Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis.

BACKGROUND: Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-ß-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human's, there have been some early drug discovery efforts towards developing potent and selective inhibitors. OBJECTIVE: Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors. CONCLUSION: Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

Subgram-Scale Synthesis of Biomass Waste-Derived Fluorescent Carbon Dots in Subcritical Water for Bioimaging, Sensing, and Solid-State Patterning.

Fluorescent carbon dots (FCDs) have received considerable attention because of the great potential for a wide range of applications, from bioimaging to optoelectronic devices. In this work, we reported the synthesis of nitrogen-doped FCDs with an average size of 2 nm in a subcritical water apparatus by using biomass waste (i.e., expired milk) as the precursor. The obtained FCDs were highly dispersed in aqueous solution because of the presence of O-containing functional groups on their surfaces. Under the excitation of ultraviolet and blue light, the FCDs exhibited excitation wavelength-dependent fluorescence in the emission range of 400-550 nm. The FCDs could be easily taken up by HeLa cells without additional surface functionalization, serving as fluorescent nanoprobes for bioimaging. The applications of FCDs as sensing agents for the detection of Fe3+, solid-state fluorescent patterning, and transparent hybrid films were also performed, demonstrating their potential for solid-state fluorescent sensing, security labeling, and wearable optoelectronics.

Improving the dissolution properties of curcumin using dense gas antisolvent technology.

The dissolution properties of curcumin are notoriously poor and hinder its bioavailability. To improve its dissolution properties, curcumin has been formulated with methyl-ß-cyclodextrin and polyvinylpyrrolidone by the atomized rapid injection solvent extraction (ARISE) system. The compounds were co-precipitated from organic solutions using carbon dioxide at 30°C and 95bar as the antisolvent. Curcumin formulations were also produced by physical mixing and freeze drying for comparative purposes. The morphology, crystallinity, solid state molecular interactions, apparent solubility and dissolution profiles of samples were observed. The results indicate that the ARISE process is effective in the preparation of curcumin micro-composites with enhanced dissolution profiles compared to unprocessed material and products from physical mixing and freeze drying.

Formation of nanocarrier systems by dense gas processing.

Nanocarrier systems, such as liposomes, polymersomes, and micelles, find applications in the delivery of a wide range of compounds, including targeted delivery of pharmaceuticals. Nanocarrier systems have the ability to increase the bioavailability, reduce toxicity, and avoid undesirable interactions of active pharmaceutical ingredients. In this work, a novel dense gas technique known as depressurization of an expanded solution into aqueous media (DESAM) was used to produce different types of nanocarrier systems. The effects of using different types of dense gases and different operating temperatures were investigated. Encapsulation of hydrophilic compounds in the vesicles (liposomes and polymersomes) was also studied. The highest encapsulation efficiencies in liposomes and polymersomes achieved were 10.2 and 9.7%, respectively. The DESAM process was also able to reduce the residual solvent content in the product to 2.2% (v/v), which is significantly lower than the solvent residual levels reported for conventional processing.

Application of a dense gas technique for sterilizing soft biomaterials.

Sterilization of soft biomaterials such as hydrogels is challenging because existing methods such as gamma irradiation, steam sterilization, or ethylene oxide sterilization, while effective at achieving high sterility assurance levels (SAL), may compromise their physicochemical properties and biocompatibility. New methods that effectively sterilize soft biomaterials without compromising their properties are therefore required. In this report, a dense-carbon dioxide (CO(2) )-based technique was used to sterilize soft polyethylene glycol (PEG)-based hydrogels while retaining their structure and physicochemical properties. Conventional sterilization methods such as gamma irradiation and steam sterilization severely compromised the structure of the hydrogels. PEG hydrogels with high water content and low elastic shear modulus (a measure of stiffness) were deliberately inoculated with bacteria and spores and then subjected to dense CO(2) . The dense CO(2) -based methods effectively sterilized the hydrogels achieving a SAL of 10(-7) without compromising the viscoelastic properties, pH, water-content, and structure of the gels. Furthermore, dense CO(2) -treated gels were biocompatible and non-toxic when implanted subcutaneously in ferrets. The application of novel dense CO(2) -based methods to sterilize soft biomaterials has implications in developing safe sterilization methods for soft biomedical implants such as dermal fillers and viscosupplements.

Particle formation of budesonide from alcohol-modified subcritical water solutions.

Recently, subcritical water (SBCW: water that has been heated to a temperature between 100°C and 200°C at pressures of up to 70bar) has been used to dissolve several hydrophobic pharmaceutical compounds (Carr et al., 2010a). Furthermore, a number of active pharmaceutical ingredients (APIs) have been rapidly precipitated from SBCW solutions (Carr et al., 2010b,c). It is possible to alter the precipitate morphology by altering the processing variables; including the SBCW-API solution injection temperature and adding impurities (such as pharmaceutical excipients, e.g. lactose) to the precipitation chamber. The work presented in this article demonstrates that the morphology of pharmaceutical particles can be tuned by adding organic solvents (ethanol and methanol) to the SBCW-API solutions. Particle morphology has also been tuned by adding different pharmaceutical excipients (polyethylene glycol 400 and lactose) to the precipitation chamber. Different morphologies of pharmaceutical particles were produced, ranging from nanospheres of 60nm diameter to 5µm plate particles. Budesonide was used as the model API in this study. Two experimental products were spray dried to form dry powder products. The aerodynamic particle size of the powder was established by running the powder through an Andersen Cascade Impactor. It has been shown that the drug particles produced from the SBCW micronization process, when coupled with a spray drying process, are suitable for delivery to the lungs.

The development of a dense gas solvent exchange process for the impregnation of pharmaceuticals into porous chitosan.

The aim of this study was to prepare stable formulations of poorly water-soluble drugs in amorphous forms to enhance their dissolution rates, promote the bioavailability, minimize the dosage, thereby theoretically decreasing their side effects. A dense gas solvent exchange process was developed for the impregnation of poorly water-soluble drugs such as camptothecin and griseofulvin into a chitosan matrix. The amount of drug impregnated was measured by UV-spectrophotometery and gravimetric techniques. Pore characteristics and the crystallinity of the drugs in the impregnated chitosan were measured. Homogenous nano-sized pores with thin walls were formed in chitosan using the dense gas solvent exchange process. The method was efficient for the impregnation of a drug into chitosan. Results of XRD, Fourier transform infrared spectroscopy and differential scanning calorimetry demonstrated that as a result of interaction between chitosan and the drug, both camptothecin and griseofulvin were in amorphous forms after processing. The dissolution rate of processed griseofulvin was increased threefold due to the hydrophilic properties of chitosan and its interaction with the drug. A new approach was developed for promoting drug bioavailability that has the potential to decrease the required dose and side effects, particularly for chemotherapeutic drugs with narrow therapeutic index.

The depressurization of an expanded solution into aqueous media for the bulk production of liposomes.

A new dense gas process for the formation of liposomes has been developed: depressurization of an expanded solution into aqueous media (DESAM). The technique provides a fast and simple process for bulk liposome formation. As an alternative to current dense gas technologies, the DESAM process reduces the pressure requirements for liposome formation. Liposomes with diameters between 50 and 200 nm were formed. For all samples produced using ethanol as the solvent, the average effective diameter ranged from 119 to 207 nm. When chloroform was used as the solvent, the average effective diameter increased to 387 nm. The residual solvent volume fraction in the liposomal product was less than 4% v/v, which is approximately one-quarter of the value reported for some other dense gas liposome formation methods. The liposomal samples were stored after formation at 5 degrees C for up to 8 months, with the average effective diameter and polydispersity increasing by only 13% and 7%, respectively, indicating high stability of the formulations.

Conventional and dense gas techniques for the production of liposomes: a review.

The aim of this review paper is to compare the potential of various techniques developed for production of homogenous, stable liposomes. Traditional techniques, such as Bangham, detergent depletion, ether/ethanol injection, reverse-phase evaporation and emulsion methods, were compared with the recent advanced techniques developed for liposome formation. The major hurdles for scaling up the traditional methods are the consumption of large quantities of volatile organic solvent, the stability and homogeneity of the liposomal product, as well as the lengthy multiple steps involved. The new methods have been designed to alleviate the current issues for liposome formulation. Dense gas liposome techniques are still in their infancy, however they have remarkable advantages in reducing the use of organic solvents, providing fast, single-stage production and producing stable, uniform liposomes. Techniques such as the membrane contactor and heating methods are also promising as they eliminate the use of organic solvent, however high temperature is still required for processing.

Increasing the dissolution rate of itraconazole processed by gas antisolvent techniques using polyethylene glycol as a carrier.

PURPOSE: The purpose of this study was to increase the dissolution rate of the poorly water soluble, antifungal drug Itraconazole. METHODS: Itraconazole was successfully micronized using both the gas antisolvent (GAS) and aerosol solvent extraction systems (ASES) using Acetone as the solvent. The affects of operating conditions such as temperature, pressure and solvent choice on variables such as morphology, particle size and dissolution were investigated. The influence of temperature in the range 25 to 40 degrees C and pressure between 90 and 190 bar were investigated. RESULTS: Solvent choice was found to have the largest affect on particle production, with acetone found to be the optimal solvent choice when compared with dimethyl formamide (DMF), tetrahydrofuran (THF) and dichloromethane (DCM). Itraconazole particles with an average particle size of 6.9 microm were formed at the optimal ASES processing conditions of 40 degrees C and 190 bar. More significantly, in the first 100 minutes of dissolution 71.1% of the dense gas processed itraconazole was dissolved compared with 52.5% of Sporonox (the commercially available formulation) and 14.6% of the unprocessed material. Additional studies demonstrated that the formation of an itraconazole/PEG composite resulted in a 6-fold increase in dissolution rate in the first 100 min, to 89.8%, when compared to the unprocessed material. CONCLUSIONS: Using ASES, microparticles of itraconazole were produced with an increased dissolution rate compared with raw material and commercially available product.

Dense gas processing of polymeric controlled release formulations.

Dense gas techniques, which utilize the properties of fluids in the vicinity of their critical points, are of increasing interest in the processing of pharmaceuticals. It is generally known that dense gases can be used for extractions, chromatographic separations and chemical synthesis due to their liquid-like solvation power and gas-like mass-transfer properties. The processes can be conducted at moderate operating conditions and are thus suitable for many heat-labile compounds such as proteins, biocompatible polymers and pharmaceuticals. Recent applications of dense gas techniques include micronization, crystallization of high-purity particles, sterilization and preparation of microencapsulated drug formulations. The following paper provides a brief overview of dense gases and various processing techniques to fabricate polymeric drug-loaded formulations for controlled release purposes.

Dense gas processing of micron-sized drug formulations incorporating hydroxypropylated and methylated beta-cyclodextrin.

PURPOSE: Because of their importance in pharmaceutical applications, hydroxypropyl-beta-cyclodextrin and methyl-beta-cyclodextrin have been selected to study the formation of micronized complexes incorporating active pharmaceutical ingredients (APIs) and cyclodextrins (CDs) by dense gas (DG) processing. METHODS: A single-step DG technique was used as an alternative to conventional methods for the manufacturing of API/CD complexes. The DG technology is highly attractive in the pharmaceutical industry because of its potential to generate micronized particles with controlled particle size distributions at moderate operating conditions. The effect of the aerosol solvent extraction system (ASES) processing on the dissolution performance of naproxen (NPX) was examined. RESULTS: The CDs were produced as microspheres smaller than 3 microm. The coprecipitation of each CD with NPX resulted in the production of microparticles with enhanced dissolution rates. CONCLUSIONS: The ASES was operated under mild conditions and generated micron-sized spherical particles that could be of particular interest in formulations for pulmonary delivery. Particular advantages of the technique are that (1) nontoxic solvents are used, and (2) it is suitable for the processing of thermally labile compounds. The proposed process can create opportunities to improve current administration routes and exploit novel delivery systems for drug formulations incorporating CDs.

Application of dense gas techniques for the production of fine particles.

The feasibility of using dense gas techniques such as rapid expansion of supercritical solutions (RESS) and aerosol solvent extraction system (ASES) for micronization of pharmaceutical compounds is demonstrated. The chiral nonsteroidal anti-inflammatory racemic ibuprofen is soluble in carbon dioxide at 35 degrees C and pressures above 90 bar. The particle size decreased to less than 2 microm while the degree of crystallinity was slightly decreased when processed by RESS. The dissolution rate of the ibuprofen (a poorly water-soluble compound) was significantly enhanced after processing by RESS. The nonsteroidal anti-inflammatory drug Cu2(indomethacin)4L2(Cu-Indo); (L = dimethylformamide [DMF]), which possessed very low solubility in supercritical CO2, was successfully micronized by ASES at 25 degrees C and 68.9 bar using DMF as the solvent and CO2 as the antisolvent. The concentration of solute dramatically influenced the precipitate characteristics. The particles obtained from the ASES process were changed from bipyramidal to spherical, with particle size less than 5 microm, as the concentration increased from 5 to 100 mg/g. A further increase in solute concentration to 200 mg/g resulted in large porous spheres, between 20 and 50 micron, when processing Cu-Indo by the ASES method. The dissolution rate of the micronized Cu-Indo was significantly higher than the commercial product.

Inactivation of bacteria and spores by pulse electric field and high pressure CO2 at low temperature.

The common methods for inactivation of bacteria involve heating or exposure to toxic chemicals. These methods are not suitable for heat-sensitive materials, food, and pharmaceutical products. Recently, a complete inactivation of many microorganisms was achieved with high-pressure carbon dioxide at ambient temperature and in the absence of organic solvent and irradiation. The inactivation of spores with CO(2) required long residence time and high temperatures, such as 60 degrees C. In this study the synergistic effect of pulsed electric field (PEF) in combination with high-pressure CO(2) for inactivation was investigated. The bacteria Escherichia coli, Staphylococcus aureus, and Bacillus cereus were suspended in glycerol solution and treated in the first step with PEF (up to 25 KV/cm) and then with high-pressure CO(2) not higher than 40 degrees C and 200 bar. The inactivation efficiency was determined by counting the colony formation units of control and sample. Samples of the cells subjected to PEF treatment alone and in combination with CO(2) treatment were examined by scanning electron microscopy to determine the effect of the processes on the cell wall. Experimental results indicate that the viability decreased with increasing electrical field strength and number of pulses. A further batch treatment with supercritical CO(2) lead to complete inactivation of bacterial species and decreased the count of the spores by at least three orders of magnitude, the inactivation being enhanced by an increase of contact time between CO(2) and the sample. A synergistic effect between the pulsed electric field and the high-pressure CO(2) was evident in all the species treated. The new low temperature process is an alternative for pasteurization of thermally labile compounds such as protein and plasma and minimizes denaturation of important nutrient compounds in the liquid media.

Generation of fine powders of recombinant human deoxyribonuclease using the aerosol solvent extraction system.

PURPOSE: To investigate the feasibility of using the Aerosol Solvent Extraction System (ASES) to produce fine powders of recombinant human deoxyribonuclease (rhDNase), lysozyme-lactose and rhDNase-lactose powders from aqueous based solutions. METHODS: The ASES technique using high pressure carbon dioxide modified with ethanol or ethanol and triethylamine was used for the generation of rhDNase powders and protein-lactose powders from aqueous based solutions. Particle size, morphology, size distributions, crystallinity, and powder aerosol performance were measured. The biochemical integrity of the processed rhDNase was assessed by testing the monomer content and the degree of deamidation. RESULTS: RhDNase precipitated as spherical particles in the size range between 50 and 500 nm. The primary nano-sized particles were agglomerated to micron-sized clumps of particles during the precipitation process. The median particle size and the fine particle fraction were functions of the operating temperature and the nozzle system used. RhDNase was substantially denatured in the ASES process using carbon dioxide modified with ethanol as anti-solvent. However almost complete recovery of the monomer was achieved using carbon dioxide modified with ethanol-triethylamine as an anti-solvent. Lysozyme-lactose and rhDNase-lactose powders were also precipitated as agglomerated spheres using the ASES process. The powders were amorphous except for those with lactose content higher than 45%. CONCLUSIONS: Micron-sized particles of rhDNase suitable for inhalation delivery were generated from aqueous based solutions using the modified ASES technique. The biochemical integrity of the rhDNase powder is a function of the antisolvent and the operating temperature.