WO2001025291A1 - Polymeres liant le sucre et leur utilisation - Google Patents

Polymeres liant le sucre et leur utilisation Download PDF

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Publication number
WO2001025291A1
WO2001025291A1 PCT/US2000/027392 US0027392W WO0125291A1 WO 2001025291 A1 WO2001025291 A1 WO 2001025291A1 US 0027392 W US0027392 W US 0027392W WO 0125291 A1 WO0125291 A1 WO 0125291A1
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Prior art keywords
hydrogel
crosslinked
polymer
glucose
allylamine
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PCT/US2000/027392
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English (en)
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Peter Kofinas
William Wizeman
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University Of Maryland
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Priority to AU78562/00A priority Critical patent/AU7856200A/en
Publication of WO2001025291A1 publication Critical patent/WO2001025291A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/18—Introducing halogen atoms or halogen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2810/00—Chemical modification of a polymer
    • C08F2810/20—Chemical modification of a polymer leading to a crosslinking, either explicitly or inherently

Definitions

  • the invention is in the field of polymer chemistry.
  • the invention relates to molecularly imprinted, crosslinked hydrogel polymers which bind specifically one or more sugars.
  • the invention also relates to methods for preparing the polymers and the use thereof to remove sugars from solutions and for treating certain conditions.
  • MIPs highly specific molecularly imprinted polymers
  • the methods of molecular imprinting and its potential applications have been recently reviewed (Wulff. G.. Chemtech 25:19-26 (1998); Mosbach, K.. and Ramstrom. O.. Bio-Technol - : 163-170 (1996)).
  • Promising applications for these MIPs include tailor-made separation materials, molecular recognition materials for biosensors, highly specific catalysts, and antibody mimics for quantitative assay and molecular recognition.
  • separation materials have been used in conjunction with chromatographic techniques to separate amino acids, drugs, pesticides, sugar derivatives, and other compounds (Mayes A. G.. et al.
  • MIPs have also been employed in efforts to synthesize materials for biosensor applications.
  • An especially interesting area of biosensors is quantitative glucose level monitoring. This is because nearly 6% of the population in the United States suffers from diabetes and quantitative blood glucose monitoring is an essential part of the successful management of the disease.
  • Arnold et al. (Chen. G.. et al, Nat. Biotechnol. 75:354-357 (1997)) have used molecular imprinting to produce a glucose sensitive polymer that exhibits a change in pH proportional to the glucose concentration of its environment. More recently,
  • Zambonin et.al. (Malitesta. C, et al, Anal. Chem. 77 : 1366-1370 (1999)) have explored a new technique utilizing electrosynthesized MIPs in an effort to develop a glucose sensitive material.
  • Catalysts and antibody mimics stand to benefit in similar manners from the technique of molecular imprinting.
  • the ability to construct complicated combinations of precisely placed functional groups in a mechanically stable and specifically shaped cavity allows the synthesis of materials capable of catalyzing complex reactions or of mimicking antibodies used in analytical reagents.
  • Mosbach et al. (Ansell. R. J.. et al. Clinical Chem. 42: 1506-1512 ( 1996)) has shown promising results in the area of artificial antibodies.
  • Molecular imprinting in polymers is achieved by incorporating a template, or imprint molecule into a highly crosslinked polymer matrix.
  • the imprint molecule is identical or similar, in both size and functionality, to the molecule for which the polymer is being imprinted, the target molecule. Given a polymer matrix with sufficient mechanical stability, cavities with size, shape and functionality specific to the template molecule are created upon removal of the template.
  • the template molecule is bonded to a polymerizable functional monomer or. in this case, polymer side group prior to crosslinking.
  • the bond between the template and the functional monomer or polymer can be either covalent or non- covalent.
  • Studies have shown successful results in the area of molecular imprinting via non-covalent interactions between template and functional monomer (Mayes A. G.. et al, Anal. Biochem. 222:483-488 (1994); Spivak, D., and Shea, K. J., J. Org. Chem.64:462 -4634 (1999)) as well as via covalent interactions (Wulff, G., and Schauhoff, S., J Org. Chem. 56:395-400 (1991)).
  • WO99/40990 discloses anion-binding polymers such as crosslinked polymeric hydrogel materials which display the capability to bind and therefore remove phosphate, nitrite, nitrate, perchlorate and sulfate anions from various types of wastewater effluents.
  • Diabetes mellitus affects more than 100 million people worldwide. 12 million in the USA. It is the greatest cause of blindness, amputations, and kidney failure among working-age adults in the US.
  • the development of a glucose binding drug would be particularly important in the management, diagnosis, and monitoring of Type II Diabetes by providing a basis for insulin administration at more appropriate dosages.
  • a glucose binding drug would also be appropriate for the treatment of obesity.
  • the present invention provides a polymer based pharmaceutical that is not absorbed by the small intestine and selectively binds glucose from the intestinal tract to reduce or prevent absorption into the bloodstream, thereby minimizing the potential for adverse effects. This polymeric drug provides a method of controlling serious diseases affecting a large segment of the world population, and will help in decreasing health care costs associated with treatment of diabetics and obesity.
  • Crosslinked polymer networks highly swollen with water i.e. hydrogels
  • hydrogels are a class of materials receiving increasing commercial attention in a wide range of technologies including absorbents, separations media, and controlled release of pharmaceuticals and agricultural agents.
  • Polymeric hydrogels have been developed that are capable of binding of sugars such as glucose.
  • a novel synthetic methodology has been developed to prepare specific binding sites in crosslinked polymers having a predetermined shape, as well as placement of functionalized sugar groups with a defined steric pattern.
  • an imprinting procedure has been developed with the aid of template network polymer molecules.
  • gels were prepared with holes that mimic the size and shape of sugar molecules. The molecular imprinting technique allows the gels to bind sugars more readily than other molecules, and at higher amounts, compared to non-molecularly imprinted gels.
  • the invention relates in one aspect to a molecularly imprinted, crosslinked hydrogel polymer which specifically binds one or more sugars.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the molecularly imprinted, crosslinked hydrogel polymer of the invention and a pharmaceutically acceptable carrier.
  • the invention also relates to a confection comprising the molecularly imprinted, crosslinked hydrogel polymer of the present invention and one or more sugars, e.g. sucrose.
  • the invention also relates to the molecularly imprinted, crosslinked hydrogel polymer with one or more sugars bound thereto.
  • the invention also relates to a method of preparing a molecularly imprinted, hydrogel polymer which specifically binds one or more sugars, comprising
  • the invention also relates to the surprising discovery that it is possible to increase the binding capacity of the hydrogel polymer by increasing the concentration of sugar phosphate in the reaction mixture.
  • the hydrogel polymer has a sugar binding capacity of about 0.4 to about 0.65 g per gram of hydrogel polymer at a pH of about 4-7. preferably, at pH of about 6.
  • the invention also relates to the surprising discovery that it is possible to modify the sugar binding selectivity of the hydrogel polymer by selecting particular crosslinking agents and by varying their concentration in the crosslinking reaction.
  • the invention also relates to a method for binding and/or removing one or more sugars from the gastrointestinal tract of a patient, comprising orally administering to the patient in need thereof a molecularly imprinted, crosslinked hydrogel polymer which specifically binds the one or more sugars.
  • the invention also relates to a method of treating type II diabetes or a complication thereof, comprising orally administering to a patient in need thereof a molecularly imprinted, crosslinked hydrogel polymer which specifically binds glucose.
  • a molecularly imprinted, crosslinked hydrogel polymer which specifically binds glucose.
  • the type II diabetes or complication thereof is treated.
  • the invention also relates to a method of diagnosis or monitoring the status of a patient having type II diabetes, comprising contacting a body fluid of a person suspected of having type II diabetes with a molecularly imprinted, hydrogel sugar binding polymer of the present invention, determining the amount of sugar bound thereto, thereby giving the amount of sugar in the body fluid, and relating the amount of sugar in the body fluid to a diagnosis or status of type II diabetes.
  • the invention also relates to a method of treating obesity or a complication thereof, comprising orally administering to a patient in need thereof a molecularly imprinted, crosslinked hydrogel polymer which specifically binds one or more sugars whereby obesity or complication thereof is treated.
  • the invention also relates to a method of preparing a molecularly imprinted, hydrogel polymer which specifically binds one or more sugars. comprising
  • a typical sugar binding hydrogel is made in two steps .
  • the first step is the synthesis and molecular imprinting of the gel. e.g. crosslinking an amino- containing polymer in the presence of one or more sugar phosphates.
  • the second step is the washing of the gel. which removes the imprinting component (e.g. glucose phosphate monosodium salt) and leaves the gel ready to bind the sugar.
  • the sugar-binding polymers that may be used in the practice of the invention are amino-containing polymers, e.g. crosslinked polymeric hydrogels.
  • Crosslinked polymeric hydrogels are hydrophilic polymer networks that are able to absorb large amounts of water but remain insoluble because of the presence of crosslinks, entanglements, or crystalline regions (Hassan. CM., et al , Macromolecules 50:6166-6173 (1997)).
  • Hydrogels. are a class of materials receiving increasing commercial attention in a wide range of technologies including absorbents, separations media, and controlled release of pharmaceuticals and agricultural agents (Peppas. N. A., Hydrogels in Medicine and Pharmacy, Vol 7 Fundamentals. CRC Press. Inc.. Boca Raton. FL (1986); Peppas. N.A. & Langer. R.. Science 2(55:1715-1720 (1994)).
  • Such hydrogel polymers may comprise the formula:
  • n is an integer of from about 1 to about 1000 or more, and each R. independently, is H or a lower alkyl (e.g., having between 1 and 5 carbon atoms, inclusive), alkylamino (e.g., having between 1 and 5 carbons atoms, inclusive, such as ethylamino) or aryl (e.g., phenyl) group.
  • the polymer may comprise the formula:
  • n is an integer of from about 1 to about 1000 or more
  • each R independently, is H or a lower alkyl (e.g., having between 1 and 5 carbon atoms, inclusive), alkylamino (e.g., having between 1 and 5 carbons atoms, inclusive, such as ethylamino) or aryl (e.g., phenyl) group, and each X " is an exchangeable negatively charged counter ion.
  • the polymer may comprise a first repeating unit having the formula
  • n is an integer of from about 1 to about 1000 or more
  • each R independently, is H or a lower alkyl (e.g.. having between 1 and 5 carbon atoms, inclusive), alkylamino (e.g.. having between 1 and 5 carbons atoms, inclusive, such as ethylamino) or aryl group (e.g.. phenyl).
  • each X " is an exchangeable negatively charged counter ion: and further comprising a second repeating unit having the formula ( 1 )
  • the polymer may comprise a repeating unit having the formula
  • n is an integer of from about 1 to about 1000 or more
  • R is H or a lower alkyl (e.g., having between 1 and 5 carbon atoms, inclusive), alkylamino (e.g., having between 1 and 5 carbons atoms, inclusive, such as ethylamino) or aryl group (e.g., phenyl).
  • One example of a copolymer that may be used in the practice of the invention may comprise a first repeating unit having the formula
  • n is an integer of from about 1 to about 1000 or more
  • R is H or a lower alkyl (e.g., having between 1 and 5 carbon atoms, inclusive), alkylamino (e.g.. having between 1 and 5 carbons atoms, inclusive, such as ethylamino) or aryl group (e.g., phenyl): and may further comprise a second repeating unit having the formula
  • each n independently, is an integer of from about 1 to about 1000 or more and R is H or a lower alkyl (e.g.. having between 1 and 5 carbon atoms, inclusive), alkylamino (e.g., having between 1 and 5 carbons atoms, inclusive, such as ethylamino) or aryl group (e.g., phenyl).
  • R is H or a lower alkyl (e.g.. having between 1 and 5 carbon atoms, inclusive), alkylamino (e.g., having between 1 and 5 carbons atoms, inclusive, such as ethylamino) or aryl group (e.g., phenyl).
  • the polymer may comprise a repeating group having the formula
  • n is an integer of from about 1 to about 1000 or more
  • each R, and R 2 independently, is H or a lower alkyl (e.g., having between 1 and 5 carbon atoms, inclusive), alkylamino (e.g.. having between 1 and 5 carbons atoms, inclusive, such as ethylamino) or aryl group (e.g., phenyl), and each X " is an exchangeable negatively charged counter ion.
  • At least one of the R-R 2 groups is a hydrogen group.
  • the polymer may comprise a repeating unit having the formula
  • n is an integer of from about 1 to about 1000 or more, each R, and R 2 . independently, is H, an alkyl group containing 1 to 20 carbon atoms, an alkylamino group (e.g., having between 1 and 5 carbons atoms, inclusive, such as ethylamino), or an aryl group containing 1 to 12 atoms (e.g., phenyl).
  • the polymer may comprise a repeating unit having the formula
  • n is an integer of from about 1 to 1000 or more, each R,. R 2 and R 3 . independently, is H. an alkyl group containing 1 to 20 carbon atoms, an alkylamino group (e.g.. having between 1 and 5 carbons atoms, inclusive, such as ethylamino), or an aryl group containing 6 to 12 atoms (e.g., phenyl). and each X " is an exchangeable negatively charged counter ion.
  • n may be any number, so long as the polymer functions to bind a sugar.
  • the negatively charged counter ions X " may be organic ions, inorganic ions, or combination thereof.
  • the inorganic ions suitable for use in this invention include the halides (especially chloride), carbonate, bicarbonate, sulfate. bisulfate. hydroxide, persulfate. sulfite. and sulfide.
  • Suitable organic ions include acetate, ascorbate. benzoate. citrate, dihydrogen citrate, hydrogen citrate, oxalate, succinate. tartrate. taurocholate, glycocholate. and cholate.
  • a preferred sugar binding polymer is poly(allyl amine) (PAA). or. its HC1 form.
  • PAA is a water soluble polymer which can be crosslinked by a variety of methods to produce a highly swollen hydrogel material (Kofinas, P., et al, Biomaterials 17: 1547- 1550 (1996)).
  • PAA-HC1 has the formula:
  • the molecular weight range of the anion-binding polymer may range from about 5,000 to about 100.000 g/mole. although the invention is not so limited.
  • Any molecular weight polymer that functions to bind sugars may be used in the practice of the present invention.
  • the cross-linking may be achieved by chemical or irradiation crosslinking.
  • Chemical crosslinking involves the use of a cross-linking agent which bridges two or more linear polymer chains.
  • cross- linking agents include diacrylates and dimethacrylates (e.g.. ethylene glycol diacrylate. propvleneglvcol diacrylate. butylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol dimethacrylate, butylene glycol dimethacrylate, polyethyleneglycol dimethacrylate, polyethyleneglycol diacrylate), methylene bisacrylamide. methylene bismethacrylamide. ethylene bisacrylamide. epichlorohydrin (EPI). toluene diisocyanate.
  • diacrylates and dimethacrylates e.g.. ethylene glycol diacrylate. propvleneglvcol diacrylate. butylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol dimethacrylate, butylene glycol dimethacrylate, polyethyleneglycol dim
  • the cross-linking agent is EPI or 1,2- ethylenediol diglycidyl ether (EGDE) which may prepared by reacting ethylene glycol and EPI (1 :2) or purchased from Aldrich Chemical Company, Milwaukee, WI.
  • EGDE 1,2- ethylenediol diglycidyl ether
  • GDE diglycidyl ether
  • the amount of crosslinking agent is typically between about 0.5 and about 75 weight %, and preferably between about 1 and about 10% by weight, based upon combined weight of crosslinking agent and monomer. In a most preferred embodiment, the crosslinking agent is present between about 2 and about 15% by weight.
  • Sugar phosphates that may be used in the practice of the invention include, but are not limited to glucose phosphates such as glucose- 1 -phosphate, glucose-6- phosphate and glucose 1.6-diphosphate; fructose phosphates such as fructose-6- phosphate and fructose- 1.6-diphosphate; and sucrose phosphates such as sucrose- 1 -phosphate and sucrose- 1.6-diphosphate. It is possible to prepare hydrogel polymers which are specific for a single sugar (e.g. glucose) or multiple sugars (glucose and fructose) by adding one or more sugar phosphates to the polymerization reaction. Hydrogel polymers that bind only glucose are useful for diagnosing, monitoring and treating type II diabetes and its complications. Hydrogel polymers that bind multiple sugars, e.g. glucose and fructose are useful for treating obesity and its complications.
  • glucose phosphates such as glucose- 1 -phosphate, glucose-6- phosphate and glucose 1.6
  • the polymerization reaction comprises the aqueous reaction of poly (allyl amine) (PAA) and EPI. GDE or EGDE. which serves as the crosslinking agent, in the presence of a sugar phosphate.
  • PAA poly (allyl amine)
  • GDE or EGDE.
  • the PAA used in the synthesis of the glucose binding gels has an average molecular weight that ranges from 5.000 to 100.000 g/mole. including a hydrochloric acid group ionically associated with each amine (PAA HC1). At least a portion of the HC1 groups of PAA ⁇ C1 may be neutralized with sodium hydroxide (NaOH) to provide free amine sites for the crosslinking reaction.
  • NaOH sodium hydroxide
  • glucose-6-phosphate monosodium salt is added to the reaction mixture.
  • the GPS becomes attached to the amine sites of the polymer. Approximately 1 % of the polymer ' s total available amine sites now have a GPS molecule bound to them. Some of the remaining amine sites will be used for the formation of a network, or gel. from the polymer.
  • the network is formed by the addition of a crosslinker to the polymer solution. This procedure provides free amine sites for the EPI, GDE or EGDE crosslinking reaction and immobilizes the GPS in the polymeric gel.
  • the crosslinker may be added such that approximately 25% of the polymer ' s available amine sites are used in the formation of the network.
  • the GPS is then removed from the network, e.g.
  • aqueous solutions of NaOH e.g. 0.5 to 4 N NaOH. most preferably, about 2N NaOH
  • KOH e.g. 0.5 to 4 N KOH. most preferably, about 2N KOH
  • LiOH e.g. 0.5 to 4 N LiOH. most preferably about 2N LiOH
  • the parameters influencing the structure, the sugar binding capacity and selectivity of the polymer are: 1. The density of crosslinks in the hydrogel. 2. The number of imprinted sites per gram of gel available for sugar binding.
  • crosslinking agent e.g. EPI. GDE or EGDE
  • the initial concentration and molecular weight of the PAA ' HCl may be independently varied by routine experimentation to determine their possible influence on the final sugar binding capability of the resulting polymer, and thus produce a hydrogel with optimum sugar binding capabilities.
  • the sugar binding capacity of the hydrogels may be increased significantly with increased amount of sugar phosphate.
  • the amount of glucose- 1 -phosphate was increased from about 75 mg to about 225 mg in the crosslinking reaction
  • the binding capacity of the hydrogel from a pH 3.9 solution increased from 0.436 g glucose/g dry gel to 0.639 g glucose/g dry gel.
  • the amount of monosaccharide phosphate in the crosslinking reaction is about 50-500 mg. more preferably about 100-225 mg.
  • a molar equivalent amount of a disaccharide phosphate may be substituted in place of the monosaccharide phosphate.
  • aqueous solutions of water soluble polymers When exposed to ionizing radiation, aqueous solutions of water soluble polymers become cross-linked and form hydrogels. primarily by radiolysis of water generating hydroxyl radicals, which attack the polymer chains resulting in the formation of macroradicals. These can crosslink or stabilize themselves by various processes, such as disproportionation or degradation.
  • Two types of crosslinking are distinguished: intramolecular crosslinking (between macroradicals of the same polymer molecule) and intermolecular crosslinking. When sufficient intermolecular crosslinks have formed, the solution becomes a gel at a specific gel dose.
  • reaction pH e.g the amount of NaOH used to neutralize HC1 groups of PAA-HC1
  • temperature e.g. the temperature of the polymer before irradiation.
  • the relative amounts of the polymer. NaOH and sugar phosphate may be varied in order to optimize the final morphology and sugar binding properties of the hydrogel.
  • the crosslink density which affects the mechanical properties and the solute transport through the hydrogel, can be altered by varying the total irradiation dose delivered to the aqueous polymer solution and by the number of free amine sites available for cross-linking (by varying the amount of NaOH added).
  • Electrons may be provided by a Cobalt-60 (Co-60) electron beam machine.
  • the number average molecular weight that may be used in the irradiation synthesis of the sugar binding gels may range from 5.000 to 100.000 g/mole.
  • PAA ⁇ Cl has a hydrochloric acid group ionically associated with each amine.
  • the hydrochloride groups of the PAA ⁇ Cl may be neutralized at least partially with NaOH to provide free amine sites for the irradiation crosslinking reaction. It was determined from preliminary irradiation experiments that the minimum dose required to make a PAA hydrogel was 150 kGy.
  • the PAA hydrogel batches crosslinked using the irradiation method may be synthesized as follows: 5.0 g of 50% b.v. PAA ⁇ Cl is diluted in 7.5 ml of distilled deionized water to a 20% b.v. final solution concentration.
  • the water in the petri dish is replaced with fresh water and this washing process is repeated two more times.
  • the salt-free hydrogel slabs are air-dried in an oven at 40-50 °C. The dried gel slabs are then cut into smaller pieces or ground into a powder depending on the experimental needs.
  • the invention also relates to molecularly imprinted, hydrogel polymers, e.g. obtained by crosslinking poly(allylamine) with epichlorohvdrin. ethvleneglycol diglycidyl ether or glycerol diglycidyl ether or by irradiation in the presence of a sugar phosphase such as glucose-6-phosphate.
  • the invention also relates to a method of removing a sugar from a sugar in water solution comprising
  • the invention further comprises:
  • the invention also relates to a method for binding and/or removing one or more sugars from the gastrointestinal tract of a patient, comprising orally administering to the patient in need thereof a molecularly imprinted, crosslinked hydrogel polymer which specifically binds the sugar(s).
  • the invention also relates to a method of treating type II diabetes or a complication thereof, comprising orally administering to a human in need thereof a molecularly imprinted, crosslinked hydrogel polymer which specifically binds one or more sugars whereby the type II diabetes or complication thereof is treated.
  • the hydrogel polymer is crosslinked and specifically binds glucose.
  • Complications of type II diabetes that may be treated according to the present invention include kidney failure, blindness, infections, stroke, diabetic neuropathy and hypertension. All diabetics need to check their sugar levels on a regular basis.
  • the development of an implantable glucose sensor for diabetic patients provides an alternative to the present discrete methods of glucose determination that are based on intermittent blood sampling. Continuous glucose sensing is particularly important for the detection and management of hypoglycemia, by providing a method for early detection of the condition and a basis for insulin administration at more appropriate dosages.
  • An implantable sensor may also be used in parallel with other existing or potential forms of insulin replacement, such as transplantation or hybrid islet devices.
  • the sugar binding hydrogel polymer of the invention responds to changing blood glucose levels and can be used as an implantable subcutaneous glucose sensor.
  • the sensing mechanism involves direct glucose binding to the hydrogel polymer, which results in a pH change detectable by a pH sensor.
  • pH change may be detected with an electrochemical sensor such as disclosed in U.S. Patent No. 5.858.186 or piezoelectric senor such as disclosed in U.S. Patent No. 5.658,732.
  • Alternative sensing methods can be used, such as fluorescence sensing, magnetic sensing, or sensing glucose levels from the saliva.
  • the invention also relates to a method of diagnosis or monitoring the status of a patient having type II diabetes, comprising contacting a body fluid of a person suspected of having type II diabetes with a molecularly imprinted.
  • hydrogel sugar binding polymer of the present invention determining the amount of sugar bound thereto, thereby giving the amount of sugar in the body fluid, and relating the amount of sugar in the body fluid to a diagnosis or status of type II diabetes.
  • hydrogel polymer of the invention examples include blood, serum, cerebrospinal fluid, urine and saliva.
  • body fluids examples include blood, serum, cerebrospinal fluid, urine and saliva.
  • the amount of hydrogel polymer, the time and temperature of incubation, as well as other conditions may be varied depending on various factors including the type of fluid being tested, the concentration of glucose and the like. Other such steps as washing, stirring, shaking, filtering and the like may of course be added as is customary or necessary for the particular fluid.
  • Standard solutions of glucose may be prepared which can be used to prepare a standard curve with concentration of glucose on the abscissa and the detection signal on the ordinate.
  • the results obtained from a fluid sample containing glucose may be interpolated from such a plot to give the concentration of glucose.
  • the invention also relates to a method of treating obesity or a complication thereof, comprising orally administering to a human in need thereof a molecularly imprinted, crosslinked hydrogel polymer which specifically binds one or more sugars whereby obesity or complication thereof is treated.
  • Complications of obesity include myocardial infarction, stroke, and hypertension.
  • patient includes any mammalian patient to which the hydrogel polymers may be administered to achieve a beneficial effect. Foremost amount such mammals are humans as well as nonhuman primates, sheep, horses, cattle, goats, pigs. dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice.
  • compositions comprising the molecularly imprinted, hydrogel polymers are administered in therapeutically effective amounts.
  • a therapeutically effective amount is that amount which produces a desired therapeutic result or ameliorates a condition being treated.
  • a therapeutically effective amount is an amount effective to reduce the level of free sugar able to be absorbed by the gastrointestinal tract.
  • the hydrogel polymers may be prepared for oral administration by methods well known in the pharmaceutical arts.
  • the hydrogel polymers may be administered alone or in admixture with a pharmaceutically acceptable carrier.
  • the carrier may be a solid, semi-solid or liquid material that acts as a vehicle, excipient or medium for the polymer.
  • the compositions may be in the form of tablets, pills, syrups, aerosols, soft or hard gelatin capsules, sterile packaged powders and the like.
  • suitable carriers include without limitation gum acacia, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone. cellulose, methyl cellulose, methylhydroxybenzoate. propylhydroxybenzoate. and talc.
  • the dry gel can be mixed with solid food comprising one or more sugars, e.g. sucrose, to give a confection, and marketed as a "candy bar for diabetics."
  • the dry gel does not have any sugar bound substantially thereto but is admixed with the candy bar.
  • the dry gel is present in a portion of the confection or candy bar apart from a substantial part of the sugar.
  • the gel may be on the inside of the confection while the sugar will be substantially on the outside of the confection. The gel will only interact with the sugar in the candy bar when it is in contact with the stomach fluids, and will bind at least part of the sugar present in the candy bar, before it gets into the blood and affects blood sugar levels.
  • the candy bar will still have a sweet taste, but the amount of sugar present in it will not affect the blood sugar level of the diabetic.
  • the following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered which are obvious to those skilled in the art are within the spirit and scope of the invention.
  • aqueous poly(allylamine) 25 % by weight solution 20 ml of aqueous poly(allylamine) 25 % by weight solution are added to a 50 ml beaker. The polymer solution is then stirred at a moderate speed. 1.5 mL of aqueous glucose phosphate monosodium salt solution, with a concentration of 0.05 grams per mL, is added directly to the stirring polymer. This solution is covered and allowed to stir at a moderate speed for 2 hours. Next. 2.675 ml of
  • the gel is allowed to stand covered at room temperature for at least 20 hours so that the maximum amount of crosslinking can occur. Washing Procedure
  • the gel is cut into squares by drawing a knife through the gel in a grid pattern.
  • the resulting gel pieces are typically about 4 mm square and 4mm thick.
  • the gel is transferred to a 250 ml beaker, and the beaker is filled with 175 ml of 2 Molar sodium hydroxide solution. This mixture is then stirred slowly for
  • NaOH is removed from the gel using the following method: The beaker is filled with enough distilled water to cover the entire gel ( «200 ml). The gel is stirred at a moderate speed for 2 hours, changing the distilled water even' 20 minutes. The pH of the distilled water at the end of the final wash should be below 7.0. In the event that the pH is higher than 7.0, the stirring and changing of the water is continued until the pH falls below 7.0. Lastly, as much solution as possible is poured off of the gel. while retaining all of the gel, and the gel is transferred to a clean petri dish or other container, and air dried in an oven at 50 degrees Celsius.
  • the method used for determining glucose concentration is an enzymatic, colorimetric test produced by
  • a glucose test solution with a pH of 3.9 and a glucose concentration of 50 mg / ml was prepared using distilled water, hydrochloric acid, and glucose. This .92-
  • the solution simulates the conditions found in the beginning of the small intestine after the consumption of an amount of sugar approximately equal to that found in two candy bars.
  • the dried gel in the amount of 2.00 grams, was placed in a 50 ml beaker with 20 ml of the test solution. This solution was then stirred for 4 hours to allow the polymer gel to hydrate completely and bind the glucose from the test solution. The solution was then filtered to remove all remaining gel in order to prevent interference with the final glucose concentration test. The final glucose concentration of the solution was 6.3 mg /ml.
  • the binding capacity of the gel produced in this experiment was found to be 0.436 grams of glucose per gram of dried gel.
  • the molecularly imprinted polymers were prepared using the polymer PAA-HC1 with an weight average molecular weight of 15.000 g/mole and a polydispersity, determined by gel permeation chromatography. of 2.7.
  • the imprint molecule was glucose phosphate mono-sodium salt (GPS).
  • the crosslinkers used to synthesize the MIPs were EPI, EGDE. and GDE. All compounds were obtained from Sigma- Aldrich and were used without further purification. All experiments were performed in aqueous solution, using water from an Elix water purification system by Millipore, model # ZLXS6003Y.
  • the crosslinked. molecularly imprinted PAA-HC1 networks were prepared by the aqueous reaction of 25% w/v solution of linear PAA-HC1 chains and EPI,
  • a typical MIP hydrogel was synthesized as follows: 25% w/v aqueous PAA-HCl solution was mixed with GPS and allowed to stir for 2 hours to ensure complete association of the imprint molecule with the polymer. A portion of the PAA-HC1 amines sites were then neutralized for crosslinking by adding NaOH under stirring. The solution was allowed to stir continuously for 20 minutes before adding one of the three aforementioned crosslinkers. Upon addition of the cross-linker, the polymer solution was stirred until just before complete gelation, in order to maximize the homogeneity of the polymers.
  • the polymerizing solution was poured into a petri dish so that, upon gelation, the MIP hydrogel would set in the form of a slab. After gelation, the polymer was allowed to sit overnight to allow complete crosslinking. The polymer was then cut into 4mm squares and washed in 1 M aqueous NaOH solution for at least 24 hours to remove the GPS imprint. The polymers were repeatedly washed with de-ionized water to remove any remaining NaOH. Finally, the completely washed gels were dried under air in a 50° C oven.
  • Preliminary quantitative analyses were performed to ensure that the imprint binding and removal techniques were effective. In order to determine the effectiveness of the techniques it was necessary to establish an accurate, quantitative detection method for the imprint, GPS. By quantitative determination of total phosphorus concentration, GPS concentration could be calculated by simple molar equivalence. Total phosphorus concentration was determined spectrophotometrically, using a Hach D2010 spectrophotometer and Hach's method 8190. Hach method 8190 is an acid persulfate digestion method used to determine total phosphorus. Newly synthesized hydrogels. still containing the GPS imprint, were placed in de-ionized water and allowed to equilibrate for 48 hours.
  • a filtered aliquot was then taken and diluted appropriately for the Hach total phosphorus test.
  • the pH of the diluted sample was checked to ensure that it was between 6.5 and 7.5, adjusted if necessary using NaOH or HC1, and tested for total phosphorus using the Hach method for total phosphorus.
  • Less than 2% of the GPS was released from the MIPs after equilibrating in water.
  • This same polymer was then placed in a 1 M NaOH solution and equilibrated for 48 hours to remove the bound GPS.
  • a filtered aliquot was then taken, diluted appropriately for the Hach total phosphorus test.
  • the sample's pH was then adjusted using HC1 so that it was between 6.5 and 7.5. This solution was then tested for total phosphorus. Imprint removal by this technique was shown to be as high as 99% with 5% error associated with the Hach total phosphorus test itself.
  • the de-ionized water washes that followed were performed in 1-2 hour intervals, 3-4 times per day. while monitoring the effluent wash pH.
  • the effluent wash water after an overnight equilibration period with the gel. was no longer basic it was determined that the polymers were free of excess NaOH.
  • 5 days of washing the MIPs as described were required to remove all of the NaOH.
  • the molar ratio of compounds used in a typical MIP hydrogel synthesis is given by:
  • Binding capacities of the hydrogels were determined via batch reactor studies.
  • the dried polymers were added to a 50 mg/mL aqueous solution of either glucose or fructose.
  • the 50 mg/mL concentration was intended to mimic the sugar concentration likely to be found in the stomach and duodenum after the consumption of a soft drink or sugary snack. This assumes a stomach volume of 1 liter and a consumption of 50 grams of sugar.
  • the test solution and the MIP or NIP being tested was then allowed to equilibrate while stirring slowly for 240 minutes, whereupon filtered aliquots of the solution were taken to determine the remaining concentration of sugar in the test solution.
  • Equilibrium binding capacities were calculated using the following equation: g sugar bound) ⁇ C I - C f g( MIP) m MIP
  • the glucose and fructose binding capacities of the MIPs presented here were determined using the equilibration tests previously described.
  • Table 1 shows the binding capacities in four different polymers which use ethylene glycol diglycidyl ether, EGDE. as the crosslinking agent.
  • the first three MIPs listed are imprinted with increasing amounts of GPS.
  • the imprint quantity given in the table are mole percents with respect to monomer units of the polymer.
  • the last polymer listed is a NIP and was used as a control sample to determine the effectiveness of the imprinting procedure.
  • Glucose binding in these MIPs was shown as high as 0.56 g of glucose per g of dried polymer gel. Data from similar studies with MIPs synthesized using EPI and GDE as the crosslinking agents are also shown.
  • Table 2 shows the glucose and fructose binding capacity in four different polymers crosslinked with EPI. Again, the first three MIPs in the table contain different amounts of imprint while the last is a NIP control. These polymers were capable of the highest degree of glucose binding at 0.58 g of glucose per g of dried MIP.
  • Table 3 is organized in the same fashion as the preceding tables and summarizes the glucose and fructose binding in the MIPs crosslinked with GDE.
  • Poly (allylamine hydrochloride) chains have an HC1 ionically associated with each of the side chain primary (NH 2 ) groups along the polymer chain backbone.
  • amine sites must be freed of the associated HC1.
  • imprinting no extra steps were necessary since the phosphate group of the GPS is highly negatively charged. This allows the GPS to displace the previously associated HC1 and bond to the amine itself.
  • crosslinking step a portion of the HC1 groups of the PAA-HC1 were neutralized with NaOH to provide free NH 2 sites for the crosslinking reactions.
  • the completion of this neutralization reaction can be monitored by temperature since the neutralization of the HC1 groups is an exothermic reaction. In other words, when the temperature of the mixture returns to the initial temperature, the reaction is complete. For total solution volumes of approximately 25 mL it was determined that 20 minutes is a sufficient reaction time for complete neutralization. For syntheses with total solution volumes of approximately 25 mL, the observed reaction times for complete gelation of EPI, EGDE. and GDE MIPs were 35, 8, and 6 minutes respectively. The differing gelation times are functions of the reaction kinetics and conversion required for gelation for each crosslinker. In order to remove the imprint molecule from the MIPs after crosslinking, it is necessary to break the ionic interaction between the GPS and the amine groups of the polymer. This was accomplished by washing the hydrogel in an aqueous solution of sodium hydroxide for at least 24 hours.
  • crosslinker is also key to forming a suitable imprint in a polymer network. Because of the relatively large amount ( 13 mole% with respect to monomer) of crosslinker used to synthesize these MIPs, the crosslinker must be in intimate contact with the imprint molecule in the MIP network. It is likely that the crosslinker plays a key role in establishing the shape and dimensions of the resulting cavities.
  • the glucose binding observed in the MIPs crosslinked with EGDE behaves as one would expect if the factors affecting MIP design discussed previously are considered. As the amount of imprint present during synthesis increases, so does the binding capacity of the MIPs. This increase in binding capacity is a result of an increase in the number of cavities through which glucose can "pass freely. " The first column of Table 1 clearly shows this trend. In addition to an increase in glucose binding with increasing imprint concentration, we also observed a decrease in the binding capacity of fructose. This trend is an indicator of specificity for glucose. Ideally, as the concentration of imprint increases, so does the number of cavities specific to glucose.
  • the concentration of the test solution should remain constant as both solvent and solute pass freely into the polymer network. This is analogous to simply removing a small volume of the test solution.
  • the test solution's solvent will be absorbed by the polymer while the solute is excluded. This will result in an eventual increase in the concentration of target molecule in the test solution.
  • the MIPs using EPI as a crosslinker were imprinted for glucose and so glucose was bound via appropriately shaped cavities.
  • MIPs and NIPs crosslinked with GDE do not follow either of the trends observed in the polymers crosslinked with EGDE or EPI.
  • Table 3 shows that as the concentration of imprint increases, both the glucose and fructose binding decrease. It is important to note that GDE crosslinked polymers had very poor mechanical stability when compared to the polymers produced by EGDE and EPI .

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Abstract

La présente invention concerne des polymères hydrogels réticulés qui lient spécifiquement un ou plusieurs sucres. L'invention concerne également des procédés de préparation d'un polymère hydrogel qui lie spécifiquement un ou plusieurs sucres. L'invention a également pour objet des procédés servant à lier et/ou retirer un ou plusieurs sucres du tractus gastro-intestinal d'un patient, consistant en l'administration par voie orale à un patient d'un polymère hydrogel réticulé qui lie spécifiquement un ou plusieurs sucres. L'invention concerne également une méthode de diagnostic, de contrôle ou de traitement du diabète de type II ou d'une complication de celui-ci, consistant en l'administration par voie orale à un patient d'un polymère hydrogel réticulé qui lie spécifiquement un ou plusieurs sucres. L'invention concerne également une méthode de traitement de l'obésité ou d'une complication de ladite obésité, consistant en l'administration par voie orale à un patient d'un polymère hydrogel réticulé qui lie spécifiquement un ou plusieurs sucres.
PCT/US2000/027392 1999-10-07 2000-10-05 Polymeres liant le sucre et leur utilisation WO2001025291A1 (fr)

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WO2002085377A1 (fr) * 2001-04-18 2002-10-31 Genzyme Corporation Methode pour reduire le glucose dans le serum
WO2003011308A1 (fr) * 2001-07-30 2003-02-13 Mitsubishi Pharma Corporation Medicaments destines a l'amelioration de l'hyperglycemie post-prandiale
EP1440986A1 (fr) 2003-01-15 2004-07-28 DSM Fine Chemicals Austria Nfg GmbH & Co KG Procédé de séchage en continu de polymères contenant des groupes N- ou amino ou ammonium ou ammonium spirobicyclique
US7261880B2 (en) 2001-04-18 2007-08-28 Genzyme Corporation Methods of treating Syndrome X with aliphatic polyamines
EP1923064A3 (fr) * 2001-04-18 2008-08-20 Genzyme Corporation Utilisation des aminopolymères pour réduire le glucose sérique
US8138289B2 (en) 2007-03-27 2012-03-20 University Of Maryland, College Park Imprinted polymeric materials for binding various targets such as viruses
CN104923191A (zh) * 2015-05-18 2015-09-23 昆明理工大学 一种替代模板的分子印迹吸附萃取搅拌棒的制备方法
CN105566555A (zh) * 2015-12-17 2016-05-11 三诺生物传感股份有限公司 一种胰岛素抗体印迹聚合物及其制备方法、应用

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7261880B2 (en) 2001-04-18 2007-08-28 Genzyme Corporation Methods of treating Syndrome X with aliphatic polyamines
WO2002085377A1 (fr) * 2001-04-18 2002-10-31 Genzyme Corporation Methode pour reduire le glucose dans le serum
JP2010090172A (ja) * 2001-04-18 2010-04-22 Genzyme Corp 血清グルコースの減少方法
JP2004535384A (ja) * 2001-04-18 2004-11-25 ジェンザイム コーポレーション 血清グルコースの減少方法
AU2002257145B2 (en) * 2001-04-18 2005-06-02 Genzyme Corporation Method for lowering serum glucose
US7229613B2 (en) 2001-04-18 2007-06-12 Genzyme Corporation Method for lowering serum glucose
EP1923064A3 (fr) * 2001-04-18 2008-08-20 Genzyme Corporation Utilisation des aminopolymères pour réduire le glucose sérique
WO2003011308A1 (fr) * 2001-07-30 2003-02-13 Mitsubishi Pharma Corporation Medicaments destines a l'amelioration de l'hyperglycemie post-prandiale
US7879869B2 (en) 2001-07-30 2011-02-01 Mitsubishi Tanabe Pharma Corporation Drugs for ameliorating postcibal hyperglycemia
EP1440986A1 (fr) 2003-01-15 2004-07-28 DSM Fine Chemicals Austria Nfg GmbH & Co KG Procédé de séchage en continu de polymères contenant des groupes N- ou amino ou ammonium ou ammonium spirobicyclique
US8138289B2 (en) 2007-03-27 2012-03-20 University Of Maryland, College Park Imprinted polymeric materials for binding various targets such as viruses
CN104923191A (zh) * 2015-05-18 2015-09-23 昆明理工大学 一种替代模板的分子印迹吸附萃取搅拌棒的制备方法
CN105566555A (zh) * 2015-12-17 2016-05-11 三诺生物传感股份有限公司 一种胰岛素抗体印迹聚合物及其制备方法、应用
CN105566555B (zh) * 2015-12-17 2018-05-04 三诺生物传感股份有限公司 一种胰岛素抗体印迹聚合物及其制备方法、应用

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