Classifications

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  • A61K9/0065—Forms with gastric retention, e.g. floating on gastric juice, adhering to gastric mucosa, expanding to prevent passage through the pylorus
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
  • G01N33/743—Steroid hormones
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

• This disclosure relates, inter alia, to methods of detecting and quantifying bile acids from saliva.
• This disclosure provides assays for the detection and quantitation of bile acids from saliva.
• the method can be used, for example, in the identification of subjects that may be receptive to the therapeutic compositions and methods described herein.
• the assay can also be used to monitor the progress of the therapies described herein.
• a method for detecting and quantifying bile acids from saliva from a human patient comprising: collecting saliva from said patient and determining the bile acid levels in the saliva using liquid chromatography with tandem mass spectrometry.
• E02 The method of E01, wherein the human patient is being treated with an enteric coated gastro- retentive oral dosage form in the form of a tablet of a bile acid sequestrant dispersed in a polymeric matrix.
• E05 The method of E04, wherein the one or more filler or compressing agent is selected from microcrystalline cellulose, butylated hydroxy toluene, colloidal silicon dioxide, lactose, starch, maltodextrins, magnesium stearate, diacetylated monoglycerides, hypromellose, and dibasic calcium phosphate.
• the one or more filler or compressing agent is selected from microcrystalline cellulose, butylated hydroxy toluene, colloidal silicon dioxide, lactose, starch, maltodextrins, magnesium stearate, diacetylated monoglycerides, hypromellose, and dibasic calcium phosphate.
• Ell The method of E10, wherein the dose of 1,500 mg is administered as either 2 tablets, each tablet having 750 mg of the bile acid sequestrant or as 3 tablets, each tablet having 500 mg of the bile acid sequestrant, twice per day.
• [E15] A method of monitoring progress of GERD, wherein samples of a subject who is being given a bile acid lowering or sequestering agent is monitored, and a reduction in bile acid levels is indicative of effective therapy.
• Fig. 1 shows the LC/MS/MS profile of an internal standard of 'spiked' bile acids.
• FIG. 2 shows a standard curve for the quantitation of GCA.
• FIG. 3 shows the level of total conjugated bile acids in normal subjects as well as GERD patients on PPI therapy before and after a hearty meal.
• FIG. 4 shows graphically when sampling saliva ⁇ 2 hrs post meal, correlation is seen between saliva bile acid positive and Bilitec positive results.
• FIG. 5 shows that using saliva samples collected ⁇ 2 hrs post meal, the threshold to reach 100% sensitivity is 13 nM.
• FIG. 6 shows that using saliva samples collected ⁇ 2 hrs post meal, the threshold to reach 100% specificity is 37 nM.
• FIG. 7 shows efficacy results in saliva bile positive subgroup compared with overall population.
• FIG. 7, left panel: %Chg from BL to W8 in WHSS; Overall Population: Diff 6.7.
• FIG. 7, right panel: %Chg from BL to W8 in WRFS; Overall Population: Diff 14.4.
• FIG. 8 shows when sampling saliva ⁇ 2 hrs post meal, correlation is seen between saliva bile DCA positive and Bilitec positive results, similar trend to total bile acids.
• FIG. 9 shows that using saliva samples collected ⁇ 2 hrs post meal, the DCA threshold to reach 80% sensitivity /specificity is 2 nM.
• FIG. 10 shows efficacy results in saliva bile DCA positive subgroup compared with overall population.
• patient and “patient” are used interchangeably.
• a patient or a subject may be a human patient or a human subject.
• PEG-7M refers to polyethylene oxide CAS Number 25322-68-3, approximate molecular weight 300,000 (PEG-7M) (PolyoxTM WSR N-750).
• polyoxTM WSR N-750 refers to polyethylene oxide CAS Number 25322-68-3, approximate molecular weight 300,000.
• gastro-retentive dosage form denotes dosage forms which are designed to be retained in the upper gastrointestinal tract for a prolonged period of time (generally, at least 4 hours) during which they release the drug on a controlled basis.
• Bile reflux occurs when bile, a digestive fluid produced in the liver, flows upward (refluxes) from the small intestine into the stomach and then into the esophagus. Bile reflux often accompanies acid reflux, and together they may cause inflammation of the esophageal lining and potentially increased risk of esophageal cancer. See AJG (1999) 94(12):3649-3650. Bile reflux may also affect the stomach, causing inflammation (gastritis, which, if untreated, can lead to peptic ulcers). Bile reflux can be difficult to distinguish from acid reflux because the signs and symptoms are similar, and the two conditions frequently occur at the same time.
• bile reflux inflames the stomach, often causing a gnawing or burning pain in the upper abdomen.
• Other signs and symptoms may include: frequent heartburn, i.e., a burning sensation in the chest that sometimes spreads to the throat along with a sour taste in the mouth; nausea; vomiting bile; a cough; or hoarseness.
• Bile acids are steroid acids found predominantly in the bile of mammals. They are produced in the liver by the oxidation of cholesterol and they and are stored in gallbladder and secreted into the intestine in the form of salts. They act as surfactants, emulsifying lipids and assisting with the absorption and digestion of dietary fat and cholesterol.
• the principal bile acids are: cholic acid, chenodeoxycholic acid, deoxycholic acid, taurocholic acid, and glycocholic acid.
• the chemical distinctions between different bile acids are small, depending only on the presence or absence of hydroxyl groups on positions 3, 7, and 12. In humans, the most prevalent bile acids are cholic acid and chenodeoxycholic acid, and their conjugates with taurine and glycine (glycocholate and taurocholate). Some mammals synthesize predominantly deoxycholic acid.
• Bile acids play an important role in the digestive process. However, the prolonged presence or excess of bile acids in the stomach and esophagus can result in toxic effects on regional tissues.
• Disorders and/or symptoms that are believed to be associated with bile reflux, either alone or in combination with acid reflux, include, for instance, heartburn, indigestion, dyspepsia, erosive esophagitis, peptic ulcer, gastric ulcer, esophageal ulcers, esophagitis, laryngitis, pharyngitis, coarse or hoarse voice, and GERD-related pulmonary dysfunction such as coughing and/or asthma.
• GERD gastroesophageal reflux disease
• Barrett's esophagus esophageal cancer
• gastritis gastritis
• GERD is a generic term encompassing diseases with various digestive symptoms such as pyrosis, acid regurgitation, obstructed admiration, aphagia, pectoralgia, permeating feeling and the like sensibility caused by reflux in the esophagus and stagnation of gastric contents, duodenal juice, pancreatic juice and the like.
• the term covers both reflux esophagitis in which erosion and ulcers are endoscopically observed, and esophageal regurgitation-type non-ulcer dyspepsia (NUD) in which no abnormality is endoscopically observed.
• NUD esophageal regurgitation-type non-ulcer dyspepsia
• a persistent GERD patient is a patient who does not respond to PPL [0026]
• a hiatal hernia may contribute to causing GERD and can happen in people of any age.
• Other factors that may contribute to GERD include, but are not limited to, alcohol use, being overweight, pregnancy, smoking, Zollinger-Ellison syndrome, hypercalcemia, and scleroderma.
• certain foods can be associated with reflux events, including, citrus fruits, chocolate, drinks with caffeine, fatty and fried foods, garlic and onions, mint flavorings, spicy foods, and tomato-based foods, like spaghetti sauce, chili, and pizza.
• the inner mucosa of the esophagus is lined with non-keratinized stratified squamous epithelium arranged in longitudinal folds. Damage to the lining of the esophagus causes the normal squamous cells lining the esophagus to turn into a type of cell not usually found in humans, called specialized columnar cells. That conversion of cells in the esophagus by the acid reflux is known as Barrett's Esophagus. Although people who do not have heartburn can have Barrett's esophagus, it is found about three to five times more often in people with this condition.
• Barrett's esophagus does not cause symptoms itself and is important only because it seems to precede the development of a particular kind of cancer— esophageal adenocarcinoma.
• the risk of developing adenocarcinoma is 30 to 125 times higher in people who have Barrett's esophagus than in people who do not. This type of cancer is increasing rapidly in white men. This increase may be related to the rise in obesity and GERD.
• Barrett's esophagus has no cure, short of surgical removal of the esophagus, which is a serious operation. Surgery is recommended only for people who have a high risk of developing cancer or who already have it. Most physicians recommend treating GERD with acid-blocking drugs, since this is sometimes associated with improvement in the extent of the Barrett's tissue. However, this approach has not been proven to reduce the risk of cancer. Treating reflux with a surgical procedure for GERD also does not seem to cure Barrett's esophagus.
• Several different experimental approaches are under study. One attempts to see whether destroying the Barrett's tissue by heat or other means through an endoscope can eliminate the condition. This approach, however, has potential risks and unknown effectiveness.
• Esophageal cancer can occur almost anywhere along the length of the esophagus, but it frequently starts in the glandular cells closest to the stomach (adenocarcinoma). Because esophageal cancer may not be diagnosed until it's quite advanced, the outlook for people with the disease is often poor. The risk of cancer of the esophagus is increased by long-term irritation of the esophagus, such as with smoking, heavy alcohol intake, and Barrett's esophagitis. Thus, there is a link between esophageal cancer and bile reflux and acid reflux. In animal models, bile reflux alone has been shown to cause cancer of the esophagus.
• bile reflux Unlike acid reflux, bile reflux usually cannot be controlled by changes in diet or lifestyle. Instead, bile reflux is most often managed with certain medications or, in severe cases, with surgery. Neither solution is uniformly effective, however, and some people continue to experience bile reflux even after treatment. [0031] Numerous medications are used to treat heartburn and indigestion.
• the main therapies employed in the treatment of GERD and upper GI tract disorders include agents for reducing the stomach acidity, such as by using the histamine H2-receptor antagonists or proton pump inhibitors (PPIs).
• PPIs proton pump inhibitors
• H2 blockers are drugs that inhibit the production of acid in the stomach.
• Exemplary histamine H2-receptor antagonists include, for example, cimetidine (as sold under the brand-name TAGAMET HB®), famotidine (as sold under the brand-name PEPCID AC®), nizatidine (as sold under the brand- name AXID AR®), and ranitidine (as sold under the brand-name ZANTAC 75®). Both types of medication are effective in treating heartburn caused by acid reflux and usually eliminate symptoms within a short period of time.
• PPIs act by inhibiting the parietal cell H + /K + ATPase proton pumps responsible for acid secretion from these cells.
• PPIs such as omeprazole and its pharmaceutically acceptable salts are disclosed, for example, in EP 05129, EP 124495 and U.S. Pat. No. 4,255,431.
• PPIs have notable limitations. For example, patients who are non-responsive to treatment with PPI inhibitor alone may be non-responsive because even though the PPI is decreasing acid reflux from the stomach, bile acid from the duodenum is still present. Also, some patients with GERD are not fully responsive.
• This disclosure provides methods for the detection and quantitation of bile acids from fluid samples from a patient, including a human patient.
• the fluid samples can include samples taken from a subject, including urine, saliva, esophageal aspirations, serum or the like.
• the method provides a highly sensitive, non-invasive assay to detect and quantitate bile acid levels in saliva.
• elevated bile acid levels in the saliva of a subject is associated with bile acid reflux and, therefore, indicates that the subject may be amenable to therapy using a bile acid sequestrant composition.
• a method for the identification of patients receptive to the therapeutic compositions comprises collecting saliva, then quantitating the bile acid levels in the saliva, and determining whether the subject has an elevated bile acid level in the saliva.
• a sample is deemed to have an elevated bile acid level when the concentration of total bile acids exceeds 50 ⁇ /L, for example, at least 75 ⁇ /L, at least 100 ⁇ /L, at least 150 ⁇ /L, at least 200 ⁇ /L, at least 250 ⁇ /L, at least 300 ⁇ /L, or higher.
• a sample is deemed to have an elevated bile acid level when the concentration of total bile acids exceeds 13nM. In some embodiments, a sample is deemed to have an elevated bile acid level when the concentration of total bile acids exceeds 37nM.
• saliva samples are taken at least 2 hours after the subject has had the last meal, to eliminate the spike in bile acid levels that shortly follows a meal. But in some embodiments, saliva samples are taken within 2 hours after the subject has had the last meal. Subjects thus identified to have an elevated level of total bile acids are then administered with the gastric-retentive bile acid sequestrant composition. [0037] In another aspect, therapeutic progress can be monitored using the detection methods described herein.
• the method comprises obtaining saliva samples from a subject who is being treated using a gastric -retentive bile acid sequestrant composition (or other agents for treating GERD) and determining the bile acid levels in the saliva samples.
• a reduction in total bile acid levels as the course of therapy progresses is an indication of successful reduction in bile acid reflux.
• the total bile acid levels are monitored during the course of therapy to determine whether the levels fall below a threshold level, which serves as an indication of successful therapy.
• the threshold level is 300 ⁇ /L or less, for example, 250 ⁇ /L or less, 200 ⁇ /L or less, 150 ⁇ /L or less, 100 ⁇ /L or less, 75 ⁇ /L or less, 50 ⁇ /L or less, 30 ⁇ /L or less. In some embodiments, the threshold level is 13nM.
• the threshold level is 37nM.
• saliva samples are taken at least 2 hours after the subject has had the last meal, to eliminate the spike in bile acid levels that shortly follows a meal. But in some embodiments, saliva samples are taken within 2 hours after the subject has had the last meal. Subjects thus identified to have an elevated level of total bile acids are then administered with the gastric-retentive bile acid sequestrant composition as described elsewhere. To minimize variability, it is best to collect the saliva samples in as consistent a manner as possible, taking into account the time since the last dose of gastric-retentive bile acid sequestrant composition, the time since last dose of PPI, time of day, etc.
• a method of titrating an optimal dose of a gastric-retentive bile acid sequestrant composition comprises administering a first dose of a gastric- retentive bile acid sequestrant composition, then obtaining a saliva sample from the subject. The subject is then provided with a second dose, and a second saliva sample is obtained. The subject can optionally be administered a third dose, after which a third saliva sample is obtained.
• the total bile acid levels of the saliva samples are determined using the method described herein. The lowest dose that yields saliva total bile acid levels below a threshold level is deemed to be the optimal dose.
• the threshold level is 300 ⁇ /L or less, for example, 250 ⁇ /L or less, 200 ⁇ /L or less, 150 ⁇ /L or less, 100 ⁇ /L or less, 75 ⁇ /L or less, 50 ⁇ /L or less, 30 ⁇ /L or less.
• the saliva may be collected from a subject by any suitable method known in the art.
• the bile acid levels may also be determined by any suitable method known in the art, such as, for example and without limitation, liquid chromatography with tandem mass spectrometry (LC -MS/MS).
• This disclosure provides a method for detecting and quantifying bile acids from saliva from a human patient, comprising: collecting saliva and determining the bile acid levels in the saliva using liquid chromatography with tandem mass spectrometry.
• the level of individual bile acid can be determined by correlating the levels determined for that bile acid in a standard curve.
• the human patient is being treated with a gastro-retentive oral dosage form comprising a bile acid sequestrant.
• the human patient is being treated with another suitable active agent.
• the human patient is being treated with an enteric coated gastro- retentive oral dosage form in the form of a tablet of a bile acid sequestrant.
• the bile acid sequestrant is dispersed in a polymeric matrix.
• the polymeric matrix consist essentially of poly(alkylene)oxide.
• the gastro-retentive oral dosage form comprises one or more filler or compressing agent selected from microcrystalline cellulose, butylated hydroxytoluene, colloidal silicon dioxide, lactose, starch, maltodextrins, magnesium stearate, diacetylated monoglycerides, hypromellose, and dibasic calcium phosphate.
• the tablet has a tablet core and is coated with an enteric coating, which in certain further embodiments is a polyvinyl alcohol based enteric coating (such as Opadry® II 85F), for prolonged retention of the bile acid sequestrant in the stomach of the patient.
• an enteric coating which in certain further embodiments is a polyvinyl alcohol based enteric coating (such as Opadry® II 85F), for prolonged retention of the bile acid sequestrant in the stomach of the patient.
• the human patient is also administered a pharmaceutical composition comprising a PPL
• Bile acid sequestrants include, for example, cholestyramine (i.e., QUESTRAN®,
• Colesevelam or colesvelam HC1 (may be referred to herein jointly as colesevelam) is an orally administered, nonabsorbed, nondigestible polymer that binds bile acids in the GI tract.
• the bile acid sequestrant is administered to a patient at 500 mg, 700 mg, 750 mg, 1,000 mg, 1,400 mg, 1,500 mg, 2,000 mg, 2,100 mg, or more.
• the bile acid sequestrant is administered to a patient, one dose per day, two dose per day, or 3 dose per day. In certain embodiments, the bile acid sequestrant is administered to a patient as three 500 mg tablets twice per day.
• the human patient has symptomatic GERD not completely responsive to proton pump inhibitors (PPI), and is being treating by a therapeutically effective amount of an enteric coated gastro-retentive oral dosage form in the form of a tablet of colesevelam or colesevelam hydrochloride a dispersed in a polymeric matrix comprising of or consisting essentially of polyethylene oxide CAS Number 25322-68-3, approximate molecular weight 300,000 (PEG-7M) and, in certain further embodiments, one or more filler or compressing agent selected from
• the tablet is coated with a polyvinyl alcohol based enteric coating, for prolonged retention of the bile acid sequestrant in the stomach of the patient in a dose of 1,500 mg twice daily; wherein: prior to administering said enteric coated gastro-retentive, oral dosage form in the form of a tablet of a bile acid sequestrant, the patient was not completely responsive to other treatments, including, in some embodiments, individually optimized, standard-labeled dose daily PPI therapy for a minimum of 8 weeks.
• the patient has erosive esophagitis.
• the patient is treated by said enteric coated gastro-retentive, oral dosage form in the form of a tablet of a bile acid sequestrant for eight weeks (eight treatment weeks) or more.
• the dosage form is retained in the stomach until it is substantially or completely disintegrated.
• the human patient is administered the enteric coated gastro- retentive oral dosage form in the form of a tablet of a bile acid sequestrant in an amount effective to reduce the saliva bile acid levels by at least 10%, when measured more than 2 hours after a meal.
• the subject is administered with the composition in an amount effective to reduce the saliva total bile acid levels by at least 15%, for example, at least 20%, at least 30%, at least 40%, at least 50% or more, when compared with levels prior to administration of said composition when measured more than 2 hours after a meal.
• the human patient is administered an enteric coated gastro- retentive oral dosage form in the form of a tablet of a bile acid sequestrant in an amount effective to reduce the saliva bile acid levels to 200 ⁇ /L or below when measured more than 2 hours after a meal.
• the subject is administered with the composition in an amount effective to reduce the saliva total bile acid levels to 150 ⁇ /L or below, 100 ⁇ /L or below, 75 ⁇ /L or below, 50 ⁇ /L or below, or lower, when measured more than 2 hours after a meal.
• the bile acid sequestrant is colesevelam or colesevelam
• each dose of the enteric coated gastro-retentive oral dosage form in the form of a tablet for prolonged retention of the bile acid sequestrant in the stomach of the patient is in a dose of 500 mg, 700 mg, 750 mg, 1,000 mg, 1,400 mg, 1,500 mg, 2,000 mg, 2,100 mg, or more. In certain further embodiments, the dose is administered twice per day.
• a dose may be several dosage forms (tablets) disclosed herein or only one. In certain embodiments, two tablets are administered to the patient twice per day. In other embodiments, three tablets are administered to the patient twice per day.
• an ingredient of this polymeric matrix is at least one hydrophilic, water-swellable, erodible, or soluble polymer, which may generally be described as an
• osmopolymer "hydrogel” or “water-swellable” polymer. More than one of such polymers may be combined in a dosage form of the invention to achieve gastric-retention as well as the desired erosion rate.
• Polymers suitable for achieving the desired gastro-retentive and sustained-release profiles of the dosage forms used in the methods disclosed herein have the property of swelling as a result of imbibing water from the gastric fluid, and gradually eroding over a time period of several hours. Since erosion of the polymer results from the interaction of the fluid with the surface of the dosage form, erosion initiates more or less simultaneously with the swelling process. While erosion and swelling may occur at the same time, the rate for achieving maximum swelling should be faster than the rate the dosage form fully erodes to achieve the desired release profile.
• Such polymers may be linear, branched, or cross linked.
• the polymers may be homopolymers or copolymers.
• the polymer is a polyalkylene oxide.
• at least one of the one or more hydrophilic polymers is a polyethylene oxide (PEO).
• the at least one hydrophilic polymer is a polyethylene oxide having a molecular weight of about 300,000 Daltons.
• Polyethylene oxide is a polyethylene oxide polymer that has a wide range of molecular weights.
• PEO is a linear polymer of ⁇ substituted ethylene oxide and has a wide range of viscosity- average molecular weights.
• Non-limiting examples of commercially available PEOs and their approximate molecular weights are: POLYOX ® NF, grade WSR coagulant, approximate molecular weight 5 million; POLYOX ® grade WSR 301, approximate molecular weight 4 million; POLYOX ® grade WSR 303, approximate molecular weight 7 million; POLYOX ® grade WSR N60-K, approximate molecular weight 2 million; POLYOX ® grade WSR N- 80K, approximate molecular weight 200,000; PolyoxTM WSR N-750 (INCI name: PEG-7M), which is a polymer of ethylene oxide, approximate molecular weight 300,000 (polyethylene oxide CAS Number 25322-68-3, approximate molecular weight 300,000 (PEG-7M)).
• the polyethylene oxide is present in the unit dosage form in an amount ranging from 40 weight percent ratio to 75 weight percent ratio. In some embodiments, the polyethylene oxide is present in the unit dosage form in an amount ranging from 40 weight percent ratio to 60 weight percent ratio. In some embodiments, the polyethylene oxide is present in the unit dosage form in an amount ranging from 45 weight percent ratio to 55 weight percent ratio. In some embodiments, the poly(ethylene)oxide is present in the unit dosage form in an amount ranging from 45 weight percent ratio to 60 weight percent ratio. In some embodiments, the polyethylene oxide is present in the unit dosage form in an amount ranging from 40 weight percent ratio to 50 weight percent ratio.
• the poly(ethylene)oxide is present in the unit dosage form in an amount ranging from 50 weight percent ratio to 60 weight percent ratio. In some embodiments, the poly(ethylene)oxide is present in the unit dosage form in an amount ranging from 47 weight percent ratio to 53 weight percent ratio.
• the poly(alkylene)oxide is polyethylene oxide CAS Number 25322- 68-3, approximate molecular weight 300,000 (PEG-7M) (Poly oxTM WSR N-750).
• PEG- 7M herein refers to polyethylene oxide CAS Number 25322-68-3, approximate molecular weight 300,000 (PEG-7M) (PolyoxTM WSR N-750).
• the polyalkylene oxide is polyethylene oxide CAS Number 25322-68-3, approximate molecular weight 300,000 (PEG-7M) (PolyoxTM WSR N-750) at about 30% to about 46% to about 60% w/w of the tablet core weight.
• the tablets have a core, which in turn is coated to become a coated tablet.
• the poly(alkylene) oxide has approximate molecular weight of 300,000 Daltons. In certain embodiments, the poly(alkylene)oxide yields viscosity of 600 to 1,000 at moderate addition levels.
• the at least one hydrophilic polymers of the dosage form include a cellulose.
• the polymers may be synthetic polymers derived from vinyl, acrylate, methacrylate, urethane, ester and oxide monomers.
• they can be derivatives of naturally occurring polymers such as polysaccharides (e.g. chitin, chitosan, dextran and pullulan; gum agar, gum arabic, gum karaya, locust bean gum, gum tragacanth, carrageenans, gum ghatti, guar gum, xanthan gum and scleroglucan), starches (e.g.
• cellulosics are cellulose polymer that has been modified by reaction of at least a portion of the hydroxyl groups on the saccharide repeat units with a compound to form an ester-linked or an ether-linked substituent.
• the cellulosic ethyl cellulose has an ether linked ethyl substituent attached to the saccharide repeat unit, while the cellulosic cellulose acetate has an ester linked acetate substituent.
• the cellulosics for the erodible matrix comprises aqueous-soluble and aqueous-erodible cellulosics can include, for example, methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxy ethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate (CB), cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethylhydroxy ethylcellulose (EHEC).
• MEC methylethyl cellulose
• CMC carboxymethyl cellulose
• CMEC hydroxy ethyl cellulose
• HPC hydroxypropyl cellulose
• CA cellulose propionate
• CB cellulose but
• the cellulosics comprises various grades of low viscosity (MW less than or equal to 50,000 Daltons, for example, the Dow MethocelTM series E5, E15LV, E50LV and K100LY) and high viscosity (MW greater than 50,000 Daltons, for example, E4MCR, EIOMCR, K4M, K15M and K100M and the MethocelTM K series) HPMC.
• low viscosity MW less than or equal to 50,000 Daltons
• high viscosity MW greater than 50,000 Daltons
• E4MCR, EIOMCR, K4M, K15M and K100M and the MethocelTM K series HPMC.
• Other commercially available types of HPMC include the Shin Etsu Metolose 90SH series.
• erodible matrix material examples include, but are not limited to, pullulan, polyvinyl pyrrolidone (povidone), polyvinyl alcohol, polyvinyl acetate, glycerol fatty acid esters, polyacrylamide, polyacrylic acid, copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT®, Rohm America, Inc., Piscataway, New Jersey) and other acrylic acid derivatives such as
• the hydrophilic polymer is used as a binder in the unit dosage form and is selected from povidone, starch, hydroxypropylcellulose, and hydroxypropylmethylcellulose.
• the tablets used in the methods disclosed herein comprise a core and an enteric coating.
• the enteric coating surrounding the core may be applied using standard coating techniques. Materials used to form the enteric coating may be dissolved or dispersed in organic or aqueous solvents and may include one or more of the following: methacrylic acid copolymers;
• shellac hydroxypropylmethylcellulose phthalate; polyvinyl acetate phthalate;
• enteric coating polymers hydroxypropylmethylcellulose trimellitate; carboxymethylcellulose; cellulose acetate phthalate; or other suitable enteric coating polymers.
• the pH at which the enteric coat will dissolve can be controlled by the polymer or combination of polymers selected and/or ratio of pendant groups. For example, dissolution characteristics of the coating can be altered by the ratio of free carboxyl groups to ester groups.
• Enteric coating layers may also contain pharmaceutical plasticizers such as: triethyl citrate; dibutyl phthalate; triacetin; polyethylene glycols; polysorbates; acetylated glycerides, etc. Additives such as dispersants, colorants, anti-adhering, taste-masking and anti-foaming agents may also be included.
• the enteric coating is a polyvinyl alcohol (PVA)-based coating composition such as Opadry ® II 85 supplied by Colorcon.
• Opadry Enteric is a platform of fully formulated delayed release coating systems from Colorcon.
• the gastro -retentive dosage forms can be prepared by any suitable process. Methods of making the dosage forms and tablets used in the methods disclosed herein are known. See U.S. Patent No. 9,205,094 and WO2014/113377.
• the patient prior to this treatment, the patient has not been completely responsive to other treatments, including individually optimized, standard-labeled dose daily PPI therapy for a minimum of 8 weeks prior to this treatment.
• the human patients may have a disease selected from heartburn, indigestion, dyspepsia, erosive esophagitis, peptic ulcer, gastric ulcer, esophageal ulcers, esophagitis, laryngitis, pharyngitis, coarse voice, gastroesophageal reflux disease (GERD), Barrett's esophagus, gastric cancer, esophageal cancer (e.g., adenocarcinoma), and gastritis and GERD-related pulmonary dysfunction, instead of, or in addition to, patients with symptomatic GERD not completely responsive to proton pump inhibitor.
• GERD gastroesophageal reflux disease
• Barrett's esophagus gastric cancer
• esophageal cancer e.g., adenocarcinoma
• gastritis and GERD-related pulmonary dysfunction instead of, or in addition to, patients with symptomatic GERD not completely responsive to proton pump inhibitor.
• an enteric coated oral dosage form described herein further comprises butylated hydroxytoluene (BHT).
• BHT butylated hydroxytoluene
• the disclosed oral dosage form comprises about 0.01 mg to about 1.5 mg of BHT.
• the disclosed oral dosage form comprises at least about 0.06% BHT by weight per tablet core; the 0.06% BHT are added to the formlation .
• formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J P harm Sci Technol 52:238-311.
• enteric coated gastro-retentive, oral dosage forms in the form of a tablet are intended for oral delivery to a patient.
• the dosage form may additionally contain suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids and other suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids and other suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids and other suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids and other suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release aids and other suitable diluents, glidants, lubricants, acidulants, stabilizers, fillers, binders, plasticizers or release
• the dosage form comprises one or more of microcrystalline cellulose (at between 1 - 10% w/w of the tablet core), butylated hydroxytoluene oxide (at between 0.01 - 0.10% w/w of the tablet core), colloidal silicon oxide (at between 1.0 - 2.5% w/w of the tablet core) and magnesium stearate (at between 0.1 - 1.0% w/w of the tablet core).
• the enteric coating is a polyvinyl alcohol (PVA)-based coating composition, such as Opadry® II 85 supplied by Colorcon.
• Opadry Enteric is a platform of fully formulated delayed release coating systems from Colorcon.
• the tablets are coated with 1-4% Opadry® II 85F w/w of the coated tablet.
• the one or more filler or compressing agent of the oral dosage form comprising a bile acid sequestrant is microcrystalline cellulose at 1-10% w/w of the tablet, butylated hydroxytoluene at 0.01 to 0.10% w/w of the tablet, colloidal silicon dioxide at 1-5% w/w of the tablet, and/or magnesium stearate at 0.1 to 1.0% w/w of the tablet.
• the one or more filler or compressing agent is microcrystalline cellulose at 5.4% w/w of the tablet, butylated hydroxytoluene at 0.06 w/w of the tablet, colloidal silicon dioxide at 2.0 % w/w of the tablet, and/or magnesium stearate at 0.5% w/w of the tablet.
• the enteric coating of the oral dosage form comprising a bile acid sequestrant is a polyvinyl alcohol based enteric coating.
• the enteric coating of the oral dosage form comprising a bile acid sequestrant is a polyvinyl alcohol based enteric coating is Opadry II 85F.
• the enteric coating of the oral dosage form comprising a bile acid sequestrant is a polyvinyl alcohol based enteric coating is Opadry II 85F at 1-5% w/w of the tablet.
• the enteric coating is a polyvinyl alcohol based enteric coating is Opadry II 85F at 3% w/w of the tablet.
• the PEG-7M (Poly oxTM WSR N-750) is at about 30 to about 60 % w/w of the tablet. In further embodiments, the PEG-7M (Poly oxTM WSR N-750) is at about 46 % w/w of the tablet.
• the methods disclosed herein may be used to treat patients using combination therapy, comprising administering a gastric -retentive oral dosage forms comprising at least one bile acid sequestrant in combination with one or more additional therapeutic agents.
• a gastric -retentive oral dosage forms comprising at least one bile acid sequestrant in combination with one or more additional therapeutic agents.
• the active agents may be administered separately or in conjunction.
• the administration of one agent may be prior to, concurrent to, or subsequent to the administration of the other agent.
• therapies e.g., prophylactic and/or therapeutic agents
• the methods further comprise administering to the patient simultaneously, separately, or sequentially, one or more proton pump inhibitors (PPI).
• PPI proton pump inhibitors
• the PPI is administered QD (once-per-day).
• the methods further comprise administering simultaneously, separately or sequentially, one or more acid pump antagonists.
• the methods further comprise administering simultaneously, separately, or sequentially one or more agents chosen from an antacid, a histamine H2-receptor antagonist, a ⁇ -aminobutyric acid- ⁇ (GABA-B) agonist, a prodrug of a GABA-B agonist, and a protease inhibitor.
• agents chosen from an antacid, a histamine H2-receptor antagonist, a ⁇ -aminobutyric acid- ⁇ (GABA-B) agonist, a prodrug of a GABA-B agonist, and a protease inhibitor are examples of agents chosen from an antacid, a histamine H2-receptor antagonist, a ⁇ -aminobutyric acid- ⁇ (GABA-B) agonist, a prodrug of a GABA-B agonist, and a protease inhibitor.
• the two or more agents in the combination therapy can be administered simultaneously, they need not be.
• administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks.
• Combination therapy can also include two or more administrations of one or more of the agents used in the combination.
• PPI drugs are substituted benzimidazole compounds that specifically inhibit gastric acid secretion by affecting the H + /K + ATPase enzyme system (the proton pump). These drugs, for example esomeprazole, are rapidly absorbed and have very short half-lives. However, they exhibit prolonged binding to the H + /K + ATPase enzyme. The anti-secretory effect reaches a maximum in about 4 days with once-daily dosing. Because of these characteristics, patients beginning PPI therapy do not receive maximum benefit of the drug and healing may not begin for up to 5 days after therapy begins when PPIs are used alone for initial therapy of upper GI tract disorders.
• Proton pump inhibitors are potent inhibitors of gastric acid secretion, inhibiting H + /K + ATPase, the enzyme involved in the final step of hydrogen ion production in the parietal cells.
• the term proton pump inhibitor includes, but is not limited to, omeprazole (as sold under the brand-names PRILOSEC®, LOSEC, or ZEGERID®), lansoprazole (as sold under the brand-name PREVACID®, ZOTON®, or INHIBITOL®), rabeprazole (as sold under the brand-name RABECID®, ACIPHEX®, or PARIET®), pantoprazole (as sold under the brand-name PROTONIX®, PROTIUM®, SOMAC®, or PANTOLOC®), tenatoprazole (also referred to as benatoprazole), and leminoprazole, including isomers, enantiomers and tauto
• the proton pump inhibitor is omeprazole, either in racemic mixture or only the (-) enantiomer of omeprazole (i.e. esomeprazole), as set forth in U.S. Pat. No. 5,877,192, hereby incorporated by reference.
• Omeprazole is typically administered in a 20 mg dose/day for active duodenal ulcer for 4-8 weeks; in a 20 mg dose/day for gastro-esophageal reflux disease (GERD) or severe erosive esophagitis for 4-8 weeks; in a 20 mg dose/twice a day for treatment of Helicobacter pylori (in combination with other agents); in a 60 mg dose/day for active duodenal ulcer for 4-8 weeks and up to 120 mg three times/day, and in a 40 mg dose/day for gastric ulcer for 4-8 weeks.
• the dose of proton pump inhibitor is sub-therapeutic.
• Lansoprazole is typically administered about 15-30 mg/day; rabeprazole is typically administered 20 mg/day and pantoprazole is typically administered 40 mg/day. However, any therapeutic or sub-therapeutic dose of these agents is considered within the scope of the present disclosure.
• Acid pump antagonists acting by K(+)-competitive and reversible (as opposed to irreversible PPIs) binding to the gastric proton pump, which is the final step for activation of acid secretion in the parietal cell.
• One class of APAs are imidazopyridines.
• BY841 was selected from this class and is chemically a (8-(2-methoxycarbonylamino-6-methyl-phenylmethylamino)-2,3-dimethyl- imidazo [1,2-a] -pyridine).
• BY841 proved to be superior to both ranitidine and omeprazole by rapidly elevating intragastric pH up to a value of 6. The duration of this pH elevation in the dog was dose-dependent. Using both acid output and continuous 24-hr pH measurements, a pronounced antisecretory effect of BY841 has been found. Actually, a single 50 mg oral dose of BY841 immediately elevated intragastric pH to about 6.
• Examples of some APAs include, but are not limited to: BY-841 (Prumaprazole), Sch-28080, YJA-20379-8, YJA-20379-1, SPI-447, SK&F-97574, AU-2064, SK&F-96356, T-330, SK&F-96067, SB-641257A (YH-1885, Revaprazan hydrochloride, RevanexR), CS-526, R-105266, Linaprazan, Sorapraza, DBM-819, KR-60436, RQ-00000004 (RQ-4) and YH-4808.
• Other agents include: histamine H2 receptor blockers, motility agents (gastroprokinetics), antacids, antiulcerative agents, ⁇ -aminobutyric acid- ⁇ (GABA-B) agonists, prodrugs of GABA-B agonists, GCC agonists and/or protease inhibitors.
• Non-limiting examples of these additional agents include: cinitapride, cisapride, fedotozine, loxiglumide, alexitol sodium, almagate, aluminum hydroxide, aluminum magnesium silicate, aluminum phosphate, azulene, basic aluminum carbonate gel, bismuth aluminate, bismuth phosphate, bismuth subgallate, bismuth subnitrate, calcium carbonate, dihydroxyaluminum aminoacetate, dihydroxy aluminum sodium carbonate, ebimar, magaldrate, magnesium carbonate hydroxide, magnesium hydroxide, magnesium oxide, magnesium peroxide, magnesium phosphate (tribasic), magnesium silicates, potassium citrate, sodium bicarbonate, aceglutamide aluminum complex, acetoxolone, aldioxa, arbaprostil, benexate hydrochloride, carbenoxolone, cetraxate, cimetidine, colloidal bismuth subcitrate, ebrotidine, ecabet
• trypsin and chymotrypsin inhibitors can include tissue-factor-pathway inhibitor; a-2 antiplasmin; serpin a-1 antichymotrypsin family members; gelin; hirustasin; eglins including eglin C; inhibitors from Bombyx mori (see; e.g.; JP 4013698 A2 and JP 04013697 A2; CA registry No.
• hirudin and variants thereof secretory leukocyte protease inhibitor (SLPI); a-1 anti-trypsin; Bowman-Birk protease inhibitors (BBIs); chymotrypsin inhibitors represented by CAS registry Nos.
• SLPI secretory leukocyte protease inhibitor
• BBI Bowman-Birk protease inhibitors
• chymotrypsin inhibitors represented by CAS registry Nos.
• Any additional suitable agents may be administered to the patient.
• Example 1 Development of a quantitative method for the detection of bile acids from human saliva
• Method In general terms, the method disclosed herein is a simple, quantitative and noninvasive method for the detection of bile acids from fluids, including saliva.
• saliva samples are processed and subjected to LC/MS/MS analysis, and compared with control samples containing internal samples.
• Collection of saliva saliva is readily collected from a subject using a collection device, for example, the SalivaBio Oral Swab (Salimetrics, Carlsbad CA), etc.
• Samples are centrifuged at 3200 rpm for 5 min at 4°C. The supernatant is transferred to a new tube and lyophilized. The dried sample is reconstituted in 50 ⁇ of a solution of 50% (v/v) methanol.
• FIG. 1 shows the LC/MS/MS profile of an internal standard of 'spiked' bile acids provided in Table 2.
• Glycocholic acid was used to develop a standard curve.
• GCA standards of 0, 0.5, 1.0, 5, 10, 50, 100, 500, and 1000 ng/ml were prepared from 10X stocks in methanol, diluted into blank human saliva (two replicates), or in solvent.
• Table 18 shows a close correlation between input and observed concentrations.
• FIG. 2 shows the correlation between samples in saliva vs. in solvent. The method described herein is highly sensitive, allowing for the detection of bile acids with a limit of quantitation of 0.001 ⁇ /L.
• GERD patients were on proton pump inhibitor (PPI) standard therapy and received a PPI dose the morning of the visit to the clinic.
• Saliva samples were collected for 2 minutes (min) at the following time points:_Fasted state in the morning; Various times after eating a hearty breakfast: 1 hour (hr) after meal; 2 hrs after meal; 3 hrs after meal; 4 hrs after meal.
• FIG. 3 shows the time course of BA levels in normal subjects as well as GERD patients on PPI therapy before and up to 4 hours after a hearty meal.
• the BA levels in both groups are roughly comparable, similar in levels to previously reported values (See, for example, De Corso et al. (2007) Ann. Surgery 245, p.880-885), with an elevation in salivary bile acid at 1-2 hours after meal. It is likely, that refractory patients experiencing bile acid reflux (patients with was not part of this pilot study.
• Example 2 DETERMINATION OF CONCENTRATIONS OF BILE ACIDS IN HUMAN SALIVA SAMPLES USING A LIQUID CHROMATOGRAPHIC-TANDEM MASS SPECTROMETRIC METHOD
• the objective of this study was to determine concentrations of bile acids (cholic acid, chenodeoxycholic acid, glycocholic acid, deoxycholic acid, glycodeoxycholic acid, lithocholic acid, taurodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid) in human saliva samples using a qualified LC -MS/MS method.
• bile acids cholic acid, chenodeoxycholic acid, glycocholic acid, deoxycholic acid, glycodeoxycholic acid, lithocholic acid, taurodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid
• BQL Below quantitation limit; CV: Coefficient of variation; Dil: Dilution; ID: Identification; IS: Internal standard; LC-MS/MS: Liquid chromatography with tandem mass spectrometry; N:
• MPA Mobile phase A
• MPB Mobile phase B
• NA Not applicable
• QC Quality control
• SD Standard deviation
• Std Standard.
• Saliva was collected from human volunteers and then pooled. Aliquots of pooled saliva were stripped of endogenous bile acids by treatment with 2 mg/mL of cholestyramine resin (Sigma Lot No. 1425455V) for 1 hour at 37°C followed by centrifugation. The treatment/centrifugation cycle was repeated four times for a total of five cycles. After the final treatment, the saliva was pooled for use. The cholestyramine-treated saliva was used for preparation of calibration standards and for quality control samples.
• cholestyramine resin Sigma Lot No. 1425455V
• Calibration standards were prepared at concentrations of 0.500, 0.750, 1.00, 2.00, 5.00, 10.0, 50.0, 75.0 and 100 ng/mL cholic acid, glycocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid in blank matrix, 5.00, 7.50, 10.0, 20.0, 50.0, 100, 500, 750 and 1000 ng/mL chenodeoxycholic acid in blank matrix and 10.0, 20.0, 50.0, 100, 500, 750 and 1000 ng/mL lithocholic acid in blank matrix. Standards were prepared in small volumes on the day of sample extraction and were analyzed in duplicate in each analytical run.
• Quality control samples were prepared containing cholic acid, glycocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid at 1.50 ng/mL (QC-Low), 8.00 ng/mL (QC-Mid) and 80.0 ng/mL (QC- High) and chenodeoxycholic acid and lithocholic acid at 15.0 ng/mL (QC Low), 80.0 ng/mL (QC- Mid) and 800 ng/mL (QC-High) in blank matrix. Quality control samples were prepared in small volumes on the day of sample extraction.
• a lOO ⁇ L aliquot of sample (calibration standards, quality controls, blanks, and study samples) was transferred into a 96 well plate, according to a pre-defined layout.
• the plates were covered, vortex-mixed and then centrifuged for 5 minutes at 3200 rpm.
• Supematants 350 ⁇ L each were transferred into the corresponding wells of a clean 96 well plate and evaporated to dryness under nitrogen in a Turbovap set to 40°C. The dried residue in each well was reconstituted with 75 ⁇ . of 50:50 (v:v) methanol:water.
• the LC system used was a CTC PAL Autosampler along with Agilent 1260 series pumps.
• Hypersil Gold, 1.9 ⁇ column (50 x 2.1 mm) was used and maintained at 40°C during analysis. The gradient and mobile phases used are shown below. The flow rate was 0.500 mL/min and the injection volume was 10 ⁇ L.
• the detector was an Applied Biosystems Sciex API-5500 triple quadrupole mass spectrometer.
• the instrument was equipped with an electrospray ionization source in positive-ion mode and the analytes were monitored in the multiple -reaction-monitoring scan mode.
• Ql and Q3 were operated with unit resolution.
• MS/MS transition masses used for the bile acids and internal standard are listed below.
• At least 75% of the calibration standards are within ⁇ 30% of their nominal concentrations.
• At least two-thirds of the total number of quality control samples (excluding dilution QCs) and at least 50% of the QC replicates per level are within ⁇ 30% of their nominal concentrations.
• For the dilution QC, at least two-thirds of the replicates are within ⁇ 30% of the nominal concentration.
• Results for deoxycholic acid concentrations in human saliva samples are reported in Table 13. For some samples, there was no significant separation between chenodeoxycholic acid and deoxycholic acid due to matrix effects. The samples were diluted by a factor of 5 with blank matrix (cholestyramine-treated human saliva) prior to extraction and were reanalyzed in Batch 04. After the diluted samples were analyzed, matrix effects were observed for 18 samples. The reported concentration for these samples may not accurately reflect true concentration. Back-calculated concentrations for the calibration standards are reported in Table 14. Results for batch acceptance quality controls are reported in Table 15.
• Glycocholic acid 0.500 to 100 ng/mL Deoxycholic acid 0.500 to 100 ng/mL
• Electrospray ionization (positive-ion mode) Multiple-reaction-monitoring scan mode
• BQL2 Below quantitation limit (25.0 ng/niL); Batch 04, diluted 5x with blank matrix prior to extraction.
• the reassay result does not match original result (BQL).
• Matrix effects no significant separation between chenodeoxycholic acid and deoxycholic acid.

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