Autophagy-Inducing Inhalable Co-crystal Formulation of Niclosamide-Nicotinamide for Lung Cancer Therapy.

Niclosamide (NIC), an anthelminthic drug, is found to be promising in overcoming the problem of various types of drug-resistant cancer. In spite of strong anti-proliferative effect, NIC shows low aqueous solubility, leading to poor bioavailability. To overcome this limitation, and enhance its physicochemical properties and pharmacokinetic profile, we used co-crystallization technique as a promising strategy. In this work, we brought together the crystal and particle engineering at a time using spray drying to enhance physicochemical and aerodynamic properties of co-crystal particle for inhalation purpose. We investigated the formation and evaluation of pharmaceutical co-crystals of niclosamide-nicotinamide (NIC-NCT) prepared by rapid, continuous and scalable spray drying method and compared with conventional solvent evaporation technique. The newly formed co-crystal was evaluated by XRPD, FTIR, Raman spectroscopy and DSC, which showed an indication of formation of H bonds between drug (NIC) and co-former (NCT) as a major binding force in co-crystal development. The particle geometry of co-crystals including spherical shape, size 1-5 µm and aerodynamic properties (ED, 97.1 ± 8.9%; MMAD, 3.61 ± 0.87 µm; FPF, 71.74 ± 6.9% and GSD 1.46) attributes suitable for inhalation. For spray-dried co-crystal systems, an improvement in solubility characteristics (≥ 14.8-fold) was observed, relative to pure drug. To investigate the anti-proliferative activity, NIC-NCT co-crystals were investigated on A549 human lung adenomas cells, which showed a superior cytotoxic activity compared with pure drug. Mechanistically, NIC-NCT co-crystals enhanced autophagic flux in cancer cell which demonstrates autophagy-mediated cell death as shown by confocal microscopy. This technique could help in improving bioavailability of drug, hence reducing the need for high dosages and signifying a novel paradigm for future clinical applications.

Alginate Microspheres Elicit Innate M1-Inflammatory Response in Macrophages Leading to Bacillary Killing.

Particulate drug delivery systems (PDDS) have been broadly explored as platforms for delivery of drugs, enzymes, cells, and vaccines for pharmaceutical applications. Studies suggest that microspheres (MS) can stimulate innate immune cells even without a drug payload; however, less is known regarding how they impact host cells in dealing with the bacillary infection. We examined the role of drug-free inhalable alginate microspheres (A-MS) on phagocytosis efficiency and subsequent immune cell activation in Escherichia coli-infected THP-1-derived macrophages. Alginate particles have been widely investigated as carriers for prolonged delivery of bioactive (i.e., drugs, diagnostics, and vaccines). A-MS were fabricated by industry scalable spray-congealing process using divalent cation-induced gelification. E. coli-infected macrophages (multiplicity of infection (MOI 1:10) were treated with drug-free A-MS, where we found a consistent moderate reduction in bacillary viability. Particles were more efficiently and rapidly phagocytized by infected macrophages as compared with normal macrophage cells. Subsequently, A-MS induced markers of M1 macrophage responses and stimulated the processing and secretion of pro-inflammatory cytokines (IL-6, IL-12). It also notably augmented the generation of reactive oxygen species (ROS) and nitric oxide (NO) in infected cells. Results illustrate that, the blank A-MS (without a drug payload) able to moderately check the growth of intracellular E. coli (without significant cytotoxicity) by modulating the M1 inflammatory response by host cells. This "added value" can be utilized in the design and development of therapeutic system with the additional advantage of immune-modulatory activity, in addition to serving as a drug carrier.

Novel pre-clinical methodologies for pharmacokinetic drug-drug interaction studies: spotlight on "humanized" animal models.

Poly-therapy is common due to co-occurrence of several ailments in patients, leading to the elevated possibility of drug-drug interactions (DDI). Pharmacokinetic DDI often accounts for severe adverse drug reactions in patients resulting in withdrawal of drug from the market. Hence, the prediction of DDI is necessary at pre-clinical stage of drug development. Several human tissue and cell line-based in vitro systems are routinely used for screening metabolic and transporter pathways of investigational drugs and for predicting their clinical DDI potentials. However, ample constraints are associated with the in vitro systems and sometimes in vitro-in vivo extrapolation (IVIVE) fail to assess the risk of DDI in clinic. In vitro-in vivo correlation model in animals combined with human in vitro studies may be helpful in better prediction of clinical outcome. Native animal models vary remarkably from humans in drug metabolizing enzymes and transporters, hence, the interpretation of results from animal DDI studies is difficult. With the advent of modern molecular biology and engineering tools, novel pre-clinical animal models, namely, knockout rat/mouse, transgenic rat/mouse with humanized drug metabolizing enzymes and/or transporters and chimeric rat/mouse with humanized liver are developed. These models nearly simulate human-like drug metabolism and help to validate the in vivo relevance of the in vitro human DDI data. This review briefly discusses the application of such novel pre-clinical models for screening various type of DDI along with their advantages and limitations.

New Generation Smart Drug Delivery Systems for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is the most common form of the chronic inflammatory autoimmune disease characterized by chronic synovitis, synovial proliferation, and cellular infiltration. Further, it leads to bone erosion, destruction of articular cartilage, intense joint pain, swelling, and a high rate of disability, causing an immense load on human health. If the disease is identified early on, and the patient has continuous and timely treatment, many patients can achieve remission. Although research in RA has made considerable progress, conventional therapies are still the most popular treatment options for most people with RA. But, conventional therapies are hampered by various drawbacks, including higher doses, low solubility and permeability, poor bioavailability, a high level of first-pass metabolism, adaptive treatment tolerance (ATT), and long-term drug use. These drawbacks can result in severe side effects and drug toxicity in patients. Advances in polymer science and the application of nanotechnology in drug delivery systems have provided new possibilities in the treatment of RA by developing new-generation smart drug delivery systems (SDDSs). The shortcomings of non-specific drug distribution and uncontrollable drug release by traditional delivery systems have motivated the creation of next-generation SDDSs. These new smart drug delivery treatment methods have significantly changed the course of RA. Such systems can improve drug delivery by virtue of their multi-functionality and targeting capabilities. The ultimate objective of next-generation SDDSs is to deliver medication at the optimal time with precise dosage and efficiency and specificity to the targeted site (such as cells, tissues, and organs), which can aid patients to adhere better to their therapy. This review highlights and discusses the various next-generation SDDSs along with the conventional treatment options available for RA management.

Enema based therapy using liposomal formulation of low molecular weight heparin for treatment of active ulcerative colitis: New adjunct therapeutic opportunity.

Ulcerative colitis (UC) is an idiopathic bowel disease involving chronic inflammation and ulcers in colon and implicates severe epithelial damage with disruption in colon homeostasis. Presently existing treatments possess serious concerns like off target effects and adverse reactions, drug inactivation, poor absorption and other complications resulting in poor bioavailability. In context of high risk of thrombotic events in UC patients, heparin can offer appreciable benefits in UC management due to its remarkable anti-coagulating properties, its ability to intervene inflammatory pathways and acceleration of wound healing process. However, oral administration of heparin being impractical due to harsh gastric acidic environment and heparin degradation, conventional heparin administration is done via intravenous route. Present study was designed to formulate, characterize and evaluate sustained release heparin formulation in mice model of experimental colitis. Heparin liposomes (HLp) were formulated by solvent evaporation and extrusion process and possessed hydrodynamic diameter of 242 ± 4.3 nm. Size, shape and surface morphology was confirmed by TEM, SEM and AFM micrographs while encapsulation efficiency and loading of heparin in optimized HLp were 59.61% and 12.27%, respectively. HLp enema administration ameliorated gross disease indices like body weight, colon length, stool consistency, fecal occult blood. Further, anti-inflammatory efficacy of HLp was established in histopathological analysis where HLp appreciably restored protective mucin layer, colon epithelial mucosal histoarchitecture and considerably attenuated mast cell infiltration in colon epithelia. Overall, results of this study indicate that HLp demonstrated an appreciable therapeutic efficacy in experimental colitis and these results are attributed to their ability to suppress inflammation.

Efficient, enzyme responsive and tumor receptor targeting gelatin nanoparticles decorated with concanavalin-A for site-specific and controlled drug delivery for cancer therapy.

The tumor targeting and stimuli responsiveness behavior of intelligent drug delivery systems imparts effective therapeutic delivery and decreases the toxicity of conventional chemotherapeutic agents in off-target organs. To achieve the receptor targeting and smart drug release, several strategies have been employed to engineer nano-carrier with stimulus sensitivity. In this work, mannose receptor-targeted and matrix metalloproteinase (MMP) responsive gelatin nanoparticles were developed and assessed for its receptor targeting and "on-demand" controlled drug delivery in lung cancer therapeutics. MMPs are protease enzymes and over-expressed in tumorous tissues in all the stages of cancer. The cancer cells also have over-expressed mannose receptors on the cell surface. The surface decoration of gelatin nanoparticles with concanavalin A (con-A) tends to bind with mannose moiety of cell surface glycoproteins which enhances the cancer cell-specific higher uptake of nanoparticles. Gelatin nanoparticles have attracted significant attraction in recent years as a potential drug carrier because of its good biocompatibility and versatile physicochemical properties desirable to deliver the drug. Cisplatin was complexed with the gelatin matrix (CG-NP) to evaluate stimuli responsiveness with the lung cancer cells and its release pattern. In this smart inhalable delivery system, cisplatin loaded gelatin nanoparticles were surface decorated with con-A (CCG-NP). In tumorous cells, con-A coating is expected to enhance mannose receptor-specific cellular internalization of CCG-NP, and subsequently high level of MMP in tumor tissues would help to release cisplatin in response and ensures controlled drug release. The synthesized CCG-NP has shown enzyme triggered drug release and favorable endocytosis after incubation of 12 h compare to uncoated nanoparticles. The efficacy of CCG-NP significantly increased in presence of MMP-2 enzyme in lung cancer cell line A549 cells. It also significantly enhanced reactive oxygen species generation, cell cycle arrest in S and G2/M phase, and apoptosis in cancer cells. Therefore, inhalable CCG-NP promises a pragmatic approach to construct a receptor targeting and an "on-demand" drug delivery system to efficiently deliver the drug at the tumor site only.

In Vitro Anti-tumoral and Anti-bacterial Activity of an Octamolybdate Cluster-Based Hybrid Solid Incorporated with a Copper Picolinate Complex.

Inorganic drugs, especially polyoxometalate-based hybrids, are expected to be developed as promising future metallodrugs. Herein, an organic-inorganic hybrid solid based on pyridine-2-carboxylic acid or picolinic acid (pic), [(Cu(pic)2)2(Mo8O26)]·8H2O (1), was synthesized. A single-crystal structure of a solid possesses a discrete ß-type octamolybdate cluster that supramolecularly aggregates with a {Cu2(pic)4}4- complex and eight lattice water molecules. The study indicates that the solid is stable in aqueous medium and less toxic toward normal cell lines. The in vitro anti-bacterial and anti-tumor properties of the solid 1 were investigated. The results of the anti-tumor action against various human cancer cell lines, namely, lung (A549), breast (MCF-7), and liver (HepG2) cancer cells suggest that this ß-octamolybdate-based solid yielded the lowest IC50 value reported so far among octamolybdate anion-based hybrid solids, i.e., 24.24 µM for MCF-7, 21.56 µM for HepG2, and 25 µM for A549, indicating significant anti-cancer activity. The cell cycle analysis further reveals the observed anti-tumor effect to be governed by the arrest of breast cancer cells in the G2/M phase while that of lung and liver cancer cells in the S phase of the cell cycle. A fluorescence quenching study suggests the binding interaction between solid and ctDNA, which in turn induces apoptosis and necrosis pathways leading to cancer cell death. This is also the first study of {Mo8O26}4- cluster-based solids as an anti-bacterial agent against Escherichia coli, and it was found to be very effective with a minimal inhibitory concentration value of ∼135 µg/mL, which is the lowest so far reported for any octamolybdate-based solid.

Matrix Metalloproteinase-Responsive Mesoporous Silica Nanoparticles Cloaked with Cleavable Protein for "Self-Actuating" On-Demand Controlled Drug Delivery for Cancer Therapy.

The tumour site-specific stimulus responsiveness of smart drug delivery systems gives a unique system for effective therapeutic delivery with reduced toxic effects of conventional chemotherapeutic drugs. In this work, matrix metalloproteinase-2 (MMP-2)-responsive mesoporous silica nanoparticles (MSNs) were synthesized and assessed for "self-actuating" on-demand controlled drug delivery for cancer therapy. MMPs are members of protease enzymes that are generally overexpressed in cancerous tissues in all stages of cancer. MSNs have attracted significant consideration as a potential delivery system because of their robust and versatile physicochemical properties suitable to deliver the therapeutic payload. Cisplatin (Cis) was used as a model drug, which was incorporated into MSNs to evaluate targeting of lung cancer cells and their release kinetics. In this delivery system, collagen was coated on the surface of Cis-loaded MSNs (Cis-MSN) to form a capping layer, resulting in collagen-coated MSNs (Cis-col-MSN). Under normal cell conditions, a collagen-capping coat efficiently forbids the release of Cis molecules from Cis-col-MSN. The tumor microenvironment would lead to augmented drug release because of the uncapping of collagen from MSN pores due to the presence of overexpressed MMP-2 enzyme and the ensuing controlled drug release. MMP-responsive experiments have shown augmented enzyme triggered drug release. The cellular uptake and cytocompatibility studies in A549 adenocarcinomic lung cancer cell lines demonstrated that this nanocarrier could be efficiently endocytosed in 24 h and have shown favorable biocompatibility with the cells. Cytotoxicity results of Cis-col-MSN demonstrated dose-dependent toxicity. The efficacy of the Cis-col-MSN significantly enhanced with the supplementation of MMP-2 enzyme with increasing concentrations in the cell culture milieu. The efficacy of formulation was attributed to significantly enhance reactive oxygen species, cell cycle arrest, and apoptosis. It is expected that Cis-col-MSN promises a pragmatic approach to constructing an "on-demand" smart drug delivery system to deliver a therapeutic payload at the tumor site only.

Inhalation Delivery of Host Defense Peptides (HDP) using Nano- Formulation Strategies: A Pragmatic Approach for Therapy of Pulmonary Ailments.

Host defense peptides (HDP) are small cationic molecules released by the immune systems of the body, having multidimensional properties including anti-inflammatory, anticancer, antimicrobial and immune-modulatory activity. These molecules gained importance due to their broad-spectrum pharmacological activities, and hence being actively investigated. Presently, respiratory infections represent a major global health problem, and HDP has an enormous potential to be used as an alternative therapeutics against respiratory infections and related inflammatory ailments. Because of their short half-life, protease sensitivity, poor pharmacokinetics, and first-pass metabolism, it is challenging to deliver HDP as such inside the physiological system in a controlled way by conventional delivery systems. Many HDPs are efficacious only at practically high molar-concentrations, which is not convincing for the development of drug regimen due to their intrinsic detrimental effects. To avail the efficacy of HDP in pulmonary diseases, it is essential to deliver an appropriate payload into the targeted site of lungs. Inhalable HDP can be a potentially suitable alternative for various lung disorders including tuberculosis, Cystic fibrosis, Pneumonia, Lung cancer, and others as they are active against resistant microbes and cells and exhibit improved targeting with reduced adverse effects. In this review, we give an overview of the pharmacological efficacy of HDP and deliberate strategies for designing inhalable formulations for enhanced activity and issues related to their clinical implications.

Targeted Pulmonary Delivery of the Green Tea Polyphenol Epigallocatechin Gallate Controls the Growth of Mycobacterium tuberculosis by Enhancing the Autophagy and Suppressing Bacterial Burden.

Growing rates of tuberculosis (TB) superbugs are alarming, which has hampered the progress made to-date to control this infectious disease, and new drug candidates are few. Epigallocatechin gallate (EGCG), a major polyphenolic compound from green tea extract, shows powerful efficacy against TB bacteria in in vitro studies. However, the therapeutic efficacy of the molecule is limited due to poor pharmacokinetics and low bioavailability following oral administration. Aiming to improve the treatment outcomes of EGCG therapy, we investigated whether encapsulation and pulmonary delivery of the molecule would allow the direct targeting of the site of infection without compromising the activity. Microencapsulation of EGCG was realized by scalable spray-freeze-drying (SFD) technology, forming free-flowing micrometer-sized microspheres (epigallocatechin-3-gallate-loaded trehalose microspheres, EGCG-t-MS) of trehalose sugar. These porous microspheres exhibited appropriate aerodynamic parameters and high encapsulation efficiencies. In vitro studies demonstrated that EGCG-t-MS exhibited dose- and time-dependent killing of TB bacteria inside mouse macrophages by cellular mechanisms of lysosome acidification and autophagy induction. In a preclinical study on TB-infected Balb/c mice model (4 weeks of infection), we demonstrate that the microencapsulated EGCG, administered 5 days/week for 6 weeks by pulmonary delivery, showed exceptional efficacy compared to oral treatment of free drug. This treatment approach exhibited therapeutic outcomes by resolution of inflammation in the infected lungs and significant reduction (P < 0.05) in bacterial burden (up to ∼2.54 Log10 CFU) compared to untreated control and orally treated mice groups. No pathological granulomas, lesions, and inflammation were observed in the histopathological investigation, compared to untreated controls. The encouraging results of the study may pave the avenues for future use of EGCG in TB therapeutics by targeted pulmonary delivery and lead to its translational success.

Dynamic mucus penetrating microspheres for efficient pulmonary delivery and enhanced efficacy of host defence peptide (HDP) in experimental tuberculosis.

Pulmonary drug delivery system is increasingly gaining popularity for several lung diseases including tuberculosis(TB) due to its ability to attain high drug concentrations at the site of infection and to minimize systemic toxicity. In TB therapy, the efficacy of the antibiotics decreases and bacteria becomes resistant in course of time due to the formation of several barriers like lung-mucus and biofilms around the microorganism. The conventional inhalable microparticles(MP) are majorly trapped in dense mucin mess network and quickly cleared by mucocilliary clearance. In this study, we determined whether the anti-TB activity of drug-loaded inhalable polymeric microparticles could be synergized with the mucus-penetrating and biofilm disrupting properties. Mucus-penetrating-microparticles(NAC/PLGA-MPP) were developed combining the benefits of anti-TB drug with host defence peptides(HDP). IDR-1018 peptide was encapsulated with/without an anti-TB drug in N-acetyl cysteine(NAC) decorated porous PLGA microspheres. Aerodynamic parameters(MMAD-3.79 ± 1.04 µm, FPF-52.9 ± 5.11%) were optimized for the finest deposition and targeting inside the lungs. The multiple-tracking-technique(MPT) results indicate that the coating of NAC on porous PLGA-MS dramatically increased (4.1fold) the particle transit through the mucus barrier. Designed inhalable NAC/PLGA-MPP do not adhere to lung mucus, disrupt the bacterial biofilm and provide uniform drug delivery to lungs after pulmonary delivery. The formulation was evaluated for activity against M.tb in macrophage cultures and in mice model infected with a low-dose bacterial (~100 CFU) aerosol. The inhalation of NAC/PLGA-MPP encapsulated with IDR-1018 significantly reduced (p < .05) bacterial load (up to ~3.02LogCFU/ml) and inflammation in lungs in a mouse model of TB compared to untreated and blank treated animals in 6 weeks of daily dose. The histopathological results validate the compelling chemotherapeutic outcome of inhaled formulations. This data supports the harnessing potential of mucus penetrating inhalable drug delivery systems as a vehicle for targeted lung delivery. This "value-added" inhalable formulation could be beneficial for resistant TB therapeutics when used as an "adjunct" to existing DOTS (Directly observed treatment, short-course) therapy.

Heparin-Encapsulated Metered-Dose Topical "Nano-Spray Gel" Liposomal Formulation Ensures Rapid On-Site Management of Frostbite Injury by Inflammatory Cytokines Scavenging.

The critical time window between the incidence of frostbite injury and the initiation of treatment in remote snowbound areas is a determining factor for an effective therapeutic response. It is an emergency condition and challenging to treat due to the poor vascularity of affected body parts, and it requires immediate action. In addition to cold trauma-induced tissue damage, the inflammatory mediators majorly contribute to pathologic aggravations. We have designed and evaluated a topical "nano-spray gel (NSG)" formulation, which is based on a combination of liposomal heparin sodium (Hp) and ibuprofen (Ibu) for rapid relief of frostbite injury in extremely low temperatures. The scientific literature suggests that heparin is associated with rapid endothelial cell repair, normalizing blood circulation in capillaries, and has a potential role in wound healing. Hp-containing liposomes were prepared by the extruder method, which suitably formulated an ibuprofen-containing gel to obtain a nano-Spray formulation (HLp-Ibu-NSG) applicable for topical delivery. A single spray puff of the formulation delivers ∼154 mg of the gel, which corresponds to ∼205 U of heparin. In this study, heparin liposomes exhibited significant healing of wound in vitro (scratch assay, fibroblast cells) and in vivo (wound healing in Sprague Dawley rats) at a low dose. In the rat model of frostbite injury, the HLp-Ibu-NSG formulation demonstrated significant reduction in the wound area (up to ∼96%) and improvement of histopathology in 14 days as compared to the control groups. No edema and erythema were detected post-treatment of HLp-Ibu-NSG in the affected area. The underlying mechanism was delineated as a modulation of the inflammatory cytokine (IL-6, TNF-α, IL-10, IL-4) mediators at the wound site and blood circulation to foster frostbite healing. Future clinical studies on the nano-spray gel are required to evaluate its efficacy for the treatment of frostbite symptoms. The instant on-site application of this formulation might be helpful in saving extremities of soldiers, mountaineers, and pilgrims having frostbite.

Mycobactericidal activity of some micro-encapsulated synthetic Host Defense Peptides (HDP) by expediting the permeation of antibiotic: A new paradigm of drug delivery for tuberculosis.

Resistance to anti-Tuberculosis (anti-TB) drugs is primarily due to unique intrinsic resistance mechanisms that mycobacterium possess. The most important determinant of resistance is a peculiar hydrophobic and multi-layered mycobacterial cell-wall structure with mycolic-acid and wax-D, which restricts permeability of both hydrophobic and hydrophilic drugs into bacteria. In this study, it was supposed that Host Defense peptides (HDP) which are known to permeabilize bacterial membranes may, therefore, help anti-TB antibiotics to target internal sites in bacteria. To test this hypothesis, we examined the effect of suboptimal concentration (10 µg/ml) of selected microencapsulated-HDP (Ub2-MS, K4-MS, and Aurein1.2-MS) with a standard anti-TB drug (Isoniazid, INH, 3 µg/ml). We also examined the combined effect of different concentrations of HDP-MS with a suboptimal concentration of anti-TB drug (INH, 1.5 µg/ml) which showed additive efficacy. A number of cationic HDP were encapsulated in inhalable microspheres (HDP-MS) and characterized for physicochemical and aerodynamic properties. These peptides were further evaluated for molecular mass by MALDI-TOF and random coil in its secondary structure as determined by circular dichroism. The anti-mycobacterial kinetics of selected HDP-MS (Ub2-MS, K4-MS, and Aurein1.2-MS) was evaluated against virulent Mycobacterium tuberculosis (Mtb), both alone and in conjunction with anti-TB drug (INH). HDP-MS exhibited up to ∼3.02 and ∼3.41-log decrease in CFU as compared to blank-MS (drug free) and untreated control group in 96 h. The combination of HDP-MS with a suboptimal concentration of INH (1.5 µg/ml) showed superior antibiotic activity against Mtb. Our findings show that the enhanced efficacy is due to augmentation of membrane permeation by HDP which expedited the entry of TB drug into apparently the impermeant mycobacterial membrane which further enhances the effective efficacy of the drug. This phenomenon can reduce the need for high dosages and represents a novel paradigm for potential clinical applications.

Lysosomal targeting strategies for design and delivery of bioactive for therapeutic interventions.

Lysosomes are of particular interest for the design and delivery of pH-dependent pro-drugs, enhancing selectivity and developing strategies to inhibit drug degradation inside the cells. There is great potential to bring intracellular drug delivery and distribution using nanotherapeutic approaches to target lysosomes for therapeutic interventions. Lysosomal targeting strategies involve two contrasting facets. One aspect is to directly target therapeutics to the lysosome through receptor-mediated endocytosis and the other facet involves strategies, which ensure escape from the lysosome in order to prevent their degradation, so that therapeutics may remain intact and available in the cytosol for their further action. It provides a unique opportunity to explore novel treatment strategies and design future drugs for the effective treatment of lysosome-related diseases especially lysosomal storage disorders (LSD), cancer, inflammatory, neurodegenerative conditions (Parkinson's, Alzheimer's and Huntington's diseases) and autoimmune diseases. In this review, we illustrate the fundamentals of membrane trafficking, subcellular organisation, strategies to target lysosomes and its implications for the advance design of efficient drug delivery vectors for safe and effective therapies.

Nano-encapsulated HHC10 host defense peptide (HDP) reduces the growth of Escherichia coli via multimodal mechanisms.

The eradication of several pathogenic drug resistant "Superbug" such as Escherichia coli became difficult especially in chronic infections using existing antibiotics due to the emergence of antibiotic resistance. Owing to their unique antibacterial properties, host defense peptides (HDP) have gained significant attention to combat colonization of bacteria. This study aims designing delivery systems for HHC10 peptide to target bacteria inside the cells might be a promising approach by protecting from degradation, controlling the release, enhancing the susceptibility of target microbes and improving bioavailability. Nano-formulated HHC10 was evaluated for its efficacy (CFU assay) and possible mechanism of action (membrane interaction and apoptosis) against E. coli. Dose-dependent inhibition of E. coli growth is observed for nano-encapsulated and bare HHC10 and encapsulated form remain non-toxic to macrophage mouse cells (RAW264.6) up to 20 µM. Mechanistic analyses using transmission electron microscopy and flow cytometry techniques revealed that bactericidal activity of HHC10-NP progresses via a multimodal mechanism of bacterial cell death by cell-membrane lysis on direct interaction with bacteria while through induction of the apoptotic death pathway inside the host cells. These results offer an insight on future strategies for the development and application of antimicrobial peptides as antibacterial alternatives. Controlled delivery of HHC10 peptide from PLGA-NP kills bacteria by two different mechanism: (i) direct killing: HHC10 disintegrate the cell membrane of bacteria by electrostatic interactions and (ii) indirect killing: induction of apoptosis in bacteria infect cells.

Reclaiming hijacked phagosomes: Hybrid nano-in-micro encapsulated MIAP peptide ensures host directed therapy by specifically augmenting phagosome-maturation and apoptosis in TB infected macrophage cells.

TB-Superbugs have emerged as one of the most challenging global health threat due to the decrease in effectiveness of conventional antibiotics. Meanwhile, Host defense peptides (HDP) have evolved as an alternative to classical therapeutics with lesser susceptibility of resistance. We describe the potential of nano-encapsulated synthetic Magainin-I analog peptide (MIAP) as Host Directed Therapy against TB. Micron-sized inhalable platform "Porous Nanoparticle Aggregates Particles (PNAP)" with nano-scale physiognomies were developed to improve the delivery of MIAP-peptide to the lungs and enhance its stability. This particle engineering enabled more control over aerodynamic characteristics and bioactive release. Antimicrobial and mechanistic studies were carried out against virulent H37Rv TB bacteria. These MIAP-PNAP nano-assemblies demonstrated dose and time dependent antibacterial action against virulent M.tb for at least 96 h, with up to ∼3.03-log CFU reduction in numbers of viable bacteria compared to untreated group. These MIAP-PNAP at concentration of 50 µM and above showed significant antibacterial effects on M.tb after 48-96 h of incubation. Mechanistically, MIAP nano-formulation enhanced host defense mechanism by averting bacteria-induced inhibition of phagosomal-lysosome fusion (Lysostracker) and apoptosis (Annexin-FITC) as shown by confocal microscopy and flow-cytometry. Encapsulated MIAP may serve for adjunctive host-directed TB therapy which may also synergizes the efficacy of standard anti-TB drugs.

Preclinical pharmacokinetics and ADME characterization of a novel anticancer chalcone, cardamonin.

Cardamonin (CRD), a chalconoid obtained from several medicinal plants of Zingiberaceae family, had shown promising potential in cancer prevention and therapy. For further development and better pharmacological elucidation, we performed a series of in vitro and in vivo studies to characterize its preclinical pharmacokinetics. The study samples were analyzed using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high performance liquid chromatography-ultra violet (HPLC-UV) methods. CRD is partially soluble (<10 µM) and possess high permeability (>0.2 × 10-4 cm/sec). It is moderately bound to plasma proteins (<50%). It shows partitioning in red blood cell (RBC) compartment with the partition coefficient between RBCs and plasma (KRBC/P ) of 0.95 at 0 min to 1.39 at 60 min, indicating significant but slow RBC uptake. In mice, CRD is poorly absorbed after oral administration with 18% oral bioavailability. It possesses high clearance, short mean residence time, and high volume of distribution in mice. It exhibited multiple peak phenomena both after oral and intravenous administration and is excreted both as conjugated and unchanged CRD in bile. It is majorly excreted in faeces and negligibly in urine. The preclinical absorption, distribution, metabolism, and excretion data are expected to succour the future clinical investigations of CRD as a promising anticancer agent. Copyright © 2016 John Wiley & Sons, Ltd.

HPLC-MS-MS Method Development and Validation of Antileishmanial Agent, S010-0269, in Hamster Serum.

A rapid, sensitive and simple high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the antileishmanial agent, S010-0269, in hamster serum. A Discovery HS C-18 column (5 µm, 50 × 4.6 mm) maintained at 40°C was utilized for chromatographic separation with mobile phase [acetonitrile: aqueous ammonium acetate (0.01 M) buffer (85:15, v/v)] at a flow rate of 0.6 mL/min. The method requires low serum volume (20 µL) with a run time of 3.5 min. Excellent linear relationships (r ≥ 0.99) were obtained between the measured and added concentration over a range of 1-200 ng/mL. Validation parameters (accuracy, specificity, precision, recovery, matrix effect and stability) were assessed as per FDA guidelines. The precision and accuracy were acceptable as indicated by relative standard deviation ranging from 2.3 to 13.6% and bias values ranging from 1.5 to 6.5%, respectively. Moreover, the compound was found stable in hamster serum even after 30 days of storage at -80°C and being subjected to two freeze-thaw cycles. The validated method was successfully applied to the pharmacokinetic study after 10 mg/kg oral dose of S010-0269 in hamsters.