HER2 Kinase-Targeted Breast Cancer Therapy: Design, Synthesis, and In Vitro and In Vivo Evaluation of Novel Lapatinib Congeners as Selective and Potent HER2 Inhibitors with Favorable Metabolic Stability.

HER2 kinase as a well-established target for breast cancer (BC) therapy is associated with aggressive clinical outcomes; thus, herein we present structural optimization for HER2-selective targeting. HER2 profiling of the developed derivatives demonstrated potent and selective inhibitions (IC50: 5.4-12 nM) compared to lapatinib (IC50: 95.5 nM). Favorably, 17d exhibited minimum off-target kinase activation. NCI-5-dose screening revealed broad-spectrum activities (GI50: 1.43-2.09 µM) and 17d had a remarkable selectivity toward BC. Our compounds revealed significant selective and potent antiproliferative activities (∼20-fold) against HER2+ (AU565, BT474) compared to HER2(-) cells. At 0.1 IC50, 15i, 17d, and 25b inhibited pERK1/2 and pAkt by immunoblotting. Furthermore, 17d demonstrated potent in vivo tumor regression against the BT474 xenograft model. Notably, a metastasis case was observed in the vehicle but not in the test mice groups. CD-1 mice metabolic stability assay revealed high stability and low intrinsic clearance of 17d (T1/2 > 145 min and CLint(mic) < 9.6 mL/min/kg).

Novel rotavirus VP7 typing assay using a one-step reverse transcriptase PCR protocol and product sequencing and utility of the assay for epidemiological studies and strain characterization, including serotype subgroup analysis.

Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in approximately 92% of samples (3, 17, 19).

Analysis of varicella zoster virus attenuation by evaluation of chimeric parent Oka/vaccine Oka recombinant viruses in skin xenografts in the SCIDhu mouse model.

Varicella-zoster virus (VZV) is the only human herpes virus for which a vaccine has been licensed. A clinical VZV isolate, designated the parent Oka (pOka) strain was passed in human and non-human fibroblasts to produce vaccine Oka (vOka). The pOka and vOka viruses exhibit similar infectivity in cultured cells but healthy susceptible individuals given vaccines derived from vOka rarely develop the cutaneous vesicular lesions characteristic of varicella. Inoculation of skin xenografts in the SCIDhu mouse model of VZV pathogenesis demonstrated that vOka had a reduced capacity to replicate in differentiated human epidermal cells in vivo (Moffat, J.F., Zerboni, L., Kinchington, P.R., Grose, C., Kaneshima, H., Arvin A.M., 1998a. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J Virol. 72:965-74). In order to investigate the attenuation of vOka in skin, we made chimeric pOka and vOka recombinant viruses from VZV cosmids. Six chimeric pOka/vOka viruses were generated using cosmid sets that incorporate linear overlapping fragments of VZV DNA from cells infected with pOka or vOka. The cosmid sets consist of pOka and vOka DNA segments that have identical restriction sites. As expected, the growth kinetics and plaque morphologies of the six chimeric pOka/vOka viruses were indistinguishable in vitro. However, the chimeric viruses exhibited varying capacities to replicate when evaluated in skin xenografts in vivo. The presence of ORFs 30-55 from the pOka genome was sufficient to maintain wild-type infectivity in skin. Chimeric viruses containing different vOka components retained the attenuation phenotype, suggesting that vOka attenuation is multi-factorial and can be produced by genes from different regions of the vOka genome.

Protective efficacy of intranasal cold-adapted influenza A/New Caledonia/20/99 (H1N1) vaccines comprised of egg- or cell culture-derived reassortants.

Live, cold-adapted, temperature-sensitive (ca/ts) Russian influenza A vaccines are prepared in eggs by a 6:2 gene reassortment of the ca/ts donor strain A/Leningrad/134/17/57 (H2N2) (Len/17) with a current wild-type (wt) influenza A strain contributing hemagglutinin (HA) and neuraminidase (NA) genes. However, egg-derived reassortant vaccines are potentially more problematic to manufacture in large quantities than vaccines from cell-based procedures. To compare egg- and cell culture-derived reassortant vaccines, we prepared in Madin Darby canine kidney (MDCK) cells two cloned, ca/ts reassortants (25M/1, 39E/2) derived from Len/17 and a wt reference strain A/New Caledonia/20/99 (H1N1) (NC/wt). Both 25M/1 and 39E/2 reassortants preserved the ca/ts phenotype and mutations described for internal genes of the A/Len/17 parent. When compared to a commercial, egg-derived ca/ts Russian A/17/NC/99/145 (H1N1) New Caledonia vaccine (NC/145), the MDCK-derived reassortant 39E/2 vaccine conferred similar levels of protection in ferrets challenged i.n. with 7 x 10(10) pfu of NC/wt. In a dose-ranging study, the protective vaccine dose for 50% of ferrets (PD50) was less than 1.2 x 10(4) pfu for the 25M/1 vaccine derived by recombination and amplification in MDCK cells. Clonal isolates of ca/ts influenza A/New Caledonia/20/99 (H1N1) obtained by recombination and amplification entirely in MDCK cells can be highly protective i.n. vaccines.