NZ626619B2 - Extended-release formulation for reducing the frequency of urination and method of use thereof - Google Patents

Abstract

Disclosed the use of a pharmaceutical composition comprising acetaminophen in combination with either (1) one or more antidiuretic agents (e.g. desmopressin), or (2) one or more spasmolytics, or (3) an antimuscarinic agent selected from the group consisting of oxybutynin, solifenacin, darifenacin and atropine, in the manufacture of a medicament for treating nocturia in a subject in need thereof, wherein said pharmaceutical composition is formulated in an extended-release formulation and wherein said acetaminophen is formulated for administration at a daily dose of 1 mg to 2000 mg. d atropine, in the manufacture of a medicament for treating nocturia in a subject in need thereof, wherein said pharmaceutical composition is formulated in an extended-release formulation and wherein said acetaminophen is formulated for administration at a daily dose of 1 mg to 2000 mg.

Description

PCT/U82012/051888 EX'I‘EN‘DEI»RELEASE FORMULATION FOR REDUCING THE FREQUENCY OF URINATION AND METHOD OF USE F [(1001] This application claims the priority oiliS‘. Patent Application Serial No. 13/487348, tiled on June 4. 2012, US. Patent Application Serial No. 13/424,000, filed on March 19, 2012, and US. Patent Application Serial No. 13/343,332, filed on January 4, 2012.

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

BACKGROUND 10003] The detrusor muscle is a layer of the urinary bladder wall made of smooth muscle fibers arranged in spiral, longitudinal, and circular bundles. When the bladder is stretched, this s the parasympathetic nervous system to contract the dctrusor muscle.

This encourages the bladder to expel urine through the urethra. [0004! For the urine to exit the bladder, both the autonomically controlled internal sphincter and the voluntarily lled external sphincter must be opened. Problems with these muscles can lead to incontinence. 1f the amount of urine reaches 10 % of the urinary bladdcr's absolute capacity, the voluntary sphincter becomes involuntary and the urine will be ejected instantly. {0005] The human adult urinary bladder usually holds about 300-350 ml of urine (the g volume), but a full adult bladder may holdup to about l000 ml (the absolute volume), varying among individuals. As urine accumulates, the ridges produced by g of the wall of the bladder (rugae) n and the wall ol‘the bladder thins as it stretches, allowing the r to store larger amounts ol‘ urine without a significant rise in internal pressure.

In most duals, the desire to e usually starts when the volume of urine in the bladder reaches around 200 ml. At this stage it is easy For the subject, il‘desired, to resist the urge to urinate. As the bladder continues to fill, the desire to urinate becomes er and harder to ignore. Eventually, the bladder will fill to the point where the urge to e s overwhelming, and the subject will no longer be able to ignore it. In some i nt‘lividuals, this desire to urinate starts when the bladder is less than 100% full in relation to its working volume. Such increased desire to urinate may interfere with normal activities, including the ability to sleep for sufficient uninterrupted periods of rest. In some cases, this increased desire to urinate may be associated with medical conditions such as benign prostate hyperplasia or prostate cancer in men, or pregnancy in women. r, increased desire to urinate also occurs in individuals, both male and female, who are not affected by another 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 ction 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 One aspect of the present application relates to a method for reducing the frequency of urination. The method comprises stering to a subject in need thereof an effective amount of a pharmaceutical composition comprising acetaminophen, wherein the pharmaceutical composition is formulated in an extended-release formulation and wherein said acetaminophen is administered at a daily dose of 1 mg to 2000 mg. The method can be used for the treatment of ia.

Another aspect of the present ation 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 pharmaceuti cal composition comprising: a first component formulated for ate-release; and a second component ated for extended-release, wherein the first component and/or the second component comprise acetaminophen, and wherein said acetaminophen is administered at a daily dose of 50 mg to 2000 mg. The method can be used for the treatment of ia.

Another aspect of the present application relates to a pharmaceutical composition comprising: acetaminophen; an antidiuretic agent, a nd a pharmaceutically acceptable carrier, wherein the ceutical composition is formulated for extendedrelease , and wherein the dosage of said acetaminophen is 50 mg to 2000 mg. [0010A] In one aspect, the present invention provides use of a pharmaceutical composition comprising acetaminophen in ation with either (1) one or more antidiuretic agents, or (2) one or more spasmolytics, or (3) an antimuscarinic agent selected from the group consisting of oxybutynin, nacin, darifenacin and atropine, 6047.1 8766425_1 (GHMatters) P97099.NZ in the manufacture of a medicament for treating nocturia in a subject in need thereof, wherein said pharmaceutical composition is formulated in an extended-release formulation and wherein said acetaminophen is formulated for administration at a daily dose of 1 mg to 2000 BRIEF DESCRIPTION OF GS Figure 1A and 1B are diagrams showing that analgesics te expression of co-stimulatory molecules by Raw 264 hage cells in the absence (Figure 1A) or WAS:186047.1 8766425_1 (GHMatters) P97099.NZ presence (Figure 1B) of LPS. Cells were cultures for 24 hrs in the presence of analgesic alone or together with Salmonella typhimurium LPS (0.05 ). Results are mean relative % of CD40+CD80+ cells.

DETAILED 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 thorough understanding of the present invention. However, it will be apparent to one skilled in the art that these specific details are not required to practice the invention. ptions 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 st possible 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 ive of anti—inflammatory compounds. Exemplary analgesic and/or anti-inflammatory agents, compounds or drugs include, but are not limited to, the ing substances: non-steroidal anti-inflammatory drugs (NSAIDs), salicylates, aspirin, salicylic acid, methyl salicylate, diflunisal, salsalate, olsalazine, sulfasalazine, para- aminophenol derivatives, acetanilide, acetaminophen, phenacetin, fenamates, mic acid, meclofenamate, sodium meclofenamate, heteroaryl acetic acid derivatives, tolmetin, ketorolac, diclofenac, nic acid tives, ibuprofen, en sodium, naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin; enolic acids, oxicam derivatives, piroxicam, meloxicam, tenoxicam, ampiroxicam, droxicam, pivoxicam, pyrazolon derivatives, phenylbutazone, oxyphenbutazone, antipyrine, aminopyrine, dipyrone, , xib, xib, nabumetone, apazone, indomethacin, ac, etodolac, ylphenyl propionic acid, lumiracoxib, etoricoxib, parecoxib, valdecoxib, tiracoxib, etodolac, darbufelone, dexketoprofen, fenac, licofelone, bromfenac, loxoprofen, pranoprofen, piroxicam, nimesulide, cizolirine, 3-formylaminomethylsulfonylaminophen0xy-4H- l -benzopyran- 5482038_I (GHMatlm) P97099 N2 PCT/U52012/051888 4—one, meloxicam, lomoxicam, d-indobufen, mofezolac. amtolmctin, pranoprofen: tolfenaniic acid, [lurbiprot‘em suprot‘en. zin. aaltoprolen, alminoprofen, tiaprofenie acid, pharmacological salts thereof, hydrates thereof, and solvates thereof.

As used herein, the terms “coxib” and “COX inhibitor" refer to a composition of compounds that is capable ofinhi‘oiting the activity or expression Z enzymes or is capable ol'inhibiting or reducing the severity, including pain and ng, of a severe inflammatory response. {0016] The urinary bladder has two important functions: storage of urine and emptying. Storage of urine occurs at low pressure, which implies that the detrusor muscle relaxes during the filling phase. Emptying, of the bladder requires a coordinated contraction of the detrusor muscle and relaxation of the sphincter muscles ol‘the urethra. Disturbances of the e on may result in lower urinary tract symptoms, such as urgency, frequency. and urge inence. the components ol'thc overactive bladder me. The overactive bladder syndrome, which may be due to ntary contractions of the smooth muscle of the bladder (detrusor) during the storage phase, is a common and underrcportcd problem, the prevalence of which has only recently been assessed. [0017} One aspect of the present application relates to a method for reducing the frequency of urination by administering to a person in need thereof a phannaceutieal composition formulated in an extended-release formulation. The pharmaceutical composition comprises one or more analgesic agents and, optionally, one or more scarinic agents,onc or more urctic 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), timc~relea5e (TR), controlled-release (CR), modified release (MR), or continuous~ release (CR), is a mechanism used in medicine tablets or capsules to dissolve slowly and release the active ingredient over time. The advantages nded-relcasc s 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 ol‘the drug in the bloodstream, thus extending the duration of the drug action. For example, an extended—release analgesic may allow a person to sleep h the night without getting up for the bathroom.

In one embodiment, the ceutical composition is formulated for extendednelease by ing the active ingredient in a matrix of insoluble substancet's) such as acrylics or chitin. An extendcd~r‘clcase form is designed to release the analgesic compound at a predetermined rate by ining a constant drug level for a specific period ol'time.

PCT/U520 1 21051888 This can be achieved through a variety of formulations, including, but not limited to liposotncs and drug—polymer conjugates, such as liydrogels.

An extended-release formulation can be designed to release the active agents at a predetemtincd rate so as to maintain a nt drug level for a specified, extended period ot‘time, 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 i hour following administration or following a lag period associated with delayed-release of the drug. {0021] in certain preferred embodiments, the active agents are released over a time interval of between about 2 to about 30 hours. Alternatively, the active agents may be released 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 stration. in some embodiments, the extended—release ation ses an active core comprised clone or more inert particles, each in the form of a bead, pellet, pill, granular particle, microcapsule, microspherc, microgranule, nanocapsule, or nanosplterc coated on its surfaces with dings in the form of e.g., a drug-containing coating or film-Forming composition using, for example, fluid bed techniques or other ologies known to those of skill in the art. The inert particle can be of various sizes, so long as it is large enough to remain poorly dissolved. Alternatively, the active core may be prepared by granulating and milling and/or by extrusion and spheronization of a polymer composition containing the drug substance.

The active agents may be introduced to the. inert carrier by techniques known to one skilled in the art. such as drug layering, powder g, extrusiou/sphcronization. roller compaction or granulation. The amount of drug in the core will depend on the dose that is required, and typically varies from about 5 to 90 weight %. lly, the polymeric coating on the active core will be from about l to 50% based on the weight n t‘ the coated particle, depending on the lag time required and/or the polymers and coating solvents chosen.

Those skilled in the art will be able to select an appropriate amount ol’ drug for coating onto or incorporating into the core to e the desired dosage. In one ment, the ve core may be a sugar sphere or a buffer crystal or an encapsulated buffer crystal such as calcium carbonate, sodium bicarbonate, fumaric acid, tartaric acid, etc. which alters the microenvironmcnt of the drug to tate its release. {0024] Extended—release fomtulations may e a variety ol’extended—release W0 20131103390 PCT/U52012/051888 coatings or mechanisms Facilitating the gradual release of active agents overtime. in some embodiments, the extended~relcase agent comprises a polymer controlling release by ution controlled release. in a particular embodiment, the active agent(s) are incorporated in a matrix sing an insoluble polymer and drug particles or granules coated with ric materials ot‘varying thickness. The polymeric material may comprise a lipid barrier comprising a waxy material, such as carnauba wax, beeswax, spermaccti wax, candellila wax, c wax. cocoa butter, etostearyl alcohol, partially enated vegetable oils, ceresin, paraffin wax, ne, myristyl alcohol, stcaiyl alcohol, cetyl alcohol and stearic acid, along with surfactants, such as polyoxyethylene sorbitan monool ante. When contacted with an aqueous medium, such as biological fluids, the polymer g emulsifies or erodes alter a predetermined lag-time depending on the thickness of the polymer coating.

The lag time is independent of gastrointestinal motility, pl-l, or gastric residence. {0025] 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 rs, pH—depcndcnt polymers. and/or pl-l‘independent polymers. 10026] ln one ment, the extended—release formulation comprises a water e or water-5i 'ellable rnatrix-lorming polymer, Optionally containing one or more Solubility—enhancing excipicnts and/or release—promoting agents. Upon solubilization of the water soluble polymer, the active agent(s) ve (if soluble) and lly diffuse through the hydrated portion of the matrix. The gel layer grows with time as more water permeates into the core of the matrix, increasing the thickness of the gel layer and ing a diffusion barrier to drug release. As the outer layer becomes fully hydrated, the polymer chains become completely relaxed and can no longer maintain the integrity of the gel layer, g to disentanglement and erosion ol‘the outer hydrated polynter on the surface of the matrix.

Water continues to penetrate towards the core through the gel layer, until it has been completely eroded. Whereas soluble drugs are released by this combination 0 l" diffusion and n mechanisms, erosion is the inant mechanism for insoluble drugs, regardless of dose. {0027] Similarly, water—swellable polymers typically hydrate and swell in biological fluids forming a homogenotts matrix structure that maintains its shape during drug release and serves as a carrier for the drug, solubility enhancers and/or release promoters. The initial matrix polymer hydration phase results in slow-release ot‘the drug (lag phase). Once the water swellable polymer is Fully hydrated and n, water within the matrix can similarly W0 2013/103390 PCT/U52012/051888 dissolve the drug substance and allow for its diffusion out h the matrix coating.

Additionally, the porosity ol’the matrix can be increased due to the leaching out of pH—dependent e promoters so as to release the drug at a faster rate The rate of the drug release then becomes constant and is a function of drug diffusion through th e. hydrated polymer gel. The release rate from the matrix is dependent upon s factors, including polymer type and level; drug solubility and dosa; polymer: drug ratio; tiller type and level; polymer to tiller ratio; particle size g and polymer; and porosity and shape of the. matrix. 10029] Exemplary hydrophilic andi'or water-sweliable, matrix forming polymers include, but are not d to, cellulosic polymers, including hydroxyalkyl celluloscs and ear‘ooxyalkyl celluloses, such as hydroxypropylmethylcellulosc (HPMC), hydroxypropylcellulosc (HPC), hydroxyethylcellulose (HEC), methylcellulose (MC), earboxymethylcellulosc (CMC ), powdered ose such as microCiystallinc cellulose, cellulose acetate, ethylcellulose, salts thereof, and combinations f; alginates, gums, including heteropolysaceharide gums and homopolysaccharide gums, such as xanthan, tragacanth, pectin, , karaya, alginates, agar, guar, hydroxypropyl guar, veegum, carragcenan, locust bean gum, gellan gum, and derivatives thereofrom; acrylic resins, including polymers and copolymers of acrylic acid. methacrylic acid, methyl acrylate and methyl methacrylate and cross—linked polyaciylic acid derivatives such as ers (cg, CARBOPOI..®, such as ing CIARBOPOL‘E’ 71G NF, available in various molecular weight grades from Noveon, lnc., Cincinnati, OH): carageenan; polyvinyl acetate (cg, KOLLEDONQ‘ SR); polyvinyl pyrrolidone and its derivatives such as crospovidonc; polyethylene oxides; and polyvinyl alcohol. Preferred hydrophilic and swellable polymers include the cellulosic polymers, especially HPMC. {0030] The. extended-release formulation may further se at least one hinder that is capable of crossulinking the hydrophilic compound to form a hydrophiiie polymer matrix (is, a gel matrix) in an aqueous medium, including biological lluids.

Exemplary binders e lysaccharidcs, such as galactomunnan gums, guar gum, ypropyl guar gum, hydroxypropylcellulose (llPC; sag, Klucel EXP) and locust bean gum. In other embodiments, the binder is an alginic acid derivative, HPC or microcrystallized cellulose (MCC‘). Other binders include, but are not limited to, starches, microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose and polyvinylpyrrolidone. in one embodiment, the introduction method is drug layering by spraying a PCT/U52012/051888 suspension of active agent(s') and a binder onto the inert carrier.

The binder may be t in the bead formulation in an amount of from about 0. % to about 1 % by weight, and preferably of from about 0.2% to about 10% by . in some embodiments, the hilie polymer matrix may further include an ionic polymer, 3 non-ionic polymer, or water-insoluble hydrophobic polymer to provide a stronger gel layer and/"or reduce pore quantity and ions in the matrix so as to slow diffusion and n rates and concomitant release of the active agent(s). This may additionally suppress the initial burst effect and produce a more steady, “zero order release" of active agent(s).

Exemplary ionic polymers for slowing dissolution rate e both anionic and cationic polymers. Exemplary anionic polymers e, for example, sodium earboxymethy[cellulose (Na CMC), sodium alginale, polymers of acrylic acid or carbomcrs (eg, OLW 934, 9-40, 974? NF); e polymers, such as polyvinyl acetate phthalate (PVAP), methacrylie acid copolymers (e.g., EUDRAGI‘I‘W l.ll)0, L 30D 55, t and FS 30D), hypromellose acetate succinale (AQUAT '); and xanthan gum.

Exemplary cationic polymers include, for example, dimethylaminoethyl methacrylate eopolymer (8.33., EUDRAGI'T?‘ E 100‘). incorporation of anionic polymers, particularly enterie polymers, is useful for developing a pl-lsiudependent release profile for weakly basic drugs as ed to hydrophilic polymer alone. {0036} Exemplary nic polymers for slowing dissolution rate, include, for example, hydroxypropylcellulose (HPC) and polyethylene oxide (PEO) (ag, POLYOXTM) {0037] Exemplary hydrophobic polymers include ethyleellulose (tag, ETHOCEL"‘M, SURELEASE“), cellulose acetate, methacrylic acid copolymers (gag, lZIJDRAGl‘l‘Tc NE 30D), ammonio-methacrylatc eopolymers (tag, EUDRAGIT‘PC RL 100 or PO R8100), polyvinyl acetate. glyceryl inonostearate, fatty acids, such as aeetyl tributyl citrate, and combinations and derivatives thereof.

The ble polymer can be incorporated in the formulation in proportion from I% to 50% by weight, preferably from 5% to 40% by weight, most preferably from 59 o to 20% by weight. The swellable rs and binders may be incorporated in the formulation either prior to or after granulation. The polymers can also be dispersed in organic solvents or hydro—alcohols and sprayed during granulation.

Exemplary release~promoting agents include pit-dependent enteric polymers that remain intact at, pH value lower than about 4.0 and ve at. pH values higher than 4.0, 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 liuu'ted to, arylie acid cepolymers, methaerylic acid—methyl methacrylate eopelymers (e.g., EUDRAGI'I"R L100 (Type A). EUDlMCEl'l‘® S 100 (Type B), Rohm GmbH. Germany: methaerylie acidetliyl aerylate copolymers (e.g., EUDRAGlT'E £10065. (Type (I) and IEZIJIDRAGI'TR’ L30D—55 copolymer dispersion, Rohm Gmbl-l, Germany); copolymers of methaerylic acid~methyl methaerylate and methyl metltacrylate (EUDRAGITE' PS); tel-polymers of methacrylie acid, rylate, and ethyl acrylate: cellulose acetate phtltalates (CAP); hydroxypropyl methyleellulose phthalate (HPMCP) (tag, HP~55, PIP-50‘ HP~SSS, Shinetsu Chemical, ; polyvinyl acetate phthalates (PVAP) (ego EC‘ZE', OPADRY'R‘ e white OY-P—717l); polywinylbutymte acetate; cellulose acetate succinates (CA8); hydroxypropyl methyleellulose acetate suecinate (l-lPMCAS), eg, l-lPMCAS LF Grade, MP Grade, HF Grade, including AQOA’rl‘ LF and AQOA"i"‘-“ MF (Shin-Etsu al. Japan); Shinetsu Chemical, Japan); shellac (mg, l\rl/1.RCOATm 125 & MARCO/VFW 125N); vinyl acetate-maleic anhydride copolymer; styrene-maleie monoester copolymer; carboxymethyl ethylcellulose (CMEC, Freund Corporation, Japan}; cellulose acetate phthalates (CAP) (rag. /~\('\)UA'l”lE-JRICR‘); cellulose acetate litates (CAT); and mixtures of two or more thereof at weight ratios between about 2:1 to about 5:1. such as, for instance, a mixture or EUDRAGIT‘" 1., 100-55 and EUDRAGITl“ s 100 at a weight ratio of about 3:1 to about 2:1, or a e of EUDRAGI'I‘I'Q‘ L 30 D-SS and EUDRAGl'l'fi PS at a weight ratio ot" about 3tl to about 5: l. 10040] 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 metltacrylie acid capolymers. These copolymers are anionic polymers based on mcthaetjylic acid and methyl metltacrylate and, preferably. have a mean molecular weight of about 85,000. A ratio of free carboxyl groups to methylnesteritied carboxyl groups in these copolymers may range, for example. from 1:1 to 1:3, 6. g. around lzl or 1:2. Such polymera are. sold under the. trade name Eudragit‘ such as the Eudragit 1.. series tag, it L 12.5”: Eudragil L 12.5l”, Eudragil L100®, Eudragit L 100-553, Eudragit L.— 300'3', Eudragit 1,30 17—55%, the it 5“ series e.g., t s 12.5", Eudragit 3 12.599 it 3100*: The release ers are not limited to pH dependent rs. Other hydropltilie molecules that dissolve rapidly and leach out of the dosage form quickly leaving a porous structure can be also be used for the same purpose.

The release-promoting agent can be. incorporated in an amount from 10% to 909/5, preferably from 20% to 809/0 and most. preferably from 30% to 70% by weight of the PCT/U52012/051888 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 dispersed or dissolved in an appropriate solvent, and dispersed during granulation. {0042] in some embodiments, the matrix may include a combination of release promoters and solubility enhancers. The lity ers can be ionic and non-ionic surfactants, cxing agents, liydrophilic rs, pl-l modifiers, such as acidifying agents and alkalinizing agents, as well as les that increase the solubility ofpoorly soluble drug through molecular entrapment. Several solubility enhancers can be ed simultaneously. {0043] Solubility enhancers may include surface active agents such as sodium docusatei sodium Iauryl sulfate, sodium stearyl fumarate, 'I'weens'g and Spans (PEO modified sorbitan monoesters and fatty acid sorbitan esters), polylcthylene oxide)—polypropylenc oxide-poly(ethylene oxide) block copolymers (aka l’l..URONlCSW); complexing agents such as low lar weight polyvinyl pyrrolitlone and low molecular weight hydroxypropyl methyl cellulose; les that aid solubility by molecular ment such as eyelodextrins, and pH modifying agents, including acidifying agents such as citric acid, fumuric acid, tartaric acid, and hydrochloric acid; and zing agents such as meglumine and sodium hydroxide.

Solubility enhancing agents typically constitute from 1% to 80% by weight, pre ‘erably from 1% to 60%, more preferably from £94) to 50%. of the dosage form and can be ineorpera ted 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, solubilizers can be sprayed as solutions with or without a binder.

In some embodiments, the extended-release formulation comprises a polymeric matrix that can e for release of the drug after a certain time independent of the pH. For purposes of the t invention, “pH independent” is d as having characteristics (eg, dissolution) which are substantially unaffected by pl-l. pi-l independent polymers are often referred to in the context of “time~controlled” or “time—dependent" release profiles. 100461 A pl'l independent polymer may be used to coat the active agent and/or provide a polymer for a hydrophilie matrix in the extended-release coating thereover. The pH independent polymer may be water-insoluble or water soluble. Exemplary water PCT/U820] 88 insoluble pH independent polymers include, but are not limited to, neutral methacrylic acid esters with a small portion ethylammonioethyl methacrylate chloride (cg, EUDl'tAGlTk' RS and EUDRAGIT'E RL; neutral ester dispersions without any fitnetional groups (fig, moment NE30D and EUDR.AG[T®NE30); osic polymers, such as ellulose, hydroxyl ethyl cellulose, cellulose acetate or mixtures and other pH independent coating products. Exemplary water soluble pH independent polymers include yalkyl cellulose others, such as hydroxypropyl methylcellulose C), and hydroxypropyl cellulose (HPC‘); polyvinylpyrrolidone (PVP), methylccllulosc, C)PADRY®arrib, gear gum, xanthan gum, gum , hydroxyethyl cellulose and ethyl acrylttte and methyl methacrylate copolymer dispersion or combinations thereof. ln one embodiment, the extended—release l‘onnulation ses a water- insoluble water-permeable polymeric coating or matrix comprising one or more water- ble watcr-penneable lilmvforming over the active core. The coating may additionally include one or more water soluble polymers and/or one or more plasticizers. The water- insoluble polymer coating comprises a barrier coating for release of active agents in the core, wherein lower molecular weight (viscosity) grades exhibit faster release rates as ed to higher viscosity grades. in preferred embodiments, the water—insoluble ornting polymers include one or more alkyl cellulose others. such as ethyl cclluloses and mixtures thereof. tag, ethyl cellulose grades PRIOO, PR45, PRZO, PRIO and PR7: ETl~lOCEI..'/’§, Dow).

An exemplary water-soluble polymer Such as polyvinylpyrrolidonc (POVlDONEtF/i, hydroxypropyl methylcellulose, hydroxypropyl cellulose and mixtures thereof, {0050! In some embodiments, the watervinsoluble polymer provides suitable properties (era. extended~release characteristics, mechanical properties, and coating properties) without the need for a plasticizer. For example, gs comprising polyvinyl acetate (PVA), neutral copolymers ofacrylateimethacrylz'tte esters such as commercially ble Eudragit NE3OD from Evonik industries, ethyl cellulose in combination with hydroxypropylcellulose, waxes, etc. can be applied without plasticizers. |005l1 ln yet another embodiment, the water-insoluble polymer matrix may further include a plasticizer. The amount of plasticizer required depends upon the cizer, the properties of the water—insoluble r, and the ultimate desired properties of the coating.

Suitable levels of plasticizer range from about 1% to about 20%, from about 3% to about %, about 3% to about 5%, about 7% to about 10%, about 12% to about l5%, about 17% to PCT/U82012/051888 about 20%, or about 1%, about %, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about. 10%. about l5%, or about 20% by weight relative to the total weight of the coating, inclusive of all ranges and sub-ranges therebctwccn.

Exemplary plasticizers include, but are not limited to, triaeetin, acetylated monoglyceride, oils {caster oil, hydrogenated castor oil, rape seed oil, sesame oil, olive oil, etc); citrate esters, tricthyl citrate, acetyltricthyl citrate acctyltributyl citrate, tributyl citrate, acetyl tri—n-butyl citrate, diethyl phtltalate, dibut‘yl phthalate, dioctyl phthalate, methyl n, propyl paraben, pi'opyl paraben, butyl parabert, diethyl te, dibutyl sebacate, glyceroltributyrate, tuted cerides and glycerides. rnonoacctylated and diacctylated glyceridcs tag, MYVACE'I“ 9-45), yl monostearate, glycerol tributyratc, polysorbate 80, polyethyleneglycol (such as PEG-400i}, PEG-400), propyleneglyeol, l,Zmropylcneglycol, glycerin, sorbitol, diethyl oxalate, diethyl malatc, l funiaratc, diethylmalonate, dibutyl succinatc, fatty acids, glycerin, sorbitol, dicthyl oxalate, diethyl malate. dicthyl maleato, diethyl fumarate, diethyl succinate, dietltyl te, dioctyl ate. dibutyl sehaeate, and mixtures thereof. The plasticizer can have surfactant properties, such that it can act, as a release modifier, For example, nonnionic ents such at Brij 58 (polyoxyethylene (‘20) cetyl , and the like, can be used. {0053] eiaers can be high boiling point organic solvents used to impart flexibility to otherwise hard or brittle polymeric materials and can affect the release profile for the active agends). Plasticizers generally cause a reduction in the cohesive intermolecular forces along the polymer chains resulting in various s in polymer properties including a reduction in tensile strength, and se in elongation and a reduction in the glass transition or softening temperature ot‘the polymer. The amount and choice of the plasticizer can affect the ss oft-1 tablet, for e, and can even affect its dissolution or disintegration characteristics, as well as its physical and chemical stability, Certain plasticizers can se the elasticity and/or pliability ofa coat, thereby decreasing the coat‘s brittleness. in another embodiment, the extended-release formulation comprises a combination ofat least two gel-forming polymers, including at least one nie gel~ forming polymer and/or at least one anionic gel~forming polymer. The gel fomted by the combination of gel-forming polymers provides controlled release, such that when the formulation is ingested and comes into contact with the gastrointestinal fluids, the polymers t. the surface hydrate to form a viscous gel layer. Because of the high viscosity, the viscous layer dissolves away only gradually, ng the material below to the same PCT/U82012/051888 process. The mass thus dissolves away , thereby slowly releasing the active ingredient into the gastrointestinal fluids. The combination ol‘at least two gel—forming polymers enables properties ofthe resultant gel, such as viscosity, to be manipulated in order to provide the desired release profile. (0055] in a particular embodiment. the formulation comprises at least one non-ionic gel-forming polymer and at least one anionic gel-forming polymer. in another ment, the atiou comprises two different non-ionic gel-forming polymers. in yet another embodiment, the Formulation comprises a combination of nomionie gel—forming polymers of the same chemistry, but having different solubilities, viscositics, and/or lar weights ( for example a combination of hydroxyproplyl methylcellulose of different viscosity grades, Such as l-lPMC K100 and HPMC K 15M or HPMCI K 100M). {0056] Exemplary anionic gel forming polymers include, but are not limited to, sodium carboxymethylccllulose (Na CMC), carboxymethyl ose (CMC), anionic polysaccharides such as sodium alginate, alginic acid, pectin, polyglucuronic acid (poly-nt— and ~B~l ,Aloglucuronic acid), lacturonic acid (pectic acid), chondroitin sulfate, carrageenan, furcellaran, anionic gums such as xanthan gum, polymers of acrylic acid or carbomers (Carbopol'fi 93-4, 940, 9741’ NF), Carbotiol'lg copolymers, a l’cmulcng” polymer, poly tarbophil, and others. [0057 l Exemplary non-ionic gel-forming polymers include, but are not limited to, l‘ovidone (PVP: polyvinyl pyrrolidone}. polyvinyl alcohol, copolymer of PVP and polyvinyl e, HPC xypmpyl cellulose), HPMC (hydroxypropyl methylcellulose), hydroxyethyl cellulose, hydroxymethyl cellulose, gelatin. polyethylene oxide, acacia, dcxtrin, , polyhydroxyethylmetliacrylate (l’llBMA), water e nonionic polymethaerylates and their copolymers, modified cellulose, modified polysaccharides, nonionic gums, nonionic polysaccharides and/er mixtures thereof.

The formulation may optionally comprise an entcric r as described above, and/or at least one excipient, such as a tiller, a binder (as bed above), a disintegrant, anther a flow aid or glidant. {0059] Exemplary lillers include but are not d to, lactose, e, fructose, e, dicalcium phosphate, sugar alcohols also known as "sugar polyol" such as sorbitol, manitol, lactitol, xylitol, isomalt, crytltritol, and hydrogenated starch hydrolysatcs (a blend of several sugar alcohols), corn starch, potato starch, sodium carboxymethycellulose, cthylcellulose and cellulose acetate, c polymers, or a mixture thereof.

Exemplary binders, include but are not limited to, water—soluble hydrophilic PCT/U82012/051888 polymers, such as one (PV 1’: polyvinyl pyrrolidone), copovidone (a copolymcr of polyvinyl pyrrolidone and polyvinyl acetate), low molecular weight HPC (hydroxypropyl cellulose) low molecular weight HPMC‘ (hydroxypropyl methylcellulosc), low molecular weight carboxy methyl cellulose, ethylcelltilose, gelatin, polyethylene oxide, acacia, dextrin, ium aluminum silicate, , and polymethacrylates such as Iiudragit NE 309, Eudragit RL, Eudragit RS, Eudragit E, polyvinyl acetate, and enteric polymers, or mixtures Exemplary disintegrants include but are not limited to low-substituted carboxymcthyl cellulose sodium, crosptwitlone (cross—linked polyvinyl pyrrolidone'), sodium carboxymcthyl starch (sodium starch glycolaie), Cl‘OSS—linkcd sodium carboxymethyl cellulose (Croscamiellose), pregelatinizcd starch (starch l5l}0), microcrystalline cellulose, water insoluble starch. calcium carboxymetltyl cellulose, low substituted hydroxypropyl cellulose, and magnesium or aluminum silicate. {0062} Exemplary glidants include but are not limited to. magnesium, silicon dioxide. talc, starch, titanium dioxide. and the like. {0063] in yet another embodiment, the extended-release formulation is formed by coating 21 water soluble/dispersible drug—containing particle, such as a head or head population therein (as described above), with a coating material, and, optionally, a pore former and other excipients. The coating material is preferably selected from a group comprising cellulosic polymers, such as cthylcellulose (sag. SLJR.El.,EA.SE®), methylcellulose, hydroxypropyl cellulose, hydroxypropylmcthyl cellulose, ose acetate, and cellulose acetate phthalate; nyl alcohol; c rs such as rylates. polyntethacrylates and mers thereof. and other water—based or t-based coating materials. The release-controlling coating for a given bead population may be controlled by at least one parameter of the release controlling coating, such as the nature of the coating, coating level, type and concentration of a pore former, process parameters and ations f. Thus, changing a parameter, such as a pore former concentration, or the ions 01" the curing, allows for changes in the release of active agent(s) from any given bead population, thereby ng for selective adjustment ol‘the ation to a prc~detcnnined release profile. {0064] 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 d from the coating in the environment of use. Exemplary pore forming agents include, but are not d to, organic compounds such as mono~, oligo~, and polysaccharit‘les including sucrose, glucose, fructose, mannitol, mannose, galactose, sorbitol, an, dextran; polymers soluble in the environment ol’use such as water-soluble ltydrophilic polymers, yalkylcclluloscs, earboxyalkylcelluloses, hydroxypropylmethylcellulose, ose , acrylic resins, polyvinylpyrrolidone, erossdinkcd polyx inylpyrrolidone, polyethylene oxide, Carbowaxes, Carbopol, and the like, diols, polyols, polyhydric alcohols, polyalkylene glycols, polyethylene glycols, polypropylene glycols, or block polymers thereof, polyglycols, poly(a~t'2)alkylenediols; inorganic compounds such as alkali metal salts, lithium carbonate, sodium chloride, sodium bromide, potassium de, potassium sulfate, potassium phosphate, sodium acetate, sodium e, suitable calcium salts, combination thereof, 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 antitbants. [0066} In some embodiments, the coated particles or beads may additionally include an "overcoat,” to provide, cg, moisture protection, static charge reduction, taste-masking. flavoring, coloring, andx'or polish or other cosmetic appeal to the beads. Suitable coating materials for such an overcoat are known in the art, and include, but are not limited to, cellulosic polymers such as hydroxypropylmethylcellulose, hydroxypropylccllulosc and microcrystnlline cellulose, or ations thercoltfor example, various OPADRYQO coating materials). {0067] The coated particles or beads may additionally contain enhancers that may be exemplified by, but not limited to, lity ers, dissolution ers, absorption enhancers, permeability enhancers, stabilizers, xing 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 tion otheads or as a powder. in yet another embodiment, the enhancerts) may be contained in a te layer on coated panicles either under or above the release lling coating. in other embodiments, the extended-release formulation is fomiulated to release the active agends) by an osmotic mechanism. By way 01‘ example, a capsule may be formulated with a single osmotic unit or it may incorporate 2, 3, 4, 5, or 6 push-pull units encapsulated within a hard gelatin capsule, whereby each bilaycr push pull unit ns an osmotic push layer and a drug layer, both surrounded by a semi-penneable membrane. One or more orifices are drilled through the membrane next to the drug layer. This membrane may be additionally covered with a rill-dependent enteric coating to prevent release until W0 03390 PCT/U82012/051888 aftcr gastric emptying. The gelatin capsule dissolves immediately after ingestion. As the push pull ) enter the small intestine, the enteric coating breaks clown, which then allows fluid to flow through the semi—permeable membrane, swelling the osmotic push tment to force to force drugs out through the orificet’s) at a rate precisely controlled by the rate of water ort through the semi-permeable membrane. e of drugs can occur over a constant rate for up to 24 hours or more. |0069l The osmotic push layer comprises one or more osmotic agents creating the driving force for transport of water through the seminpcrmcable membrane into the core of the delivery vehicle. One class of osmotic agents includes water—swellable ophilic polymers, also referred to as “osmopolymcrs” and gels," including, but not d to. hydrophilic vinyl and acrylic polymers, polysacchani‘les such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol ), poly(2~ hydroxycthyl iylatc}, polytacrylic) acid, polymicthacrylic) acid, polywinylpyirolidonc (l’V P), crosslinked PVl’, polyvinyl alcohol ONA), PVA/PVP copolymers, l’VA/PVP copolymers with hobic monomers such as methyl methaciylate and vinyl acetate, hydrophilic polyurethanes containing large PIEO blocks. sodium croscarmellose, carragcenan, hydroxyethyl ose (’HEC’I), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (ll PMC), carboxynicthyl cellulose (CMC) and carboxycthyl, cellulose (CBC), sodium alginate, polycarbophil, gelatin, xanthan gum, and sodium starch atc. l0070] 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 osmogcns include, but are not limited to, inorganic salts, such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate. potassium ates. sodium carbonate, sodium sullitc. lithium sulfate, potassium chloride, and sodium sulfate: sugars, such as dextrose, fructose, glucose, inositol, lactose. maltose, mannitol, rafflnose, sorbitol, sucrose, trchalose, and xylitol; organic acids, such as ascorbic acid, benzoic acid, hnnaric acid, citric. acid, maloic acid, sebacic acid, sorbic acid, adipic acid, cdetic acid, glutamic acid, p~toluencsulfonic acid, succinic acid, and tartaric acid; urea; and es thereof. [007l] Materials useful in forming the semipermeable membrane include various grades of cs, vinyls, others, polyamidcs, polyesters, and cellulosic derivatives that are water~permeable and water-insoluble at physiologically nt plls, or are susceptible to being rendered water-insoluble by chemical alteration, such as crosslinking. [0072} In some embodiments, the extended-release formulation may comprise a PCT/U82012/051888 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 microtlora containing biodegradable enzymes breaking down, for example. the polysaccharide coatings to release the drug contents in a controlled, time-dependent manner, Exemplar-y polysaccharide coatings may e, for example, amylose, arabinogalactan, chitosan, chondroitin sulfate, cyclodextn'n, dextran, gear gum, i xylan, and ations or derivatives rom. |00731 in some embodiments, the ceutical composition is formulated for delayed extended~releaso As used herein, the term "delayed~releasc" refers to a medication that does not immediately disintegrate and release the active ient(s) into the body. In some embodiments, the term "delayed extended-release" is used with reference to a drug l‘omtulation having a release profile in which there is a predetermined delay in the e of the drug following administration. in some embodiments, the delayed extended~release formulation includes an extended—release formulation coated with an c g, which is a barrier applied to oral medication that prevents release cation before it reaches the small intestine. Delayed—release formulations, such as enteric coatings, prevent drugs having an irritant effect on the stomach, such as n, 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 pl-i environment (intestine's pl-l 5.5 and above) where they do not degrade, and give their desired action. {0074] The term “pulsatile release” is a type 0 f delayed-release, which is used herein with reference to a drug formulation that provides rapid and transient release of the drug within a short time period immediately after a predetermined lag period, thereby producing a "pulsed" plasma profile of the drug after drug administration. Formulations may he. designed to provide a single pulsatilc e or multiple pulsatilc releases at predetermined time intervals ing administration.

A delayed-release or pttlsatile e formulation generally comprises one or more elements covered with a barrier coating, which dissolves, erodes or ruptures following a specified tag phase. In some embodiments, the pliannaceutieai composition of the present application is fomtulated for extended-release or delayed cxtcnc‘led—release and comprises 100% ofthe total dosage ofa given active agent stered in a single unit dose. In other embodiments, the pharmaceutical composition comprises an extended/dclayed-rcleasc component and an immediate-release ent. In some embodiments, the immediate- release component and the extended/delayedwreleztse component contain the same active ingredient. in other embodiments, the immediaie~release component and the 2012/051888 extended/dclaycd-release component contain different active ingredients (eg, an sic in one component and an antimuscarinie agent in another component). In some embodiments, the. first and second components each contains an analgesic selected from the group consisting ot'aspirin, ibuproten, naproxcn sodium, indomethacin, nabumetone, and acetaminophen. in other ments, the extended/delayed—release component is coated with an enteric coating. in other embodiments, the innnediate-releasc component and/or the extended/delayed-release component further comprises an antimusearinic agent selected from the group consisting of oxybutynin, solilcnacin, dari fenacin and ne. 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/(leidyed-release component liirther ses an antidiuretic agent, an antimuscarinic agent or both. in other embodiments, the treatment method includes administering to a subject. a ic at least 8 hours prior to a target time, such as e, and administering to the subject the pharmaceutical composition comprising the immediate-release component and/or the extended/delayed—rclease component within 2 hours prior to the target time. in other embodiments, the “immediate—release" component provide about 5.— 50% ot' the total dosage of the active agent(s) and the ded~release” component provides 50-95% of the total dosage of the active agent(s) to be delivered by the phannaeeutical tomtuiation. For example, the immediate-release component may provide about 20409-6, or about 2 %, 25%, 3 %. 35%, about 4094;, of the total dosage of the active agent(s) to be delivered by the pharmaceutical fonnulation. The extendedwelcasc component provides about 60%;, 65%, 709/“, 75% or 80% ot’the total dosage of the active s) to be delivered by the fomiulation. In some ments, the cxtended~releasc component tiirther comprises a r coating to delay the release of the active agent.

A barrier coating for delayed-release may consist of a variety of different materials, depending on the objective. In addition, a ibmtulation may comprise a plurality of barrier coatings to facilitate release in a temporal manner. The coating may be a sugar coating, a tilm coating (cg, based on hydroxypropyl methyleellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose. acrylate copolymers, hylene glycols and/or polyvinyipyrrolidone), or a coating based on rylic acid copolymer, cellulose acetate phthalate, hydroxypropyl celluiose phthnlatc, hydroxypropyl methylcellulose acetate succinntc, polyvinyl acetate phthalatei shellac, and/or ellulose. Furthermore, the lation may additionally include a time PCT/U82012l051888 delay material such as, for example, glycetyl monostearate or glycetyl distearate. in some embodiments, the delayed, extendedmrelease formulation includes an enteric coating comprised one or more. polymers facilitating release of active agents in proximal or distal s ol" the gastrointestinal tract. As used herein, the term “enteric polymer coating" is a coating comprising of one or more polymers having a pl-l dependent or pH-independent release . Typically the coating resists dissolution in the acidic medium ol‘the stomach, but dissolves or erodes in more distal regions ol’ the gastrointestinal tract, such as the small intestine or colOn. An enterie polymer coating typically resists releases ot‘the active agents until some time after a gastric emptying lag period of about 3—4 hours after administration. |0tl791 pH dependent enteric coatings comprises one or more till-dependent or pl-l« sensitive rs that maintain their structural ity at low pl—l, as in the stomach, but dissolve in higher pH nments in more distal regions of the gastrointestinal tract, such as the small intestine, where the drug contents are released. For purposes of the t invention, “pH dependent” is defined as having characteristics (eg, dissolution) which vary according to environmental pl-l. Exemplary til-Independent polymers include, but are not limited to, methacarylic acid ntcrs, methaciylie acidmtcthyl methacrylate copolyntcrs (tag, EUDRAGIT® 1.100 (Type A), EUDRAGIT® 8100 (Type B), Rohm Csth 1, (Ilermany: methaCtylic acid-ethyl aeiylate mers (e.g., EUDRAGl'l‘m LlOO-SS (Type C) and EUDRAGIT“ [3.0055 contilymer dispersion, Rohm Gmbl-l, Germany); copolymers ot~ methaerylic acid-methyl methacrylatc and methyl methaerylate (EUDIUXGI’I‘TZ’ FS); tetpolymcrs ol‘methacrylic acid, methaciylate, and ethyl acrylatc; cellulose acetate phthalates (CAP); hydroxypropyl niethyleellolose phthalate (l-lPMCl’) (e.g., 5t HP~50, l-lP-SSS, su Chemical, Japan); polyvinyl acetate plitltalatcs (PVAP) (e. g, JC90, OPADRYCO enteric white ()Y-l’—‘7l 71); ose acetate succinates (CA3); hydroxypropyl mctltylcellulose acetate ate (HI’MC‘AS), egz, Hl’MCAS LF Grade, MF Grade, l-lF Grade, including AQOAT‘P' LlT and AQOAT‘E' MF Etsu Chemical, Japan); Shinetsu Chemical, Japan): shellac (tag, l\/larc.oatm 125 & tTM 125M; ymethyl ethylcellulose (CMEC, Fretmd Corporation, Japan), ose acetate phthalates (CAP) (6.2g.

AQUA'l‘ERICI‘K); cellulose acetate trimellitates (CAT); and mixtures of two or more thereof at weight ratios between about 2:} to about 5:], such as, for instance, a mixture of IELH‘)RAGl‘l‘® L 100—55 and IEUDRAGI‘T'R‘ S l00 at a weight ratio ot~ about 3: l to about 2: l El FS or a mixture ofEUDRAGIT'E' L 30 D-SS and EUDRAGIT at a weight ratio ofabout‘ 3:1 to about 5: l.

PCT/U82012/051888 ependent polymers typically exhibit a characteristic pH optimum for dissolutitm. In some embodiments, the pll—depcndent polymer exhibits a pll 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 plLdepcndcnt r exhibits n pl-{ m “215.0,ol‘25.5,0126001265. or t’)f27.(). {0081] in certain embodiment, the coating methodology employs the blending of one or more pl-Ldependent and one or more pl l‘independent polymers. The ng ol‘pl-L dependent and tall-independent polymers can reduce the release rate ol’active ients once the soluble polymer has reached its optimum pH. of solubilizatitm, in some embodiments, a "time-controlled” or “timc»dcpendcnt” release profile can be ed using a water insoluble capsule body ning one or more active agents, wherein the capsule body closed at one end with an insoluble, but permeable and 'ble hydrogcl plug. Upon contact with gastrointestinal fluid or dissolution medium, the plug , pushing itselt‘out of the capsule and releasing the drugs alter a pre—determined lag time, which can be controlled by e.g.. the position and dimensions of the plug. The capsule body may be r coated with an outer pl-l-dependent enteric coating keeping the capsule intact until it reaches the small intestine. le plug materials include, for example, polyntethacrylates, credible compressed polymers {rag l-lPMC, polyvinyl alcohol). congealed melted polymer (e.g., glycoryl mono oleate) and enzymatically controlled credible polymers (rig, polysaccharides, such as aniylose, arabinogalactan, an, eliondroitin sulfate, cyclodextrin, dcxtran, guar gum, pectin and xylan). 10083] ln other embodiments, capsules or bilayercd tablets may be formulated to contain a dmg-coritaining core, covered by a swelling layer, and an outer insoluble, but semi~ permeable polymer coating or membrane. The lag time prior to rupture can be controlled by the permeation and mechanical properties of the polymer coating and the swelling behavior of the swelling layer. Typically, the swelling layer comprises one or more swelling agents, such as swellable hydrophilie polymers that swell and retain v later in their structures. ary water ble materials to be used in the d-release coating include, but are not limited to, polyethylene oxide (having e.g., an average lar weight between l,000,000 to 7,000,000, such as POLYOX‘bl, metliylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulosc; polyalkylene oxides having a weight average molecular weight of 100,000 to 6,000,000, ing but not limited to methylene oxide), poly(butylcrte oxide); poly(hydroxyall(y1 methacrylatc) having a molecular weight of from 25,000 to 5,000,000; poly(vinyl)nlcobol. having a low aoetal residue, which is cross PCT/U52012/051888 linked with glyoxal, formaldehyde or glutaraldohyde and having a degree of polymerization of from 200 to 30,000; es of methyl cellulose, cross-linked agar and ymetliyl cellulose; hydrogel forming copolymers produced by forming a dispersion ot‘a finely divided copolymer ol‘maleic anhydride with styrene, ethylene, propylene, butylene or isobutylenc cross-linked with from 0.001 to 0.5 moles of saturated cross-linking agent per mole ot‘maleie anyhydride in the copolymer; CARBOPOL'E‘ acidic carboxy polymers having a molecular weight ot‘450,000 to 4,000,000: CYANAM ER” polyacrylamides; cross-linked water swellablc indenemaloicanhydrido polymers; GOODRITEQ rylic acid having a molecular weight of 80,000 to 200,000; starch graft copolymers; AQUA~KEEPS® acrylate polymer polysaccharides composed of condensed glucose units such as dicster cross-linked polyglucan; carbomers having a viscosity ot'3,000 to 60,000 mPa as 210596-196 w/v aqueous on; cellulose others such as hydroxypropyicellulosc having a viscosity of ahout l000— 7000 mPa s as a 1% wfw aqueous solution (2:30 C): ypropyl cellulose having a viscosity ofabout l000 or higher, preferably 2,500 or higher to a maximum of25,000 ml’a as a 2% w/v aqueous solution; polyvinylpyrrolidone having a viscosity of about. 300~700 mPa s as a 10% w/v aqueous solution 3120" 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 ol‘a water insoluble r membrane ("such as ethyl cellulose. EC) ning ined micropores at the bottom ol'the body and the amount of a swellable ent, such as low substituted hydroxypropyl cellulose (L-HI’C) and sodium glycolatc. After oral administration, 61 fluids permeate through the orcs, causing swelling of the swellablc excipients, which produces an inner pressure disengaging the capsular components, including a first capsule body containing the swellable materials. a second capsule body containing the drugs. and an outer cap attached to the first e body.

The c layer may further comprise anti—tackiness agents, such as talc or glyceryl monostearate and/or plasticizers. The enteric layer may further comprise one or more plasticizers including, but not limited to, triethyl citrate. aeetyl triethyl citrate, acctyltributyl citrate, polyethylene glycol acetylated monoglyccridcs, glycerin, triacetin, propylene glycol, phtlialate esters (cg, diethyl phthalate, l phthalate), titanium dioxide, tonic oxides, castor oil, ol and dibutyl sebacatc. {0087] in another ment, the delay release formulation employs a water- permeable but insoluble l‘ilm coating to e the active ingredient and an c agent.

As water from the gut slowly diffuses through the film into the core, the core swells until the WO 03390 2012/051888 film bursts, thereby ing the active ingredients. The film coating maybe adjusted to permit various rates ofwater permeation or release time. in r embodiment. the delay release formulatiou employs a water. impermeable tablet coating whereby water enters h a controlled aperture in the coating until the core . When the tablet bursts, the drug contents are released immediately or over a longer period oftime. These and other techniques may be modified to allow for a pre» determined lag period before release oi‘drugs is initiated. [0089} In another embodiment, the active agents are delivered in a. formulation to provide both delayed-release and extended-release (delayetl-stistained). The term “delayed~ extended-release" is used herein with reference to a drug formulation providing pulsatile release ive agents at a pre-detemiined time or lag period lbllowing administration, which is then followed by extended-release of the active agents thereafter.

In some embodiments, itnmediate~relea5e, extended—release, d—release, or delayed-extended-release formulations comprises an active core comprised ol‘one or more inert particles, each in the form ot‘a bead. pellet, pill, ar particle, microcapsule, microsphere. microgranuie, nanocapsule, or herc coated on its surfaces with drugs in the form of eg, a drug-containing film-forming composition using, for e. fluid bed techniques or other methodologies known to those of skill in the art. The inert. particle can be of various sizes, so long as it is large enough to remain poorly dissolved. Alternatively, the active core may be prepared by granulating and milling and/or by extrusion and spheronization of a polymer composition containing the drug substance. l0091] The amount of drug in the core will depend on the dose that is ed, and typically varies from about 5 to 90 weight "/It Generally, the polymeric coating on the active core will be from about 1 to 50% based on the weight of the coated particle, depending on the lag time and type of release profile required andfor the polymers and coating solvents chosen.

Those skilled in the art will be able to select an approiiriate amount of drug for coating onto or incorporating into the core to e the desired dosage. In one embodiment, the inactive core may be a sugar sphere or a buffer crystal or an encapsulated buffer crystal such as calcium carbonate, sodium bicarbonate, fumarie acid, tartaric acid, etc. which alters the microenvironmcnt ol‘lhe drug to facilitate its release.

In some embodiments, for example, delayed—release or delaycd~extended~ release compositions may formed by coating a water soluble/diSpersible drug-containing particle, such as a bead, with a mixture of a water insoluble polymer and an cnterie polymen wherein the water insoluble polymer and the enteric polymer may be present. at a weight ratio 2012/051888 of from 4:1 to 1:1, and the total weight ofthe coatings is l0 to 60 weight % based on the total weight or" the coated beads. The drug layered beads may ally include an inner dissolution rate controlling membrane ylcellulose. The composition ot‘the outer layer, as well as the individual weights ol’the inner and outer layers ol’the polymeric membrane are optimized for achieving desired circadian rhythm release profiles for a given active, which are predicted based on in vitro/in vivo correlations. |0093l ln other embodiments the Formulations may comprise a mixture ediate- release drugwcontaining particles without a dissolution rate controlling polymer membrane and delayed-extended-release beads exhibiting, for example. a lag time 01‘2-4 hours following oral administration, thus ing a two-pulse release profile. in some embodiments, the active core is coated with one or more layers of dissolution rate-controlling polymers to obtain d release profiles with or without a lag time. An inner layer membrane can largely control the rate ol‘dntg release following imbibition of" water or body fluids into the core, while the outer layer membrane can provide for a d lag time (the period ot’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 e 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 comprise an enteric polymer. as described above, and a water insoluble polymer at it) to 50 weight ‘36. The ratio of water insoluble polymer to enterie polymer may vary from 4:1 to l:2, preferably the polymers are present at a ratio of about 1:1.

The water insoluble polymer typically used is ethylccllulose. [0096; Exemplary water insoluble polymers e ethylcellulose, polyvinyl acetate (Kollicoal SR#OD from BASF), neutral copolymers based on ethyl aerylate and methylmethacrylate, copolymers of acrylic and methaeiylic acid esters with quaternary ammonium groups such as EUDRAGI'I‘l NE, RS and RS3OD, R1. or RLSOD and the like. ary water soluble polymers include low molecular weight. ll i‘M C, l-IPC, methylcellulose, polyethylene glycol (PEG of molecular weigbt>3000) at a thickness ranging l‘rom l weight ‘Vo up to 10 weight. % depending on the solubility of the. active in water and the t or latex suspension based g formulation used. The water insoluble r to water soluble polymer may typically vary from 95:5 to 60:40, preferably from 80:20 to 65:35. {0097} ln some embodiments, r‘xMBliRLI'I'E"M lRPt’i‘) resin is used as an extended- e carrier. AMBERLITETM lRP69 is an insoluble, strongly acidic, sodium form cation exchange resin that is suitable as r for cationic (basic) substances. In other PCT/U82012/051888 embodiments, l’_)UOtI'_lrl‘E"'M A1’143/ 1093 resin is used as an ed-release carrier.

DUOUTETM APl43/ltl93 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 API43/ltl93 resin provides a means for binding medicinal agents onto an insoluble polymeric matrix. Extended-release is achieved through the formation ol‘rcsin-drug complexes (drug resin-ates). The drug is ed from the resin in viva as the drug reaches equilibrium with the high electrolyte concentrations, which are typical ol’thc gastrointestinal tract. More hydrophobic drugs will usually elute from the resin at a lower rate, owing to hydrophobic interactions with the aromatic stmcture of the cation exchange system. {0099} Preferably, the fonnulatitms are designed with release profiles to limit interference with restful sleep, wherein the fomtulation releases the medicine when the individual would normally be awakened by an urge to urinate. For example, consider an individual who begins sleeping at l 1 PM and is normally awakened at 12:30 A M, 3:00 AM, and 6:00 AM to urinate. A delayed—release vehicle could deliver the medicine at 12:15 AM. thereby delaying the need to urinate for perhaps 2-3 hours. By further including an onal extended-release profile or additional pulsatilc releases, the need to wake up to e may be reduced or ated altogether. [01001 The pharmaceutical composition may be administered daily or administered on an as needed basis. In n ments, the ceutical conipositimi is administered to the subject prior to bedtime. In some embodiments, the pharmaceutical composition is administered ately before bedtime. in some embodiments, the pharmaceutical composition is administered within about two hours before e, 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 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 embodiment. the phannaceutical composition is administered less titan one hour before e. In still another embodiment, the pharmaceutical composition is administered immediately before bedtime. ably, the pharmaceutical composition is administered orally. Suitable compositions for oral administration include. but are not d to: tablets. coated tablets, dragecs, capsules, powders, granulates and soluble tablets, and liquid Forms, for e. suspensions, sions or solutions.

PCT/U52012/051888 Most emetic 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) pH. ”therefore, an enteric coated pill will not dissolve in the acidic juices ot‘the stomach (pH --3), but. they will in the alkaline (pH 74)) environment present in the small intestine. Examples ot‘enteric coating materials include, but are not d to, methyl acrylate—mothttcrylic acid copolymcrs, cellulose acetate succinate, liydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phtlialate (PVAP), methyl methacrylate-methaciylic acid copolymers, sodium alginate and stearic acid.

[OIOZl in some embodiments, the pharmaceutical composition is orally administered from a variety ot‘drug formulations designed to provide delayed-release. Delayed oral dosage forms include, for example, tablets, capsules, caplets, and may also se a pltll‘t lity of granules, beads, powders or pellets that may or may not be encapsulated. Tablets and capsules represent the most convenient oral dosage Forms, in which case solid pharmaceutical carriers are employed. lit a delayed—release ation, one or more barrier coatings may be applied to pellets, tablets, or capsules to facilitate slow dissolution and concomitant release s into the intestine. lly, the barrier coating contains One or more poiylllCl‘S encasing, surrounding, or forming a layer, or membrane around the therapeutic composition or active core. [0104} In some embodiments, the active agents are delivered in a fomtulation to provide delayed-release at a pre—dctemtincd time following administration. The delay may be up to about l0 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. 30105} in other embodiments, the delayed-release is caused by an osmotic mechanism. By way ofexamplc, a capsule may be formulated with a single osmotic unit or it may incorporate 2, 3, 4, 5, or 6 pushapull units encapsulated within a hard gelatin e, whereby each hilayer push pull unit contains an osmotic push layer and a drug layer, both surrounded by a ermeable membrane. One or more orifices are. d through the membrane next to the drug layer. This membrane may be additionally covered with a pl-l— ent enteric coating to prevent release until alter c ng. The gelatin capsule dissolves immediately after ingestion. As the push pull unit(s) enter the small intestine, the enteric g breaks down, which then allows lluid to flow through the somi—pemtcable membrane, swelling the c push compartment to force to force drugs out through the PCT/U52012/051888 orifice(s) at a rate precisely centrolled by the rate of water transport h the semipermeable membrane. Release 0 t‘ drugs can occur over a constant rate for up to 24 hours or more. [0 [06] The osmotic push layer cempriscs one or more c agents ng the driving force for transport of water through the semiapermeable membrane into the core of the delivery vehicle. One class ot‘osmotie agents includes water-swellable hydrophilic polymers, also referred to as "osntopolymers" and "hydrogels," including, but not limited to, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene oxide (PRO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2- hydroxyethyl methacrylatc}, polyt'acrylic) acid, poly(methaciylic) acid, polyvinylpyn'olidonc (PVP), erosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers, PVA/PVP eopolymcrs with hydrophobic monomers such as methyl methacrylatc and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, sodium croscamtcllose, cairagcenan, ltydroxyethyl ose (REC), hydroxypropyl cellulose (HPC), hydroxypmpyl methyl cellulose (HPMC‘), earboxymethyl cellulose (CMC) and carboxycthyl, cellulose (CEC‘), sodium alginate, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate. £0107} Another class of osmotic agents es osmogens, which are capable of imbibing water to . an osmotic pressure gradient across the semi-permeable ne.

Exemplary osmogcns include, but are not limited to, inorganic salts, such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, ium e, potassium ates, sodium carbonate, sodium sulfitc, lithium sulfate, potassium chloride, and sodium sulfate; sugars, such as se. fructose, glucose, inositol, lactose, e, ol, rat‘l'mose, sorbitol, sucrose. trelialose, and l; organic acids, such as ascorbic acid, benzoie acid, liiman‘c acid, citric acid, malcic acid, sebacic acid, sorbic acid. adipic acid, edetic acid, glutamic acid, p-toluenesulfonic acid, succinic acid. and tartaric acid; urea: and mixtures thereof. als useful in forming the semipcmieable membrane include s grades of acrylics, vinyls, ethcrs, polyamidcs, polyesters, and eellulosic derivatives that are water~pcrmeahle and water—insoluble at physiologically relevant oils, or are susceptible to being rendered ttt‘ater—insoluble by chemical tion, such as crosslinking. [0109} In another embodiment, the delay release formulation employs a water~ impermeable tablet coating whereby water enters through a controlled aperture in the coating until the core bursts. When the tablet bursts, the drug contents are released immediately or Over a longer period of time. These and other techniques may be modified to allow for a pre- 2012/051888 determined lag period before release of drugs is initiated.

Various coating techniques may be applied to granules, beads, powders or s, tablets. capsules or combinations thereof containing active agents to produce rcnt and distinct release profiles. in some embodiments, the pliamiaceutical composition is in a tablet or capsule form containing a single coating layer. In other embodiments, the pharmaceutical ition is in a tablet or e form containing multiple coating layers. till I ll In some ments, the pharmaceutical composition comprises a plurality oi" active. ients selected from the group consisting of analgesics, searinic agents. antidiurctics and spasmolyties. Examples ot‘spasmolytics include, but are not limited to, earisoprodol, benzodiachines, baclofen, cyclohcnzaprinc, metaxalone, methoearbamol, elonidine, clonidine analog, and dantrolene. ln some embodiments, the phannaeeutieal composition comprises one or more analgesics. In other ments, the pharmaceutical composition comprises ( 1) one or more analgesics, and ('2) one or more other active ingredients selected from the group consisting of anti muscarinic agents, antidiuretics and spasmolyties. In another embodiment the pharmaceutical composition comprises ( 1) one or two analgesics and (2) one or two antimusearinic agents. In another ment, the pharmaceutical composition comprises (I) one or two analgesics and (2) one or two antit'liureties. In another embodiment, the phamtaceutical compositim ses (1') one or two analgesics and (2) one or two spasmolytics. in yet another embodiment, the phannaceutical composition comprises (i) one or two analgesics, (2) one or two antimuscarinie agents, and (3) one or two antidiurctics, l0112] in one embodiment, the plurality ot’activc 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 ingredients are formulated for both immediate—release and extended-release (eg, a first portion of each active ingredient is fomtulated for immediate—release and a second portion of each active ingredient is [‘omtulated for ed-release). in yet other ment, some of the plurality of active ingredients are formulated for ate-release and some of the plurality of active ingredients are l'ormulated For extended—release tag, active ingredients A. B, C are formulated for immediate-release and active ingredients C‘ and D are formulated for extended—release). In some other embodiments, the immediate-release component and/or the extendedwrelease component is r coated with a delayed—release coating, such as an cnteric g. [0113} In certain embodiments, the pharmaceutical composition comprises an W0 2013/]03390 PCT/U52012/051888 immediate-rcicase component and an extended-release component. The inimcdiatc—reiease component. may comprise one or more active ients selected from the group consisting of analgesics, antimuscarinic agents, antidiuretics and spasmolytics. The cxtcndcdureicasc component may comprise one or more active ingredients selected from the group consisting of analgesics, searinic agents, antidiuretics and spasmolytics. t n some embodiments, the immediate-release component and the. extended-release component have exactly the same active ingredients. in other embodiments. the immediate—release component and the extended—release component have different active ingredients. In yet other embodiments, the innnediate-release component and the extendetl~release component have one or more common active ingredients. in some other ments, the immediate-release component and/or the extended-release ent is further coated with a delayed-reiease coating, such as an c coating. [0114} In one embodiment, the eeuticai composition comprises two active ingredients tag, two analgesic agents. or a mixture of one analgesic agent and one antimuseurinic agent or antiuretie or spasmolylic), formulated for immediate-release at about the same time. In another embodiment, the pharmaceutical composition comprises two active ingredients, ated for extended-release at about the same time. In another embodiment, the pharmaceutical ctmiposition comprises two active ingredients fomiuluted as two extended-release components, each ing a different extended-release profile. For example, a first extended—release component releases a first active ingredient at a firSL release rate. and a second extended-release component releases a second active ingredient at a second release rate. in another embodiment, the pharmaceutical composition comprises two active ingredients formulated as two delayed-release components, each providing a different deiaycd~relcasc profile. For example, a first ed~rclease ent releases a first active ingredient at a first time point and a second delayed-release component releases a second active ingredient at a second time point. In another embodiment, the pharmaceutical composition comprises two active ingredients, one is formulated for ate-release and the other is formulated for extended-release. it)! 15] in other embodiments, the phamiaceutieal con'tposition comprises two active ingredients (e. 3;. two analgesic agents, or a mixture of one analgesic agent and one antimuscarinic agent or etie or olytic) formulated for intmediate~rclease, and (2) two active ingredients (e.g._. two analgesic agents, or a mixture of one analgesic agent and one antimuscntinic agent or antiuretic or spasmolytic) ated for extendedwrelease. In other embodiments, the pharmaceutical ition comprises three active ingredients formuiated 2012/051888 for immediate-release, and (2} three active ingredients formulated for extended-release. In other embodiments, the pharmaceutical cornposition comprises {our active ingredients ated for immediate-release, and (2) four active ingredients formulated for extended- release. in these embodiments, the active ingredient(s) in the immediate—release component can be the same as. or different from, the active ingredient(s) in the. extended-release component. In some other embodiments, the immediate-release component and/or the extended~release ent is further coated with a delayed-release coating, such as an cntcrie coating.

The term "in-unediate~release" is used herein with reference to a drug formulation that does not contain a dissolution rate controlling al. There is substantially no delay in the release ofthe active agents ing administration of an immediate-release formulation. An immediate—release g may e suitable materials immediately dissolving following administration so as to release the drug contents therein. Excmplaty immediate-release coating materials include gelatin, polyvinyl alcohol polyethylene glycol (P\"A~PEG) mers (Lag, KOLLICOATE) and various others materials known to those skilled in the art.

An immediate-release composition may comprise l00% of the total dosage of a given active agent stered in a single unit: dose. tively, an innnediaten‘elease component may be included as a component in a combined release profile formulation that may provide about l% to about 50% ol'the total dosage of the active age-nits) to be delivered by the pharmaceutical formulation. For example, the immediate-release component may provide at least about 5%, or about 10% to about 3 %, or about 45% to about 50% of the total dosage of the active agent(s) to be delivered by the l‘onnulation. In alternate embodiments, the immediate-release component provides about 2, 4, 5, IO, 15, 20, 25, 30, 35, 40, 45 or 50% ot‘the total dosage of the active agenti's) to be delivered by the formulation. in some embodiments, the immediate-release or delayed-release formulation comprises an active core sed of one or more inert les, each in the form ol’a bead, , pill, granular particle, microcapsule, microspltere, microgranule. nanocapsule, or nanosphere coated on its surfaces with drugs in the form oleg . a drug—containing tilm« forming composition using. for example, fluid bed techniques or other methodologies known to those of skill in the rut. The inert particle can be of various sizes, so lOng as it is large enough to remain poorly dissolved. Alternatively, the active core may be prepared by granulating and milling and/or by extrusion and spheronization of a polymer composition containing the drug substance.

W0 2013/]03390 PCT/U82012/051888 The amount of drug in the core will depend on the dose that is required. and typically varies from about 5 to 90 weight %. Generally, the polymeric coating on the active core will be from about 1 to 50% based on the weight of the coated particle, depending on the lag time and type of release e required and/or the polymers and coating solvents chosen.

Those skilled in the art will be able to select an riate amount of drug for g onto or incorporating into the core to achieve the desired dosage. In one embodiment, the inactive core may be a sugar sphere or a bullier crystal or an encapsulated buffer crystal such as calcium carbonate, sodium bicarbonate, l‘umuric acid, tartaric acid, (are. which alters the microenvironment of the drug to facilitate its release. in some embodiments, the delayed—release formulation is formed by coating a water soluble/dispersible drug-containing particle, such as a head, with a mixture ot’a water insoluble r and an enteric polymer, wherein the water insoluble polymer and the enteric polymer may be present at a weight ratio of from 4:1 to 1:1, and the total weight of the coatings is It) to 60 weight % based on the total weight of the coated beads. The drug layered beads may optionally e an inner dissolution rate controlling membrane ol‘ ethyleellnlose. The composition ofthe outer layer, as well as the dual weights ofthe inner and outer layers ofthe polymeric membrane are optimized for achieving desired circadian rhythm release profiles For a given , which are predicted based on in vino/in t-r'vo correlations. [012i] In other embodiments the fonnulations comprise a mixture ol" immediate- rclease drug-containing les without a dissolution rate controlling polymer membrane and delayetl~release beads exhibiting, for example. a lag time of 3-4 hours following oral administration, thus providing a two-pulse e profile. in yet other embodiments the formulations comprise a mixture oftwo types ol‘delayed—releasc beads: 21 first type that exhibits a lag time of l-3 hours and a second type that exhibits a lag time of 4-6 hours. [0122} in some embodiments, the active core is coated with one or more layers of dissolution ntrolling polymers to obtain desired release profiles with or without 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 ofwater or body fluids into the core). The inner layer membrane may comprise a water ble polymer, or a mixture of water insoluble and water soluble rs. {0123} The polymers suitable for the outer membrane, which largely controls the lag time 0!" up to 6 hours may comprise an enteric polymer, as described above, and a water PCT/U82012/051888 insoluble r at a thickness of 10 to 50 weight %. The ratio of water ble r to enteric polymer may vary from 4:1 to 1:2, preferably the polymers are present at a ratio of about [21. The water insoluble polymer typically used is cthylcellulosc. [0l24] Exemplary water insoluble polymers e etltylcellul05e, polyvinyl acetate (Kollicoat SR#0D from lilASl-‘l. neutral copolymers based on ethyl acrylate and mothyltncthaerylatc. mers of c and methacrylic acid esters with quaternary ammonium groups such as EUDRAGl'I‘f”L N131, RS and RS300, RL or R1001) and the like.

Exemplary water soluble polymers include low molecular weight HPMC, HPC, methylcellulose, polyethylene glycol (PEG of molecular weight>3000) at a thickness ranging from 1 weight % up to 10 weight % depending on the solubility of the active in water and the solvent or latex suspension based coating fonnulation 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. 10125} 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 begin: sleeping at l l PM and is ly awakened at 12:30 AM, 3:00 AM. and 6:00 AM to urinate. A delayed. ed-release vehicle could deliver the medicine at l2: 15 A M, y delaying the need to urinate for perhaps 2—3 hours.

The pharmaceutical composition may be administered daily or administered on an as needed basis. in certain embodiments, the ceutical composition is administered to the subject prior to e. in some embodiments, the pharmaceutical composition is administered immediately before bedtime. in some embodiments, the pharmaceutical composition is stered within about two hours before bedtime, preferably within about one hour before e. 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 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 e. In a still further embodiment, the pharmaceutical composition is administered less than one hour before bedtime. In still another ment, the pharmaceutical composition is administered immediately before bedtime. Preferably, the phamiaccutieal composition is administered orally. {0127} The appropriate dosage (“therapeutically effective amount”) of the active agentfs) in the immediate-release component or the extendedn‘eleasc: component will depend, PCT/U82012/051888 for example, the severity and course of the condition, the mode. of administration, the bioavailability of the particular s), the age and weight of the t, the patient's clinical history and response to the active s), discretion ot‘the physician, etc.

As a general proposition, the therapeutically effective amount ofthe active agent(s'} in the immediate-release component, the extended-release component or the delayed-extended-release component is administered in the range 01‘ about 100 iig/kg body weight/clay to about 100 mg/kg body weight/day whether by one or more administrations. In some embodiments, the range h active agent administered daily is from about 100 pig/kg body weight/day to about 50 mg/kg body weight/day, 100 gig/kg body ’day to about 10 mg/kg body weight/day, 100 ttg/kg body weight/day to about 1 mg/kg body /day, 100 gig/kg body weight/day to about 10 mg/kg body weight/day, 500 ng/lcg body weight/day to about 100 mg/kg body weight/day, 500 ttg/kg body weight/day to about 50 mg/ltg body weight/day, 500 Jug/kg body weight." day to about 5 mg/ltg body weight," day, l mgfkg body weight/day to about 100 mg/kg body /day, 1 mg/kg body weight/day to about. 50 mU g body weight/ day, l mg/‘kg body weight/day to about 10 mgjkg body weight/day, 5 mg/kg body weight/dose to about 100 rug/kg body weiglib’day, 5 mgfkg 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 mykg body weight/day. [01291 The active agent(s) described herein may be ed in an immediate-release ent or an extended-release component, a delayed-extended—release con'tpontmt 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, l 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 3000 mg, 250 mg to 500 mg, 500 mg to 1000 mg, 500 mg to 3000 mg. As expected, the dosage will be dependant on the condition, size, age and condition of the patient. [0130} in some embodiments, the. pharmaceutical composition comprises a single analgesic agent. In one embodiment, the single analgesic agent is n. In another embodiment, the single analgesic agent is ibuprofen. In another embodiment, the 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 PCT/U52012/051888 embodiment, the single analgesic agent is acetaminophen. {0131] 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 pharmaceutical composition comprises salicylic acid, fen, 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 01‘5 mg to 2000 mg, 20 mg to 2000 mg, 5 mg to 1000 mg, 20 mg to 1000 mg, 5.0 mg to 500 mg, 100 mg to 500 mg, 25.0 mg to 500 mg, 250 mg to 1000 mg or 5.00 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 ses a pair of sic agents. Examples ofsueli paired sic agents include, but are not limited to, acetylsalieylic acid and ibuprofen, acetylsalicylic acid and cn sodium. acctylsalicylic acid and nabumetone, acetylsalicylic acid and acetaminophen, acetylsalicyiic acid and indomcthanein, ibuprofen and naproxen sodium, ibuprofen and nabumetone, ibuprofen and acetaminophen, ibuprofen and indomcthancin, naproxen sodium and nabumetone, naproxen sodium and acetaminophen, naproxcn sodium and indomethancin, nabumctone and acetaminophen, nabumetone and indomethancin, and acetaminophen and indomethanein. The paired analgesic agents are mixed at a weight ratio in the range 0170.1 :1 to 10: 1, 0.12:1 to 5:1 or 03:1 to 3:1, with a combined dose in the range ol‘S mg to 2000 mg, 20 mg to 2000 mg, 300 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, 200mg 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 ment, the paired analgesic agents are mixed at a weight ratio of 1:1. [01331 In some other embodiments, the phamtacentical composition of the present application further comprises One or more scarinic agents. Examples of the antimuscarinic agents include, but are not limited to, ynin, solil‘enacin, darit‘enaein, l‘esotcrodine. toltcrodine. trospium and atropine. The daily dose oi'antimuscarinic agent is in the range of001 mg to 100 mg, ().l 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, l0 mg to 25 mg, 0.01 mg to 10 mg, 0.1 mg to mg, 1 mg to 10 mg, 10 mg to l00 mg and 10mg to '25 mg. 20] 2/051888 [0134} in certain embodiments. the pharmaceutical ition ses an analgesic agent selected from the group consisting ot’cetylsalicylie acid, ibuprofen. naproxcn sodium, nabumetone, acetaminophen and indomethancin, and an antimusearinic agent selected from the group consisting ofoxybutynin, solii‘enacin, darit‘enacin and atropine. {0135] Another aspect of the present application relates to a method for reducing the frequency of urination by administering to a person in need thereot’a pharmaceutical composition formulated in an immediate-release formulation. The phannaceutical composition comprises a ity of analgesic agents and/or antimuscarinic agents. 10l361 In n embodiments, the pharmaceutical composition comprises two or more analgesic agents. In other embodiments, the ceutical composition comprises one or more analgesic agents and one or more antirnuscarinic agents. The centical composition may be formulated into a tablet, capsule, . powder, granulatc, liquid, gel or emulsion form. Said liquid, gel or emulsion may be ingested by the subject in naked form or contained within a capsule. in certain embodiments, the analgesic agent is selected from the group consisting ot‘salicylates. aspirin. salicylic acid, methyl iate, diilunisal, saisalate, olsalazine, sulfasalazine, paraoaminophcnol derivatives. ilide, acetaminophen, phcnacetin, t‘enamates, metenamic acid, meclol‘enamate, sodium meclol‘enamate, heteroaryl acetic acid derivatives. tolmetin. kctorolac, diclofcnac, propionic acid derivatives, ibuprofen. naproxen sodium, naproxen, t’enoproi‘en, ketoproi'cn, prot‘en, oxaprozin; enolic acids. oxicam derivatives, piroxicann meloxieam, tenoxicam. ampiroxicam, droxicam, pivoxicam. pyrazolon derivatives, phenylbutazone, oxyphenbutazonc, antipyrine, aminopyrine. dipyrone, . ceiecoxib. rofecoxib, nabumetone, e, nimesulide. indomethacin. sulindac. etodoiac, diilunisal and isobutylphenyl pi‘opionie acid. The antimuscarinic agent is selected from the group ting oi‘oxybutynin, solifenacin. daritenacin and atropine. f0138] in some embodiments, the phamtaceutical composition ses a single analgesic agent and a single antimuscarinic 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 ment, the single sic agent is indomethaein. in another embodiment, the singie analgesic agent is nabumctone. In another embodiment, the single analgesic agent is acetaminophen. The analgesic agent and anti-muscarinic agent may be given at doses in the ranges described above. [089} Another aspect ol‘thc present application relates to a method for treating PCT/U82012/051888 noeturia by administering to a subject in need thereof (1) one or more analgesic agent and {2) one or more uretic agents. In certain embodiments, the antidiuretic agent(s) act. to: (1) increase vasopressin secretion; (2) se vasopressin receptor activation; (3) reduce secretion of atrial natriuretie peptide (ANP) or C-type natriuretie peptide (CNP); or (4) reduce ANP and/or (3N P receptor activation.

Exemplary antidiurctic agents include, but are not limited to, antidiurctic hormone ( ADI-i), angiotensin ll, aldosterone, vasopressin, vasopressin analogs (cg, desmopressin argipressin, lypressin, felyprcssim omipressin, terlipressin); vasopressin receptor agonists, atrial natriuretic peptide (AN?) and (TI-type natriuretic peptide (CNP) receptor (Le, Nl’Rl, 'NPRZ, NI’R3) antagonists lag. HS— l42—l, isatin, [Asu7,23‘]b-ANl’-(7« 28)], anantin, a cyclic peptide from omyces escens, and 30 l 2 n'ionoclonal antibody); somatostatin type 2 receptor nists (cg, somatostatin), and pharmaccutieally~acccptable derivatives, analogs, saitsi hydrates, and solvatcs thereof. {0141] in certain embodiments, the one or more sic agent and one or more antidiurctic agents are formulated for extended—release.

IBMZ} Another aspect of the present application relates to a method for reducing the frequency of ion by administering to a person in need thereof a first pharmaceutical composition sing a diuretic, followed with a second ceutical composition comprising one or more analgesic agents. "the first pharmaceutical composition is dosed and formulated to have a diuretic effect within 6 hours ofadministration and is stered at least 8 hours prior to bedtime. The second ceutical composition is administered within 2 hours prior to bedtime. The first pharmaceutical composition is formulated for immediate-release and the second pharmaceutical composition is formulated for extended— releasc or delayed, extended-release.

Examples ot‘diut‘etics include, but are not limited to, acidifying salts, such as CflClg and NHqu; arginine vasoprcssin receptor 2 antagonists such as ainphotericin B and m citrate: aquaretics, such as rod and .lunipe; Na-l-l exchanger antagonists. such as dopamine; carbonic anhydrase inhibitors: such as aeetazolamide and dorzolantide; loop diuretics, such as bumetaeide, ethacrynic acid, i‘urosemide and torsemide; c diuretics, such as glucose and mannitol; potassiumvsparing diuretics, such as amiloride, spironolactone, erene, potassium canrcnoatc; thiazidcs, such as bendroflumethiazide and hydrochlorothiazide: and nes, such as caffeine. theophylline and omine. [0144} In some embodiment, the second pharmaceutical composition further comprises one or more antimuscarinic agents. Examples of the antimuscarinic agents include, W0 2013/]03390 but are not limited to, oxybutynin, solifcnacin, darifcnacin, fesotcrodine, tolterodinc, trospiuin and atropine, 10145] Another aspect ofthe present application relates to a method for treating nocturia by administering to a person in need l‘a tirst phamtaeeutical composition comprising a diuretic, followed with a second pharmaceutical composition comprising one or more analgesic . The lirst 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 phamiaceutieal ition is formulated for extended-release. or delayed, extended-release, and is administered within 2 hours prior to bedtime. [0146i Examples of diuretics include, but are not limited to. acidifying salts, such as CaClg and Nl-iqu; arginine vasopressin receptor ‘2 antagonists, such as amphotericin B and lithium e; aquaretics, such as Goldenrod and Junipe; Na~H exchanger nists, such as dopamine; carbonic anhydra se tors, such as acctazolamide and dorzolamidc: loop diuretics, such as bumetanide, ethacrynic acid, furosemide and torsemide; osmotic diuretics, such as glucose and mannitol; potassium-sparing diuretics, such as amiloridc, spironolaetone, triamterene, ium canrenoate; thiazides, such as bendroflumethiazide and hydrochlorothiazide; and xanthines, such as caffeine, theophylline and thcobromine. {0147] in some embodiments, the second pharmaceutical composition further comprises one or more antimusearinic agents. Examples of the antimuscarinic agents include. but are not limited to, oxybutynin, solifenacin, darilcnacin, l‘esoterodine, tolterodine, trospium and atropine. The second pharmaceutical composition may be formulated in immediatevrelease formulation or d-release formulation, in some other embodiments, the second pharmaceutical composition further ses one or more antidiuretic agents. In some other ments, the second ceutical composition further comprises one or more spasmolytics.

Another aspect of the t ation relates to a method for reducing the frequency of urination by administering to a subject in need thereof, two or more analgesic agents altematively to prevent the development of drug resistance. In one. embodiment, the method comprises administering a lirst analgesic agent for a first period ot" time and then stering a second sic agent for a second period ot‘time. 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 ed—release or delayed, cxtendedweleasc. in one embodiment. the first analgesic agent is acetaminophen, the second analgesic agent is ibuproten and the PCT/U82012/051888 third analgesic agent is naproxcn sodium. The length of each period may vary depending on the t’s response to each analgesic agent. in some embodiments, each period lasts from 3 days to three weeks. in another ment, the first, second and third analgesic are all ated for extent‘led—rcleasc or delayed, extended-release.

Another aspect of the present application relates to a phamiaceutical composition comprising a plurality ol‘active ingredients and a pharmaceutically acceptable carrier, wherein at least one of the plurality of active ingredients is formulated for extended- relcasc or delayed, extended-release. In some embodiments, the plurality ofacrivc ingredients comprises one or more analgesics and one or more uretie agents. In other embodiments, the plurality of active ingredients comprises one or more analgesics and one or more nntidiuretic agents. In other embodiments, the plurality ofactivo ingredients comprises one or more analgesics, one or more antidiuretic agents and an antimuscarinie agent. The antimuscarinic agent may be selected from the group consisting ol‘oxybutynin, solil‘enacin, dnl'ifenacin and atropine. In other embodiments, the ceotical composition comprises two different sics selected from the group ting of cctylsalicylic acid, ibuprofen, naproxen sodium, nabumetone, acetaminophen and indomethancin. in yet other embodiments, the phamiaceutical ition comprises one analgesic selected from the group consisting ot‘cetylsalicylie acid, ibuprofen. naproxen sodium, nabumetone, acetaminophen and indomethancin; and an antimuscarinic agent selected from the group ting ol‘oxyhutynin, solil'enacin, daril‘enacin and atropine. in other embodiments, the pharmaceutical composition ofth present application further comprises one or more spasmolytics. es of spasmolytics include, but are not limited to. cariSOprodol, henzodiazepines. baclofen, cyclobenzaprine, metaxalone, methocarbamol, clonidinc, elonidine analog, and dantrolcnc. In some embodiments, the spasmolytics is used at a daily dose of 1 mg to 1000 mg, l mgto l00 mg, l0 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, .0 mg to l000 mg, 50 mg to 800 mg, 50 mg to 200 mg, 100 mg to 800 mg, [00 mg to 500 mg, 200 mg to 800 mg, and 200 mg to 500 mg. "l"he spasmolytics may be ated, alone or together with other active icnds) in the pharmaceutical composition, for ate- relense, extended-release, delayed—extended-release or cornbinations thereof. [0151} As used herein, "pliarmaccutically able carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sweeteners and the like. The phannaceutically able carriers may be prepared from a wide range of materials ing, but. not limited to, PCT/U52012/051888 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 eutic compositions is contemplated. [0152} 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 referenee. [0153i Twenty eer subjects, hoth male and female were enrolled, each of which experienced premature urge or desire to urinate, interfering with their y to sleep fora sufficient period of time to feel adequately rested. Each subject ingested 400~800 mg of ibuprofen as a single dose prior to bedtime. At least l4 subjects reported that they were able to rest better because they were not being ed as frequently by the urge to urinate. [0154} Several subjects reported that after several weeks of nightly use of ibuprofen. the benefit ot‘less frequent urges to urinate was no longer being realized. However, all of these subjects r reported the return of the benefit after several days of abstaining from taking the s.

EXAMPLE 2: EFFECT OF ANALGESJC AGENTS. BO'l‘ULlNUM NEURO‘I‘OXIN AND SCARINK‘ AGENTS ON MACROPl-IAGE RESPONSES TO INFLAMMATORY AND NON-I‘NFLAMMATORY STlMU Ll Experimental Design This study is designed to determine the dose and in win-o efficacy of analgesics and antimusearinic agents in lling macrophage response to inflammatory and non- matory stimuli mediated by COX?! and prostaglandins (POE, l’Gl-l, are). it establishes ne (dese and kinetic) responses to inflammatory and non-inflammatory effectors in bladder cells. Briefly, cultured cells are exposed to analgesic agents and/or antimuscarinic agents in the absence or presence of various effectors. {0156} The effectors include: lipopolysaecharide (LPS), an inflammatory agent and (30x2 inducer, as inflammatory stimuli; carhachol or acetyleholine, a ator of smooth muscle contraction, as flammatory stimuli; botulinurn neurotoxin A, a known inhibitor ofacetylcholine release, as positive control; and arachidonic acid (AA), gamma nic acid (DGLA) or pentaenoic acid (EPA) as precursors of prostaglandins, which are produced following the sequential oxidation of AA, DGLA or EPA inside the cell by cyclooxygenases PCT/U52012/051888 (COX1 and COX?) and temiinal prostaglandin synthases. [0157) The analgesic agents include: Salicylates such as aspirin, iso—butyhprognnoic- phenoiic acid derivative (ibuprofen) such as Advil~ Motrin, Nuprin, and Medipren, naproxen sodium such as Aleve, Anaprox, Antalgin, Feminax Ultra, Flanaxfi lnza, Midol Extended Relief. Nalgesin, Naposin, Naprelan, Naprogesic, Naprosyn, Naprosyn sion, tit"- Naprosyn, Narocin, Proxem Synttex and Xenobid, acetic acid tive such as indomethacin (tndocin),l ~nnphthaleneacetic acid derivative such as nabumetone or relation, N~ncetyl—para—nminophcnol (APA P) derivative such as acetaminophen or paracetamol ol) and Celecoxib. [0158! The antimuscarinic agents include: oxybutynin, solifenacin, darifenacin and atropine. |0159l Macrophages are subjected to short term ( l ~2 hrs) or long term (24—48 hrs) stimulation of with: 1) Each analgesic agent alone at various doses. (2} Each anaigesic agent at s doses in the presence of LPS. (3) Each analgesic agent at various doses tn the ce of carbachol or acetylcholine. (4) Each analgesic agent at various doses in the presence of AA, DGLA, or EPA. (5.) Betulinum neurctoxin A alone at various doses. (ti) Botulinum neurotoxin A at various doses in the ce of LI’S. (7) Botulinuni oxin A at various doses in the presence ot'ca rbachol or acetylcholine. (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 scarinic agent at various doses in the presence ofLPS. (_ l 1) Each antimuscartnic agent at various doses in the presence of carbachol er ucetylcholine. (1'2) {Each antimuscarinic agent at various doses in the presence ot‘AA, DCSLA, or EPA. 10160} The cells are. then analyzed for the release of l’GHg, I’GE, I)(:VEQ, aeydin, Thromboxane, , line, TNFwa, the COX2 activity, the production ot’cAMP and CG MP, the production oI‘IL- 1B, lL-(i, "l‘NF—a and COXZ mRNA, and surface expression of €080, CD86 and Ml-IC class ll molecules.

Materials and Melbodv Macrophage cells E0l61'l Murine RAW264.7 or 3 774 macrophage cells (obtained from A'I'CC) were used in this study. Cells were maintained in a culture medium containing RPM] 1640 mented with 10 % fetal bovine serum , 15 mM HEPES, 2 lel l.,~gltztarninc, 100 WO 03390 PCT/U52012/051888 U/ml penicillin, and 100 ug ./ ml of streptomycin. Cells were cultured at 37° C in a 5 % C03 atmosrihere and split (passages) once a week.

In virm treatment of macrophage cells with sics {0162! RAW26-47 macrophage cells were seeded in 96-well plates at a cell density of 1.5x105 cells per well in [00 ul ofthe culture medium. The cells were treated with (l) various concentrations ofanalgesie (acetaminophen, aspirin, ibuprophen or naproxen), (2) various concentrations ol‘lipopolysaecharide (LPS), which is an ci‘feCLor of inflammatory stimuli to macrophage cells, (3) various concentrations of carbachol or acctylcholine, which are effectors intlammatory stimuli, (4) analgesic and LPS or (5) analgesic and earbachol or acetylcholine. Briefly, the sics were dissolved in li'BS—t‘ree culture medium (in, RPMI 1640 mented with 15 mM HEPES, 2 mM L~glutamine, 100 U ml penicillin, and l00 tug .‘ ml of streptomycin), and diluted to desired concentrations by Serial dilution with the same medium. For cells treated with analgesic in the absence PS, 50 ul of analgesic on and 50 til of FBS-ft‘ee culture medium were added to each well. For cells tr *ated with analgesic in the presence ot‘I..PS, 50 ul ol‘ analgesic solution and 50 ill of LPS t from Saimone/la {whimurimm in PBS-free culture medium were added to each well.

All ions were tested in duplicates. ltllt’r3l After '24 or 48 hours ofeullure, 150 ul of culture supernatants were collected, spun down for '2 min at 8000 rpm at 4°C to remove cells and debris and stored at ~70°C for analysis ol'eytokine responses by EIJSA. The cells were ted and washed by centrifugation (5 min at 1,500 rpm at 40C) in 500 ul of Phosphate buffer (PBS). Half of the 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 cu~stimulatory molecule expression {OM43 For flow cytometry is, hages were diluted in NH) til of l7ACS buffer (phosphate buffered saline (PBS) with % bovine serum albumin (BSA) and 0.01% NaNgl and stained 30 min at 4°C by addition of Fl'l‘C—conjugared anti-CD40, PPS—conjugated D80, PIE-conjugated anti~CD86 antibody, anti MHC class 11 (l-Ad) PE (BD Bieseience). Cells were then washed by centritiigation (5 min at l,500 rpm at 4°C) in 300 til of FAC‘S buffer. After a second wash, cells were re-suspended in 200 at of FACS buffer and the tage ol‘cells expressing a given marker (single positive), or a combination. of markers (double positive) were analyzed with the aid of an Aecuri C6 flow cytometer (BD Bioscienees).

PCT/U82012/051888 Analysis of cytokine responses by ELISA {0!65] Culture supematants were subjected to cytolrine~speciiic ELI-SA to determine lL-l B, lL-6 and 'I’NF-a responses in cultures of macrophages treated with analgesic, LPS alone or a combination of LPS and analgesic. The assays were med on Nune MaxiSorp Immunoplates (None) coated ght with l00 _ul ofanti-mouse il.—6, TNF-ot mAbs (BD Bioscicnces) or II..-lB mAb (R621) Systems) in (H M sodium bicarbonate buffer {pl-l 9.5).

Al‘ter two washes with PBS (200 til per well), 200 pl ot‘PBS 3% BSA were added in each well (blocking) and the plates incubated for 2 hours at. room temperature. Plates were washed again two times by addition of 200 til per well, 100 til ot‘cytokine standards and serial dilutions of culture supernatants were added in duplicate and the plates were incubated ght at 4°C. Finally, the plates were washed twice and ted with 100 pl oi" secondary biotinylatcd anti-mouse il.~6, TN For. mAbs (BD Biosciences) or ”.rifi (R&D Systems) followed by peroxidase-labelled goat anti»biotin mAb (Vector Laboratories). The colorimetric on was developed by the addition ot‘2,.2‘-azino-bis (3)- ethylbenzylthiazolinc—6—sulfonic acid (ABTS) substrate and lng; (Sigma) and the absorbence measured at 415 nm with a Victorwv multilabel plate reader (Perkinlilmer).

Determination of COXZ activity and the tion of CAMP and eGMP {0166; The COXZ activity in the cultured macrophages is determined by sequential itive ELISA (R&D Systems). The production of cAMP and (GM? is ined by the CAMP assay and chMP assay. These assays are perfonned routinely in the art.

Matt; Table 1 summarizes the experiments perforated with Raw 264 macrophage cell line and main findings in terms ofthc effects ofanalgcsics on cell surface expression of costimulatoxy molecules CD40 and CD80. Expression of these molecules is stimulated by COXZ and in llammatory s and thus, was evaluated to determine functional consequences of inhibition of COXZ. [0168} As shown in Table 2. acetaminophen, aspirin, ibuprophen and naproxen inhibit basal expression of co—stimulatory molecules CD40 and CD80 by macrophages at all the tested doses (to, 5x 105nm, 5x 10‘ nM, 5x 103 nM, 5x 102 nM, 50 nM and 5 nM), except for the. highest dose (112., 5): l06 nM), which appears to enhance, rather than inhibit, expression of the 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 (m, 5 uM). This finding supports the notion that a controlled release of small doses WO 03390 PCT/USZ012/051888 of analgesic may be preferable to acute delivery oflargc doses. The experiment also revealed that acetaminophen, aepirin, ibuprophen and naproxen have a r inhibitory effect on LPS induced expression ofCD40 and CD80.

Table l. Summary ol‘cxpcrimcms -'LPSSalmone'lhi(mlumnrimn Acetaminophen lbuprophen Naproxcn TESTS _ Dose responses (0,150,500, 5x103, 5x104, 5x105. NW") 113% X (5 rig/ml l Dose responses X (50 nsymL (0, 5 , 50, 500. 5x103. leO“, 5x10“, 5x10“) 117% X (M100 ng'mL) Characicrization 0factivafionistimulaloiy status. Flow cytomcti'y analysis of CD40, CD80, CD86 and MHC class II ors of‘mflammatory rssponscs: ELISA analysis oflL—ll‘l, lL—(i, TNT-i3, Dose analgesic (HM) 5x {0° 4ixlO° \x10 5);“) 500 5 ............................ ................

‘ .. N I -+ N t h r .\ ~~~~~~ >- 148‘) iri)‘ (‘D40+CD80++.':.:4'."1'.i Ibuproplicn CD40‘C'DSO Nl'J“ (a.4 77 ...__.~.,. “0‘8”“?._._.._ Napmxen CD4U"CD80 Acetaminophen CD40CD80 n CD40? [380+ lbuprcphen CD4U'°CD80" Napmxcn C {MDX‘DXO’r "’ ND: not done (toxicity) PCT/U32012/051888 10169} Table 3 summarizes the results of several studies that measured serum levels of analgesxc after oral therapeutic doses in adult . As shown in Table 3, the maximum serum levels ot'aualgesrc after an oral therapeutic dose are in the range of 104 to 10" nM. ore, the doses ol'analgesic tested in vitro in Table 2 cox-er the range of concentrations achievable in viva in humans.

Table 3. Serum levels of analgesic in human blood after oral therapeutic doses Maximum serum Analgesic dmg Molecular i levels after oral References weight therapeutic doses -_._..-...—.. .......... “nun—"m...“._........................W.~_-_...._...—..._.~.M~7.2xio‘- * BMC al Pharmacology 20:0. 10:10 1.19.er 05 * Anacsth we Care. 201l‘ 39:242 Aspirin 181.66 ‘ 30~100 1.65x10‘ — * Disposilmri ofTo.ific Drugs and Chemicals (Acctylsalicylic acid) 5.5):105 in Man. 8111 Edition, Biomedical Public. i Foster City, CA, 2008, pp. 22-25 l ”‘ J Lab Clm Med. 2084 .lun;i()3:869 ibuprofen 206.29 * 'liriical PharmacologyZOlO, 10:10 [AdviL Motrin} * J Clin Phanmcol. 2001, 41:330 Naproxen 230.26 * J Clin Pharmacol. 2001, 4l 2330 (Alcx'c) l-LXAhIlPll-Z 3: [El-“FELT OI? ANALGIESIC AGlgN l'S. I.INUM NEL'RO‘I‘OXIN AND ANTIMUSCARINIC AGENTS ON MOUSE R SMOOTH MUSCLE CELL RESPONSES TO lNl‘LAlVllVlATORY AND NON-IN FLAMIVL’VI‘ORY S'I‘IMULI fixQerime/ira! Design {0170] 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 COX? and PGE2 responses. [0171} The effectors, analgesic agents and antimuscarinic agents are bed in Example 2.

PCT/U82012/051888 {0172: Primary culture of mouse bladder smooth muscle cells are subjected to short term (l 42 hrs) or long term (24—48 hrs) stimulation ol‘with: t' 1) Each sic agent alone at various doses. (2) Each analgesic agent at various doses in the presence of LPS. (3) Each analgesic agent at s doses in the presence of carbaehol or acetylcholine. (4) Each analgesic agent at various doses in the presence of AA, DCiLA, or EPA. (5) Botulinum neurotoxin A alone at various doses. (6) Botulinum oxin A at various doses in the presence ot‘I..PS. (7) Botulinum neurotoxin A at various doses in the presence of hol or acetyleholine. (8} Botulinum oxin A at various doses in the presence of AA, DGLA, or EPA (9) Each antimuscarinic agent alone at s doses. (3 0) Each antimuscarinic agent at various closes in the presence of LPS. (1 1) Each searinie agent at various doses in the presence ofcarbachol or acetylcholine. ('12) Each antimuscarinic agent at various doses in the presence of AA, DGLA. or EPA. 50173} The cells are then analyzed for the release of PGHgi PGE~ PG 13; Prostacydin, Thromboxane, ll..-l l}, lie-6, TNF-a, the COX2 activity, the production ol‘eA MP and COMP, the production ot‘lL—lfi, iL-6, o. and COXZ mRVA, and surface expression of CD80‘ CD86 and MHC class ll molecules.

Materials and drier/20d? Isolation and purification of mouse bladder cells Bladder cells were removed from euthanized animals (3578Mo mice (8- l 2 weeks old) and cells were isolated by enzymatic ion followed by purification on a Pereoll gradient. Briefly, bladders from l0 mice were minced with scissors to line slurry in ml of digestion buffer (RPMI 1640, 2% fetal bovine serum, 0.5 mg/ml collagenase~ 30 rig/ml . Bladder slurries were enzymatically digested for 30 minutes at 373C.

Undigested fragments were further dispersed through a cell~trainer. The cell suspension was pelleted and added to a discontinue 20%, 40% and 75% Percell gradient for purification on mononuclear cells. liiach experiment used 50~60 bladders. {0175] After washes in RPMI 16-40. bladder cells were resuspended RPMI 1640 supplemented with lt) % letal bovine serum, l5 mM l-lEPlES, 2 mM l_,-glutamine._ 100 Ufml 2012/051888 penicillin, and mo pg ml of streptomycin and seedcd in clear—bottom black 96-well cell culture microculture plates at a cell density of 3x l 0“ cells per well in 100 pl. Cells were cultured at 37” C in a 5 % CO; atmosphere.

In vitro treatment of cells with analgesics Bladder cells were treated with analgesic solutions (50 itl/ well) either alone or together with carbachol ( Ill-Molar, 50 ul/ well), as an example of non-intiammatmy stimuli, or lipopolysaecharide (LPS) mone/la cvp/n'murizrm (l ug/ml, 50 ul/ well), as an example ot‘non-inllammatory stimuli. When no other effectors were added to the cells, 50 ul of RPMI 1640 without fetal bovine serum were added to the wells to adjust the final volume to 200 ul. {0177] 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 (POE?) responses by ELISA. Cells were fixed, pomieahilized and blocked for detection of xygenase-Z (COX2) using a l‘luorogenie substrate. in selected experiment cells were stimulated 12 hours in vitro for analysis of (TOXZ responses is of COXZ responses [0&781 COX)? responses were analyzed by a {L‘elLBased ELISA using Human/mouse total COXZ assay (R&D Systems), following the instructions ol‘tlic manufacturer.

Briefly, ailer cells fixation and pet‘meabilixalion. a mouse anti—total COX2 and a rabbit anti- total GAPDH were added to the wells of the cleanhottom black 96-well cell culture microculture . After incubation and washes, an llRP—conjugate anti—mouse IgCl and an AP—conjugated anti—rabbit lgG were added to the wells. Following another incubation and set of , the HRP- and AP-lluorogcnie substrates were added. Finally, a Victor® V multilabel plate reader kinl'jlmer) was used to read the fluorescence emitted at 600 nm (COXZ fluorescence) and 450 nm (GAPDH fluorescence). s are expressed as relative levels of total COXZ as determined by relative fluorescence unit (RFUS) and nomtalized to the housekeeping, protein GAPDH.

Analysis of PGE2 responses [0179} l’rostaglandin E2 responses were analyzed by a sequential itive ELISA (R&D Systems). More specifically, culture supemalants or PGE2 standards were added to the wells of a 96~wcll polystyrene microplatc coated with a goat anti—mouse polyclonal antibody.

After one hour incubation on a late shaker, an HRP-conjugated PGEZ was added and plates incubated for an additional two hours at room temperature. The plates were then washed and l-lRl’ substrate solution added to each well. The color was allowed to develop for min and the reaction stopped by addition sulfuric acid before reading the plate at 450 em with wavelength correction at 570 nm. Results are expressed as mean pgi’ml of P652.

Other assays [01801 The release of PGHg, l’CilE), l’rostacydin, ’l‘hromboxane, lie-ill, IL-6r and TN 1?- 0t, the tion ol'cAMP and cGMP, the production oflL-l ll, lL—o, TNF—a and COX2 mRNA, and surface sion ol‘CIJSO, C086 and MllC‘ class lI molecules are determined as bed in e 2.

Analgesics inhibit COX2 responses of mouse bladder cells to an inflammatory stimuli {DISH Several analgesics (acetaminophen, aspirin, ibuprofen and naproxen) were tested on mouse bladder cells at the concentration ofS MA or 5.0 MA to determine whether the analgesics could induce COXZ responses. Analysis of 24—hour es showed that none ol‘the analgesics tested induced COXZ responses in mouse bladder cells in virm. [0182} The effect of these analgesics on the COXZ responses of mouse bladder cells to carbachol or LPS stimulation in viiro was also tested. As ted in Table l, the dose of hol tested has no significant effect on C‘OXZ levels in mouse bladder cells. On the other hand, LPS significantly increased total COXZ levels. Interestingly, acclall‘tll‘topilelt, aspirin, ibuprofen and naproxen could all suppress the effect of Ll’S on COX2 levels. The suppressive effect ol‘the analgesic was seen when these drugs were tested at either 5 pM or 50 ttM (”fable 4).

Table 4. COXZ expression by mouse r cells after in vino stimulation and treatment with analgesic Stimuli Analgesic Total COXZ levels (Normalized RFlls} LPS (1 itg/ml) “LPS (1 itg/ml) Acetaminophen (5 uM) LPS (1 rig/ml) Aspirin (5 uM) 240 i 17 LPS (lug/ml) Ibuprofen (5 mm.) 253 :t 32 [PS (lug/ml) Naproxen (5 itM') 284 i l l PCT/U52012/051888 LPS (lugml) Acetaminophen (50 MW) 243il5 LPS (lug/ml) Aspirin (50 MA) 2583:2l LPS (lug/ml) Ibuprofen (50 MW) .266le Li’s (lug/ml) Naproxen (50 MA) 279t23 Analgesics inhibit PGEZ responses of mouse bladder cells to an inflammatory stimuli [0l83l The secretion of PGE2 in e supernatants of mouse bladder cells was measured to detennine the biological significance of the alteration of mouse bladder cell COXZ levels by analgesics. As shown in Table 5, PGEZ was not detected in the e supernatants of unstimulated bladder cells or r cells cultured in the ce of carbnchol. Consistent with COXZ responses described above, ation of mouse bladder cells with LPS d the secretion of high levels of PGEB. Addition of the analgesics acetaminophen, aspirin, ibuprofen and naproxcn suppressed the effect ot’LPS on PGE2 secretion and no difference was seen between the responses ol‘cetls treated with the 5 or 50 ttM dose of analgesic, Table 5. PG 352 secretion by mouse bladder cells after in vitro stimulation and treatment with analgesic Stimuli Analgesic PGEZ levels (pg/ml) None Acetaminophen (5 41M) LPS (1 itg/ml) Aspirin (5 uM) LPS (lug/ml) Ibuprofen (5 tiMD LPS l) Naproxen (5 uM) LPS (lug/ml) Acetaminophen (50 uM) LPS (lug/ml) Aspirin (50 uM) LPS (lug/ml) Ibuprofen (50 uM) LPS (l ) Naproxeu (50 nM) 588 i- 37 10184} In summary, these data show that the analgesics alone at 5 uM or 50 tiM do not induce COXZ and PGEZ responses in mouse bladder cells. The analgesics at 5. iiM or 50 WO 03390 uM, however, icantly inhibit C’OXZ and P652 responses of mouse bladder cells stimulated in vitro with LPS (3 lag/ml). No cant effect of analgesics was observed on COX2 and PGE2 responses of mouse bladder cells stimulated with earbaehol (1 mM).

EXAMPLE 4: EFFECT OF ANALGE lC AGENTS BOTUI..lNUM NEUROTOXIN AND AN'l‘lh/lUSCARlNIC‘ AGENTS 0N MOUSE BLADDER SMOOTH MUSCLE CELL CONTRACTION.

Experimental" Desifrn Cultured mouse or rat bladder smooth muscle cells and mouse or rat bladder smooth muscle tissue are exposed to inflammatory stimuli and non-inllan‘imatory stimuli in the presence of analgesic agent and/or antimuscarinic agent at various concentrations. The stimuli~indueed muscle contraction is measured to evaluate the inhibitory effect of the analgesic agent and/or antimusearinie agent. [0186} The effectors, analgesic agents and antimuscarinie agents are described in Example 2. {0187] Primary culture of mouse r smooth muscle cells are subjected to short term (1-2 hrs) or long term (24—48 hrs} stimulation 0 l‘ with: (1) Each analgesic agent alone at various doses. (2) Each analgesic agent at s doses in the ce ot‘I..PS. (3) Each analgesic agent at various doses in the presence of ltol or acetyleholine. ("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 ofLPS. (7) Botulinum neurotoxin A at various dOSeS in the presence of carbaehol or eholine. (8) Botulinum neurotoxin A at s doses in the presence of AA, DGLA, or EPA. (9) Each antimuscarinic agent alone at various doses. (l0) Each antimusearinic agent at various doses in the presence of Ll’S, (ll) Each antimuscarinie agent at various doses in the presence ol‘carbachol or aeetylcholine. (12) Each usearinie agent at various doses in the presence of AA, DGLA, or EPA.

Materials and Methods {0188} Primary mouse bladder cells are isolated as described in Example 3. In PCT/U82012/051888 selected experiments, cultures of bladder tissue are used. Bladder smooth muscle cell contractions are recorded with a Grass polygraph (Quincy Mass, US A).

EXAMPLE 5: EFFECT OF ORAL ANALGESlC AGENTS AND AN’l'lMUSCARINlC AGENTS ON COXZ AND PGE2 RESPONSES OF MOUSE BLADDER SMOOTH MUSCLE (TELLS.

Exgge/‘imenm! design- |()189] Normal mice and mice with over active bladder syndrome are given oral doses ot" aspirin, naproxen sodium, Ibuprofen, lndocin, nabumetone, Tylenol, Celecoxib, oxybutynin, solifenacin, daril’enacin, atropine and combinations thereof. Control groups include untreated normal mice and untreated GAB mice without over active bladder syndrome. Thirty (30) min alter last. doses, the bladders are collected and stimulated ex vivo with carbachol or acetylcholine. In selected ments. the bladders are treated with num ncurotoxin A before stimulation with carbaehol. Animals are maintained in metabolic cages and frequency (and volume) of urination are evaluated. Bladder outputs are ined by ring water intake and cage litter weight. Serum PGHg, PGE, POE-3,, cydin, mboxane, lL-lli, IL—(i, 'l‘NF-a, CAMP, and cCiMl’ levels are determined by ELISA. CD80, CD86, MHC class It expression in whole blood cells are determined by flow cytometry.

At the end of the experiment, animal are euthanized and ex vii-'0 r contractions are recorded with a Grass polygraph. Portions of bladders are fixed in formalin, and COXZ responses are analyzed by immunohistoehemistiy.

E 6: EFFECT OF ANALGESIC AGENTS BOTULlNUM NEUROTOXIN AND AN‘I‘llVlUSCARINIC‘ AGENTS ON HUMAN BLADDER SMOOTH MUSCLE CELL RE PONSES TO lNFLAMMAT RY AND NON—INFLAMMATORY STIMULI tttantnt.£2555;ng [0191} This study is designed to characterize how the optimal doses of sic determined in Examples l-S all'ect human bladder smooth muscle cells in cell culture or tissue cultures, and to s whether different classes of analgesics can synergizc to more efficiently inhibit (“OX2 and PGEZ responses. |{)l92] The effectors, analgesic agents and antimuscarinic agents are described in Example 2.

Human bladder smooth muscle cells are ted to short term (1-2 hrs.) or long term (24—48 hrs) stimulation of with: (1') Each analgesic agent alone at various doses.

PCT/U82012/051888 (3) Each analgesic agent at s doses in the presence of LPS. (3) Each analgesic agent at s doses in the presence of carbachol or acetylcholine. (4) Each analgesic agent at various doses in the preSence ot‘AA, DGI..A, or EPA. (53) 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 hol or acctylcholine. (8) Botulimtm neurotoxin A at s doses in the ce ol‘AA, DGl.,A, or EPA. (9) Each antimuscarinie agent alone at various doses. (10) Each antimuscarinic agent at various doses in the presence of LPS. ('11) Each antimuscarinic agent at various doses in the presence of carbachol or acctylcholine. (12:) Each antimuscarinic agent at various doses in the presence of AA, {)GLA, or EPA. $0194} The cells are then analyzed for the release of POI-lg, PGlE, l’GlEig, Prostaeydin, ’l‘hromboxanc, lL- l B, lL—ti, TNF—o, the COXZ activity, the production ot’cAMP and COMP, the production of IL—l J3, ll..-6, TN Fax and COXZ mRNA, and surface expression ot’CD80, CD86 and MHC class ll les.

EXA MPLE 7: EFFECT OF ANALGESIC AGENTS. BOTUUNUM NEUROT )XIN AND ANTIMUSCARINlC AGENTS ON HUMAN BLADDER SMO TH MUSCLE CELL CONTRACTION.

Experimental Design ed human bladder smooth muscle cells 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 ction is measured to evaluate the inhibitory effect of the. analgesic agent and/or antimuscarinic agent. {0196} The effectors, analgesic agents and antimuscarinic agents are described in Example 2. l0l97} Human r smooth muscle cells are subjected to short term (L2 hrs) or long term (24—48 hrs) stimulation ol‘with: (1) Each analgesic agent alone at various doses. (2} Each analgesic agent at various doses in the ce of LPS. (3') Each analgesic agent at various doses in the presence of curbaehol or PCT/U82012/051888 acetylcholine. (4) Each analgesic agent at various doses in the presence of AA, DGLA, or EPA. l5) Botulinum neurotoxin A alone at various doses. (6) Botulimim oxin A at various doses in the presence ot’l..PS. (7) Botulinum neurotoxin A at s doses in the presence of carbachol or acctylcholinc. ( 8) Botulinum neurotoxin A at various doses in the presence of AA. .A, or EPA. (9) Each antimuscarinie agent alone at various doses. (l0) Each antimuscarinic agent at various doses in the presence of LPS. (1 1:} Each antimuscarinic agent at van’ous doses in the presence of carbachol or acotylcholine. ('12) Each antimuscarinic agent at various doses in the presence of AA, DGLA, or EPA. {01981 Bladder smooth muscle cell contractions are recorded with a Grass polygraph (Quincy Mass, USA).

EXAMPLE?) 8: EFFECT OF ANAUIEESIC AGENTS 0N NORMAL l'iUMAN BLADDER SMOOTH MUSCLE CELL RESPONSES TO MA'I‘ORY AND NON EXPERIMENTAL DESIGN Culture of normal human bladder smooth muscle cells [0199} Normal human bladder smooth muscle cells were isolated by enzymatic digestion from macroscopically nomtal pieces of human bladder. Cells V\ ere expended in l'lfI‘O by culture at 37° C in a 5 ”/6 C()3 atmosphere in RPMI 1640 supplemented with it) % fetal bovine serum, 15 mM HEPES, 2 mM L—glutaminc, 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 , 2 ng/ml fibroblast growth lactor. and 5 rig/ml insulin.

Treatment of normal human bladder smooth muscle cells with analgesics in vitro 10200} r smooth muscle cells 'Lrypsiuized and seeded in microculture plates at a cell density ot‘3x ll)4 cells per well in 100 pl were treated with sic solutions (5.0 ah" well) either alone or together carbachol (lO—Molar, 50 ul/ well), as an example of non— inl‘lammatory stimuli, or lysaccharide (LI’S) of Salmonella {whine/ring; l tag/ml, 50 W0 20131103390 PCT/U82012/051888 ul/ well). as an example ol’non-inl‘lannnatory stimuli. When no other effectors were added to the cellse 50 pl ot‘RPMI l640 without fetal bovine serum were added to the wells to : the final volume to 200 pl. {0201} After 24 hours ofculture, lSO pl ofcul'ture 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 staglandin E2 (PGEZ) ses by ELISA. Cells were fixed, permeabilized and blocked for detection of COXZ using a fluorogenic substrate. In selected experiment cells were stimulated l2 hours in vitro for is Z, PGEZ and cytokine responses.

Analysis of COXE, PGEZ and cytokine responses [0202} COXZ and l’GELZ responses were analyzed as described in e 3.

Cytokine responses were analyzed as described in Example 2 RESULTS {0203} A ics inhibit COX2 responses qfnorma/ human bladder smooth muse/e cells In inflammamijv and mm~ iI-z/Iammatmy stimuli — AnalySts o l‘ cells and culture supematants allcr 24 hours of cultures showed that none of the analgesics tested alone induced COXZ responses in normal human bladder smooth muscle cells. l-lowever, as summarized in Table 0, carbachol induced low, but significant COXZ responses in normal human bladder smooth muscle cells. On the other hand. l..PS treatment. resulted in higher levels of COX2 responses in normal human bladder smooth muscle cells. AcetaminOphen, n, ibuprofen and naproxen could all suppress the effect ol‘carbachol and LPS on COXZ levels. The suppressive effect of the analgesics was seen on Ll’S—indueed responses when these drugs were tested at either 5. pM or 50 itM.

Table 6. COXZ expression by normal human r smooth muscle cells after in vitro stimulation with inflammatory and non- inflammatory stimuli and treatment with analgesic Total coxz levels Total coxz levels Stimuli Analgesic: {Normalized RFUs) (Normalized RFUs) subject 1 subject 2 (Tarbachol 10' M Acetaminophen (50 uM) C‘al‘bacltol IO'3 M l Aspirin(50 MM) Cnrbachol It)"J Vt Ibuprofen (50 ,uM) hol 10“ M Nuproxen (SO ttM} Carbacliol ll)”3 M Acetaminophen [50 uM) WO 03390 PCT/U52012/051888 LPS ( l 0 pig/ml) ; None LPS ( l0 iiglml) ' Acetaminophen (5 tiM) LPS (10 ,ug/ml) Aspirin (5 1.1M) LPS ( l 0 gig/ml) ibuprofen {5 in“) [PS (‘10 ) Naproxcn {5 ii'vi LPS (10 tigl‘ml) : Acetaminophen [50 MM) LPS ( l 0 tigv’ml} i Aspirin (50 tiM) LPS (1 o tiglml) lbnprofcu (so oM} LPS (10 itg/ml) : Napi'oxcn(50t1M) Data are cxprcsscd as mean icatcs l0204] Jinalgesics inhibit POE?.. responses oj‘normal human bladdersmoorl; muse/6’ cells to inflammatory and non— inflammatory stimuli - Consistent with the induction of COX2 responses described above. both ho} and LPS induced production of P052 by normal human bladder smooth muscle cells. Acetaminophen, aspirin, ibuprofen and naproxcn were also found to suppicss the lPSinduced PtjE2 ses at cithorS _uM or 50 iiM (Table 7).

Table 7. POE-,2 secretion by normal hum-an bladder smooth muscle cells after in vitro stimulation with inflammatoxy and non- inflammatory stimuli and treatment with analgesic Stimuli Analgesic PGEZ levels" (pg/ml) 'PGEZ levels (pg/ml) Subject I Subject7 Cnrbachol 10'M Acetaminophen (50 fiMl Carbachol 10" M Aspirin (50 in“) Carbachol 10" ‘vi Ibuprofen (50 ns‘vf) Cnrbachol 10" M cn (50 51M) Carbzichol 10‘3 VI Acetaminophen ('50 ,uM) LPS (10 gig/ml) Acetaminophen (5. NM) LPS (10 lttg/ml) n (5 NA) LPS (in try/ml) fcn (5 HM) LPS (10 tiglml) Naproxcn (5 pM ms ( l o ngjmn Acetaminophen (50 nM} Ll-‘S (l0 ng/ml) Aspirin (50 old) LPS (‘10 tiglml) Ibuprofen (50 nM) LPS (10 ng/ml) en {50 p311) Data are cxpresscd as mean of duplicates Analgesics inhibit cyrokine responses ofnormal human bladder cells 10 an PCT/U52012/051888 atory i - Analysis of cells and culture supcmatants aficr 24 hours of es showed that none of the analgesics tested alone induced ll..—6 or TNFo ion in normal human r smooth muscle cells. As shown in Tables 8 and 9, the doses ol‘carhachol tested induced low, but significant 'l‘NFa and IL-(i responses in normal human bladder smooth muscle cells. On the other hand, LPS treatment resulted in massive induction of these proinfiammatory cytolcincs. Acetaminophen, n, ibuprofen and naproxen suppress the effect ofcarbachol and LPS on 'I‘NFo and lL—(i responses. the suppressive effect of the analgesics on LPS-induccd responses was seen when these drugs were tested at either 5 pm or 50 ,ttM.

Table 8. ’l‘NI—‘a sccrctiou by normal human bladder smooth muscle cells after in vitro stimulation with atory and non- atory stimuli and treatment with analgesic Stimuli Analgesic "t‘Nth (pg/ml) ” 'l'NFa {pg/ml) Subject 1 Subject 2 Camacho! IO”3 M None 350 (‘arbachol 10‘3 M Acetaminophen (50 uM) I33 Carbacltol 10'3 M Aspirin {50 tiM) [ l0 Cnrbztchol 10‘J \4 Ibuprofen (50 tiM) J46 C2trbachol 10 \4 Nztptoxtm (50 “VD 139 I. PS (10 ttg/nil} Acetaminophen (5 Mail LPS (l0 pig/ml) Aspirin (5 51V!) LPS (l0 ttgfml) Ibuprofen (5 uM) LPS (10 tag/ml) Naproxen (5 ttM LI’S (l0 ttg/ml) Acetaminophen (SO 3.1M) LPS {10 ttg/ml) Aspirin (50 ttM) LI-‘S (l0 uglml) Ibuprofen (50 pM) LPS (10 tlg/ml) NNaproxen (50 14M) 'rData arc expressed as mean of dupltt.ates Table 9. IL-6 secretion by normal human bladder smooth muscle cclls after in vitro stimulation with inflammatory and non- inflammatory stimuli and treatment with analgesic PCT/U82012/051888 Analgesic lL-6 (pg/ml) ' lL—tS {pg/ml) Subject 1 Subject 2 Cai'bnchol 10 M None Carbaeltol 10 M Acetaminophen (in mi) Carbzlchol " It) \l n ('50 nM) L'zlrbaehol 10 N1 Ibuprofen (50 ttM) Carhttcliol 10‘1 VI en (50 pM) Ll—‘S (10 gig/ml) Acetaminophen (5 pM) LPS (10 pg ml) Aspirin (5 well 2199 L?S (l0 pg ml) Ibuprofen (5 uVl) 2063 LPS (i0 ttg ml) cn t5 plat 2077 LPS (10 ttg: ml) Acct:iminophen (50 le LPS (10 ug/ml) Aspirin {50 ttM) -k 2010 LPS (l0 ttg ml) Ibuprofen (50 ttVl) ‘ " l99l IFS (l0 tignil) Napi‘oxcn (50it“) 2028 7‘lDam are expressed lib mein of dupltcits: [0206! Primary nomial human bladder smooth muscle cells were isolated, cultured and evaluated for their ses to analgesics 1n the presence ol‘non-inl‘laimnatory (carbachol) and inl‘lammateiy (LPS) stimuli. The goal of this study was to determine whether or not normal human bladder smooth muscle cells recapitulate the observations previouely made with murine r cells. [0207} The abovc»deeeribed ment will be ed with analgesic agents and/or antimuscai‘mic agents in delayed-release, OI extended-release formulation or delayed-antl- extended—release fommlations. {0208] The above description it; for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those OthOUS ations and variations of it which will become apparent to the skilled worker upou reading the description. it is intended. however, that all such obvious modifications and variations be ed within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

Claims (10)

WHAT IS D IS:

1. Use of a pharmaceutical composition comprising acetaminophen in combination with either (1) one or more antidiuretic agents, or (2) one or more spasmolytics, or (3) an antimuscarinic agent selected from the group consisting of ynin, nacin, darifenacin and atropine, in the manufacture of a medicament fo r treating nocturia in a subject in need thereof, wherein said pharmaceutical composition is formulated in an ed-release formulation and wherein said inophen is formulated for administration at a daily dose of 1 mg to 2000 mg.

2. The use of the pharmaceutical ition of Claim 1, wherein said acetaminophen is formulated for administration at a daily dose of 50 mg to 1000 mg.

3. The use of the pharmaceutical composition of Claim 1, wherein said acetaminophen is formulated for administration at a daily dose of 500 mg to 2000 mg.

4. The use of the pharmaceutical composition of Claim 1, wherein said acetaminophen is formulated for administration at a daily dose of 100 mg to 1500 mg.

5. The use of the pharmaceutical composition of Claim 1, wherein said acetaminophen is formulated for administration at a daily dose of 250 mg to 1000 mg.

6. The use of the pharmaceutical composition of any one of Claims 1-5, wherein said pharmaceutical composition is formulated in an ed-release formulation by embedding said acetaminophen in a matrix of ble substance(s).

7. The use of the pharmaceutical composition of any one of Cl aims 1-5, wherein said extended-release formulation comprises a polymer controlling release by dissolution controlled release.

8. The use of the pharmaceutical composition of any one of Cl aims 1-5, wherein said extended-release formulation comprises a water soluble or swellable matrix-forming polymer.

9. The use of the pharmaceutical composition of any one of Claims 1-8, wherein said pharmaceutical composition is coated with an enteric coating.

10. The use of the pharmaceutical composition of any one of Claims 1-9, wherein said ceutical composition is formulated for oral administration. WAS:186047.1