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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Department of Pharmaceutics, School of Pharmaceutical Science, Peking University Health Science Center, Beijing, People's Republic of China
Received July 5, 2003; accepted November 5, 2003.
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Abstract |
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). The dissociation constant of the hirudin-thrombin complex was determined to be as 6.3 x 10–11mol/l (Chang, 1983). Hirudin holds many advantages over the commonly used anticoagulants such as heparin. It does not need a cofactor for the interaction with the enzyme; neither is it inactivated by platelet factors. Also, it has no toxicity or antigenicity. These characteristics make hirudin a promising candidate for proteinase inhibitor therapy in clinical states associated with thrombosis and disseminated intravascular coagulation (Nowak and Markwardt, 1991). It has become possible to produce biologically active peptide and proteins that are therapeutically applicable such as hirudin by means of recombinant DNA technique. Generally, recombinant hirudin has three variants: rHV1, rHV2, and rHV3. All of them have a high degree of homology (13 variable positions only), but in contrast to the natural molecule they lack a sulfate group at Tyr63. They all have a similar effect of anticoagulation as natural hirudin (Dolt et al., 1985; Scharf et al., 1989; Vindigni et al., 1994). Most studies on hirudin are concerned with the pharmacological evaluations and chose the intravenous injection as the administration route. In clinic, frequent injections of hirudin are needed for the purpose of therapy; therefore, patients experience great discomfort. To improve the quality of life of such patients, an alternative method of administration is desired. Oral administration is the most convenient route, and development of an oral formulation for hirudin would be of great value. rHV2 has been proved has the effect of anticoagulation when administered to the duodenal of rats (Wang et al., 1999). But there is no information on the GI absorption of rHV2 administered by direct oral administration. The oral delivery of biologically active peptides and proteins is a very attractive but tough job. There are two main barriers to the GI absorption of peptide drugs: one is the poor membrane permeability that results from their macromolecular weight and hydrophilic characteristics, and another is the degradation of peptides by proteases in the GI lumen and mucosa (Lee, 1990). Scientists have made numerous attempts in this field for decades and investigated methods to enhance the absorption of peptides and proteins, as well as the mechanisms of their absorption. In the studies reported, most of the model drugs are small peptides containing fewer than five amino acids (Berteloot et al., 1981; Shen and Xu, 2000), or endogenous peptides and proteins, such as insulin (Cheeseman and Johnston, 1982; Stoll et al., 2000), insulin like growth factor I (Due et al., 1985; Toshikiro et al., 1996; Xu and Wang, 1996; Donovan et al., 1997; Hosny et al., 1997; Nakagawa et al., 1997; Uchiyama et al., 1999) and epithelial growth factor (Rao et al., 1990; Adachi et al., 1993; Shen and Xu, 1998). Few of the studies were concerned with exogenous therapeutic peptides and proteins with large molecular weight besides calcitonin (Tozaki et al., 1998) and cyclosporin A (Drewe et al., 1992; Augustijns et al., 2000). Although many approaches have been made to enhance the absorption of peptides and proteins, the absorption mechanisms of biologically active peptides and proteins in the GI tract have not yet been firmly established. In the present article, we chosen rHV2 as the model drug and rats as the model animals and then studied the possibility of oral administration of rHV2 and the possible mechanisms of its GI absorption. The improvements of absorption in rats after oral administration of rHV2 by using several protease inhibitors were also investigated. |
Materials and Methods |
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-cyclodextrin (HP--CD) was supplied by Xi'an Deli Biological and Chemical Engineering Co., Ltd. (Xi'an, China). 2,4-Dinitrophenol (DNP) was purchased from Beijing Chemical reagent factory (Beijing, China). All other chemicals were analytical grade and used as received. Animals. Male Sprague-Dawley rats weighing 270 to 300 g were provided by Vital Laboratory Animal Center (Beijing, China). All care and handling of animals were performed with the approval of Institutional Authority for Laboratory Animal Care.
Preparation of Luminal Contents and Mucosal Subcellular Fractions. The luminal contents of the GI tract were prepared according to the method of Asada et al. (1994). Rats were euthanized with an overdose intraperitoneal injection of urethane, and the GI tract was excised. The luminal contents of the stomach were collected by flushing with 15 ml of artificial gastric juice (pH 1.2), and the contents of the jejunum (20 cm below the ligament of Treitz), proximal and distal part of ileum (40 cm above the ileocecal junction, each segment was 20 cm long), were collected by flushing with 15 ml of phosphate-buffer solution (PBS, pH 7.4). Mucosal subcellular fractions of the small intestine were prepared according to the method of Bai and Chang (1995), with slight modification. The intestinal mucosa of each segment was scraped off, suspended in 0.3 M sucrose buffer (pH 7.0), and then homogenized. The homogenate was centrifuged at 100,000g at 4°C for 1 h to separate each fraction. The resulting supernatant (15 ml) was used as the cytosol fraction, and the pellet was divided into two fractions. One was resuspended with 15 ml of sucrose buffer (0.3 M, pH 7.0) as a brush-border membrane fraction, whereas the other was resuspended with 15 ml of acetate/NaOH buffer (1 M, pH 4.5) as a lysosomal fraction.
Degradation Studies. The degradation of 125I-rHV2 in the luminal contents and mucosal subcellular fraction was examined according to the method of Bai and Chang (1995). The incubation mixture consisted of 50 mM Tris/HCl buffer (pH 7.5) for luminal content of small intestine, or artificial gastric juice (pH 1.2) for luminal content of stomach, or 1 M acetate/NaOH buffer (pH 4.5) for subcellular fraction, as well as 125 mM NaCl, 1% BSA, 8 µg/ml of 125I-rHV2, some adjuvant (0.8 mg/ml of bacitracin, 10 mg/ml of casein, 20 mg/ml of HP--CD, or 2.4 mg/ml SDCh) and a luminal content or a subcellular fraction. The experiment of 125I-rHV2 degradation was performed at 37°C. Samples were taken at 0, 1, 5, 10, 15, and 30 min and determined by trichloroacetic acid (TCA)-precipitable method.
Oral Administration Experiments. Rats were fasted for 16 h before the experiments, with free access to water. Oral administration of 125I-rHV2 alone or with bacitracin or casein was carried out by using a curved bulb-ended gavage tube. 125I-rHV2 dissolved in saline solution was administered to rats at the dose of 1.0 mg/kg. When coadministration with bacitracin or casein their doses were 4.0 and 10.0 mg/kg, respectively. The animals were anesthetized with an intraperitoneal injection of urethane (1.5 g/kg). Blood samples (0.5 ml) were periodically taken from the orbit venous plexus at 0, 15, 30, 60, 120, 240, and 360 min using a heparinized capillary tube and estimated by TCA-precipitable method. In another experiment, unlabeled rHV2 was administered to rats at doses of 5.0 mg/kg, whereas bacitracin and casein were 4.0 and 10.0 mg/kg, respectively. The blood sample was anticoagulated with 3.8% trisodium citrate solution in a ratio of 8.25:1.75 (v/v), and the rHV2 in the plasma was determined by chromogenic assay. For the calculation of bioavailability in the two sets of experiments, 125I-rHV2 (0.5 mg/kg) and rHV2 (1.0 mg/kg) solution were given intravenously to rats by tail veins, respectively.
In Situ Loop Experiments. The rats were anesthetized with an intraperitoneal injection of urethane (1.5 g/kg). Anesthesia was maintained with additional urethane as needed throughout the studies. The absorptions of 125I-rHV2 from the jejunum, and proximal and distal part of ileum were examined by an in situ loop method (Bai and Chang, 1995). Having been washed out the luminal contents, the anesthetized rats were administered the drug solution at the same doses as those used in the oral administration study. Blood samples (0.5 ml) were periodically taken from the orbital venous plexus at 0, 15, 30, 60, 120, 240, and 360 min and determined by TCA-precipitable method.
In Vitro Everted Sac Experiments. Rats were euthanized with an overdose of intraperitoneal injection of urethane. The segment of jejunum was excised after washing the luminal side with 20 ml of PBS (pH 7.4) (Toshikiro et al., 1996). The isolated jejunum was everted and cut into 5-cm segments. Half a milliliter of the PBS was introduced into the serosal side, and both ends of the segment were ligated. The resulting everted sac was placed in the 10 ml of PBS (pH 7.4) incubation media, which contained 100 µg/ml rHV2 and 10 mg/ml casein with or without adjuvant (1 mM poly-L-lysine, 1 mM carbopol 941, 1 mM DNP, or 0.1 mM colchicine) and was bubbled with 95% O2, 5% CO2 throughout the experiment. The incubation was performed for 30 min at 37°C, and then the everted sac was twice immersed in 20 ml of the fresh PBS (pH 7.4, 4°C) for 5 min to remove the nonspecifically adsorbed rHV2 on the mucosal surface. To remove the rHV2 bound on the membrane, the everted sac was immersed in the ice-cold basic washing solution (0.05 M NaOH/0.5 M NaCl, 10 ml) for 10 min. After recovery of the serosal solution, the tissue was homogenized and mixed with 4% acetic acid. The mixture was centrifuged at 2000g for 10 min. The rHV2 in the resulting supernatant and in the serosal solution were determined by chromogenic assay.
Assay Procedures. The 125I-rHV2 in the blood or GI luminal contents was determined by TCA precipitation method (Toshikiro et al., 1996). For GI luminal contents, 200 µl of 15% TCA was added to 200 µl of the incubation mixture, as for plasma, 1 ml of 15% TCA was added to the mixed solution after 1 ml of Krebs-Henseleit bicarbonate buffer (119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3) containing 5% BSA was added to 50 µl of each plasma sample. Afterward, the mixtures were centrifuged at 2000g for 15 min and then the radioactivity in the precipitate was counted by a gamma-counter (SNB-69513; Rihuan Instrument Factory, Shanghai, China).
Chromogenic assay was performed according to Groetsch et al. (1991) with a little modification. The blood samples were denatured by heating for 15 min at 65°C and then 0.05 ml of HCl (1 M) was put into 0.5-ml blood sample. The samples were neutralized after cooling by addition of 0.05 ml NaOH (1 M) and then centrifuged at 3000g for 10 min. Supernatant (100 µl) was added to 200 µl of Tris buffer (50 mM, containing 154 mM NaCl and 0.12 U of thrombin), and incubated at 37°C for 5 min. Afterward, 16 µl of chromozym TH (1.9 mM) was added into the incubation mixture and kept on incubating for another 10 min at 37°C. The reaction is terminated by addition of 200 µl of acetic acid (33%) and then the absorbance of the sample was read at 405 nm with a spectrophotometer (TU-1901; Puxi Versatile Instrument Co., Beijing, China).
Data Analysis. The plasma concentration of rHV2 was calculated by dividing the total volume of plasma isolated. The plasma concentration-time data were analyzed noncompartmentally on the basis of the statistical moment theory. The area under the curve0
360 was estimated by the trapezoidal method and the bioavailability was calculated by comparing the area under the curve0360 obtained after intraintestinal or oral administration to that obtained after intravenous injection. Statistical evaluations were performed using the Student's t test. Differences with a p value less than 0.05 were considered significant.
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Results |
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Table 1 also shows the degrading activity in the intestinal mucosa. Degrading activity in a subfraction of the intestinal mucosa is nearly the same as in GI contents, except that the activity in the cytosol fraction is a little lower than in other parts of the subfraction. There was no site difference through the entire small intestine.
Oral Administration Experiments. The plasma concentration-time curves of TCA-perceptible radioactivity after oral administration of 125I-rHV2 (1.0 mg/kg) with or without bacitracin (4.0 mg/kg) and casein (10.0 mg/kg) are shown in Fig. 2. The bioavailability of rHV2 without adjuvant was 21.23 ± 3.73%. When coadministered with bacitracin or casein, the bioavailability was increased to 37.41 ± 3.55% (p < 0.05) and 29.23 ± 3.08% (p > 0.05), respectively.
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To further confirm the above-mentioned absorption results obtained from the 125I-rHV2 oral administrations, chromogenic assay of rHV2 in the plasma was also performed after oral administration of unlabeled rHV2. The plasma concentration-time curves obtained are shown in Fig. 3. The bioavailability values of rHV2 administered alone, with bacitracin or casein were 6.99 ± 0.32%, 12.70 ± 1.00% (p < 0.01), and 8.36 ± 1.02% (p > 0.05), respectively.
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In Situ Loop Experiments. The site difference of rHV2 absorption in the intestine was also examined by an in situ loop method. There was no significant variance of absorption in different segments of small intestine. The bioavailabilities of rHV2 in various parts of the intestine are expressed in Fig. 4, and the values range from 6.33 to 8.98%. The rank order of the absorption was proximal ileum 
distal ileum > jejunum with no statistical differences, whether with or without adjuvant (p > 0.05).
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In Vitro Everted Sac Experiments. To clarify the transport mechanism of rHV2 across the GI tract, we examined the effect of variant inhibitors on the uptake and transport of rHV2 across the jejunal mucosa. The results are summarized in Table 2. The transport of rHV2 to the serosal side was significantly inhibited by low temperature, DNP (an uncoupler of oxidative phosphorylation), carbopol 941 (a polyanion), and colchicines (an inhibitor of microtubular assembly), but not by poly-L-lysine (a polycation). The amount of rHV2 found in the intestinal tissue was also decreased, and the total amount of rHV2 taken in and transported across the GI tract tended to be inhibited by these treatments, except for the addition of poly-L-lysine.
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Discussion |
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). Wang et al. (1995) also proved that rHV2 retained most of its anticoagulation activity after incubated with trypsin, pepsin, or chymotrypsin, but it can be degraded by pepsin. In the above-mentioned studies only one kind of protease acts with hirudin in each experiment; however, the effects of various digestive enzymes in the GI tract should be more complex, especially in small intestine. Therefore, the luminal contents and mucosal subcellular fractions were used in our investigation to simulate the complicated surroundings in GI tract.
It was indicated from the degradation experiments that rHV2 was degraded rapidly by the GI tract luminal contents and mucosal subcellular fraction. The rHV2 concentration decreased below 50% of the initial within 30 min. The significant inhibitory effects of bacitracin and casein on rHV2 degradation have also been noticed. Xian et al. (1995) reported that casein can protect IGF-I from degradation in the stomach or duodenal flushing, but the mechanisms of this protection are not clear. The inhibitory effect of HP--CD and SDCh are relatively weak compared with bacitracin and casein, although some studies suggested that DM--CD and SDCh have the ability of inhibiting the enzyme activities (Kakemi et al., 1970; Hovgaard and Brondsted, 1995). Regardless, it seems possible to use some protease inhibitors to protect rHV2 from the degradation by digestive enzymes in GI tract.
The bioavailability of oral administration of 125I-rHV2 in rats is 21.23 ± 3.73%, estimated by TCA-precipitable method, which is not consistent with the value of 6.99 ± 0.32% determined by chromogenic assay. The variance was due to the different methods used and to the two methods' advantages and drawbacks. TCA-precipitable method is more sensitive and could prove the transport of the intact rHV2 molecules into the body circulation, whereas the chromogenic assay could demonstrate at least the absorption of biologically active segments of rHV2 molecules into the blood of the animals. With coadministration of rHV2 with bacitracin or casein, the bioavailability was increased markedly, no matter which of the two analytical methods was applied.
The results of in situ loop indicate that the absorption of rHV2 is more slow than in that of the oral administration. This finding may be related to the route of administration and the state of the animals. In the perfusion process, the drug solution was pumped into intestinal tract slowly, whereas in oral administration the drug solution was administered very rapidly. Another reason might be that the animals used in the in situ loop experiment were under anesthesia, and their systemic circulation was much slower than that of the normal states (Holzer et al., 2003). Therefore, the absorption of drug in the in situ loop experiment was also slow. On the other hand, no significant differences in the rHV2 absorption among different parts of small intestine were found, suggesting that rHV2 has no preference on the absorption sites. Coadministration with bacitracin or casein could attain a small increment in bioavailability of rHV2, possibly due to the fact that intestinal tract was washed well and cleared of most digestive enzymes before perfusion studies.
Furthermore, as demonstrated in the experiments using the everted sac of jejunum, the uptake of rHV2 was inhibited by polyanion (carbopol 941) and low temperature but not by polycation (poly-L-lysine). The significant decrease of rHV2 transport under 4°C suggested that the transport was energy-dependent, so the transport routine may be related to transcellular way. The polylysine was reported to bind anionic sites of gall bladder epithelial cell membranes, thereby producing morphological changes such as collapse of the microvillar structure, membrane folding from the apical border into the terminal web, or "fused" membranes with pentilaminar substructure (Quinton and Phillpot, 1973). These changes limited the absorption by transcellular way. Madara (1989) reported that polylysine could interact with anionic components of the glycoproteins on the surface of the epithelial cells. Moreover, the interiors of the tight junction (pores) are highly hydrated and negative charged. An alteration in the relative concentration of specific ion species in the pore would result in substantial changes in tight junction resistance, which might lead to loosening or opening of the pore and enhancing the absorption by paracellular routine.
Presuming rHV2 was absorbed by endocytosis, the transcellular way would be blocked, whereas the paracellular routine opened after polylysine was added. These two adverse effects on the epithelial cells would result in no increase of the rHV2 transport. Polylysine and rHV2 are being oppositely charged and might interact with one another, so the amount of transport of rHV2 in the mucosal tissue has a little decrease. The results of our experiments are consistent with the above-mentioned presumption, so it seems logical to deduce that rHV2 was absorbed mainly by endocytosis.
The transport of rHV2 was inhibited by DNP and colchicine. DNP is an uncoupler of oxidative phosphorylation, whereas colchicine is an inhibitor of microtubular assembly. Both of them can damage the functions of epithelial cells and inhibit the transport of rHV2 through transcellular way.
It was reported that carbopol showed a clear effect on opening of intercellular junctions, thereby enhancing the paracellular permeability for hydrophilic macromolecules (Luesen et al., 1996). Carbopol also displays strong mucoadhesive properties (Bai et al., 1995; Mortazavi, 1995) and may therefore be able to localize its enzyme inhibiting and absorption enhancing activities to a confined area in the intestinal tract. However, it was showed in this study that carbopol inhibited the rHV2 transport. The possible reason is that a stagnant layer formed by a highly viscous carbopol solution and repellence between both negatively charged carbopol and rHV2 may prevent rHV2 molecules from getting close to epithelial cell membrane, thus leading to the decrease of transport of rHV2 across the membrane.
Toshikiro demonstrated that rhIGF-I could be absorbed into systemic circulation (Toshikiro et al., 1996). The molecular weight of rhIGF-I is 7000 Da, which is almost equal to that of rHV2. The transport mechanism of rhIGF-I was inferred to be absorptive-mediated endocytosis. It was supposed from the above-mentioned results that rHV2 has the similar absorption mechanism as rhIGF-I, i.e., endocytosis might be the main route by which rHV2 molecules were absorbed into the circulation.
In conclusion, the maximum degradation was found in the stomach and distal ileum, slightly higher than that in proximal ileum, jejunum, and mucosal subfraction. Some enzyme inhibitors, such as bacitracin or casein, could inhibit the degradation to a certain degree. The bioavailability after oral administration of rHV2 to rats varies and is dependent on the analytical method, and some of the enzyme inhibitor could enhance rHV2 oral absorption. There is no site difference in rHV2 absorption in different segments of the small intestine. The possible transport mechanism of rHV2 across the GI tract is concerned with the endocytosis process.
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Footnotes |
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ABBREVIATIONS: rHV, recombinant hirudin; GI, gastrointestinal; IGF-I, insulin-like growth factor I; HP--CD, hydroxypropyl--cyclodextrin; DNP, 2,4-dinitrophenol; PBS, phosphate-buffered solution; SDCh, deoxycholic acid sodium salt; TCA, trichloroacetic acid.
Address correspondence to: Dr. Qiang Zhang, Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Health Science Center, 38 XueYuan Rd., Beijing 100083, P.R. China. E-mail: zqdodo{at}mail.bjmu.edu.cn
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Adachi I, Liu HX, Horikoshi I, Ueno M, and Sato H (1993) Possibility of lymphatic absorption of epidermal growth factor from intestine. Yakugaku-Zasshi 113: 256–263.
Asada H, Douen T, Mizukoshi Y, Fujita T, Murakami M, Yamamoto A, and Muranishi S (1994) Stability of acyl derivatives of insulin in the small intestine: relative importance of insulin association characteristics in aqueous solution. Pharm Res (NY) 11: 1115–1120.
Augustijns PF, Brown SC, Willard DH, Consler TG, Annaert PP, Hendren RW, and Bradshaw TP (2000) Hydration changes implicated in the remarkable temperature-dependent membrane permeation of cyclosporin A. Biochemistry 39: 7621–7630.[Medline]
Bai JP, Chang LL, and Guo JH (1995) Effects of polyacrylic polymers on the luminal proteolysis of peptide drugs in the colon. J Pharm Sci 84: 1291–1294.[Medline]
Bai JPF and Chang LL (1995) Transepithelial transport of insulin. I. Insulin degradation by insulin-degrading enzyme in small intestinal epithelium. Pharm Res (NY) 12: 1171–1175.
Berteloot A, Khan AH, and Ramaswamy K (1981) Characteristics of dipeptide transport in normal and papain-treated brush border membrane vesicles from mouse intestine. Biochim Biophys Acta 649: 179–188.[Medline]
Chang JY (1983) The functional domain of hirudin, a thrombin-specific inhibitor. FEBS Lett 164: 307–313.[CrossRef][Medline]
Chang JY (1991) Stability of hirudin, a thrombin-specific inhibitor. J Biol Chem 266: 10839–10843.
Cheeseman CI and Johnston G (1982) Glycy-L-leucine transport in the rat small intestine. Can J Physiol Pharmcol 60: 1177–1184.[Medline]
Dolt J, Seemuller U, and Maschler R (1985) The complete covalent structure of hirudin. Biol Hoppe Seyler 366: 379–385.
Donovan SM, Chao JC, Zijlstra RT, and Odle J (1997) Orally administered iodinated recombinant human insulin-like growth factor-I (125I-rhIGF-I) is poorly absorbed by the newborn piglet. J Pediatr Gastroenterol Nutr 24: 174–182.[CrossRef][Medline]
Drewe J, Beglinger C, and Kissel T (1992) The absorption site of cyclosporin in the human gastrointestinal tract. Br J Clin Pharmacol 33: 39–43.[Medline]
Due C, Linnet K, Langeland Johansen N, and Olsson L (1985) Analysis of insulin receptors on heterogeneous eukaryotic cell populations with fluorochrome-conjugated insulin and fluorescence-activated cell sorter. Advantages and limitations to the 125I-labelled insulin methodology. Diabetologia 28: 749–755.[Medline]
Groetsch H, Damm D, Ben Youssef R, and Haertel D (1991) Comparison of two different methods for the determination of rDNA-hirudin in plasma samples: HPLC vs chromogenic thrombin substrate. Thromb Res 64: 273–277.[Medline]
Holzer A, Winter W, Greher M, Reddy M, Stark J, Donner A, Zimpfer M, and Illievich VM (2003) A comparison of propofol and sevoflurane anesthesia: effects on aortic blood flow velocity and middle cerebral artery blood flow velocity. Anaesthesia 58: 217–222.[Medline]
Hosny EA, Ghilzai NM, and Elmazar MM (1997) Promotion of oral insulin absorption in diabetic rabbits using pH-dependent coated capsules containing sodium cholate. Pharm Acta Helv 72: 203–207.[Medline]
Hovgaard L and Brondsted H (1995) Drug delivery studies in Caco-2 monolayers. IV. Absorption enhancer effects of cyclodextrin. Pharm Res (NY) 12: 1328–1332.
Kakemi K, Sezaki H, Konishi R, Kinura T, and Okita A (1970) Effect of bile salts on the gastrointestinal absorption of drugs. II. Mechanism of the enhancement of the intestinal absorption of sulfaguanidine by bile salts. Chem Pharm Bull 18: 1034–1039.
Lee VH (1990) Protease inhibitors and penetration enhancers as approaches to modify peptide absorption. J Controlled Release 13: 213–223.
Luesen HL, de-Leeuw BJ, Langemeer MWE, de Boer AG, Verhoef JC, and Junginger HE (1996) Mucoadhesive polymers in peroral peptide drug delivery. VI. Carbomer and chitosan improve the intestinal absorption of peptide drug buserelin in vivo. Pharm Res (NY) 13: 1668–1672.
Madara JL (1989) Loosening of the tight junctions. J Clin Investig 83: 1089–1094.
Markwardt F (1970) Hirudin as an inhibitor of thrombin, in Methods in Enzymology 19: Proteolytic Enzyme (Perlmann GE and Lorand L eds) pp 924–932, Academic Press, New York.
Mortazavi SA (1995) An in vitro assessment of mucus/mucoadhesive interactions. Int J Pharm 124: 173–182.[CrossRef]
Nakagawa Y, Oxford GE, Ishibashi K, Yamamoto H, Maeda N, Bowen E, Brayer J, and Humphreys Beher MG (1997) Gastrointestinal absorption of insulin-like growth factor in the mouse in the absence of salivary insulin-like growth factor binding protein. Biochem Pharmacol 53: 233–240.[Medline]
Nowak G and Markwardt F (1991) Hirudin in disseminated intravascular coagulation. Haemostasis 21(Suppl 1): 142–148.
Quinton PM and Phillpot CW (1973) A role for anionic sites in epithelial architecture. Effect of cationic polymers on cell membrane structure. J Cell Biol 56: 787–796.
Rao RK, Koldovsky O, Korc M, Pollack PF, Wright S, and Davis TP (1990) Processing and transfer of epidermal growth factor in developing rat jejunum and ileum. Peptides 11: 1093–1102.[CrossRef][Medline]
Scharf M, Engels J, and Tripier D (1989) Primary structure of new isohirudin. FEBS Lett 225: 105–110.
Shen WH and Xu RJ (1998) Stability and distribution of orally administered epidermal growth factor in neonatal pigs. Life Sci 63: 809–820.[CrossRef][Medline]
Shen WH and Xu RJ (2000) Gastrointestinal stability and absorption of insulin in suckling pigs. Comp Biochem Physiol A Mol Integr Physiol 125: 389–401.[Medline]
Stoll BR, Leipold HR, Milstein S, and Edwards DA (2000) A mechanistic analysis of carrier-mediated oral delivery of protein therapeutics. J Controlled Release 64: 217–228.[Medline]
Toshikiro K, Yusuke M, Misako O, Seiji O, and Kazytaja H (1996) Gastrointestinal absorption of recombinant human insulin-like growth factor in rats. J Pharmacol Exp Ther 283: 611–618.
Tozaki H, Odoriba T, Iseki T, Taniguchi T, Fujita T, Murakami M, Muranishi S, and Yamamoto A (1998) Use of protease inhibitors to improve calcitonin absorption from the small and large intestine in rats. J Pharm Pharmacol 50: 913–920.[Medline]
Uchiyama T, Sugiyama T, Quan YS, Kotani A, Okada N, Fujita T, Muranishi S, and Yamamoto A (1999) Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers: their intestinal mucosal toxicity and absorption-enhancing mechanism of n-lauryl--D-maltopyranoside. J Pharm Pharmacol 51: 1241–1250.[CrossRef][Medline]
Vindigni V, Filippis VD, and Zanotti G (1994) Probing the structure of hirudin from hirudinaria manillensis by limited proteolysis: isolation, characterization and thrombin-inhibitory properties of N-terminal fragments. Eur J Biochem 226: 323–333.[Medline]
Wang ZH, Han YM, and Lu YF (1999) Studies on the effects of anticoagulant and anti-thrombin after duodenal administration of recombinant hirudin in rats. Chin J Hematol 20: 483–484.
Wang ZH, Lu YF, and Han YM (1995) Stability of recombinant hirudin-HV2. Chin Biochem J 11: 321–326.
Xian CJ, Shoubridge CA, and Read LC (1995) Degradation of IGF-I in the adult rat gastrointestinal tract is limited by a specific antiserum or the dietary protein casein J Endocrinol 146: 215–225.
Xu RJ and Wang T (1996) Gastrointestinal absorption of insulinlike growth factor-I in neonatal pigs. J Pediatr Gastroenterol Nutr 23: 430–437.[CrossRef][Medline]
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), +bacitracin (
), +casein (
), and +HP-
