The goal of this study was to determine safety, tolerability and pharmacokinetics of 2ME2 in patients with solid tumors and determine maximum tolerated dose (MTD). The following hypotheses were tested: 1) 2ME2 will be well tolerated in clinic when given orally and will have quantifiable effects on the ex vivo markers of angiogenesis and apoptosis; 2) 2ME2 will exhibit linear pharmacokinetics; 3) Plasma protein binding will be extensive and linear; 4) Sulfation will be the major metabolic pathway for 2ME2.This was a phase I dose escalation study. Twenty patients with refractory solid tumors were enrolled. 2ME2 was administered orally starting at 400 mg bid with dose escalation upto 3000 mg bid. Pharmacokinetic sampling was done up to 50 hours after single oral dose for characterization of pharmacokinetics and plasma drug concentrations which were determined by liquid chromatography tandem mass-spectrometry [LC/MS/MS, LOQ: 1ng/mL]. Circulating plasma concentrations were very low at all dose levels with high interindividual pharmacokinetic variability. Median plasma half-life was about 1-2 days. The unphysiologically high oral CL/F and Vd/F reflect low oral bioavailability of 2ME2. There was no dose proportional increase in Cmax or AUClast. There were no dose limiting toxicities at highest dose level, therefore MTD was not defined. The trial was closed due to extremely low plasma concentrations of 2ME2 achieved. Hepatic in vitro metabolism studies showed that 2ME2 was metabolized by CYP 450 enzymes (CYP 1A1, 1A2, 3A4, 3A5 and 2E1) to four major metabolites. Hepatic phase II metabolism studies revealed two major glucuronide metabolites of 2ME2. Sulfation did not play a major role in metabolism of 2ME2. Total in-vivo hepatic clearance was estimated as 862 mL/min, primarily due to glucuronidation. Less than 0.01 % of total administered dose of 2ME2 was excreted unchanged in urine, and about 1% was excreted as glucuronides. Plasma protein binding of 2ME2 was studied using equilibrium dialysis. Mean unbound fraction of 2ME2 (fu) in plasma of patients and healthy human volunteers was 0.019 ± 0.0043 and 0.027 ± 0.0019 respectively. Binding was concentration-independent and unaffected by presence of 2-methoxyestrone. 2ME2 binds to albumin, a1-acid glycoprotein (AAG) and sex-hormone binding globulin (SHBG).

Prostate cancer is the most prevalent non-cutaneous cancer among men. Since the 19th century when Virchow first introduced the concept of inflammation in cancer, chemokines and their receptors have garnered a lot of interest. Chemokine receptor CCR5 has been especially implicated in many disease states and recently found to be over expressed in prostate cancer cell lines. Anibamine, a natural CCR5 antagonist discovered in 2004, has been found to have significant anti-prostate cancer activity at micromolar level. To optimize this compound and also discover a novel pharmacophore, exploration of the original structure was carried out. Significant modifications were made to the side chain in the original structure and ten different analogues were prepared by altering the original synthetic route. While cytotoxicity assay proved the compounds to be non toxic to normal cells, anti-proliferation assay displayed that having a bulky, hydrophobic group in the side chain of the parent compound is essential for the activity. Looking at this data, the third generation of analogues can be prepared that might generate a better lead compound for the treatment of prostate cancer.

The analysis of biological samples in clinical or research settings often requires measurement of analytes from complex and limited matrices. Immunoaffinity separations in miniaturized formats offer selective isolation of target analytes with minimal reagent consumption and reduced analysis times. A prototype capillary-based microfluidic system has been developed for immunoaffinity separations in biological matrices with laser-induced fluorescence detection of labeled antigens or antibodies. The laboratory-constructed device was assembled from two micro syringe pumps, a microchip mixer, a micro-injector, a diode laser with fused-silica capillary flow cell, and a separation capillary column. The columns were prepared from polymer tubing and packed under negative pressure with a stationary phase that consisted of biotinylated antibodies attached to streptavidin-silica beads. A custom software program controlled the syringe pumps to perform step gradient elution and collected the signal as chromatograms. The system performance was evaluated with flow accuracy, mixer proportioning, pH gradient generation, and assessment of detectability. A direct labeling/direct capture immunoaffinity separation of C-reactive protein (CRP) was demonstrated in simulated serum. CRP, a biomarker of inflammation and cardiovascular disease risk assessment, was fluorescently labeled in a one-step reaction and directly injected into the system. A quadratic calibration model was selected and precision and accuracy were reported. Parathyroid hormone was also analyzed by the direct capture approach, but displayed nonspecific binding of human plasma matrix components that limited the useful assay range. Capillary sandwich assays of CRP in human serum and cerebrospinal fluid were performed using both capture and detection antibodies. The detection antibody was labeled and purified offline to minimize signal from labeled matrix components. Four parameter logistic functions were used to model the data and precision and accuracy were evaluated. During the study, 250 nL injection volumes 2.0 µL/min flow rates were employed, minimizing sample and reagent consumption. The microfluidic system was capable of separating antigens from biological matrices and is potentially portable for patient point-of-care settings. Additionally, the flexible design of the separation capillary allows for the analysis of different clinical markers by changing the antibodies and the low assay volume requirements could lead to less invasive patient sampling techniques.LabVIEW version 7 or later is required to open the attached files.

Objective: To evaluate the level of agreement between two self-reported medication adherence scales and prescription refill records in older adults. Design: Cross-sectional study Setting: Imperial Plaza; a retirement community located in Richmond, Virginia. Participants: 32 independent-living older adults, taking anti-hypertensive medications and filling their prescriptions at on-site Plaza Professional Pharmacy were recruited in the study. Methods: Participants’ 6 months refill records were obtained and Medication Possession Ration (MPR) was calculated. Participants were interviewed using Morisky Medication Adherence Scale (MMAS) & Brief Medication Questionnaire (BMQ). Kappa statistics was used to evaluate the level of agreement. Results: Poor level of agreement was found between refill records and MMAS (k=-0.004), refill records and BMQ belief screen (k=-0.09), regimen screen (k=-0.09), and recall screen (k =-0.004). Strong agreement was found between MMAS and BMQ regimen screen (k=0.79) and recall screen (k=0.87) respectively. Conclusion: Self-reported measure of adherence exhibited poor agreement with prescription refill records.

The type II topoisomerase subfamily of enzymes has been clinically targeted by the widely used, broad-spectrum quinolone class of antibacterials. Due to emerging drug-resistant strains of bacteria, the quinolones’ effectiveness is threatened. The natural product simocyclinone D8 (SD8) has shown the ability to inhibit the type II topoisomerase, DNA gyrase, even when mutated to be resistant to the quinolones. In order to determine the pharmacophore required for SD8 binding to DNA gyrase, 16 compounds were synthesized. These compounds were then tested by surface plasmon resonance for their ability to inhibit the DNA – DNA gyrase binding interaction. It was found that three compounds were able to inhibit the DNA – DNA gyrase binding interaction, while another showed partial inhibition of the interaction. From this data, a minimum pharmacophore was able to be determined. The pharmacophore required a coumarin scaffold bonded to a carboxylic acid group through an approximately 15 Å hydrocarbon linker. Functional supercoiling assays determined that while the compounds were able to bind the enzyme, the binding did not inhibit DNA gyrase’s ability to supercoil DNA.

Glycosaminoglycans (GAGs) are a family of linear bio-polysaccharides, which are heterogeneously modified with negatively charged sulfate and carboxylate groups. They are located on every cell surface, extracellular matrix or intracellular space in the body. GAGs are composed of alternating units of an amino sugars (glucosamine or galactosamine) and hexuronic acid/hexose (iduronic acid, glucoronic acid/ or galactose), which are linked by glycosidic bonds with different geometries. In recent years, GAGs have attracted considerable interest. GAGs play vital roles in fundamental biological processes, such as hemostasis, angiogenesis, cell signaling, growth and differentiation. Thus, GAGs contribute to a number of diseases such as thrombosis, cancer, inflammation, osteoarthritis and degenerative diseases. One of the most studied GAGs is heparin, which is widely used as an anticoagulant and is also implicated in other biological processes. Despite its extensive clinical use, heparin continues to suffer from major problems, such as life threatening hemorrhagic complications and heparin-induced thrombocytopenia. These activities originate from the large number of glycan sequences generated during its biosynthesis. Many different enzymes act in a non-template fashion to produce heparin chains with various chain lengths, sulfation and acetylation patterns. Their inherent heterogeneity, complexity and highly anionic nature have seriously limited the development of tools for rapid identification of sequences critical for many biological activities. A RPIP-UPLC MS protocol was developed to separate and characterize structures of heparin oligosaccharides prepared through enzymatic cleavage process and chemoenzymatic synthesis. In designing such protocol, several UPLC and MS parameters were considered. An efficient separation of each oligosaccharide mixture was achieved with different ion-pairing reagents. Yet, the structural elucidation of the multiple chromatographic peaks was hindered by the heterogeneity inherent in these mixtures even with supposedly defined standards. In order to elucidate the structures of these complex molecules, we utilized a strategy, which exploits the ease with which sulfate groups can be fragmented during MS ionization. A systematic increment of the MS cone voltage was employed starting from a minimum dissociation voltage, where the intact molecule is preserved, to a high enough dissociation voltage, where the only core oligosaccharide backbone was retained. This allowed identifying the number of sulfate groups and the core structures. However, positions of substituents were difficult to pinpoint because of the phenomenal number of possibilities. Such analysis would require tandem MS/MS–based approaches.

Due to the development of multidrug resistance in Streptococcus pneumoniae, research has begun to define new drug targets for pneumonia therapy. Different research groups have identified a lipoprotein, PsaA that is important for pneumonia virulence. PsaA is a manganese transporter that is required for bacterial virulence and growth. We have employed computer modeling to virtually screen a small-molecule database for inhibition of PsaA function by targeting the metal binding pocket, performing receptor-based virtual screening and molecular docking and scoring to identify potential inhibitors of PsaA function. We have developed an assay for screening compounds, including the use of a PsaA mutant, testing of multiple compounds, and identification of compounds that inhibit Streptococcus pneumoniae growth at concentrations less than 20 μM. We experimentally tested the effect on Mn uptake and their PsaA dependence for 42 compounds, but these experiments suggested that these compounds were affecting bacterial growth by a different mechanism.

The complex pathobiologic mechanisms of emphysema are not fully understood, leaving this deadly disease without effective pharmacotherapy for a cure. This project hypothesized that the sulfated dehydropolymer of caffeic acid (CDSO3) exhibits Fe2+ chelation-based hypoxia inducible factor-1a (HIF-1a) up-regulatory protective activities against in vitro emphysematous cell death and for in vivo reversal of emphysema induced with SU5416, a vascular endothelial growth factor blocker. Using in vitro chromogenic competitive inhibition assays, CDSO3 was shown to chelate Fe2+ (IC50 of 23 µM), but not Fe3+ ions. The trypan blue exclusion and lactate dehydrogenase assays were then employed to examine the cytoprotective activities of CDSO3 against inflammatory, oxidative, elastolytic, and apoptotic cell death using alveolar macrophages, epithelial and endothelial cells. CDSO3 at 10 µM produced significant protective activities against these emphysematous cell deaths by 50-154 %. These protective effects were opposed by the addition of the HIF-1a inhibitors, CAY10585 and echinomycin, and excess Fe2+, but not Fe3+, ions. Emphysema was then induced in rats following a subcutaneous injection of SU5416 at 20 mg/kg, after which CDSO3 at 60 µg/kg was administered to the lungs 3 times/week for two weeks. Treadmill exercise endurance (EE) was measured to assess the functional impairment, while lung tissues were removed for morphological assessments of alveolar airspace enlargement (MLI) and destruction (DI), as well as to measure protein levels using Western blot. SU5416 significantly impaired EE, MLI, and DI by 81 %, 47 %, and 5-fold, compared to the healthy animals, and these were significantly reversed by CDSO3 by 66, 74, and 87 %. CDSO3 treatment did not change the lung cytoplasmic expression of histone deacetylase 2 (HDAC2), HIF-1a, or a pro-apoptotic marker, BAX. However, induction with SU5416 significantly reduced VEGF expression by 52 % and increased cleaved caspase-3 expression by 1.5-fold, compared to the healthy animals, while CDSO3 normalized the expressions of both proteins in these emphysematous animals. However, when CDSO3 was pre-mixed with excess Fe2+, the reversal activities of CDSO3 were diminished. In conclusion, this study has demonstrated the Fe2+ chelation-based HIF-1a up-regulatory dependent in vitro and in vivo lung repairing efficacies for CDSO3 in emphysema.

Book Summary: The Handbook of Nonprescription Drugs: An Interactive Approach to Self-Care contains the most authoritative information on nonprescription drug pharmacotherapy, nutritional supplements, medical foods, nondrug and preventive measures, and complementary therapies. The 18th edition shows students and practitioners how to assess and triage a patient's medical complaints. And it provides FDA-approved dosing information for nonprescription medications along with evidence-based research on the efficacy and safety of over-the-counter, herbal, and homeopathic medications.

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