Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/10—Laxatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/12—Antidiarrhoeals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents

Definitions

• This invention is generally in the field of treating obesity and regulating food intake.
• this invention relates to compositions and methods of regulating food intake in which trichothecenes, derivatives and analogs thereof, or purinergic compounds are administered to alter gut motility and thereby satiety.
• the invention also relates to methods of screening for derivatives or analogs of trichothecenes and also for agonists and antagonists of purinergic receptors that are useful for regulating food intake.
• CNS central nervous system
• Currently available anti-obesity drugs work for the most part by targeting central nervous system (CNS) pathways to induce appetite suppression.
• CNS central nervous system
• Such drugs have a number of CNS-related side effects, such as anxiety, and there is the potential for chronic health problems such as hypertension, cardiovascular disease, and diabetes.
• Another current approach to treating obesity is to control appetite by using "bulk" products, which are ingested instead of normal food.
• Such bulk products have the problem of altering nutritional status in that the bulk product does not contain the necessary range of desirable nutrients.
• the individual who ingests a bulk product may refuse to consume any food, even desirable nutrients.
• Drugs that suppress appetite are among the least desirable means to treat obesity because weight is usually regained once administration of such drugs is halted. Furthermore, serious undesirable side effects, including increased risk of diseases such as primary pulmonary hypertension may limit the use of such drugs. For example, the appetite suppressants fenfiuramine and dexfenfluramine were recently pulled off the market by their manufacturers because of a potential for serious adverse effects on the lungs and heart.
• Another type of obesity treatment that has emerged recently is the use of drugs that interfere with fat absorption from the small intestine.
• a drug may, for example, inhibit pancreatic enyzmes used for fat digestion. Undigested fat is then passed through the intestines and excreted. Decreasing fat absorption can result in oily stool, oily spotting of undergarments, intestinal gas, frequent bowel movements, and decrease absorption of fat- soluble nutrients such as vitamins A, D, and E.
• This invention provides methods of treating obesity and controlling food intake in humans and other animals.
• the invention is based on the discovery of how mycotoxin trichothecenes produce food or feed refusal in humans and other vertebrate animals and also on the elucidation of the neural circuitry regulating patterns of gut motor activity ("gut motility"), which propels food through the organs of the gut.
• gut motility regulating patterns of gut motor activity
• the methods of treatment described herein involve administering a compound that affects the pattern of gut motility, that is, the pattern of contractions, relaxations, and quiescence of the smooth muscle tissue of the organs of the gut. Stimulating the "fed pattern" of gut motor activity signals satiety, that is, a feeling of fullness, which shortens the time an individual spends eating or feeding.
• compounds that stimulate the fed pattern of gut motility are useful in methods of treatment where the goal is to limit food intake, as in treating obesity.
• Compounds that stimulate the "fasting pattern” or prolong or prevent the onset of the fed pattern of gut motility will tend to increase eating or feeding time because satiety is not signaled to the body.
• Such compounds are particularly useful in methods of increasing weight gain in animals, such as livestock and poultry raised as commercial sources of meat.
• Methods of treating obesity comprise administering an effective amount of a trichothecene mycotoxin, or derivative thereof, which stimulates the fed pattern of gut motility.
• the methods of treating obesity comprise administering to an individual a trichothecene from the nivalenol-related group of structurally related compounds consisting of nivalenol; 4-deoxynivalenol ("DON", C 15 H 2 o ⁇ 6); trichothecolon; trichothecin; 3-acetyldeoxynivalenol ("3-acetyl DON", C 1 H 22 O 7 ); 7-acetyldeoxynivalenol; 3,15-diacetyldeoxynivalenol; 4-acetylnivalenol (fusarenon-X); 4,15- diacetylnivalenol.
• DON-based derivatives are also useful in the preferred methods of the invention, including DON carbonate (i.e., 3 -hydroxy- 12,13 -epoxy-9-tricothecin-8-one- 7,15 carbonate, C ⁇ H ⁇ O?); 3-acetyl-DON carbonate (i.e., 3-acetoxy-12,13-epoxy-9- tricothecin-8-one-7,15 carbonate, C ⁇ 8 H 2 oO 8 ); 3-acetyl-DON benzylidene acetal (i.e., 3- acetoxy-7,15-benzylidene-12,13-epoxy-9-tricothecin-8-one, C 24 H 2 6 ⁇ 8 ); DON-benzylidene acetal (i.e., 3-hydroxy-7,15-benzylidene-12,13-epoxy-9-tricothecin-8-one, C22H 2 O 7 ); isopropylidine DON (i.e., 3-hydroxy-7,
• the methods of treating obesity comprise administering a trichothecene, such as DON or a DON-based derivative, to an individual at a dose which is non-toxic and non-emetic, but which stimulates the fed pattern of gut motility in the individual.
• a trichothecene such as DON or a DON-based derivative
• the trichothecene, or derivative thereof may be administered by any of a variety of routes, including orally or parenterally.
• methods of treating obesity comprise administering a trichothecene analog, which is a compound that functions like a trichothecene to stimulate the fed pattern of gut motility.
• Trichothecene analogs may be structurally related to or structurally distinct from trichothecenes.
• trichothecene analogs may be derived from a trichothecene such as DON or may be any of a variety of compounds, including inorganic compounds, organic compounds, amino acids, peptides, polypeptides, proteins, nucleotides, nucleic acids, carbohydrates, lipids, and combinations thereof, which have the ability to stimulate fed pattern gut motility.
• the invention provides compositions and methods for regulating gut motility and treating obesity in an individual.
• Such methods of the invention comprise administering to the individual a compound that binds to and stimulates P XI purine receptors (purinoceptors), which are present in smooth muscle of gut tissues and are directly involved in regulating the fed pattern of gut motility.
• P XI purine receptors purinoceptors
• an agonist of the P 2 ⁇ purine receptor is a purinergic compound that binds the receptor to stimulate the fed pattern of gut motility.
• stimulating the fed pattern of the gut motor activity with a purinergic compound signals satiety and thereby shortens feeding time and decreases food intake.
• the agonist of the P 2 ⁇ t receptor that is useful in treating obesity according to the invention is a "non-desensitizing" agonist of the purine receptor, in that molecules of the agonist are able to bind the P2X1 receptor and to stimulate the P2 ⁇ t mediated fed pattern of gut motility, without eventually blocking or inactivating the receptor.
• the non-desensitizing agonist of the P 2 ⁇ t receptor is a structural analog of ATP or of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (“TNP-ATP”) for use in methods of stimulating the fed pattern of gut motility and for treating obesity.
• the invention provides compositions and methods for increasing weight in an individual.
• Such methods comprise administering to the individual a desensitizing agonist or an antagonist, such as TNP-ATP, of the P 2 ⁇ t receptor.
• a desensitizing agonist or an antagonist compound useful in such methods of the invention binds and blocks the P2X ⁇ receptor, and thereby inhibits or prevents the fed pattern of gut motility and/or prolongs the fasting pattern of gut motility, which in turn increases feeding time and food intake.
• Such methods are particularly useful in raising commercial livestock and poultry for market.
• methods are provided for identifying a compound that stimulates or inhibits (prevents) the fed pattern of gut motility by directly recording the pattern of gut motility in vivo in a test subject during administration and metabolism of the compound in the subject.
• Such methods may be used to test the regulatory activity of a compound, such as a known or new trichothecene compound, a trichothecene derivative compound, a trichothecene analog compound, an agonist of the P2 ⁇ t receptor, or an antagonist of the P 2 xt receptor.
• the ability of a compound to regulate (stimulate or inhibit) patterns of gut motility can be measured using an in vitro gut bath assay, an ex vivo gut organ assay, or an in vivo assay.
• Trichothecene and purinergic compounds, and derivatives or analogs thereof, that are identified by such screening methods as capable of stimulating the fed pattern of gut motility may be used to treat obesity according to the methods of the invention, whereas compounds that inhibit fed pattern of gut motility may be used to increase food uptake, as in promoting weight gain in livestock and poultry for commercial markets.
• Figure 1 shows a schematic diagram of the in vivo set up of Krantis et al. (Can. J. Physiol. Pharmacol, 74: 894-903 (1996)), employed for recording gastrointestinal motility in anaesthetized experimental animals, such as a rat (1).
• foil strain gauges (2) are shown attached (e.g., with a glue) to selected sites of gut organs, for example, the serosal surface of the gastric antrum (3), proximal duodenum (4), or distal ileum (5), along the longitudinal muscle layer.
• Wire leads are attached to an IBM computer data acquisition system (6).
• Figure 2 shows a schematic representation of the neural pathways controlling fed and fasting patterns of gut motility in the duodenum and ileum.
• the arrangement of cholinergic (ACh), nitrergic (NO), and purinergic (ATP) neurons is shown together with the different receptor targets and/or inputs: mus. (cholinergic muscarinic), 5-HT 3 (serotonergic), nic. (cholinergic nicotinic), P 2 ⁇ (purinergic).
• a plus sign (“+”) indicates a stimulatory input between neurons, and stimulation and contraction at smooth muscle of the gut; a minus sign (“-”) indicates an inhibitory input.
• DON deoxynivalenol, stimulator of gut hyperactivity (fed pattern) and satiety.
• NO nitric oxide, which is a non-adrenergic, non-cholinergic (NANC) inhibitory transmitter in the proximal duodenum and which is also an inhibitory transmitter of the propagatory P 2 ⁇ -purinergic and cholinergic (muscarinic, mus.) motor activity in duodenum and ileum.
• NANC non-adrenergic, non-cholinergic
• ATP adenosine triphosphate, de-sensitizing agonist of purinergic receptors, such as, P 2 ⁇ receptors.
• ACh acetyl choline, the cholinergic chemical signal that binds at muscarinic (mus.) receptors to excite motor neurons.
• 5-HT 5-hydroxytryptamine (serotonin), binds to 5-HT3 (serotonergic) receptors on neurons and is the major transmitter of enteric interneurones mediating neurogenic stimulation of NANC relaxations and cholinergic contractions of the smooth muscle of the gut.
• nic. cholinergic nicotinic receptor of neurons.
• Figures 3 A and 3B show diagrammatic chemical structures of 4-deoxynivalenol (DON) and related derivative compounds.
• Figure 3 A shows diagrammatic chemical structures for DON (Ci5H 2 oO 6 ); 3-acetyl-DON (C 17 H 22 O 7 ); isopropylidine DON (C 18 H 2 4O 6 , designated EN139491); and isopropylidine-3-acetyl-DON (C 2 oH 26 ⁇ 7 , designated EN139492).
• Figure 3B shows diagrammatic chemical structures for DON carbonate (C ⁇ H ⁇ O?, designated EN139494); 3-acetyl-DON carbonate (C 18 H 20 O 8 , designated EN139495); 3- acetyl-DON benzylidene acetal (C 24 H 26 ⁇ 8 , designated EN139496); and DON-benzylidene acetal (C 22 H 24 O , designated EN139497).
• "Ph” represents a phenyl group;
• OAc represents an acetyl group.
• Figure 4 shows a recording of the spontaneous motor activity of the rat gastric antrum in a control animal showing the oscillatory appearance of contractile and relaxant responses.
• FIG. 5 shows an example of a recording of the in vivo motility pattern of the rat duodenum control activity.
• the spontaneous in vivo motility pattern of the duodenum control activity (no DON) consists of periodic "grouped" (G) and "intergroup” (I) activity.
• Vertical marks indicate time (t) at 0, 30, 120, and 150 minutes after start of recording.
• Figures 6A-6D show data from a quantitative analysis of the effects of L-NAME and ⁇ , ⁇ -methylene ATP on frequency (Freq) and amplitude (Amp) of DON-induced relaxations in the rat duodenum ( Figure 6A, frequency, and Figure 6B, amplitude) and ileum ( Figure 6C, frequency, and Figure 6D, amplitude).
• Figures 7A-7D show results of a quantitative analysis of the effects of the 5-HT 3 receptor antagonist, granisetron, on spontaneous and DON-induced activity in the rat duodenum.
• Granisetron i.v. or i.a., broadly spaced diagonal bars
• selectively attenuated the frequency (Freq) and amplitude (Amp) of "grouped" relaxations (n 6)
• contractions (n 3)
• Figure 7C, frequency, and Figure 7D, amplitude it did not alter the stereotypic DON induced hyperactivity (compare closely spaced diagonal bars (DON alone) with filled bars (DON + granisetron)).
• Control "grouped" activity open bars).
• Figure 8 is a bar graph showing the effects (as percent of control) of ⁇ , ⁇ -methylene ATP on DON-enhanced motor activity for contractions and relaxations of the duodenum in piglets.
• Control represents a group of piglets that received no DON and no ⁇ , ⁇ -methylene ATP.
• DON represents a group of piglets that received DON only (1 mg-kg “1 ).
• ⁇ , ⁇ - methylene ATP + DON represents a group of piglets that received intra-arterial injection of , ⁇ -methylene ATP (300 ⁇ g/kg, i.a.) during DON (1 mg/kg) enhanced motor activity of the duodenum. Control group values were set as 100%.
• Open bars represent average (4 piglets) amplitude of relaxations. Filled bars represent average (5 piglets) frequency of relaxations. Open cross-hatched bars represent average (3 piglets) amplitude of contractions. Closely spaced cross-hatched bars represent average (2 piglets) frequency of contractions, " ⁇ " indicates p ⁇ 0.05 compared to control, " ⁇ ” indicates p ⁇ 0.05 compared to DON enhanced activity.
• Figure 9 is a bar graph showing the effects (as percent of control) of ⁇ , ⁇ -methylene ATP on DON-enhanced motor activity (contractions and relaxations) of the ileum in piglets.
• Control represents the group of piglets that received no DON and no ⁇ , ⁇ -methylene ATP.
• DON represents the group of piglets that received DON only (10 mg/kg).
• ⁇ , ⁇ -methylene ATP + DON represents the group of piglets that received intra-arterial injection of ⁇ , ⁇ - methylene ATP (300 ⁇ g/kg, i.a.) during DON (10 mg/kg) enhanced motor activity of the ileum.
• Control group values were set as 100%. All other values are per cent of control values.
• Open bars represent average (4 piglets) amplitude of relaxations. Filled bars represent average (5 piglets) frequency of relaxations. Open cross-hatched bars represent average (3 piglets) amplitude of contractions. Closely spaced cross-hatched bars represent average (2 piglets) frequency of contractions, " ⁇ " indicates p ⁇ 0.05 compared to control, " ⁇ ” indicates p ⁇ 0.05 compared to DON enhanced activity.
• Figure 10 shows a schematic representation of the arrangement of cholinergic, nitrergic, GAB Aergic, purinergic and VTPergic neural elements within the proposed tonic and modulatory pathways controlling spontaneous motor activity in the rat duodenum and ileum.
• a circuit pathway having GABAergic and nitrergic interneurones (NO) with cholinergic nicotinic input (nic.) (far right) is not present in the ileum.
• V P vasoactive intestinal peptide, which is an activator of nitrergic prejunctional inhibition of motor innervations.
• Figure 11 shows an example of a recording of the control spontaneous motor activity of the rat gastric antrum (site SI) and proximal duodenum (site DI).
• Typical fasting pattern of gut motor activity in the duodenum (recorded at duodenal site D2) and the gastric antrum (SI) are shown prior to administration of 3-acetyl DON treatment. Following injection (vertical arrow) of 3-acetyl DON (lOmg/kg, i.v.) the motor activity changed into a typical fed pattern motor activity lasting approximately 40 minutes. "MMC" is the "grouped" activity portion of fasting pattern of gut motility.
• Figure 13 shows the effects of intravenously administered 3-acetyl DON at 10 mg/kg of body weight on spontaneous motor activity in the rat gastric antrum (SI) and duodenum (D2). Within 60 minutes, the control motor pattern recovered (see recording after 130 min). "MMC" is the "grouped" activity portion of the fasting pattern of gut motility.
• Figure 14 shows typical in vivo recordings of the motor activity in the rat duodenum and gastric antrum (SI) illustrating the action of the compound EN139491 (lOmg/kg bw, i.v.) on "fasting pattern” motor activity. The top panel shows 20 minutes of normal fasting pattern motor activity prior to administration of compound.
• Figure 16 shows bar graphs of the effect on the amplitude of the relaxation component of gut motor activity by administration of the EN139491 DON derivative compound (10 mg-kg "1 i.v.) during spontaneous fasting pattern motor activity recorded in vivo from the proximal duodenum (DI) and gastric antrum of Halothane anaesthetized male Sprague
• Figure 17 shows bar graphs as described in Figure 16, except that the effect of administration of EN139491 on gut motor activity on the frequency of the relaxation component of gut motor activity is shown.
• Figure 18 shows bar graphs as described in Figure 16, except that the effect of administration of EN139491 on gut motor activity on the amplitude of the contraction component of gut motor activity is shown.
• Figure 19 shows bar graphs as described in Figure 16, except that the effect of administration of EN 139491 on gut motor activity on the frequency of the contraction component of gut motor activity is shown.
• Figure 20 shows a typical in vivo recording of the motor activity in the rat duodenum (DI andD2 duodenal recording sites) and gastric antrum (SI antral recording site) illustrating the action of the DON derivative compound EN139492 (at lOmg-kg "1 , i.v.) on the fasting pattern of gut motor activity.
• the typical fasting pattern motor activity is evident in the recording prior to administration of EN139492.
• the duodenum (DI and D2) displayed a typical pattern of low frequency spontaneous motor activity together with propagating "grouped" motor activity (MMCs), and the gastric antrum displayed a typically rhythmic motor activity.
• FIG. 21 shows a typical in vivo recording showing the effects of intravenously injected P 2 ⁇ purinoceptor antagonist TNP-ATP (3.5 mg-kg "1 ) on spontaneous motor activity in the duodenum (at site DI) of a Halothane anaesthetized male Sprague Dawley rat.
• Figure 22 shows a typical in vivo recording showing the effects of intravenously injected TNP-ATP (3.5 mg/kg) on DON-induced (10 mg/kg bw, i.v., vertical arrow above DON) fed pattern motor activity in the rat duodenum (site DI).
• TNP-ATP inhibitory effect on DON-induced fed pattern occurred within 1 minute of injection with TNP-ATP (vertical arrow above TNP-ATP).
• Figure 23 shows the effects of intravenously administered TNP-ATP (3.5 mg/kg) on DON-induced fed pattern motor activity in the rat gastric antrum (SI) and duodenum (D2). Within 60 minutes, the control motor pattern recovered. The boxed portions of the recording show the initial actions of TNP-ATP at each site.
• Figure 24 shows bar graphs of the amplitude of the relaxation component of gut motor activity recorded in vivo at the proximal duodenum (site DI) in Halothane-anaesthetized male Sprague Dawley rats.
• the amplitude of the relaxation component of DON-induced (10 mg/kg, i.v.) gut hyperactivity in the absence of TNP-ATP (open bar) and presence of TNP- ATP (3.5 mg/kg, i.v.) (checkered bar) is shown.
• the amplitude of the relaxation component of "grouped" MMC (diagonal bar) and the control "intergroup” activity (filled bar) is also shown.
• the amplitude of the relaxation component is expressed as the percent of the control "intergroup” relaxation amplitude, which was set at 100%. Asterisk indicates a significant (p ⁇ 0.05) difference compared to control "intergroup” motor activity.
• Each bar graph is the mean ⁇ SEM of data compiled from in vivo recordings obtained from 5-8 Sprague Dawley rats. Each animal was its own control. DI represents the recording obtained from a strain gauge positioned at 10 mm distal to the pyloric sphincter.
• Figure 25 shows bar graphs of the frequency of the relaxation component of gut motor activity recorded in vivo at the proximal duodenum (site DI) in Halothane-anaesthetized male Sprague Dawley rats.
• the frequency of the relaxation component of DON-induced (10 mg/kg, i.v.) hyperactivity in the absence of TNP-ATP (open bar) and presence of TNP-ATP (3.5 mg/kg, i.v.) (cross-hatched bar) is shown.
• the frequency of the relaxation component of "grouped" MMC (diagonal bar) and the control "intergroup” (filled bar) activity is also shown.
• the frequency of the relaxation component is expressed as the percent of the control "intergroup” relaxation frequency, which was set at 100%. Asterisk, statistics, and recording conditions of experiment as in Figure 24.
• Figure 26 shows bar graphs of the amplitude of the contraction component of gut motor activity recorded in vivo at the proximal duodenum (site DI) in Halothane- anaesthetized male Sprague Dawley rats.
• the amplitude of the contraction component of DON-induced (10 mg/kg, i.v.) gut hyperactivity in the absence of TNP-ATP (open bar) and presence of TNP-ATP (3.5 mg/kg, i.v.) (cross-hatched bar) is shown.
• the amplitude of the contraction component of "grouped” MMC (diagonal bar) and the control "intergroup” activity (filled bar) is also shown.
• the amplitude of the contraction component is expressed as the percent of the control "intergroup” contraction amplitude, which was set at 100%. Asterisk, statistics, and recording conditions of experiment as in Figure 24.
• Figure 27 shows bar graphs of the frequency of the contraction component of gut motor activity recorded in vivo at the proximal duodenum (site DI) in Halothane- anaesthetized male Sprague Dawley rats.
• the frequency of the contraction component of DON-induced (10 mg/kg, i.v.) hyperactivity in the absence of TNP-ATP (open bar) and presence of TNP-ATP (3.5 mg-kg "1 , i.v.) (cross-hatched bar) is shown.
• the frequency of the contraction component of "grouped” MMC (diagonal bar) and the control "intergroup” (filled bar) activity is also shown.
• the frequency of the contraction component is expressed as the percent of the control "intergroup” contraction frequency, which was set at 100%. Asterisk, statistics, and recording conditions of experiment as in Figure 24.
• This invention provides compositions and methods for treating obesity and regulating food intake by modulating the motor activity of gut organs in humans and other vertebrate animals. These methods are based on the discovery that trichothecene compounds, such as 4- deoxynivalenol (DON), stimulate the pattern of contractions and relaxations in organs of the gut that normally occur when food is ingested. Stimulation of this "fed pattern" of gut motility signals satiety, that is, the feeling of fullness, which is an important factor that affects the time that an individual spends eating. Trichothecenes, such as DON, act at a site outside the organs of the gut and send a signal, which is sent down neural pathways leading to the smooth muscle of the gut organs.
• trichothecene compounds such as 4- deoxynivalenol (DON)
• DON 4- deoxynivalenol
• gut refers to the gastrointestinal tract consisting of the stomach, small intestine, and large intestine.
• gut motility or “gut motor activity” refers to the motor behavior of the smooth muscle in the gastrointestinal organs (stomach, small intestine, and large intestine) of humans and other animals which activity consists of periods of alternating muscular contractions and relaxations, as well as periods of quiescence or relatively little activity.
• the frequency and amplitude of muscular contractions and relaxations of the small intestine become heightened when food is ingested in order to propel food aborally (forward) into the intestines for nutrient extraction and absorption (see, "fed pattern” of gut motility, below).
• Other patterns of gut motility may occur depending on the presence or absence of food in various parts of the gut organs.
• the proximal portion of a particular gut organ may exhibit motor behavior that differs from the activity in a distal portion of the organ, such as in the case of the duodenum (the beginning portion of the small intestine) and the ileum (the terminal portion of the small intestine).
• the "fed pattern”, “fed pattern activity”, and “segmentation” are synonymous and refer to the continuous pattern of contractions and relaxations of the small intestine of the gut in an animal, including humans, that normally occurs as the result of ingesting food.
• the fed pattern of gut motility propels ingested food through the gut for nutrient extraction and absorption, and eventually excretion of unabsorbed material as waste.
• the fed pattern of gut motor activity typically begins within minutes of ingesting food and is responsible for signaling satiety, that is, the feeling of fullness. Thus, satiety from the fed pattern of gut motility normally informs an individual that eating can be ended.
• Satiety is sensed by an individual via the fed pattern of gut motility long before the brain has an opportunity to analyze the nutrient content of the blood (a separate process that takes place hours after food has been consumed and that is responsible for signaling cravings for specific nutrients, such as, proteins, carbohydrates, salt, and fats, which are maintained at specific levels for health).
• the fed pattern is both characteristic and different for each organ and even different sites in the same organ of the gut.
• the fed pattern is characterized by a continuous series of contractions and relaxations of the smooth muscle, which mixes intestinal contents, propels food aborally into the intestines, and delays anterograde propulsion to enhance substrate absorption (Lundgren et al., Dig. Dis. Sci., 34: 264-283 (1989)).
• Krantis et al. can. J. Physiol.
• the fed pattern of gut motor activity in the duodenum is a characteristic intense pattern of hyperactivity whereas simultaneously in the gastric antrum, the fed pattern is characteristized as a measurable suppression or decrease in recorded tissue motor activity.
• This fed pattern activity replaces the "fasting pattern" of gut motor activity (see, below), which occurs after food has been propelled through the gut for nutrient extraction.
• Fed pattern motility is activated primarily by peripheral autonomic ganglia via primarily vagal inputs and also, but to a lesser extent, is controlled by the central nervous system (CNS) (see, Yoshida et al., J. Pharmacol. Exp.
• “Fasting pattern” or “fasting cyclic motor pattern” of gut motor activity refers to the motor behavior of the gut in the absence of ingested food matter or before ingesting food, when no ingested material is present for propulsion from the stomach and into intestines.
• the fasting pattern of gut motor activity is characterized by alternating periods of spontaneous, irregular contractions and relaxations ("grouped” activity) and relatively quiescent periods ("intergroup” activity).
• the fasting pattern is characterized by random contractile and/or relaxant motor activity or a generally quiescent state. Ingestion of food matter interrupts the fasting pattern of gut motility and stimulates the continuous activity of the fed pattern of gut motility.
• MMC cyclic motor behavior
• MMCs are associated with interdigestive propulsion of intestinal contents and involve sequential activation of excitatory and inhibitory neurons to propagate cycles of contractions and relaxations that originate in the stomach and terminate at the ileum.
• An MMC cycle consists of three distinct phases: phase I is a quiescent phase; phase II is a period of irregular spiking of activity, and phase III is a short period of rapid spike bursts of activity.
• MMCs provide a basic intrinsic motor pattern, which functions as a "housekeeper" of the small intestine.
• the highly propulsive phase III motor activity of each MMC cycle sweeps the intestinal lumen, clearing it of remnants to prevent bacterial overgrowth, back flow, and the accumulation of intestinal secretions (Caenepeel et al., Dig. Dis. Set, 34: 1180-1184 (1989)).
• gut motility may comprise both contractions and relaxations of smooth muscle.
• the "grouped" activity of the fasting pattern of intestinal gut motility appears to correspond to the same type of motor activity classically ascribed to phase III of MMCs.
• the presence of food in the intestinal lumen induces a switch from the fasting pattern to the fed pattern of gut motor activity.
• the method of Krantis et al. has also enabled the discovery of the mode of action of compounds called trichothecenes or trichothecene mycotoxins on gut motility.
• trichothecene 4-deoxynivalenol acts at sites outside the gut to stimulate the fed pattern of gut motility, which characteristically occurs after ingesting food and which signals satiety, that is, the sensation of fullness.
• This invention provides a method of treating obesity that takes advantage of the ability of trichothecene compounds to induce the fed pattern of gut motility and satiety.
• Methods of treating obesity described herein comprise administering a trichothecene or similar acting compound, which stimulates the fed pattern of gut motility and, thereby, satiety. Sensing fullness, the individual is thus given a signal to stop eating. When circulating levels of the administered compound decrease, satiety will decline and the individual may continue to eat or feed.
• This invention also provides methods of regulating food intake by administering an agonist or antagonist of the P 2 ⁇ purine receptor (purinoceptor), which mediates grouped relaxations of gut tissue.
• a trichothecene such as DON, and derivatives and analogs thereof, which stimulates the fed pattern of gut motility, actually acts at a site outside the gut. From that remote site of action, a signal travels down neural pathways to smooth muscle cells of the gut that express P2 ⁇ t purine receptors, which are involved in regulating the fed pattern of gut motility (see, Fig. 2).
• a compound that binds and affects the P 2X ⁇ purinoceptor is acting at the terminal portion of the neural pathway, whereas DON or other such trichothecenes act upstream.
• one group of compounds useful in the methods described herein consists of analogs of adenosine triphosphate (ATP) which may act as agonists or as antagonists of the P 2 ⁇ t purinoceptor.
• ATP adenosine triphosphate
• certain types of agonists of the P 2 ⁇ i purinoceptor bind the receptor and stimulate the fed pattern of gut motility.
• Such agonists of the P 2 ⁇ i purinoceptor may be used in lieu of trichothecenes in methods of treating obesity.
• An antagonist of the P 2 ⁇ purinoceptor is a compound that binds and blocks the receptor, thereby switching off or attenuating the fed pattern.
• Such P 2 ⁇ i receptor antagonists suppress or prevent fed pattern and satiety and, thus, may be used to prolong eating time and promote weight gain.
• Trichothecene compounds Useful in the Invention Historically, trichothecene compounds were identified as one of the toxic secondary metabolites produced by various fungi that can contaminate crops, hence the designation trichothecene mycotoxins. Animals, including humans, that ingest such contaminated crops may experience a variety of pathological symptoms of mycotoxicosis, such as vomiting, diarrhea, hemorrhagic lesions in internal organs, alimentary toxic aleukia (ATA), agranolocytosis, aplastic anemia, necrotic angina, inflammation of mucous membranes, refusal to eat, convulsions, sepsis, and in some cases, death (see, for example, Ueno, "Trichothecene Mycotoxins: Mycology, Chemistry, and Toxicology," in Advances in Nutritional Research 1980, 3: 301-353 (1980)).
• mycotoxicosis such as vomiting, diarrhea, hemorrhagic lesions in internal organs, alimentary toxic aleuk
• trichothecene mycotoxin or “trichothecene” refers to a member of a group of sesquite ⁇ enoid family of chemical compounds based on the non-olefinic parent or core compound trichothecane. All trichothecenes are modified sesquiterpenes, and contain an olefinic (double) bond (hence, trichothecewe) between carbon atoms at positions 9 and 10 (C-9, C-10), and an epoxy ring formed between carbon atoms at positions 12 and 13 (C-12, C-13). Thus, trichothecenes are also characterized as 12,13-epoxytrichothecene compounds.
• the group of "nivalenol-related" trichothecenes includes such naturally-occurring trichothecene mycotoxins as 4-deoxynivalenol (DON), trichothecolon, trichothecin, 3-acetyldeoxynivalenol (3-acetyl-DON), 7-acetyldeosynivalenol, 3,15- diacetyldeoxynivalenol, 4-acetylnivalenol (fusarenon-X), and 4, 15 -diacetylnivalenol.
• DON 4-deoxynivalenol
• trichothecolon trichothecin
• 3-acetyldeoxynivalenol 3-acetyldeoxynivalenol (3-acetyl-DON)
• 7-acetyldeosynivalenol 3,15- diacetyldeoxynivalenol
• nivalenol differs from DON in that nivalenol contains a hydroxyl group at C-4, whereas DON lacks the hydroxyl group ("4-deoxy") at position 4.
• DON is nevertheless considered as one of the least potent trichothecenes with respect to sub-lethal toxicosis (see, for example, Prelusky et al., Arch. Environ. Contam. Toxicol, 22: 36-40 (1992); Friend et al., Can. J. Anim. Sci., 66: 765-775 (1986); Ueno, in Developments in Food Science IV. Trichothecenes. chemical, biological, and toxicological aspects (Elsevier, Amsterdam, 1983), pp. 135-146).
• DON is also non-mutagenic as determined using a hepatocyte-mediated mutation assay with V79 Chinese hamster lung cells (Rogers and Heroux-Metcalf, Cancer Lett., 20: 29-35 (1983)) or a skin tumorigenesis Sencar mouse model (Lambert et al., Food Chem. Toxicol, 33: 217-222 (1995)).
• the cellular toxicity is not mediated by alteration in deoxyribonucleic acid (DNA) synthesis or repair (Bradlaw et al., Food Chem. Toxicol, 23: 1063-1067 (1985); Robbana-Barnat et al., Toxicology, 48: 155-166 (1988)).
• DON appears to undergo no extensive liver metabolism and is readily and predominantly eliminated in the urine.
• the derivatives deoxynivalenol glucuranide and de- epoxide DON have also been found in urine, apparently as the result of metabolism by microbes in the gut of animals that have received DON (see, for example, Worrell et al., Xenobiotica, 19: 25-32 (1989); Lake et al., Food Chem. Toxicol, 25: 589-592 (1987)).
• DON and other trichothecenes operate by stimulating responses outside the gut which have a pronounced effect on the muscular activity of the gut.
• DON does not act directly on the smooth muscle or other structures of gut tissues and involves no harmful effects on gut tissues to achieve its effects on gut motility. Accordingly, DON and the other nivalenol-related trichothecene compounds are particularly well suited for use in the methods described herein for treating obesity. Trichothecenes that are not structurally related to DON and the other nivalenol-related compounds, may also be used in methods of treating obesity provided they also stimulate the fed pattern of gut motility and at doses that do not result in any of the undesirable or severe symptoms of clinical mycotoxicosis.
• Trichothecenes and derivatives thereof useful in the invention may be produced biologically from fungal cultures or by chemical synthesis.
• a variety of soil fungi that have been found contaminating and growing on cereal grains and other crops produce trichothecenes as secondary metabolites.
• Such fungi include species of Fusarium, Tricothecium, Trichoderma, Myrothecium, Cylindrocarpon, and Stachybotrys (see, Ueno,
• various trichothecenes useful in the methods described herein may be produced and extracted from fungal cultures using standard culture and production techniques (see, for example, Ehrlich et al., Biochim. Biophys. Acta, 932: 206-213 (1987); Ueno, 1980 (supra) and references cited therein).
• DON is an abundant, natural contaminant of corn and wheat.
• DON and other tricothecenes may also be isolated from contaminated crops. Alternatively, they can be isolated from the Brazilian shrubs Baccharis magapotomica and cordfolia (Kupchan et al., J. Org. Chem., 42: 4221-4225 (1977)).
• the invention provides new derivatives of DON that can be synthesized from DON or 3-acetyl-DON as described in the Examples section below.
• fungi that produce trichothecenes may also be used to modify pre-existing trichothecenes.
• bio-transformation of DON and its derivatives has been undertaken in a variety of laboratories employing bacteria (Shima et al., Appl. Environ. Microbiol, 63: 3825- 3830 (1997)) or strains of Fusarium.
• bacteria Shia et al., Appl. Environ. Microbiol, 63: 3825- 3830 (1997)
• strains of Fusarium For example, F. roseum maintained in peptone- supplemented medium converts 3-acetyldeoxynivalenol to DON (Yoshizawa et al., Appl. Microbiol, 29: 54-58 (1975)).
• F. nivale can acetylate DON at the carbon at position 3 to give 3-acetyl-DON.
• these strains can deacetylate 7, 15-diacetyl-DON to give
• the chemistry of the tricothecenes is well known so that various trichothecene compounds may be synthesized by chemical or biochemical procedures.
• the tricothecenes are sesquite ⁇ ene alcohols or esters chemically related by the tetra-cyclic 12,13- epoxytricothec-9-ene skeleton (Williams, Arch. Environ Contam. Toxicol, 18: 374-387 (1989)).
• trichothecenes related to 4-DON can also be prepared using the trichothecene T-2 toxin (4 ⁇ ,15-diacetoxy-3 ⁇ -hydroxy-8 ⁇ -[3- methylbutyryloxy]-12,13 epoxytrichothec-9-ene) as starting material, since it is produced abundantly by F. tricinctum and is easy to modify at the C-3 and C-8 positions (Ehrlich et al., Appl. Environ. Microbiol, 50: 914-919 (1985); Udell et al., Z. Naturfarsch, 44: 660-668 (1989)).
• T-2 toxin 4 ⁇ ,15-diacetoxy-3 ⁇ -hydroxy-8 ⁇ -[3- methylbutyryloxy]-12,13 epoxytrichothec-9-ene
• T-2 Removal of the C-3 hydroxyl of T-2 involves initial conversion of T-2 to the 3- phenylthionocarbonate and then reduction of this intermediate with tri-n-butyltin hydride to give 3-deoxy-T-2.
• This approach has been used by others to prepare 3-deoxyanguidine and 4-deoxyverrucarol (Schuda et al., J. Nat. Prod., 47: 514-519 (1984)). It has also been shown that to generate the C-8 oxo-functionality (that is, trichothecenes related to DON), T-2 and deoxy-T-2 are oxidized with selenium dioxide (Bamburg et al., Tetrahedron, 24: 3329-3336 (1968)).
• THP- 7-DON tetrahydropyranyl-7-DON
• DIDON 3, 7-dideoxynivalenol
• Oxidation is not possible for preparation of 7-DON from T-2 tetraol because of competing ring cleavage reactions and the low solubility of T-2 tetraol in methylene chloride, which is used as the reaction solvent.
• MnO 2 oxidation is the only possible method, since the acetic acid used as the solvent for selenium dioxide oxidation, would remove the tetrahydropyranyl group.
• the side products in the MnO 2 oxidations are trichothecene, with 15-carboxaldehyde functionality.
• DON-related trichothecene compounds can be identified using mass spectra, NMR (nuclear magnetic resonance) spectroscopy, infra-red spectroscopy, anisaldehyde staining, and TLC (thin layer chromatography), to detect the presence of one or more various structural features of DON or other trichothecenes. All DON-related trichothecenes should have NMR spectra showing the expected AB coupling pattern from the C-13a and C-13b protons, and a proton at 6.5 ppm for C-10 (Cole and Cox, Handbook of Toxic Fungal Metabolites (Academic Press, New York, 1981), pp. 152-263).
• the carbonyl group at position 8 absorbs at 1660 - 1680 cm "1 . This confirms that the trichothecene retains the alpha, beta-unsaturated ketone functionality.
• the mass spectral data for the acetylated analogs of DON should show the parent ion and fragment ions anticipated by loss of acetyl or acetic acid during the process.
• Each trichothecene used in the methods described herein is preferably purified until it migrates as a discrete spot on thin layer chromatography (TLC). Homogeneity can be further assessed by using high pressure liquid chromatography (HPLC) in which the particular trichothecene should elute as a single peak.
• HPLC high pressure liquid chromatography
• GC-MS gas chromatography and mass spectroscopy analysis has also been useful in assessing purity, for example, as in showing a single peak for each type of species of purified acetylated trichothecene (Cole and Cox, 1981, supra).
• the invention also encompasses compounds in which one or more of the above-described structural features of DON, or another trichothecene, has been modified or even eliminated to make a derivative compound that may also be used in the compositions and methods of the invention.
• DON may be used in the methods of the invention, such as treating obesity, but must be used at a dose that stimulates the fed pattern of gut motility without causing any of the other undesirable side effects, including emesis (vomiting), one of the clinical symptoms of mycotoxicosis.
• the pharmaceutically acceptable dose of a trichothecene such as DON can be determined using standard methods, it would be desirable to produce a modification in a trichothecene chemical structure to yield a structurally-related compound, which is even more benign with respect to possible untoward side effects.
• Such a “benign trichothecene” is a derivative of the original trichothecene and is expected to be comparable or more potent than the original trichothecene in stimulating the fed pattern of gut motility, but with fewer or no unwanted side effects.
• a preferred derived trichothecene e.g., DON derivative
• DON a preferred derived trichothecene will exhibit one or more improved properties and will be preferred over a known trichothecene, such as DON, in methods of treating obesity.
• DON various structural features provide sites on the compound that are particularly attractive candidates for modification to form DON derivatives.
• DON is a relatively small molecule and has a limited number of sites available for modification to alter activity of this compound. Such sites include the unsaturated bond between C-9 and C-10, the presence of the 12,13 epoxy ring, the presence of hydroxyl or other goups on the structural nucleus of the trichothecene, and the occurrence of hydroxyl or other substituents at C-3, C-4, and C-15 (see, Figure 3A).
• space-filling molecular models reveal several features of the trichothecene nucleus that provide additional information in considering which site(s) to modify in providing a useful derivative of DON.
• Oxygen substituents in the A-ring make this side of the molecule more hydrophilic than when no substituent is present or when, like in T-2 toxin, an isovaleroxy side chain is present.
• the presence of hydroxyl groups at appropriate positions on the nucleus modifies biological activity. For example, the difference between 4- deoxynivalenol (DON) and nivalenol is the presence of a hydroxyl group at C-4 in DON.
• Examples of derivatives of the trichothecene DON useful in the compositions and methods of the invention include compounds referred herein as 3-acetyl-DON (C 1 H 2 O 7 ); isopropylidine DON (3-hydroxy-7,15-isopropylidine-12,13-epoxy-9-tricothecin-8-one, C 18 H 24 O 6 , designated EN 139491); isopropylidine-3-acetyl-DON (3-acetoxy-7,15- isopropylidine-12,13-epoxy-9-tricothecin-8-one, C 20 H 26 O 7 , designated EN 139492); DON carbonate (3-hydroxy-12,13-epoxy-9-tricothecin-8-one-7,15 carbonate, C ⁇ H ⁇ O ?
• EN 139494 3-acetyl-DON carbonate (3-acetoxy-12,13-epoxy-9-tricothecin-8-one-7,15 carbonate, C 18 H 20 O 8 , designated EN139495); DON benzylidene acetal (3-hydroxy-7,15- benzylidene- 12, 13-epoxy-9-tricothecin-8-one, C 22 H 24 O 7 , designated EN139497); and 3- acetyl-DON benzylidene acetal (3-acetoxy-7,15-benzylidene-12,13-epoxy-9-tricothecin-8- one, C 24 H 2 6O 8 , designated EN139496) (see, Figures 3A and 3B).
• These compounds may also serve as "parent" compounds or starting materials that may be further modified to yield additional new DON derivative compounds, which, preferably, exhibit one or more improved properties that will make the new derivative compound preferred over the parent compound or other known trichothecenes for use in compositions and methods for regulating gut motility described herein. It is also understood that an alternative to DON or other trichothecene need not be a structurally related, derivative compound, as any compound that regulates gut motility at a dose having minimal or no untoward side effects may be useful in the methods described herein.
• a trichothecene analog is any compound that mimics one or more of the characteristic and desirable biochemical activities of a trichothecene, whether or not the compound has structural characteristics of a trichothecene.
• trichothecene analogs useful in the methods of the invention regulate gut motility by acting outside the gut, in the periphery.
• trichothecene analogs which are useful in the methods described herein for treating obesity act outside the gut to stimulate the fed pattern of gut motility and, thereby, signal satiety to stop eating.
• trichothecene analogs may be structurally related or chemically derived from DON or another trichothecene (see above); an inorganic molecule; an organic molecule unrelated to the trichothecenes; biomolecules, such as nucleotides, nucleic acids, peptides, polypeptides, proteins, carbohydrates, lipids; or combinations thereof. Whether a particular compound is a trichothecene analog according to the invention may be determined using one or more methods of screening for trichothecene activities described herein.
• the P 2 ⁇ purinoceptor is a neurotransmitter receptor present in smooth muscle of the gut and is involved in the control of gut motility (see, Examples, Fig. 2).
• Adenosine triphosphate (ATP) is a naturally occurring ligand of the P 2X1 receptor. In the duodenum and ileum of the small intestine, stimulation of purinergic motor neurons releases ATP.
• ATP initially acts as an agonist to the P 2 ⁇ i purinoceptor in that the first ATP molecule to bind to a P 2 ⁇ t purinoceptor on a smooth muscle cell signals an inhibition of the smooth muscle, which then relaxes.
• relaxation is a component of gut motility that can be detected and measured using the method of Krantis et al. (1996).
• a molecule of ATP appears to remain bound to the P 2 ⁇ t purinoceptor and thereby desensitizes the muscle to additional relaxation by ATP because other ATP molecules cannot bind the receptor to signal additional relaxation events (Smits et al., Br. J. Pharmacol, 303: 695-703 (1996)).
• ATP is, therefore, a "desensitizing" agonist, which prevents any further relaxations, which are critical in both the fed pattern and fasting pattern of gut motility.
• ATP is capable of binding to all types of purinergic receptors.
• the synthetic ATP analog, ⁇ , ⁇ -methylene ATP is also a desensitizing agonist, but is specific for P 2 ⁇ species of purinoceptors.
• a non-desensitizing agonist of the P 2 ⁇ receptor has receptor binding properties that are necessary to provide continual stimulation of relaxations for gut motor activity.
• a non-desensitizing agonist of the P 2 ⁇ i receptor is a compound that binds but does not block the receptor. Each molecule of a non-desensitizing agonist is able to bind the P 2 ⁇ t receptor, evoke a relaxation, and then dissociate to be replaced by another of its kind, which in turn signals another relaxation, and so on.
• non-desensitizing agonists of the P 2 ⁇ i receptor are able to stimulate relaxation events as long as molecules of the non- desensiting agonist are available for binding to the P 2 ⁇ i receptor.
• Non-desensitizing agonists of the P 2 ⁇ i receptor stimulate the fed pattern of gut motility.
• non-desensitizing agonists of the P2 ⁇ i receptor are chemical alternatives to using DON, other trichothecenes, or trichothecene analogs in the methods of treating obesity described herein.
• a desensitizing agonist (see, above) or an antagonist of the P 2 xt receptor blocks the receptor and attenuates gut motility.
• Such compounds may be used to prevent or inhibit the fed pattern of gut motility and, thereby, inhibit satiety. Inhibiting satiety will promote longer eating or feeding time because the feeling of fullness is not evoked.
• an antagonist such as 2',3 , -O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate ("TNP-ATP"), or a desensitizing agonist, such as ⁇ , ⁇ -methylene ATP, that acts at V 2 ⁇ receptors in gut tissue is useful in methods of prolonging eating time and increasing weight gain.
• TNP-ATP 2',3 , -O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate
• a desensitizing agonist such as ⁇ , ⁇ -methylene ATP
• the ATP analog TNP-ATP is a P 2 ⁇ purinoceptor antagonist that has been used in vitro as a P2X subtype selective antagonist (whole tissue IC 5 0 in ⁇ M range) to determine the role of P 2 ⁇ i and ⁇ 2x31 homomeric and P 2 ⁇ 2/ 3 heteromeric purinoceptors (see, Lewis et al., Br. J. Pharmacol, 124: 1463-1466 (1998); Virginio et al., ?/. Pharmacol, 53: 969-973 (1998)).
• P 2 ⁇ 3 receptors are reported to be expressed only on sensory neurones (Evans et al., Semin. Neurosci., 8: 217-223 (1996)).
• the TNP-ATP antagonist is useful to show the direct involvement of the P 2 ⁇ t subtype purinoceptor in regulating the fed pattern of gut motility in vivo. Since TNP-ATP is able to prevent or inhibit the fed pattern of gut motility induced by DON, other trichothecene compounds, and derivatives thereof, this P 2 ⁇ t antagonist may, itself, be used in methods of the invention for prolonging onset of satiety and for increasing food uptake. Such methods are particularly useful for preparing livestock and poultry for market. Furthermore, TNP-ATP may be used as a parent molecule for producing derivative compounds having enhanced or improved properties affecting gut motility.
• Another class of compounds that may serve as a source of agonists or antagonists of the P 2 ⁇ ! receptor useful in this invention are anthroquinone-sulfonic acid derivatives originally described by Bohme et al. (Chromatogr., 69: 209-213 (1972)). Such derivatives may be viewed as ATP analogs and include a triazinyl moiety, which has been shown to antagonize certain ATP-mediated actions in the guinea pig (see, Kerr and Krantis, Proc. Austr. Physiol. Soc, 10: 156P (1979)). It is expected that those anthroquinone-sulfonic acid derivatives that are capable of binding the P 2 ⁇ t receptor and regulating (that is, either stimulating or inhibiting) patterns of gut motility may be useful in various methods described herein.
• Another approach for developing non-desensitizing agonists of the P 2xl receptor that are useful in treating obesity is to develop compounds from sulfonyl ureas, for example, by replacing the triphosphate moiety of the parent compound (adenosine 5 '-tetrahydrogen triphosphate) with unique, innovative acidic functionalities that are known to mimic the charge distribution in diphosphate or triphosphate, but that have never before been combined with the adenosine molecule.
• the adenosine-SO 2 -NH-CO moiety is available for combinatorial chemistry on a polymer base (Chiron Technologies).
• Compounds that bind the P 2 xi receptor and stimulate the fed pattern of gut motility are useful in methods of treating obesity according to the invention.
• tricothecenes bind to the 60S subunit of the eukaryotic ribosome and, thereby, interfere with peptidyltransferase.
• the degree of structural substitution on the trichothecene sesquite ⁇ ene affects the binding characteristics to the peptidyltransferase and hence the degree of inhibition of this enzyme (Erlich et al., 1987; Rotter et al., 1996).
• Cell cultures as described above can be employed to test or screen compounds of unknown activity as possible candidate compounds useful in the compositions and methods of treatment described herein.
• Such cell-based testing and screening methods are particularly useful to test and characterize various trichothecene or derivative compounds of unknown activity, such as newly synthesized or discovered compounds having structural features of known trichothecenes, such as DON or other nivalenol-related trichothecenes.
• Other compounds that are not structurally related to known trichothecenes may also be screened using such cell cultures.
• each test compound may be compared to one or more standard preparations of a known trichothecene, such as DON, which is typically prepared in stock solutions (10 ⁇ g/ml) in dimethyl sulfoxide. The concentration of dimethyl sulfoxide is adjusted so that it is always 1% (v/v) or less during incubations with the cells. Tricothecenes are generally stable for up to one year at room temperature (27°C).
• candidate compounds may also be screened and characterized using the method of Krantis et al. (1996), which uses miniaturized foil strain gauges and a computerized data analysis system to precisely and simultaneously record relaxations and contractions of smooth muscle in the gut. This is a means of screening compounds directly for their effects on gut motility.
• the method of Krantis et al. (1996) is able to provide an actual recording of the effect of a compound on fed and fasting patterns of gut motility in vivo, ex vivo, or in vitro (see, Examples 1 and 2).
• Such methods of screening or identifying a new compound for use in the compositions and methods of the invention may also comprise comparing the effect that a candidate compound has on gut motor activity with the effect that a known trichothecene, such as DON, has on gut motor activity.
• Candidate compounds can also be tested or screened for the ability to bind or block the P 2 ⁇ i subtype of purine receptor, which is the purinergic receptor expressed on smooth muscle and particularly involved in controlling the relaxation component of gut motility in the small intestine.
• P 2 -purinoceptors see, for example, Virginio et al., Mol. Pharmacol, 53:969- 973 (1998); Humphrey et al., Naunyn Schmiedeberg's Arch. Pharmacol, 352:585-596 (1995); Bo et al., Br. J.
• Agonists of P 2 -purinoceptors also present problems, since their binding affinities are only slightly higher than their affinities for other ATP binding proteins, and they are subject to hydrolysis by nucleotide hydrolyzing enzymes.
• [ 3 H]-labeled ⁇ , ⁇ -methylene ATP has been used as a radioligand for P 2 ⁇ -purinoceptors in preparations of urinary bladder and vas deferens smooth muscle.
• agonist binding affinities follow those observed in intact tissues.
• Enteric smooth muscle expressing P 2xl receptors can be dissociated, and the isolated smooth muscle cells maintained in primary culture. These cultures can be used in a binding assay for lead or candidate compounds that are able to act as agonists or antagonists of the P 2xl receptor.
• embryonic kidney 293 cells which express the P 2x ⁇ receptors may be used (Virginio et al, Mol. Pharmacol ⁇ 53:969-973 (1998)). This receptor subtype is also expressed in platelets and megakaryoblastic cell lines (Vial et al., Thromb. Haemost., 78: 1500-1504 (1997)), as well as in HL60 cells (Buell et al, Blood, 87: 2659-2664 (1996)).
• any cell, including recombinantly modified cells, that expresses the P 2 ⁇ t receptors in culture may be useful in screens for agonists or antagonists of the P 2 ⁇ t receptor.
• the P 2 1 purine receptor has been purified (Valera et al., Nature, 371: 516-519
• a purified P 2X1 receptor may be attached by any of a variety of linking agents to a solid substrate, such as the surface of a well in a microtiter plate, a resin particle, or the surface of an assay chip. Such arrangements allow very small quantities of compounds to be tested for the ability to bind to the receptor. Furthermore, the robotic technology that is available for screening samples in microtiter plates and assay chips permits hundreds or thousands of compounds to be accurately and continuously screened in hours with minimal supervision by the skilled practitioner.
• Lead or candidate compounds identified as having an activity in one of the above screening methods can be further evaluated using an in vitro assay, ex vivo gut organ assay, and/or an in vivo assay for gut motility, for example, by the method of Krantis et al. (1996) (see, Example 1).
• an in vitro gut organ bath assay portions of a gut organ, for example, segments of the duodenum, jejunum, and ileum of the small intestine, are excised from an animal and placed in a physiological maintenance medium, such as Krebs solution at physiological body temperature. Individual gut segments are usually mounted to record circular muscle activity, preferably at two attachment points.
• a compound may be injected, mixed, or applied to the extricated gut organ segments, and the effect on the organ's motility measured.
• gut organ assay the gut organs of an anesthetized animal are exposed, but maintained intact and at physiological conditions.
• a test or lead compound may then be conveniently applied (topically) directly on the organ, and the effect on the organ's motility monitored.
• a compound may be injected into or ingested by an animal, and the effect on gut motility measured directly.
• Sources of compounds to be tested or screened for use in the compositions and methods described herein include, without limitation, small molecule collections, combinatorial libraries, growth media or cell extracts from fungal, bacterial, and various eukaryotic cell cultures or fermentations, and biological fluids, tissues, and serum samples from humans and other animals.
• compositions which are used in methods of treating obesity.
• Other compositions of this invention are formulated for administering to animals to promote weight gain, which is especially useful in raising commercial livestock and poultry for market.
• Humans and other vertebrate animals have the same basic gut neurophysiology with respect to controlling gut motility.
• animals that can be treated using the methods described herein include, without limitation, humans and other primates, swine, cattle, sheep, birds (poultry and other birds), horses, cats, dogs, and rodents, including hamsters, guinea pigs, rats, and mice.
• Both pharmaceutical compositions and compositions for administration to other animals described herein contain an effective amount of a compound to achieve the desired effect on gut motility without significant or undesirable side effects.
• obesity is treated by administering to a human or other animal an effective amount of DON or other trichothecene, trichothecene derivative, trichothecene analog, or non-desensitizing agonist of the P 2X1 purinoceptor to stimulate or activate the fed pattern of gut motility and, thereby, signal satiety.
• a P 2 ⁇ i purinoceptor antagonist such as TNP-ATP
• a desensitizing agonist such as ⁇ , ⁇ -methylene ATP
• compositions may be in any of a variety of forms particularly suited for the intended mode of administration, including solid, semi-solid or liquid dosage forms, for example, tablets, lozenges, pills, capsules, powders, suppositories, liquids, powders, aqueous or oily suspensions, syrups, elixirs, and aqueous solutions.
• the pharmaceutical composition is in a unit dosage form suitable for single administration of a precise dosage, which may be a fraction or multiple of a dose which is calculated to produce the desired affect on gut motility.
• compositions will include, as noted above, an effective amount of the selected compound in combination with a pharmaceutically acceptable carrier and/or buffer, and, in addition, may include other medicinal agents or pharmaceutical agents, carriers, diluents, fillers and formulation adjuvants, or combinations thereof, which are non- toxic, inert, and pharmaceutically acceptable.
• a pharmaceutically acceptable buffer such as a phosphate buffered saline may be used.
• pharmaceutically acceptable is meant a material that is not biologically, chemically, or in any other way, incompatible with body chemistry and metabolism and also does not adversely affect any other component that may be present in the pharmaceutical composition.
• conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
• Pharmaceutically acceptable liquid compositions can, for example, be prepared by dissolving or dispersing an active compound that regulates gut motility as described herein and optimal pharmaceutical adjuvants in an excipient, such as, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
• the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, triethanolamine oleate.
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, triethanolamine oleate.
• Standard methods of preparing dosage forms are known, or will be apparent, to those skilled in this art (see, for example, Remington's Pharmaceutical Sciences (Martin, E.W. (ed.) latest edition Mack Publishing Co., Easton, PA).
• a dose is prepared that does not result in emesis (vomiting).
• Such sub-emetic doses are readily determinable as has been demonstrated in animal studies (see, Examples 1 and 2).
• the primary active ingredient of a composition of this invention is a compound which is a trichothecene, trichothecene analog, an agonist of the P 2x ⁇ receptor, or an antagonist of the P 2xl receptor that affects (modulates) gut motility.
• Trichothecenes such as DON are clearly capable of exerting their activity on gut motility when ingested.
• a preferred composition of this invention is formulated for oral administration. Such compounds may also be administered parenterally, for example, by intravenous, intramuscular, or intraperitoneal injection.
• compositions of the invention may be formulated as fine powders or granules containing of the compound that affects gut motility and may also contain diluting, dispersing, and/or surface active agents.
• Compositions for oral administration may also be presented in water or in a syrup as a solution or suspension, in pills, tablets, capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included.
• Binders and lubricants may also be used in compositions for oral administration. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.
• Parenteral administration is generally a method of injection.
• Injectable preparations can be prepared in conventional forms, either liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
• a compound useful in regulating gut motility may be injected intravenously in a pharmaceutically acceptable buffer.
• a compound useful in regulating gut motility may be injected intravenously in a pharmaceutically acceptable buffer.
• such a compound may alternatively be prepared as a bolus, which may contain a mordant for gradual release from an injection site.
• One approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained (see, for example, U.S. Patent No. 3,710,795).
• the exact, effective amount of a compound useful in regulating gut motility in the compositions and methods described herein will vary from subject to subject, depending on the age, weight and general condition of the subject, the degree of obesity being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount for an ideal dose applicable to all individuals. However, it is expected that generally a trichothecene such as DON will be used or tested in a range of 0.01 - 100 mg/kg body weight. Furthermore, the useful dosage selected for a particular individual will be a sub-emetic dose, that is, a dose that does not evoke vomiting in that individual.
• compositions For commercial pharmaceutical compositions, it is understood that a pharmaceutically effective and suitable amount of trichothecene, trichothecene derivative, trichothecene analog, P 2 ⁇ t receptor agonist, or receptor antagonist will be determined, in the case of human use, by the healthcare professional in studies acceptable to the standards of the United States Food and Drug Administration (or comparable agency). For use in animals, an appropriate composition will be determined and formulated according to the standards and practices for commercial livestock feed or veterinary medicine.
• Example 1 The following example shows that DON acts at sites outside the gut and interferes with specific intrinsic neural pathways of the stomach and small intestine, giving rise to altered patterns of motor activity. These findings show that DON induces loss of appetite (as illustrated by feed refusal in animals) and support a method of inducing such loss of appetite.
• Normal gastrointestinal motility is dependent on the intrinsic (enteric) neural networks of the gut wall, with modulatory inputs from the periphery and central nervous system (CNS).
• the intrinsic circuitry coordinates reflex activity, such as the peristaltic reflex, or complex patterned motor activity, such as interdigestive migrating myoelectric complexes (MMCs) that occur within the fasting pattern and segmentation driving fed pattern.
• MMCs interdigestive migrating myoelectric complexes
• Rats were anesthetized using a Halothane (4%) in oxygen mixture. Rats were maintained under Halothane (2%) anesthesia on a heated scavenging table to preserve body temperature at 37°C.
• the right carotid artery was exposed and cannulated using PE50 tubing to monitor blood pressure via a pressure transducer (P23ID, Gould Statham, OH) connected to an LBM PC data acquisition system.
• the jugular vein was cannulated (PE 50 tubing) to allow for intravenous (i.v.) injections.
• Foil strain gauges Showa type Nl 1, Durham Instruments, Pickering, ON
• All foil strain gauges were oriented parallel to the longitudinal muscle layer since this affords the most sensitive setting for recording circumferential motor activity.
• Wire leads from the foil strain gauges were exteriorized and attached to an LBM PC data acquisition system via a 3-channel interface box.
• a schematic diagram of this method for recording gut motility is shown in Figure 1. After completion of the surgery, the rats were turned over to the prone position, and Halothane was maintained at 1% for the remainder of the experiment.
• Ex vivo organ preparations were made using the same surgical procedures described above, except rats were left in the supine position to maintain the foil gauge attachment sites exposed. This allowed for local administration of drugs directly onto the serosal surface of the gut. Regular application of warmed saline kept the exposed gut segments moist.
• Gut organs for use in in vitro gut organ baths were prepared according to McKay and Krantis (Can. J. Physiol. Pharmacol, 69: 199-204 (1991)). Rats were euthanized, and 4-5 cm segments of the proximal duodenum, jejunum, and ileum were quickly removed, carefully cleared of contents, any mesenteric attachments dissected away, and then placed in an organ bath containing Krebs solution of the following composition (mM): Na + (151.0), K + (4.6), Mg 2+ (0.6), Ca 2+ (2.8), Cl “ (134.9), HCO 3 ' (24.9), H 2 PO 4 " (1.3), SO 4 2" (0.6), and glucose (7.7).
• mM Na + (151.0), K + (4.6), Mg 2+ (0.6), Ca 2+ (2.8), Cl “ (134.9), HCO 3 ' (24.9), H 2 PO 4 " (1.3), SO 4 2" (0.6), and glucose (7.7).
• This solution was maintained at 37° C and continuously gassed with 95% O 2 : 5% CO 2 to give a pH 7.4.
• Individual gut segments were then mounted horizontally to record circular muscle activity at two attachment points on the mesenteric border 25 mm apart, each opposite a frog heart clip tethering the segment to the bottom of the organ bath, and each connected to Grass isometric force transducers by thin polyester string. Mechanical activity detected by the transducers was monitored directly by a MacLab Macintosh data acquisition system (Apple Co ⁇ ., Toronto, Ontario).
• the motor activity recorded in the in vivo and ex vivo experiments was acquired, digitized, and stored by an IBM data acquisition system that calculated, in addition to other variables, the amplitude and frequency of motor responses (Krantis et al., 1996). Qualified responses were marked depending on their capacity to satisfy a set of six numerical parameters (separately for contraction and relaxation), based on user-defined threshold values that must be satisfied within limited time periods. These parameters were continuously monitored over sequential two minute periods and adjusted as deemed necessary to efficaciously mark motor responses with 95-100% accuracy. The data were then output and organized into tabular form for statistical analysis.
• ⁇ , ⁇ -methylene adenosine triphosphate ⁇ , ⁇ -methylene ATP, 300 mg/kg
• N- ⁇ -nitro-L-arginine methyl ester L-NAME, 10 mg/kg
• BRL 43694 granisetron, 80 mg/kg
• pentolinium 5 x 10 "5 M
• hexamethonium 18 mg/kg, s.c
• the concentrations of drugs applied in the organ bath preparations (in vitro) or topically in the ex vivo experiments were: carbachol (0.5 mM), papaverine (10 mM), ATP (0.5 mM), DMPP (50 mM), 3-APS (0.5 mM) (Sigma Chemical Company, Toronto, ON) and DON (20 mM). Drug volumes were never more than 1% of the bath volume.
• spontaneous motor activity of the distal ileum consisted primarily of randomly occurring contractions and/or relaxations of relatively high amplitude and low frequency.
• DON administered systemically in a 1 or 2 mg-kg "1 bolus, did not affect control motor activity. However, at 10 mg-kg '1 , DON disrupted spontaneous motor patterns, as described below. Treatment with 20 mg-kg '1 of DON did not yield significantly greater effects.
• Ex vivo preparations exhibited patterned motor activity similar to that of the in vivo preparations.
• the vitality of the gut regions examined was verified by observing predictable responses to pharmacological stimuli that are known to act directly on smooth muscle, such as, papaverine (10 mM), which relaxes smooth muscle, and carbachol (0.5 mM), which induces cholinergic muscarinic receptor mediated contractions.
• Topically applied DON did not interfere with the action of these drugs.
• the gut segments exhibited relaxant responses to the putative non-andrenergic, non-cholinergic (NANC) inhibitory transmitter ATP (0.5 mM) and to neural stimulation, using the GABA A receptor agonist 3-APS (0.5 mM) or the nicotinic receptor agonist DMPP (50 mM).
• NANC non-andrenergic
• GABA A receptor agonist 3-APS 0.5 mM
• DMPP nicotinic receptor agonist
• L-NAME In anesthetized rats exhibiting spontaneous motor activity, the nitric oxide (NO) synthase inhibitor, L-NAME, attenuates NO-mediated "intergroup” relaxations and enhances "grouped” activity of the duodenum (unpublished observations). Therefore, the effect of L-NAME on the action of DON was examined.
• Systemically administered L- NAME (10 mg-kg “1 , n 5), did not alleviate (p>0.05) DON induced hyperactivity of frequency and amplitude of relaxations in the duodenum (see, Figure 6 A, frequency, and Figure 6B, amplitude).
• L-NAME In the ileum, L-NAME always enhanced both the frequency and amplitude of relaxations of spontaneous motor activity to the same level as with the presence of DON alone (see, Figure 6C, frequency, and Figure 6D, amplitude).
• Nicotinic receptors Cholinergic nicotinic mechanisms are fundamentally involved in the control of intestinal motility (see, Furness and Costa, Neurosci., 5: 1-20 (1980); Gershon, Ann. Rev. Neurosci., 4: 227-272 (1981)).
• DON The action of DON was maximal with 10 mg/kg of DON; this dose is comparable to other studies using rodents, where up to 40 mg/kg (i.v.) of DON was used to induce alterations in feeding (Rapely et al., Lab. Anim. Sci, 38: 5041 (1988)).
• a progressive tolerance to DON was evident in the rats examined.
• DON-induced hyperactivity in the small intestine lasted up to 60 minutes, then full restoration of control motor patterns followed. Subsequently, responsiveness to routinely applied pharmacological stimuli was maintained; except for DON, which was ineffective after proximate successive applications, characteristic of the development of tachyphylaxis to DON. However, this tachyphylaxis was not sustained, possibly due to the high rate of DON detoxification in rats (Prelusky et al., Fund. Appl. Toxicol, 10: 276-286 (1988)).
• segmentation is characterized by narrow annular contractions inte ⁇ osed between relaxations in the small intestine, and reduced motility in the gastric antrum.
• the fed pattern functions to mix intestinal contents and delay anterograde propulsion to enhance substrate abso ⁇ tion (Lundgren et al, Dig. Dis. Sci., 34: 264-283 (1989)).
• Fed pattern motility is activated by peripheral autonomic ganglia via primarily vagal inputs and is controlled, to a lesser extent, by the CNS (Chung et al, Can. J. Physiol.
• NO nitric oxide
• L-NAME 10 mg/kg, i.v.
• Nicotinic receptor blockade abolished both spontaneous and DON induced motor activity in the small intestine, which explains results from a several previous studies.
• Nicotinic ganglionic transmissions are known to mediate cholinergic stimulation of both excitatory and inhibitory intramural neurons that modulate and process enteric neural signals (Bornstein et al, Clin. Exp. Pharmacol. Physiol, 21: 441-452 (1994), Gershon, Ann. Rev. Neurosci., 4: 227-272 (1981)).
• enteric neural signals Bosset et al, Clin. Exp. Pharmacol. Physiol, 21: 441-452 (1994), Gershon, Ann. Rev. Neurosci., 4: 227-272 (1981)
• cholinergic neurons mediate all motility patterns of the gastrointestinal tract, including the peristaltic reflex and MMCs. Therefore, treatment with nicotinic antagonists would effectively block most if not all enteric neural circuits.
• 5-HT 3 sites on vagal afferents are not likely to be involved in DON actions related to gut motor activity, since DON was ineffective when applied directly onto the exposed gut of a whole animal 5-HT 3 receptors are also localized to myenteric neurons that are involved in the enteric circuits regulating the interdigestive motor pattern (Hoyer,
• Neuropsychopharmacol 3: 371-383 (1990), Yoshida et al, 1991), and these neurons may occur within the enteric pathway(s) targeted by DON.
• Example 2 The effects of DON on spontaneous motor activity of the gastrointestinal tract in swine in vivo: involvement of enteric P2 ⁇ -purinoceptors.
• This example demonstrates that the trichothecene DON affects gut motility by acting at a site in the peripheral nervous system and that the affect of DON may be counteracted by the ⁇ 2 % ⁇ purinoceptor desensitizing agonist, ⁇ , ⁇ -methylene ATP, which binds with high affinity to the V 2 ⁇ purinoceptor on gut tissue.
• Ketamine is a dissociative anaesthetic, which causes an increase in blood pressure and skeletal tone, and the trachea will be stiff.
• a cataleptic sedation is produced with a lack of awareness of the surroundings.
• salivary secretions are increased and hence airway obstruction is a hazard; yet atropine cannot be used.
• Anesthesia was induced using a Halothane-oxygen mixture by application of a face mask.
• Topical pharyngeal anaesthesia was provided using 1-2 doses of lidocaine aerosol (10 mg per dose, Xylocain, Sigma). Animals were then intubated, and a surgical plane of anesthesia was achieved using Halothane (3-4%) in oxygen (200 ml/min) via a closed non-rebreathing circuit.
• a catheter was inserted into a superficial ear vein for electrolyte replacement (0.9% saline) and intra venous drug injections.
• the femoral artery was also cannulated for intra-arterial drug injections.
• PE 205 tubing was fed in a retrograde direction such to place the tip of the cannula at the level of the superior mesenteric artery.
• Blood pressure was also monitored through this arterial catheter by way of a pressure transducer (P23ID, Gould Statham, OH, USA) connected to an online IBM data acquisition system.
• the animals were then subjected to a laparotomy, and foil strain gauges (Showa type Nl 1, Durham Instruments, Pickering, ON) were affixed, using Vet Bond glue as described in Example 1, onto the serosa of the gastrointestinal tract.
• One strain gauge was placed on the gastric antrum (5-10 cm distal to the pylorus); a second gauge was placed on the anti- mesenteric border of the proximal duodenum (2-10 cm from the pylorus), and a final gauge was attached onto the serosa of the distal ileum (2-10 cm distal to the cecum). All three foil strain gauges were oriented parallel to the axis of the longitudinal muscle. Leads from the strain gauges were exteriorized and attached to the LBM data acquisition system, via an interface box. Following completion of the surgery, the pigs were turned over to their side, and a light plane of anesthesia was maintained for the remainder of the experiment by 1-2% Halothane.
• Motor activity was continuously recorded from all foil strain gauges simultaneously using data acquisition software (AD 1000 analog to digital conversion card, Real Time Devices Inc., Dr. Frank Johnson, Institute of Medical Engineering, University of Ottawa) and an LBM compatible computer. Qualified motor responses were selected based on their capacity to satisfy two sets (for contractions and relaxations) of six numerical values. These values defined threshold duration and magnitude parameters that efficaciously marked motor activity based on the user's visual inspection of the recordings. The user is able to continuously monitor these parameters over sequential two minute periods and adjust the values as deemed necessary to efficaciously mark motor responses within 95-100 % accuracy.
• the data acquisition software output the frequency, amplitude, area, time to peak and duration of both contractile and relaxant motor responses.
• Spontaneous motor activity consisted of an irregular pattern of contractile and/or relaxant motor activity (Table 1). Occasionally, activity resembles MMCs, consisting of phase III propagatory type activity ("grouped" activity) and quiescent periods, were evident. The duration of the "grouped” activity was approximately 5 minutes, however the cycle length could not be accurately determined, since the "grouped" activity did not arise more than 2 or 3 times during the control period, which only lasted up to 2 hours in our experiments. MMCs in fasted pigs are known to have a cycle length of 70-115 minutes.
• the "grouped" activity consisted of relatively high amplitude, high frequency relaxations and contractions: frequency of contractions: 11.9 ⁇ 0.5 events/min; amplitude of contractions: 0.08 ⁇ 0.01 g; frequency of relaxations: 12.9 ⁇ 0.8 events/min; amplitude of relaxations: 0.07 ⁇ 0.01 g.
• DON increased (p ⁇ 0.05) the frequency and amplitude of the spontaneous motor activity by 182 ⁇ 40 % and 206 ⁇ 38 % respectively; this effect lasted up to 30 minutes before recovering to the control pattern.
• This differential action of DON was not obviously correlated to either the dose injected or the route of administration. Furthermore, neither the dose injected nor the route of administration induced any changes to the mean arterial blood pressure, which continued a steady level for the duration of the control and DON treatment periods.
• DON Systemically administered DON induced a dose-dependent increase in the ileal motor activity.
• the dose-response effect was evident in both frequency and amplitude parameters of the motor activity.
• maximal effects of DON occurred at a dose equal to or greater than 1 mg/kg, where both the frequency, as well as the amplitude, of the contractile and relaxant spontaneous motor activity were significantly (p ⁇ 0.05) increased.
• the frequency and amplitude of the present motor activity started to decrease, however, two hours after the initial injection of DON, the motor activity was still significantly higher than control.
• ⁇ , ⁇ -methylene ATP (175 ⁇ gkg, i.a.) usually induced a small phasic relaxation of the duodenum.
• DON induced hyperactivity did not appear to be affected.
• ⁇ , ⁇ -methylene ATP significantly decreased the amplitude, but not the frequency, of the DON induced relaxations and contractions (see, Fig. 8).
• Example 3 Non-adrenergic, non-cholinergic (NANC) control of interdigestive motor activity in the rat small intestine, in vivo.
• NANC non-adrenergic, non-cholinergic
• the migrating motor complex is associated with interdigestive propulsion of intestinal contents, and like peristalsis, involves sequential activation of excitatory and inhibitory pathways.
• the neural circuitry underlying peristalsis comprises excitatory (primarily cholinergic) and inhibitory non-adrenergic, non-cholinergic (NANC) motor neurons innervating the gastrointestinal smooth muscle, as well as excitatory and inhibitory interneurones (Costa and Brookes, Am. Gastroenterol, 89: S129-S137 (1994)).
• excitatory primarily cholinergic
• NANC non-cholinergic
• "Grouped” activity was typified by an intense period (approximately 2-4 min) of contractile and/or relaxant motor activity that propagated caudally at a rate of 3.4 ⁇ 0.6 cm-min "1 , in a manner reminiscent of MMCs.
• the "intergroup” activity consisted of randomly occurring, low amplitude, low frequency relaxations and/or contractions.
• Spontaneous motor activity of the ileum consisted of either relaxations (50% of all animals tested) or contractions only (30% of all animals tested); in the remaining experiments, contractile and relaxant motor activity occurred together.
• the predominance of one type of motor response is thought to be indicative of the intrinsic tone of the smooth muscle; where tissue with high tone shows mainly relaxant activity, whilst tissue with low tone more readily shows contractions.
• tissue with high tone shows mainly relaxant activity
• tissue with low tone more readily shows contractions.
• the spontaneous ileal relaxations and contractions occurred at a relatively low frequency, and were of relatively high amplitude.
• the ileum displayed periodic bursts of high frequency motor responses comparable to phase III MMC activity.
• the substituted derivatives of ATP, ⁇ , ⁇ -methylene ATP and methyl-S ATP have differential affinities for P ⁇ - and P 2 ⁇ -purinoceptors, respectively (Burnstock and Kennedy, Gen. Pharmacol, 16: 433-440 (1985)). Tissues develop tachyphylaxis following prolonged exposure to these agents, and thus in this manner it was possible to discriminate between P 2 ⁇ and P 2 ⁇ receptor-mediated responses.
• the effects of ⁇ , ⁇ -methylene ATP on spontaneous duodenal contractions were variable and could not be analyzed.
• Proximate rechallenge with ⁇ , ⁇ -methylene ATP did not elicit another response, indicative of the development of tachyphylaxis.
• Spontaneous contractions were not affected by ⁇ , ⁇ -methylene ATP treatment.
• ATP exhibits multiple enteric neural functions, since in addition to mediating P 2 ⁇ - purinoceptor dependent relaxations in the duodenum and ileum, ATP via P 2Y -purinoceptors can stimulate NO-mediated non-propagating "intergroup" relaxations in the duodenum (Glasgow et al, Am. J. Physiol, 276 (Gastrointest. Liver Physiol, 38): G889-G896 (1998)).
• the P2 ⁇ -purinoceptor agonist methyl-S-ATP
• inhibited spontaneous ileal relaxations In contrast to ⁇ , ⁇ -methylene ATP, methyl-S-ATP did not evoke an ileal relaxation upon injection.
• P 2 ⁇ -purinoceptors are not present on the smooth muscle, or else are not active within the inhibitory motor innervation(s) of the ileum.
• the data support the view that in the rat ileum, P 2 ⁇ -purinoceptors are involved in the activation of pathways mediating tonic inhibition of the purinergic NANC motor neurons targeting P 2 ⁇ purinoceptors.
• P 2 Y-purinoceptors may be present on nitrergic intemeurones subserving tonic inhibition, or on other intemeurones within this prejunctional input.
• nitrergic and purinergic intemeurones may also represent the same population, since ATP and NO synthase are co-localized in myenteric neurons in the rat ileum (Belai and Bumstock, Cell Tiss. Res., 278: 197-200 (1994)).
• VIP is a NANC inhibitory transmitter in numerous gut regions (Bojo et al, Eur. J. Pharmacol, 236: 443-448 (1993); Mule et al, J. Auton. Pharmacol, 12: 81-88 (1992).
• VIP evoked a transient relaxation in the rat duodenum.
• the subsequent development of tachyphylaxis to VIP inhibited the contractile and relaxant "intergroup" activity and enhanced the "grouped” motor activity.
• the data indicate that the initial VlP-evoked relaxations are dependent on NO and sensitive to VIP desensitization.
• VIPergic intemeurones must be targeting direct motor innervations (the nitrergic and cholinergic motor neurons) of the "intergroup” activity, as well as the nitrergic prejunctional modulatory inputs of the "grouped” activity.
• VLP did not evoke a relaxation upon injection, it is unlikely that VIPergic neurons mediate direct inhibitory input to the ileal smooth muscle (Smits et al, Br. J.
• VLP plays a major role in the tonic inhibition of circular muscle motor activity via an inhibitory neural action (Fox-Threlkeld et al, Peptides, 12: 1039-1045 (1991)).
• the data support the view that VIP targets the NO-dependent prejunctional modulation of the purinergic inhibitory motor innervations in both the duodenum and ileum.
• These purinergic motor pathways specifically generate the propagatory motor activity of the small intestine.
• VIP specifically inhibits phase III activity of MMCs, and VLP antagonists initiate phase III activity (Hellstrom and Ljung, Neurogastroenterol Motil, 8: 299-306 (1996).
• the conclusion is that VLP simultaneously stimulates excitatory motor inputs and inhibitory nitrergic prejunctional inputs of the purinergic motor neurons in the rat ileum.
• This coordinated inhibition and disinhibition is mediated by the inhibitory nitrergic inputs, and it is precisely the control of these prejunctional neuronal pathways, along with the tonically active motor pathways, which generates cyclical (interdigestive) motility patterns upon an existing baseline of motor activity.
• the GABAergic/nitrergic combination pathway circuit is not present in the ileum.
• DON-based derivatives This example demonstrates the ability of DON-based derivatives to induce the fed pattern of gut motility in a manner similar to DON.
• DON derivatives were selected for a comparative study with DON in rats using methods described in the previous examples for testing DON and recording its effect on gut motility.
• One of the representative DON derivatives was 3-acetyl DON (C 1 H 22 ⁇ 7 ).
• isopropylidine DON i.e., 3- hydroxy-7,15-isopropylidine-12,13-epoxy-9-tricothecin-8-one, having the formula C 18 H O6, designated EN139491
• isopropylidine-3-acetyl-DON i.e., 3-acetyoxy-7,15-isopropylidine- 12,13-epoxy-9-tricothecin-8-one, having the formula C 20 H 26 O 7 , designated EN139492
• DON carbonate i.e., 3-hydroxy-12,13-epoxy-9-tricothecin-8-one-7,15 carbonate, having the formula C 16 H 18 O 7 , designated EN139494
• 3-acetyl-DON carbonate i.e., 3-acetoxy-12, 13- epoxy-9-tricothecin-8-one-7, 15 carbonate, having the formula C ⁇ 8 H 20 O 8 , designated EN139495
• isopropylidine DON
• This compound was prepared in 99% yield starting with 20 mg of 3-acetyl-DON, 0.023 ml of pyridine, and 10 mg of triphosgene.
• the product was obtained as a white solid after silica gel chromatography using 7:3 ethyl acetate-hexane mixture as eluent.
• DON abruptly attenuated the gastric antral (site SI) motor activity and induced a sustained hyperactivity in the duodenum (proximal duodenal site DI).
• site SI gastric antral
• duodenum proximal duodenal site DI
• the control motor pattern recovered.
• tricothecene-induced hyperactivity was characterized by an initial high frequency motor activity where the amplitude although larger than "intergroup" responses was smaller than the MMC motor activity. This is shown in Figure 11, where this initial period lasted 3-10 minutes; thereafter the amplitude increased to MMC levels.
• Figure 12 shows the time to onset of action (1 min) and duration (40 ⁇ 4 min) of effects were similar to those for DON.
• Figure 13 shows the effects of intravenously administered 3-acetyl DON on spontaneous motor activity in the rat gastric antrum (SI) and proximal duodenum (D2). Within 60 minutes, the control motor pattern recovered.
• FIG. 14 shows typical in vivo recordings of the motor activity in the rat duodenum (DI) and gastric antrum (SI) illustrating the action of the compound EN139491 on the fasting pattern of gut motor activity.
• DI rat duodenum
• SI gastric antrum
• the top panel of the recording shows 20 minutes of normal fasting pattern motor activity without any drug treatment.
• the duodenum displayed a typical pattern of low frequency spontaneous motor activity together with propagating motor activity (MMC).
• the gastric antrum displayed a typically rhythmic motor activity.
• the second panel of the recording shows activity at the time of injection with EN139491. Within 30 seconds of injection, a long lasting (40-60 min) hyperactivity in the duodenum and a simultaneous and parallel attenuation of motor activity in the gastric antrum developed.
• This EN139491 induced motor activity was typical of fed pattern motor activity. Recovery of fasting pattern motor activity is shown in the bottom panel of the recording in Figure 14.
• Figure 15 shows the recording of the effects that EN139491 had on duodenal motor activity recorded at D2 (i.e., 1.5 cm distal to the DI strain gauge).
• the recording of the induction and duration of fed pattern at D2 by EN139491 as shown in Figure 15 was similar to the results recorded at duodenal site DI as shown in Figure 14.
• EN139491 were comparable to those seen in the spontaneous "grouped” MMC activity of the gut. Likewise, both amplitude ( Figure 18) and frequency ( Figure 19) of the contraction component of the fed pattern induced by EN 139491 were comparable to those seen in the spontaneous "grouped” MMC activity of the gut. These results indicated that the fed pattern induced by EN139491 had the same features as the fed pattern induced by DON.
• FIG. 20 shows a typical in vivo recording of the motor activity at the rat duodenum DI and D2 sites and the gastric antrum SI site illustrating the action of EN 139492 on the fasting pattern of gut motor activity.
• the top panel of the recording shows more than 40 minutes of normal fasting pattern motor activity in the absence of DON or a DON derivative.
• the duodenum displayed a typical pattern of low frequency spontaneous motor "intergroup" activity together with propagating
• the gastric antrum displayed a typically rhythmic motor activity. Within 30 seconds of injection, a long lasting hyperactivity in the duodenum was initiated and a simultaneous and parallel attenuation of motor activity in the gastric antrum developed.
• the DON derivative EN139492 is able to induce a fed pattern of gut motor activity that is comparable to the fed pattern induced by DON, the structural parent of EN139491 and EN139492.
• DON carbonate EN 139494
• 3-acetyl DON carbonate EN139495
• Intravenous injection of the tricothecene based derivative EN139495 (10 mg-kg "1 ) induced a typical in vivo fed pattern motor activity in the gut recorded from the proximal duodenum (D ) and gastric antrum (S ⁇ of halothane-anaesthetized male Sprague Dawley rats, n 4.
• a proximate reapplication of DON or its derivatives within 90 minutes of the first injection was typically without effect. After 120 minutes, DON and its derivatives were again effective and could induce the fed pattern of gut motor activity.
• the foregoing results demonstrate the effectiveness of DON and a familiar derivative of DON, 3-acetylated DON (which is reported to have the lowest toxicity of all the tricothecenes), to induce a fed pattern of gut motor activity was tested.
• two new DON-based derivatives (EN139491 and EN139492) were synthesized and shown to be capable of inducing a fed pattern of gut motor activity in a manner at least comparable to DON.
• Example 5 Effects of the selective P 2 ⁇ -2X3 purinoceptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine triphosphate (TNP-ATP).
• TNP-ATP adenosine triphosphate
• the pharmacology of the intrinsic motor inhibitory innervation of the rat and porcine gastroduodenum was characterized using specific nitric oxide (NO) synthesis inhibitors and inhibitors of purinoceptor-mediated responses, such as the general P 2 receptor antagonist, suramin, and the general P 2 ⁇ agonist, ⁇ , ⁇ -methylene ATP, and the P 2 ⁇ agonist, methyl-thiol-ATP.
• NO nitric oxide
• V 2 i receptors are reported to be expressed only on sensory neurones (Evans and Suprenant, Semin. Neurosci., 8: 217-223 (1996)).
• This study represents the first use of the P2 ⁇ -selective antagonist TNP-ATP in vivo. The aim was to determine the role of P2xt receptors in control of patterned gastrointestinal motor behaviour and test directly the hypothesis that the P2X! purinoceptor subtype mediates DON-induced hyperactivity in the gut.
• TNP-ATP had no discernible effects on these parameters throughout an experiment, which often lasted up to 6hrs.
• TNP-ATP had no effect on spontaneous gastric motor activity (data not shown).
• TNP-ATP injected intravenously as a single bolus significantly and specifically affected spontaneous duodenal relaxations.
• the actions of TNP-ATP were barely observable.
• 4.5 and 5 mg/kg appeared to be supramaximal doses and the effectiveness inconsistent. Occasionally, there were also some non-specific actions on gut motor activity at these higher doses.
• TNP-ATP adminstered at 3.5 mg/kg was found to be reproducibly effective and specific in its actions. This was chosen for subsequent evaluation in the model.
• Typical in vivo recordings showing the effects of intravenously injected TNP-ATP (3.5 mg-kg "1 ) on spontaneous motor activity in the rat duodenum (at duodenal site DI) is shown in Figure 21.
• TNP-ATP did not evoke any response upon injection. However, within 1 minute of injection, MMC related relaxations were reduced. "Intergroup" motor activity was not significantly affected. There was recovery of MMC related relaxant motor activity to within 90% of control level within 30 minutes of TNP-ATP injection.
• TNP-ATP intravenously injected TNP-ATP (3.5 mg-kg '1 ) on DON-induced fed pattern motor activity in the rat stomach and duodenum are presented in Figures 22 (recording duodenal site DI) and 23 (recording duodenal site D2 and gastric antral site SI). Consistent with all other experiments, TNP-ATP did not evoke any response upon injection. However, within 1 minute of injection, the effects of DON (10 mg-kg '1 , i.v.) were significantly reduced. This inhibitory action of TNP-ATP consisted of an initial profound effect (up to 80% inhibition) lasting approximately 5 minutes, followed by a longer period of less profound (up to 40% inhibition), but significant antagonism of DON actions.
• FIG. 24- 27 The ability of TNP-ATP to counter DON actions is shown graphically in Figures 24- 27.
• the bar graphs of Figures 24-27 show the effects of intravenous treatment with TNP-ATP on DON-induced relaxations and contractions recorded from the proximal duodenum (DI).
• the effects of TNP-ATP on the amplitude ( Figure 24) and frequency ( Figure 25) of DON-induced relaxations were compared to the amplitude and frequency of the relaxation component of "grouped" MMC and the control "intergroup” motor activity, which was set as 100%.
• TNP-ATP The effectiveness of the selective P2X1 purinoceptor antagonist TNP-ATP was evaluated.
• An intravenous injection of a single bolus of this antagonist rapidly and specifically attenuated MMC related relaxations; indicative of the involvement of V 2 ⁇ purinoceptors in gut motor activity.
• Intravenous injection with a single bolus injection of TNP-ATP reduced (transiently) the DON-induced fed pattern in a dose-dependent manner.

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• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Plant Substances (AREA)

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• 2000
  • 2000-07-06 IL IL14731200A patent/IL147312A0/xx unknown
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  • 2000-07-06 BR BR0012246-7A patent/BR0012246A/pt not_active IP Right Cessation
  • 2000-07-06 CN CN00811624A patent/CN1370066A/zh active Pending
  • 2000-07-06 CA CA002374358A patent/CA2374358A1/en not_active Abandoned
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  • 2000-07-06 WO PCT/CA2000/000790 patent/WO2001001968A2/en not_active Application Discontinuation
  • 2000-07-06 EP EP00945476A patent/EP1196164A2/en not_active Withdrawn
  • 2000-07-06 AU AU59568/00A patent/AU763751B2/en not_active Ceased
  • 2000-07-06 JP JP2001518668A patent/JP2003507472A/ja active Pending
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• 2002
  • 2002-01-04 NO NO20020038A patent/NO20020038L/no not_active Application Discontinuation

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