Current Pharmaceutical Biotechnology - Volume 10, Issue 6, 2009

The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA- 122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMBprobes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.

Phosphorodiamidate morpholino oligomers (PMO), which have a neutral chemistry, are extensively being used as tools for selective inhibition of gene expression in cell culture models and are currently in human clinical trials. Unlike phosphorothioates (PS ODN) and other charged oligonucleotides, little is known about the uptake characteristics of neutral oligomers. The purpose of this study was to understand the kinetics of PMO transport in cells and correlate with antisense activity. In contrast to primary cells and some transformed cell lines which were uptake permissive, established cancer cell lines showed very poor uptake with an occasional diffuse intracellular pattern. Differential PMO uptake was also observed in immune cells, with dendritic cells and monocytes showing highest uptake compared to T and B cells. In addition, PMO localization was observed to be heterogeneous within a population of uptake permissive cells. Unassisted PMO delivery targeting specific genes was correlated with functional antisense efficacy in experiments showing correction of pre-mRNA missplicing and inhibition of target enzyme activity in cells in culture. PMO internalization in uptakepermissive cells was identified to be specific, saturable, and energy-dependent, suggesting a receptor mediated uptake mechanism. Understanding PMO transport should facilitate the design of more effective synthetic antisense oligomers as therapeutic agents.

We present the strong fluorescence effect, a new 392 nm emission peak appearing after binding of a naphtolurea inhibitor XIIa to the enzyme epoxide hydrolase (EH), along with the quenching of the EH tryptophan fluorescence. We have studied the quenching of the 392-nm peak (attributed to XIIa bound inside the active center of the enzyme) of the mixture EH+XIIa by various strong transparent inhibitors (competing with XIIa for binding to EH), and measured the corresponding values of the Stern-Volmer constants, K(mix)SV. Strong EH inhibitors demonstrate different replacement behavior which can be used to distinguish them. We further demonstrate a novel fluorescent assay which allows to distinguish highly potent inhibitors and to visualize the strongest among them. We generated our assay calibration curve based on the quenching data, by plotting quenching strength K(mix)SV versus inhibiting strength, IC50 values. We used moderate inhibitors for the assay plot generation. We then applied this curve to determine IC50 values for several highly potent inhibitors, with IC50 values at the limit of the IC50 detection sensitivity by colorimetric enzyme assay. IC50 values determined from our quenching assay show correlation with IC50 values determined in the literature by more sensitive radioactive- based assay and allow differentiating the inhibitors potency in this group. To our knowledge, this is the first inhibitor assay of such kind. Chemical inhibition of EH is an important technology in the treatment of various cardiovascular diseases, therefore, this tool may play a crucial role in discovering new inhibitor structures for therapeutic EH inhibition.

A method for generating poly(lactic-co-glycolic acid) and polycaprolactone biodegradable particles of different size and shape using a jet generated in an electric field is elucidated. These particles are suitable for use as drug carriers and the method can be developed into a mass production route. The effect of different parameters such as applied voltage, collecting distance, flow rate and polymer concentration on inducing size and shape differences in these particles was studied. It was found that the flow rate, polymer concentration and collecting distance have a significant impact on the size of the generated particles and by changing the collecting distance a systematic reduction in the particle size by at least an order of magnitude (10μm-100nm) can be achieved. By using a high polymer concentration (30 wt. %) the shape and surface morphology of these particles can also be controlled from spherical to fibrous, and smooth to irregular, respectively, which presently is an interesting strategy and concept in drug delivery. This method is very useful as a onestep generator of different sizes of drug carriers with morphological variations.

The purpose of this study was to dramatically enhance the solubility (> 400 fold) and stability of a therapeutic protein (Fibroblast Growth Factor 20) and to perform detailed biophysical characterization for the optimization of its formulation. The solubility of FGF-20 strongly depends on pH, arginine concentration and anions present in a buffer system. In the absence and presence of arginine, solubility was higher at lower pH (5 ≤ pH ≤ 6) and then decreased steadily with a minimum solubility at around pH 6.3 and plateaus at around pH 7.5 respectively. For a given pH, the protein was most soluble in arginine-sulfate. The solubility of FGF-20 increases with an increase in arginine-sulfate concentration for a given pH. However, a salting out effect was observed at higher arginine-sulfate concentration. Polysorbate-80 did not have any striking effect on solubility and no effect on thermal stability, but it significantly prevented the loss of protein under agitated conditions. Thermal stability of FGF-20 measured by DSC was increased with an increase in argininesulfate concentration (at least up to 0.5M). A sturdy dependence of thermal stability on pH was observed with about a 15°C increase in Tm (melting temperature) at pH 7.0 in comparison to pH 5.0. From the DSC data, approximate stability curves were generated and cold denaturation temperatures were predicted. Denaturant induced unfolding studies provided better insight of FGF-20 in different solution conditions in terms of structure and stability than the DSC data. An inverse relationship of solubility and thermal stability was observed in the pH range of 5.0 to 8.5 at a fixed arginine concentration and is consistent with Linderstrom-Lange’s smeared model. A direct correlation between solubility and thermal stability was observed at different arginine concentrations for a fixed pH. The effect of arginine on the solubility and stability of FGF-20 was dominated by the preferential binding interaction.

Tryparedoxin peroxidase (TryP) is a key enzyme of the trypanothione-dependent metabolism for removal of oxidative stress in leishmania. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. Inhibitors of this enzyme are presumed to be antilesihmania drugs and structural studies are prerequisite of rational drug design. We have constructed three dimensional structure of TryP of Leishmania infantum using comparative modeling. Structural analysis reveals several interesting features. Moreover, it shows remarkable structural difference with human host glutathione peroxidase, an enzyme involved in similar function and TryP from Leishmania major.

Solid tumors have several barriers that may limit drug penetration and provide inherent mechanisms of resistance. Therefore, the evaluation of antineoplastic agents should base on tumor drug exposure and antitumor activity. Owing to selective access to the extracellular space of the tumor, which acts as target site for most antineoplastic drugs, monitoring drug disposition and corresponding antitumor activity, microdialysis(MD), a probe-based sampling technique, satisfies regulatory requirements for pharmacokinetic- pharmacodynamic (PK-PD) studies. MD is a powerful tool for PK-PD studies of oncological drugs, which would allow better understanding of exposure-response relationships and contributes to the research and development of oncological drugs. Recent progresses in the development of MD for estimating PK-PD studies of oncological drugs are summarized in this review. Special preclinical application examples of MD for PK-PD studies of oncological drugs organized by types of antineoplastic drugs and informations collected are also described. In conclusion, the role of MD in preclinical PK-PD studies of oncological drugs has been confirmed and it has provided a strong foundation for further clinical research.

Natural products from plants are of major pharmaceutical and therapeutic importance, several of which are often obtained from the underground parts of the concerned plants. Deviation from standard rules in modern medicines, where instead of a single isolated fraction, a group of naturally occurring components exerts the desired therapeutic effect, was noted in case of Picroliv or Kutkin of Picrorhiza kurroa. “Picroliv” mainly a glucoside, is one such compound, normally obtained from 3 - 4 years old roots and rhizomes of an endangered medicinal plant - Picrorhiza kurroa (kutki) and constitute an important component of many Indian herbal preparations, used mainly for the treatment of a variety of liver ailments. It is an iridoid glycoside mixture containing 60% picroside I and kutkoside in the ratio of 1:1.5. Picroliv has shown efficacy comparable to silymarin in rodent models of galactosamine, paracetamol, thioacetamide and CCl4 induced hepatic damage. Picroliv has also shown cholerectic effect in rats and anti-cholestatic effect in rats, guinea pigs and cats treated with paracetamol and ethinyl estradiol. It has also anti-viral and immune-stimulant activities and is devoid of any significant CNS and CVS, autonomic and other systemic activity. Because of its apparent ability as a strong hepatoprotective and immune-modulatory compound, it is in high demand in both national and international markets. The review discusses the potential of Picrorhiza in various hepatic diseases as well as the chemistry and activity of individual compound of crude drug Picroliv.