Definitions

• Sorbents for Removing Toxicants from Blood or Plasma and Method of producing same Technical Field
• the present invention relates to sorbents for removing toxicants from blood or plasma, and also for a method of producing such sorbents.
• Blood represents the most important body fluid which circulates through all organs and tissues thus supplying them with oxygen and numerous required organic and inorganic compounds and transporting carbon dioxide and harmful metabolites towards the lung and excretion organs. Blood contributes to maintaining constant temperature of the body and specific osmotic and acid-base equilibria in the organs. It carries information messengers from one organ to the others and synchronizes their cooperation. Due to the presence of the antibodies, antitoxins and hydrolysing enzymes as well as the ability of leukocytes to encapsulate bacteria and small particles, blood exerts an important protecting function.
• Blood consists of formulate elements (erythrocytes, leukocytes, platelets and others) and plasma.
• the latter contains thousands of proteins, glycoproteins, peptides, hormones and other biologically active compounds which regulate the activity of all organs. Any disease would alter composition and properties of blood since the malfunctioning organ would release wrong metabolites into blood circulation, causing harmful intoxication of other organs and the body as a whole. By removing the intoxicants from blood, general condition of a patent can be improved significantly.
• the body contains about 70 ml blood per 1 kilogram of body weight, so that, on the average, about 5 1 blood in a man or 4 1 in a woman have to be forced to pass 2 to 4 times through the purification device. With the flow rate through the device of about 100 - 150 ml/min, the whole procedure would last for few hours.
• Hemoperfusion involves the passage of the contaminated blood over a solid surface of a detoxicant particulate mass that separates the contaminant by sorption or by ion exchange.
• Another procedure, plasma perfusion involves separation of blood cells prior to contacting plasma with the sorbent.
• treated blood or both cells and treated plasma have to be returned to the patient's blood circulation system.
• selective sorbents can be employed which inco ⁇ orate ligands specially designed to attract and bind the target species.
• sorbents such as Protein-A
• removal of circulating toxins and tumor antigens e.g., a-fetoprotein associated with hepatic cancer, carcinoembrionic antigen associated with various carcinomas, thioesterase or cytokeratins associated with breast cancer, and the like
• sorbents such as immobilized monoclonal antibodies and specific immobilized ligands
• removal of protein bound toxins and drugs e.g., in the case of psychotomimetic or narcotic drug overdose
• Hemoperfusion and plasma perfusion on non-specific activated carbon-type sorbents was shown to be extremely helpful in treatment of schizophrenia (Kinney, U.S. Patent 4,300,551 , 1981 ), multiple sclerosis (SU 1466-754-A, 1989), treatment of rhesus-conflict in obstetrics SU 1533-697-A, 1989), for detoxication of organism of patients who have undergone extensive surgery (SU 1487-909-A, 1989).
• Bodden U.S Patent 5,068,662, December 1991
• high concentrations of anti-cancer agents can be perfused through a body organ containing a tumor and then removed from the organ with effluent blood.
• the contaminated blood is then transported to an extracorporeal circuit, purified from contaminations and returned to the body.
• This permits safe infusion of greater than usual concentrations of chemotherapeutic agents and delivering lethal doses of the agents to the tumor while preventing toxic levels of the agents from entering the body's general circulation.
• the process is applicable to the treatment of a number of tumors such as those of kidney, pancreas, bladder, pelvis and, in particular, the liver.
• chemotherapeutic agents for use in the practice are Adriamycin (doxorubicin), fluorinated pyrimidines (5- fluorouracyl 5-FU or floxuridine FURD), cisplatin, Mytomycin C, cyclophosphamide, methotrexate, vincristine, Bleomycin, FAMT, and any other anti-cancer agent.
• Blood detoxication most effectively can be achieved by hemoperfusion through a cartridge with a non-specific sorbent, for example, activated carbon, able to clear the blood from the above antineoplastic agents.
• the biological defense system of blood may be activated and react in several ways: the blood may coagulate to form a clot,
• Clark U.S. Patent 4,048,064, September 1977 describes formation of a semipermeable polymeric coating on the carbon particles by polymerization of various hydrophilic monomers, in particular hydroxyethylmethacrylate (HEMA) and acrylamide. Moreover, he includes heparin into the coating polymer, in order to minimize complement activation and aggregation of platelets.
• Nakashima, et al. U.S. Patent 4,171 ,283, October 1979 suggests to add an epoxy moiety containing comonomer, which allows post-crosslinking of the polymeric coat formed, thus enhancing the mechanical stability of the coating.
• thin hydrophilic polymeric coatings were found to "fall apart", whereas thick coatings retarded diffusion and deteriorated sorption properties of the carbon.
• thin integral membranes on the surface of the adsorbent can be better prepared from hydrophobic, insoluble in water polymer, like polystyrene, polysulfone, polyether urethane which are coated in an amount of 0.1 -1 % of the weight of matrix from a solution in an organic solvent which is added to the sorbent and then evaporated under vacuum in a rotovap.
• hydrophobic membranes prevent diffusion of toxins into the sorbent bead.
• an additional, water-soluble polymer for example polyethyleneglycol
• polyethyleneglycol has to be added to the system during the coating procedure (in an amount of 0.5-5% of the weight of the membrane- forming polymer), which is then eluted with water, thus leaving pores of about 20 angstroms in diameter in the main hydrophobic coating membrane.
• activated carbon was coated with a polyelectrolyte complex prepared from a polycation (DEAE-cellulose) and heparin and precipitated on the surface of carbon beads (Valueva, et al., SU 844-569, 1981 ).
• porous polymeric hydrophobic materials can serve as non-selective adsorbents; they are, however, less examined.
• Most powerful polymeric adsorbing materials are the hypercrosslinked styrene polymers introduced by Davankov and Tsyurupa in 1969 (Davankov, et al., SU 299165, 1969; U.S. Patent 3,728,467, April 1973; Reactive Polymers, 13, 27-42, 1990). These polymers are prepared by an extensive crosslinking of polystyrene chains with rigid bifunctional crosslinking reagents in the presence of large amounts of a good, from the thermodynamic point of view, solvent. The initial polystyrene is either dissolve in of highly swollen with this solvent.
• the final product represents a low density polymer displaying a large inner surface area (of about 1 ,000 m 2 /g), and the ability to swell with any liquid, including water.
• the hypercrosslinked polystyrene exhibits outstanding adso ⁇ tion properties with respect to organic compounds dissolved in aqueous media (Rosenberg, et al., Reactive Polymers, 1 , 175-183, 1983; Davankov, et al., Reactive Polymers, in press, 1995). This stimulated Schwachula et al.
• a sorbent for removing toxicants from blood of plasma which has a plurality of beads of hypercrosslinked polystyrene-type resin, which beads have a surface modified so as to prevent adsorption of large proteins and platelets and to minimize activation of blood complement system, without affecting noticeably the accessability of the inner adsorption space of the beads for small and middle-size toxicant molecules.
• the sorbent When the sorbent is formed and produced in accordance with the present invention, the sorbent has a good biocompatibility.
• a sorbent for removing toxicants from blood or plasma which comprises a sorbent for removing toxicants from blood of plasma, which has a plurality of beads of hypercrosslinked polystyrene-type resin, which beads have a surface modified so as to prevent adsorption of large proteins and platelets and to minimize activation of blood complement system, without affection noticeably the accessability of the inner adsorption space of the beads for small and middle-size toxicant molecules.
• a method of producing of the sorbent comprises the steps of modifying the surface of the beads of the hypercrosslinked polystyrene-type resin, such that adsorption of large proteins and platelets is prevented and activation of blood complement system, without affecting noticeably the accessibility of the inner adsorption space of the beads for small and middle-size toxicant molecules.
• Modification protocols of the beads surface are identical for microporous and biporous materials.
• groups of phosphatidylcholine are formed on the surface of polystyrene beads, without a preliminary grafting ofthe hydrophilic copolymer suggested by Ishihara, et al. - Second approach consists of depositing heparin on the surface of the polystyrene beads. This can be done in several ways, including (I) chemical covalent binding of heparin to the polystyrene chains on the surface of beads, or (ii) electrostatic adsorption of heparin molecules, which are negatively charged, to positively charged ionogenic groups introduced into the surface layer of the beads. Heparin inhibits activation of the blood complement system and prevents formation of cloths.
• Still another approach consists of binding long hydrophilic polymer chains on the beads surface, which should prevent contacts between blood proteins and cells with the hydrophobic polystyrene surface.
• the fourth approach is depositing high molecular weight fluorinated polyalkoxyphosphazene on the outer surface of the beads.
• Phosphazene represents the best biocompatible polymeric material. Modification of the sorbent surface consists in contacting the polystyrene beads with an appropriate amount of a solution of the polyphosphazene in an organic solvent. Due to the ability of the hypercrosslinked polystyrene to strongly swell with the solvent, the latter appears completely incorporated into the beads after a short period of time, whereas the dissolved polyphosphazene remains deposited on the surface of beads. The solvent incorporated into the beads is then removed by heating the beads under reduced pressure.
• the chemical modification of the surface of sorbent beads which is the case in the first three of the above modification approaches, is facilitated by the remarkable peculiarity of the hypercrosslinked polystyrene, namely, that the reactive functional groups of the polymer are predominantly located on its surface.
• the hypercrosslinked polystyrene is generally prepared by crosslinking polystyrene chains with large amounts of bifunctional compounds, in particular, those bearing two reactive chloromethyl groups.
• the latter alkylate, in a two step reaction, two phenyl groups of neighboring polystyrene chains according to Friedel-Crafts reaction with evolution of two molecules of HC1 and formation of a cross bridge.
• the three-dimensional network formed acquires rigidity. This property gradually reduces the rate of the second step of the crosslinking reaction, since the reduced mobility of the pending second functional group of the initial crosslinking reagent makes it more and more difficult to find an appropriate second partner for the alkylation reaction. This is especially characteristic of the second functional groups which happen to be exposed to the surface of the bead.
• the largest portion, if not the majority of the groups, are located on the surface of the bead (or on the surface of large pores). This circumstance makes it possible to predominantly modify the surface of the polymer beads by involving the above chloromethyl groups into various chemical reactions which are subject of the present invention.
• the examples and associated preparation protocols illustrate the modification of the surface of microporous and biporous hypercrosslinked polystyrene beads prepared by an extensive crosslinking of corresponding styrene-divinylbenzene coppolymers using monochlorodimethyl ether as the bifunctional reagent or using other conventional chloromethylation and post-crosslinking protocols.
• the content of residual pending chloromethyl groups in the polystyrene beads amounts to 0.5 - 1.0% CL for the microporous and up to 7% for biporous materials.
• the beads of the initial material should preferably be spherical and smooth to minimize possible damages to hematocytes.
• the sorbents prepared in accordance with this invention are charged to a column or cartridge for service.
• the column should preferably be provided with an inlet and an outlet designed to allow easy connection with the blood circuit, and with two porous filters set between the inlet and the sorbent layer, and between the sorbent layer and the outlet.
• the column may be made of a biocompatible material, glass, polyethylene, polypropylene, polycarbonate, polystyrene. Of these, polypropylene and polycarbonate are preferred materials, because the column packed with the sorbent can be sterilized (e.g., autoclave and x-ray sterilization) before use.
• the column or cartridge is then filled with a 1 % solution of human serum albumin in normal saline and stored at 4°C.
• the column is washed with 0.9% NaC1 solution to which has been added a suitable anticoagulant, such as ACD-A containing heparin in an effective amount.
• a suitable anticoagulant such as ACD-A containing heparin in an effective amount.
• ACD-A a suitable anticoagulant
• Blood taken from a patient is first separated through a separation membrane, by centrifugation or the like into hemocytes and plasma, the plasma thus separated is then forced to pass through the column packed with the sorbent of this invention to remove toxicants from the plasma; then, the clarified plasma from the column is mixed with the hemocytes separated above, and the mixture is returned to the blood vessels of the patient.
• the modified hypercrosslinked polystyrene-type sorbents of the present invention are intended to replace in hemoperfusion and plasma perfusion procedures all kinds of activated carbons.
• the new material is mechanically stable and does not release fines causing embolia; it is much more hemocompatible, exhibits higher sorption capacities toward a broad range of blood toxicants, and can, in principle, be regenerated and reused.
• modified hypercrosslinked polystyrene sorbents of this invention extends to substances with molecular weights of between 100 and20,000 daltons.
• the maximum adsorption is of molecules
• SUBST ⁇ TI ⁇ SHEET (RULE 26) with weight of between 300 and 1 ,500 daltons, identified clinically as "medium molecules", which are present in abnormal quantities in ureamic and many others patients and are incompletely removed by conventional hemodialysis procedures.
• Such compounds as creatinine, barbiturate, phenobarbital, sodium salicylate, amphetamines, morphine sulfate, meprobamate, glutethimide, etc. can be effectively and rapidly removed from the blood using both microporous and biporous sorbents. (To avoid removal of useful drugs from blood during hemoperfusion on the new sorbents, the latter can be previously saturated with the corresponding drug to an appropriate level).
• the biporous sorbents also shows an excellent ability to absorb cytochrom C and beta-2-microglobulin(molecular weight of about 20,000 daltons) as well as vitamin B12.
• the reaction mixture was then heated for 10 hrs at 80°C, the polymer was filtrated and carefully washed with aceton, a mixture of aceton with 0.5 N HCL, 0.5 N HC1 and water until no chlorine ions were detected in the filtrate.
• the product dried in vacuum represented microporous hypercrosslinked polystyrene. It contained 0.65% pendant unreacted chlorine and displayed an inner surface area as high as 980 m 2 /g.
• the product dried in vacuum represented biporous hypercrosslinked polystyrene and contained 3.88% pendent unreacted chlorine.
• the above extensive crosslinking resulted in the increase of its inner surface area from 120 to 1 ,265 m 2 /g.
• the mixture was heated to 60°C for 4 hrs, kept at ambient temperature for 15 hrs, provided with 5 ml dry pyridine and, after additional 5 hrs, washed carefully with distilled water and rinsed with ethanol.
• the resin was kept in ethanol at 5°C before use.
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Example 2
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• the product of reacting the initial biporous polymer with 2-ethanol amine according to Example 1 was washed with 0.5 1 0.1 N HCl and water, provided with 5ml of aqueous heparin solution (5,000 U/ml) and kept for 15
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• the heparin absorbed on the polymer according to Example 5 was bonded covalently by treating the polymer for 4 hrs with an aqueous solution of glutare dialdehyde (2.0 ml of a 25% solution for 1 g of the wet polymer).
• the pendant aldehyde groups were coupled then with L-aspartic acid (0.2 g
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• the heparin absorbed on the polymer according to Example 5 was bonded covalently by washing the polymer with 500 ml dry methanol, 200 ml dry dioxane and treating it for 5 hrs with a solution of 0.1 g hexamethylene diisocyanate in 3 ml dioxane (for 1 g polymer).
• the polymer was filtered, washed with dioxane and the pendant isocyanate groups coupled with L- aspartic acid by treating the polymer with 1 g tris-trimethylsilyl derivative of L-Asp in 3 ml heptane for 15 hrs at ambient temperature.
• the polymer was washed with heptane, methanol, 0.1 N NaOH and water and kept in ethanol at 5°C before use. Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Example 10 1 g of the product of reacting the initial biporous polymer with 2- ethanol amine and activating it with glutare dialdehyde according to Example 8 was treated with 2 ml aqueous solution of 0.16 g polyethylene glycol (molecular weight 20,000) for 3 days at ambient temperature and then carefully washed with water. Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Example 11 1 g of the product of reacting the initial biporous polymer with 2- ethanol amine and activating it with hexamethylene diisocyanate according to example 9 was treated with 2 ml aqueous solution of 0.16 g polyethylene glycol (molecular weight 20,000) for 3 days at ambient temperature and then carefully washed with water.
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• biporous hypercrosslinked polymer 4 g was allowed to swell with 16 ml of an 8% solution of NaOH in ethylene glycol and then heated to 180°C for 5 hrs, in order to substitute the residual chloromethyl groups with ethylene glycol groups.
• the polymer was washed with ethanol, water, aceton and dried under vacuum.
• 2 g of dry polymer, swollen with dry dioxane, were activated with hexamethylene diisocyanate as described in Example 9, washed with dry dioxane and supplied with a solution of 1.2 g polyethylene glycol (molecular weight 40,000) in 10 ml dry dimethyl sulfoxide, heated at
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Example 13 2 g of the ethylene glycol-modified polymer prepared according to
• Example 12 were activated with glutare dialdehyde according to the procedure described in Example 8 and treated with a solution of 1.2 g polyethylene glycol (molecular weight 40,000) in 10 ml water for 1 day at ambient temperature. The polymer was washed then with ethanol and water. Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Example 14 To 3 g of dry biporous polymer, swollen with dry benzene, were added 15 ml of a solution containing 8 g alcoholate of polyethylene glycol (molecular weight 12,000) in dry benzene and the mixture was boiled under an argon atmosphere and adding small pieces of sodium as long as the latter dissolved in the reaction mixture (about 10 hrs). After additional two days at room temperature, the polymer was carefully washed with ethanol.
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Example 15 According to the procedure described in Example 14, 1 g of the polymer were treated with 1 g of the alcoholate of polyethylene glycol of lower molecular weight (6,000).
• Example 16 According to the procedure described in Example 14, 1 g of the polymer were treated with 1 g of the alcoholate of polyethylene glycol of lower molecular weight (6,000).
• Microporous hypercrosslinked polymer was modified by exactly the same procedure.
• Example 18 0.2 g chitosan were dissolved in 6ml concentrated acetic acid and added to 2 g of dry biporous polymer. After 2 hrs, 10 ml of cold 30% NaOH solution were slowly added to the above mixture, the polymer was separated from the reaction mixture, rinsed with water, dehydrated with methanol, dried and heated to 80°C with 10 ml of a solution of 0.1 g Nal in a dioxane- methanol mixture (5:1 , vol/vol) for 8 hrs, in order to accomplish alkylation of the chitosan amino groups by chloromethyl groups of the polymer. The final product was washed with aqueous acetic acid and then ethanol. Microporous hypercrosslinked polymer was modified exactly the same procedure.

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