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Sp1 polypeptides, modified sp1 polypeptides and uses thereof

a technology of sp1 and polypeptide, which is applied in the direction of peptide sources, peptide/protein ingredients, drug compositions, etc., can solve the problems of insufficient soluble sp1 in aqueous solution, adverse side effects in therapeutic concentration, and many drugs employed to treat diseases, etc., to achieve the effect of enhancing the solubility of a substance and enhancing the solubility of the substance in the solution

Inactive Publication Date: 2014-06-26
YISSUM RES DEV CO OF THE HEBREWUNIVERSITY OF JERUSALEM LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

SP1 polypeptides effectively stabilize and solubilize drugs, allowing for targeted and controlled release, reducing toxicity and improving therapeutic efficacy while maintaining stability under harsh conditions.

Problems solved by technology

However, U.S. patent application Ser. No. 10 / 233,409 do not teach, nor imply, the use of native SP1, or SP1 variants as carriers for and means of controlled release of, agents (therapeutic, cosmetic, diagnostic, conductive, etc) reversibly complexed therewith.
Many drugs employed to treat diseases are either insufficiently soluble in aqueous solutions or have adverse side effects in therapeutic concentrations.
Thus, many medical applications suffer from a lack of suitable methods for efficiently delivery of effective concentrations of drugs to a target cell or tissue in an organism (e.g., mammal) in need of treatment.
Poor solubility, causing difficulty in achieving a convenient pharmaceutical format, as hydrophobic drugs may precipitate in aqueous media.
However, the use of excipients for solubilization such as Cremphor (the solubilizer for paclitaxel in Taxol) is also associated with toxicity.
Lack of selectivity for target tissues, leading to toxicity to normal tissues, severely restricting the amount of drug that can be administered, as in the case of the cardiac toxicity of doxorubicin.
Unfavorable pharmacokinetics, such as rapid renal clearance, rapid breakdown of the drug in vivo, or loss of activity at physiological conditions (e.g. loss of activity of camptothecins at physiological pH), can also lead to heightened dosing or a frequent administration regimen.
Tissue damage on extravasation of cytotoxic drugs, leading to tissue damage (i.e. necrosis caused by free paclitaxel).
A number of approaches have resolved some of these issues in specific cases, but there is yet no general solution to the problems of drug delivery.
None of these approaches, however, offers a general solution for all cases of drug delivery problems.
Control of particle size in micellar, liposomal, and polymeric nanoparticulate systems remains a serious problem.
The inability of currently available drug delivery systems to incorporate all of the functions required for delivery into a single system is another problem with for example, micelles, nanoparticulate systems and targeted systems.
Yet further, the release rate and storage life, especially of micelles and liposomes, is difficult to control and unpredictable, and amphiphylic components can produce toxic effects.

Method used

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  • Sp1 polypeptides, modified sp1 polypeptides and uses thereof
  • Sp1 polypeptides, modified sp1 polypeptides and uses thereof
  • Sp1 polypeptides, modified sp1 polypeptides and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Structure of the SP1 Protein

[0257]SDS gel electrophoresis analysis of both native SP1 and its recombinant form shows that SP1 appears in two forms: a monomer (12.4-kDa) which appears when the sample is boiled in the gel application buffer in the presence of SDS and in an oligometric form (116-kDa protein) which appear when SP1 is not boiled prior to application on PAGE (see Wang et al 2002, Dgany 2004, Wang et al 2006, U.S. patent application Ser. No. 10 / 443,209). Several methods have been employed to demonstrate that SP1 in solution forms a dodecamer with a molecular weight of 150 kDa. Equilibrium analytical ultracentrifugation was employed to analyze the SP1 oligomeric state. As SP1 concentration approached zero, the measured molecular mass of the SP1 particles in solution (144 kDa at 5.6 μM monomer concentration) approached the value calculated for a dodecamer (148 kDa).

[0258]SP1 was subjected to MALDI-TOF-MS. The data revealed 12 protein peaks, of which the first (12338 Da) was ...

example 2

SP1 is a Boiling- and Denaturing-Stable, and Protease-Resistant Molecule

[0268]The stability of the SP1 complex in the presence of SDS was examined by incubating purified SP1 with SDS at different molar ratios and at different temperatures. Dissociation of the SP1 complex to monomers required a molar ratio greater than 600:1 (SDS:SP1-monomer) accompanied by boiling before loading onto the gel. Without boiling, even at a ratio of 3467 to 1, SP1 remained as a complex on SDS-PAGE. Incubation of SP1 with 1734-fold SDS (1%) at temperatures of 80° C. or lower did not cause significant disassociation of the complex (Wang 2006).

[0269]SP1 protein exhibits exceptional heat stability, which allows partial extraction of crude cellular preparations by heat treatment, as mentioned in Example 1 hereinabove. FIG. 6 shows the degree of purity achieved by heat treatment.

[0270]Differential scanning calorimetry study of SP1 indicates in a Tm of 107° C. for SP1. These results further support our previous...

example 3

SP1 Variants

[0274]SP1 variants can be constructed to enhance, or otherwise alter SP1 stability, capabilities for oligomerization and / or binding and / or complexing with other molecules and / or ability to form inter-subunit disulfide bonds and / or change the dimension of the central cavity. Numerous SP1 variant proteins were constructed to investigate the effects of specific alterations on properties of SP1.

[0275]The N-terminal segment points toward the solvent and apparently is not involved in protein folding or stability. Therefore it was predicted that N-terminus mutations would not alter protein structure or its stability. In agreement with this prediction all the N-terminus mutants including a mutant protein carrying deletion of the entire N-terminus Δ2-6 assembled into a stable complex (FIG. 3). Further, it was surprisingly uncovered that when the tumor recognition peptides CRGD (SEQ ID NO:5) and RGDC (SEQ ID NO:6) and RGD loop (SEQ ID NO:10) were inserted into the SP1 N-terminus, ...

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Abstract

SP1 and modified SP1 variant polypeptides capable of forming reversible molecular associations with substances, compositions-of-matter comprising same, and uses thereof are provided.

Description

RELATED APPLICATIONS[0001]This application is a division of U.S. patent application Ser. No. 11 / 988,314 filed on Jan. 4, 2008, which is a National Phase of PCT Patent Application No. PCT / IL2006 / 000795 having International Filing Date of Jul. 9, 2006. The contents of the above applications are all incorporated herein by reference.SEQUENCE LISTING STATEMENT[0002]The ASCII file, entitled 57189SequenceListing.txt, created on Feb. 23, 2014, comprising 215,552 bytes, submitted concurrently with the filing of this application is incorporated herein by referenceFIELD AND BACKGROUND OF THE INVENTION[0003]The present invention relates to denaturant and protease stable proteins, modified derivatives thereof, and uses thereof. More particularly, the present invention relates to the use of novel denaturant-stable, protease resistant, homo-oligomeric proteins, also referred to herein as stable proteins (SPs), and derivatives thereof designed for complexing, release and delivery of other molecules...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/415
CPCC07K14/415B82Y30/00A61K47/64A61K47/6929A61K38/17A61P43/00Y02A50/30
Inventor WOLF, AMNONPOUNY, YEHONATHANMARTON, IRADGANY, ORALTMAN, ARIESHOSEYOV, ODED
Owner YISSUM RES DEV CO OF THE HEBREWUNIVERSITY OF JERUSALEM LTD