Effect of Counterions on Physicochemical Properties of Prazosin Salts

This study evaluated the effect of counterions on the physicochemical properties of prazosin salts. Salt forms of prazosin, namely, mesylate, besylate, tosylate, camsylate, oxalate, and maleate, were prepared and compared with the marketed anhydrous and polyhydrate forms of prazosin hydrochloride. Physicochemical characterization was performed in the order of crystallinity, hygroscopicity, solubility, and stability to select the optimal salt(s). Permeability study in Caco-2 cell lines and in vivo bioavailability study in rat model were investigated to ascertain their biopharmaceutical advantage. All salt forms were crystalline, nonhygroscopic (except the anhydrous hydrochloride salt), and had solubility in the range of 0.2 to 1.6 mg/ml. All salts were physically and chemically stable at 40°C/75% relative humidity, but degraded in UV-visible light, except the anhydrous hydrochloride salt. Prazosin mesylate was selected as the optimal salt, as it possessed higher solubility, permeability, and bioavailability, compared to the commercial hydrochloride salts. Hydrochloride salt is reported to have poor bioavailability that is partially attributed to its low solubility and extensive common-ion effect in the gastric region. Factors like hydrophilicity of the counterion, hydration state of the salt, and melting point of the salt contribute to the physicochemical properties of the salts. This study has implications in the selection of an optimal salt form for prazosin, which is suitable for further development.     

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening.     

Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT-phosphoprotein interaction

Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (the C-terminal portion of BRCA1, a key tumor suppressor protein with various functions) and phosphorylated proteins (Abraxas/BACH1/CtIP), implicated in DNA damage response and repair pathways, should prove to be useful in studying BRCA1's role in cancer and in potentially sensitizing tumors to chemotherapeutic agents. We developed and miniaturized to a 1536-well format and 3-mul final volume a pair of fluorescence polarization (FP) assays using fluorescein- and rhodamine-labeled pBACH1 fragment. To minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members all were assayed against both the fluorescein- and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm. Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS, we were able to confidently select a number of actives for further studies.     

Solid State Characterization of Commercial Crystalline and Amorphous Atorvastatin Calcium Samples

Atorvastatin calcium (ATC), an anti-lipid BCS class II drug, is marketed in crystalline and amorphous solid forms. The objective of this study was to perform solid state characterization of commercial crystalline and amorphous ATC drug samples available in the Indian market. Six samples each of crystalline and amorphous ATC were characterized using X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis, Karl Fisher titrimetry, microscopy (hot stage microscopy, scanning electron microscopy), contact angle, and intrinsic dissolution rate (IDR). All crystalline ATC samples were found to be stable form I, however one sample possessed polymorphic impurity, evidenced in XRPD and DSC analysis. Amongst the amorphous ATC samples, XRPD demonstrated five samples to be amorphous ‘form 27’, while, one matched amorphous ‘form 23’. Thermal behavior of amorphous ATC samples was compared to amorphous ATC generated by melt quenching in DSC. ATC was found to be an excellent glass former with T_g/T_m of 0.95. Residual crystallinity was detected in two of the amorphous samples by complementary use of conventional and modulated DSC techniques. The wettability and IDR of all amorphous samples was found to be higher than the crystalline samples. In conclusion, commercial ATC samples exhibited diverse solid state behavior that can impact the performance and stability of the dosage forms.     

Hypoxia-cultured human adipose-derived mesenchymal stem cells are non-oncogenic and have enhanced viability,motility, and tropism to brain cancer

Adult human adipose-derived mesenchymal stem cells (hAMSCs) are multipotent cells, which are abundant, easily collected, and bypass the ethical concerns that plague embryonic stem cells. Their utility and accessibility have led to the rapid development of clinical investigations to explore their autologous and allogeneic cellular-based regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer properties, among others. hAMSCs are typically cultured under ambient conditions with 21% oxygen. However, physiologically, hAMSCs exist in an environment of much lower oxygen tension. Furthermore, hAMSCs cultured in standard conditions have shown limited proliferative and migratory capabilities, as well as limited viability. This study investigated the effects hypoxic culture conditions have on primary intraoperatively derived hAMSCs. hAMSCs cultured under hypoxia (hAMSCs-H) remained multipotent, capable of differentiation into osteogenic, chondrogenic, and adipogenic lineages. In addition, hAMSCs-H grew faster and exhibited less cell death. Furthermore, hAMSCs-H had greater motility than normoxia-cultured hAMSCs and exhibited greater homing ability to glioblastoma (GBM) derived from brain tumor-initiating cells from our patients in vitro and in vivo. Importantly, hAMSCs-H did not transform into tumor-associated fibroblasts in vitro and were not tumorigenic in vivo. Rather, hAMSCs-H promoted the differentiation of brain cancer cells in vitro and in vivo. These findings suggest an alternative culturing technique that can enhance the function of hAMSCs, which may be necessary for their use in the treatment of various pathologies including stroke, myocardial infarction, amyotrophic lateral sclerosis, and GBM.Mesenchymal stem cells (MSCs) are multipotent cells, isolated from the bone marrow, adipose tissue, and muscle, among others. They are clonally expansive, with the capacity to differentiate into osteocytes, adipocytes, and chondrocytes.^{1, 2} MSCs are widely studied for their regenerative potential, tissue preservation capabilities, anti-inflammatory properties, and anticancer therapeutic potential.^{3, 4, 5} MSCs can serve as vehicles for delivering effective targeted therapy to primary brain cancer and metastatic cancer.^{6, 7, 8}Notwithstanding aggressive treatment of primary brain cancer (glioblastoma (GBM)) with surgical resection, chemotherapy, and radiotherapy, the median survival following diagnosis is 14.6 months.^{9, 10, 11, 12, 13, 14, 15} GBM-targeted therapy using neural stem cells and MSCs as vehicles for therapeutic agents is a promising strategy.¹⁶ MSCs seem to be the ideal stem cells, as they are autologous, easily collected, and easily re-implanted.^{17, 18} The most commonly used MSCs are bone marrow-derived MSCs (BM-MSCs) and human adipose-derived MSCs (hAMSCs). Compared with BM-MSCs, hAMSCs are easier to obtain.^{19, 20}Despite the potential utility of hAMSCs, their use is hampered by their low concentration within tissues.^{21, 22} Thus, in vitro expansion of hAMSCs is necessary. Compared with BM-MSCs, hAMSCs are more genetically and morphologically stable in long-term culture.^{19, 20} Nevertheless, current culturing conditions for both BM-MSCs and hAMSCs show a progressive decrease in viability and proliferative ability, and an increase in senescence ratio for these stem cells with time.^{23, 24, 25, 26, 27, 28, 29} Typically, hAMSCs are cultured under ambient conditions with 21% oxygen in vitro.³⁰ However, physiologically, hAMSCs exist at much lower oxygen tensions, between 1 and 14%.^{31, 32} As a result of the limitations of culturing under normoxia, we investigated the effect of hypoxia on intraoperatively obtained hAMSCs by assessing proliferation, survival, differentiation, tumor formation, tumor tropism, and migration in vitro and in vivo in a rodent model with a human brain cancer. hAMSCs have been reported to transform into tumor-associated fibroblasts (TAFs), which can potentially support tumor growth and promote malignant phenotypes.^{33, 34} Yet, no studies have reported on the changes that may occur in hypoxia-cultured hAMSCs after they are exposed to brain cancer, both in vitro and in vivo. An understanding of the effects of hypoxia on hAMSCs³⁵ is critical for its potential therapeutic applications including in the treatment of brain tumors, stroke, neuro-degenerative diseases such as multiple sclerosis, and dementia (Figure 1a).Open in a separate windowFigure 1Primary human adipose-derived cells cultured in hypoxia (hAMSCs-H) and normoxia (hAMSCs-N) are both MSCs but normoxia-cultured cells show increased signs of senescence, such as increased area and elongated morphology, compared with hypoxia-cultured cells. (a) hAMSCs were isolated from human fat tissue and cultured in hypoxic (1.5% oxygen) or normoxic (21% oxygen) conditions in vitro. The viability, mobility, tumor tropism, safety, and tumorigenic potential were subsequently compared in vitro and in vivo. (b) Differentiation assay. hAMSCs were cultured in control media and differentiation media for 3 weeks, 10 days after the second passage. Three different stains were performed to assess differentiation capabilities (scale bar, 100 μm). (c) Flow cytometric analysis was performed to confirm the absence of CD31-, CD34-, and CD45-positive cells in both cell cultures. In addition, primary hAMSC cultures expressed high levels of CD73, CD90, and CD105, both in hypoxic and normoxic culture conditions at day 10 after passage 2. (d) Representative images of cell morphologies of hAMSCs on 2D surface (scale bar, 200 μm). (e) Schematic of 3D-nanopatterned surface used to assess morphology and motility. (f) Images of cell morphologies of hAMSCs on 3D-nanopatterned surface (scale bar, 200 μm). (g–j) The length, width, area, and length-to-width ratio were measured and compared after cell aligned on the nanopattern surface. Error bars represent S.E.M. *P<0.05, **P<0.01, N.S., not significant     

A methodology for rapid detection of Salmonella typhimurium using label-free electrochemical impedance spectroscopy

A pathogen detection methodology based on Bayesian decision theory has been developed for rapid and reliable detection of Salmonella typhimurium. The methodology exploits principles from statistical signal processing along with impedance spectroscopy in order to analytically determine the existence of pathogens in the target solution. The proposed technique is validated using a cost-effective and portable immunosensor. This device uses label-free, electrochemical impedance spectroscopy for pathogen detection and has been demonstrated to reliably detect pre-infectious levels of pathogen in sample solutions. The detection process does not entail any pathogen enrichment procedures. The results using the proposed technique indicate a detection time of approximately 6min (5min for data acquisition, 1min for analysis) for pathogen concentrations in the order of 500CFU/ml. The detection methodology presented here has demonstrated high accuracy and can be generalized for the detection of other pathogens with healthcare, food, and environmental implications. Furthermore, the technique has a low computational complexity and uses a minimal data-set (only 30 data-samples) for data analysis. Hence, it is ideal for use in hand-held pathogen detectors.     

Metabolomics of plant saponins: bioprospecting triterpene glycoside diversity with respect to mammalian cell targets

One of the goals of cancer chemotherapy and prevention is the discovery of compounds that are relatively selective to tumor cells and, therefore, have reduced effects on normal cell growth. In previously published studies, it was shown that certain triterpene saponins (called avicins) from a desert tree, Acacia victoriae, are selectively toxic to tumor cells at very low doses (IC(50): 0.2 microg/mL for Jurkat cells). To extend this research to human clinical studies, we needed to find a reliable supply of avicins and have developed a transformed "hairy root" culture as a means of biomass production. Protocols were optimized for A. victoriae micropropagation; after a boiling water treatment, A. victoriae seeds were maintained under in vitro conditions on defined media. Embryo-axis explants from shoot tips were removed and infected with Agrobacterium rhizogenes Conn (R 1000) for hairy root induction. Plasmid integration was confirmed by PCR analysis with a primer set for a segment of the rol B gene. Culture conditions have been optimized for root biomass production, and various inducers have been investigated for enhancement of avicin production. Hairy root cultures were compared with intact pod tissue from field-grown sources for avicin content following partial purification of triterpene glycosides and HPLC separation of the secondary metabolites. From bioassays of the collected HPLC fractions, we have identified putative triterpene "metabolic clusters" with enhanced activity against tumor cells. This now provides a system for both production of clinical trial lots of active samples, but also a means to correlate structure of individual triterpene glycosides with specific cellular target activity in mammalian cells.