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WO2001078715A1 - Procede de traitement de la maladie d'alzheimer - Google Patents

Procede de traitement de la maladie d'alzheimer Download PDF

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Publication number
WO2001078715A1
WO2001078715A1 PCT/US2001/012496 US0112496W WO0178715A1 WO 2001078715 A1 WO2001078715 A1 WO 2001078715A1 US 0112496 W US0112496 W US 0112496W WO 0178715 A1 WO0178715 A1 WO 0178715A1
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Prior art keywords
apoe
arginine
cells
disease
active agent
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English (en)
Inventor
Carol A. Colton
Meggan Czapiga
Michael P. Vitek
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Georgetown University
Duke University
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Georgetown University
Duke University
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Priority to AU2001255439A priority Critical patent/AU2001255439A1/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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group

Definitions

  • the present invention concerns methods of treating Alzheimer's disease by the administration of inhibitors of cationic amino acid uptake, particularly arginine uptake inhibitors.
  • Alzheimer's disease is a chronic neurodegenerative disease characterized by neuritic plaques in the patient's brain which contain apolipoprotein-E, fibrillar amyloid-Beta peptide, dystrophic neurites and activated microglia [Perlmutter, L. et al., Neurosci. Lttrs. 119, 32-36 (1990)].
  • activated • microglia release oxyradicals such as superoxide anion and nitric oxide (NO) in response to a variety of factors.
  • oxyradicals such as superoxide anion and nitric oxide (NO)
  • M. Vitek and C. Colton, PCT Application PCT/US98/20412, titled Modulation of Nitric Oxide Production describes a method of treating cells that carry at least one APOE4 allele.
  • the method comprises increasing nitric oxide levels in the cells (e.g., by administering an exogenous source of nitric oxide to the cells) by an amount sufficient to combat the decrease of nitric oxide level associated with the presence of the APOE4 allele.
  • the method is useful for, among other things, treating patients afflicted with Alzheimer's disease.
  • U.S. Patent No. 5,935,781 to Poirier describes the treatment of Alzheimer's disease with cholinomimetic drugs.
  • U.S. Patent No. 5,523,295 to Fasman describes the treatment of Alzheimer's disease by administering a silicon compound capable of interaction between aluminum and ⁇ -amyloid or neurofilament proteins.
  • a particular aspect of the present invention is a method of treating Alzheimer's disease in a subject in need thereof, comprising administering to the subject an arginine uptake inhibitor in a treatment effective amount.
  • Administering steps herein may be carried out chronically or acutely (or situationally) by any suitable route, including but not limited to parenteral and oral administration, nasal and inhalation administration, etc.
  • Arginine uptake inhibitors this term including the pharmaceutically acceptable salts thereof, are sometimes referred to as "active compounds” or “active agents” herein.
  • a still further aspect of the present invention is the use of such active agents for the preparation of a medicament for the treatment of Alzheimer's disease in a subject in need thereof.
  • a still further aspect of the present invention is a composition comprising, in combination, a first active agent for treating Alzheimer's disease and a second (i.e. different) active agent for treating Alzheimer's disease, wherein said first active agent is an arginine uptake inhibitor.
  • the composition may further comprise a pharmaceutically acceptable carrier.
  • the second active agent may be any suitable active agent, including but not limited to cholinomimetic drugs, nonsteroidal anti- inflammatory agents, histamine H2 receptor blocking agents, silicon compounds capable of interaction between aluminum and ⁇ -amyloid or neurofilament protein, and proteinase inhibitors.
  • the second active agent may be a centrally active anticholinesterase such as tacrine.
  • the composition may be provided in any suitable form, such as an oral dosage composition.
  • Alzheimer's disease as used herein is intended to encompass all types of Alzheimer's disease, including sporadic and familial AD, as well as late onset and early onset AD
  • the present invention may be used to treat Alzheimer's disease (AD) in patients previously diagnosed with AD, or in patients considered to be at risk for AD.
  • AD Alzheimer's disease
  • Risk of AD may be determined from family history, early cognitive examination, the detection of one or more (e.g., two) Apolipoprotein E4 alleles (ApoE4) in the subject as described in U.S. Patent No. 5,508,167 to Roses, or any other suitable technique (see, e.g., U.S. Patent No. 5,297,562 to Potter; U.S. Patent No. 5,972,638 to Tanzi et al.). (The disclosures of all U.S. Patent references cited herein are to be incorporated by reference herein in their entirety).
  • treat refers to any type of treatment or prevention that imparts a benefit to a patient afflicted with a disease or at risk of developing the disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the disease, delay the onset of symptoms or slow the progression of symptoms, etc.
  • treatment also includes prophylactic treatment of the subject to prevent the onset of symptoms.
  • prevention are not necessarily meant to imply cure or complete abolition of symptoms.
  • pharmaceutically acceptable means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
  • the active compounds of the present invention may optionally be administered in conjunction with other, different, active compounds useful in the treatment of the disorders or conditions described herein (e.g., Tacrine).
  • the other compounds may be administered concurrently.
  • concurrently means sufficiently close in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more administrations occurring before or after each other).
  • the present invention is primarily concerned with the treatment of human subjects, but the invention may also be carried out on animal subjects, particularly mice, such as those described in U.S. Patent No. 5,767,337 to Roses et al., for drug screening, testing and development purposes.
  • Any arginine uptake inhibitor can be used to carry out the present invention, including any inhibitor of the class of cationic amino acid transporters (CAT inhibitors) , particularly CAT3 inhibitors.
  • CAT inhibitors cationic amino acid transporters
  • arginine uptake inhibitor that can be used in carrying out the present invention are the arginine analogs, which may also be characterized as nitric oxide synthase inhibitors, as described in U.S. Patent No. 5,585,402 to Moncada et al., and U.S. Patent No. 5,028,627 to Kilbourn et al.
  • Such compounds include, but are not limited to, L-N G -substituted arginine or L- N G N ⁇ - disubstituted arginine, particularly where the substitution is (or substitutions are) a nitro, amino, alkyl, hydroxyalkyl, or alkenyl substitutent (the term "alkyl” herein preferably referring to C1-C4 loweralkyl) replacing a hydrogen of a guanidino amino group.
  • L-NMMA also known as N(G)-monomethyl-L- arginine
  • Sigma Chemical Company Limited Fancy Road, Poole, Dorset BH17 7NH, England.
  • arginine uptake inhibitor that can be used to carry out the present invention is the guanylhydrazone arginine uptake inhibitors described in U.S. Patent No. 5,854,289 to Bianchi et al.
  • the first genera of guanylhydrazone arginine uptake inhibitors consists of compounds having the formula:
  • X 2 GhyCH-, GhyCCH 3 - or H-;
  • X 2 GhyCH-, GhyCCH 3 - or H-
  • X l s X'j and X' 2 GhyCH- or GhyCCH 3 -
  • X 2 GhyCH-, GhyCCH 3 .
  • the second major genus consists of compounds of the formula:
  • the active compounds disclosed herein can, as noted above, be prepared and administered in the form of their pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart excessive toxicological effects.
  • examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfomc acid, polygalacturonic acid
  • subjects administered the active compounds described above may also be administered other active compounds for the treatment of Alzheimer's disease.
  • the combined benefit of the two different active compounds administered together may be additive, synergistic, or even less than additive so long as the combined treatment benefit to the patient is greater than the treatment benefit of either active agent given alone.
  • active agents useful in the treatment of Alzheimer's disease include, but are not limited to, cholinomimetic compounds (see, e.g., U.S. Patent No. 5,935,781 to Poirier et al.), nonsteroidal anti-inflammatory agents and histamine H2 receptor blocking agents (see, e.g., U.S. Patent No. 5,643,960), silicon compounds capable of interaction between aluminum and ⁇ -amyloid or neurofilament proteins (see U.S. Patent No. 5,523,295 to Fasman), estrogen replacement therapy agents (e.g., estrogen or estrogen receptor agonists) protease inhibitors (see, e.g., U.S. Patents Nos. 6,017,887; 6,015,879; 5,596,241; and 5,714,471).
  • cholinomimetic compounds see, e.g., U.S. Patent No. 5,935,781 to Poirier et al.
  • a particularly preferred additional or supplemental active agent for administering is combination with active compounds described herein is a centrally active anticholinesterase such as tacrine (see, e.g., U.S. Patent No. 5,698,224; 5,576,022; and 4,816,248), both of which may be administered orally, and both of which may be administered orally in a single combination dosage form.
  • a centrally active anticholinesterase such as tacrine
  • compositions The active compounds described above may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9 th Ed. 1995).
  • the pharmaceutical formulation contains in combination both a first active agent (or a pharmaceutically acceptable salt) and a second, different active agent (e.g., tacrine) for treating Alzheimer's disease, as described above, the pharmaceutical formulations are novel formulations.
  • Other active agents can be included in the formulation in their known and recommended amounts.
  • the active compound (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient.
  • the carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight of the active compound.
  • One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory ingredients.
  • the formulations of the invention include those suitable for oral, rectal, nasal, inhalation (e.g., to the lungs), buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), topical (i.e., both skin and mucosal surfaces, including airway surfaces) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.
  • Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion.
  • Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s).
  • Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.
  • Formulations suitable for oral administration may be controlled release or osmotic dosage forms, as described in U.S. Patents Nos. 5,576,022 and 5,698,244.
  • Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
  • Formulations of the present invention suitable for nasal, parenteral, or inhalation administration comprise sterile aqueous and non-aqueous injection solutions of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents.
  • the formulations may be presented in unit dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for-injection immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile composition comprising an active compound, in a unit dosage form in a sealed ' container.
  • the compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject.
  • the unit dosage form typically comprises from about 10 mg to about 10 grams of the compound or salt.
  • a sufficient amount of emulsifying agent that is physiologically acceptable may be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier.
  • emulsifying agent is phosphatidyl choline.
  • Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.
  • Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or bis ⁇ tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.
  • the present invention provides liposomal formulations of the active compounds disclosed herein.
  • the technology for forming liposomal suspensions is well known in the art.
  • the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same may be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes.
  • the lipid layer employed may be of any conventional composition and may either contain cholesterol or may be cholesterol-free.
  • the salt may be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced may be reduced in size, as through the use of standard sonication and homogenization techniques.
  • the liposomal formulations containing the compounds disclosed herein may be lyophilized to produce a lyophilizate which may be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • a pharmaceutically acceptable carrier such as water
  • compositions may be prepared from the compounds disclosed herein, or salts thereof, such as aqueous base emulsions.
  • the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound or salt thereof.
  • Particularly useful emulsifying agents include phosphatidyl cholines, and lecithin.
  • the pharmaceutical compositions may contain other additives, such as pH-adjusting additives.
  • useful pH-adjusting agents include acids, such as hydrochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate.
  • the compositions may contain microbial preservatives.
  • Useful microbial preservatives include methylparaben, propylparaben, and benzyl alcohol. The microbial preservative is typically employed when the formulation is placed in a vial designed for multidose use.
  • the pharmaceutical compositions of the present invention may be lyophilized using techniques well known in the art.
  • the present invention provides pharmaceutical formulations comprising the active compounds (including the pharmaceutically acceptable salts thereof), in pharmaceutically acceptable carriers for oral, rectal, topical, buccal, nasal, inhalation (e.g., to the lungs) parenteral, intramuscular, intradermal, or intravenous, and transdermal administration.
  • the therapeutically effective dosage of any one active agent will vary somewhat from compound to compound, and patient to patient (depending upon the age and condition of the subject), and will depend upon factors such as the age and condition of the patient and the route of delivery. Such dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art.
  • the dosage may be via injection at 70 or 15 milligrams to 2 or 2.5 milligrams per day.
  • the dosage may be as a unit containing 5 mg or 10 mg to 200 or 500 mg of the active agent.
  • the dosage is preferably by injection, such as in the form of an infusion so that between 5 and 250 mg/Kg of L-NMMA is administered per day.
  • L-NMMA may also be administered by intraveneous bolus in which case the maximum dose per bolus may be 20 mg/Kg and preferably 10 mg/Kg, the total amount being administered by this method being between about 5 and 250 mg/Kg.
  • the dose may be on sufficient to achieve a serum concentration of from about .5 ⁇ m or 5 ⁇ m to about 10 ⁇ m or 100 ⁇ M.
  • a daily parenteral dosage of about 0.01 or 0.1 to about 1 or 10 mg/Kg may be used.
  • Administration may be carried out on a chronic or acute basis.
  • the active agent may (for example) be given as a single dosage as above, or daily in the above dosages for a period of 2 days 5 days.
  • the daily dosage will be given at least 3, 4 or 5 times a week (e.g., seven days a week) for a period of at least two weeks, at least a month, at least two months, or even at least six months or more.
  • a chronic dosage regimen is completed the patient may be reevaluated and the administration continued or modified as necessary.
  • other active agents are administered in combination with the arginine uptake inhibitor, they may be given in their known or recommended amounts.
  • apolipoprotein E plays a role in the immune system in addition to a role in the transport of lipids and the regulation of cholesterol flux.
  • Treatment of macrophages with apoE enhances their production of nitric oxide (NO), a critical mediator of cellular processes and a major component of the constitutive immune response.
  • NO nitric oxide
  • Using a mouse macrophage cell line we demonstrate that apoE induction of NO depends on pre-treatment of the cells with the lymphokine priming agents, IFN ⁇ or IFN ⁇ .
  • PIPC polycytidylic acid
  • LPS lipopolysaccharide
  • Apolipoprotein E is the protein component of a lipid-protein complex and is a member of a larger family of apoproteins which transport lipids throughout the body. Although apoE is found in plasma, it is a primary component of lipid and cholesterol metabolism in the CNS and is particularly important during synthesis and repair of glial and neuronal membranes. ApoE expression is increased during the response of the CNS to injury and has been observed to promote neuron sprouting (5,18,23,34,42,52,56). The function of apoE, however, extends beyond regulation of lipid transport and the supply of lipids for membrane repair.
  • apoE serves as a direct antioxidant. Since oxidative phenomena are part of the innate immune response mediated by tissue macrophages and are a critical part of the response to tissue injury (1,6,22,45), regulation of the local tissue redox environment by. apoE may have significant implications to the repair process.
  • apoE increases the production of nitric oxide (NO) from human monocyte derived macrophages (MDM) (16). Although frequently considered to be a pro-oxidant, NO can also reduce membrane lipid peroxidation and reduce the uptake of oxidized lipids (7,33,40). Macrophages are a major source of NO in both the periphery and in the CNS and are stimulated to produce NO by immune or inflammatory factors (1,20,45). NO production by the activated macrophage, thus, may be an important link between apoE and the response of the tissue to injury or inflammation. This link may have clinical relevance since chronic neurodegenerative diseases such as Alzheimer's disease (AD) involve inflammation and oxidative stress (3,21,39,50,57).
  • AD Alzheimer's disease
  • Delipidated apoE purified from human serum was obtained from Chemicon International, Inc. (Temcula, CA) or ICN Pharmaceuticals, Inc. (Costa Mesa, CA) and was used immediately on dilution. Since mixed human seum was the source of this apoE and since 78% of the population carries the APOE3 allele (55), it is likely that apoE3 is the predominant isoform in this source.
  • VLDL, ⁇ ⁇ DL, LDL or oxidized LDL were purchased from Intracell (Rockyille, MD) and stored at 4°C under nitrogen until used. ApoE content of each of the lipid stocks was tested using a Western blot with recombinant human apoE 3 as the standard.
  • Human apoE3 was isolated from conditioned media (CM) of primary astrocyte cultures of mice made transgenic for human apoE3 on a mouse apoE knockout background.
  • the apoE was purified as described (26) and was a generous gift from Dr. David Holtzman, St. Louis University Medical School, St. Louis, MO.
  • Poly I:C (K+ salt) was purchased from Sigma Chemical Co. (St. Louis, MO).
  • LPS (055:B5) was obtained from Calbiochem (San Diego,CA) and recombinant murine IFN'y was obtained from GibCo-BRL (Rockville, MID).
  • the iNOS antibody (#06-295) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY).
  • the antibody for CAT2b was a generous gift of Drs. Kakuda and MacLeod, University of California, San Diego. All tissue culture media and supplies were purchased from GibCo-BRL (Rockville, MD). Cultures: The mouse macrophage cell line (RAW 264.7) was obtained from ATCC, Inc. (Reston, NA) and cultured in DMIEM containing 10% heat inactivated fetal calf serum (ECS), 2 mM glutamine, 100 U/mi pen-strep and 1 mM HEPES buffer. Cells were grown in a humidified 95% air, 5% oxygen atmosphere at 37°C until confluent.
  • ECS heat inactivated fetal calf serum
  • the cells Prior to the experiment, the cells were removed from the flask by trypsiization, spun at 1000 g to pellet and resuspended in growth media. The cells were plated into the center wells of 96 well dishes at an initial density of 40,000 cells per well and allowed to recover overnight. In some cases, an increased number of cells were plated into 6 well dishes for PCR or Western blot analysis.
  • Nitric oxide production was determined by measuring the supernatant levels of nitrite, the stable biological oxidation product of NO, using the Griess reaction as described previously (4,3 7).
  • RAW cells were plated into 96 well dishes and the media in each well was replaced with serum-free media in the presence and absence of putative activators of the inducible nitric oxide synthase (iNOS).
  • the supematants were then collected at various time points, transferred to a 96 well plate and assayed for NO by the addition of Griess reagents to each well.
  • the OD was read at 550 nm and the values of nitrite for the samples were obtained from a standard curve using sodium nitrite as the standard.
  • the protein content of the wells was determined using the Pierce BCA assay (Rockford, IL) with bovine serum albumin as the standard. Data are presented as nmoles of nitrite/40,000 cells or as nmoles of nitrite/O g protein. A minimum of 3 wells of cells were assayed per experimental condition for at least 3 different culture groups. Passage number was 10-30. Statistical analysis was done using an ANOVA with the Bonferroni correction or with the unpaired Students t test. Arginine uptake: RAW 247.6 were plated into 96 well plates ( — 8x10 4 cells per well) and allowed to adhere overnight. Cells were then primed with IFN- ⁇ (100 U/mi) for 8 hours in cell culture media.
  • the culture media was aspirated and discarded.
  • the cells were then treated for 4 hrs with fresh serum-free media containing 10 ⁇ M arginine plus the various treatment conditions. This value of arginine was chosen because it more closely represents extracellular arginine levels in the CNS (17).
  • media were either discarded or saved for nitrite analysis.
  • the cells were washed with warm (37°C) uptake buffer containing in mM: NaCl, 137; KC1, 2.7; CaCl 2 , 1.5; KH 2 PO 4 , 1.2; MgSO 4 , 1.0; HEPES, 20; adjusted to pH 7.4.
  • L-[ 3 H] arginine (1 Ci/ml, New England Nuclear) uptake was measured by incubating the cell monolayers for 5 minutes at 37°C in the presence of increasing concentrations of unlabeled L-arginine (1-100 ⁇ M). After the 5 min incubation, the plate was inverted to remove any buffer containing excess tracer, and the plate was placed on a tray of melting ice. Ice-cold buffer containing 10 mM unlabeled substrate was then added (0.1 ml). The buffer was removed by inversion of the plate and blotting on a paper towel. The washing procedure was repeated twice more for a total of three washes. Radioactivity in 1.0% Triton X- 100 extracts is then determined by liquid scintillation counting.
  • Non-specific uptake of L-[ 3 H]arginine was found by repeating the above procedures at 4°C. Values of non-specific uptake at 4°C were subtracted from the values at 37°C. A portion of the cell digest was used for the Bio- Rad protein measurement. Uptake was then calculated and expressed as counts per minute per Og protein. Statistical analysis was done using an unpaired Student's t test. Western blot analysis: Cell lysates from treated and untreated RAW cells were analyzed by Western blot for immunoreactivity to iNOS. Protein concentration of each cell lysate sample was determined using the Pierce BCA assay. Sample buffer was then added to equal protein concentrations from the cell samples and the mixtures boiled for 5 minutes.
  • apolipoprotein E apolipoprotein E
  • IZFN ⁇ primed conditions
  • the resultant NO production was determined by measuring nitrite concentrations in the cell supernatants.
  • apoE produced an increase in nitrite when RAW cells were pretreated with recombinant murine IFN ⁇ and then treated with apoE for an additional 24 hours.
  • the response to apoE increased with increasing concentrations of JIIFNy used during pretreatment and increased with increasing dose of apoE.
  • PIPC polyinosinic:polycytidylic acid
  • LPS lipopolysachharide
  • CM Conditioned media
  • IFN - primed RAW cells were treated for 24 hours with varying concentrations of VLDL, HDL and LDL purified from human serum.
  • Supernatant nitrite levels significantly decreased in VLDL-treated RAW cells (10 and 50 Og/ml) compared to untreated controls (Table 1).
  • No change in nitrite was seen in either LDL or HDL treated cells.
  • Treatment with oxidized LDL (10 Og/ml) resulted in a significant increase in NO production compared to untreated cells.
  • NO production can also be regulated by intracellular arginine levels, the substrate for iNOS (10,20,45).
  • Arginine uptake in macrophages is dependent on the activity of system y+ transporters, also known as cationic amino acid transporters (CAT) (2,36,44,51).
  • CAT cationic amino acid transporters
  • H 3 -arginine uptake was measured in either 10 OM or 100 OM arginine-containing media for 5 mins.
  • treatment with PIPC alone significantly enhanced arginine uptake compared to untreated controls in cells equilibrated with 10 or 100 OM arginine.
  • treatment with PIPC in the presence of apoE significantly enhanced arginine uptake over untreated controls and PIPC alone. This effect was seen in cells equilibrated with 10 or 100 OM arginine in the media. No significant change in arginine uptake was observed in cells treated with apolipoprotein B (apoB) plus PIPC. Discussion:
  • IFN then, 'sets the stage' for further modulation of NO production by apoE.
  • apoE potentiated the effect of known iNOS induction agents such as LPS, a bacterial coat component, or PIIPC, a viral mimetic.
  • LPS low-density polypeptide
  • PIIPC a viral mimetic
  • the effect of apoE thus, is additive with other agents that induce HMOS and stimulate macrophage NO production.
  • the effect is specific to apoE since treatment of the RAW cells with apoB or boiled apoE did not alter supernatant nitrite levels.
  • apoE from plasma differs from GINS-derived apoE (30)
  • R W cells were treated with apoE purified from mixed human serum and with apoE3 purified from astrocyte conditioned media of primary cultures prepared from transgenic mice which express human apoE3 (26). Since astrocytes are the primary source for apoE in the GINS, apoE3 isolated from the conditioned media of these astrocytes is likely to represent the form of apoE3 found in human CINS (14,26,30).
  • production of NO by RAW cells was increased by both the circulating and the astrocyte CM-derived form of apoE.
  • a major difference between circulating apoE and GINS -derived apoE is the form and type of lipid associated with the apoprotein.
  • LaDu et al (30) have demonstrated that GINS-derived apoE is associated with discoidal HDL-like particles rather than the spherical HDL particles found in the cerebral spinal fluid or elsewhere in the circulation (14,3 0,3 2).
  • Fagan et al (26) have further shown that human apoE3 from astrocyte conditioned media co-localizes with an HDL particle, in contrast to circulating apoE3 which is associated with VLDL (55,56). Both HDL-associated and VLDL-associated apoE3 alters neurite outgrowth in neuronal cultures (23,32).
  • both VLDL and I ⁇ DL-associated apoE3 can serve as a ligand for the lipoprotein receptor-related protein (23,32).
  • LDL and VLDL lipid component of the apolipoprotein could alter NO production.
  • No effect on NO production was observed in RAW cells exposed to LDL or HDL, suggesting that neither of these lipids is critical to the action of apoE on the macrophage.
  • VLDL depressed NO production.
  • IFN treatment of RAW cells has been shown to stimulate VLDL uptake, thus potentially decreasing extracellular concentrations of the lipid (29).
  • ApoE-induced regulation of NO production can occur at several points in the pathway.
  • the most common mechanism for regulation of macrophage NO is via the induction of the NOSII gene by a variety of suitable mediators and the consequent expression of iNOS protein (20,45).
  • our Western blot analysis suggested that PIPC -mediated iNOS expression was not altered by apoE treatment.
  • Riddell et al (25) recently demonstrated that inhibition of platelet aggregration by apoE is mediated by NO.
  • Arginine transport was considered a likely candidate since arginine availability is rate limiting for NO production in macrophages and other cells (10,20). Macrophage arginine uptake is mediated by a family of Na+-independent cationic transporters known as the system y+ transporters or, more recently, as the cationic transporters (CAT) (51).
  • CAT cationic transporters
  • CAT2b is associated with activated macrophages, including RAW cells and is induced by LPS or other inflammatory mediators (11-13,44,51).
  • Kakuda et al (13)and Stevens et al (24) have further demonstrated that CAT2 expression is tightly linked with iNOS expression and NO production, suggesting that this transporter is a major factor in the pathway for NO synthesis during the immune response.
  • arginine uptake was significantly increased by PIPC in immune-activated RAW cells and that apoE treatment of the cells in the presence of PIPC significantly increased argiine uptake over PIPC alone.
  • apoE ability of apoE to modulate arginine transport and consequently, the production of NO is a novel regulatory mechanism for this important apolipoprotein.
  • the coupling of CAT transporters, NO and apoE is likely to be an essential component of a macrophage 's response to a variety of stressful conditions and may underlie a basic component of the repair process after injury.
  • the importance of the interrelationship between immune activation, apoE arid NO production by macrophages is supported by studies on chronic inflammatory diseases such as atherosclerosis and Alzheimer's disease (AD). All stages of atherosclerotic plaques contain immune reactive cells which are IFN positive, suggesting lymphokine- mediated activation (29).
  • ApoE also appears to be involved, since mice made deficient in apoE are prone to artherosclerosis and can be rescued by replacement of macrophage-specific apoE (8,3 5). Futhermore, production of NO by macrophages in the plaques reduces uptake of oxidized lipids, serving to limit the formation of foam cells, a characteristic lesion of artherosclerosis (9,15). A similar association between apoE, macrophages and immune activation can be found in the CNS in senile plaques, a characteristic neuropathological lesion in AD. Immune reactive cells (microglia and astrocytes) and apoE have been localized to the plaque (28,31,43,50).
  • inicroglia in AD demonstrate a positive reaction for nitrotyrosine
  • an indicator or the presence of NO (19,39)but also microglia from APOE4-positive patients demonstrate a higher degree of activation compared to patients expressing apoE3 (43). Lymphokine activation is also likely since IFN has been localized to senile plaques in AD (3 1 ,48,49).
  • IFN can substitute for IFN in promoting apoE's modulation of NO production.
  • our data provide a direct link between NO production by immune activated macrophages and apoE and demonstrate that the CAT transporter is an integral part of this link.
  • Figure 1 ApoE induction of NO is dependent on pre-treatment with interferon- ⁇ (IFN ⁇ ).
  • IFN ⁇ interferon- ⁇
  • RAW cells were pre-treated with growth media containing 10 to 1000 U/ml IFN ⁇ for 12 hours or remained untreated. The media was then replaced with serum free media containing 10 to 100 nM human apoE isolated from human blood (apoE) and supernatant nitrite levels were measured at 24 hours. Data points represent the average ⁇ SEM nitrite value ( ⁇ moles/40,000 cells) for a minimum of 3 wells for 3 different culture groups.
  • FIG. 1 *P ⁇ 0.05 compared to 100 IFN ⁇ alone; ** p ⁇ 0.003 compared to IFN ⁇ alone
  • FIG. 2 ApoEp enhances the effect of PIIPC on NO production by RAW cells.
  • Panel A RAW cells were pretreated with 100 U/mL IFN ⁇ for 12 hours, followed by treatment for 24 hours with serum free media containing PIPC alone (0.5 ⁇ g/ml) or PIPC plus varying concentrations of apoE.
  • Panel B Supernatant nitrite levels were measured in IFN ⁇ pretreated RAW cells stimulated with varying concentrations of PIPC in the presence or absence of apoE (50 nM).
  • Panel B CAT2 (67 kD immunoreactive band) expression in ⁇ lFN 100 U/mL) pre-treated RAW cells treated with Poly I:C or the inactive reverse polyribonucleotide, Poly C:I.
  • LANES A molecular weight standards; B - untreated; C — 1 ⁇ g/ml Poly I:C; D — 10 ug/ml Poly I:C; E — 50 ug/ini Poly C:I. Blot shown is a representative blot from 3 different blots.
  • FIG. 5 ApoE increases arginine uptake.
  • IFN ⁇ -pretreated RAW cells remained untreated or were treated with 0.5 ⁇ g/mi PIPC, 0.5 ⁇ g/mi plus 50 nM apoE or 0.5 ⁇ g/mi PIPC plus 50 nM ApoB.
  • Nitric oxide protects against alkyl peroxide mediated cytotoxicity: Further insights into the role nitric oxide plays in oxidative stress. Arch. Biochem. Biophys.
  • mice are a model of lipoprotein oxidation in atherogenesis. Demonstration of oxidation -specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Atheroscler. Thromb. 14: 605-616, 1994. 9. Jessup, W. and R. Dean. Autoinhibition of murine macrophage-mediated oxidation of low-density lipoprotein by nitric oxide synthesis. Atherosclerosis 101 :145-155, 1993. 10. Wu, G. and S. M. Morris,Jr. Arginine metabolism: nitric oxide and beyond. Biochem. 1. 336: 1-17, 1998.
  • Apolipoprotein E from atherosclerosis to
  • Apolipoprotein- E containing high density lipoprotein promotes neurite outgrowth and is a ligand for the low density lipoprotein receptor-related protein. J Biol. Chem. 271: 30121-30125, 1996.
  • Mahley, R. Apolipoprotein E Cholesterol transport protein with expanding role in cell biology. Science 240: 622-63 0, 1988.

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Abstract

La présente invention concerne des procédés de traitement de la maladie d'Alzheimer avec des inhibiteurs de la captation d'arginine, ainsi que des formulations pharmaceutiques utiles pour mettre en oeuvre lesdits procédés.
PCT/US2001/012496 2000-04-17 2001-04-16 Procede de traitement de la maladie d'alzheimer Ceased WO2001078715A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2041078A4 (fr) * 2006-06-23 2016-06-01 The Feinstein Inst Medical Res INHIBITEURS DE L'AGRÉGATION DES Abeta ET DE LA SYNUCLÉINE

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5028627A (en) * 1989-09-13 1991-07-02 Cornell Research Foundation, Inc. Method of using arginine derivatives to inhibit systemic hypotension associated with nitric oxide production or endothelial derived relaxing factor
US5585402A (en) * 1992-12-23 1996-12-17 Glaxo Wellcome Inc. Nitric oxide synthase inhibitors
US5854289A (en) * 1994-01-21 1998-12-29 The Picower Institute For Medical Research Guanylhydrazones and their use to treat inflammatory conditions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5028627A (en) * 1989-09-13 1991-07-02 Cornell Research Foundation, Inc. Method of using arginine derivatives to inhibit systemic hypotension associated with nitric oxide production or endothelial derived relaxing factor
US5585402A (en) * 1992-12-23 1996-12-17 Glaxo Wellcome Inc. Nitric oxide synthase inhibitors
US5854289A (en) * 1994-01-21 1998-12-29 The Picower Institute For Medical Research Guanylhydrazones and their use to treat inflammatory conditions

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2041078A4 (fr) * 2006-06-23 2016-06-01 The Feinstein Inst Medical Res INHIBITEURS DE L'AGRÉGATION DES Abeta ET DE LA SYNUCLÉINE

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