U.S. patent number RE42,012 [Application Number 11/821,512] was granted by the patent office on 2010-12-28 for compositions and methods for enhancing receptor-mediated cellular internalization.
This patent grant is currently assigned to Massachusetts Institute of Technology, The Penn State Research Foundation. Invention is credited to Daniel R. Deaver, David A. Edwards, Robert S. Langer.
United States Patent |
RE42,012 |
Deaver , et al. |
December 28, 2010 |
Compositions and methods for enhancing receptor-mediated cellular
internalization
Abstract
Compositions and methods for improving cellular internalization
of one or more compounds are disclosed. The compositions include a
compound to be delivered and a biocompatible viscous material, such
as a hydrogel, lipogel, or highly viscous sol. The composition also
include, or are administered in conjunction with, an enhancer in an
amount effective to maximize expression of or binding to receptors
and enhance RME of the compound into the cells. This leads to high
transport rates of compounds to be delivered across cell membranes,
facilitating more efficient delivery of drugs and diagnostic
agents. Compositions are applied topically orally, nasally,
vaginally, rectally, and ocularly. The enhancer is administered
with the composition or separately, either systemically or
preferably locally. The compound to be delivered can also be the
enhancer.
Inventors: |
Deaver; Daniel R. (Franklin,
MA), Edwards; David A. (Boston, MA), Langer; Robert
S. (Newton, MA) |
Assignee: |
The Penn State Research
Foundation (University Park, PA)
Massachusetts Institute of Technology (Cambridge,
MA)
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Family
ID: |
22293496 |
Appl.
No.: |
11/821,512 |
Filed: |
June 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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08810275 |
Mar 3, 1997 |
5985320 |
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60103117 |
Oct 5, 1998 |
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60012721 |
Mar 4, 1996 |
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Reissue of: |
09412821 |
Oct 5, 1999 |
06387390 |
May 14, 2002 |
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Current U.S.
Class: |
424/435; 424/464;
424/423; 424/434; 424/451 |
Current CPC
Class: |
A61K
9/0034 (20130101); A61K 31/56 (20130101); A61K
48/0008 (20130101); A61P 35/00 (20180101); A61K
31/715 (20130101); A61P 15/08 (20180101); A61K
9/06 (20130101); A61P 5/00 (20180101); A61K
31/573 (20130101); A61K 47/38 (20130101); A61K
9/0046 (20130101); A61K 48/0083 (20130101); A61K
9/006 (20130101); A61K 9/0043 (20130101); A61K
9/0031 (20130101); A61K 48/00 (20130101); A61K
9/0073 (20130101); Y10S 977/906 (20130101) |
Current International
Class: |
A61F
13/02 (20060101) |
Field of
Search: |
;424/435 |
References Cited
[Referenced By]
U.S. Patent Documents
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EP |
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EP |
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EP |
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1090492 |
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Nov 1967 |
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GB |
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1 090 492 |
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Nov 1967 |
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GB |
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WO 83/01198 |
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Apr 1983 |
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WO |
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WO 86/02553 |
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May 1986 |
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WO |
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WO 87/02576 |
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May 1987 |
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WO |
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WO 89/05149 |
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Jun 1989 |
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WO |
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WO |
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WO 96/10335 |
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Apr 1996 |
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WO |
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WO 97/32572 |
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Sep 1997 |
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WO |
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|
Primary Examiner: Azpuru; Carlos A
Attorney, Agent or Firm: Pabst Patent Group LLP
Government Interests
.Iadd.GOVERNMENT SUPPORT
This invention was made with government support under Hatch Act
Project No. PEN03466, awarded by the United States Department of
Agriculture (USDA). The Government has certain rights in the
invention..Iaddend.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
.[.Priority is claimed.]. .Iadd.This application is a reissue of
U.S. Ser. No. 09/412,821, filed Oct. 5, 1999, now U.S. Pat. No.
6,387,390, which claims priority .Iaddend.to U.S. provisional
application Ser. No. 60/103,117, filed Oct. 5, 1998.Iadd., and
which is a continuation-in-part of U.S. Ser. No. 08/810,275, filed
Mar. 3, 1997, now U.S. Pat. No. 5,985,320, which claims priority to
U.S. provisional application Ser. No. 60/012,721, filed Mar. 4,
1996.Iaddend..
Claims
We claim:
.[.1. A method for delivering an agent to cells at a site where
uptake is desired comprising administering to the cells at the site
where uptake is desired a composition comprising (a) a viscous
material having an apparent viscosity between 10 and 2000 Poise and
approximately the same apparent viscosity, at a shear stress of
between approximately 1 and 200 Pascal at a strain rate
approximately that of endocytosis, as the cytosolic fluid of the
cell to which the agent is to be delivered, (b) a compound to be
delivered, and (c) an enhancer in an amount effective to enhance
expression of or binding to receptors eliciting receptor-mediated
endocytosis on the cells at the site where uptake is
desired..].
2. .[.The.]. .Iadd.A .Iaddend.method .[.of claim 1.]. .Iadd.for
delivering an agent to cells at a site where uptake is desired
comprising administering to the cells at the site where uptake is
desired a composition comprising (a) a viscous material having an
apparent viscosity between 10 and 200 Poise and approximately the
same apparent viscosity, at a shear stress of between approximately
1 and 200 Pascal at a strain rate approximately that of
endocytosis, as the cytosolic fluid of the cell to which the agent
is to be delivered, (b) a compound to be delivered, and (c) an
enhancer in an amount effective to enhance expression of or binding
to receptors eliciting receptor-mediated endocytosis on the cells
at the site where uptake is desired, .Iaddend.wherein the enhancer
is .[.a steroid.]. .Iadd.selected from the group consisting of
steroids and hormones, and wherein the enhancer is different from
the compound to be delivered.Iaddend..
3. The method of claim .[.1.]. .Iadd.2 .Iaddend.wherein the cells
to which the compound is to be delivered are in the nose, rectum,
mouth, ear, eye, or lungs.
4. The method of claim .[.1.]. .Iadd.2 .Iaddend.wherein the
composition is administered topically.
5. The method of claim .[.1.]. .Iadd.2 .Iaddend.wherein the
enhancer is administered systemically.
6. The method of claim .[.1.]. .Iadd.2 .Iaddend.wherein the
composition is administered to the vaginal mucosa.
7. The method of claim .[.1.]. .Iadd.2 .Iaddend.wherein the
compound to be delivered is selected from the group consisting of
proteins, peptides, carbohydrates, .[.nuceilc.]. .Iadd.nucleic
.Iaddend.acid molecules, and chemotherapeutic agents.
.[.8. The method of claim 7 wherein the enhancer is selected from
the group consisting of hormones, glucocorticoids, and other
molecules specifically binding to a receptor on a cell surface to
induce endocytosis..].
9. The method of claim .[.8.]. .Iadd.2 .Iaddend.wherein the
enhancer is a reproductive hormone.
10. The method of claim .[.1.]. .Iadd.2 .Iaddend.wherein the
enhancer is a glucocorticoid.
11. The method of claim 9 wherein the reproductive hormone is
selected from the group consisting of progesterone, estradiol, and
combinations thereof.
12. The method of claim .[.1.]. .Iadd.2 .Iaddend.wherein the
viscous material is selected from the group consisting of
hydrogels, lipogels and sols.
13. The method of claim 12 wherein the hydrogel is selected from
the group consisting of celluloses, polyalkyleneoxide,
polyvinylpyrrolidone, dextrans, alginates, agaroses, gelatin,
hyaluronic acid, trehalose, polyvinyl alcohol, and copolymers and
blends thereof.
.[.14. A composition for delivering an agent to cells at a site
where uptake is desired comprising: (a) a viscous fluid having an
apparent viscosity between 10 and 2000 Poise and approximately the
same apparent viscosity, at a shear stress of between approximately
1 and 200 Pascal at a strain rate approximately that of
endocytosis, as the cytosolic fluid of the cell to which the agent
is to be delivered, (b) a compound to be delivered, and (c) an
enhancer in an amount effective to enhance expression of or binding
to receptors eliciting receptor-mediated endocytosis on the cells
at the site where uptake is desired..].
15. The composition of claim .[.14.]. .Iadd.17 .Iaddend.in a
formulation suitable for administration to mucosa of tissue
selected from group consisting of the nose, the rectum, .Iadd.the
vagina, .Iaddend.the mouth, the ear, the eye, and the lungs.
16. The composition of claim .[.14.]. .Iadd.17 .Iaddend.wherein the
compound to be delivered is selected from the group consisting of
proteins, peptides, carbohydrates, nucleic acid molecules, and
chemotherapeutic agents.
17. .[.The.]. .Iadd.A .Iaddend.composition .[.of claim 14.].
.Iadd.for delivering an agent to cells at a site where uptake is
desired comprising: (a) a viscous fluid having an apparent
viscosity between 10 and 200 Poise and approximately the same
apparent viscosity, at a shear stress of between approximately 1
and 200 Pascal at a strain rate approximately that of endocytosis,
as the cytosolic fluid of the cell to which the agent is to be
delivered, (b) a compound to be delivered, and (c) an enhancer in
an amount effective to enhance expression of or binding to
receptors eliciting receptor-mediated endocytosis on the cells at
the site where uptake is desired, .Iaddend.wherein the enhancer is
selected from the group consisting of hormones and
.[.glucocorticoids.]. .Iadd.steroids, and wherein the enhancer is
different from the compound to be delivered.Iaddend..
18. The composition of claim .[.14.]. .Iadd.17 .Iaddend.wherein the
hormone is a reproductive hormone.
19. The composition of claim .[.14.]. .Iadd.17 .Iaddend.wherein the
viscous material is selected from the group consisting of
hydrogels, lipogels and sols.
20. The composition of claim 19 wherein the hydrogel is selected
from the group consisting of celluloses, polyalkyleneoxide,
polyvinylpyrrolidone, dextrans, alginates, agaroses, gelatin,
hyaluronic acid, trehalose, polyvinyl alcohol, and copolymers and
blends thereof.
21. A kit for delivering a compound to cells comprising: a first
composition comprising a viscous fluid and the compound to be
delivered, wherein the viscous fluid has an apparent viscosity
between 10 and .[.2000.]. .Iadd.200 .Iaddend.Poise and
approximately the same apparent viscosity, at a shear stress of
between approximately 1 and 200 Pascal at a strain rate
approximately that of endocytosis, as the cytosolic fluid of the
cell to which the agent is to be delivered, and a second
composition comprising an enhancer .Iadd.selected from the group
consisting of hormones and steroids .Iaddend.in an amount effective
to enhance expression of receptors eliciting receptor-mediated
endocytosis on the cells, thereby to enhance receptor mediated
endocytosis of the agent into the cells.Iadd., wherein the enhancer
is different from the compound to be delivered.Iaddend..
22. The kit of claim 21 wherein the second composition is in a
formulation suitable for topical or systemic administration.
Description
BACKGROUND OF THE INVENTION
The compositions and methods of use described herein generally are
in the field of materials and methods for enhancing cellular
internalization.
It is often difficult to deliver compounds, such as proteins,
peptides, genetic material, and other drugs and diagnostic
compounds intracellularly because cell membranes often resist the
passage of these compounds. Various methods have been developed to
administer agents intracellularly. For example, genetic material
has been administered into cells in vivo, in vitro, and ex vivo
using viral vectors, DNA/lipid complexes, and liposomes. While
viral vectors are efficient, questions remain regarding the safety
of a live vector and the development of an immune response
following repeated administration. Lipid complexes and liposomes
appear less effective at transfecting DNA into the nucleus of the
cell and potentially may be destroyed by macrophages in vivo.
Proteins and peptides are typically administered by parenteral
administration, or, in some cases, across the nasal mucous
membrane. Uptake of drugs administered topically is frequently
poor, and degradation frequently occurs when drugs are administered
orally. For example, hormones such as gonadotropin releasing
hormone ("GnRH") and its analogs have been administered to humans
in an attempt to increase fertility by increasing systemic levels
of luteinizing hormone ("LH"). When given often, low doses of
native GnRH have been shown to induce follicular development and
ovulation. These drugs are typically administered via an indwelling
catheter into the abdominal cavity. An external pump is attached to
the catheter which injects the peptide at frequent intervals. This
method of administration is extremely invasive and undesirable.
Also, the method is prohibitively expensive for use in animals.
It has recently been demonstrated that, by embedding individual
cell populations in hydrogel media of macroscopic viscosity similar
to that characteristic of cell cytoskeleta, the rate of
receptor-mediated endocytosis can be significantly enhanced
(Edwards, et al., Proc. Natl. Acad. Sci. U.S.A. 93:1786-91 (1996);
PCT US97/03276 by Massachusetts Institute of Technology and
Pennsylvania State University Foundation). This enhancement effect
appears to reflect a fluid-mechanical origin of receptor-mediated
endocytosis, involving the rapid expansion of plasma membrane in
the vicinity of a receptor cluster leading to an invaginating
membrane motion that is sensitive to the viscous properties of the
extracellular environment (Edwards, et al., Proc. Natl. Acad. Sci.
U.S.A. 93:1786-91 (1996); Edwards, et al., Biophys. J. 71:1208-14
(1996)).
It has been found, however, that the delivery of compounds via a
receptor-mediated route into the systemic circulation by
noninvasively delivering the compound in a
"rheologically-optimized" hydrogel may be inconsistent or poorly
reproducible. It would be advantageous to better understand the
role of RME in uptake of compounds in order to develop improved
methods of delivery of compounds, such as drugs,
intracellularly.
The binding of ligands or assembly proteins to surface receptors of
eucaryotic cell membranes has been extensively studied in an effort
to develop better ways to promote or enhance cellular uptake. For
example, binding of ligands or proteins has been reported to
initiate or accompany a cascade of nonequilibrium phenomena
culminating in the cellular invagination of membrane complexes
within clathrin-coated vesicles (Goldstein, et al., Ann. Rev. Cell
Biol. 1:1-39 (1985); Rodman, et al., Curr. Op. Cell Biol. 2:664-72
(1990); Trowbridge, Curr. Op. Cell Biol. 3:634-41 (1991); Smythe,
et al., J. Cell Biol. 108:843-53 (1989); Smythe, et al., J. Cell
Biol. 119:1163-71 (1992); and Schmid, Curr. Op. Cell Biol. 5:621-27
(1993)). This process has been referred to as receptor-mediated
endocytosis ("RME"). Beyond playing a central role in cellular
lipid trafficking (Pagano, Curr. Op. Cell Biol. 2:652-63 (1990)),
RME is the primary means by which macromolecules enter eucaryotic
cells.
An effective strategy for enhancing the uptake of cytotoxic and
therapeutic drugs involves exploiting the rapidity and specificity
of transmembrane transport via receptor-mediated endocytosis
(Goldstein, et al., Ann. Rev. Cell Biol. 1:1-39 (1985)) by
targeting receptors on the plasma membranes of endothelial (Barzu,
et al., Biochem. J. 15;238(3):847-854 (1986); Magnusson & Berg,
Biochem. J. 257:65-56 (1989)), phagocytic (Wright & Detmers,
"Receptor-mediated phagocytosis" in The Lung: Scientific
Foundations (Crystal, et al., eds.), pp. 539-49 (Ravens Press,
Ltd., New York, N.Y.(1991)); and tumor cells, as well as cells of
other tissues. Receptor targeting has, however, not been championed
as a means of avoiding intravenous injection of hard-to-absorb
macromolecules, probably because macromolecules often degrade prior
to reaching receptors in the gastrointestinal tract following oral
administration, and do not appear to require receptor-mediation to
permeate across the alveolar epithelium following inhalation. Other
noninvasive macromolecular drug delivery strategies either do not
expose receptors to the topical environment, for example
transdermal delivery, or have been less extensively explored, such
as nasal delivery (Illum, et al., Int. J. Pharm. 39:189-99 (1987)),
vaginal delivery, or ocular delivery.
It is therefore an object of the present invention to provide
compositions and methods for enhancing intracellular delivery of
bioactive and/or diagnostic agents, especially steroidal compounds
and materials which are endocytosed by a receptor-mediated
mechanism.
SUMMARY OF THE INVENTION
Compositions and methods for improving cellular internalization of
one or more compounds using a receptor mediated mechanism are
disclosed. The compositions include a compound to be delivered and
a biocompatible viscous material, such as a hydrogel, lipogel, or
highly viscous sol, and are administered subsequent to or with
steroid or other material binding to the receptor at the site of
application to enhance uptake (referred to as an "enhancer"). By
controlling the apparent viscosity of the viscous materials, the
rates of endocytosis, including nonspecific "pinocytosis" and
specific RME, are increased. The rate of endocytic internalization
is increased when the ratio of the apparent viscosities of
cytosolic and extracellular media approaches unity. The composition
includes, or is co-administered with, the enhancer, usually a
steroid or other molecule binding to receptors at the site of
application in an amount effective to maximize binding to the
receptors or expression of receptors and enhance RME of the
compound into the cells. This leads to high transport rates of
compounds to be delivered across cell membranes, facilitating more
efficient delivery of drugs and diagnostic agents.
Preferred viscous materials are hydrogels, lipogels (gels with
nonaqueous fluid interstices) and highly viscous sols. The apparent
viscosity of the composition is controlled such that it lies in the
range of between 0.1 and 2000 Poise, preferably between 7 and 1000
Poise, and most preferably between 2 and 200 Poise. Compounds to be
delivered include those that can be attached, covalently or
noncovalently, to a molecule that either stimulates RME or
pinocytosis by binding to receptors on the plasma membrane, binds
specifically to receptors that undergo RME or pinocytosis
independently of this binding (i.e., which are themselves
"enhancers") or at least can be associated chemically or physically
with other molecules or "carriers" that themselves undergo RME or
pinocytosis. Exemplary compounds to be delivered include proteins
and peptides, nucleotide molecules, saccharides and
polysaccharides, synthetic chemotherapeutic agents, and diagnostic
compounds. The examples demonstrate the roles of estrogen and
progesterone in vaginal delivery of peptide hormones. Peptide
transport into the systemic circulation is strongly
steroid-dependent, with most efficient transport of reproductive
hormones occurring after estradiol and progesterone pretreatment,
when hormone receptors are maximally expressed. Preferred steroids
include steroidal hormones such as estrogen and progesterone and
glucocorticoids.
The compositions are applied to cell membranes to achieve high
rates of transport of the compound to be delivered across those
membranes, relative to when non-viscous fluids are used with the
enhancers or the viscous fluids are used alone. Compositions are
applied topically orally, nasally, vaginally, rectally, and
ocularly. The enhancer is administered systemically or, more
preferably, locally. Compositions can be applied by injection via
catheter, intramuscularly, subcutaneously, and intraperitoneally.
Compositions can also be administered to the pulmonary or
respiratory system, most preferably in an aerosol.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a and 1b are graphs showing serum responses to iv injection
and vacinal administration of vasopressin and leuprolide acetate.
FIG. 1a shows serum cortisol response to iv injection and vaginal
administration of vasopressin. Vasopressin was injected
intravenously (5 .mu.g dose) through a jugular catheter. The
peptide was delivered vaginally in 5 ml of aqueous solution (200
.mu.g dose). Standard errors are based on n=6. FIG. 1b shows Serum
LH response to iv injection and vaginal administration of leuprole
acetate ("LHRH analog"). LHRH analog was injected intravenously (5
.mu.g dose) into through a jugular catheter. The peptide was
delivered vaginally in 5 ml of aqueous solution (200 .mu.g dose).
Standard errors are based on n=6.
FIG. 2 is a graph showing bioavailability of LHRH analog following
vaginal administration as a function of methyl cellulose
("methocel") concentration. Bioavailability is determined relative
to intravenous injection (FIG. 1b) and is based on LH response. The
administered dose of LHRH analog was 200 .mu.g in 5 ml of methocel
solution. Results are based on animals that responded to vaginal
treatment, with standard error computed on the basis of n.gtoreq.4.
In all cases, less than 50% of treated animals responded with LH
levels greater than 3 ng/ml for more than one sampling point, with
sampling times of 0, 30, 60, 90, 120, 180, 240, 360, and 480
min.
FIG. 3 is a graph showing the percent of responding animals to LHRH
analog vaginal delivery as a function of (simulated) stage of the
estrous cycle. Stage of estrous cycle was simulated by delivering
estradiol for two weeks to ovariectomized ewes (anestrus phase),
followed by two weeks of delivery of estradiol and progesterone
(mid-luteal phase), followed by a period of 48 h after progesterone
withdrawal (follicular phase). In each simulated phase, 10, 40, or
200 mg of LHRH analog were delivered vaginally in 5 ml of aqueous
or methocel solution to groups of six ewes. Responding animals were
defined as those treated animals with LH serum values exceeding 3
ng/ml for two or more sampling points, with sampling times of 0,
30, 60, 60, 120, 180, 240, 360, and 480 min.
FIG. 4 is a graph showing serum LH response to vaginal
administration of LHRH analog. LHRH analog was administered
vaginally in ovariectomized ewes during the simulated mid-luteal
phase in 5 ml of aqueous or methocel (1.75% methyl cellulose)
solution (40 .mu.g dose). Standard errors are based on n=6.
FIG. 5 is a graph of plasma LH concentration versus day of DES
treatment.
FIG. 6 is a graph of plasma LH concentration versus time following
administration of DES, in combination with progesterone alone or
progesterone and estradiol.
FIG. 7 is a graph of percentage of maximum LH response versus day
of DES treatment for progesterone-primed ewes.
FIG. 8 is a graph of basal plasma LH concentration versus day of
DES treatment for progesterone-primed ewes.
FIG. 9 is a graph of percentage of maximum LH response versus day
of DES treatment for progesterone-primed ewes.
DETAILED DESCRIPTION OF THE INVENTION
Compositions and methods for intracellular delivery of compounds in
a iscous solution enhancing uptake are described. Cellular
internalization is enhanced (1) by increasing the rate of
receptor-mediated endocytosis by controlling the viscosity of the
solution containing the compound to be delivered and (2) by
co-administration of an enhancer (such as a steroid) in an amount
effective to maximize expression of or binding to receptors
involved in endocytosis mediated uptake. The compositions include
one or more bioactive or diagnostic compounds and a fluid with an
apparent viscosity approximately equal to the apparent viscosity of
the cytosolic fluid in the cell to which the composition is
administered, and optionally, the enhancer. The enhancer can be
delivered in the same formulation or separately, before or after
administration of the compounds to be delivered to the site where
they are to be delivered. Alternatively, the compound can be
administered in the viscous carrier solution at a time selected to
maximize relevant steroidal levels, for example, administered
vaginally during estrus.
Preferably, the compound binds to or otherwise interacts with
receptors on the surface of the cell to which it is to be
delivered. If the compound does not itself bind to or interact with
receptors on the cell surface, it can be administered in a viscous
fluid that also includes a carrier for the compound. The carrier
contains ligands that bind to or otherwise interact with cell
surface receptors, which allows compounds that do not bind to or
otherwise interact with cell surface receptors to participate in
RME.
Compositions
The binding of ligands or assembly proteins to surface receptors of
eucaryotic cell membranes initiates or accompanies a cascade of
nonequilibrium phenomena culminating in the cellular invagination
of membrane complexes within clathrin-coated vesicles. This process
is known as receptor-mediated endocytosis (RME). RME is the primary
means by which several types of bioactive molecules, particularly
macromolecules, enter eukaryotic cells.
Research by others has primarily focused on the identification and
biochemical characterization of the early and later stages of RME,
ranging from formation of a clathrin coated pit to snap-off of a
coated vesicle. Determination of the compositions and methods for
intracellularly administering compounds described herein involved
focusing on a different aspect of RME, the process in which a
membrane depression is initially formed at the outset of RME (i.e.
the mechanism by which a spontaneous thrust of the cell membrane
toward the cytosol occurs). This process is referred to herein as
the `nucleation stage` of RME. This terminology is intended to
emphasize that the driving force for the spontaneous thrust of the
membrane toward the cytosol is related to energy liberated by one
or more of many possible exothermic membrane-binding reactions,
i.e., receptor-ligand binding, that precede or accompany formation
of a membrane depression.
Cell membranes are bound from without by extracellular fluid and
from within by cytosolic fluid. The inter- and extracellular fluids
possess different physical properties, such as density and fluid
viscosity, whose values extend up to the membrane surface where
they undergo discontinuities. The membrane itself possesses unique
equilibrium and nonequilibrium properties. An important property
when considering intracellular delivery is the membrane tension
(the free energy of the membrane per unit surface area). Membrane
tension is generally uniform and positive at an equilibrium
membrane and can be measured by routine micropipet experiments.
Most reported membrane tension values have been gathered for red
blood cells, and range from 4 dyne/cm to 0.01 dyne/cm. By contrast,
the interfacial tension of an air/water interface is 73 dyne/cm.
Membrane tension can vary from point to point on the membrane
surface as a consequence of various stimuli, such as non-uniform
heating of the membrane, membrane chemical reactions and membrane
compositional changes. These variations can give rise to membrane
and bulk-fluid motion, tenned Marangoni convection. This motion is
characterized for the most part by cytosolic and extracellular
(apparent) viscosities.
Exothermic reactions can occur on the cell membrane, due to
ligand-recptor binding, adaptor-membrane binding, clathrin-membrane
binding, a combination of these binding reactions, and other
membrane reactions. The exothermic reactions cause the membrane
tension (energy per membrane area), at least momentarily, to be
diminished at the point where the reaction occurred. As the
membrane tension is lowered, the configurational and intermolecular
potential energies of membrane-bound molecular complexes are also
lowered.
The cell membrane tension is spatially nonuniform as a consequence
of the exothermic reactions (i.e., membrane complex formation),
resulting in membrane motion. This motion will possess a
substantial component toward the cell cytosol so long as the
cytosolic viscosity exceeds that of the extracellular fluid.
This membrane motion causes membrane deformation, an event resisted
by the membrane tension. When the differences between the apparent
viscosities of the cytosolic fluid and the extracellular fluid are
extremely large, membrane deformation is strongly resisted and the
initial thrust of the membrane is damped. However, as the
differences between the apparent viscosities of the cytosolic fluid
and the extra-cellular fluid become extremely small, membrane
deformation becomes progressively rapid.
Accordingly, the rate of endocytosis can be increased by adjusting
the viscosity of the extracellular fluid so that it is
approximately the same as that of the cytosolic fluid, as described
by PCT/US97/03276. If the viscosity of the extra-cellular fluid is
appreciably higher or lower than that of the cytosolic fluid, the
rate of endocytosis decreases. This was shown experimentally in
Example 1 and FIG. 3 of PCT US97/03276, in which the ratio of
compounds that were internalized to those remaining on the surface
(In/Sur) increased as the viscosity of the extracellular fluid
increased, to a point at which the viscosity approached that of the
cytosolic fluid. Aboye that value, the ratio decreased.
Clustering of membrane complexes is favorable for rapid
internalization. The rate of internalization can be increased in
proportion to the magnitude of binding energy. This is due, in
part, to the specificity of receptors to particular ligands and/or
adaptor proteins.
Clustering of complexes occurs in the vicinity of pits to which
clathrin triskelions absorb from the cytosolic side of the cell
membrane and subsequently polymerize to form a clathrin coat. Some
clustering has also been observed in the vicinity of caveolac, or
non-clathrin-coated pits. The membrane-tension depression occurring
within the vicinity of an evolving pit, originating in the process
of membrane complexation, is directly proportional to the number of
membrane complexes formed within that pit. In general, clustered
complexes have been found to internalize substances more rapidly
than nonclustered complexes.
The magnitudes of apparent viscosity difference and receptor
clustering have each been found to alter the rate of RME. Membrane
tension can also be manipulated to influence the rate of RME.
Increasing the membrane tension `hardens` the cell membrane, making
cell membrane depression increasingly prohibitive. This phenomenon
has been commented upon by Sheetz, M.P. and Dai, J. (1995),
presented at the 60th Annual Cold Spring Harbor Symposium on
Protein Kinases, Cold Spring Harbor, N.Y., on the basis of studies
that show an increased rate of endocytosis for neuronal growth
cones coinciding with membrane tension lowering.
Accordingly, the rate of internalization can be increased by a)
adjusting the viscosity of the extracellular fluid to approximate
that of the cytosolic fluid; b) forming complexes of the material
to be internalized; and c) reducing membrane tension. Compositions
and methods for increasing the rate of endocytosis are described in
detail below.
A. Viscous Hydrogels
Suitable viscous fluids for use in intracellularly administering
compounds include biocompatible hydrogels, lipogels, and highly
viscous sols.
A hydrogel is defined as a substance formed when an organic polymer
(natural or synthetic) is cross-linked via covalent, ionic, or
hydrogen bonds to create a three-dimensional open-lattice structure
which entraps water molecules to form a gel. Examples of materials
which can be used to form a hydrogel include polysaccharides,
proteins and synthetic polymers. Examples of polysaccharides
include celluloses such as methyl cellulose, dextrans, and
alginate. Examples of proteins include gelatin and hyaluronic acid.
Examples of synthetic polymers include both biodegradeable and
non-degradeable polymers (although biodegradeable polymers are
preferred), such as polyvinyl alcohol, polyacrylamide,
polyphosphazines, polyacrylates, polyethylene oxide, and
polyalkylene oxide block copolymers ("POLOXAMERS.TM.") such as
PLURONICS.TM. or TETRONICS.TM. (polyethylene oxide-polypropylene
glycol block copolymers).
In general, these polymers are at least partially soluble in
aqueous solutions, such as water, buffered salt solutions, or
aqueous alcohol solutions. Several of these have charged side
groups, or a monovalent ionic salt thereof. Examples of polymers
with acidic side groups that can be reacted with cations are
polyphosphazenes, polyacrylic acids, poly(meth) acrylic acids,
polyvinyl acetate, and sulfonated polymers, such as sulfonated
polystyrene. Copolymers having acidic side groups formed by
reaction of acrylic or methacrylic acid and vinyl ether monomers or
polymers can also be used. Examples of acidic groups are carboxylic
acid groups, sulfonic acid groups, halogenated (preferably
fluorinated) alcohol groups, phenolic OH groups, and acidic OH
groups.
Examples of polymers with basic side groups that can be reacted
with anions arc polyvinyl amines, polyvinyl pyridine, polyvinyl
imidazole, polyvinylpyrrolidone and some imino substituted
polyphosphazenes. The ammonium or quaternary salt of the polymers
can also be formed from the backbone nitrogens or pendant imino
groups. Examples of basic side groups are amino and imino
groups.
Alginate can be ionically cross-linked with divalent cations, in
water, at room temperature, to form a hydrogel matrix. An aqueous
solution containing the compound to be delivered can be suspended
in a solution of a water soluble polymer, and the suspension can be
formed into droplets which are configured into discrete
microcapsules by contact with multivalent cations. Optionally, the
surface of the microcapsules can be crosslinked with polyamino
acids to form a semipermeable membrane around the encapsulated
materials.
The polyphosphazenes suitable for cross-linking have a majority of
side chain groups which are acidic and capable of forming salt
bridges with di- or trivalent cations. Examples of preferred acidic
side groups are carboxylic acid groups and sulfonic acid groups.
Hydrolytically stable poly-phosphazenes are formed of monomers
having carboxylic acid side groups that are crosslinked by divalent
or trivalent cations such as Ca.sup.2+ or Al.sup.3+. Polymers can
be synthesized that degrade by hydrolysis by incorporating monomers
having imidazole, amino acid ester, or glycerol side groups. For
example, a polyanionic poly[bis(carboxylatophenoxy)] phosphazenc
(PCPP) can be synthesized, which is crosslinked with dissolved
multivalent cations in aqueous media at room temperature or below
to form hydrogel matrices.
Methods for the synthesis of the polymers described above are known
to those skilled in the art. See, for example Concise Encyclopedia
of Polymer Science and Polymeric Amines and Ammonium Salts,
(Goethals, ed.) (Pergamen Press, Elmsford, N.Y. 1980). Many of
these polymers are commercially available.
Preferred hydrogels include aqueous-filled polymer networks
composed of celluloses such as methyl cellulose, dextrans, agarose,
polyvinyl alcohol, hyaluronic acid, polyacrylamide, polyethylene
oxide and polyoxyalkylene polymers ("poloxamers"), especially
polyethylene oxide-polypropylene glycol block copolymers, as
described in U.S. Pat. No. 4,810,503. Several poloxamers are
commercially available from BASF and from Wyandotte Chemical
Corporation as "Pluronics". They are available in average molecular
weights of from about 1100 to about 15,500.
As used herein, lipogels are gels with nonaqueous fluid
interstices. Examples of lipogels include natural and synthetic
lecithins in organic solvents to which a small amount of water is
added. The organic solvents include linear and cyclic hydrocarbons,
esters of fatty acids and certain amines (Scartazzini et al. Phys.
Chem. 92:829-33 (1988)).
As defined herein, a sol is a colloidal solution consisting of a
liquid dispersion medium and a colloidal substance which is
distributed throughout the dispersion medium. A highly viscous sol
is a sol with a viscosity between approximately 0.1 and 2000
Poise.
Other useful viscous fluids include gelatin and concentrated sugar
(such as sorbitol) solutions with a viscosity between approximately
0.1 and 2000 Poise.
The apparent viscosity of the extracellular fluid (the composition)
must be approximately equal to the viscosity of the cytosolic fluid
in the cell to which the compounds are to be administered. One of
skill in the art can readily determine or reasonably estimate of
the viscosity of the cytosolic fluid using a viscometer and
measuring the applied stress divided by measured strain rate at the
applied stress that corresponds to the stress the cell membrane
imparts upon the cytosolic and extracellular fluids during
endocytosis. Methods for measuring the cytosolic viscosity include
micropipette methods (Evans & Young, Biophys. J., 56:151-160
(1989)) and methods involving the motion of membrane-linked
colloids (Wang et al., Science, 260:1124-26 (1993). Typical cytosol
viscosities, measured by these techniques, range from approximately
50-200 Poise. Once this value is measured, the viscosity of the
composition can be adjusted to be roughly equal to that viscosity,
particularly when measured via routine methods at the applied
stress that corresponds to the stress the cell membrane imparts
upon the cytosolic and extracellular fluids during endocytosis.
The viscosity can be controlled via any suitable method known to
those of skill in the art. The method for obtaining a viscous
composition with the desired apparent viscosity is not particularly
limited since it is the value of the apparent viscosity relative to
the target cells which is critical. The apparent viscosity can be
controlled by adjusting the solvent (i.e., water) content, types of
materials, ionic strength, pH, temperature, polymer or
polysaccharide chemistry performed on the materials, and/or
external electric, ultrasound, or magnetic fields, among other
parameters.
The apparent viscosity of the compositions is controlled such that
it lies in the range of between 0.1 and 2000 Poise, preferably
between 7 and 1000 Poise, and most preferably between 2 and 200
Poise. The apparent viscosity can be measured by a standard
rheometer using an applied stress range of between 1 and 1000
Pascals, preferably between 1 and 500 Pascals, and most preferably
between 1 and 100 Pascals. Further, the viscosity of the
compositions is controlled so that the quotient of (apparent
viscosity of the cytosol of the target cells-apparent viscosity of
the composition) and the apparent viscosity of the cytosol of the
target cells is between approximately -0.1 and 0.3, preferably
between approximately 0 and 0.3, more preferably between
approximately 0 and 0.1, and most preferably between approximately
0 and 0.05.
The composition can be administered as an only slightly viscous
formulation that becomes more viscous in response to a condition in
the body, such as body temperature or a physiological stimulus,
like calcium ions or pH, or in response to an externally applied
condition, such as ultrasound or electric or magnetic fields. An
example is a temperature sensitive poloxamer which increases in
viscosity at body temperature.
The following are examples of suitable concentration ranges:
Methocel solutions in the range of between 1.0 and 2.0% (w/w),
polyvinyl alcohol solutions between 5 and 15%, pluronic acid
solutions between 15 and 20% and trehalose solutions between 1 and
5%.
B. Enhancers
Compounds that can be attached, covalently or noncovalently, to a
molecule that either stimulates receptor-mediated endocytosis (RME)
or pinocytosis by binding to receptors on the plasma membrane,
binds specifically to receptors that undergo RME or pinocytosis
independently of this binding, or at least can be associated
chemically or physically with other molecules or "carriers" that
themselves undergo RME or pinocytosis, are referred to as enhancers
for intracellular delivery. Examples include steroids such as
estradiol and progesterone, and some glucocorticoids.
Glucocorticoids such as dexamethasone, cortisone, hydrocortisone,
prednisone, and others are routinely administered orally or by
injection. Other glucocorticoids include beclomethasone,
dipropianate, betamethasone, flunisolide, methyl prednisone, para
methasone, prednisolone, triamcinolome, alclometasone, amcinonide,
clobetasol, fludrocortisone, diflurosone diacetate, fluocinolone
acetonide, fluoromethalone, flurandrenolide, halcinonide,
medrysone, and mometasone, and pharmaceutically acceptable salts
and mixtures thereof. Other compounds also bind specifically to
receptors on cell surfaces. Many hormone specific receptors are
known. These can all be used to enhance uptake. Selection of
molecules binding to receptors which are predominantly found on a
particular cell type or which are specific to a particular cell
type can be used to impart selectivity of uptake.
The enhancer is preferably administered at a time and in an amount
effective to maximize expression of receptors, and consequently
receptor mediated internalization of the compound. The enhancer can
itself be the compound to be delivered.
C. Compounds to be Delivered
As noted above, the compound to be delivered may be the same as or
different from the enhancer. The enhancer can be administered as
part of the formulation containing the compound to be delivered or
prior to or as part of a different formulation. The enhancer may be
administered systemically, followed by administration of the
compound to be delivered directly to the site where uptake is to
occur.
Compounds to be delivered include proteins and peptides, nucleic
acid molecules including DNA, RNA, antisense oligonucleotides,
triplex forming materials, ribozymes, and guide sequences for
ribozymes, carbohydrates and polysaccharides, lipids, and other
synthetic organic and inorganic molecules. Preferred bioactive
compounds include growth factors, antigens, antibodies or antibody
fragments, and genes such as genes useful for treatment of cystic
fibrosis, A1A deficiency and other genetic deficiencies.
Preferred hormones includes peptide-releasing hormones such as
insulin, luteinizing hormone releasing hormone ("LHRH"),
gonadotropin releasing hormone ("GnRH"), deslorelin and leuprolide
acetate, oxytocin, vasoactive intestinal peptide (VIP), glucagon,
parathyroid hormone (PTH), thyroid stimulating hormone, follicle
stimulating hormone, growth factors such as nerve growth factor
(NGF), epidermal growth factor (EGF), vascular endothelial growth
factor (VEGF). insulin-like growth factors (IGF-I and IGF-II),
fibroblast growth factors (FGFs), platelet-derived endothelial cell
growth factor (PD-ECGF), transforming growth factor beta
(TGF-.beta.), and keratinocyte growth factor (KGF). Other materials
which can be delivered include cytokines such as tumor necrosis
factors (TFN-.alpha. and TNF-.beta.), colony stimulating factors
(CSFs), interleukin-2, gamma interferon, consensus interferon,
alpha interferons, beta interferon; attachment peptides such as
RGD; bioactive peptides such as renin inhibitory peptides,
vasopressin, detirelix, somatostatin, and vasoactive intestinal
peptide; coagulation inhibitors such as aprotinin, heparin, and
hirudin; enzymes such as superoxide dismutase, neutral
endopeptidase, catalase, albumin, calcitonin, alpha-1-antitrypsin
(A1A), deoxyribonuclease (DNAase), lectins such as concanavalin A,
and analogues thereof.
Diagnostic agents can also be delivered. These can be administered
alone or coupled to one or more bioactive compounds as described
above. The agents can be radiolabeled, fluorescently labeled,
enzymatically labeled and/or include magnetic compounds and other
materials that can be detected using x-rays, ultrasound, magnetic
resonance imaging ("MRI"), computed tomography ("CT"), or
fluoroscopy.
D. Carriers for Compounds to be Delivered
The compounds to be delivered and/or enhancers can optionally be
incorporated into carriers, which are then dispersed in a viscous
fluid with an apparent viscosity approximately equal to the
cytosolic fluid of the cell to which the compounds are to be
delivered. Exemplary carriers include viruses, liposomes, lipid/DNA
complexes, micelles, protein/lipid complexes, and polymeric
nanoparticles or microparticles.
The carrier must be small enough to be effectively endocytosed.
Suitable carriers possess a characteristic dimension of less than
about 200 nm, preferably less than about 100 nm, and more
preferably, are less than about 60 nm.
The carrier must be able to bind to a cell surface receptor. If the
carrier does not naturally bind, it is well known in the art how to
modify carriers such that they are bound, ionically or covalently,
to a ligand (i.e., LHRH) that binds to a cell surface receptor. For
example, U.S. Pat. No. 5,258,499 to Konigsberg et al. describes the
incorporation of receptor specific ligands into liposomes, which
are then used to target receptors on the cell surface.
The use of carriers can be important when the compound to be
delivered does not bind to or otherwise interact with cell surface
receptors. The compound can be incorporated into a carrier which
contains a ligand or other moiety which binds to or interacts with
cell surface receptors. Then, due to the binding of or interaction
with the receptor to the cell surface and the apparent viscosity of
the composition, the carrier (and encapsulated compound) is
intracellularly delivered by endocytosis.
The use of carriers can be particularly important for
intracellularly delivering nucleic acid molecules. In one
embodiment, nucleic acid molecules are encapsulated in a liposome,
preferably a cationic liposome, that has a receptor-binding ligand,
such as LHRB, on its surface. The liposome is then dispersed in a
viscous fluid. When the composition is administered, the liposomes
are endocytosed by the cell, and the nucleic acid molecules are
released from the liposome inside the cell.
E. Compositions for Lowering or Raising Membrane Tension
The efficiency of the method can be increased by lowering the
membrane tension. Suitable methods for lowering membrane tension
include including a biocompatible surface active agent in the
hydrogel, performing exothermic reactions on the cell surface
(i.e., complex formnation), and applying an external field to the
cell surface. Suitable biocompatible surface active agents include
surfactin, trehalose, fatty acids such as palmitin and oleic acid,
polyethylene glycol, hexadecanol, and phospholipids such as
phosphatidylcholines and phosphatidylglycerols. Suitable
complex-forming chemical reactions include the reaction of
receptor-binding ligands with cell surface receptors for these
ligands, exothermic reactions such as occur between sodium
salicylate and salicylic acid, and neutralization reactions as
between hydrochloric acid and ammonia (Edwards et al. 1996 Biophys.
J. 71, 1208-1214). External fields that can be applied to a cell
surface to reduce membrane tension include ultrasound, electric
fields, and focused light beams, such as laser beams.
The rate of cellular internalization can also be increased by
causing the clustering of receptors on the cell membrane. This can
be accomplished, for example, by creating zones on the membrane
where the membrane tension is relatively high, causing the membrane
fluid to flow toward the zone of high membrane tension. This flow
can carry receptors localized in the membrane toward each other,
causing them to cluster.
Methods of Administration
In a preferred embodiment, the compound to be delivered and/or the
enhancer are contained in the same formulation for simultaneous
administration. Alternatively, the composition and steroid are
provided as parts of a kit, for separate administration. As shown
in the examples, the enhancer may be a hormone such as estradiol or
progesterone, administered systemically, while the compound to be
delivered is administered topically at a site where delivery is
enhanced by the hormone, such as the vaginal mucosa.
The compositions can be applied topically to the vagina, rectum,
nose, eye, ear, mouth and the respiratory or pulmonary system.
Preferably, the compositions are applied directly to the cells to
which the compound is to be delivered, usually in a topical
formulation. The enhancer can be administered simultaneously with
or after administration of the composition including the viscous
gel and agent to be delivered. The administration schedule (e.g.,
the interval of time between administering the enhancer and
administering the gel composition) can be readily selected by one
of skill in the art to maximize receptor expression and/or binding
before exposure of the cell surface to the agent to be
delivered.
The compositions are particularly advantageous for gene delivery
and hormone therapy By delivering a composition containing peptides
such as GnRH or its analogues across the vaginal or nasal
membranes, the compositions can be used to treat a variety of human
hormone-based disorders.
The dosage will be expected to vary depending on several factors,
including the patient, the particular bioactive compound to be
delivered, and the nature of the condition to be a treated, among
other factors. One of skill in the art can readily determine an
effective amount of the bioactive compound or compounds to
administer to a patient in need thereof.
The method involves administering the composition to cells to
enhance the rate of transport across the cell membranes, relative
to the rate of delivery when non-viscous fluids are used in
combination with enhancer or when viscous fluids are used without
enhancer. Examples of methods of administration include oral
administration, as in a liquid formulation or within solid foods,
topical administration to the skin or the surface of the eye,
intravaginal administration, rectal administration, intranasal
administration, and administration via inhalation. When the
composition is administered orally or by inhalation, it is
preferred that it is administered as a dry powder that includes a
swellable hydrogel that is designed to swell to an appropriate
viscosity after delivery to the desired location. After inhalation,
for example, the hydrogel absorbs water to obtain the desired
viscosity and then delivers agents to the respiratory system. When
administered orally, a hydrogel can be selected that does not
absorb water under conditions present in the upper gastrointestinal
tract, but which does absorb water under conditions present in the
lower gastrointestinal tract (i.e., at a pH greater than about
6.5). Such hydrogels are well known to those of skill in the art.
The use of such compositions can optimize the delivery of agents to
the lower gastrointestinal tract.
Applications for the Compositions and Methods
The methods and compositions described herein are useful in a
variety of therapeutic and diagnostic applications for humans and
other animals. Preferred applications include the treatment of
infertility and disease, such as cancer. The compositions can be
used in various hormone replacement therapies as well. In a
preferred method of use, viscous compositions arc used to deliver
progesterone vaginally to induce secretory transformation of the
endometrium and promote development of pregnancy.
The compositions and methods of use thereof described herein will
be more clearly understood with reference to the following
non-limiting examples.
EXAMPLE 1
Peptide Transport Across the Vaginal Epithelium of Sheep
This study was intended to examine the relevance of control of the
apparent viscosity of the extracellular fluid/cytosolic fluid to
the enhancement of peptide drug delivery into the body via a
noninvasive route by examining peptide transport across the vaginal
epithelium of sheep.
A. Receptor-mediated Transport of a Peptide, No Exogenous
Steroid
First, a peptide that undergoes receptor-mediated transport across
the vaginal epithelium was identified by studying the permeation of
peptides of varying molecular weight in a sheep model. Peptides
were delivered vaginally to sheep in 5 ml of aqueous or methocel
solutions with typical peptide concentrations of 10-40 .mu.g/ml. A
group of 18 intact ewes were utilized for these experiments. For
each study, sheep were randomly assigned to a treatment group. In
GNRH studies where each animal received all possible treatment
combinations, each animal was assigned to an initial treatment
group at random and subsequently randomly to each of the remaining
treatment groups. A minimum of 5 days (typically 10 or more days)
was allowed between experiments on a given animal, to provide
sufficient time for complete recovery of pituitary responsiveness
to the highest doses of the GnRH agonist used. A 16G 150 mm jugular
catheter (Abbocath-T, Abbott Laboratories, Chicago, Ill.) was
inserted and blood samples collected at 0, 30, 60, 90, 120, 180,
240, 360, 480 and 1440 min. after treatment. Luteinizing hormone
(LH) levels were determined.
The bioavailabilities of vasopressin (1084 Da), salmon calcitonin
(3416 Da), and insulin (5786 Da) all were found to be less than
0.1% following vaginal administration in an aqueous buffer.
Leuprolide acetate [luteinizing hormone releasing hormone (LHRH)
analog] (1209 Da), however, exhibited high bioavailability
(2.6.+-.0.9%) based on biological response, even though its
molecular weight is slightly larger than that of vasopressin. A
comparison of the biological response to vasopressin and leuprolide
acetate is shown in FIGS. 1a and 1b. Vasopressin administered by
intravenous injection leads to high systemic cortisol levels within
the first hour following treatment. However no detectable change in
systemic cortisol levels was observed following vaginal
administration (FIG. 1a). In contrast, LHRH analog produced
significant luteinizing hormone (LH) response following intravenous
injection and following vaginal administration (FIG. 1b). The near
coincidence of peak serum LH concentrations following injection and
vaginal administration indicated rapid internalization of
leuprolide acetate, characteristic of a receptor-mediated route of
transport.
B. Enhancement of Transport using a Viscous, Balanced Carrier
LHRH analog was placed in methyl cellulose solutions ("methocels")
of varying apparent viscosity. Studies of transferrin-mediated
endocytosis on single cells have shown peak endocytosis rates at
methyl cellulose concentrations between 1.25 and 1.75%, at which
concentrations the methocels exhibit apparent viscosities in a
range typical of intracellular viscosities (Evans & Yeung,
Biophys. J. 56:151-60 (1989)). First, 200 .mu.g of leuprolide
acetate in 5 ml of aqueous solutions with methocel weight
concentrations varying between 0% and 3.0% were vaginally
administered. LHRH analog bioavailability was found to increase as
methocel concentration increased to 1.75%, then to fall at higher
methocel concentration (FIG. 2), mirroring a trend observed for
receptormediated endocytosis with single cells (Edwards, et al.,
Proc. Natl. Acad. Sci. U.S.A. 93:1786-91 (1996)). This appears to
suggest that transfer of LHRH analog into the systemic circulation
is rate-limited by endocytic transfer from the apical side of the
vaginal epithelium, which can itself be controlled by the viscosity
of the methocel solution within which it is administered, for the
fluid-mechanical reasons described above and in Edwards, et al.,
Proc. Natl. Acad. Sci. U.S.A. 93:1786-91(1996).
The enhanced bioavailability of LHRH also coincides with a
longer-term release into the systemic circulation at the optimal
methocel concentrations. This suggests the possibility of a
diffusion-controlled delivery process, rather than an active,
endocytic-controlled process; that is, increasing hydrogel
viscosity might be related to diminished rate of peptide diffusion
through the hydrogel to the vaginal epithelium. To test this
hypothesis, the efficacy of a second, physically cross-linked
hydrogel that was believed would not enhance endocytosis, but whose
apparent viscosity (in the range of hydrogel concentrations
0.0-5.5%) was similar to that of the methocels (in the range
0.0-3.0%) was examined. It was anticipated that the physically
cross-linked structure of the "control" hydrogel would prevent its
deformation with (and entry into) invaginating sites on the
epithelial membrane, hence impeding, rather than enhancing,
endocytic uptake.
The results showed that when 5 ml of solution containing the
physically cross-linked control gel was administered vaginally, the
bioavailability of LHRH analog diminished with increasing
concentration of the hydrogel in the range of 0.0-5.5%.
Importantly, the duration of LHRH analog delivery also diminished
with increasing control gel concentration, which is an unexpected
effect if LHRH analog delivery is passive-diffusion controlled.
To determine whether membrane damage might explain the results
shown in FIG. 2, vasopressin was vaginally administered in methocel
solutions of. 1.5 and 1.75%. Identical to the saline vaginal
administration (FIG. 1a), no detectable changes in concentrations
of cortisol were observed when vasopressin was administered with
the methocel solutions, indicating that the barrier properties of
the membrane to passive transport remain intact.
C. Determination of Role of Steroids in Uptake and Transport
Biological response to vaginal LHRH analog administration exhibited
a bimodal distribution in the studies (see FIG. 2), with
approximately 30% of animals showing little or no response at all.
No such bimodal response was observed when LHRH analog was
administered by intravenous injection (FIG. 1b), indicating that
the source of the bimodal response resides in the vaginal
absorption pathway. It was therefore hypothesized that the
responsiveness of animals to LHRH analog vaginal delivery varied
with steroid-dependent hormone receptor expression (estrous cycle).
To test this hypothesis, a group of ewes was ovariectomized and
administered estradiol and progesterone to mimic the animals'
estrous cycle. Ewes were pre-medicated with atropine (0.02 mg/lb)
and Telazol (R) (2 mg/lb) intramuscularly. After induction of
recumbency, thipental (5% in water) was administered intravenously
to induce sufficient anesthesia to permit endotracheal intubation.
Anesthesia was maintained using halothane in oxygen at 1-2 liters
per minute. Ovaries were removed through a mid-line incision.
Within 24 h of surgery, a 1.5 cm silicone implant of estradiol
(Compudose 200, Elanco, Ind.) was inserted into the left ear to
provide a basal level of estradiol. The anestrus state was
simulated after two weeks of estradiol delivery following surgery.
Experiments were performed in the simulated anestrus state (i.e.
two weeks after surgery) as described above. Immediately following
the last blood sample, a progesterone-releasing intravaginal device
(CIDR-G, Carter Hold Harvey Plastic Products, Hamilton, New
Zealand) was inserted.
In a parallel study, an alternative progesterone-releasing device
(Snychro-Mate-B, Sanofi Animal Health, Overland Park, Kans.) was
placed in the left ear. The mid-luteal phase was simulated after
permitting a 10 day intravaginal or ear progesterone treatment.
Experiments were performed in the simulated mid-luteal phase as
described above.
The progesterone-releasing (vaginal or ear) implant was removed and
the follicular phase was simulated by allowing a time lapse of 48
h. Experiments were performed in the follicular phase as described
above.
Next, 200 .mu.g of leuprolide acetate in 5 ml of aqueous solutions
was vaginally administered during simulated anestrus (estradiol
only), mid-luteal (estradiol and progesterone), and follicular (48
h after progesterone withdrawal) phases, as described above. When
LHRH analog was delivered in aqueous solutions with or without
1.75% methocel, it was found that less than 50% of animals
responded during estrogen replacement without progesterone. In
contrast, 100% of animals responded when treated with progesterone
and estradiol (FIG. 3). This same trend also was observed for the
other LHRH analog doses. That is, a reproducible response was
observed in all animals only during progesterone and estradiol
treatment, when the animals can be expected to express maximal
numbers of hormone receptors.
This confirms the hypothesis that the LH response of animals
depended on steroid milieu, as is consistent with the hypothesis of
uptake of LHRH analog occurs via a receptor-mediated route.
Three doses of LHRH analog: 10, 40, and 100 .mu.g, were
administered. It was found that during progesterone and estradiol
treatment, the highest and lowest doses resulted in LH responses
that were either saturated (maximal LH response with and without
methocel) or undetectable, presumably due to the sigmoidal
dose-response nature of LHRH analog treatment. The results of the
intermediate dose-response study are shown in FIG. 4. The 1.75%
methocel administration results in a bioavailability of 6% compared
to 10% for the case of the 0% methocel. These results agree with
the results of the uncontrolled animal study (FIG. 2) (minus
non-responders).
A significant finding of this study is that LHRH analog delivery
across the vaginal mucosa is receptor-mediated, with a
reproducibility that can be increased by controlling the stage of
the estrous cycle. This approach to be peptide delivery can be
further improved by controlling the viscous properties of the
medium that contacts the vaginal mucosa, and from which the peptide
transfers. Unlike the single-cell systems where a similar
phenomenon has been observed, the vaginal mucosa includes a mucus
barrier that is itself highly viscous and presumably combines with
the administered hydrogel to create a mixture of artificial and
physiological hydrogels whose net viscous properties act to control
the rate of vesicle formation along the apical epithelial membrane.
While the exact nature of this mixed gel remains unclear (as, for
example, the administration of estradiol and progesterone changes
the rheological properties of the mucus lining, potentially
providing an alternative interpretation of the results observed in
FIG. 3), that hydrogel-enhancement of receptor-mediated transport
can be achieved at vaginal mucosa suggests that a similar
enhancement can achieved at other drug delivery sites, such as at
the nasal mucosa.
The ability to enhance the delivery of LHRH analog into the
systemic circulation by delivering LHRH analog in a
"rheologically-optimized" hydrogel should help to make noninvasive
LHRH analog therapies (such as the treatment of endometriosis or
prostate cancer) more viable than at present. Recognition that it
enters the body via a receptor-mediated route can further lead to
hormonal-control strategies to minimize irreproducibility. Finally,
the chemical attachment of LHRH analog to other molecules or
nanoparticulate carriers that are too large to cross epithelial
barriers of the body at a therapeutically relevant rate yet
sufficiently small to enter an endocytic vesicle should make it
possible to use LHRH analog as a kind of locomotive to propel other
molecules, vesicle, or particles into the body without the need for
injection.
EXAMPLE 2
Steroid and GnRH Transport
Studies were conducted to assess the involvement of steroids in
modulating the transport of GNRH across the vaginal mucosa. The
main objectives were to confirm the need for steroids in vaginal
GnRH transport, to determine if treatment with both progesterone
and estradiol were necessary, and to demonstrate down-regulation of
LH secretion with daily administration of GnRH agonist.
A. Chronic Vaginal Dosing of DES to Suppress LH Secretion
The objective was to determine if chronic vaginal dosing with 200
.mu.g of deslorelin ("DES") in gel would be able to suppress
secretion of LH. Lower doses of DES will result in the
down-regulation of the anterior pituitary gland in sheep.
Ovariectomized sheep were used for the study, since they secrete
high levels of LH in the absence of ovarian steroids. The sheep
were dosed daily with DES in 5 mL of gel, or gel only for 17 days.
A single blood sample was collected by jugular venipuncture. Plasma
was collected and assayed for LH.
Results from this study are shown in FIG. 4. There were no
differences in the average concentrations between the DES and
control groups over the course of the study. LH secretion appears
to have been slightly suppressed between days 6 and 12 of the
study. However, a much greater inhibition in LH secretion was
expected if significant amounts of DES were crossing the vaginal
mucosa. An earlier study demonstrated that estrous cycles could be
inhibited with daily vaginal administration of GnRH agonist. In
this study, however, all the animals had intact ovaries. Previous
studies have followed LH release after a single dosing. It has been
shown that sheep treated with estradiol and progesterone respond
much better in terms of the percentage of animals exhibiting LH
release, the magnitude of the LH release, and a reduction in the
variance of the response. Therefore, it is likely that treatment
with progesterone, either alone or in combination with estradiol,
was required for the GARH agonist to be transported across the
vaginal mucosa.
B. Determination of Roles of Progesterone and Estradiol in Vaginal
Transport
This study was conducted to determine if progesterone alone, or
progesterone in combination with estradiol, is required to ensure
GnRH transport across the vaginal epithelium. In past studies,
estradiol was given as an implant inserted in the outer ear, which
is a common method of administering estradiol to animals. However,
it results in high and variable estradiol concentrations in jugular
blood. It would be helpful to eliminate this steroid from the
animal model, if possible, since delivering estradiol using
GnRH-based technologies is of interest.
Ewes (n=12) were treated with progesterone using a vaginal CIDR
device. In addition, six sheep received a 15 mm silastic implant of
estradiol. Five days later, all sheep were treated vaginally with
200 .mu.g of DES in gel. Blood samples were taken every 30 minutes
for 2 hours following treatment and then every 60 minutes for an
additional four hours.
All ewes in this experiment responded with a robust discharge of LH
following GNRE treatment. There was a distinct difference in the
pattern of LH release between the two groups (FIG. 13). The peak LH
occurred earlier in ewes treated only with progesterone (120
minutes) than in ewes treated with progesterone and estradiol (240
minutes).
The difference in the timing of the peak LH between the groups is
nearly identical to those differences between ovariectomized and
ovariectomized plus estradiol treated ewes (see Deaver et al.,
Domest. Anim. Endocrinol. 4(2):95-102 (1987)). Thus, it is likely
that the difference in the patterns of LH release is attributable
to estradiol's effects on pituitary responsiveness to GnRH and not
the vaginal uptake mechanism. Furthermore, if the latter were
correct, then the lag time between treatment and vaginal transfer
of GnRiRH into the circulatory system would be on the order of 7-80
minutes.
C. Effect of Vaginal Administration of DES in Progesterone-primed
Ewes
The objective of this study was to determine if daily vaginal
administration of DES in progesterone-primed ewes would cause a
reduction in basal LH secretion and loss of pituitary
responsiveness of GnRH. Six ovariectomized ewes were used for the
study. Vaginal CIDR devices containing progesterone were inserted.
Twenty-four hours later, ewes were dosed daily with 200 .mu.g of
DES (GnRH agonist) in 5 mL of gel. Each day, blood samples were
collected at 0 and 120 minutes post-treatment. These times were
selected in order to evaluate changes in basal secretion of LH
(time 0) and the peak LH response following GnRH administration
(time 120).
The change in LH between 0 and 120 minutes was greatest in 3 of 6
ewes on the first day of DES administration. In the fourth ewe, a
robust release of LH was observed following the first DES
treatment, but the increase in LH was even higher following the
second treatment with DES. When the differential in LH release
following the first treatment with DES is assigned a value of 100%
and the change in responsiveness plotted against time (FIG. 7), it
is clear that continued daily treatment significantly reduced
pituitary responsiveness to DES. In addition, significant decreases
in basal LH also occurred in these animals (FIG. 8).
In the two remaining animals, the change in LH secretion continued
to increase 5 to 7 days following the initiation of DES treatment.
However, once the maximum response was achieved,
pituitary-responsiveness to DES rapidly declined (FIG. 16; Ewe 9).
A reasonable interpretation of these data is that insufficient DES
was transported over the first several days to initiate the
down-regulation phenomena. However, once the transport mechanism
became optimized, adequate transport of DES was achieved to
down-regulate LH release from the anterior pituitary gland.
D. Direct Vaginal Administration of DES Enhanced Uptake
An attempt was made to treat ewes with progesterone using the
systemic administration of a depot form of progesterone and ear
implants of a synthetic progestogen. Pituitary release of LH was
poor following the vaginal administration of DES in gel. When LH
release in these animals was not observed, the gel from the same
preparations used vaginally was injected subcutaneously. LH release
was then obtained, confirming that the DES/gel preparations
contained biologically active material. Given the consistent
responses obtained in earlier experiments and those obtained more
recently using the CIDR delivery system, it was concluded that the
progesterone (generally) should be applied directly to the vagina
in order to achieve sufficient local concentrations. Given that
luteal phase sheep also respond well, systemic administration of
greater amounts of progesterone than used here, in formulations
that will maintain consistently high concentrations in the blood,
should also work.
E. Controls Show Uptake is Selective.
Another study provided information about the role of the silastic
CIDR device itself in the delivery process. The study was based on
concern that the CIDR might be damaging the vaginal mucosa,
allowing for GnRH transport by a non-selective mechanism. CIDR
devices were inserted into six ewes. Five days later, all ewes were
treated vaginally with DES. Five of the six ewes had a robust
release of LH. After the initial dosing, three ewes were treated
with DES and three with gel alone every day for 18 days. At the end
of the 18-day treatment period, all ewes were again treated with
DES in gel. This time, none of the ewes displayed a robust release
of LH.
Review of the protocol and notes showed that the CIDR devices,
which were designed to release progesterone only over a 10 to
12-day period, were not changed mid-way through the trial as
initially planned. Consequently, by the time the second DES
administration was given, the CIDR devices had been in place for
approximately 24 days. The ewes therefore were no longer receiving
adequate amounts of progesterone locally to maintain the vaginal
transport system. This study showed that the CIDR per se does not
facilitate vaginal uptake of GnRH.
Conclusions
Based on the outcome of all experiments, local short periods of
progesterone treatment activates a mechanism for transporting GnRH
agonist across the vaginal mucosa in sufficient amounts to acutely
cause the release of LH and the down-regulation of LH release with
repeated dosing.
Local exposure of the vagina to progesterone is preferred for the
transport of GnRH agonist across the mucosal membrane. Systemic
administration of either progesterone or synthetic progestogens is
not preferred for achieving adequate priming of the vaginal mucosa
for GnRH transport. Intravaginal devices used for administration of
progesterone do not appear to directly effect vaginal transport of
GnRH.
Approximately 50% of ewes will have significant transport after
only 24 hours of exposure to progesterone, and essentially 100% of
the ewes will transport GnRH after four days of exposure.
Co-administration of estradiol will alter the time course of LH
release in progesterone treated ewes, which likely is due to a
direct effect on the anterior pituitary gland. The lag time is
about 70-80 minutes between vaginal administration of GNRH and the
transport of sufficient amounts of GNRH agonist into the blood to
cause LH release. Chronic administration of GnRH agonist across the
vaginal mucosa will reduce the basal secretion of LH and
down-regulate the ability of the anterior pituitary gland to
respond to GnRH agonist.
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. The
references cited herein are hereby incorporated by reference.
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