Buprenorphine loaded PLGA microparticles: Characterization of a sustained-release formulation

Buprenorphine is a short-acting analgesic drug. Its use in veterinary medicine requires several injections per day to alleviate pain in animals. We, therefore, designed a poly-lactic-co-glycolic acid (PLGA) based depot formulation of the opioid to prolong the analgesic effect. We characterized our novel microparticulate depot formulation with emphasis on a potential future product development in the present work. Microparticles showed low residual moisture levels and did meet bacterial endotoxin requirements defined by the European Pharmacopeia. Depot formulation showed physicochemical stability over 6 months at 4 °C, indicating sufficient shelf life. Reconstituted formulation stored at 4 °C showed unchanged release properties for 24 h. Terminal sterilization by x-rays with a dose of 30 kGy revealed the sensitivity of buprenorphine towards radiation, resulting in substantial drug degradation. However, microparticle properties were not affected. Our experiments indicate that sterilization of the final product is not possible, therefore requiring aseptic manufacturing protocols. In conclusion, our novel buprenorphine depot formulation based on PLGA microparticles can be stored for at least 6 month as a lyophilisate and can be used for at least 24 h after reconstitution. Pilot experiments suggest that scale-up and a future aseptic production should be feasible. We conclude that the novel depot formulation shows promising attributes, a prerequisite for future industrial production and commercialization

Design and in vivo evaluation of a microparticulate depot formulation of buprenorphine for veterinary use

Buprenorphine is a frequently used analgetic agent in veterinary medicine. A major drawback, however, is the short duration of action requiring several daily administrations. We therefore designed a poly-lactic-co-glycolic acid (PLGA) based microparticulate drug formulation for sustained parenteral drug release. Particles were designed to allow for a fast onset of action and a duration of the analgesic effect of at least two days in laboratory mice. Microparticles were produced using a solvent evaporation technique. Release rate was dependent on polymer type and particle size. Spherical particles used for subsequent animal studies had a mean size of 50 µm and contained 4.5% of buprenorphine. Drug release was characterized by an initial burst release of 30% followed by complete release over seven days. In vivo pharmacokinetic experiments in female C57BL/6 J mice confirmed prolonged exposure in plasma and brain tissue and correlated with the pharmacological effect in the hot plate assay or after minor abdominal surgery. No adverse side effects with respect to food and water intake, body weight, local tolerability, or nesting behavior were observed. Our formulation is an attractive alternative to established immediate release formulations. A use for prolonged pain management in laboratory animals is proposed

Incorporation of phosphatidylserine improves efficiency of lipid based gene delivery systems

The essential homeostatic process of dead cell clearance (efferocytosis) is used by viruses in an act of apoptotic mimicry. Among others, virions leverage phosphatidylserine (PS) as an essential "eat me" signal in viral envelopes to increase their infectivity. In a virus-inspired biomimetic approach, we demonstrate that PS can be incorporated into non-viral lipid nanoparticle (LNP) pDNA/mRNA constructs to enhance cellular transfection. The inclusion of the bioactive PS leads to an increased ability of LNPs to deliver nucleic acids in vitro to cultured HuH-7 hepatocellular carcinoma cells resulting in a 6-fold enhanced expression of a transgene. Optimal PS concentrations are in the range of 2.5 to 5% of total lipids. PS-decorated mRNA-LNPs show a 5.2-fold enhancement of in vivo transfection efficiency as compared to mRNA-LNPs devoid of PS. Effects were less pronounced for PS-decorated pDNA-LNPs (3.2-fold increase). Incorporation of small, defined amounts of PS into gene delivery vectors opens new avenues for efficient gene therapy and can be easily extended to other therapeutic systems

Targeting of hepatocytes using vector-conjugated liposomes : evaluation of targeting strategies

The need for specific targeting strategies towards hepatocytes stems from the lack of efficient therapeutic options to treat numerous serious liver diseases. Moreover, various genetic disorders, such as α1-antitrypsin deficiency and hemophilia A and B, are depending on an efficient gene delivery to defined cells, such as hepatocytes, preferentially avoiding viral vectors. Since the asialoglycoprotein receptor is primarily expressed by liver parenchymal cells, it offers a potential target for a cell specific delivery system. First, the binding of various vectors was analyzed, using the human hepatocellular carcinoma cell line HepG2 as an in vitro model. While the uptake of D-galactose as a monomer was non-specific, the glycoprotein asialofetuin was analyzed as an alternative vector, which represents the desialated derivative of fetuin, containing multi-antennary galactose- terminating glycan residues. Next to a pronounced cellular accumulation, the uptake was markedly inhibited in the presence of an excess of free asialofetuin, indicating specific endocytosis through the asialoglycoprotein receptor. Therefore, asialofetuin was selected as an ideal vector for the further development of a drug delivery system targeting liver parenchymal cells. Asialofetuin was covalently attached to pegylated liposomes, yielding a highly monodisperse preparation with a particle size below 100 nm. A subsequently incubation with HepG2 cells resulted in a specific endocytosis of the vesicles, providing an experimental proof of concept for targeting hepatocytes in vitro. The delivery and intracellular accumulation in HepG2 cells were investigated by incorporating various organic dyes and fluorescent semiconductor nanocrystals, also known as quantum dots, into liposomes. The cellular uptake of asialofetuin-conjugated liposomes, loaded with quantum dots, resulted in a bright fluorescent signal, which was impaired by the need for a specific photoactivation prior to fluorescence analysis. Despite their challenging optical properties, quantum dots are valuable fluorochromes for further optimization of drug targeting strategies. Finally, a proof of principle for a hepatocyte specific delivery was provided in vivo, by intravenously injecting rats with asialofetuin-conjugated and pegylated liposomes, which were taken up by the liver parenchymal cells. In contrast, accumulation in hepatocytes was reduced by co-injecting free asialofetuin and conventional liposomes were uniquely engulfed by Kupffer cells. Summarized, asialofetuin-conjugated pegylated liposomes represent a novel approach, combining desialated glycoproteins, which exhibit a high affinity towards the asialoglycoprotein receptor, with long circulating vesicles, for a specific targeting of liver parenchymal cells. This concept represents a most promising strategy for a hepatocyte specific drug delivery system and gives the opportunity for further studies, such as the isolated utilization of glycans only, to avoid immunogenic reactions. These targeting strategies can be used to deliver drugs to diseased tissues or organs within our body. This reflects our interests to modulate the pharmacokinetics of drugs using specific formulation strategies. Two additional pharmacokinetic investigations of pharmaceutical relevant substances were published in peer-reviewed journals. One study addresses the risk of physical drug interactions of ceftriaxone with calcium in human plasma, and the second one discusses the interaction potential of high doses of resveratrol with various cytochrome P450 isoenzymes. These studies are presented in the section “Appendix”, to separate them from the drug targeting approach of hepatocytes using liposomal formulations

Manufacturing of Liposomes: A Direct Comparison of Extrusion and Microfluidics Protocols

Liposomal formulations are frequently used for oral, topical, or parenteral drug administration. However, liposome manufacturing and industrial scale-up remains a challenge, in particular if it comes to the preparation of liposome populations with a homogenous size distribution. Therefore, extrusion through filter membranes with defined pore size is traditionally used during the preparation of small unilamellar liposomes. Microfluidics is considered to be an alternative manufacturing method. Lipids, solvents and excipients are thereby passively mixed using a microfluidics device. While the microfluidic approach is highly scalable, most of the traditional liposome preparation protocols rely on extrusion. It was therefore the aim of the present study to compare liposomal formulations with identical composition, which were prepared using either extrusion or microfluidics protocols. Liposomal formulations produced by both methods were analyzed using dynamic light scattering (DLS) to compare size, polydispersity, and ζ-potential. Our results indicate significant differences between liposomal preparations obtained using the two manufacturing methods. We conclude that the two preparation methods should not be used interchangeably

Isolation of Multiantennary N-Glycans from Glycoproteins for Hepatocyte Specific Targeting via the Asialoglycoprotein Receptor

The asialoglycoprotein receptor (ASGPR) expressed on parenchymal liver cells specifically binds multivalent carbohydrates from desialylated glycoproteins. This feature offers a huge potential for hepatocyte specific drug targeting. In this study, multiantennary N-glycans from asialofetuin have been isolated to actively target the asialoglycoprotein receptor on human hepatocellular carcinoma cells in vitro

Drug interaction potential of resveratrol

Resveratrol is a naturally occurring polyphenol that is often used as a food supplement. Many positive health effects, including cardio protection, tumor suppression, and immune modulation, are associated with the intake of resveratrol. Resveratrol is well tolerated in healthy subjects without any comedication. However, supplemental doses of resveratrol in the range of 1 g/day or above by far exceed the natural intake through food. Whether resveratrol-drug interactions can be harmful in patients taking additional medications remains unknown. Recent in vivo studies and clinical trials indicate a possible drug-drug interaction potential using high-dosage formulations. In this review, the known in vitro and in vivo effects of resveratrol on various cytochrome P450 (CYP) isoenzymes are summarized. They are discussed in relation to clinically relevant plasma concentrations in humans. We conclude that resveratrol may lead to interactions with various CYPs, especially when taken in high doses. Aside from systemic CYP inhibition, intestinal interactions must also be considered. They can potentially lead to reduced first-pass metabolism, resulting in higher systemic exposure to certain coadministrated CYP substrates. Therefore, patients who ingest high doses of this food supplement combined with additional medications may be at risk of experiencing clinically relevant drug-drug interactions

Hepatocyte targeting using pegylated asialofetuin-conjugated liposomes

Background and purpose: The hepatocyte asialoglycoprotein receptor mediates uptake of desiaylated glycoproteins by receptor-mediated endocytosis. This work explores a hepatocyte-specific targeting strategy using asialofetuin (AF) covalently coupled to pegylated liposomes. Methods: AF was conjugated to the distal end of polyethylene glycol-functionalized phospholipids. Chemical modification of AF did not interfere with its receptor interaction. AF-liposomes had a size of less than 130 nm, were judged to be monodisperse and were labelled with fluorescent organic dyes or loaded with quantum dots. Results: In vitro, binding and cellular uptake of fluorescent AF-liposomes by HepG2 hepatocellular carcinoma cells were reduced at low temperature and could be competitively inhibited by an excess of unbound AF. Hepatocyte-specific targeting and internalization of AF-liposomes in vivo was confirmed in the rat and could be competitively inhibited by co-injection of unbound AF. In contrast, non-pegylated liposomes accumulated in cells of the reticuloendothelial system such as hepatic Kupffer cells and spleen after intravenous administration. Conclusion: We conclude that the use of AF-conjugated, pegylated liposomes is a promising strategy to avoid the reticuloendothelial system and specifically target hepatocytes via the asialoglycoprotein receptor in vitro as well as in vivo

Buprenorphine loaded PLGA microparticles: Characterization of a sustained-release formulation

Buprenorphine is a short-acting analgesic drug. Its use in veterinary medicine requires several injections per day to alleviate pain in animals. We, therefore, designed a poly-lactic- co -glycolic acid (PLGA) based depot formulation of the opioid to prolong the analgesic effect. We characterized our novel microparticulate depot formulation with emphasis on a potential future product development in the present work. Microparticles showed low residual moisture levels and did meet bacterial endotoxin requirements defined by the European Pharmacopeia. Depot formulation showed physicochemical stability over 6 months at 4 °C, indicating sufficient shelf life. Reconstituted formulation stored at 4 °C showed unchanged release properties for 24 h. Terminal sterilization by x-rays with a dose of 30 kGy revealed the sensitivity of buprenorphine towards radiation, resulting in substantial drug degradation. However, microparticle properties were not affected. Our experiments indicate that sterilization of the final product is not possible, therefore requiring aseptic manufacturing protocols. In conclusion, our novel buprenorphine depot formulation based on PLGA microparticles can be stored for at least 6 month as a lyophilisate and can be used for at least 24 h after reconstitution. Pilot experiments suggest that scale-up and a future aseptic production should be feasible. We conclude that the novel depot formulation shows promising attributes, a prerequisite for future industrial production and commercialization