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WO1993003734A1 - Method for lowering blood lipid levels - Google Patents

Method for lowering blood lipid levels Download PDF

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Publication number
WO1993003734A1
WO1993003734A1 PCT/US1992/006828 US9206828W WO9303734A1 WO 1993003734 A1 WO1993003734 A1 WO 1993003734A1 US 9206828 W US9206828 W US 9206828W WO 9303734 A1 WO9303734 A1 WO 9303734A1
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Prior art keywords
lipid
mimetic
pro
drug
animal
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PCT/US1992/006828
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French (fr)
Inventor
Georges H. Van Den Berghe
Harry E. Gruber
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Gensia, Inc.
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Priority to EP92918495A priority Critical patent/EP0599987A4/en
Publication of WO1993003734A1 publication Critical patent/WO1993003734A1/en
Priority to NO940580A priority patent/NO940580L/en

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A61P3/06—Antihyperlipidemics

Definitions

  • This invention relates to methods for treating animals having elevated serum lipid levels, e.g. , animals suffering fromhypertriglyceridemia , hypercholesterolemia , atherosclerosis or obesity.
  • Mild hypertriglyceridemia without much elevation of cholesterol (e.g. , Type IV hyperlipoproteinemia of Fredrickson) , is quite common in several disorders, including uncontrolled diabetes mellitus, renal failure, systemic lupus erythematosus, alcoholism, obesity (Schaeffer & Levy, 312 New England J. Medicine 1300, 1985), and as recently reported, AIDS (Grunfeld et al., 90 Am J. Med. 154, 1991). Mild hypertriglyceridemia can be aggravated by stress and various medications, such as estrogens, oral contraceptives, beta-blockers and thiazides.
  • various medications such as estrogens, oral contraceptives, beta-blockers and thiazides.
  • Mild hypertriglyceridemia is generally due to an increase in very low density lipoproteins (VLDLs) , caused by an increase in lipid synthesis, as well as a decrease in catabolism.
  • VLDLs very low density lipoproteins
  • the association of mild hypertriglyceridemia with atherosclerosis is less well established than that of hypercholesterolemia with atherosclerosis (Schwandt, 11 Eur. Heart J. Suppl. H 38, 1990) .
  • One reason for this may be the very high intraindividual variability of fasting triglyceride levels (Brenner & Heiss, 11 Eur. Heart J. 1054, 1990).
  • Mild hypertriglyceridemia should be treated, particularly in diabetics (Lewis et al., 72 J. Clin. Endocrinol. Metab. 934, 1991).
  • bile-acid sequestrants e.g. , cholestyramine, colestipol
  • One of their side-effects is an increase in triglycerides.
  • Stimulators of lipoprotein lipase act by increasing the activity of lipoprotein lipase, and thereby decrease the plasma VLDL and triglyceride levels. They are useful in treatment of hypertriglyceridemia, although not all patients respond. In addition, they may induce an increase in total cholesterol, and in LDL cholesterol.
  • Inhibitors of triglyceride and VLDL synthesis act by decreasing lipolysis in adipose tissue, and consequently the supply of fatty acids available for esterification into triglycerides in the liver.
  • Niacin nicotinic acid
  • a major problem with niacin is intense flushing and pruritus due to prostaglandin release. It also has an hyperglycemic effect which renders insulin adjustment in diabetics necessary.
  • Gemfibrozil (a derivative of pentenoic acid) stimulates lipoprotein lipase and the synthesis of HDL.
  • HMG-CoA reductase e.g.. mevastatin, lovastatin
  • HMG-CoA reductase rate-limiting enzyme
  • HMG-CoA reductase inhibitors can produce muscle abnormalities in humans and corneal opacities in experimental animals, but have not been found to produce serious side-effects (Grundy, 319 New England J. Medicine 24, 1988).
  • This invention relates to a novel means for decreasing the level of triglycerides, cholesterol and other related lipids in human or other animal plasma.
  • the method is based upon the finding that AICAriboside onophosphate (ZMP) , and related analogs (which are structural mimetics of AMP) , are effective in reducing the amount of synthesis of these lipids.
  • ZMP AICAriboside onophosphate
  • AMP- activated protein kinase which regulates the activity of enzymes that control the synthesis of fatty acids, cholesterol, and a lipase which may in turn effect the action of lipolytic hormones.
  • the compounds may block the action of lipolytic hormones on adipose tissue hormone-sensitive lipase, and thereby prevent release of fatty acids for export to liver for re- esterification into triglycerides.
  • administration of these compounds to an animal having elevated serum lipid levels is effective to lower such lipid levels, and thus is a treatment for hypertriglyceridemia, hypercholesterolemia and obesity.
  • the invention features a method for treating an animal, e.g.. one having an elevated serum lipid level.
  • the method may include the step of identifying an animal having such an elevated serum lipid level.
  • the method includes introducing into that animal a lipid-lowering amount of an AMP mimetic, or pro-drug (a compound which can be administered (e.g., orally) to generate an AMP mimetic in vivo. e.g.. it includes compounds which upon administration are activated to produce the AMP mimetic, e.g..
  • elevated is meant to encompass a level of lipid which is above an accepted normal range for that lipid in the animal, or which is known to be associated with a pathologic process. Such levels can be measured by any standard means, for example, they can be measured chemically, biochemically, or even by study of the symptoms of an animal which may reflect an elevated lipid level. Such symptoms may include disorders which are commonly associated with elevated lipid levels, such as diabetes mellitus, renal failure, atherosclerosis, heart disease, stroke, etc.. as discussed above. Thus, to the extent that an animal may not be specifically diagnosed as having an elevated lipid level, it is appropriate in this invention to treat animals which have a significant potential of having such elevated lipid levels.
  • the term "identif ing” includes identifying those animals which have such a significant potential.
  • the phrase "significant potential” includes those disorders which are commonly recognized by those skilled in the art as being associated with elevated lipid levels. For example, it can be concluded that an elevated serum lipid level is present in obese persons (i.e.. those having excess adipose tissue) or those with the effects of elevated lipids (e.g.. those having atherosclerosis or atherosclerosis-related complications, such as transient ische ic attacks, strokes, heart attacks, angina, and peripheral vascular diseases) .
  • lipid is meant to include any of a large number of lipids present in the serum of an animal, including (as discussed above) but not limited to cholesterol, triglycerides, lipoproteins, low density lipoproteins, very low density lipoproteins, and chylomicrons.
  • the method for treating includes introducing the desired AMP mimetic or pro-drug by any standard methodology, including transdermal, injection into muscle tissue, or blood stream, or by oral or other parenteral administration.
  • a “lipid-lowering amount” includes an amount which is effective over a period of several hours or days to lower the serum lipid level in a way which can be detected either chemically, biochemically, or by a change in the appearance or symptoms of the patient. That is, "lipid lowering” means a lowering of the level of lipid in a clinically significant manner, well known to those of ordinary skill in the art.
  • AMP mimetics or pro-drugs are well known to those in the art and include, e.g.. AICAriboside (5'-amino-4- imidazolecarboxamide riboside) , AICAribotide (ZMP) , and analogs thereof, and any pro-drugs which can be used to produce such AICAriboside, ZMP, and analogs thereof, within an animal body, or tubercidin (4-amino-l- ⁇ -D- ribofuranosylpyrrolo[2,3-d]pyrimidine) , tubercidin base, tubercidin monophosphate, and prodrugs thereof, which can produce AMP mimetics within a body of the animal being treated.
  • AICAriboside 5'-amino-4- imidazolecarboxamide riboside
  • ZMP AICAribotide
  • any pro-drugs which can be used to produce such AICAriboside, ZMP, and analogs thereof, within an animal body
  • tubercidin
  • pro-drugs and related analogs described in PCT Application WO 90/09163, published August 23, 1990 are potentially useful in this invention.
  • Such mimetics can be synthesized by the methods described in this publication.
  • base is meant a compound which when phosphoribosylated is a nucleotide and serves as an AMP mimetic.
  • AMP-mimetics or pro-drugs which are suitable for lipid-lowering within an animal can be readily identified by any standard biochemical test, e.g.. drug pharmacokinetics, measurement of lipid levels in biological fluids, or lipid metabolism, either in vivo or in vitro. Examples of such tests are described below.
  • AMP mimetics or pro-drugs are used in such tests, and those useful in this invention provide results similar to or better than AICAriboside.
  • Other examples of such tests are provided by Davies et al., 186 Eur. J. Biochem. 123, 1989 and Carling et al., 186 Eur. J. Biochem. 129, 1989.
  • the pro-drugs and related drugs such as those described in PCT 90/09163, can be readily screened to determine whether they are useful in the method of this invention.
  • AMP-activated protein kinases are well known to those in the art. For example, one embodiment is described in numerous publications by Dr. Hardie and his colleagues, see. Hardie et al., 14 Trends Biochem. Sci. 20, 1989; Munday et al., 175 Eur. J. Biochem. 31, 1988; Davies et al., 187 Eur. J. Biochem. 183, 1990; Sim and Hardie, 233 FEBS LETTERS 294, 1988; Munday et al., 235 FEBS LETTERS 144, 1988; Carling et al., 186 Eur. J. Biochem. 129, 1989; and Davies et al., 186 Eur. J. Biochem. 123, 1989.
  • This invention provides a simple way in which several lipid synthesis pathways can be simultaneously inhibited. This inhibition can be produced by administration of a single drug, as discussed above, and thus has significant advantages over prior art methods of lowering lipid synthesis which require administration of several drugs. In addition, the inhibition by a drug of this invention also causes inhibition of hepatic synthesis of triglycerides, which had not to date been possible.
  • the method of this invention is advantageous since it specifically inhibits activity of liver-specific proteins, and has little or no effects in certain other tissues, such as cornea and muscle tissues.
  • the drugs are also advantageous since they control both lipid and cholesterol biosynthesis, and are potentially far more potent than existing drugs.
  • Fig. 1 is a schematic representation of two lipid synthesis pathways in animals
  • Fig. 2 is a graph showing the effect of AICAriboside on synthesis of fatty acids from endogenous substrates
  • Fig. 3 is a graph showing the dose effect of AICAriboside on synthesis of fatty acids from glucose
  • Fig. 4 is a graph showing the dose effect on AICAriboside on synthesis of fatty acids from lactate;
  • Fig. 5 is a graph showing the effect of AICAriboside on synthesis of fatty acids from leucine;
  • Fig. 6 is a graph showing the effect of AICAriboside on synthesis of fatty acids from 2-ketoisocaproate
  • Fig. 7 is a graph showing the effect of AICAriboside on acetyl-CoA carboxylase activity in isolated hepatocytes
  • Fig. 8 is a graph showing the effect of AICAriboside on incorporation of tritiated water into lipids.
  • Fig. 9 is a graph showing the effect of AICAriboside on the non-saponifiable lipid fraction.
  • Fig. 10 is a graph showing the effect of IP injection of AICAriboside (500 g/kg) on rat liver HMG-CoA reductase activity.
  • Figs. 11 and 12 are graphs showing AMP-stimulated protein kinase activity relative to ZMP and AMP concentrations.
  • Fig. 13 is a histogram showing AMP-stimulated protein kinase activity by AMP and other reagents.
  • Fig. 1 the following is a brief description of the various biochemical pathways, and enzymes involved therein, which are related to the utility of this invention.
  • triglycerides are synthesized mainly in the liver, from fatty acids either newly synthesized by the liver itself, or coming from lipolysis in adipose tissue.
  • Hepatic synthesis of fatty acids includes the conversion of various substrates into acetyl-CoA, e.g.. glucose and other monosaccharides, such as fructose and galactose, lactate, pyruvate, ketogenic amino acids and their keto derivatives.
  • Acetyl-CoA is converted into malonyl-CoA by acetyl-CoA carboxylase.
  • Malonyl-CoA is subsequently elongated into long-chain fatty acyl-CoA, which is esterified into triglycerides with glycerol 3-phosphate. These triglycerides are exported toward peripheral tissues, including adipose tissue, in the form of very-low-density lipoproteins. Lipoprotein lipase, located at the outer surface of peripheral cells, hydrolyzes the triglycerides to fatty acids and glycerol which can be taken up by the cells.
  • Acetyl-CoA carboxylase is the rate-limiting enzyme of hepatic fatty acid synthesis. Its activity is controlled by citrate, which is stimulatory, and by long-chain fatty acyl-CoA, which is inhibitory. Acetyl-CoA carboxylase is moreover regulated by reversible phosphorylation/ dephosphorylation. The dephosphorylated form of acetyl- CoA carboxylase is active, and the phosphorylated form inactive.
  • a variety of protein kinases including cAMP- dependent protein kinase and protein kinase C, are able to phosphorylate acetyl-CoA carboxylase in vitro.
  • acetyl-CoA is first converted into acetoacetyl-CoA, and thereafter into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) .
  • HMG-CoA reductase is the rate limiting step of cholesterol synthesis.
  • An ensuing 9 step sequence leads from mevalonate to cholesterol.
  • HMG-CoA reductase is located inside the smooth endoplasmic reticulum. Expression of the enzyme is regulated by the cholesterol level: excess cholesterol decreases the synthesis of the protein and increases its degradation.
  • HMG-CoA reductase is regulated by phosphorylation/dephosphorylation.
  • Purified HMG-CoA reductase can be phosphorylated and inactivated in vitro by several protein kinases, including protein kinase C, a Ca ⁇ /calmodulin-dependent protein kinase, and AMP-activated protein kinase (Hardie et al., 14 Trends Biochem. Sci. 20, 1989) .
  • AMP- activated protein kinase phosphorylates HMG-CoA reductase in vivo (Clarke & Hardie, 9 EMBO Journal 2439, 1990).
  • AMP-activated protein kinase belongs to a group of enzymes which, using ATP, phosphorylate proteins at serine or occasionally threonine residues. Best known in this group are cyclic AMP-dependent protein kinase (cA-
  • Protein kinases are components of the transduction mechanisms whereby hormones and other factors regulate physiological functions. Their action elicits conformational changes that modify either the catalytic activity of enzymes or the function of other regulatory proteins. The conformational changes induced by phosphorylation can be reversed by protein phosphatases, of which several types have been characterized in recent years.
  • AMP-PK phosphorylates three enzymes, each catalyzing a key regulatory step of lipid metabolism: [1] acetyl-CoA carboxylase, the rate-limiting step of fatty acids synthesis, which is most active in liver; [2] HMG-CoA reductase, the first committed step of cholesterol synthesis, also predominantly located in liver; and [3] hormone-sensitive lipase, the enzyme that controls the release of fatty acids from triglycerides in adipose tissue (Hardie et al., 14 Trends Biochem. Sci 20, 1989).
  • AMP-PK In accordance with its role in lipid metabolism, the highest activities of AMP-PK are found in liver and lactating mammary gland, which are very active in both fatty acid and cholesterol synthesis (Davies et al. , 186 Eur. J. Biochem. 123, 1989). Lower activities exist in tissues which have an active fatty acid metabolism, namely, adipose tissue, adrenal cortex, lung, macrophages and heart. The tissues with the lowest activity of AMP-PK are brain and muscle, two tissues in which rates of lipid synthesis are very low, at least in adults.
  • AMP-PK has been purified 4800-fold from rat liver, to a specific activity of 1.25 ⁇ mol/min/mg (Carling et al., 186 Eur. J. Biochem. 129, 1989) . Although the preparation did not display a single band on an electrophoretic gel, the fact that its specific activity was comparable with that of other protein kinases suggests that it was approaching homogeneity. Molecular mass of subunits is estimated at 63 kDa, and of the holoenzyme at 100 + 30 kDa, indicating that native AMP-PK might be a dimer. Mos investigations of the catalytic properties of AMP-PK have been performed with acetyl-CoA carboxylase as substrate.
  • AMP-PK In the absence of AMP, AMP-PK has a Km of 86 ⁇ M for ATP and of 1.9 ⁇ M for acetyl-CoA carboxylase. AMP increases Vmax 3- to 6-fold, without significantly modifying Km for either ATP or acetyl-CoA carboxylase.
  • the sensitivity to AMP depends on the concentration of ATP: at 0.2 mM ATP, half-maximal stimulation by AMP is observed at 1.4 ⁇ M; at the near-physiological ATP concentration of 2 mM, half-maximal stimulation requires 14 ⁇ M AMP.
  • Some AMP analogues are reported ineffective as substituting for AMP (Carling et al., 186 Eur. J. Biochem. 129, 1989).
  • 8-bromoadenosine 5-monophosphate is a weak stimulator at low concentrations, but it is an inhibitor at high concentrations.
  • the regulatory concentrations of AMP are an order of magnitude lower than those measured in acid extracts of liver. However, the latter have often been claimed to be artefactual, resulting from degradation of ATP. Calculations based on equilibrium constants and nuclear magnetic resonance studies have led to estimates that the free concentration of liver AMP may be around 1 ⁇ M. Any increase in AMP within this range would thus potently stimulate AMP-PK.
  • AMP-PK has been shown to inactivate acetyl-CoA carboxylase in a cell-free system by phosphorylating Ser- 79 of the protein (Hardie et al., 14 Trends Biochem. Sci 20, 1989). Addition of glucagon to intact cells, namely isolated rat hepatocytes and adipocytes, also results in the phosphorylation of Ser-79. In a cell-free system, however, cA-PK, which as a rule mediates the actions of hormones that elevate cyclic AMP, phosphorylates a different serine, namely Ser-77.
  • AMP-PK inactivates HMG-CoA reductase by phosphorylating Ser-872 of the enzyme (Clarke & Hardie, 9 EMBO Journal 2439, 1990). This phosphorylation is most likely responsible for the inactivation of HMG-CoA reductase known to occur when special precautions (e.g.. freeze-clamping) are not taken to avoid a rise of the concentration of AMP after removing liver tissue.
  • special precautions e.g. freeze-clamping
  • AMP-PK phosphorylates Ser-565 of hormone-sensitive lipase. This phosphorylation inhibits subsequent phosphorylation and activation of hormone-sensitive lipase by cA-PK (Garton et al., 179 Eur. J. Biochem 249, 1989). Phosphorylation of hormone-sensitive lipase by AMP-PK might thus block the action of lipolytic hormones (and release of free fatty acids and glycerol from fat cells) which act by way of cyclic AMP and cA-PK.
  • AMP-PK has also been reported to be itself regulated by phosphorylation, which activates the enzyme, and by dephosphorylation which inactivates the enzyme. Nanomolar concentrations of fatty acyl-CoA were shown to stimulate the 'AMP-PK kinase', thus activating AMP-PK. Since the latter activation will result in inactivation of acetyl-
  • CoA carboxylase it provides a mechanism whereby fatty acyl-CoA can exert feed-back inhibition on fatty acid synthesis.
  • the studies of the AMP-PK system indicate that it plays an important role in regulating the levels of fatty acids and cholesterol in the body. That
  • AMP-PK acts on both acetyl-CoA carboxylase and HMG-CoA reductase most likely explains why hepatic fatty acid and cholesterol synthesis are regulated in parallel in several situations (e.g.. both synthetic pathways peak at the same time of the day, both are inhibited by diets high in polyunsaturated fatty acids) .
  • AICAriboside As described in detail below, we have discovered that the addition of the nucleoside, AICAriboside, to suspensions of isolated rat hepatocytes provokes an inactivation of both acetyl-CoA carboxylase and HMG-CoA reductase. AICAriboside is efficiently converted by phosphorylation into the corresponding nucleotide, AICAribotide or ZMP, in isolated rat hepatocytes (Vincent et al. , 40 Diabetes 1259, 1991, data not shown) and in vivo (data not shown) .
  • ZMP can also be formed from AICA base administration followed by in vivo phosphoribosylation.
  • ZMP can also be formed from AICA base administration followed by in vivo phosphoribosylation.
  • 3 H 2 0 results in the labelling of NADPH. Utilization of the latter by fatty acid synthetase results in the formation of labelled fatty acids and triglycerides. Besides entering fatty acid synthesis, 3 H 2 0 can also enter the biosynthesis of cholesterol (at the level of HMG-CoA reductase) .
  • Example 1 Effect of AICAriboside on fatty acid synthesis with endogenous substrates
  • fatty acid synthesis can proceed from endogenous substrates, glycogen, and ketogenic amino acids. This endogenous fatty acid synthesis is completely inhibited by 500 ⁇ M
  • AICAriboside inhibits fatty acid synthesis from 15 mM glucose in a dose-dependent fashion (Fig. 3) . Half maximal inhibition is obtained with about 50 ⁇ M AICAriboside.
  • AICAriboside inhibits fatty acid synthesis from lactate 10 M/pyruvate 1 mM (Fig. 4) .
  • Fatty acid synthesis from lactate/pyruvate seems to be slightly less sensitive to AICAriboside than that from glucose (half- maximal inhibition with about 75 ⁇ M AICAriboside) .
  • Example 4 Effect of AICAriboside on fatty acid synthesis with ketogenic amino acids
  • Ketogenic amino acids enter fatty acid synthesis at the level of acetyl-CoA, thus bypassing the transport of pyruvate into the mitochondria, and the enzymes pyruvate dehydrogenase and pyruvate carboxylase.
  • ketogenic amino acids are thought to be first deaminated in muscle, and to be transported thereafter to the liver in the form of ketoacids.
  • fatty acid synthesis from leucine was compared with that from its transamination product, 2-ketoisocaproic acid.
  • fatty acid synthesis with both substrates is completely inhibited by AICAriboside 500 ⁇ M.
  • Example 5 Effect of AICAriboside on the activity of acetyl-CoA carboxylase
  • Acetyl-CoA carboxylase is the limiting step of fatty acid synthesis. It is interconvertible by phosphorylation/dephosphorylation, the active form being dephosphorylated. Acetyl-CoA carboxylase is activated by a dephosphorylating phosphatase and inactivated by several kinases (including cAMP-dependent protein kinase, under the influence of glucagon) . In addition, inactivation of acetyl-CoA carboxylase by an AMP-activated protein kinase has been reported by Dr. Hardie's group. Fig. 7 shows that acetyl-CoA carboxylase (assay performed by the method of Bijleveld & Geelen, 918 Bioc. Biop. Acta 274, 1987) is inactivated by the addition of AICAriboside 500 ⁇ M. This suggests that ZMP acts at the level of the AMP-activated protein.
  • Example 6 Effects on the incorporation of 3 H-,0 in the non-saponifiable lipid fraction
  • ZMP by activating AMP-activated protein kinase, inactivates both acetyl-CoA carboxylase and HMG-CoA reductase, the limiting enzymes of, respectively, fatty acid and cholesterol synthesis.
  • HMG-CoA reductase is the limiting step of cholesterol synthesis. It is interconvertible by phosphorylation/dephosphorylation, the active form being dephosphorylated. HMG-CoA reductase is activated by the dephosphorylating phosphatases, protein phosphatase 2A and 2C, and inactivated by several protein kinases. These include Ca 2 +/calmodulin-dependent protein kinase, protein kinase C, and as also described by Dr. Hardie's group. AMP-activated protein kinase. Fig.
  • Example 8 Effect of ZMP on the activity of AMP-activated protein kinase
  • Dr. Hardie and co-workers have set up a specific and sensitive assay of AMP-activated protein kinase. It is based on the incorporation of radioactivity from [y- 32 P]ATP into a 15-amino acid peptide, termed the SAMS peptide, designed after the sequence of acetyl-CoA carboxylase surrounding Ser79, the site which is phosphorylated exclusively by the AMP-activated protein kinase.
  • SAMS peptide 15-amino acid peptide
  • Fig. 11 shows that in this assay, ZMP stimulates up to nearly 8-fold the activity of rat liver AMP-activated protein kinase, partially purified up to the DEAE-Sepharose step as described by Davies et al. (Eur. J. Biochem. 186: 123-8, 1989). Half-maximal stimulation is obtained at 0.6 mM, and maximal stimulation at 4mM ZMP.
  • Fig. 12 shows that 2mM ZMP overrides the stimulatory effect of concentrations of AMP below 0.1 mM, but is not additive and even slightly inhibitory at higher concentrations of AMP, suggesting that both nucleotides bind to the same site.
  • Fig. 12 shows that 2mM ZMP overrides the stimulatory effect of concentrations of AMP below 0.1 mM, but is not additive and even slightly inhibitory at higher concentrations of AMP, suggesting that both nucleotides bind to the same site.

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Abstract

Method for treating an animal having an elevated serum lipid level. The method includes the steps of identifying an animal having such an elevated serum lipid level, and introducing into that animal a lipid-lowering amount of an AMP mimetic which stimulates AMP-activated protein kinase.

Description

DESCRIPTION
Method for Lowering Blood Lipid Levels
Related Applications
This application is a continuation-in-part of U.S. Serial No. 07/748,944, filed August 23, 1991, which is a continuation-in-part of U.S. Serial No. 07/446,979, filed January 18, 1990, which is a continuation-in-part of U.S. Serial No. 301,453, filed January 24, 1989, and of U.S. Serial No. 408,107, filed September 15, 1989, which is a continuation-in-part of U.S. Serial 301,222, filed January 24, 1989. The content of these applications, including their drawings, are hereby incorporated by reference.
Field of the Invention
This invention relates to methods for treating animals having elevated serum lipid levels, e.g. , animals suffering fromhypertriglyceridemia , hypercholesterolemia , atherosclerosis or obesity.
Background of the Invention
Mild hypertriglyceridemia, without much elevation of cholesterol (e.g. , Type IV hyperlipoproteinemia of Fredrickson) , is quite common in several disorders, including uncontrolled diabetes mellitus, renal failure, systemic lupus erythematosus, alcoholism, obesity (Schaeffer & Levy, 312 New England J. Medicine 1300, 1985), and as recently reported, AIDS (Grunfeld et al., 90 Am J. Med. 154, 1991). Mild hypertriglyceridemia can be aggravated by stress and various medications, such as estrogens, oral contraceptives, beta-blockers and thiazides. Mild hypertriglyceridemia is generally due to an increase in very low density lipoproteins (VLDLs) , caused by an increase in lipid synthesis, as well as a decrease in catabolism. The association of mild hypertriglyceridemia with atherosclerosis is less well established than that of hypercholesterolemia with atherosclerosis (Schwandt, 11 Eur. Heart J. Suppl. H 38, 1990) . One reason for this may be the very high intraindividual variability of fasting triglyceride levels (Brenner & Heiss, 11 Eur. Heart J. 1054, 1990). Nevertheless, most physicians agree that mild hypertriglyceridemia should be treated, particularly in diabetics (Lewis et al., 72 J. Clin. Endocrinol. Metab. 934, 1991).
Marked to severe hypertriglyceridemia is found in association with hypercholesterolemia in hyperlipoproteine ias type I, III, and V, and is most often due to an increase in both VLDLs and chylomicrons. The association of an elevation of total serum cholesterol, particularly in combination with increased low density lipoproteins (LDL) and decreased high density lipoproteins (HDL) , with a higher incidence of atherosclerosis and coronary heart disease is well established. In recent years, increasing evidence has also been presented that correction of hyperocholesterolemia decreases the subsequent incidence of coronary artery disease (Havel, 81 J. Clin. Invest. 1653, 1988). Obesity is defined as a condition caused by an excessive amount of adipose tissue. Present evidence indicates that obesity can be caused not only by an excessive intake of food, but also by impairment of the mechanisms that control the normal proportion of adipose tissue (about 10% for men and 25% for women) . In accordance with the central position of the liver in the build-up of triglycerides, obesity is commonly accompanied with moderate to severe hypertriglyceridemia.
Current therapy of hypertriglyceridemia and hypercholesterolemia includes specific diets and four main classes of drugs (Lopes-Virella & Colwell, 3 Diabetes/Metabolism Reviews. 691, 1987; and Grundy, 319 New England J. Medicine 24, 1988).
For example, bile-acid sequestrants (e.g. , cholestyramine, colestipol) increase the fecal excretion of bile acids, and lead to an increase in hepatic cholesterol catabolism. They are useful for treatment of isolated hypercholesterolemia with increased LDL. One of their side-effects is an increase in triglycerides.
Stimulators of lipoprotein lipase (e.g.. clofibrate) act by increasing the activity of lipoprotein lipase, and thereby decrease the plasma VLDL and triglyceride levels. They are useful in treatment of hypertriglyceridemia, although not all patients respond. In addition, they may induce an increase in total cholesterol, and in LDL cholesterol.
Inhibitors of triglyceride and VLDL synthesis (e.g.. niacin and gemfibrozil) act by decreasing lipolysis in adipose tissue, and consequently the supply of fatty acids available for esterification into triglycerides in the liver. Niacin (nicotinic acid) decreases both serum triglycerides and LDL cholesterol, and may increase HDL cholesterol. A major problem with niacin is intense flushing and pruritus due to prostaglandin release. It also has an hyperglycemic effect which renders insulin adjustment in diabetics necessary. Gemfibrozil (a derivative of pentenoic acid) stimulates lipoprotein lipase and the synthesis of HDL. It is markedly efficient in lowering triglycerides and in increasing HDL, but its effect on LDL is variable. Inhibitors of HMG-CoA reductase (e.g.. mevastatin, lovastatin) reduce cholesterol in plasma by inhibiting the rate-limiting enzyme (i.e.. HMG-CoA reductase) of cholesterol synthesis. In addition, they reduce LDL, apparently by increasing the expression of LDL receptors on the surface of the liver cells. They also raise HDL in some patients. The efficacy of HMG-CoA reductase inhibitors in hypertriglyceridemia is unclear. HMG-CoA reductase inhibitors can produce muscle abnormalities in humans and corneal opacities in experimental animals, but have not been found to produce serious side-effects (Grundy, 319 New England J. Medicine 24, 1988).
Summary of the Invention
This invention relates to a novel means for decreasing the level of triglycerides, cholesterol and other related lipids in human or other animal plasma. The method is based upon the finding that AICAriboside onophosphate (ZMP) , and related analogs (which are structural mimetics of AMP) , are effective in reducing the amount of synthesis of these lipids. These compounds have their effect by stimulating a protein kinase (AMP- activated protein kinase) which regulates the activity of enzymes that control the synthesis of fatty acids, cholesterol, and a lipase which may in turn effect the action of lipolytic hormones. For example, the compounds may block the action of lipolytic hormones on adipose tissue hormone-sensitive lipase, and thereby prevent release of fatty acids for export to liver for re- esterification into triglycerides. Thus, administration of these compounds to an animal having elevated serum lipid levels is effective to lower such lipid levels, and thus is a treatment for hypertriglyceridemia, hypercholesterolemia and obesity.
Thus, in a first aspect, the invention features a method for treating an animal, e.g.. one having an elevated serum lipid level. The method may include the step of identifying an animal having such an elevated serum lipid level. The method includes introducing into that animal a lipid-lowering amount of an AMP mimetic, or pro-drug (a compound which can be administered (e.g., orally) to generate an AMP mimetic in vivo. e.g.. it includes compounds which upon administration are activated to produce the AMP mimetic, e.g.. esters which can be cleaved, or nucleosides which can be phosporylated or bases which can be phosphoribosylated to form the AMP- mimetic) of an AMP mimetic, which stimulates AMP-activated protein kinase.
The term "elevated" is meant to encompass a level of lipid which is above an accepted normal range for that lipid in the animal, or which is known to be associated with a pathologic process. Such levels can be measured by any standard means, for example, they can be measured chemically, biochemically, or even by study of the symptoms of an animal which may reflect an elevated lipid level. Such symptoms may include disorders which are commonly associated with elevated lipid levels, such as diabetes mellitus, renal failure, atherosclerosis, heart disease, stroke, etc.. as discussed above. Thus, to the extent that an animal may not be specifically diagnosed as having an elevated lipid level, it is appropriate in this invention to treat animals which have a significant potential of having such elevated lipid levels. Thus, the term "identif ing" includes identifying those animals which have such a significant potential. Those skilled in the art will recognize that the phrase "significant potential" includes those disorders which are commonly recognized by those skilled in the art as being associated with elevated lipid levels. For example, it can be concluded that an elevated serum lipid level is present in obese persons (i.e.. those having excess adipose tissue) or those with the effects of elevated lipids (e.g.. those having atherosclerosis or atherosclerosis-related complications, such as transient ische ic attacks, strokes, heart attacks, angina, and peripheral vascular diseases) .
By "lipid" is meant to include any of a large number of lipids present in the serum of an animal, including (as discussed above) but not limited to cholesterol, triglycerides, lipoproteins, low density lipoproteins, very low density lipoproteins, and chylomicrons. The method for treating includes introducing the desired AMP mimetic or pro-drug by any standard methodology, including transdermal, injection into muscle tissue, or blood stream, or by oral or other parenteral administration.
A "lipid-lowering amount" includes an amount which is effective over a period of several hours or days to lower the serum lipid level in a way which can be detected either chemically, biochemically, or by a change in the appearance or symptoms of the patient. That is, "lipid lowering" means a lowering of the level of lipid in a clinically significant manner, well known to those of ordinary skill in the art.
AMP mimetics or pro-drugs are well known to those in the art and include, e.g.. AICAriboside (5'-amino-4- imidazolecarboxamide riboside) , AICAribotide (ZMP) , and analogs thereof, and any pro-drugs which can be used to produce such AICAriboside, ZMP, and analogs thereof, within an animal body, or tubercidin (4-amino-l-β-D- ribofuranosylpyrrolo[2,3-d]pyrimidine) , tubercidin base, tubercidin monophosphate, and prodrugs thereof, which can produce AMP mimetics within a body of the animal being treated. For example, those pro-drugs and related analogs described in PCT Application WO 90/09163, published August 23, 1990, are potentially useful in this invention. Such mimetics can be synthesized by the methods described in this publication. By "base" is meant a compound which when phosphoribosylated is a nucleotide and serves as an AMP mimetic. AMP-mimetics or pro-drugs which are suitable for lipid-lowering within an animal can be readily identified by any standard biochemical test, e.g.. drug pharmacokinetics, measurement of lipid levels in biological fluids, or lipid metabolism, either in vivo or in vitro. Examples of such tests are described below. Potentially useful AMP mimetics or pro-drugs are used in such tests, and those useful in this invention provide results similar to or better than AICAriboside. Other examples of such tests are provided by Davies et al., 186 Eur. J. Biochem. 123, 1989 and Carling et al., 186 Eur. J. Biochem. 129, 1989. Generally, in in vitro tests it is determined whether the AMP mimetic or pro-drug enhances enzymatic activity of an AMP-activated protein kinase. Thus, the pro-drugs and related drugs, such as those described in PCT 90/09163, can be readily screened to determine whether they are useful in the method of this invention.
AMP-activated protein kinases are well known to those in the art. For example, one embodiment is described in numerous publications by Dr. Hardie and his colleagues, see. Hardie et al., 14 Trends Biochem. Sci. 20, 1989; Munday et al., 175 Eur. J. Biochem. 31, 1988; Davies et al., 187 Eur. J. Biochem. 183, 1990; Sim and Hardie, 233 FEBS LETTERS 294, 1988; Munday et al., 235 FEBS LETTERS 144, 1988; Carling et al., 186 Eur. J. Biochem. 129, 1989; and Davies et al., 186 Eur. J. Biochem. 123, 1989. This invention provides a simple way in which several lipid synthesis pathways can be simultaneously inhibited. This inhibition can be produced by administration of a single drug, as discussed above, and thus has significant advantages over prior art methods of lowering lipid synthesis which require administration of several drugs. In addition, the inhibition by a drug of this invention also causes inhibition of hepatic synthesis of triglycerides, which had not to date been possible.
The method of this invention is advantageous since it specifically inhibits activity of liver-specific proteins, and has little or no effects in certain other tissues, such as cornea and muscle tissues. The drugs are also advantageous since they control both lipid and cholesterol biosynthesis, and are potentially far more potent than existing drugs. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments The drawings will first briefly be described.
Drawings
Fig. 1 is a schematic representation of two lipid synthesis pathways in animals;
Fig. 2 is a graph showing the effect of AICAriboside on synthesis of fatty acids from endogenous substrates;
Fig. 3 is a graph showing the dose effect of AICAriboside on synthesis of fatty acids from glucose;
Fig. 4 is a graph showing the dose effect on AICAriboside on synthesis of fatty acids from lactate; Fig. 5 is a graph showing the effect of AICAriboside on synthesis of fatty acids from leucine;
Fig. 6 is a graph showing the effect of AICAriboside on synthesis of fatty acids from 2-ketoisocaproate;
Fig. 7 is a graph showing the effect of AICAriboside on acetyl-CoA carboxylase activity in isolated hepatocytes;
Fig. 8 is a graph showing the effect of AICAriboside on incorporation of tritiated water into lipids; and
Fig. 9 is a graph showing the effect of AICAriboside on the non-saponifiable lipid fraction.
Fig. 10 is a graph showing the effect of IP injection of AICAriboside (500 g/kg) on rat liver HMG-CoA reductase activity.
Figs. 11 and 12 are graphs showing AMP-stimulated protein kinase activity relative to ZMP and AMP concentrations.
Fig. 13 is a histogram showing AMP-stimulated protein kinase activity by AMP and other reagents. Lipid Synthesis Pathways
Referring to Fig. 1, the following is a brief description of the various biochemical pathways, and enzymes involved therein, which are related to the utility of this invention.
In humans, triglycerides are synthesized mainly in the liver, from fatty acids either newly synthesized by the liver itself, or coming from lipolysis in adipose tissue. Hepatic synthesis of fatty acids includes the conversion of various substrates into acetyl-CoA, e.g.. glucose and other monosaccharides, such as fructose and galactose, lactate, pyruvate, ketogenic amino acids and their keto derivatives. Acetyl-CoA is converted into malonyl-CoA by acetyl-CoA carboxylase. Malonyl-CoA is subsequently elongated into long-chain fatty acyl-CoA, which is esterified into triglycerides with glycerol 3-phosphate. These triglycerides are exported toward peripheral tissues, including adipose tissue, in the form of very-low-density lipoproteins. Lipoprotein lipase, located at the outer surface of peripheral cells, hydrolyzes the triglycerides to fatty acids and glycerol which can be taken up by the cells.
Acetyl-CoA carboxylase is the rate-limiting enzyme of hepatic fatty acid synthesis. Its activity is controlled by citrate, which is stimulatory, and by long-chain fatty acyl-CoA, which is inhibitory. Acetyl-CoA carboxylase is moreover regulated by reversible phosphorylation/ dephosphorylation. The dephosphorylated form of acetyl- CoA carboxylase is active, and the phosphorylated form inactive. A variety of protein kinases, including cAMP- dependent protein kinase and protein kinase C, are able to phosphorylate acetyl-CoA carboxylase in vitro. However, strong evidence has been presented that, in intact cells, AMP-activated protein kinase phosphorylates and inactivates acetyl-CoA carboxylase (see. Hardie et al., 14 Trends Biochem. Sci. 20, 1989). Cholesterol is synthesized by nearly all tissues in humans, but the liver makes one of the largest contributions to the body's cholesterol pool. In the plasma, cholesterol is transported mainly in the form of low-density lipoproteins (LDL) , which result from the conversion of VLDL after these have released their triglycerides to peripheral tissues. Reverse transport of cholesterol, i.e. , from tissues back to the liver for catabolism and excretion, is postulated to occur via high- density lipoproteins. Similar to that of fatty acids, synthesis of cholesterol also proceeds from acetyl-CoA, which is first converted into acetoacetyl-CoA, and thereafter into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) . The subsequent reduction of HMG-CoA into mevalonate, catalyzed by HMG-CoA reductase, is the rate limiting step of cholesterol synthesis. An ensuing 9 step sequence leads from mevalonate to cholesterol. HMG-CoA reductase is located inside the smooth endoplasmic reticulum. Expression of the enzyme is regulated by the cholesterol level: excess cholesterol decreases the synthesis of the protein and increases its degradation. Similar to acetyl- CoA carboxylase, HMG-CoA reductase is regulated by phosphorylation/dephosphorylation. Purified HMG-CoA reductase can be phosphorylated and inactivated in vitro by several protein kinases, including protein kinase C, a Ca^/calmodulin-dependent protein kinase, and AMP-activated protein kinase (Hardie et al., 14 Trends Biochem. Sci. 20, 1989) . Recently, evidence has been presented that AMP- activated protein kinase phosphorylates HMG-CoA reductase in vivo (Clarke & Hardie, 9 EMBO Journal 2439, 1990).
AMP-activated Protein Kinase
AMP-activated protein kinase (AMP-PK) belongs to a group of enzymes which, using ATP, phosphorylate proteins at serine or occasionally threonine residues. Best known in this group are cyclic AMP-dependent protein kinase (cA-
PK) , Ca^/calmodulin-dependent protein kinases, and protein kinase C. Protein kinases are components of the transduction mechanisms whereby hormones and other factors regulate physiological functions. Their action elicits conformational changes that modify either the catalytic activity of enzymes or the function of other regulatory proteins. The conformational changes induced by phosphorylation can be reversed by protein phosphatases, of which several types have been characterized in recent years. AMP-PK phosphorylates three enzymes, each catalyzing a key regulatory step of lipid metabolism: [1] acetyl-CoA carboxylase, the rate-limiting step of fatty acids synthesis, which is most active in liver; [2] HMG-CoA reductase, the first committed step of cholesterol synthesis, also predominantly located in liver; and [3] hormone-sensitive lipase, the enzyme that controls the release of fatty acids from triglycerides in adipose tissue (Hardie et al., 14 Trends Biochem. Sci 20, 1989). In accordance with its role in lipid metabolism, the highest activities of AMP-PK are found in liver and lactating mammary gland, which are very active in both fatty acid and cholesterol synthesis (Davies et al. , 186 Eur. J. Biochem. 123, 1989). Lower activities exist in tissues which have an active fatty acid metabolism, namely, adipose tissue, adrenal cortex, lung, macrophages and heart. The tissues with the lowest activity of AMP-PK are brain and muscle, two tissues in which rates of lipid synthesis are very low, at least in adults.
AMP-PK has been purified 4800-fold from rat liver, to a specific activity of 1.25 μmol/min/mg (Carling et al., 186 Eur. J. Biochem. 129, 1989) . Although the preparation did not display a single band on an electrophoretic gel, the fact that its specific activity was comparable with that of other protein kinases suggests that it was approaching homogeneity. Molecular mass of subunits is estimated at 63 kDa, and of the holoenzyme at 100 + 30 kDa, indicating that native AMP-PK might be a dimer. Mos investigations of the catalytic properties of AMP-PK have been performed with acetyl-CoA carboxylase as substrate. In the absence of AMP, AMP-PK has a Km of 86 μM for ATP and of 1.9 μM for acetyl-CoA carboxylase. AMP increases Vmax 3- to 6-fold, without significantly modifying Km for either ATP or acetyl-CoA carboxylase. The sensitivity to AMP depends on the concentration of ATP: at 0.2 mM ATP, half-maximal stimulation by AMP is observed at 1.4 μM; at the near-physiological ATP concentration of 2 mM, half-maximal stimulation requires 14 μM AMP. Some AMP analogues are reported ineffective as substituting for AMP (Carling et al., 186 Eur. J. Biochem. 129, 1989). However, 8-bromoadenosine 5-monophosphate is a weak stimulator at low concentrations, but it is an inhibitor at high concentrations. The regulatory concentrations of AMP are an order of magnitude lower than those measured in acid extracts of liver. However, the latter have often been claimed to be artefactual, resulting from degradation of ATP. Calculations based on equilibrium constants and nuclear magnetic resonance studies have led to estimates that the free concentration of liver AMP may be around 1 μM. Any increase in AMP within this range would thus potently stimulate AMP-PK.
AMP-PK has been shown to inactivate acetyl-CoA carboxylase in a cell-free system by phosphorylating Ser- 79 of the protein (Hardie et al., 14 Trends Biochem. Sci 20, 1989). Addition of glucagon to intact cells, namely isolated rat hepatocytes and adipocytes, also results in the phosphorylation of Ser-79. In a cell-free system, however, cA-PK, which as a rule mediates the actions of hormones that elevate cyclic AMP, phosphorylates a different serine, namely Ser-77. This suggests that, in contradiction with generally accepted knowledge, the action of glucagon on acetyl-CoA carboxylase is not mediated by cA-PK but by AMP-PK. Recently, to resolve this contradiction, it has been proposed that, in vivo. cyclic AMP and cA-PK exert their inactivating effect on acetyl-CoA carboxylase not be a direct phosphorylation of the enzyme, but by an indirect mechanism, namely inhibition of the dephosphorylation of Ser-79 by protein phosphatase 2A (Cohen & Hardie, 1094 Bioc. Biop. Acta 292, 1991) .
AMP-PK inactivates HMG-CoA reductase by phosphorylating Ser-872 of the enzyme (Clarke & Hardie, 9 EMBO Journal 2439, 1990). This phosphorylation is most likely responsible for the inactivation of HMG-CoA reductase known to occur when special precautions (e.g.. freeze-clamping) are not taken to avoid a rise of the concentration of AMP after removing liver tissue.
AMP-PK phosphorylates Ser-565 of hormone-sensitive lipase. This phosphorylation inhibits subsequent phosphorylation and activation of hormone-sensitive lipase by cA-PK (Garton et al., 179 Eur. J. Biochem 249, 1989). Phosphorylation of hormone-sensitive lipase by AMP-PK might thus block the action of lipolytic hormones (and release of free fatty acids and glycerol from fat cells) which act by way of cyclic AMP and cA-PK.
AMP-PK has also been reported to be itself regulated by phosphorylation, which activates the enzyme, and by dephosphorylation which inactivates the enzyme. Nanomolar concentrations of fatty acyl-CoA were shown to stimulate the 'AMP-PK kinase', thus activating AMP-PK. Since the latter activation will result in inactivation of acetyl-
CoA carboxylase, it provides a mechanism whereby fatty acyl-CoA can exert feed-back inhibition on fatty acid synthesis. Taken together, the studies of the AMP-PK system indicate that it plays an important role in regulating the levels of fatty acids and cholesterol in the body. That
AMP-PK acts on both acetyl-CoA carboxylase and HMG-CoA reductase most likely explains why hepatic fatty acid and cholesterol synthesis are regulated in parallel in several situations (e.g.. both synthetic pathways peak at the same time of the day, both are inhibited by diets high in polyunsaturated fatty acids) .
As described in detail below, we have discovered that the addition of the nucleoside, AICAriboside, to suspensions of isolated rat hepatocytes provokes an inactivation of both acetyl-CoA carboxylase and HMG-CoA reductase. AICAriboside is efficiently converted by phosphorylation into the corresponding nucleotide, AICAribotide or ZMP, in isolated rat hepatocytes (Vincent et al. , 40 Diabetes 1259, 1991, data not shown) and in vivo (data not shown) . (ZMP can also be formed from AICA base administration followed by in vivo phosphoribosylation.) Taken together these data indicate that AMP-activated protein kinase can be activated by ZMP, which displays striking structural similarities with AMP. This discovery of pharmacological stimulators of AMP- activated protein kinase provides an unique means to decrease concomitantly the synthesis of fatty acids and of cholesterol in the liver. The discovery thus opens new perspectives for the treatment of hypertriglyceridemia and hypercholesterolemia and particularly of their combined treatment, which often occurs and remains difficult to manage with presently available drugs (Havel, 81 J. Clin. Inves. 1653, 1988) .
Examples
The following are specific non-limiting examples of the effects of AICAriboside on synthesis of lipids.
Methods
Experiments were performed in isolated hepatocytes prepared from normally fed rats. Measurements of fatty acid synthesis were performed by incubation of the cells with 3H20 as described by Harris, 169 Arch. Biochem. Biophys. 168, 1975. Following incubation, two fractions were prepared. (1) A saponifiable lipid fraction, which contains the fatty acids (both the free fatty acids and those derived from the hydrolysis of triglycerides by the procedure) . (2) A non-saponifiable lipid fraction, which contains mainly cholesterol, but also other steroids, terpenes, prostaglandins , also the ketone bodies (minimal in the fed state) .
In this method, addition of 3H20 results in the labelling of NADPH. Utilization of the latter by fatty acid synthetase results in the formation of labelled fatty acids and triglycerides. Besides entering fatty acid synthesis, 3H20 can also enter the biosynthesis of cholesterol (at the level of HMG-CoA reductase) .
Example 1: Effect of AICAriboside on fatty acid synthesis with endogenous substrates
In isolated hepatocytes from fed rats, fatty acid synthesis can proceed from endogenous substrates, glycogen, and ketogenic amino acids. This endogenous fatty acid synthesis is completely inhibited by 500 μM
AICAriboside (Fig. 2).
Example 2: Effect of AICAriboside on fatty acid synthesis with glucose
AICAriboside inhibits fatty acid synthesis from 15 mM glucose in a dose-dependent fashion (Fig. 3) . Half maximal inhibition is obtained with about 50μM AICAriboside.
Example 3: Effect of AICAriboside on fatty acid synthesis with lactate/pyruvate
AICAriboside inhibits fatty acid synthesis from lactate 10 M/pyruvate 1 mM (Fig. 4) . Fatty acid synthesis from lactate/pyruvate seems to be slightly less sensitive to AICAriboside than that from glucose (half- maximal inhibition with about 75 μM AICAriboside) . Example 4: Effect of AICAriboside on fatty acid synthesis with ketogenic amino acids
Ketogenic amino acids enter fatty acid synthesis at the level of acetyl-CoA, thus bypassing the transport of pyruvate into the mitochondria, and the enzymes pyruvate dehydrogenase and pyruvate carboxylase. In vivo. ketogenic amino acids are thought to be first deaminated in muscle, and to be transported thereafter to the liver in the form of ketoacids. Thus, fatty acid synthesis from leucine was compared with that from its transamination product, 2-ketoisocaproic acid. As shown in Figs. 5 and 6, fatty acid synthesis with both substrates is completely inhibited by AICAriboside 500 μM.
Example 5: Effect of AICAriboside on the activity of acetyl-CoA carboxylase
Acetyl-CoA carboxylase is the limiting step of fatty acid synthesis. It is interconvertible by phosphorylation/dephosphorylation, the active form being dephosphorylated. Acetyl-CoA carboxylase is activated by a dephosphorylating phosphatase and inactivated by several kinases (including cAMP-dependent protein kinase, under the influence of glucagon) . In addition, inactivation of acetyl-CoA carboxylase by an AMP-activated protein kinase has been reported by Dr. Hardie's group. Fig. 7 shows that acetyl-CoA carboxylase (assay performed by the method of Bijleveld & Geelen, 918 Bioc. Biop. Acta 274, 1987) is inactivated by the addition of AICAriboside 500 μM. This suggests that ZMP acts at the level of the AMP-activated protein.
Example 6: Effects on the incorporation of 3H-,0 in the non-saponifiable lipid fraction
In all experiments described above, incorporation of 3H20 also occurs in the non-saponifiable lipid fraction, although to a smaller extent than in the saponifiable lipid fraction. In all experiments also, AICA riboside inhibits the incorporation of 3H20 in the non-saponifiable fraction. Figs. 8 and 9 illustrate results in an experiment with 15 mM glucose. These data indicate that the synthesis of cholesterol is inhibited by AICAriboside. Hardie has shown that AMP-activated protein kinase inactivates HMG-CoA reductase. Similarly, ZMP, by activating AMP-activated protein kinase, inactivates both acetyl-CoA carboxylase and HMG-CoA reductase, the limiting enzymes of, respectively, fatty acid and cholesterol synthesis.
Example 7: Effect of AICAriboside on the activity of HMG-CoA reductase
HMG-CoA reductase is the limiting step of cholesterol synthesis. It is interconvertible by phosphorylation/dephosphorylation, the active form being dephosphorylated. HMG-CoA reductase is activated by the dephosphorylating phosphatases, protein phosphatase 2A and 2C, and inactivated by several protein kinases. These include Ca2+/calmodulin-dependent protein kinase, protein kinase C, and as also described by Dr. Hardie's group. AMP-activated protein kinase. Fig. 10 shows that the intraperitoneal Injection of AICArboside at the dose of 500 mg/kg inactivates HMG-CoA reductase (assay performed by the method of Easom & Zammit, Biochem. J. 220: 733-8 & 739-45, 1984) in rats in vivo. This suggests that ZMP acts at the level of the AMP-activated protein kinase.
Example 8: Effect of ZMP on the activity of AMP-activated protein kinase
Dr. Hardie and co-workers (Davies et al., Eur. J. Biochem. 186: 123-8, 1989) have set up a specific and sensitive assay of AMP-activated protein kinase. It is based on the incorporation of radioactivity from [y-32P]ATP into a 15-amino acid peptide, termed the SAMS peptide, designed after the sequence of acetyl-CoA carboxylase surrounding Ser79, the site which is phosphorylated exclusively by the AMP-activated protein kinase. Fig. 11 shows that in this assay, ZMP stimulates up to nearly 8-fold the activity of rat liver AMP-activated protein kinase, partially purified up to the DEAE-Sepharose step as described by Davies et al. (Eur. J. Biochem. 186: 123-8, 1989). Half-maximal stimulation is obtained at 0.6 mM, and maximal stimulation at 4mM ZMP. Fig. 12 shows that 2mM ZMP overrides the stimulatory effect of concentrations of AMP below 0.1 mM, but is not additive and even slightly inhibitory at higher concentrations of AMP, suggesting that both nucleotides bind to the same site. Fig. 13 shows that the property of ZMP to stimulate rat liver AMP-activated protein kinase is shared by a number of other AMP analogs, namely tubercidin monophosphates, dAMP and Ara-AMP. The succinylated derivative of ZMP, SAICAR, has no effect. Cyclic ZMP and cyclic AMP have little or slightly inhibitory effect.
Other embodiments are within the following claims.

Claims

Claims
1. A method for treating an animal, comprising the step of: introducing into said animal a lipid-lowering amount of an AMP mimetic or pro-drug which stimulates AMP- activated protein kinase.
2. A method for treating an animal having an elevated serum lipid level, comprising the steps of: identifying an animal having an elevated serum lipid level, and introducing into said animal a lipid-lowering amount of an AMP mimetic or pro-drug which stimulates AMP- activated protein kinase.
3. The method of claim 2, wherein said identifying comprises chemically or biochemically measuring the serum lipid level of said animal and comparing said lipid level to a known desired maximum level of said lipid, wherein a serum lipid level above said maximum level indicates said elevated serum lipid level.
4. The method of claim 1, wherein said lipid is cholesterol.
5. The method of claim 1, wherein said lipid is triglyceride.
6. The method of claim 1, wherein said lipid is lipoprotein.
7. The method of claim 1, wherein said lipid is very low density lipid.
8. The method of claim 1, wherein said lipid is a chylomicron.
9. The method of claim 1, wherein said lipid is a low density lipoprotein.
10. The method of claim 1 , wherein said AMP mimetic is ZMP.
11. The method of claim 1, wherein said AMP mimetic pro-drug is AICAriboside.
12. The method of claim 1, wherein said AMP mimetic prodrug is AICA base.
13. The method of claim 1, wherein said AMP mimetic is an analog of ZMP.
14. The method of claim 1, wherein said AMP mimetic pro-drug is an analog of AICAriboside.
15. The method of claim 1 wherein said AMP mimetic pro-drug is an analog of AICA base.
16. The method of claim 1, wherein said AMP mimetic is a pro-drug of AICAriboside.
17. The method of claim 1, wherein said AMP mimetic is an analog of AMP.
18. The method of claim 1 , wherein said AMP mimetic is an analog of adenine.
19. The method of claim 1 , wherein said AMP mimetic is an analog of adenosine.
20. The method of claim 1 , wherein said AMP mimetic pro-drug is an analog of adenosine.
21. The method of claim 1, wherein said animal suffers from a disease chosen from atherosclerosis, hyperlipidemia, hypercholesteremia, hypertriglyceridemia, coronary artery disease, transient ischemic attacks, stroke, angina pectoris, peripheral vascular disease and diabetes.
22. The method of claim 1, wherein said AMP mimetic pro-drug is a nucleoside which can be phosphorylated to form an AMP mimetic in vivo.
23. The method of claim 1, wherein said AMP mimetic pro-drug is a purine analog which can be phosphoribosylated to form an AMP mimetic in vivo.
24. The method of claim 1, wherein said AMP mimetic or AMP mimetic prodrug is selected from the group consisting of: tubercidin base, tubercidin nucleoside, tubercidin nucleotide, and analogs and prodrugs of said base, nucleoside and nucleotide.
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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
KR20160060137A (en) * 2013-09-26 2016-05-27 에너지네시스 바이오메디컬 컴퍼니 리미티드 Compound for activating ampk and uses thereof
WO2020050935A2 (en) 2018-08-06 2020-03-12 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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WO2003037371A3 (en) * 2001-10-31 2004-10-28 Univ Bruxelles Amp-kinase agonists or adenosine pro-drugs as immuno-stimulating agents
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)
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
EP3769766A1 (en) * 2013-09-26 2021-01-27 Energenesis Biomedical Co., Ltd. Adenine for activating ampk and their medical uses
EP3050567A1 (en) * 2013-09-26 2016-08-03 Energenesis Biomedical Co., Ltd. Compound for activating ampk and uses thereof
EP3050567A4 (en) * 2013-09-26 2017-04-05 Energenesis Biomedical Co., Ltd. Compound for activating ampk and uses thereof
KR102101465B1 (en) * 2013-09-26 2020-04-17 에너지네시스 바이오메디컬 컴퍼니 리미티드 Compound for activating ampk and uses thereof
KR20200041392A (en) * 2013-09-26 2020-04-21 에너지네시스 바이오메디컬 컴퍼니 리미티드 Compound for activating ampk and uses thereof
KR20160060137A (en) * 2013-09-26 2016-05-27 에너지네시스 바이오메디컬 컴퍼니 리미티드 Compound for activating ampk and uses thereof
KR102248806B1 (en) * 2013-09-26 2021-05-10 에너지네시스 바이오메디컬 컴퍼니 리미티드 Compound for activating ampk and uses thereof
WO2020050935A2 (en) 2018-08-06 2020-03-12 Skylark Bioscience Llc Amp-activated protein kinase activating compounds and uses thereof
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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EP0599987A1 (en) 1994-06-08
CA2115675A1 (en) 1993-03-04
NO940580L (en) 1994-04-25
IL102895A0 (en) 1993-01-31
AU2486192A (en) 1993-03-16
MX9204880A (en) 1993-04-01
EP0599987A4 (en) 1995-02-01
NO940580D0 (en) 1994-02-21

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