U.S. patent application number 15/042923 was filed with the patent office on 2016-08-25 for host defense protein (hdp) mimetics for prophylaxis and/or treatment of inflammatory diseases of the gastrointestinal tract.
This patent application is currently assigned to CELLCEUTIX CORPORATION. The applicant listed for this patent is CELLCEUTIX CORPORATION. Invention is credited to Krishna MENON.
Application Number | 20160243117 15/042923 |
Document ID | / |
Family ID | 56689106 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160243117 |
Kind Code |
A1 |
MENON; Krishna |
August 25, 2016 |
Host Defense Protein (HDP) Mimetics For Prophylaxis And/Or
Treatment Of Inflammatory Diseases Of The Gastrointestinal
Tract
Abstract
The present invention provides methods for treating and/or
preventing inflammatory diseases of the gastrointestinal tract with
one or more compounds, or pharmaceutically acceptable salts
thereof, disclosed herein, and the use of compositions comprising
the same.
Inventors: |
MENON; Krishna; (Beverly,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CELLCEUTIX CORPORATION |
BEVERLY |
MA |
US |
|
|
Assignee: |
CELLCEUTIX CORPORATION
BEVERLY
MA
|
Family ID: |
56689106 |
Appl. No.: |
15/042923 |
Filed: |
February 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62118950 |
Feb 20, 2015 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 31/506 20130101; A61K 31/167 20130101; A61P 1/04 20180101;
A61P 29/00 20180101; A61K 45/06 20130101; A61P 1/00 20180101 |
International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 45/06 20060101 A61K045/06; A61K 31/167 20060101
A61K031/167 |
Claims
1. A method of prophylaxis and/or treatment of inflammatory
diseases of the gastrointestinal tract in a mammal comprising
administering to the mammal in need of such prophylaxis and/or
treatment a therapeutically effective amount of a compound selected
from PMX-30063 (brilacidin) and PMX-60056 (delparantag) and
pharmaceutically acceptable salts thereof.
2. A method according to claim 1, wherein the disease is
inflammatory bowel disease, ulcerative colitis, collagenous
colitis, lymphocytic colitis, Crohn's disease, or irritable bowel
syndrome.
3. A method according to claim 1 wherein said compound is
administered together with an antibiotic.
4. A method according to claim 7, wherein the disease is
inflammatory bowel disease, ulcerative colitis, collagenous
colitis, lymphocytic colitis, Crohn's disease, or irritable bowel
syndrome.
Description
CROSS REFERENCE APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application 62/118,950 filed Feb. 20, 2015, and incorporates the
same by reference.
[0002] The present invention relates to the use of host defense
protein (HDP) mimetics, including brilacidin (PMX-30063) and
delparantag (PMX-60056), and the pharmaceutically acceptable salts
thereof, and pharmaceutical compositions thereof in preventing and
treating inflammatory diseases of the gastrointestinal tract.
BACKGROUND OF THE INVENTION
[0003] Inflammatory diseases of the gastrointestinal tract involves
chronic inflammation of all or part of the digestive tract.
Inflammatory diseases of the gastrointestinal tract is not a single
disorder. It is the term for a group of disorders that cause
prolonged inflammation of the digestive tract. Such a condition can
be chronic, sub-chronic or acute and can be mild, moderate or
severe according to the condition. Many diseases are included in
this umbrella term. The inflammation of the digestive tract in all
parts of the digestive tract, irrespective of the anatomical area
is included in this treatment. The main symptom of active disease
is usually constant diarrhea mixed with blood, of gradual
onset.
[0004] The digestive tract is composed of the mouth, esophagus,
stomach, small intestine, large intestine, colon, rectum, and anus.
It is responsible for breaking down food, extracting the nutrients,
and removing any unusable material and waste products. Inflammation
anywhere along the digestive tract is included in this treatment
process. The treatment includes all these conditions:
[0005] Ulcerative colitis (UC) is an inflammatory bowel disease
(IBD) that causes long-lasting inflammation and sores (ulcers) in
the innermost lining of the large intestine (colon) and rectum. The
predominant symptom is diarrhea, associated with blood in the
stool, occasionally with fever and abdominal pain. The onset may be
insidious or acute (mild-60%, moderate to severe-25%,
fulminant-15%). A severe attack may be accompanied by dilation of
the colon, known as toxic megacolon, which is associated with
significant morbidity and mortality. The complications of
ulcerative colitis are massive hemorrhage, stricture formation,
fulminant colitis (toxic megacolon) and colon cancer. Ulcerative
colitis begins in the rectum and may proceed proximally to involve
either a segment of colon or the entire colon; 60% to 75% of
ulcerative colitis patients have no disease proximal to the
sigmoid. Pancolitis occurs in 20% of patients. Ulcerative colitis
has an incidence of 1 to 20 cases per 100,000 individuals per year,
and a prevalence of 8 to 246 per 100,000 individuals. Ulcerative
colitis is classified according to the location of inflammation and
severity of symptoms: [0006] Ulcerative proctitis--Inflammation is
confined to the area closest to the anus (rectum), and rectal
bleeding may be the only sign of the disease. This form of
ulcerative colitis tends to be the mildest. [0007] Ulcerative
proctosigmoiditis--Inflammation involves the rectum and sigmoid
colon (lower end of the colon). Signs and symptoms include bloody
diarrhea, abdominal cramps and pain, and an inability to move the
bowels in spite of the urge to do so (tenesmus). [0008] Left-sided
colitis--Inflammation extends from the rectum up through the
sigmoid and descending colon. Signs and symptoms include bloody
diarrhea, abdominal cramping and pain on the left side, and
unintended weight loss. [0009] Pancolitis--Pancolitis often affects
the entire colon and causes bouts of bloody diarrhea that may be
severe, abdominal cramps and pain, fatigue, and significant weight
loss. [0010] Acute severe ulcerative colitis--Previously called
fulminant colitis, this rare form of colitis affects the entire
colon and causes severe pain, profuse diarrhea, bleeding, fever and
inability to eat.
[0011] Collagenous colitis and lymphocytic colitis also are
considered inflammatory bowel diseases but are usually regarded
separately from classic inflammatory bowel disease.
[0012] Crohn's disease is also an inflammatory bowel disease that
causes inflammation of the lining of the digestive tract. In
Crohn's disease, inflammation often spreads deep into affected
tissues. The inflammation can involve different areas of the
digestive tract--the large intestine, small intestine or both.
Three major patterns of disease distribution are ileocecal (40%),
small intestine (30%) and colon (25%). It is much less common to
have involvement of the esophagus, stomach and duodenum. The most
common symptoms are diarrhea, abdominal pain and weight loss. The
disease is often present for months or years prior to diagnosis. In
children, growth retardation may be one of major sign of indication
of disease. The presence of fistula, abscess and fissures, which
are commonly called as perianal disease is a distinguishing factor
from ulcerative colitis. Crohn's disease is also a remitting and
relapsing disease like ulcerative colitis: more than 60% of
patients will require surgery within 10 years, 70% of patients will
have endoscopic recurrence within one year of surgery, and 50% of
patients will have symptomatic recurrence within 4 years. In
Crohn's disease, the inflammation is more commonly focal, which
leads to bowel wall thickening, becoming edematous and fibrotic,
and the mesentery may become infiltrated with fat. The major
complications of are stenosis, extensive ileal disease, extensive
mucosal damage, fistulae, urinary calcium oxalate stones and
carcinoma, while massive hemorrhage is less common. Crohn's disease
may involve inflammation in different parts of the digestive tract
in different people. The most common areas affected are the last
part of the small intestine (ileum) and the colon. Inflammation may
be confined to the bowel wall, which can lead to narrowing from
inflammation or scarring or both (fibrostenosis), or may tunnel
through the bowel wall (fistula). Narrowing may lead to a blockage
(obstruction). Obstructions, stenosis and fistulas are not
associated with ulcerative colitis.
[0013] Irritable bowel syndrome (IBS) is another disease that
affects the digestive tract characterized by chronic abdominal
pain, bloating, and diarrhea or constipation. IBS has no known
specific cause, but can occur after an infection or stress. There
is no cure, but treatments include dietary changes, medication,
acupuncture, psychotherapy, and herbal remedies such as peppermint
oil. Medications include antidepressants such as clozapine or
olanzapine, laxatives, antidiarrheal, serotonin antagonists (5HT3),
such as ondansetron, clozapine or ondansetron, or serotonin
reuptake inhibitors (SSRIs), anti-spasmodics, such as hyoscyamine
or dicyclomine, proton pump inhibitors (PPIs), magnesium aluminum
silicates, alverine citrate drugs and rifaximin. IBS affects about
15% of the US population.
[0014] Inflammatory Bowel Disease
[0015] The exact causes of IBD are not yet fully understood.
Accumulating evidence suggests that the immune response has long
been involved in pathogenesis of IBD.
[0016] The intestinal microbiome consists of the microorganisms
that inhabit the gut. Host-microbiome interactions can be mutually
beneficial or can be deleterious, inciting intestinal inflammation.
The intestinal epithelium at the interface between the intestinal
microbiome and the lymphoid tissue associated with the
gastrointestinal system plays a critical role in shaping the
mucosal immune response. Intestinal epithelial cells are a physical
barrier against excessive entry of bacteria and other antigens from
the intestinal lumen into the circulation. Additional defenses
against bacterial invasion consist of specialized epithelial cells,
including goblet cells and Paneth cells. Goblet cells regulate the
production of mucus and factors that contribute to epithelial
repair and regulation of inflammation. Paneth cells secrete
antimicrobial peptides such as .alpha.-defensins. Intestinal mucus
overlies the epithelium, thereby limiting contact between bacteria
and epithelial cells. In inflammatory bowel disease, however, the
inflammatory response often results in continued epithelial injury,
which causes erosion, ulcerations, and decrease in the production
of defensin. The result is increased exposure to intestinal
microbiota and amplification of inflammatory response.
[0017] The intestinal lamina propria contains a complex population
of immune cells that balance the requirement for immune tolerance
of luminal microbiota with the need to defend against the pathogen,
excessive entry of luminal microbiota, or both. The hallmark of
active inflammatory bowel disease is a pronounced infiltration into
the lamina propria of innate immune cells (neutrophils,
macrophages, dendritic cells, and natural killer T cells) and
adaptive immune cells (T cells and B cells). Increased numbers and
activation of these cells in the intestinal mucosa elevate local
levels of TNF-.alpha., interleukin-1.beta., interleukin-6 (IL-6),
interferon-gamma (IFN-.gamma.), and cytokines of the
interleukin-23-Th17 pathway.
[0018] The proinflammatory cytokine TNF-alpha has been identified
as playing a pivotal role in the inflammatory cascade that causes
chronic inflammation, as observed in IBD. Levels of circulating
IL-6 are elevated in several inflammatory diseases including
Crohn's disease. IL-6 is key modulator of inflammatory response.
Influencing the production of this cytokine can change the balance
of effector CD4+ T cell subsets and induce B cell antibody
production. Moreover, given that IL-6 is mostly produced from
innate cells such as macrophages, neutrophils and mast cells, it is
a strategic bridge between the innate and the adaptive system.
[0019] There are no curative therapeutic treatments for IBD. The
only cure for UC is surgical removal of the large intestine, which
reduces quality of life. To allay symptoms, dietary and lifestyle
changes are important. Anti-inflammatory steroids are commonly used
but they can also induce severe side effects. One anti-inflammatory
used for IBD is mesalazine (also known as mesalamine or
5-aminosalicyclic acid), but it is more effective in UC than in
Crohn's disease. Immunomodulators such as azathioprine,
methotrexate, infliximab, adalimumab, certolizumab and natalizumab
are also used for Crohn's disease. Long term use of antibiotics is
somewhat effective for Crohn's disease, but is not effective in UC.
Chronic use of antibiotics carries the risk of developing
drug-resistant microbes. Some individuals resort to probiotics,
fish oil, acupuncture, or homeopathic treatments to try to
alleviate symptoms. In addition to the effects on the digestive
system, IBD can also lead to nutrient deficiency, iritis, uveitis,
skin rashes, arthritis, primary sclerosing cholangitis, ankylosing
spondylitis, pyoderma gangrenosum, and erythema nodosum.
Inflammatory bowel disease affects approximately 1.4 million
Americans, and its peak onset is in persons 15 to 30 years of
age.
[0020] Development of Host Defense Protein Mimetic for Inflammatory
Diseases
[0021] Host defense peptides were originally studied for their
direct antimicrobial activities and have also been found to exhibit
multifaceted immunomodulatory activities. Despite the large
diversity observed in HDPs, they generally adopt highly conserved
amphipathic topologies in which the hydrophilic and hydrophobic
side chains segregate into distinctly opposing regions or faces of
the molecule. An example of a molecule with amphipathic structure
is magainin 2. Magainins were first discovered in the African
clawed frog [Zasloff M. Magainins, a class of antimicrobial
peptides from Xenopus skin: isolation, characterization of two
active forms, and partial cDNA sequence of a precursor. PNAS
84:5449-5453 (1987)].
[0022] Biological macromolecules, including proteins and RNA,
generally adapt unique folded conformations that are responsible
for their remarkable properties. Until recently, the process of
folding was considered a mystery, but as the fields of protein
folding, RNA structure and molecular organization have evolved, it
has become increasing possible to design non-biological molecules
that fold into unique structures. To mimic natural proteins,
investigators have synthesized oligomers by sequentially coupling
individual monomer units to provide homogeneous linear molecule of
entirely uniform sequence and chain length. Oligomers that fold
into well-defined secondary structure have come to be foldamers
(Hill D J, et al., Chem. Rev. 2001, 101, 3893-4012; Home W S, et
al., Acc. Chem. Res. 2008, 41, 1399-1408; Patch J A, Barron A E, J.
Am. Chem. Soc. 2003, 125, 12092-12093). The structural simplicity
and relative ease of synthesis of many foldamers allows them to be
used as three-dimensional scaffolds for molecular recognition.
[0023] An example of the design, synthesis, and antimicrobial
activity of arylamide polymers and oligomers is presented in Tew et
al. (Tew et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 5110-5114),
which is incorporated herein by reference in its entirety. These
compounds, including brilacidin (PMX-30063) and delparantag
(PMX-60056), were synthesized to mimic naturally occurring
antimicrobial peptides. Both PMX-30063 and PMX-60056 have similar
spatio-topology which mimic the HDP structure in space.
[0024] Numerous studies with linear and cyclic peptides have
strongly supported the hypothesis that their physicochemical
properties, rather than any precise sequence, are responsible for
their ability to selectively disrupt membranes. Therefore, a series
of non-peptidic analogues of the HDPs (HDP mimetics) has been
developed and evaluated for their potential antibacterial activity.
Optimization of both total charge and the hydrophobic content
proved to be particularly important to the design of compounds that
are highly active and nontoxic in animals. Host defense proteins
(HDP) are key components of innate immune systems and play dual
roles: rapid microbial killing and subsequent immune modulation.
PMX-30063 [N.sup.4,
N.sup.6-bis(2-((R)-pyrrolidin-3-yloxy)-3-((4-carbamoylbutyl)
guanidine)-5-(trifluoromethyl)phenyl)pyrimidine-4,6-dicarboxamide
tetrahydrochloride salt, molecular formula:
C.sub.40H.sub.50F.sub.6N.sub.14O.sub.6.4 HCl, USAN name:
brilacidin] and PMX-60056
[Tetra-[(L)-lysyl-5-amino-o-methylsalicylamide, molecular formula:
C.sub.56H.sub.84Cl.sub.5N.sub.13O.sub.12.5 HCl] are non-peptide
mimics of HDP that have distinct advantages over proteins for
pharmaceutical uses. These HDP mimetics demonstrated rapid
bactericidal activity as well as anti-inflammatory and
immunomodulatory effects (Som A, Navasa N, Percher A, Scott R W,
Tew G N, Anguita. Identification of Synthetic Host Defense Peptide
Mimics That Exert Dual Antimicrobial and Anti-Inflammatory
Activities. Clin and Vaccine Immunol. 2012, 19:1784-1791:
Scorciapino M A, Rinaldi A C. Antimicrobial peptidomimetics:
reinterpreting nature to deliver innovative therapeutics. Patricia
Mendez-Samperio. Front. Immunol 2012, Vol 3, Article 171;
Peptidomimetics as a new generation of antimicrobial agents:
current progress. Infection and Drug Resistance 2014; 7:229-237). A
wide range of immunomodulatory functions have been defined for HDP
that result in net suppression of potentially harmful
proinflammatory response (Hilchie A L, et al., Nat. Chem. Biol.
2013, 9, 761-768). Their diverse immunomodulatory capability
includes the modulation of pro- and anti-inflammatory response
(Mansour S C, et al., Trends in Immunology 2014, 35, 443-450) and
acting as immunomodulators in both innate and adaptive immune
response (Wong J H, et al Curr Protein Pept Sci. 2013, 14,
504-514). Although the anti-inflammatory function of HDP was known,
the molecular mechanism of action of HDP was poorly understood. The
present inventors hypothesized that HDP may be functioning through
the cyclic AMP/cyclic GMP pathways in suppression of
proinflammatory response. The present inventors tested this
hypothesis and found that the Host Defense Protein (HDP) mimetics
PMX-30063 and PMX-60056 inhibit phosphodiesterase (PDE) in vitro,
as detailed herein. Phosphodiesterase is a family of enzymes that
catalyze the breakdown of signaling molecule cyclic AMP/or cyclic
GMP. cAMP and cGMP are ubiquitous secondary-messenger signaling
molecules produced by a large family of cyclases that participate
in a multitude of signaling processes.
[0025] PDE inhibitors have shown anti-inflammatory activity in a
variety of preclinical models (Martinez A, Gil C. Expert opinion on
therapeutic patents 2014, 24, 1311-1321). PDE4 has received
particular attention due to the fact that all of the inflammatory
and immunomodulatory cells not only express PDE4, but also that
specific functions of these cells are broadly inhibited by
selective PDE4 inhibitors. PDE4 is a predominant phosphodiesterase
expressed in neutrophils, T cells and macrophages. PDE4 inhibitors
reduce neutrophil chemotaxis, recruitment and activation; inhibit
the activation of CD4+ and CD8+ T cells; and inhibit monocytes
chemotaxis (Tamimi A, et al. Resp. Med 2012, 106, 319-328). The
discovery by the present inventors that PMX-30063 and PMX-60056 are
PDE inhibitors, discussed below, indicates that these compounds
described herein are expected to be useful in the treatment of
inflammatory diseases of the gastrointestinal tract and should be
further investigated in a clinical study as discussed in Example
19.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 illustrates that PMX-30063 inhibited PDE4 with an
IC50 in the 3 .mu.M range (n=5).
[0027] FIG. 2 illustrates that PMX-60056 inhibited PDE4 with an
IC50 in the 3 .mu.M range (n=5).
[0028] FIG. 3 illustrates that PMX-30063 inhibited PDE3 at an IC50
of 1.5.+-.0.2 .mu.M (n=4).
[0029] FIG. 4 illustrates that PMX-60056 inhibited PDE3 at an IC50
of 3 .mu.M.
[0030] FIG. 5 illustrates that PMX-30063 inhibited the LPS-induced
TNF-.alpha. production in rat macrophages.
[0031] FIG. 6 illustrates that PMX-60056 inhibited the LPS-induced
TNF-.alpha. production in rat macrophages.
[0032] FIG. 7 illustrates that PMX-30063 inhibited MCP-1 induction
after LPS stimulation of rat macrophages with a minimum of a 25%
decrease in MCP-1 levels at 0.5 .mu.M.
[0033] FIG. 8 illustrates that PMX-60056 inhibited MCP-1 induction
after LPS stimulation of rat macrophages with a minimum of 25%
decrease in MCP-1 levels at 0.5 .mu.M.
[0034] FIG. 9 illustrates that after LPS stimulation of rat
macrophages a 50% decrease in MMP-9 levels at a 12.5 .mu.M
concentration of PMX-30063 was observed.
[0035] FIG. 10 illustrates that PMX-30063 inhibited IL-6 induction
after LPS stimulation of rat macrophages with about a 50% decrease
in IL-6 levels at a 0.5 .mu.M of PMX-30063 being observed.
[0036] FIG. 11 illustrates that when PMX-30063 was evaluated for
plasma and small intestine concentration following 10 mg/kg given
orally or 5 mg/kg given IV in male Balb/c mice, the peak
concentration of PMX60073 given IV was 48,415 ng/mL, whereas, peak
concentration in plasma was 33.7 ng/mL when given PO.
[0037] FIG. 12 illustrates that when PMX-30063 was evaluated for
plasma and small intestine concentration following 10 mg/kg given
orally or 5 mg/kg given IV in male Balb/c mice (Study 16009-12001),
after PO administration, the peak concentration in the small
intestine tissue was 38,941 ng per gram of tissue.
[0038] FIG. 13 illustrates the intestine/plasma concentration ratio
following oral administration of PMX-30063 calculated based on data
derived from FIGS. 12 and 13.
[0039] FIG. 14 illustrates that in an in vivo ulcerative colitis
model, intestine weights were reduced, but not significantly,
compared to untreated controls following rectal administration of
PMX-30063.
[0040] FIG. 15 illustrates that a dose dependent decrease in
ulcerative colitis score following rectal administration of
PMX-30063 was observed; however, only in animals treated with 400
mg/kg was the score significantly reduced compared to untreated
controls; and that animals treated with 5-ASA showed no significant
efficacy.
SUMMARY OF THE INVENTION
[0041] The present invention relates to methods of prophylaxis
and/or treatment of inflammatory diseases of the gastrointestinal
tract in a mammal comprising administering to the mammal in need of
such prophylaxis and/or treatment a therapeutically effective
amount of a compound selected from brilacidin (PMX-30063) and
delparantag (PMX-60056) and pharmaceutically acceptable salts
thereof. In one embodiment, brilacidin and delparantag are
administered together. In another embodiment, the inflammatory
disease is inflammatory bowel disease ulcerative colitis,
collagenous colitis, lymphocytic colitis, Crohn's disease, or
irritable bowel syndrome. In another embodiment, said compound is
administered together with an antibiotic other than brilacidin or
delparantag.
[0042] The present invention also relates to the use of
pharmaceutical compositions for treatment of inflammatory diseases
of the gastrointestinal tract comprising a therapeutically
effective amount of a compound selected from brilacidin and
delparantag and pharmaceutically acceptable salts thereof and a
pharmaceutically acceptable carrier. Diseases include, but are not
limited to inflammatory bowel disease ulcerative colitis,
collagenous colitis, lymphocytic colitis, Crohn's disease, and
irritable bowel syndrome. In one embodiment, the pharmaceutical
composition comprises both brilacidin and delparantag. In another
embodiment, the composition comprises brilacidin or delparantag and
an antibiotic other than brilacidin. In another embodiment, the
composition comprises brilacidin or delparantag and is administered
together with an antibiotic other than brilacidin.
[0043] The present invention also provides active compounds, or
pharmaceutical compositions comprising the same, for use in the
preparation of a medicament for prophylaxis and/or treatment of
inflammatory diseases of the gastrointestinal tract in a patient.
In one embodiment, the pharmaceutical composition comprises both
brilacidin and delparantag. In another embodiment, the composition
comprises an antibiotic other than brilacidin.
[0044] The structural formulae of brilacidin and delparantag are
shown below.
##STR00001##
[0045] The present invention also provides pharmaceutical
compositions for prophylaxis and treatment of inflammatory diseases
of the gastrointestinal tract in a mammal comprising an effective
amount of one or more of the compounds described above, or one or
more salts thereof, and a pharmaceutically acceptable carrier.
Suitable compositions include, but are not limited to, oral
non-absorbed compositions. Suitable compositions also include, but
are not limited to saline, water, cyclodextrin solutions, and
buffered solutions of pH 3-9.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The starting materials, which are required to prepare the
compound brilacidin and the pharmaceutically acceptable salts
thereof, are commercially available in bulk. The compound
brilacidin and the salts are prepared by
[0047] a) reacting (R)-(-)-N-Boc-3-pyrrolidinol with
2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene in the presence of
potassium ter-butoxide to form a compound having Formula I
##STR00002##
[0048] b) reacting the compound of Formula I with an alcohol and a
transition metal catalyst in the presence of hydrogen to form a
compound of Formula II
##STR00003##
[0049] c) adding the compound of Formula II and
pyrimidine-4,6-dicarboxylic acid in the presence of
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in
pyridine to form a compound of Formula III
##STR00004##
[0050] d) reacting the compound of Formula III with
5-(carbobenzoxyamino)valeric acid to form a compound of Formula
IV
##STR00005##
[0051] e) reducing the resultant compound of formula IV in the
presence of an alcohol, a transition metal catalyst, and hydrogen
to afford formula V
##STR00006##
[0052] f) reacting the resultant compound of V with di-N-Boc
pyrazole in the presence of base to provide compound of formula
VI;
##STR00007##
and
[0053] deprotecting the compound of Formula VI using acid to
produce PMX-30063 (brilacidin); and, if desired, preparing a
pharmaceutically acceptable salt.
[0054] The compound delparantag and the pharmaceutically acceptable
salts thereof, are prepared by (a) removing the Cbz groups from a
compound of Formula VII
##STR00008##
[0055] or a pharmaceutically acceptable salt thereof, using
hydrogen gas and transitional metal catalyst to form the
delparantag, or pharmaceutically acceptable salt thereof, and (b)
optionally isolating the delparantag or pharmaceutically acceptable
salt thereof and if desired preparing a pharmaceutically acceptable
salt from the compound delparantag.
[0056] Examples of suitable hydrogenation/hydrogenolysis conditions
that can be used in step a) include those conditions known in the
art of synthetic organic chemistry. For example, H.sub.2 gas and a
transitional metal catalysts such as Pd--C (5-10%), Pd(OH).sub.2,
Platinum metal and Raney-Nickel can be used The reaction can be
carried out at a suitable temperature, for example, ambient
temperature (about 20-25.degree. C.) or up to a temperature at
which the solvent in the reaction mixture is at reflux.
[0057] PMX-60056, or a pharmaceutically acceptable salt thereof,
can be isolated (including purification) by various techniques
known in the art. For example, in some cases it might be desired to
isolate the reaction product by filtration and subsequent
precipitation of the product from the filtrate or crystallization
For another example, in some cases it might be desired to isolate
the reaction product by extraction with an appropriate solvent or
mixture of solvents, for example diethyl ether or ethyl acetate,
and subsequent chromatography on silica gel such as
[0058] 3-mercaptopropyl ethylsufided silica gel or by trituration
with an appropriate solvent such as methylene chloride, methanol or
a mixture of solvents. The recrystallization can be performed with
a solvent, or with a mixture of solvents. In some embodiments,
isolation of product includes removal of transitional metal
catalyst from the reaction product and levels of metal catalyst can
be determined by a suitable method such as Inductively Coupled
Plasma (ICP). The purity of the isolated (or purified) product can
be determined by a suitable method such as using HPLC.
[0059] The starting materials; methyl 5-amino-2-methoxybenzoate and
Boc-Lys(Cbz)-OH are commercially available, and can be easily
obtained from commercial suppliers for the preparation of compound
formula VII.
[0060] In some embodiments, the compound of Formula VII, or
pharmaceutically acceptable salt thereof, used in step a) can be
prepared by:
[0061] c) removing the Boc group to from a compound of Formula
VIII:
##STR00009##
[0062] or pharmaceutically acceptable salt thereof, to form the
compound of Formula VII or pharmaceutically acceptable salt
thereof.
[0063] Removal of the Boc group can be carried out by using a
suitable reagent such as an acid (e.g., H.sub.3PO.sub.4, TFA, HCl,
TsOH, or H.sub.2SO.sub.4) or TMSOTf/2,6-lutidine or a solution of
reagent, in a suitable polar or halogenated solvent such as THF,
EtOAc, dioxane, dioxane, water, or CH.sub.2Cl.sub.2 or a mixture of
any two or more of these solvents at a suitable temperature for
example, ambient temperature (about 20-25.degree. C.). The reaction
product of step c) can be isolated as either a salt of the compound
of Formula VII or free base, neutralizing with NaOH as a base to
neutralize the acid salt.
[0064] In some embodiments, the compound of Formula VIII, or
pharmaceutically acceptable salt thereof, used in step c) can be
prepared by:
[0065] d) reacting a compound of Formula IX:
##STR00010##
[0066] or a pharmaceutically acceptable salt thereof with a
compound of Formula X:
##STR00011##
[0067] or pharmaceutically acceptable salt thereof.
[0068] The reaction of step d) can be carried out in the presence
of a coupling reagents such as dimethylamino)phosphonium
hexafluorophosphate (BOP),
[0069] 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU),
[0070] O-(7-azabenzotriazol l-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU),
[0071] 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,
dicyclohexylcarbodimide (DCC), N,N'-diisopropylcarbodiimide (DIC),
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(Py-BOP), N,N'-carbonyldiimidazole (CDI), N-hydroxybenzotriazole
(HOBt), 1H-Benzotriazolium
1-[bis(dimethyl-amino)methylene]-5-chloro-hexafluorophosphate
(1-),3-oxide (HCTU), a suitable 1,3,5-triazine derivative (see, for
example, Kaminski, Tetrahedron Letters, 1985, 26, 2901-2904;
examples of suitable 1,3,5-triazine derivatives include, but are
not limited to, 2,4,6-trichloro-1,3,5-triazine,
2-chloro-4,6-diphenoxy-1,3,5-triazine;
2-chloro-4,6-dibenzyloxy-1,3,5-triazine;
2-chloro-4,6-dimethoxy-1,3,5-triazine;
2,4-dichloro-6-phenoxy-1,3,5-triazine;
2,4-dichloro-6-benzyloxy-1,3,5-triazine; or
2,4-dichloro-6-methoxy-1,3,5-triazine), and a mixture of two or
more thereof. If desired, the coupling reagent in step d) includes
a mixture of EDAC and HOBt and an organic base, to form the
compound of Formula VIII, or pharmaceutically acceptable salt
thereof.
[0072] The coupling reagent in step d) is chosen from those that
prevent racemization of any chiral center present in the reactants
(and/or reaction products) (see, Konig et al., Chem. Ber., 1970,
103, 788; listing HOBt as such a coupling reagent). The coupling
reaction can be carried out in the presence of a suitable base.
Examples of suitable bases include, but are not limited to,
triethylamine (TEA), diisopropylethylamine (DIEA),
N-methylmorpholine (NMM), N--N-dimethylaminopyridine (DMAP),
pyridine, and imidazole.
[0073] The reaction in step d) can be carried out in a suitable
solvent such as a polar solvent, for example, an ether, (e.g.,
tetrahydrofuran (THF), a halogenated solvent (such as
dichloromethane (DCM) or chloroform), or a mixture of suitable
solvents at a suitable temperature, for example, ambient
temperature (20-25.degree. C.)) or up to a temperature at which the
solvent in the reaction mixture is at reflux. The reaction product
of step d) can be isolated (including purification) by any suitable
techniques known in the art.
[0074] The compound of Formula X, or pharmaceutically acceptable
salt thereof, used in step d) can be prepared by:
[0075] e) reacting a compound of Formula IX:
##STR00012##
[0076] or, pharmaceutically acceptable salt thereof, with ammonia
or an ammonia producing reagent, to form a compound of Formula
XI:
##STR00013##
[0077] or pharmaceutically acceptable salt thereof; and
[0078] f) removing the Boc group from the compound of Formula XI,
or pharmaceutically acceptable salt thereof, to form the compound
of Formula X, or pharmaceutically acceptable salt thereof.
[0079] The coupling reaction of step e) is carried out in the
presence of a coupling reagent and an organic base. Suitable
coupling reagents and organic bases are known in the art.
[0080] Ammonia (either neat or in a solvent such as water or
dioxane) may be used in step e). An ammonia producing reagent (such
as NH.sub.4Cl) may be used.
[0081] Removal of the Boc group in step f) can be carried out by
using a suitable acid reagent (e.g., H.sub.3PO.sub.4, TFA, HCl,
TsOH, or H.sub.2SO.sub.4) or a solution of reagent in a solvent
(HCl-dioxane, HCl-ethyl acetate.
[0082] The compound of Formula VIII, or pharmaceutically acceptable
salt thereof, used in the present invention can be prepared by:
[0083] g) hydrolyzing a compound of Formula XII:
##STR00014##
[0084] or pharmaceutically acceptable salt thereof, in the presence
of a suitable base such as (e.g., LiOH, NaOH, KOH, Ba(OH).sub.2)
and metal carbonate (e.g., Na.sub.2CO.sub.3, K.sub.2CO.sub.3, and
Cs.sub.2CO.sub.3), to form the compound of Formula IX.
[0085] The compound of Formula XII, or pharmaceutically acceptable
salt thereof, used in the present invention can be prepared by:
[0086] h) reacting a compound of Formula XIII:
##STR00015##
[0087] or pharmaceutically acceptable salt thereof, with a compound
of Formula XIV:
##STR00016##
[0088] or pharmaceutically acceptable salt thereof, to form the
compound of Formula XII, or pharmaceutically acceptable salt
thereof.
[0089] The coupling reaction of step h) may be carried out in the
presence of a coupling reagent and an organic base, where suitable
coupling reagents and organic bases are known in the art. In some
embodiments, the coupling reaction of step h) is carried out in the
presence of a coupling reagent. In some embodiments, the coupling
reagent in step h) includes a mixture of EDAC and HOBt.
[0090] In some embodiments, the organic base in step h) is NMM.
[0091] The compound of Formula XIII or pharmaceutically acceptable
salt thereof, used in the present invention can be prepared by:
[0092] i) hydrolyzing a compound of Formula XV:
##STR00017##
[0093] or pharmaceutically acceptable salt thereof, in the presence
of a base, to form a compound of Formula XIV:
##STR00018##
and
[0094] j) removing the Boc group from a compound of Formula XIV, or
pharmaceutically acceptable salt thereof, to form the compound of
Formula XV, or pharmaceutically acceptable salt thereof. Removal of
the Boc group can be carried out by using a suitable reagent or
suitable reagents, such as an acid (e.g., H.sub.3PO.sub.4, TFA,
HCl, TsOH, or H.sub.2SO.sub.4) or TMSOTf/2,6-lutidine. An acid
(e.g., TsOH) is used for removal of the Boc Group.
[0095] Examples of suitable hydrolyzing bases in step i) include,
but are not limited to, metal hydroxide (e.g., LiOH, NaOH, KOH,
Ba(OH).sub.2) and metal carbonate (e.g., Na.sub.2CO.sub.3,
K.sub.2CO.sub.3, and Cs.sub.2CO.sub.3). In some embodiments, the
base in step i) is LiOH.
[0096] In some embodiments, the compound of Formula XV, or
pharmaceutically acceptable salt thereof, used in the present
invention can be prepared by:
[0097] k) reacting a compound of Formula XVI:
##STR00019##
[0098] or pharmaceutically acceptable salt thereof, with a compound
of Formula XVII:
##STR00020##
[0099] or pharmaceutically acceptable salt thereof, to form the
compound of Formula XV, or pharmaceutically acceptable salt
thereof.
[0100] In some embodiments, the coupling reaction of step k) is
carried out in the presence of a coupling reagent and an organic
base. Suitable coupling reagents and organic bases are known in the
art. In some embodiments, the coupling reaction of step k) is
carried out in the presence of a coupling reagent. In some
embodiments, the coupling reagent in step k) is a mixture of EDAC
and HOBt. In some embodiments, the organic base in step k) is
NMM.
[0101] Compounds of the invention can be synthesized by solid-phase
synthetic procedures well known to those of skill in the art (see,
Tew et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 5110-5114; Barany
et al., Int. J. Pept. Prot. Res., 1987, 30, 705-739; Solid-phase
Synthesis: A Practical Guide, Kates, S. A., and Albericio, F.,
eds., Marcel Dekker, New York (2000); and Dorwald, F. Z., Organic
Synthesis on Solid Phase: Supports, Linkers, Reactions, 2nd Ed.,
Wiley-VCH, Weinheim (2002)).
[0102] As used herein, the term "about" means .+-.5% of the value
it describes. For example, about 100 means from 95 to 105.
[0103] As used herein, "isolated" means that compounds are
separated from other components of a synthetic organic chemical
reaction mixture, such as by conventional techniques, and are
purified.
[0104] As used herein, the term "mammal" means a rodent (i.e., a
mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a
horse, a pig, or a human. In some embodiments, the mammal is a
human.
[0105] As used herein, the term "purified" means that, when
isolated, the isolate contains at least 90%, at least 95%, at least
98%, or at least 99% of the desired compound I by weight of the
isolate.
[0106] As used herein, the phrase "pharmaceutically acceptable
salt(s)" includes, but is not limited to, salts of acidic or basic
groups. Suitable examples of salts include, for example,
hydrochloric acid and triflouroacetic acid salts.
[0107] In some embodiments, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. The term "carrier" refers to a diluent, adjuvant, or
excipient with which a compound selected from PMX-30063 and
PMX-60056 and the pharmaceutically acceptable salts thereof
(hereinafter also referred to as active compounds) is administered.
Such pharmaceutical carriers can be liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. The pharmaceutical carriers can also be saline, gum
acacia, gelatin, starch paste, talc, keratin, colloidal silica,
urea, and the like. In addition, auxiliary, stabilizing,
thickening, lubricating and coloring agents can be used. When
administered to a human, the active compounds and pharmaceutically
acceptable carriers can be sterile. Water is a suitable carrier
when the compound is administered intravenously. Saline solutions
and aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical carriers also include excipients such as starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The present compositions, if desired, can
also contain minor amounts of wetting or emulsifying agents, or pH
buffering agents.
[0108] The compositions described herein can take the form of a
solution, suspension, emulsion, tablet, pill, pellet, capsule,
capsule containing a liquid, powder, sustained-release formulation,
suppository, aerosol, spray, or any other form suitable for use.
Examples of suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, A. R. Gennaro (Editor) Mack
Publishing Co.
[0109] The active compounds are formulated in accordance with
routine procedures as a pharmaceutical composition adapted for
administration to humans. Typically, the active compounds are
administered as solutions in sterile isotonic aqueous buffer. Where
necessary, the compositions can also include a solubilizing agent.
Compositions for intravenous administration may optionally include
a local anesthetic such as lidocaine to ease pain at the site of
the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachet indicating the
quantity of active agent. Where the compound of the invention is to
be administered by infusion, it can be dispensed, for example, with
an infusion bottle containing sterile pharmaceutical grade water or
saline. Where the active compound is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0110] The active compounds, and compositions comprising the same,
can be administered orally. Compounds and compositions for oral
delivery can be in the form of, for example, tablets, lozenges,
aqueous or oily suspensions, granules, powders, emulsions,
capsules, syrups, or elixirs. Orally administered compositions can
contain one or more optional agents, for example, sweetening agents
such as fructose, aspartame or saccharin; flavoring agents such as
peppermint, oil of wintergreen, or cherry; coloring agents; and
preserving agents, to provide a pharmaceutically palatable
preparation. Moreover, where in tablet or pill form, the
compositions may be coated to delay disintegration and absorption
in the gastrointestinal tract thereby providing a sustained action
over an extended period of time. Selectively permeable membranes
surrounding an osmotically active driving compound are also
suitable for orally administered active compounds. Oral
compositions can include standard vehicles such as mannitol,
lactose, starch, magnesium stearate, sodium saccharine, cellulose,
magnesium carbonate, etc. Such vehicles are suitably of
pharmaceutical grade.
[0111] The pharmaceutical compositions can be in unit dosage form.
In such form, the composition can be divided into unit doses
containing appropriate quantities of the active component. The unit
dosage form can be a packaged preparation, the package containing
discrete quantities of the preparations, for example, packeted
tablets, capsules, and powders in vials or ampules. The unit dosage
form can also be a capsule, cachet, or tablet itself, or it can be
the appropriate number of any of these packaged forms.
[0112] The following non-limiting Examples illustrate the
compositions and methods disclosed herein and the preparation of
the compounds disclosed herein.
[0113] Brilacidin and Delparantag in the Treatment of Inflammatory
Diseases of the Gastrointestinal Tract
[0114] The compounds brilacidin (PMX-30063) and delparantag
(PMX-60056), and the pharmaceutically acceptable salts thereof,
hereinafter also referred to as the active compounds, may be
administered for the treatment of inflammatory diseases of the
gastrointestinal tract in any conventional manner by any route
where they are active. Administration can be systemic, rectal, or
oral. For example, administration can be, but is not limited to,
parenteral, subcutaneous, intravenous, intramuscular,
intraperitoneal, transdermal, oral or buccal routes, or by depot
injections or implants. Thus, modes of administration for these
compounds (either alone or in combination with other
pharmaceuticals) can be, but are not limited to, sublingual,
injectable (including short-acting, depot, implant and pellet forms
injected subcutaneously or intramuscularly), or by use of rectal
suppositories, intrauterine devices, and transdermal forms such as
patches and creams. The selection of the specific route of
administration and the dose regimen is to be adjusted or titrated
by the clinician according to methods known to the clinician to
obtain the desired clinical response. The amount of the compounds
of the invention to be administered is that amount which is
therapeutically effective. The dosage to be administered will
depend on the characteristics of the subject being treated, e.g.,
the particular animal treated, age, weight, health, types of
concurrent treatment, if any, and frequency of treatments, and can
be easily determined by one of skill in the art (e.g., by the
clinician). The amount of a compound described herein that will be
effective in the treatment and/or prevention of inflammatory
diseases of the gastrointestinal tract will depend on the nature
and severity of the inflammatory disease, and can be determined by
standard clinical techniques. In addition, in vitro or in vivo
assays may optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the compositions will
also depend on the route of administration, and the seriousness of
the disorder, and should be decided according to the judgment of
the practitioner and each patient's circumstances. However, a
suitable dosage range for oral administration is, generally, from
about 0.001 milligram to about 1000 milligrams per kilogram body
weight. In some embodiments, the oral dose is from about 0.01
milligram to 100 milligrams per kilogram body weight, from about
0.01 milligram to about 70 milligrams per kilogram body weight,
from about 0.1 milligram to about 50 milligrams per kilogram body
weight, from 0.5 milligram to about 20 milligrams per kilogram body
weight, or from about 1 milligram to about 10 milligrams per
kilogram body weight. In some embodiments, the oral dose is about 5
milligrams per kilogram body weight. For oral administration, the
active compounds may be administered in a tablet form containing
100 mg per tablet or in liquid form by dissolving water to a
concentration of 1 to 10 mg/mL. The resulting formulation is a
clear colorless solution at pH 7. The active compounds may be given
by daily doses until the condition has resolved. For-rectal
administration, 25 mg or 50 mg is given as a retention enema in a
60 mL sterile solution. The enema is given either once daily at
bedtime or twice daily in the morning and at bedtime for 6 weeks.
If brilacidin (PMX-30063) and delparantag (PMX-60056) are
administered in a single pharmaceutical composition or concurrently
the total daily dose of the two compounds will generally be
comparable to the amounts set forth above for the daily dose of a
single compound.
[0115] The total daily dose may be administered in single or
divided doses. The present invention also encompasses sustained
release compositions. These dosages are based on an average human
subject having a weight of about 65 kg to 70 kg. The physician will
readily be able to determine doses for subjects whose weight falls
outside this range, such as infants and the elderly.
[0116] The pharmaceutical compositions and/or formulations
containing one or both of the active compounds and a suitable
carrier can be solid dosage forms which include, but are not
limited to, tablets, capsules, cachets, pellets, pills, powders and
granules; topical dosage forms which include, but are not limited
to, solutions, powders, fluid emulsions, fluid suspensions,
semi-solids, ointments, pastes, creams, gels and jellies, and
foams; and parenteral dosage forms which include, but are not
limited to, solutions, suspensions, emulsions, and dry powder;
comprising an effective amount of a compound of the invention. It
is also known in the art that the active ingredients can be
contained in such formulations with pharmaceutically acceptable
diluents, fillers, disintegrants, binders, lubricants, surfactants,
hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers,
humectants, moisturizers, solubilizers, preservatives and the like.
The means and methods for administration are known in the art and
an artisan can refer to various pharmacologic references for
guidance (see, for example, Modern Pharmaceutics, Banker &
Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman's The
Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan
Publishing Co., New York (1996)). Descriptions of pharmaceutical
compositions and methods for their preparation may be found, for
example, in `Remington's Pharmaceutical Sciences`, 19th Edition
(Mack Publishing Company, 1995).
[0117] In some embodiments, the active compounds can be used with
agents including, but not limited to, topical analgesics (e.g.,
lidocaine). The active compounds may also be administered together
with antibiotics. Examples of such antibiotics are amoxicillin,
ampicillin, azlocillin, bacitracin, carbenicillin, cefaclor,
cefamandole, cefazolin, cefmetazole, cefoperazone, cefotaxime,
cefsulodin, cefiriaxone, cephalexin, cephalosporin C, cephalothin,
cephradine, cloxacillin, D-cycloserine, dicloxacillin,
D-penicillamine, econazole, ethambutol, lysostaphin, moxalactam,
nafcillin, nikkomycin Z, nitrofurantoin, oxacillin, penicillic,
penicillin G, phenethicillin, phenoxymethylpenicillinic acid,
phosphomycin, pipemidic acid, piperacillin, ristomycin, and
vancomycin; amikacin, anisomycin, apramycin, azithromycin,
blasticidine S, brefeldin A, butirosin, chloramphenicol,
chlortetracycline, clindamycin, clotrimazole, cycloheximide,
demeclocycline, dibekacin, dihydrostreptomycin, doxycycline,
duramycin, emetine, erythromycin, fusidic acid, G 418, gentamicin,
helvolic acid, hygromycin B, josamycin, kanamycin, kirromycin,
lincomycin, meclocycline, mepartricin, midecamycin, minocycline,
neomycin, netilmicin, nitrofurantoin, nourseothricin, oleandomycin,
oxytetracycline, paromomycin, puromycin, rapamycin, ribostamycin,
rifampicin, rifamycin, rosamicin, sisomicin, spectinomycin,
spiramycin, streptomycin, tetracycline, thiamphenicol,
thiostrepton, tobramycin, tunicamycin, tylosin, viomycin, and
virginiamycin; camptothecin, 10-deacetylbaccatin III, azacytidine,
7-aminoactinomycin D, 8-quinolinol, 9-dihydro-13-acetylbaccatin
III, aclarubicin, actinomycin D, actinomycin I, actinomycin V,
bafilomycin A1, bleomycin, capreomycin, chromomycin, cinoxacin,
ciprofloxacin, cis-diammineplatinum(II) dichloride, coumermycin A1,
L(+)-lactic acid, cytochalasin B, cytochalasin D, dacarbazine,
daunorubicin, distamycin A, doxorubicin, echinomycin, enrofloxacin,
etoposide, flumequine, formycin, fumagillin, ganciclovir,
gliotoxin, lomefloxacin, metronidazole, mithramycin A, mitomycin C,
nalidixic acid, netropsin, nitrofurantoin, nogalamycin, nonactin,
novobiocin, ofloxacin, oxolinic acid, paclitaxel, phenazine,
phleomycin, pipemidic acid, rebeccamycin, sinefungin,
streptonigrin, streptozocin, succinylsulfathiazole, sulfadiazine,
sulfadimethoxine, sulfaguanidine purum, sulfamethazine,
sulfamonomethoxine, sulfanilamide, sulfaquinoxaline, sulfasalazine,
sulfathiazole, trimethoprim, tubercidin, 5-azacytidine, cordycepin,
and formycin A; 2-mercaptopyridine, 4-bromocalcimycin A23187,
alamethicin, amphotericin B, calcimycin A23187, chlorhexidine,
clotrimazole, colistin, econazole, hydrocortisone, filipin,
gliotoxin, gramicidin A, gramicidin C, ionomycin, lasalocid A,
lonomycin A, monensin,
N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, narasin,
nigericin, nisin, nonactin, nystatin, phenazine, pimaricin,
polymyxin B, DL-penicillamine, polymyxin B, praziquantel,
salinomycin, surfactin, and valinomycin; (+)-usnic acid,
(.+-.)-miconazole, (S)-(+)-camptothecin, 1-deoxymannojirimycin,
2-heptyl-4-hydroxyquinoline N-oxide, cordycepin,
1,10-phenanthroline, 6-diazo-5-oxo-L-norleucine, 8-quinolinol,
antimycin, antipain, ascomycin, azaserine, bafilomycin, cerulenin,
chloroquine, cinoxacin, ciprofloxacin, mevastatin, concanamycin A,
concanamycin C, coumermycin A1, L(+)-lactic acid, cyclosporin A,
econazole, enrofloxacin, etoposide, flumequine, formycin A,
furazolidone, fusaric acid, geldanamycin, gliotoxin, gramicidin A,
gramicidin C, herbimycin A, indomethacin, irgasan, lomefloxacin,
mycophenolic acid, myxothiazol,
N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, nalidixic acid,
netropsin, niclosamide, nikkomycin, N-methyl-1-deoxynojirimycin,
nogalamycin, nonactin, novobiocin, ofloxacin, oleandomycin,
oligomycin, oxolinic acid, piericidin A, pipemidic acid, radicicol,
rapamycin, rebeccamycin, sinefungin, staurosporine, stigmatellin,
succinylsulfathiazole, succinylsulfathiazole, sulfadiazine,
sulfadimethoxine, sulfaguanidine, sulfamethazine,
sulfamonomethoxine, sulfanilamide, sulfaquinoxaline, sulfasalazine,
sulfathiazole, triacsin C, trimethoprim, and vineomycin A1.
[0118] The active compounds may also be administered together with
antidepressants such as clozapine or olanzapine; laxatives;
antidiarrheal agents; serotonin antagonists (5-HT3) such as
ondansetron, clozapine or ondansetron; serotonin reuptake
inhibitors (SSRIs); anti-spasmodics such as hyoscyamine or
dicyclomine; proton pump inhibitors (PPIs); magnesium aluminum
silicates; alverine citrate drugs; rifaximin; anti-inflammatory
agents such as steroids, mesalazine (mesalamine or
5-aminosalicyclic acid); immunomodulators such as azathioprine,
methotrexate, infliximab, adalimumab, certolizumab, or
natalizumab.
[0119] The active compounds can be formulated for parenteral
administration by injection, such as by bolus injection or
continuous infusion. The compounds can be administered by
continuous infusion subcutaneously over a period of about 15
minutes to about 24 hours. Formulations for injection can be
presented in unit dosage form, such as in ampoules or in multi-dose
containers, with an added preservative. The compositions can take
such forms as suspensions, solutions or emulsions in oily or
aqueous vehicles, and can contain formulatory agents such as
suspending, stabilizing and/or dispersing agents.
[0120] For oral administration, the active compounds can be
formulated readily by combining these compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the compounds of the invention to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for oral ingestion by a patient to be
treated. Pharmaceutical preparations for oral use can be obtained
by, for example, adding a solid excipient, optionally grinding the
resulting mixture, and processing the mixture of granules, after
adding suitable auxiliaries, if desired, to obtain tablets or
dragee cores. Suitable excipients include, but are not limited to,
fillers such as sugars, including, but not limited to, lactose,
sucrose, mannitol, and sorbitol; cellulose preparations such as,
but not limited to, maize starch, wheat starch, rice starch, potato
starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and
polyvinylpyrrolidone (PVP). If desired, disintegrating agents can
be added, such as, but not limited to, the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Dragee cores can be provided with suitable coatings. For
this purpose, concentrated sugar solutions can be used, which can
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments can be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0121] Pharmaceutical preparations which can be used orally
include, but are not limited to, push-fit capsules made of gelatin,
as well as soft, sealed capsules made of gelatin and a plasticizer,
such as glycerol or sorbitol. The push-fit capsules can contain the
active ingredients in admixture with filler such as lactose,
binders such as starches, and/or lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In soft capsules,
the active compounds can be dissolved or suspended in suitable
liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols. In addition, stabilizers can be added. All
formulations for oral administration should be in dosages suitable
for such administration. For buccal administration, the
compositions can take the form of, such as, tablets or lozenges
formulated in a conventional manner.
[0122] The active compounds can also be formulated in rectal
compositions such as suppositories or retention enemas, such as
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0123] The active compounds can also be formulated as a depot
preparation. Such long acting formulations can be administered by
implantation (for example subcutaneously or intramuscularly) or by
intramuscular injection. Depot injections can be administered at
about 1 to about 6 months or longer intervals. Thus, for example,
the compounds can be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0124] In transdermal administration, the active compounds, for
example, can be applied to a plaster, or can be applied by
transdermal, therapeutic systems that are consequently supplied to
the organism.
[0125] The pharmaceutical compositions of the active compounds also
can comprise suitable solid or gel phase carriers or excipients.
Examples of such carriers or excipients include, but are not
limited to, calcium carbonate, calcium phosphate, various sugars,
starches, cellulose derivatives, gelatin, and polymers such as
polyethylene glycols.
[0126] The present invention also provides compounds of the
invention, or compositions comprising the same, for use in
prophylaxis and/or treatment of inflammatory diseases of the
gastrointestinal tract in a patient. The present invention also
provides compounds of the invention, or compositions comprising the
same, for use in prophylaxis and/or treatment of inflammatory
diseases of the gastrointestinal tract. The present invention also
provides compounds of the invention, or compositions comprising the
same, for use in preparation of a medicament for prophylaxis and/or
treatment of inflammatory diseases of the gastrointestinal tract in
a patient.
[0127] The present invention also provides methods for prophylaxis
and/or treatment of inflammatory diseases of the gastrointestinal
tract in an animal comprising administering to the animal in need
thereof an effective amount of a compound of the invention. The
present invention also provides methods for prophylaxis and/or
treatment of inflammatory diseases of the gastrointestinal tract in
an animal comprising administering to the animal in need thereof a
composition of the invention. The present invention also provides
methods for prophylaxis and treatment of inflammatory diseases of
the gastrointestinal tract comprising administering to the animal
an effective amount of a compound or salt of the invention.
[0128] The present invention also provides active compounds or
compositions comprising the same, for use in prophylaxis and/or
treatment of inflammatory diseases of the gastrointestinal tract in
a patient. The present invention also provides active compounds, or
compositions comprising the same, for use in preparation of a
medicament for prophylaxis and/or treatment of inflammatory
diseases of the gastrointestinal tract in a patient.
[0129] The structures depicted herein may omit necessary hydrogen
atoms to complete the appropriate valency. Thus, in some instances
a carbon atom or nitrogen atom may appear to have an open valency
(i.e., a carbon atom with only two bonds showing would implicitly
also be bonded to two hydrogen atoms; in addition, a nitrogen atom
with a single bond depicted would implicitly also be bonded to two
hydrogen atoms). For example, "--N" would be considered by one
skilled in the art to be "--NH.sub.2." Thus, in any structure
depicted herein wherein a valency is open, one or more hydrogen
atoms, as appropriate, is implicit, and is only omitted for
brevity.
[0130] Anti-Inflammatory Activities of PMX-30063 (Brilacidin) and
PMX-60056 (Delparantag)
[0131] The studies discussed in the Examples demonstrate the
anti-inflammatory activity of PMX-30063 and PMX-60056: inhibition
of phosphodiesterase PDE4 in a PDE-Glo phosphodiesterase assay;
inhibition of PDE3 in a PDE-Glo phosphodiesterase assay; inhibition
of TNF-.alpha. of lipopolysaccharide (LPS) induced TNF-.alpha.
production in NR8383 rat macrophages; inhibition of LPS induced
Monocyte Chemoattractant Protein-1 (MCP-1) release in NR8383 rat
macrophages; inhibition of LPS induced matrix-metalloproteinase-9
(MMP-9) release in NR8383 rat macrophage (PMX-30063 only);
inhibition of LPS induced IL-6 release in NR8383 rat macrophages
(PMX-30063 only). Following oral administration, PMX-30063 is taken
up by the small intestine but <0.5% enters the circulation; a
great advantage for treatment of intestinal epithelium with low
risk of systemic toxicity. In an in vivo ulcerative colitis model,
intestine weights were reduced, but not significantly, compared to
untreated controls following rectal administration of PMX-30063. A
dose dependent decrease in ulcerative colitis score was
observed.
[0132] The present inventors hypothesize that PMX-30063 and
PMX60056, as HDP mimetics, may be functioning through the cyclic
AMP/cyclic GMP pathways in suppression of proinflammatory response.
PDE4 is a predominant phosphodiesterase expressed in neutrophils, T
cells and macrophages and PDE4 inhibitors reduce neutrophil
chemotaxis, recruitment and activation, inhibit the activation of
CD4+ and CD8+ T cells, and inhibit monocytes chemotaxis. Hence,
PDE4 has a broad range of anti-inflammatory effects on various key
effector cells that may be involved in ulcerative colitis and
Crohn's disease. It is also recognized that use of PDE3 inhibitors
can provide clinical benefit to patients' inflammatory diseases. It
has shown that combining inhibitors of PDE3 and PDE4 provides
greater benefits compared with inhibiting either PDE alone (Rieder
et al. PLoS One 2013 2013; 8(2):e56867. doi: 10.1371/journal.pone.
0056867. Epub 2013 Feb. 28).
[0133] Methods
[0134] Set forth below is a discussion of the research methods
referred to in the Examples with references to the figures that are
part of the present application.
[0135] Method for FIG. 1.
[0136] Phosphodiesterase inhibition assays of PDE4 were performed,
using PMX-30063. The PDE-Glo phosphodiesterase assay (Promega,
Madison, Wis., USA Catalog No. V1361) was performed using 8 ng of
PDE4B, 1 .mu.M cAMP substrate and PMX-30063. The compounds and
PDE4B (BPS Biosciences, San Diego, Calif.) were mixed and
pre-incubated at room temperature for 15 minutes. Substrate was
added and the reaction was incubated for 7 minutes at room
temperature. Data are presented as luminescence units (RLU).
[0137] Method for FIG. 2.
[0138] Phosphodiesterase inhibition assays of PDE4 were performed,
using PMX-60056. The PDE-Glo phosphodiesterase assay (Promega,
Madison, Wis., USA Catalog No. V1361) was performed using 8 ng of
PDE4B (BPS Biosciences, San Diego, Calif.), 1 .mu.M cAMP substrate
and PMX-30063. The compounds and PDE4B were mixed and pre-incubated
at room temperature for 15 minutes. Substrate was added and the
reaction was incubated for 7 minutes at room temperature. Data are
presented as luminescence units (RLU).
[0139] Method for FIG. 3.
[0140] Phosphodiesterase inhibition assays of PDE3 were performed
using PMX-30063. The PDE-Glo phosphodiesterase assay (Promega,
Madison, Wis., USA Catalog No. V1361) was performed according to
manufacturer's instruction using 2.75 ng of PDE3A, 1 .mu.M cAMP
substrate and PMX-30063. The compounds and PDE3A were mixed and
pre-incubated at room temperature for 15 minutes. Substrate was
added and the reaction was incubated for 7 minutes at room
temperature. Data are presented as luminescence units (RLU).
[0141] Method for FIG. 4.
[0142] Phosphodiesterase inhibition assays of PDE3 were performed
using PMX-60056. The PDE-Glo phosphodiesterase assay (Promega,
Madison, Wis., USA Catalog No. V1361) was performed according to
manufacturer's instruction using 2.75 ng of PDE3A, 1 .mu.M cAMP
substrate and PMX60056. The compounds and PDE3A were mixed and
pre-incubated at room temperature for 15 minutes. Substrate was
added and the reaction was incubated for 7 minutes at room
temperature. Data are presented as luminescence units (RLU).
[0143] Method for FIG. 5.
[0144] TNF-alpha assays were performed using PMX-30063. NR8383
(CRL-2192, ATCC, Manassas, Va.) rat macrophage cells were
pretreated with PMX-30063 for 45 minutes followed by treatment with
1 .mu.g/ml Lipopolysaccharides (LPS) from E. coli (Sigma, St.
Louis, Mo.) for 8 hours. TNF-.alpha. concentrations in the
supernatants were determined by ELISA using an immunoassay kit
specific for rat TNF-.alpha. (R&D Systems, Minneapolis, Minn.)
according to manufacturer's instructions.
[0145] Method for FIG. 6.
[0146] TNF-alpha assays were performed using PMX-60056. NR8383 rat
macrophage cells were pretreated with PMX-60056 for 45 minutes
followed by treatment with 1 .mu.g/ml LPS from E. coli (Sigma, St.
Louis, Mo.) for 8 hours. TNF-.alpha. concentrations in the
supernatants were determined by ELISA using an immunoassay kit
specific for rat TNF-.alpha. (R&D Systems, Minneapolis, Minn.)
according to manufacturer's instructions.
[0147] Method for FIG. 7.
[0148] MCP-1 assays were performed using PMX-30063. Rat macrophages
(NR8383) were pretreated with PMX-30063 with concentrations shown
for 45 minutes, followed by 1 .mu.g/ml LPS treatment from E. coli
(Sigma, St. Louis, Mo.) for 8 hours. After 8 hours, supernatants
were collected for MCP-1 measurement by ELISA. MCP-1 was measured
using an immunoassay kit according to manufacturer's instructions
(Thermo Scientific, Rockford, Ill.).
[0149] Method for FIG. 8.
[0150] MCP-1 assays were performed using PMX-60056. Rat macrophages
(NR8383) were pretreated with PMX30063 with concentrations shown
for 45 minutes, followed by 1 .mu.g/ml LPS treatment from E. coli
(Sigma, St. Louis, Mo.) for 8 hours. After 8 hours, supernatants
were collected for MCP-1 measurement by ELISA. MCP-1 was measured
using an immunoassay kit according to manufacturer's instructions
(Thermo Scientific, Rockford, Ill.).
[0151] Method for FIG. 9.
[0152] MMP-9 assays were performed using PMX-30063. Rat macrophages
(NR8383) were pretreated with PMX-30063 with concentrations shown
for 45 minutes, followed by 1 .mu.g/ml LPS treatment from E. coli
(Sigma, St. Louis, Mo.) for 8 hours. After 8 hours, supernatants
were collected for MMP9 measurement by ELISA using immunoassay kit
(R&D Systems, Minneapolis, Minn.) according to manufacturer's
instructions.
[0153] Method for FIG. 10.
[0154] IL-6 release assays were performed using PMX-30063. Rat
macrophages (NR8383) were pretreated with PMX-30063 with
concentrations shown for 45 minutes, followed by 1 g/ml LPS
treatment from E. coli (Sigma, St. Louis, Mo.) for 8 hours. After 8
hours, supernatants were collected for IL-6 measurement by ELISA
using immunoassay kit (R&D Systems, Minneapolis, Minn.)
according to manufacturer's instructions.
[0155] Method for FIG. 11.
[0156] PMX-30063 was evaluated for plasma concentration following
oral (PO) or intravenous (IV) administration in male Balb/c mice
(Study 16009-12001). The test article, PMX-30063, was corrected for
salt form but no adjustment was made for purity. PMX-30063 was
dissolved in dissolved in half volume of water, then is added half
volume of 2.times. saline to yield nominal concentration of 1 mg/mL
for oral administration.
[0157] The resulting formulation was clear colorless solution (pH
7) and was stored at room temperature until administered. The
formulated solution was clear and colorless until dosing was
completed. The concentration of PMX-30063 in dosing solution was
confirmed by HPLC-UV with accuracy of 95.5%.
[0158] A total of 60 male Balb/c mice, approximately 4-7 weeks of
age at receipt with body weight of 18.0 g to 21.9 g were used in
this study. The test article, PMX-30063, was administered orally at
10 mg/kg in 10 mL/kg or intravenously at 5 mg/kg at 5 mL/kg volume
via a single bolus administration.
[0159] Three mice in each group were used for blood at each time
point at post-dose at 5 minutes, 15 minutes. 30 minutes, 1, 2, 4,
8, and 24 hours for Groups 1-2. Blood samples (at least 300 .mu.L
sample) were collected via cardiac puncture after euthanasia by
carbon dioxide inhalation at appropriate time points. Samples were
placed in tubes containing K.sub.2-EDTA, and then centrifuged at
approximately 8,000 rpm for 6 minutes at 4.degree. C. and the
resulting plasma were separated and stored frozen at approximately
-80.degree. C.
[0160] The pharmacokinetic (PK) analysis were conducted by
Medicilon Preclinical Research (Shanghai) LLC. The PK parameters
were determined by the Study Director for the test article from
mean concentration-time data in the test species. A
non-compartmental module of WinNonlin.TM. Professional 5.2 was used
to calculate parameters. Any BLQs (LLOQ=2.5 ng/mL for plasma and
LLOQ=500 ng/g for small intestine) were replaced with a value of
"0", and the mean value and its standard deviation (SD) are
calculated with these replaced values.
[0161] Plasma samples (50 .mu.L) were transferred to centrifuge
tube, then 250 .mu.L IS solution (50 ng/mL Carvedilol) was added to
it. After vortexing for 1 minute and centrifuging for 5 minutes at
15,000 rpm, 100 .mu.L aliquots of supernatant were transferred to
glass autosampler vials.
[0162] Method for FIG. 12.
[0163] PMX-30063 was also evaluated for extent of tissue
distribution following oral (PO) or intravenous (IV) administration
(PO) in male Balb/c mice (Study 16009-12001). PMX-30063 was
prepared and administered as described in FIG. 11. The small
intestine with content of each animal for were harvested and placed
per animal per tissue into a tube. The small intestine with content
samples were snap frozen in dry ice and then stored at -80.degree.
C. until bioanalysis. All the samples were labeled with detailed
information such as study number, animal number, matrix, and time
points of collection and date of collection. In addition, the extra
animals obtained for the study, but not placed on study were used
for collection of small intestine with content. The resulting small
intestine with content samples were then applied to the development
of the bioanalytical method and sample bioanalysis in this study.
Bioanalytical analysis was performed on samples by LC-MS/MS.
[0164] For analyzing the contents of the small intestine, small
intestine samples were homogenized by adding saline (1 g small
intestine: 5 mL saline). Small intestine homogenates (50 .mu.L)
were transferred to tubes and 250 .mu.L internal standard (IS)
working solution (50 ng/mL Carvedilol) was added to each sample.
After vortexing for 1 minute and centrifuging for 5 minutes at
15,000 rpm, 100 .mu.L aliquots of supernatant were transferred to
glass autosampler vials.
[0165] Method for FIG. 13.
[0166] Intestine/plasma concentration ratio following oral
administration of PMX-30063 was calculated based on data derived
from FIGS. 11 and 12. The ratios versus time are shown.
[0167] Method for FIG. 14.
[0168] PMX-30063 was evaluated for efficacy in an ulcerative
colitis (UC) model. Balb/c mice were fasted for 24 hours.
Ulcerative colitis was induced by injecting 200 .mu.L of 4% acetic
acid into the rectum. Four days later, animals were treated once
daily for 4 days with PMX-30063 at either 100 mg/kg, 200 mg/kg or
400 mg/kg intrarectally. Another group of animals were treated with
5-ASA (5-aminosalicylic acid or esalamine), and another received no
treatment. Seven days after the first dose, five cm of intestine
were cleaned in cold saline then weighed.
[0169] Method for FIG. 15.
[0170] PMX-30063 was evaluated for efficacy in an ulcerative
colitis (UC) model. Balb/c mice were fasted for 24 hours.
Ulcerative colitis was induced by injecting 200 .mu.L of 4% acetic
acid into the rectum. Four days later, animals were treated once
daily for 4 days with PMX-30063 at either 100 mg/kg, 200 mg/kg or
400 mg/kg intrarectally. Another group of animals were treated with
5-ASA (5-aminosalicylic acid or esalamine), and another received no
treatment. Seven days after the first dose the colon was examined
visually for ulcerative colitis and scored according to the Table
below.
TABLE-US-00001 Ulcerative Colitis Score Observation 0 no damage 1
localized damage with ulcers 2 linear ulcers without severe
inflammation 3 linear ulcers with inflammation at one point 4 sores
or inflammation at two or more points 5 large ulcer or inflammation
more than 1 centimeter
[0171] In order that the invention disclosed herein may be more
efficiently understood, examples are provided below. It should be
understood that these examples are for illustrative purposes only
and are not to be construed as limiting the invention in any
manner. Throughout these examples standard techniques, were carried
out using commercially available reagents, except where otherwise
noted. Brilacidin and delparantag, which were tested as described
in the Examples, were prepared by their respective suppliers by the
methods described in Examples 1 and 2 respectively, based on the
respective disclosures in U.S. patent application Ser. No.
13/661,466 filed Oct. 26, 2012 and U.S. Pat. No. 8,354,556 issued
Jan. 15, 2013. Brilacidin was obtained from Johnson Matthey Pharma
Services, Devens, Mass. and Delparantag was obtained from Ricerca
Biosciences, Concord, Ohio.
[0172] The following abbreviations have been used for common
solvents: THF, tetrahydrofuran; DMA, dimethyacetamide; DMSO,
dimethylsulphoxide; DMF, dimethylformamide; EtOAc, ethyl acetate;
TFA, trifluoroacetic acid; DCM, dichloromethane; MTBE,
t-butylmethyl ether.
EXAMPLES
Example 1
Preparation of Brilacidin (PMX-30063)
##STR00021## ##STR00022##
[0174] Step 1:
[0175] N-Boc-3-pyrrolidinol (2.2 kg) is dissolved in
tetrahydrofuran (11.2 kg) and cooled to 10.degree. C. Then
potassium tert-butoxide (1.5 kg) is added, followed by addition of
a solution of 2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene (3.0
kg) in t-butylmethyl ether (5.1 kg). The resulting mixture is
stirred for 16 hours at 10-17.degree. C. and t-butylmethyl ether
(10.7 kg) and water (15.6 kg) are then added. The organic layer is
separated and washed by brine and evaporated to dryness. The crude
product on crystallization with ethanol/water twice gives 2.17 kg
(46.3%) of
(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylic
acid tert-butyl ester (3) with expected HPLC purity of about
96.4%.
[0176] Step 2:
[0177] 2.2 kg of compound 3 is dissolved in methanol (6.1 kg), and
then 5% Pd--C (294 g) is added under nitrogen. The resulting
reaction mixture is stirred under hydrogen at 10-15 psi for 98
hours. Reaction progress is monitored by HPLC. The reaction mixture
is filtered through a Celite pad and filtrate is concentrated to
afford 1.715 kg (89.5%) of
(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylic
acid tert-butyl ester (4) with expected HPLC purity of about
96.2%.
[0178] Step 3:
[0179] Compound 4 (1.6 kg) is coupled with
pyrimidine-4,6-dicarboxylic acid (383 g) in the presence of
1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimide hydrochloride
(1.29 kg), in pyridine, under inert atmosphere, at ambient
temperature. After 25 hours, the reaction mixture is diluted in
water (92 kg), a solid is separated out, which is collected by
filtration and dried at 37-40.degree. C., and crude compound is
purified by trituration with ethyl acetate/heptanes for three
times. Yield: 1.34 kg (70%/o), expected Purity about: 87.5%.
[0180] Step 4:
[0181] The solution of 1.07 kg of DMAP in 16.9 kg anhydrous
pyridine is cooled to 0.degree. C. with ice bath. 1050 g of thionyl
chloride is added slowly and the temperature is kept below
15.degree. C. Once the solution reaches 5.degree. C.
5-N-(carbobenzoxyamino)valeric acid (2.2 kg) is added and
temperature is kept below 15.degree. C. The solution is cooled to
5.degree. C., and followed by addition of compound 5 (2.5 kg) and
stirring is continued for 22 minutes before the temperature is
brought up to room temperature. The resulting reaction mixture is
stirred for 21 hours, then ethyl acetate (17.3 kg) is added. The
organic layer is washed with 1.2N sodium hydroxide, then with
sodium chloride and dried over sodium sulfate. The organic layer is
separated and evaporated to dryness, and resulting residue on
trituration with toluene gives crude compound 6, which is purified
by silica gel chromatography using methanol/ethyl acetate as
eluent. Compound 6 is further purified by recrystallization from
dichloromethane/toluene mixture. Yield: 1.47 kg (30%), expected
HPLC purity: about 97.6%.
[0182] Step 5 and 6:
[0183] Compound 6 (1.47 kg) is dissolved in methanol (11.6 kg),
then 10% Pd--C (143 g) is added under nitrogen atmosphere. The
resulting reaction mixture is stirred 2.5 hours under hydrogen at
ambient pressure, and then catalyst is removed by filtration. To
the filtrate, IN HCl (2 L), triethylamine (420 g) and
di-boc-guanylpyrazole (0.70 g) are added in order. The resulting
reaction mixture is stirred at room temperature for 102 minutes.
Then it is evaporated, followed by dilution with ethyl acetate
(10.7 kg). The organic layer washed with sodium chloride solution,
dried over sodium sulfate and evaporated to dryness. The crude
compound 8 is purified by column chromatography with 1.9 kg of
silica gel and ethyl acetate/dichloromethane to
methanol/dichloromethane. Yield: 1.19 kg (62.3%), expected HPLC
purity: about 96.4%.
[0184] Step 7:
[0185] Compound 8 (1.17 kg) is dissolved in ethyl acetate ((21.5
kg) and 281 g of water is added. HCl gas is added to the solution
while the temperature is kept below 45.degree. C.
[0186] After 5 hours, the reaction is found to be complete. The
solid (PMX-30063, brilacidin) is collected by filtration. Further
purification of PMX-30063 is done by trituration of above solid
with methanol/THF. Yield: 696 g (84.1%), expected HPLC purity:
about 98.6%.
Example 2
Preparation of Delparantag
##STR00023## ##STR00024## ##STR00025##
[0187] Step 1: Preparation of Compound 11
[0188] A mixture of compound 9 (1665 g, 4.379 mol, 1.0 eq),
compound 10 (817 g, 4.51 mol, 1.03 eq), and N-hydroxybenzotriazole
(651 g, 4.82 mol, 1.1 eq) in 14.0 L of (dichloromethane) is treated
with NMM (N-methyl morpholine) (885 g, 8.76 mol, 2.0 eq), followed
by a portion wise addition of
N-(3-dimethyaminopropyl)-N'-ethylcarbodiimide hydrochloride (923 g,
4.82 mol, 1.10 eq). The reaction is run at 20.degree. C. and the
reaction progress is monitored by in-process HPLC. After the
reaction is completed, the reaction mixture is processed by
standard extraction procedures to afford compound 11 (2192 g, 92.1%
yield). An HPLC analysis is expected to show the purity of compound
11 to be about 97-98%. A chiral HPLC method is expected to show
that the enantiomeric purity of compound 11 is maintained (from
compound 9) during Step 1. No undesired enantiomer is expected to
be detected.
Step 2A: Preparation of Compound 13
[0189] A mixture of compound 1-3 (1250 g, 2.30 mol), THF (13.8 L),
and methanol (9.4 L) is cooled to 10.degree. C. and treated
dropwise over 30 minutes with 4 molar equivalents of lithium
hydroxide delivered as a 5% solution in water. The reaction mixture
is warmed up to room temperature with stirring and the progress is
monitored by in-process HPLC. After the reaction is completed, the
pH of the reaction mixture is neutralized with aqueous HCl,
partially concentrated, acidified with aqueous HCl, and extracted
with ethyl acetate. Compound 1-4 (1175 g, 96.5% yield) is obtained
for which HPLC analysis is expected to show a purity of about 96%.
A chiral HPLC method is expected to show that the enantiomeric
purity of compound 13 is maintained (from compound 11) during Step
2A. No undesired enantiomer is expected to be detected.
Step 2B: Preparation of Compound 12
[0190] A solution of compound 1-3 (2556 g, 4.70 mol) in DCM (15.0
L) is treated with p-toluenesulfonic acid (1073 g, 5.6 mol, 1.2
eq): and the mixture is heated to 40.degree. C. The reaction
progress is monitored by in-process HPLC. After the reaction is
completed, the reaction mixture is cooled to room temperature,
treated with an aqueous sodium bicarbonate solution, and then
processed by standard extraction procedures to afford compound 12
(2065 g, 99% yield), the purity of this product is expected to be
about 96.4% by HPLC analysis.
Step 3: Preparation of Compound 14
[0191] A mixture of compound 12 (1030 g, 2.32 mol, 1.05 eq), HOBt
(601 g, 4.45 mol, 2.0 eq), and NMM (670 g, 6.63 mol, 3.0 eq) in
chloroform (17.6 L) is treated with a solution of EDAC (511 g, 2.65
mol, 1.2 eq) in chloroform (2.0 L). This mixture is treated by drop
wise addition of a solution of compound 13 (1170 g, 2.21 mol, 1.0
eq) and NMM (337 g, 3.33 mol, 1.5 eq) in chloroform (4.2 L) and the
resultant reaction mixture is stirred at 20-25.degree. C. The
reaction progress is monitored by the in-process HPLC method. After
the reaction is completed, the reaction mixture is processed by
standard extraction procedures. The solid foam obtained shows
excess weight, and a purity of approximately 88% by HPLC analysis.
The solid foam obtained is subjected to crystallization from
heptane/EtOAc. Compound 14 (1287 g, 61% yield) is obtained and its
purity, determined by HPLC analysis, is expected to be about
97.2%.
Step 4: Preparation of Compound 15
[0192] A mixture of compound 14 (2516 g, 2.63 mol), THF (16.6 L),
and methanol (10.9 L) is cooled to 10.degree. C. and treated drop
wise over 45 minutes with 4 equivalents of LiOH delivered as a 5%
solution in water. The reaction mixture is warmed up to room
temperature with stirring, and the reaction progress is monitored
by in-process HPLC. After the reaction is completed, the reaction
mixture is neutralized with aqueous HCl, partially concentrated,
acidified with aqueous HCl, and extracted with EtOAc. Compound 15
(quantitative yield, 2813 g of the crude product) is obtained and
its purity, determined by HPLC analysis, is expected to be about
-94.7%. The crude product is directly used in the next step without
further purification.
Step 5: Preparation of Compound 16
[0193] A solution of compound 15 [1490 g of the crude product
prepared in Step 4 above, assumed to be the equivalent of 1297 g
(1.38 mol) of pure compound 15] in chloroform (13.0 L) is cooled to
10.degree. C. and treated with ethyl chloroformate (302 g, 2.78
mol, 2.0 eq) in one portion followed by drop wise addition of DIEA
(357 g, 2.76 mol, 2.0 eq.) while monitoring the internal
temperature. The reaction mixture is warmed up to ambient
temperature with stirring. The reaction progress is monitored to
show a complete conversion to the reactive mixed anhydride
intermediate by HPLC analysis of a sample that is quenched by 0.5 M
ammonia in dioxane and assessed for formation of compound 16 and
the consumption of compound 15. After complete conversion of the
acid 15 to the anhydride intermediate, the reaction mixture is
cooled to 0.degree. C. and treated through a bubbler with ammonia
gas (151 g, 8.8 mol, 6.4 eq.) while monitoring the internal
temperature. The reaction progress is monitored by in-process HPLC.
After the reaction is completed, the reaction mixture is quenched
with water and processed by standard extraction procedures.
Compound 16 (quantitative yield, 1322 g of the crude product) is
obtained and its purity, determined by HPLC analysis, is expected
to be about 93.2%. The crude product is directly used in the next
step without further purification.
Step 6: Preparation of Compound 17
[0194] A solution of compound 16 (1322 g of the crude product
prepared in Step 5 above, assumed to be the equivalent of 1298 g
(1.38 mol) of pure compound 16) in DCM (4.4 L) is cooled to
0.degree. C. and treated drop wise with trifluoroacetic acid (2.1
L, 28 mol, 20 eq.) while maintaining the internal temperature to be
below about 10.degree. C. The reaction mixture is warmed up to
ambient temperature with stirring. The reaction progress is
monitored by in-process HPLC. After the reaction is completed, the
reaction mixture is rapidly cooled to -20.degree. C. then quenched
by addition over 30 minutes to a rapidly stirred -5.degree. C.
mixture of NaOH (22 eq.) in water (9.6 L) and DCM (4.5 L). The
addition rate is such that the internal temperature of the mixture
is maintained at below about 10.degree. C. The quenched reaction
mixture is processed by standard extraction procedures to afford
compound 17 (1152 g, 99% yield), and its purity, determined by HPLC
analysis, is expected to be about 85.0%.
Step 7: Preparation of Compound 18
[0195] A mixture of compound 15 (981 g, 1.04 mol, 1.00 eq),
compound 17 (894 g, 1.06 mol, 1.02 eq), and HOBt (288 g, 2.1 mol,
2.0 eq) in chloroform (17.9 L) is treated with a solution of EDAC
(240 g, 1.25 mol, 1.2 eq) in chloroform (2.2 L) followed by an
addition of NMM (161 g, 1.6 mol, 1.5 eq.). The reaction mixture is
stirred at 20-25.degree. C. and the reaction progress is monitored
by in-process HPLC. After the reaction is completed, the reaction
mixture is processed by standard extraction procedures to afford
compound 18 (quantitative yield, 1840 g of crude product) as a
solid. The purity of the crude product 18 is expected to be
determined to be 80.0% by HPLC analysis. The crude product is
subjected to a first recrystallization from 2-propanol/methanol
followed by a second recrystallization from chloroform/2-propanol
to afford a purified compound 18 (1280 g, 69.8% yield), and its
purity, determined by HPLC analysis, is expected to be about
95.1%.
Step 8: Preparation of Compound 19
[0196] A mixture of DCM (3.1 L), THF (3.1 L), and phosphoric acid
(5323 g, 85%, 46.2 mol, 65 eq.) is prepared and the purified
compound 18 prepared in Step 7 (1248 g, 0.707 mol) is added portion
wise over 30 minutes. The reaction mixture is stirred at
20-25.degree. C. and the reaction progress is monitored by
in-process HPLC. After the reaction is completed, the reaction
mixture is quenched with aqueous NaOH (the pH of the reaction
mixture is adjusted to 8-9) and processed by standard extraction
procedures to afford compound 19 (quantitative yield, 1323 g of
crude product). The purity of the crude product is expected to be
determined to be about 90.5% by HPLC analysis. The crude product 19
is purified by silica gel chromatography. The purification process
uses 30 g of silica gel (230-400 mesh) per gram of the crude
product 19. 1% methanol/DCM to 10% methanol/DCM (in gradient) is
used as elution solvents. After the chromatography, of 460 g
(390/a) of purified compound 19 is obtained. The purity of the
purified compound 19, determined by HPLC analysis, is expected to
be about 97.5%.
Step 9: Preparation of Delparantag (PMX-60056)
[0197] A mixture of the purified compound 19 prepared by Step 8
(417 g, 0.251 mol), 10 wt % palladium on carbon (167 g), methanol
(16.7 L), and HCl (5.0 eq., in a 7.2 weight/o aqueous solution) is
subjected to hydrogen gas at 70 psi pressure. The reaction mixture
is agitated at 25.degree. C. and the reaction progress is monitored
by in-process HPLC. After the reaction is completed, the reaction
mixture is filtered and concentrated by co-distillation with
acetonitrile to afford a solid product, which is slurried in
tert-butylmethyl ether (MTBE), filtered, and dried to afford
delparantag. Yield: 300 g (91%) (as a penta HCl), expected HPLC
purity: about 97.9%
[0198] Purification of Delparantag:
[0199] Impure delparantag (274 g, 0.209 mol) is dissolved in
methanol (13.9 L), and subsequently treated with 28 g of
3-mercaptopropyl ethyl sulfided silica gel and stirred for 90
minutes. The mixture is filtered and concentrated by
co-distillation with acetonitrile to afford a solid product, which
is slurried in MTBE, filtered, and dried. This purification process
is repeated one more time on the purified product obtained
previously (266 g, 0.203 mol) and the second purification process
results in 219 g of delparantag. Expected HPLC purity:
97.9%.degree., Pd content: 2.7 ppm.
Example 3
PMX-30063 Inhibits PDE4A
[0200] Phosphodiesterase type 4 (PDE4) is predominant
phosphodiesterase expressed in neutrophils, T cells and
macrophages. PDE inhibitors show broad spectrum of
anti-inflammatory effects in almost all inflammatory cells. PDE4
inhibitors, block the degradative action of PDE4 on cAMP, thereby
increasing intracellular levels of cAMP levels which mediate
phosphorylation of protein kinases. PDE4 inhibitors reduce
neutrophil chemotaxis, recruitment and activation; inhibit the
activation of CD4+ and CD8+ T cells; and inhibit monocytes
chemotaxis. Therefore, inhibition of PDEs is expected to have a
therapeutic effect in inflammatory diseases such as inflammatory
diseases of the gastrointestinal tract. To test if PMX-30063 can
inhibit PDE4 phosphodiesterase, inhibition assays of PDE4 were
performed, using PMX-30063. The PDE-Glo phosphodiesterase assay was
performed according to the Method described for FIG. 1, using 8 ng
of PDE4B, 1 .mu.M cAMP substrate and PMX-30063. Data are presented
as luminescence units (RLU).
[0201] PMX-30063 inhibited PDE4 with an IC.sub.50 in the 3 .mu.M
range (n=5) (FIG. 1). This is the first report of an HDP mimetic
inhibiting a PDE. Since in various animal models, inhibition of
PDE4 demonstrates pronounced anti-inflammatory effects, inhibition
of PDE4 by PMX-30063 may have a broad range of anti-inflammatory
effects on various key effector cells involved in inflammatory
diseases of the gastrointestinal tract.
Example 4
PMX-60056 Inhibits PDE4A
[0202] Since PMX-30063 inhibited PDE4, it was decided to also assay
PMX-60056 for inhibition of PDE4 activity as well. Therefore,
phosphodiesterase inhibition assays of PDE4 were performed with
PMX-60056.
[0203] The PDE-Glo phosphodiesterase assay was performed according
to the Method described for FIG. 2, using 8 ng of PDE4B, 1 .mu.M
cAMP substrate PMX-60056. Data are presented as luminescence units
(RLU).
[0204] PMX-60056 inhibited PDE4 with an IC.sub.50 in the 3 .mu.M
range (n=5) (FIG. 2). Since in various animal models PDE4
inhibitions show pronounced anti-inflammatory effects, inhibition
of PDE4 by PMX-60056 may have a broad range of anti-inflammatory
effects on various key effector cells involved in inflammatory
diseases of the gastrointestinal tract.
Example 5
PMX-30063 Inhibits PDE3A
[0205] Phosphodiesterase is a family of enzymes that catalyze the
breakdown of signaling molecule cyclic AMP/or cyclic GMP. cAMP and
cGMP are ubiquitous secondary-messenger signaling molecules
produced by a large family of cyclases that participate in a
multitude of signaling processes. The present inventors
hypothesized that HDP may be functioning through the cyclic
AMP/cyclic GMP pathways in suppression of proinflammatory response.
PDE3 inhibitors block degradation of both cAMP and cGMP which leads
to an increase of intracellular cAMP/cGMP concentrations.
Therefore, phosphodiesterase inhibition assays of PDE3 were
performed with PMX-30063.
[0206] The PDE-Glo phosphodiesterase assay was performed according
to the Method described for FIG. 3 using 2.75 ng of PDE3A, 1 .mu.M
cAMP substrate and PMX-30063. The compounds and PDE3A were mixed
and pre-incubated at room temperature for 15 minutes. Substrate was
added and the reaction was incubated.
[0207] PMX-30063 inhibited PDE3 at an IC.sub.50 of 1.5.+-.0.2 .mu.M
(n=4) (FIG. 3). Thus, PMX-30063 acts as both a PDE3 and PDE4
inhibitor as single molecule. Combining the functions of PDE4 and
PDE3 inhibition, PMX-30063 can function as an antimicrobial and an
anti-inflammatory. Additive and/or synergistic effects are produced
when multiple PDEs are inhibited concurrently (Rieder et al. PLoS
One 2013 2013; 8(2):e56867. doi: 10.1371/journal.pone.0056867. Epub
2013 Feb. 28). This is expected to reduce inflammation, as occurs
in inflammatory diseases of the gastrointestinal tract.
Example 6
PMX-60056 Inhibits PDE3A
[0208] Since PMX-30063 inhibited PDE3, it was decided to also assay
PMX-60056 for inhibition of PDE3 activity as well. Therefore,
phosphodiesterase inhibition assays of PDE3 were performed with
PMX-60056. The PDE-Glo phosphodiesterase assay was performed
according to the Method described for FIG. 4 using 2.75 ng of
PDE3A, 1 .mu.M cAMP substrate and PMX-60056. The compounds and
PDE3A were mixed and pre-incubated at room temperature for 15
minutes. Substrate was added and the reaction was incubated.
[0209] PMX-60056 inhibited PDE3 at an IC.sub.50 of 3 uM (FIG. 4).
Thus, PMX-60056 acts as both a PDE3 and PDE4 inhibitor as single
molecule. Combining the functions of PDE4 and PDE3 inhibition,
PMX-60056 can function as an antimicrobial and an
anti-inflammatory. Additive and/or synergistic effects are produced
when multiple PDEs are inhibited concurrently. This is expected to
reduce inflammation as occurs in inflammatory diseases of the
gastrointestinal tract.
Example 7
PMX-30063 Inhibits TNF-.alpha.
[0210] The intestinal lamina propria contains a complex population
of immune cells that balance the requirement for immune tolerance
of luminal microbiota with the need to defend against the pathogen,
excessive entry of luminal microbiota, or both. The hallmark of
active inflammatory bowel disease is a pronounced infiltration into
the lamina propria of innate immune cells (neutrophils,
macrophages, dendritic cells, and natural killer T cells) and
adaptive immune cells (T cells and B cells). Increased numbers and
activation of these cells in the intestinal mucosa elevate local
levels of TNF-.alpha., interleukin-1.beta., interleukin-6 (IL-6),
interferon-gamma (IFN-.gamma.), and cytokines of the
interleukin-23-Th17 pathway.
[0211] The proinflammatory cytokine TNF-alpha has been identified
as playing a pivotal role in the inflammatory cascade that causes
chronic intestinal inflammation in inflammatory diseases of the
gastrointestinal tract. TNF-.alpha. is a key mediator of
neutrophilic inflammation in inflammatory diseases of the
gastrointestinal tract. Anti-TNF-alpha antibody has been shown to
mitigate this inflammatory process. TNF-alpha inhibitors have been
shown to induce apoptosis of TNF-alpha producing immune cells,
reducing the production of a variety of downstream proinflammatory
cytokines from these and other cells. Hence its inhibition has
potential to target multiple components of inflammatory diseases of
the gastrointestinal tract. Therefore, the TNF-.alpha. inhibition
assay was performed with PMX-30063.
[0212] The TNF-.alpha. inhibition assay was performed according to
the Method described for FIG. 5. NR8383 rat macrophage cells were
pretreated with PMX-30063 for 45 minutes followed by treatment with
1 .mu.g/ml LPS for 8 hours. TNF-.alpha. concentrations in the
supernatants were determined by ELISA using an immunoassay kit
specific for rat TNF-.alpha. (R&D Systems).
[0213] PMX-30063 inhibited the LPS induced TNF-.alpha. production
in NR8383 rat macrophages (CRL-2192, ATCC) by about 50% at 0.5
.mu.M PMX-30063 (FIG. 5). As an anti-inflammatory HDP, PMX-30063
reduces the levels of TNF alpha, which may be very effective for
treatment of inflammatory diseases of the gastrointestinal
tract.
Example 8
PMX-60056 Inhibits TNF-.alpha.
[0214] Since PMX-30063 inhibited TNF-.alpha., it was decided to
also assay PMX-60056 for inhibition of TNF-.alpha. activity as
well. The TNF-.alpha. inhibition assay was performed according to
the Method described for FIG. 6. NR8383 rat macrophage cells
(CRL-2192, ATCC) were pretreated with PMX-60056 for 45 minutes
followed by treatment with 1 .mu.g/ml LPS for 8 hours. TNF-.alpha.
concentrations in the supernatants were determined by ELISA using
an immunoassay kit specific for rat TNF-.alpha. (R&D
Systems).
[0215] PMX-60056 inhibited the LPS induced TNF-.alpha. production
in NR8383 rat macrophages by more than 50% at 62.5 nM PMX-60056
(FIG. 6). As an anti-inflammatory HDP, PMX-60056 reduces the levels
of TNF alpha, an activity which may be very effective for treatment
of inflammatory diseases of the gastrointestinal tract.
Example 9
PMX-30063 Inhibits Monocyte Chemoattractant Protein-1
[0216] MCP-1 is produced by a variety of cells including dendritic
cells, macrophages, endothelial cells and fibroblasts, and its
expression is upregulated after exposure to inflammatory stimuli
such as IL-1 and TNF-alpha. MCP-1 was originally identified as
monocyte-specific chemoattractant but was later on shown to act on
T cells, mast cells, basophils, and natural killer cells. Elevation
of MCP-1 is observed in mucosal tissue from patients with Crohn's
disease and ulcerative colitis and also in experimental models of
colitis. Since MCP-1 binds to C--C Chemokine Receptor type 2
(CCR2), and MCP-1 can induce T cell and monocytic migration, this
chemokine contributes to recruitment of these cells in inflammatory
diseases of the gastrointestinal tract and plays an important role
in the induction of the inflammatory response. Therefore, the MCP-1
inhibition assay was performed with PMX-30063.
[0217] The MCP-1 inhibition assay was performed according to the
Method described for FIG. 7. When NR8383 rat macrophage cells
CRL-2192, ATCC) were pretreated with PMX-30063 for 45 minutes, we
observed a strong inhibition of MCP-1 induction after LPS (1
.mu.g/ml) stimulation for 8 hours (FIG. 7).
[0218] A minimum of 25% decrease in MCP-1 levels at 0.5M for
PMX-30063 was observed. These results further demonstrate the
potent anti-inflammatory effects of PMX-30063.
Example 10
PMX-60056 Inhibits Monocyte Chemoattractant Protein-1
[0219] Since PMX-30063 inhibited MCP-1, it was decided to assay
PMX-60056 for inhibition of MCP-1 activity as well. The MCP-1
inhibition assay was performed according to the Method described
for FIG. 8. When NR8383 rat macrophage cells (CRL-2192, ATCC) were
pretreated with PMX-60056 for 45 minutes, we observed a strong
inhibition of MCP-1 induction after LPS (1 .mu.g/ml) stimulation
for 8 hours (FIG. 8).
[0220] A minimum of 25% decrease in MCP-1 levels at 0.5M for
PMX-60056 was observed. These results further demonstrate the
potent anti-inflammatory effects of PMX-60056.
Example 11
PMX-30063 Inhibits Matrix-Metalloproteinase-9
[0221] Matrix metalloproteinase (MMP-9) has been shown to be
involved in the pathogenesis of inflammatory diseases such as
inflammatory diseases of the gastrointestinal tract. Inappropriate
expression and excessive activity of MMPs has been implicated in
the tissue destructive processes associated with inflammatory
diseases of the gastrointestinal tract. Chronic inflammation is
orchestrated by inflammatory cells which release proinflammatory
and destructive mediators such as elastases, proteases,
interleukin-8 (IL-8), leukotriene B-4 (LTB4), TNF alpha, and MMPs
that attract more inflammatory cells [Gueders, M. M., Foidart, J.
M., Noel, A. & Cataldo, D. D. Matrix metalloproteinases (MMPs)
and tissue inhibitors of MMPs in the respiratory tract: potential
implications in asthma and other lung diseases. European Journal of
Pharmacology 533, 133-144, (2006); Hurst, J. R. & Wedzicha, J.
A. The biology of a chronic obstructive pulmonary disease
exacerbation. Clinics in chest medicine 28, 525-536, (2007)]. These
proinflammatory cytokines lead to prolonged cycles of chronic
inflammation. Therefore, the MMP-9 inhibition assay was performed
with PMX-30063.
[0222] The MMP-9 inhibition assay was performed according to the
Method described for FIG. 9. Levels of MMP-9 activity from
supernatants of NR8383 rat macrophages CRL-2192, ATCC) pretreated
with PMX-30063 for 45 minutes followed by LPS (1 .mu.g/ml)
induction for 8 hours were determined.
[0223] A 50% decrease in MMP-9 levels at a 12.5 .mu.M concentration
of PMX-30063 was observed (FIG. 9). These results further
demonstrate the potent anti-inflammatory effects of PMX-30063.
Example 12
PMX-30063 Inhibits IL-6 Induction
[0224] The intestinal lamina propria contains a complex population
of immune cells that balance the requirement for immune tolerance
of luminal microbiota with the need to defend against the pathogen,
excessive entry of luminal microbiota, or both. The hallmark of
active inflammatory bowel disease is a pronounced infiltration into
the lamina propria of innate immune cells (neutrophils,
macrophages, dendritic cells, and natural killer T cells) and
adaptive immune cells (T cells and B cells). Increased numbers and
activation of innate immune cells (neutrophils, macrophages,
dendritic cells, and natural killer T cells) and adaptive immune
cells (T cells and B cells). Increased numbers and activation of
these cells in the intestinal mucosa elevate local levels of
TNF-.alpha., interleukin-1.beta., interleukin-6 (IL-6),
interferon-gamma (IFN-.gamma.), and cytokines of the
interleukin-23-Th17 pathway.
[0225] Influencing the production of IL-6 can change the balance of
effector CD4+ T cell subsets and induce B cell antibody production.
Moreover, given that IL-6 is mostly produced from innate cells such
as macrophages, neutrophils and mast cells, it is a strategic
bridge between the innate and the adaptive system. IL-6 has been
shown to be key player in chronic inflammation. Levels of
circulating IL-6 are elevated in several inflammatory diseases
including Crohn's disease. Expression of IL-6 is enhanced at the
site of inflammation and blockade of IL-6 and IL-6 signaling is
effective at prevention and treatment in models of inflammatory
disease like inflammatory diseases of the gastrointestinal tract.
Therefore, the inhibition of IL-6 induction assay was performed
with PMX-30063.
[0226] The inhibition of IL-6 induction assay was performed
according to the Method described for FIG. 10. Pretreatment for 8
hours with PMX-30063 inhibited the LPS (1 .mu.g/ml) induced IL-6
production in NR8383 rat macrophages (CRL-2192, ATCC) by about
500/% at 0.5 .mu.M of PMX-30063 (FIG. 10), an activity which may be
very effective for treatment of inflammatory diseases of the
gastrointestinal tract.
[0227] Summary of Anti-Inflammatory Activity for PMX-30063 and
PMX-60056 (FIGS. 1-10)
[0228] As an anti-inflammatory agent, PMX-30063 reduced the levels
of TNF-.alpha., MCP-1, MMP-9, and IL-6. PMX-60056 also reduced the
levels of TNF-.alpha., and MCP-1. The anti-inflammatory functions
of PMX-30063 and PMX-60056 may be mediated by reducing several
proinflammatory pathways and regulating the intracellular
concentration of cyclic nucleotide and its signaling pathways
consequently effecting a myriad of biological responses in chronic
inflammatory diseases such as inflammatory diseases of the
gastrointestinal tract.
In Vivo Distribution Study Demonstrates PMX-30063 Given Orally
Remains Primarily in the Small Intestine
Example 13
Concentration of PMX-30063 in the Plasma Following Intravenous or
Oral Administration to Mice
[0229] To evaluate the extent of distribution into the plasma
following administration of PMX-30063, a study was conducted to
measure PMX-30063 plasma concentrations following 10 mg/kg given PO
or 5 mg/kg given IV in male Balb/c mice (Study 16009-12001)
according to the Method described for FIG. 11.
[0230] The plasma concentration versus time curves for PMX-30063
following IV or PO administration is shown (FIG. 11). The peak
concentration of PMX-30063 given IV was 48,415.+-.7803 ng/mL,
whereas when given PO, peak concentration in plasma was
33.7.+-.8.56 ng/mL. This demonstrates that less than 0.1% of
PMX-30063 that is administered orally enters the circulation which
greatly reduces the risk of systemic toxicity.
Example 14
Concentration of PMX-30063 in the Small Intestine Following Oral
Administration to Mice
[0231] The concentration of PMX-30063 in small intestine following
PO administration of 10 mg/kg was conducted according the Method
described for FIG. 12. At 1 hour, the concentration in the small
intestine peaked at 38,941.+-.4703 ng/gram of tissue (FIG. 12).
This demonstrates that PMX-30063 when given orally enters into the
small intestine tissues where it can exert its anti-inflammatory
effects at the local level.
Example 15
Ratio of Intestine to Plasma Concentration Following Oral
Administration of PMX-30063
[0232] The intestine to plasma concentration ratio following oral
administration of PMX-30063 was calculated based on data derived
from FIGS. 11 and 12 according to the Method described for FIG.
13.
[0233] In the first hour, these ratios ranged from 2243 to 4323
demonstrating that less than 0.1% of PMX-30063 initially enters the
circulation following oral administration. Over 4 hours, still less
than 0.5% enters the circulation following oral administration
(FIG. 13).
[0234] Pharmacokinetics parameters of PMX-30063 in the plasma and
small intestine of male mice following intravenous and oral
administration of PMX-30063 are shown in the Table below. These
data also demonstrate that total exposure (AUC) in the blood is
less than 0.5% upon oral administration.
TABLE-US-00002 AUC(0-t) AUC(0-.infin.) MRT(0-.infin.) t1/2 Tmax Vz
CL Cmax F* ng/mL*h ng/mL*h H h h L/kg L/h/kg ng/mL % Plasma: IV (5
mg/kg) 97668 97818 2.86 2.76 0.0830 0.204 0.0511 48415 PO (10
mg/kg) 111 113 2.68 1.26 2.00 NA NA 33.7 0.0579 AUC(0-t)
AUC(0-.infin.) MRT(0-.infin.) t1/2 Tmax Vz CL Cmax ng/g*h ng/g*h H
h h L/kg L/h/kg ng/g Intestine: PO (10 mg/kg) 86089 89525 1.52
0.790 1.00 NA NA 38941
[0235] FIG. 11, FIG. 12, FIG. 13 and the Table demonstrate that
with oral administration, PMX-30063 is taken up by the tissues in
the small intestine but <0.5% enters the circulation which
offers a great advantage for treatment of intestinal epithelium
with low risk of systemic toxicity.
Example 16
Effect of PMX-30063 on Intestine Weights In Vivo in an Ulcerative
Colitis Model
[0236] PMX-30063 was evaluated for in vivo efficacy in an
ulcerative colitis (UC) model according to the Method described for
FIG. 14. Briefly, UC was induced by injecting 4% acetic acid into
the rectum. Four days later, animals were treated once daily for 5
days with PMX-30063 at either 100 mg/kg, 200 mg/kg or 400 mg/kg
intrarectally, or with 5-ASA, or no treatment.
[0237] Intestine weights were significantly reduced by 17% at the
400 mg/kg dose compared to untreated controls (p=0.02) (FIG. 14,
Table below). Since UC causes inflammation of tissues, it was
expected that the intestine weights would decrease upon treatment
with PMX-30063.
Example 17
PMX-30063 Demonstrated In Vivo Efficacy in an Ulcerative Colitis
Model
[0238] PMX-30063 was evaluated for efficacy in an ulcerative
colitis (UC) model according to the Method described for FIG.
15.
[0239] A dose dependent decrease in ulcerative colitis score in
animals was observed (FIG. 15). Treatment with PMX-30063 at 100
mg/kg reduced the median UC score by 33% and at 200 and 400 mg/kg
further reduced the UC score by 67%, but not significantly (Table
below). In animals treated with 5-ASA, the UC score was also
reduced by 67%, but not significantly. Maximum weight loss was 12%
among all groups.
TABLE-US-00003 Day 7 p value Student's t test intestine weight
ulcerative colitis score 100 mg/kg vs. untreated 0.29 0.97 200
mg/kg vs. untreated 0.34 0.21 400 mg/kg vs. untreated 0.02 0.08 100
mg/kg vs. 5-ASA 0.11 0.27 200 mg/kg vs. 5-ASA 0.14 0.95 400 mg/kg
vs. 5-ASA 0.54 0.35
[0240] Therefore, this preliminary study suggested that PMX-30063
given intrarectally may be effective in reducing the clinical
symptoms of ulcerative colitis while being well-tolerated. We
hypothesize that PMX-30063, as an HDP mimetic may be functioning
through the cyclic AMP/cyclic GMP pathways in suppression of
proinflammatory response. PDE4 is a predominant phosphodiesterase
expressed in neutrophils, T cells and macrophages and PDE4
inhibitors reduce neutrophil chemotaxis, recruitment and
activation, inhibits the activation of CD4+ and CD8+ T cells, and
inhibits monocytes chemotaxis. Hence, PDE4 has a broad range of
anti-inflammatory effects on various key effector cells that may be
involved in ulcerative colitis, Crohn's disease and other
inflammatory bowel diseases.
SUMMARY OF BIOLOGICAL DATA
[0241] In summary, demonstrating anti-inflammatory activity,
PMX-30063 (brilacidin) reduced the levels of TNF-.alpha., MCP-1,
MMP-9, and IL-6. PMX-60056 (delparantag) also reduced the levels of
TNF-.alpha., and MCP-1. The anti-inflammatory functions of
PMX-30063 and PMX-60056 may be mediated by reducing several
proinflammatory pathways and regulating the intracellular
concentration of cyclic nucleotide and its signaling pathways
consequently effecting a myriad of biological responses in chronic
inflammatory diseases such as inflammatory diseases of the
gastrointestinal tract.
[0242] PMX-30063 has both antimicrobial and anti-inflammatory
effects so it can be used when both infection and inflammation are
present. It can also be used to treat inflammation when there is no
infection. PMX-60056 may be used together with PMX-30063 or with
another antibiotic when both infection and inflammation are
present. PMX-30063 and/or PMX-60056 may be used when infection is
absent but inflammation is present or to provide prophylaxis
against inflammation. The use of PMX-30063 and/or PMX-60056 for
infections that may result in inflammation would provide
prophylaxis that could prevent inflammation and thus break a
potential vicious cycle between chronic bacterial colonization,
inflammation, and epithelial damage. PMX-30063 and PMX-60056 have
the potential to prevent the induction and progression of
inflammatory diseases of the gastrointestinal tract, unlike current
therapies which have limited efficacy in inhibiting chronic
inflammation, do not reverse the pathology of disease, and fail to
modify the factors that initiate and drive the long-term
progression of disease.
Example 18
Proposed Clinical Study
[0243] A Phase 2 Open Label, Multicenter Study to Assess the
Efficacy and Safety of Rectally Administered Brilacidin (PMX-30063)
for Induction of Remission in Subjects With Active Mild to Moderate
Ulcerative Proctitis (UP) or Ulcerative Proctosigmoiditis
(UPS).
[0244] At time of screening for enrollment, subjects who meet
endoscopic enrollment criteria will have two (2) rectal and two (2)
sigmoid biopsies obtained for possible future use in analysis of
efficacy results (biopsy results are not required for enrollment).
All subjects will receive brilacidin administered per rectum.
Assignment to treatment groups will be sequential at each
participating site. No randomization will be performed. At any one
site, no more than 50% of enrolled subjects may have UPS.
[0245] PMX-30063 (brilacidin) will be administered rectally, in
water for injection (WFI) as a retention enema, at a dose of A) 25
mg in 60 mL once daily at bedtime B) 50 mg in 60 mL once daily at
bedtime, C) 25 mg in 60 mL twice daily morning and at bedtime, or
D) 50 mg in 60 mL twice daily morning and at bedtime for 6 weeks.
As a proof of concept study, approximately 10 subjects for each arm
will be enrolled into the study. During the study, eligible
subjects will be allowed to maintain previously established oral
5-ASA treatment at doses up to 4.8 grams per day. Periodic safety
monitoring, including physical examinations, vital signs,
laboratory testing, and recording of AEs and concomitant
medications, will be performed during the study.
[0246] The primary objective is to assess the frequency of clinical
and endoscopic remission after 6 weeks of treatment with PMX-30063
administered per rectum in subjects with active UP or UPS based on
the Modified Mayo Disease Activity Index (MMDAI) score. The primary
efficacy measure is the percentage of patients achieving remission,
defined as an endoscopy score .ltoreq.1, rectal bleeding score=0,
and improvement or no change from baseline in stool frequency
subscales of the MMDAI at week 6.
[0247] Secondary objectives are to evaluate the safety of
brilacidin when administered per rectum and to estimate the
statistical power for subsequent trial(s) in this indication. Key
secondary outcomes include: [0248] Percentage of subjects with
clinical response [0249] Percentage of subjects achieving a rectal
bleeding MMDAI subscale score of 0 [0250] Percentage of subjects
with an endoscopy MIMDAI subscale score <1 at week 6 [0251]
Change in fecal calprotectin [0252] Change in serum C-reactive
protein (CRP) [0253] Change in serum IL-6 [0254] Improvement in
health related Quality of Life (QOL) [0255] Pharmacokinetics
data
* * * * *