3Faculty of Pharmaceutical Sciences, Tokyo University of Pharmacy and Life Science, Tokyo, ... 4College of Pharmacy, University of Michigan, Michigan, USA.
Drug Metab. Pharmacokinet. 22 (1): 26–32 (2007).
Regular Article Characteristics of Gastrointestinal Absorption of DX-9065a, a New Synthetic Anticoagulant Yoshimine FUJII1,*, Masayuki TAKAHASHI1, Hiromi MORITA2, Hiroshi KIKUCHI1, Yukihiko ARAMAKI3 and Gordon L. AMIDON4 1Drug
Metabolism & Physicochemistry Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan 2Chemical Technology Research Laboratories, Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan 3Faculty of Pharmaceutical Sciences, Tokyo University of Pharmacy and Life Science, Tokyo, Japan 4College of Pharmacy, University of Michigan, Michigan, USA Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk
Summary: DX-9065a, a newly synthesized anticoagulant that selectively inhibits factor Xa, is a zwitterion and has characteristics of high water solubility and low lipophilicity. We predicted the fraction absorbed (Fa) of DX-9065a to be approximately 15–35z in humans, based on the boundary layer theory using the intestinal perfusion method in rats. However, human oral bioavailability was 2–3z in clinical trials, and the result of actual human bioavailability was lower than that of the predicted Fa. Thus, in this report, the reason for low oral bioavailability of DX-9065a was examined by in vitro and in vivo experiments. The factors aŠecting oral bioavailability of DX-9065a were not the hepatic ˆrst-pass eŠect, degradation of the drug in intestinal ‰uid, nor the interaction of the drug with the intestinal mucin. Furthermore, no eŠect of P-gp eŒux was observed. Oral absorption of the drug in rats with bile duct ligation was signiˆcantly higher than that in normal rats with bioavailability of 17 and 3z, respectively. It was conˆrmed that bile acids inhibited DX-9065a absorption because DX-9065a interacted with bile acids to form insoluble complexes. The results suggest that the complex formation of DX-9065a with bile acids in the intestinal tract is an important factor aŠecting absorption of DX-9065a.
Key words: DX-9065a; anticoagulant; factor Xa inhibitor; membrane permeability; rat; bioavailability; bile acid Introduction Warfarin and heparin are widely used as clinical antithrombotic therapy, however, their adverse eŠects limit the clinical use of these agents. When low molecular weight heparin was tested as an oral anticoagulant, no detectable anticoagulant activity was observed following oral administration in humans.1) Argatroban,2) a direct thrombin inhibitor can also only be administered by injection. DX-9065a (Fig. 1), a newly synthesized anticoagulant, selectively inhibits factor Xa3) and shows an anticoagulant eŠect following oral administration to rats.4,5) DX-9065a has three polar groups in its molecule, that is, an amidino group, an acetoimidoyl group and a carboxyl group. DX-9065a has high water solubility (above 600 mg W mL) and low lipophilicity (log D6.8＝ －3.0). DX-9065a might only be absorbed via the
Fig. 1.
Chemical structure of DX-9065a.
paracellular pathway, since DX-9065a exists in the ionized form under intestinal pH conditions. Furthermore, it must have low a‹nity for lipid membranes because of its low lipophilicity. Based on these characteristics, high oral bioavailability is not expected for DX-9065a.6) However, some types of cephalosporins, such as cephalexin, exhibit high bioavailability despite their being zwitterion compounds, because their absorp-
Received; May 25, 2006, Accepted; July 12, 2006 *To whom correspondence should be addressed : Yoshimine FUJII, Drug Metabolism & Physicochemistry Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo, 134–8630, Japan. Tel. ＋81-3-3680-0151, Fax. ＋81-3-5696-8228, E-mail: fujiic4b＠daiichipharm.co.jp
26
Characteristics of Gastrointestinal Absorption of DX-9065a
tion might involve carrier-mediated transport.7) With drug development, it is important to predict human oral drug absorption before starting clinical trials. Amidon et al. established an useful method to predict the human fraction absorbed based on the boundary layer theory using rat intestinal perfusion.8) In the present study, we predicted human oral bioavailability of DX-9065a using this method, and elucidated the DX-9065a absorption mechanism by in vitro and in vivo experiments. Materials and Methods Materials: DX-9065as(＋)-(2S)-2-[4-[[(3S)-1-acetoimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-[7-amidino-2naphtyl]propanoic acid hydrochloride pentahydratet, and (2RS)-(3-(7-amidinonaphth-2-yl)-2-(4-(1-ethyl-carbon-imidoylpiperidin-4-yloxy) phenyl)) propionic acid were synthesized at Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan), and the latter was used as the internal standard for high-performance liquid chromatography (HPLC) analysis. [14C] PEG-4000 was purchased from DuPont NEN (Boston, MA, USA). Bile salts (cholate, deoxycholate, taurocholate, glycocholate, chenodeoxycholate, taurodeoxycholate) were purchased as sodium salts from Sigma Chemical Co. (St. Louis, MO, USA). HIONIC-FLUOR, a scintillation cocktail, was purchased from Packard Instrument Company, Inc. (Meriden, CT, USA). All other materials and solvents were of analytical or the highest grade available. Animals: Male Sprague-Dawley rats weighing 230–400 g were fasted for 16 h prior to experiments, but had free access to water. All animal experiments were conducted with the approval of the Animal Experiment Ethics Committee of Daiichi Pharmaceutical Co., Ltd. and the University Committee on the Use and Care of animals of the University of Michigan. Rat intestinal single-pass perfusion: Rats intestinal perfusion was performed according to the method of Hu et al.10) Brie‰y, after washing the cannulated jejunum (15 cm) and ileum (15 cm) with 15 mL of saline, the jejunum and ileum segments were perfused with 0.1 and 1 mM of DX-9065a dissolved in 10 mM MES buŠer solution of pH 6.5 or 10 mM phosphate buŠer solution of pH 7.5, containing 140 mM sodium chloride, 5 mM potassium chloride and 0.01z PEG-4000 with a tracer amount of [14C] PEG-4000, at a ‰ow rate of 0.123 mL W min using the infusion pump. Following perfusion for 30 min to attain to the steady state, the perfused solution was collected in 10 min intervals for a total of 60 min. The DX-9065a concentration in the pefrusate was measured by the HPLC method, and the [14C] PEG-4000 radioactivity was measured with a liquid scintillation counter (Model LS 6000TA, Beckman, Fullerton, CA, USA). Prediction of human Fa: Unbiased membrane
27
parameters are estimated using a boundary layer model developed by Amidon et al.8,9) The regular permeability parameters are converted to dimensionless ones by multiplying R W D, where R is the radius of the intestine, and D is an estimate of the aqueous diŠusion coe‹cient of a solute in the perfusate. The dimensionless eŠective permeability, P*eŠ, and intrinsic wall permeability, P*w, were calculated based on their theory. Brie‰y, P*eŠ is calculated based on the results of the rat intestinal perfusion experiment:
P*eŠ＝
1－ C m W C0 4Gz
(1)
where C0 and Cm are inlet and outlet perfusate concentration, respectively, and Cm was compensated by [14C] PEG-4000 concentration. Graetz number, Gz, is
Gz＝
pDL 2Q
(2)
where L is the length of the intestine, and Q is the ‰uid ‰ow rate. The aqueous permeability, P*aq, can be estimated from the ˆlm model approximation to the boundary layer result: *
Paq－1＝AGz1 W3
(3)
0.004ÃGzÃ0.01
A＝10Gz＋1.01
(4)
0.01ºGzÃ0.03
A＝4.5Gz＋1.065
(5)
0.03ºGz
A＝2.5Gz＋1.125
(6)
P*w is then calculated as follows: P*w＝
P*eŠ 1－P*eŠ W P*aq
(7)
The solution for the ratio of outlet (Cm) to inlet (C0) concentration shows the fraction dose absorption (Fa):
Fa＝1－
Cm C0
(8)
Equation (8) can be converted to the following:
Fa＝1－e－4GzP*eŠ
(9)
Gz of the human intestine is estimated to be approximately 0.5, the intestinal length (L) is 500 cm, the ‰ow rate (Q) is 0.5 mL W min, and the water phase diŠusive min. constant (D) of 3×10－4 cm2 W Consequently, Eq. (9) becomes Fa＝1－e－2P*eŠ
(10)
If the assumption is made that P*aq is not limiting, Eq. (10) becomes *
Fa＝1－e－2Pw
(11)
Intravenous and portal vein administration to rats: Rats were anesthetized with sodium pentobarbital (30
28
Yoshimine FUJII, et al.
mg W kg) by intraperitoneal injection. Following midabdominal incision, DX-9065a solution in saline (0.1 mg W 0.2 mL W head) was administered by injection into the left jugular vein or the portal vein. Blood samples were withdrawn at 1, 5, 15, 30, 60, 120 and 240 min after administration from the right jugular vein under ether anesthesia. Stability in the intestinal ‰uid: Intestinal ‰uid was collected from the small intestine of rats using the method of Takada et al.11) with some modiˆcations. Rat small intestine was removed under ether anesthesia, and 20 cm of the jejunum was washed out with saline. For stability experiments, 0.5 mL of a DX-9065a saline solution (0.5 or 5 mg W mL) was added to 4.5 mL of the intestinal ‰uid or 50 mM phosphate buŠer (pH 6.8) and C for up to 180 min. Two hundred incubated at 379 microliter samples were then withdrawn from the incubated mixture, and stored at 49C until HPLC analysis. Everted and non-everted sac experiment: Everted sac experiment was performed according to the method of Niibuchi et al.12) with some modiˆcations. Brie‰y, the cannulated small intestine was then perfused with modiˆed Ringer's solution12) at a rate of 2.0 mL W min for 60 min. Intestinal mucus was removed by perfusion with Ringer's solution containing 0.02z sodium deoxycholate (w W v). Following surgical excision of a 20 cm portion of small intestine from 10 cm below the pylorus, the everted or non-everted intestine was applied to Wiseman's perfusion apparatus, which was maintained at 379 C or 49C, and 60 mL of DX-9065a Ringer's solution (0.42 mg W mL) was added to mucosal side and 20 mL of Ringer's solution to the serosal side, respectively. In the experiments examining the eŠect of sodium deoxycholate perfusion on DX-9065a transport, a 0.042 mg W mL of DX-9065a solution was used. Both sides of the intestinal membrane were gassed with 5z CO2 in oxygen. After perfusion, a 200 mL portion of perfusate was withdrawn from the serosal side, and stored at 49 C until HPLC analysis. Rat oral bioavailability study: Rat bile duct ligation was prepared using the following method. Rats were anesthetized with ether, and midabdominal incision was performed. The bile duct was ligated with surgical thread, and the abdomen was then closed. After recovering from anesthesia, rats were allowed a recovery period of approximately 1 h. DX-9065a (0.5 mg W 0.5 mL W head in saline solution) was orally administered to normal rats and rats ligated bile duct by gastric tube. Blood samples were taken at 0.5, 1, 2, 4 and 8 h after administration from the jugular vein under ether anesthesia. Bioavailability of DX-9065a after oral administration was calculated from the AUC ratio after oral and intravenous administration of DX-9065a. Equilibrium dialysis: DX-9065a was dissolved in
isotonic phosphate buŠer (pH 6.8) at a concentration of 0.2–20 mg W mL, and the bile components were dissolved in the same buŠer at a concentration of 20 mg W mL. Then 0.4 mL of DX-9065a buŠer solution was added to 0.4 mL of each bile component solution and dialyzed C for 20 h. against the same buŠer solution at 379 HPLC assay: DX-9065a concentration in plasma, perfusate, and samples from equilibrium dialysis was assayed by HPLC. HPLC (Waters 600E system, Milford, MO, USA,) was equipped with ultraviolet (UV) detector (Model 484, Waters) set at 240 nm and a reversed-phase column (L-column ODS, 4.6 mm i.d.× 150 mm length, Chemicals Inspection and Testing Institute, Tokyo, Japan) was used. The mobile phase consisted of pH 6.8 phosphate buŠer and acetonitrile (100:14) containing 5 mM octylamine. Samples were min. The DX-9065a coneluted at a ‰ow rate of 1.0 mL W centration was determined from the peak area using a calibration curve. Solid-phase extraction method was used to clean plasma samples suitable for HPLC analysis. A Sep-Pak Plus C18 Cartridge (Waters) was conditioned with 10 mL of methanol and 10 mL of distilled water. A mixed solution of 0.2 mL of the rat plasma, 0.1 mL of the internal standard solution and 5.7 mL distilled water was applied to the column. After washing with 10 mL distilled water, DX-9065a and IS were eluted with 6 mL of methanol containing 1z acetic acid. The elution medium was evaporated with a centrifuge evaporator. The dried residue was redissolved with 100 mL of the mobile phase, and 20 mL of the solution was injected into the HPLC. Data analysis: Pharmacokinetic parameters were calculated using the SAG-CP statistical analysis and graphics program (ASMedica, Tokyo, Japan) for data obtained following oral administration. Results Estimation of human oral absorption: The intrinsic wall permeability (P*w) obtained from rat intestinal single-perfusion experiments is shown in Table 1. The P*w values were within a range of 0.07 to 0.21 in the jejunum and ileum, and the predicted fraction absorbed (Fa) of DX-9065a calculated from Eq. (11) was approximately 15–35z in humans. In‰uence of hepatic ˆrst-pass eŠect on DX-9065a absorption: To examine the low bioavailability of DX-9065a, we focused on the hepatic ˆrst-pass eŠect for oral absorption of DX-9065a. After intravenous injection, the plasma concentration-time proˆle of DX-9065a was compared with that of portal vein administration in rats (Fig. 2). Plasma DX-9065a concentration-time proˆles for intravenous and portal vein administration were similar at all sampling times, suggesting that the bioavailability of DX-9065a is not
Characteristics of Gastrointestinal Absorption of DX-9065a Table 1. intestine
29
Intrinsic wall permeability (P*w) of DX-9065a in rat
Jejunum
Ileum
DX-9065a concentration
pH 6.5
pH 7.5
pH 7.5
0.1 mM 1 mM
0.206±0.119 0.067±0.053
0.102±0.066 0.122±0.088
0.104±0.061 0.080±0.029
Data are shown as the mean±S.E., n＝4.
Fig. 3. Stability of DX-9065a in rat intestinal ‰uid at 379C at 50 () mL of DX-9065a concentration. The data is expressed and 500 () mg W as a percentage of the initial value. Each value represents the mean± S.E. (n＝3).
Fig. 2. Plasma concentration proˆles after intravenous () or head) in rats. Each portal vain () injection of DX-9065a (0.1 mg W value represents the mean±S.E. (n＝5).
aŠected by the hepatic ˆrst-pass eŠect. Intestinal metabolism was also evaluated by incubation of DX-9065a with the intestinal homogenate. As the result, no metabolism was observed in 30 min (Data not shown). Stability of DX-9065a in intestinal ‰uid: The stability of DX-9065a against digestive enzymes was examined by incubating DX-9065a in intestinal ‰uid at 379 C. As shown in Fig. 3, no degradation of DX-9065a was found with incubation in intestinal ‰uid for up to 180 min. In vitro intestinal perfusion: The permeability of DX-9065a in isolated intestine with and without washing oŠ intestinal mucus by deoxycholate perfusion was determined, to examine the interaction between DX-9065a and mucus components. The intestinal permeability of DX-9065a did not change, irrespective of washing oŠ mucus or not (Fig. 4). In an attempt to clarify the mechanism of transport of DX-9065a, permeability across everted and noneverted intestine was determined at 379C. As shown in Fig. 5, there were no diŠerences in time course of DX-9065a transport across everted and non-everted intestine. No changes in DX-9065a transport across the intestine were observed under the condition of 49 C, either. In vivo absorption in rats with bile duct ligation:
Fig. 4. The permeability of DX-9065a across everted intestine perfused with () or without () 0.02z deoxycholate. Each value represents the mean±S.E. (n＝3).
Plasma concentration-time proˆles of DX-9065a after oral administration to rats with or without bile duct ligation are shown in Fig. 6, and the pharmacokinetic parameters are summarized in Table 2. AUC after intravenous administration of DX-9065a (0.1 mg W head) was 1713 ng･h W mL in rats (average, n＝5). The absolute bioavailability of DX-9065a after oral administration was approximately 3z in normal rats. Absorption was signiˆcantly higher in rats with bile duct ligation than in normal rats, and Cmax and AUC were approximately 2.5-fold and 6-fold greater, respectively, in the former than the latter. Approximately 17z bioavailability was obtained in rats with bile duct ligation. Interaction between DX-9065a and bile components: The interaction between DX-9065a and bile components was examined in the equilibrium dialysis experiment. In a preliminary experiment, it was conˆrmed that the time to attain equilibrium of DX-9065a concentration was
30
Yoshimine FUJII, et al.
over 16 h at 379C, and that no degradation during incubation, or adsorption to the apparatus, occurred under the conditions used in the present experiment (data not shown). As shown in Fig. 7, the equilibrium
Fig. 5. The permeability of DX-9065a across everted intestine (), non-everted intestine () at 379C, and everted intestine at 49C ($). The data is expressed as the cumulative amount of DX-9065a that crossed the intestinal membrane. Each value represents the mean± S.E. (n＝3).
concentration of DX-9065a was decreased in the presence of rat bile and almost all of the bile acids. An especially large decrease in DX-9065a concentration was observed with the addition of deoxycholate derivatives, such as deoxycholate, chenodeoxycholate and taurodeoxycholate. Moreover, the magnitude of decrease was increased with increasing DX-9065a
Fig. 6. Plasma concentration proˆles after oral administration of head) in normal () and bile duct ligated () rats. DX-9065a (0.5 mg W Each value represents the mean±S.E. (n＝7).
Fig. 7. The eŠect of bile components (10 mg W mL) on the equilibrium concentration of DX-9065a after 20 h of dialysis at 379C. DX-9065a mL: , 1 mg W mL: , 10 mg W mL: . Values are percentages of the equilibrium concentration of DX-9065a. Each value concentration 0.1 mg W represents the mean±S.E. (n＝3). Table 2.
PK parameters after oral administration of DX-9065a (0.5 mg W head) in normal and bile duct ligated rats.
Normal rat Bile duct ligated rat
AUC0–8 h ng･h W mL
Cmax ng W mL
MRT h
Bioabailability z
241±90 1405±156*
121±36 304±32
1.3±0.3 3.8±0.2
2.8±1.1 16.4±1.8
Data are shown as the mean±S.E., n＝7. *pº0.01, signiˆcantly diŠerent from normal rat.
Characteristics of Gastrointestinal Absorption of DX-9065a
concentration. With deoxycholate, chenodeoxycholate and taurodeoxycholate, precipitation occurred in the presence of 10 mg W mL DX-9065a. Discussion DX-9065a is a zwitterion compound that has a carboxyl group and two amino groups; pKa: 3.3, 11.2 and 13.3. Therefore, DX-9065a exists in ionized form at intestinal pH, indicating that DX-9065a does not have a high degree of membrane permeability. The low lipophilicity of DX-9065a makes it far from suitable for intestinal absorption.13) The molecular weight, lipophilicity and bioavailability of DX-9065a are similar to those of certain peptide drugs such as thyrotropinreleasing hormone (TRH) analogues.14) The low oral bioavailability of azetirelin, a TRH analogue, was due to its high degree of hydrophilicity rather than to degradation in the gastrointestinal tract.15) It thus appears that the physicochemical properties of DX9065a are in part responsible for its poor intestinal permeability. The boundary layer theory is very useful for prediction of the human oral bioavailability of compounds that are absorbed not only by passive diŠusion, but also by active transport.9) In this study, we attempted to predict the oral bioavailability of DX-9065a using the method of rat intestinal perfusion. The results of this experiment suggested that the predicted Fa of DX-9065a after oral administration was approximately 15–35z in humans, however, the actual bioavailability (2–3z in clinical trials) was signiˆcantly lower than the predicted value. These ˆndings suggest that gastrointestinal absorption of DX-9065a is inhibited by a certain factor. The following factors were considered: 1) rate-limited dissolution, 2) the ˆrst-pass eŠect, 3) degradation in the gastrointestinal tract, 4) binding with the intestinal mucus, 5) in‰uence of the eŒux system, P-gp etc., 6) interaction with components of intestinal ‰uid. Factor 1) was negligible, because of the high solubility of DX-9065a. Consequently, the other inhibiting factors of DX-9065a absorption were evaluated in detail. The plasma concentration proˆles of DX-9065a following intravenous and portal vein injection were compared to estimate the hepatic ˆrst-pass eŠect. The mean plasma concentration proˆles obtained following diŠerent administration routes showed no diŠerences. This ˆnding demonstrated that the low bioavailability of DX-9065a was not due to the hepatic and intestinal ˆrst-pass eŠect. DX-9065a was not degraded in rat intestinal ‰uids within 3 h. Furthermore, DX-9065a did not interact with the intestinal mucin. Levine and Pelikan reported that quaternary ammonium compounds interacted with intestinal mucin and formed a nonabsorbable complex.16) In addition, Niibuchi et al. found that the intestinal absorption of gentamicin and
31
cephaloridin was signiˆcantly increased in intestine treated with surfactant to wash oŠ the intestinal mucin.11) In the present study, intestinal absorption of DX-9065a was not increased following washing oŠ the mucin by surfactant treatment. These ˆndings indicated that degradation in the gastrointestinal tract and interaction with the intestinal mucin did not aŠect oral absorption of DX-9065a. Consequently, factors 2), 3) and 4) did not attribute to the low bioavailability of DX-9065a. To clarify the mechanism of membrane permeability of DX-9065a, in-vitro perfusion experiments using rat jejunum were performed. No signiˆcant diŠerences were observed in the permeability of DX-9065a between everted and non-everted intestine. The permeability of DX-9065a in everted intestine was not altered, even at a low temperature (49C), either. It was conˆrmed that DX-9065a transport across the small intestine was not saturated at the tested concentration range. These ˆndings suggest that the mechanism of intestinal absorption of DX-9065a was passive diŠusion, and that it was not aŠected by the eŒux systems, P-gp etc. Consequently, factor 5) was not considered to be a factor of low bioavailability of DX-9065a. Bile salts are known to increase the intestinal absorption of some poorly water-soluble drugs.17) In contrast, the bioavailability of DX-9065a after oral administration was enhanced in rats with bile duct ligation. This ˆnding suggests that oral absorption of DX-9065a is inhibited by bile. A few studies have reported a reduction in drug absorption due to bile. Neomycin and kanamycin were reported to form precipitates with bile.18) Furthermore, it has been reported that the absorption of b-adrenergic blocking agents such as nadolol19) and atenolol20) is inhibited by bile salts. In an attempt to elucidate the details of the interaction between DX-9065a and bile salts, equilibrium dialysis experiments were performed. A reduction in the equilibrium concentration of DX-9065a was observed in the presence of bile acids. It is possible that macromolecular complexes were formed by the intermolecular interaction between DX-9065a and bile acid, since they could not across the cellulose membrane used in these experiments. In conclusion, the actual human bioavailability was lower than the predicted human Fa. Our ˆndings indicate that the low oral bioavailability of DX-9065a is due to interaction with bile components in the intestinal tract. These ˆndings suggest that human oral bioavailability of DX-9065a may be enhanced by inhibition of the interaction of DX-9065a and bile acids in the intestinal tract. References 1)
Dryjski, M., Schneider, D. E., Mojaverian, P., Kuo, B.
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