Cell-penetrating peptides

Abstract

A vector that includes a cell-penetrating peptide linked to a therapeutic agent. The therapeutic agent can be a cytotoxic agent, an anti-viral agent, an anti-bacterial agent, or an anti-parasitic agent.

Claims

1. A vector comprising a penetrating peptide consisting of the amino acid sequence VKKKKIKAEIKI (SEQ ID NO:2) coupled to a therapeutic agent.

2. The vector of claim 1, wherein the vector is a chimeric peptide construct.

3. The vector of claim 1, wherein the therapeutic agent is selected from the group consisting of a cytotoxic agent, an anti-viral agent, an anti-bacterial agent, and an anti-parasitic agent.

4. The vector of claim 3, wherein the cytotoxic agent is a pro-apoptotic peptide.

5. The vector of claim 4, wherein the penetrating peptide is fused to the pro-apoptotic peptide, and the vector is 23 to 70 amino acids in length.

6. The vector of claim 4, wherein the penetrating peptide is fused to the pro-apoptotic peptide at the C-terminus of the penetrating peptide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph that shows stability of the mutated peptides analyzed by Proteominer fractionation and Maldi-Tof. The ratio of intensity of each pick is represented, relative to its own control. The R residue was mutated to K (Mut1-DPT-C9h), N (Mut2-DPT-C9h) or A (Mut3-DPT-C9h). Round: control DPT-C9h peptide (SEQ ID NO:6), Square: Mut1-DPT-C9h, Triangle: Mut2-DPT-C9h, Diamond: Mut3-DPT-C9h.

(2) FIG. 2 shows an analysis of apoptosis by annexin-V-FITC staining of breast cancer cell line HBCx-12A was treated for 24h with 100 M of the control and mutated peptides.

(3) FIG. 3 shows biodistribution of Cy5DPT-C9h and Cy5Mut3DPT-C9h. Mice were grafted in interscapular by luminal breast cancer xenograft HBCx-3. They received one intraperitoneal injection of DPT-C9h or Mut3DPT-C9h labelled with Cy5 at 5 mg/kg. Control mouse received the control excipient (glucose 5%). Mice were imaged between 0 (prior to injection) and 168h after injection. Fluorescence of the tumors was calculated and normalized by using Living Image software.

EXAMPLES

Example 1

Design and Characterization of Mutated Non-Degradable DPT-C9h Penetrating Peptides

(4) 1.1. Materials and Methods

(5) Peptide Synthesis and Sequence

(6) Peptides were synthesized in an automated multiple peptide synthesizer with solid phase procedure and standard Fmoc chemistry. The purity and composition of the peptides were confirmed by reverse phase HPLC and by amino acid analysis.

(7) Analysis Peptide Stability in Human Serum

(8) Analysis of peptides degradation was done by Proteominer and Maldi-Tof as previously described.

(9) 1.2. Results

(10) TABLE-US-00013 (SEQ ID NO: 6) DPT-C9h is VKKKKIKREIKI-YVETLDDIFEQWAHSEDL,

(11) The R residue was mutated to K (Mut1-DPT-C9h), N (Mut2-DPT-C9h) or A (Mut3-DPT-C9h).

(12) FIG. 1 shows that Mut3-DPC-C9h peptide is not degraded upon 24h of contact with the human serum. In addition, the other mutants showed a higher stability compared to control peptide (DPT-C9h).

Example 2

Effect of Mutated DPT-C9h on Apoptosis

(13) 2.1. Materials and Methods

(14) Cells

(15) Human breast cancer HBCx-12A, cell line has been isolated from primary human cancer xenografts and was cultured in RPMI medium supplemented with 10% of FCS.

(16) Detection of Apoptosis by Annexin-V-FITC Staining

(17) Apoptotic cells were detected using Annexin-V (-FITC from BD biosciences) as described by the manufacturer. Briefly, the cells were washed in 1 binding buffer, centrifugated and then resuspended in 200 l of 1 binding buffer containing Annexin V-FITC (0.1 g/ml) and PI (0.5 g/ml). After incubation at room temperature in the dark for 10 min, cells were analyzed by flow cytometry. Data acquired by FACSCalibur (BD biosciences) were analyzed with Cellquest Pro software.

(18) 2.2. Results

(19) The inventors have then analyzed whether the mutated peptides retain the capacity to induce apoptosis. The breast cancer cell line HBCx-12A was treated for 24h with 100 M of the control and mutated peptides. Apoptosis was analyzed by annexin-V-FITC staining. As shown in FIG. 2, the analyzed peptides induce similar levels of apoptosis. The same result was observed when using the cell lines HBC-x3 and HBCx-17.

Example 3

Biodistribution of Mut3DPT-C9h in Tumors

(20) 4.1. Materials and Methods

(21) Peptide Synthesis and Sequence

(22) Peptides (DPT-C9h and Mut3DPT-C9h) were synthesized as described above. The fluorochrome Cy5 was added during the synthesis of the peptide.

(23) Fluorescence Assays

(24) Mice were IP (intraperitonally)-injected with the peptide Cy5DPT-C9h or Mut3DPT-C9h (5 mg/kg) and then analyzed at different times after injection.

(25) Fluorescence imaging was performed with the IVIS imaging system (IVIS 100, Caliper Life Sciences, USA). Mice were anesthetized upon analysis. Imaging acquisition time was from 1 s to 10 s, depending on the fluorescence signal. Analysis was performed using software Living Image V. 2.50 (Caliper Life Sciences).

(26) 4.2. Results

(27) Biodistribution of Mut3DPT-C9h and DPT-C9h in the breast cancer xenograft models.

(28) The inventors were interested in analyzing and comparing the biodistribution of both peptides. FIG. 3 shows the biodistribution of Cy5DPT-C9h and Cy5Mut3DPT-C9h in a breast cancer xenograft model. Mice were intraperitonally (IP) injected and biodistribution analyzed at different times upon injection.

(29) FIG. 3 shows that 6 h after IP injection, the inventors were able to detect both peptides in the tumor. The maximal peak of detection of Cy5 Mut3DPT-C9h is detected 23h after injection, slightly decreasing the intensity of the fluorescence after this time. Finally, considerable level of Cy5Mut3DPT-C9h was detected 168h after injection. The maximum level of Cy5DPT-C9h fluorescence was detected 6h after injection, slightly decreasing after this period of time. Low level of Cy5DPT-C9h was detected upon 168h of treatment.

(30) Taken together, these results show that Cy5-labelled DPT-C9h and Cy5-labelled Mut3DPT-C9h reach the tumor. More importantly, Cy5-labelled Mut3DPT-C9h showed to be more stable that the original peptide, DPT-C9h.

(31) The mutated peptide Mut3DPT-C9h shows a biodistribution in the tumor more sustained than the original peptide (DPT-C9h) since we are able to detect the fluorescence of the Cy5 fluorochrome longer that the fluorescence of DPT-C9h.

(32) This new property will allow to reduce the dose of peptide injected as well as the schedule of administration. In summary, the new mutants have a clear new advantage compared to control peptide and have a new characteristic since they are not degradable by serum proteases.

REFERENCES

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