Enhanced antithrombotic effects of unfractionated heparin in rats after repeated oral doses and its relationship to endothelial heparin concentration.

BACKGROUND AND PURPOSE: An oral, single dose of 7.5 mg kg(-1) of unfractionated heparin (UFH) reduces thrombosis by 50% in a rat model of venous thrombosis. As long-term use is required clinically, our objectives were to study the antithrombotic effects following repeated oral UFH administration. EXPERIMENTAL APPROACH: Bovine lung UFH was administered by oral gavage to rats in 3 doses of 7.5 mg kg(-1) each 12, 24, 48, and 72 h apart; and in 3 or 15 doses of 1 mg kg(-1) every 48 h. The last dose was given immediately after thrombus initiation where 10% formalin in methanol was applied to the jugular vein. The vessel was examined for thrombosis 4 h later. Amounts of heparin in tissue and endothelium, and plasma anticoagulant activity were measured. KEY RESULTS: When 3 x 7.5 mg kg(-1) heparin was given, thrombotic incidence was most reduced at 48 h dose-intervals and was significantly less than single dose treatment. There was a negative correlation between endothelial heparin content and thrombotic incidence, but not anticoagulant activity. When 3 doses of 1 mg kg(-1) every 48 h were given, thrombotic incidence was similar to single dose treatment. When 15 doses were given, total thrombotic incidence was less than for 3 doses and was similar to that after s.c. administration. CONCLUSIONS AND IMPLICATIONS: Antithrombotic activity increased with repeated doses of oral UFH, with antithrombotic effects similar to s.c. administration. Antithrombotic activity was related to heparin on endothelium.

Antithrombotic efficacy in a rat model of the low molecular weight heparin, reviparin sodium, administered by the oral route.

Previous studies in rats show that unfractionated heparin and the low molecular weight heparin logiparin have a dose-dependent antithrombotic effect and are found in endothelium and plasma when administered orally. Objectives of the present study were to determine if similar evidence of absorption could be observed with oral reviparin sodium. Thrombosis incidence was determined 4 h after application of 10% formalin in methanol to the exposed jugular vein. A dose-dependent antithrombotic effect was observed when 0.01 to 7.5 mg/kg (20 rats/group) was administered by stomach tube immediately following thrombus initiation. Thrombotic incidence was also significantly reduced when 0.025 mg/kg was given 4 and 2 h prior to, immediately after, and 2 and 3 h following thrombus initiation. Reviparin was recovered from endothelium and plasma in trace amounts at all doses. At 0.025 mg/kg, peak aortic endothelial reviparin concentrations were found at 1 and 2 h and peak plasma anti-Xa activity was detected at 2 h. Trace amounts of plasma TFPI were found only at 8 h after administration. Dose-dependent antithrombotic activity and recovery from endothelium and plasma support the hypothesis that orally administered reviparin sodium is absorbed.

Tissue distribution and antithrombotic activity of unlabeled or 14C-labeled porcine intestinal mucosal heparin following administration to rats by the oral route.

Distribution and antithrombotic activity of orally administered unfractionated porcine heparin were studied. [14C]Heparin was prepared by de-N-acetylation of porcine mucosal heparin followed by re-N-acetylation, using [14C]acetic anhydride. [14C]Heparin and (or) cold heparin (60 mg/kg) were administered by stomach tube to male Wistar rats. Blood, all levels of gut and gut contents, liver, lung, spleen, kidney, and aortic and vena caval endothelium were collected under deep anesthesia at 3, 6, 15, 30, and 60 min and 4 and 24 h (6 rats/group) after administration. Urine and feces were collected at 24 h, using metabolic cages. In three additional rats, drugs were administered in gelatin capsules. Tissues listed above and tongue, esophagus, trachea, brain, heart, thymus, bile ducts, vena caval and aortic walls, ureters, bladder, samples of muscle, skin, hair, and bone marrow were collected at 24 h. Radioactivity and chemical heparin, measured by agarose gel electrophoresis, were observed in all tissues examined as well as gut washes, plasma, urine, and feces. Radiolabel recovered was confirmed to be heparin by autoradiograms of gradient polyacrylamide electrophoretic gels. [14C]Heparin and chemical heparin in gut tissue suggest a transit time of 4 h. Porcine or bovine heparin (7.5 mg/kg), administered by stomach tube, decreased the incidence of thrombosis induced by applying 10% formalin in 65% methanol to the exposed jugular vein of rats. Heparin isolation from non-gut tissue, endothelium, urine, and plasma and the observed antithrombotic effect are consistent with oral bioavailability.

Antithrombotic activity of orally administered low molecular weight heparin (Logiparin) in a rat model.

Previous studies in rats demonstrated that orally administered, unfractionated bovine lung heparin is absorbed and has a dose-dependent antithrombotic effect. The objective of this study was to determine if an oral low molecular weight heparin had a similar antithrombotic effect in the same model. Thrombosis was induced in rats by application of 10% formalin in 65% methanol to the exposed jugular vein. Immediately following, saline, unfractionated heparin (3.3-60 mg/kg) or the low molecular weight heparin, Logiparin (0.025-15 mg/kg; 20-30 rats per group) was placed in the stomach and 4 h later the jugular vein was inspected for a thrombus. Compared to saline, oral Logiparin reduced the incidence of thrombosis at all doses with a dose-dependent effect suggested. A significant increase was observed in the activated partial thromboplastin time and in plasma heparin concentrations, determined by Accuclot Heptest and anti-factor Xa chromogenic assay for rats given oral Logiparin versus saline. A dose-dependent increase in plasma heparin concentration was observed when estimated by the anti-Xa chromogenic assay. Heparin was recovered in 9% of aortic endothelial samples when > or = 0.8 mg/kg Logiparin was administered. A 50% reduction in thrombosis was observed at 0.1 mg/kg for oral Logiparin versus 7.5 mg/kg for unfractionated bovine lung heparin indicating that oral Logiparin is an effective antithrombotic agent at doses lower than unfractionated heparin. Orally administered low molecular weight heparin may be useful for the prevention and treatment of thrombosis.

Orally administered dextran sulfate is absorbed in HIV-positive individuals.

Preliminary in vivo studies suggested that oral dextran sulfate was poorly absorbed, but investigations were limited by inadequate methods for measuring the drug in the body. To determine absorption in HIV-positive subjects, hydrogenated dextran sulfate, average molecular weight 8000 (Usherdex 8), was orally administered in a short-term (single dose, 4 g/day for 5 days, 7 subjects) and in a long-term study (1 g, 4 times per day for 29 to 335 days, 8 subjects), which was a continuation of the short-term study with the inclusion of an additional subject. When an agarose gel electrophoresis technique with toluidine blue staining was used, the drug was recovered from plasma (67%, peak 2.2 microg/mL) and circulating peripheral blood lymphocyte (PBL) samples (50%, peak 333 microg/L blood) obtained at 5 and 15 minutes and 1, 3, 6, and 24 hours after the first day's dose and from plasma (56%) and PBL samples (38%) obtained 5 minutes after administration on 4 subsequent days in the short-term study. In the long-term study, the drug was found in plasma (67%, peak 2.4 microg/mL) and PBL samples (25%, peak 126 microg/L blood) obtained at monthly visits within 4 hours of the last dose. The drug was found in all urine samples from all subjects in both studies (short-term study, 24-hour samples up to 4 days after the final dose; long-term study, monthly samples within 4 hours of the last dose). In the long-term study, bone marrow preparations from 3 subjects showed metachromatic inclusions present in reticular cells when the cells were stained with toluidine blue, indicating the presence of sulfated polyanions. A significant rise in activated partial thromboplastin time and a drop in platelet count (P < .025) were demonstrated, with thrombocytopenia developing in 3 patients. Mild-to-moderate gastrointestinal disturbances were experienced by 6 subjects in the short-term study and by all subjects in the long-term study. One subject experienced mild central nervous system symptoms in the short-term study. These results indicate that dextran sulfate is absorbed after oral administration; therefore, further studies on its efficacy, particularly in the early stages of the disease, along with additional observations on its toxicity, are warranted.

Antithrombotic activity of oral unfractionated heparin.

Although heparin is believed to be poorly absorbed orally, we recently demonstrated that oral heparin rapidly enters the circulation, with most of the drug being taken up by endothelium. To determine the effective antithrombotic dose of oral heparin, we induced thrombosis by applying 10% formalin in 65% methanol to exposed rat jugular vein. Saline or heparin, at doses ranging from 3.25 to 60 mg/kg, was immediately placed in the stomach; 4 h later, the vein was inspected for a thrombus. A dose-dependent decrease in thrombosis was observed with oral heparin. Although there was little change in anticoagulant activity as measured by the activated partial thromboplastin time (APTT) of plasma samples taken 4 h after administration, a significant dose effect was demonstrated by regression analysis. Heparin could be demonstrated chemically in 52% of plasma samples and in 38% of aortic or vena caval endothelial samples. A significant dose effect was observed in aortic endothelial heparin concentrations, with amounts 1,000-fold that determined in plasma. These results indicate that oral heparin exhibits antithrombotic activity in a dose-dependent manner, with low levels in plasma.

The heparin target organ--the endothelium. Studies in a rat model.

Plasma levels of the antithrombotic drug heparin, as estimated by coagulation tests, are a poor indicator of antithrombotic effectiveness. The interaction of heparin with endothelium is a poorly studied but important factor in the clinical activity of heparin. This study describes the interaction of heparin with endothelium, following intragastric administration. The concentrations of heparin in endothelium and plasma were determined by gel electrophoresis following administration of heparin to rats by various routes. Heparin concentrations in endothelium versus plasma were approximately 100 times greater following intravenous or ex vivo administration and more than 1000 times greater when administered by intrapulmonary, subcutaneous, intraperitoneal and intragastric routes indicating that the route of administration affects the distribution of the drug. At 2.4 and 6 min after intravenous administration, 88 and 51% respectively of the administered dose was found associated with endothelium. Heparin was rapidly absorbed following intragastric administration and could be detected associated with endothelium at 2.4 min. At 6 min less than 1% of the administered dose was found in plasma, and 45% was associated with endothelium. These results show that endothelium is the main site of heparin distribution. Heparins could also be detected in cellular and pericellular fractions of cultured porcine aortic endothelial cells when 125I-heparin was added to medium. Bound radioactivity was released to medium from both cellular and pericellular fractions suggesting that heparin taken up by endothelium can be released. Intragastric administration of heparin and dextran sulphates significantly prevented thrombus formation in a rat model of thrombosis without significant changes in activated partial thromboplastin times.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence from endothelium of gastric absorption of heparin and of dextran sulfates 8000.

Heparin, hydrogenated dextran sulfate 8000 (Usherdex 8), and dextran sulfate 8000 were administered to rats, and the total drug was separated and determined in endothelium and plasma. A large amount of each drug was recovered from endothelium 2.4 and 6 minutes after intravenous injection. This accounted for the drug missing from plasma. The drugs in water were placed in the stomach by catheter. All three drugs were recovered from the endothelium and identified unchanged by electrophoresis and specific staining. The amounts that were recovered at 2.4 and 6 minutes were equivalent to most of the drug administered. Thus heparin, Usherdex 8, and dextran sulfate 8000 enter the body immediately on oral administration. At longer time intervals after intravenous and oral administration, much of each drug was not demonstrable in endothelium by the method used. Some drug could be detected in endothelium after 4 hours. After oral administration, plasma levels of each drug were rarely more than 0.5% of the dose. Formalin-alcohol was applied to the jugular veins of anesthetized rats to produce a thrombus, (see Blake et al. J Clin Path 1959;12:118-22) and the drugs were immediately introduced into the stomach. Four hours later the injured veins were inspected for thrombi. Incidence of thrombotic plug was 80% in rats that received saline solution, 4% with Usherdex 8, 0% with dextran sulfate 8000, and 0% with heparin. Usherdex 8, dextran sulfate 8000, and heparin demonstrate low, moderate, and high in vitro anticoagulant activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of absolute amounts of heparin and of dextran sulfate in plasma in microgram quantities.

Heparin and dextran sulfates 8000 are separated from citrated plasma by absorption on epichlorohydrin triethanolamine cellulose columns followed by elution with 1.1 and 1.4 mol/L NaCl in 0.05 mol/L glycine-HCl buffer. The eluate is desalted with Sephadex G25-40, dried, and dissolved in water. A 1 microliters sample is applied to an agarose gel slide. After electrophoresis, the slide is fixed and stained with toluidine blue. The sulfated polysaccharide band(s) is identified by relative electrophoretic migration. The total amount of drug is estimated by matching its optical density with that of a band on one of a set of slides with graded amounts of heparin or dextran sulfate. The reaction with toluidine blue measures the total polyelectrolyte, not just the small proportion of the drug with anticoagulant activity. Pooled normal plasma showed a trace of chondroitin and no heparin. Recovery of heparin and hydrogenated dextran sulfate that was added to pooled normal plasma was complete (lowest concentration tested was 10 micrograms/ml); however, recovery for unhydrogenated dextran sulfate declined consistently by 9 micrograms/ml for concentrations below 50 micrograms/ml, setting a limit for its recovery. Plasma samples taken from patients for coagulation tests were examined by this procedure, and in so doing, steps were ascertained to improve the procedure for routine use. Results were compared with values for prothrombin time and activated partial thromboplastin times obtained on the same samples by the clinical laboratory. Because the procedure provides an independent parameter for measurement in patients who have received heparin therapy, insight into different patient responses to the drug is therefore possible. With minor modifications, the procedure can be used for heparans, dermatans, and chondroitins, because it allows identification and microscale quantitation on the basis of charge, molecular weight, and carbohydrate structure.

Heparin and sulfated mucopolysaccharides--a micro system for quantitative differentiation and determination.

Heparin (Hep), hyaluronic acid, chondroitins (sulfate) A, B, and C, and heparins (sulfate) A, B, C, and D were subjected to microelectrophoresis in barbital-agarose gel, fixed with cetylpyridinium chloride and stained with toluidine blue. The optical densities of the resulting bands were compared with optical densities obtained upon reaction with azure A in aqueous solution and with the carbazole reagent. A linear relation was obtained between optical density and concentration of purified sulfated mucopolysaccharide (SMP). Less than 1 microgram of Hep and 2 microgram of other SMPs are required for measurement by electrophoresis, while about 30 microgram of each is required with the carbazole reagent. The optical density of a mixture of SMPs was equal to the sum of the densities for the individual SMPs upon microelectrophoresis. It was demonstrated that the individual SMPs in mixtures were distinguishabed by reaction with specific enzymes and by changes in migration in agarose with barbital, phthalate, ethylenediamine, or propanediamine buffers, permitting ready demonstration and quantitation of various SMP species. Examples are shown of the application of the procedure to measure the total SMPs and individual SMPs in tissue extracts. The method is sensitive, reproducible, flexible, and measures quantities 1/30th of those measured colorimetrically, yet is relatively unaffected by protein, carbohydrate, or inorganic electrolytes.