CA1338395C - Pharmaceutical containing tissue protein pp4, a process for the pasteurization of pp4, and the use of pp4 - Google Patents

Abstract

An agent for the therapy or prophylaxis of disturbances of hemostasis, which contains tissue protein PP4, a pro-cess for the purification of PP4 by means of a hydro-phobic adsorbent which is insoluble in water, and a pro-cess for the pasteurization of a solution containing PP4 in the presence of calcium ions and of at least one mono-or oligosaccharide or sugar alcohol and, where approp-riate, of an amino acid, and the use of PP4, are des-cribed.

Description

~ I

Pharmaceutical Cont~;n;ng Tissue Protein PP4, a Process for the Pasteurization of PP4 and the Use of PP4 The invention relates to an agent containing tissue pro-tein PP4 for ;nhibiting blood clotting, to a process for the preparation of PP4 and a process for the pasteur;za-tion thereof, and to the use of PP4.

The glycoprotein PP4, uhich is found, for example, in placenta, is described in DE 33 15 000 A 1 (US Patent 4,507,229). PP4 has a molecular ~eight of 35,000 ~ 5,000 Dalton and an ;soelectr;c range of pH 4.85 ~ 0.15.

Anticoagulant prote;ns can be d;v;ded into several groups on the basis of their mode of action. Both the group of proteinase inhibitors such as, for example, antithrombin III, and that of the proteinases, for example activated protein C, are associated ~ith some disadvantageous pro-perties for anticoagulant therapy, because they either do not act unt;l the act;vated clott;ng enzyme stage or inact;vate clotting factors by proteolysis thereof, "consum;ng" them.

Hence the object of the invent;on ~as to make available an agent ~hich inh;b;ts blood clott;ng and is able to prevent blood clotting ~ithout resulting in consumption of clotting factors. - -DE 3S 33 516 A 1, (pages 6 to 8) describes anticoagulantproteins ~hich are reported to have an isoelectric range of 4.4 - 4.6. These proteins are isolated from the intima of major vessels, for example the aorta or umbi-lica~ cord.

There is like~ise a descr;pt;on, in Acta Obst. Gynaec. ~ -Jpn. 36, No. 12, 2583-2592 (1984), of an ant;coagulant protein from placenta wh;ch has a molecular weight of 45,000 Dalton. Moreover, the properties of PP4 are des-cribed as follows in DE 33 15 000 A1 (US Patent 4,507,229):
an electrophoretic mobility in the range between that of alpha1 and alpha2 globulins; a sedimentation coefficient S20 w of 3.3 + 0.2 S; an extinction coefficient E1% m (280 nm) of 5.9 + 0.6; a carbohydrate content of 2.4 + 0.94X (9/100 9) (mannose 0.3 + 0.2%, galactose 0.4 + 0.2%, xylose 0.1 + 0.04%, glucose 0.2 + 0.1%, glucos-amine 1.0 + 2X, neuraminic acid 0.4 + 0.2%) and the following amino acid composition:

Residues per 100 residues Coefficient Amino acid (mol-X) of variation Lysine 6.95 1.14 Histidine 0.97 17.4 Arginine 5.44 1.77 Aspartic acid 11.41 1.68 Threonine 6.78 2.40 Serine 6.21 2.26 Glutamic acid 12.25 0.43 Proline 1.96 6.20 Glycine 6.68 3.83 Alanine 7.92 1.67 Cystine 1/2 0.77 19.5 ~aline 5.34 3.80 Methionine 1.98 6.00 Isoleucine 5.21 2.23 Leucine 11.50 0.45 Tyrosine 3.55 4.21 Phenylalanine 4.07 3.77 Tryptophan 0.93 23.9 -It has been found, surprisingly, that this glycoprotein PP4 has anticoagulant properties.
Thus the invention relates to the use of the glycoprotein ~ ~ 3 - 13383 95 PP4 for inhibiting blood clotting.

The invention also relates to an agent for the therapy or prophylaxis of disturbances of hemostasis, ~hich con-ta;ns an effective amount of PP4 and a pharmaceutically suitable veh;cle and/or stabilizer.

In a solution, an agent of this type preferably contains 0.001 - 100 mg of PP4 per ml of solution. It is admin;s-tered in a dose of, for example, û.001 - 50 mg per kg of body weight once or several times a day as a bolus or as a continuous drip.

A PP4-containing agent of this type is preferably pre-pared either in the form of a lyophilisate, ~here approp-riate ~ith stabilizers and additives such as, for example, albumin, gelat;n, salts, sugars and/or amino ac;ds, or ;n the form of an aqueous solution ~h;ch ;s ;soton;c ~;th blood and conta;ns, ~here appropr;ate, a bacter;cide and stabilizers such as, for example, albumin, gelatin, salts, sugars and/or amino acids. The solution can be administered, for example, by parenteral injection or perfusion.

It is also possible to use in the sense according to the invention a PP4 which has been prepared by gene manipula-tion.

Relatively large amounts of PP4 are needed for use as pharmaceutical. Th;s ;s ~hy a straightfor~ard process for isolation from tissues is desirable.

$t has been found, surpr;singly, that PP4 from a t;ssue extract or a solution can be bound to a hydrophobic carrier matrix and then, by stepped or gradient elution, can have impurities very extensiveLy removed.

Hence the invention relates to a process for the purifi-cation of PP4, which comprises contacting a solution _ 4 _ 13~8395 containing PP4, preferably a t;ssue extract contain;ng PP4, ~ith a hydrophobic adsorbent ~hich is insoluble in water, removing the adsorbent from the supernatant solu-tion, ~here appropriate ~ashing ~ith a buffer, and elut-ing the PP4.

It is also possible, before or after this process, to carry out, ~here appropriate, other kno~n purification processes such as ion exchange chromatography, gel fil-tration or adsorption onto calcium phosphate or hydroxy-1û apatite.

In a preferred procedure for purifying PP4, the solutioncontaining PP4 is, at a pH of 5.5 - 9.5, a temperature of 0C to 40C and after addition of salts, for example after addition of 50 - 250 9 of ammonium sulfate/l of solution, magnesium sulfate or 0.5 - 3 mol/l NaCl, LiCl or KCl, contacted ~ith a hydrophobic adsorbent ~hich is insoluble in ~ater, for example a carrier material ~hich has aliphatic, aromatic or araliphatic groups. Examples of carrier materials ~hich can be used are RSepharose, agarose or RFractogel. The aliphatic groups ~hich are preferably used are alkyl groups having 1 to 10 carbon atoms, for example the butyl or octyl radical, as ~ell as, for example, optionally substituted phenyl or phenyl-alanyl radicals or benzyl or substituted benzyl radicals.
The adsorbent is then removed from the solution, ~here appropriate ~ashed ~ith a solution ~hich contains, for example, 50 - 250 g/l ammonium sulfate or 0.5 - 3 mol/l NaCl, LiCl or KCl, and the PP4 is eluted using a liquid of relatively lo~ ionic strength, where appropriate by 3û means of a gradient.

In a particularly preferred embodiment, the solution con-taining PP4 is, at a pH of 6 - 8 and after addition of a solution containing 144 - 243 9 of ammonium sulfate/l, contacted ~ith, for exampLe, phenyl- or phenylalanyl-RSepharose or agarose, but is very particularly prefer-ably contacted at pH 6.5 - 7.8, and after addition of a ~ 13~8~95 solution contain;ng 170 - 200 9 of ammonium sulfate/l, with phenyl-RSepharose. The adsorbent is then removed from the solut;on, washed w;th, for example, a solut;on which contains 176 - 196 9 of ammonium sulfate/l of solu-tion, and the PP4 is eluted by means of a solution whichcontains 114 - 144 9 of ammon;um sulfate/l of solut;on.

It is furthermore exped;ent, because of the r;sk of transmission of diseases (hepatitis, AIDS), for biologi-cal therapeutics which are ;ntended for use ;n humans to be heated to k;ll ;nfectious organisms. However, this usually means loss of act;v;ty of the b;olog;cal act;ve substances.

It has been found, surpr;singly, that an aqueous solut;on of PP4 can be heated ;n the presence of calc;um ;ons and of at least one mono- or ol;gosacchar;de or sugar alcohol and, where appropriate, an amino ac;d, there be;ng very extens;ve retent;on of the ant;coagulant property.

Hence the ;nvent;on also relates to a process for the pasteur;zat;on of a solut;on contain;ng PP4, wh;ch com-prises heat;ng this solution in the presence of calc;um;ons and of at least one mono- or oligosaccharide or sugar alcohol and, where appropriate, an amino ac;d under conditions such that pathogenic organisms are killed.

In a preferred procedure for the pasteurizat;on of a solution containing PP4, the pH of the solution ;s adjus-ted to 5.0 - 9.5, and 30 - 150 9/100 ml of solut;on of at least one mono- or oligosaccharide or sugar alcohol and 0.005 - 2 mol/l calcium ions and, where appropr;ate, 0.1 - 1.5 mol/l of an am;no ac;d are added. The calcium ;ons can be introduced in the form of, for example, cal-cium salts, for example as CaCl2 or calcium acetate.

In a very part;cularly preferred embodiment, 100 - 150 9 of sucrose/100 ml of solution and 30 - 100 mmol/l CaCl2 are added to a solution wh;ch conta;ns PP4 and has a pH

~ 1338395 of 6.5 - 8.5. After the ingred;ents have dissolved it is possible to heat the solution, for example, at 40 -80C for 1 to 20 hours, but preferably at 55 - 65C
for 8 - 15 hours. The content of PP4 should not be below 20 ~g/ml of solution. After the heat;ng, it is possible to process the solution further, for example by concen-tration, d;alysis, sterile filtration and/or lyophiliza-tion.

A product obtained by the preparation and pasteurization processes described above is particularly distinguished by the very extensive removal of impurities from PP4, and its anticoagulant action being very extensively present.

A PP4 pharmaceutical can be manufactured in the custom-ary manner using a physiologically acceptable and pharma-ceutically suitable vehicle in solid or liquid form and,where appropriate, further additives such as stabilizers and auxiliaries.

Thus the invention also relates to a process for the preparation of a PP4-containing pharmaceutical for the therapy and/or prophylaxis of disturbances of hemostasis, which comprises preparation of a PP4-containing solution, to which, where appropriate, additives and stabilizers, such as, for example, albumin, gelatin, salts, sugars and/or amino acids are added, and converting this solu-tion to a dry form, ~here appropriate a lyophilisate.

The examples which follow are intended to illustrate theinvention.

Example 1 Isolation of PP4 from placental tissue, and the pasteurization thereof 2 kg of deep-frozen placenta ~ere homogenized in a tissue mincer. The homogenizate was ~ashed 3 times with 1 liter of physiological NaCl solution each time. The placental tissue was removed from the supernatant solution by centrifugation. The washed placental homogenizate was suspended in 3 liters of RTriton X 100-containing buffer A ~20 mmol/l tris.HCl, 50 mmol/l NaCl, 2 9/100 ml RTr;ton X 100, pH 7.4) and the suspension was stirred overn;ght. The extract was removed by centrifugation, and the tissue was extracted once more w;th 2 liters of buffer A overnight. The two extracts were combined ~4.8 liters) and diluted with buffer B (20 mmol/l tris.
HCl, 50 mmol/l NaCl, pH 7.5) to 15 l. 0.75 9 of DEAE-RSephadex A 50/l of solution was added, and the suspen-sion was then stirred for 1 hour, and the adsorbate was removed from the solution, washed with 40 liters of buffer B and then w;th 600 ml of buffer B wh;ch contained 100 mmol/l NaCl, and PP4 was eluted with buffer B which contained 500 mmol/l NaCl. 176 g/l ammonium sulfate were added to the eluate (600 ml), and the mixture was treated with 300 ml of phenyl-Rsepharose either in a batch pro-cess or on a column.

The phenyl-RSepharose was equilibrated in buffer B wh;ch contained 3/10 of the saturation concentration of ammon-ium sulfate. After washing with the same buffer, PP4 was eluted with buffer B which contained 1/5 of the satura-tion concentration of ammonium sulfate. The PP4 wasprecipitated by increas;ng the ammon;um sulfate concen-tration to 85 9/100 ml or ~as concentrated by means of ultrafiltration and further purified by gel filtration on ACA 54. The ACA 54 column (3 x 100 cm) was equilibra-ted in buffer B ~hich conta;ned 500 mmoL/l NaCl. Thefractions containing PP4 were combined.

~here necessary, this can be followed by a pasteurizat;on as described in Example 3. However, the heating can also be carried out at another stage, such as the DEAE-RSepha-dex A50 eluate or the eluate from the phenyl-RSepharose treatment.

~ - 8 - 13~8395 Example 2 Determination of the anticoagulant activity of PP4 Modified prothrombin time (PT) and modified partial thromboplastin time (PTT) determinations ~ere used to determine the anticoagulant act;vity of PP4 prepared as ;n Example 1.

a) Modified PT determination 150 yl of buffer A (20 mmol/l tris.HCl, 142 mmol/l NaCl, pH 7.5), 25 ul of sample or buffer B (20 mmol/l tris.HCl, 500 mmol/l NaCl, pH 7.5) and 25 )Jl of thromboplastin solution were added to 50 ,ul of stand-ard human plasma. After incubation at 37C for three m;nutes, clotting ~as started by addition of 250 yl of a solution C containing CaCl2 (10 mmol/l tris.HCl, 80 mmol/l NaCl, 20 mmol/l CaCl2, pH 7.9) and the clotting time was determined in a Schnitger-6ross coagulometer. The clotting time of the control con-taining buffer B uas 60 sec.

Dependence of the clotting time in the modified PT
determination on the content of PP4 in the solution PP4 concentration (yg/ml) 0 5 10 15 20 modified PT (sec) 60 68 89 111 132.5 b) Modified PTT determination 25 yl of sample and 75 yL of buffer A \~hich contained 100 mmol/l NaCl ~ere added to 100 ul of standard human plasma, the mixture was incubated at 37C for 3 min, 50 ,ul of RPathromtin reagent solution ~ere added, incubation ~as continued at 37C for 6 min, and then 50 yl of kaolin suspension ~ere mixed in and, after a further 2 min at 37C, the clotting ~as started by addition of 100 yl of Cacl2-solution C.

-- 133839~
_ 9 _ The sample had previously been dialyzed against buffer A which contained 500 mmol/l NaCl. The contents of one conta;ner of RPathromtin reagent solution ~ere dissolved in 1 ml of 9 g/l NaCl solution. The clotting time ~as determined in a Schnitger-Gross coagulometer.
The clotting time of the control containing buffer A
~as 70 sec.

Dependence of the clotting time in the modified PTT
determination on the content of PP4 in the solution PP4 concentration (,ug/ml) O 13 17.7 26 modified PTT (sec) 70 84 98 129 Example 3 Pasteurization of a solution containing PP4 500 ml of a solution containing PP4 (100,ug of PP4/ml) were dialyzed against 20 mmol/l tris.HCl, 150 mmol/l NaCl, pH 7.5. Then 100 9 of sucrose 100 ml of solu-tion ~ere ~eighed in. Once the sucrose had completely dissolved, 50 mmol/l CaCl2 were added. The solution can, after the heating (60C/10 h), be dialyzed against 20 mmol/l tri-Na citrate, 60 mmol/l NaCl, 10 g/l glycine, pH 7.0, and sterilized by filtration and lyophilized.

Pasteurization of PP4 (100 ug/ml) in solution - effect of calcium ions on the stability of PP4 Modified PT (sec) before heating heating ~ithout heating ~ith calcium 50 mmol/l calcium

Claims (10)

1. An agent for the therapy or prophylaxis of disturbances of hemostasis, containing an effective amount of PP4 and a pharmaceutically suitable vehicle and/or a stabilizer.

2. An agent as claimed in claim 1, which is in the form of an aqueous solution which is isotonic with blood.

3. An agent as claimed in claim 2, which contains a bactericide.

4. An agent as claimed in claim 1, which is in the form of a lyophilisate.

5. An agent as claimed in claim 4, which contains additives and stabilizers.

6. An agent as claimed in claim 1, in liquid form and containing 0.001 to 100 mg of PP4 per ml.

7. An agent as claimed in claim 1, in lyophilized form.

8. A process for the pasteurization of a solution containing PP4, which comprises heating this solution in the presence of calcium ions and of at least one mono- or oligosaccharide or sugar alcohol under conditions such that pathogenic organisms are killed.

9. A process as claimed in claim 7, wherein the solution is heated in the presence of calcium ions and of at least one mono-or oligosaccharide, or sugar alcohol, and an amino acid.

10. The use of the tissue protein PP4 for inhibiting blood clotting.