U.S. patent application number 11/562913 was filed with the patent office on 2007-07-12 for a device for enhanced epithelial permeation of y2 receptor-binding peptides.
This patent application is currently assigned to Nastech Pharmaceutical Company Inc.. Invention is credited to Gordon Brandt, Mary S. Kleppe, Conor J. MacEvilly, Steven C. Quay.
Application Number | 20070161563 11/562913 |
Document ID | / |
Family ID | 34886593 |
Filed Date | 2007-07-12 |
United States Patent
Application |
20070161563 |
Kind Code |
A1 |
Quay; Steven C. ; et
al. |
July 12, 2007 |
A DEVICE FOR ENHANCED EPITHELIAL PERMEATION OF Y2 RECEPTOR-BINDING
PEPTIDES
Abstract
A pharmaceutical device comprising a composition comprising an
aqueous solution of PYY(3-36), a cyclodextrin, and a compound
selected from phosphatidylcholine or diglyceride, wherein the
cyclodextrin and the compound selected from phosphatidylcholine or
diglyceride are present in an amount sufficient to enhance
epithelial permeation, and wherein the composition is present in a
container; and an actuator fluidly connected to the container,
wherein the actuator has a tip which defines a passage through
which the solution is ejected to produce a spray of the
solution.
Inventors: |
Quay; Steven C.;
(Woodinville, WA) ; Brandt; Gordon; (Issaquah,
WA) ; Kleppe; Mary S.; (Snohomish, WA) ;
MacEvilly; Conor J.; (Seattle, WA) |
Correspondence
Address: |
NASTECH PHARMACEUTICAL COMPANY INC
3830 MONTE VILLA PARKWAY
BOTHELL
WA
98021-7266
US
|
Assignee: |
Nastech Pharmaceutical Company
Inc.
|
Family ID: |
34886593 |
Appl. No.: |
11/562913 |
Filed: |
November 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10780325 |
Feb 17, 2004 |
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11562913 |
Nov 22, 2006 |
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10745069 |
Dec 23, 2003 |
7186691 |
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10780325 |
Feb 17, 2004 |
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10322266 |
Dec 17, 2002 |
7166575 |
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10745069 |
Dec 23, 2003 |
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60493226 |
Aug 7, 2003 |
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60501170 |
Sep 8, 2003 |
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60510785 |
Oct 10, 2003 |
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60517290 |
Nov 4, 2003 |
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60518812 |
Nov 10, 2003 |
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Current U.S.
Class: |
514/5.2 ; 514/23;
514/53; 514/54; 514/58; 514/61; 514/738; 604/500 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 9/0043 20130101; A61P 25/00 20180101; A61K 38/22 20130101;
A61P 3/10 20180101; A61P 9/00 20180101; A61K 31/724 20130101; A61K
31/715 20130101; A61P 3/04 20180101; A61P 15/00 20180101; A61K
31/7012 20130101; A61K 31/70 20130101; A61K 31/045 20130101; A61P
9/10 20180101; A61P 35/00 20180101; A61P 19/02 20180101 |
Class at
Publication: |
514/012 ;
514/054; 514/058; 514/023; 514/061; 514/053; 604/500; 514/738 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61K 31/70 20060101 A61K031/70; A61K 31/7012 20060101
A61K031/7012; A61K 31/715 20060101 A61K031/715; A61K 31/045
20060101 A61K031/045; A61K 31/724 20060101 A61K031/724 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 17, 2003 |
US |
PCT/US03/40538 |
Claims
1. A pharmaceutical device comprising: a. a composition comprising
an aqueous solution of PYY(3-36), a cyclodextrin, and a compound
selected from phosphatidylcholine or diglyceride, wherein the
cyclodextrin and the compound selected from phosphatidylcholine or
diglyceride are present in an amount sufficient to enhance
epithelial permeation, and wherein the composition is present in a
container; b. an actuator fluidly connected to the container,
wherein the actuator has a tip which defines a passage through
which the solution is ejected to produce a spray of the
solution.
2. The device of claim 1, wherein the phosphatidylcholine is
L-.alpha.-phosphatidylcholine didecanoyl (DDPC).
3. The device of claim 1, wherein the cyclodextrin is selected from
the group consisting of hydroxypropyl-.beta.-cyclodextrin,
sulfobutylether-.beta.-cyclodextrin, and
methyl-.beta.-cyclodextrin.
4. The device of claim 3, wherein the cyclodextrin is
methyl-.beta.-cyclodextrin.
5. The device of claim 1, wherein the solution has a pH of from
about 3 to about 6.
6. The device of claim 5, wherein the pH is 5.0.+-.0.5.
7. The device of claim 1, the composition further comprising a
polyol.
8. The device of claim 7, wherein the polyol is selected from the
group consisting of sucrose, mannitol, sorbitol, lactose,
L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, trehalose,
D-galactose, lactulose, cellobiose, gentibiose, glycerin and
polyethylene glycol.
9. The device of claim 7, wherein the polyol is sorbitol.
10. The device of claim 7, further comprising a chelating
agent.
11. The device of claim 10, wherein the chelating agent is ethylene
diamine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid
(EGTA).
12. The device of claim 10, wherein the chelating agent is ethylene
diamine tetraacetic acid (EDTA).
13. The device of claim 7, further comprising one or more sustained
release-enhancing agent(s).
14. The device of claim 13, wherein the sustained release-enhancing
agent is polyethylene glycol (PEG).
15. The device of claim 1, wherein the PYY(3-36) is present in
solution at a concentration of at least 200 .mu.g/mL.
16. The device of claim 1, wherein the actuator produces a spray
having a volume of between 20 and 200 microliters per
actuation.
17. The device of claim 1, wherein the spray is comprised of
droplets, and wherein less than 10% of the droplets are smaller
than 10 microns in size.
18. The device of claim 17, wherein less than 5% of the droplets
are smaller than 10 microns in size.
19. The device of claim 17, wherein less than 1% of the droplets
are smaller than 10 microns in size.
20. The device of claim 17, wherein the droplets are between 25 and
700 micrometers in size.
21. The device of claim 1, wherein each spray from the actuator
contains at least 100 micrograms of PYY(3-36).
22. The device of claim 1, wherein the spray has a spray pattern
having major and minor axes of from 10 to 50 mm when measured at a
height of from 0.5 cm to 10 cm from the actuator tip.
23. The device of claim 1, wherein the spray is an aerosol with a
spray pattern ellipticity ratio of from 1.00 to 1.40 when measured
at a height of from 0.5 cm to 10 cm from the actuator tip.
24. The device of claim 23, wherein the ellipticity ratio is from
1.00 to 1.30.
25. The device of claim 23, wherein the ellipticity ratio is from
1.15 to 1.25.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional claiming the benefit under
35 U.S.C. .sctn. 120 of U.S. patent application Ser. No.
10/780,325, filed Feb. 17, 2004, which is a continuation
application and claims priority under 35 U.S.C. .sctn.120 of
co-pending U.S. patent application Ser. No. 10/745,069 filed Dec.
23, 2003, which is a continuation-in-part of U.S. patent
application Ser. No. 10/322,266, filed Dec. 17, 2002, and claims
priority under 35 U.S.C. .sctn.119 (e) of U.S. Provisional
Application No. 60/493,226, filed Aug. 7, 2003, U.S. Provisional
Application No. 60/501,170, filed Sep. 8, 2003, U.S. Provisional
Application No. 60/510,785, filed Oct. 10, 2003, U.S. Provisional
Application No. 60/517,290, filed Nov. 4, 2003; U.S. Provisional
Application No. 60/518,812, filed on Nov. 10, 2003; and
PCT/US03/40538, filed on Dec. 17, 2003; the entire contents of
these applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The teachings of all the references cited in the present
specification are incorporated in their entirety by reference.
[0003] Obesity and its associated disorders are common and very
serious public health problems in the United States and throughout
the world. Upper body obesity is the strongest risk factor known
for type-2 diabetes mellitus, and is a strong risk factor for
cardiovascular disease. Obesity is a recognized risk factor for
hypertension, arteriosclerosis, congestive heart failure, stroke,
gallbladder disease, osteoarthritis, sleep apnea, reproductive
disorders such as polycystic ovarian syndrome, cancers of the
breast, prostate, and colon, and increased incidence of
complications of general anesthesia. It reduces life-span and
carries a serious risk of co-morbidities above, as well disorders
such as infections, varicose veins, acanthosis nigricans, eczema,
exercise intolerance, insulin resistance, hypertension
hypercholesterolemia, cholelithiasis, orthopedic injury, and
thromboembolic disease. Obesity is also a risk factor for the group
of conditions called insulin resistance syndrome, or "Syndrome
X."
[0004] It has been shown that certain peptides that bind to the Y2
receptor when administered peripherally to a mammal induce weight
loss. The Y2 receptor-binding peptides are neuropeptides that bind
to the Y2 receptor. Neuropeptides are small peptides originating
from large precursor proteins synthesized by peptidergic neurons
and endocrine/paracrine cells. Often the precursors contain
multiple biologically active peptides. There is great diversity of
neuropeptides in the brain caused by alternative splicing of
primary gene transcripts and differential precursor processing. The
neuropeptide receptors serve to discriminate between ligands and to
activate the appropriate signals. These Y2 receptor-binding
peptides belong to a family of peptides including peptide YY (PYY),
neuropeptide Y (NPY) and pancreatic peptide (PP).
[0005] NPY is a 36-amino acid peptide and is the most abundant
neuropeptide to be identified in mammalian brain. NPY is an
important regulator in both the central and peripheral nervous
systems and influences a diverse range of physiological parameters,
including effects on psychomotor activity, food intake, central
endocrine secretion, and vasoactivity in the cardiovascular system.
High concentrations of NPY are found in the sympathetic nerves
supplying the coronary, cerebral, and renal vasculature and have
contributed to vasoconstriction. NPY binding sites have been
identified in a variety of tissues, including spleen, intestinal
membranes, brain, aortic smooth muscle, kidney, testis, and
placenta.
[0006] Neuropeptide Y (NPY) receptor pharmacology is currently
defined by structure activity relationships within the pancreatic
polypeptide family. This family includes NPY, which is synthesized
primarily in neurons; PYY, which is synthesized primarily by
endocrine cells in the gut; and PP, which is synthesized primarily
by endocrine cells in the pancreas. These approximately 36 amino
acid peptides have a compact helical structure involving a
"PP-fold" in the middle of the peptide. Specific features include a
polyproline helix in residues 1 through 8, a .beta.-turn in
residues 9 through 14, an .alpha.-helix in residues 15 through 30,
an outward-projecting C-terminus in residues 30 through 36, and a
carboxyl terminal amide, which appears to be critical for
biological activity. The peptides have been used to define at least
five receptor subtypes known as Y1, Y2, Y3, Y4 and Y5. Y1 receptor
recognition by NPY involves both N- and C-terminal regions of the
peptide; exchange of Gln.sup.34 with Pro.sup.34 is fairly well
tolerated. Y2 receptor recognition by NPY depends primarily upon
the four C-terminal residues of the peptide
(Arg.sup.33-Gln.sup.34-Arg.sup.35-Tyr.sup.36-NH.sub.2) preceded by
an amphipathic an .alpha.-helix ; exchange of Gln.sup.34 with
Pro.sup.34 is not well tolerated. One of the key pharmacological
features which distinguish Y1 and Y2 is the fact that the Y2
receptor (and not the Y1 receptor) has high affinity for the NPY
peptide carboxyl-terminal fragment NPY-(13-36) and the PYY fragment
PYY(22-36).
[0007] It has been shown that a 36 amino acid peptide called
Peptide YY(1-36) [PYY(1-36)] [YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY,
SEQ ID NO: 1]. when administered peripherally by injection to an
individual produces weight loss and thus can be used as a drug to
treat obesity and related diseases, Morley, J., Neuropsychobiology
21:22-30, 1989. It was later found that to produce this effect PYY
bound to a Y2 receptor, and the binding of a Y2 agonist to the Y2
receptor caused a decrease in the ingestion of carbohydrate,
protein and meal size, Leibowitz, S. F., et al., Peptides
12:1251-1260, 1991. An alternate molecular form of PYY is PYY(3-36)
IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY [SEQ ID NO: 2], Eberlein,
Eysselein, et al., Peptides 10:797-803, 1989. This fragment
constitutes approximately 40% of total PYY-like immunoreactivity in
human and canine intestinal extracts and about 36% of total plasma
PYY immunoreactivity in a fasting state to slightly over 50%
following a meal. It is apparently a dipeptidyl peptidase-IV (DPP4)
cleavage product of PYY. PYY3-36 is reportedly a selective ligand
at the Y2 and Y5 receptors, which appear pharmacologically unique
in preferring N-terminally truncated (i.e., C-terminal fragments
of) NPY analogs. It has also been shown that a PYY fragment having
only residues 22-36 will still bind to the Y2 receptor. However, if
any of the carboxyl terminus of the peptide is cleaved, the peptide
looses its ability to bind to the Y2 receptor. Hence a PYY agonist
is a peptide, which has a partial sequence of full-length PYY and
is able to bind to a Y2 receptor in the arcuate nucleus of the
hypothalamus. Hereinafter the term PYY refers to full-length PYY
and any fragment of PYY that binds to a Y2 receptor.
[0008] It is known that PYY and PYY3-36 can be administered by
intravenous infusion or injection to treat life-threatening
hypotension as encountered in shock, especially that caused by
endotoxins (U.S. Pat. No. 4,839,343), to inhibit proliferation of
pancreatic tumors in mammals by perfusion, parenteral, intravenous,
or subcutaneous administration, and by implantation (U.S. Pat. No.
5,574,010) and to treat obesity (Morley, J., Neuropsychobiology
21:22-30, 1989, and U.S. Patent Application No. 20020141985). It is
also claimed that PYY can be administered by parenteral, oral,
nasal, rectal and topical routes to domesticated animals or humans
in an amount effective to increase weight gain of said subject by
enhancing gastrointestinal absorption of a sodium-dependent
cotransported nutrient (U.S. Pat. No. 5,912,227). However, for the
treatment of obesity and related diseases, including diabetes, the
mode of administration has been limited to intravenous IV infusion
with no effective formulations optimized for alternative
administration of PYY3-36. None of these prior art teachings
provide formulations that contain PYY or PYY(3-36) combined with
excipients designed to enhance mucosal (i.e., nasal, buccal, oral)
delivery nor do they teach the value of endotoxin-free Y2-receptor
binding peptide formulations for non-infused administration. Thus,
there is a need to develop formulations and methods for
administering PYY3-36.
[0009] The generation of aerosol formulations can enhance
absorption of formulations on mucosal (nasal, buccal, oral, vaginal
and rectal) surfaces as well as skin surfaces. Review: O'Riordan,
T. G., Formulations and Nebulizer Performance. Respir Care, 2002
November; 47(11):1305-12; discussion 1312-3.
[0010] However, the physical forces associated with droplet
formation often destroys or denatures proteins and peptides. For
example, recombinant human deoxyribonuclease (rhDNase) was
substantially denatured during processing as shown by the
significantly reduced monomer content. Similarly, albumin was
affected by processing and only 50-75% of the monomer was retained
compared with 86% in the original material. Bustami, R. T.; Chan,
H. K.; Dehghani F.; Foster, N. R., "Generation of micro-particles
of proteins for aerosol delivery using high pressure modified
carbon dioxide," Pharm Res. 17(11): 1360-6, November 2000.
[0011] The physical stability of a peptide hormone human growth
hormone (hGH) formulation upon exposure to air/water interfaces
(with vortex mixing) has been investigated. The effect of this
stress on the formation of soluble and insoluble aggregates has
been studied. The aggregates were characterized and quantified by
size exclusion-HPLC and UV spectrophotometry. Vortex mixing of hGH
solutions (0.5 mg/mL) in phosphate buffer, pH 7.4, for just 1 min
caused 67% of the drug to precipitate as insoluble aggregates.
These aggregates were noncovalent in nature. Katakam, M.; Bell, L.
N.; Banga, A. K., J. Pharm. Sci. 84(6):713-6, June 1995.
SUMMARY OF THE INVENTION
[0012] The present invention fulfills the foregoing needs and
satisfies additional objects and advantages by providing novel,
effective methods and compositions for mucosal, especially
intranasal, delivery of a Y2 receptor-binding peptide such as PYY,
Pancreatic Peptide (PP) and NPY, to treat obesity, induce satiety
in an individual and to promote weight-loss in an individual and
prevent or cure diabetes. In certain aspects of the invention, the
Y2 receptor-binding peptide is delivered in formulations to the
intranasal mucosa so as to be able to increase the concentration of
the Y2 receptor-binding peptide by at least 5 pmol, preferably by
at least 10 pmol, in the blood plasma of a mammal when a dose of
the formulations of the Y2 receptor agonist is administered
intranasally. Furthermore preferred formulations would be able to
raise the concentration of the Y2 receptor-binding peptide in the
plasma of a mammal by 10 pmol, preferably 20 pmol, when the Y2
receptor-binding peptide is administered intranasally. When 150
.mu.g is administered intranasally the preferred formulation would
be able to raise the concentration of the Y2 receptor agonist in
the plasma of the mammal by at least 40 pmol per liter of plasma.
When 200 .mu.g of the Y2 receptor-binding peptide is administered
intranasally, the formulations of the present invention induce at
least 80 pmol, per liter of plasma increase of the Y2
receptor-binding peptide. In preferred embodiments, the elevated
concentrations of the Y2-receptor-binding peptide remains elevated
in the plasma of the mammal for at least 30 minutes, preferably at
least 60 minutes following a single intranasal dose of the Y2
receptor-binding peptide.
[0013] Preferably the Y2 receptor-binding peptide is a PP, PYY or
NPY peptide and the mammal is a human. In a most preferred
embodiment the Y2 receptor-binding peptide is a PYY peptide,
preferably PYY(3-36) and the mammal is human.
[0014] The present invention is also related to a Y2
receptor-binding peptide formulation that is able to raise the
concentration of the Y2 receptor-binding peptide in the blood
plasma of a mammal by at least 5 pM when a dose containing at least
50 .mu.g of the Y2 receptor-binding peptide is administered to the
mammal. In preferred embodiments, the elevated concentrations of
the Y2-receptor-binding peptide remains elevated in the plasma of
the mammal for at least 30 minutes, preferably at least 60 minutes
following a single intranasal dose of the Y2 receptor-binding
peptide.
[0015] The present invention is also related to a Y2
receptor-binding peptide formulation that is able to raise the
concentration of the Y2 receptor-binding peptide in the
blood-plasma of a mammal by at least 20 pM when a dose containing
at least 100 pg of the Y2 receptor-binding peptide is administered
to the mammal. In preferred embodiments, the elevated
concentrations of the Y2-receptor-binding peptide remains elevated
in the plasma of the mammal for at least 30 minutes, preferably at
least 60 minutes following a single intranasal dose of the Y2
receptor-binding peptide.
[0016] The present invention is also related to a Y2
receptor-binding peptide formulation that when administered
intranasally to a mammal is able to raise the concentration of the
Y2 receptor-binding peptide in blood plasma of the mammal by at
least 30 pM when a dose containing at least 150 .mu.g of the Y2
receptor-binding peptide is administered. In preferred embodiments,
the elevated concentrations of the Y2-receptor-binding peptide
remains elevated in the plasma of the mammal for at least 30
minutes, preferably at least 60 minutes following a single
intranasal dose of the Y2 receptor-binding peptide. Preferably the
mammal is a human.
[0017] The present invention is also related to a Y2
receptor-binding peptide formulation that when administered
intranasally to a mammal is able to raise the concentration of the
Y2 receptor-binding peptide by at least 60 pM when a dose
containing at least 200 .mu.g is administered to the mammal. In
preferred embodiments, the elevated concentrations of the
Y2-receptor-binding peptide remains elevated in the plasma of the
mammal for at least 30 minutes, preferably at least 60 minutes
following a single intranasal dose of the Y2 receptor-binding
peptide. Preferably the mammal is a human.
[0018] The present invention is also directed to an intranasal
formulation of a Y2 receptor-agonist that is substantially free of
proteins or polypeptides that stabilize the formulation. In
particular, the preferred formulation is free of such proteins as
albumin, and collagen-derived proteins such as gelatin.
[0019] In other aspects of the present invention a transmucosal Y2
receptor-binding peptide formulation is comprised of a Y2
receptor-binding peptide, water and a solubilizing agent having a
pH of 3-6.5. In a preferred embodiment, the solubilization agent is
a cyclodextrin.
[0020] In another embodiment of the present invention a
transmucosal Y2 receptor-binding peptide formulation is comprised
of a Y2 receptor-binding peptide, water, a solubilizing agent,
preferably a cyclodextrin, and at least one polyol, preferably 2
polyols. In alternate embodiments the formulation may contain one
or all of the following: a chelating agent, a surface-acting agent
and a buffering agent.
[0021] In another embodiment of the present invention the
formulation is comprised of a Y2 receptor-binding peptide, water,
chelating agent and a solubilization agent.
[0022] In another embodiment of the present invention the
formulation is comprised of a Y2 receptor-binding peptide, water
and a chelating agent having a pH of 3-6.5.
[0023] In another embodiment of the present invention the
formulation is comprised of a Y2 receptor-binding peptide, water,
chelating agent and at least one polyol, preferably two polyols.
Additional embodiments may include one or more of the following: a
surface-active agent, a solubilizing agent and a buffering
agent.
[0024] In another embodiment of the present invention the
formulation is comprised of a Y2 receptor-binding peptide, water,
and at least two polyols, such as lactose and sorbitol. Additional
agents, which can be added to the formulation, include, but are not
limited to, a solubilization agent, a chelating agent, one or more
buffering agents and a surface-acting agent.
[0025] The enhancement of intranasal delivery of a Y2
receptor-binding peptide agonist according to the methods and
compositions of the invention allows for the effective
pharmaceutical use of these agents to treat a variety of diseases
and conditions in mammalian subjects.
[0026] The present invention fills this need by providing for a
liquid or dehydrated Y2 receptor-binding peptide formulation
wherein the formulation is substantially free of a stabilizer that
is a polypeptide or a protein. The liquid PYY formulation is
comprised of water, PYY and at least one of the following additives
selected from the group consisting of polyols, surface-active
agents, solubilizing agents and chelating agents. The pH of the
formulation is preferably 3 to about 7.0, referably 4.5 to about
6.0, most preferably about 5.0.+-.0.03.
[0027] Another embodiment of the present invention is an aqueous Y2
receptor-binding formulation of the present invention is comprised
of water, a Y2 receptor-binding peptide, a polyol and a
surface-active agent wherein the formulation has a pH of about 3.0
to about 6.5, and the formulation is substantially free of a
stabilizer that is a protein or polypeptide.
[0028] Another embodiment of the present invention is an aqueous Y2
receptor-binding peptide formulation comprised of water, Y2
receptor-binding peptide, a polyol and a solubilizing agent wherein
the formulation has a pH of about 3.0 to about 6.5, and the
formulation is substantially free of a stabilizer that is a protein
or polypeptide.
[0029] Another embodiment of the present invention is an aqueous Y2
receptor-binding peptide formulation comprised of water, Y2
receptor-binding peptide, a solubilizing agent and a surface-active
agent wherein the formulation has a pH of about 3.0 to about 6.5,
and the formulation is substantially free of a stabilizer that is a
protein or polypeptide.
[0030] Another embodiment of the invention is a aqueous Y2
receptor-binding peptide formulation comprised of water, a Y2
receptor-binding peptide, a solubilizing agent, a polyol and a
surface-active agent wherein the formulation has a pH of about 3.0
to about 6.5, and the formulation is substantially free of a
stabilizer that is a protein or polypeptide.
[0031] In another aspect of the present invention, the stable
aqueous formulation is dehydrated to produce a dehydrated Y2
receptor-binding peptide formulation comprised of Y2
receptor-binding peptide and at least one of the following
additives selected from the group consisting of polyols,
surface-active agents, solubilizing agents and chelating agents,
wherein said dehydrated Y2 receptor-binding peptide formulation is
substantially free of a stabilizer that is a protein or polypeptide
such as albumin, collagen or collagen-derived protein such as
gelatin. The dehydration can be achieved by various means such as
lyophilization, spray-drying, salt-induced precipitation and
drying, vacuum drying, rotary evaporation, or supercritical
CO.sub.2 precipitation.
[0032] In one embodiment, the dehydrated Y2 receptor-binding
peptide is comprised of Y2 receptor-binding peptide, a polyol and a
solubilizing agent, wherein the formulation is substantially free
of a stabilizer that is a protein.
[0033] In another embodiment, the dehydrated Y2 receptor-binding
peptide formulation is comprised of a Y2 receptor-binding peptide,
a polyol, and a surface-active agent wherein the Y2
receptor-binding peptide formulation is substantially free of a
stabilizer that is a protein or polypeptide.
[0034] In another embodiment, the dehydrated Y2 receptor-binding
peptide formulation is comprised of a Y2 receptor-binding peptide,
a surface-active agent, and a solubilizing agent wherein the Y2
receptor-binding peptide formulation is substantially free of a
stabilizer that is a protein or polypeptide.
[0035] In another embodiment of the present invention, the
dehydrated Y2 receptor-binding peptide formulation is comprised of
a Y2 receptor-binding peptide, a polyol, a surface-active agent and
a solubilizing agent wherein the Y2 receptor-binding peptide
formulation is substantially free of a stabilizer that is a protein
or polypeptide.
[0036] Any solubilizing agent can be used but a preferred one is
selected from the group consisting of
hydroxypropyl-.beta.-cyclodextran,
sulfobutylether-.beta.-cyclodextran, methyl-.beta.-cyclodextrin and
chitosan.
[0037] Generally a polyol is selected from the group consisting of
lactose, sorbitol, trehalose, sucrose, mannose and maltose and
derivatives and homologs thereof.
[0038] A satisfactory surface-active agent is selected from the
group consisting of L-.alpha.-phosphatidylcholine didecanoyl
(DDPC), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80),
polyethylene glycol (PEG), cetyl alcohol, polyvinylpyrolidone
(PVP), polyvinyl alcohol (PVA), lanolin alcohol, and sorbitan
monooleate.
[0039] In a preferred formulation, the Y2 receptor-binding peptide
formulation is also comprised of a chelating agent such as ethylene
diamine tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid
(EGTA). Also a preservative such as chlorobutanol or benzylkonium
chloride can be added to the formulation to inhibit microbial
growth.
[0040] The pH is generally regulated using a buffer such as sodium
citrate and citric acid, and sodium acetate and acetic acid. An
alternative buffer would be acetic acid and sodium acetate or
succinic acid and sodium hydroxide.
[0041] The preferred Y2 receptor-binding peptide is a PYY, PP or
NPY peptide, preferably a PYY(3-36) peptide.
[0042] The present invention also comprehends a formulation wherein
the concentration of the Y2 receptor-binding peptide is 0.1-15.0
mg/mL, preferably 1.0-2 mg/mL and the pH of the aqueous solution is
3.0-6.5 preferably about 5.0.+-.0.3.
[0043] The present invention further includes Y2 receptor-binding
peptide formulation wherein the concentration of the polyol is
between about 0.1% and 10% (w/v) and additionally wherein the
concentration of the polyol is in the range from about 0.1% to
about 3% (w/v).
[0044] The instant invention also includes a formulation, wherein
the concentration of the surface-active agent is between about
0.00001% and about 5% (w/v), and wherein the concentration of the
surface-active agent is between about 0.0002% and about 0.1%
(w/v).
[0045] The instant invention also includes a formulation, wherein
the concentration of the solubilzation agent is 1% -10% (w/v), and
wherein the concentration of the solubilizing agent is 2% to 5%
(w/v).
[0046] The finished solution can be filtered and freeze-dried,
lyophilized, using methods well known to one of ordinary skill in
the art, and by following the instructions of the manufacturer of
the lyophilizing equipment. This produces a dehydrated Y2
receptor-binding peptide formulation substantially free of a
stabilizer that is a protein.
[0047] In another embodiment of the present invention, a Y2
receptor-binding peptide formulation is comprised of an Y2
receptor-binding peptide and a pharmaceutically acceptable carrier
wherein the Y2 receptor-bind peptide formulation has at least 1%,
preferably 3% and most preferably at least 6% higher permeation in
an in vitro tissue permeation assay than a control formulation
consisting of water, sodium chloride, a buffer and the Y2
receptor-binding peptide, as determined by the transepithelial
electrical resistance assay shown in Examples 2 & 7. In a
preferred embodiment, the Y2 receptor-binding formulation is
further comprised of at least one excipient selected from the group
consisting of a surface-active agent, a solubilization agent, a
polyol, and a chelating agent. Preferably the Y2 receptor-binding
peptide is a PYY peptide, an NPY peptide or a PP peptide.
[0048] In another embodiment of the present invention a Y2
receptor-binding petide formulation is provided that is capable of
raising the concentration of the Y2 receptor-binding peptide in the
plasma of a mammal by at least 5 preferably 10, 20 40, 60, 80 or
more pmoles per liter of plasma when 100 .mu.L of the formulation
is administered intranasally to said mammal.
[0049] In exemplary embodiments, the enhanced delivery methods and
compositions of the present invention provide for therapeutically
effective mucosal delivery of the Y2 receptor-binding peptide
agonist for prevention or treatment of obesity and eating disorders
in mammalian subjects. In one aspect of the invention,
pharmaceutical formulations suitable for intranasal administration
are provided that comprise a therapeutically effective amount of a
Y2 receptor-binding peptide and one or more intranasal
delivery-enhancing agents as described herein, which formulations
are effective in a nasal mucosal delivery method of the invention
to prevent the onset or progression of obesity or eating disorders
in a mammalian subject. Nasal mucosal delivery of a therapeutically
effective amount of a Y2 receptor-binding peptide agonist and one
or more intranasal delivery-enhancing agents yields elevated
therapeutic levels of the Y2 receptor-binding peptide agonist in
the subject and inhibits food intake in the mammalian subject,
reducing symptoms of obesity or an eating disorder.
[0050] The enhanced delivery methods and compositions of the
present invention provide for therapeutically effective mucosal
delivery of a Y2 receptor-binding peptide for prevention or
treatment of a variety of diseases and conditions in mammalian
subjects. Y2 receptor-binding peptide can be administered via a
variety of mucosal routes, for example by contacting the Y2
receptor-binding peptide to a nasal mucosal epithelium, a bronchial
or pulmonary mucosal epithelium, the oral buccal surface or the
oral and small intestinal mucosal surface. In exemplary
embodiments, the methods and compositions are directed to or
formulated for intranasal delivery (e.g., nasal mucosal delivery or
intranasal mucosal delivery).
[0051] In one aspect of the invention, pharmaceutical formulations
suitable for intranasal administration are provided that comprise a
therapeutically effective amount of a Y2 receptor-binding peptide
agonist and one or more intranasal delivery-enhancing agents as
described herein, which formulations are effective in a nasal
mucosal delivery method of the invention to prevent the onset or
progression of obesity, diabetes, cancer, or malnutrition or
wasting related to cancer in a mammalian subject, or to alleviate
one or more clinically well-recognized symptoms of obesity, as well
as treating Alzheimer's disease, colon carcinoma, colon
adenocarcinoma, pancreatic carcinoma, pancreatic adenocarcinoma,
breast carcinoma.
[0052] In another aspect of the invention, pharmaceutical
formulations and methods are directed to administration of a Y2
receptor-binding peptide agonist in combination with vitamin E
succinate. A Y2 receptor-binding peptide agonist in combination
with vitamin E succinate may be administered to alleviate symptoms
or prevent the onset or lower the incidence or severity of cancer,
for example, colon adenocarcinoma, pancreatic adenocarcinoma, or
breast cancer.
[0053] In another aspect of this invention, it was surprisingly
found that the use of endotoxin-free Y2 receptor binding peptides,
for example PYY(3-36), produced increased mucosal delivery compared
to peptides in which endotoxin is not removed. The use of
endotxin-free Y2 receptor peptides in pharmaceutical formulations
is thus enabled for administration by non-infusion routes,
including mucosal delivery, nasal, oral, pulmonary, vaginal,
rectal, and the like.
[0054] The foregoing mucosal Y2 receptor-binding peptide
formulations and preparative and delivery methods of the invention
provide improved mucosal delivery of a Y2 receptor-binding peptide
to mammalian subjects. These compositions and methods can involve
combinatorial formulation or coordinate administration of one or
more Y2 receptor-binding peptides with one or more mucosal
delivery-enhancing agents. Among the mucosal delivery-enhancing
agents to be selected from to achieve these formulations and
methods are (A) solubilization agents; (B) charge modifying agents;
(C) pH control agents; (D) degradative enzyme inhibitors; (E)
mucolytic or mucus clearing agents; (F) ciliostatic agents; (G)
membrane penetration-enhancing agents (e.g., (i) a surfactant, (ii)
a bile salt, (iii) a phospholipid or fatty acid additive, mixed
micelle, liposome, or carrier, (iv) an alcohol, (v) an enamine,
(iv) an NO donor compound, (vii) a long-chain amphipathic molecule,
(viii) a small hydrophobic penetration enhancer; (ix) sodium or a
salicylic acid derivative; (x) a glycerol ester of acetoacetic
acid, (xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) a
medium-chain fatty acid, (xiii) a chelating agent, (xiv) an amino
acid or salt thereof, (xv) an N-acetylamino acid or salt thereof,
(xvi) an enzyme degradative to a selected membrane component,
(xvii) an inhibitor of fatty acid synthesis, (xviii) an inhibitor
of cholesterol synthesis; or (xiv) any combination of the membrane
penetration enhancing agents of (i)-(xviii)); (H) modulatory agents
of epithelial junction physiology, such as nitric oxide (NO)
stimulators, chitosan, and chitosan derivatives; (I) vasodilator
agents; (J) selective transport-enhancing agents; and (K)
stabilizing delivery vehicles, carriers, supports or
complex-forming species with which the Y2 receptor-binding peptide
(s) is/are effectively combined, associated, contained,
encapsulated or bound to stabilize the active agent for enhanced
mucosal delivery.
[0055] In various embodiments of the invention, a Y2
receptor-binding peptide is combined with one, two, three, four or
more of the mucosal delivery-enhancing agents recited in (A)-(K),
above. These mucosal delivery-enhancing agents may be admixed,
alone or together, with the Y2 receptor-binding peptide, or
otherwise combined therewith in a pharmaceutically acceptable
formulation or delivery vehicle. Formulation of a Y2
receptor-binding peptide with one or more of the mucosal
delivery-enhancing agents according to the teachings herein
(optionally including any combination of two or more mucosal
delivery-enhancing agents selected from (A)-(K) above) provides for
increased bioavailability of the Y2 receptor-binding peptide
following delivery thereof to a mucosal surface of a mammalian
subject.
[0056] Thus, the present invention is a method for suppressing
apetite, promoting weight loss, decreasing food intake, or treating
obesity and/or diabetes in a mammal comprising transmucosally
administering a formulation comprised of a Y2 receptor-binding
peptide, such that when at 50 .mu.g of the Y2 receptor is
administered transmucosally to the mammal the concentration of the
Y2 receptor-binding peptide in the plasma of the mammal increases
by at least 5 pmol, preferably at least 10 pmol per liter of
plasma. Examples of such formulations are described above.
[0057] The present invention further provides for the use of a Y2
receptor-binding peptide for the production of medicament for the
transmucosal, administration of a Y2 receptor-binding peptide for
suppressing apetite, promoting weight loss, decreasing food intake,
or treating obesity in a mammal such that when about 50 .mu.g of
the Y2 receptor is administered transmucosally to the mammal the
concentration of the Y2 receptor-binding peptide in the plasma of
the mammal increases by at least 5 pmol per liter of plasma. When
100 .mu.g of the Y2 receptor-binding peptide is administered
intranasally to the mammal, the concentration of the Y2 receptor
agonist increases by at least 20 pmol per liter of plasma in the
mammal. When 150 .mu.g is administered intranasally, the
concentration of the Y2 receptor-binding peptide in blood plasma of
the mammal increases by at least 30 pM. When 200 .mu.g is
administered intranasally, the concentration of the Y2
receptor-binding peptide in blood plasma of the mammal increases by
at least 60 pM. In preferred embodiments, the elevated
concentrations of the Y2-receptor-binding peptide remains elevated
in the plasma of the mammal for at least 30 minutes, preferably at
least 60 minutes following a single intranasal dose of the Y2
receptor-binding peptide. Preferably the mammal is a human.
[0058] A mucosally effective dose of peptide YY within the
pharmaceutical formulations of the present invention comprises, for
example, between about 0.001 pmol to about 100 pmol per kg body
weight, between about 0.01 pmol to about 10 pmol per kg body
weight, or between about 0.1 pmol to about 5 pmol per kg body
weight. In further exemplary embodiments, dosage of peptide YY is
between about 0.5 pmol to about 1.0 pmol per kg body weight. In a
preferred embodiment an intranasal dose will range from 50 .mu.g to
400 .mu.g, preferably 100 .mu.g to 200 .mu.g, most preferably about
100 .mu.g to 150 .mu.g. The pharmaceutical formulations of the
present invention may be administered one or more times per day
(for example, before a meal), or 3 times per week or once per week
for between one week and at least 96 weeks or even for the life of
the individual patient or subject. In certain embodiments, the
pharmaceutical formulations of the invention are administered one
or more times daily, two times daily, four times daily, six times
daily, or eight times daily.
[0059] Intranasal delivery-enhancing agents are employed which
enhance delivery of peptide YY into or across a nasal mucosal
surface. For passively absorbed drugs, the relative contribution of
paracellular and transcellular pathways to drug transport depends
upon the pKa, partition coefficient, molecular radius and charge of
the drug, the pH of the luminal environment in which the drug is
delivered, and the area of the absorbing surface. The intranasal
delivery-enhancing agent of the present invention may be a pH
control agent. The pH of the pharmaceutical formulation of the
present invention is a factor affecting absorption of peptide YY
via paracellular and transcellular pathways to drug transport. In
one embodiment, the pharmaceutical formulation of the present
invention is pH adjusted to between about pH 3.0 to 6.5. In a
further embodiment, the pharmaceutical formulation of the present
invention is pH adjusted to between about pH 3.0 to 5.0. In a
further embodiment, the pharmaceutical formulation of the present
invention is pH adjusted to between about pH 4.0 to 5.0. Generally,
the pH is 5.0.+-.0.3.
[0060] The instant invention also describes the suprising ability
to successfully aerosolize the Y2 receptor binding compound,
PYY(3-36), from an aqueous formulation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1 shows the stability of PYY3-36 at high temperature
(40.degree. C.) at various pHs from 3.0 to 7.4.
[0062] FIG. 2 shows the data for TEER of permeability
enhancers.
[0063] FIG. 3 shows the cell viabilities of candidate PYY
formulations.
[0064] FIG. 4 shows the cytotoxic effects of candidate
formulations. In FIGS. 2-4
[0065] EN1=PBS pH 5.0
[0066] EN2=L-Arginine (10% w/v)
[0067] EN3=Poly-L-Arginine (0.5% w/v)
[0068] EN4=Gamma-Cyclodextrin (1% w/v)
[0069] EN5=Alpha-Cyclodextrin (5% w/v)
[0070] EN6=Methyl-Beta-Cyclodextrin (3% w/v)
[0071] EN7=n-Capric Acid Sodium (0.075% w/v)
[0072] EN8=Chitosan (0.5% w/v)
[0073] EN9=L-Alpha-phosphatidylcholine didecanyl (3.5% w/v)
[0074] EN10=S-Nitroso-N-acetylpenicillamine, (0.02% w/v)
[0075] EN11=Palmotoyl-DL-Carnitine (0.5% w/v)
[0076] EN12=Pluronic-127 (0.3% w/v)
[0077] EN13=Sodium Nitroprusside (0.3% w/v)
[0078] EN14=Sodium Glycocholate (1% w/v)
[0079] FIG. 5 shows the synergistic contributions of the various
components on drug permeation. In FIG. 5 EN1 is DDPC, EN2 is
methyl-.beta.-cyclodextrin, and EX1 is EDTA.
[0080] FIG. 6 shows the PYY3-36 in the plasma of rats, the square
represent a dose of 4.1 .mu.g/kg, the triangle represents a dose of
41 .mu.g/kg, and the circle represent a dose of 205 .mu.g/kg.
[0081] FIG. 7 shows dose linearity following intranasal
administration PYY3-36 in rats as Cmax-Cbas pg/mL v. dose as
.mu.g/kg.
[0082] FIG. 8 shows dose linearity following intranasal
administration of PYY3-36 in rats as AUC v. dose as .mu.g/kg.
[0083] FIG. 9 shows the average plasma concentration of PYY v. time
in minutes in three human volunteers who were each administered 20
.mu.g of PYY(3-36) intranasally.
[0084] FIG. 10 shows the average plasma concentration of PYY v.
time in minutes in three human volunteers who were each
administered 50 .mu.g of PYY(3-36) intranasally.
[0085] FIG. 11 shows the average plasma concentration of PYY v.
time in minutes in three human volunteers who were each
administered 100 .mu.g of PYY(3-36) intranasally.
[0086] FIG. 12 shows the average plasma concentration of PYY v.
time in minutes in three human volunteers who were each
administered 150 .mu.g of PYY3-36 intranasally.
[0087] FIG. 13 shows the average plasma concentration of PYY v.
time in minutes in three human volunteers who were each
administered 200 .mu.g of PYY(3-36) intranasally.
[0088] FIG. 14 shows PYY plasma concentration as pmol/L v. time for
five groups of healthy human volunteers who received intranasal
PYY(3-36). The doses were 200 .mu.g, 150 .mu.g, 100 .mu.g, 50 .mu.g
and 20 .mu.g of PYY3-36.
[0089] FIG. 15 shows the dose linearity Cmax of PYY in pg/mL vs.
dose of PYY(3-36) administered to human volunteers.
[0090] FIG. 16 shows the dose linearity PYY mean AUC in pg/mL vs.
dose of PYY(3-36) administered to human volunteers.
[0091] FIG. 17 shows the visual analog scale (VAS) vs. dose of
PYY(3-36) administered to the human volunteers. The question was:
"How hungry are you?" The lower the score the less hungry an
individual was on a 100 point scale.
[0092] FIG. 18 shows the visual analog scale (VAS) vs. dose of
PYY(3-36) administered to the human volunteers. The question was:
"How much could you eat?" The lower the score the less hungry an
individual was on a 100 point scale.
[0093] FIG. 19 shows the visual analog scale (VAS) vs. dose of
PYY(3-36) administered to the human volunteers. The question was:
"How full do you feel?" The lower the score the less full an
individual was on a 100 point scale.
[0094] FIG. 20 shows the per cent permeation of PYY(3-36)
containing endotoxin vs. endotoxin-free PYY(3-36).
[0095] FIG. 21A shows a nasal spray pump/actuator that is not
engaged.
[0096] FIG. 21B shows the nasal spray pump/actuator that is engaged
and expelling a spray plume.
[0097] FIG. 22 shows an example of a spray pattern of a PYY nasal
spray of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0098] As noted above, the present invention provides improved
methods and compositions for mucosal delivery of Y2
receptor-binding peptide to mammalian subjects for treatment or
prevention of a variety of diseases and conditions. Examples of
appropriate mammalian subjects for treatment and prophylaxis
according to the methods of the invention include, but are not
restricted to, humans and non-human primates, livestock species,
such as horses, cattle, sheep, and goats, and research and domestic
species, including dogs, cats, mice, rats, guinea pigs, and
rabbits.
[0099] In order to provide better understanding of the present
invention, the following definitions are provided:
Y2 Receptor-binding Peptides
[0100] The Y2 receptor-binding peptides used in the mucosal
formulations of the present invention include the pancreatic
polypeptide family." as used herein, is comprised of three
naturally occurring bioactive peptide families, PP, NPY, and PYY.
Examples of Y2 receptor-binding peptides and their uses are
described in U.S. Pat. No. 5,026,685; U.S. Pat. No. 5,574,010; U.S.
Pat. No. 5,604,203; U.S. Pat. No. 5,696,093; U.S. Pat. No.
6,046,167; Gehlert, et al., Proc Soc Exp Biol Med 218:7-22, 1998;
Sheikh, et al., Am. J. Physiol. 261:701-15, 1991; Fournier, et al.,
Mol. Pharmacol. 45:93-101, 1994; Kirby, et al., J. Med. Chem.
38:4579-4586, 1995; Rist, et al., Eur. J. Biochem. 247:1019-1028,
1997; Kirby, et al., J. Med. Chem. 36:3802-3808, 1993; Grundemar,
et al., Regulatory Peptides 62:131-136, 1996; U.S. Pat. No.
5,696,093 (examples of PYY agonists), U.S. Pat. No. 6,046,167.
According to the present invention a Y2 receptor-binding peptide
includes the free bases, acid addition salts or metal salts, such
as potassium or sodium salts or the peptides Y2 receptor-binding
peptides that have been modified by such processes as amidation,
glycosylation, acylation, sulfation, phosphorylation, acetylation
and cyclization, (U.S. Pat. No. 6,093,692; and U.S. Pat. No.
6,225,445 and pegylation.
Peptide YY Agonists
[0101] As used herein, "PYY" refers to PYY(1-36) in native-sequence
or in variant form, as well as derivatives, fragments, and analogs
of PYY from any source, whether natural, synthetic, or recombinant.
The PYY must be comprised at least the last 15 amino acid residues
or analogoues thereof of the PYY sequence,PYY(22-36) (SEQ ID NO:
3). Other PYY peptides, which may be used are PYY(1-36) (SEQ ID NO:
1) PYY(3-36) SEQ ID NO: 2) PYY(4-36 )(SEQ ID NO:4) PYY(5-36) (SEQ
ID NO: 5), PYY(6-36) (SEQ ID NO:6), PYY(7-36) (SEQ ID NO:7)
PYY(8-36) (SEQ ID NO: 8), PYY9-36 (SEQ ID NO: 9) PYY(10-36) (SEQ ID
NO: 10), PYY(11-36) (SEQ ID NO: 11), PYY(12-36) (SEQ ID NO: 12),
PYY(13-36) (SEQ ID NO:13), PYY(14-36) (SEQ ID NO: 14), PYY(15-36)
(SEQ ID NO: 15), PYY(16-36) (SEQ ID NO: 16), PYY(17-36) (SEQ ID NO:
17), PYY(18-36) (SEQ ID NO: 18), PYY(19-36) (SEQ ID NO: 19),
PYY(20-36) (SEQ ID NO: 20) and PYY(21-36) (SEQ ID NO: 21). These
peptides typically bind to the Y receptors in the brain and
elsewhere, especially the Y2 and/or Y5 receptors. Typically these
peptides are synthesized in endotoxin-free or pyrogen-free forms
although this is not always necessary.
[0102] Other PYY peptides include those PYY peptides in which
conservative amino acid residue changes have beem made, for
example, site specific mutation of a PYY peptide including
[Asp.sup.15] PYY(15-36) (SEQ ID NO: 90), [Thr.sup.13] PYY(13-36)
(SEQ ID NO: 91), [Val.sup.12] PYY(12-36)(SEQ ID NO: 92),
[Glu.sup.11] PYY(11-36) (SEQ ID NO: 93), [Asp.sup.10] PYY(10-36)
(SEQ ID NO: 94), [Val.sup.7] PYY(7-36) (SEQ ID NO: 95), [Asp.sup.6]
PYY(6-36) (SEQ ID NO: 96), [Gln.sup.4] PYY(4-36) (SEQ ID NO: 97),
[Arg.sup.4] PYY(4-36) (SEQ ID NO: 98), [Asn.sup.4] PYY(4-36) (SEQ
ID NO: 99), [Val.sup.3] PYY(3-36) (SEQ ID NO: 100) and [Leu.sup.3]
PYY(3-36) (SEQ ID NO: 101). Other PYY peptides include those
peptides in which at least two conservative amino acid residue
changes have been made including [Asp.sup.10, Asp.sup.15]
PYY(10-36) (SEQ ID NO: 102), [Asp.sup.6, Thr.sup.13] PYY(6-36) (SEQ
ID NO: 103), [Asn.sup.4, Asp.sup.15] PYY(4-36) (SEQ ID NO: 104) and
[Leu.sup.3, Asp.sup.10] PYY(3-36) (SEQ ID NO: 105. Also included
are analogues of a PYY for example those disclosed in U.S. Pat.
Nos. 5,604,203 and 5,574,010; Balasubramaniam, et al., Peptide
Research 1:32, 1988; Japanese Patent Application No. 2,225,497,
1990; Balasubramaniam, et al., Peptides 14:1011, 1993; Grandt, et
al., Reg. Peptides 51:151, 1994; PCT International Application No.
94/03380, U.S. Pat. Nos. 5, 604,203 and 5,574,010. These peptides
typically bind to the Y receptors in the brain and elsewhere,
especially the Y2 and/or Y5 receptors. Typically these peptides are
synthesized in endotoxin-free or pyrogen-free forms although this
is not always necessary.
[0103] PYY agonists include rat PYY (SEQ ID NO: 72) and the amino
terminus truncated forms corresponding to the human, pig PYY (SEQ
ID NO: 73) and the amino terminus truncated forms corresponding to
the human and guinea pig PYY (SEQ ID NO: 74) and the amino terminus
truncated forms corresponding to the human.
[0104] According to the present invention a PYY peptide also
includes the free bases, acid addition salts or metal salts, such
as potassium or sodium salts of the peptides, and PYY peptides that
have been modified by such processes as amidation, glycosylation,
acylation, sulfation, phosphorylation, acetylation, cyclization and
other well known covalent modification methods. These peptides
typically bind to the Y receptors in the brain and elsewhere,
especially the Y2 and/or Y5 receptors. Typically these peptides are
synthesized in endotoxin-free or pyrogen-free forms although this
is not always necessary.
Neuropeptide Y Agonists
[0105] NPY is another Y2 receptor-binding peptide. NPY peptides
include full-length NPY(1-36) (SEQ ID NO: 22) as well as well as
fragments of NPY(1-36), which have been truncated at the amino
terminus. To be effective in binding the Y2 receptor, the NPY
agonist should have at least the last 11 amino acid residues at the
carboxyl terminus, i.e., be comprised of NPY(26-36) (SEQ ID NO:
23). Other examples of NPY agonists that bind to the Y2 receptor
are NPY(3-36) (SEQ ID NO: 24), NPY(4-36) (SEQ ID NO: 25), NPY(5-36)
(SEQ ID NO: 26), NPY(6-36) (SEQ ID NO: 27), NPY(7-36) (SEQ ID NO:
28), NPY(8-36) (SEQ ID NO: 29), NPY(9-36) (SEQ ID NO: 30),
NPY(10-36) (SEQ ID NO: 31), NPY(11-36) (SEQ ID NO: 32), NPY(12-36)
(SEQ ID NO: 33), NPY(13-36) (SEQ ID NO: 34), NPY(14-36) (SEQ ID NO:
35), NPY(15-36) (SEQ ID NO: 36), NPY(16-36) (SEQ ID NO: 37),
NPY(17-36) (SEQ ID NO: 38), NPY(18-36) (SEQ ID NO: 39), NPY(19-36)
(SEQ ID NO: 40), NPY(20-36) (SEQ ID NO: 41), NPY(21-36) (SEQ ID NO:
42), NPY(22-36) (SEQ ID NO: 43), NPY(23-36) (SEQ ID NO: 44),
NPY(24-36) (SEQ ID NO: 45) and NPY(25-36) (SEQ ID NO: 46).
[0106] Other NPY agonists include rat NPY (SEQ ID NO: 75) and the
amino terminus truncated forms from NPY(3-36) to NPY(26-36) as in
the human form, rabbit NPY (SEQ ID NO: 76) and the amino terminus
truncated forms from NPY(3-36) to NPY(26-36) as in the human form,
dog NPY (SEQ ID NO: 77) and the amino terminus truncated forms
NPY(3-36) to NPY(26-36) as in the human form, pig NPY (SEQ ID NO:
78) and the amino terminus truncated forms from NPY(3-36) to
NPY(26-36) as in the human form, cow NPY (SEQ ID NO: 79) and the
amino terminus truncated forms from NPY(3-36) to NPY26-36 as in the
human form, sheep NPY (SEQ ID NO:80) and the amino terminus
truncated forms from NPY(3-36) to NPY(26-36) as in the human form
and guinea pig (SEQ 81) and the amino terminus truncated forms from
NPY(3-36) to NPY(26-36) as in the human form.
[0107] According to the present invention a NPY peptide also
includes the free bases, acid additoin salts or metal salts, such
as potassium or sodium salts of the peptides, and NPY peptides that
have been modified by such processes as amidation, glycosylation,
acylation, sulfation, phosphorylation, acetylation, cyclization and
other known covalent modification methods. These peptides typically
bind to the Y receptors in the brain and elsewhere, especially the
Y2 and/or Y5 receptors. Typically these peptides are synthesized in
endotoxin-free or pyrogen-free forms although this is not always
necessary.
Pancreatic Peptide
[0108] Pancreatic Peptide (PP) and PP agonist also bind to the Y2
receptor. Examples of the PP agonists are the full-length PP(1-36)
(SEQ ID NO: 47) and a number of PP fragments, which are truncated
at the amino-terminus. To bind to the Y2 receptor the PP agonist
must have the last 11 amino acid residues at the carboxyl-terminus,
PP(26-36), (SEQ ID NO: 48). Examples of other PP, which bind to the
Y2 receptor, are PP(3-36) (SEQ ID NO: 49), PP(4-36) (SEQ ID NO:
50), PP(5-36) (SEQ ID NO: 51), PP(6-36) (SEQ ID NO: 52), PP(7-36)
(SEQ ID NO: 53), PP(8-36) (SEQ ID NO: 54), PP(9-36) (SEQ ID NO:
55), PP(10-36) (SEQ ID NO: 56), PP(11-36) (SEQ ID NO: 57),
PP(12-36) (SEQ ID NO: 58), PP(13-36) (SEQ ID NO: 59), PP(14-36)
(SEQ ID NO: 60), PP(15-36) (SEQ ID NO: 61), PP(16-36) (SEQ ID NO:
62), PP(17-36) (SEQ ID NO: 63), PP(18-36) (SEQ ID NO: 64),
PP(19-36) (SEQ ID NO: 65), PP(20-36) (SEQ ID NO: 66), PP(21-36)
(SEQ ID NO: 67), PP(22-36) (SEQ ID NO: 68), PP(23-36) (SEQ ID NO:
69), PP(24-36) (SEQ ID NO: 70) and PP(25-36) (SEQ ID NO: 71).
[0109] Other PP agonists include sheep PP (SEQ ID NO: 82) and the
amino terminus truncated forms from PP(3-36) to PP(26-36) as in the
human form, pig PP (SEQ ID NO: 83) and the amino terminus truncated
forms from PP(3-36) to PP(26-36) as in the human form, dog PP (SEQ
ID NO: 84) and the amino terminus truncated forms PP(3-36) to
PP(26-36) as in the human form, cat PP (SEQ ID NO: 85) and the
amino terminus truncated forms from PP(3-36) to PP(26-36) as in the
human form, cow PP (SEQ ID NO: 86) and the amino terminus truncated
forms from PP(3-36) to PP(26-36) as in the human form, rat PP (SEQ
ID NO:87) and the amino terminus truncated forms from PP(3-36) to
PP(26-36) as in the human form, mouse (SEQ 88) and the amino
terminus truncated forms from PP(3-36) to PP(26-36) as in the human
form, and guinea pig PP (SEQ ID NO: 89).
[0110] According to the present invention a PP peptide also
includes the free bases, acid additoin salts or metal salts, such
as potassium or sodium salts of the peptides, and PP peptides that
have been modified by such processes as amidation, glycosylation,
acylation, sulfation, phosphorylation, acetylation, cyclization,
and other known covalent modification methods. These peptides
typically bind to the Y receptors in the brain and elsewhere,
especially the Y2 and/or Y5 receptors. Typically these peptides are
synthesized in endotoxin-free or pyrogen-free forms although this
is not always necessary.
Mucosal Delivery Enhancing Agents
[0111] "Mucosal delivery enhancing agents" are defined as chemicals
and other excipients that, when added to a formulation comprising
water, salts and/or common buffers and Y2 receptor-binding peptide
(the control formulation) produce a formulation that produces a
significant increase in transport of Y2 receptor-binding peptide
across a mucosa as measured by the maximum blood, serum, or
cerebral spinal fluid concentration (C.sub.max) or by the area
under the curve, AUC, in a plot of concentration versus time. A
mucosa includes the nasal, oral, intestional, buccal,
bronchopulmonary, vaginal, and rectal mucosal surfaces and in fact
includes all mucus-secreting membranes lining all body cavities or
passages that communicate with the exterior. Mucosal delivery
enhancing agents are sometimes called carriers.
Endotoxin-free Formulation
[0112] "Endotoxin-free formulation" means a formulation which
contains a Y2-receptor-binding peptide and one or more mucosal
delivery enhancing agents that is substantially free of endotoxins
and/or related pyrogenic substances. Endotoxins include toxins that
are confined inside a microorganism and are released only when the
microorganisms are broken down or die. Pyrogenic substances include
fever-inducing, thermostable substances (glycoproteins) from the
outer membrane of bacteria and other microorganisms. Both of these
substances can cause fever, hypotension and shock if administered
to humans. Producing formulations that are endotoxin-free can
require special equipment, expert artisians, and can be
significantly more expensive than making formulations that are not
endotoxin-free. Because intravenous administration of NPY or PYY
simultaneously with infusion of endotoxin in rodents has been shown
to prevent the hypotension and even death associated with the
administration of endotoxin alone (U.S. Pat. No. 4,839,343),
producing endotoxin-free formulations of these therapeutic agents
would not be expected to be necessary for non-parental
(non-injected) administration.
Non-infused Administration
[0113] "Non-infused administration" means any method of delivery
that does not involve an injection directly into an artery or vein,
a method which forces or drives (typically a fluid) into something
and especially to introduce into a body part by means of a needle,
syringe or other invasive method. Non-infused administration
includes subcutaneous injection, intramuscular injection,
intraparitoneal injection and the non-injection methods of delivery
to a mucosa.
Treatment and Prevention of Obesity
[0114] As noted above, the instant invention provides improved and
useful methods and compositions for nasal mucosal delivery of a Y2
receptor-binding peptide to prevent and treat obesity in mammalian
subjects. As used herein, prevention and treatment of obesity mean
prevention of the onset or lowering the incidence or severity of
clinical obesity by reducing food intake during meals and/or
reducing body weight during administration or maintaining a reduced
body weight following weight loss or before weight gain has
occurred.
[0115] The instant invention provides improved and useful methods
and compositions for nasal mucosal delivery of Y2 receptor-binding
peptide to regions of the brain, for example, the hypothalamus or
the proopiomelanocortin (POMC) and NPY arcuate neurons, to prevent
and treat obesity in mammalian subjects. The Y2 receptor-binding
peptide can also be administered in conjunction with a Y1 receptor
antagonist such as dihyropyridine.
Methods and Compositions of Delivery
[0116] Improved methods and compositions for mucosal administration
of Y2 receptor-binding peptide to mammalian subjects optimize Y2
receptor-binding peptide dosing schedules. The present invention
provides mucosal delivery of Y2 receptor-binding peptide formulated
with one or more mucosal delivery-enhancing agents wherein Y2
receptor-binding peptide dosage release is substantially normalized
and/or sustained for an effective delivery period of Y2
receptor-binding peptide release ranges from approximately 0.1 to
2.0 hours; 0.4 to 1.5 hours; 0.7 to 1.5 hours; or 0.8 to 1.0 hours;
following mucosal administration. The sustained release of Y2
receptor-binding peptide achieved may be facilitated by repeated
administration of exogenous Y2 receptor-binding peptide utilizing
methods and compositions of the present invention.
Compositions and Methods of Sustained Release
[0117] Improved compositions and methods for mucosal administration
of Y2 receptor-binding peptide to mammalian subjects optimize Y2
receptor-binding peptide dosing schedules. The present invention
provides improved mucosal (e.g., nasal) delivery of a formulation
comprising Y2 receptor-binding peptide in combination with one or
more mucosal delivery-enhancing agents and an optional sustained
release-enhancing agent or agents. Mucosal delivery-enhancing
agents of the present invention yield an effective increase in
delivery, e.g., an increase in the maximal plasma concentration
(C.sub.max) to enhance the therapeutic activity of
mucosally-administered Y2 receptor-binding peptide. A second factor
affecting therapeutic activity of Y2 receptor-binding peptide in
the blood plasma and CNS is residence time (RT). Sustained
release-enhancing agents, in combination with intranasal
delivery-enhancing agents, increase C.sub.max and increase
residence time (RT) of Y2 receptor-binding peptide. Polymeric
delivery vehicles and other agents and methods of the present
invention that yield sustained release-enhancing formulations, for
example, polyethylene glycol (PEG), are disclosed herein. The
present invention provides an improved Y2 receptor-binding peptide
delivery method and dosage form for treatment of symptoms related
to obesity, colon cancer, pancreatic cancer, or breast cancer in
mammalian subjects.
[0118] Within the mucosal delivery formulations and methods of the
invention, the Y2 receptor-binding peptide is frequently combined
or coordinately administered with a suitable carrier or vehicle for
mucosal delivery. As used herein, the term "carrier" means a
pharmaceutically acceptable solid or liquid filler, diluent or
encapsulating material. A water-containing liquid carrier can
contain pharmaceutically acceptable additives such as acidifying
agents, alkalizing agents, antimicrobial preservatives,
antioxidants, buffering agents, chelating agents, complexing
agents, solubilizing agents, humectants, solvents, suspending
and/or viscosity-increasing agents, tonicity agents, wetting agents
or other biocompatible materials. A tabulation of ingredients
listed by the above categories, can be found in the U.S.
Pharmacopeia National Formulary, 1857-1859, 1990. Some examples of
the materials which can serve as pharmaceutically acceptable
carriers are sugars, such as lactose, glucose and sucrose; starches
such as corn starch and potato starch; cellulose and its
derivatives such as sodium carboxymethyl cellulose, ethyl cellulose
and cellulose acetate; powdered tragacanth; malt; gelatin; talc;
excipients such as cocoa butter and suppository waxes; oils such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,
corn oil and soybean oil; glycols, such as propylene glycol;
polyols such as glycerin, sorbitol, mannitol and polyethylene
glycol; esters such as ethyl oleate and ethyl laurate; agar;
buffering agents such as magnesium hydroxide and aluminum
hydroxide; alginic acid; pyrogen free water; isotonic saline;
Ringer's solution, ethyl alcohol and phosphate buffer solutions, as
well as other non toxic compatible substances used in
pharmaceutical formulations. Wetting agents, emulsifiers and
lubricants such as sodium lauryl sulfate and magnesium stearate, as
well as coloring agents, release agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the compositions, according to
the desires of the formulator. Examples of pharmaceutically
acceptable antioxidants include water soluble antioxidants such as
ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium
metabisulfite, sodium sulfite and the like; oil-soluble
antioxidants such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,
alpha-tocopherol and the like; and metal-chelating agents such as
citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,
tartaric acid, phosphoric acid and the like. The amount of active
ingredient that can be combined with the carrier materials to
produce a single dosage form will vary depending upon the
particular mode of administration.
[0119] Within the mucosal delivery compositions and methods of the
invention, various delivery-enhancing agents are employed which
enhance delivery of Y2 receptor-binding peptide into or across a
mucosal surface. In this regard, delivery of Y2 receptor-binding
peptide across the mucosal epithelium can occur "transcellularly"
or "paracellularly." The extent to which these pathways contribute
to the overall flux and bioavailability of the Y2 receptor-binding
peptide depends upon the environment of the mucosa, the
physico-chemical properties the active agent, and on the properties
of the mucosal epithelium. Paracellular transport involves only
passive diffusion, whereas transcellular transport can occur by
passive, facilitated or active processes. Generally, hydrophilic,
passively transported, polar solutes diffuse through the
paracellular route, while more lipophilic solutes use the
transcellular route. Absorption and bioavailability (e.g., as
reflected by a permeability coefficient or physiological assay),
for diverse, passively and actively absorbed solutes, can be
readily evaluated, in terms of both paracellular and transcellular
delivery components, for any selected Y2 receptor-binding peptide
within the invention. For passively absorbed drugs, the relative
contribution of paracellular and transcellular pathways to drug
transport depends upon the pKa, partition coefficient, molecular
radius and charge of the drug, the pH of the luminal environment in
which the drug is delivered, and the area of the absorbing surface.
The paracellular route represents a relatively small fraction of
accessible surface area of the nasal mucosal epithelium. In general
terms, it has been reported that cell membranes occupy a mucosal
surface area that is a thousand times greater than the area
occupied by the paracellular spaces. Thus, the smaller accessible
area, and the size- and charge-based discrimination against
macromolecular permeation would suggest that the paracellular route
would be a generally less favorable route than transcellular
delivery for drug transport. Surprisingly, the methods and
compositions of the invention provide for significantly enhanced
transport of biotherapeutics into and across mucosal epithelia via
the paracellular route. Therefore, the methods and compositions of
the invention successfully target both paracellular and
transcellular routes, alternatively or within a single method or
composition.
[0120] As used herein, "mucosal delivery-enhancing agents" include
agents which enhance the release or solubility (e.g., from a
formulation delivery vehicle), diffusion rate, penetration capacity
and timing, uptake, residence time, stability, effective half-life,
peak or sustained concentration levels, clearance and other desired
mucosal delivery characteristics (e.g., as measured at the site of
delivery, or at a selected target site of activity such as the
bloodstream or central nervous system) of Y2 receptor-binding
peptide or other biologically active compound(s). Enhancement of
mucosal delivery can thus occur by any of a variety of mechanisms,
for example by increasing the diffusion, transport, persistence or
stability of Y2 receptor-binding peptide, increasing membrane
fluidity, modulating the availability or action of calcium and
other ions that regulate intracellular or paracellular permeation,
solubilizing mucosal membrane components (e.g., lipids), changing
non-protein and protein sulfhydryl levels in mucosal tissues,
increasing water flux across the mucosal surface, modulating
epithelial junctional physiology, reducing the viscosity of mucus
overlying the mucosal epithelium, reducing mucociliary clearance
rates, and other mechanisms.
[0121] As used herein, a "mucosally effective amount of Y2
receptor-binding peptide" contemplates effective mucosal delivery
of Y2 receptor-binding peptide to a target site for drug activity
in the subject that may involve a variety of delivery or transfer
routes. For example, a given active agent may find its way through
clearances between cells of the mucosa and reach an adjacent
vascular wall, while by another route the agent may, either
passively or actively, be taken up into mucosal cells to act within
the cells or be discharged or transported out of the cells to reach
a secondary target site, such as the systemic circulation. The
methods and compositions of the invention may promote the
translocation of active agents along one or more such alternate
routes, or may act directly on the mucosal tissue or proximal
vascular tissue to promote absorption or penetration of the active
agent(s). The promotion of absorption or penetration in this
context is not limited to these mechanisms.
[0122] As used herein "peak concentration (C.sub.max) of Y2
receptor-binding peptide in a blood plasma", "area under
concentration vs. time curve (AUC) of Y2 receptor-binding peptide
in a blood plasma", "time to maximal plasma concentration
(t.sub.max) of Y2 receptor-binding peptide in a blood plasma" are
pharmacokinetic parameters known to one skilled in the art.
Laursen, et al., Eur. J. Endocrinology 135:309-315, 1996. The
"concentration vs. time curve" measures the concentration of Y2
receptor-binding peptide in a blood serum of a subject vs. time
after administration of a dosage of Y2 receptor-binding peptide to
the subject either by intranasal, intramuscular, subcutaneous, or
other parenteral route of administration. "C.sub.max" is the
maximum concentration of Y2 receptor-binding peptide in the blood
serum of a subject following a single dosage of Y2 receptor-binding
peptide to the subject. "t.sub.max" is the time to reach maximum
concentration of Y2 receptor-binding peptide in a blood serum of a
subject following administration of a single dosage of Y2
receptor-binding peptide to the subject.
[0123] As used herein, "area under concentration vs. time curve
(AUC) of Y2 receptor-binding peptide in a blood plasma" is
calculated according to the linear trapezoidal rule and with
addition of the residual areas. A decrease of 23% or an increase of
30% between two dosages would be detected with a probability of 90%
(type II error .beta.=10%). The "delivery rate" or "rate of
absorption" is estimated by comparison of the time (t.sub.max) to
reach the maximum concentration (C.sub.max). Both C.sub.max and
t.sub.max are analyzed using non-parametric methods. Comparisons of
the pharmacokinetics of intramuscular, subcutaneous, intravenous
and intranasal Y2 receptor-binding peptide administrations were
performed by analysis of variance (ANOVA). For pair wise
comparisons a Bonferroni-Holmes sequential procedure was used to
evaluate significance. The dose-response relationship between the
three nasal doses was estimated by regression analysis. P<0.05
was considered significant. Results are given as mean values
+/-SEM.
[0124] While the mechanism of absorption promotion may vary with
different mucosal delivery-enhancing agents of the invention,
useful reagents in this context will not substantially adversely
affect the mucosal tissue and will be selected according to the
physicochemical characteristics of the particular Y2
receptor-binding peptide or other active or delivery-enhancing
agent. In this context, delivery-enhancing agents that increase
penetration or permeability of mucosal tissues will often result in
some alteration of the protective permeability barrier of the
mucosa. For such delivery-enhancing agents to be of value within
the invention, it is generally desired that any significant changes
in permeability of the mucosa be reversible within a time frame
appropriate to the desired duration of drug delivery. Furthermore,
there should be no substantial, cumulative toxicity, nor any
permanent deleterious changes induced in the barrier properties of
the mucosa with long-term use.
[0125] Within certain aspects of the invention,
absorption-promoting agents for coordinate administration or
combinatorial formulation with Y2 receptor-binding peptide of the
invention are selected from small hydrophilic molecules, including
but not limited to, dimethyl sulfoxide (DMSO), dimethylformamide,
ethanol, propylene glycol, and the 2-pyrrolidones. Alternatively,
long-chain amphipathic molecules, for example, deacylmethyl
sulfoxide, azone, sodium laurylsulfate, oleic acid, and the bile
salts, may be employed toenhance mucosal penetration of the Y2
receptor-binding peptide. In additional aspects, surfactants (e.g.,
polysorbates) are employed as adjunct compounds, processing agents,
or formulation additives to enhance intranasal delivery of the Y2
receptor-binding peptide. Agents such as DMSO, polyethylene glycol,
and ethanol can, if present in sufficiently high concentrations in
delivery environment (e.g., by pre-administration or incorporation
in a therapeutic formulation), enter the aqueous phase of the
mucosa and alter its solubilizing properties, thereby enhancing the
partitioning of the Y2 receptor-binding peptide from the vehicle
into the mucosa.
[0126] Additional mucosal delivery-enhancing agents that are useful
within the coordinate administration and processing methods and
combinatorial formulations of the invention include, but are not
limited to, mixed micelles; enamines; nitric oxide donors (e.g.,
S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4--which are
preferably co-administered with an NO scavenger such as
carboxy-PITO or doclofenac sodium); sodium salicylate; glycerol
esters of acetoacetic acid (e.g., glyceryl-1,3-diacetoacetate or
1,2-isopropylideneglycerine-3-acetoacetate); and other
release-diffusion or intra- or trans-epithelial
penetration-promoting agents that are physiologically compatible
for mucosal delivery. Other absorption-promoting agents are
selected from a variety of carriers, bases and excipients that
enhance mucosal delivery, stability, activity or trans-epithelial
penetration of the Y2 receptor-binding peptide. These include,
inter alia, cyclodextrins and .beta.-cyclodextrin derivatives
(e.g., 2-hydroxypropyl-.beta.-cyclodextrin and
heptakis(2,6-di-O-methyl-.beta.-cyclodextrin). These compounds,
optionally conjugated with one or more of the active ingredients
and further optionally formulated in an oleaginous base, enhance
bioavailability in the mucosal formulations of the invention. Yet
additional absorption-enhancing agents adapted for mucosal delivery
include medium-chain fatty acids, including mono- and diglycerides
(e.g., sodium caprate--extracts of coconut oil, Capmul), and
triglycerides (e.g., amylodextrin, Estaram 299, Miglyol 810).
[0127] The mucosal therapeutic and prophylactic compositions of the
present invention may be supplemented with any suitable
penetration-promoting agent that facilitates absorption, diffusion,
or penetration of Y2 receptor-binding peptide across mucosal
barriers. The penetration promoter may be any promoter that is
pharmaceutically acceptable. Thus, in more detailed aspects of the
invention compositions are provided that incorporate one or more
penetration-promoting agents selected from sodium salicylate and
salicylic acid derivatives (acetyl salicylate, choline salicylate,
salicylamide, etc.); amino acids and salts thereof (e.g.,
monoaminocarboxlic acids such as glycine, alanine, phenylalanine,
proline, hydroxyproline, etc.; hydroxyamino acids such as serine;
acidic amino acids such as aspartic acid, glutamic acid, etc; and
basic amino acids such as lysine etc--inclusive of their alkali
metal or alkaline earth metal salts); and N-acetylamino acids
(N-acetylalanine, N-acetylphenylalanine, N-acetylserine,
N-acetylglycine, N-acetyllysine, N-acetylglutamic acid,
N-acetylproline, N-acetylhydroxyproline, etc.) and their salts
(alkali metal salts and alkaline earth metal salts). Also provided
as penetration-promoting agents within the methods and compositions
of the invention are substances which are generally used as
emulsifiers (e.g., sodium oleyl phosphate, sodium lauryl phosphate,
sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene
alkyl ethers, polyoxyethylene alkyl esters, etc.), caproic acid,
lactic acid, malic acid and citric acid and alkali metal salts
thereof, pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic
acid esters, N-alkylpyrrolidones, proline acyl esters, and the
like.
[0128] Within various aspects of the invention, improved nasal
mucosal delivery formulations and methods are provided that allow
delivery of Y2 receptor-binding peptide and other therapeutic
agents within the invention across mucosal barriers between
administration and selected target sites. Certain formulations are
specifically adapted for a selected target cell, tissue or organ,
or even a particular disease state. In other aspects, formulations
and methods provide for efficient, selective endo- or transcytosis
of Y2 receptor-binding peptide specifically routed along a defined
intracellular or intercellular pathway. Typically, the Y2
receptor-binding peptide is efficiently loaded at effective
concentration levels in a carrier or other delivery vehicle, and is
delivered and maintained in a stabilized form, e.g., at the nasal
mucosa and/or during passage through intracellular compartments and
membranes to a remote target site for drug action (e.g., the blood
stream or a defined tissue, organ, or extracellular compartment).
The Y2 receptor-binding peptide may be provided in a delivery
vehicle or otherwise modified (e.g., in the form of a prodrug),
wherein release or activation of the Y2 receptor-binding peptide is
triggered by a physiological stimulus (e.g., pH change, lysosomal
enzymes, etc.). Often, the Y2 receptor-binding peptide is
pharmacologically inactive until it reaches its target site for
activity. In most cases, the Y2 receptor-binding peptide and other
formulation components are non-toxic and non-immunogenic. In this
context, carriers and other formulation components are generally
selected for their ability to be rapidly degraded and excreted
under physiological conditions. At the same time, formulations are
chemically and physically stable in dosage form for effective
storage.
Peptide and Protein Analogs and Mimetics
[0129] Included within the definition of biologically active
peptides and proteins for use within the invention are natural or
synthetic, therapeutically or prophylactically active, peptides
(comprised of two or more covalently linked amino acids), proteins,
peptide or protein fragments, peptide or protein analogs, and
chemically modified derivatives or salts of active peptides or
proteins. A wide variety of useful analogs and mimetics of Y2
receptor-binding peptide are contemplated for use within the
invention and can be produced and tested for biological activity
according to known methods. Often, the peptides or proteins of Y2
receptor-binding peptide or other biologically active peptides or
proteins for use within the invention are muteins that are readily
obtainable by partial substitution, addition, or deletion of amino
acids within a naturally occurring or native (e.g., wild-type,
naturally occurring mutant, or allelic variant) peptide or protein
sequence. Additionally, biologically active fragments of native
peptides or proteins are included. Such mutant derivatives and
fragments substantially retain the desired biological activity of
the native peptide or proteins. In the case of peptides or proteins
having carbohydrate chains, biologically active variants marked by
alterations in these carbohydrate species are also included within
the invention.
[0130] As used herein, the term "conservative amino acid
substitution" refers to the general interchangeability of amino
acid residues having similar side chains. For example, a commonly
interchangeable group of amino acids having aliphatic side chains
is alanine, valine, leucine, and isoleucine; a group of amino acids
having aliphatic-hydroxyl side chains is serine and threonine; a
group of amino acids having amide-containing side chains is
asparagine and glutamine; a group of amino acids having aromatic
side chains is phenylalanine, tyrosine, and tryptophan; a group of
amino acids having basic side chains is lysine, arginine, and
histidine; and a group of amino acids having sulfur-containing side
chains is cysteine and methionine. Examples of conservative
substitutions include the substitution of a non-polar (hydrophobic)
residue such as isoleucine, valine, leucine or methionine for
another. Likewise, the present invention contemplates the
substitution of a polar (hydrophilic) residue such as between
arginine and lysine, between glutamine and asparagine, and between
threonine and serine. Additionally, the substitution of a basic
residue such as lysine, arginine or histidine for another or the
substitution of an acidic residue such as aspartic acid or glutamic
acid for another is also contemplated. Exemplary conservative amino
acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. By aligning a peptide or protein analog
optimally with a corresponding native peptide or protein, and by
using appropriate assays, e.g., adhesion protein or receptor
binding assays, to determine a selected biological activity, one
can readily identify operable peptide and protein analogs for use
within the methods and compositions of the invention. Operable
peptide and protein analogs are typically specifically
immunoreactive with antibodies raised to the corresponding native
peptide or protein.
[0131] An approach for stabilizing solid protein formulations of
the invention is to increase the physical stability of purified,
e.g., lyophilized, protein. This will inhibit aggregation via
hydrophobic interactions as well as via covalent pathways that may
increase as proteins unfold. Stabilizing formulations in this
context often include polymer-based formulations, for example a
biodegradable hydrogel formulation/delivery system. As noted above,
the critical role of water in protein structure, function, and
stability is well known. Typically, proteins are relatively stable
in the solid state with bulk water removed. However, solid
therapeutic protein formulations may become hydrated upon storage
at elevated humidities or during delivery from a sustained release
composition or device. The stability of proteins generally drops
with increasing hydration. Water can also play a significant role
in solid protein aggregation, for example, by increasing protein
flexibility resulting in enhanced accessibility of reactive groups,
by providing a mobile phase for reactants, and by serving as a
reactant in several deleterious processes such as beta-elimination
and hydrolysis.
[0132] Protein preparations containing between about 6% to 28%
water are the most unstable. Below this level, the mobility of
bound water and protein internal motions are low. Above this level,
water mobility and protein motions approach those of full
hydration. Up to a point, increased susceptibility toward
solid-phase aggregation with increasing hydration has been observed
in several systems. However, at higher water content, less
aggregation is observed because of the dilution effect.
[0133] In accordance with these principles, an effective method for
stabilizing peptides and proteins against solid-state aggregation
for mucosal delivery is to control the water content in a solid
formulation and maintain the water activity in the formulation at
optimal levels. This level depends on the nature of the protein,
but in general, proteins maintained below their "monolayer" water
coverage will exhibit superior solid-state stability.
[0134] A variety of additives, diluents, bases and delivery
vehicles are provided within the invention that effectively control
water content to enhance protein stability. These reagents and
carrier materials effective as anti-aggregation agents in this
sense include, for example, polymers of various functionalities,
such as polyethylene glycol, dextran, diethylaminoethyl dextran,
and carboxymethyl cellulose, which significantly increase the
stability and reduce the solid-phase aggregation of peptides and
proteins admixed therewith or linked thereto. In some instances,
the activity or physical stability of proteins can also be enhanced
by various additives to aqueous solutions of the peptide or protein
drugs. For example, additives, such as polyols (including sugars),
amino acids, proteins such as collagen and gelatin, and various
salts may be used.
[0135] Certain additives, in particular sugars and other polyols,
also impart significant physical stability to dry, e.g.,
lyophilized proteins. These additives can also be used within the
invention to protect the proteins against aggregation not only
during lyophilization but also during storage in the dry state. For
example sucrose and Ficoll 70 (a polymer with sucrose units)
exhibit significant protection against peptide or protein
aggregation during solid-phase incubation under various conditions.
These additives may also enhance the stability of solid proteins
embedded within polymer matrices.
[0136] Yet additional additives, for example sucrose, stabilize
proteins against solid-state aggregation in humid atmospheres at
elevated temperatures, as may occur in certain sustained-release
formulations of the invention. Proteins such as gelatin and
collagen also serve as stabilizing or bulking agents to reduce
denaturation and aggregation of unstable proteins in this context.
These additives can be incorporated into polymeric melt processes
and compositions within the invention. For example, polypeptide
microparticles can be prepared by simply lyophilizing or spray
drying a solution containing various stabilizing additives
described above. Sustained release of unaggregated peptides and
proteins can thereby be obtained over an extended period of
time.
[0137] Various additional preparative components and methods, as
well as specific formulation additives, are provided herein which
yield formulations for mucosal delivery of aggregation-prone
peptides and proteins, wherein the peptide or protein is stabilized
in a substantially pure, unaggregated form using a solubilization
agent. A range of components and additives are contemplated for use
within these methods and formulations. Exemplary of these
solubilization agents are cyclodextrins (CDs), which selectively
bind hydrophobic side chains of polypeptides. These CDs have been
found to bind to hydrophobic patches of proteins in a manner that
significantly inhibits aggregation. This inhibition is selective
with respect to both the CD and the protein involved. Such
selective inhibition of protein aggregation provides additional
advantages within the intranasal delivery methods and compositions
of the invention. Additional agents for use in this context include
CD dimers, trimers and tetramers with varying geometries controlled
by the linkers that specifically block aggregation of peptides and
protein. Yet solubilization agents and methods for incorporation
within the invention involve the use of peptides and peptide
mimetics to selectively block protein-protein interactions. In one
aspect, the specific binding of hydrophobic side chains reported
for CD multimers is extended to proteins via the use of peptides
and peptide mimetics that similarly block protein aggregation. A
wide range of suitable methods and anti-aggregation agents are
available for incorporation within the compositions and procedures
of the invention.
Charge Modifying and pH Control Agents and Methods
[0138] To improve the transport characteristics of biologically
active agents (including Y2 receptor-binding peptide, other active
peptides and proteins, and macromolecular and small molecule drugs)
for enhanced delivery across hydrophobic mucosal membrane barriers,
the invention also provides techniques and reagents for charge
modification of selected biologically active agents or
delivery-enhancing agents described herein. In this regard, the
relative permeabilities of macromolecules is generally be related
to their partition coefficients. The degree of ionization of
molecules, which is dependent on the pK.sub.a of the molecule and
the pH at the mucosal membrane surface, also affects permeability
of the molecules. Permeation and partitioning of biologically
active agents, including Y2 receptor-binding peptide and analogs of
the invention, for mucosal delivery may be facilitated by charge
alteration or charge spreading of the active agent or
permeabilizing agent, which is achieved, for example, by alteration
of charged functional groups, by modifying the pH of the delivery
vehicle or solution in which the active agent is delivered, or by
coordinate administration of a charge- or pH-altering reagent with
the active agent.
[0139] Consistent with these general teachings, mucosal delivery of
charged macromolecular species, including Y2 receptor-binding
peptide and other biologically active peptides and proteins, within
the methods and compositions of the invention is substantially
improved when the active agent is delivered to the mucosal surface
in a substantially un-ionized, or neutral, electrical charge
state.
[0140] Certain Y2 receptor-binding peptide and other biologically
active peptide and protein components of mucosal formulations for
use within the invention will be charge modified to yield an
increase in the positive charge density of the peptide or protein.
These modifications extend also to cationization of peptide and
protein conjugates, carriers and other delivery forms disclosed
herein. Cationization offers a convenient means of altering the
biodistribution and transport properties of proteins and
macromolecules within the invention. Cationization is undertaken in
a manner that substantially preserves the biological activity of
the active agent and limits potentially adverse side effects,
including tissue damage and toxicity.
Degradative Enzyme Inhibitory Agents and Methods
[0141] Another excipient that may be included in a trans-mucosal
preparation is a degradative enzyme inhibitor. Exemplary
mucoadhesive polymer-enzyme inhibitor complexes that are useful
within the mucosal delivery formulations and methods of the
invention include, but are not limited to:
Carboxymethylcellulose-pepstatin (with anti-pepsin activity);
Poly(acrylic acid)-Bowman-Birk inhibitor (anti-chymotrypsin);
Poly(acrylic acid)-chymostatin (anti-chymotrypsin); Poly(acrylic
acid)-elastatinal (anti-elastase);
Carboxymethylcellulose-elastatinal (anti-elastase);
Polycarbophil--elastatinal (anti-elastase); Chitosan--antipain
(anti-trypsin); Poly(acrylic acid)--bacitracin (anti-aminopeptidase
N); Chitosan--EDTA (anti-aminopeptidase N, anti-carboxypeptidase
A); Chitosan--EDTA--antipain (anti-trypsin, anti-chymotrypsin,
anti-elastase). As described in further detail below, certain
embodiments of the invention will optionally incorporate a novel
chitosan derivative or chemically modified form of chitosan. One
such novel derivative for use within the invention is denoted as a
.beta.-[1.fwdarw.4]-2-guanidino-2-deoxy-D-glucose polymer
(poly-GuD).
[0142] Any inhibitor that inhibits the activity of an enzyme to
protect the biologically active agent(s) may be usefully employed
in the compositions and methods of the invention. Useful enzyme
inhibitors for the protection of biologically active proteins and
peptides include, for example, soybean trypsin inhibitor,
pancreatic trypsin inhibitor, chymotrypsin inhibitor and trypsin
and chrymotrypsin inhibitor isolated from potato (solanum tuberosum
L.) tubers. A combination or mixtures of inhibitors may be
employed. Additional inhibitors of proteolytic enzymes for use
within the invention include ovomucoid-enzyme, gabaxate mesylate,
alphal-antitrypsin, aprotinin, amastatin, bestatin, puromycin,
bacitracin, leupepsin, alpha2-macroglobulin, pepstatin and egg
white or soybean trypsin inhibitor. These and other inhibitors can
be used alone or in combination. The inhibitor(s) may be
incorporated in or bound to a carrier, e.g., a hydrophilic polymer,
coated on the surface of the dosage form which is to contact the
nasal mucosa, or incorporated in the superficial phase of the
surface, in combination with the biologically active agent or in a
separately administered (e.g., pre-administered) formulation.
[0143] The amount of the inhibitor, e.g., of a proteolytic enzyme
inhibitor that is optionally incorporated in the compositions of
the invention will vary depending on (a) the properties of the
specific inhibitor, (b) the number of functional groups present in
the molecule (which may be reacted to introduce ethylenic
unsaturation necessary for copolymerization with hydrogel forming
monomers), and (c) the number of lectin groups, such as glycosides,
which are present in the inhibitor molecule. It may also depend on
the specific therapeutic agent that is intended to be administered.
Generally speaking, a useful amount of an enzyme inhibitor is from
about 0.1 mg/ml to about 50 mg/ml, often from about 0.2 mg/ml to
about 25 mg/ml, and more commonly from about 0.5 mg/ml to 5 mg/ml
of the of the formulation (i.e., a separate protease inhibitor
formulation or combined formulation with the inhibitor and
biologically active agent).
[0144] In the case of trypsin inhibition, suitable inhibitors may
be selected from, e.g., aprotinin, BBI, soybean trypsin inhibitor,
chicken ovomucoid, chicken ovoinhibitor, human pancreatic trypsin
inhibitor, camostat mesilate, flavonoid inhibitors, antipain,
leupeptin, p-aminobenzamidine, AEBSF, TLCK (tosyllysine
chloromethylketone), APMSF, DFP, PMSF, and poly(acrylate)
derivatives. In the case of chymotrypsin inhibition, suitable
inhibitors may be selected from, e.g., aprotinin, BBI, soybean
trypsin inhibitor, chymostatin, benzyloxycarbonyl-Pro-Phe-CHO,
FK-448, chicken ovoinhibitor, sugar biphenylboronic acids
complexes, DFP, PMSF, .beta.-phenylpropionate, and poly(acrylate)
derivatives. In the case of elastase inhibition, suitable
inhibitors may be selected from, e.g., elastatinal,
methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK), BBI,
soybean trypsin inhibitor, chicken ovoinhibitor, DFP, and PMSF.
[0145] Additional enzyme inhibitors for use within the invention
are selected from a wide range of non-protein inhibitors that vary
in their degree of potency and toxicity. As described in further
detail below, immobilization of these adjunct agents to matrices or
other delivery vehicles, or development of chemically modified
analogues, may be readily implemented to reduce or even eliminate
toxic effects, when they are encountered. Among this broad group of
candidate enzyme inhibitors for use within the invention are
organophosphorous inhibitors, such as diisopropylfluorophosphate
(DFP) and phenylmethylsulfonyl fluoride (PMSF), which are potent,
irreversible inhibitors of serine proteases (e.g., trypsin and
chymotrypsin). The additional inhibition of acetylcholinesterase by
these compounds makes them highly toxic in uncontrolled delivery
settings. Another candidate inhibitor,
4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF), has an
inhibitory activity comparable to DFP and PMSF, but it is markedly
less toxic (4-Aminophenyl)-methanesulfonyl fluoride hydrochloride
(APMSF) is another potent inhibitor of trypsin, but is toxic in
uncontrolled settings. In contrast to these inhibitors,
4-(4-isopropylpiperadinocarbonyl)phenyl 1,
2,3,4,-tetrahydro-1-naphthoate methanesulphonate (FK-448) is a low
toxic substance, representing a potent and specific inhibitor of
chymotrypsin. Further representatives of this non-protein group of
inhibitor candidates, and also exhibiting low toxic risk, are
camostat mesilate (N,N'-dimethyl
carbamoylmethyl-p-(p'-guanidino-benzoyloxy)phenylacetate
methane-sulphonate).
[0146] Yet another type of enzyme inhibitory agent for use within
the methods and compositions of the invention are amino acids and
modified amino acids that interfere with enzymatic degradation of
specific therapeutic compounds. For use in this context, amino
acids and modified amino acids are substantially non-toxic and can
be produced at a low cost. However, due to their low molecular size
and good solubility, they are readily diluted and absorbed in
mucosal environments. Nevertheless, under proper conditions, amino
acids can act as reversible, competitive inhibitors of protease
enzymes. Certain modified amino acids can display a much stronger
inhibitory activity. A desired modified amino acid in this context
is known as a `transition-state` inhibitor. The strong inhibitory
activity of these compounds is based on their structural similarity
to a substrate in its transition-state geometry, while they are
generally selected to have a much higher affinity for the active
site of an enzyme than the substrate itself. Transition-state
inhibitors are reversible, competitive inhibitors. Examples of this
type of inhibitor are .alpha.-aminoboronic acid derivatives, such
as boro-leucine, boro-valine and boro-alanine. The boron atom in
these derivatives can form a tetrahedral boronate ion that is
believed to resemble the transition state of peptides during their
hydrolysis by aminopeptidases. These amino acid derivatives are
potent and reversible inhibitors of aminopeptidases and it is
reported that boro-leucine is more than 100-times more effective in
enzyme inhibition than bestatin and more than 1000-times more
effective than puromycin. Another modified amino acid for which a
strong protease inhibitory activity has been reported is
N-acetylcysteine, which inhibits enzymatic activity of
aminopeptidase N. This adjunct agent also displays mucolytic
properties that can be employed within the methods and compositions
of the invention to reduce the effects of the mucus diffusion
barrier.
[0147] Still other useful enzyme inhibitors for use within the
coordinate administration methods and combinatorial formulations of
the invention may be selected from peptides and modified peptide
enzyme inhibitors. An important representative of this class of
inhibitors is the cyclic dodecapeptide, bacitracin, obtained from
Bacillus licheniformis. In addition to these types of peptides,
certain dipeptides and tripeptides display weak, non-specific
inhibitory activity towards some protease. By analogy with amino
acids, their inhibitory activity can be improved by chemical
modifications. For example, phosphinic acid dipeptide analogues are
also `transition-state` inhibitors with a strong inhibitory
activity towards aminopeptidases. They have reportedly been used to
stabilize nasally administered leucine enkephalin. Another example
of a transition-state analogue is the modified pentapeptide
pepstatin, which is a very potent inhibitor of pepsin. Structural
analysis of pepstatin, by testing the inhibitory activity of
several synthetic analogues, demonstrated the major
structure-function characteristics of the molecule responsible for
the inhibitory activity. Another special type of modified peptide
includes inhibitors with a terminally located aldehyde function in
their structure. For example, the sequence
benzyloxycarbonyl-Pro-Phe-CHO, which fulfills the known primary and
secondary specificity requirements of chymotrypsin, has been found
to be a potent reversible inhibitor of this target proteinase. The
chemical structures of further inhibitors with a terminally located
aldehyde function, e.g., antipain, leupeptin, chymostatin and
elastatinal, are also known in the art, as are the structures of
other known, reversible, modified peptide inhibitors, such as
phosphoramidon, bestatin, puromycin and amastatin.
[0148] Due to their comparably high molecular mass, polypeptide
protease inhibitors are more amenable than smaller compounds to
concentrated delivery in a drug-carrier matrix. Additional agents
for protease inhibition within the formulations and methods of the
invention involve the use of complexing agents. These agents
mediate enzyme inhibition by depriving the intranasal environment
(or preparative or therapeutic composition) of divalent cations,
which are co-factors for many proteases. For instance, the
complexing agents EDTA and DTPA as coordinately administered or
combinatorially formulated adjunct agents, in suitable
concentration, will be sufficient to inhibit selected proteases to
thereby enhance intranasal delivery of biologically active agents
according to the invention. Further representatives of this class
of inhibitory agents are EGTA, 1,10-phenanthroline and
hydroxychinoline. In addition, due to their propensity to chelate
divalent cations, these and other complexing agents are useful
within the invention as direct, absorption-promoting agents.
[0149] As noted in more detail elsewhere herein, it is also
contemplated to use various polymers, particularly mucoadhesive
polymers, as enzyme inhibiting agents within the coordinate
administration, multi-processing and/or combinatorial formulation
methods and compositions of the invention. For example,
poly(acrylate) derivatives, such as poly(acrylic acid) and
polycarbophil, can affect the activity of various proteases,
including trypsin, chymotrypsin. The inhibitory effect of these
polymers may also be based on the complexation of divalent cations
such as Ca.sup.2+ and Zn.sup.2+. It is further contemplated that
these polymers may serve as conjugate partners or carriers for
additional enzyme inhibitory agents, as described above. For
example, a chitosan-EDTA conjugate has been developed and is useful
within the invention that exhibits a strong inhibitory effect
towards the enzymatic activity of zinc-dependent proteases. The
mucoadhesive properties of polymers following covalent attachment
of other enzyme inhibitors in this context are not expected to be
substantially compromised, nor is the general utility of such
polymers as a delivery vehicle for biologically active agents
within the invention expected to be diminished. On the contrary,
the reduced distance between the delivery vehicle and mucosal
surface afforded by the mucoadhesive mechanism will minimize
presystemic metabolism of the active agent, while the covalently
bound enzyme inhibitors remain concentrated at the site of drug
delivery, minimizing undesired dilution effects of inhibitors as
well as toxic and other side effects caused thereby. In this
manner, the effective amount of a coordinately administered enzyme
inhibitor can be reduced due to the exclusion of dilution
effects.
[0150] Exemplary mucoadhesive polymer-enzyme inhibitor complexes
that are useful within the mucosal formulations and methods of the
invention include, but are not limited to:
Carboxymethylcellulose-pepstatin (with anti-pepsin activity);
Poly(acrylic acid)-Bowman-Birk inhibitor (anti-chymotrypsin);
Poly(acrylic acid)-chymostatin (anti-chymotrypsin); Poly(acrylic
acid)-elastatinal (anti-elastase);
Carboxymethylcellulose-elastatinal (anti-elastase);
Polycarbophil--elastatinal (anti-elastase); Chitosan--antipain
(anti-trypsin); Poly(acrylic acid)--bacitracin (anti-aminopeptidase
N); Chitosan--EDTA (anti-aminopeptidase N, anti-carboxypeptidase
A); Chitosan--EDTA--antipain (anti-trypsin, anti-chymotrypsin,
anti-elastase).
Mucolytic and Mucus-Clearing Agents and Methods
[0151] Effective delivery of biotherapeutic agents via intranasal
administration must take into account the decreased drug transport
rate across the protective mucus lining of the nasal mucosa, in
addition to drug loss due to binding to glycoproteins of the mucus
layer. Normal mucus is a viscoelastic, gel-like substance
consisting of water, electrolytes, mucins, macromolecules, and
sloughed epithelial cells. It serves primarily as a cytoprotective
and lubricative covering for the underlying mucosal tissues. Mucus
is secreted by randomly distributed secretory cells located in the
nasal epithelium and in other mucosal epithelia. The structural
unit of mucus is mucin. This glycoprotein is mainly responsible for
the viscoelastic nature of mucus, although other macromolecules may
also contribute to this property. In airway mucus, such
macromolecules include locally produced secretory IgA, IgM, IgE,
lysozyme, and bronchotransferrin, which also play an important role
in host defense mechanisms.
[0152] The coordinate administration methods of the instant
invention optionally incorporate effective mucolytic or
mucus-clearing agents, which serve to degrade, thin or clear mucus
from intranasal mucosal surfaces to facilitate absorption of
intranasally administered biotherapeutic agents. Within these
methods, a mucolytic or mucus-clearing agent is coordinately
administered as an adjunct compound to enhance intranasal delivery
of the biologically active agent. Alternatively, an effective
amount of a mucolytic or mucus-clearing agent is incorporated as a
processing agent within a multi-processing method of the invention,
or as an additive within a combinatorial formulation of the
invention, to provide an improved formulation that enhances
intranasal delivery of biotherapeutic compounds by reducing the
barrier effects of intranasal mucus.
[0153] A variety of mucolytic or mucus-clearing agents are
available for incorporation within the methods and compositions of
the invention. Based on their mechanisms of action, mucolytic and
mucus clearing agents can often be classified into the following
groups: proteases (e.g., pronase, papain) that cleave the protein
core of mucin glycoproteins; sulfhydryl compounds that split
mucoprotein disulfide linkages; and detergents (e.g., Triton X-100,
Tween 20) that break non-covalent bonds within the mucus.
Additional compounds in this context include, but are not limited
to, bile salts and surfactants, for example, sodium deoxycholate,
sodium taurodeoxycholate, sodium glycocholate, and
lysophosphatidylcholine.
[0154] The effectiveness of bile salts in causing structural
breakdown of mucus is in the order
deoxycholate>taurocholate>glycocholate. Other effective
agents that reduce mucus viscosity or adhesion to enhance
intranasal delivery according to the methods of the invention
include, e.g., short-chain fatty acids, and mucolytic agents that
work by chelation, such as N-acylcollagen peptides, bile acids, and
saponins (the latter function in part by chelating Ca.sup.2+ and/or
Mg.sup.2+ which play an important role in maintaining mucus layer
structure).
[0155] Additional mucolytic agents for use within the methods and
compositions of the invention include N-acetyl-L-cysteine (ACS), a
potent mucolytic agent that reduces both the viscosity and
adherence of bronchopulmonary mucus and is reported to modestly
increase nasal bioavailability of human growth hormone in
anesthetized rats (from 7.5 to 12.2%). These and other mucolytic or
mucus-clearing agents are contacted with the nasal mucosa,
typically in a concentration range of about 0.2 to 20 mM,
coordinately with administration of the biologically active agent,
to reduce the polar viscosity and/or elasticity of intranasal
mucus.
[0156] Still other mucolytic or mucus-clearing agents may be
selected from a range of glycosidase enzymes, which are able to
cleave glycosidic bonds within the mucus glycoprotein.
.alpha.-amylase and .beta.-amylase are representative of this class
of enzymes, although their mucolytic effect may be limited. In
contrast, bacterial glycosidases which allow these microorganisms
to permeate mucus layers of their hosts.
[0157] For combinatorial use with most biologically active agents
within the invention, including peptide and protein therapeutics,
non-ionogenic detergents are generally also useful as mucolytic or
mucus-clearing agents. These agents typically will not modify or
substantially impair the activity of therapeutic polypeptides.
Ciliostatic Agents and Methods
[0158] Because the self-cleaning capacity of certain mucosal
tissues (e.g., nasal mucosal tissues) by mucociliary clearance is
necessary as a protective function (e.g., to remove dust,
allergens, and bacteria), it has been generally considered that
this function should not be substantially impaired by mucosal
medications. Mucociliary transport in the respiratory tract is a
particularly important defense mechanism against infections. To
achieve this function, ciliary beating in the nasal and airway
passages moves a layer of mucus along the mucosa to removing
inhaled particles and microorganisms.
[0159] Ciliostatic agents find use within the methods and
compositions of the invention to increase the residence time of
mucosally (e.g., intranasally) administered Y2 receptor-binding
peptide, analogs and mimetics, and other biologically active agents
disclosed herein. In particular, the delivery these agents within
the methods and compositions of the invention is significantly
enhanced in certain aspects by the coordinate administration or
combinatorial formulation of one or more ciliostatic agents that
function to reversibly inhibit ciliary activity of mucosal cells,
to provide for a temporary, reversible increase in the residence
time of the mucosally administered active agent(s). For use within
these aspects of the invention, the foregoing ciliostatic factors,
either specific or indirect in their activity, are all candidates
for successful employment as ciliostatic agents in appropriate
amounts (depending on concentration, duration and mode of delivery)
such that they yield a transient (i.e., reversible) reduction or
cessation of mucociliary clearance at a mucosal site of
administration to enhance delivery of Y2 receptor-binding peptide,
analogs and mimetics, and other biologically active agents
disclosed herein, without unacceptable adverse side effects.
[0160] Within more detailed aspects, a specific ciliostatic factor
is employed in a combined formulation or coordinate administration
protocol with one or more Y2 receptor-binding peptide proteins,
analogs and mimetics, and/or other biologically active agents
disclosed herein. Various bacterial ciliostatic factors isolated
and characterized in the literature may be employed within these
embodiments of the invention. Ciliostatic factors from the
bacterium Pseudomonas aeruginosa include a phenazine derivative, a
pyo compound (2-alkyl-4-hydroxyquinolines), and a rhamnolipid (also
known as a hemolysin). The pyo compound produced ciliostasis at
concentrations of 50 .mu.g/ml and without obvious ultrastructural
lesions. The phenazine derivative also inhibited ciliary motility
but caused some membrane disruption, although at substantially
greater concentrations of 400 .mu.g/ml. Limited exposure of
tracheal explants to the rhamnolipid resulted in ciliostasis, which
was associated with altered ciliary membranes. More extensive
exposure to rhamnolipid was associated with removal of dynein arms
from axonemes.
Surface Active Agents and Methods
[0161] Within more detailed aspects of the invention, one or more
membrane penetration-enhancing agents may be employed within a
mucosal delivery method or formulation of the invention to enhance
mucosal delivery of Y2 receptor-binding peptide proteins, analogs
and mimetics, and other biologically active agents disclosed
herein. Membrane penetration enhancing agents in this context can
be selected from: (i) a surfactant, (ii) a bile salt, (iii) a
phospholipid additive, mixed micelle, liposome, or carrier, (iv) an
alcohol, (v) an enamine, (vi) an NO donor compound, (vii) a
long-chain amphipathic molecule (viii) a small hydrophobic
penetration enhancer; (ix) sodium or a salicylic acid derivative;
(x) a glycerol ester of acetoacetic acid (xi) a clyclodextrin or
beta-cyclodextrin derivative, (xii) a medium-chain fatty acid,
(xiii) a chelating agent, (xiv) an amino acid or salt thereof, (xv)
an N-acetylamino acid or salt thereof, (xvi) an enzyme degradative
to a selected membrane component, (xvii) an inhibitor of fatty acid
synthesis, or (xviii) an inhibitor of cholesterol synthesis; or
(xix) any combination of the membrane penetration enhancing agents
recited in (i)-(xviii).
[0162] Certain surface-active agents are readily incorporated
within the mucosal delivery formulations and methods of the
invention as mucosal absorption enhancing agents. These agents,
which may be coordinately administered or combinatorially
formulated with Y2 receptor-binding peptide proteins, analogs and
mimetics, and other biologically active agents disclosed herein,
may be selected from a broad assemblage of known surfactants.
Surfactants, which generally fall into three classes: (1) nonionic
polyoxyethylene ethers; (2) bile salts such as sodium glycocholate
(SGC) and deoxycholate (DOC); and (3) derivatives of fusidic acid
such as sodium taurodihydrofusidate (STDHF). The mechanisms of
action of these various classes of surface-active agents typically
include solubilization of the biologically active agent. For
proteins and peptides which often form aggregates, the surface
active properties of these absorption promoters can allow
interactions with proteins such that smaller units such as
surfactant coated monomers may be more readily maintained in
solution. Examples of other surface-active agents are
L-.alpha.-Phosphatidylcholine Didecanoyl (DDPC) polysorbate 80 and
polysorbate 20. These monomers are presumably more transportable
units than aggregates. A second potential mechanism is the
protection of the peptide or protein from proteolytic degradation
by proteases in the mucosal environment. Both bile salts and some
fusidic acid derivatives reportedly inhibit proteolytic degradation
of proteins by nasal homogenates at concentrations less than or
equivalent to those required to enhance protein absorption. This
protease inhibition may be especially important for peptides with
short biological half-lives.
Degradation Enzymes and Inhibitors of Fatty Acid and Cholesterol
Synthesis
[0163] In related aspects of the invention, Y2 receptor-binding
peptide proteins, analogs and mimetics, and other biologically
active agents for mucosal administration are formulated or
coordinately administered with a penetration enhancing agent
selected from a degradation enzyme, or a metabolic stimulatory
agent or inhibitor of synthesis of fatty acids, sterols or other
selected epithelial barrier components, U.S. Pat. No. 6,190,894.
For example, degradative enzymes such as phospholipase,
hyaluronidase, neuraminidase, and chondroitinase may be employed to
enhance mucosal penetration of Y2 receptor-binding peptide
proteins, analogs and mimetics, and other biologically active agent
without causing irreversible damage to the mucosal barrier. In one
embodiment, chondroitinase is employed within a method or
composition as provided herein to alter glycoprotein or glycolipid
constituents of the permeability barrier of the mucosa, thereby
enhancing mucosal absorption of Y2 receptor-binding peptide
proteins, analogs and mimetics, and other biologically active
agents disclosed herein.
[0164] With regard to inhibitors of synthesis of mucosal barrier
constituents, it is noted that free fatty acids account for 20-25%
of epithelial lipids by weight. Two rate-limiting enzymes in the
biosynthesis of free fatty acids are acetyl CoA carboxylase and
fatty acid synthetase. Through a series of steps, free fatty acids
are metabolized into phospholipids. Thus, inhibitors of free fatty
acid synthesis and metabolism for use within the methods and
compositions of the invention include, but are not limited to,
inhibitors of acetyl CoA carboxylase such as
5-tetradecyloxy-2-furancarboxylic acid (TOFA); inhibitors of fatty
acid synthetase; inhibitors of phospholipase A such as gomisin A,
2-(p-amylcinnamyl)amino-4-chlorobenzoic acid, bromophenacyl
bromide, monoalide, 7,7-dimethyl-5,8-eicosadienoic acid,
nicergoline, cepharanthine, nicardipine, quercetin,
dibutyryl-cyclic AMP, R-24571, N-oleoylethanolamine,
N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphostidyl serine,
cyclosporine A, topical anesthetics, including dibucaine,
prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid,
W-7, trifluoperazine, R-24571 (calmidazolium),
1-hexadocyl-3-trifluoroethyl glycero-sn-2-phosphomenthol (MJ33);
calcium channel blockers including nicardipine, verapamil,
diltiazem, nifedipine, and nimodipine; antimalarials including
quinacrine, mepacrine, chloroquine and hydroxychloroquine; beta
blockers including propanalol and labetalol; calmodulin
antagonists; EGTA; thimersol; glucocorticosteroids including
dexamethasone and prednisolone; and nonsteroidal antiinflammatory
agents including indomethacin and naproxen.
[0165] Free sterols, primarily cholesterol, account for 20-25% of
the epithelial lipids by weight. The rate limiting enzyme in the
biosynthesis of cholesterol is 3-hydroxy-3-methylglutaryl (HMG) CoA
reductase. Inhibitors of cholesterol synthesis for use within the
methods and compositions of the invention include, but are not
limited to, competitive inhibitors of (HMG) CoA reductase, such as
simvastatin, lovastatin, fluindostatin (fluvastatin), pravastatin,
mevastatin, as well as other HMG CoA reductase inhibitors, such as
cholesterol oleate, cholesterol sulfate and phosphate, and
oxygenated sterols, such as 25-OH--and 26-OH--cholesterol;
inhibitors of squalene synthetase; inhibitors of squalene
epoxidase; inhibitors of DELTA7 or DELTA24 reductases such as
22,25-diazacholesterol, 20,25-diazacholestenol, AY9944, and
triparanol.
[0166] Each of the inhibitors of fatty acid synthesis or the sterol
synthesis inhibitors may be coordinately administered or
combinatorially formulated with one or more Y2 receptor-binding
peptide proteins, analogs and mimetics, and other biologically
active agents disclosed herein to achieve enhanced epithelial
penetration of the active agent(s). An effective concentration
range for the sterol inhibitor in a therapeutic or adjunct
formulation for mucosal delivery is generally from about 0.0001% to
about 20% by weight of the total, more typically from about 0.01%
to about 5%.
Nitric Oxide Donor Agents and Methods
[0167] Within other related aspects of the invention, a nitric
oxide (NO) donor is selected as a membrane penetration-enhancing
agent to enhance mucosal delivery of one or more Y2
receptor-binding peptide proteins, analogs and mimetics, and other
biologically active agents disclosed herein. Various NO donors are
known in the art and are useful in effective concentrations within
the methods and formulations of the invention. Exemplary NO donors
include, but are not limited to, nitroglycerine, nitropruside, NOC5
[3-(2-hydroxy-1-(methyl-ethyl)-2-nitrosohydrazino)-1-propanamine],
NOC 12 [N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine],
SNAP [S-nitroso-N-acetyl-DL-penicillamine], NORI and NOR4. Within
the methods and compositions of the invention, an effective amount
of a selected NO donor is coordinately administered or
combinatorially formulated with one or more Y2 receptor-binding
peptide proteins, analogs and mimetics, and/or other biologically
active agents disclosed herein, into or through the mucosal
epithelium.
Agents for Modulating Epithelial Junction Structure and/or
Physiology
[0168] The present invention provides pharmaceutical composition
that contains one or more Y2 receptor-binding peptide proteins,
analogs or mimetics, and/or other biologically active agents in
combination with mucosal delivery enhancing agents disclosed herein
formulated in a pharmaceutical preparation for mucosal
delivery.
[0169] The permeabilizing agent reversibly enhances mucosal
epithelial paracellular transport, typically by modulating
epithelial junctional structure and/or physiology at a mucosal
epithelial surface in the subject. This effect typically involves
inhibition by the permeabilizing agent of homotypic or heterotypic
binding between epithelial membrane adhesive proteins of
neighboring epithelial cells. Target proteins for this blockade of
homotypic or heterotypic binding can be selected from various
related junctional adhesion molecules (JAMs), occludins, or
claudins. Examples of this are antibodies, antibody fragments or
single-chain antibodies that bind to the extracellular domains of
these proteins.
[0170] In yet additional detailed embodiments, the invention
provides permeabilizing peptides and peptide analogs and mimetics
for enhancing mucosal epithelial paracellular transport. The
subject peptides and peptide analogs and mimetics typically work
within the compositions and methods of the invention by modulating
epithelial junctional structure and/or physiology in a mammalian
subject. In certain embodiments, the peptides and peptide analogs
and mimetics effectively inhibit homotypic and/or heterotypic
binding of an epithelial membrane adhesive protein selected from a
junctional adhesion molecule (JAM), occludin, or claudin.
[0171] One such agent that has been extensively studied is the
bacterial toxin from Vibrio cholerae known as the "zonula occludens
toxin" (ZOT). This toxin mediates increased intestinal mucosal
permeability and causes disease symptoms including diarrhea in
infected subjects. Fasano, et al., Proc. Nat. Acad. Sci., U.S.A.
8:5242-5246, 1991. When tested on rabbit ileal mucosa, ZOT
increased the intestinal permeability by modulating the structure
of intercellular tight junctions. More recently, it has been found
that ZOT is capable of reversibly opening tight junctions in the
intestinal mucosa. It has also been reported that ZOT is capable of
reversibly opening tight junctions in the nasal mucosa. U.S. Pat.
No. 5,908,825.
[0172] Within the methods and compositions of the invention, ZOT,
as well as various analogs and mimetics of ZOT that function as
agonists or antagonists of ZOT activity, are useful for enhancing
intranasal delivery of biologically active agents--by increasing
paracellular absorption into and across the nasal mucosa. In this
context, ZOT typically acts by causing a structural reorganization
of tight junctions marked by altered localization of the junctional
protein ZO1. Within these aspects of the invention, ZOT is
coordinately administered or combinatorially formulated with the
biologically active agent in an effective amount to yield
significantly enhanced absorption of the active agent, by
reversibly increasing nasal mucosal permeability without
substantial adverse side effects.
Vasodilator Agents and Methods
[0173] Yet another class of absorption-promoting agents that shows
beneficial utility within the coordinate administration and
combinatorial formulation methods and compositions of the invention
are vasoactive compounds, more specifically vasodilators. These
compounds function within the invention to modulate the structure
and physiology of the submucosal vasculature, increasing the
transport rate of Y2 receptor-binding peptide, analogs and
mimetics, and other biologically active agents into or through the
mucosal epithelium and/or to specific target tissues or
compartments (e.g., the systemic circulation or central nervous
system.).
[0174] Vasodilator agents for use within the invention typically
cause submucosal blood vessel relaxation by either a decrease in
cytoplasmic calcium, an increase in nitric oxide (NO) or by
inhibiting myosin light chain kinase. They are generally divided
into 9 classes: calcium antagonists, potassium channel openers, ACE
inhibitors, angiotensin-II receptor antagonists, .alpha.-adrenergic
and imidazole receptor antagonists, .beta.1-adrenergic agonists,
phosphodiesterase inhibitors, eicosanoids and NO donors.
[0175] Despite chemical differences, the pharmacokinetic properties
of calcium antagonists are similar. Absorption into the systemic
circulation is high, and these agents therefore undergo
considerable first-pass metabolism by the liver, resulting in
individual variation in pharmacokinetics. Except for the newer
drugs of the dihydropyridine type (amlodipine, felodipine,
isradipine, nilvadipine, nisoldipine and nitrendipine), the
half-life of calcium antagonists is short. Therefore, to maintain
an effective drug concentration for many of these may require
delivery by multiple dosing, or controlled release formulations, as
described elsewhere herein. Treatment with the potassium channel
opener minoxidil may also be limited in manner and level of
administration due to potential adverse side effects.
[0176] ACE inhibitors prevent conversion of angiotensin-I to
angiotensin-II, and are most effective when renin production is
increased. Since ACE is identical to kininase-II, which inactivates
the potent endogenous vasodilator bradykinin, ACE inhibition causes
a reduction in bradykinin degradation. ACE inhibitors provide the
added advantage of cardioprotective and cardioreparative effects,
by preventing and reversing cardiac fibrosis and ventricular
hypertrophy in animal models. The predominant elimination pathway
of most ACE inhibitors is via renal excretion. Therefore, renal
impairment is associated with reduced elimination and a dosage
reduction of 25 to 50% is recommended in patients with moderate to
severe renal impairment.
[0177] With regard to NO donors, these compounds are particularly
useful within the invention for their additional effects on mucosal
permeability. In addition to the above-noted NO donors, complexes
of NO with nucleophiles called NO/nucleophiles, or NONOates,
spontaneously and nonenzymatically release NO when dissolved in
aqueous solution at physiologic pH. In contrast, nitro vasodilators
such as nitroglycerin require specific enzyme activity for NO
release. NONOates release NO with a defined stoichiometry and at
predictable rates ranging from <3 minutes for diethylamine/NO to
approximately 20 hours for diethylenetriamine/NO (DETANO).
[0178] Within certain methods and compositions of the invention, a
selected vasodilator agent is coordinately administered (e.g.,
systemically or intranasally, simultaneously or in combinatorially
effective temporal association) or combinatorially formulated with
one or more Y2 receptor-binding peptide, analogs and mimetics, and
other biologically active agent(s) in an amount effective to
enhance the mucosal absorption of the active agent(s) to reach a
target tissue or compartment in the subject (e.g., the liver,
hepatic portal vein, CNS tissue or fluid, or blood plasma).
Selective Transport-Enhancing Agents and Methods
[0179] The compositions and delivery methods of the invention
optionally incorporate a selective transport-enhancing agent that
facilitates transport of one or more biologically active agents.
These transport-enhancing agents may be employed in a combinatorial
formulation or coordinate administration protocol with one or more
of the Y2 receptor-binding peptide proteins, analogs and mimetics
disclosed herein, to coordinately enhance delivery of one or more
additional biologically active agent(s) across mucosal transport
barriers, to enhance mucosal delivery of the active agent(s) to
reach a target tissue or compartment in the subject (e.g., the
mucosal epithelium, liver, CNS tissue or fluid, or blood plasma).
Alternatively, the transport-enhancing agents may be employed in a
combinatorial formulation or coordinate administration protocol to
directly enhance mucosal delivery of one or more of the Y2
receptor-binding peptide proteins, analogs and mimetics, with or
without enhanced delivery of an additional biologically active
agent.
[0180] Exemplary selective transport-enhancing agents for use
within this aspect of the invention include, but are not limited
to, glycosides, sugar-containing molecules, and binding agents such
as lectin binding agents, which are known to interact specifically
with epithelial transport barrier components. For example, specific
"bioadhesive" ligands, including various plant and bacterial
lectins, which bind to cell surface sugar moieties by
receptor-mediated interactions can be employed as carriers or
conjugated transport mediators for enhancing mucosal, e.g., nasal
delivery of biologically active agents within the invention.
Certain bioadhesive ligands for use within the invention will
mediate transmission of biological signals to epithelial target
cells that trigger selective uptake of the adhesive ligand by
specialized cellular transport processes (endocytosis or
transcytosis). These transport mediators can therefore be employed
as a "carrier system" to stimulate or direct selective uptake of
one or more Y2 receptor-binding peptide proteins, analogs and
mimetics, and other biologically active agent(s) into and/or
through mucosal epithelia. These and other selective
transport-enhancing agents significantly enhance mucosal delivery
of macromolecular biopharmaceuticals (particularly peptides,
proteins, oligonucleotides and polynucleotide vectors) within the
invention. Lectins are plant proteins that bind to specific sugars
found on the surface of glycoproteins and glycolipids of eukaryotic
cells. Concentrated solutions of lectins have a `mucotractive`
effect, and various studies have demonstrated rapid receptor
mediated endocytocis (RME) of lectins and lectin conjugates (e.g.,
concanavalin A conjugated with colloidal gold particles) across
mucosal surfaces. Additional studies have reported that the uptake
mechanisms for lectins can be utilized for intestinal drug
targeting in vivo. In certain of these studies, polystyrene
nanoparticles (500 nm) were covalently coupled to tomato lectin and
reported yielded improved systemic uptake after oral administration
to rats.
[0181] In addition to plant lectins, microbial adhesion and
invasion factors provide a rich source of candidates for use as
adhesive/selective transport carriers within the mucosal delivery
methods and compositions of the invention. Two components are
necessary for bacterial adherence processes, a bacterial `adhesin`
(adherence or colonization factor) and a receptor on the host cell
surface. Bacteria causing mucosal infections need to penetrate the
mucus layer before attaching themselves to the epithelial surface.
This attachment is usually mediated by bacterial fimbriae or pilus
structures, although other cell surface components may also take
part in the process. Adherent bacteria colonize mucosal epithelia
by multiplication and initiation of a series of biochemical
reactions inside the target cell through signal transduction
mechanisms (with or without the help of toxins). Associated with
these invasive mechanisms, a wide diversity of bioadhesive proteins
(e.g., invasin, internalin) originally produced by various bacteria
and viruses are known. These allow for extracellular attachment of
such microorganisms with an impressive selectivity for host species
and even particular target tissues. Signals transmitted by such
receptor-ligand interactions trigger the transport of intact,
living microorganisms into, and eventually through, epithelial
cells by endo- and transcytotic processes. Such naturally occurring
phenomena may be harnessed (e.g., by complexing biologically active
agents such as Y2 receptor-binding peptide with an adhesin)
according to the teachings herein for enhanced delivery of
biologically active compounds into or across mucosal epithelia
and/or to other designated target sites of drug action.
[0182] Various bacterial and plant toxins that bind epithelial
surfaces in a specific, lectin-like manner are also useful within
the methods and compositions of the invention. For example,
diptheria toxin (DT) enters host cells rapidly by RME. Likewise,
the B subunit of the E. coli heat labile toxin binds to the brush
border of intestinal epithelial cells in a highly specific,
lectin-like manner. Uptake of this toxin and transcytosis to the
basolateral side of the enterocytes has been reported in vivo and
in vitro. Other researches have expressed the transmembrane domain
of diphtheria toxin in E. coli as a maltose-binding fusion protein
and coupled it chemically to high-Mw poly-L-lysine. The resulting
complex was successfully used to mediate internalization of a
reporter gene in vitro. In addition to these examples,
Staphylococcus aureus produces a set of proteins (e.g.,
staphylococcal enterotoxin A (SEA), SEB, toxic shock syndrome toxin
1 (TSST-1) which act both as superantigens and toxins. Studies
relating to these proteins have reported dose-dependent,
facilitated transcytosis of SEB and TSST-I in Caco-2 cells.
[0183] Viral haemagglutinins comprise another type of transport
agent to facilitate mucosal delivery of biologically active agents
within the methods and compositions of the invention. The initial
step in many viral infections is the binding of surface proteins
(haemagglutinins) to mucosal cells. These binding proteins have
been identified for most viruses, including rotaviruses, varicella
zoster virus, semliki forest virus, adenoviruses, potato leafroll
virus, and reovirus. These and other exemplary viral hemagglutinins
can be employed in a combinatorial formulation (e.g., a mixture or
conjugate formulation) or coordinate administration protocol with
one or more of the Y2 receptor-binding peptide, analogs and
mimetics disclosed herein, to coordinately enhance mucosal delivery
of one or more additional biologically active agent(s).
Alternatively, viral hemagglutinins can be employed in a
combinatorial formulation or coordinate administration protocol to
directly enhance mucosal delivery of one or more of the Y2
receptor-binding peptide proteins, analogs and mimetics, with or
without enhanced delivery of an additional biologically active
agent.
[0184] A variety of endogenous, selective transport-mediating
factors are also available for use within the invention. Mammalian
cells have developed an assortment of mechanisms to facilitate the
internalization of specific substrates and target these to defined
compartments. Collectively, these processes of membrane
deformations are termed `endocytosis` and comprise phagocytosis,
pinocytosis, receptor-mediated endocytosis (clathrin-mediated RME),
and potocytosis (non-clathrin-mediated RME). RME is a highly
specific cellular biologic process by which, as its name implies,
various ligands bind to cell surface receptors and are subsequently
internalized and trafficked within the cell. In many cells the
process of endocytosis is so active that the entire membrane
surface is internalized and replaced in less than a half hour. Two
classes of receptors are proposed based on their orientation in the
cell membrane; the amino terminus of Type I receptors is located on
the extracellular side of the membrane, whereas Type II receptors
have this same protein tail in the intracellular milieu.
[0185] Still other embodiments of the invention utilize transferrin
as a carrier or stimulant of RME of mucosally delivered
biologically active agents. Transferrin, an 80 kDa
iron-transporting glycoprotein, is efficiently taken up into cells
by RME. Transferrin receptors are found on the surface of most
proliferating cells, in elevated numbers on erythroblasts and on
many kinds of tumors. The transcytosis of transferrin (Tf) and
transferrin conjugates is reportedly enhanced in the presence of
Brefeldin A (BFA), a fungal metabolite. In other studies, BFA
treatment has been reported to rapidly increase apical endocytosis
of both ricin and HRP in MDCK cells. Thus, BFA and other agents
that stimulate receptor-mediated transport can be employed within
the methods of the invention as combinatorially formulated (e.g.,
conjugated) and/or coordinately administered agents to enhance
receptor-mediated transport of biologically active agents,
including Y2 receptor-binding peptide proteins, analogs and
mimetics.
Polymeric Delivery Vehicles and Methods
[0186] Within certain aspects of the invention, Y2 receptor-binding
peptide proteins, analogs and mimetics, other biologically active
agents disclosed herein, and delivery-enhancing agents as described
above, are, individually or combinatorially, incorporated within a
mucosally (e.g., nasally) administered formulation that includes a
biocompatible polymer functioning as a carrier or base. Such
polymer carriers include polymeric powders, matrices or
microparticulate delivery vehicles, among other polymer forms. The
polymer can be of plant, animal, or synthetic origin. Often the
polymer is crosslinked. Additionally, in these delivery systems the
Y2 receptor-binding peptide, analog or mimetic, can be
functionalized in a manner where it can be covalently bound to the
polymer and rendered inseparable from the polymer by simple
washing. In other embodiments, the polymer is chemically modified
with an inhibitor of enzymes or other agents which may degrade or
inactivate the biologically active agent(s) and/or delivery
enhancing agent(s). In certain formulations, the polymer is a
partially or completely water insoluble but water swellable
polymer, e.g., a hydrogel. Polymers useful in this aspect of the
invention are desirably water interactive and/or hydrophilic in
nature to absorb significant quantities of water, and they often
form hydrogels when placed in contact with water or aqueous media
for a period of time sufficient to reach equilibrium with water. In
more detailed embodiments, the polymer is a hydrogel which, when
placed in contact with excess water, absorbs at least two times its
weight of water at equilibrium when exposed to water at room
temperature, U.S. Pat. No. 6,004,583.
[0187] Drug delivery systems based on biodegradable polymers are
preferred in many biomedical applications because such systems are
broken down either by hydrolysis or by enzymatic reaction into
non-toxic molecules. The rate of degradation is controlled by
manipulating the composition of the biodegradable polymer matrix.
These types of systems can therefore be employed in certain
settings for long-term release of biologically active agents.
Biodegradable polymers such as poly(glycolic acid) (PGA),
poly-(lactic acid) (PLA), and poly(D,L-lactic-co-glycolic acid)
(PLGA), have received considerable attention as possible drug
delivery carriers, since the degradation products of these polymers
have been found to have low toxicity. During the normal metabolic
function of the body these polymers degrade into carbon dioxide and
water. These polymers have also exhibited excellent
biocompatibility.
[0188] For prolonging the biological activity of Y2
receptor-binding peptide, analogs and mimetics, and other
biologically active agents disclosed herein, as well as optional
delivery-enhancing agents, these agents may be incorporated into
polymeric matrices, e.g., polyorthoesters, polyanhydrides, or
polyesters. This yields sustained activity and release of the
active agent(s), e.g., as determined by the degradation of the
polymer matrix. Although the encapsulation of biotherapeutic
molecules inside synthetic polymers may stabilize them during
storage and delivery, the largest obstacle of polymer-based release
technology is the activity loss of the therapeutic molecules during
the formulation processes that often involve heat, sonication or
organic solvents.
[0189] Absorption-promoting polymers contemplated for use within
the invention may include derivatives and chemically or physically
modified versions of the foregoing types of polymers, in addition
to other naturally occurring or synthetic polymers, gums, resins,
and other agents, as well as blends of these materials with each
other or other polymers, so long as the alterations, modifications
or blending do not adversely affect the desired properties, such as
water absorption, hydrogel formation, and/or chemical stability for
useful application. In more detailed aspects of the invention,
polymers such as nylon, acrylan and other normally hydrophobic
synthetic polymers may be sufficiently modified by reaction to
become water swellable and/or form stable gels in aqueous
media.
[0190] Absorption-promoting polymers of the invention may include
polymers from the group of homo- and copolymers based on various
combinations of the following vinyl monomers: acrylic and
methacrylic acids, acrylamide, methacrylamide, hydroxyethylacrylate
or methacrylate, vinylpyrrolidones, as well as polyvinylalcohol and
its co- and terpolymers, polyvinylacetate, its co- and terpolymers
with the above listed monomers and
2-acrylamido-2-methyl-propanesulfonic acid (AMPS.RTM.). Very useful
are copolymers of the above listed monomers with copolymerizable
functional monomers such as acryl or methacryl amide acrylate or
methacrylate esters where the ester groups are derived from
straight or branched chain alkyl, aryl having up to four aromatic
rings which may contain alkyl substituents of 1 to 6 carbons;
steroidal, sulfates, phosphates or cationic monomers such as
N,N-dimethylaminoalkyl(meth)acrylamide,
dimethylaminoalkyl(meth)acrylate,
(meth)acryloxyalkyltrimethylammonium chloride,
(meth)acryloxyalkyldimethylbenzyl ammonium chloride.
[0191] Additional absorption-promoting polymers for use within the
invention are those classified as dextrans, dextrins, and from the
class of materials classified as natural gums and resins, or from
the class of natural polymers such as processed collagen, chitin,
chitosan, pullalan, zooglan, alginates and modified alginates such
as "Kelcoloid" (a polypropylene glycol modified alginate) gellan
gums such as "Kelocogel", Xanathan gums such as "Keltrol",
estastin, alpha hydroxy butyrate and its copolymers, hyaluronic
acid and its derivatives, polylactic and glycolic acids.
[0192] A very useful class of polymers applicable within the
instant invention are olefinically-unsaturated carboxylic acids
containing at least one activated carbon-to-carbon olefinic double
bond, and at least one carboxyl group; that is, an acid or
functional group readily converted to an acid containing an
olefinic double bond which readily functions in polymerization
because of its presence in the monomer molecule, either in the
alpha-beta position with respect to a carboxyl group, or as part of
a terminal methylene grouping. Olefinically-unsaturated acids of
this class include such materials as the acrylic acids typified by
the acrylic acid itself, alpha-cyano acrylic acid, beta
methylacrylic acid (crotonic acid), alpha-phenyl acrylic acid,
beta-acryloxy propionic acid, cinnamic acid, p-chloro cinnamic
acid, 1-carboxy-4-phenyl butadiene-1,3, itaconic acid, citraconic
acid, mesaconic acid, glutaconic acid, aconitic acid, maleic acid,
fumaric acid, and tricarboxy ethylene. As used herein, the term
"carboxylic acid" includes the polycarboxylic acids and those acid
anhydrides, such as maleic anhydride, wherein the anhydride group
is formed by the elimination of one molecule of water from two
carboxyl groups located on the same carboxylic acid molecule.
[0193] Representative acrylates useful as absorption-promoting
agents within the invention include methyl acrylate, ethyl
acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate,
isobutyl acrylate, methyl methacrylate, methyl ethacrylate, ethyl
methacrylate, octyl acrylate, heptyl acrylate, octyl methacrylate,
isopropyl methacrylate, 2-ethylhexyl methacrylate, nonyl acrylate,
hexyl acrylate, n-hexyl methacrylate, and the like. Higher alkyl
acrylic esters are decyl acrylate, isodecyl methacrylate, lauryl
acrylate, stearyl acrylate, behenyl acrylate and melissyl acrylate
and methacrylate versions thereof. Mixtures of two or three or more
long chain acrylic esters may be successfully polymerized with one
of the carboxylic monomers. Other comonomers include olefins,
including alpha olefins, vinyl ethers, vinyl esters, and mixtures
thereof.
[0194] Other vinylidene monomers, including the acrylic nitriles,
may also be used as absorption-promoting agents within the methods
and compositions of the invention to enhance delivery and
absorption of one or more Y2 receptor-binding peptide proteins,
analogs and mimetics, and other biologically active agent(s),
including to enhance delivery of the active agent(s) to a target
tissue or compartment in the subject (e.g., the liver, hepatic
portal vein, CNS tissue or fluid, or blood plasma). Useful alpha,
beta-olefinically unsaturated nitriles are preferably
monoolefinically unsaturated nitriles having from 3 to 10 carbon
atoms such as acrylonitrile, methacrylonitrile, and the like. Most
preferred are acrylonitrile and methacrylonitrile. Acrylic amides
containing from 3 to 35 carbon atoms including monoolefinically
unsaturated amides also may be used. Representative amides include
acrylamide, methacrylamide, N-t-butyl acrylamide, N-cyclohexyl
acrylamide, higher alkyl amides, where the alkyl group on the
nitrogen contains from 8 to 32 carbon atoms, acrylic amides
including N-alkylol amides of alpha, beta-olefinically unsaturated
carboxylic acids including those having from 4 to 10 carbon atoms
such as N-methylol acrylamide, N-propanol acrylamide, N-methylol
methacrylamide, N-methylol maleimide, N-methylol maleamic acid
esters, N-methylol-p-vinyl benzamide, and the like.
[0195] Yet additional useful absorption promoting materials are
alpha-olefins containing from 2 to 18 carbon atoms, more preferably
from 2 to 8 carbon atoms; dienes containing from 4 to 10 carbon
atoms; vinyl esters and allyl esters such as vinyl acetate; vinyl
aromatics such as styrene, methyl styrene and chloro-styrene; vinyl
and allyl ethers and ketones such as vinyl methyl ether and methyl
vinyl ketone; chloroacrylates; cyanoalkyl acrylates such as
alpha-cyanomethyl acrylate, and the alpha-, beta-, and
gamma-cyanopropyl acrylates; alkoxyacrylates such as methoxy ethyl
acrylate; haloacrylates as chloroethyl acrylate; vinyl halides and
vinyl chloride, vinylidene chloride and the like; divinyls,
diacrylates and other polyfunctional monomers such as divinyl
ether, diethylene glycol diacrylate, ethylene glycol
dimethacrylate, methylene-bis-acrylamide, allylpentaerythritol, and
the like; and bis (beta-haloalkyl) alkenyl phosphonates such as
bis(beta-chloroethyl) vinyl phosphonate and the like as are known
to those skilled in the art. Copolymers wherein the carboxy
containing monomer is a minor constituent, and the other vinylidene
monomers present as major components are readily prepared in
accordance with the methods disclosed herein.
[0196] When hydrogels are employed as absorption promoting agents
within the invention, these may be composed of synthetic copolymers
from the group of acrylic and methacrylic acids, acrylamide,
methacrylamide, hydroxyethylacrylate (HEA) or methacrylate (HEMA),
and vinylpyrrolidones which are water interactive and swellable.
Specific illustrative examples of useful polymers, especially for
the delivery of peptides or proteins, are the following types of
polymers: (meth)acrylamide and 0.1 to 99 wt. % (meth)acrylic acid;
(meth)acrylamides and 0.1-75 wt % (meth)acryloxyethyl
trimethyammonium chloride; (meth)acrylamide and 0.1-75 wt %
(meth)acrylamide; acrylic acid and 0.1-75 wt %
alkyl(meth)acrylates; (meth)acrylamide and 0.1-75 wt % AMPS.RTM.
(trademark of Lubrizol Corp.); (meth)acrylamide and 0 to 30 wt %
alkyl(meth)acrylamides and 0.1-75 wt % AMPS.RTM.; (meth)acrylamide
and 0.1-99 wt. % HEMA; (metb)acrylamide and 0.1 to 75 wt % HEMA and
0.1 to 99%(meth)acrylic acid; (meth)acrylic acid and 0.1-99 wt %
HEMA; 50 mole % vinyl ether and 50 mole % maleic anhydride;
(meth)acrylamide and 0.1 to 75 wt % (meth)acryloxyalky dimethyl
benzylammonium chloride; (meth)acrylamide and 0.1 to 99 wt % vinyl
pyrrolidone; (meth)acrylamide and 50 wt % vinyl pyrrolidone and
0.1-99.9 wt % (meth)acrylic acid; (meth)acrylic acid and 0.1 to 75
wt % AMPS.RTM. and 0.1-75 wt % alkyl(meth)acrylamide. In the above
examples, alkyl means C.sub.1 to C.sub.30, preferably C.sub.1 to
C.sub.22, linear and branched and C.sub.4 to C.sub.16 cyclic; where
(meth) is used, it means that the monomers with and without the
methyl group are included. Other very useful hydrogel polymers are
swellable, but insoluble versions of poly(vinyl pyrrolidone)
starch, carboxymethyl cellulose and polyvinyl alcohol.
[0197] Additional polymeric hydrogel materials useful within the
invention include (poly) hydroxyalkyl (meth)acrylate: anionic and
cationic hydrogels: poly(electrolyte) complexes; poly(vinyl
alcohols) having a low acetate residual: a swellable mixture of
crosslinked agar and crosslinked carboxymethyl cellulose: a
swellable composition comprising methyl cellulose mixed with a
sparingly crosslinked agar; a water swellable copolymer produced by
a dispersion of finely divided copolymer of maleic anhydride with
styrene, ethylene, propylene, or isobutylene; a water swellable
polymer of N-vinyl lactams; swellable sodium salts of carboxymethyl
cellulose; and the like.
[0198] Other gelable, fluid imbibing and retaining polymers useful
for forming the hydrophilic hydrogel for mucosal delivery of
biologically active agents within the invention include pectin;
polysaccharides such as agar, acacia, karaya, tragacenth, algins
and guar and their crosslinked versions; acrylic acid polymers,
copolymers and salt derivatives, polyacrylamides; water swellable
indene maleic anhydride polymers; starch graft copolymers; acrylate
type polymers and copolymers with water absorbability of about 2 to
400 times its original weight; diesters of polyglucan; a mixture of
crosslinked poly(vinyl alcohol) and poly(N-vinyl-2-pyrrolidone);
polyoxybutylene-polyethylene block copolymer gels; carob gum;
polyester gels; poly urea gels; polyether gels; polyamide gels;
polyimide gels; polypeptide gels; polyamino acid gels; poly
cellulosic gels; crosslinked indene-maleic anhydride acrylate
polymers; and polysaccharides.
[0199] Synthetic hydrogel polymers for use within the invention may
be made by an infinite combination of several monomers in several
ratios. The hydrogel can be crosslinked and generally possesses the
ability to imbibe and absorb fluid and swell or expand to an
enlarged equilibrium state. The hydrogel typically swells or
expands upon delivery to the nasal mucosal surface, absorbing about
2-5, 5-10, 10-50, up to 50-100 or more times fold its weight of
water. The optimum degree of swellability for a given hydrogel will
be determined for different biologically active agents depending
upon such factors as molecular weight, size, solubility and
diffusion characteristics of the active agent carried by or
entrapped or encapsulated within the polymer, and the specific
spacing and cooperative chain motion associated with each
individual polymer.
[0200] Hydrophilic polymers useful within the invention are water
insoluble but water swellable. Such water-swollen polymers as
typically referred to as hydrogels or gels. Such gels may be
conveniently produced from water-soluble polymer by the process of
crosslinking the polymers by a suitable crosslinking agent.
However, stable hydrogels may also be formed from specific polymers
under defined conditions of pH, temperature and/or ionic
concentration, according to know methods in the art. Typically the
polymers are cross-linked, that is, cross-linked to the extent that
the polymers possess good hydrophilic properties, have improved
physical integrity (as compared to non cross-linked polymers of the
same or similar type) and exhibit improved ability to retain within
the gel network both the biologically active agent of interest and
additional compounds for coadministration therewith such as a
cytokine or enzyme inhibitor, while retaining the ability to
release the active agent(s) at the appropriate location and
time.
[0201] Generally hydrogel polymers for use within the invention are
crosslinked with a difunctional cross-linking in the amount of from
0.01 to 25 weight percent, based on the weight of the monomers
forming the copolymer, and more preferably from 0.1 to 20 weight
percent and more often from 0.1 to 15 weight percent of the
crosslinking agent. Another useful amount of a crosslinking agent
is 0.1 to 10 weight percent. Tri, tetra or higher multifunctional
crosslinking agents may also be employed. When such reagents are
utilized, lower amounts may be required to attain equivalent
crosslinking density, i.e., the degree of crosslinking, or network
properties that are sufficient to contain effectively the
biologically active agent(s).
[0202] The crosslinks can be covalent, ionic or hydrogen bonds with
the polymer possessing the ability to swell in the presence of
water containing fluids. Such crosslinkers and crosslinking
reactions are known to those skilled in the art and in many cases
are dependent upon the polymer system. Thus a crosslinked network
may be formed by free radical copolymerization of unsaturated
monomers. Polymeric hydrogels may also be formed by crosslinking
preformed polymers by reacting functional groups found on the
polymers such as alcohols, acids, amines with such groups as
glyoxal, formaldehyde or glutaraldehyde, bis anhydrides and the
like.
[0203] The polymers also may be cross-linked with any polyene,
e.g., decadiene or trivinyl cyclohexane; acrylamides, such as
N,N-methylene-bis (acrylamide); polyfunctional acrylates, such as
trimethylol propane triacrylate; or polyfunctional vinylidene
monomer containing at least 2 terminal CH.sub.2 <groups,
including, for example, divinyl benzene, divinyl naphthlene, allyl
acrylates and the like. In certain embodiments, cross-linking
monomers for use in preparing the copolymers are polyalkenyl
polyethers having more than one alkenyl ether grouping per
molecule, which may optionally possess alkenyl groups in which an
olefinic double bond is present attached to a terminal methylene
grouping (e.g., made by the etherification of a polyhydric alcohol
containing at least 2 carbon atoms and at least 2 hydroxyl groups).
Compounds of this class may be produced by reacting an alkenyl
halide, such as allyl chloride or allyl bromide, with a strongly
alkaline aqueous solution of one or more polyhydric alcohols. The
product may be a complex mixture of polyethers with varying numbers
of ether groups. Efficiency of the polyether cross-linking agent
increases with the number of potentially polymerizable groups on
the molecule. Typically, polyethers containing an average of two or
more alkenyl ether groupings per molecule are used. Other
cross-linking monomers include for example, diallyl esters,
dimethallyl ethers, allyl or methallyl acrylates and acrylamides,
tetravinyl silane, polyalkenyl methanes, diacrylates, and
dimethacrylates, divinyl compounds such as divinyl benzene,
polyallyl phosphate, diallyloxy compounds and phosphite esters and
the like. Typical agents are allyl pentaerythritol, allyl sucrose,
trimethylolpropane triacrylate, 1,6-hexanediol diacrylate,
trimethylolpropane diallyl ether, pentaerythritol triacrylate,
tetramethylene dimethacrylate, ethylene diacrylate, ethylene
dimethacrylate, triethylene glycol dimethacrylate, and the like.
Allyl pentaerythritol, trimethylolpropane diallylether and allyl
sucrose provide suitable polymers. When the cross-linking agent is
present, the polymeric mixtures usually contain between about 0.01
to 20 weight percent, e.g., 1%, 5%, or 10% or more by weight of
cross-linking monomer based on the total of carboxylic acid
monomer, plus other monomers.
[0204] In more detailed aspects of the invention, mucosal delivery
of Y2 receptor-binding peptide, analogs and mimetics, and other
biologically active agents disclosed herein, is enhanced by
retaining the active agent(s) in a slow-release or enzymatically or
physiologically protective carrier or vehicle, for example a
hydrogel that shields the active agent from the action of the
degradative enzymes. In certain embodiments, the active agent is
bound by chemical means to the carrier or vehicle, to which may
also be admixed or bound additional agents such as enzyme
inhibitors, cytokines, etc. The active agent may alternately be
immobilized through sufficient physical entrapment within the
carrier or vehicle, e.g., a polymer matrix.
[0205] Polymers such as hydrogels useful within the invention may
incorporate functional linked agents such as glycosides chemically
incorporated into the polymer for enhancing intranasal
bioavailability of active agents formulated therewith. Examples of
such glycosides are glucosides, fructosides, galactosides,
arabinosides, mannosides and their alkyl substituted derivatives
and natural glycosides such as arbutin, phlorizin, amygdalin,
digitonin, saponin, and indican. There are several ways in which a
typical glycoside may be bound to a polymer. For example, the
hydrogen of the hydroxyl groups of a glycoside or other similar
carbohydrate may be replaced by the alkyl group from a hydrogel
polymer to form an ether. Also, the hydroxyl groups of the
glycosides may be reacted to esterify the carboxyl groups of a
polymeric hydrogel to form polymeric esters in situ. Another
approach is to employ condensation of acetobromoglucose with
cholest-5-en-3beta-ol on a copolymer of maleic acid. N-substituted
polyacrylamides can be synthesized by the reaction of activated
polymers with omega-aminoalkylglycosides: (1)
(carbohydrate-spacer)(n)-polyacrylamide, `pseudopolysaccharides`;
(2) (carbohydrate
spacer)(n)-phosphatidylethanolamine(m)-polyacrylamide,
neoglycolipids, derivatives of phosphatidylethanolamine; (3)
(carbohydrate-spacer)(n)-biotin(m)-polyacrylamide. These
biotinylated derivatives may attach to lectins on the mucosal
surface to facilitate absorption of the biologically active
agent(s), e.g., a polymer-encapsulated Y2 receptor-binding
peptide.
[0206] Within more detailed aspects of the invention, one or more
Y2 receptor-binding peptide, analogs and mimetics, and/or other
biologically active agents, disclosed herein, optionally including
secondary active agents such as protease inhibitor(s), cytokine(s),
additional modulator(s) of intercellular junctional physiology,
etc., are modified and bound to a polymeric carrier or matrix. For
example, this may be accomplished by chemically binding a peptide
or protein active agent and other optional agent(s) within a
crosslinked polymer network. It is also possible to chemically
modify the polymer separately with an interactive agent such as a
glycosidal containing molecule. In certain aspects, the
biologically active agent(s), and optional secondary active
agent(s), may be functionalized, i.e., wherein an appropriate
reactive group is identified or is chemically added to the active
agent(s). Most often an ethylenic polymerizable group is added, and
the functionalized active agent is then copolymerized with monomers
and a crosslinking agent using a standard polymerization method
such as solution polymerization (usually in water), emulsion,
suspension or dispersion polymerization. Often, the functionalizing
agent is provided with a high enough concentration of functional or
polymerizable groups to insure that several sites on the active
agent(s) are functionalized. For example, in a polypeptide
comprising 16 amine sites, it is generally desired to functionalize
at least 2, 4, 5, 7, and up to 8 or more of the sites.
[0207] After functionalization, the functionalized active agent(s)
is/are mixed with monomers and a crosslinking agent that comprise
the reagents from which the polymer of interest is formed.
Polymerization is then induced in this medium to create a polymer
containing the bound active agent(s). The polymer is then washed
with water or other appropriate solvents and otherwise purified to
remove trace unreacted impurities and, if necessary, ground or
broken up by physical means such as by stirring, forcing it through
a mesh, ultrasonication or other suitable means to a desired
particle size. The solvent, usually water, is then removed in such
a manner as to not denature or otherwise degrade the active
agent(s). One desired method is lyophilization (freeze drying) but
other methods are available and may be used (e.g., vacuum drying,
air drying, spray drying, etc.).
[0208] To introduce polymerizable groups in peptides, proteins and
other active agents within the invention, it is possible to react
available amino, hydroxyl, thiol and other reactive groups with
electrophiles containing unsaturated groups. For example,
unsaturated monomers containing N-hydroxy succinimidyl groups,
active carbonates such as p-nitrophenyl carbonate, trichlorophenyl
carbonates, tresylate, oxycarbonylimidazoles, epoxide, isocyanates
and aldehyde, and unsaturated carboxymethyl azides and unsaturated
orthopyridyl-disulfide belong to this category of reagents.
Illustrative examples of unsaturated reagents are allyl glycidyl
ether, allyl chloride, allylbromide, allyl iodide, acryloyl
chloride, allyl isocyanate, allylsulfonyl chloride, maleic
anhydride, copolymers of maleic anhydride and allyl ether, and the
like.
[0209] All of the lysine active derivatives, except aldehyde, can
generally react with other amino acids such as imidazole groups of
histidine and hydroxyl groups of tyrosine and the thiol groups of
cystine if the local environment enhances nucleophilicity of these
groups. Aldehyde containing functionalizing reagents are specific
to lysine. These types of reactions with available groups from
lysines, cysteines, tyrosine have been extensively documented in
the literature and are known to those skilled in the art.
[0210] In the case of biologically active agents that contain amine
groups, it is convenient to react such groups with an acyloyl
chloride, such as acryloyl chloride, and introduce the
polymerizable acrylic group onto the reacted agent. Then during
preparation of the polymer, such as during the crosslinking of the
copolymer of acrylamide and acrylic acid, the functionalized active
agent, through the acrylic groups, is attached to the polymer and
becomes bound thereto.
[0211] In additional aspects of the invention, biologically active
agents, including peptides, proteins, nucleosides, and other
molecules which are bioactive in vivo, are conjugation-stabilized
by covalently bonding one or more active agent(s) to a polymer
incorporating as an integral part thereof both a hydrophilic
moiety, e.g., a linear polyalkylene glycol, a lipophilic moiety
(see, e.g., U.S. Pat. No. 5,681,811). In one aspect, a biologically
active agent is covalently coupled with a polymer comprising (i) a
linear polyalkylene glycol moiety, and (ii) a lipophilic moiety,
wherein the active agent, linear polyalkylene glycol moiety, and
the lipophilic moiety are conformationally arranged in relation to
one another such that the active therapeutic agent has an enhanced
in vivo resistance to enzymatic degradation (i.e., relative to its
stability under similar conditions in an unconjugated form devoid
of the polymer coupled thereto). In another aspect, the
conjugation-stabilized formulation has a three-dimensional
conformation comprising the biologically active agent covalently
coupled with a polysorbate complex comprising (i) a linear
polyalkylene glycol moiety, and (ii) a lipophilic moiety, wherein
the active agent, the linear polyalkylene glycol moiety and the
lipophilic moiety are conformationally arranged in relation to one
another such that (a) the lipophilic moiety is exteriorly available
in the three-dimensional conformation, and (b) the active agent in
the composition has an enhanced in vivo resistance to enzymatic
degradation.
[0212] In a further related aspect, a multiligand conjugated
complex is provided which comprises a biologically active agent
covalently coupled with a triglyceride backbone moiety through a
polyalkylene glycol spacer group bonded at a carbon atom of the
triglyceride backbone moiety, and at least one fatty acid moiety
covalently attached either directly to a carbon atom of the
triglyceride backbone moiety or covalently joined through a
polyalkylene glycol spacer moiety (see, e.g., U.S. Pat. No.
5,681,811). In such a multiligand conjugated therapeutic agent
complex, the alpha' and beta carbon atoms of the triglyceride
bioactive moiety may have fatty acid moieties attached by
covalently bonding either directly thereto, or indirectly
covalently bonded thereto through polyalkylene glycol spacer
moieties. Alternatively, a fatty acid moiety may be covalently
attached either directly or through a polyalkylene glycol spacer
moiety to the alpha and alpha' carbons of the triglyceride backbone
moiety, with the bioactive therapeutic agent being covalently
coupled with the gamma-carbon of the triglyceride backbone moiety,
either being directly covalently bonded thereto or indirectly
bonded thereto through a polyalkylene spacer moiety. It will be
recognized that a wide variety of structural, compositional, and
conformational forms are possible for the multiligand conjugated
therapeutic agent complex comprising the triglyceride backbone
moiety, within the scope of the invention. It is further noted that
in such a multiligand conjugated therapeutic agent complex, the
biologically active agent(s) may advantageously be covalently
coupled with the triglyceride modified backbone moiety through
alkyl spacer groups, or alternatively other acceptable spacer
groups, within the scope of the invention. As used in such context,
acceptability of the spacer group refers to steric, compositional,
and end use application specific acceptability characteristics.
[0213] In yet additional aspects of the invention, a
conjugation-stabilized complex is provided which comprises a
polysorbate complex comprising a polysorbate moiety including a
triglyceride backbone having covalently coupled to alpha, alpha'
and beta carbon atoms thereof functionalizing groups including (i)
a fatty acid group; and (ii) a polyethylene glycol group having a
biologically active agent or moiety covalently bonded thereto,
e.g., bonded to an appropriate functionality of the polyethylene
glycol group. Such covalent bonding may be either direct, e.g., to
a hydroxy terminal functionality of the polyethylene glycol group,
or alternatively, the covalent bonding may be indirect, e.g., by
reactively capping the hydroxy terminus of the polyethylene glycol
group with a terminal carboxy functionality spacer group, so that
the resulting capped polyethylene glycol group has a terminal
carboxy functionality to which the biologically active agent or
moiety may be covalently bonded.
[0214] In yet additional aspects of the invention, a stable,
aqueously soluble, conjugation-stabilized complex is provided which
comprises one or more Y2 receptor-binding peptide proteins, analogs
and mimetics, and/or other biologically active agent(s)+ disclosed
herein covalently coupled to a physiologically compatible
polyethylene glycol (PEG) modified glycolipid moiety. In such
complex, the biologically active agent(s) may be covalently coupled
to the physiologically compatible PEG modified glycolipid moiety by
a labile covalent bond at a free amino acid group of the active
agent, wherein the labile covalent bond is scissionable in vivo by
biochemical hydrolysis and/or proteolysis. The physiologically
compatible PEG modified glycolipid moiety may advantageously
comprise a polysorbate polymer, e.g., a polysorbate polymer
comprising fatty acid ester groups selected from the group
consisting of monopalmitate, dipalmitate, monolaurate, dilaurate,
trilaurate, monoleate, dioleate, trioleate, monostearate,
distearate, and tristearate. In such complex, the physiologically
compatible PEG modified glycolipid moiety may suitably comprise a
polymer selected from the group consisting of polyethylene glycol
ethers of fatty acids, and polyethylene glycol esters of fatty
acids, wherein the fatty acids for example comprise a fatty acid
selected from the group consisting of lauric, palmitic, oleic, and
stearic acids.
Storage of Material
[0215] In certain aspects of the invention, the combinatorial
formulations and/or coordinate administration methods herein
incorporate an effective amount of peptides and proteins which may
adhere to charged glass thereby reducing the effective
concentration in the container. Silanized containers, for example,
silanized glass containers, are used to store the finished product
to reduce adsorption of the polypeptide or protein to a glass
container.
[0216] In yet additional aspects of the invention, a kit for
treatment of a mammalian subject comprises a stable pharmaceutical
composition of one or more Y2 receptor-binding peptide compound(s)
formulated for mucosal delivery to the mammalian subject wherein
the composition is effective to alleviate one or more symptom(s) of
obesity, cancer, or malnutrition or wasting related to cancer in
said subject without unacceptable adverse side effects. The kit
further comprises a pharmaceutical reagent vial to contain the one
or more Y2 receptor-binding peptide compounds. The pharmaceutical
reagent vial is composed of pharmaceutical grade polymer, glass or
other suitable material. The pharmaceutical reagent vial is, for
example, a silanized glass vial. The kit further comprises an
aperture for delivery of the composition to a nasal mucosal surface
of the subject. The delivery aperture is composed of a
pharmaceutical grade polymer, glass or other suitable material. The
delivery aperture is, for example, a silanized glass.
[0217] A silanization technique combines a special cleaning
technique for the surfaces to be silanized with a silanization
process at low pressure. The silane is in the gas phase and at an
enhanced temperature of the surfaces to be silanized. The method
provides reproducible surfaces with stable, homogeneous and
functional silane layers having characteristics of a monolayer. The
silanized surfaces prevent binding to the glass of polypeptides or
mucosal delivery enhancing agents of the present invention.
[0218] The procedure is useful to prepare silanized pharmaceutical
reagent vials to hold Y2 receptor-binding peptide compositions of
the present invention. Glass trays are cleaned by rinsing with
double distilled water (ddH.sub.2O) before using. The silane tray
is then be rinsed with 95% EtOH, and the acetone tray is rinsed
with acetone. Pharmaceutical reagent vials are sonicated in acetone
for 10 minutes. After the acetone sonication, reagent vials are
washed in ddH.sub.2O tray at least twice. Reagent vials are
sonicated in 0.1M NaOH for 10 minutes. While the reagent vials are
sonicating in NaOH, the silane solution is made under a hood.
(Silane solution: 800 mL of 95% ethanol; 96 L of glacial acetic
acid; 25 mL of glycidoxypropyltrimethoxy silane). After the NaOH
sonication, reagent vials are washed in ddH.sub.2O tray at least
twice. The reagent vials are sonicated in silane solution for 3 to
5 minutes. The reagent vials are washed in 100% EtOH tray. The
reagent vials are dried with prepurified N.sub.2 gas and stored in
a 100.degree. C. oven for at least 2 hours before using.
Bioadhesive Delivery Vehicles and Methods
[0219] In certain aspects of the invention, the combinatorial
formulations and/or coordinate administration methods herein
incorporate an effective amount of a nontoxic bioadhesive as an
adjunct compound or carrier to enhance mucosal delivery of one or
more biologically active agent(s). Bioadhesive agents in this
context exhibit general or specific adhesion to one or more
components or surfaces of the targeted mucosa. The bioadhesive
maintains a desired concentration gradient of the biologically
active agent into or across the mucosa to ensure penetration of
even large molecules (e.g., peptides and proteins) into or through
the mucosal epithelium. Typically, employment of a bioadhesive
within the methods and compositions of the invention yields a two-
to five-fold, often a five- to ten-fold increase in permeability
for peptides and proteins into or through the mucosal epithelium.
This enhancement of epithelial permeation often permits effective
transmucosal delivery of large macromolecules, for example to the
basal portion of the nasal epithelium or into the adjacent
extracellular compartments or a blood plasma or CNS tissue or
fluid.
[0220] This enhanced delivery provides for greatly improved
effectiveness of delivery of bioactive peptides, proteins and other
macromolecular therapeutic species. These results will depend in
part on the hydrophilicity of the compound, whereby greater
penetration will be achieved with hydrophilic species compared to
water insoluble compounds. In addition to these effects, employment
of bioadhesives to enhance drug persistence at the mucosal surface
can elicit a reservoir mechanism for protracted drug delivery,
whereby compounds not only penetrate across the mucosal tissue but
also back-diffuse toward the mucosal surface once the material at
the surface is depleted.
[0221] A variety of suitable bioadhesives are disclosed in the art
for oral administration, U.S. Pat. Nos. 3,972,995; 4,259,314;
4,680,323; 4,740,365; 4,573,996; 4,292,299; 4,715,369; 4,876,092;
4,855,142; 4,250,163; 4,226,848; 4,948,580; U.S. Pat. No. Reissue
33,093, which find use within the novel methods and compositions of
the invention. The potential of various bioadhesive polymers as a
mucosal, e.g., nasal, delivery platform within the methods and
compositions of the invention can be readily assessed by
determining their ability to retain and release Y2 receptor-binding
peptide, as well as by their capacity to interact with the mucosal
surfaces following incorporation of the active agent therein. In
addition, well known methods will be applied to determine the
biocompatibility of selected polymers with the tissue at the site
of mucosal administration. When the target mucosa is covered by
mucus (i.e., in the absence of mucolytic or mucus-clearing
treatment), it can serve as a connecting link to the underlying
mucosal epithelium. Therefore, the term "bioadhesive" as used
herein also covers mucoadhesive compounds useful for enhancing
mucosal delivery of biologically active agents within the
invention. However, adhesive contact to mucosal tissue mediated
through adhesion to a mucus gel layer may be limited by incomplete
or transient attachment between the mucus layer and the underlying
tissue, particularly at nasal surfaces where rapid mucus clearance
occurs. In this regard, mucin glycoproteins are continuously
secreted and, immediately after their release from cells or glands,
form a viscoelastic gel. The luminal surface of the adherent gel
layer, however, is continuously eroded by mechanical, enzymatic
and/or ciliary action. Where such activities are more prominent or
where longer adhesion times are desired, the coordinate
administration methods and combinatorial formulation methods of the
invention may further incorporate mucolytic and/or ciliostatic
methods or agents as disclosed herein above.
[0222] Typically, mucoadhesive polymers for use within the
invention are natural or synthetic macromolecules which adhere to
wet mucosal tissue surfaces by complex, but non-specific,
mechanisms. In addition to these mucoadhesive polymers, the
invention also provides methods and compositions incorporating
bioadhesives that adhere directly to a cell surface, rather than to
mucus, by means of specific, including receptor-mediated,
interactions. One example of bioadhesives that function in this
specific manner is the group of compounds known as lectins. These
are glycoproteins with an ability to specifically recognize and
bind to sugar molecules, e.g. glycoproteins or glycolipids, which
form part of intranasal epithelial cell membranes and can be
considered as "lectin receptors."
[0223] In certain aspects of the invention, bioadhesive materials
for enhancing intranasal delivery of biologically active agents
comprise a matrix of a hydrophilic, e.g., water soluble or
swellable, polymer or a mixture of polymers that can adhere to a
wet mucous surface. These adhesives may be formulated as ointments,
hydrogels (see above) thin films, and other application forms.
Often, these adhesives have the biologically active agent mixed
therewith to effectuate slow release or local delivery of the
active agent. Some are formulated with additional ingredients to
facilitate penetration of the active agent through the nasal
mucosa, e.g., into the circulatory system of the individual.
[0224] Various polymers, both natural and synthetic ones, show
significant binding to mucus and/or mucosal epithelial surfaces
under physiological conditions. The strength of this interaction
can readily be measured by mechanical peel or shear tests. When
applied to a humid mucosal surface, many dry materials will
spontaneously adhere, at least slightly. After such an initial
contact, some hydrophilic materials start to attract water by
adsorption, swelling or capillary forces, and if this water is
absorbed from the underlying substrate or from the polymer-tissue
interface, the adhesion may be sufficient to achieve the goal of
enhancing mucosal absorption of biologically active agents. Such
`adhesion by hydration` can be quite strong, but formulations
adapted to employ this mechanism must account for swelling which
continues as the dosage transforms into a hydrated mucilage. This
is projected for many hydrocolloids useful within the invention,
especially some cellulose-derivatives, which are generally
non-adhesive when applied in pre-hydrated state. Nevertheless,
bioadhesive drug delivery systems for mucosal administration are
effective within the invention when such materials are applied in
the form of a dry polymeric powder, microsphere, or film-type
delivery form.
[0225] Other polymers adhere to mucosal surfaces not only when
applied in dry, but also in fully hydrated state, and in the
presence of excess amounts of water. The selection of a
mucoadhesive thus requires due consideration of the conditions,
physiological as well as physico-chemical, under which the contact
to the tissue will be formed and maintained. In particular, the
amount of water or humidity usually present at the intended site of
adhesion, and the prevailing pH, are known to largely affect the
mucoadhesive binding strength of different polymers.
[0226] Several polymeric bioadhesive drug delivery systems have
been fabricated and studied in the past 20 years, not always with
success. A variety of such carriers are, however, currently used in
clinical applications involving dental, orthopedic,
ophthalmological, and surgical uses. For example, acrylic-based
hydrogels have been used extensively for bioadhesive devices.
Acrylic-based hydrogels are well suited for bioadhesion due to
their flexibility and nonabrasive characteristics in the partially
swollen state, which reduce damage-causing attrition to the tissues
in contact. Furthermore, their high permeability in the swollen
state allows unreacted monomer, un-crosslinked polymer chains, and
the initiator to be washed out of the matrix after polymerization,
which is an important feature for selection of bioadhesive
materials for use within the invention. Acrylic-based polymer
devices exhibit very high adhesive bond strength. For controlled
mucosal delivery of peptide and protein drugs, the methods and
compositions of the invention optionally include the use of
carriers, e.g., polymeric delivery vehicles, that function in part
to shield the biologically active agent from proteolytic breakdown,
while at the same time providing for enhanced penetration of the
peptide or protein into or through the nasal mucosa. In this
context, bioadhesive polymers have demonstrated considerable
potential for enhancing oral drug delivery. As an example, the
bioavailability of 9-desglycinamide, 8-arginine vasopressin (DGAVP)
intraduodenally administered to rats together with a 1% (w/v)
saline dispersion of the mucoadhesive poly(acrylic acid) derivative
polycarbophil, was 3-5-fold increased compared to an aqueous
solution of the peptide drug without this polymer.
[0227] Mucoadhesive polymers of the poly(acrylic acid)-type are
potent inhibitors of some intestinal proteases. The mechanism of
enzyme inhibition is explained by the strong affinity of this class
of polymers for divalent cations, such as calcium or zinc, which
are essential cofactors of metallo-proteinases, such as trypsin and
chymotrypsin. Depriving the proteases of their cofactors by
poly(acrylic acid) was reported to induce irreversible structural
changes of the enzyme proteins which were accompanied by a loss of
enzyme activity. At the same time, other mucoadhesive polymers
(e.g., some cellulose derivatives and chitosan) may not inhibit
proteolytic enzymes under certain conditions. In contrast to other
enzyme inhibitors contemplated for use within the invention (e.g.,
aprotinin, bestatin), which are relatively small molecules, the
trans-nasal absorption of inhibitory polymers is likely to be
minimal in light of the size of these molecules, and thereby
eliminate possible adverse side effects. Thus, mucoadhesive
polymers, particularly of the poly(acrylic acid)-type, may serve
both as an absorption-promoting adhesive and enzyme-protective
agent to enhance controlled delivery of peptide and protein drugs,
especially when safety concerns are considered.
[0228] In addition to protecting against enzymatic degradation,
bioadhesives and other polymeric or non-polymeric
absorption-promoting agents for use within the invention may
directly increase mucosal permeability to biologically active
agents. To facilitate the transport of large and hydrophilic
molecules, such as peptides and proteins, across the nasal
epithelial barrier, mucoadhesive polymers and other agents have
been postulated to yield enhanced permeation effects beyond what is
accounted for by prolonged premucosal residence time of the
delivery system. The time course of drug plasma concentrations
reportedly suggested that the bioadhesive microspheres caused an
acute, but transient increase of insulin permeability across the
nasal mucosa. Other mucoadhesive polymers for use within the
invention, for example chitosan, reportedly enhance the
permeability of certain mucosal epithelia even when they are
applied as an aqueous solution or gel. Another mucoadhesive polymer
reported to directly affect epithelial permeability is hyaluronic
acid and ester derivatives thereof. A particularly useful
bioadhesive agent within the coordinate administration, and/or
combinatorial formulation methods and compositions of the invention
is chitosan, as well as its analogs and derivatives. Chitosan is a
non-toxic, biocompatible and biodegradable polymer that is widely
used for pharmaceutical and medical applications because of its
favorable properties of low toxicity and good biocompatibility. It
is a natural polyaminosaccharide prepared from chitin by
N-deacetylation with alkali. As used within the methods and
compositions of the invention, chitosan increases the retention of
Y2 receptor-binding peptide proteins, analogs and mimetics, and
other biologically active agents disclosed herein at a mucosal site
of application. This mode of administration can also improve
patient compliance and acceptance. As further provided herein, the
methods and compositions of the invention will optionally include a
novel chitosan derivative or chemically modified form of chitosan.
One such novel derivative for use within the invention is denoted
as a .beta.-[1.fwdarw.4]-2-guanidino-2-deoxy-D-glucose polymer
(poly-GuD). Chitosan is the N-deacetylated product of chitin, a
naturally occurring polymer that has been used extensively to
prepare microspheres for oral and intra-nasal formulations. The
chitosan polymer has also been proposed as a soluble carrier for
parenteral drug delivery. Within one aspect of the invention,
o-methylisourea is used to convert a chitosan amine to its
guanidinium moiety. The guanidinium compound is prepared, for
example, by the reaction between equi-normal solutions of chitosan
and o-methylisourea at pH above 8.0.
[0229] The guanidinium product is -[14]-guanidino-2-deoxy-D-glucose
polymer. It is abbreviated as Poly-GuD in this context (Monomer
F.W. of Amine in Chitosan=161; Monomer F.W. of Guanidinium in
Poly-GuD=203).
[0230] One exemplary Poly-GuD preparation method for use within the
invention involves the following protocol. [0231] Solutions: [0232]
Preparation of 0.5% Acetic Acid Solution (0.088N): [0233] Pipette
2.5 mL glacial acetic acid into a 500 mL volumetric flask, dilute
to volume with purified water. [0234] Preparation of 2N NaOH
Solution: [0235] Transfer about 20 g NaOH pellets into a beaker
with about 150 mL of purified water. Dissolve and cool to room
temperature. Transfer the solution into a 250-mL volumetric flask,
dilute to volume with purified water. [0236] Preparation of
O-methylisourea Sulfate (0.4N urea group equivalent): [0237]
Transfer about 493 mg of O-methylisourea sulfate into a 10-mL
volumetric flask, dissolve and dilute to volume with purified
water. [0238] The pH of the solution is 4.2 [0239] Preparation of
Barium Chloride Solution (0.2M): [0240] Transfer about 2.086 g of
Barium chloride into a 50-mL volumetric flask, dissolve and dilute
to volume with purified water. [0241] Preparation of Chitosan
Solution (0.06N amine equivalent): [0242] Transfer about 100 mg
Chitosan into a 50 mL beaker, add 10 mL 0.5% Acetic Acid (0.088 N).
Stir to dissolve completely. [0243] The pH of the solution is about
4.5 [0244] Preparation of O-methylisourea Chloride Solution (0.2N
urea group equivalent): [0245] Pipette 5.0 mL of O-methylisourea
sulfate solution (0.4 N urea group equivalent) and 5 mL of 0.2M
Barium chloride solution into a beaker. A precipitate is formed.
Continue to mix the solution for additional 5 minutes. Filter the
solution through 0.45 m filter and discard the precipitate. The
concentration of O-methylisourea chloride in the supernatant
solution is 0.2 N urea group equivalents. [0246] The pH of the
solution is 4.2. [0247] Procedure: [0248] Add 1.5 mL of 2 N NaOH to
10 mL of the chitosan solution (0.06N amine equivalent) prepared as
described in Section 2.5. Adjust the pH of the solution with 2N
NaOH to about 8.2 to 8.4. Stir the solution for additional 10
minutes. Add 3.0 mL O-methylisourea chloride solution (0.2N urea
group equivalent) prepared as described above. Stir the solution
overnight. [0249] Adjust the pH of solution to 5.5 with 0.5% Acetic
Acid (0.088N). [0250] Dilute the solution to a final volume of 25
mL using purified water. [0251] The Poly-GuD concentration in the
solution is 5 mg/mL, equivalent to 0.025 N (guanidium group).
[0252] Additional compounds classified as bioadhesive agents for
use within the present invention act by mediating specific
interactions, typically classified as "receptor-ligand
interactions" between complementary structures of the bioadhesive
compound and a component of the mucosal epithelial surface. Many
natural examples illustrate this form of specific binding
bioadhesion, as exemplified by lectin-sugar interactions. Lectins
are (glyco) proteins of non-immune origin which bind to
polysaccharides or glycoconjugates.
[0253] Several plant lectins have been investigated as possible
pharmaceutical absorption-promoting agents. One plant lectin,
Phaseolus vulgaris hemagglutinin (PHA), exhibits high oral
bioavailability of more than 10% after feeding to rats. Tomato
(Lycopersicon esculeutum) lectin (TL) appears safe for various
modes of administration.
[0254] In summary, the foregoing bioadhesive agents are useful in
the combinatorial formulations and coordinate administration
methods of the instant invention, which optionally incorporate an
effective amount and form of a bioadhesive agent to prolong
persistence or otherwise increase mucosal absorption of one or more
Y2 receptor-binding peptide proteins, analogs and mimetics, and
other biologically active agents. The bioadhesive agents may be
coordinately administered as adjunct compounds or as additives
within the combinatorial formulations of the invention. In certain
embodiments, the bioadhesive agent acts as a `pharmaceutical glue`,
whereas in other embodiments adjunct delivery or combinatorial
formulation of the bioadhesive agent serves to intensify contact of
the biologically active agent with the nasal mucosa, in some cases
by promoting specific receptor-ligand interactions with epithelial
cell "receptors", and in others by increasing epithelial
permeability to significantly increase the drug concentration
gradient measured at a target site of delivery (e.g., liver, blood
plasma, or CNS tissue or fluid). Yet additional bioadhesive agents
for use within the invention act as enzyme (e.g., protease)
inhibitors to enhance the stability of mucosally administered
biotherapeutic agents delivered coordinately or in a combinatorial
formulation with the bioadhesive agent.
Liposomes and Micellar Delivery Vehicles
[0255] The coordinate administration methods and combinatorial
formulations of the instant invention optionally incorporate
effective lipid or fatty acid based carriers, processing agents, or
delivery vehicles, to provide improved formulations for mucosal
delivery of Y2 receptor-binding peptide proteins, analogs and
mimetics, and other biologically active agents. For example, a
variety of formulations and methods are provided for mucosal
delivery which comprise one or more of these active agents, such as
a peptide or protein, admixed or encapsulated by, or coordinately
administered with, a liposome, mixed micellar carrier, or emulsion,
to enhance chemical and physical stability and increase the half
life of the biologically active agents (e.g., by reducing
susceptibility to proteolysis, chemical modification and/or
denaturation) upon mucosal delivery.
[0256] Within certain aspects of the invention, specialized
delivery systems for biologically active agents comprise small
lipid vesicles known as liposomes. These are typically made from
natural, biodegradable, non-toxic, and non-immunogenic lipid
molecules, and can efficiently entrap or bind drug molecules,
including peptides and proteins, into, or onto, their membranes.
The attractiveness of liposomes as a peptide and protein delivery
system within the invention is increased by the fact that the
encapsulated proteins can remain in their preferred aqueous
environment within the vesicles, while the liposomal membrane
protects them against proteolysis and other destabilizing factors.
Even though not all liposome preparation methods known are feasible
in the encapsulation of peptides and proteins due to their unique
physical and chemical properties, several methods allow the
encapsulation of these macromolecules without substantial
deactivation.
[0257] A variety of methods are available for preparing liposomes
for use within the invention, U.S. Pat. Nos. 4,235,871, 4,501,728,
and 4,837,028. For use with liposome delivery, the biologically
active agent is typically entrapped within the liposome, or lipid
vesicle, or is bound to the outside of the vesicle.
[0258] Like liposomes, unsaturated long chain fatty acids, which
also have enhancing activity for mucosal absorption, can form
closed vesicles with bilayer-like structures (so called
"ufasomes"). These can be formed, for example, using oleic acid to
entrap biologically active peptides and proteins for mucosal, e.g.,
intranasal, delivery within the invention.
[0259] Other delivery systems for use within the invention combine
the use of polymers and liposomes to ally the advantageous
properties of both vehicles such as encapsulation inside the
natural polymer fibrin. In addition, release of biotherapeutic
compounds from this delivery system is controllable through the use
of covalent crosslinking and the addition of antifibrinolytic
agents to the fibrin polymer.
[0260] More simplified delivery systems for use within the
invention include the use of cationic lipids as delivery vehicles
or carriers, which can be effectively employed to provide an
electrostatic interaction between the lipid carrier and such
charged biologically active agents as proteins and polyanionic
nucleic acids. This allows efficient packaging of the drugs into a
form suitable for mucosal administration and/or subsequent delivery
to systemic compartments.
[0261] Additional delivery vehicles for use within the invention
include long and medium chain fatty acids, as well as surfactant
mixed micelles with fatty acids. Most naturally occurring lipids in
the form of esters have important implications with regard to their
own transport across mucosal surfaces. Free fatty acids and their
monoglycerides which have polar groups attached have been
demonstrated in the form of mixed micelles to act on the intestinal
barrier as penetration enhancers. This discovery of barrier
modifying function of free fatty acids (carboxylic acids with a
chain length varying from 12 to 20 carbon atoms) and their polar
derivatives has stimulated extensive research on the application of
these agents as mucosal absorption enhancers.
[0262] For use within the methods of the invention, long chain
fatty acids, especially fusogenic lipids (unsaturated fatty acids
and monoglycerides such as oleic acid, linoleic acid, linoleic
acid, monoolein, etc.) provide useful carriers to enhance mucosal
delivery of Y2 receptor-binding peptide, analogs and mimetics, and
other biologically active agents disclosed herein. Medium chain
fatty acids (C6 to C12) and monoglycerides have also been shown to
have enhancing activity in intestinal drug absorption and can be
adapted for use within the mocosal delivery formulations and
methods of the invention. In addition, sodium salts of medium and
long chain fatty acids are effective delivery vehicles and
absorption-enhancing agents for mucosal delivery of biologically
active agents within the invention. Thus, fatty acids can be
employed in soluble forms of sodium salts or by the addition of
non-toxic surfactants, e.g., polyoxyethylated hydrogenated castor
oil, sodium taurocholate, etc. Other fatty acid and mixed micellar
preparations that are useful within the invention include, but are
not limited to, Na caprylate (C8), Na caprate (C10), Na laurate
(C12) or Na oleate (C18), optionally combined with bile salts, such
as glycocholate and taurocholate.
Pegylation
[0263] Additional methods and compositions provided within the
invention involve chemical modification of biologically active
peptides and proteins by covalent attachment of polymeric
materials, for example dextrans, polyvinyl pyrrolidones,
glycopeptides, polyethylene glycol and polyamino acids. The
resulting conjugated peptides and proteins retain their biological
activities and solubility for mucosal administration. In alternate
embodiments, Y2 receptor-binding peptide proteins, analogs and
mimetics, and other biologically active peptides and proteins, are
conjugated to polyalkylene oxide polymers, particularly
polyethylene glycols (PEG). U.S. Pat. No. 4,179,337.
[0264] Amine-reactive PEG polymers for use within the invention
include SC-PEG with molecular masses of 2000, 5000, 10000, 12000,
and 20 000; U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000;
T-PEG-12000; and TPC-PEG-5000. PEGylation of biologically active
peptides and proteins may be achieved by modification of carboxyl
sites (e.g., aspartic acid or glutamic acid groups in addition to
the carboxyl terminus). The utility of PEG-hydrazide in selective
modification of carbodiimide-activated protein carboxyl groups
under acidic conditions has been described. Alternatively,
bifunctional PEG modification of biologically active peptides and
proteins can be employed. In some procedures, charged amino acid
residues, including lysine, aspartic acid, and glutamic acid, have
a marked tendency to be solvent accessible on protein surfaces.
Other Stabilizing Modifications of Active Agents
[0265] In addition to PEGylation, biologically active agents such
as peptides and proteins for use within the invention can be
modified to enhance circulating half-life by shielding the active
agent via conjugation to other known protecting or stabilizing
compounds, for example by the creation of fusion proteins with an
active peptide, protein, analog or mimetic linked to one or more
carrier proteins, such as one or more immunoglobulin chains.
Formulation and Administration
[0266] Mucosal delivery formulations of the present invention
comprise Y2 receptor-binding peptide, analogs and mimetics,
typically combined together with one or more pharmaceutically
acceptable carriers and, optionally, other therapeutic ingredients.
The carrier(s) must be "pharmaceutically acceptable" in the sense
of being compatible with the other ingredients of the formulation
and not eliciting an unacceptable deleterious effect in the
subject. Such carriers are described herein above or are otherwise
well known to those skilled in the art of pharmacology. Desirably,
the formulation should not include substances such as enzymes or
oxidizing agents with which the biologically active agent to be
administered is known to be incompatible. The formulations may be
prepared by any of the methods well known in the art of
pharmacy.
[0267] Within the compositions and methods of the invention, the Y2
receptor-binding peptide proteins, analogs and mimetics, and other
biologically active agents disclosed herein may be administered to
subjects by a variety of mucosal administration modes, including by
oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal
delivery, or by topical delivery to the eyes, ears, skin or other
mucosal surfaces. Optionally, Y2 receptor-binding peptide proteins,
analogs and mimetics, and other biologically active agents
disclosed herein can be coordinately or adjunctively administered
by non-mucosal routes, including by intramuscular, subcutaneous,
intravenous, intra-atrial, intra-articular, intraperitoneal, or
parenteral routes. In other alternative embodiments, the
biologically active agent(s) can be administered ex vivo by direct
exposure to cells, tissues or organs originating from a mammalian
subject, for example as a component of an ex vivo tissue or organ
treatment formulation that contains the biologically active agent
in a suitable, liquid or solid carrier.
[0268] Compositions according to the present invention are often
administered in an aqueous solution as a nasal or pulmonary spray
and may be dispensed in spray form by a variety of methods known to
those skilled in the art. Preferred systems for dispensing liquids
as a nasal spray are disclosed in U.S. Pat. No. 4,511,069. The
formulations may be presented in multi-dose containers, for example
in the sealed dispensing system disclosed in U.S. Pat. No.
4,511,069. Additional aerosol delivery forms may include, e.g.,
compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers,
which deliver the biologically active agent dissolved or suspended
in a pharmaceutical solvent, e.g., water, ethanol, or a mixture
thereof.
[0269] Nasal and pulmonary spray solutions of the present invention
typically comprise the drug or drug to be delivered, optionally
formulated with a surface-active agent, such as a nonionic
surfactant (e.g., polysorbate-80), and one or more buffers. In some
embodiments of the present invention, the nasal spray solution
further comprises a propellant. The pH of the nasal spray solution
is optionally between about pH 3.0 and 6.0, preferably 5.0.+-.0.3.
Suitable buffers for use within these compositions are as described
above or as otherwise known in the art. Other components may be
added to enhance or maintain chemical stability, including
preservatives, surfactants, dispersants, or gases. Suitable
preservatives include, but are not limited to, phenol, methyl
paraben, paraben, m-cresol, thiomersal, chlorobutanol,
benzylalkonimum chloride, and the like. Suitable surfactants
include, but are not limited to, oleic acid, sorbitan trioleate,
polysorbates, lecithin, phosphotidyl cholines, and various long
chain diglycerides and phospholipids. Suitable dispersants include,
but are not limited to, ethylenediaminetetraacetic acid, and the
like. Suitable gases include, but are not limited to, nitrogen,
helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs),
carbon dioxide, air, and the like.
[0270] Within alternate embodiments, mucosal formulations are
administered as dry powder formulations comprising the biologically
active agent in a dry, usually lyophilized, form of an appropriate
particle size, or within an appropriate particle size range, for
intranasal delivery. Minimum particle size appropriate for
deposition within the nasal or pulmonary passages is often about
0.5 .mu. mass median equivalent aerodynamic diameter (MMEAD),
commonly about 1 .mu. MMEAD, and more typically about 2 .mu. MMEAD.
Maximum particle size appropriate for deposition within the nasal
passages is often about 10 .mu. MMEAD, commonly about 8 .mu. MMEAD,
and more typically about 4 .mu. MMEAD. Intranasally respirable
powders within these size ranges can be produced by a variety of
conventional techniques, such as jet milling, spray drying, solvent
precipitation, supercritical fluid condensation, and the like.
These dry powders of appropriate MMEAD can be administered to a
patient via a conventional dry powder inhaler (DPI), which rely on
the patient's breath, upon pulmonary or nasal inhalation, to
disperse the power into an aerosolized amount. Alternatively, the
dry powder may be administered via air-assisted devices that use an
external power source to disperse the powder into an aerosolized
amount, e.g., a piston pump.
[0271] Dry powder devices typically require a powder mass in the
range from about 1 mg to 20 mg to produce a single aerosolized dose
("puff"). If the required or desired dose of the biologically
active agent is lower than this amount, the powdered active agent
will typically be combined with a pharmaceutical dry bulking powder
to provide the required total powder mass. Preferred dry bulking
powders include sucrose, lactose, dextrose, mannitol, glycine,
trehalose, human serum albumin (HSA), and starch. Other suitable
dry bulking powders include cellobiose, dextrans, maltotriose,
pectin, sodium citrate, sodium ascorbate, and the like.
[0272] To formulate compositions for mucosal delivery within the
present invention, the biologically active agent can be combined
with various pharmaceutically acceptable additives, as well as a
base or carrier for dispersion of the active agent(s). Desired
additives include, but are not limited to, pH control agents, such
as arginine, sodium hydroxide, glycine, hydrochloric acid, citric
acid, etc. In addition, local anesthetics (e.g., benzyl alcohol),
isotonizing agents (e.g., sodium chloride, mannitol, sorbitol),
adsorption inhibitors (e.g., Tween 80), solubility enhancing agents
(e.g., cyclodextrins and derivatives thereof), stabilizers (e.g.,
serum albumin), and reducing agents (e.g., glutathione) can be
included. When the composition for mucosal delivery is a liquid,
the tonicity of the formulation, as measured with reference to the
tonicity of 0.9% (w/v) physiological saline solution taken as
unity, is typically adjusted to a value at which no substantial,
irreversible tissue damage will be induced in the nasal mucosa at
the site of administration. Generally, the tonicity of the solution
is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2,
and most often 3/4 to 1.7.
[0273] The biologically active agent may be dispersed in a base or
vehicle, which may comprise a hydrophilic compound having a
capacity to disperse the active agent and any desired additives.
The base may be selected from a wide range of suitable carriers,
including but not limited to, copolymers of polycarboxylic acids or
salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with
other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.),
hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl
alcohol, polyvinylpyrrolidone, cellulose derivatives such as
hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural
polymers such as chitosan, collagen, sodium alginate, gelatin,
hyaluronic acid, and nontoxic metal salts thereof. Often, a
biodegradable polymer is selected as a base or carrier, for
example, polylactic acid, poly(lactic acid-glycolic acid)
copolymer, polyhydroxybutyric acid, poly(hydroxybutyric
acid-glycolic acid) copolymer and mixtures thereof. Alternatively
or additionally, synthetic fatty acid esters such as polyglycerin
fatty acid esters, sucrose fatty acid esters, etc. can be employed
as carriers. Hydrophilic polymers and other carriers can be used
alone or in combination, and enhanced structural integrity can be
imparted to the carrier by partial crystallization, ionic bonding,
crosslinking and the like. The carrier can be provided in a variety
of forms, including, fluid or viscous solutions, gels, pastes,
powders, microspheres and films for direct application to the nasal
mucosa. The use of a selected carrier in this context may result in
promotion of absorption of the biologically active agent.
[0274] The biologically active agent can be combined with the base
or carrier according to a variety of methods, and release of the
active agent may be by diffusion, disintegration of the carrier, or
associated formulation of water channels. In some circumstances,
the active agent is dispersed in microcapsules (microspheres) or
nanocapsules (nanospheres) prepared from a suitable polymer, e.g.,
isobutyl 2-cyanoacrylate and dispersed in a biocompatible
dispersing medium applied to the nasal mucosa, which yields
sustained delivery and biological activity over a protracted
time.
[0275] To further enhance mucosal delivery of pharmaceutical agents
within the invention, formulations comprising the active agent may
also contain a hydrophilic low molecular weight compound as a base
or excipient. Such hydrophilic low molecular weight compounds
provide a passage medium through which a water-soluble active
agent, such as a physiologically active peptide or protein, may
diffuse through the base to the body surface where the active agent
is absorbed. The hydrophilic low molecular weight compound
optionally absorbs moisture from the mucosa or the administration
atmosphere and dissolves the water-soluble active peptide. The
molecular weight of the hydrophilic low molecular weight compound
is generally not more than 10000 and preferably not more than 3000.
Exemplary hydrophilic low molecular weight compound include polyol
compounds, such as oligo-, di- and monosaccarides such as sucrose,
mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose,
D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose,
gentibiose, glycerin and polyethylene glycol. Other examples of
hydrophilic low molecular weight compounds useful as carriers
within the invention include N-methylpyrrolidone, and alcohols
(e.g. oligovinyl alcohol, ethanol, ethylene glycol, propylene
glycol, etc.). These hydrophilic low molecular weight compounds can
be used alone or in combination with one another or with other
active or inactive components of the intranasal formulation.
[0276] The compositions of the invention may alternatively contain
as pharmaceutically acceptable carriers substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, etc. For solid compositions, conventional
nontoxic pharmaceutically acceptable carriers can be used which
include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, magnesium carbonate, and the like.
[0277] Therapeutic compositions for administering the biologically
active agent can also be formulated as a solution, microemulsion,
or other ordered structure suitable for high concentration of
active ingredients. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. Proper
fluidity for solutions can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of a desired
particle size in the case of dispersible formulations, and by the
use of surfactants. In many cases, it will be desirable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the biologically active agent can be
brought about by including in the composition an agent which delays
absorption, for example, monostearate salts and gelatin.
[0278] In certain embodiments of the invention, the biologically
active agent is administered in a time-release formulation, for
example in a composition which includes a slow release polymer. The
active agent can be prepared with carriers that will protect
against rapid release, for example a controlled release vehicle
such as a polymer, microencapsulated delivery system or bioadhesive
gel. Prolonged delivery of the active agent, in various
compositions of the invention can be brought about by including in
the composition agents that delay absorption, for example, aluminum
monosterate hydrogels and gelatin. When controlled release
formulations of the biologically active agent is desired,
controlled release binders suitable for use in accordance with the
invention include any biocompatible controlled-release material
which is inert to the active agent and which is capable of
incorporating the biologically active agent. Numerous such
materials are known in the art. Useful controlled-release binders
are materials that are metabolized slowly under physiological
conditions following their intranasal delivery (e.g., at the nasal
mucosal surface, or in the presence of bodily fluids following
transmucosal delivery). Appropriate binders include but are not
limited to biocompatible polymers and copolymers previously used in
the art in sustained release formulations. Such biocompatible
compounds are non-toxic and inert to surrounding tissues, and do
not trigger significant adverse side effects such as nasal
irritation, immune response, inflammation, or the like. They are
metabolized into metabolic products that are also biocompatible and
easily eliminated from the body.
[0279] Exemplary polymeric materials for use in this context
include, but are not limited to, polymeric matrices derived from
copolymeric and homopolymeric polyesters having hydrolysable ester
linkages. A number of these are known in the art to be
biodegradable and to lead to degradation products having no or low
toxicity. Exemplary polymers include polyglycolic acids (PGA) and
polylactic acids (PLA), poly(DL-lactic acid-co-glycolic acid)(DL
PLGA), poly(D-lactic acid-coglycolic acid)(D PLGA) and
poly(L-lactic acid-co-glycolic acid)(L PLGA). Other useful
biodegradable or bioerodable polymers include but are not limited
to such polymers as poly(epsilon-caprolactone),
poly(epsilon-aprolactone-CO-lactic acid),
poly(.epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy
butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels such as
poly(hydroxyethyl methacrylate), polyamides, poly(amino acids)
(i.e., L-leucine, glutamic acid, L-aspartic acid and the like),
poly (ester urea), poly (2-hydroxyethyl DL-aspartamide), polyacetal
polymers, polyorthoesters, polycarbonate, polymaleamides,
polysaccharides and copolymers thereof. Many methods for preparing
such formulations are generally known to those skilled in the art.
Other useful formulations include controlled-release compositions
e.g., microcapsules, U.S. Pat. Nos. 4,652,441 and 4,917,893, lactic
acid-glycolic acid copolymers useful in making microcapsules and
other formulations, U.S. Pat. Nos. 4,677,191 and 4,728,721, and
sustained-release compositions for water-soluble peptides, U.S.
Pat. No. 4,675,189.
[0280] Sterile solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders, methods of preparation include vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The prevention of the action of
microorganisms can be accomplished by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like.
[0281] Mucosal administration according to the invention allows
effective self-administration of treatment by patients, provided
that sufficient safeguards are in place to control and monitor
dosing and side effects. Mucosal administration also overcomes
certain drawbacks of other administration forms, such as
injections, that are painful and expose the patient to possible
infections and may present drug bioavailability problems. For nasal
and pulmonary delivery, systems for controlled aerosol dispensing
of therapeutic liquids as a spray are well known. In one
embodiment, metered doses of active agent are delivered by means of
a specially constructed mechanical pump valve, U.S. Pat. No.
4,511,069.
Dosage
[0282] For prophylactic and treatment purposes, the biologically
active agent(s) disclosed herein may be administered to the subject
in a single bolus delivery, via continuous delivery (e.g.,
continuous transdermal, mucosal, or intravenous delivery) over an
extended time period, or in a repeated administration protocol
(e.g., by an hourly, daily or weekly, repeated administration
protocol). In this context, a therapeutically effective dosage of
the Y2 receptor-binding peptide may include repeated doses within a
prolonged prophylaxis or treatment regimen that will yield
clinically significant results to alleviate one or more symptoms or
detectable conditions associated with a targeted disease or
condition as set forth above. Determination of effective dosages in
this context is typically based on animal model studies followed up
by human clinical trials and is guided by determining effective
dosages and administration protocols that significantly reduce the
occurrence or severity of targeted disease symptoms or conditions
in the subject. Suitable models in this regard include, for
example, murine, rat, porcine, feline, non-human primate, and other
accepted animal model subjects known in the art. Alternatively,
effective dosages can be determined using in vitro models (e.g.,
immunologic and histopathologic assays). Using such models, only
ordinary calculations and adjustments are typically required to
determine an appropriate concentration and dose to administer a
therapeutically effective amount of the biologically active
agent(s) (e.g., amounts that are intranasally effective,
transdermally effective, intravenously effective, or
intramuscularly effective to elicit a desired response).
[0283] The actual dosage of biologically active agents will of
course vary according to factors such as the disease indication and
particular status of the subject (e.g., the subject's age, size,
fitness, extent of symptoms, susceptibility factors, etc), time and
route of administration, other drugs or treatments being
administered concurrently, as well as the specific pharmacology of
the biologically active agent(s) for eliciting the desired activity
or biological response in the subject. Dosage regimens may be
adjusted to provide an optimum prophylactic or therapeutic
response. A therapeutically effective amount is also one in which
any toxic or detrimental side effects of the biologically active
agent are outweighed in clinical terms by therapeutically
beneficial effects. A non-limiting range for a therapeutically
effective amount of an Y2 agonist within the methods and
formulations of the invention is 0.7 .mu.g/kg to about 25 .mu.g/kg.
To promote weight loss, an intranasal dose of Y2 receptor-binding
peptide is administered at dose high enough to promote satiety but
low enough so as not to induce any unwanted side-effects such as
nausea. A preferred intranasal dose of PYY.sub.3-36 is about 1
.mu.g-10 .mu.g/kg weight of the patient, most preferably from about
1.5 .mu.g/kg to about 3 .mu.g/kg weight of the patient. In a
standard dose a patient will receive 50 .mu.g to 1600 .mu.g, more
preferably about between 75 .mu.g to 800 .mu.g, most preferably 100
.mu.g, 150 .mu.g, 200 .mu.g to about 400 .mu.g. Alternatively, a
non-limiting range for a therapeutically effective amount of a
biologically active agent within the methods and formulations of
the invention is between about 0.001 pmol to about 100 pmol per kg
body weight, between about 0.01 pmol to about 10 pmol per kg body
weight, between about 0.1 pmol to about 5 pmol per kg body weight,
or between about 0.5 pmol to about 1.0 pmol per kg body weight.
Dosages within this range can be achieved by single or multiple
administrations, including, e.g., multiple administrations per day,
daily or weekly administrations. Per administration, it is
desirable to administer at least one microgram of the biologically
active agent (e.g., one or more Y2 receptor-binding peptide
proteins, analogs and mimetics, and other biologically active
agents), more typically between about 10 .mu.g and 5.0 mg, and in
certain embodiments between about 100 .mu.g and 1.0 or 2.0 mg to an
average human subject. For certain oral applications, doses as high
as 0.5 mg per kg body weight may be necessary to achieve adequate
plasma levels. It is to be further noted that for each particular
subject, specific dosage regimens should be evaluated and adjusted
over time according to the individual need and professional
judgment of the person administering or supervising the
administration of the permeabilizing peptide(s) and other
biologically active agent(s). An intranasal dose of a PYY will
range from 50 .mu.g to 1600 .mu.g of PYY, preferably 75 .mu.g to
800 .mu.g, more preferably 100 .mu.g to 400 .mu.g with a most
preferred dose being between 100 .mu.g to 200 .mu.g with 150 .mu.g
being a dose that is considered to be highly effective. Repeated
intranasal dosing with the formulations of the invention, on a
schedule ranging from about 0.1 to 24 hours between doses,
preferably between 0.5 and 24.0 hours between doses, will maintain
normalized, sustained therapeutic levels of Y2 receptor-binding
peptide to maximize clinical benefits while minimizing the risks of
excessive exposure and side effects. This dose can be administered
several times a day to promote satiety, preferably one half hour
before a meal or when hunger occurs. The goal is to mucosally
deliver an amount of the Y2 receptor-binding peptide sufficient to
raise the concentration of the Y2 receptor-binding peptide in the
plasma of an individual to mimic the concentration that would
normally occur postpradially, i.e., after the individual has
finished eating.
[0284] Dosage of Y2 agonists such as PYY may be varied by the
attending clinician or patient, if self administering an over the
counter dosage form, to maintain a desired concentration at the
target site.
[0285] In an alternative embodiment, the invention provides
compositions and methods for intranasal delivery of Y2
receptor-binding peptide, wherein the Y2 receptor-binding peptide
compound(s) is/are repeatedly administered through an intranasal
effective dosage regimen that involves multiple administrations of
the Y2 receptor-binding peptide to the subject during a daily or
weekly schedule to maintain a therapeutically effective elevated
and lowered pulsatile level of Y2 receptor-binding peptide during
an extended dosing period. The compositions and method provide Y2
receptor-binding peptide compound(s) that are self-administered by
the subject in a nasal formulation between one and six times daily
to maintain a therapeutically effective elevated and lowered
pulsatile level of Y2 receptor-binding peptide during an 8 hour to
24 hour extended dosing period.
Kits
[0286] The instant invention also includes kits, packages and
multicontainer units containing the above described pharmaceutical
compositions, active ingredients, and/or means for administering
the same for use in the prevention and treatment of diseases and
other conditions in mammalian subjects. Briefly, these kits include
a container or formulation that contains one or more Y2
receptor-binding peptide proteins, analogs or mimetics, and/or
other biologically active agents in combination with mucosal
delivery enhancing agents disclosed herein formulated in a
pharmaceutical preparation for mucosal delivery.
[0287] The intranasal formulations of the present invention can be
administered using any spray bottle or syringe. An example of a
nasal spray bottle is the, "Nasal Spray Pump w/Safety Clip,
Pfeiffer SAP #60548, which delivers a dose of 0.1 mL per squirt and
has a diptube length of 36.05 mm. It can be purchased from Pfeiffer
of America of Princeton, N.J. Intranasal doses of a Y2
receptor-binding peptide such as PYY can range from 0.1 .mu.g/kg to
about 1500 .mu.g/kg. When administered in as an intranasal spray,
it is preferable that the particle size of the spray are between
10-100 .mu.m (microns) in size, preferably 20-100 .mu.m in
size.
[0288] To promote weight loss, an intranasal dose of a Y2
receptor-binding peptide PYY is administered at dose high enough to
promote satiety but low enough so as not to induce any unwanted
side-effects such as nausea. A preferred intranasal dose of a Y2
receptor-binding peptide such as PYY(3-36) is about 3 .mu.g -10
.mu.g/kg weight of the patient, most preferably about 6 .mu.g/kg
weight of the patient. In a standard dose a patient will receive 50
.mu.g to 800 .mu.g, more preferably about between 100 .mu.g to 400
.mu.g, most preferably 150 .mu.g to about 200 .mu.g. The a Y2
receptor-binding peptide such as PYY(3-36) is preferably
administered at least ten minutes to one hour prior to eating to
prevent nausea but no more than about twelve to twenty-four hours
prior to eating. The patient is dosed at least once a day
preferably before every meal until the patient has lost a desired
amount of weight. The patient then receives maintenance doses at
least once a week preferably daily to maintain the weight
loss."
[0289] As is shown by the data from the following examples, when
administered intranasally to humans using the Y2 receptor-binding
peptide formulation of the present invention, PYY(3-36) was found
to reduce appetite. The examples also show that for the first time
post-prandial physiological levels of a PYY peptide could be
reached through an intranasal route of administration using the Y2
receptor-binding peptide formulations of the present invention in
which PYY(3-36) was the Y2 receptor-binding peptide.
Aerosal Nasal Administration of PYY
[0290] We have discovered that the Y2 receptor-binding peptides can
be administered intranasally using a nasal spray or aerosol. This
is surprising because many proteins and peptides have been shown to
be sheared or denatured due to the mechanical forces generated by
the actuator in producing the spray or aerosol. In this area the
following definitions are useful. [0291] 1. Aerosol--A product that
is packaged under pressure and contains therapeutically active
ingredients that are released upon activation of an appropriate
valve system. [0292] 2. Metered aerosol--A pressurized dosage form
comprised of metered dose valves, which allow for the delivery of a
uniform quantity of spray upon each activation. [0293] 3. Powder
aerosol--A product that is packaged under pressure and contains
therapeutically active ingredients in the form of a powder, which
are released upon activation of an appropriate valve system. [0294]
4. Spray aerosol--An aerosol product that utilizes a compressed gas
as the propellant to provide the force necessary to expet the
product as a wet spray; it generally applicable to solutions of
medicinal agents in aqueous solvents. [0295] 5. Spray--A liquid
minutely divided as by a jet of air or steam. Nasal spray
drugproducts contain therapeutically active ingredients dissolved
or suspended in solutions or mixtures of excipients in
nonpressurized dispensers. [0296] 6. Metered spray--A
non-pressurized dosage form consisting of valves that allow the
dispensing of a specified quantity of spray upon each activation.
[0297] 7. Suspension spray--A liquid preparation containing solid
particles dispersed in a liquid vehicle and in the form of course
droplets or as finely divided solids. The fluid dynamic
characterization of the aerosol spray emitted by metered nasal
spray pumps as a drug delivery device ("DDD"). Spray
characterization is an integral part of the regulatory submissions
necessary for Food and Drug Administration ("FDA") approval of
research and development, quality assurance and stability testing
procedures for new and existing nasal spray pumps.
[0298] Thorough characterization of the spray's geometry has been
found to be the best indicator of the overall performance of nasal
spray pumps. In particular, measurements of the spray's divergence
angle (plume geometry) as it exits the device; the spray's
cross-sectional ellipticity, uniformity and particle/droplet
distribution (spray pattern); and the time evolution of the
developing spray have been found to be the most representative
performance quantities in the characterization of a nasal spray
pump. During quality assurance and stability testing, plume
geometry and spray pattern measurements are key identifiers for
verifying consistency and conformity with the approved data
criteria for the nasal spray pumps.
Definitions
[0299] Plume Height--the measurement from the actuator tip to the
point at which the plume angle becomes non-linear because of the
breakdown of linear flow. Based on a visual examination of digital
images, and to establish a measurement point for width that is
consistent with the farthest measurement point of spray pattern, a
height of 30 mm is defined for this study.
[0300] Major Axis--the largest chord that can be drawn within the
fitted spray pattern that crosses the COMw in base units (mm).
[0301] Minor Axis--the smallest chord that can be drawn within the
fitted spray pattern that crosses the COMw in base units (mm).
[0302] Ellipticity Ratio--the ratio of the major axis to the minor
axis.
[0303] D.sub.10--the diameter of droplet for which 10% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m).
[0304] D.sub.50--the diameter of droplet for which 50% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m), also known as the mass median diameter.
[0305] D.sub.90--the diameter of droplet for which 90% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m).
[0306] Span--measurement of the width of the distribution, The
smaller the value, the narrower the distribution. Span is
calculated as (D.sub.90-D.sub.10)/D.sub.50.
[0307] % RSD--percent relative standard deviation, the standard
deviation divided by the mean of the series and multiplied by 100,
also known as % CV.
[0308] FIGS. 21A and 21B show a nasal spray device 10 before
engagement (FIG. 21A) and after engagement (FIG. 21B). The nasal
spray bottle 10 is comprised of a bottle 12 into which is the nasal
Y2 receptor-binding peptide formulation is placed, and an actuator
14, which when actuated or engage forces a spray plume, 16, of the
Y2 receptor-binding peptide out of the spray bottle, 12, through
the actuator, 14. A spray pattern is determined by taking a
photograph of a cross-section of the spary plume 16 above a
predetermined height, 18, of the plume. The spray plume also has
angle of ejection, 20, as it leaves actuator, 14. A spray pattern
of spray plume 16 is shown on FIG. 22. Spray pattern 22, is
elliptical and has a major axis, 24, and a minor axis 26.
[0309] The following examples are provided by way of illustration,
not limitation.
EXAMPLE 1
[0310] An exemplary formulation for enhanced nasal mucosal delivery
of peptide YY following the teachings of the instant specification
was prepared and evaluated as follows: TABLE-US-00001 TABLE 1
Peptide YY Formulation Composition Peptide YY.sub.3-36 Formu- Per
100 ml lations Sample Mucosal Delivery Enhancing Agent A 60 .mu.g
Phosphate-buffered saline (0.8%) pH 7.4 (Control 1) B 60 .mu.g
Phosphate-buffered saline (0.8%) pH 5.0 (Control 2) C 60 .mu.g
L-Arginine (10% w/v) D 60 .mu.g Poly-L-Arginine (0.5% w/v) E 60
.mu.g Gamma-Cyclodextrin (1% w/v) F 60 .mu.g .alpha.-Cyclodextrin
(5% w/v) G 60 .mu.g Methyl-.beta.-Cyclodextrin (3% w/v) H 60 .mu.g
n-Capric Acid Sodium (0.075% w/v) I 60 .mu.g Chitosan (0.5% w/v) J
60 .mu.g L-.alpha.-phosphatidylcholine didecanyl (3.5% w/v) K 60
.mu.g S-Nitroso-N-Acetyl-Penicillamine (0.5% w/v) L 60 .mu.g
Palmotoyl-DL-Carnitine (0.02% w/v) M 60 .mu.g Pluronic-127 (0.3%
w/v) N 60 .mu.g Sodium Nitroprusside (0.3% w/v) O 60 .mu.g Sodium
Glycocholate (1% w/v) P 60 .mu.g F1: Gelatin, DDPC, MBCD, EDTA F 1
L-.alpha.-phosphatidylcholine didecanyl (0.5% w/v) Methyl .beta.
Cyclodextrin (3% w/v) EDTA (0.1% w/v, Inf. Conc. 0.5 M) Gelatin
(0.5% w/v)
EXAMPLE 2
Nasal Mucosal Delivery--Permeation Kinetics And Cytotoxicity
[0311] 1. Organotypic Model
[0312] The following methods are generally useful for evaluating
nasal mucosal delivery parameters, kinetics and side effects for
peptide YY within the formulations and method of the invention, as
well as for determining the efficacy and characteristics of the
various intranasal delivery-enhancing agents disclosed herein for
combinatorial formulation or coordinate administration with peptide
YY.
[0313] Permeation kinetics and cytotoxicity are also useful for
determining the efficacy and characteristics of the various mucosal
delivery-enhancing agents disclosed herein for combinatorial
formulation or coordinate administration with mucosal
delivery-enhancing agents. In one exemplary protocol, permeation
kinetics and lack of unacceptable cytotoxicity are demonstrated for
an intranasal delivery-enhancing agent as disclosed above in
combination with a biologically active therapeutic agent,
exemplified by peptide YY.
[0314] The EpiAirway system was developed by MatTek Corp (Ashland,
Mass.) as a model of the pseudostratified epithelium lining the
respiratory tract. The epithelial cells are grown on porous
membrane-bottomed cell culture inserts at an air-liquid interface,
which results in differentiation of the cells to a highly polarized
morphology. The apical surface is ciliated with a microvillous
ultrastructure and the epithelium produces mucus (the presence of
mucin has been confirmed by immunoblotting). The inserts have a
diameter of 0.875 cm, providing a surface area of 0.6 cm.sup.2. The
cells are plated onto the inserts at the factory approximately
three weeks before shipping. One "kit" consists of 24 units. [0315]
A. On arrival, the units are placed onto sterile supports in 6-well
microplates. Each well receives 5 mL of proprietary culture medium.
This DMEM-based medium is serum free but is supplemented with
epidermal growth factor and other factors. The medium is always
tested for endogenous levels of any cytokine or growth factor,
which is being considered for intranasal delivery, but has been
free of all cytokines and factors studied to date except insulin.
The 5 mL volume is just sufficient to provide contact to the
bottoms of the units on their stands, but the apical surface of the
epithelium is allowed to remain in direct contact with air. Sterile
tweezers are used in this step and in all subsequent steps
involving transfer of units to liquid-containing wells to ensure
that no air is trapped between the bottoms of the units and the
medium. [0316] B. The units in their plates are maintained at
37.degree. C. in an incubator in an atmosphere of 5% C02 in air for
24 hours. At the end of this time the medium is replaced with fresh
medium and the units are returned to the incubator for another 24
hours.
[0317] 2. Experimental Protocol--Permeation Kinetics [0318] A. A
"kit" of 24 EpiAirway units can routinely be employed for
evaluating five different formulations, each of which is applied to
quadruplicate wells. Each well is employed for determination of
permeation kinetics (4 time points), transepithelial resistance,
mitochondrial reductase activity as measured by MTT reduction, and
cytolysis as measured by release of LDH. An additional set of wells
is employed as controls, which are sham treated during
determination of permeation kinetics, but are otherwise handled
identically to the test sample-containing units for determinations
of transepithelial resistance and viability. The determinations on
the controls are routinely also made on quadruplicate units, but
occasionally we have employed triplicate units for the controls and
have dedicated the remaining four units in the kit to measurements
of transepithelial resistance and viability on untreated units or
we have frozen and thawed the units for determinations of total LDH
levels to serve as a reference for 100% cytolysis. [0319] B. In all
experiments, the nasal mucosal delivery formulation to be studied
is applied to the apical surface of each unit in a volume of 100
.mu.L, which is sufficient to cover the entire apical surface. An
appropriate volume of the test formulation at the concentration
applied to the apical surface (no more than 100 .mu.L is generally
needed) is set aside for subsequent determination of concentration
of the active material by ELISA or other designated assay. [0320]
C. The units are placed in 6 well plates without stands for the
experiment: each well contains 0.9 mL of medium which is sufficient
to contact the porous membrane bottom of the unit but does not
generate any significant upward hydrostatic pressure on the unit.
[0321] D. To minimize potential sources of error and avoid any
formation of concentration gradients, the units are transferred
from one 0.9 mL-containing well to another at each time point in
the study. These transfers are made at the following time points,
based on a zero time at which the 100 .mu.L volume of test material
was applied to the apical surface: 15 minutes, 30 minutes, 60
minutes, and 120 minutes. [0322] E. In between time points the
units in their plates are kept in the 37.degree. C. incubator.
Plates containing 0.9 mL medium per well are also maintained in the
incubator so that minimal change in temperature occurs during the
brief periods when the plates are removed and the units are
transferred from one well to another using sterile forceps. [0323]
F. At the completion of each time point, the medium is removed from
the well from which each unit was transferred, and aliquotted into
two tubes (one tube receives 700 .mu.L and the other 200 .mu.L) for
determination of the concentration of permeated test material and,
in the event that the test material is cytotoxic, for release of
the cytosolic enzyme, lactic dehydrogenase, from the epithelium.
These samples are kept in the refrigerator if the assays are to be
conducted within 24 hours, or the samples are subaliquotted and
kept frozen at -80.degree. C. until thawed once for assays.
Repeated freeze-thaw cycles are to be avoided. [0324] G. In order
to minimize errors, all tubes, plates, and wells are prelabeled
before initiating an experiment. [0325] H. At the end of the 120
minute time point, the units are transferred from the last of the
0.9 mL containing wells to 24-well microplates, containing 0.3 mL
medium per well. This volume is again sufficient to contact the
bottoms of the units, but not to exert upward hydrostatic pressure
on the units. The units are returned to the incubator prior to
measurement of transepithelial resistance.
[0326] 3. Experimental Protocol--Transepithelial Resistance [0327]
A. Respiratory airway epithelial cells form tight junctions in vivo
as well as in vitro, restricting the flow of solutes across the
tissue. These junctions confer a transepithelial resistance of
several hundred ohms.times.cm2 in excised airway tissues; in the
MatTek EpiAirway units, the transepithelial resistance (TER) is
claimed by the manufacturer to be routinely around 1000
ohms.times.cm2. We have found that the TER of control EpiAirway
units which have been sham-exposed during the sequence of steps in
the permeation study is somewhat lower (700-800 ohms.times.cm2),
but, since permeation of small molecules is proportional to the
inverse of the TER, this value is still sufficiently high to
provide a major barrier to permeation. The porous membrane-bottomed
units without cells, conversely, provide only minimal transmembrane
resistance (5-20 ohms.times.cm2). [0328] B. Accurate determinations
of TER require that the electrodes of the ohmmeter be positioned
over a significant surface area above and below the membrane, and
that the distance of the electrodes from the membrane be
reproducibly controlled. The method for TER determination
recommended by MatTek and employed for all experiments here employs
an "EVOM".TM. epithelial voltohmmeter and an "ENDOHM".TM. tissue
resistance measurement chamber from World Precision Instruments,
Inc., Sarasota, Fla. [0329] C. The chamber is initially filled with
Dulbecco's phosphate buffered saline (PBS) for at least 20 minutes
prior to TER determinations in order to equilibrate the electrodes.
[0330] D. Determinations of TER are made with 1.5 mL of PBS in the
chamber and 350 .mu.L of PBS in the membrane-bottomed unit being
measured. The top electrode is adjusted to a position just above
the membrane of a unit containing no cells (but containing 350
.mu.L of PBS) and then fixed to ensure reproducible positioning.
The resistance of a cell-free unit is typically 5-20
ohms.times.cm.sup.2 ("background resistance"). [0331] E. Once the
chamber is prepared and the background resistance is recorded,
units in a 24-well plate which had just been employed in permeation
determinations are removed from the incubator and individually
placed in the chamber for TER determinations. [0332] F. Each unit
is first transferred to a petri dish containing PBS to ensure that
the membrane bottom is moistened. An aliquot of 350 .mu.L PBS is
added to the unit and then carefully aspirated into a labeled tube
to rinse the apical surface. A second wash of 350 .mu.L PBS is then
applied to the unit and aspirated into the same collection tube.
[0333] G. The unit is gently blotted free of excess PBS on its
exterior surface only before being placed into the chamber
(containing a fresh 1.5 ML aliquot of PBS). An aliquot of 350 .mu.L
PBS is added to the unit before the top electrode is placed on the
chamber and the TER is read on the EVOM meter. [0334] H. After the
TER of the unit is read in the ENDOHM chamber, the unit is removed,
the PBS is aspirated and saved, and the unit is returned with an
air interface on the apical surface to a 24-well plate containing
0.3 mL medium per well. [0335] I. The units are read in the
following sequence: all sham-treated controls, followed by all
formulation-treated samples, followed by a second TER reading of
each of the sham-treated controls. After all the TER determinations
are complete, the units in the 24-well microplate are returned to
the incubator for determination of viability by MTT reduction.
[0336] 4. Experimental Protocol--Viability by MTT Reduction
[0337] MTT is a cell-permeable tetrazolium salt which is reduced by
mitochondrial dehydrogenase activity to an insoluble colored
formazan by viable cells with intact mitochondrial function or by
nonmitochondrial NAD(P)H dehydrogenase activity from cells capable
of generating a respiratory burst. Formation of formazan is a good
indicator of viability of epithelial cells since these cells do not
generate a significant respiratory burst. We have employed a MTT
reagent kit prepared by MatTek Corp for their units in order to
assess viability. [0338] A. The MTT reagent is supplied as a
concentrate and is diluted into a proprietary DMEM-based diluent on
the day viability is to be assayed (typically the afternoon of the
day in which permeation kinetics and TER were determined in the
morning). Insoluble reagent is removed by a brief centrifugation
before use. The final MTT concentration is 1 mg/mL. [0339] B. The
final MTT solution is added to wells of a 24-well microplate at a
volume of 300 .mu.L per well. As has been noted above, this volume
is sufficient to contact the membranes of the EpiAirway units but
imposes no significant positive hydrostatic pressure on the cells.
[0340] C. The units are removed from the 24-well plate in which
they were placed after TER measurements, and after removing any
excess liquid from the exterior surface of the units, they are
transferred to the plate containing MTT reagent. The units in the
plate are then placed in an incubator at 37.degree. C. in an
atmosphere of 5% CO.sub.2 in air for 3 hours. [0341] D. At the end
of the 3-hour incubation, the units containing viable cells will
have turned visibly purple. The insoluble formazan must be
extracted from the cells in their units to quantitate the extent of
MTT reduction. Extraction of the formazan is accomplished by
transferring the units to a 24-well microplate containing 2 mL
extractant solution per well, after removing excess liquid from the
exterior surface of the units as before. This volume is sufficient
to completely cover both the membrane and the apical surface of the
units. Extraction is allowed to proceed overnight at room
temperature in a light-tight chamber. MTT extractants traditionally
contain high concentrations of detergent, and destroy the cells.
[0342] E. At the end of the extraction, the fluid from within each
unit and the fluid in its surrounding well are combined and
transferred to a tube for subsequent aliquotting into a 96-well
microplate (200 .mu.L aliquots are optimal) and determination of
absorbance at 570 nm on a VMax multiwell microplate
spectrophotometer. To ensure that turbidity from debris coming from
the extracted units does not contribute to the absorbance, the
absorbance at 650 nm is also determined for each well in the VMax
and is automatically subtracted from the absorbance at 570 nm. The
"blank" for the determination of formazan absorbance is a 200 .mu.L
aliquot of extractant to which no unit had been exposed. This
absorbance value is assumed to constitute zero viability. [0343] F.
Two units from each kit of 24 EpiAirway units are left untreated
during determination of permeation kinetics and TER. These units
are employed as the positive control for 100% cell viability. In
all the studies we have conducted, there has been no statistically
significant difference in the viability of the cells in these
untreated units vs cells in control units which had been sham
treated for permeation kinetics and on which TER determinations had
been performed. The absorbance of all units treated with test
formulations is assumed to be linearly proportional to the percent
viability of the cells in the units at the time of the incubation
with MTT. It should be noted that this assay is carried out
typically no sooner than four hours after introduction of the test
material to the apical surface, and subsequent to rinsing of the
apical surface of the units during TER determination.
[0344] 5. Determination of Viability by LDH Release
[0345] While measurement of mitochondrial reductase activity by MTT
reduction is a sensitive probe of cell viability, the assay
necessarily destroys the cells and therefore can be carried out
only at the end of each study. When cells undergo necrotic lysis,
their cytotosolic contents are spilled into the surrounding medium,
and cytosolic enzymes such as lactic dehydrogenase (LDH) can be
detected in this medium. An assay for LDH in the medium can be
performed on samples of medium removed at each time point of the
two-hour determination of permeation kinetics. Thus, cytotoxic
effects of formulations which do not develop until significant time
has passed can be detected as well as effects of formulations which
induce cytolysis with the first few minutes of exposure to airway
epithelium. [0346] A. The recommended LDH assay for evaluating
cytolysis of the EpiAirway units is based on conversion of lactate
to pyruvate with generation of NADH from NAD. The NADH is then
reoxidized along with simultaneous reduction of the tetrazolium
salt INT, catalyzed by a crude "diaphorase" preparation. The
formazan formed from reduction of INT is soluble, so that the
entire assay for LDH activity can be carried out in a homogenous
aqueous medium containing lactate, NAD, diaphorase, and INT. [0347]
B. The assay for LDH activity is carried out on 50 .mu.L aliquots
from samples of "supernatant" medium surrounding an EpiAirway unit
and collected at each time point. These samples were either stored
for no longer than 24 h in the refrigerator or were thawed after
being frozen within a few hours after collection. Each EpiAirway
unit generates samples of supernatant medium collected at 15 min,
30 min, 1 h, and 2 h after application of the test material. The
aliquots are all transferred to a 96 well microplate. [0348] C. A
50 .mu.L aliquot of medium which had not been exposed to a unit
serves as a "blank" or negative control of 0% cytotoxicity. We have
found that the apparent level of "endogenous" LDH present after
reaction of the assay reagent mixture with the unexposed medium is
the same within experimental error as the apparent level of LDH
released by all the sham-treated control units over the entire time
course of 2 hours required to conduct a permeation kinetics study.
Thus, within experimental error, these sham-treated units show no
cytolysis of the epithelial cells over the time course of the
permeation kinetics measurements. [0349] D. To prepare a sample of
supernatant medium reflecting the level of LDH released after 100%
of the cells in a unit have lysed, a unit which had not been
subjected to any prior manipulations is added to a well of a 6-well
microplate containing 0.9 mL of medium as in the protocol for
determination of permeation kinetics, the plate containing the unit
is frozen at -80.degree. C., and the contents of the well are then
allowed to thaw. This freeze-thaw cycle effectively lyses the cells
and releases their cytosolic contents, including LDH, into the
supernatant medium. A 50 .mu.L aliquot of the medium from the
frozen and thawed cells is added to the 96-well plate as a positive
control reflecting 100% cytotoxicity. [0350] E. To each well
containing an aliquot of supernatant medium, a 50 .mu.L aliquot of
the LDH assay reagent is added. The plate is then incubated for 30
minutes in the dark. [0351] F. The reactions are terminated by
addition of a "stop" solution of 1 M acetic acid, and within one
hour of addition of the stop solution, the absorbance of the plate
is determined at 490 nm. [0352] G. Computation of percent cytolysis
is based on the assumption of a linear relationship between
absorbance and cytolysis, with the absorbance obtained from the
medium alone serving as a reference for 0% cytolysis and the
absorbance obtained from the medium surrounding a frozen and thawed
unit serving as a reference for 100% cytolysis.
[0353] 6. ELISA Determinations
[0354] The procedures for determining the concentrations of
biologically active agents as test materials for evaluating
enhanced permeation of active agents in conjunction with coordinate
administration of mucosal delivery-enhancing agents or
combinatorial formulation of the invention are generally as
described above and in accordance with known methods and specific
manufacturer instructions of ELISA kits employed for each
particular assay. Permeation kinetics of the biologically active
agent is generally determined by taking measurements at multiple
time points (for example 15 min., 30 min., 60 min. and 120 min)
after the biologically active agent is contacted with the apical
epithelial cell surface (which may be simultaneous with, or
subsequent to, exposure of the apical cell surface to the mucosal
delivery-enhancing agent(s)).
[0355] The procedures for determining the concentrations of peptide
YY neuropeptide Y, and pancreatic peptide in blood serum, central
nervous system (CNS) tissues or fluids, cerebral spinal fluid
(CSF), or other tissues or fluids of a mammalian subject may be
determined by immunologic assay for peptide YY neuropeptide Y, and
pancreatic peptide. The procedures for determining the
concentrations of peptide YY neuropeptide Y, and pancreatic peptide
as test materials for evaluating enhanced permeation of active
agents in conjunction with coordinate administration of mucosal
delivery-enhancing agents or combinatorial formulation of the
invention are generally as described above and in accordance with
known methods and specific manufacturer instructions for
radioimmunoassay (RIA), enzyme immunoassay (EIA), and antibody
reagents for immunohistochemistry or immunofluorescence for peptide
YY neuropeptide Y, and pancreatic peptide. Bachem AG (King of
Prussia, Pa.).
[0356] EpiAirway.TM. tissue membranes are cultured in phenol red
and hydrocortisone free medium (MatTek Corp., Ashland, Mass.). The
tissue membranes are cultured at 37.degree. C. for 48 hours to
allow the tissues to equilibrate. Each tissue membrane is placed in
an individual well of a 6-well plate containing 0.9 mL of serum
free medium. 100 .mu.L of the formulation (test sample or control)
is applied to the apical surface of the membrane. Triplicate or
quadruplicate samples of each test sample (mucosal
delivery-enhancing agent in combination with a biologically active
agent, peptide YY) and control (biologically active agent, peptide
YY, alone) are evaluated in each assay. At each time point (15, 30,
60 and 120 minutes) the tissue membranes are moved to new wells
containing fresh medium. The underlying 0.9 mL medium samples is
harvested at each time point and stored at 4.degree. C. for use in
ELISA and lactate dehydrogenase (LDH) assays.
[0357] The ELISA kits are typically two-step sandwich ELISAs: the
immunoreactive form of the agent being studied is first "captured"
by an antibody immobilized on a 96-well microplate and after
washing unbound material out of the wells, a "detection" antibody
is allowed to react with the bound immunoreactive agent. This
detection antibody is typically conjugated to an enzyme (most often
horseradish peroxidase) and the amount of enzyme bound to the plate
in immune complexes is then measured by assaying its activity with
a chromogenic reagent. In addition to samples of supernatant medium
collected at each of the time points in the permeation kinetics
studies, appropriately diluted samples of the formulation (i.e.,
containing the subject biologically active test agent) that was
applied to the apical surface of the units at the start of the
kinetics study are also assayed in the ELISA plate, along with a
set of manufacturer-provided standards. Each supernatant medium
sample is generally assayed in duplicate wells by ELISA (it will be
recalled that quadruplicate units are employed for each formulation
in a permeation kinetics determination, generating a total of
sixteen samples of supernatant medium collected over all four time
points). [0358] A. It is not uncommon for the apparent
concentrations of active test agent in samples of supernatant
medium or in diluted samples of material applied to the apical
surface of the units to lie outside the range of concentrations of
the standards after completion of an ELISA. No concentrations of
material present in experimental samples are determined by
extrapolation beyond the concentrations of the standards; rather,
samples are rediluted appropriately to generate concentrations of
the test material which can be more accurately determined by
interpolation between the standards in a repeat ELISA. [0359] B.
The ELISA for a biologically active test agent, for example,
peptide YY, is unique in its design and recommended protocol.
Unlike most kits, the ELISA employs two monoclonal antibodies, one
for capture and another, directed towards a nonoverlapping
determinant for the biologically active test agent, e.g., peptide
YY, as the detection antibody (this antibody is conjugated to
horseradish peroxidase). As long as concentrations of peptide YY
that lie below the upper limit of the assay are present in
experimental samples, the assay protocol can be employed as per the
manufacturer's instructions, which allow for incubation of the
samples on the ELISA plate with both antibodies present
simultaneously. When the peptide YY levels in a sample are
significantly higher than this upper limit, the levels of
immunoreactive peptide YY may exceed the amounts of the antibodies
in the incubation mixture, and some peptide YY which has no
detection antibody bound will be captured on the plate, while some
peptide YY which has detection antibody bound may not be captured.
This leads to serious underestimation of the peptide YY levels in
the sample (it will appear that the peptide YY levels in such a
sample lie significantly below the upper limit of the assay). To
eliminate this possibility, the assay protocol has been modified:
[0360] B.1. The diluted samples are first incubated on the ELISA
plate containing the immobilized capture antibody for one hour in
the absence of any detection antibody. After the one hour
incubation, the wells are washed free of unbound material. [0361]
B.2. The detection antibody is incubated with the plate for one
hour to permit formation of immune complexes with all captured
antigen. The concentration of detection antibody is sufficient to
react with the maximum level of peptide YY which has been bound by
the capture antibody. The plate is then washed again to remove any
unbound detection antibody. [0362] B.3. The peroxidase substrate is
added to the plate and incubated for fifteen minutes to allow color
development to take place. [0363] B.4. The "stop" solution is added
to the plate, and the absorbance is read at 450 nm as well as 490
nm in the VMax microplate spectrophotometer. The absorbance of the
colored product at 490 nm is much lower than that at 450 nm, but
the absorbance at each wavelength is still proportional to
concentration of product. The two readings ensure that the
absorbance is linearly related to the amount of bound peptide YY
over the working range of the VMax instrument (we routinely
restrict the range from 0 to 2.5 OD, although the instrument is
reported to be accurate over a range from 0 to 3.0 OD). The amount
of peptide YY in the samples is determined by interpolation between
the OD values obtained for the different standards included in the
ELISA. Samples with OD readings outside the range obtained for the
standards are rediluted and run in a repeat ELISA.
Results
[0363] Measurement of Transepithelial Resistance by TER Assay
[0364] After the final assay time points, membranes were placed in
individual wells of a 24 well culture plate in 0.3 mL of clean
medium and the trans epithelial electrical resistance (TER) was
measured using the EVOM Epithelial Voltohmmeter and an Endohm
chamber (World Precision Instruments, Sarasota, Fla.). The top
electrode was adjusted to be close to, but not in contact with, the
top surface of the membrane. Tissues were removed, one at a time,
from their respective wells and basal surfaces were rinsed by
dipping in clean PBS. Apical surfaces were gently rinsed twice with
PBS. The tissue unit was placed in the Endohm chamber, 250 .mu.gL
of PBS added to the insert, the top electrode replaced and the
resistance measured and recorded. Following measurement, the PBS
was decanted and the tissue insert was returned to the culture
plate. All TER values are reported as a function of the surface
area of the tissue.
[0365] The final numbers were calculated as: TER of cell
membrane=(Resistance (R) of Insert with membrane-R of blank
Insert).times.Area of membrane (0.6 cm.sup.2).
[0366] Exemplary peptide YY formulation, Formulation P, showed the
greatest decrease in cell membrane resistance. (Table 2). The
results indicate that the exemplary formulation (e.g., Formulation
P) reduces the resistance of the membrane to less than 1% of the
control at the concentrations tested. The values shown are the
average of three replicates of each formulation. Formulations A and
B are controls prepared by reconstituting peptide YY (Bachem A G,
King of Prussia, Pa.) containing 60 .mu.pg peptide Y.sub.3-36 in
100 ml of phosphate buffered saline (PBS) at pH 7.4 or 5.0. Peptide
YY without mucosal delivery enhancers did not decrease the
resistance.
[0367] The results indicate that an exemplary formulation for
enhanced intranasal delivery of peptide YY (e.g., Formulation P)
decreases cell membrane resistance and significantly increases
mucosal epithelial cells permeability. The exemplary formulations
will enhance intranasal delivery of peptide YY to the blood serum
or to the central nervous system tissue or fluid. The results
indicate that these exemplary formulations when contacted with a
mucosal epithelium yield significant increases in mucosal
epithelial cell permeability to peptide YY. TABLE-US-00002 TABLE 2
Influence of Pharmaceutical Formulations Comprising Peptide YY and
Intranasal Delivery-Enhancing Agents on Transepithelial Resistance
(TER) of EpiAirway Cell Membrane Mucosal Delivery Enhancing
Formulation Agent % TER A PBS pH 7.4 (Control 1) 100 B PBS pH 5.0
(Control 2) 100 C L-Arginine (10% w/v) 47.88 D Poly-L-Arginine
(0.5% w/v) 3.96 E Gamma-Cyclodextrin 91.67 (1% w/v) F
Alpha-Cyclodextrin (5% w/v) 88.91 G Methyl-.beta.-Cyclodextrin
97.51 (3% w/v) H n-Capric Acid Sodium 47.72 (0.075% w/v) I Chitosan
(0.5% w/v) 4.77 J L-.alpha.-phosphatidylcholine 0.49 didecanyl
(3.5% w/v) K S-Nitroso-N-Acetyl- 44.35 Penicillamine (0.5% w/v) L
Palmotoyl-DL-Carnitine 1.76 (0.02% w/v) M Pluronic-127 (0.3% w/v)
97.57 N Sodium Nitroprusside 92.41 (0.3% w/v) O Sodium Glycocholate
14.25 (1% w/v) P F1: Gelatin, DDPC, MBCD, 0.65 EDTA
Permeation Kinetics as Measured by ELISA Assay
[0368] The effect of pharmaceutical formulations of the present
invention comprising peptide YY and intranasal delivery-enhancing
agents on the permeation of peptide YY across the EpiAirway.TM.
Cell Membrane (mucosal epithelial cell layer) is measured as
described above. The results are shown in Table 3. Permeation of
peptide YY across the EpiAirway.TM. Cell Membrane is measured by
ELISA assay.
[0369] For the exemplary intranasal formulations (e.g., Formulation
P) of the present invention, the greatest increase in peptide YY
permeation occurred in Formulation P as shown in Table 3. The
procedure uses an ELISA assay to determine the concentration of
biologically active peptide YY that has permeated the epithelial
cells into the surrounding medium over multiple time points. The
results show increased permeation of peptide YY in Formulation P
compared to Formulation A or B (peptide YY control formulation; 60
.mu.g peptide YY.sub.3-36 in 100 ml of phosphate buffered saline
(PBS) at pH 7.4 or 5.0; Bachem A G, King of Prussia, Pa.). On
average the cumulative increase in permeation at 120 minutes using
Formulation P exemplary intranasal formulation is about 1195 fold
greater than Formulations A or B controls. TABLE-US-00003 TABLE 3
Influence of Pharmaceutical Formulations Comprising Peptide YY and
Intranasal Delivery-Enhancing Agents on Permeation of Peptide YY
through EpiAirway Cell Membrane by ELISA Assay. Formulation Fold
Peptide YY.sub.3-36 % Permeation at Time Points (min) Total %
Increase in (60 .mu.g/100 ml) 0 15 30 60 120 Permeation
Permeability A PBS pH 7.4 (Control 1) 0 0.00171 0.00096 0.00451
0.00327 0.01 1 B PBS pH 5.0 (Control 2) 0 0.00093 0.00048 0.00042
0.00367 0.01 1 C L-Arginine (10% w/v) 0 0.00119 0.00277 0.00685
0.00566 0.02 2 D Poly-L-Arginine (0.5% w/v) 0 0.00324 0.01587
0.10395 0.49656 0.62 62 E Gamma-Cyclodextrin (1% w/v) 0 0.00017
0.00042 0.00028 0.0035 0 1 F .alpha.-Cyclodextrin (5% w/v) 0
0.00031 0.000745 0.00147 0.0031 0.01 1 G Methyl-.beta.-Cyclodextrin
(3% w/v) 0 0.00028 0.00038 0.00059 0.01028 0.01 1 H n-Capric Acid
Sodium (0.075% w/v) 0 0.0004 0.00131 0.00448 0.00821 0.01 1 I
Chitosan (0.5% w/v) 0 0.00086 0.01098 0.09749 0.82126 0.93 93 J
L-.alpha.-phosphatidylcholine 0 0.00934 0.02 0.08507 1.9642 2.08
208 didecanyl (3.5% w/v) K S-Nitroso-N-Acetyl- 0 0.00074 0.0032
0.0688 0.90432 0.98 98 Penicillamine (0.5% w/v) L
Palmotoyl-DL-Carnitine (0.02% w/v) 0 0.00378 0.03422 0.15141
1.31011 1.5 150 M Pluronic-127 (0.3% w/v) 0 0.00025 0.00027 0.00066
0.00395 0.01 1 N Sodium Nitroprusside (0.3% w/v) 0 0.00171 0.00114
0.00079 0.05492 0.05 5 O Sodium Glycocholate (1% w/v) 0 0.00325
0.00313 0.09023 0.70214 0.8 80 P F1 Gelatin, DDPC, MBCD, EDTA 0
0.05864 1.3972 2.9799 7.519 11.95 1195
MTT Assay: The MTT assays were performed using MTT-100, MatTek
kits. 300 mL of the MTT solution was added into each well. Tissue
inserts were gently rinsed with clean PBS and placed in the MTT
solution. The samples were incubated at 37.degree. C. for 3 hours.
After incubation the cell culture inserts were then immersed with
2.0 mL of the extractant solution per well to completely cover each
insert. The extraction plate was covered and sealed to reduce
evaporation. Extraction proceeds overnight at RT in the dark. After
the extraction period was complete, the extractant solution was
mixed and pipetted into a 96-well microtiter plate. Triplicates of
each sample were loaded, as well as extractant blanks. The optical
density of the samples was then measured at 550 nm on a plate
reader (Molecular Devices).
[0370] The MTT assay on an exemplary formulation for enhanced nasal
mucosal delivery of peptide YY following the teachings of the
instant specification (e.g., Formulation P) compared to control
formulation (Formulations A or B) are shown in Table 4. The results
for formulations comprising peptide YY and one or more intransal
delivery enhancing agents, for example, Formulation P (experiment
performed in three replicates) indicate that there is minimal toxic
effect of this exemplary embodiment on viability of the mucosal
epithelial tissue. TABLE-US-00004 TABLE 4 Influence of
Pharmaceutical Formulations Comprising Peptide YY and Intranasal
Delivery-Enhancing Agents on the Viability of EpiAirway Cell
Membrane as shown by % MTT Formulations Treatment % MTT A PBS pH .4
(Control1) 100 B PBS pH 5.0 (Control 2) 100 C L-Arginine (10% w/v)
91.54 D Poly-L-Arginine (0.5% w/v) 79.39 E Gamma-Cyclodextrin (1%
w/v) 100 F .alpha.-Cyclodextrin (5% w/v) 96.63 G
Methyl-.beta.-Cyclodextrin (3% w/v) 100 H n-Capric Acid Sodium 100
(0.075% w/v) I Chitosan (0.5% w/v) 100 J
L-.alpha.-phosphatidylcholine 94.25 didecanyl (3.5% w/v) K
S-Nitroso-N-Acetyl- 97.64 Penicillamine (0.5% w/v) L
Palmotoyl-DL-Carnitine 91.77 (0.02% w/v) M Pluronic-127 (0.3% w/v)
100 N Sodium Nitroprusside 100 (0.3% w/v) O Sodium Glycocholate (1%
w/v) 100 P F1: Gelatin, DDPC, MBCD, 88.75 EDTA
[0371] LDH Assay: The LDH assay on an exemplary formulation for
enhanced nasal mucosal delivery of peptide YY following the
teachings of the instant specification (e.g., Formulation P) are
shown in Table 5. The results for three replicates of Formulation P
indicate that there is minimal toxic effect of this exemplary
embodiment on viability of the mucosal epithelial tissue.
TABLE-US-00005 TABLE 5 Influence of Pharmaceutical Formulations
Comprising Peptide YY and Intranasal Delivery-Enhancing Agents on
the Viability of EpiAirway Cell Membrane as shown by % Dead Cells
(LDH Assay) Formulations Treatment % dead cells A PBS pH .4
(Control1) 1.0 B PBS pH 5.0 (Control 2) 1.1 C L-Arginine (10% w/v)
0.8 D Poly-L-Arginine (0.5% w/v) 1.4 E Gamma-Cyclodextrin (1% w/v)
0.8 F .alpha.-Cyclodextrin (5% w/v) 0.7 G
Methyl-.beta.-Cyclodextrin (3% w/v) 0.8 H n-Capric Acid Sodium 1.3
(0.075% w/v) I Chitosan (0.5% w/v) 0.7 J
L-.alpha.-phosphatidylcholine 1.2 didecanyl (3.5% w/v) K
S-Nitroso-N-Acetyl- 0.7 Penicillamine (0.5% w/v) L
Palmotoyl-DL-Carnitine 0.8 (0.02% w/v) M Pluronic-127 (0.3% w/v)
1.0 N Sodium Nitroprusside 0.6 (0.3% w/v) O Sodium Glycocholate (1%
w/v) 0.8 P F1: Gelatin, DDPC, MBCD, 2.0 EDTA
EXAMPLE 3
Formulation P (Peptide YY) of the Present Invention in Combination
with Triamcinolone Acetonide Corticosteroid Improves Cell
Viability
[0372] The present example provides an in vitro study to determine
the permeability and reduction in epithelial mucosal inflammation
of an intranasally administered peptide YY, for example, human
peptide YY, in combination with a steroid composition, for example,
triamcinolone acetonide, and further in combination with one or
more intranasal delivery-enhancing agents. The study involves
determination of epithelial cell permeability by TER assay and
reduction in epithelial mucosal inflammation as measured by cell
viability in an MTT assay by application of an embodiment
comprising peptide YY and triamcinolone acetonide.
[0373] Formulation P (see Table 1 above) is combined in a
formulation with triamcinolone acetonide at a dosage of 0.5, 2.0,
5.0, or 50 .mu.g. Normal dose of triamcinolone acetonide,
(Nasacort.RTM., Aventis Pharmaceuticals) for seasonal allergic
rhinitis, is 55 .mu.g per spray. Formulation P in combination with
triamcinolone acetonide corticosteroid improves cell viability as
measured by the MTT assay, while maintaining epithelial cell
permeability as measured by TER and ELISA assays.
[0374] According to the methods and formulations of the invention,
measurement of permeability of Formulation P in the presence or
absence of triamcinolone acetonide is performed by transepithelial
electrical resistance (TER) assays in an EpiAirway.TM. cell
membrane. TER assays of Formulation P plus triamcinolone acetonide
at a concentration of 0.5, 2.0, 5.0, or 50 .mu.g per spray indicate
that peptide YY permeability did not decrease and was equal to
permeability of Formulation P alone. Formulation P plus
triamcinolone acetonide at a triamcinolone acetonide concentration
between 0 and 50 .mu.g per spray is typically, at least 10-fold to
100-fold greater than permeability of Formulations A or B (peptide
YY control).
[0375] According to the methods and formulations of the invention,
measurement of permeability of Formulation P in the presence or
absence of triamcinolone acetonide is performed by ELISA assay in
an EpiAirway.TM. cell membrane. Similar to the TER assay above,
ELISA assay of Formulation P plus triamcinolone acetonide at a
concentration of 0.5, 2.0, 5.0, or 50 .mu.g per spray indicate that
peptide YY permeability did not decrease and was equal to
permeability of Formulation P alone. Formulation P plus
triamcinolone acetonide at a triamcinolone acetonide concentration
between 0 and 50 .mu.g per spray is typically greater than
permeability of Formulations A or B (peptide YY control).
[0376] According to the methods and formulations of the invention,
MTT assay measured cell viability of Formulation P in the presence
or absence of triamcinolone acetonide. Typically, addition of
triamcinolone acetonide (at a concentration of 0.5, 2.0, 5.0, or 50
.mu.g per spray) to Formulation P improves cell viability compared
to Formulation P in the absence of triamcinolone acetonide.
[0377] Addition of triamcinolone acetonide to Formulation P
increases cell viability and maintains epithelial permeability as
measured by TER assay comparable to Formulation P in the absence of
triamcinolone acetonide.
[0378] Reduction in epithelial mucosal inflammation of an
intranasally administered peptide YY is accomplished with an
intranasal formulation of peptide YY in combination with one or
more steroid or corticosteroid compound(s) typically high potency
compounds or formulations, but also in certain cases medium
potency, or low potency compounds or formulations. Overall potency
(equivalent dosages) of high, medium, and low potency steroids are
given. Typically, an intranasal formulation of peptide YY in
combination with a high potency steroid composition includes, but
is not limited to, betamethasone (0.6 to 0.75 mg dosage), or
dexamethasone (0.75 mg dosage). In an alternative formulation, an
intranasal formulation of peptide YY in combination with a medium
potency steroid composition includes, but is not limited to,
methylprednisolone (4 mg dosage), triamcinolone (4 mg dosage), or
prednisolone (5 mg dosage). In a further alternative formulation,
an intranasal formulation of peptide YY in combination with a low
potency steroid composition includes, but is not limited to
hydrocortisone (20 mg dosage) or cortisone (25 mg dosage).
EXAMPLE 4
Preparation of a PYY Formulation Free of a Stabilizer that is a
Protein
[0379] A PYY formulation suitable for intranasal administration of
PYY, which was substantially free of a stabilizer that is a protein
was prepared having the formulation listed below. [0380] 1. About
3/4 of the water was added to a beaker and stirred with a stir bar
on a stir plate and the sodium citrate was added until it was
completely dissolved. [0381] 2. The EDTA was then added and stirred
until it was completely dissolved. [0382] 3. The citric acid was
then added and stirred until it was completely dissolved. [0383] 4.
The methyl-.beta.-cyclodextrin was added and stirred until it was
completely dissolved. [0384] 5. The DDPC was then added and stirred
until it was completely dissolved. [0385] 6. The lactose was then
added and stirred until it was completely dissolved. [0386] 7. The
sorbitol was then added and stirred until it was completely
dissolved. [0387] 8. The chlorobutanol was then added and stirred
until it was completely dissolved. [0388] 9. The PYY 3-36 was added
and stirred gently until it dissolved. [0389] 10. 11 Check the pH
to make sure it is 5.0.+-.0.25. Add dilute HCl or dilute NaOH to
adjust the pH.
[0390] 11. Add water to final volume. TABLE-US-00006 TABLE 6
Reagent Grade Vendor mg/mL % Cholorbutanol, anhydrous NF Spectrum
5.0 0.50 Methyl-.beta.-Cyclodextrin Sigma 45 4.5
L-.alpha.-Phosphatidylcholine Sigma 1 0.1 Didecanoyl Edetate
Disodium USP Dow 1 0.1 Chemicals Sodium Citrate, Dihydrate USP
Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.86 0.086
.alpha.-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82
PYY 3-36 GMP Bachem 1 0.1 Purified Water
[0391] Formulation pH 5+/-0.25 [0392] Osmolarity.about.250
EXAMPLE 5
[0393] A second formulation was prepared as above, except the
concentration of PYY 3-36 was 15 mg/mL as shown below in Table 7.
TABLE-US-00007 TABLE 7 Reagent Grade Vendor mg/mL % Cholorbutanol,
anhydrous NF Spectrum 5.0 0.50 Methyl-.beta.-Cyclodextrin Sigma 45
4.5 L-.alpha.-Phosphatidylcholine Sigma 1 0.1 Didecanoyl Edetate
Disodium USP Dow 1 0.1 Chemicals Sodium Citrate, Dihydrate USP
Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.86 0.086
.alpha.-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82
PYY 3-36 GMP Bachem 15 0.1 Purified Water Formulation pH 5 +/-
0.25
EXAMPLE 6
Determination of Optimal pH of PYY
[0394] Determination of PYY.sub.3-36 stability v's pH at 40.degree.
C. for 5 days
[0395] A. Protocol for Formulating PYY (3-36)/pH stability study
samples [0396] Osmolarity: Target 250 mM [0397] Using a
Citrate/Sodium citrate, tri-basic buffer, 10 mM Osmolarity =
.times. no . .times. particles .times. molarity = .times. ( 1 + 3 )
.times. 10 .times. .times. mM = 50 .times. .times. mM ##EQU1##
[0398] Therefore bring osmolarity to 250 mM with 100 mM NaCl (2
particles)
[0399] B. Made Up Stability Samples as Follows (3500 .mu.) [0400]
Final Concentration [0401] Citrate buffer 1400 .mu.L 25 mM (of
required final pH) 10 mM [0402] PYY 700 .mu.l 1.5 mg/mL 300 ug/ml
[0403] Chlorobutanol 350 .mu.l 2.5%, 0.25% [0404] NaCl, 350 .mu.l
1.0 M, 100 mM [0405] Check pH and adjust if required [0406] Q.S. to
3500 .mu.L with water [0407] Procedure: [0408] 120 .mu.l sample in
200 ul sialanized inserts in autosampler vials [0409] 3 pulls/time
point [0410] Samples incubated at 40.degree. C. for 5 days
[0411] C. Comparison of Target and Actual Final pH of Stability
Mixtures TABLE-US-00008 Target pH Actual pH 3.0 2.99 3.5 3.47 4.0
3.90 4.5 4.42 5.0 4.90 7.0 7.38
[0412] D. HPLC Procedure [0413] Column: Waters C18 Bondapak 10
.mu.m 4.6.times.300 mm [0414] HPLC system: Waters Alliance 2690
[0415] Detector: Waters 2487 Dual wavelength at 220 nm [0416] Flow
rate: 1 ml/mim [0417] Injection volume: 30 .mu.L [0418] Column
temp. 30.degree. C. [0419] Mobile phases: [0420] Buffer A: 0.1%
TFA, 1% acetonitrile in water [0421] Buffer B: 0.11% TFA in
acetonitrile
[0422] Gradient: TABLE-US-00009 Time (mins) % A % B 0 75 25 17 42
58 19 75 25 28 75 25
[0423] E. Results and Conclusions:
[0424] Results indicate that under the particular conditions used
in this study, that the optimal pH for stability is 4.90. There is
an increase in stability from 76 to 87% with increasing pH from
2.99 to 4.90.
[0425] At higher pH, i.e., 7.38 there is a large drop in the
stability of PYY(3-36) with only 15% of time zero remaining.
EXAMPLE 7
Intranasal Formulation Development
[0426] Peptides and proteins are relatively fragile molecules
compared to low-molecular-weight therapeutics. The objective of the
formulation development phase was to identify a candidate
formulation suitable for intranasal delivery. In order to achieve
this goal, numerous candidates were tested in order to identify a
formulation with acceptable drug stability, delivery across the
nasal mucosa, toxicity and preservative effectiveness.
[0427] Initially, the effect of pH was examined. FIG. 1 shows the
stability of PYY 3-36 at high temperature (40.degree. C.) at
various pHs from 3.0 to 7.4. At physiological pH, there was
substantial loss of drug at elevated temperature. Best stability
was achieved at about pH 5.0. This pH was chosen for further
formulation optimization.
[0428] To further optimize stability, various stabilizing agents
were tested for their ability to facilitate passage of drug across
the nasal mucosa. The enhancers tested were chosen based on their
ability to open tight junctions with limited cellular toxicity. To
accomplish this, a primary human epithelial cell model (EpiAirway,
MatTek, Inc., Ashland Mass.) was employed. This cell line forms a
pseudo-stratified columnar epithelial cell layer with tight
junctions similar to the respiratory epithelium found in the nose.
Drug formulations were placed on the apical side of the tissue
layer, and drug quantitation carried out for the basal media. The
extent of tight junction opening was measured by decrease in the
transepithelial electrical resistance (TEER). Cell viability and
cytotoxicity were monitored by MTT and LDH assays, respectively.
Data from a representative screening experiment are depicted in
FIGS. 2-5.
[0429] FIG. 2 shows the data for TEER. In some cases there was
little or no decrease in TEER compared to the control, indicating
tight junctions which remain closed. In other cases there was a
substantial drop in TEER indicating tight junction opening. The
results demonstrate that the in vitro cell model is capable of
discriminating the ability of different formulations to open the
tight junctions.
[0430] In the candidate formulations tested the cell viabilities
(FIG. 3; MTT) were good and cyctotoxicities (FIG. 4; LDH) were
low.
[0431] In total, over 200 different formulations were tested,
reflecting the high-throughput nature of the in vitro screening
model. Using all the available data, a multivariate analysis was
conducted to elucidate the effect each formulation component
exerted on each of the 7 output variables (drug permeability,
osmolality, stability at refrigerated and accelerated conditions,
TEER, and MTT and LDH assays). The multivariate analysis consisted
of an initial analysis of each formulation component for some level
of correlation with output parameters (p<0.1). With the subset
identified, either a linear regression or stepwise logistic
selection model was used. The results suggest that one excipient
correlated to osmolality and toxicity (r.sup.2=0.91 and 0.27,
respectively), two correlated to PYY.sub.3-36 permeation
(r.sup.2=0.44) three affected stability (r.sup.2=0.24), and five
impacted paracellular resistance (r.sup.2=0.55). The best
formulations determined by this process increased at least 30-75
fold the PYY.sub.3-36 transport compared to simple buffer
solutions.
[0432] Based on these analyses, an optimized PYY.sub.3-36
formulation was selected for further development. This optimized
formulation contained two stabilizers, two permeation enhancers,
one chelating agent, and one preservative in a sodium acetate
buffer, pH 5.0. This formulation passed the USP Preservative
Effectiveness Test. The synergistic contributions of the various
components on drug permeation is presented in FIG. 5. Compared to
simple buffer formulations at the same osmolality, the optimized
formulation exhibits more than 100-fold increased drug
permeation.
[0433] Finally, pre-clinical and clinical batches of the optimized
formulation were prepared and placed on stability at 5.degree. C.
and 25.degree. C. in the final product packaging. Preliminary data,
depicted in Table 8 reveal that storage for up to two months at
either 5.degree. C. or 25.degree. C. results in 90% or better
peptide retention. TABLE-US-00010 TABLE 8 5.degree. C. 25.degree.
C. Time point % PYY % PYY 0 100.0 100.0 3 days 99.8 100.1 7 days
102.6 98.4 10 days 103.3 101.7 2 weeks 101.4 97.9 3 weeks 100.3
95.4 1 month 100.5 96.3 1.5 onths 100.1 90.6 2 months 99.8 92.3
[0434] In summary, our process of formulation development has
produced a PYY.sub.3-36 formulation with suitable drug stability,
delivery across the nasal mucosa, toxicity and preservative
effectiveness, which enables delivery of a 4 kD peptide.
Preclinical Studies
[0435] To date, a series of six preclinical studies in rats,
rabbits, and dogs have been completed. Plasma PYY.sub.3-36 levels
in all species were determined by a validated proprietary
radioimmunoassay method.
[0436] Bioavailability (the molar fraction of drug identified in
plasma divided by the amount administered nasally) in rats was
determined to be approximately 6%, and in rabbits is approximately
8%. These values may understate the true bioavailability, as any
peptide degradation in plasma before sampling, or degradation after
sampling despite the presence of a proteinase inhibitor, will
decrease the measured bioavailability.
[0437] Nasal toxicity has been evaluated in rat and rabbit models
for up to 14 consecutive days at doses 50.times. the expected human
clinical dose on a mg/kg basis. There were no microscopic or gross
pathological findings related to the test article. There were no
clinical observations.
[0438] Systemic toxicity following intravenous administration was
evaluated in rat and rabbit models. At IV doses up to approx
160.times. the expected human dose (400 ug/kg in the rat and 205
ug/kg in the rabbit) there were no test article related microscopic
or macroscopic findings.
[0439] Cardiovascular toxicity was assessed in the anesthetized dog
model in a dose ranging study design. The highest dosage, an
infusion of PYY.sub.3-36 up to 24ug/kg over 60 minutes corresponded
to 33.times. the expected human dose on a body surface area basis.
The resultant plasma levels, 30 ng/mL, was approximately 380.times.
the basal canine plasma level. At this plasma level, there was no
effect on arterial blood pressure, femoral blood flow, or QTc and
only minor changes in heart rate (increase from 123 to 148 bpm
mean) and respiratory rate (decrease from 54 to 36) were noted.
[0440] Pharmacokinetic data was collected in these preclinical
studies. From one study in rats, the plasma levels following
intranasal administration at various doses are shown in FIGS. 6, 7
and 8. FIG. 6 shows PYY.sub.3-36 is seen in the plasma within 5
minutes, peak plasma concentrations (Tmax) are reached in 10-15
minutes, and the terminal elimination half life is approx 15
minutes. Both C.sub.max and AUC.sub.0-t are linear with respect to
intranasal dose.
Clinical Studies
[0441] A dose ranging clinical trial has been initiated with the
goal of establishing safety, PK, and bioavailability of the
intranasal formulation of PYY.sub.3-36. To date, patients have been
enrolled in the first two of five dose cohorts. One patient
reported a taste in the back of his throat; there have been no
other adverse events to date.
Conclusion
[0442] Formulation, preclinical, and initial clinical work have
begun on an intranasal formulation of PYY.sub.3-36. The approach to
formulation has resulted in a more than one hundred fold increase
in transmembrane permeability of this 4 kD peptide with no increase
in cellular toxicity. Preclinical studies have demonstrated a
considerable safety margin for nasal, cardiovascular, and systemic
toxicity for PYY.sub.3-36. On the basis on the ongoing dose ranging
clinical studies, chronic administration weight loss studies are
planned.
EXAMPLE 8a
Clinical Protocol
Nasal Absorption of Intranasal Peptide YY.sub.3-36 (PYY.sub.3-36)
in Healthy Human Subjects Object of the Present Study:
[0443] The object of the present study was to evaluate the
absorption of intranasally administered PYY3-36 into the blood
stream from the nose. This was a phase I, in clinic, single dose,
doses escalation study involving fasted, normal, healthy male and
female volunteers. Ascending doses of intranasal PYY3-36 were
evaluated between 20 .mu.g to 200 .mu.g to evaluate safety, nasal
tolerance and absorption of PYY3-36. Assessment of appetite
sensation in each individual was also evaluated.
[0444] PYY.sub.3-36 was administered to 15 healthy humans divided
into 5 Groups of 3 individuals each.
Group I
[0445] The first group was administered by an intranasal spray 20
.mu.g of PYY.sub.3-36 in a 0.1 ml solution. Group II [0446] The
second group received intranasally 50 .mu.g of PYY.sub.3-36 in a
0.1 ml solution. Group III [0447] The third group received
intranasally 100 .mu.g of PYY.sub.3-36 in a 0.1 ml solution. Group
IV [0448] The fourth group received intranasally 150 .mu.g of
PYY.sub.3-36 in a 0.1 ml solution. Group V [0449] The fifth group
received intranasally 200 .mu.g of PYY.sub.3-36 in a 0.1 ml
solution.
[0450] Blood samples were taken collected and the plasma
concentrations of PYY were determined at 0 (i.e., pre-dose), 5,
7.5, 10, 15, 20, 30, 45, 60 minutes post-dose. The subjects were
then fed and a blood sample taken and the concentration of PYY was
determined 30 minutes postprandial. Plasma concentrations of
PYY.sub.3-36 were determined using a validated analytical
procedure.
[0451] For each subject, the following PK parameters were
calculated, whenever possible, based on the plasma concentrations
of PYY.sub.3-36, according to the model independent approach:
[0452] C.sub.max Maximum observed concentration. [0453] t.sub.max
Time to maximum concentration. [0454] AUC.sub.0-t Area under the
concentration-time curve from time 0 to the time of last measurable
concentration, calculated by the linear trapezoidal rule. The
following parameters were calculated when the data permits accurate
estimation of these parameters: [0455] AUC.sub.0-.infin. Area under
the concentration-time curve extrapolated to infinity, calculated
using the formula: AUC 0 - .infin. = AUC 0 - t + C t K e ##EQU2##
[0456] where C.sub.t is the last measurable concentration and
K.sub.e is the apparent terminal phase rate constant. [0457]
K.sub.e Apparent terminal phase rate constant, where K.sub.e is the
magnitude of the slope of the linear regression of the log
concentration versus time profile during the terminal phase. [0458]
t.sub.1/2 Apparent terminal phase half-life (whenever possible),
where t.sub.1/2=(ln2)/K.sub.e. PK calculations were performed using
commercial software such as WinNonlin (Pharsight Corporation,
Version 3.3, or higher). The results are shown in the graphs below.
Discussion and Conclusion
[0459] Background:
[0460] Each dosing group included three subjects who were dosed
intranasally once with a formulation of this invention that
contained a specified dose of synthetic, pyrogen-free human
PYY.sub.3-36. Five dosing groups were organized, with escalating
doses of PYY.sub.3-36 in the formulation. Blood samples were drawn
at specified intervals into blood collection tubes that contained
lithium heparin (to inhibit coagulation) and aprotinin (to preserve
PYY.sub.3-36). Plasma from each blood sample was collected by
centrifugation and stored in frozen aliquots. One frozen aliquot of
each blood sample was shipped to Nastech Analytical Services and
arrived frozen. Each sample was stored frozen until assayed for PYY
concentration by radioimmunoassay (RIA).
[0461] Observations:
[0462] Group 1: This group of subjects was dosed with 20 micrograms
of PYY.sub.3-36. Plasma PYY concentrations for the subjects varied
from a minimum of "less than 20 pg/ml" (below the lower limit of
quantitation of the radioimmunoassay) to a maximum of 159 pg/ml.
The trends of concentrations observed are not consistent with
significant absorption of drug into the blood of the subjects
studied.
[0463] Group 2: This group of subjects was dosed with 50 micrograms
of PYY.sub.3-36. Plasma PYY concentrations for the subjects varied
from a minimum of 50 pg/ml to a maximum of 255 pg/ml. The trends of
concentrations observed are consistent with significant absorption
of drug into the blood of the subjects studied.
[0464] Group 3: This group of subjects was dosed with 100
micrograms of PYY 3-36. Plasma PYY concentrations for the subjects
varied from a minimum of 87 pg/ml to a maximum of 785 pg/ml. The
trends of concentrations observed are consistent with significant
absorption of drug into the blood of the subjects studied.
[0465] Group 4: This group of subjects was dosed with 150
micrograms of PYY 3-36. Plasma PYY concentrations for the subjects
varied from a minimum of 45 pg/ml to a maximum of 2022 pg/ml. The
trends of concentrations observed are consistent with significant
absorption of drug into the blood of the subjects studied.
[0466] Group 5: This group of subjects was dosed with 200
micrograms of PYY 3-36. Plasma PYY concentrations for the subjects
varied from a minimum of 48 pg/ml to a maximum of 1279 pg/ml. The
trends of concentrations observed are consistent with significant
absorption of drug into the blood of the subjects studied.
[0467] These results are consistent with a dose dependent
absorption of PYY.sub.3-36.
Additional Observations and Data:
[0468] Summary of Findings: [0469] At intranasal doses of 50 ug-200
ug, there is dose dependent plasma uptake of PYY. [0470] The
duration of elevated plasma concentrations is considerably longer
than would have been predicted, with an elimination half-life
calculated at 55 minutes. [0471] Cmax and AUC 0-t show good
linearity with dose. [0472] There is considerable inter-subject
variability at a given dose. [0473] Surprisingly, this study failed
to detect postprandial elevation of PYY although the quantity of
food actually eaten was not measured and if too little was eaten
could explain the observations. [0474] Visual-analog scale hunger
questions suggest decreased hunger with increasing doses of PYY.
[0475] Nausea and lightheadedness appear to be related to very high
plasma concentration of PYY.
[0476] Notes: [0477] In some cases pMol/L are used as the PYY
measurement units; in other analyses, pg/mL are used. The
conversion factor is pmol/L*4.05=pg/mL. [0478] In some cases, the
150-minute time point is displayed in plots. Strictly speaking,
this is a postprandial datapoint, and may therefore confound PK
evaluation. However, an unexpected finding described in more detail
below is that the 30-minute postprandial timepoint is no different
from the baseline value. PYY Plasma Concentrations:
[0479] The PYY assay described in this specification has been
validated for its own PYY plasma concentration assay. Using this
assay, samples from each timepoint were assayed in triplicate. Note
that out of the 180 datapoints, 3 (1.6%) appear to be biologically
implausible "outliers." The data throughout this preliminary
analysis use a dataset in which these three datapoints were
removed. TABLE-US-00011 TABLE 9 Descriptive PK parameters:
Calculated PK parameters include: Tmax Cmax AUC 0-last AUC 0-inf
T1/2 (min) (pg/mL) (min * pg/mL) (min * pg/mL) (min) 20 60 47 3850
50 18 89 4960 12379 112 100 32 342 26535 41102 27 150 23 530 31659
39476 39 200 23 683 34823 48618 42
[0480] Examination of the mean PK plots suggests a dose response
from 50-200 ug doses, but that the 20 .mu.g dose is in the noise.
Therefore, many of the subsequent analyses will be based on data
only from the 50-200 ug doses. We also propose that, because PYY is
an endogenous molecule, the AUC 0-t is more relevant than AUC
0-inf. TABLE-US-00012 Tmax and T1/2 (elimination half life): Tmax
T.sub.1/2 (min) (min) Mean values for 50-200 ug dose groups: 24
55
[0481] The Tmax of 24 minutes is typical for a nasal product. The
elimination half life of 55 minutes is considerably longer than
would have been expected. Literature references indicated a
t.sub.1/2 typically of 5-10 minutes. The elimination half-life may
also be affected by some continued uptake from the nasal mucosa
occurring after the Tmax and by formulation components that effect
peptide metabolism. Alternatively, because the assay described in
this specification employs an extraction procedure, the assay will
capture both free and protein-bound PYY, whereas an assay that does
not use an extraction may assay primarily the free fraction.
[0482] From this analysis of mean VAS change from baseline (mean of
10, 30, and 60 minute values minus baseline) vs dose, one
observes:
[0483] For VAS Q1 "How hungry do you feel?" subjects were less
hungry after receiving higher PYY doses. For VAS Q3, "How much do
you think you can eat?" subjects thought they could eat less after
receiving higher doses of PYY. However, for VAS Q2 "How full do you
feel?" subjects felt less full after receiving higher doses of PYY.
This suggests that the sensation follow PYY administration does not
include fullness, bloating, or gastric hypercontractility.
EXAMPLE 8b
[0484] TABLE-US-00013 PYY Human Administration and Weight Loss The
following PYY Nasal formulation was made. Reagent Grade Vendor Cat
# Lot # F.W. mg/ml % Cholorbutanol, anhydrous NF Spectrum CH123
RI1646 177.46 2.5 0.25 Methyl-.beta.-Cyclodextrin Sigma C-4555
81K1179 45 4.5 L-.alpha.-Phosphatidylcholine Sigma P-7081 55H8377
565.7 1 0.1 Didecanoyl Edetate Disodium USP Dow 1034N-00269-2 372.2
1 0.1 (EDTA) Chemicals Sodium Citrate, Dihydrate USP Spectrum S0165
RH1056 294.1 1.6 0.16 Citric Acid, Anhydrous USP Sigma C-1857
062K003 192.13 0.9 0.09 PYY(3-36), endotoxin-free Phoenix 059-02
420338 4049.71 2 0.2 Purified Water
[0485] Formulation pH 5+/-0.25
[0486] One or two sprays were administered daily to a human subject
over 10 day period and a weight loss of 2.5 pounds was recorded.
During periods ranging from 10 minutes to 12 hours after
administration the subject recorded reduced hunger.
EXAMPLE 9
Buccal Formulation of PYY3-36
Prophetic
[0487] Bilayer tablets are prepared in the following manner. An
adhesive layer is prepared by weighing 70 parts by weight
polyethylene oxide (Polyox 301N; Union Carbide), 20 parts by weight
polyacrylic acid (Carbopol 934P; B.F. Goodrich), and 10 parts by
weight of a compressible xylitol/carboxymethyl cellulose filler
(Xylitab 200; Xyrofin). These ingredients are mixed by rolling in a
jar for 3 minutes. The mixture is then transferred to an
evaporating dish and quickly wet granulated with absolute ethanol
to a semi-dough-like consistency. This mass is immediately and
rapidly forced through a 14 mesh (1.4 mm opening) stainless steel
screen, to which the wet granules adhered. The screen is covered
with perforated aluminum foil, and the wet granules are dried
overnight at 30.degree. C. The dried granules are removed from the
screen and then passed through a 20 mesh (0.85 mm opening) screen
to further reduce the size of the granules. Particles that do not
pass through the 20 mesh screen are ground briefly with a mortar
and pestle to minimize the amount of fines and then passed through
the 20 mesh screen. The resulting granules are then placed in a
mixing jar, and 0.25 parts by weight stearic acid and 0.06 parts by
weight mint flavor (Universal Flavors) are added and blended to the
granules. The final percentages by weight of the ingredients are
thus 69.78% polyethylene oxide, 9.97% compressible
xylitol/carboxymethyl cellulose filler, 19.94% polyacrylic acid,
0.25% stearic acid, and 0.06% mint flavor. A 50 mg amount of this
mixture is placed on a 0.375 inch diameter die and precompressed on
a Carver Press Model C with 0.25 metric ton pressure for a 3 second
dwell time to form the adhesive layer.
[0488] The active layer is prepared by weighing 49.39 parts by
weight of mannitol, 34.33 parts by weight of hydroxypropyl
cellulose (Klucel, L. F.; Aqualon, Wilmington, Del.) and 15.00
parts by weight of sodium taurocholate (Aldrich, Milwaukee, Wis.),
and mixing by rolling in a jar for 3 minutes. The mixture is then
transferred to an evaporating dish and quickly wet granulated with
absolute ethanol to a semi-dough-like consistency. This mass is
immediately and rapidly forced through a 14 mesh stainless steel
screen, to which the wet granules adher. The screen is covered with
perforated aluminum foil, and the granules dried at 30.degree. C.
The dried granulation is then passed sequentially through 20, 40
(0.425 mm opening), and 60 (0.25 mm opening) mesh screens to reduce
particle size further. Particles that do not pass through a screen
are briefly ground with a mortar and pestle to minimize fines and
then passed through the screen. The screened particles were
weighed, and then 0.91 parts by weight of PYY3-36 and 0.06 parts by
weight of FD&C yellow #6HT aluminum lake dye are sequentially
blended with the dry granulation by geometric dilution. The dyed
granulation is then placed in a mixing jar and blended with 0.25
parts by weight magnesium stearate (lubricant) and 0.06 parts by
weight mint flavor by rolling for 3 minutes. A 50 mg sample of this
material is placed on top of the partially compressed adhesive
layer and both layers are then compressed at 1.0 ton pressure for a
3 second dwell time to yield a bilayer tablet suitable for buccal
delivery.
[0489] This procedure results in a gingival tablet wherein the
active layer contains 0.91% by weight of PYY3-36, 15% by weight of
NaTC, and 84.09% by weight of filler, lubricant, colorant,
formulation aids, or flavoring agents.
EXAMPLE 10
[0490] A study was conducted comparing the ability of
endotoxin-free PYY(3-36) (SEQ ID NO: 2) vs non-endotoxin-free
PYY(3-36) to permeate the bronchial epithelium according of to the
procedure of Example 1. It was determined that about twice the
amount of enodotoxin-free PYY(3-36) permeated the bronchial
epithelium as compared to PYY(3-36) formulation that contained
endotoxin.
[0491] Both formulations contained Chlorobutanol 2.5 mg/ml, 2.0
mg/ml of DDPC, 10 mg/ml of albumin, 1 mg/ml of EDTA (edetate
disodium) and 45 mg/ml of M-B-CD. One formulation contained
endotoxin-free PYY(3-36) and the other formulation contained 70 EUs
or greater of endotoxin.
[0492] The average MTT of the PYY(3-36) formulation containing
endotoxin was 91.72% while the endotoxin-free PYY(3-36) formulation
had an average MTT of 100.16%.
[0493] The average permeation of the PYY(3-36) formulation
containing endotoxin was 5.36%, while the average permeation of the
endotoxin-free PYY(3-36) formulation was 10.75%.
[0494] A number of known mucosal delivery enhancing excipients can
be effectively combined with endotoxin-free Y2 receptor binding
peptides, especially endotoxin-free PYY3-36, and can be used to
improve non-infusion formulations, especially oral delivery. Such
excipients are contained in the following patent applications that
are incorporated by reference: U.S. Patent Application Nos.
20030225300; 20030198658; 20030133953; 20030078302; 20030045579;
20030012817; 20030012817; 20030008900; 20020155993; 20020127202;
20020120009; 20020119910; 20020065255; 20020052422; 20020040061;
20020028250; 20020013497; 20020001591; 20010039258;
20010003001.
Oral Formulation of a Y2 Receptor-Binding Peptide
[0495] An oral formulation of a Y2 receptor-binding peptide can be
prepared according to the following procedure. A preferred
formulation for oral delivery contains approximately 0.5 mg/kg
endotoxin-free PYY and between 100 and about 200 mg/kg of one or
more mucosal delivery enhancing excipients.
Prophetic
EXAMPLE 11
Preparation of N-cyclohexanoylphenylalanine Aldehyde:
[0496] Phenylalanine methyl ester (1 g., 0.0046 moles) is dissolved
in pyridine 5 mL. Cyclohexanoyl chloride (0.62 mL) is added and the
mixture is stirred for 2 hours. The reaction mixture is poured onto
hydrochloric acid (1N) and crushed ice. The aqueous mixture is
extracted twice with toluene. The combined toluene extracts are
concentrated in vacuo to give 1.1 g of crude
N-cyclohexanoylphenylalanine methyl ester.
[0497] N-Cyclohexanoylphenylalanine methyl ester (0.5 g) is
dissolved in ethylene glycol dimethyl ether (20 mL). The solution
is cooled to 70.degree. C. and diisobutylaluminum hydride (2.04 mL
of a 1.5M solution in toluene) is added. The resulting reaction
mixture is stirred at -70.degree. C. for 2 hours. The reaction is
quenched by dropwise addition of 2N hydrochloric acid. The mixture
is extracted with cold ethyl acetate. The ethyl acetate solution is
washed with brine and dried over sodium sulfate. Concentration in
vacuo furnishes a white solid, which is purified by silica gel
chromatography. .sup.1H NMR (300 MHz, DMSO-d6): 9.5 (s, 1H), 8.2
(d, 1H), 7.2 (m, 5H), 4.2 (m, 1H), 3.2 (d, 1H), 2.7 (d, 1H), 2.1
(m, 1H), 1.6 (br.m, 4H), 1.2 (br.m, 6H). R (KBr): 3300, 3050, 2900,
2850, 2800, 1700, 1600, 1500 cm-.sup.1.
[0498] Mass Spec.: M+1 m/e 261.
EXAMPLE 12
Preparation of N-acetylphenylalanine Aldehyde
[0499] N-Acetylphenylalanine methyl ester (4.2 g, 19 mmol) is
dissolved in ethylene glycol dimethyl ether. The solution is cooled
to -70.degree. C. and diisobutylaluminum hydride (25.3 mL of a 1.5M
solution in toluene, 39 mmol) is added. The resulting reaction
mixture is stirred at -70.degree. C. for 2 hours. The reaction is
quenched by addition of 2N hydrochloric acid. The mixture is
extracted 4 times with cold ethyl acetate and 4 times with toluene.
The extracts are combined, washed with brine and dried over
magnesium sulfate. Concentration in vacuo followed by silica gel
chromatography furnishes 2.7 g of a white solid. The NMR is as
reported in the literature, Biochemistry 18:921-926, 1979.
EXAMPLE 13
Preparation of 3-acetamido-4-(p-hydroxy)phenyl-2-butanone
(N-acetyltyrosinone)
[0500] A mixture of tyrosine (28.9 g, 16 mmol), acetic anhydride
(97.9 g, 96 mmol) and pyridine (35 g, 16 mmol) are heated to
100.degree. C. for 1 hour. The reaction mixture is concentrated in
vacuo to furnish a yellow oil. The oil is distilled at reduced
pressure to furnish 29.9 g or an oil.
[0501] .sup.1H NMR (DMSO-d6): NMR (d6-DMSO); 8.2 (d, 1H), 7.3 (d,
2H), 7.0 (d, 2H), 4.4 (m, 1H), 3.1 (dd, 1H), 2.7 (dd, 1H), 2.3 (s,
3H), 1.8 (s, 3H)
EXAMPLE 14
Preparation of 3-acetamido-7-amino-2-butanone
(N-acetyllysinone)
[0502] Following the procedure of Example 3 lysine is converted to
N-acetyllysinone.
[0503] .sup.1H NMR (DMSO-d6): 8.1 (d, 1H), 7.8 (br.m. 1H), 4.1 (m,
1H), 3.0 (m, 2H), 2.0 (s, 3H), 1.9 (s, 3H) and 1.3 (br.m, 6H).
EXAMPLE 15
Preparation of 3-acetamido-5-methyl-2-butanone
(N-acetylleucinone)
[0504] Following the procedure of Example 3 leucine is converted to
N-acetylleucinone. .sup.1H NMR (DMSO-d6): 8.1 (d, 1H), 4.2 (m, 1H),
2.0 (s, 3H), 1.8 (s, 3H), 0.8 (d, 6H).
EXAMPLE 16
Modification of 4-(4-aminophenyl)butyric Acid Using Benzene
Sulfonyl Chloride
[0505] 4-(4-Aminophenyl)butyric acid, (20 g 0.11 moles) is
dissolved in 110 mL of aqueous 2N sodium hydroxide solution. After
stirring for about 5 minutes at room temperature, benzene sulfonyl
chloride (14.2 mL, 0.11 moles) is added dropwise into the amino
acid solution over a 15 minute period. After stirring for about 3
hours at room temperature the mixture is acidified to pH 2 by
addition of hydrochloric acid. This furnishes a light brown
precipitate which is isolated by filtration. The precipitate is
washed with warm water and dried. The melting point is
123-25.degree. C.
[0506] If necessary, the modified amino acids can be purified by
recrystallization and/or chromatography.
EXAMPLE 17
Modification of 4-amindbenzoic Acid Using Benzene Sulfonyl
Chloride
[0507] Following the procedure of Example 6 4-aminobenzoic acid is
converted to 4-(phenylsulfonamido)benzoic acid.
EXAMPLE 18
Modification of 4-aminophenylacetic Acid, 4-aminohippuric Acid, and
4-aminomethylbenzoic Acid Using Benzene Sulfonyl Chloride
[0508] Following the procedure of Example 6, 4-aminophenylacetic
acid, 4-aminohippuric acid, and 4-amino-methylbenzoic acid are
converted to 4-(phenylsulfonamido)phenylacetic acid,
4-(phenylsulfonamido)hippuric acid, and
4-(phenylsulfonamidomethyl)benzoic acid respectively.
EXAMPLE 19
Modification of Amino Acids with Benzene Sulfonyl Chloride
[0509] A mixture of sixteen amino acids are prepared prior to
chemical modification. The constituents of the mixture are
summarized in the Table below. 65 grams of the amino acid mixture
(total concentration of [--NH.sub.2] groups=0.61 moles) is
dissolved in 760 mL of 1N sodium hydroxide solution (0.7625
equivalents) at room temperature. After stirring for 20 minutes,
benzene sulfonyl chloride (78 ml, 1 eq.) is added over a 20 minute
period. The reaction mixture is then stirred for 2.5 hours, without
heating. As some precipitation may occur, additional NaOH solution
(2N) may be added to the solution until it reaches pH 9.3. The
reaction mixture is stirred overnight at room temperature.
Thereafter, the mixture is acidified using dilute hydrochloric acid
(38%, 1:4) and a cream colored material precipitates out. The
resulting precipitate is isolated by decantation and dissolved in
sodium hydroxide (2N). This solution is then reduced in vacuo to
give a yellow solid, which is dried on the lyophilizer.
TABLE-US-00014 TABLE 10 Amino Acid Composition No. of Moles of No.
of Weight % of Total Each Amino Moles of - Amino Acid (g) Weight
Acid (.times.10.sup.-2) [--NH.sub.2] Thr 2.47 3.8 2.07 2.07 Ser
2.25 3.46 2.1 2.1 Ala 4.61 7.1 5.17 5.17 Val 4.39 6.76 3.75 3.75
Met 0.53 0.82 0.35 0.35 Ile 2.47 3.8 0.36 0.36 Leu 3.86 5.94 2.95
2.95 Tyr 1.03 1.58 0.56 0.56 Phe 4.39 6.76 0.27 0.27 His 2.47 3.8
1.6 3.2 Lys 4.94 7.6 3.4 6.8 Arg 5.13 7.9 2.95 5.90 Glutamine 9.87
15.18 6.76 13.42 Glutamic Acid 9.87 15.18 6.70 6.70 Asparagine 3.32
5.11 2.51 5.02 Aspartic Acid 3.32 5.11 2.50 2.50
EXAMPLE 20
Modification of a Mixture of Five Amino Acids Using Benzene
Sulfonyl Chloride
[0510] An 86.1 g (0.85 moles of NH.sub.2) mixture of amino acids
(see Table below) is dissolved in 643 mL (1.5 eq.) of aqueous 2N
sodium hydroxide solution. After stirring for 30 minutes at room
temperature, benzene sulfonyl chloride (108 ML, 0.86 moles) is
added portionwise into the amino acid solution over a 15 minute
period. After stirring for 2.5 hours at room temperature, the pH of
the reaction mixture (pH 5) is adjusted to pH 9 with additional 2N
sodium hydroxide solution. The reaction mixture is stirred
overnight at room temperature. Thereafter, the pH of the reaction
mixture is adjusted to pH 2.5 by addition of dilute aqueous
hydrochloric acid solution (4:1, H.sub.2O: HCl) and a precipitate
of modified amino acids is formed. The upper layer is discarded and
the resulting yellow precipitate is isolated by decantation, washed
with water and dissolved in 2N sodium hydroxide (2N). The solution
is reduced in vacuo to give a yellow solid, which is lyophilized
overnight. TABLE-US-00015 TABLE 11 Moles of Amino Moles of Amino
Acid Acid (.times.10.sup.-2) [--NH.sub.2] .times. 10.sup.-2 Valine
7.5 7.5 Leucine 10.7 10.5 Phenylalanine 13.4 13.4 Lysine 21.0 42.0
Arginine 6.0 12.0
EXAMPLE 21
Modification of a Mixture of Five Amino Acids Using Benzoyl
Chloride
[0511] An 86 g (0.85 moles of NH.sub.2) mixture of amino acids (see
Table in Example 20) is dissolved in 637 mL (1.5 eq.) of aqueous 2N
sodium hydroxide solution. After stirring for 10 minutes at room
temperature, benzoyl chloride (99 mL, 0.85 moles) is added
portionwise into the amino acid solution over a 10 minute period.
After stirring for 2.5 hours at room temperature, the pH of the
reaction mixture (pH 12) is adjusted to pH 2.5 using dilute
hydrochloric acid (4:1, H.sub.2O: HCl) and a precipitate of
modified amino acids is formed. After settling for 1 hour, the
resulting precipitate is isolated by decantation, washed with water
and dissolved in sodium hydroxide (2N). This solution is then
reduced in vacuo to give crude modified amino acids as a white
solid (expected yield 220.5 g).
EXAMPLE 22
Modification of L-valine Using Benzene Sulfonyl Chloride
[0512] L-Valine (50 g, 0.43 mol) is dissolved in 376 mL (0.75 eq.)
of aqueous 2N sodium hydroxide by stirring at room temperature for
10 minutes. Benzene sulfonyl chloride (68.7 mL, 0.38 mol, 1.25 eq.)
is then added to the amino acid solution over a 20 minute period at
room temperature. After stirring for 2 hours at room temperature, a
precipitate appears. The precipitate is dissolved by adding 200 mL
of additional 2N sodium hydroxide solution. After stirring for an
additional 30 minutes, dilute aqueous hydrochloric acid solution
(4:1, H.sub.2O: HCl) is added until the pH of the reaction mixture
reaches 2.6. A precipitate of modified amino acids formed and is
recovered by decantation. This material is dissolved in 2N sodium
hydroxide and dried in vacuo co to give a white solid. Expected
yield of crude modified amino acids is 84.6 g, 77%).
EXAMPLE 23
Modification of Phenylalanine Methyl Ester Using Hippuryl
Chloride
[0513] L-Phenylalanine Methyl Ester Hydrochloride (15 g, 0.084
mole) is dissolved in dimethylformamide (DMF) (100 mL) and to this
is added pyridine (30 mL). A solution of hippuryl chloride (16.6 g,
0084 moles in 100 mL DMF) is immediately added to the amino acid
ester solution in two portions. The reaction mixture is stirred at
room temperature overnight. The reaction mixture is then reduced in
vacuo and dissolved in 1N aqueous sodium hydroxide. The solution is
heated at 70.degree. C. for 3 hours in order to hydrolyze the
methyl ester to a free carboxyl group. Thereafter, the solution is
acidified to pH 2.25 using dilute aqueous hydrochloric acid
solution (1:3 HCl/H.sub.2O). A gum-like precipitate is formed and
this is recovered and dissolved in 1N sodium hydroxide. The
solution is reduced in vacuo to afford an expected 18.6 g of crude
modified amino acid product. After recrystallization from
acetonitrile, pure modified phenylalanine (expected yield 12 g) is
recovered as a white powder. m.p. 223-225.degree. C.
EXAMPLE 24
Preparation of Dosing Solutions of PYY(3-36)
[0514] In a test tube 568 mg of acetyl phenylalanine aldehyde, 132
mg of carbomethoxy phenylalanylleucine and 100 mg
acetyl-Phe-Leu-Leu-Arg aldehyde are added to 2.9 ml of 15% ethanol.
The solution is stirred and NaOH (1.0 N) is added to raise the pH
to 7.2. Water is added to bring the total volume to 4.0 mL. The
sample had a carrier concentration of 200 mg/mL. PYY(3-36) (800
.mu.g) is added to the solution. The total PYY3-36 concentration is
200 .mu.g/mL.
[0515] Following a similar procedure a second solution having 668
mg of acetyl phenylalanine aldehyde and 132 mg of carbomethoxy
phenylalanylleucine as the carrier composition and a third solution
having as the carrier acetyl phenylalanine aldehyde are prepared.
Each solution had an endotoxin-free PYY(3-36) concentration of 200
.mu.g/mL.
EXAMPLE 25
Preparation of Modified Amino Acid/PYY(3-36) Compositions
Preparation of Modified Amino Acid Microspheres Containing
Encapsulatedendotoxin-free PYY3-36.
[0516] The modified amino acid mixture, prepared in accordance with
Example 9, is dissolved at 40.degree. C. in distilled water (pH
7.2) at a concentration of 100 mg/ml. The solution is then filtered
with a 0.2 micron filter and the temperature is maintained at
40.degree. C. PYY3-36 (Bachem) is dissolved in an aqueous solution
of citric acid (1.7N) and gelatin (5%) at a concentration of 150
mg/ml. This solution is then heated to 40 C. The two heated
solutions are then mixed 1:1 (v/v). The resulting microsphere
suspension is then filtered with glass wool and centrifuged for 50
minutes at 1000 g. The pellet is resuspended with 0.85N citric acid
to a volume 5 to 7 fold less than the original volume. PYY3-36
concentration of the resuspended pellet is determined by HPLC.
Additional microspheres are made according to the above procedure
without PYY3-36. These "empty microspheres" are used to dilute the
encapsulated salmon PYY3-36 microsphere preparation to a final
dosing suspension, if needed.
Preparation of a Soluble Modified Amino Acid Carrier/PYY3-36
System
[0517] A soluble amino acid dosing preparation containing PYY3-36
is prepared by dissolving the modified amino acid material in
distilled water (pH 8) to an appropriate concentration. The
solution is heated to 40.degree. C. and then filtered with a 0.2
micron filter. PYY3-36, also dissolved in distilled water, is then
added to the modified amino acid solution prior to oral
administration.
Pulmonary Delivery of PYY3-36 (Prophetic)
[0518] The carrier compounds, prepared as described below may be
used directly as a delivery carrier by simply mixing one or more
compound or salt, poly amino acid or peptide with an endotoxin-free
Y2 receptor-binding peptide for pulmonary delivery.
[0519] The administration mixtures are prepared by mixing an
aqueous solution of the carrier with an aqueous solution of the
active ingredient, just prior to administration. Alternatively, the
carrier and the biologically or chemically active ingredient can be
admixed during the manufacturing process. The solutions may
optionally contain additives such as phosphate buffer salts, citric
acid, acetic acid, gelatin, and gum acacia.
[0520] A number of known pulmonary delivery methods can use
endotoxin-free Y2 receptor-binding peptides, especially PYY3-36, to
improve the delivery of PYY to the lungs. The following
non-limiting patent applications are incorporated herein by
reference for pulmonary delivery: U.S. Patent Application Nos.
20030223939; 20030215514; 20030215512; 20030209243; 20030203036;
20030198601; 20030183228; 200301885765; 20030150454; 20030124193;
20030094173.
EXAMPLE 26
Preparation of Carriers
Preparation of 2-(4-(N-salicyloyl)aminophenyl) propionic acid
(Carrier B)
[0521] A slurry of 58.6 g (0.355 mol) of 2-(4-aminophenyl)propionic
acid and 500 ml of methylene chloride is treated with 90.11 ml
(77.13 g. 0-710 mol) of trimethylsilyl chloride and is heated to
reflux for 120 min. The reaction mixture is cooled to 0.degree. C.
and treated with 184.44 ml (107.77 g, 1.065 mol) of triethylamine.
After stirring for 5 minutes, this mixture is treated with a
solution of 70.45 g (0.355 mol) of 0-acetylsalicyloyl chloride and
150 ml of methylene chloride. The reaction mixture is warmed to
25.degree. C. and stirred for 64 hr. The volatiles are removed in
vacuo. The residue is stirred in 2N aqueous sodium hydroxide for
one hour and acidified with 2 M aqueous sulfuric acid. The solid is
recrystallized twice from ethanol/water to give a tan solid.
Isolation by filtration results in an expected yield of 53.05 g
(52% yield) of 2-(4-(N-salicyloyl)aminophenyl)propionic acid.
Properties. Solubility: 200 mg/m: 200 mg+350 ..mu.L 2N NaOH+650
..mu.L H.sub.2O-pH-7.67. Analysis: C, 67.36; H, 5.3; N, 4.91.
Preparation of Sodium 2-(4-(N-salicyloyl)aminophenyl)propionate
(Sodium Salt of Carrier B)
[0522] A solution of 53.05 g (0.186 mol) of
2-(4-(N-salicyloyl)aminophenyl-)propionic acid and 300 ml of
ethanol is treated with 7.59 g (0.190 mol) of NaOH dissolved in 22
ml of water. The reaction mixture is stirred for 30 min at
25.degree. C. and for 30 min at 0.degree. C. The resulting pale
yellow solid is isolated by filtration to give 52.61 g of sodium
2-(4-(N-salicyloyl)aminophenyl)propionate. Properties. Solubility:
200 mg/ml clear solution, pH=6.85. Analysis C, 60.45; H, 5.45; N,
3.92; Na, 6.43. Melting point 236-238.degree. C.
Preparation of the Sodium Salt of Carrier C
[0523] A 2L round bottom flask equipped with a magnetic stirrer and
a reflux condenser is charged with a suspension of
3-(4-aminophenyl)propio-nic acid (15.0 g. 0.084 moles. 1.0 equiv.)
in dichloromethane (250 ml). Chlorotrimethylsilane (18.19 g, 0.856
moles, 2.0 equiv.) is added in one portion, and the mixture is
heated to reflux for 1.5 h under argon. The reaction is allowed to
cool to room temperature and is placed in an ice bath (internal
temperature <10.degree. C.). The reflux condenser is replaced
with an addition funnel containing triethylamine (25.41 g, 0.251
moles, 3.0 equiv.). The triethylamine is added dropwise over 15
min, and a yellow solid forms during the addition. The funnel is
replaced by another addition funnel containing a solution of
2,3-dimethoxybenzoylchlo-ride (I 8.31 g, 0.091 moles, 1.09 equiv.)
in dichloromethane (100 mL). The solution is added dropwise over 30
nm. The reaction is stirred in the ice bath for another 30 min and
at ambient temperature for 3 h. The dicholoromethane is evaporated
in vacuo to give a brown oil. The brown oil is cooled in an ice
bath, and an ice-cold solution of saturated sodium bicarbonate (250
ml) is added. The ice bath is removed, and the reaction is stirred
1 h to afford a clear brown solution. The solution is acidified
with concentrated HCl and stored at ca SC for 1 hour. The mixture
is extracted with dichloromethane (3.times. 100 mL), dried over
sodium sulfate, the salts filtered off and the dichloromethane
removed in vacuo. The resulting solid is recrystallized from 50%
ethyl acetate/water (v/v) to afford Carrier C acid as off white
needles (25.92 g. 90%). Analysis for C.sub.19H.sub.21NO.sub.5: C,
66.46; H, 6.16; N, 4.08. mp=99-102.degree. C.
[0524] 12 grams of the Carrier C acid is dissolved in ethanol, 75
mL, with warming. To this solution a 8.5 M Sodium hydroxide (1.02
molar equivalents, 1.426 grams in 4.5 mL water) solution is added.
The mixture is stirred for 15 minutes. Approximately three quarters
of the ethanol is remove in vacuo and n-heptane, 100 mL, is added
to the resulting oil causing a precipitate to form. The solids are
dried in vacuo at 50.degree. C. Analysis:
[0525] C.sub.19H.sub.20NO.sub.5Na0.067H.sub.2O: C, 62.25; H, 5.54;
N, 3.82; Na, 6.27.
Preparation of N-(4-methylsalicyloyl)-8-aminocaprylic acid (Carrier
D)
[0526] (a) Preparation of Oligo(4-methylsalicylate)
[0527] Acetic anhydride (32 mL, 34.5 g, 0.338 mol, 1.03 eq),
4-methylsalicylic acid (50 g, 0.329 mmol, 1.00 eq), and xylenes
(100 mL) are added to a 1 L, four-neck flask fitted with a magnetic
stir bar, a thermometer, and a condenser. The flask is placed in a
sand bath and heating of the cloudy white mixture begun. The
reaction mixture clears to a yellow solution around 90.degree. C.
Most of the volatile organics (xylenes and acetic acid) are
distilled into the Dean-Stark trap over three hours
(135-146.degree. C.). Distillation is continued for another hour (a
total of 110 mL distilled), during which the pot temperature slowly
rises to 204.degree. C. and the distillate slows to a trickle. The
residue is poured off while still hot into an aluminum tray. Upon
cooling a brittle yellow glass forms. The solid is ground to a fine
powder. The oligo(4-methylsalicylate) received is used without
further purification.
[0528] (b) Preparation of N-(4-methylsalicyloyl)-8-aminocaprylic
acid
[0529] A 7M solution of potassium carbonate (45 mL, 43.2 g, 0.313
mol, 0.95 eq), 8-aminocaprylic acid (41.8 g, 262 mol, 798 eq), and
water (20 mL) are added to a 1 L round bottom flask equipped with a
magnetic stir bar, condenser, and an addition fuel. The white
cloudy mixture is treated with a solution of
oligo(4-methylsalicylate) (44.7 g, 0.329 mmol 1.0 eq) and dioxane
(250 mL), added over thirty minutes. The reaction mixture is heated
to 90.degree. C. for 3 hours (at which time the reaction is
determined to have finished, by HPLC). The clear orange reaction
mixture is cooled to 30.degree. C. and acidified to pH=2 with 50%
aqueous sulfuric acid (64 g). The resulting solid is isolated by
filtration. The white solid is recrystallized from 1170 mL of 50%
ethanol-water. The solid is recovered by filtration and is dried
over 18 hours in a 50.degree. C. vacuum oven. The
N-(4-methylsalicyloyl)-8-ami-nocaprylic acid is isolated as a white
solid (30.88 g, 52%); mp=113-114.degree.. Analysis:
[0530] C.sub.6H.sub.23NO.sub.4: C, 65.51; H, 7.90; N, 4.77.
[0531] An aqueous solution of PYY(3-36) is then prepared and mixed
with one or more of the carrier to produce a PYY(3-36) composition,
which then can be sprayed into the lungs. A suitable concentration
of PYY3-36 for the resultant composition should be about 400
.mu.g/mL. See U.S. Patent Application No. 20030072740.
EXAMPLE 27
Total Extraction Radioimmunoassay for the Determination of the
concentration of PYY in Plasma
1.0 Introduction:
[0532] A radioimmunoassay was developed to measure the
concentration of Human Peptide YY(3-36) (hPYY) in human plasma.
Samples are collected with anticoagulant (EDTA) and protease
inhibitor (aprotinin) and frozen. The assay is a four day process.
Samples, controls, and standards are extracted in alcohol and dried
on Day 1. All samples are reconstituted and mixed with a polyclonal
rabbit antiserum directed against hPYY on Day 2. Iodinated hPYY is
added on Day 3. Specific precipitating agents (Goat anti-Rabbit IgG
and Normal Rabbit Serum) are added on Day 4. Bound tracer is
separated from free tracer by centrifugation, and the bound tracer
is counted in the gamma counter. Concentration is calculated by
interpolation of a standard curve and assay performance is
controlled with Quality Control samples.
2.0 Materials:
[0533] 2.1 Peninsula PYY kit (Peninsula Laboratories, Cat. No.
S-2043-0001) [0534] 2.2 Reagent Alcohol (Fisher Inc., Cat. No.
A995-4) (or equivalent) [0535] 2.3 Stripped human plasma (with
Lithium Heparin, fasted, pooled) Golden West Biologics Inc. (Cat.
No., SD1020-H) (Analytical SOP # A-003) [0536] 2.4 Ice Baths
(Fisher, Cat No. 11-676-36) (or equivalent) [0537] 2.5 Disposable
10 mL pipets (Fisher Cat. No. 13-678-11LE) (or equivalent) [0538]
2.6 Standard Synthetic Human PYY from Nastech QC (3-36) (Bachem
Cat. No. H8585) [0539] 2.7 Distilled Water (Milli-Q Millipore, Cat.
No. ZMQ56VFT1) (or equivalent) [0540] 2.8 Triton X-100 (Sigma, Cat.
No. T-9284) (or equivalent) [0541] 2.9 Aluminum Foil (Fisher, Cat.
No. 01-213-3) (or equivalent) [0542] 2.10 Aprotinin (ICN
Biomedicals Inc. Cat. No. 190779) (or equivalent) [0543] 2.11
12.times.75 mm tubes (Evergreen Scientific, Cat. No. 214-2023-010)
(or equivalent) [0544] 2.12 12.times.75 mm tube caps (Evergreen
Scientific, Cat. No. 300-2912-G20) (or equivalent) [0545] 2.13 1.5
mL microfuge tubes (Fisher, Cat. No. 05-402-25) (or equivalent)
[0546] 2.14 3.0 Instruments: [0547] 3.1 Wallac WIZARD 1470
Automatic Gamma Counter (Perkin Elmer, Model No. 1470-002) (or
equivalent) [0548] 3.2 Isotemp Basic Freezer, -70.degree. C.
(Kendro Laboratory Products, Model No. C90-3A31) (or equivalent)
[0549] 3.3 CentriVap Concentrator (Labconco, Cat. No. 7810000) (or
equivalent) [0550] 3.4 VX-2500 Multi-tube Vortexer (VWR, Cat. No.
58816-115) (or equivalent) [0551] 3.5 Marathon 21000R Centrifuge
(Fisher, Cat. No. 04-977-21000R) (or equivalent) [0552] 3.6
Swinging bucket rotor (Fisher, Cat. No. 04-976-006) (or equivalent)
[0553] 3.7 Motorized pipet-aid (Fisher, Cat. No. 13-681-15E) (or
equivalent) [0554] 3.8 Eppendorf Micropipette [0555] 3.8.1 2
.mu.L-20 .mu.L (Fisher, Cat. No. 21-371-6) (or equivalent) [0556]
3.8.2 20 .mu.L-200 .mu.L (Fisher, Cat. No. 21-371-10) (or
equivalent) [0557] 3.8.3 100 .mu.L-1000 .mu.L (Fisher, Cat. No.
21-371-13) (or equivalent) [0558] 3.9 Eppendorf Repeating Pipettor
(Fisher, Cat. No. 21-380-9) (or equivalent) [0559] 3.10 Eppendorf
Repeating Pipettor Combi-tips [0560] 3.10.1 2.5 mL (Fisher, Cat.
No. 21-381-331) (or equivalent) [0561] 3.10.2 25 mL (Fisher, Cat.
No. 21-381-115) (or equivalent) [0562] 3.11 Positive displacement
pipet (Fisher, Cat. No. 21-169-10A) (or equivalent) 4.0 Procedure
DAY 1 [0563] 4.1 Thaw necessary reagents and samples for the assay.
Prepare RIA buffer to 1X concentration (RIAB) if sufficient amount
is not available. [0564] 4.2 Prepare standard curve samples in
pooled stripped human plasma. Prepare as follows if using a
starting concentration of 12.8 .mu.g/mL. [0565] 4.2.1 Add 990 .mu.L
RIAB to tube O. [0566] 4.2.2 Add 990 .mu.L pooled plasma to tube A.
[0567] 4.2.3 Add 500 .mu.L pooled plasma to tubes B-H. [0568] 4.2.4
Add 10 .mu.L 12.8 .mu.g/mL Standard to tube O. Vortex. [0569] 4.2.5
Add 10 .mu.L solution from tube O to tube A. Vortex. [0570] 4.2.6
Add 500 .mu.L solution from tube A to tube B. Vortex. [0571] 4.2.7
Add 500 .mu.L solution from tube B to tube C. Vortex. [0572] 4.2.8
Repeat dilutions as in 4.2.7 through tube H. (See Diagram #1)
[0573] 4.3 Dilute unknown human plasma samples to be tested if
necessary. Samples should be diluted in pooled stripped human
plasma. [0574] 4.4 Add 1.2 mL of cold alcohol to empty tubes for
NSB, TB, all Standards, QC samples, and human plasma samples to be
tested. [0575] 4.5 Add 400 .mu.L of pooled stripped human plasma to
NSB and TB tubes. Cap, Vortex. [0576] 4.6 Add 400 .mu.L of each
prepared Standard sample from 4.2.5 to 4.2.8 to respective standard
curve tubes H-A (See Diagram #1). Cap, Vortex. [0577] 4.7 Add 400
.mu.L of QC samples to respective tubes. Cap, Vortex. [0578] 4.8
Add 400 .mu.L of each sample to be tested its respective tube. Cap,
Vortex. [0579] 4.9 Incubate all samples on ice for 30-60 minutes.
[0580] 4.10 Turn on the cold-trap switch on the Concentrator.
[0581] 4.11 Centrifuge all tubes at 3000 rpm, 4.degree. C. for 15
minutes. [0582] 4.12 Transfer 1.3 mL of supernatant from each
sample to a new set of empty tubes. Store in an ice bath or at
2-8.degree. C. if not spun immediately. [0583] 4.13 Place samples
in the Concentrator. [0584] 4.14 Samples should spin for two hours
at 40.degree. C., then at ambient temperature for a total of 5
hours or until dry. [0585] 4.15 Remove dried samples, cover and
store overnight at 2-8.degree. C. DAY 2 [0586] 4.16 Remove the
dried tubes from the 2-8.degree. C. cooler. [0587] 4.17 Add 100
.mu.L of 4.times. RIA buffer concentrate to each tube. [0588] 4.18
Add 100 .mu.L of 0.6% TX100 to each tube. (Attachment #1) Vortex
for a minimum of 30 seconds to ensure all extracts are fully
reconstituted. [0589] 4.19 Incubate all samples on ice for 30-60
minutes. [0590] 4.20 Add 200 .mu.L of distilled water to each tube.
Vortex. [0591] 4.21 Transfer 100 .mu.L of each sample extract to
respective tube. [0592] Note: NSB, TB, TC, Standard Curve samples,
and QCs are typically run in triplicate, requiring three tubes per
sample. Human plasma samples many be tested in any variation (up to
three replicates) depending on sample availability. [0593] 4.22
Prepare Rabbit anti-PYY as described in the Peninsula Laboratories
kit insert. [0594] 4.23 Add 100 .mu.L RIAB to each NSB tube. [0595]
4.24 Add 200 .mu.L RIAB to each TC tube. [0596] 4.25 Add 100 .mu.L
Rabbit anti-PYY to all remaining tubes. Vortex. [0597] 4.26 Cover
with foil and store overnight at 2-8.degree. C. DAY 3 [0598] 4.27
Remove the tubes from the 2-8.degree. C. cooler. [0599] 4.28
Prepare .sup.125I-Peptide YY tracer (Attachment #2). [0600] 4.29
Add 100 .mu.L of prepared tracer to all tubes. Cap and vortex.
[0601] 4.30 Store overnight at 2-8.degree. C. DAY 4 [0602] 4.31
Remove the tubes from the 2-8.degree. C. cooler. [0603] 4.32
Prepare Goat anti-Rabbit IgG serum (GARGG) and Normal Rabbit Serum
(NRS) as described in the Peninsula Laboratories kit insert. [0604]
4.33 Add 100 .mu.L GARGG to each tube (except TC tubes). [0605]
4.34 Add 100 .mu.L NRS to each tube (except TC tubes). Vortex.
[0606] 4.35 Incubate 90-120 minutes at room temperature. [0607]
4.36 Add 500 .mu.L RIAB to tubes to be centrifuged immediately
(except TC tubes). Vortex. [0608] Note: 500 .mu.L RIAB should be
added to tubes just prior to centrifugation. Only add RIAB to the
number of tubes that are ready to be centrifuged. 500 .mu.L RIAB
should be added to additional tubes when they are ready to be
centrifuged. [0609] 4.37 Centrifuge tubes (containing 500 .mu.L
RIAB) at 3000 rpm at 4.degree. C., for 15 minutes. Do not
centrifuge TC tubes. [0610] 4.38 Aspirate supernatant from
centrifuged tubes. [0611] 4.39 Place tubes in designated black
racks for counting on the Gamma counter. The first rack should have
the appropriate Program number attached. All racks that follow
should contain no program number. Samples should be added in the
following order: [0612] 4.39.1 NSB tubes [0613] 4.39.2 TB tubes
[0614] 4.39.3 TC tubes [0615] 4.39.4 Standard tubes (increasing
concentration) [0616] 4.39.5 QC samples (3 concentrations) [0617]
4.39.6 Unknown human samples [0618] 4.39.7 QC samples (3
concentrations) [0619] 4.40 Place an empty black rack with the Stop
label attached after all samples tobe counted. [0620] 4.41 Press
`Start` on the Gamma Counter keypad to start counting. [0621] 4.42
Press `E` for enter on the Gamma Counter keypad to display CPM
results. 5.0 Evaluation of Results [0622] 5.1 The following
guidelines are applied to the identification and rejection of
outliers in the assay. In order for a result to qualify as an
outlier and not be included in the final calculation of results,
all of the following conditions must be met. [0623] 5.1.1 QCs and
unknown samples: [0624] 5.1.1.1% CV of all replicates must be great
than 20%. [0625] 5.1.1.2 There must be at least three results to
evaluate. [0626] 5.1.1.3 The difference between the suspected
outlier and the result next closest in value must be greater than
20%. [0627] 5.1.1.4 The difference between the high and low
remaining results must be less than 20%. [0628] 5.1.2 Standard
Curve samples: [0629] 5.1.2.1% CV of all replicates much be greater
than 15%. [0630] 5.1.2.2 There must be at least three results to
evaluate. [0631] 5.1.2.3 The difference between the suspected
outlier and the result next closest in value must be greater than
15%. [0632] 5.1.2.4 The difference between the high and low
remaining results must be less than 15%. 6.0 Assay Specifications
[0633] 6.1 QC samples are prepared at the following concentrations.
Two QCsamples at each concentration are tested in an assay. Four of
the six QC samples tested must be within the following ranges (+30%
of nominal concentration). At least one of the two QCs tested at
any concentration must be within range of the assay for data to be
acceptable. [0634] 6.1.1 QC1 (100 pg/mL) 70-130 pg/mL [0635] 6.1.2
QC2 (200 pg/mL) 140-260 pg/mL [0636] 6.1.3 QC3 (500 pg/mL) 350-650
pg/mL [0637] 6.2 Standard curve parameter requirements TBD.
[0638] PYY RIA Standard: TABLE-US-00016 Tube designation
Concentration of Standard A 1280 pg/mL B 640 pg/mL C 320 pg/mL D
160 pg/mL E 80 pg/mL F 40 pg/mL G 20 pg/mL H 10 pg/mL
Attachment #1 [0639] 0.6% TX-100 [0640] Reagent: 0.6% TX-100 [0641]
Materials: Milli-Q Distilled Water [0642] TX-100 [0643]
Preparation: 1) Measure 50 mL of Milli-Q Distilled Water [0644] 2)
Add 300 .mu.L of TX-100 using positive displacement pipet [0645] 3)
Mix well. Attachment #2 [0646] .sup.125I -Peptide PYY Tracer [0647]
Reagent: 1251-Peptide PYY Tracer [0648] Materials: 1.times. RIA
Buffer [0649] .sup.125I-Peptide PYY [0650] Preparation: 1)
Reconstitute tracer with 1 mL of 1.times. RIA Buffer. [0651] 2)
Measure the quantity of the tracer on the Gamma Counter. Transfer
10 .mu.L of reconstituted tracer to a tube. Place it in a black
rack for the Gamma Counter with Program #30 attached. [0652] 3)
Place rack on the Gamma Counter with the Stop rack behind it.
[0653] 4) Press `Start" to begin counting, then `E` to view CPM
results. [0654] 5) Determine amount of tracer (X .mu.L) to prepare
and RIAB (Y mL) needed as follows: X .times. .times. .mu.L = ( 5
.times. .times. .mu.L ) .times. ( cpm .times. .times. value )
.times. ( # .times. .times. tubes + 10 ) ( cpm .times. .times. from
.times. .times. stock .times. .times. solution ) ##EQU3## X .times.
.times. mL = ( 0.1 ) .times. ( # .times. .times. tubes + 10 )
##EQU3.2## [0655] 6) Combine X .mu.L of .sup.125I-Peptide YY with Y
mL of RIAB. Mix well.
EXAMPLE 28
Preparation of an NPY Formulation Free of a Stabilizer that is a
Protein
[0656] A PYY formulation suitable for intranasal administration of
NPY, which is substantially free of a stabilizer that is a protein
is prepared having the formulation listed below. [0657] 1. About
3/4 of the water is added to a beaker and stirred with a stir bar
on a stir plate and the sodium citrate is added until it is
completely dissolved. [0658] 2. The EDTA is then added and stirred
until it is completely dissolved. [0659] 3. The citric acid is then
added and stirred until it is completely dissolved. [0660] 4. The
methyl-.beta.-cyclodextrin is added and stirred until it is
completely dissolved. [0661] 5. The DDPC is then added and stirred
until it is completely dissolved. [0662] 6. The lactose is then
added and stirred until it is completely dissolved. [0663] 7. The
sorbitol is then added and stirred until it is completely
dissolved. [0664] 8. The chlorobutanol is then added and stirred
until it is completely dissolved. [0665] 9. The NPY(3-36) is added
and stirred gently until it dissolved. [0666] 10. Check the pH to
make sure it is 5.0.+-.0.25. Add dilute HCl or dilute NaOH to
adjust the pH.
[0667] 11. Add water to final volume. TABLE-US-00017 TABLE 12
Reagent Grade Vendor mg/mL % Cholorbutanol, anhydrous NF Spectrum
5.0 0.50 Methyl-.beta.-Cyclodextrin Sigma 45 4.5
L-.alpha.-Phosphatidylcholine Sigma 1 0.1 Didecanoyl Edetate
Disodium (EDTA) USP Dow 1 0.1 Chemicals Sodium Citrate, Dihydrate
USP Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.86 0.086
.alpha.-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82
NPY(3-36) GMP Bachem 1 0.1 Purified Water Formulation pH 5 +/- 0.25
Osmolarity .about.250
EXAMPLE 29
[0668] A second formulation is prepared as above, except the
concentration of NPY(3-36) is 15 mg/mL as shown below in Table 13.
TABLE-US-00018 TABLE 13 Reagent Grade Vendor mg/ml % Cholorbutanol,
anhydrous NF Spectrum 5.0 0.50 Methyl-.beta.-Cyclodextrin Sigma 45
4.5 L-.alpha.-Phosphatidylcholine Sigma 1 0.1 Didecanoyl Edetate
Disodium USP Dow 1 0.1 Chemicals Sodium Citrate, Dihydrate USP
Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.86 0.086
.alpha.-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82
NPY(3-36) GMP Bachem 15 0.1 Purified Water Formulation pH 5 +/-
0.25
EXAMPLE 30
Preparation of Pancreatic Pep etide (PP) Formulation Free of a
Stabilizer that is a Protein
[0669] A PYY formulation suitable for intranasal administration of
PP, which is substantially free of a stabilizer that is a protein
is prepared having the formulation listed below. [0670] 1. About
3/4 of the water is added to a beaker and stirred with a stir bar
on a stir plate and the sodium citrate is added until it is
completely dissolved. [0671] 2. The EDTA is then added and stirred
until it is completely dissolved. [0672] 3. The citric acid is then
added and stirred until it is completely dissolved. [0673] 4. The
methyl-.beta.-cyclodextrin is added and stirred until it is
completely dissolved. [0674] 5. The DDPC is then added and stirred
until it is completely dissolved. [0675] 6. The lactose is then
added and stirred until it is completely dissolved. [0676] 7. The
sorbitol is then added and stirred until it is completely
dissolved. [0677] 8. The chlorobutanol is then added and stirred
until it is completely dissolved. [0678] 9. The PP(3-36) is added
and stirred gently until it dissolved. [0679] 10. 11 Check the pH
to make sure it is 5.0.+-.0.25. Add dilute HCl or dilute NaOH to
adjust the pH.
[0680] 11. Add water to final volume. TABLE-US-00019 TABLE 14
Reagent Grade Vendor mg/mL % Cholorbutanol, anhydrous NF Spectrum
5.0 0.50 Methyl-.beta.-Cyclodextrin Sigma 45 4.5
L-.alpha.-Phosphatidylcholine Sigma 1 0.1 Didecanoyl Edetate
Disodium USP Dow 1 0.1 Chemicals Sodium Citrate, Dihydrate USP
Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.86 0.086
.alpha.-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82
PP(3-36) GMP Bachem 1 0.1 Purified Water Formulation pH 5 +/- 0.25
Osmolarity .about.250
EXAMPLE 31
[0681] A second formulation is prepared as above, except the
concentration of PP(3-36) is 15 mg/mL as shown below in Table 15.
TABLE-US-00020 TABLE 15 Reagent Grade Vendor mg/ml % Cholorbutanol,
anhydrous NF Spectrum 5.0 0.50 Methyl-.beta.-Cyclodextrin Sigma 45
4.5 L-.alpha.-Phosphatidylcholine Sigma 1 0.1 Didecanoyl Edetate
Disodium USP Dow 1 0.1 Chemicals Sodium Citrate, Dihydrate USP
Spectrum 1.62 0.162 Citric Acid, Anhydrous USP Sigma 0.86 0.086
.alpha.-Lactose monohydrate Sigma 9 0.9 Sorbitol Sigma 18.2 1.82
PP(3-36) GMP Bachem 15 0.1 Purified Water Formulation pH 5 +/-
0.25
EXAMPLE 32
[0682] This example describes a pharmaceutical composition product
comprising an aqueous solution formulation of a Y2 receptor binding
compound at a concentration sufficient to produce therapeutically
effective plasma concentrations and an actuator to produce an
aerosol of said solution, wherein the spray pattern ellipticity
ratio of said aerosol is between 1.00 and 1.40 when measured at a
height of between 0.5 cm and 10 cm distance from the actuator
tip.
[0683] Suprisingly a PYY(3-36) formulation of the instant
specification can be aerosolized and still be therapeutically
effective (as shown in Example 8a). The volume of the aerosol can
be between about 5 microliters and 1.0 ml, preferably between 20
and 200 microliters.
[0684] This test method describes the procedure for characterizing
plume geometry of Y2 receptor binding compound nasal solution
formulations using the SprayView NSP system. The plume geometry is
characterized using a SprayView High Speed Optical Spray
Characterization System (SprayView NSP) with Integrated SprayView
NSx actuation station (Image Therm Engineering, Inc., Sudbury,
Mass.) according to the methods described in U.S Pat. No. 6,665,421
and U.S. Patent Application Publication No. 20030018416 published
Jan. 23, 2003.
[0685] Using the formulation of table 14 or placebo the spray
characterization and droplet size of the formulation in both a 1 mL
and a 3 mL bottle both having a nasal Spray Pump w/Safety Clip,
Pfeiffer SAP #60548, which delivers a dose of 0.1 mL per squirt and
has a diptube length of 36.05 mm.
[0686] The droplet size data are shown in the following table.
[0687] Droplet Size for Nasal Spray Bottle and Pfeiffer SAP #60548
TABLE-US-00021 % < 10 D.sub.10 D.sub.50 D.sub.90 Span micrometer
1 mL Y2 Receptor 33.36 229.21 704.66 3.23 0.29 binding compound
(PYY)
[0688] TABLE-US-00022 3 mL Y2 receptor 23.26 92.31 610.46 6.60 0.59
binding compound (PYY)
[0689] Below are listed the spray pattern and plume geometry
results TABLE-US-00023 Ellipticity Pattern Spray Pattern MajorAxis
MinorAxis Ratio Dmin Dmax Ovality Area active 1 mL 25.0 21.1 1.2
20.1 26.4 1.3 419.4 3 mL 26.5 22.6 1.2 22.1 29.1 1.3 468.2
[0690] TABLE-US-00024 Plume Geometry Angle Width active 1 mL 48.5
27.1 3 mL 44.8 25.0
Although the foregoing invention has been described in detail by
way of example for purposes of clarity of understanding, it will be
apparent to the artisan that certain changes and modifications are
comprehended by the disclosure and may be practiced without undue
experimentation within the scope of the appended claims, which are
presented by way of illustration not limitation.
* * * * *