HK1089450B - Method and carrier complexes for delivering molecules to cells - Google Patents

Description

Method and carrier complex for delivering molecules to cells

This application claims priority from U.S. provisional application serial No. 60/467,516 filed on 1/5/2003. The specification of U.S. provisional application serial No. 60/467,516 is incorporated herein by reference in its entirety.

The invention was made with government support from the national institute for drug abuse with approval number P01-DA-08924. The united states government has certain rights in this invention.

Background

Biological cells are generally highly selective for molecules that are allowed to pass through the cell membrane. Thus, the delivery (or transport) of compounds, such as small molecules and biomolecules, into cells is often limited by the physical properties of the compounds. The small molecules and biomolecules may be, for example, compounds having pharmaceutical activity.

The inability of such molecules (including macromolecules such as proteins and nucleic acids) to be delivered into cells in vivo has been a barrier to the use of a large number of potentially effective compounds in therapy, prophylaxis and/or diagnosis. In addition, because of the lack of ability to deliver compounds efficiently into cells in vivo, many compounds that appear promising in vitro have been abandoned as potential drugs.

Many reports have addressed the problem of delivering compounds to cells by covalently attaching the compounds to the "protein transduction structural thresholds" (PTDs). Schwarze et al (TrendsPharmacol Sci.2000; 21: 45-8) and U.S. Pat. No. 6,221,355 to Dowdy disclose several PTDs capable of crossing the lipid bilayer of cells in a concentration-dependent manner. The PTDs disclosed include PTDs derived from the HIV-1 tat protein, from the Drosophila homotype transcription factor encoded by the antennapedia (abbreviated as ANTP) gene, and from the herpes simplex virus VP22 transcription factor. The length of the HIV-1 tat PTD is eleven amino acids, the length of the ANTP PTD is sixteen amino acids, and the length of the VP22 PTD is 34 amino acids.

However, recently published data show that these PTDs enter cells via energy-dependent endocytosis. Thus, the "PTD-cargo" complex is included within vesicles of cellular endosomes, but is absent from, for example, the cytoplasm of the cell. Thus, the "PTD-cargo" complex must be released from the vesicles of the endosome in order to be biologically active (Richard et al, J biol. chem.2003; 278: 585-. In addition, recent reports have shown that PTDs are toxic to cells.

Thus, there is a need for peptides that are capable of passing through the lipid membrane of a cell in an energy-independent, non-endocytotic manner. In addition, to avoid the generally well-known immune response to larger peptides, there is a need for smaller, peptidase-resistant peptides. Finally, it is also important that the peptide vector is not toxic to cells.

Disclosure of Invention

These needs are met by the present invention which provides a method for delivering molecules to cells. The method comprises contacting a carrier complex with the cell, wherein the carrier complex comprises the molecule and an aromatic-cationic peptide, and wherein the aromatic-cationic peptide comprises:

(a) at least one net positive charge;

(b) a minimum of three amino acids;

(c) a maximum of ten amino acids;

(d) minimum number of net positive charges (p)_m) The relationship with the total number of amino acid residues (r) is that, wherein 3p_mIs the maximum number less than or equal to r + 1; and

(e) minimum number of aromatic groups (a) and total number of net positive charges (p)_t) Wherein 3a is less than or equal to p_tMaximum of +1, except when a is 1, p_tAnd may be 1.

In another embodiment, the invention provides a carrier complex comprising a molecule and an aromatic-cationic peptide, wherein the aromatic-cationic peptide comprises:

(a) at least one net positive charge;

(b) a minimum of three amino acids;

(c) a maximum of ten amino acids;

(d) minimum number of net positive charges (p)_m) The relationship with the total number of amino acid residues (r) is that, wherein 3p_mIs the maximum number less than or equal to r + 1; and

(e) minimum number of aromatic groups (a) and total number of net positive charges (p)_t) Wherein 3a is less than or equal to p_tMaximum of +1, except when a is 1, p_tAnd may be 1.

In another embodiment, the invention provides a method for delivering a molecule to a cell. The method comprises contacting a molecule and an aromatic-cationic peptide with a cell, wherein the aromatic-cationic peptide comprises:

(a) at least one net positive charge;

(b) a minimum of three amino acids;

(c) a maximum of ten amino acids;

(d) minimum number of net positive charges (p)_m) The relationship with the total number of amino acid residues (r) is that, wherein 3p_mIs the maximum number less than or equal to r + 1; and

(e) minimum number of aromatic groups (a) and total number of net positive charges (p)_t) Wherein 3a is less than or equal to p_tMaximum of +1, except when a is 1, p_tAnd may be 1.

Drawings

FIG. 1. Capo-2 intracellular peptide uptake. [³H][Dmt¹]DALDA (A) and [2 ]¹⁴C]Time course of uptake of Gly-Sar (B). The Caco-2 cell is contacted with the polypeptide at 37 ℃ or 4 ℃³H][Dmt¹]DALDA (250nM, 47Ci/mmol) or [2 ]¹⁴C]Gly-Sar (50. mu.M, 56.7mCi/mmol) was incubated for 1 hour. Radioactivity was then measured in the lysed cells. (C) Will 2³H][Dmt¹]The accumulation of DALDA was pickled. Caco-2 cell and³H][Dmt¹]DALDA was incubated at 37 ℃ for 1 hour. Before cell lysis, the cells are acid washed to remove radioactivity (material) bound to the cell surface. (D) [ Dmt¹]Concentration of DALDA vs [ Dmt¹]Effect of DALDA uptake. Cells were incubated at 37 ℃ with a range of concentrations (1. mu.M-3 mM) [ Dmt ]¹]DALDA was incubated for 1 hour. All ofData for (d) are expressed as the mean of three independent monolayers ± s.e. Where error bars (errorbar) are not evident, they are smaller than the marker sets.

FIG. 2. intracellular [2 ] pH and DEPC vs. Caco-2³H][Dmt¹]DALDA (A and C) and [ solution ]¹⁴C]Effect of uptake of Gly-Sar (B and D). Reacting Caco-2 cells with [2 ] at 37 ℃ and different pH³H][Dmt¹]DALDA (250nM, 47Ci/mmol) or [2 ]¹⁴C]Gly-Sar (50. mu.M, 56.7mCi/mmol) was incubated for 1 hour (A and B). Contacting the cell with [ alpha ], [³H][Dmt¹]DALDA (250nM, 47Ci/mmol) or [2 ]¹⁴C]Cells were preincubated with 0.2mM DEPC for 10 min at 25 ℃ before 1 hour (C and D) incubation of Gly-Sar (50. mu.M, 56.7 mCi/mmol). All data are expressed as the mean of three independent monolayers ± s.e.

FIG. 3 (A) ([ alpha ]³H][Dmt¹]Uptake of DALDA in different cell lines. Contacting the cell with the polynucleotide at 37 ℃³H][Dmt¹]DALDA (250nM, 47Ci/mmol) was incubated for 1 hour. Prior to cell lysis, cells were acid washed to remove cell surface bound radioactivity. Data are shown representing acid-resistant radioactivity and are expressed as the mean of three independent monolayers ± s.e. (B) 2 [ alpha ]³H][Dmt¹]Specific binding of DALDA to cell membranes. A cell membrane prepared from SH-SY5Y cell and Caco-2 cell is contacted with the cell membrane at 25 ℃³H][Dmt¹]DALDA (15-960pM) was incubated for 1 hour. By 1. mu.M unlabeled [ Dmt ]¹]Inclusion of DALDA assessed non-specific binding. Free radioligand was separated from bound radioligand by rapid filtration. No specific binding to Caco-2 cells was seen. For SH-SY5Y cells, K_dThe value is 118pM (range 87-149), and B_maxThe value was 96fmol/mg protein.

FIG. 4 (A) ([ alpha ]³H][Dmt¹]DALDA (packed column) and [ solution ]¹⁴C]Leakage of Gly-Sar (open column). Used at 37 ℃ or 4 ℃³H][Dmt¹]DALDA (250nM, 47Ci/mmol) or [2 ]¹⁴C]Gly-Sar (50. mu.M, 56.7mCi/mmol) pre-loaded Caco-2 cells for 1 hour. Subsequently washing the cells with a culture mediumIncubate with medium at 37 ℃ or 4 ℃ for 1 hour. Radioactivity was measured in both the medium and the cell lysate, and the data is expressed as the percentage of peptide that leaked into the medium. (B) DEPC pair [ alpha ], [ alpha ] propylene³H][Dmt¹]The effect of DALDA leakage. Used in³H][Dmt¹]Cells were preincubated with 0.2mM DPC for 10 min at 25 ℃ before DALDA loading. (C) Isopacedine, a p-glycoprotein inhibitor, p- ([ alpha ], [ beta ] -a³H][Dmt¹]Effects of leakage (C) and uptake (D) of DALDA.

Figure 5³H][Dmt¹]DALDA and [2 ]¹⁴C]Gly-Sar transport across Caco-2 monolayers. Caco-2 cells (2X 10)⁵) Inoculating on microporous membrane in rotary hole cell culture container. By mixing³H][Dmt¹]DALDA or [2 ]¹⁴C]Gly-Sar was added to the top compartment to determine the transport of the peptide from the top to the bottom side, and 20- μ l aliquots were removed from the top and bottom side compartments at different times after addition of the peptide to determine radioactivity.

Fig. 6 [ Dmt¹，dnsDap⁴]DALDA and [ Dmt¹，atnDap⁴]Cellular uptake of DALDA. Caco-2 cells were incubated with 0.1. mu.M [ Dmt ] at 37 deg.C¹，dnsDap⁴]DALDA was incubated for 15 min. Cells were subsequently washed and covered with PBS. Microscopic observation was performed at room temperature over 10 minutes. Excitation was performed at 340nm and emission was measured at 520 nm. The fluorescence that appears spreads throughout the cytoplasm but is completely excluded from the nucleus. Absence of vesicle concentration at 37 ℃ indicates non-endocytic uptake.

FIG. 7. Mass spectrometric confirmation of binding of the three peptides to the crosslinker SMCC. SMCC (1. mu.g) and peptide (5. mu.g) were dissolved together in 2ml PBS, incubated at room temperature for 30 minutes, and then stored at 4 ℃. An aliquot of the sample was mixed with a matrix (3-hydroxypyridine carboxylic acid (HPA) saturated in 50% acetonitrile, 10mg/ml ammonium citrate) in a ratio of 1:10 and then spotted onto a stainless steel target plate. The samples were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS). The molecular weights of the peptides and their respective SMCC conjugates are shown on the mass spectrum.

FIG. 8. peptide increases uptake of beta-galactosidase (. beta. -Gal) into N₂A neuroblastoma cell capacity. Mixing cells (N)₂A neuroblastoma cells or Caco-2) were plated in 96-well plates (2X 10)⁴Individual cells/well) followed by incubation of the cells with β -galactosidase or with peptide-bound (via SMCC) β -galactosidase at 37 ℃ for 1 hour. The cells were then washed 4 times with phosphate buffer. Cells were then stained with a beta-galactosidase staining system (Roche) at 37 ℃ for at least 2 hours, and then examined under a microscope. (A) When Caco-2 cells were incubated with β -galactosidase, no β -galactosidase uptake was observed. (B) The presence of blue cells indicates intracellular association of [ Dmt ] with Caco-2¹]Uptake of DALDA conjugated β -galactosidase. (C) Caco-2 intracellular and [ D-arginine-Dmt-lysine-phenylalanine-NH₂]Increased uptake of bound β -galactosidase. (D) Intracellular reaction of Caco-2 with [ Phe¹]Uptake of DALDA-bound β -galactosidase was increased. Binding of β -galactosidase to SMCC alone did not increase uptake.

FIG. 9. and [ Dmt¹]Co-incubation of DALDA-SMCC conjugate conjugates increased the uptake of Green Fluorescent Protein (GFP) into Huh7 cells. Washing of Huh7 cells (1X 10) with DMEM⁶Individual cells/well) followed by incubation of the cells with 0.5ml of DMEM for 60 minutes at 37 ℃, the DMEM: contains only 3. mu.g GFP (A); containing 3. mu.g GFP and 40. mu.l [ Dmt ]¹]DALDA (B); or 3. mu.g GFP and 40. mu.l [ Dmt ] conjugated to SMCC¹]DALDA (C). Subsequently 2ml of cell culture medium was added to the cells and incubated in the cell incubator for an additional 24 hours. After incubation, cells were washed four times in cell culture medium, followed by observation of GFP remaining in viable cells by confocal laser scanning microscopy. Excitation was performed at 340nm and emission was measured at 520 nm. The top panel represents the image of GFP passing through the 0.8 μm thick central horizontal optic portion of Huh7 cells. The bottom panel represents a contrasting image of different interfaces within the same area.

Fig. 10.[ Dmt¹]Conjugation of DALDA to RNA oligomers. By using gamma-³²P-ATP and polynucleotide kinase phosphorylate synthetic RNA oligomers (40 nucleotides in length) at the 5' end. The product was purified by gel electrophoresis. In the presence of 1mg of EDC (N- [ 3-dimethylaminopropyl-N' -ethylcarbodiimide)]) 500,000cpm gel-purified RNA oligomer with [ Dmt ] when present¹]DALDA conjugated. The product of the conjugation reaction ([ Dmt ]¹]DALDA-RNA oligomer) was analyzed on a 15% polyacrylamide urea gel with RNA oligomers alone.

FIG. 11 [ Dmt ]¹]DALDA-[³²P]The conjugate of the RNA oligomer was taken up into Caco-2 cells. Caco-2 cells (1X 10)⁶) Three washes in DMEM medium and preincubation for 5 minutes in DMEM. Subsequently, the cells were incubated with [ Dmt ]¹]DALDA-[³²P]Conjugate of RNA oligomer or control RNA (approximately 20,000cpm) was incubated at 37 ℃ for 60 minutes. After incubation, cells were washed, lysed, and radioactivity in the cell lysate was determined. [ Dmt¹]DALDA-[³²P]Uptake of RNA conjugate conjugates is higher than uptake of RNA alone (>) Three times more (fig. 11).

FIG. 12 Effect of peptide-SMCC conjugate on increasing uptake of RNA oligomers into Huh7 cells. (A) Effect of time on cellular uptake of RNA oligomers. Washing of Huh7 cells (1X 10) with DMEM⁶Individual cells/well), followed by incubating the cells with 1.0ml of DMEM comprising the individual [ DMEM ] at 37 ℃ for 15 or 60 minutes³²P]An RNA oligomer (single strand, 11 bases, 100,000cpm), or comprises [ alpha ], [ beta ], [ alpha³²P]RNA oligomer and 40. mu.l [ Dmt ]¹]DALDA-SMCC conjugate. The cells were then washed four times in DMEM and once in sodium acetate solution to remove non-specific binding before incubating them in lysis buffer for 30 minutes, and then the retained radioactivity was determined. RNA oligomer and [ Dmt¹]The uptake of RNA oligomers was increased by co-incubation of DALDA-SMCC at 37 ℃, 10-fold after 15 min incubation and 20-fold after 60 min incubation. (B) Effect of temperature on uptake of RNA oligomers by cells. At 4 deg.C [ Dmt¹]DALDA-SMCC is carriedThe ability for high RNA uptake is less, although it will still increase uptake by 10-fold. (C) RNA uptake by cells was increased by different peptide-SMCC conjugate conjugates. Washing of Huh7 cells (1X 10) with DMEM⁶Individual cells/well), followed by incubating the cells with 1.0ml of DMEM comprising the individual [ DMEM ] at 37 ℃ for 15 minutes³²P]An RNA oligomer, or a nucleic acid comprising³²P]RNA oligomer and 40ml peptide-SMCC conjugate. All three peptide-SMCC conjugate conjugates increased RNA uptake.

FIG. 13. and [ Dmt¹]Co-incubation of DALDA-SMCC conjugate increased uptake of two different lengths of RNA. Will [ Dmt¹]DALDA conjugated to SMCC and confirmed by mass spectrometry. Reacting an 11-mer RNA oligomer and a 1350-mer RNA with [ Dmt ] at room temperature¹]The DALDA-SMCC conjugate was mixed for 15 minutes. Washing of Huh7 cells (1X 10) with DMEM⁶Individual cells/well) followed by 5% CO at 37 ℃₂The cells were then incubated with 1ml of DMEM containing RNA alone (-100,000 cpm), or [ Dmt ] for 60 minutes¹]DALDA-SMCC conjugate mixed RNA. The cells were then washed four times in DMEM and once in sodium acetate solution to remove non-specific binding. The washed cells were incubated in lysis buffer for 30 minutes, followed by counting the radioactivity retained. Compared to incubation with RNA alone, [ Dmt¹]Co-incubation of the DALDA-SMCC conjugate increased uptake of the 11-mer RNA by 22-fold and increased uptake of the 1350-mer RNA by 3-fold.

FIG. 14 DNA oligomer with [ Dmt¹]Conjugation of DALDA. SMCC (1. mu.g) and [ Dmt ]¹]DALDA (5. mu.g) was dissolved together in 2ml PBS, incubated at room temperature for 30 minutes, and then mixed with the deprotected 3' -thiol DNA oligomer at 4 ℃ for 24 hours. After incubation, an aliquot of the sample was mixed with a matrix (3-hydroxypyridine carboxylic acid (HPA) saturated in 50% acetonitrile, 10mg/ml ammonium citrate) in a ratio of 1:10 and spotted onto a stainless steel target plate. The sample (A) was analyzed by MALDI-TOF MS. 3' -thiol group DNA oligomer and [ Dmt ] have been found¹]Of DALDA-DNA covalent complexesMolecular weights were 6392 and 7171, respectively. Use of gamma-³²P-ATP phosphorylates conjugated and unconjugated oligomers at the 5' -end, and the products of the kinase reaction were analyzed on a 15% polyacrylamide urea gel and gel-purified for cellular uptake studies (B).

FIG. 15. and [ Dmt¹]Cellular uptake of DALDA conjugated DNA oligomers. Conjugation of 3' -thiol-modified 20-matrix DNA to [ Dmt ] Using SMCC¹]DALDA, and confirmation of conjugate formation by mass spectrometry. The conjugated and unconjugated DNA oligomers were labeled with a radioisotope at their 5' -ends with 32P and gel purified. Flushing of nerves N with DMEM₂A(1×10⁶Individual cells/well) cells, then at 37 ℃ and 5% CO₂The cells were incubated for 2 hours or 19 hours with 1ml of DMEM with or without [ Dmt ] conjugated to DNA oligomers (-100,000 cpm)¹]DALDA. The cells were then washed four times in DMEM and once in sodium acetate solution to remove non-specific binding. The cells were then incubated in lysis buffer for 30 minutes, and the retained radioactivity was then determined. The Y-axis represents the uptake of DNA as a percentage of the total radioactivity.

Fig. 16 [ Dmt¹]DALDA was not toxic to the cultured cells. Let nerve N₂A cells and [ Dmt¹]DALDA (1nM to 10 μ M) was incubated and the viability of the cells was determined by MTT assay.

Fig. 17 [ Dmt¹]The DALDA-SMCC conjugate did not cause apoptosis in Huh7 cells. Huh7 cells (1X 10)⁶Individual cells/well) were washed three times in DMEM and 1ml of fresh medium was added. Subsequently, 50. mu.l of PBS [ Dmt [ ] was added¹]DALDA-SMCC conjugate (1mM) or PBS only (control) was added to the cell culture medium and incubated at 37 ℃ and 5% CO₂And incubated for 24 hours. After incubation, 1 μ l of Hoechst dye that can stain apoptotic nuclei was added to the cells and incubated for an additional 15 minutes. By usingCell culture medium (without pH indicator) washing cells removed excess Hoechst dye, and then compared using a fluorescence microscope (excitation at 350nm, emission measured at 461 nm) via [ Dmt [)¹]DALDA-SMCC conjugate treated cells and control cells.

Detailed Description

The present invention is based on the surprising discovery by the inventors that: some carrier complexes comprising at least one molecule and an aromatic cationic peptide are capable of crossing the cell membrane in an energy independent mechanism and delivering the molecule into the cell.

Aromatic cationic peptides

The aromatic-cationic peptides used in the present invention have a net positive charge as described below, are water-soluble and highly polar. The peptide comprises a minimum of three amino acids, preferably a minimum of four amino acids, covalently linked by peptide bonds.

The maximum number of amino acids present in the aromatic-cationic peptide is ten, preferably about eight, most preferably about six. Most preferably, the number of amino acids present in the peptide is about four. The term "about" as used to define the maximum number of amino acids means either increasing or decreasing by one amino acid.

The amino acid of the aromatic-cationic peptide used in the present invention may be any amino acid. As used herein, the term "amino acid" refers to any organic molecule that includes at least one amino group and at least one carboxyl group. Preferably, at least one amino group is located in the alpha position relative to the carboxyl group.

The amino acid may be naturally occurring. Naturally occurring amino acids include, for example, the twenty most common amino acids that are typically present in proteins, i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).

Other naturally occurring amino acids include, for example, amino acids that are synthesized in metabolic processes unrelated to protein synthesis. For example, mammals metabolize synthetic amino acids during the process of producing urine: ornithine.

The aromatic-cationic peptides used in the present invention optionally comprise one or more non-naturally occurring amino acids. In a particular embodiment, the peptide has no naturally occurring amino acids.

Non-naturally occurring amino acids are amino acids that are not normally synthesized in the normal metabolic processes of the organism and do not occur naturally in proteins.

Furthermore, the non-naturally occurring amino acids used in the present invention are preferably not recognized by common proteases. Thus, non-naturally occurring amino acids are preferably resistant to common proteases, and more preferably are not sensitive to common proteases.

Non-naturally occurring amino acids can be present at any position of the peptide. For example, the non-naturally occurring amino acid can be at the N-terminus, C-terminus, and/or any one or more positions between the N-terminus and C-terminus.

For example, a non-naturally occurring amino acid can include an alkyl group, an aryl group, or an alkaryl group. Some examples of alkyl amino acids include: alpha-aminobutyric acid, beta-aminobutyric acid, gamma-aminobutyric acid, delta-aminopentanoic acid, and epsilon-aminocaproic acid. Some examples of aryl amino acids include ortho-, meta-, and para-aminobenzoic acid. Some examples of alkylaryl amino acids include o-, m-, and p-aminophenylacetic acid, and gamma-phenyl-beta-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives of naturally occurring amino acids. Derivatives of naturally occurring amino acids can include, for example, the addition of one or more chemical groups to the naturally occurring amino acid.

For example, one or more chemical groups may be added to one or more of the 2 ', 3 ', 4 ', 5 ', or 6 ' positions of the aromatic ring of a phenylalanine or tyrosine residue, or one or more of the 4 ', 5 ', 6 ', or 7 ' positions of the benzo ring of a tryptophan residue. The group can be any chemical group that can be added to an aromatic ring. Some examples of such groups include: branched or unbranched C₁-C₄Alkyl radicals, e.g. methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or tert-butyl, C₁-C₄Hydrocarbyloxy (i.e. alkylene oxide), amino, C₁-C₄Alkylamines (e.g. methylamino) and C₁-C₄Dialkylamines (e.g., dimethylamino), nitro, hydroxy, halo (i.e., fluoro, chloro, bromo, or iodo). Some specific examples of non-naturally occurring derivatives of naturally occurring amino acids include: norvaline (Nva), norleucine (Nle), and hydroxyproline (Hyp).

Another example of modification of amino acids in the peptide used in the present invention is derivatization (derivatization) of the carboxyl group of an aspartic acid or glutamic acid residue in the peptide. One example of derivatization is amination with ammonia or with primary or secondary amines, for example methylamine, ethylamine, dimethylamine or diethylamine. Another example of derivatization includes esterification using, for example, methanol or ethanol.

Another such modification includes modification of the amino group of a lysine, arginine or histidine residue. For example, such an amino group may be acylated. Some suitable acyl groups include, for example, any of the above C₁-C₄Benzoyl or alkanoyl of alkyl groups, such as acetyl or propionyl.

Non-naturally occurring amino acids can generally be of the L-form (L-), the D-form (D-), or mixtures thereof. Examples of suitable non-naturally occurring amino acids also include dextrorotatory (D-) forms of any of the naturally occurring L-amino acids described above, as well as L-and/or D-non-naturally occurring amino acids. In this regard, it should be noted that: although D-amino acids have been found to be present in certain peptide antibiotics that are synthesized using means other than the normal ribosomal protein synthesizer of the cell, they are not normally present in proteins. As used herein, such D-amino acids refer to non-naturally occurring amino acids.

To minimize sensitivity to proteases, the peptides used in the present invention should have less than five, preferably less than four, more preferably less than three, and most preferably less than two adjacent L-amino acids recognized by common proteases, whether naturally occurring or non-naturally occurring. In one embodiment, the peptide has only D-amino acids, and no L-amino acids.

If the peptide comprises an amino acid sequence that is sensitive to a protease, then at least one of the amino acids is preferably a non-naturally occurring D-amino acid, thereby rendering it protease resistant. Examples of protease-sensitive sequences include two or more adjacent basic amino acids that can be cleaved by common proteases, such as endopeptidases and trypsin. Examples of basic amino acids include arginine, lysine and histidine.

It is important that the peptide has a minimum number of net positive charges at physiological pH compared to the total number of amino acid residues in the aromatic-cationic peptide. The minimum number of net positive charges at physiological pH is hereinafter denoted as (p)_m). The total number of amino acid residues in the peptide is denoted as (r).

The minimum number of net positive charges described below are all at physiological pH. The term "physiological pH" as used herein refers to the normal pH within the cells of the tissues and organs of the mammalian body. For example, the physiological pH of humans is normally close to 7.4, but the normal physiological pH of mammals can be any pH from about 7.0 to about 7.8.

As used herein, "net charge" refers to the difference between the number of positive charges and the number of negative charges carried by the amino acids present in the peptide. In the present specification, it is understood that the net charge is measured at physiological pH. Naturally occurring amino acids that have a positive charge at physiological pH include L-lysine, L-arginine, and L-histidine. Naturally occurring amino acids having a negative charge at physiological pH include L-aspartic acid and L-glutamic acid.

Typically, peptides have a positively charged N-terminal amino group and a negatively charged C-terminal carboxyl group. At physiological pH the charges cancel each other out. As an example of calculating the net charge, peptide: tyrosine-arginine-phenylalanine-lysine-glutamic acid-histidine-tryptophan-arginine (Tyr-Arg-Phe-Lys-Glu-His-Trp-Arg) has one negatively charged amino acid (i.e., glutamic acid) and four positively charged amino acids (i.e., two arginine residues, one lysine, and one histidine). Thus, the above peptides have three net positive charges.

In one embodiment of the invention, the aromatic-cationic peptide has a minimum number of net positive charges (p) at physiological pH_m) The relationship with the total number of amino acid residues (r) is: wherein 3p_mIs the maximum number less than or equal to r + 1. In this particular embodiment, the minimum number of net positive charges (p)_m) The relationship with the total number of amino acid residues (r) is as follows:

(r)	3	4	5	6	7	8	9	10
									(p)	1	1	2	2	2	3	3	3

in another embodiment, the aromatic-cationic peptide has a minimum number of net positive charges (p)_m) The relationship with the total number of amino acid residues (r) is: wherein 2p is_mIs the maximum number less than or equal to r + 1. In this particular embodiment, the minimum number of net positive charges (p)_m) The relationship with the total number of amino acid residues (r) is as follows:

(r)	3	4	5	6	7	8	9	10
									(p)	2	2	3	3	4	4	5	5

in one embodiment, the number of net positive charges (p)_m) Is equal to the number of amino acid residues (r). In another preferred embodiment, the peptide hasThere are three or four amino acid residues and at least one net positive charge, preferably at least two net positive charges, and more preferably at least three net positive charges.

Aromatic cationic peptides have a total number of positive charges (p) compared to the net_t) This is also important. The minimum number of aromatic groups is hereinafter denoted as (a).

Naturally occurring amino acids having an aromatic group include these amino acids: histidine, tryptophan, tyrosine, and phenylalanine. For example, hexapeptides: lysine-glutamine-tyrosine-arginine-phenylalanine-tryptophan have two net positive charges (provided by lysine and arginine residues) and three aromatic groups (provided by tyrosine, phenylalanine, and tryptophan residues).

In one embodiment of the invention, the aromatic-cationic peptides used in the methods of the invention have a minimum number of aromatic groups (a) and a total number of net positive charges at physiological pH (p)_t) The relationship between them is: wherein 3a is less than or equal to p_tMaximum of +1, except when p_tWhen the number is 1, a may be 1. In this embodiment, the minimum number of aromatic groups (a) and the total number of net positive charges (p)_t) The relationship between them is as follows:

(p)	1	2	3	4	5	6	7	8	9	10
											(a)	1	1	1	1	2	2	2	3	3	3

in another embodiment, the aromatic-cationic peptide has a minimum number of aromatic groups (a) and a total number of net positive charges (p)_t) The relationship between them is: wherein 2a is less than or equal to p_tMaximum of + 1. In this embodiment, the minimum number of aromatic amino acid residues (a) and the total number of net positive charges (p)_t) The relationship between them is as follows:

(p)	1	2	3	4	5	6	7	8	9	10
											(a)	1	1	2	2	3	3	4	4	5	5

in another embodiment, the number of aromatic groups (a) and the total number of net positive charges (p)_t) Are equal.

The carboxyl group, especially the terminal carboxyl group of the C-terminal amino acid, is preferably amidated with, for example, ammonia to form a C-terminal amide. Alternatively, the terminal carboxyl group of the C-terminal amino acid may be amidated with a primary or secondary amine. The primary or secondary amine may be, for example, an alkyl group, especially a branched or straight chain C₁-C₄Alkyl, or arylamine. Accordingly, the amino acid at the C-terminal end of the peptide may be converted into an amide group, an N-methylamide group, an N-ethylamide group, an N, N-dimethylamide group, an N, N-diethylamide group, an N-methyl-N-ethylamide group, an N-phenylamide group or an N-phenyl-N-ethylamide group.

In addition, the free carboxyl group of an amino acid residue having more than one carboxyl group (e.g., asparagine residue, glutamine residue, aspartic acid residue, and glutamic acid residue), whether present at any position, may also be amidated. Amidation at these sites can be carried out using ammonia or any of the primary or secondary amines described above.

In a particular embodiment, the aromatic-cationic peptide used in the method of the invention is a tripeptide having two net positive charges and at least one aromatic amino acid. In a specific embodiment, the aromatic-cationic peptide used in the method of the invention is a tripeptide having two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides used in the methods of the invention include, but are not limited to, the following examples of peptides:

lysine-dextro arginine-tyrosine-NH₂，

Phenylalanine-dextro-arginine-histidine,

right handed tyrosineacid-Tryptophan-lysine-NH₂，

Tryptophan-D-lysine-tyrosine-arginine-NH₂，

Tyrosine-histidine-d-glycine-methionine,

phenylalanine-arginine-dextro-histidine-aspartic acid,

tyrosine-dextro arginine-phenylalanine-lysine-glutamic acid-NH₂，

Methionine-tyrosine-dextrolysine-phenylalanine-arginine,

dextro histidine-glutamic acid-lysine-tyrosine-dextro phenylalanine-arginine,

lysine-D-glutamine-tyrosine-arginine-D-phenylalanine-tryptophan-NH₂，

Phenylalanine-dextro arginine-lysine-tryptophan-tyrosine-dextro arginine-histidine,

glycine-dextro phenylalanine-lysine-tyrosine-histidine-dextro arginine-tyrosine-NH₂，

valine-D-lysine-histidine-tyrosine-D-phenylalanine-serine-tyrosine-arginine-NH₂，

Tryptophan-lysine-phenylalanine-dextro aspartic acid-arginine-tyrosine-dextro histidine-lysine,

lysine-tryptophan-dextro tyrosine-arginine-asparagine-phenylalanine-tyrosine-dextro histidine-NH₂，

Threonine-glycine-tyrosine-arginine-dextro-histidine-phenylalanine-tryptophan-dextro-histidine-lysine,

aspartic acid-D-tryptophan-lysine-tyrosine-D-histidine-phenylalanine-arginine-D-glycine-lysineAmino acid-NH₂，

D-histidine-lysine-tyrosine-D-phenylalanine-glutamic acid-D-aspartic acid-D-histidine-D-lysine-arginine-tryptophan-NH₂And an

Alanine-dextro phenylalanine-dextro arginine-tyrosine-lysine-dextro tryptophan-histidine-dextro tyrosine-glycine-phenylalanine.

In a particularly preferred embodiment, the aromatic-cationic peptide has the formula: tyrosine-dextro arginine-phenylalanine-lysine-NH₂(this abbreviation is denoted by DALDA for convenience). DALDA has a structure consisting of amino acids: three net positive charges provided by tyrosine, arginine, and lysine, and the three positive charges provided by the amino acids: phenylalanine and tyrosine. The tyrosine in DALDA may be a modified derivative of tyrosine, such as 2 ', 6' -dimethyltyrosine, to produce a compound of the formula 2 '6' -Dmt-D-Arg-Phe-Lys-NH₂(i.e., Dmt)¹-DALDA). Other modified tyrosine derivatives include 2' -methyl tyrosine (Mmt); n, 2 ', 6' -trimethyltyrosine (Tmt); and 2 '-hydroxy-6' -methyl tyrosine (Hmt).

In another preferred embodiment, the amino acid at the N-terminus of DALDA may be phenylalanine or a derivative thereof. Aromatic cationic peptide having phenylalanine at the N-terminus of formula: phenylalanine-dextro-arginine-phenylalanine-lysine-NH₂(i.e., Phe)¹-DALDA). Preferred derivatives of phenylalanine include: 2 ' -Methylphenylalanine (Mmp), 2 ', 6 ' -dimethylphenylalanine (Dmp), N, 2 ', 6 ' -trimethylphenylalanine (Tmp), and 2 ' -hydroxy-6 ' -methylphenylalanine (Hmp).

In another embodiment, Dmt¹The amino acid sequence of DALDA was rearranged so that Dmt was not located at the N-terminus. Examples of such aromatic-cationic peptides have the formula: d-arginine-2 '6' Dmt-lysineAmino acid-phenylalanine-NH₂。

Any particular peptides mentioned herein, such as those mentioned above and those mentioned below, for example, the peptides mentioned in table 1, including Dmt¹-DALDA、DALDA、Phe¹-DALDA, d-arginine-2 '6' Dmt-lysine-phenylalanine-NH₂And their derivatives may further include functional analogs. If the analogue is reacted with Dmt¹-DALDA、DALDA、Phe¹-DALDA, or d-arginine-2 '6' Dmt-lysine-phenylalanine-NH₂Having the same function, such a peptide is considered to be Dmt¹-DALDA、DALDA、Phe¹-DALDA, or d-arginine-2 '6' Dmt-lysine-phenylalanine-NH₂A functional analog of (a). The analog can be, for example, Dmt¹-DALDA、DALDA、Phe¹-DALDA, or d-arginine-2 '6' Dmt-lysine-phenylalanine-NH₂The substituted variant of (1), wherein one or more amino acids are substituted with another amino acid.

Dmt¹-DALDA、DALDA、Phe¹-DALDA, or d-arginine-2 '6' Dmt-lysine-phenylalanine-NH₂Suitable substitution variants of (a) include conservative amino acid substitutions. Amino acids can be grouped according to their physicochemical properties as follows:

(a) non-polar amino acids: alanine (a) serine (S) threonine (T) proline (P) glycine (G);

(b) acidic amino acids: asparagine (N) aspartic acid (D) glutamic acid (E) glutamine (Q);

(c) basic amino acids: histidine (H) arginine (R) lysine (K);

(d) hydrophobic amino acids: methionine (M) leucine (L) isoleucine (I) valine (V); and

(e) aromatic amino acids: phenylalanine (F) tyrosine (Y) tryptophan (W) histidine (H).

Substitutions (replacements) of amino acids in peptides by another amino acid of the same group are referred to as conservative substitutions. Conservative substitutions help to preserve the physicochemical properties of the original peptide. Conversely, substitution of an amino acid in a peptide with a different set of additional amino acids is generally more likely to alter the physicochemical properties of the original peptide.

Examples of analogs useful in the practice of the present invention include, but are not limited to, the aromatic-cationic peptides shown in tables 1 and 2.

TABLE 1

Amino acid C-terminal

Position 1 position 2 position 3 position 4 position 5 (if present) modification

Tyrosine dextral arginine phenylalanine lysine NH₂

Tyrosine dextral arginine phenylalanine ornithine NH₂

Tyrosine dextral arginine phenylalanine Dab NH₂

Tyrosine dextral arginine phenylalanine Dap NH₂

2 '6' Dmt D-arginine phenylalanine lysine NH₂

2 '6' Dmt D-arginine phenylalanine lysine cysteine NH₂

2 '6' Dmt D-arginine phenylalanine lysine-NH (CH)₂)₂-NH-dns NH₂

2 '6' Dmt D-arginine phenylalanine lysine-NH (CH)₂)₂-NH-atn NH₂

2 '6' Dmt D-arginine phenylalanine dns lysine NH₂

2 '6' Dmt D-citrulline phenylalanine lysine NH₂

2 '6' Dmt D-citrulline phenylalanine Ahp NH₂

2 '6' Dmt D-arginine phenylalanine ornithine NH₂

2 '6' Dmt D-arginine phenylalanine Dab NH₂

2 '6' Dmt D-arginine phenylalanine Dap NH₂

2 '6' Dmt D-arginine phenylalanine Ahp (2-aminoheptanoic acid) NH₂

Bio-2 '6' Dmt D-arginine phenylalanine lysine NH₂

3 '5' Dmt D-arginine phenylalanine lysine NH₂

3 '5' Dmt D-arginine phenylalanine ornithine NH₂

3 '5' Dmt D-arginine phenylalanine Dab NH₂

3 '5' Dmt D-arginine phenylalanine Dap NH₂

Tyrosine dextro arginine tyrosine lysine NH₂

Tyrosine dextro arginine tyrosine ornithine NH₂

Tyrosine dextral arginine tyrosine Dab NH₂

Tyrosine dextro-arginine tyrosine Dap NH₂

2 '6' Dmt D-arginine tyrosine lysine NH₂

2 '6' Dmt D-arginine tyrosine ornithine NH₂

2 '6' Dmt D-arginine tyrosine Dab NH₂

2 '6' Dmt D-arginine tyrosine Dap NH₂

2 '6' Dmt D-arginine 2 '6' Dmt lysine NH₂

2 '6' Dmt D-arginine 2 '6' Dmt ornithine NH₂

2 '6' Dmt D-arginine 2 '6' Dmt Dab NH₂

2 '6' Dmt D-arginine 2 '6' Dmt Dap NH₂

3 '5' Dmt D-arginine 3 '5' Dmt arginine NH₂

3 '5' Dmt D arginine 3 '5' Dmt lysine NH₂

3 '5' Dmt D-arginine 3 '5' Dmt ornithine NH₂

3 '5' Dmt D-arginine 3 '5' Dmt Dab NH₂

Tyrosine D-lysine phenylalanine Dap NH₂

Tyrosine dextral lysine phenylalanine arginine NH₂

Tyrosine D-lysine phenylalanineLysine NH₂

Tyrosine D-lysine phenylalanine ornithine NH₂

2 '6' Dmt D-lysine phenylalanine Dab NH₂

2 '6' Dmt D-lysine phenylalanine Dap NH₂

2 '6' Dmt D-lysine phenylalanine arginine NH₂

2 '6' Dmt D-lysine phenylalanine lysine NH₂

3 '5' Dmt D-lysine phenylalanine ornithine NH₂

3 '5' Dmt D-lysine phenylalanine Dab NH₂

3 '5' Dmt D-lysine phenylalanine Dap NH₂

3 '5' Dmt D-lysine phenylalanine arginine NH₂

Tyrosine dextro-lysine tyrosine lysine NH₂

Tyrosine dextro-lysine tyrosine ornithine NH₂

Tyrosine dextro-lysine tyrosine Dab NH₂

Tyrosine dextro-lysine tyrosine Dap NH₂

2 '6' Dmt D-lysine tyrosine lysine NH₂

2 '6' Dmt D-lysine tyrosine ornithine NH₂

2 '6' Dmt D-lysine tyrosine Dab NH₂

2 '6' Dmt D-lysine tyrosine Dap NH₂

2 '6' Dmt D lysine 2 '6' Dmt lysine NH₂

2 '6' Dmt D-lysine 2 '6' Dmt Ornithine NH₂

2 '6' Dmt D-lysine 2 '6' Dmt Dab NH₂

2 '6' Dmt D-lysine 2 '6' Dmt Dap NH₂

2 '6' Dmt D-arginine phenylalanine dnsDap NH₂

2 '6' Dmt D-arginine phenylpropylAmino acid atnDap NH₂

3 '5' Dmt D lysine 3 '5' Dmt lysine NH₂

3 '5' Dmt D-lysine 3 '5' Dmt Ornithine NH₂

3 '5' Dmt D-lysine 3 '5' Dmt Dab NH₂

3 '5' Dmt D-lysine 3 '5' Dmt Dap NH₂

Tyrosine dextral lysine phenylalanine arginine NH₂

Tyrosine D-ornithine phenylalanine arginine NH₂

Tyrosine dextro Dab phenylalanine arginine NH₂

Tyrosine dextro Dap phenylalanine arginine NH₂

2 '6' Dmt D-arginine phenylalanine arginine NH₂

2 '6' Dmt D-lysine phenylalanine arginine NH₂

2 '6' Dmt D-Ornithine Phe Arg NH₂

2 '6' Dmt D-Dab Phe arginine NH₂

3 '5' Dmt D-Dap phenylalanine arginine NH₂

3 '5' Dmt D-arginine phenylalanine arginine NH₂

3 '5' Dmt D-lysine phenylalanine arginine NH₂

3 '5' Dmt D-Ornithine Phe Arg NH₂

Tyrosine dextro lysine tyrosine arginine NH₂

Tyrosine D-ornithine arginine NH₂

Tyrosine dextro Dab tyrosine arginine NH₂

Tyrosine dextro Dap tyrosine arginine NH₂

2 '6' Dmt D-arginine 2 '6' Dmt arginine NH₂

2 '6' Dmt D lysine 2 '6' Dmt arginine NH₂

2 '6' Dmt D-ornithine2 '6' Dmt arginine NH₂

2 '6' Dmt dextro Dab 2 '6' Dmt arginine NH₂

3 '5' Dmt dextro Dap 3 '5' Dmt arginine NH₂

3 '5' Dmt D-arginine 3 '5' Dmt arginine NH₂

3 '5' Dmt D lysine 3 '5' Dmt arginine NH₂

3 '5' Dmt D-Ornithine 3 '5' Dmt arginine NH₂

Mmt D-arginine phenylalanine lysine NH₂

Mmt D-arginine phenylalanine ornithine NH₂

Mmt D-arginine phenylalanine Dab NH₂

Mmt D-arginine phenylalanine Dap NH₂

Tmt D-arginine phenylalanine lysine NH₂

Tmt D-arginine phenylalanine ornithine NH₂

Tmt right handArginine phenylalanine Dab NH₂

Tmt D-arginine phenylalanine Dap NH₂

Hmt D-arginine phenylalanine lysine NH₂

Hmt D-arginine phenylalanine ornithine NH₂

Hmt D-arginine phenylalanine Dab NH₂

Hmt D-arginine phenylalanine Dap NH₂

Mmt D-lysine phenylalanine lysine NH₂

Mmt D-lysine phenylalanine ornithine NH₂

Mmt D-lysine phenylalanine Dab NH₂

Mmt D-lysine phenylalanine Dap NH₂

Mmt D-lysine phenylalanine arginine NH₂

Tmt D-lysine phenylalanine lysine NH₂

Tmt D-lysine phenylalanine ornithine NH₂

Tmt D-lysine phenylalanine Dab NH₂

Tmt D-lysine phenylalanine Dap NH₂

Tmt D-lysine phenylalanine arginine NH₂

Hmt D-lysine phenylalanine lysine NH₂

Hmt D-lysine phenylalanine ornithine NH₂

Hmt D-lysine phenylalanine Dab NH₂

Hmt D-lysine phenylalanine Dap NH₂

Hmt D-lysine phenylalanine arginine NH₂

Mmt D-lysine phenylalanine arginine NH₂

Mmt D-Ornithine Phe arginine NH₂

Mmt D-Dab phenylalanine arginine NH₂

Mmt D-Dap phenylalanine arginine NH₂

Mmt D-arginine phenylalanine arginine NH₂

Tmt D-lysine phenylalanine arginine NH₂

Tmt D-Ornithine phenylalanine arginine NH₂

Tmt D-Dab phenylalanine arginine NH₂

Tmt D-Dap phenylalanine arginine NH₂

Tmt D-arginine phenylalanine arginine NH₂

Hmt D-lysine phenylalanine arginine NH₂

Hmt D-Ornithine phenylalanine arginine NH₂

Hmt dextro Dab phenylalanine arginine NH₂

Hmt dextro Dap phenylalanine arginine NH₂

Hmt D-arginine phenylalanine arginine NH₂

Dab ═ diaminobutyric acid

Dap ═ diaminopropionic acid

Dmt ═ dimethyl tyrosine

Mmt ═ 2' -methyl tyrosine

Tmt ═ N, 2 ', 6' -trimethyltyrosine

Hmt-2 '-hydroxy, 6' -methyl tyrosine

dnsDap ═ beta-dansyl-L-alpha, beta-diaminopropionic acid

atnDap ═ beta-anthranoyll-alpha, beta-diaminopropionic acid

Biotin (Bio ═ biotin)

TABLE 2

Amino acid C-terminus

Position 1 position 2 position 3 position 4 modification

D-arginine Dmt lysine phenylalanine NH2

D-arginine Dmt phenylalanine lysine NH₂

D-arginine phenylalanine lysine Dmt NH₂

D-arginine phenylalanine Dmt lysine NH₂

D-arginine lysine Dmt phenylalanine NH₂

D-arginine lysine phenylalanine Dmt NH₂

Phenylalanine lysineDmt D-arginine NH₂

Phenylalanine lysine dextro arginine Dmt NH₂

Phenylalanine dextro-arginine Dmt lysine NH₂

Phenylalanine dextro-arginine lysine Dmt NH₂

Phenylalanine dextro-arginine phenylalanine lysine NH₂

Phenylalanine Dmt dextro-arginine lysine NH₂

Phenylalanine Dmt lysine dextro arginine NH₂

Lysine phenylalanine dextro-arginine Dmt NH₂

Lysine phenylalanine Dmt dextro arginine NH₂

Lysine Dmt D arginine phenylalanine NH₂

Lysine Dmt phenylalanine dextro arginine NH₂

Lysine dextro arginine phenylalanine Dmt NH₂

Lysine dextro arginine Dmt phenylalanine NH₂

D-arginine Dmt-D-arginine phenylalanine NH₂

D-arginine Dmt-D-arginine Dmt NH₂

D-arginine Dmt-D-arginine tyrosine NH₂

D-arginine Dmt-D-arginine tryptophan NH₂

Tryptophan-D-arginine-phenylalanine-lysine NH₂

Tryptophan-D-arginine-tyrosine-lysine NH₂

Tryptophan D-arginine Tryptophan lysine NH₂

Tryptophan-D-arginine-Dmt-lysine-NH₂

D-arginine tryptophan lysine phenylalanine NH₂

D-arginine tryptophan phenylalanine lysine NH₂

D-arginine tryptophan lysine Dmt NH₂

D-arginine tryptophan Dmt lysine NH₂

D-arginine lysine tryptophan phenylalanine NH₂

D-arginine lysine tryptophan Dmt NH₂

Cha D-arginine phenylalanine lysine NH₂

Alanine D-arginine phenylalanine lysine NH₂

Cha ═ cyclohexyl

The amino acids in the peptides shown in tables 1 and 2 may be in the L-configuration or the D-configuration.

More cationic peptides can be found in U.S. provisional application No. 60/444,777 filed on 4/2/2003, which is incorporated herein by reference.

Molecule

The molecule may be a biomolecule or a small molecule. Preferably, the biomolecule or small molecule is a molecule having pharmaceutical activity. As used herein, a molecule having pharmaceutical activity is any molecule capable of exerting a beneficial effect in vivo.

A biomolecule is any molecule comprising a nucleic acid or amino acid sequence and having a molecular weight greater than 450. Such nucleic acid and amino acid sequences are referred to herein as "poly (poly) nucleotides" and "poly (poly) amino acids", respectively.

Biomolecules include polynucleotides, peptide nucleotides, and polyamines, such as peptides, polypeptides, and proteins. Examples of biomolecules having pharmaceutical activity include: endogenous peptides (e.g., vasopressin, glutathione), proteins (e.g., interferon), hormones (e.g., human growth hormone), enzymes (e.g., alpha-galactosidase), antibodies (e.g., anti-beta-amyloid antibodies, which can be used to treat alzheimer's disease), nerve growth factors (e.g., nerve growth factor NGF, brain derived nerve factor BDNF), cytokines (e.g., platelet derived growth factor PDGF, vascular endothelial growth factor VEGF), and oligonucleotides.

The oligonucleotide may comprise a polynucleotide of any sequence, such as DNA or RNA. The DNA and RNA sequences may be single-stranded or double-stranded. For example, DNA encoding proteins that facilitate cell survival during stress may be combined with the polypeptides of the invention. Examples of such proteins include heat shock proteins (e.g., hsp60, hsp70, etc.).

Examples of single stranded RNA molecules include: ribozymes, RNA decoys (decoys), external guide sequences for ribozymes, antisense RNA, and mRNA. For a review of these single-stranded RNA molecules, see Sullenger et al (Nature 2002, 418: 252-247). The descriptions of these single-stranded DNA molecules, as well as the descriptions of diseases and disorders that can be treated with ribozymes, RNA decoys, external guide sequences for ribozymes, antisense RNA and mRNA molecules, disclosed by sulllenger are incorporated herein by reference.

An example of a double-stranded RNA is an RNA interfering molecule (i.e., an RNAi, e.g., an siRNA, (i.e., a small interfering RNA)). The siRNA may be any known to those skilled in the art.

The siRNA can, for example, be sufficiently complementary to mRNA to inhibit transcription of a protein associated with a disease, disorder, or condition. Examples of such proteins include, for example, beta-amyloid associated with alzheimer's disease and ras protein associated with cancer.

Alternatively, the siRNA may, for example, be sufficiently complementary to RNA produced by a virus. The RNA produced by the virus may be any RNA that is generally required for viral infection of the host cell, survival of the virus, and/or propagation of the virus. Examples of such RNAs include: internal ribosome entry sites, RNA-dependent polymerase initiation sites, and (RNA's) encoding viral envelope proteins, viral nucleases, and viral proteases.

Examples of viruses include, for example, hepatitis viruses such as hepatitis A (A), B (B), C (C), human immunodeficiency virus, EB virus, cytomegalovirus, and human papilloma virus.

Sirnas against viral RNA are known to those skilled in the art. For example, sirnas against hepatitis C virus RNA are known to those skilled in the art, see Randall et al, PNAS, 2003, 100: 235-240.

The molecule may be a small molecule. The small molecules include: organic compounds, organometallic compounds, salts of organic compounds and organometallic compounds, monosaccharides, amino acids, and nucleotides. Small molecules may further include molecules that are not considered biomolecules as long as their molecular weight is no greater than 450. Thus, the small molecule can be a lipid, oligosaccharide, oligopeptide, and oligonucleotide, and derivatives thereof, having a molecular weight of 450 or less.

It is emphasized that the small molecules may have any molecular weight. They are called small molecules simply because they are not called biomolecules and generally have a molecular weight of less than 450. Small molecules include compounds found in nature, as well as synthetic compounds. Examples of small molecules with pharmaceutical activity include: antibiotics (e.g., tetracycline, penicillin, erythromycin), cytotoxic agents (e.g., streptomycin, doxorubicin), and antioxidants (e.g., vitamin E, vitamin C, beta-carotene).

Carrier complex

At least one of the above molecules, and at least one of the above aromatic-cationic peptides, are bound (associated) to form a carrier complex. The molecule and aromatic cationic peptide can be combined by any method known to those skilled in the art. Suitable types of bonding include chemical bonding and physical bonding. Chemical bonding includes, for example, bonding by covalent bonding and bonding by coordinate bonding. Physical binding includes, for example, binding by hydrogen bonding, dipolar interaction, van der waals force, electrostatic interaction, hydrophobic interaction, and aromatic stacking (aromatic stacking).

The type of bond between the molecule and the aromatic-cationic peptide typically depends on, for example, the available functional groups on the molecule and the available functional groups on the aromatic-cationic peptide.

For physical or chemical bonding, the functional group on the molecule is typically bonded to the functional group on the aromatic-cationic peptide. Optionally, the functional group on the aromatic-cationic peptide is associated with a functional group on the molecule.

The molecule and the functional group on the aromatic cationic peptide may be directly bound. For example, a functional group (e.g., a thiol group) on a molecule is bound to a functional group (e.g., a thiol group) on an aromatic cationic peptide to form a disulfide.

Alternatively, the functional groups may be bound by a crosslinking agent (e.g., a linking agent). Some examples of crosslinking agents are described below. The cross-linking agent may be attached to either the molecule or the aromatic-cationic peptide.

The linker may or may not affect the number of net charges of the aromatic-cationic peptide. The linker generally does not affect the net charge of the aromatic-cationic peptide. Each amino group, if any, present in the linker will affect the net positive charge of the aromatic-cationic peptide. Each carboxyl group, if any, present in the linker will affect the net negative charge of the aromatic-cationic peptide.

The number of molecules or aromatic-cationic peptides in the carrier complex is limited by the ability of the peptide to accommodate multiple molecules or the ability of the molecule to accommodate multiple peptides. For example, steric hindrance may hinder the ability of the peptide to accommodate, inter alia, macromolecules. Alternatively, steric hindrance may hinder the ability of the molecule to accommodate relatively large (e.g., seven, eight, nine, or ten amino acids in length) aromatic-cationic peptides.

The number of molecules or aromatic-cationic peptides in the carrier complex is also limited by the number of functional groups present on each other. For example, the maximum number of molecules that bind to a peptide depends on the number of functional groups present on the peptide. Alternatively, the maximum number of peptides bound to a molecule depends on the number of functional groups present on the molecule.

In a particular embodiment, the carrier complex comprises at least one molecule, and preferably at least two molecules, bound to the aromatic-cationic peptide. Relatively large peptides (e.g., eight, ten amino acids in length) comprising multiple (e.g., 3, 4, 5, or more) functional groups can be conjugated to multiple (e.g., 3, 4, 5, or more) molecules.

In another embodiment, the carrier complex comprises at least one aromatic-cationic peptide, and preferably at least two aromatic-cationic peptides, bound to a molecule. For example, a molecule comprising multiple (e.g., 3, 4, 5, or more) functional groups can be conjugated to multiple (e.g., 3, 4, 5, or more) peptides.

In another embodiment, the carrier complex comprises an aromatic-cationic peptide bound to a molecule.

In a particular embodiment, the carrier complex comprises at least one molecule chemically bound (e.g., conjugated) to at least one aromatic-cationic peptide. The molecule may be chemically bound to the aromatic-cationic peptide by any method known to those skilled in the art. For example, the functional group on the molecule can be directly linked to a functional group on the aromatic-cationic peptide. Some examples of suitable functional groups include, for example, amino, carboxyl, thiol, maleimide, isocyanate, isothiocyanate, and hydroxyl groups.

The molecule may also be chemically bound to the aromatic cationic peptide by means of a crosslinking agent, such as dialdehyde, carbodiimide, bismaleimide, and the like. The cross-linking agent may, for example, be obtained from Pierce Biotechnology, Inc. of Rockford, Ill. Pierce Biotechnology, Inc., under URLhttp:// www.piercenet.com/products/browse. cfmfldID ═ 26436A16-60A0-4A56-85F7-213A50830440, can provide assistance. Additional crosslinking agents include platinum crosslinking agents described in U.S. patent nos. 5,580,990, 5,985,566, and 6,133,028 of Kreatech Biotechnology b.v. amsterdam, the netherlands.

The functional group of the molecule may be different from the functional group of the peptide. For example, if a thiol group is present on the molecule, such as in β -galactosidase or in 5 '-and/or 3' -terminal thiol group modified DNA and RNA oligonucleotides, the molecule can be crosslinked to a peptide via the 4-amino group of lysine via the crosslinking reagent SMCC (i.e., succinimide 4- (N-maleimidomethyl) cyclohexyl-1-carboxylate) from pierce biotechnology, e.g., [ Dmt [ Dmt ] ]¹]DALDA (see example 10 below). In another example, the 4-amino group of lysine of DALDA may be modified by the crosslinker EDC from Pierce Biotechnology (i.e., (N- [ 3-dimethylaminopropyl-N' -ethylcarbodiimide)]) Direct conjugation binds to the alpha-phosphate group at the 5' -end of RNA or DNA (see example 13 below).

Alternatively, the functional groups of the molecule and the peptide may be the same. Homobifunctional (homobifunctional) crosslinkers can generally be used to crosslink the same functional groups. Examples of homobifunctional crosslinking agents include: such homobifunctional crosslinkers are also available from Pierce Biotechnology, Inc., EGS (i.e., ethylene glycol bis [ succinimidyl succinate ]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3, 3 ' -dithio [ thiosuccinimidyl propionate ]), DPDBP (i.e., 1, 4-bis- [3 ' - (2 ' -pyridylthio) -propionamido ] butane, and BMH (i.e., di-maleimidohexane).

In order to chemically bind the molecule and peptide, the molecule, peptide, and cross-linking agent are typically mixed together. The order of addition of the molecule, peptide, and crosslinker is not critical. For example, the peptide may be mixed with the cross-linking agent first, followed by addition of the molecule. Alternatively, the molecule may be mixed with the cross-linking agent first, followed by addition of the peptide. Optimally, the molecule may be mixed with the peptide first, followed by the addition of the cross-linking agent.

The chemically bound carrier complex can deliver the molecule to a cell. In some examples, the molecule does not separate from the aromatic-cationic peptide when acting intracellularly. For example, if the aromatic-cationic peptide does not interfere with the catalytic site of the molecule, it is not necessary to separate the molecule from the aromatic-cationic peptide (see example 11 below).

In other embodiments, it may be advantageous to separate the molecule from the aromatic compound. The website of the above-mentioned Pierce Biotechnology company also provides the person skilled in the art with the aid of selecting suitable cross-linking agents which can be separated, for example, by intracellular enzymes. Thus, the molecule can be separated from the aromatic-cationic peptide. Examples of linkers that can be separated include: SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-a- [ 2-pyridyldithio ] toluene, Sulfo-LC-SPDP (i.e., thiosuccinimidyl 6- (3- [ 2-pyridyldithio ] -propionamido) hexanoate), LC-SPDP (i.e., succinimidyl 6- (3- [ 2-pyridyldithio ] -propionamido) hexanoate), Sulfo-LC-SPDP (i.e., thiosuccinimidyl 6- (3- [ 2-pyridyldithio ] -propionamido) hexanoate), SPDP (i.e., N-succinimidyl 3- [ 2-pyridyldithio ] -propionamidohexanoate), and AEDP (i.e., 3- [ (2-aminoethyl) dithio ] -propionic acid & HCl).

In another embodiment, the carrier complex comprises at least one molecule physically bound to at least one aromatic-cationic peptide. The molecule may be physically bound to the aromatic-cationic peptide by any method known to those skilled in the art.

For example, the aromatic-cationic peptide can be mixed with the molecule by any method known to those skilled in the art. The order of mixing is not important. For example, the molecule may be physically mixed with the modified or unmodified aromatic-cationic peptide by any method known to those skilled in the art. Alternatively, the modified or unmodified aromatic-cationic peptide may be physically mixed with the molecule by any method known to those skilled in the art.

For example, the aromatic-cationic peptides and molecules can be placed in a container and agitated, for example, by shaking the container, to mix the aromatic-cationic peptides and molecules.

The aromatic-cationic peptide can be modified by any method known to those skilled in the art. For example, the aromatic-cationic peptide may be modified by means of a cross-linking agent or functional groups as described above. The linker may or may not affect the number of net charges of the aromatic-cationic peptide. Typically, the linker does not affect the net charge of the aromatic-cationic peptide. Each amino group, if any, present in the linker will affect (increase) the net positive charge of the aromatic-cationic peptide. Each carboxyl group, if any, present in the linker will affect the net negative charge of the aromatic-cationic peptide.

For example, [ Dmt [ ]¹]DALDA can be modified by the 4-amino group of lysine by the cross-linking reagent SMCC from Pierce Biotechnology (i.e., succinimide 4- (N-maleimidomethyl) cyclohexyl-1-carboxylate) (see example 10 below). To form the carrier complex, the modified aromatic-cationic peptide is typically formed first and then mixed with the molecule.

One advantage of physically bound carrier complexes is that: the molecule does not require removal of aromatic-cationic peptides when it functions intracellularly, such as those carrier complexes in which the molecule is chemically bound to an aromatic-cationic peptide. Furthermore, dissociation of the complex is not necessary if the aromatic-cationic peptide does not interfere with the catalytic site of the molecule (see example 12 below).

Synthesis of aromatic-cationic peptides

The peptides used in the methods of the invention may be chemically synthesized by any method known in the art. Suitable Methods for synthesizing the protein include, for example, by Stuart and Young in "Solid Phase Peptide Synthesis", "second edition, Pierce Chemical Company (1984), and in" Solid Phase Peptide Synthesis "," Methods Enzymol,289，those described in academic Press, Inc, New York (1997).

Mode of administration

In one embodiment, the invention relates to a method of delivering a molecule to a cell. The method comprises contacting the molecule and the aromatic-cationic peptide with a cell. The molecule and aromatic-cationic peptide can be contacted with the cell by any method known to those skilled in the art. For example, cells can be incubated with molecules and aromatic-cationic peptides in vitro. In one aspect, the cell and the aromatic-cationic peptide can be in the form of a carrier complex, such as those described above, comprising a chemically-bound or physically-bound molecule and the aromatic-cationic peptide.

In another embodiment, a method of delivering a molecule to a cell comprises contacting a carrier complex with the cell. The molecule is delivered to the cell by contacting the cell with a carrier complex comprising the molecule and an aromatic-cationic peptide. The carrier complex may be contacted with the cell by any method known to those skilled in the art.

For example, cells can be incubated with the carrier complex in vitro. The cell may be any cell. The cells may be derived from plants, animals, or bacteria. Examples of plant cells include cells of the genus Abutia. Examples of cells of bacteria include Saccharomyces and Lactobacillus. Animal cells include mammalian cells such as nerve cells, renal epithelial cells, kidney cells, vascular endothelial cells, glial cells, intestinal epithelial cells, and liver cells. An example of a vascular endothelial cell is a blood brain barrier endothelial cell.

Alternatively, the carrier complex may be administered to a mammal in vivo. An effective amount of the carrier complex, preferably a pharmaceutical composition, can be administered to the mammal in need thereof by any of a variety of well-known methods for administering pharmaceutical compounds.

The carrier complex may be administered systemically or locally. In a particular embodiment, the carrier complex is administered intravenously. For example, the carrier complex may be administered via a rapid bolus intravenous injection. Preferably, however, the carrier complex is administered intravenously at a constant rate.

The carrier complex may be administered topically to a tissue of a mammal. For example, by injection into tissue that is readily accessible by the injector. For example, if the carrier complex comprises a cytotoxic agent to be delivered to a tumor in a mammal, preferably, the tumor is susceptible to local administration. Such tumors include, for example, skin cancer and breast cancer.

The carrier complex may also be administered orally, topically, intranasally, intramuscularly, subcutaneously, or transdermally. In a preferred embodiment, the transdermal administration of the carrier complex is by iontophoresis, wherein the carrier complex is delivered transdermally by an electric current.

Other routes of administration include intracerebroventricular or intrathecal administration. Intraventricular administration involves administration into the ventricular system of the brain. Intrathecal administration involves administration into the subarachnoid space of the brain or spinal cord. Thus intraventricular or intrathecal administration may be preferred for those diseases or pathologies affecting organs or tissues of the central nervous system.

The carrier complex used in the method of the invention may be administered to the mammal by sustained release means, as is known in the art. Sustained release administration is a method of drug delivery that allows the drug to reach a certain level over a specific period of time. This level is typically determined by serum concentration.

Any dosage form known in the pharmaceutical art is suitable for administration of the carrier complex. For oral administration, liquid or solid dosage forms may be used. Some examples of dosage forms include tablets, capsules, pills, lozenges, elixirs, suspensions, syrups, wafers (wafers), chews (chewing gum), and the like. The peptide may be mixed with a suitable pharmaceutical carrier (vehicle) or excipient as will be appreciated by those skilled in the art. Examples of carriers and excipients include starches, emulsions, sugars, certain types of clays, gums, lactic acid, stearic acid or its salts, including magnesium or calcium stearate, talc, vegetable fats or oils, gums and glycols.

For systemic administration, intracerebroventricular administration, intrathecal administration, topical administration, intranasal administration, subcutaneous administration, or transdermal administration, the dosage form of the carrier complex may utilize conventional diluents, carriers, or excipients, and the like, as are known in the art, which may be used to deliver the carrier complex. For example, the dosage form may include one or more of the following: stabilizers, surfactants, preferably non-ionic surfactants, and optionally salts and/or buffers. The carrier complex may be delivered in the form of an aqueous solution, or in lyophilized form.

The stabilizer may be, for example, an amino acid such as, for example, glycine; or oligosaccharides such as, for example, sucrose, tetrasaccharides, lactose or dextran. Alternatively, the stabilizer may also be a sugar alcohol, such as, for example, mannitol; or a combination thereof. Preferably, the stabilizer or combination of stabilizers constitutes from about 0.1% to about 10% by weight of the carrier composite.

The surfactant is preferably a nonionic surfactant, such as a polysorbate. Some examples of suitable surfactants include: tween 20, tween 80; polyethylene glycol or polyoxyethylene polyoxypropylene glycol, such as Pluronic F-68 from about 0.001% (w/v) to about 10% (w/v).

The salt or buffer may be any salt or buffer such as, for example, sodium chloride, or sodium/potassium phosphate, respectively. Preferably, the buffer maintains the pH of the pharmaceutical composition in the range of about 5.5 to about 7.5. The salt and/or buffer also serve to maintain osmolarity at a level suitable for administration to a human or animal. Preferably, the salt or buffer is present in a roughly isotonic concentration of about 150mM to about 300 mM.

The components of the carrier composite used in the method of the invention may also comprise one or more conventional additives. Some examples of such additives include: solubilizers such as, for example, glycerol; antioxidants, such as, for example, benzalkonium chloride (a mixture of quaternary ammonium compounds, known as "quats"), benzyl alcohol, chloroxanthone, or chlorobutanol; anesthetics, such as, for example, morphine derivatives; or an isotonic agent, such as those mentioned above. As a precaution to further prevent oxidation or other spoilage, the pharmaceutical composition may be stored under nitrogen gas in vials sealed with impermeable stoppers.

The mammal can be any mammal, including, for example, livestock, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals such as rats, mice and rabbits. In a preferred embodiment, the mammal is a human.

Applications of

Since the carrier complex is capable of crossing cell membranes in an energy-independent mechanism, it can be used in many applications both in vivo and in vitro.

The carrier complex can, for example, be used in vitro as a research tool. For example, the carrier complex can deliver a molecule, such as a protein, into a cell in order to study the functional role of the molecule. Such molecules include, for example, cellular signaling proteins (e.g., nuclear factor NF-. kappa.B, kinases such as JAK).

Another application in vitro includes, for example, the delivery of a marker, such as β -galactosidase, into a cell, such as a stem cell, hematopoietic cell, or embryonic cell, in order to determine the progeny (lineage) of the cell.

Other applications in vitro include, for example, the delivery of a detectable antibody into a cell in order to determine the presence of a particular protein within the cell.

The carrier complex also has therapeutic use in vivo. For example, the aromatic-cationic peptide can be used to deliver antisense polynucleotides into cells of a mammal in order to down-regulate the overexpression of the protein. In addition, the aromatic-cationic peptides can be used to deliver oligonucleotides that can be used for RNA interference (RNAi).

RNAi, as used herein, refers to cellular mechanisms used to regulate the expression of genes or viral or bacterial replication. This mechanism involves introducing double-stranded RNA (e.g., siRNA) into the product (usually RNA) of the target gene.

The blood brain barrier has a particular selectivity. Thus, another application in vivo involves the delivery of molecules across the blood-brain barrier. Such molecules may include, for example, antibodies to beta-amyloid in the treatment of alzheimer's disease patients.

A typical problem associated with chemotherapeutic agents is reaching a sufficient level within the cell. For example, the chemotherapeutic agent may be too large or not aromatic and not sufficient to cross the cell membrane. Thus, yet another application in vivo involves the delivery of chemotherapeutic agents, such as the cytotoxic agents described above, into cells.

Examples

Example 1: materials and methods

Drugs and chemicals. [ Dmt¹]DALDA and [2 ]³H][Dmt¹]DALDA (47Ci/mmol) was synthesized as described previously (Schiller et al, Eur.J.Med.chem.2000, 35: 895-. [¹⁴C]Gly-Sar (56.7mCi/mmol) and [2 ], [ solution of³H][D-Ala²，N-Me-Phe⁴，Gly⁵-ol]Enkephalins (50Ci/mmol) were purchased from Amersham biosciences (Piscataway, N.J.). All other drugs and chemicals were from Sigma-Aldrich (st louis, missouri).

And (5) culturing the cells. All cell lines were from the American Type Culture Collection (manassas town, va) and supplies required for cell Culture were from Invitrogen (carlsbad, ca). Caco-2 cells were grown in MEM, while SH-SY5Y, HEK293, and Huh7 cells were grown in Dulbecco's modified Eagle medium. Growth medium was supplemented with 10% fetal bovine serum, 200. mu.g/ml penicillin, and 100. mu.g/ml streptomycin sulfate. CRFK cells were grown in MEM + 10% horse serum, non-essential amino acids, and penicillin/streptomycin. All cell lines were maintained at 37 ℃ with 95% air and 5% CO₂In the humid gas of (c).

Peptide uptake experiments. Internalization of the peptide was first studied using Caco-2 cells and subsequently confirmed using SH-SY5Y, HEK293, and CRFK cells. Monolayers of cells on collagen-coated 12-well plates (5X 10)⁵Individual cells/well) were grown for 3 days. On day 4, the cells were washed twice with preheated HBSS, followed by contacting the cells with a solution containing 250nM [2 ], [ 1 ]³H][Dmt¹]DALDA or 50. mu.M [ alpha ]¹⁴C]0.2ml HBSS of Gly-Sar was incubated for various times to 1 hour. In a separate experiment, at unlabeled [ Dmt¹]DALDA (1. mu.M-3 mM) presentIn the case of (2), the cells are contacted with the same concentration of [2 ]³H][Dmt¹]DALDA was incubated at 37 ℃ for 1 hour. To investigate uptake at 4 ℃, the cells were contacted with [ [ alpha ], [ alpha ] ]³H][Dmt¹]DALDA or [2 ]¹⁴C]Cells were placed on ice for 20 min before incubation with Gly-Sar. After the end of this incubation period, cells were washed four times with HBSS and 0.2ml of 0.1n NaOH with 1% SDS was added to each well. The cell contents were then transferred to scintillation vials and the radioactivity was counted. Aliquots of cell lysates were used to determine protein content by the method of Bradford (Bio-Rad, Hercules, Calif.). To distinguish between intrinsic and surface-bound radioactivity, an acid wash step was included. Cells were incubated with 0.2ml0.2M acetic acid/0.05M NaCl on ice for 5 minutes before cell lysis.

Leakage experiments of peptides derived from CaCo-2 cells. Monolayer Caco-2 cells on 12-well plates (5X 10)⁵Individual cells/well) were grown for 3 days. On day 4, use at 37 ℃³H][Dmt¹]DALDA or [2 ]¹⁴C]Gly-Sar preloads (preloads) the cells for 1 hour. The cells were then washed four times with 1ml of ice-cold incubation solution to stop uptake, and subsequently incubated with 0.5ml of MEM at 37 ℃ or 4 ℃ for 1 hour to determine the leakage of peptide from the cells into the incubation medium. The amount of radioactivity was determined in the cell lysate and incubation medium. To examine the role P-glycoprotein plays in peptide uptake and leakage from cells, [ Dmt ] was also determined in the presence of 100. mu.M verapamil¹]Uptake and leakage of DALDA.

Translocation of peptides across Caco-2 monolayers. Monolayers of Caco-2 cells were prepared as described previously (Irie et al, J.Pharmacol. exp. Ther.2001, 298: 711-717). Caco-2 cells (2X 10)⁵) Seeded on a microporous membrane filter (24mm, 0.4 μm) in a rotating well cell culture vessel (Corning Glassworks, Corning, N.Y.). Each rotating well vessel was filled with 1.5ml of medium in its top compartment and 2.5ml of medium in its bottom compartment. The cell monolayer was given fresh medium every 1 to 2 days and used for transport experiments on day 28. By mixing 0.2. mu.M [ sic ]³H][Dmt¹]DALDA or 100. mu.M [ alpha ]¹⁴C]Gly-Sar was added to the top compartment to determine the transport of peptide from the top compartment to the bottom compartment, and 50- μ l aliquots were removed from the top and bottom compartments at different times after peptide addition to determine the radioactive counts.

The apparent permeability coefficient was calculated according to the following formula: p_appX/(t · a · Co), where X/t is the uptake rate in the receiving compartment and a is the diffusion area (4.72 cm)²) And Co is the initial concentration in the donor compartment.

Laser confocal scanning microscope. Uptake of aromatic-cationic peptides into cells was confirmed by laser confocal scanning microscopy (CLSM) using two fluorescent peptides: [ Dmt¹，dnsDap⁴]DALDA (Dmt-D-arginine-phenylalanine-dnsDap-NH)₂Wherein dnsDap is beta-dansyl-1-alpha, beta-diamino-propionic acid) and [ Dmt [ (. beta. -hydroxy-propionic acid) ]¹，atnDap⁴]DALDA (Dmt-D-arginine-phenylalanine-atnDap-NH₂Wherein atnDap is β -anthranoyll- α, β -diaminopropionic acid). Caco-2 cells or SH-SY5Y cells were grown as described above and then plated on (35-mm) glass chassis (MatTek, Ashland, Mass.) for two days. This medium was then removed and the cells were incubated with 1ml HBSS containing 0.1 μ M fluorescent peptide for 15 minutes at 4 ℃ or 37 ℃. The cells were then washed three times with ice-cold HBSS, followed by covering the cells with 200 μ l PBS, followed by microscopic examination at room temperature within 10 minutes with a confocal laser scanning microscope (Nikon, tokyo, japan) with a C-apochromatic lens 63 x/1.2W correction objective. [ Dmt¹，dnsDap⁴]DALDA and [ Dmt¹，atnDap⁴]The excitation/emission wavelengths of DALDA were set at 340/520nm and 320/420nm, respectively. For z-axis optical sectioning, 5-10 frames are cut every 2.0 μm.

Radioligand binding experiments using cell membranes. [³H][Dmt¹]The specific binding of DALDA to cell surface receptors was determined using membranes prepared from Caco-2 and SH-SY5Y cells. After 4 days of culture, cells were washed 2 times with PBS bufferAnd then scraping the cells. Cells were centrifuged at 500g for 5 minutes, and the pellet was then stored at-80 ℃. Cells were homogenized in ice-cold 50mM Tris-HCl buffer (5. mu.g/ml leupeptin, 2. mu.g/ml chymotrypsin inhibitor, 10. mu.g/ml bestatin, and 1mM EGTA, pH 7.4). The homogenate was centrifuged at 36,000g for 20 minutes. The pellet was resuspended in 50mM Tris-HCl. An aliquot of the membrane homogenate (. about.140. mu.g protein) and [ alpha ], [ alpha ] protein³H][Dmt¹]DALDA (15-960pM) was incubated for 60 minutes. Non-specific binding was achieved by mixing in 1. mu.M unlabelled [ Dmt ]¹]DALDA for evaluation. Free radioligand was separated from bound radioligand by rapid filtration through a GF/B filter (Whatman, medstein, uk) with a cell harvester (Brandel corporation, gaithersburg, maryland). The filters were washed three times with 10ml Tris buffer, followed by determination of radioactivity by liquid scintillation counting. Binding affinity (K) was determined using non-linear regression (GraphPad Software, san Diego, Calif.)_d) And number of receptors (B)_max)。

Conjugation of proteins to [ Dmt¹]DALDA. [ Dmt ] Using crosslinker SMCC (succinimide 4- (N-maleimidomethyl) cyclohexyl-1-carboxylate) (Pierce)¹]DALDA is cross-linked to β -galactosidase (recombiant e. SMCC with amine-containing molecules ([ Dmt¹]Lys of DALDA⁴) Reacting to form a stable amide bond. Its maleimide terminus can then be conjugated to a thiol-containing compound to form a thioether linkage (bioconjugate techniques by Greg T. Hermanson, Academic Press, Page 234-. Beta-galactosidase contains a large number of free thiols in its native state. Uptake of beta-galactosidase provides convenient reading with X-gal. Briefly, 1ml of 5X 10 are introduced at room temperature^-3M[Dmt¹]DALDA was mixed with 1mg SMCC in phosphate buffer for 1 hour. This will form an "activated peptide". The "activated peptide" was diluted 1:10 with phosphate buffer. 1mg of beta-galactosidase was added to 1ml of the 1:10 "activated peptide" and mixed at 4 ℃ for 2 hours or overnight。

Will [ Dmt¹]DALDA was linked to the cross-linker SMCC and confirmed by mass spectrometry. SMCC (1. mu.g) and [ Dmt ]¹]DALDA (5. mu.g) was dissolved together in 2ml PBS, incubated at room temperature for 30 minutes, and then stored at 4 ℃. An aliquot of this sample was mixed with a matrix (3-hydroxypyridine carboxylic acid (HPA) saturated in 50% acetonitrile, 10mg/ml ammonium citrate) in a ratio of 1:10 and then spotted onto a stainless steel target plate. Analysis was performed in forward reflectance mode by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Applied Biosystems (Voyager DE Pro)).

Binding of RNA to [ Dmt¹]DALDA, and confirmed by gel electrophoresis. By using gamma-³²P-ATP and polynucleotide kinase phosphorylate synthetic RNA oligomers (40 nucleotides in length) at the 5' end. The product was purified by gel. In the presence of 1mg of EDC (N- [ 3-dimethylaminopropyl-N' -ethylcarbodiimide)]) In the presence of 500,000cpm of gel-purified RNA oligomer with [ Dmt¹]DALDA binds. The combined product ([ Dmt ]¹]DALDA-RNA oligomer) were analyzed separately on a 15% polyacrylamide urea gel.

Binding of DNA to [ Dmt¹]DALDA, and confirmed by mass spectrometry. SMCC (1. mu.g) and [ Dmt ]¹]DALDA (5 μ g) was dissolved together in 2ml PBS, incubated at room temperature for 30 minutes, and mixed with the deprotected 3' -thiol DNA oligomer at 4 ℃ for 24 hours. After incubation, an aliquot of this sample was mixed with a matrix (3-hydroxypyridine carboxylic acid (HPA) saturated in 50% acetonitrile, 10mg/ml ammonium citrate) in a ratio of 1:10 and spotted onto a stainless steel target plate. Samples were analyzed by MALDI-TOFMS.

By physically mixing RNA and [ Dmt ]¹]The DALDA-SMCC conjugate forms a carrier complex. Preparation of [ Dmt ] as described above¹]A DALDA-SMCC conjugate. The RNA molecules were conjugated to [ Dmt ] at room temperature before use in cellular uptake studies¹]The DALDA-SMCC conjugate was mixed in PBS for 15 minutes.

By physically mixing the protein and [ Dmt ]¹]The DALDA-SMCC conjugate forms a carrier complex. [ Dmt ] prepared as described above¹]A DALDA-SMCC conjugate. The protein molecules (i.e., green fluorescent protein, GFP) were incubated with [ Dmt ] at room temperature prior to use in cellular uptake studies¹]The DALDA-SMCC conjugate was mixed for 15 minutes.

[Dmt¹]Experiments in which the DALDA-RNA conjugate was taken up into cells. By using gamma-³²P-ATP and polynucleotide kinase phosphorylate the synthesized RNA oligomer at the 5' end and the product is purified by gel electrophoresis. 500,000cpm of gel-purified RNA oligomer was reacted with [ Dmt ] in the presence of 1mg of N- (3-dimethylaminopropyl-N' -Ethylcarbodiimide) (EDC)¹]DALDA binding (coupling). Caco-2 cells (1X 10)⁶) Three washes in DMEM medium and preincubation for 5 minutes in DMEM. The cells were then incubated with [ Dmt ] at 37 ℃¹]DALDA-[³²P]The RNA oligomer conjugate or unconjugated RNA (approximately 20,000cpm) was incubated for 60 minutes. After incubation, the cells were washed three times in DMEM, diluted with lysis buffer and subsequently assayed for radioactivity in the cell lysate.

RNA is in the presence of [ Dmt¹]The DALDA-crosslinker conjugate was mixed and taken up into the cell experiment. Washing of Huh7 cells (1X 10) with DMEM⁶Individual cells/well), followed by incubating the cells with 1.0ml of DMEM containing only [2 ], [2 ] at 37 ℃ or 4 ℃ for 60 minutes³²P]An RNA oligomer, or a nucleic acid comprising³²P]RNA oligomer and 40. mu.l [ Dmt ]¹]A DALDA-SMCC conjugate. Cells were then washed four times in DMEM and once in sodium acetate solution to reduce non-specific binding prior to incubation in lysis buffer for 30 minutes, followed by measurement of radioactivity in the cell lysate.

[Dmt¹]Experiments in which the DALDA-protein conjugate was taken up into cells. Mixing cells (N)₂A neuroblastoma cells or Caco-2) were plated in 96-well plates (2X 10)⁴Individual cells/well), followed by contacting the cells with beta-galactosidase cross-linked [ Dmt ] at 37 ℃¹]DALDA or incubation with β -galactosidase alone for 1 hour. Cells were then washed 4 times with PBS. Cells were then stained with a beta-galactosidase staining system (Roche) at 37 ℃ for at least 2 hours, and then examined under a microscope.

Protein with [ Dmt¹]Experiments in which DALDA-SMCC conjugate was taken up into cells after incubation. Washing of Huh7 cells (1X 10) with DMEM⁶Individual cells/well) followed by incubation of the cells with 0.5ml of DMEM for 60 minutes at 37 ℃, the DMEM: contains only 3. mu.g of Green Fluorescent Protein (GFP) (A); containing 3. mu.g GFP and 40. mu.l [ Dmt ]¹]DALDA (B); or 3. mu.g GFP and 40. mu.l [ Dmt ] bound to SMCC¹]DALDA (C). 2ml of cell culture medium was then added to the cells and incubated in the cell culture incubator for an additional 24 hours. After incubation, cells were washed four times in cell culture medium, followed by observation of GFP remaining in viable cells by confocal laser scanning microscopy. Excitation was performed at 340nm and emission was measured at 520 nm.

And (4) carrying out apoptosis experiments. Apoptosis was determined by staining apoptotic nuclei with Hoechst dye (Molecular Probes, ukin, oregon). Hoechst dye was loaded into cell culture and incubated for 15 minutes. Excess Hoechst dye was removed by washing the cells with cell culture medium (without pH indicator) and then the cells were examined using a fluorescence microscope (excitation at 350nm and emission at 461nm (measured)).

Example 2: [ Dmt ¹ ]DALDA and Gly-Sar are taken into Caco-2 cells Time course.

When 2³H][Dmt¹]When DALDA was incubated with Caco-2 cells at 37 ℃, the expression of the cell lysate was observed as early as 5 minutes³H][Dmt¹]DALDA, and reached a steady state level by 30 minutes (fig. 1A). After 1 hour of incubation, the recovered cell lysate³H][Dmt¹]The total amount of DALDA accounted for about 1% of the total drug. In contrast, under the same experimental conditions, [ alpha ], [ beta ]¹⁴C]Gly-Sar continued to increase at 5 to 45 minutes (FIG. 1B). It is believed that the measured radioactivity may reflect [ Dmt¹]Level of DALDA, since [ Dmt ] has been previously confirmed¹]DALDA can prevent the decrease of peptidase (Szeto et al, J.Pharmacol.exp.Ther., 2001, 298: 57-61). To determine whether the measured radioactivity is associated with the cell membrane, the cells were acid washed to remove surface binding. FIG. 1C shows that 80.8% of³H][Dmt¹]DALDA is resistant to acid washing and is therefore judged to be present inside the cell. It has been found that [ Dmt¹]Uptake of DALDA was concentration-dependent over a wide range of concentrations (fig. 1D).

Example 3: pH to [ Dmt ¹ ]Temperature dependence of uptake of DALDA and Gly-Sar Dependence and influence.

When incubated at 4 ℃³H][Dmt¹]Uptake of DALDA was slower than at 37 ℃, but reached 76.5% at 45 minutes (fig. 1A) and 86.3% at 1 hour (fig. 1A). Compared with the above solution, the product¹⁴C]The uptake of Gly-Sar was completely eliminated at 4 deg.C (FIG. 1B). It is known that the uptake of Gly-Sar by PEPT1 is pH dependent and that uptake is optimal at pH6.0 (Terada et al, 1999, am.J.Physiol.276: G1435-G1441). This was confirmed in our study (fig. 2B). In contrast, when the pH is changed from 4.0 to 7.4, [2 ]³H][Dmt¹]Uptake of DALDA did not change (fig. 2A). This lack of temperature and pH dependence indicates [ Dmt ] in Caco-2 cells¹]Uptake of DALDA was not mediated via PEPT1 (transporter 1 for peptide).

Example 4: DEPC to [ Dmt¹]Influence of DALDA and Gly-Sar uptake.

To further show that PEPT1 does not involve [ Dmt¹]Transport of DALDA, we studied DEPC (diethyl pyrocarbonate; 0.2mM) for [2 ], [³H][Dmt¹]DALDA and [2 ]¹⁴C]Effect of Gly-Sar uptake. DEPC is a modification reagent for histidine residues, which has been shown to inhibit Caco-2 intracellular PEPT1(Terada et al, FEBS. Lett., 1996, 394: 196-200). Addition of DEPC to the incubation Medium significantly inhibited the [2 ]¹⁴C]Gly-Sar uptake (FIG. 2D). Surprisingly, not only does DEPC not inhibit³H][Dmt¹]DALDA is taken in, and it is naturally expressed³H][Dmt¹]DALDA uptake increased 34-fold (fig. 2C).

Example 5: [ Dmt ¹ ]Internalization of DALDA in different cell types.

To prove [ Dmt¹]Internalization of DALDA is not limited to Caco-2 cells, and we compared [ Dmt ] in a number of different cell lines¹]Internalization of DALDA. An acid wash step is added to distinguish the intrinsic radioactivity (acid resistant) from the surface bound radioactivity (acid sensitive). FIG. 3A compares the level of acid-tolerant radioactivity in Caco-2 cells, SH-SY5Y cells, HEK293 cells, and CRFK cells. Result display³H][Dmt¹]DALDA is taken up abundantly in all cell types.

Example 6: and 2 ³ H][Dmt ¹ ]Radioligand binding experiments with DALDA.

To determine [ Dmt¹]Whether internalization of DALDA is via a receptor-mediated mechanism, we have performed radioligand (, [ solution ])³H][Dmt¹]DALDA) was tested in combination with membranes prepared from Caco-2 cells and SH-SY5Y cells. FIG. 3B shows [2 ]³H][Dmt¹]The specific binding of DALDA to SH-SY5Y cell membrane. The calculated Kd value was 118pM (range 87-149), and the calculated B_maxThe value was 96fmol/mg protein (range 88-104). This corresponds to the values obtained using recombinant human μ -opioid receptors expressed on chinese hamster ovary cells (g. -m.zhao and h.h.szeto, unpublished data). No specificity of high affinity to Caco-2 cell membranes was observedSexual association (fig. 3B). HEK293 cells are known to lack opioid receptors (Blake et al, J.biol.chem., 1997, 272: 782-790).

Example 7: [ Dmt ¹ ]Leakage of DALDA and Gly-Sar from Caco-2 cells.

2 in Caco-2 cell³H][Dmt¹]DALDA in<After 30 minutes incubation, a steady state level was reached, indicating that the rate of leakage of the peptide from the cells at this time was equal to the rate of uptake. To determine Gly-Sar and [ Dmt¹]The amount of leakage of DALDA from the cells, using [2 ]¹⁴C]Gly-Sar or [2 ]³H][Dmt¹]DALDA preloads Caco-2 cells, which were then replaced with fresh medium containing no peptide. FIG. 4A shows that 39% of the protein is found in the medium after 1 hour at 37 ℃¹⁴C]Gly-Sar。[¹⁴C]The leakage of Gly-Sar was significantly reduced at 4 ℃.2 [ alpha ] derived from Caco-2 cell³H][Dmt¹]The leakage of DALDA was much faster, with 80% of the peptide leaking into the medium at 1 hour (fig. 4A). And 2³H][Dmt¹]The internalization of DALDA is reversed (FIG. 1A), temperature for [2 ]³H][Dmt¹]Leakage of DALDA from the cells had a significant impact (fig. 4A). In DEPC-treated cells, [ Dmt [ Dmt ] ]¹]The leakage of DALDA is reduced (fig. 4B). Caused by DEPC³H][Dmt¹]The reduction in the amount of DALDA leakage and the presence of DEPC³H][Dmt¹]A significant increase in DALDA uptake (fig. 2C) was consistent. On the other hand³H][Dmt¹]The leakage of DALDA was not affected by verapamil, an inhibitor of P-glycoprotein (fig. 4C). The therapeutic pair of heteroprolidine³H][Dmt¹]Cellular uptake of DALDA also had no effect (fig. 4D).

If at [ Dmt¹]After the DALDA-protein conjugate is taken up by the cells, it is cleaved by enzymes, [ Dmt¹]DALDA leaks out of the cell while the protein cargo is still intracellular [ Dmt ]¹]It is advantageous for DALDA to leak out of the cells.

Example 8: [ Dmt ¹ ]Trans-cellular transport of DALDA and Gly-Sar

Caco-2 monolayers grown in a transwell (transwell) were used in the study³H][Dmt¹]DALDA and [2 ]¹⁴C]Transport of Gly-Sar from top to bottom side. Figure 5 shows the values of [ alpha ], [ beta ]¹⁴C]Gly-Sar and [2 ]³H][Dmt¹]Transport of DALDA. In 60 minutes³H][Dmt¹]The percentage of DALDA transferred from the top to the bottom side (10.4%) was compared with that transferred¹⁴C]The percentage of Gly-Sar (11.9%) was comparable. According to the calculation, [ Dmt¹]The apparent permeability coefficient of DALDA is 1.24X 10^-5cm/s and the apparent permeability coefficient of Gly-Sar is 1.26X 10^-5cm/s.

Example 9: the cellular uptake of aromatic-cationic peptides was observed using CLSM.

To observe the uptake and pattern of cellular internalization of aromatic-cationic peptides, two fluorescent peptides ([ Dmt) were studied with CLSM¹，dnsDap⁴]DALDA and [ Dmt¹，atnDap⁴]DALDA). FIG. 6 shows the interaction of Caco-2 cells with 0.1. mu.M [ Dmt¹，dnsDap⁴]After incubation of DALDA at 37 ℃ for 15 minutes, internalization of the fluorescent peptide into Caco-2 cells. The fluorescence appeared to diffuse throughout the cytoplasm without significant vesicular distribution, indicating that the uptake of the peptide was not involved in endocytosis and that the peptide was not enclosed within the endosome. It was also noted that the peptide was completely excluded from the nucleus. In SH-SY5Y cells and 0.1 mu M [ Dmt ]¹，atnDap⁴]After 30 min incubation of DALDA at 4 ℃, [ Dmt¹，atnDap⁴]DALDA internalizes into SH-SY5Y cells, clearly supporting an energy-independent, endocytosis-independent uptake mechanism, since endocytosis is an energy-dependent process.

Example 10: the peptide was attached to the cross-linker SMCC and confirmed by mass spectrometry And (4) identifying.

SMCC (1. mu.g) and 5. mu.g of [ Dmt [ [ Dmt ]¹]DALDA，[Phe¹]DALDA, or [ D-arginine-Dmt-lysine-phenylalanine-NH₂]Dissolved together in 2ml PBS, incubated at room temperature for 30 minutes, and then stored at 4 ℃. An aliquot of this sample was mixed with a matrix (3-hydroxypyridine carboxylic acid (HPA) saturated in 50% acetonitrile, 10mg/ml ammonium citrate) in a ratio of 1:10 and then spotted onto a stainless steel target plate. The samples were analyzed in forward reflectance mode by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Applied Biosystems (Voyager DE Pro)). The molecular weights of the peptides and their respective peptide-SMCC conjugate conjugates are shown on the mass spectrum (fig. 7).

Example 11: the peptide bound to the protein cargo carries the protein cargo into the fine And (4) cells.

Various peptides were cross-linked to β -galactosidase (recombiant e.coli, Sigma-Aldrich) using the cross-linking agent smcc (pierce). SMCC with amine-containing molecules ([ Dmt¹]Lys of DALDA⁴) Reacting to form a stable amide bond. The formation of the peptide-SMCC conjugate was confirmed by mass spectrometry (fig. 7). Its maleimide terminal may then be conjugated to a thiol-containing compound to form a thioether bond (B bioconjugate Techniques of Greg T. Hermanson, Academic Press, p. 234-237). Beta-galactosidase contains a large number of free thiols in its native state. Uptake of beta-galactosidase provides convenient reading with X-gal.

Briefly, 1ml of 5X 10 was added at room temperature^-3M[Dmt¹]DALDA、[Phe¹]DALDA, or [ D-arginine-Dmt-lysine-phenylalanine-NH₂]Mixed with 1mg SMCC in phosphate buffer for 1 hour. This forms an "activated peptide". The "activated peptide" was diluted 1:10 with phosphate buffer. 1mg of beta-galactosidase was added to 1ml of the 1:10 "activated peptide" and mixed at 4 ℃ for 2 hours or overnight。

Mixing cells (N)₂A neuroblastoma cells or Caco-2) were plated in 96-well plates (2X 10)⁴Individual cells/well) and subsequently contacting the cells with beta-galactosidase or with [ Dmt ] at 37 ℃¹]DALDA-crosslinked beta-galactosidase, [ Phe ]¹]DALDA or [ D-arginine-Dmt-lysine-phenylalanine-NH₂]Incubate for 1 hour. The cells were then washed 4 times with phosphate buffer. Cells were then stained with a beta-galactosidase staining system (Roche) at 37 ℃ for at least 2 hours, and then examined under a microscope.

When Caco-2 cells were incubated with β -galactosidase, no β -galactosidase uptake was observed (fig. 8A). The presence of blue cells indicates intracellular association of [ Dmt ] with Caco-2¹]Uptake of DALDA conjugated β -galactosidase (fig. 8B). In the presence of beta-galactosidase and [ D-arginine-Dmt-lysine-phenylalanine-NH₂](FIG. 8C) or [ Phe¹]Upon conjugate binding of DALDA (fig. 8D), increased uptake of β -galactosidase was also found. Conjugation of β -galactosidase to SMCC alone did not increase uptake.

In the utilization of nerve N₂Similar results were obtained for A cells and CHO cells (Chinese hamster ovary cells).

Example 12: and [ Dmt ¹ ]Incubation of the DALDA-SMCC conjugate increased green color Uptake of fluorescent protein (GFP) into Huh7 cells.

Washing of Huh7 cells (1X 10) with DMEM⁶Individual cells/well) followed by incubation of the cells with 0.5ml of DMEM for 60 minutes at 37 ℃, the DMEM: contains only 3. mu.g GFP; containing 3. mu.g GFP and 40. mu.l [ Dmt ]¹]DALDA; or 3. mu.g GFP and 40. mu.l [ Dmt ] bound to SMCC¹]DALDA. 2ml of cell culture medium was then added to the cells and incubated in the cell culture incubator for an additional 24 hours. After incubation, cells were washed four times in cell culture medium, followed by observation of GFP remaining in viable cells by confocal laser scanning microscopy. At 340nmExcitation was performed and emission was measured at 520 nm.

Fig. 9 (top panel) represents an image of GFP passing through a 0.8 μm thick central horizontal optic portion of Huh7 cells. Fig. 9 (bottom panel) represents a comparative image of different interfaces within the same area.

GFP and [ Dmt¹]Incubation with DALDA showed: the green fluorescence in the cell cytoplasm was moderately increased compared to incubation with GFP alone (fig. 9B). No green fluorescence was observed in the nuclei. GFP and [ Dmt¹]Incubation with DALDA-SMCC conjugate showed even more GFP uptake (fig. 9C). These data show that: [ Dmt¹]DALDA can increase the uptake of a protein into a cell only by physical mixing of the modified peptide with the protein, without the need for chemical binding between the peptide and the protein.

Example 13: [ Dmt ¹ ]Binding of DALDA to RNA cargo.

Use of gamma-³²P-ATP phosphorylates synthetic RNA oligomers (40 nucleotides in length) at the 5' end. The product was purified by gel. In the presence of 1mg of EDC (N- [ 3-dimethylaminopropyl-N' -ethylcarbodiimide)]) In the presence of 500,000 counts/min of gel-purified RNA oligomer at 10mM [ Dmt¹]DALDA was conjugated in the reaction. Will bind to the product of the reaction ([ Dmt¹]DALDA-RNA oligomer) was analyzed on a 15% polyacrylamide urea gel with control RNA oligomer alone. Two separate bands on the gel represent the RNA oligomer alone and [ Dmt¹]Conjugate of DALDA-RNA oligomer (fig. 10).

Example 14: [ Dmt ¹ ]The conjugate of DALDA-RNA oligomer is taken up Caco-2 cells.

Caco-2 cells (1X 10)⁶) Washing in DMEM MediumThree times and preincubated in DMEM for 5 minutes before addition of oligomer. Subsequently, [ Dmt [ ] is¹]Conjugate or unconjugated RNA (each approximately 20,000 counts/minute) of DALDA-RNA oligomer was added to the cell culture medium and incubated at 37 ℃ for 60 minutes.

After incubation, the reaction medium was removed, the cells were washed four times with DMEM and once in sodium acetate solution to reduce non-specific binding. Finally, cells were incubated in lysis buffer for 30 minutes, followed by radioactivity determination in the cell lysate.

Caco-2 cells showed: to [ Dmt¹]Uptake of the conjugate of DALDA-RNA oligomer was more than three times greater than uptake of the unconjugated conjugated RNA oligomer alone (fig. 11). Thus, [ Dmt¹]DALDA can facilitate the passage of RNA oligomers across cell membranes.

Example 15: RNA and [ Dmt ¹ ]Hybrid enhancement of DALDA-SMCC linkers The RNA is taken into the cells.

By combining RNA with [ Dmt ]¹]The DALDA-SMCC conjugate was physically mixed to form the carrier complex. By mixing [ Dmt ] as described below¹]Preparation of [ Dmt ] by mixing DALDA with crosslinker SMCC¹]A DALDA-SMCC conjugate. Prior to use in a cellular uptake study, the single-stranded 11-mer³²P]RNA oligomer and [ Dmt¹]The DALDA-SMCC conjugate was mixed for 15 minutes at room temperature.

Washing of Huh7 cells (1X 10) with DMEM⁶Individual cells/well), followed by incubating the cells with 1.0ml of DMEM containing only [2 ], [2 ] at 37 ℃ or 4 ℃ ]³²P]An RNA oligomer (. about.100,000 cpm), or comprises³²P]RNA oligomer and 40. mu.l [ Dmt ]¹]A DALDA-SMCC conjugate. The cells were then washed four times in DMEM and once in sodium acetate solution to remove non-specific binding before incubating them in lysis buffer for 30 minutes, and then the retained radioactivity was determined.

RNA oligomerizationObject and [ Dmt¹]Incubation of DALDA-SMCC at 37 ℃ increased uptake of RNA oligomers as a function of time (fig. 12A). At one hour, the uptake of RNA oligomers is [ Dmt¹]The DALDA-SMCC conjugate increased by-20 fold in the presence compared to RNA incubation alone. Uptake of RNA by [ Dmt¹]DALDA-SMCC was significantly improved (fig. 12B). These data show that: may not be subjected to AND [ Dmt ]¹]Chemical conjugation of DALDA increased RNA uptake. Uptake at 4 ℃ increased the energy-independent non-endocytosis process with [ Dmt¹]DALDA is consistent with its ability to enter the cell membrane by passive diffusion.

Except that [ Dmt¹]DALDA-SMCC, with [ Phe ]¹]Incubation of the DALDA-SMCC conjugate also increased uptake of the 11-mer RNA oligomer. Figure 12C shows an increase in RNA uptake, incubated with three different peptide-SMCC conjugates for 15 minutes at 37 ℃.

As shown in FIG. 13, [ Dmt ]¹]Incubation with DALDA-SMCC conjugate also promoted cellular uptake of much larger RNA molecules (1350-mers), although not as much for smaller oligomers.

Example 16: [ Dmt ¹ ]Binding of DALDA to DNA oligomers.

SMCC (1. mu.g) and [ Dmt ]¹]DALDA (SS 002; 5. mu.g) was dissolved together in 2ml PBS, incubated at room temperature for 30 minutes and subsequently mixed with the deprotected 3' -thiol DNA oligomer at 4 ℃ for 24 hours. After incubation, an aliquot of the sample was mixed with a matrix (3-hydroxypyridine carboxylic acid (HPA) saturated in 50% acetonitrile, 10mg/ml ammonium citrate) in a ratio of 1:10 and spotted onto a stainless steel target plate.

Confirmation of DNA- [ Dmt ] by MALDI-TOF MS¹]Formation of DALDA conjugates. 3' -thiol group DNA oligomer and [ Dmt ] have been found¹]The molecular weights of the DALDA-DNA covalent complexes were 6392 and 7171, respectively (fig. 14A).

Example 17: [ Dmt ¹ ]The conjugate of DALDA-DNA oligomer is taken into the fine And (4) cells.

Conjugation of 3' -thiol-modified 20-mer DNA to [ Dmt ] Using SMCC¹]DALDA, and confirmation of binder formation by mass spectrometry. By using³²P bound and unbound DNA oligomers were labeled with a radioisotope at their 5' -end (fig. 14B).

Flushing of nerves N with DMEM₂A(1×10⁶Individual cells/well) cells, then at 37 ℃ and 5% CO₂The cells were incubated for 2 hours or 19 hours with 1ml of DMEM with or without [ Dmt ] bound to DNA oligomers (-100,000 cpm)¹]DALDA. The cells were then washed four times in DMEM and once in sodium acetate solution to reduce non-specific binding. The cells were then incubated in lysis buffer for 30 minutes, and the retained radioactivity was then determined.

After 19 hours of incubation with [ Dmt ]¹]DALDA-bound DNA was taken up more than unbound DNA (FIG. 15), indicating binding to [ Dmt [ [ Dmt ]¹]The binding of DALDA can increase DNA uptake.

Example 18: the peptides and peptide-SMCC conjugates were not toxic to cells.

Neither the peptide nor the peptide-SMCC conjugate was toxic to the cultured cells. By [ Dmt¹]DALDA (1nM to 10. mu.M) treatment for 24 hours had no effect on cell viability by affecting N₂MTT assay of A cells, SH-SY5Y cells or Caco-2 cells (MTS assay, Promega, Madison, Wis.) (FIG. 16). P- [ D-arginine-Dmt-lysine-phenylalanine-NH₂]The same study also showed no effect on cell viability.

Incubation of cultured cells with peptide-SMCC conjugate also had no effect on cell viability as measured by trypan blue uptake. Trypan blue is taken up only by cells with increased cell membrane permeability. In DMEM, with 1ml of fresh medium, or containing 50. mu.l of 1mM [ Dmt¹]DALDA-SMCC conjugate, [ dextro-arginine-Dmt-lysine-phenylalanine-NH₂]-SMCC conjugate, or [ Phe¹]Huh7 cells (1X 10) in DALDA-SMCC conjugate⁶) Washed three times and at 37 ℃ and 5% CO₂And incubated for 24 hours. The cells were then washed three times with DMEM, and then 1ml of 0.4% trypan blue was added to the cells for 2 minutes. Excess dye was removed by washing the cells in cell culture medium and the cells were examined by light microscopy.

Examination of the cells by light microscopy showed: cells incubated with medium alone showed minimal trypan blue uptake. At and [ Dmt¹]DALDA-SMCC, [ D-arginine-Dmt-lysine-phenylalanine-NH₂]-SMCC, or [ Phe¹]No increase in trypan blue uptake was observed in DALDA-incubated cells. In contrast, cells incubated with DEPC (diethylpyrocarbonate) produced a significant increase in trypan blue uptake.

Cultured cells with [ Dmt¹]Incubation with DALDA-SMCC conjugate also did not cause apoptosis in cultured Huh7 cells. Huh7 cells (1X 10)⁶Individual cells/well) were washed three times in DMEM and 1ml of fresh medium was added. Subsequently, 50. mu.l of the modified [ Dmt ] PBS was added¹]DALDA (1mM) or PBS only (control) was added to the cell culture medium and incubated at 37 ℃ and 5% CO₂And incubated for 24 hours. After incubation, 1ml of Hoechst dye (Molecular Probes, ewing, oregon) that can stain apoptotic nuclei was added to the cells and incubated for an additional 15 minutes. Excess Hoechst dye was removed by washing the cells with cell culture medium (without pH indicator) and then compared by [ Dmt ] using a fluorescence microscope (excitation at 350nm, emission measured at 461 nm)¹]DALDA-SMCC conjugate treated cells and control cells. Apoptosis is shown by the concentration of fluorescence in the nucleus. FIG. 17 shows that: through [ Dmt ]¹]The level of apoptosis in DALDA-SMCC treated Huh7 cells was the same as the level of apoptosis in control cells.

Claims (87)

1. Use of a carrier complex for the manufacture of a medicament for delivering a molecule to a cell, wherein the carrier complex comprises the molecule and an aromatic-cationic peptide, wherein the aromatic-cationic peptide has the formula:

2 ', 6' -Dmt-D-arginine-phenylalanine-lysine-NH₂，

D-arginine-2 ', 6' Dmt-lysine-phenylalanine-NH₂Or is or

phenylalanine-dextro-arginine-phenylalanine-lysine-NH₂。

2. The use of claim 1, wherein the molecule is a small molecule.

3. The use of claim 2, wherein the small molecule is a pharmaceutically active molecule.

4. Use according to claim 3, wherein the pharmaceutically active molecule is an antibiotic.

5. The use of claim 3, wherein the pharmaceutically active molecule is a cytotoxic agent.

6. The use of claim 5, wherein the cytotoxic agent is streptomycin.

7. The use of claim 5, wherein the cytotoxic agent is doxorubicin.

8. Use according to claim 3, wherein the pharmaceutically active molecule is an antioxidant.

9. Use according to claim 8, wherein the antioxidant is vitamin E.

10. Use according to claim 8, wherein the antioxidant is vitamin C.

11. Use according to claim 8, wherein the antioxidant is β -carotene.

12. The use of claim 1, wherein the molecule is a biomolecule.

13. The use of claim 12, wherein the biomolecule is a polyamino acid.

14. The use of claim 12, wherein the biomolecule is a pharmaceutically active molecule.

15. The use of claim 14, wherein the pharmaceutically active molecule is an endogenous peptide or protein.

16. Use according to claim 14, wherein the pharmaceutically active molecule is an enzyme.

17. The use of claim 14, wherein the pharmaceutically active molecule is an antibody.

18. The use of claim 14, wherein the pharmaceutically active molecule is a nerve growth factor.

19. The use of claim 14, wherein the pharmaceutically active molecule is a cytokine.

20. The use of claim 14, wherein the pharmaceutically active molecule is a polynucleotide.

21. Use according to claim 14, wherein the pharmaceutically active molecule is an oligonucleotide.

22. The use of claim 21, wherein the oligonucleotide is RNA.

23. The use of claim 22, wherein the RNA is double-stranded RNA.

24. The use of claim 23, wherein the double stranded RNA is siRNA.

25. The use of claim 21, wherein the oligonucleotide is DNA.

26. The use of claim 21, wherein the oligonucleotide is a single-stranded RNA.

27. The use of claim 26, wherein the single-stranded RNA is messenger RNA.

28. The use of claim 21, wherein the oligonucleotide is a ribozyme.

29. The use of claim 21, wherein the oligonucleotide is an antisense RNA.

30. The use of claim 21, wherein the oligonucleotide is an external guide sequence for a ribozyme.

31. The use of claim 21, wherein the oligonucleotide is an RNA decoy.

32. The use of claim 12, wherein the biomolecule is a polyamino acid.

33. The use of claim 1, wherein the cell is a bacterial cell.

34. The use of claim 1, wherein the cell is a plant cell.

35. The use of claim 1, wherein the cell is an animal cell.

36. The use of claim 35, wherein the animal cell is a mammalian cell.

37. The use of claim 35, wherein the cell is a neural cell.

38. The use of claim 35, wherein the cell is a renal epithelial cell.

39. The use of claim 35, wherein the cell is an intestinal epithelial cell.

40. The use of claim 35, wherein the cell is a vascular endothelial cell.

41. The use of claim 35, wherein said vascular endothelial cells are blood brain barrier endothelial cells.

42. The use of claim 35, wherein the cell is a glial cell.

43. The use of claim 35, wherein the cell is a hepatocyte.

44. The use of claim 1, wherein the aromatic-cationic peptide comprises a linker.

45. The use of claim 1, wherein the molecule comprises a linker.

46. Use according to claim 1, wherein the molecule and the aromatic-cationic peptide are chemically bound.

47. The use of claim 1, wherein the molecule and aromatic-cationic peptide are physically bound.

48. A carrier complex comprising a molecule and an aromatic-cationic peptide, wherein the aromatic-cationic peptide has the formula:

2 ', 6' -Dmt-D-arginine-phenylalanine-lysine-NH₂，

D-arginine-2 ', 6' Dmt-lysine-phenylalanine-NH₂Or is or

phenylalanine-dextro-arginine-phenylalanine-lysine-NH₂。

49. A carrier complex according to claim 48, wherein the molecule is a small molecule.

50. A carrier complex according to claim 48, wherein the small molecule is a biomolecule.

51. A carrier complex according to claim 50, wherein the biomolecule is a polyamino acid.

52. A carrier complex according to claim 49, wherein the small molecule is a pharmaceutically active molecule.

53. A carrier complex according to claim 52, wherein the pharmaceutically active molecule is an antibiotic.

54. A carrier complex according to claim 52, wherein the pharmaceutically active molecule is a cytotoxic agent.

55. A carrier complex according to claim 54, wherein the cytotoxic agent is streptomycin.

56. A carrier complex according to claim 54, wherein the cytotoxic agent is doxorubicin.

57. A carrier complex according to claim 52, wherein the pharmaceutically active molecule is an antioxidant.

58. A carrier complex according to claim 57, wherein the antioxidant is vitamin E.

59. A carrier complex according to claim 57, wherein the antioxidant is vitamin C.

60. A carrier complex according to claim 57, wherein the antioxidant is β -carotene.

61. A carrier complex according to claim 48, wherein the molecule is a biomolecule.

62. A carrier complex according to claim 61, wherein the biomolecule is a pharmaceutically active molecule.

63. A carrier complex according to claim 62, wherein the pharmaceutically active molecule is an endogenous peptide or protein.

64. A carrier complex according to claim 62, wherein the pharmaceutically active molecule is an enzyme.

65. A carrier complex according to claim 62, wherein the pharmaceutically active molecule is an antibody.

66. A carrier complex according to claim 62, wherein the pharmaceutically active molecule is a nerve growth factor.

67. A carrier complex according to claim 62, wherein the pharmaceutically active molecule is a cytokine.

68. A carrier complex according to claim 62, wherein the pharmaceutically active molecule is a polynucleotide.

69. A carrier complex according to claim 62, wherein the pharmaceutically active molecule is an oligonucleotide.

70. A carrier complex according to claim 69, wherein the oligonucleotide is RNA.

71. A carrier complex according to claim 70, wherein the RNA is a double stranded RNA.

72. A carrier complex according to claim 71, wherein the double stranded RNA is siRNA.

73. A carrier complex according to claim 69, wherein the oligonucleotide is DNA.

74. A carrier complex according to claim 69, wherein the oligonucleotide is a single stranded RNA.

75. A carrier complex according to claim 74, wherein the single stranded RNA is messenger RNA.

76. A carrier complex according to claim 69, wherein the oligonucleotide is a ribozyme.

77. A carrier complex according to claim 69, wherein the oligonucleotide is an antisense RNA.

78. A carrier complex according to claim 69, wherein the oligonucleotide is an external guide sequence for a ribozyme.

79. A carrier complex according to claim 69, wherein the oligonucleotide is an RNA decoy.

80. A carrier complex according to claim 61, wherein the biomolecule is a polyamino acid.

81. A carrier complex according to claim 48, wherein the aromatic-cationic peptide comprises a linker.

82. A carrier complex according to claim 48, wherein the molecule comprises a linker.

83. A carrier complex according to claim 48, wherein the molecule and aromatic-cationic peptide are chemically bound.

84. A carrier complex according to claim 48, wherein the molecule and aromatic-cationic peptide are physically bound.

85. A method of delivering a molecule to a cell in vitro, the method comprising contacting a molecule and an aromatic-cationic peptide with the cell in vitro, wherein the aromatic-cationic peptide has the formula:

2 ', 6' -Dmt-D-arginine-phenylalanine-lysine-NH₂，

D-arginine-2 ', 6' Dmt-lysine-phenylalanine-NH₂Or is or

phenylalanine-dextro-arginine-phenylalanine-lysine-NH₂。

86. The method of claim 85, wherein said molecule and aromatic-cationic peptide are chemically bound.

87. The method of claim 85, wherein the molecule and aromatic-cationic peptide are physically bound.