Glucocorticosteroids in Nano-Sterically Stabilized Liposomes Are Efficacious for Elimination of the Acute Symptoms of Experimental Cerebral Malaria

Cerebral malaria is the most severe complication of Plasmodium falciparum infection, and a leading cause of death in children under the age of five in malaria-endemic areas. We report high therapeutic efficacy of a novel formulation of liposome-encapsulated water-soluble glucocorticoid prodrugs, and in particular beta-methasone hemisuccinate (BMS), for treatment of experimental cerebral malaria (ECM), using the murine P. berghei ANKA model. BMS is a novel derivative of the potent steroid beta-methasone, and was specially synthesized to enable remote loading into nano-sterically stabilized liposomes (nSSL), to form nSSL-BMS. The novel nano-drug, composed of nSSL remote loaded with BMS, dramatically improves drug efficacy and abolishes the high toxicity seen upon administration of free BMS. nSSL-BMS reduces ECM rates in a dose-dependent manner and creates a survival time-window, enabling administration of an antiplasmodial drug, such as artemisone. Administration of artemisone after treatment with the nSSL-BMS results in complete cure. Treatment with BMS leads to lower levels of cerebral inflammation, demonstrated by changes in cytokines, chemokines, and cell markers, as well as diminished hemorrhage and edema, correlating with reduced clinical score. Administration of the liposomal formulation results in accumulation of BMS in the brains of sick mice but not of healthy mice. This steroidal nano-drug effectively eliminates the adverse effects of the cerebral syndrome even when the treatment is started at late stages of disease, in which disruption of the blood-brain barrier has occurred and mice show clear signs of neurological impairment. Overall, sequential treatment with nSSL-BMS and artemisone may be an efficacious and well-tolerated therapy for prevention of CM, elimination of parasites, and prevention of long-term cognitive damage

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 50 and 10mg/kg NSSL-MPS in the acute EAE mice model.

<p>SJL mice were treated by IV injections on days 10, 12, 14 post-immunization with saline (control) (◆), 10mg/kg NSSL-MPS (▲) or 50mg/kg NSSL-MPS (■).</p

Effect of membrane lipid composition (EPC:Chol:PEG-DSPE or DMPC:DPPC:Chol:PEG-DSPE) on TMN retention in NSSL.

<p>% Free TMN was determined by ESR for both lipid compositions, EPC:Chol:PEG-DSPE (■) and DMPC:DPPC:Chol:PEG-DSPE (</p><p></p><p><mi>■</mi></p><p></p>) in vitro at 5°C <b>(A)</b>, 25°C <b>(B)</b>, and 37°C <b>(C)</b> during 4.5 months.<p></p

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

<p>SJL/J mice (n = 10) were treated by IV injections every other day starting on day 8 with: EPC:Chol:PEG-DSPE NSSL-TMN 8.5 mg/kg BW (■), DMPC:DPPC:Chol:PEG-DSPE NSSL-TMN 8.5mg/kg BW (▲), and dextrose 5% (control) (●).</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

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