U.S. patent application number 17/046753 was filed with the patent office on 2021-11-04 for ceramide-like lipid-based delivery vehicles and uses thereof.
This patent application is currently assigned to Children's Medical Center Corporation. The applicant listed for this patent is Children's Medical Center Corporation. Invention is credited to Daniel JF Chinnapen, Richard I. Duclos, Wayne I. Lencer.
Application Number | 20210338823 17/046753 |
Document ID | / |
Family ID | 1000005910309 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210338823 |
Kind Code |
A9 |
Lencer; Wayne I. ; et
al. |
November 4, 2021 |
CERAMIDE-LIKE LIPID-BASED DELIVERY VEHICLES AND USES THEREOF
Abstract
Provided herein, in some aspects, are delivery vehicles
comprising a ceramide and an agent to be delivered attached to the
ceramide. In some embodiments, the ceramide does not comprise a
fatty acid (i.e., is a sphingosine). In some embodiments, the
ceramide comprises a fatty acid. In some embodiments, the ceramide
is a glycoceramide. In some embodiments, the agent is attached to
the ceramide covalently (e.g., via a linker). In some embodiments,
the agent to be delivered is a therapeutic agent. The ceramide is
able to deliver the agent to a cell or to a cellular compartment,
as well as across the musical barrier. In some embodiments, agents
delivered using the ceramide described herein exhibit longer
half-life, compared to agents delivered alone. Methods of
delivering a therapeutic agent to a subject for treating a disease
using the ceramide delivery vehicle are also provided.
Inventors: |
Lencer; Wayne I.; (Jamaica
Plain, MA) ; Chinnapen; Daniel JF; (Quincy, MA)
; Duclos; Richard I.; (Quincy, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Medical Center Corporation |
Boston |
MA |
US |
|
|
Assignee: |
Children's Medical Center
Corporation
Boston
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20210030880 A1 |
February 4, 2021 |
|
|
Family ID: |
1000005910309 |
Appl. No.: |
17/046753 |
Filed: |
April 12, 2019 |
PCT Filed: |
April 12, 2019 |
PCT NO: |
PCT/US2019/027281 PCKC 00 |
371 Date: |
October 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62656474 |
Apr 12, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/543 20170801;
A61K 47/65 20170801; A61K 31/7105 20130101; A61K 9/1617
20130101 |
International
Class: |
A61K 47/54 20060101
A61K047/54; A61K 47/65 20060101 A61K047/65; A61K 9/16 20060101
A61K009/16; A61K 31/7105 20060101 A61K031/7105 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grants
Nos. R37 DK048106, RO1 DK104868, R21 DK090603, and P30 DK034854,
awarded by the National Institutes of Health. The government has
certain rights in the invention.
Claims
1. A delivery vehicle comprising a ceramide and an agent to be
delivered, wherein the ceramide: (a) does not comprise a fatty
acid; or (b) comprises a fatty acid of C1-C28; and wherein the
agent is attached to the ceramide.
2. The delivery vehicle of claim 1, wherein the ceramide is a
ceramide analog.
3. The delivery vehicle of claim 2, wherein the ceramide analog is
selected from the group consisting of: 2-hydroxy-ceramide,
diene-deoxy-ceramide, dihydroceramide, dihydroceramide phosphate,
o-acyl-ceramide, ceramide phosphate, sphinganine, and
methyl-sphingosine.
4. The delivery vehicle of claim 2, wherein the ceramide analog
comprises an unsaturated hydrocarbon chain attached to ornithine,
tyrosine, glycine, leucine, proline, glutamine, or taurine.
5. The delivery vehicle of any one of claims 1-4, wherein the
ceramide is a glycoceramide.
6. The delivery vehicle of claim 5, wherein the glycoceramide
comprises a sugar selected from the group consisting of: glucose,
galactose, fructose, and GalNac.
7. The delivery vehicle of claim 6, wherein the agent to be
delivered is attached to the sugar.
8. The delivery vehicle of any one of claims 1-4, wherein no sugar
is attached to the ceramide.
9. The delivery vehicle of claim 8, wherein the ceramide is a
sphingosine.
10. The delivery vehicle of claim 8 or claim 9, wherein the agent
to be delivered is attached to the primary or secondary hydroxyl
group of the ceramide.
11. The delivery vehicle of any one of claims 1-10, wherein the
agent to be delivered is attached to the ceramide via a linker.
12. The delivery vehicle of claim 11, wherein the linker is a
pseudo-glycopeptide linker.
13. The delivery vehicle of claim 12, wherein the
pseudo-glycopeptide linker comprises at least one sugar attached to
an amino acid backbone.
14. The delivery vehicle of claim 13, wherein the at least one
sugar is selected from the group consisting of: glucose, galactose,
and N-Acetylgalactosamine.
15. The delivery vehicle of claim 14, wherein the at least one
sugar is attached to the amino acid backbone via a serine side
chain.
16. The delivery vehicle of claim 11, wherein the linker is a
cleavable linker.
17. The delivery vehicle of claim 16, wherein the cleavable linker
comprises an ester linkage.
18. The delivery vehicle of claim 17, wherein the cleavable linker
is a peptide linker comprising an ester linkage.
19. The delivery vehicle of claim 16, wherein the cleavable linker
comprises a cleavage motif for an endosomal protease.
20. The delivery vehicle of claim 19, wherein the endosomal
protease is furin or matriptase.
21. The delivery vehicle of claim 11, wherein the linker is a
disulfide linkage.
22. The delivery vehicle of any one of claims 1-21, wherein the
ceramide comprises a fatty acid of C1-C6.
23. The delivery vehicle of claim 22, wherein the ceramide
comprises a fatty acid of C4.
24. The delivery vehicle of claim 22, wherein the ceramide
comprises a fatty acid of C6.
25. The delivery vehicle of any one of claims 22-24, wherein the
fatty acid has no double bonds between two carbon atoms.
26. The delivery vehicle of any one of claims 1-21, wherein the
ceramide comprises a fatty acid of C7-C28.
27. The delivery vehicle of claim 22, wherein the ceramide
comprises a fatty acid of C8.
28. The delivery vehicle of claim 26 or 27, wherein the fatty acid
has at least one cis double bonds between two carbon atoms.
29. The delivery vehicle of claim 28, wherein the at least one cis
double bond is in C1-C18 region.
30. The delivery vehicle of claim 26, wherein the fatty acid
comprises a chemical moiety in C1-C18 region.
31. The delivery vehicle of any one of claims 1-21, wherein the
ceramide does not comprise a fatty acid.
32. The delivery vehicle of any one of claims 1-31, wherein the
agent to be delivered is selected from the group consisting of
proteins, peptides, nucleic acids, polysaccharides and
carbohydrates, lipids, glycoproteins, small molecules, synthetic
organic and inorganic drugs exerting a biological effect when
administered to a subject, and combinations thereof.
33. The delivery vehicle of any one of claims 1-32, wherein the
agent to be delivered is a therapeutic agent.
34. The delivery vehicle of claim 33, wherein the therapeutic agent
is an anti-inflammatory agent, a vaccine antigen, a small molecule
drug, an anti-cancer drug or chemotherapeutic drug, a clotting
factor, a hormone, a steroid, a cytokine, an antibiotic, an
antibody, a ScFv, a nanobody, a vaccine adjuvant, an agent that
induces immune tolerance, or a drug for the treatment of a
cardiovascular disease, an infectious disease, an autoimmune
disease, allergy, a blood disorder, a metabolic disorder, a skin
disease, an eye disease, a lysosomal storage disease, or a
neurological disease.
35. The delivery vehicle of any one of claims 1-31, wherein the
agent to be delivered is a protein or a peptide.
36. The delivery vehicle of claim 35, wherein the protein or
peptide is a vaccine antigen.
37. The delivery vehicle of claim 35, wherein the protein or
peptide is an antibody, a ScFv, or a nanobody.
38. The delivery vehicle of claim 35, wherein the protein or
peptide is an enzyme.
39. The delivery vehicle of claim 35, wherein the protein or
peptide is a hormone.
40. The delivery vehicle of claim 35, wherein the protein or
peptide is a neurotransmitter.
41. The delivery vehicle of claim 35, wherein the protein or
peptide is GLP-1, or a functional fragment thereof.
42. The delivery vehicle of claim 35, wherein the protein or
peptide is Exendin-4, or a functional fragment thereof.
43. The delivery vehicle of claim 33, wherein the therapeutic agent
comprises GLP-1 or a functional fragment thereof, and Exendin-4 or
a functional fragment thereof.
44. The delivery vehicle of claim 33, wherein the therapeutic agent
comprises a ligand for a cell receptor.
45. The delivery vehicle of claim 44, wherein the cell receptor is
a growth factor receptor, a G-protein coupled receptor, or a
toll-like receptor.
46. The delivery vehicle of claim 33, wherein the therapeutic agent
is a nucleic acid.
47. A ceramide-therapeutic agent complex comprising a ceramide and
an agent to be delivered, wherein the ceramide: (a) does not
comprise a fatty acid; or (b) comprises a fatty acid of C1-C28; and
wherein the agent is attached to the ceramide.
48. The ceramide-therapeutic agent complex of claim 47, wherein the
ceramide is a ceramide analog.
49. The ceramide-therapeutic agent complex of claim 48, wherein the
ceramide analog is selected from the group consisting of:
2-hydroxy-ceramide, diene-deoxy-Cer, dihydroceramide,
dihydroceramide phosphate, o-acyl-ceramide, ceramide phosphate,
sphinganine, and methyl-sphingosine.
50. The delivery vehicle of claim 48, wherein the ceramide analog
comprises an unsaturated hydrocarbon chain attached to ornithine,
tyrosine, glycine, leucine, proline, glutamine, or taurine.
51. The ceramide-therapeutic agent complex of any one of claims
47-50, wherein the ceramide is a glycoceramide.
52. The ceramide-therapeutic agent complex of claim 51, wherein the
glycoceramide comprises a sugar selected from the group consisting
of: glucose, galactose, fructose, and GalNAc.
53. The ceramide-therapeutic agent complex of claim 52, wherein the
agent to be delivered is attached to the sugar.
54. The ceramide-therapeutic agent complex of any one of claims
47-50, wherein no sugar is attached to the ceramide.
55. The ceramide-therapeutic agent complex of claim 54, wherein the
ceramide is a sphingosine.
56. The ceramide-therapeutic agent complex of claim 54 or claim 55,
wherein the agent to be delivered is attached to the primary or
secondary hydroxyl group of the ceramide.
57. The ceramide-therapeutic agent complex of any one of claims
47-56, wherein the agent to be delivered is attached to the
ceramide via a linker.
58. The ceramide-therapeutic agent complex of claim 57, wherein the
linker is a pseudo-glycopeptide linker.
59. The ceramide-therapeutic agent complex of claim 58, wherein the
pseudo-glycopeptide linker comprises at least one sugar attached to
an amino acid backbone.
60. The ceramide-therapeutic agent complex of claim 59, wherein the
at least one sugar is selected from the group consisting of:
glucose, galactose, and N-Acetylgalactosamine.
61. The ceramide-therapeutic agent complex of claim 60, wherein the
at least one sugar is attached to the amino acid backbone via a
serine side chain.
62. The ceramide-therapeutic agent complex of claim 57, wherein the
linker is a cleavable linker.
63. The ceramide-therapeutic agent complex of claim 62, wherein the
cleavable linker comprises an ester linkage.
64. The ceramide-therapeutic agent complex of claim 63, wherein the
cleavable linker is a peptide linker comprising an ester
linkage.
65. The ceramide-therapeutic agent complex of claim 62, wherein the
cleavable linker comprises a cleavage motif for an endosomal
protease.
66. The ceramide-therapeutic agent complex of claim 65, wherein the
endosomal protease is furin or matriptase.
67. The ceramide-therapeutic agent complex of claim 57, wherein the
linker is a disulfide linkage.
68. The ceramide-therapeutic agent complex of any one of claims
47-67, wherein the ceramide comprises a fatty acid of C1-C6.
69. The ceramide-therapeutic agent complex of claim 68, wherein the
ceramide comprises a fatty acid of C4.
70. The ceramide-therapeutic agent complex of claim 68, wherein the
ceramide comprises a fatty acid of C6.
71. The ceramide-therapeutic agent complex of any one of claims
68-70, wherein the fatty acid has no double bonds between two
carbon atoms.
72. The ceramide-therapeutic agent complex of any one of claims
47-67, wherein the ceramide comprises a fatty acid of C7-C28.
73. The ceramide-therapeutic agent complex of claim 72, wherein the
ceramide comprises a fatty acid of C8.
74. The ceramide-therapeutic agent complex of claim 72 or 73,
wherein the fatty acid has at least one cis double bonds between
two carbon atoms.
75. The ceramide-therapeutic agent complex of claim 74, wherein the
at least one cis double bond is in C1-C18 region.
76. The ceramide-therapeutic agent complex of claim 72, wherein the
fatty acid comprises a chemical moiety in C1-C18 region.
77. The ceramide-therapeutic agent complex of any one of claims
47-67, wherein the ceramide does not comprise a fatty acid.
78. The ceramide-therapeutic agent complex of any one of claims
47-77, wherein the therapeutic agent is selected from the group
consisting of proteins, peptides, nucleic acids, polysaccharides
and carbohydrates, lipids, glycoproteins, small molecules,
synthetic organic and inorganic drugs exerting a biological effect
when administered to a subject, and combinations thereof.
79. The ceramide-therapeutic agent complex of claim 78, wherein the
therapeutic agent is an anti-inflammatory agent, a vaccine antigen,
a small molecule drug, an anti-cancer drug or chemotherapeutic
drug, a clotting factor, a hormone, a steroid, a cytokine, an
antibiotic, an antibody, a ScFv, a nanobody, a vaccine adjuvant, an
agent that induces immune tolerance, or a drug for the treatment of
cardiovascular disease, an infectious disease, an autoimmune
disease, allergy, a blood disorder, a metabolic disorder, a skin
disease, an eye disease, a lysosomal storage disease, or a
neurological disease.
80. The ceramide-therapeutic agent complex of any one of claims
47-77, wherein the therapeutic agent is a protein or a peptide.
81. The ceramide-therapeutic agent complex of claim 80, wherein the
protein or peptide is a vaccine antigen.
82. The ceramide-therapeutic agent complex of claim 80, wherein the
protein or peptide is an antibody, a ScFv, or a nanobody.
83. The ceramide-therapeutic agent complex of claim 80, wherein the
protein or peptide is an enzyme.
84. The ceramide-therapeutic agent complex of claim 80, wherein the
protein or peptide is a hormone.
85. The ceramide-therapeutic agent complex of claim 80, wherein the
protein or peptide is a neurotransmitter.
86. The ceramide-therapeutic agent complex of claim 80, wherein the
protein or peptide is GLP-1, or a functional fragment thereof.
87. The ceramide-therapeutic agent complex of claim 80, wherein the
protein or peptide is Exendin-4, or a functional fragment
thereof.
88. The ceramide-therapeutic agent complex of claim 80, wherein the
therapeutic agent comprises GLP-1 or a functional fragment thereof,
and Exendin-4 or a functional fragment thereof.
89. The ceramide-therapeutic agent complex of claim 79, wherein the
therapeutic agent comprises a ligand for a cell receptor.
90. The ceramide-therapeutic agent complex of claim 89, wherein the
cell receptor is a growth factor receptor, a G-protein coupled
receptor, or a toll-like receptor.
91. The ceramide-therapeutic agent complex of claim 79, wherein the
therapeutic agent is a nucleic acid.
92. A composition comprising the delivery vehicle of any one of
claims 1-46, or the ceramide-therapeutic agent complex of any one
of claims 47-91.
93. The composition of claim 92, further comprising a
pharmaceutically acceptable carrier.
94. A method of delivering an agent into a cell, across a mucosal
surface, or across an endothelial barrier, the method comprising
contacting the delivery vehicle of any one of claims 1-46 with the
cell, the mucosal surface, or the endothelial lumenal surface,
under conditions appropriate for uptake of the delivery vehicle or
the agent into the cell or absorption of the delivery vehicle or
the agent across the mucosal surface or the endothelial
surface.
95. A method of delivering an agent into a cell, across a mucosal
surface, or across an endothelial barrier, the method comprising
contacting the ceramide-therapeutic complex of any one of claims
47-91, with the cell or the mucosal surface or the endothelial
lumenal surface, under conditions appropriate for uptake of the
ceramide-therapeutic agent complex or the agent into the cell or
absorption of the ceramide-therapeutic agent complex or the agent
across the mucosal surface or the endothelial barrier.
96. A method of delivering an agent into a cell, across a mucosal
surface, or across an endothelial barrier, the method comprising
contacting the composition of any one of claim 92 or claim 93, with
the cell, the mucosal surface, or the endothelial lumenal surface,
under conditions appropriate for uptake of the composition or the
agent into the cell or absorption of the composition or the agent
across the mucosal surface or the endothelial barrier.
97. A method of delivering an agent into a cells, across a mucosal
surface or an endothelial barrier in a subject, the method
comprising administering to the subject the delivery vehicle of any
one of claims 1-46, the ceramide-therapeutic agent complex of any
one of claims 47-91, or the composition of claim 92 or claim
93.
98. A method of enhancing the half-life of an agent in a subject,
the method comprising administering to the subject the delivery
vehicle of any one of claims 1-46, the ceramide-therapeutic agent
complex of any one of claims 47-91, or the composition of claim 92
or claim 93.
99. A method of treating a disease or condition in a subject in
need thereof, the method comprising administering to the subject an
effective amount of the delivery vehicle of any one of claims 1-46,
the ceramide-therapeutic agent complex of any one of claims 47-91,
or the composition of claim 92 or claim 93, wherein the effective
amount is an amount sufficient to ameliorate/reduce the extent to
which the disease or condition occurs in the subject.
100. The method of any one of claims 97-99, wherein the delivery
vehicle, the ceramide-therapeutic agent complex, or the composition
is administered parenterally.
101. The method of claim 100, wherein the delivery vehicle, the
ceramide-therapeutic agent complex, or the composition is
administered intravenously, intramuscularly, intradermally,
subcutaneously, intrathecally, intraperitoneally, intraarterially,
intracardiacally, intraosseously, intraocularly, intravitreally,
intranasally, or intrapleurally.
102. The method of any one of claims 97-99, wherein the delivery
vehicle, the ceramide-therapeutic agent complex, or the composition
is administered nonparenterally.
103. The method of claim 102, wherein the delivery vehicle, the
ceramide-therapeutic agent complex, or the composition is
administered orally, sublingually, topically, rectally, or via
inhalation.
Description
RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application No. 62/656,474, filed Apr.
12, 2018, and entitled "CERAMIDE-LIKE LIPID-BASED DELIVERY VEHICLES
AND USES THEREOF," the entire contents of which are incorporated
herein by reference.
BACKGROUND
[0003] One of the major challenges for applying protein and peptide
biologies to clinical medicine is the lack of rational and
efficient methods to circumvent epithelial and endothelial cell
barriers separating large molecules from target tissues. In the
case of epithelial cells lining mucosal surfaces, the pathway for
absorption of large solutes (e.g., biologies) is by transcytosis--a
process of transcellular endosome trafficking that connects one
surface of the cell with the other.
SUMMARY
[0004] The present disclosure, in some aspects, relates to using
ceramides (e.g., naturally occurring ceramides and ceramide analogs
or ceramide-like molecules) as delivery vehicles to deliver agents
(e.g., therapeutic agents) into cells or across epithelial and/or
endothelial barriers. The sorting of the agents via different
endocytic pathways relate to the structure of the ceramide. In some
embodiments, the ceramides are used to deliver agents to a targeted
site, e.g., to treat a disease in a subject.
[0005] Some aspects of the present disclosure provide delivery
vehicles comprising a ceramide and an agent to be delivered,
wherein the ceramide: (a) does not contain a fatty acid; or (b)
comprises a fatty acid of C1-C28; and wherein the agent is attached
to the ceramide. In some embodiments, the ceramide is a ceramide
analog. In some embodiments, the ceramide analog is selected from
the group consisting of: 2-hydroxy-ceramide, diene-deoxy-ceramide,
dihydroceramide, phytosphingosine, dihydroceramide phosphate,
o-acyl-ceramide, ceramide phosphate, sphinganine, and
methyl-sphingosine. In some embodiments, the ceramide analog
comprises an unsaturated hydrocarbon chain attached to ornithine,
tyrosine, glycine, leucine, praline, glutamine, or taurine.
[0006] In some embodiments, the ceramide is a glycoceramide. In
some embodiments, the glycoceramide comprises a sugar selected from
the group consisting of: glucose, galactose, fructose, and GalNac.
In some embodiments, the agent to be delivered is attached to the
sugar. In some embodiments, no sugar is attached to the ceramide.
In some embodiments, the ceramide is a sphingosine. In some
embodiments, the agent to be delivered is attached to the primary
hydroxyl group of the ceramide. In some embodiments, the agent to
be delivered is attached to the secondary hydroxyl group of the
ceramide.
[0007] In some embodiments, the agent to be delivered is attached
to the ceramide via a linker. In some embodiments, the linker is a
pseudo-glycopeptide linker. In some embodiments, the
pseudo-glycopeptide linker comprises at least one sugar attached to
an amino acid backbone. In some embodiments, the at least one sugar
is selected from the group consisting of: glucose, galactose, and
N-Acetylgalactosamine. In some embodiments, the at least one sugar
is attached to the amino acid backbone via a serine side chain.
[0008] In some embodiments, the linker is a cleavable linker. In
some embodiments, the cleavable linker comprises an ester linkage.
In some embodiments, the cleavable linker is a peptide linker
comprising an ester linkage. In some embodiments, the cleavable
linker comprises a cleavage motif for an endosomal protease. In
some embodiments, the endosomal protease is furin or matriptase. In
some embodiments, the linker is a disulfide linkage.
[0009] In some embodiments, the ceramide comprises a fatty acid of
C1-C6. In some embodiments, the ceramide comprises a fatty acid of
C4. In some embodiments, the ceramide comprises a fatty acid of C6.
In some embodiments, the fatty acid has no double bonds between two
carbon atoms. In some embodiments, the ceramide comprises a fatty
acid of C7-C28. In some embodiments, the ceramide comprises a fatty
acid of C8. In some embodiments, the fatty acid has at least one
cis double bonds between two carbon atoms. In some embodiments, the
at least one cis double bond is in C1-C18 region. In some
embodiments, the fatty acid comprises a chemical moiety in C1-C18
region. In some embodiments, the ceramide does not comprise a fatty
acid.
[0010] In some embodiments, the ceramide comprises a fatty acid of
C1-C12.
[0011] In some embodiments, the agent to be delivered is selected
from the group consisting of proteins, peptides, nucleic acids,
polysaccharides and carbohydrates, lipids, glycoproteins, small
molecules, synthetic organic and inorganic drugs exerting a
biological effect when administered to a subject, and combinations
thereof. In some embodiments, the agent to be delivered is a
therapeutic agent. In some embodiments, the therapeutic agent is an
anti-inflammatory agent, a vaccine antigen, a small molecule drug,
an anti-cancer drug or chemotherapeutic drug, a clotting factor, a
hormone, a steroid, a cytokine, an antibiotic, an antibody, a ScFv,
a nanobody, a vaccine adjuvant, or a drug for the treatment of a
cardiovascular disease, an infectious disease, an autoimmune
disease, allergy, a blood disorder, a metabolic disorder, a skin
disease, an eye disease, a lysosomal storage disease or a
neurological disease.
[0012] In some embodiments, the agent to be delivered is a protein
or a peptide. In some embodiments, the protein or peptide is a
vaccine antigen. In some embodiments, the protein or peptide is an
antibody, a ScFv, or a nanobody. In some embodiments, the protein
or peptide is an enzyme. In some embodiments, the enzyme is a
lysosomal replacement enzyme. In some embodiments, the protein or
peptide is a hormone. In some embodiments, the protein or peptide
is a neurotransmitter. In some embodiments, the protein or peptide
is GLP-1, or a functional fragment thereof. In some embodiments,
the protein or peptide is Exendin-4, or a functional fragment
thereof. In some embodiments, the therapeutic agent comprises GLP-1
or a functional fragment thereof, and Exendin-4 or a functional
fragment thereof. In some embodiments, the therapeutic agent
comprises a ligand for a cell receptor. In some embodiments, the
cell receptor is a growth factor receptor, a G-protein coupled
receptor, or a toll-like receptor.
[0013] In some embodiments, the therapeutic agent is a nucleic
acid.
[0014] Other aspects of the present disclosure provide
ceramide-therapeutic agent complexes comprising a ceramide and an
agent to be delivered, wherein the ceramide: (a) does not comprise
a fatty acid; or (b) comprises a fatty acid of C1-C28; and wherein
the agent is attached to the ceramide.
[0015] In some embodiments, the ceramide is a ceramide analog. In
some embodiments, the ceramide analog is selected from the group
consisting of: 2-hydroxy-ceramide, diene-deoxy-ceramide,
dihydroceramide, phytosphingosine, dihydroceramide phosphate,
o-acyl-ceramide, ceramide phosphate, sphinganine, and
methyl-sphingosine. In some embodiments, the ceramide analog
comprises an unsaturated hydrocarbon chain attached to ornithine,
tyrosine, glycine, leucine, praline, glutamine, or taurine.
[0016] In some embodiments, the ceramide is a glycoceramide. In
some embodiments, the glycoceramide comprises a sugar selected from
the group consisting of: glucose, galactose, fructose, and GalNAc.
In some embodiments, the agent to be delivered is attached to the
sugar. In some embodiments, no sugar is attached to the ceramide.
In some embodiments, the ceramide is a sphingosine. In some
embodiments, the agent to be delivered is attached to the primary
hydroxyl group of the ceramide. In some embodiments, the agent to
be delivered is attached to the secondary hydroxyl group of the
ceramide.
[0017] In some embodiments, the agent to be delivered is attached
to the ceramide via a linker. In some embodiments, the linker is a
pseudo-glycopeptide linker. In some embodiments, the
pseudo-glycopeptide linker comprises at least one sugar attached to
an amino acid backbone. In some embodiments, the at least one sugar
is selected In some embodiments, the at least one sugar is attached
to the amino acid backbone via a serine side chain.
[0018] In some embodiments, the linker is a cleavable linker. In
some embodiments, the cleavable linker comprises an ester linkage.
In some embodiments, the cleavable linker is a peptide linker
comprising an ester linkage. In some embodiments, the cleavable
linker comprises a cleavage motif for an endosomal protease. In
some embodiments, the endosomal protease is furin or matriptase. In
some embodiments, the linker is a disulfide linkage.
[0019] In some embodiments, the ceramide comprises a fatty acid of
C1-C6. In some embodiments, the ceramide comprises a fatty acid of
C4. In some embodiments, the ceramide comprises a fatty acid of C6.
In some embodiments, the fatty acid has no double bonds between two
carbon atoms. In some embodiments, the ceramide comprises a fatty
acid of C7-C28. In some embodiments, the ceramide comprises a fatty
acid of C8. In some embodiments, the fatty acid has at least one
cis double bonds between two carbon atoms. In some embodiments, the
at least one cis double bond is in C1-C18 region. In some
embodiments, the fatty acid comprises a chemical moiety in C1-C18
region. In some embodiments, the ceramide does not comprise a fatty
acid.
[0020] In some embodiments, the ceramide comprises a fatty acid of
C1-C12.
[0021] In some embodiments, the therapeutic agent is selected from
the group consisting of proteins, peptides, nucleic acids,
polysaccharides and carbohydrates, lipids, glycoproteins, small
molecules, synthetic organic and inorganic drugs exerting a
biological effect when administered to a subject, and combinations
thereof. In some embodiments, the therapeutic agent is an
anti-inflammatory agent, a vaccine antigen, a small molecule drug,
an anti-cancer drug or chemotherapeutic drug, a clotting factor, a
hormone, a steroid, a cytokine, an antibiotic, an antibody, a ScFv,
a nanobody, a vaccine adjuvant, or a drug for the treatment of
cardiovascular disease, an infectious disease, an autoimmune
disease, allergy, a blood disorder, a metabolic disorder, a skin
disease, an eye disease, a lysosomal storage disease, or a
neurological disease. In some embodiments, the therapeutic agent is
a protein or a peptide. In some embodiments, the protein or peptide
is a vaccine antigen. In some embodiments, the protein or peptide
is an antibody, a ScFv, or a nanobody. In some embodiments, the
protein or peptide is an enzyme. In some embodiments, the enzyme is
a lysosomal storage enzyme. In some embodiments, the protein or
peptide is a hormone. In some embodiments, the protein or peptide
is a neurotransmitter. In some embodiments, the protein or peptide
is GLP-1, or a functional fragment thereof. In some embodiments,
the protein or peptide is Exendin-4, or a functional fragment
thereof. In some embodiments, the therapeutic agent comprises GLP-1
or a functional fragment thereof, and Exendin-4 or a functional
fragment thereof. In some embodiments, the therapeutic agent
comprises a ligand for a cell receptor. In some embodiments, the
cell receptor is a growth factor receptor, a G-protein coupled
receptor, or a toll-like receptor.
[0022] In some embodiments, the therapeutic agent is a nucleic
acid.
[0023] Further provided herein are compositions comprising the
delivery vehicle, or the ceramide-therapeutic agent complex
described herein. In some embodiments, the composition further
comprises a pharmaceutically acceptable carrier.
[0024] Other aspects of the present disclosure provide methods of
delivering an agent into a cell, across a mucosal surface, or
across an endothelial barrier the method comprising contacting the
delivery vehicle the ceramide-therapeutic complex with the cell,
the mucosal surface, or the endothelial lumenal surface, under
conditions appropriate for uptake of the delivery vehicle or the
agent into the cell or absorption of the delivery vehicle or the
agent across the mucosal surface or endothelial barrier.
[0025] Other aspects of the present disclosure provide methods of
delivering an agent into a cell or across a mucosal or endothelial
surface, the method comprising contacting the composition described
herein with the cell, the mucosal surface, or the endothelial
lumenal surface, under conditions appropriate for uptake of the
composition or the agent into the cell or absorption of the
composition or the agent across the mucosal surface or the
endothelial barrier.
[0026] Other aspects of the present disclosure provide methods of
delivering an agent into a cells, across a mucosal surface, or
across an endothelial barrier in a subject, the method comprising
administering to the subject the delivery vehicle, the
ceramide-therapeutic agent, or the composition described
herein.
[0027] Other aspects of the present disclosure provide methods of
enhancing the half-life of an agent in a subject, the method
comprising administering to the subject the delivery vehicle, the
ceramide-therapeutic agent, or the composition described
herein.
[0028] Other aspects of the present disclosure provide methods of
treating a disease or condition in a subject in need thereof, the
method comprising administering to the subject an effective amount
of the delivery vehicle, the ceramide-therapeutic agent, or the
composition described herein, wherein the effective amount is an
amount sufficient to ameliorate/reduce the extent to which the
disease or condition occurs in the subject. In some embodiments,
the delivery vehicle, the ceramide-therapeutic agent complex, or
the composition is administered parenterally. In some embodiments,
the delivery vehicle, the ceramide-therapeutic agent complex, or
the composition is administered intravenously, intramuscularly,
intradermally, subcutaneously, intrathecally, intraperitoneally,
intraarterially, intracardiacally, intraosseously, intraocularly,
intravitreally, intranasally or intrapleurally. In some
embodiments, the delivery vehicle, the ceramide-therapeutic agent
complex, or the composition is administered nonparenterally. In
some embodiments, the delivery vehicle, the ceramide-therapeutic
agent complex, or the composition is administered orally,
sublingually, topically, rectally, or via inhalation. For delivery
across tight endothelial barriers, in some embodiments, the
ceramide-therapeutic agent complex is delivered intravenously,
intramuscularly, or subcutaneously.
[0029] The summary above is meant to illustrate, in a non-limiting
manner, some of the embodiments, advantages, features, and uses of
the technology disclosed herein. Other embodiments, advantages,
features, and uses of the technology disclosed herein will be
apparent from the Detailed Description, the Drawings, the Examples,
and the Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The accompanying drawings are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component that is illustrated in various figures is represented by
a like numeral. For purposes of clarity, not every component may be
labeled in every drawing. In the drawings:
[0031] FIG. 1. Schematic for trafficking pathway for delivery of
peptide or protein drugs across tight epithelial and endothelial
barriers.
[0032] FIG. 2. Schematic of reporter peptide (amino acid sequence:
GSGYGRGSGK, SEQ ID NO: 1) fused to the glycoceramide GM1.
[0033] FIG. 3. Structure function studies on GM1 ceramide domain.
Non-native GM1 species with amplified transcytosis across
epithelial barriers in vitro were identified. Time course studies
and dose dependency of fusion molecules compared to peptide alone
are shown.
[0034] FIGS. 4A-4D. In vivo mouse studies. Absorption from
intestine into blood for GM-fusion molecules 15 and 30 minutes
after gastric gavage (FIG. 4A), and absorption from intestine into
liver for GM-fusion molecules 1 hour after gastric gavage (FIG. 4B)
are shown. Peptide alone was not absorbed. Time course for
absorption into blood was plotted (FIG. 4C). N=3 independent
studies. Schematic of the GM-fusion molecule is shown in FIG.
4D.
[0035] FIGS. 5A-5B. Transcytosis of the incretin hormone GLP-1 when
fused to the GM1 transport vehicles across model epithelial
barriers in vitro. GLP-1 incretin function is retained after fusion
to the oligosaccharide domain of GM1 (FIG. 5B) (half-log loss in
activity). When tested in vitro, GLP-1 was transported across
epithelial barriers by the short-chain GM1-species nearly 100-fold
above that observed for the peptide alone (FIG. 5A).
[0036] FIG. 6. In vivo intestinal absorption of GLP-1-GM1 fusion
molecules delivered by gastric gavage (oral) to mice corrected
serum glucose levels after glucose challenge (glucose tolerance
test) at about the same dose administered by intraperitoneal
injection (IP). N=6 independent experiments.
[0037] FIG. 7. Chemical synthesis of peptide onto ceramide and
glucosylceramide. The primary hydroxyl (circle) located on the
ceramide (or on sugar of Glc-Cer) are oxidized to form an aldehyde
catalyzed by copper bromide,
2,2,6,6-Tetramethyl-piperidin-1-yl)oxyl (TEMPO) and 2,2 bipyridine.
The reaction is enhanced in the presence of the base potassium
tert-butoxide (KOtBu). This allows for the reaction to aminooxy
containing peptides to form stable bonds (box).
[0038] FIG. 8. GM1 oligosaccharide domain structure and replacement
with approximated peptide-sugar linkers. The structure of the GM1
headgroup (Left) with the sialic acid site for coupling to cargo
molecules (blue line). Linker peptides are synthesized via
precursor building blocks of serine attached to different sugar
types. Three different linkers are designed to test the minimal
structure required.
[0039] FIG. 9. Optimization of a furin cleavage motif for
incorporation into the GM1-cargo linker peptide. A fluorescence
energy transfer (FRET) based assay was designed by appending paired
fluorophores at each end of the peptide to be tested. Cleavage of
the peptide by recombinant purified furin was measured as increase
in fluorescence (decrease in FRET quenching) in vitro, using buffer
alone as negative control (no cleavage) and high dose trypsin as
positive control (the furin motif can be cleaved by other serine
proteases). The motif termed FRET 7 was most efficient.
[0040] FIG. 10. Linkage to ceramide without sugars led to
transcytosis across epithelial barriers in vitro. Linkage to fatty
acid alone (dodecyl-C12) did not. Peptide alone did not cross the
epithelial barrier, as expected.
[0041] FIG. 11. Oxidation of C2-Ceramide. A method called
"Parikh-Doering" oxidation was used to oxidize the head group of
ceramide to a reactive aldehyde in organic solvent.
[0042] FIG. 12. Reaction of peptide containing aminooxy to the
aldehyde group. The "LC9" (D-isomer) linker peptide was coupled
using oxime-mediated reductive amination. LC9-sequence:
[alkyne]-[lys-bio]-GSGYGRGSG-[lys-aoa].
[0043] FIGS. 13A-13D. HPLC chromatograms of the products generated
in FIG. 12. (FIG. 13A) Control peptide. (FIG. 13B) Crude reaction
of ceramide to peptide. (FIG. 13C) After purification. (FIG. 13D)
Mass spectrometry analysis confirmed the presence of peptide linked
to ceramide (2+ and 3+ charged species).
[0044] FIG. 14. Copper click reaction of Alexa Fluor 488 to
ceramide-peptide conjugate.
[0045] FIG. 15. HPLC chromatograms of the ceramide-peptide
conjugated prepared in FIG. 14.
[0046] FIG. 16. Investigating the role of double bond positioning
& hydrocarbon chain length of the ceramide fatty acid in lipid
packing and subsequent differential endosomal sorting. GM1 isoforms
with identical oligosaccharide head groups but ceramides of
different endogenous structure, by systematically increasing length
and double bond position (02:0 to C26:1) were synthesized.
Partially adopted from Arumugam et al., Essays in Biochemistry
57(1): 109-119, incorporated herein by reference.
[0047] FIG. 17 Functionalization of GM1 species.
[0048] FIG. 18. Intracellular lipid trafficking--plasma membrane
depletion dynamics of GM1 shows transport to the lysosome for some
GM1 species.
[0049] FIG. 19. Live cell direct fluorescence images of different
GM1 species in sorting endosomes and sorting endosome tubules.
Table quantifies degree of entry into sorting tubules for the
different GM1 species.
[0050] FIG. 20. Intracellular lipid sorting-transcytosis and
retrograde trafficking. Shows how the position of the double bond
influences entry into endosome sorting tubules that traffic across
the cell by transcytosis or retrograde into the ER.
[0051] FIG. 21. Confocal fluorescence images of transcytosis in
MDCK polarized cells. Only the short chain GM1 C12:0 species were
transcytosed.
[0052] FIG. 22. Knockdown or inhibition of cellular machinery
involved in transcytosis by Dyngo-4a chemical inhibition (for
Dynamin, left panel), and gene knockdown by siRNA for Exocyst2 for
the exocyst complex (right panel).
[0053] FIG. 23. Immunostaining images of C6-GM1 peptide or peptide
alone transported across mouse nasal epithelium by 2-photon
microscopy (left) and analysis of blood by streptavidin pull-down
assay (right).
[0054] FIGS. 24A-24H. Ceramide analogs. (FIG. 24A)
N-acyloxyacyl-ornitine. (FIG. 24B) Cerilipin. (FIG. 24C) Brominated
mololipids. R1, R2=C14 to C20 fatty acid. (FIG. 24D) iso-3-hydroxy
heptadecanoic acid-containing lipid. (FIG. 24E) Lipstatin. (FIG.
24F) N-stearoyl proline. (FIG. 24G) Volicitin. (FIG. 24H) N-acyl
Taurine.
[0055] FIGS. 25A-25C. Confocal fluorescence microscopy images of
lysosomal transport of GM1-peptides. (FIG. 25A) A schematic of
endosomal sorting in human microvascular endothelial cells (HMECs).
(FIG. 25B) Images HMECs incubated either with C16:0-GM1-peptide
(top panels), or peptide alone (bottom panel). Cells were treated
with 2 .mu.M fluorescently green labeled compound for 1 hour for
continuous uptake on coverslips, and imaged in the presence of
Lysotracker-Red (red). The C16-GM1 lipid colocalized mostly to
lysosomes (yellow puncta), whereas the peptide alone did not enter
into cells. (FIG. 25C) C12-GM1 (green) was incubated for continuous
uptake for 1 hour with HMEC cells and colocalized to lysosomes
(yellow), in addition to plasma membrane localization, indicating
that this lipid can be sorted both the recycling and lysosomal
pathways. The merged image on the left is zoomed in and displayed
as Merged, green, and red channels on the right. Scale bars=10
.mu.m.
[0056] FIG. 26 shows the nasal bioavailability of a peptide fused
to GM1-6. Apparent bioavailability for peptide fused to GM1-C6:0
was 24.4% compared to the same molecule injected intraperitoneally.
Bioavailability of peptide alone was 1.3% compared to the for
peptide fused to GM1-C6:0 injected intraperitoneally.
[0057] FIG. 27 shows the structure of a panel of ceramides and
ceramide analogs (Diene-deoxy-Cer and Dihydro-Cer.
[0058] FIG. 28 shows transcytosis of Glc-Cer-C8 and Cer-C6 across
MDCKII cells. Efficiency of transcytosis is greater than for
peptides fused to GM1 or GM3 species
[0059] FIG. 29 shows transcytosis of Glc-Cer-C8 and Cer-C6 across
T84 intestinal cells. Efficiency of transcytosis is greater than
for peptides fused to GM1 or GM3 species
[0060] FIG. 30 shows transcytosis of peptides fused to
ceramide-like molecules. They are as efficient as the
ceramide-alone vehicles. FIG. also shows that low temperature
(4.degree. C.) blocks transport of the indicated ceramides or
ceramide analogs across the MDCKII cell layer, indicating that the
transportation is via transcytosis.
[0061] FIG. 31 shows that peptide fused to ceramide-C2, transports
across MDCK monolayer. Peptide fusion to a C12 fatty acid alone,
did not transport across MDCK cells by transcytosis.
[0062] FIGS. 32A-32C show that serum half-life is strongly
prolonged by fusion to GM1-C4:0. (FIG. 32A) Validation of the
fusion molecule in mice. (FIG. 38B) Serum levels of intravenously
administered GM1-peptide fusion or peptide alone 1 day after
administration. (FIG. 32C) Serum levels of intravenously
administered GM1-peptide fusion or peptide alone 1 day after
administration.
[0063] FIG. 33 shows that several ceramides or ceramide analogs can
transport across MDCK cells and the transport is via
transcytosis.
[0064] FIG. 34 shows the structures of a panel of
glycosphingolipids, ceramides or ceramide analogs to be conjugated
to cargos.
[0065] FIG. 35 shows ceramides, linker-ceramides, LC9-linker
ceramides, and AF488-LC9-linker ceramides that were tested.
[0066] FIG. 36 shows LC9 oximes.
[0067] FIG. 37 shows AF488-LC9 oximes.
[0068] FIG. 38 shows that ceramide C6 (Cer0C6) is absorbed via the
nose in mice 15 minutes after dosing.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0069] Delivery of biologically active molecules across tight
mucosal epithelial barriers is a major challenge preventing
application of most therapeutic peptides for oral drug delivery.
The sorting endosome sorts cargo into four separate pathways: the
lysosome pathway, the retrograde pathway to Golgi and ER, the
recycling pathway back to the plasma membrane, and (in polarized
cells like epithelial and endothelial cells) into the transcytotic
pathway that connects one cell surface with the other, allowing for
adsorption. The pathways are distinct and do not intersect with
each other (e.g., as described in Saslowsky et al., J Biol Chem.
Sep. 6; 288(36):25804-9, incorporated herein by reference). It is
possible that lipid sorting into these different pathways may be
regulated by the molecular shape of the lipid allowing them to move
into highly-curved membrane buds and tubules that serve the
recycling, retrograde and transcytotic pathways. Additionally,
there can be specific sorting into the different pathways via
distinct sorting mechanisms.
[0070] Glycosphingolipids are present within the outer membrane
leaflet of cell membranes. They contain a ligand-binding
oligosaccharide domain that faces the extracellular space, and a
ceramide domain that anchors the lipid in the membrane bilayer.
Ceramides consist of a sphingosine chain (typically C18:1 or C20:1)
coupled to a fatty acid that can have diverse structures. The
oligosaccharide domain prevents lipid flip-flop between membrane
leaflets, causing all the glycosphingolipids to be distributed
among intracellular compartments only by vesicular trafficking.
Sorting of proteins and certain sphingolipids to various
intracellular compartments of eukaryotic cells depends on movement
of membranes through the secretory and endocytic pathways by
vesicular carriers. For proteins, this occurs according to multiple
and hierarchically ordered sorting determinants structurally
encoded within the protein itself or within the structure of an
associated receptor or chaperone. Methods of using
glycosphingolipids isoforms containing a ceramide that comprises
fatty acids of different structures (e.g., different fatty acid
chain length, with or without double bonds) to deliver an agent
(e.g., a therapeutic agent) into a cell or across a mucosal barrier
have been described (e.g., in U.S. Pat. No. 9,457,097, incorporated
herein by reference).
[0071] Simplifying the lipid carrier may simplify their synthesis,
amplify their activity in transport of biologies, and promote their
clinical translation. It is known that ceramides alone (e.g.,
without the oligosaccharide group, or simply glycoceramides) can
flip flop from one membrane leaflet to another (e.g., as described
in Lopez-Montero et al., Biochim Biophys Acta. July; 1798(7):
1348-56, incorporated herein by reference). Provided herein, in
some aspects, are the use of ceramides (e.g., ceramides alone or
glycoceramides) for trafficking agents (e.g., therapeutic agents)
intracellularly or across epithelial or endothelial barriers.
[0072] Accordingly, some aspects of the present disclosure provide
delivery vehicles comprising a ceramide and an agent to be
delivered, wherein the ceramide: (a) does not comprise a fatty
acid; or (b) comprises a fatty acid of C1-C28; and wherein the
agent is attached to the ceramide. A "delivery vehicle" refers to a
molecule or system that delivers an agent (e.g., a therapeutic
agent) to a desired location, e.g., without limitation, to enter a
cell or to reach a desired cellular compartment (e.g., the
endoplasmic reticulum), to reach a desired part in a subject (e.g.,
an organ), or to reach a diseased site in a subject (e.g., a tumor
site). In some embodiments, the delivery vehicle includes the agent
to be delivered. In some embodiments, the delivery vehicle is
associated with (or attached to) the agent to be delivered. In
these situations, complexes comprising the delivery vehicle and the
agent to be delivered are formed and termed herein a
"ceramide-agent complex." In some embodiments, the agent is a
therapeutic agent and the complex comprising the delivery vehicle
and the therapeutic agent is herein termed a "ceramide-therapeutic
agent complex."
[0073] A "ceramide," as used herein, refers to a molecule
comprising a sphingosine core structure. A sphingosine is an amino
alcohol with an unsaturated hydrocarbon chain that is typically
18-carbon or 20-carbon in length, which forms a primary part of
sphingolipids (e.g., ceramides). The unsaturated hydrocarbon chain
is attached to the amino acid serine to form the sphingosine. The
term "ceramide" encompasses natural ceramides and ceramide analogs
(e.g., synthetic or natural ceramide analogs). In some embodiments,
the ceramide is a sphingolipid composed of sphingosine and a fatty
acid. In some embodiments, the ceramide of the present disclosure
is a ceramide analog. For example, the ceramide of the present
disclosure may contain additional chemical moieties appended to a
natural ceramide or contain modifications compared to a natural
ceramide. Non-limiting examples of ceramide analogs that may be
used in accordance with the present disclosure include:
2-hydroxy-ceramide, diene-deoxy-ceramide, dihydroceramide,
dihydroceramide phosphate, o-acyl-ceramide, ceramide phosphate,
sphinganine, and methyl-sphingosine.
[0074] In some embodiments, the ceramides of the present disclosure
encompass ceramide analogs with the core structure built upon amino
acids other than serine (i.e., having a core structure other than
sphingosine). For example, in some embodiments, the ceramide
described herein comprises amino acid backbones containing
ornithine (e.g., N-acyloxyacyl-ornitine, and bacterial cerilipin,
as described in Kawai et al., FEMS Immunol Med Microbiol 1999, 23,
67 and Tahara et al., Agric Biol Chem 1976, 40, 243, incorporated
herein by reference), tyrosine (e.g., Brominated mololipids from
sea sponge as described in Ross et al., J Nat Prod 2000, 63, 501,
incorporated herein by reference), glycine (e.g., iso-3-hydroxy
heptadecanoic acid-containing lipid from Cytophaga johnsonae, as
described in Kawazoe et al., J Bacteriol 1991, 173, 5470,
incorporated herein by reference), leucine (e.g., Lipstatin, which
is an inhibitor of pancreatic protease, as described in Weibel et
al., J Antibiot 1987, 1081, incorporated herein by reference),
proline (e.g., N-stearoyl praline as described in Sivasamy et al.,
JAOCS 2001, 78, 897, incorporated herein by reference), glutamine
(e.g., Volicitin, N-(17-hydroxylinolenoyl)-1-glutamine, as
described in Pare et al., PNAS 1998, 95, 13971, incorporated herein
by reference) or taurine (e.g., N-acyl taurine, as described in
Saghatelian et al., Biochemistry 2006, 45, 9007, incorporated
herein by reference). Non-limiting, exemplary structures of these
ceramide analogs are provided in FIGS. 24A-24G.
[0075] A "fatty acid" is a carboxylic acid with an aliphatic chain,
which is either saturated or unsaturated. Fatty acids that have
double bonds between backbone carbons are known as unsaturated.
Fatty acids without double bonds between backbone carbons are known
as saturated. The length of a fatty acid chain, is herein referred
to using the number of backbone carbons atoms in the fatty acid
chain. For example, a fatty acid chain with X number of backbone
carbons is expressed as CX herein, wherein X is an integer. In some
embodiments, X is 0, meaning the ceramide does not comprise a fatty
acid. A ceramide that does not have a fatty acid is a sphingosine
(also referred to as a "lyso-ceramide" herein). When a sphingosine
is referred to herein, it also means the molecule does not have a
sugar moiety. In some embodiments, X is an integer between 1-30
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In some
embodiments, the ceramide of the present disclosure comprises a
fatty acid of C1-C28 (e.g., C1, C2, C3, C4, C5, C6, C1, C8, C9,
C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22,
C23, C24, C25, C26, C27, or C28) in length. The number of double
bonds between backbone carbons in a fatty acid chain with X number
of backbone carbons is expressed as CX:Y, wherein Y is the number
of double bonds between backbone carbons and is an integer. For
example, a fatty acid chain with 20 backbone carbons and 1 double
bond is expressed as "C20:1" herein. In some embodiments, Y is 0,
meaning the fatty acid does not contain a double bond between
backbone carbons (i.e., a saturated fatty acid). The ceramide chain
of the present disclosure, in some embodiments, contains one or
more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) double bonds
between backbone carbons. In some embodiments, the double bond is a
cis-double bond. A "cis-double bond" refers to an isoform of a
double bond formed between two carbon atoms. In addition to the
double bond, other chemical groups (e.g., --H, --CH3, --COOH) also
form bonds with the carbon atom involved in the cis-double bond,
and "cis" indicates that the chemical groups other than --H are on
the same side of the carbon chain. One skilled in the art is
familiar with these terms.
[0076] In some embodiments, the ceramide comprises a fatty acid of
C1-C12 (e.g., for transcytosis applications). In some embodiments,
the ceramide comprises a fatty acid of 03-C28 (e.g., for
non-transcytosis applications).
[0077] In some embodiments, the ceramide comprises a fatty acid of
C1-C6 (e.g., C1, C2, C3, C4, C5, or C6) in length. In some
embodiments, the fatty acid of C1-C6 has no double bond between two
carbon atoms (e.g., any of the backbone carbons). In some
embodiments, the ceramide comprises a fatty acid chain of C4. In
some embodiments, the ceramide comprises a fatty acid chain of C6.
In some embodiments, the fatty acid of C1-C6 comprises at least one
cis-double bond (e.g., 1, 2, or more cis double bond).
[0078] In some embodiments, the ceramide comprises a fatty acid of
C7-C28 (e.g., C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17,
C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, or C28) in
length. In some embodiments, the ceramide comprises a fatty acid
chain of C8. In some embodiments, the fatty acid of C7-C28
comprises at least one cis-double bonds between two carbon atoms
(e.g., any of the backbone carbons). For example, the fatty acid of
C7-C28 may comprise 1-10 cis-double bonds. In some embodiments, the
fatty acid of C7-C28 comprises 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4,
1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8,
3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8,
5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-10, or 9-10
cis-double bonds. In some embodiments, the fatty acid of C7-C28
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cis-double bonds.
In some embodiments, the fatty acid is C7-C16 and comprises at
least one cis-double bonds. In some embodiments, the fatty acid is
C17-C28 and comprises at least one cis-double bonds in C1-C18
region of the fatty acid. For example, the at least one cis-double
bonds may be in C1-C18, C1-C17, C1-C16, C1-C15, C1-C14, C1-C13,
C1-C12, C1-C11, C1-C10, C1-C9, C1-C8, C1-C7, C1-C6, C1-C5, C1-C4,
C1-C3, C1-C2, C2-C18, C2-C17, C2-C16, C2-C15, C2-C14, C2-C13,
C2-C12, C2-C11, C2-C10, C2-C9, C2-C8, C2-C7, C2-C6, C2-C5, C2-C4,
C2-C3, C3-C18, C3-C17, C3-C16, C3-C15, C3-C14, C3-C13, C3-C12,
C3-C11, C3-C10, C3-C9, C3-C8, C3-C7, C3-C6, C3-C5, C3-C4, C4-C18,
C4-C17, C4-C16, C4-C15, C4-C14, C4-C13, C4-C12, C4-C11, C4-C10,
C4-C9, C4-C8, C4-C7, C4-C6, C4-C5, C5-C18, C5-C17, C5-C16, C5-C15,
C5-C14, C5-C13, C5-C12, C5-C11, C5-C10, C5-C9, C5-C8, C5-C7, C5-C6,
C6-C18, C6-C17, C6-C16, C6-C15, C6-C14, C6-C13, C6-C12, C6-C11,
C6-C10, C6-C9, C6-C8, C6-C7, C7-C18, C7-C17, C7-C16, C7-C15,
C7-C14, C7-C13, C7-C12, C7-C11, C7-C10, C7-C9, C7-C8, C8-C18,
C8-C17, C8-C16, C8-C15, C8-C14, C8-C13, C8-C12, C8-C11, C8-C10,
C8-C9, C9-C18, C9-C17, C9-C16, C9-C15, C9-C14, C9-C13, C9-C12,
C9-C11, C9-C10, C10-C18, C10-C17, C10-C16, C10-C15, C10-C14,
C10-C13, C10-C12, C10-C11, C11-C18, C11-C17, C11-C16, C11-C15,
C11-C14, C11-C13, C11-C12, C12-C18, C12-C17, C12-C16, C12-C15,
C12-C14, C12-C13, C13-C18, C13-C17, C13-C16, C13-C15, C13-C14,
C14-C18, C14-C17, C14-C16, C14-C15, C15-C18, C15-C17, C15-C16,
C16-C18, C16-C17, or C17-C18 region of the fatty acid. In some
embodiments, the fatty acid is C17-C28 and the at least one
cis-double bonds are in C1-C16 region of the fatty acid. In some
embodiments, the fatty acid is C17-C28 and the at least one
cis-double bonds are in C1-C14 region of the fatty acid. In some
embodiments, the fatty acid of C7-C28 has no double bond between
two carbon atoms (e.g., any of the backbone carbons).
[0079] In some embodiments, the fatty acid of C7-C28 comprises one
or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemical
moieties (e.g., bulky chemical moieties) appended (e.g., covalently
attached) to the fatty acid chain. Non-limiting examples of
chemical moieties that may be appended to the C7-C28 fatty acid
chain of the ceramide described herein include: branched
methylation or acylation, bulky non-polar fluorophores such as
BODIPY, aromatic rings, sterols, prenylation, halogenation (e.g.,
fluorination), and any compound that deviates from the linear
structure of a fully saturated hydrocarbon chain. In some
embodiments, the fatty acid is C7-C16 and comprises at least one
chemical moieties (e.g., bulky chemical moieties) appended to the
fatty acid chain. In some embodiments, the fatty acid is C17-C28
and comprises at least one chemical moieties (e.g., bulky chemical
moieties) appended to the fatty acid chain in C1-C18 region of the
fatty acid. For example, the at least one chemical moieties (e.g.,
bulky chemical moieties) may be appended in C1-C18, C1-C17, C1-C16,
C1-C15, C1-C14, C1-C13, C1-C12, C1-C11, C1-C10, C1-C9, C1-C8,
C1-C7, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C18, C2-C17, C2-C16,
C2-C15, C2-C14, C2-C13, C2-C12, C2-C11, C2-C10, C2-C9, C2-C8,
C2-C7, C2-C6, C2-C5, C2-C4, C2-C3, C3-C18, C3-C17, C3-C16, C3-C15,
C3-C14, C3-C13, C3-C12, C3-C11, C3-C10, C3-C9, C3-C8, C3-C7, C3-C6,
C3-C5, C3-C4, C4-C18, C4-C17, C4-C16, C4-C15, C4-C14, C4-C13,
C4-C12, C4-C11, C4-C10, C4-C9, C4-C8, C4-C7, C4-C6, C4-C5, C5-C18,
C5-C17, C5-C16, C5-C15, C5-C14, C5-C13, C5-C12, C5-C11, C5-C10,
C5-C9, C5-C8, C5-C7, C5-C6, C6-C18, C6-C17, C6-C16, C6-C15, C6-C14,
C6-C13, C6-C12, C6-C11, C6-C10, C6-C9, C6-C8, C6-C7, C7-C18,
C7-C17, C7-C16, C7-C15, C7-C14, C7-C13, C7-C12, C7-C11, C7-C10,
C7-C9, C7-C8, C8-C18, C8-C17, C8-C16, C8-C15, C8-C14, C8-C13,
C8-C12, C8-C11, C8-C10, C8-C9, C9-C18, C9-C17, C9-C16, C9-C15,
C9-C14, C9-C13, C9-C12, C9-C11, C9-C10, C10-C18, C10-C17, C10-C16,
C10-C15, C10-C14, C10-C13, C10-C12, C10-C11, C11-C18, C11-C17,
C11-C16, C11-C15, C11-C14, C11-C13, C11-C12, C12-C18, C12-C17,
C12-C16, C12-C15, C12-C14, C12-C13, C13-C18, C13-C17, C13-C16,
C13-C15, C13-C14, C14-C18, C14-C17, C14-C16, C14-C15, C15-C18,
C15-C17, C15-C16, C16-C18, C16-C17, or C17-C18 region of the fatty
acid. In some embodiments, the fatty acid is C17-C28 and the at
least one chemical moieties (e.g., bulky chemical moieties) are
appended in C1-C16 region of the fatty acid. In some embodiments,
the fatty acid is C17-C28 and the at least one chemical moieties
(e.g., bulky chemical moieties) are appended in C1-C14 region of
the fatty acid.
[0080] In some embodiments, any of the ceramide of the present
disclosure (e.g., a ceramide with or without a fatty acid chain)
further comprises a sugar (e.g., a glycoceramide). In some
embodiments, the ceramide comprises a fatty acid chain of C1-C6 and
further comprises a sugar. In some embodiments, the ceramide
comprises a fatty acid chain of C7-C28 and further comprises a
sugar. In some embodiments, the ceramide does not comprise a fatty
acid chain (i.e., a sphingosine or a lyso-ceramide) and further
comprises a sugar. A ceramide that does not contain a fatty acid
chain but comprises a sugar is also referred to herein as a
"glycosphingosine" or a "lyso-glycoceramide."
[0081] A "glycoceramide" refers to a ceramide comprising a sugar
attached to its primary hydroxyl group (FIG. 7). In some
embodiments, the sugar is a simple sugar. A "simple sugar" is a
monosaccharide made up of single sugar molecules. Non-limiting
examples of simple sugars include: glucose, fructose, and
galactose. Thus, in some embodiments, the ceramide of the present
disclosure is a glucose-ceramide (Glc-Cer), a fructose ceramide, or
a galactose ceramide. In some embodiments, the sugar is an
oligosaccharide. In some embodiments, the ceramide is a
glycoceramide and the agent to be delivered is attached to the
sugar of the glycoceramide.
[0082] In some embodiments, the ceramide of the present disclosure
does not comprise a sugar. In some embodiments, the agent is
attached to the ceramide (e.g., to the primary hydroxyl group of
the ceramide, see FIG. 7).
[0083] The agent may be attached to the ceramide by any methods
known in the art. In some embodiments, the agent is attached
non-covalently, e.g., without limitation, by van der Waals forces,
hydrophobic interaction, hydrogen bond interaction, or ionic
interactions.
[0084] In some embodiments, the agent is attached covalently. For
example, in some embodiments, the ceramide (e.g., the primary
hydroxyl group of the ceramide) or the sugar of the glycoceramide
may be functionalized with a reactive chemical group. One example
of such reactive group is a "click chemistry handle." Click
chemistry is a chemical approach introduced describes chemistry
tailored to generate substances quickly and reliably by joining
small units together. See, e.g., Kolb, Finn and Sharpless
Angewandte Chemie International Edition (2001) 40: 2004-2021;
Evans, Australian Journal of Chemistry (2007) 60: 384-395).
Exemplary coupling reactions (some of which may be classified as
"Click chemistry") include, but are not limited to, formation of
esters, thioesters, amides (e.g., such as peptide coupling) from
activated acids or acyl halides; nucleophilic displacement
reactions (e.g., such as nucleophilic displacement of a halide or
ring opening of strained ring systems); azide-alkyne Huisgon
cycloaddition; thiol-yne addition; imine formation; and Michael
additions (e.g., maleimide addition). Non-limiting examples of a
click chemistry handle include an azide handle, an alkyne handle,
or an aziridine handle. Azide is the anion with the formula N3-. It
is the conjugate base of hydrazoic acid (HN3). N3- is a linear
anion that is isoelectronic with CO2, NCO--, N2O, NO2+ and NCF.
Azide can be described by several resonance structures, an
important one being --N.dbd.N+=N--. An alkyne is an unsaturated
hydrocarbon containing at least one carbon-carbon triple bond. The
simplest acyclic alkynes with only one triple bond and no other
functional groups form a homologous series with the general
chemical formula CnH2n-2. Alkynes are traditionally known as
acetylenes, although the name acetylene also refers specifically to
C2H2, known formally as ethyne using IUPAC nomenclature. Like other
hydrocarbons, alkynes are generally hydrophobic but tend to be more
reactive. Aziridines are organic compounds containing the aziridine
functional group, a three-membered heterocycle with one amine group
(--NH--) and two methylene bridges (.about.CH2-). The parent
compound is aziridine (or ethylene imine), with molecular formula
C2H5N.
[0085] Other non-limiting, exemplary reactive groups include:
acetals, ketals, hemiacetals, and hemiketals, carboxylic acids,
strong non-oxidizing acids, strong oxidizing acids, weak acids,
acrylates and acrylic acids, acyl halides, sulfonyl halides,
chloroformates, alcohols and polyols, aldehydes, alkynes with or
without acetylenic hydrogen amides and imides, amines, aromatic,
amines, phosphines, pyridines, anhydrides, aryl halides, azo,
diazo, azido, hydrazine, and azide compounds, strong bases, weak
bases, carbamates, carbonate salts, chlorosilanes, conjugated
dienes, cyanides, inorganic, diazonium salts, epoxides, esters,
sulfate esters, phosphate esters, thiophosphate esters borate
esters, ethers, soluble fluoride salts, fluorinated organic
compounds, halogenated organic compounds, halogenating agents,
aliphatic saturated hydrocarbons, aliphatic unsaturated
hydrocarbons, hydrocarbons, aromatic, insufficient information for
classification, isocyanates and isothiocyanates, ketones, metal
hydrides, metal alkyls, metal aryls, and silanes, alkali metals,
nitrate and nitrite compounds, inorganic, nitrides, phosphides,
carbides, and silicides, nitriles, nitro, nitroso, nitrate, nitrite
compounds, organic, non-redox-active inorganic compounds,
organometallics, oximes, peroxides, organic, phenolic salts,
phenols and cresols, polymerizable compounds, quaternary ammonium
and phosphonium salts, strong reducing agents, weak reducing
agents, acidic salts, basic salts, siloxanes, inorganic sulfides,
organic sulfides, sulfite and thiosulfate salts, sulfonates,
phosphonates, organic thiophosphonates, thiocarbamate esters and
salts, and dithiocarbamate esters and salts. The agent to be
attached to the ceramide (e.g., via the reactive chemical group)
may contain a corresponding chemical group that reacts with the
oligosaccharide or ceramide, thus resulting in covalent attachment.
For example, an agent that is a protein or polypeptide can be
coupled via its N- or C-terminus, or via an endogenous residue
(e.g., lysine) by chemical cross-linking.
[0086] In some embodiments, the agent is attached to the ceramide
via a linker. In some embodiments, the linker is a
pseudo-glycopeptide linker (e.g., see FIG. 8). A
"pseudo-glycopeptide linker" refers to linkers comprising at least
one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) sugar
(carbohydrate) covalently attached an amino acid backbone (the
peptide) via side chains of the amino acids in the backbone.
Non-limiting examples of sugars that may be attached to the amino
acid backbone include: fructose, glucose, galactose, sialic acid,
and N-Acetylgalactosamine (GalNAc). In some embodiments, the sugar
in the pseudo-glycopeptide linker is attached to the amino acid
backbone via the side chain of a serine, threonine, or asparagine.
For pseudo-glycopeptides that contain more than one sugars, the
sugars may be attached to the amino acid backbone via the same or
different amino acid side chain. In some embodiments, the amino
acid backbone of the pseudo-glycopeptide linker comprises 1-30
amino acids. For example, the amino acid backbone of the
pseudo-glycopeptide linker may comprise 1-30, 1-25, 1-20, 1-15,
1-1-, 1-5, 5-10, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20,
10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 amino acids. In
some embodiments, the amino acid backbone of the
pseudo-glycopeptide linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30 amino acids. In some embodiments, the amino acid
backbone of the pseudo-glycopeptide linker comprises 1-10 amino
acids. For example, the amino acid backbone of the
pseudo-glycopeptide linker may comprise 1-10, 1-9, 1-8, 1-7, 1-6,
1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10,
3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10,
5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10,
8-10, or 9-10 amino acids. In some embodiments, the amino acid
backbone of the pseudo-glycopeptide linker comprises 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 amino acids. In some embodiments, the amino acid
backbone of the pseudo-glycopeptide linker comprises more than 10
amino acids (e.g., 11-30 amino acids). For example, the amino acid
backbone of the pseudo-glycopeptide linker may comprise 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
30 amino acids.
[0087] In some embodiments, the linker is a peptide linker. In some
embodiments, the agent is a protein or a peptide. In such
instances, one or more of the amino acids of the protein or peptide
may be modified to include a chemical entity such as a carbohydrate
group, a hydroxyl group, a phosphate group, a farnesyl group, an
isofarnesyl group, a fatty acid group, a linker for attaching to
the oligosaccharide.
[0088] In some embodiments, the linker is a cleavable linker. A
"cleavable linker" refers to a linker that can be cleaved by a
chemical agent or an enzyme and thus release the agent from the
ceramide carrier. In some embodiments, the cleavable linker
comprises an ester linkage (e.g., the linker can be a peptide
linker comprising an ester linker). In some embodiments, the ester
linkage can be cleaved by an esterase (e.g., leukocyte esterase
whose level is elevated at sites of inflammation, or
carboxylesterase hCE-2 that is specific to gastrointestinal
endosomes).
[0089] In some embodiments, the cleavable linker comprises a
cleavage motif for an endosomal protease. An "endosomal protease"
refers to a protease that is present in endosomes. It is herein
interchangeably referred to as a "lysosomal protease." Endosomal
proteases belong to the aspartic, cysteine, or serine proteinase
families of hydrolytic enzymes. Endosomal proteases are expressed
ubiquitously, and in a tissue- or cell type-specific manner and are
usually detected within all vesicles of the endocytic pathway.
Reference and classification of endosomal proteases is available in
the art. For example, lists of known endosomal proteases can be
found in the MERGES database (merops.sanger.ac.uk). In some
embodiments, the endosomal protease is furin or matriptase. Furin
is a calcium-dependent serine endoprotease that can efficiently
cleave precursor proteins at their paired basic amino acid
processing sites. Furin cleaves proteins downstream of a basic
amino acid target sequence (canonically, Arg-X-(Arg/Lys)-Arg').
Matriptase is a trypsin-like integral-membrane serine peptidase and
cleaves substrates with Arg or Lys at the P1 position and prefers
small side-chain amino acids, such as Ala and Gly, at the P2
position.
[0090] In some embodiments, the cleavable linker comprises a
disulfide linkage. A "disulfide linkage" is also referred to as a
disulfide bond or S--S bond, which is a covalent bond derived from
two thiol groups. Disulfide bonds can be formed by oxidation of
sulfhydryl groups and can be cleaved via reduction (e.g., via using
reductants such as tris(2-carboxyethyl)phosphine (TCEP),
2-Mercaptoethanol (.beta.-ME) or dithiothreitol (DTT)).
[0091] The ceramides described herein are able to act as delivery
vehicles to deliver an agent across cell membrane or across mucosal
barrier and direct intracellular trafficking of the agent. For
example, in some embodiments, the ceramide-agent complex are
directed by the ceramide to a desired intracellular location, e.g.,
the endoplasmic reticulum (ER). In some embodiments, the
ceramide-agent complex is directed by the ceramide away from
degradative pathways (e.g., lysosome). As such, in some
embodiments, the cellular half-life of the agent is prolonged when
the agent is part of the ceramide-agent complex, compared to when
the agent is delivered into cells alone. In some embodiments, the
cellular half-life of the agent is prolonged by at least 20% when
the agent is part of the ceramide-agent complex, compared to when
the agent is delivered into cells alone. In some embodiments, the
cellular half-life of the agent is prolonged by at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 100%, at least 2 fold, at
least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold,
at least 40 fold, at least 50 fold, at least 60 fold, at least 70
fold, at least 80 fold, at least 90 fold, at least 100 fold, at
least 500 fold, at least 1000 fold or more, when the agent is part
of the ceramide-agent complex, compared to when the agent is
delivered into cells alone. In some embodiments, the cellular
half-life of the agent is prolonged by 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 2 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40
fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 500
fold, 1000 fold or more, when the agent is part of the
ceramide-agent complex, compared to when the agent is delivered
into cells alone.
[0092] In some embodiments, the agent is delivered across an
epithelial or endothelial barrier (e.g., a mucosal barrier) by
transcytosis, when the agent is attached to the ceramide to form a
ceramide-agent complex. Mucosal barrier is composed of compact
epithelial cell lining (e.g., in the stomach or in the intestines).
The intestinal mucosal barrier, also referred to as intestinal
barrier, refers to the property of the intestinal mucosa that
ensures adequate containment of undesirable luminal contents within
the intestine while preserving the ability to absorb nutrients. The
gastric mucosal barrier is the property of the stomach that allows
it to safely contain the gastric acid required for digestion.
[0093] In some embodiments, the agent is not able to cross an
epithelial or endothelial barrier (e.g., a mucosal barrier) alone
and is able to cross mucosal barriers in complex with the ceramides
described herein. In some embodiments, the delivery of the agent
across an epithelial or endothelial barrier (e.g., a mucosal
barrier) is enhanced (e.g., by at least 20%) when the agent is in
complex with the ceramides described herein, compared to when the
agent is delivered alone. For example, the delivery of the agent
across an epithelial or endothelial barrier (e.g., a mucosal
barrier) is enhanced by at least 20%, at least 30%, at least 40%,
at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 100%, at least 2 fold, at least 5 fold, at least 10
fold, at least 20 fold, at least 30 fold, at least 40 fold, at
least 50 fold, at least 60 fold, at least 70 fold, at least 80
fold, at least 90 fold, at least 100 fold, at least 500 fold, at
least 1000 fold or more, when the agent is in complex with the
ceramides described herein, compared to when the agent is delivered
alone. In some embodiments, the delivery of the agent across an
epithelial or endothelial barrier (e.g., a mucosal barrier) is
enhanced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 fold, 5
fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70
fold, 80 fold, 90 fold, 100 fold, 500 fold, 1000 fold or more, when
the agent is in complex with the ceramides described herein,
compared to when the agent is delivered alone.
[0094] Other aspects of the present disclosure provide agents that
may be delivered by the ceramides described herein. The agent may
be any bioactive agent or therapeutic agent. A "therapeutic agent"
refers to an agent that has therapeutic effects to a disease or
disorder. The complex between the ceramide and the therapeutic
agent is referred to herein as the "ceramide-therapeutic agent
complex." A therapeutic agent may be, without limitation, proteins,
peptides, nucleic acids, small molecules drugs, polysaccharides and
carbohydrates, lipids, glycoproteins, small molecules, synthetic
organic and inorganic drugs exerting a biological effect when
administered to a subject, and combinations thereof. In some
embodiments, the therapeutic agent is an anti-inflammatory agent, a
vaccine antigen, a vaccine adjuvant, an antibody, a ScFv, a
nanobody, and enzyme, an anti-cancer drug or chemotherapeutic drug,
a clotting factor, a hormone, a steroid, a cytokine, an antibiotic,
or a drug for the treatment of cardiovascular disease, an
infectious disease, an autoimmune disease, allergy, a blood
disorder, a metabolic disorder, a skin disease, an eye disease, a
lysosomal storage disease, or a neurological disease. In some
embodiments, the therapeutic agent is a protein or a peptide. In
some embodiments, the protein or peptide is glucagon-like peptide-1
(GLP-1), or a functional fragment thereof. In some embodiments, the
protein or peptide is Exendin-4, or a functional fragment
thereof.
[0095] "Glucagon-like peptide-1 (GLP-1)" is a 30 amino acid long
peptide hormone deriving from the tissue-specific posttranslational
processing of the proglucagon gene. It is produced and secreted by
intestinal enteroendocrine L-cells and certain neurons within the
nucleus of the solitary tract in the brainstem upon food
consumption. The initial product GLP-1(1-37) is susceptible to
amidation and proteolytic cleavage which gives rise to the two
truncated and equipotent biologically active forms, GLP-1
(7-36)amide and GLP-1(7-37). Active GLP-1 composes two
.alpha.-helices from amino acid position 13-20 and 24-35 separated
by a linker region. GLP-1 possesses several physiological
properties that make it (and its functional analogs) a subject of
intensive investigation as a potential treatment of diabetes
mellitus. Further, GLP-1 is has the ability to decrease blood sugar
levels in a glucose-dependent manner by enhancing the secretion of
insulin. Thus, GLP-1 has been associated with numerous regulatory
and protective effects. GLP-1-based treatment has been associated
with weight loss and lower hypoglycemia risks, two very important
aspects of a life with diabetes.
[0096] "Exendin-4" is a peptide agonist of the glucagon-like
peptide (GLP) receptor that promotes insulin secretion. Exendin-4
binds to the intact human Glucagon-like peptide-1 receptor (GLP-1R)
in a similar way to GLP-1 and bears a 50% amino acid homology to
GLP-1. Exendin-4 facilitates glucose control via augmentation of
pancreas response (i.e. increases insulin secretion) in response to
eating meals, suppressing pancreatic release of glucagon in
response to eating, reducing rate of gastric emptying, suppressing
appetite, and reducing liver fat content. In some embodiments, the
therapeutic agent is a fusion protein comprise GLP-1 or a
functional fragment thereof and Exendin-4 or a functional fragment
thereof.
[0097] In some embodiments, the therapeutic agent is a vaccine
antigen. A "vaccine antigen" is a molecule or moiety that, when
administered to a subject, activates or increases the production of
antibodies that specifically bind the antigen. In some embodiments,
an antigen is a protein or a polysaccharide. Antigens of pathogens
are well known to those of skill in the art and include, but are
not limited to parts (coats, capsules, cell walls, flagella,
fimbriae, and toxins) of bacteria, viruses, and other
microorganisms. A vaccine typically comprises an antigen, and is
intentionally administered to a subject to induce an immune
response in the recipient subject. The antigen may be from a
pathogenic virus, bacteria, or fungi.
[0098] Examples of pathogenic virus include, without limitation:
Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1
(also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and
other isolates, such as HIV-LP; Picornaviridae (e.g., polio
viruses, hepatitis A virus; enteroviruses, human coxsackie viruses,
rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause
gastroenteritis); Togaviridae (e.g., equine encephalitis viruses,
rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis
viruses, yellow fever viruses); Coronaviridae (e.g.,
coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,
rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae
(e.g., parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus); Orthomyxoviridae (e.g., influenza
viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,
phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever
viruses); Reoviridae (e.g., reoviruses, orbiviurses and
rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);
Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses,
polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV), herpes viruses'); Poxviridae (variola
viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g.,
African swine fever virus); and unclassified viruses (e.g., the
etiological agents of Spongiform encephalopathies, the agent of
delta hepatitis (thought to be a defective satellite of hepatitis B
virus), the agents of non-A, non-B hepatitis (class 1=internally
transmitted; class 2=parenterally transmitted (i.e., Hepatitis C);
Norwalk and related viruses, and astroviruses).
[0099] Examples of pathogenic bacteria include, without limitation:
Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,
Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M.
intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis, Listeria
monocytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic spp.), Streptococcus pneumoniae,
pathogenic Campylobacter sp., Enterococcus sp., Haemophilus
influenzae, Bacillus anthracis, Corynebacterium diphtheriae,
Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium
perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella
pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponema pallidum,
Treponema pertenue, Leptospira, and Actinomyces israelii.
[0100] Examples of pathogenic fungi include, without limitation:
Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans. Other infectious organisms (i.e., protists) include:
Plasmodium falciparum and Toxoplasma gondii.
[0101] In some embodiments, the therapeutic agent is an agent that
induces immunological tolerance. Immunologic tolerance is a state
of immune unresponsiveness specific to a particular antigen or set
of antigens induced by previous exposure to that antigen or set. In
some embodiments, the immunologic tolerance is oral tolerance. Oral
tolerance is the state of local and systemic immune
unresponsiveness that is induced by oral administration of
innocuous antigen such as food proteins. In some embodiments, the
therapeutic agent is an agent for induce immunological tolerance
for the treatment of allergy or autoimmune disease (e.g., multiple
sclerosis).
[0102] Other non-limiting examples of agents that may be delivered
using the ceramides described herein are provided.
[0103] Non-limiting, exemplary chemopharmaceutically compositions
that may be used in the liposome drug delivery systems of the
present disclosure include, Actinomycin, All-trans retinoic acid,
Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin,
Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide,
Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin,
Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine,
Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Mechlorethamine,
Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin,
Paclitaxel, Pemetrexed, Tempo side, Tioguanine, Topotecan,
Valrubicin, Vinblastine, Vincristine, Vindesine, and
Vinorelbine.
[0104] Examples of antineoplastic compounds include, without
limitation: nitrosoureas, e.g., carmustine, lomustine, semustine,
strepzotocin; Methylhydrazines, e.g., procarbazine, dacarbazine;
steroid hormones, e.g., glucocorticoids, estrogens, progestins,
androgens, tetrahydrodesoxycaricosterone, cytokines and growth
factors; Asparaginase.
[0105] Examples of immunoactive compounds include, without
limitation:: immunosuppressives, e.g., pyrimethamine,
trimethopterin, penicillamine, cyclosporine, azathioprine;
immunostimulants, e.g., levamisole, diethyl dithiocarbamate,
enkephalins, endorphins.
[0106] Examples of antimicrobial compounds include, without
limitation: antibiotics, e.g., beta lactam, penicillin,
cephalosporins, carbapenims and monobactams, beta-lactamase
inhibitors, aminoglycosides, macrolides, tetracyclins,
spectinomycin; Antimalarials, Amebicides, Antiprotazoal,
Antifungals, e.g., amphotericin beta or clotrimazole, antiviral,
e.g., acyclovir, idoxuridine, ribavirin, trifluridine, vidarbine,
gancyclovir.
[0107] Examples of parasiticides include, without limitation:
antihalmintics, Radiopharmaceutics, gastrointestinal drugs.
[0108] Examples of hematologic compounds include, without
limitation: immunoglobulins; blood clotting proteins; e.g.,
antihemophilic factor, factor IX complex; anticoagulants, e.g.,
dicumarol, heparin Na; fibrolysin inhibitors, tranexamic acid.
[0109] Examples of cardiovascular drugs include, without
limitation: peripheral antiadrenergic drugs, centrally acting
antihypertensive drugs, e.g., methyldopa, methyldopa HCl;
antihypertensive direct vasodilators, e.g., diazoxide, hydralazine
HCl; drugs affecting renin-angiotensin system; peripheral
vasodilators, phentolamine; antianginal drugs; cardiac glycosides;
inodilators; e.g., amrinone, milrinone, enoximone, fenoximone,
imazodan, sulmazole; antidysrhythmic; calcium entry blockers; drugs
affecting blood lipids; ranitidine, bosentan, rezulin.
[0110] Examples of respiratory drugs include, without limitation:
sypathomimetic drugs: albuterol, bitolterol mesylate, dobutamine
HCl, dopamine HCl, ephedrine SO, epinephrine, fenfluramine HCl,
isoproterenol HCl, methoxamine HCl, norepinephrine bitartrate,
phenylephrine HCl, ritodrine HCl; cholinomimetic drugs, e.g.,
acetylcholine Cl; anticholinesterases, e.g., edrophonium Cl;
cholinesterase reactivators; adrenergic blocking drugs, e.g.,
acebutolol HCl, atenolol, esmolol HCl, labetalol HCl, metoprolol,
nadolol, phentolamine mesylate, propanolol HCl; antimuscarinic
drugs, e.g., anisotropine methylbromide, atropine SO4, clinidium
Br, glycopyrrolate, ipratropium Br, scopolamine HBr.
[0111] Examples of neuromuscular blocking drugs include, without
limitation: depolarizing, e.g., atracurium besylate, hexafluorenium
Br, metocurine iodide, succinylcholine Cl, tubocurarine Cl,
vecuronium Br; centrally acting muscle relaxants, e.g.,
baclofen.
[0112] Examples of neurotransmitters and neurotransmitter agents
include, without limiation: acetylcholine, adenosine, adenosine
triphosphate, amino acid neurotransmitters, e.g., excitatory amino
acids, GABA, glycine; biogenic amine neurotransmitters, e.g.,
dopamine, epinephrine, histamine, norepinephrine, octopamine,
serotonin, tyramine; neuropeptides, nitric oxide, K+ channel
toxins,
[0113] Examples of antiparkinson drugs include, without limiation:
amaltidine HCl, benztropine mesylate, e.g., carbidopa.
[0114] Examples of diuretic drugs include, without limitation:
dichlorphenamide, methazolamide, bendroflumethiazide,
polythiazide.
[0115] Examples of uterine, antimigraine drugs include, without
limitation: carboprost tromethamine, mesylate, methysergide
maleate.
[0116] Examples of hormones include, without limitation: pituitary
hormones, e.g., chorionic gonadotropin, cosyntropin, menotropins,
somatotropin, iorticotropin, protirelin, thyrotropin, vasopressin,
lypressin; adrenal hormones, e.g., beclomethasone dipropionate,
betamethasone, dexamethasone, triamcinolone; pancreatic hormones,
e.g., glucagon, insulin; parathyroid hormone, e.g.,
dihydrochysterol; thyroid hormones, e.g., calcitonin etidronate
disodium, levothyroxine Na, liothyronine Na, liotrix,
thyroglobulin, teriparatide acetate; antithyroid drugs; estrogenic
hormones; progestins and antagonists, hormonal contraceptives,
testicular hormones; gastrointestinal hormones: cholecystokinin,
enteroglycan, galanin, gastric inhibitory polypeptide, epidermal
growth factor-urogastrone, gastric inhibitory polypeptide,
gastrin-releasing peptide, gastrins, pentagastrin, tetragastrin,
motilin, peptide YY, secretin, vasoactive intestinal peptide,
sincalide.
[0117] Examples of enzymes include, without limitation: lysosomal
storage enzymes, hyaluronidase, streptokinase, tissue plasminogen
activator, urokinase, PGE-adenosine deaminase, oxidoreductases,
transferases, polymerases, hydrolases, lyases, synthases,
isomerases, and ligases, digestive enzymes (e.g., proteases,
lipases, carbohydrases, and nucleases). In some embodiments, the
enzyme is selected from the group consisting of lactase,
beta-galactosidase, a pancreatic enzyme, an oil-degrading enzyme,
mucinase, cellulase, isomaltase, alginase, digestive lipases (e.g.,
lingual lipase, pancreatic lipase, phospholipase), amylases,
cellulases, lysozyme, proteases (e.g., pepsin, trypsin,
chymotrypsin, carboxypeptidase, elastase), esterases (e.g. sterol
esterase), disaccharidases (e.g., sucrase, lactase,
beta-galactosidase, maltase, isomaltase), DNases, and RNases.
[0118] Examples of intravenous anesthetics include, without
limitation: droperidol, etomidate, fetanyl citrate/droperidol,
hexobarbital, ketamine HCl, methohexital Na, thiamylal Na,
thiopental Na.
[0119] Examples of antiepileptics include, without limitation,
carbamazepine, clonazepam, divalproex Na, ethosuximide,
mephenytoin, paramethadione, phenytoin, primidone.
[0120] Examples of peptides and proteins that may be used as
therapeutic agents include, without limiation: ankyrins, arrestins,
bacterial membrane proteins, clathrin, connexins, dystrophin,
endothelin receptor, spectrin, selectin, cytokines; chemokines;
growth factors, insulin, erythropoietin (EPO), tumor necrosis
factor (TNF), neuropeptides, neuropeptide Y, neurotensin,
transforming growth factor alpha, transforming growth factor beta,
interferon (IFN), and hormones, growth inhibitors, e.g., genistein,
steroids etc; glycoproteins, e.g., ABC transporters, platelet
glycoproteins, GPIb-IX complex, GPIIb-IIIa complex, vitronectin,
thrombomodulin, CD4, CD55, CD58, CD59, CD44, lymphocye
function-associated antigen, intercellular adhesion molecule,
vascular cell adhesion molecule, Thy-1, antiporters, CA-15-3
antigen, fibronectins, laminin, myelin-associated glycoprotein,
GAP, GAP-43, Exendin-4, and GLP-1.
[0121] Examples of cytokines and cytokine receptors include,
without limitation: interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16, IL-17, IL-18, IL-1 receptor, IL-2 receptor, IL-3 receptor,
IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8
receptor, IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12
receptor, IL-13 receptor, IL-14 receptor, IL-15 receptor, IL-16
receptor, IL-17 receptor, IL-18 receptor, lymphokine inhibitory
factor, macrophage colony stimulating factor, platelet derived
growth factor, stem cell factor, tumor growth factor beta, tumor
necrosis factor, lymphotoxin, Fas, granulocyte colony stimulating
factor, granulocyte macrophage colony stimulating factor,
interferon-alpha, interferon-beta, interferon-gamma.
[0122] Examples of growth factors and protein hormones include,
without limitation: erythropoietin, angiogenin, hepatocyte growth
factor, fibroblast growth factor, keratinocyte growth factor, nerve
growth factor, tumor growth factor-alpha, thrombopoietin, thyroid
stimulating factor, thyroid releasing hormone, neurotrophin,
epidermal growth factor, VEGF, ciliary neurotrophic factor, LDL,
somatomedin, insulin growth factor, insulin-like growth factor I
and II.
[0123] Examples of chemokines include, without limitation: ENA-78,
EEC, GRO-alpha, GRO-beta, GRO-gamma, HRG, LIE, IP-10, MCP-1, MCP-2,
MCP-3, MCP-4, MIP-1 alpha, MIP-1beta, MIG, MDC, NT-3, NT-4, SCF,
LIE, leptin, RANTES, lymphotactin, eotaxin-1, eotaxin-2, TARC,
TECK, WAP-1, WAP-2, GCP-1, GCP-2; alpha-chemokine receptors: CXCR1,
CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7; beta-chemokine receptors:
CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7.
[0124] In some embodiments, antibodies that may be delivered using
the delivery vehicle described herein target antigens including,
without limitation: (a) anti-cluster of differentiation antigen
CD-1 through CD-166 and the ligands or counter receptors for these
molecules; (b) anti-cytokine antibodies, e.g., anti-IL-1 through
anti-IL-18 and the receptors for these molecules; (c) anti-immune
receptor antibodies, antibodies against T cell receptors, major
histocompatibility complexes I and II, B cell receptors, selectin
killer inhibitory receptors, killer activating receptors, OX-40,
MadCAM-1, Gly-CAM1, integrins, cadherens, sialoadherens, Fas,
CTLA-4, Fc.gamma.-receptors, Fcalpha-receptors,
Fc.epsilon.-receptors, Fc.mu.-receptors, and their ligands; (d)
anti-metalloproteinase antibodies, e.g., collagenase, MMP-1 through
MMP-8, TIMP-1, TIMP-2; anti-cell lysis/proinflammatory molecules,
e.g., perforin, complement components, prostanoids, nitron oxide,
thromboxanes; and (e) anti-adhesion molecules, e.g.,
carcioembryonic antigens, lamins, fibronectins.
[0125] Non-limiting, exemplary antibodies and fragments thereof
include: bevacizumab (AVASTIN.RTM.), trastuzumab (HERCEPTIN.RTM.),
alemtuzumab (CAMPATH.RTM., indicated for B cell chronic lymphocytic
leukemia), gemtuzumab (MYLOTARG.RTM., hP67.6, anti-CD33, indicated
for leukemia such as acute myeloid leukemia), rituximab
(RITUXAN.RTM.), tositumomab (BEXXAR.RTM., anti-CD20, indicated for
B cell malignancy), MDX-210 (bispecific antibody that binds
simultaneously to HER-2/neu oncogene protein product and type I Fc
receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab
(OVAREX.RTM., indicated for ovarian cancer), edrecolomab
(PANOREX.RTM.), daclizumab (ZENAPAX.RTM.), palivizumab
(SYNAGIS.RTM., indicated for respiratory conditions such as RSV
infection), ibritumomab tiuxetan (ZEVALIN.RTM., indicated for
Non-Hodgkin's lymphoma), cetuximah (ERBITUX.RTM.), MDX-447, MDX-22,
MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CD1), IOR EGF/R3,
celogovab (ONCOSCINT.RTM. OV103), epratuzumab (LYMPHOCIDE.RTM.),
pemtumomab (THERAGYN.RTM.) and Gliomab-H (indicated for brain
cancer, melanoma). Other antibodies and antibody fragments are
contemplated and may be used in accordance with the disclosure. In
some embodiments, the therapeutic agent is a nanobody. A "nanobody"
is a therapeutic protein based on single-domain antibody fragments
that contain the unique structural and functional properties of
naturally-occurring heavy chain only antibodies. In some
embodiments, the nanobody is anti-inflammatory. In some
embodiments, the nanobody is against pathogenic agents (e.g.,
anthrax).
[0126] In some embodiments, the therapeutic agent is a ligand for a
cell receptor (e.g., without limitation, a growth factor receptor,
a G-protein coupled receptor, or a toll-like receptor).
[0127] A regulatory protein may be, in some embodiments, a
transcription factor or a immunoregulatory protein. Non-limiting,
exemplary transcriptional factors include: those of the NFkB
family, such as Rel-A, c-Rel, Rel-B, p50 and p52; those of the AP-1
family, such as Fos, FosB, Fra-1, Fra-2, Jun, JunB and JunD; ATF;
CREB; STAT-1, -2, -3, -4, -5 and -6; NFAT-1, -2 and -4; MAE;
Thyroid Factor; IRE; Oct-1 and -2; NF-Y; Egr-1; and USF-43, EGR1,
Sp1, and E2F1.
[0128] Examples of antiviral agents include, without limitation:
reverse transcriptase inhibitors and nucleoside analogs, e.g. ddI,
ddC, 3TC, ddA, AZT; protease inhibitors, e.g., Invirase, ABT-538;
inhibitors of in RNA processing, e.g., ribavirin.
[0129] Other non-limiting examples of known therapeutics which may
be delivered by coupling to a ceramide described herein
include:
[0130] (a) Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil,
Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime, VePesid,
Paraplatin, Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS,
Estrace, Glucophage (Bristol-Myers Squibb);
[0131] (b) Ceclor, Lorabid, Dynabac, Prozac, Darvon, Permax,
Zyprexa, Humalog, Axid, Gemzar, Evista (Eli Lily);
[0132] (c) Vasotec/Vaseretic, Mevacor, Zocor, Prinivil/Prinizide,
Plendil, Cozaar/Hyzaar, Pepcid, Prilosec, Primaxin, Noroxin,
Recombivax HB, Varivax, Timoptic/XE, Trusopt, Proscar, Fosamax,
Sinemet, Crixivan, Propecia, Vioxx, Singulair, Maxalt, Ivermectin
(Merck & Co.);
[0133] (d) Diflucan, Unasyn, Sulperazon, Zithromax, Trovan,
Procardia XL, Cardura, Norvasc, Dofetilide, Feldene, Zoloft,
Zeldox, Glucotrol XL, Zyrtec, Eletriptan, Viagra, Droloxifene,
Aricept, Lipitor (Pfizer);
[0134] (e) Vantin, Rescriptor, Vistide, Genotropin,
Micronase/Glyn./Glyb., Fragmin, Total Medrol, Xanax/alprazolam,
Sermion, Halcion/triazolam, Freedox, Dostinex, Edronax, Mirapex,
Pharmorubicin, Adriamycin, Camptosar, Remisar, Depo-Provera,
Caverject, Detrusitol, Estring, Healon, Xalatan, Rogaine (Pharmacia
& Upjohn);
[0135] (f) Lopid, Accrupil, Dilantin, Cognex, Neurontin, Loestrin,
Dilzem, Fempatch, Estrostep, Rezulin, Lipitor, Omnicef, FemHRT,
Suramin, Clinafloxacin (Warner Lambert).
[0136] Non-limiting examples of therapeutic agents for eye diseases
include: Anti-infective drugs (e.g., Aciclovir, Chloramphenicol,
Ciprofloxacin, Gentamicin, Neomycin, Polymyxin B);
Anti-inflammatory drugs (e.g., Betamethasone, Dexamethasone,
Emedastine, Nedocromil sodium, Prednisolone, Sodium cromoglicate);
Artificial tears (e.g., Carmellose, Hydroxyethylcellulose,
Hypromellose, Polyvinyl alcohol); and Mydriatics (e.g., Atropine,
cyclopentolate, Phenylephrine).
[0137] Further non-limiting examples of therapeutic agents which
may be delivered by the ceramide-therapeutic agent complex of the
present invention may be found in: Goodman and Gilman's The
Pharmacological Basis of Therapeutics. 9th ed. McGraw-Hill 1996,
incorporated herein by reference.
[0138] The delivery vehicle comprising a ceramide and an agent to
be delivered, or a ceramide-agent complex (e.g., a
ceramide-therapeutic agent) complex may be formulated into
pharmaceutical compositions. In some embodiments, the
pharmaceutical composition further comprises a pharmaceutically
acceptable carrier. "Pharmaceutically acceptable" refers to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk ratio.
A "pharmaceutically acceptable carrier" may be a pharmaceutically
acceptable material, composition or vehicle, such as a liquid or
solid filler, diluent, excipient, solvent or encapsulating
material, involved in carrying or transporting the subject agents
from one organ, or portion of the body, to another organ, or
portion of the body. Each carrier must be "acceptable" in the sense
of being compatible with the other ingredients of the formulation
and not injurious to the tissue of the patient (e.g.,
physiologically compatible, sterile, physiologic pH, etc.). The
term "carrier" denotes an organic or inorganic ingredient, natural
or synthetic, with which the active ingredient is combined to
facilitate the application. The components of the pharmaceutical
compositions also are capable of being co-mingled with the
molecules of the present disclosure, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy. Some examples of
materials which can serve as pharmaceutically-acceptable carriers
include: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and
its derivatives, such as sodium carboxymethyl cellulose,
methylcellulose, ethyl cellulose, microcrystalline cellulose and
cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin;
(7) lubricating agents, such as magnesium stearate, sodium lauryl
sulfate and talc; (8) excipients, such as cocoa butter and
suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
(10) glycols, such as propylene glycol; (11) polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)
esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)
buffering agents, such as magnesium hydroxide and aluminum
hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)
isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)
pH buffered solutions; (21) polyesters, polycarbonates and/or
polyanhydrides; (22) bulking agents, such as polypeptides and amino
acids (23) serum component, such as serum albumin, HDL and LDL;
(22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic
compatible substances employed in pharmaceutical formulations.
Wetting agents, coloring agents, release agents, coating agents,
sweetening agents, flavoring agents, perfuming agents, preservative
and antioxidants can also be present in the formulation.
[0139] The pharmaceutical compositions may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well-known in the art of pharmacy. The term "unit dose"
when used in reference to a pharmaceutical composition of the
present disclosure refers to physically discrete units suitable as
unitary dosage for the subject, each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
diluent; i.e., carrier, or vehicle.
[0140] The formulation of the pharmaceutical composition may
dependent upon the route of administration. Injectable preparations
suitable for parenteral administration or intratumoral,
peritumoral, intralesional or perilesional administration include,
for example, sterile injectable aqueous or oleaginous suspensions
and may be formulated according to the known art using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution,
suspension or emulsion in a nontoxic parenterally acceptable
diluent or solvent, for example, as a solution in 1,3 propanediol
or 1,3 butanediol. Among the acceptable vehicles and solvents that
may be employed are water, Ringer's solution, U.S.P. and isotonic
sodium chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic
mono- or di-glycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables. The injectable
formulations can be sterilized, for example, by filtration through
a bacterial-retaining filter, or by incorporating sterilizing
agents in the form of sterile solid compositions which can be
dissolved or dispersed in sterile water or other sterile injectable
medium prior to use.
[0141] Compositions suitable for oral administration may be
presented as discrete units, such as capsules, tablets, lozenges,
each containing a predetermined amount of the anti-inflammatory
agent. Other compositions include suspensions in aqueous liquids or
non-aqueous liquids such as a syrup, elixir or an emulsion.
[0142] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the anti-inflammatory agent,
increasing convenience to the subject and the physician. Many types
of release delivery systems are available and known to those of
ordinary skill in the art. They include polymer base systems such
as poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono- di- and tri-glycerides;
hydrogel release systems; sylastic systems; peptide based systems;
wax coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the anti-inflammatory agent is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in
which an active component permeates at a controlled rate from a
polymer such as described in U.S. Pat. Nos. 3,832,253, and
3,854,480. In addition, pump-based hardware delivery systems can be
used, some of which are adapted for implantation.
[0143] Use of a long-term sustained release implant may be
particularly suitable for treatment of chronic conditions.
Long-term release, are used herein, means that the implant is
constructed and arranged to delivery therapeutic levels of the
active ingredient for at least 30 days, and preferably 60 days.
Long-term sustained release implants are well-known to those of
ordinary skill in the art and include some of the release systems
described above.
[0144] In some embodiments, the pharmaceutical compositions used
for therapeutic administration must be sterile. Sterility is
readily accomplished by filtration through sterile filtration
membranes (e.g., 0.2 micron membranes). Alternatively,
preservatives can be used to prevent the growth or action of
microorganisms. Various preservatives are well known and include,
for example, phenol and ascorbic acid. The pharmaceutical
composition ordinarily will be stored in lyophilized form or as an
aqueous solution if it is highly stable to thermal and oxidative
denaturation. The pH of the preparations typically will be about
from 6 to 8, although higher or lower pH values can also be
appropriate in certain instances.
[0145] Other aspects of the present disclosure provide methods of
delivering an agent (e.g., a therapeutic agent) into a cell or
across a mucosal surface or an endothelial barrier, the method
comprising contacting the delivery vehicle, the ceramide-agent
complex (e.g., the ceramide-therapeutic agent complex), or the
pharmaceutical composition comprising the delivery vehicle or the
ceramide-agent complex (e.g., the ceramide-therapeutic agent
complex) with the cell, the mucosal surface, or the endothelial
lumenal surface, under conditions appropriate for uptake of the
delivery vehicle or the agent into the cell or absorption of the
delivery vehicle or the agent across the mucosal surface or the
endothelial barrier (e.g., via transcytosis). In some embodiments,
the delivery vehicle, the ceramide-agent complex, or the
pharmaceutical composition comprising the delivery vehicle or the
ceramide-agent complex (e.g., the ceramide-therapeutic agent
complex) are administered to a subject.
[0146] Methods of delivering agents (e.g., therapeutic agents) to
different organs via intravenous infusion of the delivery vehicle
or the ceramide-agent complex are also provided. Such organs may
be, for example, without limitation, skeleton, joints, muscles,
tendons, various types of glands, esophagus, stomach, small
intestine, large intestine, liver, pancreas, pharynx, larynx,
trachea, bronchi, lungs, diaphragm, kidneys, ureters, bladder,
urethra, ovaries, uterus, prostate, heart, lymph nodes, bone
marrow, thymus, spleen, brain, and spinal cord. In some
embodiments, the delivery vehicle or the ceramide-agent complex is
delivered across an endothelial barrier (e.g., the endothelial
barrier in the heart or brain) when it reaches the organ after
intravenous infusion. In some embodiments, the intravenously
infused delivery vehicle or the ceramide-agent complex are
delivered to different organs and are sorted into intracellular
compartments such as the lysosome (e.g., for lysosomal replacement
therapies) or ER (e.g., for addressing protein folding problems).
Delivery to liver after intravenous infusion of the delivery
vehicle or the ceramide-agent complex greatly enhances (e.g., by at
least 20%) the delivery of the agent to the liver, compared to
delivering the agent alone.
[0147] Methods of enhancing the half-life of an agent in a subject
are provided, the method comprising administering to the subject
the delivery vehicle, the ceramide-therapeutic agent complex, or
the composition described herein.
[0148] Methods of treating a disease or condition in a subject in
need thereof are provided, the method comprising administering to
the subject an effective amount of the delivery vehicle, the
ceramide-therapeutic agent complex, or the composition described
herein, wherein the effective amount is an amount sufficient to
ameliorate/reduce the extent to which the disease or condition
occurs in the subject. The disease may be any disease that can be
treated by the agents described herein. In some embodiments, the
disease is infection, allergy, autoimmune diseases (e.g., multiple
sclerosis), liver diseases, lung diseases, neurological diseases,
eye diseases or cancer.
[0149] An "effective amount" is the amount necessary or sufficient
to have a desired effect in a subject. The effective amount will
vary with the particular condition being treated, the age and
physical condition of the subject being treated, the severity of
the condition, the duration of the treatment, the nature of the
concurrent therapy (if any), the specific route of administration
and other factors within the knowledge and expertise of the health
care practitioner. For example, an effective amount could be that
amount necessary to eliminate a tumor, cancer, or bacterial, viral
or fungal infection. This amount will vary from individual to
individual and can be determined empirically using known methods by
one of ordinary skill in the art.
[0150] The delivery vehicle, the ceramide-agent complex, or the
pharmaceutical composition comprising the delivery vehicle or the
ceramide-agent complex (e.g., the ceramide-therapeutic agent
complex) may be administered by any route. Routes of administration
include enteral routes, such as oral and any other means by which
the gastrointestinal tract is involved, and parenteral routes, such
as by injection (subcutaneous, intravenous, intramuscular
injection) or infusion (typically by intravenous route). In some
embodiments, the administration is done intravenously,
intramuscularly, intradermally, subcutaneously, intrathecally,
intraperitoneally, intraarterially, intracardiacally,
intraosseously, intraocularly, intravitreally, intrapleurally,
intranasally, or injection into the joint. The injection can be in
a bolus or a continuous infusion. In some embodiments, the
administration is done nonparenterally (e.g., done orally,
sublingually, topically, rectally, via inhalation, nasally, as eye
drops, as eye patches, to the cervix, or to the skin). For delivery
across tight endothelial barriers, in some embodiments, the
ceramide-therapeutic agent complex is delivered intravenously,
intramuscularly, or subcutaneously.
[0151] Methods of treating a disease or disorder are also provided.
The delivery vehicle, the ceramide-agent complex, or the
pharmaceutical composition comprising the delivery vehicle or the
ceramide-agent complex (e.g., the ceramide-therapeutic agent
complex) may be administered to a subject who has, has had or is
susceptible to developing one or more conditions/diseases that
require or would benefit from treatment. For example, the
compositions described herein may be used to treat, prevent or
ameliorate immune system deficiencies, infectious diseases (viral,
fungal, bacterial or parasitic), autoimmune diseases, diabetes,
blood disorders, cancers, metabolic disorders, allergies,
inflammatory bowel disease and skin disorders. In addition,
gangliosides attached to antigen can be administered to stimulate a
subject's response to a vaccine. The antigen is selected from the
group consisting of: an antigen that is characteristic of a
pathogen, an antigen that is characteristic of an autoimmune
disease, an antigen that is characteristic of an allergen and an
antigen that is characteristic of a tumor. In some embodiments, the
disease or disorder to be treated is diabetes. In some embodiments,
the disease or disorder is infection, e.g., by a pathogenic virus,
bacteria, or fungi. In some embodiments, the disease or disorder is
cancer.
[0152] Immune system deficiencies include any disease or disorder
in which a subject's immune system is not functioning normally or
in which it would be useful to boost the subject's immune response,
for example to eliminate a tumor or cancer (e.g. tumors of the
brain, lung (e.g. small cell and non-small cell), ovary, breast,
prostate, colon, as well as other carcinomas and sarcomas) or an
infection in a subject.
[0153] Examples of autoimmune diseases include, without limitation:
Addison's disease, diabetes mellitus (type 1), Graves' disease,
interstitial cystitis, lupus erythematous, multiple sclerosis and
Hashimoto's thyroiditis. Allergic conditions include eczema,
allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria
(hives) and food allergies, and other atopic conditions.
[0154] Non-limiting, exemplary cancers include: neoplasms,
malignant tumors, metastases, or any disease or disorder
characterized by uncontrolled cell growth such that it would be
considered cancerous. The cancer may be a primary or metastatic
cancer. Cancers include, but are not limited to, adult and
pediatric acute lymphoblastic leukemia, acute myeloid leukemia,
adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer
of the appendix, astrocytoma, basal cell carcinoma, bile duct
cancer, bladder cancer, bone cancer, biliary tract cancer,
osteosarcoma, fibrous histiocytoma, brain cancer, brain stem
glioma, cerebellar astrocytoma, malignant glioma, glioblastoma,
ependymoma, medulloblastoma, supratentorial primitive
neuroectodermal tumors, hypothalamic glioma, breast cancer, male
breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoid
tumor, carcinoma of unknown origin, central nervous system
lymphoma, cerebellar astrocytoma, malignant glioma, cervical
cancer, childhood cancers, chronic lymphocytic leukemia, chronic
myelogenous leukemia, acute lymphocytic and myelogenous leukemia,
chronic myeloproliferative disorders, colorectal cancer, cutaneous
T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer,
Ewing family tumors, extracranial germ cell tumor, extragonadal
germ cell tumor, extrahepatic bile duct cancer, intraocular
melanoma, retinoblastoma, gallbladder cancer, gastric cancer,
gastrointestinal stromal tumor, extracranial germ cell tumor,
extragonadal germ cell tumor, ovarian germ cell tumor, gestational
trophoblastic tumor, glioma, hairy cell leukemia, head and neck
cancer, hepatocellular cancer, Hodgkin lymphoma, non-Hodgkin
lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway
glioma, intraocular melanoma, islet cell tumors, Kaposi sarcoma,
kidney cancer, renal cell cancer, laryngeal cancer, lip and oral
cavity cancer, small cell lung cancer, non-small cell lung cancer,
primary central nervous system lymphoma, Waldenstrom
macroglobulinema, malignant fibrous histiocytoma, medulloblastoma,
melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous
neck cancer, multiple endocrine neoplasia syndrome, multiple
myeloma, mycosis fungoides, myelodysplastic syndromes,
myeloproliferative disorders, chronic myeloproliferative disorders,
nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,
neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic
cancer, parathyroid cancer, penile cancer, pharyngeal cancer,
pheochromocytoma, pineoblastoma and supratentorial primitive
neuroectodermal tumors, pituitary cancer, plasma cell neoplasms,
pleuropulmonary blastoma, prostate cancer, rectal cancer,
rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma,
uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small
intestine cancer, squamous cell carcinoma, squamous neck cancer,
supratentorial primitive neuroectodermal tumors, testicular cancer,
throat cancer, thymoma and thymic carcinoma, thyroid cancer,
transitional cell cancer, trophoblastic tumors, urethral cancer,
uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
choriocarcinoma, hematological neoplasm, adult T-cell leukemia,
lymphoma, lymphocytic lymphoma, stromal tumors and germ cell
tumors, or Wilms tumor. In some embodiments, the cancer is lung
cancer, breast cancer, prostate cancer, colorectal cancer, gastric
cancer, liver cancer, pancreatic cancer, brain and central nervous
system cancer, skin cancer, ovarian cancer, leukemia, endometrial
cancer, bone, cartilage and soft tissue sarcoma, lymphoma,
neuroblastoma, nephroblastoma, retinoblastoma, or gonadal germ cell
tumor.
[0155] As used herein, the term "treating" refers to the
application or administration of the delivery vehicle, the
ceramide-therapeutic agent complex, or the composition comprising
such to a subject in need thereof. "A subject in need thereof",
refers to an individual who has a disease, a symptom of the
disease, or a predisposition toward the disease, with the purpose
to cure, heal, alleviate, relieve, alter, remedy, ameliorate,
improve, or affect the disease, the symptom of the disease, or the
predisposition toward the disease.
[0156] A "subject" to which administration is contemplated refers
to a human (i.e., male or female of any age group, e.g., pediatric
subject (e.g., infant, child, or adolescent) or adult subject
(e.g., young adult, middle-aged adult, or senior adult)) or
non-human animal. In some embodiments, the non-human animal is a
mammal (e.g., rodent (e.g., mouse or rat), primate (e.g.,
cynomolgus monkey or rhesus monkey), commercially relevant mammal
(e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird
(e.g., commercially relevant bird, such as chicken, duck, goose, or
turkey)). The non-human animal may be a male or female at any stage
of development. The non-human animal may be a transgenic animal or
genetically engineered animal.
[0157] In some embodiments, the subject is a companion animal (a
pet). "A companion animal," as used herein, refers to pets and
other domestic animals. Non-limiting examples of companion animals
include dogs and cats; livestock such as horses, cattle, pigs,
sheep, goats, and chickens; and other animals such as mice, rats,
guinea pigs, and hamsters. In some embodiments, the subject is a
research animal. Non-limiting examples of research animals include:
rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or
non-human primates.
[0158] In some embodiments, a "subject in need thereof" refers to a
subject that needs treatment of a disease described herein.
[0159] Alleviating a disease includes delaying the development or
progression of the disease, or reducing disease severity.
Alleviating the disease does not necessarily require curative
results. As used therein, "delaying" the development of a disease
means to defer, hinder, slow, retard, stabilize, and/or postpone
progression of the disease. This delay can be of varying lengths of
time, depending on the history of the disease and/or individuals
being treated. A method that "delays" or alleviates the development
of a disease, or delays the onset of the disease, is a method that
reduces probability of developing one or more symptoms of the
disease in a given time frame and/or reduces extent of the symptoms
in a given time frame, when compared to not using the method. Such
comparisons are typically based on clinical studies, using a number
of subjects sufficient to give a statistically significant
result.
[0160] "Development" or "progression" of a disease means initial
manifestations and/or ensuing progression of the disease.
Development of the disease can be detectable and assessed using
standard clinical techniques as well known in the art. However,
development also refers to progression that may be undetectable.
For purpose of this disclosure, development or progression refers
to the biological course of the symptoms. "Development" includes
occurrence, recurrence, and onset. As used herein "onset" or
"occurrence" of a disease includes initial onset and/or
recurrence.
[0161] Conventional methods, known to those of ordinary skill in
the art of medicine, can be used to administer the isolated
polypeptide or pharmaceutical composition to the subject, depending
upon the type of disease to be treated or the site of the disease.
This composition can also be administered via other conventional
routes, e.g., administered orally, parenterally, by inhalation
spray, topically, rectally, nasally, buccally, vaginally or via an
implanted reservoir. The term "parenteral" as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intraarticular, intraarterial, intrasynovial, intrasternal,
intrathecal, intralesional, and intracranial injection or infusion
techniques. In addition, it can be administered to the subject via
injectable depot routes of administration such as using 1-, 3-, or
6-month depot injectable or biodegradable materials and
methods.
[0162] Some of the embodiments, advantages, features, and uses of
the technology disclosed herein will be more fully understood from
the Examples below. The Examples are intended to illustrate some of
the benefits of the present disclosure and to describe particular
embodiments, but are not intended to exemplify the full scope of
the disclosure and, accordingly, do not limit the scope of the
disclosure.
EXAMPLES
Example 1. Test if the Oligosaccharide Domain of GM1 is Essential
for Intracellular Trafficking and Cargo Transport
[0163] Mucosal surfaces represent vast areas where host tissues are
separated from the environment only by a delicate but highly
effective single layer of columnar epithelial cells, joined by
tight junctions that are impermeable to proteins and even small
peptides. Proteins non-specific ally taken up directly into the
epithelial cell by endocytosis are generally transported to
lysosomes for degradation. So far, the lack of rational and
efficient methods to circumvent this barrier has prevented the
application of most therapeutic proteins and peptides for mucosal
drug delivery.
[0164] Endothelial cells also form vast and highly restrictive
single cell thick barriers that separate most tissues from the
blood stream. Except for the vasculature in the intestine,
glomerulus, and liver, the healthy non-inflamed endothelial barrier
strongly limits the permeability of large molecules; thus
preventing access of many protein-based biologies to cells of many
tissues--even when the therapeutic proteins are applied
intravenously.
[0165] The pathway for large solutes (e.g., protein and peptide
biologies) to cross simple epithelial and endothelial barriers with
highly resistant intercellular tight junctions is by
transcytosis--a process of transcellular membrane trafficking that
connects one surface of polarized cells with the other (FIG. 1). It
has been described that the GM1 glycosphingolipid crosses
epithelial barriers by transcytosis and the structure of its
ceramide domain dictates transport through this pathway. GM1
species containing non-native "short chain" fatty acids in the
ceramide domain was shown to allow for more efficient transport
across simple epithelial barriers, and some can release from the
cell membrane back into solution after transcytosis. It was also
found that the "short-chain" GM1 glycosphingolipids can carry
therapeutic peptides (GLP-1) across the intestinal epithelial
barrier to deliver them systemically. These in vivo studies show
absorption far surpassing the best currently achieved for oral
delivery of biologies.
[0166] It was found in non-polarized cells that GM1-ceramides
containing "kinked" cis-unsaturated C18:1 or C16:1 fatty acids, or
non-native "short chain" C12:0 to C4:0 fatty acids, enter the
sorting/recycling endosome of epithelial cells for transport to
various intracellular destinations: including the recycling pathway
and retrograde pathway to Golgi and ER. These lipids do not traffic
into the late endosome-lysosome pathway. In contrast, the
GM1-sphingolipids with long saturated fatty acid chains (C16:0 or
longer) sort efficiently into late endosomes and lysosomes (FIG.
19). The results are consistent with models for lipid sorting by
molecular shape and membrane domains.
[0167] In polarized epithelial cells, another sorting event emerges
from the sorting endosome and leads into the transcytotic pathway.
This pathway allows for the short- or unsaturated GM1-ceramides to
cross the cell to the opposite cell surface, thus breeching the
epithelial barrier by transcytosis (FIG. 1). It has been found that
GM1 glycolipids can carry the 80 kDa protein cholera toxin fully
across polarized monolayers of intestinal epithelial cells in
culture, implicating a function for this pathway in protein
absorption across the intestine in vivo.
[0168] Based on these results, the idea that the glycosphingolipids
may be harnessed as trafficking vehicles for biologic drug delivery
was tested. A all D-isomer amino acid reporter-peptide carrying a
reactive aminooxy C-terminal residue for coupling to sialic acid of
the glycoceramide GM1, a biotin group for rapid and high affinity
isolation, and an alkyne group for coupling to cargo (e.g., for
detection via Alexa Fluor 488) was developed (FIG. 2).
[0169] Structure-function studies showed that certain novel
short-chain ceramide species allowed for transcellular transport
(transcytosis) of the pep tide-GM1 fusion molecule across
epithelial monolayers in vitro as assessed biochemically (FIG. 3)
and morphologically (FIG. 21). Studies using chemical inhibitors
and genetic depletion of genes required for transcytosis showed
that the pep tide-GM1 fusion molecules cross the epithelial
monolayers by passing through the cell by transcytosis, and not
around the cell by paracellular "leak" (FIG. 22).
[0170] When applied orally (by gastric gavage) to mice in vivo, the
pep tide-GM1 fusion molecules were absorbed across the intestine
into the circulation as assessed in blood and in liver tissue
(FIGS. 4A-4D). Uncoupled peptides, however, are not absorbed. The
absorption of the pep tide-GM1 fusion molecules in the nasal
epithelium was observed, as assessed biochemically in blood, and
morphologically by imaging (FIG. 23). Translational studies were
carried out using the incretin hormone GLP-1 as cargo. Human GLP1
is a cleavage product of pro glucagon produced in pancreatic or
intestinal enteroendocrine cells. Native GLP-1 possesses several
physiological properties on glucose metabolism that make it an
effective treatment for diabetes mellitus type II. There also are
receptors for GLP-1 in the brain, and evidence that the molecule
may operate in appetite control, suggesting other clinical
applications. In this case, transport across the epithelial barrier
is almost 100-fold above the GLP-1 peptide alone. Fusion of the
peptide hormone to the lipid carrier causes a very modest loss of
hormone function as assessed by dose response in bioassay shown in
panel to right (fusion molecule in RED is about a half-log less
potent but still functional in picomolar range). It was found that
GLP-1 incretin function is retained after fusion to the
oligosaccharide domain of GM1 (FIG. 5A) (half-log loss in
activity). When tested in vitro, GLP-1 was transported across
epithelial barriers by the short-chain GM1-species nearly 100-fold
above that observed for the peptide alone (FIG. 5B).
[0171] Consistent with the in vivo studies using the reporter-pep
tide-GM1 fusion molecules, it was found that when applied to mice
via gastric gavage, GLP-1-GM1 fusion molecules were absorbed and
had effects over glucose metabolism, evidenced by reduced time to
normalize blood sugar in glucose tolerance tests (FIG. 6). An assay
was recently developed to confirm biochemically that the GLP-1-GM1
fusion molecules are absorbed into the blood (not shown). The GLP-1
peptide alone is not absorbed and has no detectable effect on
glucose metabolism.
[0172] Another structural feature of GM1 that might play a role in
trafficking is the extracellular oligosaccharide head group.
Further described herein are studies designed to test if the
oligosaccharide domain of GM1 is essential for intracellular
trafficking and cargo transport. Simplifying the lipid carrier may
be important to clinical translation. It is tested herein if a
simple glycoceramide easily harvested from buttermilk can be used
in place of GM1, and if the oligosaccharide domain of GM1 can be
eliminated completely, thus defining a simplified core delivery
molecule that can be fully and pragmatically synthesized.
[0173] Simplifying the glycoceramide structure would have two major
significances. First, this would allow for total chemical synthesis
of the molecules and permit easier translation to the clinic.
Second, structural elements of the oligosaccharide head group that
affect trafficking may be identified, enhancing the understanding
of the structure-activity relationship for the glycosphingolipids,
and aiding in design of new peptide linkers that recapture the lost
functionalities.
[0174] Some functionality of the sugar head groups are anticipated.
At minimum, the oligosaccharide domain of GM1 is expected to
function by trapping the ceramide in the outer membrane leaflet,
preventing flip-flop and rendering the molecule dependent on
membrane trafficking for distribution within and across cells, or
by shaping the molecule. Such functions, however, might be
reproduced simply by attachment of peptide or protein cargo to the
ceramide domain. Still, the oligosaccharide head group of GM1 might
also participate in sorting reactions that drive GM1 trafficking.
If so, it may be possible to design peptide linkers that recover
such trafficking functions. The testing of the oligosaccharide
domain and accommodation to its loss are the topics of this
aim.
[0175] Initially, two simplified structural analogs of GM1 are
tested: glucosylceramide (Glc-Cer--one sugar residue), and ceramide
alone (no sugars). These lipids are commercially available with
different ceramide structures (Avanti Polar Lipids). Because
ceramide (Cer) and Glc-Cer lack a sialic acid, this necessitates an
alternative chemical strategy to link peptides onto the ceramide
headgroup. All lipids are coupled to the reporter peptide using
copper-catalyzed "click" chemistry. Because Cer and Glc-Cer lack a
sialic acid, this necessitates an alternative chemical strategy to
link peptides onto the ceramide headgroup. Details of the chemical
reactions are shown in FIG. 7. The primary hydroxyl located on the
ceramide (or on glucose ring of Glc-Cer) are oxidized to react with
peptides containing aminooxy reactive groups as in established
method (12, 13). All compounds are confirmed for mass by MALDI mass
spectrometry (or LC-MS) and NMR.
[0176] The transcytosis of Glc-Cer and Cer are tested in vitro
using polarized cell lines T84, Caco2 and MDCK. Testing the new
peptide-ceramide fusion molecules for transcytosis using the same
methods described in FIG. 5. Due to a possibility of being less
soluble, the lipids are complexed to BSA (1:1) as described in
Pagano et al. (43).
[0177] If reporter peptide fusions to ceramide alone mimic the
GM1-trafficking platform, it can be concluded that the sugar groups
are dispensable, except perhaps for blocking the sphingolipid from
membrane flip-flop to the inner leaflet, a function presumably
accommodated by the peptide-linker and cargo. If the
oligosaccharide domain contributes decisively to trafficking
function (and thus are required), peptide linkers that would
replace the functionality of the oligosaccharide domains in
trafficking can be designed. Due to the inherent difficulty in
synthesizing complex O-linked oligosaccharides onto lipids,
pseudo-glycopeptide linkers are synthesized using backbone amino
acids coupled to glucose, galactose or galactose-N-acetyl residues
via serine side chains (44). FMoc-protected building block amino
acids containing monosaccharides are commercially available and
will be used for solid phase peptide synthesis of short linear
peptides containing up to 4-5 sugar residues each (FIG. 8).
Addition of the sugar groups may reproduce the overall "bulk" and
strong hydrophilicity imparted by the endogenous GM1
oligosaccharide, or function to display glucose or galactose as
ligands for extracellular lectins that could participate in the
lipid sorting events--such as that proposed for galectin 3 in
non-clathrin mediated endocytosis (41). When necessary, additional
peptides are synthesized in a branched manner (as observed with the
natural sialic acid residue), or cyclized to alter headgroup
geometry to approximate the GM1 oligosaccharide. The transcytosis
assay can be performed in a medium throughput manner, thus assess
many different peptides constructs together.
[0178] If glycoceramide (single sugar) mimics results with the
GM1-lipids, but ceramide alone does not, it can be concluded that
the sugar groups contribute to glycosphingolipid sorting primarily
by anchoring the molecules in the outer-leaflet of the membrane. In
both cases, it is possible that simplified sphingolipids can
substitute for GM1 (or with redesigned peptide-sugar linkers).
[0179] The ability of ceramide alone or Glc-Cer to function as the
GM1-lipid platform allow more rapid translation to clinical
application, as these molecules can be synthesized or readily
purified from milk fats.
Example 2. Test if the Peptide Linker Between GM1 and Cargo can be
Designed to Release the Cargo after or During Transcytosis
[0180] Described herein is the design of a delivery vehicle that
releases the cargo after transport. For instance, fusion of GM1 to
the smaller peptide hormone GLP-1 causes a 3 to 8-fold loss of
function. While still highly potent (pM activity) and
physiologically relevant, the GLP-1-GM1 fusion molecule is still
less potent than the native peptide. Here, two approaches to design
the peptide linker so that it can release cargo from GM1 after
uptake into the cell or after arrival in the basolateral endosome
were tested. One approach involved the incorporation of an ester
linkage in the peptide linker (as target for esterases increased at
sites of inflammation, e.g., leukocyte esterase, or
carboxylesterase hCE-2 that is specific to gastrointestinal
endosomes) (1, 45); and the other involved incorporation of a
cleavage motif for the endosomal protease furin (2-4).
[0181] Different furin cleavage motifs were tested for activity
within the context of short peptide linker. The assay was designed
as high-throughput using fluorescence energy transfer (FRET) by
appending paired fluorophores at each end of the peptide to be
tested. FIG. 9 shows identification of an optimal sequence (FRET 7)
for further testing. This motif was incorporated into the linker
system described herein and the peptide is synthesized, purified,
and validated by mass spec in large quantities. It is ready for
coupling to cargo and GM1 for testing in vitro and in vivo.
[0182] The FRET 7 furin cleavage motif or a cleavable ester linkage
are incorporated into the reporter-peptide linker, and also with
GLP-1 incorporated as cargo. The GM1-C12:0 species are used as a
fusion partner because this lipid does not readily release from
membranes and will provide a more sensitive approach to test this
technology. The efficiency for cleavage are first tested in vitro
using recombinant esterases or recombinant furin and analyzed by
HPLC. If cleavage is confirmed, the fusion molecules are tested for
efficiency of cargo transport across highly resistant T84
epithelial barriers (as described in FIG. 5). Control cargos are
fused to GM1-C12:0 using non-cleavable linkers. The fusion
molecules are applied to apical surfaces of T84 cells in the
presence of serine protease inhibitors, BSA as serine-protease
decoys, and ester-linked peptide decoys. Rates of transepithelial
transport will be compared using non-cleavable peptide linkers as
controls. An increase in transepithelial transport (or cytosolic
delivery of cargo) above that achieved by the non-cleavable peptide
linkers suggests that cleavable linker technologies are
feasible.
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Example 3. Fatty Acid Length & Double Bond Positioning Dictate
Endosomal Sorting of Glycosphingolipids
[0228] The ceramide structure plays a decisive role in determining
the cellular fate of glycosphingolipids after endocytosis-explained
by their propensity to sort into highly curved tubules and buds
emerging from the sorting endosome. It was tested herein whether
glycosphingolipid sorting depends on molecular shape or on assembly
into nano-domains (or both). GM1 species with saturated long chain
fatty acids might a higher propensity to self-assemble with
cholesterol into nano-domains. These domains might be recognized by
cellular factors and sorted into the degradative pathway;
preventing entering the recycling endosomal tubules.
[0229] To investigate the role of double bond positioning &
hydrocarbon chain length of the ceramide fatty acid in lipid
packing and subsequent differential endosomal sorting, GM1 isoforms
with identical oligosaccharide head groups but ceramides of
different endogenous structure were synthesized, by systematically
increasing length and double bond position (C12:0 to C26:1).
[0230] The GM1 species with different ceramide structures were
functionalized with a D-amino acid reporter peptide, containing a
biotin and fluorophore group. Alternatively, a fluorophore was
directly onto the sialic acid of the sugar head group. Lipids were
purified by HPLC and structures validated by LC/MS (FIG. 17).
[0231] Surface GM1 at steady state depends on lipid-sorting into
highly curved tubules of the recycling pathway and this can be
detected using fluorescent avidin probes. GM1 with saturated longer
chain (>C14:0) fatty acid or long chain unsaturated fatty acid
(>C24:1) are depleted from the plasma membrane, presumably
sorted to the lysosome, suggesting inefficient entry into sorting
tubules. GM1 containing a ceramide with C14:0 or C24:1 fatty acid
have intermediate phenotype (FIG. 18).
[0232] Fluorescently-labeled GM1 in sorting tubules emerging from
early endosomes (labeled with dextran and TFnR) was measured
directly in A431 cells and quantified. GM1 with short (<C14:0)
or unsaturated (<C24:1) fatty acids escaped the degradative
pathway and enter recycling endosomal tubules. Depletion of
cellular cholesterol by MbCD releases GM1 species with long
(>C14:0, .gtoreq.C24:1) fatty acids into recycling endosomal
tubules (FIG. 19).
[0233] Two additional endocytic pathways depend upon sorting into
highly curved tubules-transcytosis and retrograde, providing other
quantitative measures for GM1 entry into sorting tubules.
Transcytosis of individual GM1 species were measured in polarized
MDCK cells detecting apical to basolateral transport of GM1 with a
fluorescent avidin probe. Retrograde trafficking of individual GM1
species was measured using cAMP after intoxification by cholera
toxin as read out in GM1 negative cell lines. The toxin must
traffic retrograde to the ER to induce cAMP. GM1 with longer chain
fatty acids (.gtoreq.C14:0) or ceramides containing a
mono-unsaturated fatty acid (.gtoreq.C22:1) inefficiently enter
sorting tubules and (presumably) are retained in the lysosomal
pathway (FIG. 20). The position of the unsaturated bond suggests a
decisive factor may be interaction with cholesterol.
Methods of Examples 1-3
Synthesis of Peptide-GM1 Conjugates
[0234] A short reporter peptide (all-D isomer) was synthesized and
chemically linked onto the headgroup of ceramide lipid containing a
C2:0 fatty acid tail. The chemistry to do this was very difficult
due to the nature of the components and insolubility on standard
solvents. Many different approaches had been tried a strategy that
worked well was found. The 3-step synthetic approach is described
herein.
Step 1: Oxidation of C2-Ceramide.
[0235] A method called "Parikh-Doering" oxidation was used to
oxidize the headgroup of ceramide to a reactive aldehyde in organic
solvent. Reaction mixture contains: (1) SO3Py: 190-400 mg/mL in dry
DMSO; (2) C8 glucosylceramide: 0.2-0.6M solution in dry DMSO with
Triethylamine; and (3) Triethylamine: 7-17 equivalents. The
resulting product was purified by solvent extraction and dried in a
speed vac (FIG. 11).
Step 2: Reaction of peptide containing aminooxy to the aldehyde
group The "LC9" (D-isomer) linker peptide was coupled using
oxime-mediated reductive amination (FIG. 12) in 80% DMF in 20% PBS
pH6.9 in 500 .mu.F. The reaction mixture contained 471 nmoles
peptide, 1,642 nmoles oxidized ceramide, and 1 .mu.F aniline. After
overnight incubation, 5 mM sodium cyanoborohydride was added to
reduce the oxime bond formed and to make it a permanently stable
bond.
[0236] The resulting crude peptide-ceramide conjugates or purified
conjugates (60% yield) were analyzed by HPLC (FIGS. 13A-13C). The
products were also analyzed by mass spectrometry to confirm the
presence of the peptide-ceramide conjugates (FIG. 13D).
Step 3: Copper Click Reaction of Alexa Fluor 488 to
Ceramide-Peptide Conjugate
[0237] Alexa Fluorophore containing an azide reactive group was
reacted to the peptide-ceramide conjugate via the N-terminal alkyne
using Huisgen copper-catalyzed cycloaddition chemistry (FIG. 14).
Briefly, Ceramide-peptide conjugate at 186 nmoles were reacted to
279 nmoles fluorophore in 50 mM Tris-C1 pH8, containing 100 mM
ascorbate, 5 mM copper sulfate, 0.06 mM TBTA and 1 mM TCEP. The
products were analyzed and purified by semi-preparative HPLC (FIG.
15). Final molecular weight of the produce was 2,425.1 Da
Transcytosis in MDCK Cells
[0238] The in vitro transport (transcytosis) of the pep tide-GM1
conjugates across a monolayer of MDCK cells, and in vivo transport
across an intestinal or nasal barrier into the blood are assessed.
The peptide alone are used as negative controls, and the GM1
analogs as positive controls. C6, and C12 fatty acid analogs of
ceramide were also included in the experiments as direct
comparisons to the GM1 and GM3 species.
[0239] MDCK cells were seeded in 0.33 cm.sup.2 inserts 3 days prior
in media containing HBSS and 100 mM defatted BSA. Electrical
resistance was checked on day of experiment.
Test Samples
[0240] 1) peptide Control [0241] 2) GM1-C6:0-peptide [0242] 3)
Peptide-C12 fatty acid [0243] 4) Ceramide-C2-
Media Preparation:
[0244] Make: 27 mL T84-SF PEGS 1% dfBSA (add 270 mg dfBSA to 27 mL)
Make: 500 uL.times.50 samples=25 mL T84-SF PLUS 0.1% dfBSA (add 25
mg to 25 mL)
Transwell Assay
[0245] 1) Check electrical resistance by EVOM. [0246] 2) Prepare
stock solutions above. [0247] 3) Wash cells apical and basolateral
with HBSS, without FBS. (dunk method) [0248] 4) EVOM the cells.
[0249] 5) Replace apical with HBSS+0.1 uM dfBSA, basolateral with
HBSS+1% dfBSA. [0250] 6) Incubate again for 20 minutes. Re-check
EVOM. [0251] 7) Wait another 20 minutes and remove/replace apical
with 200 .quadrature.L compound. [0252] 8) Continue incubation for
3 hours total. [0253] 9) Remove apical media to tubes. [0254] 10)
Replace with apical media and recheck EVOM final. [0255] 11) Remove
basolateral media to eppendorf tubes on ice.
Pull-down Assay:
[0255] [0256] 1) Washed Stretavidin Magnetic beads with TBS-T
4.times., to remove azide. [0257] 2) Resuspend beads in 800 uL TBS
then aliquoted 50 uL out to each tube [0258] 3) Add 1000 .mu.L to
streptavidin beads overnight at 4.degree. C. with rotation O/N
covered in foil. [0259] 4) Remove media and wash 3.times.TBS-T.
[0260] 5) Elute by addition of 220 ul 95% Formamide, 10 mM EDTA 0.4
mg/ml biotin--2 min @65 C [0261] 6) Pipetted 100 uL of each sample
.times.2 on 96 well plate [0262] 7) Fluorescence was read out on a
microplate reader for Fluorescein channel, and against a standard
curve.
Example 4. Pharmacokinetics and Bioavailability of Peptide-GM1-C6:0
Fusion
[0263] The bioavailability of the peptide-GM1-C6:0 fusion molecule
when applied nasally to mice and the pharmacokinetics are shown in
FIG. 26 (means of two independent experiments). The dose was 2.5
nmol/kg for 7 week old C57BL/6J with an intranasal administration
volume of 10 .mu.L and an intraperitoneal injection volume of 200
.mu.L. Nasal bioavailability of C6-GM1 is 24.4%, and nasal
bioavailability of the peptide alone is 1.3%. The 24% absorption
compared to intraperitoneal injection of the same molecule is high
for mucosal absorption of therapeutic peptides.
[0264] Next, the bioavailability and pharmacokinetics of a peptide
fused to GM-C4:0 was tested in vivo in rat. The fusion molecule was
validated in mice before the experiment (FIG. 32A). The peptide
alone or the peptide-GM1-C4:0 conjugated was administered to rat
via gastric route or via intravenous injection. The result showed
that there was no adsorption of gastric peptide-GM1-C4:0 conjugate.
However, serum half-life of the peptide was strongly prolonged by
fusing the peptide to GM1-C4:0, which is consistent with protection
against degradation or excretion by GM1-dependent trafficking into
recycling endosomes of endothelial and other cell types (liver,
lymphocytes, spleen, etc.). The results showed that, on day one
after intravenous injection, the level of peptide-GM1-C4 fusion in
rat serum was 5.6 nmol/kg and the level of peptide alone in rat
serum was 10.8 nmol/kg (FIG. 32B). On day two after intravenous
injection, the level of peptide-GM1-C4 fusion in rat serum was 14.4
nmol/kg and the level of peptide alone in rat serum was 12.8.8
nmol/kg (FIG. 32C).
Example 5. Ceramides and Analogs as Delivery Vehicles
[0265] A panel of ceramides and ceramide analogs were synthesized
(FIG. 27). Transcytosis of the ceramides and ceramide analogs in
MDCKII cells was evaluated, and the apparent permeability
coefficient (Papp) values are shown in FIG. 28. Transcytosis of the
ceramides and ceramide analogs in T84 intestinal cells was also
evaluated, and the apparent permeability coefficient (Papp) values
are shown in FIG. 29. The result shows that glucoceramide and
ceramide alone are functional as delivery platforms and exhibited
even more efficient than GM1.
[0266] Cer-C6 was tested along with a 4.degree. C. temperature
block to demonstrate evidence for the mechanism of transport by
transcytosis. The ceramide analog Diene C6 along (structure shown
in FIG. 27) was also tested in the same experiment. The results
show that the low temperature reduced the uptake of Cer-C6 and the
Diene C6 ceramide analog by MDCK II cells, indicating that the
transport was via transcytosis with a 4.degree. C. temperature
block (FIG. 30). This experiment was repeated with ceramides of
different fatty acid chain length (C4, C6, C8 (C8 results not
shown)), and with or without a sugar moiety. The results show that
ceramide Cer-C6:0 carrier (without sugar) and the ceramide analog
diene carrier (C6:0) is effective in transporting across the MDCKII
cells via transcytosis, with the Cer-C6:0 being more effective. The
glucoceramide C8:0 carriers also effectively transported via
transcytosis (data now shown).
[0267] Next, a ceramide with a C2 fatty acid chain and without a
sugar moiety (Cer-C2) was tested. Peptide fused to Cer-C2 was able
to transport across monolayers of MDCKII cells in a transcytosis
assay (FIG. 31). The GM1-C6:0 molecule was used as a positive
control, and a peptide fused to a fatty acid dodecyl-C12 was used
as a negative control. Dodecyl-C12 did not enable transport across
MDCKII cells by transcytosis (FIG. 31).
[0268] A repeat experiment of transcytosis in MDCK cells is shown
in FIG. 33. Papp at 37.degree. C. was paired with 4.degree. C. to
test for transport by transcytosis (and not paracellular leak).
Another independent experiment that shows the ceramide alone
Cer-C6:0 carrier is effective. The ceramide-like diene carrier
(C6:0) also works, but is less effective compared to the
ceramide-C6:0 species. The glucoceramide C8:0 carriers also work
and are probably as effective as ceramide C6:0 alone. Both are more
effective than the original fusion molecules using GM1 C6:0.
[0269] Further, whether a ceramide can be absorbed via the nose is
tested. A reporter peptide was conjugated to a ceramide-C6 (Cer-C6)
was administered to C57BL/6J mice (n=1, from Jackson Labs) at a
dose of 2 nmol/kg. GM1-C6 conjugated to the same reported peptide
was used as control. A total of 5 .mu.l per nostril was
administered over 5 minutes under isoflurane. Blood was collected
via cardiac puncture 15 minutes post dose and the amount of the
conjugate was evaluated by pulling down with magnetic streptavidin
beads followed by elution and plate fluorescence measurement. The
data showed that ceramide-C6-reporter peptide conjugate can be
absorbed via nose in mice, and that the peptide conjugated to
Cer-C6 is absorbed more efficiently than peptide conjugated to
GM1-C6 (FIG. 38).
Example 6. Synthesis of Peptide-Ceramide Conjugates as a Platform
for Protein Drug Delivery
[0270] Diene-analogs (due to dehydration) and dihydro-analogs of
ceramide have been underlined for clarity in this disclosure.
Oximes may be a mix of cis and trans geometric isomers. Oximes have
been arbitrarily drawn as cis-isomers in FIG. 35 and as
trans-isomers (likely the predominant isomer) in FIG. 36 and FIG.
37. Some oximes have been reduced to the corresponding
O-alkylhydroxylamine analogs. (Data not shown). Reactions were run
in Wheaton vials with triangular magnets unless otherwise stated.
Chromatography on flash silica gel 60 (230-400 mesh) at 35.degree.
C. unless otherwise stated. Analytical thin layer chromatography
(TLC) utilized silica gel 60 on glass plates that were visualized
by UV 254, and by phosphoric acid charring on a hot plate at
240.degree. C. HPLC purifications on RP-C3 columns eluting with
acetonitrile/water containing 0.1% formic acid. Mass Spectral data
from an Agilent Technologies 6120 quadripole LC-MS.
Linker Chemistry and "Diene"-Analog of Ceramide (Dehydration
Compound)
[0271]
(2S,3R,4E)-1-(O-Triphenylmethyl)-2-(N-hexanoylamino)-4-octadecene-1-
,3-diol [1-trityl-C6-ceramide 2 (R.dbd.--C.sub.5H.sub.11)]. To a
vigorously stirred solution of C6-ceramide 1
(R.dbd.--C.sub.5H.sub.11) (30.3 mg, 7.62.times.10.sup.-5 mol),
ethyldiisopropylamine (18 .mu.L, 13 mg, 1.0.times.10.sup.-4 mol),
and DMAP (1.1 mg, 9.0.times.10.sup.-6 mol) in 500 .mu.L of dry
CH.sub.2Cl.sub.2, was added trityl chloride (23.4 mg,
8.4.times.10.sup.-5 mol) in 200 .mu.L of CH.sub.2Cl.sub.2 at room
temperature over 5 min. The reaction mixture was stirred for 3
days. The mixture was concentrated and chromatographed to give
1-trityl-C6-ceramide 2 (R.dbd.--C.sub.5H.sub.11) (32.0 mg,
5.00.times.10.sup.-5 mol, 66%) as a clear and colorless viscous
liquid that was homogeneous by TLC (90:10:0.1 CHCl.sub.3/EtOAc/TEA
R.sub.f 0.42): .sup.1H NMR (DMSO-d.sub.6).
[0272]
(2S,3R,4E)-3-O-Benzoyl-1-(O-triphenylmethyl)-2-(N-hexanoylamino)-4--
octadecene-1,3-diol [1-trityl-3-benzoyl-C6-ceramide 3
(R.dbd.--C.sub.5H.sub.11)]. To a vigorously stirred solution of
1-trityl-C6-ceramide 2 (R.dbd.--C.sub.5H.sub.11) (9.5 mg,
1.5.times.10.sup.-5 mol), ethyldiisopropylamine (13.1 .mu.L, 9.7
mg, 7.5.times.10.sup.-5 mol, 500 mol %), and catalytic DMAP in 500
.mu.L of dry toluene, was added benzoyl chloride (2.5 mg,
1.8.times.10.sup.-5 mol). After 1 day, additional
ethyldiisopropylamine (13.1 .mu.L) and benzoyl chloride (2.5 mg)
were added. The reaction was filtered through a plug of 0.2 g
silica gel eluting with CHCl.sub.3/EtOAc/TEA (97:3:0.1
CHCl.sub.3/EtOAc/TEA). The filtrate containing product was
chromatographed (97:3:0.1 CHCl.sub.3/EtOAc/TEA) to give
1-trityl-3-benzoyl-C6-ceramide 3 (R.dbd.--C.sub.5H.sub.11) (10.0
mg, 1.34.times.10.sup.-5 mol, 89%) as a clear and colorless viscous
liquid that was homogeneous by TEC (97:3:0.1 CHCl.sub.3/EtOAc/TEA
R.sub.f 0.45): .sup.1H NMR (DMSO-d.sub.6).
[0273]
(2S,3R,4E)-3-O-Benzoyl-2-(N-hexanoylamino)-4-octadecene-1,3-diol
[3-benzoyl-C6-ceramide 4a (R.dbd.--C.sub.5H.sub.11)]. To a stirred
solution of l-trityl-3-benzoyl-C6-ceramide 3
(R.dbd.--C.sub.5H.sub.11) (10. mg, 1.3.times.10.sup.-5 mol) in 2 mL
of CH.sub.2Cl.sub.2/MeOH (1:1) was added a solution of
p-toluenesulfonic acid monohydrate (3.8 mg, 2.0.times.10.sup.-5
mol) in 0.5 mL of MeOH. After 3 days, CH.sub.2Cl.sub.2 was added
and the solution washed twice with 1 mL portions of 8.5 mg/mL
aqueous NaHCO.sub.3. The organic phase was then washed with
H.sub.2O, dried over Na.sub.2SO.sub.4, and chromatographed (96:4
CH.sub.2Cl.sub.2/MeOH) to give 3-benzoyl-C6-ceramide 4a
(R.dbd.--C.sub.5H.sub.11) (6.5 mg, 1.3.times.10.sup.-5 mol, 100%)
as a white solid that was nearly homogeneous by TEC (96:4
CH.sub.2Cl.sub.2/MeOH, R.sub.f 0.20): .sup.1H NMR (DMSO-d.sub.6);
mass spectrum C.sub.31H.sub.52NO.sub.4.sup.+: m/z calculated 502.4,
observed 502.5.
Linker
[0274]
(2S,3R,4E)-3-O-Benzoyl-1-O-(2-(2-(2-(4-formylbenzamido)ethoxy)ethox-
y)ethan-1-carbamoyl)-2-(N-hexanoylamino)-4-octadecene-1,3-diol
[1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate 6a
(R.dbd.--C.sub.5H.sub.11)]. To a stirred solution of triphosgene
(5.9 mg, 2.0.times.10.sup.-5 mol, 20 eq) in 0.2 mL of anhydrous
CH.sub.2Cl.sub.2 was added dropwise over 2 min a solution of
3-benzoyl-C6-ceramide 4a (R.dbd.--C.sub.5H.sub.11) (1.5 mg,
3.0.times.10.sup.-5 mol, 1 eq) in 0.2 mL of anhydrous
CH.sub.2Cl.sub.2 containing ethydiisopropylamine (22 .mu.L, 16 mg,
3.0.times.10.sup.-5 mol, 43 eq). After 2 h, the reaction mixture
was distilled using a stream of nitrogen gas blowing over the
reaction mixture and out through a solution of aqueous ammonium
hydroxide/H.sub.2O/i-PrOH (1:1:1). The residue was redissolved in
0.1 mL of anhydrous CH.sub.2Cl.sub.2 to give a light yellow
solution. To this solution of crude chloroformate 5a
(R.dbd.--C.sub.5H.sub.11) was added rapidly dropwise a freshly
prepared solution of Ald-PEG2-ammonium trifluoracetate (3.9 mg,
9.9.times.10.sup.-6 mol, 3.3 eq, Broadpharm, CAS 2055013-56-2) in
0.1 mL of dry CH.sub.2Cl.sub.2 containing ethydiisopropylamine (12
.mu.L, 8.9 mg, 6.9.times.10.sup.-5 mol, 7 eq). After 1 h 15 min,
the solvent was removed to give a yellow semi-solid that was
chromatographed (96:4:0.1 CHCl.sub.3/EtOAc/TEA) to give a nearly
quantitative yield of 1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate
6a (R.dbd.--C.sub.5H.sub.11) as a faint yellow semisolid that was
nearly homogeneous by TLC (96:4:0.1 CHCl.sub.3/EtOAc/TEA, R.sub.f
0.18): .sup.1H NMR (DMSO-d.sub.6).
Linker
[0275] The linker already has aldehyde function.
Coupling with LC9 to Give the Oxime.
[0276] LC9-oxime-PEG2-carbamoyl-C6-ceramide-3-benzoate 8a
(R.dbd.--C.sub.5H.sub.11). To a solution of
1-(Ald-PEG2-carbamoyl)-C6-ceramide-3-benzoate 6a
(R.dbd.--C.sub.5H.sub.11) (1.5 mg, 1.9.times.10.sup.-6 mol) and LC9
(2.7 mg, 1.9.times.10.sup.-6 mol) in 800 .mu.L of DMF was added 1
.mu.L of aniline and the reaction stirred overnight. HPLC
purification of the reaction mixture gave
LC9-oxime-PEG2-carbamoyl-C6-ceramide-3-benzoate 8a
(R.dbd.--C.sub.5H.sub.11); mass spectrum
C.sub.106H.sub.163N.sub.24O.sub.4S.sup.+: m/z calculated for
(M+2H).sup.+2 1118.6, observed 1118.9.
[0277] The "click" reaction with AF488-azide which is a
copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to
give the corresponding predominantly 1,4-substituted-1,2,3-triazole
(as drawn in FIG. 37, though may contain some 1,5-substituted
triazole). AF488-LC9-oxime-PEG2-carbamoyl-C6-ceramide-3 benzoate 9a
(R.dbd.--C.sub.5H.sub.11). mass spectrum m/z calculated for
(M+2H).sup.+2, observed.
Diene
Oxidation of Primary Alcohol to Aldehyde.
[0278] To a solution of C6-ceramide-3-benzoate 4a
(R.dbd.--C.sub.5H.sub.11) (1 mg, 2.times.10.sup.-6 mol) in 0.2 mL
of dry CH.sub.2Cl.sub.2, was added a 0.30 M solution of Dess-Martin
periodinane (13 .mu.L, 4.times.10.sup.-6 mol) dropwise over 1 min
during which time the solution remained homogeneous. After 10 min,
a vortex-mixed solution of H.sub.2O (0.04 .mu.L, 0.04 mg,
2.times.10.sup.-6 mol) in 40 .mu.L of CH.sub.2Cl.sub.2 was added
dropwise over 10 min during which time the reaction mixture became
cloudy. The reaction mixture was vigorously stirred for an
additional 1.5 h. The reaction mixture was then washed with 0.2 mL
of 1:1 saturated aqueous NaHCO.sub.3/15% Na.sub.2S.sub.2O.sub.3,
dried over Na.sub.2SO.sub.4, filtered through a 0.45 .mu.m GHP
filtration cartridge, and the solvent removed to give a light
yellow viscous oil that contained the corresponding aldehyde by TLC
(90:10:0.1 CHCl.sub.3/EtOAc/TEA, R.sub.f 0.66) and was used
immediately.
Diene
[0279] Coupling with LC9 to Give the Oxime.
[0280] LC9-oxime-C6-ceramide-diene 10 (R.dbd.--C.sub.5H.sub.11). To
a solution of the crude aldehyde (0.5 mg, 1.times.10.sup.-6 mol)
and LC9 (1.7 mg, 1.2.times.10.sup.-6 mol) in 1 mL of DMF was added
1 .mu.L of aniline and the reaction stirred overnight. HPLC
purification of the reaction mixture gave
LC9-oxime-C6-ceramide-diene analog 10 (R.dbd.--C.sub.5H.sub.11)
(0.4 mg, 2.times.10.sup.-7 mol): mass spectrum
C.sub.84H.sub.137N.sub.22O.sub.20S.sup.+: m/z calculated for
(M+2H).sup.+2 903.5, observed 903.8, calculated for (M+3H).sup.+3
602.7, observed 603.1.
Diene
[0281] The "click" reaction with AF488-azide which is a
copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to
give the corresponding predominantly 1,4-substituted-1,2,3-triazole
(as drawn in FIG. 37, though may contain some 1,5-substituted
triazole). AF488-LC9-oxime-C6-ceramide-diene analog
13(R.dbd.--C.sub.5H.sub.11). A solution of
LC9-oxime-C6-ceramide-diene analog 10 (R.dbd.--C.sub.5H.sub.11)
(0.4 mg, 2.times.10.sup.-7 mol), Alexa Fluor.RTM. 488 (0.38 mg,
4.4.times.10.sup.-7 mol),
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
(6.times.10.sup.-8 mol), tris(2-carboxyethyl)phosphine
(1.times.10.sup.-6 mol), ascorbic acid (lx 10.sup.-4 mol),
Cu(II)SO.sub.4 (5.times.10.sup.-6 mol), in 1 mL of 8:2 DMSO/Tris
buffer pH 8, was stirred overnight protecting from light. The
reaction mixture was centrifuged, and the colored precipitate was
purified by HPLC to give AF488-LC9-oxime-C6-ceramide-diene analog
13 (R.dbd.--C.sub.5H.sub.11) (14 .mu.g, 5.7.times.10.sup.-9 mol,
2.8% yield); mass spectrum
C.sub.111H.sub.163N.sub.28O.sub.30S.sub.3.sup.+: m/z calculated for
(M+2H).sup.+2 1232.6, observed 1232.5, calculated for (M+3H).sup.+3
822.1, observed 822.2.
AF488-LC9-Oxime-C6 Ceramide
C6
[0282]
(2S,3R,4E)-3-O-Triethylsilyl-2-(N-hexanoylamino)-4-octadecene-1,3-d-
iol [3-TES-C6-ceramide 4b (R.dbd.--C.sub.5H.sub.11)]. To a stirred
solution of C6-ceramide 1 (R.dbd.--C.sub.5H.sub.11) (30.3 mg,
7.62.times.10.sup.-5 mol), ethyldiisopropylamine (150 .mu.L, 111
mg, 8.5.times.10.sup.-4 mol), and DMAP (9.5 mg, 7.8.times.10.sup.-5
mol) in 1.5 mL of dry CH.sub.2Cl.sub.2, was added triethylsilyl
chloride (12 .mu.L, 11 mg, 7.3.times.10.sup.-5 mol) dropwise at
room temperature over 2 min. The reaction mixture was stirred for
1.5 h. The mixture was concentrated and chromatographed (90:10:0.1
CHCl.sub.3/EtOAc/TEA) first eluting 1,3-diTES-C6-ceramide
R.dbd.--C.sub.5H.sub.11) followed by 1-TES-C6-ceramide
(R.dbd.--C.sub.5H.sub.11), then the desired 3-TES-C6-ceramide 4b
(R.dbd.--C.sub.5H.sub.11) as the minor product (3.7 mg,
7.2.times.10.sup.-6 mol, 9.4%) as a clear and colorless viscous
liquid that was homogeneous by TLC (90:10:0.1 CHCl.sub.3/EtOAc/TEA
R.sub.f 0.15): .sup.1H NMR (DMSO-d.sub.6); mass spectrum
C.sub.30H.sub.62NO.sub.3Si.sup.+: m/z calculated 512.5, observed
512.4.
C6
[0283] Step One is oxidation of primary alcohol to aldehyde. To a
solution of C6-ceramide-3-TES 4b (R.dbd.--C.sub.5H.sub.11) (2.2 mg,
4.3.times.10.sup.-6 mol) in 0.2 mL of dry CH.sub.2Cl.sub.2, was
added a 0.30 M solution of Dess-Martin periodinane (22 .mu.L,
5.7.times.10.sup.-6 mol, 1.5 eq) dropwise over 1 min during which
time the solution remained homogeneous. After 10 min, a
vortex-mixed solution of H.sub.2O (0.13 .mu.L, 0.13 mg,
7.2.times.10.sup.-6 mol) in 130 .mu.L of CH.sub.2Cl.sub.2 was added
dropwise over 10 min during which time the reaction mixture became
cloudy. The reaction mixture was vigorously stirred for an
additional 15 min. The reaction mixture was then washed with 0.2 mL
of 1:1 saturated aqueous NaHCO.sub.3/15% Na.sub.2S.sub.2O.sub.3,
dried over Na.sub.2SO.sub.4, filtered through a 0.45 .mu.m GHP
filtration cartridge, and the solvent removed to give a light
yellow viscous oil that contained the corresponding aldehyde by TEC
(90:10:0.1 CHCl.sub.3/EtOAc/TEA, R.sub.f 0.62) and was used
immediately.
C6
[0284] Step Two is the coupling with LC9 to give the oxime.
LC9-oxime-C6-ceramide-3-TES 11a (R.dbd.--C.sub.5H.sub.11). To a
solution of the crude aldehyde (2 mg, 4.times.10.sup.-6 mol) and
LC9 (6.2 mg, 4.3.times.10.sup.-6 mol) in 1 mL of DMF was added 1
.mu.L of aniline and the reaction stirred overnight. HPLC
purification of the reaction mixture gave
LC9-oxime-C6-ceramide-3-TES 11a (R.dbd.--C.sub.5H.sub.11); mass
spectrum C.sub.90H.sub.153N.sub.22O.sub.21SSi.sup.+: m/z calculated
for (M+2H).sup.+2 969.6, observed 970.5, calculated for
(M+3H).sup.+3 646.7, observed 647.2.
C6
[0285] Step Three is removal of the TES. LC9-oxime-C6-ceramide lib
(R.dbd.--C.sub.5H.sub.11). A solution of
LC9-oxime-C6-ceramide-3-TES 11a (R.dbd.--C.sub.5H.sub.11) in 80:20
acetic acid/H.sub.2O was stirred for two hours. Solvents were
removed by lyophylization. HPLC purification of the reaction
mixture gave LC9-oxime-C6-ceramide lib (R.dbd.--C.sub.5H.sub.11)
(1.0 mg, 5.5.times.10.sup.-7 mol); mass spectrum
C.sub.84H.sub.139N.sub.22O.sub.21S.sup.+: m/z calculated for
(M+2H).sup.+2 912.5, observed 912.7, calculated for (M+3H).sup.+3
608.7, observed 609.1.
C6
[0286] Step Four is the "click" reaction with AF488-azide which is
a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction)
to give the corresponding predominantly
1,4-substituted-1,2,3-triazole (as drawn in FIG. 37, though may
contain some 1,5-substituted triazole).
[0287] AF488-LC9-oxime-C6-ceramide 14b (R.dbd.--C.sub.5H.sub.11). A
solution of LC9-oxime-C6-ceramide lib (R.dbd.--C.sub.5H.sub.11)
(1.0 mg, 5.5.times.10.sup.-7 mol), Alexa Fluor.RTM. 488 (0.71 mg,
8.2.times.10.sup.-7 mol),
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
(6.times.10.sup.-8 mol), tris(2-carboxyethyl)phosphine (lx
10.sup.-6 mol), ascorbic acid (lx 10.sup.-4 mol), Cu(II)SO.sub.4
(5.times.10.sup.-6 mol), in 1 mL of 8:2 DMSO/Tris buffer pH 8, was
stirred overnight protected from light. The reaction mixture was
centrifuged, and the colored precipitate was purified by HPLC to
give AF488-LC9-oxime-C6-ceramide 14a (R.dbd.--C.sub.5H.sub.11) (100
.quadrature.g, 6.2 10.sup.-8 mol), mass spectrum
C.sub.111H.sub.165N.sub.28O.sub.31S.sub.3.sup.+: m/z calculated for
(M+2H).sup.+2 1241.1, observed 1241.6, calculated for (M+3H).sup.+3
827.7, observed 828.2.
AF488-LC9-Oxime-C18 Ceramide
C18
[0288]
(2S,3R,4E)-3-O-Triethylsilyl-2-(N-octadecanoylamino)-4-octadecene-1-
,3-diol [3-TES-C18-ceramide 4b (R.dbd.--C.sub.17H.sub.35)]. To a
stirred solution of C18-ceramide 1 (R.dbd.--C.sub.17H.sub.35) (41.2
mg, 7.28.times.10.sup.-5 mol), ethyldiisopropylamine (150 .mu.L,
111 mg, 8.5.times.10.sup.-4 mol), and DMAP (12.7 mg,
1.04.times.10.sup.-5 mol) in 1.5 mL of dry CH.sub.2Cl.sub.2, was
added triethylsilyl chloride (14 .mu.L, 13 mg, 8.6.times.10.sup.5
mol) dropwise at room temperature over 2 min. The reaction mixture
was stirred for 1.5 h. The mixture was concentrated and
chromatographed (90:10:0.1 CHCl.sub.3/EtOAc/TEA) first eluting
1,3-diTES-C18-ceramide R.dbd.--C.sub.17H.sub.35) followed by
1-TES-C18-ceramide (R.dbd.--C.sub.17H.sub.35), then the desired
3-TES-C18-ceramide 4b (R.dbd.--C.sub.17H.sub.35) as the minor
product (2.5 mg, 3.7.times.10.sup.-6 mol, 5.1%) as an amorphous
white solid that was homogeneous by TLC (90:10:0.1
CHCl.sub.3/EtOAc/TEA R.sub.f 0.15): NMR (DMSO-d.sub.6).
C18
[0289] Step One is oxidation of primary alcohol to aldehyde. To a
solution of 3-TES-C18-ceramide 4b (R.dbd.--C.sub.17H.sub.35) (2.5
mg, 3.7.times.10.sup.-6 mol) in 0.2 mL of dry CH.sub.2Cl.sub.2, was
added a 0.30 M solution of Dess-Martin periodinane (19 .mu.L,
5.7.times.10.sup.-6 mol, 1.5 eq) dropwise over 1 min during which
time the solution became cloudy. After 10 min, a vortex mixed
solution of H.sub.2O (0.11 .mu.L, 0.11 mg, 6.1.times.10.sup.-6 mol)
in 110 .mu.L of CH.sub.2Cl.sub.2 was added dropwise over 10 min.
The reaction mixture was vigorously stirred for an additional 15
min. The reaction mixture was then washed with 0.2 mL of 1:1
saturated aqueous NaHCO.sub.3/15% Na.sub.2S.sub.2O.sub.3, dried
over Na.sub.2SO.sub.4, filtered through a 0.45 .mu.m GHP filtration
cartridge, and the solvent removed to give yellow viscous oil that
was highly homogeneous aldehyde by TLC (90:10:0.1
CHCl.sub.3/EtOAc/TEA, R.sub.f 0.59) and was used immediately.
C18
[0290] Step Two is the coupling with LC9 to give the oxime with
protecting group removal. LC9-oxime-C18-ceramide 11e
(R.dbd.--C.sub.17H.sub.35). To a solution of the crude aldehyde
(2.5 mg, 3.7.times.10.sup.-6 mol) and LC9 (5.3 mg,
3.7.times.10.sup.-6 mol) in 1 mL of DMF was added 1 .mu.L of
aniline and the reaction stirred overnight. HPLC purification of
the reaction mixture gave only a small amount of
LC9-oxime-C18-ceramide-3-TES 11d (R.dbd.--C.sub.17H.sub.35), but
most of the TES had fallen off and gave LC9-oxime-C18-ceramide 11e
(R.dbd.--C.sub.17H.sub.35) (3.9 mg, 2.0.times.10.sup.-6 mol, 54%
yield) as a white solid: .sup.1H NMR (DMSO-d.sub.6); mass spectrum
C.sub.96H.sub.163N.sub.22O.sub.21S.sup.+: m/z calculated for
(M+2H).sup.+2 996.6, observed 996.9, calculated for (M+3H).sup.+3
664.7, observed 665.1.
C18
[0291] Finally, the "click" reaction with AF488-azide which is a
copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC Reaction) to
give the corresponding predominantly 1,4-substituted-1,2,3-triazole
(as drawn in FIG. 37, though may contain some 1,5-substituted
triazole).
[0292] AF488-LC9-oxime-C18-ceramide 14b (R.dbd.--C.sub.17H.sub.35).
A solution of LC9-oxime-C18-ceramide 11e (R.dbd.--C.sub.17H.sub.35)
(0.64 mg, 5.5.times.10.sup.-7 mol), Alexa Fluor.RTM. 488 (0.71 mg,
4.9.times.10.sup.-7 mol),
tris[(1-benzyl-177-1,2,3-triazol-4-yl)methyl]amine
(6.times.10.sup.-8 mol), tris(2-carboxyethyl)phosphine
(1.times.10.sup.-6 mol), ascorbic acid (lx 10.sup.-4 mol),
Cu(II)SO.sub.4 (5.times.10.sup.-6 mol), in 1 mL of 8:2 DMSO/Tris
buffer pH 8, was stirred overnight protected from light. The
reaction mixture was centrifuged, and the colored precipitate
purified by HPLC to give AF488-LC9-oxime-C18-ceramide 14b
(R.dbd.--C.sub.17H.sub.35); mass spectrum
C.sub.123H.sub.189N.sub.28O.sub.31S.sub.3.sup.+: m/z calculated for
(M+2H).sup.+2 1325.7, observed 1325.6, calculated for (M+3H).sup.+3
884.1, observed 884.8.
AF488-LC9-Oxime-Dihydroceramide
Dihydro
[0293]
(2S,3R)-3-O-Triethylsilyl-2-(N-hexanoylamino)-octadecane-1,3-diol
[3-TES-C6-dihydroceramide 7b (R.dbd.--C.sub.5H.sub.11)]. To a
stirred solution of C6-dihydroceramide 7a (R.dbd.--C.sub.5H.sub.11)
(27.8 mg, 6.96.times.10.sup.-5 mol), ethyldiisopropylamine (280
.mu.L, 208 mg, 1.61.times.10.sup.-3 mol), and DMAP (12 mg,
9.8.times.10.sup.-5 mol) in 1.5 mL of dry CH.sub.2Cl.sub.2, was
added triethylsilyl chloride (14 .mu.L, 13 mg, 8.6.times.10.sup.-5
mol) dropwise at room temperature over 2 min. The reaction mixture
was stirred for 1.5 h. The mixture was concentrated and
chromatographed (90:10:0.1 CHCl.sub.3/EtOAc/TEA) first eluting
1,3-diTES-C6-dihydroceramide (R.dbd.--C.sub.5H.sub.11) followed by
1-TES-C6-dihydroceramide (R.dbd.--C.sub.5H.sub.11), then the
desired 3-TES-C6-dihydroceramide 7b (R.dbd.--C.sub.5H.sub.11) as
the minor product (2.7 mg, 5.2.times.10.sup.-6 mol, 7.5%) as a
white waxy solid that was homogeneous by TEC (90:10:0.1
CHCl.sub.3/EtOAc/TEA R.sub.f 0.14): NMR (DMSO-d.sub.6).
Dihydro
[0294] Step One is oxidation of primary alcohol to aldehyde. To a
solution of C6-dihydroceramide-3-TES 7b (R.dbd.--C.sub.5H.sub.11)
(2 mg, 4.times.10.sup.-6 mol) in 0.2 mL of dry CH.sub.2Cl.sub.2,
was added a 0.30 M solution of Dess-Martin periodinane (13 .mu.L,
3.9.times.10.sup.-6 mol, 1.5 eq) dropwise over 1 min during which
time the solution remained homogeneous. After 10 min, a
vortex-mixed solution of H.sub.2O (0.079 .mu.L, 0.079 mg,
4.4.times.10.sup.-6 mol) in 10 .mu.L of CH.sub.2Cl.sub.2 was added
dropwise over 10 min during which time the reaction mixture became
cloudy. The reaction mixture was vigorously stirred for an
additional 30 min. The reaction mixture was then washed with 0.2 mL
of 1:1 saturated aqueous NaHCO.sub.3/15% Na.sub.2S.sub.2O.sub.3,
dried over Na.sub.2SO.sub.4, filtered through a 0.45 .mu.m GHP
filtration cartridge, and the solvent removed to give a light
yellow viscous oil that contained the corresponding aldehyde by TEC
(90:10:0.1 CHCl.sub.3/EtOAc/TEA, R.sub.f 0.71) and was used
immediately.
Dihydro
[0295] Step Two is the coupling with LC9 to give the oxime with
protecting group removal. LC9-oxime-C6-dihydroceramide 12b
(R.dbd.--C.sub.5H.sub.11). To a solution of the crude aldehyde (2
mg, 4.times.10.sup.-6 mol) and LC9 (5.6 mg, 3.9.times.10.sup.-6
mol) in 1 mL of DMF was added 1 .mu.L of aniline and the reaction
stirred overnight. HPLC purification of the reaction mixture gave
only a small amount of protecting group removed
LC9-oxime-C6-dihydroceramide 12b (R.dbd.--C.sub.5H.sub.11); mass
spectrum C.sub.84H.sub.141N.sub.22O.sub.21S.sup.+: m/z calculated
for (M+2H).sup.+2 913.5, observed 913.7, calculated for
(M+3H).sup.+3 609.4, observed 609.7.
[0296] All publications, patents, patent applications, publication,
and database entries (e.g., sequence database entries) mentioned
herein, e.g., in the Background, Summary, Detailed Description,
Examples, and/or References sections, are hereby incorporated by
reference in their entirety as if each individual publication,
patent, patent application, publication, and database entry was
specifically and individually incorporated herein by reference. In
case of conflict, the present application, including any
definitions herein, will control.
Equivalents and Scope
[0297] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the embodiments described herein. The scope of the
present disclosure is not intended to be limited to the above
description, but rather is as set forth in the appended claims.
[0298] Articles such as "a," "an," and "the" may mean one or more
than one unless indicated to the contrary or otherwise evident from
the context. Claims or descriptions that include "or" between two
or more members of a group are considered satisfied if one, more
than one, or all of the group members are present, unless indicated
to the contrary or otherwise evident from the context. The
disclosure of a group that includes "or" between two or more group
members provides embodiments in which exactly one member of the
group is present, embodiments in which more than one members of the
group are present, and embodiments in which all of the group
members are present. For purposes of brevity those embodiments have
not been individually spelled out herein, but it will be understood
that each of these embodiments is provided herein and may be
specifically claimed or disclaimed.
[0299] It is to be understood that the disclosure encompasses all
variations, combinations, and permutations in which one or more
limitation, element, clause, or descriptive term, from one or more
of the claims or from one or more relevant portion of the
description, is introduced into another claim. For example, a claim
that is dependent on another claim can be modified to include one
or more of the limitations found in any other claim that is
dependent on the same base claim. Furthermore, where the claims
recite a composition, it is to be understood that methods of making
or using the composition according to any of the methods of making
or using disclosed herein or according to methods known in the art,
if any, are included, unless otherwise indicated or unless it would
be evident to one of ordinary skill in the art that a contradiction
or inconsistency would arise.
[0300] Where elements are presented as lists, e.g., in Markush
group format, it is to be understood that every possible subgroup
of the elements is also disclosed, and that any element or subgroup
of elements can be removed from the group. It is also noted that
the term "comprising" is intended to be open and permits the
inclusion of additional elements or steps. It should be understood
that, in general, where an embodiment, product, or method is
referred to as comprising particular elements, features, or steps,
embodiments, products, or methods that consist, or consist
essentially of, such elements, features, or steps, are provided as
well. For purposes of brevity those embodiments have not been
individually spelled out herein, but it will be understood that
each of these embodiments is provided herein and may be
specifically claimed or disclaimed.
[0301] Where ranges are given, endpoints are included. Furthermore,
it is to be understood that unless otherwise indicated or otherwise
evident from the context and/or the understanding of one of
ordinary skill in the art, values that are expressed as ranges can
assume any specific value within the stated ranges in some
embodiments, to the tenth of the unit of the lower limit of the
range, unless the context clearly dictates otherwise. For purposes
of brevity, the values in each range have not been individually
spelled out herein, but it will be understood that each of these
values is provided herein and may be specifically claimed or
disclaimed. It is also to be understood that unless otherwise
indicated or otherwise evident from the context and/or the
understanding of one of ordinary skill in the art, values expressed
as ranges can assume any subrange within the given range, wherein
the endpoints of the subrange are expressed to the same degree of
accuracy as the tenth of the unit of the lower limit of the
range.
[0302] Where websites are provided, URL addresses are provided as
non-browser-executable codes, with periods of the respective web
address in parentheses. The actual web addresses do not contain the
parentheses.
[0303] In addition, it is to be understood that any particular
embodiment of the present disclosure may be explicitly excluded
from any one or more of the claims. Where ranges are given, any
value within the range may explicitly be excluded from any one or
more of the claims. Any embodiment, element, feature, application,
or aspect of the compositions and/or methods of the disclosure, can
be excluded from any one or more claims. For purposes of brevity,
all of the embodiments in which one or more elements, features,
purposes, or aspects is excluded are not set forth explicitly
herein.
* * * * *