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WO1996038166A1 - Proteine amyloide serique a - Google Patents

Proteine amyloide serique a Download PDF

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Publication number
WO1996038166A1
WO1996038166A1 PCT/CA1996/000404 CA9600404W WO9638166A1 WO 1996038166 A1 WO1996038166 A1 WO 1996038166A1 CA 9600404 W CA9600404 W CA 9600404W WO 9638166 A1 WO9638166 A1 WO 9638166A1
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WO
WIPO (PCT)
Prior art keywords
saa
hdl
ligand
cholesterol
macrophages
Prior art date
Application number
PCT/CA1996/000404
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English (en)
Inventor
Robert Kisilevsky
Original Assignee
Queen's University At Kingston
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/458,054 external-priority patent/US6004936A/en
Application filed by Queen's University At Kingston filed Critical Queen's University At Kingston
Priority to AU61182/96A priority Critical patent/AU6118296A/en
Publication of WO1996038166A1 publication Critical patent/WO1996038166A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to means for potentiating the collection of cholesterol from inflammatory or atherosclerotic sites, having the capability of being used to treat hypercholesterolemia and improving atherosclerotic conditions. More specifically, the present invention provides therapeutic methods which potentiate the ability to transfer macrophage cholesterol to a natural transport mechanism for subsequent excretion.
  • Serum levels of cholesterol and atherosclerosis are significant topics addressed by health care professionals as they relate to cardiac disease, as well as other circulatory and systemic diseases. There is a great interest in the medical field with regard to the reduction of serum cholesterol and the reversal of an atherosclerotic condition.
  • Serum amyloid A is an apolipoprotein which is present on high density lipoprotein (HDL) only during inflammatory states.
  • SAA was discovered approximately 15 years ago in the course of studies examining serum for potential precursors to the inflammation-associated AA form of amyloid. It has been determined that the AA peptide responsible for the inflammation-associated amyloid fibril represented a fragment of the SAA protein 12,25 Based on amino acid sequencing of SAA in the preparation, cloning, and identification of genes possessing the information for this protein 12,20 ⁇ jt became apparent that SAA was not a single protein, but rather a family of several related proteins.
  • the present invention provides means for potentiating the efflux of macrophage cholesterol, thereby providing a means for therapeutically reducing cholesterol at atherosclerotic sites. This potentiating effect leads to reversal of an atherosclerotic condition.
  • a method of potentiating the release and collection of cholesterol from inflammatory or atherosclerotic sites in vivo by increasing the affinity of high-density lipoprotein for macrophages by administering to a patient an effective amount of a composition comprising a compound selected from the group consisting of native serum amyloid A (SAA) and a ligand having SAA properties thereby increasing the affinity of high density lipoprotein (HDL) for macrophages and potentiating release and collection of cholesterol.
  • SAA native serum amyloid A
  • HDL high density lipoprotein
  • the invention features a method of potentiating the release and collection of cholesterol from inflammatory or atherosclerotic sites in vivo comprising: administering to a subject a nucleic acid construct encoding a peptide selected from the group consisting of SAA and a ligand having SAA properties, under conditions such that the construct is incorporated into cells of the subject and the peptide is expressed in the subject, thereby increasing the affinity of high density lipoprotein (HDL) for macrophages and potentiating release and collection of cholesterol.
  • HDL high density lipoprotein
  • the invention provides a method of reversing an atherosclerotic condition; the method comprises administering to a patient in need thereof an effective amount of a composition comprising a compound selected from the group consisting of native serum amyloid A (SAA) and a ligand having SAA properties, thereby increasing the affinity of high density lipoprotein (HDL) for macrophages and potentiating release and collection of cholesterol, such that an atherosclerotic condition is reversed.
  • SAA native serum amyloid A
  • HDL high density lipoprotein
  • the invention features a pharmaceutical composition, comprising a nucleic acid construct encoding a peptide selected from the group consisting of SAA and a ligand having SAA properties, in a pharmaceutically acceptable carrier.
  • FIGURE 1 illustrates a representative binding curve of ⁇ l- ⁇ DL (10 ⁇ g/ml) to normal-hepatocytes as a function of time;
  • FIGURE 2A shows saturation binding curves of HDL for hepatocytes from various physiological conditions
  • FIGURE 2B shows saturation binding curves of HDL/SAA for hepatocytes from various physiological conditions
  • FIGURE 3 A shows saturating binding curves of HDL for peritoneal macrophages from various physiological conditions, the individual points representing the experimental data and the solid lines representing the curves of best fit, which provided the values of the parameters set forth in the result sections, the insets representing Scatchard plots;
  • FIGURE 3B shows saturating binding curves of HDL/SAA for peritoneal macrophages from various physiological conditions, the individual points representing the experimental data and the solid lines representing the curves of best fit, which provided the values of the parameters set forth in the result sections, the insets representing Scatchard plots;
  • FIGURE 4 A shows the inhibition of ⁇ l-HDL binding (10 ⁇ g/ml) to macrophages by increasing concentrations of its unlabelled counterpart (filled circles), or unlabelled HDL/SAA (open triangles);
  • FIGURE 4B shows the inhibition of ⁇ I-HDL binding (10 ⁇ g/ml) to macrophages by increasing concentrations of its unlabelled counterpart (open triangles), or unlabelled HDL (filled circles);
  • FIGURE 5 is a schematic representation of HDL function during inflammatory states
  • FIGURE 6 illustrates the elution profile of apoA-I liposomes (second peak) on Sephacryl S-200; a similar profile was seen with SAA liposomes;
  • FIGURE 7 are graphs demonstrating the effect of different concentrations of liposomes on cholesterol efflux, open circles are controls of medium only, closed circles are liposomes without protein, open squares are liposomes with apoA-I and closed triangles are liposomes with SAA; and
  • FIGURE 8 is a graph of cholesterol efflux as a function of the PC/protein ratio of liposomes, SAA (open symbols), apoA-I (closed symbols), PC at 200 ⁇ g/ml (circles), 400 ⁇ g/ml (triangles) and 800 ⁇ g/ml (squares).
  • the present invention provides a method of potentiating the collection of macrophage cholesterol by increasing the affinity of high-density lipoprotein for macrophages, exposing the macrophage to HDL, and potentiating the release of macrophage cholesterol to the reverse cholesterol transport mechanism.
  • the present invention provides the initial discovery of the ability to alter and significantly increase the affinity of HDL for the macrophage using SAA. This increased affinity is biochemically directly related to increased capacity of HDL to collect macrophage cholesterol for subsequent excretion.
  • the affinity of HDL for macrophages is increased by binding serum amyloid-A (SAA) or a ligand having serum SAA binding activity to HDL.
  • SAA serum amyloid-A
  • SAA has been shown to be made by the liver and associates with HDLl- ⁇ However, it has been shown that SAA is secreted from the liver apparently prior to any association with HDL 4. This indicates that by administering SAA alone, it will associate spontaneously with HDL. Further, SAA has been shown in vitro to spontaneously associate with HDL and displace some HDL-apolipoprotein- ⁇ 1.
  • SAA exists in several different native forms, of which some are amyloidogenic, and some are non-amyloidogenic.
  • murine SAAj and S AA2 are forms of amyloid protein which circulate in the plasma at approximately equal concentrations, but only SAA2 is deposited as amyloid 1 ⁇ , 32
  • in vitro assays of the fibrillogenic properties of several synthetic peptides corresponding to human and murine protein AA segments have shown that amyloidogenicity of AA proteins may reside primarily in the N-terminal fragment.33
  • Such an SAA is preferred since it is possible that administration of an amyloidogenic form of SAA could lead to undesirable amyloid formation as a side effect.
  • ligand having serum SAA binding activity to HDL means a moiety other than native SAA which is capable of binding to HDL and which is capable of increasing the affinity of HDL for macrophages.
  • a ligand can be derived by isolating the active site of SAA on HDL binding, SAA having been sequenced, cDNA being derived, and the genes being cloned ,20,25 Similarly, the active site on SAA for macrophage binding may be determined. Hence, state of the art modeling to derive the active site would result in derivation of a ligand having SAA activity.
  • Ligands useful in the invention may also be identified by testing proposed ligands for the ability to bind to HDL, and the ability to increase the affinity of HDL for macrophages, as described below.
  • SAA/HDL complex refers to a complex formed by the association of SAA or a ligand having SAA properties, as described above, with HDL, either in vivo or in vitro.
  • ligands will not significantly increase amyloid formation in vivo.
  • Amyloid formation in vivo can be assayed according to the methods described in, for example, R. Kisilevsky et al., (1995) Nature Med. 1:143-148, or U.S. Patent Application No. 08/403,230, the contents of both of which are hereby incorporated by reference.
  • Particularly preferred ligands include non-amyloidogenic peptides derived from SAA.
  • non-amyloidogenic peptides may be derived from native SAA by cleavage, for example by chemical or enzymatic methods well known in the art, or by other modifications, for example, alkylation or acylation, which may also be by well-known chemical or enzymatic methods.
  • a ligand can be obtained by coupling of cleavage fragments obtained from native SAA by any of the methods described hereinbefore.
  • Non- amyloidogenic peptides may also be derived by de novo synthesis, including synthesis by chemical or biochemical methods, and may include natural and non-natural amino acids.
  • ligands may be produced by manipulation of the cDNA coding for SAA by techniques of molecular biology, incorporation of the modified cDNA in an appropriate vector, and expression of the recombinant protein in an appropriate host cell, to produce recombinant SAA proteins which contain more, fewer, or altered amino acid residues.
  • the choice of expression vectors and hosts will be routine for the skilled artisan.
  • ligands include non-amyloidogenic peptides which comprise unnatural amino acids, including amino acids of unnatural configuration.
  • the present invention also encompasses peptidomimetic ligands, including "retro-inverso" peptides (see, e.g., U.S. Patent 4,522,752 to Sisto et al), "peptoids” (see, e.g., Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367), and other peptidomimetics known in the art.
  • the choice of a preferred ligand may be made according to considerations such as pharmacokinetics, pharmacodynamics, solubility, efficacy, ease of synthesis, ease of dosing, and the like.
  • non-amyloidogenic ligands having SAA properties can be identified by chemical (e.g., solution or solid-phase) or biochemical (e.g., phage-display) synthesis of combinatorial libraries (e.g., of peptides or peptoids) and screening of the resulting libraries according to known techniques.
  • chemical e.g., solution or solid-phase
  • biochemical e.g., phage-display
  • Administration of the SAA/HDL complex can be accomplished by various means, such as infusion of a solution including the SAA/HDL complex so as to provide an amount of the complex systemically to effectively induce macrophage cholesterol efflux.
  • the serum amyloid A or a ligand having SAA properties is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners.
  • the "effective amount" for purposes herein is thus determined by such considerations as are known in the art.
  • the serum amyloid A or a ligand having SAA properties can be administered in various ways. They can be administered as the compound or as a pharmaceutically acceptable salt and can be administered alone or in combination with pharmaceutically acceptable carriers.
  • the compounds can be administered orally or parenterally including intravenous, intraperitoneal, intranasal and subcutaneous administration. Implants of the compounds are also useful.
  • the patient being treated is a warm-blooded animal and, in particular, mammals including man.
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions.
  • various additives which enhance the stability, sterility, and isotonicity of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example, sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.
  • Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.
  • a pharmacological formulation of the SAA or ligands having SAA properties can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
  • An implant suitable for use in the present invention can take the form of a pellet which slowly dissolves after being implanted or a biocompatible delivery module well known to those skilled in the art. Such well known dosage forms and modules are designed such that the active ingredients are slowly released over a period of several days to several weeks.
  • Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4.,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
  • a pharmacological formulation of the SAA or ligands having SAA properties utilized in the present invention can be administered orally to the patient.
  • Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable.
  • Known techniques which deliver the SAA or ligands having SAA properties orally or intravenously and retain the biological activity are preferred.
  • the SAA or ligands having SAA properties can be administered initially by intravenous injection to bring blood levels to a suitable level. The levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used.
  • the quantity of SAA or ligands having SAA properties to be administered will vary for the patient being treated and will vary from about 2 mg/kg of body weight to 100 mg/kg of body weight per day and preferably will be from 2 mg/kg to 20 mg/kg per day.
  • a nucleic acid construct which encodes an SAA protein or a peptide having SAA properties is administered to a subject under conditions under which the construct is incorporated into cells of the subject and the peptide is expressed in the subject.
  • This approach is generally referred to as “gene therapy”, and has the advantage of providing a long-term supply of the gene product without need for periodic dosing.
  • Approaches to gene therapy include insertion of the subject nucleic acid construct (the "gene") in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaP ⁇ 4 precipitation carried out in vivo or in vitro.
  • lipofectin liposomes
  • derivatized e.g. antibody conjugated
  • a preferred approach for in vivo introduction of nucleic acid encoding one of the subject proteins into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product.
  • a viral vector containing nucleic acid e.g. a cDNA
  • Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid.
  • molecules encoded within the viral vector e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
  • a major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
  • the development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.
  • recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the subject SAA peptides or proteins, rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al.
  • retroviruses examples include pLJ, pZIP, pWE and pEM, which are well known to those skilled in the art.
  • suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al.
  • retroviral-based vectors by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and W094/11524).
  • strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al.
  • Coupling can be in the form of the chemical cross-linking with a protein or other chemical moiety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single-chain antibody/env fusion proteins).
  • a protein or other chemical moiety e.g. lactose to convert the env protein to an asialoglycoprotein
  • fusion proteins e.g. single-chain antibody/env fusion proteins
  • retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the SAA (or SAA ligand) gene of the retroviral vector.
  • non-viral methods can also be employed to cause expression of a SAA protein or a peptide having SAA properties in the tissue of an animal.
  • Most non viral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non- viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject SAA-gene by the targeted cell.
  • Exemplary gene delivery systems of this type include liposomal derived systems, poly ⁇ lysine conjugates, and artificial viral envelopes.
  • a gene encoding one of the subject SAA proteins or a peptide having SAA properties can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
  • lipofection of neuroglioma cells can be carried out -lo ⁇ using liposomes tagged with monoclonal antibodies against glioma-associated antigen (Mizuno et al. (1992) Neurol. Med. Chir. 32:873-876).
  • the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as poly ⁇ lysine (see, for example, PCT publications WO93/04701, W092/22635, WO92/20316, W092/19749, and WO92/06180).
  • a gene binding agent such as poly ⁇ lysine
  • the subject SAA-gene construct can be used to transfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g. poly-lysine (see U.S. Patent 5,166,320).
  • the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g. Chen et al. (1994) PNAS 91 : 3054-3057).
  • the pharmaceutical preparation can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals, and can be adapted for release of viral particles through the manipulation of the polymer composition and form.
  • biocompatible polymers including hydrogels
  • biodegradable and non-degradable polymers can be used to form an implant for the sustained release of an the viral particles by cells implanted at a particular target site.
  • Such embodiments of the present invention can be used for the delivery of an exogenously purified virus, which has been incorporated in the polymeric device, or for the delivery of viral particles produced by a cell encapsulated in the polymeric device.
  • the following experimental data demonstrate the capacity of HDL/SAA administration to significantly shift the HDL cholesterol carrying capacity towards the macrophage.
  • the data specifically demonstrates the effect of SAA to reduce HDL's affinity for normal hepatocytes by a factor of 2.
  • the HDL/SAA complex had a 3-to-4 fold higher affinity for macrophages then HDL alone.
  • a profound effect on this finding is the further finding that the number of binding sites for HDL/SAA increased on macrophages during inflammation, while decreasing on hepatocytes.
  • the data further provide competition experiments showing that there is a specific SAA binding site on macrophages.
  • Binding studies were conducted between various concentrations of HDL, HDL/SAA, and fixed numbers of normal hepatocytes or peritoneal macrophages. Similar binding studies were also conducted with hepatocytes and macrophages obtained at several time points after the induction of inflammation. Binding curves were constructed between macrophages or hepatocytes ii different physiological states and various concentrations of HDL or HDL/SAA.
  • Collagenase Type 1
  • BSA fatty acid free bovine serum albumin
  • Nitex nylon membrane filters were purchased from B & SH Thompson Company limited, Ville Mont Royale, Quebec.
  • William's medium and RPMI medium were bought from Gibco Incorporated, Grand Island, New York.
  • mice All mice were of the CD/1 strain and six-eight weeks old, purchased from Charles Rivers, Montreal, Quebec. Some animals were treated with a subcutaneous injection of 0.5 ml of 2% AgNO to produce a sterile subcutaneous inflammatory reaction as described previously by Kisilevsky et al 1 ".
  • Hepatocytes Hepatocytes were isolated from six-eight week old CD/1 mice by liver perfusion. The cells were collected in William's medium which had been pregassed with 95% O2 and 5% CO2 for 15 minutes, washed twice in William's medium and centrifuged for five minutes at room temperature at 150 x g. The cells were resuspended in William's medium containing 5 mM HEPES and 2% BSA, counted, and diluted to a concentration of 6x1 cells/ml. They were kept on ice 0-4°C for ligand binding studies. Hepatocytes were also collected from mice 24 and 72 hours after subcutaneous injections of AgNO ⁇ .
  • Peritoneal macrophages were collected either from normal mice or mice treated with AgNO3 as described above. The mice were sacrificed by cervical dislocation, the peritoneal cavity filled with 5 ml of cold RPMI 1640 medium containing 0.5% BSA and massaged gently by hand. The peritoneal wash containing macrophages was withdrawn with the same syringe and filtered through a Nitex filter (100 ⁇ m) into a 50 ml centrifuge tube kept in an ice bath. Washings from 10-12 mice were collected in each tube, centrifuged twice at 300 x g for 10 minutes and resuspended in cold RPMI 1640 containing 5 mM HEPES and 2% BSA. Following a cell count, the concentration was adjusted to the desired cell concentration and the diluted suspension kept cold before use in ligand binding experiments. Preparation of Lipoproteins
  • mice Under anesthesia (sodium nembutal 6 mg/kg), mice were exsanguinated from the retroorbital sinus into a small quantity of EDTA which was used as an anticoagulant. Following centrifugation to remove the cells an HDL fraction was prepared from the EDTA treated plasma of normal mice, and those receiving the AgN ⁇ 3 24 hours earlier. These plasma samples were fractionated by floatation in KBr27,3 . After removing the low density lipoprotein (density 1.006-1.063), HDL and HDL/SAA were collected from the top layers of plasma whose density was adjusted to 1.21 with KBr. The collected lipoprotein was overlaid with KBr (density +1.21) and recentrifuged. The washed sample was dialyzed for 24 hours against EDTA saline (10 mM EDTA). The protein content was determined by the standard Lowry techniques ⁇ 1.
  • the apolipoproteins were fractionated on Sephacryl S-200 in 10% formic acid.
  • the apoA-I and SAA peaks were pooled, diluted with three volumes. of distilled water, lyophilized, delipidated ⁇ , and stored at -20° until ready for use.
  • Liposomes containing apoA-I or SAA were prepared using egg phosphatidyl-choline (PC) and the sodium cholate dispersal technique 1". After removing any large aggregates by centrifugation, the liposomes were filtered on a 1.5x50 cm Sepharose C1-4B column, liposomes of uniform size were eluted as a single peak (see Figure 6), with larger complexes emerging in the void column. Following concentration, the liposomes were dialyzed overnight, at 0-4°C, against RPMI 1640 (Gibco), and the protein and phosphate content of the particles were determined-' ,21 _
  • High density lipoprotein was iodinated with Na ⁇ 5j using the iodine monochloride method, and purified by ion exchange chromatography. Iodination was done at pH 10, and greater than 95% of the radioactivity was found to be protein bound. On electrophoresis in 12% polyacrylamide gels containing 0.1% SDS and beta mercaptoethanol, followed by autoradiography, only 125 ⁇ _ a p 0 -I and 125l-apoA-II were detectable in the HDL preparations, while apoA-1, SAA, and apoE were detected in the HDL/SAA preparations. In HDL, more than 95% of the protein was represented by apoA-1 , while in the HDL/SAA preparations approximately equal quantities of apoA-I and SAA were detected.
  • the cells were washed free of unbound label by centrifuging a known volume of cell suspension through a layer of equal volumes of medium and phthalate mixture, in a conical microvial in a microfuge.
  • the supernatant containing the incubation medium and the separating oil were removed by aspiration and the remaining liquid was drained.
  • BQ is the background in the absence of added ligand
  • FIG. 2 shows saturation binding curves of HDL (panel A) and HDL/SAA (panel B) for hepatocytes from various physiological conditions.
  • Log plots of ligand concentrations have been used to better illustrate binding at low concentrations.
  • the individual points represent the experimental data.
  • the solid lines represent the curves of best fit employing the polynomial for a single class of binding sites described above.
  • the parameters were obtained from the curves of best fit.
  • the insets represent Scatchard plots. Each such experiment was performed in triplicate. The spread of results and their statistical analyses are presented in Table 1.
  • Panel A A representative curve of HDL binding to normal hepatocytes
  • Panel B A representative curve of HDL/SAA binding to hepatocytes 72 hours after inducing inflammation
  • Figure 3 shows saturation binding curves of HDL (panel A) and HDL/SAA (panel B) for peritoneal macrophages from various physiological conditions.
  • the individual points represent the experimental data.
  • the solid lines represent the curves of best fit, which provided the values of the parameters listed below.
  • the insets represent Scatchard plots. Each such experiment was performed in triplicate. The spread of results and their statistical analyses are presented in Table 2.
  • Panel A A representative curve of HDL binding to normal macrophages
  • Panel B A representative curve of HDL/SAA binding to normal macrophages
  • Peritoneal macrophages were collected from normal animals as described previously 1 ⁇ . After culturing for 24 hours in RPMI 1640 and 10% FCS, the cells were washed three times in fresh RPMI 1640 and loaded with 3 H-cholesterol as described by Delamatre et al. 5
  • the labelling medium (RPMI 1640) contained 1% FCS, 2 mg/ml BSA, 6 ⁇ g/ml PC, 2.4 ⁇ g/ml unesterified cholesterol, 2 ⁇ g/ml Sandoz 58035 as an ACAT inhibitor, and 0.5 ⁇ Ci/ml of 3 H-cholesterol (NEN-NET139).
  • the binding of the ligands (HDL/SAA) to cells was initially done as a function of time to determine the time needed for the labelled ligand to reach maximum equilibrium binding.
  • the labelled ligand at a concentration of 10 ⁇ g/ml was added to either macrophages or hepatocytes (6 x 10"/ml) which had been precooled on ice.
  • the ligand and cell suspension were incubated for three hours, mixing constantly in a rotator. Aliquots of the cell suspension (100 ⁇ l) were taken at different time points and the cells and attached ligand pelleted through oil as described above.
  • Figure 1 demonstrates a representative binding curve of *l25 ⁇ _ HDL to mouse hepatocytes over a period of three hours. Similar results were obtained for HDL/SAA. These experiments were repeated on at least three occasions using separate preparations of cells and ligand. An equilibrium state of binding was reached within 90 minutes. Peritoneal macrophages gave very similar results. A binding time of two hours was therefore used in all subsequent experiments. Specificity of binding was demonstrated by competition experiments in the presence of a hundred fold excess unlabelled ligand which prevented the binding of its corresponding labelled partner. Competition binding studies between labelled HDL and "cold" HDL/SAA and the converse, were also performed.
  • FIGS. 2A and 2B Representative examples of saturation binding curves of hepatocytes for varying concentrations of HDL, and for varying concentrations of HDL/SAA are shown in FIGS. 2A and 2B respectively. Scatchard plots of these data are presented as the insets in FIGS. 2A and 2B.
  • FIGS. 3A and 3B Similar binding curves of HDL and HDL/SAA for peritoneal macrophages are shown in FIGS. 3A and 3B respectively.
  • HDL/SAA's affinity for hepatocytes continued to increase with time.
  • the Kd dropped from 32 nM to 17 nM and 6 nM (assuming an average molecular weight of HDL/SAA of 175 kDa) at 24 and 72 hours respectively.
  • a physiologic change is probably taking place in the hepatocyte during inflammation increasing its affinity for HDL/SAA.
  • a potential mechanism involving apolipoprotein E (apoE) is presented below.
  • the hepatocyte B max for HDL did not increase at 24 hours but was two fold higher at 72 hours. In contrast, with HDL/SAA there was relatively little change in B max at 24 hours but a significant drop occurred at 72 hours.
  • Table 2 contains a summary and statistical analysis of the Kd's and B max for HDL's and HDL/SAA's interaction with peritoneal macrophages.
  • the affinity of HDL/SAA for normal macrophages was three to four fold higher than HDL alone. Twenty-four and 72 hours into an inflammatory reaction, there was no change in HDL's affinity or B max for HDL. Here again there may be an underestimate of the B max at the 24 hour interval as the macrophages are harvested from animals with a high level of SAA.
  • the extent to which endogenous SAA would be a confounding problem in the macrophage binding studies would be far less important than with hepatocytes, since it has been shown that peritoneal macrophages contain little SAA even during inflammation 3 .
  • HDL/SAA consistently had a higher affinity for macrophages than HDL, regardless of time period into the inflammatory reaction at which one examined the macrophages.
  • the corresponding figures are: for HDL, an increase from 160,000 to 325,000, and for HDL/SAA, a decrease from 400,000 to 95,000.
  • the net effect of both the changes in affinity and numbers of binding sites is a significant shift in HDL cholesterol carrying capacity towards the macrophage.
  • HDL has a higher apoA-1 content then HDL/SAA. If HDL/SAA was also binding to macrophages solely through apoA-1, then one would expect less unlabelled HDL, than HDL/SAA, to be effective in reducing labelled HDL/SAA binding by 50%. However, HDL was far less
  • SUBSTITUTE SHEET (RULE 1 26) effective a competition than HDL/SAA. To achieve a 50% reduction of HDL/SAA binding, approximately 200 ⁇ g/ml HDL were required.
  • cytokines liberated by activated inflammatory cells serve as signals to a variety of cells and organs.
  • IL-1, IL-6 and tumor necrosis factor induce the expression of acute phase proteins, among them SAA.
  • SAA in turn associated with HDL as shown herein serves to preferentially direct HDL to macrophages and probably other reticuloendothelial cells (RES). This process would not necessarily depend on the appearance of a new SAA receptor on these cells, although such an event is not excluded. The receptor might already exist on these cells. Its ligand, SAA, would appear only when necessary, addressing HDL to such cells at the time of greatest need, i.e., inflammation.
  • RES reticuloendothelial cells
  • SAA may be displaced and released near RES cells and (if an amyloidogenic SAA) might therefore become available for amyloid formation at these anatomic sites.
  • the entire process is illustrated schematically in Figure 5. It is one that ensures an efficient and directed lipid/cholesterol transport mechanism during inflammation. Further, from this postulated role emerges the physiologic reason for the specific anatomic localization of inflammation- type amyloidosis.
  • HDL/SAA In response to cytokines released by activated inflammatory cells, SAA is secreted by the liver, and binds to HDL, primari'-- HDL3.
  • the HDL/SAA particle has a higher affinity for reticuloendothelial cells such as 1 macrophages, than does HDL alone.
  • macrophages during inflammation develop increased numbers of binding sites for HDL/SAA.
  • hepatocytes lose binding sites for HDL/SAA.
  • a net shift of reverse cholesterol carrying capacity towards macrophage-type cells thus occurs during inflammation.
  • these cells On arrival of HDL/SAA, these cells release apoE and cholesterol, a process which likely displaces the SAA.
  • the HDL/apoE/cholesterol complex is transported to sites for receptor mediated uptake/use or excretion.
  • SAA Binds Preferentially to Endogenous HDL in vivo
  • Native SAA was labelled with radioactive iodine 1 5j us i n g a conventional iodine monochloride technique. Aliquots of the labelled SAA were injected intravenously into CDl mice which, 20 minutes later, were sacrificed and exsanguinated. EDTA was added to the blood as an anticoagulant. Blood from five mice was pooled and, after centrifugation, plasma was collected. Differential centrifugation in potassium bromide at density 1.063 was performed to separate a VLDL and LDL fraction (very low and low density lipoprotein) followed by differential centrifugation in potassium bromide at density 1.21 to separate an HDL fraction. The radioactivity of each fraction was determined: percent of SAA recovered in VLDL/LDL was 7%; in HDL, it was 83.1%; all remaining SAA was 9.9%.
  • SAA serum amyloid
  • HDL high density lipoproteins
  • Figure 6 illustrates the elution profile of apoA-I liposomes (second peak) on Sephacryl S-200. A similar profile was seen with SAA liposomes.
  • Figure 7 demonstrates that the rate of efflux was a function not only of the concentration of liposomes, but also the nature of the liposome.
  • SAA liposomes were more effective in eliciting cholesterol from macrophages than equivalent concentrations of apoA-I liposomes.
  • SAA liposomes were consistently more effective as cholesterol acceptors than their apoA-I counterparts.
  • HDL/SAA provide a relevant and significant therapeutic mechanism for removing cholesterol from macrophages at atherosclerotic sites.
  • Kds are expressed in micrograms/milliliter ⁇ SEM of three experiments.
  • D B max are expressed as ng/10° cells ⁇ SEM of three experiments.
  • Kd values can be converted to M units assuming an average molecular weight for HDL or HDL/SAA of 175 kilodalcons.
  • Inflammation 72 hour 1.7 ⁇ 0.1 1.8 ⁇ 0.4 NS a Kds are expressed in micrograms/milliliter ⁇ SEM of three experiments, b ⁇ max are expressed as ng/10° cells ⁇ SEM of three experiments. C NS, not significant.
  • Kd values can be converted to M units assuming an average molecular weight for HDL or HDL/SAA of 175 kilodaltons.

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Abstract

L'invention porte sur un procédé permettant de potentialiser la libération et le recueil du cholestérol à partir de sites inflammatoires ou athéroscléreux in vivo. Le procédé consiste à augmenter l'affinité des lipoprotéines de haute densité pour les macrophages par administration à un patient d'une dose efficace d'une composition comprenant un composé choisi dans le groupe constitué d'amyloïde sérique A natif (SAA) et d'un ligand aux propriétés SAA. Il en résulte une augmentation de l'affinité des lipoprotéines de haute densité (HDL) pour les macrophages et une potentialisation de la libération et du recueil du cholestérol.
PCT/CA1996/000404 1995-06-01 1996-06-03 Proteine amyloide serique a WO1996038166A1 (fr)

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US08/458,054 US6004936A (en) 1992-05-29 1995-06-01 Method of use of serum amyloid a protein

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004111084A3 (fr) * 2003-06-12 2005-07-14 Univ Kingston Compositions et methodes de traitement de l'atherosclerose
US7674772B2 (en) 2006-03-31 2010-03-09 Queen's University At Kingston Compositions and methods for treating atherosclerosis
US7700544B2 (en) 2003-06-12 2010-04-20 Queens's University At Kingston Compositions and methods for treating atherosclerosis
EP2810695A1 (fr) * 2013-06-06 2014-12-10 Ceva Sante Animale Compositions et procédé de commande d'infections chez des mammifères non humains à l'aide de protéines de phase aiguë
CN109957003A (zh) * 2019-04-15 2019-07-02 南京立顶生物科技有限公司 一种稳定的saa突变体及其在疾病检测中的应用
CN114470160A (zh) * 2022-03-18 2022-05-13 山东农业大学 病毒复制抑制剂及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024530A1 (fr) * 1992-05-29 1993-12-09 Queen's University At Kingston Proteine de serum amyloide a

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1993024530A1 (fr) * 1992-05-29 1993-12-09 Queen's University At Kingston Proteine de serum amyloide a

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KISILEVSKY, R. ET AL: "Serum amyloid A influences the efflux of cholesterol from macrophages", AMYLOID AMYLOIDOSIS 1993, PROC. INT. SYMP. AMYLOIDOSIS, 7TH (1994), MEETING DATE 1993, 115-18. EDITOR(S): KISILEVSKY, R., 1994, XP000603315 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004111084A3 (fr) * 2003-06-12 2005-07-14 Univ Kingston Compositions et methodes de traitement de l'atherosclerose
US7291590B2 (en) 2003-06-12 2007-11-06 Queen's University At Kingston Compositions and methods for treating atherosclerosis
US7700544B2 (en) 2003-06-12 2010-04-20 Queens's University At Kingston Compositions and methods for treating atherosclerosis
EP2270035A3 (fr) * 2003-06-12 2011-03-09 Queen's University At Kingston Peptides améliorant l'activité CEH, compositions pharmaceutiques comprenant ces peptides et leur utilisation dans le traitement de l'athérosclérose
US8703698B2 (en) 2003-06-12 2014-04-22 The University Of Chicago Compositions and methods for treating atherosclerosis
US7674772B2 (en) 2006-03-31 2010-03-09 Queen's University At Kingston Compositions and methods for treating atherosclerosis
EP2810695A1 (fr) * 2013-06-06 2014-12-10 Ceva Sante Animale Compositions et procédé de commande d'infections chez des mammifères non humains à l'aide de protéines de phase aiguë
WO2014195413A1 (fr) * 2013-06-06 2014-12-11 Ceva Sante Animale Compositions et procédés de lutte contre des infections chez des mammifères non humains en utilisant des protéines de phase aiguë
CN105407972A (zh) * 2013-06-06 2016-03-16 法国诗华动物保健公司 使用急性期蛋白在非人类哺乳动物中控制感染的组合物和方法
CN105407972B (zh) * 2013-06-06 2021-12-24 法国诗华动物保健公司 使用急性期蛋白在非人类哺乳动物中控制感染的组合物和方法
CN109957003A (zh) * 2019-04-15 2019-07-02 南京立顶生物科技有限公司 一种稳定的saa突变体及其在疾病检测中的应用
CN114470160A (zh) * 2022-03-18 2022-05-13 山东农业大学 病毒复制抑制剂及其应用
CN114470160B (zh) * 2022-03-18 2023-08-25 山东农业大学 病毒复制抑制剂及其应用

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