eEF-2 kinase dictates cross-talk between autophagy and apoptosis induced by Akt Inhibition, thereby modulating cytotoxicity of novel Akt inhibitor MK-2206

Inhibition of the survival kinase Akt can trigger apoptosis, and also has been found to activate autophagy, which may confound tumor attack. In this study, we investigated regulatory mechanisms through which apoptosis and autophagy were modulated in tumor cells subjected to Akt inhibition by MK-2206, the first allosteric small molecule inhibitor of Akt to enter clinical development. In human glioma cells, Akt inhibition by MK-2206 or siRNA-mediated attenuation strongly activated autophagy, whereas silencing of eukaryotic elongation factor-2 (eEF-2) kinase, a protein synthesis regulator, blunted this autophagic response. Suppression of MK-2206-induced autophagy by eEF-2 silencing was accompanied by a promotion of apoptotic cell death. Similarly, siRNA-mediated inhibition of eEF-2 kinase potentiated the efficacy of MK-2206 against glioma cells. Together, these results showed that blunting autophagy and augmenting apoptosis by inhibition of eEF-2 kinase could modulate the sensitivity of glioma cells to Akt inhibition. Our findings suggest that targeting eEF-2 kinase may reinforce the antitumor efficacy of Akt inhibitors such as MK-2206.     

Antisperm contraceptive vaccines: where we are and where we are going?

This is a review of current status and future perspectives on the development of antisperm contraceptive vaccines (CV) and immunocontraceptives. The development of antisperm CV is an exciting proposition. There is a strong rationale and recent data indicating that this proposition can translate into reality. The search for novel sperm-specific antigens/genes, that can be used for CV, continues using various recent developing technologies. Various approaches of proteomics, genomics, reproductive biology, mucosal immunity and vaccinology and several novel technologies such as gene knockout technology, phage display technology, antibody engineering, differential display technique, subtractive hybridization, and hybridoma technology are being used to delineate sperm-specific antigens and construct CV. Various sperm antigens/genes have been delineated, cloned, and sequenced from various laboratories. Vaccination with these sperm antigens (recombinant/synthetic peptide/DNA) causes a reversible contraceptive effect in females and males of various animal species, by inducing a systemic and local antisperm antibody response. The efficacy is enhanced by combination vaccination, including peptides based on various sperm antigens. Several human novel scFv antibodies with unique complementarity-determining regions (CDRs), that react with specific well-defined fertility-related sperm antigens, have been synthesized. These human infertility-related antibodies may find application in the development of novel immunocontraceptives. Besides finding the novel sperm antigens, the present and future focus is on enhancing the immunogenicity, bioefficacy, and on obliterating the inter-individual variability of the immune response, and proceeding for primate and human clinical trials. Multi-epitope vaccines combining sperm proteins involved in various steps of fertilization cascade have been found to enhance the immunogenicity and bioefficacy of the contraceptive effect. The in vitro synthesis of infertility-related human scFv antibodies may provide unique once-a-month immunocontraceptives, the first of its kind, for human use. The multi-epitope CV and preformed engineered human antibodies of defined specificity may obliterate the concern related to inter-individual variability of the immune response.     

Identification of novel SRY mutations and SF1 (NR5A1) changes in patients with pure gonadal dysgenesis and 46,XY karyotype

Primary amenorrhea due to 46,XY disorders of sexual development (DSD) is complex with the involvement of several genes. Karyotyping of such patients is important as they may develop dysgerminoma and molecular analysis is important to identify the underlying mechanism and explore the cascade of events occurring during sexual development. The present study was undertaken for the genetic analysis in seven patients from five families presenting with primary amenorrhea and diagnosed with pure gonadal dysgenesis. Karyotyping was done and the patients were screened for underlying changes in SRY, desert hedgehog (DHH), DAX1 (NR0B1) and SF1 (NR5A1) genes, mutations in which are implicated in DSD. All the patients had 46,XY karyotype and two novel SRY mutations were found. In Family 1 (Patient S1.1) a missense mutation c.294G>A was seen, which results in a stop codon at the corresponding amino acid (Trp98X) and in Family 2 (Patients S2.1, S2.2 and S2.3), a missense mutation c.334G>A (Glu112Leu) was identified in all affected sisters. Both mutations were seen to occur in the conserved high mobility group box of SRY gene. One heterozygous change c.427G>A resulting in Glu143Lys in DHH gene in one patient and two heterozygous changes in the intronic region of SF1 (NR5A1) gene (c.244+80G>A+ c.1068-20C>T) in another patient were noted. One individual did not show changes in any of the genes analyzed. These results reiterate the importance of SRY and others, such as SF1 (NR5A1) and DHH, that are involved in the cascade of events leading to sex determination and also their role in sex reversal.     

Decision rules and associated sample size planning for regional approval utilizing multiregional clinical trials

Multiregional clinical trials provide the potential to make safe and effective medical products simultaneously available to patients globally. As regulatory decisions are always made in a local context, this poses huge regulatory challenges. In this article we propose two conditional decision rules that can be used for medical product approval by local regulatory agencies based on the results of a multiregional clinical trial. We also illustrate sample size planning for such trials.     

Design and evaluation of moxifloxacin hydrochloride ocular inserts

The objective of the present investigation was to prepare and evaluate ocular inserts of moxifloxacin. An ocular insert was made from an aqueous dispersion of moxifloxacin, sodium alginate, polyvinyl alcohol, and dibutyl phthalate by the film casting method. The ocular insert (5.5 mm diameter) was cross-linked by CaCl2 and was coated with Eudragit S-100, RL-100, RS-100, E-100 or L-100. The in vitro drug drainage/permeation studies were carried out using an all-glass modified Franz diffusion cell. The drug concentration and mucoadhesion time of the ocular insert were found satisfactory. Cross-linking and coating with polymers extended the drainage from inserts. The cross-linked ocular insert coated with Eudragit RL-100 showed maximum drug permeation compared to other formulations.