U.S. patent application number 15/277830 was filed with the patent office on 2017-02-16 for bacterial phospholipase inhibitors as modulator of colonic bacterial flora.
The applicant listed for this patent is Universitaetsklinikum Heidelberg. Invention is credited to Wolfgang STREMMEL.
Application Number | 20170043026 15/277830 |
Document ID | / |
Family ID | 50389882 |
Filed Date | 2017-02-16 |
United States Patent
Application |
20170043026 |
Kind Code |
A1 |
STREMMEL; Wolfgang |
February 16, 2017 |
BACTERIAL PHOSPHOLIPASE INHIBITORS AS MODULATOR OF COLONIC
BACTERIAL FLORA
Abstract
The present invention relates to the field of gastroenterology
and, more particular, to the field of intestinal diseases. More
specifically, it concerns uses and methods for the treatment of
inflammatory bacterial diseases of the intestine. In particular, it
relates to diseases that are associated with bacterial invasion of
the intestinal mucus, including, inflammatory bowel diseases, and
infectious bacterial diseases. Therefore, the present invention
provides agents, a pharmaceutical composition and a kit for
treating said diseases. It further relates to a use and a method
for treating invasive bacterial diseases of the large
intestine.
Inventors: |
STREMMEL; Wolfgang;
(Heidelberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Universitaetsklinikum Heidelberg |
Heidelberg |
|
DE |
|
|
Family ID: |
50389882 |
Appl. No.: |
15/277830 |
Filed: |
September 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15129814 |
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PCT/EP2015/056326 |
Mar 25, 2015 |
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15277830 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/552 20170801;
A61K 45/06 20130101; A61K 47/55 20170801; A61K 47/554 20170801;
A61P 1/00 20180101; A61K 47/544 20170801 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 45/06 20060101 A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2014 |
EP |
14162028.6 |
Claims
1.-15. (canceled)
16. A method of treatment of an inflammatory bacterial disease of
an intestine in a subject, the method comprising the step of: (a)
administering to the subject an effective amount of a
pharmaceutical composition comprising a lysophospholipid-conjugate;
wherein the lysophospholipid-conjugate inhibits a bacterial
phospholipase.
17. The method according to claim 16, wherein the bacterial
phospholipase is selected from the group consisting of
phospholipase A.sub.2, phospholipase A.sub.1, phospholipase B,
phospholipase C, and phospholipase D.
18. The method according to claim 16, wherein the inflammatory
bacterial disease of the intestine is invasive.
19. The method according to claim 16, wherein the intestine is the
large intestine.
20. The method according to claim 16, wherein the subject is a
mammal.
21. The method according to claim 16, wherein the disease is
selected from the group consisting of appendicitis,
pseudoappendicits, ulcerative colitis, Crohn's disease,
enterohemorrhagic colitis, pseudomembranous colitis, collagenous
colitis, lymphocytic colitis, ischaemic colitis, diversion colitis,
microscopic colitis, Behcet's disease, indeterminate colitis,
diverticulitis, megacolon, toxic megacolon, and enterocolitis.
22. The method according to claim 16, wherein the
lysophospholipid-conjugate comprises a lysophospholipid chemically
coupled to a bile acid.
23. The method according to claim 22, wherein the bile acid is
ursodeoxycholate or deoxycholate.
24. The method according to claim 16, wherein the
lysophospholipid-conjugate comprises lysophosphatidylethanolamine
or lysophosphatidylcholine.
25. The method according to claim 16, wherein the method further
comprises the step of: (b) administering to the subject one or more
agents selected from the group consisting of an antibiotic, an
anti-inflammatory agent, an immunosuppressive agent, and an
anti-diarrheal agent.
26. The method according to claim 25, wherein the one or more
agents are administered prior to, simultaneously, or after step
(a).
27. The method according to claim 16, wherein the pharmaceutical
composition further comprises a pharmaceutically acceptable
carrier, diluent or excipient.
28. The method according to claim 25, wherein the
lysophospholipid-conjugate and the one or more agents are
administered as combination therapy.
29. A method of manufacturing a pharmaceutical composition for the
treatment of an inflammatory bacterial disease of an intestine in a
subject, the method comprising the step of: (a) combing a
lysophospholipid-conjugate and a pharmaceutically acceptable
carrier, diluent or excipient; wherein the
lysophospholipid-conjugate inhibits a bacterial phospholipase.
30. A pharmaceutical composition for the treatment of an
inflammatory bacterial disease of an intestine in a subject, the
pharmaceutical composition comprising: (i) a
lysophospholipid-conjugate; and (ii) a pharmaceutically acceptable
carrier, diluent or excipient; wherein the
lysophospholipid-conjugate inhibits a bacterial phospholipase.
31. The pharmaceutical composition according to claim 30, wherein
the lysophospholipid-conjugate is chemically coupled to a bile
acid.
32. The pharmaceutical composition according to claim 31, wherein
the bile acid is ursodeoxycholate or deoxycholate.
33. The pharmaceutical composition according to claim 30, wherein
the lysophospholipid-conjugate comprises
lysophosphatidylethanolamine or lysophosphatidylcholine.
34. The pharmaceutical composition according to claim 30, wherein
the pharmaceutical composition further comprises: (iii) one or more
agents selected from the group consisting of an antibiotic, an
anti-inflammatory agent, an immunosuppressive agent, and an
anti-diarrheal agent.
35. The pharmaceutical composition according to claim 30, wherein
the bacterial phospholipase is selected from the group consisting
of phospholipase A.sub.2, phospholipase A.sub.1, phospholipase B,
phospholipase C, and phospholipase D.
Description
BACKGROUND
[0001] The intestine is a vital part of the gastrointestinal tract
(GIT) that primarily functions to absorb nutrients and water from
ingested food and drinks. In consequence, it is constantly exposed
to digestive juices, containing enzymes and other agents that act
to break down nutrients, dietary antigens and potentially
pathogenic microorganisms from the external environment. In
addition, the intestine houses the gut flora, myriads of
microorganisms from about 500 different species, including
bacteria, archaea and eukaryotes.
[0002] The mucosa as the innermost layer of the GIT is the
interface that is exposed to and interacts with the external
environment and the luminal content of the intestine. It consists
of epithelial cells forming crypts and villi, subepithelial tissue
and lymph nodes (lamina propria), and underneath a continuous sheet
of smooth muscle cells (muscularis mucosae). The entire mucosa
rests on the submucosa, which consists of a variety of inflammatory
cells, lymphatics, autonomic nerve fibers, and ganglion cells. The
mucus layer lining the mucosal epithelial cells is the first
defensive barrier that protects the underlying mucosa from the
entrance of harmful substances or pathogens. Mucus is a complex
viscous composition, which typically forms layers surrounding the
intestinal lumen. It consists of large glycoproteins, called
mucins, which are secreted by specialized epithelial cells and
serve as a scaffold for the mucus gel, further containing salts,
lipids and proteins (Johansson, et al., 2011) (Turner, 2009). The
mucus layer converts the hydrophilic epithelial surface into a
hydrophobic "closing seal" that interfaces with luminal contents
(Hicks, et al., 2006) (Hills, 2002). Phosphatidylcholines (PC) bind
to the negatively charged mucins with their positively charged
headgroups while their hydrophobic acyl chains extend to the lumen,
thus establishing a high surface tension which normally helps to
exclude bacteria from this compartment (Willumeit, et al., 2007)
(Guo, et al., 1993).
[0003] However, under certain circumstances, bacteria somehow
overcome the mucosal barrier of the intestine and elicit intestinal
inflammation. Inflammatory bacterial diarrhea is a significant
health problem in both developing and developed regions of the
world that particularly affects children, elderly persons, and
immunosuppressed individuals. The standard treatment is mostly
restricted to antibiotics. However, antibiotic therapy is often
leveraged by bacterial antibiotic resistances. In addition,
antibiotics often elicit severe side effects and can unbalance the
gut flora, which may lead to follow-up infections caused by other
pathogenic microorganisms.
[0004] Intestinal inflammation is also a characteristic of
inflammatory bowel diseases (IBD). These chronic, relapsing
diseases have been linked to a dysregulated immune response to
components of the gut flora. IBD have therefore long been
classified as "autoimmune" diseases and are typically treated with
anti-inflammatory agents, e.g., corticosteroids,
immunosuppressives, antibiotics or even surgical approaches in
those who are non-responders to medical treatment. The
disadvantages of antibiotic treatment have already been elucidated,
and many anti-inflammatory and immunosuppressive agents, too, evoke
severe side effects. Surgery should, of course, be the last resort
for treatment of IBD (Triantafillidis, et al., 2011).
[0005] The exact mechanisms whereby bacteria disrupt and enter the
intestinal mucus and the underlying mucosa are still the subject of
ongoing research. Despite the differences regarding disease
etiology and pathogenesis, the present inventors have identified
one common factor shared by many inflammatory intestinal diseases:
Bacteria or bacterial antigens cross the natural mucosal barrier
and reach the underlying mucosa, where an inflammatory response
arises. This understanding lead to the idea to implement treatment
at the beginning, i.e. by preventing bacterial invasion of the
mucus barrier. And, for the first time, it is herein suggested to
do so by inhibiting bacterial phospholipase activity.
[0006] Phospholipases (PL) are abundant throughout the prokaryotic
and eukaryotic kingdom and constitute a heterogenous group of
diverse lipolytic enzymes that share the ability to hydrolyze one
or more ester linkages in phospholipids. PL are typically
classified based on their site of action; whether they cleave in
the hydrophobic diacylglycerol moiety (PLA) or in the polar head
group of the amphipathic phospholipid (PLC and PLD). PLAs can be
further defined by their positional specificity, i.e. preference
for the acyl group attached to position 1 or 2 of the glycerol
backbone, as PLA1 and PLA.sub.2, respectively; PLBs have both PLA1
and PLA.sub.2 activity, i.e., little or no positional specificity
(Istivan & Coloe, 2006).
[0007] Relatively few studies have elucidated the role of bacterial
PL, in particular bacterial PLA.sub.2, in host-pathogen
interactions. It has been acknowledged that the action of bacterial
PLA.sub.2 results in the accumulation of free fatty acids and
lysophospholipids, which are known to destabilize (host) membranes.
Few data suggesting a role in pathogenesis for bacterial PLA,
including PLA from Vibrio parahaemolytics, Ricksettia prowazekil
and Campylobacter coli, have been linked to hemolytic activity due
to the accumulation of lysophospholipids (Schmiel & Miller,
1999) (Istivan & Coloe, 2006). It has further been speculated
that bacterial PLA might promote bacterial survival and growth by
disrupting innate immune cells, thereby hampering the host's immune
defenses, and by providing nutrients in the form of fatty acids for
biosynthesis or metabolism. Another option raised was that Yersinia
enterocolitica PLA may stimulate the pro-inflammatory arachnidonic
acid cascade by releasing fatty acids, including arachidonic acid,
from the glycerol backbone of phospholipids (Schmiel & Miller,
1999). Other studies gave a contradictory picture for bacterial
PLA. For example, the injection of Salmonella newport into ligated
ileal loops induced similar levels of fluid accumulation,
desquamation, and mononuclear cell infiltration as the injection of
bacteria. In contrast, a PLA mutant strain of Vibrio cholerae was
reported to induce similar amounts of fluid accumulation in rabbit
ligated ileal loops compared to the parent strain. (for review, see
(Schmiel & Miller, 1999) (Istivan & Coloe, 2006)).
[0008] Even though antibiotics, anti-inflammatory,
immunosuppressive and anti-diarrheal agents are available for the
treatment of inflammatory bacterial diseases of the intestine,
there is still a need for alternative or additional drugs for
combating such diseases, because of bacterial resistances to
antibiotics, the potential damage of the host's gut flora and the
adverse effects.
[0009] The technical problem underlying the present invention is to
comply with this need. The solution is set out in the claims and
the description in aspects and embodiments of the present invention
that follow as well as illustrated by the figures and exemplified
in the appended examples. To date no therapeutics targeting the
detrimental effects of bacterial PLA in diseases affecting the
intestine have been developed; as their role of has largely been
ignored.
[0010] The present inventors, for the first time, present an
approach to treat various inflammatory bacterial diseases of the
intestine by inhibiting bacterial phospholipases (PL), in
particular bacterial PLA.sub.2. Of note, therapeutic approaches
targeting PLA in inflammatory diseases, and i.a. inflammatory bowel
diseases, have focused on inhibiting endogenous host
PLA--presumably because host PLA are implicated in a variety of
signaling pathways and mechanisms; many of which are of relevance
in the pathogenesis of inflammatory diseases (see (Linkous &
Yazlovitskaya, 2010), (Murakami, et al., 2011)). For example, one
function of host soluble PLA.sub.2 (sPLA.sub.2) is antimicrobial
defense through degradation of bacterial membrane phospholipids
(see (Murakami, et al., 2011) for review). An increased host
cytoplasmic PLA.sub.2 (cPLA.sub.2) activity has also been reported
in tissues infected with Mycobacterium tuberculosis, Pseudomonas
aeruginosa, Listeria monocytogenes and Helicobacter pylori (see
(Linkous & Yazlovitskaya, 2010) for review).
[0011] Accordingly, several host PLA.sub.2 inhibitors have been
developed to inhibit or decrease host PLA.sub.2 activity. For
example, WO9944604 provides an inhibitor of human non-pancreatic
sPLA.sub.2 for the treatment of inflammatory diseases, including
inflammatory bowel diseases. The approach however ignores the role
of gut bacteria implicated in IBD, and provides an
anti-inflammatory agent to disrupt the host's immune overreaction
to the gut flora, rather than targeting harmful effects of the gut
flora itself. Similar approaches have been adopted by, e.g.,
US2005075345, GB2346885, EP0465913, U.S. Pat. No. 6,110,933.
[0012] Interfering with host PLA.sub.2 activity, however, poses
risks. Host PLA.sub.2 are abundant and fulfill a plethora of
functions. Hence, drugs inhibiting host PLA.sub.2 activity are
predestined for eliciting systemic side effects, e.g. by impeding
membrane maintenance, signaling and immune defense mechanisms. In
fact, targeting host PLA.sub.2 may even exacerbate bacterial
intestinal diseases, as host secreted PLA.sub.2 reportedly
participate in host defense by destroying bacterial membranes.
Instead of providing just another anti-inflammatory agent that
attenuates the host's response to bacterial challenge, the present
inventors were the first to specifically target bacterial PL, in
particular bacterial PLA.sub.2 activity of harmful bacteria,
thereby avoiding an impairment of host PLA function. This clearly
renders the approach superior to state-of-the-art therapeutics for
the treatment of inflammatory bacterial diseases of the
intestine.
[0013] In contrast to any other approach revealed in the prior art,
the present inventors have developed a way to effectively target a
key event in the pathogenesis of inflammatory bacterial diseases of
the intestine: the invasion of the protective intestinal mucus
barrier. As of this writing, efforts have been put into inhibiting
host PLA to decrease inflammation and tissue destruction in a
variety of inflammatory diseases. The present inventors were the
first to understand that bacterial PL, in particular bacterial
PLA.sub.2, are crucial for the pathogenesis of a vast number of
inflammatory bacterial diseases affecting the intestine, and,
importantly, that inhibiting bacterial PL, in particular bacterial
PLA.sub.2, activity offers an elegant solution to the task of
providing a therapy that acts adversely neither on the patient, nor
on his gut flora.
[0014] In addition, the present invention provides
lysophospholipid-conjugates as inhibitors of bacterial PL, in
particular bacterial PLA.sub.2, and, consequently, as potent
therapeutics for the treatment of a vast number of inflammatory
bacterial diseases of the intestine. Clearly, the ability of
lysophospholipid-conjugates to interfere with bacterial PLA could
not be foreseen.
[0015] UDCA-LPE, being one exemplary lysophospholipid-conjugate
according to the present invention, had initially been designed for
the delivery of the phospholipid-precursor LPE to the steatotic
liver, which typically exhibits low PC/LCP levels that have been
linked to an increased PLA.sub.2 activity (Chamulitrat, et al.,
2009). The hepatoprotective effects of UDCA-LPE have been linked to
the inhibition of hepatic PLA.sub.2 which, on the one hand, results
in disintegration of the fatty acid uptake complex and, on the
other hand, suppression of the cytosolic generation of
lysophosphatidylcholine (LPC), which in turn results in
deactivation of JNK1, a common promoter of fatty acid influx,
inflammation and apoptosis (Stremmel & Staffer, 2012)
(Stremmel, et al., 2012). Of note, phospholipases are a very
diverse group of enzymes. For example, although all, eukaryotic and
prokaryotic, sPLA.sub.2s share the same catalytic mechanism, there
is considerable variation in their sequence identity and structure
(Nevalainen, et al., 2012). Hence, the finding that UDCA-LPE
interferes with hepatic PLA.sub.2 can certainly not be expanded to
bacterial PLA. The finding that lysophospholipid-conjugates can
effectively decrease bacterial PLA activity and therefore exhibit a
significant potential for the treatment of intestinal diseases
associated with inflammation and, eventually, bacterial invasion of
the mucosal barrier of the intestine, therefore clearly came as a
surprise. The present invention offers a new, unexpected way to
treat inflammatory intestinal diseases that acts more specific, is
less toxic and more convenient than state-of-the-art methods.
SUMMARY
[0016] The present inventors have surprisingly discovered that
lysophospholipid-conjugates, e.g. the bile-acid phospholipid
conjugate UDCA-LPE, act as potent inhibitors of bacterial
phospholipase, in particular bacterial PLA.sub.2. However, also
other bacterial phospholipases, such as phospholipase C (PLC), were
shown to be inhibited by, e.g. UDCA-LPE. Bacterial PL, particularly
bacterial PLA.sub.2, have been suggested to play an important role
in the pathogenesis of many diseases affecting the intestine,
because they can disrupt the protective mucus lining of the
intestinal tract (Sawai, 2000). When bacteria penetrate the mucus,
an inflammatory response can be triggered in the underlying mucosa.
Lysophospholipid-conjugates of the present invention, such as
UDCA-LPE, may preferably consist of two tolerable substrates that
naturally occur in the host, and preferably exert fewer side
effects than conventional antibiotic or anti-inflammatory
therapeutics. In addition, because the lysophospholipid-conjugates
of the present invention specifically inhibit harmful bacterial PL,
in particular PLA activity, they are less toxic for the beneficial
bacteria of the gut flora, and are therefore superior to other
antibiotic agents that currently represent the standard for
treating many acute inflammatory bacterial diseases of the
intestine. Patients with chronic inflammatory diseases affecting
the intestine are likewise thought to benefit from treatment with
lysophospholipid-conjugates. For example, in inflammatory bowel
diseases (IBD), bacterial PLA.sub.2 activity of resident gut
bacteria is thought to promote the chronic inflammatory response
that is typically associated with IBD. The present inventors were
the first to acknowledge bacterial PL, in particular bacterial
PLA.sub.2, activity as a common underlying causative or
contributing factor of various acute and chronic inflammatory
diseases of the intestine, and to recognize that inhibitors of
bacterial PL, in particular bacterial PLA.sub.2, are potential
therapeutics for the treatment of said diseases.
[0017] In fact, the prior art did neither explicitly nor implicitly
disclose or teach that bacterial phospholipases are to be inhibited
for the treatment of inflammatory bacterial diseases of the
intestine in a subject. Though phospholipase inhibitors may have
been administered to subjects in the prior art (see Krimsky, 2003,
US 2007/0117779 or WO 2012/073245), there is no teaching that a
bacterial phospholipase should be inhibited with the aim of
treating inflammatory bacterial diseases of the intestine in a
subject, particularly invasive inflammatory bacterial diseases of
the intestine in a subject. Indeed, the etiology of inflammatory
diseases of the intestine that were treated with phospholipase
inhibitors in the prior art was and is unknown or incompletely
understood. As such, the finding of the present inventor that
bacterial phospholipases are to be inhibited allows for a selective
treatment regimen in that bacterial phospholipases are to be
targeted, thereby allowing the treatment of inflammatory bacterial
diseases of the intestine in a subject.
[0018] It is an established principle that a sharp line must be
drawn between what is in fact made available, and what remains
hidden or otherwise has not been made available. In the present
case, the prior art failed to make technically available what the
present inventor found and what is thus described and reflected
herein: inhibition of a bacterial phospholipase paves the way for
the treatment of inflammatory bacterial diseases of the intestine
in a subject, particularly of invasive inflammatory bacterial
diseases of the intestine in a subject. Indeed, as shown in the
Examples, bacterial phospholipases, such as PLA.sub.2 or PLC, are
significantly inhibited. Given the involvement of bacterial
phospholipases in the invasiveness of bacteria in the intestine, it
is apparent that the inhibition of bacterial phospholipases seems
to be a promising way of treating inflammatory bacterial diseases
of the intestine in a subject.
[0019] Thus, in a first aspect, the present invention relates to an
inhibitor of bacterial PLA.sub.2, PLA.sub.1, PLB, PLC and/or PLD
for use in a method of treatment of inflammatory bacterial diseases
of the intestine.
[0020] In addition, the present inventors pioneered in presenting
lysophospholipid-conjugates as potent inhibitors of PL, in
particular bacterial PLA.sub.2.
[0021] Accordingly, in a second aspect the present invention
relates to lysophospholipid-conjugates for use in a method of
treatment of inflammatory bacterial diseases of the intestine in a
subject.
[0022] The subject is a mammal, for example a mouse, rat, guinea
pig, hamster, rabbit, dog, cat, or primate. Preferably, the subject
is a human.
[0023] Without wishing to be bound by a specific theory, it is
speculated that bacterial PL, in particular bacterial PLA.sub.2,
promote bacterial invasion of the protective mucus layer lining the
intestinal tract, and thereby allow bacterial access to the
underlying mucosa, where an inflammatory response can arise. Hence,
the present invention further relates to the use of
lysophospholipid-conjugates for use in a method of treatment of
inflammatory bacterial diseases of the intestine in a subject,
wherein the inflammatory bacterial diseases are invasive bacterial
diseases.
[0024] The mammalian intestine comprises two segments: The small
intestine and the large intestine. Many intestinal bacterial
diseases particularly affect the large intestine. Therefore, in
another aspect, the present invention comprises inhibitors of
bacterial PL, in particular bacterial PLA.sub.2, preferably
lysophospholipid-conjugates, for use in a method of treatment of
inflammatory diseases of the intestine, wherein the intestine is
the large intestine.
[0025] It is envisaged that bacterial PL inhibitors, in particular
bacterial PLA.sub.2 inhibitors, preferably
lysophospholipid-conjugates, can be used for the treatment of a
broad spectrum of inflammatory bacterial diseases of the intestine,
including acute and chronic inflammatory diseases. It is further
envisaged that inhibitors of the invention, and in particular
lysophospholipid-conjugates, can be used to treat such diseases
when bacterial PLA activity may be a causative or a contributing
factor. Such diseases include, but are not limited to,
appendicitis, pseudoappendicits, ulcerative colitis, Crohn's
disease, enterhemorrhagic colitis, pseudomembranous colitis,
collagenous colitis, lymphocytic colitis, ischaemic colitis,
diversion colitis, microscopic colitis, Behcet's disease,
indeterminate colitis, diverticulitis, megacolon, toxic megacolon,
enterocolitis, and caecitis.
[0026] The lysophospholipid-conjugate preferably comprises a
lysophospholipid chemically coupled to a carrier. Said carrier is
preferably a bile acid. Suitable carrier bile acids include
ursodeoxycholate (UDCA) and deoxycholate (DCA).
[0027] Lysophospholipids as part of the lysophospholipid-conjugate
of the present invention can be, preferably,
lysophosphatidylcholine (LPC) or lysophosphatidylethanolamine
(LPE).
[0028] It is further envisaged that the inhibitor of bacterial PL,
in particular bacterial PLA.sub.2, which is preferably a
lysophospholipid-conjugate, of the present invention is
administered together with one or more agents selected from the
group of antibiotics, anti-inflammatory agents, immunosuppressive
agents and anti-diarrheal agents. The term "administered together"
comprises administration of the one or more agents prior to,
simultaneously, or after inhibitor of the invention.
[0029] It is preferred that the inhibitor of the invention, and in
particular the lysophospholipid-conjugate of the present invention
inhibits bacterial PL, in particular bacterial PLA.sub.2.
[0030] In a further aspect, the present invention further relates
to a pharmaceutical composition comprising
lysophospholipid-conjugates and a pharmaceutical carrier, excipient
or diluent. Said pharmaceutical composition is preferably for the
treatment of inflammatory bacterial diseases of the intestine. Said
pharmaceutical composition can further optionally comprise one or
more agents selected from the group of antibiotics,
immunosuppressive agents, anti-inflammatory agents and
anti-diarrheal agents. In a further aspect, the present invention
relates to a kit comprising a lysophospholipid-conjugate, for the
treatment of inflammatory bacterial diseases of the intestine. Said
kit can further optionally comprise one or more agents selected
from the group of antibiotics, immunosuppressive agents,
anti-inflammatory agents and anti-diarrheal agents, optionally
together with a pharmaceutical carrier, excipient or diluent.
[0031] In yet another aspect, the present invention also relates to
a method of treatment of inflammatory bacterial diseases of the
intestine in a subject in need thereof that comprises a step of
administering a therapeutically effective amount of an inhibitor of
bacterial PL, in particular bacterial PLA.sub.2, preferably a
lysophospholipid-conjugate to said subject. Said method can further
comprise administering one or more agents selected from the group
of antibiotics, anti-inflammatory agents, immunosuppressive agents
and anti-diarrheal agents. The agent can be administered prior to,
simultaneously, or after the inhibitor of the invention.
[0032] In yet another aspect, the present invention also relates to
the use of an inhibitor of bacterial PL, in particular bacterial
PLA.sub.2, which is preferably a lysophospholipid-conjugate, for
the manufacture of a pharmaceutical composition for the treatment
of inflammatory bacterial diseases of the intestine in a
subject.
[0033] In yet another aspect, the present invention relates to the
use of an inhibitor of bacterial PL, in particular bacterial
PLA.sub.2, which is preferably a lysophospholipid-conjugate, for
the treatment of inflammatory bacterial diseases of the large
intestine in a subject.
[0034] Also, the present invention relates to a method for the
production of a pharmaceutical composition for the treatment of
inflammatory bacterial diseases of the intestine in a subject,
comprising mixing an inhibitor of bacterial PL, in particular
bacterial PLA.sub.2, which is preferably a
lysophospholipid-conjugate, with a pharmaceutically acceptable
carrier, diluent or excipient.
[0035] It must be noted that as used herein, the singular forms
"a", "an", and "the", include plural references unless the context
clearly indicates otherwise. Thus, for example, reference to "a
reagent" includes one or more of such different reagents and
reference to "the method" includes reference to equivalent steps
and methods known to those of ordinary skill in the art that could
be modified or substituted for the methods described herein.
[0036] Unless otherwise indicated, the term "at least" preceding a
series of elements is to be understood to refer to every element in
the series. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention.
[0037] The term "and/or" wherever used herein includes the meaning
of "and", "or" and "all or any other combination of the elements
connected by said term".
[0038] The term "about" or "approximately" as used herein means
within 20%, preferably within 10%, and more preferably within 5% of
a given value or range. It includes, however, also the concrete
number, e.g., about 20 includes 20.
[0039] The term "less than" or "greater than" includes the concrete
number. For example, less than 20 means less than or equal to.
Similarly, more than or greater than means more than or equal to,
or greater than or equal to, respectively.
[0040] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integer or step. When used herein the term
"comprising" can be substituted with the term "containing" or
"including" or sometimes when used herein with the term
"having".
[0041] When used herein "consisting of" excludes any element, step,
or ingredient not specified in the claim element. When used herein,
"consisting essentially of" does not exclude materials or steps
that do not materially affect the basic and novel characteristics
of the claim.
[0042] In each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms.
[0043] It should be understood that this invention is not limited
to the particular methodology, protocols, material, reagents, and
substances, etc., described herein and as such can vary. The
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention, which is defined solely by the claims.
[0044] All publications and patents cited throughout the text of
this specification (including all patents, patent applications,
scientific publications, manufacturer's specifications,
instructions, etc.), whether supra or infra, are hereby
incorporated by reference in their entirety. Nothing herein is to
be construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention. To the
extent the material incorporated by reference contradicts or is
inconsistent with this specification, the specification will
supersede any such material.
DESCRIPTION OF THE FIGURES
[0045] FIG. 1 For determination of phospholipase A.sub.2 activity
according to Bhat et al. (1993), 10 .mu.l of aliquots of certain
isolated bacterial strains prepared from fresh stool samples were
incubated with arachidonoyl-thio phosphatidylcholine for 1 hour at
25.degree. C. in a Ca.sup.2+ free buffer as described herein. The
reaction was terminated by 5,5-dithio-bis-2-nitrobenzoic acid.
A.sub.405 was measured and specific PLA.sub.2 activity expressed as
.mu.mol LPCmg.sup.-1 proteinh.sup.-1
[0046] FIG. 2 Phospholipase A.sub.2 activity in Streptomyces
violaceoruber. It is apparent that bacterial phospholipase A2 is
inhibited by an inhibitor of the present invention.
[0047] FIG. 3 Phospholipase C activity in Clostridium perfringens
and Bacilllus cereus. It is apparent that bacterial phospholipase
A2 is inhibited by an inhibitor of the present invention.
DETAILED DESCRIPTION
[0048] The mucus lines the interface between the gut epithelium and
the luminal contents and represents the first line of defense of
the intestinal tract. Without wishing to be bound by a specific
theory, it is supposed that the activity of bacterial
phospholipases, in particular bacterial phospholipases A.sub.2, is
implicated in various inflammatory intestinal diseases. For
example, bacterial PL, in particular bacterial PLA.sub.2, can
disrupt mucus phospholipids and thereby promote bacterial invasion
of the mucus--a scenario that can occur during the course of
bacterial infections of the intestine, and which is thought to
result in an acute inflammatory response in the intestine. Attempts
to control bacterial infections with common antibiotics remain
problematic, because bacterial antibiotic resistances are common
and still on the rise. In addition, the use of, e.g., broad
spectrum antibiotics can unbalance the gut flora, leading for
example to overgrowth of harmful bacteria and a reduction of the
body's ability to ferment carbohydrates and metabolize bile acids,
which often results in antibiotic-associated diarrhea (AAD).
Further, antibiotics can cause undesirable side effects in the
patient, including abdominal pain, nausea, or hypersensitivity
reactions. Bacterial invasion of the intestinal mucus poses a
problem not only in infectious bacterial diseases, but is also
thought to be of relevance for inflammatory bowel diseases (IBD),
which are linked to a dysregulated chronic immune response to the
gut flora. IBD are currently treated with corticosteroids or
immunosuppressive agents that can elicit severe side effects in the
patient. When no response to medical treatment occurs, surgery can
be a last option. Controlling bacterial invasion, while at the same
time avoiding the use of drugs that are poorly tolerated or even
harmful for the patient as well as for beneficial bacteria of the
gut flora is therefore a key challenge in the treatment of many
diseases affecting the intestine.
[0049] The present inventors have strikingly discovered that
lysophospholipid-conjugates acting as inhibitors of PLA.sub.2, can
reduce the activity of bacterial PLA.sub.2, enzymes that are
suggested as important bacterial virulence factors in the
pathogenesis of various inflammatory bacterial diseases of the
intestine. It is thought that PLA.sub.2 are crucial for bacterial
invasion of the mucus and the underlying mucosa. The present
inventors have developed the idea that inhibiting bacterial
PLA.sub.2 activity can be a key step in treating a broad spectrum
of inflammatory intestinal diseases which are caused or contributed
to by bacteria. Accordingly, in a first aspect, the present
invention relates to an inhibitor of bacterial PL, in particular
bacterial PLA.sub.2, for use in a method of treatment of
inflammatory bacterial diseases of the intestine in a subject.
Whether or not a particular agent is capable of inhibiting
bacterial PL, in particular bacterial PLA.sub.2, can be determined
by the skilled person, e.g., by the methods described herein.
[0050] In principle, in order to identify potential inhibitor of
bacterial PL, in particular bacterial PLA.sub.2, the person skilled
in the art can use the three dimensional structure of bacterial PL
or its active site in order to predict which compounds might be
inhibitors. Various methods for determining possible ligands have
been reviewed by Anderson, 2003. In general, once the target
structure has been determined, e.g., by X-ray crystallography, NMR,
or homology modeling, computer algorithms can be used to position
compounds or fragments thereof from a database into a selected
region of the structure. These compounds can be scored and ranked
based on their steric and electrostatic interactions with the
target site. Such compounds are useful for example as a lead for
the development of further analogues, which in turn may have an
enhanced inhibitory potential or otherwise beneficial therapeutic
properties. On the other hand, the selected compound may bind to a
site of the target other than known ligands. Lead compounds can be
improved using the 3-D structure of the complex of the lead
compound and its biological target. The activity of the selected
compound can further be tested with the biochemical assays
described herein.
[0051] The crystal structure and the tertiary structure of secreted
prokaryotic PLA.sub.2 (EC 3.1.1.4) from Streptomyces violaceoruber
A-2688 have been determined by NMR and X-ray analyses (Matoba, et
al., 2002) (Matoba & Sugiyama, 2003). It is envisaged that the
3-D structure of S. violaceoruber allows an evaluation of bacterial
PLA.sub.2 inhibitors. Accordingly, computer-aided methods can be
used to identify candidate inhibitors for bacterial PLA.sub.2. Said
methods are further classified into at least three categories:
inspection, virtual screening, and de novo generation. In the first
category, inspection, known molecules that bind the site, such as
substrates or cofactors, are modified to become inhibitors based on
maximizing complementary interactions in the target site.
Initially, the crystal structure is solved in the presence of a
substrate, cofactor, or drug lead. Then, modifications to direct
the small molecule toward being a potent inhibitor are designed in
silico based on the interactions of the molecule with the target
site. The newly designed compounds are then scored for binding
using evaluative scoring algorithms available in virtual screening
methods.
[0052] In virtual screening, databases of available small molecules
are docked into the region of interest in silico and scored based
on predicted interactions with the site, e.g. shape complementarity
or estimated interaction energy. For de novo generation small
fragments of molecules, such as benzene rings, carbonyl groups,
amino groups, etc., are positioned in the site, scored, and linked
in silico. Some programs, e.g., LUDI, are capable of docking
fragments of compounds as well as entire compounds, and can thus be
used for virtual screening and de novo generation, respectively.
Suitable screening tools that can be used to find bacterial
PLA.sub.2 inhibitors include DOCK, FlexX, FlexE, LUDI and Legend
(see Anderson (2003) for other suitable programs).
[0053] In view of the fact that bacterial PLA.sub.2 was
crystallized, the known bacterial PLA.sub.2 crystal can also be
used in X-ray crystallography-driven screening technique that
combines the steps of lead identification, structural assessment,
and optimization such as described for example in Nienaber, et al.,
(2000). This crystallographic screening method (named CrystaLEAD)
has been used to sample large compound libraries and detecting
ligands by monitoring changes in the electron density map of the
crystal relative to the unbound form. The electron density map
yields a high-resolution picture of the ligand-enzyme complex that
provides key information to a structure-directed drug discovery
process. The bound ligand is directly visualized in the electron
density map. Ligands that bind way off the target site may be
eliminated. The above described methods can be combined with
state-of-the-art laboratory data collection facilities including
CCD detectors and data acquisition robotics.
[0054] The above-mentioned methods can be used to assess the
inhibitory potential of a given compound on bacterial PLA.sub.2,
and/or to identify candidate bacterial PLA.sub.2 inhibitors from a
library. Once a compound has been identified as a candidate
inhibitor by the above methods, its inhibitory effect on bacterial
PLA.sub.2 may be tested by the method described in the appended
example.
[0055] Analogous methods can be employed in order to find
inhibitors of bacterial phospholipases A.sub.1, B, C and D.
[0056] It is preferred that the inhibitors of the invention act on
bacterial phospholipases, but not on host phospholipases.
[0057] Some bacterial phospholipase inhibitors, such as UDCA-LPE,
stay within the intestinal lumen of the host and do not enter/are
not absorbed by the host mucosal epithelial cells in a considerable
amount. Thus, such inhibitors primarily act on bacterial PL, in
particular bacterial PLA.sub.2, but not on host PL.
[0058] It is also conceivable that the bacterial phospholipase
inhibitors selectively inhibit bacterial phospholipases, but not
host phospholipases, A "host" is a subject affected by the
condition to be treated with the inhibitor of the invention. The
term "host PL", in particular "host PLA.sub.2" includes without
limitation secreted and cytosolic forms of PL, in particular
PLA.sub.2.
[0059] The inhibitory effect of a specific compound on host PL,
such as host PLA.sub.2, can easily be determined by the skilled
person by using routine methods known in the art. Various assays,
including enzyme assays and cell-based assays, for testing host
PLA.sub.2 activity have been described, e.g., by Ono, et al.,
(2002). Exemplary enzyme assays for assessing, e.g., human
cytosolic PLA.sub.2 activity, include, without limitation, the
phosphatidylcholine (PC)/Triton assay according to Street, et al.,
(1993), modified by Ono et al. (2002), the chromogenic assay
according to Reynolds, et al., (1994) and the PC/DOG assay
according to Kramer, et al., (1991). Human sPLA.sub.2 activity can
for example be measured using diheptanoyl thio-PC as a substrate
according to Reynolds, et al., (1992). In principle, all methods
provide phospholipid-substrates and assess their conversion due to
PLA activity.
[0060] Many mammalian PLAs are commercially available and can be
used in the above-mentioned assays to determine the inhibitory
potential of a specific compound. For example, recombinant
secretory human PLA.sub.2 can be purchased from, e.g., R&D
systems and Cayman Chemicals, or can be produced in, e.g., E. coli
following the protocol of Zhang, et al., (2011).
[0061] Cell-based assays for assessing human PLA.sub.2 activity
employ, for example, thrombin-stimulated platelets,
calcium-ionophore-stimulated monocytes (e.g., THP-1, see Ono et al.
2002) or interleukin-1.alpha. stimulated human mesangial cells.
Fatty acids including arachnidonic acid, prostaglandin or
leukotriene from stimulated cells are extracted, and quantified,
e.g. by HPLC or by using radioimmunoassay kits or enzyme
immunoassay kits.
[0062] Ready-to-use kits for determining enzyme activity of
mammalian PLA in the presence of a candidate inhibitor are also
commercially available, e.g., from Cayman Chemicals (sPLA.sub.2
(Type V) Inhibitor Screening Assay Kit) and Invitrogen
(EnzChek.RTM. Phospholipase A.sub.2 Assay Kit).
[0063] However, it is also envisaged that the inhibitors of the
invention inhibit bacterial PLA.sub.1, PLB, PLC and/or PLB. Methods
for assessing the inhibitory potential of a specific compound on
bacterial PLA.sub.1, PLB, PLC and/or PLB are available in the prior
art.
[0064] In particular, the present inventors have discovered that
lysophospholipid-conjugates are potent bacterial phospholipase
A.sub.2 inhibitors. Phospholipase A.sub.2 is thought to play an
important role in intestinal inflammation associated with bacteria.
Thus, in one aspect, the present invention relates to
lysophospholipid-conjugates for the treatment of inflammatory
bacterial diseases of the intestine.
[0065] The novel treatment according to the present invention is
envisaged for a mammal, which can be, for instance, a mouse, rat,
guinea pig, hamster, rabbit, dog, cat, or primate. Preferably, the
subject is a human.
[0066] The present inventors were the first to provide
lysophospholipid-conjugates for the treatment of inflammatory
bacterial diseases of the intestine as described herein. The term
"lysophospholipid-conjugate" as used herein refers to
lysophospholipids that are chemically coupled to a carrier. The
term "chemically coupled" means, e.g., covalently coupled. However,
any other chemical bond is also conceivable. Lysophospholipids
(LPL) are naturally derived from phospholipids, such as
phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidic acid (PA), phosphatidylserine (PS),
phosphatidylinositol (PI) or other phospholipids. Lysophospholipids
can, for example, be the result of phospholipase A-type enzymatic
activity on phospholipids. Phospholipids are typically composed of
two fatty acids, a glycerol unit, a phosphate group and a polar
molecule such as, e.g., choline in phosphatidylcholine, or
ethanolamine in phosphatidylethanolamine. In contrast,
lysophospholipids typically comprise only one fatty acid. In
general, any lysophospholipid can be used in the
lysophospholipid-conjugate according to the present invention.
Suitable lysophospholipids include, but are not limited to,
lysophosphatidate, lysophosphatidylethanolamine,
lysophosphatidylcholine, lysophosphatidylserine,
lysophosphatidylinositol, lysophosphatidylinositolphosphate,
lysophosphatidylinositolbisphosphate,
lysophosphatidylinositoltriphosphate. In one preferred embodiment,
the lysophospholipid is lysophosphatidylethanolamine or
lysophosphatidylcholine. It is also envisaged that a
phospholipid-conjugate can be used for the treatment of the
diseases described herein. The phospholipid could, for example, be
phosphatidic acid (phosphatidate), phosphatidylethanolamine (PE),
phosphatidylcholine (PC), phosphatidylserine (PS),
phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP),
phosphatidylinositol bisphosphate (PIP2) or phosphatidylinositol
triphosphate (PIP3).
[0067] Suitable carriers are, for example, bile acids. However, it
is also envisaged that other carriers may be used.
[0068] In general, any lysophospholipid and any carrier can be
selected to yield the lysophospholipid-conjugate of the invention.
The lysophospholipid-conjugate should preferably have capabilities
that are similar to the capabilities of the
lysophospholipid-conjugate which are evaluated in the appended
examples. For example, the lysophospholipid-conjugate of the
present invention is preferably capable of inhibiting bacterial PL,
in particular bacterial PLA.sub.2 activity. The skilled
practitioner readily knows how to determine the inhibitory effect
of a specific lysophospholipid-conjugate on bacterial an PL, in
particular bacterial PLA.sub.2, by methods known in the art, e.g.,
by the method described by Bhat et al. (1993) and in the appended
examples.
[0069] The selected carrier can be, or can be derived from, a
naturally occurring component. Bile acids are steroid acids that
are naturally found in the mammalian bile. Primary bile acids, such
as, e.g. cholate, are naturally synthesized in the liver and
secreted into the lumen of the intestine, where intestinal bacteria
chemically convert them to form the secondary bile acids such as,
e.g., deoxycholic acid. Suitable bile acids include, but are not
limited to, cholic acid, chenodeoxycholic acid, deoxycholic acid,
lithocholic acid, glycocholic acid, taurocholic acid,
ursodeoxycholic acid. In one preferred embodiment, the bile acid is
ursodeoxycholic acid (UDCA) or deoxycholic acid (DCA). It is also
envisaged that chemically modified bile acids can be used as
carriers in the present invention.
[0070] In view of the above, preferred embodiments of the invention
thus involve the use of UDCA-LPE, UDCA-LPC, DCA-LPE and DCA-LPC for
the treatment of the diseases described herein. However, as already
mentioned, other carriers can be used as well to yield the
lysophospholipid-conjugates of the present invention. It is in
particular envisaged that UDCA-LPE, UDCA-LPC, DCA-LPE and DCA-LPC
are formulated in their acidic (i.e., protonated) form.
[0071] By inhibiting bacterial PL, in particular bacterial
PLA.sub.2, it is contemplated the inhibitors of the invention,
which are preferably lysophospholipid-conjugates, prevent or
decrease bacterial invasion of the intestinal mucus, an event that
may eventually result in inflammation of the underlying mucosa.
Accordingly, it is envisaged that the inhibitor of the invention,
preferably a lysophospholipid-conjugate, is used for treating
inflammatory bacterial diseases of the intestine, preferably in a
mammal. The inflammatory bacterial diseases to be treated according
to the present invention can in particular be invasive inflammatory
bacterial diseases.
[0072] The mammalian intestine is the part of the alimentary canal
extending from the pyloric sphincter of the stomach to the anus. It
consists of two parts: the small intestine and the large intestine.
It is to be noted that "diseases of the intestine" is to be
understood herein as diseases affecting primarly the intestine, but
not necessarily limited to the intestine, i.e. the expression also
comprises diseases that affect further parts of the body besides
the intestine, for example another part of the gastrointestinal
tract. In humans, the small intestine is further subdivided into
the duodenum, jejunum, and ileum. The "large intestine" is the
posterior section of the intestine, and consists of four regions:
the cecum, colon, rectum, and anus. The colon the longest segment
of the large intestine and houses a large proportion of the gut
flora. Many diseases of the intestine affect the large intestine,
which in turn often involve the colon.
[0073] The expression "invasion" when used herein is to be
understood in its broadest sense and, in the context of the present
invention, includes one or more of the following: bacterial
damaging, disrupting, penetrating and/or crossing of the intestinal
mucus. It can further involve bacterial spread in the invaded
mucus, and possibly the underlying mucosa. Accordingly, "invasive"
means associated with bacterial damage, disruption, penetration,
crossing of and/or bacterial spread in the intestinal mucus and/or
mucosa. Without wishing to be bound by a specific theory, it is
assumed that bacterial PL, in particular bacterial PLA.sub.2,
promote bacterial invasion of the intestinal mucus and are pivotal
factors in the pathogenesis of many inflammatory bacterial diseases
of the intestine.
[0074] The mucus lining the intestinal tract has an essential
barrier function in the intestine. In the large intestine, it is
organized in two layers: an inner, stratified mucus layer that
adheres to the underlying epithelial cells; and an outer,
nonattached layer. The inner mucus layer is dense and does usually
not allow bacteria to penetrate, thus keeping the mucosal
epithelial cell surface free from bacteria. The inner mucus layer
transitions the outer layer, which harbors the gut flora.
Accordingly, bacterial invasion in the large intestine involves
invasion of both mucus layers, in particular the inner mucus layer.
Mucus typically comprises mucus glycoproteins (also referred to as
mucins), that serve as a scaffold for the mucus gel. The mucus gel
further typically comprises water, inorganic salts, lipids and
further proteins (Johansson, et al., 2011). Phosphatidylcholines
are thought to be relevant mucus components. Without wishing to be
bound by a specific theory, it is speculated that
phosphatidylcholines bind to the mucins with their polar
hydrophilic headgroup and extend their hydrophobic fatty acid tails
towards the lumen, thereby constituting a hydrophobic barrier that
usually prevents bacteria from invading the mucus.
[0075] It is further suggested that bacterial PL, in particular
bacterial PLA.sub.2, may play a pivotal role in bacterial invasion
of the intestinal mucus by cleaving mucus phospholipids and thereby
compromising the protective features of the mucus. For example,
cleavage of mucus phosphatidylcholines may result in distortion of
its hydrophobic, exclusive features. Bacterial PL, in particular
bacterial PLA.sub.2, may further, in a second step, impair the
membrane integrity of the mucosal epithelial cells once they have
crossed the mucus barrier.
[0076] Bacterial phospholipases are a group of enzymes that
catalyze the cleavage of phospholipids and are classified in four
major groups (A, B, C and D) based on the site of cleavage of their
substrates. Bacterial phospholipases A (PLA) and B (PLB) hydrolyze
a fatty acid from the phospholipid glycerol backbone, thereby
yielding a lysophospholipid. PLA can be further defined by their
positional preference for the acyl group attached to position 1 or
2 of the phospholipid glycerol backbone as PLA.sub.1 and PLA.sub.2,
respectively. The present inventors have recognized that bacterial
PLA.sub.2 are involved in the bacterial invasion of the intestinal
mucus which can ultimately result in acute or chronic inflammation,
thus contributing to the pathogenesis of a plethora of inflammatory
intestinal diseases. The present inventors have further
acknowledged that lysophospholipid-conjugates can be used to
inhibit bacterial bacterial PL, in particular bacterial PLA.sub.2,
activity. Hence, the lysophospholipid-conjugates according to the
present invention preferably inhibit bacterial PLA, and more
preferably they inhibit bacterial PLA.sub.2.
[0077] It is also conceivable that the lysophospholipid-conjugates
of the invention, for example UDCA-LPE or any other
lysophospholipid-conjugate, inhibit bacterial phospholipases
PLA.sub.1, PLB, PLC or PLD. Without wishing to be bound by a
specific theory, it is speculated that UDCA-LPE may bind to a
common specific enzymatic pocket residing in several types of
phospholipases which may result in inhibition of the enzymatic
activity.
[0078] The term "PL" is used herein to refer to phospholipase and
applies both for the singular and the plural form. The term "PLA"
is used herein to refer to phospholipase A, and applies both for
the singular and the plural form. The same is applicable for
"PLA.sub.1" and "PLA.sub.2", respectively. Notably, unless
indicated otherwise, when the terms "PLA", "PLA.sub.1", "PLA.sub.2"
are used herein they refer to bacterial phospholipases A, A.sub.1
and A.sub.2, respectively.
[0079] In general, bacterial invasion and/or inflammation can occur
at any time point during the course of the disease. E.g., when
bacteria penetrate the intestinal mucus and bacterial antigens
reach the underlying mucosa, an inflammatory response can be
triggered. Inflammatory bacterial diseases of the intestine
comprise, i.a., inflammatory bowel diseases (IBD). IBD are a group
of chronic diseases typically associated with inflammation of the
intestinal tract. The etiology of IBD is not completely understood,
however, pathogenesis has been linked to a dysregulated immune
response to elements of the intestinal tract. IBD are therefore
also referred to as autoimmune disorders. Meanwhile, there is
evidence suggesting that chronic inflammation in IBD is, at least
in part, caused by an overreaction of the host's immune system to
the gut flora. The present inventors have suggested that bacterial
PL, and in particular bacterial PLA.sub.2, contribute to IBD
pathogenesis, e.g. by consuming mucus PC below a borderline level
in an individual that already exhibits decreased mucus PC levels.
When the mucus barrier is impaired, bacteria can penetrate the
mucus and evoke an inflammatory response the underlying mucosa. IBD
are typically characterized by periods of clinical exacerbation and
remission, with periods of improvement followed by relapse. It is
envisaged that lysophospholipid-conjugates of the invention can be
used for the treatment of IBD during any of the aforementioned
phases. IBD comprise, but are not limited to, Crohn's disease (CD)
and ulcerative colitis (UC). Other diseases such as collagenous
colitis, lymphocytic colitis, ischaemic colitis, diversion colitis,
BehGet's disease and indeterminate colitis are also sometimes
classified as IBD. Treatment of said diseases with inhibitors of
bacterial PL, and in particular bacterial PLA.sub.2, preferably
lysophosphatidyl-conjugates, is also envisaged.
[0080] The use of the inhibitors of the invention, which are,
preferably, lysophospholipid-conjugates, is however not restricted
to the treatment of inflammatory bowel diseases. Other diseases
that are envisaged for lysophospholipid-conjugate treatment
according to the present invention include bacterial infectious
diseases, also referred to herein as bacterial infections, of the
intestine. Such diseases can occur when pathogenic bacteria enter
the intestine and penetrate the mucus, which typically results in
inflammation. Further, potentially pathogenic bacteria can already
be present in the gut, and, for example as a result of an
unbalanced gut flora that fails to suppress their excessive growth,
become prevalent. This scenario can arise, e.g., after the use of
broad-spectrum antibiotics that act on and interfere with the gut
flora. Examples for pathogenic bacteria that can cause an
inflammatory infection of the intestine include, but are not
limited to, Shigella, Salmonella, Campylobacter, Clostridium
difficile, and Escherichia coli species. It is suggested that such
pathogenic bacteria may use bacterial PL, in particular bacterial
PLA.sub.2, to penetrate the protective mucus, which typically
results in inflammation. Thus, infectious inflammatory bacterial
diseases, such as (infectious) colitis, enterocolitis and
pseudomembranous colitis, are also envisaged for
lysophospholipid-conjugate treatment.
[0081] In general, the use of and the inhibitors of the invention,
in particular, lysophospholipid-conjugates, is further thought to
be of benefit in the treatment of any intestinal diseases that is
associated with inflammation, but may not initially be caused by
bacteria. For example, when the intestinal mucosa is inflamed, it
is thought that preventing the detrimental action of bacterial PL,
in particular bacterial PLA.sub.2, on the protective mucus barrier
by the use of the inhibitors of the invention, in particular,
lysophospholipid-conjugates, can help to avoid and/or reduce
additional tissue damage and inflammation.
[0082] The finding that lysophospholipid-conjugates such as
UDCA-LPE can inhibit bacterial PL, in particular bacterial
PLA.sub.2, activity and, therefore, prevent bacteria from invading
the intestinal mucus, is, in itself, highly relevant. However,
without wishing to be bound by a specific theory, it is further
speculated that treatment with the inhibitors of the invention and,
in particular, lysophospholipid-conjugates might even favor
harmless and/or beneficial bacteria of the gut flora and thereby
shift the bacterial spectrum in the intestine towards more
tolerable, less invasive bacterial strains. The inhibition of
bacterial phospholipases may therefore be selective for specific
bacterial strains whereas other strains may not be affected.
Therefore a selection process for specific non-invasive bacterial
species could be assumed, while pathogenic or tissue-destructing
bacterial species carrying phospholipases are reduced. This could
be for example important with regard to chronic inflammatory
diseases of the intestine, such as IBD, wherein a gut flora
exhibiting a low phospholipase activity could potentially outgrow
more invasive bacterial species, and prevent re-colonization with
invasive species by competitive exclusion, i.e., by consuming
nutrients and space. This might break the cycle of recurrent mucus
invasion and dysregulated immune response. The selection of
specific bacterial strains could further be used to modulate the
milieu within the intestinal lumen by inhibiting or stimulating the
production of bacterial derived products which have an impact on
human metabolism. This could be used, e.g. to suppress bacterial
derived ammonium generation in small and large intestine which has
an impact for induction of hepatic encephalopathy in end stage
disease, liver failure or portal hypertension. Thus, these
conditions can be treated. Moreover, the generation of the
proatherogenic metabolites of phosphorylcholine-containing food
constituents or drugs, e.g. trimethylamine-N-oxide (TMAO) could be
suppressed by the employed bacterial phospholipase inhibitors such
as UDCA-LPE, thereby treating rheumatoid arthritis (RA), since TMAO
is suspected to be involved in the etiology of RA.
[0083] In contrast to anti-inflammatory and immunosuppressive drugs
commonly prescribed for IBD, the inhibitors of the invention and,
in particular, lysophospholipid-conjugates, may protect the
integrity of the intestinal mucus as a first line defense
mechanism, instead of suppressing the host's immune response to an
ongoing bacterial invasion.
[0084] Accordingly, lysophospholipid-conjugates can be used for the
treatment of diseases selected from the group including, but not
limited to, appendicitis, pseudoappendicits, ulcerative colitis,
Crohn's disease, enterhemorrhagic colitis, pseudomembranous
colitis, collagenous colitis, lymphocytic colitis, ischaemic
colitis, diversion colitis, microscopic colitis, Behcet's disease,
indeterminate colitis, diverticulitis, megacolon, toxic megacolon,
enterocolitis and caecitis.
Pharmaceutically Acceptable Salts
[0085] For the purpose of the invention, the inhibitors as defined
herein also include the pharmaceutically acceptable salt(s)
thereof. The phrase "pharmaceutically acceptable salt(s)", as used
herein, means those salts of inhibitors of bacterial PL, in
particular bacterial PLA.sub.2, inhibitors that are safe and
effective for the desired administration form. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0086] The use of salt formation as a means of varying the
properties of pharmaceutical compounds is well known and well
documented. Salt formation can be used to increase or decrease
solubility, to improve stability or toxicity and to reduce
hygroscopicity of a drug product. There are a wide range of
chemically diverse acids and bases, with a range of pKa values,
molecular weights, solubilities and other properties, used for this
purpose. Of course, any counter-ions used in pharmaceuticals must
be considered safe, and several lists of pharmaceutically approved
counter-ions exist, which vary depending on the source. Approved
salt formers can e.g. be found in the Handbook of Pharmaceutical
Salts (Stahl P H, Wermuth C G, editors. 2002. Handbook of
pharmaceutical salts: Properties, selection and use.
Weinheim/Zurich: Wiley-VCH/VHCA.). Thus, the present invention also
comprises the use of pharmaceutically acceptable salts of the
bacterial PLA inhibitors of the invention for the treatment of
inflammatory bacterial diseases of the intestine in a subject.
Chemical Modification
[0087] It is envisaged that bacterial PLA inhibitors can be
provided in any chemical form that ensures that it reaches the site
of action, i.e., the intestine.
[0088] The bacterial PLA inhibitors of the invention may be
chemically modified. Generally, all kind of modifications of
bacterial PLA inhibitors are comprised by the present invention as
long as they do not reduce or abolish the advantageous capabilities
and/or the therapeutic effect of the bacterial PLA inhibitors
described herein. In the context with the present invention the
term "therapeutic effect" in general refers to the desirable or
beneficial impact of a treatment, e.g. amelioration or remission of
the disease manifestations. The term "manifestation" of a disease
is used herein to describe its perceptible expression, and includes
both clinical manifestations, hereinafter defined as indications of
the disease that may be detected during a physical examination
and/or that are perceptible by the patient (i.e., symptoms), and
pathological manifestations, meaning expressions of the disease on
the cellular and/or molecular level.
[0089] The therapeutic effect of the uses and methods described
herein is additionally detectable by all methods and approaches
that are established for indicating a therapeutic effect in the
treatment of inflammatory bacterial diseases of the intestine.
Methods for monitoring the therapeutic effect of bacterial PLA
inhibitors include, but are not limited to, assessing the presence
blood in stool, the number and species of gut bacteria, evaluating
symptoms like fever, abdominal pain, and diarrhea, using endoscopic
methods or noninvasive imaging techniques to assess the redness,
swelling, mucus condition, and assessing inflammation, e.g., by
obtaining tissue samples and screen for inflammatory cytokines,
chemokines or others and numbers and types of inflammatory
cells.
[0090] Additionally or alternatively it is also possible to
evaluate the general appearance of the respective patient (e.g.,
fitness, well-being) which will also aid the skilled practitioner
to evaluate whether a therapeutic effect has been elicited.
[0091] The skilled person is aware of numerous other ways which are
suitable to observe a therapeutic effect of the bacterial PLA
inhibitors of the present invention.
[0092] Thus, a further embodiment of the present invention is the
use of bacterial PLA inhibitors which are chemically modified.
Treatment
[0093] The term "treatment" in all its grammatical forms includes
therapeutic or prophylactic treatment of inflammatory bacterial
diseases of the intestine. A "therapeutic or prophylactic
treatment" comprises prophylactic treatments that aim at the
complete prevention of clinical and/or pathological manifestations
or therapeutic treatment which that aims at amelioration or
remission of clinical and/or pathological manifestations. The term
"treatment" thus also includes the amelioration or prevention of
inflammatory bacterial diseases of the intestine.
Dose
[0094] The exact dose of bacterial PLA inhibitors will depend on
the purpose of the treatment (e.g. remission maintenance vs. acute
flare of disease), and will be ascertainable by one skilled in the
art using known techniques. As is known in the art and described
above, adjustments for route of administration, age, body weight,
general health, sex, diet, time of administration, drug interaction
and the severity of the condition may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
Composition
[0095] Bacterial PLA inhibitors, which are preferably
lysophospholipid-conjugates, can also be used as part of a
pharmaceutical composition. Thus, a further aspect of the invention
a pharmaceutical composition comprising lysophospholipid-conjugates
for the treatment of inflammatory bacterial diseases of the
intestine. It is to be acknowledged that the embodiments described
in the context of the use of bacterial PLA inhibitors and
lysophospholipid-conjugates are equally applicable to the
pharmaceutical composition of the invention, mutatis mutandis. The
pharmaceutical composition may further comprise a pharmaceutically
acceptable excipient, carrier or diluent. Processes known per se
for producing medicaments are for example indicated in Forth,
Henschler, Rummel (1996) Allgemeine und spezielle Pharmakologie und
Toxikologie, Urban & Fischer.
[0096] Pharmaceutical compositions of the invention comprise a
therapeutically effective amount of lysophospholipid-conjugates and
can be formulated in various forms, e.g. in solid, liquid, gaseous
or lyophilized form and may be, inter alia, in the form of an
ointment, a cream, transdermal patches, a gel, powder, a tablet,
solution, an aerosol, granules, pills, suspensions, emulsions,
capsules, syrups, liquids, elixirs, extracts, tincture or fluid
extracts or in a form which is particularly suitable for topical or
oral administration.
[0097] By "therapeutically effective amount" is meant an amount of
lysophospholipid-conjugate that elicits a therapeutic effect as
described herein.
[0098] In general, coated dosage forms, e.g., for oral
administration, may be a single unit system or a multi-particulate
system, and each of these may be a single-layer product or a
multi-layer product. In either case, the coating can be applied to
a variety of solid core formulations such as tablets, capsules,
mini tablets, pellets, granules or the like.
[0099] Systems for transdermal delivery are fabricated as
multi-layered polymeric laminates in which a drug reservoir or a
drug-polymer matrix is sandwiched between two polymeric layers: an
outer impervious backing layer that prevents the loss of drug
through the backing surface and an inner polymeric layer that
functions as an adhesive and/or rate-controlling membrane.
Transdermal drug delivery systems comprise different systems such
as the reservoir systems, microreservoir systems, and the
combination of reservoir and matrix-dispersion systems.
[0100] Reservoir-based drug delivery systems can be used, e.g., for
oral, dermal and implantable delivery systems. In the reservoir
system, the drug reservoir is embedded between an impervious
backing layer and a rate-controlling membrane. The drug releases
only through the rate-controlling membrane, which can be
microporous or non-porous. In the drug reservoir compartment, the
drug can be in the form of a solution, suspension, or gel or
dispersed in a solid polymer matrix. On the outer surface of the
polymeric membrane a thin layer of drug-compatible, hypoallergenic
adhesive polymer can be applied. In the Matrix systems and
Drug-in-adhesive system the drug reservoir is formed by dispersing
the drug in an adhesive polymer and then spreading the medicated
polymer adhesive by solvent casting or by melting the adhesive (in
the case of hot-melt adhesives) onto an impervious backing layer.
On top of the reservoir, layers of unmedicated adhesive polymer are
applied. In the Matrix-dispersion system the drug is dispersed
homogeneously in a hydrophilic or lipophilic polymer matrix. This
drug-containing polymer disk then is fixed onto an occlusive
baseplate in a compartment fabricated from a drug-impermeable
backing layer. Instead of applying the adhesive on the face of the
drug reservoir, it is spread along the circumference to form a
strip of adhesive rim. The drug delivery system is a combination of
reservoir and matrix-dispersion systems. The drug reservoir is
formed by first suspending the drug in an aqueous solution of
water-soluble polymer and then dispersing the solution
homogeneously in a lipophilic polymer to form thousands of
unleachable, microscopic spheres of drug reservoirs. The
thermodynamically unstable dispersion is stabilized quickly by
immediately cross-linking the polymer in situ.
[0101] Rectal applications can be compounded in many forms. Liquid
rectal medicine solutions are given by enema. Creams, lotions and
ointments are applied externally or inserted internally using an
applicator. Suppositories might be prepared by mixing medicine with
a wax-like substance to form a semi-solid, bullet-shaped form that
will melt after insertion into the rectum.
[0102] Intraperitoneal injection or IP injection is the injection
of a substance into the peritoneum (body cavity). A further form of
administration of an inventive composition is the topic
administration, for instance in form of an ointment or cream. Such
an ointment or cream may additionally comprise conventional
ingredients, like carriers or excipients as described herein.
Lysophospholipid-conjugates can also be used in sprays, for example
for inhalation. Lysophospholipid-conjugates may also be added to
foods.
[0103] The pharmaceutical composition may be administered with a
pharmaceutically acceptable carrier, excipient or diluent to a
patient, as described herein. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency
or other generally recognized pharmacopoeia for use in animals, and
more particularly in humans. Accordingly, the pharmaceutical
composition may further comprise a pharmaceutically acceptable
carrier, diluent or excipient. Generally all carriers are suitable
that are pharmaceutically acceptable and enable a release of the
lysophospholipid-conjugate at the desired site of action. The
person skilled in the art knows which type of carrier is suitable
depending on, e.g., the chosen administration route and the target
site. For example, carriers in the context with e.g. a rectal
application are e.g. multi matrix systems using methacrylic acid
copolymers. If e.g. the desired site of action is the colon and the
compound as described herein is applied orally the carrier has to
be resistant to gastric acid in order to enable a release of the
compound as described herein in the colon.
[0104] Exemplary pharmaceutically acceptable carriers that are
suitable for formulating the composition include (biodegradable)
liposomes; microspheres made of the biodegradable polymer
poly(D,L-lactic-coglycolic acid (PLGA), albumin microspheres;
synthetic polymers (soluble); nanofibers, protein-DNA complexes;
protein conjugates; erythrocytes; or virosomes. Various carrier
based dosage forms comprise solid lipid nanoparticles (SLNs),
polymeric nanoparticles, ceramic nanoparticles, hydrogel
nanoparticles, copolymerized peptide nanoparticles, nanocrystals
and nanosuspensions, nanocrystals, nanotubes and nanowires,
functionalized nanocarriers, nanospheres, nanocapsules, liposomes,
lipid emulsions, lipid microtubules/microcylinders, lipid
microbubbles, lipospheres, lipopolyplexes, inverse lipid micelles,
dendrimers, ethosomes, multicomposite ultrathin capsules,
aquasomes, pharmacosomes, colloidosomes, niosomes, discomes,
proniosomes, microspheres, microemulsions and polymeric
micelles.
[0105] Other suitable pharmaceutically acceptable carriers and
excipients are inter alia described in Remington's Pharmaceutical
Sciences, 15th Ed., Mack Publishing Co., New Jersey (1991) and
Bauer et al., Pharmazeutische Technologie, 5.sup.th Ed.,
Govi-Verlag Frankfurt (1997).
[0106] A variety of routes are applicable for administration of the
lysophospholipid-conjugate of the invention, including, but not
limited to, orally, topically, transdermally, subcutaneously,
intravenously, intraperitoneally, intramuscularly or intraocularly.
Of course, any other route may readily be chosen by the person
skilled in the art if desired.
[0107] In one preferred embodiment, the
lysophospholipid-conjugation of the invention is administered
orally. Accordingly, the lysophospholipid-conjugate, which
preferably released within the intestinal tract, is formulated such
that it allows an enteric coating. An enteric coating as used
herein refers in general to a coating that controls the location in
the digestive system where a drug is released.
[0108] Lysophospholipid-conjugates can inhibit bacterial PLA
activity, and are therefore considered as a potential drugs for
treating inflammatory bacterial diseases of the intestine. In one
embodiment, lysophospholipid-conjugates are delivered to and
released in the colon. It is within the knowledge of the person
skilled in the art to select a pharmaceutical carrier, excipient or
diluent that can be used to formulate a delivery system such as,
e.g., a colon-targeted drug delivery system.
[0109] Suitable delivery systems for intestinal drug delivery, and
in particular colonic delivery, have been reviewed by, e.g., Jain
and Jain (2008) and Van den Mooter (2006). In general, suitable
formulations for intestinal delivery comprise delayed release
dosage forms that may be designed to provide a "burst release" or a
sustained/prolonged release once they reach the target site. The
person skilled in the art is aware that the proper selection of a
suitable formulation approach is dependent on several factors, for
example pathology of the disease, physicochemical and
biopharmaceutical properties of the drug and the desired release
profile of the active ingredient. Systems that are particularly
suitable for intestinal delivery include prodrugs, pH-dependent
delivery systems, time release/delayed systems, microbial-triggered
systems, and pressure-dependent systems.
[0110] Generally, a prodrug is a composed of a drug and a carrier
which are chemically coupled to each other. Upon administration,
the moiety preferably maintains its integrity while passing the
non-target intestinal parts until it reaches its target, such as,
e.g., the colon. On reaching its final destination, the prodrug is
then converted into the parent drug molecule. Site-specific drug
delivery through site-specific prodrug activation can be
accomplished by utilizing a specific property of the target site,
e.g. a low pH or the presence of specific enzymes, including host
enzymes as well as enzymes of the bacterial gut flora. E.g., when
targeting drugs to the colon, a prodrug can be used that is
converted into the parental drug via the action of bacterial
enzymes of the gut flora, such as azoreductase, glycosidase,
polysaccharidases, or cyclodextrinase. This can also be referred to
as "microbial-triggered" delivery. Accordingly, prodrug approaches
including azo bond prodrugs, glysoside conjugates, glucuronide
conjugates, cyclodextrin conjugates, dextran conjugates and amino
acid conjugates can be used to target the compound of the invention
to the target site. More specifically, pectin, guar gum, inulin,
locus bean gum, glucomannan, chitosan, chondroitin sulfate,
hyaluronic acid, alginates, dextran, starch, amylase, cellulose,
cyclodextrin, curdlan, and sclereoglucan conjugates or mixtures
thereof, optionally comprising other polymers, can be used.
[0111] The rationale underlying pH-dependent intestinal delivery
systems is the use of coating agents that dissolve only at a
certain pH range that can be found in a specific part of the
intestine. Thus, pH dependent delivery systems exploit the rising
of the pH from the stomach to the large intestine. For the purpose
of targeting drugs to, e.g., the colon, tablets, capsules or the
like can be coated with a pH-dependent polymer that is insoluble at
low pH but soluble at neutral or slightly alkaline pH. This would
thus preferably release the drug in the colon. Conversely, when the
target site lies in the stomach, the person skilled in the art will
select a pH-dependent polymer that is insoluble at a high pH but
dissolves in the acidic environment of the stomach. Exemplary
pH-dependent polymers include, but are not limited to, derivatives
of acrylic acid and cellulose such as polyvinyl acetate phtalathe
(PVAP, e.g., Coateric.RTM.), cellulose acetate trimellitate (CAT),
hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropyl
methylcellulose acetate succinate (HPMCAS), methylacrylic acid
copolymer (e.g., Eudragit.RTM.), and cellulose acetate phthalate
(CAP, e.g., Aquateric.RTM.). Eudragit.RTM. coating is preferred for
the delivery of lysophospholipid-conjugates.
[0112] Time-dependent delivery systems can also be applied in order
to deliver lysophospholipid-conjugates to the intestine. These
systems are in general designed to resist the environment of the
non-target sites of the intestine and to undergo a silent phase of
a predetermined time duration, after which site-specific release of
the drug takes place. For example, for colon-targeted drug
delivery, the silent phase is the transit time from the mouth to
the terminal ileum. Examples for time-dependent delivery systems
include, but are not limited to, Pulsincap.RTM. and the delayed
release osmotic dosage form Oros CT.RTM.. Other time-dependent drug
delivery systems comprise multiple coated oral dosage forms and
enteric-coated time-release press-coated tablets (ETP tablets).
[0113] Another approach is the use of pressure-controlled drug
delivery capsules (PCDC) that rely on the physiological luminal
pressure in the intestine which results from peristalsis for drug
release.
[0114] Other systems for site-specific drug delivery, in particular
to the colon, include the CODES.RTM. technology, and the use of
recombinant bacteria, e.g., Lactobacillus sporogenes, as a live
vector system that have been genetically engineered to colonize a
specific part of the intestine and produce the desired drug
there.
[0115] The pharmaceutical composition of the present invention may
further comprise one or more additional agents. Preferably, said
agents are therapeutically effective for treatment of inflammatory
bacterial diseases of the intestine and are selected from the group
of antibiotics, immunosuppressive agents, anti-inflammatory agents,
and anti-diarrheal agents. Of course, the person skilled will
select agents that are therapeutically effective for the treatment
of the specific inflammatory bacterial disease of the intestine to
be addressed.
[0116] The term "antibiotics" is used herein to refer to chemical
substances that kill and/or inhibit growth of certain
microorganisms, in particular certain bacteria. Antibiotics may be
broad-spectrum, i.e. active against a wide range of microorganisms
or narrow-spectrum, i.e. active against one specific microorganism,
or one specific class of microorganisms. Either one may be used
within the pharmaceutical composition or the kit according to the
present invention. The term "antibiotics" refers to natural
antibiotics (i.e., naturally produced by a microorganism),
semisynthetic antibiotics (i.e., natural antibiotic derivatives
that have been chemically modified) and fully synthetic antibiotics
(i.e., antibiotics that are not of natural origin) can be used in
the context of the present invention. Suitable antibiotics for the
use according to the present invention include, but are not limited
to, penicillins such as penicillin G, penicillin V, benzathine
penicillin, methicillin, oxacillin, nafcillin, cloxacillin,
dicloxacillin, ampicillin, amoxicillin, carbenicillin, and
ticarcillin, mezlocillin, piperacillin, amoxicillin, azlocillin,
flucloxacillin; cephalosporins such as cefazolin, cefadroxil,
cephalothin, cephalexin, cefazolin, cephalothin, cephaloclor,
cefamandole, cefoxitin, cefprozil, cefuroxime, cefdinir,
cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftibuten,
ceftizoxime, ceftaroline fosamil, ceftobiprole, ceftriaxone,
cefixime, ceftazidime, cefepime; aminoglycosides such as amikacin,
gentamicin, kanamycin, neomycin, netilmicin, tobramycin,
paromomycin, spectinomycin; glycopeptides such as teicoplanin,
vancomycin, telavancin; lincosamides such as clindamycin,
lincomycin; lipopetides such as daptomycin; macrolides such as
azithromycin, clarithromycin, dirithromycin, erythromycin,
roxithromycin, troleandomycin, telithromycin, spiramycin;
monobactams such as aztreonam; nitrofurans such as furazolidone,
nitrofurantoin; oxazolidonones such as linezolid, posizolid,
radezolid, torezolid; ansamycins such as geldanamycin, herbimycin;
carbacephems such as loracarbef; carbapenems such as ertapenem,
doripenem, iminipem, meropenem; polypeptides such as bacitracin,
colistin, polymyin B; quinolones such as ciprofloxacin, enoxacin,
gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic
acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,
sparfloxacin, temafloxacin; sulfonamides such as mafenide,
sulfacetamide, sulfadiazine, silver sulfadiazine sulfadimethoxine,
sulfamethizole, sulfamethoxazole, sulfasalazine, sulfisoxazole;
tetracyclines such as demeclocycline, doxycycline, minocycline,
oxytetracycline, tetracycline; broxyquinoline, acetarsol,
nifuroxazide, nifurzide, metronidazole.
[0117] Other agents that can be comprised within the pharmaceutical
composition of the invention include anti-inflammatory agents,
immunosuppressive agents, and anti-diarrheal agents.
[0118] "Anti-inflammatory agents" inhibit or reduce inflammation,
e.g., by inducing the production of anti-inflammatory mediators
and/or inhibiting the production of pro-inflammatory mediators.
Suitable anti-inflammatory agents for use according to the present
invention include glucocorticoids, e.g. cortisone, hydrocortisone
(cortisol), prednisone, prednisolone, methylprednisolone,
dexamethasone, betamethasone, triamcinolone, beclometasone,
fludrocortisone acetate, deoxycorticosterone actetate, budesonide,
tixocortol. Immunosuppressive agents inhibit or prevent activity of
the immune system, e.g., by inhibiting lymphocyte proliferation.
Exemplary immunosuppressive agents suitable for use according to
the present invention include, e.g. 5-aminosalicylate (5-ASA),
mesalazine, sulfasalazine, olsalazine, balsalazine, azathioprine,
mycophenolate, methotrexate, cyclosporine. In addition, the
pharmaceutical composition of the invention may further comprise
other agents that are hereinafter referred to as "anti-diarrheal
agents". Diarrhea is often associated with inflammatory diseases of
the intestine. The following agents can be applied to provide
relief from diarrhea and are thus are suitable for the treatment of
inflammatory bacterial diseases of the intestine: flavonoids,
prebiotics, intestinal adsorbents, e.g., charcoal preparations,
bismuth preparations, pectin, kaolin, crospovidone, attapulgite,
diosmectite, and combinations or derivatives thereof,
antipropulsives, e.g., diphenoxylate, opium, loperamide, difenoxin,
loperamide oxide, morphine, and combinations or derivatives
thereof, cholestyramine, cholestipol, heparin, albumin tannate,
ceratonia, calcium compounds, racecadotril, aluminium salicylates,
zinc oxide, and oral rehydration salts. Monoclonal antibodies and
antibody fragments, e.g., infliximab, natalizumab, can also be used
as additional agents in the pharmaceutical composition of the
present invention.
[0119] Further, probiotics can be used. Probiotics are living
microorganisms that upon ingestion in specific numbers have a
beneficial effect, e.g., by inhibiting pathogenic bacteria,
producing cytokines, exerting anti-inflammatory effects or enhance
the digestion and absorption of food. Exemplary probiotics include
Lactobacillus and Bifidobacterium species, Saccharomyces boulardi,
and E. coli Nissle bacteria.
Kit
[0120] It is also envisaged by the present invention that
lysophospholipid-conjugates can be used as part of a kit.
Accordingly, in a further aspect, the present invention also
relates to a kit comprising lysophospholipid-conjugates for use in
a method of treatment of inflammatory bacterial diseases of the
intestine.
[0121] The kit may be a kit of two or more parts, and comprises
lysophospholipid-conjugates and optionally a pharmaceutically
acceptable carrier, diluent or excipient. The components of the kit
may be contained in a container or vials. The kit may further
comprise one or more agents selected from the group of antibiotics,
immunosuppressive agents and anti-inflammatory agents. Suitable
agents have been described in the context of the pharmaceutical
composition of the invention and are equally applicable for the
kit, mutatis mutandis. It is envisaged that the agents are applied
simultaneously, or sequentially, or separately with respect to the
administration of the lysophospholipid-conjugate. The present
invention further encompasses the application of the agents via
different administration routes.
[0122] Therefore, suitable agents for use in the kit further
comprise, e.g., intravenously administered glucocorticoids for
simultaneous, or sequential, or separate use with an orally
administered lysophospholipid-conjugate.
[0123] In general, it is envisaged that the
lysophospholipid-conjugate of the present invention and the one or
more additional agents described herein, when provided in form of
the pharmaceutical composition or the kit of the present invention,
are used for combination therapy.
Method of Treatment
[0124] Another aspect of the present invention is a method of
treatment of inflammatory bacterial diseases of the intestine in a
subject in need thereof, comprising administering a therapeutically
effective amount of an inhibitor of bacterial PL, in particular
bacterial PLA.sub.2, which is preferably a
lysophospholipid-conjugate, to said subject. The person skilled in
the art will acknowledge that the embodiments described herein in
the context of the use of the bacterial phospholipase A inhibitor,
the pharmaceutical composition and the kit of the present invention
are also applicable to the method of treatment, mutatis
mutandis.
[0125] The method according to the present invention may further
comprise additional steps that are suitable for treating
inflammatory bacterial diseases of the intestine. For example,
lysophospholipid-conjugate treatment may be combined with a
specific diet, e.g., a restricted or low fibre diet. It is also
envisaged that the method of treatment according to the present
invention further comprises administering one or more agents
selected from the group of antibiotic, anti-inflammatory,
immunosuppressive and anti-diarrheal agents. Said agents can be
administered prior to, simultaneously, or after the inhibitor of
bacterial PL, in particular bacterial PLA.sub.2, which is
preferably a lysophospholipid-conjugate.
[0126] In another aspect, the present invention provides a method
of producing of a pharmaceutical composition comprising a
lysophospholipid-conjugate. The use of lysophospholipid-conjugates
for the preparation of a pharmaceutical composition is another
aspect of the present invention.
[0127] A better understanding of the present invention and of its
advantages will be had from the following examples, offered for
illustrative purposes only. The examples are not intended to limit
the scope of the present invention in any way.
Example 1
Preparation of Bacterial Lysates
Cultivation
[0128] Suspensions of the respective bacterial strain in 0.9% NaCl
were prepared in Sarstedt tubes and adjusted to 1.0
(3.times.10.sup.8 CFU/ml) with opacimeter (Densimat.RTM. or
Densicheck.RTM.). Subsequently, a BHI (brain heart infusion,
purchased from Merck) was inoculated with 500 .mu.l of the
suspension and incubated at 37.degree. C. aerob or in an airtight
jar with BD Gas Pak.TM. for 16 hours overnight on a shaker.
Incubation with Phospholipase Inhibitor
[0129] BHI was centrifuged for 10 min at 3000 g. The pellet was
resuspended in 1 ml
[0130] PBS and PL-inhibitor (UDCA-LPE, 100 .mu.M; 5 .mu.l stock (20
mM) in 1 ml) or solvent control (Ethanol) and incubated for 1 hour
on the shaker.
Preparation of Bacterial Lysates
[0131] The bacterial suspensions were washed twice in 2.times.PBS
(centrifugation for 10 min at 3000 g). The pellet was resuspended
in 150 .mu.l 1.5% Triton X-100 (in PBS). Subsequently, 1.5 ml
Eppendorf tubes were filled with sterile glass beads in the lower
third of the cone and the bacterial suspension was transferred to
the Eppendorf tubes and put on ice.
[0132] Then, the suspension was homogenized 4.times.1 min in the
bead beater (Biospec.RTM.), and put on ice for 1 min after every
homogenization step. The suspension was then incubated for 60 min
on the shaker on ice and centrifuged for 15 min at 10 000 g and
4.degree. C. The supernatant was then removed, put on ice and used
for determination of the protein concentration (according to
Pierce, Thermo Scientific) and determination of PLA activity.
Example 2
Determination of PLA.sub.2 Activity
[0133] PLA.sub.2 activity was determined as described by Bhat, et
al. (1993).
Reagents
2.times.PLA.sub.2 Assay Buffer
TABLE-US-00001 [0134] Ca.sup.2+-dependent_Pl,A2
Ca.sup.2+-independent PLA.sub.2 300 mM NaCl 300 mM NaCl 0.5% Triton
X100 0.5% Triton X100 60% glycerol 60% glycerol 20 mM CaCl.sub.2 4
mM EGTA 160 mM HEPES, pH 7.4 10 mM HEPES, pH 7.4 then added with 2
mg/ml BSA then added with 2 mg/ml BSA for assay for assay
(typically 5 mg BSA/ (typically 5 mg BSA/10 ml buffer) 10 ml
buffer)
cPLA.sub.2 Colour Reagent
[0135] 5,5'dithiobis-2-nitrobenzoic acid stock (DTNB, FW=396.35
g/m01).fwdarw.25 mM DTNB in 0.5 M Tris (pH 8.0), 4.75 mM EGTA
Assay
[0136] For substrate preparation, 125 .mu.g substrate arachidonyl
PC (AAPC) was pipetted into Eppendorf reaction tubes (therefore,
the volume to pipet from AAPC stocks in ethanol as supplied by
Cayman Chemicals was calculated). The aliquots were dried with
speed vac for 10-15 min at 30.degree. C. and stored at -20.degree.
C. Before next use, the pellet was airdried under nitrogen to
remove residual ethanol.
[0137] Immediately before performing the assay, 125 .mu.g substrate
pellet was resuspended in 95 .mu.l 2.times.PLA.sub.2 buffer by
vortexing. 95 .mu.l H.sub.2O was added and mixed well by vortexing,
thereby yielding 1.times.PLA.sub.2 buffer=190 .mu.l. 10 .mu.l of
the sample was pipetted into a 96 well plate and add 190 .mu.l of
substrate solution was added. The plate was shaken for 30 sec to
mix and covered with a plate cover. The plate was incubated at room
temperature for 1 hour. Subsequently 10 .mu.l of 25 mM DTNB stock
was added into each well to terminate the reaction, the plate was
shaken and incubated for 5 min at room temperature.
[0138] Finally, the absorption was read at 405 nm and 595 nm.
Calculation
[0139] A unit of PLA.sub.2 is defined as a nmol of product formed
by 1 ml sample in 1 h incubation. PLA.sub.2 activity was determined
using the following formula: PLA.sub.2 activity (unit)=(OD405
nm-OD.sub.595nm).times.78.62.times.50
[0140] Wherein 78.62 is the amount of product (nmol) producing
OD.sub.405 nm of 1.0 in 0.2 ml, and 50 is the correction factor for
10 .mu.l of sample to 1 ml.
[0141] AAPC at 125 .mu.g per sample in 200 .mu.l gives 1 mM final.
One may also use 62.5 .mu.g per sample giving 0.5 mM final
concentration (to save material for low activity samples or for
sorcening purposes).
CITED LITERATURE
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Design. Chem & Biol, Volume 10, pp. 787-797. [0143] Bhat, M.
K., Mueller-Harvey, I., Summer, I. G. & Goodenough, P. W.,
1993. Simplified methods for the synthesis of
2-hexadeconylthio-1-ethylphosphorylcholine and for the
determination of phospholipase A2 activity. Biochim Biophys Act,
Volume 1166, pp. 244-250. [0144] Chamulitrat, W. et al., 2009. Bile
Salt-Phospholipid Conjugate Ursodeoxycholyl
Lysophosphatidylethanolamide as a Hepatoprotective Agent.
Hepatology, 50(1), pp. 143-154. [0145] Chamulitrat, W. et al.,
2012. Hepatoprotectant ursodeoxycholyl lysophosphatidylethanolamide
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