HK1234997A1 - Pharmaceutical formulation for reducing frequency of urination and method of use thereof - Google Patents

Description

Pharmaceutical formulation for reducing frequency of urination and method of use thereof

Technical Field

The present invention relates generally to methods and compositions for inhibiting bladder smooth muscle, and in particular for reducing frequency of urination.

Background

The detrusor muscle is a layer of the bladder wall composed of smooth muscle fibers arranged in helical, longitudinal and annular bundles. When the bladder is stretched, this indicates that the parasympathetic nervous system contracts the detrusor muscle. This causes the bladder to drain urine through the urethra.

In order for urine to leave the bladder, both the automatically controlled internal sphincter and the autonomously controlled external sphincter must be opened. Problems with these muscles can lead to incontinence. If the amount of urine reaches 100% of the absolute capacity of the bladder, the voluntary sphincter will become involuntary and the urine will be discharged immediately.

Adult bladders typically hold about 300 to 350 milliliters of urine (working volume), but full adult bladders can hold up to about 1000 milliliters (absolute volume) of urine, with variation between individuals. As urine accumulates, the folds (rugae) created by the folds of the bladder wall flatten and the bladder wall thins as it stretches, allowing the bladder to store larger amounts of urine without a significant rise in internal pressure.

In most individuals, the urine will typically begin to urinate when the volume of urine in the bladder reaches about 200 milliliters. At this stage, the individual is prone to resist the urge to urinate, if desired. As the bladder continues to fill, the urine changes stronger and becomes more difficult to ignore. Eventually, the bladder will fill to the point where the urge to urinate cannot be resisted, at which point the individual will no longer be able to stand alone.

In some individuals, this desire begins to develop when the urine in the bladder reaches less than 100% of its working volume. This enhanced urinalysis can interfere with normal activities, including sleep ability during substantially uninterrupted rest periods. In some cases, this enhanced urinary sensation may be associated with a medical condition, such as benign prostatic hyperplasia in men or prostate cancer or pregnancy in women. However, increased urinary willingness also occurs in male and female individuals unaffected by other medical conditions.

In some individuals, such as children, involuntary urination (e.g., bedwetting) may occur due to lack of control over the bladder muscle. In other individuals, involuntary urination (e.g., urinary incontinence) may occur due to an underlying medical condition.

Accordingly, there is a need for compositions and methods for treating male and female individuals suffering from undesirable frequency of urination.

Disclosure of Invention

One aspect of the invention relates to a method of reducing the frequency of urination in an individual. The method comprises administering to an individual having a disease that causes undesired frequency of urination an effective amount of a pharmaceutical composition comprising one or more prostaglandin pathway (prostagladin pathway) inhibitors.

Another aspect of the invention relates to a pharmaceutical composition for treating a disease that results in an undesired frequency of urination. The pharmaceutical composition comprises one or more prostaglandin pathway inhibitors and a pharmaceutically acceptable carrier.

Drawings

Fig. 1A and 1B are graphs showing that analgesics modulate the expression of Raw264 macrophages on co-stimulatory molecules in the absence (fig. 1A) or presence (fig. 1B) of Lipopolysaccharide (LPS). The cells were cultured for 24 hours (hr) in the presence of an analgesic alone or in combination with Salmonella typhimurium LPS (0.05. mu.g/ml). Results are the average relative% of CD40+ CD80+ cells.

Detailed Description

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

The term "Prostaglandin (PG)" as used herein refers to a group of lipid compounds derived from fatty acids by enzymatic action (enzymaticailly) and having various physiological effects in animals, such as regulating contraction and relaxation of smooth muscle tissues. Each prostaglandin contains 20 carbon atoms and includes a 5-carbon ring. Examples of prostaglandins include prostaglandin E1(PGE1), prostaglandin E₂(PGE₂) Prostaglandin D₂Prostaglandin I₂(PGI₂Prostacyclin) and prostaglandin F_2α(PGF_2α)。

The term "Prostaglandin (PG) pathway inhibitor" as used herein refers to an agent that interacts directly or indirectly with one or more components involved in the synthesis of PG or the effect of PG on a target tissue and interferes with the level of prostaglandin or its ultimate effect on the target tissue. PG pathway inhibitors include, but are not limited to, PG inhibitors, prostaglandin transporter (PGT) inhibitors, and prostaglandin receptor (PGR) inhibitors. However, the term "PG pathway inhibitor" does not include the analgesics defined below.

The term "PG inhibitor" as used herein includes, but is not limited to, inhibitors of PG synthesis and inhibitors of PG activity. The term "PG synthesis inhibitor" as used herein refers to an agent that inhibits prostaglandin production, such as agents that inhibit the expression or activity of phospholipase a2, prostaglandin synthase, and tissue-specific isomerases, as well as synthetases such as thromboxane (thromboxane) synthase, PGF synthase, cytosolic PG synthase (cpgs), prostaglandin I synthase (PGIS), and microsomal PGES enzyme (mPGES). An example of a PG synthesis inhibitor includes flunixin meglumine (flunixin meglumine). The term "inhibitor of PG synthesis" or "PG synthesis inhibitors" as used herein does not include analgesics as defined below.

The term "inhibitor of PG activity" as used herein refers to an agent that antagonizes the action of the prostaglandin itself in any way. Agents that interfere only with the synthesis of prostaglandins, such as by interfering with the action of prostaglandin synthase, but not with the action of prostaglandins, are not included within the definition of PG activity inhibitors used in the present specification.

The term "PGT inhibitor" as used herein refers to an agent that inhibits the expression or activity of a PG transporter, such as the ATP dependent multi-drug resistance (MDR) transporter-4 (ATP dependent multi-drug resistance transporter-4) or other MDR channels (mdrchchannels) such as ABCC1, ABCC2, ABCC3, ABCC6, ABCG2 and ABCB 11. Examples of PGT inhibitors that inhibit PGT activity include, but are not limited to, compounds that inhibit MDR membrane pumps, such as triazine compounds (triazine compounds), verapamil (verapamil), and calcium channel blockers; channels include quinidine, ketoconazole, itraconazole, azithromycin, valspodar, cyclosporin, elacridar, fumagillin-C, gefitinib and erythromycin. Examples of PGT inhibitors that inhibit PGT expression include, but are not limited to, agents that control transcription of MDR genes by targeting the promoter region and/or transcription factors that bind to the promoter or other gene control regions. The term "PGR inhibitor" as used herein refers to an agent that inhibits the activity or expression of a PGR. In some embodiments, the PGR comprises the E prostanoid receptor (EP) 1, EP2, EP3, and EP4 subtypes of the PGE receptor; PGD receptor (DP 1); PGF receptor (FP); PGI receptor (IP) and thrombolipoprotein receptor (TP). Both two other isoforms of human TP (TP α and TP β) and two other isoforms of FP (FPA and FPB) and eight EP3 variants were produced via selective splicing, which differed only at the C-terminal tail. In some embodiments, the PGR further comprises a G protein-coupled receptor (G protein-coupled receptor), which is referred to as a chemokine receptor-homologous molecule (CRHME). In other embodiments, the PGR includes all receptors that activate the rhodopsin-like 7 transmembrane-G protein-coupled receptor (rhodopsin-like 7-transmembrane-transducing-mapping G protein-coupled receptor).

Examples of inhibitors of PGR activity include, but are not limited to, anti-PGR antibodies and any agent that inhibits the G-protein coupled receptor (G-protein coupled receptor) signaling pathway. Inhibitors of PGR expression include agents that inhibit PGR expression at the transcriptional, translational or post-transcriptional level. Examples of PGR expression inhibitors include, but are not limited to, anti-pgrsirnas and mirnas.

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

The term "analgesic" as used herein refers to an agent, compound or drug that is used to relieve pain and includes anti-inflammatory compounds. Exemplary analgesic and/or anti-inflammatory agents, compounds, or drugs include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), salicylates (salicylates), aspirin (aspirin), salicylic acid (salicylic acid), methyl salicylate (methyl salicylate), diflunisal (diflunisal), salsalates (salsalate), olsalazine (olsalazine), sulfasalazine (sulfasalazine), para-aminophenol (para-aminophenol) derivatives, acetanilide (acetanilide), acetaminophen (acetaminophen), phenacetin (phenacetin), fenamic acid (fenamate), mefenamic acid (mefenamic acid), meclofenamic acid (meclofenamate), sodium meclofenamate (sodium mefenamate), heteroaryl acetic acid (heteroacetic), mefenamic acid (ketofenamic acid), diclofenac (ketoprofen) derivatives, ibuprofen (diclofenac), ibuprofen (ibuprofen) derivatives, ibuprofen (ketoprofen), ibuprofen (diclofenac), ibuprofen (ketoprofen) derivatives, ibuprofen (ketoprofen (ibuprofen (diclofenac), ibuprofen (ketoprofen) derivatives), ibuprofen (ketoprofen) derivatives, ibuprofen (ketoprofen), ibuprofen (ketoprofen) derivatives, diclofenac (ketoprofen), ibuprofen (ketoprofen) derivatives, flufenamic acid (ketoprofen), flufenamic acid (ketoprofen, flufenamic acid, flufena, Naproxen (naproxen), fenoprofen (fenoprofen), ketoprofen (ketoprofen), flurbiprofen (flurbiprofen), oxaprozin (oxaprozin); enolic acid (enoolic acids), oxicam (oxicam) derivatives, piroxicam (piroxicam), meloxicam (meloxicam), tenoxicam (tenoxicam), ampiroxicam (ampiroxicam), droxicam (droxicam), pyrivoxicam (pivoxicam), pyrazolone (pyrazolon) derivatives, phenylbutazone (phenylbutazone), oxyphenbutazone (oxyphenbutazone), amphetaline (antipyrine), aminopyrroline (amidopyrine), analgin (dipyrone), coxibs (coxibs), celecoxib (celecoxib), rofecoxib (rofecoxib), nabumetone (nabumetone), apazone (apazone), indomethacin (indomethacin), sulindac (sulindac), etodolac (oxyphenbutazone), etodolac (ketoprofen) (etodolac), etodolac (ketoprofen) (etoricide), etodolac (ketoprofen) (etoricide), etoricide (ketoprofen) (etoricide), etoricide (ketoprofen) (etoricide), etoricide (ketoprofen) (etoricide), etoricide (ketoprofen) (ketoprofen, Aceclofenac (aceclofenac), lincomone (licofelone), bromfenac (bronfnac), loxoprofen (loxoprofen), pranoprofen (pranoprofen), piroxicam (piroxicam), nimesulide (nimesulide), cimetizoline (cizolirine), 3-carboxamido-7-methanesulfonamido-6-phenoxy-4H-1-benzopyran-4-one (3-formamido-7-methysullfurylamino-6-phenoxy-4H-1-benzopyran-4-one), meloxicam (meloxicam), lornoxicam (loxoxicam), prad-indobufen (d-indobufen), moxazonic acid (mofezofen), tolmetin (amycetin), loxoprofen (loxoprofen), loxoprofen (oxaprofen), loxoprofen (loxoprofen), ibuprofen (loxoprofen, Amiprofen (alminoprofen), tiaprofenic acid (tiaprofenic acid), pharmacological salts thereof, hydrates thereof and solvates thereof.

The term "coxib" as used herein refers to a compound or combination of compounds that inhibits the activity or expression of COX1 and COX2 enzymes.

The term "derivative" as used herein refers to a chemically modified compound, wherein the modification is conventionally recognized by a chemist of ordinary skill, such as an ester or amide of an acid, or a protecting group, such as benzyl for an alcohol or thiol or tert-butyloxycarbonyl (tert-butyloxycarbonyl) for an amine.

The term "analog" as used herein refers to a compound that comprises a chemically modified form of a particular compound or class thereof and retains the pharmaceutically and/or pharmacologically active characteristics of that compound or class.

The term "pharmaceutically acceptable salt" as used herein refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, basic or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid (stearic acid), lactic acid, malic acid (malic acid), tartaric acid, citric acid, ascorbic acid, pamoic acid (pamoic acid), maleic acid (maleic acid), hydroxycitric acid (hydroxymaleic acid), phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid (sulfamic acid), 2-acetoxybenzoic acid (2-acetoxybenzoic acid), fumaric acid (fumaric acid), toluenesulfonic acid, methanesulfonic acid, ethane disulfonic acid (ethanesulfonic acid), oxalic acid (oxaloic acid), isethionic acid (isethionic acid), and the like.

The phrase "pharmaceutically acceptable" as used herein pertains to the use of compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "immediate-release" is used herein to refer to a pharmaceutical formulation that does not contain dissolution rate controlling materials. The release of the active agent is not substantially delayed after administration of the immediate release formulation. The immediate release coating may comprise a suitable material that dissolves upon administration to release the drug contents therein. In some embodiments, the term "immediate release" is used to mean a pharmaceutical formulation that releases an active ingredient within less than 10 minutes (min), 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, or 120 minutes after administration to a patient.

The term "extended-release" (also known as sustained-release; SR), sustained-action (SA), timed-release (TR), controlled-release (CR), modified-release (MR) or continuous-release (CR)) as used herein refers to a mechanism for a medical tablet or capsule that slowly dissolves over time and releases an active ingredient. An advantage of extended release tablets or capsules is that they can often be taken less frequently than immediate release formulations of the same drug and that they maintain a more stable level of drug in the bloodstream, thereby extending the duration of drug action and reducing the peak amount of drug in the bloodstream. In some embodiments, the term "extended release" refers to a release profile in which the active ingredient in a tablet or capsule is released continuously or in pulses over a period of time (period) of 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours after administration to a patient.

The term "delayed-release" as used herein refers to a drug release profile in which the release of the active ingredient of a pharmaceutical composition is delayed or delayed for a given period of time (e.g., 1,2, 3, 4, or 5 hours, or after the stomach) after administration of the pharmaceutical composition.

The term "delayed-extended-release" as used herein refers to a drug release profile in which the release of the active ingredient of the pharmaceutical composition is delayed or delayed for a given period of time (e.g., a lag period of 1,2, 3, 4, or 5 hours, or after the stomach) after administration of the pharmaceutical composition. Once release is initiated, the active ingredient is slowly released over time (e.g., over a period of 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours), either continuously or in pulses.

Method for reducing frequency of urination

One aspect of the invention relates to a method of reducing the frequency of urination by administering to an individual suffering from a disease that causes undesired frequency of urination an effective amount of a pharmaceutical composition. The pharmaceutical composition comprises one or more PG pathway inhibitors and a pharmaceutically acceptable carrier. Diseases that lead to undesirable frequency of urination include, but are not limited to, nocturia (nocturia), overactive bladder (overactive bladder), urinary incontinence (urinary incontinence) and bed wetting (bed wetting).

In some embodiments, the PG inhibitor is an inhibitor of PG synthase. In other embodiments, the PG inhibitor is an inhibitor of PG activity. Examples of inhibitors of PG activity include, but are not limited to, agents that block binding of PG to any of its receptors (EP1, EP2, EP3, EP4, DP1, DP2, FP2, IP and TP). Examples of these types of inhibitors include, but are not limited to, the IP receptor inhibitor RO3244019, the EP1 receptor antagonist ONO-85-39, the dual receptor antagonist AH6809 of EP1 and EP2, and the EP4 antagonist RQ-15986 developed by Roche (Roche). In certain embodiments, the one or more PG pathway inhibitors comprise a PGT inhibitor. In some embodiments, the PGT inhibitor is an inhibitor of PGT activity. Examples of inhibitors of PGT activity include, but are not limited to, antibodies against PGT, and any compound known to inhibit the ATP-dependent multidrug resistance transporter-4 or associated MDR pump that is shown to transport PG. In other embodiments, the PGT inhibitor is a PGT expression inhibitor. Examples of PGT expression inhibitors include, but are not limited to, anti-PGT sirnas, antisense RNAs targeting PGT mrnas, and agents that control gene transcription by affecting DNA methylation and/or chromatin modification (chromatin modification).

In some embodiments, the one or more PG pathway inhibitors comprise an inhibitor that targets the COX active site and the POX active site contained in both COX1 and COX 2. In other embodiments, the one or more PG pathway inhibitors comprise an inhibitor of PGE₂Inhibitors of the pathway.

In certain embodiments, the one or more PG pathway inhibitors comprise a PGR inhibitor. PGR is a G-protein-coupled receptor (G-protein-coupled receptors) containing seven transmembrane domains. Examples of PGRs include EP1, EP2, EP3, EP4, DP1, DP2, FP, IP1, IP2, CRTH2 and TP receptors. In some embodiments, the one or more PG pathway inhibitors comprise an inhibitor that inhibits any of the PG receptors listed above. In some embodiments, the PGR inhibitor is an inhibitor of PGR activity. Examples of inhibitors of PGR activity include, but are not limited to, antibodies against PGR. In some embodiments, the PGR inhibitor is an inhibitor of PGE2 receptor activity, such as an inhibitor of EP1 activity, an inhibitor of EP2 activity, an inhibitor of EP3 activity, or an inhibitor of EP4 activity.

In other embodiments, the PGR inhibitor is a PGR expression inhibitor. Examples of PGR expression inhibitors include, but are not limited to, anti-PGR sirnas, antisense RNAs targeting PGR mRNA, or agents that control gene transcription by affecting DNA methylation and/or chromatin modification. In some embodiments, the PGR expression inhibitor is a PGE2 receptor expression inhibitor, such as an EP1 expression inhibitor, an EP2 expression inhibitor, an EP3 expression inhibitor, or an EP4 expression inhibitor. In some embodiments, the one or more PG pathway inhibitors comprise a small molecule inhibitor. The term "small molecule inhibitor" as used herein refers to an inhibitor having a molecular weight of 1000 daltons (dalton) or less than 1000 daltons.

In some embodiments, the PG pathway inhibitor comprises short interfering rna (sirna). sirnas are double-stranded RNAs that can be engineered to induce sequence-specific post-transcriptional gene silencing (gene silencing) of mrnas corresponding to components of the PG pathway. siRNA employs an RNA interference (RNAi) mechanism for the purpose of "silencing" the expression of genes such as the targeted PGE2 receptor gene. This "silencing" was initially observed in the context of transfecting double stranded rna (dsrna) into cells. Upon entry, the dsRNA was found to be cleaved by an RNase-III like enzyme Dicer into double-stranded small interfering RNA (siRNA) of 21 to 23 nucleotides in length containing a 2-nucleotide overhang at its 3' end. In the ATP-dependent step, sirnas are integrated into a multiple unit RNAi-induced silencing complex (RISC) that signals for AGO 2-mediated cleavage of mRNA complements, which in turn leads to subsequent degradation by cellular exonucleases (cellular exonucleases).

In some embodiments, the PG pathway inhibitor comprises a synthetic siRNA or other species of small RNA that targets PG synthase RNA, PGT RNA, or PGR RNA in the target cell/tissue. Synthetically produced sirnas structurally mimic the type of siRNA normally processed by the enzyme Dicer in cells. Synthetically produced sirnas can incorporate any chemical modification into the RNA structure known to enhance siRNA stability and functionality. For example, in some cases, the siRNA may be synthesized as a Locked Nucleic Acid (LNA) modified siRNA. LNA is a nucleotide analog containing a methylene (methylene) bridge connecting the 2 '-oxygen of the ribose sugar to the 4' carbon. The bicyclic structure locks the furanose ring (furanose ring) of the LNA molecule into a 3'-endo configuration (3' -endo formation), thereby structurally mimicking standard RNA monomers.

In other embodiments, the PG pathway inhibitor comprises an expression vector engineered to transcribe short double-stranded hairpin-like RNAs (shrnas) that are processed to target sirnas within the cell. May be used, for example, of AmbionsiRNA construction kit and GENESUPPRESSOR of Imgenex^TMConstruction kit andinvitrogen's BLOCK-IT^TMInducible RNAi plasmids and lentiviral vectors (lentivirus vectors) clone the shRNA in a suitable expression vector. Synthetic sirnas and shrnas can be designed using well-known algorithms and synthesized using conventional DNA/RNA synthesizers.

In some embodiments, the secondary active agent comprises an antisense oligonucleotide or polynucleotide capable of inhibiting expression of a PG pathway component. The antisense oligonucleotide or polynucleotide may comprise a DNA backbone, an RNA backbone, or chemical derivatives thereof. In one embodiment, the antisense oligonucleotide or polynucleotide comprises a single stranded antisense oligonucleotide or polynucleotide targeted for degradation. In certain embodiments, the anti-inflammatory agent comprises a single-stranded antisense oligonucleotide complementary to an mRNA sequence of a PG pathway component. The single stranded antisense oligonucleotides or polynucleotides may be produced synthetically or may be expressed by suitable expression vectors. Antisense nucleic acids are designed to bind to the mRNA sense strand (sense strand) via complementary binding to promote RNase H activity, which results in mRNA degradation. The antisense oligonucleotides are preferably chemically or structurally modified to increase nuclease stability and/or enhance binding.

In some embodiments, antisense oligonucleotides are modified to produce oligonucleotides with unconventional chemistry or backbone additions or substitutions, including, but not limited to, Peptide Nucleic Acids (PNA), Locked Nucleic Acids (LNA), morpholino (morpholino) backbone nucleic acids, methylphosphonates (methylphosphonates), doubly stable stilbene or pyrenyl terminated caps (duplex stabilizing stilbene or pyrenyl caps), phosphorothioates (phosphothioates), phosphoramidates (phosphoamides), phosphotriesters (phosphotriesters), and analogs thereof. For example, a modified oligonucleotide may combine or replace one or more naturally occurring nucleotides with an analog; modification between nucleotides introduces, for example, uncharged linkages (e.g., methyl phosphates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.); the modification incorporates an intercalator (e.g., acridine, psoralen, etc.), a chelator (e.g., a metal, radioactive metal, boron, oxidative metal, etc.), or an alkylating agent and/or a modified linkage (e.g., alpha-mutameric nucleic acids, etc.).

In some embodiments, the single stranded oligonucleotide is internally modified to include at least one neutral charge in its backbone. For example, the oligonucleotide may comprise a methylphosphonate backbone or a Peptide Nucleic Acid (PNA) that is complementary to a specific target sequence. These modifications have been found to prevent or reduce helicase-mediated helication. The use of uncharged probes also increases the rate of hybridization to polynucleotide targets in a sample by mitigating repulsion of negatively charged nucleic acid strands in typical hybridizations.

PNA oligonucleotides are uncharged nucleic acid analogs in which the phosphodiester backbone is polyamide-displaced, making PNA polymers with 2-aminoethyl-glycine (2-aminoethyl-glycine) units held together by amide linkages. PNAs were synthesized using the same Boc or Fmoc chemistry as used in standard peptide synthesis. The bases (adenine, guanine, cytosine and thymine) are linked to the backbone via methylene carboxy linkages (methylene carboxy linkages). Thus, PNA is acyclic (acyclic), achiral (achiral) and neutral. Other properties of PNAs are increased specificity and melting temperature compared to nucleic acids, ability to form triple helices, stability at acidic pH, inability to be recognized by cellular enzymes such as nucleases, polymerases, etc.

The methylphosphonate-containing oligonucleotide is a neutral DNA analog containing a methyl group substituted for one of the non-bonded phosphoryl oxides. Oligonucleotides with methylphosphonate linkages were first reported to inhibit protein synthesis via antisense blockade of translation.

In some embodiments, the phosphate backbone in the oligonucleotide may contain phosphorothioate linkages or phosphoramidate. Combinations of these oligonucleotide linkages are also within the scope of the invention.

In other embodiments, the oligonucleotide may contain a backbone of modified sugars joined by phosphodiester internucleotide linkages. The modified sugars may include furanose (furanose) analogues including, but not limited to, 2-deoxyribofuranosides (2-deoxyribofuranosides), α -D-arabinofuranosides (α -D-arabinofuranosides), α -2'-deoxyribofuranosides (α -2' -deoxyribofuranosides) and 2',3' -dideoxy-3'-aminoribofuranosides (2',3'-dideoxy-3' -aminoribofuranosides). In alternative embodiments, the 2-deoxy- β -D-ribofuranosyl (2-deoxy- β -D-ribofuranosyl groups) may be replaced by other sugars (e.g., β -D-ribofuranosyl). In addition, β -D-ribofuranoses may be present in which the 2-OH of the ribose moiety is alkylated with C1-6 alkyl (2- (O- -C1-6 alkyl) ribose) or with C2-6 alkenyl (2- (O- -C2-6 alkenyl) ribose) or replaced with fluoro (2-fluororibose).

The relevant oligomer-forming saccharides (oligomer-forming sugar) include those used in Locked Nucleic Acids (LNA) as described above. Exemplary LNA oligonucleotides include modified bicyclic monomeric units having a 2'-O-4' -C methylene bridge, as described in U.S. patent No. 6,268,490.

Chemically modified oligonucleotides may also include 2 '-sugar modifications, 5-pyrimidine modifications (e.g., 5- (N-benzylcarboxamide) -2' -deoxyuridine (5- (N-benzylcarboxyyamide) -2'-deoxyuridine), 5- (N-isobutylcarboxamide) -2' -deoxyuridine (5- (N-isobutrylcarboxyyamide) -2'-deoxyuridine), 5- (N- [2- (1H-indol-3-yl) ethyl ] carboxamide) -2' -deoxyuridine (5- (N- [2- (1H-indole-3-yl) ethyl ] carboxamide) -2'-deoxyuridine, 5- (N- [1- (3-trimethylammonium) propyl ] carboxamide) -2' -deoxyuridine chloride (5- (N- [1- (3-trimethylalamonium) propyll ] carboxyyamide) -2' -deoxyuridinehloride), 5- (N-naphthylcarboxamide) -2' -deoxyuridine (5- (N-napthylcarboxamido-2 ' -deoxyuridine), 5- (N- [1- (2,3-dihydroxypropyl) ] carboxamide) -2' -deoxyuridine) (5- (N- [1- (2,3-dihydroxypropyl) ] carboxyyamide) -2' -deoxyuridine), 8-position purine modification, modification of exocyclic amine (exocyclic amine), substitution of 4-thiouridine (4-thiouridine), substitution of 5-bromo-or 5-iodo-uracil, methylation, unusual base-pairing combinations such as isocytidine (isocytidine) and isoguanidine (isogluanidine), and analogs thereof.

In some embodiments, the one or more PG pathway inhibitors comprise a ribozyme (ribozyme) that inhibits expression of a PG pathway component. Ribozymes are nucleic acid molecules that catalyze intramolecular or intermolecular chemical reactions. Thus, ribozymes are catalytic nucleic acids. Ribozymes preferably catalyze intermolecular reactions. There are many different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions, which are based on ribozymes found in natural systems, such as hammerhead ribozymes (hammerhead ribozymes), hairpin ribozymes (hairpin ribozymes), and tetrahymena ribozymes (tetrahymena ribozymes). There are also a number of ribozymes that are not found in natural systems, but which have been re (de novo) engineered to catalyze specific reactions. Preferred ribozymes cleave RNA or DNA substrates and more preferably cleave RNA substrates such as mRNA of PG pathway components. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to a target substrate and then cleaving. This recognition is usually based primarily on canonical or atypical base pair interactions. This property makes ribozymes a particularly good candidate for targeted specific cleavage of nucleic acids, since the recognition of the target substrate is based on the target substrate sequence.

In some embodiments, the one or more PG pathway inhibitors comprise a triple helix forming oligonucleotide capable of inhibiting expression of a PG pathway component. Triple helix forming oligonucleotides (TFOs) are molecules that can interact with double and/or single stranded nucleic acids, including coding and non-coding regions in genomic DNA targets. When TFO interacts with the target region, a so-called triple helix structure is formed in which triple-stranded DNA forms a complex that relies on Watson-Crick base pairing with Hoogsteen. TFO can bind target regions with high affinity and specificity. In some preferred embodiments, the triple helix forming molecule is less than 10^-6、10^-8、10^-10Or 10^-12Binds to the target molecule. Exemplary TFOs for use in the present invention include PNAs, LNAs, and LNA-modified PNAs, such as Zorro-LNAs.

In some embodiments, the one or more PG pathway inhibitors comprise an External Guide Sequence (EGS). An External Guide Sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex. This complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target selected mRNA molecules. RNAse P facilitates intracellular processing of transfer RNA (tRNA). Can be prepared by using a nucleic acid sequence that results in target RNA: the EGS complex mimics the EGS of the native tRNA substrate, recruiting bacterial RNAse P to cleave virtually any RNA sequence. Similarly, eukaryotic EGS/RNAse P-directed RNA cleavage can be used to cleave a desired target in eukaryotic cells.

In other embodiments, the one or more PG pathway inhibitors comprise a biomolecule (biomolecule). The term "biomolecule" as used herein is any molecule produced by a living organism, including large macromolecules such as proteins, polysaccharides, lipids and nucleic acids, and small molecules such as primary metabolites, secondary metabolites and natural products.

In other embodiments, the one or more PG pathway inhibitors comprise a targeted neutralizing agent. The term "target neutralizing agent" as used herein refers to an antibody, antibody fragment or any other non-antibody peptide or synthetic binding molecule, such as an aptamer (aptamer) or synthetic antibody (synbody), that is capable of specifically binding a component of the PG pathway, either directly or indirectly, to interfere with the ultimate effect of the prostaglandin on the target tissue.

The target neutralizing agent can be generated by any conventional method for generating high affinity binding ligands, including SELEX, phage display, and other methods, including combinatorial chemistry and/or high throughput methods known to those skilled in the art.

Aptamers are nucleic acid variants of antibodies comprising a class of oligonucleotides that can form specific three-dimensional structures that exhibit high affinity binding to a variety of cell surface molecules, proteins, and/or macromolecular structures. Aptamers are typically identified by in vitro (in vitro) selection methods, sometimes referred to as "EXponential enrichment of Ligands by ectopic evolution" or "SELEX". SELEX generally begins with a collection of very large numbers of random polynucleotides, which is generally limited to targeting one aptamer ligand per molecule. Aptamers are typically small nucleic acids in the range of 15 to 50 bases in length that fold into defined secondary and tertiary structures, such as stem loops (stemloops) or G-quartets (G-quatets).

Aptamers can be chemically linked or conjugated to the above-described nucleic acid inhibitors to form targeted nucleic acid inhibitors, such as aptamer-siRNA chimeras. The aptamer-siRNA chimera contains a target portion in the form of an aptamer linked to siRNA. When aptamer-siRNA chimeras are used, it is preferred to use cell internalizing aptamers. Upon binding to specific cell surface molecules, the aptamer can promote internalization into the cell where the nucleic acid inhibitor acts. In one embodiment, both the aptamer and the siRNA comprise RNA. The aptamer and siRNA can comprise any nucleotide modification as further described herein. Aptamers preferably comprise target moieties that specifically direct binding to cells (e.g., lymphocytes, epithelial cells, and/or endothelial cells) that express chemokine- (chemokine-), cytokine- (cytokine-), and/or receptor target genes.

Synthetic antibodies (synbodies) consist of synthetic antibodies (synthetic antibodies) generated in libraries comprising random peptide strings screened for binding to target proteins of interest.

Target neutralizers (including aptamers and synthetic antibodies) can be engineered to be 10^-10To 10^-12The Kd of the molar concentration (M) binds very tightly to the target molecule. In some embodiments, the target neutralizer comprises less than 10^-6Less than 10^-8Less than 10^-9Less than 10^-10Or less than 10^-12The Kd of M binds to the target molecule.

In certain embodiments, the one or more PG pathway inhibitors are polynucleotides comprising a polynucleotide encoding and adapted to express a PGT inhibitor and/or a PGR inhibitor. In other embodiments, the one or more PG pathway inhibitors are expression vectors comprising a coding PGT inhibitor and/or PGR inhibitor and adapted to express the PGT inhibitor and/or PGR inhibitor.

In some embodiments, the PG pathway inhibitor is an engineered protein containing TALE sequences or engineered Zinc fingers (Zinc fingers) directed against genes encoding any component of the PG pathway. Such TALEs or zinc fingers can be designed to bind directly to a gene and inhibit its expression by cleaving the gene, altering its nucleotide sequence, or tethering a suppressor protein to the gene to silence it.

In some embodiments, the PG pathway inhibitor is produced using a CRISPR/CAS system. In this strategy, a specific guide molecule (guide molecule) is designed for the gene sequence of each PG pathway gene and introduced into cells or tissues using the delivery system described above (viruses, plastids, etc.). Operation of the CRISPR/CAS system will modify the DNA sequence of the gene such that the PG pathway gene is deleted or inhibits the ability to express RNA.

In some embodiments, the PG pathway inhibitor is capable of turning off transcription of one or more PG pathway genes by targeting chromatin-associated enzymes that post-translationally modify tissue proteins in chromatin. Examples of such enzymes are, but are not limited to, histone deacetylases (histones deacetylases), histone demethylases (histones acetyltransferases), histone methyltransferases (histones) and helicases (helicases).

In some embodiments, the PG pathway inhibitor targets the gene encoding each component by altering the DNA methylation state of the gene. Compounds that target the TET family of DNA demethylases (demethylases) and DNA methyltransferases (DNMT1, DNMTa and DNMTb) can alter expression of RNA from any gene in the PG pathway.

The expression vectors of the invention comprise a polynucleotide encoding a PG pathway inhibitor or a portion thereof. The expression vector also includes one or more regulatory sequences operably linked to the expressed polynucleotide. The regulatory sequences are selected based on the type of host cell. One skilled in the art will appreciate that the design of an expression vector will depend on factors such as the choice of host cell and the level of expression desired.

In some embodiments, the expression vector is a plastid vector. In other embodiments, the expression vector is a viral vector. Examples of viral vectors include, but are not limited to, retroviral (retroviruses), lentiviral (lentiviruses), adenoviral (adeno-associated viruses), AAV (adeno-associated viruses), herpes virus (herpes virus), or alphaviral (alphavirus) vectors. The viral vector may also be a astrovirus (astrovirus), coronavirus (coronavirus), orthomyxovirus (orthomyxovirus), papovavirus (papovavirus), paramyxovirus (paramyxovirus), parvovirus (paravorus), picornavirus (picornavirus), poxvirus (poxvirus) or togavirus (togavirus) vector. When used in mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are from polyoma Virus (polyoma), adenovirus type 2 (adenovirus 2), cytomegalovirus (cytomegalovirus), and Simian Virus 40(Simian Virus 40). In some embodiments, the expression vector contains tissue-specific regulatory elements. Delivery of expression vectors includes, but is not limited to, direct infection with a viral vector, exposure of target tissue to polycationic condensed DNA (polycationic condensed DNA) linked or unlinked to a killed virus, ligand-linked DNA, gene gun, ionizing radiation (ioningradiation), nuclear charge (nuclear acid) neutralization or fusion with cell membranes. Naked plasmid (nakedplasmid) or viral DNA may also be used. Biodegradable latex beads can be used to improve absorption efficiency. This process can be further improved by treating the beads to increase their hydrophobicity. Liposome (liposome) based methods can also be used to introduce plastids or viral vectors into target tissues.

In some embodiments, the pharmaceutical composition further comprises one or more active ingredients selected from the group consisting of: analgesics, antimuscarinic agents, antidiuretic agents, spasmolytics, phosphodiesterase type 5 inhibitors (PDE5 inhibitors) and zolpidem (zolpidem).

Examples of antimuscarinic agents include, but are not limited to oxybutynin (oxybutynin), solifenacin (solifenacin), darifenacin (darifenacin), fesoterodine (fesoterodine), tolterodine (tolterodine), trospium (trospium), atropine (atropine), and tricyclic antidepressants (tricyclicalcepressants). Examples of antidiuretic agents include, but are not limited to, antidiuretic hormone (ADH), vasopressin II (angiotensin II), aldosterone (aldosterone), vasopressin (vasopressin), vasopressin analogs (e.g., desmopressin (desmopressin), arginine vasopressin (argipressin), lysine vasopressin (lypressin), benzene vasopressin (felpressin), ornithine vasopressin (orniprpressin), terlipressin (terlipressin)), vasopressin receptor agonists, atrial natriuretic peptide (atriuretic peptide, ANP), and C-type natriuretic peptide (C-type natriuretic, CNP) receptors (i.e., NPR1, NPR2, and NPR HS 32) antagonists (e.g., 39142-1, isatin (isantin), [ 7,23' ] b-28- (ANP) ], monoclonal anti-angiotensin G2, and Streptomyces peptide (Streptomyces 3), Streptomyces peptide (Streptomyces 3), and Streptomyces peptide (Streptomyces sp) Somatostatin type 2 receptor antagonists (e.g., somatostatin), pharmaceutically acceptable derivatives, and analogs, salts, hydrates, and solvates thereof. Examples of antispasmodics include, but are not limited to, myotonin (carisoprodol), benzodiazepines (benzodiazepines), baclofen (baclofen), cyclobenzaprine (cyclobenzapine), metaxalone (metaxalone), methocarbamol (clonidine), clonidine analogs, and dantrolene (dantrolene). Examples of PDE5 inhibitors include, but are not limited to, tadalafil (tadalafil), sildenafil (sildenafil), and vardenafil (vardenafil).

The pharmaceutical composition may be formulated for immediate release, extended release, delayed release, or a combination thereof.

In some embodiments, the pharmaceutical composition is formulated for immediate release.

In other embodiments, the pharmaceutical composition is formulated for extended release by embedding the active ingredient in a matrix of a non-soluble substance such as acrylic resins (acrylics) or chitin (chitin). The extended release form is designed to release the active ingredient at a predetermined rate by maintaining a constant drug level over a specified period of time. This can be achieved via a variety of formulations including, but not limited to, liposomes and drug-polymer conjugates, such as hydrogels (hydrogels).

The extended release formulation may be designed to release the active ingredient at a predetermined rate, for a specified duration of time, such as up to about 24 hours, about 22 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, or about 2 hours after administration or after a lag period associated with delayed release of the active ingredient. Constant active ingredient levels can be maintained by continuous release of the active ingredient or pulsed release of the active ingredient.

In certain embodiments, the active ingredient in the extended release formulation is released over a time interval of about 2 hours to about 12 hours. Alternatively, the active ingredient may be released within about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 hours, about 11 hours, or about 12 hours. In yet other embodiments, the active ingredient in the extended release formulation is released over a period of about 5 to about 8 hours after administration.

In some embodiments, the extended release formulation comprises an active core (active core) composed of one or more inert particles, each in the form of beads, pills, tablets, granules, microcapsules (microcapsules), microspheres (microspheres), microgranules (microspheres), nanocapsules (nanocapsules), or nanospheres (nanospheres), coated on its surface with a drug in the form of a drug-containing coating or film-forming composition using, for example, fluid bed techniques (fluidized bed techniques) or other methods known to those skilled in the art. The inert particles can be of various sizes as long as they are large enough to remain insoluble. Alternatively, the active core may be prepared by granulating (milling) and grinding and/or by extruding and spheronizing (spheronization) a polymer composition containing the drug substance. The term "drug" as used herein refers to the active ingredient of a pharmaceutical composition.

The active ingredient may be incorporated into the inert carrier by techniques known to those skilled in the art, including, for example, drug layering (drug layering), powder coating, extrusion/spheronization, roller compaction (roller compaction), or granulation. The amount of active ingredient in the core will depend on the dosage required and typically varies from about 1 to 100 wt%, about 5 to 100 wt%, about 10 to 100 wt%, about 20 to 100 wt%, about 30 to 100 wt%, about 40 to 100 wt%, about 50 to 100 wt%, about 60 to 100 wt%, about 70 to 100 wt%, or about 80 to 100 wt%.

Generally, depending on the desired lag time (lag time) and/or the polymer and coating solvent selected, the polymer coating on the active core is about 1 to 50% based on the weight of the coated particle. One skilled in the art will be able to select the appropriate amount of drug for coating onto or incorporation into the core to achieve the desired dosage. In one embodiment, the inactive core may be a sugar sphere or a buffer crystal or an encapsulated buffer crystal, such as calcium carbonate, sodium bicarbonate, fumaric acid, tartaric acid, etc., which alters the microenvironment of the drug to facilitate its release.

Extended release formulations may utilize various extended release coatings or mechanisms that facilitate the gradual release of the active agent over time. In some embodiments, the extended release agent comprises a polymer that controls release by dissolution controlled release. In one particular embodiment, the active agent is incorporated into a matrix comprising a non-soluble polymer and drug particles or particles coated with polymeric materials of varying thicknesses. The polymeric material may comprise a lipid barrier (lipid barrier) comprising waxy materials (such as carnauba wax, beeswax, spermaceti wax, candelilla wax, shellac wax, cocoa butter, cetostearyl alcohol, partially hydrogenated vegetable oils, ceresin, paraffin wax, ceresin, myristyl alcohol, stearyl alcohol, cetyl alcohol, stearic acid, and stearic acid) and surfactants such as polyoxyethylene sorbitan monooleate. Upon contact with an aqueous medium, such as a biological fluid, the polymer coating emulsifies or erodes after a predetermined lag time, depending on the thickness of the polymer coating. This lag time is independent of gastrointestinal motility (motility), pH or gastric retention (gastric retention).

In other embodiments, the extended release agent comprises a polymeric matrix that achieves diffusion-controlled release. The matrix may comprise one or more hydrophilic and/or water swellable matrix-forming polymers, pH dependent polymers and/or pH independent polymers.

In one embodiment, the extended release formulation comprises a water-soluble or water-swellable matrix-forming polymer, optionally with one or more dissolution enhancing agents and/or release promoting agents. As the water-soluble polymer dissolves, the active agent dissolves (if soluble) and gradually diffuses through the hydrated portion of the matrix. As more water penetrates into the core of the matrix, the gel layer grows with time, increasing the thickness of the gel layer and providing a diffusion barrier that retards drug release. When the outer layer is fully hydrated, the polymer chains relax completely and the integrity of the gel layer can no longer be maintained, resulting in disentanglement (discrete) and etching (erosion) of the outer hydrated polymer on the substrate surface. The water continues to penetrate through the gel layer towards the core until it has been completely etched. Although soluble drugs are released by a combination of diffusion and erosion mechanisms, erosion is the primary mechanism for insoluble drugs, independent of dose.

Similarly, water-swellable polymers typically hydrate and swell in biological fluids to form a homogeneous matrix structure that retains its shape during drug release and acts as a carrier for the drug, solubility-enhancing agent, and/or release-enhancing agent. The initial matrix polymer hydration phase results in a slow release of the drug (lag phase). Once the water-swellable polymer is fully hydrated and swollen, the water in the matrix can likewise dissolve the drug substance and allow it to diffuse out through the matrix coating.

In addition, the porosity (porosity) of the matrix may be increased by leaching (learningout) of the pH-dependent release promoter, thereby releasing the drug at a faster rate. The rate of drug release then becomes constant and is a function of drug diffusion through the hydrated polymer gel. The rate of release from the matrix depends on various factors including the type and level of polymer, the solubility and dosage of the drug, the ratio of polymer to drug, the type and level of filler, the ratio of polymer to filler, the particle size of drug to polymer, and the porosity and shape of the matrix.

Exemplary hydrophilic and/or water swellable matrix-forming polymers include, but are not limited to, cellulosic polymers, including hydroxyalkyl celluloses (hydroxyalkylcelluloses) and carboxyalkyl celluloses (carboxylkylcelluloses), such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), Methylcellulose (MC), carboxymethyl cellulose (CMC); powdered cellulose, such as microcrystalline cellulose (microcrystalline cellulose), cellulose acetate (cellulose acetate), ethyl cellulose (ethylcellulose), salts thereof, and combinations thereof; alginate (alginate); gums, including heterologous polysaccharide gums (heteropolysaccharide gums) and homologous polysaccharide gums (homopolysaccharide gums), such as xanthan gum (xanthohan), tragacanth gum (tragacanth), pectin, acacia gum (acacia), karaya gum (karaya), alginates, agar, guar gum (guar), hydroxypropyl guar (hydroxypropyl guar), veegum (veegum), carrageenan (carrageenans), locust bean gum (locustbean gum), gellan gum (gellan gum), and derivatives thereof; acrylic resin (acrylic resin) comprisingPolymers and copolymers of acrylic acid (acrylic acid), methacrylic acid (methacrylic acid), methyl acrylate (methyl acrylate), and methyl methacrylate (methyl methacrylate); and cross-linked polyacrylic acid (cross-linked polyacrylic acid) derivatives, such as carbomers (carbomers) (e.g.,including various molecular weight grades available from Noveon, Inc., Cincinnati, OH71G NF), carrageenan; polyvinyl acetate (polyvinyl acetate) (e.g.,SR); and polyvinylpyrrolidone (polyvinylpyrrolidones) and derivatives thereof, such as crospovidone (crospovidone), polyethylene oxide (polyethyleneoxide), and polyvinyl alcohol (polyvinyl alcohol). Preferred hydrophilic and water swellable polymers include cellulosic polymers, especially HPMC.

The extended release formulation may further comprise at least one binder capable of crosslinking the hydrophilic compound in an aqueous medium, including biological fluids, to form a hydrophilic polymer matrix (i.e., a gel matrix).

Exemplary binders include homologous polysaccharides, such as galactomannan gums

(galctomann gums), guar gum, hydroxypropyl cellulose (HPC; e.g. Klucel EXF) and locust bean gum. In other embodiments, the binder is an alginic acid (acid) derivative, HPC, or microcrystalline cellulose (MCC). Other binders include, but are not limited to, starch, microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.

In one embodiment, the method of incorporation is a drug layering process by spraying a suspension of the active agent and a binder onto an inert carrier.

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

In some embodiments, the hydrophilic polymer matrix may further comprise an ionic polymer, a non-ionic polymer, or a non-water soluble hydrophobic polymer to provide a stronger gel layer and/or to reduce the number and size of pores in the matrix, thereby slowing the diffusion and erosion rates of the active agent and the concomitant release of the active agent. This in addition may also suppress the initial burst effect and result in a more stable "zero order release" of the active agent.

Exemplary ionic polymers for slowing the rate of dissolution include anionic and cationic polymers. Exemplary anionic polymers include, for example, sodium carboxymethylcellulose (Na CMC); sodium alginate, acrylic acid or carbomer (e.g.,934. 940, 974P NF); enteric (enteric) polymers such as polyvinyl acetate phthalate (PVAP), methacrylic acid copolymers (e.g.,l100, L30D 55, a and FS 30D) and hypromellose acetate succinate (AQUAT HPMCAS); and xanthan gum. Exemplary cationic polymers include, for example, dimethylaminoethyl methacrylate copolymer (e.g.,e100) In that respect The introduction of anionic polymers (especially enteric polymers) helped to develop a pH-independent release profile of weakly basic drugs relative to hydrophilic polymers alone.

Exemplary nonionic polymers for slowing dissolution rates include, for example, hydroxypropyl cellulose (HPC) and polyethylene oxide (PEO) (e.g., POLYOX)^TM)。

Exemplary hydrophobic polymers include ethylcellulose (e.g., ETHOCEL)^TM、) Cellulose acetate, methacrylic acid copolymers (e.g.,NE30D), ammonium-methacrylate copolymers (e.g.,RL 100 or PO RS 100), polyvinyl acetate, glycerol monostearate (glyceryl monostearate), fatty acids such as acetyl tributyl citrate (acetyl tributylate), and combinations and derivatives thereof.

The swellable polymer may be incorporated in the formulation in a proportion of from 1% to 50% by weight, preferably from 5% to 40% by weight, optimally from 5% to 20% by weight. The swellable polymers and binders may be incorporated into the formulation before or after granulation. The polymer may also be dispersed in an organic solvent or hydro-alcohol and sprayed during granulation.

Exemplary release promoters include pH-dependent enteric polymers that remain intact at pH values below about 4.0 and dissolve at pH values above 4.0, preferably above 5.0, optimally about 6.0, and which are believed to contribute to the present invention as release promoters. Exemplary pH-dependent polymers include, but are not limited to, methacrylic acid copolymers; methacrylic acid-methyl methacrylate copolymer (e.g.,l100 (type A),S100 (type B), rowam GmbH, Germany (Rohm GmbH, Germany)), methacrylic acid-ethyl acrylate copolymer (methacrylic acid-ethyl acrylate copolymer) (for example,l100-55 (type C) andL30D-55 copolymer dispersion, Rohm GmbH, Germany); copolymer of methacrylic acid-methyl methacrylate and methyl methacrylate(s) (ii)FS); terpolymers of methacrylic acid, methacrylate ester and ethyl acrylate, Cellulose Acetate Phthalate (CAP); hydroxypropylmethylcellulose phthalate (HPMCP) (e.g., HP-55, HP-50, HP-55S, Japan shin-Etsu Chemical, Japan)); polyvinyl acetate phthalate (PVAP) (e.g.,enteric white OY-P-7171); polyvinyl butyrate acetate, Cellulose Acetate Succinate (CAS); hydroxypropyl methylcellulose acetate succinate (HPMCAS) (e.g., HPMCAS LF grade, MF grade, and HF grade, includingLF andMF, Shin-Etsu Chemical, Japan), shellac (shellac) (e.g., MARCOAT)^TM125 and MARCOAT^TM125N); vinyl acetate-maleic anhydride copolymer (vinyl acetate-maleic anhydride copolymer), styrene-maleic acid monoester copolymer (styrene-maleic acid ester copolymer), carboxymethyl ethyl cellulose (carboxy ethyl cellulose, CMEC, french Corporation); cellulose Acetate Phthalate (CAP) (e.g.,) Cellulose Acetate Trimellitate (CAT), and a weight ratio of two or more thereof in the range of about 2: 1 to about 5: 1, such as a weight ratio of about 3: 1 to about 2: 1 ofL100-55 anda mixture of S100, or a weight ratio of about 3: 1 to about 5: 1 ofL30D-55 and(iii) a mixture of FS.

These polymers may be used alone or in combination, or together with polymers other than the above-mentioned polymers. The preferred enteric pH-dependent polymer is a pharmaceutically acceptable methacrylic acid copolymer. These copolymers are anionic polymers based on methacrylic acid and methyl methacrylate and preferably have an average molecular weight of about 50,000 to 200,000, preferably about 135,000. The ratio of free carboxyl groups to methyl esterified carboxyl groups in the copolymer can be, for example, in the range of 1: 1 to 1: 3, for example about 1: 1 or 1: 2. the release promoter is not limited to pH-dependent polymers. Other hydrophilic molecules that dissolve rapidly and leach out of the dosage form quickly leaving a porous structure may also serve the same purpose.

In some embodiments, the matrix may include a combination of a release enhancer and a solubility enhancer. Solubility enhancers can be ionic and non-ionic surfactants, complexing agents, hydrophilic polymers, and pH modifiers (such as acidifying and basifying agents) as well as molecules that increase the solubility of poorly soluble drugs via molecular entrapment (entralpriment). Several solubility enhancers may be used simultaneously.

Solubility enhancers may include surfactants such as sodium docusate (sodium docusate); sodium lauryl sulfate (sodium lauryl sulfate); sodium stearyl fumarate (sodium stearyl fumarate);and Spans (PEO modified sorbitan monoesters (sorbitan monoester) and sorbitan fatty acid esters (fattyaced sorbitan ester)); poly (ethylene oxide) -polyoxypropylene-poly (ethylene oxide) block copolymer (also known as PLURONICS)^TM) (ii) a Complexing agents, such as low molecular weight polyvinylpyrrolidone and low molecular weight hydroxypropyl methylcellulose; molecules that aid solubility via molecular entrapment, such as cyclodextrins; and pH modifiers including acidifying agents (such as citric acid, fumaric acid, tartaric acid, and hydrochloric acid) and alkalinizing agents (such as meglumine (meglumine) and sodium hydroxide).

Solubility enhancers typically comprise 1% to 80% by weight, 1% to 60% by weight, 1% to 50% by weight, 1% to 40% by weight, and 1% to 30% by weight of the dosage form, and may be incorporated in various ways. They may be incorporated into the formulation in dry or wet form prior to granulation. It may also be added to the formulation after the remaining materials have been granulated or otherwise processed. During granulation, the solubility enhancing agent may be sprayed in the form of a solution with or without a binder.

In one embodiment, the extended release formulation comprises a water-insoluble water-permeable polymer coating or matrix formed on an active core comprising one or more water-insoluble water-permeable film-forming polymers. The coating may additionally comprise one or more water-soluble polymers and/or one or more plasticizers (plastisizers). The water-insoluble polymer coating comprises a barrier coating for the release of the active agent in the core, wherein the lower molecular weight (viscosity) grade exhibits a faster release rate compared to the higher viscosity grade.

In some embodiments, the water-insoluble film-forming polymer includes one or more alkyl cellulose ethers, such as ethyl cellulose and mixtures thereof (e.g., ethyl cellulose grades PR100, PR45, PR20, PR10, and PR 7;Dow)。

in some embodiments, the water-insoluble polymer provides suitable properties (e.g., extended release characteristics, mechanical properties, and coating properties) without the need for a plasticizer. For example, coatings comprising polyvinyl acetate (PVA), neutral copolymers of acrylate/methacrylate (such as Eudragit NE30D available from the winning industrial group (Evonik Industries)), combinations of ethylcellulose and hydroxypropylcellulose, waxes, and the like, can be applied without plasticizers.

In yet another embodiment, the water insoluble polymer matrix may further comprise a plasticizer. The amount of plasticizer required depends on the plasticizer, the nature of the water insoluble polymer and the final desired properties of the coating. Suitable levels of plasticizer are in the range of about 1% to about 20%, about 3% to about 5%, about 7% to about 10%, about 12% to about 15%, about 17% to about 20% by weight, or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20% by weight, relative to the total weight of the coating, including all ranges and subranges therebetween.

Exemplary plasticizers include, but are not limited to, triacetin (triacetin), acetylated monoglycerides (acetylated monoglycerides), oils (castor oil, hydrogenated castor oil, grape seed oil, sesame oil, olive oil, etc.), citric acid esters, triethyl citrateEsters, acetyl triethyl citrate (acetyltriethyl citrate), acetyl tributyl citrate, acetyl tri-n-butyl citrate, diethyl phthalate, dibutyl phthalate, dioctyl phthalate, methyl paraben, propyl paraben, butyl paraben, diethyl sebacate, dibutyl sebacate, glyceryl tributyrate (glycerol tributyrate), substituted triglycerides and glycerides, monoacylated (monoacylated), and diacetylated (diacetylated) glycerides (e.g.,9-45), glyceryl monostearate, glyceryl tributyrate, polysorbate 80, polyethylene glycols (such as PEG-4000 and PEG-400), propylene glycol, 1, 2-propylene glycol, glycerin, sorbitol (sorbitol), diethyl oxalate, diethyl malate, diethyl fumarate (diethyl fumarate), diethyl malonate, dibutyl succinate, fatty acids, diethyl maleate, diethyl succinate, dioctyl phthalate, dibutyl sebacate, and mixtures thereof. The plasticizer may have surfactant properties such that it can act as a release modifier. For example, nonionic detergents such as Brij 58 (polyoxyethylene (20) cetyl ether) and the like may be used.

Plasticizers can be high boiling point organic solvents used to impart flexibility to rigid or brittle (brittle) polymeric materials, and can affect the release profile of the active agent. Plasticizers generally result in a decrease in intermolecular cohesion along the polymer chain, thereby causing various changes in the polymer properties. Such changes include, but are not limited to, a decrease in tensile strength (tensilestrength) with an increase in elongation and a decrease in the glass transition temperature (trnasition) or softening temperature of the polymer. The amount and choice of plasticizer can influence, for example, the hardness of the tablet and can even influence its dissolution or disintegration (disintegration) characteristics and its physical and chemical stability. Certain plasticizers can increase the elasticity and/or flexibility (pliability) of the coating, thereby reducing the brittleness of the coating.

In another embodiment, the extended release formulation comprises a combination of at least two gel forming polymers, including at least one non-ionic gel forming polymer and/or at least one anionic gel forming polymer. The gel formed by the combination of gel-forming polymers provides controlled release such that when the formulation is ingested and contacted with gastrointestinal fluids, the polymers closest to the surface hydrate to form a viscous gel layer. Due to the high viscosity, the viscous layer only gradually dissolves away in the same process, exposing the underlying material. The mass thus dissolves away slowly, thereby slowly releasing the active ingredient into the gastrointestinal fluids. The combination of at least two gel forming polymers allows the properties, such as viscosity, of the resulting gel to be manipulated to provide a desired release profile.

In a particular embodiment, the formulation comprises at least one non-ionic gel-forming polymer and at least one anionic gel-forming polymer. In another embodiment, the formulation comprises two different non-ionic gel-forming polymers. In yet another embodiment, the formulation comprises a combination of non-ionic gel-forming polymers (e.g., a combination of hydroxypropyl methylcellulose with different viscosity grades, such as HPMC K100 with HPMC K15M or HPMC K100M) having the same chemistry, but different solubilities, viscosities, and/or molecular weights.

Exemplary anionic gel-forming polymers include, but are not limited to, sodium carboxymethylcellulose (Na CMC), carboxymethylcellulose (CMC), anionic polysaccharides such as sodium alginate, alginic acid, pectin, polyglucuronic acid (polyglucuronic acid) (poly- α -and- β -1,4-glucuronic acid (poly- α -and- β -1,4-glucuronic acid)), polygalacturonic acid (polygalacturonic acid) (pectic acid), chondroitin sulfate, carrageenan (carrageenans), furcellaran (furcellaran), anionic gums (such as xanthan gum (xanthan gum)), acrylic acid, or carbomer (carbomer), (CMC), and (I, N934. 940, 974P NF) polymer,A copolymer,Polymers, polycarbophil (polycarbophil), and others.

Exemplary nonionic gel-forming polymers include, but are not limited to, Povidone (PVP: polyvinylpyrrolidone), polyvinyl alcohol, copolymers of PVP and polyvinyl acetate, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose, hydroxymethyl cellulose, gelatin, polyethylene oxide, gum arabic, dextrin, starch, Polyhydroxyethylmethacrylate (PHEMA), water-soluble nonionic polymethacrylates (polymethacrylates) and copolymers thereof, modified cellulose, modified polysaccharides, nonionic gums, nonionic polysaccharides, and/or mixtures thereof.

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

Exemplary fillers include, but are not limited to, lactose, glucose, fructose, sucrose, dibasic calcium phosphate, sugar alcohols (also known as "sugar polyols") (such as sorbitol, mannitol, lactitol (lactitol), xylitol, isomalt (isomalt), erythritol, and hydrogenated starch hydrolysates (blends of several sugar alcohols)), corn starch, potato starch, sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate, enteric polymers, or mixtures thereof.

Exemplary binders include, but are not limited to, water-soluble hydrophilic polymers such as povidone (PVP: polyvinylpyrrolidone), copovidone (copolymer of polyvinylpyrrolidone and polyvinyl acetate), low molecular weight hydroxypropyl cellulose (HPC), low molecular weight hydroxypropyl methylcellulose (HPMC), low molecular weight carboxymethyl cellulose, ethyl cellulose, gelatin, polyethylene oxide, acacia, dextrin, magnesium aluminum silicate (maganesium silicate), and starch, and polymethacrylates such as Eudragit NE30D, Eudragit RL, Eudragit RS, Eudragit E, polyvinyl acetate, enteric polymers, or mixtures thereof.

Exemplary disintegrants include, but are not limited to, low substituted sodium carboxymethylcellulose, crospovidone (crospovidone), sodium carboxymethyl starch (sodium carboxymethyl starch) (sodium starch glycolate), Croscarmellose sodium (Croscarmellose), pregelatinized starch (starch 1500), microcrystalline cellulose, non-water soluble starch, calcium carboxymethylcellulose, low substituted hydroxypropyl cellulose, and magnesium or aluminum silicate.

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

In yet another embodiment, the extended release formulation is formed by coating water-soluble/dispersible drug-containing particles, such as beads or bead populations (as described above) therein, with a coating material and optionally a pore former and other excipients. The coating material is preferably selected from the group comprising: cellulosic polymers, such as ethyl cellulose (e.g.,) Methyl cellulose, hydroxypropyl methyl cellulose, cellulose acetate and cellulose acetate phthalate; polyvinyl alcohol; acrylic polymers such as polyacrylates, polymethacrylates, and copolymers thereof; and other water-based or solvent-based coating materials. The controlled release coating for a given bead population may be controlled by at least one parameter of the controlled release coating, such as the nature of the coating, the level of coating, the type and concentration of pore formers, processing parameters, and combinations thereof. Thus, varying parameters (such as pore former concentration or curing conditions) results in a change in the release of active agent from any given bead population, thereby selectively adjusting the formulation to a predetermined release profile.

Pore formers suitable for use in the controlled release coatings herein can be organic or inorganic and include materials that are soluble, extractable or leachable from the coating in the environment of use. Exemplary pore formers include, but are not limited to, organic compounds such as monosaccharides, oligosaccharides, and polysaccharides (including sucrose, glucose, fructose, mannitol, mannose, galactose, sorbitol, pullulan (pullulan), and polyglucose (dextran)); polymers that are soluble in the environment of use, such as water-soluble hydrophilic polymers, hydroxyalkyl celluloses, carboxyalkyl celluloses, hydroxypropyl methyl celluloses, cellulose ethers, acrylic resins, polyvinyl pyrrolidones, cross-linked polyvinyl pyrrolidones, polyethylene oxides, carbomer waxes (carbopax), carbopols (Carbopol) and their analogs, glycols, polyols, polyhydroxyl alcohols (polyhydroxy alcohols), polyalkylene glycols (polyalkylene glycols), polyethylene glycols (polyethylene glycols), polypropylene glycols or block polymers thereof, polyglycols (polyglycols) and poly (alpha-omega) alkylene glycols (poly (alpha-omega) alkylene glycols); and inorganic compounds such as alkali metal salts, lithium carbonate, sodium chloride, sodium bromide, potassium chloride, potassium sulfate, potassium phosphate, sodium acetate, sodium citrate, suitable calcium salts, combinations thereof, and the like.

The controlled release coating may further comprise other additives known in the art, such as plasticizers, anti-tack agents, glidants (or flow aids), and anti-foaming agents. In some embodiments, the coated particles or beads may additionally include an "overcoat" to provide, for example, moisture protection, static charge reduction, taste masking, flavoring, coloring, and/or polishing or other enhancements to the beads. Suitable coating materials for such an outer coating are known in the art and include, but are not limited to, cellulosic polymers such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, and microcrystalline cellulose or combinations thereof (e.g., variousA coating material).

The coated particles or beads may additionally contain an enhancer, which may be exemplified by, but not limited to, solubility enhancers, absorption enhancers, permeability enhancers, stabilizers, complexing agents, enzyme inhibitors, p-glycoprotein inhibitors, and multidrug resistance protein inhibitors. Alternatively, the formulation may also contain an enhancer separate from the coated particles, for example in a separate bead population or in powder form. In yet another embodiment, an enhancer may be contained in a separate layer on the coated particles below or above the controlled release coating.

In other embodiments, the extended release formulation is formulated to release the active agent by an osmotic mechanism. For example, the capsule may be formulated to have a single osmotic unit or it may incorporate 2,3, 4, 5 or 6 push-pull units (push-pull units) encapsulated in a hard gelatin capsule, whereby each dual layer push-pull unit contains an osmotic push layer (osmotic push layer) and a drug layer (drug layer), both surrounded by a semipermeable membrane. One or more holes are drilled into the membrane adjacent to the drug layer. Such a membrane may additionally be covered with a pH-dependent enteric coating, so that release does not take place until after gastric emptying. The gelatin capsule dissolves immediately upon ingestion. Upon entry of the push-pull unit into the small intestine, the enteric coating disintegrates and fluid can subsequently be forced out through the semipermeable membrane, swelling the osmotic push compartment to drive the drug out through the small orifice at a precisely controlled rate of water transport through the semipermeable membrane. The release of the drug may be at a constant rate for up to 24 hours or more.

The osmotic push layer includes one or more osmotic agents, thereby creating a driving force for water transport through the semipermeable membrane into the core of the delivery vehicle (delivery vehicle). One class of osmotic agents includes water-swellable hydrophilic polymers, also known as "osmopolymers" and "hydrogels," which include, but are not limited to, hydrophilic ethylene and acrylic acid polymers, polysaccharides (e.g., calcium alginate), polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly (2-hydroxyethyl methacrylate), poly (propylene) acid, poly (methacrylic) acid, polyvinylpyrrolidone (PVP), crosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers containing hydrophobic monomers such as methyl methacrylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, crosslinked sodium carboxymethylcellulose, carrageenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), polyvinyl acetate (PEO), polyvinyl acetate (PVP) and/PVA copolymers (PVA and/PVP) polymers, Carboxymethyl cellulose (CMC) and carboxyethyl cellulose (CEC), sodium alginate, polycarbophil, gelatin, xanthan gum and sodium starch glycolate.

Another class of osmotic agents includes osmogens (osmogens) that are capable of absorbing water to affect the osmotic pressure gradient across a semi-permeable membrane. Exemplary osmogens include, but are not limited to, inorganic salts such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, potassium phosphate, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, and sodium sulfate; sugars such as dextrose (dextrose), fructose, glucose, inositol (inositol), lactose, maltose, mannitol, raffinose (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, tetraacetic acid (ethylenediamine acid), glutamic acid, p-toluenesulfonic acid, succinic acid, and tartaric acid; urea; and mixtures thereof.

Materials that may be used to form the semipermeable membrane include various grades of acrylic, vinyl, ether, polyamide, polyester, and cellulose derivatives that are water permeable and water insoluble at physiologically relevant pH or are readily rendered water insoluble by chemical changes (e.g., crosslinking).

In some embodiments, the extended release formulation comprises a polysaccharide coating that is erosion resistant in both the stomach and intestine. The polymer is only degradable in the colon, which contains a large microbiota containing biodegradable enzymes degrading e.g. polysaccharide coatings to release the drug content in a controlled time dependent manner. Exemplary polysaccharide coatings may include, for example, amylose, arabinogalactan (arabinogalactan), chitosan (chitosan), chondroitin sulfate, cyclodextrin, polyglucose, guar gum, pectin, wood gum (xylan), and combinations or derivatives thereof.

In some embodiments, the pharmaceutical composition is formulated for delayed-release or delayed-extended-release. In some embodiments, the delayed extended release formulation comprises an extended release formulation coated with an enteric coating, which is a barrier coated on the oral drug to prevent release of the drug prior to reaching the small intestine. Delayed release formulations (e.g., enteric coatings) prevent dissolution in the stomach of drugs that have a gastric irritating effect (e.g., aspirin). The term "enteric coating" as used herein comprises a coating of one or more polymers having a pH-dependent or pH-independent release profile. Enteric coated pellets do not dissolve in the acidic liquid of the stomach (pH about 3), but they will dissolve in the alkaline (pH 7 to 9) environment of the small intestine or colon. Enteric polymer coatings generally resist release of the active agent until a period of gastric emptying lag of about 3 to 4 hours after administration.

These coatings also serve to protect the acid labile drug from exposure to gastric acid, but rather deliver it to an alkaline pH environment (pH 5.5 and above of the intestine) where it does not degrade and produce its desired effect. The term "pulsatile-release" is a delayed release, which as used herein refers to a drug formulation that provides a rapid and short-lived release of the drug within a short period of time, immediately after a predetermined lag period, thereby producing a "pulsatile" plasma distribution of the drug after administration of the drug. The formulation may be designed to provide a single burst or multiple bursts at predetermined time intervals after administration, or a burst (e.g., 20% to 60% of the active ingredient) followed by an extended release (e.g., continuous release of the remaining active ingredient) for a period of time. Delayed release or pulsatile release formulations typically comprise one or more ingredients coated with a barrier coating that dissolves, erodes or ruptures after a specified lag time.

Barrier coatings for delayed release can be composed of a variety of different materials depending on the target. In addition, the formulation may include multiple barrier coatings to facilitate release in a transient manner. The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycol, and/or polyvinylpyrrolidone) or a coating based on methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac (shellac), and/or ethylcellulose. Furthermore, the formulation may additionally include a time delay material, such as glyceryl monostearate or glyceryl distearate.

In some embodiments, the delayed-extended release formulation includes an enteric coating comprising one or more polymers that facilitate release of the active agent in the proximal or distal regions of the gastrointestinal tract. The pH-dependent enteric coating comprises one or more pH-dependent or pH-sensitive polymers that maintain their structural integrity at low pH (e.g., in the stomach) but dissolve and release the drug contents in the higher pH environment of the more remote regions of the gastrointestinal tract (e.g., the small intestine). For the purposes of the present invention, "pH dependent" is defined as having a characteristic that varies (e.g., dissolves) according to the pH of the environment. Exemplary pH-dependent polymers have been described above. The pH-dependent polymer generally exhibits a characteristic pH optimum for dissolution. In some embodiments, the pH-dependent polymer exhibits a pH optimum between about 5.0 and 5.5, between about 5.5 and 6.0, between about 6.0 and 6.5, or between about 6.5 and 7.0. In other embodiments, the pH-dependent polymer exhibits a pH optimum of ≧ 5.0, ≧ 5.5, ≧ 6.0, ≧ 6.5 or ≧ 7.0.

In certain embodiments, the coating process employs blending of one or more pH-dependent polymers with one or more pH-independent polymers. Blending of the pH-dependent polymer with the non-pH-dependent polymer can reduce the release rate of the active ingredient when the soluble polymer reaches its optimal pH for dissolution.

In some embodiments, a profile of "delayed release" or "delayed-extended release" may be obtained using a water-insoluble capsule containing one or more active agents, wherein one end of the capsule is closed with an insoluble, but permeable and swellable hydrogel plug (plug). Upon contact with gastrointestinal fluids or dissolution media, the plug swells, pushes itself out of the capsule and releases the drug after a predetermined lag time, which may be controlled by, for example, the position and size of the plug. The capsule body may be further coated with an external pH-dependent enteric coating to keep the capsule intact until it reaches the small intestine. Suitable plug materials include, for example, polymethacrylates, erodible compressed polymers (e.g., HPMC, polyvinyl alcohol), coagulated (gelled) molten polymers (e.g., glycerol monooleate), and enzymatically controlled erodible polymers (e.g., polysaccharides such as amylose, arabinogalactan, chitosan, chondroitin sulfate, cyclodextrin, polydextrose, guar gum, pectin, and xylan gum).

In other embodiments, the capsule or bilayer tablet may be formulated to contain a drug-containing core (drug-containing core) surrounded by a swelling layer and an outer insoluble, but semipermeable, polymeric coating or membrane. The lag time before rupture can be controlled by the permeability and mechanical properties of the polymer coating and the swelling behavior of the swollen layer. Typically, the swelling layer comprises one or more swelling agents, such as a swellable hydrophilic polymer that swells and retains water in its structure.

Exemplary water-swellable materials for use in the delayed release coating include, but are not limited to, polyethylene oxide (having an average molecular weight of, for example, between 1,000,000 and 7,000,000, such as) (ii) a Methyl cellulose; hydroxypropyl cellulose; hydroxypropyl methylcellulose; polyalkylene oxides having a weight average molecular weight of 100,000 to 6,000,000, including, but not limited to, poly (methylene oxide), poly (butylene oxide), poly (hydroxyalkyl methacrylate) having a molecular weight of 25,000 to 5,000,000, poly (hydroxyalkyl methacrylate) having a molecular weight of 100,000 to 6,000,000Poly (vinyl) alcohol having low acetal residues (low) and crosslinked with glyoxal, formaldehyde or glutaraldehyde and having a degree of polymerization of between 200 and 30,000; a mixture of methylcellulose, cross-linked agar and carboxymethylcellulose; hydrogel-forming copolymers produced by forming a dispersion of a finely divided copolymer of maleic anhydride (maleic anhydride) and styrene, ethylene, propylene, butylene or isobutylene, crosslinked with 0.001 to 0.5 moles of a saturated crosslinking agent per mole of maleic anhydride in the copolymer; having a molecular weight of 450,000 to 4,000,000An acidic carboxyl polymer;polyacrylamide; cross-linked water-swellable indene maleic anhydride (indenemaleicahydride) polymers; having a molecular weight of 80,000 to 200,000Polyacrylic acid; starch graft copolymers (starch graft copolymers); consisting of condensed glucose unitsAcrylate polymer polysaccharides such as diester cross-linked polydextrose (ester cross-linked polyglucan); carbomer in the form of an aqueous solution having a mass to volume (w/v) ratio of from 0.5% to 1% and a viscosity of from 3,000 to 60,000 millipascal-seconds (mPa's); cellulose ethers, such as hydroxypropyl cellulose, having a viscosity of about 1000 to 7000 mpa.s, and being in the form of a 1% weight/weight aqueous solution (25 ℃); hydroxypropyl methylcellulose having a viscosity of about 1000 or more, preferably 2,500 or more, to a maximum of 25,000 mpa-sec, in the form of a 2% w/v aqueous solution; polyvinylpyrrolidone having a viscosity of about 300 to 700 mpa · s at 20 ℃ and being in the form of a 10% w/v aqueous solution; and combinations thereof.

Alternatively, the release time of the drug may be controlled by a disintegration lag time (disintegration lag time), which depends on the balance between the tolerance and thickness of a water-insoluble polymer film (e.g., ethylcellulose, EC) containing predetermined micropores at the bottom of the body and the amount of swelling excipients (e.g., low-substituted hydroxypropylcellulose (L-HPC) and sodium glycolate). After oral administration, GI fluid permeates through the micropores causing swelling of the swelling excipient, which creates an internal pressure that disengages a capsule assembly comprising a first capsule body comprising the swelling material, a second capsule body comprising the drug, and an outer cover attached to the first capsule body.

The delayed release coating may further comprise an anti-tack agent, such as talc and glyceryl monostearate. The delayed release coating may further comprise one or more plasticizers, including, but not limited to, triethyl citrate, acetyl tributyl citrate, polyethylene glycol acetylated monoglycerides (polyethylene glycol acetylated monoglycerides), glycerol, triacetin, propylene glycol, phthalates (e.g., diethyl phthalate, dibutyl phthalate), titanium dioxide, iron oxide, castor oil, sorbitol, and dibutyl sebacate.

In another embodiment, the delayed release formulation employs a water permeable but non-soluble membrane coating to encapsulate the active ingredient and osmotic agent. When water from the intestinal tract slowly diffuses through the membrane into the core, the core swells until the membrane bursts, thereby releasing the active ingredient. The membrane coating can be tailored to achieve various water permeation rates or release times.

In another individual embodiment, the delayed release formulation employs a water impermeable tablet coating whereby water undergoes controlled pore passage into the coating until the core bursts. When the tablet bursts, the drug content is released immediately or over a longer period of time. This and other techniques may be modified to allow for a predetermined lag time before drug release begins.

In another embodiment, the active agent is delivered in a formulation to provide delayed release and extended release (delayed-extended release). The term "delayed-extended release" as used herein relates to pharmaceutical formulations providing a pulsatile release of an active agent at a predetermined time or lag phase after administration followed by an extended release of the active agent.

In some embodiments, an immediate release, extended release, delayed release, or delayed-extended release formulation comprises an active core composed of one or more inert particles, each in the form of a bead, pill, tablet, granule, microcapsule, microsphere, microparticle, nanocapsule, or nanosphere, the surface of which is coated with a drug, for example, in the form of a drug-containing film-forming composition (drug-containing film-forming composition) using, for example, fluid bed techniques or other methods known to those skilled in the art. The inert particles can be of various sizes as long as they are large enough to remain insoluble. Alternatively, the active core may be prepared by granulating and milling and/or by extruding and spheronizing a polymer composition containing the drug substance.

The amount of drug in the core will depend on the dosage required and typically ranges from about 1 to 100 wt.%, about 5 to 100 wt.%, about 10 to 100 wt.%, about 20 to 100 wt.%, about 30 to 100 wt.%, about 40 to 100 wt.%, about 50 to 100 wt.%, about 60 to 100 wt.%, about 70 to 100 wt.%, or about 80 to 100 wt.%. Generally, depending on the lag time and type of release profile desired and/or the polymer and coating solvent selected, the polymer coating on the active core will be about 1 to 50% based on the weight of the coated particle. One skilled in the art will be able to select the appropriate amount of drug for coating onto or incorporation into the core to achieve the desired dosage. In one embodiment, the inactive core may be a sugar sphere or a buffer crystal or an encapsulated buffer crystal, such as calcium carbonate, sodium bicarbonate, fumaric acid, tartaric acid, etc., which alters the microenvironment of the drug to facilitate its release.

In some embodiments, for example, delayed-release or delayed-extended release compositions may be formed by coating water-soluble/dispersible drug-containing particles (e.g., beads) with a mixture of water-insoluble polymers and enteric polymers, wherein the water-insoluble polymers and enteric polymers may be present in a ratio of 4: 1 to 1: 1, and the total weight of the coating is from 10 to 60 wt% based on the total weight of the coated beads. The drug laminated beads may optionally include an ethylcellulose membrane that controls the internal dissolution rate. The composition of the outer layer of the polymeric membrane and the respective weights of the inner and outer layers are optimized and predicted based on in vitro/in vivo correlations to achieve a desired circadian rhythm (circadian rhythm) release profile for a given activity.

In other embodiments, the formulation may comprise a mixture of immediate release drug-containing particles (without a dissolution rate controlling polymer film) and delayed-extended release beads that exhibit a lag time of 2 to 4 hours, e.g., after oral administration, thereby providing a two-pulse release profile.

In some embodiments, the active core is coated with one or more layers of controlled dissolution rate polymers to achieve a desired release profile with or without lag time. The inner membrane provides a large degree of control over the rate of release of the drug after water or body fluid has been imbibed into the core, while the outer membrane provides the desired lag time (the period of no or minimal drug release after water or body fluid has been imbibed into the core). The inner film may comprise a water insoluble polymer or a mixture of water insoluble and water soluble polymers.

Polymers suitable for the outer film that largely control the lag time of up to 6 hours may comprise an enteric polymer as described above and 10 to 50 wt% of a water insoluble polymer. The ratio of water insoluble polymer to enteric polymer may be in the range of 4: 1 to 1: 2, these polymers are preferably present in a ratio of about 1: a ratio of 1 exists. The water-insoluble polymer generally used is ethyl cellulose.

Exemplary water insoluble polymers include ethyl cellulose, polyvinyl acetate (kollicoat sr #0D from BASF), neutral copolymers based on ethyl acrylate and methyl methacrylate, with quaternary ammoniumCopolymers of acrylic and methacrylic esters of radicals, e.g.NE, RS and RS30D, RL or RL30D, and the like. Exemplary water-soluble polymers include low molecular weight HPMC, HPC, methylcellulose, polyethylene glycol (molecular weight)>3000 PEG) in a thickness ranging from 1% up to 10% by weight, depending on the solubility of the active agent in water and the solvent or latex suspension based coating formulation used. The ratio of water insoluble polymer to water soluble polymer may generally be in the range of 95: 5 to 60: 40. preferably 80: 20 to 65: 35, may be varied within the range of 35. In some embodiments, AMBERLITE is used^TMIRP69 resin served as an extended release carrier. AMBERLITE^TMIRP69 is an insoluble, strongly acidic, sodium-form cation exchange resin suitable as a carrier for cationic (basic) substances. In other embodiments, DUOLITE is used^TMAP143/1093 resin as an extended release carrier. DUOLITE^TMAP143/1093 is an insoluble, strongly basic, anion exchange resin that is suitable as a carrier for anionic (acidic) materials. When used as a pharmaceutical carrier, AMBERLITE^TMIRP69 or/and DUOLITE^TMThe AP143/1093 resin provides a means for binding the agent to the insoluble polymeric matrix. Extended release is achieved via the formation of resin-drug complexes (drug resinates). When the drug reaches equilibrium with high electrolyte concentrations, which are typical of the gastrointestinal tract, the drug is released from the resin in vivo. More hydrophobic drugs will generally dissolve away from the resin at a lower rate due to hydrophobic interactions with the aromatic structure of the cation exchange system.

In some embodiments, the pharmaceutical composition is formulated for oral administration. Oral dosage forms include, for example, tablets, capsules, and caplets and may also comprise a plurality of particles, beads, powders, or pills, which may or may not be encapsulated. Tablets and capsules represent the most convenient oral dosage forms, in which case solid pharmaceutical carriers are employed.

In delayed release formulations, the pill, tablet or capsule may be coated with one or more barrier coatings to promote slow dissolution of the drug while releasing it into the intestinal tract. The barrier coating typically comprises one or more polymers that surround, enclose, or form a layer or film around the therapeutic composition or active core. In some embodiments, the active agent is delivered in a formulation to provide a 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 more.

Various coating techniques can be applied to the active agent-containing particles, beads, powders or pills, tablets, capsules, or combinations thereof to produce different and unique release profiles. In some embodiments, the pharmaceutical composition is in the form of a tablet or capsule containing a single layer coating. In other embodiments, the pharmaceutical composition is in the form of a tablet or capsule containing a multilayer coating. In some embodiments, the pharmaceutical compositions of the present invention are formulated for extended release or delayed extended release of 100% of the active ingredient.

In other embodiments, the pharmaceutical compositions of the invention are formulated for dual-stage extended-release (two-phase extended-release) or delayed dual-stage extended-release (delayed two-phase extended-release), characterized in that the "immediate-release" component is released within two hours of administration and the "extended-release" component is released within a period of 2 to 12 hours. In some embodiments, the "immediate release" component provides about 1% to 90% of the total dose of active agent to be delivered from the pharmaceutical formulation, while the "extended release" component provides 10% to 99% of the total dose of active agent to be delivered from the pharmaceutical formulation. For example, the immediate release component may provide about 10% to 90%, or about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the total dose of active agent to be delivered by the pharmaceutical formulation. The extended release component provides about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the total dose of active agent to be delivered by the formulation. In some embodiments, the immediate release component and the extended release component contain the same active ingredient. In other embodiments, the immediate release component and the extended release component contain different active ingredients (e.g., one PG pathway inhibitor in one component and another PG pathway inhibitor in another component). In some embodiments, the immediate release component and the extended release component each comprise a PG pathway inhibitor and an analgesic agent selected from the group consisting of aspirin, ibuprofen, naproxen sodium, indomethacin, nabumetone, and acetaminophen. In other embodiments, the immediate release component and/or the extended release component further comprise one or more additional active agents selected from the group consisting of antimuscarinic agents, antidiuretic agents, spasmolytics, phosphodiesterase (PDE 5) type inhibitors, and zolpidem.

In some embodiments, the pharmaceutical composition comprises a plurality of active ingredients selected from the group consisting of PG pathway inhibitors, analgesics, antimuscarinic agents, antidiuretic agents, spasmolytics, PDE5 inhibitors, and zolpidem. Examples of antimuscarinic agents include, but are not limited to oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine, trospium, atropine, and tricyclic antidepressants. Examples of antidiuretic agents include, but are not limited to, antidiuretic hormone (ADH), vasopressin II, aldosterone, vasopressin analogs (e.g., desmopressin, arginine vasopressin, lysine vasopressin, benzene lysine vasopressin, ornithine vasopressin, terlipressin), vasopressin receptor agonists, atrial Natriuretic Peptide (ANP) and C-type natriuretic peptide (CNP) receptors (i.e., NPR1, NPR2, and NPR3) antagonists (e.g., HS-142-1, isatin, [ Asu7,23' ] b-ANP- (7-28) ], ansamitene (a cyclic peptide from Streptomyces coelicolor), and 3G12 monoclonal antibody), somatostatin type-2 receptor antagonists (e.g., somatostatin), pharmaceutically acceptable derivatives, and analogs, salts, hydrates, and solvates thereof. Examples of antispasmodics include, but are not limited to, myotonin, benzodiazepines, baclofen, cyclobenzaprine, metaxalone, methocarbamol, clonine analogs, and dantrolene. Examples of PDE5 inhibitors include, but are not limited to, tadalafil, sildenafil, and vardenafil.

In some embodiments, the pharmaceutical composition comprises a plurality of active ingredients comprising (1) one or more PG pathway inhibitors and (2) one or more other active ingredients selected from the group consisting of analgesics, antimuscarinic agents, antidiuretic agents, spasmolytics, PDE5 inhibitors, and zolpidem. In some embodiments, the plurality of active ingredients are formulated for immediate release. In other embodiments, the plurality of active ingredients are formulated for extended release. In other embodiments, the plurality of active ingredients are formulated for delayed release. In other embodiments, the plurality of active ingredients are formulated for immediate release and extended release (e.g., a first portion of each active ingredient is formulated for immediate release and a second portion of each active ingredient is formulated for extended release). In still other embodiments, some of the active ingredients are formulated for immediate-release and some of the active ingredients are formulated for extended-release (e.g., active ingredient A, B, C is formulated for immediate-release and active ingredients C and D are formulated for extended-release). In some other embodiments, the plurality of active ingredients are formulated for delayed-extended release.

In certain embodiments, the pharmaceutical composition comprises an immediate release component and an extended release component. The immediate release component may comprise one or more active ingredients selected from the group consisting of PG pathway inhibitors, analgesics, antimuscarinic agents, antidiuretic agents, spasmolytics, PDE5 inhibitors, and zolpidem. The extended release component may comprise one or more active ingredients selected from the group consisting of PG pathway inhibitors, analgesics, antimuscarinic agents, antidiuretic agents, spasmolytics, PDE5 inhibitors, and zolpidem. In some embodiments, the immediate release component and the extended release component have exactly the same active ingredient. In other embodiments, the immediate release component and the extended release component have different active ingredients. In still other embodiments, the immediate release component and the extended release component have one or more active ingredients in common. In some other embodiments, the immediate release component and/or the extended release component are further coated with a delayed release coating (e.g., an enteric coating). In other embodiments, the pharmaceutical composition comprises two or more active ingredients formulated as two extended-release components, each providing a different extended-release profile. For example, the first extended-release component releases the first active ingredient at a first release rate and the second extended-release component releases the second active ingredient at a second release rate.

In some embodiments, the pharmaceutical composition comprises an immediate release component and a delayed release component. In other embodiments, the pharmaceutical composition comprises two or more active ingredients formulated as two delayed-release components, each providing a different delayed-release profile. For example, the first delayed-release component releases the first active ingredient at a first point in time and the second delayed-release component releases the second active ingredient at a second point in time.

The components of the combined release profile formulation (e.g., a formulation having an immediate release component in combination with an extended release component, an immediate release component in combination with a delayed release component, an immediate release component, a delayed release component in combination with an extended release component, two or more delayed release components, or two or more extended release components) can contain the same active ingredient or different active ingredients. In some embodiments, the immediate release component may provide from about 1% to about 80% of the total dose of active agent to be delivered by the pharmaceutical formulation. In some embodiments, the combined release profile formulation contains an immediate release component and the immediate release component provides from about 1% to about 90%, from about 10% to about 90%, from about 20% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, or from about 80% to about 90% of the total dose of each active ingredient to be delivered from the formulation. In alternative embodiments, the immediate release component provides up to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total dose of the various ingredients to be delivered by the formulation.

In some embodiments, the pharmaceutical formulation comprises an active core composed of one or more inert particles, each in the form of a bead, pill, tablet, granule, microcapsule, microsphere, microgranule, nanocapsule, or nanosphere, the surface of which is coated with a drug in the form of, for example, a drug-containing film-forming composition using, for example, fluid bed techniques or other methods known to those skilled in the art. The inert particles can be of various sizes as long as they are large enough to remain insoluble. Alternatively, the active core may be prepared by granulating and milling and/or by extruding and spheronizing a polymer composition containing the drug substance.

The amount of drug in the core will depend on the desired dosage and will generally vary from about 5 to 90% by weight. Generally, depending on the lag time and type of release profile desired and/or the polymer and coating solvent selected, the polymer coating on the active core will be about 1 to 50% based on the weight of the coated particle. One skilled in the art will be able to select the appropriate amount of drug for coating onto or incorporation into the core to achieve the desired dosage. In one embodiment, the inactive core may be a sugar sphere or a buffer crystal or an encapsulated buffer crystal, such as calcium carbonate, sodium bicarbonate, fumaric acid, tartaric acid, etc., which alters the microenvironment of the drug to facilitate its release.

In some embodiments, the pharmaceutical composition comprises a delayed release component formed by coating water-soluble/dispersible drug-containing particles (e.g., a bead) with a mixture of a water-insoluble polymer and an enteric polymer, wherein the water-insoluble polymer and the enteric polymer may be present in a ratio of 4: 1 to 1: 1, and the total weight of the coating is from 10 to 60 wt% based on the total weight of the coated beads. The drug layered beads may optionally include an ethylcellulose membrane that controls the internal dissolution rate. The composition of the outer layer of the polymeric film and the respective weights of the inner and outer layers are optimized and predicted based on in vitro/in vivo correlations to achieve a desired circadian release profile for a given activity.

In other embodiments, the formulation comprises a mixture of immediate release drug-containing particles (polymeric film without controlled dissolution rate) and delayed release beads that exhibit a lag time of 2 to 4 hours after, for example, oral administration, thereby providing a bi-pulse release profile. In still other embodiments, the formulation comprises a mixture of two types of delayed release beads: the first type exhibits a lag time of 1 to 3 hours and the second type exhibits a lag time of 4 to 6 hours. In still other embodiments, the formulation comprises a mixture of two types of release beads: the first type exhibits immediate release, while the second type exhibits a lag time of 1 to 4 hours, followed by extended release.

In some embodiments, the formulations are designed to have a release profile such that a portion of the active ingredient (e.g., 10% to 80%) is released immediately or within two hours after administration, while the remainder is released over an extended period of time (e.g., over a period of 2 to 24 hours). In other embodiments, the formulations are designed to have a release profile such that one active ingredient (e.g., an analgesic) is released immediately or within two hours after administration, while one or more other active ingredients (e.g., PG pathway inhibitors) are released over an extended period of time (e.g., over a period of 2 to 24 hours).

The pharmaceutical composition may be administered daily or on an as-needed basis. The pharmaceutical composition may be administered orally, intravenously, or intramuscularly. In a preferred embodiment, the pharmaceutical composition is administered orally. In other embodiments, the pharmaceutical composition is administered by retrograde perfusion (retrograde perfusion) through the urinary tract. In other embodiments, the pharmaceutical composition is administered by direct injection into the bladder muscle.

In some embodiments, the pharmaceutical composition is administered twice a day or three times a day on a daily basis. In other embodiments, the pharmaceutical composition is administered every other day, every 3 days, every 4 days, every 5 days, every 6 days, every week, every 2 weeks, every 3 weeks, every month, every 2 months, or every 3 months.

In some embodiments, the pharmaceutical composition is administered at bedtime. In some embodiments, the pharmaceutical composition is administered within about two hours prior to bedtime, preferably within about one hour prior to bedtime. In another embodiment, the pharmaceutical composition is administered about 2 to 4 hours prior to bedtime. In yet another embodiment, the pharmaceutical composition is administered at least 4 hours prior to bedtime.

The appropriate dosage ("therapeutically effective amount") of the active ingredient in the immediate-release component, the extended-release component, the delayed-release component, or the delayed-extended-release component will depend, for example, on the severity and course of the disease, the mode of administration, the bioavailability (biavailability) of the particular ingredient, the age and weight of the patient, the clinical history and response to the active agent of the patient, the judgment of the physician, and the like.

As a general proposition, a therapeutically effective amount of the PG pathway inhibitor in the immediate-release component, the delayed-release component, the extended-release component, or the delayed-extended-release component is administered in the range of about 1 microgram per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, whether by one or more administrations. In some embodiments, the range of single or multiple daily administration of each active agent is from about 1 microgram per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, from 1 microgram per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, from 1 microgram per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, from 1 microgram per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, from 1 microgram per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, from 1 microgram per kilogram of body weight per day to about 300 micrograms per kilogram of body weight per day, from 1 microgram per kilogram of body weight per day to about 100 micrograms per kilogram of body weight per day, from 1 microgram per kilogram of body weight per day to about 30 micrograms per kilogram of body weight per day, from 1 microgram per kilogram of body weight per day to about 10 micrograms per, 10 micrograms per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, 10 micrograms per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, 10 micrograms per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, 10 micrograms per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 10 micrograms per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, 10 micrograms per kilogram of body weight per day to about 300 micrograms per kilogram of body weight per day, 10 micrograms per kilogram of body weight per day to about 100 micrograms per kilogram of body weight per day, 10 micrograms per kilogram of body weight per day to about 30 micrograms per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 300 micrograms per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 100 micrograms per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 300 micrograms per kilogram of body weight per day, 300 micrograms per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, 300 micrograms per kilogram of body weight per, 300 micrograms per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 300 micrograms per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, 1 milligram per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, 1 milligram per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, 1 milligram per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, 1 milligram per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 3 mg per kg body weight per day to about 100 mg per kg body weight per day, 3 mg per kg body weight per day to about 30 mg per kg body weight per day, 3 mg per kg body weight per day to about 10 mg per kg body weight per day, 10 mg per kg body weight per day to about 100 mg per kg body weight per day, 10 mg per kg body weight per day to about 30 mg per kg body weight per day, or 30 mg per kg body weight per day to about 100 mg per kg body weight per day.

As a general proposition, a therapeutically effective amount of the analgesic agent in the immediate-release component, the delayed-release component, the extended-release component, or the delayed-extended-release component is administered in the range of about 10 micrograms per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, whether by one or more administrations. In some embodiments, the range of single or multiple daily administration of each active agent is from about 10 micrograms per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, from 10 micrograms per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, from 10 micrograms per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, from 10 micrograms per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, from 10 micrograms per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, from 10 micrograms per kilogram of body weight per day to about 300 micrograms per kilogram of body weight per day, from 10 micrograms per kilogram of body weight per day to about 100 micrograms per kilogram of body weight per day, from 10 micrograms per kilogram of body weight per day to about 30 micrograms per kilogram of body weight per day, from 30 micrograms per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, from 30, 30 micrograms per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 300 micrograms per kilogram of body weight per day, 30 micrograms per kilogram of body weight per day to about 100 micrograms per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, 100 micrograms per kilogram of body weight per day to about 300 micrograms per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, and a combination thereof, 300 micrograms per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, 300 micrograms per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, 300 micrograms per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 300 micrograms per kilogram of body weight per day to about 1 milligram per kilogram of body weight per day, 1 milligram per kilogram of body weight per day to about 100 milligrams per kilogram of body weight per day, 1 milligram per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, 1 milligram per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, 1 milligram per kilogram of body weight per day to about 3 milligrams per kilogram of body weight per day, 3 milligrams per kilogram of body weight per day to about 30 milligrams per kilogram of body weight per day, 3 milligrams per kilogram of body weight per day to about 10 milligrams per kilogram of body weight per day, 10 milligrams per kilogram of body weight per day to about 100 milligrams per kilogram of, From 10 mg per kg body weight per day to about 30 mg per kg body weight per day or from 30 mg per kg body weight per day to about 100 mg per kg body weight per day.

The analgesic described herein may be included in an immediate-release component or an extended-release component, a delayed-extended-release component, or a combination thereof for daily oral administration in a single dose or a combined dose range of 1 mg to 2000 mg, 1 mg to 1000 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 10 mg, 1 mg to 3 mg, 3 mg to 2000 mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 2000 mg, 10 mg to 1000 mg, 10 mg to 100 mg, 10 mg to 30 mg, 30 mg to 2000 mg, 30 mg to 1000 mg, 30 mg to 300 mg, 30 mg to 100 mg, 100 mg to 2000 mg, 100 mg to 1000 mg, 1 mg to 10 mg, 10 mg to 100 mg, 1 mg to 2000 mg, or a combination thereof, 100 mg to 300 mg, 300 mg to 2000 mg, 300 mg to 1000 mg or 1000 mg to 2000 mg. As expected, the dosage will depend on the disease, size, age and condition 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 agent is ibuprofen. In another embodiment, the single analgesic agent is naproxen or naproxen sodium. In another embodiment, the single analgesic agent is indomethacin. In another embodiment, the single analgesic agent is nabumetone. In another embodiment, the single analgesic agent is acetaminophen.

In other embodiments, the pharmaceutical composition comprises a pair of analgesics. Examples of such pair analgesics include, but are not limited to, acetylsalicylic acid and ibuprofen, acetylsalicylic acid and naproxen sodium, acetylsalicylic acid and nabumetone, acetylsalicylic acid and acetaminophen, acetylsalicylic acid and indomethacin (indomethancin), ibuprofen and naproxen sodium, ibuprofen and nabumetone, ibuprofen and acetaminophen, ibuprofen and indomethacin, naproxen sodium and nabumetone, naproxen sodium and acetaminophen, naproxen sodium and indomethacin, nabumetone and acetaminophen, nabumetone and indomethacin, and acetaminophen and indomethacin. The pair of analgesics was mixed at a ratio of 0.1: 1 to 10: 1. 0.2: 1 to 5: 1 or 0.3: 1 to 3: 1, in a weight ratio within the range of 1. In one embodiment, the pair of analgesics is present in a ratio of 1: 1 by weight ratio.

In some other embodiments, the pharmaceutical compositions of the present application further comprise one or more antimuscarinic agents. Examples of antimuscarinic agents include, but are not limited to oxybutynin, solifenacin, darifenacin, fesoterodine, tolterodine, trospium, atropine, and tricyclic antidepressants. A daily dose of the antimuscarinic agent in the range of 1 microgram to 300 milligrams, 1 microgram to 100 milligrams, 1 microgram to 30 milligrams; 1 microgram to 10 milligrams, 1 microgram to 3 milligrams, 1 microgram to 1 milligram, 1 microgram to 300 micrograms, 1 microgram to 100 micrograms, 1 microgram to 30 micrograms, 1 microgram to 10 micrograms, 1 microgram to 3 micrograms, 3 microgram to 100 milligrams, 3 microgram to 30 milligrams; 3 micrograms to 10 milligrams, 3 micrograms to 3 milligrams, 3 micrograms to 1 milligram, 3 micrograms to 300 micrograms, 3 micrograms to 100 micrograms, 3 micrograms to 30 micrograms, 3 micrograms to 10 micrograms, 10 micrograms to 300 milligrams, 10 micrograms to 100 milligrams, 10 micrograms to 30 milligrams; 10 micrograms to 10 milligrams, 10 micrograms to 3 milligrams, 10 micrograms to 1 milligram, 10 micrograms to 300 micrograms, 10 micrograms to 100 micrograms, 10 micrograms to 30 micrograms, 30 micrograms to 300 milligrams, 30 micrograms to 100 milligrams, 30 micrograms to 30 milligrams; 30 micrograms to 10 milligrams, 30 micrograms to 3 milligrams, 30 micrograms to 1 milligram, 30 micrograms to 300 micrograms, 30 micrograms to 100 micrograms, 100 micrograms to 300 milligrams, 100 micrograms to 100 milligrams, 100 micrograms to 30 milligrams; 100 micrograms to 10 milligrams, 100 micrograms to 3 milligrams, 100 micrograms to 1 milligram, 100 micrograms to 300 micrograms, 300 micrograms to 300 milligrams, 300 micrograms to 100 milligrams, 300 micrograms to 30 milligrams; 300 micrograms to 10 milligrams, 300 micrograms to 3 milligrams, 300 micrograms to 1 milligram, 1 milligram to 300 milligrams, 1 milligram to 100 milligrams, 1 milligram to 30 milligrams, 1 milligram to 3 milligrams, 3 milligrams to 300 milligrams, 3 milligrams to 100 milligrams, 3 milligrams to 30 milligrams, 3 milligrams to 10 milligrams, 10 milligrams to 300 milligrams, 10 milligrams to 100 milligrams, 10 milligrams to 30 milligrams, 30 milligrams to 300 milligrams, 30 milligrams to 100 milligrams, or 100 milligrams to 300 milligrams.

In some other embodiments, the pharmaceutical compositions of the present application further comprise one or more antidiuretic agents. Examples of antidiuretic agents include, but are not limited to, antidiuretic hormone (ADH), vasopressin II, aldosterone, vasopressin analogs (e.g., desmopressin, arginine vasopressin, lysine vasopressin, benzene lysine vasopressin, ornithine vasopressin, and terlipressin), vasopressin receptor agonists, atrial Natriuretic Peptide (ANP) and C-type natriuretic peptide (CNP) receptors (i.e., NPR1, NPR2, and NPR3) antagonists (e.g., HS-142-1, isatin, [ Asu7,23' ] b-ANP- (7-28) ], ansamitene (a cyclic peptide from Streptomyces coelicolor), and 3G12 monoclonal antibody), somatostatin type-2 receptor antagonists (e.g., somatostatin), pharmaceutically acceptable derivatives, and analogs, salts, hydrates, and solvates thereof. In some embodiments, the one or more antidiuretic agents comprise desmopressin. In other embodiments, the one or more antidiuretic agents is desmopressin. Daily doses of antidiuretic agent are in the range of 1 microgram to 300 mg, 1 microgram to 100 mg, 1 microgram to 30 mg; 1 microgram to 10 milligrams, 1 microgram to 3 milligrams, 1 microgram to 1 milligram, 1 microgram to 300 micrograms, 1 microgram to 100 micrograms, 1 microgram to 30 micrograms, 1 microgram to 10 micrograms, 1 microgram to 3 micrograms, 3 microgram to 100 milligrams, 3 microgram to 30 milligrams; 3 micrograms to 10 milligrams, 3 micrograms to 3 milligrams, 3 micrograms to 1 milligram, 3 micrograms to 300 micrograms, 3 micrograms to 100 micrograms, 3 micrograms to 30 micrograms, 3 micrograms to 10 micrograms, 10 micrograms to 300 milligrams, 10 micrograms to 100 milligrams, 10 micrograms to 30 milligrams; 10 micrograms to 10 milligrams, 10 micrograms to 3 milligrams, 10 micrograms to 1 milligram, 10 micrograms to 300 micrograms, 10 micrograms to 100 micrograms, 10 micrograms to 30 micrograms, 30 micrograms to 300 milligrams, 30 micrograms to 100 milligrams, 30 micrograms to 30 milligrams; 30 micrograms to 10 milligrams, 30 micrograms to 3 milligrams, 30 micrograms to 1 milligram, 30 micrograms to 300 micrograms, 30 micrograms to 100 micrograms, 100 micrograms to 300 milligrams, 100 micrograms to 100 milligrams, 100 micrograms to 30 milligrams; 100 micrograms to 10 milligrams, 100 micrograms to 3 milligrams, 100 micrograms to 1 milligram, 100 micrograms to 300 micrograms, 300 micrograms to 300 milligrams, 300 micrograms to 100 milligrams, 300 micrograms to 30 milligrams; 300 micrograms to 10 milligrams, 300 micrograms to 3 milligrams, 300 micrograms to 1 milligram, 1 milligram to 300 milligrams, 1 milligram to 100 milligrams, 1 milligram to 30 milligrams, 1 milligram to 3 milligrams, 3 milligrams to 300 milligrams, 3 milligrams to 100 milligrams, 3 milligrams to 30 milligrams, 3 milligrams to 10 milligrams, 10 milligrams to 300 milligrams, 10 milligrams to 100 milligrams, 10 milligrams to 30 milligrams, 30 milligrams to 300 milligrams, 30 milligrams to 100 milligrams, or 100 milligrams to 300 milligrams.

In other embodiments, the pharmaceutical compositions of the present application further comprise one or more spasmolytic agents. Examples of antispasmodics include, but are not limited to, myotonin, benzodiazepines, baclofen, cyclobenzaprine, metaxalone, methocarbamol, clonine analogs, and dantrolene. In some embodiments, the spasmolytic is in the range of 0.1 mg to 1000 mg, 0.1 mg to 300 mg, 0.1 mg to 100 mg, 0.1 mg to 30 mg, 0.1 mg to 10 mg, 0.1 mg to 3 mg, 0.1 mg to 1 mg, 0.1 mg to 0.3 mg, 0.3 mg to 1000 mg, 0.3 mg to 300 mg, 0.3 mg to 100 mg, 0.3 mg to 30 mg, 0.3 mg to 10 mg, 0.3 mg to 3 mg, 0.3 mg to 1 mg, 1 mg to 1000 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to 30 mg, 1 mg to 10 mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 1000 mg, 10 mg to 300 mg, 10 mg to 100 mg, 10 mg to 30 mg, 0.1 mg to 10 mg, 0.1 mg, 0.3 mg to 1 mg, 0.3 mg to 10 mg, or a A daily dose of 30 mg to 300 mg, 30 mg to 100 mg, 100 mg to 1000 mg, 100 mg to 300 mg or 300 mg to 1000 mg.

In other embodiments, the pharmaceutical compositions of the present application further comprise one or more PDE5 inhibitors. Examples of PDE5 inhibitors include, but are not limited to, tadalafil, sildenafil, and vardenafil. In some embodiments, the one or more PDE5 inhibitors comprise tadalafil. In other embodiments, the one or more PDE5 inhibitors is tadalafil. In some embodiments, the PDE5 inhibitor is administered in an amount of 0.1 mg to 1000 mg, 0.1 mg to 300 mg, 0.1 mg to 100 mg, 0.1 mg to 30 mg, 0.1 mg to 10 mg, 0.1 mg to 3 mg, 0.1 mg to 1 mg, 0.1 mg to 0.3 mg, 0.3 mg to 1000 mg, 0.3 mg to 300 mg, 0.3 mg to 100 mg, 0.3 mg to 30 mg, 0.3 mg to 10 mg, 0.3 mg to 3 mg, 0.3 mg to 1 mg, 1 mg to 1000 mg, 1 mg to 300 mg, 1 mg to 100 mg, 1 mg to 10 mg, 1 mg to 3 mg, 3 mg to 1000 mg, 3 mg to 300 mg, 3 mg to 100 mg, 3 mg to 30 mg, 3 mg to 10 mg, 10 mg to 1000 mg, 10 mg to 300 mg, 10 mg to 100 mg, 10 mg to 30 mg, 0.1 mg to 1 mg, 0.3 mg to 10 mg, or a combination thereof, A daily dose of 30 mg to 300 mg, 30 mg to 100 mg, 100 mg to 1000 mg, 100 mg to 300 mg or 300 mg to 1000 mg.

In some other embodiments, the pharmaceutical composition of the present application further comprises zolpidem. The daily dose of zolpidem is in the range of 100 micrograms to 100 milligrams, 100 micrograms to 30 milligrams, 100 micrograms to 10 milligrams, 100 micrograms to 3 milligrams, 100 micrograms to 1 milligram, 100 micrograms to 300 micrograms, 300 micrograms to 100 milligrams, 300 micrograms to 30 milligrams, 300 micrograms to 10 milligrams, 300 micrograms to 3 milligrams, 300 micrograms to 1 milligram, 1 milligram to 100 milligrams, 1 milligram to 30 milligrams, 1 milligram to 10 milligrams, 1 milligram to 3 milligrams, 10 milligrams to 100 milligrams, 10 milligrams to 30 milligrams, or 30 milligrams to 100 milligrams.

The antimuscarinic agent, antidiuretic agent, spasmolytic, zolpidem, and/or PDE5 inhibitor may be formulated in a pharmaceutical composition for immediate release, extended release, delayed-extended release, or a combination thereof, alone or with other active ingredients.

In certain embodiments, the pharmaceutical composition is formulated for extended release and comprises (1) an analgesic selected from the group consisting of acetylsalicylic acid, ibuprofen, naproxen sodium, nabumetone, acetaminophen, and indomethacin and (2) a PDE5 inhibitor, such as tadalafil.

The pharmaceutical composition may be formulated in the form of tablets, capsules, dragees (dragees), powders, granules, liquids, gels or emulsions. The liquid, gel or emulsion may be ingested by the individual in naked form (as in naked form) or contained in a capsule.

In some embodiments, the pharmaceutical composition comprises a single analgesic and a single PDE5 inhibitor. In one embodiment, the single analgesic is aspirin. In another embodiment, the single analgesic agent is ibuprofen. In another embodiment, the single analgesic agent is naproxen or naproxen sodium. In another embodiment, the single analgesic agent is indomethacin. In another embodiment, the single analgesic agent is nabumetone. In another embodiment, the single analgesic agent is acetaminophen. In another embodiment, the single PDE5 inhibitor is tadalafil. The analgesic and the PDE5 inhibitor may be administered at dosages within the ranges set forth above.

In some embodiments, the pharmaceutical composition comprises, individually or in combination, one or more analgesic agents in an amount of between 10 to 1000 mg, 10 to 800 mg, 10 to 600 mg, 10 to 500 mg, 10 to 400 mg, 10 to 300 mg, 10 to 250 mg, 10 to 200 mg, 10 to 150 mg, 10 to 100 mg, 30 to 1000 mg, 30 to 800 mg, 30 to 600 mg, 30 to 500 mg, 30 to 400 mg, 30 to 300 mg, 30 to 250 mg, 30 to 200 mg, 30 to 150 mg, 30 to 100 mg, 100 to 1000 mg, 100 to 800 mg, 100 to 600 mg, 100 to 400 mg, 100 to 250 mg, 300 to 1000 mg, 300 to 800 mg, 300 to 600 mg, 300 to 400 mg, 400 to 1000 mg, 400 to 800 mg, 400 to 600 mg, 600 to 1000 mg, 600 to 800 mg, or 800 to 1000 mg, wherein the composition is formulated for extended release, the release profile is wherein the one or more analgesic agents are released continuously over a period of 2 to 12 hours or 5 to 8 hours.

In some embodiments, the composition is formulated for extended release with a release profile in which at least 90% of the one or more analgesic agents are released continuously over a period of 2 to 12 hours or 5 to 8 hours.

In some embodiments, the composition is formulated for extended release with a release profile in which the one or more analgesic agents are released continuously over a period of 5, 6, 7, 8, 10, or 12 hours. In some embodiments, the pharmaceutical composition further comprises an antimuscarinic agent, an antidiuretic, a spasmolytic, zolpidem, or a PDE5 inhibitor.

In other embodiments, the composition is formulated for extended release with a release profile in which the analgesic is released at a steady rate over a period of 2 to 12 hours or 5 to 8 hours. In other embodiments, the composition is formulated for extended release with a release profile in which the analgesic is released at a steady rate over a period of 5, 6, 7, 8, 10, or 12 hours. As used herein, a "steady rate over a period of time" is defined as a release profile in which the release rate at any point during a given period of time is within 30% to 300% of the average release rate over that given period of time. For example, if 80 mg of aspirin is released at a steady rate over an 8 hour period, the average release rate during this period is 10 mg/hour, and the actual release rate at any time during this period is in the range of 3 mg/hour to 30 mg/hour (i.e., within 30% to 300% of the average release rate of 10 mg/hour over the 8 hour period). In some embodiments, the pharmaceutical composition further comprises an antimuscarinic agent, an antidiuretic, a spasmolytic, zolpidem, or a PDE5 inhibitor.

In some embodiments, the analgesic agent is selected from the group consisting of aspirin, ibuprofen, naproxen sodium, naproxen, indomethacin, nabumetone, and acetaminophen. In one embodiment, the analgesic is acetaminophen. The pharmaceutical compositions are formulated to provide stable release of small amounts of the analgesic agent to maintain effective drug concentrations in the blood, thereby reducing the total amount of drug in a single dose as compared to an immediate release formulation.

In some other embodiments, the pharmaceutical composition comprises, individually or in combination, one or more analgesic in an amount of between 10 to 1000 mg, 10 to 800 mg, 10 to 600 mg, 10 to 500 mg, 10 to 400 mg, 10 to 300 mg, 10 to 250 mg, 10 to 200 mg, 10 to 150 mg, 10 to 100 mg, 30 to 1000 mg, 30 to 800 mg, 30 to 600 mg, 30 to 500 mg, 30 to 400 mg, 30 to 300 mg, 30 to 250 mg, 30 to 200 mg, 30 to 150 mg, 30 to 100 mg, 100 to 1000 mg, 100 to 800 mg, 100 to 600 mg, 100 to 400 mg, 100 to 250 mg, 300 to 1000 mg, 300 to 800 mg, 300 to 600 mg, 300 to 400 mg, 400 to 1000 mg, 400 to 800 mg, 400 to 600 mg, 600 to 1000 mg, 600 to 800 mg, or 800 to 1000 mg, wherein the analgesic is formulated for extended release, characterized by a biphasic release profile, wherein 20% to 60% of the analgesic is released within 2 hours of administration and the remainder is released continuously or at a steady rate over a period of 2 to 12 hours or 5 to 8 hours. In yet another embodiment, the analgesic is formulated for extended release with a biphasic release profile, wherein 20%, 30%, 40%, 50% or 60% of the analgesic is released within 2 hours of administration and the remainder is released continuously or at a steady rate over a period of 2 to 12 hours or 5 to 8 hours. In one embodiment, the analgesic agent is selected from the group consisting of aspirin, ibuprofen, naproxen sodium, naproxen, indomethacin, nabumetone, and acetaminophen. In one embodiment, the analgesic is acetaminophen. In another embodiment, the analgesic is acetaminophen. In some embodiments, the pharmaceutical composition further comprises an antimuscarinic agent, an antidiuretic, a spasmolytic, zolpidem, and/or a PDE5 inhibitor. In some embodiments, the antimuscarinic agent, antidiuretic agent, spasmolytic, zolpidem, and/or PDE5 inhibitor are formulated for immediate release.

Another aspect of the invention relates to a method of reducing frequency of urination by preventing development of drug resistance by alternating administration of two or more PG pathway inhibitors to an individual in need thereof. In one embodiment, the method comprises administering a first PG pathway inhibitor for a first period of time and subsequently administering a second PG pathway inhibitor for a second period of time. In another embodiment, the method further comprises administering a third PG pathway inhibitor for a third period of time. The first PG pathway inhibitor, the second PG pathway inhibitor, and the third PG pathway inhibitor are different from each other and can be formulated for immediate release, extended release, delayed release, or a combination thereof.

Another aspect of the invention relates to a method of treating nocturia by administering a diuretic to a subject in need thereof, followed by administration of the pharmaceutical composition of the present application. The diuretic is dosed and formulated to have a diuretic effect (diuretic effect) within 6 hours of administration and is administered at least 8 or 7 hours prior to bedtime. The pharmaceutical compositions of the present application are formulated for extended release or delayed extended release and are administered within 2 hours prior to bedtime.

Examples of diuretics include, but are not limited to, acidifying salts, such as CaCl₂And NH₄Cl; arginine vasopressin receptor 2 antagonists such as amphotericin b (amphotericin b) and lithium citrate; drainage agents (aquatics), e.g. autumnEucheuma (golden) and Juniper (Juniper); Na-H exchange antagonists (Na-H exchangeable antaconists), such as dopamine (dopamine); carbonic anhydrase (carbonic anhydrase) inhibitors such as acetamidine (acetazolamide) and dorzolamide (dorzolamide); loop diuretics (loop diuretics) such as bumetanide (bumetanide), ethacrynic acid (ethacrynic acid), furosemide (furosemide) and torasemide (torsemide); osmotic diuretics such as glucose and mannitol; potassium-sparing diuretics (potassium-sparing diuretics), such as amiloride, spironolactone, triamterene, potassium canrenoate; thiazines (thiazides), such as bendroflumethiazide (bendroflumethiazide) and hydrochlorothiazide (hydrochlorothiazide); and xanthines (xanthines), such as caffeine (caffeine), theophylline (theophylline), and theobromine (theobromine).

Another aspect of the present application relates to a method of reducing frequency of urination, comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition of the present application and an effective amount of botulinum toxin (botulin toxin).

In some embodiments, the botulinum toxin is administered by injection into the bladder muscle and the pharmaceutical composition of the present application is administered orally to the individual. In some embodiments, the injecting step comprises injecting 10 to 200 units of botulinum toxin at 5 to 20 sites in the bladder muscle at an injection dose of 2 to 10 units per site. In one embodiment, the injecting step comprises injecting botulinum toxin at 5 locations in the bladder muscle at an injection dose of 2 to 10 units per location. In another embodiment, the injecting step comprises injecting botulinum toxin at 10 locations in the bladder muscle at an injection dose of 2 to 10 units per location. In another embodiment, the injecting step comprises injecting botulinum toxin at 15 locations in the bladder muscle at an injection dose of 2 to 10 units per location. In yet another embodiment, the injecting step comprises injecting botulinum toxin at 20 sites in the bladder muscle at an injection dose of 2 to 10 units per site. In some embodiments, the injecting step is repeated every 3 months, 4 months, 6 months, 8 months, 10 months, or 12 months.

The invention is further illustrated by the following examples, which should not be construed as limiting the invention. The contents of all references, patents, and published patent applications cited in this disclosure are hereby incorporated by reference.

Example 1: use of ibuprofen to inhibit urination

20 volunteer subjects (including males and females) were recruited, each experiencing excessive urination or an urge to urinate early enough to interfere with their inability to sleep for a sufficient period of time to get full rest. Each individual ingested 400 to 800 mg of ibuprofen in a single administration prior to bedtime. At least 14 individuals reported that they had better rest because they were not frequently awakened by urine.

Several individuals reported that the benefit of reduced frequency of urination was no longer felt after several weeks of overnight ibuprofen use. However, all of these individuals further reported that benefit recovered several days after discontinuing this dose. Recent tests have demonstrated that similar results can be achieved at much lower doses without any benefit from the subsequent reduction.

Example 2: analgesic, botulinum neurotoxin, and antimuscarinic agents in response to inflammatory and non-inflammatory stimuli to macrophages Influence of (2)

Design of experiments

This study was designed to determine the dose and in vitro efficacy of analgesics and antimuscarinics in controlling macrophage response to inflammatory and non-inflammatory stimuli mediated by COX2 and prostaglandins (PGE, PGH, etc.). A baseline (dose and kinetics) response to inflammatory and non-inflammatory effectors (effectors) in bladder cells is established. Briefly, cultured cells are exposed to an analgesic and/or antimuscarinic agent in the absence or presence of various effectors.

The effectors include: lipopolysaccharide (LPS), an inflammatory agent, and COX2 inducer as inflammatory stimuli; carbachol (carbachol) or acetylcholine, a stimulator of smooth muscle contraction, as a non-inflammatory stimulus; botulinum neurotoxin a, a known inhibitor of acetylcholine release, as a positive control; and Arachidic Acid (AA), gamma linolenic acid (DGLA), or eicosapentaenoic acid (EPA), which are precursors of prostaglandins produced by continuous oxidation of AA, DGLA or EPA inside cells by cyclooxygenase (COX1 and COX2) and terminal prostaglandin synthase.

Analgesics include: salicylates, such as aspirin; isobutyl-propionic acid-phenolic acid derivatives (ibuprofen), such as javay (Advil), methylene (Motrin), naprin (Nuprin), and medypren (Medipren); naproxen sodium, such as alevir (Aleve), ananaprox (Anaprox), antagon (Antalgin), Ultra-finas (femimax Ultra), finas (Flanax), pizza (Inza), mido extension release (midolextedrelease), nateglinide (Nalgesin), empoassin (napoln), naperlan (napelan), naproxen (naprogegesic), naproxen (naproxen), naproxen suspension, EC-naproxen, nafroxen (Narocin), plen (Proxen), suflox (Synflex), and xinobid (Xenobid); acetic acid derivatives, such as indomethacin (Indocin); 1-naphthaleneacetic acid (1-naphthaleneacetic acid) derivatives, such as nabumetone or rilifen; n-acetyl-para-aminophenol (APAP) derivatives, such as acetaminophen or acetaminophen (Tylenol); and Celecoxib (Celecoxib).

Antimuscarinic agents include oxybutynin, solifenacin, darifenacin, and atropine.

Macrophages are stimulated either short-term (1 to 2 hours) or long-term (24 to 48 hours) with the following agents:

(1) each analgesic is stimulated individually at various doses.

(2) Each analgesic was stimulated at various doses in the presence of LPS.

(3) Each analgesic is stimulated at various doses in the presence of carbachol or acetylcholine.

(4) Each analgesic was stimulated at various doses in the presence of AA, DGLA or EPA.

(5) Botulinum neurotoxin a was stimulated individually at various doses.

(6) Botulinum neurotoxin a was stimulated in the presence of LPS at various doses.

(7) Botulinum neurotoxin a is stimulated in the presence of carbachol or acetylcholine at various doses.

(8) Botulinum neurotoxin A is stimulated in the presence of AA, DGLA or EPA at various doses.

(9) Each antimuscarinic agent is separately stimulated at various doses.

(10) Each antimuscarinic agent is stimulated in the presence of LPS at various doses.

(11) Each antimuscarinic agent is stimulated at various doses in the presence of carbachol or acetylcholine.

(12) Each antimuscarinic agent is stimulated at various doses in the presence of AA, DGLA, or EPA.

Cells were subsequently analyzed for PGH₂Release of (1); release of PGE; PGE₂Release of prostacyclin, release of thromboxane, release of IL-1 β, release of IL-6, release of TNF- α, COX2 activity, production of cAMP and cGMP, production of IL-1 β, IL-6, TNF- α, and COX2mRNA, and surface expression of CD80, CD86, and MHC class II molecules.

Materials and methods

Macrophage cell

Murine RAW264.7 or J774 macrophages (obtained from ATCC) were used in this study. Maintaining the cells in a culture medium containing a culture medium supplemented with 10% Fetal Bovine Serum (FBS),HEPES at 15 millimolar (mM), L-glutamine at 2 millimolar (L-glutamine), penicillin at 100 units/ml (U/ml), and streptomycin at 100 microgram/ml (μ g/ml) in RPMI 1640 medium. At 5% CO₂The cells were cultured at 37 ℃ in an atmosphere of (atmosphere) and the cells divided (subcultured) once a week.

Treating macrophages with analgesics in vitro

RAW264.7 macrophages were incubated in 100 microliters of medium at 1.5 × 10 per well⁵Cell density of individual cells was seeded (seed) in 96 well culture plates. Cells were treated with each of the following: (1) various concentrations of analgesic (acetaminophen, aspirin, ibuprofen, or naproxen), (2) various concentrations of Lipopolysaccharide (LPS), which is an effector of inflammatory stimulation of macrophages, (3) various concentrations of carbachol or acetylcholine, which is an effector of non-inflammatory stimulation, (4) analgesic and LPS, or (5) analgesic and carbachol or acetylcholine. Briefly, the analgesic was dissolved in FBS-free medium (i.e., RPMI 1640 supplemented with 15 mm HEPES, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μ g/ml streptomycin) and diluted to the desired concentration by serial dilution (serial dilution) using the same medium. For cells treated with the analgesic in the absence of LPS, 50 microliters of the analgesic solution and 50 microliters of FBS-free medium were added to each well. For cells treated with the analgesic in the presence of LPS, 50 microliters of the analgesic solution and 50 microliters of LPS (from Salmonella typhimurium) in FBS-free medium were added to each well. All conditions were tested in duplicate.

After 24 or 48 hours of incubation, 150 microliters of culture supernatant was collected, centrifuged at 8,000 revolutions per minute (spin down) at 4 ℃ for 2 minutes to remove cells and debris and stored at-70 ℃ in preparation for analysis of cytokine response by ELISA. Cells were collected and washed by centrifugation (4 ℃, 1,500 rpm, 5 minutes) in 500 μ l of Phosphate Buffered Saline (PBS). Half of the cells were then snap frozen in liquid nitrogen and stored at-70 ℃. The remaining cells were stained with fluorescent monoclonal antibodies and analyzed by flow cytometry.

Flow cytometry analysis of costimulatory molecule expression

For flow cytometry analysis, macrophages were washed in 100 microliters of FACS buffer (with 2% Bovine Serum Albumin (BSA) and 0.01% NaN₃Phosphate buffered saline) and stained at 4 ℃ for 30 minutes by adding FITC-bound anti-CD 40 antibody, PE-bound anti-CD 80 antibody, PE-bound anti-CD 86 antibody, anti-mhc class ii (I-Ad) PE (bd bioscience). The cells were then washed by centrifugation (1,500 rpm, 5 minutes at 4 ℃) in 300. mu.l of FACS buffer. After the second wash, the cells were resuspended in 200 μ l FACS buffer and the percentage of cells expressing a given marker (single positive) or combination of markers (double positive) was analyzed by means of Accuri C6 flow cytometry (BD Biosciences).

Analysis of cytokine response by ELISA

Cytokine-specific ELISA was performed on culture supernatants to determine IL-1 β, IL-6, and TNF- α responses in macrophage cultures treated with analgesics, LPS alone, or a combination of LPS and analgesics 100 microliters of anti-mouse IL-6(anti-mouse IL-6), TNF- α monoclonal antibody (TNF- α mAb, BD Biosciences), or IL-1 β monoclonal antibody (IL-1 β mAb, R) in 0.1 molar sodium bicarbonate buffer (pH 9.5)&D Systems) overnight coated Nunc MaxiSorp immunolates (Nunc) analysis after two washes with PBS (200. mu.l per well), 200. mu.l PBS (3% BSA) was added per well (blocking) and the plates were incubated at room temperature for 2 hours, 200. mu.l PBS was added per well and the plates were washed twice, two replicates of serial dilutions of 100. mu.l cytokine standards and culture supernatant were added and the plates were incubated overnight at 4 ℃(BD Biosciences) or IL-1 β (R)&D Systems) followed by incubation with peroxidase-labeled goat anti-biotin monoclonal antibody (peroxidase-labeled goat anti-biotin mAb, Vector Laboratories). By adding 2,2 '-azoxy-bis (3) -ethylbenzylthiazoline-6-sulfonic acid (2, 2' -azino-bis (3) -ethylbenzylthiazoline-6-sulfonic acid, ABTS) substrate and H₂O₂(Sigma) for colorimetric reaction (colorimetric reaction) at 415 nmV Multi-labeled plate reader (Multilabel plate reader, Perkinelmer) to measure absorbance.

Determination of COX2 Activity and production of cAMP and cGMP

The activity of COX2 in cultured macrophages was determined by sequential competitive ELISA (R & D Systems). cAMP and cGMP production was determined by cAMP and cGMP assays. The analysis is performed by means conventional in the art.

Table 1 summarizes the experiments performed with the Raw264 macrophage strain and the main findings on the effect of analgesics on the cell surface expression of the co-stimulatory molecules CD40 and CD 80. The expression of this molecule was stimulated by COX2 and inflammatory signals, and was therefore assessed to determine the functional outcome of inhibition of COX 2.

As shown in table 2, except for the highest dose (i.e., 5 × 10)⁶Nanomolar (nM) molarity), acetaminophen, aspirin, ibuprofen, and naproxen were present at all doses tested (i.e., 5 × 10⁵nM mole, 5 × 10⁴nM mole, 5 × 10³nM mole, 5 × 10²nM moles, 50nM moles, and 5nM moles) inhibits basal expression of costimulatory molecules CD40 and CD80 by macrophages, while the highest dose appears to enhance rather than inhibit expression of costimulatory molecules. As shown in FIGS. 1A and 1B, inhibitory effects on CD40 and CD50 expression were observed at analgesic doses as low as 0.05 nanomolar (i.e., 0.00005 micromolar (μ M)) dosesAnd (5) fruit. This finding supports the idea that controlled release of small doses of analgesic is better than acutely delivered large doses. This experiment also showed that acetaminophen, aspirin, ibuprofen, and naproxen had similar inhibitory effects on LPS-induced CD40 and CD80 expression.

TABLE 1 summary of the experiments

TABLE 2 summary of the main findings

ND: not performed (toxicity)

Table 3 summarizes the results of several studies measuring analgesic serum levels after oral treatment doses in adults. As shown in Table 3, the maximum serum level of analgesic after oral treatment was 10⁴To 10⁵In nM molar range. Thus, the analgesic doses for the in vitro (in vitro) test in table 2 encompass the range of concentrations that are achievable in humans (in vivo).

TABLE 3 serum levels of analgesics in human blood after oral therapeutic dose

Example 3: analgesic, botulinum neurotoxin, and antimuscarinic agents on inflammatory stimulation and noninflammatory effects of mouse bladder smooth muscle cells Effect of the sexual stimulation response

Design of experiments

This study was designed to characterize how the optimal dose of analgesic as determined in example 2 affects bladder smooth muscle cells in cell or tissue culture and to address whether different classes of analgesics can act synergistically to more effectively inhibit COX2 and PGE2 responses.

Effectors, analgesics, and antimuscarinic agents are described in example 2.

Primary cultures of mouse bladder smooth muscle cells were stimulated either short-term (1 to 2 hours) or long-term (24 to 48 hours) with the following agents:

(1) each analgesic is stimulated individually at various doses.

(2) Each analgesic was stimulated at various doses in the presence of LPS.

(3) Each analgesic is stimulated at various doses in the presence of carbachol or acetylcholine.

(4) Each analgesic is stimulated at various doses in the presence of AA, DGLA, or EPA.

(5) Botulinum neurotoxin a was stimulated individually at various doses.

(6) Botulinum neurotoxin a was stimulated in the presence of LPS at various doses.

(7) Botulinum neurotoxin a is stimulated in the presence of carbachol or acetylcholine at various doses.

(8) Botulinum neurotoxin A is stimulated in the presence of AA, DGLA or EPA at various doses.

(9) Each antimuscarinic agent is separately stimulated at various doses.

(10) Each antimuscarinic agent is stimulated in the presence of LPS at various doses.

(11) Each antimuscarinic agent is stimulated at various doses in the presence of carbachol or acetylcholine.

(12) Each antimuscarinic agent is stimulated at various doses in the presence of AA, DGLA, or EPA.

Cells were subsequently analyzed for PGH₂Release of (1); release of PGE; PGE₂Release of prostacyclin, release of thromboxane, release of IL-1 β, release of IL-6, release of TNF- α, COX2 activity, production of cAMP and cGMP, production of IL-1 β, IL-6, TNF- α, and COX2mRNA, and surface expression of CD80, CD86, and MHC class II molecules.

Materials and methods

Isolation and purification of mouse bladder cells

Bladder cells were removed from euthanized C57BL/6 mice (8 to 12 weeks old) and cells were isolated by enzymatic digestion followed by purification on Percoll gradients. Briefly, bladders from 10 mice were minced into a fine slurry with scissors in 10 ml of digestion buffer (RPMI 1640, 2% fetal bovine serum, 0.5 mg/ml collagenase, 30 μ g/ml deoxyribonuclease (Dnase)). The bladder slurry was enzymatically digested at 37 ℃ for 30 minutes. The undigested fragments were further dispersed by cell-trainer (cell-train). The cell suspension was pelleted and added to discrete 20%, 40% and 75% Percoll gradients for purification of monocytes. 50 to 60 bladders were used for each experiment.

After washing in RPMI 1640, bladder cells were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum, 15 millimolar (mM) HEPES, 2 millimolar l-glutamine, 100 units/ml penicillin and 100 micrograms/ml streptomycin at 100 microliters per well of 3 × 10⁴The cell density of individual cells was seeded in 96-well cell culture microplates with a black transparent bottom. At 37 ℃ at 5%CO₂Culturing the cells in the atmosphere of (2).

In vitro treatment of cells with analgesics

Bladder cells were treated with either an analgesic solution (50 microliters per well) alone or with carbachol (10 Molar (Molar) mols, 50 microliters per well) as an example of a non-inflammatory stimulus, or with Lipopolysaccharide (LPS) of salmonella typhimurium (1 microgram/ml, 50 microliters per well) as an example of an inflammatory stimulus. When no other effectors were added to the cells, 50 microliters of RPMI 1640 without fetal bovine serum was added to the wells to adjust the final volume to 200 microliters.

After 24 hours of incubation, 150 microliters of culture supernatant was collected and centrifuged at 8,000 rpm for 2 minutes at 4 ℃ to remove cells and debris and stored at-70 ℃ in preparation for analysis of prostaglandin E2(PGE2) response by ELISA. Cells were fixed, permeabilized, and blocked to detect cyclooxygenase-2 (COX2) using a fluorogenic substrate. In selected experiments, cells were stimulated in vitro for 12 hours to analyze the response of COX 2.

Analysis of COX2 reactions

Human/mouse Total COX2immunoassay (human/mouse Total COX2 immunological, R) was used by cell-based ELISA according to the manufacturer's instructions&D Systems) to analyze the reaction of COX 2. Briefly, after cells were fixed and permeabilized, wells of a 96-well cell culture microplate with a black clear background were supplemented with mouse anti-total COX2(mouse anti-total COX2) and rabbit anti-total GAPDH (rabbitant-total GAPDH). After incubation and washing, HRP-conjugated anti-mouse IgG (HRP-conjugated anti-mouse IgG) and AP-conjugated anti-rabbit IgG (AP-conjugated anti-rabbit IgG) were added to the wells. After another incubation and another set of washes, HRP fluorogenic substrate and AP fluorogenic substrate are added. Finally, useV multiple-tag plate reader (Perkinelmer) to read at 600 nm (COX2 fluorescence)) And emission fluorescence at 450 nm (GAPDH fluorescence). Results are expressed as relative levels of COX2 determined by Relative Fluorescence Units (RFU) and referenced to housekeeping protein (GAPDH).

Analysis of PGE2 response

The response of prostaglandin E2 was analyzed by continuous competitive ELISA (R & D Systems). More specifically, culture supernatants or PGE2 standards were added to wells of 96-well polystyrene microplates coated with goat anti-mouse polyclonal antibodies (goat anti-mouse antibodies). After one hour incubation on a microplate shaker, HRP-conjugated PGE2(HRP-conjugated PGE2) was added and the plates were incubated at room temperature for an additional two hours. The plates were then washed and HRP substrate solution was added to each well. The reaction was stopped by addition of sulfuric acid after development for 30 minutes and reading of the plate at 450 nm with wavelength correction of 570 nm. Results are expressed as the average picograms/milliliter of PGE 2.

Other analysis

The release of PGH2 was determined as described in example 2; release of PGE, release of prostacyclin; release of coagulosin; release of IL-1 beta; release of IL-6; and TNF-alpha release; production of cAMP and cGMP; production of IL-1 β, IL-6, TNF- α, and COX2 mRNA; and surface expression of CD80, CD86, and MHC class II molecules.

Results

Analgesics inhibit COX2 responses of mouse bladder cells to inflammatory stimuli

Several analgesics (acetaminophen, aspirin, ibuprofen, and naproxen) were tested at 5 micromolar (μ M) or 50 micromolar (M) concentrations on mouse bladder cells to determine whether the analgesics could induce a COX2 response. Analysis of the 24 hour culture showed that none of the tested analgesics induced a COX2 response in vitro to mouse bladder cells.

The effect of the analgesic on the mouse bladder cell response to carbachol or LPS stimulated COX2 in vitro was also tested. As shown in table 1, the doses of carbachol tested had no significant effect on COX2 levels in mouse bladder cells. LPS on the other hand, significantly increased total COX2 levels. Interestingly, acetaminophen, aspirin, ibuprofen, and naproxen all inhibited the effect of LPS on COX2 levels. The inhibitory effect of the analgesic was seen when the drug was tested at 5 micromolar or 50 micromolar (table 4).

TABLE 4 COX2 expression in mouse bladder cells following in vitro stimulation and treatment with analgesics

Analgesics inhibit PGE2 response of mouse bladder cells to inflammatory stimuli

The secretion of PGE2 in the culture supernatant of mouse bladder cells was measured to determine the biological significance of altering mouse bladder cell COX2 levels by analgesics. As shown in table 5, PGE2 was not detected in the culture supernatant of unstimulated bladder cells or bladder cells cultured in the presence of carbachol. In agreement with the COX2 response described above, LPS stimulation of mouse bladder cells elicited secretion of high levels of PGE 2. The addition of analgesics (acetaminophen, aspirin, ibuprofen, and naproxen) inhibited the effect of LPS on PGE2 secretion, and no difference was seen between the cellular responses treated with 5 or 50 micromolar doses of the analgesic.

TABLE 5 PGE2 secreted by mouse bladder cells following in vitro stimulation and treatment with analgesics

Taken together, these data show that analgesics at 5 micromolar (μ M) or 50 micromolar concentrations alone do not induce COX2 and PGE2 responses in mouse bladder cells. However, 5 micromolar or 50 micromolar analgesic significantly inhibited both COX2 and PGE2 responses in vitro in LPS (1 microgram/ml) (μ g/ml) stimulated mouse bladder cells. No significant effect of the analgesic on COX2 and PGE2 responses in mouse bladder cells stimulated with carbachol (1 millimolar (mM)) was observed.

Example 4: effect of analgesics, botulinum neurotoxins, and antimuscarinic agents on contraction of mouse bladder smooth muscle cells

Design of experiments

The cultured mouse or rat bladder smooth muscle cells and mouse or rat bladder smooth muscle tissue are exposed to inflammatory and non-inflammatory stimuli in the presence of various concentrations of analgesic and/or antimuscarinic agents. The muscle contraction induced by the stimulation is measured to assess the inhibitory effect of the analgesic and/or antimuscarinic agent.

Effectors, analgesics, and antimuscarinic agents are described in example 2.

Primary cultures of mouse bladder smooth muscle cells were stimulated either short-term (1 to 2 hours) or long-term (24 to 48 hours) with the following agents:

(1) each analgesic is stimulated individually at various doses.

(2) Each analgesic was stimulated at various doses in the presence of LPS.

(3) Each analgesic is stimulated at various doses in the presence of carbachol or acetylcholine.

(4) Each analgesic is stimulated at various doses in the presence of AA, DGLA, or EPA.

(5) Botulinum neurotoxin a was stimulated individually at various doses.

(6) Botulinum neurotoxin a was stimulated in the presence of LPS at various doses.

(7) Botulinum neurotoxin a is stimulated in the presence of carbachol or acetylcholine at various doses.

(8) Botulinum neurotoxin A is stimulated in the presence of AA, DGLA or EPA at various doses.

(9) Each antimuscarinic agent is separately stimulated at various doses.

(10) Each antimuscarinic agent is stimulated in the presence of LPS at various doses.

(11) Each antimuscarinic agent is stimulated at various doses in the presence of carbachol or acetylcholine.

(12) Each antimuscarinic agent is stimulated at various doses in the presence of AA, DGLA, or EPA.

Materials and methods

Primary mouse bladder cells were isolated as described in example 3. In selected experiments, bladder tissue cultures were used. Bladder smooth muscle cell contractions were recorded with a Grass variegated penetrometer (Grass polygraph, Quincy Mass, USA).

Example 5: effect of oral analgesics and antimuscarinics on mouse bladder smooth muscle cell COX2 and PGE2 responses

Design of experiments

Administering to normal mice and mice with overactive bladder (overactive bladder syndrome) an oral dose of aspirin, naproxen sodium, ibuprofen, indomethacin, nabumetone, tylenol, celecoxib, oxybutynin, solifenacin, darifenacin, atropine, and combinations thereof. Control groups included untreated normal mice and untreated OAB mice with overactive bladder. 30 minutes after the last dose was given, the bladder was collected and stimulated with carbachol or acetylcholine ex vivo (ex vivo). In selected experiments, the bladder was treated with botulinum neurotoxin A prior to stimulation with carbachol. Animals were kept in metabolic cages (metablic cales) and their frequency (and volume) of urination was assessed. By monitoring water intake and cage litterTo determine bladder output. Determination of serum PGH by ELISA₂、PGE、PGE₂The levels of prostacyclin, thromboxane, IL-1 β, IL-6, TNF- α, cAMP, and cGMP the expression of CD80, CD86, and MHC class II in whole blood cells was determined by flow cytometry.

At the end of the experiment, animals were euthanized and isolated bladder contractions were recorded with Grass multiple wave-graph. Bladder sections were fixed in formalin (formalin) and COX2 responses were analyzed by immunohistochemistry.

Example 6: analgesic, botulinum neurotoxin, and antimuscarinic agents on inflammatory stimulation and noninflammation of human bladder smooth muscle cells Effect of the sexual stimulation response

Design of experiments

This study was designed to characterize how the optimal dose of analgesic as determined in examples 1 to 5 affects human bladder smooth muscle cells in cell culture or tissue culture and to address whether different classes of analgesics can act synergistically to more effectively inhibit COX2 and PGE2 responses.

Effectors, analgesics, and antimuscarinic agents are described in example 2.

Human bladder smooth muscle cells were stimulated either short-term (1 to 2 hours) or long-term (24 to 48 hours) with the following agents:

(1) each analgesic is stimulated individually at various doses.

(2) Each analgesic was stimulated at various doses in the presence of LPS.

(3) Each analgesic is stimulated at various doses in the presence of carbachol or acetylcholine.

(4) Each analgesic is stimulated at various doses in the presence of AA, DGLA, or EPA.

(5) Botulinum neurotoxin a was stimulated individually at various doses.

(6) Botulinum neurotoxin a was stimulated in the presence of LPS at various doses.

(7) Botulinum neurotoxin a is stimulated in the presence of carbachol or acetylcholine at various doses.

(8) Botulinum neurotoxin A is stimulated in the presence of AA, DGLA or EPA at various doses.

(9) Each antimuscarinic agent is separately stimulated at various doses.

(10) Each antimuscarinic agent is stimulated in the presence of LPS at various doses.

(11) Each antimuscarinic agent is stimulated at various doses in the presence of carbachol or acetylcholine.

(12) Each antimuscarinic agent is stimulated at various doses in the presence of AA, DGLA, or EPA.

Cells were subsequently analyzed for PGH₂Release of (1); release of PGE; PGE₂Release of prostacyclin, release of thromboxane, release of IL-1 β, release of IL-6, release of TNF α, COX2 activity, production of cAMP and cGMP, production of IL-1 β, IL-6, TNF α, and COX2mRNA, and surface expression of CD80, CD86, and MHC class II molecules.

Example 7: effect of analgesics, botulinum neurotoxins, and antimuscarinics on human bladder smooth muscle cell contraction

Design of experiments

Cultured human bladder smooth muscle cells are exposed to inflammatory and non-inflammatory stimuli in the presence of various concentrations of analgesic and/or antimuscarinic agents. The muscle contraction induced by the stimulation is measured to assess the inhibitory effect of the analgesic and/or antimuscarinic agent.

Effectors, analgesics, and antimuscarinic agents are described in example 2.

Human bladder smooth muscle cells were stimulated either short-term (1 to 2 hours) or long-term (24 to 48 hours) with the following agents:

(1) each analgesic is stimulated individually at various doses.

(2) Each analgesic was stimulated at various doses in the presence of LPS.

(3) Each analgesic is stimulated at various doses in the presence of carbachol or acetylcholine.

(4) Each analgesic is stimulated at various doses in the presence of AA, DGLA, or EPA.

(5) Botulinum neurotoxin a was stimulated individually at various doses.

(6) Botulinum neurotoxin a was stimulated in the presence of LPS at various doses.

(7) Botulinum neurotoxin a is stimulated in the presence of carbachol or acetylcholine at various doses.

(8) Botulinum neurotoxin A is stimulated in the presence of AA, DGLA or EPA at various doses.

(9) Each antimuscarinic agent is separately stimulated at various doses.

(10) Each antimuscarinic agent is stimulated in the presence of LPS at various doses.

(11) Each antimuscarinic agent is stimulated at various doses in the presence of carbachol or acetylcholine.

(12) Each antimuscarinic agent is stimulated at various doses in the presence of AA, DGLA, or EPA.

Bladder smooth muscle cell contractions were recorded with Grass multiple wave-writing apparatus (Quincy Mass, USA).

Example 8: effect of analgesics on Normal human bladder smooth muscle cell responses to inflammatory and non-inflammatory stimuli

Design of experiments

Culturing normal human bladder smooth muscle cells

Normal human bladder smooth muscle cells were isolated by enzymatic digestion from visually normal human bladder sections. Propagating cells in vitro by: at 5% CO₂In RPMI 1640 supplemented with 10% fetal bovine serum, 15 mmol HEPES, 2 mmol l-glutamine, 100 units/ml (U/ml) penicillin and 100 mg/ml streptomycin, and the cells were separated by trypsin treatment, followed by subculturing in a new culture flask once a week at 37 ℃. In the first week of culture, 0.5 ng/ml epidermal growth factor (epidermal growth factor), 2 ng/ml fibroblast growth factor (fibroblast growth factor) and 5. mu.g/ml insulin (insulin) were added to the medium.

Treatment of normal human bladder smooth muscle cells with analgesics in vitro

Trypsin treated (trypsinized) and at 100 microliter per well 3 × 10 microliter was treated with an analgesic solution (50 microliter per well) alone or with carbachol (10 Molar, 50 microliter per well) as an example of a non-inflammatory stimulus, or with Lipopolysaccharide (LPS) of salmonella typhimurium (1 microgram/ml, 50 microliter per well) as an example of an inflammatory stimulus⁴Cell density of individual cells bladder smooth muscle cells were seeded in microplates. When no other effectors were added to the cells, 50 microliters of RPMI 1640 without fetal bovine serum was added to the wells to adjust the final volume to 200 microliters.

After 24 hours of incubation, 150 microliters of culture supernatant was collected, centrifuged at 8,000 rpm at 4 ℃ for 2 minutes to remove cells and debris, and stored at-70 ℃ for analysis of prostaglandin E2(PGE2) response by ELISA. Cells were fixed, permeabilized and blocked, and COX2 was detected using a fluorescent substrate. In selected experiments, cells were stimulated in vitro for 12 hours to analyze COX2, PGE2, and cytokine responses.

Analysis of COX2, PGE2 and cytokine response

The COX2 and PGE2 reactions were analyzed as described in example 3. Cytokine responses were analyzed as described in example 2.

Results

Analgesics inhibited normal human bladder smooth muscle cells from reacting to COX2 with inflammatory and non-inflammatory stimuli-analysis of cells and culture supernatants after 24 hours of culture showed that none of the analgesics tested alone induced a COX2 response in normal human bladder smooth muscle cells. However, as summarized in table 6, carbachol induced a low but significant COX2 response in normal human bladder smooth muscle cells. LPS treatment, on the other hand, resulted in higher levels of COX2 responses in normal human bladder smooth muscle cells. Acetaminophen, aspirin, ibuprofen, and naproxen all inhibited the effect of carbachol and LPS on COX2 levels. The inhibitory effect of the analgesic on LPS-induced responses was seen when the drug was tested at 5 micromolar or 50 micromolar.

TABLE 6 COX2 expression in normal human bladder smooth muscle cells following in vitro stimulation with inflammatory and non-inflammatory stimuli and treatment with analgesics

^#Data are presented as mean of duplicate experiments

Analgesics inhibited the response of normal human bladder smooth muscle cells to PGE2 in inflammatory and non-inflammatory stimuli-consistent with the induction of the COX2 response described above, both carbachol and LPS induce PGE2 in normal human bladder smooth muscle cells. Acetaminophen, aspirin, ibuprofen, and naproxen were also found to inhibit LPS-induced PGE2 responses at either 5 micromolar or 50 micromolar (table 7).

TABLE 7 PGE2 expression in normal human bladder smooth muscle cells following in vitro stimulation with inflammatory and non-inflammatory stimuli and treatment with analgesics

^#Data are presented as mean of duplicate experiments

Analgesics inhibited the cytokine response of normal human bladder cells to inflammatory stimuli-analysis of cells and culture supernatants after 24 hours in culture showed that none of the analgesics tested induced IL-6 or TNF α secretion in normal human bladder smooth muscle cells alone. As shown in tables 8 and 9, the carbachol doses tested induced low but significant TNF α and IL-6 responses in normal human bladder smooth muscle cells. On the other hand, LPS treatment results in the induction of large amounts of the proinflammatory cytokines. Acetaminophen, aspirin, ibuprofen, and naproxen suppress the effects of carbachol and LPS on TNF α and IL-6 responses. The inhibitory effect of the analgesic on LPS-induced responses was seen when the drug was tested at 5 micromolar or 50 micromolar.

TABLE 8 TNF α secretion from normal human bladder smooth muscle cells following in vitro stimulation with inflammatory and non-inflammatory stimuli and treatment with analgesics

^#Data are presented as mean of duplicate experiments

TABLE 9 IL-6 secretion from normal human bladder smooth muscle cells following in vitro stimulation with inflammatory and non-inflammatory stimuli and treatment with analgesics

^#Data are presented as mean of duplicate experiments

Primary normal human bladder smooth muscle cells were isolated, cultured, and evaluated for their response to analgesics in the presence of non-inflammatory (carbachol) and inflammatory (LPS) stimuli. The objective of this study was to determine whether normal human bladder smooth muscle cells recapitulate the observations previously made with murine bladder cells.

The above experiments were repeated for the analgesic and/or antimuscarinic agent in a delayed release formulation or an extended release formulation or a delayed-extended release formulation.

The above description is intended to teach one of ordinary skill in the art how to practice the invention and is not intended to detail all those obvious modifications and variations of the invention that will become apparent to the skilled worker upon reading this description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention which is defined by the following claims. The claims are intended to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context clearly indicates to the contrary.

Claims (31)

1. A method of reducing the frequency of urination in an individual, comprising: administering to an individual having a disease that results in undesired frequency of urination an effective amount of a pharmaceutical composition comprising one or more prostaglandin pathway inhibitors.

2. The method of claim 1, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin activity.

3. The method of claim 1, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin synthesis.

4. The method of claim 1, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin transporter activity.

5. The method of claim 1, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin transporter expression.

6. The method of claim 1, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin receptor activity.

7. The method of claim 1, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin receptor expression.

8. The method of claim 1, wherein the pharmaceutical composition further comprises an analgesic.

9. The method of claim 8, wherein the analgesic is acetaminophen.

10. The method of claim 1, wherein the pharmaceutical composition is formulated for immediate release, delayed release, or extended release.

11. The method of claim 1, wherein the pharmaceutical composition is administered orally.

12. The method of claim 1, wherein the pharmaceutical composition is administered by direct injection into bladder muscle.

13. The method of claim 1, wherein the disorder that results in undesirably high frequency of urination is nocturia.

14. The method of claim 1, wherein the disease that results in undesirably high frequency of urination is overactive bladder.

15. The method of claim 1, wherein the disorder that causes undesirable frequency of urination is urinary incontinence.

16. The method of claim 1, wherein the disease that causes undesirable frequency of urination is bedwetting.

17. A pharmaceutical composition for treating a disease that results in undesired frequency of urination, comprising:

one or more prostaglandin pathway inhibitors; and

a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin activity.

19. The pharmaceutical composition of claim 17, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin synthesis.

20. The pharmaceutical composition of claim 17, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin transporter activity.

21. The pharmaceutical composition of claim 17, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin transporter expression.

22. The pharmaceutical composition of claim 17, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin receptor activity.

23. The pharmaceutical composition of claim 17, wherein the one or more prostaglandin pathway inhibitors comprise an inhibitor of prostaglandin receptor expression.

24. The pharmaceutical composition of claim 17, further comprising an analgesic.

25. The pharmaceutical composition of claim 24, wherein the analgesic is acetaminophen.

26. The pharmaceutical composition of claim 24, further comprising an antimuscarinic agent, an antidiuretic, a spasmolytic or zolpidem.

27. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is formulated for immediate-release.

28. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is formulated for delayed release.

29. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is formulated for extended release.

30. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition is further coated with an enteric coating.

31. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is formulated for oral administration.