KR20180054656A - Delayed-release formulations, methods of manufacture and uses - Google Patents

Abstract

Disclosed are a composition for reducing the frequency of urination and a method for producing the composition. One method of manufacture comprises forming a first mixture comprising a first active ingredient comprising at least one analgesic agent, coating the first mixture with a first delay-release coating to form a first component, Forming a second mixture comprising a second active ingredient comprising an analgesic agent; coating the second mixture with a second delay-release coating to form a second component; And mixing with the second component to form a third mixture.

Description

Delayed-release formulations, methods of manufacture and uses

This application claims priority from U.S. Serial No. 14 / 842,539, filed September 1, 2015. The entirety of the above application is incorporated by reference.

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

The detrusor muscle is a layer of bladder wall made of smooth muscle fibers arranged in bundles of spiral, longitudinal and circular. When the bladder is stretched, it signals the parasympathetic nervous system and contracts the detrusor. This allows the bladder to drain urine through the urethra.

In order for the urine to be excreted in the bladder, both the autonomously regulated sphincter and the spontaneously regulated external sphincter must be opened. Problems with these muscles can lead to incontinence. When the amount of urine reaches 100% of the absolute capacity of the bladder, the spontaneous sphincter becomes involuntary and the urine is immediately discharged.

An adult's bladder can usually hold about 300-350 ml of urine (working volume), but a full adult bladder can store about 1000 ml (absolute volume), which varies from person to person. As the urine accumulates, the wall of the bladder (the wrinkle, rugae) folds and ridges, and as the bladder stretches, the wall of the bladder becomes thinner, allowing the bladder to store more urine without significant increase in internal pressure.

In most individuals, the urge to urinate usually begins when the volume of urine within the bladder reaches about 200 ml. At this stage, the subject can easily cope with the urge to urinate if desired. As the bladder continues to fill, the urge to urinate becomes stronger and more difficult to ignore. Eventually, the bladder is filled to the point where the urination urge becomes overwhelming, and the subject can no longer ignore it. In some individuals, the urge to urinate begins when the bladder is less than 100% of the working volume. This increased need for urination may interfere with normal activities, including sleeping ability, during an uninterrupted enough break. In some cases, this increased need for urination may be associated with medical conditions such as benign prostatic hyperplasia in men, prostate cancer, or pregnancy in women. However, increased urination needs occur individually in both men and women who are not affected by other diseases.

Accordingly, there is a need for compositions and methods for the treatment of male and female subjects suffering from a urge to urinate even when the bladder is less than 100% associated with the operating volume of the bladder. The compositions and methods are needed for inhibition of muscle contraction such that the urination volume is initiated in the subject when the volume of urine in the bladder exceeds about 100% of the bladder working volume.

One aspect of the present invention relates to a method of making a pharmaceutical composition for reducing urinary frequency. The method includes forming a first mixture comprising a first active ingredient comprising at least one analgesic agent; Coating the first mixture with a first delay-release coating to form a first component, wherein the first delay-release coating is applied to the first delay- Releasing the mixture; Forming a second mixture comprising a second active ingredient comprising at least one analgesic agent; Coating said second mixture with a second delay-release coating to form a second component, said second delay-release coating releasing said second mixture with a delay time of 4-6 hours after oral administration ; And mixing the first component with the second component to form a third mixture.

In another embodiment, the method comprises forming a first mixture comprising a first active ingredient comprising at least one analgesic agent; Said first delay-release coating releasing said first mixture with a delay time of 1-4 hours after direct contact with bodily fluids; Coating the core structure with a second mixture to form a coated core structure, wherein the second mixture comprises a second active ingredient comprising at least one analgesic; And coating the coated core structure with a second delay-release coating to form a dual-coated core structure, wherein the second delay-release coating is applied to the second mixture at a delay time of 1-4 hours after oral administration, Lt; / RTI >

Another aspect of the present application relates to a pharmaceutical composition prepared using the methods of the present application.

Figures 1A and 1B are graphs showing that analgesics control the expression of co-stimulatory molecules by Raw 264 macrophages in the absence (Figure 1A) or presence (Figure IB) of LPS. Cells were treated with analgesics alone or with Salmonella typhimurium LPS (0.05 μg / ml) for 24 h. The results are expressed as an average relative percentage of CD40 + CD80 + cells.

The following detailed description is presented to enable a person skilled in the art to make and use the invention. For purposes of explanation, specific nomenclature is provided to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that such details are not required to practice the present invention. The description of the specific application is provided only as a representative embodiment. The present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

The term " effective amount " as used herein means the amount necessary to achieve a selected result.

The term " analgesic " as used herein refers to a medicament, compound or drug used to alleviate pain and includes anti-inflammatory compounds. Exemplary analgesic and / or anti-inflammatory drugs, compounds or drugs include, but are not limited to, the following: non-steroidal anti-inflammatory drugs (NSAIDs), salicylate, aspirin, salicylic acid, Or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the at least one compound is selected from the group consisting of at least one compound selected from the group consisting of atorvastatin, atorvastatin, Arylacetic acid derivatives, tolmetin, ketorolac, diclofenac, propionic acid derivatives, ibuprofen, naproxen sodium, naproxen, fenoprofen, ketoprofen, fluorevifrophen, oxaprozin; Wherein the at least one compound is selected from the group consisting of at least one selected from the group consisting of at least one selected from the group consisting of at least one selected from the group consisting of benzoic acid, Wherein the active ingredient is selected from the group consisting of paclitaxel, paclitaxel, paclitaxel, paclitaxel, paclitaxel, paclitaxel, paclitaxel, But are not limited to, thiazepine, thiazepine, thiazepine, thiazepine, thiazepine, thiazepine, thiazepine, thiazepine, thiazepine, -7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one, meloxicam, loroxicam, d-indofuran, mofezolek, amolol methine, Methic acid, fluvifrophen, surpopen, oxaprozin, zalto propen, alminopropene, thiopropenoic acid, their pharmaceutical salts, their Cargo, and their solvates.

As used herein, the terms " coxib " and " COX inhibitor " can inhibit the activity or expression of a COX2 enzyme or inhibit the severity of a severe inflammatory response, including pain and swelling Or reduce the amount of the compound.

The bladder has two important functions: the storage and emptying of the urine. Storage of urine occurs at low pressure, which means that the detrusor relaxes during the filling phase. Emptying the bladder requires coordinated shrinkage of the detrusor and relaxation of the sphincter of the urethra. Failure of the storage function can cause lower urinary tract symptoms such as urgency, incidence, and impulsive incontinence. Overactive bladder syndrome due to involuntary contraction of the smooth muscle of the bladder (detrusor) during the storage phase is a common, unreported problem, and the epidemic has only recently been evaluated.

One aspect of the present invention relates to a method of reducing the frequency of urination by administering a delayed-release formulated pharmaceutical composition to a person in need thereof. The pharmaceutical composition comprises one or more analgesic agents and, optionally, one or more antimuscarinic agents.

The term " delayed-release " as used herein means a drug that releases the active ingredient (s) into the body without immediate degradation. In some embodiments, the term " delayed release " is used with respect to a drug formulation having a release profile with a predetermined delay in release of the drug after administration. In some embodiments, the delayed-release formulation comprises a barrier coating applied to an oral medicament to prevent release of the drug prior to reaching the small intestine. Delayed-release formulations, such as enteric coatings, prevent gastrointestinal irritants such as aspirin from dissolving in the stomach. This coating protects acid labile drugs from acid exposure of the stomach and delivers them to a basic pH environment (pH above the gut) that can give the desired action without degrading them.

The term " pulsatile release " is a type of delay-release that provides rapid and instantaneous release of a drug within a short period of time immediately after a predetermined delay time, thereby providing a " pulsed " plasma profile of the drug after drug administration It is used herein with respect to drug preparations. The agent may be designed to provide single beating release or multiple beating release at predetermined time intervals after administration.

Most enteric coatings work by being present on the surface and are stable at high acidic pH in the stomach, but rapidly collapse at low acidic (relatively more basic) pH. Thus, intestinally coated pills are not soluble in gastric acidic juices (pH ~ 3), but are soluble in the alkaline (pH 7-9) environment of the small intestine. Examples of enteric coating materials include, but are not limited to, methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate (hiropromellose acetate succinate), polyvinylacetate Phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, sodium alginate and stearic acid, but are not bonded thereto.

In some embodiments, the pharmaceutical compositions are administered orally in a variety of drug formulations designed to provide delayed-release. The delayed oral dosage form may include, for example, tablets, capsules, caplets, and also a plurality of granules, beads, powders or pellets that are not encapsulated or encapsulated. Tablets and capsules represent the easiest oral dosage form, in which case solid pharmaceutical carriers are used.

In delayed-release formulations, the one or more barrier coatings may be applied to pellets, tablets or capsules to slow the dissolution and subsequent release of the drug in the small intestine. Typically, the barrier coating contains one or more polymers that wrap, surround, or form a layer around the pharmaceutical composition or active core, or the membrane.

In some embodiments, the active agent is delivered to the formulation to provide delayed release at a predetermined time after administration. The delay may be up to about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, or even longer.

The delayed-release composition comprises 100% of the total dose of active agent administered in a single unit dose. Alternatively, the immediate-release component can be included as a component of the combined release profile formulation, which can provide about 30-95% of the total dose of activator (s) delivered by the pharmaceutical formulation have. For example, the immediate-release component can provide at least about 5-70%, or about 50% of the total dose of activator (s) delivered by the formulation. In an alternate embodiment, the delay-release type component is selected from the group consisting of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 (%) of the total dose of activator (s) , 90 or 95%, respectively.

Delay-release formulations generally include a barrier coating that delays the release of the active ingredient (s). The barrier coating may be made of a variety of different materials depending on its purpose. Additionally, the formulation may include a plurality of barrier coatings to facilitate release in a temporary manner. The coating may be applied to a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, carboxymethylcellulose, acrylic copolymer, polyethylene glycol and / or polyvinylpyrrolidone) Or a coating based on methacrylic acid, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinylacetate phthalate, shellac, and / or ethylcellulose. In addition, the formulation may additionally comprise a time delay material, for example, glyceryl monostearate or glyceryl distearate.

In some embodiments, the delayed-release formulation comprises an enteric coating comprising at least one polymer that facilitates release of the active agent in a proximal or distal region of the gastrointestinal tract. The term " enteric polymer coating " as used herein is a coating comprising one or more polymers having a pH-dependent or pH-independent release profile. Generally, the coating is not dissolved in the acidic medium of the stomach, but is dissolved or eroded in the more distal regions of the gastrointestinal tract, such as the small intestine or colon. The enteric polymer coating generally inhibits release of the active agent until after a delay of gastric emptying of about 3 to 4 hours after administration.

The pH-dependent intestinal coating maintains its structural integrity at low pH, such as the stomach, but retains one or more pH-dependent or pH-dependent conditions that dissolve in higher pH environments of the distal region of the gastrointestinal tract, such as the small intestine where the drug content is released. Sensitive polymer. For purposes of the present invention, " pH dependent " is defined as having properties (e.g., dissolution) that vary depending on the pH environment. Examples of pH- dependent polymers include methacrylic acid copolymers, methacrylic acid-methyl methacrylate copolymer (e.g., EUDRAGIT ^® L100 (type A), EUDRAGIT S100 ^® (type B), Rohm GmbH, Germany); Methacrylic acid-ethyl acrylate copolymer (e.g., EUDRAGIT ^® L100-55 (type C) and EUDRAGIT ^® L30D-55 copolymer dispersion, Rohm GmbH, Germany); Methacrylic acid-methyl methacrylate and methyl methacrylate copolymers of acrylate (EUDRAGIT ^® FS); Terpolymers of methacrylic acid, methacrylate and ethyl acrylate; Cellulose acetate phthalate (CAP); Hydroxypropyl methylcellulose phthalate (HPMCP) (e.g., HP-55, HP-50, HP-55S, Shinetsu Chemical, Japan); Polyvinyl acetate phthalate (PVAP) (e.g., COATERIC ^® , OPADRY ^® enteric white OY-P-7171); Cellulose acetate succinate (CAS); Hydroxypropyl methyl cellulose acetate succinate (HPMCAS), e.g., HPMCAS Grade LF, MF Grade, AQOAT ^® LF and MF AQOAT ^® Grade HF containing (Shin-Etsu Chemical, Japan); Shellac (e.g., Marcoat ^TM 125 and Marcoat ^TM 125N); Carboxymethyl ethylcellulose (CMEC, Freund Corporation, Japan), cellulose acetate phthalate (CAP) (e.g., AQUATERIC ^®); Cellulose acetate trimellitate (CAT); And those of the two at least about 2: 1 to about 5: 1 were mixed in a weight ratio between the mixture, for example, EUDRAGIT ^® L 100-55 and EUDRAGIT ^® S 100 to about 3: 1 to about 2: 1 , Or mixtures of EUDRAGIT ^® L 30 D-55 and EUDRAGIT ^® FS at a weight ratio of about 3: 1 to about 5: 1, but are not combined therewith.

pH-dependent polymers generally exhibit an optimum pH that is characteristic of dissolution. In some embodiments, the pH-dependent polymer exhibits an optimum pH 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 another embodiment, the pH-dependent polymer exhibits an optimum pH of ≥5.0, ≥5.5, ≥6.0, ≥6.5, or ≥7.0.

In other embodiments, the enteric coating may comprise one or more pH-independent polymers. These polymers provide release of the drug after a period of time, independent of pH. For the purposes of the present invention, " pH independent " is defined as having properties (e.g., dissolution) that are substantially unaffected by pH. pH-independent polymers are often referred to in the context of " time-controlled " or " time-dependent " release profiles.

The pH-independent polymer may be water-insoluble or water-soluble. Water-insoluble, pH-independent polymers include, for example, neutral methacrylic acid esters with small portions of trimethylammonioethyl methacrylate chloride (e.g., EUDRAGIT ^® RS and EUDRAGIT ^® RL); Neutral ester dispersions with no action (for example, EUDRAGIT ^® NE30D and EUDRAGIT ^® NE30); But are not limited to, cellulosic polymers such as ethylcellulose, hydroxyethylcellulose, cellulose acetate or mixtures thereof and other pH-independent coating products. The water-soluble, pH-independent polymer includes, for example, OPADRY ^® amb.

In some embodiments, the pH-independent polymer may include one or more polysaccharide coatings that are resistant to erosion in both the stomach and intestines. Such polymers can be degraded only in a colon containing large microflora containing, for example, biodegradable enzymes that degrade polysaccharide coatings, and release the drug content in a controlled, time-dependent manner .

In certain embodiments, the coating method utilizes at least one pH-dependent and at least one blend of pH-independent polymers. When the soluble polymer reaches the optimum pH of solubilization, blending of the pH-dependent and pH-independent polymers can reduce the rate of release of the active ingredient.

In some embodiments, a "time-controlled" or "time-dependent" release profile can be obtained using a water-insoluble capsule body containing one or more active agents, the capsule body being insoluble but permeable and swellable, One end is closed. Upon contact with gastrointestinal fluids or dissolution media, the plug expands and pushes itself out of the capsule, releasing the drug after a predetermined delay time, which can be adjusted, for example, by the position and size of the plug . The capsule body may further be coated with an external pH-dependent intestinal coating that maintains the capsule intact until it reaches the small intestine. Suitable plug materials include, for example, polymethacrylates, erodible compressed polymers (e.g., HPMC, polyvinyl alcohol), cogealed melted polymers (e.g., glyceryl mono (Such as amylose, arabinogalactan, chitosan, chondroitin sulfate, cyclodextrin, dextran, guar gum, polysaccharides such as pectin and xylyl), as well as enzymatically controlled erodable polymers do.

In other embodiments, the capsule or bilayer tablet contains a drug-containing core that is formulated and covered with a swellable layer, and an external insoluble, but semi-permeable polymeric coating or film. The delay time before rupture can be controlled by the permeation and mechanical properties of the polymer coating and the swelling behavior of the swollen layer. Generally, the swell layer comprises at least one swelling agent, such as a swellable hydrophilic polymer that can expand and retain water in its structure.

Examples of the water-swellable material may include polyethylene oxide (e. G., Those having an average molecular weight between 1,000,000 to 7,000,000, such as POLYOX ^®), methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose; Polyalkylene oxides having an average molecular weight of 100,000 to 6,000,000, including but not limited to poly (methylene oxide), poly (butylene oxide); Poly (hydroxyalkyl methacrylates) having a molecular weight of from 25,000 to 5,000,000; Poly (vinyl) alcohols having a low acetal moiety cross-linked with glyoxal, formaldehyde or glutaraldehyde and having a degree of polymerization of from 200 to 30,000; Methylcellulose, a mixture of cross-linked agar and carboxymethylcellulose; The copolymer produced by forming a dispersion of a finely divided copolymer of cross-linked styrene, ethylene, propylene, butylene or isobutylene and maleic anhydride in a proportion of from 0.001 to 0.5 mol of a saturated crosslinking agent per mole of maleic anhydride in the copolymer ≪ / RTI > CARBOPOL ^占 acid carboxy polymer having a molecular weight of 450,000 to 4,000,000; CYANAMER ^® polyacrylamide; Cross-linked water swellable indene maleic anhydride polymer; GOODRITE ^® polyacrylic 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 a combined de-ester-cross-linked poly-glucan; A carbomer having a viscosity of 3,000 to 60,000 mPa as an aqueous solution of 0.5% -1% w / v; Cellulose ethers such as hydroxypropylcellulose having a viscosity of about 1000-7000 mPa as a 1% w / v aqueous solution (25 캜); Hydroxypropylmethylcellulose having a viscosity of at least about 1000, preferably at least about 2,500 and at most about 25,000 mPa as a 2% w / v aqueous solution; Polyvinylpyrrolidone having a viscosity of about 300-700 mPa as a 10% w / v aqueous solution at 20 占 폚; And combinations thereof, but are not limited thereto.

The enteric layer further comprises an anti-tackiness agent such as talc or glyceryl monostearate and / or a plasticizer. Wherein the enteric layer is selected from the group consisting of triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, polyethylene glycol acetylated monoglycerides, glycerin, triacetin, propylene glycol, phthalate esters (e.g., diethyl phthalate, ), Titanium dioxide, ferric oxide, castor oil, sorbitol and dibutyl sebacate, but are not bound thereto.

In another embodiment, the delayed release formulation is a water permeable, but insoluble film coating that wraps the active ingredient and the osmotic agent. As the water slowly diffuses from the gut into the core through the film, the core expands until the film ruptures, thereby releasing the active ingredient. The film coating can be adjusted to allow for various rates of water permeation or release.

Alternatively, the release time of the drug may be controlled by a predetermined amount of micropore at the bottom of the body, and a water-insoluble polymer membrane containing an amount of a swollen excipient such as low substituted hydroxypropylcellulose (L-HPC) and sodium glycolate (Such as ethyl cellulose, EC), and the degradation delay time due to the balance of thickness. After oral administration, the gastric juice penetrates through the pores to cause swelling of the swellable excipient, which results in a first capsule body containing a swellable substance, a second capsule body containing the drug, and a second capsule body attached to the first capsule body Creating an internal pressure that separates the components in the capsule, including the cap.

In other embodiments, the extended-release formulation is formulated to release the active (s) by an osmotic mechanism. By way of example, the capsule may comprise 2, 3, 4, 5, or 6 push-pull units that are formulated as a single osmotic unit or encapsulated within a hard gelatin capsule, and each double- The unit comprises an osmotic push layer and a drug layer surrounded by a semi-permeable membrane. One or more orifices are drilled through the membrane next to the drug layer. This membrane may additionally be covered with a pH-dependent enteric coating to prevent release until gastric drainage is complete. The gelatin capsules dissolve immediately after ingestion. When the push-pull unit (s) enter the small intestine, the enteric coating collapses, causing the fluid to flow through the semi-permeable membrane, and the osmotic push compartment expands to a rate that is precisely controlled by the rate of water movement through the anti- It is forced through the orifice (s) to exit the drug. Release of the drug may occur at a constant rate of up to 24 hours.

The osmotic push layer comprises at least one osmotic agent that creates a driving force to cause water to migrate through the semi-permeable membrane into the core of the vehicle. One class of osmotic agents includes water-swellable hydrophilic polymers, also termed "osmopolymers" and "hydrogels ", which include hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene oxide (PEO) (PEG), polypropylene glycol (PPG), poly (2-hydroxyethyl methacrylate), poly (acrylic acid), poly (methacrylic acid), polyvinylpyrrolidone (PVP) , Polyvinyl alcohol (PVA), PVA / PVP copolymers, PVA / PVP copolymers with hydrophobic monomers such as methyl methacrylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, sodium croscarmellose, carrageenan, Hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropylmethyl cellulose (HPMC), carboxymethyl cellulose (CMC), carboxyethyl cellulose (CEC ), Sodium alginate, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate, but are not bound thereto.

Another class of osmotic agents includes osmogen which imbibes water to affect the osmotic gradient across the semi-permeable membrane. Examples of osmogens include inorganic salts such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, potassium phosphate, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, and sodium sulfate; Sugars such as dextrose, fructose, inositol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, trehalose, and xylitol; Organic acids such as ascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid, sebacic acid, sorbic acid, adipic acid, edetic acid, glutamic acid, p-toluenesulfonic acid, succinic acid and tartaric acid; Urea; And mixtures thereof, but are not bound thereto.

Substances useful for forming a semipermeable membrane are those that are water permeable and water-insoluble at physiologically relevant pHs, or that have various grades of acrylic, vinyl, ether, polyamide, polyester , And cellulose derivatives.

In another embodiment, the delayed release formulation utilizes a water-impermeable tablet coating whereby water enters through the controlled hole until the core is ruptured. When the tablets are ruptured, the drug content is released immediately or over a longer period of time. These and other techniques may be modified to allow a predetermined delay period before the release of the drug is initiated.

Various coating techniques may be applied to the active agent-containing granules, beads, powders or pellets, tablets, capsules or combinations thereof to produce different and distinctly different release profiles. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule containing a single coating layer. In another embodiment, the pharmaceutical composition is in the form of a tablet or capsule containing multiple coating layers.

In some embodiments, the pharmaceutical composition comprises a plurality of active ingredients selected from the group consisting of analgesics, antimuscarinics, antidiuresis, and antisuns. Examples of antispasmodic agents include, but are not limited to, carisoprodol, benzodiazepine, baclofen, cyclobenzaprine, metocarbone, methocarbamol, clonidine, clonidine analogs, and dantrolene. In some embodiments, the pharmaceutical composition comprises one or more analgesics. In another embodiment, the pharmaceutical composition comprises one or more other active ingredients selected from the group consisting of (1) one or more analgesics, and (2) antimuscarinics, antidiabetics and antispasmodics. In another embodiment, the pharmaceutical composition comprises (1) one or two analgesics and (2) one or two antimuscarinic agents. In another embodiment, the pharmaceutical composition comprises (1) one or two analgesics, and (2) one or two anti-diuretics. In another embodiment, the pharmaceutical composition comprises (1) one or two analgesics and (2) one or two antispasmodics. In another embodiment, the pharmaceutical composition comprises (1) one or two analgesics, (2) one or two antimuscarinic agents, and (3) one or two antidiuretic agents do.

In one embodiment, the plurality of active ingredients is formulated in an immediate-release form. In another embodiment, the plurality of active ingredients is formulated in a delay-release form. In another embodiment, the plurality of active ingredients is formulated as both immediate-release and delayed-release forms (e. G., The first portion of each active ingredient is formulated into an immediate-release form, The second portion of the component is formulated in a delay-release form). In another embodiment, a portion of the plurality of active ingredients is formulated in an immediate-release form, and a portion of the plurality of active ingredients is formulated in a delay-release form (e.g., the active ingredients A, B, Ready-to-release type, and active ingredients C and D are formulated in a delay-release form.

In certain embodiments, the pharmaceutical composition comprises an immediate-release component and a delay-release component. The immediate-release component may comprise one or more active ingredients selected from the group consisting of analgesics, antimuscarinics, antidiuresis, and antispasmodics. The delayed-release component may comprise one or more active ingredients selected from the group consisting of analgesics, antimuscarinics, antidiuretics and antispasmodics. In some embodiments, the immediate-release component and the delay-release component have exactly the same active ingredient. In another embodiment, the sustained-release component and the delayed-release component have different active components. In another embodiment, the immediate-release component and the delay-release component have one or more common active components.

In one embodiment, the pharmaceutical composition comprises two active ingredients formulated for immediate immediate release, such as two analgesics, or one analgesic and one antimuscarinic or antidiuretic agent, Or a mixture of antispasmodic agents). In another embodiment, the pharmaceutical composition comprises two active ingredients formulated to be delayed-released at about the same time. In another embodiment, the pharmaceutical compositions comprise two active ingredients formulated in two delay-release forms, each providing a different delay-release profile. For example, a first delay-release component emits a first active component at a first time and a second delay-release component emits a second active component at a second time. In another embodiment, the pharmaceutical composition comprises two active ingredients, one of which is formulated to be immediate-release and the other to be delayed-release.

In another embodiment, the pharmaceutical composition comprises two active ingredients formulated in an immediate-release form (e.g., two analgesic agents, or one analgesic agent and one antimuscarinic agent or antidiuretic agent, (2) a combination of two active ingredients formulated in a delayed-release form (e.g., two analgesics, or one analgesic and one antimuscarinic or antidiuretic or antispasmodic ≪ / RTI > In another embodiment, the pharmaceutical composition comprises three active ingredients formulated in an immediate-release form, and (2) three active ingredients formulated in a delayed-release form. In another embodiment, the pharmaceutical composition comprises four active ingredients formulated in an immediate-release form, and (2) four active ingredients formulated in a delayed-release form. In such embodiments, the active ingredient (s) in the immediate-release type component may be the same as or different from the active ingredient (s) in the delay-release type component.

The term " immediate-release " as used herein means a drug formulation that does not include a dissolution rate modulating agent. There is no substantial delay in the release of the active agent after administration of the immediate-release formulation. Immediate-release coatings may include suitable materials that are readily dissolved to release the drug content after administration. Immediate-release coating and examples of the material, include gelatin, polyvinyl alcohol polyethylene glycol (PVA-PEG) various other materials known to those skilled in the copolymer (e.g., KOLLICOAT ^®), and the art.

The immediate-release composition may comprise 100% of the total dose of active agent administered in a single unit dose. Alternatively, the immediate-release component can be provided as a component of a combined release profile formulation that can provide from about 1% to about 50% of the total dose of activator (s) delivered by the pharmaceutical formulation . For example, the immediate-release component may comprise at least about 5%, or from about 10% to about 30%, or from about 45% to about 50% of the total dose of activator (s) Can be provided. The remainder of the active agent (s) may be delivered in a delayed-release formulation. In an alternative embodiment, the immediate-release component comprises about 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the total dose of activator (s) delivered by the formulation to provide. The delayed release component provides about 90, 85, 80, 75, 70, 65, 60, 55 or 50% of the total dose of activator (s) delivered by the formulation.

In some embodiments, the immediate-release or delayed-release formulations comprise an active core comprised of one or more inert particles, each of which can be administered orally or parenterally, for example, using a fluid bed technique or other technique known in the art Pellets, pills, granular particles, microcapsules, microspheres, microparticles, nanocapsules, or nanospheres in the form of a drug-containing coating or film-forming composition. The inert particles can be of various sizes as long as they are large enough to maintain poor dissolution. Alternatively, the active core may be prepared by granulating and milling the drug-containing polymer composition and / or by extrusion and spheronization.

The amount of drug in the core may be dependent upon the dose required, and typically varies from about 5 to 90% by weight. Generally, the polymer coating on the active core will range from about 1 to 50%, based on the weight of the coated particles, depending on the type of retardation and release profile desired and / or the type of polymer and coating solvent selected. Those skilled in the art will be able to select the appropriate amount of drug for inclusion in the coating or core on the core to achieve the desired dosage. In one embodiment, the inactive core may be an encapsulated buffer crystal such as sucrose or buffer crystals or calcium carbonate, sodium bicarbonate, fumaric acid, tartaric acid, etc., which alters the microenvironment of the drug to facilitate drug release.

In some embodiments, the delayed-release formulation can be formed by coating a water soluble / water dispersible drug-containing particle, such as a bead, with a mixture of a water insoluble polymer and an enteric polymer, wherein the water insoluble polymer and the enteric polymer comprise 4 : 1 to 1: 1, and the total weight of the coating is 10 to 60% by weight, based on the total weight of the coated beads. The drug layer beads may optionally comprise an internal dissolution rate controlling membrane of ethyl cellulose. The composition of the outer layer, as well as the composition of the outer layer, as well as the individual weight of the inner and outer layers of the polymeric membrane, are optimized to achieve the desired biological cyclic rhythmic release profile for a given activity, ≪ / RTI >

In other embodiments, the formulation may include immediate-release drug-containing particles free of dissolution rate controlling polymeric film and delayed-release beads that exhibit a delay time of, for example, 2-4 hours after oral administration, Thus providing two pulse emission profiles. In another embodiment, the formulation comprises a mixture of two delay-emitting beads: a first type indicative of a delay time of 1-3 hours and a second type indicative of a 4-6 delay time.

In some embodiments, the active core is coated with one or more layers of a dissolution rate-regulating polymer to obtain a desired release profile with or without delay time. The outer layer membrane can provide the desired delay time (the period during which the drug is released or some drug released after the absorption of water or body fluids into the core) while the inner layer membrane releases drug Can be controlled greatly. The inner layer film may comprise a water insoluble polymer, or a mixture of water insoluble and water soluble polymers.

Suitable polymers for the outer membrane which greatly regulate a delay time of up to 6 hours can comprise an enteric polymer and a water-insoluble polymer in a thickness of 10 to 50% by weight, as described above. The ratio of the water-insoluble polymer to the polymer can vary from 4: 1 to 1: 2, and preferably the polymers are present in a ratio of about 1: 1. The water-insoluble polymer generally used is ethylcellulose.

Examples of water-insoluble polymers include ethylcellulose, polyvinyl acetate (Kollicoat SR # 0D from BASF), neutral copolymers based on ethyl acrylate and methyl methacrylate, EUDRAGIT ^® NE, RS and RS30D, RL or RL30D 4 < / RTI > ammonium groups, and copolymers of methacrylic acid esters. Examples of the water-soluble polymer include low molecular weight HPMC, HPC, methyl cellulose, polyethylene glycol (molecular weight of PEG> 100 wt%) which is in the range of 1 wt% to 10 wt%, depending on the active solubility in a solvent or latex suspension based on water and the coating formulation used. 3000). The water-insoluble polymer to water-soluble polymer may generally vary from 95: 5 to 60:40, preferably 80:20 to 65:35.

Preferably, the formulation is designed as a release profile that combines to interfere with a comfortable sleep, and the formulation generally releases the drug when the subject wakes up due to a urination urge. For example, consider sleeping at 11 PM and waking up at 12:30 AM, 3:00 AM, and 6:00 AM in general and urinate. The delayed, extended-release means may deliver the drug to 12:15 AM, thereby delaying the urination challenge for perhaps 2-3 hours.

The pharmaceutical composition may be administered daily or as needed. In certain embodiments, the pharmaceutical composition is administered to a subject prior to bedtime. In some embodiments, the pharmaceutical composition is administered just before bedtime. In some embodiments, the pharmaceutical composition is administered within about 2 hours before sleeping, preferably within about 1 hour before sleeping. In a further embodiment, the pharmaceutical composition is administered about 2 hours before bedtime. In a still further embodiment, the pharmaceutical composition is administered at least 2 hours prior to bedtime. In yet another embodiment, the pharmaceutical composition is administered about 1 hour before bedtime. In a further embodiment, the pharmaceutical composition is administered at least one hour prior to bedtime. In a still further embodiment, the pharmaceutical composition is administered less than one hour before bedtime. In another embodiment, the pharmaceutical composition is administered just before bedtime. Preferably, the pharmaceutical composition is administered orally.

("Therapeutically effective amount") of the active agent (s) in the immediate-release component or the prolonged-release component will depend on, for example, the severity and stage of the condition, the mode of administration, The bioavailability, the age and weight of the patient, the clinical history of the patient, the response to the active agent (s), and the judgment of the practitioner.

As generally suggested, a therapeutically effective amount of the active (s) in the immediate-release component or delay-release component is from about 100 μg / kg body weight / day to about 100 mg / kg body weight / Day. ≪ / RTI > In some embodiments, the daily dosage range of each active agent is about 100 μg / kg body weight / day to about 50 mg / kg body weight / day, 100 μg / kg body weight / day to about 10 mg / kg body weight / kg body weight / day to about 1 mg / kg body weight / day, 100 μg / kg body weight / day to about 10 mg / kg body weight / day, 500 μg / kg body weight / day to about 100 mg / 1 mg / kg body weight / day to about 100 mg / kg body weight / day, 500 mg / kg body weight / day to about 5 mg / kg body weight / Kg body weight per day to about 50 mg / kg body weight per day, from 1 mg / kg body weight / day to about 10 mg / kg body weight / day, from 5 mg / kg body weight / day to about 100 mg / kg body weight / day, Day to about 50 mg / kg body weight / day, from 10 mg / kg body weight / day to about 100 mg / kg body weight / day, and from 10 mg / kg body weight / day to about 50 mg / kg body weight / Day.

The active agent (s) described herein may be administered in an immediate-release or delayed-release component or a combination thereof in an amount of from 1 mg to 2000 mg, from 5 mg to 2000 mg, from 10 mg to 2000 mg, from 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 From 5 mg to 500 mg, from 5 mg to 500 mg, from 1 mg to 500 mg, from 1 mg to 500 mg, from 1 mg to 200 mg, from 5 mg to 1000 mg, from 5 mg to 500 mg, Wherein the amount of the compound of the present invention is 200 mg to 10 mg to 1000 mg, 10 mg to 500 mg, 10 mg to 200 mg, 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, 250 mg to 1000 mg, , 500 mg to 1000 mg, 500 mg to 2000 mg, or in a combined dose for daily oral administration. As expected, the dosage may vary depending on the condition, size, and age of the patient.

In some embodiments, the pharmaceutical composition comprises a single analgesic. In one embodiment, the single analgesic is aspirin. In another embodiment, the single analgesic is ibuprofen. In another embodiment, the single analgesic is naproxen sodium. In another embodiment, the single analgesic is indomethacin. In another embodiment, the single analgesic agent is nabumetone. In another embodiment, the single analgesic is acetaminophen.

In some embodiments, the single analgesic agent is administered in an amount of from 1 mg to 2000 mg, from 5 mg to 2000 mg, from 20 mg to 2000 mg, from 5 mg to 1000 mg, from 20 mg to 1000 mg, from 50 mg to 500 mg, , 250 mg to 500 mg, 250 mg to 1000 mg, or 500 mg to 1000 mg per day. In certain embodiments, the pharmaceutical composition comprises acetylsalicylic acid, ibuprofen, naproxen sodium, indomethacin, nabumetone, or acetaminophen as a single analgesic agent, wherein the analgesic agent comprises 5 mg to 2000 mg, 20 mg to 2000 mg, Orally administered in a daily dose ranging from 1 mg to 1000 mg, 20 mg to 1000 mg, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg, 250 mg to 1000 mg or 500 mg to 1000 mg. In some embodiments, the second analgesic agent is administered in an amount of from 1 mg to 2000 mg, from 5 mg to 2000 mg, from 20 mg to 2000 mg, from 5 mg to 1000 mg, from 20 mg to 1000 mg, from 50 mg to 500 mg, , 250 mg to 500 mg, 250 mg to 1000 mg, or 500 mg to 1000 mg per day.

In another embodiment, the pharmaceutical composition comprises a pair of analgesic agents. Examples of such a pair of analgesic agents include acetylsalicylic acid and ibuprofen, acetylsalicylic acid and naproxen sodium, acetylsalicylic acid and nabumetone, acetylsalicylic acid and acetaminophen, acetylsalicylic acid and indomethacin, ibuprofen and naproxen sodium, ibuprofen and nabumetone, ibuprofen And acetaminophen, ibuprofen and indomethacin, naproxen sodium and napromethone, naproxen sodium and acetaminophen, naproxen sodium and indomethacin, nabumetone and acetaminophen, nabumetone and indomethacin, and acetaminophen and indomethacin However, it is not combined with it. Wherein the pair of analgesics are mixed in a weight ratio ranging from 0.1: 1 to 10: 1, from 0.2: 1 to 5: 1 or from 0.3: 1 to 3: 1 and the combined dose ranges from 5 mg to 2000 mg, 20 From 200 mg to 2000 mg, from 500 mg to 2000 mg, from 5 mg to 1500 mg, from 20 mg to 1500 mg, from 100 mg to 1500 mg, from 200 mg to 1500 mg, from 500 mg to 2000 mg, 500 mg to 1000 mg, 500 mg to 1500 mg, 250 mg to 1000 mg, 250 mg to 1000 mg, 500 mg to 1000 mg, 5 mg to 1000 mg, 20 mg to 1000 mg, 100 mg to 1000 mg, , 1000 mg to 1500 mg, and 1000 mg to 2000 mg. In one embodiment, the pair of analgesics are mixed in a weight ratio of 1: 1.

In some other embodiments, the pharmaceutical composition of the present application further comprises at least one antimuscarinic agent. Examples of such antimuscarinic agents include, but are not limited to, oxybutynin, solifenacin, dipalpanacin, fezerodine, tolterodine, trospium and atropine. The daily dose of the antimuscarinic agent may be 0.01 mg to 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 10 mg to 100 mg, 0.01 mg to 25 mg, 0.1 mg to 25 mg, 1 mg to 25 mg , 10 mg to 25 mg, 0.01 mg to 10 mg, 0.1 mg to 10 mg, 1 mg to 10 mg, 10 mg to 100 mg and 10 mg to 25 mg.

Another aspect of the present invention relates to a method of reducing the frequency of urination by administering a pharmaceutical composition formulated with an immediate-release formulation to a subject in need thereof. In some embodiments, the pharmaceutical composition comprises at least one analgesic agent and at least one antimuscarinic agent. In another embodiment, the pharmaceutical composition comprises one or more analgesics and one or more anti-diuretics. In another embodiment, the pharmaceutical composition comprises at least one analgesic agent, at least one antimuscarinic agent, and at least one antidiuretic agent.

In another embodiment, the pharmaceutical composition of the present application further comprises one or more antispasmodics. Examples of antispasmodic agents include, but are not limited to, carisoprodol, benzodiazepine, baclofen, cyclobenzaprine, metocarbone, methocarbamol, clonidine, clonidine analogs, and dantrolene. In some embodiments, the antispasmodic agent is selected from the group consisting of 1 mg to 1000 mg, 1 mg to 100 mg, 10 mg to 1000 mg, 10 mg to 100 mg, 20 mg to 1000 mg, 20 mg to 800 mg, 20 mg to 500 mg, The present invention provides a method for the treatment and / or prophylaxis of diabetic nephropathy, comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound selected from the group consisting of 20 mg to 200 mg, 50 mg to 1000 mg, 50 mg to 800 mg, 50 mg to 200 mg, 100 mg to 800 mg, 100 mg to 500 mg, 200 mg to 800 mg, Lt; / RTI > The antispasmodic may be formulated into pharmaceutical compositions either alone or in combination with other active ingredient (s), for immediate release, delayed release or combinations thereof.

In certain embodiments, the pharmaceutical composition comprises two or more analgesics. In another embodiment, the pharmaceutical composition comprises at least one analgesic agent and at least one antimuscarinic agent and / or an anti-diuretic agent. The pharmaceutical composition may be formulated into tablets, capsules, sugar solutions, powders, granules, liquids, gels or emulsions. The liquid, gel or emulsion may be contained in a naked form or in a capsule to be ingested by the subject.

In certain embodiments, the analgesic agent is selected from the group consisting of salicylate, aspirin, salicylic acid, methyl salicylate, dipulose, salsarate, olsalazine, sulfasalazine, para-aminophenol derivatives, acetanilide, acetaminophen, But are not limited to, fenamate, mefenamic acid, meclofenamate, sodium methophenemate, heteroaryl acetic acid derivatives, tolmetin, ketorolac, diclofenac, propionic acid derivatives, ibuprofen, naproxen sodium, naproxen, fenoprofen, , Flurbiprofen, oxaprofen; Wherein the at least one compound is selected from the group consisting of at least one selected from the group consisting of at least one selected from the group consisting of at least one selected from the group consisting of benzoic acid, Wherein the active agent is selected from the group consisting of acyclovir, coxivir, celecoxib, rofecoxib, nabumetone, apazone, nimesulide, indomethacin, sulindac, etodolac, dipyridicar and isobutylphenylpropionic acid. The antimuscarinic agents are selected from the group consisting of oxybutynin, solifenacin, dipalpanacin, and atropine.

In some embodiments, the pharmaceutical composition comprises a single analgesic and a single antimuscarinic agent. In another embodiment, the single analgesic is aspirin. In another embodiment, the single analgesic is ibuprofen. In another embodiment, the single analgesic is naproxen sodium. In another embodiment, the single analgesic is indomethacin. In another embodiment, the single analgesic agent is nabumetone. In another embodiment, the single analgesic is acetaminophen. The analgesic agent and the antimuscarinic agent can be dosed within the above-mentioned range.

Another aspect of the present invention relates to a method of treating nocturnal enuresis by administering (1) one or more analgesics and (2) one or more anti-diuretics to a subject in need thereof. In certain embodiments, the antidiuretic agent (s) is / are: (1) increased vasopressin secretion; (2) increased vasopressin receptor activation; (3) decrease secretion of ANP (atrial natriuretic peptide) or CNP (C-type natriuretic peptide); Or (4) reduce ANP and / or CNP receptor activation.

Examples of antidiuretic agents include, but are not limited to, antidiuretic hormone (ADH), angiotensin II, aldosterone, vasopressin, vasopressin analogs (eg, desmopressin, arzipesin, refresin, pelifrenic, ornifresin, God); An antagonist (e.g., HS-142-1, isatin, and the like) of a vasopressin receptor agonist, atrial natriuretic peptide (ANP) and a C-type natriuretic peptide (CNP) receptor (i.e., NPR1, NPR2, NPR3) [Asu7,23 '] b-ANP- (7-28)], anantine, cyclic peptides from Streptomyces coerulescens, and 3G12 monoclonal antibodies); But are not limited to, somatostatin type 2 receptor antagonists (for example, somatostatin), and pharmaceutically-acceptable derivatives, analogs, salts, hydrates and solvates thereof.

In certain embodiments, the one or more analgesic agents and one or more antidiuretic agents are formulated in a delayed-release form.

Another aspect of the present application relates to a method of reducing the frequency of urination by administering to a subject in need thereof a first pharmaceutical composition comprising a diuretic and then administering a second pharmaceutical composition comprising at least one analgesic will be. The first pharmaceutical composition is dosed and formulated to have a diuretic effect within 6 doses of administration and is administered at least 8 hours before bedtime. The second pharmaceutical composition is administered within 2 hours before sleeping.

Examples of diuretics include acidifying salts such as CaCl ₂ and NH ₄ Cl; Arginine vasopressin receptor 2 antagonists such as amphotericin B and lithium citrate; Aquaretics such as Goldenrod and Junipe; Na-H exchange antagonists such as dopamine; Carbonic anhydrase inhibitors such as acetazolamide and dorzolamide; Loop diuretics such as bumetanide, ethacrynic acid, furosemide, and torsemide; Osmotic diuretics such as glucose and mannitol; Potassium-sparing diuretics such as amylolide, spironolactone, triamterene, potassium carenoate; Thiazides such as benzofluorometazide and hydrochlorothiazide; And xanthines such as caffeine, theophylline and theobromine, but are not bound thereto.

In some embodiments, the second pharmaceutical composition further comprises at least one antimuscarinic agent. Examples of such antimuscarinic agents include, but are not limited to, oxybutynin, solifenacin, dipalpanacin, fezerodine, tolterodine, trospium and atropine. The second pharmaceutical composition may be formulated into an immediate-release formulation or a delayed-release formulation. In one embodiment, the first pharmaceutical composition is formulated in an immediate-release form, and the second pharmaceutical composition is formulated in a delay-release form.

In some other embodiments, the second pharmaceutical composition further comprises one or more antidiuretic agents.

Another aspect of the present application is directed to a pharmaceutical composition comprising a plurality of active ingredients and a pharmaceutically acceptable carrier. In some embodiments, the plurality of active ingredients comprises two or more analgesic agents. In another embodiment, the plurality of active ingredients comprises at least one analgesic agent and at least one antimuscarinic agent. In another embodiment, the plurality of active ingredients comprises at least one analgesic agent and at least one antidiuretic agent. In another embodiment, the plurality of active ingredients comprises at least one analgesic agent, at least one antidiuretic agent, and at least one antimuscarinic agent. In another embodiment, at least one of the plurality of active ingredients is formulated in a delay-release form.

In some embodiments, the pharmaceutical composition comprises two analgesic agents selected from the group consisting of acetylsalicylic acid, ibuprofen, naproxen sodium, nabumetone, acetaminophen, and indomethacin. In another embodiment, the pharmaceutical composition comprises one or more analgesics selected from the group consisting of acetylsalicylic acid, ibuprofen, naproxen sodium, nabumetone, acetaminophen and indomethacin; And an antimuscarinic agent selected from the group consisting of oxybutynin, solifenacin, diperanacin and atropine.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sweetening agents and the like. The pharmaceutically acceptable carrier may be prepared from, but is not bound to, a wide range of materials including flavors, sweeteners, buffers and sorbents necessary to prepare certain therapeutic compositions. The use of such media and preparations in conjunction with pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use in a therapeutic composition is contemplated.

Another aspect of the present invention relates to a method of reducing the frequency of urination by administering to a subject in need thereof two or more analgesic agents that prevent progression of drug resistance. In one embodiment, the method comprises administering a first analgesic agent for a first period of time followed by a second analgesic agent for a second period of 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 are selected from the group consisting of salicylate, aspirin, salicylic acid, methyl salicylate, dipyrunicar, salsarate, olsalazin, sulfasalazine, para-aminophenol derivatives, , Acetaminophen, phenacetin, phenamate, mefenamic acid, meclofenamate, sodium mecophenamate, heteroaryl acetic acid derivatives, tolmetin, ketorolac, diclofenac, propionic acid derivatives, ibuprofen, naproxen sodium, Norfropen, ketoprofen, fluorevifrophen, oxaprofen; Wherein the at least one compound is selected from the group consisting of at least one selected from the group consisting of at least one selected from the group consisting of at least one selected from the group consisting of benzoic acid, And may be selected from the group consisting of acyclovir, acyclovir, coxivir, celecoxib, rofecoxib, nabumetone, apazone, nimesulide, indomethacin, sulindac, etodolac, and isobutylphenylpropionic acid.

In one embodiment, the first analgesic is acetaminophen, the second analgesic is ibuprofen, and the third analgesic is naproxen sodium. The length of each period may vary depending on the response of the subject to each analgesic agent. In some embodiments, each period lasts from 3 days to 3 weeks. In another embodiment, the first, second and third analgesics are formulated in a delay-release form.

Another aspect of the present application is a pharmaceutical composition comprising: (1) a first component comprising one or more analgesics, wherein the first component is formulated to delay-release at a delay time of 1-4 hours after oral administration; And (2) a second component comprising one or more analgesic agents, wherein the second component is formulated to delay-release at a delay time of 4-6 hours after oral administration.

In some embodiments, the one or more analgesic agents in the first component or the second component are selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen.

In some embodiments, the second component comprises an analgesic agent such as acetaminophen in an amount of 5-2000 mg.

In some embodiments, the pharmaceutical composition is coated with enteric coating.

In some embodiments, the first component further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the second component further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, both the first and second components further comprise an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the second component is coated with a blend of a water insoluble polymer (WIP): enteric polymer (EP) ratio of 4: 1 to 1: 2 and an enteric polymer.

In some embodiments, the second component is coated with multiple coating layers.

In some embodiments, the second component is covered with a swellable layer and an outer, insoluble but semi-transparent polymeric coating. In some related embodiments, the swell layer is selected from the group consisting of hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymer, polyethylene glycol, polyvinylpyrrolidone, Acid copolymers, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinylacetate phthalate, shellac, and ethylcellulose.

In some embodiments, the second component formulation comprises a water insoluble capsule body that is insoluble but permeable and swellable at one end closed with a hydrogel plug, the plug comprising a polymeric material selected from the group consisting of polymethacrylate, ≪ / RTI > and an enzymatically controlled erodible polymer.

Another aspect of the present application is a pharmaceutical composition comprising (1) a first component comprising an analgesic agent such as acetaminophen in an amount of 5-2000 mg, said first component being formulated in an immediate-release form; And (2) a second component comprising one or more analgesic agents, said second component being formulated to delay-release at a delay time of 1-4 hours after oral administration; And (2) a third component comprising at least one analgesic agent, wherein the third component is formulated to delay-release at a delay time of 4-6 hours after oral administration.

In some embodiments, the one or more analgesic agents in the second component or the third component or both are selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen .

In some embodiments, the second component comprises an analgesic agent such as acetaminophen in an amount of 5-2000 mg.

In some embodiments, the second component is coated with an enteric coating.

In some embodiments, the third component comprises an analgesic, such as acetaminophen, in an amount of 5-2000 mg.

In some embodiments, the first component further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the second component further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the third component further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the first and second components each comprise an agent selected from the group consisting of an antimuscarinic agent, an anti-diuretic agent, and an antispasmodic agent.

In some embodiments, the first and third components each comprise an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the second and third components each comprise a formulation selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the first, second, and third components each comprise an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the second component is coated with an enteric coating.

In some embodiments, the second or third component is coated with a water insoluble polymer (WIP): enteric polymer (EP) ratio of from 4: 1 to 1: 2 and an enteric polymer.

In some embodiments, the third component is coated with multiple coating layers.

Another aspect of the present application is a pharmaceutical composition comprising (1) a first component comprising acetaminophen in an amount of 5-2000 mg, said first component being formulated in an immediate-release form; And (2) a second component comprising one or more analgesic agents, said second component being formulated to delay-release at a delay time of 1-4 hours after oral administration; And (2) a third component comprising one or more analgesic agents, wherein said first component is formulated to delay-release at a delay time of 4-6 hours after oral administration, Wherein said pharmaceutical composition is to reduce the frequency of urination in a patient in need thereof.

In some embodiments, the one or more analgesics in the second component or the third component or both are selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen .

Manufacturing method

Another aspect of the present invention relates to a method of making a delayed-release pharmaceutical composition for reducing urinary frequency. In some embodiments, the method comprises forming a first mixture comprising a first active ingredient comprising at least one analgesic agent; Coating said first mixture with a first delay-release coating to form a first component, said first delay-release coating releasing said first mixture at a delay time of 1-4 hours after oral administration ; Forming a second mixture comprising a second active ingredient comprising at least one analgesic agent; Coating said second mixture with a second delay-release coating to form a second component, said second delay-release coating releasing said second mixture with a delay time of 4-6 hours after oral administration ; And mixing the first component with the second component to form a third mixture.

In some embodiments, the first, second and / or third mixture comprises a pharmaceutically acceptable carrier, such as an excipient.

In some embodiments, the one or more analgesic agents in the second component, third component, or both are selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen.

In some embodiments, at least one of the first and second active ingredients contains from 5 mg to 2000 mg of an analgesic such as acetaminophen. In some embodiments, the first active ingredient contains from 5 mg to 2000 mg of an analgesic such as acetaminophen. In another embodiment, the second active ingredient contains from 5 mg to 2000 mg of an analgesic such as acetaminophen. In another embodiment, the first and second active ingredients each comprise from 5 mg to 2000 mg of an analgesic such as acetaminophen.

In some embodiments, at least one of the first and second active ingredients further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent. In some embodiments, the first active ingredient further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent. In another embodiment, the second active ingredient further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent and an antispasmodic agent. In another embodiment, the first and second active ingredients each comprise a formulation selected from the group consisting of antimuscarinics, antidiuresis, and antispasmodics.

In some embodiments, the first delay-release coating is an enteric coating. In some embodiments, the second delay-release coating comprises a blend of a water insoluble polymer (WIP): enteric polymer (EP) ratio of from 4: 1 to 1: 2 and an enteric polymer. In some embodiments, the second delay-release coating comprises multiple coating layers.

In some embodiments, the second delay-release coating comprises a swellable layer and an exterior insoluble but semi-transparent polymeric coating. In a related embodiment, the swell layer is selected from the group consisting of hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymer, polyethylene glycol, polyvinylpyrrolidone, methacrylic acid Cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinylacetate phthalate, shellac, and ethylcellulose.

In some embodiments, the second component formulation comprises a water insoluble capsule body that is insoluble but permeable and swellable at one end closed with a hydrogel plug, the plug comprising a polymeric material selected from the group consisting of polymethacrylate, ≪ / RTI > and an enzymatically controlled erodible polymer.

In some embodiments, the method further comprises compressing or molding the third mixture into a tablet form.

In some embodiments, the third mixture is formed by mixing the first component and the second component with a third component, and the third component comprises a third active ingredient comprising at least one analgesic agent do. In some embodiments, the one or more analgesics are selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen.

In some embodiments, the first, second, and third active ingredients each comprise one or more analgesic agents. In some embodiments, the one or more analgesics are selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen. In some embodiments, the first, second, and third active ingredients each comprise 5-2000 mg of acetaminophen. In some embodiments, one of the first, second, and third active ingredients comprises 5-2000 mg of acetaminophen. In some embodiments, two of the first, second, and third active ingredients comprise 5-2000 mg of acetaminophen.

In another embodiment, the method comprises forming a first mixture comprising a first active ingredient comprising at least one analgesic agent; Coating said first mixture with a first delay-release coating to form a core structure, said first delay-release coating releasing said first mixture with a delay time of 1-4 hours after direct contact with bodily fluids that; Coating the core structure with a second mixture to form a coated core structure, wherein the second mixture comprises a second active ingredient comprising at least one analgesic; And coating the coated core structure with a second delay-release coating to form a dual-coated core structure, wherein the second delay-release coating is applied to the second mixture at a delay time of 1-4 hours after oral administration, Lt; / RTI > In some embodiments, the first mixture is not in direct contact with bodily fluids until the second mixture is released.

In some embodiments, the at least one analgesic agent in the first active ingredient or the second active ingredient is selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen.

In some embodiments, the second delay-release coating is an enteric coating.

In some embodiments, both the first active ingredient or the second active ingredient, or both, further comprise an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent.

In some embodiments, the first active ingredient or the second active ingredient, or both, comprise acetaminophen.

In some embodiments, the method further comprises coating the double-coated core structure with a third coating, wherein the third coating comprises a third active component comprising at least one analgesic agent. In some embodiments, the one or more analgesics in the third active ingredient is selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen. In some embodiments, the third active ingredient comprises from 5 mg to 2000 mg of an analgesic. In some embodiments, the third active ingredient comprises 5 mg to 2000 mg of acetaminophen.

In some related embodiments, the third active ingredient further comprises an agent selected from the group consisting of an antimuscarinic agent, an antidiuretic agent, and an antispasmodic agent. In another related embodiment, the first, second and third active ingredients each comprise acetaminophen.

In some embodiments, the first delay-release coating comprises a pH-independent polymer containing one or more polysaccharides, wherein the pH-independent polymer is resistant to erosion in both the stomach and intestines, have.

In another embodiment, the first delay-release coating, or the second delay-release coating, or both, comprises a water permeable and water insoluble film coating to provide the core structure or the active Components and osmotic agent. As the water slowly diffuses from the interior through the water-insoluble film into the core structure, the core expands until the film ruptures, thereby releasing the active component. The film coating can be adjusted to allow for various rates of water permeation or release. As mentioned earlier, the release time of the drug is controlled by the presence of water-insoluble polymeric membranes (ethylcellulose (L-HPC), hydroxypropylcellulose , ≪ / RTI > EC), and the degradation delay time according to the balance of the thickness. After oral administration, the gastric fluid penetrates through the pores, causing swelling of the swelling excipient, which creates an internal pressure that separates the components in the capsule.

In another embodiment, the drug is released by an osmotic mechanism. In some embodiments, the first delay-release coating, or the second delay-release coating, or both, comprise a semi-transmissive film. In some embodiments, the semi-permeable membrane is additionally covered with a pH-dependent enteric coating to prevent release until after gastric emptying.

The present invention is further illustrated by the following examples, which should not be construed as combining. All references, patents and published patent applications cited throughout this application are incorporated herein by reference.

Example 1: Suppression of urination urge

A total of 20 men and women who experienced premature urge urination or urge to urinate had their sleep interrupted for a sufficient period of time to get enough rest. Each subject ingested 400-800 mg of ibuprofen in a single dose before bedtime. At least 14 subjects reported that they were able to take a break because they had not been awakened often due to urge incontinence.

Some subjects reported that after a few weeks of ingesting ibuprofen at night, the effect of feeling less urge urge was no longer felt. However, all of these subjects further reported that the effects reappear a few days after stopping their use.

Example 2: Effect of botulinum neurotoxin and antimuscarinic agent as analgesic agents in macrophage response to inflammatory and non-inflammatory stimuli

Experimental Design

This experiment confirmed the dose and in vitro efficacy of analgesics and antimuscarinics that control macrophage responses to inflammatory and non-inflammatory stimuli mediated by Cox-2 and prostaglandins (PGE, PGH, etc.) Respectively. This established a baseline response (dose and exercise) to inflammatory and non-inflammatory effectors in bladder cells. Briefly, the cultured cells are exposed to analgesics and / or antimuscarinics in the absence or presence of various reactors.

The reactor includes: LPS (lipopolysaccharide), an inflammatory agent and a Cox2 inducer as an inflammatory stimulant; Carbachol or acetylcholine, a smooth muscle contraction stimulator, as a non-inflammatory stimulant; As a positive control, botulinum neurotoxin A known as acetylcholine release inhibitor; , Arachidonic acid (AA), DGLA (gamma (DGLA)), which are prostaglandin precursors produced by continuous oxidation of AA, DGLA or EPA in cells by cyclooxygenases (COX1 and COX2) and terminal prostaglandin synthases linolenic acid or EPA (eicosapentaenoic acid).

Such painkillers include: salicylates such as aspirin; Isobutyl propane phenolic acid derivatives such as adbill, motrin, nuprin and medifrene (ibuprofen); Nemo Progressive, EC-I, Neprean, Nafrogen, Nafrogen, Nafrogen, Nafrogen, Nafrogen, Naproxen sodium, such as prozin, narocin, proxen, synplex and genovid; Acetic acid derivatives such as indomethacin (india); 1-naphthalene acetic acid derivatives such as nabumetone or reraphen; N-acetyl-para-aminophenol (APAP) derivatives such as acetaminophen or paracetamol (Tylenol); And celecoxib.

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

Macrophages were stimulated for short (1-2 hours) or long (24-48 hours)

(1) Various doses of each analgesic agent alone.

(2) each analgesic at various doses in the presence of LPS.

(3) Various analgesic agents in various doses in the presence of carbachol or acetylcholine.

(4) Various analgesic agents at various doses in the presence of AA, DGLA, or EPA.

(5) Various doses of botulinum neurotoxin A alone.

(6) Various doses of botulinum neurotoxin in the presence of LPS.

(7) Various doses of botulinum neurotoxin in the presence of carbachol or acetylcholine.

(8) Various doses of botulinum neurotoxin A. in the presence of AA, DGLA, or EPA.

(9) Various doses of each antimuscarinic agent alone.

(10) Various doses of each antimuscarinic agent in the presence of LPS.

(11) Various doses of each antimuscarinic agent in the presence of carbachol or acetylcholine.

(12) Various doses of each antimuscarinic agent in the presence of AA, DGLA, or EPA.

These cells are then used for the release of PGH ₂ , PGE, PGE ₂ , Prostacydin, Thromboxane, IL-1β, IL-6 and TNF-α, production of COX2 activity, cAMP and cGMP, IL -1β, IL-6, TNF-α and COX2 mRNA and surface expression of CD80, CD86 and MHC class II molecules.

Macrophage

Murine RAW264.7 or J774 macrophage cells (obtained from ATCC) were used in this experiment. Cells were cultured in media containing RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 15 mM HEPES, 2 mM L-glutamine, 100 U / ml penicillin and 100 ug / ml streptomycin. Cells were cultured at 37 ° C in 5% CO ₂ and subcultured once a week.

In vitro  Analgesic treatment of macrophages

RAW 264.7 macrophages were plated in 96-well plates at a density of ^1.5 x ¹⁰ cells per well in 100 μl of the culture. Cells were exposed to various concentrations of LPS, which are (1) various concentrations of analgesics (acetaminophen, aspirin, ibuprofen or naproxen), (2) various concentrations of LPS as reactors for inflammatory stimuli to macrophages, and (3) Carbachol or acetylcholine, (4) analgesics and LPS or (5) analgesics and carbachol or acetylcholine. Briefly, analgesics were dissolved in FBS-free medium (i.e., RPMI 1640 supplemented with 15 mM HEPES, 2 mM L-glutamine, 100 U / ml penicillin and 100 ug / ml streptomycin) . For experiments on cells treated with analgesics in the absence of LPS, 50 진 of analgesic solution and 50 F of FBS-free medium were added to each well. For experiments on cells treated with analgesics in the presence of LPS, 50 [mu] l of analgesic solution and 50 [mu] l of LPS (isolated from Salmonella typhimurium ) in FBS-free media were added to each well. All conditions were duplicated.

After incubation for 24 or 48 hours, 150 占 퐇 of the culture supernatant was obtained, and the cells and debris were removed by centrifugation at 4 占 폚 for 8 minutes at 8,000 rpm for 2 minutes, and analyzed for cytokine response by ELISA using -70 ≪ / RTI > To the obtained cells, 500 μl of PBS (phosphate buffer) was added, followed by centrifugation (at 4 ° C for 5 minutes at 1,500 rpm). Half of the cells were frozen in liquid nitrogen and stored at -70 ° C. The remaining cells were stained with a fluorescent monoclonal antibody and analyzed by flow cytometry.

Flow cytometry analysis of co-stimulatory molecule expression

For flow cytometry analysis, macrophages were diluted in 100 μl of FACS buffer (PBS with 2% BSA and 0.01% NaN ₃ ) and incubated with FITC-conjugated anti-CD40 antibody, PE-conjugated anti-CD80 antibody, PE- Conjugated anti-CD86 antibody, anti-MHC class II (IA ^d ) PE (BD Bioscience) was added and stained for 30 minutes at 4 ° C. Then, 300 F of FACS buffer was added to the cells, and the cells were washed by centrifugation (at 1,500 rpm for 5 minutes at 4 캜). After a second wash, the cells were resuspended in 200 [mu] l of FACS buffer and the percentage of cells expressing the desired marker (single positive) or the percentage of cells expressing the combined marker (double positive) were measured using an Accuri C6 flow cytometer Bioscience).

Analysis of cytokine response using ELISA

The IL-1β, IL-6 and TNF-α responses were confirmed by cytokine-specific ELISA in culture supernatants obtained by culturing macrophages treated with analgesics, LPS alone or in combination with LPS and analgesics. Nunc MaxiSorp Immunoplates (Nunc) coated overnight with 100 μl of IL-6, TNF-α mAbs (BD Biosciences) or IL-1β mAb (R & D Systems) in 0.1 M sodium bicarbonate buffer Analysis. After washing twice with PBS (200 μl per well), 200 μl of PBS 3% BSA was added (blocked) to each well and the plate was incubated at room temperature for 2 hours. 200 [mu] l per well was added, the plate was washed twice again, 100 [mu] l of cytokine standard and serial dilutions of the culture supernatant were added, and the plate was incubated overnight at 4 [deg.] C. Finally, the plate was washed twice and incubated with 100 μl of secondary biotin-conjugated anti-mouse IL-6, TNFα mAbs (Biosciences) or IL-1β (R & D Systems), followed by peroxidase- And incubated with anti-biotin mAb (Vector Laboratories). The color reaction was confirmed by the addition of 2,2'-azino-bis (3) -ethylbenzylthiazoline-6-sulfonic acid (ABTS) substrate and H ₂ O ₂ (Sigma), and a Victor ^® V multi-label plate reader (PerkinElmer ) Was measured at 415 nm.

Determination of COX2 activity and production of cAMP and cGMP

The COX2 activity of cultured macrophages was determined by the COX2 activation assay. Production of cAMP and cGMP was determined by cAMP assay and cGMP assay. Such assays are generally performed in the art.

result

Table 1 summarizes the experimental and major experimental results of the Raw 264 macrophage cell line in terms of the effect of analgesics on the cell surface expression of the co-stimulatory molecules CD40 and CD80. Expression of these molecules was stimulated by COX2 and inflammatory signals, thus evaluating the expression of the molecules to determine the functional outcome of inhibition of COX2.

Experimental summary Control group LPS
Salmonella typhimurium Acetaminophen aspirin Ibuprofen Naproxen TESTS One X 2 X Dose response
(0, 5, 50, 1000) ng / ml 3 X Dose response
(0, 5, 50, 500, 5 x 10 ³ , 5 x 10 ⁴ , ⁵ x 10 ⁵ , ⁵ x 10 ⁶ ) nM 4 X X (5 ng / ml)
X (50 ng / ml
X (1000 ng / ml) Dose response
(0, 5, 50, 500, 5.10 ³ , 5.10 ⁴ , 5.10 ⁵ , 5.10 ⁶ ) nM ANALYSIS a Activation / Stimulation Characteristics: Flow cytometry analysis of CD40, CD80, CD86 and MHC class II b Mediator of inflammatory response: IL-1β, IL-6, TNF-α ELISA analysis

As shown in Table 2, acetaminophen, aspirin, ibuprofen and naproxen, the highest dose (i.e., 5x 10 ⁶ nM) except all tested doses (that ^{is, 5x 10 5 nM, 5x 10} 4 nM, 5x 10 ³ nM, 5x10 ² nM, 50 nM, and 5 nM) inhibited macrophage-mediated basal expression of the co-stimulatory molecules CD40 and CD80, and at the highest dose the expression of co-stimulatory molecules appeared to be enhanced rather than inhibited . As shown in Figures 1A and 1B, this inhibitory effect on CD40 and CD50 expression was also observed at analgesic doses as low as 0.05 nM (i.e., 0.00005 [mu] M). This supports that controlled release of low doses of analgesic may be preferable to acute delivery of large doses. In addition, these experiments show that acetaminophen, aspirin, ibuprofen, and naproxen exhibit similar inhibitory effects on LPS-induced expression of CD40 and CD80.

Summary of Key Experimental Results Reactor % positivity Negative control group LPS
5 ng / ml Analgesic dose (nM) 5x10 ⁶ 5x10 ⁵ 5x10 ⁴ 5x10 ³ 500 50 5 CD40 ⁺ CD80 ⁺ 20.6 77.8 Acetaminophen CD40 ⁺ CD80 ⁺ 63 18 12 9.8 8.3 9.5 7.5 aspirin CD40 ⁺ CD80 ⁺ 44 11 10.3 8.3 8 10.5 7.5 Ibuprofen CD40 ⁺ CD80 ⁺ ND * 6.4 7.7 7.9 6.0 4.9 5.8 Naproxen CD40 ⁺ CD80 ⁺ 37 9.6 7.7 6.9 7.2 6.8 5.2 Analgesics and LPS Acetaminophen CD40 ⁺ CD80 ⁺ 95.1 82.7 72.4 68.8 66.8 66.2 62.1 aspirin CD40 ⁺ CD80 ⁺ 84.5 80 78.7 74.7 75.8 70.1 65.7 Ibuprofen CD40 ⁺ CD80 ⁺ ND 67 77.9 72.9 71.1 63.7 60.3 Naproxen CD40 ⁺ CD80 ⁺ 66.0 74.1 77.1 71.0 68.8 72 73

* ND: not done (toxicity)

Table 3 summarizes the results of several studies that measured serum concentrations of analgesics after ingesting an oral dose of treatment. As shown in Table 3, the maximum serum concentration of analgesic after oral treatment administration ranged from 10 ⁴ to 10 ⁵ nM. Thus, the doses of the analgesic agents tested in vitro of Table 2 include a range of concentrations achievable in vivo in humans.

Serum concentration of human blood analgesic after oral administration painkiller Molecular Weight The maximum serum concentration after oral administration Reference Mg / L nM Acetaminophen
(Tylenol) 151.16 11-18 7.2 x ^{10 4} -1.19 x 10 ⁵ * BMC Clinical Pharmacology.2010, 10:10
* Anaesth Intensive Care. 2011, 39: 242 aspirin
(Acetylsalicylic acid) 181.66 30-100 1.65x10 ⁵ -5.5x10 ⁵ * Disposition of Toxic Drugs and Chemicals in Man , 8th Edition, Biomedical Public, Foster City, CA, 2008, pp. 22-25

* J Lab Clin Med. 1984 Jun; 103: 869 Ibuprofen
(Adville, Motrin) 206.29 24-32 1.16 x 10 ⁵ -1.55 x 10 ⁵ * BMC Clinical Pharmacology 2010, 10:10
* J Clin Pharmacol. 2001, 41: 330 Naproxen
(Allebe) 230.26 Up to 60 maximum
2.6x10 ⁵ * J Clin Pharmacol. 2001, 41: 330

Example 3: Effect of painkillers, botulinum neurotoxins and antimuscarinics on bladder smooth muscle cell response to inflammatory and non-inflammatory stimuli

Experimental Design

This experiment characterizes the effect of the optimal dose of analgesic agent determined in Example 2 on cell culture or tissue cultured bladder smooth muscle cells and shows that different types of analgesics can produce synergies that more effectively inhibit COX2 and PGE2 responses To verify whether or not it is.

The reactor, analgesic agent and antimuscarinic agent were described in Example 2.

Primary cultures of mouse bladder smooth muscle cells were stimulated for short (1-2 hours) or long (24-48 hours)

(1) Various doses of each analgesic agent alone.

(2) each analgesic at various doses in the presence of LPS.

(3) Various analgesic agents in various doses in the presence of carbachol or acetylcholine.

(4) Various analgesic agents at various doses in the presence of AA, DGLA, or EPA.

(5) Various doses of botulinum neurotoxin A alone.

(6) Various doses of botulinum neurotoxin in the presence of LPS.

(7) Various doses of botulinum neurotoxin in the presence of carbachol or acetylcholine.

(8) Various doses of botulinum neurotoxin A. in the presence of AA, DGLA, or EPA.

(9) Various doses of each antimuscarinic agent alone.

(10) Various doses of each antimuscarinic agent in the presence of LPS.

(11) Various doses of each antimuscarinic agent in the presence of carbachol or acetylcholine.

(12) Various doses of each antimuscarinic agent in the presence of AA, DGLA, or EPA.

These cells are then used for the release of PGH ₂ , PGE, PGE ₂ , Prostacydin, Thromboxane, IL-1β, IL-6 and TNF-α, production of COX2 activity, cAMP and cGMP, IL -1β, IL-6, TNF-α and COX2 mRNA and surface expression of CD80, CD86 and MHC class II molecules.

Materials and methods

Isolation and Purification of Murine Bladder Cells

Bladder was isolated from euthanized C57BL / 6 mice (8-12 weeks of age), enzymatically digested, and then purified by Percoll gradient to separate cells. Briefly, 10 bladder mice were scraped into 10 ml of digestion buffer (RPMI 1640, 2% fetal bovine serum, 0.5 mg / ml collagenase, 30 / / The bladder slurry was enzymatically digested at 37 ° C for 30 minutes. The undegraded pieces were further dispersed through a cell-trainer. The cell suspension was pooled and discontinuous 20%, 40% and 75% Percoll gradient for mononuclear cell purification was added. Each experiment used 50-60 blad- ers.

After washing with RPMI 1640, bladder cells were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES, 2 mM L-glutamine, 100 U / ml penicillin and streptomycin 100 / / Were dispensed into clear-bottom black 96-well cell culture micro-culture plates at a density of 3 x ¹⁰ cells per well. The cells were cultured at 37 ° C in 5% CO ₂ .

In vitro  Cellulite treatment

Bladder cells were treated with analgesic solution (50 [mu] l / well) alone or with LPS (1 [mu] g / ml) of Salmonella typhimurium as a noninflammatory stimulus, , 50 [mu] l / well) were treated together. If no other reactors for the cells were added, 50 μl of RPMI 1640 medium without fetal bovine serum was added to the wells to final volume to 200 μl.

24 hours of incubation, the obtained culture supernatant of 150 ㎕, centrifuged for 2 minutes, and 8,000 rpm at 4 ℃ to remove the cells and debris and, ℃ -70 for analysis of PEG ₂ (Prostaglandin E2) reaction by ELISA Lt; / RTI > To detect COX2 (Cyclooxygenase-2), cells were fixed and permeabilized and blocked using a fluorescent substrate. In selected experiments, cells were stimulated in vitro for 12 hours to analyze the COX2 response.

Analysis of COX2 response

The COX2 response was analyzed by cell-based ELISA using human / mouse whole COX2 immunoassay (R & D Systems) according to the manufacturer's instructions. Briefly, after cell fixation and permeabilization, mouse anti-total COX2 and rabbit anti-total GAPDH were added to the wells of clear-bottom black 96-well cell culture microculture plates. After incubation and washing, HRP-conjugated anti-mouse IgG and AP-conjugated anti-rabbit IgG were added to the wells. After further incubation and washing, HRP- and AP- fluorescent substrates were added. Finally, the fluorescence was read using a Victor V multi-label plate reader ^® (PerkinElmer) emitted from the 600 nm (COX2 fluorescence) and 450 nm (fluorescent GAPDH). Results were expressed as relative levels of total COX2 determined by RFU (relative fluorescence unit) and normalized to GAPDH, a housekeeping protein.

Analysis of PGE2 Reaction

Prostaglandin E2 responses were analyzed using sequential competitive ELISA (R & D Systems). More specifically, the culture supernatant or PGE2 standard was added to a 96-well polystyrene microplate coated with a goat anti-mouse polyclonal antibody. After incubation in a microplate stirrer for 1 hour, HRP-conjugated PGE2 was added and the plate was incubated for an additional 2 hours at room temperature. The plate was then washed and HRP substrate solution was added to each well. The reaction was stopped for 30 minutes and the reaction was stopped by addition of sulfuric acid, followed by wavelength correction at 570 nm and reading at 450 nm. The results are expressed as the average pg / ml of PGE2.

Other analysis

As described in Example 2, the release of PGH ₂ , PGE, PGE ₂ , Prostacydin, Thromboxane, IL-1β, IL-6 and TNF-α, production of cAMP and cGMP , IL-1β, IL-6, TNF-α and COX2 mRNA and surface expression of CD80, CD86 and MHC class II molecules.

result

Analgesics inhibit the COX2 response of murine bladder cells to inflammatory stimuli

Various analgesics (acetaminophen, aspirin, ibuprofen and naproxen) at concentrations of 5 μM or 50 μM were tested in mouse bladder cells to determine whether analgesics could induce a COX2 response. After 24-hour incubation, the results showed that any analgesics treated did not induce a COX2 response in in vitro mouse bladder cells.

We also examined the effect of these analgesics on the COX2 response of mouse bladder cells to carbachol or LPS stimulation in vitro . As shown in Table 1, the dose of carbachol tested does not have a significant effect on COX2 levels in mouse bladder cells. On the other hand, LPS significantly increased total COX2 levels. Interestingly, acetaminophen, aspirin, ibuprofen, and naproxen were all able to inhibit the effect of LPS on COX2 levels. The inhibitory effect of the analgesic was shown when these drugs were 5 [mu] M or 50 [mu] M (Table 4).

COX2 expression by stimulated and analgesic-treated murine bladder cells in vitro Stimulation painkiller Total COX2 levels (Normalized RFUs) None None 158 ± 18 Carbachol (mM) None 149 ± 21 LPS (1 [mu] g / ml) None 420 ± 26 LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml) Acetaminophen (5 μM)
Aspirin (5 μM)
Ibuprofen (5 μM)
Naproxen (5 μM) 275 ± 12
240 ± 17
253 ± 32
284 ± 11 LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml) Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM) 243 ± 15
258 ± 21
266 ± 19
279 ± 23

Analgesics inhibit the PGE2 response of murine bladder cells to inflammatory stimuli

The secretion of PGE2 in the culture supernatants of murine bladder cells was measured to confirm the biological significance of changes in murine bladder cell COX2 levels by analgesics. As shown in Table 5, PGE2 was not detected in the culture supernatant of blast cell cultured in the presence of unstimulated bladder cells or carbachol. Consistent with the COX2 response described above, stimulation of mouse bladder cells with LPS induced a high level of secretion of PGE2. Addition of the analgesic agents acetaminophen, aspirin, ibuprofen, and naproxen suppressed the effects of LPS on PGE2 secretion and showed no difference between the cell responses treated with 5 or 50 μM doses of analgesics.

PGE2 secretion by stimulated and analgesic-treated murine bladder cells in vitro Stimulation painkiller PGE2 levels (pg / ml) None None <20.5 Carbachol (mM) None <20.5 LPS (1 [mu] g / ml) None 925 ± 55 LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml) Acetaminophen (5 μM)
Aspirin (5 μM)
Ibuprofen (5 μM)
Naproxen (5 μM) 619 ± 32
588 ± 21
593 ± 46
597 ± 19 LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml)
LPS (1 [mu] g / ml) Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM) 600 ± 45
571 ± 53
568 ± 32
588 ± 37

In summary, these data show that 5 μM or 50 μM of analgesic alone treatment in murine bladder cells does not induce COX2 and PGE2 responses. However, 5 μM or 50 μM of analgesic in the in vitro stimulated murine bladder cells with LPS (1 μg / ml) significantly inhibited the COX2 and PGE2 responses. No significant effects of analgesics on COX2 and PGE2 responses were observed in murine bladder cells stimulated with carbachol (1 mM).

Example 4: Effect of painkillers, botulinum neurotoxins and antimuscarinics on contraction of bladder smooth muscle cells

Experimental Design

Cultured mouse or rat bladder smooth muscle cells and mouse or rat bladder smooth muscle tissue were exposed to inflammatory stimuli and non-inflammatory stimuli in the presence of various concentrations of analgesic and / or antimuscarinic agents. Stimulation-induced muscle contraction was measured to assess the inhibitory effect of analgesics and / or antimuscarinics.

The reactor, analgesic agent and antimuscarinic agent were described in Example 2.

The primary cultures of mouse bladder smooth muscle cells were stimulated for a short period (1-2 hours) or for a prolonged period (24-48 hours) as follows:

(1) Various doses of each analgesic agent alone.

(2) each analgesic at various doses in the presence of LPS.

(3) Various analgesic agents in various doses in the presence of carbachol or acetylcholine.

(4) Various analgesic agents at various doses in the presence of AA, DGLA, or EPA.

(5) Various doses of botulinum neurotoxin A alone.

(6) Various doses of botulinum neurotoxin in the presence of LPS.

(7) Various doses of botulinum neurotoxin in the presence of carbachol or acetylcholine.

(8) Various doses of botulinum neurotoxin A. in the presence of AA, DGLA, or EPA.

(9) Various doses of each antimuscarinic agent alone.

(10) Various doses of each antimuscarinic agent in the presence of LPS.

(11) Various doses of each antimuscarinic agent in the presence of carbachol or acetylcholine.

(12) Various doses of each antimuscarinic agent in the presence of AA, DGLA, or EPA.

Materials and methods

As described in Example 3, early mouse bladder cells were isolated. In selected experiments, cultured bladder tissue was used. Bladder smooth muscle cell contractions were recorded with a Grass polygraph (Quincy Mass, USA).

Example 5: Effect of oral analgesics and antimuscarinics on COX2 and PGE2 responses of bladder smooth muscle cells

Experimental Design:

It has been reported that mice that are in normal mouse and over active bladder syndrome (OAB) are exposed to aspirin, naproxen sodium, ibuprofen, india, nabumetone, tylenol, celecoxib, oxybutynin, A combination of these was taken orally. The control group was a normal mouse without any treatment and an OAB mouse without any treatment. After 30 minutes, the final dose, to obtain a bladder in vitro with carbachol or acetylcholine (ex vivo) stimulated. In selected experiments, the bladder was treated with botulinum neurotoxin A prior to carbaco stimulation. The experimental animals were placed in a metabolic cage and the frequency (and amount) of urination was measured. The output of the bladder was determined by monitoring the water intake and the weight of the dirt in the cage. Levels of serum PGH ₂ , PGE, PGE ₂ , prostasidine, thromboxane, IL-1β, IL-6, TNF-α, cAMP, and cGMP were confirmed by ELISA. Expression of CD80, CD86, and MHC class II in whole blood cells was confirmed by flow cytometry.

At the end of the experiment, animals were euthanized and in vitro bladder contractions were recorded with a Grass polygraph. A portion of the bladder was fixed in formalin and analyzed for COX2 response by immunohistochemistry.

Example 6: Effect of analgesic, botulinum neurotoxin and antimuscarinic agents on human bladder smooth muscle cell response to inflammatory and non-inflammatory stimuli

Experimental Design

This experiment characterizes the effect of the optimal dose of analgesic agent determined in Examples 1 to 5 on cell culture or tissue culture of human bladder smooth muscle cells and shows synergistic effects that different types of analgesics inhibit the COX2 and PGE2 responses more efficiently It is designed to confirm whether or not it can be put out.

The reactor, analgesic agent and antimuscarinic agent were described in Example 2.

Human bladder smooth muscle cells were stimulated for a short period (1-2 hours) or for a prolonged period (24-48 hours) as follows:

(1) Various doses of each analgesic agent alone.

(2) each analgesic at various doses in the presence of LPS.

(3) Various analgesic agents in various doses in the presence of carbachol or acetylcholine.

(4) Various analgesic agents at various doses in the presence of AA, DGLA, or EPA.

(5) Various doses of botulinum neurotoxin A alone.

(6) Various doses of botulinum neurotoxin in the presence of LPS.

(7) Various doses of botulinum neurotoxin in the presence of carbachol or acetylcholine.

(8) Various doses of botulinum neurotoxin A. in the presence of AA, DGLA, or EPA.

(9) Various doses of each antimuscarinic agent alone.

(10) Various doses of each antimuscarinic agent in the presence of LPS.

(11) Various doses of each antimuscarinic agent in the presence of carbachol or acetylcholine.

(12) Various doses of each antimuscarinic agent in the presence of AA, DGLA, or EPA.

Thereafter, the cells were stimulated with the release of PGH ₂ , PGE, PGE ₂ , prostasidine, thromboxane, IL-1 ?, IL-6 and TNF- ?, COX2 activity, production of cAMP and cGMP, , NF- alpha and COX2 mRNA production, and surface expression of CD80, CD86 and MHC class II molecules.

Example 7: Effect of painkillers, botulinum neurotoxins and antimuscarinics on the contraction of human bladder smooth muscle cells

Experimental Design

Cultured human bladder smooth muscle cells were exposed to inflammatory stimuli and non-inflammatory stimuli in the presence of various concentrations of analgesic and / or antimuscarinic agents. Stimulation-induced muscle contraction was measured to assess the inhibitory effect of analgesics and / or antimuscarinics.

The reactor, analgesic agent and antimuscarinic agent were described in Example 2.

Human bladder smooth muscle cells were stimulated for a short period (1-2 hours) or for a prolonged period (24-48 hours) as follows:

(1) Various doses of each analgesic agent alone.

(2) each analgesic at various doses in the presence of LPS.

(3) Various analgesic agents in various doses in the presence of carbachol or acetylcholine.

(4) Various analgesic agents at various doses in the presence of AA, DGLA, or EPA.

(5) Various doses of botulinum neurotoxin A alone.

(6) Various doses of botulinum neurotoxin in the presence of LPS.

(7) Various doses of botulinum neurotoxin in the presence of carbachol or acetylcholine.

(8) Various doses of botulinum neurotoxin A. in the presence of AA, DGLA, or EPA.

(9) Various doses of each antimuscarinic agent alone.

(10) Various doses of each antimuscarinic agent in the presence of LPS.

(11) Various doses of each antimuscarinic agent in the presence of carbachol or acetylcholine.

(12) Various doses of each antimuscarinic agent in the presence of AA, DGLA, or EPA.

Bladder smooth muscle cell contractions were recorded with a Grass polygraph (Quincy Mass, USA).

Example 8 Effect of Analgesics on Normal Human Bladder Smooth Muscle Cell Response to Inflammatory and Non-Saline Neutral Signals

Experimental Design

Culture of Normal Human Bladder Smooth Muscle Cells

Normal human bladder smooth muscle cells were isolated using enzymatic degradation from normal tissue of human bladder. 10% fetal bovine serum, 15 mM HEPES, 2 mM of L- glutamine, 100 U / ㎖ penicillin and streptomycin in vitro cells in 5% CO _2, 37 ℃ in a medium containing 100 ㎍ / ㎖ the addition of RPMI 1640 after incubation in Nancy, it processes the once a week off trypsin the cells were subcultured to dispensing a new flask. In the first week of culture, 0.5 ng / ml EGF (epidermal growth factor), 2 ng / ml FGF (fibroblast growth factor) and 5 / / ml insulin were added to the culture medium.

In vitro  Analgesic treatment of normal human bladder smooth muscle cells

Human bladder smooth muscle cells were treated with trypsin and then dispensed into a micro-culture plate at a density of 3 x ¹⁰ cells / 100 μl per well. Analgesics solution to the cells alone handle (50 ㎕ / well), or non - LPS (1 ㎍ / ㎖ of Salmonella typhimurium as an inflammatory stimulus, - an inflammatory stimulus carbachol (10-Molar, 50 ㎕ / well), or non- 50 [mu] l / well) were treated together. If no other reactors for the cells were added, 50 μl of RPMI 1640 medium without fetal bovine serum was added to the wells to final volume to 200 μl.

After 24 hours of culture, 150 占 퐇 of the culture supernatant was obtained, and the cells and debris were removed by centrifugation at 4 占 폚 and 8,000 rpm for 2 minutes, and analyzed at -70 占 폚 for analysis of PEG2 (Prostaglandin E2) Respectively. To detect COX2 (Cyclooxygenase-2), cells were fixed and permeabilized and blocked using a fluorescent substrate. In selected experiments, cells were stimulated in vitro for 12 hours to analyze COX2, PGE2 and cytokine response.

Analysis of COX2, PGE2 and cytokine responses

As described in Example 3, the COX2 and PGE2 reactions were analyzed. The cytokine response was analyzed as described in Example 2.

result

Analgesics inhibit the COX2 response of normal human bladder smooth muscle cells to inflammatory and noninflammatory stimuli - Analysis of cell and culture supernatants after 24 hours of incubation shows that any analgesic administered alone has a COX2 response in normal human bladder smooth muscle cells . However, as summarized in Table 6, carbachol induced a weak but significant COX2 response in normal human bladder smooth muscle cells. On the other hand, LPS treatment resulted in a high COX2 response in normal human bladder smooth muscle cells. Acetaminophen, aspirin, ibuprofen, and naproxen were all able to inhibit the effects of carbachol and LPS on COX2 levels. The inhibitory effect of the analgesic showed LPS-induced response when these drugs were 5 [mu] M or 50 [mu] M.

COX2 expression by normal human bladder smooth muscle cells stimulated by inflammatory and noninflammatory stimuli in vitro and treated with analgesics
Stimulation
painkiller Total COX2 level ^#
(Normalized RFUs)
Target 1 Total COX2 level
(Normalized RFUs)
Target 2 None None 230 199 Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM)
Acetaminophen (50 μM) 437
298
312
309
296 462
310
297
330
354 LPS (10 [mu] g / ml) None 672 633 LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml) Acetaminophen (5 μM)
Aspirin (5 μM)
Ibuprofen (5 μM)
Naproxen (5 μM) 428
472
417
458 457
491
456
501 LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml) Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM) 399
413
427
409 509
484
466
458

# Data represents the average of duplicate experiments.

Analgesic-inflammatory and non-inhibiting the normal PGE2 response of the human bladder smooth muscle cells to the inflammatory stimuli, consistent with the induction of the above-described COX2 reaction, both carbachol and LPS induces the production of PGE2 by normal human urinary bladder smooth muscle cells Respectively. In addition, acetaminophen, aspirin, ibuprofen and naproxen at a dose of 5 μM or 50 μM inhibited the LPS-induced PGE2 response (Table 7).

PGE2 secretion by normal human bladder smooth muscle cells stimulated by inflammatory and noninflammatory stimuli in the test tube and treated with analgesics Stimulation painkiller PGE2 level ^#  (pg / ml)
Target 1 PGE2 levels (pg / ml)
Target 2 None None <20.5 <20.5 Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM)
Acetaminophen (50 μM) 129
76
89
84
77 104
62
59
73
66 LPS (10 [mu] g / ml) None 1125 998 LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml) Acetaminophen (5 μM)
Aspirin (5 μM)
Ibuprofen (5 μM)
Naproxen (5 μM) 817
838
824
859 542
598
527
506 LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml) Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM) 803
812
821
819 540
534
501
523

# Data represents the average of duplicate experiments.

Analgesics inhibit the cytokine response of normal human bladder smooth muscle cells to inflammatory stimuli -Analysis of the cell and culture supernatants after 24 hours of incubation shows that any analgesic agent that has been treated alone does not inhibit IL-6 or TNFα And did not induce the secretion. As shown in Tables 8 and 9, carbachol induced a weak but significant TNFa and IL-6 response in normal human bladder smooth muscle cells. On the other hand, LPS treatment resulted in the mass induction of these proinflammatory cytokines. Acetaminophen, aspirin, ibuprofen and naproxen inhibited the effects of carbachol and LPS on TNFa and IL-6 responses. The inhibitory effect of the analgesic showed LPS-induced response when these drugs were 5 [mu] M or 50 [mu] M.

TNFα secretion by normal human bladder smooth muscle cells stimulated with inflammatory and non-inflammatory stimuli in vitro and treated with analgesics Stimulation painkiller TNF? (Pg / ml)  ^#
Target 1 TNF? (Pg / ml)
Target 2 None None <5 <5 Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M None
Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM) 350
138
110
146
129 286
164
142
121
137 LPS (10 [mu] g / ml) None 5725 4107 LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml) Acetaminophen (5 μM)
Aspirin (5 μM)
Ibuprofen (5 μM)
Naproxen (5 μM) 2338
2479
2733
2591 2267
2187
2288
2215 LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml) Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM) 2184
2266
2603
2427 2056
2089
1997
2192

# Data represents the average of duplicate experiments.

IL-6 secretion by normal human bladder smooth muscle cells stimulated with inflammatory and non-inflammatory stimuli in the test tube and treated with analgesics Stimulation painkiller IL-6 (pg / ml)  ^#
Target 1 IL-6 (pg / ml)
Target 2 None None <5 <5 Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M
Carbachol 10 ^-3 M None
Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM) 232
119
95
107
114 278
135
146
118
127 LPS (10 [mu] g / ml) None 4838 4383 LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml) Acetaminophen (5 μM)
Aspirin (5 μM)
Ibuprofen (5 μM)
Naproxen (5 μM) 2012
2199
2063
2077 2308
2089
2173
2229 LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml)
LPS (10 [mu] g / ml) Acetaminophen (50 μM)
Aspirin (50 μM)
Ibuprofen (50 μM)
Naproxen (50 μM) 2018
1987
2021
2102 1983
2010
1991
2028

# Data represents the average of duplicate experiments.

Early normal human bladder smooth muscle cells were isolated and cultured to assess response to analgesics in the presence of non-inflammatory (carbachol) and inflammatory (LPS) stimuli. The purpose of this experiment is to confirm whether normal human bladder smooth muscle cells reproduce the observation results in the prior murine bladder cells.

The above-mentioned experiments were performed using analgesics of delayed-release, extended-release, or delayed-and-extended-release formulations and / or anti- It can be repeated zero.

The above description is intended to teach those skilled in the art how to practice the invention and is not intended to be exhaustive of all obvious modifications and variations that may become apparent to those skilled in the art upon a reading of the above description. However, all such obvious modifications and variations are intended to be included within the scope of the present invention as defined by the following claims. The claims are intended to include the claimed elements and steps in any order effective for achieving the intended purpose, unless the context otherwise requires.

Claims (33)

Forming a first mixture comprising a first active ingredient comprising at least one analgesic agent;
Coating the first mixture with a first delay-release coating to form a first component, wherein the first delay-release coating has a delay time of 1-4 hours after oral administration Releasing the first mixture;
Forming a second mixture comprising a second active ingredient comprising at least one analgesic agent;
Coating said second mixture with a second delay-release coating to form a second component, said second delay-release coating releasing said second mixture with a delay time of 4-6 hours after oral administration ; And
Mixing the first component with the second component to form a third mixture
&Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof. The method according to claim 1,
Wherein the at least one analgesic agent comprised in the first active component and the second active component is selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone and acetaminophen. A method for preparing a pharmaceutical composition. The method according to claim 1,
Wherein the first delay-release coating is an enteric coating. The method according to claim 1,
Wherein the first active ingredient, or the second active ingredient, or both, further comprises a medicament selected from the group consisting of an antimuscarinic agent, an anti-diuretic agent, and an antispasmodic agent. Way. The method according to claim 1,
Wherein the second delay-release coating comprises a blend of a water insoluble polymer (WIP): an insoluble polymer having an enteric polymer (EP) ratio of from 4: 1 to 1: 2 and an enteric polymer, &Lt; / RTI &gt; The method according to claim 1,
Wherein the second delay-release coating comprises multiple coating layers. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein the second delay-release coating comprises a swellable layer and an insoluble, semi-permeable outer polymeric coating. 8. The method of claim 7,
The swelling layer may be formed of at least one selected from the group consisting of hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymer, polyethylene glycol, polyvinylpyrrolidone, methacrylic acid copolymer, cellulose acetate A method for manufacturing a pharmaceutical composition for reducing urination frequency, which comprises a material selected from the group consisting of phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinylacetate phthalate, shellac and ethylcellulose . The method according to claim 1,
Wherein the second delay-release coating comprises a water-insoluble capsule body that is insoluble but permeable and swellable with one end closed with a hydrogel plug, the plug comprising a polymeric material selected from the group consisting of polymethacrylate, erodible compressed polymers, Wherein the composition comprises a material selected from the group consisting of a molten polymer and an enzymatically controlled erodible polymer. The method according to claim 1,
&Lt; / RTI &gt; further comprising compressing said third mixture into tablets. The method according to claim 1,
Wherein the third mixture is formed by mixing the first component and the second component with a third component and the third component comprises a third active component comprising at least one analgesic agent. A method for preparing a pharmaceutical composition for reducing urinary frequency. A pharmaceutical composition prepared by the method of claim 1. Forming a first mixture comprising a first active ingredient comprising at least one analgesic agent;
Coating the first mixture with a first delay-release coating to form a core structure, wherein the first delay-release coating is applied to the first delay- Releasing the mixture;
And coating the core structure with a second mixture to form a coated core structure, wherein the second mixture comprises one or more compounds selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen Comprising a second active ingredient comprising an analgesic; And
Coating the coated core structure with a second delay-release coating to form a dual-coated core structure, wherein the second delay-release coating is applied to the second mixture at a delay time of 1-4 hours after oral administration Releasing
&Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof. 14. The method of claim 13,
Wherein the second delay-release coating is an enteric coating, wherein the second delay-release coating is an enteric coating. 14. The method of claim 13,
Wherein the at least one analgesic agent in the first active ingredient and the second active ingredient is selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone and acetaminophen. &Lt; / RTI &gt; 14. The method of claim 13,
Wherein the first active ingredient or the second active ingredient, or both, further comprises a medicament selected from the group consisting of an antimuscarinic agent, an anti-diuretic agent and an antispasmodic agent. 14. The method of claim 13,
Coating the dual coated core structure with a third coating, wherein the third coating is selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone, and acetaminophen Or more of the analgesic agent. &Lt; Desc / Clms Page number 24 &gt; 18. The method of claim 17,
Wherein the third active ingredient further comprises a medicament selected from the group consisting of an antimuscarinic agent, an anti-diuretic agent and an antispasmodic agent. 14. A pharmaceutical composition prepared by the method of claim 13. A first component comprising at least one analgesic selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone and acetaminophen, said first component having a delay time of 1 to 4 hours Having been formulated for delayed release; And
A second component comprising at least one analgesic selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone and acetaminophen, said second component having a delay time of 4-6 hours Having been formulated for delayed release
&Lt; / RTI &gt; 21. The method of claim 20,
Wherein the pharmaceutical composition is coated with an enteric coating. 21. The method of claim 20,
Wherein the first component and / or the second component further comprises a medicament selected from the group consisting of an antimuscarinic agent, an antidiuretic agent and an antispasmodic agent. 21. The method of claim 20,
Wherein the second component is coated with a blend of a water insoluble polymer (WIP): enteric polymer (EP) ratio of 4: 1 to 1: 2 and an enteric polymer. 21. The method of claim 20,
Wherein the second component is coated with multiple coating layers. 21. The method of claim 20,
Wherein the second component is coated with a swellable layer and an insoluble but semi-permeable outer polymer coating. 21. The method of claim 20,
The swelling layer may be formed of at least one selected from the group consisting of hydroxypropylmethylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymer, polyethylene glycol, polyvinylpyrrolidone, methacrylic acid copolymer, cellulose acetate A material selected from the group consisting of phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinylacetate phthalate, shellac, and ethylcellulose. 21. The method of claim 20,
Said second component formulation comprising a water insoluble capsule body closed at one end with an insoluble but permeable and swellable hydrogel plug, said plug comprising a polymeric material selected from the group consisting of polymethacrylate, an erodible crimped polymer, a condensed molten polymer and an enzyme Wherein the erodible polymer comprises a material selected from the group consisting of erodible and controlled erodible polymers. A first component comprising acetaminophen in an amount of 5-2000 mg, said first component being formulated for immediate-release; And
A second component comprising at least one analgesic selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone and acetaminophen, said second component having a delay time of 1 to 4 hours Having been formulated for delayed release; And
A third component comprising at least one analgesic selected from the group consisting of aspirin, ibuprofen, naproxen, naproxen sodium, indomethacin, nabumetone and acetaminophen, said third component having a delay time of 4-6 hours Having been formulated for delayed release
&Lt; / RTI &gt; 29. The method of claim 28,
Wherein the second component is coated with an enteric coating. 29. The method of claim 28,
Wherein the first component and / or the second component and / or the third component further comprises a medicament selected from the group consisting of an antimuscarinic agent, an anti-diuretic agent and an antispasmodic agent. 31. The method of claim 30,
Wherein the second component is coated with an enteric coating. 29. The method of claim 28,
Wherein the second component or the third component is coated with a water insoluble polymer (WIP) and an enteric polymer (EP) in a ratio of WIP: EP of from 4: 1 to 1: 2. 29. The method of claim 28,
Wherein the third component is coated with multiple coating layers.

Images