Pharmaceutical preparation containing plazmogeneratora

 

(57) Abstract:

The invention relates to medicine. A new pharmaceutical preparation containing plazmogeneratora, phosphate buffer, sugar, tranactional acid and surfactant. The proposed structure is well-tolerated for veins, contains the active substance in high enough concentrations, provides good solubility and stability of plazmogeneratora. 6 table.

The invention relates to pharmaceutical preparations, containing as active substances plazmogeneratora or derivatives thereof, and the respective pharmaceutical forms application in the form of liofilizatow or injection or for infusion solutions.

Human tissue plasmogenerator (t-PA) has great therapeutic value in the dissolution of blood clots, such as heart attacks heart. t-PA causes the dissolution of blood clots by activating plasminogen to plasmin. The plasmin in turn dissolves fibrin, the main protein component of the matrix clotted blood.

Natural t-PA is composed of several functional domains, F, E, K1, K2 and p Domain P coderjoe getting plazmogeneratora (PA), in particular, t-PA or t-PA-muteena in eukaryotic and prokaryotic cells, are already known. While t-PA derived from prokaryotes in contrast to natural t-PA is synthesized in neglikolizirovanny.

As plazmogeneratora fundamentally good in the sense of the invention are, in particular recombinante derived derived t-PA, which essentially include those of natural proteins, which are responsible for fibrinolysis of clots. This can also be used and such derived t-PA, which have deletions or deputies of individual or several amino acids in the sequence t-PA.

According to the invention can be applied, for example, the following plazmogeneratora: t-PA (for example, alteplase), LY210825 (CR derived from cell lines of the Syrian hamster ("Syrian hamster"). Girc. 1990, 82,930-940); FE3x and FEK1 (CCR, Blood 1988, 71, 216-219); FEK1 (K2P from C127 mouse cells, J. Cardiovasc. Pharmacol. 1990, 16, 197-209); E-6010 (Jap. Circ. J. 1989, 53, p. 918); t-PA variants (Thromb. Haemost., 1989, 62, p. 542); K2P and D-CR (Thromb. Haemost., 1989, 62, p. 393); MB-1018 (FK2K2P, Thromb. Haemost., 1989, 62, p. 543); FK2P (FASEB J., 1989, 3, A1031, abstract 4791); 1x (Circulation, 1988, 4, 11-15); KIK2P (Thromb. Res., 1988, 50, 33-41); FK1K2P (J. Biol. Chem., 1988, 263, 1599-1602); TNK-variant t-PA (WO 93/24635); bat-PA (Witt et al., Blood 1992; 79: 1213-1217 and Mullot et al., Arterioscler. Thrombos. 1992; 12: 212-221). In frequent the domain ("P"). For example, in this respect, it should be called t-PA-mutein "r-PA" type CR, which is described in EP 0382174 (WO 90/09437).

The present invention relates, in particular, to plazmogeneratora type K2P, K1K2P, FK1K2P and FK2K2P, as they are described in the following sources: Protein Engineering 5(1), 93-100 (1992); DE-OS-3923339.1; Circ. 1990, 82, 930-940; J. Cardiovasc. Pharmacol. 1990, 16, 197-203; Blood 1988, 71, 216-219; J. Biol. Chem. 1988, 263, 1599-1602; Thromb. Haemost. 1989, 62, S. 543. Especially used recombinant plazmogeneratora type K2P described in EP-A-0382174 and Protein Engineering Vol. 5(1), S. 93-100 (1992). Other t-PA-mutiny of this type are described in the following patent applications: U.S. 4970159; EP-A-0196920; EP-A-0207589; AU 61804; EP-A-0231624; EP-A-0289508; JP63 133988; EP-A-0234051; EP-A-0263172; EP-A-0241208; EP-A-0292009; EP-A-0297066; EP-A-0302456; EP-A-0379890.

In the prior art it is known that the sugar content has a significant influence on the solubility and aggregation of proteins (J. Biol. Chem. 263 (1988), 8832-8837). Thus, in European patent EP-B-458-950, it was found that, for example, neglikolizirovanny recombinant plasmogenerator with the composition of the domains K2P has significantly worse solubility than glycolytically t-PA derivatives. Neglikolizirovanny plazmogeneratora, such as r-PA, as a rule, dissolve only slightly in commonly used to solubilize FR the thief NaCl. For use as a therapeutic agent required, however, to plazmogeneratora was present in sufficiently high concentrations, preferably in concentrations up to 10 mg/ml

From European patent EP-A-0217379 it is known that the solubility of t-PA from prokaryotes can be increased by using a neutral or slightly alkaline arginine compounds. The disadvantage of this method is, however, that the good solubility of t-PA from prokaryotes can be achieved only at very high concentrations of arginine.

Other herbal formulations plazmogeneratora or their derivatives are known from WO 90/01333, WO 89/050347, WO 90/08557, EP 0297294, EP 0156169 and EP 0228862.

Thus, in patent WO 90/01333 (Invitron) describes the combination of lysine, histidine, arginine with citrate for t-PA derived from bacteria. Citrate is applied in an amount of 5 mmol/l, lysine, histidine, arginine in the amount of 150 mmol/l at pH 6. Also add albumin.

In WO 89/050347 (Invitron) describes the combination of arginine (20-200 mmol/l) and citrate (20-80 mmol/l) at pH 5-8.

WO 90/08557 (Genetics Institute) discloses the combination of creatinine with the addition of various substances, such as histidine, arginine, Proline, betaine, Collin, imidazole, tryptophan, C is Orascom patent EP 0297294 (Behring) revealed a combination of at least two amino acids such as lysine, ornithine, arginine, transactiona acid and other additives at pH 5-10.

European patent EP 0156169 (Asahi) describes ornithin and/or lysine, possibly with the addition of citrate, glycine, or phosphate, and EP 0228862 (Genentech) discloses a composition containing arginine chloride, or without, as well as detergent or without.

Alternative structures with r-PA in the presence of lysine or lysine analogues in solution, superyoung citric acid, are described in EP 0458950 (Boehringer Mannheim). More recent studies suggest, however, that the solubility of r-PA in these compounds is not yet fully satisfied. It was found that although the solubility and can be improved by increasing the concentration of citrate, however, such compositions are not compatible with the veins or only conditionally compatible. Along with this, the high salt concentration and the associated low glass transition temperature (Tg') lead to the fact that the lyophilization these compounds have to spend at temperatures below minus 45oC and minus 50oC. These temperatures can be implemented technically often expensive and require expensive controls and control systems for the measurement and regulation of optimal conditions for lyophilization. the onin industrial production using the old lyophilization installation do not possess the necessary complex measuring and regulating equipment, and these units, therefore, can guarantee, as a rule, only a working temperature of about minus 45oC.

The objective of the invention is the preparation of the composition of plazmogeneratora and their derivatives, which is well-tolerated for veins, contains the active substance in high enough concentrations and provides good solubility of the active substance, and the stability of the PA in the lyophilisate for an extended period of time should be preserved. In addition, the aim of the invention is to develop compositions that can be reliably dried and scale of industrial production than should be well reproducible quality freeze-dried product.

This task according to the invention is solved by a pharmaceutical preparation containing plasmogenerator, and the product contains as pharmaceutical additives of at least one sugar and tranactional acid. The drug in dried form can persist for an extended period of time. Water injection solution obtained by reconstitution, also the and the pH of the solution set to a value between 5.5-6.5. As other additives, pharmaceutical preparation may contain the usual buferiruemoi substances or surfactants (anionic, cationic or neutral surfactants).

As an example, for plazmogeneratora (PA), is suitable in the sense of the present invention, used is described in detail in European patent application EP-A-0382174 plasmogenerator K2P (BM 06.022). It consists of Kringel (K2) and proteasome domain (P) human t-PA and due to their expression is presented in the cells: Escherichia coli in neglikolizirovanny form.

As sugar compositions according to the invention contain preferably mono - or disaccharides. Of particularly suitable disaccharides sucrose, trehalose, maltose or lactose. Monosaccharides are, in particular, galactose or appropriate amino sugar, such as galactosamine. Preferably used is not restored sugar is sucrose or trehalose. The concentration of sugar in aqueous injectable solution is typically 40-100 mg/ml, preferably from 50 to 70 mg/ml

As buffer substances, the pharmaceutical preparations can contain commonly used for these purposes, substances presenting a strong sour salt is a salt of phosphoric acid, tartaric acid, malic acid and the like. Also we are talking about the amino acids. The inventive preparations preferably contain phosphate buffer. The concentration of phosphate buffer in the prepared injection solution is, in particular, 50-300 mmol/l, preferably 80-220 mmol/l and particularly preferably 130-170 mmol/l as phosphate buffer is used as a rule, acid disodium or dailypost that phosphoric acid is brought to the appropriate pH value.

As agents of the dissolution of the preparations according to the invention contain tranactional acid (TES). The concentration of tranactional acid in ready for injection aqueous solution is preferably 1-50 mmol/l, preferably 8-12 mmol/L. Particularly advantageous to use a concentration of 10 mmol/L. it has been Unexpectedly found that transactiona acid increases the solubility of plazmogeneratora, in particular neglikolizirovanny of plazmogeneratora, in the aquatic environment. For example, in the case of a derived plazmogeneratora r-PA solubility increased at least 1.5 times in comparison with a solution not containing tranactional acid. Preferably the factor of increasing the solubility lies in the area of 2-3. Atonalities, as with the ability of increasing concentrations of plazmogeneratora in applied solutions can be significantly reduced the amount of water necessary to obtain, in General, achieved energy savings and reduced production costs. In particular, this reduces the time of drying in the production process.

As surface-active substances can be used nonionic, anionic or cationic surfactant, preferably, however, nonionic, such as Tween 80 or Tween 20. The concentration of surfactants ranges from 0.005 to 0.1% (weight/vol.), preferably of 0.01%.

Suitable for the inventive pharmaceutical preparation is a pH between 5.5 and about 6.5, preferably pH of 5.8 to 6.2.

The inventive preparations contain the active substance in concentrations up to 10 mg/ml, preferably from 3 to 5 mg/ml

The pharmaceutical preparations according to the invention are applied in the form of injection or infusion solutions. This is achieved by the fact that you are getting ready for injection solution containing the composition according to the invention. However, it is also possible to prepare pharmaceutical preparations in the form of liofilizatow. Then these measures, water). As injection medium for preparations according to the invention is applied mainly water, which optionally may contain conventional isotonic additives, such as, for example, some physiological concentration of NaCl.

The subject invention is also a method of obtaining a pharmaceutical preparation containing plazmogeneratora or derivatives thereof with a pH from 5.5 to 6.5, and their application to pharmaceutical preparations according to the invention.

In addition, the subject invention is the use of a specific mixture of pharmaceutical excipients consisting of a group of sugar and tranactional acid for long-lasting stabilization of plasmaweather. Particularly favorable for achieving long-term stability during storage the combination of excipients sugar, phosphate buffer, transactiona acid and surfactant.

Stability studies show that the preparations according to the invention is stable in solution at 4oC at least 30 days. The stability of the active substance in lyophilizate according to the invention is at 4oC from two to five years. Person the burden excellent stability, which makes them largely insensitive to temperature fluctuations, which can occur, in particular in regions with a hot climate, in particular the violation of the continuous cooling process. In effect often long delivery routes in the case of pharmaceutical dosage forms from manufacturer's drugs to the end user in different countries, it cannot be excluded that the drugs unintentionally and also, perhaps, for a long time, exposed to temperature fluctuations, leading to loss of activity of the protein, and thereby at least calling into question therapeutic success of their application. Dosage forms according to the invention, however, is largely insensitive to such temperature fluctuations. In the preliminary stability studies were able to show that the drugs, also at temperatures 35oC, they were subjected to during the period of time of 30 days, not have any significant deterioration in the stability of the protein. Therefore, it can be assumed that the preparations when stored at a temperature refrigerating Cabinet will have stability from two to five years the military solution to obtain liofilizatow relatively high and lies between -33oC and -40oC, so that the reproducibility of the quality of the product liofilizatow is also guaranteed at industrial scale with sufficient reliability. The relatively high glass transition temperature, in particular, because favorable, due to a technical or unforeseen increases in temperature during the often very long time lyophilization risk of unintended thawing of frozen solutions less likely. In cases where the glass transition temperature is below -40oC and during lyophilization process this temperature is exceeded, which in particular takes place on lyophilization facilities operating at temperatures near -45oC, an undesirable excess of the glass transition temperature can lead to adverse changes in frozen solution. Good quality product then it is not guaranteed. Thus, in particular, proteinogram of liofilizatow may occur loss of activity or the formation of aggregates. Another advantage of the preparations according to the invention with a glass transition temperature from -33oC -40oC is that minimized the cost of energy on the process of freeze-drying, as the process Ego of the invention a relatively high glass transition temperature for frozen solutions are achieved in particular, when as a phosphate buffer for lyophilization the desired solution is applied potassium salt, for example disubstituted or one-deputizing potassium phosphate. In particular, in this regard, suitable disubstituted phosphate potassium concentration of 20-40 mg/ml, preferably 20-30 mg/ml, the temperature of vitrification can be reinforced by the addition of sucrose. Sucrose added for this purpose mainly in the concentration of 60-90 mg/ml, in particular 60-80 mg/ml, Especially preferably apply a solution containing about 26 mg/ml disubstituted phosphate potassium (K2HPO412H2O) 70 mg/ml sucrose. The pH of a solution is preferably set with 85% phosphate acid (approx. 12 mg/ml) to the value 6. Then, the solution contains about 0.5-5 mg/ml, preferably 1-2 mg/ml of tranactional acid, in particular about 1.6 mg/ml in Addition, may also contain surfactants, such as Tween 80, which is used preferably in a concentration of 0.01-0.3 mg/ml, in particular about 0.1 mg/ml

Another advantage of liofilizatow according to the invention is that they have a relatively small residual moisture after lyophilisation. Estato is for liofilizatow amount value above 6%. Low residual moisture helps to ensure that the drugs have better storage stability. Drugs with higher residual moisture often lead to instability of the protein, which becomes noticeable due to the loss of biological activity or the formation of aggregates.

Another advantage of the preparations according to the invention is that upon receipt of liofilizatow on the basis of increasing the solubility deglycosylation of plazmogeneratora achieved by the addition of tranactional acid, in particular in the case of Malinov type K2P, K1K2P or P, it can be assumed smaller volumes (for example approx. 5 ml in a single dosage form) freeze fluids, lyophilization reduced considerably in comparison with used so far to obtain liofilizatow solutions (for example, about 10 ml per dosage form). Preferably originate from solutions containing the active substance in approximately twice higher concentration compared with the finished water for injection dosage forms, so that instead of the usual still volumes of solutions in 10 ml can be applied in a water volume of 5 ml.

Studies venous perenosimost following examples run which do not limit the invention.

Example 1:

Turbidity after mechanical stress/measure light scattering preparations according to the invention

r-PA (BM 06.022) brought to the protein concentration (CFR) 6 mg/ml (ultrafiltration through a membrane Amicon YM-10) and deliberately against the specified buffer. Then the samples were mounted on the concentration of CFR= 4 mg/ml and a) were left unchanged, (b) was added to 0.01% Tween 80 and (c) was added 0,0-1% Tween 20.

Loading of samples was carried out respectively for 10 to Whirl Mix (Janke &Kunkel IKA-Labortechnik, the maximum number of revolutions). Then samples were incubated for 2 minutes at room temperature.

The light diffusing unloaded and loaded samples was measured fluorometrically (Ex. 360 nm, Em.: 360 nm; bandwidth Ex.: 3 nm; bandwidth Em.: 10 nm; measurement interval: 10 seconds for 3 minutes).

Example 2:

Storage of the compositions according to the invention containing r-PA (BM 06.022) TES/sucrose,// the repeated freezing and thawing

r-PA were dialyzed against specified in the table. 1 buffer (without Twen 30), was established on the concentration of CFR= 4 mg/ml, was added Tween 80 to p = 0,01%, portionable and sterilized. Froze, smo is Roma control, thawed (15 minutes at 25oC in a water bath). Each time he took one sample for analysis (CFRand amidopirina activity). The remaining samples again deep frozen at -20 or -70oC. At the end of the series was determined by the activity of the control group not subjected to stress.

The result: As can be seen from table 1, r-PA may without loss of activity frozen or thaw at least eight times.

Example 3:

The stability of r-PA in solution

Comparison of different compounds with CFR= 4 mg/ml

r-PA (BM 06.022) focused through the membrane 10 to 5 mg/ml were dialyzed against specified in table 2 buffer (without Tween 80). The dialysates were installed on CFR= 4 mg/ml, was added Tween 80 0.01%, was portionately and kept at -20oC and at 4oC. After 7, 14, 20, and 30 days was determined amylolyticus activity and the concentration of protein loaded samples.

Results: Samples were stored at -20oC and 4oC remain unchanged after 30 days (see table. 2).

Example 4:

The stability of r-PA in solution

Comparison of different compounds WITHFR= 6 mg/ml

r-PA (BM 06.022) deliberately during the night against the following buffer and is rcii and kept for 30 days at -20oC and 4oC. After 1, 2, 5, 9, 15 and 29 days, respectively, was determined activity and protein content(see table. 3).

Results: From the data table. 4 it follows that the active substance BM 06.022 in the said composition is stable at -20oC and 4oC at least 29 days.

Example 5:

The solubility of r-PA in the compositions according to the invention

r-PA (BM 06.022) focused through YM 10 to 6 mg/ml were dialyzed against nienazwane buffer. The dialysates then concentrated through the membrane Amicon YM 10 until, until turbidity. After centrifugation of the samples of the supernatant was kept for five days at 4oC. At the beginning and at the end of storage was determined amidopirina activity and CFR.

Results: the solubility of r-PA is about 10 mg/ml With a maximum protein concentration of the samples at 4oC can be maintained without changing amylolyticus activity at least 5 days (see table. 5).

Example 6

Preparation of liquid dosage forms

The following applications have been received as the solution before lyophilization:

The composition of the solution before lyophilization:

Solution A:

BM 06.022 4 mg/ml

Na2HPO412H2O - 53,72 mg/ is pH 6 - 0.1 mg/ml

Solution B:

BM 06.022 4 mg/ml

K2HPO412H2O - to 26.2 mg/ml

H3PO485% - 11.6 mg/ml

Transactiona acid - 1.6 mg/ml

Sucrose - 70 mg/ml

Tween 80, pH 6 0.1 mg/ml

Getting liofilizatow:

After sterile filtration of aliquots of 5 ml were collected in the appropriate flask with a capacity of 20 ml each. The lyophilization was carried out as follows: the filled flask was placed in the freeze dryer and freeze at temperatures from -40oC to -50oC (the temperature of the plates) for 10 hours at atmospheric pressure. Then the camera was created by the vacuum value from p = 0.01 to 0.1 mbar. The temperature of the plates was then set so that the temperature of the product at any time was below the respective glass transition temperature. After all the ice tabletirovanne, the temperature of the plates is increased to a value of 20-40oC and then spent an additional drying in vacuum. Residual moisture was determined by conventional methods (Karl Fisher), and it was the solution to A 6%, from a solution B - 3%. Storing liofilizatow for studies of stability occurs at a temperature of 4oC (temperature refrigerating Cabinet), 20oC (room and concentration of protein, the stability of liofilizatow after a storage time of four to twelve weeks to complete.

Reconstitute of liofilizatow for use:

Lyophilizate was filled with water for injection purposes to 10 ml This corresponds to a dilution in two times compared to the initial volume of the solution prior to lyophilization.

Example 7:

The test of public acceptability of the compositions according to the invention

Prepare two valid and two idle control solutions listed in table 6 compositions and administered to rabbits intravenously. The applied amount is 0.5 ml/animal, for each of the four test solutions used five animals.

Result: the Response of the animals to the injection and histological data analysis show that all applied subjects solutions are well tolerated.

Pharmaceutical drug-based plazmogeneratora and conventional additives, characterized in that it contains

Plasmogenerator - 3 - 10 mg/ml

Sugar - 40 - 100 mg/l

Phosphate buffer - 50 - 300 mmol/l

Transactiona acid - 1 - 50 mmol/l

Detergent (surfactant) - 0,1%

at pH values from 5.5 to 6.5.

 

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