Influence of chemical and structural features of low molecular weight heparins (LMWHs) on skin penetration S. Franzè, C.G.M. Gennari, P. Minghetti, F. Cilurzo * Department of Pharmaceutical Sciences – Università degli Studi di Milano, Via Giuseppe Colombo 71, 20133 Milan, Italy A R T I C L E I N F O Article history: Received 16 December 2014 Received in revised form 30 January 2015 Accepted 1 February 2015 Available online 2 February 2015 Keywords: LMWHs Human epidermis Skin penetration Circular dichroism Chain flexibility A B S T R A C T Low molecular weight heparins (LMWHs) are obtained from unfractionated heparin (UFH) through different depolymerization methods (DM), which produce compounds having specific chemical features and biological activity. It is then supposed that LMWHs also exhibit different skin permeability properties. The current work aimed to get an insight on the in vitro passive diffusion through human epidermis of six commercially available LMWHs in comparison with UFH. The in vitro studies were performed using Franz diffusion cells. Heparins samples were assayed measuring the anti-factor Xa activity. Circular dichroism was used to evaluate the effect of the counter-ion (sodium or calcium) on the chain flexibility. The penetrated amounts after 24 h (Q 24 ) of sodium LMWHs were related to M w by an exponential relationship (R = 0.758). The flux resulted dependent by DM following the rank order: b-elimination (8–11 mIU/cm 2 h range) > deaminative cleavage (5–7 mIU/cm 2 h range) > radical depo- lymerization (0.1 mIU/cm 2 h). Finally, the calcium ion, reducing the chain flexibility, significantly affected the Q 24 (0.001  0.000 and 0.157  0.049 IU/cm 2 for calcium and sodium nadroparin, respectively). Both the lower M w and the introduction of new residues at the chain ends improved the skin penetration of LMWHs with respect to UFH (Q 24 = 0.001  0.001 IU/cm 2 ), with bemiparin and enoxaparin being the most interesting compounds. ã 2015 Elsevier B.V. All rights reserved. 1. Introduction Low molecular weight heparins (LMWHs) are anticoagulant and antithrombotic drugs, mainly used in the prevention and treatment of deep vein thrombosis (DVT) after major surgical procedures. They have almost one third of the molecular mass of unfractionated heparin (UFH) and exhibit a lower affinity for plasma proteins thus providing more predictable effects, better pharmacokinetic parameters and safety profile (e.g. lower risk of bleeding and heparin-induced thrombocytopenia) (Hirsh, 1995). Therefore, they are currently administered to outpatients (Hirsh and Raschke, 2004) replacing in clinical practice the use of UFH. LMWHs available on the market are obtained by four different depolymerization strategies of UFH, namely deaminative cleavage, chemical or enzymatic b-elimination and oxidative cleavage with radical agents. Depending on the specific reaction and the cleavage points along the UFH chain, LMWHs vary for average molecular weight (M w ) and molecular weight distribution, degree of sulfation, and peculiar terminal end residues, which are artifacts of the production process. As an example, LMWHs obtained by deaminative cleavage with nitrous acid present a 2,5-anhydro mannose residue at the reducing end, whereas b-elimination in alkaline conditions produces 4-enopyranose uronate residues at non reducing ends and N-sulfoglucosamine at the reducing end (Table 1) (Guerrini and Bisio, 2012; Linhardt and Gunay, 1999). LMWHs obtained by different depolymerization methods are then dissimilar in structure, and exhibit distinct physical, chemical and biological properties (Minghetti et al., 2013). In the last decade, several efforts were made to propose non- invasive alternatives to subcutaneous route of administration (Motlekar and Youan, 2006). The poor intestinal absorption together with the degradation of heparins both in liver and in gastrointestinal tract make the oral administration not feasible (Qi et al., 2004). Transdermal delivery may represent a valid alternative, even if LMWHs diffusivity through the skin is limited by large M w , hydrophilicity and negative charge (Gomaa et al., 2012). These issues along with the possible strategies to enhance LMWHs permeation through the skin can be easily deduced by literature data (Lanke et al., 2009a). However, since these strategies were * Corresponding author. Tel.: +39 02 503 24635; fax: +39 02 503 24657. E-mail address: francesco.cilurzo@unimi.it (F. Cilurzo). http://dx.doi.org/10.1016/j.ijpharm.2015.02.001 0378-5173/ ã 2015 Elsevier B.V. All rights reserved. International Journal of Pharmaceutics 481 (2015) 79–83 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsev ier.com/locate /ijpharm