U.S. patent application number 10/126845 was filed with the patent office on 2003-09-25 for conjugates of membrane translocating agents and pharmaceutically active agents.
Invention is credited to Houghten, Richard, Lambkin, Imelda, O'Mahony, Daniel, Pinilla, Clemencia.
Application Number | 20030181367 10/126845 |
Document ID | / |
Family ID | 29248426 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030181367 |
Kind Code |
A1 |
O'Mahony, Daniel ; et
al. |
September 25, 2003 |
Conjugates of membrane translocating agents and pharmaceutically
active agents
Abstract
Membrane translocation peptides, compositions comprising them,
chimeric molecules comprising them, and methods of using them to
achieve transmembrane transport of various agents.
Inventors: |
O'Mahony, Daniel;
(Blackrock, IE) ; Lambkin, Imelda; (Sutton,
IE) ; Pinilla, Clemencia; (Cardiff, CA) ;
Houghten, Richard; (Solana Beach, CA) |
Correspondence
Address: |
CAESAR, RIVISE, BERNSTEIN, COHEN & POKOTILOW, LTD.
ATTN: ELAN
12TH FLOOR, SEVEN PENN CENTER
1635 MARKET STREET
PHILADELPHIA
PA
19103-2212
US
|
Family ID: |
29248426 |
Appl. No.: |
10/126845 |
Filed: |
April 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10126845 |
Apr 19, 2002 |
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09671089 |
Sep 27, 2000 |
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60156246 |
Sep 27, 1999 |
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Current U.S.
Class: |
514/1.2 ;
514/19.9 |
Current CPC
Class: |
A61K 9/1271 20130101;
A61K 38/17 20130101; C07K 7/08 20130101; C07K 14/50 20130101; A61K
9/5153 20130101; C07K 2319/00 20130101; B82Y 5/00 20130101; C07K
14/665 20130101; A61K 47/62 20170801; C12N 15/88 20130101; A61K
9/5138 20130101; A61K 48/00 20130101; A61K 47/65 20170801; A61K
47/64 20170801; A61K 47/6935 20170801; A61K 38/28 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Claims
We claim:
1. A composition comprising a translocating peptide, said
translocating peptide selected from the group consisting of a
transport peptide, an extended peptide comprising said transport
peptide, and a transport-active fragment of at least 4 amino acids
of said transport peptide, wherein said transport peptide is
selected from the group consisting of an L-peptide, a d-peptide,
and a retroinverted peptide, and wherein said L-peptide has an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 2-13, and 15-24, wherein said d-peptide has an amino acid
sequence selected from the group consisting of SEQ ID NOS. 102-124
corresponding to the d-forms of L-peptides of SEQ ID NOS. 2-24, and
wherein the retroinverted peptide has an amino acid sequence
selected from the group consisting of a peptide of SEQ ID NOS.
202-224, corresponding to retroinverted forms of L-peptides of SEQ
ID NOS: 2-24.
2. A composition of claim 1, wherein said L-peptide has an amino
acid sequence selected from the group consisting of SEQ ID NOS:
2-4, 16, 23 and 24. wherein said d-peptide has an amino acid
sequence selected from the group consisting of SEQ ID NOS. 102-104,
116, 123 and 124 corresponding to the d-forms of L-peptides of SEQ
ID NOS. 2-4,16, 23 and 24, wherein the retroinverted peptide has an
amino acid sequence selected from the group consisting of a peptide
of SEQ ID NOS. 202-204, 216, 223 and 224, corresponding to
retroinverted forms of an L-peptides of SEQ ID NOS: 2-4, 16, 23 and
24.
3. A composition of claim 1, wherein said transport peptide is
partially or completely cyclic.
4. A composition of claim 3 wherein any fragment of said transport
peptide is also partially or completely cyclic.
5. A composition of claim 4 where in the wherein said L-peptide has
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 5-13; wherein said d-peptide has an amino acid sequence
selected from the group consisting of SEQ ID NOS. 105-113
corresponding to the d-forms of L-peptides of SEQ ID NOS. 5-13,
wherein the retroinverted peptide has an amino acid sequence
selected from the group consisting of a peptide of SEQ ID NOS.
205-213, corresponding to retroinverted forms of L-peptides of SEQ
ID NOS: 5-13.
6. A composition of claim 1, wherein said translocating peptide is
an extended peptide of a transport peptide.
7. A composition of claim 6 wherein the extended peptide is not
more than 100 amino acids in length.
8. A composition of claim 7 wherein the extended peptide is not
more than 50 amino acids in length.
9. A composition of claim 1 wherein the translocating peptide is a
transport peptide.
10. A composition of claim 1 where in the transport-active fragment
is at least 6 amino acids of a transport peptide.
11. A composition of claim 1 where in the transport-active fragment
is at least 8 amino acids of a transport peptide.
11A. A composition of claim 1 wherein the translocating peptide is
selected from the group consisting of Elan 094, Elan178, Elan207,
and Elan208.
12. A composition of claim 1 wherein the carboxyl end group of the
translocating peptide has been modified to create an amide
group.
13. A composition comprising a translocating peptide, said
translocating peptide selected from the group consisting of a
transport peptide, an extended peptide comprising said transport
peptide, and a transport-active fragment of at least 4 amino acids
of said transport peptide, said transport peptide being an
L-peptide that has an amino acid sequence SEQ ID NO: 14 blocked at
its carboxyl end with an amide group and wherein any of said
fragments is also blocked at its carboxyl end with an amide
group.
14. The composition of claims 1 or 13, further comprising an active
agent, wherein the translocating peptide is complexed to an active
agent to form a translocating peptide-active agent complex.
15. The composition of claims 1 or 13, further comprising an active
particle, wherein the translocating peptide is complexed to the
active particle to form a translocating peptide-active particle
complex.
16. A method for enhancing movement of an active agent across a
lipid membrane, comprising using a translocating peptide-active
agent complex, wherein the translocating peptide enhances movement
of the active agent across the lipid membrane.
17. A method for enhancing movement of an active particle across a
lipid membrane, comprising using a translocating peptide-active
particle complex, wherein the translocating peptide enhances
movement of the active particle across the lipid membrane.
18. A method for identifying a compound having enhanced ability to
transport an active agent across a lipid membrane, wherein the
compound competes with the translocating peptide for transport of
an fMLP across a membrane selected from the group consisting of a
cell membrane, an intracellular membrane, the apical and basal
membranes of an epithelial cell layer.
19. The method of claim 18, wherein the epithelial cell layer is a
polarized epithelial cell layer.
20. A method for treating a pathological disorder in an animal,
comprising orally administering to the animal in need of such
treatment a complex selected from the group consisting of a
translocating peptide-active agent complex and a translocating
peptide-active particle complex, wherein an amount of the active
agent effective to treat the pathological disorder is moved across
the gastrointestinal epithelium of the animal into the
circulation.
21. A chimeric polypeptide comprising (A) a translocating peptide
of claims 1 or 13, (B) a translocatable peptide, and (C) an amino
acid linker sequence that directly links the translocating peptide
to the translocatable peptide, wherein said translocatable peptide
is between 3 and 200 amino acids, and wherein said amino acid
linker sequence is between 1 and 20 amino acids.
22. A chimeric peptide of claim 21 wherein said translocatable
peptide is between 3 and 30 amino acids.
23. A chimeric peptide of claim 21 wherein the translocatable
peptide is an opioid peptide.
24. A chimeric peptide of claim 21 wherein said linker sequence is
not more than 7 amino acids.
25. A chimeric peptide of claim 24 wherein said linker sequence is
not more than 3 amino acids.
26. A chimeric peptide of claim 25 wherein said linker sequence is
1 amino acids.
27. A chimeric peptide of claim 26 wherein said at least 50% of the
amino acids in the linker sequence are lysines.
28. A chimeric peptide of claim 26 wherein said at least 80% of the
amino acids in the linker sequence are lysines.
29. A chimeric peptide of claim 26 wherein all of the amino acids
in the linker sequence are lysines.
30. A method of delivering a chimeric peptide to the blood, said
method comprising orally administering a chimeric peptide of claim
21.
31. A nucleic acid molecule coding for a translocating peptide of
claim 1.
32. A nucleic acid molecule of claim 31 wherein the translocating
peptide is an L-form peptide.
33. A nucleic acid molecule coding for a chimeric protein of claim
21.
34. A nucleic acid molecule of claim 33 wherein the chimerica
peptide is an L-form peptide.
35. A chimeric constructs comprising (A) a translocating peptide of
claims 1 or 13, (B) a translocatable peptide, and (C) an non-amino
acid linker that directly links the translocating peptide to the
translocatable peptide, wherein said translocatable peptide is
between 3 and 200 amino acids.
36. A method of delivering a chimeric construct to a site within a
person, said method comprising administering a chimeric construct
of claim 35, said site being selected from the group consisting of
a tissue, a fluid, a cell, and a sub-cellular compartment.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 09/671,089 filed Sep. 27, 2000, incorporated by reference
herein in its entirety, and also claims the benefit of U.S.
provisional application serial No. ______ filed Apr. 30, 2001 with
the title "Lipid-Comprising Drug Delivery Complexes and Method for
Their Production", incorporated herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to peptides, which enhance
uptake of a pharmaceutically active agent into a cell, into or out
of an intracellular compartment, and across a cell layer. More
particularly, the present invention relates to membrane
translocating peptides, thereof and to the nucleotide sequences
coding therefor, which enhance uptake of a pharmaceutically active
agent into a cell, into or out of an intracellular compartment, and
across a cell layer either directly or from a pharmaceutically
active agent loaded particle.
BACKGROUND OF THE INVENTION
[0003] The epithelium lining the gastrointestinal tract
(hereinafter, "GIT") is a major barrier to absorption of orally
administered pharmaceutically active agents (hereinafter, "active
agents"). Absorption across the GIT epithelium can be by
transcellular transport through the cells and by paracellular
transport between the cells. Transcellular transport includes, but
is not limited to, receptor-mediated, transporter-mediated,
channel-mediated, pinocytotic and endocytotic mechanisms and to
diffusion. Paracellular transport includes, but is not limited to,
movement through tight junctions. Of particular interest is the
development of non-invasive methods for enhancing uptake of active
agents across the GIT epithelium into the body (Evers, P.
Developments in Drug Delivery: Technology and Markets, Financial
Times Management Report, 1995).
[0004] To develop non-invasive methods, phage display libraries
have been used to identify specific peptide sequences, which bind
preferentially to specific GIT membrane receptor, transporter,
channel, pinocytotic or endocytotic target pathways (hereinafter,
"targeting peptides") within the GIT. Included among the target
pathways, which have been screened with phage display libraries,
are the GIT membrane transporters HPT1, hPEPT1, D2H and hSI. HPT1
and hPEPT1 transport dipeptides and tripeptides. D2H transports
neutral and basic amino acids and is a transport activating protein
for a range of amino acid translocases. hSI is involved in sugar
metabolism and comprises 9% of the brush border protein in the
jejunum. Specific peptide sequences, which interact with the HPT1,
hPEPT1, D2H and hSI membrane transporters have been identified in
the following 4 applications, each of which is incorporated herein
in its entirety: U.S. patent applications Ser. Nos. 09/079,819,
09/079,723 and 09/079,678, and PCT application, PCT/US98/10088,
published as WO 98/51325.
[0005] Non-target pathway based assays have been used to identify
peptides with inherent cell membrane translocating properties.
These cell membrane translocating peptides interact directly with
and penetrate the lipids of cell membranes (Fong et al. Drug
Development Research 33:64, 1994). The central hydrophobic h-region
of the signal sequence of Kaposi's fibroblast growth factor,
AAVLLPVLLAAP (SEQ ID NO: 1) is considered to be a membrane
translocating peptide. This peptide (SEQ ID NO: 1) has been used as
a carrier to deliver various short peptides (<25 mer), through
the lipid bilayer, into living cells in order to study
intracellular protein functions and intracellular processes (Lin et
al. J. Biol. Chem. 271:5305,1996; Liu et al. Proc. Natl. Acad. Sci.
USA 93:11819, 1996; Rojas et al. J. Biol. Chem. 271:27456, 1996;
Rojas et al. Biochem. Biophys. Res. Commun. 234:675,1997). A 41-kDa
glutathione S-transferase fusion protein containing SEQ ID NO: 1
(GST-Grbs-SH.sub.2fused to SEQ ID NO: 1) has been shown to be
imported into NIH 3T3 fibroblasts and to inhibit epidermal growth
factor induced EGFR-Grb2 association and MAP kinase activation
(Rojas et al. Nature Biotechnology 16:370, 1998). However, these
studies do not address the use of membrane translocating peptides
to enhance active agent uptake into a cell, into and out of an
intracellular compartment, or across a cell layer when the active
agent is complexed to a membrane translocating peptide or when the
active agent is incorporated into a particle and the particle is
modified with (hereinafter, "complexed to") a membrane
translocating peptide.
[0006] The ability to enhance movement of an active agent across a
cell membrane is important because, although an active agent can be
administered to an animal by a variety of routes including, but not
limited to, oral, nasal, mucosal, topical transdermal, intravenous,
intramuscular, intraperitoneal, intrathecal and subcutaneous, oral
administration is the preferred route. Nasal, mucosal, topical and
transdermal administration depend on drug absorption through the
mucosa or skin into the circulation. Intravenous administration can
result in adverse effects from rapid accumulation of high
concentrations of drug, in patient discomfort and in infection at
the injection site. Intramuscular administration can cause pain at
the injection site. Subcutaneous administration is not suitable for
large volumes or for irritating substances. Although oral
administration is the preferred route, many active agents are not
absorbed efficiently across the GIT epithelium. This results from
enzymatic degradation of active agents within the lumen of the GIT,
from the limited permeability of the GIT epithelium to active
agents, from the large molecular size of active agents and from the
hydrophilic properties of active agents (Fix, JA. J. Pharmac. Sci.
85:1282, 1996). To develop an oral formulation, an active agent
must be protected from enzymatic digestion within the lumen of the
GIT, presented to the absorptive epithelial cells of the GIT in an
effective concentration and "moved" across the epithelium in an
apical to basolateral direction.
[0007] Therefore, because of the advantages of oral drug
administration, there is a need for delivery systems, which protect
orally ingested active agents from enzymatic degradation within the
lumen of the GIT and which promote the absorption of orally
ingested active agents into and across the epithelial cells lining
the GIT.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention fulfills the above-noted needs by
providing membrane translocating peptides (hereinafter referred to
interchangeably either as "MTLPs" or "translocating peptides") or
nucleotide sequences coding therefore, MTLP-active agent complexes
and MTLP-active particle complexes, wherein the MTLP enhances
movement of the active agent or the active particle across a lipid
membrane. More particularly, the present invention provides a MTLP,
MTLP-active agent complexes and MLTP-active particle complexes,
wherein the MTLP enhances movement of the active agent or of the
active particle into a cell, into and out of an intracellular
compartment and across a cell layer in an animal, including a
human. Methods of making and methods of using MTLPs, MTLP-active
agent complexes and MTLP-active particle complexes also are
included.
[0009] Compositions and Their Peptides
[0010] More precisely, in a first general aspect, the invention is
a composition comprising a translocating peptide, said
translocating peptide selected from the group consisting of a
transport peptide, an extended peptide comprising said transport
peptide, and a transport-active fragment of at least 4 amino acids
of said transport peptide, wherein said transport peptide is
selected from the group consisting of an L-peptide, a d-peptide,
and a retroinverted peptide, and
[0011] wherein said L-peptide has an amino acid sequence selected
from the group consisting of SEQ ID NOS: 2-13, and 15-24,
[0012] wherein said d-peptide has an amino acid sequence selected
from the group consisting of SEQ ID NOS. 102-124 corresponding to
the d-forms of L-peptides of SEQ ID NOS. 2-24, and
[0013] wherein the retroinverted peptide has an amino acid sequence
selected from the group consisting of a peptide of SEQ ID NOS.
202-224, corresponding to retroinverted forms of L-peptides of SEQ
ID NOS: 2-24.
[0014] A specific embodiment of the composition (first general
aspect) is one wherein terminal or near-terminal lysines play a
role:
[0015] the L-peptide has an amino acid sequence selected from the
group consisting of SEQ ID NOS: 2-4, 16, 23 and 24.
[0016] the d-peptide has an amino acid sequence selected from the
group consisting of SEQ ID NOS. 102-104, 116, 123 and 124
corresponding to the d-forms of L-peptides of SEQ ID NOS. 2-4, 16,
23 and 24, and
[0017] the retroinverted peptide has an amino acid sequence
selected from the group consisting of a peptide of SEQ ID NOS.
202-204, 216, 223 and 224 corresponding to retroinverted forms of
an L-peptides of SEQ ID NOS: 2-4, 16, 23 and 24.
[0018] In another specific embodiment of the composition, the
transport peptide is partially or completely cyclic. In a related
embodiment, any fragment of the transport peptide is also partially
or completely cyclic. Cyclic peptides of particular interest are
those in which
[0019] the L-peptide has an amino acid sequence selected from the
group consisting of SEQ ID NOS: 5-13;
[0020] the d-peptide has an amino acid sequence selected from the
group consisting of SEQ ID NOS. 105-113 corresponding to the
d-forms of L-peptides of SEQ ID NOS. 5-13, and
[0021] the retroinverted peptide has an amino acid sequence
selected from the group consisting of a peptide of SEQ ID NOS.
205-213, corresponding to retroinverted forms of L-peptides of SEQ
ID NOS: 5-13.
[0022] In another particular embodiment of the composition, the
translocating peptide is an extended peptide of a transport
peptide. Preferably, the extended peptide is not more than 100
amino acids in length, more preferably not more than 50 amino acids
in length.
[0023] In a further embodiment of the composition, the
translocating peptide is a transport peptide.
[0024] It is preferred that the transport-active fragment is at
least 6 amino acids, more preferably 8 amino acids of a transport
peptide.
[0025] In preferred embodiments, the carboxyl end group of the
translocating peptide is one that has been modified to create an
amide group.
[0026] Closely related to the above compositoins is one comprising
a translocating peptide, said translocating peptide selected from
the group consisting of a transport peptide, an extended peptide
comprising said transport peptide, and a transport-active fragment
of at least 4 amino acids of said transport peptide, said transport
peptide being an L-peptide that has an amino acid sequence SEQ ID
NO: 14 blocked at its carboxyl end with an amide group and wherein
any of said fragments is also blocked at its carboxyl end with an
amide group.
[0027] The foregoing compositions can, for example, further
comprise an active agent, wherein the translocating peptide is
complexed to an active agent to form a translocating peptide-active
agent complex.
[0028] Additionally, the compositions can, for example, further
comprise an active particle, wherein the translocating peptide is
complexed to the active particle to form a translocating
peptide-active particle complex.
[0029] Chimeric Peptides
[0030] Chimeric polypeptides comprising the translocating peptides
are also part of the invention. Specifically such polypeptides
comprise (A) a translocating peptide of this invention, (B) a
translocatable peptide, and (C) an amino acid linker sequence that
directly linkd the translocating peptide to the translocatable
peptide, wherein said translocatable peptide is between 3 and 200
amino acids, and wherein said amino acid linker sequence is between
1 and 20 amino acids.
[0031] In particular embodiments of the chimeric peptides, the
translocatable peptide is between 3 and 30 amino acids.
[0032] In other embodiments, the translocatable peptide is an
opioid peptide (examples of which are listed elsewhere herein).
[0033] In some particular embodiments, the linker sequence is not
more than 7 amino acids, preferably not more than 3 amino acids. In
some useful embodiments, the linker sequence is 1 amino acid.
[0034] In some preferred embodiments least 50% of the amino acids
in the linker sequence are lysines. More preferably at least 80% of
the amino acids in the linker sequence are lysines. Most preferably
all of the amino acids in the linker sequence are lysines.
[0035] Chimeric Constructs
[0036] Closely related to the chimeric peptides of this invention
are chimeric constructs. Such constructs comprise (A) a
translocating peptide of this invention, (B) a translocatable
peptide, and (C) a non-amino acid linker that directly links the
translocating peptide to the translocatable peptide, wherein said
translocatable peptide is between 3 and 200 amino acids.
[0037] Preferred non-amino acid linkers are those that have a
molecular weight of less than 1000 (more preferably less than
500).
[0038] Preferred non-amino acid linkers are those that provide the
chimeric construct at least 50% (more preferably at least 100%) of
the stability in SIF as a single lysine linker does for the
corresponding chimeric peptide when the translocating peptide is
Elan207 and the translocatable peptide is the kappa opioid peptide
(at 37.degree. C. for 1 hour, where stability is indicated by
retention of the structure of the chimeric structure or
peptide.)
[0039] Preferred non-amino acid linkers are those that provide the
chimeric construct with an IC50 that is not more than twice that of
the corresponding chimeric peptide when the translocating peptide
is Elan207 and the translocatable peptide is the kappa opioid
peptide and the IC50 is measured in the radio-labelled kappa
peptide rat brain homogenate assay described herein.
[0040] Two or more of the above preferred aspects for a non-amino
acid linker is even more preferable.
[0041] Examples of non-amino acid linkers are:
[0042] Hydrocarbon chains which can include both unsubstituted and
substituted alkyl, aryl, or macrocyclic R groups. Alkyl is intended
to mean any straight, branched, saturated, unsaturated or cyclic
C1-20 alkyl group. Aryl is intended to mean any aromatic cyclic
hydrocarbon based on a six-membered ring. Macrocycle refers to R
groups containing at least one ring containing more than seven
carbon atoms. Substituted is intended to mean any alkyl, aryl, or
macrocyclic groups in which at least one carbon atom is covalently
bonded to any functional groups comprising the atoms H. C, N, O, S,
F, Cl, Br and I. For details see Application PCT/US00/23440
published as WO 01/01/13957, pages 5-7 of which are incorporated by
reference herein in their entirety).
[0043] Additional possible linkers are summarized in PCT
application/US99/13660 published as WO 99/67284, especially pages
21-23;
[0044] Some linkers are well-suited for situations where cleavage
of the linker is desired only at specific sites in a person, for
example, specific tissue, specific fluid, specific cells, or
specific sub-cellular compartments. Examples of where some of the
linkers display such cleavage specificity is denoted in parentheses
after those linkers, as follows:
[0045] Amide (amidase sensitive)
[0046] Carbamate (stable in plasma, triggered release)
[0047] Disulphide (stable in plasma, reduced in cell compartments,
reduced during crossing of BBB)
[0048] Ester (pH sensitive, esterase sensitive)
[0049] Carbonate (pH sensitive, non-specific enzymatic
degradation)
[0050] Methods of the Invention
[0051] Related to the compositions of the invention are methods
that utilize them.
[0052] One method of the invention is one that delivers a chimeric
peptide to the blood, said method comprising orally administering a
chimeric peptide of the invention.
[0053] Another method of the invention is a method of delivering a
chimeric construct to a site within a person, said method
comprising administering a chimeric construct of this invention,
said site being selected from the group consisting of a tissue, a
fluid, a cell, and a sub-cellular compartment.
[0054] Another method of the invention is for enhancing movement of
an active agent across a lipid membrane, which comprises using a
translocating peptide-active agent complex, wherein the
translocating peptide enhances movement of the active agent across
the lipid membrane.
[0055] Another method of the invention is one for enhancing
movement of an active particle across a lipid membrane, which
comprises using a translocating peptide active particle complex,
wherein the translocating peptide enhances movement of the active
particle across the lipid membrane.
[0056] Still another method of the invention is one for identifying
a compound having enhanced ability to transport an active agent
across a lipid membrane, wherein the compound competes with the
translocating peptide for transport across a membrane selected from
the group consisting of a cell membrane, an intracellular membrane,
the apical and basal membranes of an epithelial cell layer. In a
particular embodiment, the epithelial cell layer is a polarized
epithelial cell layer.
[0057] Another method of the invention is one for treating a
pathological disorder in an animal, comprising orally administering
to the animal in need of such treatment a complex selected from the
group consisting of a translocating peptide-active agent complex
and a translocating peptide-active particle complex, wherein an
amount of the active agent effective to treat the pathological
disorder is moved across the gastrointestinal epithelium of the
animal into the circulation.
[0058] MTLPs of the present invention are capable of displaying one
or more known functional activities associated with a full-length
MTLP. Such functional activities include, but are not limited to,
the ability to interact with a membrane and the ability to compete
for transport of a reporter drug molecule (fMLP) across epithelial
cells including, but not limited to, polarized, differentiated
human derived Caco-2 cells. Additional functional activities
include, but are not limited to, antigenicity, which includes, but
is not limited to, the ability to bind to an anti-MTLP antibody and
the ability to compete with a MTLP for interaction with a membrane;
and, immunogenicity, which includes, but is not limited to, the
ability to stimulate antibody generation.
[0059] Methods of making a MTLP-active agent complex include, but
are not limited to, covalent coupling of a MTLP and an active agent
and noncovalent coupling of a MTLP and an active agent. Methods of
making a MTLP-active particle complex include, but are not limited
to, incorporating an active agent into a particle including, but
not limited to, a nanoparticle, a microparticle, a capsule, a
liposome, a non-viral vector system and a viral vector system. The
MTLP can be complexed to the active particle by methods including,
but not limited to, adsorption to the active particle, noncovalent
coupling to the active particle and covalent coupling, either
directly or via a linker, to the active particle, to the polymer or
polymers used to synthesize the active particle, to the monomer or
monomers used to synthesize the polymer, and to other components
comprising the active particle.
[0060] The present invention also includes the nucleotide
sequences, which code for the MTLPs. Methods of making nucleotide
sequences include, but are not limited to, recombinant means.
[0061] MTLPs, MTLP-active agent complexes and MTLP-active particle
complexes can be used alone, in combination with or conjugated to
other molecules including, but not limited to, molecules that bind
to target pathways, to nuclear uptake pathways and to endosomal
pathways, molecules that enable mucoadhesion, molecules that
facilitate diffusion across lipid membranes or through water filled
pores and molecules that regulate or direct intra-cellular
trafficking. That is, by using different mechanisms simultaneously,
active agent bioavailability may be enhanced.
[0062] Related inventions are the use of translocating peptides
(i.e., MTLPs) in the following:
[0063] a composition comprising a translocating peptide-active
particle complex, wherein the particle is a microparticle;
[0064] a composition comprising a translocating peptide-active
particle complex, wherein the particle is a nanoparticle;
[0065] a composition comprising a translocating peptide-active
particle complex, wherein the particle is a liposome;
[0066] a composition comprising a viral DNA particle, wherein the
viral particle is modified to express a translocating peptide on
its surface;
[0067] a composition comprising a viral DNA particle, wherein the
viral particle is complexed to a translocating peptide following
virus production and purification;
[0068] a composition comprising a viral DNA particle, wherein the
viral particle is complexed to a translocating peptide following
virus production in and purification from a mammalian cell; and
[0069] a composition comprising a non-viral based gene delivery
system, wherein the non-viral based gene delivery system is
complexed to a translocating peptide.
[0070] Further related inventions are the use of translocating
peptides in the following methods:
[0071] a method to enhance the movement of an active agent across a
lipid membrane;
[0072] a method to enhance the uptake of an active agent into a
cell;
[0073] a method to enhance the uptake of an active agent across a
cell layer;
[0074] a method to enhance the uptake of an active agent into an
epithelial cell;
[0075] a method to enhance the uptake of an active agent across an
epithelial cell layer;
[0076] a method to enhance the uptake of an active agent across the
epithelial cell layer lining the GIT into the circulation of an
animal;
[0077] a method to enhance the movement of an active particle
across a lipid membrane;
[0078] a method to enhance the uptake of an active particle into a
cell;
[0079] a method to enhance the uptake of an active particle across
a cell layer;
[0080] a method to enhance the uptake of an active particle into an
epithelial cell;
[0081] a method to enhance the uptake of an active particle across
an epithelial cell layer;
[0082] a method to enhance the uptake of an active particle across
the epithelial cell layer lining the GIT into the circulation of an
animal;
[0083] a method to provide intracellular gene delivery by a
non-viral based gene delivery system;
[0084] a method to provide intracellular gene delivery by a
non-viral based gene delivery system, wherein the non-viral based
gene delivery system is complexed to a translocating peptide;
[0085] a method to provide a rapid screening method to identify
translocating peptides, which retain the essential functional
activity of the full-length translocating peptide;
[0086] a method to provide cell-based screens for assaying the
functional activity of; and
[0087] a method to provide cell-based screens for characterizing
the properties of a translocating peptide.
[0088] Another aspect of the present invention is a method to
provide a method for diagnosing a pathological disorder by oral
administration of an amount of a translocating peptide-active agent
complex, wherein the active agent is a diagnostic agent, such that
the systemic concentration of the diagnostic agent is effective to
diagnose the pathological disorder.
[0089] Another aspect of the present invention is a method to
provide a method for preventing a pathological disorder by oral
administration of a translocating peptide-active agent complex,
wherein the active agent is a prophylactic agent, such that the
systemic concentration of the prophylactic agent is effective to
prevent the pathological disorder.
[0090] Another aspect of the present invention is a method for
treating a pathological disorder by oral administration of a
translocating peptide-active agent complex, wherein the active
agent is a therapeutic agent, such that the systemic concentration
of the therapeutic agent is effective to treat the pathological
disorder.
[0091] Another aspect of the present invention is a method to
provide a method for diagnosing a pathological disorder by oral
administration of a translocating-active particle complex, wherein
the active particle contains a diagnostic agent, such that the
systemic concentration of the diagnostic agent is effective to
diagnose the pathological disorder.
[0092] Another aspect of the present invention is a method to
provide a method for preventing a pathological disorder by oral
administration of a a translocating peptide-active particle
complex, wherein the active particle contains a prophylactic agent,
such that the systemic concentration of the prophylactic agent is
effective to prevent the pathological disorder.
[0093] Another aspect of the present invention is a method to
provide a method for treating a pathological disorder by oral
administration of a a translocating peptide-active particle
complex, wherein the active particle contains a therapeutic agent
such that the systemic concentration of the therapeutic agent is
effective to treat the pathological disorder.
[0094] Other objectives, features and advantages of the present
invention will become apparent after a review of the following
detailed description of the disclosed embodiments and the appended
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] FIG. 1 shows the hydropathy plot for ZElan094 (16 mer) (SEQ
ID NO: 2);
[0096] FIG. 2 shows the systemic blood insulin levels following in
vivo delivery of insulin from a ZElan094-insulin nanoparticle
complex and from HAX42-, PAX2- and P31-insulin nanoparticle
complexes in the open loop rat model. Each point is the mean of 6-7
animals;
[0097] FIG. 3 shows the systemic blood glucose levels following in
vivo delivery of insulin from a ZElan094-insulin nanoparticle
complex and from HAX42-, PAX2- and P31-insulin nanoparticle
complexes in the open loop rat model. Each point is the mean of 6-7
animals;
[0098] FIG. 4 shows the transport of the reporter drug .sup.3H-fMLP
across Caco-2 monolayers in the presence of the MTLPs ZElan094,
178, 187 and the targeting peptide ZElan022;
[0099] FIG. 5 shows the transport of the reporter drug .sup.3H-fMLP
across Caco-2 monolayers in the presence of increasing
concentrations of the MTLP ZElan094.
[0100] FIG. 6 shows the transport of .sup.3H-Kappa peptide
conjugates across Caco-2 monolayers.
DETAILED DESCRIPTION OF THE INVENTION
[0101] The present invention relates to novel membrane
translocating peptides (MTLPs or, alternatively, "translocating
peptides") to nucleotide sequences coding therefor, to MTLP-active
agent complexes and to MTLP-active particle complexes, wherein the
MTLP enhances movement of the active agent or of the active
particle across a membrane. More particularly, the present
invention relates to novel MTLPs, to nucleotide sequences coding
therefore, to MTLP-active agent complexes and to MTLP-active
particle complexes, wherein the MTLP enhances movement of the
active agent in the MTLP-active agent complex, of the active agent
in the MTLP-active particle complex and of the active particle in
the MTLP active-particle complex into a cell, into and out of an
intracellular compartment and across a cell layer in an animal,
including a human. Methods of making and methods of using MTLPs
also are included.
[0102] The present invention also provides methods for diagnosing,
preventing or treating a pathological disorder in an animal in need
of diagnosis, prevention or treatment of a pathological disorder by
administrating to the animal an amount of a MTLP-active agent
complex or of a MTLP-active particle complex, such that the
systemic concentration of the active agent is effective to
diagnose, prevent or treat the pathological disorder.
[0103] An "active agent", as used herein, includes any diagnostic,
prophylactic or therapeutic agent that can be used in an animal,
including a human.
[0104] An "active particle", as used herein is a particle into
which one or more active agents have been loaded.
[0105] A membrane translocating peptide, as used herein, is a
peptide which interacts directly with and penetrates the lipids of
a physiological membrane.
[0106] A "MTLP", as used herein, is a general term that refers to
any translocating peptides refered to herein. Specific MTLPs where
sequences are described herein can be part of larger peptides or
polypeptides, all which, in turn, are MTLPs. Transport-active
fragements of MTLPs are also MTLPs.
[0107] A "transport-active fragment" of a translocating peptide is
one that increases the plasma .sup.3H bioavailability of a Kappa
peptide by 30%, compared to the Kappa peptide above after
intraduodenal installation in the Wistar rat model described herein
in Example 15.
[0108] The terms "peptide" and "polypeptide" are used to some
extent interchangebly herein and no precise size demarcation
between the two is intended.
[0109] A "composition comprising a translocating peptide" could
include not only homogeneous composition consisting only of
particular peptide, but also compositions that contain additional
components, including end-group moities that are covalently linked
to the amino or carboxyl end of such translocating peptides.
Specific examples of such in end-group moieties, such as amide,
dansyl and biotin groups, are provided herein.
[0110] The term "translocating peptide" is used for convenience in
phrasing claims that refer to a group of various possible transport
peptides. No biochemical difference in function between
translocating and transport peptides is intended.
[0111] "Complexed to", as used herein, includes adsorption,
non-covalent coupling and covalent coupling of a MTLP to an active
agent or to an active particle.
[0112] A "MTLP-active agent complex", as used herein, includes one
or more MTLPs complexed to an active agent.
[0113] A "MTLP-active particle complex", as used herein, includes
one or more MTLPs complexed to an active particle.
[0114] The active agent used depends on the pathological condition
to be diagnosed, prevented or treated, the individual to whom it is
to be administered, and the route of administration. Active agents
include, but are not limited to, imaging agents, antigens,
antibodies, oligonucleotides, antisense oligonucleotides, genes,
gene correcting hybrid oligonucleotides, aptameric
oligonucleotides, triple-helix forming oligonucleotides, ribozymes,
signal transduction pathway inhibitors, tyrosine kinase inhibitors,
DNA-modifying agents, therapeutic genes, systems for therapeutic
gene delivery, drugs and other agents including, but not limited
to, those listed in the United States Pharmacopeia and in other
known pharmacopeias
[0115] Drugs include, but are not limited to, peptides, proteins,
hormones and analgesics, cardiovascular, narcotic, antagonist,
chelating, chemotherapeutic, sedative, anti-hypertensive,
anti-anginal, anti-migraine, anti-coagulant, anti-emetic
anti-neoplastic and anti-diuretic agents Hormones include, but are
not limited to, insulin, calcitonin, calcitonin gene regulating
protein, atrial natriuretic protein, colony stimulating factor,
erythropoietin (EPO), interferons, somatotropin, somatostatin,
somatomedin, luteinizing hormone releasing hormone (LHRH), tissue
plasminogen activator (TPA), growth hormone releasing hormone
(GHRH), oxytocin, estradiol, growth hormones, leuprolide acetate,
factor VIII, testosterone and analogs thereof. Analgesics include,
but are not limited to, fentanyl, sufentanil, butorphanol,
buprenorphine, levorphanol, morphine, hydromorphone, hydrocodeine,
oxymorphone, methadone, lidocaine, bupivacaine, diclofenac,
naproxen, paverin, and analogs thereof. Anti-migraine agents
include, but are not limited to heparin, hirudin, and analogs
thereof. Anti-coagulant agents include, but are not limited to,
scopolamine, ondansetron, domperidone, etoclopramide, and analogs
thereof. Cardiovascular, anti-hypertensive and vasodilator agents
include, but are not limited to, diltiazem, clonidine, nifedipine,
verapamil, isosorbide-5-mononitrate, organic nitrates,
nitroglycerine and analogs thereof. Sedatives include, but are not
limited to, benzodiazeines, phenothiozines and analogs thereof.
Narcotic antagonists include, but are not limited to, naltrexone,
naloxone and analogs thereof. Chelating agents include, but are not
limited to deferoxamine and analogs thereof. Anti-diuretic agents
include, but are not limited to, desmopressin, vasopressin and
analogs thereof. Anti-neoplastic agents include, but are not
limited to, 5-fluorouracil, bleomycin, vincristine, procarbazine,
temezolamide, CCNU, 6-thioguanine, hydroxyurea and analogs
thereof.
[0116] An active agent can be formulated in neutral or salt form.
Pharmaceutically acceptable salts include, but are not limited to,
those formed with free amino groups; those formed with free
carboxyl groups; and, those derived from sodium, potassium,
ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine,
2-ethylaminoethanol, histidine and procaine. An active agent can be
loaded into a particle prepared from pharmaceutically acceptable
ingredients including, but not limited to, soluble, insoluble,
permeable, impermeable, biodegradable or gastroretentive polymers
or liposomes. Such particles include, but are not limited to,
nanoparticles, biodegradable nanoparticles, microparticles,
biodegradable microparticles, nanospheres, biodegradable
nanospheres, microspheres, biodegradable microspheres, capsules,
emulsions, liposomes, micelles and viral vector systems.
[0117] MTLPs have functional activities. Such functional activities
include, but are not limited to, enhancing uptake of an active
agent into a cell, into and out of an intracellular compartment and
across a cell layer and competing with the full-length peptide in
enhancing uptake of an active agent into a cell, across a cell
layer or into and out of an intracellular compartment.
[0118] Examples of MTLPs of the present invention include, but are
not limited, to those containing as primary amino acid sequences,
all or part of the amino acid sequences substantially as depicted
in Table 1
1TABLE 1 MTLPs Amino acid sequences SEQUENCE + K(.epsilon.-dansyl)
ELAN NO. SEQ ID NO. H-K(.epsilon.-dansyl)KKAAAVLLPVLLAAP-NH2
ZElan094 2 H-KKAAAVLLPVLLAAP-FITC-LC-NH2 FElan094 3
H-K(.epsilon.-dansyl)KKA- AAVLLPVLLAAPREDL-NH2 ZElan094R 4
H-K(.epsilon.-dansyl)KKCAAVLLPVLL- AAPC-NH2 ZElan176 5
H-K(.epsilon.-dansyl)CAAVLLPVLLAAC-NH2 ZElan177 6
H-K(.epsilon.-dansyl)KKCAAVLLPVLLAC-NH2 ZElan178 7
H-K(.epsilon.-dansyl)CAAVLLPVLLC-NH2 ZElan179 8
H-K(.epsilon.-dansyl)CAAVLLPVLC-NH2 ZElan180 9
H-K(.epsilon.-dansyl)CAVLLPVLLAAPC-NH2 ZElan181 10
H-K(.epsilon.-dansyl)CVLLPVLLAAPC-NH2 ZElan182 11
H-K(.epsilon.-dansyl)CLLPVLLAAPC-NH2 ZElan183 12
H-K(.epsilon.-dansyl)CLPVLLAAPC-NH2 ZElan184 13
H-K(.epsilon.-dansyl)AAVLLPVLLAAP-NH2 ZElan185 14
H-K(.epsilon.-dansyl)AAVLLPVLLAA-NH2 ZElan186 15
H-K(.epsilon.-dansyl)KKAAVLLPVLLA-NH2 ZElan187 16
H-K(.epsilon.-dansyl)AAVLLPVLL-NH2 ZElan188 17
H-K(.epsilon.-dansyl)AAVLLPVL-NH2 ZElan189 18
H-K(.epsilon.-dansyl)AVLLPVLLAAP-NH2 ZElan190 19
H-K(.epsilon.-dansyl)VLLPVLLAAP-NH2 ZElan191 20
H-K(.epsilon.-dansyl)LLPVLLAAP-NH2 ZElan192 21
H-K(.epsilon.-dansyl)LPVLLAAP-NH2 ZElan193 22
H-K(.epsilon.-dansyl)AAVLLPVLLAAKKKRKA-NH2 Zelan204N 23
H-K(.epsilon.-dansyl)KKKRKAAAAVLLPVLLA-NH2 ZElanN204 24 [Underline
denotes cyclisation]
[0119] An L-peptide that has an amino acid sequence of SEQ ID NO: 2
would be KKAAAVLLPVLLAAP. A composition that comprises ZElan094,
comprises an L-peptide of SEQ ID NO: 2, and further comprises both
a K(.epsilon.-dansyl) group and an amide group.
[0120] The 16 residue hydrophobic peptide ZElan094 (SEQ ID NO: 2)
is related in sequence to the 12 residue hydrophobic peptide
sequence AAVLLPVLLAAP (SEQ ID NO: 1) (Rojas et al. Nature
Biotechnology 16:370, 1998). However, the 16 residue ZElan094
differs from the 12 residue SEQ ID NO: 1 in that it has four
additional amino acid residues, KKKA, at the N-terminus and a
blocking amide at the C-terminus. These N-terminus and C-terminus
modifications are designed to enhance the solubility and the in
vivo stability of the MTLP, respectively. The NH.sub.2 terminus
alanine also may contribute to the alpha helical properties of the
peptide.
[0121] The MTLPs of the present invention include peptides
comprising all of or a fragment of ZElan094 or having at least 4 of
the contiguous amino acids of ZElan094. The MTLPs of the present
invention also include sequences that are substantially homologous
to regions of ZElan094. Preferably these show at least 70%, 80% or
90% identity over an identical size sequence.
[0122] It is understood that a person in the art can make chemical
changes in the MTLPs without significantly altering their
activity.
[0123] Examples of nucleic acid sequences, which encode the peptide
sequences of the MTLPs ZElan094, Felan 094, ZElan 094R, 176-193,
204N and N204 (SEQ ID NOS: 2-24) are provided in Table 2 (SEQ ID
NOS: 25-47). However, due to the degeneracy of nucleotide coding
sequences, different nucleotide sequences, which encode
substantially the same amino acid sequence, may be used. That is, a
nucleotide sequence, altered by substitution of a different codon,
can encode a functionally equivalent amino acid to produce a silent
change.
[0124] MTLPs may be synthesized using chemical methods (U.S. Pat.
Nos. 4,244,946, 4,305,872 and 4,316,891; Merrifield et al. J. Am.
Chem. Soc. 85:2149, 1964; Vale et al. Science 213:1394, 1981; Marki
et al. J. Am. Chem. Soc. 103:3178, 1981); recombinant DNA methods
(Maniatis, Molecular Cloning, a Laboratory Manual, 2d ed. Cold
Spring Harbor Laboratory, Cold Spring Harbor N.Y., 1990); viral
expression or other methods known to those skilled in the art.
[0125] Chemical methods include, but are not limited to, solid
phase peptide synthesis. Briefly, solid phase peptide synthesis
consists of coupling the carboxyl group of the C-terminal amino
acid to a resin and successively adding N-alpha protected amino
acids. The protecting groups may be any known in the art. Before an
amino acid is added to the growing peptide chain, the protecting
group of the previous amino acid is removed (Merrifield J. Am.
Chem. Soc. 85:2149 1964; Vale et al. Science 213:1394, 1981; Marki
et al. J. Am. Chem. Soc. 103:3178,1981). The synthesized peptides
are then purified by methods known in the art.
2TABLE 2 MTLPs nucleic acid sequence SEQ ID ZElan NO: NO: Sequence
25 94 AARAARAARGCNGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNCCN 26 Felan094
AARAARAARGCNGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNCCN 27 094R
AARAARAARGCNGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNCCNMGNGARGAYYTN 28
176 AARAARAARTGYGCNGCNGTNYTNY- TNCCNGTNYTN YTNGCNGCNCCNTGY 29 177
AARAARAARTGYGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNTGY 30 178
AARAARAARTGYGCNGCNGTNYTNYTNCCNGTNYT NYTNGCNTGY 31 179
AARTGYGCNGCNGTNYTNYTNCCNGTNYTNYTNTGY 32 180
AARTGYGCNGCNGTNYTNYTNCCNGTNYTNTGY 33 181
AARTGYGCNGTNYTNYTNCCNGTNYTNYTNGCNGCN CCNTGY 34 182
AARTGYGTNYTNYTNCCNGTNYTNYTNGCNGCNCCN TGY 35 183
AARTGYYTNYTNCCNGTNYTNYTNGCNGCNCCNTGY 36 184
AARTGYYTNCCNGTNYTNYTNGCNGCNCCNTGY 37 185
AARGCNGCNGTNYTNYTNCCNGTNYTNYTNGCNGCN CCN 38 186
AARGCNGCNGTNYTNYTNCCNGTNYTNYTNGCNGCN 39 187
AARAARAARGCNGCNGTNYTNYTNCCNGTNYTNYTN GCN 40 188
AARGCNGCNGTNYTNYTNCCNGTNYTNYTN 41 189
AARGCNGCNGTNYTNYTNCCNGTNYTNYTN 42 190
AARGCNGTNYTNYTNCCNGTNYTNYTNGCNGCNCCN 43 191
AARGTNYTNYTNCCNGTNYTNYTNGCNGCNCCN 44 192
AARYTNYTNCCNGTNYTNYTNGCNGCNCCN 45 193 AARYTNCCNGTNYTNYTNGCNGCNCCN
46 204N AARGCNGCNGTNYTNYTNCCNGTNYTNYTNGCNGCN AARAARAARMGNAARGCN 47
N204 AARAARAARAARMGNAARGCNGCNGCNGCNGTNYTN YTNCCNGTNYTNYTNGCN
[0126] Preferably, solid phase peptide synthesis is done using an
automated peptide synthesizer such as, but not limited to, an
Applied Biosystems Inc. (ABI) model 431A using the "Fastmoc"
synthesis protocol supplied by ABI. This protocol uses
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetr- amethyluronium
hexafluorophosphate (HBTU) as coupling agent (Knorr et al. Tet.
Lett. 30:1927,1989). Syntheses can be carried out on 0.25 mmol of
commercially available 4-(2',
4'-dimethoxyphenyl-(9-fluorenyl-ethoxycarbo- nyl)-aminomethyl)
phenoxy polystyrene resin (Rink H. Tet. Lett. 28:3787, 1987). Fmoc
amino acids (1 mmol) are coupled according to the Fastmoc protocol.
N-methylpyrrolidone (NMP) is used as solvent, with HBTU dissolved
in N,N-dimethylformamide (DMF). The following side chain protected
Fmoc amino acid derivatives are used: FmocArg(Pmc)OH;
FmocAsn(Mbh)OH; FmocAsp(tBu)OH; FmocCys(Acm)OH; FmocGlu(tBu)OH;
FmocGln(Mbh)OH; FmocHis(Tr)OH; FmocLys(Boc)OH; FmocSer-(tBu)OH;
FmocThr(tBu)OH; FmocTyr(tBu)OH. (Abbreviations:
Acm:acetamidomethyl; Boc:tert-butoxycarbonyl; tBu:tert-butyl; Fmoc:
9-fluorenylmethoxy-carbony- l; Mbh:4,4'-dimethoxybenzhydryl;
Pmc:2,2,5,7,8-pentamethyl-chro-man-6-sulf- onyl; Tr:5 trityl.)
[0127] At the end of each synthesis, the amount of peptide is
assayed by ultraviolet spectroscopy. A sample of dry peptide resin
(about 3-10 mg) is weighed, then 20% piperidine in DMA (10 ml) is
added. After 30 min sonication, the UV (ultraviolet) absorbance of
the dibenzofulvene-piperidine adduct (formed by cleavage of the
N-terminal Fmoc group) is recorded at 301 nm. Peptide substitution
(in mmol/g) is calculated according to the equation: 1 Substitution
= A .times. v .times. 1000 7800 .times. w
[0128] where A is the absorbance at 301 nm, v the ml of 20%
piperidine in DMA, 7800 the extinction coefficient
(mol/dm.sup.3/cm) of the dibenzofulvene-piperidine adduct, and w
the mg of peptide resin sample. The N-terminal Fmoc group is
cleaved using 20% piperidine in DMA, then acetylated using acetic
anhydride and pyridine in DMA. The peptide resin is thoroughly
washed with DMA, CH.sub.2C.sub.12 and diethyl ether.
[0129] Methods used for cleavage and deprotection (King et al. Int.
J. Peptide Protein Res. 36:255, 1990) include, but are not limited
to, treating the air-dried peptide resin with ethylmethyl-sulfide
(EtSMe), ethanedithiol (EDT) and thioanisole (PhSMe) for
approximately 20 min and adding 95% aqueous trifluoracetic acid
(TFA). Approximately 50 ml of these reagents are used per gram of
peptide resin in a ratio of TFA:EtSMe:EDT:PhSme (10:0.5:0.5:0.5).
The mixture is stirred for 3 h at RT under an N.sub.2 atmosphere,
filtered and washed with TFA (2.times.3 ml). The combined filtrate
is evaporated in vacuo and anhydrous diethyl ether is added to the
yellow/orange residue. The resulting white precipitate is isolated
by filtration. Purification of the synthesized peptides is done by
standard methods including, but not limited to, ion exchange,
affinity, sizing column and high performance liquid chromatography,
centrifugation or differential solubility.
[0130] Recombinant DNA methods for expressing peptides are well
known to those skilled in the art and include expression in a
biological system including, but not limited to a mammalian system,
an insect system, a plant system and a viral system (Maniatis, T.
Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1990). For example, a MTLP
can be expressed by a virus, by a virus fused to a viral coat
protein, a viral capsid protein or a viral surface protein.
Further, MTLP-viral protein complexes can be expressed in mammalian
hosts or in helper viruses used to produce the virus of
interest.
[0131] In the production of a gene encoding an extended version of,
or a fragmentof a full-length peptide, care should be taken to
ensure that the modified gene remains within the same translational
reading frame uninterrupted by translational stop signals in the
gene region where the desired activity is encoded.
[0132] Further, phage display vectors including, but not limited
to, bacteriophage M13 or bacteriophage Fd can be modified to
express a MTLP fused to the gene III protein product or gene VII
protein product of the bacteriophage. A library of sequences coding
for MTLPs or potential MTLPs can be created including, but not
limited to, alanine scan positional mutants, successive random
positional scanning mutants and sequences derived therefrom as, for
example, those shown in Table 1, can be cloned in-frame to either
gene II or gene VII of the bacteriophage. The phage display library
can then be screened to identify new MTLPs having enhanced ability
to transport active agents or active particles across
membranes.
[0133] Chimeric or fusion peptides include, without limitation,
those comprising a MTLP or multiple repeats thereof, preferably
consisting of at least one domain or motif of the full-length
peptide sequence or a portion thereof joined at its amino-terminus,
at its carboxy-terminus or at an internal site via a peptide bond
to an amino acid sequence of a different peptide. Methods for
producing chimeric peptides include, but are not limited to,
recombinant expression of a nucleic acid including the MTLP coding
sequence joined in-frame to the coding sequence of a different
peptide. Using methods known in the art, the nucleic acid sequences
encoding the desired amino acid sequences are ligated to each other
in the proper order and the chimeric product is expressed. For
example, chimeric genes comprising portions of MTLP nucleic acid
fused to any heterologous protein-encoding nucleic acid may be
constructed. Alternatively, chimeric MTLPs may be synthesized using
techniques including, but not limited to, a peptide
synthesizer.
3 Opioid peptides include those contained in the
corticotropin-lipoportein precursor, the proenkaphalin A precursor,
and the beta-neoendorphin-dynorphin precursor, as follows:
COLI_HUMAN (P01189) Corticotropin-lipotropin precursor
(Pro-opiomelanocortin) (POMC) Contains: NPP; Melanotropin gamma
(Gamma-MSH); Corticotropin (Adrenocorticotropic hormone) (ACTH);
Melanotropin alpha (Alpha-MSH); Corticotropin-like intermediary
peptide (CLIP); Lipotropin beta (Beta-LPH); Lipotropin gamma
(Gamma-LPH); Melanotropin beta (Beta-MSH); Beta-endorphin; and Met-
enkephalin]. PENK_HUMAN (P01210) Proenkephalin A precursor
contains: Met-enkephalin;and Leu-enkephalin. NDDB_HUMAN (P01213)
Beta-neoendorphin-dynorphin precursor (Proenkephalin B)
(Preprodynorphin) contains: Beta-neoendorphin; Dynorphin;
Leu-Enkephalin; Rimorphin; and Leumorphin]. Their amino acid
sequences as obtained from the SWISSPROT database are as follows:
LOCUS COLI_HUMAN 267 aa linear PRI 01-MAR-2002 DEFINITION
Corticotropin-lipotropin precursor (Pro-opiomelanocortin) (POMC)
[Contains: NPP; Melanotropin gamma (Gamma-MSH); Corticotropin
(Adrenocorticotropic hormone) (ACTH); Melanotropin alpha
(Alpha-MSH); Corticotropin-like intermediary peptide (CLIP);
Lipotropin beta (Beta-LPH); Lipotropin gamma (Gamma-LPH);
Melanotropin beta (Beta-MSH); Beta-endorphin; Met-enkephalin].
ACCESSION P01189 PID g116880 VERSION P01189 GI:116880 DBSOURCE
swissprot: locus COLI_HUMAN, accession P01189; SOURCE human. Region
27..102 = NPP Region 77..87 ="MELANOTROPIN GAMMA." Region 105..134
="?" Region 138..150 =MELANOTROPIN ALPHA." Region 138..176
="CORTICOTROPIN." Region 156..176 ="CORTICOTROPIN-LIKE INTERMEDIARY
PEPTIDE." Region 179..234 ="LIPOTROPIN GAMMA." Region 179..267
="LIPOTROPIN BETA." Region 217..234 ="MELANOTROPIN BETA." Region
237..241 ="MET-ENKEPHALIN." Region 237..267 ="BETA-ENDORPHIN."
ORIGIN 1 mprsccsrsg alllalllqa smevrgwcle ssqcqdltte snllecirac
kpdlsaetpm 61 fpgngdeqpl tenprkyvmg hfrwdrfgrr nssssgssga
gqkredvsag edcgplpegg 121 peprsdgakp gpregkrsys mehfrwgkpv
gkkrrpvkvy pngaedesae afplefkrel 181 tgqrlregdg pdgpaddgag
aqadlehsll vaaekkdegp yrmehfrwgs ppkdkryggf 241 mtseksqtpl
vtlfknaiik naykkge -----------------------------
---------------------------------------------- LOCUS PENK_HUMAN 267
aa linear PRI 15-JUL-1999 DEFINITION PROENKEPHALIN A PRECURSOR
[CONTAINS: MET-ENKEPHALIN; LEU-ENKEPHALIN]. ACCESSION P01210 PID
g129770 VERSION P01210 GI:129770 DBSOURCE swissprot: locus
PENK_HUMAN, accession P01210; SOURCE human. Region 100..104
="MET-ENKEPHALIN 1." Region 107..111 ="MET-ENKEPHALIN 2." Region
136..140 ="MET-ENKEPHALIN 3." Region 186..193
="MET-ENKEPHALIN-ARG-GLY-LEU." Region 210..214 ="MET-ENKEPHALIN 4."
Region 230..234 ="LEU-ENKEPHALIN." Region 261..267 ="MET-EN
KEPHALIN-ARG-PHE." ORIGIN 1 marfltlctw llllgpglla tvraecsqdc
atcsyrlvrp adinflacvm ecegklpslk 61 iwetckellq lskpelpqdg
tstlrenskp eeshllakry ggfmkryggf mkkmdelypm 121 epeeeangse
llakryggfm kkdaeeddsl anssdllkel letgdnrers hhqdgsdnee 181
evskryggfm rglkrspqle deakelqkry ggfmrrvgrp ewwmdyqkry ggflkrfaea
241 lpsdeegesy skevpemekr yggfmrf //
----------------------------------------------------------------
LOCUS PENK_HUMAN 267 aa linear PRI 15-JUL-1999 DEFINITION
PROENKEPHALIN A PRECURSOR [CONTAINS: MET-ENKEPHALIN
LEU-ENKEPHALIN]. ACCESSION P01210 PID g129770 VERSION P01210
GI:129770 DBSOURCE swissprot: locus PENK_HUMAN, accession P01210;
SOURCE human. Region 100..104 ="MET-ENKEPHALIN 1." Region 107..111
="MET-ENKEPHALIN 2." Region 136..140 ="MET-ENKEPHALIN 3." Region
186..193 ="MET-ENKEPHALIN-ARG-GLY-LEU." Region 210..214
="MET-ENKEPHALIN 4." Region 230..234 ="LEU-ENKEPHALIN." Region
261..267 ="MET-ENKEPHALIN-ARG-PHE." ORIGIN 1 marfltlctw llllgpglla
tvraecsqdc atcsyrlvrp adinflacvm ecegklpslk 61 iwetckellq
lskpelpqdg tstlrenskp eeshllakry ggfmkryggf mkkmdelypm 121
epeeeangse ilakryggfm kkdaeeddsl anssdllkel letgdnrers hhqdgsdnee
181 evskryggfm rglkrspqle deakelqkry ggfmrrvgrp ewwmdyqkry
ggflkrfaea 241 lpsdeegesy skevpemekr yggfmrf //
----------------------------------------------------------
[0134] The SwissProt database records for accession numbers P01189,
P01210, and P01213 as they appeared on Apr. 17, 2002 are
incorporated herein by reference in their entireties.
[0135] Additional opioid peptides include, but are not limited
to:
[0136] boc-Tyr-Tic
[0137] cy-[Tyr-Tic]
[0138] Tyr-D-Tic
[0139] Tyr-D-Tic-NH2
[0140] Tyr-cy-[D-Cys-Phe-D-Pen]
[0141] Tyr-D-Tic-Phe-Phe-NH2
[0142] Tyr-Tic-Phe-Phe
[0143] Tyr-cy-[D-Pen-Ala-Phe-D-Pen]
[0144] Tyr-cy-[D-Pen-D-Ala-Phe-D-Pen]
[0145] Tyr-cy-[D-Pen-Gly-Phe-D-Pen]
[0146] Tyr-cy-[D-Pen-Ser-Phe-Pen]
[0147] Tyr-D-Pen-Gly-Phe-DMPT
[0148] Tyr-D-Ala-Gly-Phe-D-Leu
[0149] Tyr-D-Nle-Gly-Phe-NleS
[0150] Tyr-Gly-Gly-Phe-Leu
[0151] Tyr-Gly-Gly-Phe-Met
[0152] Tyr-D-The-Gly-Phe-Leu-Thr
[0153] N,N-diallyl-(O-t-butyl)-Tyr-Aib-Aib-Phe-Leu-O-Me
[0154] Tyr-cy-[D-Pen-Gly-Phe-D-Pen]-Nle-Gly-NH2
[0155] Tyr-Gly-Gly-Phe-NH-NH-Phe-Gly-Gly-Tyr
[0156] [Leu]-enkephalin
[0157] Where the following abbreviations are used:
[0158] Pen--penicilamine
[0159] Nle--nor-leucine (CH3-CH2-CH2-CH2-CH-(NH2)COOH)
[0160] NleS--CH3-CH2-CH2-CH2-CH-(NH2)SO3H
[0161] Tic--tetrahydroisoquinoline-3-carboxylic acid
[0162] Aib--alpha-aminoisobutryric acid
[0163] cy--cyclo
[0164] MTLPs may be linked to other molecules including, but not
limited to, detectable labels, adsorption facilitating molecules,
toxins or solid substrata by methods including, but not limited to,
the use of homobifunctional and heterobifunctional cross-linking
molecules (Carlsson et al. Biochem. J. 173:723, 1978; Cumber et al.
Methods in Enzymology 112:207, 1978; Jue et al. Biochem.
17:5399,1978; Sun et al. Biochem. 13:2334, 1974; Blattler et al.
Biochem. 24:1517, 1985; Liu et al. Biochem. 18:690, 1979; Youle and
Neville Proc. Natl. Acad. Sci. USA 77:5483,1980; Lerner et al.
Proc. Natl. Acad. Sci. USA 78:3403.1981; Jung and Moroi Biochem.
Biophys. Acta 761:162 1983; Caulfield et al. Biochem. 81:7772,
1984; Staros Biochem. 21:3950, 1982; Yoshitake et al. Eur. J.
Biochem. 101:395, 1979; Yoshitake et al. J. Biochem. 92:1413, 1982;
Pilch and Czech J. Biol. Chem. 254:3375, 1979; Novick et al. J.
Biol. Chem. 262:8483. 1987; Lomant and Fairbanks J. Mol. Biol.
104:243, 1976; Hamada and Tsuruo Anal. Biochem. 160:483,1987;
Hashida et al J. Applied Biochem. 6:56,1984; Means and Feeney
Bioconjugate Chem. 1:2,1990).
[0165] MTLPs may be used as immunogens to generate antibodies which
immunospecifically bind the immunogen. Such antibodies include, but
are not limited to polyclonal, monoclonal, chimeric, single chain
Fab fragments, F(ab').sub.2 fragments and Fab expression libraries.
Uses of such antibodies include, but are not limited to,
localization, imaging, diagnosis, treatment and treatment efficacy
monitoring. For example, antibodies or antibody fragments specific
to a domain of a MTLP, such as a dansyl group or some other epitope
introduced into the peptide, can be used to identify the presence
of the MTLP, to bind the MTLP to the surface of a particle, to
quantitate the amount of the MTLP on a particle, to measure the
amount of the MTLP in a physiological sample, to
immunocytochemically localize the MTLP in a cell or tissue sample,
to image the MTLP after in vivo administration and to purify the
MTLP by immunoaffinity column chromatography.
[0166] The functional activity of a MTLP can be determined by
suitable in vivo or in vitro assays known to those skilled in the
art. These include, but are not limited to, immuno-,
immunoradiometric-, immunodiffusion- and immunofluorescence assays
and to western blot analysis.
[0167] A MTLP functions to target an active agent or an active
particle to a cell, intracellular compartment, or cell layer and to
enhance the uptake of the active agent or of the active particle
into a cell, into and out of an intracellular compartment and
across a cell layer. Cells include, but are not limited to,
epithelial, endothelial and mesothelial cells, unicellular
organisms and plant cells. Cell layers include epithelial,
endothelial and mesothelial cell layers such as, but not limited
to, the gastrointestinal tract, pulmonary epithelium, blood brain
barrier and vascular endothelium. Preferably the cell is an
epithelia, cell and the cell layer is an epithelial cell layer.
Most preferably, the cell is a GIT epithelial cell and the cell
layer is the GIT epithelial cell layer. Intracellular compartments
include, but not limited to, nuclear, mitochondrial, endoplasmic
reticular and endosomal compartments. MTLPs can be used to enhance
the uptake of an active agent or active particle that regulates or
directs intra-cellular trafficking. Further, MTLPs can be used to
enhance intracellular gene delivery. That is, a gene or plasmid DNA
is encapsulated or complexed within a cationic lipid polymer system
and the surface of the cationic lipid polymer system is complexed
with an MTLP or with a targeting peptide. Alternatively, a plasmid
DNA is condensed, the condensate is complexed with cationic lipids
and the surface of the cationic lipids is complexed with an MTLP or
with a targeting peptide.
[0168] Methods used to complex a MTLP to an active agent
(MTLP-active agent complex) include, but are not limited to,
covalent coupling of a MTLP and an active agent, either directly or
via a linking moiety, noncovalent coupling of a MTLP and an active
agent and generation of a fusion protein, wherein a MTLP is fused
in-frame to an active agent including, but not limited to a
therapeutic protein.
[0169] Methods used to complex a MTLP to an active agent loaded
particle (MTLP-active particle complex) include, but are not
limited to, adsorption to the active particle, noncovalent coupling
to the active particle; covalent coupling, either directly or via a
linker, to the active particle, to the polymer or polymers used to
synthesize the active particle, to the monomer or monomers used to
synthesize the polymer; and, to any other component comprising the
active particle. Further, MTLPs can be complexed to a slow-release
(controlled release) particle or device (Medical Applications of
Controlled Release, Langer & Wise (eds.), CRC Press, Boca
Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product
Design and Performance, Smolen and Ball (eds.), Wiley, New York,
1984; Ranger et al. J. Macromol. Sci. Rev. Macromol. Chem. 23:61,
1983; Levy et al. Science 228:190, 1985; During et al. Ann. Neurol.
25:351, 1989; Howard et al. J. Neurosurg. 71:105 1989).
[0170] Methods used for viral based gene delivery systems include,
but are not limited to, vectors modified at the nucleic acid level
to express a MTLP on the surface of a viral particle and mammalian
cells or helper viruses, which express MTLP-virus fusion proteins
that are incorporated into a viral vector.
[0171] The present invention also provides pharmaceutical
formulations, comprising a therapeutically effective amount of a
MTLP-active agent complex or of a MTLP-active particle complex and
a pharmaceutically acceptable carrier (Remington's Pharmaceutical
Sciences by E. W. Martin). The term "pharmaceutically acceptable"
includes, but is not limited to, carriers approved by a regulatory
agency of a country or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. The term "carrier" refers
to a diluent, adjuvant, excipient, or vehicle with which the
MTLP-active agent complex or the MTLP-active particle complex is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical formulation is administered orally. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The formulation, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions include, but are not limited to, solutions,
suspensions, emulsion, tablets, pills, capsules, powders and
sustained-release formulations. The formulation can be a
suppository, with traditional binders and carriers such as
triglycerides. Oral formulation can include standard carriers
including, but not limited to, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose
and magnesium carbonate. Such formulations will contain a
therapeutically effective amount of the active agent or of the
active agent loaded into a particle, together with a suitable
amount of carrier so as to provide the form for proper
administration to an individual in need of the active agent.
[0172] Any route known in the art may be used to administer a
MTLP-active agent complex or a MTLP-active particle complex,
including but not limited, to oral, nasal, topical, mucosal,
intravenous, intraperitoneal, intradermal, intrathecal,
intramuscular, transdermal and osmotic. Preferably, administration
is oral, wherein the MTLP enhances uptake of the active agent into
a GIT epithelial cell and across the GIT epithelium into the
circulation. The precise amount of active agent to be administered
for the diagnosis, prevention or treatment of a particular
pathological condition will depend on the pathological disorder,
the severity of the pathological disorder, the active agent used
and the route of administration. The amount of active agent to be
administered and the schedule of administration can be determined
by the practitioner using standard clinical techniques. In
addition, in vitro assays may optionally be employed to help
identify optimal ranges for active agent administration.
[0173] The following examples will serve to further illustrate the
present invention without, at the same time, however, constituting
any limitation thereof. On the contrary, it is to be clearly
understood that resort may be had to various other embodiments,
modifications, and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the present invention
and/or the scope of the appended claims.
EXAMPLE 1
[0174] Peptide Synthesis
[0175] The membrane translocating peptides ZElan094, 204N and 204
and the targeting peptides HAX42, PAX2, P31 and Sni34 (U.S. patent
applications Ser. Nos. 09/079,819, 09/079,723 and 09/079,678) were
synthesized chemically using a fmoc synthesis protocol (Anaspec,
Inc., San Jose, Calif.). A dansyl group was added at the N-terminus
of each sequence in order to enable the detection of the peptide
with anti-dansyl antibody (Table 1).
[0176] The physical characteristics of Zelan094 (SEQ ID NO: 2) are
shown in Table 3.
4TABLE 3 Physical characteristics of ZElan 094 (SEQ ID NO: 2) Mass
(M+H+): 1838.03 Solubility 1 mg/ml water Appearance white powder
HPLC purity >95% Kyle-Doolittle Hydropathy Plot
EXAMPLE 2
Preparation of MTLP-active Particle Complexes and of Targeting
Peptide-Active Particle Complexes
[0177] Active particles were prepared from a polymer using a
coacervation method. Preferably, particle size is between about 5
nm and 750 .mu.m, more preferably between about 10 nm and 500 .mu.m
and most preferably between about 50 nm and 800 nm. MTLPs or
targeting peptides were complexed to the particles using various
methods known to those skilled in the art.
[0178] The following is a general method for preparation of
coacervated particles.
[0179] Phase A
[0180] A polymer agent, a surface-active agent, a
surface-stabilizing agent, a surface-modifying agent or a
surfactant is dissolved in water (A). Preferably the agent is a
polyvinyl alcohol (hereinafter "PVA") or a derivative thereof
having a % hydrolysis of about 50-100 and a molecular weight range
of about 500-500,000 kDa. More preferably the agent is a PVA having
a % hydrolysis of 80-100 and a molecular weight range of about
10,000-150,000 kDa. The mixture (A) is stirred under low shear
conditions at 10-2000 rpm and, more preferably, at 100-600 rpm. The
pH and ionic strength of the solution may be modified using salts,
buffers or other modifying agents. The viscosity of the solution
may be modified using polymers, salts, or other viscosity modifying
agents.
[0181] Phase A may include agents such as, but not limited to,
emulsifying agents, detergents, solubilizing agents, wetting
agents, foaming agents, antifoaming agents, flocculents and
defloculents. Examples include, but are not limited to, anionic
surface agents such as sodium dodecanoate, sodium
dodecyl-(lauryl)sulphate, sodium dioctyl-sulphosuccinate,
cetostearyl alcohol, stearic acid and its salts such as magnesium
stearate and sodium stearate, sodium dodecyl-benzene sulphonate,
sodium cholate triethanolamine; cationic surface agents such as
hexadecyl trimethyl ammonium bromide (cetrimide), dodecyl
pyridinium iodide, dodecyl pyridinium chloride; non-ionic surface
agents such as hexaoxyethylene monohexadecyl ether, polysorbates
(Tweens), sorbitan esters (Spans), Macrogol ethers, Poloxalkols
(Poloxamers), PVA, PVP, glycols and glycerol esters, fatty alcohol
poly glycol ethers, dextrans, higher fatty alcohols; and,
amphoteric surface agents such as N-dodecyl alanine, lecithin,
proteins, peptides, polysaccharides, semisynthetic polysaccharides,
sterol-containing substances, and finely divided solids such as
magnesium hydroxide and montmorillonite clays.
[0182] Phase B
[0183] A polymer is dissolved in a water miscible organic solvent
to form the organic phase (B). Preferably the organic phase is an
acetone-ethanol mixture in ratios from 0:100 acetone:ethanol to
100:0 acetone:ethanol depending upon the polymer used. Other
polymers, peptides, sugars, salts, natural-polymers, synthetic
polymers or other agents may be added to the organic phase (B) to
modify the physical and chemical properties of the resultant
particle product.
[0184] The polymers may be soluble, permeable, impermeable,
biodegradable or gastroretentive. They may be a mixture of natural
or synthetic polymers and copolymers. Such polymers include, but
are not limited to, polylactides, polyglycolides, DL, L and D forms
of poly(lactide-coglycolides) (PLGA), copolyoxalates,
polycaprolactone, polyester-amides, polyorthoesters,
polyanhydrides, polyalkyl-cyano-acrylates, polyhydroxy-butyrates,
polyurethanes, albumin, casein, citosan derivatives, gelatin,
acacia, celluloses, polysaccharides, alginic acid, polypeptides and
the like, copolymers thereof, mixtures thereof, enantiomeric forms
thereof, stereoisomers thereof and any MTLP conjugate thereof.
Synthetic polymers include, but are not limited to, alkyl
celluloses, hydroxyalkyl celluloses, cellulose ethers- cellulose
esters, nitrocelluloses, acrylic and methacrylic acids and esters
thereof, dextrans, polyamides, polycarbonates, polyalkylenes,
polyalkylene glycols, polyalkylene oxides, polyalkylene
terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl
esters, polyvinyl halides, polyvinylpyrrolidones, polysiloxanes,
polyurethanes and copolymers thereof.
[0185] Phase C
[0186] Phase B is stirred into phase A at a continuous rate.
Solvent is evaporated, preferably by increasing the temperature
over ambient and/or by using a vacuum pump. The resultant particles
are in the form of a suspension (C) An active agent may be added
into phase A or into phase B. Active agent loading may be in the
range 0-90% w/w. An MTLP or a targeting peptide may be added into
phase C. MTLP and targeting peptide loading may be in the range
0-90% W/W.
[0187] Phase D
[0188] The particles (D) are separated from the suspension (C)
using standard colloidal separation techniques including, but not
limited to, centrifugation at high `g` force, filtration, gel
permeation chromatography, affinity chromatography or charge
separation. The liquid phase is discarded and the particles (D) are
re-suspended in a washing solution such as, but not limited to,
water, salt solution, buffer or organic solvent. The particles are
separated from the washing liquid using standard colloidal
separation techniques and are washed two or more times. A MTLP or
targeting peptide may be used to wash the particles or,
alternatively, may be dissolved in the final wash. The particles
are dried.
[0189] A secondary layer of polymers, peptides sugars, salts,
natural and/or biological polymers or other agents may be deposited
onto the preformed particulate core by any suitable method known in
the art. The dried particles can be further processed by, for
example, tableting, encapsulating or spray drying. The release
profile of the particles formed may be varied from immediate to
controlled or delayed release depending on the formulation used
and/or desired.
EXAMPLE 3
Bovine Insulin Loaded-MTLP Coated Nanoparticles--MTLP Added in the
Final Wash
[0190] Fast acting bovine insulin (28.1 lU/mg) was incorporated
into polylactide-co-glycolide (PLGA, Boehringer Ingelheim,
Indianapolis, Ind.) at a theoretical loading of 300 IU of
insulin/210 mg of nanoparticles and the nanoparticles were coated
with the dansylated ZElan094 (SEQ ID NO: 2).
5 COMPONENT AMOUNT PLGA RG504H (Lot # 250583) 2 g Acetone 45 mls
Ethanol 5 mls PVA (5% w/v) (13-23 kDa, 98% hydrolysis) 400 mls
Bovine Insulin (Lot # 86HO674) 100 mg ZElan094 (SEQ. ID NO: 2) 10
mg/50 ml dH20
[0191] Preparation:
[0192] 1. Water was heated to near boiling, PVA was added to 5% w/v
and the solution was stirred until cool (phase A).
[0193] 2. Acetone and ethanol were mixed to form the organic phase
(phase B).
[0194] 3. PLGA was added to the acetone and ethanol (step 2) and
dissolved by stirring (phase B).
[0195] 4. An IKA.TM. reactor vessel was set at 25.degree. C. Phase
A (step 1) was added into the reactor vessel and stirred at 400
rpm.
[0196] 5. Bovine insulin was added into the stirring phase A (step
4).
[0197] 6. Using clean tubing and a green needle, phase B (step 3)
was slowly dripped into the stirring solution (step 5) using a
peristaltic pump set at 40.
[0198] 7. The solvent was evaporated by opening the IKA.TM. reactor
vessel ports and stirring overnight at 400 rpm to form a suspension
(phase C).
[0199] 8. The suspension, phase C (step 7) was centrifuged in a
XL90 centrifuge at 12,500 to 15,000 rpm for 25 to 40 minutes at
4.degree. C.
[0200] 9. The supernatant was discarded, the particle "cake" broken
up, and the particles (phase D) washed twice in 200 ml of dH.sub.20
by centrifugation in an XL90 centrifuge at 12,500 to 15,000 rpm for
10-15 minutes at 4.degree. C. The dansylated ZElan094 (SEQ ID NO:
2) was added into the final wash.
[0201] 10. The supernatant was decanted, the `cake` broken up and
the particles dried in a vacuum oven. The dried particles were
ground, placed in a securitainer and analyzed. Insulin loading was
5% or 50 mg insulin/g particles. Insulin potency, determined in
HPLC, was 51.4 mg/g. Scanning electron microscopy showed discrete,
reasonably spherical particles of about 300-400 nm in diameter.
EXAMPLE 4
Bovine Insulin Loaded-MTLP Coated Nanoparticles--MTLP Added to
Phase C
[0202] Fast acting bovine insulin (28.1 lU/mg) was incorporated
into PLGA nanoparticles at a theoretical loading of 300 IU of
insulin/210 mg of nanoparticles and the nanoparticles were coated
with the MTLP ZElan094 (SEQ ID NO: 2).
6 COMPONENT AMOUNT PLGA RG504H (Lot # 250583) 2 g Acetone 45 mls
Ethanol 5 mls PVA (5% w/v) (13-15 kDa, 98% hydrolysis) 400 mls
Bovine Insulin (Lot #. 86HO674) 100 mg ZElan094 (SEQ. ID NO: 2) 10
mg/50 ml dH20
[0203] Preparation:
[0204] See steps 1-4 of Example 3.
[0205] Step 5.
[0206] Insulin and ZElan094 were added to the stirring PVA
solution.
[0207] See steps 6-9 of Example 3.
[0208] The particles (step 9) were ground, placed in a securitainer
and anlayzed.
EXAMPLE 5
Bovine Insulin Loaded-MTLP Coated Nanoparticles--MTLP Added 1 Hour
Prior to Centrifugation
[0209] Fast acting bovine insulin (28.1 Iu/mg) was incorporated
into PLGA nanoparticles at a theoretical loading of 300 IU of
insulin/210 mg of nanoparticles and the nanoparticles were coated
with dansylated ZElan094 (SEQ ID NO: 2).
7 COMPONENT AMOUNT PLGA RG504H (Lot # 250583) 2 g Acetone 45 mls
Ethanol 5 mls PVA (5% w/v) (13-15 kDa, 98% hydrolysis) 400 mls
Bovine Insulin (Lot #. 86HO674) 100 mg ZElan094 (SEQ. ID NO: 2) 10
mg/50 ml dH20
[0210] Preparation.
[0211] See steps 1-7 of Example 3.
[0212] Step 8.
[0213] ZElan094 was added to the stirring particle suspension.
After 1 hr, the suspension was centrifuged at 12,500-14,000 rpm for
20 to 40 min at 4.degree. C.
[0214] See steps 9-10 of Example 3.
EXAMPLE 6
Bovine Insulin Loaded-MTLP Nanoparticles--MTLP Conjugated
Polymer
[0215] Fast acting bovine insulin is incorporated into
PLGA-dansylated ZElan094 (SEQ ID NO: 2) conjugate nanoparticles at
a theoretical loading of 300 IU of insulin/210 mg of nanoparticles
as follows.
[0216] Component
[0217] PLGA RG504H (Lot# 250583)
[0218] RG504H-ZElan094 (SEQ ID NO: 2) conjugate
[0219] Acetone
[0220] Ethanol
[0221] PVA (5%w/v) (13-15 kDa, 98% hydrolysis)
[0222] Bovine Insulin
[0223] Preparation is as in steps 1-10 of Example 3, except that in
step 3 RG504H and RG504H-ZElan094 conjugate are added to phase B
(step 2).
EXAMPLE 7
Bovine Insulin Loaded-Target Peptide Coated Nanoparticles
[0224] Fast acting bovine insulin (28.1 lU/mg) was incorporated
into PLGA nanoparticles at a theoretical loading of 300 IU of
insulin/210 mg of nanoparticles and the nanoparticles were coated
with the targeting peptides dansylated ZElan 011, 055, 091, 101,
104, 128, 129 and 144 (U.S. patent application Ser. Nos.
09/079,819, 09/079,723 AND 09/079,678).
8 COMPONENT AMOUNT PLGA RG504H (Lot # 250583) 2 g Acetone 45 ml
Ethanol 5 ml PVA (5% w/v) (13-15 kDa, 98% hydrolysis) 400 ml Bovine
Insulin (Lot #. 86HO674) 100 mg ZElan011, 055, 091, 101, 104, 128,
129 and 144 10 mg/50 ml dH20
[0225] (U.S. patent application Ser. Nos. 09/079,819, 09/079,723
AND 09/079,678, AND PCT APPLICATION PCT/US98/10088, PUBLISHED AS WO
98/51325))
[0226] Preparation:
[0227] See steps 1-10 of Example 3.
[0228] Insulin loading was 5% or 50 mg insulin/g particles.
EXAMPLE 8
[0229] Animal Studies
[0230] In vivo oral insulin bioavailability from MTLP-insulin
particle complexes (Example 3) and from targeting peptide-insulin
particle complexes (Example 7) were assessed in the open loop rat
model.
[0231] Fifty-nine Wistar rats (300-350 g) were fasted for 4 hours
and were anaesthetized by intramuscular injection of 0.525 ml of
ketamine (100 mg/ml)+0.875 ml of acepromazine maleate-BP (2 mg/ml)
15 to 20 minutes prior to administration of MTLP-insulin particle
complexes or of targeting peptide-insulin particle complexes. The
rats were divided into 9 groups, each group containing 6 or 7
animals. Approximately 200 mg of MTLP-insulin (300 IU) particle
complexes, suspended in 1.5 ml of PBS, were injected
intra-duodenally at 2-3 cm below the pyloris of each of 6 rats
(Group 5). Approximately 200 mg of targeting peptide-insulin (300
IU) particle, complexes, suspended in 1.5 ml of PBS, were injected
intra-duodenally at 2-3 cm below the pyloris of each of 6-7 rats
(Groups 1-4 and 6-9). The study groups are( shown in Table 4.
9TABLE 4 Study Groups GROUP # # OF RATS PEPTIDE ZELAN NO SEQ ID
NO.sup.a: 1 6 HAX42 091 50 2 7 PAX2 144 53 3 7 PAX2 129 54 4 6 P31
101 52 5 6 MTLP 094 48 6 7 PAX2 128 55 7 7 PAX2 104 56 8 7 HAX42
011 49 9 7 PAX2 055 51 .sup.a(U.S. PATENT APPLICATIONS NOS.
09/079,819, 09/079,723 AND 09/079,678, AND PCT APPLICATION
PCT/US98/10088 PUBLISHED AS WO 98/51325)
[0232] Systemic blood was sampled from the tail vein (0.4 ml) of
each rat at 0 minutes and at 15, 30, 45, 60 and 120 minutes after
intra-duodenal administration of the ZElan094-insulin particle
complexes or of the targeting peptide-insulin particle complexes.
Blood glucose in each sample was measured using a Glucometer
(Bayer; 0.1 to 33.3 .mu.m/mol/L). The blood was centrifuged and the
plasma was retained. Plasma insulin was assayed in duplicate using
a Phadeseph RIA Kit (Pharmacia, Piscataway, N.J.; 3 to 240
.mu.U/ml).
[0233] FIG. 2 shows the plasma insulin levels following
intra-duodenal administration of ZElan094-insulin particle
complexes (Group 5) and of targeting peptide ZElan091l-(Group 1),
144-(Group 2), 129-(Group 3), 101-(Group 4), 128-(Group 6),
104-(Group 7) and 011-(Group 8) insulin particle complexes. As
shown in FIG. 2, during the 60 minutes following intra-duodenal
administration, ZElan094-insulin particles complexes provided the
most potent enhancement of insulin delivery followed byt ZElan055-,
129- and 094-, 101-, 128-, 091- and 144, and 011-insulin particle
complexes. These data show that the plasma insulin levels obtained
using MTLP-insulin particle complexes, were greater than those
obtained using the targeting peptide-insulin particle
complexes.
[0234] To ensure that the insulin delivered from the MTLP-insulin
particle complexes and from the targeting peptide-insulin particle
complexes was bioactive, blood glucosea levels were measured. As
shown in FIG. 3, during the 20 minutes following intra-duodenal
administration, blood glucose levels fell from between about
6.0-9.5 mmol/L to about 4.5-7.0 mmol/L and remained significantly
below control values (PBS) for at least 60 minutes. There was no
significant differences in blood glucose levels among the animals
receiving the MTLP-insulin particle complexes and the animals
receiving the targeting peptide-insulin particle complexes at 60
minutes and at 120 minutes;. These data show that insulin delivered
from the dansylated ZElan094-insulin particle complexes and from
the dansylated Zelan011, 055, 091, 144, 129, 101, 129, 128 and
104-insulin particle complexes remained bioactive. Further, these
data show that insulin delivered from MTLP-insulin particle
complexes enabled a significant and long lasting decrease in blood
glucose levels.
EXAMPLE 9
Preparation of DNA Containing Liposomes and of DNA Containing MTLP
Coated Liposomes
[0235] DNA containing liposomes and DNA containing MLTP coated
liposomes were, prepared as follows:
[0236] Solution 1
[0237] Twelve nmol lipofectamine (Gibco BRL, Rockville, Md.),
.+-.0.6 mg of protamine sulphate, was prepared in a final volume of
75 ml optiMEM.
[0238] Solution 2
[0239] One mg of pHM6lacZ DNA (Boehringer Mannheim) was prepared in
a final volume of 75 ml optiMEM. The reporter plasmid pHM6lacZ
contains the lacZ gene, which codes for bacterial
.beta.-galactosidase.
[0240] Solution 3
[0241] Solution 1 and Solution 2 were combined and incubated for 15
minutes at RT to enable complex formation.
[0242] Solution 4
[0243] ZElan094, 204N or 204 (SEQ ID Nos: 2, 23, 24) were added to
Solution 3 to a final concentration of 100 mM and incubated for 5
minutes at RT. Six-hundred ml of optiMEM was added and the solution
was mixed gently.
[0244] The DNA containing liposomes and the DNA containing MTLP
coated liposom a complexes were analyzed in scanning electron
microscopy (SCM) or in transmission electron microscopy (TEM) to
confirm complex liposome formation and by zeta potential analysis
to confirm surface charge properties.
EXAMPLE 10
Delivery of DNA From Liposomes and from MTLP-Liposomes into Caco-2
Cells
[0245] DNA delivery into Caco-2 cells from liposomes and from MTLP
coated liposomes was calculated as .beta.-galactosidase expression
per mg of total protein in the, cell supernatant.
.beta.-galactosidase expression was determined using the Boehringer
Mannheim chemiluminescence kit. Protein was determined using the
Pierce Micro bichinconate (BCA) protein assay.
[0246] Caco-2 cells were plated at 1.times.10.sup.5 cells/well in 1
ml of culture media and incubated at 37.degree. C. in 5% CO.sub.2
overnight. The cells were washed twice in 0.5 ml of optiMEM.
ZElan094, 204N or 204 (SEQ ID NOS: 2, 23, 24) (Solution 4, Example
9) were each added to triplicate wells (250 .mu.l/well) of the
washed cells and incubated for 4 h at 37.degree. C. After 4 h, 250
.mu.l of optiMEM containing 2.times.fetal calf serum was added and
the cells were incubated for an additional 20 h at 37.degree. C. At
24 h post-transfection, the cells were lysed with Boehringer
Mannheim Lysis Buffer. The lysate was centrifuged for 2 min at
14,000 rpm in an Eppendorf Centrifiguge and the supernatant was
collected.
[0247] Table 5 shows relative .beta.-galactosidase expression per
mg of total protein using ZElan094, ZElan204N and ZElan204 (SEQ ID
NOS: 2, 23, 24) coated liposomes as the DNA delivery particles.
10TABLE 5 .beta.-galactosidase expression in Caco-2 cells
EXPERIMENTS 1 2 Lipofectamine + DNA (control) 100% 100%
Lipofectamine + DNA + protamine (control) 90% 162% Lipofectamine +
DNA + protamine + ZElan094 387% 260% Lipofectamine + DNA +
protamine + ZElan204N 495% 217% Lipofectamine + DNA + protamine +
ZElanN204 176% 122%
[0248] The MLTPs ZElan094, 204N and N204 (SEQ ID NOS: 2, 23 and 24)
coated liposomes delivered more DNA into the Caco-2 cells than did
the lipofectamine+DNA and lipofectamine+DNA+protamine control
liposomes. Moreover, as indicated by b-galactosidase expression,
the ZElan094 derivative ZElan204N, which is modified at the
C-terminus by the addition of a nuclear localisation sequence
(NLS), was most effective in enhancing both delivery of DNA into
and expression of DNA within Caco-2 cells. The MTLP ZElan094 and
its derivatives, in combination with cationic lipids and DNA
condensing agents, enhanced both the targeting of genes to cells
and the subsequent uptake of the genes by the cells.
[0249] As MTLPs enhance uptake of both active-agents and
active-particles into cells, MTLPs including, but not limited to,
ZElan094 and ZElan 204N, can be used as coating agents on polymer
based particle systems and on liposome based particle systems as
active agent and active particle delivery systems. Further, MTLPs
also can be used as coating agents on viral vector based particle
systems including, but not limited to, adenovirus, adeno-associated
virus, lentivirus, and vaccinia virus. In such systems, the virus
itself may code for the MTLP, wherein the DNA sequence coding for
the MTLP has been cloned in frame to one or more genes which code
for one or more, viral capsid protein or for one or more viral
surface proteins. Alternatively, the surface, of the virus used for
gene delivery may be modified with a MTLP following virus;
production and purification from a cell including, but not limited
to, a mammalian cell.
EXAMPLE 11
Effects of MTLPs and of the Targeting Peptides on Substrate
Transport Across a Cell Layer
[0250] The effect of the MTLPs ZElan094, ZElan178 and ZElan187 (SEQ
ID NOS: 2, 7 and 16) and of the targeting peptide ZElan022 (U.S.
patent application Ser. Nos. 09/079,819, 09/079,723 AND 09/079,678,
AND PCT APPLICATION PCT/US98/10088, PUBLISHED AS WO 98/51325) on
the transport of the dipeptide .sup.14C-gly-sar and of the reporter
molecule .sup.3H-fMLP across Caco-2 monolayers was determined. The
Caco-2 monolayers were grown on Transwell-Snapwells. Cell viability
was determined by measuring TEER of the Caco-2 monolayers during
each experiment. No significant drop in TEER was measured. Cell
permeability was determined by measuring mannitol flux across the
Caco-2 monolayers during each experiment. No increase in mannitol
flux was measured in the presence of the MTLP ZElan094.
[0251] The flux of the dipeptide .sup.14C-gly-sar and of the
reporter molecule .sup.3H-fMLP across the Caco-2 monolayers in the
absence and in the presence of the MTLPs ZElan094, Elan178 and
ZElan187 (SEQ ID NOS: 2, 7 and 16) and of the targeting peptide
ZElan022 (U.S. patent application Ser Nos. 09/079,819, 09/079,723
AND 09/079,678), AND PCT APPLICATION PCT/US98/10088, PUBLISHED AS
WO 98/51325 was measured over 2 h, and reduction in the
permeability coefficient was; determined in the presence of cold
substrates.
[0252] As shown in Table 6, the MTLPs ZElan 094,178 and 187
inhibited transport of the reporter molecule .sup.3H-fMLP (FIG. 4),
but did not inhibit transport of the dipeptide .sup.14C-gly-sar.
The targeting peptide ZElan 022 inhibited transport of the reporter
molecule .sup.3-fMLP (FIG. 4). The ability of the MTLPs ZElan094,
178 and 187 to compete for the transport of fMLP across polarised
Caco-2 cells indicates that this novel transport assay can be used
to screen derivatives, fragments, motifs, analogs and
peptidomimetics of ZElan094 and small organic molecules
functionally similar to ZElan094 to identify those having improved
transport characteristics.
11TABLE 6 Transport studies % inhibition .sup.3H-fMLP % inhibition
ZElan N0: SEQ ID NO: transport .sup.14C-gly-sar transport 094 2
77.2 NS 178 7 71.5 NS 187 16 84.5 NS 022 U.S. PATENT 00.0
APPLICATIONS NOS. 09/079,819, 09/079,723 AND 09/079,678, , AND PCT
APPLICATION PCT/US98/10088, PUBLISHED AS WO 98/51325
[0253] NS: no significant difference between experimental (+MTLP)
and control cells (-MTLP) in the transport of radiolabeled
drug.
[0254] Moreover, that the MTLPs inhibited transport of the reporter
molecule .sup.3H-fMLP, but did not inhibit transport of the
dipeptide .sup.14C-gly-sar suggest that their effect on fMLP
transport is not due to a generalized perturbation of the membranes
in polarized epithelial cells. Further, as fMLP is known to play a
role in inflammation in the GIT, MTLPs, which decrease transport of
fMLP across Caco-2 monolayers, may have a therapeutic role in
preventing local inflammation by decreasing the chemoattractant
effect of fMLP in the GIT.
EXAMPLE 12
Effect of Increasing Concentrations of an MTLP on the Transport of
.sup.3H-fMLP Across c Cell Layer
[0255] Caco-2 monolayers were grown and tested for viability as in
Example 11. Transport of .sup.3H-fMLP across Caco-2 monolyers was
measured in the presence from 0 to 200 .mu.g/ml of the MTLP
ZElan094. As shown in FIG. 5, the MTLP ZElan094 inhibited
.sup.3H-fMLP transport even at the lowest concentration (13 mg/ml
or 7.1 ml) tested. This indicates that the MTLP ZElan094 is a
potent inhibitor of fMLP transport across an epithelial cell
layer.
EXAMPLE 13
Stability of MTLPs in Simulated Intestinal Fluid
[0256] MTLPs ZElan094 and ZElan207 (SEQ. ID NO: 2 AND 102)were
dissolved in water and mixed with simulated intestinal fluid pH 6.8
containing porcine derived pancreatin (SIF+Pancreatin) at
37.degree. C. The mixtures were incubated for up to 60 minutes at
37.degree. C., with aliquots taken at designated time points. The
reaction was quenched with quenching solution after the relevant
time points in order to halt the reaction between the SIF and
ligand.
[0257] The composition of Simulated Intestinal Fluid was as
follows:
[0258] Amylase 25 USP Units
[0259] Lipase 2.0 USP units
[0260] Protease 25 USP units
[0261] (Sigma P8096)
[0262] Approximately 1 mg of ligand was dissolved in 1 ml of water.
This standard stock solution of ligand was used to prepare the
solutions containing Ligand+SIF+Pancreatin. 50 .mu.L of ligand
solution was mixed with volumes of SIF+Pancreatin in separate
eppendorfs, as follows:
12TABLE 7 Volume of Ligand Solution Volume Temperature (.mu.L) of
SIF (.mu.L) (.degree. C.) Time (minutes) 50 100 37 5 50 100 37 10
50 100 37 30 50 100 37 60 50 200 37 5 50 200 37 10 50 200 37 30 50
200 37 60
[0263] Two different volumes of SIF were utilised in order to
monitor if increasing the SIF:ligand ratio had an effect on the
extent and rate of degradation. At the appropriate time point the
reaction between the ligand and SIF was stopped by pipetting 100
.mu.L of the mixture into 500 .mu.L of quenching solution
(Acetonitrile: Water 30:70). 20 .mu.L of the mixture was injected
onto the HPLC system.
[0264] HPLC Experimental
13 Column: Jupiter C.sub.18 RP, 5 .mu.m, 300.ANG., 250 .times. 4.6
mm, TCD #188 Mobile phase: A: 10% acetonitrile in 0.1%
trifluoroacetic acid in water B: 0.1% trifluoroacetic acid in
acetonitrile Flow rate: 1.0 ml/min Temperature: ambient Injection
20 .mu.l volume: Detector .lambda.: 220 nm Run time: 38 minutes
[0265] Control samples of ligand in water and in quenching solution
were prepared to check for recovery of ligand in the absence of
SIF+Pancreatin.
[0266] No recovery for ligand ZELAN094 (SEQ ID NO 2) was obtained
at any of the time points. Degradation products were seen in the
chromatogram.
[0267] No result table is presented for ZELAN094 as no recovery was
obtained for an,y of the time points. This is demonstrated by the
disappearance of the peptide peak. This ligand is therefore
degraded almost immediately on contact with SIF medium. Degradation
products appear at retention times 12.258, 13.55 and 14.067
minutes.
[0268] The controls demonstrated that the peptide is not degraded
at 37.degree. C. over timea when SIF+Pancreatin are not present.
Also, the quenching solution does not affect the recovery. It
should be remembered that while the HPLC method used above has not
been optimised as a stability-indicating assay, the disappearance
of the ligand peaks and appearance of new component peaks were
clearly visible.
[0269] The recovery of ZELAN207 (SEQ ID NO 102) from SIF solutions
is tabulated below.
14TABLE 8 Stability of peptide ZELAN 207 (Lot# 11243) in presence
of SIF(USP) pH 6.8 at 37 C Actual Theoretical Time Std Std Conc
Drug point Std area precision Conc Sample Drug Conc % Sample (min)
(mean) (% CV) (ug/ml) Area (ug/ml) (ug/ml) Recovery Peptide + 100
uL SIF 5 2012290 0.9 60.63 1727236 52.04 53.9 96.6 10 2012290 0.9
60.63 1742290 52.49 53.9 97.4 30 2012290 0.9 60.63 1571456 47.35
53.9 87.8 60 2012290 0.9 60.63 1747950 52.67 53.9 97.7 Peptide +
200 uL SIF 5 2013467 1.0 60.63 1062099 31.98 32.3 99.0 10 2013467
1.0 60.63 1032597 31.09 32.3 96.3 30 2013467 1.0 60.63 995036 29.96
32.3 92.8 60 2013467 1.0 60.63 1035460 31.18 32.3 96.5 Pep +
H2O(100 ul) + Q* 60 2016394 1.1 60.63 1810419 54.44 53.9 101.0 Pep
H2O(100 ul) + H2O 60 2016394 1.1 60.63 1806474 54.32 53.9 100.8 *Q
= quenching solution
[0270] Incubation with SIF for up to 1 hour allowed recovery of
>90% of the parent peptide indicating that D-amino acid
substitution substantially increased the stability profile for the
peptide.
EXAMPLE 14
Evaluation of MTLPs for Delivery of a Model Opioid Peptide In Vitro
(Caco-2) and In Vivo (Intraduodenal; Conscious Rat Model)
[0271] A. Opioid Peptide Stability in SIF
[0272] A model D-form opioid peptide (H-ffir-NH2; kappa receptor
specific; molecular weight 581 Da) was evaluated for stability in
SIF.
[0273] Pancreatin (Fisher Scientific) was dissolved at 1 mg/ml in
1.times.phosphate buffer solution. The pH of the solution was
adjusted to 7.5 with 0.01 M NaOH and it was heated in a waterbath
to 37.degree. C. Two dry peptide samples were weighed out. One
sample was dissolved in phosphate buffer solution at 1.0 mg/ml as a
control. The second sample was dissolved in SIF at 1.0 mg/ml for
stability analysis.
[0274] HPLC Analysis Conditions
[0275] RP-HPLC analysis with C-18 short column (Betasil column, 5
.mu.m (50.times.3 mm) PN: 055-701-3) with the following
gradient:
15 Time (min) Solvents Mixture 0 95% water-5% acetonitrile (0.05%
TFA) 0 95% water-5% acetonitrile (0.05% TFA) 0 5% water-95%
acetonitrile (0.05% TFA) linear solvent gradient 8 5% water-95%
acetonitrile (0.05% TFA)
[0276] Diode array detector on the system, 214 nm used for
analysis
[0277] Peptide control initially injected
[0278] Solution in SIF injected at 0, 1, 3, and 24 hours
[0279] LCMS Analysis
[0280] Analyzed control and 24 hour sample by LCMS.
[0281] The kappa peptide, H-ffir-NH2, was very stable over the 24
hour period (see Table 9). Mass spectrometry confirmed this result
with only one compound detectable initially and after 24 hours. The
kappa peptide was selected as a suitable model drug for further
evaluation in targeting studies.
16TABLE 9 Control Initial 1 hr 24 hr H-ffgr-NH2 RT Area RT Area RT
Area RT Area 0.467 347767 0.467 336841 0.467 360462 0.467 330859
2.133 249457 2.117 285901 2.333 2.317 11107788 2.317 11691106 2.317
11215885 Sequence H- f f i r --NH2 % Compound 1 hr 105.25% 24 hr
100.97%
[0282] B. Synthesis and Evaluation of Kappa Peptide Conjugates In
Vitro
[0283] Conjugates of the kappa peptide, H-ffir-NH2, and various
MTLPs (ELAN094, ELAN207, ELAN208 & ELAN 178) were synthesised
in various formats to determine optimal conjugation strategies for
subsequent synthesis of batches for in vivo evaluation. Structural
formats included:
[0284] i) C-terminal or N-terminal opioid peptide,
[0285] ii) Conjugated with I without a lysine linker,
[0286] iii) Unlabelled I biotin labelled.
[0287] The integrity of the opioid peptide was evaluated post
conjugation to assess suitability for inclusion in further studies.
Opioid activity was assessed in vitro using a competition assay
(competition for binding of a radiolabelled kappa peptide to rat
brain homogenates). Results are expressed as IC50 values i.e. the
concentration of conjugate which inhibits binding of the
radiolabelled ligand by 50%.
17TABLE 10 Day 1 Day 5 Day 82 Cmpnd # M.W. IC50 nM IC50 nM IC50 nM
IC50 nM IC50 nM IC50 nM P10- 110 H-ffirk(kkaaavllpvllaap-NH-e)-NH2
2166 52.1 81.8 181.8 25.9 17.1 19704.9 P10- 114
H-kkaaavllpvllaapk(ffir-NH-e)-NH2 2166 3.7 19.9 1.0 0.8 0.8 P10-
118 H-ffirkkaaavllvllaap-NH2 2038 113.1 63.0 302.5 177.1 38.8
H-ffir-NH2 14.0 3.3 4 1.7 3.9
[0288] In Table 10, 3 Elan207 (SEQ ID NO 102) conjugates are
compared to the H-ffir-NH2 peptide control (unconjugated). The
three conjugates are 1) H-ffir-NH2 conjugated to N-terminal of 207
through a lysine linker (P10-110), 2) H-ffir-NH2 conjugated to
C-terminal of 207 through a lysine linker (P10-114) and 3)
H-ffir-NH2 conjugated to N-terminal of 207 directly i.e. no linker
(P10-118). Assays were performed on 3 separate occasions
(duplicates on day 5 and triplicates on day 82). Opioid activity is
significantly reduced by conjugation as described in 1) and 3).
Using 2) i.e. C-terminus and lysine linker the opioid activity is
retained, or even enhanced suggesting the need for the linker amino
acid and/or conformation induced in this format. The assay is cell
based and subject to some variation--however the trend remains
consistent throughout.
[0289] IC50 values indicated that optimal opioid activity was
retained post conjugation of the opioid peptide to the C-terminus
of the ELAN207 MTLP through a lysine linker. Note that the opioid
activity may have been enhanced by addition of ELAN207.
[0290] In Table 11, ELAN094, 178, 207 & 208 (SEQ ID NOs 2, 7,
102 & 202) biotin labelled conjugates are compared to the
H-ffir-NH2 peptide control (unconjugated).
18 TABLE 11 ELAN No. MW IC50 nM IC50 nM P37-114
H-K(biotin-LC)KKAAAVLLPVLLAAPK(ffir-NH-e)-NH2 94 2633 0.4 7.9
P37-116 H-K(biotin-LC)KKCAAVLLPVLLACK(ffir-NH-e)-NH2 178 2598 0.2
11.7 P34-154 H-K(biotin-LC)kkaaavllpvllaapk(ffir-NH-- e)-NH2 207
2633 1.1 12.4 P37-115 H-K(biotin-LC)paallvpllvaaakkK(ff-
ir-NH-e)-NH2 208 2633 9.2 9.6 H-ffir-NH2 1.7 3.9
[0291] IC50 values indicated that:
[0292] i) the ELAN207 opioid peptide conjugate (P34-154) retained
activity post biotin labelling
[0293] ii) ELAN094 (P37-114), 178 (P37-116) & 208 (P37-115)
opioid peptide conjugates, with C-terminal opioid peptide and
lysine linker, exhibited activity equivalent to that observed with
ELAN207 (P34-154).
[0294] Peptide conjugates P37-114 (ELAN094) and P34-154 (ELAN207)
were synthesised in a tritium labelled format for in vivo
studies.
EXAMPLE 15
[0295] C. Synthesis and Evaluation of Tritiated Kappa Peptide
Conjugates In Vitro
[0296] Conjugates of the kappa peptide, H-ffir-NH2, and MTLPs
ELAN094 and ELAN207 (SEQ ID NOs 2, 102), were synthesised using
standard peptide synthesis protocols. Two phenylalanine residues on
the kappa peptide were labelled by tritium exchange and
radio-peptide purity was assessed by RP-HPLC. Peptide purity of
>95% and specific activities of 42-54 Ci/mmol were achieved.
[0297] The tritiated opioid peptide conjugates were evaluated for
permeability through differentiated Caco-2 cell monolayers i.e. to
assess the integrity of the membrane translocating peptide moiety
Both the kappa peptide conjugates and the kappa peptide control
exhibited permeability coefficients in the 10.sup.-6 cm/sec range,
indicating that they would be suitable candidates for oral
bioavailability evaluation in vivo. No significant increase in
permeability was detectable when the ZELAN094 or ZELAN207 peptide
conjugates were compared to the kappa peptide control (n=5).
Highest transport values were obtained in the first 30 min followed
by a 2-4 fold decrease at 60 min. The significance of these
findings is unclear. The data may be indicative of dual
binding/uptake events occurring through i) a kappa receptor
specific mechanism and ii) direct membrane interaction through
lipid interaction of the membrane permeable peptide.
[0298] FIG. 6 shows results on the transport of tritiated kappa
peptide conjugates and kappa peptide control across differentiated
Caco-2 cell monolayers.
[0299] D. Evaluation of Tritiated Kappa Peptide Conjugates In
Vivo
[0300] The possible enhancing effects of ELAN094 and ELAN207 MTLPs
(SEQ ID NOs 2, 102) on intestinal absorption of the kappa peptide
were examined in a conscious rat model.
[0301] Experimental:
[0302] The study was non-randomised, parallel group design. Wistar
rats within the 250-350 g weight range were used. All animals were
fasted for a period of 16 h prior to study initiation. Water was
available at all times.
[0303] Treatment Regimen:
[0304] Group 1 (n=6)
[0305] Intravenous injection of 10 .mu.Ci of .sup.3H-kappa
peptide-Kaffiralin-1 (plain kappa peptide) (tail vein
injection).
[0306] Group 3 (n=6)
[0307] Intraduodenal instillation of 100 .mu.Ci of .sup.3H-kappa
peptide-Elan094 (Analysed). The Elan094 ligand contains a membrane
translocating sequence.
[0308] Group 4 (n=6)
[0309] Intraduodenal instillation of 100 .mu.Ci of .sup.3H-kappa
peptide-Elan207 (Analysed). The Elan207 ligand contains a membrane
translocating sequence.
[0310] Group 10 (n=6)
[0311] Intraduodenal instillation of 100 .mu.Ci of .sup.3H-kappa
peptide-Kaffiralin-1.
[0312] .sup.3H Bio-analysis: Plasma Samples
[0313] The plasma (100-250 .mu.l) was made up to 1 ml with BTS-450
(an organic tissue solubiliser) and incubated for 1 hour. Ten ml of
Scintillation fluid was added and the radioactivity was
measured.
[0314] Results:
[0315] The study was a non-randomised, parallel group biostudy
designed to evaluates the systemic bioavailability of tritium
labelled kappa peptide conjugates after intra-duodenal instillation
in the conscious rat model. Each rat only received one treatment
and the plasma samples were analysed for tritium content. The
calculation of AUClast, Amax, tmax, Volume of Distribution (V d)
and Clearance was based on base line corrected data. The absolute
bioavailability was calculated using potency corrected data.
[0316] Stock solutions of the tritiated ligand were administered to
the rats on different dates. These stock solutions were analysed in
order to correct for potency. Data analysed on one particular day
suggested a discrepancy in the bioavailability for Group 4 and a
repeat of Group 4. It was decided to re-analyse the stock solution
administered to Group 4 (repeat). The potency correction factor for
all treatments, including the re-analysis of Group 4(repeat), are
summarised below:
[0317] Potency Correction Analysis:
19TABLE 12 Theoritical Actual % of Potency Day Value Group Value
theo Corr. received uCi/ml No Ligand dpm/100 ul dpm/ml uCi/ml dose
Factor 1 303 10 Kappa-peptide 74811764 748117640 336.990 111.22
0.899 6 303 10 Kappa-peptide 47279266 472792660 212.970 70.29 1.423
13 303 4 Elan 207 83174222 831742220 374.659 123.65 0.809 21 303 4
Elan 207 83186452 831864520 374.714 123.67 0.809 27 50 1
Kappa-peptide 12839706 128397060 57.837 115.67 0.865 48 303 3
Elan094 32985636 329856360 148.584 49.04 2.039 62 303 4(Repeat)
Elan207 44012732 440127320 198.256 65.43 1.528 113 303 4(Repeat)
Elan207 88311270 883112700 397.799 131.29 0.762
[0318] Day numbers are unrelated to those in Table 10.
[0319] The absolute bioavailability and mean pharmacokinetic
parameters are summarized below.
[0320] Absolute .sup.3H Bioavailability (%)
[0321] The absolute .sup.3H bioavailability for the treatments in
rank order were the following: for Treatment 3--100 .mu.Ci
.sup.3H-Kappa peptide-Elan094 (ID) was 46.48.+-.6.24% (CV 13.4%),
for Treatment 4--100 .mu.Ci .sup.3H-Kappa peptide Elan207 (ID)
(Repeat) was 16.60.+-.2.50% (15.1%), for Treatment 4--100 .mu.Ci
.sup.3H-Kappa peptide Elan207 (ID) (original) was 11.52.+-.0.96%
(CV 8.3%), and in the absence of delivery enhancer ligand Treatment
10--100 .mu.Ci .sup.3H-Kappa peptide Kaffiralin-1 (ID) was
7.94.+-.1.92% (CV 24.1%). This represents approximately a 6 fold
increase in delivery with Treatment 3--100 .mu.Ci .sup.3H-Kappa
peptide-Elan094 (ID), approx. 2 fold increase with Treatment 4--100
.mu.Ci .sup.3H-Kappa peptide Elan207 (I D) (Repeat). A similar
delivery was observed with Treatment 4--100 .mu.Ci .sup.3H-Kappa
peptide Elan207 (ID) (original) as compared to the absolute .sup.3H
bioavailability of Kappa-peptide in the absence of enhancer ligand
Treatment 10--100 .mu.Ci .sup.3H-Kappa peptide Kaffiralin-1
(ID).
[0322] .sup.3H AUClast (dpm/ml.h)
[0323] The AUClast for the ID treatments in rank order were as
follows: for Treatment 3--100 .mu.Ci .sup.3H-Kappa peptide-Elan094
(ID) was 260642.53.+-.35010.58 dpm/ml.h (CV 13.4%), for Treatment
4--100 .mu.Ci 3H-Kappa peptide Elan207 (ID) (Repeat) was
249021.32.+-.37503.78 dpm/ml.h (CV 15.1%), for Treatment 4--100
.mu.Ci .sup.3H-Kappa peptide-Elan207 (ID) (original) was
162743.70.+-.13549.10 dpm/ml.h (CV 8.3%), for Treatment 1--10
.mu.Ci .sup.3H Kappa Peptide-Kaffiralin-1 (IV) was
132184.24.+-.13288.32 dpm/ml.h (CV 10.1%), while in the absence of
ligand Treatment 10--100 .mu.Ci .sup.3H-Kappa peptide Kaffiralin-1
(ID) was 73098.43.+-.9493.88 dpm/ml.h (CV % 13.0). .sup.3H Amax
(dpm/ml)
[0324] The maximum radioactivity following administration of the ID
treatments in order of magnitude were as follows: for Treatment
1--10 .mu.Ci .sup.3H Kappa Peptide-Kaffiralin-1 (IV) was
903603.03.+-.186855.31 dpm/ml (CV 20.7%), for Treatment 4--100
.mu.Ci .sup.3H-Kappa peptide Elan207 (ID) (Repeat) was
201464.40.+-.44854.19 dpm/ml (CV 22.3%), for Treatment 3--100
.mu.Ci .sup.3H-Kappa peptide-Elan094 (ID) was 158512.67.+-.18907.79
dpm/ml (CV % 11.9), for Treatment 4--100 .mu.Ci .sup.3H-Kappa
peptide-Elan207 (ID) (original) was 100041.17.+-.6274.57 dpm/ml (CV
% 6.3), for Kappa-peptide alone Treatment 10--100 .mu.Ci
.sup.3H-Kappa peptide Kaffiralin-1 (ID) was 70925.33.+-.23631.28
dpm/ml (CV % 33.3).
[0325] H tmax (h)
[0326] The time to reach maximum radioactivity was 0.08.+-.0.00 h
for all ID treatments.
[0327] Volume of Distribution (ml)
[0328] The observed volume of distribution for Trt 1 10 .mu.Ci
3H-Kappa peptide-Kaffiralin-1 (IV) was 963.87.+-.255.65 ml (CV
26.5%).
[0329] Clearance (mi/h)
[0330] The observed clearance for Trt 1 10 .mu.Ci 3H-Kappa
peptide-Kaffiralin-1 (IV) was 142.85.+-.17.24 ml/h (CV 12.1%).
[0331] The Elan207 MTLP (SEQ ID NO 102) showed absolute .sup.3H
bioavailability in excess of 1.5 fold increase respectively in
comparison to administration of the kappa peptide control.
Following re-analysis of the stocks for Group 4 (repeat), the
recalculated absolute bioavailability was comparable to that
observed for Group 4 (original). The correlation of this repeat
data, together with the observed stability of the ELAN207 and kappa
peptides in simulated intestinal fluid, would suggest that the
observed radioactivity is associated with presence of the intact
radio-peptide conjugate in the plasma. The radioactivity profiles
will be correlated with LCMS analysis of plasma samples exhibiting
high tritium counts for verification.
[0332] The Elan094 MTLP (SEQ ID NO 2) showed absolute .sup.3H
bioavailability in excess of approximately 5.8 fold compared to
administration of the kappa peptide control. Note that the inherent
instability of the ELAN094 peptide in simulated intestinal fluid
would suggest that the radioactivity profile should be interpreted
with caution in this instance. It is possible that the parent
ELAN094 kappa peptide conjugate has deteriorated in vivo and thus,
the observed radioactivity may not be associated with presence of
the intact radio-peptide conjugate in the plasma.
[0333] A rapid delivery of .sup.3H kappa-peptide was detected in
all treatments with observed tmax of 0.08 h for all treatments.
This suggests that the kappa peptide rapidly crossed the
gastrintestinal barriers into the systemic circulation. It should
be noted that as the absorption was very rapid, the AUC might be
underestimated due to the fact that the maximum radioactivity was
measured in the first sampling point (5 minutes after dosing).
[0334] 5.sup.3H Bio-analysis: Tissue Samples
[0335] The tissue was weighed and a representative sample, (ca 0.1
g), was removed to a scintillation vial. The tissue was solublized
with 1 ml of BTS-450 (an organic tissue solubliser). Ten ml of
scintillation fluid was added and the radioactivity was measured.
The radioactivity was expressed as dpm/initial weight of
tissue.
[0336] Results:
[0337] The results are summarized in Table 14.
20TABLE 14 Summary Table % of administered dose (Mean .+-. SD - CV
%) (Potency corrected data) Group 1 Group 4 Group 10 10 .mu.Ci 3H-
Group 3 100 .mu.Ci 3H- 100 .mu.Ci 3H- Kappa 100 .mu.Ci 3H- Kappa
Kappa peptide- Kappa peptide-Elan peptide- Kaffiralin-1
peptide-Elan094 207 Kaffiralin-1 % of Admin. (IV) (IDV) (ID) (ID)
Dose n = 6 n = 6 n = 6 n = 6 *Total 34.43 .+-. 13.36 46.00 .+-.
19.91 40.91 .+-. 23.32 61.79 .+-. 16.93 Recovery (%) (CV %) 38.8
43.3 57.0 27.4 Catheter -- 0.20 .+-. 0.10 0.14 .+-. 0.09 0.06 .+-.
0.05 (%) (CV %) -- 49.3 63.4 88.8 Plasma 0.16 .+-. 0.03 0.42 .+-.
0.08 0.10 .+-. 0.01 0.06 .+-. 0.01 (%) (CV %) 18.9 17.7 9.2 11.6
Liver 6.66 .+-. 1.77 0.37 .+-. 0.06 0.47 .+-. 0.20 0.63 .+-. 0.37
(%) (CV %) 26.6 17.3 41.9 57.9 Kidney 0.51 .+-. 0.08 0.12 .+-. 0.07
0.17 .+-. 0.07 0.16 .+-. 0.17 (%) (CV %) 16.1 54.1 40.6 109.6 GI
Tissue 1.62 .+-. 0.41 11.61 .+-. 11.27 4.08 .+-. 2.47 7.72 .+-.
4.98 (%) (CV %) 25.4 97.1 60.4 64.5 GI contents 23.50 .+-. 11.04
23.57 .+-. 21.57 26.34 .+-. 17.00 37.31 .+-. 15.75 (%) (CV %) 47.0
91.50 64.5 42.2 GI Washing 1.98 .+-. 1.01 9.70 .+-. 5.24 9.60 .+-.
16.65 15.85 .+-. 5.00 (%) (CV %) 51.1 54.1 173.4 31.5 Total GI
27.10 .+-. 11.93 44.89 .+-. 19.94 40.03 .+-. 23.23 60.87 .+-. 16.74
(% recovery from GI tissue, GI contents & GI wash) (CV %) 44.0
44.4 58.0 27.5 Summation of radioactivity recovered from catheter,
plasma, all tissues, GI contents & GI washing expressed as a
percentage of administered dose
[0338] The tissue distribution following IV administration in rank
order were as follows: GI contents>Liver>GI Washing>GI
Tissue>Kidney>Plasma. Assuming the .sup.3H label remained
attached to the Kappa-peptide, this data suggests that following IV
administration the administered .sup.3H Kappa-peptide-Kaffiralin-1
had widely distributed throughout the rat and was concentrated
mainly in the GI, particularly in GI contents. The concentration in
GI contents may possibly be due to biliary excretion. However only
34.43.+-.13.36% (CV 38.8%) of administered dose had been recovered
at t=6 h suggesting some Kappa-peptide had been excreted or had
possibly distributed to other sites.
[0339] % Recovered in Catheter
[0340] The % of administered dose recovered in the catheter, for
the various treatments, ranged from 0.06-0.39%. This data suggests
that a negligible amount of the administered dose is lost in the
catheter.
[0341] % Recovered in Plasma
[0342] The % of administered dose recovered in plasma was
calculated at t=6 h. The % of administered dose ranged from
0.06-0.42% for the various treatments. The rank order for the
various treatments were as follows: for Treatment 3--100 .mu.Ci
.sup.3H-Kappa peptide-Elan094 (ID) was 0.42.+-.0.08% (17.7%), for
Treatment 1--10.quadrature.Ci .sup.3H Kappa peptide-Kaffiralin-1
(IV) was 0.16.+-.0.03% (CV 18.9%), for Treatment 4--100 .mu.Ci
.sup.3H-Kappa peptide Elan207 (ID) (original) was 0.10.+-.0.01% (CV
9.2%), and in the absence of delivery enhancer ligand Treatment
10--100 .mu.Ci .sup.3H-Kappa peptide Kaffiralin-1 (ID) the % of
administered dose was 0.06.+-.0.01% (CV 11.6%). This rank order
correlates identically with that observed for absolute
bioavailability reported in the data pack for Biostudy 1000003 for
the various treatments.
[0343] % Recovered in Liver
[0344] The % of administered dose recovered in the liver ranged
from 0.26-6.66%. Following IV administration, Treatment 1--10
.mu.Ci .sup.3H Kappa peptide-Kaffiralin-1 (IV), the % recovery was
6.66.+-.1.77% (CV 26.6%). This recovery far exceeded the amount
recovered in liver tissue for any other treatments.
[0345] % Recovered in Kidney Tissue
[0346] The % of administered dose recovered in kidney ranged from
0.12-0.51% for the various treatments. This data suggests a
negligible amount of administered dose of .sup.3H Kappa-peptide had
accumulated in kidney tissue at t=6 h.
[0347] % Recovered in GI Tract
[0348] The % of administered dose recovered in the GI tract ranged
from 27.10-89.26%. The GI tract can be further sub divided into GI
tissue, GI contents and GI Washing, the range for each was
1.62-11.97%, 23.50-65.68% and 1.98-25.84% respectively. The data
presented as % of administered dose recovered in the GI Tract
represents the summation of 6 segments of GI tissue. Within the GI
tract the highest levels of radioactivity were associated with the
latter segments (refer to raw data table). The recovered
radioactivity associated with GI segment 6 and its corresponding GI
contents were higher than that observed for other segments.
[0349] In summary, the greatest % of administered dose of .sup.3H
Kappa-peptide-ligand conjugates were recovered primarily in the GI
tract and more specifically in GI contents. Assuming that the
.sup.3H label remained attached to the Kappa-peptide, not all the
administered dose was recovered suggesting that the
Kappa-peptide-ligand-conjugates may have been excreted or had
distributed to other sites within the rat. It must be noted that
other potential sites for distribution, such as CNS tissue, weren't
analysed in this study. These measurements are of .sup.3H
Kappa-peptide only and not of intact conjugate.
[0350] The present invention is not to be limited in scope by the
sepcific embodiment, described herein. Various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications are intended to fall
within the scope of the appended claims.
* * * * *