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Methods and compositions for enhancing transport across biological membranes

a biological membrane and transport technology, applied in the direction of biocide, drug composition, peptide/protein ingredients, etc., can solve the problems of affecting the therapeutic utility of high-charge molecules, affecting the use of tat proteins, and affecting the transport of high-charge molecules. , to achieve the effect of promoting the transport of agents and enhancing the transport of selected compounds

Inactive Publication Date: 2006-05-25
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] The present invention includes, in one aspect, a method for enhancing transport of a selected compound across a biological membrane. In the method, a biological membrane is contacted with a conjugate containing a biologically active agent that is covalently attached to at least one transport polymer. The conjugate is effective to promote transport of the agent across the biological membrane at a rate that is greater than the trans-membrane transport rate of the biological agent in non-conjugated form.
[0015] The transport-enhancing polymers are exemplified, in a preferred embodiment, by peptides in which arginine residues constitute the subunits. Such a polyarginine peptide may be composed of either all D-, all L- or mixed D- and L-arginines, and may include additional amino acids. More preferably, at least one, and preferably all of the subunits are D-arginine residues, to enhance biological stability of the polymer during transit of the conjugate to its biological target.
[0019] The agent may be linked to the polymer by a linking moiety, which may impart conformational flexibility within the conjugate and facilitate interactions between the agent and its biological target. In one embodiment, the linking moiety is a cleavable linker, e.g., containing a linker group that is cleavable by an enzyme or by solvent-mediated cleavage, such as an ester, amide, or disulfide group. In another embodiment, the cleavable linker contains a photocleavable group.

Problems solved by technology

Highly charged molecules in particular experience difficulty in passing across membranes.
Thus, while biotechnology has made available a greater number of potentially valuable therapeutics, bioavailability considerations often hinder their medicinal utility.
However, use of the tat protein has certain disadvantages, including unfavorable aggregation and insolubility properties.
However, slightly shorter versions of this peptide (15 residues) are not effectively taken up by cells (Derossi et al.

Method used

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  • Methods and compositions for enhancing transport across biological membranes
  • Methods and compositions for enhancing transport across biological membranes
  • Methods and compositions for enhancing transport across biological membranes

Examples

Experimental program
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Effect test

example 1

[0172] Peptide Synthesis

[0173] Peptides were synthesized using solid phase techniques on an Applied Biosystems Peptide synthesizer using FastMOC™ chemistry and commercially available Wang resins and Fmoc protected amino acids, according to methods well known in the art (Bonifaci et al., Aids 9:995-1000). Peptides were purified using C4 or C18 reverse phase HPLC columns, and their structures were confirmed using amino acid analysis and mass spectrometry.

example 2

[0174] Fluorescence Assays

[0175] Fluorescent peptides were synthesized by modification of the amino terminus of the peptide with aminocaproic acid followed by reaction with fluorescein isothiocyanate in the presence of (2-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate / N-hydroxy benzotriazole dissolved in N-methyl pyrrolidone. The products were purified by gel filtration.

[0176] Suspension cells (106 / mL) were incubated for varying times, at 37° C., 23° C., or 4° C., with a range of concentrations of peptides or conjugates in PBS pH 7.2 containing 2% fetal calf serum (PBS / FCS) in 96 well plates. After a 15 minute incubation, the cells were pelleted by centrifugation, washed three times with PBS / FCS containing 1% sodium azide, incubated with trypsin / EDTA (Gibco) at 37° C. for five minutes, then washed twice more with PBS / FCS / NaN3. The pelleted cells were resuspended in PBS containing 2% FCS and 0.1% propidium iodide and analyzed on a FACScan (Becton Dickinson, M...

example 3

[0177] Tat Basic Peptide Versus Poly-Arg Peptides

[0178] Uptake levels of the following polypeptides were measured by the method in Example 2: (1) a polypeptide comprising HIV tat residues 49-57 (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg=SEQ ID NO: 1), (2) a nonamer of L-Lys residues (K9, SEQ ID NO:2), and (3) homo-L or homo-D-polypeptides containing four to nine Arg residues (SEQ ID NO:3-8 and 12-17). Results a re shown in FIGS. 1A-1C.

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Abstract

Methods and compositions for transporting drugs and macromolecules across biological membranes are disclosed. In one embodiment, the invention pertains to a method for enhancing transport of a selected compound across a biological membrane, wherein a biological membrane is contacted with a conjugate containing a biologically active agent that is covalently attached to a transport polymer. In a preferred embodiment, the polymer consists of from 6 to 25 subunits, at least 50% of which contain a guanidino or amidino sidechain moiety. The polymer is effective to impart to the attached agent a rate of trans-membrane transport across a biological membrane that is greater than the rate of trans-membrane transport of the agent in non-conjugated form.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. Ser. No. 10 / 338,348, filed Jan. 7, 2003, which is a continuation of U.S. Ser. No. 09 / 396,194, filed Sep. 14, 1999, which is a divisional of U.S. Ser. No. 09 / 083,259, filed May 21, 1998, now U.S. Pat. No. 6,306,993, which claims priority to U.S. provisional application Ser. No. 60 / 047,345, filed May 21, 1997. The aforementioned patent and applications are incorporated herein by reference.GOVERNMENT INTEREST [0002] This invention was made with the support of NIH grant number CA 65237. Accordingly, the U.S. Government has certain rights in this invention.FIELD OF THE INVENTION [0003] The present invention is directed to methods and compositions that are effective to enhance transport of biologically active agents, such as organic compounds, polypeptides, oligosaccharides, nucleic acids, and metal ions, across biological membranes. BACKGROUND OF THE INVENTION [0004] The plasma membranes of cells pr...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/17A61K31/785A61K31/711A61K38/00A61K47/42A61K47/48A61P43/00C07B61/00C07D305/14
CPCA61K31/785A61K47/48238A61K47/48315A61K48/00C07B2200/11C07D305/14C40B40/00A61K47/62A61K47/645A61P43/00
Inventor ROTHBARD, JONATHANWENDER, PAUL
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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