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    • 专利摘要:
    The invention relates to polyelectrolytes and their use in the separation of blood proteins. …<??>Blood coagulation factors such as Factor VIII are separated from admixture with other blood proteins without producing activation of said coagulation factor by contacting with a water-insoluble, cross-linked polyelectrolyte copolymer of (a) C2-18 unsaturated monomer and (b) C4-12 unsaturated polycarboxylic acid or anhydride, in which 2-100% of the carboxyl sites are substituted with amineimides and substantially all the free anhydrides are blocked with alkoxyalkylamine to form alkoxyalkylimide units.
    公开号:SU1082338A3
    申请号:SU782640948
    申请日:1978-07-24
    公开日:1984-03-23
    发明作者:Эдвард Фильдс Джосеф；Джэксен Слокомб Роберт
    申请人:Монсанто Компани (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to the fractionation of blood, in particular, to methods for isolating a specific VIII factor. The process of blood coagulation is a complex physiological event involving the interaction of many substances contained in ordinary whole blood. It is known that in some organisms certain factors are absent or are contained in too small a quantity related to the blood coagulation mechanism. In patients suffering from the classic form of hemophilia, antihemophilic factor A (AGF, USH factor) is insufficiently contained. In the body of patients suffering from hemophilia B, the plasma-thromboplastin component (PTK, factor IX) is absent in the blood. A small number of people suffering from hemophilia also lack the so-called von Willebrand factor related to the USh factor. There are a number of other factors that are known: an important role in the mechanism of blood coagulation, the absence of which can also lead to bleeding disorders, this is, for example, factors II, UE and X. The last three factors, together with factor IX, are often called factors prothrombin complex. In the system of development of modern blood storage programs, including the collection and storage of large quantities of blood and its components, working out proper preservation systems is very acute. Starting from the period of the Second World War, blood was stored in a solution of sodium citrate and dextrose citrate, known as ACD-bPood. The problem of blood preservation is greatly simplified if the bottom is reduced to preserving individual blood components, since it is easier to achieve the conditions required for this component than for whole blood. Moreover, it is completely unnecessary and even harmful to introduce into the patient's organism a greater number of blood components than is required. Thus, those suffering from hemophilia and requiring certain blood clotting factors are best introduced only by these factors or, at least, by a purified Concentrate of these factors. The fractionation of blood coagulation factors, in particular the VIII factor and factors related to the prothrombin complex is known. Among the various compounds that are used in this fractionation, for example, barium sulfate, aluminum hydroxide, polyethylene glycol, rivanol- (6.9 -diamino-2-ethoxyacridine) glycine, DEAE-cellulose and DEAE-Sephadex ij. However, this method is multistage and therefore time consuming. The closest in technical essence to the present invention is a method for isolating various compounds. blood, including coagulation factor, from serum or blood plasma using a non-soluble, cross-cell polyselectrolyte. The method involves contacting the source liquid with water-insoluble cross-linked polyelectrolyte - a copolymer of maleic acid or its anhydride and an unsaturated monomer selected from the group consisting of ethylene, styrene and isobutylene, with a degree of substitution of carboxyl or anhydride groups with aminoimides selected from the group consisting of of the lower alkylaminone-lower alkylimide and lower alkylimino di (lower alkylimide), where the alkyl contains 1-5 carbon atoms, equal to 2-100% of the total number of these carboc yl or anhydride groups in the copolymer, followed by elution. The isolation process is based on the adsorption interaction of the isolated protein product and the polyelectrolyte used, having electrostatically charged groups in its structure. However, when fractionating the USh factor by certain poly-electrolytes in a known manner, it was found that the use of isolated blood factors in standard experiments for determining the clotting time causes a significant reduction in the clotting time. This preactivation of the isolated component, the USh factor, represents a serious disadvantage in the use of this material in clinical practice. Such premature activation is also observed in dogs after intravenous administration of this factor into the dog's body. Reactions include the following symptoms: change in behavior — the dog spins in its cage for 24 hours; hemolysis reaction, which is manifested in urinary hemoglobin release; a marked reduction in the number of platelets, while simultaneously increasing blood pressure. This premature activation was found to be related to the structure and composition of the adsorbing polyelectrolyte, which may contain free hydroxyl groups capable of forming hydrogen bonds. So, poly-
    the electrolyte may contain the following structural polymeric zyen:
    sh
    Sshivan} sh, and group
    where R is alkyl with 1-5 carbon atoms;
    Z is a bivalent hydrocarbon residue with 2-18 carbon atoms. According to a known method, the polyelectrolyte may contain 2-100% amino-imide bonds, and the remaining carboxyl groups in anhydride form. Residual non-modified polymeric anhydride units transform into neutral groups or units by reacting unreacted anhydride units with compounds such as alkylamines, amino alcohols and alcohols. In the structural formula given, functional amino-imide bonds are shown at G, cross-linking bonds at III, and the remaining carboxyl groups in anhydride form are shown in the state of conversion to neutral groups by blocking or interaction with an amine alcohol at II, causing formation of oxyalkyl imide units.
    In spite of the indicated blocking of residual anhydride groups with an amine alcohol at II, it was found that the free hydroxyl groups introduced in this polymer into the polymer skeleton as oxyalkylimide groups at II (see Formula) contribute to the above-mentioned extremely active activation of VIII fraction fractionated by this type polyelectrolyte.
    It is assumed that the activation
    Factor VIII can be partially associated with the presence of plasma zymogens or enzymes from zymogen activation in an isolated fraction of VIII factor. These zymogens like
    5 is known to be activated by negatively charged surfaces or hydroxylated surfaces that can form hydrogen bonds, such as collagen. These polymers containing free hydroxyls are capable of forming intra- and interchain bonds with proteins through the formation of negatively charged BOHs.
    The aim of the invention is to establish the preactivation of the USh factor in the isolation process.
    This goal is achieved by the method of isolating a specific VUS factor from serum or plasma of red by contacting the initial liquid with a water-insoluble cross-linked polyelectrolyte copolymer of maleic acid or its anhydride and an unsaturated monomer selected from the group consisting of ethylene, styrene and isobutylene. with a degree of substitution of carboxyl or anhydride groups with amino-imides selected from the group
    0 consisting of a lower alkyl-aminone-lower alkylimide and lower alkyl imino- (lower alkyl) imide, where alkyl contains 1-5 carbon atoms, equal to 2-10% of the total number of these carboxyl or
    anhydride groups in the copolymer, followed by elution, the above copolymer being reacted with an alkoxyalkylamine, in which the alkoxy group and alkyl contain 1-5 carbon atoms, taken in an amount of 50-85 mol%.
    Typically, said polymer is reacted with methoxypropyl Min or methoxy ethylamine.
    The interaction of the source liquid with polyelectrolyte is carried out at a pH of 5.5-9.5. Thus, it is highly preferred to convert unreacted anhydride units during the preparation of alkoxyalkyl imide units using alkoxyalkylamine, for example, methoxypropylamine. This anhydride blocking can be carried out in any sequence of polyelectrolyte synthesis.
    I
    Polymers of type EAMK (ethylene anhydride of maleic acid or the acid itself) based on carboxylic acids or their anhydrides, in particular olefinic polymers based on maleic acid or its anhydride of the specified type. Copolymers are obtained by reacting ethylene or another unsaturated monomer or their mixtures with acid anhydride in the presence of a peroxide catalyst in a solvent based on an aliphatic or aromatic hydrocarbon that dissolves the monomers, but does not dissolve the resulting intermediate polymer. Benzene, toluene, xylene, chlorinated benzene and the like should be referred to the corresponding solvents. Benzoyl peroxide is usually the preferred catalyst. Other peroxides: acetyl, butyryl, dithitic butyl, lauryl, etc. or one of a variety of aeosalysts is also satisfactory due to their solubility in organic solvents. The copolymer contains mainly equimolar amounts of the olefin and anhydride residue. It has a degree of polymerization of about 8-10000, preferably about 100-5000 at mol. mass of about 1000-1000000, preferably about 10000SOOOOO. The properties of the polymer, such as molecular weight, are controlled by the choice of catalyst and the adjustment of one or more variables, such as the ratio of reagents, temperature, catalyst concentration, or the addition of chain transfer regulating agents such as druzopropylbenzene, propionic acid, alkyl aldehydes and etc.
    After the initial polymer EAMK has been formed, it is aggregated by heating in an inert organic solvent at a temperature of 155-160 ° C, but below the softening point of the polymer, for about 15 minutes. As a result of this aggregation, the filterability of the quality during drying and the physical properties of the polymer used in the production of polyelectrolyte are improved without significantly reducing its ability to absorb protein.
    However, for the proposed method, this aggregation does not play a significant role.
    The polymer originally aggregated, whether aggregated or not aggregated, is a cross-linked product, substituted by aminoimide groups in a sequence optimizing the properties of the product, which is accomplished by proper distribution of specific groups within the particles themselves.
    These groups are generally basic and may be aliphatic or aromatic. Preferred aliphatic unbranched rpynnaMH are di-lower alkylamino-lower alkyl imidines or lower alkyl-imine di-lower alkyl imide.
    The starting copolymers of anhydrides and another monomer can be converted to carboxyl-containing copolymers by reacting with water and their salts, C1MMONIUM, alkali and alkaline earth metals and alkylamine salts by reacting with alkali and alkaline earth compounds, amines or ammonium. Other useful derivatives of these polymers include alkyl or other esters, alkylamides, dialkylamides, phenylalkylamides or phenylamides, obtained by reacting carboxyl groups on the polymer chain with selected amines or alkyl, or phenylalkyl alcohol, as well as amino esters, oxyamides and oxyethers. wherein the functional groups are separated by alkylene, phenyl, phenylalkyl, phenylalkylphenyl or alkylphenylalkyl or other aryl groups. A link containing amines or amino salts, including quaternary salt groups, is usually obtained by reacting carboxyl groups of their anhydride precursors, where possible, with polyfunctional amines, such as dimethylaminopropylamino, at elevated temperatures to form an imide bond with the vicinal carboxyl groups. Such additional free amino groups can then be converted to their simple or quaternary salts. Partial imides of the starting polymer, for example of the EAMK type containing carboxylic acid or its anhydride, are obtained by heating a limited amount of secondary or tertiary amino lower alkyl amine with an anhydride or carboxyl-containing form of the polymer in an appropriate solvent {for example, xylene) at a temperature of about 140150 C until no more water is removed. This reaction simultaneously gives imide groups according to the amount of amine added; anhydrite groups are again formed for the residue of polymer units. Thus, imide-polymer products are formed, for which the content of 2-100% imide bonds is characteristic, and if residual carboxyl groups are formed, then in the form of anhydrides. Alternatively, the partial amide polymer product can be converted to a partial imide product by heating the partial amide polymer product in vacuum at 140-150 ° C until the water returns. Such an imide polymer product as well. contains comparable amounts of imide and anhydride groups depending on the number of amide groups originally contained in the starting amide polymer product. Partial secondary or tertiary amino lower alkylamides of the starting polymer, for example, type EAMA containing carboxylic acid or its anhydride, are obtained by reacting the polymer with a limited amount of the selected amine, suspended in a solvent such as benzene or hexane, to form a derivative of the polymer / containing partial amide and acid anhydride, or its corresponding amidcarboxylate derivative. The number of amide groups depends on the amount of amine used in comparison with the amount of polymer used. Such amide-polymer products usually contain 2-100% of the groups, with the remaining carboxyl groups being in the form of acid or anhydride groups. If necessary, blocking and deblocking can be carried out: Nie amine level of the reagent used in the preparation of amines or imine. Residual unmodified polymer units can be converted to neutral groups or units by reacting with certain non-functional groups, such as alkylamines, amino alcohols, and alcohols. Alternatively, an additional cationic character can be achieved in the polymer by introducing monomers that impart a basic or cationic character, such as C-vinylpyridines, vinylamine, various amino-substituted vinylbenzenes (or toluenes, etc.), amino-containing acrylates (or methacrylates, etc.) , vinylimidazole and similar monomers of the same kind. Thus, in any case, the polymer product will contain residual active reactive groups, which may belong to various types, including mixtures, however, these active or reactive groups or residual sites inside the polymer will in some way contain a certain percentage basic groups for the purpose of giving the polymer the necessary basicity. In particular, preferred polymers that meet these requirements and properties are selected from the group consisting of copolymers containing ethylene maleic acid or its anhydride, styrene maleic acid or its anhydride, methylpentene maleic acid or its anhydride, and isobutylene maleic acid or its anhydride. As can be seen from the above, the main groups of the poly-polyionic or polyampholytic electrolyte (PE) have an imide character, including the di-alkylamino-lower alkylimide groups obtained by reacting the di-alkylamino-lower-alkylimide with the carboxyl groups of the previously obtained polymer or the polymerization of unsaturated olefin with unsaturated olefin acid or its anhydride, containing also previously obtained imide groups in an amount of not less than a part of the unsaturated reagent - polycarbonate howl acid. Imide groups can be crosslinked by a polymer using lower alkylimino-5c - (lower alkylamine) which, during crosslinking by the interaction between the final amino groups of the crosslinking agent and the carboxyl groups in the polymer chain, forms imido groups on both
    ends of the crosslinking chain to form the desired lower alkylimino-Su (lower alkylimide) bonds. Other groups, such as di-alkylamino-lower alkylamide groups, from which the required imide groups can be obtained by heating at elevated temperatures, can also be present. Dimethyl alkyl-amino lower alkyl ester groups can be present, as well as other groups, if the required percentage amounts of imide groups of the required type are present in the polyelectrolyte molecule, as well as the residual acid groups of the initial unsaturated acid or its anhydride if the polyelectrolyte is a polyampholyte. Acid and imide groups do not necessarily have to be present in the polyelectrolyte, they can be present in the form of their simple derivatives, for example, salt.
    Alicyclic or aromatic groups which may be substituted on aggregated polymers of the type EAMK, for example, include amino lower alkyl pyridine, piperidine, piperazine, picoline, pyrrolidine, morpholine and imidazole.
    These groups can be substituted on an aggregated polymer in a manner similar to amines with an aliphatic chain, but using, for example, the following compounds instead of cyclic amines:
    2-aminopyridine;
    2-amino-4-methylpyridine;
    2-amino-6-methylpyridine;
    2- (2-Aminoethyl) -pyridine;
    4- (Aminoethyl) -piperidine;
    3-Amino-N-ethylpiperidine;
    N- (2-Aminoethyl) -piperidine;
    N- (2-aminomethyl) -1-ethylpyrrolidine;
    3-picolylamine;
    4-picolylamine;
    2- (Aminoethyl) -1-ethylpyrrolidine;
    N- (3-Aminopropyl) -2-pyrrolidine;
    N- (2-Aminoethyl) -morpholine;
    N- (3-Aminopropyl) -morpholine;
    4-imidazole.
    Anhydride blocking groups can be introduced in any desired sequence during the preparation of the above polyelectrolytes. Preferably, the nx is introduced after cross-linking and replacement with the desired amino-amide functional group. Thus, any free anhydride group can be blocked by using an excess of a blocking agent. .
    The most preferred blocking agents are methoxy.
    propylamine and methoxyethylamine. Anhydride blocking agents, such as oxypropylamine and hydroxyethylamine, are ineffective in preventing the pre-activation of blood coagulation factors fractionated by polyelectrolytes. Other agents characteristic for blocking the hydroxyl group may find use instead:
    0 alkylating (for example, diazomethane), acylating (for example, acetyl chloride) and esterifying into esters (for example, acetic acid) agents,
    5 Example. In a 5 liter flask equipped with a reflux condenser, a Dean-Stark trap, a stirrer, a tank for adding reagent, a thermometer and equipment
    Q for nitrogen purge load
    193.05 g of a copolymer of the type EAMK containing ethylene and maleic anhydride (1.5 mol, anhydride base) and 2,700 ml of xylene.
    5 The mixture was stirred at a speed of 200 rpm using a stirrer equipped with 6.5 inches of blades and heated to reflux temperature, which ranged from 135-139 ° C depending on the water content in the AMOC and azeotropic removal of water during the period reflux. The slurry is treated with reflux for 60 minutes with full age reflux at 135 ° C.
    5 After one hour, the reactor is cooled to 125 ° C. under a nitrogen atmosphere by adding 7.66 g (0.075 mol) of dimethylaminopropylamine (DMAP). The mixing is continued for 1 hour.
    0 at 125 ° C without reflux, after which 10.89 g (0.075 mol) of methylimino-bos-propylamine (MIBPA) is added, after which the entire mixture is stirred for another 1 h at.
    5 The mixture is then heated under reflux (134 ° C) and maintained at this temperature for 7 hours, removing the water continuously with azeotropic distillation. Final temperature
    138 ° C. A total of 5.3 ml of water is collected. The temperature of the sludge is reduced to 115 ° C, then adding 127.02 g (1.42 mol) of methoxypropylamine (MOPA). The mixture is stirred for 1 h without refluxing at 115-J18c, after which the temperature is raised to 120 ° C at the time of the onset of xylene-water azeotropic reflux. Reflux is maintained with bh, removed by continuous azeotropic distillation of water at a final temperature of 138 ° C and yield of 23.6 ml of water.
    The product is isolated as the free amine by the following method.
    The specified target slurry was filtered hot (100 ° C), the cake was again dissolved in 2700 ml of a mixture (3: 1) of xylene with ethanol, stirred for 1 hour under reflux and filtered. This step is repeated a second time using an extraction mixture of 3: 1 xylene with alcohol. It is then filtered again, the pulp is redissolved in xylene (2700 ml) and stirred for 1 hour at reflux temperature, followed by filtration. Extraction with xylene is reversed again, the target cake is again dissolved in 2700 ml of hexane for 1 hour at room temperature and filtered. The extraction with genesan is repeated 3 more times, after which the target cake is dried in air in a roller mill with a rotating perforated bottom for 1 hour and passed through a 100 mesh screen without crushing and dried under vacuum overnight at 50 ° C. 165 g of fine product passing through a 100 mesh opening and 14 g of a rougher product are not obtained. passing through these holes. The thin product was tested by detecting the presence of 8.09% nitrogen and was immediately dispersed in O, 04-molar brine without difficulty. The pH of such a dispersion (0.2 g of product in 20 ml of O, 04-molar brine) is 8.10. Example2. In order to process as the hydrochloride salt, the target reaction slurry of example 1 is filtered hot, after which it is again dissolved in reverse; using a 3: 1 mixture of xiolol and alcohol twice, again, two are refluxed times in xylene as in Example 1 and then extracted twice at room temperature for 1 hour each time in portions
    Table 2700 ml of acetone. The filtered product is converted into the hydrochloride by re-dissolving in 2700 ml of acetone, followed by the gradual addition of 14 ml of concentrated 12N with stirring over 10 minutes. hydrochloric acid. Then add 2 more hours at room temperature. The filtered product is then immediately washed (in the case of sludge with stirring) three times with 10 liters of deionized water for 2 hours each time and finally filtered. The salt remaining on the filter is re-dissolved 4 times in 2700 ml of acetone (each time for 1 hour) to remove water, filtered, air-dried for 30 minutes. and then in a vacuum oven at 55 ° C. The dried end product is passed through a sieve without crushing, with 95% of the product passing through 100 mesh openings; then bottled. Analysis: 7.79% nitrogen and 2.62% chlorine - ionic chloride. 186 g of product are obtained in the form of salt, passing through 100 mesh openings. It is dissolved in a 0.04 molar brine (5 g per 120 ml); The pH of the dispersion is 3.85. Example 3: Example 1 is repeated, changing the composition of the product by adding different amounts of amines of Example 1. The procedure is the same as Example 1, but in no case is water added to the amines to facilitate the process. . All products are treated as free amines, as in Example 1. The first 11-hour reflux period of the EAMA in xylene sludge is carried out with a full reflux condenser as in Example 1 (without removing water) or according to the table (with azeotropic reflux distillation of all water) The results of the synthesis in examples 1-3 are shown in table 13.
    During run 8, MIBPA is added in two consecutive portions, each for a 1-hour reflux period, as a two-step cross-linking variant before removing water from the condensation reaction from DMAPA and MIBPA.
    EXAMPLE 4 If the initial 1-hour period of refluxing of the EAMA in ethylene is carried out under azeotropic distillation conditions, i.e. Having removed all the water from these reagents before the subsequent amine reactions, 40 then the latter proceed with a slowdown. As a result, the total completion time of the process reactions is increased. By adding small amounts of water with various “1” amines to this problem 45
    14
    1082338
    Continued table. one
    can be solved. Using the method of Example 1, several runs were carried out, but at runs 7, 8, 10 and 11, water was removed from refluxing EAMA in xylene for the first hour, as in Example 3, (Table 1). In these runs, water is additionally added with 3-amines. All runs are treated as the free amine of Example 1. The results of the synthesis of Example 4 are given in Table 2.
    table 2
    MIBPA is added before DMAPA; in all other cases, vice versa; HMDA is hexamethylenediamine, used as a crosslinking agent instead of MIBPA.
    Example 5. The equipment of example 1. Download 193,05 g (1.5 mol EAMK and 2700 ml of xylene, heating all with reflux condenser (137 ° C with stirring at 200 rpm, after which the reflux is kept for 1 h. During this time all the water is continuously removed from the mixture by azeotropic distillation. In the Dean-Stark trap, the total amount of water is collected (1.6 mp. After an hour, the sludge is cooled to add 7.66 g (0.075 mol) DMAPA and keeping the mixture for 1 hour at 125 ° C, after which a mixture of 21.70 g (0.15 mol) of MIBPA and 1.5 ml of water is added. The mixture is kept for another 1 h at 125 ° C, then it is heated with a reverse chill nickname (and kept at this temperature, water is continuously removed with a Dean-Stark trap,
    0.5
    206 1.0 207 2.0 214 4.0 219
    Continued table. 2
    within 15 h, collecting a total of b, 3 ml of water at a final temperature of 138 ° C. The slurry is cooled to 125 ° C, then 113.65 g (1.25 mol) of MOPA is added, keeping the resulting slurry for another 1 hour at. The temperature is then flashed until it continues to remove water by azeotropic reflux for 6 hours to obtain 21.5 ml. The product was converted to the hydrochloride salt according to the method of Example 2. 4 runs were performed using different amounts of concentrated 12N. hydrochloric acid to determine the effect of excess and insufficient amounts of hydrochloric acid on the properties of the product.
    The results of the synthesis in example 5 are given in table 3.
    Table 3
    8.24
    1.77
    1.70 8.10 3.00 2.76 8.22 3.32 8.21
    3.37 3.40
    Example The method of Example 5 is repeated, except that after the interaction of DMAPA and MIBPA, 85.77 g (1.27 mol) of MOEA (methoxyethylamine) are added instead of methoxypropylamine (MOPA). Get
    Example 7. The method of Example 1 is used to obtain products in which the excess anhydride groups remaining after the interaction of DMAPA and MIBPA are fully reacted with either hydroxyethylamine or 3-hydroxypropylamine or 2-hydroxypropylamine (Table 5). Products are also obtained as free amines (using 585 (113 g) -7 (15.25 g) 81 (85.2 g) Ethanolamine 781 (85.2 g) - Note
    2 products, one in the form of free amine by treatment with the methods of example 1, the other in the form of the hydrochloric acid salt by treatment with the methods of example 2. The results of the synthesis of example 6 are given in table 4.
    Table 4
    methods of example 1), and as a salt of 5 hydrochloric acid (using the methods of example 2). The first hour of the aggregation period of EAMA in xylene under reflux is carried out with aerootropic removal of water, in each case MIBPA is added as a mixture with 1.5 ml of water.
    The results are shown in table.5. Table 5 e: All molar ratios of the compositions in this and other examples in terms of amine; Since the crosslinking agent interacts at both ends, its molar amounts are twice as large as those calculated on an anhydride basis. she was 7.75 1.85 Free 8.63. amine her, 49 2.57
    Example A whole range of polyelectrolytes is obtained in which the content of the crosslinking agent (MIBPA) is maintained at 5 mol%, and the blocking amine / reacting with an excess of anhydride groups, in each case is ethanolamine at a concentration of 85 mol%. The functional unit that performs the potential cationic function is contained in an amount of 5 mol-0, but the nature and structure of the functional unit is varied by using 16 different reactive amines.
    In all cases, equipment is used, EAMA and xylene according to example 1. The sludge is heated to 9095 ° C by adding 10.89 g {0.075 mol MIBPA with stirring for 1 hour at
    Dimethylaminostilamine
    Diethylaminoethylamine
    Dimethylaminopropylamine
    Diethylaminopropylamine
    Dioxyethylaminopropylamine
    Dibutylaminopropylamine
    2-amino-5-diethylaminopentane
    2-aminomethyl-1-ethylpyrrolidine
    Z-Amino-Y-ethylpiperidine
    N-2-Aminoethylpiperidine
    Z-Aminopropyl-2-pyrrolidone
    N-2-Aminoethylmorpholine
    N-3-Aminopropylmorpholine
    N-2-Aminbethylpiperazine
    2 {2-Aminoethyl) pyridine
    N-Phenylethylenediamine
    Э5®С. Then 0.075 mol of the functional amine is added and stirred for 1 hour at 95 ° C. The slurry is then heated under reflux (134 ° C), completely removing water by azeotropic distillation at a final temperature of 139 ° C. Then the slurry is cooled to 95 ° C by adding S7.05 g of hydroxyethylamine and stirring the slurry for 1 h at. Then the slurry temperature
    0 is raised to 134-C, completely removing water from the reaction by azeotropic distillation to a final temperature of 139-140 ° C. The target slurry is filtered in hot form and processed 5 is melted in the form of free amine according to the method of Example 1, dried, crushed in a ball mill, and 100 mesh is passed through the holes. The results of the synthesis are given in table 6.
    Table 6
    Run amines 1-7 are examples of a dialkylamino imide substitution in a polyelectrolyte, while run amines 8-15 are a glitch example of a retrocyclic aminoalkyl imide substitution. For evaluation, 3 of them are treated as the hydrochloric acid salt. The method of example 1 is used and, when converted to salt, work according to the method of example 2. Dimethylaminopropylamine (run 3 above) is chosen as an example of dialkylamino-aminopropyl-85-hydroxyethyl-3-amino-N-ethylpiperidine
    The same 5 5-3-Aminopropil-85-Oxyethylamine, morpholine
    5 Same
    PRI me R 9. Upon receipt of the hydrochloride salts of polyelectrolytes of general composition (5 mol.% Of a MIBP cross-linking agent, 5 mol.% Of three different functional aminoalkyl imide units and 85 mol.% Of nonfunctional imide units) work according to the method of examples 1 and 2 ( see tab.7).
    I'll try it on. The methoxypropylimide derivatives of a copolymer containing styrene and maleic anhydride are obtained as in Example 5. The equipment is the same as in Example 1. nepBOHa4anbHiTo mixture 101.1 g
    (0.5 mol) of styrene-maleic anhydride and 2.7 l of xylene are heated under reflux at 135 ° C, azeotropically distill the water for 1 h and then cooled to 125 ° C; 2.55 g then added
    (0.025 mol) DMPA, heating the mixture without refluxing refrigerator 1 h.
    alkylimide substitution, and 3-aminoN-ethylpiperidine and N-3-aminopropylmorpholine (runs 9 and 13 above) as an example of a heterocyclic C1-aminoalkylimide substitution. In all three cases, polyelectrolytes in the form of a salt are obtained using hydroxyethylamine and methoxypropylamine as a tertiary amine, which interacts with all the excess anhydride groups. These products are given in table. 7
    Table 7
    1.81
    236
    8.41
    amine
    85-Methoxypro 186
    2.62 7.79 pilamine
    1.54
    85 Oxythylamine 238
    7.37
    85-Methoxypropyl-244 8.19 1.20 amine
    85-Methoxypro- 234 7.58 1.59 Pilamine
    Then a mixture of 2.91 g (0.025 mol) of hexamethylenediamine (SCCA) and 1.0 ml of water is added, continuing to heat at reflux 1.4. After an hour, 2.55 g DMAPA (0.025 mol) was added again and the mixture was heated at 125 ° C for 1 hour. The slurry is then heated to 135 ° C under reflux for 4 hours with azeotropic distillation of water (1.25 ml collected). After cooling to 125 ° C, 40.56 g of methoxypropylamine is added, then the sludge is heated to 125 ° C for 1 hour. The slurry is then heated under reflux () with azeotropic distillation for 6 hours, thereby removing 8.2 ml of water using a Dean-Stark trap. The product is obtained as the hydrochloride salt using the method of Example 2 and 5.0 ml of concentrated 12N. hydrochloric acid. Target 5 washed product is 113.0 g 250 8.55 1.68
    and analysis contains 5.27% nitrogen and 1.00% chlorine.
    Example 11. Methylesipropylimide derivatives of a copolymer containing isobutyl anhydride of maleic acid, as in Example 5, are obtained using the equipment of Example 1. The initial mixture of 154.16 g (1.0 mol) of maleic acid and 2.7 l of xylene is treated with reflux at 135 ° C with azeotropic removal of water for 1 h, then cool to. Thereafter, 5.11 g (0.05 mol) of DMAPA is added, heating the slurry for 1 hour at 120125 ° C without a reflux condenser. To this sludge, 7, .26 g (0.05 mol) of MIBPA and 1, O ml of water are added, after which the mixture is again heated for 1 hour at 120-125 ° C without a reflux condenser. Thereafter, the slurry is heated under reflux (135 ° C), removing water by azeotropic distillation for 4 hours. The final temperature is 137 ° C, the amount of water taken is 1.20 ml. After the temperature of the slurry has decreased to 120, 84.7 g of methoxypropylamine is added, the mixture is then heated without reflux at 120-125 ° C for 1 hour. After that, the temperature of the slurry is raised to reflux (135c) and the azeotropic distillate is removed for 6 hours. tie. The final temperature, the amount of water collected is 16.3 ml. The product is obtained as a hydrochloride salt using the method of Example 2 with 10.0 ml of concentrated 12N. hydrochloric acid. The target washed and dried product is 186 g and contains 6.44% nitrogen and 1.25% chlorine.
    Example12. A 5 L flask equipped with a reflux condenser, a Dean-Stark trap, a stirrer, a tank for adding reagent, a thermometer and equipment for purification with nitrogen gas, was charged with 193.05 g of a copolymer of the type EAMK containing ethylene anhydride maleic acid (1.5 mol. in terms of anhydride) and 2700 ml of xylene. The mixture is stirred at 200 rpm using a paddle stirrer (6.5-inch) with heating to reflux temperature. At this temperature, the sludge is kept for 60 minutes with a full reflux of n 135 ° C. After 1 hour, the reactor is cooled to 125 ° C under nitrogen, then a mixture of 10.89 g of MYBPA (0.075 mol) and 1.5 ml of water is added. The mixture is then heated under reflux (134-sec) and kept for 1 h at this temperature, continuously removing water azeotropically until the end
    Noah temperature 137 ° C. The temperature of the reaction mixture is again lowered to 125-s under nitrogen atmosphere, then a mixture of 153.3 g (1.5 mol) of DMAP and 4.5 ml of water is added. Then heated to 133-s, keeping at this temperature for 1-10 minutes before the start of reflux. Water is formed as a result of the reaction. Stirring and heating with reverse
    Refrigerator is continued until the azeotropic distillation of water is completed. The final temperature is 139C.
    To obtain the hydrochloride salt, the sludge is filtered hot, re-solution of the cake in 2700 ml of a 3: 1 mixture of xylene and alcohol, stirring for 1 hour under reflux and filter in a hot form. This procedure is repeated on refluxing again for 2 hours and a third time for 3 hours, and after each run it is filtered in a hot form. The obtained extracted product is again dissolved in 2700 ml.
    5 acetone for 1 hour at room temperature and filtered, after which the cake is again dissolved in 2700 ml of acetone for 1 hour at room temperature and filtered. This extracted meal is dissolved in 2,700 ml of alcohol at room temperature and converted into the hydrochloride salt by adding 112 ml of concentrated 12N over 10 minutes. the hydrochloride salt with stirring at room temperature for 2 hours. The filtered product is then washed (with the presence of sludge with stirring) three times with 10 liters of deionized water (each time 2 hours) and filtered. The salt cake was dissolved, 4 times in 2700 ml of acetone, one hour each time to remove water, filtered, air-dried for 30 minutes and in a vacuum oven at 55 ° C overnight. The target product consists of 333 g of product (not crushed) passing through the openings of 100 mesh, containing 10.65% of nitrogen and 13.03% of chlorine in the form of chloride ion.
    Example 13. Using the equipment and method of example 1, a polyelectrolyte sample sample is prepared containing only links of the MIBPA and MOPA cross-linking agents, i.e. without DMAPA or other functional amines.
    A mixture of 193.05 g of EAMA and 2700 ml of xylene is heated under reflux for 1 hour to remove water at 135 ° C in an amount of 1.2 ml. This slurry is cooled to 125 ° C by adding 5 then 13.37 g (0.150 mol) of MOPA
    with stirring at 125 ° C for 1 hour. After this, in two steps, add; MIBP - snachbsha in an amount of 21.79 g (0.150 mol) and 1.5 MP water with stirring for 1 hour without reflux at 125 C, followed by raising the temperature to 135 ° C and removal of water azeotropically under reflux for 4 hours and was removed. 7.4 ml of water are used. After cooling to 125 s, a second portion of MIBPA is added in the amount of 21.79 g (0.150 mol) and 1.5 ml of water with heating the slurry at 125 ° C for 1 hour without refluxing the condenser. At 183s, a second process of water removal is carried out with azeotropic refluxing, removing 5.7 ml for 7 hours. The slurry is again cooled to 125 seconds by adding 60.23 g of MOPA, stirring at 125 ° C for 1 hour and, aeootropically, water is removed under reflux ( 135-138 ° C) for 8 h in the amount of 16.2 ml. The target slurry is filtered hot and processed as a free amine using the method of Example 1. The product consists of 260 g of material containing 8.57% nitrogen.
    Example 14. The factor VIII is adsorbed to form a clot from human blood plasma by various polyelectrolytes of examples 1-13, eluting and emitting factor VIII from the polymer complex.
    Human blood plasma used for fractionation studies is a fresh frozen plasma from whole blood donors. The total protein content of such a plasma varies between 60-80 g per liter, with higher concentrations occurring in summer rather than in winter.
    The units obtained are either O-positive or A-positive in type.
    Clotting factor IX can be removed first from plasma prior to the adsorbing factor VIII. The process will include the following stages:
    1. Thaw out 5 units of fresh frozen plasma in a water bath at 37 ° C.
    2. Place thawed plasma in a polyethylene or polypropylene chemical beaker. Measure out 1 l
    into a polyethylene graduated cylinder and poured into a 2-liter polyethylene beaker.
    3. Add 0.35-0.50 g of the polyelectrolyte of Example 12 and adjust the pH to 8.0 with 1.0 mol sodium hydroxide with a magnetic stirrer. Hindered for 20 minutes, maintaining a pH of 8.0.
    4. Filtered on a Buchner funnel (12.5 cm) using paper Whatman No. 54 in a polyethylene bottle containing a 4-liter bottomless flask on a glass
    5 plate. The filtrate is stored.
    5. Scrape the filtered polymer from the filter paper into a 100 ml polyethylene chemical beaker, wash the filter paper with 20 ml of distilled water, and add the polymer to the beaker. 5 minutes, stirred with a magnetic stirrer. Filter through Whatman paper number 1 in a Buchner funnel.
    5 (4.25 cm) in the same way as in a plastic cup.
    6. Connect the filtrates of steps 4 and 5 as plasma without factor IX, of which the ush factor can be
    Q is deleted in the following steps.
    A 10 ml aliquot of this plasma without factor IX was used to pass through the sieve during the subsequent fractionation of the VIII factor.
    5 With appropriate equipment modifications, the same general method is used for removing factor VIII from 1-liter portions of undiluted plasma that does not contain factor IX.
    7. Disperse 0.50 g using -. polyelectrolyte in 10 ml
    154-molar brine, adjusted to pH 4.0 or 1.0 n., Or 1.0 mol p5 citric acid. Citric acid is preferable due to the stabilizing effect on the factor VIII. in case of using amine form of polyelectrolyte pH
    0 should be adjusted with hydrochloric or citric acid. When applying the previously prepared hydrochloric acid salt form, the pH will approximate 5 s to 4, O. All subsequent procedures using 10 ml aliquots are carried out in polypropylene or polycarbonate centrifuges with a capacity of 50 ml. Move
    n very small magnetic rods.
    8. Centrifuge the dispersion of stage 7 for 5 minutes at
    2000 rpm and eject the supernatant.
    9. Add 10 ml of 0.154 molar brine and carefully adjust the pH to 5.8 with 0.1 or 1.0 molar sodium hydroxide.
    10. Centrifuge the dispersion.
     steps of 8 five minutes at 2000 rpm and eject the supernatant.
    11. Carefully adjust the pH of the plasma without factor IX, step 6, flo 5.8, with a 1.0 molar monoacid.
    12. Add 10 ml aliquots of plasma of step 11 to the precipitated polyelectrolyte of step 10. Stir for 20 minutes, maintaining the pH at 5
    13. Centrifuged for 5 min at 2000 rpm.
    14. An appropriate sample of the floated product of step 13 for biopoops is selected according to the non-adsorbed USh factor (see the method of example 15). Throw away the pop-up product.
    15. Add 10 ml of O, 154-molar brine of the polymer complex based on the VIII factor 13, stir 5 minutes, centrifuge and eject the supernatant.
    16. Add 10 ml of 1.7 molar brine to a polymer complex based on factor VIII (from step 15), adjust pH to 6.0 with 0.10 molar citric acid in order to elute factor VIII.
    17. Stir for 20 minutes, maintaining the pH at 6.0.
    18. Centrifuge for 5 minutes at 2000 rpm and take an appropriate sample from the supernatant to conduct biopoids to determine the secreted factor VIII. Throw away the pop-up product.
    Using appropriately modified instruments and equipment for filtering and minor deviations from the washing and elution processes, the USh factor is fractionated in a volume of 1 liter (from step 6) using 60 g of polyelectrolyte per liter of plasma. Again, on the filtrate of stage 14, a bioassay is carried out to determine the factor Y (not adsorbed), and on the filtrate stage 18 - to isolate the factor VIII.
    Example 15. In order to compare the yields of the described resins with respect to the isolation of the USh factor from undiluted human blood plasma by the procedure of Example 14 and the evaluation of the activation level of the USH enzyme thus obtained, two experiments were conducted.
    Isolation or exit of the USh factor.
    As a modification of the experience of obtaining thromboplastin (TST), a two-step bioassay for measuring the factor VIII is carried out. Basically, in this experiment, the time required for the formation of clots of standard plasma substrate (fresh, depleted platelets) after separation of the average level of clotting from the product by means of time-controlled addition of clotting factors from other sources is measured. Facto 1Y required for normal
    time coagulated and in the TST experiment in vitro (12-20 s), these are factors XII, XI, IX, XIII, U, phospholipid and calcium chloride. These factors are obtained from initial mixtures of calcium chloride, human serum for factors XII, XI and IX, cattle serum for factor Y and chloroform extract of the human brain for phospholipid using a time scale, after which platelet-depleted plasma is added. The time of formation of clots of the target plasma substrate is measured automatically. Before the last addition of the plasma substrate, fresh plasma control is added (the same plasma as fractionation of USH factor) or target eluents of USH factor in the appropriate dilution as the measured source of USH factor.
    Standard curves are compiled using standardized plasma with a known content of factor VIII with a content of 1 unit of factor VIII per ml. Standard curves are plotted on millimeter paper in coordinates: the time of formation of crycTKQB in seconds against units of activity of USh factor per ml (dilution of the indicated plasma control). From these standard curves, the content of VH factor in units / ml is obtained for the test samples.
    Standard curves are compiled for daily runs in determining factor VIII.
    The whole experience is in determining the time and temperature using a BBL Fibrometer Fibro System TM fibrometer, supplied by the BBL Division of Becton, Dickinson & Co., Cockeysvil1e, Mary Band and equipped with two .ThermaE Prep Bofck heating units and an automatic time control pipette.
    Bio-test for the activation of the USh factor.
    The test is designed to measure the relative time of activation of the USh factor outside plasma conditions as a modification of the described test for partial thromboplastin time. In this test, only phospholipid and calcium chloride are added to the substrate depleted with platelets. Clot formation time is measured on the indicated fibrometer by adding appropriate diluted solutions of USh factor to the plasma substrate. Inactivated control (plasma) in this test gives a clotting time of 190 s. More
    fast clotting (150 s) indicates activation or the presence of activating enzymes. In each activation detection run, two fast clotting controls are used, as well as plasma control and buffer.
    The bio-testing for excretion is carried out with the product remaining from steps 14 and 18 (example 14), while the activation tests are carried out with the product remaining from step 18. All remaining products are appropriately diluted in an imidazole buffer to give the desired brine with a molarity below 0.08.
    Example 16. Output of the USh factor and activation of the products obtained in the examples using 10 ml of the plasma fractionation product of Example 14.
    The wide spectrum of preparation in examples 1-13 of the products is tested for fractionation of the USh factor and activation by the method of example 14, subjecting them to bio-tests on these two pas
    Free amine
    Citric acid
    ,four
    --0.4
    pH adjusted to 5.8
    HC1
    2 2
    0.8 0.5
    0.4 run 2
    0.5
    0.4
    The method given in Example 15. The following results are valid for fractionation using 10 ml of plasma and 0.1-0.8 g of PE per 10 ml of plasma. By pre-adjusting the pH of the polyelectrolytes prepared and used as amines (step 7, example 14), a salt is obtained in situ during the fractionation process, using hydrochloric or citric acid in this way.
    As indicated in Example 14, the plasma varies from mixture to mixture, as well as in winter or summer, relative to the total protein content. Different plasma units used for these studies, namely, the content of the VIII factor, vary from 0.8 to 1.3 units per ml (example 15, experiment 1). The plasma coagulation time according to the activation test (example 15, experiment 2) varies between 185 and 240 s.
    The test results are shown in table.8.
    Table
    0.72 299 No 0.56 270 -0, 62 273 regulation
    Without prior citric acid, the pH is adjusted to 4.0.
    259
    No 272 There are 104 113
    0.53 98
    3 run 1 Free amine 0.5 --- 2 - - 0.4 3 - - 0.6 - 4 0.6 5 - - 0.6 s -
    0.6
    -. -
    7
     I I I
    - - 0,5
    8 0.6
    -four
    9
    - -
    ten
       I I
     I I
    eleven
    1 Free amine
    2- - 0.5
     0.5
    4-- 0.5
    5 Free amine 0.5
    ,five
    ,four
    8- -0,5
    ,five
    0.5
    run 1 HCg
     I I 0,5
    - - 0,5
      one.
    0.5 0.5
    rogon 2 -
    Continuation of table.8
    198
    0.49
    Not
    oh acid
    - -
    270 0.56
     I I 308 0.65
    acid acid
      I - "324 0.50 0.90 317
    Without trial
    0.60 340 No
    Citric Acid 0.78 269-0, 80 317 - Without test
    Without trial
    Not
    Citric acid
     I I
    Citric acid
     1 I
    Not
    Without regulation
      I

       -
    There are I I I 2 3 4 run 5 Free amine 0.5 - s. 7 - "- t 8 9 O, 5 carried - 10 - I .. 11 -I - 12 13 -. I 14 15 16 Without test Lemon i Lemon Without test - Lemon tani 0,54 272 No acid 0,, 64 279 - acid 0,59 244 ani. II 0,43 252 andslot
    Example17. Two samples of USh factor, obtained using the polyelectrolyte of Examples 1 and 7 (run 2), compare the relatively toxic effects by intravenous infusion to beagle dogs.
    Continuation of table.8
    Experiments were performed on animals weighing 8–9 kg; CS factor is obtained in sterilized water (10 units per ml). Single doses of 100 units (100 ml) were administered to each dog. The results shown in Table 9 were obtained.
    371082338
    Table 9
    Twists in
    Normalmal cell
    Normal - Significantly lower (1-24 h).
    Without change- 60%
    neniy below (1-24 h)
    NormalPulse
    Unchanged (1-24 h)
    Red shach
    ricky
    Also
    38
    Continued table. 9
    Without changes
    Normal (1-24 h)
    A femoral catheter connected to a recording physiograph for measuring pulse and blood pressure.
    According to hemocytometer. By counter CouEter. Technicon Run
    SMAC (18 biopsies). The technical and economic efficiency of the method consists in obtaining a specific factor of VIII without prior activation. The time of clotting when using the specific factor of lumbarum selected by this method is significantly increased, which ensures the possibility of effective use of this blood component.
    权利要求:
    Claims (4)
    [1]
    1. METHOD FOR ISOLATING A SPECIFIC FACTOR VIII from serum or plasma by contacting the initial liquid with a water-insoluble cross-linked polyelectrolyte — a copolymer of maleic acid or its anhydride and an unsaturated monomer selected from the group consisting of ethylene, styrene and isobutene carboxylic or anhydride groups with aminoimides selected from the group consisting of lower alkylamino lower alkylimide and lower alkyliminodiamines (lower alkylimide), where the alkyl contains 1-5 carbon atoms carbon dioxide, equal to 2-10% of the total amount of the indicated carboxyl or anhydride groups in the copolymer, followed by elution, characterized in that, in order to eliminate the preliminary activation of factor VIII in the isolation process, the copolymer is used, which is reacted with alkoxyalkylamine, in which the alkoxy group and alkyl contain 1-5 carbon atoms, taken in an amount of 50-85 mol.%.
    [2]
    2. The method according to π.
    [3]
    3. The method of pop. 1, characterized in that the specified polymer is used, subjected to interaction with methoxyethylamine.
    [4]
    4. The method according to p.
    SU „„ 1082338 A
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    同族专利:
    公开号 | 公开日
    ATA531678A|1980-12-15|
    AU519848B2|1981-12-24|
    ES471858A1|1979-02-01|
    HU182537B|1984-02-28|
    IT7826003D0|1978-07-21|
    RO74902A|1982-04-12|
    AU3824278A|1980-01-24|
    CA1133191A|1982-10-05|
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    JPS6241212B2|1987-09-02|
    PT68336A|1978-08-01|
    EP0000650A3|1979-04-04|
    DE2861003D1|1981-11-26|
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    IL55192D0|1978-09-29|
    JPS5464623A|1979-05-24|
    EP0000650B1|1981-09-02|
    AT363189B|1981-07-10|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US81891877A| true| 1977-07-25|1977-07-25|
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