DESCRIPTION
Pharmaceutical Composition for Transnasal Administration
TECHNICAL FIELD
The present invention relates to a pharmaceutical composition to allow
migration of a hydrophilic bioactive substance from the nasal cavity into blood while
maintaining the activity thereof.
BACKGROUND ART
In addition to low molecular weight hydrophobic drugs which have been
mainly employed so far, hydrophilic bioactive substances such as peptides and
nucleic acids have recently appeared in clinical sites. They are employed as
pharmaceuticals and exhibit remarkable therapeutic effects. However, up to now, in
most cases, their administration methods are limited to usage as injection solutions.
This is because hydrophilic substances are incapable of passing through the epithelial
cell layer of mucosa, unlike known drugs. Since the cells in the epithelial cell layer
are closely bound to each other to prevent invasion of foreign substances into the
living body, substances having hydrophilicity can hardly pass through the layer, so
that oral administration, which is a generally-used administration method of drugs, is
incapable of allowing the substances to act inside the body.
Since administration of a drug by injection is a heavy burden on a patient and
a physician, particularly in cases where the therapy is frequent and continues for a
long time, various methods have been studied to enable administration of such a
hydrophilic bioactive substance by a method other than injection.
In oral administration which is widely used as a general method for
administration of a drug, there are a number of attempts for technologies to allow
absorption of a hydrophilic bioactive substance. In particular, to improve
permeability of the intestinal epithelial cell layer which is a major region of
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absorption when oral administration is carried out, attempts have been made using
surfactants, epithelial cell-adhesive substances, cell penetrating peptides and the like.
"Cell penetrating peptide" is a general term for peptides having a property to
migrate from outside a cell to inside the cell without destroying the cell membrane.
Well-known examples of the peptides include various types of peptides such as
oligoarginine having a stretch of arginine; Tat, a peptide of the HIV-1 virus (Patent
Literature 1); and penetratin having the same amino acid sequence as shown in SEQ
ID NO:l of the present invention (Patent Literatures 2 and 3). Various peptides are
included therein, and examples thereof include those characterized by simple
basicity; those having a hydrophobic domain; those characterized by amphiphilicity
of the primary structure or secondary structure of the peptides; and those having an
uncertain mechanism. Using these peptides, researches on their migration
capabilities into the cell, and their uses, as vehicles, for delivery of genes linked to
them into the cell have been extensively carried out.
Further, studies aiming to promote penetration through the epithelia cell layer
and to promote absorption upon oral administration using such a property have been
carried out (Patent Literatures 4 and 5). However, since the absorption promotion
effects of the cell penetrating peptides used in these studies are based on evaluation
with rinsed intestinal tract and the like, the actual effects upon oral administration are
not clear. Further, it is difficult to realize stable absorption because of digestive
degradation in stomach and changes in the environment in the gastrointestinal tract
by ingestion, so that practical application of oral administration of a hydrophilic
bioactive substance has not been achieved.
As a method other than oral administration to realize the absorption, an
administration method employing the nasal mucosal layer has been devised.
Between nasal mucosa and mucosa of the gastrointestinal tract, there are differences
in the types of the cells constituting the mucosal layer and differences in properties
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of individual cells, and degradative enzymes existing in the lumens show various
differences such as differences in their types and amounts, so that technologies other
than those for promotion of absorption in the gastrointestinal tract are demanded for
promotion of transnasal absorption.
Administration can be easily carried out through nasal mucosa, and nasal
mucosa has properties suitable for absorption of drugs because of developed blood
vessels and lymph vessels under it. Further, it is considered that, since the drug
absorbed from the nasal cavity directly passes into the general circulation after the
absorption, the first-pass effect by liver can be avoided, so that the nasal cavity is
considered to be effective as an administration site for drugs prone to be metabolized
in the gastrointestinal tract and/or liver. However, since, as in the other absorption
sites, nasal mucosa has low permeabilities for hydrophilic bioactive substances, and
since the area of the absorption site is small, an absorption-promoting technology is
required to realize absorption of hydrophilic bioactive substances from the nasal
cavity.
There are examples of clinical tests with transnasal formulations using
surfactants as absorption-promoting agents by a plurality of drug makers so far, but
all of the tests were given up because of strong irritation to nasal mucosa. Further, as
a method which does not employ a surfactant, promotion of absorption using a
peptide has been attempted at the research phase, and an attempt to promote
transnasal absorption by direct linking of a peptide to a drug is disclosed in Patent
Literature 6. However, since this technology requires chemical modification of a
drug, it necessarily has many problems to be solved such as decrease in the
pharmacological activity of the drug, changes in the pharmacokinetics, increase in
the production cost and antigenicity of the administered drug.
Possibilities of promotion of transnasal absorption by penetratin or a
modified product thereof are mentioned in Patent Literature 2 and 3, but since these
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require covalent bond between the drug to be allowed to permeate the mucosa and
the modified product of penetratin, they are technologies different from the present
invention wherein a hydrophilic bioactive substance and penetratin or a modified
product thereof are not covalently bonded to each other. It is known that efficient
occurrence of desorption from the cell, in addition to migration from outside the cell
to inside the cell, is required to realize promotion of nasal absorption using a cell
penetrating peptide, and me absorbability upon absorption through nasal mucosa is
affected by various factors other than cell permeability such as degradability of the
peptide by various degradative enzymes existing in the nasal mucosal tissue.
Therefore, necessity for confirmation, by experiments using a model animal or the
like, of whether or not the cell penetrating peptide has permeability through nasal
mucosa is common general technical knowledge of those skilled in the art. However,
since Patent Literatures 2 and 3 do not disclose experimental verification on nasal
mucosal permeability of penetratin and the modified product thereof, it is not easy
for those skilled in the art to infer from Patent Literature 2 and 3 that penetratin and
the modified product thereof have nasal mucosal permeability.
There is also an attempt to administer oligoarginine which is not covalently
linked to the drug as a nasal absorption-promoting substance together with the drug
(Patent Literature 7), but this requires a high concentration of oligoarginine and its
effect has been demonstrated with only a single model drug, fluorescently-labeled
dextran, so that permeability of a peptide hydrophilic bioactive substance is
unknown.
Thus, a highly-practical technology to enable high efficiency nasal absorption
of hydrophilic bioactive substances has not been discovered yet.
[Patent Literature 1] JP 10-33186 A
[Patent Literature 2] Japanese Translated PCT Patent Application Laid-open
No. 2002-530059
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[Patent Literature 3] Japanese Translated PCT Patent Application Laid-open
No. 2002-519392
[Patent Literature 4] JP 2006-257074 A
[Patent Literature 5] JP 2008-7448 A
[Patent Literature 6] WO 2004/037859
[Patent Literature 7] JP 10-95738 A
DISCLOSURE OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
The present invention aims to provide a pharmaceutical composition to allow
migration of a nasally administered hydrophilic bioactive substance into blood.
MEANS FOR SOLVING THE PROBLEMS
To overcome the above-described problems, the present inventors studied a
method to improve absorption efficiency of a hydrophilic bioactive substance which
has a low migration capability through mucosa into blood under normal conditions,
and discovered that a pharmaceutical composition comprising a hydrophilic bioactive
substance and: (a) a peptide having the amino acid sequence shown in SEQ ID NO: 1,
with the proviso that a C-terminal amidated peptide is excluded; (b) a peptide having
the same amino acid sequence as shown in SEQ ID NO: 1 except that one or several
amino acids are deleted, substituted and/or added, the peptide having nasal mucosal
permeability, with the proviso that a C-terminal amidated peptide is excluded; or (c)
a peptide having an amino acid sequence represented by the reverse sequence of (a)
or (b), the peptide having nasal mucosal permeability, with the proviso that a C-
terminal amidated peptide is excluded; is effective and can realize highly efficient
migration of the hydrophilic bioactive substance into blood upon nasal
administration. That is, the present invention has the following constitution.
(1) A pharmaceutical composition for nasal administration comprising a
hydrophilic bioactive substance and any one of (a) to (c) below, with the proviso that
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a C-terminal amidated peptide is excluded:
(a) a peptide having the amino acid sequence shown in SEQ ID NO:l;
(b) a peptide having the same amino acid sequence as shown in SEQ ID
NO:l except that one or several amino acids are deleted, substituted and/or added,
the peptide having nasal mucosal permeability; and
(c) a peptide having an amino acid sequence represented by the reverse
sequence of (a) or (b), the peptide having nasal mucosal permeability.

(2) The pharmaceutical composition for nasal administration according to (1),
wherein the peptide in (b) has the same amino acid sequence as shown in SEQ ID
NO:l except that one or several basic amino acids are substituted to another/other
basic amino acid(s) or added, the peptide having nasal mucosal permeability.
(3) The pharmaceutical composition for nasal administration according to (1)
or (2), wherein the peptide in (b) has the amino acid sequence shown in any one of
SEQ ID NOs:3 to 6.
(4) The pharmaceutical composition for nasal administration according to any
one of (1) to (3), wherein the peptide in (c) has the amino acid sequence shown in
SEQ ID NO:2.
(5) The pharmaceutical composition for nasal administration according to any
one of (1) to (4), containing any one of the peptides in (a) to (c) at a concentration of
0.2 to 2.0 mM.
(6) The pharmaceutical composition for nasal administration according to any
one of (1) to (5), wherein the hydrophilic bioactive substance is a peptide.
(7) The pharmaceutical composition for nasal administration according to any
one of (1) to (6), wherein the hydrophilic bioactive substance is insulin or interferon
P-
EFFECT OF THE INVENTION
By the present invention, migration of a nasally-administered hydrophilic
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bioactive substance into blood is possible, and simpler and more patient-oriented
pharmacotherapy is possible compared to the conventional administration method by
injection.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the absorption promotion effect on nasally administered insulin,
obtained by using the peptide of SEQ ID NO:l or SEQ ID NO:7: blood level.
Fig. 2 shows the absorption promotion effect on nasally administered insulin,
obtained by using the peptide of SEQ ID NO.l or SEQ ID NO:7: blood glucose
level.
Fig. 3 shows the absorption promotion effect on nasally administered insulin,
obtained by using the peptide of SEQ ID NO:l or SEQ ID NO:7: bioavailability.
Fig. 4 shows the absorption promotion effect on nasally administered insulin,
obtained by using the peptide of SEQ ID NO:l: concentration dependency.
Fig. 5 shows the absorption promotion effect on nasally administered
fluorescently labeled dextran, obtained by using the peptide of SEQ ID NO: 1.
Fig. 6 shows the absorption promotion effect on nasally administered
interferon p, obtained by using the peptide of SEQ ID NO:l.
Fig. 7 shows the absorption promotion effect on nasally administered insulin,
obtained by using the peptides of SEQ ID NOs:l to 6: blood glucose level.
Fig. 8 shows the absorption promotion effect on nasally administered insulin,
obtained by using the peptides of SEQ ID NOs: 1 to 6: blood level.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention relates to a pharmaceutical composition for nasal
administration to allow migration of a hydrophilic bioactive substance from the nasal
cavity into blood, which pharmaceutical composition contains a hydrophilic
bioactive substance as a pharmacologically active component and a specific peptide
having nasal mucosal permeability that are blended independently and not covalently
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linked to each other. Here, "hydrophilic bioactive substance" in the present
invention means a physiologically active substance which is characteristically
hydrophilic. "Hydrophilic" herein means having high solubility into water, and a
substance which dissolves into water in an amount of 1 ug or more per 1 ml of water
is defined as hydrophilic. "Physiologically active substance" means substances in
general which act on a living body and cause changes in the living body, and
examples thereof include proteins which bind to receptors on specific cells and
enzymes having affinities to substances in the living body. Further, it may be a
substance which does not cause a direct reaction with a substance in a living body
and includes substances capable of being administered to a living body for medical
use, such as dextran which is used as an alternative to blood plasma to increase
blood.
"Peptide" in the present invention means a substance having a structure
wherein amino acids are linked to each other by peptide bonds. Among peptides,
those having a high molecular weight are generally called proteins, but in the present
specification, these proteins are also called peptides without being limited by their
molecular weights. Further, glycoproteins having a sugar chain linked to a protein,
and derivatives having a chemical modification such as polyethylene glycolation
(PEGylation) are also included in the peptides in the present invention.
Details of the pharmaceutical composition for nasal administration of the
present invention will now be described.
In the present invention, the peptide which is contained together with a
hydrophilic bioactive substance and has the amino acid sequence shown in SEQ ID
NO:l is the peptide generally called penetratin. Penetratin is a peptide having cell
permeability found at the DNA-binding site of the peptide in Drosophila called
Antennapedia. The present inventors newly found that nasal administration of
penetratin, with the proviso that a C-terminal amidated peptide is excluded, together
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with a hydrophilic bioactive substance allows migration of the hydrophilic bioactive
substance into blood at a high rate in a living body due to the excellent nasal mucosal
permeability of penetratin, thereby completing the present invention.
For the peptide having the amino acid sequence of SEQ ID NO: 1 used in the
present invention, deletion, substitution and/or addition of one or several amino
acid(s) is/are acceptable as long as the difference(s) is/are within the range wherein
the nasal mucosal permeability required in the present invention is retained as the
entire peptide, with the proviso that a C-terminal amidated peptide is excluded. For
example, cases where a basic amino acid(s) in the peptide is/are substituted with
another or other several basic amino acid(s), cases where a hydrophilic amino acid(s)
is/are substituted with another or other plurality of hydrophilic amino acid(s), and
cases where a hydrophobic amino acid(s) is/are substituted with another or other
plurality of hydrophobic amino acid(s) in the peptide do not change the properties of
the entire peptide, and therefore these are accepted without problems. In particular,
cases where one or several basic amino acid(s) is/are substituted in or added to the
amino acid sequence shown in SEQ ID NO:l are preferably accepted. The number
of the deletion(s), substitution(s) and/or addition(s) of the above-described amino
acids is preferably small, and preferably 1 to 5 amino acid(s), more preferably 1 to 3
amino acid(s), still more preferably 1 amino acid is/are involved therein. Here, in the
present invention, "hydrophobic amino acid" means an amino acid selected from the
group consisting of leucine, isoleucine, tryptophan, phenylalanine, valine and
alanine, and "hydrophilic amino acid" means an amino acid selected from the group
consisting of serine, threonine, aspartic acid, glutamic acid, lysine, arginine and
histidine. "Basic amino acid" means an amino acid selected from the group
consisting of lysine, arginine and histidine. Preferred examples of the peptide
having the same amino acid sequence as shown in SEQ ID NO: 1 except that one or
several amino acids are deleted, substituted and/or added, which peptide has nasal
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mucosal permeability include the peptide having the amino acid sequences shown in
any one of SEQ ID NOs:3 to 6.
In the present invention, even if the peptide contained together with a
hydrophilic bioactive substance is a peptide represented by the reverse sequence of
either a peptide having the amino acid sequence shown in SEQ ID NO:l which
exhibits an effective transnasal mucosal permeability or a peptide having the same
amino acid sequence as shown in SEQ ID NO:l except that a part of amino acids are
deleted, substituted and/or added, it is acceptable as long as the difference is within
the range wherein the nasal mucosal permeability required in the present invention is
retained as the entire peptide, with the proviso that a C-terminal amidated peptide is
excluded. Here, the peptide represented by the reverse sequence means that the
sequence of the amino acids constituting the peptide is reversed. For example, when
the sequence of the amino acids in a peptide from the N terminus to the C terminus is
arginine, glutamine, isoleucine and lysine, the reversed peptide thereof has the amino
acid sequence of, from the N terminus to the C terminus, lysine, isoleucine,
glutamine and arginine. Preferred examples thereof include the peptide represented
by the reverse sequence of a peptide having the amino acid sequence shown in SEQ
IDNO:l(SEQIDNO:2).
In the present invention, as the amino acids constituting the peptide contained
together with a hydrophilic bioactive substance, naturally occurring L-amino acids as
well as non-naturally occurring amino acids such as a derivative produced by
modification of a part of a naturally occurring amino may be used. For example,
since D-amino acids are hardly degraded by proteases, they may be effectively used,
and therefore the amino acid sequence of the peptide may be either partially or
entirely constituted by D-amino acids, and appropriately selected depending on the
hydrophilic bioactive substance administered together with the peptide. In cases
where the hydrophilic bioactive substance has negative charges as the whole, the
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total amino acid sequence is preferably constituted by L-amino acids. For example,
when the hydrophilic bioactive substance is insulin, the total amino acid sequence is
preferably constituted by L-amino acids. In cases where the hydrophilic bioactive
substance does not have charges, or in cases where it has positive charges, the total
amino acid sequence is preferably constituted by D-amino acids. For example, in the
case of interferon P having positive charges, the total amino acid sequence is
preferably constituted by D-amino acids.
In the present invention, the peptide contained together with a hydrophilic
bioactive substance may be prepared by a conventional method for synthesizing a
peptide, and may also be prepared, for example, by introduction of a gene encoding
the amino acid sequence of the peptide into a microorganism such as E. coli, animal
cells, insect cells or the like and allowing expression of the gene. Further, the
peptide may also be obtained by degradation of a protein having the amino acid
sequence of the peptide occurring naturally. For example, the peptide having the
amino acid sequence shown in SEQ ID NO:l has partially the same sequence as the
Antennapedia protein in Drosophila, and it may also be prepared by protease
treatment of the protein occurring naturally.
In the present invention, either a single type or a plurality of types of the
peptide(s) may be contained together with a hydrophilic bioactive substance, and a
single type of the peptide is preferred. The concentration thereof is not limited, and
as shown in Examples, it is preferably 0.2 to 2 mM. A concentration of 0.5 to 2 mM
is more preferred since remarkable nasal permeation of a hydrophilic bioactive
substance is observed therewith. The concentration of a peptide in the present
invention means the concentration of the peptide upon its administration to the nasal
cavity, and in cases where the pharmaceutical composition of the present invention is
a solution, it means the concentration in the solution, and in the case of a solid, it
means the concentration obtained when the dose of the composition per
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administration is restored to 40 uJ of a solution which is a standard volume for a
single nasal administration.
Specific examples of the hydrophilic bioactive substance used in the present
invention include peptides, sugar chains and nucleic acids, and it is preferably a
peptide. Specific examples of the peptide include antibodies, peptide hormones and
cytokines, and it is preferably a peptide hormone or a cytokine. Examples of the
peptide hormone include insulin, calcitonin, parathyroid hormone, growth hormone,
GLP-1 and Exendin-4; and examples of the cytokine include interferons, interleukins
and G-CSF; and it is preferably insulin or interferon p. Each of these hydrophilic
bioactive substances may be either a naturally-occurring substance or a derivative
produced by modification of a part of the structure of the sequence thereof. Further,
it may be a derivative produced by a chemical modification such as polyethylene
glycolation (PEGylation).
The nasal absorption of a hydrophilic bioactive substance in the present
invention means migration of the hydrophilic bioactive substance administered to the
nasal cavity, from the nasal cavity into blood. Its result can be confirmed by
observation of increase in the blood level of the hydrophilic bioactive substance or
expression of pharmacological activity thereof. The blood level of the hydrophilic
bioactive substance can be measured by a method conventionally used by those
skilled in the art, such as an immunoassay. The pharmacological activity can be
measured by using as an index, in the case of an enzyme, its enzyme activity; and in
the case of a substance which acts on a receptor on the cell, its ability to change a
function of the target cell or the amount of production of a marker substance. For
example, the pharmacological activity of insulin can be measured by using as an
index the blood glucose level of the animal to which insulin was administered.
Whether or not the absorption is promoted can be confirmed by comparison
between: the ratio of the amount of migration of the drug into blood with respect to
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the amount of the drug administered observed when the hydrophilic bioactive
substance, which is a pharmacologically active component, was solely administered
to the nasal cavity; and the ratio of the amount of migration of the drug into blood
with respect to the amount of the drug administered observed when the
pharmaceutical composition of the present invention was also administered. If the
latter rate is higher, the absorption can be confirmed to have been promoted. To
allow exertion of a sufficient function as an actual pharmaceutical agent, the
bioavailability value, which indicates the percentage of the AUC (area under the
blood concentration versus time curve) value with respect to the AUC upon
administration of the same amount of the hydrophilic bioactive substance by
injection, of the pharmaceutical composition is preferably not less than 10%, more
preferably not less than 20%.
The pharmaceutical composition for nasal administration of the present
invention may also contain a pharmaceutically acceptable carrier and/or additive.
Examples of such a carrier and additive include water, pharmaceutically acceptable
organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, 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.
The pharmaceutical composition for nasal administration of the present
invention may be used in various forms such as a solution, solid or powder, but in
view of stability and ease of handling, a form of solid or powder produced by a
method such as freeze-drying is preferred.
The method for administering the pharmaceutical composition for nasal

administration of the present invention to animals (including human) is not restricted
in terms of its specific form. For example, the composition in a dry state or in the
form of a solution may be administered as it is; the composition may be filled in a
capsule together with a vehicle and then administered; or the composition in a dry
state may be once dissolved or dispersed in water and then administered.
The dose and the number of doses upon administration of the pharmaceutical
composition for nasal administration of the present invention to a living body are
appropriately selected depending on the dosage form, age and body weight of the
patient, and severity of the symptoms, and the composition may be normally
administered at a dose within the range of 0.0001 to 50 mg, preferably within the
range of 0.001 to 20 mg per adult per day based on the weight of the hydrophilic
bioactive substance contained.
EXAMPLES
Example 1: Nasal Administration of Insulin

A prescribed amount of insulin (Wako Pure Chemicals) powder was scaled
and placed into a 1.5 ml tube (Eppendorf), followed by dissolving it in 0.1 N HC1
and then addition of the same amount of 0.1 N NaOH to prepare an insulin solution.
Penetratin (SEQ ID NO:l; synthesis thereof was entrusted to Sigma Genosys)
or oligoarginine (SEQ ID NO:7; synthesis thereof was entrusted to Sigma Genosys)
having an amino acid sequence constituted entirely by L-amino acids or by D-amino
acids was dissolved in PBS and combined with the above-mentioned insulin solution
to prepare, for each administration experiment, 40 u.1 of a mixed solution containing
insulin (10 IU/kg) and 0.5 mM of each peptide.
Male SD rats having a body weight of about 200 g were fasted for 24 hours
and anesthetized by intraperitoneal injection of 50 mg/kg of pentobarbital, followed
by incision of the cervical part to expose trachea. A polyethylene tube
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(INTRAMEDIC PE205, Clay Adams) was inserted into trachea, and esophagus was
partially incised and a tube having the same diameter was carefully inserted from the
incised part of the esophagus to choanae without damaging tissues. The tip of the
tube to be inserted into the choanae was preliminarily tightly sealed with absorbent
cotton and an adhesive. To prevent leakage of the drug solution, the nasopalatine
duct in maxilla opening into the oral cavity was closed with a synthetic adhesive
(Aran Alpha A; manufactured by Daiichi Sankyo Company, Limited). From the
jugular vein, 0.25 ml of blood was collected before and 5, 10, 15, 30, 60, 120, 180
and 240 minutes after administration of the prepared mixed solution of insulin and
the peptide, or only insulin, followed by centrifugation to separate blood plasma and
measuring the plasma insulin concentration by EIA kit (Levis). The bioavailability
was calculated by comparison with data obtained by subcutaneous administration of
insulin.

Changes in the blood insulin level with time are shown in Fig. 1, and changes
in the blood glucose level are shown in Fig. 2. Rats to which only insulin was
nasally administered hardly exhibited increase in the blood insulin level, while in rats
to which penetratin or oligoarginine was administered together with insulin,
migration of insulin into blood was observed from immediately after the
administration. The highest maximum blood level was achieved with L-penetratin,
and this was followed in order by D-penetratin, D-oligoarginine and L-oligoarginine
(Fig. 1). Decrease in the blood glucose level, which is a pharmacological activity
caused by migration of insulin into blood, was observed, and decrease in the blood
glucose level reflecting the blood insulin level was confirmed (Fig. 2).
The bioavailability was 0.8% in rats to which only insulin was administered,
while in the cases of simultaneous administration of insulin and L- or D-
oligoarginine, the bioavailability (BA) was 1.1% for L-oligoarginine and 2.0% for D-
15

oligoarginine, and in the cases of simultaneous administration of insulin and
penetratin, the BA was 3.4% for D-penetratin and 7.1% for L-penetratin (Fig. 3),
showing that penetratin has a higher insulin absorption promotion effect compared to
oligoarginine.
Example 2: Nasal Administration of Insulin and Its Dependency on Peptide
Concentration

L-penetratin (SEQ ID NO:l), which showed the highest efficiency of the
absorption promotion effect in Example 1, was similarly evaluated in the same
manner as in Example 1 except that its concentration in the solution to be
administered was changed to 0.2 mM, 0.5 mM, 1 mM or 2 mM.

While the bioavailability in the case of administration of only insulin was
1.7%, the bioavailabilities in the cases of administration of 0.2 mM, 0.5 mM, 1 mM
and 2 mM L-penetratin were 15.1%, 17.9%, 28.4% and 50.7%, respectively. Thus,
the absorption efficiency of insulin was improved dependently on the concentration
of penetratin (Fig. 4).
Example 3: Nasal Administration of Fluorescently-labeled Dextran

A fluorescently-labeled dextran FD-4 (Molecular probes, Inc.) was diluted
with PBS to provide a 4 mg/ml solution. Using 40 uJ of this solution, the evaluation
was carried out in the same manner as in Example 1. The FD concentration in blood
was quantified by fluorometry. The bioavailability was calculated by comparison
with the case where the same amount of FD-4 was intravenously administered.

The bioavailability was 4.9% in the rats to which only FD-4 was
administered, 17% in the case of usage of L-penetratin, and 36% in the case of usage
16

of D-penetratin. The bioavailabilities in the rats to which L-oligoarginine and D-
oligoarginine were administered were 12.4% and 12.4%, respectively. Thus,
penetratin exhibited a higher absorption promotion effect compared to oligoarginine
(Fig. 5).
Example 4: Nasal 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
obtain a 6,000,000 IU/ml solution, and a 100 ul aliquot of the resulting solution was
taken. To this aliquot, 566 ul of PBS supplemented with Tween 20 was added to
obtain a 900,000 IU/ml solution. Each of D-penetratin and L-penetratin (SEQ ID
NO:l) was scaled, and 40 ul of the interferon p solution was added thereto such that
the final concentration of 0.5 mM or 2 mM was attained, to obtain a mixed solution
of interferon P and penetratin, which was then evaluated in the same manner as in
Example 1. The concentration of interferon p was measured by "Human Interferon P
ELISA Kit" manufactured by Kamakura Techno-Science Inc., and the bioavailability
was calculated by comparison of its plasma concentration with that of the case where
the same amount of interferon p was intravenously administered.

By addition of penetratin together with interferon p, migration of interferon P
into blood was observed. The bioavailability was 6.1% for 0.5 mM L-penetratin,
11.0% for 0.5 mM D-penetratin, and 22.0% for 2 mM D-penetratin (Fig. 6).
Example 5: Nasal Administration of Insulin

A prescribed amount of insulin (Wako Pure Chemicals) powder was scaled
and placed into a 1.5 ml tube (Eppendorf), followed by dissolving it in 0.1 N HC1
and then addition of the same amount of 0.1 N NaOH to prepare an insulin solution.
17

Each of the peptides having an amino acid sequence constituted entirely by
L-amino acids (SEQ ID NOs:l to 6; synthesis thereof was entrusted to Sigma
Genosys) was dissolved in PBS and combined with the above-mentioned insulin
solution to prepare, for each administration experiment, 40 ul of a mixed solution
containing insulin (1 lU/kg) and 0.5 mM of each peptide. The prepared solutions
were subjected to the evaluation in the same manner as in Example 1.

Changes in the blood glucose level with time are shown in Fig. 7; changes in
the blood insulin level with time are shown in Fig. 8; and various parameters
obtained by the evaluation results are shown in Table 1. The increase in the blood
insulin level could be hardly observed in rats to which only insulin was nasally
administered, while simultaneous administration of each of the peptides of SEQ ID
NO:l to 6 with insulin caused migration of insulin into blood from immediately after
the administration, leading to the decrease in the blood glucose level. The
bioavailability (BA) calculated based on the changes in the blood insulin level was
2.3% in rats to which only insulin was administered, while it was 4.4 to 20.1% in the
cases with the peptides of SEQ ID NOs:l to 6. The pharmaceutical availability (PA)
calculated based on the changes in the blood glucose level was 4.7% in the case of
administration of only insulin, but 15.7% to 37.5% in the cases with the peptides of
SEQIDNOs:lto6.
[Table 1]
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INDUSTRIAL APPLICABILITY
By the present invention, hydrophilic bioactive substances which have been
administered so far as injection solutions can be nasally administered, and therefore
drugs which largely improve the pain and the inconvenience of patients can be
provided. Improvement of the pain and the inconvenience of hospital visits of
patients caused by these injection solutions may not only realize patient-oriented
health care in clinical sites but also drastically change the conventional concept of
formulation, leading to creation of epoch-making formulations.

WE CLAIM:
1. A pharmaceutical composition for nasal administration comprising a
hydrophilic bioactive substance and any one of (a) to (c) below, with the proviso that
a C-terminal amidated peptide is excluded:
(a) a peptide having the amino acid sequence shown in SEQ ID NO.l;
(b) a peptide having the same amino acid sequence as shown in SEQ ID
NO:l except that one or several amino acids are deleted, substituted and/or added,
said peptide having nasal mucosal permeability; and
(c) a peptide having an amino acid sequence represented by the reverse
sequence of (a) or (b), said peptide having nasal mucosal permeability.

2. The pharmaceutical composition for nasal administration according to claim
1, wherein said peptide in (b) has the same amino acid sequence as shown in SEQ ID
NO: 1 except that one or several basic amino acids are substituted by another/other
basic amino acid(s) or added, said peptide having nasal mucosal permeability.
3. The pharmaceutical composition for nasal administration according to claim
1 or 2, wherein said peptide in (b) has the amino acid sequence shown in any one of
SEQ ID NOs:3 to 6.
4. The pharmaceutical composition for nasal administration according to any
one of claims 1 to 3, wherein said peptide in (c) has the amino acid sequence shown
in SEQ ID NO:2.
5. The pharmaceutical composition for nasal administration according to any
one of claims 1 to 4, containing any one of said peptides in (a) to (c) at a
concentration of 0.2 to 2.0 mM.
6. The pharmaceutical composition for nasal administration according to any
one of claims 1 to 5, wherein said hydrophilic bioactive substance is a peptide.
7. The pharmaceutical composition for nasal administration according to any

one of claims 1 to 6, wherein said hydrophilic bioactive substance is insulin or
interferon p.

By a pharmaceutical composition for nasal administration comprising a
hydrophilic bioactive substance and any one of (a) to (c) below, with the proviso that
a C-terminal amidated peptide is excluded, a hydrophilic bioactive substance having
a low transmucosal absorption capability which has conventionally been able to be
administered by only injection can be nasally administered. Such a pharmaceutical
composition is useful for improvement of the pain and the inconvenience of patients
caused by administration by injection.
(a) A peptide having the amino acid sequence shown in SEQ ID NO.1.
(b) A peptide having the same amino acid sequence as shown in SEQ ID
NO:1 except that one or several amino acids are deleted, substituted and/or added,
the peptide having nasal mucosal permeability.
(c) A peptide having an amino acid sequence represented by the reverse
sequence of (a) or (b), the peptide having nasal mucosal permeability.