JPS6157289B2 - - Google Patents

Description

[Detailed description of the invention]

The invention particularly relates to compositions or products based on compounds of the plasminogen type, preferably of human origin, to medicaments containing these compositions or products, and to these compositions or products. The present invention relates to a method for producing and purifying a composition containing a water-soluble plasminogen-type compound extracted from placental pulp. Compositions or products based on compounds of the water-soluble plasminogen type, in particular of placenta origin, have already been described in the parent patent application. The parent patent application further describes a method for preparing such compositions from placental pulp, which is prepared by mechanically pulping frozen placenta and thawing the resulting pulp. Thawing is practically complete when the temperature of the pulp exceeds 1 degree Celsius, and preferably at a temperature of around 4 degrees Celsius, the placental blood is separated, especially by centrifugation, and the insoluble pulp is collected. The insoluble pulp therefore contains what was present in the placenta, especially placental tissue and fibrin pseudoclumps. According to the method described in the parent patent application, the extraction of plasminogen-type compounds contained in the pulp is carried out in a solution having a pH that is capable of keeping the extracted plasminogen in solution, especially from 5 to 10. This is done by disaggregating the pulp in the presence of plasminogen activation inhibitors, especially epsilon-amino acids, in a solution with a pH between 100 and 1000, preferably neutral, and the resulting solution removes residual Removal of the pulp yields a solution containing the desired plasminogen type compound. At least the majority of the activation inhibitors and proteins associated with the plasminogen-type compounds in the solution may be removed by conventional methods, particularly those described in the parent patent application. Thus, plasminogen with a plasmin content higher than 4 CAU/mg (casein breakdown units per mg) and a plasmin content that is usually less than 0.04 CAU/mg and can therefore be considered negligible. It has become possible to produce water-soluble compositions or products of the type. In particular, the plasminogen content of the compositions currently obtained by carrying out the process described in the parent patent application, expressed in microkatals, is between 2.3 and 2.6 microkatals. For convenience, the activity of 1 microcatalyst of plasminogen is 1 micromole of N per second at 37 degrees C after activation with urokinase.
This corresponds to the amount of plasmin obtained from plasminogen that can hydrolyze -acetylglycine-L-lysine (the ester decomposition activity of plasmin is measured using a PH-stat). Analysis of compositions such as those obtained by the method of the parent patent application shows that they contain various compounds of the plasminogen type. In particular, according to the electrofocal method, it showed eight protein peaks, with isoelectric points ranging from 6.00 to 8.25. Determination of the amino-terminal amino acids of various compounds of the plasminogen type contained in compositions of the above-mentioned type shows, inter alia, the method of GROS and LABOUESSE (Europ. J. Biochem., 1969, 7, p. 453). ), it is derived from total plasminogen called "glutamyl-plasminogen".
It is found to contain a high content of "preactivated plasminogen" or "partially" proteolyzed plasminogen. Incidentally, glutamyl plasminogen has the same amino acid NH ₂ -terminus as plasminogen in the circulating blood, and the preactivated plasminogen is more NH It results from the loss of at least one peptide fragment containing a terminal amino acid consisting of glutamic acid with a free _2- group. These plasminogen-type compounds appear to be the same type as those that appear transiently during proteolysis of original plasminogen to plasmin due to the action of plasminogen activators or plasmin itself. It looks like Thus, "preactivated plasminogen" or "partially proteolyzed plasminogen" may be used to refer to certain plasminogen-type compounds of the present invention.
The term was used. It is often observed that the pre-activated plasminogen contained in the parent patent product is predominately peptides having an amino terminal amino acid composed of methionine. This substance will be referred to as methionyl plasminogen in the following description. Thus, the product or composition of the parent patent application may contain, for example, 40% to 60% methionyl plasminogen. As described in the parent patent application, the above compositions are water soluble. They are also stable due to their negligible, if not absent, plasmin content. Furthermore, the compositions produced by the process described in the parent patent application, especially when carried out on an industrial scale, contain significant amounts of other types of preactivated plasminogen, especially free amino groups. It has been found that the terminal amino acid containing can contain a plasminogen-type compound (lysyl plasminogen) consisting of lysine. The present invention provides compositions with a higher content of "preactivated plasminogen," particularly water-soluble compositions in which almost all of the plasminogen-type constituents are in the preactivated type; It concerns their manufacturing method. More specifically, the present invention relates to a composition in which all of the components are adapted to become completely and stably fixed to the fibrin mass, particularly under the conditions described below. This is in contrast to "glutamyl plasminogen" or circulating plasminogen, which is fixed to fibrin in small amounts under the same conditions as above and does not remain bound to fibrin in any case. . Finally, the invention provides that starting solutions containing compounds of the plasminogen type, in which a part of the constituents of the plasminogen type are already in an activated state or can be brought into such a state, It also relates to a method of manufacturing a composition as described above. The water-soluble composition of the present invention, which consists essentially of a plasminogen-type compound that can be activated by plasmin, has the following characteristics. Substantially free of glutamyl-plasminogen and plasmin. The above compound is a pre-activated zymogen,
In particular, it consists essentially of a glutamyl plasminogen derivative produced by glutamyl plasminogen losing at least one peptide fragment containing a terminal amino acid consisting of glutamic acid with a free _NH2 group. Essentially, all of these compounds can be bound to the fibrin mass and remain bound even after washing the mass under the conditions described below. These compounds have an isoelectric point of 6.7. higher, especially between 7 and 9. The method used to measure the degree of immobilization of plasminogen compounds on fibrin is the method developed by VAIREL (EG) to measure low plasmin activity (Prod. Pharm, 1970, 5, 25, 347).
−353 pages) is applied. The principle is as follows. In a test tube, 0.4 cc of human blood plasma is redeposited to form a clot. The mass is washed through an isotonic salt solution so that it is no longer lysed in the presence of the plasminogen activator. This mass is brought into contact for one period with a solution of the plasminogen to be tested. It is then washed again with isotonic salt solution to extract unbound plasminogen. The presence of bound plasminogen on fibrin is determined by the presence of a plasminogen activator [20 CTA units/cc (CTA units are from USA, the Commitee on Thrombolytic
agents, the standard unit of urokinase activity adopted by the National Heart Institute) or
150 SK units/cc of urokinase for 2 c.c.
Determination is made by melting the lump after contacting it with the [use]. Preferably, the compositions of the invention are of placenta origin. The invention even more particularly relates to compositions consisting essentially of preactivated plasminogen, and in particular to compositions consisting essentially of methionyl-plasminogen. Alternatively, it further relates to compositions containing lysyl-plasminogen as a major constituent. More specifically, it relates to compositions consisting essentially of methionyl plasminogen, lysyl plasminogen, and possibly valyl plasminogen. The method of the invention for obtaining the composition of the invention comprises:
Fibrin and a solution containing a plasminogen-type compound, preferably derived from placenta, essentially free of plasminogen activation inhibitors, and partially preactivated, are added to the fibrin. The amount of preactivated plasminogen contained in the solution is reduced at temperatures between 0 and 40 degrees C, in particular at temperatures of the order of plus 4 degrees C, at preferably neutral pH between The moieties are allowed to contact the fibrin for a sufficient time to effectively bind, and the fibrin is washed with a medium, preferably of the type used to predissolve the plasminogen-type compounds in the starting solution described above, to remove unbound proteins. and then contacting the fibrin with a solution of a plasminogen activation inhibitor to elute the preactivated plasminogen bound to the fibrin. These preactivated plasminogens can then be harvested or harvested in purified form by removing the inhibitor. A preferred starting solution for use in the process of the invention consists of a solution of a compound of the plasminogen type as obtained using the process of the invention of the parent patent application. In particular, those derived from placental pulp are preferred. These compositions, as mentioned above, in fact already contain a significant proportion of preactivated plasminogen and, moreover, are virtually free of plasmin. However, the starting solution used can of course also be obtained from other sources of plasminogen, in particular blood plasma. However, it should be understood that the plasminogen-type compounds they contain, in particular "glutamyl plasminogen", must be partially preactivated by known methods. Methods are known for the purification of plasminogen, in particular from such sources, whereby the original plasminogen contained in these sources is consequently partially proteolyzed during purification. The fibrin used is advantageously fibrin from which plasmatic contaminants, especially traces of residual plasminogen, have been removed beforehand; such fibrin is not affected by the plasminogen activator. In an advantageous embodiment of the process according to the invention, the buffer is preferably chromatographed on a fibrin column equilibrated with a solution of the same nature and composition as the constituent medium of the starting solution containing the plasminogen to be purified. That is, the starting plasminogen solution to be purified is passed through the fibrin column, which is then washed with the above-mentioned medium in the absence of plasminogen, so that the eluate no longer contains protein; Plasminogen bound to fibrin is eluted through a solution containing a plasminogen activation inhibitor. Fibrin can be obtained by any known method, in particular by adding thrombin to fibrinogen, especially human fibrinogen. Alternatively, it can be obtained by dissolving fibrinogen in blood plasma and redepositing calcium while stirring the blood plasma. The resulting fibrin is ground into a powder, preferably a fine powder. Milling is accomplished by homogenizing a suspension of fibrin in isotonic sodium chloride solution (isotonic salt solution). Advantageously, the resulting micronized fibrin is freed of plasma-derived or other contaminants, in particular residual plasminogen, which the fibrin is likely to contain, by repeated washing several times. A solution containing a plasminogen activation inhibitor is used at least once during washing. For example, the fibrin is washed each time with 4 to 6 volumes of aqueous solution, at least once using a 0.5 M aqueous epsilon aminocaproic acid solution. In this way, fibrin that is not affected by the activator, such as urokinase solution (especially
About 1 c.c. of solution containing 20 CTA units of urokinase
Fibrin is obtained which is not affected by incubation for 24 hours in (used at a rate of 500 mg of fibrin) for 24 hours. Next, a column is prepared using this fibrin. The column is then equilibrated with a solution buffered to a pH of 6 to 9, preferably 7, and with an ionic strength equal to or less than 2. As much as possible
Use 0.9% sodium chloride solution. For a solution containing 1 mg of a plasminogen type compound, use a fibrin column of 1 to 6 c.c., especially 4 c.c., and calculate the amount of plasminogen solution to be purified that passes through the column per hour. Approximately 1 to 100% of column capacity, preferably 25 to 50
%, for example 25%, a good extraction yield of preactivated plasminogen in such solutions is achieved. The plasminogen solution at the top of the column preferably contains 1 to 10 microcatalytes of plasminogen per cc. After running this solution through the column, the column is washed with a volume of buffer solution at least equal to the volume of the column. The end point of elution of unbound protein is checked by known methods, for example, by optical density in the wavelength range of 280 nm. Of note, the binding of pre-activated plasminogen to fibrin, in particular the binding of methionyl- and lysyl-plasminogen, is sufficiently selective at the conditions specified above to withstand this washing with buffer solutions. It is stable and stable. Elution of plasminogen-type compounds immobilized on fibrinogen can be carried out with solutions of any known plasminogen inhibitors. Advantageously, it is carried out in a solution containing epsilon aminocaproic acid at a concentration of 0.005M or more, for example 0.005M, in an isotonic salt solution. Of course, other plasminogen activation inhibitors may also be used. Examples are trans-4-amino-methyl-cyclohexanecarboxylic acid and para-amino-methylbenzoic acid. Compounds of the plasminogen type can then be collected from the eluate using known methods, such as those described in the parent patent application. Epsilon aminocaproic acid is
For example, it may be removed by dialysis against distilled water, by passage through suitable molecular sieves, or by precipitation of the protein using, for example, alcohol or ammonium sulfate. Advantageously, the eluted protein is
Selectively precipitate by adding alcohol to obtain a GL water-alcohol mixture. The final product can be lyophilized. Starting from a placenta-derived plasminogen solution as obtained by the method described in the parent patent application,
A composition containing mainly methionine or lysine as the _-NH2 terminal amino acid is obtained, which does not contain any "glutar-plasminogen". The isoelectric point of the plasminogen type component of the resulting composition is higher than 6.7. In particular, between 7 and 9. Plasminogen is selectively and stably bound to the fibrin clot and remains fixed to the fibrin clot even after washing with a neutral PH buffered solution free of plasminogen activation inhibitors. In another advantageous embodiment of the invention, it is also possible to immobilize plasminogen-type compounds on fibrin, wash the fibrin, and then elute the retained plasminogen by a method other than chromatography. It is. In particular, it can be carried out by the batch method. For example, fibrin is suspended in a solution of plasminogen to be purified, and when fixation of plasminogen is completed, fibrin holding plasminogen is separated and collected. The fibrin is then washed and resuspended in a solution containing a plasminogen activation inhibitor, and finally the fibrin is separated to obtain a solution containing the desired preactivated plasminogen. Of course, the operation time, pH and temperature may be within the ranges described above. It should be noted that methionyl plasminogen and lysyl plasminogen differ from each other by only a few, no more than 5 amino acids. More specifically, lysyl plasminogen appears to be derived from methionyl plasminogen by the loss of a very small peptide fragment formed from the few amino acids mentioned above. In particular, they range from the initial step of extracting crude plasminogen extract from placental pulp according to the method described in the parent patent to the final purification step to obtain a highly purified composition of plasminogen-type compounds. This occurs either as a result of the extended storage of the extract at some intermediate stage, or as a result of the handling of large quantities of raw materials when the entire process is carried out on an industrial scale, which increases the time required for each step. Other features of the invention will become clearer from the following description, which exemplifies one of the methods of the invention.
Note that this is merely shown as an example. Example (1) Extraction and purification of plasminogen-type compounds from placenta The starting placenta, membranes and placental cords are immediately frozen and stored at -20°C as soon as they are supplied. The placenta is mechanically ground in the frozen state into pieces 1 to 5 mm long and then thawed by stirring in 2 volumes of isotonic salt solution. When the temperature of the suspension reaches +6°C, centrifuge.
Discard the liquid supernatant (placental blood, which contains hemoglobin and plasma proteins, but virtually no plasminogen). The placental tissue and, to a lesser extent, the sediment or pulp formed by the fibrin clot are twice the volume.
Disaggregate in an isotonic salt solution of 0.05M epsilon-aminocaproic acid at +4°C for 2 hours and centrifuge again. The extracted plasminogen type compounds are present in the supernatant liquid. Precipitate plasminogen by adding enough ammonium sulfate to achieve 40% saturation at pH 7.0. The proenzyme contained in the precipitate was determined by Deutsch (D) and Merz (ET).
Purification by affinity chromatography according to the method described in Science, 1970, 170, pages 1095-1096. Aprotinine
(Kunitz inhibitor, pancreatic extract, sold under the name INIPROL)] is added at each step of purification to avoid degradation of plasminogen-type compounds due to proteolysis by contaminating plasmin. Regardless of the initial amount of placenta processed (5, 50, 200 or 1000 Kg), in all cases a preparation of plasminogen-type compounds with a specific activity of 2.4 to 2.6 microcatalytes per milligram in the dry state is obtained. It will be done. Analysis of the amino-terminal amino acids by the method of Gros and Labouesse, already shown, reveals the presence of high amounts of lysyl-plasminogen and methionyl-plasminogen. The remainder of the plasminogen-type compounds is mostly glutamyl-plasminogen. Typical compositions are shown in Table 1 below. Table 1 Amount of amino terminal amino acids (%) Methionine 55 Glutamic acid 25 Lysine 20 Valine Trace (2) Preparation of fibrin column Dissolve 9 g of human fibrinogen in 200 c.c. of human blood with stirring for 3 hours. This blood clot is recalcified by adding 30 ml of 2% CaCl ₂ aqueous solution. After confirming the suspicion,
The resulting clot is left at room temperature for 36 hours to allow the factors in the blood plasma to complete their action and to improve the "solidity" of the fibrin. The clots are then chopped and homogenized in 80 c.c. isotonic salt solution (ULTRA
5 at maximum speed in a TURAX type homogenizer
). The fibrin particles were then placed on a Büchner with 3 liters of isotonic salt solution, 1 liter of 0.5M epsilon-aminocaproic acid isotonic salt solution, 1 liter of isotonic salt solution, 2M at pH 7.2.
Wash sequentially with 1 liter of NaCl solution and 2 liters of isotonic salt solution. The fibrin obtained is free of plasmatic contaminants and is insoluble in 5M urea solution. In particular, plasminogen activators (2 CTA units/cc urokinase at a rate of 2 c.c.
No lysis occurs even after contact treatment with 150 SK units/cc streptokase for 24 hours). The resulting fibrin was then placed in a column 2.5 cm in diameter and 45 cm in height and equilibrated for 48 hours with an isotonic salt solution of pH 7.2 flowing at a rate of 8 c.c./hour. (3) Extraction of the preactivated plasminogen component from the plasminogen-type compound preparation obtained in (1) 60 mg of the plasminogen-type compound preparation was added to an isotonic salt solution of 30 c.c. Dissolve and place on top of the column. The column is washed to remove unbound protein with the same isotonic salt solution until no protein is present in the eluate. Absence of protein is 280n in the wash fraction
Check by measuring the optical density in m (nanometers). Bound plasminogen is eluted with the above isotonic salt solution plus a 0.05M concentration of epsilon-aminocaproic acid. The elution fraction is approximately 75% of the protein in the initial preparation.
and consists of a mixture of plasminogens containing methionine and lysine as amino-terminal amino acids. Does not contain glutamyl plasminogen. Electrofocalization shows that the fractions contain proteins with isoelectric points of 7.70; 7.65; 8.00; 8.12 and 8.75. The eluted protein is selectively precipitated by adding alcohol to the obtained eluate to form a water-alcohol mixture at 35 degrees GL. All operations are performed at approximately 4 degrees Celsius unless otherwise noted. The product may bind to the fibrin mass, particularly the fibrin mass from which plasminogen has first been removed. It should be noted that fibrin clots that do not contain any plasminogen cannot be melted in the presence of a plasminogen activator under the conditions described above. However, it has been observed that these same masses, after contact with the above-mentioned products, become capable of melting under the action of the plasminogen activator. On the other hand, these same masses, from which the plasminogen has first been removed, do not exhibit the above-mentioned effects when contacted with blood plasma containing the native plasminogen glutamyl plasminogen under at least the same conditions as with the compounds of the invention. Under no conditions can it be melted. The test results will be shown later in Table 2, but here the test conditions are shown in the column labeled "Test Properties." Aprotein is added when plasminogen is dissolved in a buffer solution to avoid decomposition of plasminogen by contaminating plasmin and also to avoid dissolution of fibrin by the same plasmin. Test No. 1 shows that the original plasminogen in blood plasma does not remain adsorbed to the fibrin produced during pseudocoagulation. On the other hand, plasminogen extracted from the placenta (Test No. 2) remains adsorbed to the fibrin, and the amount adsorbed is a function of the amount brought into contact with the blood clot. (Test No. 2, 3, 4) Contains preactivated plasminogen. The preparation obtained is particularly useful because it can be completely bound to the fibrin clot, contrary to glutamyl-plasminogen, which tends to dissolve in blood plasma and does not remain inside the clot in any case. It is the main ingredient of many medicines. Selective binding of plasminogen preparations to fibrin clots facilitates fibrin clot melting after complete plasminogen activation, allowing for better improvements in thrombotic disease treatment. In general, the plasminogen-based compositions of the present invention are utilized in the treatment of thrombosis and pathological fibrin deposition, particularly the following clinical conditions: (a) Acute respiratory distress in neonates and preterm infants where there is complete or partial deficiency of plasminogen. This syndrome is generally caused by fibrin deposits in the alveoli. (b) Ambalism and venous thrombosis. (c) Micro-amberism, cerebral in nature. (d) Acute, subacute and chronic conditions in which intravascular pseudosolids tend to spread, localize and diffuse. Merely by way of example, administration of placental plasminogen can range from 1,000 to
It is administered by continuous intravenous infusion or slow intravenous injection of 1200 CAU of plasminogen (for a boy weighing 60 kg). In infants, administer by slow infusion over 24 hours using a 9/1000 isotonic salt solution buffered to PH 7.4 at a dose of 2000 CAU every 6 hours. An advantageous administration form for the preparations of the invention includes plasminogen dissolved in a pharmaceutically acceptable vehicle, particularly physiological serum, preferably glucose.

[Table] Additional Relationships The present invention provides a method for selectively adsorbing and eluting only preactivated plasminogen, which is advantageous for therapeutic purposes, with fibrin, thus preactivating plasminogen that does not contain native plasminogen. The present invention provides a composition containing only converted plasminogen. On the other hand, the original invention patent number
No. 1316337 (Special Publication No. 60-42207) describes plasminogen whose terminal amino acid is glutamic acid (original plasminogen) and preactivated plasminogen lacking the peptide chain containing this terminal amino acid. This method extracts plasminogen-type compounds that contain both. Therefore, the present invention is an invention in which the main parts of the essential parts of the original invention are the main parts of the essential parts of the original invention, and it achieves the same purpose as that invention. Therefore, there is an additional relationship pursuant to the provisions of Article 31, Paragraph 1 of the Patent Act.

Claims (1)

[Scope of Claims] 1 (i) It essentially does not contain glutamyl-plasminogen and plasmin; (ii) the plasminogen-type compound (i) is capable of forming a compound from the amino terminal of glutamyl-plasminogen to the amino terminal thereof; consists essentially of preactivated plasminogen-type compounds resulting from the loss of glutamic acid-containing peptides; remains immobilized even after washing with a solution containing water-soluble composition. 2. The composition according to item 1 above, which is derived from placenta. 3. The composition according to any one of the above items 1 and 2, characterized in that the compound of the plasminogen type contains methionyl-plasminogen. 4 The above compound of plasminogen type is lysyl-
characterized by containing plasminogen,
The composition according to any one of items 1 to 3 above. 5. The composition according to any one of items 3 and 4 above, characterized in that it also contains valyl-plasminogen. 6. Composition according to any one of the above items 1 to 4, characterized in that said compound of plasminogen type consists essentially of methionyl-plasminogen and lysyl-plasminogen. 7. An aqueous solution containing a plasminogen-type compound that is substantially free of plasminogen activation inhibitors and that is partially preactivated is PH
After the preactivated plasminogen-type compound is brought into contact with fibrin in steps 6 to 9 to bind to fibrin, unbound proteins are washed away, and then the preactivated plasminogen bound to fibrin is removed. eluting the gene-type compound with an aqueous solution containing a plasminogen activation inhibitor, and removing the inhibitor from the eluate. A method for producing a water-soluble composition. 8. The method according to item 7 above, wherein the aqueous solution used as the starting solution is derived from placenta. 9. The method according to item 7 above, wherein the fibrin used is free of plasma impurities, particularly residual plasminogen, and is not affected by a plasminogen activator. 10. The method according to any one of items 7 to 9 above, wherein the aqueous starting solution is brought into contact with fibrin at a pH around neutrality. 11 Mix the aqueous starting solution with fibrin at 0°C.
A method according to any one of the preceding clauses 7 to 9, wherein the contacting is carried out at 40°C, in particular at about 4°C. 12. The method according to any one of items 7 to 9 above, wherein unbound protein is washed away in a medium similar to that of the aqueous starting solution. 13 Place the plasminogen to be purified onto a fibrin column, preferably a fibrin column pre-equilibrated with a buffer of the same nature and composition as that used for the solution containing the plasminogen to be purified. The containing solution is passed through, the column is washed with the same buffer, and finally,
7 above, characterized in that the bound plasminogen-type compounds are eluted by chromatography comprising passing a solution of the plasminogen activation inhibitor through a fibrin column. The method described in any one of paragraphs 9 to 9. 14 The ionic strength of the buffer solution used is between 0 and 2.
The amount of fibrin used should be between about 1 to about 5 c.c., preferably 3 c.c., per milligram of plasminogen to be purified; The containing buffer solution is passed through the column at a flow rate of between about 1 and 100% by volume, preferably between 25 and 50% by volume, per hour of the volume of fibrin in the column and the plasminol. Item 13 above, characterized in that after binding the plasminogen-type compound to fibrin, the plasminogen-type compound is eluted with a solution of a plasminogen activation inhibitor at a concentration equal to or greater than 0.005M. Method described. 15. Suspend fibrin in a solution containing the plasminogen to be purified, separate and collect the fibrin containing plasminogen, and once binding is completed, wash the fibrin and remove the plasminogen activation inhibitor. 7 to 9 above, characterized in that the solution containing preactivated plasminogen is collected after separating the fibrin.
The method described in any one of the preceding paragraphs.