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Uses of protein precursors as prodrugs

A protein and precursor technology, applied in the field of drug delivery and protein engineering, can solve the problems of final product composition and size heterogeneity

Inactive Publication Date: 2015-09-09
沈维强
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the major obstacle to chemically conjugating two domains together is that the final product can be heterogeneous in composition and size, which is unacceptable for therapeutic use

Method used

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  • Uses of protein precursors as prodrugs
  • Uses of protein precursors as prodrugs
  • Uses of protein precursors as prodrugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Expression and Characterization of PI-Tf Recombinant Fusion Protein

[0041] Using molecular cloning, the preproinsulin sequence (NM_000207) fused in frame with the Tf sequence (NM_001063) was engineered into the pcDNA3.1(+) expression vector (Invitrogen, CA) ( figure 1 ). A plasmid containing the preproinsulin-Tf fusion gene was transiently transfected into HEK 293 cells by polyethyleneimine-mediated DNA transfection. Serum-free conditioned medium was collected, concentrated using a lab-scale Tangential Flow Filtration System (Millipore, MA) and then ultrafiltered using a Centricon (Millipore, MA). The PI-Tf fusion protein was characterized and quantified by Western blotting using anti-Tf antibody (Sigma, MO) and anti-insulin (pro) antibody (Abeam, MA). Anti-Tf and anti-insulin (pro) western blots showed the presence of a major band with a molecular weight of about 89 kD, which indicated that the PI-Tf fusion protein was successfully expressed and secreted into the...

Embodiment 2

[0043] Enhanced inhibitory effect of PI-Tf fusion protein on hepatic glucose production in H4IIE hepatoma cells

[0044] Rat hepatoma H4IIE cells were cultured in high glucose DMEM containing 10% fetal bovine serum. After confluence, cells were treated with different drugs for 24 hours at 37°C. Cells were washed twice with phosphate buffered saline. Glucose production medium consisting of serum-free, glucose-free, phenol red-free DMEM supplemented with 2 mM sodium pyruvate and 40 mM LD-lactate was added to the cells and incubated for an additional 3 hours. The supernatant was harvested and the glucose concentration was measured with the Amplex Red Glucose / Glucose Oxidase Kit (Invitrogen, CA) [5]. Cells were lysed in 1M NaOH, and protein amounts were determined with BCA (Thermo Scientific, IL).

[0045] Proinsulin and insulin showed comparable inhibitory effects on glucose production, with IC 50 The values ​​were 1441.3±641.6 pM and 1093.9±105.6 pM, respectively (Fig. 3A)...

Embodiment 3

[0047] Liver cancer cells convert PI-Tf to insulin-Tf fusion protein

[0048] Rat hepatoma H4IIE cells (ATCC, VA) were treated with PI-Tf fusion protein in DMEM medium and incubated at 37°C. Media were collected at various times and subjected to insulin-specific and proinsulin-specific radioimmunoassays (Millipore, MA). Concentrations of proinsulin and insulin were obtained based on standard curves from radioimmunoassays. After up to 24 hours of treatment in H4IIE cells, insulin-containing material was continuously produced in PI-Tf fusion protein-treated samples, but not in proinsulin-treated samples ( Figure 4 ). The resulting insulin-containing material was shown to be insulin-Tf rather than released insulin because the two moieties are linked by a stable peptide bond. The conversion efficiency of PI-Tf to insulin-Tf was estimated to be 8.8% when the dose of PI-Tf was 10 nM, and 21.6% when the dose of PI-Tf was 1 nM. These results demonstrate that the prohormone fusi...

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Abstract

The present invention provides compositions useful as prodrugs and methods for making the same. The compositions include a fusion protein having a first delivery domain and a second protein precursor domain linked together via a linker sequence. The delivery domain is a protein capable of facilitating entry to a target cells via the endocytotic pathway, such as transferrin. The protein precursor is a prohormone or a profactor, such as proinsulin. Methods of this invention include the steps of selecting a protein suitable as the delivery domain, constructing a vector to encode the fusion protein, and expressing the fusion protein in a suitable expression host. Also disclosed is a method for targeted-delivery of prodrugs to livers and a method of reducing hepatic glucose production.

Description

[0001] Cross References to Related Applications [0002] This application is a continuation-in-part of U.S. Patent Application 13 / 174,520, filed June 30, 2011, which has the benefit of U.S. Provisional Application 61 / 361,248, filed July 2, 2010, and is hereby incorporated in its entirety Both are incorporated herein by reference. [0003] Statement Regarding Federally Funded Research and Development [0004] This invention was made with Government support under Contract GM 063647 awarded by NIH and HIGMS. The government has certain rights in this invention. technical field [0005] The invention belongs to the fields of drug delivery and protein engineering. More specifically, the present invention relates to methods and compositions for the delivery of protein-based therapeutic agents, such as prohormones or profactors, that do not require in vitro chemical or proteolytic processing to produce therapeutically effective drugs. Background technique [0006] Many biologica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K38/28
CPCA61K38/00A61K47/483C07K14/62C07K2319/74C07K2319/33A61K47/644
Inventor 沈维强王燕J·克兰克尔
Owner 沈维强
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