U.S. patent application number 14/536703 was filed with the patent office on 2015-03-05 for conjugates of a phospholipid and a drug for the treatment of inflammatory bowel disease.
The applicant listed for this patent is Ben-Gurion University of the Negev Research and Development Authority. Invention is credited to Arik DAHAN.
Application Number | 20150065462 14/536703 |
Document ID | / |
Family ID | 45446141 |
Filed Date | 2015-03-05 |
United States Patent
Application |
20150065462 |
Kind Code |
A1 |
DAHAN; Arik |
March 5, 2015 |
CONJUGATES OF A PHOSPHOLIPID AND A DRUG FOR THE TREATMENT OF
INFLAMMATORY BOWEL DISEASE
Abstract
Conjugates of drugs suitable for use in the treatment of
inflammatory bowel disease and phospholipids, and their use in the
treatment of inflammatory bowel disease, are disclosed. The
disclosed conjugates serve as targeted prodrugs which are suitable
for oral administration, and which are capable of releasing the
drug selectively at the diseased tissue upon activation by
PLA.sub.2.
Inventors: |
DAHAN; Arik; (ModiIn,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ben-Gurion University of the Negev Research and Development
Authority |
Beer-Sheva |
|
IL |
|
|
Family ID: |
45446141 |
Appl. No.: |
14/536703 |
Filed: |
November 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13990457 |
May 30, 2013 |
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PCT/IL2011/050037 |
Dec 1, 2011 |
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14536703 |
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61418892 |
Dec 2, 2010 |
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Current U.S.
Class: |
514/78 ;
558/172 |
Current CPC
Class: |
A61K 9/0053 20130101;
A61P 1/00 20180101; A61K 47/544 20170801; A61K 31/606 20130101;
A61K 31/166 20130101 |
Class at
Publication: |
514/78 ;
558/172 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 31/166 20060101 A61K031/166 |
Claims
1. A method of treating an inflammatory bowel disease in a subject
in need thereof, the method comprising orally administering to the
subject a therapeutically effective amount of a conjugate which
comprises a drug useful in the treatment of the inflammatory bowel
disease covalently linked to a phospholipid.
2. The method of claim 1, wherein the conjugate is administered as
a liquid oral formulation.
3. The method of claim 1, wherein said drug useful in the treatment
of inflammatory bowel disease is selected from the group consisting
of a drug that comprises 5-ASA, tacrolimus and methotrexate.
4. The method of claim 1, wherein said phospholipid is a
phosphoglycerol.
5. The method of claim 4, wherein said drug is linked to position
sn-2 of said phosphoglycerol.
6. The method of claim 1, wherein said drug is linked to said
phospholipid via a bridging unit.
7. The method of claim 6, wherein said bridging unit is an alkylene
being from 3 to 6 carbon atoms in length.
8. The method of claim 1, wherein said conjugate has a general
Formula: ##STR00009## wherein: A is an alkylene chain being 3-30
carbon atoms in length; X is a bridging unit; D is said drug
suitable for use in the treatment of said inflammatory bowel
disease; and R is selected from the group consisting of
--P(.dbd.O)(ORa)(ORb), phosphoryl choline, phosphoryl ethanolamine,
phosphoryl serine, phosphoryl cardiolipin, phosphoryl inositol,
ethylphosphocholine, phosphorylmethanol, phosphorylethanol,
phosphorylpropanol, phosphorylbutanol,
phosphorylethanolamine-N-lactose,
phosphoethanolamine-N-[methoxy(propylene glycol)],
phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,
pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,
dinitrophenyl-phosphoethanolamine and phsophoglycerol, wherein Ra
and Rb are each independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl and aryl.
9. The method of claim 8, wherein said drug comprises 5-ASA.
10. The method of claim 9, wherein X is an alkylene chain being 3-6
carbon atoms in length.
11. The method of claim 9, wherein R is phosphoryl choline.
12. A pharmaceutical composition comprising, as an active
ingredient, a conjugate which comprises a drug useful in the
treatment of the inflammatory bowel disease covalently linked to a
phospholipid, and a pharmaceutically acceptable carrier, the
composition being formulated for oral administration and is being
packaged in a packaging material and identified in print, in or on
said packaging material, for use in the treatment of said
inflammatory bowel disease.
13. The composition of claim 12, being formulated as a liquid oral
formulation.
14. The composition of claim 13, wherein said drug useful in the
treatment of inflammatory bowel disease is selected from the group
consisting of a drug that comprises 5-ASA, tacrolimus and
methotrexate.
15. The composition of claim 13, wherein said phospholipid is a
phosphoglycerol.
16. The composition of claim 15, wherein said drug is linked to
position sn-2 of said phosphoglycerol.
17. The composition of claim 13, wherein said drug is linked to
said phospholipid via a bridging unit.
18. The composition of claim 17, wherein said bridging unit is an
alkylene being from 3 to 6 carbon atoms in length.
19. The composition of claim 13, wherein said conjugate has a
general Formula: ##STR00010## wherein: A is an alkylene chain being
3-30 carbon atoms in length; X is a bridging unit; D is said drug
suitable for use in the treatment of said inflammatory bowel
disease; and R is selected from the group consisting of
--P(.dbd.O)(ORa)(ORb), phosphoryl choline, phosphoryl ethanolamine,
phosphoryl serine, phosphoryl cardiolipin, phosphoryl inositol,
ethylphosphocholine, phosphorylmethanol, phosphorylethanol,
phosphorylpropanol, phosphorylbutanol,
phosphorylethanolamine-N-lactose,
phosphoethanolamine-N-[methoxy(propylene glycol)],
phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,
pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,
dinitrophenyl-phosphoethanolamine and phsophoglycerol, wherein Ra
and Rb are each independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl and aryl.
20. The composition of claim 19, wherein said drug comprises
5-ASA.
21. The composition of claim 20, wherein X is an alkylene chain
being 3-6 carbon atoms in length.
22. The composition of claim 20, wherein R is phosphoryl
choline.
23. An oral liquid dosage form comprising a conjugate which
comprises a drug useful in the treatment of the inflammatory bowel
disease covalently linked to a phospholipid, and a pharmaceutically
acceptable carrier.
24. The dosage form of claim 23, wherein said drug useful in the
treatment of inflammatory bowel disease is selected from the group
consisting of a drug that comprises 5-ASA, tacrolimus and
methotrexate.
25. The dosage form of claim 23, wherein said phospholipid is a
phosphoglycerol.
26. The dosage form of claim 25, wherein said drug is linked to
position sn-2 of said phosphoglycerol.
27. The dosage form of claim 23, wherein said conjugate has a
general Formula: ##STR00011## wherein: A is an alkylene chain being
3-30 carbon atoms in length; X is a bridging unit; D is said drug
suitable for use in the treatment of said inflammatory bowel
disease; and R is selected from the group consisting of
--P(.dbd.O)(ORa)(ORb), phosphoryl choline, phosphoryl ethanolamine,
phosphoryl serine, phosphoryl cardiolipin, phosphoryl inositol,
ethylphosphocholine, phosphorylmethanol, phosphorylethanol,
phosphorylpropanol, phosphorylbutanol,
phosphorylethanolamine-N-lactose,
phosphoethanolamine-N-[methoxy(propylene glycol)],
phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,
pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,
dinitrophenyl-phosphoethanolamine and phsophoglycerol, wherein Ra
and Rb are each independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl and aryl.
28. The dosage form of claim 27, wherein said drug comprises
5-ASA.
29. The dosage form of claim 28, wherein X is an alkylene chain
being 3-6 carbon atoms in length.
30. The dosage form of claim 28, wherein R is phosphoryl
choline.
31. A conjugate comprising a phospholipid having attached thereto
5-aminosalicylic acid (5-ASA).
32. The conjugate of claim 31, wherein said conjugate has a general
Formula: ##STR00012## wherein: A is an alkylene chain being 3-30
carbon atoms in length; X is a bridging unit or absent; D is 5-ASA;
and R is selected from the group consisting of
--P(.dbd.O)(ORa)(ORb), phosphoryl choline, phosphoryl ethanolamine,
phosphoryl serine, phosphoryl cardiolipin, phosphoryl inositol,
ethylphosphocholine, phosphorylmethanol, phosphorylethanol,
phosphorylpropanol, phosphorylbutanol,
phosphorylethanolamine-N-lactose,
phosphoethanolamine-N-[methoxy(propylene glycol)],
phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,
pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,
dinitrophenyl-phosphoethanolamine and phsophoglycerol, wherein Ra
and Rb are each independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl and aryl.
33. An oral liquid dosage form comprising the conjugate of claim
31.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/990,457 filed on May 30, 2013, which is a
National Phase of PCT Patent Application No. PCT/IL2011/050037
having International filing date of Dec. 1, 2011, which claims the
benefit of priority under 35 USC .sctn.119(e) of U.S. Provisional
Patent Application No. 61/418,892 filed on Dec. 2, 2010. The
contents of the above applications are all incorporated by
reference as if fully set forth herein in their entirety.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention, in some embodiments thereof, relates
to pharmacology and, more particularly, but not exclusively, to a
novel methodology for the treatment of inflammatory bowel
diseases.
[0003] Inflammatory bowel disease, or IBD, is a collective term
encompassing related, but distinct, chronic inflammatory disorders
of the gastrointestinal tract, such as Crohn's disease, ulcerative
colitis (UC), indeterminate colitis, microscopic colitis and
collagenous colitis, with Crohn's disease and ulcerative colitis
being the most common diseases. Ulcerative colitis is confined to
the large intestine (colon) and rectum, and involves only the inner
lining of the intestinal wall. Crohn's disease may affect any
section of the gastrointestinal tract (e.g., mouth, esophagus,
stomach, small intestine, large intestine, rectum and anus) and may
involve all layers of the intestinal wall. Both diseases, as well
as other IBDs, are characterized by abdominal pain and cramping,
diarrhea, rectal and/or intestinal bleeding, weight loss and fever.
The symptoms of these diseases are usually progressive, and
sufferers typically experience periods of remission followed by
severe flare-ups. Less frequent, but also possible, IBD symptoms
reflect mucosal inflammation of other sections of the GI tract,
such as duodenitis, jejunitis and proctitis.
[0004] The goal of IBD therapy is to reduce the extent and symptoms
of the inflammation, rather than to actually cure the disease
(Kesisoglou and Zimmermann, 2005). Aminosalicylates and
corticosteroids are the traditional mainstays of IBD therapy.
Immunomodulators (e.g. 6-mercaptopurine, its prodrug azathioprine,
tacrolimus), antibiotics, and biological response modulators
(infliximab) are also commonly used.
[0005] The most commonly used medications to treat IBD are
anti-inflammatory drugs such as the salicylates. Preparations of
salicylate are effective in treating mild to moderate disease and
can also decrease the frequency of disease flares when the
medications are taken on a prolonged basis. Examples of salicylates
include sulfasalazine, olsalazine, and mesalamine. Particularly,
sulfasalazine and related drugs having the bioactive
5-amino-salicylic acid (5-ASA) moiety are widely used to control
moderate IBD symptoms and to maintain remission. All of these
medications are given orally in high doses for maximal therapeutic
benefit. However, treatments with these medications is typically
accompanied with adverse side effects such as nausea, dizziness,
changes in blood chemistry (including anemia and leukopenia), skin
rashes and drug dependence.
[0006] Corticosteroids are more potent and faster-acting
anti-inflammatory drugs in the treatment of IBD, as compared with
salicylates. Prednisone, for example, is a corticosteroid commonly
used in the treatment of severe cases of IBD. Nevertheless,
potentially serious side effects limit the use of corticosteroids
to patients with more severe disease. Side effects of
corticosteroids usually occur upon long term use and include
thinning of the bone and skin, infections, diabetes, muscle
wasting, rounding of faces, psychiatric disturbances, and, on rare
occasions, destruction of hip joints.
[0007] In cases where IBD patients do not respond to salicylates or
corticosteroids, medications that suppress the immune system,
namely immunosuppressants, are used. Examples of immunosuppressants
include azathioprine and 6-mercaptopurine. However, as
immunosuppressants may render the patient immuno-compromised and
susceptible to other diseases, the use thereof in the treatment of
IBD is not recommended.
[0008] IBD presents a challenging target for drug delivery (Klotz
and Schwab, 2005). Because the original disease pathogenesis and
some of the major pathological manifestations are confined to the
GIT tissue, an ideal delivery strategy for IBD should result in an
elevated concentration of the therapeutic entity in the diseased
intestinal tissue with minimal systemic exposure. For example,
studies have shown that for 5-aminosalicylic acid (5-ASA), the
therapeutic effect in IBD is directly correlated with the drug
concentration in the diseased intestinal mucosa (see, FIG. 1)
(Frieri et al., 2000).
[0009] Current delivery strategies in the treatment of IBD are
based on either a delayed release formulation or a chemical
modification of the drug molecule. Delayed release of the drug is
typically achieved with polymer coating. For instance, Asacol.RTM.
is an Edugarit S-coated 5-ASA formulation in which 5-ASA is
designed to be released at pH>7, which is found in the ileum and
further; Pentasa.RTM. is an ethylcellulose-coated 5-ASA formulation
in which 5-ASA is continuously release over several hours. The
chemical modification approach is focused mainly on increasing
first-pass hepatic metabolism and thereby reducing systemic drug
levels (e.g., budesonide), or linking the drug (mainly 5-ASA) to a
carrier via azo bond to reduce the absorption of the complex in the
small intestine and thereby targeting the colon; in the colon,
bacterial azo-reductases are able to liberate the free drug from
the complex, effectively leading to colonic drug targeting (e.g.
sulfasalazine, olsalazine and balsalazide).
[0010] All these strategies, however, target a region of the
intestine (typically the colon), and not the actual diseased tissue
itself. This is disadvantageous with respect to drug therapy and
patient care, as it essentially leads to a waste of significant
portion of the administered dose, and to increased chances of side
effects. Additionally, it excludes the use of these drug products
in cases where the inflammation is outside of the particular
targeted region, e.g., the small intestine in CD patients.
[0011] PLA.sub.2 (phospholipases A.sub.2) represent a family of
enzymes that catalyze the hydrolysis of the sn-2 fatty acyl bond of
phospholipids (PL), to liberate a free fatty acid and a
lysophospholipid (see, FIG. 2). At least 19 enzymes with PLA.sub.2
activity have been identified to date; 10 isozymes are secreted
from cells (sPLA.sub.2), and the others are cytosolic enzymes
(cPLA.sub.2), however, by definition, all of them hydrolyze the
ester bond at the so-2 position of PL (Murakami and Kudo, 2002;
Touqui and Alaoui-El-Azher, 2001). It has been reported that
sPLA.sub.2 enzymes do not demonstrate any specific fatty acid
selectivity (Kurz and Scriba, 2000; Laye and Gill, 2003).
[0012] In the past decade, PLA.sub.2 expression in the inflamed
tissue of IBD patients, both CD and UC, has been consistently
reported to be significantly elevated. In CD, significantly
increased gene expression of PLA.sub.2 was found in both the small
and the large intestinal mucosa with active inflammation (Haapamaki
et al., 1999a), as well as significantly higher PLA.sub.2 mRNA
levels and activity in ileal mucosa from CD patients than from
controls (FIG. 3A) (Lilja et al., 1995). In the protein level, the
mass concentration of group II PLA.sub.2 protein was found to be
significantly higher in colonic mucosa of CD patients compared to
control (Haapamaki et al., 1998; Minami et al., 1994), and the
increased level was correlated with the degree of the inflammatory
activity in the intestinal wall (Haapamaki et al., 1998). PLA.sub.2
activity in CD patients was measured as well, and was reported to
be significantly higher (about 5-folds) than that in control
subjects, with an association to the degree of inflammation (see,
for example, FIG. 3B) (Minami et al., 1994). The situation was not
different in UC; significantly increased gene expression of
PLA.sub.2 was found in inflamed large intestinal mucosa of UC
patients compared to control, with an association between the
PLA.sub.2 mRNA levels and the degree of inflammation (Haapamaki et
al., 1997). The concentration of PLA.sub.2 protein in the colonic
mucosa of UC patients was found to be significantly higher compared
with control (Haapamaki et al., 1999b; Minami et al., 1994), and
increased activity in the diseased tissue of UC patient was evident
as well (FIG. 3C) (Minami et al., 1994; Peterson et al., 1996).
Overall, these data clearly indicate that PLA.sub.2 levels are
elevated in the diseased IBD tissue, and support the theory that
PLA.sub.2 is involved in the local and generalized pathological
processes of IBD, CD and UC.
[0013] The present inventors have previously disclosed an
exploitation of the esterase enzyme phospholipase A.sub.2
(PLA.sub.2) to mechanistically target drug molecules to diseased
cells. Thus, the present inventors have designed and investigated a
series of conjugates of a phospholipid and the non-steroidal
anti-inflammatory drug indomethacin and of a phospholipid and
valproic acid, differing in the length of the carbonic linker
between the PL and the drug moiety (Dahan et al., 2007; Dahan et
al., 2008).
[0014] WO 91/16920 discloses lipid derivatives of anti-inflammatory
drugs, including aspirin, other salicylates and other non-steroidal
anti-inflammatory drugs (NSAIDs), which serve as phospholipid
prodrugs that are activated by digestive enzymes such as
phospholipase A2 and other phospholipases and lysophospholipases,
so as to release the drug and provide a steady level of the drug in
the bloodstream while reducing toxicity. According to the teachings
of WO 91/16920, the phospholipid prodrugs are useful in treating
chronic inflammatory diseases such as rheumatoid arthritis and
osteoarthritis.
[0015] WO 00/31083 discloses phospholipid derivatives of NSAIDs in
which the drug is covalently linked to a phospholipid moiety via a
bridging group, and which release the NSAID upon enzymatic cleavage
at the diseases site. According to the teachings of WO 00/31083,
the bridging group is designed to be sensitive to cleavage by
phospholipases such as PLA2 that are specifically elevated at the
site of the disease.
[0016] Additional background art includes Dahan and Amidon, Am. J.
Physiol. Gastrointest. Liver Physiol. 2009; Dahan et al., J.
Control. Release, 2007; Dahan et al., J. Control. Release, 2008.
Dahan and Hoffman, Drug Metab. Dispos., 2007; Dahan and Hoffman,
European Journal of Pharmaceutical Sciences, 2005; Dahan and
Hoffman, European Journal of Pharmaceutical Sciences, 2006; Dahan
and Hoffman, Pharmaceutical Research., 2006; Dahan et al., Drug
Metab. Dispos., 2009; and Dvir et al., CNS Drug Rev. 2007.
SUMMARY OF THE INVENTION
[0017] While the prior art teaches phospholipid derivatives of
anti-inflammatory drugs which are activated by phospholipases so as
to release the drug at the diseased site, the prior art fails to
teach phospholipid derivatives that can serve as a targeted drug
delivery system in the treatment of inflammatory bowel diseases
(IBD).
[0018] The prior art further fails to teach a targeted drug
delivery system that can deliver an active drug to the small
intestine, for the treatment of, for example, Crohn's disease in
patients with lesions in the small intestine.
[0019] The prior art further fails to teach a targeted drug
delivery system for the treatment of IBD, which is suitable to be
administered as an oral liquid dosage form.
[0020] The commonly used drug products for treating IBDs commonly
require high daily doses, at least partially to ineffective
targeting to the diseased tissue.
[0021] The present inventors have now designed a novel methodology
for targeting IBD drugs to diseased tissues. This novel methodology
enables the treatment of IBD patients with inflamed tissues at the
small intestine, and further enables the treatment of IBD patients
while utilizing lower daily doses and optionally liquid oral
formulations, thus increasing patient's compliance.
[0022] According to an aspect of some embodiments of the present
invention there is provided a method of treating an inflammatory
bowel disease in a subject in need thereof, the method comprising
orally administering to the subject a therapeutically effective
amount of a conjugate which comprises a drug useful in the
treatment of the inflammatory bowel disease covalently linked to a
phospholipid.
[0023] According to some embodiments of the invention, the
conjugate is formulated as a liquid oral formulation.
[0024] According to an aspect of some embodiments of the present
invention there is provided a use of a conjugate which comprises a
drug useful in the treatment of the inflammatory bowel disease
covalently linked to a phospholipid in the manufacture of a
medicament for treating the inflammatory bowel disease, the
medicament being formulated for oral administration.
[0025] According to some embodiments of the invention, the
medicament is formulated as a liquid oral formulation.
[0026] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising a drug useful in
the treatment of the inflammatory bowel disease being covalently
linked to a phospholipid, the conjugate being identified for use in
the treatment of the inflammatory bowel disease via oral
administration of the conjugate.
[0027] According to some embodiments of the invention, the
conjugate is formulated as a liquid oral formulation.
[0028] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition
comprising, as an active ingredient, a conjugate which comprises a
drug useful in the treatment of the inflammatory bowel disease
covalently linked to a phospholipid, and a pharmaceutically
acceptable carrier, the composition being formulated for oral
administration and is being packaged in a packaging material and
identified in print, in or on the packaging material, for use in
the treatment of the inflammatory bowel disease.
[0029] According to some embodiments of the invention, the
composition is being formulated as a liquid oral formulation.
[0030] According to an aspect of some embodiments of the present
invention there is provided an oral liquid dosage form comprising a
conjugate which comprises a drug useful in the treatment of the
inflammatory bowel disease covalently linked to a phospholipid, and
a pharmaceutically acceptable carrier.
[0031] According to some embodiments of the invention, the
inflammatory bowel disease is selected from the group consisting of
Crohn's disease and ulcerative colitis.
[0032] According to some embodiments of the invention, the
inflammatory bowel disease is manifested in the small
intestine.
[0033] According to some embodiments of the invention, the drug
useful in the treatment of inflammatory bowel disease is selected
from the group consisting of a salicylate, a corticosteroid, an
immunomodulator, an antibiotic and a biological response
modulator.
[0034] According to some embodiments of the invention, the drug
useful in the treatment of inflammatory bowel disease comprises
5-ASA.
[0035] According to some embodiments of the invention, the drug
useful in the treatment of inflammatory bowel disease is selected
from the group consisting of a drug that comprises 5-ASA,
tacrolimus and methotrexate.
[0036] According to some embodiments of the invention, the
phospholipid is a phosphoglycerol.
[0037] According to some embodiments of the invention, the drug is
linked to position sn-2 of the phosphoglycerol.
[0038] According to some embodiments of the invention, the drug is
linked to the phospholipid directly.
[0039] According to some embodiments of the invention, the drug is
linked to the phospholipid via a bridging unit.
[0040] According to some embodiments of the invention, the bridging
unit comprises an alkylene being from 1 to 20 carbon atoms in
length.
[0041] According to some embodiments of the invention, the alkylene
is being from 3 to 6 carbon atoms in length.
[0042] According to some embodiments of the invention, the
conjugate has a general Formula:
##STR00001##
wherein:
[0043] A is an alkylene chain being 3-30 carbon atoms in
length;
[0044] X is the bridging unit or absent;
[0045] D is the drug suitable for use in the treatment of the
inflammatory bowel disease; and
[0046] R is selected from the group consisting of
--P(.dbd.O)(ORa)(ORb), phosphoryl choline, phosphoryl ethanolamine,
phosphoryl serine, phosphoryl cardiolipin, phosphoryl inositol,
ethylphosphocholine, phosphorylmethanol, phosphorylethanol,
phosphorylpropanol, phosphorylbutanol,
phosphorylethanolamine-N-lactose,
phosphoethanolamine-N-[methoxy(propylene glycol)],
phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,
pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,
dinitrophenyl-phosphoethanolamine and phsophoglycerol, wherein Ra
and Rb are each independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl and aryl.
[0047] According to some embodiments of the invention, the drug
comprises 5-ASA.
[0048] According to some embodiments of the invention, the drug is
selected from the group consisting of a drug that comprises 5-ASA,
a mercaptopurine such tacrolimus and methotrexate.
[0049] According to some embodiments of the invention, X is an
alkylene chain being 3-6 carbon atoms in length.
[0050] According to some embodiments of the invention, wherein R is
phosphoryl choline.
[0051] According to some embodiments of the invention, A is an
alkylene chain being 15 or 17 carbon atoms in length.
[0052] According to an aspect of some embodiments of the present
invention there is provided a process of preparing a conjugate
which comprises a phospholipid having attached thereto a drug
suitable for the treatment of an inflammatory bowel disease, the
process comprising covalently coupling the drug to a
lysophospholipid.
[0053] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising a phospholipid
having attached thereto 5-amino salicylic acid (5-ASA).
[0054] According to some embodiments of the invention, the
conjugate has a general Formula:
##STR00002##
wherein:
[0055] A is an alkylene chain being 3-30 carbon atoms in
length;
[0056] X is a bridging unit or absent;
[0057] D is 5-ASA; and
[0058] R is selected from the group consisting of
--P(.dbd.O)(ORa)(ORb), phosphoryl choline, phosphoryl ethanolamine,
phosphoryl serine, phosphoryl cardiolipin, phosphoryl inositol,
ethylphosphocholine, phosphorylmethanol, phosphorylethanol,
phosphorylpropanol, phosphorylbutanol,
phosphorylethanolamine-N-lactose,
phosphoethanolamine-N-[methoxy(propylene glycol)],
phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,
pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,
dinitrophenyl-phosphoethanolamine and phsophoglycerol, wherein Ra
and Rb are each independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl and aryl.
[0059] According to some embodiments of the invention, X is an
alkylene chain being 3-6 carbon atoms in length.
[0060] According to some embodiments of the invention, R is
phosphoryl choline.
[0061] According to some embodiments of the invention, A is an
alkylene chain being 15 or 17 carbon atoms in length.
[0062] According to some embodiments of the invention, there is
provided an oral liquid dosage form comprising the PL-5-ASA
conjugate as described herein.
[0063] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0065] In the drawings:
[0066] In the drawings:
[0067] FIG. 1 (Background Art) is graph demonstrating a correlation
between IBD mucosal tissue concentrations of 5-ASA and soluble
interleukin 2 receptor (sIL-2R) (Frieri et al 2000 Gut);
[0068] FIG. 2 (Background Art) illustrates the chemical structure
of an exemplary phospholipid (the phosphoglycerol lecithin), with
the arrow indicating PLA.sub.2 site of action;
[0069] FIG. 3A (Background Art) presents a Northern blot analysis
of group II PLA.sub.2 mRNA in ileal mucosa from four patients, two
controls (a and b) and two CD patients (c and d) (Lilja et al 1995
Gut); and
[0070] FIGS. 3B and 3C (Background Art) present bar graphs showing
PLA.sub.2 activity in the colonic mucosa of control and CD patients
(FIG. 3B) and UC patient (FIG. 3C) (Minami et al 1994 Gu).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0071] The present invention, in some embodiments thereof, relates
to pharmacology and, more particularly, but not exclusively, to a
novel methodology for the treatment of inflammatory bowel
diseases.
[0072] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0073] The methodology described herein is aimed at targeting the
drug of interest to the diseased tissue per se, in order to improve
drug therapy and patient care in IBD, as is detailed
hereinbelow.
[0074] Based on the findings that PLA.sub.2 is overexpressed in the
GIT of patients suffering from IBDs, the present inventors have
realized that previously described phospholipid derivatives of
anti-inflammatory drugs, when administered orally to IBD patients,
would release the drug in the GIT, rather than in the blood system.
The present inventors have further realized that the overexpression
of PLA.sub.2 in the GIT of patients suffering from IBDs can be
exploited for targeted delivery of anti-inflammatory drugs for
treating the IBD.
[0075] The present inventors have thus realized that conjugates of
a phospholipid and an IBD drug (also referred to herein,
interchangeably, as PL-drug conjugates) can be designed and
utilized so as to improve therapeutic index and patient care in
IBD, based on the following underlying basis.
[0076] Since the activation of a PL-drug conjugate as disclosed
herein is PLA.sub.2-mediated, the increased levels of this enzyme
in the diseased tissue leads to increased free drug in the actual
diseased tissues, essentially accompanied by decreased drug levels
in non-diseased tissues, resulting in extended therapeutic index
and improved drug therapy. In fact, it has been realized that the
use of conjugates of a phospholipid and an IBD drug provides a
"sink effect" since the conjugate acts as a prodrug which is
continuously targeted to the diseased tissue and is continuously
released in the diseased tissue, as a result of the overexpression
of PLA.sub.2. Since no accumulation of the prodrug is effected at
the diseased tissue, systemic absorption of the drug is reduced,
and thus, adverse side effects are reduced. In addition, the amount
of free drug at the diseased tissue is increased, and overall, the
therapeutic index (efficacy vs. toxicity) is improved.
[0077] Notably, the prodrug is targeted to diseased tissues and not
to a diseased site, where both diseased and healthy tissues are
present, thus avoiding adverse side effects.
[0078] It is to be noted that at least some of the currently
available IBD drug products are utilized in very high daily oral
doses, at least partially due to the poor targeting of the
available formulations. Products containing 5-ASA, for example,
represent some of the highest oral dose/day medications on the
market, with a maximum daily dose of up to 4.8 grams, which
sometimes correspond to eight tablets and even more. The targeted
delivery of the drug to the inflamed tissue by the disclosed
conjugates thus decreases the required dose, leading to improved
convenience and better patient compliance.
[0079] In addition, unlike the currently available IBD drug
products, the conjugates described herein enable specific delivery
to diseased tissue throughout the entire gastrointestinal tract
(GIT), including the small intestine. It is to be noted that
currently available IBD drug products, and 5-ASA products in
particular, deliver the drug to the colon, and hence are not
effective in treating patients having inflammatory lesions in other
tissues, and particularly in small intestine tissues. This is
particularly advantageous in the treatment of Crohn's disease
patients that have such lesions, and for which current drug
therapies are ineffective.
[0080] The use of the conjugates described herein further provides
for targeted release of the drug without formulation manipulations
and hence enables to utilize liquid formulations instead of solid
dosage forms (as described hereinabove in the context of
formulation manipulations made in order to achieve targeted
delivery to certain areas in the GIT). Since the targeting
methodology described herein is not formulation-dependent but
integral to the PL-drug conjugate molecule, it is possible to
formulate it in a liquid oral dosage form without affecting the
targeting abilities. This is of great benefit in, for example,
pediatric and elderly populations, for whom swallowing of a solid
dosage form is impossible/painful. This advantage becomes even more
important in light of the large size of most of oral IBD products
available on the market.
[0081] Finally, the methodology disclosed herein is
drug-independent, as it depends on the conjugation to a
phospholipid. Thus, any drug of interest for the treatment of IBD
can be utilized and is encompassed by the embodiments of the
present invention.
[0082] Embodiments of the present invention thus pertain to a novel
use of a conjugate of a phospholipid and an IBD drug (PL-drug
conjugates), as described herein. While some of the PL-drug
conjugates have been previously described in the art, none was
described in the context of the methodologies described herein.
Embodiments of the present invention also relate to those
conjugates of a phospholipid and an IBD drug, which have not been
previously described, including, but not limited to, a conjugate of
a phospholipid (e.g., a glycerophosphate) and an IBD drug that
comprises or consists of 5-ASA, a conjugate of a phospholipid
(e.g., a glycerophosphate) and a mercaptopurine such tacrolimus,
and a conjugate of a phospholipid (e.g., a glycerophosphate) and
methotrexate, each further comprising, optionally, a bridging unit,
as described herein.
[0083] For any of the aspects of embodiments of the present
invention, there are provided conjugates, each comprising a drug
useful in the treatment of the inflammatory bowel disease being
covalently linked to a phospholipid.
[0084] As used herein, the phrase "a drug useful in the treatment
of an inflammatory bowel disease", which is also referred to herein
interchangeably as "a drug useful in the treatment of an IBD" or as
"an IBD drug", or simply as "drug", encompasses any drug that has a
therapeutic effect on an inflammatory bowel disease, as the latter
is defined herein. This phrase encompasses commercially available
drugs, as well as drugs that are under development or that has been
suggested as useful in the treatment of an IBD. This phrase further
encompasses any pharmaceutically acceptable salt of these drugs,
known prodrugs of these drugs, and derivatives of these drugs, and
any crystalline form of these drugs (including amorphous form), as
these terms are defined herein, all of which are such that exhibit
a therapeutic effect on an IBD.
[0085] Representative examples of IBD drugs include, but are not
limited, salicylates (e.g., aminosalicylates such as drugs
comprising 5-amino-salicylic acid (5-ASA), for example,
sulfasalazine, olsalazine, and mesalamine); a cortocisteroid (e.g.,
Prednisone); an immunomodulator (e.g., 6-mercaptopurine, its
prodrug azathioprine, tacrolimus); an antibiotic; and a biological
response modulator (e.g., infliximab); as well as some anti-cancer
agents (e.g., paclitaxel, methotrexate and cyclosporine).
[0086] Exemplary drugs include, but are not limited to, 5-ASA,
methotrexate, paclitaxel, tacrolimus, colchicine, cyclosporine,
Azathioprine, mercaptopurine, Beclomethasone Dipropionate,
Infliximab, Traficet-EN, and any other drug for treating an
inflammatory bowel disease which is commercially available or is
currently under research.
[0087] According to some embodiments of the invention, the drug
useful in the treatment of inflammatory bowel disease comprises
5-ASA.
[0088] By "a drug that comprises 5-ASA" are encompassed 5-ASA
itself (e.g., mezalazine) and compounds that are metabolized to
release 5-ASA (e.g., sulfasalazine).
[0089] Exemplary drugs that comprise 5-ASA include, but are not
limited to, mezalazine, sulfasalazine, olsalazine and
balsalazide.
[0090] As discussed hereinabove 5-ASA is the mainstay of treatment
in IBD. Further, since a correlation has been found between the
5-ASA levels in the inflamed tissue and the therapeutic effect of
the drug (see, FIG. 1), an augmented efficacy, as exhibited by the
disclosed conjugates, is particularly beneficial.
[0091] According to some embodiments of the invention, the drug
useful in the treatment of inflammatory bowel disease is
methotrextae.
[0092] According to some embodiments of the invention, the drug
useful in the treatment of inflammatory bowel disease is a
mercatopurine such as, for example, tacrolimus.
[0093] As used herein, a "phospholipid" describes compounds that
comprise a lipid moiety and a phosphate moiety. Commonly available
phospholipids are those belonging to the glycerophospholipid class
(also known as phosphoglycerols or as diacylglyceride phosphates),
including, but not limited to, phosphatidic acid (phosphatidate)
(PA), phosphatidylethanolamine (cephalin) (PE), phosphatidylcholine
(lecithin) (PC), phosphatidylserine (PS), and phosphoinositides
such as, for example, phosphatidylinositol (PI),
phosphatidylinositol phosphate (PIP), phosphatidylinositol
bisphosphate (PIP2) and phosphatidylinositol triphosphate (PIP3);
and those belonging to the phosphosphingolipids class (which are
derived from sphongosine), including, but not limited to, ceramide
phosphorylcholine (sphingomyelin) (SPH), ceramide
phosphorylethanolamine (Cer-PE), and ceramide
phosphorylglycerol.
[0094] Phosphoglycerols have a glycerolic backbone to which are
attached two fatty acyl groups at positions sn-1 and sn-2, and one
phosphate moiety at position sn-3.
[0095] Phosphosphingolipids have a sphingosine backbone which
comprises one unsaturated fatty acyl, and to which are attached one
fatty acyl via an amide bond and one phosphate moiety.
[0096] According to some embodiments of the invention, the
phospholipid is phosphoglycerol.
[0097] According to some embodiments of the invention, the drug is
linked to position sn-2 of the phosphoglycerol. Such phospholipids
are efficient substrates of PLA.sub.2.
[0098] In some embodiments, the IBD drug is linked to an available
phosphglycerol (e.g., lechitin, lysolechitin, phsophinositol, and
the like) by replacing the fatty acyl or acyl at position sn-2 of
the phosphoglycerol by the drug moiety.
[0099] In these embodiments, the phospholipid portion of the
conjugate comprises a fatty acyl as present in the phosphoglycerol
prior to conjugation, preferably at position sn-1, and a phosphate
moiety as present in the phosphglycerol prior to conjugation, at
position sn-3.
[0100] The term "acyl" as used herein describes a --C(.dbd.O)--R
moiety, typically derived from the corresponding carboxylate
(R'--O--C(.dbd.O)--R). The term "fatty acyl" as used herein
describes an acyl derived from fatty acid, such that R is a high
hydrocarbon chain, typically being at least 2 or at least 3 carbon
atoms in length, or from 6 to 30 carbon atoms in length. The
hydrocarbon chain can be saturated or unsaturated, the latter can
comprise one or more double and/or triple bonds.
[0101] In some embodiments, the hydrocarbon chain in a fatty acyl
comprises an odd number of carbon atoms, thus being derived from a
fatty acid.
[0102] Representative examples of fatty acyls are those which
comprise a hydrocarbon chain that is derived from palmitic acid,
and as such are 15 carbon atoms in length, or from stearic acid,
and as such are of 17 carbon atoms in length.
[0103] In some embodiments, the drug is linked to the phospholipid
via an ester bond.
[0104] As used herein, an "ester bond" describes a --O--C(.dbd.O)--
bond, which is typically formed by reacting a hydroxyl group and a
carboxylate or carbonyl group.
[0105] In some embodiments, the conjugate comprises a phospholipid
that is derived from a glycerophospholipid (a phosphoglycerol), as
described herein, in which the IBD drug is attached at position
sn-2 of the glycerolic backbone via an ester bond (replacing a
fatty acyl of the phosphoglycerol at that position), whereby the
other fatty acyl, at position sn-1, and the phosphate moiety at
position sn-3 are the same as in the phosphoglycerol.
[0106] According to some embodiments of the invention, the IBD drug
is linked to the phospholipid directly.
[0107] In some embodiments, the IBD drug is linked to the
phospholipid directly, via an ester bond, as defined herein. Such
conjugates are typically formed by utilizing IBD drugs that have a
carboxylate group (e.g., a carboxylic acid group) or by utilizing
derivatives of IBD drugs which have been modified to include a
carboxylate group or any other reactive group that can form a bond
with the hydroxyl group of the phospholipid (see, Example 1
hereinbelow).
[0108] According to some embodiments of the invention, the drug is
linked to the phospholipid via a bridging unit.
[0109] As used herein, a "bridging unit", which is also referred to
herein interchangeably as a linking unit, a linking moiety or
simply as a linker, is a moiety that is attached to the
phospholipid at one end thereof and to the IBD drug at another end
thereof, and thus links the IBD drug to the phospholipid.
[0110] The bridging unit is typically derived from a suitable
precursor, namely, a bifunctional compound that has a first and a
second reactive group, as described in detail in Example 1 in the
Examples section that follows.
[0111] In some embodiments, the bridging unit is desired in cases
where the IBD drug utilized does not have a functional group that
can form a covalent bond (e.g., an ester bond) with the
phospholipid. In these embodiments, the bridging unit can be seen
as providing a chemical functionality for attaching the IBD drug to
the phospholipid.
[0112] In some embodiments, the bridging unit is desired for
obviating steric hindrance of the ester bond that is to be
subjected to enzymatic hydrolysis (e.g., by PLA.sub.2).
[0113] Thus, in some embodiments, the desired length of the
bridging unit depends, in part, on the size of the linked drug,
such that, for example, for bulky drugs, relatively long (e.g.
higher than 2 and even higher than 6 carbon atoms) moieties are
desired, whereby for relatively small drug molecules, moieties
being of 2-6 or from 3-5 carbon atoms in length suffice.
[0114] In some embodiments, the bridging unit is 1 carbon atom, 2
carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6
carbon atoms in length, and can be even of 30 carbon atoms or more,
in length.
[0115] Exemplary bridging units include, but are not limited to,
hydrocarbons being from 1 to 30 carbon atoms in length, optionally
interrupted by one or more heteroatoms; amino acids; and/or
peptides (e.g., short peptides being of 2-10 amino acids).
[0116] As used herein, the term "hydrocarbon" encompasses any
moiety that comprises carbons and hydrogens covalently linked, in a
form of anyone or a combination of alkyl, alkenyl, cycloalkyl,
alkynyl and aryl, such that a backbone chain linking the IBD drug
to the phospholipid is from 1 to 30 carbon atoms in length, or from
1 to 20, or from 1 to 15, or from 2 to 20, or from 2 to 15, or from
3 to 30, or from 3 to 20, or from 3 to 15, or from 2 to 10, or from
3 to 10, or from 3 to 6, or from 3 to 5, carbon atoms in
length.
[0117] According to some embodiments of the invention, the bridging
unit comprises an alkylene being from 1 to 20 carbon atoms in
length, or from 2 to 20 carbon atoms in length, or from 3 to 20
carbon atoms in length, or from 3 to 10 carbon atoms in length, or
from 3 to 6, or from 3 to 5 carbon atoms in length.
[0118] As used herein, the term "alkylene" describes a hydrocarbon
that is comprised of an alkyl chain, which can be represented by
--(CRcRd)n-, wherein Rc and Rd can be the same or different in any
unit of the n units in the chain, and each can independently be
hydrogen or any of the substituents described herein for an
"alkyl".
[0119] Alternatively, the bridging unit comprises or is derived
from an amino acid or a short peptide (e.g., di- or
tri-peptides).
[0120] Any of the naturally occurring or non-naturally occurring
amino acids, and peptides made therefrom, are contemplated.
[0121] In some embodiments, the bridging unit is selected such that
upon enzymatic cleavage of the bond connecting it to the
phospholipid, the released moiety, which contains the IBD drug, can
exert its therapeutic effect.
[0122] In some embodiments, the released moiety is the IBD drug
having the bridging unit attached thereto (whereby the bridging
unit terminates with a functional group generated by the cleavage),
and such a moiety is capable of exerting a therapeutic effect
comparable to that of the IBD drug per se (namely, the bridging
unit does not interfere with the therapeutic activity of the IBD
drug).
[0123] In some embodiments, the released moiety is the IBD drug
having the bridging unit attached thereto (whereby the bridging
unit terminates with a functional group generated by the cleavage),
and such a moiety is capable of undergoing cleavage under
physiological conditions (e.g., a pH-dependent cleavage or an
enzymatic cleavage by e.g., esterases, amidases or proteolytic
enzymes), so as to release the IBD drug.
[0124] In some embodiments, the released moiety is the IBD drug
having the bridging unit attached thereto (whereby the bridging
unit terminates with a functional group generated by the cleavage),
and such a moiety is capable of undergoing self-immolation under
physiological conditions as described supra, to thereby release the
IBD drug.
[0125] In some embodiments, the bridging moiety is selected such
that upon release of the IBD drug, by cleavage or self-immolation
of the IBD drug having the bridging unit attached thereto (whereby
the bridging unit terminates with a functional group generated by
the cleavage), a biocompatible moiety is formed (e.g., a metabolite
such as, for example, an amino acid or a peptide, urea, and the
like).
[0126] The PL-drug conjugates described herein can be collectively
represented by the following general Formula:
##STR00003##
wherein:
[0127] A is saturated or unsaturated, substituted or unsubstituted
hydrocarbon chain being 2-30 carbon atoms in length;
[0128] X is a bridging unit, as described herein, or absent;
[0129] D is the drug suitable for use in the treatment of said
inflammatory bowel disease (an IBD drug), as described herein;
and
[0130] R is selected from the group consisting of
--P(.dbd.O)(ORa)(ORb), phosphoryl choline, phosphoryl ethanolamine,
phosphoryl serine, phosphoryl cardiolipin, phosphoryl inositol,
ethylphosphocholine, phosphorylmethanol, phosphorylethanol,
phosphorylpropanol, phosphorylbutanol,
phosphorylethanolamine-N-lactose,
phosphoethanolamine-N-[methoxy(propylene glycol)],
phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,
pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,
dinitrophenyl-phosphoethanolamine and phsophoglycerol, wherein Ra
and Rb are each independently selected from the group consisting of
hydrogen, alkyl, cycloalkyl and aryl.
[0131] In some embodiments, D is a drug that comprises 5-ASA, as
defined herein.
[0132] In some embodiments, D is a mercaptopurine such as
tacrolimus.
[0133] In some embodiments, D is methotrexate.
[0134] In some embodiments, A is a hydrocarbon chain derived from a
fatty acid, as defined herein for a fatty acyl.
[0135] In some embodiments, A is a hydrocarbon chain derived from a
fatty acid, as defined herein for a fatty acyl, and R is selected
from the group consisting of phosphate, phosphoryl choline,
phosphoryl serine, phosphoryl enthanolamine, and a phosphoryl
inositol.
[0136] In some of these embodiments, X can be absent or can be an
alkylene of from 3 to 6 carbon atoms.
[0137] In some embodiments, A is a hydrocarbon chain derived from a
fatty acid, as defined herein for a fatty acyl, R is selected from
the group consisting of phosphate, phosphoryl choline, phosphoryl
serine, phosphoryl enthanolamine, and a phosphoryl inositol, and D
is a drug that comprises 5-ASA, as defined herein.
[0138] In some embodiments, A is a hydrocarbon chain derived from a
fatty acid, as defined herein for a fatty acyl, R is selected from
the group consisting of phosphate, phosphoryl choline, phosphoryl
serine, phosphoryl enthanolamine, and a phosphoryl inositol, and D
is a mercaptopurine such as tacrolimus.
[0139] In some embodiments, A is a hydrocarbon chain derived from a
fatty acid, as defined herein for a fatty acyl, R is selected from
the group consisting of phosphate, phosphoryl choline, phosphoryl
serine, phosphoryl enthanolamine, and a phosphoryl inositol, and D
is methotrexate.
[0140] In some of these embodiments, X can be absent or can be an
alkylene of from 3 to 6 carbon atoms.
[0141] In some embodiments, A is a hydrocarbon chain derived from a
fatty acid, as defined herein for a fatty acyl, and D is a drug
that comprises 5-ASA, as defined herein.
[0142] In some of these embodiments, X can be absent or can be an
alkylene of from 3 to 6 carbon atoms.
[0143] In some embodiments, A is a hydrocarbon chain derived from a
fatty acid, as defined herein for a fatty acyl, and D is a
mercaptopurine such as tacrolimus.
[0144] In some of these embodiments, X can be absent or can be an
alkylene of from 3 to 6 carbon atoms.
[0145] In some embodiments, A is a hydrocarbon chain derived from a
fatty acid, as defined herein for a fatty acyl, and D is
methotrexate.
[0146] In some of these embodiments, X can be absent or can be an
alkylene of from 3 to 6 carbon atoms.
[0147] According to some embodiments, there is provided a conjugate
that comprises a drug that comprises 5-ASA as defined herein, being
covalently linked to a phospholipid, as defined herein.
[0148] In some of these embodiments, the drug is attached to the
phospholipid as defined herein, either directly, or via a bridging
unit, as defined herein.
[0149] According to some embodiments, there is provided a conjugate
that comprises any of the IBD drugs as defined herein, being
covalently linked to a phospholipid, as defined herein.
[0150] In some of these embodiments, the drug is attached to the
phospholipid as defined herein, either directly, or via a bridging
unit, as defined herein.
[0151] In some embodiments, the drug is a mercaptopurine such as
tacrolimus.
[0152] In some embodiments, the drug is methotrexate.
[0153] According to an aspect of some embodiments of the present
invention there is provided a process of preparing a conjugate
which comprises a phospholipid having attached thereto a drug
suitable for the treatment of an inflammatory bowel disease,
essentially as described herein. The process is effected by
covalently coupling that drug to the phospholipid. In some
embodiments, the process is effected by first coupling to the
phospholipid a precursor compound for generating the bridging
moiety, and then coupling to the obtained phospholipid, having this
precursor compound coupled thereto, the IBD drug. Further details
regarding the process, according to these embodiments, are
described under Example 1 in the Examples section that follows.
[0154] In any of the embodiments and aspects of the present
invention, the PL-drug conjugate can also be in a form of a
pharmaceutically acceptable salt thereof, a hydrate or a solvate
thereof, and be in any crystalline form, including an amorphous
form.
[0155] The term "solvate" refers to a complex of variable
stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on),
which is formed by a solute (the conjugate as described herein) and
a solvent, whereby the solvent does not interfere with the
biological activity of the solute. Suitable solvents include, for
example, ethanol, acetic acid and the like.
[0156] The term "hydrate" refers to a solvate, as defined
hereinabove, where the solvent is water or an aqueous solution.
[0157] The phrase "pharmaceutically acceptable salt" refers to a
charged species of the parent compound and its counter ion, which
is typically used to modify the solubility characteristics of the
parent compound and/or to reduce any significant irritation to an
organism by the parent compound, while not abrogating the
biological activity and properties of the administered compound. An
example, without limitation, of a pharmaceutically acceptable salt
would be a hydroxyl anion (O.sup.-) and a cation such as, but not
limited to, ammonium, sodium, potassium and the like. Another
example, without limitation, of a pharmaceutically acceptable salt
would be an ammonium cation and an acid addition salt thereof.
Examples of acid addition salts include, but are not limited to,
hydrochloric acid addition salt, sulfuric acid addition salt
(sulfate salt), acetic acid addition salt, ascorbic acid addition
salt, benzenesulfonic acid addition salt, camphorsulfonic acid
addition salt, citric acid addition salt, maleic acid addition
salt, methanesulfonic acid addition salt, naphthalenesulfonic acid
addition salt, oxalic acid addition salt, phosphoric acid addition
salt, succinic acid addition salt, sulfuric acid addition salt,
tartaric acid addition salt, and toluenesulfonic acid addition
salt.
[0158] The conjugates described herein can be seen as a prodrug. As
used in the art, the term "prodrug" refers to an agent, which is
converted into the active compound (the active drug) in vivo.
Prodrugs are typically useful for facilitating the administration
of the parent drug. In embodiments of the present invention, the
conjugate serves as a targeted prodrug, which releases the active
drug at a selected tissue (e.g., an inflamed tissue in the GI
tract).
[0159] As discussed hereinabove, the PL-drug conjugates as
described herein can be advantageously utilized in the treatment of
an inflammatory bowel disease.
[0160] According to an aspect of some embodiments of the present
invention there is provided a method of treating an inflammatory
bowel disease in a subject in need thereof, the method comprising
administering to the subject a therapeutically effective amount of
a conjugate which comprises a drug useful in the treatment of the
inflammatory bowel disease covalently linked to a phospholipid, as
described herein. According to some embodiments of the present
invention, administering the described conjugate is effected
orally. According to some embodiments of the invention, the
conjugate is utilized as a pharmaceutical composition that
comprises the conjugate and optionally further comprises a
pharmaceutical acceptable carrier, as described herein.
[0161] According to an aspect of some embodiments of the present
invention there is provided a use of a conjugate which comprises a
drug useful in the treatment of the inflammatory bowel disease
covalently linked to a phospholipid in the manufacture of a
medicament for treating said inflammatory bowel disease. According
to some embodiments of the present invention, the medicament is
formulated for oral administration. According to some embodiments
of the invention, the conjugate is utilized as a pharmaceutical
composition that comprises the conjugate and optionally further
comprises a pharmaceutical acceptable carrier, as described
herein.
[0162] According to an aspect of some embodiments of the present
invention there is provided a conjugate which comprises a drug
useful in the treatment of the inflammatory bowel disease being
covalently linked to a phospholipid, and which is identified for
use in the treatment of the inflammatory bowel disease. In some
embodiments, the conjugate is identified for use via oral
administration of the conjugate. According to some embodiments of
the invention, the conjugate is utilized as a pharmaceutical
composition that comprises the conjugate and optionally further
comprises a pharmaceutical acceptable carrier, as described
herein.
[0163] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition
comprising, as an active ingredient, a conjugate which comprises a
drug useful in the treatment of the inflammatory bowel disease
covalently linked to a phospholipid, and a pharmaceutically
acceptable carrier. According to some embodiments of the present
invention, the composition is formulated for oral administration.
According to some embodiments of the present invention, the
pharmaceutical composition is packaged in a packaging material and
identified in print, in or on said packaging material, for use in
the treatment of said inflammatory bowel disease.
[0164] As used herein, the phrase "inflammatory bowel disease
(IBD)" refers to a disorder or disease characterized by
inflammatory activity in the GI tract. Examples of IBDs include,
without limitation, Crohn's disease (both distal and proximal),
ulcerative colitis, indeterminate colitis, microscopic colitis,
collagenous colitis, idiopathic inflammation of the small and/or
proximal intestine, irritant bowel syndrome, and IBD-related
diarrhea.
[0165] In some embodiments, the IBD is manifested in the small
intestine. By "manifested in the small intestine" it is meant that
at least a portion of the inflamed tissues in the diseased subject
are found in the small intestine.
[0166] IBDs that can be manifested in the small intestine include,
but are not limited to, Crohn's disease and ulcerative colitis.
[0167] In some embodiments, the IBD is Crohn's disease. It is to be
noted that while IBDs such as, for example, colitis, typically
involve inflammation in the large intestine (colon) and/or the
ileum, Crohn's disease often involves inflammation in certain
regions of the small intestine. Targeting the small intestine is a
difficult task to achieve, as described in detail hereinabove and
to date, none of the currently available IBD drug therapies can
target the small intestine.
[0168] In some embodiments, the IBD is associated with
overexpression of PLA.sub.2 at an inflamed tissue of the GI
tract.
[0169] In some embodiments, the inflamed tissue is in the small
intestine.
[0170] It is to be noted that since, in some embodiments, the
PL-drug conjugates disclosed herein are designed to release the
drug at an inflamed tissue in the GI tract which is associated with
overexpression of PLA.sub.2, the conjugate of choice can comprise
any IBD drug that is suitable for treating the inflammation at this
inflamed tissue.
[0171] As discussed hereinabove, the conjugates described herein
are characterized by an improved therapeutic index, at least
compared with the IBD drug when used in a non-conjugated form.
[0172] As used herein and known in the art, a "therapeutic index"
describes the ratio between the toxic dose of a drug and the
therapeutic dose of the drug. This ratio is often defined as
LD.sub.50:ED.sub.50. A desirable therapeutically active agent would
have an ED.sub.50 value much higher that the LD.sub.50 value.
[0173] The "sink" effect and other effects exhibited by the
disclosed conjugates, as discussed supra provide for an improved
efficacy and a reduced toxicity (reduced adverse side effected) of
the drug.
[0174] In some embodiments, the conjugates described herein are
characterized by a therapeutic index that is significantly
improved, e.g., improved by at least 10%, or by at least 50%, or
even by 100%, as compared to the IBD drug product in which the drug
is utilized as a non-conjugated form.
[0175] In any of the aspects described herein in the context of
treatment of an IBD, the conjugates are utilized in an amount
effective to achieve the intended purpose, namely, in a
therapeutically effective amount. A therapeutically effective
amount means an amount of compounds presented herein effective to
prevent, alleviate or ameliorate symptoms of the disorder, or
prolong the survival of the subject being treated.
[0176] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0177] For any of the conjugates as presented herein, the
therapeutically effective amount or dose can be estimated initially
from activity assays in animals. Such information can be used to
more accurately determine useful doses in humans.
[0178] Toxicity and therapeutic efficacy of the compounds presented
herein can be determined by standard pharmaceutical procedures in
experimental animals, e.g., by determining the EC.sub.50 (the
concentration of a compound where 50% of its maximal effect is
observed) and the LD.sub.50 (lethal dose causing death in 50% of
the tested animals) for a subject compound. The data obtained from
these activity assays and animal studies can be used in formulating
a range of dosage for use in human.
[0179] Dosage amount and interval may be adjusted individually to
provide plasma levels of the compounds presented herein which are
sufficient to maintain the desired effects, termed the minimal
effective concentration (MEC). The MEC will vary for each
preparation, but can be estimated from activity data; e.g., the
concentration of the conjugates necessary to achieve 50-90%
remission of an inflamed tissue. Dosages necessary to achieve the
MEC will depend on individual characteristics and route of
administration. HPLC assays or bioassays can be used to determine
plasma concentrations.
[0180] Dosage intervals can also be determined using the MEC value.
Preparations should be administered using a regimen, which
maintains plasma levels above the MEC for 10-90% of the time,
preferable between 30-90% and most preferably 50-90%.
[0181] Depending on the severity and responsiveness of the
condition to be treated, dosing can also be a single or chronic
periodic administration, with course of periodic treatment lasting
from several days to several weeks or until sufficient amelioration
is effected during the periodic treatment or substantial diminution
of the disorder state is achieved for the periodic treatment.
[0182] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0183] In some embodiments, the "sink" effect and other effects
exhibited by the disclosed conjugates, as discussed supra, enables
to use lower daily doses of the conjugate, as compared to a
non-conjugated IBD drug product.
[0184] In some embodiments, a therapeutically effective amount of
the conjugate described herein is lower by at least 10%, at least
20%, at least 30% at least 40% and even by 50%, compared to a
corresponding IBD in a non-conjugates form.
[0185] A therapeutically effective amount of the conjugate
described herein can range from 0.1 to 100 mg/kg body/day,
including any value within this range. As noted hereinabove, the
conjugates described herein are advantageously utilized as oral
formulations.
[0186] For oral administration, the conjugate or a pharmaceutical
composition or a medicament containing same can be formulated
readily by combining the conjugate with pharmaceutically acceptable
carriers well known in the art. Such carriers enable the conjugate
to be formulated as tablets, pills, dragees, capsules, liquids,
gels, syrups, slurries, suspensions, and the like, for oral
ingestion by a patient.
[0187] Hereinafter, the term "pharmaceutically acceptable carrier"
refers to a carrier or a diluent that does not cause significant
irritation to an organism and does not abrogate the biological
activity and properties of the administered compound. Examples,
without limitations, of carriers are: propylene glycol, saline,
emulsions and mixtures of organic solvents with water, as well as
solid (e.g., powdered) and gaseous carriers.
[0188] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of a compound. Examples, without limitation, of
excipients include calcium carbonate, calcium phosphate, various
sugars and types of starch, cellulose derivatives, gelatin,
vegetable oils and polyethylene glycols.
[0189] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences" Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0190] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0191] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more pharmaceutically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
compounds presented herein into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0192] Pharmacological preparations for oral use can be made using
a solid excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0193] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0194] Pharmaceutical compositions which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0195] In some embodiments, the conjugate or the composition or
medicament containing same are formulated as a liquid formulation
for oral administration.
[0196] The phrase "liquid formulation for oral administration" is
also referred to herein interchangeably as "an oral liquid
formulation" or as an "an oral liquid dosage form" and describes a
formulation of the conjugate as described herein which is in a
liquid form and which can be administered by swallowing the
liquid.
[0197] By "liquid form" it is meant that a substantial portion of
the formulation is liquid. This expression encompasses a solution,
in which the conjugate is dissolved or solubilized, a dispersion or
suspension of small particles of the conjugate within a liquid
solution or an emulsion.
[0198] Exemplary liquid dosage forms include solutions, syrups,
liquids, slurries, suspensions, emulsions and the like.
[0199] In some embodiments, a liquid dosage form comprises an
aqueous carrier.
[0200] Aqueous carrier for e.g., solutions or, may contain
substances, which increase the viscosity of the solution or
suspension, such as sodium carboxymethyl cellulose, sorbitol or
dextran. Optionally, the suspension may also contain suitable
stabilizers or agents, which increase the solubility of the
conjugates, to allow for the preparation of highly concentrated
solutions.
[0201] In some embodiments, a liquid dosage form comprises an oily,
lipophilic carrier vehicle.
[0202] Exemplary lipophilic solvents or vehicles include fatty oils
such as sesame oil, or synthetic fatty acids esters such as ethyl
oleate, triglycerides or liposomes.
[0203] In some embodiments, a liquid dosage form comprises a
flavoring agent.
[0204] In some embodiments, a liquid dosage form comprises one or
more of a carrier, a co-solvent, a surfactant, a cyclodextrin,
and/or a self-emulsifying drug delivery system, including
nanosystems.
[0205] According to an aspect of some embodiments of the present
invention, there is provided an oral liquid dosage form which
comprises any of the conjugates described herein.
[0206] In some embodiments, there is provided an oral liquid dosage
form which comprises a conjugate as described herein, in which the
IBD drug comprises 5-ASA, as described herein.
[0207] Any of the compositions and formulations described herein
may, if desired, be presented in a pack or dispenser device, such
as an FDA (the U.S. Food and Drug Administration) approved kit,
which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as, but not limited to a blister pack or a
pressurized container (for inhalation). The pack or dispenser
device may be accompanied by instructions for administration. The
pack or dispenser may also be accompanied by a notice associated
with the container in a form prescribed by a governmental agency
regulating the manufacture, use or sale of pharmaceuticals, which
notice is reflective of approval by the agency of the form of the
compositions for human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a compound according to the present
embodiments, formulated in a compatible pharmaceutical carrier may
also be prepared, placed in an appropriate container, and labeled
for treatment of an IBD, as is detailed hereinabove.
[0208] It is expected that during the life of a patent maturing
from this application many relevant drugs for treating IBD will be
developed and the scope of the term "a drug suitable for use in the
treatment of IBD" is intended to include all such new drugs a
priori.
[0209] As used herein the term "about" refers to .+-.10%.
[0210] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0211] The term "consisting of" means "including and limited
to".
[0212] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0213] The word "exemplary" is used herein to mean "serving as an
example, instance or illustration". Any embodiment described as
"exemplary" is not necessarily to be construed as preferred or
advantageous over other embodiments and/or to exclude the
incorporation of features from other embodiments.
[0214] The word "optionally" is used herein to mean "is provided in
some embodiments and not provided in other embodiments". Any
particular embodiment of the invention may include a plurality of
"optional" features unless such features conflict.
[0215] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0216] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0217] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0218] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0219] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition or substantially preventing the appearance
of clinical or aesthetical symptoms of a condition.
[0220] As used herein throughout, the term "alkyl" refers to a
saturated aliphatic hydrocarbon including straight chain and
branched chain groups. Preferably, the alkyl group has 1 to 20
carbon atoms. Whenever a numerical range; e.g., "1-20", is stated
herein, it implies that the group, in this case the alkyl group,
may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up
to and including 20 carbon atoms. More preferably, the alkyl is a
medium size alkyl having 1 to 10 carbon atoms. The alkyl group may
be unsubstituted or substituted, as long as the substituent does
not interfere with the performance and/or intended use of the
compound. When substituted, the substituent group can be, for
example, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl,
heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,
thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide,
phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea,
thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,
C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as
these terms are defined herein.
[0221] A "cycloalkyl" group refers to an all-carbon monocyclic or
fused ring (i.e., rings which share an adjacent pair of carbon
atoms) group wherein one of more of the rings does not have a
completely conjugated pi-electron system. Examples, without
limitation, of cycloalkyl groups are cyclopropane, cyclobutane,
cyclopentane, cyclopentene, cyclohexane, cyclohexadiene,
cycloheptane, cycloheptatriene, and adamantane. A cycloalkyl group
may be unsubstituted or substituted, as long as the substituent
does not interfere with the performance and/or intended use of the
compound. When substituted, the substituent group can be, for
example, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,
thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide,
phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea,
thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,
C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as
these terms are defined herein.
[0222] An "alkenyl" group refers to an alkyl group which consists
of at least two carbon atoms and at least one carbon-carbon double
bond.
[0223] An "alkynyl" group refers to an alkyl group which consists
of at least two carbon atoms and at least one carbon-carbon triple
bond.
[0224] An "aryl" group refers to an all-carbon monocyclic or
fused-ring polycyclic (i.e., rings which share adjacent pairs of
carbon atoms) groups having a completely conjugated pi-electron
system. Examples, without limitation, of aryl groups are phenyl,
naphthalenyl and anthracenyl. The aryl group may be unsubstituted
or substituted, as long as the substituent does not interfere with
the performance and/or intended use of the compound. When
substituted, the substituent group can be, for example, alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic,
halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,
thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl,
phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,
N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as these
terms are defined herein.
[0225] A "heteroaryl" group refers to a monocyclic or fused ring
(i.e., rings which share an adjacent pair of atoms) group having in
the ring(s) one or more atoms, such as, for example, nitrogen,
oxygen and sulfur and, in addition, having a completely conjugated
pi-electron system. Examples, without limitation, of heteroaryl
groups include pyrrole, furane, thiophene, imidazole, oxazole,
thiazole, pyrazole, pyridine, pyrimidine, indole, indolenine,
quinoline, isoquinoline and purine. The heteroaryl group may be
unsubstituted or substituted, as long as the substituent does not
interfere with the performance and/or intended use of the compound.
When substituted, the substituent group can be, for example, alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic,
halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,
thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl,
phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,
N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as these
terms are defined herein.
[0226] A "heteroalicyclic" group refers to a monocyclic or fused
ring group having in the ring(s) one or more atoms such as
nitrogen, oxygen and sulfur. The rings may also have one or more
double bonds. However, the rings do not have a completely
conjugated pi-electron system. The heteroalicyclic may be
unsubstituted or substituted, as long as the substituent does not
interfere with the performance and/or intended use of the compound.
When substituted, the substituted group can be, for example, lone
pair electrons, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,
thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano,
nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl,
urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl,
N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,
sulfonamido, and amino, as these terms are defined herein.
Representative examples are piperidine, piperazine,
tetrahydrofuran, tetrahydropyran, morpholine and the like.
[0227] A "hydroxy" group refers to an --OH group.
[0228] An "azide" group refers to a --N.dbd.N.sup.+.dbd.N.sup.-
group.
[0229] An "alkoxy" group refers to both an --O-alkyl and an
--O-cycloalkyl group, as defined herein.
[0230] An "aryloxy" group refers to both an --O-aryl and an
--O-heteroaryl group, as defined herein.
[0231] A "thiohydroxy" or "thiol" group refers to a --SH group.
[0232] A "thioalkoxy" group refers to both an --S-alkyl group, and
an --S-cycloalkyl group, as defined herein.
[0233] A "thioaryloxy" group refers to both an --S-aryl and an
--S-heteroaryl group, as defined herein.
[0234] A "carbonyl" group refers to a --C(.dbd.O)--R' group, where
R' is defined as hereinabove.
[0235] A "thiocarbonyl" group refers to a --C(.dbd.S)--R' group,
where R' is as defined herein.
[0236] A "C-carboxy" group refers to a --C(.dbd.O)--O--R' groups,
where R' is as defined herein.
[0237] An "O-carboxy" group refers to an R'C(.dbd.O)--O-- group,
where R' is as defined herein.
[0238] An "oxo" group refers to a .dbd.O group.
[0239] A "carboxylate" or "carboxyl" encompasses both C-carboxy and
O-carboxy groups, as defined herein.
[0240] A "carboxylic acid" group refers to a C-carboxy group in
which R' is hydrogen.
[0241] A "thiocarboxy" or "thiocarboxylate" group refers to both
--C(.dbd.S)--O--R' and --O--C(.dbd.S)R' groups.
[0242] An ester bond refers to a --O--C(.dbd.O)-- bond.
[0243] A "halo" group refers to fluorine, chlorine, bromine or
iodine.
[0244] A "sulfinyl" group refers to an --S(.dbd.O)--R' group, where
R' is as defined herein.
[0245] A "sulfonyl" group refers to an --S(.dbd.O).sub.2--R' group,
where R' is as defined herein.
[0246] A "sulfonate" group refers to an --S(.dbd.O).sub.2--O--R'
group, where R' is as defined herein.
[0247] A "sulfate" group refers to an --O--S(.dbd.O).sub.2--O--R'
group, where R' is as defined as herein.
[0248] A "sulfonamide" or "sulfonamido" group encompasses both
S-sulfonamido and N-sulfonamido groups, as defined herein.
[0249] An "S-sulfonamido" group refers to a
--S(.dbd.O).sub.2--NR'R'' group, with each of R' and R'' as defined
herein.
[0250] An "N-sulfonamido" group refers to an
R'S(.dbd.O).sub.2--NR'' group, where each of R' and R'' is as
defined herein.
[0251] An "O-carbamyl" group refers to an --OC(.dbd.O)--NR'R''
group, where each of R' and R'' is as defined herein.
[0252] An "N-carbamyl" group refers to an R'OC(.dbd.O)--NR''--
group, where each of R' and R'' is as defined herein.
[0253] A "carbamyl" or "carbamate" group encompasses O-carbamyl and
N-carbamyl groups.
[0254] A carbamate bond describes a --O--C(.dbd.O)--NR'-- bond,
where R' is as described herein.
[0255] An "O-thiocarbamyl" group refers to an --OC(.dbd.S)--NR'R''
group, where each of R' and R'' is as defined herein.
[0256] An "N-thiocarbamyl" group refers to an R'OC(.dbd.S)NR''--
group, where each of R' and R'' is as defined herein.
[0257] A "thiocarbamyl" or "thiocarbamate" group encompasses
O-thiocarbamyl and N-thiocarbamyl groups.
[0258] A thiocarbamate bond describes a --O--C(.dbd.S)--NR'-- bond,
where R' is as described herein.
[0259] A "C-amido" group refers to a --C(.dbd.O)--NR'R'' group,
where each of R' and R'' is as defined herein.
[0260] An "N-amido" group refers to an R'C(.dbd.O)--NR''-- group,
where each of R' and R'' is as defined herein.
[0261] An "amide" group encompasses both C-amido and N-amido
groups.
[0262] An amide bond describes a --NR'--C(.dbd.O)-- bond, where R'
is as defined herein.
[0263] A "urea" group refers to an --N(R')--C(.dbd.O)--NR''R'''
group, where each of R' and R'' is as defined herein, and R''' is
defined as R' and R'' are defined herein.
[0264] A "nitro" group refers to an --NO.sub.2 group.
[0265] A "cyano" group refers to a --C.ident.N group.
[0266] The term "phosphonyl" or "phosphonate" describes a
--P(.dbd.O)(OR')(OR'') group, with R' and R'' as defined
hereinabove.
[0267] The term "phosphate" describes an --O--P(.dbd.O)(OR')(OR'')
group, with each of R' and R'' as defined hereinabove.
[0268] A "phosphoric acid" is a phosphate group is which each of R
is hydrogen.
[0269] The term "phosphinyl" describes a --PR'R'' group, with each
of R' and R'' as defined hereinabove.
[0270] The term "thiourea" describes a --N(R')--C(.dbd.S)--NR''--
group, with each of R' and R'' as defined hereinabove.
[0271] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0272] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0273] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
Example 1
Chemical Syntheses
[0274] General Procedure:
[0275] The PL-drug conjugates disclosed herein are prepared
according to published procedures (see, for example, Dvir et al.,
2008 and Kurz and Scriba, 2000).
[0276] In an exemplary general procedure, a lysophospholipid, the
(IBD) drug of interest and, optionally, a suitable starting
material for providing the linker of interest (the bridging
moiety), are used as starting materials.
[0277] The term "lysophospholipid" as used herein encompasses a
derivative of phosphatidic acid in which one of the fatty acid
chains has been removed, leaving at the corresponding position a
hydroxyl group.
[0278] The term "phosphatidic acid", as known in the art, is used
to collectively represent fatty acid derivatives of
glycerophosphates, which are composed of a glycerol backbone to
which 1 mole of phosphoric acid is attached via an ester bond at
the terminal 3-hydroxyl group (position sn-3) and 2 moles of fatty
acids are attached via an ester bond at the other two hydroxyl
groups (at positions sn-1 and sn-2). Conjugation of the fatty acids
to the glycerolic backbone results in fatty acyl moieties at
positions sn-1 and sn-2.
[0279] A "phosphatidic acid" can be represented as follows:
##STR00004##
[0280] wherein:
[0281] A.sub.1 and A.sub.2 are each independently a saturated or
unsaturated hydrocarbon chain being at least 3 carbon atoms in
length, as defined herein; and
[0282] Ra and Rb are each independently hydrogen, alkyl,
cycloalkyl, aryl, aminoalkyl, hydroxyalkyl, and the like, or are
selected such that the group --P(.dbd.O)(ORa)(ORb) represents a
phosphoryl choline, phosphoryl ethanolamine, phosphoryl serine,
phosphoryl cardiolipin, phosphoryl inositol, ethylphosphocholine,
phosphorylmethanol, phosphorylethanol, phosphorylpropanol,
phosphorylbutanol, phosphorylethanolamine-N-lactose,
phosphoethanolamine-N-[methoxy(propylene glycol)],
phosphoinositol-4-phosphate, phosphoinositol-4,5-biposphonate,
pyrophosphate, phosphoethanolamine-diethylenetriamine-pentaacetate,
dinitrophenyl-phosphoethanolamine and phosphoglycerol.
[0283] Phosphatidic acids that comprise some of the above-indicated
phosphoryl groups can be referred to, for example, as phosphatidyl
choline, phosphatidyl ethanolamine, phosphatidyl serine,
phosphatidyl cardiolipin, phosphatidyl inositol, phosphatidyl ethyl
phosphocholine, phosphatidyl methanol, phosphatidyl ethanol,
phosphatidyl propanol, phosphatidyl butanol, phosphatidyl
ethanolamine-N-lactose, phosphatidyl
ethanolamine-N-[methoxy(propylene glycol)], phosphatidyl
inositol-4-phosphate, phosphatidyl inositol-4,5-biposphonate,
phosphatydil ethanolamine-diethylenetriamine-pentaacetate, and
dinitrophenyl-phosphatidyl ethanolamine.
[0284] Exemplary lysophospholipids include derivatives of any of
the above-described phosphatidic acids, in which the ester bond at
position sn-1 or sn-2 has been cleaved into a free fatty acid and a
hydroxy group at the respective position of the glycerol
backbone.
[0285] Lysophospholipids having a hydroxyl group at position sn-1
can be represented by:
##STR00005##
[0286] wherein A.sub.2, Ra and Rb are as defined herein.
[0287] Lysophospholipids having a hydroxyl group at position sn-2
can be represented by:
##STR00006##
[0288] wherein A.sub.1, Ra and Rb are as defined herein.
[0289] Lysophospholipids having a hydroxyl group at position sn-2,
as described herein, are preferred. Such lysophopholipids are also
referred to as 1-acyl-2-lyso-phosphoglycerol. An exemplary
lysophospholipid is lysolecithin (derived from phosphatidyl
choline).
[0290] The drug of interest includes any of the drugs suitable for
the treatment of an IBD, as described herein, including, for
example, 5-ASA, tacrolimus and methotrexate.
[0291] The linker of interest can be any of the bridging moieties
(or linking moieties) as described herein.
[0292] A suitable starting material (precursor) for providing the
linker of interest is preferably a bi-functional compound having at
one end a first reactive group for forming a bond with the hydroxyl
group of the lysophospholipid, and at another end a second reactive
group for forming a bond with the drug of interest.
[0293] As used herein, the phrase "reactive group", which refers to
both the first and the second reactive groups, describes a chemical
moiety that is capable of undergoing a chemical reaction that
typically leads to a bond formation. The bond, according to some
embodiments of the present invention, is preferably a covalent
bond. Chemical reactions that lead to a bond formation include, for
example, nucleophilic and electrophilic substitutions, nucleophilic
and electrophilic addition reactions, cycloaddition reactions,
rearrangement reactions and any other known organic reactions that
involve a reactive group.
[0294] Representative examples of suitable reactive groups
according to some embodiments of the present invention include,
without limitation, amine, halide, acyl-halide, sulfonate,
sulfoxides, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,
thioaryloxy, cyano, nitro, azo, isocyanate, sulfonamide,
carboxylate, thiocarbamate, urea, thiourea, carbamate, amide,
guanyl, guanidine and hydrazine, as these terms are defined
hereinabove.
[0295] In some embodiments, the first and/or second reactive
group(s) include, but are not limited to, halide, amine,
carboxylate, thiol, alkoxy, aryloxy, thioalkoxy, thioaryloxy,
acyl-halide, and anhydride.
[0296] In some embodiments, the first reactive group is selected so
as to form an ester bond with the hydroxyl group of the
lysophospholipid. Such an ester bond is cleavable by PLA.sub.2.
Suitable first reactive groups therefore include, but are not
limited to, carboxylates, acyl halides and anhydrides.
[0297] In some embodiments, the second reactive group is selected
so as to form a bond with the drug of interest and hence is
selected so as to form a bond with a functional group of the drug.
In some embodiments, the bond is stable (non-cleavable) under
physiological conditions. Exemplary bonds include, without
limitation, ester bond, amide bond and thioester bond. Thus, for
example, in cases where the drug of interest has a functional group
such as amine, thiol or hydroxyl, the second reactive group can be
a carboxylate, an anhydride or an acyl halide. In cases where the
drug of interest has a functional group such as a carboxylate, the
second reactive group can be amine, thiol or hydroxyl. Any
"couples" of a second reactive group of the precursor of the
bridging moiety and a functional group on the drug of interest,
which are compatible one with another by means of forming a bond
therebetween, are contemplated.
[0298] In an exemplary procedure, where the drug is conjugated
directly to the lysophospholipid, the drug of interest preferably
comprises a carboxylate (e.g., a carboxylic acid) or,
alternatively, is modified so as to comprise a carboxylate. The
carboxylate can further be modified to a more reactive group, such
as an acyl halide. In some embodiments, prior to being reacted with
the lysophospholipid, the carboxylate group on the drug is
converted to an acyl chloride or an anhydride, so as to facilitate
the reaction. In some embodiments, the reaction is performed in the
presence of a coupling agent suitable for esterification (e.g.,
DCC).
[0299] In another exemplary procedure, where the drug is conjugated
to the lysophospholipid via a liker, a bifunctional compound
selected for providing the linker of interest is reacted with a
suitable protecting group, so as to have the second reactive group
protected, and the protected compound is reacted with the
lysophospholipid. In some embodiment, the protected compound is
converted to an anhydride prior to the reaction. Upon the reaction,
a protected moiety of the compound is conjugated to the
lysophospholipid, and this intermediate is further reacted with the
drug by (i) removing the protecting group, so as to generate the
second reactive group; and (ii) reacting with the drug of interest
so as to form the desired bond, as described hereinabove.
[0300] In any of the above-described general procedures, the
chemical structure of obtained product is verified using common
analytical procedures (e.g., NMR measurements, such .sup.1H,
.sup.13C and .sup.31P NMR spectra, elemental analysis, IR
spectroscopy, mass spectrometry and/or UV spectrometry. The purity
of the product is determined by high-performance liquid
chromatography and/or thin layer chromatography.
[0301] If required, purification of the obtained product is
performed by common procedures such as, for example, column
chromatography, recrystallization, and the like.
[0302] Preparation of a Conjugate of 5-ASA and Lysolecithin (Having
a 5-Carbon Linker):
[0303] A conjugate of 5-ASA and lysolecithin, having the following
structure:
##STR00007##
2-(5-(5-amino-2-hydroxybenzamido)pentanoyloxy)-3-(palmitoyloxy)propyl
2-(trimethylammonio)ethyl phosphate
[0304] was prepared according to Scheme 1 below.
##STR00008##
[0305] Preparation of Compound 1:
[0306] To a solution of Lysophosphatidyl choline (100 mg, 0.2 mmol)
and Fmoc-N-aminohexanoic acid (83 mg, 0.24 mmol) in
chloroform/dichloromethane 1:4 v/v (5 ml) were added DMAP (5 mg,
0.041 mmol) and DCC (37.08 mg, 0.18 mmol). The mixture was stirred
at room temperature for 2 hours. The extract was successively
washed with 0.1N HCl, saturated NaHCO.sub.3 and water and the
solvents were thereafter removed under reduced pressure.
[0307] Preparation of Compound 2:
[0308] The Fmoc group was removed using 25% piperidine in DMF (6
minutes). Compound 2 was obtained after crystallization with
methanol:water (4:1 v/v) and recrystallization with acetone.
[0309] Preparation of Compound 3:
[0310] Compound 3 was prepared by mixing Compound 2 (200 mg, 1.52
mmol) and 5-ASA (302.59 mg, 1.97 mmol) with DCC (344.98 mg, 1.67
mmol) in THF at 0.degree. C. and stirring the obtained mixture for
1 hour at 0.degree. C., following by stirring at room temperature
for 2 hours. The obtained DCU precipitate was removed at the end of
the procedure using methanol:water (4:1 v/v).
[0311] The product structure is verified as described
hereinabove.
[0312] Preparation of a Series of PL-5-ASA Conjugates:
[0313] Following the above-described procedures, a series of
conjugates of 5-ASA and various lysophopholipids is prepared. For
each lysophopsholipid utilized, the 5-ASA is attached in the
absence of a linker and in presence of various linkers, as
described herein.
[0314] The disclosed series of PL-5-ASA conjugates is then utilized
in various activity studies, for identifying the most potent
conjugate for an indicated condition.
Example 2
Degradation by PLA.sub.2
[0315] PLA.sub.2 belongs to a family of enzymes that catalyze the
hydrolysis of the sn-2 fatty acyl bond of phospholipids to liberate
free fatty acid and lysophospholipid (Kudo and Murakami 2002).
[0316] Cleavage of the phospholipid drug conjugates as described
herein, in the presence of a PLA.sub.2 from is determined as
follows:
[0317] A mixture of solutions of a phospholipid-drug conjugate
(e.g., a PL-5-ASA conjugate as described herein) in ethanol,
L-.alpha.-phosphatidylcholine and di-o-hexadecyl in Chloroform:MeOH
(1:1 v/v), and of phosphatidyl choline (in Chloroform:MeOH 1:1),
are evaporated under a stream of nitrogen. Reaction buffer (e.g.,
containing 300 mM NaCl, 10 mM CaCl.sub.2, 25 mM Tris-HCl pH 7.4) is
added, and the lipid mixture is sonicated for 10 minutes in a
sonication bath, transferred to ice for a few minutes, sonicated
for additional 10 minutes, and transferred again to ice.
[0318] PLA.sub.2 is thereafter added to the tubes, and the obtained
solutions are incubated for 1.5 hours at 25.degree. C.
[0319] Two types of controls are used: reaction mixture without
PLA.sub.2; and a reaction mixture that contains PLA.sub.2 but do
not contain lipids.
Example 3
Intestinal Permeability
[0320] The intestinal permeability of the conjugates is assessed to
ensure that the intact PL-5-ASA conjugate is not absorbed. The
intestinal permeability is evaluated using transepithelial
permeability studies across Caco-2 cell monolayers, as previously
described [see, for example, Dahan et al., Am. J. Physiol.
Gastrointest. Liver Physiol. 2009].
[0321] Briefly, 5.times.10.sup.4 cells/cm.sup.2 are seeded onto
collagen-coated membranes (12-well Transwell plate, 0.4-.mu.m pore
size, 12-mm diameter; Corning Costar, Cambridge, Mass.) and are
allowed to grow for 21 days. Mannitol and Lucifer yellow
permeabilities are assayed for each batch of Caco-2 monolayers
(n=3), and transepithelial electrical resistance (TEER)
measurements are performed on all monolayers (Millicell-ERS
epithelial Voltohmmeter; Millipore, Bedford, Mass.). Monolayers
with apparent mannitol and Lucifer yellow permeability
<3.times.10.sup.7 cm/s and TEER values >300 .OMEGA.cm2 are
used for all studies. On the experiment day, the monolayers are
rinsed and incubated for 20 minutes with a blank transport buffer.
The transport buffer contains 1 mM CaCl.sub.2, 0.5 mM
MgCl.sub.2.6H2O, 145 mM NaCl, 3 mM KCl, 1 mM NaH.sub.2PO.sub.4, 5
mM D-glucose, and 5 mM MES. Similar pH is used in both apical (AP)
and basolateral (BL) sides (pH 6.5) to maintain constant degree of
ionization in both AP-BL and BL-AP direction experiments and to
avoid possible influence of this factor on the permeability across
the cells.
[0322] Following the 20-minutes incubation, the drug-free transport
buffer is removed from the donor side (AP in the AP-BL-direction
studies or BL in the BL-AP-direction studies) and replaced by a
drug uptake buffer solution (pH 6.5), with or without the tested
conjugate. Throughout the experiment, the transport plates are kept
in a shaking incubator (50 revolution/minute) at 37.degree. C.
Samples are taken from the receiver side at various time points up
to 120 minutes (100 .mu.l from BL side or 70 .mu.l from AP side),
and similar volumes of blank buffer are added following each sample
withdrawal. At the last time point (120 minutes), a sample is taken
from the donor side as well to confirm mass balance. Samples are
immediately assayed for conjugate content. All Caco-2 monolayers
are checked for confluence by measuring the TEER before and after
the transport study (TEER values remained steady throughout the
experiment).
[0323] The intestinal permeability is further evaluated by
measuring in-situ intestinal perfusions in a rat model, as
previously described [see, for example, Dahan et al., European
Journal of Pharmaceutical Sciences. 36:320-329 (2009), which is
incorporated by reference as if fully set forth herein.
[0324] Briefly, rats are anesthetized with an intra-muscular
injection of 1 ml/kg of ketamine-xylazine solution (9%:1%,
respectively) and placed on a heated surface maintained at
37.degree. C. (Harvard Apparatus Inc., Holliston, Mass.). The
abdomen is opened by amidline incision of 3-4 cm. A proximal
jejunal (13.5.+-.1.6 cm average distance of the inlet from the
pylorus) or distal ileal segment (14.7.+-.1.8 cm average distance
of the inlet from the cecum) of approximately 10 cm is carefully
exposed and cannulated on two ends with flexible PVC tubing (2.29
mm i.d., inlet tube 40 cm, outlet tube 20 cm, Fisher Scientific
Inc., Pittsburgh, Pa.). Care is taken to avoid disturbance of the
circulatory system, and the exposed segment is kept moist with
37.degree. C. normal saline solution. The perfusate is incubated in
a 37.degree. C. water bath to maintain temperature, and a perfusion
solution containing 10 m MMES buffer, pH 6.5, 135 mM NaCl, 5 mM
KCl, and 0.1 mg/ml phenol red with an osmolarity of 290 mosm/l is
pumped through the intestinal segment (Watson MarlowPumps 323S,
Watson-MarlowBredel Inc., Wilmington, Mass.). The isolated segment
is rinsed with blank perfusion buffer, pH 6.5 at a flow rate of 0.5
ml/min in order to clean out any residual debris.
[0325] At the start of the study, perfusion solution containing the
tested conjugate is perfused through the intestinal segment at a
flow rate of 0.2 ml/min. Phenol red is added to the perfusion
buffer as a nonabsorbable marker for measuring water flux.
[0326] The concentrations of the conjugates used in the perfusion
studies are determined by dividing the highest prescribed dose by
250 ml, the standard volume for a glass of water advised to be
taken with the dose and hence the accepted minimal gastric volume,
in order to represent the maximal drug concentration present in the
intestinal segment, and were within their intrinsic solubility
reported at pH 6.5 (Avdeef and Berger, 2001; Avdeef et al., 2000).
The perfusion buffer is first perfused for 1 hour, in order to
assure steady state conditions (as also assessed by the inlet over
outlet concentration ratio of phenol red which approaches 1 at
steady state). Following reaching to steady state, samples are
taken in 10 minutes intervals for 1 hour (10, 20, 30, 40, 50, and
60 min). All samples including perfusion samples at different time
points, original drug solution, and inlet solution taken at the
exit of the syringe are immediately assayed by HPLC. Following the
termination of the experiment, the length of the perfused
intestinal segment is accurately measured.
Example 4
In Vivo Studies
[0327] PL-5-ASA conjugates which exhibit high activation by
PLA.sub.2 and low intestinal permeability as an intact conjugate
are selected for in vivo studies.
[0328] In vivo studies are performed in IBD (UC and CD) models in
rats.
[0329] Any available IBD rat model can be used. In an exemplary
model, mice are lightly anesthetized with halothane, and a 3.5F
catheter is inserted intrarectally 4 cm from the anus. To induce
colitis, 100 .mu.L of 3 mg TNBS (Sigma Chemical Co, St. Louis, Mo.)
in 50% ethanol (to break the intestinal epithelial barrier) are
slowly administered into the lumen via the catheter filled to a
1-mL syringe. Control mice receive 50% ethanol alone (100
.mu.L).
[0330] Quantification of PLA.sub.2 is performed by Western blotting
with a monoclonal antibody against PLA.sub.2, as described in, for
example, Dahan et al., Drug Metab Dispos., 2009; or otherwise by
quantification of PLA2 gene expression, protein content and
enzymatic activity, as described, for example, in Haapamaki et al.
(1999a), in Lilja et al. (1995) and/or in Minami et al. (1994), all
of which are incorporated by reference as if fully set forth
herein.
[0331] Pharmacokinetic evaluation of the conjugates activity in the
rat model is performed as described, for example, in Dahan and
Hoffman 2005, and Dahan and Hoffman 2006, which are incorporated by
reference as if fully set forth herein. Pharmacodynamic studies are
performed by the SSD rat model for IBD.
[0332] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0333] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
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