Patent Application
20100279918
The present invention relates to chimeric constructs between cancer-homing peptides and cell-penetrating peptides coupled to anticancer drugs and/or diagnostic agents for cancer treatment and/or diagnostic purposes. These constructs result in breast cancer cell homing and breast cancer cell penetration both in vitro and in vivo.
By definition, cell-penetrating peptides (CPP) consist of less than 30 amino acids and have a net positive charge1; 2. CPPs internalize in living animal cells in vitro3; 4 and in vivo5; 6 in both, endocytotic, or seemingly receptor- or energy-independent manner7; 8 with subsequent re-evaluation of the translocation mechanisms for some CPPs9; 10; 11. There are several classes of CPPs with various origins, from totally protein-derived CPPs via chimeric CPPs to completely synthetic CPPs. The mechanism of internalization of CPPs is not yet characterized in detail, but the uptake seems to be different between the classes with a different element of endocytosis12; 13. pVEC (SEQ ID NO:6) is an 18-amino acid long cell-penetrating peptide derived from murine vascular endothelial cadherin (amino acids 615-632)14; 15.
The plasma membrane is impenetrable for most polar hydrophilic macromolecules as proteins and oligonucleotides. Those molecules internalize into animal cells mainly via endocytosis. Several endocytotic mechanisms have been described16 among which the clathrin-dependent receptor-mediated endocytosis is used for internalization of ligand-receptor complexes17. Most common procedures to deliver polar hydrophilic macromolecules into cells are electroporation and microinjection, but unfortunately the molecules can be delivered only in in vitro systems by these methods18; 19.
Blood vessels in normal individual tissues and different tumor tissues distinguish from each other due to the expression of different molecular markers20; 21. In tumors both blood and lymphatic vessels differ from normal vessels22. Several peptides have been isolated by combining ex vivo and in vivo phage display for homing to tumors23; 24; 25; 26. Drug therapy for treating cancer is limited by a narrow therapeutic index. By coupling an anti-cancer drug to the homing motif it can be possible to direct the drug to the cancer cells23; 27; 28.
The present invention provides chimeric constructs between cancer-homing peptides and cell-penetrating peptides (CPPs) coupled to anticancer drugs and/or diagnostic agents for cancer treatment and/or diagnostic purposes.
By coupling a cancer-homing peptide to a CPP the construct will be able to internalize targeted cells. Additionally coupling or conjugating an anticancer drug to the cancer-homing-CPP the drug can be transported into breast cancer cells. This targeting can efficient the therapy while reducing side effects. When a diagnostic agent is coupled to the cancer-homing-CPP, the construct can be used for diagnostic purposes for detection of breast cancer cells by use of an appropriate method adapted for detection of the marker. When a diagnostic agent is coupled to a cancer-homing-CPP that carries an anticancer drug, the internalized construct can be used to monitor the effect of the anticancer drug on the breast cancer cells by use of an appropriate method adapted for detection of the marker.
One aspect of the invention is directed to a construct comprising a cancer-homing peptide, an optional linker and a cell-penetrating peptide coupled to an anticancer drug and/or a marker.
In an embodiment of the invention the cancer-homing peptide is selected from a linear pentapeptide CREKA (SEQ ID NO:1), AREKA (SEQ ID NO: 23) or CREKAD (SEQ ID NO: 23), and a cyclic nonapeptide CPGPEGAGC (SEQ ID NO:2), wherein the two cysteines are cyclized.
The optional linker can be an amino acid or peptide that does not interfere with the respective functions of the cancer-homing peptide and cell-penetrating peptide.
In another embodiment of the invention the cell-penetrating peptide is selected from the following peptide sequences:
wherein the peptides are C-terminal free acids unless stated otherwise.
In a further embodiment of the invention, the anticancer drug is selected from alkylating agents, such as 4-[4-Bis(2-chloroethyl)amino)phenyl]butyric acid (Chlorambucil, further referred to as Cbl) or 3-[4-(Bis(2-chloroethyl)amino)phenyl]-L-alanine (Melphalan), antimetabolites, such as N-[4-(N-(2,4-Diamino-6-pteridinylmethyl)methylamino)-benzoyl]-L-glutamic acid (Methotrexate) and cytotoxic antibiotics, such as (8S,10S)-10-[(3-Amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione (Doxorubicin).
In yet another embodiment of the invention the diagnostic agent is a fluorescent label, such as a fluoresceinyl label.
In a preferred embodiment of the invention, the construct is the peptide CPGPEGAGC-LLIILRRRIRKQAHAHSK-amide (SEQ ID NO:21).
In another preferred embodiment of the invention the construct is the peptide CREKA-LLIILRRRIRKQAHAHSK-amide (SEQ ID NO:22).
Another aspect of the invention is directed to a method of delivering an anticancer drug and/or a diagnostic agent into a cancer cell comprising administration of a construct according to the invention in vivo to a subject expected to have breast cancer cells, such as breast cancer cells, or in vitro to a tissue sample or cell culture.
The invention will now be illustrated with reference to the drawings and the experiments.
In this study, two cancer homing peptides, a cyclic nonapeptide CPGPEGAGC (SEQ ID NO:2) (cyclic between the two cysteines)25 and a linear pentapeptide CREKA (SEQ ID NO:1), have been coupled to the N-terminus of the cell-penetrating peptide pVEC14 (SEQ ID NO:6) in order to achieve the synergistic properties of both components, i.e. the cancer cell targeting together with cellular penetration of the construct. A well-known anticancer drug, chlorambucil (
Peptides (Table 1) were synthesized automatically (model 431A; Applied Biosystems) on solid support p-methylbenzhydrylamin resin (Neosystem) (substitution 1.16 mmol/g) generating C-terminally amidated peptides according to manufacturers instructions. Stepwise coupling reactions were performed with t-Boc-protected amino acids (Neosystem), 1-hydroxybenzotriazole, N,N′-dicyclohexylcarbodiimide (4:4:4 eq, 35 min, RT) followed by N-terminal deprotection of the t-Boc-group with TFA:dichloromethane (1:1) (14 min, RT).
For cellular uptake and in vivo studies the peptides were labeled N-terminally with 5(6)-carboxyfluorescein (Molecular Probes) (3 eq) in N,N′-diisipropylcarbodiimide (3 eq) and hydroxybenzotriazole (3 eq) in dimethyl sulfoxide:dimethylformamide (1:2) in dark overnight.
For cytotoxicity studies chlorambucil (4 eq) (Sigma-Aldrich) was coupled to N-terminal amino group of the peptides with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (4 eq), 1-hydroxybenzotriazole (4 eq) and diisopropylethylamine (8 eq) in dimethylformamide for 30 min.
Deprotection of the dinitrophenyl group was performed by treating the peptides with thiophenol:dimethylformamide (1:4) (1 h, RT). Final cleavage of the peptides from the resin was performed in hydrofluoric acid (1 h, 0° C.) in the presence of p-cresol and p-thiocresol.
Fluoresceinyl labeled or chlorambucil coupled CPGPEGAGC-amide (SEQ ID NO:2) (in text and figures referred to as PEGA) and CPGPEGAGC-pVEC-amide (SEQ ID NO:21) (in text and figures referred to as PEGA-pVEC) were incubated overnight in dimethyl sulfoxide generating disulfide bridges between cysteines.
Peptides (Table 3) were synthesized automatically (Syro2000 Multiple Peptide Synthesizer; MultiSynTech GmbH) on solid support RINK amide methylbenzhydrylamin resin (Neosystem) (substitution 0.85 mmol/g) generating C-terminally amidated peptides according to manufacturers instructions. Stepwise coupling reactions were performed with Fmoc-protected amino acids (MultiSynTech GmbH), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, diisopropylethylamine (4:8:8 eq, 30 min, RT) followed by N-terminal deprotection of the Fmoc-group with 20% piperidine in DMF (v/v) (20 min, RT).
For in vivo studies the peptides were labeled N-terminally with 5(6)-carboxyfluorescein (Molecular Probes) (3 eq) in N,N′-diisipropylcarbodiimide (3 eq) and hydroxybenzotriazole (3 eq) in dimethyl sulfoxide:dimethylformamide (1:2) in dark overnight.
Final cleavage of the peptides from the resin was performed using TFA-scavengers cocktail. Standard reagent K content was modified to prevent irreversible modification of product by linker. The cocktail mixture contained 95% TFA, 2.5% water, 2.5% TIS (sometimes with addition of 2.5% thioanisole).
The peptides (Table 1 and Table 3) were purified by RP-HPLC (Discovery C-18 HPLC column, 25 cm, 21.2 mm, 5 μm) using a gradient of acetonitrile/water with 0.1% TFA (50 min, 2 ml/min). The identity and quality of the purified products were verified by matrix assisted laser desorption ionization time-of-flight mass-spectrometer (prOTOF™ 2000 MALDI O-TOF, PerkinElmerSCIEX). The mass-spectra were acquired in positive ion reflector mode using α-cyano-4-hydroxycinnamic acid as a matrix (Sigma-Aldrich) (10 mg/ml, 7:3 acetonitrile:water, 0.1% TFA).
The human breast cancer cell line MDA-MB-231 and the human breast cancer cell line MCF-7 (ATCC via LGC, Sweden) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, sodium pyruvate (1 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) and 1% nonessential amino acids. The human breast cancer cell line MDA-MB-435 was maintained in DMEM supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 μg/ml). All cells were incubated at 37° C. in 5% CO2.
MCF-7 and MDA-MB-231 cells (150 000 cells/well) were seeded on a 12 well plate two days before the experiment. The cells were washed with HEPES buffered krebs ringer solution (HKR) (2×1 ml) and exposed to a drug solution (500 μl, 5 μM) for 30 min at 37° C. The cells were washed with HKR (2×1 ml) and treated with trypsin-EDTA (200 μl) for 5 min at 37° C. HKR (1 ml) was added and the cells were transferred to eppendorf tubes and centrifuged (1000×g) for 5 min at 4° C. Cell pellet was lysed with NaOH (300 μl, 0.1 M) for 1 h at 4° C. and centrifuged (10000×g) for 10 min at 4° C. The fluorescence (494/518 nm) was measured with SPEKTRAmax® GEMINI XS spectrofluorometric plate reader (Molecular Devices).
MCF-7 and MDA-MB-231 cells (20 000 cells/well) were seeded two days before the experiment on a Lab-Tek® 8-well chamber slides. The cells were washed with HKR (2×400 μl) and exposed to drug solution (400 μl, 5 μM) for 45 min at 37° C. The cells were washed with HKR (3×400 μl) where after the pictures were taken with UltraView ERS Confocal Live Cell Imager microscope (Zeiss, PerkinElmer).
MDA-MB-435 cells (10 000 cells/well) were seeded one day before the experiment on a Lab-Tek® 8-well chamber slides. The cells were washed with serum-free DMEM (2×300 μl) and exposed to drug solution (300 μl, 10 μM or 20 μM) for 1 h or 4 h at 37° C. The cells were washed five times (5×300 μl) with PBS, fixed with 4% PFA at room temperature for 15 minutes and washed two times with PBS. The slides were mounted in Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame), and observed under a Radiance MP confocal microscope.
MCF-7 and MDA-MB-231 cells (20 000 cells/well) were seeded in a 48 well plate two days before the experiment. The cells were exposed to drug in different concentration in full media containing 1% FBS for 3 h where after 10% FBS containing full media was added to achieve 5% FBS containing media and then incubated 45 or 69 h at 37° C. The plate was cooled to room temperature and CellTiter-Glo® Reagent (Promega) was added. The plate was incubated for 10 min at room temperature where after the luminescence was measured.
To produce tumors, nude BALB/c mice were orthotopically injected with 1×106 MDA-MB-435 tumor cells into mammary fat pad. The tumor bearing mice were used for homing analysis of peptides when the tumor size reached about 10 mm. Tissue distribution of fluoresceinyl labeled peptides was studied by intravenously injecting the peptides (100 μM in 200 μl PBS) into tumor-bearing mice. The injected peptides were allowed to circulate 2 or 24 h, and the mice were perfused with 4% paraformaldehyde through the left ventricle of heart. Tissues were dissected and frozen in OCT embedding medium (Tissue-Tek, Elkhart, Ind.). Frozen sections were prepared for immunohistological analysis.
Treatment of Mice Bearing MDA-MB-435 Tumors with CBL-CREKA-pVEC (SEQ ID NO:1) Peptide Conjugates
The orthotopic xenografted breast tumors were established by injecting 1×106 MDA-MB-435 human breast cancer cells into the mammary fat pad of nude Balb/c mice. Treatment started when mean tumor volumes reached about 100 mm3. Mice with size-matched tumors were randomized into five groups (eight animals per group): Cbl-CREKA-pVEC (SEQ ID NO:22), Cbl plus CREKA-pVEC, Cbl-CREKA (SEQ ID NO:1), Cbl-pVEC (SEQ ID NO:6) and PBS. Mice were treated intravenously with 15 μg of Cbl-equivalent/mouse every other day for seven times. Tumor size was measured every three days. The mice were monitored for weight loss. The animal experiments reported here were approved by the Animal Research Committee of Burnham Institute for Medical Research.
To determine the cell-penetrating properties of the homing peptides alone and homing-CPP chimeras the uptake of the fluoresceinyl labeled peptides was investigated quantitatively (
To determine that the concentration (10 μM) used for pVEC (SEQ ID NO:6) and its conjugates for uptake studies are not toxic, the toxicity of different concentrations of pVEC was measured (
Coupling a cytotoxic agent to the homing conjugate could direct the drug into tumors. Chlorambucil is a cytotoxic agent that acts inside the cell. It alkylates DNA and prevents proliferation of cancer cells and may also induce apoptosis. The ability of the chimeric peptides to internalize chlorambucil as measured by the ability of the conjugates to kill tumor cells was investigated in two different cell lines, MCF-7 and MDA-MB-231. Cbl-CREKA-pVEC (SEQ ID NO:22) improved Cbl toxicity both in MCF-7 (IC50 value 10.5 μM) (
Homing Specificity of Chimeric pVEC (SEQ ID NO:6) Peptides
To evaluate the homing specificity of the CREKA-pVEC (SEQ ID NO:22), the mice bearing MDA-MB-435 tumors were intravenously injected with 100 μM of fluoresceinyl labeled CREKA-pVEC chimeric peptide (SEQ ID NO:22) or an equimolar amount of fluoresceinyl labeled CREKA (SEQ ID NO:1) or fluoresceinyl labeled pVEC (SEQ ID NO:6) as controls. The results showed that the chimeric peptide strongly homes to tumors, but not to control organs such as liver, heart, skin, kidney, gut and brain. The pattern of tissue distribution of this chimeric peptide is similar to that of CREKA peptide (SEQ ID NO:1) alone except the chimeric peptide is weakly positive in lung and spleen (
To study the association of CREKA-pVEC peptides (SEQ ID NO:22) with the vasculature, fluoresceinyl labeled chimeric or control peptides were intravenously injected into MDA-MB-435 tumor bearing mice, and peptide localization was compared to blood vessel markers localized with anti-MECA32 antibody. The chimeric peptide showed substantial co-localization with the MECA-32 (
A chimeric peptide composed of pVEC (SEQ ID NO:6) coupled to another tumor-homing peptide, CPGPEGAGC25 (SEQ ID NO:2) was also studied. This chimeric peptide, PEGA-pVEC (SEQ ID NO:21), showed less clear increase of tumor specificity than the CREKA (SEQ ID NO:22) conjugate, but similar to was observed with the CREKA (SEQ ID NO:22) conjugates, the homing peptide appeared to reduce the accumulation of the peptide in non-tumor tissues (
The tissue distribution and localization of CPGPEGAGC-pVEC (referred as PEGA-pVEC) (SEQ ID NO:21) were analyzed by using the same method as described above
A partial structure-activity analysis (SAR) was performed with the CREKA peptide. One amino acid at a time was replaced with alanine, except that the alanine in the original peptide was converted to a D-alanine (Table 3). Fluoresceinyl labeled peptides (100 μM) were intravenously injected into the mice bearing MDA-MB-435 tumors. After 24 h circulation, tumors and various control organs were dissected for histology analysis. The results show that the REK motif is critical for CREKA peptide (SEQ ID NO:1) homing to MDA-MB-435 tumors (
Treatment of Mice Bearing MDA-MB-435 Tumors with cbl-CREKA-pVEC Peptide Conjugates (SEQ ID NO:22)
To determine whether the CREKA-pVEC peptides (SEQ ID NO:22) could be used to improve the therapeutic index of cancer chemotherapeutics, we coupled them with chlorambucil. The Cbl-CREKA-pVEC conjugates were used to treat mice bearing MDA-MB-435 xenograft tumors.
The previous report showed that the ED15d dose of Cbl in tumor mice is 15 mg/Kg (about 300 μg per mouse for 15 days)29. Because we expected the Cbl conjugates to be more effective than the free drug, the tumor mice were treated with drug conjugates at a dose of Cbl-equivalent of 15 μg per mouse every other day for 14 days and were then observe. As shown in
The cyclic peptide CPGPEGAGC-amide (SEQ ID NO:2) and the linear peptides CREKA-amide (SEQ ID NO:1), AREKA-amide (SEQ ID NO: 23) and CREKAD-amide (SEQ ID NO: 23) are not cell penetrating by themselves. By conjugating the peptides to the cell penetrating peptide pVEC, LLIILRRRIRKQAHAHSK-amide (SEQ ID NO:6), they can be transported inside the cell in vitro and in vivo. The conjugates CPGPEGAGC-LLIILRRRIRKQAHAHSK-amide (SEQ ID NO:21) and CREKA-LLIILRRRIRKQAHAHSK-amide (SEQ ID NO:22) internalize into three different cell lines MCF-7, MDA-MB-231 and MDA-MB-435. The homing specificity of CREKA-pVEC (SEQ ID NO:22) to tumor is better than that of PEGA-pVEC (SEQ ID NO:21) (Table 2). The REK motif is critical for CREKA (SEQ ID NO:1) homing to tumor in vivo. The treatment of mice bearing MDA-MB-435 tumor with Cbl-CREKA-pVEC (SEQ ID NO:22) conjugates resulted in a significant inhibition of tumor growth compared with control groups (p<0.01).
It should be understood that the invention is not limited to the specifically disclosed examples and that modifications there are within the scope of this invention. The teachings of the cited literature are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/SE2007/000268 | 3/20/2007 | WO | 00 | 5/5/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60783396 | Mar 2006 | US |
