Pharmacokinetics comparison of two pegylated interferon alfa formulations in healthy volunteers.

BACKGROUND: Several countries have used pegylation technology to improve the pharmacokinetic properties of essential drugs. Recently, a novel interferon alfa-2b protein conjugated to four-branched 12 kDa polyethylene glycol molecules was developed jointly between Cuba and Brazil. The aim of this study was to compare the pharmacokinetic properties of BIP48 (pegylated interferon alfa-2b from Bio-Manguinhos/Fiocruz, Brazil) to those of PEGASYS® (commercially available pegylated interferon alfa-2a from Roche Pharmaceutical). METHODS: This phase I, single-centre, randomized, double-blind crossover trial enrolled 31 healthy male volunteers aged 19 to 35 who were allocated to two stages, either side of a 5-week wash-out period, with each arm lasting 14 consecutive days after subcutaneous administration of 180 µg of one formulation or the other (study or comparator). The main outcome variable was serum pegylated interferon concentrations in 15 samples collected during the course of the study and tested using an enzyme immunoassay. RESULTS: There were no differences between formulations in terms of magnitude or absorption parameters. Analysis of time parameters revealed that BIP48 remained in the body significantly longer than PEGASYS® (Tmax: 73 vs. 54 h [p = 0.0010]; MRT: 133 vs. 115 h [p = 0.0324]; ke: 0.011 vs. 0.013 h(-1) [p = 0.0153]; t1/2: 192 vs. 108 h [p = 0.0218]). CONCLUSION: BIP48 showed the expected pharmacokinetic profile for a pegylated product with a branched molecular structure. Compared to PEGASYS®, the magnitude absorption was similar, but time parameters were consistent with slower elimination. Further studies should be conducted to evaluate the clinical implications of these findings. A phase II-III repeated-dose clinical trial is ongoing to study these findings in patients with chronic hepatitis C virus infection. TRIAL REGISTRATION: This study is registered on the ClinicalTrials.gov platform (accession number NCT01889849 ). This trial was retrospectively registered in June 2013.

PCA3 long noncoding RNA modulates the expression of key cancer-related genes in LNCaP prostate cancer cells.

Prostate cancer antigen 3 (PCA3) is a prostate-specific long noncoding RNA (lncRNA) involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. To further comprehend the mechanisms by which PCA3 modulates LNCaP cell survival, we characterized the expression patterns of several cancer-related genes, including those involved in epithelial-mesenchymal transition (EMT) and AR cofactors in response to PCA3 silencing. We also aimed to develop a strategy to stably silence PCA3. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) was used to knock down PCA3 in LNCaP cells. The expression of 84 cancer-related genes, as well as those coding for AR cofactors and EMT markers, was analyzed by quantitative real-time PCR (qRT-PCR). LNCaP-PCA3 silenced cells differentially expressed 16 of the 84 cancer genes tested, mainly those involved in gene expression control and cell signaling. PCA3 knockdown also induced the upregulation of several transcripts coding for AR cofactors and modulated the expression of EMT markers. LNCaP cells transduced with lentivirus vectors carrying an shRNA sequence targeting PCA3 stably downregulated PCA3 expression, causing a significant drop (60 %) in the proportion of LNCaP cells expressing the transgene. In conclusion, our data provide evidence that PCA3 silencing modulates the expression of key cancer-related genes, including those coding for AR cofactors and EMT markers. Transducing LNCaP cells with an shRNA sequence targeting PCA3 led to loss of viability of the cells, supporting the proposal of PCA3 knockdown as a putative therapeutic approach to inhibit PCa growth.

Generation of monoclonal antibodies against human recombinant interferon beta using genetic immunization with simultaneous expression of IgM and IgG isotypes.

Monoclonal antibodies (MAbs) against human recombinant interferon beta (hrIFNbeta) were generated by genetic immunization (GI). In order to test two viral promoters frequently used in mammalian expression plasmid vectors, mice were inoculated four times by intramuscular injection, without adjuvant, with 100 microg of either pcDNA 3.1hrIFNbeta or pZeoSV2IFNbeta containing the entire human interferon beta gene and under the control of, respectively, human cytomegalovirus (HCMV) immediate-early promoter or early SV-40 enhancer/promoter. Only serum samples from mice immunized with pZeoSV2IFNbeta were positive to anti-hrIFNbeta. The spleens of the immunized mice were fused with myeloma Sp2/0 cells and the hybridoma clones generated screened by an in house enzyme-linked immunosorbent assay (ELISA). Fourteen MAbs were selected as reactive with hrIFNbeta. Western blot analysis was performed and only one recognized the 18 kDa isoform (non-glycosylated) of hrIFNbeta. All MAbs were subjected to antibody isotype characterization with a commercial ELISA and showed unusual profile with simultaneous expression of both IgM and IgG2a isotypes. This observation is further supported by RT-PCR amplification of the IgM CH4 domain using total RNA from hybridomas.

Evaluation of murine monoclonal antibodies targeting different epitopes of the hepatitis B virus surface antigen by using immunological as well as molecular biology and biochemical approaches.

The hepatitis B virus surface protein (HBsAg) displays the major B cells antigenic determinants that can induce protective immunity and prevent the hepatitis B virus (HBV) infection, a major health problem. A panel of murine monoclonal antibodies against the HBsAg (MAb anti-HBs), raised after mice immunization with a pool of plasma of hepatitis chronic carriers, has been established. Mainly using simple immunological tools such as enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, we could trace the location of the epitopes on the HBsAg determinants. We also report the use of two specific methodology approaches based on molecular biology and biochemical techniques such as, respectively, cloning and expression of preS1 major neutralizing epitope of the HBsAg in Escherichia coli and ELISA accomplished to chemical reduction with dithiothreitol (DTT), which were able to complete the MAb anti-HBs characterization. Our results showed that the majority of the MAbs anti-HBs were directed to the HBV common determinant a. One MAb recognizes a discontinuous epitope present in all forms of the HBsAg when evaluated by Western blot.