The different interactions between viral proteins and cellular host proteins are required for efficient replication of HIV-1. Various reports implicated host cellular proteins as a key factor that either interact directly with HIV-1 integrase (IN) or get involved in the integration process of virus resulting in the modulation of integration step. Polypyrimidine tract binding protein and associated splicing factor (PSF) has diverse functions inside the cell such as transcriptional regulation, DNA repair, acts as nucleic acids binding protein and regulate replication and infectivity of different viruses. The protein binding study identified the association of host protein PSF with HIV-1 integrase. The siRNA knockdown (KD) of PSF resulted in increased viral replication in TZM-bl cells, suggesting PSF has negative influence on viral replication. The quantitative PCR of virus infected PSF knockdown TZM-bl cells showed more integrated DNA and viral cDNA as compared to control cells. We did not observe any significant difference between the amount of early reverse transcription products as well as infectivity of virus in the PSF KD and control TZM-bl cells. Molecular docking study supported the argument that PSF hinders the binding of viral DNA with IN. In an attempt to study the host interacting protein of IN, we have identified a new interacting host protein PSF which is a splicing factor and elucidated its role in integration and viral replication. Experimental as well as in silico analysis inferred that the host protein causes not only change in the integration events but also targets the incoming viral DNA or the integrase-viral DNA complex. The role of PSF was also investigated at early reverse transcript production as well as late stages. The PSF is causing changes in integration events, but it does not over all make any changes in the virus infectivity. MD trajectory analyses provided a strong clue of destabilization of Integrase-viral DNA complex occurred due to PSF interaction with the conserved bases of viral DNA ends that are extremely crucial contact points with integrase and indispensable for integration. Thus our study emphasizes the negative influence of PSF on HIV-1 replication.

Co-infecting pathogens have been speculated to influence Human Immunodeficiency Virus (HIV) disease progression. Herpes Simplex Virus Type-2 (HSV-2), another sexually transmitted pathogen, is commonly observed in individuals with HIV-1. Some clinical studies have observed an increase in HIV-1 viral copy number in HSV-2 co-infected individuals. In vitro studies have also demonstrated an increase in the expression of HIV-1 co-receptors on immune cells infected with HSV-2. Although both the viruses show distinctive persistent infection, the influence of HSV-2 on HIV-1 is poorly understood. Here we present a comparative analysis of primary CD4+ T-cells and four different T-cell lines (PM-1, CEM CCR5+, MOLT4 CCR5+, and A3R5.7) to assess the influence of HSV-2 co-infection on HIV-1 replication in vitro. Cell lines indicating significant changes in HIV-1 viral copy number [CEM CCR5+ (0.61 Log10), A3R5.7 (0.78 Log10)] were further evaluated for the infectivity of HIV-1 virions and the changes in gene expression profiles of HSV-2/HIV-1 co-infected and mono-infected cells, which were further confirmed by qPCR. Significant changes in NUP, MED, and VPS mRNA expression were observed in the gene expression profiles in co-infected CEM CCR5+ and A3R5.7 cells. In both cell lines, it was observed that the WNT signaling, PI3 kinase, apoptosis, and T-cell activation pathways were negatively affected in co-infected cells. The data suggest that HSV-2 infection of T-cells may influence the expression of genes that have been previously shown to affect HIV-1 replication in vitro. This idea needs to be explored further to identify anti-viral targets for HSV-2 and HIV-1.

The objective of the study was to evaluate the effect of formulation factors, such as type of drug and particulate properties of a drug, and processing variables, i.e. jacket temperature, impeller speed, and scale, on granulation kinetics the of hot-melt granulation (HMG) process. Two model active pharmaceutical ingredients (API) Ro-A and indomethacin were selected for this evaluation using poloxamer 188 as a meltable binder. The effect of solid-state properties of API was investigated for Ro-A, whereas the binder properties were maintained constant. General factorial design was used to investigate the effect of independent process variables, impeller speed and jacket temperature using impeller motor power consumption as response variable. Consistent granulation could be developed for Ro-A by optimizing the binder level and impeller speed, however, the addition of third excipient was necessary for indomethacin. The granulation rate was related to the bulk density and the surface area of the drug. The jacket temperature affected overall granulation time but had no significant effect on the granulation kinetics, suggesting that faster heating rate is desirable for optimal productivity. A significant increase in the granulation rate was observed with increase in impeller speed. The effect of impeller speed was further confirmed at 5 L and 25 L scale. From the formulation prospective, the critical factors were the level of binder, inherent binding properties of the API, the solid-state properties of API and binder. From processing perspectives, the impeller speed had a significant effect on the granulation kinetics.

The hot melt extrusion (HME) is a novel technology to prepare pharmaceutical dosage forms by embedding a drug in a polymer while shaping the composite material to form a pharmaceutical product. The use of polymeric carriers generally requires the incorporation of a plasticizer into the formulation to improve the processing conditions of certain high molecular weight polymers. With liquid plasticizers, non uniform mixing, unstable mass flow, loss and evaporation of plasticizers during high temperature processing may lead to variable process and product performance. To overcome the shortcomings of liquid plasticizers, it is important to evaluate solid-state plasticizers as HME processing aid. Therefore, objective of this study was to judiciously select solid-state plasticizers (SSP) and evaluate of their impact on HME process and product performance including, the physicochemical stability. Indomethacin (INM) and Eudragit E PO (EPO) were selected as the model drug and the polymer, respectively. Stearic acid, glyceryl behenate and PEG 8000 were selected as SSP using solubility parameter. Various binary (polymer: plasticizer) and ternary (drug: polymer: plasticizers) physical mixtures were prepared and evaluated for thermal and rheological evaluation for HME process. The various INM/EPO/plasticizer mixtures were processed using twin screw extruders. Percentage-torque was monitored as a response variable from the processing aspects and the extrudates were characterized using differential scanning calorimetry, powder x-ray diffraction, Fourier Transform Infrared Spectroscopy, solubility, chemical assay and intrinsic dissolution rate studies. The physicochemical stability of the extrudates were monitored at 40°C/75% RH for 1 month and 30°C/60% RH for 6 months in open HDPE bottles. All plasticizers were effective in reducing the percentage-torque even at the lower concentrations. Plasticizers at lower concentrations appeared to have no effect on product performance at lover concentrations. The formulation containing higher concentration of plasticizer (>5%) had adverse effect on the Tg but had no effect on the PXRD or IDR suggesting that mechanism of solid-state stabilization of amorphous INM may not be sensitive to the mobility of the system. Judicious use of plasticizer may help in HME processing without affecting the solid state stability of extruded formulations.