U.S. patent application number 11/931201 was filed with the patent office on 2009-05-28 for intranasal administration of active agents to the central nervous system.
Invention is credited to Johanna Bentz, Beth Hill, Lisbeth Illum.
Application Number | 20090136505 11/931201 |
Document ID | / |
Family ID | 40591743 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090136505 |
Kind Code |
A1 |
Bentz; Johanna ; et
al. |
May 28, 2009 |
Intranasal Administration of Active Agents to the Central Nervous
System
Abstract
Pharmaceutical compositions and methods for delivering a
polypeptide to the central nervous system of a mammal via
intranasal administration are provided. The polypeptide can be a
catalytically active protein or an antibody, antibody fragment or
antibody fragment fusion protein. The polypeptides are formulated
with one or more specific agents.
Inventors: |
Bentz; Johanna; (Mountain
View, CA) ; Hill; Beth; (Mountain View, CA) ;
Illum; Lisbeth; (Nottingham, GB) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
40591743 |
Appl. No.: |
11/931201 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11342058 |
Jan 27, 2006 |
|
|
|
11931201 |
|
|
|
|
60655809 |
Feb 23, 2005 |
|
|
|
Current U.S.
Class: |
424/139.1 ;
424/130.1; 424/94.1 |
Current CPC
Class: |
A61P 21/04 20180101;
A61K 47/34 20130101; C07K 16/18 20130101; A61P 3/10 20180101; A61P
3/00 20180101; A61P 9/00 20180101; A61K 38/50 20130101; A61K 47/26
20130101; A61K 51/086 20130101; A61P 25/28 20180101; A61K 47/28
20130101; A61P 21/00 20180101; A61K 47/6851 20170801; C07K 14/685
20130101; A61P 43/00 20180101; A61P 1/14 20180101; A61P 25/08
20180101; A61P 25/00 20180101; A61K 47/6811 20170801; A61P 37/06
20180101; C07K 2319/30 20130101; A61P 3/04 20180101; A61K 9/0043
20130101; A61K 51/088 20130101; A61K 38/34 20130101; A61P 25/14
20180101; A61P 25/16 20180101; A61P 25/20 20180101 |
Class at
Publication: |
424/139.1 ;
424/130.1; 424/94.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/43 20060101 A61K038/43 |
Claims
1. A pharmaceutical composition comprising: a) a catalytically
active peptide chain or a peptide chain comprising an antibody Fc
or Fab fragment; and b) a permeation enhancer in a concentration
sufficient to enhance intranasal administration of the
catalytically active peptide chain or the peptide chain comprising
the antibody Fc or Fab fragment to the central nervous system of an
animal.
2. The pharmaceutical composition of claim 1 wherein the
catalytically active peptide chain is an enzyme.
3. The pharmaceutical composition of claim 1 wherein the peptide
chain comprising the antibody Fc or Fab fragment is a whole
antibody.
4. The pharmaceutical composition of claim 1 wherein the peptide
chain comprising the antibody Fc or Fab fragment is a
mimetibody.
5. The pharmaceutical composition of claim 1 in liquid form.
6. The pharmaceutical composition of claim 1 in powder or
lyophilized form.
7. A pharmaceutical composition comprising: a) a catalytically
active peptide chain or a peptide chain comprising an antibody Fc
or Fab fragment; and b) about 0.1 to about 1 g of chitosan
glutamate or corresponding amounts of another chitosan salt per 100
ml of the pharmaceutical composition; wherein the diluent is an
aqueous buffer at standard state.
8. The pharmaceutical composition of claim 7 comprising about 0.5 g
of chitosan glutamate per 100 ml of the pharmaceutical
composition.
9. A pharmaceutical composition comprising: a) a catalytically
active peptide chain or a peptide chain comprising an antibody Fc
or Fab fragment; and b) from about 0.125 g to about 1.0 g of a
compound selected from the group consisting of
1-O-n-dodecyl-beta-D-maltopyranoside,
1-O-n-decyl-beta-D-maltopyranoside,
1-O-n-tetradecyl-beta-D-maltopyranoside, and
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate per
100 ml of the pharmaceutical composition; wherein the diluent is an
aqueous buffer at standard state.
10. The pharmaceutical composition of claim 9 comprising about
0.125 g to about 0.5 g of a compound selected from the group
consisting of 1-O-n-dodecyl-beta-D-maltopyranoside,
1-O-n-decyl-beta-D-maltopyranoside,
1-O-n-tetradecyl-beta-D-maltopyranoside, and
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate per
100 ml of the pharmaceutical composition.
11. A pharmaceutical composition comprising: a) a catalytically
active peptide chain or a peptide chain comprising an antibody Fc
or Fab fragment; and b) from about 5 ml to about 20 ml of propylene
glycol per 100 ml of the pharmaceutical composition; wherein the
diluent is an aqueous buffer at standard state and the propylene
glycol is at standard state.
12. The pharmaceutical composition of claim 11 comprising from
about 10 ml to about 20 ml of propylene glycol per 100 ml of the
pharmaceutical composition.
13. A pharmaceutical composition comprising: a) a catalytically
active peptide chain or a peptide chain comprising an antibody Fc
or Fab fragment; and b) about 1 g to about 10 g of heptakis
(2,6-di-O-methyl)-beta-cyclodextrin per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state.
14. The pharmaceutical composition of claim 13 comprising about 5 g
of heptakis (2,6-di-O-methyl)-beta-cyclodextrin per 100 ml of the
pharmaceutical composition.
15. A pharmaceutical composition comprising: a) a catalytically
active peptide chain or a peptide chain comprising an antibody Fc
or Fab fragment; and b) about 1 g to about 5 g of
1,2-didecanoyl-sn-glycero-3-phosphocholine per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state and the
1,2-didecanoyl-sn-glycero-3-phosphocholine is emulsified in the
aqueous buffer.
16. The pharmaceutical composition of claim 15 comprising about 2 g
of 1,2-didecanoyl-sn-glycero-3-phosphocholine per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state and the
1,2-didecanoyl-sn-glycero-3-phosphocholine is emulsified in the
aqueous buffer.
17. A pharmaceutical composition comprising: a) a catalytically
active peptide chain or a peptide chain comprising an antibody Fc
or Fab fragment; and b) about 0.1 g to about 1 g of a compound
selected from the group consisting of sodium glycocholate hydrate,
taurocholic acid sodium salt hydrate, and sodium
tauroursodeoxycholate per 100 ml of the pharmaceutical composition;
wherein the diluent is an aqueous buffer at standard state.
18. The pharmaceutical composition of claim 17 comprising about 1 g
of a compound selected from the group consisting of sodium
glycocholate hydrate, taurocholic acid sodium salt hydrate, and
sodium tauroursodeoxycholate per 100 ml of the pharmaceutical
composition; wherein the diluent is an aqueous buffer at standard
state.
19. A pharmaceutical composition comprising: a) a catalytically
active peptide chain or a peptide chain comprising an antibody Fc
or Fab fragment; and b) from about 1 ml to about 10 ml of
tetrahydrofurfuryl-polyethyleneglycol per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state and the
tetrahydrofurfuryl-poyethylenglycol is at standard state.
20. The pharmaceutical composition of claim 1, 7, 9, 11, 13, 15, 17
or 19 wherein the peptide chain comprising an antibody Fc fragment
binds to a melanocortin 4 receptor comprising the amino acid
sequence shown in SEQ ID NO: 2.
21. The pharmaceutical composition of claim 1, 7, 9, 11, 13, 15, 17
or 19 wherein the peptide chain comprising an antibody Fc fragment
comprises the amino acid sequence shown in SEQ ID NO: 3.
22. A method of delivering a catalytically active peptide chain or
a peptide chain comprising an antibody Fc or Fab fragment to the
central nervous system of an animal comprising: a) providing the
pharmaceutical composition of claim 1, 7, 9, 11, 13, 15, 17 or 19;
and b) administering the pharmaceutical composition to the nasal
cavity of an animal; whereby the catalytically active peptide chain
or the peptide chain comprising the antibody Fc or Fab fragment
enters the central nervous system of the animal.
23. A method of delivering a peptide chain comprising an antibody
Fc fragment to the central nervous system of an animal comprising:
a) providing the pharmaceutical composition of claim 1, 7, 9, 11,
13, 15, 17 or 19 wherein the peptide chain comprising the antibody
Fc fragment binds to a melanocortin 4 receptor comprising the amino
acid sequence shown in SEQ ID NO: 2; and b) administering the
pharmaceutical composition to the nasal cavity of an animal;
whereby the peptide chain comprising the antibody Fc fragment
enters the central nervous system of the animal.
24. A method of delivering a peptide chain comprising an antibody
Fc fragment to the central nervous system of an animal comprising:
a) providing the pharmaceutical composition of claim 1, 7, 9, 11,
13, 15, 17 or 19 wherein the peptide chain comprising the antibody
Fc fragment comprises the amino acid sequence shown in SEQ ID NO:
3; and b) administering the pharmaceutical composition to the nasal
cavity of an animal; whereby the peptide chain comprising the
antibody Fc fragment enters the central nervous system of the
animal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/655,809, filed Feb. 23, 2005 and U.S. Utility
application Ser. No. 11/342,058 filed Jan. 1, 2006, both of which
are incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0002] The subject matter described herein relates to methods and
compositions for intranasal administration of active agents to the
central nervous system of a mammal.
BACKGROUND
[0003] Delivery of drugs to the central nervous system (CNS)
remains a challenge, despite recent advances in drug delivery and
knowledge of mechanisms of delivery of drugs to the brain. For
example, CNS targets are poorly accessible from the peripheral
circulation due to the blood-brain barrier (BBB), which provides an
efficient barrier for the diffusion of most, especially polar,
drugs into the brain from the circulating blood. Attempts to
circumvent the problems associated with the BBB to deliver drugs to
the CNS include: 1) design of lipophilic molecules, as lipid
soluble drugs with a molecular weight of less than 600 Da readily
diffuse through the barrier; 2) binding of drugs to transporter
molecules which cross the BBB via a saturable transporter system,
such as transferrin, insulin, IGF-1, and leptin; and 3) binding of
drugs to polycationic molecules such as positively-charged proteins
that preferentially bind to the negatively-charged endothelial
surface (See, e.g., Illum, Eur. J. Pharm. Sci. 11:1-18 (2000) and
references therein; W. M. Partridge. "Blood-brain barrier drug
targeting: the future of brain drug development", Mol. Interv.
3(2):90-105 (2003); W. M. Partridge et al., "Drug and gene
targeting to the Brain with molecular Trojan horses", Nature
Reviews-Drug Discovery 1:131-139 (2002)).
[0004] The intranasal route has been explored as a non-invasive
method to circumvent the BBB for transport of drugs to the CNS.
Although intranasal delivery to the CNS has been demonstrated for a
number of small molecules and some peptides and smaller proteins,
there is little evidence demonstrating the delivery of protein
macromolecules to the CNS via intranasal pathways, presumably due
to the larger size and varying physico-chemical properties unique
to each macromolecule or class of macromolecules, that may hinder
direct nose-to-brain delivery.
[0005] The primary physical barrier for intranasal delivery is the
respiratory and olfactory epithelia of the nose. It has been shown
that the permeability of the epithelial tight junctions in the body
is variable and is typically limited to molecules with a
hydrodynamic radius less than 3.6 A; permeability is thought to be
negligible for globular molecules with a radius larger than 15 A
(B. R. Stevenson et al., Mol. Cell. Biochem. 83, 129-145 (1988)).
Therefore, the size of the molecule to be administered is
considered an important factor in achieving intranasal transport of
a macromolecule to the central nervous system. Fluorescein-labeled
dextran, a linear molecule having a dextran molecular weight of 20
kD can be delivered to cerebrospinal fluid from the rat nasal
cavity, however 40 kDa dextran cannot (Sakane et al, J. Pharm.
Pharmacol. 47, 379-381 (1995)). It has also been reported that an
infectious organism, such as a virus, can enter the brain through
the olfactory region of the nose (S. Perlman et al., Adv. Exp. Med.
Biol, 380:73-78 (1995)).
[0006] In published delivery studies to date, intranasal delivery
efficiency to the CNS has been very low and the delivery of large
globular macromolecules, such as antibodies and their fragments,
has not been demonstrated. Yet, because antibodies, antibody
fragments, and antibody fusion molecules are potentially useful
therapies for treating disorders having a CNS target, e.g.,
Alzheimer's disease, Parkinson's disease, multiple sclerosis,
stroke, epilepsy, and metabolic and endocrine disorders, it is
desirable to provide a method for delivering these large
macromolecules to the CNS non-invasively.
BRIEF SUMMARY
[0007] An aspect of the invention is a pharmaceutical composition
comprising a catalytically active peptide chain or a peptide chain
comprising an antibody Fc or Fab fragment; and a permeation
enhancer in a concentration sufficient to enhance intranasal
administration of the catalytically active peptide chain or peptide
chain comprising an antibody Fc or Fab fragment to the central
nervous system of an animal.
[0008] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc or Fab fragment; and about
0.1 to about 1 g of chitosan glutamate per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state.
[0009] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and from about
0.125 g to about 1 g of a compound selected from the group
consisting of 1-O-n-dodecyl-beta-D-maltopyranoside,
1-O-n-decyl-beta-D-maltopyranoside,
1-O-n-tetradecyl-beta-D-maltopyranoside, and
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate per
100 ml of the pharmaceutical composition; wherein the diluent is an
aqueous buffer at standard state.
[0010] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and from about 5
ml to about 20 ml of propylene glycol per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state and the propylene glycol is at standard
state.
[0011] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and about 5 g of
heptakis (2,6-di-O-methyl)-beta-cyclodextrin per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state.
[0012] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and about 2 g of
1,2-didecanoyl-sn-glycero-3-phosphocholine per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state and the
1,2-didecanoyl-sn-glycero-3-phosphocholine is emulsified in the
aqueous buffer.
[0013] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and about 0.1 to
about 1 g of a compound selected from the group consisting of
sodium glycocholate hydrate, taurocholic acid sodium salt hydrate,
and sodium tauroursodeoxycholate per 100 ml of the pharmaceutical
composition; wherein the diluent is an aqueous buffer at standard
state.
[0014] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and from about 1
ml to about 10 ml of tetrahydrofurfuryl polyethylenglycol per 100
ml of the pharmaceutical composition; wherein the diluent is an
aqueous buffer at standard state and the
tetrahydrofurfuryl-polyethylenglycol is at standard state.
[0015] These and other aspects and embodiments will be apparent
from the description, drawings, and sequences herein.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 is a graph showing the distribution of
.sup.125I-.alpha.-melanocyte stimulating hormone
(.sup.125I-.alpha.-MSH) mimetibody in rats 25 minutes (open bars)
and 5 hours (dotted bars) after intranasal administration of
.sup.125I-.alpha.-MSH mimetibody, as more fully described in
Example 1.
[0017] FIG. 2 is a graph showing the blood concentration of
.sup.125I-.alpha.-MSH mimetibody, in nmol, after intranasal
(diamonds) or intravenous (squares) administration of
.sup.125I-.alpha.MSH mimetibody to rats, as a function of time post
delivery, in minutes, as more fully described in Example 1.
[0018] FIG. 3 is a graph comparing the distribution of
.sup.125I-.alpha.-MSH mimetibody in the central nervous system and
peripheral tissues of rats after either intranasal (open bars) or
intravenous (dofted bars) administration of .sup.125I-.alpha.-MSH
mimetibody, as more fully described in Example 1.
[0019] FIGS. 4A-4D show computer-generated autoradiographs of
coronal sections of rat brains 25 minutes after administration of
.sup.125I-.alpha.-MSH mimetibody either intranasally (FIGS. 4A, 4C)
or intravenously (FIGS. 4B, 4D), as more fully described in Example
1.
[0020] FIG. 5 is a graph showing the reduction of cumulative food
intake in rats, in grams, 24 hours after intranasal treatment with
.alpha.-MSH mimetibody at varying doses, in nmol.
[0021] FIG. 6 is a graph showing the percentage reduction in
cumulative food intake in rats, as a function of time, in hours,
after intranasal treatment with .alpha.-MSH mimetibody at a dose of
2.5 nmol (diamonds), 6.25 nmol (squares), 25 nmol (triangles), or
50 nmol (circles),
[0022] FIG. 7 is a bar graph showing the cumulative food intake in
rats, in grams, at the indicated times post treatment with
.alpha.-MSH mimetibody (open bars) or saline (dofted bars)
administered intranasally.
[0023] FIG. 8 is a bar graph showing increased antibody fragment
delivery to central nervous system tissues in isoflurane
anesthetized rats after nasal administration of hFc formulations
comprising 5% (v/v) tetrahydrofurfuryl-polyethylenglycol or 1%
(w/v) sodium glycocholate hydrate.
[0024] FIG. 9 is a bar graph showing that nasal administration of
formulations comprising 1% (w/v) sodium glycocholate hydrate
permits delivery of biologically active, high molecular weight
enzymes to central nervous system tissues in isoflurane
anesthetized animals.
[0025] FIG. 10 is a bar graph showing increased antibody fragment
delivery to central nervous system tissues in isoflurane
anesthetized rats after nasal administration of hFc formulations
comprising from 0.125% (w/v) to 0.5% (w/v) of
1-O-n-dodecyl-beta-D-maltopyranoside (designated A3),
1-O-n-decyl-beta-D-maltopyranoside (designated A1),
1-O-n-tetradecyl-beta-D-maltopyranoside (designated A5), and
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate
(designated B3).
[0026] FIG. 11 is a schematic of representative peptide chains
comprising antibody Fc fragments. Selected peptide chain features
are noted.
DETAILED DESCRIPTION
[0027] For the purposes of promoting an understanding of the
subject matter herein, reference will now be made to preferred
embodiments and specific language will be used to describe the
same. It will nevertheless be understood that no limitation of the
scope of the invention is thereby intended, such alterations and
further modifications of the subject matter, and such further
applications of the principles as illustrated herein, being
contemplated as would normally occur to one skilled in the art to
which the subject matter relates.
[0028] It has been discovered that globular protein molecules, such
as an antibody fragment linked to a therapeutic peptide or protein,
can be delivered directly to the central nervous system of a
mammal, thereby bypassing the blood-brain barrier. Accordingly,
methods of delivering a therapeutic composition to the central
nervous system of a mammal are provided. The methods are
advantageous in treating a wide variety of diseases or conditions.
Methods of treatment are therefore also provided.
[0029] Methods of delivering therapeutic compositions to the
central nervous system, including the brain and spinal cord and
cervical nodes, of a mammal by a non-systemic route, e.g., by a
route other than one which delivers or otherwise affects the body
as a whole are provided. The delivery method therefore allows for
localized and targeted delivery of the therapeutic compositions to
the brain via the nasal passage. Consequently, the method relates
to delivery of the compositions by a route other than intravenous,
intramuscular, transdermal, intraperitoneal, or similar route which
delivers the composition through, for example, the blood
circulatory system. It has been discovered that antibody fragments
conjugated or otherwise linked to a therapeutic polypeptide may be
delivered to the central nervous system, including the brain and
spinal cord and cervical nodes, of a mammal by administration of
the fusion molecule intranasally.
[0030] As used herein, the term "polypeptide" intends a polymer of
amino acids and does not refer to a specific length of a polymer of
amino acids. Thus, for example, the terms peptide, oligopeptide,
protein, and enzyme are included within the definition of
polypeptide. This term also includes post-expression modifications
of the polypeptide, for example, glycosylations, acetylations,
phosphorylations, and the like. In some instances, the terms
protein, peptide, and polypeptide are used interchangeably.
[0031] The compositions are applied intranasally such that the
compositions will be transported to the brain directly, such as by
a non-systemic route. Accordingly, methods of delivering
therapeutic compositions to the central nervous system of a mammal
are provided herein. Methods of treating a disorder responsive to
treatment by application of a therapeutic composition to the
central nervous system of a mammal are also provided and described
below.
[0032] A. Composition Components
[0033] The therapeutic composition for intranasal delivery is a
fusion polypeptide comprised of polypeptide and an antibody or
antibody fragment. In one embodiment, the polypeptide is
biologically active and preferably causes or otherwise brings about
a particular biological effect, such as a therapeutic effect.
Various example of polypeptides are given below. The polypeptide is
linked to an antibody or antibody fragment directed against an
endogenous target. The antibody or antibody fragment, in addition
to having binding affinity for a cellular target, may be
biologically active to cause a therapeutic effect. Together the
polypeptide and the attached antibody or antibody fragment comprise
a therapeutic compound or therapeutic fusion polypeptide, that can
be formulated as desired for intranasal delivery. As will be
illustrated below, the increased size and/or hydrophilicity of the
fusion polypeptide, relative to the individual components, reduces
the blood bioavailability of the polypeptide while allowing
delivery to the central nervous system, thus improving drug
targeting while reducing systemic exposure and associated side
effects.
[0034] i. Antibody or Antibody Fragment
[0035] The antibody or antibody fragment in the therapeutic fusion
compound may be selected to serve as a targeting agent, to provide
a biologically desired effect, or both. The antibody or antibody
fragment may be a polyclonal or a monoclonal antibody, and
exemplary antibodies and fragments, sources of and preparation of
the same, are now described.
[0036] Polyclonal antibodies may be obtained by injecting a desired
antigen into a subject, typically an animal such as a mouse, as
well established in the art. The antigen is selected based on the
disorder to be treated. For example, in treating Alzheimer's
disease, the antigen may be .beta.-amyloid protein or peptides
thereof. In treating cancer, the antigen may be a tumor-associated
antigen, such as various peptides known to the art, including, for
example, interleukin-13 receptor-.alpha. (for malignant
astrocytoma/glioblastoma multiforme as discussed in Joshi, B. H. et
al., Cancer Res. 60:1168-1172 (2000)), BF7/GE2 (microsomal epoxide
hydrolase; mEH) (for treatment of tumors with abnormal mEH
expression as discussed in Kessler, R. et al., Cancer Res.
60:1403-1409 (2000)), tyrosinase-related protein-2 (TRP-2) (for
treatment of glioblastoma multiforme), MAGE-1, 3 or 6 (for
medulloblastomas) and MAGE-2 (for glioblastoma multiforme) (both as
discussed in Scarcella, D. L., et al., Clin. Cancer Res., 5:331-341
(1999)), and survivin (for medulloblastomas as described in Bodey,
B. B., In Vivo, 18(6)713-718 (2004)). For treatment of neurotrauma
to suppress inflammation such as in spinal cord injury and acute
brain injury, the antigen may be -TNF-alpha and various
interleukins, including interleukin-1.quadrature.. The antigen,
along with an adjuvant such as Freund's complete adjuvant, may be
injected into the subject multiple times subcutaneously or
intraperitoneally.
[0037] Another method to increase the immunogenicity of the antigen
is to conjugate or otherwise link the antigen to a protein that is
immunogenic in the particular species which will produce the
antibodies. For example, the antigen may be conjugated to
polytuftsin (TKPR40), a synthetic polymer of the natural
immunomodulator tuftsin, which has been shown to increase the
immunogenicity of synthetic peptides in mice (Gokulan K. et al.,
DNA Cell Biol. 18(8):623-630 (1999)). The method of conjugation may
involve use of a bifunctional or derivatizing agent, such as
maleimidobenzoyl sulfosuccinimide ester for conjugation through
cysteine residues, N-hydroxysuccinimide for conjugation through
lysine residues, glutaradehyde or succinic anhydride.
[0038] After a sufficient period of time after the initial
injection, such as, for example, about one month, the animals may
be boosted with a fraction of the original amount of peptide
antigen, such as 1/10 the amount, and may then be bled about 7 to
14 days later and the antibodies may be isolated from the blood of
the animals by standard methods known to the art, including
affinity chromatography using, for example, protein A or protein G
sepharose; ion-exchange chromatography, hydroxylapatite
chromatography or gel electrophoresis. Antibody purification
procedures may be found, for example, in Harlow, D. and Lane E.,
Using Antibodies: A Laboratory Manual, Cold Springs Harbor
Laboratory Press, Woodbury, N.Y. (1998); and Subramanian, G.,
Antibodies: Production and Purification, Kluwer Academic/Plenum
Publishers, New York, N.Y. (2004).
[0039] Non-human antibodies may be humanized by a variety of
methods. For example, hypervariable region sequences in the
non-human antibodies may be substituted for the corresponding
sequences of a human antibody as described, for example, in Jones
et al., Nature, 321:522-525 (1986); Reichmann et al., Nature,
332:323-327 (1988) and Verhoeyen et al., Science, 239:1534-1536
(1988). As the antibody is intended for human therapy, it is
preferable to select a human variable domain for guidance in making
a humanized antibody, in order to reduce the antigenicity of the
antibody. In order to accomplish this, the sequence of the variable
domain of the non-human antibody may be screened against a library
of known human variable domain sequences. The human variable domain
sequence which is the closest match to that of the animal is
identified and the human framework region within it is utilized in
the human antibody as described, for example, in Sims et al., J.
Immunol, 151:2296-2308 (1993) and Chothia et al., J. Mol. Biol.,
196:901-917 (1987).
[0040] The antibody may be a full length antibody or a fragment.
The full length antibody or fragment may be modified to allow for
improved stability of the antibody or fragment and to modulate
effector function, such as binding to an Fc receptor. This may be
achieved, for example, by utilizing human or murine isotypes, or
variants of such molecules such as IgG4 with Ala/Ala mutations, to
lose effector function and yet still maintain IgG structure. The
antibody fragment may be a monomer or a dimer, and includes Fab,
Fab', F(ab')2, Fc, or an Fv fragment. These fragments may be
produced, for example, by proteolytic degradation of the intact
antibody. For example, digestion of intact antibodies with papain
results in two Fab fragments. Treatment of intact antibodies with
pepsin provides a F(ab')2 fragment. The F(ab')2 fragment is a dimer
of Fab, which is a light chain joined to VH-CH1 by a disulfide
bond. The F(ab)'2 may be reduced under mild conditions to break the
disulfide linkage in the hinge region, thereby converting the
(Fab').sub.2 dimer into a Fab' monomer. The Fab' monomer is
essentially a Fab fragment with part of the hinge region (see
Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993),
for a more detailed description of other antibody fragments).
[0041] Many fragments, including those that have the Fc portion,
can also be produced by recombinant DNA technology methods known to
the art.
[0042] A wide variety of antibodies may be used to obtain the
antibody fragments utilized in the compositions for intranasal
delivery to the central nervous system described herein. Exemplary
antibodies include IgG, IgM, IgA, IgD, and IgE. Subclasses of these
antibodies may also be used to obtain the antibody fragments.
Exemplary subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
The antibody fragments may be obtained by proteolytic degradation
of the antibodies which may be produced as previously discussed
herein. In one embodiment, the antibody fragment is utilized to
increase the half-life of the polypeptide, and antibodies may be
isolated from a subject without immunization and may be isolated by
antibody isolation procedures previously described herein. Antibody
fragments may alternatively be produced by recombinant DNA methods
as previously described herein, in order to produce chimeric or
fusion polypeptides. For example, a fusion molecule may be produced
utilizing a plasmid encoding the respective proteins to generate
the mimetibody, which includes the antibody fragment and the
therapeutic polypeptide.
[0043] Antibodies, antibody fragments or antibody fragments linked
to polypeptides, or biologically active portions thereof, may be
purified by affinity purification including use of a Protein A
column and size exclusion chromatography utilizing, for example,
Superose columns. Purification methods are well known in the art.
Specific monoclonal antibodies may be prepared by the technique of
Kohler and Milstein, Eur. J. Immunol., 6:511-519 (1976) and
improvements and modifications thereof. Briefly, such methods
include preparation of immortal cell lines capable of producing
desired antibodies. The immortal cell lines may be produced by
injecting the antigen of choice into an animal, such as a mouse,
harvesting B cells from the animal's spleen and fusing the cells
with myeloma cells to form a hybridoma. Colonies may be selected
and tested by routine procedures in the art for their ability to
secrete high affinity antibody to the desired epitope. After the
selection procedures, the monoclonal antibodies may be separated
from the culture medium or serum by antibody purification
procedures known to the art, including those procedures previously
described herein.
[0044] Alternatively, antibodies may be recombinantly produced from
expression libraries by various methods known in the art. For
example, cDNA may be produced from ribonucleic acid (RNA) that has
been isolated from lymphocytes, preferably from B lymphocytes and
preferably from an animal injected with a desired antigen. The
cDNA, such as that which encodes various immunoglobulin genes, may
be amplified by the polymerase chain reaction (PCR) and cloned into
an appropriate vector, such as a phage display vector. Such a
vector may be added to a bacterial suspension, preferably one that
includes E. coli, and bacteriophages or phage particles may be
produced that display the corresponding antibody fragment linked to
the surface of the phage particle. A sublibrary may be constructed
by screening for phage particles that include the desired antibody
by methods known to the art, including, for example, affinity
purification techniques, such as panning. The sublibrary may then
be utilized to isolate the antibodies from a desired cell type,
such as bacterial cells, yeast cells or mammalian cells. Methods
for producing recombinant antibodies as described herein, and
modifications thereof, may be found, for example, in Griffiths, W.
G. et al., Ann. Rev. Immunol., 12:433-455 (1994); Marks, J. D. et
al., J. Mol. Biol, 222:581-597 (1991); Winter, G. and Milstein, C.,
Nature, 349:293-299 (1991); and Hoogenboom, H. R. and Winter, G.,
J. Mol. Biol., 227(2):381-388 (1992).
[0045] Human antibodies may also be produced in transgenic animals.
For example, homozygous deletion of the antibody heavy chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production
such that transfer of a human germ-line immunoglobulin gene array
into such mutant mice results in production of human antibodies
when immunized with antigen. See, e.g., Jakobovits et al., Proc.
Natl. Acad. Sci. USA, 90:2551-2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); U.S. Pat. Nos. 5,545,806; 5,569,825;
5,591,669; 5,545,807 and PCT publication WO 97/17852.
[0046] ii. Polypeptide
[0047] As noted above, the antibody or antibody fragment is linked
to a polypeptide. Preferably, the polypeptide is one that may bind
to a region of the central nervous system. The polypeptide is
further preferably one that has a beneficial effect on the central
nervous system, and includes one that has a beneficial effect on
functions regulated by the central nervous system of a mammal, such
as for therapeutic purposes. The polypeptide may exert its effects
by binding to, for example, cellular receptors in various regions
of the brain. As one example, in order for .alpha.-melanocyte
stimulating hormone (.alpha.-MSH) to exert its effect in body
weight reduction, it binds to the melanocortin 4 receptor (MCR-4)
on neurons in the hypothalamus. As a further example, in order for
erythropoietin (EPO), active EPO fragments or EPO analogs to
improve neurologic function after stroke or acute brain injury, it
has to bind to neuronal receptors, e.g., on hippocampal cells,
astrocytes, or similar cells.
[0048] A wide variety of proteins or peptides may be utilized. The
polypeptides may have a molecular weight of about 200 Daltons to
about 200,000 Daltons, but are typically about 300 Daltons to about
100,000 Daltons.
[0049] In one embodiment, the polypeptide and antibody or antibody
fragment, after attachment, have a combined molecular weight of
greater than about 25 kDa, more preferably of greater than about 30
kDa, still more preferably of greater than about 40 kDa.
[0050] In another embodiment, the polypeptide has a molecular
weight of less than about 25 kDa and is hydrophobic.
[0051] A wide variety of therapeutic proteins, or biologically
active portions thereof, may be linked or otherwise attached to the
antibody fragments that may be utilized in the methods described
herein. The proteins are preferably in the form of peptides. The
specific therapeutic peptide selected will depend on the disease or
condition (collectively referred to as "disorder") to be treated.
For neurodegenerative disorders, such as, for example, Alzheimer's
disease, Parkinson's disease and Huntington's disease, or other
disease involving loss of locomotion or cognitive function such as
memory, neuroprotective or neurotrophic agents are preferred. The
neuroprotective or neurotrophic agent may be one that promotes
neuronal survival, stimulates neurogenesis and/or synaptogenesis,
rescues hippocampal neurons from beta-amyloid-induced neurotoxicity
and/or reduces tau phosphorylation. Examples of agents suitable for
treating such neurodegenerative disorders, and neurological
disorders, include leutenizing hormone releasing (LHRH) and
agonists of LHRH, such as deslorelin; neurotrophic factors, such as
those from the neurotrophin family, including nerve growth factor
(NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 and
neurotrophin-4/5; the fibroblast growth factor family (FGFs),
including acidic fibroblast growth factor and basic fibroblast
growth factor; the neurokine family, including ciliary neurotrophic
factor, leukemia inhibitory factor, and cardiotrophin-1; the
transforming growth factor-.beta. family, including transforming
growth factor-.beta.-1-3 (TGF-betas), bone morphogenetic proteins
(BMPs), growth/differentiation factors such as growth
differentiation factors 5 to 15, glial cell line-derived
neurotrophic factor (GDNF), neurturin, artemin, activins and
persephin; the epidermal growth factor family, including epidermal
growth factor, transforming growth factor-.alpha. and neuregulins;
the insulin-like growth factor family, including insulin-like
growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2);
the pituitary adenylate cyclase-activating polypeptide
(PACAP)/glucagons superfamily, including PACAP-27, PACAP-38,
glucagons, glucagons-like peptides such as GLP-1 and GLP-2, growth
hormone releasing factor, vasoactive intestinal peptide (VIP),
peptide histidine methionine, secreting and glucose-dependent
insulinotropic polypeptide; and other neurotrophic factors,
including activity-dependent neurotrophic factor and
platelet-derived growth factors (PDGFs). Such agents are also
suitable for treating acute brain injury, chronic brain injury
(neurogenesis) and neuropsychologic disorders, such as
depression.
[0052] In the case of stroke treatment, the therapeutic agent may
be one that protects cortical neurons from nitric oxide-mediated
neurotoxicity, promotes neuronal survival, stimulates neurogenesis
and/or synaptogenesis and/or rescues neurons from glucose
deprivation. Examples of such agents include the neurotrophic
factors previously described herein, active fragments thereof, as
well as erythropoietin (EPO), analogs of EPO, such as carbamylated
EPO, and active fragments of EPO. Examples of EPO analogs that may
be used include those known to the skilled artisan and described,
for example, in U.S. Pat. Nos. 5,955,422 and 5,856,298. Peptide
growth factor mimetics of, and antagonists to, for example, EPO,
granulocyte colony-stimulating factor (GCSF), and thrombopoietin
useful in the invention can be screened for as reviewed by K.
Kaushansky, Ann. NY Acad. Sci., 938:131-138 (2001) and as described
for EPO mimetic peptide ligands by Wrighton et al., Science,
273(5274); 458-450 (1996). The mimetics, agonists and antagonists
to the peptide growth factors, or other peptides or proteins
described herein, may be shorter in length than the peptide growth
factor or other polypeptide that the mimetic, agonist or antagonist
is based on.
[0053] Therapeutic polypeptides for treatment of eating disorders,
such as for prevention of weight loss (anorexia) and weight gain
(obesity), include melanocortin receptor (MCR) agonists and
antagonists. Suitable MCR agonists include .alpha.-melanocyte
stimulating hormone (.alpha.-MSH) as well as beta and gamma-MSH,
and derivatives thereof, including amino acids 1 to 13 of human
.alpha.-MSH (SEQ ID NO:1 SYSMEHFRWGKPV) and specifically receptor
binding amino acid sequence 4-10, as in adrenocorticotropic hormone
(MSH/ACTH.sub.4-10), melanocortin receptor-3 (MCR3) or melanocortin
receptor 4 (MCR4) agonists, such as melanotan II (MT II), a potent
non-selective MCR agonist, MRLOB-0001 and active fragments of the
peptides and/or proteins. Other peptides for obesity treatment
include hormone peptide YY (PYY), especially amino acids 3 to 36 of
the peptide, leptin and ghrelin, ciliary neurotrophic factor or
analoqs thereof, glucagon-like peptide-1 (GLP-1), insulin mimetics
and/or sensitizers, leptin, leptin analogs and/or sensitizers and
dopaminergic, noradrenergic and serotinergic agents.
[0054] Corresponding MCR antagonists regulating body weight
homeostasis include endocannabinoid receptor antagonists, fatty
acid synthesis receptor inhibitors, ghrelin antagonists,
melanin-concentrating hormone receptor antagonists, PYY receptor
antagonists and tyrosine phosphatase-1B inhibitors (J. Korner et
al., J. Clin. Invest., 111:565-570 (2003)). MCR antagonists, such
as Agouti signaling protein (ASIP) and Agouti-related protein
(AGRP), which are endogenous MCR3 and MCR4 antagonists, and their
peptoid variants and mimetics may be used to control body weight
homeostasis and to treat eating disorders such as anorexia (Y K
Yang et al., Neuropeptides, 37(6):338-344 (2003); DA Thompson et
al, Bioorg Med Chem. Lett., 13:1409-1413 (2003); and C. Chen et al,
J. Med. Chem., 47(27):6821-30 (2004)).
[0055] The previously mentioned peptide hormones and analogs
thereof that bind to melanocortin receptors (MCRs) may also be
useful to control inflammation and improve male and female sexual
dysfunction (A. Catania et al., Pharmacol Rev, 56(1): 1-29
(2004)).
[0056] The therapeutic protein for treatment of endocrine
disorders, such as diabetes mellitus includes, for example,
glucagon-like peptide 1 (GLP-1); peptides from the GLP-1 family,
including pituitary adenylate cyclase-activating polypeptide
(PACAP), vasoactive intestinal peptide (VIP), exendin-3 and
exendin-4; and insulin-like growth factor (IGF-1), IGF binding
protein 3 (IGFBP3) and insulin, and active fragments thereof.
[0057] The therapeutic polypeptide for treatment of sleep
disorders, such as insomnia, includes growth hormone releasing
factor, vasopressin, and derivatives of vasopressin, including
desmopressin, glypressin, ornipressin and ternipressin; Included
are peptide variants and mimetic peptide ligands that bind to the
same receptor targets resulting in either the same/similar or the
opposite biological response. The therapeutic protein for treatment
of autoimmune disorders, such as multiple sclerosis, includes
interferons, including .beta.-interferon, and transforming growth
factor .beta.'s.
[0058] The therapeutic polypeptide for treatment of psychiatric
disorders, such as schizophrenia, includes neuregulin-1, EPO,
analogs of EPO, such as carbamylated EPO, and active fragments of
EPO and EPO mimetics as previously described herein. Various
neurotrophic factors and regulatory peptide hormones, such as
brain-derived neurotrophic factor (BDGF) and insulin, may be used
to treat depression, and psychoendocrinologic and metabolic
disorders.
[0059] The therapeutic polypeptide for treatment of lysosomal
storage disorders of the brain includes, for example, lysosomal
enzymes.
[0060] The therapeutic polypeptide for treatment of eating
disorders such as anorexia includes, for example, melanocortin
receptor (MCR) antagonists such as Agouti signaling protein (ASIP)
and Agouti related protein (AGRP).
[0061] The therapeutic polypeptides may be human polypeptides,
although the polypeptides may be from other species or may be
synthetically or recombinantly produced. The original amino acid
sequence may also be modified or reengineered such as for improved
potency or improved specificity (e.g. eliminate binding to multiple
receptors) and stability.
[0062] Therapeutic polypeptides utilized herein may also be
mimetics, such as molecules that bind to the same receptor but have
amino acid sequences that are non-homologous to endogenous human
peptides. For example, the agonist and antagonists, including
agonists and antagonists of melanocortin receptor, growth hormone
releasing factor receptor, vasopressin receptor, hormone peptide YY
receptor, a neuropeptide Y receptor, or erythropoietin receptor,
may include natural amino acids, such as the L-amino acids or
non-natural amino acids, such as D-amino acids. The amino acids in
the polypeptide may be linked by peptide bonds or, in modified
peptides, including peptidomimetics, by non-peptide bonds (J. Zhang
et al., Org. Lett., 5(17): 3115-8 (2003)).
[0063] Polypeptide mimetics, and receptor agonists and antagonists
can be selected and produced utilizing high throughput screening
known to the art for specific biological function and receptor
binding. The availability of such methods allows rapid screening of
millions of randomly produced organic compounds and peptides to
identify lead compounds for further development. Strategies used to
screen libraries of small molecules and peptides and the success in
finding mimetics and antagonists, e.g., for/to EPO, GCSF and
thrombopoietin, are reviewed by K. Kaushansky, Ann. NY Acad. Sci.,
938:131-138 (2001).
[0064] A wide variety of modifications to the amide bonds which
link amino acids may be made to the agonists and antagonists
described herein, and such modifications are well known in the art.
For example, such modifications are discussed in general reviews,
including in Freidinger, R. M. "Design and Synthesis of Novel
Bioactive Peptides and Peptidomimetics" J. Med. Chem., 46:5553
(2003), and Ripka, A. S., Rich, D. H. "Peptidomimetic Design" Curr.
Opin. Chem. Biol., 2:441 (1998). Many of the modifications are
designed to increase the potency of the peptide by restricting
conformational flexibility.
[0065] For example, the agonists and antagonists may be modified by
including additional alkyl groups on the nitrogen or alpha-carbon
of the amide bond, such as the peptoid strategy of Zuckerman et al,
and the alpha modifications of, for example Goodman, M. et. al.
(Pure Appl. Chem., 68:1303 (1996)). The amide nitrogen and alpha
carbon may be linked together to provide additional constraint
(Scott et al., Org. Letts., 6:1629-1632 (2004)).
[0066] iii. Linkages
[0067] The polypeptide is linked to the antibody or antibody
fragment to form the therapeutic compound for delivery. The
antibody or antibody fragment, in one embodiment, increases the
stability of the polypeptide, thereby increasing its half life in
vivo, including in the nasal cavity and the central nervous system
of a mammal. The combined polypeptide-antibody fragment compound is
also referred to herein as a "mimetibody". In this section,
approaches for linking the two moieties is described.
[0068] The antibody fragment and polypeptide may be linked to each
other by methods known to the art, and typically through covalent
bonding. The linking or conjugation method may include use of amino
acid linkers, including use of glycine and serine. The fragment and
polypeptide may be conjugated or otherwise linked by cross-linking
or other linking procedures know to the art and discussed, for
example, in Wong, S. S., Chemistry of Protein Conjugation and
Cross-Linking, CRC Press, Boca Raton, Fla. (1991). For example, the
polypeptides may be conjugated utilizing homo-bifunctional and/or
hetero-bifunctional or multifunctional cross-linkers known to the
art. Examples of cross-linking agents include carbodiimides, such
as EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride); imidoesters, N-hyroxysuccinimide-esters,
maleimides, pyridyl disulfides, hydrazides and aryl azides. Several
points of attachment between the active agent polypeptide and the
antibody fragment are envisioned, including linkage of the
N-terminus of the peptide to the C-terminus of the antibody
fragment. The polypeptide may, alternatively, be attached at its
C-terminus to the N-terminus of the antibody fragment. Conjugation
may further be via cysteine or other amino acid residues or via a
carbohydrate functional moiety of the antibody.
[0069] iv. Formulation of the Therapeutic Polypeptide-Antibody
Compound
[0070] The active agent polypeptide in the therapeutic composition
may be mixed with a pharmaceutically-acceptable carrier or other
vehicle. The carrier may be a liquid suitable, for example, for
administration as nose drops or as a nose spray, and includes
water, saline or other aqueous or organic and preferably sterile
solution. The carrier may be a solid, such as a powder, gel or
ointment and may include inorganic fillers such as kaolin,
bentonite, zinc oxide, and titanium oxide; viscosity modifiers,
antioxidants, pH adjusting agents, lyoprotectants and other
stability enhancing excipients, including sucrose, antioxidants,
chelating agents; humectants such as glycerol, and propylene
glycol; and other additives which may be incorporated as necessary
and/or desired.
[0071] Where the therapeutic compound is administered as a gel or
ointment, the carrier may include suitable solid, such as a
pharmaceutically acceptable base material known for use in such
carriers, including, for example, natural or synthetic polymers
such as hyaluronic acid, sodium alginate, gelatin, corn starch, gum
tragacanth, methylcellulose, hydroxyethylcellulose,
carboxymethylcellulose, xanthan gum, dextrin, carboxymethylstarch,
polyvinyl alcohol, sodium polyacrylate, methoxyethylene maleic
anhydride copolymer, polyvinylether, polyvinylpyrrolidone; fats and
oils such as beeswax, olive oil, cacao bufter, sesame oil, soybean
oil, camellia oil, peanut oil, beef fat, lard, and lanolin; white
petrolatum; paraffins; hydrocabon gel ointments; fatty acids such
as stearic acid; alcohols such as cetyl alcohol and stearyl
alcohol; polyethylene glycol; and water.
[0072] Where the therapeutic compound is administered as a powder,
the carrier may be a suitable solid such as oxyethylene maleic
anhydride copolymer, polyvinylether, polyvinylpyrrolidone polyvinyl
alcohol; polyacrylates, including sodium, potassium or ammonium
polyacrylate; polylactic acid, polyglycolic acid, polyvinyl
alcohol, polyvinyl acetate, carboxyvinyl polymer,
polyvinylpyrrolidone, polyethylene glycol; celluloses, including
cellulose, microcrystalline cellulose, and .alpha.-cellulose;
cellulose derivatives, including methyl cellulose, ethyl cellulose,
hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl
methyl cellulose, sodium carboxymethyl cellulose and ethylhydroxy
ethyl cellulose; dextrins, including alpha-, beta- or
.gamma-cyclodextrin, dimethyl-beta.-cyclodextrin; starches,
including hydroxyethyl starch, hydroxypropyl starch, carboxymethyl
starch; polysaccharides, including dextran, dextrin and alginic
acid; hyaluronic acid; pectic acid; carbohydrates, such as
mannitol, glucose, lactose, fructose, sucrose, and amylose;
proteins, including casein, gelatin, chitin and chitosan; gums,
such as gum arabic, xanthan gum, tragacanth gum and glucomannan;
phospholipids and combinations thereof.
[0073] The particle size of the powder may be determined by
standard methods in the art, including screening or sieving through
appropriately sized mesh. If the particle size is too large, the
size can be adjusted by standard methods, including chopping,
cutting, crushing, grinding, milling, and micronization. The
particle size of the powders typically range from about 0.05 .mu.m
to about 100 .mu.m. The particles are preferably no larger than
about 400 .mu.m.
[0074] The compositions may further include agents which improve
the mucoadhesivity, nasal tolerance, or the flow properties of the
composition, mucoadhesives, absorption enhancers, odorants,
humectants, and preservatives. Suitable agents which increase the
flow properties of the composition when in an aqueous carrier
include, for example, sodium carboxymethyl cellulose, hyaluronic
acid, gelatin, algin, carageenans, carbomers, galactomannans,
polyethylene glycols, polyvinyl alcohol, polyvinylpyrrolidone,
sodium carboxymethyl dextran and xantham gum. Suitable absorption
enhancers include bile salts, phospholipids, sodium
glycyrrhetinate, sodium caprate, ammonium tartrate,
gamma.aminolevulinic acid, oxalic acid, malonic acid, succinc acid,
maleic acid and oxaloacetic acid. Suitable humectants for aqueous
compositions include, for example, glycerin, polysaccharides and
polyethylene glycols. Suitable mucoadhesives include, for example,
polyvinyl pyrrolidone polymer.
[0075] Another aspect of the invention is pharmaceutical
compositions that may further include permeation enhancer agents
that enhance delivery of protein substances to the central nervous
system via intranasal administration. The protein substances can be
whole antibodies, antibody fragments known to those skilled in the
art such as Fab fragments or Fc-containing protein substances such
as mimetibodies. Further, the protein substance can be a
catalytically active protein such as an enzyme. The protein
substances administered are useful for prophylactic, therapeutic or
diagnostic purposes.
[0076] Absorption enhancers facilitate the transport of molecules
through the mucosa, which includes the mucous, and the epithelial
cell membrane. A variety of absorption enhancer classes have been
described, including mucoadhesives, ciliary beat inhibitors, mucous
fluidizers, membrane fluidizers, and tight junction modulators. In
the present invention, membrane fluidizers and tight junction
modulators most effectively increase the delivery of protein
substances, deposited on the nasal epithelium, to the CNS.
[0077] Tight junctions are intercellular structures that restrict
paracellular transport between the apical and basolateral sides of
an epithelial layer or between aspects of certain specialized cells
including neurons, activated immune cells, and some endothelial
cells. The permeability of the tight junctions varies between
tissues in the body; tight junctions generally limit transport to
those molecules with a hydrodynamic radius less than 3.6 A and
disallow significant transport of molecules with a radius larger
than 15 A (Stevenson, B. R. et al., Mol. Cell Biochem 83:129-145
(1988)). See also Illum L, J. Pharm. Pharmacol, 56:3-17 (2004).
[0078] Tight junctions are composed of integrated transmembrane and
intracellular proteins and the adjacent membranous microdomains.
Tight junction permeability is modulated by intra- and
extra-cellular stimuli that modify tight junction proteins or
lipids thereby changing their physical interactions with each other
and leading to either more restricted or less restricted
paracellular transport. Some chemicals have been shown to mimic
these intra- and extra-cellular stimuli to induce temporary changes
in either the proteins or lipids in the tight junctions to result
in reversible increases in paracellular transport. Including these
chemicals as excipients in formulations allows increased amounts of
a molecule to pass through the nasal epithelium or between the
axons and adjacent cells to enter paracellular and perineurial
spaces (extracellular spaces).
[0079] The functionality of such membrane modifiers can be measured
by their barrier disruption potential (reduction in TEER) and
visualized by microscopic methods (e.g., fluorescence light
microscope, electron microscope). The functionalities include
extraction or fluidization of lipid bilayers or disruption of cell
membrane protein interactions, e.g., via perturbation, disruption
of intermolecular ionic forces such as hydrogen bonding or specific
binding. This can be achieved via solvents such as propylene
glycol, glycofurol and glycerol or bile salts and ionic or nonionic
surfactants such as glycocholate, taurocholate and
tauroursodeoxycholate and alkylglycosides such as decyl, dodecyl
and tetradecyl maltosides or lipids such as
lysophopsphatidylcholine (LPC),
1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and fatty acids
such as oleic acid or charged compounds such as chitosan and others
as typically described in the art.
[0080] The enhancers as described in Example 7 and listed in Table
A herein are representative examples of membrane modifiers and
include chitosan glutamate, n-dodecyl-beta-D-maltopyranoside,
n-decyl-beta-D-maltopyranoside,
n-tetradecyl-beta-D-maltopyranoside,
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate,
propylene glycol, heptakis(2,6-di-O-methyl)-beta-cyclodextrin,
1,2-didecanoyl-sn-glycero-3-phosphocholine, sodium glycocholate
hydrate, taurocholic acid sodium salt hydrate, sodium
tauroursodeoxycholate, and tetrahydrofurfuryl polyethylenglycol.
Naming conventions, sources and other relevant information for
these compounds is provided in Table A.
TABLE-US-00001 TABLE A Purity by Mass of Compound in Compound
Preparation & Other Characteristics Shorthand Source of of the
Designation or Name Compound Compound for Compound Compound Name(s)
Preparation Preparation chitosan chitosan glutamate NovaMatrix A
chitosan USA d.b.a. preparation in FMC which 75-90% BioPolymer of
the acetyl AS groups are (Sandvika, deacetylated. Norway)
Typically, the molecular weight for chitosan glutamate in the
PROTASAN UP G 213 is in the 20,0000 to 60,0000 g/mol range
(measured as a chitosan acetate). Intravail A3 n-dodecyl-beta-D-
Inalco SpA .gtoreq.99% maltopyranoside; (Milano, Italy) dodecyl
maltoside (C12 chain length) Intravail A5 n-tetradecyl-beta-D-
Inalco SpA .gtoreq.99% maltopyranoside; (Milano, Italy) tetradecyl
maltoside; (C14 chain length) TDM Intravail B3 beta-D- Anatrace,
98.90% fructopyranosyl-alpha- Inc. glucopyranoside (Maumee,
monododecanoate; OH) sucrose monododecanoate; dodecanoyl sucrose;
lauroyl sucrose Intravail A1 n-decyl-beta-D- Aegis N/A
maltopyranoside (C10 Therapeutics, chain length) LLC (San Diego,
CA) propylene glycol propylene glycol Dow 99.50% Chemical (Midland,
MI) di-methyl-beta- heptakis(2,6-di-O- Sigma N/A cyclodextrin
methyl)-beta- (St. Louis, cyclodextrin MO) DDPC 1,2-didecanoyl-sn-
Sigma 99% glycero-3- (St. Louis, phosphocholine MO) sodium
glycocholate sodium glycocholate Sigma >97% hydrate (St. Louis,
MO) sodium taurocholate taurocholic acid Aldrich 97% sodium salt
hydrate (St. Louis, MO) tauroursodeoxycholate sodium Sigma 90%
tauroursodeoxycholate (St. Louis, MO) glycofurol
Tetrahydrofurfuryl- Alchymars N/A polyethylenglycol (Milano, Italy)
ether
[0081] The term "peptide chain" means a molecule comprising at
least two amino acid residues linked by a peptide bond to form a
chain. Large peptide chains of more than 50 amino acids may be
referred to as "polypeptides" or "proteins." Small peptide chains
of less than 50 amino acids may be referred to as "peptides."
[0082] The term "catalytically active" means a molecule that is
capable of increasing the rate of a chemical reaction. Such
chemical reactions may include synthetic, decomposition,
combustion, single displacement, double displacement, acid-base
equilibrium, reduction-oxidation, or other physical processes such
as particle release (e.g. ion release) or other changes that are
thermodynamically favorable. Examples of "catalytically active"
peptide chains include enzymes, such as beta-lactamases or
lysosomal enzymes, and allosteric effectors. Those skilled in the
art will recognize other such catalytically active peptide
chains.
[0083] The term "antibody Fc fragment" means a peptide chain
comprising a portion of an immunoglobulin heavy chain C.sub.H2
constant region peptide chain and immunoglobulin heavy chain
C.sub.H3 constant region peptide chains sufficient to bind protein
A. Such constant region peptide chains may be derived from antibody
heavy chains of any isotype, such as IgG.sub.1, and may also be
referred to as an "Fc domain" (see e.g. FIG. 11) or "Fc peptide
chain." An antibody Fc fragment may be an individual peptide chain
comprising both an immunoglobulin heavy chain C.sub.H2 constant
region peptide chain and an immunoglobulin heavy chain C.sub.H3
constant region peptide chain sufficient to bind protein A (e.g.
single chain antibody) or an association of two such individual
peptide chains (e.g. antibody or mimetibody). "Peptide chains
comprising an antibody Fc fragment" represent a genus of molecules
that includes, for example, antibody molecules comprising Fc
domains, Fc fusion peptide chains comprising Fc domains, and
mimetibody peptide chains comprising Fc domains (FIG. 11). The term
"antibody Fc fragment" can be used to describe unaggregated Fc
peptide chains, aggregated Fc peptide chains, or both aggregated
and unaggregated Fc peptide chains.
[0084] The term "emulsion" means a mixture of at least two
immiscible substances such that one substance (the dispersed phase)
is dispersed in the other (the continuous phase). A compound is
said to be "emulsified" when it is present in an emulsion,
typically in the dispersed phase.
[0085] The invention provides liquid pharmaceutical compositions
comprising various formulations useful and acceptable for nasal
administration to an animal or human patient. Such pharmaceutical
compositions are prepared using aqueous buffers at "standard state"
as the diluent, and in some aspects of the invention liquid
compounds such as propylene glycol or
tetrahydrofurfuryl-polyethylenglycol ether at standard state. The
liquid pharmaceutical compositions of the invention can be prepared
using routine methods well known to those of ordinary skill in the
art. For example, the aqueous buffer component of the
pharmaceutical composition may be provided first followed by the
addition of an appropriate mass or volume of the other components
of the pharmaceutical composition at "standard state." The desired
amount of a catalytically active peptide chain or a peptide chain
comprising an antibody Fc or Fab fragment may then be added. Last,
the volume of the pharmaceutical composition is adjusted to the
desired final volume under "standard state" conditions using
aqueous buffer as the diluent. Those skilled in the art will
recognize a number of other methods suitable for the preparation of
the claimed pharmaceutical compositions and would also recognize
that the pharmaceutical compositions of the invention could be
prepared in powder or lyophilized form.
[0086] Aqueous buffers suitable for use in the pharmaceutical
compositions and methods of the invention are physiologically
acceptable for nasal administration. The pH of such buffers must be
physiologically acceptable for nasal administration and compatible
with the nasal delivery of a catalytically active peptide chain or
a peptide chain comprising an antibody Fc or Fab fragment to the
central nervous system. The pH of such an aqueous buffer may be
between pH 4.0 and pH 8.0 with a pH of about 6.0 to 7.6 being
preferred. Phosphate, acetate, borate, phthalate and amino acid
based aqueous buffers such as histidine based buffers are examples
of physiologically acceptable buffers. Phosphate buffered saline
(PBS) with a pH of 7.4 at "standard state" is one example of an
aqueous buffer suitable for use in the pharmaceutical compositions
of the invention and is a preferred aqueous buffer. PBS comprises
0.138 M NaCl, 0.0027 M KCl with a pH of 7.4 "standard state." Those
skilled in the art will recognize other aqueous buffers suitable
for the preparation of the claimed pharmaceutical compositions and
for use in the methods of the invention.
[0087] The claimed pharmaceutical compositions may be aqueous
solutions or suspensions, such as emulsions, comprising the
indicated mass, or volume, of each constituent per unit of water
volume or having an indicated pH at "standard state." As used
herein, the term "standard state" means a temperature of 25.degree.
C.+/-2.degree. C. and a pressure of 1 atmosphere. The term
"standard state" is not used in the art to refer to a single art
recognized set of temperatures or pressure, but is instead a
reference state that specifies temperatures and pressure to be used
to describe a solution or suspension with a particular composition
under the reference "standard state" conditions. This is because
the volume of a solution is, in part, a function of temperature and
pressure. Those skilled in the art will recognize that
pharmaceutical compositions equivalent to those disclosed here can
be produced at other temperatures and pressures. Whether such
pharmaceutical compositions are equivalent to those disclosed here
should be determined under the "standard state" conditions defined
above (e.g. 25.degree. C.+/-2.degree. C. and a pressure of 1
atmosphere). Additionally, the pharmaceutical compositions of the
invention are described in terms of the mass, or volume at standard
state, of a given component per a 100 ml volume of the
pharmaceutical composition. As those in the art will recognize,
this is simply one way of facilitating a description of the
pharmaceutical compositions of the invention and should not be
construed as limiting the claims (e.g. to a 100 ml volume of the
pharmaceutical composition).
[0088] Importantly, the pharmaceutical compositions of the
invention may contain component masses "about" a certain value
(e.g. "about 1 g of sodium tauroursodeoxycholate") per unit volume
of the pharmaceutical composition or have pH values about a certain
value. A component mass or volume present in a pharmaceutical
composition or pH value is "about" a given numerical value if the
catalytically active peptide chain or peptide chain comprising an
antibody Fc or Fab fragment present in the pharmaceutical
composition is biologically active while such peptide chains are
present in the pharmaceutical composition or after such peptide
chains have been removed from the pharmaceutical composition (e.g.
by dilution or delivery). Stated differently, a value, such as a
component mass value, volume value or pH value, is "about" a given
numerical value when the catalytic activity of a catalytically
active peptide chain or the binding activity of the peptide chain
comprising an antibody Fc or Fab fragment is maintained and
detectable after placing the isolated antibody in the
pharmaceutical composition.
[0089] Alternatively, a value, such as a component mass value,
volume value or pH value, is "about" a given numerical value when
the value is within a range that includes values that are
uncorrected for the purity by, mass or volume, of the component
compound in a given compound preparation and values that have been
corrected for the purity, by mass or volume, of a component
compound in a given compound preparation. For example, if a given
compound preparation is 90% pure by mass for a given component
compound values "about" 1 g would include 0.9 g, 1 g, and 1.1 g
since these values are in the range described above. This reflects
the practical fact that for many compound preparations the actual
purity by mass or volume of a given compound in the preparation is
not known with absolute certainty, but is instead only know within
the limits of the assay techniques used. Furthermore, due to the
heterogenous nature of some compounds such as polymers and hydrates
it is often difficult to describe the purity of the compound by
mass because the mass of the compounds in the preparation, such as
polymers, or the mass and stoichiometry of hydrate in the
preparation is variable or undetermined.
[0090] Last, a value such as the molecular mass in Dalton based
units is "about" a given numerical value when the value is within a
range that includes the standard deviation observed when using a
technique, such a SDS-PAGE, for measuring the molecular mass of a
peptide chain. For example, if the mean molecular mass in Daltons
of a peptide chain as measure by SDS-PAGE is 25,000 Daltons with a
standard deviation of (+/-) 5,000 Daltons then values "about"
25,000 Daltons are those in the range 20,000 Daltons; 25,000
Daltons; and 30,000 Daltons.
[0091] One aspect of the invention is a pharmaceutical composition
comprising a catalytically active peptide chain or a peptide chain
comprising an antibody Fc or Fab fragment; and about 0.1 to about
1.0 g, such as 0.5 g, of chitosan glutamate or corresponding
amounts of another chitsoan salt per 100 ml of the pharmaceutical
composition; wherein the diluent is an aqueous buffer at standard
state. Chitosan salts, such as chitosan glutamate, useful in the
pharmaceutical compositions and methods of the invention includes
chitosans with different molecular weights including for example
chitosans with molecular weights of 50,000 g/mol to 60,000 g/mol
and which are approximately 90% deacetylated. Other chitosan salts
such as, for example, chitosan chloride and others recognized by
those of ordinary skill in the art may also be used in the
compositions and methods of the invention.
[0092] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc or Fab fragment; and from
about 0.125 g to about 1 g, such as about 0.5 g or less, of a
compound selected from the group consisting of
n-dodecyl-beta-D-maltopyranoside, n-decyl-beta-D-maltopyranoside,
n-tetradecyl-beta-D-maltopyranoside, and
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate per
100 ml of the pharmaceutical composition; wherein the diluent is an
aqueous buffer at standard state.
[0093] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc or Fab fragment; and from
about 5 ml to about 20 ml, such as about 10 ml to about 20 ml, of
propylene glycol per 100 ml of the pharmaceutical composition;
wherein the diluent is an aqueous buffer at standard state and the
propylene glycol is at standard state.
[0094] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and about 1 g to
about 10 g, such as about 5 g, of heptakis
(2,6-di-O-methyl)-beta-cyclodextrin per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state.
[0095] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and about 1 g to
about 5 g, such as about 2 g, of
1,2-didecanoyl-sn-glycero-3-phosphocholine per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state and the
1,2-didecanoyl-sn-glycero-3-phosphocholine is emulsified in the
aqueous buffer. Methods for emulsification such as sonication, the
use of stabilizing surfactants and the like are well known in the
art and are suitable for preparing the pharmaceutical compositions
of the invention comprising
1,2-didecanoyl-sn-glycero-3-phosphocholine in emulsion.
[0096] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc fragment; and about 01 g to
about 1 g, such as about 1 g, of a compound selected from the group
consisting of sodium glycocholate hydrate, taurocholic acid sodium
salt hydrate, and sodium tauroursodeoxycholate per 100 ml of the
pharmaceutical composition; wherein the diluent is an aqueous
buffer at standard state. Other salts of these compounds which will
be readily recognized by those of ordinary skill in the art may
also be used in the compositions and methods of the invention.
[0097] Another aspect of the invention is a pharmaceutical
composition comprising a catalytically active peptide chain or a
peptide chain comprising an antibody Fc or Fab fragment; and from
about 1 ml to about 10 ml of tetrahydrofurfuryl-polyethylenglycol
per 100 ml of the pharmaceutical composition; wherein the diluent
is an aqueous buffer at standard state and the
tetrahydrofurfuryl-poyethylenglycol is at standard state.
[0098] In another embodiment of the pharmaceutical composition of
the invention, the peptide chain comprises an antibody Fc fragment
that binds to a melanocortin 4 receptor comprising the amino acid
sequence shown in SEQ ID NO: 2. Such binding can be assayed by a
variety of well known techniques such as, for example, ELISAs,
Western blots, BIACORE.TM. (GE Healthcare, Piscataway, N.J.)
instrumentation based techniques and the like.
[0099] In another embodiment of the pharmaceutical compositions of
the invention, the peptide chain comprising an antibody Fc fragment
comprises the amino acid sequence shown in SEQ ID NO: 3.
[0100] In one embodiment of this pharmaceutical composition the
aqueous buffer is phosphate buffered saline (PBS) at about pH
7.4.
[0101] B. Nasal Delivery
[0102] The term "nasal cavity" means the large air-filled space
within an animal's nose.
[0103] The term "central nervous system" means the part of the
nervous system in vertebrates that comprises the brain and spinal
cord, to which sensory impulses are transmitted and from which
motor impulses pass out, and which coordinates the activity of the
entire nervous system. In particular, the term "central nervous
system" includes the olfactory bulb, left brain hemisphere, right
brain hemisphere, cerebellum, and brain stem as well as their
substructures. It also may include the cervical nodes, superficial
and axillary nodes.
[0104] The term "animal" means any member of the kingdom Animalia
which has a central nervous system and which normally has an air
filled nasal cavity and includes humans.
[0105] The therapeutic composition, comprised of an antibody or
antibody fragment linked to a polypeptide, may be administered by a
wide variety of methods, and some exemplary methods are provided
below. Absorption of the fusion polypeptide once introduced into
the nasal cavity may occur via absorption across the olfactory
epithelium, which is found in the upper one-third of the nasal
cavity. Absorption may also occur across the nasal respiratory
epithelium, which is innervated with trigeminal nerves, in the
lower two-thirds of the nasal cavity. The trigeminal nerves also
innervate the conjunctive, oral mucosa, and certain areas of the
dermis of the face and head, and absorption after intranasal
administration of the fusion polypeptide from these regions may
also occur.
[0106] One exemplary formulation for intranasal delivery of the
fusion polypeptide is a liquid preparation, preferably an aqueous
based preparation, suitable for application as drops into the nasal
cavity. For example, nasal drops can be instilled in the nasal
cavity by tilting the head back sufficiently and apply the drops
into the nares. The drops may also be inhaled through the nose.
[0107] Alternatively, a liquid preparation may be placed into an
appropriate device so that it may be aerosolized for inhalation
through the nasal cavity. For example, the therapeutic agent may be
placed into a plastic bottle atomizer. In one embodiment, the
atomizer is advantageously configured to allow a substantial amount
of the spray to be directed to the upper one-third region or
portion of the nasal cavity. Alternatively, the spray is
administered from the atomizer in such a way as to allow a
substantial amount of the spray to pass the nasal valve and to be
directed to the upper one-third region or portion of the nasal
cavity. By "substantial amount of the spray" it is meant herein
that at least about 50%, further at least about 70%, but preferably
at least about 80% or more of the spray passes the nasal valve and
is directed to the upper and distal portion of the nasal cavity
with about 10% or more reaching the upper third of the nasal
cavity.
[0108] Additionally, the liquid preparation may be aerosolized and
applied via an inhaler, such as a metered-dose inhaler. One example
of a preferred device is that disclosed in U.S. Pat. No. 6,715,485
to Djupesland, and which involves a bi-directional delivery
concept. In using the device, the end of the device having a
sealing nozzle is inserted into one nostril and the patient or
subject blows into the mouthpiece. During exhalation, the soft
palate closes due to positive pressure thereby separating the nasal
and oral cavities. The combination of closed soft palate and sealed
nozzle creates an airflow in which drug particles are released
entering one nostril, turning 180 degrees through the communication
pathway and exiting through the other nostril, thus achieving
bi-directional flow.
[0109] The fusion polypeptide can also be delivered in the form of
a dry powder, as in known in the art. An example of a suitable
device is the dry powder nasal delivery device marketed under the
name DIRECTHALER.TM. nasal, and which is disclosed in PCT
publication No. 96/222802. This device also enables closing of the
passage between the nasal and oral cavity during dose delivery.
Another device for delivery of a dry or liquid preparation is the
device sold under the trade designation OPTINOSE.TM..
[0110] One embodiment of the invention is a method of delivering a
catalytically active peptide chain or a peptide chain comprising an
antibody Fc or Fab fragment to the central nervous system of an
animal comprising providing a a permeation enhancer in a
concentration sufficient to enhance intranasal administration of
the catalytically active peptide chain or peptide chain comprising
an antibody Fc or Fab fragment to the central nervous system of an
animal; and administering the pharmaceutical composition to the
nasal cavity of an animal; whereby the catalytically active peptide
chain or a peptide chain comprising an antibody Fc or Fab fragment
enters the central nervous system of the animal. A pharmaceutical
composition of the invention can be administered to the nasal
cavity of an animal in the form of nasal drops, aerosol
preparations, and the like as discussed above. A cannula or other
device, such as the DIRECTHALER.TM. or OPTINOSE.TM. devices, can be
used in the methods of the invention to facilitate the dispersion
and nasal delivery of the pharmaceutical compositions of the
invention. In the methods of the invention the animal can be a
rodent, primate, human or other animal with a nasal cavity.
[0111] Another embodiment of the invention is a method of
delivering a catalytically active peptide chain or a peptide chain
comprising an antibody Fc or Fab fragment to the central nervous
system of an animal comprising providing a pharmaceutical
composition of the invention; and administering the pharmaceutical
composition to the nasal cavity of an animal; whereby the
catalytically active peptide chain or the peptide chain comprising
an antibody Fc or Fab fragment enters the central nervous system of
the animal.
[0112] Another embodiment of the invention is a method of
delivering a peptide chain comprising an antibody Fc fragment to
the central nervous system of an animal comprising providing a
pharmaceutical composition of the invention wherein the peptide
chain comprising the antibody Fc fragment binds to a melanocortin 4
receptor comprising the amino acid sequence shown in SEQ ID NO: 2;
and administering the pharmaceutical composition to the nasal
cavity of an animal; whereby the peptide chain comprising the
antibody Fc fragment enters the central nervous system of the
animal.
[0113] Another embodiment of the invention is a method of
delivering a peptide chain comprising an antibody Fc fragment to
the central nervous system of an animal comprising providing a
pharmaceutical composition of the invention wherein the peptide
chain comprising the antibody Fc fragment comprises the amino acid
sequence shown in SEQ ID NO: 3; and administering the
pharmaceutical composition to the nasal cavity of an animal;
whereby the catalytically active peptide chain or a peptide chain
comprising the antibody Fc fragment enters the central nervous
system of the animal.
[0114] C. Methods of Treatment
[0115] In yet another aspect, methods of treatment are provided.
The treatment methods may advantageously be utilized to treat a
disorder in a mammal that is amenable to treatment by
administration of a therapeutic agent to the central nervous
system, such as the brain and/or spinal cord. That is, the disorder
is one where the symptoms decrease or are otherwise eliminated, the
rate of progression of the disorder decreases, and/or the disorder
is eliminated by an agent that acts on the central nervous
system.
[0116] In one embodiment, a method includes administering to the
nasal cavity of a mammal, such as to cells and/or tissue in a
region or portion of the nasal cavity of a mammal occupied by the
superior turbinates, a therapeutically effective amount of an
antibody fragment linked or otherwise conjugated to a
polypeptide.
[0117] The method may be used to treat a wide variety of disorders.
Suitable disorders include, for example, neurological and
neurodegenerative disorders such as Alzheimer's disease,
Parkinson's disease, and Huntington's disease, as well as other
disorders known to the art that cause a loss of memory, such as
multi-infarct dementia, Creutzfeldt-Jakob disease, Lewy body
disease, normal pressure hydrocephalus and HIV dementia; or a loss
of locomotion, such as stroke, amyotropic lateral sclerosis,
myasthenia gravis and Duchenne dystrophy; endocrine, metabolic or
energy balance disorders, such as obesity, diabetes and sleeping
disorders, including insomnia; autoimmune disorders, such as
multiple sclerosis; anorexia and treatment of acute injury from
stroke or spinal cord injuries.
[0118] In one embodiment, a method of delivering a therapeutic
composition to the central nervous system of a mammal includes
administering the composition to the mammal intranasally,
preferably to olfactory and/or trigeminal nerve endings, cells and
nasal epithelium in a region of the nasal cavity located in the
superior turbinates. This region or area is typically located in,
but is not limited to, the upper one-third portion of the nasal
cavity.
[0119] Although not being limited to any theory by which the method
achieves its advantageous results, the agents that are applied
intranasally according to the methods described herein may reach
the brain directly by an extracellular or intracellular pathway.
See, e.g., Thorne, R. G. et al., Neuroscience, 127:481-496 (2004).
Intracellular pathways include transport through olfactory sensory
neurons. This may involve, for example, absorptive or
receptor-mediated endocytosis into olfactory sensory neurons and
subsequent transport to olfactory bulb glomeruli. As another
example, such transport may involve intraneuronal transport within
the trigeminal nerve such that the composition is delivered to
trigeminal ganglion and parts of the trigeminal brainstem nuclear
complex, such as the subnucleus caudalis. In such intracellular
pathways, the therapeutic agent may first be transported though
nasal mucosa. Although antibody fragments that include the Fc
portion (constant region) of an immunoglobulin may also be
delivered by one of the aforementioned routes, one of the delivery
routes may include being taken up by cells in the nasal mucosal
epithelium having neonatal Fc receptors (FcRn) which may, depending
on the mechanism, facilitate or hinder transport of the composition
across the olfactory epithelium.
[0120] Extracellular pathways of entry of the composition into the
central nervous system via the nasal cavity include direct entry
into the cerebrospinal fluid, entry into the CNS parenchyma through
extracellular spaces and channels, tracts or compartments
associated with the olfactory system, such as the peripheral
olfactory system, including the system that connects the nasal
passages with the olfactory bulbs and rostral brain areas; and
entry into the CNS parenchyma through extracellular spaces and
channels, tracts or compartments associated with the trigeminal
system, such as the peripheral trigeminal system, including the
system connecting the nasal passages with the brainstem and spinal
chord (Thorne, R. G. et al., Neuroscience 127:481-496 (2004)).
Direct transport as used herein includes transport via one or more
of the non-systemic pathways described herein.
[0121] Transport of the composition directly to the central nervous
system by one or more of the mechanisms described herein allows the
blood-brain barrier to be bypassed and overcomes the associated
challenges and disadvantages surrounding systemic transport of
agents to the central nervous system. Additionally, transporting
the compositions by the methods described herein may allow less of
the composition to be used as a greater proportion of the
administered dose reaches the central nervous system target. In the
case of administration of agents that are endogenously produced in
the subject treated, the physiologic effects are typically
comparable to the endogenous agent.
[0122] A therapeutically effective amount of the therapeutic
composition is provided. As used herein, a therapeutically
effective amount of the composition is the quantity of the
composition required to achieve a specific therapeutic effect. For
example, the amount is typically that required to reach a specified
or desired clinical endpoint, such as a decrease in the progression
of the disorder, a lessening of the severity of the symptoms of the
disorder and/or elimination of the disorder. This amount will vary
depending on the time of administration, the route of
administration, the duration of treatment, the specific composition
used and the health of the patient as known in the art. The skilled
artisan will be able to determine the optimum dosage.
[0123] By intranasally administering the compositions by the
methods described herein, it is realized that a smaller amount of
the composition may be administered compared to systemic
administration, including intravenous, oral, intramuscular,
intraperitoneal, transdermal, etc. The amount of active agent
and/or compositions required to achieve a desired clinical endpoint
or therapeutic effect when intranasally administered as described
herein may be less compared to systemic administration.
Additionally, upon administering the compositions intranasally in
the delivery and treatment methods described herein, about 5-fold
to about 500-fold, and further about 10-fold to about 100-fold,
less systemic exposure may be obtained compared to administration
of the same amount systemically. Furthermore, at least about
5-fold, further at least about 10-fold, preferably at least about
20-fold and further at least about 50-fold less systemic exposure
may be obtained compared to administration of the same amount
systemically. In determining the therapeutic effectiveness of the
compositions, clinical endpoints known to the art for the
particular disorder may be monitored. For example, suitable
clinical endpoints for dementia and Alzheimers' disease include,
for example, improved cognitive function, decreases in memory loss,
language deterioration, confusion, restlessness and mood swings;
and improved ability to mentally manipulate visual information as
determined by standard methods.
[0124] Suitable clinical endpoints for Huntington's disease include
a decrease in uncontrolled movements, and an improvement or no
further decrease of intellectual faculties.
[0125] Suitable clinical endpoints for Parkinson's disease include,
for example, a decrease in the characteristic tremor (trembling or
shaking) of a limb, especially when the body is at rest, an
increase in movement (to help overcome bradykinesia), improved
ability to move (to help overcome akinesia), less rigid limbs,
improvement in a shuffling gait, and an improved posture
(correcting the characteristic stooped posture). Such clinical
endpoints may be observed by standard methods. Other suitable
clinical endpoints include a decrease in nerve cell degeneration
and/or no further decline in nerve cell degeneration and may be
observed, for example, by brain imaging techniques, including
computer assisted tomography (CAT) scanning, magnetic resonance
imaging methods, or similar methods known to the art.
[0126] Suitable clinical endpoints for obesity include, for
example, a decrease in body weight, body fat, food intake or an
increase in lean body mass, metabolic rate or a combination
thereof.
[0127] Suitable clinical endpoints for sleep disorders, such as
insomnia, include, for example, an improvement in the ability to
sleep, and especially improved rapid eye movement (REM) sleep.
[0128] Suitable clinical endpoints for autoimmune disorders such as
multiple sclerosis include, for example, a decrease in the number
of brain lesions, increased extremity strength or a decreased in
tremors or paralysis of extremities. Decreases in the number of
brain lesions may be observed by brain imaging techniques
previously described herein. Other suitable clinical endpoints
include a decrease in inflammation of nervous tissue which may be
determined by, for example, lumbar puncture techniques and
subsequent analysis of cerebrospinal fluid known to the art.
[0129] In individuals who have experienced a stroke, a suitable
clinical endpoint includes an increase in blood flow in the
affected blood vessel as determined by computer tomographic methods
as known in the art and as described, for example, in Nabavi, D.
G., et al., Radiology 213:141-149 (1999). A further clinical
endpoint includes a decrease in numbness in the face, arm or leg;
or a decrease in the intensity of a headache associated with the
stroke. Yet another clinical endpoint includes a decrease in the
cell, tissue or organ damage or death due to the stroke. Such
decrease in cell or tissue damage may be assessed by brain imaging
techniques previously described herein, or similar methods known to
the art.
[0130] Suitable clinical endpoints in neuropsychologic disorders
such as schizophrenia include, for example, improvements in
abnormal behavior, and a decrease in hallucinations and/or
delusions.
[0131] The patient or subject treated according to the methods of
the present invention is typically one in need of such treatment,
including one that has a particular disorder amenable to treatment
by such methods. The patient or subject is typically a mammal, such
as a human, although other mammals may also be treated.
EXAMPLES
[0132] Reference will now be made to specific illustrative
examples. It is to be understood that the examples are provided to
illustrate preferred embodiments and that no limitation to the
scope is intended thereby. Additionally, all documents cited herein
are indicative of the level of skill in the art and are hereby
incorporated by reference in their entirety.
Example 1
Brain Distribution of .alpha.-Melanocyte Stimulating Hormone
Mimetibody after Intranasal Administration
[0133] This example shows that an .alpha.-melanocyte stimulating
hormone mimetibody (.alpha.-MSH mimetibody) is transported to
various regions in the brain and was detected at about 25 minutes
after intranasal administration while reducing systemic exposure
according to the methods of the present invention. The example
further shows that the .alpha.-MSH mimetibody delivered to the
brain is retained in the brain for at least up to 5 hours
post-delivery.
Methods
[0134] An .alpha.-MSH mimetibody was prepared, to serve as a model
and exemplary therapeutic compound to illustrate the claimed
method. The .alpha.-MSH mimetibody is a homo-dimeric fusion
molecule that consists of the therapeutic .alpha.-MSH polypeptide,
identified herein as SEQ ID NO:1, and the Fc portion of the human
immunoglobulin G1 (IgG1) monoclonal antibody. The engineered fusion
polypeptide was produced using recombinant DNA methods.
[0135] The .alpha.-MSH mimetibody was iodinated by Amersham
Biosciences's Iodine-125 Custom Labeling Services using the
Chloramine T method. .sup.125I-labeled .alpha.-MSH mimetibody,
together with unlabeled .alpha.-MSH mimetibody as a cold carrier,
was intranasally or intravenously administered to eight
anesthetized rats (Sprague Dawley, 200-250 g). Intranasal drug
administration was performed in the fume hood behind a
lead-impregnated shield. Each rat was placed on its back on a
heating pad with a 37.degree. C. rectal probe; the rat's head was
slightly elevated by rolled-up 4.times.4 gauze. The unlabeled
mimetibody, dissolved in PBS, was spiked with 39 .mu.Ci of
.sup.125-I labeled .alpha.-MSH mimetibody. A total volume of 100
.mu.l containing approximately 13 nmol or 0.8 mg of .alpha.-MSH
mimetibody was administrated in 10 .mu.l nose drops to alternating
nares every two minutes over a 15-20 minute time period to young
male rats while under anesthesia and lying on their back. For
intravenous administration, .sup.125I-labeled .alpha.-MSH
mimetibody was delivered as a bolus injection through the tail vein
in a total volume of 0.5 ml (diluted in saline). Rats were
administered either a full dose (equivalent to intranasal) or
1/10th of the intranasal dose (0.08 mg or 1.3 nmol .alpha.-MSH
mimetibody containing 39 .mu.Ci). Blood samples were taken every 5
minutes up to 25 minutes. At about 27 minutes or 5 hours after the
beginning of drug administration, the rats were perfused to remove
blood-borne label and fixed.
[0136] The distribution of .sup.125I-labeled .alpha.-MSH mimetibody
in the CNS and peripheral organs was assessed following intranasal
or intravenous delivery in rats. Tissue pieces from the brain,
organs and peripheral tissues were carefully excised, weighed and
gamma-counted. Concentrations of .alpha.-MSH mimetibody were
assessed using either gamma counting (quantitative analysis) or by
autoradiography of coronal brain section (qualitative analysis).
The nanomolar concentration in each tissue piece and in the blood
was determined based on the amount of counts per tissue weight and
specific activity of the radio-labeled protein.
Results
[0137] As seen in FIG. 1, the .sup.125I-labeled .alpha.-MSH
mimetibody can be detected in various CNS tissues after intranasal
delivery into young male rates within 25 minutes after
administration. FIG. 1 further shows that most of the
.sup.125I-labeled .alpha.-MSH mimetibody is retained at 5 hours
post-intranasal delivery, suggesting that the half-life of
.alpha.-MSH mimetibody is greater than 5 hours. It is more
specifically seen that the .sup.125I-labeled .alpha.-MSH mimetibody
reached the hypothalamus, the target site for action of the
.alpha.-MSH peptide (binding to MCR4 on hypothalamic neurons). In
addition, the hypothalamus (3 nM of mimetibody) is targeted with
intranasal delivery although there is significant delivery to all
brain regions, especially the medulla, pons and frontal cortex
[0138] Table 1 further compares the distribution of
.sup.125I-labeled .alpha.-MSH mimetibody administered intranasally
and intravenously.
TABLE-US-00002 TABLE 1 Distribution of .alpha.-MSH Mimetibody After
Intranasal and Intravenous Delivery Average Concentration of
.alpha.MSH- Mimetibody (nM) Intranasal Intravenous Tissue (13 n
mol) (1.3 n mol) blood sample 1 (5 min) 0.5 +/- 0.1 33.4 +/- 2.97
blood sample 2 (10 min) 1.6 +/- 0.2 35.5 +/- 3.18 blood sample 3
(15 min) 2.9 +/- 0.4 32.1 +/- 2.83 blood sample 4 (20 min) 4.6 +/-
0.7 34.1 +/- 3.0.4 blood sample 5 (25 min) 5.4 +/- 0.8 28.2 +/-
2.59 olfactory epithelium 17.1 +/- 1.6 1.8 +/- 0.16 olfactory bulb
16.2 +/- 5 0.2 +/- 0.02 trigeminal nerve 19.1 +/- 3.4 0.5 +/- 0.03
frontal cortex 1.3 +/- 0.3 0.2 +/- 0.02 Medulla 1.8 +/- 0.4 0.1 +/-
0.01 Hypothalamus 3.0 .+-. 0.4 0.4 .+-. 0.06 Liver 1.8 +/- 1.0 18.9
+/- 1.59 Kidney 3.3 +/- 0.5 5.7 +/- 0.58 Spleen 1.2 +/- 0.2 3.9 +/-
0.76
[0139] Intravenous delivery also targets the hypothalamus. However,
despite the 13.5 higher blood exposure (AUC) with intravenous
administration (see Table 1 and FIGS. 1 and 2), intranasal
administration results in greater CNS delivery. Delivery of the
peptide to the hypothalamus, frontal cortex, and medulla were 7.5,
6.5 and 18 fold higher, respectively, with intranasal than
intravenous administration.
[0140] Table 2 shows the relative effectiveness of intranasal
(i.n.) and intravenous (i.v.) delivery by comparing various ratios
of polypeptide tissue concentrations. Specifically, the ratio of
polypeptide concentration in the hypothalamus to polypeptide
concentration in the blood at 25 minutes post delivery is shown in
Table 2, for both intranasal and intravenous delivery. The ratio of
polypeptide concentration in the hypothalamus to polypeptide
concentration in the liver at 25 minutes post delivery is also
shown in Table 2, for both intranasal and intravenous delivery.
Intranasal delivery was significantly more effective, as evidenced
by the 48 and 75 fold ratios, to deliver the polypeptide to the
hypothalamus than was intravenous delivery.
TABLE-US-00003 TABLE 2 Relative effectiveness of intranasal and
intravenous delivery in targeting the hypothalamus Ratio i.n.* i.v*
(i.n.)/(i.v.)
[polypeptide].sub.hypothalamus/[polypeptide].sub.blood 0.558 0.012
48 [polypeptide].sub.hypothalamus/[polypeptide].sub.liver 1.640
0.022 75 *i.n. = intranasal; i.v. = intravenous
[0141] The data in Table 1 and FIG. 2 also show that systemic
exposure of the .sup.125I-labeled .alpha.-MSH mimetibody was low
when administered intransally. An intranasal one-tenth the amount
of the intravenous dose resulted in a 13.5-fold lower systemic
exposure, based on the blood AUC (intravenous)/AUC (intranasal)
ratio, and a 10.5-fold lower exposure based on a ratio of liver
protein concentration when dosed intravenously to the liver protein
concentration when dosed intranasally. Further. a consistent depot
of the mimetibody (17.1+/-uM) was created in the olfactory
epithelium across the 14 animals (see Table 1 above), and olfactory
and trigeminal pathway concentrations of the test protein were
similar upon intranasal administration indicating that the protein
travels to the CNS via the olfactory and trigeminal neural
pathways. Comparing equal intranasal and intravenous doses,
systemic exposure was about 96-fold lower based on blood AUC
(i.v.)/AUC (i.n.) ratio with approximately equal amounts of protein
delivered to the CNS and hypothalamus.
[0142] FIG. 3 shows that delivery of the .sup.125I-labeled
.alpha.-MSH mimetibody to the central nervous system is unlikely to
be secondary through the blood. For example, as seen in FIG. 3,
when rats are exposed to a 10-fold higher dosage of
.sup.125I-labeled .alpha.-MSH mimetibody by intranasal
administration compared to intravenous administration, there was a
higher accumulation of the .sup.125I-labeled .alpha.-MSH mimetibody
in the central nervous system by intranasal administration.
[0143] FIGS. 4A-4D show computer-generated autoradiographs of
coronal sections of the rat brains 25 minutes after administration
of .sup.125I-.alpha.-MSH mimetibody intranasally (FIGS. 4A, 4C) or
intravenously (FIGS. 4B, 4D). The darkened area in the
autoradiographs corresponds to the regions of high image intensity,
which correlates to regions of fusion polypeptide delivery. As seen
in FIGS. 4A, 4C, which correspond to the animals treated
intranasally, the highest image intensities were observed in the
olfactory tracts, hypothalamus, and frontal cortex. These images
confirm findings from quantitative measurements.
Example 2
Dose-Dependent Reduction in Cumulative Food Intake in Normal Rats
after Intranasal Administration of Alpha-MSH
[0144] This example shows that intranasal administration of a
single dose of the N-acetylated .alpha.-melanocyte stimulating
hormone
(Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2, SEQ ID
NO:1, supplied by Phoenix Pharmaceuticals, INC) was sufficient to
achieve a dose dependent, pharmacodynamic response; specifically, a
reduction of cumulative food intake, with an ED.sub.50 at 24 hours
of 6-7 nmol.
Methods
[0145] Two groups of nine rats each were assembled. In a cross-over
design, each week one group was dosed with a phosphate buffered
saline (PBS) vehicle and the other group was dosed with .alpha.-MSH
peptide; the following week the treatment administered to each
group was reversed. Prior to the study, the light cycle was slowly
reversed, within a 2 weeks acclimation period. Rats were fasted for
24 hours prior to each experiment (water was always available), and
received anesthesia 30 minutes prior to the beginning of the dark
cycle (or the period of lights off). A single dose of drug ranging
from 2.5 to 50 nmols or phosphate-saline buffered vehicle control
was intranasally administered during anesthesia over approximately
20 minutes, similar to the procedure set forth in Example 1. Rats
were placed on their backs on a heating pad and monitored until
they become active, and then were placed in their cages with
pre-weighed amounts of food. Food intake measurements were taken at
2, 4, 8, 24, 48 and 72 hours. Water intake and body weight were
determined at 24 and 48 hours post-dosing.
Results and Conclusions
[0146] As seen in FIG. 5, intranasal .alpha.-MSH peptide reduces
cumulative food intake dose dependently between 2.5-25 nmols at 24
hours with an ED.sub.50 at 6-7 nmols.
[0147] As shown in FIG. 6, a single dose of 25-50 nmol was
maximally effective in reducing percent cumulative food intake. The
25 nmol dose reduced cumulative food consumption by 30% at 2 hours,
by 18% at 8 hours, and by 9% at 24 hours. Water consumption and
body weight remained unchanged. This study shows a dose dependent
pharmacodynamic effect of a polypeptide after intranasal
administration to a mammal.
Example 3
Reduction in Cumulative Food Intake in Normal Rats after Intranasal
Administration of Alpha-MSH Mimetibody
[0148] This example shows that intranasal administration of a
single dose of 25 nmols (5 mg/kg) of the .alpha.-MSH mimetibody is
sufficient to reduce cumulative food intake significantly at 8 and
24 hours. Water consumption and body weight remained unchanged.
Methods
[0149] The study protocol and methods used were the same as
described in Example 2. The total number of rats was 14.
Results and Conclusions
[0150] As seen in FIG. 7, a single dose of 25 nmol of intranasally
delivered alpha-MSH mimetibody had a significant effect on
decreasing cumulative food intake at 8 and 24 hours, with a
non-statistically significant trend toward reduction at 48 and 72
hours. The significance at the later time points was likely lost
due to the relatively small number of animals used in the study
(n=14). The study shows that a 62 kDa large protein, like the
.alpha.-MSH mimetibody, can be delivered to the CNS via the nasal
route of administration.
[0151] While a number of exemplary aspects and embodiments have
been discussed above, those of skill in the art will recognize
certain modifications, permutations, additions and sub-combinations
thereof. It is therefore intended that the following appended
claims and claims hereafter introduced are interpreted to include
all such modifications, permutations, additions and
sub-combinations as are within their true spirit and scope.
Example 4
Nasal Administration of Formulations Comprising 5% (v/v)
Tetrahydrofurfuryl-Polyethylenglycol or 1% (w/v) Sodium
Glycocholate Hydrate Increase Antibody Fragment Delivery to Central
Nervous System Tissues
[0152] Nasal administration of a Homo sapiens derived antibody Fc
fragment (hFc) to central nervous system (CNS) tissues was
increased, relative to controls, by inclusion of either 5% (v/v)
tetrahydrofurfuryl-polyethylenglycol or 1% (w/v) sodium
glycocholate hydrate in the hFc formulation (FIG. 8).
[0153] A rat model was used for evaluating intranasal drug delivery
to the brain. Male Sprague-Dawley rats were anesthetized with 40 mg
of sodium pentobarbital (NEMBUTAL.RTM.) per kg of animal body
weight administered intraperitoneally (i.p.) or 3.0% isoflurane
inhalant to facilitate nasal administration of the liquid
formulations comprising hFc. Anesthetized rats were placed on their
backs and their necks were supported with a neck pillow to elevate
their heads at which point 60 .mu.L of the liquid formulations were
administered drop-wise to alternating nostrils of each animal every
2 minutes as described by Thorne and Frey (Thorne et al, 127
Neuroscience 481 (2004)). A heating pad was used to maintain body
temperature of anesthesized animals.
[0154] 25 minutes after start of nasal administration of the hFc
formulations animals were perfused via cardiac puncture with
phosphate buffered saline (PBS) containing protease inhibitors (1
protease inhibitor cocktail tablet/Roche per 10 ml of PBS) and 5 nM
EDTA tetrasodium salt. Brains and other tissues were dissected and
collected for analysis of hFc content. Blood was collected prior to
the study, which was typically one day prior to nasal
administration of the hFc formulations, and prior to the start of
perfusion. Tissues were homogenized in a homogenization buffer
containing protease inhibitors (1 protease inhibitor cocktail
tablet/Roche per 10 ml of PBS), 5 nM EDTA tetrasodium salt, and 1%
(v/v) Tween 20. Homogenized tissue samples or blood were then
centrifuged and supernatants were collected for hFc assays. hFc
concentrations were determined in tissue homogenate and blood
plasma samples, using standard ELISA assay techniques specific for
Homo sapiens antibody Fc chains of the IgG1 isotype. hFc is a Homo
sapiens antibody fragment comprising a Fc fragment of the IgG1
isotype and has a molecular weight of 52,862 Dal and was purified
using standard techniques after expression by eukaryotic cells.
[0155] Control hFc formulations comprised 30 mg/ml hFc in PBS at pH
7.4. Tetrahydrofurfuryl-polyethylenglycol comprising hFc
formulations contained 5% (v/v; e.g. 5 ml
tetrahydrofurfuryl-polyethylenglycol diluted to 100 ml PBS buffer)
tetrahydrofurfuryl-polyethylenglycol in PBS buffer at pH 7.4 and 30
mg/ml hFc. Sodium glycocholate hydrate comprising hFc formulations
contained 1% (w/v; e.g. 1 g of 97% pure sodium glycocholate hydrate
in 100 ml of PBS buffer diluent) sodium glycocholate hydrate in PBS
buffer at pH 7.4 and 30 mg/ml hFc.
[0156] Percent (w/v) and percent (v/v) values reported herein are
uncorrected for the purity by mass or volume of a given compound in
a formulation. For example, 100 ml of a 1% (w/v) sucrose solution
made from a 97% pure sucrose preparation would contain only 0.97
gram of sucrose in 100 ml. This reflects the practical fact that
for many compound preparations the actual purity by mass or volume
of a given compound in the preparation is not known with absolute
certainty, but is instead only known within the limits of the assay
techniques used. Furthermore, due to the heterogenous nature of
some compounds such as polymers and hydrates it is often difficult
to describe the purity of the compound by mass because the mass of
the compounds in the preparation, such as polymers, or the mass and
stoichiometry of hydrate in the preparation is variable or
undetermined.
[0157] Sodium glycocholate hydrate was from Sigma-Aldrich (St.
Louis, Mo.) and had a purity of greater than 97% by mass (Table 3).
"Sodium glycocholate" is a shorthand designation or nomenclature
used in the Figures, and Tables to refer to "sodium glycocholate
hydrate."
[0158] Tetrahydrofurfuryl-polyethylenglycol was from Alchymars SpA
(Milano, Italy) (Table 3). "Glycofurol" is a shorthand designation
or nomenclature used in the Figures and Tables to refer to
"tetrahydrofurfuryl polyethylenglycol."
[0159] As seen in FIG. 8, nasal administration of hFc formulations
comprising 5% (v/v) tetrahydrofurfuryl-polyethylenglycol or 1%
(w/v) sodium glycocholate hydrate to isoflurane anesthetized
animals resulted in hFc concentrations in CNS tissues that were
higher than those in isoflurane anesthetized animals receiving the
control hFc formulation.
[0160] In FIG. 8 "sodium glycocholate" is used to designate "sodium
glycocholate hydrate" and "glycofurol" is used to designate
"tetrahydrofurfuryl-polyethylenglycol." Abbreviations used in FIG.
8 are as follows: "iso"=isoflurane; "pento"=pentobarbital;
OB=olfactory bulb; LH=Ieft brain hemisphere; RH=right brain
hemisphere; CB=cerebellum; BS=brain stem; TN=trigeminal nerve.
Error bars represent standard errors for a given data set. Mean
values are indicated above off-scale bars as necessary. The number
of animals in each group is as indicated in FIG. 8.
Example 5
Nasal Administration of Formulations Comprising 1% (w/v) Sodium
Glycocholate Hydrate Permits Delivery of Biologically Active, High
Molecular Weight Enzymes to Central Nervous System Tissues
[0161] Nasal administration of a biologically active, high
molecular weight enzyme to central nervous system (CNS) tissues was
achieved (FIG. 9). As shown in FIG. 9, delivery of the 32,000 Da
beta-lactamase enzyme to CNS tissues was increased, relative to
controls, by the inclusion of 1% (w/v) sodium glycocholate hydrate
in the nasally administered formulations comprising this enzyme
(FIG. 9).
[0162] Formulations comprising the beta-lactamase enzyme were
nasally administered to anesthetized rats using the methods
described in Example 4 above; 60 .mu.L of formulation was
administered. Tissue and plasma samples were also prepared as
described in Example 4. Beta-lactamase enzyme activity and
concentrations were determined in tissue homogenate and blood
plasma samples, using standard fluorescent substrate based assay
techniques.
[0163] Beta-lactamase is an enzyme that hydrolyzes the beta-lactam
ring in certain antibiotics containing beta-lactam rings (e.g.
penicillin). Beta-lactamase has a molecular weight of about 32,000
Da.
[0164] Control beta-lactamase formulations comprised 20 mg/ml
beta-lacatamase in PBS at pH 7.4. Sodium glycocholate hydrate
comprising beta-lactamase formulations contained either 1% (w/v;
e.g. 1 g of 97% pure sodium glycocholate hydrate in 100 ml of PBS
buffer diluent) sodium glycocholate hydrate or 0.1% sodium
glycocholate hydrate in PBS buffer at pH 7.4 and 20 mg/ml
beta-lactamase. Percent (w/v) and percent (v/v) values reported
herein are uncorrected for the purity by mass or volume of a given
compound in a formulation.
[0165] Sodium glycocholate hydrate was from Sigma (St. Louis, Mo.)
and had a purity of greater than 97% by mass (Table 3). "Sodium
glycocholate" is a shorthand designation or nomenclature used in
the Figures and Tables to refer to "sodium glycocholate
hydrate."
[0166] As seen in FIG. 9, nasal administration of a biologically
active, high molecular weight enzyme to central nervous system
(CNS) tissues was achieved. Furthermore, nasal administration of
beta-lactamase formulations comprising 0.1-1% (w/v) sodium
glycocholate hydrate to isoflurane anesthetized animals resulted in
beta-lactamase concentrations in CNS tissues that were higher than
those in isoflurane anesthetized animals receiving the control
beta-lactamase formulation in PBS only.
[0167] Abbreviations and compound designations used in FIG. 9 are
the same as described for FIG. 8 in Example 4 above. Error bars
represent standard deviations for a given data set. Mean values are
indicated in FIG. 9 above off-scale bars as necessary. P-values
used for statistical analyses are indicated in FIG. 9. The number
of animals in each group is as indicated in FIG. 9.
Example 6
Nasal Administration of Formulations Comprising from 0.125% (w/v)
to 0.5% (w/v) of N-Decyl-Beta-D-Maltopyranoside,
N-Dodecyl-Beta-D-Maltopyranoside,
N-Tetradecyl-Beta-D-Maltopyranoside, or
Beta-D-Fructopyranosyl-Alpha-Glucopyranoside Monododecanoate
Increase Antibody Fragment Delivery to Central Nervous System
Tissues
[0168] Nasal administration of a Homo sapiens derived antibody Fc
fragment (hFc) to central nervous system (CNS) tissues was
increased, relative to controls, by inclusion of 0.125% (w/v) to
0.5% (w/v) of n-decyl-beta-D-maltopyranoside,
n-dodecyl-beta-D-maltopyranoside,
n-tetradecyl-beta-D-maltopyranoside, or
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate in the
hFc formulation (FIG. 10).
[0169] A rat model was used for evaluating intranasal drug delivery
to the brain. Male Sprague-Dawley rats were anesthetized with 3.0%
isoflurane inhalant to facilitate nasal administration of the
liquid formulations comprising hFc. Anesthetized rats were placed
on their backs and their necks and head were supported with a
platform to elevate their heads at a 45.degree. angle. A
micro-cannula was then inserted 1 cm into the right nostril of the
animals and a 50 .mu.L bolus of the liquid formulations was
administered over the span of 1 minute. A heating pad was used to
maintain body temperature of anesthesized animals.
[0170] 20 minutes after nasal administration of the hFc
formulations, animals were perfused via cardiac puncture with
phosphate buffered saline (PBS) containing protease inhibitors (1
Roche protease inhibitor cocktail tablet per 10 ml of PBS) and 5 nM
EDTA tetrasodium salt. Brains, blood and other tissues were then
dissected, collected, and analyzed for hFc content as described in
Example 4 above.
[0171] Control hFc formulations comprised 36 mg/ml hFc in PBS at pH
7.4. n-decyl-beta-D-maltopyranoside comprising hFc formulations
contained 0.125% (w/v) to 0.5% (w/v) of
n-decyl-beta-D-maltopyranoside in PBS buffer at pH 7.4 and 36 mg/ml
hFc. n-dodecyl-beta-D-maltopyranoside comprising hFc formulations
contained 0.125% (w/v) to 0.5% (w/v) of
n-dodecyl-beta-D-maltopyranoside in PBS buffer at pH 7.4 and 36
mg/ml hFc. n-tetradecyl-beta-D-maltopyranoside comprising hFc
formulations contained 0.125% (w/v) to 0.5% (w/v) of
n-tetradecyl-beta-D-maltopyranoside in PBS buffer at pH 7.4 and 36
mg/ml hFc. Beta-D-fructopyranosyl-alpha-glucopyranoside
monododecanoate comprising hFc formulations contained 0.125% (w/v)
to 0.5% (w/v) of beta-D-fructopyranosyi-alpha-glucopyranoside
monododecanoate in PBS buffer at pH 7.4 and 36 mg/ml hFc. Percent
(w/v) and percent (v/v) values reported herein are uncorrected for
the purity by mass or volume of a given compound in a
formulation.
[0172] n-decyl-beta-D-maltopyranoside (Table 3) was obtained from
Aegis Therapeutics, LLC (San Deigo, Calif.).
n-dodecyl-beta-D-maltopyranoside was obtained from Inalco SpA
(Milano, Italy) and had a purity of greater than 99% by mass (Table
3). n-tetradecyl-beta-D-maltopyranoside was from Inalco SpA
(Milano, Italy) and had a purity of greater than 99% by mass.
Beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate was
from Anatrace, Inc. (Maumee, Ohio) and had a purity of 98.90% by
mass.
[0173] As seen in FIG. 10, nasal administration of hFc formulations
comprising 0.125% (w/v) to 0.5% (w/v) of
n-decyl-beta-D-maltopyranoside, n-dodecyl-beta-D-maltopyranoside,
1-O-n-tetradecyl-beta-D-maltopyranoside, or
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate to
isoflurane anesthetized animals resulted in hFc concentrations in
CNS tissues that were higher than those in isoflurane anesthetized
animals receiving the control hFc formulation.
[0174] In FIG. 10, the designation "A1" is used for
n-decyl-beta-D-maltopyranoside formulations. "A3" is used for
n-dodecyl-beta-D-maltopyranoside formulations. "A5" is used for
n-tetradecyl-beta-D-maltopyranoside. "B3" is used for
beta-D-fructopyranosyl-alpha-glucopyranoside monododecanoate
formulations. Otherwise, abbreviations and designations used in
FIG. 10 are the same as described for FIG. 8 in Example 4 above.
Error bars represent standard errors for a given data set. Mean
values are indicated in FIG. 10 above off-scale bars as necessary.
The number of animals in each group is as indicated in FIG. 10.
Example 7
Nasal Administration of Formulations that Increase Antibody
Fragment Delivery to Central Nervous System Tissues
[0175] Nasal administration of a Homo sapiens derived antibody Fc
fragment (hFc) to central nervous system (CNS) tissues was
increased, relative to control hFc formulations, by inclusion of
the compounds listed in Table 3 below in the hFc formulation.
TABLE-US-00004 TABLE 3 Antibody hFc formulations that increase
antibody fragment delivery to central nervous system tissues,
relative to control hFc formulations, in isoflurane anesthetized
animals. Compound Identity and Quantity in PBS Mean Fold Increase*
in Brain (pH 7.4) Diluent Based Formulations Delivery Efficiency of
hFc after Containing 36 mg/ml hFc Nasal Administration of Compound
Identity Quantity Formulation chitosan glutamate 0.5% (w/v) 6.1**
(n = 23) n-dodecyl-beta-D- 0.5% (w/v) 7.6*** (n = 21)
maltopyranoside n-decyl-beta-D- 0.5% (w/v) 4.3 (n = 7)
maltopyranoside propylene glycol 10-20% (v/v) 4.5**** (n = 13)
heptakis(2,6-di-O- 5% (w/v) 3.1 (n = 8) methyl)-beta- cyclodextrin
1,2-didecanoyl-sn- 2% (w/v) 9.6 (n = 6) glycero-3- emulsion in
phosphocholine 20% v/v soy bean oil, 2% v/v glycerol sodium
glycocholate 1% (w/v) 17.6 (n = 15) hydrate taurocholic acid 1%
(w/v) 14 (n = 6) sodium salt hydrate Sodium 1% (w/v) 4.0 (n = 6)
taurourso- deoxycholate *represents mean fold increase in brain
delivery efficiency (DE) of nasally administered hFc relative to
control formulation (PBS; pH 7.4 and 36 mg/ml hFc) **4 studies,
range 3-11 fold increase ***4 studies, range 6-9 fold increase
****10 and 20% PG, n = 6, range 4-10 fold increase *****3 studies,
range 16-19 fold increase
[0176] Formulations comprising the compounds identified in Table 3
above were nasally administered to anesthetized rats using the
methods described in Example 6 above; a 50 .mu.L of bolus of each
formulation was administered. Animals were perfused as described in
Example 6 above. Brains, blood and other tissues were then
dissected, collected, and analyzed for hFc content as described in
Example 4 above.
[0177] Control hFc formulations comprised 36 mg/ml hFc in PBS at pH
7.4.
[0178] The formulations described in Table 3 above contained each
identified compound preparation in the quantity indicated in the
Table. The purity by mass of compounds in compound preparations and
their source is as indicated in Table A above. Percent (w/v) and
percent (v/v) values reported herein are uncorrected for the purity
by mass or volume of a given compound in a formulation.
Formulations were made using PBS at pH 7.4 as the diluent and
contained a final hFc concentration of 36 mg/ml.
[0179] As seen in Table 4, nasal administration of hFc formulations
comprising 0.5% (w/v) chitosan glutamate, 0.5%
(w/v)-n-dodecyl-beta-D-maltopyranoside, 0.5% (w/v)
n-decyl-beta-D-maltopyranoside, 10% to 20% (v/v) propylene glycol,
5% (w/v) heptakis(2,6-di-O-methyl)-beta-cyclodextrin, 2% (w/v)
1,2-didecanoyl-sn-glycero-3-phosphocholine, 1% (w/v) sodium
glycocholate hydrate; 1% (w/v) taurocholic acid sodium salt
hydrate, or 1% (w/v) sodium tauroursodeoxycholate to isoflurane
anesthetized animals resulted in hFc concentrations in CNS tissues
that were increased relative to those in isoflurane anesthetized
animals receiving the control hFc formulation in PBS only. Mean
fold increase values and the number of animals in each study group
are indicated in Table 3.
[0180] The present invention now being fully described, it will be
apparent to one of ordinary skill in the art that changes and
modifications can be made thereto without departing from the scope
and spirit of the appended claims.
Sequence CWU 1
1
3113PRTHomo sapiens 1Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys
Pro Val1 5 102331PRTHomo sapiens 2Val Asn Ser Thr His Arg Gly Met
His Thr Ser Leu His Leu Trp Asn1 5 10 15Arg Ser Ser Tyr Arg Leu His
Ser Asn Ala Ser Glu Ser Leu Gly Lys20 25 30Gly Tyr Ser Asp Gly Gly
Cys Tyr Glu Gln Leu Phe Val Ser Pro Glu35 40 45Val Phe Val Thr Leu
Gly Val Ile Ser Leu Leu Glu Asn Ile Leu Val50 55 60Ile Val Ala Ile
Ala Lys Asn Lys Asn Leu His Ser Pro Met Tyr Phe65 70 75 80Phe Ile
Cys Ser Leu Ala Val Ala Asp Met Leu Val Ser Val Ser Asn85 90 95Gly
Ser Glu Thr Ile Ile Ile Thr Leu Leu Asn Ser Thr Asp Thr Asp100 105
110Ala Gln Ser Phe Thr Val Asn Ile Asp Asn Val Ile Asp Ser Val
Ile115 120 125Cys Ser Ser Leu Leu Ala Ser Ile Cys Ser Leu Leu Ser
Ile Ala Val130 135 140Asp Arg Tyr Phe Thr Ile Phe Tyr Ala Leu Gln
Tyr His Asn Ile Met145 150 155 160Thr Val Lys Arg Val Gly Ile Ile
Ile Ser Cys Ile Trp Ala Ala Cys165 170 175Thr Val Ser Gly Ile Leu
Phe Ile Ile Tyr Ser Asp Ser Ser Ala Val180 185 190Ile Ile Cys Leu
Ile Thr Met Phe Phe Thr Met Leu Ala Leu Met Ala195 200 205Ser Leu
Tyr Val His Met Phe Leu Met Ala Arg Leu His Ile Lys Arg210 215
220Ile Ala Val Leu Pro Gly Thr Gly Ala Ile Arg Gln Gly Ala Asn
Met225 230 235 240Lys Gly Ala Ile Thr Leu Thr Ile Leu Ile Gly Val
Phe Val Val Cys245 250 255Trp Ala Pro Phe Phe Leu His Leu Ile Phe
Tyr Ile Ser Cys Pro Gln260 265 270Asn Pro Tyr Cys Val Cys Phe Met
Ser His Phe Asn Leu Tyr Leu Ile275 280 285Leu Ile Met Cys Asn Ser
Ile Ile Asp Pro Leu Ile Tyr Ala Leu Arg290 295 300Ser Gln Glu Leu
Arg Lys Thr Phe Lys Glu Ile Ile Cys Cys Tyr Pro305 310 315 320Leu
Gly Gly Leu Cys Asp Leu Ser Ser Arg Tyr325 3303245PRTArtificial
SequenceSynthetic alpha-MSH mimetibody without signal sequence
which comprises an antibody Fc fragment peptide chain and which
binds Homo sapiens MCR4 and other melanocortin receptors. 3Ser Tyr
Ser Cys Glu His Phe Arg Trp Cys Lys Pro Val Gly Gly Gly1 5 10 15Gly
Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Ala20 25
30Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr35
40 45Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val50 55 60Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val65 70 75 80Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser85 90 95Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu100 105 110Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser115 120 125Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro130 135 140Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln145 150 155 160Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala165 170
175Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr180 185 190Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu195 200 205Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser210 215 220Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser225 230 235 240Leu Ser Leu Gly
Lys245
* * * * *