U.S. patent application number 11/425237 was filed with the patent office on 2006-12-07 for pharmaceutical formulations and ligands for use therein; mimetics for uea-1.
Invention is credited to Christa Hamashin, Richard Houghten, Imelda Lambkin, Daniel O'Mahony, Lisa Osthues-Spindler, Clemencia Pinilla, Amy Schink.
Application Number | 20060276628 11/425237 |
Document ID | / |
Family ID | 26973111 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060276628 |
Kind Code |
A1 |
Houghten; Richard ; et
al. |
December 7, 2006 |
Pharmaceutical Formulations and Ligands for Use Therein; Mimetics
for UEA-1
Abstract
UEA-1 Mimetics, pharmaceutical formulations comprising them, and
their uses as targeting agents for therapeutic and diagnostic
purposes.
Inventors: |
Houghten; Richard; (Solana
Beach, CA) ; Pinilla; Clemencia; (Cardiff, CA)
; Lambkin; Imelda; (Sutton, IE) ; O'Mahony;
Daniel; (Blackrock, IE) ; Hamashin; Christa;
(San Diego, CA) ; Schink; Amy; (San Diego, CA)
; Osthues-Spindler; Lisa; (Julian, CA) |
Correspondence
Address: |
SYNNESTVEDT & LECHNER LLP;ELAN CORPORATION PLC
1101 MARKET STREET
SUITE 2600
PHILADELPHIA
PA
19107-2950
US
|
Family ID: |
26973111 |
Appl. No.: |
11/425237 |
Filed: |
June 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10187550 |
Jul 2, 2002 |
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11425237 |
Jun 20, 2006 |
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60302822 |
Jul 2, 2001 |
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60302868 |
Jul 3, 2001 |
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Current U.S.
Class: |
530/331 ;
564/152 |
Current CPC
Class: |
A61K 47/54 20170801 |
Class at
Publication: |
530/331 ;
564/152 |
International
Class: |
C07K 5/06 20060101
C07K005/06; C07C 237/02 20060101 C07C237/02 |
Claims
1. A pharmaceutical formulation comprising a pharmaceutical agent
and a bioadhesive ligand, said bioadhesive ligand comprising a
organocyclic moiety, said organocyclic moiety a polyhydroxy- or
polyalkoxy-substituted moiety.
2. The pharmaceutical formulation of claim 1 wherein the
bioadhesive ligand is covalently or noncovalently bound to a
carrier entity comprising the pharmaceutical agent.
3. The pharmaceutical formulation of claim 2, wherein the carrier
entity is selected from the group consisting of a nanoparticle, a
microparticle, and a liposome.
4. The pharmaceutical formulation of claim 1 wherein the backbone
of the organocyclic moiety comprises a backbone ring that consists
of 5 to 7 atoms.
5. The pharmaceutical formulation of claim 4 wherein the ring
backbone of 5 to 7 atoms is unsaturated.
6. The pharmaceutical formulation of claim 4 wherein all the atoms
of the ring backbone are carbon atoms.
7. The pharmaceutical formulation of claim 4 where the backbone of
the organocyclic moiety is identical to that of a radical selected
from the group consisting of phenyl, napthyl, cyclohexyl, benzyl,
benzoyl, pyridine, and dihydrobenzopyran.
8. The pharmaceutical formulation of claim 4 wherein the
organocyclic moiety is one in which two or more hydroxyl groups are
substituted in a radical selected from the group consisting of
phenyl, napthyl, cyclohexyl, benzyl, benzoyl, pyridine, and
dihydrobenzopyran.
9. The pharmaceutical formulation of claim 8 wherein the 2 to 4
hydroxyls are substituted in each ring backbone of the selected
radical.
10. The pharmaceutical formulation of claim 8 wherein the selected
radical is a galloyl radical.
11. The pharmaceutical formulation of claim 1 wherein the
organocyclic moiety is covalently linked to a scaffold moiety.
12. The pharmaceutical formulation of claim 11 wherein the
bioadhesive ligand comprises two or more organocyclic moieties
linked by a scaffold moiety.
13. The pharmaceutical formulation of claim 12 wherein the shortest
ring-to-ring length along the scaffold and between the two
organocyclic moieties is from 1 to 20 atoms.
14. The pharmaceutical formulation of claims 11 wherein the
scaffold moiety comprises a moiety selected from the group
consisting of amino acids, guanidines, hydantoins, thiohydantoins,
thioureas, cathechins, acylamines, dicyclicamines, tricyclicamines,
and saccharides.
15. The pharmaceutical formulation of claim 13, wherein the
scaffold moiety comprises an amino acid.
16. The pharmaceutical formulation of claim 15 wherein a
trihydroxyphenyl or trimethoxyphenyl moiety is linked to the amino
acid.
17. The pharmaceutical formulation of claims 15 wherein the amino
acid is lysine.
18. The pharmaceutical formulation of claim 15 wherein the scaffold
moiety comprises a peptide comprising at least 2 amino acids.
19. The pharmaceutical formulation of claim 15 wherein a galloyl
moiety is linked to both of the at least 2 amino acids.
20. The pharmaceutical formulation of claim 18 wherein the at least
two amino acids are both lysines.
21. The pharmaceutical formulation of claim 14 wherein the scaffold
moiety selected is an acylamine.
22. The pharmaceutical formulation of claim 21 wherein the
acylamine is of the structure X--NH--(C.dbd.O)--Y, where X
comprises a linear backbone comprising at least two atoms selected
from the group, C and N, and wherein Y comprises an organocyclic
moiety.
23. The pharmaceutical formulation of claim 20 wherein the
organocylic moiety is linked to the (C.dbd.O) group of the
acylamine either directly or by a linker moiety backbone that does
not exceed 10 atoms.
24. The pharmaceutical formulation of claim 22 wherein the R group
of at least one amino acid is linked to the X moiety of the
acylamine, said amino acid selected from the group consisting of
D-Norleucine, L-norleucine, D-tyrosine, L-tyrosine,
D-cyclohexylalanine, D-cyclohexylalanine, D-arginine, and
L-arginine.
25. The pharmaceutical formulation of claim 22 wherein an
organocyclic moiety is linked to the X moiety of the acyl amine,
said organocyclic moiety selected from the group consisting of
D-napthylmethyl, L-napthylmethyl, and L-p-chloro-benzyl.
26. The pharmaceutical formulation of claim 22 wherein the Y moiety
is selected from the group consisting of 3,4,5-trihydroxyphenyl,
3,4,5-trimethoxyphenyl, 4-biphenylmethyl, and
4-ethyl-4-biphenylmethyl.
27. The pharmaceutical formulation of claim 22 wherein
--(C.dbd.O)--Y is a galloyl group.
28. The pharmaceutical formulation of claim 22 wherein X comprises
2 to 10 organocylic moieties, each linked to the linear backbone
either directly or by a linker moiety backbone that does not exceed
10 atoms.
29. The pharmaceutical formulation of claim 1 wherein the
bioadhesive ligand comprises a compound selected from the group
consisting of those compounds specified in Tables 1, 2, 3, 4, 5, 6,
7A, 7B, and 8.
30. The compound selected from the group consisting of those
compounds listed in Tables 1, 2, 3, 4, 5, 6, 7, 7A, 7B, and 8.
31-38. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application Ser. No. 60/302,822, filed Jul. 2, 2001 and U.S.
provisional application Ser. No. 60/302,868, filed Jul. 3, 2001,
both of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] This invention has to do with pharmaceutical formulations,
and particularly pharmaceutical formulations suitable for enteral
administration, notably oral dosage forms. The invention has
particular reference to adapting a pharmaceutical formulation with
a view to improving the take-up of pharmaceutically-active
ingredient such as a drug or vaccine through the body's epithelial
layer, especially the enterocytes lining the lumenal side of the
gastrointestinal tract (GIT). An aspect of the invention relates to
the identification, preparation and use of categories of compounds,
including novel compounds, able to be incorporated in a
pharmaceutical formulation to enhance the transport of
pharmaceutically-active ingredients through the epithelial layer.
Such compounds include novel compounds useful for other purposes as
mimetics of certain naturally-occuring compounds.
[0003] This invention also has to do with compositions and methods
useful in the diagnosis and prognosis of disease states, in
particular involving the imaging e.g. by staining, or marking, of
certain tissue cell types from the human or animal body in order to
help establish the status or condition of the tissue. In particular
in one aspect the compositions disclosed herein are applied to the
investigation or evaluation of cells of the gastrointestinal tract
(GIT). This may be for assessment of a suspected or known condition
of inflammation, neoplasia, dysplasia or other abnormal and perhaps
malignant cell transformation in the cells concerned. Diseases of
particular interest include colon carcinoma, ulcerative colitis and
Crohn's Disease.
[0004] In another aspect the composition and methods disclosed
herein are applicable to the evaluation of the disposition of blood
vessels in human or animal tissue, wherever malignant or
non-malignant.
BACKGROUND TO THE DRUG/VACCINE DELIVERY ASPECTS OF THE
INVENTION
[0005] It is well known that the effect of a
pharmaceutically-active ingredient administered to the body depends
greatly on the administration route. Ideally of course one wants to
create a concentration of the active ingredient localised at the
affected site, but there is seldom a practical way of achieving
this directly. For many drugs parenteral administration (e.g.
intravenous, subcutaneous, intramuscular) is most effective but it
has well known limitations and disadvantages. These include the
risk of adverse effects from local high concentrations of drug
substance in the body, the risk of infection at injection sites and
in general a measure of discomfort or inconvenience tending to
reduce patient compliance. Patient compliance is very important
where a drug is to be routinely self-administered.
[0006] Other routes exploit drug transport across epithelial
barriers, e.g. buccal, nasal, vaginal, rectal and intestinal. Among
these, enteral and particularly oral administration is by far the
most convenient and favoured by patients. However enteral drug
delivery is notoriously problematic because of the very indirect
route by which the active ingredient enters the system. To show a
therapeutic effect an orally-administered drug must survive the
acidic environment of the stomach and then cross the epithelial
barrier i.e. the gut lining in order to enter the circulation or
interact with the immune system.
[0007] A number of published and practical proposals exist for
coating and/or encapsulating pharmaceutically-active ingredients in
excipients which allow the active substance to pass through the
stomach and survive until they reach the target region of the GIT.
One formulation type of particular current interest is the so
called microparticles and nanoparticles, made of bioerodible or
biodegradable polymeric excipients which can retain and protect the
active substance as it travels along the GIT and then be absorbable
through the gut wall, after which the particles should break down
in the bloodstream and release the active ingredient to exert its
therapeutic effect.
[0008] In practice however it has been found that bioavailability
with these formulations is nevertheless much lower than with
parenteral routes and also highly variable from one patient to
another. This is generally regarded as being because of the
difficulty in getting the active substance, in its
bioerodible/biodegradable encapsulation where present, across the
gut wall with its mucosal layer and highly selective epithelial
cells.
[0009] Particular challenges in this respect arise in relation to
the pharmaceutical use of biological or biotechnology products such
as hormones and enzymes. These are generally macromolecular, e.g.
proteins, peptides, genes, pieces of DNA, DNA vaccines, antisense
oligonucleotides etc. Their large molecular size makes it difficult
for them to cross the epithelium. Their stability in the GIT is
poor because of the action of acids and enzymes. The
bioavailability via the oral route is therefore a very low
percentage, which is doubly problematic having in mind such drugs'
scarcity and expense. Currently only parenteral administration is
usable, with its attendant disadvantages. It would be highly
desirable to improve the bioavailability of these macromolecular
drugs and vaccines via other routes.
[0010] Various proposals have been published relating to means for
giving drug-active particles a positive affinity to the gut wall so
that whatever transepithelial mechanism operated would have a
persistent presence of the active substance to work on, and/or some
biochemical incentive to promote cellular uptake of the active.
[0011] Some work has been done on this and it has been pointed out
and shown that various lectins--naturally-occurring protein
substances with specific affinities for certain sugar
residues--will bind specifically to model enterocyte-type cell
lines. This is because the enterocyte surface displays
oligosaccharide moieties. It has therefore been proposed to use
lectins as carriers for oral drug delivery, particularly taking
into account that non-toxic plant lectins are already in the human
diet. Reference is made to the following publications. F. Gabor et
al, Journal of Controllable Release 55 (1998), pp 131-142: N.
Foster et al, Vaccine 16, No. 5 (1998), pp 536-541: C. M. Lehr et
al, Pharm Res. 12 (1992) pp 547-553, and other articles on related
themes.
[0012] Despite these interesting results, the use of lectins to
promote "bioadhesion" of drug substances in the GIT remains
problematic, because such large protein molecules are liable to
degradation and loss of activity both in the gut under the action
of enzymes and during processing to prepare formulations. This
large size, together with potential immunogenicity and cytotoxicity
effects, limits the use of lectins per se as targeting agents to
deliver drugs and vaccines to and across the human GIT.
[0013] The present inventors have carried out very extensive
investigations with a view to identifying, testing and preparing
alternative substances showing an affinity for epithelial cells,
and hence a "bioadhesive" capacity making them useful as moieties,
ingredients or coatings in enterally-administered pharmaceutical
formulations.
BACKGROUND TO THE DIAGNOSTIC AND PROGNOSTIC ASPECTS OF THE
INVENTION
[0014] It has been noted that alteration and/or upregulation of
surface sugar residues in the intestinal mucosa have been
associated with malignant transformation, dysplastic changes and
extensive colitis. For example, tissues from ulcerative colitis and
Crohn's disease patients exhibited altered distribution of Ulex
europaeus I (UEA1) labelling sites (Yoshioka et al, 1989). The
expression of lectin-binding sites on human intestinal goblet mucin
was specifically altered in these conditions, thus possibly
providing an alternative approach to the assessment of neoplastic
risk in these diseases (Yoshioka et al, 1989). Patterns of UEA1 and
Dolichos biflorus agglutinin (DBA) in carcinomas of the large
intestine were also altered when compared to normal mucosa and
adenomas (Iwakawa et al, 1996)
[0015] UEA1 is a lectin protein of approximately 60 kDa derived
from furze (Ulex europaeus) that is known to bind to fucose
residues and in particular is known to bind to epithelial
cells.
[0016] We have done a large amount of work investigating the
properties of UEA1 and in identifying and synthesising other
molecules which mimic UEA1, in the sense that they share to a
lesser or greater degree (a greater degree, in some cases) the
characteristic binding activity of UEA1 to epithelial cells, but
because of their simpler molecular structure may enjoy any of
higher stability, reduced cost, easier labelling or the possibility
of use in multivalent forms. These other molecules, referred to in
what follows as "UEA1 mimetics" may have any of a variety of
organomolecular structures. They may be peptides, peptidomimetics
and/or small organic molecules. A variety (non-limiting) of such
molecules and methods of identifying and preparing them are
discussed later below.
[0017] We have confirmed the effectiveness of UEA1 and of its
mimics in binding to human intestinal tissue sections. In view of
the relationships noted above between various disease states, we
put forward the first aspect of the present invention which is
methods and compositions for assessing the status of GIT cells by
means of imaging, using UEA1 or a UEA1 mimetic as a localisation
agent which binds characteristically to epithelial cells.
[0018] A second aspect of the invention relates to the fact that
UEA1 binds specifically to the vascular endothelium of various
human tissues irrespective of the blood group type or secretive
status of the tissue. UEA1 staining of blood vessels has been
evaluated in various studies of malignant and nonmalignant tissues.
For example, most vessels in malignant and nonmalignant tissues of
bladder, prostate and testis were readily identified (Fujime et al,
1984). UEA1 visualized the endothelia of blood vessels with equal
intensity, sensitivity, and reliability in normal brain and in
tumour tissue with neovascularization (Weber et al, 1985). While
large, medium, and small vessels were equally well demonstrated by
UEA1 and antibodies against FVIII/RAG, capillaries and endothelial
sprouts were stained more consistently and intensely by UEA1. UEA1
was also a specific and sensitive marker for the endothelial cells
in benign vascular lesions (Miettinen et al, 1983). UEA1 also
stained many neoplastic cells of endothelial sarcomas. Melanomas,
anaplastic carcinomas, and other types of sarcomas were
negative.
[0019] Since UEA1 stains blood vessels of both normal and tumour
tissues with equal intensity it is not an obvious tumour
vasculature-specific targeting agent. However we observe that UEA1
staining has potential application in studying distribution of
vessels in relation to various normal and pathological events.
Since blood vessel invasion is one of the most important diagnostic
and prognostic parameters used by pathologists in the evaluation of
neoplastic conditions, UEA1 and mimetics thereof such as peptides,
peptidomimetics and/or small organic molecules which mimic UEA1
have value in establishing the diagnosis of lymphovascular
involvement.
[0020] Thus, the use of UEA1 and its mimetics as disclosed herein
in the diagnosis/prognosis of conditions by observation of vascular
involvement and corresponding compositions which may be adapted for
imaging as in the first aspect of the above is a further aspect of
the invention.
SUMMARY OF THE INVENTION
[0021] In one general aspect, the invention is a pharmaceutical
formulation comprising a pharmaceutical agent and a bioadhesive
ligand, said bioadhesive ligand comprising an organocyclic (C,N, O
and/or S) moiety, said organocyclic moiety a polyhydroxy- or
polyalkoxy-substituted moiety (at least 2 hydroxy or 2 alkoxy
groups, respectively.) Polyhydroxy-substituted organocyclic
moieties are preferred. For polyalkoxy-substituted organocyclic
moieties, C.sub.1-C.sub.5 alkoxys are preferred, and
C.sub.1-C.sub.3 alkoxys are more preferred, where for example a
C.sub.2 alkoxy is ethoxy.
[0022] The ligand may be bound, either covalently or noncovalently,
to a carrier entity comprising the pharmaceutical agent. The ligand
is preferably bound to the surface of the carrier.
[0023] In embodiments of particular interest, the carrier entity is
selected from the group consisting of a nanoparticle, a
microparticle, and a liposome.
[0024] In some preferred embodiments, the backbone of the
organocyclic moiety comprises a backbone ring that consists of 5 to
7 atoms. (For purposes herein, benzene has a backbone of 6 carbon
atoms in a single ring, naphthalene a backbone of 10 carbon atoms
and has two backbone rings, each consisting of 6 carbon atoms, the
two rings sharing two carbon atoms). The backbone ring of 5 to 7
atoms may be unsaturated (i.e., aromatic). In some highly preferred
embodiments, all the atoms of the ring backbone are carbon
atoms.
[0025] Preferred backbones for the organocyclic moiety are those
identical to that of a radical selected from the group consisting
of phenyl, napthyl, cyclohexyl, benzyl, benzoyl, pyridine and
dihydrobenzopyran. It is particularly preferred that such a
backbone is substituted with 2 or more hydroxy radicals, most
preferably 2 to 4 hydroxy radicals. Highly preferred organocylcic
moieties are galloyl or trimethoxyphenyl radicals.
[0026] In a highly preferred set of embodiments, the organocyclic
moiety is covalently linked to a scaffold moiety. In one such
embodiment, the bioadhesive ligand comprises two or more
organocyclic moieties linked by a scaffold moiety. Among the
preferred constructs are those wherein the shortest ring-to-ring
length along the scaffold and between the two organocyclic moieties
is from 1 to 20 atoms. (To illustrate, in compound 2, described
below, the shortest ring-to-ring length between the napthyl and
chloro-phenyl radicals is 6, between the napthyl and biphenyl
radicals it is 5.)
[0027] A preferred group of scaffold moieties comprises a moiety
selected from the group consisting of amino acids, guanidines,
hydantoins, thiohydantoins, thioureas, cathechins, acylamines,
dicyclicamines, tricyclicamines, and saccharides. A scaffold
comprising an amino acid is highly preferred, as are ones
comprising a peptide of at least 1 amino acids (preferably 2 to 50,
more preferably 2 to 20, most preferably 2 to 6 amino acids). It is
also highly preferred that a trihydroxybenzoyl or trimethoxybenzyl
moiety be linked (either directly or via a linker) to the amino
acid or amino acids through the amide functionality. Lysine is a
highly preferred amino acid for purposes of building the
scaffold.
[0028] Preferably X comprises 2 to 10 organocylic moieties, each
linked to the linear backbone either directly or by a linker moiety
backbone that does not exceed 10 atoms.
[0029] Another preferred scaffold moiety is an acylamine. Preferred
acylamines are those of the structure X--NH--(C.dbd.O)--Y, where X
comprises a linear backbone comprising at least two atoms
(preferably 2 to 20) selected from the group, C and N. More
preferably X comprises 2 to 10 organocylic moieties, each linked to
the linear backbone either directly or by a linker moiety backbone
that does not exceed 10 atoms.
[0030] Y preferably comprises a polyhydroxy or polymethoxy
organocyclic moiety. It is particularly preferred that such an
organocylic moiety is linked to the (C.dbd.O) group of the
acylamine either directly or by a linker moiety backbone that does
not exceed 10 atoms. In a preferred set of embodiments, the Y
moiety is selected from the group consisting of
3,4,5-trihydroxyphenyl, 3,4,5-trimethoxyphenyl, 4-biphenylmethyl,
and 4-ethyl-4-biphenylmethyl. In one highly preferred embodiment,
--(C.dbd.O)--Y is a galloyl group.
[0031] In one set of preferred embodiments, the R group (e.g., the
cyclohexylmethyl moiety of cyclohexylalanine) of at least one amino
acid is linked to the X moiety of the acylamine, said amino acid
selected from the group consisting of D-Norleucine, L-norleucine,
D-tyrosine, L-tyrosine, D-cyclohexylalanine, L-cyclohexylalanine,
D-arginine, and L-arginine. An organocyclic moiety can be linked to
the X moiety of the acyl amine, said organocyclic can for example
be selected from the group consisting of D-napthylmethyl,
L-napthylmethyl, and L-p-chloro-benzyl.
[0032] As regards examples of specific bioadhesive ligands, the
bioadhesive ligand comprises a compound selected from the group
consisting of those compounds specified in Tables 1-6, 7A, 7B, and
8, below. (The foregoing takes into account, for example, that the
R1, R2, and R3 compounds in the individual columns in Tables 1, 2,
and 3, were the compounds used to generate the somewhat smaller R1,
R2, and R3 moieties in the chemical diagram (equivalent to
--NH--CH2-CHR1-N(-)--CH2-CHR2-NH--CO--R3) immediately preceding
those tables). As a result, the appearance of Nap-ala for
napthylalanine as an R1 in Table 1 indicates that the corresponding
R1 radical in the chemical diagram is napthylmethyl- and the
appearance of 3,4,5-trimethoxybenzoic acid as an R3 in Table 1
indicates that the corresponding R3 radical in the chemical diagram
is 3,4,5, trimethoxyphenyl. An analogous situation will be seen to
exist for other Tables)
[0033] As to Table 4, it is preferred that the ligand comprises a
compound that has a Single Tier Assay Avg % inhibition at 250.0
(.mu.g/ml) of at least 30, more preferably at least 20.
[0034] As to Table 6, it is preferred that the ligand comprises a
compound that has a Single Tier Assay IC50(50 ug/ml) less than 250,
preferably less than 100, most preferably less than 30.
[0035] As to Table 7(a) it is preferred that the bioadhesive ligand
comprises a 2-copy structure specified in Table 7(A) that has a 2nd
Tier IC50 value (uM) of 350uM or less, preferably less than 200 uM,
more preferably less than 100 uM.
[0036] As to Table 7(b) it is preferred that the bioadhesive ligand
comprises a compound that is a 4-copy structure specified as having
a 2nd Tier IC50 value (uM) of 250 uM or less, more preferably less
than 200 uM, even preferably less than 50 uM, most preferably less
than 3 uM.
[0037] As to Table 8, it is preferred that the bioadhesive ligand
comprises a compound that has an IC 50 (uM), in a 2nd tier assay,
that is less than 150, preferably less than 15.
[0038] The tested compounds described in Tables 1, 2, 3, 4, 5, 6,
7, 7A, 7B, and 8 are themselves aspects of the invention.
[0039] In another general aspect, the invention is a method of
administering a pharmaceutical formulation to an organism having an
intestinal epithelium (preferably a mammal, most preferably a
human), said method comprising administering a pharmaceutical
formulation of Claim 1. In one set of embodiments of interest, the
bioadhesive ligand is covalently or noncovalently bound (preferably
on the surface) to a carrier comprising the pharmaceutical
agent.
[0040] The present inventors have carried out very extensive
investigations with a view to identifying, testing and preparing
alternative substances showing an affinity for epithelial cells,
and hence a "bioadhesive" capacity making them useful as moieties,
ingredients or coatings in enterally-administered pharmaceutical
formulations.
[0041] We screened a number of combinatorial libraries, including
both peptide and non-peptide organic molecules, in competitive
assays with the lectin UEA-1, a lectin protein of approximately 60
kDa derived from furze (Ulex europaeus) that is known to bind to
fucose residues and in particular is known to bind to epithelial
cells.
[0042] What we have found is that cyclic organic groups having two
or more and preferably three or more hydroxy or hydroxy-bearing
substituents can be binding-active moieties with respect to
epithelial cells including epithelial cells of the intestinal
tract. Organic compounds having such binding-active moieties, and
particularly having two or more of them on an organic skeleton or
"scaffold", can be used as bioadhesive ligands in pharmaceutical
formulations.
[0043] The cyclic group may be carbocyclic or heterocyclic. It may
be aromatic, non-aromatic, fused aromatic or fused partly-aromatic
ring systems.
[0044] Polyhydroxy-substituted aromatic groups are preferred, e.g.
diols, triols, tetrols etc of phenyl and related aryl ring systems
e.g. naphthyl, or also alicyclics such as cyclohexenyl. The phenyl
or related ring may be joined to a molecular skeleton or scaffold
as a benzyl or benzoyl group, or the equivalent for the related
ring systems.
[0045] Hydroxy groups on the ring may be vicinal.
[0046] In particular we have found good results with
trihydroxyphenyl groups, which may be linked to a scaffold as
trihydroxybenzyl or benzoyl.
[0047] The most preferred binding-active moiety that we have found
is based on a 3,4,5-hydroxyphenyl group which may be joined to a
scaffold e.g. via an amide or other acyl link, so that it
constitutes a galloyl (3,4,5-hydroxybenzoyl) group.
[0048] Preferably the hydroxy groups take the --OH form, although
thiol analogues and masked e.g. alkoxylated forms may also be
useful.
[0049] As referred to above, we have found that good results are
achieved when two or more, and preferably three or more,
binding-active moieties as specified above are provided on an
organomolecular scaffold or skeleton. A wide variety of options
exist for this scaffold but of course it is preferably biologically
compatible in the sense that it will not break down to harmful
substances, and generally preferably contains nothing other than
carbon, nitrogen, oxygen, sulphur and hydrogen. It may be linear,
branched, cyclic or any combination of these.
[0050] Preferably the scaffold consists of hydrocarbon entities
linked via functional groups. Suitable functional groups are
preferably selected from but not limited to amino, amido, acyl,
ether, ester, carboxylic acid and urea linkages.
[0051] In view of their established biological acceptability,
molecular scaffolds based on or comprising amino acid units, and/or
analogues or derivatives thereof, are preferred. The scaffold may
be or comprise an amino acid, peptide, oligopeptide (preferably
from 2 to 10 and more preferably from 1 to 6 amino acids)
substituted with one or more and preferably plural of the
binding-active moieties mentioned above. Natural or synthetic amino
acids may be used in the scaffold.
[0052] Non-peptide scaffolds are also possible. The skilled person
is already aware of peptidomimetic molecules and molecular
frameworks of established effectiveness, and these include, among
various types of molecules using the functional groups and linkages
mentioned above, molecules comprising heterocyclic rings,
guanidines, hydantoins, thiohydantoins, thioureas, catechins,
acylamines, saccharides and so forth.
[0053] Where the scaffold comprises an amino acid, peptide,
oligopeptide or analogue thereof at least one binding-active moiety
may be linked at the C-terminal of the scaffold.
[0054] The scaffold may provide a linear or cyclic backbone from
which the binding-active groups are branched, optionally via branch
spacer chains such as hydrocarbon chains.
[0055] Links between the binding-active moieties and the scaffold
may be via amino, amido, acyl, ether, alkylene, alkenylene or other
suitable functionalities, or any combination of these.
[0056] While many of the binding-active compounds (ligands)
proposed herein are believed to be novel, it is also possible to
use existing compounds and analogues thereof such as tannic acid
and the other tannins, which in general feature plural galloyl
substituents on a sugar substrate. For these known substances, this
is a newly-proposed use and formulation.
[0057] Considering the ligand compound as a whole (one or more
binding-active moieties plus any scaffold) or multimers thereof its
molecule is designed in line with conventional biochemical practice
so as to be sufficiently stable in the enteric tract. By comparison
with the lectins as previously discussed, the molecular weight of
the ligand may be low and this, together with a suitable chemical
make up, can provide stability, reduction in the potential for
immunogenicity and cytotoxicity, as well as facilitating the
manufacture and processing of synthetic ligands. A preferred
molecular weight is less than 5000, preferably less than 2000 or
1500, but does not exclude multimers thereof.
[0058] However it should be noted that the present proposals also
comprehend the possibility of providing the binding-active moieties
whose effectiveness has been disclosed here on other types of
molecule. For example they may be provided as substituents or
grafts on a polymeric excipient used in the pharmaceutical
formulation, such as a biodegradable polymer. The ligand molecule
as a whole may be covalently or non-covalently bound on or into the
pharmaceutical formulation. Similarly, the ligand molecule as a
whole may be covalently or non-covalently bounds to a drug or
antigen or adjuvant.
[0059] The novel pharmaceutical formulations exploiting these
binding-active moieties and ligand compounds are one aspect of the
invention. The use of the binding-active moieties and ligand
compounds to enhance drug delivery in an enteric e.g. oral
formulation is another aspect.
[0060] The ligand compounds proposed herein are for the most part
novel, and in themselves, as UEA1 mimics, are an aspect of the
invention claimed here.
[0061] The corresponding methods are also aspects of the invention
claimed here, namely methods comprising the synthesis of the novel
binding compounds, and methods of preparing pharmaceutical
formulations comprising incorporating the ligand compounds--whether
novel or not--into the formulation by blending, binding, coating or
by other means.
[0062] In particular embodiments of the invention, one of the
aforementioned bioadhesive ligands is covalently or non-covalently
bound to a carrier entity comprising a pharmaceutical agent. For
example, the carrier entity is selected from the group consisting
of a nanoparticle, microparticle and liposome. It is preferred that
the carrier entity have a largest dimension that is in the range of
10 nm to 500 .mu.m, as discussed in more detail elsewhere herein.
In particular embodiments of the invention, the pharmaceutical
agent is a drug or therapeutic agent. In other specific
embodiments, the pharmaceutical agent is a pathogen antigen.
[0063] Certain aspects of the invention involve the use of the
bioadhesive ligands to target delivery of pharmaceutical
agents.
[0064] In one aspect, the invention is a method of administering a
pharmaceutical agent to an organism having intestinal epithelium,
said method comprising contacting said intestinal epithelium with
one of the aforementioned bioadhesive ligands that is covalently,
or non-covalently bound to, a carrier entity. In preferred the
embodiments, the organism is a mammal. Most preferably, the mammal
is a human.
[0065] In particular embodiments of the method, the carrier entity
is from the group consisting of a nanoparticle, microparticle or
liposome. Preferably, the carrier entity has its major dimension in
the range of 10 nm to 500 .mu.m. In preferred embodiments, the
carrier entity drug-loaded or drug-encapsulated. The preferred
route of administration for delivery of the ligand-carrier entity
is the oral route. Other possible routes are the rectal,
subcutaneous, intramuscular and intravenous routes.
[0066] As used herein, the term "carrier entity" is defined as a
particle or droplet that can carry a pharmaceutical agent. A
microparticle is defined as a particle whose major dimension in the
range 1 to 5 .mu.m, most preferably in the range 1 to 3 .mu.m. A
nanoparticle is defined as a particle whose major dimension is less
than 1.mu., preferably in the range 1 nm to 500 nm, most preferably
in the range 10 nm to 500 nm.
[0067] As used herein, the major dimension of a spherical particle
is its diameter, or a rod shaped particle, its length. For other
particles, it is the longest dimension possible for the
particle.
[0068] Nano- and microparticles that are loaded with, or
encapsulate, pharmaceutical agents, can be coated with the
bioadhesive ligands, such as those of the present invention, that
target intestinal epithelium tissue. The coating can be effected by
covalent or non-covalent bonding. The covalent bonding can be
achieved by adsorption or any other coating process. In either
case, the bonding can be made to completed particles or to particle
components that subsequently form part of the particles.
[0069] Biodegradable Particles are Preferred.
[0070] Pharmaceutical agents can, in the alternative, be directly
linked a bioadhesive ligand.
[0071] A "pharmaceutical agent" is a therapeutic or diagnostic
agent. Therapeutic agents are those that are administered either to
treat an existing disease or prophylactically to protect against a
potential future disease. Diagnostic agents are any agents that are
administered as part of a diagnostic procedure.
[0072] Examples of therapeutic agents are drugs, genes,
gene-delivery vectors, and antigens/vaccines.
[0073] Drugs include, for example, analgesics, anti-migraine
agents, anti-coagulant agents, anti-emetic agents, cardiovascular
agents, anti-hypertensive agents, narcotic antagonists, chelating
agents, anti-anginal agents, chemotherapy agents, sedatives,
anti-neoplastics, prostaglandins and antidiuretic agents, antisense
oligonucleotides, gene-correcting hybrid oligonucleotides,
ribozymes, RNA interference (RNAi) oligonucleotides, silencing RNA
(siRNA) oligonucleotides, aptameric oligonucleotides and
triple-helix forming oligonucleotides, DNA vaccines, adjuvants,
recombinant viruses.
[0074] Examples of gene-delivery vectors are DNA molecules, viral
vectors (E.g. adenovirus, adeono-associated virus, retroviruses,
herpes simplex virus, and sindbus virus), and cationic lipid-coated
DNA and DNA-dendrimers.
[0075] Drugs include conventional small molecule drugs, proteins,
oligopeptides, peptides, and glycoproteins.
[0076] Examples of drugs are as insulin, calcitonin, calcitonin
gene regulating protein, atrial natriuretic protein, colony
stimulating factor, betaseron, erythropoietin (EPO), interferons
(E.g. .alpha., .beta. or .gamma. interferon), somatropin,
somatotropin, somatostatin, insulin-like growth factor
(somatomedins), luteinizing hormone releasing hormone (LHRH),
tissue plasminogen activator (TPA), growth hormone releasing
hormone (GHRH), oxytocin, estradiol, growth hormones, leuprolide
acetate, factor VIII and interleukins (E.g. interleukin-2).
Representative drugs also include: analgesics (E.g. fentanyl,
sufentanil, butorphanol, buprenorphine, levorphanol, morphine,
hydromorphone, hydrocodone, oxymorphone, methadone, lidocaine,
bupivacaine, diclofenac, naproxen and paverin); anti-migraine
agents (E.g. sumatriptan and ergot alkaloids); anti-coagulant
agents (E.g. heparin and hirudin); anti-emetic agents (E.g.
scopolamine, ondansetron, domperidone and metoclopramide);
cardiovascular agents, anti-hypertensive agents and vasodilators
(E.g. diltizem, clonidine, nifedipine, verapamil,
isosorbide-5-mononitrate, organic nitrates and agents used in
treatment of heart disorders); sedatives (E.g. benzodiazepines and
phenothiozines); narcotic antagonists (E.g. naltrexone and
naloxone); chelating agents (E.g. deferoxamine); anti-diuretic
agents (E.g. desmopressin and vasopressin); anti-anginal agents
(E.g. nitroglycerine); anti-neoplastics (E.g. 5-fluorouracil and
bleomycin); prostaglandins; and chemotherapy agents (E.g.
vincristine).
[0077] Examples of antigens that are therapeutic agents are tumor
antigens, pathogen antigens and allergen antigens. A vaccine
preparation will contain at least one antigen. "Pathogen antigens"
are those characteristic of pathogens, such as antigens derived
from viruses, bacteria, parasites or fungi.
[0078] Examples of important pathogens include vibrio choleras,
enterotoxigenic E. Coli, rotavirus, Clostridium difficile, Shigella
species, Salmonella typhi, parainfluenza virus, influenza virus,
Streptococcus mutans, Plasmodium falciparum, Staphylococcus aureus,
rabies virus and Epstein-Barr virus.
[0079] Viruses in general include the following families:
picronaviridae; caliciviridae, togaviridae; flaviviridae;
coronaviridae; rhabodviridae; filoviridae; paramyxoviridae;
orthomyxoviridae; bunyaviridae; arenaviridae; reoviridae;
retroviridae; hepadnaviridae; parvoviridae; papovaviridae;
adenoviridae; herpesviridae and poxyviridae.
[0080] Bacteria in general include but are not limited to: P.
aeruginosa; E. coli; Klebsiella sp.; Serratia sp; Pseudomanas sp.;
P. cepacia; Acinetobacter sp.; S. epidermis; E. faecalis; S.
pneumonias; S. aureus; Haemophilus sp.; Neisseria sp.; N.
meningitidis; Bacterodies sp.; Citrobacter sp.; Branhamella sp.;
Salmonelia sp.; Shigella sp.; S. Lesteria sp., Pasteurella
multocida; Streptobacillus sp.; S. pyogenes; Proteus sp.;
Clostridium sp.; Erysipelothrix sp.; Spirillum sp.; Fusospirocheta
sp.; Treponema pallidum; Borrelia sp.; Actinomycetes; Mycoplasma
sp.; Chlamydia sp.; Rickettsia sp., Spirchaeta; Legionella sp.;
Mycobacteria sp.; Urealplasma sp.; Streptomyces sp.; Trichomoras
sp.; and P. mirabilis
[0081] Parasites include but are not limited to: Plasmodium
falciparum, P. vivax, P. ovale, P. malaria; Toxoplasma gondii;
Leishmania mexicana, L. tropica, L. major, L. aethiopica, L.
donovani, Trypanosoma cruzi, T. brucei, Schistosoma mansoni, S.
haematobium, S. japonium; Trichinella spiralis; Wuchereria
bancrofti; Brugia malayli; Entamoeba histolytica; Enterobus
vermiculoarus; Taenia solium, T. saginata, Trichomonas vaginitis,
T. hominis, T. tenax; Giardia lamblia; Cryptosporidium parvum;
Pneumocytis carinii, Babesia bovis, B. divergens, B. microti,
Isospore belli, L. hominis; Dientamoeba fragiles; Onchocerca
volvulus; Ascaris lumbricoides; Necator americanis; Ancylostoma
duodenale; Strongyloides stercoralis; Capillaria philippinensis;
Angiostrongylys cantonensis; Hymenolepis nan; Diphyllobothrium
latum; Echinococcus granulosus, E. multilocularis; Paragonimus
westermani, P. caliensis; Chlonorchis sinensis; Opisthorchis
felineas, G. Viverini, Fasciola hepatica, Sarcoptes scabiei,
Pediculus humanus; Phtirius pubis; and Dermatobia hominis.
[0082] Fungi in general include but are not limited to:
Crytpococcus neoformans; Blastomyces dematitidis; Aiellomyces
dermatitidis Histoplasfrai capsulatum; Coccidiodes immitis; Candids
species, including C. albicans, C. tropicalis, C. parapsilosis, C.
guilliermondii and C. krusei, Aspergillus species, including A.
fumigatus, A. flavus and A. niger, Rhizopus species; Rhizomucor
species; Cunnighammella species; Apophysomyces species, including
A. saksenaea, A. mucor and A. absidia; Sporothrix schenckii,
Paracoccidioides brasiliensis; Pseudallescheria boydii, Torulopsis
glabrata; and Dermatophyres species.
[0083] Antigens that are allergens can be haptens, or antigens
derived from pollens, dust, molds, spores, dander, insects and
foods. Specific examples include the urusiols of Toxicodendron
species and the sesquiterpenoid lactones.
[0084] Examples of adjuvants: Freund's Complete Adjuvant, Freund's
Incomplete Adjuvant, Hunter's Titermax, Gerbu Adjuvant, Ribi's
Adjuvant, Montanide ISA Adjuvant, Aluminum Salt Adjuvants and
Nitrocellulose adsorbed protein.
[0085] In another general aspect we have confirmed the
effectiveness of UEA1 and of its mimics in binding to human
intestinal tissue sections. In view of the relationships noted
above between various disease states, we put forward the first
aspect of the present invention which is methods and compositions
for assessing the status of GIT cells by means of imaging, using
UEA1 or a UEA1 mimetic as a localisation agent which binds
characteristically to epithelial cells. Relevant disease states
include any of those mentioned above, for example, colon carcinoma,
ulcerative colitis and Crohn's Disease. The UEA1 or UEA1 mimetic
localisation agent may be exploited for diagnostic/prognostic
imaging in any of a variety of ways and these may in themselves be
conventional. For example the UEA1 and the UEA1 mimetic may be used
in an immunoassay procedure with an antibody therefor or other
specific binding substance, and an imaging agent or agents (e.g. a
colour staining test kit) for providing a characteristic
image/colour when reacted with the antibody or other specific
binding substance.
[0086] Alternatively UEA1 or UEA1 mimetic may be directly labelled,
e.g. biotinylated or by some other means, so that its bound
presence on the test cells can be verified by reaction with avidin
or the appropriate other imaging substance(s) or test for the type
of label used. Other possibilities include NMR imaging and
radiolabelling.
[0087] Compositions for the present purpose may comprise the UEA1
or UEA1 mimetics, which may be labelled, as part of a
diagnostic/prognostic imaging kit including any necessary
complementary binding substances and imaging media.
[0088] The use of UEA1 and its mimetics as disclosed herein in the
diagnosis/prognosis of conditions by observation of vascular
involvement and corresponding compositions which may be adapted for
imaging as in the first aspect of the above is a further aspect of
the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0089] FIG. 1 Synthesis of compounds having four copies of gallic
acid.
[0090] FIG. 2 Surface binding and uptake of MSI35-2 gallic acid
mimetic coated particles.
[0091] FIG. 3 Surface binding and uptake of UEA1 coated
particles.
[0092] FIG. 4 Surface binding & uptake of biocytin mimetic
coated particles.
[0093] FIG. 5 MSI 35: Biotinylated 2-copy and 4-copy
structures.
[0094] FIGS. 6 and 7. Stained human tissue samples.
[0095] FIG. 8 Endothelial cell permeability assay.
DETAILED DESCRIPTION
[0096] There now follows a detailed description of those aspects of
our experimental work leading to the identification of the
particular binding-active moieties disclosed herein, ligand
compounds bearing them, verification of their activity and various
examples of ligand compounds embodying the invention.
[0097] The search for small molecular weight ligands capable of
binding surface receptors of epithelial cells began with the
screening of various combinatorial libraries, which contained both
peptides and non-peptide (organic) molecules. These libraries were
synthesized in positional scanning format in which oligopeptide
mixture sets comprise one predetermined residue at a single
predetermined position of the oligopeptide chain (Pinilla et al.
U.S. Pat. No. 5,556,762, Pinilla et al. 1992. BioTechniques. Vol
13, No 6).
[0098] The epithelial cell binding ability of compounds and
mixtures obtained from synthetic combinatorial libraries was
determined by competition assays. These assays were set up to
measure the inhibition of binding of biotinylated UEA-1 to membrane
preparations of the epithelial cell line Caco-2 (colon carcinoma
cells-2), by compounds and mixtures of the combinatorial libraries.
This cell line is a conventional model for epithelial cells. The
ability of a compound or compounds to inhibit UEA-1 binding would
suggest that this chemical itself was binding to fucose residues on
the surface of epithelial cells and was hence a potential
ligand.
[0099] Two competition inhibition assays were used in this case,
namely single tier and two tier assays. These assays differed from
each other in that in the single tier assay, the mixtures/compounds
and the biotinylated UEA-1 were incubated together with the Caco-2
cell membrane preparations, while in case of the two-tier assay,
the mixture/compounds were allowed to incubate alone with the cell
membranes in the absence of biotinylated UEA-1, which was added in
the next step. Addition of the extra step in the two-tier assay was
to ensure that the compounds inhibiting the binding of biotinylated
UEA-1 to the Caco-2 cell membranes were doing so by themselves
binding to surface receptors on the Caco-2 cells and not to the
biotinylated UEA-1. Each assay will be described in detail.
Preparation of Caco-2 Cell Membrane (P100) and Cytosolic (S100)
Fractions:
[0100] 1. Confluent Caco-2 cell monolayers (grown in 75 cm.sup.2
flasks for up to 1 week at 37.degree. C. and 5% CO.sub.2) were
washed twice in Dulbecco's PBS (DPBS). [0101] 2. Cell monolayers
were treated with 10 mM EDTA-DPBS for 5-10 min at 37.degree. C. and
cells were harvested by centrifugation at 1000 rpm for 5 min.
[0102] 3. Cells were washed 3 times in DPBS. [0103] 4. The cell
pellet was resuspended in 3 volumes of ice cold HED buffer (20 mM
HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 1 mM
phenylmethylsulphonyl fluoride (PMSF)) and the cells were allowed
to swell for 5 min on ice. [0104] 5. The cells were homogenised for
30 sec. [0105] 6. The homogenates were centrifuged in hard walled
tubes at 40,000 rpm for 45 min at 4.degree. C. [0106] 7. The
supernatant (S100) was removed and the pellet (P100) was
resuspended in HEDG buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5
mM dithiothreitol, 100 mM NaCl, 10% glycerol, 1 mM PMSF): 3 volumes
of buffer were added, the pellet was resuspended and centrifuged at
1000 rpm for 2 min. The supernatant was removed and stored on ice.
The procedure was repeated adding the second supernatant to the
first. [0107] 8. The protein concentration was determined using
e.g. a Bio-Rad protein assay. [0108] 9. All fractions were stored
at -80.degree. C. prior to use. Single Tier Assays: [0109] 1.
96-well microtiter plates were coated with membrane preparations of
Caco-2 cells either by allowing them to incubate on the plates for
21/2 hours at room temperature or by incubating them overnight at
4.degree. C. 100 .mu.l of 10 .mu.g/ml (in 0.05 M carbonate buffer,
pH 9.6) of membrane preparation was added to each well. [0110] 2.
The plates were flicked out, patted dry and blocked with bovine
serum albumin (BSA)/DPBS (200 .mu.l/well) for 1 to 4 hours at room
temperature after which they were washed three times in water.
[0111] 3. 50 .mu.l/well (in 1.5% BSA/DPBS) of the
mixtures/compounds from the combinatorial libraries were added to
the wells. Control wells were also set up in which the Caco-2 cell
membrane preparations were incubated with unconjugated UEA-1. A 1
in 4 dilution series of this control compound was set up starting
from 0.04 .mu.g/ml to 160 .mu.g/ml (50 .mu.l/well in 1.5%
BSA/DPBS). [0112] 4. Biotinylated UEA-1 at a final concentration of
1 .mu.g/ml (50 .mu.l in 1.5% BSA/DPBS) was added to each well and
the plates were left to incubate overnight at 4.degree. C. [0113]
5. Following overnight incubation the plates were washed thoroughly
(3-6 times). The plates were flicked out, patted dry and
biotinylated UEA-1 was detected with commercial Streptavidin
conjugated to horseradish peroxidase (HRP) (CalBiochem). This
reagent was added to each well at a 1:5000 dilution prepared in
1.5% BSA-DPBS such that 100 .mu.l of the reagent was added to each
well to achieve the final concentration. The plates were left to
incubate for an hour at room temperature. [0114] 6. The plates were
then washed 3-6 times and the biotin-streptavidin binding was
detected by adding an HRP substrate OPD (orthophenyl diamine) to
each well. 100 .mu.l/well of this substrate at a final
concentration of 1.6 mg/ml was added to each well. Before adding to
the wells, this substrate was activated by addition of 50 .mu.l of
3% H.sub.2O.sub.2 per plate and the reaction was allowed to develop
in the dark. [0115] 7. The reaction was stopped by adding 50 .mu.l
of 4 N H.sub.2SO.sub.4 to each well after approximately 5-10
minutes or when sufficient colour had developed. The OD.sub.490
(absorbance at 490 nm) of each well was measured using a
conventional 96 well plate reading spectrophotometer. Two Tier
Assays: [0116] 1. 96 well plates were coated with Caco-2 cell
membrane preparations in the same manner as described in the single
tier assay. [0117] 2. Plates were flicked out, patted dry and
blocked with 1.5% BSA-DPBS (200 .mu.l/well) for 1-4 hours at room
temperature and then washed three times in water. [0118] 3. The
compounds/mixtures from various combinatorial libraries were then
added to individual wells (100 .mu.l/well in 1.5% BSA-DPBS).
Control wells were also set up containing a range of concentrations
of purified UEA-1 which were set up as 1 in 4 dilutions starting
from 0.04 .mu.g/ml to 160 .mu.g/ml. 100 .mu.l at these final
concentrations were added to each well. [0119] 4. The plates were
then left to incubate overnight, a step that would allow the
mixtures and compounds to interact with the Caco-2 cell membranes
without the presence of the competitor (biotinylated UEA-1) itself.
[0120] 5. Following overnight incubation, the plates were washed
thoroughly in water 3-6 times. [0121] 6. 100 .mu.l/well of
biotinylated UEA-1 at a final concentration of 1 .mu.g/ml in 1.5%
BSA-DPBS was added to each well and the plates were allowed to
incubate for two hours at room temperature. [0122] 7. After washing
the plates 3-6 times, biotin was detected by use of commercial
Streptavidin conjugated to HRP, which was added to each well at a
1:5000 dilution prepared in 1.5% BSA/DPBS such that 100 .mu.l of
the reagent was added to each well to achieve the final
concentration. The plates were left at room temperature for an
hour. [0123] 8. After 3-6 washes, 100 .mu.l/well of OPD (final
concentration 1.6 mg/ml) activated by H.sub.2O.sub.2 was added to
each well and the reaction was allowed to develop in the dark.
[0124] 9. The reaction was stopped by adding 50 .mu.l of 4 N
H.sub.2SO.sub.4 after approximately 10-15 minutes or when
sufficient colour had developed. [0125] 10. The absorbance at 490
nm of each plate was measured by spectrophotometry. Results:
[0126] The results are illustrated as the percentage inhibitory
activity or the IC.sub.50 (the concentration of the compound at
which 50% inhibition of UEA-1 was reported). The absorbance at 490
nm of the unconjugated UEA-1 controls (1:4 dilutions: 160 .mu.g/ml
to 0.04 .mu.g/ml) was used to set up a standard curve. The highest
concentration of unconjugated UEA-1 was 160 .mu.g/ml and wells
containing this level of protein showed little or no colour change,
as high levels of previously incubated UEA-1 bound to the majority
of UEA-1 binding sites on the Caco-2 membrane preparations, thereby
leaving no sites for biotinylated UEA-1 (added later) to bind to
and hence no biotinylated UEA-1 was detected.
[0127] Wells containing 0.04 .mu.g/ml of unconjugated UEA-1 showed
high absorbance at 490 nm as low concentration of UEA-1 meant that
most UEA-1 receptors were left unbound, which allowed biotinylated
UEA-1 to bind to these receptors on the Caco-2 cell membranes, thus
resulting in high absorbance of these wells at 490 nm. The
absorbance at 160 .mu.g/ml was taken as 100% inhibition and the
absorbance at the other end of the scale (0.01 .mu.g/ml) was taken
as 0% inhibition. The percentage inhibition of each compound or
mixtures of compounds was estimated from similar binding curves.
The IC.sub.50 values of the active compounds was determined using
serial dilutions of each compound.
[0128] Using combinatorial chemistry, a large number of diverse
chemical compounds, both peptide and non-peptide (organic) referred
to as libraries were tested in single and two tier assays. Each
library comprises a common scaffold or framework. Compounds of each
library are synthesized by arranging a wide range of side chains
and groups both branched and linear in different sequences on the
scaffold backbone. In a different library the same elements can be
arranged in a similar manner or in a different manner on another
type of scaffold (for reference see Meyer et al., U.S. Pat. No.
5,859,190, Houghten, U.S. Pat. No. 4,631,211, Pinilla, U.S. Pat.
No. 5,556,762). By way of example, some of the organic backbones
used in combinatorial chemistry are listed below. The libraries
screened in order to identify the active compounds of the invention
were not limited to the backbone structures defined below.
##STR1##
[0129] Amongst the wide range of libraries tested, mixtures from
thiohydantoin based, N-6-acylamino bicycylic guanidine based,
N-acylamine based and polyphenylurea based libraries showed high
inhibitory activity. To narrow the search, individual compounds
from these active mixtures were deconvoluted and each tested for
inhibitory activity. Results revealed that individual purified
compounds from most libraries showed inhibitory activity. These
included compounds from the N-acylamine-based libraries. A
structure of such acylamines is e.g. as represented by formula A.
##STR2##
[0130] The acylamines were synthesized on solid phase resin. Here
R1 and R2 groups were derived from amino acids that were coupled
using conventional tBOC chemistry which involves blocking the N
terminus of each incoming amino acid by BOC (N-tertbutoxyl
carbonyl) to avoid its participation in the reaction. The N
terminus is unblocked once the amino acid is attached. In case of
synthesis of acylamines, the amide bonds of the amino acids were
first methylated and then reduced to amines. The N-terminus of the
developing chain was acylated with a carboxylic acid adding R3. The
amine seen on the left side of the molecule was derived from the
solid phase mBHA resin which has an amino group extending out that
reacts with the carboxy terminus of the incoming amino acid.
Cleavage of this amine from the solid support during incubation
with hydrogen fluoride results in the release of this amino group
thus forming the amino terminus of the acylamine.
[0131] The scheme is shown below. ##STR3##
[0132] Tables 1, 2 and 3 show the inhibitory activity of compounds
belonging to three N-acylamine-based libraries. TABLE-US-00001
TABLE 1 50 .mu.g/ml 250 .mu.g/ml 50 .mu.g/ml 250 .mu.g/ml Av % Av %
Av % Av % R1 R2 R3 Inhibition Inhibition Inhibition Inhibition 5.
Nap-ala Nap-Ala 3,4,5-Trimethoxybenzoic Acid 16 54 39 51 8. Nap-ala
Nap-ala 4-Biphenylacetic Acid 59 85 0 50 10. Nap-ala Nap-ala
3,4,5-Trimethoxybenzoic Acid -65 41 24 66 20. Nap-Ala Nap-ala
3,4,5-Trimethoxybenzoic Acid -121 24 52 75 23. pCl-F Nap-Ala
4-Biphenylacetic Acid 52 21 39 59 25. pCl-F Nap-Ala
3,4,5-Trimethoxybenzoic Acid -173 66 65 63 26. pCl-F Nap-ala
4-Ethyl-4-Biphenylcarboxylic Acid -116 -2 39 56 Av %
Inhibition:--Average Percent Inhibition
[0133] TABLE-US-00002 TABLE 2 Two Tier Assay N-acyl triamine
Library TP1012 50 ug/ml 250 ug/ml R1 R2 R3 Avg % Inhib Avg % Inhib
86 D-Nle D-chAla Gallic acid 61.61308 76.22587 125 L-Leu L-Phe
Gallic acid 61.02335 77.78063 87 D-Nle D-Arg(Tsl) Gallic acid
55.81319 65.64472 84 L-Tyr(Brz) L-chAla Gallic acid 54.9682
70.64093 92 L-Nle D-chAla Gallic acid 54.8899 67.69989 90 D-Nle
L-chAla Gallic acid 53.65788 69.94 91 L-Nle L-Tyr(Brz) Gallic acid
53.05769 65.97437 94 L-Nle L-Arg(Tsl) Gallic acid 51.51322 66.36993
78 D-Tyr L-chAla Gallic acid 47.10349 61.0039 93 L-Nle D-Arg(Tsl)
Gallic acid 46.83358 58.5174 85 D-Nle L-Tyr(Brz) Gallic acid
46.51721 66.21152 96 L-Nle L-chAla Gallic acid 42.93428 65.78116 74
D-Tyr D-chAla Gallic acid 42.91734 57.91947 144 L-pF-Phe L-pF-Phe
a,a,a-(Trifluoro-m-Tolyl)acetic acid 42.26244 66.71332 89 D-Nle
D-Tyr Gallic acid 42.12294 65.78345 95 L-Nle D-Tyr Gallic acid
41.98644 66.4157 141 L-pF-Phe L-pF-Phe 3,4 DichloroPhenylacetic
acid 41.43191 72.63565 75 D-Tyr D-Arg(Tsl) Gallic acid 40.14659
51.4412 88 D-Nle L-Arg(Tsl) Gallic acid 38.0323 59.11027 82
L-Tyr(Brz) L-Arg(Tsl) Gallic acid 37.9772 55.92339 Av %
Inhibition:--Average Percent Inhibition
[0134] TABLE-US-00003 TABLE 3 Single Tier Two Tier Acylamine
Library MSI 22 Assay Assay R1 R2 R3 62.5 .mu.g/ml 250 .mu.g/ml 62.5
.mu.g/ml 250 .mu.g/ml L-NapAla L-pCl- Gallic Acid 31.5 46.9 26.7
54.7 Phe D-Nve D-chAla Gallic Acid 22.0 47.8 20.6 48.5 D-Tyr(Et) D-
Gallic Acid 27.2 48 30 61 Arg(Tos) D-Tyr(Et) L-chAla Gallic Acid
8.5 33.3 31.8 52.7 D-Nve D-Val Gallic Acid 13.7 46 12.1 29.8 D-Tyr-
L-chAla 3,4,5-Trimethoxy benzoic -0 -11.7 39.7 58.9 (BrZ) Acid
D-Napala L-pCl- Gallic Acid -14.7 7.4 23.4 43.3 Phe D-Napala D-Val
Gallic Acid 4.7 26.8 24.2 46.9 L-NapAla D-Napala Gallic Acid -3 7
22.0 44.6 D-Tyr(Et) L-chAla 3,4,5-Trimethoxy benzoic -9 -55.7 49.8
43.8 Acid Av % Inhibition:--Average Percent Inhibition
[0135] Compounds in these libraries have the same N-acylamine
scaffold but differ in the arrangement of side groups on each
scaffold. Library TPI 1066 (Table 1) for example contains compounds
with aromatic functionalities, library TPI 1012 (Table 2) contains
compounds with aromatic and non aromatic functionalities are
arranged on a N-acylamine based scaffold, and compounds from
library MSI 22 (Table 3) are a combination of the first two
libraries, in that the N-acylamine scaffold has both aromatic
groups and amino acids attached to it; results are illustrated as
the average percent inhibition by 50 .mu.g/ml or 250 .mu.g/ml of
these compounds. Note that the compounds from MSI 22 were tested at
doubling dilutions. Results are illustrated as the average percent
inhibition by 62.5 .mu.g/ml or 250 .mu.g/ml of these compounds.
Structures of some of the synthetic compounds tested from TPI 1066
are shown below: ##STR4## ##STR5##
[0136] From the acylamine libraries, we found that compounds
bearing cyclic groups having hydroxy or hydroxy bearing
substituents at position R3 were the most active inhibitors of
biotinylated UEA-1 binding (Table 2). In case of the library TPI
1012, compounds with the following groups at positions R1, R2 and
R3 showed the most activity. TABLE-US-00004 ##STR6## ##STR7##
[0137] In the light of these results, further libraries were
synthesized using a range of carboxylic acids with the intention of
assessing if multiple copies of polyhydroxyaryl groups such as
galloyl groups and some other cyclic groups increased the
inhibitory activity of the compounds. These compounds were
synthesized on a lysine scaffold in which carboxylic acids were
coupled to amines on both alpha and epsilon positions. Two
different constructs with each carboxylic acid were made: a `two
copy` construct represented as formula (C) and a `four copy`
construct represented as formula(B). ##STR8##
[0138] A range of carboxylic acids were used to synthesize these
two copy and four copy constructs. Structures of the acids attached
to lysine scaffolds of this library are given below. ##STR9##
[0139] By way of reference example, the reaction scheme for
synthesis of compounds carrying four copies of gallic acid on a
lysine scaffold is now described. The typical synthesis of such
compounds involved solid phase organic chemistry methodology. This
reaction scheme is illustrated in FIG. 1. [0140] 1. A quantity of
100 mg mBHA (methylbenzhydryl amine, sub. 0.8 mmol/g) derivatized
polystyrene resin was contained within a polypropylene mesh packet
called a "tea-bag". A mesh of this nature has previously been
disclosed in Houghten, R. A., Proc Nat Acad Sci. USA. 1985, 82,
5131. The resin functionality, methyl benzhydrylamine, is the site
of attachment for the first step in the synthesis. The mesh packet
containing the resin was contained in a polyethylene bottle. The
resin was washed three times with 5 ml methylene chloride and
neutralized three times with 5 ml of 5%
diisopropylethylamine/methylene chloride solution.
Fmoc-D-Lysine(Boc)-OH (6 eq.) was then coupled (step 1 of FIG. 1)
for two hours in the presence of HOBt (1-hydroxybenzotriazole, 6
eq.)and DIPCDI (diisopropylcarbodiimide, 6 eq.) in DMF
(dimethylformamide) to afford compound A (FIG. 1). All reagents are
utilized in six-fold excess to assure complete acylation. This
mixture was shaken with the resin for two hours. The excess
reagents were then washed out with DMF and methylene chloride.
[0141] 2. The t-Boc protecting group was removed (Step 2 of FIG. 1)
with 5 ml 55% trifluoroacetic acid/methylene chloride solution for
30 minutes. The resin was then washed with methylene chloride,
isopropanol and again with methylene chloride. [0142] 3. The Fmoc
group was then removed (Step 3 of FIG. 1) with a 5 ml solution of
20% piperidine in DMF for 30 minutes. Excess base was removed by
washing three times with DMF. [0143] 4. The coupling procedure was
repeated (Step 4 of FIG. 1) as described above to couple
Fmoc-D-Lysine(Boc)-OH to both the .alpha. and .epsilon. amino
positions producing compound B (FIG. 1). [0144] 5. Again, the t-Boc
and Fmoc groups were removed (Steps 5 and 6). [0145] 6. The mesh
packet and resin were next immersed in a solution of gallic acid
(3,4,5-trihydroxybenzoic acid, 6 eq.), HOBt (6 eq.) and DIPCDI (6
eq.) in DMF. [0146] 7. The reaction mixture was shaken overnight
(Step 7). All couplings were tested by the Kaiser test to verify
completeness of the reaction. [0147] 8. Following the coupling of
gallic acid, a treatment was necessary to remove esters that formed
due to the phenolic nature of gallic acid. These esters were
hydrolyzed in the final cleavage from the resin, however they
remained as an undesired side product, complicating the
post-synthesis purification process. The tea-bag was treated with a
solution of 2 ml Hydrazine in 15 ml of 10% Methanol/90% Dioxane and
shaken overnight (Step 8). The bag was finally washed with dioxane
three times. [0148] 9. The compound was cleaved from the resin
(Step 9) by hydrofluoric acid with 5% anisole as a scavenger. This
reaction was kept at 0.degree. C. for 90 minutes followed by a
stream of nitrogen to remove excess HF. Following extraction with
95% acetic acid/5% water and lyophilization, the desired product C
(FIG. 1) was obtained.
[0149] The biotinylated UEA-1 binding inhibitory activity of these
compounds (Library N78) is shown in Table 4. TABLE-US-00005 TABLE 4
INHIBITORY ACTIVITY OF INDIVIDUAL COMPOUNDS CONTAINING MULTIPLE
COPIES OF CARBOXYLIC ACIDS. Gallic acid D-Lys 76.1 80.4 83.1 87.7
Thioproline D-Lys Bis boc -28.2 -35.5 -0.2 -29.4
3,4-(Methylenedioxy)- D-Lys Bis boc -6.2 -15.3 26.6 36.3
Phenylacetic Acid Rhodanine 3-acetic acid D-Lys Bis boc -6 32.5 4.8
16.5 Gallic Acid D-Lys Bis boc 66.6 51.3 87 91 Thioproline D-Lys
Bis boc -10.9 -27.2 2.3 4.1 SD:--Standard Deviation Av %
Inhibition:--Average Percent Inhibition
[0150] As seen in Table 4, the polyhydroxyphenyl constructs were
significantly more active than the individual monohydroxyphenyl
compounds in inhibiting biotinylated UEA-1 binding to Caco-2 cell
membranes. Then there was the question whether or not the presence
or nature of the scaffolds upon which these groups were arranged
contributed to the inhibitory activity of these compounds. In order
to investigate this, firstly a range (library MSI 26) of
commercially available compounds having aromatic groups with one or
more hydroxy groups, such as gallic acid, and other related
compounds were tested in the inhibition assays. The structures of
compounds tested in this experiment (Table 5) are shown below.
##STR10## ##STR11## TABLE-US-00006 TABLE 5 Two Tier Assay Single
Tier Assay Avg % Inhibition Avg % Inhibtion Library MSI 26 62.5
.mu.g/ml 250 .mu.g/ml 62.5 .mu.g/ml 250 .mu.g/ml Methyl 3,5,4- -11
-8 1 4.5 trihydroxybenzoate Lauryl gallate 20.4 47.9 39.1 22.5
Gallocatechin gallate 7 14.1 54.6 33.6 Epigallocatechin -12 -10
10.1 0 3,4,5- 10.7 41.4 41.7 21 trihydroxybenzamide
Isopropylgallate -7 8.9 10.8 3.9 Dilazep dihydrochloride -25 -25
14.7 10.4 Methyl syringate -10 0.2 17.4 8.4 4-hydroxybenzamide -1
4.2 -3 8.4 3,5-dihydroxybenzamide -7 -2 -1 2.4 3,5-dihydroxybenzoic
-5 -7 0 1.1 Acid Av % Inhibition:- Average Percent Inhibition
[0151] As seen in Table 5, the rather low (although appreciable)
inhibitory activity of the various polyhydroxyphenyl containing
compounds suggested preferability of substantial molecular
scaffolds in binding to the UEA-1 receptor. In addition, compounds
with multiple polyhydroxyphenyl groups showed greater inhibitory
activity. The results suggested the effect should be optimised by
having multiple active side groups on scaffolds.
[0152] Therefore, a range of polyhydroxy aryl groups such as
galloyl and other cyclic groups, as shown below, were attached to
different lysine scaffolds. ##STR12##
[0153] Both branched and linear skeletons as shown below were
synthesized in this library which preferably contained up to four
or more hydroxyl moieties. The resulting compounds (library MSI 27)
were tested in single and two tier assays the results of which are
shown in Table 6. ##STR13## ##STR14## ##STR15##
[0154] Analysis of the biotinylated UEA-1 binding inhibitory
activity of these linear and branched compounds shows that the
activity of the compounds increased with the increase in the number
of gallic acid groups (structures of the active compounds are shown
below).
[0155] Compounds with other carboxylic acids attached as side
groups on the lysine scaffolds were not as active (Table 6).
TABLE-US-00007 TABLE 6 Inhibitory activity of lysine scaffolds both
linear or branched with a variation of gallic acid constructs
Single Tier Two Tier Assay Assay Cpd Library MSI 27 IC50 IC50 # R
Comment MW (ug/ml) uM (ug/ml) uM 6 Gallic acid 2 copy structure
449.41 18.97 42.21 12.97 28.86 2 3,4-Dimethoxybenzoic acid 2 copy
structure 473.52 128.20 270.74 96.44 203.67 5
4-ethoxycarbonyl-3,5-dimethoxybenzoic acid 2 copy structure 505.52
168.10 332.53 130.70 258.55 1 Syringic acid 2 copy structure 505.52
250.00 494.54 250.00 494.54 8 3,5-dihydroxybenzoic acid 2 copy
structure 417.41 125.00 299.47 171.90 411.83 4
3,4,5-trimethoxybenzoic acid 2 copy structure 533.57 250.00 468.54
250.00 468.54 3 3,4,5-triethoxybenzoic acid 2 copy structure 617.72
250.00 404.71 250.00 404.71 7 4-Hydroxybenzoic acid 2 copy
structure 385.41 250.00 648.66 250.00 648.66 13
4-ethoxycarbonyl-3,5-dimethoxybenzoic acid 1 copy structure(N-term
acetylated) 367.40 89.92 244.75 73.30 199.51 14 Gallic acid 1 copy
structure(N-term acetylated) 339.35 129.70 382.20 91.52 269.69 15
4-Hydroxybenzoic acid 1 copy structure(N-term acetylated) 307.35
111.40 362.45 86.81 282.45 10 3,4-Dimethoxybenzoic acid 1 copy
structure(N-term acetylated) 351.41 250.00 711.43 125.00 355.71 9
Syringic acid 1 copy structure(N-term acetylated) 367.41 125.00
340.22 101.20 275.45 16 3,5-dihydroxybenzoic acid 1 copy
structure(N-term acetylated) 323.35 250.00 773.16 108.30 334.93 11
3,4,5-triethoxybenzoic acid 1 copy structure(N-term acetylated)
423.51 250.00 590.31 250.00 590.31 12 3,4,5-trimethoxybenzoic acid
1 copy structure(N-term acetylated) 381.43 250.00 655.43 250.00
655.43 22 Gallic acid 4 copy structure 1008.98 17.61 17.45 2.86
2.84 17 Syringic acid 4 copy structure 1121.20 145.80 130.04 95.95
85.58 18 3,4-Dimethoxybenzoic acid 4 copy structure 1057.20 111.30
105.28 90.21 85.33 21 4-ethoxycarbonyl-3,5-dimethoxybenzoic acid 4
copy structure 1121.19 162.50 144.94 114.90 102.48 19
3,4,5-triethoxybenzoic acid 4 copy structure 1345.60 250.00 185.79
250.00 185.79 24 3,5-dihydroxybenzoic acid 4 copy structure 944.98
250.00 264.56 250.00 264.56 20 3,4,5-trimethoxybenzoic acid 4 copy
structure 1177.30 250.00 212.35 250.00 212.35 23 4-Hydroxybenzoic
acid 4 copy structure 880.98 250.00 283.77 250.00 283.77 30 Gallic
acid Linear 3 copy structure 729.69 25.89 35.48 8.36 11.45 26
3,4-Dimethoxybenzoic acid Linear 3 copy structure 765.86 125.00
163.22 85.62 111.80 29 4-ethoxycarbonyl-3,5-dimethoxybenzoic acid
Linear 3 copy structure 813.85 111.50 137.00 93.43 114.80 27
3,4,5-triethoxybenzoic acid Linear 3 copy structure 982.16 250.00
254.54 250.00 254.54 25 Syringic acid Linear 3 copy structure
813.86 250.00 307.18 250.00 307.18 28 3,4,5-trimethoxybenzoic acid
Linear 3 copy structure 855.93 250.00 292.08 250.00 292.08 31
4-Hydroxybenzoic acid Linear 3 copy structure 633.69 250.00 394.51
250.00 394.51 32 3,5-dihydroxybenzoic acid Linear 3 copy structure
681.69 250.00 366.74 250.00 366.74 37
4-ethoxycarbonyl-3,5-dimethoxybenzoic acid Linear 2 copy
structure(N-term acetylated) 675.73 82.46 122.03 54.27 80.31 38
Gallic acid Linear 2 copy structure(N-term acetylated) 619.62 87.78
141.67 54.27 87.59 33 Syringic acid Linear 2 copy structure(N-term
acetylated) 675.73 97.85 144.81 72.17 106.80 34
3,4-Dimethoxybenzoic acid Linear 2 copy structure(N-term
acetylated) 643.73 121.40 188.59 87.01 135.17 36
3,4,5-trimethoxybenzoic acid Linear 2 copy structure(N-term
acetylated) 703.78 250.00 355.22 125.00 177.61 39 4-Hydroxybenzoic
acid Linear 2 copy structure(N-term acetylated) 555.62 250.00
449.95 158.60 285.45 35 3,4,5-triethoxybenzoic acid Linear 2 copy
structure(N-term acetylated) 787.93 250.00 317.29 250.00 317.29 40
3,5-dihydroxybenzoic acid Linear 2 copy structure(N-term
acetylated) 587.62 250.00 425.45 250.00 425.45 46 Gallic acid
Linear 4 copy structure 1009.97 14.92 14.77 2.00 1.98 41 Syringic
acid Linear 4 copy structure 1122.19 89.77 80.00 83.26 74.19 42
3,4-Dimethoxybenzoic acid Linear 4 copy structure 1058.19 107.00
101.12 113.80 107.54 45 4-ethoxycarbonyl-3,5-dimethoxybenzoic acid
Linear 4 copy structure 1122.18 154.20 137.41 136.30 121.46 43
3,4,5-triethoxybenzoic acid Linear 4 copy structure 1346.59 250.00
185.65 250.00 185.65 48 3,5-dihydroxybenzoic acid Linear 4 copy
structure 945.97 250.00 264.28 250.00 264.28 44
3,4,5-trimethoxybenzoic acid Linear 4 copy structure 1178.29 250.00
212.17 250.00 212.17 47 4-Hydroxybenzoic acid Linear 4 copy
structure 881.97 250.00 283.46 250.00 283.46 54 Gallic acid Linear
3 copy structure(N-term acetylated) 899.90 6.85 7.61 3.00 3.33 50
3,4-Dimethoxybenzoic acid Linear 3 copy structure(N-term
acetylated) 936.07 77.09 82.36 42.79 45.71 53
4-ethoxycarbonyl-3,5-dimethoxybenzoic acid Linear 3 copy
structure(N-term acetylated) 1200.25 90.15 75.11 114.80 95.65 49
Syringic acid Linear 3 copy structure(N-term acetylated) 984.07
118.00 119.91 108.80 110.56 56 3,5-dihydroxybenzoic acid Linear 3
copy structure(N-term acetylated) 851.90 125.00 146.73 125.00
146.73 52 3,4,5-trimethoxybenzoic acid Linear 3 copy
structure(N-term acetylated) 1026.14 174.80 170.35 226.70 220.93 55
4-Hydroxybenzoic acid Linear 3 copy structure(N-term acetylated)
803.90 250.00 310.98 250.00 310.98 51 3,4,5-triethoxybenzoic acid
Linear 3 copy structure(N-term acetylated) 1152.37 250.00 216.95
250.00 216.95 IC 50 = concentration of compound at which inhibition
of binding is 50%
[0156] A second batch of the MSI 27 library of compounds was
synthesized and the biotinylated UEA-1 binding inhibitory activity
of compounds from the new batch (called MSI 40) was compared with
their chemically identical counterparts from MSI 27. As shown in
Tables 7(a) and 7(b), chemically identical compounds from both
batches behaved in a similar manner. In addition, compounds from
these libraries having gallic acid as their active binding moiety
were compared with compounds where other carboxylic acids were
attached as side groups on the lysine linear or branched scaffolds.
As seen in Tables 7(a), (b) compounds having gallic acid as their
side groups had low IC 50 values which suggested that the UEA-1
receptor binding inhibitory activity of these compounds was related
to the specific gallic acid structure rather than being a general
feature of carboxylic acids. ##STR16## ##STR17## ##STR18##
##STR19##
[0157] From these data, it appears that having more than one
polyhydroxy aryl group contributes to good inhibition. Therefore, a
range of such compounds was synthesized where up to eight--it could
of course be more--galloyl polyhydroxy side groups were attached to
lysine and other scaffolds, examples of which are shown below. The
inhibitory activity of these compounds is shown in Table 8.
TABLE-US-00008 TABLE 8 Structure Synthesis Number IC 50 (ug/ml) IC
50 (uM) One copy N-Acetylated ##STR20## MST 27 #14 1.sup.st tier =
72 2.sup.nd tier = 211 1.sup.st tier = 54 2.sup.nd tier = 160 Two
copy branched ##STR21## MSI 27 #6 MSI 40 #1 1.sup.st tier = 11
2.sup.nd tier = 8 1.sup.st tier = 24 2.sup.nd tier = 17 Two copy
DAP ##STR22## MSI 34 #1 MSI 39 #1 1.sup.st tier = 1 2.sup.nd tier =
0.8 1.sup.st tier = 4 2.sup.nd tier = 2 Two copy DAB ##STR23## MSI
34 #2 MSI 39 #5 1.sup.st tier = 3 2.sup.nd tier = 2 1.sup.st tier =
6 2.sup.nd tier = 5 Two copy Orn ##STR24## MSI 34 #3 1.sup.st tier
= 2 2.sup.nd tier = 0.7 1.sup.st tier = 4 2.sup.nd tier = 2 Two
copy Phe (p-NH2) ##STR25## MSI 34 #4 1.sup.st tier = 17 2.sup.nd
tier = 18 1.sup.st tier = 35 2.sup.nd tier = 37 Two copy
N-Acetylated ##STR26## MSI 27 #38 1.sup.st tier = 54 2.sup.nd tier
= 48 1.sup.st tier = 86 2.sup.nd tier = 77 Three copy linear
##STR27## MSI 27 #30 MSI 39 #9 1.sup.st tier = 14 2.sup.nd tier = 5
1.sup.st tier = 19 2.sup.nd tier = 7 Three copy linear DAP
##STR28## MSI 34 #9 1.sup.st tier = 0.6 2.sup.nd tier = 0.5
1.sup.st tier = 0.9 2.sup.nd tier = 0.8 Three copy linear DAB
##STR29## MSI 34 #10 1.sup.st tier = 0.6 2.sup.nd tier = 0.7
1.sup.st tier = 0.9 2.sup.nd tier = 1 Three copy linear Orn
##STR30## MSI 34 #11 1.sup.st tier = 1 2.sup.nd tier = 0.8 1.sup.st
tier = 2 2.sup.nd tier = 1 Three copy linear Phe (p-NH2) ##STR31##
MSI 34 #12 1.sup.st tier = 6 2.sup.nd tier = 4 1.sup.st tier = 8
2.sup.nd tier = 5 Three copy N-Acetylated ##STR32## MSI 27 #54 MSI
39 #10 1.sup.st tier = 4 2.sup.nd tier = 2 1.sup.st tier = 5
2.sup.nd tier = 3 Three copy N-Ac DAP ##STR33## MSI 34 #13 1.sup.st
tier = 2 2.sup.nd tier = 2 1.sup.st tier = 3 2.sup.nd tier = 2
Three copy N-Ac DAB ##STR34## MSI 34 #13 1.sup.st tier = 2 2.sup.nd
tier = 1 1.sup.st tier = 2 2.sup.nd tier = 1 Three copy N-Ac Orn
##STR35## MSI 34 #15 1.sup.st tier = 8 2.sup.nd tier = 2 1.sup.st
tier = 9 2.sup.nd tier = 2 Three copy N-Ac Phe (p-NH2) ##STR36##
MSI 34 #16 1.sup.st tier = 3 2.sup.nd tier = 6 1.sup.st tier = 3
2.sup.nd tier = 6 Four copy branched ##STR37## MSI 27 #22 MSI 40 #8
1.sup.st tier = 10 2.sup.nd tier = 1 1.sup.st tier = 10 2.sup.nd
tier = 1 Four copy DAP ##STR38## MSI 34 #5 1.sup.st tier = 0.5
2.sup.nd tier = 1 1.sup.st tier = 0.5 2.sup.nd tier = 1 Two-by-Two
DAP with linker ##STR39## MSI 39 #4 1.sup.st tier = 6 2.sup.nd tier
= 0.8 1.sup.st tier = 5 2.sup.nd tier = 0.7 Four copy DAB ##STR40##
MSI 34 #6 1.sup.st tier = 0.6 2.sup.nd tier = 1 1.sup.st tier = 0.6
2.sup.nd tier = 1 Four copy Orn ##STR41## MSI 34 #7 1.sup.st tier =
0.9 2.sup.nd tier = 1 1.sup.st tier = 0.9 2.sup.nd tier = 1 Four
copy Phe (p-NH2) ##STR42## MSI 34 #8 1.sup.st tier = 4 2.sup.nd
tier = 3 1.sup.st tier = 4 2.sup.nd tier = 2 Four copy linear
##STR43## MSI 27 #46 MSI 39 #11 1.sup.st tier = 8 2.sup.nd tier =
0.6 1.sup.st tier = 8 2.sup.nd tier = 0.6 Four copy linear DAP
##STR44## MSI 34 #17 1.sup.st tier = 1 2.sup.nd tier = 0.7 1.sup.st
tier = 1 2.sup.nd tier = 0.8 Four copy linear DAB ##STR45## MSI 34
#18 1.sup.st tier = 3 2.sup.nd tier = 0.9 1.sup.st tier = 3
2.sup.nd tier = 1 Four copy linear Orn ##STR46## MSI 34 #19
1.sup.st tier = 11 2.sup.nd tier = 1 1.sup.st tier = 11 2.sup.nd
tier = 1 Four copy linear Phe (p-NH2) ##STR47## MSI 34 #20 1.sup.st
tier = 2 2.sup.nd tier = 6 1.sup.st tier = 2 2.sup.nd tier = 5 Four
copy N-Acetylated ##STR48## MSI 30 #5 MSI 39 #12 1.sup.st tier = 8
2.sup.nd tier = 3 1.sup.st tier = 7 2.sup.nd tier = 2 Four copy
N-Ac DAP ##STR49## MSI 34 #21 1.sup.st tier = 2 2.sup.nd tier = 1
1.sup.st tier = 2 2.sup.nd tier = 1 Four copy N-Ac DAB ##STR50##
MSI 34 #22 1.sup.st tier = 3 2.sup.nd tier = 1 1.sup.st tier = 3
2.sup.nd tier = 1 Four copy N-Ac Orn ##STR51## MSI 34 #23 1.sup.st
tier = 6 2.sup.nd tier = 1 1.sup.st tier = 5 2.sup.nd tier = 1 Four
copy N-Ac Phe (p-Nh2) ##STR52## MSI 34 #24 1.sup.st tier = 3
2.sup.nd tier = 9 1.sup.st tier = 2 2.sup.nd tier = 7 Five copy
linear ##STR53## MSI 30 #5 1.sup.st tier 75 2.sup.nd tier = 12
1.sup.st tier = 64 2.sup.nd tier = 10 Five copy N-Acetylated
##STR54## MSI 30 #8 1.sup.st tier = 70 2.sup.nd tier = 15 1.sup.st
tier = 48 2.sup.nd tier = 10 Six copy linear ##STR55## MSI 30 #10
1.sup.st tier = 23 2.sup.nd tier = 5 1.sup.st tier = 14 2.sup.nd
tier = 3 Six copy N-Acetylated ##STR56## MSI 30 #11 1.sup.st tier =
13 2.sup.nd tier = 2 1.sup.st tier = 7 2.sup.nd tier = 1 Seven copy
linear ##STR57## MSI 30 #13 1.sup.st tier = 9 2.sup.nd tier = 3
1.sup.st tier = 5 2.sup.nd tier = 1 Seven copy N-Acetylated
##STR58## MSI 30 #14 1.sup.st tier = 17 2.sup.nd tier = 9 1.sup.st
tier = 8 2.sup.nd tier = 4 Eight copy linear ##STR59## MSI 30 #16
1.sup.st tier = 19 2.sup.nd tier = 8 1.sup.st tier = 9 2.sup.nd
tier = 4 Eight copy branched ##STR60## MSI 30 #17 1.sup.st tier = 5
2.sup.nd tier = 5 1.sup.st tier = 2 2.sup.nd tier = 2 ##STR61##
Note: The Lysine scaffolds are synthesized from Fmoc-D-Lys(Boc)-OH
whereas other scaffolds (DAP, DAB etc) are synthesized from the L
derivatives. R = 3,4,5-trihydroxybenzoyl
Pharmaceutical Formulations
[0158] As regards the kinds of pharmaceutical formulations to which
the invention relates, it will be understood from the above that
they may in general be any kind of formulation which is to be
applied to the body's epithelium, but more particularly will
generally be enteric formulations and most preferably oral
formulations. Typically these consist of or comprise solids, such
as capsules, tablets, powders, emulsions, such as microemulsions
and other types thereof and suspensions. Preferred embodiments
include controlled release oral formulations, in which the
pharmaceutically-active ingredient is encapsulated in a
biodegradable polymeric body or coating, e.g. by means of a solvent
evaporation method. Coatings of this kind are known in the art, for
example the polylactide polymer coatings discussed in our
WO-A-00/12124 and elsewhere. There is no particular limit on the
type of biodegradable polymer that may be used.
[0159] The encapsulated pharmaceutically-active material is
preferably in the form of small particles, particularly
microparticles or nanoparticles. For example, it may be a
particulate formulation in which at least 50% of the particles are
smaller than 5 .mu.m, or more preferably in which as least 50% of
the particles are smaller than 600 nm. Microparticulate and
nanoparticulate compositions of this type, comprising drug-active
material encapsulated in biodegradable polymer, are known as such
to the skilled person: see e.g. WO-A-00/12124 and
WO-A-96/31202.
[0160] The present ligand compound may be bound to, coated onto or
blended with the drug formulation in any appropriate manner using
physical and chemical techniques appropriate to the compounds
concerned. Typically these consist of, but are not limited to,
passive adsorption, direct conjugation during one step synthesis
(e.g. ligand-peptide drug synthesis on standard columns or in
solution), covalent coupling (e.g. amino group of ligand to
carboxylate modified drug or delivery system using standard
methodologies such as carbodiimide), and biotin-streptavidin
interaction (e.g. using biotinylated ligand and streptavidin
modified drug or delivery system).
[0161] As is also known practice, the particulate formulation may
be given an "enteric coating" to protect it against gastric fluids
so that the particles can pass intact into the intestine.
Development of a Whole Cell Binding Assay for the Characterization
of Binding Affinities of the Lectin Mimetics:
[0162] A whole cell binding assay was developed for the
characterization of binding affinities of various lectin mimetics
of UEA-1. This assay was developed to allow structure activity
analysis of these UEA-1 mimetics in order to identify functional
groups that enhance activity using whole cells in solution.
Methods:
[0163] Caco-2 cells were analysed by flow cytometry for binding of
both biotinylated UEA-1 and a biotinylated lectin mimetic of UEA-1
using a streptavidin FITC probe. While clear binding (positives) of
biotinylated UEA-1 at concentrations as low as 1.0 .mu.g/ml was
seen, binding of the lectin mimetics of UEA-1 was negative even at
concentrations of up to 65 .mu.M. In order to amplify the signal, a
FITC-avidin D sandwich protocol was used. In this method, after
binding of the biotinylated compounds to the cells, a series of
FITC-avidin D/anti-avidin D/FITC-avidin D stainings were performed,
which resulted in a several fold increase in fluorescent
signal.
Results:
[0164] The sandwich protocol described above increased the
fluorescent signal as evidenced by the ability to measure a
biotinylated UEA-1 sample of 0.02 .mu.g/ml. Previously, the lower
limit had been 1.0 .mu.g/ml with the streptavidin-FITC probe. The
nucleic acid stain 7-actinomycin D (7-AAD), a fluorescent dye for
dead cells, was used to exclude these cells from analysis as they
were known to bind non specifically to FITC-avidin D. Binding of
biotinylated lectin mimetics of UEA-1 was demonstrated at both 50
.mu.m and 10 .mu.m concentrations.
[0165] Approximately, 15% of the population of Caco-2 cells tested
were FL1 (FITC) positive, FL3(7-AAD)negative at both concentrations
after subtraction of background fluorescence. The streptavidin FITC
stain was unable to detect these concentrations of the
compound.
CONCLUSION
[0166] A whole cell binding assay has been developed for the
characterization of binding affinities of small molecule lectin
mimetics of UEA-1. This will allow structure activity analysis of
these mimetics to identify those functional groups that enhance
activity using whole cells in solution. This assay will aid
selection of small molecule mimics of UEA1 for further studies in
vivo and is an independent novel aspect herein.
Evaluation of the Ability of Gallic Acid UEA 1 Mimetic Compounds to
Mediate Delivery of a Model Particle System In Vivo:
[0167] The ability of compounds having gallic acid side chains to
mimic UEA-1 was assessed in vivo in mouse models. Here, binding and
uptake by M cells of particles coated with these compounds was
assessed.
Methods:
[0168] Biotinylated ligand MSI 35-2 (4 copies of gallic acid;
lysine scaffold) (see FIG. 5), a UEA-1 control (Vector
Laboratories) and a biocytin control (Molecular Probes) were
adsorbed on to fluorescent Estapor streptavidin polystyrene
particles (FITC label; 0.289 .mu.m diameter) using routine
methodologies at room temperature.
[0169] Mouse intestinal loops containing one or more Peyer's
Patches were inoculated with polystyrene particle suspensions
(typically 500 .mu.l containing 5.0.times.10.sup.10 particles per
ml) and incubated for 30 min. The Peyer's patches were excised,
fixed in methanol and the M cells were counter-stained with
UEA1-rhodamine for subsequent analysis by confocal microscopy.
Stained tissues were examined on a BioRad MRC 600 confocal laser
scanning microscope equipped with an argon/krypton mixed gas laser.
The number of particles observed per total area was measured and
reported quantitatively. For details of typical procedures and full
protocols see: Foster, N., Clark, M. A., Jepson, M. A. & Hirst,
B. H. Ulex europaeus 1 lectin targets microspheres to mouse Peyer's
patch M-cells in vivo. Vaccine 1998:16(5); 536-541.
Results:
[0170] Scatter plots (log scale) for binding and uptake of MSI35-2,
UEA1 and control coated particles are illustrated as FIGS. 2-4.
[0171] FIG. 2 Surface binding and uptake of MSI35-2 gallic acid
mimetic coated particles. [0172] FIG. 3 Surface binding and uptake
of UEA1 coated particles. [0173] FIG. 4 Surface binding &
uptake of biocytin mimetic coated particles. [0174] Each point on
the scatter plot represents one image.
CONCLUSIONS
[0175] Fluorescent streptavidin polystyrene particles coated with
the biotinylated ligand MSI35-2 (4 copies of gallic acid; lysine
scaffold) exhibited binding and uptake into M-cells in vivo
comparable to or greater than UEA-1 coated control particles in a
mouse intestinal loop model. Binding and uptake into M-cells was
significantly higher than that observed using biocytin coated
control particles.
[0176] The potential for use of these lectin mimetics in oral
targeted drug delivery applications has been demonstrated using a
model particulate system. The M-cell specific nature of the mimetic
in the mouse intestinal loop model is of particular interest in the
context of vaccine delivery to antigen presenting cells.
[0177] Staining of Human Tissue Sections with UEA-1:
[0178] The application of UEA-1 as a diagnostic/prognostic
indicator of disease states was verified in human tissues.
Methods:
I. Immunohistochemistry Procedure
[0179] Using UEA1 @ 0.25 .mu.g/ml, goat anti-UEA1 @ 1:10,000
dilution and a Vector ABC-AP Kit (AK5002) with a Vector Red
substrate kit (SK-5100) microscopic analysis of human tissue
sections revealed a fuchsia-colored red deposit at the site of
ligand binding. Negative controls, performed in the absence of the
primary ligand prior to application of the goat anti-UEA1 antibody
and detection staining, revealed negligible background. Tissues
stained with a positive control antibody (i.e. CD31; to ensure that
the tissue antigens were preserved and accessible for
immunohistochemical analysis) confirmed tissue integrity.
TABLE-US-00009 II. Tissue Sources and Diagnoses Tissue Diagnosis
Sample ID Age/Sex Ileum Normal, with Peyer's 1 68 F patches Ileum
Normal, with Peyer's 2 36 F patches Colon Normal 1 26 M Colon
Normal 2 73 F Colon Carcinoma 1 Adult Colon Carcinoma 2 Adult Colon
Crohn's disease 1 25 M Colon Crohn's disease 2 31 M Colon
Ulcerative colitis 1 Adult Colon Ulcerative colitis 2 54 M
Results: [0180] FIGS. 6, 7 show images of the stained cell samples.
In the normal tissue, both the ileum and colon mucosal epithelia
showed moderately positive cytoplasmic labeling of the absorptive,
goblet, M-cell, and crypt enterocytes with UEA1. The staining
reaction was slightly more pronounced in the colon than in the
ileum and appeared strongest within cytoplasmic vacuoles,
especially along the microvillus luminal surface of the mucosal
cells. The stained material was secreted into the mucous that lines
the intervillous and surface mucosal cells. Cell membranes and/or
glycocalyx were stained, while the intercellular junctions were not
demonstrated by the staining of the ligand binding sites. Goblet
cells in both samples showed staining of fine dust-like to punctate
cytoplasmic vesicles, and almost all goblet cells had strong
staining of globular proteinaceous masses within the large
cytoplasmic mucous vacuole. The staining indicated that the ligand
binds to a specific component in the mucous or at the apical-mucous
interface on the apical surface and not mucous in general, because
some of the goblet cells, which contained mucous, did not stain.
Several macrophages were positive with what appeared to be
phagocytized debris.
[0181] Throughout the stroma of the normal and diseased ileum and
colon and the colon carcinoma, there was a moderate to marked
staining of all of the endothelial cells, and a minimal to moderate
staining of the Schwann cells surrounding nerve axons. The
Meissner's plexus and myenteric plexus and their axons were
consistently positive.
[0182] In the colon carcinoma samples the intensity of staining was
similar to that of normal colon. However, the pattern was somewhat
different. In the colon carcinoma sections the foci of cytoplasmic
staining tended to be smaller and of more uniform size and
cytoplasmic distribution. In the normal tissue the reactivity was
marked in the goblet cells, whereas in the carcinoma tissue goblet
cells were less common and did not stain as intensely. In the colon
sections, the staining reactivity grade was similar to that of the
normal samples, but it was noticeably higher than that in the
adjacent normal colon tissue in the same sample as the evaluated
neoplastic tissue.
[0183] The immunohistological evaluation of inflammatory bowel
disease with the ligand consisted of staining two cases each of
Crohn's disease and ulcerative colitis. The expression of UEA1
receptors appeared to be significantly up-regulated in all of the
mucosal enterocyte types in Crohn's disease, and appeared to be
significantly down-regulated in ulcerative colitis in all of the
colonic mucosal enterocytes.
CONCLUSIONS
[0184] This difference in intensity of UEA-1 staining and/or
patterning of the staining as between normal and afflicted cells
provides a basis for a diagnosis or prognosis in the present
techniques.
[0185] Further Staining of Human Tissue Sections with UEA-1 and
Gallic Acid UEA-1 Mimetic Compounds:
[0186] Staining of normal human tissues with UEA-1 and a gallic
acid UEA-1 mimetic was compared as an initial step to determine if
the mimetic would also be suitable for use as a
diagnostic/prognostic indicator of disease states in human
tissues.
Methods:
[0187] UEA1 and the biotinylated ligand MSI 35-2 (4 copies of
gallic acid; lysine scaffold) (see FIG. 5) were used in staining
protocols comparable to that described above. Negative controls,
performed in the absence of the primary ligand, revealed negligible
background.
Results:
[0188] Table 9 summarises the normal human tissue staining profiles
for UEA-1 and the UEA-1 mimetic. TABLE-US-00010 TABLE 9 UEA-1 UEA-1
mimetic Oesophagus +++ - Stomach +++ - Small intestine +++ ++ Large
intestine +++ +++ Pancreas +++ - Muscle +/- - Brain ++ - Kidney +++
- Liver +++ - Spleen +++ -
CONCLUSIONS
[0189] The staining profile observed using the UEA-1 mimetic on
large intestine sections was comparable to that obtained using
UEA-1 (as described above). This correlated well with the selection
procedure for the UEA-1 mimetics, which was carried out using human
Caco-2 cell membrane fractions i.e. cells which exhibit features
characteristic of colonic epithelia. The UEA-1 mimetic also
exhibited staining of small intestine sections as would be
expected. Interestingly, no gastrointestinal or other organ tissue
was stained using the UEA-1 mimetic. This was in marked contrast to
UEA-1, which stained all of the tissue types, and may be a
favourable factor in selection of the mimetic over UEA-1 for
clinical applications.
[0190] Evaluation of Binding and Uptake of the Gallic Acid UEA-1
Mimetic into Endothelial Cells:
[0191] The gallic acid UEA-1 mimetic was compared to known cell
permeable peptides in an endothelial cell permeability assay to
assess it's application in a) diagnosis/prognosis of disease states
(by monitoring vascularization) and b) in therapeutic delivery of
pharmaceutical formulations.
Methods:
[0192] 1. ECV-304 cells were seeded on round coverslips at a
concentration of 2.times.10.sup.5 cells per coverslip, on 24 well
plates, and allowed to grow to confluency. [0193] 2. The
serum-containing medium was replaced by serum-free medium (OptiMEM
with penicillin streptomycin). [0194] 3. Aqueous stock solutions of
ligands were added directly to the medium surrounding the cells to
reach a final concentration of 10 .mu.M, in 500 .mu.l of medium.
[0195] 4. One plate of samples was incubated at 37.degree. C. in 5%
CO.sub.2 enriched air, and the other at 4.degree. C. in air, both
plates were incubated for 1 hr. [0196] 5.Coverslips were then given
3.times.5 minute washes with PBS. [0197] 6.Cells were fixed and
permeabilised in 500 .mu.l of methanol at -20.degree. C. for 10
minutes. [0198] 7. A further 3.times.5 minute washes with PBS were
given. [0199] 8.Non-specific binding sites were blocked by
incubating the cells for 1 hr at room temperature in 500 .mu.l of
5% milk solution in PBS (Marvel). [0200] 9.The peptides were
stained with 500 .mu.l of 15 nM Streptavidin-FITC for 1 hr at room
temperature. [0201] 10. Cells were given final 3.times.5 minute
washes with PBS. [0202] 11. The coverslips were then removed from
the wells and dipped briefly in water, before being mounted on
glass slides with Vectashield mounting medium for fluorescence with
Dapi. Results:
[0203] See FIG. 8 for comparison of the UEA-1 mimetic to a known
cell permeable peptide and relevant controls. The UEA-1 mimetic
exhibited strong staining of the nucleus with diffuse staining
throughout the cytoplasm.
CONCLUSIONS
[0204] Uptake of the UEA-1 mimetic by endothelial cells is
suggestive of suitability of the mimetic for use as a marker of
endothelia/blood vessels, and hence, as a diagnostic or prognostic
marker in disease states.
[0205] In addition the uptake profile correlated well with that of
the SynB1 peptide which is known to mediate delivery of therapeutic
agents such as doxorubicin into cells. Further applications of the
UEA-1 mimetic in delivery of pharmaceutical formulations through
mechanisms involving direct interaction with lipid membranes (as in
the case of SynB1) will be investigated.
* * * * *