DESCRIPTION
Cell-penetrating peptides
TECHNICAL FIELD
[0001]
The present invention relates to a novel peptide having cell membrane
permeability, and a pharmaceutical composition comprising the peptide and a
hydrophilic physiologically active substance.
BACKGROUND ART
[0002]
Cells are isolated from the outside by the cell membrane, which is
impermeable to hydrophilic physiologically active substances such as proteins and
nucleic acids. Absence of methods for delivering such hydrophilic physiologically
active substances into the cell has prevented clinical use of many hydrophilic
physiologically active substances whose sites of action are located in the cells.
[0003]
Several techniques to deliver such cell-impermeable hydrophilic
physiologically active substances have been reported. The most popular method is
one which gives hydrophobicity to a hydrophilic physiologically active substance
using a lipid, and, by this, permeability through the cell membrane can be increased.
Further, a technique wherein a peptide ligand is used to increase the permeability has
also been reported.
[0004]
A peptide ligand having a property which allows its transfer from the outside
of the cell into the cell without destroying the cell membrane is called a cell-
penetrating peptide. Examples of well-known cell-penetrating peptides include
oligoarginines, wherein arginines are linked to each other; Tat (Patent Document 1),
which is derived from HIV-1 virus; and penetratin (Patent Document 2), which is a

peptide derived from Drosophila. In addition to these, various cell-penetrating
peptides, such as those characterized by simple basicity, those characterized by
amphipathicity of the primary structure or the secondary structure of the peptide, and
those having a mechanism which has not been clarified, have also been reported. In
addition to the permeability of the peptide itself into the cell, its use for delivering
into the cell a hydrophilic physiologically active substance such as a gene to which
the peptides is linked as a vehicle has been extensively studied. However, although
these peptides have been successful as reagents for research, only a small number of
peptides can be used for clinical application. Therefore, the peptides have been
studied in various ways, and, in each use, sequences with which the peptides can be
more efficiently transferred into cells are being searched.
[0005]
A part of cell-penetrating peptides are reported to be capable of promoting
permeation of a hydrophilic physiologically active substance through the mucosal
epithelial cell layer in cases where these are orally or intranasally administered
together with the hydrophilic physiologically active substance, allowing the
hydrophilic physiologically active substance to pass into the general circulation with
a high efficiency. It is thought that not all of the cell-penetrating peptides have the
permeation-promoting capacity through mucosa, and that not only cell membrane
permeability but also the intracellular dynamics, separation from the cell, and the like
are involved in the permeation-promoting capacity. Thus, it is thought that only a
part of the cell-penetrating peptides, which satisfy these conditions, have the ability
to promote transmucosal absorption of hydrophilic physiologically active substances.
However, only a small number of types of peptides are reported to have such a
function. Further, the peptides for which the transmucosal absorption-promoting
capacity has been reported are those with which the effect can be confirmed only in
cases where they are used in the forms of conjugates with particular physiologically
active substances (Patent Documents 2 and 3); and those which require, even in cases

where the possibility that the peptides can be used in the forms not requiring covalent
bonding with a drug is shown, large amounts of the cell-penetrating peptides in order
to obtain a sufficient effect (Patent Document 4). Therefore, many problems
remain to be solved before practical use of the peptides.
[0006]
In recent years, in addition to low molecular hydrophobic drugs, which have
been mainly used so far, hydrophilic physiologically active substances are drawing
attention as candidate compounds of pharmaceuticals. Although the hydrophilic
physiologically active substances have shown remarkable therapeutic effects, their
poor transmucosal absorbability has limited the method of their administration
almost only to injection. Therefore, development of a technology to allow
absorption of hydrophilic physiologically active substances through mucosa is
strongly desired for the purpose of non-injection administration of hydrophilic
physiologically active substances. In particular, the absorption-promoting
technique using a cell-penetrating peptide is expected to show a lower level of
stimulation to mucosa compared to the absorption-promoting technique using
surfactants, which has been extensively studied so far, and discovery of cell-
penetrating peptides that cause efficient promotion of absorption may lead to
development of a promising technique.
PRIOR ART DOCUMENTS
[0007]
Patent Documents
[Patent Document 1] JP 10-33186 A
[Patent Document 2] Japanese Translated PCT Patent Application Laid-open No.
2002-530059
[Patent Document 3] WO 2004/037859
[Patent Document 4] JP 10-95738 A
DISCLOSURE OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION
[0008]
The present invention aims to provide a novel cell-penetrating peptide which
can allow a hydrophilic physiologically active substance to permeate into the cell.
MEANS FOR SOLVING THE PROBLEMS
[0009]
The present inventors searched for peptide sequences having cell membrane
permeability, and discovered novel peptide sequences having the desired cell
membrane permeability. That is, the present invention has the following
constitution.
[0010]
(1) A cell-penetrating peptide which is any of (A) to (D) below:
(A) a peptide having the amino acid sequence represented by SEQ ID NO:l;
(B) a peptide having an amino acid sequence which is the same as the amino
acid sequence represented by SEQ ID NO: 1 except that one or several basic amino
acids are substituted, deleted, inserted and/or added, which peptide has cell
membrane permeability;
(C) a peptide having an amino acid sequence which is the same as the amino
acid sequence represented by SEQ ID NO:l except that 1 to 5 amino acids are
substituted, deleted, inserted and/or added, which peptide has cell membrane
permeability;
(D) a peptide having: an amino acid sequence represented by the reverse
sequence of any of (A) to (C); an amino acid sequence which is the same as the
amino acid sequence represented by the reverse sequence of (A) except that one or
several basic amino acids are substituted, deleted, inserted and/or added; or an amino
acid sequence which is the same as the amino acid sequence represented by the
reverse sequence of (A) except that 1 to 5 amino acids are substituted, deleted,
inserted and/or added; which peptide has cell membrane permeability.

[0011]
(2) The cell-penetrating peptide according to (1), wherein the peptide (B) has
the amino acid sequence represented by any of SEQ ID NOs:2 to 4, 9 to 10 and 13.
[0012]
(3) The cell-penetrating peptide according to (1) or (2), wherein the peptide
(C) has the amino acid sequence represented by any of SEQ ID NOs:12 and 15 to 30.
[0013]
(4) The cell-penetrating peptide according to (1) to (3), wherein the peptide
(D) has the amino acid sequence represented by SEQ ID NO:5 or 14.
[0014]
(5) A pharmaceutical composition comprising the cell-penetrating peptide
according to any of (1) to (4) and a hydrophilic physiologically active substance.
[0015]
(6) A pharmaceutical composition for oral administration, the pharmaceutical
composition comprising the cell-penetrating peptide according to any of (1) to (4)
and a hydrophilic physiologically active substance.
[0016]
(7) A pharmaceutical composition for intranasal administration, the
pharmaceutical composition comprising the cell-penetrating peptide according to any
of (1) to (4) and a hydrophilic physiologically active substance.
[0017]
(8) The pharmaceutical composition according to any of (5) to (7), wherein
the hydrophilic physiologically active substance is a peptide, protein or nucleic acid.
EFFECT OF THE INVENTION
[0018]
By the present invention, efficient transfer of a hydrophilic physiologically
active substance into cells is possible, and a novel pharmacotherapy targeting
molecules in the cell is possible. Further, a hydrophilic physiologically active

substance which has been able to be administered only by injection can be
administered by oral administration, intranasal administration or the like, enabling a
simple and patient-oriented pharmacotherapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019]
Fig. 1 is a graph showing transfer of cell-penetrating peptides into HeLa cells
Fig. 2 is a graph showing promotion of insulin transfer into HeLa cells by
cell-penetrating peptides.
Fig. 3 is a graph showing promotion of transfer of polystyrene beads into
HeLa cells by cell-penetrating peptides.
Fig. 4 is a graph showing the blood glucose level as an index of intranasal
absorption of insulin, which was observed when a cell-penetrating peptide was used.
Fig. 5 is a graph showing the plasma insulin level as an index of intranasal
absorption of insulin, which was observed when a cell-penetrating peptide was used.
Fig. 6 is a graph showing the bioavailability as an index of intranasal
absorption of insulin, which was observed when cell-penetrating peptides were used.
Fig. 7 is a graph showing the plasma interferon-P level as an index of
intranasal absorption of interferon-P, which was observed when cell-penetrating
peptides were used.
Fig. 8 is a graph showing the plasma exendin-4 level as an index of intranasal
absorption of exendin-4, which was observed when cell-penetrating peptides were
used.
Fig. 9 is a graph showing the blood glucose level as an index of intestinal
absorption of insulin, which was observed when cell-penetrating peptides were used.
Fig. 10 is a graph showing the plasma insulin level as an index of intestinal
absorption of insulin, which was observed when cell-penetrating peptides were used.
Fig. 11 is a graph showing LDH leakage as an index of intranasal toxicity of a
cell-penetrating peptide.

Fig. 12 shows confocal laser microscopic images showing promotion of
insulin transfer into HeLa cells by the respective cell-penetrating peptides. In each
diagram, A (upper left) shows localization of rhodamine-labeled insulin; B (upper
right) shows the cell membrane stained with DiD'Oil; C (lower left) shows a
differential interference image; and A+B (lower right) shows a superimposed image
of the image A and the image B.
Fig. 13 is a graph showing promotion of insulin transfer into HeLa cells by
cell-penetrating peptides.
Fig. 14 is a graph showing AUC as an index of intranasal absorption of
insulin, which was observed when cell-penetrating peptides were used.
Fig. 15 is a graph showing transfer of cell-penetrating peptides into HeLa
cells.
Fig. 16 is a graph showing promotion of insulin transfer into HeLa cells by
cell-penetrating peptides.
BEST MODE FOR CARRYING OUT THE INVENTION
[0020]
The present inventors newly discovered that a peptide having the amino acid
sequence shown in SEQ ID NO:l (hereinafter referred to as the peptide of SEQ ID
NO:l) or its modified, peptide is a cell-penetrating peptide, thereby completing the
present invention. The cell-penetrating peptides of the present invention will now
be described below in detail.
[0021]
The cell membrane permeability of a cell-penetrating peptide of the present
invention means a property to pass through a lipid membrane separating the inside of
the cell from the outside thereof. Whether or not a peptide having cell-membrane
permeability can be confirmed by linking a fluorescent substance to the peptide and
adding the resultant to cells, followed by observing the cells by a confocal laser
microscope or the like to see if the fluorescent substance can be detected in the cells.

Further, quantitative confirmation of permeability into cells can be carried out by
incorporating the fluorescent substance-linked peptide into the cells and
homogenizing the cells, followed by measuring the fluorescence intensity of the
homogenate using a spectrophotometer. In the present specification, in cases where
the amount of permeation of a fluorescent substance (e.g., fluorescein)-linked peptide
into cells is not less than 3 times higher than the amount of permeation of a
fluorescent substance-linked cell membrane non-permeable peptide having the amino
acid sequence represented by SEQ ID NO:6 into the cells, the former peptide is
judged to be a cell-penetrating peptide.
[0022]
Further, the cell-penetrating peptides of the present invention have a property
to give cell membrane permeability to hydrophilic physiologically active substances.
In the present specification, when the efficiency of permeation of a fluorescently
labeled hydrophilic physiologically active substance into cells is not less than 3 times
higher in cases where the fluorescently labeled hydrophilic physiologically active
substance is linked to or mixed with a peptide and brought into contact with cells
than in cases where the fluorescently labeled hydrophilic physiologically active
substance alone is brought into contact with cells, the peptide is judged to have a
property to give cell membrane permeability to the hydrophilic physiologically
active substance.
[0023]
Further, the cell-penetrating peptides of the present invention have a property
to give transmucosal absorbability (preferably intestinal absorbability or intranasal
absorbability) to hydrophilic physiologically active substances. More particularly,
even in cases where a hydrophilic physiologically active substance is hardly
absorbed by itself into the living body through mucosa, its transmucosal absorption
(preferably intestinal absorption or intranasal absorption) is promoted by bringing the
hydrophilic physiologically active substance linked to or mixed with a cell-

penetrating peptide into contact with the mucosal tissue (preferably intestinal tissue
or intranasal tissue).
[0024]
The peptide having the amino acid sequence shown in SEQ ID NO:l is a
novel peptide discovered by screening of cell-penetrating peptides. The type of the
cell through which the peptide passes through is not restricted and may be either a
prokaryotic cell or eukaryotic cell, and the peptide can preferably pass through the
cell membrane of a eukaryotic cell, preferably mammalian cell, still more preferably
through the mucosal cell membrane of a mammal. The peptide per se has cell
membrane permeability, and moreover, by covalently binding the peptide to a
hydrophilic physiologically active substance, cell membrane permeability can be
given to the hydrophilic physiologically active substance. Preferably, the peptide
can give cell membrane permeability to a hydrophilic physiologically active
substance even in cases where the peptide and the hydrophilic physiologically active
substance exist independently from each other in a composition.
[0025]
Further, a modified peptide having an amino acid sequence which is the same
as the amino acid sequence represented by SEQ ID NO: 1 except that one or several
basic amino acids are substituted, deleted, inserted and/or added is also a cell-
penetrating peptide of the present invention as long as the modified peptide has cell
membrane permeability. Here, the basic amino acid means any of the amino acids
arginine, lysine and histidine. The number of the substituted, deleted, inserted
and/or added basic amino acid(s) is preferably 1 to 7, more preferably 1 to 5, still
more preferably 1 to 3, especially preferably 1. In the amino acid sequence which
has been mutated by the substitution, deletion and/or insertion, a sequence of not less
than 3 continuous amino acids in the original amino acid sequence is preferably
conserved, and a sequence of not less than 5 continuous amino acids in the original
amino acid sequence is more preferably conserved. Further, in the case of a

modified peptide produced by substituting a basic amino acid(s) of the amino acid
sequence represented by SEQ ID NO: 1 with another/other basic amino acid(s), the
modification is most acceptable since substitution with another amino acid having
the same property is not likely to change the overall property of the peptide.
Preferred examples of the peptide having an amino acid sequence which is the same
as the amino acid sequence represented by SEQ ID NO:l except that one or several
basic amino acids are deleted, substituted and/or added, which peptide has cell
membrane permeability, include peptides having the amino acid sequences
represented by any of SEQ ID NOs:2 to 4, 9 to 10 and 13.
[0026]
Further, a modified peptide having an amino acid sequence which is the same
as the amino acid sequence represented by SEQ ID NO:l except that 1 to 5,
preferably 1 to 3, more preferably 1 to 2, still more preferably 1 amino acid(s), which
is/are not limited to a basic amino acid(s), is/are substituted, deleted, inserted and/or
added is also a cell-penetrating peptide of the present invention as long as the
modified peptide has cell membrane permeability. In the amino acid sequence
produced by substitution, deletion, insertion and/or addition of an amino acid(s) in
SEQ ID NO:l, a sequence of not less than 3 continuous amino acids in the original
amino acid sequence is preferably conserved, and a sequence of not less than 5
continuous amino acids in the original amino acid sequence is more preferably
conserved. Further, for example, in cases where a basic amino acid(s) in the amino
acid sequence represented by SEQ ID NO: 1 is/are substituted with another/other
basic amino acid(s); in cases where a hydrophilic amino acid(s) in the amino acid
sequence represented by SEQ ID NO:l is/are substituted with another/other
hydrophilic amino acid(s); and in cases where a hydrophobic amino acid(s) in the
amino acid sequence represented by SEQ ID NO: 1 is/are substituted with
another/other hydrophobic amino acid(s); the modification is most acceptable since
substitution with an amino acid having the same property is not likely to change the

overall property of the peptide. Preferred examples of the peptide having an amino
acid sequence which is the same as the amino acid sequence represented by SEQ ID
NO:l except that 1 to 5 amino acids, which is/are not limited to a basic amino acid(s),
are substituted, deleted, inserted and/or added, which peptide has cell membrane
permeability, include peptides having the amino acid sequences represented by any
of SEQ ID NOs:12 and 15 to 30.
[0027]
Further, a peptide having the amino acid sequence represented by the reverse
sequence of the peptide having the amino acid sequence represented by SEQ ID
NO:l; a peptide having an amino acid sequence represented by the reverse sequence
of an amino acid sequence which is the same as the amino acid sequence represented
by SEQ ID NO: 1 except that one or several basic amino acids are substituted, deleted,
inserted and/or added; a peptide having an amino acid sequence which is the same as
the reverse sequence of the amino acid sequence represented by SEQ ID NO:l
except that one or several basic amino acids are substituted, deleted, inserted and/or
added; a peptide having an amino acid sequence represented by the reverse sequence
of an amino acid sequence which is the same as the amino acid sequence represented
by SEQ ID NO: 1 except that 1 to 5 amino acids, which are not limited to basic amino
acids, are substituted, deleted, inserted and/or added; and a peptide having an amino
acid sequence which is the same as the reverse sequence of the amino acid sequence
represented by SEQ ID NO:l except that 1 to 5 amino acids, which are not limited to
basic amino acids, are substituted, deleted, inserted and/or added; are also cell-
penetrating peptides of the present invention as long as these have cell membrane
permeability. Here, the peptide having the reverse sequence means that the
sequence of the constituting amino acids is reversed, and, for example, when the
sequence of the amino acids from the N-terminus to the C-terminus is arginine,
glutamine, isoleucine and lysine, the reverse peptide thereof means the peptide
whose sequence of amino acids from the N-terminus to the C-terminus is lysine,

isoleucine, glutamine and arginine. Particular examples thereof include a peptide
having the amino acid sequence represented by SEQ ID NO:5 or 14, which has the
amino acid sequence represented by the reverse sequence of the peptide having the
amino acid sequence represented by SEQ ID NO:l and has cell membrane
permeability.
[0028]
As the amino acids constituting the cell-penetrating peptides of the present
invention, amino acids having the configuration of L-body, which are naturally
occurring amino acids, as well as non-naturally occurring amino acids such as
derivatives produced by partial modification of the structures of naturally occurring
amino acids may be used. For example, since amino acids having the configuration
of D-body are not likely to be degraded by proteases and hence can be effectively
used, a part of the amino acid sequence of the peptide may have the configuration of
D-body.
[0029]
The amino acids constituting the cell-penetrating peptides of the present
invention are not restricted as long as they are molecules having a carboxyl group
and an amino group irrespective of whether these naturally exist, and may also be
amino acids modified by post-translational modification normally seen in the living
body, such as hydroxylation, phosphorylation or glycosylation. The amino acid
sequence is preferably composed of naturally-occurring amino acids normally
existing in mammalian cells and/or optical isomers thereof, and examples of the
amino acid sequence include those composed of arginine (Arg), lysine (Lys), aspartic
acid (Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gin), histidine (His),
proline (Pro), tyrosine (Tyr), tryptophan (Tip), serine (Ser), threonine (Thr), glycine
(Gly), alanine (Ala), methionine (Met), cysteine (Cys), phenylalanine (Phe), leucine
(Leu), valine (Val), isoleucine (He) and/or the like.
[0030]

The cell-penetrating peptides of the present invention may be modified as
appropriate by methods known to those skilled in the art, and more particularly, these
may be derivatives chemically modified by polyethylene glycolation (PEGylation),
acetylation of the N-terminus, amidation of the C-terminus, and/or the like. It
should be noted that, in cases where a cell-penetrating peptide of the present
invention is used for the later-mentioned pharmaceutical composition, the peptide is
preferably not modified. Further, the cell-penetrating peptides of the present
invention may be either linear or circular.
[0031]
The cell-penetrating peptides of the present invention may be used in
combination with other known cell-penetrating peptides. Further, the cell-
penetrating peptides of the present invention may be used in the forms of fusion
peptides or fusion proteins prepared by fusing the peptides with other peptides or
proteins by the later-mentioned method, as long as the cell membrane permeability is
maintained.
[0032]
The cell-penetrating peptides of the present invention may be produced by
common chemical synthesis methods. Examples of the production method include
the peptide synthesis methods by the commonly used liquid phase method or solid
phase method. Examples of such peptide synthesis methods include the stepwise
elongation method, wherein each amino acid is successively bound to elongate a
chain, and the fragment condensation method, wherein fragments composed of
several amino acids are preliminarily synthesized followed by subjecting the
respective fragments to the coupling reaction. Synthesis of the cell-penetrating
peptides of the present invention may be carried out by either of these methods.
[0033]
The condensation method employed for the above peptide synthesis may also
be carried out according to various known methods. Particular examples thereof

include the azide method, mixed anhydride method, DCC method, active ester
method, oxidation-reduction method, DPPA (diphenylphosphorylazide) method,
DCC+additives (e.g., 1-hydroxybenzotriazole, N-hydroxysuccinimide andN-
hydroxy-5-norbornene-2,3-dicarboxyimide) and Woodward method. The solvent
that can be used for each of these methods may be appropriately selected from
commonly used ones which are well known to be used for such types of peptide
condensation reactions. Examples of the solvent include dimethylformamide
(DMF), dimethylsulfoxide (DMSO), hexamethylphosphoramide, dioxane,
tetrahydrofuran (THF) and ethyl acetate, and mixed solvents thereof.
[0034]
In the above-described peptide synthesis reactions, carboxyl groups of amino
acids or peptides which are not involved in the reactions may be generally protected
by esterification to form a lower-alkyl ester such as a methyl ester, ethyl ester or tert-
butyl ester; or an aralkyl ester such as a benzyl ester, p-methoxybenzyl ester or p-
nitrobenzyl ester. Further, hydroxyl groups of amino acids having functional
groups in their side chains, for example, the hydroxyl group of Tyr, may be protected
by an acetyl group, benzyl group, benzyloxycarbonyl group, tert-butyl group or the
like, but such protection is not necessarily required. Further, the guanidino group of
Arg may be protected with an appropriate protecting group such as a nitro group,
tosyl group, 2-methoxybenzenesulfonyl group, methylene-2-sulfonyl group,
benzyloxycarbonyl group, isobornyloxycarbonyl group or adamantyloxycarbonyl
group. Deprotection reactions for these protecting groups in the amino acids, the
peptides, and the finally obtained peptides of the present invention may also be
carried out according to conventional methods such as the catalytic reduction method
and methods using liquid ammonia/sodium, hydrogen fluoride, hydrogen bromide,
hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid and methanesulfonic
acid.
[0035]

Alternatively, the cell-penetrating peptides of the present invention may be
prepared by conventional methods using genetic engineering techniques. The thus
obtained cell-penetrating peptides of the present invention may be purified as
appropriate according to methods commonly used in the field of peptide chemistry,
such as those using ion-exchange resins, partition chromatography, gel
chromatography, affinity chromatography, high performance liquid chromatography
(HPLC) and the countercurrent distribution method.
[0036]
Alternatively, the cell-penetrating peptides of the present invention may be
used in the forms of nucleic acids encoding the peptides. Particular examples of the
nucleic acids include, but are not limited to, plasmid vectors, virus vectors,
phagemids and transposons which contain recombinant nucleic acids encoding fusion
proteins with the peptides of the present invention and which allow expression of
these peptides. Further, the recombinant nucleic acids are preferably used for, for
example, industrial production of a cell-penetrating peptide of the present invention,
or a fusion protein with a cell-penetrating peptide of the present invention;
introduction of an expression vector encoding a cell-penetrating peptide of the
present invention into living body-derived cells removed from the body; and
administration of a cell-penetrating peptide of the present invention to the body to
make cells in the body express the cell-penetrating peptide of the present invention or
a fusion protein therewith. In particular, the recombinant nucleic acids are
preferably used for methods of in vitro production of cell-penetrating peptides of the
present invention or fusion proteins with cell-penetrating peptides of the present
invention.
[0037]
The recombinant nucleic acid means DNA or RNA which was artificially
prepared. Examples of the recombinant nucleic acid include those synthesized by
linking nucleic acids such as adenine, cytosine, guanine, thymine and/or uracil with

each other; those prepared by cleaving out a part of DNA or RNA contained in an
organism and modifying it by removal of a part of its bases, linking it with other
bases, and/or the like; and those prepared by replicating these recombinant nucleic
acids.
[0038]
Further, the present invention relates to a pharmaceutical composition
containing a cell-penetrating peptide and a hydrophilic physiologically active
substance, and a method of administration of a hydrophilic physiologically active
substance to the living body by combined use of a cell-penetrating peptide of the
present invention and the hydrophilic physiologically active substance.
[0039]
Because of not only the fact that the cell-penetrating peptides of the present
invention themselves have cell membrane permeability, but also the fact that the
peptides can give cell membrane permeability to hydrophilic physiologically active
substances, in vivo delivery of a hydrophilic physiologically active substance
through the cell membrane is possible by blending of a cell-penetrating peptide of the
present invention in a pharmaceutical composition comprising as an effective
component the hydrophilic physiologically active substance or by administration of
the hydrophilic physiologically active substance and a cell-penetrating peptide of the
present invention in combination. More particularly, since a cell-penetrating
peptide of the present invention can not only preferably pass through the mucosal
cell membrane but also give transmucosal absorbability (preferably intestinal
absorbability or intranasal absorbability) to a hydrophilic physiologically active
substance, in vivo delivery of the hydrophilic physiologically active substance
through mucosa is possible. The transmucosal absorption of a hydrophilic
physiologically active substance means that a hydrophilic physiologically active
substance administered to mucosa passes into the blood through a mucosal layer, and
its result can be confirmed by increase in the blood level of the hydrophilic

physiologically active substance or expression of the pharmacological activity. The
blood level of a hydrophilic physiologically active substance can be measured by a
method normally used by those skilled in the art, such as an immunoassay. The
pharmacological activity can be measured using as an index, in the case of an
enzyme, its enzymatic activity, or, in the case of a substance that acts on a receptor in
the cell, an ability to change a function of the target cell or the production amount of
a marker substance. For example, the pharmacological activity of insulin can be
measured using as an index the blood glucose level of the animal to which insulin
was administered.
[0040]
The cell-penetrating peptide which gives cell membrane permeability,
preferably transmucosal absorbability, to a hydrophilic physiologically active
substance is not restricted as long as the peptide is the above-mentioned cell-
penetrating peptide of the present invention, and particular examples thereof include
a peptide having the amino acid sequence represented by SEQ ID NO:l and a
modified peptide produced by substituting, deleting, inserting and/or adding 1 to 7,
preferably 1 to 5, more preferably 1 to 2 basic amino acids in the amino acid
sequence represented by SEQ ID NO:l, which modified peptide has cell membrane
permeability. In particular, examples of a cell-penetrating peptide of the present
invention which gives intranasal absorbability to a hydrophilic physiologically active
substance and is preferably used for a pharmaceutical composition for intranasal
administration include a peptide having the amino acid sequence represented by SEQ
ID NO:l and a modified peptide produced by substituting, deleting, inserting and/or
adding 1 or 2 basic amino acid(s) in the amino acid sequence represented by SEQ ID
NO: 1, which modified peptide has cell membrane permeability. Particular
examples of a peptide which is a cell-penetrating peptide of the present invention and
gives intranasal absorbability to a hydrophilic physiologically active substance;
which peptide is a modified peptide produced by substituting, deleting, inserting

and/or adding 1 or 2 basic amino acid(s) in the amino acid sequence represented by
SEQ ID NO:l; which peptide has cell membrane permeability; are shown in Table 1.
In the modified peptide, substitution of a basic amino acid(s) in the amino acid
sequence represented by SEQ ID NO:l with another/other basic amino acid(s) is
most acceptable since substitution with a peptide having the same property is not
likely to change the overall property of the peptide.
[0041]
[Table 1]

[0042]
Preferred examples of a peptide having an amino acid sequence which is the
same as the amino acid sequence represented by SEQ ID NO: 1 except that one or
several basic amino acids are deleted, substituted and/or added, which peptide gives

transmucosal absorbability to a hydrophilic physiologically active substance, include
a peptide having the amino acid sequence represented by any of SEQ ID NO:4, SEQ
ID NO:9 and SEQ ID NO: 10, and, in particular, preferred examples of a peptide
which gives intranasal absorbability to a hydrophilic physiologically active substance
include a peptide having the amino acid sequence represented by SEQ ID NO:9 or 10.
[0043]
Further, a cell-penetrating peptide of the present invention which is a
modified peptide represented by a sequence which is the same as the amino acid
sequence represented by SEQ ID NO:l except that 1 to 5, preferably 1 to 3, more
preferably 1 or 2, still more preferably 1 amino acid(s) not limited to a basic amino
acid(s) is/are substituted, deleted, inserted and/or added, which modified peptide
enables transmucosal absorption of a hydrophilic physiologically active substance,
may also be used for a pharmaceutical composition of the present invention. In the
sequence which has been mutated by the substitution, deletion and/or insertion, a
sequence of not less than 3 continuous amino acids in the original amino acid
sequence is preferably conserved, and a sequence of not less than 5 continuous
amino acids in the original amino acid sequence is more preferably conserved.
Further, substitution with another peptide having the same property, such as
substitution of a basic amino acid(s) with another/other basic amino acid(s),
substitution of a hydrophilic amino acid(s) with another/other hydrophilic amino
acid(s), and/or substitution of a hydrophobic amino acid(s) with another/other
hydrophobic amino acid(s) in the amino acid sequence represented by SEQ ID NO:l,
is the most acceptable change since the overall property of the peptide is not likely to
change in this case.
[0044]
Further, a cell-penetrating peptide of the present invention which enables
transmucosal absorption of a hydrophilic physiologically active substance, which
cell-penetrating peptide is a peptide having the amino acid sequence represented by

the reverse sequence of the peptide having the amino acid sequence represented by
SEQ ID NO:l; a peptide having an amino acid sequence represented by the reverse
sequence of an amino acid sequence which is the same as the amino acid sequence
represented by SEQ ID NO: 1 except that one or several basic amino acids are
substituted, deleted, inserted and/or added; a peptide having an amino acid sequence
which is the same as the reverse sequence of the amino acid sequence represented by
SEQ ID NO:l except that one or several basic amino acids are substituted, deleted,
inserted and/or added; a peptide having an amino acid sequence represented by the
reverse sequence of an amino acid sequence which is the same as the amino acid
sequence represented by SEQ ID NO:l except that 1 to 5 amino acids, which are not
limited to basic amino acids, are substituted, deleted, inserted and/or added; or a
peptide having an amino acid sequence which is the same as the reverse sequence of
the amino acid sequence represented by SEQ ID NO: 1 except that 1 to 5 amino acids,
which are not limited to basic amino acids, are substituted, deleted, inserted and/or
added; may also be used for a pharmaceutical composition of the present invention.
[0045]
The hydrophilic physiologically active substance means a physiologically
active substance having a property of hydrophilicity. The hydrophilicity means that
the solubility in water is high, and, in the present specification, a substance is defined
to be hydrophilic when not less than 1 u.g of the substance is soluble to 1 ml of water.
The physiologically active substance means any substance that acts on a living body
to cause change in the living body, and examples thereof include proteins which are
bound to receptors in specific cells, and enzymes having affinities to substances in a
living body. Further, the physiologically active substance may also be a substance
which does not directly react with a substance in a living body, and, for example, it
may also be a substance which can be administered to a living body for a medical
purpose, such as dextran employed for increasing blood as an alternative to plasma.
The physiologically active substance is preferably a peptide, protein or nucleic acid,

more preferably a peptide or protein, which is poorly permeable through a biological
barrier such as the cell membrane or a transmucosal constituent cell layer. Further,
glycoproteins wherein sugar chains are bound to these proteins which are hydrophilic
physiologically active substances, and protein derivatives produced by chemical
modification such as polyethylene glycolation (PEGylation) are also preferably used
as the hydrophilic physiologically active substances.
[0046]
The molecular weight of the hydrophilic physiologically active substance is
not restricted, but in cases where the molecular weight is too high, passage of the
substance through the cell membrane is sometimes prevented, so that the molecular
weight is preferably not more than 500,000, more preferably not more than 30,000.
[0047]
Particular preferred examples of the hydrophilic physiologically active
substance include parathyroid hormone (PTH), calcitonin, insulin, angiotensin,
glucagon, glucagon-like peptide (GLP-1), exendin-4, gastrin, growth hormone,
prolactin (luteotropic hormone), gonadotropin (gonadotropic hormone), cytotropic
hormone, adrenocorticotropic hormone, melanocyte-stimulating hormone,
vasopressin, oxytocin, protirelin, luteinizing hormone (LH), corticotropin,
somatropin, thyrotropin (thyroid-stimulating hormone), somatostatin (growth
hormone-stimulating factor), hypothalamic hormone (GnRH), G-CSF, erythropoietin,
HGF, EGF, VEGF, angiopoietin, interferon-a, interferon-p, interferon-y, interleukins,
superoxide dismutase (SOD), urokinase, lysozyme and vaccines, and the hydrophilic
physiologically active substance is more preferably insulin, calcitonin, parathyroid
hormone, growth hormone, interferon, interleukin, G-CSF, glucagon-like peptide
(GLP-1) or exendin-4.
[0048]
In one preferred mode of the present invention, the cell-penetrating peptide
and the hydrophilic physiologically active substance form a conjugate. The

"conjugate" herein means a state wherein not less than 2 substances can move at the
same time, and the conjugate also includes substances bound to each other by
covalent bonding, substances electrostatically bound to each other by ionic bonding,
and substances which are not bound to each other but have spatial structures by
which one of these can limit movement of the other(s), enabling the substances to
move together. For example, a particle such as a micelle, liposome or
macromolecule whose surface is modified with a peptide of the present invention and
which encloses a biologically active drug therein can be said to be forming a
"conjugate". Examples of the conjugate include one wherein a peptide of the
present invention and a hydrophilic physiologically active substance are covalently
bound to each other. Examples of such a conjugate include a pharmaceutical
composition wherein an amino group or carboxyl group, or the thiol group of
cysteine, in a protein which is a hydrophilic physiologically active substance and a
cell-penetrating peptide of the present invention are covalently bound to each other.
[0049]
In cases where a cell-penetrating peptide and a protein which is a hydrophilic
physiologically active substance are made into a conjugate, the conjugate may be
prepared as a fusion protein. The site of addition of the cell-penetrating peptide of
the present invention is not restricted, and preferably, the peptide having cell
membrane permeability is presented on the outside of the protein and does not
largely affect the activity and the function of the protein after the fusion. The cell-
penetrating peptide is preferably fused to the N-terminus or C-terminus. The type
of the protein to be fused is not restricted, but, since a drug is prevented from passing
through the cell membrane in cases where its molecular weight it too high, the
molecular weight of the protein is preferably not more than 500,000, more preferably
not more than 30,000.
[0050]
The fusion protein with a cell-penetrating peptide may be produced by a

common chemical synthesis method. Examples of the method include a method
wherein a cell-penetrating peptide of the present invention and insulin are mixed
together and bound to each other by addition of a condensing agent, and a method
using a peptide synthesizer (e.g., Applied Biosystems Medel 433). Further, the
fusion protein may be produced based on the nucleic acid sequence information
using a genetic engineering technique according to a conventional method.
Examples of the method include a method wherein a nucleic acid sequence encoding
a cell-penetrating peptide of the present invention and a protein to be fused is
incorporated into a gene expression vector having a promoter for protein expression,
followed by producing a fusion protein.
[0051]
Further, in another preferred mode of the present invention, the cell-
penetrating peptide and the hydrophilic physiologically active substance are used in a
state where these are not covalently linked to each other. Even in cases where the
cell-penetrating peptide and the hydrophilic physiologically active substance are not
forming a conjugate by covalent bonding when a pharmaceutical composition of the
present invention is administered, a desired effect can be obtained by a process
wherein the cell-penetrating peptide and the hydrophilic physiologically active
substance are mixed with each other after their administration and then the peptide
and the hydrophilic physiologically active substance form a conjugate by ionic
bonding, hydrophobic action and/or charge action. Since, in such cases, direct
modification of the hydrophilic physiologically active substance is not necessary, the
bioactivity originally retained by the hydrophilic physiologically active substance is
not affected, which is preferred.
[0052]
In the present invention, the mucosa through which transmucosal absorption
of the hydrophilic physiologically active substance is possible is not limited, and
examples thereof include mucosa of the eye, nasal cavity, hypoglottis, lung, oral

cavity, skin, vagina and intestine. The mucosa is preferably mucosa of the nasal
cavity or intestine. Further, the administration method for transmucosal absorption
of the hydrophilic physiologically active substance by the present invention is not
limited, and examples of the administration method include oral, nasal, rectal and
percutaneous administration and administration by injection. The administration
method is preferably oral, nasal or rectal administration.
[0053]
In the present invention, the dose and the number of doses for administration
of the hydrophilic physiologically active substance and the cell-penetrating peptide
to a living body may be appropriately selected depending on the hydrophilic
physiologically active substance, dosage form, age of the patient, body weight and
severity of symptoms, and usually, they are administered at a dose of 0.01 to 50 mg,
preferably 0.1 to 20 mg per adult per day in terms of the content of the hydrophilic
physiologically active substance. The blend ratio of the cell-penetrating peptide
and the hydrophilic physiologically active substance is not restricted, and the ratio is
preferably determined depending on the type of the hydrophilic physiologically
active substance blended and the mode of blending of the cell-penetrating peptide
and the hydrophilic physiologically active substance. In order to give a sufficient
action to the hydrophilic physiologically active substance, the cell-penetrating
peptide is used, in any mode of blending, in an amount preferably not less than 1
time larger, more preferably not less than 2 times larger than the molar amount of the
hydrophilic physiologically active substance which is to be allowed to act. This
means that, in cases where the cell-penetrating peptide and the hydrophilic
physiologically active substance are forming a conjugate by covalent bonding, not
less than 1 cell-penetrating peptide is bound to each hydrophilic physiologically
active substance, and, in cases where these are independently used, not less than 1
mole of the cell-penetrating peptide is contained with respect to 1 mole of the
hydrophilic physiologically active substance used.

[0054]
In the present invention, the mode of administration of the hydrophilic
physiologically active substance and the cell-penetrating peptide to an animal
(including human) is not particularly restricted. For example, these may be
administered as they are in the dry state or in the form of a solution, or may be filled
in a capsule together with a vehicle followed by administration of the capsule.
Further, those in the dry state may be once dissolved or dispersed in water and then
administered.
[0055]
Examples of the form of the pharmaceutical composition of the present
invention include powder forms composed of the cell-penetrating peptide and the
hydrophilic physiologically active substance as well as pharmaceutically acceptable
additives; liquid forms composed of a mixture with a medium, such as water, and a
pharmaceutically acceptable base other than the medium, and the like; and solid or
semisolid forms composed of a combination with a pharmaceutically acceptable base.
Examples of the base include various organic and inorganic substances commonly
used as formulation materials, such as vehicles, lubricants, binders, disintegrators,
solvents, solubilizers, suspending agents, isotonic agents, buffering agents, soothing
agents and absorption enhancers. Particular examples thereof include water,
pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol,
polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose,
sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl
starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein,
gelatin, agar, diglycerol, propylene glycol, polyethylene glycol, vaseline, paraffin,
stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose,
and surfactants acceptable as pharmaceutical additives.
[0056]
Further, the pharmaceutical composition of the present invention may be

preferably used in combination not only with simple additives but also with
advanced delivery techniques. For example, for the purpose of delivering a
pharmaceutical composition of the present invention to the intestinal tract by oral
administration, it is preferred to encapsulate the pharmaceutical composition in an
enteric capsule or in mucoadhesive hydroxypropylcellulose or Smart Hydrogel
poly(methacrylic acid) grafted with poly(ethylene glycol) P(MAA-g-EG), or the like,
in view of avoiding degradation of the peptide and the drug by digestive enzymes.
EXAMPLES
[0057]
Example 1: Evaluation of Peptide Permeability into Cells

In DMEM medium supplemented with 10% FBS, 0.2 ml of HeLa cells were
plated on a 96-well glass bottom plate, and the cells were cultured at 37°C for 48 to
72 hours to allow the cells to adhere to the bottom surface. After 3 times of
washing with 200 ul of PBS, 50 u,l of DMEM medium containing each of the
peptides of SEQ ID NOs:l to 6 labeled with fluorescein at the amino terminus
(contract synthesis by Sigma-Genosys) at a final concentration of 5 uM and FBS at a
final concentration of 10% was added to each well. The plate was incubated in a
CO2 incubator for 3 hours and then washed 3 times with DMEM medium
supplemented with 10% FBS, followed by addition of 50 ul of a lysis solution (10
mM Tris-HCl, 5 mM EDTA, 100 mM NaCl, 1% SDS, 100 ug/ml proteinase K) and
1 hour of incubation at room temperature, to decompose the cells and the peptides
incorporated into the cells. The whole lysis solution was recovered, and the amount
of fluorescein incorporated into the cells was measured using a fluorescence intensity
measuring apparatus (HORIBA FLUOROMAX-3) at an excitation wavelength of
494 nm and a fluorescence wavelength of 512 nm.
[0058]

The amount of fluorescein incorporated into the cells with respect to the
amount of fluorescein added to each well is shown in Fig. 1. For each value, the
average value and the standard error obtained by 3 times of evaluation are shown.
In contrast to the random peptide of SEQ ID NO:6, which was hardly incorporated
into the cells and hence not a cell-penetrating peptide, the fluorescein-labeled
peptides of SEQ ID NOs:l to 5, which are peptides of the present invention, were
incorporated into the cells with not less than 10 times higher efficiencies than the
random peptide of SEQ ID NO:6.
[0059]
Example 2: Evaluation of Promotion of Insulin Transfer into Cells

In DMEM medium supplemented with 10% FBS, 0.2 ml of HeLa cells were
plated on a 96-well glass bottom plate, and the cells were cultured at 37°C for 48 to
72 hours to allow the cells to adhere to the bottom surface. After 3 times of
washing with 200 ul of PBS, 50 ul of DMEM medium containing each of the
peptides of SEQ ID NOs: 1 to 6 labeled with fluorescein at the amino terminus
(contract synthesis by Sigma-Genosys) at a final concentration of 5 uM, rhodamine-
labeled human insulin at a final concentration of 100 ug/ml and 10% FBS was added
to each well. The plate was incubated in a CO2 incubator for 3 hours and then
washed 3 times with DMEM medium supplemented with 10% FBS, followed by
addition of 50 ul of a lysis solution (10 mM Tris-HCl, 5 mM EDTA, 100 mM NaCl,
1% SDS, 100 ug/ml proteinase K) and 1 hour of incubation at room temperature, to
decompose the cells and the rhodamine-labeled insulin incorporated into the cells.
The whole lysis solution was recovered, and the amount of the rhodamine-labeled
insulin incorporated into the cells was measured using a fluorescence intensity
measuring apparatus at an excitation wavelength of 555 nm and a fluorescence
wavelength of 580 nm.
[0060]

Further, in the same manner, a solution containing rhodamine-labeled human
insulin and each of the peptides of SEQ ID NOs:l to 6 was added to HeLa cells and
the resultant was the incubated, followed by 3 times of washing with 300 ul of
DMEM medium supplemented with 10% FBS, 3 hours of fixation with 4%
paraformaldehyde solution and 3 times of washing with 300 ul of PBS. Thereafter,
the cells were incubated in 5% DiD'Oil solution (Molecular probes, Inc.) overnight,
and localization of the rhodamine-labeled insulin was observed using a confocal laser
microscope (FV-1000, Olympus Corporation).
[0061]
The rhodamine-labeled insulin was prepared by the following method. In
0.5 ml of 0.1 N hydrochloric acid, 20 mg of human insulin was dissolved, and 3 ml
of PBS and 0.5 ml of 0.1 N sodium hydroxide were then added thereto to neutralize
the solution. The resulting solution was subjected to solution exchange using a
desalting column (PD-10 column), to obtain 5 ml of 50 mM NaHC03 solution
(insulin concentration, 4 mg/ml). To the resulting solution, 4 mg of NHS-
rhodamine (Pierce) dissolved in 0.5 ml of DMSO was added, and the reaction was
allowed to proceed at room temperature overnight. To the reaction solution, 0.5 ml
of 1 M Tris-HCl (pH 8) was added, and the resulting mixture was further allowed to
react at room temperature for 30 minutes, followed by desalting against water using a
PD-10 column and adding water to obtain a solution in a final volume of 10 ml (2
mg/ml in terms of the insulin concentration).
[0062]

The amount of rhodamine-labeled insulin incorporated into the cells with
respect to the amount of rhodamine-labeled insulin added to each well is shown in
Fig. 2. Each value is represented as the average value and the standard error
obtained by 3 times of evaluation. Although addition of the rhodamine-labeled
insulin alone and addition of the rhodamine-labeled insulin together with the peptide

having the random sequence shown in SEQ ID NO:6 to the cells resulted in almost
no incorporation of the rhodamine-labeled insulin into the cells, addition of the
rhodamine-labeled insulin together with the peptides of SEQ ID NOs:l to 5, which
are peptides of the present invention, resulted in incorporation of the rhodamine-
labeled insulin into the cells with not less than 50 times higher efficiencies than the
random peptide of SEQ ID NO:6.
[0063]
Results of observation using a confocal laser microscope after addition of
each of the peptides of SEQ ID NOs:l to 6 together with rhodamine-labeled insulin
are shown in Fig. 12. In contrast to the random peptide of SEQ ID NO:6, which is
not a cell-penetrating peptide and did not allow permeation of the rhodamine-labeled
insulin into the cells, the cell-penetrating peptides of SEQ ID NOs:l to 5 of the
present invention were confirmed to have allowed permeation of the rhodamine-
labeled insulin into the cells.
[0064]
Example 3: Evaluation of Promotion of Transfer of Polystyrene Beads into Cells

In DMEM medium supplemented with 10% FBS, 0.2 ml of HeLa cells were
plated on a 96-well glass bottom plate, and the cells were cultured at 37°C for 48 to
72 hours to allow the cells to adhere to the bottom surface. After 3 times of
washing with 200 ul of PBS, 50 ul of DMEM medium containing each of the
peptides of SEQ ID NOs: 1 to 6 labeled with fluorescein at the amino terminus
(contract synthesis by Sigma-Genosys) at a final concentration of 1 uM, fluorescent
polystyrene beads (Fluorospheres Carboxylated Microbeads, 0.1 um; manufactured
by Molecular probes, Inc.) at a final concentration of 1 ul/well and 10% FBS was
added to each well. The plate was incubated in a CO2 incubator for 3 hours and
then washed 3 times with DMEM medium supplemented with 10% FBS, followed
by addition of 50 ul of a lysis solution (10 mM Tris-HCl, 5 mM EDTA, 100 mM

NaCl, 1% SDS, 100 ug/ml proteinase K) and 1 hour of incubation at room
temperature, to decompose the cells. The whole lysis solution was recovered, and
the amount of fluorescent polystyrene beads incorporated into the cells was measured
using a fluorescence intensity measuring apparatus at an excitation wavelength of
580 nm and a fluorescence wavelength of 605 nm.
[0065]

The amount of fluorescent polystyrene beads incorporated into the cells with
respect to the amount of fluorescent polystyrene beads added to each well is shown
in Fig. 3. For each value, the average value and the standard error obtained by 3
times of evaluation are shown. Although addition of the fluorescent polystyrene
beads alone and addition of the beads together with the random peptide of SEQ ID
NO:6, which is not a cell-penetrating peptide, to the cells resulted in almost no
incorporation of the beads into the cells, addition of the beads together with the
peptides of SEQ ID NOs:l to 5, which are peptides of the present invention, resulted
in recovery of not less than 0.7% of the fluorescent polystyrene beads added.
[0066]
Example 4: Intranasal Administration of Insulin

A specific amount of insulin (Wako Pure Chemical Industries, Ltd.) powder
was placed in a 1.5-ml tube (Eppendorf) and dissolved in 0.1 N HC1, followed by
adding the same quantity of 0.1 N NaOH to prepare an insulin solution. A solution
of insulin alone, and 40 ul of a mixed solution for each administration experiment
were prepared, which mixed solution was prepared by dissolving the peptide of SEQ
ID NO:l whose total amino acid sequence is constituted by amino acids having the
configuration of L-body (contract synthesis by Sigma-Genosys) in PBS and
combining the resulting solution with the above-mentioned insulin solution such that
the mixed solution contains insulin (1 IU/kg) and each peptide at a concentration of

0.5 mM. To a male SD rat having a body weight of about 200 g which had been
fasted for 24 hours, 50 mg/kg of pentobarbital was intraperitoneally injected for
anesthetization, followed by incision of the neck to expose the trachea. A
polyethylene tube (INTRAMEDIC PE205, Clay Adams) was inserted into the
trachea, and a part of the esophagus was incised, followed by inserting a tube having
the same diameter carefully from the incised portion of the esophagus into a choana
such that tissues are not damaged. The tip of the tube to be inserted into the choana
was preliminarily sealed with absorbent cotton and an adhesive. To prevent leakage
of the drug solution, the nasopalatine canal in the maxillary region opening to the
oral cavity is closed with a synthetic adhesive ("Aron alpha A" manufactured by
Daiichi-Sankyo Company, Limited). Before the administration, and 5, 10, 15, 30,
60, 120, 180 and 240 minutes after the administration of the prepared mixture of
insulin and the peptide, or insulin alone, 0.25 ml of blood was collected from the
cervical vein and subjected to measurement of the blood glucose level using a blood
glucose level measuring apparatus "Novo Assist Plus" (Novo Nordisk Pharma Ltd.).
The remaining blood was subjected to centrifugation for separation of blood plasma,
and the plasma insulin level was measured using EIA kit (Revis). The
bioavailability was calculated by comparison with the case of subcutaneous
administration of insulin.
[0067]

Change in the blood glucose level with time after the administration is shown
in Fig. 4, and change in the blood insulin level with time after the administration is
shown in Fig. 5. For each value, the average value and the standard error obtained
by 3 or 6 times of evaluation are shown. In rats to which only insulin was
intranasally administered, increase in the blood insulin level could be hardly
confirmed, but in rats to which insulin was administered together with the peptide of
SEQ ID NO:l, transfer of insulin into the blood was observed from immediately

after the administration. Further, decrease in the blood glucose level, which is a
pharmacological activity due to transfer of insulin into the blood, was observed,
which decrease in the blood glucose level corresponded to the blood insulin level.
[0068]
Example 5: Intranasal Administration of Insulin 2

Insulin was prepared by the same method as in Example 4, and a solution of
insulin alone, and 40 uJ of a mixed solution for each administration experiment were
prepared, which mixed solution was prepared by dissolving the peptide of SEQ ID
NO:l, SEQ ID NO:7 (oligoarginine) or SEQ ID NO:8 (penetratin) whose total amino
acid sequence is constituted by amino acids having the configuration of L-body
(contract synthesis by Sigma-Genosys) in PBS and combining the resulting solution
with the above-mentioned insulin solution such that the mixed solution contains
insulin (1 IU/kg) and each peptide at a concentration of 0.5 mM. Each mixed
solution was administered to rats by the same method as described in Example 4, and
blood collection and measurement were carried out with time, thereby measuring the
plasma insulin level, followed by calculating the bioavailability by comparison with
the case of subcutaneous administration of insulin
[0069]

The average value and the standard error of the bioavailability calculated by 3
times of evaluation by administration of insulin alone or the mixed solution of insulin
and each of the peptides of SEQ ID NOs:l, 7 and 8 are shown in Fig. 6. The cell-
penetrating peptide of the present invention having the amino acid sequence
represented by SEQ ID NO: 1 showed a significantly higher bioavailability than those
of the known cell-penetrating peptides represented by SEQ ID NOs:7 and 8.
[0070]
Example 6: Intranasal Administration of Interferon p

With ice cooling, 1 ml of PBS supplemented with Tween 20 was added to
human wild-type interferon p ("Feron" manufactured by Toray Industries, Inc.) to
attain a concentration of 6,000,000 IU/ml, and 100 ul of this solution was aliquoted,
followed by adding 566 ul of PBS supplemented with Tween 20 to the aliquot to
prepare a 900,000 IU/ml solution. A solution of interferon p alone, and 40 ul of a
mixed solution for each administration experiment were prepared, which mixed
solution was prepared by dissolving the peptide of SEQ ID NO:l or 4 whose total
amino acid sequence is constituted by amino acids having the configuration of L-
body (contract synthesis by Sigma-Genosys) in PBS and combining the resulting
solution with the above-mentioned interferon p solution such that the mixed solution
contains interferon P (0.18><106 IU/kg) and each peptide at a concentration of 0.5
mM. The evaluation was carried out by the same method as described in Example
4. The plasma interferon p level was measured by "human interferon p ELISA kit"
(Kamakura Techno-Science Inc.).
[0071]

Change in the plasma interferon P level after the administration is shown in
Fig. 7. For each value, the average value and the standard error obtained by 3 times
of evaluation are shown. Although rats to which interferon p alone was intranasally
administered showed only small increase in the blood interferon p level, rats to
which interferon P was administered together with the peptide of SEQ ID NO:l or
SEQ ID NO:4 showed transfer of interferon p into the blood from immediately after
the administration.
[0072]
Example 7: Intranasal Administration of Exendin-4

A solution of exendin-4 (contract synthesis by Sigma-Genosys) alone, and 40

ul of a mixed solution for each administration experiment were prepared, which
mixed solution was prepared by dissolving the peptide of SEQ ID NO:l or SEQ ID
NO:4 whose total amino acid sequence is constituted by amino acids having the
configuration of L-body (contract synthesis by Sigma-Genosys) in PBS and
combining the resulting solution with the above-mentioned exendin-4 solution such
that the mixed solution contains exendin-4 (0.25 mg/kg) and each peptide at a
concentration of 0.5 mM. The evaluation was carried out by the same method as
described in Example 4. The plasma exendin-4 level was measured by the
sandwich ELISA (enzyme-linked immunosorbent assay) method using an anti-
exendin-4 monoclonal antibody as a solid phase, and a biotin-labeled anti-exendin-4
polyclonal antibody and streptavidin-HRP for detection.
[0073]

Change in the plasma exendin-4 level after the administration is shown in Fig.
8. For each value, the average value and the standard error obtained by 3 times of
evaluation are shown. Although rats to which only exendin-4 was intranasally
administered showed almost no increase in the blood exendin-4 level, rats to which
exendin-4 was administered together with the peptide of SEQ ID NO:l or SEQ ID
NO:4 showed transfer of exendin-4 into the blood from immediately after the
administration.
[0074]
Example 8: Intestinal Administration of Insulin

An insulin solution was prepared by the same method as in Example 4. A
solution of insulin alone, and 500 ul of a mixed solution for each administration
experiment were prepared, which mixed solution was prepared by dissolving the
peptide of SEQ ID NO:l or SEQ ID NO:4 whose total amino acid sequence is
constituted by amino acids having the configuration of L-body (contract synthesis by

Sigma-Genosys) in PBS and combining the resulting solution with the above-
mentioned insulin solution such that the mixed solution contains insulin (50 IU/kg)
and each peptide at a concentration of 0.5 mM. To a male SD rat having a body
weight of about 200 g which had been fasted for 24 hours, 50 mg/kg of pentobarbital
was intraperitoneally injected for anesthetization, followed by opening the abdomen
in the median line to expose the intestinal tract. A silicone tube was inserted at a
position 2 to 3 cm distant from the ileocecal junction toward the ileum, and a feeding
needle was inserted at a position about 10 cm upward therefrom. Further, sutures
were passed through an area of 6 cm between these. From the feeding needle, 20
ml of phosphate buffered saline (PBS) at pH 7.4 prewarmed to 37°C was fed at a
flow rate of 5 ml/min. to allow discharging of the contents, and the silicone tube was
plugged, followed by administering 1 ml of PBS prewarmed to 37°C from the
feeding needle and keeping it retained for 30 minutes. After the administration, the
feeding needle was quickly plugged and the intestinal tract was returned into the
abdominal cavity, followed by closing the site of incision with a clip and allowing
the rat to keep still. After the retention, the plugs of the feeding needle and the
silicone tube were removed, and 20 ml of PBS prewarmed to 37°C was fed into the
loop at a flow rate of 5 ml/min. to allow discharging of the contents, followed by
ligating the area of 6 cm through which the suture had been preliminarily passed, to
form a loop. To this loop, 0.5 ml of the insulin solution alone or the mixture of
insulin and the peptide of SEQ ID NO:l or SEQ ID NO:4 was administered, and the
intestinal tract was returned into the abdominal cavity, followed by closing the site of
incision with a clip and allowing the rat to keep still. The rat during the experiment
was fixed on its back on a hot plate kept at 37°C by a hot-water circulating pump to
regulate the body temperature. Before the administration, and 5, 10, 15, 30, 60, 120
and 180 minutes after, the administration, 0.25 ml of blood was collected from the
cervical vein and subjected to measurement of the blood glucose level using a blood
glucose level measuring apparatus "Novo Assist Plus" (Novo Nordisk Pharma Ltd.).

The remaining blood was subjected centrifugation for separation of blood plasma,
and the insulin level was quantified by an enzyme-linked immunosorbent assay.
Also for rats to which the same quantity of the insulin solution alone was
subcutaneously administered, the blood collection and the measurement were carried
out in the same manner.
[0075]

Change in the blood glucose level with time after the administration is shown
in Fig. 9, and change in the blood insulin level with time after the administration is
shown in Fig. 10. For each value, the average value and the standard error obtained
by 3 or 6 times of evaluation are shown. Compared to the case of administration of
insulin alone as a control, evident increase in the blood insulin level and evident
decrease in the blood glucose level were observed in the cases of combined
administration of the peptide of SEQ ID NO:l or 4 and insulin (Fig. 9 and Fig. 10).
[0076]
Example 9: Evaluation of Damage to Nasal Cavity

By the same method as in Example 4, insulin alone; insulin and the peptide of
SEQ ID NO: 1; or 5% (w/v) taurodeoxycholic acid solution was intranasally
administered. Fifteen minutes later, the nasal cavity was washed with 10 ml of PBS
warmed to 37°C at a flow rate of 2 ml/min. The washing solution was recovered,
and the LDH leaked into the solution was measured using LDH activity measuring
kit (Pointe Scientific, Inc.).
[0077]

The LDH activity in the washing solution is shown in Fig. 11. For each
value, the average value and the standard error obtained by 3 times of evaluation are
shown. Compared to the case of administration of 5% (w/v) sodium

taurodeoxycholate, release of LDH from the nasal cavity was significantly lower in
mice to which the mixed solution of the peptide of SEQ ID NO:l and insulin was
administered, and the release in the latter case was as low as in the cases where PBS
or insulin was solely administered.
[0078]
Example 10: Evaluation of Promotion of Insulin Transfer into Cells 2

Evaluation of promotion of insulin transfer into HeLa cells was carried out in
the same manner as in Example 2 except that the peptides of SEQ ID NOs:l, 6 and 9
to 11 whose amino termini are not labeled (contract synthesis by Sigma-Genosys)
were used. The incubation time in a CO2 incubator was 5 hours, and the amount of
rhodamine-labeled insulin incorporated into the cells was measured using a
fluorescence intensity measuring apparatus.
[0079]

The amount of rhodamine-labeled insulin incorporated into the cells with
respect to the amount of rhodamine-labeled insulin added to each well is shown in
Fig. 13. For each value, the average value and the standard error obtained by 3
times of evaluation are shown. In the cases where the random peptide of SEQ ID
NO:6 or 11, which had been confirmed not to be a cell-penetrating peptide, was
added together to the cells, only not more than 0.05% of the rhodamine-labeled
insulin added was incorporated into the cells, while in the cases where the peptide of
SEQ ID NO: 1 or SEQ ID NO: 9 or 10 was added together, not less than 0.7% of the
rhodamine-labeled insulin added was incorporated into the cells.
[0080]
Example 11: Intranasal Administration of Insulin 2

Insulin was prepared by the same method as in Example 4, and a solution of

insulin alone, and 40 uj of a mixed solution for each administration experiment were
prepared, which mixed solution was prepared by dissolving the peptide of SEQ ID
NO:l, 9 or 10 whose total amino acid sequence is constituted by amino acids having
the configuration of L-body (contract synthesis by Sigma-Genosys) in PBS and
combining the resulting solution with the above-mentioned insulin solution such that
the mixed solution contains insulin (1 IU/kg) and each peptide at a concentration of
0.5 mM. Each mixed solution was administered to rats by the same method as
described in Example 4, and blood collection and measurement were carried out with
time, thereby measuring the plasma insulin level, followed by calculating the AUC
(area under the blood concentration-time curve) based on change in the blood level
(uU/ml) with time (minutes).
[0081]

The average value and the standard error of AUC calculated by 3 or 6 times
of evaluation wherein insulin alone; or the mixture of the peptide of SEQ ID NO: 1
or SEQ ID NO: 9 or 10 and insulin; was administered are shown in Fig. 14. In the
cases where the cell-penetrating peptide of SEQ ID NO: 1, 9 or 10 was administered
together with insulin, a significantly higher intranasal absorption of insulin was
observed compared to the case where insulin alone was administered.
[0082]
Example 12: Evaluation of Promotion of Insulin Transfer into Cells 3

Evaluation of promotion of insulin transfer into HeLa cells was carried out in
the same manner as in Example 2 using the peptides of SEQ ID NO: 6 and 12 to 30
whose amino termini are labeled with fluorescein (contract synthesis by Sigma-
Genosys). The incubation time in a C02 incubator was 5 hours, and the amount of
rhodamine-labeled insulin incorporated into the cells was measured using a
fluorescence intensity measuring apparatus. Further, the amount of fluorescein

incorporated into the cells was measured by the same method as in Example 1.

The amount of fluorescein incorporated into the cells with respect to the
amount of fluorescein added to each well is shown in Fig. 15. For each value, the
average value and the standard error obtained by 2 times of evaluation are shown.
Although only about 0.2% of the fluorescein added was incorporated into the cells in
the case of the peptide of SEQ ID NO:6 having a random sequence, not less than
0.8% of the fluorescein added was incorporated into the cells in all the cases of the
cell-penetrating peptides of SEQ ID NOs:12 to 30 of the present invention.
[0083]
The amount of rhodamine-labeled insulin incorporated into the cells with
respect to the amount of rhodamine-labeled insulin added to each well is shown in
Fig. 16. For each value, the average value and the standard error obtained by 2
times of evaluation are shown. Only about 0.2% of the rhodamine-labeled insulin
added was incorporated into the cells in the case of the random peptide of SEQ ID
NO:6, which is not a cell-penetrating peptide, while not less than 1% was
incorporated into the cells in all the cases where the cell-penetrating peptides of SEQ
ID NOs:12 to 30 of the present invention were added together with the insulin.
INDUSTRIAL APPLICABILITY
[0084]
By the present invention, introduction of a hydrophilic physiologically active
substance into the cell, which has been difficult so far, becomes possible, and
development of a novel medical technology can be expected. More particularly,
since a formulation which enables transmucosal administration of a hydrophilic
physiologically active substance can be obtained using a cell-penetrating peptide of
the present invention, pain and inconvenience of patients, which have been caused by
conventional formulations (e.g., injection solutions) comprising hydrophilic
physiologically active substances, can be largely improved and hence patient-

oriented medical care can be realized at clinical sites. Further, the cell-penetrating
peptides of the present invention may fundamentally change the concept of
formulations comprising hydrophilic physiologically active substances, leading to
creation of epoch-making formulations.
SEQUENCE LISTING FREE TEXT
[0085]
SEQ ID NOs: 1 to 5 Cell-penetrating peptides of the present invention;
SEQ ID NO:6 Random peptide;
SEQ ID NO:7 Oligoarginine;
SEQ ID NO: 8 Penetratin
SEQ ID NOs: 9 to 10 Cell-penetrating peptides of the present invention;
SEQ ID NO: 11 Random peptide 2;
SEQ ID NOs: 12 to 30 Cell-penetrating peptides of the present invention.
SEQUENCE LISTING

We claim
1. A cell-penetrating peptide which is any of (A) to (D) below:
(A) a peptide having the amino acid sequence represented by SEQ ID NO:l;
(B) a peptide having an amino acid sequence which is the same as the amino
acid sequence represented by SEQ ID NO: 1 except that one or several basic amino
acids are substituted, deleted, inserted and/or added, which peptide has cell
membrane permeability;
(C) a peptide having an amino acid sequence which is the same as the amino
acid sequence represented by SEQ ID NO: 1 except that 1 to 5 amino acids are
substituted, deleted, inserted and/or added, which peptide has cell membrane
permeability;
(D) a peptide having: an amino acid sequence represented by the reverse
sequence of any of (A) to (C); an amino acid sequence which is the same as the
amino acid sequence represented by the reverse sequence of (A) except that one or
several basic amino acids are substituted, deleted, inserted and/or added; or an amino
acid sequence which is the same as the amino acid sequence represented by the
reverse sequence of (A) except that 1 to 5 amino acids are substituted, deleted,
inserted and/or added; which peptide has cell membrane permeability.

2. The cell-penetrating peptide according to claim 1, wherein said peptide (B)
has the amino acid sequence represented by any of SEQ ID NOs:2 to 4, 9 to 10 and
13.
3. The cell-penetrating peptide according to claim 1 or 2, wherein said peptide
(C) has the amino acid sequence represented by any of SEQ ID NOs:12 and 15 to 30.
4. The cell-penetrating peptide according to any of claims 1 to 3, wherein said
peptide (D) has the amino acid sequence represented by SEQ ID NO:5 or 14.
5. A pharmaceutical composition comprising the cell-penetrating peptide
according to any of claims 1 to 4 and a hydrophilic physiologically active substance.

6. A pharmaceutical composition for oral administration, said pharmaceutical
composition comprising the cell-penetrating peptide according to any of claims 1 to
4 and a hydrophilic physiologically active substance.
7. A pharmaceutical composition for intranasal administration, said
pharmaceutical composition comprising the cell-penetrating peptide according to any
of claims 1 to 4 and a hydrophilic physiologically active substance.
8. The pharmaceutical composition according to any of claims 5 to 7, wherein
said hydrophilic physiologically active substance is a peptide, protein or nucleic acid.

ABSTRACT

A cell-penetrating peptide described in any of (A) to (D) below gives cell
membrane permeability and further transmucosal absorbability to a physiologically
active substance:
(A) a peptide having the amino acid sequence represented by SEQ ID NO: 1;
(B) a peptide having an amino acid sequence which is the same as the amino
acid sequence represented by SEQ ID NO: 1 except that one or several basic amino
acids are substituted, deleted, inserted and/or added, which peptide has cell
membrane permeability;
(C) a peptide having an amino acid sequence which is the same as the amino
acid sequence represented by SEQ ID NO:l except that 1 to 5 amino acids are
substituted, deleted, inserted and/or added, which peptide has cell membrane
permeability;
(D) a peptide having: an amino acid sequence represented by the reverse
sequence of any of (A) to (C); an amino acid sequence which is the same as the
amino acid sequence represented by the reverse sequence of (A) except that one or
several basic amino acids are substituted, deleted, inserted and/or added; or an amino
acid sequence which is the same as the amino acid sequence represented by the
reverse sequence of (A) except that 1 to 5 amino acids are substituted, deleted,
inserted and/or added; which peptide has cell membrane permeability.