A selective cellular screening assay for B-Raf and c-Raf kinases.

The Ras/Raf signaling pathway has been recognized as an important process in cancer biology. Recently, activating mutations in the BRAF gene were reported to be present in approximately 66% of malignant melanomas as well as other malignancies such as colon cancer. Here, the authors report the development of a B-Raf-specific cellular assay to profile cell-active B-Raf inhibitors. Expression of the active B-Raf mutant (V600E) and the kinase-inactive form of its substrate, MEK1, was regulated by mifepristone, and the catalytic activity of B-Raf was monitored by following MEK1 phosphorylation. Target specificity was ensured because the phosphorylation of MEK1 was significantly inhibited when kinase-inactive B-Raf was used in place of the active kinase. A cellular c-Raf assay was similarly established to monitor the selectivity between B-Raf and c-Raf. Z' factor values were consistently above 0.50 with either kinase, indicating that assay performance was sufficiently robust for use as cellular profiling assays. The authors used this system to demonstrate that the selectivity profile of compounds targeted against B-Raf and c-Raf kinases could be quantitatively determined. This platform provides a quantitative cellular readout for a spectrum of specific inhibitors of B-Raf and c-Raf kinases that is particularly suitable for use in drug discovery.

Efficacy of a new warm moist air device on tear functions of patients with simple meibomian gland dysfunction.

PURPOSE: To evaluate the safety and efficacy of an original warm moist air device on tear functions and ocular surface of patients with simple meibomian gland dysfunction (MGD). METHODS: Fifteen patients with simple MGD and 20 healthy volunteers were recruited in an initial prospective interventional clinical trial to evaluate the safety and short-term effects of the warm moist air device. The device was applied to the eyes of the subjects for 10 minutes. Temperatures of the eye lids and corneas were measured with an infrared thermometer. Symptoms of ocular fatigue were scored using visual analog scales (VASs). Schirmer test, tear film break-up time (BUT), DR-1 tear film lipid layer interferometry, fluorescein staining, and rose bengal staining were also performed before and after the application of the eye steamer. After the initial study, another 2-week prospective clinical trial was carried out in 10 patients with MGD who received the warm moist air treatment. Ten other patients were also recruited and received warm compress treatment with hot towels for 2 weeks to evaluate the long-term effects of the warm moist air device and the warm compresses on tear film lipid layer thickness and ocular surface health. The warm moist air device and the warm compresses were applied for 10 minutes twice a day. The changes in VAS scores for symptoms, BUT values, fluorescein, and rose bengal staining scores were examined before and after each treatment during the second trial. RESULTS: VAS scores of ocular fatigue improved significantly with short- and long-term applications of the warm moist air device in both studies. The mean corneal surface and eye lid temperatures showed significant elevation within safe limits 10 minutes after the moist air application. The mean BUT prolonged significantly in the patients receiving warm moist air applications but did not change significantly in those treated with warm compresses. DR-1 tear film lipid layer interference showed evidence of lipid expression in the patients and controls, with thickening of the tear film lipid layer after 10 minutes of warm moist air device use. In the 2-week trial, tear film lipid layer thickness increased in both warm moist air device and warm compress groups, with a greater extent of increase in the warm moist air device group. CONCLUSION: Warm moist air device use provided symptomatic relief of ocular fatigue and improvement of tear stability in patients with MGD. The new warm moist air device seems to be a safe and promising alternative in the treatment of MGD.

Therapeutic effect of cevimeline on dry eye in patients with Sjögren's syndrome: a randomized, double-blind clinical study.

PURPOSE: Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by salivary and lacrimal glandular destruction leading to symptoms of dry mouth and dry eye. Dryness can also occur in the absence of glandular destruction. Patients with SS have autoantibodies that bind to muscarinic acetylcholine receptors in the exocrine glands. Recently, a muscarinic acetylcholine receptor agonist, cevimeline, has been approved for use against symptoms of dry mouth in patients with SS. In this study, the efficacy of cevimeline in improving symptoms of dry eye was examined. DESIGN: Prospective, randomized, double-blind, multi-center clinical study. METHODS: Sixty patients were randomly assigned to three groups-placebo; cevimeline, 20 mg three times daily; or cevimeline, 30 mg three times daily-and received treatment for 4 weeks. Patients were evaluated before treatment, at week 2, at the end of treatment, and at the end of a 2- to 4-week follow-up period. RESULTS: Compared with the placebo, statistically significant differences were seen with cevimeline, 20 mg three times daily, in subjective symptoms, tear dynamics, condition of the corneoconjunctival epithelium, and global improvement rating. No difference was found among the three groups regarding the safe use of the drug. CONCLUSIONS: These results indicate that cevimeline, 20 mg three times daily, is safe and effective in improving symptoms of dry eye in patients with SS. Additional studies, with larger patient populations, are needed to further assess the effectiveness of cevimeline for dry eye.

Low-concentration homogenized castor oil eye drops for noninflamed obstructive meibomian gland dysfunction.

OBJECTIVE: We developed low-concentration homogenized castor oil eye drops for the treatment of patients with noninflamed obstructive meibomian gland dysfunction (MGD), a major cause of lipid-deficiency dry eye, and assessed the safety, stability, and efficacy of the eye drops. DESIGN: Randomized, double-masked, placebo-controlled crossover clinical trial. PARTICIPANTS: Forty eyes of 20 patients with noninflamed MGD. METHODS: After a preliminary study of eye drops containing castor oil, 2% castor oil and 5% polyoxyethylene castor oil (emulsifier) were mixed to formulate homogenized oil eye drops. The patients were assigned randomly to receive oil eye drops or placebo six times daily for 2 periods of 2 weeks each. MAIN OUTCOME MEASURES: At the end of each treatment period, we assessed symptoms, tear interference grade, tear evaporation, fluorescein and rose bengal scores, tear break-up time (BUT), and meibomian gland orifice obstruction. Safety and stability tests were also performed. RESULTS: Symptom scores, tear interference grade, tear evaporation test results, rose bengal scores, tear BUT, and orifice obstruction scores after the oil eye drop period showed significant improvement compared with the results after the placebo period. No complications attributable to the eye drops were observed. The oil eye drops were stable when stored at 4 degrees C. CONCLUSIONS: The results indicate that castor oil eye drops are effective and safe in the treatment of MGD. The possible mechanisms of this treatment are improvement of tear stability as a result of lipid spreading, ease of meibum expression, prevention of tear evaporation, and the lubricating effect of the oil eye drops.