Phospholipid nanoparticles: Therapeutic potentials against atherosclerosis via reducing cholesterol crystals and inhibiting inflammation.

BACKGROUND: Atherosclerosis-related cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. Cholesterol crystals (CCs) induce inflammation in atherosclerosis and are associated with unstable plaques and poor prognosis, but no drug can remove CCs in the clinic currently. METHODS: We generated a phospholipid-based and high-density lipoprotein (HDL)-like nanoparticle, miNano, and determined CC-dissolving capacity, cholesterol efflux property, and anti-inflammation effects of miNano in vitro. Both normal C57BL/6J and Apoe-deficient mice were used to explore the accumulation of miNano in atherosclerotic plaques. The efficacy and safety of miNano administration to treat atherosclerosis were evaluated in the Ldlr-deficient atherosclerosis model. The CC-dissolving capacity of miNano was also detected using human atherosclerotic plaques ex vivo. FINDINGS: We found that miNano bound to and dissolved CCs efficiently in vitro, and miNano accumulated in atherosclerotic plaques, co-localized with CCs and macrophages in vivo. Administration of miNano inhibited atherosclerosis and improved plaque stability by reducing CCs and macrophages in Ldlr-deficient mice with favorable safety profiles. In macrophages, miNano prevented foam cell formation by enhancing cholesterol efflux and suppressed inflammatory responses via inhibiting TLR4-NF-κB pathway. Finally, in an ex vivo experiment, miNano effectively dissolved CCs in human aortic atherosclerotic plaques. INTERPRETATION: Together, our work finds that phospholipid-based and HDL-like nanoparticle, miNano, has the potential to treat atherosclerosis by targeting CCs and stabilizing plaques. FUNDING: This work was supported by the National Institutes of Health HL134569, HL109916, HL136231, and HL137214 to Y.E.C, HL138139 to J.Z., R21NS111191 to A.S., by the American Heart Association 15SDG24470155, Grant Awards (U068144 from Bio-interfaces and G024404 from M-BRISC) at the University of Michigan to Y.G., by the American Heart Association 19PRE34400017 and Rackham Helen Wu award to M.Y., NIH T32 GM07767 to K. H., Barbour Fellowship to D.L.

Overview of Humira® Biosimilars: Current European Landscape and Future Implications.

Humira® (adalimumab) by AbbVie has been the top-selling biologic drug product for the last few years - reaching nearly $20 billion in annual sales in 2018. Upon the October 2018 release of four adalimumab biosimilars into the European market, those sales began to shrink. By the end of 2019, the annual sales of Humira®, albeit still high, dipped closer to $19 billion as nearly 35% of European patients had been switched from Humira® to a biosimilar. Diminishing sales are expected to continue as the adoption of adalimumab biosimilars increases in Europe and Humira®'s patent protection is lost in the United States come 2023. In this review we discuss how impactful the availability of biosimilars has been to the European adalimumab market approximately two years after their release. We further analyze the marketed biosimilars with regards to differences in their formulation, delivery devices, biological activity, physicochemical properties, clinical trials data, and current financial foothold. More importantly, though, we highlight how "similar" these biosimilars are to Humira®. In doing so, we seek to educate the public on what they may be able to expect once adalimumab biosimilars enter the United States market in 2023.

Characterization of attributes and in vitro performance of exenatide-loaded PLGA long-acting release microspheres.

Bydureon® (Bdn) is a once-weekly injectable long-acting release (LAR) product for adults with type 2 diabetes based on PLGA microspheres encapsulating the glucagon like peptide (GLP-1) analog, exenatide. Despite its widespread use in type 2 diabetes treatment, little information has been published concerning the physical-chemical aspects and exenatide stability in this product. Here, we developed and validated methods to evaluate attributes and performance of Bdn such as particle size/size distribution and residual levels of moisture and organic solvent(s). The reverse engineering of the exenatide LAR was also performed to identify and quantify principal components in the product. Stability-indicating UPLC and LC-MS methods were applied to characterize exenatide degradation (such as oxidation, deamidation and acylation products) during in vitro release evaluation. The 55-µm volume-median Bdn microspheres slowly released the exenatidein vitroover two months with a very low initial burst release to avoid unwanted side effects. Residual organic solvent levels (methylene chloride, ethanol, heptane, and silicon oil) also met the USP criteria. Peptide acylation was the most prominent peptide reaction during both encapsulation and in vitro release, and the acylated peptide steadily increased during release relative to parent exenatide, becoming the most abundant peptide species extracted from the microspheres at later release stages. The presence of peptide impurities during the release period, which are not extractable in the polymer and likely insoluble in water, might be one potential cause for immunogenicity. Further evaluation will be needed to confirm this hypothesis. Release of peptide was minimal over the first 2 weeks before the microspheres steadily released peptide for more than 28 days. The rigorous technical approach discussed in this paper may provide critical information for both companies and the FDA for developing generic exenatide-PLGA formulations and other important PLGA microsphere products.

Analytical method development and comparability study for AmBisome® and generic Amphotericin B liposomal products.

Liposomal Amphotericin B, known as AmBisome®, is a life-saving antifungal product that sold $407 million in 2019. AmBisome® has a rather complex physical structure in that Amphotericin B (AmpB) forms a stable ionic complex with the lipid bilayer to maintain AmBisome®'s low toxicity and high stability in systemic circulation. Failed attempts to reproduce AmBisome®'s precise structure has resulted in faster drug release and higher toxicity both in vitro and in vivo. In this study, we established several analytical methodologies to quantify liposomal AmpB components, characterize thermal properties of the liposome, and determine particle size distribution, AmpB aggregation state, and drug release kinetics. We applied these methodologies together with in vitro hemolytic potential and antifungal activity tests to characterize multiple lots of AmBisome® and two generic products approved in India, Phosome® and Amphonex®. We also used Fungizone®, a micellar AmpB formulation, and "leaky" AmpB liposomes as negative controls. Our results showed that Phosome® and Amphonex® were both similar to AmBisome®, while Fungizone® and 'leaky" liposomes exhibited differences in both thermal properties and AmpB aggregation state, leading to faster drug release and higher toxicity. Due to the increased interest of the pharmaceutical industry in making generic AmBisome® and the lack of standard analytical methods to characterize liposomal AmpB products, the methodologies described here are valuable for the development of generic liposomal AmpB products.

Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann-Pick diseases.

BACKGROUND: Niemann-Pick disease type C is a fatal and progressive neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in late endosomes and lysosomes. We sought to develop new therapeutics for this disorder by harnessing the body's endogenous cholesterol scavenging particle, high-density lipoprotein (HDL). METHODS: Here we design, optimize, and define the mechanism of action of synthetic HDL (sHDL) nanoparticles. RESULTS: We demonstrate a dose-dependent rescue of cholesterol storage that is sensitive to sHDL lipid and peptide composition, enabling the identification of compounds with a range of therapeutic potency. Peripheral administration of sHDL to Npc1 I1061T homozygous mice mobilizes cholesterol, reduces serum bilirubin, reduces liver macrophage size, and corrects body weight deficits. Additionally, a single intraventricular injection into adult Npc1 I1061T brains significantly reduces cholesterol storage in Purkinje neurons. Since endogenous HDL is also a carrier of sphingomyelin, we tested the same sHDL formulation in the sphingomyelin storage disease Niemann-Pick type A. Utilizing stimulated Raman scattering microscopy to detect endogenous unlabeled lipids, we show significant rescue of Niemann-Pick type A lipid storage. CONCLUSIONS: Together, our data establish that sHDL nanoparticles are a potential new therapeutic avenue for Niemann-Pick diseases.

Characterizing release mechanisms of leuprolide acetate-loaded PLGA microspheres for IVIVC development I: In vitro evaluation.

Release testing of parental controlled release microspheres is an essential step in controlling quality and predicting the duration of efficacy. In the first of a two-part study, we examined the effect of various incubation media on release from leuprolide-loaded PLGA microspheres to understand the influence of external pH, plasticization, and buffer type on mechanism of accelerated release. PLGA 50/50 microspheres encapsulating ~5% w/w leuprolide were prepared by the double emulsion-solvent evaporation method with or without gelatin or by the self-healing encapsulation method. The microspheres were incubated at 37°C up to 56days in various media: pH5.5, 6.5, and 7.4 phosphate buffered-saline (PBS) containing 0.02% Tween 80; pH7.4 PBS containing 1.0% triethyl citrate (PBStc); and pH7.4 HEPES buffered-saline containing 0.02% Tween 80 (all media contained 0.02% sodium azide). The recovered release media and microspheres were examined for released drug, polymer molecular weight (Mw), water uptake, mass loss, and BODIPY (green-fluorescent dye) diffusion coefficient in PLGA. After the initial burst release, release of leuprolide from acid-capped PLGA microspheres appeared to be controlled initially by erosion and then by a second mechanism after day 21, which likely consists of a combination of peptide desorption and/or water-mediated breakage of pore connections. PBStc and acidic buffers accelerated degradation of PLGA and pore-network development and increased BODIPY diffusion coefficient, resulting in faster release. Release of leuprolide from the end-capped PLGA showed similar trends as found with acid capped PLGA but with a longer lag time before release. These data provide a baseline mechanistic signature of in vitro release of leuprolide for future comparison with corresponding in vivo performance, and in turn could lead to future development of rational in vitro-in vivo correlations.

Injectable controlled release depots for large molecules.

Biodegradable, injectable depot formulations for long-term controlled drug release have improved therapy for a number of drug molecules and led to over a dozen highly successful pharmaceutical products. Until now, success has been limited to several small molecules and peptides, although remarkable improvements have been accomplished in some of these cases. For example, twice-a-year depot injections with leuprolide are available compared to the once-a-day injection of the solution dosage form. Injectable depots are typically prepared by encapsulation of the drug in poly(lactic-co-glycolic acid) (PLGA), a polymer that is used in children every day as a resorbable suture material, and therefore, highly biocompatible. PLGAs remain today as one of the few "real world" biodegradable synthetic biomaterials used in US FDA-approved parenteral long-acting-release (LAR) products. Despite their success, there remain critical barriers to the more widespread use of PLGA LARproducts, particularly for delivery of more peptides and other large molecular drugs, namely proteins. In this review, we describe key concepts in the development of injectable PLGA controlled-release depots for peptides and proteins, and then use this information to identify key issues impeding greater widespread use of PLGA depots for this class of drugs. Finally, we examine important approaches, particularly those developed in our research laboratory, toward overcoming these barriers to advance commercial LAR development.