Heparin and low-molecular-weight heparin (enoxaparin) significantly ameliorate experimental colitis
Article first published online: 7 JUL 2008
Background and aims:
The anticoagulants, unfractionated heparin and low-molecular-weight heparin, demonstrated anti-inflammatory effects in animal models and in humans. Because of its dual effects, high-dose heparin was proposed as a therapeutic modality for ulcerative colitis. We investigated whether a low dose of low-molecular-weight heparin—enoxaparin (Clexane, Rhône-Poulenc Rorer, France)—ameliorates the inflammatory response in two models of experimental colitis.
Colitis was induced in rats by intrarectal administration of dinitrobenzene sulphonic acid. Enoxaparin (40, 80 and 200 μg/kg) or unfractionated heparin (100, 200 and 400 U/kg) were administered subcutaneously immediately after the induction of damage. Enoxaparin, 80 μg/kg, was also administered after induction of colitis by intrarectal administration of iodoacetamide. Rats were sacrificed 1, 3 or 7 days after induction of injury. Colonic damage was assessed macroscopically and histologically. Mucosal prostaglandin E2 generation, myeloperoxidase and nitric oxide synthase activities and tumour necrosis factor-α levels in blood were determined.
Enoxaparin and heparin significantly ameliorated the severity of dinitrobenzene sulphonic acid- and iodoacetamide-induced colitis as demonstrated by a decrease in mucosal lesion area, colonic weight and mucosal myeloperoxidase and nitric oxide synthase activities. The dose–response curve had a bell-shaped configuration: enoxaparin, 80 μg/kg, and unfractionated heparin, 200 U/kg, were the optimal doses.
Low-dose enoxaparin and unfractionated heparin ameliorate the severity of experimental colitis. This effect is related to their anti-inflammatory rather than anticoagulant properties.
Low-molecular-weight heparins are preparations made by partial hydrolysis or enzymatic degradation of unfractionated heparin.1, 2 They are considered to be as effective an anticoagulant as unfractionated heparin and to have better pharmacodynamic and pharmacokinetic profiles. Clinical data suggest that low- molecular-weight heparins are safer than unfractionated heparin.3 It was recently found that, like unfractionated heparin, low-molecular-weight heparins possess anti-inflammatory properties, independent of their anticoagulant properties.4, 5 The anti-inflammatory effects of low-molecular-weight heparins were demonstrated in both animal models of inflammation and in human diseases. Low-molecular-weight heparin significantly decreased hepatic damage in immune-mediated concanavalin-A hepatitis in mice.6 It reduced myocardial inflammation and collagen deposition in a model of chronic Coxsackie virus B3-induced myocarditis in A/J mice.7 Heparin disaccharides and low-molecular-weight heparins inhibited the delayed-type hypersensitivity reactivity and the severity of arthritis in a model of delayed-type hypersensitivity in balb/c mice and in a model of adjuvant arthritis in Lewis rats.8 The anti-inflammatory effect was achieved in low doses, i.e. 5 μg/mouse and 20 μg/rat.9 Modified heparin, with diminished anticoagulant activity, also inhibited complement activation in a guinea pig model.10 In humans, single11 or 4–6-weekly12 3 mg doses of the low-molecular-weight heparin enoxaparin (Clexane, Rhône-Poulenc Rorer, France) were effective in decreasing contact sensitivity11 and lichen planus12—T-cell-mediated disorders. The common feature of most of these models is the aberrant immune response, mainly due to imbalance in the activation of T cells. In inflammatory bowel diseases, there is also imbalance in T-cell responsiveness, whether exaggerated T-helper-1 (Th1) cytokine profile, as in Crohn’s disease, or Th2, as suggested in ulcerative colitis.13 The aetiology of inflammatory bowel disease is not yet known, and treatment is mainly empirical and non-specific, e.g. 5-aminosalicylic acid compounds, steroids and immunosuppressives. Anecdotal reports recently suggested that heparin and low-molecular-weight heparin in high, anticoagulant doses have therapeutic effects in inflammatory bowel disease, mainly in ulcerative colitis, both in severe refractory disease as well as in moderate disease.14–19 The suggested mechanism was a combination of anticoagulant and anti-inflammatory effects, based on the microcirculatory changes reported in inflammatory bowel disease, mainly in Crohn’s disease,20and on the tendency towards a hypercoagulable state21 and thromboembolic phenomena.22 Recently, monotherapy with high-dose heparin was found to be ineffective in the treatment of moderate to severe ulcerative colitis.23
In the present study, we hypothesized that, as in other inflammatory models and diseases, low-dose low- molecular-weight heparin (enoxaparin) will have a beneficial effect in a model of colitis in rats, which has many common features with inflammatory bowel disease. Such an effect was demonstrated: macroscopic damage, inflammatory mediators and cytokines were inhibited by a low, non-anticoagulant dose of enoxaparin and unfractionated heparin.
MATERIALS AND METHODS
All the studies used male Wistar rats weighing 200–250 g and fed ad libitum. The local ethics committee approved the study.
Dinitrobenzene sulphonic acid (DNBS)-induced colitis
Inflammation of the colon was induced by a single intrarectal administration of 0.25 mL of 50% ethanol containing 30 mg of DNBS. The solution was introduced by a catheter with an outer diameter of 0.3 mm placed 7 cm proximal to the anus. Treated rats received a single subcutaneous injection of enoxaparin, 40, 80 or 200 μg/kg, or unfractionated heparin, 100, 200 or 400 U/kg, immediately after induction of colitis by DNBS.
Before they were sacrificed at 1, 3 and 7 days after induction of injury, rats underwent a laparotomy and a blood sample was drawn from the descending aorta for the determination of tumour necrosis factor-α (TNF-α). The colon was isolated, placed unstretched on a ruler and a 10 cm segment of the distal colon was resected and weighed after rinsing the lumen with saline. Mucosal damage was measured macroscopically and expressed in mm2/rat. Tissue samples from the most damaged site were obtained for histological assessment, and the mucosa was scraped and samples of it were processed to determine myeloperoxidase and nitric oxide synthase activities and eicosanoid generation. All measurements were performed blindly by a researcher who was unaware of the treatment group.
Iodoacetamide (IA)-induced colitis
Colitis was induced by intrarectal instillation of 0.1 mL IA 3% in methylcellulose as described earlier.24 Treated rats received a single subcutaneous injection of enoxaparin, 40, 80 or 200 μg/kg, immediately after the induction of damage. Rats were sacrificed after 1, 3 or 7 days and damage was assessed by weight and lesion area of the distal 10 cm of colon and by myeloperoxidase activity. Nitric oxide synthase activity and prostaglandin E2 generation in mucosal scrapings were determined on day 7.
Control rats for each model were treated with saline only after damage induction.
Determination of myeloperoxidase activity
Two hundred milligrams of colonic mucosal scrapings were homogenized with a polytron in ice-cold hexadecyltrimethylammonium bromide (0.5%) in 50 mm phosphate buffer, pH 6.0. The homogenate was sonicated for 10 s, freeze–thawed three times and centrifuged for 15 min. An aliquot of the supernatant was taken for the determination of the enzyme activity, according to the method of Bradley et al.25
Determination of nitric oxide synthase activity
Nitric oxide synthase activity was monitored by the conversion of (14C)-l-arginine to citrulline, according to the method of Bush et al.26 One hundred milligrams of colonic mucosal scrapings were homogenized with a polytron for 30 s at 4 °C in 0.9 mL of ice-cold 50 mm tris-HCl, pH 7.4, containing 0.1 mm ethylenediaminetetra-acetic acid (EDTA), 0.1 mm ethyleneglycoltetra-acetic acid (EGTA), 0.5 mm dithiothreitol and 1 mm phenylmethylsulphonyl fluoride. Homogenates were centrifuged at 20 000 ×gfor 60 min at 4 °C and the supernatant was used as the source of nitric oxide synthase. Enzymatic reactions were conducted at 37 °C in 50 mm tris-HCl, pH 7.4, containing 100 μm l-arginine, 100 μm NADPH, 1 mm EGTA, 8 mm valine, 0.2–0.4 mg supernatant protein and approximately 200 000 dpm of (14C)-l-arginine HCl (296 mCi/mmol) (Amersham, UK) to a final volume of 100 μL. Enzymatic reactions were terminated by the addition of 2.0 mL of ice-cold ‘stop buffer’: 20 mm sodium acetate, pH 5.5, 1 mm citrulline, 2 mm EDTA and 0.2 mm EGTA. Citrulline was determined by applying the samples to columns (diameter, 1 cm) containing 1.0 mL of Dowex AG50W-X8, the sodium form, which had been pre-equilibrated with stop buffer. Columns were eluted with 4 × 1.0 mL of water collected into scintillation vials. Opti-fluor 10 mL was added to each vial and samples were counted in a Packard Tri-Carb liquid ion spectrometer. Citrulline was recovered in the first 4 mL of the Dowex column eluted to the extent of 96 ± 2%.
Determination of eicosanoid generation
Mucosa (150 mg) was placed in tubes containing 1.0 mL of phosphate buffer (50 mm, pH 7.4), minced with scissors and centrifuged for 60 s. The pellet was re-suspended in 1.0 mL of the buffer and incubated for 1 min in a vortex mixer, after which 10 μg of indomethacin was added and the tubes were centrifuged for 60 s. The supernatants were kept at −20 °C until radioimmunoassays were performed. Prostaglandin E2 generation was determined using a radioimmunoassay kit, as previously described.27 The capability of the mucosa to generate prostaglandin E2 was expressed as nanograms per gram wet tissue weight.
Measurement of serum TNF-α
Serum TNF-α was measured using a specific immunoassay (Amersham RPN 2734). The minimum detecs dose of the enzyme-linked immunoabsorbent assay kit is < 10 pg/mL.
Colonic segments were fixed in phosphate-buffered formaldehyde, embedded in paraffin wax and 5 μm sections were prepared in the usual manner. Tissues were stained with haematoxylin and eosin and blindly evaluated by a pathologist whose expertise was gastrointestinal pathology. Histological scoring was determined by examining each specimen for the following features of damage: depth of necrosis, extent of necrosis, inflammation, extent of inflammation and fibrosis, and allocating increasing points according to the severity of the finding (0=no damage, to 4=extensive/severe damage). The scores for each category examined were added up for each specimen to obtain the total score.
l-Arginine, l-citrulline, NADPH, valine, dithiothreitol, phenylmethylsulphonyl fluoride, EDTA, EGTA and DNBS were purchased from Sigma Israel Chemicals Ltd, Rehovot, Israel. Dowex AG50W-X8 (sodium form), 100–200 mesh, was purchased from Bio-Rad, Richmond, CA, USA. (3H)-Prostaglandin E2, TNF-α and (14C)-l-arginine were purchased from Amersham International, Buckinghamshire, UK. Enoxaparin (Clexane) was purchased from Rhône-Poulenc Rorer, France and heparin sodium was purchased from Kamada, Beit-Kama, Israel.
Data are expressed as the mean ± s.e. Statistical analysis for significant differences was performed according to Student’s t-test for unpaired data and the non-parametric Mann–Whitney U-test. A P value of < 0.05 was considered to be significant.
Intracolonic administration of DNBS/ethanol or IA resulted in extensive ulcerative damage. During laparotomies at days 1, 3 and 7, colons were thickened and adhered to surrounding organs. When cut open, red, oedematous and ulcerated regions were noticed in the exposed areas. The lesion area and colonic weight reflect the extent of these macroscopic characteristics of colitis in both models. The colons of control rats were intact.
In order to determine the optimal enoxaparin dosage, a series of dose–response experiments was performed. Figure 1 demonstrates that enoxaparin, 80 μg/kg, is the most effective dose. This was most pronounced 3 days after damage induction, when rats to which DNBS alone was introduced had a lesion area of 568 ± 55 mm2/rat, while those treated with enoxaparin, 80 μg/kg, after damage induction had a lesion area of 125 ± 43 mm2/rat (P < 0.05). Treatment with enoxaparin, 40 or 200 μg/kg, was not effective as demonstrated by lesion areas of 607 ± 36 and 821 ± 113 mm2/rat, respectively. This bell-shaped effect, with 80 μg/kg being more effective than the lower dose of 40 μg/kg and the higher dose of 200 μg/kg, was demonstrated also in myeloperoxidase activity and colonic weight (although for weight there is no statistical significance). The bell-shaped effect of enoxaparin dosage was demonstrated in the IA model as well. Three days after damage induction, the lesion area was 651 ± 104 mm2/rat, while rats treated with enoxaparin, 80 μg/kg, subcutaneously immediately after IA introduction had a lesion area of 226 ± 77 mm2/rat only. Treatment with either 40 or 200 μg/kg of enoxaparin was ineffective, as demonstrated by lesion areas of 637 ± 138 and 642 ± 162 mm2/rat, respectively, and by smaller decreases in myeloperoxidase activity (Figure 2).
Figure 1. The effect of enoxaparin dose on lesion area and myeloperoxidase activity in dinitrobenzene sulphonic acid (DNBS)- induced colitis. The dose of 80 μg/kg is more efficient than the lower and higher doses in decreasing colonic damage. Colitis was induced by intrarectal administration of 30 mg DNBS in 0.25 mL of 50% ethanol. Several doses of subcutaneous enoxaparin were injected immediately after damage induction. Rats were sacrificed on day 3, the mucosal lesion area was measured and the myeloperoxidase activity was determined. Results are the mean ± s.e. at each dosage. MPO, myeloperoxidase.
Figure 2. The effect of enoxaparin dose on lesion area and myeloperoxidase activity in iodoacetamide (IA)-induced colitis. The dose of 80 μg/kg is more efficient than the lower and higher doses in decreasing colonic damage. Colitis was induced by intrarectal administration of 0.1 mL 3% IA dissolved in 1% methyl cellulose. Several doses of subcutaneous enoxaparin were injected immediately after damage induction. Rats were sacrificed on day 3, the mucosal lesion area was measured and the myeloperoxidase activity was determined. Results are the mean ± s.e. at each time interval. MPO, myeloperoxidase.
Based on this set of experiments, a dosage of a single subcutaneous injection of 80 μg/kg enoxaparin immediately after damage induction was chosen as the treatment regimen. This treatment significantly ameliorated colonic damage in both models. Tables 1 and 2 demonstrate the effect of enoxaparin on macroscopic indices of damage in DNBS and IA models of colitis, respectively. In the DNBS model, an early beneficial effect is demonstrated. Lesion area and colonic weight are significantly decreased in the treatment group already after 24 h. This effect lasts at all time points (although the lower colonic weight on day 3 in the treated rats is not statistically significant). The decrease in macroscopic damage was accompanied by a statistically non-significant decrease in histological damage score from 11.27 ± 4.83 to 8.25 ± 4.77, 7 days after damage induction. The histological damage is demonstrated in Figure 3. The lesions in the DNBS group were severe ulcers and transmural necrosis (Figure 3A), while rats treated with enoxaparin, 80 μg/kg, after damage induction had much milder lesions in their mucosa (Figure 3B): only focal erosive colitis was demonstrated and no ulcers were noted. The protection provided by enoxaparin was accompanied by a significant decrease in colonic myeloperoxidase activity, with values on days 1 and 3 of 2.3 ± 0.4 U/g vs. 4.2 ± 0.9 U/g and 1.8 ± 0.4 U/g vs. 5.6 ± 1.2 U/g, respectively, in the treated vs. untreated rats. In the IA model, the beneficial effect is delayed: the lesion area decreases significantly to 226 ± 77 vs. 651 ± 104 mm2/rat on day 3, and the colonic weight of treated vs. untreated rats decreases significantly to 1.1 ± 0.1 vs. 1.95 ± 0.16, respectively, on day 7. Myeloperoxidase activity is lower at all time points in the treated vs. untreated rats, but a statistically significant decrease is demonstrated only on day 7.
Figure 3. Histological damage. (A) Rat colon with dinitrobenzene sulphonic acid (DNBS)-induced colitis. The damage involves severe transmural necrosis with heavy inflammatory infiltrate and severe ulcers. (B) A typical example of a rat colon with DNBS-induced colitis treated with enoxaparin. Only mild, focal erosive colitis without ulcers or necrosis is demonstrated.
As compared with the colonic nitric oxide synthase activity of normal rats (3.46 ± 0.15 nmol.min/g), the activity in the DNBS group was significantly increased at all time points, while in the enoxaparin-treated group it was significantly lower at all time points. It was most pronounced on day 3 (Table 1) when enoxaparin-treated rats had activities of 3.9 ± 0.2 nmol.min/g (n=6), while non-treated rats had activities of 24.3 ± 0.4 nmol.min/g (n=7) (P < 0.05). The significant decrease in colonic nitric oxide synthase activity in the enoxaparin-treated rats is demonstrated in Figure 4: 64% and 84% decreases are demonstrated 24 and 72 h, respectively, after damage induction. In IA colitis, mucosal nitric oxide synthase activity was higher in colitic rats (30 ± 4.4 nmol.min/g, n=18) vs. normal controls (2.7 ± 0.2 nmol.min/g, n=19). A significant decrease to 1.6 ± 0.2 nmol.min/g (n=9) was demonstrated on day 7 in treated rats (P < 0.05).
Figure 4. Colonic nitric oxide synthase activity in dinitrobenzene sulphonic acid (DNBS)-induced colitis. Colitis was induced by intrarectal administration of 30 mg DNBS in 0.25 mL of 50% ethanol. Subcutaneous enoxaparin, 80 μg/kg, was injected immediately after damage induction. Rats were sacrificed on days 1, 3 or 7 and mucosal nitric oxide synthase activity was determined. Results are the mean ± s.e. at each time interval. NOS, nitric oxide synthase.
In the DNBS model, mucosal prostaglandin E2 generation in the enoxaparin-treated rats vs. untreated rats was significantly lower on day 3, being 283 ± 38 ng/g vs. 446 ± 48 ng/g (P < 0.05). In the IA model, enoxaparin-treated rats had significantly lower prostaglandin E2 levels (33 ± 6 ng/g, n=9) vs. untreated rats (149 ± 17 ng/g,n=18) (P < 0.05) on day 7.
Tumour necrosis factor-α levels, measured in the DNBS model, were significantly lower in enoxaparin-treated vs. untreated rats on day 7, being 96 ± 11 pg/mL vs. 181 ± 38 pg/mL, respectively (P < 0.05).
In order to investigate whether the beneficial properties of enoxaparin also existed in the unfractionated preparation, heparin, 100, 200 or 400 U/kg, was injected subcutaneously into rats immediately after induction of damage by DNBS. A bell-shaped phenomenon was also demonstrated with unfractionated heparin. A dose of 200 U/kg was most beneficial, as demonstrated in Table 3. The lesion area in unfractionated heparin-treated rats (200 U/kg) vs. the DNBS alone group was significantly reduced as of day 3 (Table 4). A statistically non-significant decreased colonic weight was also demonstrated. Mucosal myeloperoxidase activity, reflecting neutrophil accumulation and activity, was significantly decreased on days 1 and 3 in the unfractionated heparin-treated vs. DNBS-induced colitic rats, similar to the results achieved in the DNBS-induced colitic rats treated with enoxaparin.
The data presented in this paper suggest that a single low dose (80 μg/kg) of the low-molecular-weight heparin preparation, enoxaparin (Clexane), significantly ameliorated experimental colitis in two murine models. Improvement was demonstrated in both macroscopic and microscopic indices of damage in rats treated with enoxaparin after colitis induction by DNBS vs. untreated rats. The macroscopic improvement was reflected in the significant decreases in lesion area and colonic weight as of day 1. The decrease in inflammatory activity in the mucosa was reflected by the significantly lower mucosal myeloperoxidase and nitric oxide synthase activities and prostaglandin E2 generation. A beneficial effect of enoxaparin, 80 μg/kg, was also demonstrated in IA-induced colitis.
The experimental approach, using a single dose of enoxaparin or unfractionated heparin, is based on previous data from both animal models of inflammation and human disease, in which once-weekly doses were sufficient to ameliorate inflammation.8, 11, 12 It is hypothesized that the mechanism involves heparin degradation products which inhibit inflammatory cell recruitment and the generation of inflammatory mediators by the interaction of minute concentrations with cell surface receptors. The concentrations of enoxaparin and unfractionated heparin used were indeed minute, and demonstrated a unique dose–response curve, 80 μg/kg of enoxaparin being more effective than the lower dose of 40 μg/kg and the higher dose of 200 μg/kg, for both the DNBS and IA models of colonic inflammation (Figures 1 & 2). A similar curve was demonstrated for unfractionated heparin in the DNBS model, in which 200 U/kg was more effective than the lower (100 U/kg) and higher (400 U/kg) doses. The phenomenon of a bell-shaped dose–response curve, where a certain optimal dose is more effective than lower and higher doses, has been reported in other models of low-molecular-weight heparins in inflammation.8, 28 While the reasons for this phenomenon are not yet known, several mechanisms may be responsible. One involves interaction between heparin fragments and their receptors: while lower doses do not, or only partially, activate the receptors, ‘ideal’ doses fully activate them, and ‘overdoses’ may either saturate or de-sensitize the receptors, thus contributing no additional effect, or may even lessen the effect by binding to functionally different receptors.
As the best results were achieved with low doses of enoxaparin (80 μg/kg) and unfractionated heparin (200 U/kg), it is unlikely that the anticoagulant property had a significant role. In studies evaluating the haemorrhagic effects of several low-molecular-weight heparins in Wistar rats, the dose of enoxaparin used (according to the manufacturers’ recommendations for high-risk situations) was 55 antifactor Xa IU/kg,29 and a significant increase in haemorrhage occurred only with doses higher than 500 antifactor Xa IU/kg.30 In the work presented, rats received a dose of only 8 antifactor Xa IU/kg. Furthermore, other authors have used low doses of low-molecular-weight heparin and heparin, or modified heparin, devoid of anticoagulant properties and have demonstrated significant anti-inflammatory properties.31, 32 Therefore, we assume that the mechanism of the anti-inflammatory property is different from that of the anticoagulant property, and is related to factors such as the number of O-sulphate groups on the molecule, chain length and charge, as has been suggested previously.31, 32
By what mechanism do heparin fragments ameliorate mucosal damage in experimental colitis? One explanation involves the reduced production of inflammatory mediators, such as eicosanoids and nitric oxide, or the down-regulation of TNF-α production by T lymphocytes and macrophages. Our results, which demonstrate a decrease in mucosal myeloperoxidase and nitric oxide synthase activities, prostaglandin E2 generation and TNF-α blood levels in DNBS rats treated with enoxaparin, support this possibility. Other authors have described decreased TNF-α levels after heparin treatment, such as in haemodialysis patients who have elevated levels of TNF-binding protein-I after exposure to heparin,33 or after heparin disaccharides, made by enzymatic cleavage of heparin by the enzyme heparinase 1, were used to treat delayed-type hypersensitivity in rodents.8, 34 The effect of enoxaparin might be similar to the effect of these disaccharides. Another possible mechanism is the inhibition of migration of inflammatory cells, such as lymphocytes and polymorphonuclears, to the point of colonic injury. The extravasation of activated leucocytes and their infiltration of adjacent inflammatory sites are associated with the secretion of extracellular matrix degrading enzymes, such as heparinase. This enzyme is able to degrade heparan sulphate moieties of the extracellular matrix,35, 36 thus generating disaccharides with immunomodulatory properties. Heparan sulphate and heparin preparations (due to their similarity to heparan sulphate) inhibit heparinase-mediated degradation of heparan sulphate moieties of the extracellular matrix37 and thus inhibit the migration of blood-borne cells to extracellular sites.31, 38 Heparin oligosaccharides were also found to bind to L- and P-selectins, another possible way of preventing immune cell migration out of blood vessels, thus preventing acute inflammation.32 Because the induction of experimental immune-mediated colitis requires extravasation and migration of activated leucocytes, heparin and low-molecular-weight heparin could decrease the severity of DNBS- and IA-induced colitis by this mechanism. The histological picture of dense inflammatory infiltrate after DNBS administration and of only scattered lymphocytes following enoxaparin treatment (Figure 3), as well as the decrease in mucosal myeloperoxidase activity, a marker of activated neutrophils, in treated vs. untreated rats, could be indirect supportive evidence for this mechanism.
A third possibility has recently been suggested: basic fibroblast growth factor is a heparin-binding, anti-ulcerogenic growth factor. The serum level is increased in active inflammatory bowel disease (mainly Crohn’s disease),39 but its protective effect is decreased due to decreased levels of an essential component of its receptor, the cell surface heparan sulphate proteoglycan, syndecan-1.40 Heparin fragments might serve as substitutes for syndecan-1, restoring the ability of the ulcerated mucosa to bind basic fibroblast growth factor and to heal.41, 42 Other authors have demonstrated the importance of this mechanism in colonic mucosal healing.43 Although this was not directly evaluated in the present work, the advantage of enoxaparin over unfractionated heparin may suggest that its binding to the fibroblast growth factor receptor is more efficient than that of unfractionated heparin, as could be expected due to its more homogeneous nature. It is most likely that the ameliorating effect of enoxaparin and unfractionated heparin in colitis is a combination of the three mechanisms described.
DNBS-induced colitis is an established model of inflammatory bowel disease in rats and has many similarities to human inflammatory bowel disease, mainly Crohn’s disease.44 In order to demonstrate a general anti-inflammatory effect, and one which is not model-dependent, we also used a model of IA-induced colitis, in which damage induction is biochemical rather than hapten-induced as in the DNBS model. The evidence of a beneficial effect of enoxaparin in both models suggests that it has anti-inflammatory effects independent of the pathogenic process. The delayed effect of enoxaparin in the IA vs. DNBS model may be due to different intrinsic properties of the two models, as damage in the DNBS model is more pronounced earlier, therefore enabling the beneficial effect to be demonstrated earlier.
To the best of our knowledge, this is the first report evaluating the effects of the low-molecular-weight heparin, enoxaparin, on DNBS- and IA-induced colitis. Other authors have examined the effects of unfractionated heparin on experimental colitis.45, 46 Fries et al. used a trinitrobenzene sulphonic acid model in the rat, but employed higher doses of heparin, i.e. 500 U/kg t.d.s., assuming that the beneficial effect was an anticoagulant one,45 therefore choosing microangiography as the main way of demonstrating an effect. In these experiments, indices of inflammation, such as colonic weight and macroscopic damage, were not significantly different when rats were treated with heparin vs. untreated rats. We demonstrated that heparin’s activity is achieved in much lower doses and that it reduces significantly the lesion area, colonic weight and myeloperoxidase activity. Korzenik et al. used high-dose heparin (1200 U/kg) to treat mice with dextran sulphate sodium colitis.46 There was no amelioration of damage with this dosage, again supporting the concept of the better effect of lower doses, as demonstrated by us.
Our results indicate that low doses of enoxaparin and heparin may be used to treat experimentally induced colitis. More studies are needed in order to determine the exact regimen required and to clarify the mechanism involved.
Fareed J, Jeske W, Hoppenstadt D, Clarizio R, Walenga JM. Are the available low-molecular weight heparin preparations the same? Semin Thromb Hemostas1996; 22(Suppl. 1): 77–91.
Fuss IJ, Neurath M, Boirivant M, et al. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J Immunol 1996; 157: 1261–70.
Gaffney PR & Gaffney A. Heparin therapy in refractory ulcerative colitis—an update. Gastroenterology 1996; 110: A913–A913 (Abstract).
Folwaczny C, Wiebecke B, Loeschke K. Unfractionated heparin in the therapy of patients with highly active inflammatory bowel disease. Gastroenterology 1999;116: G31119–G31119 (Abstract).
Korzenick JR, Robert ME, Bitton A, et al. A multi-center, randomized, controlled trial of heparin for the treatment of ulcerative colitis. Gastroenterology 1999;116: G3264–G3264 (Abstract).
Vrij AA, Schon EJ, Henker HC, Stockbrugger RW. Low molecular weight heparin (LMWH) treatment in steroid refractory ulcerative colitis (U.C).Gastroenterology 1999; 116: G3655–G3655(Abstract).
Wakefield AJ, Dhillon AP, Rowles PM, et al. Pathogenesis of Crohn’s disease: multifocal gastrointestinal infarction. Lancet 1989; ii: 1057–62.
Sharon P, Ligumsky M, Rachmilewitz D, Zor U. Role of prostaglandins in ulcerative colitis: enhanced production during active disease and inhibition by sulfasalazine. Gastroenterology 1978; 65: 638–40.
Mazner Y, Bar-Ner M, Yahalom J, Ishai-Michaeli R, Fuks Z, Vlodavsky I. Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparinase from human neutrophils. Possible role in invasion through basement membranes. J Clin Invest 1985; 76: 1306–13.
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