Preparation of polymeric micelles for use as carriers of tuberculostatic drugs

Purpose: This paper focuses on the characterization of polymeric micelle-forming tuberculostatic prodrugs and the antimycobacterial activity of these prodrugs.Method: By the condensation of hydroxymethylpyrazinamide, isoniazid and rifampin with free carboxyl groups on the copolymer poly(ethyleneglycol)-poly(aspartic acid), micelle-forming carrier-drug conjugates were obtained. These micelles were characterized by dynamic light scattering, to measure the micelle diameter; by acid-base titration, to determine the percentage of carboxylic groups occupied by the tuberculostatic; by Sudan III solubility tests, to estimate the critical micelle concentration (CMC); and visual control and spectrophotometric measurement, to determine the stability of micelles. These micelles were tested in vitro against several Mycobacterium strains.Results: As expected, the size and distribution of the micelle-forming tuberculostatic prodrugs found to be small (78.2nm, 84.2nm and 98.9 nm) while the level of the drug conjugated was high (65.02-85.7%). Furthermore, the micelles were stable in vitro, exhibiting a low level of CMC and stronger antimycobacterial activity than the original drugs.Conclusion: the results demonstrate that polymeric micelles can be used as efficient carriers for drugs, which alone, exhibit undesired pharmacokinetics, poor solubility, and low stability. The synthesized micelle-forming tuberculostatic prodrugs opens a perspective of alternative prodrugs that prolong action and decrease the toxicity of the tuberculostatic drugs of choice.

Pharmacological Evaluation and Preparation of Nonsteroidal Anti-Inflammatory Drugs Containing an N-Acyl Hydrazone Subunit

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pharmacological evaluation and preparation of nonsteroidal anti-inflammatory drugs containing an N-Acyl hydrazone subunit

A series of anti-inflammatory derivatives containing an N-acyl hydrazone subunit (4a–e) were synthesized and characterized. Docking studies were performed that suggest that compounds 4a–e bind to cyclooxygenase (COX)-1 and COX-2 isoforms, but with higher affinity for COX-2. The compounds display similar anti-inflammatory activities in vivo, although compound 4c is the most effective compound for inhibiting rat paw edema, with a reduction in the extent of inflammation of 35.9% and 52.8% at 2 and 4 h, respectively. The anti-inflammatory activity of N-acyl hydrazone derivatives was inferior to their respective parent drugs, except for compound 4c after 5 h. Ulcerogenic studies revealed that compounds 4a–e are less gastrotoxic than the respective parent drug. Compounds 4b–e demonstrated mucosal damage comparable to celecoxib. The in vivo analgesic activities of the compounds are higher than the respective parent drug for compounds 4a–b and 4d–e. Compound 4a was more active than dipyrone in reducing acetic-acid-induced abdominal constrictions. Our results indicate that compounds 4a–e are anti-inflammatory and analgesic compounds with reduced gastrotoxicity compared to their respective parent non-steroidal anti-inflammatory drugs.

Sequence of reactions leading to cytochrome-p-450 synthesis-effect of drugs

The effect of phenobarbital on the rates of the synthesis of the protein and heme moieties of cytochrome P-450 has been studied. For this purpose, cytochrome P-450 has been partially purified as its P-420 derivative and the labeled amino acid incorporation into the protein has been studied after subjecting a partially purified preparation to sodium dodecyl sulfate gel electrophoresis. The incorporation studies into the protein species after sodium dodecyl sulfate gel electrophoresis reveal that the drug primarily accelerates the rate of apoprotein synthesis followed by an increase in the rate of heme synthesis. The messenger for apocytochrome P-450 appears to be fairly stable.

Studies of the serotonin type 3A receptor and the chemical preparation of tRNA

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT₃AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.

In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT₃A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-π interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT₃AR, which establishes a binding orientation for these drugs.

In Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT₃A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-π interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.

In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes.

Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, K_B, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT₃A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.

New Catalytic Methods for the Preparation of Tryptophans and Pyrroloindolines: Total Synthesis of (+)-Naseseazines A and B and (–)-Aspergilazine A

Tryptophan and unnatural tryptophan derivatives are important building blocks for the total synthesis of natural products, as well as the development of new drugs, biological probes, and chiral small molecule catalysts. This thesis describes various catalytic methods for the preparation of tryptophan derivatives as well as their functionalization and use in natural product total synthesis.

Herein, the tandem Friedel–Crafts conjugate addition/asymmetric protonation reaction between 2-substituted indoles and methyl 2-acetamidoacrylate to provide enantioenriched trytophans is reported. This method inspired further work in the area of transition metal catalyzed arylation reactions. We report the development of the coppercatalyzed arylation of tryptamine and tryptophan derivatives. The utility of these transformations is highlighted in the five-step syntheses of the natural products (+)-naseseazine A and B. Further work on the development of a mild and general Larock indolization protocol to access unnatural tryptophans is also discussed.

Preparation of core-sheath composite nanofibers by emulsion electrospinning

Uniform core-sheath nanofibers are prepared by electrospinning a water-in-oil emulsion in which the aqueous phase consists of a poly(ethylene oxide) (PEO) solution in water and the oily phase is a chloroform solution of an amphiphilic poly(ethylene glycol)-poly(L-lactic acid) (PEGPLA) diblock copolymer. The obtained fibers are composed of a PEO core and a PEG-PLA sheath with a sharp boundary in between. By adjusting the emulsion composition and the emulsification parameters, the overall fiber size and the relative diameters of the core and the sheath can be changed. A mechanism is proposed to explain the process of transformation from the emulsion to the core-sheath fibers, i.e., the stretching and evaporation induced de-emulsification. In principle, this process can be applied to other systems to prepare core-sheath fibers in place of concentric electrospinning and it is especially suitable for fabricating composite nanofibers that contain water-soluble drugs.

Screening of Conditions for the Preparation of Poly( -Caprolactone) Nanocapsules Containing the Local Anesthetic Articaine

The local anesthetic articaine (ATC) is widely used in dentistry; however, its side effects can include paresthesia and nerve injury. Polymeric nanocapsules (PN) can be used as carriers for drugs, and help to reduce undesirable symptoms. The objective of this study was to evaluate the influence of different factors on the average size, polydispersion, and encapsulation efficiency of PN containing ATC. Poly(ε-caprolactone) (PCL) nanocapsules containing ATC were prepared by the oil-in-water emulsion/solvent evaporation method. The final ATC concentration was 2%. The preparation conditions were optimized using a central composite blocked cube-star design to investigate the influence of two variables at five levels, with 22 factorial points (–1 and +1), two replicates of the central point, 2×2 axial points (–1.414 and +1.414), and an orthogonal distribution, resulting in 10 experiments. The factors varied were the PVA concentration and the sonication time. The nanocapsules showed a satisfactory size range, a polydispersivity index less than 0.2, and high encapsulation efficiency. The values of the factors had no significant influence on either average size or polydispersion, although the encapsulation efficiency was significantly influenced by the sonication time. Improved formulations were identified using the central composite design, which revealed that the main consideration in selecting a suitable formulation was the encapsulation efficiency. Two of the formulations showed both high encapsulation efficiency and colloidal characteristics appropriate for the route of administration.

Efficient microwave hydrothermal preparation of nanocrystalline anatase TiO2 colloids

Titanium dioxide nanocrystals are an important commercial product used primarily in white pigments and abrasives, however, more recently the anatase form of TiO2 has become a major component in electrochemical and photoelectrochemical devices. An important property of titanium dioxide nanocrystals for electrical applications is the degree of crystallinity. Numerous preparation methods exist for the production of highly crystalline TiO2 particles. The majority of these processes require long reaction times, high pressures and temperatures (450–1400 °C). Recently, hydrothermal treatment of colloidal TiO2 suspensions has been shown to produce quality crystalline products at low temperatures (<250 °C). In this paper we extend this idea utilising a direct microwave heating source. A comparison between convection and microwave hydrothermal treatment of colloidal TiO2 is presented. The resulting highly crystalline TiO2 colloids were characterised using Raman spectroscopy, XRD, TEM, and electron diffraction. The results show that the microwave treatment of colloidal TiO2 gives comparable increases in crystallinity with respect to normal hydrothermal treatments while requiring significantly less time and energy than the hydrothermal convection treatment.

Preparation of future weather data to study the impact of climate change on buildings

The dynamic interaction between building systems and external climate is extremely complex, involving a large number of difficult-to-predict variables. In order to study the impact of climate change on the built environment, the use of building simulation techniques together with forecast weather data are often necessary. Since most of building simulation programs require hourly meteorological input data for their thermal comfort and energy evaluation, the provision of suitable weather data becomes critical. In this paper, the methods used to prepare future weather data for the study of the impact of climate change are reviewed. The advantages and disadvantages of each method are discussed. The inherent relationship between these methods is also illustrated. Based on these discussions and the analysis of Australian historic climatic data, an effective framework and procedure to generate future hourly weather data is presented. It is shown that this method is not only able to deal with different levels of available information regarding the climate change, but also can retain the key characters of a “typical” year weather data for a desired period.

Fumaric acid monoethyl ester-functionalized poly(D,L-Lactide)/N-vinyl-2- pyrrolidone Resins for the preparation of tissue engineering scaffolds by stereolithography

Polymer networks were prepared by photocross-linking fumaric acid monoethyl ester (FAME) functionalized, three-armed poly(D,L-lactide) oligomers using Af-vinyl-2-pyrrolidone (NVP) as diluent and comonomer. The use of NVP together with FAME-functionalized oligomers resulted in copolymerization at high rates, and networks with gel contents in excess of 90 were obtained. The hydrophilicity of the poly(D,L-lactide) networks increases with increasing amounts of NVP, networks containing 50 wt of NVP absorbed 40 of water. As the amount of NVP was increased from 30 to 50 wt , the Young's modulus after equilibration in water decreased from 0.8 to 0.2 GPa, as opposed to an increase from 1.5 to 2.1 GPa in the dry state. Mouse preosteoblasts readily adhered and spread onto all prepared networks. Using stereolithography, porous structures with a well-defined gyroid architecture were prepared from these novel materials. This allows the preparation of tissue engineering scaffolds with optimized pore architecture and tunable material properties.