Liposomal Carrier Conjugated to APP-Derived Peptide for Brain Cancer Treatment.

Brain tumors are hard to treat with the currently available therapy. The major obstacle in the treatment of brain tumors is the lack of therapeutic strategies capable to penetrate the blood-brain barrier (BBB). The BBB is an endothelial interface that separates the brain from the circulatory blood system and prevents the exposure of the central nervous system (CNS) to circulating toxins and potentially harmful compounds. Unfortunately, the BBB prevents also the penetration of therapeutic compounds into the brain. We present here a drug-delivery liposomal carrier, conjugated to a peptide inserted in the liposomal membrane, which is putatively recognized by BBB transporters. The peptide is a short sequence of 5 amino acids (RERMS) present in the amyloid precursor protein (APP). This APP-targeted liposomal system was designed specifically for transporting compounds with anti-cancer activity via the BBB into the brain in an effective manner. This drug-delivery liposomal carrier loaded with the anti-cancer compounds temozolomide (TMZ), curcumin, and doxorubicin crossed the BBB in an in vitro model as well as in vivo (mice model). In the in vitro model, the targeted liposomes crossed the BBB model fourfold higher than the non-targeted liposomes. Labeled targeted liposomes penetrated the brain in vivo 35% more than non-targeted liposomes. Treatment of mice that underwent intracranial injection of human U87 glioblastoma, with the targeted liposomes loaded with the three tested anti-cancer agents, delayed the tumor growth and prolonged the mice survival in a range of 45% -70%. It appears that the targeted liposomal drug-delivery system enables better therapeutic efficacy in a SCID mouse model of glioblastoma compared to the corresponding non-targeted liposomes and the free compounds.

Liposome-based targeting of dopamine to the brain: a novel approach for the treatment of Parkinson's disease.

Delivery of drugs into the brain is poor due to the blood brain barrier (BBB). This study describes the development of a novel liposome-based brain-targeting drug delivery system. The liposomes incorporate a diacylglycerol moiety coupled through a linker to a peptide of 5 amino acids selected from amyloid precursor protein (APP), which is recognized by specific transporter(s)/receptor(s) in the BBB. This liposomal system enables the delivery of drugs across the BBB into the brain. The brain-directed liposomal system was used in a mouse model of Parkinson's disease (PD). Intra-peritoneal (IP) administration of liposomes loaded with dopamine (DA) demonstrated a good correlation between liposomal DA dose and the behavioral effects in hemiparkinsonian amphetamine-treated mice, with an optimal DA dose of 60 µg/kg. This is significantly lower dose than commonly used doses of the DA precursor levodopa (in the mg/kg range). IP injection of the APP-targeted liposomes loaded with a DA dose of 800 µg/kg, resulted in a significant increase in striatal DA within 5 min (6.9-fold, p < 0.05), in amphetamine-treated mice. The increase in striatal DA content persisted for at least 3 h after administration, which indicates a slow DA release from the delivery system. No elevation in DA content was detected in the heart or the liver. Similar increases in striatal DA were observed also in rats and mini-pigs. The liposomal delivery system enables penetration of compounds through the BBB and may be a candidate for the treatment of PD and other brain diseases.

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

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 ß-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.

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 ß-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 ß-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.

A new liposome-based gene delivery system targeting lung epithelial cells using endothelin antagonist.

We formulated a new gene delivery system based on targeted liposomes. The efficacy of the delivery system was demonstrated in in vitro and in vivo models. The targeting moiety consists of a high-affinity 7-amino-acid peptide, covalently and evenly conjugated to the liposome surface. The targeting peptide acts as an endothelin antagonist, and accelerates liposome binding and internalization. It is devoid of other biological activity. Liposomes with high phosphatidyl serine (PS) were specially formulated to help their fusion with the endosomal membrane at low pH and enable release of the liposome payload into the cytoplasm. A DNA payload, pre-compressed by protamine, was encapsulated into the liposomes, which directed the plasmid into the cell's nucleus. Upon exposure to epithelial cells, binding of the liposomes occurred within 5-10 min, followed by facilitated internalization of the complex. Endosomal escape was complete within 30 min, followed by DNA accumulation in the nucleus 2h post-transfection. A549 lung epithelial cells transfected with plasmid encoding for GFP encapsulated in targeted liposomes expressed significantly more protein than those transfected with plasmid complexed with Lipofectamine. The intra-tracheal instillation of plasmid encoding for GFP encapsulated in targeted liposomes into rat lungs resulted in the expression of GFP in bronchioles and alveoli within 5 days. These results suggest that this delivery system has great potential in targeting genes to lungs.

Lipopolysaccharide induced protection against sulfur mustard cytotoxicity in RAW264.7 cells through generation of TNF-alpha.

Sulfur mustard (HD), a very potent alkylating agent and lipopolysacchride (LPS), are both well characterized inflammatory factors. We have found that concomitant exposure of murine macrophage cells (RAW264.7) to LPS and HD induced protection against HD induced cytotoxicity. Both HD and LPS induce release of inflammatory markers in RAW264.7 cells. However, there are marked differences in the repertoire of inflammatory factors released by the two toxins: While exposure to HD, induced a dose-dependant death of these cells, no significant change in survival rate was observed following LPS (1-100 ng/ml) exposure. Additionally, LPS elicited a robust nitric oxide (NO) and TNF-alpha secretion whereas HD was practically ineffective. Both toxins increased PGE(2) secretion in a concentration dependent manner. Treatment of HD-exposed RAW264.7 cells with anti-inflammatory drugs such as dexamethazone (5 muM), voltaren (diclofenac) (8 muM) or doxycycline (5 muM), decreased the release of cytokines but had no effect on cell viability. Simultaneous application of LPS (100 ng/ml) and HD (20-100 muM) resulted in an amelioration of HD cytotoxicity. Adding the NO generator S-nitrosoglutathione (GSNO) or inhibiting NO production using L-N(G)-monomethyl Arginine, had no effect on cell viability. Moreover, addition of PGE(2) (20 ng/ml) failed to induce any changes in cell viability under basal or HD-induced toxicity. In contrast, TNF-alpha (20 ng/ml) provided remarkable protection against HD-induced cell death. These findings strongly suggest that LPS exerts its protective action against HD toxicity through the generation of TNF-alpha and may provide better understanding of the mechanism of cytoprotection.

Inhalation exposure to sulfur mustard in the guinea pig model: clinical, biochemical and histopathological characterization of respiratory injuries.

Guinea pigs (GP) were exposed (head only) in individual plethysmographs to various concentrations of sulfur mustard vapor, determined online, using FTIR attached to flow chamber. The LCt(50) and the inhaled LD(50) were calculated at different time points post exposure. Surviving animals were monitored for clinical symptoms, respiratory parameters and body weight changes for up to 30 days. Clinical symptoms were noted at 3 h post exposure, characterized by erythematic and swelling nose with extensive mucous secretion (with or without bleeding). At 6 h post exposure most of the guinea pigs had breathing difficulties, rhonchi and dyspnea and few deaths were noted. These symptoms peaked at 48 h and were noted up to 8 days, associated with few additional deaths. Thereafter, a spontaneous healing was noted, characterized by recovery of respiratory parameters and normal weight gain with almost complete apparent healing within 2 weeks. Histopathological evaluation of lungs and trachea in the surviving GPs at 4 weeks post exposure revealed a dose-dependent residual injury in both lung and trachea expressed by abnormal recovery of the tracheal epithelium concomitant with a dose-dependent increase in cellular volume in the lungs. These abnormal epithelial regeneration and lung remodeling were accompanied with significant changes in protein, LDH, differential cell count and glutathione levels in the bronchoalveolar lavage (BAL). It is suggested that the abnormal epithelial growth and cellular infiltration into the lung as well as the continuous lung inflammation could cause recurrent lung injury similar to that reported for HD exposed human casualties.

Subchronic exposure to low-doses of the nerve agent VX: physiological, behavioral, histopathological and neurochemical studies.

The highly toxic organophosphorous compound VX [O-ethyl-S-(isoporopylaminoethyl) methyl phosphonothiolate] undergoes an incomplete decontamination by conventional chemicals and thus evaporates from urban surfaces, e.g., pavement, long after the initial insult. As a consequence to these characteristics of VX, even the expected low levels should be examined for their potential to induce functional impairments including those associated with neuronal changes. In the present study, we developed an animal model for subchronic, low-dose VX exposure and evaluated its effects in rats. Animals were exposed to VX (2.25 microg/kg/day, 0.05 LD(50)) for three months via implanted mini osmotic pumps. The rapidly attained continuous and marked whole-blood cholinesterase inhibition (approximately 60%), fully recovered 96 h post pump removal. Under these conditions, body weight, blood count and chemistry, water maze acquisition task, sensitivity to the muscarinic agonist oxotremorine, peripheral benzodiazepine receptors density and brain morphology as demonstrated by routine histopathology, remained unchanged. However, animals treated with VX showed abnormal initial response in an Open Field test and a reduction (approximately 30%) in the expression of the exocytotic synaptobrevin/vesicle associate membrane protein (VAMP) in hippocampal neurons. These changes could not be detected one month following termination of exposure. Our findings indicate that following a subchronic, low-level exposure to the chemical warfare agent VX some important processes might be considerably impaired. Further research should be addressed towards better understanding of its potential health ramifications and in search of optimal countermeasures.

Single whole-body exposure to sarin vapor in rats: long-term neuronal and behavioral deficits.

Freely moving rats were exposed to sarin vapor (34.2+/-0.8 microg/l) for 10 min. Mortality at 24 h was 35% and toxic sings in the surviving rats ranged from sever (prolonged convulsions) through moderate to almost no overt signs. Some of the surviving rats developed delayed, intermittent convulsions. All rats were evaluated for long-term functional deficits in comparison to air-exposed control rats. Histological analysis revealed typical cell loss at 1 week post inhalation exposure. Neuronal inflammation was demonstrated by a 20-fold increase in prostaglandin (PGE(2)) levels 24 h following exposure that markedly decreased 6 days later. An additional, delayed increase in PGE(2) was detected at 1 month and continued to increase for up to 6 months post exposure. Glial activation following neural damage was demonstrated by an elevated level of peripheral benzodiazepine receptors (PBR) seen in the brain 4 and 6 months after exposure. At the same time muscarinic receptors were unaffected. Six weeks, four and six months post exposure behavioral evaluations were performed. In the open field, sarin-exposed rats showed a significant increase in overall activity with no habituation over days. In a working memory paradigm in the water maze, these same rats showed impaired working and reference memory processes with no recovery. Our data suggest long lasting impairment of brain functions in surviving rats following a single sarin exposure. Animals that seem to fully recover from the exposure, and even animals that initially show no toxicity signs, developed some adverse neural changes with time.