Identification of a 3-ß-homoalanine conjugate of brusatol with reduced toxicity in mice.

Brusatol, a quassinoid natural product, is effective against multiple diseases including hematologic malignancies, as we reported recently by targeting the PI3Kγ isoform, but toxicity limits its further development. Herein, we report the synthesis of a series of conjugates of brusatol with amino acids and short peptides at its enolic hydroxyl at C-3. A number of conjugates with smaller amino acids and peptides demonstrated activities comparable to brusatol. Through in vitro and in vivo evaluations, we identified UPB-26, a conjugate of brusatol with a L- ß-homoalanine, which exhibits good chemical stability at physiological pH's (SGF and SIF), moderate rate of conversion to brusatol in both human and rat plasmas, improved mouse liver microsomal stability, and most encouragingly, enhanced safety compared to brusatol in mice upon IP administration.

Pharmacokinetic, metabolic profiling and elimination of brusatol in rats.

Brusatol, a quassinoid isolated from the traditional Chinese medicine Brucea javanica, has been reported to be an inhibitor of Nrf2 pathway and has great potential to be developed into a novel chemotherapeutic adjuvant. However, the in vivo process of brusatol has not been comprehensively explained yet. Therefore, this paper focused on the pharmacokinetic metabolism and excretion of brusatol in rats using a simple and reproducible LC-MS/MS method. The results indicated that the plasma concentration of brusatol decreased rapidly; the average cumulative excretion rate in urine was 5.82% during 24 h, and 0.71% in bile during 12 h. High-resolution mass spectrometry was applied for the identification of metabolites; as a result, four metabolites were detected and the structure was tentatively deduced on the base of the MS2 data, Compound Discoverer 2.0 and Mass Frontier 7.0 software. Hydroxylation, hydrolysis and glucuronidation were suggested as major metabolic pathways in vivo. The in vivo process and detection of metabolites of brusatol might improve the understanding of the mechanism of its anticancer effect and provide valuable information for its safety estimation, which will be essential to the new drug development.

Anticancer Activity of Pasak Bumi Root Extract (Eurycoma longifolia Jack) on Raji Cells.

<b>Background and Objective:</b> The use of the roots of the pasak bumi (<i>E. longifolia</i> Jack) to treat cancer has been studied widely, however, the scientific basis of these plants used as an anticancer drug is widely unknown. The purpose of this study was to examine the anticancer activity of ethyl acetate and non-ethyl acetate fractions of pasak bumi roots in Raji cells. <b>Materials and Methods:</b> The cytotoxicity test is using the direct cell count method with trypan blue staining. The growth inhibition is using doubling time analysis of Raji cells. Observation of the apoptotic events of Raji cells used ethidium bromide staining, while observing the expression of p53 protein in Raji cells was done by immunohistochemical staining. <b>Results:</b> The results of the cytotoxicity and doubling time test showed that the activity of the non-ethyl acetate fraction was greater than that of the roots of pasak bumi. The lower concentration of non-ethyl acetate fraction of pasak bumi roots was able to delay the multiplication time of Raji cells which was greater than that of ethyl acetate. The results of the cytotoxicity and doubling time test showed that the activity of the non-ethyl acetate fraction was greater than that of the roots of pasak bumi. <b>Conclusion:</b> It can be concluded that the ethyl acetate and non-ethyl acetate fractions of the roots of pasak bumi have cytotoxic and antiproliferative activity on Raji cells, however they cannot induce apoptosis in Raji cells. The death of Raji cells is through the mechanism of inhibiting Raji cell proliferation as evidenced by an increase in p53 protein expression.

Nifedipine inhibits oxidative stress and ameliorates osteoarthritis by activating the nuclear factor erythroid-2-related factor 2 pathway.

Nifedipine is a voltage-gated calcium channel inhibitor widely used in the treatment of hypertension. Nifedipine has been reported to have antioxidant and anti-apoptotic effects and promotes cell proliferation. However, the effects of nifedipine on oxidative stress and apoptosis in osteoarthritic (OA) chondrocytes are still unclear. In this study, we sought to investigate whether nifedipine alleviates oxidative stress and apoptosis in OA through nuclear factor erythroid-2-related factor 2 (Nrf2) activation. The cytotoxicity of nifedipine against human chondrocytes was detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit, whereas mRNA and protein expression levels were measured using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The oxidative stress level was analyzed by measuring reactive oxygen species (ROS), glutathione peroxidase (GSH-px), catalase (CAT) and superoxide dismutase (SOD) activities. The role of Nrf2 in the effect of nifedipine on OA was analyzed using an Nrf2 inhibitor brusatol (BR). The result showed that nifedipine inhibited the expression of matrix metalloprotein(MMP)-13, interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, inducible nitric oxide (NO) synthase (iNOS), and prostaglandin E2 (PGE2), as well as reduced ROS production in human OA chondrocytes, which was partially reversed by BR. Nifedipine prevented cartilage degeneration and contributed to the expression of Nrf-2 in chondrocytes. These results indicate that nifedipine inhibited inflammation and oxidative stress in chondrocytes via activation of Nrf-2/HO-1 signaling.

Cloning, characterisation, docking and expression analysis of 3-beta-hydroxysteroid dehydrogenase during ontogenetic development and annual reproductive cycles in catfish, Clarias batrachus.

Fish like higher animals, have a well-defined mechanism to produce sex steroids that play a critical role in gonadal development and maturation. In this study, we aimed to analyse the expression pattern of 3ß-HSD in different tissues, during ontogenetic development and gonadal recrudescence of Clarias batrachus. A full-length cDNA of 1617 bp including an open reading frame (ORF) of 1125 bp encoding 374 amino acids was isolated from testes of C. batrachus. The docking analysis between C. batrachus 3ß-HSD protein and eurycomanone exhibited high binding affinity toward each other with total energy of -108.292 kcal/mol and van der Waals (VDW) interaction of -84.2838 kcal/mol. The 3ß-HSD transcript level during ontogeny was detected in all the stages starting from the fertilized egg. The mature C. batrachus showed more expression of 3ß-HSD mRNA in gonads and brain while weak expression was detected in the remaining tissues analysed. The 3ß-HSD mRNA expression during annual reproductive phases of gonads was more in preparatory and pre-spawning stages than that of spawning and post-spawning phases. The mRNA expression results together suggest that 3ß-HSD plays an important role in gonadal development. Furthermore, the active binding sites on 3ß-HSD protein could be targeted in pharmacological drug designing to cope with reproductive dysfunctions in fish.