Paclitaxel delivery by micro/nano encapsulation using layer-by-layer assembly
2009-04-20: Nature Precedings

A novel formulation of paclitaxel (PTX) has been developed by providing multilayer assembly over drug loaded porous CaCO3 microparticles (CaCO3 MP) using combination of biocompatible and biodegradable polyelectrolytes (PE’s). PTX was encapsulated into the nanopores of preformed CaCO3 MP prepared by the co-precipitation method. Infrared (IR) and X-ray diffraction (XRD) provides evidences that PTX has been encapsulated into nanopores of CaCO3 MP and not crystallized on the surface. PTX loaded CaCO3 MP (CaCO3-PTX) was found to be highly stabilized against thermal decomposition as evinced by thermo gravimetric analysis (TGA) indicating decomposition at 600°C and 250°C for CaCO3-PTX and PTX respectively. The multilayer assembly over CaCO3-PTX was effectuated by alternate deposition of protamine sulfate (PRM) and sodium alginate (SA) using LBL technique followed by subsequent core removal [PTX- (PRM/SA)5]. The pay load efficiency of PTX in this system was found to be 78.98±2.14%. The developed system was further evaluated for surface morphology, size and size distribution, surface charge, core removal and layer-by-layer growth due to sequential adsorption of PE’s. The release data of PTX-(PRM/SA)5 was comparable with marketed formulation of PTX (PTX-M) and CaCO3-PTX when performed in simulated intestinal fluid (SIF pH=7.4). The release profile of PTX-(PRM/SA)5 indicates that PEs based multilayer matrix is capable to provide barrier to PTX release as it has been found to follow first order matrix diffusion kinetics with 64±4.8% release within 24 hrs. The t50% of PTX-M, CaCO3-PTX and PTX-(PRM/SA)5 was found to be 70, 90 and 480 minutes respectively. This alternative delivery system of PTX disguised in the form of LBL assembly could have immense application for the treatment of metastasized mammary glands vis-à-vis existing formulation of PTX which is by and large criticized for having certain toxic excipients to be given parentrally. Moreover, the proposed system provides ample of opportunity to modify the surface for targeted application of PTX.

Genome-wide transcription analysis of interaction between the human macrophage and Mycobacterium tuberculosis during concurrent drug administration by conventional and novel methods
2009-04-20: Nature Precedings
Awadh Bihari Yadav
Targeted drug delivery to alveolar macrophages harboring Mycobacterium tuberculosis (Mtb) holds promise of high efficacy against pulmonary tuberculosis (TB). It was investigated whether inhalable microparticles (MP) can rescue macrophages from ‘alternative’ activation induced by pathogenic Mtb in addition to achieving targeted drug delivery. A genome-wide transcription analysis (Affymetrix HG-U133 Plus 2.0 DNA microarray) of THP-1 cell line derived macrophages was undertaken after exposing them to infection with 10 MOI of MTB H37Rv at 0, 12 and 24 hours post infection. The Molecular markers of macrophage bactericidal activity were assayed in THP-1- and primary human peripheral blood mononuclear cell (PBMC)-derived macrophages, in the presence or absence of soluble anti-tuberculosis drugs, drug-containing MP and blank MP. About 1,500 genes were differentially upregulated and about 500 genes differentially downregulated in response to various modes of treatment. Variations were also observed in the kinetics of gene expression. Cluster analysis indicated activation of several pathways related to innate immune response (cytokines, chemokines, receptors and ligands), apoptosis, cytoskeleton and membrane remodeling, general metabolism and general housekeeping. Some of these results were validated at the functional level, by studying caspase activities, concentrations and time-courses of effector molecules , rates/extents of apoptosis and nitrite oxide induction. Production of cytokines and NO, apoptosis, and bacterial survival were studied as pharmacodynamic outcomes. Cytokine responses of THP-1 derived macrophages were estimated. MP reversed suppression of tumor necrosis factor (TNF) induced by infection, and transiently upregulated γ-interferon (IFN-γ). Drug-free MP surprisingly induced IFN-γ, but not TNF. Primary cells responded to MP, regardless of drug content, by upregulation of NO; but THP-1-derived cells did not respond to blank MP. About 19% of infected cells exposed to MP underwent apoptosis as compared to ~11% cells treated with soluble drugs or blank MP. Cell death induced by blank MP was caspase-independent. Only drug-containing MP induced apoptosis through caspase-8 and caspase-9. Bacterial survival after different treatments varied between individuals. In the best case, while untreated infection resulted in survival of 900±141 colony forming units (CFU), treatment with soluble drugs, drug-containing MP and blank MP respectively, reduced CFU counts to 8.5± 0.7, 3±1.4 and 102±138.6. The results suggest a role of the drug delivery system in macrophage activation as a component of therapeutic strategy against TB.

PLA Microparticles for Pulmonary Delivery of AntiTB drugs: Biodistribution study
2008-10-29: Nature Precedings
Rahul Kumar Verma
A dry powder inhalable (DPI) microparticles comprising anti-tuberculosis drugs incorporated in biodegradable polymers was developed for the treatment of pulmonary tuberculosis (P. Muttil et al. 2007). Poly L-lactic acid (PLA) microparticles incorporating a high payload of rifabutin and isoniazid were fabricated by spray drying (Buchi 190). Microparticles were composed of PLA and the drugs (rifabutin and isoniazid) at a 2:1:1 weight ratio. Microparticles of desired high encapsulation efficiency and sustained release characteristics were produced having a diameter range of 2-10 µm (Malvern Mastersizer 2000). Differential scanning calorimetry (DSC) was carried out to study drug polymer interaction. The time course of tissue biodistribution following a single inhalation dose of microparticles was evaluated. Thirty-two BALB/c mice were divided into groups of four and administered the DPI using an in-house (nose only) apparatus (Kaur et al. 2008; Verma et al. 2008). A validated HPLC method was used for determination of rifabutin and isoniazid in the lungs (target organ), liver and kidneys (major sites of toxicity) at different time-points after inhalation. A comparison was made with mice receiving free drugs (intravenous) at equivalent doses. Deposition of microparticles in lungs of mice following aerosolization was also evaluated. Pharmacokinetic parameters in different organs were calculated using WinNonlin software version 5.2. Area under the concentration-time curve observed (AUCobs), Cmax, half-life (t½) and clearance (CL) in lungs following inhalation /intravenous administration were:Rifabutin: AUCobs-96h= 1697.39 ±154.67 (187.63 ±23.93) µg/ml-1hr-1; Cmax = 33.42±3.80 (4.17±0.31) µg.ml-1; t½= 78.08±9.42 (34.00 ±3.31) and Cl= 1.16±.22 (0.68 ±0.45) ml.h-1.Isoniazid: AUCobs-24h= 566.31±123.96 (99.85 ±14.24) µg/ml-1hr-1; Cmax= 24.02±1.71 (8.16±0.93) µg.ml-1; t½= 25.88±12.16 (6.45±3.24) h; and Cl= 5.47±1.30 (0.96±0.14) ml.h-1.The relative bioavailability of both drugs incorporated in microparticles was significantly higher compared with free drugs. Peak levels of isoniazid and rifabutin in lungs (target organ) were much higher than those in the liver and kidney of mice in case of inhalation as compared to intravenous administration. Inhalation of microparticles resulted in targeting both drugs to the lungs, with the effect being more pronounced in the case of rifabutin than isoniazid. High and prolonged drug concentrations and increased AUC values (~9-fold and ~6 fold increase of rifabutin and isoniazid in case of lungs) with respect to free drugs were observed. Significant decrease in drug concentration was found in the liver and kidneys. Drug levels were maintained above the minimum inhibitory concentration (MIC) in organs through out the study after administration of encapsulated drugs. Based on favorable biodistribution kinetics, these microparticles hold great potential in reducing dosing frequency and toxicity of antituberculosis drugs.

A Targeted Therapeutic Rescues Botulinum Toxin-A Poisoned Neurons
2009-04-20: Nature Precedings
Prabhati Ray
Botulinum neurotoxin (BoNT), a Category A biothreat agent, is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. BoNT acts specifically by blocking neurotransmission primarily at peripheral nerve-muscle junctions causing severe flaccid muscle paralysis, which is fatal if proper medical care is not provided. The neurotoxin acts by specifically entering the presynaptic nerve endings where it interferes with the biochemical machinery involved in the process of neurotransmitter release, i.e., neuroexocytosis. Most serotypes of BoNT are known to remain active for weeks to months after entering the nerves, but BoNT/A is the most potent and long lasting in causing muscle paralysis. An effective medical countermeasure strategy requires developing a drug that could rescue poisoned neuromuscular synapses, and would include its efficient delivery specifically to presynaptic nerve terminals. Here we report rescuing of botulinum poisoned nerve cells by Mastoparan-7 (Mas-7), a peptide constituent of bee venom, that was delivered through a drug delivery vehicle (DDV) constructed from the non-toxic fragment of botulinum neurotoxin itself. We found that Mas-7 that was delivered into BoNT/A intoxicated cultured mouse spinal cord cells restored over 40% of stimulated neurotransmitter release. The rescue of the cell poisoning did not occur from inhibition of the endopeptidase activity of BoNT/A against its well known substrate, SNAP-25 that is mechanistically involved in the exocytosis process. Rather, Mas-7 induced a physiological host response apparently unrelated to SNAP-25, but linked to the phospholipase signal transduction pathway. In addition to providing the first effective antidote against botulism, our results open new avenues to study the mechanism of exocytosis, and also to examine an alternative cellular mechanism of botulinum neurotoxin action. An effective BoNT-based DDV can also be utilized for drug delivery against many neuronal and neuromuscular disorders.

Successful use of axonal transport for drug delivery by synthetic molecular vehicles
2008-08-07: Nature Precedings
Aaron G. Filler
We report the use of axonal transport to achieve intraneural drug delivery. We constructed a novel tripartite complex of an axonal transport facilitator conjugated to a linker molecule bearing up to a hundred reversibly attached drug molecules. The complex efficiently enters nerve terminals after intramuscular or intradermal administration and travels within axonal processes to neuron cell bodies. The tripartite agent provided 100-fold amplification of saturable neural uptake events, delivering multiple drug molecules per complex. In vivo, analgesic drug delivery to systemic and to non-targeted neural tissues was greatly reduced compared to existing routes of administration, thus exemplifying the possibility of specific nerve root targeting and effectively increasing the potency of the candidate drug gabapentin 300-fold relative to oral administration.

An intelligent liposome that may deliver drug molecules in a well controlled fashion
2009-04-20: Nature Precedings
Dumitru Popescu
The passage of molecules, especially large ones, through the cellular membrane is a very important problem for some biotechnological applications, such as drug delivery. The appearance of pores in the lipid bilayer following some controlled mechanisms may be an adequate and interesting way. Some pores, named stochastic pores, can appear due to structural and dynamic properties of lipid bilayer, but others may be favored by mechanical tension induced by different ways. Recently, a sequence of 30-40 pores was observed in the same vesicle, a pore at a time, which can appear in vesicles stretched by optical induced mechanical tension. There are two very interesting biotechnological applications that require the increase of membrane permeability: gene therapy and targeted drug delivery. In the first one, the transport of DNA fragments through cellular and nuclear membranes is required. The second application uses drug molecules encapsulated in vesicles, which have to be transported to a target place. Having reached that point, one supposes that the liposome discharges its content by its breakdown. In this paper, we will write about how a lipid vesicle has to release the drug molecules in a well-controlled fashion. Such liposomes are named pulsatory liposomes and they induce cyclic activity. We will demonstrate that this liposome may be programmed to work a certain number of cycles, settled in advance. Also, we will calculate the amount of drug delivered during each cycle. In fact, a pulsatory liposome may be conceived as a drug dose micro device, which works according to a medical prescription established a priori.