WO2001093874A1 - Procede de traitement de l'obesite et de la paralysie musculaire par des aides ergogeniques - Google Patents

Procede de traitement de l'obesite et de la paralysie musculaire par des aides ergogeniques Download PDF

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WO2001093874A1
WO2001093874A1 PCT/US2001/018812 US0118812W WO0193874A1 WO 2001093874 A1 WO2001093874 A1 WO 2001093874A1 US 0118812 W US0118812 W US 0118812W WO 0193874 A1 WO0193874 A1 WO 0193874A1
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protein kinase
activated protein
kinase activator
mammal
aicar
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William Winder
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Brigham Young University
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Priority to EP01948319A priority Critical patent/EP1427425A4/fr
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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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

Definitions

  • the present invention relates to the methods of treatment of obesity and paralyzed muscle. More specifically, the invention relates methods of treatment of obesity and paralyzed muscle through artificial activation of metabolic pathways.
  • Obesity is one of the largest health problems in the United States and is a growing concern for many health care officials. By one account more than 33 percent of adults and 20 percent of children in the United States are considered obese. Obesity is defined as having excessive amounts of body fat. Body fat (adipose tissue) is necessary for certain bodily functions. However, when body fat accumulates to excessive amounts the person is considered obese. Obesity can lead to a number of different illness including: heart disease, high blood pressure, increased cholesterol, diabetes, certain types of cancer, orthopedic problems, musculo-skeletal diseases, decreased flexibility, and difficulty breathing. Obesity can have both genetic and habitual causes. A diet high in fat combined with a sedentary lifestyle can contribute the accumulation of body fat.
  • AMPK activity has been shown to increase in skeletal muscle of rats running on the treadmill and in electrically stimulated muscle. Ruderman, N.B., et al. Am. J. Physiol. 276:E1-E18, 1999; Winder, W.W., & D.G. Hardie Am. J. Physiol. 270:E299-E304, 1996; Winder, W.W., & D.G. Hardie, Am. J. Physiol. 277:E1-10, 1999. These observations taken together suggest that AMPK activation may be involved in mediating the effect of exercise on at least some of the biochemical adaptations of muscle.
  • AMPK has recently been implicated as being important as a metabolic master switch in the muscle, controlling both fat metabolism and glucose uptake. Winder, W.W., ' & D.G. Hardie, Am. J. Physiol. 277. ⁇ 1-10, 1999. This enzyme is controlled by both allosteric and covalent mechanisms. It is activated allosterically by an increase in 5'- AMP and inhibited by ATP and creatine phosphate. Phosphorylation of AMPK by an upstream kinase (AMPKK), which is also activated by 5 '-AMP, also results in activation. A large amplification of activity can result from maximal stimulation by both mechanisms. Hardie, D.G. & D. Carling Eur. J. Biochem.
  • the invention relates to a method of treating obesity in a mammal.
  • the method includes the step of administering a therapeutically effective amount of an AMP-activated protein kinase activator to the mammal.
  • the mammal may be for example, a human, a rat, a mouse, and the like.
  • the AMP-activated protein kinase activator can be subcutaneously injected into the mammal or administered in any other manner that provides for uptake of the AMP-activated protein kinase activator into the cells of the mammal.
  • the activation of the AMP-activated protein kinase activator can produce the benefits of exercise training including the loss of body fat.
  • the invention also relates to a method of treating insulin resistance in a mammal suffering from obesity, type 2 diabetes, or muscle paralysis.
  • a therapeutically effective amount of an AMP-activated protein kinase activator is given to the mammal.
  • An AMP-activated protein kinase activator can also be used as an ergogenic aid to treat for example, paralyzed muscle or to enhance the mitochondrial eznyme content in muscle of athletes, armed forces personnel, and others where an increased mitochondrial content of muscle would be advantageous.
  • Another possible use may be for treatment of individuals requiring prolonged bed rest or inactivity which have been shown to cause regression in muscle mitochondrial content.
  • AMP-activated protein kinase can be activated allosterically by increases in the concentration of AMP or by a compound that is analogous to AMP.
  • an AMP analog such as adenosine-5'-thiomonophosphate, adenosine 5'- phosphoramidate, formycin A 5'-monophosphate, or ZMP is administered to a subject so that the AMP analog is taken into the cells of the subject. This may require modification of the analog so that it maybe transported into the cell. Because these AMP analogs are not readily transported into a cell the analog may be administered intracellularly.
  • 5-aminoimidazole-4-carboxamide ribonucleoside is an adenosine analog that is phosphorylated in muscle cells to become ZMP. This allows the 5- aminoimidazole-4-carboxamide to enter the cells and then be converted to ZMP to mimic the effect of AMP in the cell.
  • 5-aminoimidazole-4-carboxamide ribonucleoside can be administered at a dose from about 0.5 to at least about 1.0 mg/g body weight.
  • the AMP-activated protein kinase activator is administered acutely in a single dose.
  • the AMP-activated protein kinase activator is administered chronically over a period of weeks to provide an additional benefit to the subject. To better mimic the effect of exercise teaining the AMP-activated protein kinase activator can be administered intermittently for a period of time.
  • Figure 1 is a set of graphs illustrating the food intake and body weight of rats injected with AICAR.
  • Figure 2 is a set of bar graphs illustrating ⁇ -Aminolevulinate synthase and cytochrome c protein expression in rats injected with AICAR.
  • Figure 3 is a bar graph illustrating the citrate synthase activity in rats injected with AICAR or saline.
  • Figure 4 is a set of bar graphs illustrating succinate dehydrogenase and malate dehydrogenase in rats injected with AICAR or saline.
  • Figure 5 is a set of bar graphs illustrating hexokinase activity and GLUT4 in rats injected with AICAR.
  • Figure 6 is a schematic representation of the putative actions of AMPK in skeletal muscle.
  • Figure 7 is a bar graph illustrating the AMPK activity in gastrocnemius muscles from denervated rats acutely treated with saline or AICAR.
  • Figure 8 is a bar graph illustrating the AMPK activity in soleus muscles from denervated rats acutely treated with saline or AICAR.
  • Figure 9 is a bar graph illustrating the ACC specific activity for soleus muscles from denervated rats treated with saline or AICAR.
  • Figure 10 is a graph showing citrate dependence of acetyl-CoA carboxylase (ACC) in denervated and contealateral innervated gastrocnemius muscles from rats treated with AICAR.
  • ACC acetyl-CoA carboxylase
  • Figure 11 is a graph showing dose dependent response of GLUT4 levels.
  • Figure 12A is a bar graph showing GLUT4 protein content in gastrocnemius muscles.
  • Figure 12B illustrates a Western Blot showing GLUT4 protein content in gastrocnemius muscles.
  • Figure 13 is a bar graph showing the GLUT4 protein concentration in soleus muscles from saline and AICAR treated rats.
  • the invention relates to a method of treating obesity in a mammal.
  • the method of the present invention may also be used to treat insulin resistance in a mammal suffering from obesisty, type 2 diabetes, or muscle paralysis.
  • the present invention also relates to ergo genie aids that can enhance the building of muscle in response to exercise.
  • the method includes the step of administering a therapeutically effective amount of an AMP-activated protein kinase activator to the mammal.
  • therapeutically effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease being treated, i other words, therapeutically effective amount is intended to mean an amount of a compound sufficient to produce the desired pharmacological effect. It is understood that the therapeutically effective amount to be used in the treatment of obesity, insulin resistance, or as an ergogenic aid must be subjectively determined according to the type of mammal and the desired effect.
  • Variables involved include the size of the patient, the type of AMPK activator, the state of the disease, age of the patient, and response pattern of the patient.
  • the novel methods of the invention for treating, preventing or alleviating the conditions described herein comprise administering to mammals in need thereof, including humans, an effective amount of one or more compounds of this invention or a non-toxic, pharmaceutically acceptable addition salt thereof.
  • the compounds may be administered subcutaneously, orally, rectally, parenterally, or topically to the skin and mucosa.
  • many of the known AMP analogs are phosphorylated, it is difficult to get an effective amount of the analog inside a cell by injection or topical methods. Thus, it may be necessary to administer the analog directly into the muscle of the mammal by for example methods of in vivo electroporation.
  • the mammal may be for example, a human, a rat, a mouse, and the like.
  • the AMP-activated protein kinase activator can be subcutaneously injected into the mammal or administered in any other manner that provides for uptake of the AMP-activated protein kinase activator into the cells of the mammal.
  • the activation of the AMP- activated protein kinase activator can produce the benefits of exercise training including the loss of body fat.
  • AMP-activated protein kinase can be activated allosterically by increases in the concentration of AMP or by a compound that is analogous to AMP.
  • an AMP analog such as adenosine-5'-thiomonophosphate, adenosine 5'- phosphoramidate, formycin A 5'-monophosphate, or ZMP is administered to a subject so that the AMP analog is taken into the cells of the subject. This may require modification of the analog so that it may be transported into the cell. Because these AMP analogs are not readily transported into a cell the analog may be administered inteacellularly.
  • 5-aminoimidazole-4-carboxamide ribonucleoside is an adenosine analog that is phosphorylated in muscle cells to become ZMP. This allows the 5- aminoimidazole-4-carboxamide to enter the cells and then be converted to ZMP to mimic the effect of AMP in the cell.
  • 5-aminoimidazole-4-carboxamide ribonucleoside can be administered at a dose from about 0.5 to at least about 1.0 mg/g body weight. It has been shown that an effective dose in a rat is about 1.0 mg/g body weight. However, when determining the dose the treatment of a human, the dose may be higher or lower.
  • the AMP-activated protein kinase activator is administered acutely in a single dose. This provides the acute activation of AMPK and provides for a short-lived effect similar to exercising once.
  • the AMP-activated protein kinase activator can be administered chronically over a period days or weeks to provide an additional benefit to the subject. Providing a dose for a chronic period of about 28 days has been shown to give significant benefits over a single acute activation of AMPK.
  • the AMPK activator can also be administered intermittently over a period of time to better mimic the effect of exercise training.
  • Such intermittent activation can consist of activating AMPK for a period of at least one day, followed by a period of non-activation for at least one day, followed by an additional period of activation of at least one day.
  • the period of activation followed by non-activation can be repeated as needed to obtain the desired results. For example an increased effectiveness was observed when rats were intermittently injected with AICAR as follows: injection for 3 days, followed by 2 days without injection, followed by 5 days of injection, followed by two days without injection, followed by 3 days with injection.
  • 5'-aminoimidazole-4-carboxamide 1- ⁇ -D-ribofuranoside also known as AICA riboside and AICAR
  • AICA riboside and AICAR is an analogue of adenosine that is taken up into the cell and phosphorylated to form ZMP, an analog of 5'- Adenosine monophosphate (AMP).
  • AMP AMP- activated protein kinase
  • AMPK AMP- activated protein kinase
  • AMPK phosphorylates muscle acetyl-CoA carboxylase, thereby decreasing its activity and reducing the production of malonyl-CoA, an inhibitor of fatty acid oxidation in muscle.
  • GLUT4 the insulin-sensitive glucose transporter
  • hexokinase proteins which can influence the degree of insulin sensitivity. Both of these actions would be beneficial for patients who are obese.
  • AMPK activators such as AICA riboside, would be useful for treatment of obesity and of the insulin resistance associated with obesity. It has long been known that regular endurance exercise (1-2 hr of ninning, swimming, or cycling) over a period of days, weeks and months, increases mitochondria, increases the amount of insulin-sensitive glucose transporter protein (GLUT4) in muscle, and increases insulin sensitivity.
  • Conclusive data is provided showing that chronic chemical activation of the AMP-activated protein lcinase with AICA riboside results in an increase in the total quantity of GLUT4 and mitochondrial enzymes in the muscle of rats.
  • This chronic chemical activation of AMPK may prove useful in increasing insulin- sensitivity of type 2 diabetics who can not exercise, for increasing insulin sensitivity of muscle in patients confined to bed rest, and for increasing GLUT4, insulin sensitivity and muscle mitochondrial levels in individuals with muscle paralysis. It also may be useful as an ergogenic aid, producing an accelerated training effect (ie increase in mitochondrial oxidative enzymes, GLUT4 and hexokinase) on muscle.
  • AMP-activated protein kinase activators may be used to treat insulin resistance in type 2 diabetes and in individuals with muscle paralysis.
  • AMP-activated protein kinase activators such as AICA-riboside, may also be used as ergogenic aids, producing many of the intramuscular adaptations that occur with prolonged endurance exercise training.
  • AICA-riboside is the only adenosine analog that has been found useful in activating AMPK in vivo and which has been utilized to show effects of chronic activation of this kinase.
  • AICAR 5-aminoimidazole-4- carboxamide-1- ⁇ -D-ribofuranoside
  • Acetyl-CoA carboxylase activity (ACC), a target for AMPK, decreased in all three muscle types, but was lowest in the white quadriceps in response to AICAR injection, hi rats given daily subcutaneous injections of AICAR (1 mg/g) for 4 wk, activities of citrate synthase, succinate dehydrogenase and malate dehydrogenase were all increased in white quadriceps and soleus but not in red quadriceps. Cytochrome c and delta-aminolevulinic acid synthase were increased in white but not in red quadriceps. Carnitine palmitoyl-teansferase and hydroxy-acylCoA dehydrogenase were not significantly increased.
  • acetyl-CoA carboxylase is the only phosphorylation target for AMPK that has been identified in skeletal muscle.
  • AMPK is naturally activated during muscle conteaction, but may be activated artificially by inj ecting AICAR subcutaneously into rats or by exposing incubated or perfused muscle to AICAR.
  • AMP may directly activate AMPK allosterically or may activate the AMPKK (allosterically) which in turn phosphorylates/activates AMPK.
  • Creatine Phosphate CP is an allosteric inhibitor of AMPK. The decline in CP during muscle conteaction is thought to relieve inhibition of AMPK, resulting in increased activity.
  • both AMPKK and AMPK are also activated allosterically by the free AMP concenteation, which has been shown to increase in the muscle in response to conteaction. Winder, W.W., & D.G. Hardie, Am. J. Physiol. 277:E1-10, 1999.
  • AMPK is inhibited allosterically by creatine phosphate, making the decline in CP during muscle contraction an important signal for allowing activation of this enzyme. Winder, W.W., & D.G. Hardie, Am. J. Physiol. 277:E1-10, 1999.
  • These allosteric effects are lost during preparation of the AMPK extracts and can not therefore be detected with the AMPK assay. However, it is possible to obtain an estimation of the in vivo activation of AMPK by measuring the effects of phosphorylation of one of its known target proteins.
  • ACC Purified muscle acetyl-CoA carboxylase
  • AMPK activity provides evidence that AMPK activity was stimulated in all three muscle types in response to AICAR.
  • AMPK was apparently activated by predominately allosteric mechanisms (ZMP effects on AMPK) and not by phosphorylation of AMPK by AMPKK.
  • Citrate synthase, cytochrome c, delta-aminolevulinate synthase (ALA-S), and malate dehydrogenase were all significantly increased in the white region of the quadriceps muscle in response to 4 weeks AICAR injections.
  • the delta-aminolevulinate synthase activity has previously been reported to increase within 16 hours following a prolonged bout of exercise.
  • the decline in creatine phosphate accompanying contraction may be a critical component of the signal. Muscle creatine phosphate is not significantly changed in red quadriceps in response to AICAR. The time course of the decline in gastrocnemius ACC activity during electrical stimulation correlates more closely with the decline in creatine phosphate than with the measured AMPK activity (data not shown). The increase in cytosolic calcium during excitation contraction coupling and the rise in plasma fatty acids that accompany prolonged exercise bouts may also be important in inducing the increase in mitochondrial enzymes. These effects of exercise are not mimicked by AICAR inj ections .
  • AMPK pathway is activated with this calcium ionophore.
  • AMPK activity was not quantitated in that study. It is unclear at this point why the white quadriceps muscle responded with an increase in mitochondrial enzymes and the red quadriceps did not.
  • One possible interpretation is that AMPK activation is not responsible for the mitochondrial adaptations. It is also possible that it is the phosphorylated species of AMPK that is responsible for triggering increased rates of synthesis of mitochondrial enzymes. If the decline in ACC activity is a true measure of in vivo AMPK activity, it might concluded that in red quadriceps AMPK is activated allosterically by AICAR injection, but the phosphorylation state of AMPK is unchanged with respect to controls.
  • the total daily contractile activity would be expected to be greater in red quadriceps and in soleus fibers than in fibers of the white quadriceps.
  • the AMPK response to AICAR appeared to be down-regulated by the end of the four week treatment period. No significant increase was observed in AMPK activity in any of the muscle types one hour following injection of AICAR into the chronically-treated rats. The decrease in ACC activity at 0.2 mM citrate was similar however to that seen in rats at the beginning of the four week treatment regimen. The reason for this down-regulation of response is not clear. Significant liver hypertrophy was noted, increasing the probability of more rapid metabolism of the AICAR after the daily injections.
  • AMPK activity is markedly increased in denervated gastrocnemius muscles but not in denervated soleus muscles by acute injectin of AICAR. Further, AMPK activity is significantly increased in contealateral innervated AICAR-teeated gastrocnemius and soleus muscles.
  • ACC activity is used in this investigation as a reporter enzyme to indicate the in vivo action of AMPK since the AMPK activity assay is performed on ammonium sulfate precipitated homogenates that do not reflect any modulation of AMPK activity due to allosteric effects. ACC activity is markedly decreased in the denervated and contealateral innervated AICAR-teeated gastrocnemius and soleus muscles.
  • IRS-1 phosphorylation in the tibialis anterior, PI3-K activity in the tibialis anterior, and Akt-1 kinase activity in soleus and plantaris muscles are all significantly decreased in long term denervated muscles when stimulated by insulin.
  • GLUT4 translocates from microvesicles in response to insulin and contraction
  • AICAR is an adenosine analogue
  • denervation of rat hind limbs (4) activation of AMPK
  • animal care (6) western blot analysis for GLUT4; and (7) statistical analysis.
  • GLUT4 translocates from microvesicles to the sarcolemmal and T- tubular membranes in response to insulin and contraction. See Pessin JE, et al. JBiol Chem 274:2593-2596, 1999; Goodyear LJ, & Ka n . Annu Rev Med 49:235-261, 1998; Hayashi T, et al. Am J Physiol 273: E1039-E1051, 1997. GLUT4 teanslocation stimulated by conteaction uses a separate pathway than does insulin. Goodyear LJ, et al. Am J Physiol 268: E987-E995, 1995; Hayashi T, et al. Diabetes 47: 1369-1373.
  • AICAR 5-aminoimidazole-4-carboxamide-riboside
  • Denervation of rat hindlimbs is a model for glucose transport studies which produces significant muscle atrophy, insulin resistance, and also causes a decrease in the total muscle GLUT4 content. Henriksen IJ, et al. J Appl Physiol 70: 2322-2327. 1991; Burant CF, et al. Am J Physiol 247: E657-E666, 1984; Turinsky J, Am J Physiol 252 (Regulatory Integrative Comp Physiol 21): R531-R537, 1987; Block N, et al. J Clin Invest 88:1546-1552, 1991; Castello A, et al. JBiol Chem 268:14998-15003, 1993; Coderre L, et al.
  • Denervation also induces a significant decline in mRNA levels in the same three day time period suggesting teanscriptional mediation of this change in GLUT4 expression.
  • Block N et al. JClin Invest 88:1546-1552, 1991; Didyk RB, et al. Metabolism 43:1389- 1394, 1994; Jones JP, et al. J Appl Physiol 84(5):1661-1666, 1998.
  • insulin resistance occurs within three hours of denervation and decrease in muscle GLUT4 protein or GLUT4 mRNA content is not observable until two and three days after denervation.
  • the left hindlimb was sham operated to serve as a control. Both skin wounds were closed with a metal clips.
  • the rats used for the acute study were also implanted with a jugular catheter which was exteriorized on the back of the neck at the time of denervation for rapid administration of anesthesia.
  • AMPK Activation of AMPK AMPK can be activated allosterically by increases in the concenteation of AMP, but is also inhibited by ATP and is therefore sensitive to the AMP/ ATP concenteation.
  • Gorton JM et al. Current Biol 4:315-324, 1994; Salt IP, et al. Biochem J334: 177-187, 1998; Davies SP, et al. FEBSLett 377:421-425, 1995; Hawley SA, et al. JBiol Chem 271 : 27879-27887, 1996.
  • AMPK is inhibited by creatine phosphate (CP) and is likely sensitive to the CP/creatine (C) ratio .
  • AMP also serves to activate an upstream kinase, AMPKK, which in turn phosphorylates and thereby activates AMPK.
  • Davies SP et al. FEBSLett 377:421-425, 1995; Hawley SA, et al. JBiol Chem 270: 27186-27191, 1995.
  • ZMP a normal intermediate in the purine nucleotide synthetic pathway and an AMP analogue, has been shown to imitate the effects of AMP and stimulate both AMPK and AMPKK. Corton, JM, et al. Eur J
  • Muscle was ground to a powder under liquid nitrogen and homogenized (lmg muscle:9mL buffer) in HEPES buffer [25mM HEPES, ImM EDTA, ImM benzamadine, 1 mM 4-(2-aminoethyl)-benzene + sulfonyl fluoride, 1 ⁇ M leupeptin, 1 ⁇ M antitrypsin, 1 ⁇ M aprotinin, pH 7.5]. This homogenate was then diluted with water and Laemmli's buffer (ImL homogenate:2mL water: ImL buffer) immediately before loading.
  • HEPES buffer 25mM HEPES, ImM EDTA, ImM benzamadine, 1 mM 4-(2-aminoethyl)-benzene + sulfonyl fluoride, 1 ⁇ M leupeptin, 1 ⁇ M antitrypsin, 1 ⁇ M aprotinin, pH 7.5.
  • the membranes were exposed to horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Life Sciences, Arlington Heights, IL) for 1 h. at room temperature. After again washing twice with PBST and twice with PBS, the membranes were incubated in enhanced chemoluminescence-detection reagent and then visualized on enhanced chemoluminescence hyperfilm (Amersham Life Sciences).
  • GLUT4 Relative amounts of GLUT4 were then quantified using a Hewlett Packard Scan Jet 6200C and SigmaGel software (SPSS, Chicago, IL). Western blot data are expressed as arbitrary units where individual values were divided by the mean basal GLUT4 value (sham operated leg/injected with 0.9%NaCl solution).
  • Results are expressed as means ⁇ SE. Statistically significant differences between treatment groups were analyzed using Fischer's least significant difference test. Statistical significance is defined as P ⁇ 0.05.
  • Example 1 - Acute Injection of AICAR The purpose of these studies was to determine if AMPK activity was acutely increased in muscle by the AICAR treatment.
  • rats were anesthetized by intravenous injection of pentobarbital sodium (4.8 mg/100 g body wt). The superficial white and the deep red regions of the quadriceps muscles, and the soleus muscles were quickly removed and frozen with stainless steel clamps at liquid nitrogen temperature.
  • Resuspended ammonium sulfate precipitates of tissue homogenates were analyzed for AMPK activity and acetyl-CoA carboxylase activity as described previously. Li, B., et al. J Biol. Chem. 274:17534-17540, 1999; Winder, W.W., & D.G. Hardie Am. J. Physiol. 270:E299-E304, 1996.
  • This measurement of AMPK only detects increases in AMPK activity that survive ammonium sulfate precipitation of the muscle homogenate (ie increases due to phosphorylation) and does not provide information concerning allosteric control by AMP, CP, and ATP.
  • ACC activity at 0.2 mM citrate provides some indication of the in vivo activity of AMPK, since ACC is a target for phosphorylation of AMPK. ACC activity has previously been reported to decrease in response to phosphorylation by AMPK. Winder, W.W., & D.G. Hardie Am. J. Physiol. 270:E299-E304, 1996. The acute experiment was also repeated on rats treated with AICAR or saline for 4 weeks to see if the responses of AMPK and ACC to AICAR persisted for the entire treatment period. In both experiments ATP and CP were measured on neutralized perchloric acid extracts of muscle.
  • a single injection of AICAR in rats not previously treated with AICAR resulted in significant increases in AMPK activity in white quadriceps and soleus muscle but not in red quadriceps sixty minutes following the injection as seen in Table 1.
  • the activity of acetyl-CoA carboxylase at 0.2 mM citrate was markedly decreased in all three muscle types.
  • Table 1 shows the concentrations of ATP ( ⁇ mol/g), CP ( ⁇ mol/g), AMP-activated protein kinase (AMPK)(pmol/g/min) and acetyl-CoA carboxylase (ACC) (nmol/g/min at 0.2 mM citrate) in muscles of rats 60 minutes following injection of saline or AICAR.
  • AMPK AMP-activated protein kinase
  • ACC acetyl-CoA carboxylase
  • the ZMP concentration one hour after injection was 0.42 ⁇ 0.04 ⁇ mol/g in white quadriceps, 0.94 ⁇ 0.08 ⁇ mol/g in red quadriceps, and 1.15 ⁇ 0.07 ⁇ mol/g in soleus muscle. These values were in the same range as those seen in the gastrocnemius muscle 60 min following a single injection of AICAR. See Holmes, B.F., et al. J. Appl. Physiol. 87:1990-1995, 1999. ZMP was not detectable in rats injected with saline.
  • Creatine phosphate was significantly lower in the AICAR injected rats compared to saline-injected controls only in the white quadriceps prior to chronic teeatment and in soleus muscle after 4 weeks of AICAR injections. ATP was increased to a small extent by AICAR injection in red quadriceps prior to chronic treatment and in white quadriceps after chronic treatment. Table 1.
  • rats were injected subcutaneously (between 8 and 10 a.m.) with AICAR (1 mg/g) or saline vehicle daily for 28 ⁇ 1 days in succession. Beginning with the first injection, controls were pair fed with AICAR- injected rats. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium 22-25 hours after the last AICAR injection and the white and red regions of the quadriceps and the soleus muscles were removed and frozen as described above. Muscles were kept frozen at -70 °C until analyzed. Liver, heart, kidney, and fat pads were weighed.
  • GLUT4 glycogen GLUT-4, hexokinase, lactate dehydrogenase, and several mitochondrial enzymes. Hassid, W.Z., & S. Abraham Methods Enzymol. 3:35-36, 1957. GLUT4 was quantitated by western blotting as described previously using GLUT4 polyclonal antibody RalRGT,
  • cytoclirome c For determination of cytoclirome c, muscles were homogenized in 10 mM n-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 1 mM EDTA, and 250 M sucrose (HES buffer). Homogenates were centeifuged at 700g for 10 min and aliquots of the supernatant containing 100 ⁇ g protein were solubilized in Laemmli sample buffer, subjected to SDS-polyacrylamide gel electeophoresis (15% resolving gel) and then transferred to nitrocellulose. Cytochrome c was detected by incubating the nitrocellulose blots with a rabbit polyclonal antibody against rabbit heart cytochrome c (Alpha).
  • succinate dehydrogenase and lactate dehydrogenase an aliquot of the homogenate was centeifuged at 700 X g for 10 min at 4 °C. The remainder of the assays were performed on aliquots and dilutions of the mixed whole homogenate. Assays were performed by the following methods: citrate synthase, succinate dehydrogenase, the mitochondrial fraction of malate dehydrogenase, hexokinase, lactate dehydrogenase, camitine palmitoyl transferase and hydroxyacyl-CoA dehydrogenase. Srere, P.A. Methods in Enzymology 13:3-6, 1969; King, T.E.
  • Cytochrome c was also increased in white but not in red quadriceps.
  • the citrate synthase activity in red and white quadriceps and soleus muscles of rats injected with AICAR or saline (controls) for 4 weeks is shown. Values are means ⁇ SEM for 8 muscles per group. An asterisk denotes a significant difference from saline injected controls, P ⁇ 0.001. Citrate synthase was significantly increased in white quadriceps, and soleus, but not in red quadriceps.
  • Lactate dehydrogenase, hydroxyacyl-CoA dehydrogenase and camitine palmitoyl teansferase were not significantly influenced in any of the muscle types as shown in Table 3. An increase in glycogen was observed to occur in white and red quadriceps, but not in soleus the day following the last injection of AICAR.
  • LDH lactate dehydrogenase
  • CPT camitine palmitoyl-transferase
  • HADH hydroxyacyl-CoA dehydrogenase
  • citrate synthase in the white quadriceps was 15.5 ⁇ 1.3 ⁇ mol/g/min in controls vs 23.0 ⁇ 1.2 ⁇ mol/g/min in AICAR injected rats. This represented a 48% increase and was highly significant (p ⁇ 0.001). A significant increase also occurred in the red region of the quadriceps (47.0 ⁇ 2.8 ⁇ mol g/min in controls vs 57.4 ⁇ 2.5 ⁇ mol/g/min in AICAR injected, p ⁇ 0.025) in response to AICAR.
  • AMPK activity was acutely increased in denervated muscles after AICAR injection. Rats were denervated between 8:30 a.m. and 10:30 a.m. and were then given 25 g of food. Rats were handled twice during the day to accustom them to being handled. The rats were given a single subcutaneous injection 24 hours, after surgery of either AICAR (1 mg/g body weight) in sterile 0.9% NaCl or with just 0.9% NaCl, and were subsequently anesthetized intravenously. Soleus and gastrocnemius muscles from both sham and denervated sides were extracted and quick frozen in liquid nitrogen for analysis. AMPK activity and acetyl-Co A carboxylase (ACC) activity were determined as previously described.
  • AICAR acetyl-Co A carboxylase
  • the first objective was to insure that AMPK was in fact being activated in response to subcutaneous AICAR injections. Using ammonium sulfate precipitated homogenates.
  • AMPK AMP-activated protein kinase
  • the collection of ACC activity data from the entire citrate activation curve was not possible due to the limited amount of tissue from soleus muscles.
  • the maximal velocity for the reaction (V max ) as a function of increasing citrate concentration is significantly decreased (P ⁇ 0.01) from 36.7 ⁇ 5 in the saline- treated denervated gastrocnemius to 24.7 ⁇ 1.9 in the AICAR-teeated denervated gastrocnemius.
  • the citrate activation constant (K a ) was increased (P ⁇ 0.01) from 3.6 ⁇ 0.3 to 13.2 ⁇ 1.1.
  • Rats were injected either with AICAR (lmg in 0.9% NaCl/g body weight) or a 0.9%) NaCl solution ⁇ 0, ⁇ 24, and ⁇ 48 hours after denervation and were then sacrificed ⁇ 72 hours after denervation without injection. Rats were anesthetized with sodium pentobarbital (4.8 mg/100 g. body weight), soleus and gastrocnemius muscles from both sham and denervated sides were extracted, and muscles were quick frozen in liquid nitrogen and stored for analysis. Table 4 summarizes the findings relating to muscle atrophy.
  • the denervated gasteocnemius and soleus muscles from both saline-treated (0.9% NaCl) and AICAR- teeated (1 mg in 0.9 % NaCl/g body weight) were significantly atrophied ( P ⁇ .05) as compared to the contealateral, innervated controls. Furthermore, the amount of atrophy was not significantly increased or decreased in the AICAR-teeated muscles vs. the saline- teeated muscles.
  • the soleus muscles in both treatment groups lost -20% of their muscle mass after 3 days of denervation.
  • Values are means ⁇ SE. * P ⁇ 0.05 control vs. denervated.
  • Total GLUT4 increases in muscles of denervated rats in response to 3 day AICAR injection.
  • GLUT4 content of denervated gastrocnemius muscles was found to be 60.1% ⁇ 4.7 of GLUT4 content in the contealateral innervated gastrocnemius muscles.
  • GLUT4 content in denervated gasteocnemius muscles treated with AICAR was significantly increased (106.6%) ⁇ 5.5) over GLUT4 levels in denervated gastrocnemius muscles treated with saline.
  • the AICAR-teeated contralateral innervated muscles contained significantly increased levels of GLUT4 (130.1% ⁇ 7.2) when compared to saline-treated contralateral gasteocnemius muscles.
  • Rats of each teeatment group were subsequently injected with the respective AICAR solutions at ⁇ 24 and ⁇ 48 hours after denervation. Rats were killed ⁇ 72 hours after denervation without an AICAR injection and muscles were collected as described above.
  • Figure 11 shows the dose dependent response of GLUT4 in AICAR-treated denervated gastrocnemius muscles (curve B) and AICAR-treated contealateral innervated gasteocnemius muscles (curve A). Values are expressed as percent intensity of saline- treated contralateral innervated controls. Values are means ⁇ SE. + P ⁇ 0.05 vs. saline- treated denervated muscle. A # indicates P ⁇ 0.05 vs. saline-treated contralateral innervated control. For all GLUT4 data all saline-treated, contealateral, innervated muscles were normalized to 100%.
  • the lmg AICAR in 0.9%) NaCl/g body weight dose is also shown to be the only dose that significantly raises GLUT4 content in innervated gasteocnemius muscles above the GLUT4 content in saline- treated innervated gastrocnemius muscles ( P ⁇ .05).
  • chrome AMPK activation using AICAR injection in resting rats results in significant increases in ALA synthase, cytochrome c, citrate synthase, malate dehydrogenase, in white quadriceps, but not in red quadriceps.
  • Hexokinase activity was significantly increased in all three muscle types.
  • GLUT4 was increased in both red and white quadriceps, and a trend toward an increase was noted in soleus.

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Abstract

L'invention porte sur un procédé de traitement de l'obésité chez un mammifère. Le procédé consiste à administrer au mammifère une quantité efficace d'un point de vue thérapeutique d'un activateur de protéine kinase activée par AMP. Le mammifère peut être un homme, un rat, une souris et analogue. L'activateur de la protéine kinase activée par AMP peut être injecté par voie sous-cutanée ou administré d'une autre manière qui permet l'absorption dans les cellules du mammifère de l'activateur de la protéine kinase activée par AMP. L'activation de l'activateur précité peut avoir les mêmes avantages qu'un exercice d'entraînement, y compris la perte de poids. Cette invention porte également sur un procédé de traitement de la résistance à l'insuline chez un mammifère souffrant d'obésité, du diabète de type 2, de paralysie musculaire. Pour réduire la résistance à l'insuline, on donne au mammifère une quantité efficace d'un point de vue thérapeutique d'un activateur de protéine kinase activée par AMP.
PCT/US2001/018812 2000-06-09 2001-06-11 Procede de traitement de l'obesite et de la paralysie musculaire par des aides ergogeniques WO2001093874A1 (fr)

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US10/297,117 US20030212034A1 (en) 2001-06-11 2001-06-11 Method of treatment of obesity and paralyzed muscle and ergogenic aids
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004113562A1 (fr) * 2003-06-17 2004-12-29 Medical Research Council Procede de dosage de l'activite de phosphorylation lkb1
WO2007082309A2 (fr) * 2006-01-16 2007-07-19 The Board Of Regents Of The University Of Texas System Procédés et composition induisant une torpeur chez un sujet
US7560435B2 (en) 2002-03-21 2009-07-14 Advanced In Vitro Cell Technologies, S. A. Therapeutic use of riboside of 5-aminoimidazole-4-carboxamide (acadesine)
EP2234622A2 (fr) * 2007-12-28 2010-10-06 The Salk Institute for Biological Studies Méthodes améliorant les performances et le tonus musculaires
WO2012114204A3 (fr) * 2011-02-15 2013-06-27 Ecole Polytechnique Federale De Lausanne (Epfl) Epfl-Tto Procédés de traitement d'une dysfonction mitochondriale
US8895520B2 (en) 2011-10-26 2014-11-25 Universite Nice Sophia Antipolis Method for treating a human patent suffering from Myeloid Neoplasias using 5-aminoimidazole-4-carboxamide
US9192601B2 (en) 2006-12-29 2015-11-24 Salk Institute For Biological Studies Methods for enhancing muscle performance and tone
WO2020050935A2 (fr) 2018-08-06 2020-03-12 Skylark Bioscience Llc Composés activant la protéine kinase activée par l'amp et leurs utilisations
US11779590B2 (en) 2020-10-30 2023-10-10 Skylark Bioscience Llc AMP-activated protein kinase activating compounds and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658889A (en) * 1989-01-24 1997-08-19 Gensia Pharmaceuticals, Inc. Method and compounds for aica riboside delivery and for lowering blood glucose
WO2001010449A1 (fr) * 1999-08-09 2001-02-15 Trustees Of Boston University Technique permettant de maintenir l'integrite vasculaire a l'aide de l'aicar (5-amino-4-imidazole riboside) et de composes associes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5777100A (en) * 1990-08-10 1998-07-07 Gensia Inc. AICA riboside analogs
AU2001268236A1 (en) * 2000-06-06 2001-12-17 Trustees Of Boston University Use of aicar and related compounds

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658889A (en) * 1989-01-24 1997-08-19 Gensia Pharmaceuticals, Inc. Method and compounds for aica riboside delivery and for lowering blood glucose
WO2001010449A1 (fr) * 1999-08-09 2001-02-15 Trustees Of Boston University Technique permettant de maintenir l'integrite vasculaire a l'aide de l'aicar (5-amino-4-imidazole riboside) et de composes associes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAYASHI ET AL.: "Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport", DIABETES, vol. 47, no. 8, August 1998 (1998-08-01), pages 1369 - 1373, XP002948202 *
See also references of EP1427425A4 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7560435B2 (en) 2002-03-21 2009-07-14 Advanced In Vitro Cell Technologies, S. A. Therapeutic use of riboside of 5-aminoimidazole-4-carboxamide (acadesine)
JP2007520199A (ja) * 2003-06-17 2007-07-26 メディカル リサーチ カウンシル Ｌｋｂ１リン酸化活性を検定する方法
JP4651617B2 (ja) * 2003-06-17 2011-03-16 メディカル リサーチ カウンシル Ｌｋｂ１リン酸化活性を検定する方法
WO2004113562A1 (fr) * 2003-06-17 2004-12-29 Medical Research Council Procede de dosage de l'activite de phosphorylation lkb1
WO2007082309A2 (fr) * 2006-01-16 2007-07-19 The Board Of Regents Of The University Of Texas System Procédés et composition induisant une torpeur chez un sujet
WO2007082309A3 (fr) * 2006-01-16 2007-10-04 Univ Texas Procédés et composition induisant une torpeur chez un sujet
US9192601B2 (en) 2006-12-29 2015-11-24 Salk Institute For Biological Studies Methods for enhancing muscle performance and tone
EP2234622A2 (fr) * 2007-12-28 2010-10-06 The Salk Institute for Biological Studies Méthodes améliorant les performances et le tonus musculaires
EP2234622A4 (fr) * 2007-12-28 2011-03-09 Salk Inst For Biological Studi Méthodes améliorant les performances et le tonus musculaires
WO2012114204A3 (fr) * 2011-02-15 2013-06-27 Ecole Polytechnique Federale De Lausanne (Epfl) Epfl-Tto Procédés de traitement d'une dysfonction mitochondriale
US10709724B2 (en) 2011-02-15 2020-07-14 Ecole Polytechnique Federale De Lausanne (Epfl) Methods of treating mitochondrial dysfunction
US8895520B2 (en) 2011-10-26 2014-11-25 Universite Nice Sophia Antipolis Method for treating a human patent suffering from Myeloid Neoplasias using 5-aminoimidazole-4-carboxamide
WO2020050935A2 (fr) 2018-08-06 2020-03-12 Skylark Bioscience Llc Composés activant la protéine kinase activée par l'amp et leurs utilisations
JP2021534128A (ja) * 2018-08-06 2021-12-09 スカイラーク バイオサイエンス エルエルシー Ａｍｐ活性化プロテインキナーゼ活性化化合物およびその使用
JP7376951B2 (ja) 2018-08-06 2023-11-09 スカイラーク バイオサイエンス エルエルシー Ａｍｐ活性化プロテインキナーゼ活性化化合物およびその使用
US11834469B2 (en) 2018-08-06 2023-12-05 Skylark Bioscience Llc AMP-activated protein kinase activating compounds and uses thereof
US11779590B2 (en) 2020-10-30 2023-10-10 Skylark Bioscience Llc AMP-activated protein kinase activating compounds and uses thereof

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