- Email: [email protected]
Evidence for the absorption of heparin by rat stomach
Biomedicine & Pharmacotherapy 61 (2007) 68e74 www.elsevier.com/locate/biopha
Original article
Evidence for the absorption of heparin by rat stomach Linda M. Hiebert*, Sandra M. Wice, Taher Abdelhameed Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK S7N 5B4, Canada Received 1 August 2006; accepted 23 August 2006 Available online 4 December 2006
Abstract Antithrombotic activity and heparin with endothelium are observed in rats when heparin is administered by the oral route. Peak endothelial concentrations at 6 min suggest rapid absorption. To identify the site of absorption, stomach and duodenum were isolated by tying the pyloric sphincter of male Wistar rats and heparin (unfractionated bovine lung, 60 mg/kg) was administered by stomach tube or injected into the duodenum. Heparin in plasma and aortic endothelium, collected within 15 min, was determined by densitometry following agarose gel electrophoresis with toluidine blue staining. Heparin was recovered in 5 of 10 endothelial (0.136 0.068 mg/cm2) and 6 of 10 plasma (0.06 0.02 mg/ ml) samples when administered in the stomach and in 0 of 9 endothelial and 2 of 9 plasma (0.02 0.02 mg/ml) samples when injected into the duodenum. To further study heparin distribution, stomach layers were separated and analysed 15 min and 4 h following heparin administration by stomach tube. Heparin was recovered in muscle and mucosal layers as well as washes indicating that heparin passes through stomach tissue. Heparin was also recovered from the portal vein, endothelium and lung. These results indicate that heparin is absorbed following oral administration and that the stomach is an important site of absorption. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Heparin; Oral; Stomach; Absorption; Endothelium
1. Introduction Heparin, a glycosaminoglycan derived from animal tissue, is used extensively as an antithrombotic drug. Heparin is primarily administered by the intravenous and subcutaneous routes and is believed not to be effective when given orally [1]. However, our previous studies in rats have shown that orally administered unfractionated bovine lung heparin and the low molecular weight heparins, tinzaparin and reviparin, are absorbed [2e4]. These drugs are found primarily associated with endothelium with low levels in plasma and can be detected in human urine following oral administration [5]. Unfractionated heparins and low molecular weight heparins also have dose-dependent antithrombotic activity in a rat model [4,6,7].
* Corresponding author. Tel.: þ1 306 966 7358; fax: þ1 306 966 7376. E-mail address: [email protected] (L.M. Hiebert). 0753-3322/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2006.08.006
There has been considerable effort spent in developing an oral heparin [1,8] although little is known about the fate of heparin in the gastrointestinal tract. In our studies, when heparin or dextran sulphate, a similar anionic polyelectrolyte, were given by stomach tube, the drugs could be detected in plasma and endothelium as early as 2.5 min following administration with peak concentrations observed at 6 min. The almost immediate detection of heparin in the circulation and rapid onset of peak levels [2] suggested that absorption may have occurred in the stomach. In addition we have observed that 14C label can be detected in stomach tissue in amounts greater than all other tissues up to 4 h and is still detectable at 24 h after administration of 14C-labelled unfractionated heparin, also suggesting that stomach tissue may be a site of distribution [9]. The objectives of this study were to more closely observe heparin absorption in a rat model, where the stomach was isolated from the duodenum, examining plasma, endothelial and tissue concentrations after oral administration. The distribution of heparin in layers of stomach tissue was also
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
studied to better understand the movement of heparin through the stomach tissue.
69
portion was determined. Heparin was extracted from the stomach washes, each layer of the stomach, the portal vein, endothelium and from the lung.
2. Methods and materials 2.4. Harvesting endothelium 2.1. Heparin Unfractionated bovine lung heparin, 150 units/mg, Lot No. ZX320, was obtained from Upjohn Ltd., Kalamazoo, MI. Heparin was dissolved in water at a concentration of 100 mg/ml. 2.2. Animals Male Wistar rats, weighing 320.9 6.2 g, were handled and housed according to the Principles of Animal Care set by the Canadian Federation of Biological Societies. Animals were fasted overnight prior to treatment and were anaesthetized with barbital and methoxyfluorane for experimental procedures.
Endothelium was removed from blood vessels by a modified method of Hiebert and Jaques [10]. The aorta was removed, slit open, pinned to dental wax lumen side up, and rinsed in saline. Cellulose acetate paper was applied to the lumen surface and when lifted, endothelium was removed. The length and width of the imprint was measured to the nearest mm. Mean area for aortic endothelium was 2.7 0.8 cm2 ( S.D.). 2.5. Heparin extraction from endothelium Cellulose acetate paper was removed from endothelium by dissolving in cold acetone followed by centrifuging and discarding the supernatant. The process was repeated to give a dry tissue powder [10].
2.3. Experimental procedures 2.6. Heparin extraction from plasma 2.3.1. Heparin absorption in the stomach versus the duodenum To isolate the stomach from the duodenum a laparotomy was performed and the pyloric sphincter was tied tightly with a cotton thread. Heparin (60 mg/kg) was introduced into the stomach by stomach tube (10 rats) or injected into the duodenum with a 25-gauge needle (9 rats) in a volume of approximately 0.15 ml. For positive controls heparin (60 mg/kg) was given by stomach tube as described above without tying the pyloric sphincter (24 rats). Controls were given saline by stomach tube or tap water ad libitum. At 5, 10 or 15 min following administration of heparin, blood samples of approximately 10 ml (9 parts blood to 1 part 3.8% sodium citrate) were taken from the abdominal aorta and plasma was prepared. The activated partial thromboplastin time (APTT) was determined when plasma was derived from blood samples obtained with a minimum of tissue trauma. The remaining plasma was frozen for later extraction of heparin. As a source of endothelium, the thoracic aorta was removed and placed in saline. Each animal was examined for signs of internal haemorrhage and the time when the blood clotted in the body cavity was recorded. Tissues including stomach, duodenum, jejunum, ileum, colon, were frozen for later heparin extraction. 2.3.2. Heparin distribution in the stomach Bovine lung heparin (60 mg/kg) was administered by stomach tube in a volume of approximately 0.2 ml (6 rats). Control rats were given saline. At 15 min or 4 h later rats were killed under deep metofane anaesthesia and stomach, samples of the portal vein, endothelium, lung, and a blood sample were collected. The stomach lumen was washed with 10 ml distilled water (3) and 10 ml of 1.4 M Na Cl (3). The stomach was then divided into the glandular stomach and forestomach. The glandular stomach was further divided into the mucosal and muscle layer using a scalpel. The wet weight of each
Plasma extracts were prepared according to the methods of Jaques et al. [11]. Heparin was obtained by chromatography on ECTEOLA-cellulose columns with elution by 1.1 M NaCl and 1.4 M NaCl in 0.05 M glycine HCl (pH 2.9). Eluates were desalted by passing through a Sephadex G25-40 column and evaporated to dryness with an air jet. 2.7. Heparin extraction from tissues Heparin was extracted from tissue by standard methods used previously for polyanions [12]. Tissues were minced, and digested by pronase in 0.1 M Tris buffer 0.1 M CaCl2 at pH 8 at 37 C. Digests were purified by precipitating with 1% NaCl in acetone and then methanol. The precipitates were dried, dissolved in water and then analysed. 2.8. Identification and measurement of heparin Agarose gel electrophoresis was used to identify and measure heparin in all plasma, tissue and endothelial extracts. The dried powders, dissolved in suitable volumes of water, were applied to agarose gel slides. Heparin dissolved in water was used as a reference. Following electrophoresis, gels were fixed in 0.1% hexadecyltrimethylammonium bromide and air dried. Slides were stained with 0.04% Toluidine Blue in 80% acetone and background colour was removed with 1% acetic acid. Heparin was identified by electrophoretic migration compared to reference material and amounts determined by densitometry. 2.9. Data analysis All data are expressed as mean S.E.M. except for area of the aortic endothelium which is expressed as mean S.D. For
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
70
plasma and endothelial samples following administration into the stomach or duodenum, with and without tying off the pyloric sphincter, significant differences in numbers of positive samples between groups were determined by c2 for goodness of fit while significant differences in concentrations between groups were determined by the ManneWhitney one-tailed t-test. A one-tailed t-test was used to determine differences in heparin concentrations in stomach layers and washes following heparin administration for 15 min or 4 h when compared to control tissue. A two-tailed t-test was used to compare heparin concentrations at 15 min versus 4 h. 3. Results 3.1. Recovery from endothelium and plasma after oral administration To help determine the site of absorption, heparin was administered to rats by stomach tube or by injection directly into the duodenum following tying of the pyloric sphincter. Plasma and aortic endothelium were examined for heparin. Values were similar when data was collected at 5, 10 and 15 min of administration and thus results were combined. When heparin was placed in the stomach, with the pyloric sphincter tied, heparin was recovered from the plasma of six of ten rats and from endothelium of five of ten rats as shown in Table 1. When heparin was injected into the duodenum, with the pyloric sphincter tied, heparin was found in plasma of two of nine rats and but not in aortic endothelium. This compared to recovery of heparin from plasma and endothelium of 15 of 24 rats when heparin was administered by stomach tube without tying the pyloric sphincter. Heparin was not recovered from plasma samples of 23 control rats and only trace amounts of heparin were found with endothelium of 37 control rats. The plasma heparin concentration following administration into the stomach with the pyloric sphincter tied was Table 1 Heparin in plasma and endothelium following administration of heparin into the stomach or duodenum when the pyloric sphincter is tied Site of administration
Stomach with pylorus tied Duodenum with pylorus tied Stomach without pylorus tied Control a
Plasma
3.2. Recovery of heparin from gut tissue Heparin was extracted from gut tissue of rats where the pylorus was tied and heparin was administered into the stomach or duodenum to affirm the distribution of heparin in the gut. When heparin was administered into the stomach, with the pyloric sphincter tied, heparin was recovered from the stomach but not the duodenum, ileum or colon with only trace amounts found in the jejunum (Table 2). This agreed in principle with results when heparin was administered in the stomach without the pylorus tied where heparin was recovered from the stomach with only trace amounts found in the duodenum and jejunum. When heparin was injected into the duodenum it was recovered from the duodenum, jejunum, ileum with trace amounts found in the colon and stomach. Only trace amounts of heparin could be recovered from all levels of the gut when rats were given saline by stomach tube.
Endothelium
No. of samples positivec
Concentration (mg/ml)d Mean S.E.
No. of samples positivec
6/10a
0.06 0.02a
5/10a
0.1360 0.0683a
2/9a
0.02 0.02b
0/9b
0.0000 0.0000b
15/24a
2.16 1.02a
15/24a
0.1508 0.0529a
0/23
0.000 0.000
2/37
0.0016 0.0013
Concentration (mg/cm2)d Mean S.E.
Significantly different than control. Significantly different than stomach with and without pylorus tied. c Significant differences in numbers of samples positive were determined by c2 for goodness of fit. d Significant differences in concentrations determined by ManneWhitney one-tailed t-test. b
significantly greater than control values ( p ¼ 0.004), was similar to when heparin was administered in the stomach without the pyloric sphincter tied and showed a trend towards an increase compared to when it was administered in the duodenum ( p ¼ 0.05). The plasma heparin concentration, when heparin was administered into the duodenum with the pyloric sphincter tied, was similar to control values and significantly different from concentrations when heparin was administered into the stomach without the pyloric sphincter tied ( p ¼ 0.004). There was no difference in the APTT between groups (data not shown). The endothelial heparin concentration showed a similar pattern. When heparin was administered into the stomach with the pyloric sphincter tied, endothelial heparin concentrations were significantly greater than control values ( p ¼ 0.01), showed a trend towards an increase compared to when it was administered in the duodenum ( p ¼ 0.05) and was similar to when heparin was administered in the stomach without the pyloric sphincter tied. The endothelial heparin concentration, when heparin was administered into the duodenum with the pyloric sphincter tied, was similar to control values and significantly different from those when heparin was administered into the stomach without the pyloric sphincter tied ( p ¼ 0.005).
3.3. Distribution of heparin in rat stomach In order to better understand the movement of heparin through the rat stomach, stomach washes and dissected stomach layers were examined at 15 min and 4 h after administration of heparin by stomach tube (Fig. 1). When heparin content of the forestomach was examined, more heparin was found at 15 min and 4 h compared to controls ( p ¼ 0.01 and 0.024, one-tailed t-test). Although there was a reduced amount recovered at 4 h compared to 15 min, this difference did not reach significance ( p ¼ 0.073, two-tailed t-test). Similar results were observed when the stomach mucosa from the glandular portion was considered where heparin recovered at 15 min and 4 h was significantly different from controls
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
71
Table 2 Recovery of heparin from rat gut tissues 5e15 min following administration of heparin (60 mg/kg) into the stomach or duodenum with the pyloric sphincter tied Treatmenta
Stomach mg/g wet wt
Stomach with pylorus tied (10) Duodenum with pylorus tied (9) Stomach without pylorus tied (7) Control (8) a
Duodenum mg/g wet wt
Jejunum mg/g wet wt
Ileum mg/g wet wt
Colon mg/g wet wt
423.2 211.6
0.0 0.0
0.1 0.1
0.0 0.0
0.0 0.0
0.7 0.7
103.9 51.4
159.4 97.7
17.8 14.7
1.4 1.4
1418.7 455.2
0.8 0.6
0.6 0.6
0.0 0.0
0.0 0.0
3.9 3.9
4.5 1.7
0.5 0.3
3.0 1.3
1.1 0.7
Number of animals per group in parentheses.
( p ¼ 0.013 and 0.031, respectively, one-tailed t-test). There was no significant difference in heparin recovery at 4 h compared to 15 min ( p ¼ 0.59, two-tailed t-test). When the muscle layers of the glandular stomach were considered, there was significantly more heparin recovered at 15 min and 4 h after oral administration than stomach muscle from rats given saline ( p ¼ 0.011 and p ¼ 0.024, respectively one-tailed t-test). Although there was some indication that amounts recovered from the muscle layer increased with time when means were considered (Fig. 1), there was no significant difference in the concentration of heparin recovered at 15 min versus 4 h ( p ¼ 0.403, two-tailed t-test). Stomach washes were also analysed for heparin content at 15 min and 4 h after administration. Heparin recovered from stomach washes at 15 min, expressed as mg/mg wet weight of total stomach, was significantly greater than that found in
control samples and at 4 h after heparin administration ( p ¼ 0.027 and 0.044, respectively, one tailed t-test). Amounts found at 4 h did not differ significantly from controls ( p ¼ 0.124, one-tailed t-test). Amounts found in the stomach washes at 15 min and 4 h after administration accounted for 14% and 2% respectively of the original administered dose. Additional heparin was removed from the lumen with salt washes. For the 15 min treated group the average salt wash was 24.9% of the total stomach wash and 3.5% of the total original administered dose. For the 4 h group the average salt wash was 83.7% of the total stomach wash and 1.93% of the total original administered dose. The aortic endothelium, portal vein and lung were also examined for heparin in these same rats. Heparin was recovered from endothelium and the lungs at 15 min and 4 h after and from the portal vein at 15 min but not 4 h after administration (Table 3).
A. Tissue 0.4
B. Washes 3.5
Forestomach Glandular Stomach
0.35
a
Mucosa
3.0 a
Muscle 0.30 2.5
µg/mg wet weight
a 0.25
2.0 0.20 1.5 0.15
a 1.0
a
0.10
a ab
0.5
0.05 a 0
Control
15 min
4h
0
Control
15 min
4h
Fig. 1. Heparin recovered from rat forestomach and glandular stomach layers and stomach washes at different times following administration of unfractionated heparin by stomach tube. Mean S.E.M. are shown for six rats. Washes are the total heparin recovered from water and sodium chloride washes. a - Significantly greater than control values, one-tailed t-test; b- significantly different than 15 min, two-tailed t-test.
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
72
4. Discussion Heparins are commonly used for the prevention of postoperative thromboembolism, treatment of stroke, for dialysis, and for management of angina. Possible uses of heparin, based on laboratory experiments, include the prevention of atherosclerosis [13]; adjuncts to chemotherapeutic agents [14,15]; as anti-inflammatory [16] and antiviral agents [17]; for control of angiogenesis [18]; treatment of hypertension[19]; fibromyalgia [20]; and Crohn’s disease [21,22]. These possible uses would require long term repeated administration for which an oral heparin would be ideally suited. Although oral heparin is considered ineffective, results from our laboratory and others have indicated that heparin is absorbed following administration by the oral route [23e25]. This observation has been supported by evidence of oral absorption of related polyanionic compounds such as chondroitin sulphate [26] and sucrose octasulphate [27]. Despite these observations, the site and mechanism of absorption of polyanions are not understood. This is particularly challenging considering that heparin and related compounds have high charge and molecular weight and are thus considered unlikely to be absorbed. We chose the stomach as a possible site since peak endothelial concentrations were seen at 6 min after oral administration in previous studies, suggesting that absorption is occurring rapidly following placing the drug in the stomach [28]. Other studies have suggested that heparin can easily cross membranes and has been shown to be effective following intrapulmonary and dermal routes [29e31]. In the present study, we used a model where the stomach and duodenum were isolated by tying the pyloric sphincter. Our results show that, within 15 min of administration, endothelial and plasma heparin concentrations are greater when heparin is placed in the isolated stomach than when injected in the duodenum. Our analysis of the gastrointestinal tract shows that as expected in this model, heparin is confined to the stomach when administered by stomach tube but when injected into the duodenum heparin is found in the jejunum and ileum as well as the duodenum (Table 2). These observations thus suggest that the stomach is an important site of heparin absorption. These results agree with those of Sue et al who showed a significant increase in whole blood clotting time and Factor Xa inhibition 1 h after heparin administration (240e260 mg/kg) in the stomach versus the duodenum of mice [32]. We have demonstrated the same evidence of stomach absorption using one-fourth the dose (60 mg/kg) by
Table 3 Recovery of heparin from aortic endothelium, portal vein and lung following administration of heparin (60 mg/kg) to rats by stomach tube Treatment
Portal vein mg/mg wet weight
Lung mg/mg wet weight
Endothelium mg/cm2
Control Heparin 15 min Heparin 4 h
0.00 0.00 0.13 0.06 0.00 0.00
0.00 0.00 0.19 0.08 0.01 0.01
0.00 0.00 0.08 0.08 0.12 0.08
examining the endothelium as well as plasma and by measuring the chemical amount of the drug present instead of the anticoagulant response in plasma. The increased sensitivity of measurement of heparin with endothelium is not surprising considering the plasma to endothelial ratios are shown to be on the order of 1000 to 1 following heparin administration [28]. Our results suggest that the intestine is a poor absorption site for heparin since although heparin was located in the duodenum, jejunum and ileum the amount found on endothelium was less than when heparin was placed in the stomach with and without tying of the pyloric sphincter. The poor absorption of heparin in the intestine has been previously observed by others [1,33]. To further study the distribution of heparin in the stomach, heparin was administered to rats by stomach tube and layers of the stomach were examined for heparin at 15 min and 4 h after administration. These times were selected since evidence of absorption was seen within 15 min of administration in previous studies and since most of the 14C-label was seen in stomach tissue versus other parts of the gut at 4 h after administration. The dose selected was 60 mg/kg and although this dose is considered high it was to insure that chemical heparin could be recovered from the tissues in detectable concentrations. The stomach was washed first with distilled water and then with 1 M sodium chloride. Washes with sodium chloride were done to remove any heparin that was electrostatically bound to the mucosal surface. Heparin was recovered from all washes and stomach layers at 15 min and 4 h after administration. Heparin recovered from distilled water washes were 14.1% of the administered dose at 15 min and 2.3% of dose at 4 h, indicating that most of the heparin was gone from the lumen by 4 h. Heparin recovered in sodium chloride washes was 3.5% and 1.9% of the total administered dose but 25% and 84% of the total washings at 15 min and 4 h, respectively. Therefore at 4 h most of the heparin found in the stomach washings was bound to the mucosal surface and not free in the lumen. The stomach was divided into a forestomach, glandular stomach mucosa and glandular stomach muscle sections. The rat, in contrast to humans, has a forestomach (non glandular portion) adjacent to the oesophagus where the mucosa is covered with a cornified stratified squamous epithelium [34]. The mucosa of the forestomach is difficult to separate from the underlying muscle layers and therefore the forestomach was collected as a separate section containing both mucosa and muscle layers. Considerably less amounts of heparin were found in the forestomach portion than in the other layers and less was found at 4 h than 15 min, suggesting the forestomach is likely not a site of heparin uptake. In contrast, the glandular stomach in the rat is similar to that in humans and can be used as a model for human disease [35]. The glandular stomach was sectioned into two portions; a mucosal portion, that was in contact with the lumen, and the deeper muscular layer. Heparin was found in the mucosal portion at both 15 min and 4 h after administration suggesting that heparin is taken up by the mucosa. Heparin could also be recovered
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
from the muscle layers at 15 min and 4 h after administration. Since this layer is not in contact with the lumen, absorption of heparin by the stomach is supported. In general, the mean heparin concentration in the muscle layer at 4 h was twice the concentration at 15 min while the mean heparin concentration in the mucosal layer at 4 h was 4/5 that at 15 min. This suggests movement from the mucosa into the muscle. Heparin could also be found in the portal vein and lung at 15 min and from the aortic endothelium at 15 min and 4 h after placing heparin in the stomach. This agrees with previous observations and demonstrates that orally administered heparin is absorbed and rapidly moves into the circulation [9]. The continued presence of heparin in the stomach and the inability to detect chemical heparin in the portal vein and lung at 4 h suggests that after a rapid distribution phase a much slower movement of heparin occurs from the stomach tissue into the circulation. This is supported by previous studies showing that 14 C and chemical heparin levels were greatest on endothelium from 6 to 15 min following administration with only trace amounts detected at 30 min, and 1, 4, and 24 h after oral administration [9]. In conclusion, these results agree with our previous findings that heparin is absorbed following oral administration. Our observations suggest that the stomach may play an important role in the absorption of heparin and possibly other polyanions following oral administration thus absorption in the stomach should be considered when designing polyanionic compounds. Further studies are required to determine the mechanism of heparin absorption. Acknowledgment This work was supported by a grant from the Heart and Stroke Foundation of Saskatchewan. References [1] Kim SK, Vaishali B, Lee E, Lee S, Lee YK, Kumar TS, et al. Oral delivery of chemical conjugates of heparin and deoxycholic acid in aqueous formulation. Thromb Res 2006;117:419e27. [2] Jaques LB, Hiebert LM, Wice SM. Evidence from endothelium of gastric absorption of heparin and of dextran sulfates 8000. J Lab Clin Med 1991; 117(2):122e30. [3] Hiebert LM, Wice SM, Ping T. Tissue distribution of the low molecular weight heparin, tinzaparin, following administration to rats by the oral route. Biomed Pharmacother 2004;58:372e80. [4] Hiebert LM, Wice SM, Ping T, Herr D, Laux V. Antithrombotic efficacy in a rat model of the low molecular weight heparin, reviparin sodium, administered by the oral route. Thromb Haemost 2001;85:114e8. [5] Hiebert LM, Wice SM, Ping T. Increased plasma anti-Xa activity and recovery of heparin from urine suggest absorption of orally administered unfractionated heparin in human subjects. J Lab Clin Med 2005;145: 151e5. [6] Hiebert LM, Wice SM, Jaques LB. Antithrombotic activity of oral unfractionated heparin. J Cardiovasc Pharmacol 1996;28:26e9. [7] Hiebert LM. Evidence that orally administered heparins are absorbed. In: Vossoughi J, Fareed J, Mousa SA, Karanian JW, editors. Thrombosis research and treatment. Bench to bedside. Washington DC: Medical and Engineering Publishers; 2004. p. 553e63.
73
[8] Berkowitz SD, Marder VJ, Kosutic G, Baughman RA. Oral heparin administration with a novel drug delivery agent (SNAC) in healthy volunteers and patients undergoing elective total hip arthroplasty. J Thromb Haemost 2003;1:1914e9. [9] Hiebert LM, Wice SM, Ping T, Hileman RE, Capila I, Linhardt RJ. Tissue distribution and antithrombotic activity of unlabeled or 14C-labeled porcine intestinal mucosal heparin following administration to rats by the oral route. Can J Physiol Pharm 2000;78:307e20. [10] Hiebert LM, Jaques LB. The observation of heparin on endothelium after injection. Thromb Res 1976;8:195e204. [11] Jaques LB, Wice SM, Hiebert LM. Determination of absolute amounts of heparin and of dextran sulfate in plasma in microgram quantities. J Lab Clin Med 1990;115(4):422e32. [12] Jaques LB. Determination of heparin and related sulfated mucopolysaccharides. Methods Biochem Anal 1977;24:203e312. [13] Engelberg H. Actions of heparin in the atherosclerotic process. Pharmacol Rev 1996;48:327e52. [14] Engelberg H. Actions of heparin that may affect the malignant process. Cancer 1999;85(2):257e72. [15] Levine MN, Lee AY, Kakkar AK. From Trossseau to targeted therapy: new insights and innovations in thrombosis and cancer. J Thromb Haemost 2003;1:1456e63. [16] Gorski A, Wasik M, Nowaczyk M, Korczak-Kowalska G. Immunomodulating activity of heparin. FASEB J 1991;5:2287e91. [17] Bagasra O, Lischner HW. Activity of dextran sulfate and other polyanionic polysaccharides against human immunodeficiency virus. J Infect Dis 1988;158:1084e7. [18] Folkman J, Weisz PB, Joullie MM, Li WW, Ewing WR. Control of angiogenesis with synthetic heparin substitutes. Science 1989;243:1490e3. [19] Mandal AK, Lyden T, Saklayen MG. Heparin lowers blood pressure: Biological and clinical perspectives. Kidney Int 1995;47:1017e22. [20] Berg D, Berg LH, Couvaras J, Harrison H. Chronic fatigue syndrome and/or fibromyalgia as a variation of antiphospholipid antibody syndrome: an explanatory model and approach to laboratory diagnosis. Blood Coagul Fibrinolysis 1999;10:435e8. [21] Prajapati DN, Newcomer JR, Emmons J, Abu-Hajir M, Binion DG. Successful treatment of an acute flare of steroid-resistant Crohn’s colitis during pregnancy with unfractionated heparin. Inflamm Bowel Dis 2002; 8:192e5. [22] Ang YS, Mahmud N, White B, Byrne M, Kelly A, Lawler M, et al. Randomized comparison of unfractionated heparin with corticosteroids in severe active inflammatory bowel disease. Aliment Pharmacol Ther 2000; 14:1015e22. [23] Hiebert LM. Oral heparins. Clin Lab 2002;48:111e6. [24] Gorski A, Lagodzinski Z. Oral heparin prolongs survival of skin allografts. Arch Immunol Ther Exp 1991;39:557e62. [25] Engelberg H. Orally ingested heparin is absorbed in humans. Clin Appl Thromb Hemost 1995;1(4):283e5. [26] Bucsi L, Poor G. Efficacy and tolerability of oral chondroitin sulfate as a symptomatic slow-acting drug for osteoarthritis. Osteoarthritis Cartilage 1998;6:31e6. [27] Hiebert LM, Wice SM, Ping T, Hileman R, Polat T, Linhardt RJ. Tissue distribution of [14C] sucrose octasulfate following oral administration to rats. Pharm Res 2002;19:838e44. [28] Hiebert LM, Wice SM, McDuffie NM, Jaques LB. The heparin target organ e the endothelium. Studies in a rat model. Q J Med 1993;86:341e8. [29] Mahadoo J, Wright CJ. Intrapulmonary heparin as a prospective anticoagulant regimen. In: Fareed JW, Messmore HL, Fenton JW, Brinkhous KM, editors. Perspectives in hemostasis. Permagon Press; 1981. p. 233e40. [30] Bick RL, Ross ES. Clinical use of intrapulmonary heparin. Semin Thromb Hemost 1985;11:213e7. [31] de Moerloose P, Gavillet O, Salomon P, Reber G. Heparin-related activity in peripheral venous blood after percutaneous application of various glycosaminoglycan creams. Blood Coagul Fibrinol 1992;3:827e8. [32] Sue TK, Jaques LB, Yuen E. Effects of acidity, cations and alcoholic fractionation on absorption of heparin from gastrointestinal tract. Can J Physiol Pharm 1976;54:613e7.
74
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
[33] Brayden D, Creed E, O’Connell A, Leipold H, Agarwal R, LeoneBay A. Heparin absorption across the intestine: effects of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate in rat in situ intestinal instillations and in Caco-2 monolayers. Pharm Res 1997;14:1772e9. [34] Brown HR, Hardisty JF. Oral cavity, esophagus, and stomach. In: Boorman GA, Eustis SL, Elwell MR, Montgomery CA,
Mackenzie WF, editors. Pathology of the Fischer rat: reference and atlas. San Diego: Academic Press; 1990. p. 9e29. [35] Ichikawa T, Ishihara K, Kusakabe T, Hiruma H, Kawakami T, Hotta K. CGRP modulates mucin synthesis in surface mucus cells of rat gastric oxyntic mucosa. Am J Physiol Gastrointest Liver Physiol 2000;279: G82e9.
Original article
Evidence for the absorption of heparin by rat stomach Linda M. Hiebert*, Sandra M. Wice, Taher Abdelhameed Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK S7N 5B4, Canada Received 1 August 2006; accepted 23 August 2006 Available online 4 December 2006
Abstract Antithrombotic activity and heparin with endothelium are observed in rats when heparin is administered by the oral route. Peak endothelial concentrations at 6 min suggest rapid absorption. To identify the site of absorption, stomach and duodenum were isolated by tying the pyloric sphincter of male Wistar rats and heparin (unfractionated bovine lung, 60 mg/kg) was administered by stomach tube or injected into the duodenum. Heparin in plasma and aortic endothelium, collected within 15 min, was determined by densitometry following agarose gel electrophoresis with toluidine blue staining. Heparin was recovered in 5 of 10 endothelial (0.136 0.068 mg/cm2) and 6 of 10 plasma (0.06 0.02 mg/ ml) samples when administered in the stomach and in 0 of 9 endothelial and 2 of 9 plasma (0.02 0.02 mg/ml) samples when injected into the duodenum. To further study heparin distribution, stomach layers were separated and analysed 15 min and 4 h following heparin administration by stomach tube. Heparin was recovered in muscle and mucosal layers as well as washes indicating that heparin passes through stomach tissue. Heparin was also recovered from the portal vein, endothelium and lung. These results indicate that heparin is absorbed following oral administration and that the stomach is an important site of absorption. Ó 2006 Elsevier Masson SAS. All rights reserved. Keywords: Heparin; Oral; Stomach; Absorption; Endothelium
1. Introduction Heparin, a glycosaminoglycan derived from animal tissue, is used extensively as an antithrombotic drug. Heparin is primarily administered by the intravenous and subcutaneous routes and is believed not to be effective when given orally [1]. However, our previous studies in rats have shown that orally administered unfractionated bovine lung heparin and the low molecular weight heparins, tinzaparin and reviparin, are absorbed [2e4]. These drugs are found primarily associated with endothelium with low levels in plasma and can be detected in human urine following oral administration [5]. Unfractionated heparins and low molecular weight heparins also have dose-dependent antithrombotic activity in a rat model [4,6,7].
* Corresponding author. Tel.: þ1 306 966 7358; fax: þ1 306 966 7376. E-mail address: [email protected] (L.M. Hiebert). 0753-3322/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2006.08.006
There has been considerable effort spent in developing an oral heparin [1,8] although little is known about the fate of heparin in the gastrointestinal tract. In our studies, when heparin or dextran sulphate, a similar anionic polyelectrolyte, were given by stomach tube, the drugs could be detected in plasma and endothelium as early as 2.5 min following administration with peak concentrations observed at 6 min. The almost immediate detection of heparin in the circulation and rapid onset of peak levels [2] suggested that absorption may have occurred in the stomach. In addition we have observed that 14C label can be detected in stomach tissue in amounts greater than all other tissues up to 4 h and is still detectable at 24 h after administration of 14C-labelled unfractionated heparin, also suggesting that stomach tissue may be a site of distribution [9]. The objectives of this study were to more closely observe heparin absorption in a rat model, where the stomach was isolated from the duodenum, examining plasma, endothelial and tissue concentrations after oral administration. The distribution of heparin in layers of stomach tissue was also
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
studied to better understand the movement of heparin through the stomach tissue.
69
portion was determined. Heparin was extracted from the stomach washes, each layer of the stomach, the portal vein, endothelium and from the lung.
2. Methods and materials 2.4. Harvesting endothelium 2.1. Heparin Unfractionated bovine lung heparin, 150 units/mg, Lot No. ZX320, was obtained from Upjohn Ltd., Kalamazoo, MI. Heparin was dissolved in water at a concentration of 100 mg/ml. 2.2. Animals Male Wistar rats, weighing 320.9 6.2 g, were handled and housed according to the Principles of Animal Care set by the Canadian Federation of Biological Societies. Animals were fasted overnight prior to treatment and were anaesthetized with barbital and methoxyfluorane for experimental procedures.
Endothelium was removed from blood vessels by a modified method of Hiebert and Jaques [10]. The aorta was removed, slit open, pinned to dental wax lumen side up, and rinsed in saline. Cellulose acetate paper was applied to the lumen surface and when lifted, endothelium was removed. The length and width of the imprint was measured to the nearest mm. Mean area for aortic endothelium was 2.7 0.8 cm2 ( S.D.). 2.5. Heparin extraction from endothelium Cellulose acetate paper was removed from endothelium by dissolving in cold acetone followed by centrifuging and discarding the supernatant. The process was repeated to give a dry tissue powder [10].
2.3. Experimental procedures 2.6. Heparin extraction from plasma 2.3.1. Heparin absorption in the stomach versus the duodenum To isolate the stomach from the duodenum a laparotomy was performed and the pyloric sphincter was tied tightly with a cotton thread. Heparin (60 mg/kg) was introduced into the stomach by stomach tube (10 rats) or injected into the duodenum with a 25-gauge needle (9 rats) in a volume of approximately 0.15 ml. For positive controls heparin (60 mg/kg) was given by stomach tube as described above without tying the pyloric sphincter (24 rats). Controls were given saline by stomach tube or tap water ad libitum. At 5, 10 or 15 min following administration of heparin, blood samples of approximately 10 ml (9 parts blood to 1 part 3.8% sodium citrate) were taken from the abdominal aorta and plasma was prepared. The activated partial thromboplastin time (APTT) was determined when plasma was derived from blood samples obtained with a minimum of tissue trauma. The remaining plasma was frozen for later extraction of heparin. As a source of endothelium, the thoracic aorta was removed and placed in saline. Each animal was examined for signs of internal haemorrhage and the time when the blood clotted in the body cavity was recorded. Tissues including stomach, duodenum, jejunum, ileum, colon, were frozen for later heparin extraction. 2.3.2. Heparin distribution in the stomach Bovine lung heparin (60 mg/kg) was administered by stomach tube in a volume of approximately 0.2 ml (6 rats). Control rats were given saline. At 15 min or 4 h later rats were killed under deep metofane anaesthesia and stomach, samples of the portal vein, endothelium, lung, and a blood sample were collected. The stomach lumen was washed with 10 ml distilled water (3) and 10 ml of 1.4 M Na Cl (3). The stomach was then divided into the glandular stomach and forestomach. The glandular stomach was further divided into the mucosal and muscle layer using a scalpel. The wet weight of each
Plasma extracts were prepared according to the methods of Jaques et al. [11]. Heparin was obtained by chromatography on ECTEOLA-cellulose columns with elution by 1.1 M NaCl and 1.4 M NaCl in 0.05 M glycine HCl (pH 2.9). Eluates were desalted by passing through a Sephadex G25-40 column and evaporated to dryness with an air jet. 2.7. Heparin extraction from tissues Heparin was extracted from tissue by standard methods used previously for polyanions [12]. Tissues were minced, and digested by pronase in 0.1 M Tris buffer 0.1 M CaCl2 at pH 8 at 37 C. Digests were purified by precipitating with 1% NaCl in acetone and then methanol. The precipitates were dried, dissolved in water and then analysed. 2.8. Identification and measurement of heparin Agarose gel electrophoresis was used to identify and measure heparin in all plasma, tissue and endothelial extracts. The dried powders, dissolved in suitable volumes of water, were applied to agarose gel slides. Heparin dissolved in water was used as a reference. Following electrophoresis, gels were fixed in 0.1% hexadecyltrimethylammonium bromide and air dried. Slides were stained with 0.04% Toluidine Blue in 80% acetone and background colour was removed with 1% acetic acid. Heparin was identified by electrophoretic migration compared to reference material and amounts determined by densitometry. 2.9. Data analysis All data are expressed as mean S.E.M. except for area of the aortic endothelium which is expressed as mean S.D. For
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
70
plasma and endothelial samples following administration into the stomach or duodenum, with and without tying off the pyloric sphincter, significant differences in numbers of positive samples between groups were determined by c2 for goodness of fit while significant differences in concentrations between groups were determined by the ManneWhitney one-tailed t-test. A one-tailed t-test was used to determine differences in heparin concentrations in stomach layers and washes following heparin administration for 15 min or 4 h when compared to control tissue. A two-tailed t-test was used to compare heparin concentrations at 15 min versus 4 h. 3. Results 3.1. Recovery from endothelium and plasma after oral administration To help determine the site of absorption, heparin was administered to rats by stomach tube or by injection directly into the duodenum following tying of the pyloric sphincter. Plasma and aortic endothelium were examined for heparin. Values were similar when data was collected at 5, 10 and 15 min of administration and thus results were combined. When heparin was placed in the stomach, with the pyloric sphincter tied, heparin was recovered from the plasma of six of ten rats and from endothelium of five of ten rats as shown in Table 1. When heparin was injected into the duodenum, with the pyloric sphincter tied, heparin was found in plasma of two of nine rats and but not in aortic endothelium. This compared to recovery of heparin from plasma and endothelium of 15 of 24 rats when heparin was administered by stomach tube without tying the pyloric sphincter. Heparin was not recovered from plasma samples of 23 control rats and only trace amounts of heparin were found with endothelium of 37 control rats. The plasma heparin concentration following administration into the stomach with the pyloric sphincter tied was Table 1 Heparin in plasma and endothelium following administration of heparin into the stomach or duodenum when the pyloric sphincter is tied Site of administration
Stomach with pylorus tied Duodenum with pylorus tied Stomach without pylorus tied Control a
Plasma
3.2. Recovery of heparin from gut tissue Heparin was extracted from gut tissue of rats where the pylorus was tied and heparin was administered into the stomach or duodenum to affirm the distribution of heparin in the gut. When heparin was administered into the stomach, with the pyloric sphincter tied, heparin was recovered from the stomach but not the duodenum, ileum or colon with only trace amounts found in the jejunum (Table 2). This agreed in principle with results when heparin was administered in the stomach without the pylorus tied where heparin was recovered from the stomach with only trace amounts found in the duodenum and jejunum. When heparin was injected into the duodenum it was recovered from the duodenum, jejunum, ileum with trace amounts found in the colon and stomach. Only trace amounts of heparin could be recovered from all levels of the gut when rats were given saline by stomach tube.
Endothelium
No. of samples positivec
Concentration (mg/ml)d Mean S.E.
No. of samples positivec
6/10a
0.06 0.02a
5/10a
0.1360 0.0683a
2/9a
0.02 0.02b
0/9b
0.0000 0.0000b
15/24a
2.16 1.02a
15/24a
0.1508 0.0529a
0/23
0.000 0.000
2/37
0.0016 0.0013
Concentration (mg/cm2)d Mean S.E.
Significantly different than control. Significantly different than stomach with and without pylorus tied. c Significant differences in numbers of samples positive were determined by c2 for goodness of fit. d Significant differences in concentrations determined by ManneWhitney one-tailed t-test. b
significantly greater than control values ( p ¼ 0.004), was similar to when heparin was administered in the stomach without the pyloric sphincter tied and showed a trend towards an increase compared to when it was administered in the duodenum ( p ¼ 0.05). The plasma heparin concentration, when heparin was administered into the duodenum with the pyloric sphincter tied, was similar to control values and significantly different from concentrations when heparin was administered into the stomach without the pyloric sphincter tied ( p ¼ 0.004). There was no difference in the APTT between groups (data not shown). The endothelial heparin concentration showed a similar pattern. When heparin was administered into the stomach with the pyloric sphincter tied, endothelial heparin concentrations were significantly greater than control values ( p ¼ 0.01), showed a trend towards an increase compared to when it was administered in the duodenum ( p ¼ 0.05) and was similar to when heparin was administered in the stomach without the pyloric sphincter tied. The endothelial heparin concentration, when heparin was administered into the duodenum with the pyloric sphincter tied, was similar to control values and significantly different from those when heparin was administered into the stomach without the pyloric sphincter tied ( p ¼ 0.005).
3.3. Distribution of heparin in rat stomach In order to better understand the movement of heparin through the rat stomach, stomach washes and dissected stomach layers were examined at 15 min and 4 h after administration of heparin by stomach tube (Fig. 1). When heparin content of the forestomach was examined, more heparin was found at 15 min and 4 h compared to controls ( p ¼ 0.01 and 0.024, one-tailed t-test). Although there was a reduced amount recovered at 4 h compared to 15 min, this difference did not reach significance ( p ¼ 0.073, two-tailed t-test). Similar results were observed when the stomach mucosa from the glandular portion was considered where heparin recovered at 15 min and 4 h was significantly different from controls
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
71
Table 2 Recovery of heparin from rat gut tissues 5e15 min following administration of heparin (60 mg/kg) into the stomach or duodenum with the pyloric sphincter tied Treatmenta
Stomach mg/g wet wt
Stomach with pylorus tied (10) Duodenum with pylorus tied (9) Stomach without pylorus tied (7) Control (8) a
Duodenum mg/g wet wt
Jejunum mg/g wet wt
Ileum mg/g wet wt
Colon mg/g wet wt
423.2 211.6
0.0 0.0
0.1 0.1
0.0 0.0
0.0 0.0
0.7 0.7
103.9 51.4
159.4 97.7
17.8 14.7
1.4 1.4
1418.7 455.2
0.8 0.6
0.6 0.6
0.0 0.0
0.0 0.0
3.9 3.9
4.5 1.7
0.5 0.3
3.0 1.3
1.1 0.7
Number of animals per group in parentheses.
( p ¼ 0.013 and 0.031, respectively, one-tailed t-test). There was no significant difference in heparin recovery at 4 h compared to 15 min ( p ¼ 0.59, two-tailed t-test). When the muscle layers of the glandular stomach were considered, there was significantly more heparin recovered at 15 min and 4 h after oral administration than stomach muscle from rats given saline ( p ¼ 0.011 and p ¼ 0.024, respectively one-tailed t-test). Although there was some indication that amounts recovered from the muscle layer increased with time when means were considered (Fig. 1), there was no significant difference in the concentration of heparin recovered at 15 min versus 4 h ( p ¼ 0.403, two-tailed t-test). Stomach washes were also analysed for heparin content at 15 min and 4 h after administration. Heparin recovered from stomach washes at 15 min, expressed as mg/mg wet weight of total stomach, was significantly greater than that found in
control samples and at 4 h after heparin administration ( p ¼ 0.027 and 0.044, respectively, one tailed t-test). Amounts found at 4 h did not differ significantly from controls ( p ¼ 0.124, one-tailed t-test). Amounts found in the stomach washes at 15 min and 4 h after administration accounted for 14% and 2% respectively of the original administered dose. Additional heparin was removed from the lumen with salt washes. For the 15 min treated group the average salt wash was 24.9% of the total stomach wash and 3.5% of the total original administered dose. For the 4 h group the average salt wash was 83.7% of the total stomach wash and 1.93% of the total original administered dose. The aortic endothelium, portal vein and lung were also examined for heparin in these same rats. Heparin was recovered from endothelium and the lungs at 15 min and 4 h after and from the portal vein at 15 min but not 4 h after administration (Table 3).
A. Tissue 0.4
B. Washes 3.5
Forestomach Glandular Stomach
0.35
a
Mucosa
3.0 a
Muscle 0.30 2.5
µg/mg wet weight
a 0.25
2.0 0.20 1.5 0.15
a 1.0
a
0.10
a ab
0.5
0.05 a 0
Control
15 min
4h
0
Control
15 min
4h
Fig. 1. Heparin recovered from rat forestomach and glandular stomach layers and stomach washes at different times following administration of unfractionated heparin by stomach tube. Mean S.E.M. are shown for six rats. Washes are the total heparin recovered from water and sodium chloride washes. a - Significantly greater than control values, one-tailed t-test; b- significantly different than 15 min, two-tailed t-test.
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
72
4. Discussion Heparins are commonly used for the prevention of postoperative thromboembolism, treatment of stroke, for dialysis, and for management of angina. Possible uses of heparin, based on laboratory experiments, include the prevention of atherosclerosis [13]; adjuncts to chemotherapeutic agents [14,15]; as anti-inflammatory [16] and antiviral agents [17]; for control of angiogenesis [18]; treatment of hypertension[19]; fibromyalgia [20]; and Crohn’s disease [21,22]. These possible uses would require long term repeated administration for which an oral heparin would be ideally suited. Although oral heparin is considered ineffective, results from our laboratory and others have indicated that heparin is absorbed following administration by the oral route [23e25]. This observation has been supported by evidence of oral absorption of related polyanionic compounds such as chondroitin sulphate [26] and sucrose octasulphate [27]. Despite these observations, the site and mechanism of absorption of polyanions are not understood. This is particularly challenging considering that heparin and related compounds have high charge and molecular weight and are thus considered unlikely to be absorbed. We chose the stomach as a possible site since peak endothelial concentrations were seen at 6 min after oral administration in previous studies, suggesting that absorption is occurring rapidly following placing the drug in the stomach [28]. Other studies have suggested that heparin can easily cross membranes and has been shown to be effective following intrapulmonary and dermal routes [29e31]. In the present study, we used a model where the stomach and duodenum were isolated by tying the pyloric sphincter. Our results show that, within 15 min of administration, endothelial and plasma heparin concentrations are greater when heparin is placed in the isolated stomach than when injected in the duodenum. Our analysis of the gastrointestinal tract shows that as expected in this model, heparin is confined to the stomach when administered by stomach tube but when injected into the duodenum heparin is found in the jejunum and ileum as well as the duodenum (Table 2). These observations thus suggest that the stomach is an important site of heparin absorption. These results agree with those of Sue et al who showed a significant increase in whole blood clotting time and Factor Xa inhibition 1 h after heparin administration (240e260 mg/kg) in the stomach versus the duodenum of mice [32]. We have demonstrated the same evidence of stomach absorption using one-fourth the dose (60 mg/kg) by
Table 3 Recovery of heparin from aortic endothelium, portal vein and lung following administration of heparin (60 mg/kg) to rats by stomach tube Treatment
Portal vein mg/mg wet weight
Lung mg/mg wet weight
Endothelium mg/cm2
Control Heparin 15 min Heparin 4 h
0.00 0.00 0.13 0.06 0.00 0.00
0.00 0.00 0.19 0.08 0.01 0.01
0.00 0.00 0.08 0.08 0.12 0.08
examining the endothelium as well as plasma and by measuring the chemical amount of the drug present instead of the anticoagulant response in plasma. The increased sensitivity of measurement of heparin with endothelium is not surprising considering the plasma to endothelial ratios are shown to be on the order of 1000 to 1 following heparin administration [28]. Our results suggest that the intestine is a poor absorption site for heparin since although heparin was located in the duodenum, jejunum and ileum the amount found on endothelium was less than when heparin was placed in the stomach with and without tying of the pyloric sphincter. The poor absorption of heparin in the intestine has been previously observed by others [1,33]. To further study the distribution of heparin in the stomach, heparin was administered to rats by stomach tube and layers of the stomach were examined for heparin at 15 min and 4 h after administration. These times were selected since evidence of absorption was seen within 15 min of administration in previous studies and since most of the 14C-label was seen in stomach tissue versus other parts of the gut at 4 h after administration. The dose selected was 60 mg/kg and although this dose is considered high it was to insure that chemical heparin could be recovered from the tissues in detectable concentrations. The stomach was washed first with distilled water and then with 1 M sodium chloride. Washes with sodium chloride were done to remove any heparin that was electrostatically bound to the mucosal surface. Heparin was recovered from all washes and stomach layers at 15 min and 4 h after administration. Heparin recovered from distilled water washes were 14.1% of the administered dose at 15 min and 2.3% of dose at 4 h, indicating that most of the heparin was gone from the lumen by 4 h. Heparin recovered in sodium chloride washes was 3.5% and 1.9% of the total administered dose but 25% and 84% of the total washings at 15 min and 4 h, respectively. Therefore at 4 h most of the heparin found in the stomach washings was bound to the mucosal surface and not free in the lumen. The stomach was divided into a forestomach, glandular stomach mucosa and glandular stomach muscle sections. The rat, in contrast to humans, has a forestomach (non glandular portion) adjacent to the oesophagus where the mucosa is covered with a cornified stratified squamous epithelium [34]. The mucosa of the forestomach is difficult to separate from the underlying muscle layers and therefore the forestomach was collected as a separate section containing both mucosa and muscle layers. Considerably less amounts of heparin were found in the forestomach portion than in the other layers and less was found at 4 h than 15 min, suggesting the forestomach is likely not a site of heparin uptake. In contrast, the glandular stomach in the rat is similar to that in humans and can be used as a model for human disease [35]. The glandular stomach was sectioned into two portions; a mucosal portion, that was in contact with the lumen, and the deeper muscular layer. Heparin was found in the mucosal portion at both 15 min and 4 h after administration suggesting that heparin is taken up by the mucosa. Heparin could also be recovered
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
from the muscle layers at 15 min and 4 h after administration. Since this layer is not in contact with the lumen, absorption of heparin by the stomach is supported. In general, the mean heparin concentration in the muscle layer at 4 h was twice the concentration at 15 min while the mean heparin concentration in the mucosal layer at 4 h was 4/5 that at 15 min. This suggests movement from the mucosa into the muscle. Heparin could also be found in the portal vein and lung at 15 min and from the aortic endothelium at 15 min and 4 h after placing heparin in the stomach. This agrees with previous observations and demonstrates that orally administered heparin is absorbed and rapidly moves into the circulation [9]. The continued presence of heparin in the stomach and the inability to detect chemical heparin in the portal vein and lung at 4 h suggests that after a rapid distribution phase a much slower movement of heparin occurs from the stomach tissue into the circulation. This is supported by previous studies showing that 14 C and chemical heparin levels were greatest on endothelium from 6 to 15 min following administration with only trace amounts detected at 30 min, and 1, 4, and 24 h after oral administration [9]. In conclusion, these results agree with our previous findings that heparin is absorbed following oral administration. Our observations suggest that the stomach may play an important role in the absorption of heparin and possibly other polyanions following oral administration thus absorption in the stomach should be considered when designing polyanionic compounds. Further studies are required to determine the mechanism of heparin absorption. Acknowledgment This work was supported by a grant from the Heart and Stroke Foundation of Saskatchewan. References [1] Kim SK, Vaishali B, Lee E, Lee S, Lee YK, Kumar TS, et al. Oral delivery of chemical conjugates of heparin and deoxycholic acid in aqueous formulation. Thromb Res 2006;117:419e27. [2] Jaques LB, Hiebert LM, Wice SM. Evidence from endothelium of gastric absorption of heparin and of dextran sulfates 8000. J Lab Clin Med 1991; 117(2):122e30. [3] Hiebert LM, Wice SM, Ping T. Tissue distribution of the low molecular weight heparin, tinzaparin, following administration to rats by the oral route. Biomed Pharmacother 2004;58:372e80. [4] Hiebert LM, Wice SM, Ping T, Herr D, Laux V. Antithrombotic efficacy in a rat model of the low molecular weight heparin, reviparin sodium, administered by the oral route. Thromb Haemost 2001;85:114e8. [5] Hiebert LM, Wice SM, Ping T. Increased plasma anti-Xa activity and recovery of heparin from urine suggest absorption of orally administered unfractionated heparin in human subjects. J Lab Clin Med 2005;145: 151e5. [6] Hiebert LM, Wice SM, Jaques LB. Antithrombotic activity of oral unfractionated heparin. J Cardiovasc Pharmacol 1996;28:26e9. [7] Hiebert LM. Evidence that orally administered heparins are absorbed. In: Vossoughi J, Fareed J, Mousa SA, Karanian JW, editors. Thrombosis research and treatment. Bench to bedside. Washington DC: Medical and Engineering Publishers; 2004. p. 553e63.
73
[8] Berkowitz SD, Marder VJ, Kosutic G, Baughman RA. Oral heparin administration with a novel drug delivery agent (SNAC) in healthy volunteers and patients undergoing elective total hip arthroplasty. J Thromb Haemost 2003;1:1914e9. [9] Hiebert LM, Wice SM, Ping T, Hileman RE, Capila I, Linhardt RJ. Tissue distribution and antithrombotic activity of unlabeled or 14C-labeled porcine intestinal mucosal heparin following administration to rats by the oral route. Can J Physiol Pharm 2000;78:307e20. [10] Hiebert LM, Jaques LB. The observation of heparin on endothelium after injection. Thromb Res 1976;8:195e204. [11] Jaques LB, Wice SM, Hiebert LM. Determination of absolute amounts of heparin and of dextran sulfate in plasma in microgram quantities. J Lab Clin Med 1990;115(4):422e32. [12] Jaques LB. Determination of heparin and related sulfated mucopolysaccharides. Methods Biochem Anal 1977;24:203e312. [13] Engelberg H. Actions of heparin in the atherosclerotic process. Pharmacol Rev 1996;48:327e52. [14] Engelberg H. Actions of heparin that may affect the malignant process. Cancer 1999;85(2):257e72. [15] Levine MN, Lee AY, Kakkar AK. From Trossseau to targeted therapy: new insights and innovations in thrombosis and cancer. J Thromb Haemost 2003;1:1456e63. [16] Gorski A, Wasik M, Nowaczyk M, Korczak-Kowalska G. Immunomodulating activity of heparin. FASEB J 1991;5:2287e91. [17] Bagasra O, Lischner HW. Activity of dextran sulfate and other polyanionic polysaccharides against human immunodeficiency virus. J Infect Dis 1988;158:1084e7. [18] Folkman J, Weisz PB, Joullie MM, Li WW, Ewing WR. Control of angiogenesis with synthetic heparin substitutes. Science 1989;243:1490e3. [19] Mandal AK, Lyden T, Saklayen MG. Heparin lowers blood pressure: Biological and clinical perspectives. Kidney Int 1995;47:1017e22. [20] Berg D, Berg LH, Couvaras J, Harrison H. Chronic fatigue syndrome and/or fibromyalgia as a variation of antiphospholipid antibody syndrome: an explanatory model and approach to laboratory diagnosis. Blood Coagul Fibrinolysis 1999;10:435e8. [21] Prajapati DN, Newcomer JR, Emmons J, Abu-Hajir M, Binion DG. Successful treatment of an acute flare of steroid-resistant Crohn’s colitis during pregnancy with unfractionated heparin. Inflamm Bowel Dis 2002; 8:192e5. [22] Ang YS, Mahmud N, White B, Byrne M, Kelly A, Lawler M, et al. Randomized comparison of unfractionated heparin with corticosteroids in severe active inflammatory bowel disease. Aliment Pharmacol Ther 2000; 14:1015e22. [23] Hiebert LM. Oral heparins. Clin Lab 2002;48:111e6. [24] Gorski A, Lagodzinski Z. Oral heparin prolongs survival of skin allografts. Arch Immunol Ther Exp 1991;39:557e62. [25] Engelberg H. Orally ingested heparin is absorbed in humans. Clin Appl Thromb Hemost 1995;1(4):283e5. [26] Bucsi L, Poor G. Efficacy and tolerability of oral chondroitin sulfate as a symptomatic slow-acting drug for osteoarthritis. Osteoarthritis Cartilage 1998;6:31e6. [27] Hiebert LM, Wice SM, Ping T, Hileman R, Polat T, Linhardt RJ. Tissue distribution of [14C] sucrose octasulfate following oral administration to rats. Pharm Res 2002;19:838e44. [28] Hiebert LM, Wice SM, McDuffie NM, Jaques LB. The heparin target organ e the endothelium. Studies in a rat model. Q J Med 1993;86:341e8. [29] Mahadoo J, Wright CJ. Intrapulmonary heparin as a prospective anticoagulant regimen. In: Fareed JW, Messmore HL, Fenton JW, Brinkhous KM, editors. Perspectives in hemostasis. Permagon Press; 1981. p. 233e40. [30] Bick RL, Ross ES. Clinical use of intrapulmonary heparin. Semin Thromb Hemost 1985;11:213e7. [31] de Moerloose P, Gavillet O, Salomon P, Reber G. Heparin-related activity in peripheral venous blood after percutaneous application of various glycosaminoglycan creams. Blood Coagul Fibrinol 1992;3:827e8. [32] Sue TK, Jaques LB, Yuen E. Effects of acidity, cations and alcoholic fractionation on absorption of heparin from gastrointestinal tract. Can J Physiol Pharm 1976;54:613e7.
74
L.M. Hiebert et al. / Biomedicine & Pharmacotherapy 61 (2007) 68e74
[33] Brayden D, Creed E, O’Connell A, Leipold H, Agarwal R, LeoneBay A. Heparin absorption across the intestine: effects of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate in rat in situ intestinal instillations and in Caco-2 monolayers. Pharm Res 1997;14:1772e9. [34] Brown HR, Hardisty JF. Oral cavity, esophagus, and stomach. In: Boorman GA, Eustis SL, Elwell MR, Montgomery CA,
Mackenzie WF, editors. Pathology of the Fischer rat: reference and atlas. San Diego: Academic Press; 1990. p. 9e29. [35] Ichikawa T, Ishihara K, Kusakabe T, Hiruma H, Kawakami T, Hotta K. CGRP modulates mucin synthesis in surface mucus cells of rat gastric oxyntic mucosa. Am J Physiol Gastrointest Liver Physiol 2000;279: G82e9.