Combination Antifungal Therapy Involving Amphotericin B,Rapamycin and 5-Fluorocytosine Using PEG-Phospholipid Micelles

PURPOSE: Rapamycin and 5-fluorocytosine (5-FC) are antifungal agents with unique mechanisms of activity, with potential for cooperative interaction with AmB. Combination antifungal therapy involving conventional AmB has been restricted by poor physical stability and compatibility with antifungal drugs and vehicles. METHODS: AmB and rapamycin were encapsulated in 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy poly(ethylene glycol) (PEG-DSPE) micelles using a solvent evaporation method. The physical stability of micelle encapsulated AmB and rapamycin with 5-FC and saline was evaluated using dynamic light scattering (DLS). In vitro susceptibility of Candida albicans isolates to 5-FC and PEG-DSPE micelle solubilized AmB and rapamycin has been evaluated. Interactive effects have been quantified using a checkerboard layout. RESULTS: In contrast with conventional AmB, PEG-DSPE micelles encapsulating AmB and rapamycin are compatible with saline and 5-FC over 12 h. The solubilized drugs retain high level of potency in vitro. The combination of solubilized AmB and rapamycin was indifferent, as fractional inhibitory concentration (FIC) index and combination index (CI) values were approximately 1. Combinations of solubilized AmB or rapamycin with 5-FC, and the three-drug combination were moderately synergistic since the FIC index and CI values were consistent less than 1. CONCLUSIONS: These results indicate that AmB solubilized in PEG-DSPE micelles is compatible with solubilized rapamycin and 5-FC. The indifferent or moderately synergistic activity of combinations is encouraging and warrants further investigation in appropriate rodent models.     

Room temperature ionic liquids and their mixtures: Potential pharmaceutical solvents

Room temperature ionic liquids (RTILs) are organic salts which are liquids at ambient temperature. Composed of relatively large asymmetric organic cations and inorganic or organic anions, they have generated interest as ‘green’ solvents. Here we report on the solvency of alkyl imidazolium salts (PF₆⁻Br⁻Cl⁻) for poorly water-soluble model drugs, albendazole and danazol, indicating their potential application as pharmaceutical solvents/cosolvents. The solubility of albendazole, for example, is increased by more than 10,000 times by 1-butyl-3-methylimidazolium hexaflourophosphate ([bmim]PF₆⁻). Ionic liquids can be water-miscible or water-immiscible. The aqueous miscibility of a poorly water-miscible RTIL such as of [bmim]PF₆⁻ can be improved by the inclusion of a second more miscible RTIL (e.g. 1-hexyl-3-methylimidazolium bromide ([hmim]Br⁻)). The extent of improvement in water miscibility was found to correlate with the hydrophilicity of the second RTIL. This ability to modulate RTILs’ aqueous miscibility increases their usefulness as pharmaceutical solvents.     

The effect of immunosuppressive agents (FK-506, rapamycin) on renal P450 systems in rat models.

It is well known that cyclosporin, rapamycin and FK-506 (tacrolimus) are metabolized by the liver microsomal cytochrome P450 enzyme system. Although there have been reports of interaction between these drugs and the renal P450 enzyme system, differences among these immunosuppressants has not been comprehensively demonstrated. We have studied the individual capacities of these immunosuppressants to induce renal microsomal P450 enzymes similar to CYP2B4 and CYP4A2 by examining renal function in treated rats, and have correlated the results by means of biochemical, immunological and immunohistochemical assays of renal P450 enzymes. Cyclosporin caused impairment of renal function with an increase in renal-specific P450 content, but FK-506 and rapamycin did not. Laurate omega- and (omega-1)-hydroxylase activity increased in rats treated with rapamycin but decreased in those treated with FK-506. Prostaglandin A1 (PGA1) omega-hydroxylase activity increased in rats treated with FK-506 but was reduced by treatment with cyclosporin. Aminopyrine N-demethylase activity increased in rats treated with cyclosporin or FK-506, but not in those treated with rapamycin. Western-blot analysis revealed significant induction of P450, (similar to CYP2B4 of the rabbit P450 isozyme) in kidneys from rats treated with cyclosporin but not in those from rats receiving FK-506 or rapamycin. Histochemical studies clearly demonstrated a form of P450 such as CYP4A2 in the proximal tubules of rats treated with cyclosporin, but not in those of rats treated with FK-506 or rapamycin. These results show that although cyclosporin has a strong effect on renal P450 systems and induces such a system in kidney cortex (microsomal P450), FK-506 and rapamycin have no substantial effect on the induction of renal P450. These findings might clarify the nephrotoxicity induced by these immunosuppressive drugs.     

The properties of amylose-ethylcellulose films cast from organic-based solvents as potential coatings for colonic drug delivery.

The purpose of the study was to establish the physico-mechanical and digestibility properties of water-miscible organic solvent-based amylose-ethylcellulose films as potential coatings for colonic drug delivery. Free films containing different ratios of amylose to ethylcellulose were cast from the water-miscible organic solvent, ethyl lactate, in combination with the plasticiser, dibutyl sebacate. The resultant mixed films were characterised in terms of tensile strength and elasticity, polymer miscibility, permeability, and digestibility under simulated colonic conditions. Films containing higher concentrations of amylose displayed increasing weakness and softness and faster permeation to hydrogen ions compared to films with lower amylose content. No apparent miscibility was detected between the amylose and ethylcellulose, regardless of film composition. The films were found to be susceptible to digestion by bacterial enzymes within a simulated colonic environment. The extent of digestion was directly proportional to the amount of amylose present within the film. Overall, the results suggest that such amylose-ethylcellulose films could be used as coatings for drug delivery to the colon.     

哺乳动物雷帕霉素靶蛋白抑制剂的开发

哺乳动物雷帕霉素靶蛋白(mTOR)是细胞生长和代谢的中心调控因子,雷帕霉素等作为mTOR抑制剂,具有免疫抑制、抗癌等疗效,临床上应用不断拓展。本文综述mTOR的信号通路及mTOR抑制剂开发。     

Paradoxical effect of rapamycin on S6 phosphorylation in rats with or without seizures

OBJECTIVE Accumulating data have demonstrated that seizures induced by kainate or pilocarpine activate the mammalian target of rapamycin(mTOR) pathway and mTOR inhibitor rapamycin can inhibit mTOR activation which subsequently has potential anti-epileptic effects.However,a preliminary study showed a paradoxical exacerbation of increased mTOR pathway activity reflected by S6 phosphorylation when rapamycin was administrated within a short period before kainate injection.In the present study,we examined this paradoxical effect of rapamycin in more detail,both in normal rats and kainate-injected animals.METHODS The expression of mTOR signaling target both of phosphorylated and unphosphorylated forms were detected by Western Blotting analysis.Seizure onset and duration was monitored by Video.Neuronal cell death was detected by Fluoro-Jade B staining.RESULTS In normal rats,we found that rapamycin showed the expected dose-dependent inhibition of S6 phosphorylation 3~24 h after injection,while a paradoxical elevation of S6 phosphorylation was observed 1 h after rapamycin.Similarly,pretreatment with rapamycin over 10 h prior to kainate inhibits the kainate-induced mTOR activation.In contrast,rapamycin administered 1-6 h before kainate causes a paradoxical increase in the kainate-induced mTOR activation.Rats pretreated with rapamycin 1h prior to kainate showed an increase in severity and duration of seizures and more neuronal cell death as compared to vehicle treated groups.In contrast,rapamycin pretreated 10 h prior to KA had no effect on the seizures and decreased neuronal cell death.The paradoxical effect of rapamycin on S6 phosphorylation was correlated with upstream mTOR signaling and reversed by pre-treatment of perifosine,an akt inhibitor.CONCLUSION These data indicate the complexity of S6 regulation and its effect on epilepsy.Paradoxical effects of rapamycin need to be considered in clinical applications,such as potential treatments for epilepsy and other neurological disorders.     

A novel pathway regulating the mammalian target of rapamycin (mTOR) signaling

Originally discovered as an anti-fungal agent, the bacterial macrolide rapamycin is a potent immunosuppressant and a promising anti-cancer drug. In complex with its cellular receptor, the FK506-binding protein (FKBP12), rapamycin binds and inhibits the function of the mammalian target of rapamycin (mTOR). By mediating amino acid sufficiency, mTOR governs signaling to translational regulation and other cellular functions by converging with the phosphatidylinositol 3-kinase (PI3K) pathway on downstream effectors. Whether mTOR receives mitogenic signals in addition to nutrient-sensing has been an unresolved issue, and the mechanism of action of rapamycin remained unknown. Our recent findings have revealed a novel link between mitogenic signals and mTOR via the lipid second messenger phosphatidic acid (PA), and suggested a role for mTOR in the integration of nutrient and mitogen signals. A molecular mechanism for rapamycin inhibition of mTOR signaling is proposed, in which a putative interaction between PA and mTOR is abolished by rapamycin binding. Collective evidence further implicates the regulation of the rapamycin-sensitive signaling circuitry by phospholipase D, and potentially by other upstream regulators such as the conventional protein kinase C, the Rho and ARF families of small G proteins, and calcium ions. As the mTOR pathway has been demonstrated to be an important anti-cancer target, the identification of new components and novel regulatory modes in mTOR signaling will facilitate the future development of diagnostic and therapeutic strategies.     

Rapamycin, but not FK506 and GPI-1046, increases neurite outgrowth in PC12 cells by inhibiting cell cycle progression

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.     

Rapamycin inhibits polyglutamine aggregation independently of autophagy by reducing protein synthesis

Accumulation of misfolded proteins and protein assemblies is associated with neuronal dysfunction and death in several neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease (HD). It is therefore critical to understand the molecular mechanisms of drugs that act on pathways that modulate misfolding and/or aggregation. It is noteworthy that the mammalian target of rapamycin inhibitor rapamycin or its analogs have been proposed as promising therapeutic compounds clearing toxic protein assemblies in these diseases via activation of autophagy. However, using a cellular model of HD, we found that rapamycin significantly decreased aggregation-prone polyglutamine (polyQ) and expanded huntingtin and its inclusion bodies (IB) in both autophagy-proficient and autophagy-deficient cells (by genetic knockout of the atg5 gene in mouse embryonic fibroblasts). This result suggests that rapamycin modulates the levels of misfolded polyQ proteins via pathways other than autophagy. We show that rapamycin reduces the amount of soluble polyQ protein via a modest inhibition of protein synthesis that in turn significantly reduces the formation of insoluble polyQ protein and IB formation. Hence, a modest reduction in huntingtin synthesis by rapamycin may lead to a substantial decrease in the probability of reaching the critical concentration required for a nucleation event and subsequent toxic polyQ aggregation. Thus, in addition to its beneficial effect proposed previously of reducing polyQ aggregation/toxicity via autophagic pathways, rapamycin may alleviate polyQ disease pathology via its effect on global protein synthesis. This finding may have important therapeutic implications.     

Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.     

Comparative hemolytic activity of undiluted organic water-miscible solvents for intravenous and intra-arterial injection

In humans, nonaqueous solvents are administered intravascularly in two kinds of situations. They have been used in subcutaneous or intramuscular pharmaceutical formulations to dissolve water-insoluble drugs. The need for these vehicles had increased in recent years, since the drug development process has yielded many poorly water-soluble drugs. The use of water-miscible nonaqueous solvents in therefore one of the approaches for administering these products as reference solutions useful in formulation bioequivalence studies. The intravascular use of organic solvents has also gained importance owing to a new approach for the treatment of cerebral malformations using precipitating polymers dissolved in water-miscible organic solvents. At present, the solvent most commonly used for the liquid embolics to solubilize the polymers is dimethyl sulfoxide, which exhibits some local and hemodynamic toxicities. In order to find new, less toxic vehicles for pharmaceutical formulations for the intravenous and intra-arterial routes and for embolic materials, 13 water-miscible organic solvents currently used (diluted with water) for pharmaceutical applications, were evaluated in this study. Their hemolytic activity and the morphological changes induced when mixed with blood (1:99, 5:95, 10:90 solvent:blood) were estimated in vitro. From these data, the selected organic solvents could be subdivided into four groups depending on their hemolytic activity: very highly hemolytic solvents (ethyl lactate, dimethyl sulfoxide), highly hemolytic solvents (polyethylene glycol 200, acetone), moderately hemolytic solvents (tetrahydrofurfuryl alcohol, N-methyl-2-pyrrolidone, glycerol formal, ethanol, Solketal, glycofurol) and solvents with low hemolytic activity (propylene glycol, dimethyl isosorbide, diglyme).     

Biochemical characterization of transporter associated with antigen processing (TAP)-like (ABCB9) expressed in insect cells

The ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL) tagged with a histidine cluster was overexpressed, amounting to as much as 1-2% of total membrane proteins in Drosophila cell line S2. TAPL was effectively solubilized from membranes by Triton X-100, NP-40 and n-dodecyl-beta-D-maltoside. Solubilized TAPL bound ATP-agarose and adenosine 5'-diphosphate (ADP)-agarose but not adenosine 5'-monophosphate (AMP)-agarose. The binding was competed for by excess free ATP, ADP, guanosine 5'-triphosphate (GTP) and dATP but not by AMP. Pyrimidine nucleotides such as uridine 5'-triphosphate (UTP) and cytidine 5'-triphosphate (CTP) were less effective competitors, suggesting that purine nucleotide triphosphates are substrates for TAPL. The ATP-binding of TAPL required Mg(2+), and was observed at neutral pH. Chemical cross-linking experiments suggested that TAPL forms a homodimer in the membrane and under the solubilized conditions.     

Immunosuppressant rapamycin inhibits protein kinase C alpha and p38 mitogen-activated protein kinase leading to the inhibition of chondrogenesis

Immunosuppressants are now known to modulate bone metabolism, including bone formation and resorption. Because cartilage, formed by differentiated chondrocytes, serves as a template for endochondral bone formation, we examined the effects of the immunosuppressant rapamycin on the chondrogenesis of mesenchymal cells and on the cell signaling that is required for chondrogenesis, such as protein kinase C, extracellular signal-regulated kinase-1 (ERK-1), and p38 mitogen-activated protein (MAP) kinase pathways. Rapamycin inhibited the expression of type II collagen and the accumulation of sulfate glycosaminoglycan, indicating inhibition of the chondrogenesis of mesenchymal cells. Rapamycin treatment did not affect precartilage condensation, but it prevented cartilage nodule formation. Exposure of chondrifying mesenchymal cells to rapamycin blocked activation of the protein kinase C alpha and p38 MAP kinase, but had no discernible effect on ERK-1 signaling. Selective inhibition of PKCalpha or p38 MAP kinase activity, which is dramatically increased during chondrogenesis, with specific inhibitors in the absence of rapamycin blocked the chondrogenic differentiation of mesenchymal cells. Taken together, our data indicate that the immunosuppressant rapamycin inhibits the chondrogenesis of mesenchymal cells at the post-precartilage condensation stage by modulating signaling pathways including those of PKCalpha and p38 MAP kinase.     

An unexpected inhibitory effect of rapamycin against germination of spores of Bacillus brevis strain Nagano

Rapamycin is used in medicine as an immunosuppressive agent (Sirolimus; Rapamune) although discovered as an antifungal agent. It is thought not to have antibacterial activity. Surprisingly, we found that rapamycin inhibits the germination of Bacillus brevis Nagano spores, but is inactive against Bacillus brevis Nagano vegetative cells. Surprisingly rapamycin did not show antimicrobial activity against other Bacillus strains, including other gramicidin S-producing Bacillus brevis strains such as ATCC 9999 and BI-7, whether tested as spores or vegetative cells.     

Solvent injection as a new approach for manufacturing lipid nanoparticles--evaluation of the method and process parameters.

Lipid nanoparticles (LNP) can be prepared by rapidly injecting a solution of solid lipids in water-miscible solvents or a water-miscible solvent mixture into water. The aim of the present study was to evaluate the potential of this method for the preparation of LNP and the physicochemical characterization of the particles produced by this method. The results show that solvent injection is a potent and versatile approach for LNP preparation. Acetone, ethanol, isopropanol and methanol are suitable solvents in contrast to ethylacetate with which no LNP could be prepared. The obtained particle sizes (z-average) were between 80 and 300 nm depending on the preparation conditions. Up to 96.5% of the employed lipid was directly transformed into LNP. The LNP formation process seems to be diffusion controlled. Physicochemical characterization of the particles by differential scanning calorimetry (DSC), transmission electron microscopy and X-ray diffraction analysis reveals a distinct decrease in crystallinity of the colloidal lipid in comparison to the bulk lipid. Furthermore, DSC analysis of LNP hints at a delayed recrystallization of the colloidal lipid and the presence of two modifications. Therefore, a certain physical instability of the LNP has to be considered.     

Combination of rapamycin and 17-allylamino-17-demethoxygeldanamycin abrogates Akt activation and potentiates mTOR blockade in breast cancer cells

Increased Akt phosphorylation was reported in cancer cell lines and tumor tissues of patients exposed to rapamycin, a response likely contributing to the attenuated antitumor activity of rapamycin. It is, therefore, necessary to develop and validate combination strategies to reverse rapamycin-induced Akt signaling. We now report that Akt activation in response to rapamycin is abrogated by 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor. Rapamycin/17-AAG combination results in an enhanced antiproliferative activity in both MCF-7 and MDA-MB-231 breast cancer cells. In combination 17-AAG confers potent suppression of Raf-MEK-extracellular signal-regulated kinase signaling, a pathway that is otherwise not inhibited by rapamycin individually. Importantly, 17-AAG cooperates with rapamycin to block the phosphorylation of the mammalian target of rapamycin at Ser2448, as well as its downstream effectors ribosomal p70 S6 kinase and eukaryotic initiation factor 4E binding protein 1, which is accompanied by a substantial reduction in cyclins D1 and E. The potency of rapamycin/17-AAG combination is not affected by the activation of insulin-like growth factor 1 receptor signaling, which has been previously shown to diminish the antiproliferative activity of rapamycin. Rapamycin/17-AAG combination alleviates the induction of HSP90 protein, a heat shock response frequently associated with 17-AAG monotherapy. Our findings establish a mechanistic rationale for a combination approach using rapamycin and 17-AAG in the treatment of breast cancer.     

Isolation and identification of new rapamycin dihydrodiol metabolites from dexamethasoneinduced rat liver microsomes

1. Rapamycin is metabolically transformed in rat liver microsomes to 3,4- and 5,6- dihydrodiol metabolites under the influence of the cytochrome P-450 mixed function oxygenase system. These metabolites were produced from dexamethasone-induced as well as from non-induced rat liver microsomes. The comparison of the ion spray mass spectra of the 5,6-dihydrodiol with the 3,4-dihydrodiol of rapamycin shows clearly that dihydrodiols were formed in two distinct positions of rapamycin. 2. FAB mass spectra as well as electrospray mass spectra of two additional peaks isolated from the same chromatographic run confirm the presence of a 3,4-dihydrodiol metabolite of rapamycin as also strongly suggested by UV spectra.Hplc reinjection of each individualpeak always resultedinchromatograms showing a combinationof thesame three peaks and therefore are to be considered as tautomers of the 3,4-dihydrodiol of rapamycin. 3. These tautomeric conformations were found to have no immunosuppressive potency, most probably due to important structural and stereochemical modifications of the rapamycin binding domain to the binding protein (FKBP-12) and or to important metabolic structural modifications of rapamycin effector domain.     

Intranasal Rapamycin Rescues Mice from Staphylococcal Enterotoxin B-Induced Shock

Staphylococcal enterotoxin B (SEB) and related exotoxins produced by Staphylococcus aureus are potent activators of the immune system and cause toxic shock in humans. Currently there is no effective treatment except for the use of intravenous immunoglobulins administered shortly after SEB exposure. Intranasal SEB induces long-lasting lung injury which requires prolonged drug treatment. We investigated the effects of rapamycin, an immunosuppressive drug used to prevent graft rejection, by intranasal administration in a lethal mouse model of SEB-induced shock. The results show that intranasal rapamycin alone delivered as late as 17 h after SEB protected 100% of mice from lethal shock. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB. Intranasal rapamycin attenuated lung MCP-1, IL-2, IL-6, and IFNγ by 70%, 30%, 64%, and 68% respectively. Furthermore, short courses (three doses) of rapamycin were sufficient to block SEB-induced shock. Intranasal rapamycin represents a novel use of an immunosuppressant targeting directly to site of toxin exposure, reducing dosages needed and allowing a wider therapeutic window.