Heparin in vitro permeation study through human skin

The anticoagulant properties of Unfractionated Heparin (UFH) as well as its efficacy in the treatment and prevention of deep venous thrombosis (DVT), pulmonary embolism (PE) and acute arterial occlusion are well known. Superficial Venous Thrombosis (SVT) is considered a relatively benign disease usually treated with elastic stockings and nonsteroidal anti-inflammatory drugs to reduce pain and inflammation. In patients at risk of DVT or PE, subcutaneous injections of Low Molecular Weight Heparins (LMWHs) are considered a valid therapeutic option in spite of the risk of bleeding.1,2 A topical UFH spray solution (UFH-ss) available on the market (Viatromb®, Lipohep®) showed efficacy comparable to subcutaneous LMWHs in reducing both thrombus size and pain/inflammation in patients with SVT without particular safety concerns.1,3 More recently, a study of UFH-ss in the maintenance of patency of the arterovenous fistula confirmed the local activity of this topical formulation in another clinical setting.4 In spite of these evidences, an in vitro investigation based on a biological assay published in 2001 concluded that “UFH is found not to permeate human epidermal membrane which represents the major skin permeation barrier”.5 Pharmacokinetic characterization of systemically administered UFH is a difficult task. Information is available from radiolabeled products and, indirectly, from pharmacodynamic effects. For a topical UFH product the situation is even more difficult and proof of drug penetration through the skin is still an unsolved issue. Due to the high molecular weight, hydrophilic properties and high negative charge, the ability of UFH to penetrate the skin is reduced. Moreover, the complex nature of this substance and the unavailability of a sensitive physico-chemical analytical method does not allow to demonstrate that UFH permeates the skin. Among the available techniques, capillary electrophoresis is a method suitable for the analysis of intact UFH in pharmaceutical formulations, but it lacks the sensitivity required for a skin permeation study. Other approaches involving HPLC require breaking down UFH by either digestion using heparin lyases6 or chemical hydrolysis7 followed by analysis. The aim of this study was to evaluate the in vitro permeation of UFH through human epidermis. Vertical Franz diffusion cells were used to investigate the skin permeation after 8h and 24h using UFH-ss as donor phase. The cumulative amount of UFH in the receiver phase was indirectly determined by controlled hydrolysis with 2M trifluoroacetic acid at 95°C for 2h and subsequent analysis of glucosamine by a LC/MS method. The LC separation was carried out using hydrophilic interaction liquid chromatography with acetonitrile ammonium formate buffer as mobile phase. The MS detection was based on glucosamine pseudomolecular ion (m/z 180) and allows the quantification of UFH in the range from 0.05 to 1.2 I.U./mL. The dosed glucosamine at the considered time points and, consequently the UFH permeated amount corresponding to about 0.5 I.U./mL at 24h, support that UFH is able to permeate the human epidermis.