Docetaxel in cationic lipid nanocapsules for enhanced in vivo activity.

The usefulness of Docetaxel (DT) as an anti-cancer agent is limited to parenteral route owing to its very poor oral bioavailability. Thus, to improve its oral efficacy, DT was loaded in novel cationic lipid nanocapsules (DT CLNC). The DT CLNC possessed size of 130-150 nm, zeta potential of +72mV, adequate DT loading and over 95% encapsulation efficiency. TEM revealed capsular structure of DT CLNC. Lipolysis study indicated improved solubilization of DT by nanocapsules in comparison to DT solution. DT CLNC exhibited significantly higher release of DT in comparison to DT solution during in vitro permeation studies employing non-reverted rat-intestinal sac. Superior uptake of DT in zebra fishes exposed to DT CLNC resulted in greater apoptosis-based cell death as compared to those exposed to DT solution. This correlated well with the significantly superior (p < 0.05) anti-angiogenic activity of DT CLNC system over DT solution, in zebra fish model. DT CLNC also inhibited tumor growth in melanoma cell line induced tumors in C57BL/6 mice significantly, as compared to DT solution (p < 0.05). The DT CLNC system demonstrated adequate stability, with tremendous potential to improve oral efficacy of DT and can serve as an alternative to existing DT formulations available commercially for parenteral use.

Preclinical formulations: insight, strategies, and practical considerations.

A lot of resources and efforts have been directed to synthesizing potentially useful new chemical entities (NCEs) by pharmaceutical scientists globally. Detailed physicochemical characterization of NCEs in an industrial setup begins almost simultaneously with preclinical testing. Most NCEs possess poor water solubility posing bioavailability issues during initial preclinical screening, sometimes resulting in dropping out of an NCE with promising therapeutic activity. Selection of right formulation approach for an NCE, based on its physicochemical properties, can aid in improving its solubility-related absorption and bioavailability issues. The review focuses on preclinical formulations stressing upon different preclinical formulation strategies and deciphers the understanding of formulation approaches that could be employed. It also provides detailed information related to a vast pool of excipients available today, which is of immense help in designing preclinical formulations. Few examples mentioned, throw light on key aspects of preclinical formulation development. The review will serve as an important guide for selecting the right strategy to improve bioavailability of NCEs for academic as well as industrial formulation scientists.

Reducing amyloid-related Alzheimer's disease pathogenesis by a small molecule targeting filamin A.

PTI-125 is a novel compound demonstrating a promising new approach to treating Alzheimer's disease (AD), characterized by neurodegeneration and amyloid plaque and neurofibrillary pathologies. We show that the toxic signaling of amyloid-ß(42) (Aß(42)) by the α7-nicotinic acetylcholine receptor (α7nAChR), which results in tau phosphorylation and formation of neurofibrillary tangles, requires the recruitment of the scaffolding protein filamin A (FLNA). By binding FLNA with high affinity, PTI-125 prevents Aß(42)'s toxic cascade, decreasing phospho-tau and Aß aggregates and reducing the dysfunction of α7nAChRs, NMDARs, and insulin receptors. PTI-125 prevents Aß(42) signaling by drastically reducing its affinity for α7nAChRs and can even dissociate existing Aß(42)-α7nAChR complexes. Additionally, PTI-125 prevents Aß-induced inflammatory cytokine release by blocking FLNA recruitment to toll-like receptor 4, illustrating an anti-inflammatory effect. PTI-125's broad spectrum of beneficial effects is demonstrated here in an intracerebroventricular Aß(42) infusion mouse model of AD and in human postmortem AD brain tissue.

Direct interaction of taurine with the NMDA glutamate receptor subtype via multiple mechanisms.

Taurine has neuroprotective capabilities against glutamate-induced excitotoxicity through several identified mechanisms including opening of the Cl(-)channel associated with GABA(A)and glycine receptors, or a distinct Cl(-)channel. No existing work has however shown a direct interaction of taurine with the glutamate NMDA receptor. Here we demonstrate such direct interactions using electrophysiological and receptor binding techniques on rat medial prefrontal cortical (mPFC) slices and well-washed rat cortical membrane. Electrically evoked field potential responses were recorded in layer 4/5 of mPFC in the presence of picrotoxin to prevent opening of Cl(-)channels gated by GABA or taurine. Applied taurine markedly diminished evoked-response amplitude at the peak and latter phases of the response. These phases were predominantly sensitive to the NMDA antagonist, MK-801, but not the AMPA/kainate receptor antagonist CNQX. Furthermore, this taurine effect was blocked by APV pretreatment. Taurine (0.1 mM) decreased spermine-induced enhancement of specific ((3)H) MK-801 binding to rat cortical membrane in the presence of glycine, though it was ineffective in the absence of spermine. Our preliminary work shows that taurine diminished the apparent affinity of NMDA receptor to glycine in the presence of spermine. These results indicate that taurine may directly interact with the NMDA receptor through multiple mechanisms.

Increased Aß42-α7-like nicotinic acetylcholine receptor complex level in lymphocytes is associated with apolipoprotein E4-driven Alzheimer's disease pathogenesis.

BACKGROUND: The apolipoprotein E ε4 (APOE4) genotype is a prominent late-onset Alzheimer's disease (AD) risk factor. ApoE4 disrupts memory function in rodents and may contribute to both plaque and tangle formation. METHODS: Coimmunoprecipitation and Western blot detection were used to determine: 1) the effects of select fragments from the apoE low-density lipoprotein (LDL) binding domain and recombinant apoE subtypes on amyloid beta (Aß)42-α7 nicotinic acetylcholine receptor (α7nAChR) interaction and tau phosphorylation in rodent brain synaptosomes; and 2) the level of Aß42-α7nAChR complexes in matched controls and patients with mild cognitive impairment (MCI) and dementia due to AD with known APOE genotypes. RESULTS: In an ex vivo study using rodent synaptosomes, apoE141-148 of the apoE promotes Aß42-α7nAChR association and Aß42-induced α7nAChR-dependent tau phosphorylation. In a single-blind study, we examined lymphocytes isolated from control subjects, patients with MCI and dementia due to AD with known APOE genotypes, sampled at two time points (1 year apart). APOE ε4 genotype was closely correlated with heightened Aß42-α7nAChR complex levels and with blunted exogenous Aß42 effects in lymphocytes derived from AD and MCI due to AD cases. Similarly, plasma from APOE ε4 carriers enhanced the Aß42-induced Aß42-α7nAChR association in rat cortical synaptosomes. The progression of cognitive decline in APOE ε4 carriers correlated with higher levels of Aß42-α7nAChR complexes in lymphocytes and greater enhancement by their plasma of Aß42-induced Aß42-α7nAChR association in rat cortical synaptosomes. CONCLUSIONS: Our data suggest that increased lymphocyte Aß42-α7nAChR-like complexes may indicate the presence of AD pathology especially in APOE ε4 carriers. We show that apoE, especially apoE4, promotes Aß42-α7nAChR interaction and Aß42-induced α7nAChR-dependent tau phosphorylation via its apoE141-148 domain. These apoE-mediated effects may contribute to the APOE ε4-driven neurodysfunction and AD pathologies.

LeciPlex, invasomes, and liposomes: A skin penetration study.

The present study compares three vesicular systems, cationic LeciPlex, invasomes, and conventional liposomes for their ability to deliver drugs deep into the skin. Skin penetration ability of the three vesicular systems was studied for two drugs namely idebenone (antioxidant/anticancer) and azelaic acid (antiacne). All systems showed sizes in nanometer range with small polydispersity indices. Vesicular systems were characterized by CryoTEM studies to understand the differences in morphology of the vesicular systems. Ex vivo human skin penetration studies suggested a pattern in penetration of drugs in different layers of the skin: LeciPlex showed higher penetration for idebenone whereas invasomes showed higher penetration of azelaic acid. Ex vivo study using a fluorescent dye (DiI) was performed to understand the differences in the penetration behavior of the three vesicular systems on excised human skin. In vitro cytotoxicity studies on B16F10 melanoma cell lines revealed, when loaded with idebenone, LeciPlex formulations had the superior activity followed by invasomes and liposomes. In vitro antimicrobial study of azelaic acid loaded systems on Propionibacterium acne revealed high antimicrobial activity for DDAB leciplex followed by almost equal activity for invasomes and CTAB LeciPlex followed by liposomes. Whereas antiacne efficacy study in rats for azelaic acid loaded systems, invasomes exhibited the best antiacne efficacy followed by liposomes and LeciPlex.

Formulation and Characterization of Solid Dispersion Prepared by Hot Melt Mixing: A Fast Screening Approach for Polymer Selection.

Solid dispersion is molecular dispersion of drug in a polymer matrix which leads to improved solubility and hence better bioavailability. Solvent evaporation technique was employed to prepare films of different combinations of polymers, plasticizer, and a modal drug sulindac to narrow down on a few polymer-plasticizer-sulindac combinations. The sulindac-polymer-plasticizer combination that was stable with good film forming properties was processed by hot melt mixing, a technique close to hot melt extrusion, to predict its behavior in a hot melt extrusion process. Hot melt mixing is not a substitute to hot melt extrusion but is an aid in predicting the formation of molecularly dispersed form of a given set of drug-polymer-plasticizer combination in a hot melt extrusion process. The formulations were characterized by advanced techniques like optical microscopy, differential scanning calorimetry, hot stage microscopy, dynamic vapor sorption, and X-ray diffraction. Subsequently, the best drug-polymer-plasticizer combination obtained by hot melt mixing was subjected to hot melt extrusion process to validate the usefulness of hot melt mixing as a predictive tool in hot melt extrusion process.

Modes of direct modulation by taurine of the glutamate NMDA receptor in rat cortex.

Taurine is an endogenous brain substance with robust neuromodulatory and possible neuroprotective properties. Though other mechanisms of action have been reported, its interaction with the NMDA (N-methyl-D-aspartic acid) receptor is undocumented. We investigated taurine's interaction with the NMDA receptor using electrophysiological and receptor binding approaches. The effects of taurine on field potential responses in layer-5 of prelimbic cortex in rat brain slices evoked by single-pulse electrical stimulation of ventral medial cortex were determined. Picrotoxin (80 µM) was present in all control and drug solutions to block the Cl(-) channels associated with the GABA-, taurine-, and strychnine sensitive glycine- receptors. A typical response consisted of an NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-[f]-quinoxaline-7-sulfonamide)-sensitive negative wave (N1) followed by a positive wave (P1) and a broad negativity (N2), both sensitive to dl-AP5 (dl-2-amino-5-phosphonopentanoic acid) inhibition. Taurine exerted a 41.5 ± 8.3% (n = 9) voltage reduction within the late phase of N2. This taurine action was prevented by 100 µM AP5, but not by 10 µM nifedipine, supporting a direct modulation of NMDA receptor function by taurine, without requiring the involvement of the L-type Ca(2+) channel. Taurine did not alter specific [(3)H] MK-801 binding to rat cortical membranes in the presence of glycine or glutamate; but inhibited spermine-potentiated specific [(3)H] MK-801 binding to NMDA receptors by 15-20% in the presence of glycine. In addition, taurine reduced the apparent affinity of the NMDA receptor for glycine (in the presence of spermine) by 10-fold. These results show that taurine interacts directly with the NMDA receptor by multiple mechanisms.

Tamoxifen guided liposomes for targeting encapsulated anticancer agent to estrogen receptor positive breast cancer cells: in vitro and in vivo evaluation.

Tamoxifen (TMX), an estrogen receptor (ER) antagonist, incorporated at surface of liposomes loaded with Doxorubicin (DOX), was hypothesized to serve as ligand for targeting overexpressed ERs on surface and cytosol of breast cancer cells, in addition to its synergism with DOX in killing MCF-7 cells. The TMX-DOX liposomes demonstrated mean size of 188.8±2.2nm and positive potential of+47mV, both suitable for better cellular interaction. TMX-DOX liposomes sustained DOX release in vitro (25.9%) in pH 7.4 at 48h, in comparison with 64.5% DOX release at pH 5.5. In vitro cell line studies demonstrated that TMX-DOX liposomes were more cytotoxic to ER+ve MCF-7 cells as compared to DOX liposomes, DOX solution and TMX-DOX solution (P<0.05). However, there was no statistical difference in cyto-toxicity of TMX-DOX liposomes and DOX liposomes towards ER-ve MDA-MB-231 cells. Flow cytometry and confocal studies in MCF-7 cells revealed greater cell and nuclear uptake of DOX, with TMX guided liposomes as compared to DOX liposomes and DOX solution. TMX-DOX liposomes demonstrated significantly increased inhibition of MCF-7 cell based tumor growth in nude mice (P<0.05) in comparison to DOX solution and DOX liposomes, indicative of target specificity and higher DOX accumulation at tumor site.

Liposomes for targeting hepatocellular carcinoma: use of conjugated arabinogalactan as targeting ligand.

Present study investigates the potential of chemically modified (Shah et al., 2013) palmitoylated arabinogalactan (PAG) in guiding liposomal delivery system and targeting asialoglycoprotein receptors (ASGPR) which are expressed in hepatocellular carcinoma (HCC). PAG was incorporated in liposomes during preparation and doxorubicin hydrochloride was actively loaded in preformed liposomes with and without PAG. The liposomal systems with or without PAG were evaluated for in vitro release, in vitro cytotoxicity, in vitro cell uptake on ASGPR(+) cells, in vivo pharmacokinetic study, in vivo biodistribution study, and in vivo efficacy study in immunocompromised mice. The particle size for all the liposomal systems was below 200 nm with a negative zeta potential. Doxorubicin loaded PAG liposomes released significantly higher amount of doxorubicin at pH 5.5 as compared to pH 7.4, providing advantage for targeted tumor therapy. Doxorubicin in PAG liposomes showed superior cytotoxicity on ASGPR(+) HepG2 cells as compared to ASGPR(-), MCF7, A549, and HT29 cells. Superior uptake of doxorubicin loaded PAG liposomes as compared to doxorubicin loaded conventional liposomes was evident in confocal microscopy studies. Higher AUC in pharmacokinetic study and higher deposition in liver was observed for PAG liposomes compared to conventional liposomes. Significantly higher tumor suppression was noted in immunocompromised mice for mice treated with PAG liposomes as compared to the conventional liposomes. Targeting ability and superior activity of PAG liposomes is established pre-clinically suggesting potential of targeted delivery system for improved treatment of HCC.

Synthesis, characterization, and in vitro evaluation of palmitoylated arabinogalactan with potential for liver targeting.

Arabinogalactan (AG), a water soluble polysaccharide with more than 80 mol% galactose units, was hydrophobized by covalent attachment of palmitoyl chains using a base-catalyzed esterification reaction with the objective of effective amalgamation of arabinogalactan in liposomes for targeting asialoglycoprotein receptors (ASGPR) on liver parenchymal cells. Palmitoylated AG (PAG) was characterized by physico-chemical parameters, IR, (1)H NMR, and (13)C NMR and molecular weight determination by gel permeation chromatography. PAG was incorporated in liposomes and the liposomes were characterized by dynamic light scattering, optical microscopy, zeta potential, and transmission electron microscopic (TEM) techniques. The liposomal system was evaluated for acute toxicity in swiss albino mice and was found to be safe. Targeting ability of PAG was confirmed by in vitro binding affinity to Ricinus communis agglutinin (RCA(120)), a lectin specific for galactose. The liposomal system with PAG was evaluated for cytotoxicity on HepG2, MCF7, and A549 cancer cell lines. Cytotoxicity study revealed enhanced activity on ASGPR-expressive HepG2 cells as compared to MCF7.

Lipid colloidal carriers for improvement of anticancer activity of orally delivered quercetin: formulation, characterization and establishing in vitro-in vivo advantage.

Novel lipid nanocarriers, GeluPearl (GP) comprising of Precirol ATO 5 lipid nanoparticles with (GPNLC) or without oil (GPSLN), loaded with Quercetin (QR), were successfully fabricated to improve therapeutic efficacy. QR loaded GP nanoparticles were optimized to yield adequate colloidal stability, mean particle size in range of 350-380 nm and entrapment efficiency of more than 90%. GPSLN and GPNLC were characterized for morphological evaluation by virtue of cryo-TEM, surface charge, protection offered to QR against alkali mediated degradation and fluorescence studies to evaluate QR-lipid interaction. DSC analysis was performed to get insight into physical state of QR loaded in nanosystems. The in vitro release studies demonstrated sustained drug release potential of QR loaded GP. In vitro lipolysis studies confirmed that lipidic nanocarriers can improve QR solubilization. QR loaded GP nanosystems significantly (P < 0.05) reduced flank tumor volumes in C57BL/6 mice over a 22 day study period compared to QR suspension. GPSLN significantly reduced lung colonization and enhanced antimetastatic activity (P < 0.05) of drug against B16F10 melanoma cells in C57BL/6 mice as compared to QR suspension. QR loaded GPSLN and GPNLC could be effectively lyophilized without much change in particle size and drug content using 15% w/v mannitol as cryoprotectant.