Lyophilised protein formulations as a patient-centric dosage form: A contribution toward sustainability paradigm.

At present, society has embraced the fact apropos population aging and climate changes, that demand, amongst others, innovative pharmaceutical technologies, emphasising the development of patient-specific delivery systems and thus the provision of efficient and sustainable drugs. Protein drugs for subcutaneous administration, by allowing less frequent application, represent one of the most important parts of the pharmaceutical field, but their development is inevitably faced with obstacles in providing protein stability and suitable formulation viscosity. To gain further knowledge and fill the gaps in the already constructed data platform for the development of monoclonal antibody formulations, we designed a study that examines small model proteins, i.e., bovine serum albumin. The main aim of the presented work is to evaluate the effect of protein concentrations on critical quality attributes of both, pre-lyophilised liquid formulations, and lyophilised products. Through the study, the hypothesis that increasing protein concentration leads to higher viscosity and higher reconstitution time without affecting the stability of the protein was confirmed. The most important finding is that sucrose plays a key role in the lyophilisation of investigated protein, nevertheless, it can be predicted that, to ensure the beneficial effect of mannitol, its amount has to prevail over the amount of sucrose.

Development and preclinical pharmacokinetics of a novel subcutaneous thermoresponsive system for prolonged delivery of heparin.

Heparin is still widely used for treatment and prevention of thromboembolic diseases. Due to specific physicochemical properties, it requires frequent parenteral injections. In this study we present the development and in vitro evaluation of an advanced delivery system for prolonged subcutaneous release of heparin. The delivery system consisted of an in situ forming thermoresponsive poloxamer-based platform combined with pH-responsive polyelectrolyte heparin/chitosan nanocomplexes. Thermoresponsive hydrogels were tested for gelation temperature, gel dissolution and in vitro heparin release, whereas polyelectrolyte nanocomplexes were physico-chemically characterized, as well as tested for in vitro cytotoxicity and in vitro heparin release. Hydrogel combined of two poloxamers demonstrated the highest gelation temperature (28.6°C), while the addition of hydroxypropyl methylcellulose prolonged gel dissolution. On the other hand, nanocomplexes' dispersions, prepared at 1:1 heparin/chitosan mass ratio and in the concentration range 0.375-1.875mg/mL, demonstrated mean diameter <400nm and zeta potential >34mV. Pharmacokinetics of selected formulations (thermoresponsive hydrogel, nanocomplexes and a dual system consisting of nanocomplexes incorporated into thermoresponsive hydrogel) were studied in rats. Heparin plasma concentration-time profiles revealed a double-peak phenomenon, probably due to heparin diffusion inside the polymer matrix and gel dissolution. Pharmacokinetic parameters were determined by a non-linear mixed effects modeling approach. It was demonstrated that thermoresponsive hydrogel with heparin/chitosan nanocomplexes enabled the lowest absorption rate of heparin into systemic circulation and provided heparin concentration above the prophylaxis threshold for 5 days. In situ gelling thermoresponsive matrix combined with chitosan nanocomplexes present a promising delivery system for heparin, requiring less frequent administration during long-term treatment.

Prolonged subcutaneous delivery of low molecular weight heparin based on thermoresponsive hydrogels with chitosan nanocomplexes: Design, in vitro evaluation, and cytotoxicity studies.

Low molecular weight heparins (LMWHs) have risen in popularity over the past decades. Owing to their appropriate pharmacokinetic profile, they enable long-term clinical applications, e.g. prophylaxis of deep vein thrombosis. Although the administration of LMWHs is not as frequent as of heparin, it still requires once daily injection at least. In order to prolong LMWH release, and thus provide less frequent subcutaneous administration, we designed and thoroughly evaluated thermoresponsive poloxamer-based systems combined with LMWH/chitosan pH-responsive nanocomplexes. A LMWH/chitosan mass ratio of 1:2 was the most appropriate for preparation of small, homogenous and stable nanocomplexes. Thermoresponsive hydrogels were examined by gelation temperature and time, thermal analysis, gel dissolution, LMWH release, and cytotoxicity in vitro. Hydrogels' behaviour was significantly shifted by gel composition e.g. the addition of hydroxypropylmethylcellulose to poloxamer-based systems decreased gelation temperature and time (from 28.6°C to 25.1°C and from 50s to 44s, respectively), but prolonged gel dissolution and LMWH release (7 and 4 days, respectively). Prolongation of drug release was additionally achieved with incorporation of LMWH/chitosan nanocomplexes into the gelling systems. As formulations demonstrated no cytotoxicity in vitro, it may be concluded that these double-responsive platforms are promising candidates for prolonged subcutaneous LMWH delivery during long-term treatment.

Development and validation of a simple and sensitive size-exclusion chromatography method for quantitative determination of heparin in pharmaceuticals.

Heparin is widely used as an anticoagulant for the treatment and prevention of various thrombotic diseases. However, due to its high anionic charge, heterogeneity in size distribution and high polarity, its analysis is very challenging. In this paper, a novel method based on size-exclusion chromatography (SEC) for quantitative determination of intact heparin in pharmaceuticals is presented. Analyses were performed on a BioSep-SEC-S 2000 column with Larginine solution at pH 6.5 as mobile phase and UV detection at 210 nm. The proposed method was found to be selective, linear (R2>0.997) over the concentration range of 3.1 to 1222 µg mL(-1), with a limit of detection of 1.0 µg mL(-1). Intraday and inter-day precision were below 5.1% and inaccuracy expressed as bias did not exceed 6.5 %. The reported method is simple, selective, sensitive, and requires no laborious sample preparation, which makes it appropriate for routine quantitative analysis of heparin in pharmaceuticals.

Thermoresponsive polymers: insights into decisive hydrogel characteristics, mechanisms of gelation, and promising biomedical applications.

Thermally induced gelling systems have gained enormous attention over the last decade. They consist of hydrophilic homopolymers or block copolymers in water that present a sol at room temperature and form a gel after administration into the body. This article reviews the main types of thermoresponsive polymers, with special focus on decisive hydrogel characteristics, mechanisms of gelation, and biocompatibility. Promising biomedical applications are described with a focus on injectable formulations, which include solubilization of small hydrophobic drugs, controlled release, delivery of labile biopharmaceutics, such as proteins and genes, cell encapsulation, and tissue regeneration. Furthermore, combinations of thermoresponsive hydrogels and various nanocarriers as promising systems for sustained drug delivery are discussed through selected examples from the literature. Finally, there is a brief overview of current progress in nano-sized systems incorporating thermoresponsive properties.

Autoimmune reactivity of IgM acquired after oxidation.

OBJECTIVES: Redox-reactive antibodies, mainly of the IgG class, gained a wide area of interest after their autoimmune reactivity was revealed following the application of chemical and physiological oxidants. In this study, we examined the susceptibility of IgMs to oxidation and evaluated their binding to the autoantigens important in some autoimmune diseases. METHODS: IgM and IgG fractions, isolated from healthy individuals' sera, were oxidized using direct electric current or physiological oxidant hemin. Specificities towards beta-2-glycoprotein I (ß(2)-GPI), cardiolipin (CL), and rheumatoid factor were evaluated with the enzyme-linked immunosorbent assays (ELISAs). Post-translational modification was investigated by 2,4-dinitrophenylhydrazine reaction. RESULTS: Electrochemically oxidized IgM fractions exhibited altered immunoreactivity - low to medium titers in anti-CL and low positive titers in anti-ß(2)-GPI ELISA but exhibited no rheumatoid factor reactivity. Oxidized IgG and IgM fractions exhibited 2.5- and 5-fold increase in the carbonyl content, respectively. DISCUSSION: An increase in the carbonyl content along with increased immunoreactivity after oxidation suggests modifications of the IgM paratopes. These results point towards possible modifications of native IgMs to their autoimmune state despite the fact that IgMs were less susceptible to oxidation than IgGs. The importance of an individual's redox status in maintenance of autoimmune reactions was emphasized by in vitro diagnostic tests.

Structural characterization of liposomes made of diether archaeal lipids and dipalmitoyl-L-α-phosphatidylcholine.

The physicochemical properties of binary lipid mixtures of diether C(25,25) lipids and dipalmitoyl-L-α-phosphatidylcholine (DPPC) were studied using photon correlation, fluorescence and electron paramagnetic resonance spectroscopy, and transmission electron microscopy. These two types of lipids can be mixed at all molar ratios to form unilamellar and multilamellar liposomes. Fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatrien in mixed liposomes indicates that the abrupt changes in order parameter in the hydrophobic part of bilayer membranes made of DPPC lipids disappears with increasing mol%C(25,25) lipids. Electron paramagnetic resonance spectroscopy shows that at temperatures below 50 °C, the interfacial regions of membrane bilayer of mixed liposomes is more fluid than for pure DPPC liposomes, while at higher temperatures, the impact of the long isoprenoid chains on the membrane stability becomes more pronounced. Photon correlation spectroscopy and transmission electron microscopy show that mixed liposomes do not fuse or aggregate, even after 41 days at 4 °C.

The manufacturing techniques of drug-loaded polymeric nanoparticles from preformed polymers.

Over the past few decades, nanoparticle (NP) formulation has been the subject of extensive research. The choice of a suitable NP formulation technique is dependent on the physicochemical properties of the drug, such as solubility and chemical stability. Different NP manufacturing methods enable modification of the physicochemical characteristics such as size, structure, morphology and surface texture, but also affect the drug loading, drug entrapment efficiency and release kinetics. This review covers an update on the state of art of the manufacturing of polymeric NPs from preformed polymers. Both, conventional methods for NP preparation, such as spontaneous formulation and emulsification-based methods, and new approaches in NP technology are presented. A comparative analysis is given for polymer, drug and solvent nature, toxicity, purification, drug stability and scalability of the method. The information obtained allows establishing criteria for selecting a method for preparation of NPs according to its advantages and limitations.

Current view on nanosized solid lipid carriers for drug delivery to the skin.

Drug delivery into the skin is problematic because, as a natural barrier, it has a very low permeation rate. Several approaches have been made to increase the latter. Current advances in materials science and nanotechnology promise the development of new generations of drug carriers for therapeutic, diagnostic and protective purposes. We have reviewed various lipid drug delivery systems to the skin, with the main focus on solid lipid nanoparticles (SLNs). Further the article concerns possible variants in formulation of SLNs, the selection of lipid compounds and safe stabilizers, and the influence of the physicochemical properties of the drugs on distribution after loading into SLNs. In the last part the advantages of delivery into keratinocytes by SLNs over that of the free drug is addressed, focusing on decreased cytotoxicity of the incorporated drug in SLNs and controlled drug delivery to the subcellular compartments. This knowledge is important in the design and production of advanced solid lipid drug carriers. Additionally, the need for novel approaches or devices to improving permeation is also discussed in the context of moving the nanocarriers to targets deeper in the skin.

High celecoxib-loaded nanoparticles prepared by a vibrating nozzle device.

Drug delivery research has resulted in the availability of several enabling technologies for formulating poorly water-soluble compounds. In this study the vibrating nozzle device, originally used for encapsulation of drugs, cells and microorganisms, has been used to formulate nanoparticles (NP) with high loading capacity. Celecoxib was incorporated in NP of polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) and the influence of polymers, initial drug : polymer ratio and stabilizer concentration on NP size and surface properties, entrapment efficiency, drug loading and in vitro release profile were investigated. NP were in the size range of 230-270 nm, with a polydispersity index less than 0.25 and a spherical shape. The highest celecoxib loading (13% w/w) was obtained at initial ratio celecoxib : Resomer RG 502 (PLA/PGA = 50/50) of 1 : 5 and 0.1% w/w polyvinyl alcohol concentration. Thermal analysis and X-ray diffraction suggested that celecoxib was amorphous or molecularly dispersed in the polymeric matrix. The release profile exhibited an initial burst followed by sustained release. The freeze-dried NP could be completely dispersed on addition of lyoprotectants. The production of NP by the vibrating nozzle device is highly reproducible, time saving, can be performed under aseptic conditions and offers the possibility of scale-up.