U.S. patent application number 14/395807 was filed with the patent office on 2015-06-18 for peptide having cancer selective translocation function and use thereof.
This patent application is currently assigned to SNU R&DB Foundation. The applicant listed for this patent is Nano Intelligent Biomedical Engineering Corporation Co., Ltd., SNU R&DB Foundation. Invention is credited to Yoon Jung Choi, Chong-Pyoung Chung, Yoon Jeong Park, Jin Sook Suh.
Application Number | 20150165060 14/395807 |
Document ID | / |
Family ID | 50341668 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150165060 |
Kind Code |
A1 |
Park; Yoon Jeong ; et
al. |
June 18, 2015 |
PEPTIDE HAVING CANCER SELECTIVE TRANSLOCATION FUNCTION AND USE
THEREOF
Abstract
The present invention relates a peptide having cancer selective
translocation function, and the use thereof, and more particularly
to a VEGF-binding protein transduction domain (VPTD) represented as
SEQ ID NO: 1 or a heparin-binding protein transduction domain
(HPTD) represented as SEQ ID NO: 2, which bind specifically to VEGF
and heparin, which are overexpressed specifically in tumor cells or
tumor tissues, and to a conjugate comprising a drug linked to the
peptide. The peptide and the peptide-drug conjugate bind
specifically to VEGF and heparin in tumor cells or tumor tissue and
accumulate in the tumor cells or tumor tissue, and thus can be used
for the accurate diagnosis and treatment of cancer. Also, the
non-specific distribution of the peptide or the conjugate in the
body after administration is inhibited, and thus the side effects
thereof can be minimized. Accordingly, the peptide or the conjugate
is useful for the diagnosis and treatment of cancer.
Inventors: |
Park; Yoon Jeong; (Seoul,
KR) ; Chung; Chong-Pyoung; (Seoul, KR) ; Suh;
Jin Sook; (Seoul, KR) ; Choi; Yoon Jung;
(Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SNU R&DB Foundation
Nano Intelligent Biomedical Engineering Corporation Co.,
Ltd. |
Seoul
Seoul |
|
KR
KR |
|
|
Assignee: |
SNU R&DB Foundation
Seoul
KR
Nano Intelligent Biomedical Engineering Corporation Co.,
Ltd.
Seoul
KR
|
Family ID: |
50341668 |
Appl. No.: |
14/395807 |
Filed: |
September 16, 2013 |
PCT Filed: |
September 16, 2013 |
PCT NO: |
PCT/KR13/08324 |
371 Date: |
October 20, 2014 |
Current U.S.
Class: |
530/327 |
Current CPC
Class: |
A61K 49/0056 20130101;
A61K 38/00 20130101; C12Q 1/66 20130101; A61K 47/64 20170801; C07K
14/47 20130101; A61K 31/704 20130101; C12Q 1/26 20130101; C12Y
301/13 20130101; C07K 7/08 20130101; A61P 35/00 20180101; G01N
33/574 20130101; C12Q 1/58 20130101; C07K 2319/10 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 49/00 20060101 A61K049/00; A61K 31/704 20060101
A61K031/704; C07K 7/08 20060101 C07K007/08; A61K 38/46 20060101
A61K038/46 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 18, 2012 |
KR |
10-2012-0103620 |
Claims
1. A peptide having cancer selective translocation function
comprising VPTD (VEGF-binding protein transduction domain peptide)
peptide represented as SEQ ID NO: 1 or HPTD (heparin-binding
protein transduction domain) peptide represented as SEQ ID NO:
2.
2. The peptide having cancer selective translocation function of
claim 1, wherein the VPTD peptide represented as SEQ ID NO: 1 or
the HPTD peptide represented as SEQ ID NO: 2 binds specifically to
vascular endothelial growth factor (VEGF) and heparin in tumor
cells or tumor tissues.
3. The peptide having cancer selective translocation function of
claim 1, wherein the VPTD peptide represented as SEQ ID NO: 1 or
the HPTD peptide represented as SEQ ID NO: 2 is composed of D-type
or L-type amino acids and contains one or more amino acids selected
from the group consisting of arginine, lysine and histidine in an
amount of 70-80%.
4. The peptide having cancer selective translocation function of
claim 1, wherein the VPTD peptide represented as SEQ ID NO: 1 or
the HPTD peptide represented as SEQ ID NO: 2 is composed of D-type
or L-type amino acids and contains one or more amino acids selected
from the group consisting of arginine, lysine and histidine in an
amount of 70-80%.
5. A contrast agent for cancer diagnosis having cancer selective
translocation function, which comprises VPTD peptide represented as
SEQ ID NO: 1 or HPTD peptide represented as SEQ ID NO: 2 to which a
fluorescent substance is bound.
6. The contrast agent for cancer diagnosis having cancer selective
translocation function of claim 5, wherein the fluorescent
substance is selected from the group consisting of fluorescein
isothiocyanate (FITC), radioisotopes, quantum dots, MRI contrast
agents, fluorescein, tetramethylrhodamine, BODIPY, and Alexa.
7. A composition for treating cancer having cancer selective
translocation function, which comprises VPTD peptide represented as
SEQ ID NO: 1 or HPTD peptide represented as SEQ ID NO: 2.
8. A composition for enhancing cancer selective translocation,
which comprises VPTD peptide represented as SEQ ID NO: 1 or HPTD
peptide represented as SEQ ID NO: 2 as an active ingredient.
9. A peptide having cancer selective translocation function-drug
conjugate, which comprises a drug chemically linked to the
N-terminus or C-terminus of VPTD peptide represented as SEQ ID NO:
1 or HPTD peptide represented as SEQ ID NO: 2.
10. The peptide having cancer selective translocation function-drug
conjugate of claim 9, wherein the VPTD peptide represented as SEQ
ID NO: 1 or HPTD peptide represented as SEQ ID NO: 2 and the drug
are linked to each other by a cysteine.
11. The peptide having cancer selective translocation function-drug
conjugate of claim 9, wherein the chemical linkage of the drug to
the peptide is performed by any one crosslinking agent selected
from the group consisting of 1,4-bis-maleimidobutane (BMB),
1,11-bis-maleimidotetraethyleneglycol (BM[PEO]4),
1-ethyl-3-[3-dimethyl aminopropyl]carbodiimide hydrochloride (EDC),
succinimidyl-4-[N-maleimidomethylcyclohexane-1-carboxy-[6-amidocaproate]]
(SMCC) and its sulfonate (sulfo-SMCC), succimidyl
6-[3-(2-pyridyldithio)-ropionamido]hexanoate (SPDP) and its
sulfonate (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide
ester (MBS) and its sulfonate (sulfo-MBS), and
succimidyl[4-(p-maleimidophenyl) butyrate] (SMPB) and its sulfonate
(sulfo-SMPB).
12. The peptide having cancer selective translocation function-drug
conjugate of claim 9, wherein the drug is selected from the group
consisting of an antisense oligonucleotide targeting RNA of a
protein that causes a tumor or inflammatory disease, a siRNA
targeting RNA of a protein that causes a tumor or inflammatory
disease, an anticancer agent, an anticancer protein, an
anti-inflammatory agent, an anti-inflammatory protein, an
immune-enhancing protein, and a bone resorption inhibitor.
13. The peptide having cancer selective translocation function-drug
conjugate of claim 12, wherein the protein that causes a tumor or
inflammatory disease is selected from the group consisting of
vascular endothelial growth factor (VEGF), B-cell leukemia/lymphoma
2 (BCL2), epidermal growth factor receptor (EGFR), human epidermal
growth factor receptor 2 (HER2), Janus kinase (JAN), and
phosphatidylinositol-3-kinase/Akt kinase (PI3-K/AKT).
14. A composition for treating cancer comprising a peptide having
cancer selective translocation function-drug conjugate, which
comprises a drug chemically linked to the N-terminus or C-terminus
of VPTD peptide represented as SEQ ID NO: 1 or HPTD peptide
represented as SEQ ID NO: 2.
15. The composition for treating cancer of claim 14, wherein the
drug is selected from the group consisting of an anticancer agent,
an anticancer protein, an antisense oligonucleotide targeting RNA
of a protein that cause a tumor, and a siRNA targeting RNA of a
protein that cause a tumor.
16. A drug delivery system having cancer selective translocation
function, which comprises a drug linked to the N-terminus or
C-terminus of VPTD peptide represented as SEQ ID NO: 1 or HPTD
peptide represented as SEQ ID NO: 2.
17. The drug delivery system having cancer selective translocation
function of claim 16, wherein the drug is selected from the group
consisting of an antisense oligonucleotide targeting RNA of a
protein that causes a tumor or inflammatory disease, a siRNA
targeting RNA of a protein that causes a tumor or inflammatory
disease, an anticancer agent, an anticancer protein, an
anti-inflammatory agent, an anti-inflammatory protein, an
immune-enhancing protein, and a bone resorption inhibitor.
Description
TECHNICAL FIELD
[0001] The present invention relates to a peptide having cancer
selective translocation function, and the use thereof, and more
particularly to a peptide having cancer selective translocation
function, a conjugate comprising a drug such as an anticancer agent
linked to the peptide having cancer selective translocation
function, and the use thereof.
[0002] The peptide having cancer selective translocation function
of the present invention or a conjugate of the peptide and a drug
can selectively penetrate tumor cells or tumor tissues, and thus
can be widely used for the diagnosis or treatment of various
cancers.
BACKGROUND ART
[0003] For the treatment of tumors or the diagnosis and treatment
of inflammations, for example, osteoarthritis, and skin diseases,
many studies have been conducted on proteins or small materials,
which are present specifically in disease foci. Thus, many kinds of
such materials have been identified while studies on treatment with
such materials have also been actively conducted. For example, it
was shown that prostate-specific antigen (PSA) is frequently
present in prostate cancer, and matrix metalloprotease (MMP) is
highly expressed specifically in arthritis tissue or other tumor
tissues compared to that in normal tissue. Thus, such materials
have been targeted in disease research and treatment. However, if
materials that are used for the diagnosis and treatment of diseases
do not act specifically against such targets only, problems of side
effects and low image quality will be caused by non-specific
distribution of the materials. For this reason, there has been a
demand for the development of formulations that remain or act
specifically in their targets.
[0004] Meanwhile, only some small materials can enter the cytoplasm
or nucleus of live cells through the cell membrane at a very low
ratio, whereas large molecules cannot enter cells. Because most
materials prepared for therapeutic, preventive or diagnostic
purposes, each of which requires an effective amount to be
delivered into cells, are large molecules or macromolecules,
methods of delivering biologically active macromolecules into cells
without damaging the cells both in vivo and ex vivo have been
demanded.
[0005] As a result of studies conducted to satisfy this demand,
protein transduction domains (PTDs) have been suggested, and among
them, TAT protein, which is the transcription factor of human
immunodeficiency virus-1 (HIV-1), has been most frequently studied.
It was found that the TAT protein is more effective in passing
through the cell membrane when it is composed of amino acids 47 to
57 (YGRKKRRQRRR), on which positively charged amino acids are
concentrated, compared to when it is in a full-length form
consisting of 86 amino acids (Fawell, S. et al., Proc. Natl. Acad.
Sci. USA, 91:664, 1994). Other examples verifying the effects of
PTDs include a peptide having a sequence of amino acids 267 to 300
of the VP22 protein of Herpes Simplex Virus type 1 (HSV-1)
(Elliott, G. et al., Cell, 88:223, 1997), a peptide having a
sequence of amino acids 84 to 92 of the UL-56 protein of HSV-2
(GeneBank code:D1047[gi:221784]), and a peptide having a sequence
of amino acids 339 to 355 of the Antennapedia (ANTP) protein of
Drosophila sp (Schwarze, S. R. et al., Trends. Pharmacol. Sci.,
21:45, 2000). In addition, artificial peptides consisting of
positively charged amino acids also showed the effect of delivering
drugs (Laus, R. et al., Nature. Biotechnol., 18:1269, 2000).
[0006] Recently, the present inventors reported the preparation of
a low-molecular-weight protamine (LMWP) and the cell-penetrating
activity thereof, in which the low-molecular-weight protamine
(LMWP) has a peptide sequence similar to TAT, serves as a protein
transduction domain and contains a large amount of cationic amino
acids such as arginine. Particularly, the LMWP peptide is a
naturally occurring cationic peptide from protamine and is
advantageous in that it presents no toxicity concerns and can be
produced in large amounts (Park, Y. J. et al., J. Gene. Med., 700,
2003). Meanwhile, the present inventors have found that the LMWP
peptide selectively binds to vascular endothelial growth factor
(VEGF) and heparin, which are distributed specifically in tumor
tissue, and have expected that the peptide would have the effect of
selectively inhibiting tumors. Meanwhile, this LMWP peptide will
hereinafter be referred to as "VEGF-binding protein transduction
domain (VPTD) or peptide".
[0007] The present inventors have found that a VEGF-binding protein
transduction domain (VPTD) peptide represented as SEQ ID NO: 1 or a
heparin-binding protein transduction domain (HPTD) peptide
represented as SEQ ID NO: 2 binds specifically to vascular
endothelial growth factor (VEGF) and heparin in tumor cells or
tumor tissues and also accumulates selectively in tumor cells or
tumor tissues to inhibit the growth of the tumor cells, thereby
completing the present invention.
DISCLOSURE OF INVENTION
[0008] It is an object of the present invention to provide a
peptide which can minimize problems associated with side effects or
low image quality, which can occur due to the non-specific
distribution of conventional tumor diagnostic or therapeutic
agents, in which the peptide can deliver tumor diagnostic agents or
disease therapeutic agents selectively to target cells only.
[0009] Accordingly, a main object of the present invention is to
reconstitute the oxidation/reduction balance in mutant strains,
which lack each of 472 genes involved in breathing, electron
transfer and oxidation/reduction reactions, at the genome-wide
level by anaerobic fermentation, and based on the reconstitution,
select genes capable of controlling the carbon metabolic flow and
provide a mutant microorganism in which lactic acid, succinic acid
or ethanol is produced in large amounts but the production of other
organic acids is significantly reduced, and a method for producing
the mutant microorganism. Still another object of the present
invention is to use the peptide for the diagnosis or treatment of
cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows the results of measuring the tumor
cell-penetrating abilities of a VEGF-binding protein transduction
domain (VPTD) peptide represented as SEQ ID NO: 1 (VSRRRRRRGGRRRR)
and a heparin-binding protein transduction domain (HPTD)
represented as SEQ ID NO: 2. Specifically, FIG. 1a is a graphic
diagram showing the results of flow cytometry, and FIG. 1b is a set
of confocal scanning microscope images showing the observation of
the results shown in FIG. 1a.
[0011] FIG. 2 shows the results of examining the accuracy of
synthesis of the inventive VPTD peptide represented as SEQ ID NO: 1
or the inventive HPTD peptide represented as SEQ ID NO: 2 and
measuring the binding affinity between the peptides and their
targets, VEGF and heparin.
[0012] FIG. 3 shows the results of examining the tumor inhibitory
effects of the inventive VPTD peptide represented as SEQ ID NO: 1,
an anticancer agent (Dox; doxrubicin), and a conjugate of the VPTD
peptide of SEQ ID NO: 1 and the anticancer agent (Dox).
BEST MODE FOR CARRYING OUT THE INVENTION
[0013] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains.
Generally, the nomenclature used herein and the experiment methods,
which will be described below, are those well known and commonly
employed in the art.
[0014] The definition of major terms used in the present invention
is as follows.
[0015] As used herein, the term "protein transduction domain (PTD)"
refers to a cell-penetrating peptide capable of delivering drugs or
drug-containing particles into the cytoplasm or nucleus of cells.
Specifically, the term refers to a peptide that can form a covalent
bond with oligonucleotides, peptides, proteins, oligosaccharides,
polysaccharides or nanoparticles to introduce these materials into
cells without needing to use a separate receptor, carrier or
energy. The inventive VEGF-binding protein transduction domain
peptide represented as SEQ ID NO: 1 and the inventive
heparin-binding protein transduction domain peptide represented as
SEQ ID NO: 2 are also included in the scope of the PTD.
[0016] As used herein, the term "tumor cell" or "tumor tissue"
refers to an in vivo or ex vivo cell or tissue into which a drug or
a drug-containing particle are delivered by a tumor-penetrating
peptide. In other words, as used herein, the term "tumor tissue" is
meant to include in vivo cells, that is, cells that constitute the
organ or tissue of living animals or humans, or microorganisms that
are found in living animals or humans.
[0017] In one aspect, the present invention provides peptide having
cancer selective translocation function comprising VPTD
(VEGF-binding protein transduction domain peptide) peptide
represented as SEQ ID NO: 1 or HPTD (heparin-binding protein
transduction domain) peptide represented as SEQ ID NO: 2, and a
composition for enhancing cancer selective translocation, which
comprises the peptide as an active ingredient.
[0018] The VPTD peptide represented as SEQ ID NO: 1 or the HPTD
peptide represented as SEQ ID NO: 2 is characterized in that it
binds specifically to vascular endothelial growth factor (VEGF) and
heparin in tumor cells or tumor tissue (see FIG. 2). The expression
level of VEGF or heparin in tumor cells or tumor tissue is higher
than that in normal tissue, and thus the inventive peptide
represented as SEQ ID NO: 1 or 2 can bind specifically to tumor
tissue or tumor cells. Also, the VPTD peptide represented as SEQ ID
NO: 1 or the VPTD peptide represented as SEQ ID NO: 2, according to
the present invention, has the ability to penetrate cells, and thus
can bind specifically to VEGF or heparin, and then enter cells
without having to use endocytosis that is a conventional
intracellular absorption process. Thus, it can accumulate
selectively in tumor cells or tumor tissue.
[0019] The VPTD peptide represented as SEQ ID NO: 1 or the VPTD
peptide represented as SEQ ID NO: 2 may be composed of D-type or
L-type amino acids depending on in vivo stability and may contain
one or more amino acids selected from the group consisting of
arginine, lysine and histidine in an amount of 70-80%.
[0020] Examples of protein transduction domains (PTDs) having the
above-described characteristic include, in addition to the protein
transduction domains VPTD (SEQ ID NO: 1; VSRRRRRRGGRRRR) and HPTD
(SEQ ID NO: 2; CSSRKKNPNCRRH) found by the present inventors,
cationic protein transduction domains having an arginine, lysine or
histidine content of 70-80% or more, such as penetratin (SEQ ID NO:
3; RQIKIWFQNRRMKWKK), polyarginine (SEQ ID NO: 4; RRRRRRR),
polylysine (SEQ ID NO: 5; KKKKKKKKKK), a protamine fragment, and
Antennapedia (ANTP), as well as defensin-derived HBD (SEQ ID NO:
6:GKCSTRGRKCCRRKK) and TAT (SEQ ID NO: 7: YGRKKRRQRRR). Thus, the
PTDs of SEQ ID NO: 3 to SEQ ID NO: 7 may also be used as a
substitute for the peptide of SEQ ID NO: 1 or 2.
[0021] In another aspect, the present invention provides a method
of using the VPTD peptide of SEQ ID NO: 1 or the HPTD peptide of
SEQ ID NO: 2 to diagnose or treat cancer.
[0022] The VPTD peptide of SEQ ID NO: 1 or the HPTD peptide of SEQ
ID NO: 2 binds specifically to vascular endothelial growth factor
(VEGF) and heparin in tumor cells or tumor tissue and accumulates
in the tumor cells or tumor tissue, and thus the peptide can be
used for the diagnosis or treatment of cancer.
[0023] In still another aspect, the present invention provides
contrast agent for cancer diagnosis having cancer selective
translocation function, which comprises VPTD peptide represented as
SEQ ID NO: 1 or HPTD peptide represented as SEQ ID NO: 2 to which a
fluorescent substance is bound.
[0024] Because the VPTD peptide represented as SEQ ID NO: 1 or the
HPTD peptide represented as SEQ ID NO: 2 accumulates selectively in
tumor cells or tumor tissues, a conjugate comprising a fluorescent
substance bound to the peptide may be used as a contrast agent for
cancer diagnosis (see Example 3 and FIG. 1).
[0025] The fluorescent substance that is used in the present
invention may be selected from the group consisting of fluorescein
isothiocyanate (FITC), radioisotopes, quantum dots, MRI contrast
agents, fluorescein, tetramethylrhodamine, BODIPY, and Alexa, but
is not limited thereto.
[0026] In yet another aspect, the present invention provides a
composition for treating cancer having cancer selective
translocation function, which comprises VPTD peptide represented as
SEQ ID NO: 1 or HPTD peptide represented as SEQ ID NO: 2.
[0027] Because the VPTD peptide represented as SEQ ID NO: 1 or the
HPTD peptide represented as SEQ ID NO: 2 according to the present
invention shows the effect of inhibiting the growth of tumor cells
by its selective binding to VEGF and heparin, which are highly
expressed in tumor cells or tumor tissue (see Examples 4 and FIGS.
2 and 3), the composition containing the peptide may also be used
as a composition for treating cancer.
[0028] In a further aspect, the present invention provide a peptide
having cancer selective translocation function-drug conjugate,
which comprises a drug chemically linked to the N-terminus or
C-terminus of VPTD peptide represented as SEQ ID NO: 1 or HPTD
peptide represented as SEQ ID NO: 2.
[0029] The VPTD peptide represented as SEQ ID NO: 1 or the HPTD
peptide represented as SEQ ID NO: 2 and the drug may be linked to
each other by a cysteine. Specifically, the peptide-drug conjugate
is prepared by linking a drug having a thiol group to the
N-terminus or C-terminus of the peptide of SEQ ID NO: 1 or 2, which
contains a cysteine. If the above-described peptide analogue is
used as a substitute for the peptide of SEQ ID NO: 1 or 2, the
peptide-drug conjugate may also be prepared by attaching a cysteine
to the terminus of the peptide, and then reacting the peptide with
a drug. Examples of the drug having a thiol group include not only
drugs that naturally contain a thiol group, but also drugs modified
to have a thiol group.
[0030] In addition, the peptide-drug conjugate may also be prepared
by linking the peptide with the drug by a crosslinking agent.
Because the N-terminus of the protein transduction domain (PTD)
peptide has a free amino group, it is easy to form the peptide-drug
conjugate by a crosslinking agent. Examples of the crosslinking
agent that can be used in the present invention include, but not
limited to, 1,4-bis-maleimidobutane (BMB),
1,11-bis-maleimidotetraethyleneglycol (BM[PEO]4),
1-ethyl-3-[3-dimethyl aminopropyl]carbodiimide hydrochloride (EDC),
succinimidyl-4-[N-maleimidomethylcyclohexane-1-carboxy-[6-amidocap-
roate]] (SMCC) and its sulfonate (sulfo-SMCC), succimidyl
6-[3-(2-pyridyldithio)-ropionamido]hexanoate (SPDP) and its
sulfonate (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide
ester (MBS) and its sulfonate (sulfo-MBS), and
succimidyl[4-(p-maleimidophenyl) butyrate] (SMPB) and its sulfonate
(sulfo-SMPB). If the cell-penetrating peptide and a drug or a
drug-containing nanoparticle are linked to each other by an S--S
bond, the drug can be dissociated from the cell-penetrating peptide
by reductase or the like in cells. If this method is used, drugs,
proteins or other nanoparticles can be introduced into cells in an
easy and convenient manner without having to use a recombinant
vector that is constructed in a time-consuming manner, and thus the
desired therapeutic can be easily achieved.
[0031] Examples of the drug that is used in the present invention
include anticancer agents, anti-inflammatory agents, bone
resorption inhibitors, anticancer proteins, anti-inflammatory
proteins, immune-enhancing proteins, anticancer and
anti-inflammatory siRNAs, oligonucleotides, and magnetic
nanoparticles containing them. As used herein, the term "siRNA"
refers to RNA that silences the expression of the target RNA. The
target RNA is the mRNA transcribed from a gene that causes disease,
particularly a tumor or inflammation. Examples of oncogenes
include, but are not limited to, vascular endothelial growth factor
(VEGF) gene.
[0032] The aforementioned protein that causes a tumor or
inflammatory disease may be selected from the group consisting of
vascular endothelial growth factor (VEGF), B-cell leukemia/lymphoma
2 (BCL2), epidermal growth factor receptor (EGFR), human epidermal
growth factor receptor 2 (HER2), Janus kinase (JAN), and
phosphatidylinositol-3-kinase/Akt kinase (PI3-K/AKT).
[0033] In a still further aspect, the present invention provides a
drug delivery system having cancer selective translocation
function, which comprises a drug linked to the N-terminus or
C-terminus of VPTD peptide represented as SEQ ID NO: 1 or HPTD
peptide represented as SEQ ID NO: 2.
[0034] In a yet further aspect, the present invention provides a
composition for treating cancer comprising a peptide having cancer
selective translocation function-drug conjugate, which comprises a
drug chemically linked to the N-terminus or C-terminus of VPTD
peptide represented as SEQ ID NO: 1 or HPTD peptide represented as
SEQ ID NO: 2.
[0035] The composition for treating cancer according to the present
invention can be administered with a pharmaceutically acceptable
carrier. For example, for oral administration, the composition of
the present invention can comprise binders, lubricants,
disintegrants, excipients, emulsifiers, dispersions, stabilizers,
suspending agents, pigments, perfumes, etc., for injection
administration, the composition can comprises buffers,
preservatives, analgesics, emulsifiers, isotonic agents,
stabilizers, etc., and for local administration, the composition
can comprises bases, excipients, lubricants, preservatives,
etc.
[0036] The inventive composition for treating cancer can be
formulated with a pharmaceutically acceptable carrier as described
above in various manners. For example, for oral administration, the
composition of the present invention can be formulated in the form
of tablet, troche, capsule, elixir, suspension, syrup, wafer, etc.,
and for injection administration, the composition can be formulated
as a unit dosage ampoule or a multiple dosage form.
[0037] The inventive composition for treating cancer can be
administered in an effective amount for the therapeutic or
prevention purpose. The dose of the composition of the present
invention may vary depending on various factors, such as disease
type and severity, age, sex, body weight, sensitivity to drugs,
type of current therapy, mode of administration, target cell, etc.,
and may be easily determined by those of ordinary skill in the art.
The composition of the present invention may also be administered
in combination with conventional therapeutic or preventive agents
for cancer, sequentially or simultaneously with the conventional
therapeutic agents, and in single dose or multiple doses.
Preferably, with all of the factors taken into account, it is
imperative that the minimum dose required to achieve the maximum
effect without side effects be administered, which can be easily
determined by those of ordinary skill in the art. As used herein,
the term "administration" means introducing a desired material into
a patient by any suitable method. The pharmaceutical composition of
the present invention may be administered through any general
route, as long as it can reach a desired tissue. The composition of
the present invention can be administered intraperitoneally,
intravenously, intramuscularly, subcutaneously, transdermally,
orally, topically, intranasally, intrapulmonarily or intrarectally,
but is not limited thereto. In addition, the pharmaceutical
composition of the present invention may also be administered by
any device that can deliver the active ingredient into target
cells.
[0038] Particularly, although the following examples illustrated
only the anticancer protein Gelonin as a drug, it will be obvious
to those skilled in the art that the use of other anticancer
proteins, an antisense oligonucleotide against an oncogene, an
siRNA, or particles containing them, can also show tumor
therapeutic effects that are equal or similar to those of the use
of Gelonin, and that the use of an anti-inflammatory agent or an
anti-inflammatory protein as a drug can also show anti-inflammatory
effects.
EXAMPLES
[0039] Hereinafter, the present invention will be described in
further detail with reference to examples. It will be obvious to a
person having ordinary skill in the art that these examples are
illustrative purposes only and are not to be construed to limit the
scope of the present invention.
Example 1
Preparation of Target-Selective Cell/Tissue-Penetrating Peptide
[0040] Peptides were prepared by the F-moc chemistry method using
an automatic peptide synthesizer, and then the peptide moieties
were cut by resin, washed, freeze-dried, followed by purification
by liquid chromatography, thereby preparing a VPTD peptide
represented as SEQ ID NO: 1 (VSRRRRRRGGRRRR) and an HPTD peptide
represented as SEQ ID NO: 2 (CSSRKKNPNCRRH). The molecular weights
of the purified peptides were analyzed by MALDI.
Example 2
Preparation of Tumor-Targeting and Tumor-Penetrating Peptide-Drug
Conjugates
[0041] The VPTD peptide of SEQ ID NO: 1 and the HPTD peptide of SEQ
ID NO: 2, prepared in Example 1, contained the free-sulfhydryl
group of a cysteine residue, and thus chemical linkage between the
peptide and the anticancer protein RNase or doxorubicin was induced
using the free-sulfhydryl group as a chemical crosslinking agent.
The surfaces of RNase and doxorubicin (Dox) were modified to have a
thiol group attached to the carboxyl group on the surfaces. 10
molecules of peptide-SH were added to 1 molecule of particle
surface-SH and reacted at 4.degree. C. for 12 hours, and then
unreacted molecules were removed by ultrafiltration, followed by
freeze drying, thereby obtaining a conjugate of the VPTD peptide of
SEQ ID NO: 1 and RNase, a conjugate of the VPTD peptide of SEQ ID
NO: 1 and Dox, a conjugate of the HPTD peptide of SEQ ID NO: 2 and
RNase, and a conjugate of the HPTD peptide of SEQ ID NO: 2 and
Dox.
Example 3
Tumor Cell Penetration Abilities of Tumor-Targeting and
Cell-Penetrating Peptides
[0042] In order to test the tumor cell-targeting and tumor
cell-penetrating abilities of the VPTD peptide of SEQ ID NO: 1 and
the HPTD peptide of SEQ ID NO: 1, prepared in Example 1, the
termini of the prepared peptides were labeled with a fluorescent
dye, and then each of the peptides was inoculated into a tumor cell
line (MDA-MB-231, ATCC) at various concentrations.
[0043] 60 minutes after the inoculation, the fluorescence of the
cells was measured by FACS, and the results of the measurement are
shown in FIG. 1. As can be seen in FIG. 1(A), the fluorescence of
the tumor cells increased in a manner dependent on the
concentration of the peptide. FIG. 1(B) shows the results of
observing the fluorescence of the tumor cells by a confocal laser
scanning microscope in order to examine the tumor cell penetration
abilities of the VPTD peptide of SEQ ID NO: 1 and the HPTD peptide
of SEQ ID NO: 1. In FIG. 1(B), in order to demonstrate that the
stained portion is the cells, the cell nuclei were stained with
Hoechst 33342 (5 .mu.g/.mu.l), and then the cells were fixed with
10% neutral formalin solution. As a result, as can be seen in FIG.
1(B), the abilities of the peptide to penetrate the tumor cell line
increased in a manner dependent on the concentration of the
peptide.
Example 4
Measurement of Tumor Inhibitory Effects of Tumor-Targeting and
Target-Penetrating Peptide-Anticancer Agent Conjugates
[0044] In order to test the effects of the VPTD peptide of SEQ ID
NO: 1 (VSRRRRRRGGRRRR) and the HPTD peptide of SEQ ID NO: 2
(CSSRKKNPNCRRH), prepared in Example 1, and the tumor-targeting and
tumor-penetrating peptide-drug conjugates prepared in Example 2, a
tumor was induced in mice, and when the tumor grew to a
predetermined size, each of the peptides prepared in Example 1 and
the tumor-targeting and tumor-penetrating peptide-drug conjugates
prepared in Example 2 was injected into the mice. After injection,
the volume of the tumor was measured at intervals of 3-4 days for
30 days. On day 30, the mice were sacrificed, and the tumor was
extracted and photographed.
[0045] As a result, as shown in FIG. 3, in the tumor-induced mice
treated with a control, the tumor was not inhibited, whereas in the
tumor-induced mice treated with each of the VPTD peptide and the
VPTD peptide-drug (Dox) conjugate, the progression of the tumor was
significantly inhibited. This is believed to be because the
tumor-penetrating ability of the VPTD peptide of the present
invention was maximized in the tumor tissue and the VPTD peptide
did bind specifically to VEGF to inhibit vascular formation
essential for the formation and progression of tumors. To
demonstrate such results, the ability of the inventive VPTD peptide
of SEQ ID NO: 1 or the inventive HPTD peptide of SEQ ID NO: 2 to
bind to VEGF and heparin was evaluated by slot-blot analysis. As a
result, it could be seen that the VPTD peptide and the HPTD peptide
did all bind to VEGF and heparin, which are overexpressed in tumor
cells or tumor tissue.
[0046] As described above, the tumor-targeting and
tumor-penetrating peptide of the present invention and a conjugate
of the peptide and a drug break from conventional non-specific and
non-selective transduction peptides, and can maximize the effects
of diagnosis and drug therapies through optimal targeting, and the
side effects thereof in the body can be minimized. Thus, the use of
the tumor-selective and tumor-penetrating peptide according to the
present invention can present innovative disease diagnostic and
therapeutic technologies.
INDUSTRIAL APPLICABILITY
[0047] As described above, the peptide or peptide-drug conjugate of
the present invention selectively penetrates tumor cells or tumor
tissue only, and thus can be used for the diagnosis or treatment of
cancer. Conventional drugs or materials that are used in the
diagnosis or treatment of tumors can cause unexpected side effects
if they are distributed non-specifically in vivo, and in many
cases, it is difficult to achieve accurate diagnosis using the
conventional drugs or materials. However, the use of the peptide of
the present invention can solve such problems.
[0048] Although the present invention has been described in detail
with reference to the specific features, it will be apparent to
those skilled in the art that this description is only for a
preferred embodiment and does not limit the scope of the present
invention. Thus, the substantial scope of the present invention
will be defined by the appended claims and equivalents thereof.
Sequence CWU 1
1
7114PRTArtificial SequenceVPTD 1Val Ser Arg Arg Arg Arg Arg Arg Gly
Gly Arg Arg Arg Arg 1 5 10 213PRTArtificial SequenceHPTD 2Cys Ser
Ser Arg Lys Lys Asn Pro Asn Cys Arg Arg His 1 5 10 316PRTArtificial
SequencePenetratin 3Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met
Lys Trp Lys Lys 1 5 10 15 47PRTArtificial Sequencepolyarginine 4Arg
Arg Arg Arg Arg Arg Arg 1 5 510PRTArtificial Sequencepolylysine
5Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 615PRTArtificial
Sequencedefensin 6Gly Lys Cys Ser Thr Arg Gly Arg Lys Cys Cys Arg
Arg Lys Lys 1 5 10 15 711PRTArtificial SequenceHuman
immunodeficiency virus-1 7Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg 1 5 10
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