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.

The close relationship of heparin and the vessel wall.

For over 100 years heparin has attracted interest because of its anticoagulant powers. Commercial heparin has now been shown to be a mixture of over 100 different closely related sulfated polysaccharides of which only 10% activate antithrombin-III. Fifty years ago the original research teams in Toronto and Stockholm in demonstrating the clinical uses of heparin observed that antithrombotic activity did not correspond to levels of anticoagulation. It has been shown that: (a) Heparin accumulates rapidly and specifically in the endothelium against a concentration gradient of hundreds- to thousands-fold. (b) Experimental thrombosis, however produced, is accompanied by a marked decrease in the electronegative charge of the vessel wall and the charge is restored in all cases by heparin. (c) The normal electronegative charge is due to glycosaminoglycans. Heparin possesses the strongest electronegative charge of these substances and is present in the vessel wall as a component of a larger heparitin (sulfate) proteoglycan molecule. (d) Maintenance of the normal electronegative charge depends on adequate supply of oxygen (adequate blood flow). (e) Commercial heparin releases enzymes from the endothelium, lipoprotein lipase and histaminase (D.A.O.). Lipoprotein lipase changes the composition of plasma lipids and lipoproteins and histaminase provides a check for fat absorption. The release of these enzymes decrease and prevent atherosclerotic changes. (f) After administration of commercial heparin, heparin isolated from the plasma has higher antithrombin activity than that injected. The heparin taken up by the endothelium is returned with greater activity. The anticoagulant effect of administered heparin does not produce hemorrhage since this requires simultaneous occurrence of defects in the vascular factor of hemostasis (the result of stress or pituitary-adrenal imbalance) or platelet defect. Thus, clinical effectiveness of heparin is an expression of its close relationship to the vessel wall.

Drug prophylaxis in atherosclerosis.

Heparin and heparinoids constitute the one drug group shown to arrest and reverse atherosclerotic changes in rabbits on a high cholesterol/fat diet. Little use has been made of this finding because heparin has been administered by routes which produce anticoagulation and thus bleeding. Inhalation once a fortnight results in a high concentration of heparin in endothelium with low plasma concentrations. No toxicity has been demonstrated with long term heparin but this is not true for heparinoids.

Effect of intrapulmonary heparin on plasma diamine oxidase (histaminase) activity in mice.

Heparin releases diamine oxidase (DAO, histaminase) from binding sites in the intestinal vasculature. Histamine is involved in a number of pathological lesions. In this study we have examined the effect of intrapulmonary administration of heparin on plasma DAO activity in mice. For comparative purposes the same parameter was measured following the administration of an intravenous heparin regimen. The time course and dose-response were examined with the two heparin regimens. The doses of heparin were based on appropriate clinical equivalents. Both heparin regimens showed a dose dependent response (correlation coefficient r = 0.9). The dose of intrapulmonary heparin (10 mg/kg) was 12 times greater than the dose injected intravenously but the DAO response lasted 48 times longer that that obtained from the intravenous heparin regimen.

Reversal of protamine of the prolonged response to intrapulmonary heparin.

A single large dose of heparin (2000 units/kg) was administered to dogs by intratracheal instillation. Whole blood clotting times and plasma heparin concentrations were measured at intervals. At each interval the calculated dose of protamine required to neutralize the circulation heparin was given intravenously and the measurement of plasma heparin concentration repeated. The authors found that the whole blood clotting time was prolonged for 24 to 48 hours and there was a detectable concentration of heparin in the plasma for 96 hours. On each occasion the protamine eliminated the circulation heparin, but more heparin continued to enter the circulation. It is hypothesized that after rapid absorption from the lung, heparin is stored temporarily in a cellular pool throughout the body and then released into the circulation. At any given time the anticoagulant effect can be reversed by intravenously administered protamine sulfate if this should become necessary, but repeated administration would be required. Intrapulmonary heparin may have useful clinical applications but further clinical and laboratory investigations are required.

Effect of intrapulmonary heparin on lipoprotein lipase activity in mice.

The lipoprotein lipase (LPL) activity obtained from the intrapulmonary administration of 2-10 mg of heparin in mice was compared with the same parameter measured for intravenously administered heparin. The doses administered were based on appropriate clinical equivalents. A relatively large dose of intrapulmonary heparin produced a peak LPL activity which was a third of the maximum response obtained from a small dose of i.v. heparin. This was followed by a moderate LPL activity (twice the control level) which persisted for the next 4 days while the response obtained from i.v. administered heparin lasted only 2 h. Both the intrapulmonary and the i.v. administration of heparin produced dose-dependent increases in plasma LPL activity (correlation coefficient r = 0.9). This study indicates that intrapulmonary heparin causes a prolonged antilipemic effect.

Effects of long-term treatment of mice with intrapulmonary heparin.

Intrapulmonary heparin calls for the administration of a single large dose of heparin. This results in a prolonged but low grade heparinemia lasting for many days. These features are quite different the response obtained from any of the currently used heparin regimens. A toxicity study was therefore conducted on mice exposed to heparin aerosol(7ppm) for 20 min, one a week for ten weeks. No deleterious effects were observed on physical and macroscopic examinations of the internal organs as well as on histological examination of tissues from most organs of the body.