NZ732149B2 - Extended-release formulation for reducing the frequency of urination and method of use thereof - Google Patents

Abstract

Treating overactive bladder syndrome, using a dosage form comprising a immediate-release component and a extended-release component, each comprising acetaminophen and ibuprofen, each in an amount of 5 mg to 2000 mg in each component. The extended-release component may comprise an insoluble matrix, a polymer controlling release by dissolution controlled release, a water soluble or water-swellable matrix-forming polymer or a enteric coating. polymer controlling release by dissolution controlled release, a water soluble or water-swellable matrix-forming polymer or a enteric coating.

Description

TITLE EXTENDED-RELEASE FORMULATION FOR REDUCING THE FREQUENCY OF URINATION AND METHOD OF USE THEREOF This application is a divisional application ofNew Zealand Patent Application No. 721818. This application claims the priority of US. Patent Application Serial No. 13/487,348, filed on June 4, 2012, US. Patent Application Serial No. 13/424,000, filed on March 19, 2012, and US. Patent ation Serial No. 13/343,332, filed on January 4, 2012. The entirety ofthe aforementioned ations is incorporated herein by reference.

FIELD The present application generally relates to methods and compositions for inhibiting the contraction of muscles and, in particular, to methods and itions for inhibiting the contraction of smooth muscles ofthe urinary bladder.

OUND The dctrusor muscle is a layer ofthe urinary r wall made of smooth muscle fibers arranged in , longitudinal, and circular bundles. When the bladder is stretched, this signals the parasympathetic nervous system to contract the or muscle.

This encourages the bladder to expel urine through the urethra.

For the urine to exit the bladder, both the autonomically controlled internal Sphincter and the voluntarily controlled al sphincter must be opened. Problems with these muscles can lead to incontinence. Ifthe amount of urine reaches 100% ofthe urinary bladder's absolute capacity, the voluntary sphincter becomes involuntary and the urine will be ejected instantly.

The human adult urinary bladder usually holds about 0 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 ed by folding of the wall ofthe bladder (rugae) flatten and the wall ofthe bladder thins as it stretches, allowing the bladder to store larger amounts of urine without a significant rise in internal pressure. 1n 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, ifdesired, 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 s elming, and the subject will no longer be able to ignore it. In some 9075662 1 OHMnnels) 1397099 NZ 2 individuals, this desire to e starts when the r is less than 100% full in relation to its working volume. Such sed desire to urinate may ere 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 duals, both male and , who are not affected by r medical condition.

Accordingly, there exists a need for COmpositions and methods for the treatment of male and female subjects who suffer from a desire to urinate when the r is less than 100% full of urine in relation to its working volume. Said compositions and methods are needed for the inhibition of muscle contraction in order to allow in said subjects the desire to urinate to start when the volume of urine in the bladder exceeds around 100% of its working volume.

SUMMARY [0007A] In one aspect of the present invention, there is provided use of a pharmaceutical composition comprising a first component formulated for immediate-release and a second component ated for extended-release in the cture of a ment for overactive bladder syndrome in a subject in need thereof, wherein the first component and the second component each ses acetaminophen and ibuprofen, and wherein each of the acetaminophen and ibuprofen in the first and second components is present in an amount of 5 mg to 2000 mg.

One aspect of the t application s to a method for reducing the frequency of urination. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a first analgesic agent selected from the group consisting of aspirin, ibuprofen, naproxen sodium, indomethacin, nabumetone, and acetaminophen, wherein the pharmaceutical composition is formulated in an extended—release formulation and wherein said first analgesic agent is administered orally at a daily dose of 5 mg to 2000 mg. The method can be used for the treatment of nocturia.

Another aspect of the present application relates to a method for reducing the frequency of urination. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising: a first component formulated for immediate-release; and a second component formulated for extended-release, wherein the first component and the second component each comprises one or more analgesic agent selected from the group consisting of aspirin, ibuprofen, naproxen sodium, thacin, nabumetone, and acetaminophen, and wherein each of the first component and said second component is administered orally at a daily dose of5 mg to 2000 mg. The method can be used for the treatment of nocturia.

Another aspect ofthe t application relates to a pharmaceutical composition comprising: one or more analgesic agents selected from the group consisting of aspirin, ibuprofen, naproxen sodium, indomethacin, nabumetone, and acetaminophen; one or more antidiuretic agents, one or more antimuscarinic agents and/or pone or more 5_l [GHMaflm) P97099 NZ 2 spasmolytics; and a pharmaceutically acceptable carrier, n the pharmaceutical ition is formulated for extended-release.

BRIEF DESCRIPTION OF DRAWINGS Figure 1A and 18 are diagrams showing that analgesics regulate expression of eo-stimulatory molecules by Raw 264 macrophage cells in the absence (Figure 1A) or presence e 18) ofLPS. Cells were cultures for 24 hrs in the ce of analgesic alone or together with ella typhimurium LPS (0.05 pig/ml). Results are mean relative % of CD40+CD80+ cells.

ED DESCRIPTION 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 gh understanding ofthc t 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 le scope consistent with the principles and features disclosed herein.

As used herein, the term “effective amount” means an amount necessary to achieve a selected result.

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, the following substances: non-steroidal anti-inflammatory drugs (NSAIDS), salicylates, aspirin, salicylic acid, methyl salicylate, isal, salsalate, olsalazine, sulfasalazinc, para- aminophenol derivatives, acctanilide, acetaminophen, phenacetin, fenamates, mefenamic acid, mcclofenamate, sodium meclofenamate, heteroaryl acetic acid derivatives, tolmetin, ketorolac, diclofcnac, propionic acid derivatives, fen, naproxen , naproxen, fetiOprofen, ofcn, flurbiprofen, oxaprozin; enolic acids, oxicam derivatives, piroxieam, meloxicam, tenoxicam, ampiroxicam, droxicam, pivoxicam, lon derivatives, phenylbutazone, oxyphenbutazone, antipyrine, aminopyrine, dipyrone, coxibs, ccleeoxib, rofecoxib, nabumetone, apazone, thaein, sulindac, etodolac, isobutylphenyl pr0pionic acid, lumiracoxib, etorieoxib, parecoxib, oxib, tiracoxib, ctodolac, elone, dcxketoprofen, aeeclofenac, licofelone, bromfenac, lox0profen, pranoprofen, piroxicam, 9075662_l (GHMufleu) PB7085 NZ 2 nimesulide, cizolirine, 3-formylaminomethylsulfonylaminophenoxy~4H-l -benzopyran- 4-one, meloxicam, lomoxicam, d-indobufen, mofezolac, amtolmetin, pranoprofen, tolfenamic acid, flurbiprofen, suprofen, oxaprozin, rofcn, almin0profen, tiaprofenie acid, pharmacological salts thereof, hydrates thereof, and solvates thereof.

As used herein, the terms “eoxib” and "COX inhibitor” refer to a composition of compounds that is capable ofinhibiting the activity or expression of COX2 enzymes or is capable ofinhibiting or reducing the severity, including pain and swelling, ofa severe inflammatory response.

The urinary bladder has two important ons: storage of urine and emptying. Storage of urine occurs at low pressure, which implies that the detrusor muscle relaxes during the filling phase. ng ofthc r requires a coordinated contraction of the detrusor muscle and tion ofthc sphincter muscles ofthc urethra. Disturbances of the storage function may result in lower urinary tract symptoms, such as urgency, ncy, and urge incontinence, the components of the overactive bladder syndrome. The overactive bladder syndrome, which may be due to involuntary contractions ofthc smooth muscle of the bladder (detrusor) during the storage phase, is a common and underreported problem, the prevalence of which has only ly been assessed.

One aspect ofthe t application relates to a method for ng the frequency of urination by administering to a person in need thereofa pharmaceutical composition formulated in an extended—release ation. The ceutical composition ses one or more analgesic agents and, optionally, one or more antimuscarinie agents,one or more antidiuretic agents, and/or one or more spasmolytics. . The method can be used for the treatment of nocturia.

“Extended-release,” also known as sustained-release (SR), sustained-action (SA), time-release (TR), controlled—release (CR), modified release (MR), or continuous- release (CR), is a mechanism used in medicine s or capsules to dissolve slowly and release the active ingredient overtime. 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 ofthc drug action. For example, an extendedvrelease analgesic may allow a person to sleep through the night without getting up for the bathroom.

In one embodiment, the pharmaceutical composition is ated for extended-release by embedding the active ingredient in a matrix of insoluble substance(s) such as acrylics or chitin. An extended-release form is designed to e the sic compound 9075652_1 [GHMBHCIM PD7099 NZ.2 at a predetermined rate by maintaining a constant drug level for a specific period oftimc.

This can be achieved through a variety of formulations, including, but not limited to liposomes and olymer conjugates, such as hydrogels.

An extended-release formulation can be designed to release the active agents at a ermined rate so as to in a constant drug level for a specified, extended period , such as up to about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour following administration or following a lag period associated with delayed-release ofthe drug.

In certain preferred embodiments, the active agents are released over a time interval of between about 2 to about 10 hours. Alternatively, the active agents may be ed over about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 hours.

In yet other embodiments, the active agents are released over a time period between about three to about eight hours following administration.

In some embodiments, the extended-release formulation comprises an active core comprised of one or more inert particles, each in the form ofa bead, , pill, ar particle, microcapsule, microsphere, ranule, 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 ed by granulating and milling and/or by extrusion and spheronization ofa polymer ition containing the drug substance.

The active agents 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 ofdrug 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 ofthe 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 m carbonate, sodium bicarbonate, fumarie acid, tartaric acid, etc. which alters the microenvironment ofthe drug to facilitate its e. 9075662¢l [GHMIneu) P97099NZ 2 Extended-release formulations may utilize a variety ofextended-release coatings or mechanisms facilitating the gradual release of active agents over time. In some embodiments, the extended-release agent comprises a r controlling release by dissolution controlled release. In a particular embodiment, the active agent(s) are incorporated in a matrix comprising an insoluble polymer and drug particles or granules coated with polymeric als of varying thickness. The polymeric material may comprise a lipid r comprising a waxy material, such as carnauba wax, beeswax, spemtaceti wax, candellila wax, shallac wax, cocoa butter, cetostearyl alcohol, partially hydrogenated vegetable oils, ceresin, paraffin wax, ceresine, myristyl l, l l, 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 fies 0r erodes after a predetermined lag-time depending on the thickness ofthe polymer coating.

The lag time is independent of gastrointestinal motility, pH, or c residence.

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 pI-I-independent polymers.

In one embodiment, the extended—release ation comprises a water soluble or water-swellablc matrix-forming polymer, optionally containing one or more solubility-enhancing exeipients and/or release-promoting agents. Upon lization ofthe water soluble polymer, the active agent(s) dissolve (if soluble) and lly e through the hydrated portion ofthe matrix. The gel layer grows with time as more water permeates into the core ofthe 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 ity ofthe gel layer, leading to disentanglement and erosion ofthe outer hydrated polymer on the surface ofthe .

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 usion and n mechanisms, erosion is the predominant mechanism for insoluble drugs, regardless of dose. rly, 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 ers. The initial matrix polymer hydration phase results in slow—release ofthe drug (lag phase). Once the 90756611 (GHMultm' P570” NZ 2 water ble 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.

Additionally, the porosity of the matrix can be increased due to the leaching out of cndent 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 ofdrug diffusion h 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: drug ratio; filler type and level; polymer to filler ratio; particle size of drug and polymer; and porosity and shape of the matrix.

Exemplary hydrophilie and/or water-sweliable, 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), methyleellulose (MC), carboxymethyleellulose (CMC), powdered cellulose such as rystalline cellulose, cellulose acetate, ethylcellulose, salts thereof, and combinations thereof; alginates, gums, including heteropolysaccharide gums and homopolysaccharide gums, such as n, anth, pectin, acacia, karaya, alginates, agar, guar, hydroxypropyl guar, veegum, carrageenan, locust bean gum, gellan gum, and derivatives thereofrom; acrylic resins, including polymers and mers ofaerylie acid, methacrylic acid, methyl acrylate and methyl methacrylate and linked polyacrylic acid derivatives such as ers (e.g., CARBOPOL®, such as including CARBOPOL® 7lG NF, available in various molecular weight grades from Noveon, lne., nati, OH); caragccnan; polyvinyl acetate (e.g., KOLLIDON® SR); polyvinyl pyrrolidone and its derivatives such as crospovidone; polyethylene oxides; and polyvinyl alcohol. red hydrophilic and water-swellable rs include the cellulosic polymers, especially HPMC.

The extended-release formulation may fiirther comprise at least one binder that is e of cross-linking the hydrophilie compound to form a hilic polymer matrix (116., a gel matrix) in an aqueous medium, including biological fluids.

Exemplary binders include homopolysaeeharides, such as galactomannan gums, guar gum, hydroxypropyl guar gum, hydroxypropylccllulose (HPC; e.g., Klueel EXF) and locust bean gum. In other embodiments, the binder is an e 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. 9075662_1 (<3 HMonm) P970913 :42 2 In one embodiment, the introduction method is drug layering by spraying a suspension of active agent(s) and a binder onto the inert carrier.

The binder may be present in the bead formulation in an amount of from about 0.1% to about 15% by weight, and preferably of from about 0.2% to about 10% by weight.

In some embodiments, the hydrophilie 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 ofthe active agent(s). This may additionally suppress the initial burst effect and produce a more , “zero order release” of active agent(s). ary ionic polymers for slowing dissolution rate include both anionic and cationic polymers. Exemplary anionic polymers include, for example, sodium ymethyleellulose (Na CMC), sodium alginate, polymers of acrylic acid or carbomers (e.g., CARBOPOL® 934, 940, 974P NF); enteric polymers, such as polyvinyl acetate phthalate (PVAP), methacrylic acid mers (e.g., EUDRAGIT® LlOO, L 30D 55, A, and FS 30D), hypromellose acetate succinate (AQUAT HPMCAS); and xanthan gum.

Exemplary cationic rs include, for example, dimethylaminoethyl methacrylate copolymer (e.g., EUDRAGIT® E 100). Incorporation of anionic rs, particularly enteric polymers, is useful for dcchOping a pI-l-independcnt release profile for weakly basic drugs as compared to hydrophilie polymer alone. ary nic polymers for slowing dissolution rate, include, for example, hydroxypropylcellulosc (HPC) and polyethylene oxide (PEO) (e.g., POLYOXTM) Exemplary hydrophobic polymers e ethylcellulose (e.g., ETHOCELTM, SURELEASE®), cellulose acetate, methacrylic acid copolymers (e.g., EUDRAGIT® NE 30D), o—methacrylate copolymers (e.g., EUDRAGIT® RL 100 or PO RS 100), polyvinyl e, glyceryl monostearate, fatty acids, such as acctyl tributyl citrate, and combinations and derivatives f.

The swellablc polymer can be incorporated in the formulation in tion 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 rs can also be diSpersed in organic solvents or hydro-alcohols and Sprayed during granulation Exemplary release-promoting agents include endent enteric polymers that remain intact at pH value lower than about 4.0 and dissolve at pH values higher than 4.0, 9075662 I (GHMInamJ P970WNZ 2 preferably higher than 5.0, most preferably about 6.0, are considered useful as release- promoting agents for this invention. Exemplary pH-dependent polymers include, but are not limited to, arylic acid copolymers, methacrylic acid-methyl methacrylate copolymers (e.g., EUDRAGIT® L100 (Type A), EUDRAGIT® 8100 (Type B), Rohm Gmbl—l, y; rylic acid-ethyl acrylate copolymers (e.g., EUDRAGIT“ 5 (Type C) and EUDRAGIT® L30D-55 copolymer diSpcrsion, Rohm GmbH, Germany); copolymers of methacrylic acid-methyl methacrylate and methyl methacrylate (EUDRAGIT® FS); tcrpolymers of methacrylic acid, rylate, and ethyl acrylate; cellulose acetate ates (CAP); hydroxypropyl cellulose phtlialate (HPMCP) (e.g., HP—SS, HP-SO, HP-SSS, Shinetsu Chemical, Japan); polyvinyl acetate phthalates (PVAP) (e.g., COATERIC®, OPADRY® enteric white OY-P-717l); polyvinylbutyrate acetate; cellulose acetate succinates (CAS); hydroxypropyl methyleellulose acetate succinate (HPMCAS), e.g., HPMCAS LF Grade, MF Grade, HF Grade, including AQOAT® LF and AQOAT® MF (Shin-Etsu Chemical, Japan); Shinetsu Chemical, Japan); shellac (e.g., MARCOATrM 125 & MARCOATTM 125N); vinyl acetate—maleic anhydride eopolymer; styrene-maleic monoester copolymer; ymethyl ethyleellulosc (CMEC, Freund Corporation, Japan); cellulose acetate phthalates (CAP) (e.g., AQUATERIC®); ose acetate litates (CAT); and mixtures oftwo or more thereof at weight ratios between about 2:1 to about 5:1, such as, for instance, a mixture or EUDRAG1T® L 100—55 and EUDRAGIT® s 100 at a weight ratio or about 3:1 to about 2:1, or a mixture of EUDRAGIT® L 30 D-55 and EUDRAGIT® FS at a weight ratio of about 3:1 to about 5:1.

These polymers may be used either alone or in combination, or together with polymers other than those mentioned above. Preferred enterie pH-dependent rs 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 135,000. A ratio of free carboxyl groups to methyl-esterificd carboxyl groups in these copolymers may range, for example, from 1:1 to 1:3, ag. around 1:1 or 1 :2. Such polymers are sold under the trade name Eudragit® such as the it L series e,g,, Eudragit L 12.5®, Eudragit L 12.5P®, Eudragit L100®, Eudragit L 100—55®, Eudragit L- 30D®, Eudragit L-30 D-55®, the Eudragit S® series e.g., it S 12.5®, Eudragit S , Eudragit S 100®. The release promoters are not limited to pH dependent polymers. Other hydrophilie molecules that dissolve rapidly and leach out ofthe dosage form quickly leaving a porous structure can be also be used for the same purpose.

The release—promoting agent can be incorporated in an amount from 10% to 9075662-! [GHMIflcrfl P97099 NZ 2 90%, preferably from 20% to 80% and most preferably from 30% to 70% by weight ofthe dosage unit. The agent can be incorporated into the formulation either prior to or after granulation, The release-promoting agent can be added into the formulation either as a dry material, or it can be sed or dissolved in an appropriate solvent, and dispersed during granulation.

In some embodiments, the matrix may include a combination of release promoters and solubility enhancers. The solubility enhancers can be ionic and non-ionic surfactants, complexing , hydrophilic polymers, pH modifiers, such as acidifying agents and alkalinizing agents, as well as molecules that increase the solubility of poorly soluble drug through lar entrapment. Several lity ers can be utilized aneously.

Solubility enhancers may include surface active agents, such as sodium docusate, sodium lauryl sulfate, sodium stearyl fumarate, Twecns® and Spans (PEO modified sorbitan ters and fatty acid sorbitan esters), poly(ethylene oxide)-polypropylene oxide-poly(ethy|ene oxide) block copolymers (aka PLURONICSTM); xing agents such as low lar weight polyvinyl pyrrolidone and low molecular weight hydroxypropyl methyl ose; 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.

Solubility enhancing agents typically constitute from 1% to 80% by weight, preferably from 1% to 60%, more preferably from 1% to 50%, ofthe 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 ofthe materials are granulated or otherwise processed. During granulation, lizers can be sprayed as solutions with or without a binder.

In some embodiments, the extended-release ation comprises a polymeric matrix that can provide for release ofthe drug after a certain time, independent of the pH. For purposes ofthe present invention, “pl-l independent" is defined as having characteristics (e.g., dissolution) which are substantially unaffected by pH. pH independent polymers are often referred to in the context of“time-controlled” or “time-dependent” e profiles.

A pH independent polymer may be used to coat the active agent and/or provide a polymer for a hydrophilic matrix in the extended-release coating ver. The 9075562_1 (G HManen) F97U99.NZ 2 pH independent polymer may be water—insoluble or water soluble. Exemplary water insoluble pH independent polymers include, but are not d to, l methaerylie acid esters with a small ponion oftrimethylammonioethyl methacrylate chloride (e.g., EUDRAGIT® RS and EUDRAGIT® RL; neutral ester sions without any functional groups (e.g., EUDRAGIT® NE30D and EUDRAGIT® NE30); cellulosie polymers, such as cthylcellulose, hydroxyl ethyl cellulose, cellulose acetate or mixtures and other pl-I independent coating products. Exemplary water e pH independent polymers include liydroxyalkyl ose ethers, such as hydroxypropyl methyleellulose (HPMC), and hydroxypropyl cellulose (HPC); polyvinylpyrrolidone (PVP), methylcellulose, OPADRY®amb, guar gum, xanthan gum, gum arabic, ycthyl cellulose and ethyl acrylate and methyl methacrylate cepolymer dispersion or combinations thereof.

In one embodiment, the extended-release formulation comprises a water- insoluble water-permeable polymeric coating or matrix comprising one or more water- ble water-permeable film-forming over the active core. The coating may additionally include one or more water soluble rs and/or one or more plasticizers. The water- insoluble polymer coating comprises a barrier coating for e ofactive agents in the core, wherein lower molecular weight (viscosity) grades exhibit faster release rates as ed to higher viscosity grades.

In preferred embodiments, the water~insoluble film-forming polymers include one or more alkyl cellulose , such as ethyl celluloscs and mixtures thereof, (e.g., ethyl cellulose grades PRlOO, PR45, PRZO, PRIO and PR7; ETHOCEL®, Dow).

An exemplary water-soluble polymer such as polyvinylpyrrolidone (POVIDONE®), hydroxypropyl metliylcellulose, hydroxypropyl cellulose and mixtures thereof.

In some embodiments, the water-insoluble r provides suitable properties (eg, ed-release characteristics, mechanical ties, 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.

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 ofthe water-insoluble polymer, and the ultimate desired ties ofthe coating.

Suitable levels ofplasticizer range from about 1% to about 20%, from about 3% to about 9075862, I ers) PWUWANZ 2 %, 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 ofthe coating, inclusive of all ranges and sub-ranges therebetween.

Exemplary plasticizers e, but are not limited to, triacetin, acetylated monoglyceridc, oils (castor oil, hydrogenated castor oil, rape seed oil, sesame oil, olive oil, etc.); citrate esters, tricthyl citrate, acetyltricthyl citrate acetyltributyl e, tributyl citrate, acetyl tri—n-butyl citrate, diethyl phthalate, dibutyl ate, dioctyl phthalate, methyl paraben, propyl paraben, propyl paraben, butyl paraben, diethyl sebacate, l sebacate, glyceroltributyrate, substituted triglycerides and glycerides, monoacetylated and diacetylated glycerides (eg, MYVACET® 9-45), glyceryl monostcaratc, glycerol tributyrate, polysorbatc 80, polyethyleneglycol (such as PEG-4000, PEG-400), propyleneglycol, 1,2-propyleneglycol, glycerin, sorbitol, diethyl oxalate, diethyl malate, diethyl fumarate, diethylmalonatc, dibutyl succinatc, fatty acids, glycerin, sorbitol, diethyl oxalate, diethyl malate, l malcatc, diethyl fumarate, diethyl succinatc, l malonate, dioctyl phthalate, dibutyl sebaeate, and mixtures thereof. The plasticizer can have surfactant properties, such that it can act as a release modifier. For e, non-ionic ents such at Brij 5 8 (polyoxyethylene (20) cctyl ether), and the like, can be used.

Plasticizers can be high g point organic solvents used to impart flexibility to otherwise hard or brittle polymeric als 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 ing in various changes in polymer properties including a reduction in tensile strength, and increase in elongation and a reduction in the glass transition or softening temperature ofthe polymer. The amount and choice ofthe plasticizer can affect the hardness of a tablet, for example, and can even affect its dissolution or disintegration teristics, as well as its physical and chemical stability. Certain plasticizers can increase the elasticity and/or lity ofa coat, thereby decreasing the coat‘s brittleness.

In another embodiment, the extended-release formulation comprises a combination of at least two geLforming polymers, including at least one non-ionic gel- g polymer and/or at least one anionic rming polymer. The gel formed by the combination of gel-forming polymers es 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. Because ofthe high viscosity, the 9075662 1 [6HMarteu) F97059,N7_2 s 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 ation of at least two gel-forming polymers enables preperties of the resultant gel, such as viscosity, to be manipulated in order to provide the desired release profile.

In a particular embodiment, the formulation comprises at least one non-ionic gel—forming polymer and at least one anionic gel-fonning 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 of the same chemistry, but having different solubilities, viscosities, and/or molecular weights (for example a combination of hydroxyproplyl methylccllulosc of different viscosity , such as l-IPMC K100 and HPMC KlSM or HPMC KlOOM).

Exemplary anionic gel forming polymers include, but are not limited to, sodium carboxymethyicellulose (Na CMC), carboxymethyl cellulose (CMC), anionic ccharides such as sodium te, c acid, pectin, polyglucuronic acid (poly-0t- and -[5-l,4-glucuronic acid), polygalacturonic acid c acid), chondroitin sulfate, carrageenan, furcellaran, anionic gums such as xanthan gum, polymers of acrylic acid or carbomers pol® 934, 940, 974? NF), Carbopol® copolymers, a Pemulen® polymer, polycarbophil, and others.

Exemplary non-ionic gel-forming polymers include, but are not limited to, Povidone (PVP: polyvinyl pyrrolidonc), polyvinyl alcohol, mer of PVP and polyvinyl acetate, HPC (hydroxypropyl cellulose), HPMC (hydroxypropyl methylccllulose), hydroxycthyl cellulose, hydroxymethyl cellulose, n, polyethylene oxide, acacia, dextrin, starch, po]yhydroxyethylmethacrylate (PHEMA), water soluble nonionie polymethacrylates and their copolymers, d cellulose, modified polysaccharides, nonionie gums, nonionie polysaccharides and/or mixtures thereof.

The ation may optionally comprise an enteric polymer as described above, and/or at least one cxcipient, such as a filler, a binder (as described above), a disintegrant, and/or a flow aid or t.

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, t, erythritol, and hydrogenated starch hydrolysatcs (a blend of several sugar alcohols), corn , potato starch, sodium ymethycellulosc, ethylccllulose and cellulose acetate, c polymers, or a e thereof. 9075662_1 (G HMnn an) P971199 NZ 2 Exemplary binders, include but are not limited to, water-soluble hilic polymers, such as Povidone (PVP: polyvinyl pyrrolidone), copovidone (a copolymer of polyvinyl pyrrolidone and polyvinyl e), low molecular weight HPC (hydroxypropyl cellulose) low molecular weight HPMC (hydroxypropyl methylcellulose), low molecular weight carboxy methyl cellulose, ethylcellulose, gelatin, polyethylene oxide, acacia, dextrin, ium aluminum silicate, starch, and polymethacrylates such as Eudragit NE 30D, Eudragit RL, Eudragit RS, Eudragit E, polyvinyl e, and cntcrie polymers, or mixtures thereof.

Exemplary disintegrants include but are not limited to bstituted carboxymethyl cellulose sodium, crospovidone -linked polyvinyl idone), sodium carboxymethyl starch (sodium starch glycolate), cross-linked sodium carboxymethyl cellulose (Croscarmcllose), pregelatinized starch (starch 1500), microcrystalline cellulose, water insoluble starch, calcium carboxymethyl cellulose, low substituted hydroxypropyl cellulose, and magnesium or aluminum silicate.

Exemplary glidants include but are not limited to, magnesium, silicon dioxide, talc, starch, titanium dioxide, and the like.

In yet another embodiment, the extended-release formulation is formed by coating 21 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 ed from a group comprising osic polymers, such as ellulose (e.g., ASE®), inethylcellulose, hydroxypropyl ose, hydroxyprOpylmethyl cellulose, cellulose acetate, and cellulose acetate phthalate; polyvinyl alcohol; acrylic polymers such as polyacrylates, polymethacrylates and e0polymers 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 ofthe release controlling coating, Such as the nature ofthe 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 ofthe , allows for changes in the release of active agent(s) from any given bead tion, thereby ng for selective adjustment ofthe formulation to a pro-determined release profile.

Pore formers suitable for use in the release controlling g herein can be organic or inorganic agents, and include materials that can be dissolved, extracted or leached from the g in the environment of use. Exemplary pore forming agents include, but are 9075662_l (GHMancfl) PD7099 NZ 2 not limited to, organic compounds such as mono-, oligo-, and polysaccharides including sucrose, e, fructose, mannitol, mannose, galactose, ol, pullulan, n; polymers soluble in the environment of use such as water-soluble hydrophilic polymers, hydroxyalkylcelluloses, carboxyalkylcelluloses, hydroxypropylmethylcellulose, cellulose ethers, acrylic , polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, polyethylene oxide, Carbowaxcs, Carbopol, and the like, diols, polyols, polyhydric alcohols, polyalkylene glycols, polyethylene glycols, polypropylene glycols, or block polymers thereof, polyglyeols, poly(a-Q)alkylenediols; inorganic compounds such as alkali metal salts, lithium ate, sodium chloride, sodium bromide, ium chloride, potassium sulfate, potassium phOSphate, sodium acetate, sodium citrate, suitable calcium salts, combination f, and the like.

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 les or beads may additionally include an "overcoat," to provide, e.g., moisture protection, static charge reduction, taste-masking, flavoring, coloring, and/or polish or other ic 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 hydroxypropylmethylccllulose, ypropylcellulose and microcrystalline cellulose, or combinations thereof(for example, various OPADRY® coating materials).

The coated particles or beads may additionally contain enhancers that may be exemplified by, but not d to, solubility enhancers, dissolution enhancers, absorption enhancers, permeability enhancers, stabilizers, complexing agents, enzyme inhibitors, p- glycoprotein inhibitors, and multidrug resistance protein inhibitors. Alternatively, the ation 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 e controlling coating.

In other embodiments, the extended-release formulation is formulated to e 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 orate 2, 3, 4, 5, or 6 push-pull units encapsulated within a hard gelatin e, 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 d through the membrane next to the drug layer. This membrane 9075662_1 (GHMlllerl) P97099.NZ 2 may be additionally covered with a endent enteric coating to prevent release until after gastric emptying. The gelatin capsule dissolves immediately after ingestion. As the push pull unit(s) enter the small intestine, the c coating breaks down, which then allows fluid to flow through the semi-permeable membrane, swelling the osmotic push compartment to force 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.

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, hilic vinyl and acrylic polymers, polysaccharides such as m alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), - yethyl methacrylate), poly(acrylic) acid, poly(methacrylic) acid, nylpyrrolidone (PVP), crosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers, PVA/PVP mcrs with hydrophobic monomers such as methyl rylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, sodium croscarmellosc, carrageenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) and carboxyethyl, cellulose (CBC), sodium algiuatc, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate.

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 de, sodium chloride, lithium chloride, potassium sulfate, potassium phosphates, sodium carbonate, sodium sulfite, lithium sulfate, ium chloride, and sodium sulfate; sugars, such as se, fructose, glucose, inositol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, trehalose, and l; organic acids, such as ic acid, benzoic acid, fumarie 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.

Materials 1.18le in g the semipermeable membrane include various grades of acrylics, , , polyamides, polyesters, and eellulosic derivatives that are permeable and water-insoluble at physiologically relevant pHs, or are susceptible to being rendered water-insoluble by chemical alteration, such as crosslinking. 9075662_1 (GHMaRerIJ PB7059,NZ 2 In some embodiments, the extended-release formulation may comprise 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 s breaking down, for example, the ccharide coatings to e the drug contents in a controlled, time-dependent manner. Exemplary polysaeeliaride coatings may include, for example, amylose, arabinogalaetan, chitosan, chondroitin sulfate, cyclodcxtrin, dcxtran, guar gum, pectin, xylan, and ations or derivatives therefrom.

In some embodiments, the pharmaceutical composition is formulated for delayed extended-release. As used herein, the term "delayed-release" refers to a medication that does not immediately disintegrate and release the active ingredient(s) into the body. In some embodiments, the term "delayed extended-release" is used with reference to a drug formulation having a release profile in which there is a predetermined delay in the release of the drug following stration. In some ments, 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 tion before it reaches the small intestine. d—release formulations, such as enteric coatings, prevent drugs having an irritant effect on the stomach, such as aspirin, from dissolving in the stomach. Such coatings are also used to protect acid-unstable drugs from the stomaeh'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 delaycd~releasc, which is used herein with reference to a drug formulation that es rapid and transient release ofthe drug within a short time period immediately after a predetermined lag period, thereby producing a d” plasma profile ofthe drug after drug administration. Formulations may be designed to provide a single pulsatile release or multiple pulsatile es at predetermined time intervals following stration.

A delayed-release or pulsatile release ation generally comprises one or more elements covered with a barrier coating, which dissolves, erodes or ruptures following a specified lag phase. In some embodiments, the ceutical composition ofthe t application is ated for extended-release or delayed extended-release and comprises 100% ofthe total dosage ofa given active agent administered in a single unit dose. In other embodiments, the ceutical composition comprises an extended/delayed-release component and an immediate-release component. In some embodiments, the immediaterelease component and the cxtended/delayed-release component contain the same active 9075662~l lGHMIflcII) P971399 NZ 2 ingredient. In other embodiments, the ate-release component and the extended/delayed-release component contain ent active ingredients (e.g., an analgesic in one ent and an antimuscarinic agent in r component). In some embodiments, the first and second components each contains an analgesic selected from the group ting ofaspirin, ibuprofen, naproxen sodium, indomethaein, nabumetone, and acetaminophen. In other embodiments, the extended/delayed-release component is coated with an cnteric coating. In other embodiments, the immediate-release component and/or the extended/delayed-release component further comprises an antimuscarinic agent selected from the group consisting of oxybutynin, solifenacin, darifenacin and atropine. In other embodiments, the analgesic agent in each component is administered orally at a daily dose of mg - 2000 mg, 20 mg - 1000 mg, 50 mg - 500 mg or 250-1000 mg. In other embodiments, the immediate-release component and/or the extended/delayed-releasc component further comprises an antidiuretie agent, an scarinic agent or both. In other embodiments, the treatment method includes administering to a t a diuretic at least 8 hours prior to a target time, such as bedtime, and administering to the subject the ceutical composition comprising the immediate-release component and/or the extended/delayed-rclcase component within 2 hours prior to the target time.

In other embodiments, the “immediate-release” component provide about 5- 50% ofthe total dosage ofthe active agent(s) and the “extended-release” component provides 50—95% ofthe total dosage ofthe active agent(s) to be delivered by the ceutical fomiulation. For example, the immediate-release component may provide about 20-40%, or about 20%, 25%, 30%, 35%, about 40%, ofthe total dosage ofthe active s) to be delivered by the pharmaceutical formulation. The extended-release component provides about 60%, 65%, 70%, 75% or 80% ofthe total dosage ofthe active agent(s) to be delivered by the formulation. In some embodiments, the extended—release component further comprises a r coating to delay the release ofthe active agent.

A barrier coating for delayed-release may consist ofa variety of different materials, depending on the objective. In addition, a formulation may comprise a plurality of barrier gs to facilitate release in a temporal manner, The coating may be a sugar coating, a film coating (cg, based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulosc, ypropylccllulose, carboxymethylcellulose, aerylate mers, polyethylene glycols and/or polyvinylpyrrolidone), or a g based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, nyl acetate phthalate, 9075662 1 -GHMaItmr) P97099142? shellac, and/or ellulose. Furthermore, the formulation may additionally include a time delay material such as, for example, glyceryl monostearate or glyceryl rate.

In some embodiments, the delayed, extended-release ation includes an enteric coating sed one or more polymers facilitating release of active agents in proximal or distal regions ofthe gastrointestinal tract. As used herein, the term “enteric polymer coating” is a coating comprising of one or more polymers having a pH dependent or pH—independent release profile. lly the coating resists dissolution in the acidic medium ofthe h, but dissolves or erodes in more distal regions ofthe gastrointestinal tract, such as the small ine or colon. An enteric polymer coating typically s releases ofthe active agents until some time after a gastric emptying lag period of about 3-4 hours after administration. pH ent enteric coatings comprises one or more pH-dependcnt or pH- scnsitive polymers that maintain their structural integrity at low pH, as in the h, but dissolve in higher pH environments in more distal regions ofthe gastrointestinal tract, such as the small intestine, where the drug ts are ed. For purposes ofthe present invention, “pH dependent” is defined as having teristics (eg., dissolution) which vary according to environmental pH. Exemplary pI-I-dependcnt polymers include, but are not limited to, methaearylic acid copolymers, methacrylie acid-methyl methacrylatc copolymers (erg, EUDRAGIT® L100 (Type A), EUDRAGIT® 8100 (Type B), Rohm GmbH, Germany; rylie acid-ethyl acrylate copolymers (cg, EUDRAGIT® LlOO-SS (Type C) and EUDRAGIT® L30D-SS copolymer dispersion, Rohm GmbH, Germany); copolymers of methacrylie acid-methyl methacrylatc and methyl methacrylatc (EUDRAGIT® FS); terpolymers of methacrylic acid, methacrylatc, and ethyl acrylate; cellulose acetate phthalates (CAP); hydroxypropyl methylccllulosc phthalate (HPMCP) (e.g., PIP—55, PIP-50, HP-SSS, Shinetsu Chemical, Japan); polyvinyl acetate phthalates (PVAP) (eg, COATERIC", ® enteric white OY-P-7 l 71); cellulose acetate succinates (CAS); hydroxypropyl methylcellulose acetate suecinatc (HPMCAS), e.g., HPMCAS LF Grade, MF Grade, HF Grade, including AQOAT® LF and AQOAT® MF (Shin-Etsu Chemical, Japan); Shinetsu Chemical, Japan); shellac (e.g., MarcoatTM 125 & MarcoatTM 125N); carboxymethyl cthylcellulose (CMEC, Freund Corporation, Japan), cellulose acetate phthalates (CAP) (erg, AQUATERIC®); cellulose acetate trimellitates (CAT); and es oftwo or more thereof at weight ratios between about 2:1 to about 5:1, such as, for instance, a mixture of EUDRAGIT® L 100—55 and EUDRAGIT® s 100 at a weight ratio of about 3:1 to about 2: 1, or a mixture ofEUDRAGIT® L 30 D-SS and IT® rs at a weight ratio of about 3:1 9075662_l (GHMnnais) P970913 N22 to about 5:1. 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 onS .0, of25.5, of26.0, of26.5, or on7.0.

In certain embodiment, the coating methodology employs the ng of one or more pH-dependent and one or more pH-independent polymers. The blending of pH- ent and pH—independent polymers can reduce the release rate of active ingredients once the soluble polymer has reached its m pH of solubilization.

In some embodiments, a controlled” or dependent" 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 itselfout ofthe capsule and releasing the drugs after a pre—determined lag time, which can be controlled by e.g., the position and dimensions ofthe plug. The e body may be further coated with an outer endent enterie g keeping the capsule intact until it reaches the small intestine. Suitable plug materials include, for example, polymethacrylates, lc compressed rs (e.g., HPMC, polyvinyl alcohol), led melted polymer (cg, yl mono oleate) and enzymatically controlled crodiblc rs (e.g., polysaccharides, such as amylose, arabinogalactan, chitosan, chondroitin sulfate, cyclodextrin, dcxtran, guar gum, pectin and xylan).

In other embodiments, capsules or bilayered tablets may be formulated to n 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 ofthe polymer coating and the ng behavior ofthe 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.

Exemplary water swellable materials to be used in the delayedvrelease coating include, but are not limited to, polyethylene oxide (having e.g., an average molecular weight between 1,000,000 to 7,000,000, such as POLYOX®), methylcellulosc, hydroxypropyl cellulose, hydroxypropyl methylcellulose; polyalkylene oxides having a weight average molecular weight of 100,000 to 6,000,000, ing but not limited to poly(methylcne oxide), poly(butylcnc oxide); poly(hydroxy alkyl methacrylate) having a molecular weight of 9075662 1 (GHManais) P87099 NZ 2 from 25,000 to 5,000,000; poly(vinyl)alcohol, having a low acetal residue, which is cross- linked with glyoxal, dehyde or glutaraldehyde and having a degree of polymerization of from 200 to 30,000; mixtures of methyl cellulose, cross—linked agar and carboxymethyl cellulose; hydrogel forming copolymers produced by forming a dispersion ofa 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® acidic carboxy polymers having a molecular weight of 0 to 4,000,000; CYANAMER® polyacrylamides; cross-linked water swellablc indenemaleicanhydride polymers; GOODRITE® rylic acid having a molecular weight of 80,000 to 200,000; starch graft copolymers; AQUA-KEEPS® acrylatc polymer ccharides ed of condensed glucose units such as diester cross-linked poiyglucan; carbomers having a ity of 3,000 to 60,000 mPa as a % w/v aqueous solution; cellulose others such as hydroxypropylccllulose having a viscosity of about 1000- 7000 mPa s as a 1% w/w aqueous solution (25° C); ypropyl methylecllulose having a viscosity of about 1000 or higher, preferably 2,500 or higher to a maximum of 25,000 mPa as a 2% w/v aqueous solution; polyvinylpyrrolidone having a viscosity of about 300-700 mPa s as a 10% w/v aqueous solution at 20° C; and combinations thereof.

Alternatively, the release time of the drugs can be controlled by a disintegration lag time depending on the balance between the tolerability and thickness ofa water insoluble polymer membrane (such as ethyl cellulose, EC) containing predefined micropores at the bottom ofthe body and the amount ofa swellable ent, such as low substituted hydroxypropyl cellulose (L-HPC) and sodium glycolate. After oral administration, GI fluids permeate through the micropores, causing swelling ofthe swellable ents, which produces an inner re disengaging the capsular components, including a first capsule body ning the swellable als, a second capsule body containing the drugs, and an outer cap attached to the first capsule body.

The enteric layer may further comprise anti-tackincss agents, such as talc or glyceryl monostearate and/or plasticizers. The enteric layer may further comprise one or more plasticizers including, but not d to, yl citrate, acetyl triethyl citrate, acetyltributyl citrate, polyethylene glycol acetylated ycerides, glycerin, triacetin, propylene glycol, ate esters (e.g., diethyl phthalate, dibutyl phthalate), titanium dioxide, ferric oxides, castor oil, sorbitol and dibutyl sebacate. in another embodiment, the delay release fomiulation employs a water- permeablc but ble film coating to enclose the active ingredient and an osmotic agent. 9075652_1 (GHMlttcu) P971399 NZ 2 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 ed to permit various rates ofwater permeation or release time.

In r embodiment, the delay release formulation employs a water- impermeable tablet coating whereby water enters through a controlled re in the coating until the core bursts. When the tablet bursts, the drug contents are ed immediately or over a longer period oftime. These and other ques may be modified to allow for a pre- determined lag period before release ofdrugs is initiated.

In another embodiment, the active agents are delivered in a formulation to provide both delayed-release and extended-release ed-sustained). 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 ed-release ofthc active agents thereafter.

In some embodiments, immediate-release, extended-release, d—release, or dclayed-extended-release formulations comprises an active core comprised of one or more inert particles, each in the form ofa bead, pellet, pill, granular particle, microcapsule, microspliere, microgranulc, nanocapsule, or nanosphere coated on its surfaces with drugs in the form ofe.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 ed by granulating and milling and/or by extrusion and spheronization ofa polymer composition containing the drug substance.

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 g on the active core will be from about I to 50% based on the weight 0fthe 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 ment, the inactive core may be a sugar sphere or a buffer crystal or an encapsulated buffer l such as calcium carbonate, sodium bicarbonate, fumaric acid, tartaric acid, etc. which alters the microcnvironment oftlie drug to facilitate its e.

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 ofa water insoluble polymer and an enteric polymer, 9075M2 1 tGHMallcrs) P970” NZ 2 wherein the water ble polymer and the enteric polymer may be present at a weight ratio of from 4:1 to 1:1, and the total weight ofthe coatings is 10 to 60 weight % based on the total weight ofthe coated beads. The drug layered beads may Optionally include an inner dissolution rate controlling membrane of ethylcellulosc. The composition ofthe outer layer, as well as the individual weights ofthe inner and outer layers ofthe 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.

In other embodiments the formulations may comprise a mixture ofimmediate— release drug-containing particles without a ution rate controlling polymer membrane and dclayed-extended-release beads exhibiting, for example, a lag time of 2-4 hours following oral administration, thus providing a two-pulse release profile.

In some ments, the active core is coated with one or more layers of dissolution ontrolling polymers to obtain desired release profiles with or t a lag time. An inner layer membrane can largely control the rate of drug e 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.

The polymers suitable for the outer membrane, which largely controls the lag time of up to 6 hours may se an enteric r, as described above, and a water insoluble polymer at 10 to 50 weight %. The ratio of water insoluble polymer to c 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.

Exemplary water insoluble polymers include ethyleellulose, polyvinyl acetate (Kollicoat SR#0D from BASF), neutral copolymers based on ethyl acrylate and methylmethaerylate, copolymers of acrylic and methacrylic acid esters with quaternary ammonium groups such as EUDRAGIT® 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 g from 1 weight % up to 10 weight % ing on the solubility ofthe active in water and the t 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 ments, ITETM 1RP69 resin is used as an extended- e carrier. AMBERLITETM 1RP69 is an insoluble, strongly acidic, sodium form cation 9075652J (GHMalten) P97099.NZ.2 exchange resin that is suitable as carrier for cationic ) substances. In other embodiments, DUOLITETM APl43/IO93 resin is used as an extended-release carrier.

ETM APl43/1093 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 IRP69 or/and DUOLITETM AP143/l 093 resin provides a means for binding medicinal agents onto an insoluble polymeric matrix. Extended-release is achieved through the fomiation of resin-drug complexes (drug rcsinates). The drug is released from the resin in vivo as the drug s equilibrium with the high electrolyte concentrations, which are typical ofthe gastrointestinal tract. More hydrophobic drugs will y elute from the resin at a lower rate, owing to hydrophobic interactions with the aromatic ure ofthe cation exchange system.

Preferably, the formulations are designed with release profiles to limit interference with restful sleep, wherein the formulation releases the medicine when the individual would normally be awakened by an urge to urinate. For example, consider an individual who begins sleeping at 11 PM and is normally awakened at 12:30 AM, 3:00 AM, and 6:00 AM to e. A d-release vehicle could deliver the ne at 12:15 AM, thereby delaying the need to urinate for perhaps 2-3 hours. By further including an additional extended—release profile or additional pulsatile releases, the need to wake up to urinate may be reduced or eliminated altogether.

The pharmaceutical composition may be administered daily or stered on an as needed basis. In certain ments, the pharmaceutical composition is administered to the t prior to bedtime. In some embodiments, the pharmaceutical composition is administered immediately before bedtime. In some embodiments, the pharmaceutical composition is administered within about two hours before bedtime, preferably within about one hour before e. In another embodiment, the ceutical composition is administered about two hours before bedtime. In a further embodiment, the pharmaceutical composition is administered at least two hours before bedtime. In another embodiment, the pharmaceutical composition is administered about one hour before bedtime. In a further embodiment, the pharmaceutical composition is administered at least one hour before bedtime. In a still further ment, the pharmaceutical composition is administered less than one hour before bedtime. In still another embodiment, the pharmaceutical composition is administered immediately before bedtime. Preferably, the pharmaceutical composition is administered . Suitable itions for oral administration include, but are not limited to: tablets, coated tablets, dragees, capsules, powders, granulates and soluble tablets, 9075652 l tGHMallcls) P97099NZ 2 and liquid forms, for example, suspensions, dispersions or solutions.

Most enteric coatings work by presenting a surface that is stable at the highly acidic pH found in the stomach, but breaks down rapidly at a less acidic (relatively more basic) pl-l. Therefore, an enteric coated pill will not dissolve in the aeidiejuices ofthe stomach (pl-I ~3), but they will in the alkaline (pH 7-9) environment present in the small intestine. Examples of enteric coating als include, but are not limited to, methyl acrylate-methacrylie acid eopolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose ate, hydroxy pr0pyl methyl cellulose e ate (hypromellose acetate succinate), polyvinyl e phthalate (PVAP), methyl methacrylate-methacrylic acid eopolymers, sodium alginate and stearic acid.

In some embodiments, the ceutical composition is orally administered from a variety of drug formulations designed to provide d-release. Delayed oral dosage forms include, for example, tablets, capsules, eaplets, and may also comprise a plurality of granules, beads, powders or pellets that may or may not be encapsulated. s and capsules represent the most convenient oral dosage forms, in which case solid pharmaceutical carriers are employed. 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. lly, the barrier coating ns one or more polymers eneasing, nding, 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.

In other ments, the delayed-release is caused 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, y each bilayer push pull unit contains an c 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 enterie coating to prevent release until after gastric emptying. The n capsule dissolves immediately after ingestion. As the push pull unit(s) enter the small intestine, the enteric coating breaks down, which then allows fluid to flow through the semi-permeable 9075662_1 (GHMnnauJ P970” NZ.2 ne, swelling the osmotic push compartment to force 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.

The osmotic push layer comprises one or more c agents creating the driving force for transport of water through the ermeable membrane into the core of the delivery vehicle. One class ofosmotic 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 alginatc, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2- hydroxycthyl methacrylate), crylic) acid, poly(methacrylic) acid, polyvinylpyrrolidone (PVP), inkcd PVP, nyl alcohol (PVA), PVA/PVP copolymcrs, PVA PVP mers with hydrophobic rs such as methyl methacrylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, sodium croscarmcllose, carrageenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), ypropyl methyl cellulose (HPMC), carboxymcthyl cellulose (CMC) and carboxyethyl, cellulose (CEC), sodium alginatc, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate.

Another class of osmotic agents includes osmogens, which are capable of imbibing water to affect an osmotic pressure gradient across the semi-permeable membrane.

Exemplary osmogens e, but are not d to, nic salts, such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, potassium ates, sodium carbonate, sodium sulfitc, lithium e, 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 f.

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.

In another embodiment, the delay release formulation employs a water- impermeable tablet coating whereby water enters h a controlled re in the coating until the core bursts. When the tablet bursts, the drug contents are released immediately or 9075562_i (GHMIHNIJ P97099.NZ 2 over a longer period . These and other techniques may be modified to allow for a pre- determined lag period before release of drugs is initiated.

Various coating techniques may be applied to granules, beads, powders or pellets, tablets, capsules or combinations f containing active agents to produce ent and distinct release profiles. In some embodiments, the pharmaceutical composition is in a tablet or capsule form ning 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 comprises a plurality of active ingredients selected from the group consisting of analgesics, scarinic agents, antidiuretics and spasmolytics. Examples ofspasmolytics e, but are not limited to, carisoprodol, benzodiazepines, en, cyclobcnzaprine, metaxalonc, methocarbamol, ine, clonidine analog, and lcne. In some embodiments, the pharmaceutical composition comprises one or more analgesics. In other embodiments, the pharmaceutical composition comprises (1) one or more analgesics, and (2) one or more other active ingredients selected from the group consisting of antimuscarinie agents, antidiuretics and spasmoiytics. In another embodiment, the pharmaceutical composition comprises (1) one or two analgesics and (2) one or two antimuscarinic agents. In another embodiment, the pharmaceutical composition comprises (1) one or two analgesics and (2) one or two antidiuretics. In another embodiment, the pharmaceutical ition comprises (1) one or two analgesics and (2) one or two Spasmolytics. In yet another embodiment, the pharmaceutical composition comprises (1) one or two analgesics, (2) one or two antimuscarinic agents, and (3) one or two antidiuretics.

In one embodiment, the plurality of active ingredients are formulated for immediate-release. In other embodiment, the plurality of active ingredients are formulated for extended—release. In other embodiment, the plurality of active ients are formulated for both immediate—release and extended-release (tag, a first portion of each active ingredient is fomiulatcd for immediate-release and a second portion of each active ingredient is formuiated for extended-release). In yet other embodiment, some ofthe plurality of active ingredients are formulated for immediate-release and some of the plurality of active ingredients are ated for extended-release (e.g., active ingredients A, B, C are formulated for ate-release and active ingredients C and D are formulated for extended-release). In some other ments, the immediate-release component and/or the extended-release component is further coated with a delayed-release coating, such as an cnterie coating. 9075662_l (GHMuflers) P97099341 2 In certain embodiments, the pharmaceutical composition comprises an immediate-release component and an extended-release component. The ate-release component may comprise one or more active ingredients selected from the group consisting of analgesics, antimusearinic agents, antidiuretics and spasmolytics. The extended-release component may comprise one or more active ingredients selected from the group consisting of analgesics, antimusearinic agents, antidiuretics and spasmolytics. In some embodiments, the immediate—release component and the ed-release ent 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 ent and the extended-release component have one or more common active ingredients. In some other embodiments, the immediate—release component andi‘or the extended-release component is further coated with a delayed-release coating, such as an cnteric coating.

In one embodiment, the pharmaceutical ition ses two active ingredients (cg, two analgesic agents, or a mixture of one sic agent and one antimusearinic agent or antiuretic or lytic), formulated for immediate-release at about the same time. In another ment, the pharmaceutical composition comprises two active ingredients, formulated for extended-release at about the same time. In another embodiment, the pharmaceutical composition comprises two 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 ent releases a second active ingredient at a second release rate. In another embodiment, the pharmaceutical ition comprises two 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 dclaycd-releaSe component es a second active ingredient at a second time point. In another embodiment, the pharmaceutical composition comprises two active ingredients, one is formulated for ate-release and the other is formulated for extended—release.

In other embodiments, the pharmaceutical composition comprises two active ingredients (e.g., two analgesic agents, or a mixture of one analgesic agent and one antimusearinic agent or antiuretic or lytic) formulated for immediate~rclease, and (2) two active ingredients (cg, two sic agents, or a mixture of one sic agent and one antimusearinic agent or antiuretic or spasmolytie) formulated for extended-release. In other 9075562_1 (GHMutIeID) PWWS NZ 2 embodiments, the pharmaceutical ition comprises three active ingredients formulated for immediate-release, and (2) three active ingredients formulated for extended-release. In other ments, the ceutical composition comprises four active ingredients formulated for immediate-release, and (2) four active ingredients formulated for extended- rclcase. In these embodiments, the active ingredient(s) in the immediate-release component can be the same as, or different from, the active ient(s) in the extended-release component. 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 cnteric coating. [0116} The term "immediate—release" is used herein with reference to a drug formulation that does not contain a dissolution rate controlling material. There is ntially no delay in the release ofthe active agents following administration ofan immediate-release formulation. An immediate-release coating may include suitable materials immediately dissolving following administration so as to release the drug contents therein. Exemplary ate-release g materials include gelatin, polyvinyl alcohol polyethylene glycol (PVA-PEG) copolymers (e.g., KOLLICOAT®) and various others materials known to those skilled in the art.

An immediate-release composition may comprise 100% of the total dosage of a given active agent administered in a single unit dose. Alternatively, an immediate-release component may be included as a component in a combined release profile formulation that may provide about 1% to about 50% ofthe total dosage ofthe active s) to be delivered by the pharmaceutical formulation. For e, the immediate-release component may provide at least about 5%, or about 10% to about 30%, or about 45% to about 50% ofthe total dosage ofthe active agent(s) to be delivered by the formulation. In alternate embodiments, the ate-release component provides about 2, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the total dosage ofthe active agent(s) to be delivered by the formulation.

In some embodiments, the immediate-release or delayed—release formulation comprises an active core comprised ofone or more inert particles, each in the form ofa 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 filmv forming composition using, for example, fluid bed ques 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 nization ofa polymer composition 5075662'1 (GHMBllclB) P9709? NZ 2 containing the drug substance.

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 ofthe 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 d dosage. In one embodiment, the inactive core may be a sugar sphere or a buffer crystal or an encapsulated buffer crystal such as m carbonate, sodium bicarbonate, e acid, tartaric acid, etc. which alters the microenvironment ofthe drug to facilitate its release.

In some embodiments, the d-release formulation is formed by coating 3 water soluble/dispersible ontaining particle, such as a bead, with a e ofa water insoluble polymer and an enterie polymer, wherein the water insoluble polymer and the enterie polymer may be present at a weight ratio of from 4:l to 1:1, and the total weight of the coatings is 10 to 60 weight % based on the total weight ofthe coated beads. The drug layered beads may optionally include an inner dissolution rate controlling membrane of ethylcellulosc. The composition ofthe outer layer, as well as the individual weights ofthe inner and outer layers ofthe polymeric membrane are optimized for achieving desired ian rhythm e profiles for a given active, which are predicted based on in vitro/in vivo ations.

In other embodiments the formulations comprise a mixture of immediate- release drug-containing particles without a dissolution rate controlling polymer ne 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 oftwo 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 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 ing 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 e 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.

The rs suitable for the outer membrane, which largely ls the lag 9075662_1 (GHMmm) 997099141 2 time of up to 6 hours may comprise an enterie polymer, as described above, and a water insoluble polymer at a thickness of 10 to 50 weight %. The ratio ofwatcr insoluble polymer to enteric polymer may vary from 4:1 to 1:2, ably the polymers are present at a ratio of about 1:1. The water insoluble polymer typically used is ethylcellulose.

Exemplary water insoluble polymers include ethylceliulose, polyvinyl acetate (Kollicoat SR#0D from BASF), l copolymers based on ethyl acrylate and methylmethacrylate, copolymers of acrylic and methacrylic acid esters with nary um groups such as EUDRAGIT® 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 ofthe active in water and the solvent or latex suspension based g 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.

Preferably, the formulations are designed with release profiles to limit interference with restful sleep, wherein the formulation releases the ne when the individual would ly be awakened by an urge to urinate. For example, consider an individual who begins sleeping at 11 PM and is normally awakened at 12:30 AM, 3:00 AM, and 6:00 AM to urinate. A delayed, extended-release vehicle could deliver the ne at 12:15 AM, thereby delaying the need to urinate for perhaps 2-3 hours. 10126] The pharmaceutical composition may be administered daily or administered on an as needed basis. In certain embodiments, the pharmaceutical composition is administered to the subject prior to bedtime. In some ments, the pharmaceutical composition is administered immediately before 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 two hours before bedtime. In a further embodiment, the pharmaceutical composition is administered at least two hours before bedtime. In r embodiment, the pharmaceutical composition is administered about one hour before bedtime. In a further ment, the pharmaceutical composition is administered at least one hour before e. In a still further embodiment, the pharmaceutical composition is administered less than one hour before e. In still another embodiment, the pharmaceutical composition is administered immediately before e. Preferably, the pharmaceutical composition is administered .

The appropriate dosage (“therapeutically effective amount”) ofthe active 9075662_1 (GHMnttell) 997099142 2 agent(s) in the immediate-release component or the extended-release component will depend, for example, the severity and course ofthe condition, the mode of administration, the bioavailability ofthe particular agent(s), the age and weight ofthe patient, the patient's clinical history and response to the active agent(s), discretion ofthe ian, etc.

As a l proposition, the eutically effective amount ofthe active agent(s) in the immediate-release component, the ed-release component or the delayed-extended-re1ease component is administered in the range of about 100 ug/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 is from about 100 ug/kg body weight/day to about 50 mg/kg body weight/day, 100 pg/kg body weight/day to about 10 mg/kg body weight/day, 100 ug/kg body /day to about 1 mg/kg body weight/day, 100 ug/kg body weight/day to about 10 mg/kg body weight/day, 500 ug/kg body weight/day to about 100 mg/kg body weight/day, 500 pg/kg body weight/day to about 50 mg/kg body weight/day, 500 pig/kg body weight/ day to about 5 mg/kg body / day, 1 mg/kg body weight/day to about 100 mg/kg body weight/day, 1 mg/kg body weight/day to about 50 mg/kg body / day, 1 mg/kg body weight/day to about IO mg/kg body weight/day, 5 mg/kg body weight/dose to about 100 mg/kg body weight/day, 5 mg/kg body weight/dose to about 50 mg/kg body weight/day, 10 mg/kg body weight/day to about 100 mg/kg body weight/day, and 10 mg/kg body weight/day to about 50 mg/kg body weight/day.

The active agent(s) described herein may be included in an immediate-release component or an extended-release component, a delayed-cxtcnded-rclcase component or combinations thereof for daily oral administration at a single dose or combined dose range of 1 mg to 2000 mg, 5 mg to 2000 mg, 10 mg to 2000 mg, 50 mg to 2000 mg, 100 mg to 2000 mg, 200 mg to 2000 mg, 500 mg to 2000 mg, 5 mg to 1800 mg, 10 mg to 1600 mg, 50 mg to 1600 mg, 100 mg to 1500 mg, 150 mg to 1200 mg, 200 mg to 1000 mg, 300 mg to 800 mg, 325 mg to 500 mg, 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 200 mg, 5 mg to 1000 mg, 5 mg to 500 mg, 5 mg to 200 mg, 10 mg to 1000 mg, 10 mg to 500 mg, 10 mg to 200 mg, 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, 250 mg to 1000 mg, 250 mg to 500 mg, 500 mg to 1000 mg, 500 mg to 2000 mg. As expected, the dosage will be dependant on the condition, size, age and condition ofthe patient.

In some embodiments, the pharmaceutical composition ses a single analgesic agent. In one embodiment, the single analgesic agent is aspirin. In another embodiment, the single sic agent is ibuprofen. In another embodiment, the single analgesic agent is naproxen . In another embodiment, the single analgesic agent is sovsse2_1 (GHMaflcrl) 997059»: 2 indomethacin, In another embodiment, the single analgesic agent is nabumetone. In r embodiment, the single analgesic agent is acetaminOphen.

In some embodiments, the single analgesic agent is given at a daily dose of 1 mg to 2000 mg, 5 mg to 2000 mg, 20 mg to 2000 mg, 5 mg to 1000 mg, 20 mg to 1000 mg, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg, 250 mg to 1000 mg or 500 mg to 1000 mg. In certain embodiments, the ceutical composition ses acetylsalicylic acid, ibuprofen, naproxen sodium, indomethancin, nabumetone or acetaminophen as a single analgesic agent and the analgesic agent is administered orally at a daily dose in the range of5 mg to 2000 mg, 20 mg to 2000 mg, 5 mg to 1000 mg, 20 mg to 1000 mg, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg, 250 mg to 1000 mg or 500 mg to 1000 mg. In some embodiments, a second analgesic agent is given at a daily dose of 1 mg to 2000 mg, 5 mg to 2000 mg, 20 mg to 2000 mg, 5 mg to 1000 mg, 20 mg to 1000 mg, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg, 250 mg to 1000 mg or 500 mg to 1000 mg.

In other embodiments, the pharmaceutical composition comprises a pair of analgesic agents. Examples ofsueh paired analgesic agents include, but are not limited to, acetylsalicylic acid and ibuprofen, salicylic acid and naproxen sodium, acetylsalicylic acid and nabumetone, acetylsalicylic acid and acetaminophen, acetylsalicylic acid and thancin, fen and naproxen sodium, ibuprofen and nabumetone, ibuprofen and inophen, fen 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 of0.1:1 to 10:1, 02:1 to 5:1 or 0.3:1 to 3: l, with a combined dose in the range of5 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.

In some other embodiments, the pharmaceutical composition of the present application r comprises one or more antimuscarinic agents. Examples ofthe antimuscarinic agents include, but are not limited to, oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine, trOSpium and atropine. The daily dose ofantimuscarinic agent is in the range 0f0.01 mg to 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 10 mg to 100 mg, 0.01 mg to 25 mg, 0.1 mg to 25 mg, 1 mg to 25 mg, 10 mg to 25 mg, 0.01 mg to 10 mg, 0.1 mg to 9075662_l (GHManL-ia) 997099 N2 2 mg, 1 mg to 10 mg, 10 mg to 100 mg and 10 mg to 25 mg.

In certain ments, the pharmaceutical composition comprises an sic agent selected from the group consisting ofcetylsalicylic acid, ibuprofen, naproxen sodium, nabumetone, acetaminOphen and indomethancin, and an antimuscarinie agent selected from the group consisting ofoxybutynin, solifenacin, darifenacin and atropine.

Another aspect ofthe present application relates to a method for reducing the frequency of urination by administering to a person in need thereofa pharmaceutical composition formulated in an immediate-release formulation. The pharmaceutical composition comprises a ity of analgesic agents and/or antimusearinic agents.

In n embodiments, the pharmaceutical composition comprises two or more analgesic agents. In other ments, the pharmaceutical composition comprises one or more sic agents and one or more antimuscarinie agents. The pharmaceutical composition may be formulated into a 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 e.

In certain embodiments, the analgesic agent is ed from the group consisting of salicylates, aspirin, salicylic acid, methyl salicylate, diflunisal, salsalate, zinc, sulfasalazine, para-aminophenol derivatives, acetanilide, acetaminophen, etin, fenamates, mefenamic acid, meclofenamate, sodium mcelofenamate, heteroaryl acetic acid tives, tolmetin, ketorolac, diclofenae, propionic acid derivatives, ibuprofen, en sodium, naproxen, ofen, ketoprofen, flurbiprofen, oxaprozin; enolic acids, oxicam derivatives, piroxieam, meloxicam, cam, ampiroxicam, droxieam, pivoxicam, pyrazolon derivatives, phenylbutazone, oxyphenbutazone, antipyrine, aminopyrine, dipyrone, coxibs, celecoxib, rofccoxib, nabumetone, apazone, nimcsulidc, indomethaein, sulindac, etodolae, diflunisal and isobutylphenyl propionic acid. The antimuscarinic agent is selected from the group consisting of oxybutynin, solifenaein, darifenacin and atr0pine.

In some embodiments, the pharmaceutical composition comprises a single analgesic agent and a single antimusearinic agent. In one embodiment, the single analgesic agent is aspirin. In another embodiment, the single analgesic agent is ibuprofen. In another embodiment, the 5 single analgesic agent is 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 inophen. The analgesic agent and uscarinic agent may be given at doses in the ranges described above. 9075662_l [GHMaflml) 997099 NZ 2 Another aSpect of the present application relates to a method for treating nocturia by administering to a subject in need thereof(l) one or more sic agent and (2) one or more antidiuretic agents. In certain embodiments, the antidiuretic agent(s) act to: (1) increase vaSOprcssin secretion; (2) increase vasopressin receptor activation; (3) reduce secretion of atrial natriuretie peptide (ANP) or C-type natriuretic peptide (CNP); or (4) reduce ANP and/or CNP receptor activation.

Exemplary antidiuretic agents include, but are not limited to, uretic hormone (ADH), angiotensin II, aldosterone, vasopressin, vaSOprcssin analogs (e.g., ressin argipressin, lypressin, essin, omipressin, terlipressin); vasopressin receptor agonists, atrial natriuretic peptide (ANP) and C-type retic peptide (CNP) receptor (116., NPR] , NPRZ, NPR3) nists (cg, HS-l42—l, isatin, [Asu7,23']b-ANP—(7- 28)], anantin, a cyclic peptide from Streptomyces coerulescens, and 3Gl2 monoclonal antibody); somatostatin type 2 receptor antagonists (e.g., somatostatin), and pharmaceutically—aeceptable derivatives, analogs, salts, es, and solvates thereof.

In n embodiments, the one or more analgesic agent and one or more antidiurctic agents are formulated for extended-release.

Another aspect ofthc present application relates to a method for ng the frequency of urination by stering to a person in need thereofa first pharmaceutical composition comprising a diuretic, followed with a second pharmaceutical composition comprising one or more sic agents. The first pharmaceutical composition is dosed and formulated to have a diuretic effect within 6 hours of administration and is administered at least 8 hours prior to bedtime. The second pharmaceutical composition is administered within 2 hours prior to bedtime. The first pharmaceutical ition is formulated for immediate-release and the second pharmaceutical composition is formulated for extended- release or delayed, extended—release.

Examples ofdiurctics include, but are not limited to, acidifying salts, such as CaClz and NH4Cl; arginine vasopressin or 2 antagonists, such as amphotericin B and m citrate; aquaretics, such as Goldenrod and Junipe; Na-H ger antagonists, such as dopamine; carbonic anhydrase inhibitors, such as acetazolamide and dorzolamide; loop diuretics, such as nide, 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.

In some embodiment, the second pharmaceutical composition fiirther 9075662_| [GHMaflarl) 997099 NZ 2 comprises one or more antimuscarinic agents. Examples ofthe antimuscarinic agents include, but are not limited to, oxybutynin, solifenacin, darifenacin, rodine, tolterodine, trospium and atropine.

Another aspect of the present application relates to a method for treating nocturia by administering to a person in need thereofa first pharmaceutical composition sing a diuretic, followed with a second pharmaceutical composition comprising one or more analgesic . The first pharmaceutical composition is dosed and formulated to have a diuretic effect within 6 hours of administration and is administered at least 8 hours prior to bedtime. The second pharmaceutical composition is formulated for extended~release or delayed, extended-release, and is administered within 2 hours prior to bedtime.

Examples of diuretics include, but are not d to, acidifying salts, such as CaClz and NI-I4Cl; arginine vasopressin receptor 2 antagonists, such as amphotericin B and m citrate; aquaretics, such as Goldenrod and Junipe; Na-l-l exchanger antagonists, such as dopamine; carbonic anhydrase inhibitors, such as olamide and dorzolamide; loop diuretics, such as bumetanide, ynic acid, furosemide and torsemide; osmotic diuretics, such as glucose and mannitol; potassium-sparing diuretics, such as amiloride, olactone, triamterene, potassium eanrenoate; thiazides, such as bendroflumethiazide and hydrochlorothiazide; and xanthines, such as caffeine, theophylline and theobromine.

In some embodiments, the second pharmaceutical composition further comprises one or more antimuscarinic agents. Examples ofthe antimuscaiinic agents include, but are not limited to, oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine, trOSpium and atropine. The second pharmaceutical composition may be formulated in immediate-release formulation or d-release formulation. In some other embodiments, the second pharmaceutical composition further comprises one or more antidiuretic agents. In some other embodiments, the second ceutical composition r comprises one or more spasmolytics.

Another aspect ofthe present application relates to a method for reducing the frequency ofurination by stering to a subject in need thereof, two or more analgesic agents alternatively to prevent the development of drug resistance. In one embodiment, the method comprises stering a first sic agent for a first period oftime and then administering a second analgesic agent for a second period oftime. In another embodiment, the method further comprises administering a third analgesic agent for a third period of time.

The first, second and third analgesic agents are different from each other and at least one of which is formulated for extended-release or delayed, extended-release. In one embodiment, 9075652_l [GHMannn] F970” NZ.2 the first analgesic agent is acetaminophen, the second analgesic agent is ibuprofen and the third sic agent is naproxen sodium. The length of each period may vary depending on the subject’s response to each analgesic agent. In some ments, each period lasts from 3 days to three weeks. In another embodiment, the first, second and third sic are all formulated for ed-release or delayed, extended-release.

Another aspect of the present application relates to a pharmaceutical composition comprising a plurality of active ingredients and a pharmaceutically acceptable r, wherein at least one ofthe plurality of active ingredients is formulated for extended- release or delayed, extended-release. In some embodiments, the plurality of active ingredients comprises one or more analgesics and one or more antidiuretic agents. In other embodiments, the ity of active ingredients comprises one or more sics and one or more antidiuretic agents. In other embodiments, the plurality of active ingredients comprises one or more analgesics, one or more antidiuretic agents and an antimuscarinic agent. The antimuscarinic agent may be selected from the group consisting of oxybutynin, solifenaein, nacin and atropine. In other embodiments, the ceutical composition comprises two different sics selected from the group consisting of cetylsalicylic acid, ibuprofen, naproxen sodium, nabumetone, acetaminophen and indomethancin. In yet other embodiments, the pharmaceutical composition comprises one analgesic selected from the group consisting of cetylsalicylic acid, ibuprofen, naproxen sodium, nabumetonc, acetaminophen and thancin; and an antimuscarinic agent selected from the group consisting ofoxybutynin, solifenacin, darifcnacin and atropine.

In other embodiments, the ceutical composition ofthe present application further comprises one or more Spasmolytics. Examples of spasmolytics include, but are not limited to, carisoprodol, benzodiazepines, baclofen, cyclobenzaprine, metaxalone, methocarbamol, clonidine, ine analog, and dantrolene. In some embodiments, the spasmolytics is used at a daily dose of I mg to 1000 mg, 1 mg to 100 mg, 10 mg to 1000 mg, mg to 100 mg, 20 mg to 1000 mg, 20 mg to 800 mg, 20 mg to 500 mg, 20 mg to 200 mg, 50 mg to 1000 mg, 50 mg to 800 mg, 50 mg to 200 mg, 100 mg to 800 mg, 100 mg to 500 mg, 200 mg to 800 mg, and 200 mg to 500 mg. The spasmolytics may be formulated, alone or together with other active ingredient(s) in the pharmaceutical composition, for immediate- release, extended-release, delayed-extended-release or combinations thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sweeteners and the like, The pharmaceutically acceptable 9075662_l [GHMAtlom] P970951 NZ 2 carriers may be prepared from a wide range ofmaterials including, but not limited to, flavoring agents, sweetening agents and miscellaneous materials such as buffers and absorbents that may be needed in order to prepare a particular therapeutic composition. The use of such media and agents with pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

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: TION OF THE URGE TO URINATE Twenty volunteer ts, both male and female were enrolled, each of which experienced premature urge or desire to urinate, interfering with their y to sleep for a ient period oftime to feel tely . 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 e.

Several subjects reported that after several weeks of nightly use of ibuprofen, the benefit ofless nt urges to urinate was no longer being realized. However, all of these ts r reported the return ofthe benefit after several days aining from taking the dosages.

E 2: EFFECT OF ANALGESIC AGENTS BOTULINUM NEUROTOXIN AND ANTIMUSCARINIC AGENTS ON MACROPHAGE RESPONSES TO INFLAMMATORY AND NON-INFLAMMATORY STIMULI Experimema/ Design This study is designed to determine the dose and in vitro efficacy ofanalgesics and antimuscarinie agents in controlling macrophage response to inflammatory and non- nmatory 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 sic agents and/0r antimusearinie agents in the absence or presence of various effectors.

The effectors include: lipopolysaccharide (LPS), an inflammatory agent and Cox2 inducer, as inflammatory stimuli; carbachol or acetylcholine, a stimulator 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 8075662 1 els) 997099141 2 following the sequential oxidation of AA, DGLA or EPA inside the cell by eyelooxygcnases (COXl and COX2) and terminal prostaglandin synthases.

The analgesic agents include: Salicylates such as aSpirin, iso-butyl-propanoic- phenolic acid derivative ofen) such as Advil, Motrin, Nuprin, and Medipren, naproxen sodium such as Aleve, Anaprox, Antalgin, Feminax Ultra, Flanax, Inza, Midol Extended , in, Naposin, Naprelan, esic, Naprosyn, Naprosyn suspension, EC- Naprosyn, Narocin, Proxen, Synflex and Xenobid, acetic acid derivative such as indomethaein (Indocin), l-naphthaleneaeetic acid derivative such as nabumetone or relafen, N-acctyl-para~aminophenol (APAP) derivative such as acetaminophen or paracetamol (Tylenol) and Cclecoxib.

The antimuscarinic agents include: oxybutynin, solifenacin, darifenacin and atropine.

Macrophages are subjected to short term (1-2 hrs) or long term (24-48 hrs) stimulation ofwith: 1) Each analgesic agent alone at s doses. (2) Each analgesic agent at various doses in the presence of LPS. (3) Each analgesic agent at various doses in the presence of carbachol or acetylcholine. (4) Each analgesic agent at various doses in the presence of AA, DGLA, or EPA. (5) Botulinum neurotoxin A alone at s doses. (6) num neurotoxin A at various doses in the presence of LPS. (7) Botulinum neurotoxin A at various doses in the presence of carbachol or acctylcholine. (8) num neurotoxin A at various doses in the presence of AA, DGLA, or EPA. (9) Each antimuscarinic agent alone at various doses. (10) Each antimuscarinic agent at various doses in the ce of LPS. (1 1) Each antimuscarinic agent at various doses in the presence of carbachol or acetylcholine. (12) Each antimuscarinic agent at various doses in the ce of AA, DGLA, or EPA.

The cells are then analyzed for the release of PGHZ, PGE, PGEZ, cydin, Thromboxanc, IL— 1 [3, IL-6, TNF-a, the COX2 activity, the production ochMP and cGMP, the production ofIL-IB, lL-6, TNF-a and COX2 mRNA, and surface expression of CD80, CD86 and MHC class II molecules.

Materials and Methods Macrophage cells ] 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 .1 [GHMBH urn) P97I199.NZ.2 supplemented with IO % fetal bovine serum (PBS), 15 mM HEPES, 2 mM nglutamine, 100 U/ml penicillin, and 100 pg / ml of streptomycin. Cells were cultured at 37° C in a 5 % C02 atmOSphere and split ges) once a week.

In vitro treatment of macrophage cells with analgesics RAW264.7 macrOphage cells were seeded in 96-well plates at a cell density of 1.5xlO5 cells per well in 100 pl ofthe culture medium. The cells were treated with (1) various concentrations of analgesic (acetaminOphen, aspirin, ibuprophen or naproxen), (2) various concentrations of lipopolysaccharide (LPS), which is an effector of matory stimuli to macrophage cells, (3) various concentrations of earbachol or acctylcholine, which are effectors intlammatory stimuli, (4) analgesic and LPS or (5) sic and earbachol or acetyleholine. Briefly, the analgesics were dissolved in PBS-free culture medium (i.e., RPMI 1640 supplemented with 15 mM HEPES, 2 mM L-glutamine, 100 U / ml penicillin, and 100 pg / ml of streptomycin), and diluted to desired concentrations by serial dilution with the same medium. For cells treated with sic in the e of LPS, 50 pl ofanalgesie solution and 50 pl of PBS-free culture medium were added to each well. For cells treated with analgesic in the ce of LPS, 50 pl ofanalgesic solution and 50 pl of LPS (from Salmonella typhimurium) in ce e medium were added to each well.

All conditions were tested in duplicates.

After 24 or 48 hours ofculture, 150 pl ofculture supernatants were collected, Spun down for 2 min at 8,000 rpm at 4°C to remove cells and debris and stored at -70°C for analysis ofcytokine responses by ELISA. The cells were collected and washed by centrifugation (5 min at 1,500 rpm at 4°C) in 500 pl of Phosphate buffer (PBS). Half ofthe cells were then snap frozen in liquid nitrogen and stored at -70°C. The remaining cells were stained with fluorescent monoclonal antibodies and analyzed by flow cytometry.

Flow cytometry analysis of co-stimulatory molecule expression For flow cytomctry analysis, macrophages were diluted in 100 p1 of FACS buffer (phosphate buffered saline (PBS) with 2% bovine serum albumin (BSA) and 0.01% NaNg) and stained 30 min at 4°C by addition of onjugated anti-CD40, PE«conjugated D80, PIE-conjugated anti-CD86 antibody, anti MHC class II (I~Ad) PE (BD enec). Cells were then washed by centrifugation (5 min at 1,500 rpm at 4°C) in 300 pl of FACS buffer. After a second wash, cells were re-suspended in 200 pl of FACS buffer and the percentage of cells expressing a given marker (single ve), or a combination of markers (double positive) were analyzed with the aid of an Accuri C6 flow cytometer (BD 9075662_l (GHMalleIa) P97OWNZ 2 Biosciences).

Analysis of cytokine responses by ELISA Culture tants were subjected to cytokine—specific ELISA to determine IL- 1 B, IL~6 and TNF-a 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 ul of anti-mouse IL-6, TNF—a mAbs (BD Biosciences) or IL—1 B mAb (R&D Systems) in 0.1 M sodium onate buffer (pl-1 9.5).

After two washes with PBS (200 ul per well), 200 [,li of PBS 3% BSA were added in each well (blocking) and the plates incubated for 2 hours at room ature. Plates were washed again two times by addition of 200 u] per well, 100 pl of cytokine standards and serial dilutions of culture supernatants were added in duplicate and the plates were ted overnight at 4°C. Finally, the plates were washed twice and incubated with 100 ul of secondary biotinylated anti-mouse IL—6, TNFOt mAbs (BD Biosciences) or IL-IB (R&D Systems) followed by peroxidase—labelled goat anti-biotin mAb (Vector Laboratories). The colorimetric on was dcv010pcd by the addition of 2,2’-azino—bis (3)- ethylbcnzylthiazoline—6—sulfonic acid (ABTS) substrate and Egg; (Sigma) and the absorbanee measured at 415 nm with a Victor®V multilabel plate reader (PerkinElmer).

Determination of COXZ activity and the production of CAMP and eGMP The COX2 activity in the cultured macrophages is determined by sequential competitive ELISA (R&D Systems). The production ochMP and eGMP is ined by the CAMP assay and cGMP assay. These assays are performed ely in the art.

Results Table 1 summarizes the experiments performed with Raw 264 macrophage cell line and main findings in terms ofthe effects of analgesics on cell surface expression of costimulatory molecules CD40 and CD80. sion ofthese molecules is ated by COX2 and inflammatory signals and thus, was evaluated to ine functional consequences ofinhibition 0fCOX2.

As shown in Table 2, acetaminophen, aspirin, ibuprophen and naproxen inhibit basal expression of co—stimulatory les CD40 and CD80 by macrophages at all the tested doses 0.6., 5x 105 nM, 5x 104 nM, 5x 103 nM, 5x 102 nM, 50 nM and 511M), except for the highest dose (i'.e., 5x 106 nM), which appears to enhance, rather than inhibit, expression ofthe co-stimulatory molecules. As shown in Figures 1A and 18, such inhibitory effect on CD40 and CD50 expression was observed at analgesic doses as low as 0.05 nM 9075662‘l (GHManml) P971399 NZ 2 (i.e., 0.00005 ttM). This finding supports the notion that a controlled e of small doses of analgesic may be preferable to acute delivery of large doses. The experiment also revealed that acetaminophen, aspirin, ibuprOphen and naproxen have a similar inhibitory effect on LPS induced expression of CD40 and CD80.

Table 1. Summary of experiments Control ella Iyphimurium Acetaminophen n phen 2 X (0, 5,50, 1000) ng/mL Dose responses (0, 5, so, 500, 5x103, 5x104, 5x105, 5x10“) nM 4 X (5 ng/mL) Dose reSponses X (50 ng/mL (0,5 nM , 50, 500, 5x103, 5x104, 5x105, 5x10“) X (1000 ng/mL) ANALYSIS Characterization ofactivation/stimulatory status: Flow cytomctry analysis ofCD40, CD80, CD86 and MHC class II Mediators ofinflammatory responses: ELISA analysis ofIL-IB, lL—6, TNF-a Table 2. Summary of main findings Effectors % Positive ve Dose analgesic (nM) Control 5 rig/ml - U‘ 778 - w ii 00 I.» \O u: A ---11 10.3 8.3 mu] - U)..".‘“ 00 b) \l \I \l W Li! N Analgesic plus LPS 95.1 82.7 72.4 68.8 66.8 66.2 62.1 84.5 80 78.7 74.7 75.8 70,1 65.7 lbuprophen CD40+CD80+ E2O 67 77.9 72.9 71.1 O\3.7 . 9.: 74.1 i8.8 \JN \IO\ mo 9075662_l (GHMnher:} 997099 NZ 2 “‘ ND: not done (toxicity) Table 3 summarizes the results of several studies that measured serum levels ofanalgesie after oral eutic 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 104 to 105 nM.

Therefore, the doses of analgesic tested in vitro in Table 2 cover the range of concentrations achievable in vivo in humans.

Table 3. Serum levels of analgesic in human blood after oral therapeutic doses Maximum serum Analgesic drug Molecular levels after oral References weight therapeutic doses —--—— Acetaminophen ' "‘ BMC Clinical cologyl2010, 10:10 (Tylenol) * Anaesth Intensive Care. 2011, 391242 Aspirin * ition of Toxic Drugs and Chemicals lsalicylic acid) in Man, 8th Edition, Biomedical Public, Foster City, CA, 2008, pp. 22-25 * J Lab Clin Med. 1984 Jun;103:869 fen * BMC Clinical PharmacologyZOlO, 10:10 (Advil, Motrin) * J Clin Pharmacol. 2001, 412330 Naproxen * J Clin Pharmacol. 2001, 41:330 (Aleve) E 3: EFFECT OF ANALGESIC AGENTS BOTULINUM NEUROTOXIN AND ANTIMUSCARTNIC AGENTS ON MOUSE BLADDER SMOOTH MUSCLE CELL RESPONSES TO MATORY AND NON-INFLAMMATORY STIMULI Exgerimemal Design This study is designed to characterize how the optimal doses of analgesic 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.

The effectors, analgesic agents and antimuscarinic agents are described in 907 5662_l (GHMaItL-ri) 997099 NZ 2 e 2.

Primary culture of mouse bladder smooth muscle cells are subjected to short term (1-2 hrs) or long term (24-48 hrs) stimulation of with: (1) Each analgesic agent alone at various doses. (2) Each analgesic agent at s doses in the presence of LPS. (3) Each analgesic agent at various doses in the presence of carbachol or acetylcholine. (4) Each analgesic agent at various doses in the presence of AA, DGLA, or EPA. (5) Botulinum neurotoxin A alone at various doses. (6) Botulinum neurotoxin A at various doses in the presence of LPS. (7) Botulinum neurotoxin A at various doses in the presence of carbachol or acctylcholine. (8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA, or EPA. (9) Each antimusearinic agent alone at various doses. (10) Each scarinie agent at various doses in the presence of LPS. (1 1) Each antimuscarinie agent at various doses in the ce of earbaehol or acetylcholine. (12) Each antimuscarinie agent at various doses in the ce of AA, DGLA, or EPA.

The cells are then analyzed for the release of PGHZ, PGE, PGEZ, Prostacydin, Thromboxane, IL- l [3, lL-6, TNF-a, the COX2 activity, the production of CAMP and cGMP, the production oflL-IB, IL-6, TNF-a and COX2 mRNA, and surface expression of CD80, CD86 and MHC class II molecules.

Materials and Methods Isolation and purification of mouse bladder cells Bladder cells were removed from euthanized animals CS7BL/6 mice (8-12 weeks old) and cells were isolated by enzymatic ion followed by purification on a Pcrcoll gradient. Briefly, bladders from 10 mice were minced with scissors to fine slurry in ml of digestion buffer (RPMI 1640, 2% fetal bovine serum, 05 mg/ml collagenase, 30 ttg/ml DNase). Bladder slurries were enzymatically digested for 30 minutes at 37°C.

Undigested fragments were further sed h a cell—trainer. The cell suspension was ed and added to a discontinue 20%, 40% and 75% Pereoll gradient for purification on mononuclear cells. Each experiment used 50-60 bladders.

After washes in RPMI 1640, bladder cells were resuspended RPMI 1640 9075562_1 em P97059.NZ 2 supplemented with 10 % fetal bovine serum, 15 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ug ’ ml of streptomycin and seeded in clear~bottom black 96-well cell culture microculture plates at a cell density of 3x104 cells per well in 100 pl. Cells were cultured at 37° C in a 5 % C02 atmosphere.

In vitro ent of cells with analgesics r cells were treated with analgesic solutions (50 till well) either alone or together with carbachol (IO-Molar, 50 111/ well), as an example of non-inflammatory stimuli, or lipopolysaccharide (LPS) of Salmonella typhimurium (1 pg/ml, 50 pl/ well), as an example of non-inflammatory stimuli. When no other effectors were added to the cells, 50 it] of RPMI 1640 without fetal bovine serum were added to the wells to adjust the final volume to 200 pl.

After 24 hours of culture, 150 pl ofculture supernatants were collected, spun down for 2 min at 8,000 rpm at 4°C to remove cells and debris and stored at -70°C for analysis taglandin E2 (PGEZ) responses by ELISA. Cells were fixed, permeabilized and blocked for detection of Cyclooxygenase-Z (COX2) using a fluorogenic substrate. In selected experiment cells were stimulated 12 hours in vitro for analysis of COX2 responses Analysis of COX2 responses 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 bilization, a mouse otal COX2 and a rabbit anti- total GAPDH were added to the wells ofthe clear-bottom black 96—well cell culture microculture plates. After incubation and washes, an HRP-conjugated anti-mouse IgG and an AP-conjugated abbit IgG were added to the wells. Following another incubation and set of washes, the HRP- and AP-fluorogenic ates were added. Finally, a Victor® 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 oftotal COX2 as determined by relative fluorescence unit (RFUs) and normalized to the housekeeping n GAPDH.

Analysis of PGE2 responses Prostaglandin E2 responses were analyzed by a sequential competitive ELISA (R&D Systems). More specifically, culture supernatants or PGEZ standards were added to the wells ofa 96—well yrene microplate coated with a goat anti-mouse polyelonal antibody.

After one hour tion on a microplate shaker, an HRP-conjugated PGE2 was added and 9075662_I (GHMulmn) P97099iNZ ‘2 plates 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 min and the on stopped by addition 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 The release of PGHz, PGE, Prostacydin, oxane, IL-lfi, lL—6, and TNF- a, the production of cAMP and cGMP, the production ofIL-IB, lL-6, TNF-a and COX2 mRNA, and surface expression of CD80, CD86 and MHC class II molecules are determined as described in Example 2.

Analgesics inhibit COX2 responses of mouse bladder cells to an inflammatory stimuli Several analgesics (acetaminophen, aspirin, ibuprofen and naproxen) were tested on mouse bladder cells at the concentration ofS uM or 50 uM to determine whether the analgesics could induce COX2 responses. is of 24-hour cultures showed that none of the sics tested induced COX2 responses in mouse bladder cells in vitro.

The effect ofthese analgesics on the COX2 responses of mouse bladder cells to carbachol or LPS stimulation in vitro was also tested. As indicated in Table l, 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 ssive effect of the analgesic was seen when these drugs were tested at either 5 uM or 50 uM (Table 4).

Table 4. COX2 expression by mouse bladder cells after in vitro stimulation and treatment with sic Stimuli ——Analgesic Total COX2 levels LPS (lug/ml) None 420 i 26 LPS l) inophen (5 ttM) LPS (lug/ml) Aspirin (5 ttM) LPS l) Ibuprofen (5 uM)) 253 :t 32 9075662_! (Gt-(Mullen) P97099.NZ 2 LPS (lug/ml) en (5 uM) LPS (lug/ml) Acetaminophen (50 uM) LPS (lug/ml) Aspirin (50 uM) LPS (lug/ml) Ibuprofen (50 th) LPS (lug/ml) Naproxen (50 itM) sics inhibit PGEZ responses of mouse bladder cells to an inflammatory stimuli The secretion of PGE2 in culture supernatants of mouse bladder cells was measured to determine the biological significance ofthe tion of mouse bladder cell COX2 levels by analgesics. As shown in Table 5, PGE2 was not ed in the culture supernatants ofunstimulatcd bladder cells or bladder cells cultured in the presence of earbachol. Consistent with COX2 responses described above, stimulation of mouse bladder cells with LPS induced the ion ofhigh levels of PGE2. Addition ofthe analgesics acetaminophen, aspirin, fen and naproxen suppressed the effect of LPS on PGE2 secretion and no difference was seen between the ses of cells treated with the 5 or 50 uM dose of analgesic.

Table 5. PGE2 secretion by mouse bladder cells after in vitro stimulation and treatment with analgesic LPS (lug/ml) Acetaminophen (5 uM) LPS (lug/ml) Aspirin (5 uM) LPS (lug/ml) Ibuprofen (5 uM)) LPS (lug/ml) Naproxen (5 uM) LPS (lug/ml) Acetaminophen (50 uM) LPS (lug/ml) Aspirin (50 uM) LPS (lug/ml) Ibuprofen (50 ttM) LPS (lug/ml) Naproxen (50 uM) In summary, these data show that the analgesics alone at 5 uM or 50 uM do 9075662_! (GHMBroo) P87089.NZIZ not induce COX2 and PGE2 responses in mouse bladder cells. The analgesics at 5 uM or SO uM, however, significantly inhibit COX2 and PGE2 responses of mouse r cells stimulated in vitro with LPS (I pg/ml). No significant effect of analgesics was observed on COX2 and PGE2 ses ofmouse bladder cells stimulated with carbachol (1 mM).

EXAMPLE 4: EFFECT OF ANALGESIC AGENTS BOTULINUM NEUROTOXIN AND ANTIMUSCARTNIC AGENTS ON MOUSE BLADDER SMOOTH MUSCLE CELL CONTRACTION.

EXQC’FI'WGHICJ/ Design 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 stimuli-induced muscle contraction is measured to evaluate the inhibitory effect ofthe analgesic agent and/or antimuscarinic agent.

The effectors, analgesic agents and scarinie agents are described in Example 2.

Primary culture of mouse bladder smooth muscle cells are subjected to short term (1-2 hrs) or long term (24-48 hrs) ation of with: (1) Each analgesic agent alone at various doses. (2) Each analgesic agent at various doses in the presence of LPS. (3) Each analgesic agent at various doses in the presence of carbachol or acetylcholine. (4) Each sic agent at various doses in the presence of AA, DGLA, or EPA. (5) Botulinum neurotoxin A alone at various doses. (6) num neurotoxin A at s doses in the presence of LPS. (7) Botulinum neurotoxin A at various doses in the presence ofcarbachol or acetyleholine. (8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA, or EPA. (9) Each antimuscarinic agent alone at various doses. (10) Each antimuscarinic agent at various doses in the presence of LPS. (1 1) Each antimuscarinic agent at various doses in the presence of hol or acetyleholine. (12) Each antimuscarinic agent at various doses in the presence of AA, DGLA, or EPA.

Materials and 61’s 9075662_l [GHMnnals) F970” NZ 2 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).

E 5: EFFECT OF ORAL SIC AGENTS AND ANTIMUSCARINIC AGENTS ON COX2 AND PGE2 RESPONSES OF MOUSE BLADDER SMOOTH MUSCLE CELLS.

Experimema/ design.’ Normal mice and mice with over active bladder syndrome are given oral doses ofaspirin, naproxen sodium, Ibuprofen, n, nabumetone, Tylenol, Celccoxib, oxybutynin, solifenacin, darifenaein, atropine and combinations f. Control groups include untreated normal mice and untreated OAB mice without over active bladder syndrome. Thirty (30) min after last doses, the bladders are collected and ated 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. Scrum PGHZ, PGE, PGEZ, Prostacydin, Thromboxanc, IL-IB, lL-6, TNF-a, CAMP, and cGMP levels are determined by ELISA. CD80, CD86, MHC class II expression in whole blood cells are determined by flow cytometry.

At the end ofthe experiment, animal are euthanized and ex vivo bladder contractions are recorded with a Grass aph. Portions of bladders are fixed in formalin, and COX2 ses are analyzed by immunohistoehemistry.

EXAMPLE 6: EFFECT OF ANALGESIC AGENTS BOTULINUM NEUROTOXIN AND ANTIMUSCARINIC AGENTS ON HUMAN BLADDER SMOOTH MUSCLE CELL SES TO TNFLAMMATORY AND NON-INFLAMMATORY STIMULI Experimental Design ] This study is designed to characterize how the optimal doses ofanalgesie determined in Examples 1-5 affect human r smooth muscle cells in cell e or tissue cultures, and to address whether different classes of analgesics can ize to more efficiently t COX2 and PGE2 responses.

The effectors, analgesic agents and antimuscarinic agents are described in Example 2.

Human bladder smooth muscle cells are subjected to short term (1-2 hrs) or long term (24-48 hrs) stimulation of with: 9075582~1 [GHMafleflJ P970“ NZ 2 (1) Each analgesic agent alone at various doses. (2) Each analgesic agent at s doses in the presence of LPS. (3) Each analgesic agent at various doses in the presence of carbachol or acetylcholinc. (4) Each analgesic agent at various doses in the presence of AA, DGLA, or EPA. (5) num neurotoxin A alone at various doses. (6) Botulinum neurotoxin A at various doses in the presence of LPS. (7) Botulinum neurotoxin A at various doses in the presence ofcarbachol or aeetylcholine. (8) Botulinum oxin A at various doses in the presence of AA, DGLA, or EPA. (9) Each antimuscarinic agent alone at various doses. (10) Each antimuscarinic agent at various doses in the presence of LPS. (1 1) Each antimuscarinic agent at various doses in the presence of carbachol or acetylcholine. (12) Each antimuscarinic agent at various doses in the presence of AA, DGLA, or EPA.

The cells are then analyzed for the release of PGHZ, PGE, PGEz, Prostacydin, Thromboxane, IL-lB, lL-6, TNF—a, the COX2 activity, the production of CAMP and cGMP, the tion B, IL—6, TNF~a 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.

Exacrimenta/ Design Cultured human bladder smooth muscle cells are exposed to inflammatory stimuli and flammatory i in the presence of analgesic agent and/or antimuscarinic agent at various concentrations. The stimuli—induced muscle contraction is measured to evaluate the inhibitory effect ofthe analgesic agent and/0r antimuscarinic agent.

Thc effectors, analgesic agents and antimuscarinic agents are bed in Example 2.

Human bladder smooth muscle cells are subjected to short term (1-2 hrs) or long term (24-48 hrs) stimulation of with: (1) Each analgesic agent alone at s doses. (2) Each analgesic agent at various doses in the presence of LPS. 9075662_1 [GHMntteln) P971199 N11 (3) Each analgesic agent at various doses in the presence of carbachol or acetylcholine. (4) Each analgesic agent at s doses in the presence of AA, DGLA, or EPA. (5) Botulinum neurotoxin A alone at various doses. (6) Botulinum oxin A at various doses in the presence of LPS. (7) Botulinum neurotoxin A at various doses in the presence of carbachol or acctylcholine. (8) Botulinum neurotoxin A at various doses in the presence of AA, DGLA, or EPA. (9) Each antimuscarinic agent alone at various doses. (10) Each antimuscarinic agent at various doses in the presence of LPS. (1 1) Each antimusearinic agent at various doses in the presence of carbachol or acetylcholinc. (12) Each scarinic agent at various doses in the presence of AA, DGLA, or EPA.

Bladder smooth muscle cell contractions are ed with a Grass aph y 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 Normal human r smooth muscle cells were isolated by enzymatic digestion from macroscopically normal pieces of human bladder. Cells were expended in vitro by culture at 37° C in a 5 % C02 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 rcsceding 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 ug/ml insulin.

Treatment of normal human bladder smooth muscle cells with analgesics in vitro Bladder smooth muscle cells trypsinized and seeded in ulture plates at a cell density of3x104 cells per well in 100 pl were treated with analgesic solutions (50 ltlr well) either alone or together carbachol (IO-Molar, 50 111/ well), as an example of non- 9075662 1 (GHM:|10I$)PS7U§9,NZ.2 inflammatory stimuli, or lipopolysaccharidc (LPS) of Salmonella typhi/mli'ium l uglml, 50 ul/ well), as an example of non-inflammatory stimuli. When no other effectors were added to the cells, 50 pl of RPMI 1640 without fetal bovine serum were added to the wells to adjust the final volume to 200 pl.

After 24 hours of culture, 150 pl of culture supernatants were collected, spun down for 2 min at 8,000 rpm at 4°C to remove cells and debris and stored at -70°C for analysis of Prostaglandin E2 (PGEZ) responses by ELISA. Cells were fixed, permeabilized and blocked for detection of COX2 using a fluorogenic substrate. In selected experiment cells were stimulated 12 hours in vitro for analysis of COX2, PC1132 and cytokine responses.

Analysis of COX2, PGE2 and cytokine responses COX2 and PGE2 responses were analyzed as described in Example 3.

Cytokine responses were analyzed as described in Example 2 RESULTS sics inhibit COX2 ses ofnormal human bladder smooth muscle cells to inflammaiory and non- inflammatory stimuli - Analysis of cells and culture supematants after 24 hours of cultures showed that none ofthe analgesics tested alone induced COX2 responses in normal human r smooth muscle cells. However, as summarized in Table 6, carbachol induced low, but cant 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 rCSponses when these drugs were tested at either 5 uM or 50 MM.

Table 6. COX2 sion by normal human bladder smooth muscle cells after in vitro stimulation with inflammatory and non- inflammatory stimuli and ent with analgesic Total COX2 ” Total coxz levels Stimuli sic (Normalized RFUs) (Normalized RFUs) Carbachol 10‘ M Acetaminophen (50 uM) Carbachol 10'3 M Aspirin (so uM) Carbachol 10'3 M Ibuprofen (50 uM) Carbachol 10'3 M Naproxcn (50 uM) Carbachol 10'3 M inophen (50 uM) 907 5662Hl (GHM allot.) PQTOQBiNZ 2 LPS (10 pg/ml) LPS (10 ug/ml) Acetaminophen (5 uM) LPS (IO pg/ml) n (5 uM) LPS (IO ug/ml) Ibuprofen (5 uM) LPS (10 uglinl) en (5 uM LPS (10 pig/ml) Acetaminophen (50 11M) IPS (IO ug/ml) Aspirin (50 uM) LPS (10 ug/ml) Ibuprofen (50 uM) LPS (10 uglinl) Naproxen (50 uM) Data are expressed as mean ofduplicates Analgesics t PGE2 responses ofnormal human bladder smooth muscle cells to inflammatory and non— inflammatory stimuli — Consistent with the induction of COX2 responses described above, both earbachol and LPS induced production of PGE2 by normal human bladder smooth muscle cells. Acetaminophen, aspirin, ibuprofen and naproxen were also found to ss the LPS-induced PGE2 responses at either 5 uM or SO uM (Table 7).

Table 7. PGE2 secretion by normal human bladder smooth muscle cells after in vitro stimulation with atory and non- inflammatory stimuli and treatment with analgesic PGEZ levels (pg/ml) Subject 2 Carbachol 10 M Acetaminophen (50 uM) Carbachol 10" M Aspirin (50 uM) 62 Carbaehol IO‘3 M Ibuprofen (50 uM) 59 Carbachol 10‘) M Naproxen (50 uM) 73 Carbachol 10‘3 M Acetaminophen (50 uM) LPS (10 ug/ml) LPS (10 ug/ml) Acetaminophen (5 uM) LPS (IO ug/ml) Aspirin (5 uM) LPS (10 ug/ml) Ibuprofen (5 uM) LPS (10 ug/ml) en (5 uM LPS (10 uglml) Acetaminophen (50 uM) LPS (10 ug/ml) Aspirin (50 pM) LPS (10 ug/ml) Ibuprofen (50 uM) LPS (10 tip/ml) Naproxcn (SO tiM) Data are expressed as mean of duplicates 9075662_I (GHM-ners) P970” NZ. 2 Analgesics inhibit cylokine responses ofnormal human bladder cells to an inflammatory stimuli - Analysis of cells and culture supernatants after 24 hours of cultures showed that none ofthe analgesics tested alone induced lL-6 or TNFa secretion in normal human bladder smooth muscle cells. As shown in Tables 8 and 9, the doses of carbaehol tested induced low, but cant TNFor 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 TNFa and lL-6 responses. The suppressive effect ofthe analgesics on LPS-induced responses was seen when these drugs were tested at either 5 uM or 50 uM.

Table 8. TNFa secretion by normal human r smooth muscle cells after in vitro stimulation with inflammatory and non- inflammatory i and treatment with analgesic Stimuli Analgesic ” TNFa (pg/ml) g/n1l) Subject 1 Subject 2 Carbachol 10 M None Carbachol 10' M Acetaminophen (50 uM) hol 10'3 M Aspirin (50 uM) Carbachol 10’3 M Ibuprofen (50 M) Carbachol 10‘3 M Naproxcn (50 uM) LPS (10 rig/ml) LPS (10 ug/ml) Acetaminophen (5 uM) LPS (IO ug/ml) Aspirin (5 HM) LPS (10 ug/ml) Ibuprofen (5 uM) LPS (IO ) Naproxen (5 uM LPS (IO pig/ml) inophen (50 uM) LPS (10 ) Aspirin (50 1.1M) LPS (10 ug/ml) Ibuprofen (50 MM) LPS (10 rig/ml) Naproxcn (50 uM) Data are expressed as mean ofduplieates, 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 907 566 2_I [GHMaIIufi 997099 NZ] Stimuli Analgesic lL—6 (pg/ml) " IL-6 (pg/ml) Subject 1 Subject 2 Carbachol 10 M None Carbachol 10' M Acetaminophen (SO uM) Carbachol IO'JM Aspirin (50 uM) hol 10‘3 M Ibuprofen (50 uM) Carbachol 10' M Naproxen (50 uM) LPS (10 rig/ml) inophen (5 uM) 2308 LPS (10 ug/ml) n (5 tiM) LPS (10 ug/tnl) Ibuprofen (5 uM) LPS (10 ug/ml) Naproxcn (5 uM LPS (10 rig/nil) Acetaminophen (50 1.1M) LPS (10 pig/ml) Aspirin (50 uM) LPS (10 ug/ml) Ibuprofen (50 uM) LPS (IO ug/ml) Naproxen (50 uM) Data are expressed as mean ofduplicatos Primary normal human bladder smooth muscle cells were isolated, cultured and ted for their responses to analgesics in the presence of non-inflammatory chol) and inflammatory (LPS) stimuli. The goal ofthis study was to determine whether or not normal human bladder smooth muscle cells recapitulate the observations previously made with murine bladder cells.

The above-described experiment will be repeated with analgesic agents and/0r antimuscarinic agents in d-release, 0r extended-release formulation 0r delayed-and- cd-releasc formulations.

The above description is for the purpose ofteaching the 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 ofit 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 ofthe present invention, which is defined by the following . The claims are intended to cover the claimed ents and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary. 9075662”! (GHMatlevs) PSTOSSNZ 2

Claims (9)

WHAT IS CLAIMED IS:

1. Use of a pharmaceutical composition comprising a first component formulated for immediate-release and a second component formulated for extended-release in the cture of a medicament for overactive bladder syndrome in a subject in need thereof, wherein the first component and the second component each comprises acetaminophen and ibuprofen, and wherein each of the acetaminophen and ibuprofen in the first and second components is t in an amount of 5 mg to 2000 mg.

2. The use of Claim 1, wherein each of the acetaminophen and ibuprofen in the first and second components is present in an amount of 50 mg to 500 mg.

3. The use of Claim 2, wherein each of the acetaminophen and ibuprofen in the first and second components is present in an amount of 100 mg to 500 mg.

4. The use of Claim 3, n each of the acetaminophen and ibuprofen in the first and second components is present in an amount of 250 mg to 500 mg.

5. The use of Claim 1, wherein each of the acetaminopehn and ibuprofen in the first and second components is present in an amount of 250 mg to 1000 mg.

6. The use of any one of Claims 1-5, the inophen and ibuprofen in the second component are embedded in a matrix of insoluble substance(s).

7. The use of any one of Claims 1-5, wherein the second component comprises a polymer controlling release by dissolution controlled release.

8. The use of any one of Claims 1-5, n the second component comprises a water soluble or water-swellable matrix-fonning r.

9. The use of any one of Claims 1-5, wherein the second ent comprises an enteric coating. PCT/USZOIZ/051888 1/]. owmwmficd‘ ummwmficd :21 ceuaoééfifduitu m“ mmoo 808583 3.235;}? “Amwwoxmfifiliztn at n, .. .. c3 a? a: cw av ANN gazwmfixwm acoum a «mum cnx :21 Emwmfimd. :1“. we 3 mmoo 883,83“ .3 . _ own can 9% omfi 03. 23 om agsafimuv Jnomgm gonuoo ;o waxed) SII33 +0803+01703