Smart MSN-Drug-Delivery System for Tumor Cell Targeting and Tumor Microenvironment Release.

Tumor-targeted delivery and controlled release of antitumor drugs are promising strategies for increasing chemotherapeutic efficacy and reducing adverse effects. Although mesoporous silica nanoparticles (MSNs) have been known as a potential delivery system for doxorubicin (DOX), they have restricted applications due to their uncontrolled leakage and burst release from their large open pores. Herein, we engineered a smart drug-delivery system (smart MSN-drug) based on MSN-drug loading, cell membrane mimetic coating, on-demand pore blocking/opening, and tumor cell targeting strategies. The pore size of DOX-loaded MSNs was narrowed by polydopamine coating, and the pores/channels were blocked with tumor-targeting ligands anchored by tumor environment-rupturable -SS- chains. Furthermore, a cell membrane mimetic surface was constructed to enhance biocompatibility of the smart MSN-drug. Confocal microscopy results demonstrate highly selective uptake (12-fold in comparison with L929 cell) of the smart MSN-drug by HeLa cells and delivery into the HeLa cellular nuclei. Further in vitro IC50 studies showed that the toxicity of the smart MSN-drug to HeLa cells was 4000-fold higher than to the normal fibroblast cells. These exciting results demonstrate the utility of the smart MSN-drug capable of selectively killing tumor cells and saving the normal cells.

Phosphorylcholine- and cation-bearing copolymer coating with superior antibiofilm and antithrombotic properties for blood-contacting devices.

Nosocomial infections resulting from bacterial attachment on blood-contacting medical devices, as well as biofilm and thrombus formation caused by fibrin crosslinking and platelet accumulation/activation are a major health concern and may lead to severe morbidity and mortality. Therefore, there is an urgent need to develop facile and efficient surface coatings with both antibiofilm and antithrombotic properties to prevent medical-device associated infections as well as thrombus formation. In this study, the copolymers containing quaternary ammonium (QA) and phosphorylcholine (PC) groups were synthesized through traditional free-radical copolymerization. The cationic group of QA provides bactericidal properties, and the cell membrane-mimicking group of PC provides antithrombotic and antifouling properties. Long-term stability of the copolymer coating was achieved via simple dip coating. X-ray photoelectron spectroscopy and water contact angle measurement demonstrated that the QA and PC groups possessed inversion properties once in contact with water allowing for long-term stability. Scanning electron microscopy and confocal laser scanning microscopy demonstrated that the copolymer coating could maintain antibiofilm properties for one week in a nutrient-rich environment. Furthermore, the copolymer coating significantly decreased platelet adhesion/activation and did not cause hemolysis. The ex vivo blood circulation showed no thrombus formation which confirmed the excellent antithrombotic property of the copolymer coating. Such coatings that maintain high cell viability and exhibit both antibiofilm and antithrombotic properties present potential applications for blood-contacting devices.

A Biomimetic Nanoparticle to "Lure and Kill" Phospholipase†A2.

Inhibition of phospholipase A2 (PLA2) has long been considered for treating various diseases associated with an elevated PLA2 activity. However, safe and effective PLA2 inhibitors remain unavailable. Herein, we report a biomimetic nanoparticle design that enables a "lure and kill" mechanism designed for PLA2 inhibition (denoted "L&K-NP"). The L&K-NPs are made of polymeric cores wrapped with modified red blood cell membrane with two inserted key components: melittin and oleyloxyethyl phosphorylcholine (OOPC). Melittin acts as a PLA2 attractant that works together with the membrane lipids to "lure" in-coming PLA2 for attack. Meanwhile, OOPC acts as inhibitor that "kills" PLA2 upon enzymatic attack. Both compounds are integrated into the L&K-NP structure, which voids toxicity associated with free molecules. In the study, L&K-NPs effectively inhibit PLA2-induced hemolysis. In mice administered with a lethal dose of venomous PLA2, L&K-NPs also inhibit hemolysis and confer a significant survival benefit. Furthermore, L&K-NPs show no obvious toxicity in mice. and the design provides a platform technology for a safe and effective anti-PLA2 approach.

Bioinspired Surface Functionalization of Titanium Alloy for Enhanced Lubrication and Bacterial Resistance.

In clinics it is extremely important for implanted devices to achieve the property of enhanced lubrication and bacterial resistance; however, such a strategy has rarely been reported in previous literature. In the present study, a surface functionalization method, motivated by articular cartilage-inspired superlubrication and mussel-inspired adhesion, was proposed to modify titanium alloy (Ti6Al4V) using the copolymer (DMA-MPC) synthesized via free radical copolymerization. The copolymer-coated Ti6Al4V (Ti6Al4V@DMA-MPC) was evaluated by X-ray photoelectron spectroscopy, water contact angle, and Raman spectra to confirm that the DMA-MPC copolymer was successfully coated onto the Ti6Al4V substrate. In addition, the tribological test, with the polystyrene microsphere and Ti6Al4V or Ti6Al4V@DMA-MPC as the tribopair, indicated that the friction coefficient was greatly reduced for Ti6Al4V@DMA-MPC. Furthermore, the bacterial resistance test showed that bacterial attachment was significantly inhibited for Ti6Al4V@DMA-MPC for the three types of bacteria tested. The enhanced lubrication and bacterial resistance of Ti6Al4V@DMA-MPC was due to the tenacious hydration shell formed surrounding the zwitterionic charges in the phosphorylcholine group of the DMA-MPC copolymer. In summary, a bioinspired surface functionalization strategy is developed in this study, which can act as a universal and promising method to achieve enhanced lubrication and bacterial resistance for biomedical implants.

Alginate nanoparticles as non-toxic delivery system for miltefosine in the treatment of candidiasis and cryptococcosis.

INTRODUCTION AND OBJECTIVE: Previous studies indicate that miltefosine (MFS) may be an alternative as an antifungal agent; however, it presents several adverse effects. Thus, the aim of this study was to produce miltefosine-loaded alginate nanoparticles (MFS.Alg) for toxicity reduction to be used as an alternative for the treatment of cryptococcosis and candidiasis. METHODS: Alginate nanoparticles were produced using the external emulsification/gelation method, and their physicochemical and morphological characteristics were analyzed. MFS encapsulation efficiency, release assay and toxicity on red blood cells and on Galleria mellonella larvae were assessed. The antifungal activity was evaluated using in vitro and in vivo larval models of G. mellonella infected with Candida albicans (SC5314 and IAL-40), Cryptococcus neoformans H99 and Cryptococcus gattii ATCC 56990. The treatment efficacy was evaluated by survival curve, colony forming unit (CFU) counting and histopathological analysis. RESULTS: MFS.Alg nanoparticles presented a mean size of 279.1±56.7 nm, a polydispersity index of 0.42±0.15 and a zeta potential of -39.7±5.2 mV. The encapsulation efficiency of MFS was 81.70±6.64%, and its release from the nanoparticles occurred in a sustained manner. MFS in alginate nanoparticles presented no hemolytic effect and no toxicity in G. mellonella larvae. Treatment with MFS.Alg extended the survival time of larvae infected with C. albicans and C. gattii. In addition, the fungal burden reduction was confirmed by CFU and histopathological data for all groups treated with 200 mg/Kg of MFS.Alg. CONCLUSION: These results support the use of alginate-based drug delivery systems as carriers for MFS for drug toxicity reduction and control of the fungal infection in the in vivo model of G. mellonella.

Cell-Membrane-Inspired Silicone Interfaces that Mitigate Proinflammatory Macrophage Activation and Bacterial Adhesion.

Biofouling on silicone implants causes serious complications such as fibrotic encapsulation, bacterial infection, and implant failure. Here we report the development of antifouling, antibacterial silicones through covalent grafting with a cell-membrane-inspired zwitterionic gel layer composed of 2-methacryolyl phosphorylcholine (MPC). To investigate how substrate properties influence cell adhesion, we cultured human-blood-derived macrophages and Escherichia coli on poly(dimethylsiloxane) (PDMS) and MPC gel surfaces with a range of 0.5-50 kPa in stiffness. Cells attach to glass, tissue culture polystyrene, and PDMS surfaces, but they fail to form stable adhesions on MPC gel surfaces due to their superhydrophilicity and resistance to biofouling. Cytokine secretion assays confirm that MPC gels have a much lower potential to trigger proinflammatory macrophage activation than PDMS. Finally, modification of the PDMS surface with a long-term stable hydrogel layer was achieved by the surface-initiated atom-transfer radical polymerization (SI-ATRP) of MPC and confirmed by the decrease in contact angle from 110 to 20° and the >70% decrease in the attachment of macrophages and bacteria. This study provides new insights into the design of antifouling and antibacterial interfaces to improve the long-term biocompatibility of medical implants.

Leishmanicidal Activity of Withanolides from Aureliana Fasciculata var. Fasciculata.

Leishmaniasis is the generic denomination to the neglected diseases caused by more than 20 species of protozoa belonging to the genus Leishmania. The toxic and parenteral-delivered pentavalent antimonials remain to be the first-line treatment. However, all the current used drugs have restrictions. The species Aureliana fasciculata (Vell.) Sendtner var. fasciculata is a native Brazilian species parsimoniously studied on a chemical point of view. In this study, the antileishmanial activity of A. fasciculata was evaluated. Among the evaluated samples of the leaves, the dichloromethane partition (AFfDi) showed the more pronounced activity, with IC50 1.85 µg/ml against promastigotes of L. amazonensis. From AFfDi, two active withanolides were isolated, the Aurelianolides A and B, with IC50 7.61 µM and 7.94 µM, respectively. The withanolides also proved to be active against the clinically important form, the intracellular amastigote, with IC50 2.25 µM and 6.43 µM for Aurelianolides A and B, respectively. Furthermore, withanolides showed results for in silico parameters of absorption, distribution, metabolism, excretion, and toxicity (ADMET) similar to miltefosine, the reference drug, and were predicted as good oral drugs, with the advantage of not being hepatotoxic. These results suggest that these compounds can be useful as scaffolds for planning drug design.

Efficacy of Apigenin and Miltefosine Combination Therapy against Experimental Cutaneous Leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by several different species of Leishmania. Treatment of leishmaniasis involves a limited drug arsenal that is associated with severe side effects, high costs, and drug resistance. Therefore, combination therapy has emerged as a strategy to improve leishmaniasis treatment. Here, we report the interaction of miltefosine and apigenin in vitro and in vivo. Combination therapy using low doses of these two drugs results in good clinical and parasitological responses.

Chemotherapeutics of visceral leishmaniasis: present and future developments.

Treatment of Visceral Leishmaniasis (VL), a neglected tropical disease, is very challenging with few treatment options. Long duration of treatment and drug toxicity further limit the target of achieving VL elimination. Chemotherapy remains the treatment of choice. Single dose of liposomal amphotericin B (LAmB) and multidrug therapy (LAmB + miltefosine, LAmB + paromomycin (PM), or miltefosine + PM) are recommended treatment regimen for treatment of VL in Indian sub-continent. Combination therapy of pentavalent antimonials (Sbv) and PM in East Africa and LAmB in the Mediterranean region/South America remains the treatment of choice. Various drugs having anti-leishmania properties are in preclinical phase and need further development. An effective treatment and secondary prophylaxis of HIV-VL co-infection should be developed to decrease treatment failure and drug resistance.

Triggering of Eryptosis, the Suicidal Erythrocyte Death, by Perifosine.

BACKGROUND/AIMS: The alkylphospholipid perifosine is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. Side effects of perifosine include anemia. At least in theory, perifosine-induced anemia could result from stimulation of suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, increase of ceramide abundance, as well as activation of staurosporine sensitive protein kinase C and/or of SB203580 sensitive p38 kinase. The present study explored, whether perifosine induces eryptosis and, if so, whether its effect involves and/or requires Ca2+ entry, oxidative stress, ceramide and kinase activation. METHODS: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. RESULTS: A 24 hours exposure of human erythrocytes to perifosine (2.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased average forward scatter, significantly increased the percentage of shrunken erythrocytes, and significantly decreased the percentage of swollen erythrocytes. Perifosine significantly increased the percentage of hemolytic erythrocytes. Perifosine significantly increased Fluo3-fluorescence, but decreased DCFDA fluorescence and ceramide abundance. The effect of perifosine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of staurosporine (1 µM), but not by addition of SB203580 (2 µM). CONCLUSIONS: Perifosine triggers eryptosis, an effect at least in part due to Ca2+ entry and activation of staurosporine sensitive kinases.

In vitro drug susceptibility of two strains of the wildlife trypanosome, Trypanosoma copemani: A comparison with Trypanosoma cruzi.

Trypanosomes are blood protozoan parasites that are capable of producing illness in the vertebrate host. Within Australia, several native Trypanosoma species have been described infecting wildlife. However, only Trypanosoma copemani has been associated with pathological lesions in wildlife hosts and more recently has been associated with the drastic decline of the critically endangered woylie (Bettongia penicillata). The impact that some trypanosomes have on the health of the vertebrate host has led to the development of numerous drug compounds that could inhibit the growth or kill the parasite. This study investigated and compared the in vitro susceptibility of two strains of T. copemani (G1 and G2) and one strain of Trypanosoma cruzi (10R26) against drugs that are known to show trypanocidal activity (benznidazole, posaconazole, miltefosine and melarsoprol) and against four lead compounds, two fenarimols and two pyridine derivatives (EPL-BS1937, EPL-BS2391, EPL-BS0967, and EPL-BS1246), that have been developed primarily against T.cruzi. The in vitro cytotoxicity of all drugs against L6 rat myoblast cells was also assessed. Results showed that both strains of T. copemani were more susceptible to all drugs and lead compounds than T. cruzi, with all IC50 values in the low and sub-µM range for both species. Melarsoprol and miltefosine exhibited the highest drug activity against both T. copemani and T. cruzi, but they also showed the highest toxicity in L6 cells. Interestingly, both fenarimol and pyridine derivative compounds were more active against T. copemani and T. cruzi than the reference drugs benznidazole and posaconazole. T. copemani strains exhibited differences in susceptibility to all drugs demonstrating once again considerable differences in their biological behaviour.

The antileishmanial drug miltefosine (Impavido(®)) causes oxidation of DNA bases, apoptosis, and necrosis in mammalian cells.

Miltefosine was developed to treat skin cancer; further studies showed that the drug also has activity against Leishmania. Miltefosine is the first oral agent for treating leishmaniasis. However, its mechanism of action is not completely understood. We have evaluated the induction of DNA damage by miltefosine. Cytotoxicity and genotoxicity (comet assay) tests were performed on human leukocytes exposed to the drug in vitro. Apoptosis and necrosis were also evaluated. In vivo tests were conducted in Swiss male mice (Mus musculus) treated orally with miltefosine. Oxidation of DNA bases in peripheral blood cells was measured using the comet assay followed by digestion with formamidopyrimidine glycosylase (FPG), which removes oxidized guanine bases. The micronucleus test was performed on bone marrow erythrocytes. Miltefosine caused DNA damage, apoptosis, and necrosis in vitro. Mice treated with miltefosine showed an increase in the DNA damage score, which was further increased following FPG digestion. The micronucleus test was also positive.

Rescue of mitochondrial function in parkin-mutant fibroblasts using drug loaded PMPC-PDPA polymersomes and tubular polymersomes.

Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a class of steroid-like hydrophobic compounds able to rescue mitochondrial function in parkin-mutant fibroblasts. Whilst these possess therapeutic potential, the size and high hydrophobicity of some may limit their ability to penetrate the blood-brain barrier from systemic circulation, something that could be improved by novel drug formulations. In the present study, the steroid-like compounds Ursolic Acid (UA) and Ursocholanic Acid (UCA) were successfully encapsulated within nanoscopic polymersomes formed by poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-di-isopropylamino)ethyl methacrylate) (PMPC-PDPA) and separated into spherical and tubular morphologies to assess the effects of nanoparticle mediated delivery on drug efficacy. Following incubation with either morphology, parkin-mutant fibroblasts demonstrated time and concentration dependent increases in intracellular ATP levels, resembling those resulting from treatment with nascent UA and UCA formulated in 0.1% DMSO, as used in the original drug screen. Empty PMPC-PDPA polymersomes did not alter physiological measures related to mitochondrial function or induce cytotoxicity. In combination with other techniques such as ligand functionalisation, PMPC-PDPA nanoparticles of well-defined morphology may prove a promising platform for tailoring the pharmacokinetic profile and organ specific bio-distribution of highly hydrophobic compounds.

PMA and crystal-induced neutrophil extracellular trap formation involves RIPK1-RIPK3-MLKL signaling.

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside-in signaling pathway triggering NET formation is unknown. Here, we show that the receptor-interacting protein kinase (RIPK)-1-stabilizers necrostatin-1 or necrostatin-1s and the mixed lineage kinase domain-like (MLKL)-inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal- or PMA-induced NET formation in human and mouse neutrophils. These compounds do not affect PMA- or urate crystal-induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA-induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal-induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.

A novel nasal powder formulation of glucagon: toxicology studies in animal models.

BACKGROUND: Glucagon nasal powder (GNP), a novel intranasal formulation of glucagon being developed to treat insulin-induced severe hypoglycemia, contains synthetic glucagon (10% w/w), beta-cyclodextrin, and dodecylphosphocholine. The safety of this formulation was evaluated in four studies in animal models. METHODS: The first study evaluated 28-day sub-chronic toxicology in rats treated intranasally with 1 and 2 mg of GNP/day (0.1 and 0.2 mg glucagon/rat/day). The second study evaluated 28-day sub-chronic toxicology in dogs administered 20 and 40 mg of formulation/dog/day (2 and 4 mg glucagon/dog/day) intranasally. A pulmonary insufflation study assessed acute toxicology following intra-tracheal administration of 0.5 mg of GNP (0.05 mg glucagon) to rats. Local tolerance to 30 mg of GNP (equivalent to 3 mg glucagon, the final dose for humans) was tested through direct administration into the eyes of rabbits. RESULTS: There were no test article-related adverse effects on body weight and/or food consumption, ophthalmology, electrocardiography, hematology, coagulation parameters, clinical chemistry, urinalysis, or organ weights, and no macroscopic findings at necropsy in any study. In rats, direct intra-tracheal insufflation at a dose of 0.5 mg of GNP/rat (0.05 mg glucagon/rat) did not result in adverse clinical, macroscopic, or microscopic effects. In dogs, the only adverse findings following sub-chronic use were transient (<30 s) salivation and sneezing immediately post-treatment and mild to moderate reversible histological changes to the nasal mucosa. Daily dosing over 28 days in rats resulted in mild to moderate, unilateral or bilateral erosion/ulceration of the olfactory epithelium, frequently with minimal to mild, acute to sub-acute inflammation of the lamina propria at the dorsal turbinates of the nasal cavity in 2/10 males and 3/10 females in the high-dose group (0.2 mg glucagon/day). These lesions resolved completely over 14 days. Histological examination of tissues from both sub-chronic studies in dogs and rats revealed no microscopic findings. In rabbits, clinical observations noted in the GNP-treated eye and/or surrounding areas included ≥1 of the following: clear discharge, red conjunctiva, partial closure, and swelling of the peri-orbital area, which correlated with erythema and edema noted during ocular observations and grading. DISCUSSION: The studies reported here revealed no safety concerns associated with GNP in animal models. Studies published earlier have highlighted the local safety profile of intranasally administered cyclodextrins (a component of GNP). The choline group, the phosphate group, and the saturated 12-carbon aliphatic chain that are present in the dodecylphosphocholine excipient used in GNP are all present in the phospholipids and lecithins seen ubiquitously in mammalian cell membranes and are unlikely to pose safety concerns; this notion is supported by several studies conducted by the authors that revealed no safety concerns. Taken together, these results suggest that intranasal delivery of GNP holds promise as a future rescue medication for use by caregivers to treat insulin-induced hypoglycemic episodes in patients with type 1 or type 2 diabetes. CONCLUSION: This novel drug product is well tolerated in animal models.

Miltefosine is active against Sporothrix brasiliensis isolates with in vitro low susceptibility to amphotericin B or itraconazole.

Sporotrichosis is a common mycosis caused by dimorphic fungi from the Sporothrix schenckii complex. In recent years, sporotrichosis incidence rates have increased in the Brazilian state of Rio de Janeiro, where Sporothrix brasiliensis is the species more frequently isolated from patients. The standard antifungals itraconazole and amphotericin B are recommended as first-line therapy for cutaneous/lymphocutaneous and disseminated sporotrichosis, respectively, although decreased sensitivity to these drugs in vitro was reported for clinical isolates of S. brasiliensis. Here, we evaluated the activity of the phospholipid analogue miltefosine - already in clinical use against leishmaniasis - towards the pathogenic yeast form of S. brasiliensis isolates with low sensitivity to itraconazole or amphotericin B in vitro. Miltefosine had fungicidal activity, with minimum inhibitory concentration (MIC) values of 1-2 µg ml(-1). Miltefosine exposure led to loss of plasma membrane integrity, and transmission electron microscopy (TEM) analysis revealed a decrease in cytoplasmic electron density, alterations in the thickness of cell wall layers and accumulation of an electron-dense material in the cell wall. Flow cytometry analysis using an anti-melanin antibody revealed an increase in cell wall melanin in yeasts treated with miltefosine, when compared with control cells. The cytotoxicity of miltefosine was comparable to those of amphotericin B, but miltefosine showed a higher selectivity index towards the fungus. Our results suggest that miltefosine could be an effective alternative for the treatment of S. brasiliensis sporotrichosis, when standard treatment fails. Nevertheless, in vivo studies are required to confirm the antifungal potential of miltefosine for the treatment of sporotrichosis.

Toxicity evaluation of prolonged convection-enhanced delivery of small-molecule kinase inhibitors in naïve rat brainstem.

PURPOSE: Convection-enhanced delivery (CED), a local drug delivery technique, is typically performed as a single session and drug concentrations therefore decline quickly post CED. Prolonged CED (pCED) overcomes this problem by performing a long-term infusion to maintain effective drug concentrations for an extended period. The purpose of the current study was to assess the toxicity of using pCED to deliver single and multi-drug therapy in naïve rat brainstem. METHODS: Sixteen rats underwent pCED of three small-molecule kinase inhibitors in the pons. Single and multi-drug combinations were delivered continuously for 7 days using ALZET mini-osmotic pumps (model 2001, rate of 1 µl/h). Rats were monitored daily for neurological signs of toxicity. Rats were sacrificed 10 days post completion of infusion, and appropriate tissue sections were analyzed for histological signs of toxicity. RESULTS: Two rats exhibited signs of neurological deficits, which corresponded with diffuse inflammation, necrosis, and parenchymal damage on histological analysis. The remaining rats showed no neurological or histological signs of toxicity. CONCLUSION: The neurological deficits in the two rats were likely due to injury from physical force, such as cannula movement post insertion and subsequent encephalitis. The remaining rats showed no toxicity and therefore brainstem targeting using pCED to infuse single and multi-drug therapy was well tolerated in these rats.

A neurotoxic glycerophosphocholine impacts PtdIns-4, 5-bisphosphate and TORC2 signaling by altering ceramide biosynthesis in yeast.

Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-ß42 signals the intraneuronal accumulation of PC(O-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(O-16:0/2:0) is also toxic to Saccharomyces cerevisiae, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)-binding protein, was relocalized in response to PC(O-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(O-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P2 leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(O-16:0/2:0) on the distribution of PM PtdIns(4,5)P2 pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(O-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.

Miltefosine and polyhexamethylene biguanide: a new drug combination for the treatment of Acanthamoeba keratitis.

BACKGROUND: In this study, a series of compounds - miltefosine, polyhexamethylene biguanide, chlorhexidine and propamidine isethionate - and combinations of the latter three agents with miltefosine were prepared and used in a rat model for the topical treatment of Acanthamoeba keratitis. METHODS: The corneas of rats were infected with Acanthamoeba hatchetti. On the fifth day, all corneas were microscopically examined in order to determine the grade of infections. Nine groups were then prepared: miltefosine (65.12 µg/mL); chlorhexidine (0.02%); polyhexamethylene biguanide (0.02%), propamidine isethionate (0.1%), miltefosine plus chlorhexidine, miltefosine plus polyhexamethylene biguanide; miltefosine plus propamidine isethionate; infected control; and a non-infected control group. The treatment was continued for 28 days. After the treatment, the corneas were excised and used for Acanthamoeba culture to investigate the presence of Acanthamoeba growth. For the determination of cytotoxicity of the drugs on L929 cells, colorimetric assays were performed. RESULTS: The best treatment results were obtained from the polyhexamethylene biguanide plus miltefosine group; the ratio of fully recovered eyes was 28.4%. It was proven that the miltefosine-polyhexamethylene biguanide combination yielded the highest anti-acanthamoebal activity in that approximately 86% of the eyes were cleared from amoebae. The cytotoxicity values of the miltefosine and the control groups were compared with other groups and found to be statistically different (P < 0.05). CONCLUSION: This in vivo study demonstrates that a miltefosine-polyhexamethylene biguanide combination is highly effective for the treatment of Acanthamoeba keratitis.