Fabrication Principles and Their Contribution to the Superior In Vivo Therapeutic Efficacy of Nano-Liposomes Remote Loaded with Glucocorticoids

We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t1/2 ∼1 h), or a slow, zero-order release rate (t1/2 ∼ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies

20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model

The steroid hormone 20-hydroxyecdysone (20HE) is an essential signaling molecule that modulates molting response in insects and may function as a putative anabolic factor in vertebrate animals, although no mammalian 20HE receptor has been identified. Here we show that in H4IIE cell culture, 20HE treatment decreased expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), reduced glucose production, and induced Akt2 phosphorylation sensitive to the phosphoinositide-3 kinase pathway-specific inhibitor LY-294002. Daily oral administration of 20HE (10 mg/kg for 13 wk) ameliorated obesity and insulin resistance in C57BL/6J mice fed a high-fat diet and produced a significant decrease of body weight gain and body fat mass compared with nontreated animals as demonstrated by dual-energy X-ray absorptiometry analysis. In addition, plasma insulin levels and glucose tolerance were significantly lowered by 20HE treatment. These changes were accompanied by the reduced hepatic expression of PEPCK and G6Pase and increased adiponectin production by visceral fat tissue. These studies demonstrate the anti-obesity and anti-diabetic effects of 20HE and begin to elucidate its putative cellular targets both in vitro and in vivo

Nano-Drugs Based on Nano Sterically Stabilized Liposomes for the Treatment of Inflammatory Neurodegenerative Diseases

<div><p>The present study shows the advantages of liposome-based nano-drugs as a novel strategy of delivering active pharmaceutical ingredients for treatment of neurodegenerative diseases that involve neuroinflammation. We used the most common animal model for multiple sclerosis (MS), mice experimental autoimmune encephalomyelitis (EAE). The main challenges to overcome are the drugs’ unfavorable pharmacokinetics and biodistribution, which result in inadequate therapeutic efficacy and in drug toxicity (due to high and repeated dosage). We designed two different liposomal nano-drugs, i.e., nano sterically stabilized liposomes (NSSL), remote loaded with: (a) a “water-soluble” amphipathic weak acid glucocorticosteroid prodrug, methylprednisolone hemisuccinate (MPS) or (b) the amphipathic weak base nitroxide, Tempamine (TMN). For the NSSL-MPS we also compared the effect of passive targeting alone and of active targeting based on short peptide fragments of ApoE or of β-amyloid. Our results clearly show that for NSSL-MPS, active targeting is not superior to passive targeting. For the NSSL-MPS and the NSSL-TMN it was demonstrated that these nano-drugs ameliorate the clinical signs and the pathology of EAE. We have further investigated the MPS nano-drug’s therapeutic efficacy and its mechanism of action in both the acute and the adoptive transfer EAE models, as well as optimizing the perfomance of the TMN nano-drug. The highly efficacious anti-inflammatory therapeutic feature of these two nano-drugs meets the criteria of disease-modifying drugs and supports further development and evaluation of these nano-drugs as potential therapeutic agents for diseases with an inflammatory component.</p></div

Comparison of the therapeutic efficacy of EPC-based NSSL-TMN and DMPC:DPPC-based NSSL-TMN in acute EAE mice model.

<p>^a Significant difference from the control group P<0.0001</p><p>^b Significant difference from the EPC NSSL-TMN treated group P<0.001.</p><p>Comparison of the therapeutic efficacy of EPC-based NSSL-TMN and DMPC:DPPC-based NSSL-TMN in acute EAE mice model.</p

Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide conjugated NSSL-MPS in the acute EAE mice model.

<p>^a Significant difference from the control group P<0.00001</p><p>^b Significant difference from the control group P<0.001</p><p>^c Significant difference from the β-amyloid NSSL-MPS group P<0.0005</p><p>^d Significant difference from the β-amyloid NSSL-MPS group P<0.0001</p><p>^e Significant difference from the control group P<0.005</p><p>^f Significant difference from the control group P<0.0001.</p><p>Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide conjugated NSSL-MPS in the acute EAE mice model.</p

Membrane width (head-to-head distance) of the NSSL at 4°C and 37°C, with and without TMN using small angle X-ray scattering measurements.

<p>Membrane width (head-to-head distance) of the NSSL at 4°C and 37°C, with and without TMN using small angle X-ray scattering measurements.</p

Comparison of the therapeutic efficacy of NSSL-MPS and free MPS in the adoptive transfer EAE mice model.

<p>^a Significant difference from the control group P<0.000001</p><p>^b Significant difference from the NSSL-MPS group P<0.0005</p><p>^c Significant difference from the control group P<0.00005</p><p>^d Significant difference from the NSSL-MPS group P<0.005.</p><p>Comparison of the therapeutic efficacy of NSSL-MPS and free MPS in the adoptive transfer EAE mice model.</p

Comparison of passively targeted NSSL and actively targeted peptide-conjugated NSSL.

<p><b>(A)</b> Representative fluorescent microscopy images comparing brain accumulation of NSSL and their payload as is (A, A1), β-amyloid NSSL(B,B1), and ApoE NSSL (C,C1) in healthy mice brain showing an increase in the amount of actively targeted NSSL and their payload accumulating, compared to passively targeted NSSL. <b>(B)</b> Comparison of the therapeutic efficacy of passively targeted NSSL-MPS and actively targeted peptide-conjugated NSSL-MPS in the acute EAE mice model. SJL mice were treated by IV injections on days 10, 12, 14 post-immunization with saline (control) (◆), NSSL-MPS (●), Apo-E NSSL-MPS (▲) or β-amyloid NSSL-MPS (<b>■</b>). * p-value < 0.0001.</p