U.S. patent application number 10/144516 was filed with the patent office on 2002-11-21 for covalent polar lipid conjugates with biologically active compounds for use in salves.
Invention is credited to Stowell, Michael HB, Yatvin, Milton B..
Application Number | 20020173498 10/144516 |
Document ID | / |
Family ID | 27385855 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020173498 |
Kind Code |
A1 |
Yatvin, Milton B. ; et
al. |
November 21, 2002 |
Covalent polar lipid conjugates with biologically active compounds
for use in salves
Abstract
This invention herein describes a method of facilitating the
entry of drugs into cells and tissues at pharmokinetically useful
levels and also a method of targeting drugs to specific organelles
within the cell. This polar lipid/drug conjugate targeting
invention embodies an advance over other drug targeting methods
because through this method, intracellular drug concentrations may
reach levels which are orders of magnitude higher than those
achieved otherwise. Furthermore, it refines the drug delivery
process by allowing therapeutic agents to be directed to certain
intracellular structures. This technology is appropriate for use
with antiproliferative, antibiotic, antimycotic, antiviral and
antineoplastic drugs, in particular in combination with a
multiplicity of other emollients and agents to make up
topically-active substances such as salves, for rapid and efficient
introduction of such agents through the epidermis for treatment of
skin diseases and other disorders.
Inventors: |
Yatvin, Milton B.;
(Portland, OR) ; Stowell, Michael HB; (Pasadena,
CA) |
Correspondence
Address: |
DAVIDSON, DAVIDSON & KAPPEL, LLC
485 SEVENTH AVENUE, 14TH FLOOR
NEW YORK
NY
10018
US
|
Family ID: |
27385855 |
Appl. No.: |
10/144516 |
Filed: |
May 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10144516 |
May 13, 2002 |
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09415640 |
Oct 12, 1999 |
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6387876 |
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10144516 |
May 13, 2002 |
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08142771 |
Oct 26, 1993 |
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5543389 |
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08142771 |
Oct 26, 1993 |
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07911209 |
Jul 9, 1992 |
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5256641 |
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07911209 |
Jul 9, 1992 |
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07607982 |
Nov 1, 1990 |
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5149794 |
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Current U.S.
Class: |
514/178 ;
514/121; 514/179; 514/26 |
Current CPC
Class: |
A61K 9/167 20130101;
B82Y 5/00 20130101; A61K 47/544 20170801; Y10S 514/863 20130101;
A61K 47/543 20170801; A61K 47/6903 20170801; A61K 47/645 20170801;
A61K 31/00 20130101; C07H 19/10 20130101; C07H 19/06 20130101 |
Class at
Publication: |
514/178 ;
514/179; 514/26; 514/121 |
International
Class: |
A61K 031/704; A61K
031/66; A61K 031/56; A61K 031/57 |
Claims
What is claimed is:
1. A pharmaceutical composition comprising a corticosteroid drug, a
polar lipid carrier, two linker functional groups and a spacer,
wherein the spacer has a first end and a second end and wherein the
polar lipid is attached to the first end of the spacer through a
first linker functional group and the drug is attached to the
second end of the spacer through a second linker functional group,
the composition further comprising a medicinal ointment or
salve.
2. The pharmaceutical composition of claim 1 wherein the drug is
selected from the group consisting of cortisone, cortisol,
hydrocortisone, prednisone, fluorinated corticosteroids,
dexamethasone, alcloethasone, fluoroandrenolide and mometasone.
3. A pharmaceutical composition according to claim 1 wherein the
spacer allows the drug to act without being released at an
intracellular site and wherein the first linker functional group
attached to the first end of the spacer is strong and the second
linker functional group attached to the second end of the spacer is
weak.
4. A pharmaceutical composition according to claim 1 wherein the
spacer allows the facilitated hydrolytic release of the drug at an
intracellular site and wherein the first linker functional group
attached to the first end of the spacer is strong and the second
linker functional group attached to the second end of the spacer is
weak.
5. A pharmaceutical composition according to claim 1 wherein the
spacer allows the facilitated enzymatic release of the drug at an
intracellular site and wherein the first linker functional group
attached to the first end of the spacer is strong and the second
linker functional group attached to the second end of the spacer is
weak.
6. A pharmaceutical composition according to claim 1 wherein the
polar lipid is acyl carnitine, acylated carnitine, sphingosine,
ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl
ethanolamine, phospliatidyl inositol, phosphatidyl serine,
cardiolipin or phosphatidic acid.
7. A pharmaceutical composition comprising a corticosteroid drug
having a first functional linker group, and a polar lipid carrier
having a second functional linker group, wherein the drug is
covalently linked to the polar lipid carrier by a chemical bond
between the first and second functional linker groups, the
composition further comprising a medicinal ointment or salve.
8. A pharmaceutical composition according to claim 7 wherein the
first functional linker group is a hydroxyl group, a primary or
secondary amino group, a phosphate group or substituted derivatives
thereof or a carboxylic acid group.
9. A pharmaceutical composition according to claim 7 wherein the
second functional linker group is a hydroxyl group, a primary or
secondary amino group, a phosphate group or substituted derivatives
thereof or a carboxylic acid group.
10. A pharmaceutical composition according to claim 7 wherein the
polar lipid is acyl carnitine, acylated carnitine, sphingosine,
ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl
ethanolamine, phosphatidyl inositol, phosphatidyl serine,
cardiolipin or phosphatidic acid.
11. The pharmaceutical composition of claim 7 wherein the drug is
selected from the group consisting of cortisone, cortisol,
hydrocortisone, prednisone, fluorinated corticosteroids,
dexamethasone, alcloethasone, fluoroandrenolide and mometasone.
12. A method for treating a pathological condition or disease state
in cells, tissues or organs in an animal, the method comprising the
step of administering to the animal a pharmaceutical composition of
claim 1 in an acceptable carrier or formulation and in an amount
sufficient to alleviate the pathological condition or disease state
in the animal.
13. A method for treating a pathological condition or disease state
in skin of an animal, wherein the pathological condition or disease
state results from an abnormal proliferation of cells in the
animal, the method comprising the step of administering to the
animal a pharmaceutical composition according to claim 1 in an
acceptable carrier or formulation and in an amount sufficient to
alleviate the pathological condition or disease state in the
animal.
14. The method of claim 12 wherein the animal is a human.
15. A method for treating a pathological condition or disease state
in cells, tissues or organs in an animal, the method comprising
administering to the animal a pharmaceutical composition according
to claim 7 in an acceptable carrier or formulation and in an amount
sufficient to alleviate the pathological condition or disease state
in the animal.
16. A method for treating a pathological condition or disease state
in skin of an animal, wherein the pathological condition or disease
state results from an abnormal proliferation of cells in the
animal, the method comprising the step of administering to an
animal a pharmaceutical composition according to claim 7 in an
acceptable carrier or formulation and in an amount sufficient to
alleviate the pathological condition or disease state in the
animal.
17. The method of claim 15 wherein the animal is a human.
18. A pharmaceutical composition according to claim 1 wherein the
spacer is a peptide of formula (amino acid).sub.n, wherein n is an
integer between 2 and 25, and the peptide comprises a polymer of
one or more amino acids.
19. The method of claim 12, wherein the disease state is a skin
disease.
20. A pharmaceutical composition according to claim 1 wherein the
spacer is a cleavable linker moiety that is specifically, cleaved
inside a mammalian skin cell.
21. The pharmaceutical composition of claim 20 wherein the
cleavable linker moiety is chemically cleaved inside a mammalian
skin cell.
22. The pharmaceutical composition of claim 20 wherein the
cleavable linker moiety is a substrate for a protein having an
enzymatic activity said protein being specifically expressed in a
mammalian skin cell.
23. A pharmaceutical composition according to claim 7 wherein the
chemical bond linker the polar lipid and the corticosteroid drug is
specifically cleaved inside a mammalian skin cell.
24. The pharmaceutical composition of claim 23 wherein the chemical
bond is chemically cleaved inside a mammalian skin cell.
25. The pharmaceutical composition of claim 23 wherein the chemical
bond is a substrate for a protein having an enzymatic activity said
protein being specifically expressed in a mammalian skin cell.
26. The method of claim 16 wherein the animal is a human.
27. The method of claim 13, wherein the disease state is a skin
disease.
28. The method of claim 15, wherein the disease state is a skin
disease.
29. The method of claim 16, wherein the disease state is a skin
disease.
30. The method of claim 13, wherein the animal is a human.
Description
[0001] This application is a continuation of U.S. Ser. No.
08/142,771, filed Oct. 26, 1993, now U.S. Pat. No. 5,543,389,
issued Aug. 6, 1996, which is a continuation-in-part of U.S. Ser.
No. 07/07/911,209, filed Jul. 9, 1992, now U.S. Pat. No. 5,256,641,
issued Oct. 26, 1993, which is a continuation-in-part of U.S.
patent application Ser. No. 07/607,982, filed Nov. 1, 1990, now
U.S. Pat. No. 5,149,794, issued Sep. 22, 1992, each of which are
herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] A major goal in the pharmacological arts has been the
development of methods and compositions to facilitate the specific
delivery of therapeutic and other agents to the appropriate cells
and tissues that would benefit from such treatment, and the
avoidance of the general physiological effects of the inappropriate
delivery of such agents to other cells or tissues of the body. One
common example of the need for such specificity is in the field of
antiproliferative agent therapy for the treatment of skin diseases
and disorders, in which the amount of a variety of
antiproliferative agents to be safely administered topically or
locally to a patient is limited by their systemic cytotoxic
effects.
[0004] In addition, it is recognized in the medical arts that
certain subcellular organelles are the sites of pharmacological
action of certain drugs or are involved in the biological response
to certain stimuli. Specific delivery of diagnostic or therapeutic
compounds to such intracellular organelles is thus desirable to
increase the specificity and effectiveness of such clinical
diagnostic or therapeutic techniques. The invention also provides
polar lipid drug conjugates that target dermal, intradermal and
infradermal structures in skin for delivery of therapeutic agents
for the treatment of skin diseases and disorders.
[0005] A. Drug Targeting
[0006] It is desirable to increase the efficiency and specificity
of administration of a therapeutic agent to the cells of the
relevant tissues in a variety of pathological states. This is
particularly important as relates to antiproliferative agents. Such
agents typically have pleiotropic antibiotic and cytotoxic effects
that damage or destroy uninvolved cells and tissues as well as
cells and tissues comprising the pathological site. Thus, an
efficient delivery system which would enable the delivery of such
drugs specifically to the diseased or affected tissues cells would
increase the efficacy of treatment and reduce the associated "side
effects" of such drug treatments, and also serve to reduce
morbidity and mortality associated with clinical administration of
such drugs.
[0007] Numerous methods for enhancing the biological activity and
the specificity of drug action have been proposed or attempted. To
date, however, efficient or specific drug delivery remains to be
predictably achieved.
[0008] An additional challenge in designing an appropriate drug
delivery scheme is to include within the drug conjugate a
functionality which could either accelerate or reduce the rate at
which the drug is released upon arrival at the desired site. Such a
functionality would be especially valuable if it allowed
differential rates of drug release.
[0009] Medicinal salves and ointments for topical treatment
purposes are known in the prior art for the treatment of a variety
of pathological conditions. A multitude of pathological and other
conditions have been treated by topical application of many classes
of compounds in a variety of carriers, such as salves and
ointments. However, carriers used in these conventional treatments
are in no way specific for deposition of drugs, and suffer from
non-specific deposition of the antiproliferative drug into both
healthy and affected portions of the skin. Appropriate
concentrations of topically-applied antiproliferative drugs, for
example, are currently limited by the escape of the active agent(s)
into the systemic circulation, with deleterious effects on other
tissues and organs. An example of such a situation is the use of
the drug methotrexate to treat psoriasis, where the amount of
methotrexate that is capable of being topically applied is limited
by hepato- and nephrotoxicity caused by systemic escape of the
compound from the skin.
[0010] There remains a need in the art for an effective means for
delivering biologically-active compounds, specifically drugs
including antiproliferative drugs, to skin by topical
administration of salves, ointments, and the like. Advantageous
embodiments of such delivery means are formulated to efficiently
deliver the biologically-active compound to the appropriate layer
of the skin, while minimizing transit of the compound into the
systemic circulation.
BRIEF SUMMARY OF THE INVENTION
[0011] The present invention is directed to an improved method for
delivering biologically-active compounds, particularly drugs
including preferably antiproliferative, antibiotic, antimycotic,
antiviral and antineoplastic drugs, to cells comprising skin in
animals in vivo and in vitro. This delivery system achieves
specific delivery of such biologically-active compounds through
conjugating the compounds with a polar lipid carrier. This
invention has the specific advantage of facilitating the entry of
such compounds into cells via a polar lipid carrier, achieving
effective intracellular concentration of such compounds more
efficiently and with more specificity than conventional delivery
systems. The invention particularly provides pharmaceutical
composition comprising the drug/polar lipid conjugates of the
invention formulated with a medicinal ointment or salve for
treatment of a variety of skin disorders.
[0012] The invention provides compositions of matter comprising a
biologically-active compound covalently linked to a polar lipid
carrier molecule. Preferred embodiments also comprise a spacer
molecule having two linker functional groups, wherein the spacer
has a first end and a second end and wherein the lipid is attached
to the first end of the spacer through a first linker functional
group and the biologically-active compound is attached to the
second end of the spacer through a second linker functional group.
In preferred embodiments, the biologically-active compound is a
drug, most preferably an antiproliferative drug or agent, an
antibiotic drug, an antiviral drug, an antineoplastic drug or a
corticosteroid. Preferred polar lipids include but are not limited
to acyl- and acylated carmitine, sphingosine, ceramide,
phosphatidyl choline, phosphatidyl glycerol, phosphatidyl
ethanolamine, phosphatidyl inositol, phosphatidyl serine,
cardiolipin and phosphatidic, acid. Preferred biologically-active
compounds include antineoplastic and antiproliferative agents such
as methotrexate, corticosteroids, antimycotics, antibiotics and
antiviral compounds. Pharmaceutical compositions comprising the
drug/polar lipid conjugates of the invention formulated with a
medicinal ointment or salve are also provided.
[0013] The invention also provides compositions of matter
comprising a biologically-active compound covalently linked to a
lipid, most preferably a polar lipid, carrier molecule via a spacer
molecule wherein the spacer allows the biologically-active compound
to act without being released at an intracellular site. In these
embodiments of the invention, the first linker functional group
attached to the first end of the spacer is characterized as
"strong" and the second linker functional group attached to the
second end of the spacer is characterized as "weak", with reference
to the propensity of the covalent bonds between each end of the
spacer molecule to be broken.
[0014] In other embodiments of the compositions of matter of the
invention, the spacer allows the facilitated hydrolytic release of
the biologically-active compound at an intracellular site. Other
embodiments of the spacer facilitate the enzymatic release of the
biologically-active compound at an intracellular site. In
particularly preferred embodiments, the spacer functional group is
hydrolyzed by an enzymatic activity found in skin, preferably an
esterase and most preferably an esterase having a differential
expression and activity profile in different skin layers. In
additional preferred embodiments, specific release of
biologically-active compounds is achieved by enzymatic or chemical
release of the biologically-active compound by intracellular
cleavage of a cleavable linker moiety in cells infected by a
pathogenic organism or otherwise expressing a disease state (for
example, hyperplasia associated with a benign or malignant skin
condition) via an enzymatic activity specific for such a pathogenic
organism or disease state, or by extracellular cleavage of a
cleavable linker moiety via an enzymatic activity specific for a
pathogenic organism or disease state.
[0015] The invention also provides polar lipid drug conjugates that
target dermal, intradermal and infradermal structures in skin for
delivery of therapeutic agents for the treatment of skin diseases
and disorders. Specifically, the invention provides such conjugates
comprising a spacer that allows facilitated hydrolytic or enzymatic
release of the biologically-active compound at a dermal,
intradermal or infradermal site in skin.
[0016] In another embodiment of this aspect of the invention, the
spacer molecule is a peptide of formula (amino acid).sub.n, wherein
n is an integer between 2 and 25, preferably wherein the peptide
comprises a polymer of one or more amino acids.
[0017] In other embodiments of the compositions of matter of the
invention, the biologically-active compound of the invention has a
first functional linker group, and a lipid, most preferably a polar
lipid, carrier has a second functional linker group, and the
compound is covalently linked directly to the lipid carrier by a
chemical bond between the first and second functional linker
groups. In preferred embodiments, each of the first and second
functional linker groups is a hydroxyl group, a primary or
secondary amino group, a phosphate group or substituted derivatives
thereof or a carboxylic acid group.
[0018] In another aspect of the invention is provided compositions
of matter comprising a drug, most preferably an antiproliferative
drug, an antineoplastic drug, an antibiotic, an antimycotic, or an
antiviral drug, covalently linked to a polar lipid carrier
molecule. Preferred embodiments also comprise a spacer molecule
having two linker functional groups, wherein the spacer has a first
end and a second end and wherein the lipid is attached to the first
end of the spacer through a first linker functional group and the
drug is attached to the second end of the spacer through a second
linker functional group. Preferred embodiments of the invention are
provided wherein the drug is an antiproliferative agent, such as
methotrexate, an antiviral agent such as an antiherpetic agent, an
antibiotic agent such as rifampicin or streptomycin, or an
antimycotic such as econazole. Preferred polar lipids include but
are not limited to acyl- and acylated carnitine, sphingosine,
ceramide, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl
ethanolamine, phosphatidyl inositol, phosphatidyl serine,
cardiolipin and phosphatidic acid. Pharmaceutical compositions
comprising the drug/polar lipid conjugates of the invention
formulated with a medicinal ointment or salve are also
provided.
[0019] The invention also provides compositions of matter
comprising an antiproliferative agent, an antineoplastic drug, an
antibiotic, an antimycotic, or an antiviral drug, covalently linked
to a polar lipid carrier molecule via a spacer molecule, wherein
the spacer allows the drug to act without being released at an
intracellular site. In these embodiments of the invention, the
first linker functional group attached to the first end of the
spacer is characterized as "strong" and the second linker
functional group attached to the second end of the spacer is
characterized as "weak", with reference to the propensity of the
covalent bonds between each end of the spacer molecule to be
broken.
[0020] In other embodiments of the compositions of matter of the
invention, the spacer allows the facilitated hydrolytic release of
an antiproliferative drug, an antineoplastic drug, an antibiotic,
an antimycotic, or an antiviral drug, at an intracellular site.
Other embodiments of the spacer facilitate the enzymatic release of
the antiproliferative, antineoplastic, antibiotic, antimycotic or
antiviral drugs of the invention at an intracellular site. In
particularly preferred embodiments, the spacer functional group is
hydrolyzed by an enzymatic activity found in skin, preferably an
esterase and most preferably an esterase having a differential
expression and activity profile in different skin layers. In
additional preferred embodiments, specific release of the
antiproliferative, antineoplastic, antibiotic, antimycotic or
antiviral drugs of the invention is achieved by enzymatic or
chemical release of these drugs by intracellular cleavage of a
cleavable linker moiety in cells infected by a pathogenic organism
or otherwise expressing a disease state (for example, hyperplasia
associated with a benign or malignant skin condition) via an
enzymatic activity specific for such a pathogenic organism or
disease state, or by extracellular cleavage of a cleavable linker
moiety via an enzymatic activity specific for a pathogenic organism
or disease state.
[0021] The invention also provides polar lipid conjugates of of the
antiproliferative, antineoplastic, antibiotic, antimycotic or an
antiviral drugs of the invention that target dermal, intradermal
and infradermal structures in skin for delivery of therapeutic
agents for the treatment of skin diseases and disorders.
Specifically, the invention provides such conjugates comprising a
spacer that allows facilitated hydrolytic or enzymatic release of
the of such antiproliferative, antineoplastic, antibiotic,
antimycotic or an antiviral drugs at a dermal, intradermal or
infradermal site in skin.
[0022] In another embodiment of this aspect of the invention, the
spacer molecule is a peptide of formula (amino acid).sub.n, wherein
n is an integer between 2 and 25, preferably wherein the peptide
comprises a polymer of one or more amino acids.
[0023] In still further embodiments of the compositions of matter
of the invention are provided an antiproliferative drug, an
antineoplastic drug, an antibiotic, an antimycotic, or an antiviral
drug, having a first functional linker group, and a polar lipid
carrier having a second functional linker group, wherein the drug
is covalently linked directly to the polar lipid carrier by a
chemical bond between the first and second functional linker
groups. In preferred embodiments, each of the first and second
functional linker groups is a hydroxyl group, a primary or
secondary amino group, a phosphate group or substituted derivatives
thereof or a carboxylic acid group. Preferred embodiments of the
invention are provided wherein the drug is an antiproliferative
agent, such as methotrexate, an antiviral agent such as an
antiherpetic agent, an antibiotic agent such as rifampicin or
streptomycin, or an antimycotic such as econazole. Preferred polar
lipids include but are not limited to acyl- and acylated carnitine,
sphingosine, ceramide, phosphatidyl choline, phosphatidyl glycerol,
phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl
serine, cardiolipin and phosphatidic acid. Pharmaceutical
compositions comprising the drug/polar lipid conjugates of the
invention formulated with a medicinal ointment or salve are also
provided.
[0024] The invention also provides compositions of matter
comprising an antiproliferative drug, an antineoplastic drug, an
antibiotic, an antimycotic, or an antiviral drug covalently linked
to a polar lipid carrier molecule via a spacer molecule wherein the
spacer allows the drug to act without being released at an
intracellular site. In these embodiments of the invention, the
first linker functional group attached to the first end of the
spacer is characterized as "strong" and the second linker
functional group attached to the second end of the spacer is
characterized as "weak", with reference to the propensity of the
covalent bonds between each end of the spacer molecule to be
broken.
[0025] In other embodiments of the compositions of matter of the
invention, the spacer allows the facilitated hydrolytic release of
the antiproliferative, antineoplastic, antibiotic, antimycotic or
an antiviral drug at an intracellular site. Other embodiments of
the spacer facilitate the enzymatic release of a drug as provided
by the invention at an intracellular site. In particularly
preferred embodiments, the spacer functional group is hydrolyzed by
an enzymatic activity found in skin, preferably an esterase and
most preferably an esterase having a differential expression and
activity profile in different skin layers. In additional preferred
embodiments, specific release of the antiproliferative,
antineoplastic, antibiotic, antimycotic or antiviral drugs of the
invention is achieved by enzymatic or chemical release of these
drugs by intracellular cleavage of a cleavable linker moiety in
cells infected by a pathogenic organism or otherwise expressing a
disease state (for example, hyperplasia associated with a benign or
malignant skin condition) via an enzymatic activity specific for
such a pathogenic organism or disease state, or by extracellular
cleavage of a cleavable linker moiety via an enzymatic activity
specific for a pathogenic organism or disease state.
[0026] In other embodiments of the compositions of matter of the
invention, the spacer allows the facilitated enzymatic or
hydrolytic release of the antiproliferative, antineoplastic,
antibiotic, antimycotic or an antiviral drug at dermal, intradermal
and infradermal structures in skin for delivery of therapeutic
agents for the treatment of skin diseases and disorders.
[0027] In another embodiment of this aspect of the invention, the
spacer molecule is a peptide of formula (amino acid).sub.n, wherein
n is an integer between 2 and 25, preferably wherein the peptide
comprises a polymer of one or more amino acids.
[0028] A preferred embodiments of this aspect of the invention
include compositions of matter that are N-methotrexate ceramide,
methotrexate-glycylglycylglycylglycyl ceramide ester,
methotrexate-(tri-.beta.-hydroxypropionylester)-O.sup.x-ceramide
ester, methotrexate-glycylglycylglycylglycyl ceramide ester,
methotrexate-aminohexanoyl) sphingosine amide,
methotrexate-valinylvaliny- l sphingosine amide and
methotrexate-O.sup.x-ceramide ester.
[0029] Particular preferred embodiments of the polar lipid/drug
conjugates of this invention are provided as salves and other
topically or locally applied compositions comprising the drug/polar
lipid conjugates of the invention and any of a variety of
emollients or other commonly encountered components of cremes,
salves, poultices, lotions, gels or other substances well-known in
the art for applying compounds to skin and other tissues.
Appropriate formulations of such compositions comprising the drug/
polar lipid conjugates of the invention will be apparent and within
the skill of one of ordinary skill in this art to advantageously
prepare in view of the instant disclosure.
[0030] In preferred embodiments, the drug/lipid conjugates of the
invention comprise a functionality recognized by an enzymatic
activity, most preferably an esterase activity, that has a
differential pattern of expression or activity in different skin
layers. In additional preferred embodiments, specific release of
the antiproliferative, antineoplastic, antibiotic, antimycotic or
antiviral drugs of the invention is achieved by enzymatic or
chemical release of these drugs by intracellular cleavage of a
cleavable linker moiety in cells infected by a pathogenic organism
or otherwise expressing a disease state (for example, hyperplasia
associated with a benign or malignant skin condition) via an
enzymatic activity specific for such a pathogenic organism or
disease state, or by extracellular cleavage of a cleavable linker
moiety via an enzymatic activity specific for a pathogenic organism
or disease state.
[0031] As disclosed herein, the invention comprehends a polar
lipid-drug conjugate wherein the polar lipid will selectively
associate with certain biological membranes, and thereby facilitate
entry of the drug into cells and cellular organelles. In
embodiments comprising a spacer moiety, the spacer component of the
conjugates of the invention will preferably act to release the drug
from the lipid, target the conjugate to the cell, or perform other
functions to maximize the effectiveness of the drug.
[0032] This type of conjugate has numerous advantages. First, the
drug-lipid conjugates of the invention promote the intracellular
entry of a variety of potentially useful drugs at pharmokinetic
rates not currently attainable. Second, the range of targeted cell
types is not limited per se by particular, limited biological
properties of the cell (such as the number and type of specific
receptor molecules expressed on the cell surface). Third, in
contrast to traditional attempts to simply target drugs to specific
cells, this method may target drugs to specific intracellular
organelles and other intracellular compartments. Fourth, the
compositions of matter of the invention incorporate a variable
spacer region that may allow pharmacologically-relevant rates of
drug release from polar lipid carrier molecules to be engineered
into the compositions of the invention, thereby increasing their
clinical efficacy and usefulness. Thus, time-dependent drug release
and specific drug release in cells expressing the appropriate
degradative enzymes are a unique possibility using the drug-lipid
conjugates of the invention. Fifth, the conjugates of the invention
can be combined with other drug delivery approaches to further
increase specificity and to take advantage of useful advances in
the art. Sixth, the conjugates of the invention can be topically
applied to skin, and the layer of skin penetrated determined by the
formulation used. Seventh, in such formulations, the amount and
activity of the topically-applied drug can be modulated by release
via cleavage, preferably hydrolytic cleavage, of the spacer moiety,
most preferably by an enzymatic activity in skin that has a
differential pattern of expression or activity in different skin
layers.
[0033] Specific preferred embodiments of the present invention will
become evident from the following more detailed description of
certain preferred embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 depicts the synthetic scheme put forth in Example
1.
[0035] FIG. 2 depicts the synthetic scheme put forth in Example
2.
[0036] FIG. 3 depicts the synthetic scheme put forth in Example
3.
[0037] FIG. 4 depicts the synthetic scheme put forth in Example
4.
[0038] FIG. 5 depicts the synthetic scheme put forth in Example
5.
[0039] FIG. 6 depicts the synthetic scheme put forth in Example
6.
[0040] FIG. 7 depicts the synthetic scheme put forth in Example
7.
[0041] FIG. 8 depicts the synthetic scheme put forth in Example
8.
[0042] FIGS. 9A through 9D depict prodrugs tested as in Example
9.
[0043] FIG. 10 depicts the synthetic scheme put forth in Example
10.
[0044] FIGS. 11 through 19 illustrate targeting of polar
lipid-conjugated biologically active compounds to skin.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0045] The present invention provides compositions of matter and
methods for facilitating the entry into cells of
biologically-active compounds. For the purposes of this invention,
the term "biologically-active compound" is intended to encompass
all naturally-occurring or synthetic compounds capable of eliciting
a biological response or having an effect, either beneficial or
cytotoxic, on biological systems, particularly cells and cellular
organelles. These compounds are intended to include but are not
limited to all varieties of drugs, particularly antiproliferative
drugs and agents, antibacterial, fungicidal, anti-protozoal and
antiviral drugs, antineoplastic drugs, and cytotoxic and cytostatic
compounds.
[0046] Pharmaceutical compositions comprising the drug/polar lipid
conjugates of the invention formulated with a medicinal ointment or
salve are also provided. As used herein the terms "medicinal
ointment or salves" are considered equivalent. The term is intended
to encompass any of a variety of salves and other topically or
locally applied formulations known in the art, and specifically to
encompass any of a variety of emollients or other commonly
encountered components of cremes, salves, poultices, lotions, gels
or other substances well-known in the art for applying compounds to
skin and other tissues. Appropriate formulations of such
compositions comprising the drug/ polar lipid conjugates of the
invention will be apparent and within the skill of one of ordinary
skill in this art to advantageously prepare in view of the instant
disclosure.
[0047] The compositions of matter provided by the invention
comprise the biologically-active compounds of the invention
covalently linked to a polar lipid carrier. A polar lipid carrier,
as defined herein is intended to mean any polar lipid having an
affinity for, or capable of crossing, a biological membrane,
including but not limited to sphingosine, ceramide, phosphatidyl
choline, phosphatidyl glycerol, phosphatidyl ethanolamine,
phosphatidyl inositol, phosphatidyl serine, cardiolipin,
phosphatidic acid, sphingomyelin and other sphingolipids, as these
terms are understood in the art (see, Lehninger, Biochemistry, 2d
ed., Chapters 11 & 24, Worth Publishers: New York, 1975).
Additionally, certain other lipids, such as acylated carnitine,
comprise the conjugates of the invention (see Small, 1986, "From
alkanes to phospholipids," Handbook of Lipid Research: Physical
Chemistry of Lipids, Volume 4, Chapters 4 and 12, Plenum Press: New
York).
[0048] The compositions of matter of the invention may be further
comprised of a spacer moiety comprising a first end and a second
end, each end of the spacer having a functional linking group. For
the purposes of this invention, the term "spacer" or "spacer
moiety" is intended to encompass any chemical entity that links the
biologically-active compound and the polar lipid. Such spacer
moieties may be designed to facilitate the attachment of the
conjugates of the invention to a target cell, or to facilitate,
influence, modulate or regulate the release of the
biologically-active compound at the desired target site. Such
spacers may also facilitate enzymatic release at certain
intracellular sites. Spacer groups, as described herein, include,
but are not limited to aminohexanoic acid, polyglycine, polyamides,
polyethylenes, and short functionalized polymers having a carbon
backbone which is from one to about twelve carbon molecules in
length. Particularly preferred embodiments of such spacer moieties
comprise peptides of formula (amino acid).sub.n, wherein n is an
integer between 2 and 25 and the peptide is a polymer of one or
more amino acids.
[0049] The term "linker functional group" is defined herein as any
functional group for covalently binding the polar lipid carrier or
biologically-active agent to the spacer group. These groups can be
designated either "weak" or "strong" based on the stability of the
covalent bond which the linker functional group will form between
the spacer and either the polar lipid carrier or the
biologically-active compound. The weak functionalities include, but
are not limited to phosphoramide, phosphoester, carbonate, amide,
carboxyl-phosphoryl anhydride, ester and thioester. The strong
functionalities include, but are not limited to ether, thioether,
amine, amide and ester. The use of a strong linker functional group
between the spacer group and the biologically-active compound will
tend to decrease the rate at which the compound will be released at
the target site, whereas the use of a weak linker functional group
between the spacer group and the compound may act to facilitate
release of the compound at the target site. Enzymatic release is,
of course, also possible, but such enzyme-mediated modes of release
will not necessarily be correlated with bond strength in such
embodiments of the invention. Spacer moieties comprising enzyme
active site recognition groups, such as spacer groups comprising
peptides having proteolytic cleavage sites therein, are envisioned
as being within the scope of the present invention.
[0050] The drug/polar lipid conjugates of the invention are
preferably provided comprised of spacer moieties that impart
differential release properties on the conjugates related to
differential expression or activity of enzymatic activities in
different layers of skin. Biologically active agents such as
antiproliferative, antiviral or antineoplastic drugs linked to
polar lipids can be delivered to different layers and structures in
the skin based on the distinct differences in lipid handling within
the skin. Such different structures include dermal, intradermal and
infradermal regions or sections of the skin. As shown herein in
Example 11, there are important differences in the delivery of a
fluorescent marker in mouse skin, based on the polar lipid to which
it was linked via an amide bond. In addition to distribution by
lipid handling, differences in hydrolytic enzymes activity, such as
esterases and peptidases, within the skin may be employed to convey
specificity to drug distribution.
[0051] For example, hydrolysis of esters within the skin by native
esterases has been well documented. Moreover, the unique pattern of
metabolites found herein suggested that skin expresses a collection
of esterases different than other high esterases containing organs,
such as liver (Henrikus and Kampffmeyer, 1992, Xenobiotica 22:
1357-1366). Significant variation in substrate specificity among
esterases has been shown previously. For example, Heymann et al.
(1993, Chem. Biol. Interactions 87: 217-226) showed the existence
of at least three families of .beta.-type esterase, identified by
pI and all of which had activity with simple aromatic and aliphatic
esters. The rate of ester hydrolysis within skin has been found to
be significant. Boehnlein et al. (1994, Pharmaceutical Research 11:
1155-1159) showed that about half of dermally-applied retinyl
palmitate was hydrolyzed within a few minutes; similar results were
reported witch methyl salicylte. .beta.-methasone-17-valerate, a
steroid fatty acid ester, has also been reported to be hydrolyzed
at a pharmacologically-important rate by skin esterases (Kubota et
al., 1994, Dermatology 188: 13-17). In addition, smaller molecules
are delivered more efficiently to skin linkage with lipids. For
example, Buyuktimkin el al. (1993, Pharmaceutical Research
10:1632-1637) found that uptake of indomethacin and clonidine could
be enhanced by linkage to a polar lipid.
[0052] Distribution of specific esterases within the skin has not
been extensively studied. It is known that some cell types do
possesses different constituent esterases. The Langerhans cells
contain an .alpha.-naphthylacetate hydrolyzine esterase, used in
antigen recognition and processing, that is not found in other cell
types (Lipozencic et al., 1994, Eur. J. Histochem. 38: 303-310).
Ketatinocytes are also known to have high levels of chloroacetate
esterase (Katz et al., 1995, Brit. J. Dermatology 133:
842-846).
[0053] Esterase activity is also known to vary with pathological
conditions. Nonspecific esterase activity increases in cells around
the edge of many types of wounds (Dachun et al., 1992, Forensic
Science International 53: 203-213). Although not produced by skin
cells per se, esterases are known to increase in the skin due to
influx of other cell types during illness. On example is
chloroacetate esterase activity is increased in any condition
causing chronic uticaria due to mast cell infiltration (Barlow et
al., 1995, Clinical & Experimental Allergy 25: 317-322). In
another example, monocytes and macrophages found in the skin in
leprosy increase esterase activity (SivaSai et al., 1993, Int. J.
Leprosy & Other Mycobacterial Diseases 61: 259-269).
[0054] Other enzymatic activities are associated with skin.
Peptidases are found in high levels within skin tissues and the
non-dermal cell types infiltrating the skin. Expression of both
endo- and exo-peptidases and proteases varies with cell type and
pathological condition. Skin fibroblasts contain high levels of
peptidases, not seen in other more highly differentiated layers of
skin (Bou-Gharios et al., 1995, Annals of Rheumatic Disease 54:
111-116). High levels of dipeptidyl dipeptidase IV are seen in
precancerous dermatoses and basal cell carcinoma (Moehrle et al.,
1995, J. Cutaneous Pathology 22: 241-247). Increased peptidase
activity is associated with stress responses: exemplary are
apoptotic skin cells produced after UV irradiation (so-called
sunburn cells), which have high amino and endopeptidase activity:
Brown et al. (1994, J. Cellular Biochemistry 54: 320-331) have
suggested that these apoptotic keratinocytes may be essential
elements in the repair process for epithelial cells.
[0055] The current state of knowledge of these hydrolytic enzymes
in skin is not extensive relative to other organs. However,
sufficient data is available to see that there are differences in
substrate specificity and enzyme activity that can be exploited to
further refine the specificity of the polar lipid delivery vehicle.
Formulation of cleavable linkages directed at these enzymes,
combined with the larger distributive properties of the lipid
carries could provide important new ways to deliver pharmaceutical
agents to highly defined locations in the skin, dependent on either
normal skin or pathological conditions. Conjugates that
advantageously utilize the differential expression and/or activity
levels of esterases, proteases, and other enzymatic functionalities
are provided by this invention.
[0056] As provided by this invention, the specificity of the
cleavage of the linker moiety as provided by this invention is the
result of the combination of particular linker moieties selected to
be specifically cleaved inside an infected skin cell. In one
aspect, such specific cleavage is due to an chemical linkage which
is labile within the infected cell due to conditions caused by or
that result from infection of the cell with a particular pathogenic
organism. In another aspect, such specific cleavage is due to an
enzymatic activity which is produced either by the pathogen itself
or by the cell as the result of infection with said pathogen,
wherein the linkage is enzymatically cleaved by the enzymatic
activity. Similarly, specific cleavage is obtained in cells
expressing a disease or pathologic state, whereby a chemical
linkage labile to the conditions inside the diseased cell or
enzymatic cleavage due to an enzymatic activity expressed in a
diseased cell using drug/polar lipid conjugates comprising the
appropriate linking moiety comprising the labile chemical linkage
or enzyme recognition site. Differences in skin and other tissues
present extracellularly due to the expression of a disease state or
the presence of a pathogenic organism are also comprehended to be
within the scope of the invention.
[0057] Examples of such combinations resulting in specific release
of an antimicrobial drug embodiment of the invention within
infected cells include but are not limited to a urea-based linker
for use against a pathogen which produces urease (e.g.,
Mycobacteria spp. and B. pertussis); a peptide linker comprised of
(AlaAlaAlaAla).sub.n, wherein n can be an integer from 1-5, for use
against a pathogen that produces the protease oligopeptidase A
(e.g., Salmonella spp.); a peptide comprised of from 3 to about 20
amino acids comprising the sequence --Pro-Xaa-Pro--, where Xaa is
any amino acid, for use against a pathogen that produced proline
peptidase (e.g., Salmonella spp.); peptides comprising the
dipeptide MetMet or LeuAla, or peptides comprising the amino acid
sequence GSHLVEAL, HLVRALYL, VEALYLVC, or EALYLVCG, for use against
human immunodeficiency virus 1 producing a specific protease termed
HIV-1 protease; a peptide comprising the amino acid sequence:
-Ala-Xaa-Cy.sub.Acm-Tyr-Cys-Arg-Ile-Pro-Ala-Cys.sub.Acm-Ile-Ala-Gly-Asp-A-
rg-Arg-Tyr-Gly-Thr-Cys.sub.Acm-Ile-Tyr-Gln-Gly-Arg-Leu-Trp-Ala-Phe-Cys.sub-
.Acm-Cys.sub.Acm-, wherein the pathogen expresses an enzymatic
activity that specifically disables the endogenous antimicrobial
peptide defensin (e.g., Mycobacterium spp. and L. pneumophila),
(-Cys.sub.Acm-) represent cysteine residues having the sidechain
sulfur atom protected by covalent linkage to an acetamidomethyl
group (it will be recognized that embodiments of such peptides
having alternative sulfur protecting groups are also within the
scope of the disclosure herein) and Xaa is either absent or Asp
(said peptides are also useful against a pathogen such as
Legionella spp. producing a 39 kDa metalloprotease); hippurate
esters that are hydrolyzed by pathogen-specific (e.g., L.
pneumophila and Listeria spp.) hydrolase; nicotinic acid amides
cleaved by nicotinamidases, pyrazinamides cleaved by
pyrazinamidase; allolactose linkages cleaved by
.beta.-galactosidase; and allantoate linkages cleaved by
allantoicase (e.g., Mycobacterium spp.).
[0058] Thus, the present invention provides methods and
compositions of matter for facilitating the entry of
antiproliferative, antibiotic, antimycotic, antiviral and
antineoplastic agents, drugs and compounds into dermal and
epidermal cells, across mucosal membranes where appropriate, and
distributed within skin tissue for efficient delivery of such
compounds locally and topically for the treatment of animal,
preferably human, diseases and pathological conditions. The
invention provides salves, ointments, poultices and other
topically-applied embodiments of the drug/lipid conjugates of the
invention for the treatment of a variety of skin diseases and
disorders.
[0059] Among the most common dermatological complaints is
dermatitis, further differentiated into contact, seborrheic,
nummular, exfoliative stasis, and neurodermatitis. Vesicular
dermatitis, commonly referred to as eczema, is separated from other
dermatitis to reflect the chronic nature of the condition. All
forms of dermatitis are characterized by superficial inflammation,
vesiculation and localized edema. Accompanying these psoriasiform
conditions is thickening of the epidermis showing both
hyperkeratosis and parakeratosis. Topical corticosteroids are
widely prescribed for these conditions, including the Potency class
I drugs betamethasone dipropionate, clobetasol propionate,
diflorasone diacetate and flucinolone. Of particular interest in
the treatment of psoriasis is methotrexate, the use of which is
severely limited by the nephro- and hepatotoxicity. In a preferred
embodiment, the invention provides a conjugate, methotrexate
ceramide ester (ME6C), that does not concentrate in either liver or
kidney to the same extent as the free drug, and is therefore useful
in for treatment of chronic psoriasis and related conditions.
[0060] Another skin condition conventionally treated by topical
application of an antineoplastic agent is the treatment of
precancerous lesions classified as actinic keratoses. These lesions
respond to 5-fluorouracil (5-FU) topically applied in a propylene
glycol carrier. However, topical application of 5-FU is limited by
the toxicity of this compound in the systemic circulation. Topical
application can be improved by covalent, hydrolyzable linkage of
5-FU to polar lipids that is cleaved by an esterase or peptidase
activity in skin. Such compounds have the advantage of better
penetration, longer retention and controlled release of active
drug.
[0061] Superficial fungal infections by the genera Microsporum,
Trichophyton, and Epidermophyton, the so-called derrnatophyte
infections, are extremely common. Ringworm and the Tenia
infections, including candidiasis, colonize the area between the
dead skin and living layers eliciting vesicular and bullous
diseases as well as inflamed lesions of the scalp due to strong
immunological responses to the fungi. All respond, to some extent,
to topical antifungals. Topical application reduces reliance on
systemic antifungals, avoiding the hepatic toxicity produced by
administration of the currently used antifungals such as
ketoconazole, grisefulvin, ciclopixox, naftitine and other
imidizole antimycotics. By conjugating these compounds with polar
lipid carriers, these drugs are delivered to specific areas of the
skin in greater quantity and maintained for longer periods of time
as a consequence of the lipid formulation. Release of active drug
from these reservoirs is effectuated by the cleavage of
hydrolyzable bonds between the drug and the lipid carrier. By
relying on the specific partitioning effect of the polar lipids, a
greater therapeutic index is thereby achieved, thus reducing the
need for system antimycotics and reducing the risk of hepatic
toxicity. In addition, the characteristic accumulation of fluids in
eczema, or other bullus conditions, may be a limiting factor in the
delivery of water soluble antimycotics. Use of the lipid prodrugs
to effectively deliver antifungals to deeper skin structures would
improve treatment of eczema and yeast-exacerbated dermatitis.
[0062] In skin infestations and dermatitis, deep colonization or
involvement of sub-epidermal structures is common. Delivery of
drugs to deep skin structures is important for relief of these
conditions. The conjugation of compounds to polar lipids, as shown
in Example 10 below, indicates that transport and release of
medicaments through the skin is possible.
[0063] The invention provides polar lipid/drug conjugates
comprising corticosteroids, including but not limited to cortisone,
cortisol, hydrocortisone, prednisone, fluorinated corticosteroids
(such as fluocinalone and triamcinolone), dexamethasone,
alcloethasone, fluoroandrenolide and mometasone.
[0064] The invention provides polar lipid/drug conjugates
comprising antimycotic compounds including but not limited to
clotrimazole, ciclopirox, nystatin, econazole and myconixole. The
invention provides polar lipid/drug conjugates comprising
antibiotics including but not limited to penicillin and drugs of
the penicillin family of antimicrobial drugs, including but not
limited to penicillin-G, penicillin-V, phenethicillin, ampicillin,
amoxacillin, cyclacillin, bacampicillin, hetacillin, cloxacillin,
dicloxacillin, methicillin, nafcillin, oxacillin, azlocillin,
carbenicillin, meziocillin, piperacillin, ticaricillin, and
imipenim; cephalosporin and drugs of the cephalosporin family,
including but not limited to cefadroxil, cefazolin, caphalexn,
cephalothin, cephapirin, cephradine, cefaclor, cefamandole,
cefonicid, cefoxin, cefuroxime, ceforanide, cefotetan, cefmetazole,
cefoperazone, cefotaxime, ceftizoxime, ceftizone, moxalactam,
ceftazidime, and cefixime; aminoglycoside drugs and drugs of the
aminoglycoside family, including but not limited to streptomycin,
neomycin, kanamycin, gentamycin, tobramycin, amikacin, and
netilmicin; macrolide and drugs of the macrolide family,
exemplified by azithromycin, clarithromycin, roxithromycin,
erythromycin, lincomycin, and clindamycin; tetracyclin and drugs of
the tetracyclin family, for example, tetracyclin, oxytetracyclin,
democlocyclin, methacyclin, doxycyclin, and minocyclin; quinoline
and quinoline-like drugs, such as, for example, naladixic acid,
cinoxacin, norfloxacin, ciprofloxacin, ofloxicin, enoxacin, and
pefloxacin; antimicrobial peptides, including but not limited to
polymixin B, colistin, and bacatracin, as well as other
antimicrobial peptides such as defensins (Lehrer et al., 1991, Cell
64: 229-230), magainins (Zasloff, 1987, Proc. Natl. Acad. Sci. USA
84: 5449-5453), cecropins (Lee et al., 1989, Proc. Natl. Acad. Sci.
USA 86: 9159-9162 and Boman et al., 1990, Eur. J. Biochem. 201:
23-31), and others, provided as naturally-occurring or as the
result of engineering to make such peptides resistant to the action
of deactivating enzymes; other antibiotic drugs, including
chloramphenicol, vancomycin, rifampicin, metronidazole, ethambutol,
pyrazinamide, sulfonamides, isoniazid, and erythromycin.
[0065] The invention also provides polar lipid/drug conjugates of
antiviral agents, including but not limited to reverse
transcriptase inhibitors, protease inhibitors, antiherpetics such
as acyclovir and gangcyclovir, azidothymidine, cytidine
arabinoside, ribavirin, amantadine, iododeoxyuridine, poscarnet,
trifluoridine, methizazone, vidarabine and levanisole.
[0066] The invention provides polar lipid/drug conjugates of
antiproliferative and antineoplastic agents, including but not
limited to methotrexate, doxarubicin, daunarubicin, actinomycin D,
vinblastine, vincristine, colchicine and taxol.
[0067] The invention specifically provides methods for preparing
and administering such antiproliferative compounds for use in
treating pathological conditions in vivo.
[0068] Animals to be treated with polar lipid-antiproliferative
agent conjugates using the methods of the invention are intended to
include all vertebrate animals, preferably domesticated animals,
such as cattle, horses, goats, sheep, fowl, fish, household pets,
and others, as well as wild animals, and most preferably
humans.
[0069] The following Examples illustrate certain aspects of the
above-described method and advantageous results. The following
examples are shown by way of illustration and not by way of
limitation.
EXAMPLE 1
[0070] An antibiotic drug/polar lipid conjugate of the invention is
prepared by conjugating a specifically-cleavable peptide to a polar
lipid and an antibiotic drug as follows. An derivatized polar lipid
comprising unconjugated amino groups is reacted with a
proteolytically-inert peptide in which the terminal amine and any
of the constituent amino acid sidechain reactive amines are covered
by tert-butoxycarbonyl (t-Boc) protecting groups in the presence of
triphenyl phosphine as described by Kishimoto (1975, Chem. Phys.
Lipids 15: 33-36). The peptide/polar lipid conjugate is then
reacted in the presence of pyridine hydrofluoride as described by
Matsuura et al. (1976, J. Chem. Soc. Chem. Comm. xx: 451-459) to
remove the t-Boc protecting groups. The peptide/polar lipid is then
conjugated to the specifically-cleavable peptide, in which the
terminal amine and any of the constituent amino acid sidechain
reactive amines are covered by t-Boc protecting groups, as
described in the presence of triphenyl phosphine. After
deprotection of reactive amines with pyridine hydrofluoride as
described, an antibiotic drug having a reactive carboxylic acid
group is conjugated to a free amino group of the polar
lipid/peptide/specifically-cleavable peptide to yield the
antibiotic drug/polar lipid conjugate of the invention. This
reaction scheme is illustrated in FIG. 1.
EXAMPLE 2
[0071] An antiviral compound (HIV1 protease inhibitor; compound 8)
is conjugated to sphingosine as follows. Sphingosine is reacted
with 1,3 bis(trimethylsilyl)urea as described by Verbloom et al.
(1981, Synthesis 1032: 807-809) to give a trimethylsilyl derivative
of sphingosine. The sphingosine derivative is then conjugated with
a specifically-cleavable peptide in which the terminal amine and
any of the constituent amino acid sidechain reactive amines are
covered by tert-butoxycarbonyl (t-Boc) protecting groups in the
presence of diethylazo-dicarboxylate (DEAD) and triphenyl phosphine
as described by Kishimoto (1975, Chem. Phys. Lipids 15: 33-36). The
sphingosine/peptide conjugate is then reacted in the presence of
pyridine hydrofluoride as described by Matsuura et al. (1976, J.
Chem. Soc. Chem. Comm. xx: 451-459) to remove the t-Boc protecting
group, to yield the peptide covalently linked to sphingosine
through an amide bond. This reaction scheme is illustrated in FIG.
2. Sphingosine/peptide conjugates are then linked to the antiviral
compound as described in Example 1.
EXAMPLE 3
[0072] An antiviral compound (compound 8) is conjugated to ceramide
via a polyglycine spacer as follows and as illustrated in FIG. 3.
The amino terminus of polyglycine is protected by a t-Boc group.
Polyglycine is conjugated through its carboxy terminus to ceramide
forming an ester linkage, as described in Anderson et al., ibid.
The resulting compound is then conjugated through the amino
terminus of the polyglycine residue. The amino terminus of Compound
8 is also protected by a t-Boc protecting group. Conjugation with
polyglycyl-sphingosine takes place between the amino terminus of
the polyglycyl spacer moiety and the carboxy terminus of the HIV-1
protease inhibitor. This reaction is carried out in the presence of
DEAD and triphenyl phosphine as described in Examples 1 and 2.
Following this conjugation, the amino terminus of the HIV-1
protease inhibitor residue is deprotected according to the method
of Matsuura et al., ibid.
EXAMPLE 4
[0073] An antiviral compound is prepared wherein ceramide is first
conjugated to a first end of an oligomeric 3-hydroxy propanoic acid
spacer through an ester functional group, and wherein AZT is
conjugated to a second end of said polyester spacer through a
phosphodiester bond. First a polyester spacer is obtained, having a
carboxyl at a first end and a triphenylmethyl group esterified to a
second end. This spacer is conjugated to ceramide at its first end
through an ester functional linker group according to the method of
Anderson et al., ibid. This compound is then conjugated through the
second end of the spacer compound to AZT monophosphate by means of
a phosphodiester bond according to the method of Baer (1955, Can.
J. Biochem. Phys. 34: 288). In this antiviral compound, the bond
breakage between the spacer and the drug would be slow in the
absence of a phosphohydrolase. This reaction scheme is illustrated
in FIG. 4.
EXAMPLE 5
[0074] An antiviral compound wherein phosphatidic acid,
phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol,
phosphatidyl glycerol or phosphatidylethanolamine is linked through
a phosphoester linker functional group to the antiviral drug
azidothymidine (AZT). Phosphatidic acid, phosphatidyl choline,
phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol
or phosphatidyl ethanolamine is conjugated to AZT according to the
method of Salord et al. (1986, Biochim. Biophys. Acta 886: 64-75).
This reaction scheme is illustrated in FIG. 5.
EXAMPLE 6
[0075] An antiviral compound is prepared wherein aminohexanoyl
sphingosine is conjugated to AZT. Aminohexanoyl sphingosine is
conjugated with AZT according to the method of Kishimoto (1975,
Chem. Phys. Lipid 15: 33-36). This reaction scheme is illustrated
in FIG. 6 to yield aminohexanoyl sphingosine conjugated to AZT
through a phosphoramide bond.
EXAMPLE 7
[0076] An antiviral compound consisting of ceramide conjugated to
AZT-monophosphate is provided. Ceramide is reacted with
AZT-monophosphate in the presence of dicyclohexylcarbodiimide as
described in Smith and Khorana (1958, J. Amer. Chem. Soc. 80: 1141)
to yield ceramide conjugated through a phosphodiester bond to
AZT-monophosphate. This reaction scheme is illustrated in FIG.
7.
EXAMPLE 8
[0077] An antiviral compound is prepared wherein ceramide is
conjugated through an ester functional group to a first end of a
polyglycine spacer, and wherein AZT is conjugated through a
phosphoester functional group to a second end of the polyglycine
spacer. Ceramide is first conjugated through an ester functional
group to a first end of a polyglycine spacer (as described in
Example 2). The ceramide-polyglycine compound is then conjugated
through a phosphoester bond to a second end of the polyglycine
spacer to AZT monophosphate according to the method of Paul and
Anderson, ibid. This reaction scheme is illustrated in FIG. 8.
EXAMPLE 9
[0078] The effect of presenting a biologically active compound such
as a drug to mammalian cells as a prodrug covalently linked to a
polar lipid carrier moiety was determined as follows. The
antifolate drug methotrexate was conjugated with a variety of polar
lipid carriers via organic spacer moieties having specific reactive
functional groups. A representative sample of such compounds is
shown in FIGS. 9A through 9C, wherein MC represents Mtx linked to
sphingosine via an amide bond to a 6-aminohexanoic acid spacer,
ME.sub.6C represents Mtx linked to sphingosine via an ester linkage
to a 6-hydroxyhexanoic acid spacer, and MSC represents Mtx linked
to sphingosine via a salicylic acid ester linkage to a
6-aminohexanoic acid spacer. Also studied was a conjugate of
azidothymidine linked to sphingosine via an ester linkage to a
6-hydroxyhexanoic acid spacer (N-AZT-ceramide; FIG. 9D). The
compounds were tested for their growth inhibitory effects on murine
NIH 3T3 cells growing in cell culture. About one million such cells
per P100 tissue culture plate were grown in DMEM media supplemented
with 10% fetal calf serum (GIBCO, Grand island, N.Y.) in the
presence or absence of a growth-inhibitory equivalent of each
prodrug. Cell numbers were determined after 70 hours growth in the
presence or absence of the prodrug. In a second set of experiments
was included in the growth media an amount of a brain homogenate
containing an enzymatically-active esterase.
[0079] The results from these experiments are shown in Table I. As
can be seen from these data, the MC prodrug had no effect on the
growth and survival of the cells. This result did not change upon
co-incubation with the esterase-containing brain extract, which was
expected due to the nature of the drug/spacer linkage (an amide
bond). A different result was obtained with the ME.sub.6C
conjugate. The prodrug was ineffective in inhibiting cell growth or
survival in the absence of brain extract. Upon addition of the
brain extract, a significant increase in Mtx cytotoxicity was
observed. This is consistent with cleavage of the ester linkage by
the brain extract-derived esterase. A similar result was obtained
with the MCS conjugate, indicating that the brain extract esterase
activity was capable of cleaving the salicylic acid ester.
[0080] Table II shows the results of drug uptake studies performed
with the prodrug N-AZT-ceramide. Antiviral amounts of the prodrug
conjugate were added to NIH 3T3 cell cultures, and the antiviral
activity of the prodrug was found to be equivalent to the activity
of free AZT. In addition, upon removal of the prodrug,
intracellular retention of prodrug was found to be up to 15-fold
higher than free AZT (Table II) over a 23 h period.
[0081] These results indicate that for Mtx-containing conjugates,
the free drug must be released from the prodrug for biological
activity. These results suggest that specific release of this drug,
and perhaps others, can be achieved using cleavable linker moieties
that are specifically cleaved only in pathogen-infected cells.
1 TABLE I Sample.sup.1 # cells/plate.sup.2 Sample.sup.3 #
cells/plate.sup.4 Control/FBS 7.8 .times. 10.sup.6 Control/FBS 13
.times. 10.sup.6 ME.sub.6C/FBS 6.5 .times. 10.sup.6 MSC/FBS 2.1
.times. 10.sup.6 ME.sub.6C/brain 2.7 .times. 10.sup.6 MSC/brain
0.51 .times. 10.sup.6 Mtx/FBS 0.16 .times. 10.sup.6 Mtx/FBS 0.13
.times. 10.sup.6 Mtx/brain 0.09 .times. 10.sup.6 Mtx/brain 0.06
.times. 10.sup.6 Control/brain N.D. Control/brain 6.2 .times.
10.sup.6 .sup.1= cells incubated with drug/FBS or drug/brain
extract for 1 hour at 37.degree. C. .sup.2= cell growth survival
determined 70 hours after drug addition .sup.3= cells incubated
with drug/FBS or drug/brain extract for 2 hours at 37.degree. C.
.sup.4= cell growth and survival determined 72 hours after drug
addition
[0082]
2TABLE II Time.sup.1 AZT.sup.2 N-AZT-Ceramide.sup.2 0 hr. 6.49 8.45
23 hr. 0.55 7.78 .sup.1= time between the end of drug treatment and
assay for intracellular drug concentration .sup.2= nM/10.sup.6
cells
EXAMPLE 10
[0083] An antiproliferative agent is prepared wherein the
anti-proliferative drug methotrexate (Mtx) is conjugated to
sphingosine via a 6-aminocaproic acid spacer. This reaction scheme
is illustrated in FIG. 10. The primary amino and hydroxyl groups of
sphingosine are acylated by reaction with activated
N-(methotrexate)aminocaproic acid overnight at 40-50.degree. C.,
followed by base hydrolysis in 0.1N methanolic KOH. The Mtx
derivative of 6-aminocaproic acid is synthesized by activating the
carboxylic acid moiety of Mtx and reacting with 6-aminocaproic acid
for 2 days at 60-70.degree. C. This reaction is stopped under
acidic conditions to liberate anhydrides that form under these
conditions.
EXAMPLE 11
[0084] An in vivo mouse skin model system was used to demonstrate
the use of embodiments of the polar lipid conjugates of the
invention for introducing biologically-active compounds through the
epidermal layer of the skin and into the underlying skin
layers.
[0085] In these experiments, various embodiments of the polar
lipids of the invention were conjugated to a fluorescent compound,
(7-nitro-2-1,3-benzoxadiazol-4-yl)-hexanoate (NBD), conjugation
being achieved using the methods disclosed herein (Example 10). The
NBD-polar lipid conjugates were mixed with dimethylsulfoxide
(DMSO), and 20 .mu.L of a 1.7% solution of each conjugate in DMSO
were applied to shaved mouse skin and allowed to penetrate the skin
for 4 hours. After the 4 hour incubation, skin sections were
excised and prepared for light or fluorescence microscopy, using
standard histological techniques.
[0086] The results of these experiments are shown in FIGS. 11
through 19. In each Figure, the outer layer of the epidermis is
located in the upper, left-hand corner of the photomicrograph.
[0087] FIG. 11 is a photomicrograph that illustrates
hematoxylin-eosin (H&E) staining of mouse skin, observed by
light microscopy under 100.times. magnification. It was observed in
this photomicrograph that H&E staining was concentrated in the
epidermis and reticular dermis, and that the papillary dermis
remained relatively unstained.
[0088] In comparison, FIG. 12 is a fluorescence photomicrograph
that illustrates ceramide-NBD staining of mouse skin, observed by
fluorescence microscopy under 100.times. magnification. In this
Figure it was observed that the ceramide-NBD fluorescence was
carried through the stratum granulosum and epidermis. No
partitioning into keratinocytes or Langerhans cells was observed,
but distribution through the skin section appeared to be
cell-dependent, that is, fluorescence was evenly distributed
throughout the cells in the section, rather than being distributed
nonspecifically through the microscopic field of view.
[0089] FIG. 13 is a fluorescence photomicrograph that illustrates
phosphatidylcholine-NBD staining of mouse skin, observed by
fluorescence microscopy under 100.times. magnification. FIG. 14 is
a fluorescence photomicrograph that illustrates
phosphatidylethanolamine-NBD staining of mouse skin, observed by
fluorescence microscopy under 100.times. magnification, and FIG. 15
is a fluorescence photomicrograph that illustrates
phosphatidylserine-NBD staining of mouse skin, observed by
fluorescence microscopy under 100.times. magnification. Each of
these conjugates was observed to result in localized fluorescence
in the outer layers of the skin. In FIGS. 13 and 15, some staining
of specific areas below the epidermis was also observed, and the
compound of FIG. 15 was observed to penetrate into the papillary
dermis.
[0090] FIG. 16 is a fluorescence photomicrograph that illustrates
1-R{6[(7-nitro-2-1,3-benzoxadiazol-4-ethylamino)caproyl}-NBD
(termed phospho-rac-(1-glycerol)-NBD(caproyl)) staining of mouse
skin, observed by fluorescence microscopy under 100.times.
magnification. It was observed that the
phospho-rac-(1-glycerol)-NBD(caproyl) conjugate penetrated the skin
extensively, but in a pattern distinct and different from
ceramide-NBD shown in FIG. 12. It was observed that the
ceramide-NBD distributed through the cell structure of the deeper
skin layers, while the phospho-rac-(1-glycerol)-NBD(caproyl)
conjugate concentrated mainly in plasma membranes of fat cells, as
well as in some unidentified structures.
[0091] FIG. 17 is a fluorescence photomicrograph that illustrates
phosphatidylserine-NBD staining of mouse skin, observed by
fluorescence microscopy under 100.times. magnification. The
compound used in FIG. 17 differs from the phosphatidylserine-NBD
conjugate shown in FIG. 15 in that the NBD dye is conjugated to the
polar lipid via a dodecanoyl moiety in the compound of FIG. 17 and
via a caproyl moiety in the compound of FIG. 15. In contrast to the
results obtained with the compound of FIG. 15, the
dodecanoyl-conjugated NBD compound of FIG. 17 penetrated into the
cornified layer, with only minimal penetration into the
epidermis.
[0092] FIG. 18 is a fluorescence photomicrograph that illustrates
1-R(12{(7-nitro-2-1,3-benzoxadiazol-4-ethylamino)}dodecanoyl)-NBD
(termed phospho-rac-(1-glycerol)-NBD(dodecanoyl)) staining of mouse
skin, observed by fluorescence microscopy under 100.times.
magnification. This compound is related to the compound of FIG. 16,
but differs in the size of the acyl chain to which the fluorescent
NBD label is conjugated; here, it is a dodecanoyl chain, while in
FIG. 16 it is a caproyl chain. Both the compound of FIG. 16 and the
instant compound were observed to penetrate the papillary dermis
and the reticular dermis. In addition, the dodecanoyl-containing
compound of FIG. 18 was also observed to accumulate in hair
follicles.
[0093] FIG. 19 is a fluorescence photomicrograph that illustrates
phosphatidylethanolamine-NBD staining of mouse skin, observed by
fluorescence microscopy under 100.times. magnification. The
compound used in FIG. 19 differs from the
phosphatidylethanolamine-NBD conjugate shown in FIG. 14 in that the
NBD dye is conjugated to the polar lipid via a dodecanoyl moiety in
the compound of FIG. 19 and via a caproyl moiety in the compound of
FIG. 14. In contrast to the results obtained with the compound of
FIG. 14, the dodecanoyl-conjugated NBD compound of FIG. 19
penetrated into the dermis.
[0094] Since all compounds were administered in the DMSO vehicle,
lack of penetration of some compounds into some or most skin layers
discounts the possibility that the DMSO vehicle was responsible for
non-specifically carrying fluorophore into the tissue.
[0095] These results demonstrate that certain of these conjugates
showed specific partitioning into defined layers of the skin.
Ceramide-NBD, phospho-rac-(1-glycerol)-NBD(caproyl) and
phospho-rac-(1-glycerol)-NBD(do- cecanoyl) penetrated the skin to
the reticular dermis. Caproyl-conjugated
phosphatidylethanolamine-NBD, in contrast, did not penetrate beyond
the outermost layers of the epidermis, while dodecanoyl-conjugated
phosphatidylethanolamine-NBD was observed to penetrate into the
dermis. On the other hand, caproyl-conjugated
phosphatidylserine-NBD penetrated into the papillary dermis, while
dodecanoyl-conjugated phosphatidylethanolamine-NBD did not
penetrate past the cornified layer of the epidermis. These results
suggested that polar lipid composition is a determinant in the
penetrating ability of the conjugates of the invention, and
demonstrated that linkage to polar lipids produced increased
penetration of the skin by non-penetrating compounds. These results
also demonstrated that the conjugates of the invention partitioned
selectively in skin layers and cells, depending on the lipid
carrier used in the conjugate. These results further indicate that
conjugating antiproliferative compounds of the invention with polar
lipids can be used to deliver drugs to specific areas of the skin
in greater quantity and concentration than can currently be
achieved, and that such drugs can be maintained in specific areas
and cells in the skin for longer periods of time. As a consequence
of lipid-drug formulation, release of active drug from these
conjugates can be achieved by the use of hydrolyzable bonds between
drug and carrier.
[0096] The specific partitioning of the conjugates of the
invention, achieved through the use of polar lipid conjugates, also
permits a greater therapeutic index to be achieved. These
capacities of the antiproliferative drug conjugates of the
invention have important applications to the delivery of medicinal
compounds into the skin to treat a variety of pathological
conditions. Medicinal salves and ointments for topical treatment
purposes are known in the prior art for the treatment of a variety
of pathological conditions, but they suffer from non-specific
deposition of the antiproliferative drug into both healthy and
affected portions of the skin. In addition, appropriate
concentrations of topically-applied antiproliferative drugs are
currently limited by the escape of the active agent(s) into the
systemic circulation, with deleterious effects on other tissues and
organs. An example of such a situation is the use of the drug
methotrexate to treat psoriasis, where the amount of methotrexate
that is capable of being topically applied is limited by hepato-
and nephrotoxicity caused by systemic escape of the compound from
the skin.
[0097] One advantage of the methotrexate-containing embodiments of
conjugates of the invention (such as methotrexate ceramide ester,
ME.sub.6C), is that this compound does not concentrate in the liver
or kidney to the same extent as free drug, even upon escape into
the systemic circulation.
[0098] Similarly, treatment of fungal infections in the skin is
limited by systemic hepatotoxicity of many topically-applied
antifungal agents, such as ketoconazole, griseofulvin, and
ciclopixox. Specific localization of such compounds to the skin
using polar lipid-drug conjugates of the invention provides a means
of increasing the dosages of such antifungal agents that can be
topically applied. Other uses of the conjugates of the invention
include treatment of precancerous lesions with polar lipid
conjugated 5-fluorouracil.
[0099] The present invention therefore solves a problem common to
treatment of a variety of pathological conditions in skin tissue
with topically-applied salves, ointments or similar
medicaments.
[0100] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
* * * * *