Dual approach utilizing self microemulsifying technique and novel P-gp inhibitor for effective delivery of taxanes

In the present work, concomitant use of self-microemulsifying drug delivery systems (SMEDDS) and a novel third-generation P-gp inhibitor, GF120918 (elacridar), for the effective transport of taxanes (paclitaxel and docetaxel) across an in?vitro model of the intestinal epithelium and uptake into tumor cells were investigated. On the basis of solubility studies and ternary phase diagrams, different SMEDDS formulations of taxanes were prepared and characterized. In caco-2 cell permeation study, paclitaxel-loaded SMEDDS along with GF120918 showed a four-fold increase in apparent permeability, while docetaxel-loaded SMEDDS in combination with GF120918 showed a nine-fold increase in permeability, as compared to plain drug solution. Cell uptake studies on A549 cells were performed with microemulsions formed from both SMEDDS formulations loaded with rhodamine 123 dye and showed good uptake than plain dye solution. Confocal laser scanning microscopic images further confirmed the higher uptake of both SMEDDS formulations in the presence of GF120918.     

Understanding the mechanisms for metabolism-linked hemolytic toxicity of primaquine against glucose 6-phosphate dehydrogenase deficient human erythrocytes: evaluation of eryptotic pathway

Therapeutic utility of primaquine, an 8-aminoquinoline antimalarial drug, has been limited due to its hemolytic toxicity in population with glucose 6-phosphate dehydrogenase deficiency. Recent investigations at our lab have shown that the metabolites generated through cytochrome P(450)-dependent metabolic reactions are responsible for hemotoxic effects of primaquine, which could be monitored with accumulation of methemoglobin and increased oxidative stress. The molecular markers for succeeding cascade of events associated with early clearance of the erythrocytes from the circulation were evaluated for understanding the mechanism for hemolytic toxicity of primaquine. Primaquine alone though did not induce noticeable methemoglobin accumulation, but produced significant oxidative stress, which was higher in G6PD-deficient than in normal erythrocytes. Primaquine, presumably through redox active hemotoxic metabolites generated in situ in human liver microsomal metabolism-linked assay, induced a dose-dependent methemoglobin accumulation and oxidative stress, which were almost similar in normal and G6PD-deficient erythrocytes. Primaquine alone or in presence of pooled human liver microsomes neither produced significant effect on intraerythrocytic calcium levels nor affected the phosphatidyl serine asymmetry of the normal and G6PD-deficient human erythrocytes as monitored flowcytometrically with Annexin V binding assay. The studies suggest that eryptosis mechanisms are not involved in accelerated removal of erythrocytes due to hemolytic toxicity of primaquine.     

Chemotypical variations in Withania somnifera lead to differentially modulated immune response in BALB/c mice

Withania somnifera (Ashwagandha) is a plant with known ethnomedicinal properties and its use in Ayurvedic medicine in India is well documented. The present investigation reports on immunomodulatory efficacy of aqueous-ethanol extracts of roots of three selected Withania somnifera chemotypes designated as NMITLI 101R, NMITLI 118R and NMITLI 128R. Each chemotype was administered 10-100 mg/kg orally to BALB/c mice once daily for 14 days. The immunomodulatory consequences were recorded by determining the humoral immune response with the help of hemagglutination, plaque forming cell assay and cellular response by measuring delayed type hypersensitivity reaction. Additionally, other immune parameters such as proliferation of T and B cells, intracellular and secreted Th1 and Th2 cytokines along with modulation in ROS production by peritoneal macrophages were monitored after feeding with lower doses (3-30 mg/kg/day) of these three chemotypes individually. NMITLI 101R incited both humoral and cellular immune response in terms of higher number of antibody producing cells and enhanced foot pad swelling at the 10 mg dose as also dose dependent B and T cell proliferations. Levels of intracellular and secreted cytokines post-NMITLI 101R treatment illustrated generation of mixed Th1/Th2 response that remained more polarized towards Th1. This chemotype also generated maximum reactive oxygen species. NMITLI 118R provoked comparatively reduced immune response in all humoral and cellular parameters at lower doses but induced highly polarized Th1 cytokine response. In contrast, NMITLI 128R led to enhanced antibody production with minimal cellular response demonstrating marginally Th2 dominance at a lower dose. Taken together, it may therefore be concluded that there were distinct modulation in the immune response exhibited by the three chemotypes of Withania somnifera and NMITLI 101R appeared to possess a better immunostimulatory activity than the other chemotypes at lower doses.     

Immunogenicity study of recombinant human sperm-associated antigen 9 in bonnet macaque (Macaca radiata)

BACKGROUND: The human sperm-associated antigen 9 (hSPAG9) is of special interest attributing to the findings indicating that SPAG9 is an acrosomal molecule. SPAG9 is not only restricted to acrosomal compartment but also persists in equatorial segment post-acrosome reaction, which is a key location in sperm-egg interaction. METHODS AND RESULTS: Immunogenicity studies in macaques were carried out with recombinant hSPAG9 (rhSPAG9) adsorbed on alum, which resulted in high titres of anti-rhSPAG9 antibodies as determined by enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis employing anti-rhSPAG9 antibodies generated in monkeys indicated that antibodies specifically reacted with native SPAG9 from macaque and human sperm and rhSPAG9 protein. Furthermore, indirect immunofluorescence experiments demonstrated SPAG9 localization in the acrosomal compartment of macaque and human sperm. In addition, monkey antibodies against rhSPAG9 significantly inhibited the human spermatozoa adherence or penetration in zona-free hamster oocytes. CONCLUSION: These results demonstrate that rhSPAG9 adsorbed on alum is highly immunogenic in subhuman primate model and therefore represents a suitable sperm-based vaccine immunogen for fertility trials in macaque.     

WNT-5A triggers Cdc42 activation leading to an ERK1/2 dependent decrease in MMP9 activity and invasive migration of breast cancer cells

An important role for WNT-5A is implicated in a variety of tumors, including breast carcinoma. We previously showed that WNT-5A signaling inhibits migration and metastasis of breast cancer cells, and that patients with primary breast cancer in which WNT-5A was expressed have a better prognosis. Despite the fact that RhoGTPase Cdc42 is commonly associated with increased cell migration, we here show that recombinant WNT-5A activates the Cdc42 in breast cancer cells (lines MDA-MB468 and MDA-MB231) in a time-dependent manner. Activation of Cdc42 was also observed in MDA-MB468 cells that were stably transfected with a WNT-5A plasmid (MDA-MB468-5A). In all situations, increased Cdc42 activity was accompanied by decreased migration and invasion of the breast cancer cells. To explore these findings further we also investigated the effect of WNT-5A signaling on ERK1/2 activity. Apart from an initial Ca²⁺-dependent rWNT-5A-induced activation of ERK1/2, Cdc42 activity was inversely correlated with ERK1/2 activity in both rWNT-5A-stimulated parental MDA-MB468 and MDA-MB468-5A cells. We also demonstrated increased ERK1/2 activity in MDA-MB468-5A cells following siRNA knockdown of Cdc42. Consistent with these results, breast cancer cells transfected with constitutively active Cdc42 exhibited reduced ERK1/2 activity, migration and invasion, whereas cells transfected with dominant negative Cdc42 had increased ERK1/2 activity in response to rWNT-5A. To gain information on how ERK1/2 can mediate its effect on breast cancer cell migration and invasion, we next investigated and demonstrated that WNT-5A signaling and constitutively active Cdc42 both decreased matrix metalloproteinase 9 (MMP9) activity. These data indicate an essential role of Cdc42 and ERK1/2 signaling and MMP9 activity in WNT-5A-impaired breast cancer cells.     

Contraceptive coverage after medical termination of pregnancy

Abortion has become a popular method of prevention of unwanted births in India since the passage in 1972 of the Medical Termination of Pregnancy Act. About 6 million procedures are performed each year. Of concern, however, is the failure on the part of many of these abortion patients to begin contraceptive use after pregnancy termination. To determine the extent of this problem and its sociodemographic correlates, an analysis was performed of the records of the 1482 abortion patients at an Agra hospital in 1976-85. Overall, contraceptive coverage after abortion was 53%; however, this rate fluctuated from 82% in 1976-77 to 15% in 1980-81 to a high of 90% in 1984-85. The methods most often selected by these women were tubectomy (25%), Nirodh (14%), oral contraceptives (8), and the IUD (6%). Contraceptive coverage was significantly higher in the final quarter of each year, presumably as a result of the efforts of family planning personnel to meet annual targets. Contraceptive coverage after abortion showed significant, positive associations with maternal age (39% in the 15-19-year age group versus 59% among those 30-34 years old), maternal education (41% among illiterate women compared to 62% among women with a high school education), and parity (18% among women of parity versus 45% among those of parity 1 and 62% among those to parity 4). Urban women were somewhat more likely to become contraceptive acceptors after abortion; however, tubectomy acceptance was highest among older rural women. These findings suggest that young, uneducated women should be targeted for counseling about the importance of contraceptive use after the termination of an unplanned pregnancy to avoid the need for repeat abortion.     

Development and characterization of 5-FU bearing ferritin appended solid lipid nanoparticles for tumour targeting

Ferritin coupled solid lipid nanoparticles were investigated for tumour targeting. Solid lipid nanoparticles were prepared using HSPC, cholesterol, DSPE and triolien. The SLNs without ferritin which has similar lipid composition were used for comparison. SLNs preparations were characterized for shape, size and percentage entrapment. The average size of SLNs was found to be in the range 110-152 nm and maximum drug entrapment was found to be 34.6-39.1%. In vitro drug release from the formulations is obeying fickian release kinetics. Cellular uptake and IC(50) values of the formulation were determined in vitro in MDA-MB-468 breast cancer cells. In vitro cell binding of Fr-SLN exhibits 7.7-folds higher binding to MDA-MB-468 breast cancer cells in comparison to plain SLNs. Ex-vivo cytotoxicity assay on targeted nanoparticles gave IC(50) of 1.28 microM and non-targeted nanoparticles gave IC(50) of 3.56 microM. In therapeutic experiments, 5-FU, SLNs and Fr-SLNs were administered at the dose of 10 mg 5-FU/kg body weight to MDA-MB-468 tumour bearing Balb/c mice. Administration of Fr-SLNs formulation results in effective reduction in tumour growth as compared with free 5-FU and plain SLNs. The result demonstrates that this delivery system possessed an enhanced anti-tumour activity. The results warrant further evaluation of this delivery system.     

Immunogenicity and contraceptive potential of recombinant human sperm associated antigen (SPAG9)

Human sperm-associated antigen 9 (hSPAG9) is a potential target for sperm-based contraceptive vaccine in lieu of its location on the sperm acrosomal compartment and its implication in sperm-egg interaction. SPAG9 is an acrosomal molecule which is not only restricted to a specific region (domain) of the acrosome but also undergoes relocation to the equatorial region in a stage-specific manner during acrosome reaction, demonstrating its potential role in sperm-egg binding. Human SPAG9 nucleotide sequence revealed 94% identity with macaque SPAG9 and 96.8% with baboon SPAG9 over the entire sequence. The amino acid sequence comparison of human SPAG9 with macaque and baboon revealed an overall homology of 84.9% and 90.6%, respectively. The presence of a high level of homology at the amino acid and nucleotide levels indicates that SPAG9 is conserved in macaque, baboon and human sharing common function and common origin in the biological past. Immunogenicity studies were carried in rats, which demonstrated that recombinant hSPAG9 protein adsorbed on alum is highly immunogenic. Antibodies thus generated after immunization reacted with recombinant human SPAG9 (rhSPAG9) and native SPAG9 protein from human sperm in Western blot analysis. In an in vitro assay, anti-rhSPAG9 antibodies inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. Further, anti-SPAG9 antibodies inhibited the binding of human sperm to intact human oocyte as well as to matched hemi-zonae, indicating that the recombinant protein is a suitable contraceptive vaccinogen. Together these results demonstrate that the rhSPAG9 adsorbed on alum is immunogenic in nature, which is a permissible adjuvant for immunogenicity and fertility trials in non-human primates.