Nonclinical Evaluation of Novel Cationically Modified Polysaccharide Antidotes for Unfractionated Heparin

Protamine, the only registered antidote of unfractionated heparin (UFH), may produce a number of adverse effects, such as anaphylactic shock or serious hypotension. We aimed to develop an alternative UFH antidote as efficient as protamine, but safer and easier to produce. As a starting material, we have chosen generally non-toxic, biocompatible, widely available, inexpensive, and easy to functionalize polysaccharides. Our approach was to synthesize, purify and characterize cationic derivatives of dextran, hydroxypropylcellulose, pullulan and γ-cyclodextrin, then to screen them for potential heparin-reversal activity using an in vitro assay and finally examine efficacy and safety of the most active polymers in Wistar rat and BALB/c mouse models of experimentally induced arterial and venous thrombosis. Efficacy studies included the measurement of thrombus formation, activated partial thromboplastin time, bleeding time, and anti-factor Xa activity; safety studies included the measurement of hemodynamic, hematologic and immunologic parameters. Linear, high molecular weight dextran substituted with glycidyltrimethylammonium chloride groups at a ratio of 0.65 per glucose unit (Dex40-GTMAC3) is the most potent and the safest UFH inhibitor showing activity comparable to that of protamine while possessing lower immunogenicity. Cationic polysaccharides of various structures neutralize UFH. Dex40-GTMAC3 is a promising and potentially better UFH antidote than protamine.

then isolated from the solution using the freeze-drying technique. In the case of the derivatives of Dex1-GTMAC and GCD the reaction solution was neutralized with HCl to pH ~7 and then the product was precipitated in cold acetone. The precipitate was rinsed with acetone twice. The resulting product was again washed with methanol (Dex1-GTMAC) or ethanol (GCD derivatives) to completely wash out the sodium chloride and unreacted GTMAC. Purification of the products was confirmed with measurements of the conductivity of the washing liquid. The obtained Dex1-GTMAC and GCD derivatives were dried in a vacuum oven for 24h.

Synthesis of dextran (Dex) and hydroxypropylcellulose (HPC) substituted with N-acrylamidopropyl-N,N,N-trimethylammonium chloride (APTMAC)
The derivatives of dextran (Dex, 40 kDa) and hydroxypropylcellulose (HPC) substituted with APTMAC were synthesized using the general procedure described previously [1]. The experimental conditions of the synthesis are given in Table B. In a three-necked flask 1.5 g of the respective polysaccharide was dissolved in 15 mL of a solvent. The solution was degassed by bubbling with nitrogen for 30 min and a solution of the initiator was added. After 5 min 75 wt% solution of APTMAC in water was added.
The reaction mixture was heated at 70 o C for 3 h under constant mixing with a magnetic stirrer and under bubbling with nitrogen. Then, the mixture was cooled and dialyzed first against the solvent used and after 12 h against a mixture of the solvent and water. The fraction of water had been gradually increased for a week, and finally the dialysis was performed in pure water. The dialysis was carried out against water for 2 more weeks. The polymers obtained (Dex40-APTMAC and HPC-APTMAC) were isolated from the solution using the freeze-drying technique.

Synthesis of dextran (Dex) grafted with poly(allylamine hydrochloride) (PAH)
Allylamine hydrochloride was prepared by dropwise adding 4 g of allylamine to 7 ml of concentrated hydrochloric acid. Then, 1.5 g of Dex (40 kDa) was dissolved in 15 ml water. The Dex solution was degassed by bubbling with nitrogen for 30 min and a solution of the initiator (1.35 g of AAPH in 7.5 mL of degassed water) was added. After 5 min allylamine hydrochloride was added. The reaction mixture was heated at 55 o C for 3 h under constant mixing with a magnetic stirrer and bubbled with nitrogen.
The mixture was then cooled and dialyzed against water in the dialysis tubes (Mw cutoff value of 12 kDa). The obtained product (Dex40-PAH) was isolated from the solution using the freeze-drying technique.

Synthesis of dextran (Dex) grafted with poly(allylamine hydrochloride) (PAH) substituted with arginine (Arg)
The synthesis of dextran (Dex) grafted with poly(allylamine hydrochloride) (PAH) substituted with Larginine (Arg) followed that described previously [2]. To the solution of 0.4 g Dex40-PAH in 10 ml of distilled water 120 mg of EDC, 60 mg of NHS, and 2 g of Arg were added. The mixture was heated to

Synthesis of dextran substituted with spermine (Spm)
The synthesis of dextran (Dex) grafted with spermine (Spm) generally followed that described previously [3]. 0.5 g of Dex (40 kDa) was dissolved in 10 ml of DMSO followed by the addition of 1 g of CDI and 1 g of Spm dissolved in10 ml of DMSO. The reaction mixture was heated at 40°C for 2 h under constant mixing with a magnetic stirrer. Then, the mixture was cooled down and dialyzed in the dialysis tubes (Mw cutoff value of 12 kDa) against DMSO for 12 h against a mixture of DMSO and water. The fraction of water had been gradually increased for a week, and finally the dialysis had been performed against pure water for a week. The insoluble fraction was removed by filtration and the resulting polymer (Dex40-Spm) was isolated from the aqueous solution by freeze-drying.

Solubility of the polymers at physiological pH
Prior to the tests assessing the ability of the synthesized polymers to interact with heparin their solubility in PBS buffer (pH=7.4) was examined by mixing of 1 mg of the test substance and 1 ml of pH 7.4 PBS buffer. The solubility of the compound was checked visually and with a DLS measurement. All the materials, except for Dex40-PAH, were soluble at this concentration and in the DLS measurements no objects larger than 50 nm were found. Therefore, Dex40-PAH was excluded from UFH binding test and biological studies.