A Mixed Micelle Formulation for Oral Delivery of Vitamin K

Pharm Res (2016) 33:2168–2179 DOI 10.1007/s11095-016-1954-9 RESEARCH PAPER A Mixed Micelle Formulation for Oral Delivery of Vitamin K Feilong Sun 1 & Tessa C. C. Jaspers 1 & Peter M. van Hasselt 2 & Wim E. Hennink 1 & Cornelus F. van Nostrum 1 Received: 1 April 2016 / Accepted: 23 May 2016 / Published online: 31 May 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com ABSTRACT Purpose To develop a stable micellar formulation of vitamin K for oral delivery, because the commercial and clinically used formulation of vitamin K (Konakion® MM) destabilizes at gastric pH resulting in low bioavailability of this vitamin in neonates with cholestasis. Methods Mixed micelles composed of EPC, DSPE-PEG 2000 and glycocholic acid, with and without vitamin K, were prepared by a film hydration method. The influence of pH on the stability of the micelles was analyzed by dynamic light scattering (DLS). The critical micelle concentration (CMC) was determined by fluorescence spectroscopy using pyrene and the morphology was evaluated by transmission electron microscopy . Caco-2 cells were used to study the cytocompatibilty. Results Mixed micelles with mean diameters from 7.1 to 11.0 nm and a narrow size distribution (PDI < 0.2) were obtained after 3 membrane extrusion cycles. Konakion® MM formed aggregated particles at gastric pH, which was avoided through steric stabilization by introducing PEG. TEM showed that mixed micelles had a spherical size (diameter of around 10 nm) with a narrow size distribution in agreement with the DLS results. The loading capacities for vitamin K of mixed micelles with varying molar fractions of DSPE-PEG and EPC (from 0/100 to 50/50 (mol/mol)) were 10.8–5.0 w%, respectively. The mixed micelles showed good Electronic supplementary material The online version of this article (doi:10.1007/s11095-016-1954-9) contains supplementary material, which is available to authorized users. cytocompatibility at concentrations of glycocholic acid between 0.12 and 1.20 mM. Conclusions Mixed micelles with superior stability to Konakion® MM at low pH were obtained by introducing DSPE-PEG 2000. These are therefore attractive oral formulations for vitamin K. KEY WORDS Konakion® MM . Mixed micelles . PEGylation . pH stability . Vitamin K ABBREVIATIONS EPC DSPE-PEG 2000 TEM DLS CMC VKDB MM PP V Ao L FaSSGF PMS XTT Egg phosphatidylcholine 1,2-distearoyl-sn-glycero-3phosphoethanolamineN-[methoxy(polyethyleneglycol)-2000] Transmission electron microscopy Dynamic light scattering The critical micelle concentration Vitamin K deficiency bleeding Mixed micelles Packing parameter Molecular volume Polar surface cross-area Length of the hydrophobic chain Fasted state simulated gastric fluid N-Methyl dibenzopyrazine methylsulfate Sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium] -bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) * Cornelus F. van Nostrum INTRODUCTION 1 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, The Netherlands 2 Department of Pediatrics, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands Vitamin K is an essential cofactor for γ-glutamyl carboxylase, an enzyme that catalyzes the carboxylation of glutamic acid residues in a number of proteins that are involved in the blood A Mixed Micelle Formulation for Oral Delivery of Vitamin K coagulation (1). In healthy adults, bile that is secreted into the intestine facilitates the solubilization, transport and uptake of vitamin K present in food (2). Therefore, dietary deficiency of vitamin K is rare in adults, except for patients suffering from intestinal disorders and malabsorption (3). On the other hand, vitamin K deficiency does frequently occur in neonates due to limited transport of vitamin K through the placental barrier and low concentration in breast milk (main exogenous source of vitamin K in neonates) (4). This in turn is associated with the risk of life-threatening vitamin K deficiency bleeding (VKDB), which is due to the low activity of vitamin Kdependent coagulation factors (II, VII, IX, X) (5). Konakion® mixed micelles (MM) Paediatric is a formulation composed of vitamin K, egg phosphatidylcholine (EPC or lecithin) and glycocholic acid (6). It is indicated for both the prophylaxis and treatment of VKDB in neonates as well as infants. However, orally administered Konakion® MM fails to prevent VKDB in cholestatic infants due to impaired intestinal absorption of vitamin K (7), which is due to the pathophysiological conditions in the upper gastrointestinal tract of those infants. Glycocholic acid has a carboxylic acid group with a pKa of 3.8 (8), which ensures a good colloidal stability of the mixed micelles above pH > ~4. However, Konakion® MM is unstable and forms large aggregates at low pH of the stomach because of the protonation of the carboxylate group of glycocholic acid, eventually causing coalescence of the formulation (9). After stomach passage, large aggregates of lipids and coalesced vitamin K enter the duodenum, which under normal physiological conditions can subsequently be solubilized by endogenous bile salts (10,11), pancreatic lipases and elevated pH above the pKa of glycocholic acid (12,13). On the other hand, once large aggregates are formed in the stomach of neonates suffering from cholestasis, coalesced vitamin K cannot be solubilized due to the very low level of bile salts in these patients (13,14). Mixed micelles composed of phospholipids and bile salts were first described by Hoffman and Borgstrom (15) as associated amphiphilic biliary and dietary components. The hydrophobic tails of EPC molecules are positioned in the core of the mixed micelles to minimize their contact with water molecules while their polar head groups are present at the interface with water (16). It has been further shown that phospholipids self-assemble into bilayered structures in aqueous solution, whereas mixed micelles are formed in the presence of bile salts (16). These structures can solubilize poorly watersoluble drugs in the inner micellar core (17). Furthermore, mixed micelles are thermodynamically stable with sizes usually ranging from 5 to 60 nm (18). Particles with such small diameters are necessary to transport vitamin K to the surface of enterocytes and to retain mixed micelles at the base of microvilli (19). The aim of this study was to develop a mixed micellar formulation of vitamin K that is stable at low pH and 2169 therefore suitable for oral administration. Therefore, part of EPC of the Konakion® MM formulation was substituted by a PEGylated lipid (DSPE-PEG 2000). The rationale behind this idea is that PEGylated phospholipids are known to act as a steric barrier stabilizing nanostructures such as liposomes (20,21), micelles (22), disks (23) and dendritic nanocarriers (24), and will thereby enhance the stability of drug loade (...truncated)