Lishan Chen

Development of Calcitonin Salmon Nasal Spray: Similarity of Peptide Formulated in Chlorobutanol Compared to Benzalkonium Chloride as Preservative

The similarity of an intranasal salmon calcitonin (sCT) employing chlorobutanol as preservative (Calcitonin Salmon Nasal Spray) was compared to the reference listed drug (RLD) employing benzalkonium chloride as preservative (Miacalcin Nasal Spray). Various orthogonal methods assessed peptide structuring, dynamics, and aggregation state. Mass spectrometry, amino acid analysis, and N-terminal sequencing all demonstrated similarity in primary structure. Near- and far-UV circular dichroism (CD) data supported similarity in secondary and tertiary sCT structure. Nuclear magnetic resonance studies further supported similarity of three-dimensional structure and molecular dynamics of the peptide. Other methods, such as sedimentation velocity and size exclusion chromatography, demonstrated similarity in peptide aggregation state. These latter methods, in addition to reversed phase chromatography, were also employed for monitoring stability under forced degradation, and at the end of recommended shelf storage and patient use conditions. In all cases and for all methodologies employed, similarity to the RLD was observed with respect to extent of aggregation and other degradation processes. Finally, ELISA and bioassay data demonstrated similarity in biological properties. These investigations comprehensively demonstrate physicochemical similarity of Calcitonin Salmon Nasal Spray and the RLD, and should prove a useful illustration to pharmaceutical scientists developing alternative and/or generic peptide or protein products.

Peptide - Mediated Delivery of siRNA via Noncovalent Complexes and Covalent Conjugates

Introduction Improving the intracellular delivery of synthetic oligonucleotides and their analogs is an important goal in the development of small interfering RNA (siRNA) therapeutics for affecting gene expression in cell culture and in vivo. One of the major challenges to the effective use of therapeutic siRNAs is their relatively inefficient uptake by cells. siRNAs are polyanions and their in vitro and in vivo activity is improved substantially by covalent attachment or complex formation with natural peptide sequences that posses the ability to translocate across the cell membrane [1-3]. We have performed a systematic analysis of the ability of different structural classes of peptides to translocate across cell membranes and deliver siRNA into cells. Over 100 peptides were screened by mixing with siRNA to generate a noncovalent complex; the top 20 candidates with the best uptake properties were covalently conjugated to the 5’or 3’end of the siRNA sense strands via a thioether bond. The uptake efficiency of siRNA was evaluated by flow cytometry using fluorescently labeled siRNAs (FAM or Cy5) in both established and primary human and mouse cells (Fig. 1). The localization of siRNA within the cells was visualized by fluorescence microscopy. Finally, the ability of complexed or conjugated siRNA to mediate knockdown of TNF-α mRNA was measured in activated human monocytes.