは維持され, もともと 吸収率の高い

PL

P

3.5

2

3)

S9

BNPP

Isovaleryl-PL

90%と 84%寄与することが示された.

CES

in vivo

S9

K

1.7-1.8

1.5-1.6

, V

S9

CYP2B

Testosterone 16β-水酸化活性は,

3

差は, CYP2B活性の変動幅よりも小さいことが明らかとなった.

4)

90

S9

p-Nitrophenyl acetate (PNPA)

K

V

値が若干増大した. また, 一連の

PL

mRNA

mRNA

kdel

CES

CES1

CES2

mRNA

CES2

および

AY034877)

CES2

ソザイム

D50580,

CES1

B/C)

mRNA

CES1

2

CES

mRNA

CES

以上のように, 本研究では, エステル型プロドラッグの小腸での加水分解および吸収挙 動を明らかにし, さらに吸収時の加水分解に寄与する主な酵素として

CES

CES

CES

目次

第

1

……….……. 1

第

2

Isovaleryl-PL

……….…..……… 8

2-1

………..……… 8

2-2

Propranolol (PL)

……..………..……… 8

2-3 Single-pass

Propranolol

………..…….….…..…….…….. 10

2-4 Single-pass

Isovaleryl-PL

………..…….……… 12

2-4-1 Pro-moiety

Isovaleric acid

……….…..……… 15

2-4-2

…..……….. 16

2-5

PL

Isovaleryl-PL

……… 18

2-6

………..… 21

第

3

Isovaleryl-PL

in situ

……….…………. 22

3-1

………..…… 22

3-2

single-pass

Bis(p-nitrophenyl)-phosphate (BNPP)

……….. 22

3-3 BNPP

Esterase

Aminopeptidase

……….…. 25

3-4 BNPP

………..………. 26

3-5 Isovaleryl-PL

CES

………..… 27

3-6

………..… 29

第

4

CES

………..… 31

4-1

………..… 31

4-2

S9

in vitro

CES

4-3

,

S9

CES

P-450

………...… 33

4-4

S9

Valeryl-PL

PNPA

……….. 35

4-5

,

CES

………..…… 37

4-6

CES2

………..……. 39

4-7

………...… 41

第

5

CES

…….…………. 42

5-1

……….. 42

5-2

P-450

………... 43

5-3 PL

……….………… 45

5-4

CES

………. 47

5-5

KDEL

... 49

5-6

CES

….………..……. 51

5-7

KDEL

………. 53

5-8 CES

……….………. 55

5-9

………..……… 56

第

6

….………..………… 58

6-1

………..………. 58

6-2

…………...……… 58

6-3

Esterase

……….…..……….…….….. 60

6-4

……….. 61

6-5

CES

……… 62

6-6

………. 64

6-7

………..………… 65

第

7

……….……. 67

実験の部………...…… 70

謝辞

………..…… 84

参考文献………..….... 85

本論文で使用した略号一覧表

AP Apical

AUC

Area under the concentration curve of a drug in the mesenteric vein AUC

Area under the concentration curve of a drug in the luminal loop BL Basolateral

BNPP Bis-p-nitrophenyl-phosphate CES Carboxylesterase CL

Apparent absorption clearance CL

Apparent degradation clearance

FD-4 Fluorescein isothiocyanate dextran 4000

GAPDH Glyceraldehyde-3-phosphate-dehydrogenase HEPES N-2-Hydroxyethylpiperazine-N’-2-ethansulfonic acid HPLC High performance liquid chromatography

IVA Isovaleric acid K

Michaelis constant Leu-p-NA L-Leucyl-p-nitroanilide

MES 2-(N-Morpholino) ethanesulfonic acid P

Apparent permeability coefficient in in vitro P

Apparent permeability coefficient in in situ PAGE Polyacrylamide gel electrophoresis

PL Propranolol PNPA p-Nitrophenyl acetate

Q

Flow rate of luminal perfusate Q

Flow rate of vascular perfusate

v

Appearance rate of a drug in the mesenteric vein

v

Disappearance rate of a drug in the jejunal lumen

v

Appearance rate of drug in the jejunal lumen

V

Maximum velocity

本論文は, 学術雑誌に掲載された次の論文を基礎とするものである.

1) First-pass hydrolysis of a propranolol ester derivative in rat small intestine Drug Metabolism and Disposition, 34, 398-404 (2006)

Kenji Masaki, Megumi Taketani, and Teruko Imai

2) Intestinal first-pass metabolism via carboxylesterase in rat jejunum and ileum Drug Metabolism and Disposition, submitted for publication.

Kenji Masaki, Mitsuru Hashimoto, and Teruko Imai

3) The effect of age on carboxylesterase activity and expression International Journal of Pharmacetics, in preparation

Kenji Masaki, Kayoko Ohura, Mitsuru Hashimoto, and Teruko Imai

第1章 緒言

生理活性既知の薬物 (親薬物) に, 薬理不活性な置換基 (pro-moiety) を導入して, 親薬物の持つ問題点を克服する分子修飾法としてプロドラッグ化がある. 例えば, 薬物 の溶解や膜透過に起因する吸収性

,

,

,

.

,

,

(HTS)

,

,

.

プロドラッグは

,

,

.

,

.

,

,

(

)

,

.

プロドラッグの生体内変換速度が

,

,

. Scheme 1

,

,

(

)

,

k

A

k

k

.

, Fig. 1A

. Figure 1B

C

,

k

,

,

k

より速い変換速度を保つように設計されたものの中にも, 予期せぬ速い速度でプロド ラッグが代謝されることがあり, Fig. 1Cのようなプロファイルを描くことがある. この ように, プロドラッグの設計においては, 親薬物への変換速度が他の代謝速度に比べて 速いことが重要であり, 初回通過代謝のような速い速度で親薬物に復元されるプロド

ラッグが理想的なプロドラッグと言える

.

プロドラッグが初回代謝のような速度で効率よく親薬物に変換するためには

,

(Hydrolase)

.

(COOH),

(NH

)

,

.

したがって, プロドラッグの生体内変換にはこれらの結合様式を広く認識し, 加水分解 を触媒する

Carboxylesterase (CES)

CES

.

Scheme 1 Typical Disposition of Prodrug Prodrug

Metabolite A

Parent Drug Metabolite B

k

k

k

Scheme 1 Typical Disposition of Prodrug Prodrug

Metabolite A

Parent Drug Metabolite B

k

k

k

Prodrug

Prodrug

Metabolite A Metabolite A

Parent Drug Metabolite B

Parent Drug

Parent Drug Metabolite B Metabolite B k

k

k

Fig. 1 Simulation Curve of Blood Concentration after Oral Administration of Prodrug A: k

>>k

, k

>>k

(Ideal prodrug), B: k

>k

, k

>k

, C: k

>k

, k

>k

Fig. 1 Simulation Curve of Blood Concentration after Oral Administration of Prodrug

A: k

>>k

, k

>>k

(Ideal prodrug), B: k

>k

, k

>k

, C: k

>k

, k

>k

小腸上皮細胞など肝臓以外の組織における活性も比較的高いことから

CES

CES1

hCE1

(CES1A1, HU1) のアミノ酸配列に基づく相同性から,

5

れる. その系統樹を

Fig. 2

Fig. 2 Phylogenetic Tree of the Carboxylesterase Superfamily

Human CES1A1/Hu1a Human CES1A2/Hu1b Monkey CES1 Human PlacentaCES Rabbit CES1 Rat BC078681 Rat ES-10 Mouse CES MH1 Mouse TGH Hamster AT31 Felica CES1 Felica CESK1 Dog CESD1 Pig CES Pig CES1

Mouse ES22 (Egasyn) Rat ES-3 (Egasyn) Bovine-REH Mouse EST1 Mouse EST-N Rat CES 60KDa Rat HydrolaseC Rat CES RL1 Rat ES-4/HydrolaseB Mouse ES-4 Bovine CES2 Human CES2/hCE2 Rabbit CES2 Hamster AT41 Mouse BC022148 Rat CES5 Mouse BC015286 Mouse mCES2 Rat BC097486 Rat CES RL4 Rat SICES Hamster AT51 Rat CES6 Dog CAUXIN Felica CAUXIN Mouse CAUXIN Rat CAUXIN Human AcylCoA Human hCE3 Mouse ES31 Mouse AADAC Rat AADAC Human AADAC

Trival name

L07765/AB119995 AB119996 AB10633 MN016280 AF036930 BC078681 X51974 AB023631 AF378751 D50578 AB094147 AB114676 AB023629 X63323 AF064741 S8091 X81395 AY369075 Y12887 M57960 M20629 U10698 AB023630 X81825 BC013479 NM001034260 U60553 P14943 (protein) D50577 BC022148 XM341636/D50580 BC015286 BC031170/BC015290 BC097486 AB010635/AB191005 AB010632 D28566

NM144743/AY034877 AB186392 AB045377 AB186393 AF479659 AK056109 XM016735 S64130 BC054823 BC088143

NM001086 CES5A

CES5B CES3A CES4C CES4B CES4A CES2A CES1H CES1G CES1E CES1F CES1D CES1C CES1B CES1A Genebank

Fig. 2 Phylogenetic Tree of the Carboxylesterase Superfamily

Human CES1A1/Hu1a Human CES1A2/Hu1b Monkey CES1 Human PlacentaCES Rabbit CES1 Rat BC078681 Rat ES-10 Mouse CES MH1 Mouse TGH Hamster AT31 Felica CES1 Felica CESK1 Dog CESD1 Pig CES Pig CES1

Mouse ES22 (Egasyn) Rat ES-3 (Egasyn) Bovine-REH Mouse EST1 Mouse EST-N Rat CES 60KDa Rat HydrolaseC Rat CES RL1 Rat ES-4/HydrolaseB Mouse ES-4 Bovine CES2 Human CES2/hCE2 Rabbit CES2 Hamster AT41 Mouse BC022148 Rat CES5 Mouse BC015286 Mouse mCES2 Rat BC097486 Rat CES RL4 Rat SICES Hamster AT51 Rat CES6 Dog CAUXIN Felica CAUXIN Mouse CAUXIN Rat CAUXIN Human AcylCoA Human hCE3 Mouse ES31 Mouse AADAC Rat AADAC Human AADAC

Trival name

L07765/AB119995 AB119996 AB10633 MN016280 AF036930 BC078681 X51974 AB023631 AF378751 D50578 AB094147 AB114676 AB023629 X63323 AF064741 S8091 X81395 AY369075 Y12887 M57960 M20629 U10698 AB023630 X81825 BC013479 NM001034260 U60553 P14943 (protein) D50577 BC022148 XM341636/D50580 BC015286 BC031170/BC015290 BC097486 AB010635/AB191005 AB010632 D28566

NM144743/AY034877 AB186392 AB045377 AB186393 AF479659 AK056109 XM016735 S64130 BC054823 BC088143

NM001086 CES5A

CES5B CES3A CES4C CES4B CES4A CES2A CES1H CES1G CES1E CES1F CES1D CES1C CES1B CES1A Genebank

コの尿中に見出された分泌型の

CES

,

CES1

CES2

.

プロドラッグを経口投与した場合, 全身循環で高い親薬物濃度を得るためには, 肝 臓および小腸における初回代謝が重要である

(Fig. 3).

,

,

,

.

,

,

,

,

.

,

,

.

,

,

,

,

.

,

.

Fig. 3 A Model for First-pass Hydrolysis of Orally Administered Ester-type Prodrug by Hydrolase in Gastrointestinal Mucosa and Liver.

P and M represent ester-type prodrug and the metabolite (parent drug), respectively. E and T represent enzyme (hydrolase) and transporter, respectively.

Oral Administration

E E

Fig. 3 A Model for First-pass Hydrolysis of Orally Administered Ester-type Prodrug by Hydrolase in Gastrointestinal Mucosa and Liver.

P and M represent ester-type prodrug and the metabolite (parent drug), respectively. E and T represent enzyme (hydrolase) and transporter, respectively.

Oral Administration

E

E

小腸は, 管腔の粘膜に多数の輪状ヒダ, さらにその上に無数の絨毛を突出した形態 を有している. この粘膜上の突出した絨毛は, 一層の円柱上皮細胞で覆われ, この細胞 の管腔に面した側に存在する微絨毛が, 刷子縁膜を形成することで, 広大な表面積を得 ることができ, 栄養物や薬物の吸収に適した構造となっている. 小腸上皮細胞は, 隣接 細胞と密着帯あるいは細胞間接合部

(Tight junction)

,

,

(Transcellular pathway)

(Paracellular

pathway)

.

大や取り込みトランスポーターへの認識性を改善した化合物は

,

.

,

,

,

,

.

一般に

,

,

in vitro

. Table 1

,

S9

,

.

,

,

. In vitro

,

,

Caco-2

HT-29

.

,

,

,

.

,

,

,

,

in vivo

.

一方

in vivo

in situ

,

,

にトランスポーターや蠕動運動などの機能および臓器血流も加わるため, 小腸での代 謝および膜透過性を同時に評価することができる

.

, in vivo

,

in situ

in vivo

現在, in situ法は, 目的および薬物の特性の違いによって, closed loop法, recirculating 灌流法および

single-pass

3

に, closed loop法は腸管腔内に一定容量の薬液を投与する方法, recirculating 灌流法は何 度も薬液を灌流する方法, single-pass 灌流法は一度だけ灌流する方法である. いずれの 方法も一定時間での吸収率もしくは灌流前後での薬物濃度比を求めることにより, 膜 透過性を推定する. エステル型プロドラッグの腸管吸収時における加水分解を評価す るには

,

.

, closed loop

recirculating

,

,

. Single-pass

,

,

.

,

in vitro experiments Metabolism

S9 ER, Cytoplasm

Microsome ER Cytosol Cytoplasm Membrane permeability

Everted sac Ussing chamber

Caco-2 cell monolayer

in vivo experiments Oral administration Intravenous injection

in situ experiments Closed loop

Recirculating perfusion Single-pass perfusion

S9: homogenate 9,000g supernatant, ER: Endoplasmic reticulum

Merit Convenient and inexpensive method Determination of activity in each fractions

Demerit Independent functions (metabolite/membrane transport) Delocalization of metabolic enzymes

Merit Convenient and inexpensive method Demerit Low correlativeness

(absorbed fraction in human / permeability led to these technique) Merit High correlativeness

(absorbed fraction in human / permeability led to this technique) Hold cell construction

Demerit Spend much time due to culture cells.

Low expression level (Enzymes and transporters) Absence of mucin

Merit Hold original tissue construction Evaluation of whole body metabolism

Multiple functions (several transporters and enzymes) Demerit Present of species differences

NOT lead to evaluate metabolism in a organ.

Merit Hold original tissue (intestine) construction

Lead to evaluate absorption and metabolism in the intestine.

Multiple functions (several transporters and enzymes) Demerit Technical difficulties

Closed loop Recirculating perfusion Single-pass perfusion Table 1 Characteristics of Techniques for Investigation of Absorption and Metabolism in vitro experiments

Metabolism

S9 ER, Cytoplasm

Microsome ER Cytosol Cytoplasm Membrane permeability

Everted sac Ussing chamber

Caco-2 cell monolayer

in vivo experiments Oral administration Intravenous injection

in situ experiments Closed loop

Recirculating perfusion Single-pass perfusion

S9: homogenate 9,000g supernatant, ER: Endoplasmic reticulum

Merit Convenient and inexpensive method Determination of activity in each fractions

Demerit Independent functions (metabolite/membrane transport) Delocalization of metabolic enzymes

Merit Convenient and inexpensive method Demerit Low correlativeness

(absorbed fraction in human / permeability led to these technique) Merit High correlativeness

(absorbed fraction in human / permeability led to this technique) Hold cell construction

Demerit Spend much time due to culture cells.

Low expression level (Enzymes and transporters) Absence of mucin

Merit Hold original tissue construction Evaluation of whole body metabolism

Multiple functions (several transporters and enzymes) Demerit Present of species differences

NOT lead to evaluate metabolism in a organ.

Merit Hold original tissue (intestine) construction

Lead to evaluate absorption and metabolism in the intestine.

Multiple functions (several transporters and enzymes) Demerit Technical difficulties

Closed loop Recirculating perfusion Single-pass perfusion

Table 1 Characteristics of Techniques for Investigation of Absorption and Metabolism

実験動物として齧歯類を用いるときに懸念される血漿中での加水分解^{6, 7)} を回避し, 小 腸粘膜のみでの加水分解を評価することを可能にする.

医薬品開発において, 目的に応じたプロドラッグのデザインは, 親薬物への変換部 位とその速度をコントロールすることで達成されるが, この変換速度のコントロール は非常に難しく

,

,

.

,

,

,

,

,

,

.

.

,

2

,

in situ

Isovaleryl-propranolol (Isovaleryl-PL)

.

3

,

Isovaleryl-PL

in situ

,

(CES)

.

4

, CES

mRNA

,

素を

in vitro

.

5

,

および肝臓に存在する

CES

.

,

6

,

.

7

,

.

第2章 ラット小腸における

Isovaleryl-PL

2-1

経口投与されたプロドラッグが効率良く代謝されて親薬物に変換されるためには, 消化管あるいは肝臓で

,

.

,

,

.

,

Phase I, Phase II

.

,

,

.

P-450

,

,

.

,

,

single-pass

,

.

2-2

Propranolol (PL)

本研究では

,

in vitro

in situ

, β-

Propranolol (PL)

β-

2-7

,

(Table 2).

PL

,

,

,

,

(pKa 9.44),

(logD 0.38 (1.002); 1-Octanolol/pH4.0 (pH7.0) Phosphate buffer)

.

, Fig. 4

, PL

.

,

.

PL

ン環の

4, 5, 7

N-脱イソプロピル化が主代謝経路である

ことが報告されている²³⁾

.

.

PL

Caco-2

いた経細胞輸送実験²⁴⁾ や

in vivo

,

.

Compound Propranolol (PL) O-Acetyl-PL O-Propionyl-PL O-Butyryl-PL O-Valeryl-PL O-Caproyl-PL O-Enantyl-PL O-Isobutyryl-PL O-Isovaleryl-PL

O-2-methyl-n-valeryl-PL O-3-methyl-n-valeryl-PL O-Isocaproyl-PL

1 2 3 4 5 6 7 8 9 10 11

R H

COCH ₃ COCH ₂ CH ₃ COC ₂ H ₄ CH ₃ COC ₃ H ₆ CH ₃ COC ₄ H ₈ CH ₃ COC ₅ H ₁₀ CH ₃ COCH(CH ₃ ) ₂ COCH ₂ CH(CH ₃ ) ₂ COCH(CH ₃ )C ₂ H ₄ CH ₃ COCH ₂ CH(CH ₃ )CH ₂ CH ₃ COC ₂ H ₄ CH(CH ₃ ) ₂

* : Asymmetrical carbon

O N

H O

* R

. HCl

Table 2 Structure of PL Ester Derivatives

Compound Propranolol (PL) O-Acetyl-PL O-Propionyl-PL O-Butyryl-PL O-Valeryl-PL O-Caproyl-PL O-Enantyl-PL O-Isobutyryl-PL O-Isovaleryl-PL

O-2-methyl-n-valeryl-PL O-3-methyl-n-valeryl-PL O-Isocaproyl-PL

1 2 3 4 5 6 7 8 9 10 11

R H

COCH ₃ COCH ₂ CH ₃ COC ₂ H ₄ CH ₃ COC ₃ H ₆ CH ₃ COC ₄ H ₈ CH ₃ COC ₅ H ₁₀ CH ₃ COCH(CH ₃ ) ₂ COCH ₂ CH(CH ₃ ) ₂ COCH(CH ₃ )C ₂ H ₄ CH ₃ COCH ₂ CH(CH ₃ )CH ₂ CH ₃ COC ₂ H ₄ CH(CH ₃ ) ₂

Compound Propranolol (PL) O-Acetyl-PL O-Propionyl-PL O-Butyryl-PL O-Valeryl-PL O-Caproyl-PL O-Enantyl-PL O-Isobutyryl-PL O-Isovaleryl-PL

O-2-methyl-n-valeryl-PL O-3-methyl-n-valeryl-PL O-Isocaproyl-PL

1 2 3 4 5 6 7 8 9 10 11

R H

COCH ₃ COCH ₂ CH ₃ COC ₂ H ₄ CH ₃ COC ₃ H ₆ CH ₃ COC ₄ H ₈ CH ₃ COC ₅ H ₁₀ CH ₃ COCH(CH ₃ ) ₂ COCH ₂ CH(CH ₃ ) ₂ COCH(CH ₃ )C ₂ H ₄ CH ₃ COCH ₂ CH(CH ₃ )CH ₂ CH ₃ COC ₂ H ₄ CH(CH ₃ ) ₂

* : Asymmetrical carbon

O N

H O

* R

. HCl _HCl

.

Table 2 Structure of PL Ester Derivatives

2-3 Single-pass

Propranolol

本研究で用いた血管および空腸ループの同時灌流による

single-pass

Fig. 5

.

(10 cm)

0.3 mL/min

.

,

,

3.0 mL/min

3% BSA

Krebs-Henseleit bicarbonate

,

ることによって

,

.

,

,

PL

.

,

PL

PL

経時的に各サンプルを回収し, 腸間膜静脈での

PL

)

)

Fig. 6

20 min

v

v

Fig. 4 Summary of the Relationship between First-order Rate Constants for the Intestinal Absorption and Lipid Solubility of Various Drugs.

The results shown in circles, were reported by Komiya et al. (1980)

, Martin et al. (1981)

and Taylor et al. (1985)

, represent the relationship between intestinal absorption rate constants (k

) obtained by in situ jejunum loop method in rats and octanol-buffer (pH7.0) partition coefficients (log D).

Fig. 4 Summary of the Relationship between First-order Rate Constants for the Intestinal Absorption and Lipid Solubility of Various Drugs.

The results shown in circles, were reported by Komiya et al. (1980)

, Martin et al. (1981)

and Taylor

et al. (1985)

, represent the relationship between intestinal absorption rate constants (k

) obtained by in

situ jejunum loop method in rats and octanol-buffer (pH7.0) partition coefficients (log D).

Propranolol

Imipramine

9.9 min

. v

Fig. 5 Diagrammatic Representation of Rat Jejunal Single-pass Perfusion.

Vascular and luminal perfusate were Krebs-Henseleit bicarbonate buffer containing 3% BSA and 10 mM D-glucose (pH 7.4) and MES buffer containing a test drug (pH 6.5), respectively.

Q

and Q

represent flow rate of vascular and luminal perfusion, respectively.

C

and C

represent drug concentration at the entrance and exit of jejunal loop, respectively.

v

and v

represent an appearance rate of drug in the mesenteric vein and a disappearance rate of drug in the jejunal lumen, respectively.

Fig. 5 Diagrammatic Representation of Rat Jejunal Single-pass Perfusion.

Vascular and luminal perfusate were Krebs-Henseleit bicarbonate buffer containing 3% BSA and 10 mM D-glucose (pH 7.4) and MES buffer containing a test drug (pH 6.5), respectively.

Q

and Q

represent flow rate of vascular and luminal perfusion, respectively.

C

and C

represent drug concentration at the entrance and exit of jejunal loop, respectively.

v

and v

represent an appearance rate of drug in the mesenteric vein and a disappearance rate of drug in the jejunal lumen, respectively.

0 2 4 6 8 10 12

0 10 20 30 40 50 60

Time (min) Di sappe ara nce rate of PL ( v

, nm ol/m in )

B) Jejunal lumen

0 2 4 6 8 10 12

0 10 20 30 40 50 60

Time (min) Appe ara nce ra te of PL ( v

, nm ol /m in)

A) Mesenteric vein

Fig. 6 Absorption of PL in the Rat Jejunal Single-pass Perfusion.

A) An appearance rate of PL in the mesenteric vein (v

); B) a disappearance rate of PL in the jejunal lumen (v

). Rat jejunal loop (10 cm) was perfused with 300 μM PL. The perfusion flow rate was 2.8 and 0.3 mL/min for the mesenteric vein and jejunal lumen, respectively. Each point represents mean ± S.D. (n = 3).

0 2 4 6 8 10 12

0 10 20 30 40 50 60

Time (min) Di sappe ara nce rate of PL ( v

, nm ol/m in )

B) Jejunal lumen

0 2 4 6 8 10 12

0 10 20 30 40 50 60

Time (min) Di sappe ara nce rate of PL ( v

, nm ol/m in )

B) Jejunal lumen

0 2 4 6 8 10 12

0 10 20 30 40 50 60

Time (min) Appe ara nce ra te of PL ( v

, nm ol /m in)

A) Mesenteric vein

0 2 4 6 8 10 12

0 10 20 30 40 50 60

Time (min) Appe ara nce ra te of PL ( v

, nm ol /m in)

A) Mesenteric vein

Fig. 6 Absorption of PL in the Rat Jejunal Single-pass Perfusion.

A) An appearance rate of PL in the mesenteric vein (v

); B) a disappearance rate of PL in the

jejunal lumen (v

). Rat jejunal loop (10 cm) was perfused with 300 μM PL. The perfusion flow

rate was 2.8 and 0.3 mL/min for the mesenteric vein and jejunal lumen, respectively. Each point

represents mean ± S.D. (n = 3).