JPH0570607B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a plasminogen activator,
More specifically, purified plasminogen activity based on a TPA/proTPA couple characterized by culturing a proTPA-producing cell culture in a suitable medium and then recovering proTPA (and optionally TPA). The present invention relates to a method for producing a curing agent. TPA/Pro
TPA capsules are used in pharmacological preparations. As used herein, the term "TPA/pro-TPA couple" refers to tissue plasminogen activator (TPA), which can be recovered from, for example, Bowes melanoma cell tissue culture and is defined below.
and/or its precursor pro-TPA as defined below, or a combination of both. TPA is an abbreviation used in the art for tissue plasminogen activator, the double-chain plasminogen activator identified, for example, in Bowes melanoma cells. used about Pro-TPA is a TPA precursor (precursor)
of TPA), a single-chain compound that has the same molecular weight as TPA and is converted to double-chain TPA by certain enzymes. Prior art literature indicates that both single and double stranded forms have virtually the same activity. From this point of view, the present invention includes completely different findings. A plasminogen activator is a substance that generates plasmin by acting on plasminogen (plasmin precursor). Plasmin then acts on fibrin (or blood clots) to liquefy or dissolve the fibrin. Plasmin also causes the dissolution phenomenon of fibrinogen, a fibrin precursor. The aforementioned effects play an important role in the natural fibrinolytic system. Plasminogen activation also occurs in the therapeutic administration of plasminogen activators for the treatment of thrombosis or other pathologies where it is desired to generate local fibrinolytic or proteolytic activity through a plasminogen activation mechanism. agent is used. Plasminogen activators can also be used as reagents for scientific, diagnostic or pathological tests, including in vitro fibrinolysis. Two human plasminogen activators are widely used. The first activator is streptokinase, a bacterial protein that acts by a non-proteolytic mechanism. The second activator is urokinase, a protease obtained from urine or cultured kidney cells, although urokinase is known to occur in most other types of mammalian tissues. These two compounds have the therapeutic disadvantage that their action is not limited to fibrin-related plasminogen in blood clots. These compounds generally act on plasminogen in the blood circulation, resulting in extensive plasmin generation with extensive dissolution of fibrinogen, the precursor of fibrin. This leads to a bleeding condition as a result of ineffective normal aggregation of blood. Furthermore, urokinase has the disadvantage of being expensive to isolate and produce and can only be obtained in low yields by currently used methods. This disadvantage also applies to valuable uses of urokinase other than fibrinolytic therapy, limiting the availability of urokinase as a valuable commercial product for such uses. Streptokinase is a heterologous protein that elicits an immune response in patients. The resulting antibodies neutralize the action of streptokinase on plasminogen, thus reducing therapeutic efficacy. Generally known as "tissue plasminogen activator". A third plasminogen activator, herein abbreviated as "TPA", is also known to exist. This enzyme is identical or indistinguishable from the enzyme found in most human tissues and which is also a characteristic secreted product of human melanoma cells cultured in vitro. Although TPA catalyzes the formation of plasmin from plasminogen in much the same way that urokinase does, it differs from urokinase in a number of important ways. The two enzymes are scientifically different. They have different molecular weights, and one enzyme will not react with the other enzyme's antibodies. The catalytic activity of TPA, but not urokinase, is enhanced by fibrin. TPA has the important property of binding to fibrin, but urokinase does not. These facts are summarized in C. Dingeman, C. Rijken et al., “Purification
and characterization of the plasminogen
activator secreted by human melanoma cells
in culture” (J.Biological Chem., 256, No. 13,
7035-7041).
The paper also describes the production of TPA from cultured human melanoma cells as well as from normal human tissue. The purification procedure consisted of chromatography on zinc chelated agarose, concanavalin-A agarose and Sephadex G-150 in the presence of detergent. The tendency of TPA to bind to fibrin, the fibrinolytic action of TPA which is greatly enhanced in the presence of fibrin, and its relative inferiority as a plasminogen activator in the absence of fibrin when compared to urokinase and streptokinase.
The cumulative effects of TPA make it the plasminogen activator of choice for the treatment of human blood clots.
A significant degree of interaction between fibrin and TPA localizes plasmin production to the clot area and
Attenuate or avoid the effects of indiscriminate plasminogen activation as seen when using urokinase or streptokinase. Furthermore, streptokinase is an immunochemically foreign protein to humans, whereas TPA is not. W. Weimar et al., “Specific lysis of an
iliofemoral thrombis by administration of
extrinsic (tisse type) plasminogen activator”
(Lancet, November 7, 1018 (1981)) recently reported that
The clinical utility of TPA is being tested. P. Wallen et al. (Prog. Chem. Fibrinolysis
According to Thrombolysis 5 , 16-23 (1981),
TPA occurs in two different forms, one as a single-stranded form, which is converted by plasmin or trypsin into a double-stranded chain linked by disulfide bridges. The authors considered this duplex to be a degradation product of the single chain. The double strand shape is
It can be said that it is more effective in terms of fibrinolysis than the single chain form in a fibrinolysis test system (in vitro). Aprotinin prevents the conversion of single strands to double stranded form so that the single stranded form is restored as the only or predominant form. Both forms are considered tissue plasminogen activators. For this study, see Dingeman, Rijken,
A more recent publication by Hoylaerts and Collen (J.
Biol.Chem., 257.2920-2925 (1982), European patent application no. Both shapes are listed. They suggested that the single-stranded form, when adsorbed to a fibrin clot, quickly converts to the double-stranded form such that fibrinolysis "occurs primarily through the double-stranded derivative." They are,
They conclude that "this conversion does not appear to play a role in the regulation of fibrinolysis."
They found that "the two molecular shapes have the same activity (dissolution time)" and that "the catalytic effectiveness of both molecular shapes is virtually the same, so the single-chain shape is a less active precursor. , or that the double-stranded form is a less active degradation product.
Whether the above conclusion is entirely correct or not,
It is clear that both "molecular forms" have therapeutic utility as much as in vitro dissolution. Indeed, based on our recent findings, it is clear that the foregoing conclusion requires certain limitations. Although pro-TPA is truly a precursor to TPA, it is not an entirely inert precursor.
The intrinsic activity of pro-TPA is about 1/10 that of TPA, so based on this alone, pro-TPA must be converted to TPA in advance in order to express its maximum therapeutic effect, so TPA is more active than pro-TPA. It is immediately considered preferable to administer it for therapeutic purposes. In fact, European Patent Application No. 0041766 describes two compounds possibly representing TPA as well as pro-TPA, but only the latter compound is distinctive with respect to fibrinolytic, fibrinogenolytic and thrombolytic effects. This description of pro-TPA and its use as a pharmacological composition
There is no special mention regarding the use of TPA. The pharmacological method of use in which the compound exhibits its characteristics is claimed only for the double-stranded form, ie, TPA. The only published use of the substance in actual therapy uses dual-stranded TPA (Weimer et al., The Lancet,
Nov. 7, 1018-1020 (1981)). According to the above-mentioned prior art, double-stranded
There was no known method for readily producing TPA, and the single chain form referred to herein as "pro-TPA." According to the inventors' recent proposals (South African Patent Application No. 82/9168 and applications based thereon in other countries, which are not currently part of the state of the art), it is possible to A method for easy production has been proposed. It is an object of the present invention to provide a new and relatively simple method for the selective reversible adsorption of TPA and/or pro-TPA from solution, in particular for the production of purified plasminogen activators. A special object of the present invention is to optionally provide purified TPA;
but preferably purified pro-TPA and pro-TPA
The purpose of the present invention is to provide a new method for producing a purified mixture of TPA and pro-TPA. A further object is to provide a method as described above which makes it possible to separate TPA and pro-TPA from urokinase and, if desired, applied to the separation and recovery of urokinase. It is also an object to provide a formulation of TPA, if desired, but preferably pro-TPA or a mixture of both, in which pro-TPA is predominant. Based on the previously unknown properties and benefits of pro-TPA over TPA, the formulation can be beneficially used in the treatment of thrombosis and embolism as well as in the treatment of such diseases. It is a particular object to provide pharmacological preparations that also provide for the prevention of. One aspect of the present invention is a method for producing a purified plasminogen activator based on a TPA/pro-TPA couple, comprising: culturing a pro-TPA-producing cell culture in a suitable medium;
Cells are cultured in the absence of an enzyme that catalyzes the conversion of pro-TPA to TPA, or, if present, in the presence of a suitable inhibitor to inhibit said enzyme, resulting in 50 to Produce a harvest liquor with 100% pro-TPA and balance TPA, produce a harvest liquor containing dissolved pro-TPA and impurities in the seeds of Erythrina lateitusima and other Erythrina species, and produce a harvest liquor with a concentration of trypsin, plasmin and TPA. catalyzed in close contact with an affinity reagent comprising an immobilized Kunitz-type inhibitor that is an inhibitor, but does not act on urokinase, thereby selectively adsorbing pro-TPA and any TPA present from the medium, and pro-TPA and removing the TPA-loaded affinity reagent present from contact with the aqueous medium and unadsorbed impurities, desorbing the pro-TPA and any TPA present from the affinity reagent, and then recovering the desorbed pro-TPA and, if necessary, the TPA. The characteristic lies in the manufacturing method. If you use a cell culture that also produces urokinase, pro-TPA in the harvest fluid and
TPA has become impure due to urokinase.
Urokinase is removed along with unadsorbed impurities and then optionally recovered as valuable product. Erythrina Lateitsima (Erythrina)
Seeds of Latissima, broad-leaved Erythrina) and other Erythrina species contain two protease inhibitors (Francois J Joubert et al., Z. Physiol.
Chem., 362 , 531-538). These two inhibitors are
In the above literature, they are called DE-1 and DE-3. DE-1 inhibits bovine chymotrypsin but not bovine trypsin, whereas DE-3 inhibits bovine trypsin but not bovine chymotrypsin. DE-3 has properties as a Kunitz-type enzyme inhibitor and as an inhibitor of trypsin, plasmin, and TPA;
Has no effect on urokinase. DE-3 was developed by TPA after screening hundreds of enzyme inhibitors.
It was selected as the only inhibitor that can distinguish between urokinase and urokinase. If this inhibitor is immobilized, for example in a manner known per se, TPA can be selectively extracted by adsorption to form an affinity reagent, as well as a previously unknown precursor of TPA, pro-TPA. .
However, unadsorbed impurities remain in the aqueous solution. DE−
Since 3 is an inhibitor of plasmin, it is possible to inhibit pro-TPA by the action of plasmin, which is normally present in aqueous solutions suitable as TPA and/or pro-TPA sources.
Inhibits conversion to TPA. Therefore, this immobilized inhibitor has remarkable properties when used as an adsorbent for pro-TPA. A more complete chemical characterization of DE-3 is described in Joubert et al., supra. Even if urokinase is present in aqueous solution, it is not adsorbed. Therefore, TPA and/or
Affinity reagents can be used in the method of the invention even when TPA is contaminated with urokinase;
Urokinase is removed along with unadsorbed impurities.
This suggests that pro-TPA and/or pro-TPA can be obtained from multiple sources such as various tissues where TPA and/or pro-TPA occur as a mixture with urokinase.
This means that this method can be applied to recover TPA. However, a particularly preferred aqueous medium used to extract pro-TPA and optionally TPA from an aqueous medium is obtained from tissue culture of human melanoma cells containing pro-TPA and 0-50% TPA in the secreted products of the cells. It is harvest liquid. According to a preferred embodiment, pro-TPA and any TPA present are adsorbed by passing an aqueous medium containing them through an affinity reagent layer, the layer is then washed, the pro-TPA and any TPA present are desorbed, and the desorbed pro-TPA is removed. TPA and any TPA present is washed out of the layer. The layer may be in the shape of a column. Desorption is preferably about 1-3 mol/(e.g.
This is carried out using an aqueous potassium thiocyanate solution of 1.4 to 2 mol/, preferably 1.6 mol/). In this case, the compound is at a pH where TPA is stable.
Preferably approximately neutral phosphate buffered saline (e.g.
0.3 to 1 mol/) for NaCl, preferably about 0.4 mol/), preferably with a stabilizing amount of a suitable sea surfactant (e.g. 0.1% TritonX).
-100 or Tween). Desorption may be effected by chaotropic desorbents other than potassium thiocyanate. Desorption is carried out at a low pH, e.g. 2-3.5, preferably 2.8-
3 may be performed. Other suitable desorption conditions are highly concentrated saline and/or the presence of arginine and/or benzamidine. Such desorption conditions are known to those skilled in the art and will not be described in detail. TPA without pro-TPA even according to the invention
If it is desired to recover the
For example, pro-TPA in aqueous solution can be converted into plasmin or pro-TPA even if not made in an earlier method step.
By exposure to an enzyme that has an equivalent effect on
It can be easily catalytically converted to TPA.
One such enzyme, which has not yet been identified, is TPA
For the same reason that the conversion of proTPA to occurs in the presence of fibrin, e.g. Kallikrein also produces this effect, although to a lesser extent. Pro-TPA produced according to the present invention is a single-chain compound with little plasminogen activator properties, whereas TPA (molecular weight 72,000
Dalton) was found to differ from TPA in that it is a double-stranded compound and has significant plasminogen activating properties that are about 10 times more potent than pro-TPA. Pro TPA is TPA
has the same molecular weight as Due to weak activity in single-stranded and double-stranded forms
Due to the knowledge that TPA is highly active, it is the TPA form that is more effective in the blood clot lysis process in vivo, and therefore, it has been proposed in the prior art to include it in pharmacological compositions.
There has usually been a strong motivation to use plasminogen activators in the TPA form (ie, double-stranded molecular form). This idea is confirmed by the finding that pro-TPA is very weakly active. However, surprisingly, the use of TPA as an active ingredient in pharmacological compositions for the treatment of thrombosis and other treatments in which the fibrinolytic effect of plasminogen activators is exploited is more important than the use of pro-TPA forms. Experimental evidence has demonstrated that there is reason to believe in using a minogen activator (preferably comprising at least primarily pro-TPA, more preferably all pro-TPA). The reason for this is that pro-TPA has a higher affinity for adsorption to fibrin than the TPA form;
and that pro-TPA is converted to TPA very quickly upon adsorption to fibrin, and then obtains maximum desired activity exactly where it is needed, ie at the clot site. The conclusions drawn from this discovery are as follows. (a) Pro-TPA is more rapidly and completely adsorbed to the aggregated fibrin portion, ie, where the activator is actually needed. professional at that location
TPA is produced by a protease found in fibrin.
It is rapidly converted to TPA, and the full plasminogen activator activity of TPA becomes immediately available to the active part of the blood clot. (b) Pro-TPA is more selective than TPA in effecting fibrinolysis. because,
This is because pro-TPA is relatively inactive until it binds to fibrin, and becomes maximally active by being converted to TPA in situ.
TPA has approximately a 10-fold greater potential to activate plasminogen in the bloodstream rather than in the clot area, which requires a fibrinolytic effect. This considerable possibility of adsorption to the fibrin part
This is further increased by the low affinity of TPA. (c) due to severely reduced activity in the bloodstream;
We can confidently recommend the use of pro-TPA to prevent future thrombosis or embolism. (d) On the other hand, the use of relatively high dosages in acute thrombotic occlusions reduces the risk of side effects. (e) TPA has a short half-life in vivo. Professional
The properties of TPA are to make the utilization of plasminogen activators more effective. On the one hand, pro-TPA is less likely to be consumed by ineffective reactions when in the bloodstream, and on the other hand, pro-TPA is
TPA is likely to be quickly "trapped" in the clot where pro-TPA may be useful. Based on key concepts in previous findings, the present invention provides a method for treating thrombosis, embolism, or other diseases in which it is desired to generate localized fibrinolytic or proteolytic activity through a plasminogen activation mechanism. A pharmacological composition for prevention,
The pharmaceutical composition comprises 50-100% pro-TPA and 0
It comprises a plasminogen activator component designated as a TPA/pro-TPA couple consisting of ~50% TPA. The TPA/pro-TPA couple preferably consists of 30-70% pro-TPA and 0-30% TPA, more preferably 80-100% pro-TPA and 0-20% TPA. The pharmaceutical composition of the present invention may be applied to the treatment of thrombosis and embolism when thrombotic occlusion has already occurred. The present invention provides 1 to 10 mg of TPA/pro-TPA capsules.
administered intravenously to the patient in dosage units of
It is also particularly applicable to the prevention of diseases involving thrombotic occlusion characterized by the intravenous infusion of the plasminogen activating composition at a TPA/pro-TPA couplet infusion rate of 0.5-2 mg/hr. Such treatment may be administered to patients who are at risk but do not show any signs of thrombosis or embolism. Examples include bedridden and debilitated patients who are particularly prone to severe venous thrombosis and embolism due to venous cessation. Another example is a patient with unstable angina pectoris where coronary thrombotic occlusion is likely. Another example is a patient with valvular heart disease who has a thrombotic site and is at risk for cerebral embolism. As an example, some patients have areas or lesions in the pericardial cavity or large blood vessels where there is a risk of blood clot migration and cardiovascular occlusion (stroke). Examples include patients with prosthetic vascular valves, atrial fibrillation, and mildly acute bacterial endocarditis. The pharmacological compositions of the present invention are useful for the prophylactic treatment of patients receiving intravenous therapy or parenteral nutrition that are likely to result in thrombotic occlusion and/or embolization of the vein when inserting an intravenous line into the vena cava and injecting a stimulant solution. It is useful for Although continuous intravenous infusion is preferred for administration, intermittent intravenous bolus doses may be administered e.g.
It may be applied in said dosage of mg. The present invention provides various pharmaceutical compositions specifically prepared and adapted for use in the aforementioned applications. Purified TPA or pro-TPA can be prepared separately or as a mixture according to the present invention. According to the present invention, TPA can be recovered from an aqueous medium containing TPA and plasmin and/or plasminogen and a plasminogen activator. This requires inhibition of plasmin in the aqueous catalyst and removal of pro-TPA from the aqueous medium while plasmin is inhibited.
These points out that the TPA present acts as a plasminogen activator, causing the formation of plasmin, which must be inhibited. According to the invention, in the method
ProTPA is adsorbed from an aqueous medium by an affinity reagent having an affinity for TPA and comprising an immobilization inhibitor of plasmin, and then TPA is desorbed from the affinity reagent to recover proTPA in a purified state. The pro-TPA thus recovered is then sterilized, stabilized with a physiologically compatible stabilizer, and placed in the form of an aqueous solution that can be injected intravenously. The DE-3 inhibitor used in immobilized form as an affinity reagent in accordance with the present invention happens to be an inhibitor of plasmin, and plasmin is normally present in the medium in which TPA and pro-TPA are recovered, and pro-TPA is
Because it has the property of converting TPA to TPA,
The use of a DE-3 inhibitor to co-isolate TPA serves to keep pro-TPA in the pro-TPA form. According to another aspect of the invention, selectively adsorbing TPA and/or proTPA separately from or together with urokinase from an aqueous medium containing both plasminogen activators (by themselves or as precursors). is possible. This applies to the production of purified plasminogen activators comprising urokinase-free purified TPA and/or proTPA on the one hand or TPA and proTPA-free purified urokinase or both on the other hand. This produces such an aqueous medium in the seeds of Erythrina lateitsima and other Erythrina species, and immobilized Kunitz-type inhibitors of the type that are inhibitors of trypsin, plasmin and TPA, but do not act on urokinase. to selectively adsorb TPA and/or proTPA from the medium, the TPA and/or proTPA loaded affinity reagent being contacted with an affinity reagent comprising
TPA and/or pro-TPA is gone,
This is carried out by removing the urokinase from contact with the aqueous medium that still contains the urokinase and recovering the urokinase from the aqueous medium that is depleted of TPA and/or pro-TPA. The aqueous medium suitable for this method can be an aqueous extract of any mammalian tissue cells, as the medium contains both plasminogen activators, and a medium containing the secretions of such cells. It's okay. I'll say it again, it was absorbed.
The TPA and/or proTPA is desorbed, recovered, and if desired, the proTPA may be converted to TPA before or after adsorption to a selective affinity reagent. Urokinase may be recovered from the aqueous medium in any suitable manner, for example by adsorption from the aqueous medium using an affinity reagent that has an affinity for urokinase. Such an affinity reagent may be an immobilized inhibitor that does not have selectivity for TPA over urokinase. It is also possible to use suitably immobilized anti-urokinase antibodies in a manner known per se as affinity reagents. The Kunitz-type inhibitor of the present invention is produced in the seeds of Erythrina lateitsima and other Erythrina species, and contains trypsin, plasmin and
Although it is an inhibitor of TPA, it does not act on urokinase, and is immobilized, for example, by covalently bonding to a solid carrier.
For example, the inhibitor may be coupled to cyanogen bromide activated agarose by methods known to those skilled in the art. However, depending on the chemical nature of the nature of the surface to which the inhibitor is attached, other coupling agents and other solid carriers may be used. Examples include: (a) A carbodiimide coupling agent that couples a free amino group to a free carboxyl group. (b) A glutaraldehyde coupling agent that couples NH ₂ groups to agarose that has previously been converted to the aminoalkyl form. (c) periodate activity to generate an aldehyde functionality and then react with an inhibitor amine at PH 4-6 to form a Schiff base and then reduced by sodium borohydride or sodium cyanoborohydride. agarose. (d) Agarose may be converted to a hydrazide succinyl derivative, so that the carboxyl ligand may be coupled with an amine by EDC or diazotization. (e) The cellulose fibers or particles are converted to hydrazide derivatives using the method of Parikh et al., Methods in emzymology, 34 , 77-102,
editors W B Jakobi and Wilcheck
Academic Press NY). (f) The polyacrylamide particles may be attached by the direct glutaraldehyde method or by diazotization of p-aminobenzamidoethyl or hydrazide derivatives. (g) Proteins, such as albumin or trypsin or gelatin, may be bound, for example in the form of hydrazides. (h) Finally, glass particles (or other solids) which can be coated with proteins may also be used, as proteins described in South African Patent Application No. 81/4481;
and a West German patent application, which is an overseas corresponding patent.
P3224837.7, Japanese Patent Application No. 116086, British Patent Application No. 8219070, United States Patent Application
Mention may be made of gelatin, which may be coupled to the inhibitor and crosslinked by a method similar to the coupling method described in No. 392,111. Said method is known to the person skilled in the art and/or
Since it is also described in the reference cited above, it will not be described in further detail. The affinity reagents of the present invention may be used to produce TPA and/or proTPA or formulations comprising TPA and/or proTPA, eg, in a manner as detailed above. but,
Affinity reagents may also have utility as diagnostic or analytical reagents. The scope of the invention includes purified mixtures of TPA and pro-TPA and in particular a ratio of pro-TPA to TPA of 1:
as well as more than one mixture containing pro-TPA purified according to the invention. In order to obtain high ratios, e.g. 9:1 or higher, the conversion of pro-TPA to TPA may be carried out in a suitable medium in the absence of or containing such enzymes (such as serum). ) as well as a suitable inhibitor applied to inhibit the enzymes (in particular plasmin) that catalyze the conversion of proTPA to TPA by proTPA producing cell cultures (e.g. melanoma cells). It is suggested that. Suitable inhibitors are trypsin inhibitors derived from soybean or α-l-antiplasmin. Aprotinin may be used instead (as is known). Then the professional
TPA is recovered from the medium in the absence of the aforementioned catalyst or in the presence of an inhibitor. If a mixture of pro-TPA and TPA is available and it is desired to limit the catalytic activity of both to that of pro-TPA, TPA can be removed by using an inhibitor that irreversibly inhibits TPA. It is. A suitable example of this is the irreversible protease inhibitor diisopropylfluorophosphate. Inhibitors are subsequently removed by dialysis or gel chromatography. irreversibly inhibited
The TPA may remain in the mixture. The scope of the invention extends to pharmaceutical compositions comprising TPA and/or pro-TPA dissolved in a physiologically compatible infusion medium and prepared or purified by the method of the invention. The scope of the invention also extends to fibrinolytic preparations in which fibrinolysis is carried out in vitro, for example for diagnostic or pathological or general scientific purposes. TPA or Pro as the only active ingredient
TPA, or TPA as well as pro-TPA at desired ratio
It is considered an advantage of the present invention that it can be practically optionally applied to produce the aforementioned compositions comprising: The present invention will be explained in more detail by showing Examples below. Example 1 Preparation of affinity reagent: - A DE-3 inhibitor was prepared as follows: Ersulina lareisima seeds were collected and processed according to the method of Joubert et al. Grind the seeds, defatte them, and cool them at 10°C with a 0.5 mol/NaCl aqueous solution.
Extracted overnight. The extract was centrifuged and DE-3
was recovered from the supernatant by ammonium sulfate precipitation, followed by chromatography on Sephadex G50, DEA-Cellulose and DEA-Sepharose. The apparent molecular weight of the final purified product was determined by electrophoresis on a 15% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS).
It traveled as a single band of 22,000 Daltons. Purified DE-3 (26 mg) was coupled in a conventional manner to 5 ml of commercially available cyanogen bromide activated agarose (Sepharose 4b, commercially available from Pharmacia, which also teaches how to use the preparation). The affinity reagent was equilibrated against a phosphate buffer at pH 7.4 containing 0.4 mol NaCl/, 0.1% TritonX-100 (a commercial surfactant commonly used in the art) and 0.02% sodium azide stabilizer. Ta. This affinity reagent was prepared from a 5 ml cylinder made from a disposable plastic syringe.
was packed into a column. Example 2 Preparation and purification of TPA and proTPA: - A medium containing TPA and proTPA was prepared as follows. Bowes Cell RPMI−
Human melanoma cells of the cell lineage known as 7272 (available from Denver Hospital, Denver, Colorado; described in J. Biol. Chem., 256 , 7035-7041) were incubated at 10% heat inactivated (56 °C for 30 minutes) killed cow serum (FCS) and antibiotics (penicillin 300 μg/ml, streptomycin 200 mg/ml)
RPMI− supplemented with 10 μg/ml of tyrosine and
1640 was grown as an adherent monolayer in tissue culture media. Aggregated cells (approximately 4 × 10 ⁵ cells/cm ² ) were subjected to trypsin digestion and replanted at 5 × 10 ⁵ cells (100 mm dishes). After aspirating the medium, the cells were diluted with Tris-Dulbetzko saline (Na ₂ HPO ₄ 0.1 mmol/,
KCl 5 mmol/, NaCl 0.14 mol/, Tris
0.25 in a PH7.4 solution containing 24.8 mmol HCl/
% trypsin solution at 37°C for 5 minutes. Dissociated cells were dispersed by gentle pipetting and the dispersion was added to an equal volume of medium containing FCS-containing medium to neutralize proteases. Wash cells by centrifugation at 350G for 5 minutes.
Replanted in a new dish. From this stock culture, cells were grown as adherent monolayers in 75 cm ² or 150 cm ² tissue culture flasks,
Cells were grown in medium in 10% FCS until nearly confluent. To achieve a final pro-TPA:TPA ratio of 1:1 or greater, serum proteolytic enzymes (ie, plasmin) were inhibited or removed as described in more detail in Example 5 or with aprotinin. Cultures were washed once and coated with 20 ml of serum-free medium. After 24 hours, the media was collected and fresh media was added. Harvest liquid to remove whole cells and cell debris.
Centrifugation was performed at 2000 rpm for 5 minutes. TritonX solution
stabilized with −100 (0.1%) or Tween (0.1%);
Acidified with glacial acetic acid (PH 5.5-6.0) and stored at -20°C. Harvest liquid 2 was made up to 0.4 mol/NaCl and filtered through a 0.45 μm membrane. The filtered harvest was applied to the affinity column at room temperature at a flow rate of 45 ml/hr. 4
Collect the effluent at °C. The presence of plasminogen-dependent fibrinolytic activity was determined. No TPA or pro-TPA was detected. After passing the entire harvest through the column, the column was washed with 6 column volumes of phosphate buffered saline (PBS) containing 0.4 mol/NaCl and 0.1% TritonX-100. This method did not elute TPA or pro-TPA. The adsorbed protein is potassium thiocyanate
1.6 mol/, NaCl 0.4 mol/ and 0.1%
It was eluted using PBS containing TritonX-100. Protein content was determined by reading absorption at 280 nm. When potassium thiocyanate is added to the elution buffer, all plasminogen-dependent fibrinolytic activators elute and are seen as a sharp peak, which corresponds to a small peak of protein. The fraction with the highest activity was pooled and incubated at −20°C.
6-8 ml of solution was obtained which was stored at . This fraction represents 70-80% of the activity applied to the column. Fractions with lower activity are pooled separately. The total recovery activity during both reservoirs is typically 90-100%.
Potassium thiocyanate eluate is pure TPA
and a molecular weight 72000 that matches the mixture of pro-TPA
It contains a single band of Dalton's protein, the latter in different runs amounting to about 39-50% or more of the total. Yields can be improved by using the same column over and over again. Columns are not only effective at being reused over and over again, they actually get better with use. Inhibitor DE− when TPA is not in immobilized form
This is surprising since it has the property of adhering to 3. Example 3 Pharmacological formulations containing TPA and/or pro-TPA: - The pooled fraction containing TPA and/or pro-TPA was mixed with 0.3 mol of NaCl containing 0.01% by volume of Tween 80/
Dialyze against aqueous solution and store at -80°C. Before administration, adjust the concentration of TPA and pro-TPA to 75 μg/ml (for therapeutic purposes).
The administration rate of TPA was the same as pro-TPA) and adjusted to 0.3 mol/NaCl. Sterilize the solution by filtration through a 0.22 μm filter membrane. The solution is administered at a rate of 3-15 mg/24 hours, typically
Administer intravenously at 7.5 mg/24 hours. Ideally, the rate of administration would be such that during treatment, the total plasminogen activity (including precursors) level in the blood is between 0.6 and 1;
It should preferably be about 0.8 international units/ml. Treatment at such concentrations has been found to be effective in clearing blood clots;
No systemic fibrinolysis is observed. No other toxicity was observed. However, since pro-TPA is only converted to TPA after binding to fibrin, its actual activity when in the bloodstream is when all plasminogen activators are in their maximally active TPA form. It can be seen that the activity is small, about 10% of the existing activity. Furthermore, pro-TPA is adsorbed faster and more strongly by fibrin than TPA. Promiscuous reactions are further reduced. Example 4 Large scale culture of melanoma cells: - Rosswell Park Memorial Institute medium
Melanoma cells were seeded into spinner flasks filled with 1640 and 10% FCS at a concentration of 5 x ¹⁰⁵ cells/ml. The flask was kept at 37°C and stirred magnetically at 30 rpm. Media is pumped out of the flask at a rate of 1% of flask volume/hour and fresh media in equilibrium with air containing 5% _CO2 is introduced at the same rate. Keep the cells in a culture vessel packed with an outlet glass tube with glass wool. The collected medium is filtered through a Millipore filter. The collected fluid can be pasteurized and cultured according to Example 2. Example 5 Culture of pro-TPA high producing melanoma cells: - Example 2 was modified by adding sufficient soybean trypsin inhibitor or α-l-antiplasmin to inhibit serum plasmin to the culture medium. Otherwise repeat Example 2 and produce the final product.
The TPA to TPA ratio increased to approximately 9:1. Details of the experiment are as follows. Bowes cell medium contained 0.1% Triton and 10 KIU Trasylol (plasmin inhibitor). The original harvest liquid had a volume of 1960 ml. The potassium thiocyanate eluate had a volume of 20.8 ml. The recovery of TPA + pro-TPA was 100%, of which 78% was pro-TPA. When a comparative experiment was performed without Trasylol, the recovery was 98%, which was all TPA and no pro-TPA was detected. Example 6 Removal of residual TPA with a mixture of pro-TPA and TPA: - The solution of TPA and pro-TPA is brought to 10 mmol/diisopropylfluorophosphate. PH 8
Adjust to After 4 hours, TPA is permanently inhibited. The remaining inhibitor is then removed by dialysis. Example 7 Conversion of pro-TPA to TPA: - An aqueous solution containing 100 μg/ml of protein was added to plasmin.
Mix with the same volume of phosphate buffered saline containing 5 μg/ml and incubate at 20°C for 16 hours. Add sodium todecyl sulfate to a final concentration of 0.1%, and precipitate the protein with 6% trichloroacetic acid. The precipitate was washed with acetone, 1% sodium dodecyl sulfate and
Tris HCl 0.06 mol/containing 10% glycerol
Dissolves in a solution with a pH of 6.8. After this treatment, electrophoresis showed that pro-TPA was completely converted to active TPA. Example 8 Selectivity of pro-TPA to insolubilized fibrin: - Contains various proportions of pro-TPA (remaining TPA)
Regarding the sample of TPA/Pro TPA couple,
Tests were carried out under identical conditions to establish in each case the proportion of substance bound to fibrin. Pro in the sample of TPA/Pro TPA couple
The percentage of TPA (remaining TPA) was determined by measuring the activity of the samples before and after treatment with plasmin (to convert pro-TPA to TPA). Then, a correction factor of 10% was applied to correct for the fact that pro-TPA has 10% activity of TPA. 0.1% TritonX−100
When the sample was diluted in a pH 8.1 solution containing 0.1 mol/HCl of Tris-HCl, in the presence of several μg of plasminogen, it was labeled with iodine-125 and coated the bottom of the Limbro wells. Approximately 30-50% of the fibrin was released in 0.2 ml within 1 hour. Small volumes (0.2 ml) of the sample were added four times to four limbrow wells coated with fibrin labeled with radioisotope iodine. After incubation for 1 hour at 0°C, two of the wells were treated with Tris-HCl three times to remove unbound TPA.
Binding activity was then determined. This result was compared to that obtained with unwashed wells. The results are shown in Table 1. The results show that pro-TPA binds fibrin substantially more completely than TPA.

[Table] Example 9 Prophylactic treatment: - Prophylactic treatment was performed in patients at substantial risk of thrombosis or embolism (note: this procedure is contraindicated after surgery or immediately after Partem). ). Suitable patients include bedridden or debilitated patients who are prone to severe venous thrombosis and embolism due to venous cessation, unstable angina patients who are susceptible to coronary thrombotic occlusion, and patients with diseased hearts. Includes patients with cavities or large vessels, prosthetic vascular valves, atrial fibrillation, moderately acute bacterial endocarditis, and patients undergoing intravenous treatment with irritant solutions. Example 10 The plasminogen activating activity of proTPA is not zero like most zymogens, but the double-chain
It was determined that 10% of the activity after complete conversion to TPA. This result shows that Pro TPA50-100% and TPA0
Administration of a TPA/pro-TPA couple consisting of ~50% (which is related to the half-life of the substance in vivo, even at the highest concentration injections) is less than that resulting from administration of an equal volume of TPA. It results in low activity in the bloodstream. This can be understood from Table 2.

【table】

Table 2 shows the reduced potential for promiscuous plasminogen activity in the bloodstream resulting solely from the newly discovered fact that pro-TPA has an activity that is 10% of that of TPA. In addition to this, there is also the newly discovered effect that pro-TPA is selectively (ie, quickly and strongly) adsorbed from the bloodstream as soon as it reaches the fibrin portion (see Example 8).

Claims (1)

[Scope of Claims] 1. A pharmacological composition for the prevention or treatment of thrombosis, embolism or other diseases in which it is desired to produce local fibrinolytic or proteolytic activity through a plasminogen activation mechanism. , a plasminogen activator consisting of 50-100% pro-TPA and 0-50% TPA. 2. The pharmaceutical composition according to item 1, characterized in that it consists of 70-100% pro-TPA and 0-30% TPA. 3. The pharmaceutical composition according to item 1, characterized in that it consists of 80-100% pro-TPA and 0-20% TPA.