Physical characteristics and properties of estradiol softgel vaginal inserts.

OBJECTIVE: TX-004HR is a low-dose estradiol (E2) softgel vaginal insert designed to be rapidly dissolving and mucoadhesive. This report describes the physical attributes and pharmacokinetic parameters of the softgel vaginal insert evaluated for the treatment of moderate to severe dyspareunia due to menopausal vulvar and vaginal atrophy. METHODS: In vitro dissolution studies with 25-µg E2 inserts were performed and media samples were analyzed for E2 by high-performance liquid chromatography. Effects of body position on E2 bioavailability were assessed in a phase 1, randomized trial of the 25-µg softgel capsule versus a reference product in which women remained supine after dosing (n = 16), and in a substudy (n = 16) in which women were ambulatory or seated after dosing. Estradiol C max, AUC0-24, and t max were measured by high-performance liquid chromatography-tandem mass spectroscopy. A phase 2, randomized study (n = 50) of 10-µg E2 versus placebo inserts assessed timing of capsule disintegration at days 1 and 15. RESULTS: In vitro testing detected more than 80% of E2 in the dissolution medium by 15 minutes (first time point measured). In the phase 1 studies, baseline-corrected E2 plasma levels were not significantly different regardless of supine versus ambulatory/seated position after dosing: C max, 24.1 versus 34.3 pg/mL; AUC0-24, 77.6 versus 93.7 h ·â€Špg/mL; and t max, 2.1 versus 1.9 hours, respectively. In the phase 2 study, no remnants of the softgel capsule were found at day 1 (6 hours) after dosing and day 15. Vaginal discharge was minimal (1/48 women; 2.1%). CONCLUSIONS: The presented data support rapid dissolution of the softgel capsule and similar E2 pharmacokinetic parameters regardless of body position after dosing.

The role of monocarboxylate transporters in uptake of lactic acid in HeLa cells.

This study was aimed to identify the monocarboxylate transporters (MCTs) in HeLa cells and to delineate their role in transportation of L-lactic acid. The functional role of MCTs in lactic acid transport was evaluated at various mucosal pHs (4.5-7.4) or in the presence of various loading doses (0.2-2mM) of lactic acid, MCT substrates (nicotinic acid, n-butyric acid, etc.) and inhibitors (alpha-cyano-4-hydroxycinnamate and para-chloromercuribenzoic acid). The molecular properties of MCTs were characterized using reverse transcription-polymerase chain reaction (RT-PCR). The uptake rate of lactic acid by HeLa cells significantly increased from 0.353+/-0.052 to 1.103+/-0.196 micromol/mg protein as the extra-cellular pH changed from 7.4 to 4.5, indicating that activities of MCT were mediated through H(+)-linked mechanism. The uptake profile of lactic acid followed the saturable process with the K(m) value of 0.53 mM. The uptake rate of lactic acid is concentration dependent and is reduced in the presence of MCT inhibitors. MCT isoforms 1, 5 and 6 in HeLa cells were identified by RT-PCR. HeLa cell line can be used as an effective screening tool for intravaginally administered drugs targeted toward MCT.

The functional role of C-reactive protein in aortic wall calcification.

As an ongoing effort to elucidate the mechanisms involved in bioprosthetic heart valve (BHV) calcification, the role of C-reactive protein (CRP) in the tissue calcification process was investigated. The profile of calcium-associated proteins (CAP) on glutaraldehyde-preserved (0.6%) porcine aortic wall, which were subcutaneously implanted in rats for up to 8 weeks, showed a temporal appearance pattern. The total extracted amount of proteins from the control tissues implanted for 8 weeks was significantly greater than that from ethanol-treated tissues (1.78+/-0.2 vs. 1.27+/-0.18 microg/mg), indicating that the binding affinity of CAP for BHV pretreated with an anticalcification agent was significantly decreased (p<0.05). The dye Stains-All method showed that the dark-blue colored bands, representing high calcium binding and phosphorylated proteins, were stained from the extract of the control BHV at the molecular weight varying from 4 to 250 kDa, but rarely seen in the extract of BHV pretreated with ethanol. One of those proteins was exclusively immunoreactive with CRP antibody, while there was no immunoreaction in less calcified tissues. When aortic wall was exposed to an excess amount of CRP in an in vitro simulating model, the calcification rate of aortic wall increased as the concentration of CRP increased. The results of this work clearly revealed that CRP has indirect vascular effects, leading to an increased rate of aortic wall calcification.

Biocompatibility of components of a female controlled drug delivery system.

A female controlled drug delivery system (FcDDS) containing sodium dodecyl sulfate as a microbicide, ethylenediaminetetraacetic acid (EDTA) as a synergistic microbicide, and lactic acid as a pH modulator was developed as an intravaginal barrier device against sexually transmitted diseases. The host response of the vagina to the FcDDS was evaluated through biocompatibility tests including cell viability, estrogenicity, and cytotoxicity assays on HeLa cervical cells and NIH:Ovcar-3 ovarian cells. Gel electrophoresis and reverse transcriptase polymerase chain reaction assays on HeLa cervical cell lines were also performed to elucidate the effects of EDTA on the expression of particular proteins of interest. The results of the cell viability test showed no significant difference in viability of cells upon exposure to EDTA at concentrations less than 0.035% that was reported to exert spermicidal activity. EDTA at concentrations less than 0.035% did not cause any cytotoxicity. The results of reverse transcriptase polymerase chain reaction analysis revealed that EDTA induced the expression of a 67-kDa protein in HeLa cells, which was identified as elastin binding protein (a part of the elastin receptor complex). This work has demonstrated that FcDDS containing EDTA is biocompatible and safe to be used as an intravaginal barrier device.

EDTA-induced activation of Ca-regulated proteins in the vaginal mucosa.

The effects of EDTA on the expression and topologic localization of mitogen-activated protein (MAP) kinases (ERK, JNK, and p38), along with nitric oxide synthase (NOS), I-KappaB, and p53 were examined to elucidate the host response provoked by the intravaginal application of a female controlled drug delivery system (FcDDS) containing a spermicidal/microbicidal agent and EDTA. Immunohistochemical and immunoblotting studies were conducted to identify and quantitate the EDTA-inducible proteins in vaginal mucosa. The content of nitrite, which is one of the primary stable breakdown products of nitric oxide (NO), was determined to correlate the expression of NOS with NO formation in HeLa cervical carcinoma cell line. The immunohistochemical study demonstrated that the modulation of the calcium gradient by EDTA activated MAP kinases (ERK and JNK) in the rabbit vaginal mucosa. The results of Western immunoblot study demonstrated differential expression of MAP kinases (ERK and JNK) with EDTA treatment, whereas the expression of NOS and NF-KappaB was not affected by EDTA. There was no significant difference in nitrite production in the HeLa cell line upon exposure to EDTA compared with the control, which was consistent with the results of the Western blot study. The results of this work support that the regulation of MAP kinase was affected by calcium, which is controlled by chelation activity of EDTA. The specific tissue responses exerted by the loading components of a biomaterial-based system should be fully taken into consideration for its intravaginal application.