U.S. patent application number 15/638006 was filed with the patent office on 2017-10-19 for composition for reducing frequency of urination, method of making and use thereof.
The applicant listed for this patent is WELLESLEY PHARMACEUTICALS, LLC. Invention is credited to David A. DILL.
Application Number | 20170296492 15/638006 |
Document ID | / |
Family ID | 55654698 |
Filed Date | 2017-10-19 |
United States Patent
Application |
20170296492 |
Kind Code |
A1 |
DILL; David A. |
October 19, 2017 |
COMPOSITION FOR REDUCING FREQUENCY OF URINATION, METHOD OF MAKING
AND USE THEREOF
Abstract
Pharmaceutical compositions for reducing frequency of urination
are disclosed. The pharmaceutical compositions comprise one or more
prostaglandin pathway inhibitors and a pharmaceutically acceptable
carrier. Also disclosed are methods of making and using the
pharmaceutical compositions.
Inventors: |
DILL; David A.; (Newtown,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WELLESLEY PHARMACEUTICALS, LLC |
Newtown |
PA |
US |
|
|
Family ID: |
55654698 |
Appl. No.: |
15/638006 |
Filed: |
June 29, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14975332 |
Dec 18, 2015 |
|
|
|
15638006 |
|
|
|
|
14298511 |
Jun 6, 2014 |
9532959 |
|
|
14975332 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/167 20130101;
A61K 31/167 20130101; A61K 31/616 20130101; A61K 31/192 20130101;
A61K 31/7088 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/405
20130101; A61K 2300/00 20130101; A61K 31/437 20130101; A61K 31/122
20130101; A61K 31/405 20130101; A61K 31/122 20130101; A61K 31/44
20130101; A61K 31/192 20130101; A61K 45/06 20130101; A61K 31/27
20130101; A61K 31/616 20130101 |
International
Class: |
A61K 31/167 20060101
A61K031/167; A61K 31/7088 20060101 A61K031/7088; A61K 31/44
20060101 A61K031/44; A61K 31/192 20060101 A61K031/192; A61K 31/437
20060101 A61K031/437; A61K 31/27 20060101 A61K031/27; A61K 45/06
20060101 A61K045/06; A61K 31/616 20060101 A61K031/616 |
Claims
1. A method for manufacturing a pharmaceutical composition for
reducing the frequency of urination, comprising: forming a first
mixture comprising a first active ingredient formulated for
immediate release and a second active ingredient formulated for
extended release; coating the first mixture with a delayed release
coating to form a core structure; coating the core structure with a
second mixture comprising a third active ingredient formulated for
immediate release and a fourth active ingredient formulated for
extended release, wherein at least one of the first, second, third
and fourth active ingredients comprises a prostaglandin pathway
inhibitor.
2. The method of claim 1, wherein at least one of the first,
second, third and fourth active ingredients comprises analgesic
agent.
3. The method of claim 2, wherein at least one of the first,
second, third and fourth active ingredients comprises (1)
prostaglandin pathway inhibitor and (2) an analgesic agent selected
from the group consisting of aspirin, ibuprofen, naproxen, naproxen
sodium, indomethacin, nabumetone, and acetaminophen.
4. The method of claim 2, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin activity and wherein the
analgesic agent is acetaminophen.
5. The method of claim 2, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin synthesis and wherein
the analgesic agent is acetaminophen.
6. The method of claim 2, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin transporter activity or
prostaglandin transporter expression and wherein the analgesic
agent is acetaminophen.
7. The method of claim 2, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin receptor activity or
prostaglandin receptor expression and wherein the analgesic agent
is acetaminophen.
8. The method of claim 1, wherein at least one of the first,
second, third and fourth active ingredients comprises an agent
selected from the group consisting antimuscarinic agents,
antidiuretic agents and spasmolytics.
9. The method of claim 1, wherein the delayed release coating is an
enteric coating.
10. The method of claim 9, wherein the enteric coating comprises a
pH-dependent polymer.
11. The method of claim 1, wherein the delayed release coating
comprises a swelling layer covered by an outer semi-permeable
polymer layer.
12. The method of claim 1, wherein the second active ingredient, or
the fourth active ingredient or both comprise an active core
comprising an extended-release coating or a polymeric matrix
effecting diffusion controlled release.
13. A pharmaceutical composition produced by the method of claim
1.
14. A method for manufacturing a pharmaceutical composition for
reducing the frequency of urination, comprising: forming a core
structure comprising a first active ingredient formulated for
immediate release and a second active ingredient formulated for
extended release; coating the core structure with a delayed release
coating to form a coated core structure; mixing the coated core
structure with a third active ingredient formulated for immediate
release and a fourth active ingredient formulated for extended
release to form a final mixture; and preparing a dosage form with
the final mixture, wherein at least one of the first, second, third
and fourth active ingredients comprises a prostaglandin pathway
inhibitor.
15. The method of claim 14, wherein at least one of the first,
second, third and fourth active ingredients comprises an analgesic
agent.
16. The method of claim 15, wherein at least one of the first,
second, third and fourth active ingredients comprises (1)
prostaglandin pathway inhibitor and (2) an analgesic agent selected
from the group consisting of aspirin, ibuprofen, naproxen, naproxen
sodium, indomethacin, nabumetone, and acetaminophen.
17. The method of claim 15, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin activity and wherein the
analgesic agent is acetaminophen.
18. The method of claim 15, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin synthesis and wherein
the analgesic agent is acetaminophen.
19. The method of claim 15, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin transporter activity or
prostaglandin transporter expression and wherein the analgesic
agent is acetaminophen.
20. The method of claim 15, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin receptor activity or
prostaglandin receptor expression and wherein the analgesic agent
is acetaminophen.
21. The method of claim 15, wherein at least one of the first,
second, third and fourth active ingredients comprises an agent
selected from the group consisting antimuscarinic agents,
antidiuretic agents and spasmolytics.
22. A pharmaceutical composition produced by the method of claim
14.
23. A method for manufacturing a pharmaceutical composition for
reducing the frequency of urination, comprising: forming a core
structure comprising a first active ingredient formulated for
immediate release and a second active ingredient formulated for
extended release; coating the core structure with a delayed release
coating to form a coated core structure; coating the coated core
structure with a third active ingredient formulated for extended
release to form an extended-release layer coated core structure;
and coating the extended-release layer coated core structure with a
fourth active ingredient, wherein at least one of the first,
second, third and fourth active ingredients comprises a
prostaglandin pathway inhibitor.
24. The method of claim 23, wherein at least one of the first,
second, third and fourth active ingredients comprises an analgesic
agent.
25. The method of claim 24, wherein at least one of the first,
second, third and fourth active ingredients comprises (1)
prostaglandin pathway inhibitor and (2) an analgesic agent selected
from the group consisting of aspirin, ibuprofen, naproxen, naproxen
sodium, indomethacin, nabumetone, and acetaminophen.
26. The method of claim 24, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin activity and wherein the
analgesic agent is acetaminophen.
27. The method of claim 24, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin synthesis and wherein
the analgesic agent is acetaminophen.
28. The method of claim 24, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin transporter activity or
prostaglandin transporter expression and wherein the analgesic
agent is acetaminophen.
29. The method of claim 24, wherein the prostaglandin pathway
inhibitor is an inhibitor of prostaglandin receptor activity or
prostaglandin receptor expression and wherein the analgesic agent
is acetaminophen.
30. The method of claim 24, wherein at least one of the first,
second, third and fourth active ingredients comprises an agent
selected from the group consisting antimuscarinic agents,
antidiuretic agents and spasmolytics.
31. A pharmaceutical composition produced by the method of claim
23.
32. A pharmaceutical composition, comprising: a first component
comprising an immediate-release subcomponent and an
extended-release subcomponent, wherein the first component is
formulated to release the subcomponents immediately after
administration; and a second component comprising an
immediate-release subcomponent and an extended-release
subcomponent, wherein the second component is formulated for a
delayed-release of the subcomponents, wherein at least one of
subcomponents in the first component or the second component
comprises an active ingredient comprising a prostaglandin pathway
inhibitor.
33. The pharmaceutical composition of claim 32, wherein at least
one of subcomponents in the first component and the second
component comprises an active ingredient comprising an analgesic
agent.
34. The pharmaceutical composition of claim 33, wherein the active
ingredient in at least one subcomponent comprises (1) an analgesic
agent selected from the group consisting of aspirin, ibuprofen,
naproxen, naproxen sodium, indomethacin, nabumetone, and
acetaminophen, and (2) a prostaglandin pathway inhibitor.
35. The pharmaceutical composition of claim 34, wherein the
prostaglandin pathway inhibitor is an inhibitor of prostaglandin
activity and wherein the analgesic agent is acetaminophen.
36. The pharmaceutical composition of claim 34, wherein the
prostaglandin pathway inhibitor is an inhibitor of prostaglandin
synthesis and wherein the analgesic agent is acetaminophen.
37. The pharmaceutical composition of claim 34, wherein the
prostaglandin pathway inhibitor is an inhibitor of prostaglandin
transporter activity or prostaglandin transporter expression and
wherein the analgesic agent is acetaminophen.
38. The pharmaceutical composition of claim 34, wherein the
prostaglandin pathway inhibitor is an inhibitor of prostaglandin
receptor activity or prostaglandin receptor expression and wherein
the analgesic agent is acetaminophen.
39. The pharmaceutical composition of claim 32, wherein the second
component is coated with an enteric coating.
40. The pharmaceutical composition of claim 32, wherein the second
component is formulated to release the subcomponents after a lag
time of 1-4 hours following oral administration.
41. The pharmaceutical composition of claim 32, wherein the
extended-release subcomponent in the first component is formulated
to release its active ingredient over a time interval of about 2-10
hours.
42. The pharmaceutical composition of claim 41, wherein the
extended-release subcomponent in the second component is formulated
to release its active ingredient over a time interval of about 2-10
hours.
43. The pharmaceutical composition of claim 32, wherein at least
one of the subcomponents of the first or second component further
comprises an agent selected from the group consisting of
antimuscarinic agents, antidiuretic agents and spasmolytics.
44. A pharmaceutical composition, comprising: a first component
comprising an immediate-release subcomponent, wherein the
immediate-release subcomponent comprises an active ingredient
comprising one or more agents selected from the group consisting of
analgesic agents and prostaglandin pathway inhibitors, wherein the
first component is formulated to release its subcomponent
immediately after oral administration; and a second component
comprising an immediate-release subcomponent and an
extended-release subcomponent, wherein the second component is
formulated to release its subcomponent after gastric emptying of
the second component, wherein at least one of the subcomponents in
the first or the second components comprises an active ingredient
comprising one or more agents selected from the group consisting of
analgesic agents and prostaglandin pathway inhibitors.
45. The pharmaceutical composition of claim 44, wherein the
analgesic agents are selected from the group consisting of aspirin,
ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and
acetaminophen.
46. The pharmaceutical composition of claim 44, wherein the second
component is formulated to release the subcomponents after a lag
time of 1-4 hours following oral administration.
47. The pharmaceutical composition of claim 44, wherein the first
component further comprises an extended-release subcomponent,
wherein the extended-release subcomponent comprises an active
ingredient comprising one or more agents selected from the group
consisting of analgesic agents and prostaglandin pathway
inhibitors.
48. The pharmaceutical composition of claim 44, wherein the active
ingredient in the immediate-release subcomponent of the first
component, and the active ingredient in the immediate-release
subcomponent and the extended-release subcomponent of the second
component further comprise an agent selected from the group
consisting of antimuscarinic agents, antidiuretic agents and
spasmolytics.
49. A pharmaceutical composition, comprising: an immediate-release
component comprising acetaminophen and an NSAID, each in an amount
of 5-2000 mg; and an extended-release component comprising
acetaminophen and an NSAID, each in an amount of 5-2000 mg, wherein
the immediate-release component, or the extended-release component,
or both, further comprise a prostaglandin pathway inhibitor.
50. The pharmaceutical composition of claim 49, wherein the
extended-release component is further coated with a delayed-release
coating.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 14/975,332, filed Dec. 18, 2015 which is a
continuation-in-part application of U.S. application Ser. No.
14/298,511, filed on Jun. 6, 2014, now U.S. Pat. No. 9,532,959. The
entirety of the aforementioned application is incorporated herein
by reference.
FIELD
[0002] The present application generally relates to methods and
compositions for inhibiting the smooth muscles of the urinary
bladder and, in particular, to methods and compositions for
reducing the frequency of urination.
BACKGROUND
[0003] The detrusor muscle is a layer of the urinary bladder wall
made of smooth muscle fibers arranged in spiral, longitudinal, and
circular bundles. When the bladder is stretched, this signals the
parasympathetic nervous system to contract the detrusor muscle.
This encourages the bladder to expel urine through the urethra.
[0004] For the urine to exit the bladder, both the autonomically
controlled internal sphincter and the voluntarily controlled
external sphincter must be opened. Problems with these muscles can
lead to incontinence. If the amount of urine reaches 100% of the
urinary bladder's absolute capacity, the voluntary sphincter
becomes involuntary and the urine will be ejected instantly.
[0005] The human adult urinary bladder usually holds about 300-350
ml of urine (the working volume), but a full adult bladder may hold
up to about 1000 ml (the absolute volume), varying among
individuals. As urine accumulates, the ridges produced by folding
of the wall of the bladder (rugae) flatten and the wall of the
bladder thins as it stretches, allowing the bladder to store larger
amounts of urine without a significant rise in internal
pressure.
[0006] In most individuals, the desire to urinate usually starts
when the volume of urine in the bladder reaches around 200 ml. At
this stage it is easy for the subject, if desired, to resist the
urge to urinate. As the bladder continues to fill, the desire to
urinate becomes stronger and harder to ignore. Eventually, the
bladder will fill to the point where the urge to urinate becomes
overwhelming, and the subject will no longer be able to ignore
it.
[0007] In some individuals, this desire to urinate starts when the
bladder is less than 100% full in relation to its working volume.
Such increased desire to urinate may interfere with normal
activities, including the ability to sleep for sufficient
uninterrupted periods of rest. In some cases, this increased desire
to urinate may be associated with medical conditions such as benign
prostate hyperplasia or prostate cancer in men, or pregnancy in
women. However, increased desire to urinate also occurs in
individuals, both male and female, who are not affected by another
medical condition.
[0008] In some individuals, such as in children, involuntary
urination (e.g., bed wetting) may occur as a result of lack of
control to the bladder muscle. In other individuals, involuntary
urination (e.g., urinary incontinence) may occur as a result from
an underlying medical condition.
[0009] Accordingly, there exists a need for compositions and
methods for the treatment of male and female subjects who suffer
from an undesired frequency of urination.
SUMMARY
[0010] One aspect of the present application relates to a method
for manufacturing a pharmaceutical composition for reducing the
frequency of urination. In some embodiments, the method comprises
the steps of forming a first mixture comprising a first active
ingredient formulated for immediate release and a second active
ingredient formulated for extended release; coating the first
mixture with a delayed release coating to form a core structure;
coating the core structure with a second mixture comprising a third
active ingredient formulated for immediate release and a fourth
active ingredient formulated for extended release, wherein at least
one of the first, second, third and fourth active ingredients
comprises a prostaglandin pathway inhibitor. In some embodiments,
the prostaglandin pathway inhibitor is a prostaglandin (PG)
inhibitor, or a prostaglandin transporter (PGT) inhibitor, or a
prostaglandin receptor (PGR) inhibitor.
[0011] In other embodiments, the method comprises the steps of
forming a core structure comprising a first active ingredient
formulated for immediate release and a second active ingredient
formulated for extended release; coating the core structure with a
delayed release coating to form a coated core structure; mixing the
coated core structure with a third active ingredient formulated for
immediate release and a fourth active ingredient formulated for
extended release to form a final mixture; and preparing a dosage
form with the final mixture, wherein at least one of the first,
second, third and fourth active ingredients comprises a
prostaglandin pathway inhibitor.
[0012] In other embodiments, the method comprises the steps of
forming a core structure comprising a first active ingredient
formulated for immediate release and a second active ingredient
formulated for extended release; coating the core structure with a
delayed release coating to form a coated core structure; coating
the coated core structure with a third active ingredient formulated
for extended release to form an extended-release layer coated core
structure; and coating the extended-release layer coated core
structure with a fourth active ingredient, wherein at least one of
the first, second, third and fourth active ingredients comprises a
prostaglandin pathway inhibitor.
[0013] Another aspect of the present application relates to a
pharmaceutical composition for treating a condition that results in
undesired frequency of urination. In some embodiments, the
pharmaceutical composition comprises: a first component comprising
an immediate-release subcomponent and an extended-release
subcomponent, wherein the first component is formulated to release
the subcomponents immediately after administration; and a second
component comprising an immediate-release subcomponent and an
extended-release subcomponent, wherein the second component is
formulated for a delayed-release of the subcomponents, wherein at
least one of subcomponents in the first component and the second
component comprises an active ingredient comprising a prostaglandin
pathway inhibitor.
[0014] In other embodiments, the pharmaceutical composition
comprises: a first component comprising an immediate-release
subcomponent, wherein the immediate-release subcomponent comprises
an active ingredient comprising one or more agents selected from
the group consisting of analgesic agents and prostaglandin pathway
inhibitors, wherein the first component is formulated to release
its subcomponent immediately after oral administration; and a
second component comprising an immediate-release subcomponent and
an extended-release subcomponent, wherein the second component is
formulated to release its subcomponent after gastric emptying of
the second component, wherein at least one of the subcomponents in
the first and the second components comprises an active ingredient
comprising one or more agents selected from the group consisting of
analgesic agents and prostaglandin pathway inhibitors.
[0015] In other embodiments, the pharmaceutical composition
comprises: an immediate-release component comprising acetaminophen
and an NSAID, each in an amount of 5-2000 mg; and an
extended-release component comprising acetaminophen and an NSAID,
each in an amount of 5-2000 mg, wherein the immediate-release
component, or the extended-release component, or both, further
comprise a prostaglandin pathway inhibitor.
BRIEF DESCRIPTION OF DRAWINGS
[0016] FIGS. 1A and 1B are diagrams showing that analgesics
regulate expression of co-stimulatory molecules by Raw 264
macrophage cells in the absence (FIG. 1A) or presence (FIG. 1B) of
LPS. Cells were cultured for 24 hrs in the presence of analgesic
alone or together with Salmonella typhimurium LPS (0.05 .mu.g/ml).
Results are mean relative % of CD40+CD80+ cells.
DETAILED DESCRIPTION
[0017] The following detailed description is presented to enable
any person skilled in the art to make and use the invention. For
purposes of explanation, specific nomenclature is set forth to
provide a thorough understanding of the present invention. However,
it will be apparent to one skilled in the art that these specific
details are not required to practice the invention. Descriptions of
specific applications are provided only as representative examples.
The present invention is not intended to be limited to the
embodiments shown, but is to be accorded the broadest possible
scope consistent with the principles and features disclosed
herein.
[0018] As used herein, the term "prostaglandin (PG)" refers to a
group of lipid compounds that are derived enzymatically from fatty
acids and have a variety of physiological effects, such as
regulating the contraction and relaxation of smooth muscle tissue,
in the animal body. Every prostaglandin contains 20 carbon atoms,
including a 5-carbon ring. Examples of prostaglandin include,
prostaglandin E.sub.1 (PGE.sub.1), prostaglandin E.sub.2
(PGE.sub.2), Prostaglandin D.sub.2, prostaglandin I.sub.2
(PGI.sub.2, prostacyclin), and prostaglandin F.sub.2.alpha.
(PGF.sub.2.alpha.).
[0019] As used herein, the term "prostaglandin (PG) pathway
inhibitor" refers to agents that interact directly or indirectly
with one or more components involved in the synthesis or action of
PG on a target tissue, and interfere with either the levels or the
ultimate actions of prostaglandin on the target tissue. PG pathway
inhibitors include, but are not limited to, PG inhibitors,
prostaglandin transporter (PGT) inhibitors and prostaglandin
receptor (PGR) inhibitors. The term "PG pathway inhibitor,"
however, does not include the analgesics defined below.
[0020] The term "PG inhibitors," as used herein, include but are
not limited to, inhibitors of PG synthesis and inhibitors of PG
activity. The term "inhibitors of PG synthesis," as used herein,
refers to agents that inhibit the production of prostaglandin, such
as agents that inhibit the expression or activity of phospholipase
A2, the prostaglandin synthases and the tissue specific isomerases
and synthase enzymes such as: Thromboxane synthase, PGF synthase,
cytosolic PG synthase (cPGES), prostaglandin I synthase (PGIS) and
the microsomal PGES enzymes (mPGES). Examples of PG synthesis
inhibitors include flunixin meglumine. As used herein, the terms
"inhibitors of PG synthesis" and "PG synthesis inhibitors" do not
include the analgesics defined below.
[0021] The term "inhibitors of PG activity," as used herein, refers
to agents that antagonize the action of prostaglandin itself by any
means. Agents that interfere solely with the synthesis of
prostaglandins, such as by interfering with the action of
prostaglandin synthases, but which do not interfere with the action
of prostaglandins are not included within the definition of
inhibitors of PG activity as used in this specification.
[0022] The term "PGT inhibitors," as used herein, refers to agents
that inhibits the expression or the activity of PG transporters,
such as ATP dependent multi-drug resistance (MDR) transporter-4, or
other MDR channels such as ABCC1, ABCC2, ABCC3, ABCC6, ABCG2 and
ABCB11. Examples of PGT inhibitors that inhibit PGT activity
include, but are not limited to, compounds that inhibit MDR
membrane pumps, such as triazine compounds, verapamil, and calcium
channel blockers; channels include quinidines, ketoconazole,
itraconazole, azithromycin, valspodar, cyclosporine, elacridar,
fumitremorgin-C, gefitinib and erythromycin. Examples of PGT
inhibitors that inhibit PGT expression include, but are not limited
to, agents which control the transcription of the MDR genes by
targeting the promoter region and/or transcription factors which
bind to the promoter or other gene control regions. The term "PGR
inhibitors," as used herein, refers to agents that inhibits the
activity or expression of PGRs. In some embodiments, the PGRs
comprise E prostanoid receptor EP1, EP2, EP3, and EP4 subtypes of
the PGE receptor; PGD receptor (DP1); PGF receptor (FP); PGI
receptor (IP); and thromboxane receptor (TP). Two additional
isoforms of the human TP (TP.alpha. and TP.beta.) and FP (FPA and
FPB) and eight EP3 variants are generated through alternative
splicing, which differ only in their C-terminal tails. In some
embodiments, the PGRs further comprise a G protein-coupled receptor
termed chemo-attractant receptor-homologous molecule (CRHME). In
other embodiments, the PGRs include all of the receptors that
activate rhodopsin-like 7-transmembrane-spanning G protein-coupled
receptors.
[0023] Examples of PGR activity inhibitors include, but are not
limited to, anti-PGR antibodies and any agent that inhibits the
G-protein coupled receptor signaling pathway. PGR expression
inhibitors include agents that inhibit PGR expression at the
transcriptional level, translational level or post transcriptional
level. Examples of PGR expression inhibitors include, but are not
limited to, anti-PGR siRNA and mi RNAs.
[0024] As used herein, the term "an effective amount" means an
amount necessary to achieve a selected result.
[0025] As used herein, the term "analgesic" refers to agents,
compounds or drugs used to relieve pain and inclusive of
anti-inflammatory compounds. Exemplary analgesic and/or
anti-inflammatory agents, compounds or drugs include, but are not
limited to, non-steroidal anti-inflammatory drugs (NSAIDs),
salicylates, aspirin, salicylic acid, methyl salicylate,
diflunisal, salsalate, olsalazine, sulfasalazine, para-aminophenol
derivatives, acetanilide, acetaminophen, phenacetin, fenamates,
mefenamic acid, meclofenamate, sodium meclofenamate, heteroaryl
acetic acid derivatives, tolmetin, ketorolac, diclofenac, propionic
acid derivatives, ibuprofen, naproxen sodium, naproxen, fenoprofen,
ketoprofen, flurbiprofen, oxaprozin; enolic acids, oxicam
derivatives, piroxicam, meloxicam, tenoxicam, ampiroxicam,
droxicam, pivoxicam, pyrazolon derivatives, phenylbutazone,
oxyphenbutazone, antipyrine, aminopyrine, dipyrone, coxibs,
celecoxib, rofecoxib, nabumetone, apazone, indomethacin, sulindac,
etodolac, isobutylphenyl propionic acid, lumiracoxib, etoricoxib,
parecoxib, valdecoxib, tiracoxib, etodolac, darbufelone,
dexketoprofen, aceclofenac, licofelone, bromfenac, loxoprofen,
pranoprofen, piroxicam, nimesulide, cizolirine,
3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one,
meloxicam, lornoxicam, d-indobufen, mofezolac, amtolmetin,
pranoprofen, tolfenamic acid, flurbiprofen, suprofen, oxaprozin,
zaltoprofen, alminoprofen, tiaprofenic acid, pharmacological salts
thereof, hydrates thereof, and solvates thereof.
[0026] As used herein, the term "coxib" refers to a compound or
composition of compounds that is capable of inhibiting the activity
or expression of COX1 and COX2 enzymes.
[0027] As used herein, the term "derivative" refers to a chemically
modified compound wherein the modification is considered routine by
the ordinary skilled chemist, such as an ester or an amide of an
acid, or protecting groups such as a benzyl group for an alcohol or
thiol, or a tert-butoxycarbonyl group for an amine.
[0028] As used herein, the term "analogue" refers to a compound
which comprises a chemically modified form of a specific compound
or class thereof and which maintains the pharmaceutical and/or
pharmacological activities characteristic of said compound or
class.
[0029] As used herein, the term "pharmaceutically acceptable salts"
refer to derivatives of the disclosed compounds wherein the parent
compound is modified by making acid or base salts thereof. Examples
of pharmaceutically acceptable salts include, but are not limited
to, mineral or organic acid salts of basic residues such as amines,
alkali or organic salts of acidic residues such as carboxylic
acids, and the like. The pharmaceutically acceptable salts include
the conventional non-toxic salts or the quaternary ammonium salts
of the parent compound formed, for example, from non-toxic
inorganic or organic acids. For example, such conventional
non-toxic salts include those derived from inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid,
phosphoric acid, nitric acid, and the like and the salts prepared
from organic acids such as acetic acid, propionic acid, succinic
acid, glycolic acid, stearic acid, lactic acid, malic acid,
tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic
acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic
acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid,
fumaric acid, toluensulfonic acid, methanesulfonic acid, ethane
dislfonic acid, oxalic acid, isethionic acid, and the like.
[0030] As used herein, the phrase "pharmaceutically acceptable" is
used with reference to compounds, materials, compositions and/or
dosage forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problems or complications commensurate with a reasonable
benefit/risk ratio.
[0031] The term "immediate-release" is used herein with reference
to a drug formulation that does not contain a dissolution rate
controlling material. There is substantially no delay in the
release of the active agents following administration of an
immediate-release formulation. An immediate-release coating may
include suitable materials immediately dissolving following
administration so as to release the drug contents therein. In some
embodiments, the term "immediate-release" is used with reference to
a drug formulation that releases the active ingredient in less than
10 min, 20 min, 30 min, 40 min 50 min, 60 min, 90 min or 120 min
after administration into a patient.
[0032] As used herein, the term "extended-release," also known as
sustained-release (SR), sustained-action (SA), time-release (TR),
controlled-release (CR), modified release (MR), or
continuous-release (CR), refers to a mechanism used in medicine
tablets or capsules to dissolve slowly and release the active
ingredient over time. The advantages of extended-release tablets or
capsules are that they can often be taken less frequently than
immediate-release formulations of the same drug and that they keep
steadier levels of the drug in the bloodstream, thus extending the
duration of the drug action and lowering the peak amount of drug in
the bloodstream. In some embodiments, the term "extended-release"
refers to a release profile that the active ingredient in a tablet
or capsule is released over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 14, 16, 18, 20, 22 or 24 hour, either continuously or in pulses
after administration into a patient.
[0033] As used herein, the term "delayed-release" refers to a drug
release profile that the release of the active ingredient(s) of a
pharmaceutical composition is delayed or postponed for a given
period of time (e.g., 1, 2, 3, 4 or 5 hours, or after stomach)
after administration of the pharmaceutical composition.
[0034] As used herein, the term "delayed-extended-release" refers
to a drug release profile that the release of the active
ingredient(s) of a pharmaceutical composition is delayed or
postponed for a given period of time (e.g., the lag period of 1, 2,
3, 4 or 5 hours, or after stomach) after administration of the
pharmaceutical composition. Once the release starts, the active
ingredient(s) is released slowly over time (e.g., over a period of
2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or 24 hour),
either continuously or in pulses.
Method for Reducing Frequency of Urination
[0035] One aspect of the present application relates to a method
for reducing frequency of urination by administering to a subject
having a condition that results in an undesired frequency of
urination an effective amount of a pharmaceutical composition. The
pharmaceutical composition comprises one or more PG pathway
inhibitors and a pharmaceutically acceptable carrier. Conditions
that result in an undesired frequency of urination include, but are
not limited to, nocturia, overactive bladder, urinary incontinence
and bed wetting.
[0036] In some embodiments, the PG inhibitor is an inhibitor of PG
synthetase. Examples of inhibitors of PG synthesis include, but are
not limited to, inhibitors of PG synthetase. (this is redundant) In
other embodiments, the PG inhibitor is an inhibitor of PG activity.
Examples of inhibitors of PG activity include, but are not limited
to agents which block the binding of PG to any of its receptors:
EP1, EP2, EP3, EP4. DP1, DP2, FP2, IP and TP. Examples of these
types of inhibitors include, but are not limited to, the IP
receptor inhibitor developed by Roche: RO3244019, ONO-85-39 which
is an EP1 receptor antagonist, the dual EP1 and EP2 receptor
antagonist AH 6809, and the EP4 antagonist. RQ-15986. In certain
embodiments, the one or more PG pathway inhibitors comprise a PGT
inhibitor. In some embodiments, the PGT inhibitor is a PGT activity
inhibitor. Examples of PGT activity inhibitor include, but are not
limited to, anti-PGT antibodies, and any known compound which can
inhibit the ATP-dependent Multi drug resistance transporter-4 or
related MDR pumps that are shown to transport PGs. In other
embodiments, the PGT inhibitor is a PGT expression inhibitor.
Examples of PGT expression inhibitor include, but are not limited
to, anti-PGT siRNA, antisense RNAs that target PGT mRNA, and agents
which control the transcription of the gene by influencing DNA
methylation and or chromatin modification.
[0037] In some embodiments, the one or more PG pathway inhibitors
comprise an inhibitor that targets both the COX active site and the
PDX active site which are contained in both COX1 and COX2. In other
embodiments, the one or more PG pathway inhibitors comprise an
inhibitor that inhibits the PGE.sub.2 pathway.
[0038] In certain embodiments, the one or more PG pathway
inhibitors comprise a PGR inhibitor. PGRs are G-protein-coupled
receptors containing seven transmembrane domains. Examples of PGR
include, EP1, EP2, EP3, EP4, DP1, DP2, FP, IP1, IP2, CRTH2 and TP
receptors. In some embodiments, the one or more PG pathway
inhibitors comprise an inhibitor that inhibits any of the PG
receptors listed above. In some embodiments, the PGR inhibitor is a
PGR activity inhibitor. Examples of PGR activity inhibitor include,
but are not limited to, anti-PGR antibodies. In some embodiments,
the PGR inhibitor is an inhibitor of PGE2 receptor activity, such
as EP1 activity inhibitor, EP2 activity inhibitor, EP3 activity
inhibitor or EP4 activity inhibitor.
[0039] In other embodiments, the PGR inhibitor is a PGR expression
inhibitor. Examples of PGR expression inhibitor include, but are
not limited to, anti-PGR siRNA, antisense RNAs that target PGR
mRNA, or agents which control the transcription of the gene by
influencing DNA methylation and or chromatin modification. In some
embodiments, the PGR expression inhibitor is an inhibitor of PGE2
receptor expression, such as EP1 expression inhibitor, EP2
expression inhibitor, EP3 expression inhibitor or EP4 expression
inhibitor. In some embodiments, the one or more PG pathway
inhibitors comprise a small molecule inhibitor. As used herein, the
term "small molecule inhibitor" refers to inhibitors having a
molecular weight of 1000 dalton or less.
[0040] In some embodiments, the PG pathway inhibitor comprises a
short interfering RNA (siRNA). An siRNA is a double-stranded RNA
that can be engineered to induce sequence-specific
post-transcriptional gene silencing of mRNAs corresponding to a
component of the PG pathway. siRNAs exploit the mechanism of RNA
interference (RNAi) for the purpose of "silencing" gene expression
of e.g., targeted PGE.sub.2 receptor genes. This "silencing" was
originally observed in the context of transfecting double stranded
RNA (dsRNA) into cells. Upon entry therein, the dsRNA was found to
be cleaved by an RNase III-like enzyme, Dicer, into double stranded
small interfering RNAs (siRNAs) 21-23 nucleotides in length
containing 2 nucleotide overhangs on their 3' ends. In an ATP
dependent step, the siRNAs become integrated into a multi-subunit
RNAi induced silencing complex (RISC) which presents a signal for
AGO2-mediated cleavage of the complementary mRNA sequence, which
then leads to its subsequent degradation by cellular
exonucleases.
[0041] In some embodiments, the PG pathway inhibitor comprises a
synthetic siRNA or other class of small RNA targeting a PG synthase
RNA, a PGT RNA or a PGR RNA in the target cell/tissue.
Synthetically produced siRNAs structurally mimic the types of
siRNAs normally processed in cells by the enzyme Dicer.
Synthetically produced siRNAs may incorporate any chemical
modifications to the RNA structure that are known to enhance siRNA
stability and functionality. For example, in some cases, the siRNAs
may be synthesized as a locked nucleic acid (LNA)-modified siRNA.
An LNA is a nucleotide analogue that contains a methylene bridge
connecting the 2'-oxygen of the ribose with the 4' carbon. The
bicyclic structure locks the furanose ring of the LNA molecule in a
3'-endo conformation, thereby structurally mimicking the standard
RNA monomers.
[0042] In other embodiments, the PG pathway inhibitor comprises an
expression vector engineered to transcribe a short double-stranded
hairpin-like RNA (shRNA) that is processed into a targeted siRNA
inside the cell. The shRNAs can be cloned in suitable expression
vectors using kits, such as Ambion's SILENCER.RTM. siRNA
Construction Kit, Imgenex's GENESUPPRESSOR.TM. Construction Kits
and Invitrogen's BLOCK-IT.TM. inducible RNAi plasmid and lentivirus
vectors. Synthetic siRNAs and shRNAs may be designed using well
known algorithms and synthesized using a conventional DNA/RNA
synthesizer.
[0043] In some embodiments, the secondary active agent comprises an
antisense oligonucleotide or polynucleotide capable of inhibiting
the expression of a component of the PG pathway. The antisense
oligonucleotide or polynucleotide may comprise a DNA backbone, RNA
backbone or chemical derivative thereof. In one embodiment, the
antisense oligonucleotide or polynucleotide comprises a single
stranded antisense oligonucleotide or polynucleotide targeting for
degradation. In certain embodiments, the anti-inflammatory agent
comprises a single stranded antisense oligonucleotide complementary
to the mRNA sequence of a component of the PG pathway. The single
stranded antisense oligonucleotide or polynucleotide may be
synthetically produced or it may be expressed from a suitable
expression vector. The antisense nucleic acid is designed to bind
via complementary binding to the mRNA sense strand so as to promote
RNase H activity, which leads to degradation of the mRNA.
Preferably, the antisense oligonucleotide is chemically or
structurally modified to promote nuclease stability and/or
increased binding.
[0044] In some embodiments, the antisense oligonucleotides are
modified to produce oligonucleotides with nonconventional chemical
or backbone additions or substitutions, including, but not limited
to, peptide nucleic acids (PNAs), locked nucleic acids (LNAs),
morpholino backboned nucleic acids, methylphosphonates, duplex
stabilizing stilbene or pyrenyl caps, phosphorothioates,
phosphoroamidates, phosphotriesters and the like. By way of
example, the modified oligonucleotides may incorporate or
substitute one or more of the naturally occurring nucleotides with
an analog; internucleotide modifications incorporating, for
example, uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoamidates, carbamates, etc.) or charged
linkages (e.g., phosphorothioates, phosphorodithioates, etc.);
modifications incorporating intercalators (e.g., acridine,
psoralen, etc.), chelators (e.g., metals, radioactive metals,
boron, oxidative metals, etc.) or alkylators and/or modified
linkages (e.g., alpha anomeric nucleic acids, etc.).
[0045] In some embodiments, the single stranded oligonucleotides
are internally modified to include at least one neutral charge in
its backbone. For example, the oligonucleotide may include a
methylphosphonate backbone or peptide nucleic acid (PNA)
complementary to the target-specific sequence. These modifications
have been found to prevent or reduce helicase-mediated unwinding.
The use of uncharged probes may further increase the rate of
hybridization to polynucleotide targets in a sample by alleviating
the repulsion of negatively-charges nucleic acid strands in
classical hybridization.
[0046] PNA oligonucleotides are uncharged nucleic acid analogs for
which the phosphodiester backbone has been replaced by a polyamide,
which makes PNAs a polymer of 2-aminoethyl-glycine units bound
together by an amide linkage. PNAs are synthesized using the same
Boc or Fmoc chemistry as are use in standard peptide synthesis.
Bases (adenine, guanine, cytosine and thymine) are linked to the
backbone by a methylene carboxyl linkage. Thus, PNAs are acyclic,
achiral and neutral. Other properties of PNAs are increased
specificity and melting temperature as compared to nucleic acids,
capacity to form triple helices, stability at acid pH,
non-recognition by cellular enzymes like nucleases, polymerases,
etc.
[0047] Methylphosphonate-containing oligonucleotides are neutral
DNA analogs containing a methyl group in place of one of the
non-bonding phosphoryl oxygens. Oligonucleotides with
methylphosphonate linkages were among the first reported to inhibit
protein synthesis via anti-sense blockade of translation.
[0048] In some embodiments, the phosphate backbone in the
oligonucleotides may contain phosphorothioate linkages or
phosphoroamidates. Combinations of such oligonucleotide linkages
are also within the scope of the present invention.
[0049] In other embodiments, the oligonucleotide may contain a
backbone of modified sugars joined by phosphodiester
internucleotide linkages. The modified sugars may include furanose
analogs, including but not limited to 2-deoxyribofuranosides,
.alpha.-D-arabinofuranosides, .alpha.-2'-deoxyribofuranosides and
2',3'-dideoxy-3'-aminoribofuranosides. In alternative embodiments,
the 2-deoxy-.beta.-D-ribofuranose groups may be replaced with other
sugars, for example, .beta.-D-ribofuranose. In addition,
.beta.-D-ribofuranose may be present wherein the 2-OH of the ribose
moiety is alkylated with a C1-6 alkyl group (2-(O--C1-6 alkyl)
ribose) or with a C2-6 alkenyl group (2-(O--C2-6 alkenyl) ribose)
or is replaced by a fluoro group (2-fluororibose).
[0050] Related oligomer-forming sugars include those used in locked
nucleic acids (LNA) as described above. Exemplary LNA
oligonucleotides include modified bicyclic monomeric units with a
2'-O-4'-C methylene bridge, such as those described in U.S. Pat.
No. 6,268,490.
[0051] Chemically modified oligonucleotides may also include,
singly or in any combination, 2'-position sugar modifications,
5-position pyrimidine modifications (e.g.,
5-(N-benzylcarboxyamide)-2'-deoxyuridine,
5-(N-isobutylcarboxyamide)-2'-deoxyuridine,
5-(N-[2-(1H-indole-3yl)ethyl]carboxyamide)-2'-deoxyuridine,
5-(N-[1-(3-trimethylammonium) propyl]carboxyamide)-2'-deoxyuridine
chloride, 5-(N-napthylcarboxyamide)-2'-deoxyuridine,
5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2'-deoxyuridine),
8-position purine modifications, modifications at exocyclic amines,
substitution of 4-thiouridine, substitution of 5-bromo- or
5-iodo-uracil, methylations, unusual base-pairing combinations,
such as the isobases isocytidine and isoguanidine and the like.
[0052] In some embodiments, the one or more PG pathway inhibitors
comprise a ribozyme capable of inhibiting the expression of a
component of the PG pathway. Ribozymes are nucleic acid molecules
that are capable of catalyzing a chemical reaction, either
intramolecularly or intermolecularly. Ribozymes are thus catalytic
nucleic acid. It is preferred that the ribozymes catalyze
intermolecular reactions. There are a number of different types of
ribozymes that catalyze nuclease or nucleic acid polymerase type
reactions which are based on ribozymes found in natural systems,
such as hammerhead ribozymes, hairpin ribozymes and tetrahymena
ribozymes. There are also a number of ribozymes that are not found
in natural systems, but which have been engineered to catalyze
specific reactions de novo. Preferred ribozymes cleave RNA or DNA
substrates and more preferably cleave RNA substrates, such as mRNAs
of components of the PG pathway. Ribozymes typically cleave nucleic
acid substrates through recognition and binding of the target
substrate with subsequent cleavage. This recognition is often based
mostly on canonical or non-canonical base pair interactions. This
property makes ribozymes particularly good candidates for target
specific cleavage of nucleic acids because recognition of the
target substrate is based on the target substrates sequence.
[0053] In some embodiments, the one or more PG pathway inhibitors
comprise triplex forming oligonucleotide capable of inhibiting the
expression of a component of the PG pathway. Triplex forming
oligonucleotides (TFOs) are molecules that can interact with either
double-stranded and/or single-stranded nucleic acids, including
both coding and non-coding regions in genomic DNA targets. When
TFOs interact with a target region, a structure called a triplex is
formed, in which there are three strands of DNA forming a complex
dependant on both Watson-Crick and Hoogsteen base-pairing. TFOs can
bind target regions with high affinity and specificity. In
preferred embodiments, the triplex forming molecules bind the
target molecule with a Kd less than 10-6, 10-8, 10-10 or 10-12.
Exemplary TFOs for use in the present invention include PNAs, LNAs
and LNA modified PNAs, such as Zorro-LNAs.
[0054] In some embodiments, the one or more PG pathway inhibitors
comprise an external guide sequences (EGS). External guide
sequences (EGSs) are molecules that bind a target nucleic acid
molecule forming a complex. This complex is recognized by RNase P,
which cleaves the target molecule. EGSs can be designed to
specifically target an mRNA molecule of choice. RNAse P aids in
processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can
be recruited to cleave virtually any RNA sequence by using an EGS
that causes the target RNA:EGS complex to mimic the natural tRNA
substrate. Similarly, eukaryotic EGS/RNAse P-directed cleavage of
RNA can be utilized to cleave desired targets within eukaryotic
cells.
[0055] In other embodiments, the one or more PG pathway inhibitors
comprise a biomolecule. As used herein, the term "biomolecule" is
any molecule that is produced by a living organism, including large
macromolecules such as proteins, polysaccharides, lipids, and
nucleic acids, as well as small molecules such as primary
metabolites, secondary metabolites, and natural products.
[0056] In other embodiments, the one or more PG pathway inhibitors
comprise target neutralization agents. As used herein, the term
"target neutralization agent" refers to antibodies, fragments of
antibodies, or any other non-antibody peptide or synthetic binding
molecule, such as an aptamer or synbody, which is capable of
specifically binding directly or indirectly to a component of the
PG pathway so as to interfere with the ultimate actions of
prostaglandin on the target tissue.
[0057] The target neutralization agents may be produced by any
conventional method for generating high-affinity binding ligands,
including SELEX, phage display and other methodologies, including
combinatorial chemistry and/or high throughput methods known to
those of skill in the art.
[0058] An aptamer is a nucleic acid version of an antibody that
comprises a class of oligonucleotides that can form specific three
dimensional structures exhibiting high affinity binding to a wide
variety of cell surface molecules, proteins and/or macromolecular
structures. Aptamers are commonly identified by an in vitro method
of selection sometimes referred to as Systematic Evolution of
Ligands by EXponential enrichment or "SELEX." SELEX typically
begins with a very large pool of randomized polynucleotides which
is generally narrowed to one aptamer ligand per molecular target.
Typically, aptamers are small nucleic acids ranging from 15-50
bases in length that fold into defined secondary and tertiary
structures, such as stem-loops or G-quartets.
[0059] An aptamer can be chemically linked or conjugated to the
above described nucleic acid inhibitors to form targeted nucleic
acid inhibitors, such as aptamer-siRNA chimeras. An aptamer-siRNA
chimera contains a targeting moiety in the form of an aptamer which
is linked to an siRNA. When using an aptamer-siRNA chimera, it is
preferable to use a cell internalizing aptamer. Upon binding to
specific cell surface molecules, the aptamer can facilitate
internalization into the cell where the nucleic acid inhibitor
acts. In one embodiment both the aptamer and the siRNA comprises
RNA. The aptamer and the siRNA may comprise any nucleotide
modifications as further described herein. Preferably, the aptamer
comprises a targeting moiety specifically directed to binding cells
expressing the chemokine-, cytokine- and/or receptor target genes,
such as lymphoid, epithelial cell and/or endothelial cells.
[0060] Synbodies are synthetic antibodies produced from libraries
comprised of strings of random peptides screened for binding to
target proteins of interest.
[0061] Target neutralization agents, including aptamers and
synbodies, can be engineered to bind target molecules very tightly
with Kds between 10.sup.-10 to 10.sup.-12 M. In some embodiments,
the target neutralization agent binds the target molecule with a Kd
less than 10.sup.-6, less than 10.sup.-8, less than 10.sup.-9, less
than 10.sup.-10 or less than 10.sup.-12 M.
[0062] In certain embodiments, the one or more PG pathway
inhibitors comprise a polynucleotide that encodes and is adapted to
express a PGT inhibitor and/or a PGR inhibitor. In other
embodiments, the one or more PG pathway inhibitors comprise an
expression vector that encodes and is adapted to express a PGT
inhibitor and/or a PGR inhibitor.
[0063] In some embodiments, the PG pathway inhibitor is an
engineered protein containing a TALE sequence or and engineered
zinc finger directed at the gene encoding any component of the PG
pathway. This TALE or zinc finger could be designed to bind
directly to the gene and inhibit its expression by cleaving the
gene, altering its nucleotide sequence, or tethering a repressor
protein to the gene which serves to silence the gene.
[0064] In some embodiments, the PG pathway inhibitor is produced
using the CRISPR/CAS system. In this strategy a guide molecule
specific for the gene sequence of each pg pathway gene is designed
and introduced into the cell or tissue using delivery systems
described above (viruses, plasmids, etc). The action of the
CRISPR/CAS system would modify the DNA sequence of the gene such
that the PG pathway gene is deleted or inhibited in ability to
express the RNA.
[0065] In some embodiments, the PG pathway inhibitor is capable of
turning off the transcription of one or more PG pathway genes by
targeting the chromatin associated enzymes which post
translationally modify histones in chromatin. Examples of such
enzymes are (but not limited to) histone deacetylases, histone
demethylases, histone acetyltransferases, histone
methyltransferases, and helicases.
[0066] In some embodiments, the PG pathway inhibitor targets the
gene encoding each component by altering the DNA methylation status
of the gene. Compounds which target the TET family of DNA
demethylases and the DNA methyltransferases (DNMT1, DNMTa and
DNMTb) could change the expression of RNA from the any of the genes
in the PG pathway.
[0067] The expression vector of the present application comprises a
polynucleotide encoding a PG pathway inhibitor or a portion
thereof. The expression vectors also include one or more regulatory
sequences operably linked to the polynucleotide being expressed.
These regulatory sequences are selected based on the type of host
cells. It will be appreciated by those skilled in the art that the
design of the expression vector depends on such factors as the
choice of the host cells and the desired expression levels.
[0068] In some embodiments, the expression vector is a plasmid
vector. In other embodiments, the expression vector is a viral
vector. Examples of viral vectors include, but are not limited to,
retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV),
herpes virus, or alphavirus vectors. The viral vectors can also be
astrovirus, coronavirus, orthomyxovirus, papovavirus,
paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus
vectors. When used in mammalian cells, the expression vector's
control functions are often provided by viral regulatory elements.
For example, commonly used promoters are derived from polyoma,
adenovirus 2, cytomegalovirus and Simian Virus 40. In some
embodiments, the expression vector contains tissue-specific
regulatory elements. Delivery of the expression vector include, but
are not limited to, direct infection with viral vectors, exposing
target tissue to polycationic condensed DNA linked or unlinked to
killed virus, ligand linked DNA, gene guns, ionizing radiation,
nucleic charge neutralization, or fusion with cell membranes. Naked
plasmid or viral DNA can also be employed. Uptake efficiency may be
improved using biodegradable latex beads. This method can be
further improved by treating the beads to increase their
hydrophobicity. Liposome-based methods can also be used to
introduce plasmid or viral vector into the target tissue.
[0069] In some embodiments, the pharmaceutical composition further
comprises one or more active ingredients selected from the group
consisting of analgesic agents, antimuscarinic agents,
antidiuretics, spasmolytics, inhibitors of phosphodiesterase type 5
(PDE 5 inhibitors) and zolpedim.
[0070] Examples of antimuscarinic agents include, but are not
limited to, oxybutynin, solifenacin, darifenacin, fesoterodine,
tolterodine, trospium, atropine, and tricyclic antidepressants.
Examples of antidiuretics include, but are not limited to,
antidiuretic hormone (ADH), angiotensin II, aldosterone,
vasopressin, vasopressin analogs (e.g., desmopressin argipressin,
lypressin, felypressin, ornipressin, terlipressin), vasopressin
receptor agonists, atrial natriuretic peptide (ANP) and C-type
natriuretic peptide (CNP) receptor (i.e., NPR1, NPR2, and NPR3)
antagonists (e.g., HS-142-1, isatin, [Asu7,23']b-ANP-(7-28)],
anantin, a cyclic peptide from Streptomyces coerulescens, and 3G12
monoclonal antibody), somatostatin type 2 receptor antagonists
(e.g., somatostatin), pharmaceutically-acceptable derivatives, and
analogs, salts, hydrates, and solvates thereof. Examples of
spasmolytics include, but are not limited to, carisoprodol,
benzodiazepines, baclofen, cyclobenzaprine, metaxalone,
methocarbamol, clonidine, clonidine analog, and dantrolene.
Examples of PDE 5 inhibitors include, but are not limited to,
tadalafil, sildenafil and vardenafil.
[0071] The pharmaceutical composition may be formulated for
immediate-release, extended-release, delayed-release, or
combinations thereof.
[0072] In some embodiments, the pharmaceutical composition is
formulated for immediate-release.
[0073] In other embodiments, the pharmaceutical composition is
formulated for extended-release by embedding the active ingredient
in a matrix of insoluble substance(s) such as acrylics or chitin.
The extended-release form is designed to release the active
ingredient at a predetermined rate by maintaining a constant drug
level for a specific period of time. This can be achieved through a
variety of formulations, including, but not limited to, liposomes
and drug-polymer conjugates, such as hydrogels.
[0074] An extended-release formulation can be designed to release
the active ingredient at a predetermined rate so as to maintain a
constant drug level for a specified, extended period of time, such
as up to about 24 hours, about 22 hours, about 20 hours, about 18
hours, about 16 hours, about 14 hours, about 12 hours, about 10
hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours,
about 5 hours, about 4 hours, about 3 hours or about 2 hours
following administration or following a lag period associated with
delayed-release of the active ingredient. The constant active
ingredient level may be maintained by a continuous release of the
active ingredient or pulsed-release of the active ingredient.
[0075] In certain embodiments, the active ingredient in an
extended-release formulation is released over a time interval of
between about 1 to about 24 hours, or between 2 to about 12 hours.
Alternatively, the active ingredient may be released over about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10
hours, about 11 hours, about 12 hours, about 14 hours, about 16
hours, about 18 hours, about 20 hours, about 22 hours or about 24
hours. In yet other embodiments, the active ingredient in an
extended-release formulation is released over a time period between
about 5 to about 8 hours following administration.
[0076] In some embodiments, the extended-release formulation
comprises an active core comprised of one or more inert particles,
each in the form of a bead, pellet, pill, granular particle,
microcapsule, microsphere, microgranule, nanocapsule, or nanosphere
coated on its surfaces with drugs in the form of a drug-containing
coating or film-forming composition using, for example, fluid bed
techniques or other methodologies known to those of skill in the
art. The inert particle can be of various sizes, so long as it is
large enough to remain poorly dissolved. Alternatively, the active
core may be prepared by granulating and milling and/or by extrusion
and spheronization of a polymer composition containing the drug
substance. As used herein, the term "drug" refers to the active
ingredient of a pharmaceutical composition.
[0077] The active ingredient may be introduced to the inert carrier
by techniques known to one skilled in the art, such as drug
layering, powder coating, extrusion/spheronization, roller
compaction or granulation. The amount of active ingredient in the
core will depend on the dose that is required and typically varies
from about 1 to 100 weight %, about 5 to 100 weight %, about 10 to
100 weight %, about 20 to 100 weight %, about 30 to 100 weight %,
about 40 to 100 weight %, about 50 to 100 weight %, about 60 to 100
weight %, about 70 to 100 weight %, or about 80 to 100 weight
%.
[0078] Generally, the polymeric coating on the active core will be
from about 1 to 50% based on the weight of the coated particle,
depending on the lag time required and/or the polymers and coating
solvents chosen. Those skilled in the art will be able to select an
appropriate amount of drug for coating onto or incorporating into
the core to achieve the desired dosage. In one embodiment, the
inactive core may be a sugar sphere or a buffer crystal or an
encapsulated buffer crystal such as calcium carbonate, sodium
bicarbonate, fumaric acid, tartaric acid, etc. which alters the
microenvironment of the drug to facilitate its release.
[0079] Extended-release formulations may utilize a variety of
extended-release coatings or mechanisms facilitating the gradual
release of active agents over time. In some embodiments, the
extended-release agent comprises a polymer controlling release by
dissolution controlled release. In a particular embodiment, the
active agent(s) is incorporated in a matrix comprising an insoluble
polymer and drug particles or granules coated with polymeric
materials of varying thickness. The polymeric material may comprise
a lipid barrier comprising a waxy material, such as carnauba wax,
beeswax, spermaceti wax, candellila wax, shallac wax, cocoa butter,
cetostearyl alcohol, partially hydrogenated vegetable oils,
ceresin, paraffin wax, ceresine, myristyl alcohol, stearyl alcohol,
cetyl alcohol, and stearic acid, along with surfactants, such as
polyoxyethylene sorbitan monooleate. When contacted with an aqueous
medium, such as biological fluids, the polymer coating emulsifies
or erodes after a predetermined lag-time depending on the thickness
of the polymer coating. The lag time is independent of
gastrointestinal motility, pH, or gastric residence.
[0080] In other embodiments, the extended-release agent comprises a
polymeric matrix effecting diffusion controlled release. The matrix
may comprise one or more hydrophilic and/or water-swellable, matrix
forming polymers, pH-dependent polymers and/or pH-independent
polymers.
[0081] In one embodiment, the extended-release formulation
comprises a water soluble or water-swellable matrix-forming
polymer, optionally containing one or more solubility-enhancing
agents and/or release-promoting agents. Upon solubilization of the
water soluble polymer, the active agent(s) dissolves (if soluble)
and gradually diffuses through the hydrated portion of the matrix.
The gel layer grows with time as more water permeates into the core
of the matrix, increasing the thickness of the gel layer and
providing a diffusion barrier to drug release. As the outer layer
becomes fully hydrated, the polymer chains become completely
relaxed and can no longer maintain the integrity of the gel layer,
leading to disentanglement and erosion of the outer hydrated
polymer on the surface of the matrix. Water continues to penetrate
towards the core through the gel layer, until it has been
completely eroded. Whereas soluble drugs are released by this
combination of diffusion and erosion mechanisms, erosion is the
predominant mechanism for insoluble drugs, regardless of dose.
[0082] Similarly, water-swellable polymers typically hydrate and
swell in biological fluids forming a homogenous matrix structure
that maintains its shape during drug release and serves as a
carrier for the drug, solubility enhancers and/or release
promoters. The initial matrix polymer hydration phase results in
slow-release of the drug (lag phase). Once the water swellable
polymer is fully hydrated and swollen, water within the matrix can
similarly dissolve the drug substance and allow for its diffusion
out through the matrix coating.
[0083] Additionally, the porosity of the matrix can be increased
due to the leaching out of pH-dependent release promoters so as to
release the drug at a faster rate. The rate of the drug release
then becomes constant and is a function of drug diffusion through
the hydrated polymer gel. The release rate from the matrix is
dependent upon various factors, including polymer type and level,
drug solubility and dose, polymer to drug ratio, filler type and
level, polymer to filler ratio, particle size of drug and polymer,
and porosity and shape of the matrix.
[0084] Exemplary hydrophilic and/or water-swellable, matrix forming
polymers include, but are not limited to, cellulosic polymers
including hydroxyalkyl celluloses and carboxyalkyl celluloses such
as hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose
(HPC), hydroxyethylcellulose (HEC), methylcellulose (MC),
carboxymethylcellulose (CMC); powdered cellulose such as
microcrystalline cellulose, cellulose acetate, ethylcellulose,
salts thereof, and combinations thereof; alginates; gums including
heteropolysaccharide gums and homopolysaccharide gums such as
xanthan, tragacanth, pectin, acacia, karaya, alginates, agar, guar,
hydroxypropyl guar, veegum, carrageenan, locust bean gum, gellan
gum, and derivatives therefrom; acrylic resins including polymers
and copolymers of acrylic acid, methacrylic acid, methyl acrylate,
and methyl methacrylate; and cross-linked polyacrylic acid
derivatives such as Carbomers (e.g., CARBOPOL.RTM., including
CARBOPOL.RTM. 71G NF, which is available in various molecular
weight grades from Noveon, Inc., Cincinnati, Ohio), carageenan;
polyvinyl acetate (e.g., KOLLIDON.RTM. SR); and polyvinyl
pyrrolidone and its derivatives such as crospovidone, polyethylene
oxides, and polyvinyl alcohol. Preferred hydrophilic and
water-swellable polymers include the cellulosic polymers,
especially HPMC.
[0085] The extended-release formulation may further comprise at
least one binder that is capable of cross-linking the hydrophilic
compound to form a hydrophilic polymer matrix (e.g., a gel matrix)
in an aqueous medium, including biological fluids.
[0086] Exemplary binders include homopolysaccharides such as
galactomannan gums, guar gum, hydroxypropyl guar gum,
hydroxypropylcellulose (HPC; e.g., Klucel EXF), and locust bean
gum. In other embodiments, the binder is an alginic acid
derivative, HPC or microcrystallized cellulose (MCC). Other binders
include, but are not limited to, starches, microcrystalline
cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose,
hydroxypropylmethyl cellulose, and polyvinylpyrrolidone.
[0087] In one embodiment, the introduction method is drug layering
by spraying a suspension of active agent(s) and a binder onto the
inert carrier.
[0088] The binder may be present in the bead formulation in an
amount of about 0.1% to about 15% by weight and preferably of from
about 0.2% to about 10% by weight.
[0089] In some embodiments, the hydrophilic polymer matrix may
further include an ionic polymer, a non-ionic polymer, or
water-insoluble hydrophobic polymer to provide a stronger gel layer
and/or reduce pore quantity and dimensions in the matrix so as to
slow diffusion and erosion rates and concomitant release of the
active agent(s). This may additionally suppress the initial burst
effect and produce a more steady "zero order release" of active
agent(s).
[0090] Exemplary ionic polymers for slowing dissolution rate
include both anionic and cationic polymers. Exemplary anionic
polymers include, for example, sodium carboxymethylcellulose (Na
CMC); sodium alginate, polymers of acrylic acid or carbomers (e.g.,
CARBOPOL.RTM. 934, 940, 974P NF); enteric polymers such as
polyvinyl acetate phthalate (PVAP), methacrylic acid copolymers
(e.g., EUDRAGIT.RTM. L100, L 30D 55, A, and FS 30D), and
hypromellose acetate succinate (AQUAT HPMCAS); and xanthan gum.
Exemplary cationic polymers include, for example,
dimethylaminoethyl methacrylate copolymer (e.g., EUDRAGIT.RTM. E
100). Incorporation of anionic polymers, particularly enteric
polymers, is useful for developing a pH-independent release profile
for weakly basic drugs as compared to hydrophilic polymer
alone.
[0091] Exemplary non-ionic polymers for slowing dissolution rate,
include, for example, hydroxypropylcellulose (HPC) and polyethylene
oxide (PEO) (e.g., POLYOX.TM.).
[0092] Exemplary hydrophobic polymers include ethylcellulose (e.g.,
ETHOCEL.TM., SURELEASE.RTM., cellulose acetate, methacrylic acid
copolymers (e.g., EUDRAGIT.RTM. NE 30D), ammonio-methacrylate
copolymers (e.g., EUDRAGIT.RTM. RL 100 or PO RS100), polyvinyl
acetate, glyceryl monostearate, fatty acids such as acetyl tributyl
citrate, and combinations and derivatives thereof.
[0093] The swellable polymer can be incorporated in the formulation
in proportion from 1% to 50% by weight, preferably from 5% to 40%
by weight, most preferably from 5% to 20% by weight. The swellable
polymers and binders may be incorporated in the formulation either
prior to or after granulation. The polymers can also be dispersed
in organic solvents or hydro-alcohols and sprayed during
granulation.
[0094] Exemplary release-promoting agents include pH-dependent
enteric polymers that remain intact at a pH value lower than about
4.0 and dissolve at pH values higher than 4.0, preferably higher
than 5.0, most preferably about 6.0, and are considered useful as
release-promoting agents for this invention. Exemplary pH-dependent
polymers include, but are not limited to, methacarylic acid
copolymers; methacrylic acid-methyl methacrylate copolymers (e.g.,
EUDRAGIT.RTM. L100 (Type A), EUDRAGIT.RTM. S100 (Type B), Rohm
GmbH, Germany), methacrylic acid-ethyl acrylate copolymers (e.g.,
EUDRAGIT.RTM. L100-55 (Type C) and EUDRAGIT.RTM. L30D-55 copolymer
dispersion, Rohm GmbH, Germany); copolymers of methacrylic
acid-methyl methacrylate and methyl methacrylate (EUDRAGIT.RTM.
FS); terpolymers of methacrylic acid, methacrylate, and ethyl
acrylate, cellulose acetate phthalates (CAP); hydroxypropyl
methylcellulose phthalate (HPMCP) (e.g., HP-55, HP-50, HP-555,
Shinetsu Chemical, Japan); polyvinyl acetate phthalates (PVAP)
(e.g., COATERIC.RTM., OPADRY.RTM. enteric white OY-P-7171);
polyvinylbutyrate acetate, cellulose acetate succinates (CAS);
hydroxypropyl methylcellulose acetate succinate (HPMCAS) (e.g.,
HPMCAS LF Grade, MF Grade, and HF Grade, including AQOAT.RTM. LF
and AQOAT.RTM. MF, Shin-Etsu Chemical, Japan), shellac (e.g.,
MARCOAT.TM. 125 and MARCOAT.TM. 125N); vinyl acetate-maleic
anhydride copolymer, styrene-maleic monoester copolymer,
carboxymethyl ethylcellulose (CMEC, Freund Corporation, Japan);
cellulose acetate phthalates (CAP) (e.g., AQUATERIC.RTM.),
cellulose acetate trimellitates (CAT), and mixtures of two or more
thereof at weight ratios between about 2:1 to about 5:1, such as a
mixture of EUDRAGIT.RTM. L 100-55 and EUDRAGIT.RTM. S 100 at a
weight ratio of about 3:1 to about 2:1, or a mixture of
EUDRAGIT.RTM. L 30 D-55 and EUIDRAGIT.RTM. FS at a weight ratio of
about 3:1 to about 5:1.
[0095] These polymers may be used either alone or in combination,
or together with polymers other than those mentioned above.
Preferred enteric pH-dependent polymers are the pharmaceutically
acceptable methacrylic acid copolymers. These copolymers are
anionic polymers based on methacrylic acid and methyl methacrylate
and, preferably, have a mean molecular weight of about 50,000 to
200,000, preferably about 135,000. A ratio of free carboxyl groups
to methyl-esterified carboxyl groups in these copolymers may range,
for example, from 1:1 to 1:3, e.g. around 1:1 or 1:2. The release
promoters are not limited to pH dependent polymers. Other
hydrophilic molecules that dissolve rapidly and leach out of the
dosage form quickly leaving a porous structure can be also be used
for the same purpose.
[0096] In some embodiments, the matrix may include a combination of
release promoters and solubility enhancing agents. The solubility
enhancing agents can be ionic and non-ionic surfactants, complexing
agents, hydrophilic polymers, and pH modifiers such as acidifying
agents and alkalinizing agents, as well as molecules that increase
the solubility of poorly soluble drug through molecular entrapment.
Several solubility enhancing agents can be utilized
simultaneously.
[0097] Solubility enhancing agents may include surface active
agents, such as sodium docusate; sodium lauryl sulfate; sodium
stearyl fumarate; Tweens.RTM. and Spans (PEO modified sorbitan
monoesters and fatty acid sorbitan esters); poly(ethylene
oxide)-polypropylene oxide-poly(ethylene oxide) block copolymers
(aka PLURONICS.TM.); complexing agents such as low molecular weight
polyvinyl pyrrolidone and low molecular weight hydroxypropyl methyl
cellulose; molecules that aid solubility by molecular entrapment
such as cyclodextrins and pH modifying agents, including acidifying
agents such as citric acid, fumaric acid, tartaric acid, and
hydrochloric acid, and alkalizing agents such as meglumine and
sodium hydroxide.
[0098] Solubility enhancing agents typically constitute from 1% to
80% by weight, from 1% to 60% by weight, from 1% to 50% by weight,
from 1% to 40% by weight and from 1% to 30% by weight, of the
dosage form and can be incorporated in a variety of ways. They can
be incorporated in the formulation prior to granulation in dry or
wet form. They can also be added to the formulation after the rest
of the materials are granulated or otherwise processed. During
granulation, solubility enhancing agents can be sprayed as
solutions with or without a binder.
[0099] In one embodiment, the extended-release formulation
comprises a water-insoluble water-permeable polymeric coating or
matrix comprising one or more water-insoluble water-permeable
film-forming over the active core. The coating may additionally
include one or more water soluble polymers and/or one or more
plasticizers. The water-insoluble polymer coating comprises a
barrier coating for release of active agents in the core, wherein
lower molecular weight (viscosity) grades exhibit faster release
rates as compared to higher viscosity grades.
[0100] In some embodiments, the water-insoluble film-forming
polymers include one or more alkyl cellulose ethers, such as ethyl
celluloses and mixtures thereof, (e.g., ethyl cellulose grades
PR100, PR45, PR20, PR10, and PR7; ETHOCEL.RTM., Dow).
[0101] In some embodiments, the water-insoluble polymer provides
suitable properties (e.g., extended-release characteristics,
mechanical properties, and coating properties) without the need for
a plasticizer. For example, coatings comprising polyvinyl acetate
(PVA), neutral copolymers of acrylate/methacrylate esters such as
commercially available Eudragit NE30D from Evonik Industries, ethyl
cellulose in combination with hydroxypropylcellulose, waxes, etc.
can be applied without plasticizers.
[0102] In yet another embodiment, the water-insoluble polymer
matrix may further include a plasticizer. The amount of plasticizer
required depends upon the plasticizer, the properties of the
water-insoluble polymer and the ultimate desired properties of the
coating. Suitable levels of plasticizer range from about 1% to
about 20%, from about 3% to about 20%, about 3% to about 5%, about
7% to about 10%, about 12% to about 15%, about 17% to about 20%, or
about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about
7%, about 8%, about 9%, about 10%, about 15%, or about 20% by
weight relative to the total weight of the coating, inclusive of
all ranges and sub-ranges therebetween.
[0103] Exemplary plasticizers include, but are not limited to,
triacetin, acetylated monoglyceride, oils (castor oil, hydrogenated
castor oil, grape seed oil, sesame oil, olive oil, and etc.),
citrate esters, triethyl citrate, acetyltriethyl citrate
acetyltributyl citrate, tributyl citrate, acetyl tri-n-butyl
citrate, diethyl phthalate, dibutyl phthalate, dioctyl phthalate,
methyl paraben, propyl paraben, propyl paraben, butyl paraben,
diethyl sebacate, dibutyl sebacate, glyceroltributyrate,
substituted triglycerides and glycerides, monoacetylated and
diacetylated glycerides (e.g., MYVACET.RTM. 9-45), glyceryl
monostearate, glycerol tributyrate, polysorbate 80,
polyethyleneglycol (such as PEG-4000 and PEG-400), propyleneglycol,
1,2-propyleneglycol, glycerin, sorbitol, diethyl oxalate, diethyl
malate, diethyl fumarate, diethylmalonate, dibutyl succinate, fatty
acids, glycerin, sorbitol, diethyl oxalate, diethyl malate, diethyl
maleate, diethyl fumarate, diethyl succinate, diethyl malonate,
dioctyl phthalate, dibutyl sebacate, and mixtures thereof. The
plasticizer can have surfactant properties, such that it can act as
a release modifier. For example, non-ionic detergents such as Brij
58 (polyoxyethylene (20) cetyl ether), and the like, can be
used.
[0104] Plasticizers can be high boiling point organic solvents used
to impart flexibility to otherwise hard or brittle polymeric
materials and can affect the release profile for the active
agent(s). Plasticizers generally cause a reduction in the cohesive
intermolecular forces along the polymer chains resulting in various
changes in polymer properties. These changes include, but are not
limited to, a reduction in tensile strength and increase in
elongation and a reduction in the glass transition or softening
temperature of the polymer. The amount and choice of the
plasticizer can affect the hardness of a tablet, for example, and
can even affect its dissolution or disintegration characteristics,
as well as its physical and chemical stability. Certain
plasticizers can increase the elasticity and/or pliability of a
coat, thereby decreasing the coat's brittleness.
[0105] In another embodiment, the extended-release formulation
comprises a combination of at least two gel-forming polymers,
including at least one non-ionic gel-forming polymer and/or at
least one anionic gel-forming polymer. The gel formed by the
combination of gel-forming polymers provides controlled release,
such that when the formulation is ingested and comes into contact
with the gastrointestinal fluids, the polymers nearest the surface
hydrate to form a viscous gel layer. Due to the high viscosity, the
viscous layer dissolves away only gradually, exposing the material
below to the same process. The mass thus dissolves away slowly,
thereby slowly releasing the active ingredient into the
gastrointestinal fluids. The combination of at least two
gel-forming polymers enables properties of the resultant gel, such
as viscosity, to be manipulated in order to provide the desired
release profile.
[0106] In a particular embodiment, the formulation comprises at
least one non-ionic gel-forming polymer and at least one anionic
gel-forming polymer. In another embodiment, the formulation
comprises two different non-ionic gel-forming polymers. In yet
another embodiment, the formulation comprises a combination of
non-ionic gel-forming polymers with the same chemistry, but
different solubilities, viscosities, and/or molecular weights (for
example, a combination of hydroxyproplyl methylcellulose of
different viscosity grades, such as HPMC K100 and HPMC K15M or HPMC
K100M).
[0107] Exemplary anionic gel forming polymers include, but are not
limited to, sodium carboxymethylcellulose (Na CMC), carboxymethyl
cellulose (CMC), anionic polysaccharides such as sodium alginate,
alginic acid, pectin, polyglucuronic acid (poly-.alpha.- and
-.beta.-1,4-glucuronic acid), polygalacturonic acid (pectic acid),
chondroitin sulfate, carrageenan, furcellaran, anionic gums such as
xanthan gum, polymers of acrylic acid or carbomers (Carbopol.RTM.
934, 940, 974P NF), Carbopol.RTM. copolymers, a Pemulen.RTM.
polymer, polycarbophil, and others.
[0108] Exemplary non-ionic gel-forming polymers include, but are
not limited to, Povidone (PVP: polyvinyl pyrrolidone), polyvinyl
alcohol, copolymer of PVP and polyvinyl acetate, HPC (hydroxypropyl
cellulose), HPMC (hydroxypropyl methylcellulose), hydroxyethyl
cellulose, hydroxymethyl cellulose, gelatin, polyethylene oxide,
acacia, dextrin, starch, polyhydroxyethylmethacrylate (PHEMA),
water soluble nonionic polymethacrylates and their copolymers,
modified cellulose, modified polysaccharides, nonionic gums,
nonionic polysaccharides, and/or mixtures thereof.
[0109] The formulation may optionally comprise an enteric polymer
as described above and/or at least one excipient, such as a filler,
a binder (as described above), a disintegrant and/or a flow aid or
glidant.
[0110] Exemplary fillers include, but are not limited to, lactose,
glucose, fructose, sucrose, dicalcium phosphate, sugar alcohols
also known as "sugar polyol" such as sorbitol, manitol, lactitol,
xylitol, isomalt, erythritol, and hydrogenated starch hydrolysates
(a blend of several sugar alcohols), corn starch, potato starch,
sodium carboxymethycellulose, ethylcellulose and cellulose acetate,
enteric polymers, or a mixture thereof.
[0111] Exemplary binders include, but are not limited to,
water-soluble hydrophilic polymers such as Povidone (PVP: polyvinyl
pyrrolidone), copovidone (a copolymer of polyvinyl pyrrolidone and
polyvinyl acetate), low molecular weight HPC (hydroxypropyl
cellulose), low molecular weight HPMC (hydroxypropyl
methylcellulose), low molecular weight carboxy methyl cellulose,
ethylcellulose, gelatin, polyethylene oxide, acacia, dextrin,
magnesium aluminum silicate, and starch and polymethacrylates such
as Eudragit NE 30D, Eudragit RL, Eudragit RS, Eudragit E, polyvinyl
acetate, enteric polymers, or mixtures thereof.
[0112] Exemplary disintegrants include, but are not limited to,
low-substituted carboxymethyl cellulose sodium, crospovidone
(cross-linked polyvinyl pyrrolidone), sodium carboxymethyl starch
(sodium starch glycolate), cross-linked sodium carboxymethyl
cellulose (Croscarmellose), pregelatinized starch (starch 1500),
microcrystalline cellulose, water insoluble starch, calcium
carboxymethyl cellulose, low substituted hydroxypropyl cellulose,
and magnesium or aluminum silicate.
[0113] Exemplary glidants include but are not limited to,
magnesium, silicon dioxide, talc, starch, titanium dioxide, and the
like.
[0114] In yet another embodiment, the extended-release formulation
is formed by coating a water soluble/dispersible drug-containing
particle, such as a bead or bead population therein (as described
above), with a coating material and, optionally, a pore former and
other excipients. The coating material is preferably selected from
a group comprising cellulosic polymers such as ethylcellulose
(e.g., SURELEASE.RTM.), methylcellulose, hydroxypropyl cellulose,
hydroxypropylmethyl cellulose, cellulose acetate, and cellulose
acetate phthalate; polyvinyl alcohol; acrylic polymers such as
polyacrylates, polymethacrylates, and copolymers thereof; and other
water-based or solvent-based coating materials. The
release-controlling coating for a given bead population may be
controlled by at least one parameter of the release controlling
coating, such as the nature of the coating, coating level, type and
concentration of a pore former, process parameters, and
combinations thereof. Thus, changing a parameter, such as a pore
former concentration, or the conditions of the curing, allows for
changes in the release of active agent(s) from any given bead
population, thereby allowing for selective adjustment of the
formulation to a pre-determined release profile.
[0115] Pore formers suitable for use in the release controlling
coating herein can be organic or inorganic agents and include
materials that can be dissolved, extracted or leached from the
coating in the environment of use. Exemplary pore forming agents
include, but are not limited to, organic compounds such as mono-,
oligo-, and polysaccharides including sucrose, glucose, fructose,
mannitol, mannose, galactose, sorbitol, pullulan, and dextran;
polymers soluble in the environment of use such as water-soluble
hydrophilic polymers, hydroxyalkylcelluloses,
carboxyalkylcelluloses, hydroxypropylmethylcellulose, cellulose
ethers, acrylic resins, polyvinylpyrrolidone, cross-linked
polyvinylpyrrolidone, polyethylene oxide, Carbowaxes, Carbopol, and
the like, diols, polyols, polyhydric alcohols, polyalkylene
glycols, polyethylene glycols, polypropylene glycols, or block
polymers thereof, polyglycols, and
poly(.alpha.-.OMEGA.)alkylenediols; and inorganic compounds such as
alkali metal salts, lithium carbonate, sodium chloride, sodium
bromide, potassium chloride, potassium sulfate, potassium
phosphate, sodium acetate, sodium citrate, suitable calcium salts,
combination thereof, and the like.
[0116] The release controlling coating can further comprise other
additives known in the art, such as plasticizers, anti-adherents,
glidants (or flow aids) and antifoams. In some embodiments, the
coated particles or beads may additionally include an "overcoat,"
to provide, for example, moisture protection, static charge
reduction, taste-masking, flavoring, coloring, and/or polish or
other cosmetic appeal to the beads. Suitable coating materials for
such an overcoat are known in the art and include, but are not
limited to, cellulosic polymers such as
hydroxypropylmethylcellulose, hydroxypropylcellulose, and
microcrystalline cellulose or combinations thereof (for example,
various OPADRY.RTM. coating materials).
[0117] The coated particles or beads may additionally contain
enhancers that may be exemplified by, but not limited to,
solubility enhancers, dissolution enhancers, absorption enhancers,
permeability enhancers, stabilizers, complexing agents, enzyme
inhibitors, p-glycoprotein inhibitors, and multidrug resistance
protein inhibitors. Alternatively, the formulation can also contain
enhancers that are separated from the coated particles, for
example, in a separate population of beads or as a powder. In yet
another embodiment, the enhancer(s) may be contained in a separate
layer on coated particles either under or above the release
controlling coating.
[0118] In other embodiments, the extended-release formulation is
formulated to release the active agent(s) by an osmotic mechanism.
By way of example, a capsule may be formulated with a single
osmotic unit or it may incorporate 2, 3, 4, 5, or 6 push-pull units
encapsulated within a hard gelatin capsule, whereby each bilayer
push pull unit contains an osmotic push layer and a drug layer,
both surrounded by a semi-permeable membrane. One or more orifices
are drilled through the membrane next to the drug layer. This
membrane may be additionally covered with a pH-dependent enteric
coating to prevent release until after gastric emptying. The
gelatin capsule dissolves immediately after ingestion. As the push
pull unit(s) enters the small intestine, the enteric coating breaks
down, which then allows fluid to flow through the semi-permeable
membrane, swelling the osmotic push compartment to force drugs out
through the orifice(s) at a rate precisely controlled by the rate
of water transport through the semi-permeable membrane. Release of
drugs can occur over a constant rate for up to 24 hours or
more.
[0119] The osmotic push layer comprises one or more osmotic agents
creating the driving force for transport of water through the
semi-permeable membrane into the core of the delivery vehicle. One
class of osmotic agents includes water-swellable hydrophilic
polymers, also referred to as "osmopolymers" and "hydrogels,"
including, but not limited to, hydrophilic vinyl and acrylic
polymers, polysaccharides such as calcium alginate, polyethylene
oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG),
poly(2-hydroxyethyl methacrylate), poly(acrylic) acid,
poly(methacrylic) acid, polyvinylpyrrolidone (PVP), crosslinked
PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers, PVA/PVP
copolymers with hydrophobic monomers such as methyl methacrylate
and vinyl acetate, hydrophilic polyurethanes containing large PEO
blocks, sodium croscarmellose, carrageenan, hydroxyethyl cellulose
(HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl
cellulose (HPMC), carboxymethyl cellulose (CMC) and carboxyethyl,
cellulose (CEC), sodium alginate, polycarbophil, gelatin, xanthan
gum, and sodium starch glycolate.
[0120] Another class of osmotic agents includes osmogens, which are
capable of imbibing water to effect an osmotic pressure gradient
across the semi-permeable membrane. Exemplary osmogens include, but
are not limited to, inorganic salts such as magnesium sulfate,
magnesium chloride, calcium chloride, sodium chloride, lithium
chloride, potassium sulfate, potassium phosphates, sodium
carbonate, sodium sulfite, lithium sulfate, potassium chloride, and
sodium sulfate; sugars such as dextrose, fructose, glucose,
inositol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose,
trehalose, and xylitol; organic acids such as ascorbic acid,
benzoic acid, fumaric acid, citric acid, maleic acid, sebacic acid,
sorbic acid, adipic acid, edetic acid, glutamic acid,
p-toluenesulfonic acid, succinic acid, and tartaric acid; urea; and
mixtures thereof.
[0121] Materials useful in forming the semipermeable membrane
include various grades of acrylics, vinyls, ethers, polyamides,
polyesters, and cellulosic derivatives that are water-permeable and
water-insoluble at physiologically relevant pHs, or are susceptible
to being rendered water-insoluble by chemical alteration, such as
crosslinking.
[0122] In some embodiments, the extended-release formulation
comprises a polysaccharide coating that is resistant to erosion in
both the stomach and intestine. Such polymers can be only degraded
in the colon, which contains a large microflora containing
biodegradable enzymes breaking down, for example, the
polysaccharide coatings to release the drug contents in a
controlled, time-dependent manner. Exemplary polysaccharide
coatings may include, for example, amylose, arabinogalactan,
chitosan, chondroitin sulfate, cyclodextrin, dextran, guar gum,
pectin, xylan, and combinations or derivatives therefrom.
[0123] In some embodiments, the pharmaceutical composition is
formulated for delayed-release or delayed-extended-release. In some
embodiments, the delayed extended-release formulation includes an
extended-release formulation coated with an enteric coating, which
is a barrier applied to oral medication that prevents release of
medication before it reaches the small intestine. Delayed-release
formulations, such as enteric coatings, prevent drugs having an
irritant effect on the stomach, such as aspirin, from dissolving in
the stomach. As used herein, the term "enteric coating" is a
coating comprising of one or more polymers having a pH dependent or
pH-independent release profile. An enteric coated pill will not
dissolve in the acidic juices of the stomach (pH .about.3), but
they will in the alkaline (pH 7-9) environment present in the small
intestine or colon. An enteric polymer coating typically resists
releases of the active agents until sometime after a gastric
emptying lag period of about 3-4 hours after administration.
Accordingly, a formulation that releases it component "after
gastric emptying" refers to a delayed formulation that releases the
active ingredient(s) after the formulation is emptied from the
stomach and enters intestine.
[0124] Such coatings are also used to protect acid-unstable drugs
from the stomach's acidic exposure, delivering them instead to a
basic pH environment (intestine's pH 5.5 and above) where they do
not degrade and give their desired action. The term
"pulsatile-release" is a type of delayed-release, which is used
herein with reference to a drug formulation that provides rapid and
transient release of the drug within a short time period
immediately after a predetermined lag period, thereby producing a
"pulsed" plasma profile of the drug after drug administration.
Formulations may be designed to provide a single pulsatile release
or multiple pulsatile releases at predetermined time intervals
following administration, or a pulsatile release (e.g., 20-60% of
the active ingredient) followed with extended release over a period
of time (e.g., a continuous release of the remainder of the active
ingredient). A delayed-release or pulsatile release formulation
generally comprises one or more elements covered with a barrier
coating, which dissolves, erodes or ruptures following a specified
lag phase.
[0125] A barrier coating for delayed-release may consist of a
variety of different materials, depending on the objective. In
addition, a formulation may comprise a plurality of barrier
coatings to facilitate release in a temporal manner. The coating
may be a sugar coating, a film coating (e.g., based on
hydroxypropyl methylcellulose, methylcellulose, methyl
hydroxyethylcellulose, hydroxypropylcellulose,
carboxymethylcellulose, acrylate copolymers, polyethylene glycols,
and/or polyvinylpyrrolidone) or a coating based on methacrylic acid
copolymer, cellulose acetate phthalate, hydroxypropyl
methylcellulose phthalate, hydroxypropyl methylcellulose acetate
succinate, polyvinyl acetate phthalate, shellac, and/or
ethylcellulose. Furthermore, the formulation may additionally
include a time delay material such as glyceryl monostearate or
glyceryl distearate.
[0126] In some embodiments, the delayed-extended-release
formulation includes an enteric coating comprising one or more
polymers facilitating release of active agents in proximal or
distal regions of the gastrointestinal tract. pH dependent enteric
coatings comprise one or more pH-dependent or pH-sensitive polymers
that maintain their structural integrity at low pH, as in the
stomach, but dissolve in higher pH environments in more distal
regions of the gastrointestinal tract, such as the small intestine,
where the drug contents are released. For purposes of the present
invention, "pH dependent" is defined as having characteristics
(e.g., dissolution) which vary according to environmental pH.
Exemplary pH-dependent polymers have been described earlier.
pH-dependent polymers typically exhibit a characteristic pH optimum
for dissolution. In some embodiments, the pH-dependent polymer
exhibits a pH optimum between about 5.0 and 5.5, between about 5.5
and 6.0, between about 6.0 and 6.5, or between about 6.5 and 7.0.
In other embodiments, the pH-dependent polymer exhibits a pH
optimum of .gtoreq.5.0, of .gtoreq.5.5, of .gtoreq.6.0, of
.gtoreq.6.5, or of .gtoreq.7.0.
[0127] In certain embodiments, the coating methodology employs the
blending of one or more pH-dependent and one or more pH-independent
polymers. The blending of pH-dependent and pH-independent polymers
can reduce the release rate of active ingredients once the soluble
polymer has reached its optimum pH of solubilization.
[0128] In some embodiments, a "delayed-release" or
"delayed-extended-release" profile can be obtained using a water
insoluble capsule body containing one or more active agents,
wherein the capsule body closed at one end with an insoluble, but
permeable and swellable hydrogel plug. Upon contact with
gastrointestinal fluid or dissolution medium, the plug swells,
pushing itself out of the capsule and releasing the drugs after a
pre-determined lag time, which can be controlled by, for example,
the position and dimensions of the plug. The capsule body may be
further coated with an outer pH-dependent enteric coating keeping
the capsule intact until it reaches the small intestine. Suitable
plug materials include, for example, polymethacrylates, erodible
compressed polymers (e.g., HPMC, polyvinyl alcohol), congealed
melted polymer (e.g., glyceryl mono oleate), and enzymatically
controlled erodible polymers (e.g., polysaccharides, such as
amylose, arabinogalactan, chitosan, chondroitin sulfate,
cyclodextrin, dextran, guar gum, pectin and xylan).
[0129] In other embodiments, capsules or bilayered tablets may be
formulated to contain a drug-containing core, covered by a swelling
layer and an outer insoluble, but semi-permeable polymer coating or
membrane. The lag time prior to rupture can be controlled by the
permeation and mechanical properties of the polymer coating and the
swelling behavior of the swelling layer. Typically, the swelling
layer comprises one or more swelling agents, such as swellable
hydrophilic polymers that swell and retain water in their
structures.
[0130] Exemplary water swellable materials to be used in the
delayed-release coating include, but are not limited to,
polyethylene oxides (having e.g., an average molecular weight
between 1,000,000 and 7,000,000, such as POLYOX.RTM.);
methylcellulose; hydroxypropyl cellulose; hydroxypropyl
methylcellulose; polyalkylene oxides having a weight average
molecular weight of 100,000 to 6,000,000, including, but not
limited to, poly(methylene oxide), poly(butylene oxide),
poly(hydroxy alkyl methacrylate) having a molecular weight of
25,000 to 5,000,000, poly(vinyl)alcohol having a low acetal
residue, which is cross-linked with glyoxal, formaldehyde, or
glutaraldehyde, and having a degree of polymerization from 200 to
30,000; mixtures of methyl cellulose, cross-linked agar, and
carboxymethyl cellulose; hydrogel forming copolymers produced by
forming a dispersion of a finely divided copolymer of maleic
anhydride with styrene, ethylene, propylene, butylene, or
isobutylene cross-linked with from 0.001 to 0.5 moles of saturated
cross-linking agent per mole of maleic anyhydride in the copolymer;
CARBOPOL.RTM. acidic carboxy polymers having a molecular weight of
450,000 to 4,000,000; CYANAMER.RTM. polyacrylamides; cross-linked
water swellable indenemaleicanhydride polymers; GOODRITE.RTM.
polyacrylic acid having a molecular weight of 80,000 to 200,000;
starch graft copolymers; AQUA-KEEPS.RTM. acrylate polymer
polysaccharides composed of condensed glucose units such as diester
cross-linked polyglucan; carbomers having a viscosity of 3,000 to
60,000 mPas as a 0.5%-1% w/v aqueous solution; cellulose ethers
such as hydroxypropylcellulose having a viscosity of about
1000-7000 mPa s as a 1% w/v aqueous solution (25.degree. C.);
hydroxypropyl methylcellulose having a viscosity of about 1000 or
higher, preferably 2,500 or higher to a maximum of 25,000 mPas as a
2% w/v aqueous solution; polyvinylpyrrolidone having a viscosity of
about 300-700 mPas as a 10% w/v aqueous solution at 20.degree. C.;
and combinations thereof.
[0131] Alternatively, the release time of the drugs can be
controlled by a disintegration lag time depending on the balance
between the tolerability and thickness of a water insoluble polymer
membrane (such as ethyl cellulose, EC) containing predefined
micropores at the bottom of the body and the amount of a swellable
excipient, such as low substituted hydroxypropyl cellulose (L-HPC)
and sodium glycolate. After oral administration, GI fluids permeate
through the micropores, causing swelling of the swellable
excipients, which produces an inner pressure disengaging the
capsular components, including a first capsule body containing the
swellable materials, a second capsule body containing the drugs,
and an outer cap attached to the first capsule body.
[0132] The delayed-release coating layer may further comprise
anti-tackiness agents, such as talc and glyceryl monostearate. The
delayed-release coating layer may further comprise one or more
plasticizers including, but not limited to, triethyl citrate,
acetyl triethyl citrate, acetyltributyl citrate, polyethylene
glycol acetylated monoglycerides, glycerin, triacetin, propylene
glycol, phthalate esters (e.g., diethyl phthalate, dibutyl
phthalate), titanium dioxide, ferric oxides, castor oil, sorbitol,
and dibutyl sebacate.
[0133] In another embodiment, the delayed-release formulation
employs a water-permeable but insoluble film coating to enclose the
active ingredient and an osmotic agent. As water from the gut
slowly diffuses through the film into the core, the core swells
until the film bursts, thereby releasing the active ingredients.
The film coating may be adjusted to permit various rates of water
permeation or release time.
[0134] In another embodiment, the delayed release formulation
employs a water-impermeable tablet coating whereby water enters
through a controlled aperture in the coating until the core bursts.
When the tablet bursts, the drug contents are released immediately
or over a longer period of time. These and other techniques may be
modified to allow for a pre-determined lag period before release of
drugs is initiated.
[0135] In another embodiment, the active agents are delivered in a
formulation to provide both delayed-release and extended-release
(delayed-extended-release). The term "delayed-extended-release" is
used herein with reference to a drug formulation providing
pulsatile release of active agents at a pre-determined time or lag
period following administration, which is then followed by
extended-release of the active agents thereafter.
[0136] In some embodiments, immediate-release, extended-release,
delayed-release, or delayed-extended-release formulations comprises
an active core comprised of one or more inert particles, each in
the form of a bead, pellet, pill, granular particle, microcapsule,
microsphere, microgranule, nanocapsule, or nanosphere coated on its
surfaces with drugs in the form of e.g., a drug-containing
film-forming composition using, for example, fluid bed techniques
or other methodologies known to those of skill in the art. The
inert particle can be of various sizes, so long as it is large
enough to remain poorly dissolved. Alternatively, the active core
may be prepared by granulating and milling and/or by extrusion and
spheronization of a polymer composition containing the drug
substance.
[0137] The amount of drug in the core will depend on the dose that
is required and typically varies from about 1 to 100 weight %,
about 5 to 100 weight %, about 10 to 100 weight %, about 20 to 100
weight %, about 30 to 100 weight %, about 40 to 100 weight %, about
50 to 100 weight %, about 60 to 100 weight %, about 70 to 100
weight %, or about 80 to 100 weight %. Generally, the polymeric
coating on the active core will be from about 1 to 50% based on the
weight of the coated particle, depending on the lag time and type
of release profile required and/or the polymers and coating
solvents chosen. Those skilled in the art will be able to select an
appropriate amount of drug for coating onto or incorporating into
the core to achieve the desired dosage. In one embodiment, the
inactive core may be a sugar sphere or a buffer crystal or an
encapsulated buffer crystal such as calcium carbonate, sodium
bicarbonate, fumaric acid, tartaric acid, etc. which alters the
microenvironment of the drug to facilitate its release.
[0138] In some embodiments, for example, delayed-release or
delayed-extended-release compositions may formed by coating a water
soluble/dispersible drug-containing particle, such as a bead, with
a mixture of a water insoluble polymer and an enteric polymer,
wherein the water insoluble polymer and the enteric polymer may be
present at a weight ratio of from 4:1 to 1:1, and the total weight
of the coatings is 10 to 60 weight % based on the total weight of
the coated beads. The drug layered beads may optionally include an
inner dissolution rate controlling membrane of ethylcellulose. The
composition of the outer layer, as well as the individual weights
of the inner and outer layers of the polymeric membrane are
optimized for achieving desired circadian rhythm release profiles
for a given active, which are predicted based on in vitro/in vivo
correlations.
[0139] In other embodiments the formulations may comprise a mixture
of immediate-release drug-containing particles without a
dissolution rate controlling polymer membrane and
delayed-extended-release beads exhibiting, for example, a lag time
of 2-4 hours following oral administration, thus providing a
two-pulse release profile.
[0140] In some embodiments, the active core is coated with one or
more layers of dissolution rate-controlling polymers to obtain
desired release profiles with or without a lag time. An inner layer
membrane can largely control the rate of drug release following
imbibition of water or body fluids into the core, while the outer
layer membrane can provide for a desired lag time (the period of no
or little drug release following imbibition of water or body fluids
into the core). The inner layer membrane may comprise a water
insoluble polymer, or a mixture of water insoluble and water
soluble polymers.
[0141] The polymers suitable for the outer membrane, which largely
controls the lag time of up to 6 hours may comprise an enteric
polymer, as described above, and a water insoluble polymer at 10 to
50 weight %. The ratio of water insoluble polymer to enteric
polymer may vary from 4:1 to 1:2, preferably the polymers are
present at a ratio of about 1:1. The water insoluble polymer
typically used is ethylcellulose.
[0142] Exemplary water insoluble polymers include ethylcellulose,
polyvinyl acetate (Kollicoat SR#0D from BASF), neutral copolymers
based on ethyl acrylate and methylmethacrylate, copolymers of
acrylic and methacrylic acid esters with quaternary ammonium groups
such as EUDRAGIT.RTM. NE, RS and RS30D, RL or RL30D, and the like.
Exemplary water soluble polymers include low molecular weight HPMC,
HPC, methylcellulose, polyethylene glycol (PEG of molecular
weight>3000) at a thickness ranging from 1 weight % up to 10
weight % depending on the solubility of the active in water and the
solvent or latex suspension based coating formulation used. The
water insoluble polymer to water soluble polymer may typically vary
from 95:5 to 60:40, preferably from 80:20 to 65:35. In some
embodiments, AMBERLITE.TM. IRP69 resin is used as an
extended-release carrier. AMBERLITE.TM. IRP69 is an insoluble,
strongly acidic, sodium form cation exchange resin that is suitable
as carrier for cationic (basic) substances. In other embodiments,
DUOLITE.TM. AP143/1093 resin is used as an extended-release
carrier. DUOLITE.TM. AP143/1093 is an insoluble, strongly basic,
anion exchange resin that is suitable as a carrier for anionic
(acidic) substances. When used as a drug carrier, AMBERLITE.TM.
IRP69 or/and DUOLITE.TM. AP143/1093 resin provides a means for
binding medicinal agents onto an insoluble polymeric matrix.
Extended-release is achieved through the formation of resin-drug
complexes (drug resinates). The drug is released from the resin in
vivo as the drug reaches equilibrium with the high electrolyte
concentrations, which are typical of the gastrointestinal tract.
More hydrophobic drugs will usually elute from the resin at a lower
rate, owing to hydrophobic interactions with the aromatic structure
of the cation exchange system.
[0143] In some embodiments, the pharmaceutical composition is
formulated for oral administration. Oral dosage forms include, for
example, tablets, capsules, and caplets and may also comprise a
plurality of granules, beads, powders, or pellets that may or may
not be encapsulated. Tablets and capsules represent the most
convenient oral dosage forms, in which case solid pharmaceutical
carriers are employed.
[0144] In a delayed-release formulation, one or more barrier
coatings may be applied to pellets, tablets, or capsules to
facilitate slow dissolution and concomitant release of drugs into
the intestine. Typically, the barrier coating contains one or more
polymers encasing, surrounding, or forming a layer, or membrane
around the therapeutic composition or active core. In some
embodiments, the active agents are delivered in a formulation to
provide delayed-release at a pre-determined time following
administration. The delay may be up to about 10 minutes, about 20
minutes, about 30 minutes, about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, or longer.
[0145] Various coating techniques may be applied to granules,
beads, powders or pellets, tablets, capsules or combinations
thereof containing active agents to produce different and distinct
release profiles. In some embodiments, the pharmaceutical
composition is in a tablet or capsule form containing a single
coating layer. In other embodiments, the pharmaceutical composition
is in a tablet or capsule form containing multiple coating layers.
In some embodiments, the pharmaceutical composition of the present
application is formulated for extended-release or delayed
extended-release of up to 100% of the active ingredient.
[0146] In other embodiments, the pharmaceutical composition of the
present application is formulated for a two-phase extended-release
or delayed two-phase extended-release characterized by an
"immediate-release" component that is released within two hours of
administration and an "extended-release" component which is
released over a period of 2-12 hours. In some embodiments, the
"immediate-release" component provides about 1-90% of the total
dosage of the active agent(s) and the "extended-release" component
provides 10-99% of the total dosage of the active agent(s) to be
delivered by the pharmaceutical formulation. For example, the
immediate-release component may provide about 10-90%, or about 10%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85% or 90% of the total dosage of the active agent(s) to be
delivered by the pharmaceutical formulation. The extended-release
component provides about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the
total dosage of the active agent(s) to be delivered by the
formulation. In some embodiments, the immediate-release component
and the extended-release component contain the same active
ingredient. In other embodiments, the immediate-release component
and the extended-release component contain different active
ingredients (e.g., one PG pathway inhibitor in one component and
another PG pathway inhibitor in another component). In some
embodiments, the immediate-release component and the
extended-release component each contains a PG pathway inhibitor and
an analgesic selected from the group consisting of aspirin,
ibuprofen, naproxen sodium, indomethacin, nabumetone, and
acetaminophen. In other embodiments, the immediate-release
component and/or the extended-release component further comprises
one or more additional active agents selected from the groups
consisting of an antimuscarinic agent, an antidiuretic, a
spasmolytic, an inhibitor of phosphodiesterase type (PDE 5
inhibitor) and zolpidem.
[0147] In some embodiments, the pharmaceutical composition
comprises a plurality of active ingredients selected from the group
consisting of PG pathway inhibitors, analgesics, antimuscarinic
agents, antidiuretics, spasmolytics, PDE 5 inhibitors and zolpidem.
Examples of antimuscarinic agents include, but are not limited to,
oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine,
trospium, atropine, and tricyclic antidepressants. Examples of
antidiuretics include, but are not limited to, antidiuretic hormone
(ADH), angiotensin II, aldosterone, vasopressin, vasopressin
analogs (e.g., desmopressin argipressin, lypressin, felypressin,
ornipressin, terlipressin); vasopressin receptor agonists, atrial
natriuretic peptide (ANP) and C-type natriuretic peptide (CNP)
receptor (i.e., NPR1, NPR2, and NPR3) antagonists (e.g., HS-142-1,
isatin, [Asu7,23']b-ANP-(7-28)], anantin, a cyclic peptide from
Streptomyces coerulescens, and 3G12 monoclonal antibody);
somatostatin type 2 receptor antagonists (e.g., somatostatin),
pharmaceutically-acceptable derivatives, and analogs, salts,
hydrates, and solvates thereof. Examples of spasmolytics include,
but are not limited to, carisoprodol, benzodiazepines, baclofen,
cyclobenzaprine, metaxalone, methocarbamol, clonidine, clonidine
analog, and dantrolene. Examples of PDE 5 inhibitors include, but
are not limited to, tadalafil, sildenafil and vardenafil.
[0148] In some embodiments, the pharmaceutical composition
comprises a plurality of active ingredients comprising (1) one or
more PG pathway inhibitors and (2) one or more other active
ingredients selected from the group consisting of analgesics,
antimuscarinic agents, antidiuretics, spasmolytics, PDE 5
inhibitors and zolpidem. In some embodiments, the plurality of
active ingredients are formulated for immediate-release. In other
embodiments, the plurality of active ingredients are formulated for
extended-release. In other embodiments, the plurality of active
ingredients are formulated for delayed-release. In other
embodiments, the plurality of active ingredients are formulated for
both immediate-release and extended-release (e.g., a first portion
of each active ingredient is formulated for immediate-release and a
second portion of each active ingredient is formulated for
extended-release). In yet other embodiments, some of the plurality
of active ingredients are formulated for immediate-release and some
of the plurality of active ingredients are formulated for
extended-release (e.g., active ingredients A, B, C are formulated
for immediate-release and active ingredients C and D are formulated
for extended-release). In some other embodiments, the plurality of
active ingredients are formulated for delayed-extended-release.
[0149] In certain embodiments, the pharmaceutical composition
comprises an immediate-release component and an extended-release
component. The immediate-release component may comprise one or more
active ingredients selected from the group consisting of PG pathway
inhibitors, analgesics, antimuscarinic agents, antidiuretics,
spasmolytics, PDE 5 inhibitors and zolpidem. The extended-release
component may comprise one or more active ingredients selected from
the group consisting of PG pathway inhibitors, analgesics,
antimuscarinic agents, antidiuretics, spasmolytics, PDE 5
inhibitors and zolpidem. In some embodiments, the immediate-release
component and the extended-release component have exactly the same
active ingredients. In other embodiments, the immediate-release
component and the extended-release component have different active
ingredients. In yet other embodiments, the immediate-release
component and the extended-release component have one or more
common active ingredients. In some other embodiments, the
immediate-release component and/or the extended-release component
is further coated with a delayed-release coating, such as an
enteric coating. In other embodiments, the pharmaceutical
composition comprises two or more active ingredients formulated as
two extended-release components, each providing a different
extended-release profile. For example, a first extended-release
component releases a first active ingredient at a first release
rate and a second extended-release component releases a second
active ingredient at a second release rate.
[0150] In some embodiments, the pharmaceutical composition
comprises an immediate-release component and a delayed-release
component. In other embodiments, the pharmaceutical composition
comprises two or more active ingredients formulated as two
delayed-release components, each providing a different
delayed-release profile. For example, a first delayed-release
component releases a first active ingredient at a first time point,
and a second delayed-release component releases a second active
ingredient at a second time point.
[0151] The components in a combined release profile formulation
(e.g., formulations with a combination of an immediate-release
component and an extended-release component, a combination of an
immediate-release component and a delayed-release component, a
combination of an immediate-release component, a delayed-release
component, and an extended-release component, a combination of two
or more delayed-release components, or a combination of two or more
extended-release components) may contain the same active
ingredient(s) or different active ingredient(s). In some
embodiments, the immediate-release component may provide about 1%
to about 80% of the total dosage of the active agent(s) to be
delivered by the pharmaceutical formulation. In some embodiments,
the combined release profile formulation contains an
immediate-release component and the immediate-release component
provide about 1% to about 90%, about 10% to about 90%, about 20% to
about 90%, about 30% to about 90%, about 40% to about 90%, about
50% to about 90%, about 60% to about 90%, about 70% to about 90% or
about 80% to about 90% of the total dosage of each active
ingredient to be delivered by the formulation. In alternate
embodiments, the immediate-release component provides up to about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the total dosage
of each ingredient to be delivered by the formulation.
[0152] In some embodiments, the pharmaceutical formulation
comprises an active core comprised of one or more inert particles,
each in the form of a bead, pellet, pill, granular particle,
microcapsule, microsphere, microgranule, nanocapsule, or nanosphere
coated on its surfaces with drugs in the form of e.g., a
drug-containing film-forming composition using, for example, fluid
bed techniques or other methodologies known to those of skill in
the art. The inert particle can be of various sizes, so long as it
is large enough to remain poorly dissolved. Alternatively, the
active core may be prepared by granulating and milling and/or by
extrusion and spheronization of a polymer composition containing
the drug substance.
[0153] The amount of drug in the core will depend on the dose that
is required and typically varies from about 5 to 90 weight %.
Generally, the polymeric coating on the active core will be from
about 1 to 50% based on the weight of the coated particle,
depending on the lag time and type of release profile required
and/or the polymers and coating solvents chosen. Those skilled in
the art will be able to select an appropriate amount of drug for
coating onto or incorporating into the core to achieve the desired
dosage. In one embodiment, the inactive core may be a sugar sphere
or a buffer crystal or an encapsulated buffer crystal such as
calcium carbonate, sodium bicarbonate, fumaric acid, tartaric acid,
which alters the microenvironment of the drug to facilitate its
release.
[0154] In some embodiments, the pharmaceutical composition
comprises a delayed-release component formed by coating a water
soluble/dispersible drug-containing particle, such as a bead, with
a mixture of a water insoluble polymer and an enteric polymer,
wherein the water insoluble polymer and the enteric polymer may be
present at a weight ratio of 4:1 to 1:1, and the total weight of
the coatings is 10 to 60 weight % based on the total weight of the
coated beads. The drug layered beads may optionally include an
inner dissolution rate controlling membrane of ethylcellulose. The
composition of the outer layer, as well as the individual weights
of the inner and outer layers of the polymeric membrane are
optimized for achieving desired circadian rhythm release profiles
for a given active, which are predicted based on in vitro/in vivo
correlations.
[0155] In other embodiments, the formulations comprise a mixture of
immediate-release drug-containing particles without a dissolution
rate controlling polymer membrane and delayed release beads
exhibiting, for example, a lag time of 2-4 hours following oral
administration, thus providing a two-pulse release profile. In yet
other embodiments, the formulations comprise a mixture of two types
of delayed-release beads: a first type that exhibits a lag time of
1-3 hours and a second type that exhibits a lag time of 4-6 hours.
In yet other embodiments, the formulations comprise a mixture of
two types of release beads: a first type that exhibits
immediate-release and a second type that exhibits a lag time of 1-4
hours followed with extended-release.
[0156] In some embodiments, the formulations are designed with a
release profile such that a fraction of the active ingredient(s)
(e.g., 10-80%) is released immediately or within two hours of
administration, and the rest is released over an extended period of
time (e.g., over a period of 2-24 hours). In other embodiments, the
formulations are designed with a release profile such that one
active ingredient (e.g., an analgesic) are released immediately or
within two hours of administration, and one or more other active
ingredients (e.g., a PG pathway inhibitor) are released over an
extended period of time (e.g., over a period of 2-24 hours).
[0157] The pharmaceutical composition may be administered daily or
administered on an as needed basis. The pharmaceutical composition
may be administered orally, intravenously, or intramuscularly. In
preferred embodiments, the pharmaceutical composition is
administered orally. In other embodiments, the pharmaceutical
composition is administered by retrograde perfusion through the
urinary tract. In other embodiments, the pharmaceutical composition
is administered by direct injection into bladder muscle.
[0158] In some embodiments, the pharmaceutical composition is
administered daily, twice a day or three times a day. In other
embodiments, the pharmaceutical composition is administered every
other day, every 3 days, every 4 days, every 4 days, every 5 days,
every 6 days, every week, every 2 weeks, every 3 weeks, every
month, every 2 months or every 3 months.
[0159] In some embodiments, the pharmaceutical composition is
administered at bedtime. In some embodiments, the pharmaceutical
composition is administered within about two hours before bedtime,
preferably within about one hour before bedtime. In another
embodiment, the pharmaceutical composition is administered about
2-4 hours before bedtime. In a further embodiment, the
pharmaceutical composition is administered at least 4 hours before
bedtime.
[0160] The appropriate dosage ("therapeutically effective amount")
of the active ingredient(s) in the immediate-release component, the
extended-release component, the delayed-release component or
delayed-extended-release component will depend, for example, on the
severity and course of the condition, the mode of administration,
the bioavailability of the particular ingredient(s), the age and
weight of the patient, the patient's clinical history and response
to the active agent(s), discretion of the physician, etc.
[0161] As a general proposition, the therapeutically effective
amount of the PG pathway inhibitor(s) in the immediate-release
component, the delayed-release component, the extended-release
component or the delayed-extended-release component is administered
in the range of about 1 .mu.g/kg body weight/day to about 100 mg/kg
body weight/day whether by one or more administrations. In some
embodiments, the range of each active agent administered daily in a
single dose or in multiple does is from about 1 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 1 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 1 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 1 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 1 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 1 .mu.g/kg body
weight/day to about 300 .mu.g/kg body weight/day, 1 .mu.g/kg body
weight/day to about 100 .mu.g/kg body weight/day, 1 .mu.g/kg body
weight/day to about 30 .mu.g/kg body weight/day, 1 .mu.g/kg body
weight/day to about 10 .mu.g/kg body weight/day, 1 .mu.g/kg body
weight/day to about 3 .mu.g/kg body weight/day, 10 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 300 .mu.g/kg body weight/day, 10 .mu.g/kg body
weight/day to about 100 .mu.g/kg body weight/day, 10 .mu.g/kg body
weight/day to about 30 .mu.g/kg body weight/day, 30 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 300 .mu.g/kg body weight/day, 30 .mu.g/kg body
weight/day to about 100 .mu.g/kg body weight/day, 100 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 300 .mu.g/kg body weight/day, 300 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 300 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 300 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 300 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 300 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 1 mg/kg body
weight/day to about 100 mg/kg body weight/day, 1 mg/kg body
weight/day to about 30 mg/kg body weight/day, 1 mg/kg body
weight/day to about 10 mg/kg body weight/day, 1 mg/kg body
weight/day to about 3 mg/kg body weight/day, 3 mg/kg body
weight/day to about 100 mg/kg body weight/day, 3 mg/kg body
weight/day to about 30 mg/kg body weight/day, 3 mg/kg body
weight/day to about 10 mg/kg body weight/day, 10 mg/kg body
weight/day to about 100 mg/kg body weight/day, 10 mg/kg body
weight/day to about 30 mg/kg body weight/day or 30 mg/kg body
weight/day to about 100 mg/kg body weight/day.
[0162] As a general proposition, the therapeutically effective
amount of the analgesic agent(s) in the immediate-release
component, the delayed-release component, the extended-release
component or the delayed-extended-release component is administered
in the range of about 10 .mu.g/kg body weight/day to about 100
mg/kg body weight/day whether by one or more administrations. In
some embodiments, the range of each active agent administered daily
in a single dose or in multiple does is from about 10 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 10 .mu.g/kg body
weight/day to about 300 .mu.g/kg body weight/day, 10 .mu.g/kg body
weight/day to about 100 .mu.g/kg body weight/day, 10 .mu.g/kg body
weight/day to about 30 .mu.g/kg body weight/day, 30 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 30 .mu.g/kg body
weight/day to about 300 .mu.g/kg body weight/day, 30 .mu.g/kg body
weight/day to about 100 .mu.g/kg body weight/day, 100 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 100 .mu.g/kg body
weight/day to about 300 .mu.g/kg body weight/day, 300 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, 300 .mu.g/kg body
weight/day to about 30 mg/kg body weight/day, 300 .mu.g/kg body
weight/day to about 10 mg/kg body weight/day, 300 .mu.g/kg body
weight/day to about 3 mg/kg body weight/day, 300 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, 1 mg/kg body
weight/day to about 100 mg/kg body weight/day, 1 mg/kg body
weight/day to about 30 mg/kg body weight/day, 1 mg/kg body
weight/day to about 10 mg/kg body weight/day, 1 mg/kg body
weight/day to about 3 mg/kg body weight/day, 3 mg/kg body
weight/day to about 100 mg/kg body weight/day, 3 mg/kg body
weight/day to about 30 mg/kg body weight/day, 3 mg/kg body
weight/day to about 10 mg/kg body weight/day, 10 mg/kg body
weight/day to about 100 mg/kg body weight/day, 10 mg/kg body
weight/day to about 30 mg/kg body weight/day or 30 mg/kg body
weight/day to about 100 mg/kg body weight/day.
[0163] The analgesic agent(s) described herein may be included in
an immediate-release component or an extended-release component, a
delayed-release component, a delayed-extended-release component or
combinations thereof for daily oral administration at a single dose
or combined dose range of 1 mg to 2000 mg, 1 mg to 1000 mg, 1 mg to
300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 10 mg, 1 mg to 3 mg,
3 mg to 2000 mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3
mg to 30 mg, 3 mg to 10 mg, 10 mg to 2000 mg, 10 mg to 1000 mg, 10
mg to 300 mg, 10 mg to 100 mg, 10 mg to 30 mg, 30 mg to 2000 mg, 30
mg to 1000 mg, 30 mg to 300 mg, 30 mg to 100 mg, 100 mg to 2000 mg,
100 mg to 1000 mg, 100 mg to 300 mg, 300 mg to 2000 mg, 300 mg to
1000 mg or 1000 mg to 2000 mg. As expected, the dosage will be
dependent on the condition, size, age, and condition of the
patient.
[0164] In some embodiments, the pharmaceutical composition
comprises a single analgesic agent. In one embodiment, the single
analgesic agent is aspirin. In another embodiment, the single
analgesic agent is ibuprofen. In another embodiment, the single
analgesic agent is naproxen or naproxen sodium. In another
embodiment, the single analgesic agent is indomethacin. In another
embodiment, the single analgesic agent is nabumetone. In another
embodiment, the single analgesic agent is acetaminophen.
[0165] In other embodiments, the pharmaceutical composition
comprises a pair of analgesic agents. Examples of such paired
analgesic agents include, but are not limited to, acetylsalicylic
acid and ibuprofen, acetylsalicylic acid and naproxen sodium,
acetylsalicylic acid and nabumetone, acetylsalicylic acid and
acetaminophen, acetylsalicylic acid and indomethancin, ibuprofen
and naproxen sodium, ibuprofen and nabumetone, ibuprofen and
acetaminophen, ibuprofen and indomethancin, naproxen, naproxen
sodium and nabumetone, naproxen sodium and acetaminophen, naproxen
sodium and indomethancin, nabumetone and acetaminophen, nabumetone
and indomethancin, and acetaminophen and indomethancin. The paired
analgesic agents are mixed at a weight ratio in the range of 0.1:1
to 10:1, 0.2:1 to 5:1 or 0.3:1 to 3:1. In one embodiment, the
paired analgesic agents are mixed at a weight ratio of 1:1.
[0166] In some other embodiments, the pharmaceutical composition of
the present application further comprises one or more
antimuscarinic agents. Examples of the antimuscarinic agents
include, but are not limited to, oxybutynin, solifenacin,
darifenacin, fesoterodine, tolterodine, trospium, atropine, and
tricyclic antidepressants. The daily dose of antimuscarinic agent
is in the range of 1 .mu.g to 300 mg, 1 .mu.g to 100 mg, 1 .mu.g to
30 mg; 1 .mu.g to 10 mg, 1 .mu.g to 3 mg, 1 .mu.g to 1 mg, 1 .mu.g
to 300 .mu.g, 1 .mu.g to 100 .mu.g, 1 .mu.g to 30 .mu.g, 1 .mu.g to
10 .mu.g, 1 .mu.g to 3 .mu.g, 3 .mu.g to 100 mg, 3 .mu.g to 100 mg,
3 .mu.g to 30 mg; 3 .mu.g to 10 mg, 3 .mu.g to 3 mg, 3 .mu.g to 1
mg, 3 .mu.g to 300 .mu.g, 3 .mu.g to 100 .mu.g, 3 .mu.g to 30
.mu.g, 3 .mu.g to 10 .mu.g, 10 .mu.g to 300 mg, 10 .mu.g to 100 mg,
10 .mu.g to 30 mg; 10 .mu.g to 10 mg, 10 .mu.g to 3 mg, 10 .mu.g to
1 mg, 10 .mu.g to 300 .mu.g, 10 .mu.g to 100 .mu.g, 10 .mu.g to 30
.mu.g, 30 .mu.g to 300 mg, 30 .mu.g to 100 mg, 30 .mu.g to 30 mg;
30 .mu.g to 10 mg, 30 .mu.g to 3 mg, 30 .mu.g to 1 mg, 30 .mu.g to
300 .mu.g, 30 .mu.g to 100 .mu.g, 100 .mu.g to 300 mg, 100 .mu.g to
100 mg, 100 .mu.g to 30 mg; 100 .mu.g to 10 mg, 100 .mu.g to 3 mg,
100 .mu.g to 1 mg, 100 .mu.g to 300 .mu.g, 300 .mu.g to 300 mg, 300
.mu.g to 100 mg, 300 .mu.g to 30 mg; 300 .mu.g to 10 mg, 300 .mu.g
to 3 mg, 300 .mu.g to 1 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to
30 mg, 1 mg to 3 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mg to 30 mg,
3 mg to 10 mg, 10 mg to 300 mg, 10 mg to 100 mg, 10 mg to 30 mg, 30
mg to 300 mg, 30 mg to 100 mg or 100 mg to 300 mg.
[0167] In some other embodiments, the pharmaceutical composition of
the present application further comprises one or more
antidiuretics. Examples of the antidiuretics include, but are not
limited to, antidiuretic hormone (ADH), angiotensin II,
aldosterone, vasopressin, vasopressin analogs (e.g., desmopressin
argipressin, lypressin, felypressin, ornipressin, and
terlipressin), vasopressin receptor agonists, atrial natriuretic
peptide (ANP) and C-type natriuretic peptide (CNP) receptor (i.e.,
NPR1, NPR2, and NPR3) antagonists (e.g., HS-142-1, isatin,
[Asu7,23']b-ANP-(7-28)], anantin, a cyclic peptide from
Streptomyces coerulescens, and 3G12 monoclonal antibody),
somatostatin type 2 receptor antagonists (e.g., somatostatin),
pharmaceutically-acceptable derivatives, and analogs, salts,
hydrates, and solvates thereof. In some embodiments, the one or
more antidiuretics comprise desmopressin. In other embodiments, the
one or more antidiuretics is desmopressin. The daily dose of
antidiuretic is in the range of 1 .mu.g to 300 mg, 1 .mu.g to 100
mg, 1 .mu.g to 30 mg; 1 .mu.g to 10 mg, 1 .mu.g to 3 mg, 1 .mu.g to
1 mg, 1 .mu.g to 300 .mu.g, 1 .mu.g to 100 .mu.g, 1 .mu.g to 30
.mu.g, 1 .mu.g to 10 .mu.g, 1 .mu.g to 3 .mu.g, 3 .mu.g to 100 mg,
3 .mu.g to 100 mg, 3 .mu.g to 30 mg; 3 .mu.g to 10 mg, 3 .mu.g to 3
mg, 3 .mu.g to 1 mg, 3 .mu.g to 300 .mu.g, 3 .mu.g to 100 .mu.g, 3
.mu.g to 30 .mu.g, 3 .mu.g to 10 .mu.g, 10 .mu.g to 300 mg, 10
.mu.g to 100 mg, 10 .mu.g to 30 mg; 10 .mu.g to 10 mg, 10 .mu.g to
3 mg, 10 .mu.g to 1 mg, 10 .mu.g to 300 .mu.g, 10 .mu.g to 100
.mu.g, 10 .mu.g to 30 .mu.g, 30 .mu.g to 300 mg, 30 .mu.g to 100
mg, 30 .mu.g to 30 mg; 30 .mu.g to 10 mg, 30 .mu.g to 3 mg, 30
.mu.g to 1 mg, 30 .mu.g to 300 .mu.g, 30 .mu.g to 100 .mu.g, 100
.mu.g to 300 mg, 100 .mu.g to 100 mg, 100 .mu.g to 30 mg; 100 .mu.g
to 10 mg, 100 .mu.g to 3 mg, 100 .mu.g to 1 mg, 100 .mu.g to 300
.mu.g, 300 .mu.g to 300 mg, 300 .mu.g to 100 mg, 300 .mu.g to 30
mg; 300 .mu.g to 10 mg, 300 .mu.g to 3 mg, 300 .mu.g to 1 mg, 1 mg
to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 3 mg, 3 mg to 300
mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 300 mg,
10 mg to 100 mg, 10 mg to 30 mg, 30 mg to 300 mg, 30 mg to 100 mg
or 100 mg to 300 mg.
[0168] In other embodiments, the pharmaceutical composition of the
present application further comprises one or more spasmolytics.
Examples of spasmolytics include, but are not limited to,
carisoprodol, benzodiazepines, baclofen, cyclobenzaprine,
metaxalone, methocarbamol, clonidine, clonidine analog, and
dantrolene. In some embodiments, the spasmolytics is used at a
daily dose of 0.1 mg to 1000 mg, 0.1 mg to 300 mg, 0.1 mg to 100
mg, 0.1 mg to 30 mg, 0.1 mg to 10 mg, 0.1 mg to 3 mg, 0.1 mg to 1
mg, 0.1 mg to 0.3 mg, 0.3 mg to 1000 mg, 0.3 mg to 300 mg, 0.3 mg
to 100 mg, 0.3 mg to 30 mg, 0.3 mg to 10 mg, 0.3 mg to 3 mg, 0.3 mg
to 1 mg, 1 mg to 1000 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to
30 mg, 1 mg to 10 mg, 1 mg to 3 mg, 3 mg to 1000 mg, 3 mg to 300
mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 1000 mg,
10 mg to 300 mg, 10 mg to 100 mg, 10 mg to 30 mg, 30 mg to 1000 mg,
30 mg to 300 mg, 30 mg to 100 mg, 100 mg to 1000 mg, 100 mg to 300
mg, or 300 mg to 1000 mg.
[0169] In other embodiments, the pharmaceutical composition of the
present application further comprises one or more PDE 5 inhibitors.
Examples of PDE 5 inhibitors include, but are not limited to,
tadalafil, sildenafil and vardenafil. In some embodiments, the one
or more PDE 5 inhibitors comprise tadalafil. In other embodiments,
the one or more PDE 5 inhibitors is tadalafil. In some embodiments,
the PDE 5 inhibitor is used at a daily dose of 0.1 mg to 1000 mg,
0.1 mg to 300 mg, 0.1 mg to 100 mg, 0.1 mg to 30 mg, 0.1 mg to 10
mg, 0.1 mg to 3 mg, 0.1 mg to 1 mg, 0.1 mg to 0.3 mg, 0.3 mg to
1000 mg, 0.3 mg to 300 mg, 0.3 mg to 100 mg, 0.3 mg to 30 mg, 0.3
mg to 10 mg, 0.3 mg to 3 mg, 0.3 mg to 1 mg, 1 mg to 1000 mg, 1 mg
to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 10 mg, 1 mg to 3
mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mg to 30 mg,
3 mg to 10 mg, 10 mg to 1000 mg, 10 mg to 300 mg, 10 mg to 100 mg,
10 mg to 30 mg, 30 mg to 1000 mg, 30 mg to 300 mg, 30 mg to 100 mg,
100 mg to 1000 mg, 100 mg to 300 mg, or 300 mg to 1000 mg.
[0170] In some other embodiments, the pharmaceutical composition of
the present application further comprises zolpidem. The daily dose
of zolpidem is in the range of 100 .mu.g to 100 mg, 100 .mu.g to 30
mg, 100 .mu.g to 10 mg, 100 .mu.g to 3 mg, 100 .mu.g to 1 mg, 100
.mu.g to 300 .mu.g, 300 .mu.g to 100 mg, 300 .mu.g to 30 mg, 300
.mu.g to 10 mg, 300 .mu.g to 3 mg, 300 .mu.g to 1 mg, 1 mg to 100
mg, 1 mg to 30 mg, 1 mg to 10 mg, 1 mg to 3 mg, 10 mg to 100 mg, 10
mg to 30 mg, or 30 mg to 100 mg.
[0171] The antimuscarinic agents, antidiuretics, spasmolytics,
zolpidem and/or PDE 5 inhibitors may be formulated, alone or
together with other active ingredient(s) in the pharmaceutical
composition, for immediate-release, extended-release, delayed
release, delayed-extended-release or combinations thereof.
[0172] In certain embodiments, the pharmaceutical composition is
formulated for extended release and comprises (1) an analgesic
agent selected from the group consisting of cetylsalicylic acid,
ibuprofen, naproxen, naproxen sodium, nabumetone, acetaminophen,
and indomethancin and (2) a PDE 5 inhibitor, such as tadalafil.
[0173] The pharmaceutical composition may be formulated into a
tablet, an orally disintegrating tablet, capsule, dragee, powder,
granulate, liquid, gel or emulsion form. Said liquid, gel or
emulsion may be ingested by the subject in naked form or contained
within a capsule.
[0174] In some embodiments, the pharmaceutical composition
comprises a single analgesic agent and a single PDE 5 inhibitor. In
one embodiment, the single analgesic agent is aspirin. In another
embodiment, the single analgesic agent is ibuprofen. In another
embodiment, the single analgesic agent is naproxen or naproxen
sodium. In another embodiment, the single analgesic agent is
indomethacin. In another embodiment, the single analgesic agent is
nabumetone. In another embodiment, the single analgesic agent is
acetaminophen. In another embodiment, the single PDE 5 inhibitor is
tadalafil. The analgesic agent and PDE 5 inhibitor may be given at
doses in the ranges described above.
[0175] In some embodiments, the pharmaceutical composition
comprises one or more analgesic agents, individually or in
combination, in an amount between 10-1000 mg, 10-800 mg, 10-600 mg,
10-500 mg, 10-400 mg, 10-300 mg, 10-250 mg, 10-200 mg, 10-150 mg,
10-100 mg 30-1000 mg, 30-800 mg, 30-600 mg, 30-500 mg, 30-400 mg,
30-300 mg, 30-250 mg, 30-200 mg, 30-150 mg, 30-100 mg, 100-1000 mg,
100-800 mg, 100-600 mg, 100-400 mg, 100-250 mg, 300-1000 mg,
300-800 mg, 300-600 mg, 300-400 mg, 400-1000 mg, 400-800 mg,
400-600 mg, 600-1000 mg, 600-800 mg or 800-1000 mg, wherein the
composition is formulated for extended release with a release
profile in which the one or more analgesic agents are released
continuously over a period of 2-12 hours or 5-8 hours.
[0176] In some embodiments, the composition is formulated for
extended-release with a release profile in which at least 90% of
the one or more analgesic agents are released continuously over a
period of 2-12 hours or 5-8 hours.
[0177] In some embodiments, the composition is formulated for
extended release with a release profile in which the one or more
analgesic agents are released continuously over a period of 5, 6,
7, 8, 10 or 12 hours. In some embodiments, the pharmaceutical
composition further comprises an antimuscarinic agent, an
antidiuretic, a spasmolytic, zolpidem or a PDE 5 inhibitor.
[0178] In other embodiments, the composition is formulated for
extended-release with a release profile in which the analgesic
agent is released at a steady rate over a period of 2-12 hours or
5-8 hours. In other embodiments, the composition is formulated for
extended release with a release profile in which the analgesic
agent is released at a steady rate over a period of 5, 6, 7, 8, 10
or 12 hours. As used herein, "a steady rate over a period of time"
is defined as a release profile in which the release rate at any
point during a given period of time is within 30%-300% of the
average release rate over that given period of time. For example,
if 80 mg of aspirin is released at a steady rate over a period of 8
hours, the average release rate is 10 mg/hr during this period of
time and the actual release rate at any time during this period is
within the range of 3 mg/hr to 30 mg/hr (i.e., within 30%-300% of
the average release rate of 10 mg/hr during the 8 hour period). In
some embodiments, the pharmaceutical composition further comprises
an antimuscarinic agent, an antidiuretic a spasmolytic, zolpidem or
a PDE 5 inhibitor.
[0179] In some embodiments, the analgesic agent is selected from
the group consisting of aspirin, ibuprofen, naproxen sodium,
naproxen, indomethacin, nabumetone and acetaminophen. In one
embodiment, the analgesic agent is acetaminophen. The
pharmaceutical composition is formulated to provide a steady
release of small amount of the analgesic agent to maintain an
effective drug concentration in the blood such that the overall
amount of the drug in a single dosage is reduced compared to the
immediate release formulation.
[0180] In some other embodiments, the pharmaceutical composition
comprises one or more analgesic agent(s), individually or in
combination, in an amount between 10-1000 mg, 10-800 mg, 10-600 mg,
10-500 mg, 10-400 mg, 10-300 mg, 10-250 mg, 10-200 mg, 10-150 mg,
10-100 mg 30-1000 mg, 30-800 mg, 30-600 mg, 30-500 mg, 30-400 mg,
30-300 mg, 30-250 mg, 30-200 mg, 30-150 mg, 30-100 mg, 100-1000 mg,
100-800 mg, 100-600 mg, 100-400 mg, 100-250 mg, 300-1000 mg,
300-800 mg, 300-600 mg, 300-400 mg, 400-1000 mg, 400-800 mg,
400-600 mg, 600-1000 mg, 600-800 mg or 800-1000 mg, wherein the
analgesic agent(s) are formulated for extended release,
characterized by a two-phase release profile in which 20-80% of the
analgesic agent(s) are released within 2 hours of administration
and the remainder are released continuously, or at a steady rate,
over a period of 2-12 hours or 5-8 hours. In yet another
embodiment, the analgesic agent(s) is formulated for
extended-release with a two-phase release profile in which 20, 30,
40, 50 or 60% of the analgesic agent(s) are released within 2 hours
of administration and the remainder are released continuously, or
at a steady rate, over a period of 2-12 hours or 5-8 hours. In one
embodiment, the analgesic agent(s) are selected from the group
consisting of aspirin, ibuprofen, naproxen sodium, naproxen,
indomethacin, nabumetone, and acetaminophen. In one embodiment, the
analgesic agent is acetaminophen. In another embodiment, the
analgesic agent is acetaminophen. In some embodiments, the
pharmaceutical composition further comprises an antimuscarinic
agent, an antidiuretic, a spasmolytic, zolpidem and/or a PDE 5
inhibitor. In some embodiments, the antimuscarinic agent,
antidiuretic, spasmolytic, zolpidem and/or PDE 5 inhibitor is/are
formulated for immediate-release.
[0181] Another aspect of the present application relates to a
method for reducing frequency of urination by administering to a
subject in need thereof, two or more PG pathway inhibitors
alternatively to prevent the development of drug resistance. In one
embodiment, the method comprises administering a first PG pathway
inhibitor for a first period of time and then administering a
second PG pathway inhibitors for a second period of time. In
another embodiment, the method further comprises administering a
third PG pathway inhibitor for a third period of time. The first,
second, and third PG pathway inhibitors are different from each
other and may be formulated for immediate-release,
extended-release, delayed-release or combinations thereof.
[0182] Another aspect of the present application relates to a
method for treating nocturia by administering to a person in need
thereof a diuretic, followed with the pharmaceutical composition of
the present application. The diuretic is dosed and formulated to
have a diuretic effect within 6 hours of administration and is
administered at least 8 or 7 hours prior to bedtime. The
pharmaceutical composition of the present application is formulated
for extended-release or delayed, extended-release, and is
administered within 2 hours prior to bedtime.
[0183] Examples of diuretics include, but are not limited to,
acidifying salts, such as CaCl.sub.2 and NH.sub.4Cl; arginine
vasopressin receptor 2 antagonists, such as amphotericin B and
lithium citrate; aquaretics, such as Goldenrod and Juniper; Na--H
exchanger antagonists, such as dopamine; carbonic anhydrase
inhibitors, such as acetazolamide and dorzolamide; loop diuretics,
such as bumetanide, ethacrynic acid, furosemide and torsemide;
osmotic diuretics, such as glucose and mannitol; potassium-sparing
diuretics, such as amiloride, spironolactone, triamterene,
potassium canrenoate; thiazides, such as bendroflumethiazide and
hydrochlorothiazide; and xanthines, such as caffeine, theophylline
and theobromine.
[0184] Another aspect of the present application relates to a
method for reducing the frequency of urination, comprising
administering to a subject in need thereof an effective amount of
the pharmaceutical composition of the present application and an
effective amount of botulinum toxin.
[0185] In some embodiments, the botulinum toxin is administered by
injection into a bladder muscle; and orally administering to the
subject the pharmaceutical composition of the present application.
In some embodiments, the injecting step comprises injection of
10-200 units of botulinum toxin at 5-20 sites in bladder muscle
with an injection dose of 2-10 units per site. In one embodiment,
the injecting step comprises injection of botulinum toxin at 5
sites in bladder muscle with an injection dose of 2-10 units per
site. In another embodiment, the injecting step comprises injection
of botulinum toxin at 10 sites in bladder muscle at an injection
dose of 2-10 units per site. In another embodiment, the injecting
step comprises injection of botulinum toxin at 15 sites in bladder
muscle at an injection dose of 2-10 units per site. In yet another
embodiment, the injecting step comprises injection of botulinum
toxin at 20 sites in bladder muscle at an injection dose of 2-10
units per site. In some embodiments, the injecting step is repeated
every 3, 4, 6, 8, 10 or 12 months.
[0186] In some embodiments, the pharmaceutical composition
comprises one or more analgesic agents selected from the group
consisting of aspirin, ibuprofen, naproxen, naproxen sodium,
indomethacin, nabumetone and acetaminophen in an amount of 5-2000
mg per agent, and one or more spasmolytics selected from the group
consisting of carisoprodol, benzodiazepines, baclofen,
cyclobenzaprine, metaxalone, methocarbamol, clonidine, clonidine
analog, and dantrolene in a total amount of 50-500 mg, wherein the
pharmaceutical composition is formulated for extended release with
a two-phase release profile in which 20-60% of the active
ingredients are released within 2 hours of administration, and the
remainder are released continuously, or at a steady rate, in a
period of 5-24 hours, 5-8 hours, 8-16 hours or 16-24 hours.
[0187] Another aspect of the present application relates to a
pharmaceutical composition that comprises a first component having
an immediate-release subcomponent and an extended-release
subcomponent, wherein the first component is formulated to release
the subcomponents immediately after administration; and a second
component comprising an immediate-release subcomponent and an
extended-release subcomponent, wherein the second component is
formulated for a delayed-release of the subcomponents. In some
embodiments, at least one of the subcomponents in the first
component or the second component comprises an active ingredient
comprising one or more analgesic agents, and at least one of the
subcomponents in the first component or the second component
comprises an active ingredient comprising a PG pathway inhibitor,
such as a PG inhibitor, a PGT inhibitor or a PGR inhibitor. In some
embodiments, the PG pathway inhibitor is selected from the groups
consisting of inhibitors of PG activity, inhibitors of PG
synthesis, inhibitors of PGT activity, inhibitors of PGT
expression, inhibitors of PGR activity, and inhibitors of PGR
expression.
[0188] In some embodiments, each of the subcomponents in the first
component or the second component comprises an active ingredient
comprising one or more analgesic agents and/or a PG pathway
inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR
inhibitor.
[0189] In some embodiments, the one or more analgesic agents are
selected from the group consisting of aspirin, ibuprofen, naproxen,
naproxen sodium, indomethacin, nabumetone, and acetaminophen.
[0190] In some related embodiments, the immediate-release
subcomponent and the extended-release subcomponent in the first
component each comprises an active ingredient comprising one or
more analgesic agents, and/or a PG pathway inhibitor, such as a PG
inhibitor, a PGT inhibitor or a PGR inhibitor. In other
embodiments, the immediate-release subcomponent and the
extended-release subcomponent in the second component each
comprises an active ingredient comprising one or more analgesic
agents, and/or a PG pathway inhibitor, such as a PG inhibitor, a
PGT inhibitor or a PGR inhibitor.
[0191] In some embodiments, the one or more analgesic agents
comprise acetaminophen. In yet other embodiments, at least one of
the subcomponents in the first component or the second component
comprises an active ingredient comprising one or more analgesic
agents and a PG pathway inhibitor, such as a PG inhibitor, a PGT
inhibitor or a PGR inhibitor.
[0192] In some related embodiments, the second component is coated
with an enteric coating.
[0193] In some related embodiments, the second component is
formulated to release the subcomponents after a lag time of 1-4 or
2-4 hours or 4-8 hours following oral administration.
[0194] In some related embodiments, the extended-release
subcomponent in the first component is formulated to release its
active ingredient over a time interval of about 2-10 hours.
[0195] In some related embodiments, the extended-release
subcomponent in the second component is formulated to release its
active ingredient over a time interval of about 2-10 hours.
[0196] In some related embodiments, the active ingredient in the
immediate-release subcomponent and the extended-release
subcomponent in the first component further comprises an
antimuscarinic agent. In some embodiments, the active ingredient in
the immediate-release subcomponent and the extended-release
subcomponent in the second component further comprises an
antimuscarinic agent. In some embodiments, the active ingredient in
the immediate-release subcomponent and the extended-release
subcomponent in both the first and the second component further
comprises an antimuscarinic agent.
[0197] In some related embodiments, the active ingredient in the
immediate-release subcomponent and the extended-release
subcomponent in the first component further comprises an
antidiuretic agent. In some embodiments, the active ingredient in
the immediate-release subcomponent and the extended-release
subcomponent in the second component further comprises an
antidiuretic agent. In some embodiments, the active ingredient in
the immediate-release subcomponent and the extended-release
subcomponent in both the first and the second component further
comprises an antidiuretic agent.
[0198] In some related embodiments, the active ingredient in the
immediate-release subcomponent and the extended-release
subcomponent in the first component further comprises a
spasmolytic. In some embodiments, the active ingredient in the
immediate-release subcomponent and the extended-release
subcomponent in the second component further comprises a
spasmolytic. In some embodiments, the active ingredient in the
immediate-release subcomponent and the extended-release
subcomponent in both the first and the second component further
comprises a spasmolytic.
[0199] In some related embodiments, the immediate-release
subcomponent and the extended-release subcomponent in the first
component each comprises an analgesic agent, such as acetaminophen,
in an amount of 5-2000 mg. In some embodiments, the
immediate-release subcomponent and the extended-release
subcomponent in the second component each comprises an analgesic
agent, such as acetaminophen, in an amount of 5-2000 mg. In some
embodiments, the active ingredient in the immediate-release
subcomponent and the extended-release subcomponent in both the
first and the second component each comprises an analgesic agent,
such as acetaminophen, in an amount of 5-2000 mg.
[0200] In some related embodiments, the active ingredient in the
immediate-release subcomponent of the first component and the
active ingredient in the immediate-release subcomponent of the
second component both comprise an analgesic agent, such as
acetaminophen. In some embodiments, the active ingredient in the
immediate-release subcomponent of the first component and the
active ingredient in the immediate-release subcomponent of the
second component comprise different analgesic agents.
[0201] Another aspect of the present application relates to a
pharmaceutical composition that comprises a first component
comprising an immediate-release subcomponent, wherein the
immediate-release subcomponent comprises an active ingredient
comprising one or more agents selected from the group consisting of
analgesic agents and a PG pathway inhibitor, such as a PG
inhibitor, a PGT inhibitor or a PGR inhibitor, wherein the first
component is formulated to release its subcomponent immediately
after oral administration; and a second component comprising an
immediate-release subcomponent and an extended-release
subcomponent, wherein the second component is formulated to release
its subcomponent after gastric emptying, wherein the subcomponents
in the second component each comprises an active ingredient
comprising one or more agents selected from the group consisting of
analgesic agents and a PG pathway inhibitor, such as a PG
inhibitor, a PGT inhibitor or a PGR inhibitor. In some embodiments,
the PG pathway inhibitor is selected from the groups consisting of
inhibitors of PG activity, inhibitors of PG synthesis, inhibitors
of PGT activity, inhibitors of PGT expression, inhibitors of PGR
activity, and inhibitors of PGR expression.
[0202] In some embodiments, the one or more analgesic agents are
selected from the group consisting of aspirin, ibuprofen, naproxen,
naproxen sodium, indomethacin, nabumetone, and acetaminophen.
[0203] In some related embodiments, the second component is
formulated to release the subcomponents after a lag time of 2-12
hours, 2-4 hours, 2-6 hours, 2-8 hours, or 4-8 hours following oral
administration.
[0204] In some related embodiments, the active ingredient in the
immediate-release subcomponent and the extended-release
subcomponent of the second component comprises one or more
analgesic agents.
[0205] In some related embodiments, the first component further
comprises an extended-release subcomponent, wherein the
extended-release subcomponent comprises an active ingredient
comprising one or more agents selected from the group consisting of
analgesic agents and a PG pathway inhibitor, such as a PG
inhibitor, a PGT inhibitor or a PGR inhibitor. In some embodiments,
the one or more agents comprises an analgesic agent selected from
the group consisting of aspirin, ibuprofen, naproxen, naproxen
sodium, indomethacin, nabumetone, and acetaminophen.
[0206] In some embodiments, the immediate-release subcomponent and
the extended-release subcomponent in the second component each
comprises a PG pathway inhibitor, such as a PG inhibitor, a PGT
inhibitor or a PGR inhibitor.
[0207] In some related embodiments, at least one of the active
ingredients in the immediate-release subcomponent and/or the
extended-release subcomponent of the first and the second
components further comprises an agent selected from the group
consisting of antimuscarinic agents, antidiuretic agents and
spasmolytics.
[0208] In some related embodiments, the active ingredient in the
immediate-release subcomponent and/or the extended-release
subcomponent of the first component further comprises an agent
selected from the group consisting of antimuscarinic agents,
antidiuretic agents and spasmolytics.
[0209] In some related embodiments, the active ingredient in the
immediate-release subcomponent and/or the extended-release
subcomponent of the second component further comprises an agent
selected from the group consisting of antimuscarinic agents,
antidiuretic agents and spasmolytics.
[0210] Another aspect of the present application relates to a
pharmaceutical composition that comprises a first component
comprising an immediate-release subcomponent and an
extended-release subcomponent, wherein the first component is
formulated to release the subcomponents immediately after
administration; and a second component comprising an
immediate-release subcomponent and an extended-release
subcomponent, wherein the second component is formulated for a
delayed-release of the subcomponents, wherein the immediate-release
subcomponent and the extended-release subcomponent in the first
component each comprises an active ingredient comprising one or
more analgesic agents and a PG pathway inhibitor, such as a PG
inhibitor, a PGT inhibitor or a PGR inhibitor, and wherein the
immediate-release subcomponent and the extended-release
subcomponent in the second component each comprises an active
ingredient comprising one or more analgesic agents and a PG pathway
inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR
inhibitor, wherein the pharmaceutical composition reduces the
frequency of urination in patients in need thereof. In some
embodiments, the PG pathway inhibitor is selected from the groups
consisting of inhibitors of PG activity, inhibitors of PG
synthesis, inhibitors of PGT activity, inhibitors of PGT
expression, inhibitors of PGR activity, and inhibitors of PGR
expression.
[0211] In some embodiments, the one or more analgesic agents are
selected from the group consisting of aspirin, ibuprofen, naproxen,
naproxen sodium, indomethacin, nabumetone, and acetaminophen. In
some embodiments, the one or more analgesic agents comprise
acetaminophen.
[0212] In other embodiments, the pharmaceutical composition
comprises a pair of analgesic agents. Examples of such paired
analgesic agents include, but are not limited to, acetaminophen and
an NSAID, acetylsalicylic acid and ibuprofen, acetylsalicylic acid
and naproxen sodium, acetylsalicylic acid and nabumetone,
acetylsalicylic acid and acetaminophen, acetylsalicylic acid and
indomethancin, ibuprofen and naproxen sodium, ibuprofen and
nabumetone, ibuprofen and acetaminophen, ibuprofen and
indomethancin, naproxen sodium and nabumetone, naproxen sodium and
acetaminophen, naproxen sodium and indomethancin, nabumetone and
acetaminophen, nabumetone and indomethancin, and acetaminophen and
indomethancin. The paired analgesic agents are mixed at a weight
ratio in the range of 0.1:1 to 10:1, 0.2:1 to 5:1 or 0.3:1 to 3:1
with a combined dose or single dose (i.e., the dose for each
analgesic) in the range of 5 mg to 2000 mg, 20 mg to 2000 mg, 100
mg to 2000 mg, 200 mg to 2000 mg, 500 mg to 2000 mg, 5 mg to 1500
mg, 20 mg to 1500 mg, 100 mg to 1500 mg, 200 mg to 1500 mg, 500 mg
to 1500 mg, 5 mg to 1000 mg, 20 mg to 1000 mg, 100 mg to 1000 mg,
250 mg to 500 mg, 250 mg to 1000 mg, 250 mg to 1500 mg, 500 mg to
1000 mg, 500 mg to 1500 mg, 1000 mg to 1500 mg, and 1000 mg to 2000
mg. In one embodiment, the paired analgesic agents are mixed at a
weight ratio of 1:1.
[0213] Another aspect of the present application relates to a
pharmaceutical composition that comprises an immediate-release
component and an extended-release component. Each component
comprises a pair of analgesic agents as described above and a PG
pathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR
inhibitor. In some embodiments, the immediate-release component and
the extended-release component comprise different pairs of
analgesic agents. In some embodiments, the immediate-release
component and the extended-release component comprise the same pair
of analgesic agents. In some embodiments, the immediate-release
component and the extended-release component each comprises
acetaminophen and an NSAID. In some embodiments, the
immediate-release component and the extended-release component each
comprises acetaminophen and ibuprofen. In some embodiments, the
immediate-release component and the extended-release component each
consists of acetaminophen, ibuprofen and a PG pathway inhibitor,
such as a PG inhibitor, a PGT inhibitor or a PGR inhibitor. In some
embodiments, the PG pathway inhibitor is selected from the groups
consisting of inhibitors of PG activity, inhibitors of PG
synthesis, inhibitors of PGT activity, inhibitors of PGT
expression, inhibitors of PGR activity, and inhibitors of PGR
expression.
[0214] In some embodiments, the extended-release component is
formulated for extended release over a period of 0.5-24, 2-6, 6-10,
10-14, or 14-24 hours. In some embodiments, the extended-release
component is formulated for extended release over a period of about
8 hours. In some embodiments, the extended-release component is
coated with a delayed-release coating. In some embodiments, the
delayed-release coating delays the release of the extended-release
component for a period of 0.1-12, 0.5-12, 1-12, 2-12, 1-4, 2-4, 4-8
or 8-12 hours. In some embodiments, the delayed-release coating is
an enteric coating. In some embodiments, the pharmaceutical
composition with an immediate-release component and an
extended-release component is formulated into an orally
disintegrating tablet.
[0215] As used herein, the term "orally disintegrating tablet" or
"orally disintegrating formulation" refers to drug tablet or
formulation that rapidly disintegrates or dissolves in the oral
cavity. Orally disintegrating formulations differ from traditional
tablets in that they are designed to be dissolved on the tongue
rather than swallowed whole. In some embodiments, the orally
disintegrating formulations are designed to completely disintegrate
or dissolve in the oral cavity without the aid of additional water
(i.e., in saliva only) in 5, 10, 20, 30, 60, 90, 120, 180, 240 or
300 seconds.
[0216] In some embodiments, the pharmaceutical composition with an
immediate-release component and an extended-release component is
formulated into a liquid form for oral administration. Examples of
the liquid form formulation include, but are not limited to, gels,
emulsions and particle suspensions. For example, the
extended-release component may be formulated into a gel form that
solidifies in the stomach. In some embodiments, the pharmaceutical
composition with an immediate-release component and an
extended-release component is formulated into a pixie pack of
powder that can quickly melt on the tongue. In some embodiments,
the immediate-release component or the extended release component
or both further comprise one or more additional agents selected
from the group consisting of antimuscarinic agents, spasmolytics
and antidiuretic agents.
Method of Manufacture
[0217] Another aspect of the present application relates to methods
of manufacturing extended-release pharmaceutical compositions for
reducing the frequency of urination. In some embodiments, the
method comprises the steps of forming a first mixture having a
first active ingredient formulated for immediate release and a
second active ingredient formulated for extended release; coating
the first mixture with a delayed release coating to form a core
structure; and then coating the core structure with a second
mixture comprising a third active ingredient formulated for
immediate release and a fourth active ingredient formulated for
extended release. In one embodiment, at least one of the first,
second, third and fourth active ingredients comprises an analgesic
agent and at least one of the first, second, third and fourth
active ingredients comprises a PG pathway inhibitor, such as a PG
inhibitor, a PGT inhibitor or a PGR inhibitor. In some embodiments,
the PG pathway inhibitor is selected from the groups consisting of
inhibitors of PG activity, inhibitors of PG synthesis, inhibitors
of PGT activity, inhibitors of PGT expression, inhibitors of PGR
activity, and inhibitors of PGR expression.
[0218] In some embodiments, the analgesic agent is selected from
the group consisting of aspirin, ibuprofen, naproxen, naproxen
sodium, indomethacin, nabumetone and acetaminophen, and wherein at
least one of the first, second, third and fourth active ingredients
comprises 5 mg to 2000 mg of the analgesic agent.
[0219] In some embodiments, at least one of the first, second,
third and fourth active ingredients comprises (1) an analgesic
agent selected from the group consisting of aspirin, ibuprofen,
naproxen, naproxen sodium, indomethacin, nabumetone, and
acetaminophen, and (2) a PG pathway inhibitor, such as a PG
inhibitor, a PGT inhibitor or a PGR inhibitor.
[0220] In some embodiments, the at least one of the first, second,
third and fourth active ingredients comprises (1) acetaminophen,
and (2) a PG pathway inhibitor, such as a PG inhibitor, a PGT
inhibitor or a PGR inhibitor.
[0221] In some embodiments, the at least one of the first, second,
third and fourth active ingredients comprises an agent selected
from the group consisting antimuscarinic agents, antidiuretic
agents and spasmolytics.
[0222] In some embodiments, the delayed release coating is an
enteric coating. In some embodiments, the enteric coating comprises
a pH-dependent polymer. In some embodiments, the delayed release
coating comprises a swelling layer covered by an outer
semi-permeable polymer layer. In some embodiments, the delayed
release coating is formulated to release the coated material after
a lag time of 0.1-12 hours, 0.5-12 hours, 1-12 hours, 2-12 hours,
1-4 hours, 2-4 hours, 2-6 hours, 2-8 hours, 4-6 hours or 4-8 hours
after oral administration.
[0223] In some embodiments, the second active ingredient, or the
fourth active ingredient or both comprise an active core comprising
an extended-release coating or a polymeric matrix effecting
diffusion controlled release.
[0224] In some embodiments, the first mixture is prepared by mixing
the first active ingredient in liquid or powder form with the
second active ingredient, which is formulated for extended release.
As described above, the second active ingredient may be formulated
in an extended release formulation having an active core comprised
of one or more inert particles, each in the form of a bead, pellet,
pill, granular particle, microcapsule, microsphere, microgranule,
nanocapsule, or nanosphere coated on its surfaces with drugs in the
form of e.g., a drug-containing coating or film-forming composition
using, for example, fluid bed techniques or other methodologies
known to those of skill in the art. The inert particle can be of
various sizes, so long as it is large enough to remain undissolved.
Alternatively, the active core may be prepared by granulating and
milling and/or by extrusion and spheronization of a polymer
composition containing the drug substance. In some embodiments, the
active core comprises an extended-release coating or a polymeric
matrix effecting diffusion controlled release, as described in more
detail earlier. In some embodiments, the polymeric matrix is a
water soluble or water-swellable matrix. In some embodiments, the
second active ingredient is simply mixed with the first active
ingredient. Either ingredient or both ingredients may be in the
form of bead, pellet, granular particle, pill, microcapsule,
microsphere, microgranule, nanocapsule or nanosphere as a powder or
as a liquid suspension. In other embodiments, the second active
ingredient form an active core that is coated with the first active
ingredient. In some embodiments, the second active ingredient in
the first mixture is formulated to release the active ingredient
over a period of 2-4 hours, 2-6 hours, 2-8 hours or 2-10 hours.
[0225] In some embodiments, the second active ingredient is kept in
a compartment partially or completely separate from the first
active ingredient. In other embodiments, the first mixture is
formed by keeping the second active ingredient in a compartment
partially or completely separated from the first active
ingredient.
[0226] The first mixture is then coated with a delayed release
coating to form a core structure. In some embodiments, the delayed
release coating is an enteric coating. In some embodiments, the
enteric coating comprises a pH-dependent polymer that maintains its
structure integrity at low pH, such as the pH in the stomach
(normally in the range of 1.5-3.5). In some embodiments, the term
"low pH" refers to a pH value of 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0
or lower. In some embodiments, the enteric coating comprises one or
more pH-dependent polymers and one or more polysaccharides that are
resistant to erosion in both the stomach and intestine, thus
allowing the release of the first mixture only in the colon. In
some embodiments, the delayed release coating comprises two or more
layers of coating. In some embodiments, the delayed release coating
comprises a swelling layer and an outer semi-permeable polymer
layer that covers the swelling layer.
[0227] In the next step, the coated core structure is re-coated
with a second mixture that comprises a third active ingredient
formulated for immediate release and a fourth active ingredient
formulated for extended release. In some embodiments, the second
mixture is prepared by mixing the third active ingredient in liquid
or powder form with the fourth active ingredient, which is
formulated for extended release. The fourth active ingredient may
be formulated in an extended release formulation having an active
core comprised of one or more inert particles, each in the form of
a bead, pellet, pill, granular particle, microcapsule, microsphere,
microgranule, nanocapsule, or nanosphere coated on its surfaces
with drugs in the form of e.g., a drug-containing coating or
film-forming composition using, for example, fluid bed techniques
or other methodologies known to those of skill in the art. The
inert particle can be of various sizes, so long as it is large
enough to remain poorly dissolved. Alternatively, the active core
may be prepared by granulating and milling and/or by extrusion and
spheronization of a polymer composition containing the drug
substance. In some embodiments, the active core comprises an
extended-release coating or a polymeric matrix effecting diffusion
controlled release, as described in more detail earlier. In some
embodiments, the polymeric matrix is a water soluble or
water-swellable matrix. In some embodiments, the fourth active
ingredient is simply mixed with the third active ingredient. Either
ingredient or both ingredients may be in the form of bead, pellet,
granular particle, pill, microcapsule, microsphere, microgranule,
nanocapsule or nanosphere as a powder or as a liquid
suspension.
[0228] In other embodiments, the coated core structure is re-coated
first with the fourth active ingredient, and then coated with the
third active ingredient. In some embodiments, the fourth active
ingredient is formulated to release the active ingredient over a
period of 2-4 hours, 2-6 hours, 2-8 hours or 2-10 hours.
[0229] In some embodiments, the fourth active ingredient is kept in
a compartment partially or completely separated from the third
active ingredient. In other embodiments, the second mixture is
formed by keeping the fourth active ingredient in a compartment
partially or completely separated from the third active
ingredient.
[0230] In other embodiments, the method comprises the steps of
forming a core structure comprising a first active ingredient
formulated for immediate release and a second active ingredient
formulated for extended release, coating the core structure with a
delayed release coating to form a coated core structure, and mixing
the coated core structure with a third active ingredient formulated
for immediate release and a fourth active ingredient formulated for
extended release. The first, second, third and fourth active
ingredients can be the active ingredients described above. In one
embodiment, the first, second, third and fourth active ingredients
each comprises an analgesic agent and/or a PG pathway inhibitor,
such as a PG inhibitor, a PGT inhibitor or a PGR inhibitor. In some
embodiments, the PG pathway inhibitor is selected from the groups
consisting of inhibitors of PG activity, inhibitors of PG
synthesis, inhibitors of PGT activity, inhibitors of PGT
expression, inhibitors of PGR activity, and inhibitors of PGR
expression. In some embodiments, the analgesic agent is selected
from the group consisting of aspirin, ibuprofen, naproxen, naproxen
sodium, indomethacin, nabumetone and acetaminophen. In some
embodiments, the method further comprises the step of preparing a
dosage form with the final mixture. In some embodiments, the dosage
form is in a tablet form. In some embodiments, the dosage form is
in an orally disintegrating form, e.g., orally disintegrating
tablet form. In some embodiments, the dosage form is in a
beads-in-a-capsule form. In some embodiments, the dosage form is in
a liquid (e.g., emulsion) form.
[0231] In other embodiments, the method comprises the steps of
forming a core structure comprising a first active ingredient
formulated for immediate release and a second active ingredient
formulated for extended release, coating the core structure with a
delayed release coating to form a coated core structure, mixing the
coated core structure with a third ingredient formulated for
immediate release and a fourth ingredient formulated for extended
release.
[0232] Another aspect of the present application relates to a
method for manufacturing a pharmaceutical composition for reducing
the frequency of urination. The method comprises the step of
forming a core structure comprising a first active ingredient
formulated for immediate release and a second active ingredient
formulated for extended release; coating the core structure with a
delayed release coating to form a coated core structure; mixing the
coated core structure with a third active ingredient formulated for
immediate release and a fourth active ingredient formulated for
extended release to form a final mixture, and compressing the final
mixture into a tablet. In some embodiments, at least one of the
first, second, third and fourth active ingredients comprises an
analgesic agent and at least one of the first, second, third and
fourth active ingredients comprises a PG pathway inhibitor, such as
a PG inhibitor, a PGT inhibitor or a PGR inhibitor. In some
embodiments, the PG pathway inhibitor is selected from the groups
consisting of inhibitors of PG activity, inhibitors of PG
synthesis, inhibitors of PGT activity, inhibitors of PGT
expression, inhibitors of PGR activity, and inhibitors of PGR
expression.
[0233] In some embodiments, the analgesic agent is selected from
the group consisting of aspirin, ibuprofen, naproxen, naproxen
sodium, indomethacin, nabumetone and acetaminophen and wherein at
least one of the first, second, third and fourth active ingredients
comprises 5-2000 mg of the analgesic agent.
[0234] In some embodiments, the at least one of the first, second,
third, and fourth active ingredients comprises: (1) acetaminophen;
and (2) a PG pathway inhibitor, such as a PG inhibitor, a PGT
inhibitor or a PGR inhibitor.
[0235] In some embodiments, the at least one of the first, second,
third and fourth active ingredients comprises an agent selected
from the group consisting of antimuscarinic agents, antidiuretic
agents and spasmolytics.
[0236] Another aspect of the present application relates to a
method for manufacturing a pharmaceutical composition for reducing
the frequency of urination. The method comprises the steps of
forming a core structure comprising a first active ingredient
formulated for immediate release and a second active ingredient
formulated for extended release; coating the core structure with a
delayed release coating to form a coated core structure; coating
the coated core structure with a third active ingredient formulated
for immediate release to form a double-coated core structure. In
some embodiments, wherein at least one of the first, second and
third active ingredients comprises an analgesic agent and at least
one of the first, second and third active ingredients comprises a
PG pathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a
PGR inhibitor. In some embodiments, the PG pathway inhibitor is
selected from the groups consisting of inhibitors of PG activity,
inhibitors of PG synthesis, inhibitors of PGT activity, inhibitors
of PGT expression, inhibitors of PGR activity, and inhibitors of
PGR expression.
[0237] In some embodiments, the analgesic agent is selected from
the group consisting of aspirin, ibuprofen, naproxen, naproxen
sodium, indomethacin, nabumetone and acetaminophen and wherein at
least one of the first, second and third active ingredients
comprises 5-2000 mg of the analgesic agent.
[0238] In some embodiments, at least one of the first, second and
third active ingredients comprises: (1) acetaminophen; and (2) a PG
pathway inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR
inhibitor.
[0239] In some embodiments, the at least one of the first, second
and third active ingredients comprises an agent selected from the
group consisting antimuscarinic agents, antidiuretic agents and
spasmolytics.
[0240] Another aspect of the present application relates to a
method for manufacturing a pharmaceutical composition for reducing
the frequency of urination. The method comprises the steps of
forming a core structure comprising a first pair of analgesic
agents formulated for extended-release, and coating the core
structure with a coating layer comprising a second pair of
analgesics, wherein the second pair of analgesics is formulated for
immediate release and wherein either the core structure or the
coating layer or both further comprise a PG pathway inhibitor, such
as a PG inhibitor, a PGT inhibitor or a PGR inhibitor. In some
embodiments, the PG pathway inhibitor is selected from the groups
consisting of inhibitors of PG activity, inhibitors of PG
synthesis, inhibitors of PGT activity, inhibitors of PGT
expression, inhibitors of PGR activity, and inhibitors of PGR
expression.
[0241] In some embodiments, the core structure is first coated with
a delayed-release coating and then coated with a coating layer
comprising a second pair of analgesics, wherein the second pair of
analgesics is formulated for immediate release.
[0242] In some embodiments, the method comprises the steps of
forming a first mixture comprising a first pair of analgesic agents
formulated for extended-release, forming a second mixture
comprising a second pair of analgesic agents formulated for
immediate-release, and combining the first mixture and the second
mixture to form a final mixture, wherein either the first mixture
or the second mixture or both further comprise a PG pathway
inhibitor, such as a PG inhibitor, a PGT inhibitor or a PGR
inhibitor.
[0243] In some embodiments, the first mixture, the second mixture
and the final mixture are mixtures of solid materials. In some
embodiments, the final mixture is in powder or granulate form. In
some embodiments, the method further comprises the step of pressing
the final mixture into a tablet form. In some embodiments, the
final mixture is in a liquid, gel or emulsion form.
[0244] Examples of paired analgesic agents include, but are not
limited to, acetaminophen and an NSAID, acetylsalicylic acid and
ibuprofen, acetylsalicylic acid and naproxen sodium,
acetylsalicylic acid and nabumetone, acetylsalicylic acid and
acetaminophen, acetylsalicylic acid and indomethancin, ibuprofen
and naproxen sodium, ibuprofen and nabumetone, ibuprofen and
acetaminophen, ibuprofen and indomethancin, naproxen sodium and
nabumetone, naproxen sodium and acetaminophen, naproxen sodium and
indomethancin, nabumetone and acetaminophen, nabumetone and
indomethancin, and acetaminophen and indomethancin. In some
embodiments, the first pair of analgesic agents is different from
the second pair of analgesic agents. In other embodiments, the
first pair of analgesic agents is the same as the second pair of
analgesic agents. In one embodiment, the first pair of analgesic
agents and the second pair of analgesic agents are both
acetaminophen and ibuprofen.
[0245] For example, the extended-release component may be
formulated into a gel form that solidifies in the stomach. In some
embodiments, the pharmaceutical composition with an
immediate-release component and an extended-release component is
formulated into a pixie pack of powder that can quickly melt on the
tongue. In some embodiments, the pharmaceutical composition with an
immediate-release component and an extended-release component is
formulated into an orally disintegrating tablet using loose
compression tableting. In loose compression, orally disintegrating
formulation is compressed at much lower forces (4-20 kN) than
traditional tablets. In some embodiments, the orally disintegrating
formulation contains some form of sugar, such as mannitol, to
improve mouth feel. In some embodiments, the orally disintegrating
tablet is produced using lyophilized orally disintegrating
formulation.
[0246] The present invention is further illustrated by the
following example which should not be construed as limiting. The
contents of all references, patents, and published patent
applications cited throughout this application are incorporated
herein by reference.
Example 1: Inhibition of the Urge to Urinate with Ibuprofen
[0247] Twenty volunteer subjects, both male and female were
enrolled, each of which experienced a premature urge or desire to
urinate, interfering with their ability to sleep for a sufficient
period of time to feel adequately rested. Each subject ingested
400-800 mg of ibuprofen as a single dose prior to bedtime. At least
14 subjects reported that they were able to rest better because
they were not being awakened as frequently by the urge to
urinate.
[0248] Several subjects reported that after several weeks of
nightly use of ibuprofen, the benefit of less frequent urges to
urinate was no longer being realized. However, all of these
subjects further reported the return of the benefit after several
days of abstaining from taking the dosages. More recent testing has
confirmed similar results can be achieved at much lower dosages
without any subsequent diminution of benefits.
Example 2: Effect of Analgesic Agents, Botulinum Neurotoxin and
Antimuscarinic Agents on Macrophage Responses to Inflammatory and
Non-Inflammatory Stimuli
Experimental Design
[0249] This study is designed to determine the dose and in vitro
efficacy of analgesics and antimuscarinic agents in controlling
macrophage response to inflammatory and non-inflammatory stimuli
mediated by COX2 and prostaglandins (PGE, PGH, etc.). It
establishes baseline (dose and kinetic) responses to inflammatory
and non-inflammatory effectors in bladder cells. Briefly, cultured
cells are exposed to analgesic agents and/or antimuscarinic agents
in the absence or presence of various effectors.
[0250] The effectors include: lipopolysaccharide (LPS), an
inflammatory agent, and Cox2 inducer as inflammatory stimuli;
carbachol or acetylcholine, stimulators of smooth muscle
contraction as non-inflammatory stimuli; botulinum neurotoxin A, a
known inhibitor of acetylcholine release, as positive control; and
arachidonic acid (AA), gamma linolenic acid (DGLA), or
eicosapentaenoic acid (EPA) as precursors of prostaglandins, which
are produced following the sequential oxidation of AA, DGLA, or EPA
inside the cell by cyclooxygenases (COX1 and COX2) and terminal
prostaglandin synthases.
[0251] The analgesic agents include: Salicylates such as aspirin;
iso-butyl-propanoic-phenolic acid derivative (ibuprofen) such as
Advil, Motrin, Nuprin, and Medipren; naproxen sodium such as Aleve,
Anaprox, Antalgin, Feminax Ultra, Flanax, Inza, Midol Extended
Relief, Nalgesin, Naposin, Naprelan, Naprogesic, Naprosyn, Naprosyn
suspension, EC-Naprosyn, Narocin, Proxen, Synflex and Xenobid;
acetic acid derivative such as indomethacin (Indocin);
1-naphthaleneacetic acid derivative such as nabumetone or relafen;
N-acetyl-para-aminophenol (APAP) derivative such as acetaminophen
or paracetamol (Tylenol); and Celecoxib.
[0252] The antimuscarinic agents include oxybutynin, solifenacin,
darifenacin, and atropine.
[0253] Macrophages are subjected to short term (1-2 hrs) or long
term (24-48 hrs) stimulation with:
[0254] (1) Each analgesic agent alone at various doses.
[0255] (2) Each analgesic agent at various doses in the presence of
LPS.
[0256] (3) Each analgesic agent at various doses in the presence of
carbachol or acetylcholine.
[0257] (4) Each analgesic agent at various doses in the presence of
AA, DGLA, or EPA.
[0258] (5) Botulinum neurotoxin A alone at various doses.
[0259] (6) Botulinum neurotoxin A at various doses in the presence
of LPS.
[0260] (7) Botulinum neurotoxin A at various doses in the presence
of carbachol or acetylcholine.
[0261] (8) Botulinum neurotoxin A at various doses in the presence
of AA, DGLA, or EPA.
[0262] (9) Each antimuscarinic agent alone at various doses.
[0263] (10) Each antimuscarinic agent at various doses in the
presence of LPS.
[0264] (11) Each antimuscarinic agent at various doses in the
presence of carbachol or acetylcholine.
[0265] (12) Each antimuscarinic agent at various doses in the
presence of AA, DGLA, or EPA.
[0266] The cells are then analyzed for the release of PGH.sub.2;
PGE; PGE.sub.2; Prostacydin; Thromboxane; IL-1.beta.; IL-6;
TNF-.alpha.; the COX2 activity; the production of cAMP and cGMP;
the production of IL-1.beta., IL-6, TNF-.alpha., and COX2 mRNA; and
surface expression of CD80, CD86, and MHC class II molecules.
Materials and Methods
Macrophage Cells
[0267] Murine RAW264.7 or J774 macrophage cells (obtained from
ATCC) were used in this study. Cells were maintained in a culture
medium containing RPMI 1640 supplemented with 10% fetal bovine
serum (FBS), 15 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin,
and 100 .mu.g/ml of streptomycin. Cells were cultured at 37.degree.
C. in a 5% CO.sub.2 atmosphere and split (passages) once a
week.
In Vitro Treatment of Macrophage Cells with Analgesics
[0268] RAW264.7 macrophage cells were seeded in 96-well plates at a
cell density of 1.5.times.10.sup.5 cells per well in 100 .mu.l of
the culture medium. The cells were treated with (1) various
concentrations of analgesic (acetaminophen, aspirin, ibuprofen or
naproxen), (2) various concentrations of lipopolysaccharide (LPS),
which is an effector of inflammatory stimuli to macrophage cells,
(3) various concentrations of carbachol or acetylcholine, which are
effectors of non-inflammatory stimuli, (4) analgesic and LPS or (5)
analgesic and carbachol or acetylcholine. Briefly, the analgesics
were dissolved in FBS-free culture medium (i.e. RPMI 1640
supplemented with 15 mM HEPES, 2 mM L-glutamine, 100 U/ml
penicillin, and 100 .mu.g/ml of streptomycin) and diluted to
desired concentrations by serial dilution with the same medium. For
cells treated with analgesic in the absence of LPS, 50 .mu.l of
analgesic solution and 50 .mu.l of FBS-free culture medium were
added to each well. For cells treated with analgesic in the
presence of LPS, 50 .mu.l of analgesic solution and 50 .mu.l of LPS
(from Salmonella typhimurium) in FBS-free culture medium were added
to each well. All conditions were tested in duplicates.
[0269] After 24 or 48 hours of culture, 150 .mu.l of culture
supernatants were collected, spun down for 2 min at 8,000 rpm at
4.degree. C. to remove cells and debris and stored at -70.degree.
C. for analysis of cytokine responses by ELISA. The cells were
collected and washed by centrifugation (5 min at 1,500 rpm at
4.degree. C.) in 500 .mu.l of Phosphate buffer (PBS). Half of the
cells were then snapped frozen in liquid nitrogen and stored at
-70.degree. C. The remaining cells were stained with fluorescent
monoclonal antibodies and analyzed by flow cytometry.
Flow Cytometry Analysis of Co-Stimulatory Molecule Expression
[0270] For flow cytometry analysis, macrophages were diluted in 100
.mu.l of FACS buffer (phosphate buffered saline (PBS) with 2%
bovine serum albumin (BSA) and 0.01% NaN.sub.3) and stained 30 min
at 4.degree. C. by addition of FITC-conjugated anti-CD40,
PE-conjugated anti-CD80, PE-conjugated anti-CD86 antibody, anti MHC
class II (I-A.sup.d) PE (BD Bioscience). Cells were then washed by
centrifugation (5 min at 1,500 rpm at 4.degree. C.) in 300 .mu.l of
FACS buffer. After a second wash, cells were re-suspended in 200
.mu.l of FACS buffer and the percentage of cells expressing a given
marker (single positive), or a combination of markers (double
positive) were analyzed with the aid of an Accuri C6 flow cytometer
(BD Biosciences).
Analysis of Cytokine Responses by ELISA
[0271] Culture supernatants were subjected to cytokine-specific
ELISA to determine IL-1.beta., IL-6, and TNF-.alpha. responses in
cultures of macrophages treated with analgesic, LPS alone or a
combination of LPS and analgesic. The assays were performed on Nunc
MaxiSorp Immunoplates (Nunc) coated overnight with 100 .mu.l of
anti-mouse IL-6, TNF-.alpha. mAbs (BD Biosciences) or IL-1.beta.
mAb (R&D Systems) in 0.1 M sodium bicarbonate buffer (pH 9.5).
After two washes with PBS (200 .mu.l per well), 200 .mu.l of PBS 3%
BSA were added in each well (blocking) and the plates incubated for
2 hours at room temperature. Plates were washed again two times by
addition of 200 .mu.l per well, 100 .mu.l of cytokine standards and
serial dilutions of culture supernatants were added in duplicate,
and the plates were incubated overnight at 4.degree. C. Finally,
the plates were washed twice and incubated with 100 .mu.l of
secondary biotinylated anti-mouse IL-6, TNF.alpha. mAbs (BD
Biosciences), or IL-1.beta. (R&D Systems) followed by
peroxidase-labeled goat anti-biotin mAb (Vector Laboratories). The
colorimetric reaction was developed by the addition of
2,2'-azino-bis (3)-ethylbenzylthiazoline-6-sulfonic acid (ABTS)
substrate and H.sub.2O.sub.2 (Sigma) and the absorbance measured at
415 nm with a Victor.RTM. V multilabel plate reader
(PerkinElmer).
Determination of COX2 Activity and the Production of cAMP and
cGMP
[0272] The COX2 activity in the cultured macrophages is determined
by sequential competitive ELISA (R&D Systems). The production
of cAMP and cGMP is determined by the cAMP assay and cGMP assay.
These assays are performed routinely in the art.
[0273] Table 1 summarizes the experiments performed with Raw 264
macrophage cell line and main findings in terms of the effects of
analgesics on cell surface expression of costimulatory molecules
CD40 and CD80. Expression of these molecules is stimulated by COX2
and inflammatory signals and, thus, was evaluated to determine
functional consequences of inhibition of COX2.
[0274] As shown in Table 2, acetaminophen, aspirin, ibuprofen, and
naproxen inhibit basal expression of co-stimulatory molecules CD40
and CD80 by macrophages at all the tested doses (i.e.,
5.times.10.sup.5 nM, 5.times.10.sup.4 nM, 5.times.10.sup.3 nM,
5.times.10.sup.2 nM, 50 nM, and 5 nM), except for the highest dose
(i.e., 5.times.10.sup.6 nM), which appears to enhance, rather than
inhibit, expression of the co-stimulatory molecules. As shown in
FIGS. 1A and 1B, such inhibitory effect on CD40 and CD50 expression
was observed at analgesic doses as low as 0.05 nM (i.e., 0.00005
This finding supports the notion that a controlled release of small
doses of analgesic may be preferable to acute delivery of large
doses. The experiment also revealed that acetaminophen, aspirin,
ibuprofen, and naproxen have a similar inhibitory effect on LPS
induced expression of CD40 and CD80.
TABLE-US-00001 TABLE 1 Summary of experiments LPS Con- Salmonella
Aceta- Ibu- Napro- trol typhimurium minophen Aspirin profen xen
TESTS 1 X 2 X Dose responses (0, 5, 50, 1000) ng/mL 3 X Dose
responses (0, 5, 50, 500, 5 .times. 10.sup.3, 5 .times. 10.sup.4, 5
.times. 10.sup.5, 5 .times. 10.sup.6) nM 4 X X (5 ng/mL) Dose
responses X (50 ng/mL (0, 5, 50, 500, 5 .times. 10.sup.3, 5 .times.
10.sup.4, X (1000 5 .times. 10.sup.5, 5 .times. 10.sup.6) nM ng/mL)
ANALYSIS a Characterization of activation/stimulatory status: Flow
cytometry analysis of CD40, CD80, CD86, and MHC class II b
Mediators of inflammatory responses: ELISA analysis of IL-1.beta.,
IL-6, TNF-.alpha.
TABLE-US-00002 TABLE 2 Summary of main findings LPS Negative 5 Dose
analgesic (nM) Effectors % Positive Control ng/ml 5 .times.
10.sup.6 5 .times. 10.sup.5 5 .times. 10.sup.4 5 .times. 10.sup.3
500 50 5 CD40.sup.+CD80.sup.+ 20.6 77.8 Acetaminophen
CD40.sup.+CD80.sup.+ 63 18 12 9.8 8.3 9.5 7.5 Aspirin
CD40.sup.+CD80.sup.+ 44 11 10.3 8.3 8 10.5 7.5 Ibuprofen
CD40.sup.+CD80.sup.+ ND* 6.4 7.7 7.9 6.0 4.9 5.8 Naproxen
CD40.sup.+CD80.sup.+ 37 9.6 7.7 6.9 7.2 6.8 5.2 Analgesic plus LPS
Acetaminophen CD40.sup.+CD80.sup.+ 95.1 82.7 72.4 68.8 66.8 66.2
62.1 Aspirin CD40.sup.+CD80.sup.+ 84.5 80 78.7 74.7 75.8 70.1 65.7
Ibuprofen CD40.sup.+CD80.sup.+ ND 67 77.9 72.9 71.1 63.7 60.3
Naproxen CD40.sup.+CD80.sup.+ 66.0 74.1 77.1 71.0 68.8 72 73 *ND:
not done (toxicity)
[0275] Table 3 summarizes the results of several studies that
measured serum levels of analgesic after oral therapeutic doses in
adult humans. As shown in Table 3, the maximum serum levels of
analgesic after an oral therapeutic dose are in the range of
10.sup.4 to 10.sup.5 nM. Therefore, the doses of analgesic tested
in vitro in Table 2 cover the range of concentrations achievable in
vivo in humans.
TABLE-US-00003 TABLE 3 Serum levels of analgesic in human blood
after oral therapeutic doses Maximum serum levels after oral
Molecular therapeutic doses Analgesic drug weight mg/L nM
References Acetaminophen 151.16 11-18 7.2 .times. 10.sup.4-1.19
.times. BMC Clinical Pharmacology. 2010, 10: 10 (Tylenol) 10.sup.5
Anaesth Intensive Care. 2011, 39: 242 Aspirin 181.66 30-100 1.65
.times. 10.sup.5-5.5 .times. Disposition of Toxic Drugs and
Chemicals in (Acetylsalicylic acid) 10.sup.5 Man, 8th Edition,
Biomedical Public, Foster City, CA, 2008, pp. 22-25 J Lab Clin Med.
1984 Jun; 103: 869 Ibuprofen 206.29 24-32 1.16 .times.
10.sup.5-1.55 .times. BMC Clinical Pharmacology2010, 10: 10 (Advil,
Motrin) 10.sup.5 J Clin Pharmacol. 2001, 41: 330 Naproxen 230.26 Up
to Up to J Clin Pharmacol. 2001, 41: 330 (Aleve) 60 2.6 .times.
10.sup.5
Example 3: Effect of Analgesic Agents, Botulinum Neurotoxin and
Antimuscarinic Agents on Mouse Bladder Smooth Muscle Cell Responses
to Inflammatory and Non-Inflammatory Stimuli
Experimental Design
[0276] This study is designed to characterize how the optimal doses
of analgesics determined in Example 2 affect bladder smooth muscle
cells in cell culture or tissue cultures, and to address whether
different classes of analgesics can synergize to more efficiently
inhibit COX2 and PGE2 responses.
[0277] The effectors, analgesic agents and antimuscarinic agents
are described in Example 2.
[0278] Primary culture of mouse bladder smooth muscle cells are
subjected to short term (1-2 hrs) or long term (24-48 hrs)
stimulation with: [0279] (1) Each analgesic agent alone at various
doses. [0280] (2) Each analgesic agent at various doses in the
presence of LPS. [0281] (3) Each analgesic agent at various doses
in the presence of carbachol or acetylcholine. [0282] (4) Each
analgesic agent at various doses in the presence of AA, DGLA, or
EPA. [0283] (5) Botulinum neurotoxin A alone at various doses.
[0284] (6) Botulinum neurotoxin A at various doses in the presence
of LPS. [0285] (7) Botulinum neurotoxin A at various doses in the
presence of carbachol or acetylcholine. [0286] (8) Botulinum
neurotoxin A at various doses in the presence of AA, DGLA, or EPA.
[0287] (9) Each antimuscarinic agent alone at various doses. [0288]
(10) Each antimuscarinic agent at various doses in the presence of
LPS. [0289] (11) Each antimuscarinic agent at various doses in the
presence of carbachol or acetylcholine. [0290] (12) Each
antimuscarinic agent at various doses in the presence of AA, DGLA,
or EPA.
[0291] The cells are then analyzed for the release of PGH.sub.2;
PGE; PGE.sub.2; Prostacydin; Thromboxane; IL-1.beta.; IL-6;
TNF-.alpha.; the COX2 activity; the production of cAMP and cGMP;
the production of IL-1.beta., IL-6, TNF-.alpha., and COX2 mRNA; and
surface expression of CD80, CD86, and MEW class II molecules.
Materials and Methods
Isolation and Purification of Mouse Bladder Cells
[0292] Bladder cells were removed from euthanized animals C57BL/6
mice (8-12 weeks old), and cells were isolated by enzymatic
digestion followed by purification on a Percoll gradient. Briefly,
bladders from 10 mice were minced with scissors to fine slurry in
10 ml of digestion buffer (RPMI 1640, 2% fetal bovine serum, 0.5
mg/ml collagenase, 30 .mu.g/ml DNase). Bladder slurries were
enzymatically digested for 30 minutes at 37.degree. C. Undigested
fragments were further dispersed through a cell-trainer. The cell
suspension was pelleted and added to a discontinue 20%, 40%. and
75% Percoll gradient for purification on mononuclear cells. Each
experiment used 50-60 bladders.
[0293] After washes in RPMI 1640, bladder cells were resuspended
RPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES, 2
mM L-glutamine, 100 U/ml penicillin, and 100 .mu.g/ml of
streptomycin and seeded in clear-bottom black 96-well cell culture
microculture plates at a cell density of 3.times.10.sup.4 cells per
well in 100 Cells were cultured at 37.degree. C. in a 5% CO.sub.2
atmosphere.
In Vitro Treatment of Cells with Analgesics
[0294] Bladder cells were treated with analgesic solutions (50
.mu.l/well) either alone or together with carbachol (10-Molar, 50
.mu.l/well), as an example of non-inflammatory stimuli, or
lipopolysaccharide (LPS) of Salmonella typhimurium (1 .mu.g/ml, 50
.mu.l/well), as an example of non-inflammatory stimuli. When no
other effectors were added to the cells, 50 .mu.l of RPMI 1640
without fetal bovine serum were added to the wells to adjust the
final volume to 200 .mu.l.
[0295] After 24 hours of culture, 150 .mu.l of culture supernatants
were collected, spun down for 2 min at 8,000 rpm at 4.degree. C. to
remove cells and debris, and stored at -70.degree. C. for analysis
of Prostaglandin E2 (PGE.sub.2) responses by ELISA. Cells were
fixed, permeabilized, and blocked for detection of Cyclooxygenase-2
(COX2) using a fluorogenic substrate. In selected experiment cells
were stimulated 12 hours in vitro for analysis of COX2
responses.
Analysis of COX2 Responses
[0296] COX2 responses were analyzed by a Cell-Based ELISA using
human/mouse total COX2 immunoassay (R&D Systems), following the
instructions of the manufacturer. Briefly, after cells fixation and
permeabilization, a mouse anti-total COX2 and a rabbit anti-total
GAPDH were added to the wells of the clear-bottom black 96-well
cell culture microculture plates. After incubation and washes, an
HRP-conjugated anti-mouse IgG and an AP-conjugated anti-rabbit IgG
were added to the wells. Following another incubation and set of
washes, the HRP- and AP-fluorogenic substrates were added. Finally,
a Victor.RTM. V multilabel plate reader (PerkinElmer) was used to
read the fluorescence emitted at 600 nm (COX2 fluorescence) and 450
nm (GAPDH fluorescence). Results are expressed as relative levels
of total COX2 as determined by relative fluorescence unit (RFUs)
and normalized to the housekeeping protein GAPDH.
Analysis of PGE2 Responses
[0297] Prostaglandin E2 responses were analyzed by a sequential
competitive ELISA (R&D Systems). More specifically, culture
supernatants or PGE2 standards were added to the wells of a 96-well
polystyrene microplate coated with a goat anti-mouse polyclonal
antibody. After one hour incubation on a microplate shaker, an
HRP-conjugated PGE2 was added and the plates were incubated for an
additional two hours at room temperature. The plates were then
washed and HRP substrate solution added to each well. The color was
allowed to develop for 30 minutes, and the reaction stopped by the
addition of sulfuric acid before reading the plate at 450 nm with
wavelength correction at 570 nm. Results are expressed as mean
pg/ml of PGE2.
Other Assays
[0298] The release of PGH.sub.2; PGE, Prostacydin; Thromboxane;
IL-1.beta.; IL-6; and TNF-.alpha.; the production of cAMP and cGMP;
the production of IL-1.beta., IL-6, TNF-.alpha., and COX2 mRNA; and
surface expression of CD80, CD86, and MHC class II molecules are
determined as described in Example 2.
Results
Analgesics Inhibit COX2 Responses of Mouse Bladder Cells to an
Inflammatory Stimulus
[0299] Several analgesics (acetaminophen, aspirin, ibuprofen, and
naproxen) were tested on mouse bladder cells at the concentration
of 5 .mu.M or 50 .mu.M to determine whether the analgesics could
induce COX2 responses. Analysis of 24-hour cultures showed that
none of the analgesics tested induced COX2 responses in mouse
bladder cells in vitro.
[0300] The effect of these analgesics on the COX2 responses of
mouse bladder cells to carbachol or LPS stimulation in vitro was
also tested. As indicated in Table 1, the dose of carbachol tested
has no significant effect on COX2 levels in mouse bladder cells. On
the other hand, LPS significantly increased total COX2 levels.
Interestingly, acetaminophen, aspirin, ibuprofen, and naproxen
could all suppress the effect of LPS on COX2 levels. The
suppressive effect of the analgesic was seen when these drugs were
tested at either 5 .mu.M or 50 .mu.M (Table 4).
TABLE-US-00004 TABLE 4 COX2 expression by mouse bladder cells after
in vitro stimulation and treatment with analgesic Total COX2 levels
Stimulus Analgesic (Normalized RFUs) None None 158 .+-. 18
Carbachol (mM) None 149 .+-. 21 LPS (1 .mu.g/ml) None 420 .+-. 26
LPS (1 .mu.g/ml) Acetaminophen (5 .mu.M) 275 .+-. 12 LPS (1
.mu.g/ml) Aspirin (5 .mu.M) 240 .+-. 17 LPS (1 .mu.g/ml) Ibuprofen
(5 .mu.M)) 253 .+-. 32 LPS (1 .mu.g/ml) Naproxen (5 .mu.M) 284 .+-.
11 LPS (1 .mu.g/ml) Acetaminophen (50 .mu.M) 243 .+-. 15 LPS (1
.mu.g/ml) Aspirin (50 .mu.M) 258 .+-. 21 LPS (1 .mu.g/ml) Ibuprofen
(50 .mu.M) 266 .+-. 19 LPS (1 .mu.g/ml) Naproxen (50 .mu.M) 279
.+-. 23
Analgesics Inhibit PGE2 Responses of Mouse Bladder Cells to an
Inflammatory Stimulus
[0301] The secretion of PGE2 in culture supernatants of mouse
bladder cells was measured to determine the biological significance
of the alteration of mouse bladder cell COX2 levels by analgesics.
As shown in Table 5, PGE2 was not detected in the culture
supernatants of unstimulated bladder cells or bladder cells
cultured in the presence of carbachol. Consistent with COX2
responses described above, stimulation of mouse bladder cells with
LPS induced the secretion of high levels of PGE2. Addition of the
analgesics acetaminophen, aspirin, ibuprofen, and naproxen
suppressed the effect of LPS on PGE2 secretion, and no difference
was seen between the responses of cells treated with the 5 or 50
.mu.M dose of analgesic.
TABLE-US-00005 TABLE 5 PGE2 secretion by mouse bladder cells after
in vitro stimulation and treatment with analgesic. Stimulus
Analgesic PGE2 levels (pg/ml) None None <20.5 Carbachol (mM)
None <20.5 LPS (1 .mu.g/ml) None 925 .+-. 55 LPS (1 .mu.g/ml)
Acetaminophen (5 .mu.M) 619 .+-. 32 LPS (1 .mu.g/ml) Aspirin (5
.mu.M) 588 .+-. 21 LPS (1 .mu.g/ml) Ibuprofen (5 .mu.M)) 593 .+-.
46 LPS (1 .mu.g/ml) Naproxen (5 .mu.M) 597 .+-. 19 LPS (1 .mu.g/ml)
Acetaminophen (50 .mu.M) 600 .+-. 45 LPS (1 .mu.g/ml) Aspirin (50
.mu.M) 571 .+-. 53 LPS (1 .mu.g/ml) Ibuprofen (50 .mu.M) 568 .+-.
32 LPS (1 .mu.g/ml) Naproxen (50 .mu.M) 588 .+-. 37
[0302] In summary, these data show that the analgesics alone at 5
.mu.M or 50 .mu.M do not induce COX2 and PGE2 responses in mouse
bladder cells. The analgesics at 5 .mu.M or 50 .mu.M, however,
significantly inhibit COX2 and PGE2 responses of mouse bladder
cells stimulated in vitro with LPS (1 .mu.g/ml). No significant
effect of analgesics was observed on COX2 and PGE2 responses of
mouse bladder cells stimulated with carbachol (1 mM).
Example 4: Effect of Analgesic Agents, Botulinum Neurotoxin and
Antimuscarinic Agents on Mouse Bladder Smooth Muscle Cell
Contraction
Experimental Design
[0303] Cultured mouse or rat bladder smooth muscle cells and mouse
or rat bladder smooth muscle tissue are exposed to inflammatory
stimuli and non-inflammatory stimuli in the presence of analgesic
agent and/or antimuscarinic agent at various concentrations. The
stimulus-induced muscle contraction is measured to evaluate the
inhibitory effect of the analgesic agent and/or antimuscarinic
agent.
[0304] The effectors, analgesic agents, and antimuscarinic agents
are described in Example 2.
[0305] Primary cultures of mouse bladder smooth muscle cells are
subjected to short term (1-2 hrs) or long term (24-48 hrs)
stimulation with: [0306] (1) Each analgesic agent alone at various
doses. [0307] (2) Each analgesic agent at various doses in the
presence of LPS. [0308] (3) Each analgesic agent at various doses
in the presence of carbachol or acetylcholine. [0309] (4) Each
analgesic agent at various doses in the presence of AA, DGLA, or
EPA. [0310] (5) Botulinum neurotoxin A alone at various doses.
[0311] (6) Botulinum neurotoxin A at various doses in the presence
of LPS. [0312] (7) Botulinum neurotoxin A at various doses in the
presence of carbachol or acetylcholine. [0313] (8) Botulinum
neurotoxin A at various doses in the presence of AA, DGLA, or EPA.
[0314] (9) Each antimuscarinic agent alone at various doses. [0315]
(10) Each antimuscarinic agent at various doses in the presence of
LPS. [0316] (11) Each antimuscarinic agent at various doses in the
presence of carbachol or acetylcholine. [0317] (12) Each
antimuscarinic agent at various doses in the presence of AA, DGLA,
or EPA.
Materials and Methods
[0318] Primary mouse bladder cells are isolated as described in
Example 3. In selected experiments, cultures of bladder tissue are
used. Bladder smooth muscle cell contractions are recorded with a
Grass polygraph (Quincy Mass, USA).
Example 5: Effect of Oral Analgesic Agents and Antimuscarinic
Agents on Cox2 and PGE2 Responses of Mouse Bladder Smooth Muscle
Cells
Experimental Design
[0319] Normal mice and mice with over active bladder syndrome are
given oral doses of aspirin, naproxen sodium, ibuprofen, Indocin,
nabumetone, Tylenol, Celecoxib, oxybutynin, solifenacin,
darifenacin, atropine, and combinations thereof. Control groups
include untreated normal mice and untreated OAB mice with over
active bladder syndrome. Thirty (30) minutes after last doses, the
bladders are collected and stimulated ex vivo with carbachol or
acetylcholine. In selected experiments, the bladders are treated
with botulinum neurotoxin A before stimulation with carbachol.
Animals are maintained in metabolic cages and frequency (and
volume) of urination are evaluated. Bladder outputs are determined
by monitoring water intake and cage litter weight. Serum PGH.sub.2,
PGE, PGE.sub.2, Prostacydin, Thromboxane, IL-1.beta., IL-6,
TNF-.alpha., cAMP, and cGMP levels are determined by ELISA. CD80,
CD86, and MHC class II expression in whole blood cells are
determined by flow cytometry.
[0320] At the end of the experiment, animals are euthanized, and ex
vivo bladder contractions are recorded with a Grass polygraph.
Portions of bladders are fixed in formalin, and COX2 responses are
analyzed by immunohistochemistry.
Example 6: Effect of Analgesic Agents, Botulinum Neurotoxin and
Antimuscarinic Agents on Human Bladder Smooth Muscle Cell Responses
to Inflammatory and Non-Inflammatory Stimuli
Experimental Design
[0321] This study is designed to characterize how the optimal doses
of analgesic determined in Examples 1-5 affect human bladder smooth
muscle cells in cell culture or tissue cultures and to address
whether different classes of analgesics can synergize to more
efficiently inhibit COX2 and PGE2 responses.
[0322] The effectors, analgesic agents, and antimuscarinic agents
are described in Example 2.
[0323] Human bladder smooth muscle cells are subjected to short
term (1-2 hrs) or long term (24-48 hrs) stimulation with: [0324]
(1) Each analgesic agent alone at various doses. [0325] (2) Each
analgesic agent at various doses in the presence of LPS. [0326] (3)
Each analgesic agent at various doses in the presence of carbachol
or acetylcholine. [0327] (4) Each analgesic agent at various doses
in the presence of AA, DGLA, or EPA. [0328] (5) Botulinum
neurotoxin A alone at various doses. [0329] (6) Botulinum
neurotoxin A at various doses in the presence of LPS. [0330] (7)
Botulinum neurotoxin A at various doses in the presence of
carbachol or acetylcholine. [0331] (8) Botulinum neurotoxin A at
various doses in the presence of AA, DGLA, or EPA. [0332] (9) Each
antimuscarinic agent alone at various doses. [0333] (10) Each
antimuscarinic agent at various doses in the presence of LPS.
[0334] (11) Each antimuscarinic agent at various doses in the
presence of carbachol or acetylcholine. [0335] (12) Each
antimuscarinic agent at various doses in the presence of AA, DGLA,
or EPA.
[0336] The cells are then analyzed for the release of PGH.sub.2;
PGE; PGE.sub.2; Prostacydin; Thromboxane; IL-1.beta.; IL-6;
TNF.alpha.; the COX2 activity; the production of cAMP and cGMP; the
production of IL-1.beta., IL-6, TNF.alpha., and COX2 mRNA; and
surface expression of CD80, CD86, and MHC class II molecules.
Example 7: Effect of Analgesic Agents, Botulinum Neurotoxin and
Antimuscarinic Agents on Human Bladder Smooth Muscle Cell
Contraction
Experimental Design
[0337] Cultured human bladder smooth muscle cells are exposed to
inflammatory stimuli and non-inflammatory stimuli in the presence
of an analgesic agent and/or antimuscarinic agent at various
concentrations. The stimuli-induced muscle contraction is measured
to evaluate the inhibitory effect of the analgesic agent and/or
antimuscarinic agent.
[0338] The effectors, analgesic agents, and antimuscarinic agents
are described in Example 2.
[0339] Human bladder smooth muscle cells are subjected to short
term (1-2 hrs) or long term (24-48 hrs) stimulation with: [0340]
(1) Each analgesic agent alone at various doses. [0341] (2) Each
analgesic agent at various doses in the presence of LPS. [0342] (3)
Each analgesic agent at various doses in the presence of carbachol
or acetylcholine. [0343] (4) Each analgesic agent at various doses
in the presence of AA, DGLA, or EPA. [0344] (5) Botulinum
neurotoxin A alone at various doses. [0345] (6) Botulinum
neurotoxin A at various doses in the presence of LPS. [0346] (7)
Botulinum neurotoxin A at various doses in the presence of
carbachol or acetylcholine. [0347] (8) Botulinum neurotoxin A at
various doses in the presence of AA, DGLA, or EPA. [0348] (9) Each
antimuscarinic agent alone at various doses. [0349] (10) Each
antimuscarinic agent at various doses in the presence of LPS.
[0350] (11) Each antimuscarinic agent at various doses in the
presence of carbachol or acetylcholine. [0351] (12) Each
antimuscarinic agent at various doses in the presence of AA, DGLA,
or EPA.
[0352] Bladder smooth muscle cell contractions are recorded with a
Grass polygraph (Quincy Mass, USA).
Example 8: Effect of Analgesic Agents on Normal Human Bladder
Smooth Muscle Cell Responses to Inflammatory and Non Inflammatory
Signals
Experimental Design
Culture of Normal Human Bladder Smooth Muscle Cells
[0353] Normal human bladder smooth muscle cells were isolated by
enzymatic digestion from macroscopically normal pieces of human
bladder. Cells were expended in vitro by culture at 37.degree. C.
in a 5% CO.sub.2 atmosphere in RPMI 1640 supplemented with 10%
fetal bovine serum, 15 mM HEPES, 2 mM L-glutamine, 100 U/ml
penicillin, and 100 mg/ml of streptomycin and passage once a week
by treatment with trypsin to detach cells followed by reseeding in
a new culture flask. The first week of culture, the culture medium
was supplemented with 0.5 ng/ml epidermal growth factor, 2 ng/ml
fibroblast growth factor, and 5 .mu.g/ml insulin.
Treatment of Normal Human Bladder Smooth Muscle Cells with
Analgesics In Vitro
[0354] Bladder smooth muscle cells trypsinized and seeded in
microculture plates at a cell density of 3.times.10.sup.4 cells per
well in 100 .mu.l were treated with analgesic solutions (50
.mu.l/well) either alone or together carbachol (10-Molar, 50
.mu.l/well), as an example of non-inflammatory stimuli, or
lipopolysaccharide (LPS) of Salmonella typhimurium (1 .mu.g/ml, 50
.mu.l/well), as an example of non-inflammatory stimuli. When no
other effectors were added to the cells, 50 .mu.l of RPMI 1640
without fetal bovine serum were added to the wells to adjust the
final volume to 200 .mu.l.
[0355] After 24 hours of culture, 150 .mu.l of culture supernatants
were collected, spun down for 2 min at 8,000 rpm at 4.degree. C. to
remove cells and debris, and stored at -70.degree. C. for analysis
of Prostaglandin E2 (PGE.sub.2) responses by ELISA. Cells were
fixed, permeabilized, and blocked for detection of COX2 using a
fluorogenic substrate. In selected experiments, cells were
stimulated 12 hours in vitro for analysis of COX2, PGE2, and
cytokine responses.
Analysis of COX2, PGE2, and Cytokine Responses
[0356] COX2 and PGE2 responses were analyzed as described in
Example 3. Cytokine responses were analyzed as described in Example
2.
[0357] Results
[0358] Analgesics Inhibit COX2 Responses of Normal Human Bladder
Smooth Muscle Cells to Inflammatory and Non-Inflammatory
Stimuli--
[0359] Analysis of cells and culture supernatants after 24 hours of
cultures showed that none of the analgesics tested alone induced
COX2 responses in normal human bladder smooth muscle cells.
However, as summarized in Table 6, carbachol induced low, but
significant COX2 responses in normal human bladder smooth muscle
cells. On the other hand, LPS treatment resulted in higher levels
of COX2 responses in normal human bladder smooth muscle cells.
Acetaminophen, aspirin, ibuprofen, and naproxen could all suppress
the effect of carbachol and LPS on COX2 levels. The suppressive
effect of the analgesics was seen on LPS-induced responses when
these drugs were tested at either 5 .mu.M or 50 .mu.M.
TABLE-US-00006 TABLE 6 COX2 expression by normal human bladder
smooth muscle cells after in vitro stimulation with inflammatory
and non-inflammatory stimuli and treatment with analgesic Total
Total COX2 COX2 levels.sup.# levels (Normalized (Normalized RFUs)
RFUs) Stimulus Analgesic subject 1 subject 2 None None 230 199
Carbachol 10.sup.-3M None (50 .mu.M) 437 462 Carbachol 10.sup.-3M
Acetaminophen (50 .mu.M) 298 310 Carbachol 10.sup.-3M Aspirin (50
.mu.M) 312 297 Carbachol 10.sup.-3M Ibuprofen (50 .mu.M) 309 330
Carbachol 10.sup.-3M Naproxen (50 .mu.M) 296 354 LPS (10 .mu.g/ml)
None 672 633 LPS (10 .mu.g/ml) Acetaminophen (5 .mu.M) 428 457 LPS
(10 .mu.g/ml) Aspirin (5 .mu.M) 472 491 LPS (10 .mu.g/ml) Ibuprofen
(5 .mu.M) 417 456 LPS (10 .mu.g/ml) Naproxen (5 .mu.M 458 501 LPS
(10 .mu.g/ml) Acetaminophen (50 .mu.M) 399 509 LPS (10 .mu.g/ml)
Aspirin (50 .mu.M) 413 484 LPS (10 .mu.g/ml) Ibuprofen (50 .mu.M)
427 466 LPS (10 .mu.g/ml) Naproxen (50 .mu.M) 409 458 .sup.#Data
are expressed as mean of duplicates
[0360] Analgesics Inhibit PGE2 Responses of Normal Human Bladder
Smooth Muscle Cells to Inflammatory and Non-Inflammatory
Stimuli--
[0361] Consistent with the induction of COX2 responses described
above, both carbachol and LPS induced production of PGE2 by normal
human bladder smooth muscle cells. Acetaminophen, aspirin,
ibuprofen, and naproxen were also found to suppress the LPS-induced
PGE2 responses at either 5 .mu.M or 50 .mu.M (Table 7).
TABLE-US-00007 TABLE 7 PGE2 secretion by normal human bladder
smooth muscle cells after in vitro stimulation with inflammatory
and non-inflammatory stimuli and treatment with analgesic PGE2
levels.sup.# PGE2 levels (pg/ml) (pg/ml) Stimulus Analgesic Subject
1 Subject 2 None None <20.5 <20.5 Carbachol 10.sup.-3M None
129 104 Carbachol 10.sup.-3M Acetaminophen (50 .mu.M) 76 62
Carbachol 10.sup.-3M Aspirin (50 .mu.M) 89 59 Carbachol 10.sup.-3M
Ibuprofen (50 .mu.M) 84 73 Carbachol 10.sup.-3M Naproxen (50 .mu.M)
77 66 LPS (10 .mu.g/ml) None 1125 998 LPS (10 .mu.g/ml)
Acetaminophen (5 .mu.M) 817 542 LPS (10 .mu.g/ml) Aspirin (5 .mu.M)
838 598 LPS (10 .mu.g/ml) Ibuprofen (5 .mu.M) 824 527 LPS (10
.mu.g/ml) Naproxen (5 .mu.M 859 506 LPS (10 .mu.g/ml) Acetaminophen
(50 .mu.M) 803 540 LPS (10 .mu.g/ml) Aspirin (50 .mu.M) 812 534 LPS
(10 .mu.g/ml) Ibuprofen (50 .mu.M) 821 501 LPS (10 .mu.g/ml)
Naproxen (50 .mu.M) 819 523 .sup.#Data are expressed as mean of
duplicates
[0362] Analgesics Inhibit Cytokine Responses of Normal Human
Bladder Cells to Inflammatory Stimuli--
[0363] Analysis of cells and culture supernatants after 24 hours of
culture showed that none of the analgesics tested alone induced
IL-6 or TNF.alpha. secretion in normal human bladder smooth muscle
cells. As shown in Tables 8 and 9, the doses of carbachol tested
induced low, but significant TNF.alpha. and IL-6 responses in
normal human bladder smooth muscle cells. On the other hand, LPS
treatment resulted in massive induction of these proinflammatory
cytokines. Acetaminophen, aspirin, ibuprofen, and naproxen suppress
the effect of carbachol and LPS on TNF.alpha. and IL-6 responses.
The suppressive effect of the analgesics on LPS-induced responses
was seen when these drugs were tested at either 5 .mu.M or 50
.mu.M.
TABLE-US-00008 TABLE 8 TNF.alpha. secretion by normal human bladder
smooth muscle cells after in vitro stimulation with inflammatory
and non-inflammatory stimuli and treatment with analgesic
TNF.alpha. (pg/ml).sup.# TNF.alpha. (pg/ml) Stimuli Analgesic
Subject 1 Subject 2 None None <5 <5 Carbachol 10.sup.-3M None
350 286 Carbachol 10.sup.-3M Acetaminophen (50 .mu.M) 138 164
Carbachol 10.sup.-3M Aspirin (50 .mu.M) 110 142 Carbachol
10.sup.-3M Ibuprofen (50 .mu.M) 146 121 Carbachol 10.sup.-3M
Naproxen (50 .mu.M) 129 137 LPS (10 .mu.g/ml) None 5725 4107 LPS
(10 .mu.g/ml) Acetaminophen (5 .mu.M) 2338 2267 LPS (10 .mu.g/ml)
Aspirin (5 .mu.M) 2479 2187 LPS (10 .mu.g/ml) Ibuprofen (5 .mu.M)
2733 2288 LPS (10 .mu.g/ml) Naproxen (5 .mu.M 2591 2215 LPS (10
.mu.g/ml) Acetaminophen (50 .mu.M) 2184 2056 LPS (10 .mu.g/ml)
Aspirin (50 .mu.M) 2266 2089 LPS (10 .mu.g/ml) Ibuprofen (50 .mu.M)
2603 1997 LPS (10 .mu.g/ml) Naproxen (50 .mu.M) 2427 2192
.sup.#Data are expressed as mean of duplicates.
TABLE-US-00009 TABLE 9 IL-6 secretion by normal human bladder
smooth muscle cells after in vitro stimulation with inflammatory
and non-inflammatory stimuli and treatment with analgesic IL-6 IL-6
(pg/ml).sup.# (pg/ml) Stimulus Analgesic Subject 1 Subject 2 None
None <5 <5 Carbachol 10.sup.-3M None 232 278 Carbachol
10.sup.-3M Acetaminophen (50 .mu.M) 119 135 Carbachol 10.sup.-3M
Aspirin (50 .mu.M) 95 146 Carbachol 10.sup.-3M Ibuprofen (50 .mu.M)
107 118 Carbachol 10.sup.-3M Naproxen (50 .mu.M) 114 127 LPS (10
.mu.g/ml) None 4838 4383 LPS (10 .mu.g/ml) Acetaminophen (5 .mu.M)
2012 2308 LPS (10 .mu.g/ml) Aspirin (5 .mu.M) 2199 2089 LPS (10
.mu.g/ml) Ibuprofen (5 .mu.M) 2063 2173 LPS (10 .mu.g/ml) Naproxen
(5 .mu.M 2077 2229 LPS (10 .mu.g/ml) Acetaminophen (50 .mu.M) 2018
1983 LPS (10 .mu.g/ml) Aspirin (50 .mu.M) 1987 2010 LPS (10
.mu.g/ml) Ibuprofen (50 .mu.M) 2021 1991 LPS (10 .mu.g/ml) Naproxen
(50 .mu.M) 2102 2028 .sup.#Data are expressed as mean of
duplicates
[0364] Primary normal human bladder smooth muscle cells were
isolated, cultured and evaluated for their responses to analgesics
in the presence of non-inflammatory (carbachol) and inflammatory
(LPS) stimuli. The goal of this study was to determine whether or
not normal human bladder smooth muscle cells recapitulate the
observations previously made with murine bladder cells.
[0365] The above-described experiment will be repeated with
analgesic agents and/or antimuscarinic agents in delayed-release,
or extended-release formulation or delayed-and-extended-release
formulations.
[0366] The above description is for the purpose of teaching a
person of ordinary skill in the art how to practice the present
invention, and it is not intended to detail all those obvious
modifications and variations of it which will become apparent to
the skilled worker upon reading the description. It is intended,
however, that all such obvious modifications and variations be
included within the scope of the present invention, which is
defined by the following claims. The claims are intended to cover
the claimed components and steps in any sequence which is effective
to meet the objectives there intended, unless the context
specifically indicates the contrary.
* * * * *