Increased Antithrombotic Properties of Unfractionated Heparin in Rats Following Oral Administration of Multiple Doses and Its Correlation with Endothelial Heparin Concentration

Review Article

Thromb Haemost Res. 2023; 7(2): 1093.

“Increased Antithrombotic Properties of Unfractionated Heparin in Rats Following Oral Administration of Multiple Doses and Its Correlation with Endothelial Heparin Concentration”

Hari Prasad Sonwani*

Apollo College of Pharmacy, Durg CG, India

*Corresponding author: Hari Prasad Sonwani Apollo College of Pharmacy, Durg CG, India. Email: harisonwani10@gmail.com

Received: November 09, 2023 Accepted: December 23, 2023 Published: December 30, 2023

Abstract

In a rat model of venous thrombosis, a single oral dose of 7.5mg kg-1 of Unfractionated Heparin (UFH) lowers thrombosis by 50%. Since clinical long-term use is necessary, our goals were to investigate the antithrombotic effects of repeated oral UFH administration. Rats were given three doses of 7.5mg kg-1 every 12, 24, 48, and 72 hours apart—as well as three or fifteen doses of 1 mg kg—1 every 48 hours—by oral gavage of cow lung UFH. The final dosage was injecting 10% formalin in methanol into the jugular vein right after thrombus start. Four hours later, the vessel was checked for thrombosis. Heparin levels in endothelium and tissue as well as plasma anticoagulant activity were assessed. As 3×7.5 When heparin (mg kg-1) was administered, the incidence of thrombosis was notably lower during 48-hour dosing intervals than with a single dose. Endothelial heparin concentration and thrombotic incidence were negatively correlated, while anticoagulant activity was not. Treatment with three doses of 1mg kg-1 every 48 hours resulted in a thrombotic incidence comparable to that of a single dosage. The total thrombotic incidence after 15 doses was comparable to that following s.c. treatment and was lower than that of 3 doses. Oral UFH delivery resulted in increased antithrombotic activity and antithrombotic effects that were comparable to those of s.c. administration. Heparin’s antithrombotic effect on endothelium was linked to it.

Keyword: Heparin; Oral heparin; Venous thrombosis; Rats; Endothelium; Anticoagulant activity

Introduction

Heparins are essential medications that are used to stop blood clots in the veins [7]. Although lower molecular weight derivatives are injected Subcutaneously (s.c. ), Unfractionated Heparins (UFH) are provided via both intravenous and Subcutaneous (s.c.) routes. Heparin is thought to be inappropriate for absorption due to its large size and high negative charge, the low pH of the stomach, and the presence of digestive enzymes [1]. This view is also supported by the fact that there is little change in anticoagulant activity after oral treatment [8]. However, in rat venous and arterial thrombosis models, our research demonstrates that oral heparins prevent thrombosis [17,30]. The venous model showed a 50% decrease in the occurrence of stable thrombi following the gavage administration of 7.5mg-1 kg-1. Heparin may therefore be administered orally. Oral heparin would be especially helpful in situations when long-term administration is needed. It would save costs and make therapy more convenient for the patient by avoiding the hospital stay required for intravenous heparin. Additionally, it would stop haematomas and bruises that happen when s.c. administration is used. Additional heparin uses may also be possible with long-term oral heparin therapy. When administered orally, heparin has been shown to have the following positive effects: avoidance of vascular disease (Engelberg, adjuncts to chemotherapy drugs [26]. Few research have examined the consequences of oral heparin administered repeatedly without the use of delivery agents. Therefore, choosing the right dose for long-term therapy and considering the effects of repeated administration are crucial. This study sought to ascertain whether UFH accumulated in the body and whether it had an antithrombotic effect when administered repeatedly in a rat venous model. Additionally, we wanted to use a rat thrombosis model to determine the most efficacious repeated dosage schedule for oral heparin. Monitoring was also done on recovered heparin and plasma anticoagulant activity in tissues and endothelium.

Techniques

Heparin

Scientific Protein Labs, Division of Viobin Corporation, WI, USA provided the unfractionated bovine lung heparin (156. 2U mg-1), which was dissolved in water.

Animals at 3, 20, and 25 mg m. The Canadian Federation of Biological Societies' Principles of Animal Care were followed in the handling, housing, and conduct of all animal experiments. In total, 283 Wistar male rats, 305±4g (mean±standard error) in weight were acquired from Charles River in terms of the mean (s.e.m.) Prior to treatment, the animals were starved for a full night, and they were given barbital and methoxyflurane anesthesia for the experiments animal prototype. Four hours before the animals were killed; saline and the final dose of heparin were administered. When three repeating doses were administered daily, as well as during a 24-hour period before and on days 29–30, when fifteen repetitive doses were administered, rats were housed in metabolic cages with the purpose of collecting urine and faeces samples. Prior to heparin and on days 15 and 30, the animals were weighed.

Test for Thrombosis

A thrombus was started just before the final heparin dosage using a modified version of Blake et al.'s (1959) technique. The exposed jugular vein was treated with few drops of 10% formalin in 65% methanol, and the incision was then closed. The rat was anesthetized and checked for evidence of external bleeding after four hours. The wound was opened, and a cotton pledget was used to gently push the jugular vein to check for thrombi. If there was a visible thrombus in the jugular vein and there was restricted blood flow, the thrombus was graded as positive (stable thrombus). If the thrombus was visible in the jugular vein and moved when examined with a cotton pledget, the thrombus was considered unstable and the blood flow continued. If there was no visible thrombus and no disruption of blood flow, the thrombus was graded as negative. The same observer, who was not aware of the treatments, conducted the grading. By dividing the total number of events by the total number of thrombotic events, the percent incidence of both stable and unstable thrombi was determined.

Quantity of Animals Seen Receiving Therapy

Tissue collection As soon as the vessel was examined, tissue samples were taken. Samples of blood were drawn from the abdominal aorta in 3.8% sodium citrate (1 part sodium citrate to 9 parts blood). The inferior vena cava and thoracic aorta were excised and immersed in saline as a source of endothelium. Prairie Diagnostic Services, received a blood sample for complete blood counts. Next, plasma from blood samples taken with little tissue trauma was used to calculate the Activated Partial Thromboplastin Time (APTT). Extra aliquots of plasma were frozen in order to further determine the anti-Factor Xa or anti-Factor IIa activity. Lung, kidney, and liver were taken out.

After removal, the colon, ileum, jejunum, stomach, and duodenum were cleaned with distilled water. Frozen tissues and washes were used to extract heparin later on.

Heparin extraction and quantification from tissues and endothelium.

A modified version of a reported technique [15] was used to obtain endothelium. Vases were cut open, pinned lumen side up to dental wax, and then saline washed. Endothelium was removed from the lumenal surface after cellulose acetate membrane (Schleicher & Schuel Bio- Science, GmbH, Dassel, Germany) was applied.

Measuring to the nearest millimeter from the monolayer on cellulose acetate paper, the mean areas of the aorta and vena caval endothelium were 2.60±0.04 and 0.45±0.01 cm2 (mean±s.e.m.), respectively. The membrane made of cellulose acetate was taken from endothelium by centrifuging, removing the supernatant, and repeating the process twice in cold acetone. After the precipitates were allowed to air dry, 10 milliliters of pronase (derived from Streptomyces griseus, Sigma-Aldrich Canada) were added. 40mg kg-1 in 1 M Tris buffer) for 48 hours at 37 degrees Celsius (Limited, Oakville, ON, Canada). After centrifuging the digests for 10 minutes at 8000×g, the supernatant was collected and the precipitate washed twice. After washing 100 milliliters of 26.8% NaCl into the supernatant. Using five volumes of methanol and the supernatant, glycosaminoglycans were precipitated.

precipice was desiccated. Using modified techniques as previously described, heparin was recovered from tissue, gastrointestinal washes, and feces [23]. Following three consecutive dosages, saline washes were utilized to clean the gastrointestinal tissues, and the washes were collected. For the stomach, duodenum, jejunum, ileum, colon, liver, lung, and kidney, the average wet weights of tissue were 1.9, 1.8, 1.9, 1.8, 1.4, 9.1, 2.0, and 1.2 g, respectively. The average weights of the stomach, duodenum, jejunum, ileum, and colon washes were, respectively, 1.1, 0.4, 0.7, 0.8, and 2.7g. These weights were calculated by deducting the difference between the gut weights before and after washing. The total weight of excrement over a 24-hour period was 10.4g on average. Isoproterenol:petroleum ether and acetone were used to defat minced tissues, stomach washes, and excrement. (1:1), then processed at Dried precipitate was used.

According to previously published procedures, heparin was isolated from tissue, gut washes, and feces [23]. Saline was used to wash the gastrointestinal tissues after three consecutive dosages, and the washes were collected. The average tissue wet weights for the stomach, duodenum, jejunum, ileum, colon, liver, lung, and kidney were 1.9, 1.8, 1.9, 1.8, 1.4, 9.1, 2.0, and 1.2g, respectively. For stomach, duodenum, jejunum, ileum, and colon washes, the average weights were 1.1, 0.4, 0.7, 0.8, and 2.7g, respectively. These weights were calculated by deducting the difference in gut weight before and after washing. Over the course of a day, the weight of feces collected averaged 10.4g. Acetone and isoproterenol:petroleum ether were used to defat minced tissues, stomach washes, and feces and processed at (1:1).

At 405 nm, the sample's absorbance was measured. Heparin concentrations in anti-Factor Xa and anti-Factor IIa-based samples were determined by comparing the absorbance of the test samples to a standard curve that was created with known heparin addition quantities to control plasma. It was also tested using the APTT (Biopool, Ventura, CA, USA).

Data Evaluations

The mean±s.e.m. is used to express all results. Data on thrombosis are given in percentage form. The percentage of endothelial samples positive for heparin and thrombotic occurrences were compared using the w2-test for difference between proportions. Prior to analysis, the amounts of heparin in tissues and stomach washes were transformed using a logarithmic function to guarantee comparable variances among the groups. The average and as the antilog, s.e.m. are expressed; hence, positive and adverse are distinct and present in the tables and text. One-way analysis of variance and Tukey's multiple comparison test were used to analyze differences in endothelium and plasma heparin concentrations found by anticoagulant tests. When endothelium and plasma heparin concentrations were taken into account, a single dosage and 30-day controls were merged. A one-tailed t-test was used to compare variations in tissue heparin concentrations. The relationship between mean endothelium concentrations and the overall thrombotic incidence was examined using a Pearsons correlation. Po0.05 probability was regarded as substantial.

Outcomes

To find out if giving heparin orally in three consecutive doses as opposed to a single dose had an accumulative antithrombotic effect, preliminary research was conducted. The intervals between doses were adjusted to determine whether Antithrombotic activity was impacted by this. Furthermore, the effects of 15 doses of 1mg per kg administered by stomach tube, 3 doses of 1 mg per kg administered by oral gavage, and 15 doses of 1mg per kg administered by s.c. injection were compared to see if a cumulative effect could be observed after repeated dosing with oral heparin.

Antithrombogenic Properties

When compared to control rats, the incidence of stable thrombi was much lower after a single oral dose of 7.5mg kg-1 (Figure 1). The combined incidence of stable and unstable thrombi did not differ substantially from that of control rats.