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WO1997049389A1 - Produits terminaux de glycosylation avancee, 3-alkylamino-2-hydroxy-4-hydroxymethyl-cyclopentene-1-ones, et methodes d'utilisation desdits produits - Google Patents

Produits terminaux de glycosylation avancee, 3-alkylamino-2-hydroxy-4-hydroxymethyl-cyclopentene-1-ones, et methodes d'utilisation desdits produits Download PDF

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Publication number
WO1997049389A1
WO1997049389A1 PCT/US1997/011406 US9711406W WO9749389A1 WO 1997049389 A1 WO1997049389 A1 WO 1997049389A1 US 9711406 W US9711406 W US 9711406W WO 9749389 A1 WO9749389 A1 WO 9749389A1
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WO
WIPO (PCT)
Prior art keywords
advanced glycosylation
pharmaceutical composition
compound
proteins
amount
Prior art date
Application number
PCT/US1997/011406
Other languages
English (en)
Inventor
Peter C. Ulrich
Xini Zhang
Original Assignee
The Picower Institute For Medical Research
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 US08/673,217 external-priority patent/US5688653A/en
Priority claimed from US08/880,565 external-priority patent/US5814664A/en
Application filed by The Picower Institute For Medical Research filed Critical The Picower Institute For Medical Research
Priority to AU35112/97A priority Critical patent/AU3511297A/en
Priority to EP97931496A priority patent/EP1014958A1/fr
Publication of WO1997049389A1 publication Critical patent/WO1997049389A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the present invention relates generally to the aging of proteins and other amino- containing-biomolecules resulting from reaction of glucose, and particularly to the non- enzymatic glycation or glycosylation of proteins and other susceptible amine -presenting molecules and subsequent reactions leading to advanced glycosylation end products, and to methods for their use.
  • AGEs Advanced glycosylation endproducts
  • Monnier et al. recently isolated the fluorescent crosslink pentosidine from human dura collagen.
  • Pentosidine appears to form as the condensation product of lysine, arginine, and a reducing sugar precursor.
  • pentosidine may be readily produced upon incubation of the N-alpha-protected derivatives of arginine, lysine, and sugars such as ribose, glucose, fructose, ascorbate, or dehydroascorbate.
  • This compound has been theorized to react with the carbonyl moiety of the early glycosylation product of a target protein formed subsequent to the initial non-enzymatic reaction with glucose or another reducing sugar, and thereby prevent further reaction to form advanced glycosylation end products.
  • AGEs advanced glycation end-products
  • AGEs can be colored or fluorescent and which can contain electrophilic centers which can cross-link two proteins together.
  • AGEs are usually formed only in tiny amounts relative to starting materials or early glycation products, from which they must be separated for structural characterization. Often, arbitrary distinguishing criteria such as fluorescence or color must be used to determine what components may be of interest. However, in recent years it has become apparent that protein cross-linking caused by advanced glycation is due primarily to unknown structures which are not highly colored or fluorescent.
  • model advanced glycosylation endproducts have been prepared which examine the AGE formation pathway wherein the Amadori Product (AP) is doubly dehydrated to form the AP-ene-dione and proceeds further to form advanced glycation endproducts.
  • AGEs model advanced glycosylation endproducts
  • the present invention also has particular diagnostic applications as the Maillard process acutely affects several of the significant protein masses in the body, among them collagen, elastin, lens proteins, and the kidney glomerular basement membranes. These proteins deteriorate both with age (hence the application of the term "protein aging") and as one of the sequelae of diabetes melitis. Consequently, the ability to measure the amount of the formation of advanced glycosylation end products carries the promise of favorably treating significant adverse effects of aging and of diabetes at an earlier stage, and, of course, improving the quality and perhaps duration of animal life, including for instance human life.
  • This method finds particular use, among other applications, in the diagnosis of glycation-related disease and the monitoring of anti-glycation therapy or prophylactic treatment.
  • the compound 4a the pyranose form of 4
  • Reductive amination of protected aldehyde 9 with propylamine gave 10 in 95% yield.
  • Compound 10 was also obtained by treating propylamine Amadori product 8 with concentrated H 2 SO 4 in acetone in 90% yield.
  • the propyl derivative is shown in the description and schemes herein, the other lower alkyl derivatives can similarly be produced by substitution of the appropriate starting material.
  • the lower alkyl group contains from 1 to 6 carbon atoms, and is exemplified by methyl, ethyl, propyl, butyl, pentyl and hexyl, and the corresponding branched chain isomers thereof.
  • R is a lower alkyl group containing from 1 to 6 carbon atoms .
  • Scheme 4 shows how the enol of AP-ene-dione 3 can undergo internal cyclization in two ways: via attack of C-l of the enol (path A) or the amine
  • the 4-0-mono-tosylate of Amadori product 8 can be synthesized.
  • the cross-linking potential of AP-ene-dione 3 can be clarified by studies of model cross-linking reactions of 3 (via solvolysis of 4), 6, and 7 with various nucleophiles.
  • the presence of cypentodine (6) among the various solvolysis products which slowly form during incubation of propyl Amadori product 8 by itself under physiological conditions, and in the complex mixtures which form in the classical Maillard reaction conditions of incubation of glucose with amines can be utilized is the basis for the diagnostic methods of the present invention.
  • the animal host intended for treatment may have administered to it a quantity of the advanced glycosylation endproduct of formula 6, either as a free cyclic pentosidine compound or formed within the sequence of a longer peptide or peptide-like molecule, in a suitable pharmaceutical form.
  • Such administration can increase the macrophage recognition and elimination of other advanced glycosylation endproducts in the mammalian body.
  • Administration may be accomplished by known techniques, such as oral, topical and parenteral techniques such as intradermal, subcutaneous, intravenous, or intraperitoneal injection, as well as by other conventional means such as inhaled aerosols or nebulized droplets.
  • Administration of the agents may take place over an extended period of time at a dosage level of, for example, up to about 25 mg/kg.
  • compositions useful in the present invention comprise the compound of formula 6, together with carriers suitable for their intended use.
  • the findings of the present invention can also be utilized to screen for additional agents which would have utility as agents for inhibiting advanced glycosylation or glycation.
  • the measurement of the amount of the formation of the compound of formula 6 when exposed to an amount of a potential inhibitor of the advanced glycosylation reaction would enable one to assess the usefulness of an agent as a potential inhibitor of the advanced glycosylation or glycation process.
  • the compounds of formula 6 may be used in standard fashion to prepare either polyclonal or monoclonal antibodies thereto for diagnostic purposes. Such antibodies are preparable by standard procedures, and thus enable the use of diagnostic assays for assessing and monitoring the effectiveness of therapeutic regimens where AGE inhibition has been initiated. Said immunological regents directed against generic and specific structures of the present invention are also useful to detect the degree of advanced glycosylation in a sample from a subject animal, including, for example, a human being, thereby to infer degree of advanced glycosylation which has occurred in the subject, by reference to a standard.
  • Said polyclonal or monoclonal immunological reagents can optionally be included in a kit, with instructions, and, optionally, a standardized preparation of a cyclic pentosidine compound of the present invention, to facilitate such determinations all as contemplated hereunder.
  • compositions may be prepared with a pharmaceutically effective quantity of the agents or compounds of the present invention and may include a pharmaceutically acceptable carrier, selected from known materials utilized for this purpose.
  • a pharmaceutically acceptable carrier selected from known materials utilized for this purpose.
  • Such compositions may be prepared in a variety of forms, depending on the method of administration. For example, a liquid form would be utilized in the instance where administration is by intravenous or intraperitoneal injection, which liquid might be aerosolized for delivery by inhalation; while, if appropriate, tablets, capsules, etc. , may be prepared for oral administration.
  • a lotion or ointment may be formulated with the agent in a suitable vehicle, perhaps including a carrier to aid in penetration into the skin.
  • Other suitable forms for administration to other body tissues are also contemplated.
  • the animals to be treated would have administered to them a regular quantity of the pharmaceutical composition of the present invention.
  • Administration could take place, for example, daily, and an effective quantity of the agent or compound of the present invention could range up to 25 mg/kg of body weight of the animal.
  • a topical preparation may, for example, include up to 10% of the agent or composition in an ointment or lotion for application to the skin.
  • the suggested amounts are provided in fulfillment of applicants' duty to disclose the best mode for the practice of the present invention.
  • the in vivo therapeutic implications of the present invention relate to the reversal of several of the pathogenic activities associated with the aging process which have, as indicated earlier, been identified in the aging of key tissue and circulating proteins by advanced glycosylation and crosslinking through the mechanism of macrophage stimulation of the removal of the advanced glycosylation endproducts.
  • body proteins, and particularly structural body proteins such as collagen, elastin, lens proteins, nerve proteins and kidney glomerular basement membranes would all benefit in their longevity and operation from the practice of the present invention.
  • the present invention thus reduces the senescence caused by pathologies involving the entrapment of proteins by crosslinked target proteins, as exemplified, for instance, in retinopathy, cataracts, diabetic kidney disease, glomerulosclerosis, peripheral vascular disease, arteriosclerosis obliterans, peripheral neuropathy, stroke, hypertension, atherosclerosis, osteoarthritis, periarticular rigidity, loss of elasticity and wrinkling of skin, stiffening of joints, glomerulonephritis, etc. Likewise, all of these conditions are in evidence in patients afflicted with diabetes mellitus.
  • the present therapeutic method is relevant to treatment of the noted conditions in patients either of advanced age or those suffering from one of the mentioned pathologies, particularly in association with hyperglycemia, which accelerates gly cation-mediated senescence.
  • Protein crosslinking through advanced glycosylation product formation can decrease solubility of structural proteins such as collagen in vessel walls, and as well as trap serum proteins, such as lipoproteins to structural proteins such as collagen. Also, this may result in covalent trapping of extravasated plasma proteins in subendothelial matrix, and reduction in susceptibility of both plasma and matrix proteins to physiological degradation by enzymes. For these reasons, the progressive occlusion of diabetic vessels induced by chronic hyperglycemia has been hypothesized to result in part from excessive formation of glucose-derived adducts and crosslinks. Such diabetic macrovascular changes and microvascular occlusion can be effectively treated by enhancing the removal of advanced glycosylation endproducts utilizing a composition and the methods of the present invention.
  • Example 1 The compound 4a, the pyranose form of 4, was synthesized from the propylamine Amadori product 1-deoxy-l-propylamino- ⁇ -D-fructopyranose hydrogen oxalate (8) or 2,3 :4,5-di-O-isopropylidene- «We ⁇ yrfo- ⁇ -D- ⁇ rabmo-hexos-2-ulo-2,6-pyranose (9) in 77% or 81 % overall yield, respectively.
  • Antigens, and conjugated immunogens corresponding to the cyclic pentosidine-like advanced glycosylation endproducts of the present invention, including the products described in Example 1 can conveniently be prepared, either by isolation from incubation mixtures or by direct synthetic approaches.
  • the AGEs thus prepared may then be used as an immunogens to raise a variety of antibodies which recognize specific epitopes or molecular features thereof.
  • the 3-alkylamino-2- hydroxy-4-hydroxymethyl-2-cyclopenten-l-one itself is considered a hapten, which is correspondingly coupled to any of several preferred carrier proteins, including for instance keyhole limpet hemocyanin (KLH), thyroglobulin, and most preferred, bovine serum albumin (BSA), using any of a number of well-known divalent coupling reagents such as a carbodiimide like EDC, according to protocols widely circulated in the art.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the 3-alkylamino-2-hydroxy-4-hydroxymethyI-2-cyclopenten- 1 -one-like AGE of formula 6, alone or coupled to a carrier protein, may be employed in any well-recognized immunization protocol to generate antibodies and related immunological reagents that are useful in a number of applications owing to the specificity of the resulting antibodies for molecular features of the 3-alkylamino-2- hydroxy-4-hydroxymethyl-2-cyclopenten-l -one-like AGE of formula 6.
  • any of several animal species may be immunized to produce polyclonal antisera directed against the cypentodine-carrier protein conjugate, including for instance mice, rats, hamsters, goats, rabbits, and chickens.
  • the first of three of the aforesaid animal species are particularly desirable choices for the subsequent production of hybridomas secreting hapten-specific monoclonal antibodies.
  • the production of said hybridomas from spleen cells of immunized animals may conveniently be accomplished by any of several protocols popularly practiced in the art, and which describe conditions suitable for immortalization of immunized spleen cells by fusion with an appropriate cell line, e.g. a myeloma cell line.
  • Said protocols for producing hybridomas also provide methods for selecting and cloning immune splenocyte/myeloma cell hybridomas and for identifying hybridomas clones that stably secrete antibodies directed against the desired epitope(s).
  • Animal species such as rabbit and goat are more commonly employed for the generation of polyclonal antisera, but regardless of whether polyclonal antisera or monoclonal antibodies are desired ultimately, the hapten-modified carrier protein typically is initially administered in conjunction with an adjuvant such as Complete Freund's Adjuvant.
  • Immunizations may be administered by any of several routes, typically intraperitoneal, intramuscular or intradermal; certain routes are preferred in the art according to the species to be immunized and the type of antibody ultimately to be produced.
  • booster immunizations are generally administered in conjunction with an adjuvant such as alum or Incomplete Freund's Adjuvant.
  • Booster immunizations are administered at intervals after the initial immunization; generally one month is a suitable interval, with blood samples taken between one and two weeks after each booster immunization.
  • hyperimmunization schedules which generally feature booster immunizations spaced closer together in time, are sometimes employed in an effort to produce anti-hapten antibodies preferentially over anti-carrier protein antibodies.
  • the antibody titers in post-boost blood samples can be compared for hapten-specific immune titer in any of several convenient formats including, for instance, Ouchterlony diffusion gels and direct ELISA protocols.
  • a defined antigen is immobilized onto the assay well surface, typically in a 96-well microtiter plate format, followed by a series of incubations separated by rinses of the assay well surface to remove unbound binding partners.
  • the wells of an assay plate may receive a dilute, buffered aqueous solution of the hapten/carrier conjugate, preferably wherein the carrier protein differs from that used to immunize the antibody-producing animal to be tested; e.g. serum from cypentodine/KLH conjugate- immunized animal might be tested against assay wells decorated with immobilized cypentodine/BSA conjugate.
  • the assay surface may be decorated by incubation with the hapten alone.
  • the surface of the assay wells is then exposed to a solution of an irrelevant protein, such as casein, to block unoccupied sites on the plastic surfaces.
  • the well After rinsing with a neutral buffered solution that typically contains salts and a detergent to minimize non-specific interactions, the well is then contacted with one of a serial dilution of the serum prepared from the blood sample of interest (the primary antiserum in crude or purified form).
  • the extent of test antibodies immobilized onto the assay wells by interaction with the desired hapten or hapten/carrier conjugate can be estimated by any of a number of well known procedures including, for instance incubation with a commercially available enzyme-antibody conjugate, wherein the antibody portion of this secondary conjugate is directed against the species used to produce the primary antiserum; e.g.
  • the secondary antibody if the primary antiserum were raised in rabbits, a commercial preparation of anti-rabbit antibodies raised in goat and conjugated to one of several enzymes, such as horseradish peroxidase, can be used as the secondary antibody. Following procedures specified by the manufacturer, the amount of this secondary antibody can then be estimated quantitatively by the activity of the associated conjugate enzyme in an assay, typically a colorimetric assay. Many related ELISA or radioimmunometric protocols, such as competitive ELISAs or sandwich ELISAs, all of which are well-known in the art, may optionally be substituted, to identify the desired antisera of high titer; that is, the particular antiserum which gives a true positive result at high dilution (e.g. greater than 1/1000 and more preferably greater than 1/10,000).
  • Similar immunometric protocols can be used to estimate the titer of antibodies in culture supernatants from hybridomas prepared from spleen cells of immunized animals.
  • control incubations e.g. with different carrier proteins, related but structurally distinct haptens or antigens, and omitting various reagents in the immunometric procedure in order to minimize non-specific signals in the assay and to identify reliable determinations of antibody specificity and titer from false positive and false negative results.
  • the types of control incubations to use in this regard are well known.
  • the same general immunometric protocols subsequently may be employed with the antisera identified by the above procedures to be of high titer and to be directed against specific structural determinants of cypentodine-like AGEs present in biological samples, foodstuffs or other comestibles, or other amine-bearing substances and biomolecules of interest.
  • Such latter applications of the desired antibodies, whether polyclonal or monoclonal, together with instructions and optionally with other useful reagents and diluents, including, without limitation, a set of molecular standards of cypentodine-like AGEs may be provided in kit form for the convenience of the operator.

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Abstract

La présente invention concerne des produits terminaux de glycosylation avancée et particulièrement l'utilisation des nouvelles aminoréductones cyclopenténones, les 3-alkylamino-2-hydroxy-4-hydroxyméthyl-cyclopentène-1-ones. De tels produits de glycosylation avancée peuvent s'utiliser dans diverses méthodes diagnostiques et thérapeutiques.
PCT/US1997/011406 1996-06-27 1997-06-26 Produits terminaux de glycosylation avancee, 3-alkylamino-2-hydroxy-4-hydroxymethyl-cyclopentene-1-ones, et methodes d'utilisation desdits produits WO1997049389A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU35112/97A AU3511297A (en) 1996-06-27 1997-06-26 3-alkylamino-2-hydroxy-4-hydroxymethyl-2-cyclopenten-1-one advanced glycosylation endproducts and methods of use therefor
EP97931496A EP1014958A1 (fr) 1996-06-27 1997-06-26 Produits terminaux de glycosylation avancee, 3-alkylamino-2-hydroxy-4-hydroxymethyl-cyclopentene-1-ones, et methodes d'utilisation desdits produits

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US08/673,217 1996-06-27
US08/673,217 US5688653A (en) 1996-06-27 1996-06-27 3-alkylamino-2-hydroxy-4-hydroxymethyl-2-cyclopenten-1-one advanced glycosylation endproducts and methods of use therefor
US08/880,565 1997-06-23
US08/880,565 US5814664A (en) 1996-06-27 1997-06-23 3-alkylamino-2-hydroxy-4-hydroxymethyl-2-cyclopenten-1-one advanced glycosylation endproducts and methods of use therefor

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WO1997049389A1 true WO1997049389A1 (fr) 1997-12-31

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PCT/US1997/011406 WO1997049389A1 (fr) 1996-06-27 1997-06-26 Produits terminaux de glycosylation avancee, 3-alkylamino-2-hydroxy-4-hydroxymethyl-cyclopentene-1-ones, et methodes d'utilisation desdits produits

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EP (1) EP1014958A1 (fr)
AU (1) AU3511297A (fr)
WO (1) WO1997049389A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993016087A2 (fr) * 1992-02-13 1993-08-19 Torf Establishment Produits et composes de transposition d'amadori, leur procede de fabrication et leur utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993016087A2 (fr) * 1992-02-13 1993-08-19 Torf Establishment Produits et composes de transposition d'amadori, leur procede de fabrication et leur utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ZHANG ET AL: "Directed approaches to reactive maillard intermediates: formation of a novel 3-alkylamino-2-hydroxy-4-hydroxymethyl-2-cyclopenten-1-one ("cypentodine")", TETRAHEDRON LETTERS, vol. 37, no. 27, 1996, pages 4667 - 70, XP002045858 *
ZHANG ET AL: "Two cyclic AGEs from dehydration of the amadori product are recognized by polyclonal and monoclonal antibodies to AGEs", DIABETES, vol. 46, no. suppl.1, May 1997 (1997-05-01), pages 598, XP002045859 *

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EP1014958A1 (fr) 2000-07-05
AU3511297A (en) 1998-01-14

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