Stable Dried Catalase Particles Prepared by Electrospraying

Therapeutic proteins and peptides are clinically important, offering potency while reducing the potential for off-target effects. Research interest in developing therapeutic polypeptides has grown significantly during the last four decades. However, despite the growing research effort, maintaining the stability of polypeptides throughout their life cycle remains a challenge. Electrohydrodynamic (EHD) techniques have been widely explored for encapsulation and delivery of many biopharmaceuticals. In this work, we explored monoaxial electrospraying for encapsulation of bovine liver catalase, investigating the effects of the different components of the electrospraying solution on the integrity and bioactivity of the enzyme. The catalase was successfully encapsulated within polymeric particles made of polyvinylpyrrolidone (PVP), dextran, and polysucrose. The polysorbate 20 content within the electrospraying solution (50 mM citrate buffer, pH 5.4) affected the catalase loading-increasing the polysorbate 20 concentration to 500 μg/mL resulted in full protein encapsulation but did not prevent loss in activity. The addition of ethanol (20% v/v) to a fully aqueous solution improves the electrospraying process by reducing surface tension, without loss of catalase activity. The polymer type was shown to have the greatest impact on preserving catalase activity within the electrosprayed particles. When PVP was the carrier there was no loss in activity compared with fresh aqueous solutions of catalase. The optimum particles were obtained from a 20% w/v PVP or 30% w/v PVP-trehalose (1:1 w/w) solution. The addition of trehalose confers stability advantages to the catalase particles. When trehalose-PVP particles were stored at 5 °C, enzymatic activity was maintained over 3 months, whereas for the PVP-only analogue a 50% reduction in activity was seen. This demonstrates that processing catalase by monoaxial electrospraying can, under optimised conditions, result in stable polymeric particles with no loss of activity

Effects of methylphenidate: the cellular point of view

The psychostimulant methylphenidate (MPH) is the first choice of treatment in attention-deficit hyperactivity disorder and is based mainly on inhibition of dopamine transporter (DAT). Nonetheless, the complete cellular effects of MPH are still unknown. We attempted to determine whether MPH influences neurotransmitter levels, synaptic gene expression, and cell proliferation in a dose-dependent manner in rat pheochromocytoma cells (PC12) lacking DAT. PC12 were treated in a dose-dependent manner with MPH. Gene expression level of synaptotagmin (Syt) 1 and 4, syntaxin 1a (Stx1a), and synaptic vesicle glycoprotein 2C (SV2C) was measured using quantitative real-time RT-PCR. Different Neurotransmitter release was measured using high-performance liquid chromatography (HPLC). Differences in cell proliferation were evaluated via BrdU incorporation. Treatment with low-dose MPH (1-100nM) altered intra-/extracellular neurotransmitter levels, down-regulated all investigated genes as well as enhanced cell proliferation significantly. These data point to diverse effects of MPH on cell metabolism independent of inhibiting DA

Aminoacyl-tRNA-Charged Eukaryotic Elongation Factor 1A Is the Bona Fide Substrate for Legionella pneumophila Effector Glucosyltransferases

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNAPhe and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNAHis and Phe-tRNAPhe) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt's and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt's

Advanced Formulation Approaches for Proteins

Proteins and peptides are highly desirable as therapeutic agents, being highly potent and specific. However, there are myriad challenges with processing them into patient-friendly formulations: they are often unstable and have a tendency to aggregate or degrade upon storage. As a result, the vast majority of protein actives are delivered parenterally as solutions, which has a number of disadvantages in terms of cost, accessibility, and patient experience. Much work has been undertaken to develop new delivery systems for biologics, but to date this has led to relatively few products on the market. In this chapter, we review the challenges faced when developing biologic formulations, discuss the technologies that have been explored to try to overcome these, and consider the different delivery routes that can be applied. We further present an overview of the currently marketed products and assess the likely direction of travel in the next decade

Effects of methylphenidate: the cellular point of view

The psychostimulant methylphenidate (MPH) is the first choice of treatment in attention-deficit hyperactivity disorder and is based mainly on inhibition of dopamine transporter (DAT). Nonetheless, the complete cellular effects of MPH are still unknown. We attempted to determine whether MPH influences neurotransmitter levels, synaptic gene expression, and cell proliferation in a dose-dependent manner in rat pheochromocytoma cells (PC12) lacking DAT. PC12 were treated in a dose-dependent manner with MPH. Gene expression level of synaptotagmin (Syt) 1 and 4, syntaxin 1a (Stx1a), and synaptic vesicle glycoprotein 2C (SV2C) was measured using quantitative real-time RT-PCR. Different Neurotransmitter release was measured using high-performance liquid chromatography (HPLC). Differences in cell proliferation were evaluated via BrdU incorporation. Treatment with low-dose MPH (1-100nM) altered intra-/extracellular neurotransmitter levels, down-regulated all investigated genes as well as enhanced cell proliferation significantly. These data point to diverse effects of MPH on cell metabolism independent of inhibiting DA

Particulate levodopa nose-to-brain delivery targets dopamine to the brain with no plasma exposure

Levodopa (L-DOPA) is an oral Parkinson's Disease drug that generates the active metabolite - dopamine (DA) in vivo. However, oral L-DOPA exhibits low oral bioavailability, limited brain uptake, peripheral DA-mediated side effects and its poor brain bioavailability can lead to long-term complications. Here we show that L-DOPA forms stable (for at least 5 months) 300 nm nanoparticles when encapsulated within N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ). A nano-in-microparticle GCPQ-L-DOPA formulation (D50 = 7.2 µm), prepared by spray-drying, was stable for one month when stored at room and refrigeration temperatures and was capable of producing the original GCPQ-L-DOPA nanoparticles upon aqueous reconstitution. Nasal administration of reconstituted GCPQ-L-DOPA nanoparticles to rats resulted in significantly higher DA levels in the brain (Cmax of 94 ng g-1 above baseline levels 2 h post-dosing) when compared to nasal administration of L-DOPA alone, with DA being undetectable in the brain with the latter. Furthermore, nasal GCPQ-L-DOPA resulted in higher levels of L-DOPA in the plasma (a 17-fold increase in the Cmax, when compared to L-DOPA alone) with DA undetectable in the plasma from both formulations. These data provide evidence of effective delivery of DA to the brain with the GCPQ-L-DOPA formulation

Effects of methylphenidate: the cellular point of view

The psychostimulant methylphenidate (MPH) is the first choice of treatment in attention-deficit hyperactivity disorder and is based mainly on inhibition of dopamine transporter (DAT). Nonetheless, the complete cellular effects of MPH are still unknown. We attempted to determine whether MPH influences neurotransmitter levels, synaptic gene expression, and cell proliferation in a dose-dependent manner in rat pheochromocytoma cells (PC12) lacking DAT. PC12 were treated in a dose-dependent manner with MPH. Gene expression level of synaptotagmin (Syt) 1 and 4, syntaxin 1a (Stx1a), and synaptic vesicle glycoprotein 2C (SV2C) was measured using quantitative real-time RT-PCR. Different Neurotransmitter release was measured using high-performance liquid chromatography (HPLC). Differences in cell proliferation were evaluated via BrdU incorporation. Treatment with low-dose MPH (1–100 nM) altered intra-/extracellular neurotransmitter levels, down-regulated all investigated genes as well as enhanced cell proliferation significantly. These data point to diverse effects of MPH on cell metabolism independent of inhibiting DAT