Mutual inhibition between carvedilol enantiomers during racemate glucuronidation mediated by human liver and intestinal microsomes

Carvedilol is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers for treatment of angina pectoris, hypertension and chronic heart failure. We have reported that enzyme kinetic parameters for carvedilol glucuronidation by human liver microsomes (HLM) differed greatly depending on the substrate form, namely, racemic carvedilol and each enantiomer. These phenomena were thought to be caused by mutual inhibition between carvedilol enantiomers during racemate glucuronidation. The aim of this study was to clarify the mechanism of these phenomena in HLM and human intestinal microsomes (HIM) and its relevance to uridine 5'-diphosphate (UDP)-glucuronosyl transferase (UGT) 1A1, UGT2B4 and UGT2B7, which mainly metabolize carvedilol directly in phase II enzymes. HLM apparently preferred metabolizing (S)-carvedilol to (R)-carvedilol in the racemate, but true activities of HLM for both glucuronidation were approximately equal. By determination of the inhibitory effects of (S)-carvedilol on (R)-carvedilol glucuronidation and vice versa, it was shown that (R)-carvedilol glucuronidation was more easily inhibited than was (S)-carvedilol glucuronidation. UGT2B7 was responsible for (S)-carvedilol glucuronidation in HLM. Ratios of contribution to (R)-carvedilol glucuronidation were approximately equal among UGT1A1, UGT2B4 and UGT2B7. However, enzyme kinetic parameters were different between the two lots of HLM used in this study, depending on the contribution ratio of UGT2B4, in which (R)-glucuronidation was much more easily inhibited by (S)-carvedilol than was (S)-glucuronidation by (R)-carvedilol. Meanwhile, HIM preferred metabolizing (R)-carvedilol, and this tendency was not different between the kinds of substrate form.     

A novel pathway underlying the inhibitory effects of melatonin on isolated rat urinary bladder contraction

The aim of the present study was to elucidate the direct effects of melatonin on bladder activity and to determine the mechanisms responsible for the detrusor activity of melatonin in the isolated rat bladder. We evaluated the effects of melatonin on the contractions induced by phenylephrine (PE), acetylcholine (ACh), bethanechol (BCh), KCl, and electrical field stimulation (EFS) in 20 detrusor smooth muscle samples from Sprague-Dawley rats. To determine the mechanisms underlying the inhibitory responses to melatonin, melatonin-pretreated muscle strips were exposed to a calcium channel antagonist (verapamil), three potassium channel blockers [tetraethyl ammonium (TEA), 4-aminopyridine (4-AP), and glibenclamide], a direct voltage-dependent calcium channel opener (Bay K 8644), and a specific calcium/calmodulin-dependent kinase II (CaMKII) inhibitor (KN-93). Melatonin pretreatment (10(-8)~10(-6) M) decreased the contractile responses induced by PE (10(-9)~10(-4) M) and Ach (10(-9)~10(-4) M) in a dose-dependent manner. Melatonin (10(-7) M) also blocked contraction induced by high KCl ([KCl](ECF); 35 mM, 70 mM, 105 mM, and 140 mM) and EFS. Melatonin (10(-7) M) potentiated the relaxation response of the strips by verapamil, but other potassium channel blockers did not change melatonin activity. Melatonin pretreatment significantly decreased contractile responses induced by Bay K 8644 (10(-11)~10(-7) M). KN-93 enhanced melatonin-induced relaxation. The present results suggest that melatonin can inhibit bladder smooth muscle contraction through a voltage-dependent, calcium-antagonistic mechanism and through the inhibition of the calmodulin/CaMKII system.     

Genome-wide copy number analysis in primary breast cancer

INTRODUCTION: Carcinogenesis is considered to be a multistep process that may involve cumulative genomic alterations. Loss of chromosomal material would inactivate tumor suppressor genes and gain of chromosomal material has the potential to activate tumor-promoting genes. AREAS COVERED: Recent intensive studies by array comparative genomic hybridization (aCGH) have demonstrated frequent alterations in multiple regions of the genome. This suggests that these regions contain a variety of oncogenes and tumor suppressor genes associated with breast cancer development. The patterns of copy number variations (CNVs) have been suggested to be associated with breast cancer subtypes, indicating the importance of genomic instability in the development of breast cancer. EXPERT OPINION: To further clarify the complexity of gene alterations, one approach is to employ a CNV-targeted platform that harbors a large number of direct CNV markers located in the repeat-rich unstable regions of the human genome. Next generation sequencing is another approach to overcome the limitations of aCGH such as the repeat-rich regions. Genomic analysis should be combined with expression analysis to elucidate individual genes relevant to breast cancer development and progression. The elucidation of the functions of the affected genes would lead to identification of new molecular targets for breast cancer eradication.     

Pharmacokinetics and pharmacodynamics of oral tolvaptan administered in 15- to 60-mg single doses to healthy Korean men

Tolvaptan is a selective arginine vasopressin V2-receptor antagonist that is used as an aquaretic agent. This trial investigated the pharmacokinetics and pharmacodynamics of 15- to 60-mg single oral doses of tolvaptan in healthy Korean men. A dose block-randomized, placebo-controlled, double-blind, single ascending dose trial was conducted with 46 subjects receiving tolvaptan (15, 30, or 60 mg) or placebo. To determine pharmacokinetics and pharmacodynamics, blood and urine samples were collected at baseline and up to 48 hours after drug administration. Urine volume and fluid intake were measured for 24 hours starting the day before dosing (day -1) as baseline, and on the day of drug administration (day 1). Tolvaptan showed dose-linear pharmacokinetic characteristics regarding area under the concentration-time curve. Changes from baseline in 24-hour urine volume and 24-hour fluid balance correlated significantly with area under the concentration-time curve from time 0 to the last measurable time. Dose-dependent increases were observed in serum osmolality, serum sodium concentration, and free water clearance in the 4- to 8-hour interval after dosing, and these increases were maintained for at least 24 hours. Single 15- to 60-mg doses of tolvaptan exhibited linear pharmacokinetics and resulted in substantial, dose-dependent aquaresis in healthy Korean men.     

Basic investigation for classification of anticancer drugs by pharmacological effects

The most effective drugs based on the type of cancer are chosen for chemotherapy. Tumor cells can be targeted at the DNA, RNA or protein level, and most of the classical anticancer drugs interact with tumor DNA in a time-dependent manner or a concentration-dependent manner. However, it has been unclear to date whether a combination therapy is carried out by using exact classification. Thus it is necessary to reclassify a great number of anticancer drugs. We propose a new classification system based on pharmacological effects of anticancer drugs. Classification of four anticancer drugs (cisplatin, carboplatin, paclitaxel and gemcitabine) was performed by the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The four anticancer drugs were grouped by IC50 values (inhibitory concentration, 50%) in a time-dependent manner and a concentration-dependent manner. The present approach may be combined to enhance the chemosensitivity, improve the dose of cytotoxic drugs and evaluate the effects of novel anticancer drugs.     

Schedule-dependent cytotoxicity of etoposide (VP-16) and cyclophosphamide in leukemia cell line K-562

In allogeneic bone marrow transplantation (allo-BMT) in patients with leukemia, the combination of VP-16 and cyclophosphamide (CY) is commonly used for the conditioning regimen. In the present study, we demonstrated schedule-dependent cytotoxicity of VP-16 and CY in K-562 cells. K-562 cells were pretreated with low concentrations (2.5 and 5?μg/mL) of 4-hydroperoxycyclophosphamide (40487S), which is a preactivated analog of CY. It was confirmed that these concentrations did not influence cell viability. Cells subsequently exposed to 0.5-100?μg/mL of VP-16 showed reduced the viability compared to that of control cells not treated with 40487S. In contrast, there was no change in the viability of K-562 cells pretreated with low concentrations (0.5 and 1?μg/mL) of VP-16. It was confirmed that these concentrations did not influence cell viability. Viability of subsequently exposed to 1-20?μg/mL was not different from that of control cells not treated with VP-16. VP-16 caused cell cycle arrest at G?/M phase. On the other hand, 40487S arrested the cell cycle at S phase. Thymidine-synchronized cells, VP-16 showed cell cycle specificity for cell killing from early-S to mid-S phase. On the other hand, 40487S showed cell cycle-independent cytotoxicity. Exposure of cells to VP-16 after 40487S induced a greater cytotoxic effect on K-562 cells. The findings may lead to improvements in clinical combination chemotherapy.     

Prolonged Release of Bioactive Model Proteins from Anionic Microgels Fabricated with a New Microemulsion Approach

Purpose

Therapeutic proteins have become an integral part of health care. However, their controlled delivery remains a challenge. Protein function depends on a delicate three dimensional structure, which can be damaged during the fabrication of controlled release systems. This study presents a microgel-based controlled release system capable of high loading efficiencies, prolonged release and retention of protein function.

Methods

A new DMSO/Pluronic microemulsion served as a reaction template for the crosslinking of poly(acrylic acid) and oligo (ethylene glycol) to form microgels. Poly(acylic acid) molecular weights and microgel crosslinking densities were altered to make a series of microgels. Microgel capacity to capture and retain proteins of different sizes and isoelectric points, to control their release rate (over ~30 days) and to maintain the biofunctionality of the released proteins were evaluated.

Results

Microgels of different sizes and morphologies were synthesized. Loading efficiencies of 100% were achieved with lysozyme in all formulations. The loading efficiency of all other proteins was formulation dependent. Release of lysozyme was achieved for up to 30 days and the released lysozyme retained over 90% of its activity.

Conclusions

High loading efficiencies and prolonged release of different proteins was achieved. Furthermore, lysozyme’s functionality remained uncompromised after encapsulation and release. This work begins to lay the foundation for a broad platform for the delivery of therapeutic proteins.

     

Regulatory Perspectives on Strength-Dependent Dissolution Profiles and Biowaiver Approaches for Immediate Release (IR) Oral Tablets in New Drug Applications

Dissolution profile comparisons are used by the pharmaceutical industry to assess the similarity in the dissolution characteristics of two formulations to decide whether the implemented changes, usually minor/moderate in nature, will have an impact on the in vitro/in vivo performance of the drug product. When similarity testing is applied to support the approval of lower strengths of the same formulation, the traditional approach for dissolution profile comparison is not always applicable for drug products exhibiting strength-dependent dissolution and may lead to incorrect conclusions about product performance. The objective of this article is to describe reasonable biopharmaceutic approaches for developing a biowaiver strategy for low solubility, proportionally similar/non-proportionally similar in composition immediate release drug products that exhibit strength-dependent dissolution profiles. The paths highlighted in the article include (1) approaches to address biowaiver requests, such as the use of multi-unit dissolution testing to account for sink condition differences between the higher and lower strengths; (2) the use of a single- vs. strength-dependent dissolution method; and (3) the use of single- vs. strength-dependent dissolution acceptance criteria. These approaches are cost- and time-effective and can avoid unnecessary bioequivalence studies.     

Synthesis and characterization of G5 PAMAM dendrimer containing daunorubicin for targeting cancer cells

A multifunctional dendritic nanodevice containing folate (FA) as the targeting molecule and daunorubicin (DNR) as the chemotherapeutic drug has been designed and synthesized. The primary amino groups on the surface of the generation 5 poly(amidoamine) dendrimer (G5 dendrimer) were acetylated partially. FA and DNR were conjugated to the remaining primary amino groups on the G5 dendrimer through amide and ester linkage, respectively, to generate the difunctional dendritic device. The synthetic approach of G5 PAMAM dendrimer containing daunorubicin and folic acid would be useful for targeting the overexpressed membrance-associated folate receptors of KB human carcinoma cell line and discreating them effectively.