Pharmaceutical for subcutaneous or intramuscular administration containing polypeptides

Liquid pharmaceuticals for subcutaneous (sc) or intramuscular (im) administration containing a polypeptide and at least one amino acid, a process for the preparation of such a pharmaceutical and the use of an amino acid, which is suitable for sc or im administration, in a solution of a polypeptide are described.

The invention relates to a liquid pharmaceutical for subcutaneous (sc) or 
intramuscular (im) administration containing a polypeptide and at least 
one amino acid, a process for the preparation of such a pharmaceutical and 
the use of an amino acid, which is suitable for sc or im administration, 
in a solution of a polypeptide. 
The presentation forms of proteins to be used therapeutically are with a 
few exceptions limited to intravenous administration. Oral administration 
of polypeptides is in many cases unsuccessful, as the polypeptides are 
degraded in the gastrointestinal tract. It has already been attempted with 
the aid of various methods, for example by micro encapsulation in 
liposomes, to develop orally administrable presentation forms of 
polypeptides. Until now, however, these methods were only suitable for low 
molecular weight polypeptides. 
It has therefore been attempted to administer the polypeptides sc or im. 
However, in this case it turns out that sc or im administration, in 
comparison to classical iv administration, offers a distinctly poorer 
bioavailability or can be affected by local intolerability reactions. 
However, sc and im administration forms offer potentially important 
advantages in comparison to iv administration. Among these are included, 
in particular, the possibility of self-treatment for the patients and a 
longer-lasting action of the therapeutic. 
In WO 86/06962 and EP-A-0,292,908, methods for sc or im administration of 
proteins in the presence of hydroxylamine or methylamine are described. 
However, it is known that these substances can only be used in a 
restricted manner owing to their limited tolerability. 
The object of the present invention was therefore to make available an 
agent containing a polypeptide, which is suitable for sc or im 
administration.

This object is achieved according to the invention in that at least one 
amino acid, or a salt, derivative or homolog of an amino acid, is 
incorporated in the polypeptide-containing solution. Preferably, a D- or 
L-amino acid is incorporated. 
It has surprisingly been shown that the addition of such a substance 
drastically improves the bioavailability and tolerability of polypeptides 
administered sc or im and thus makes possible their sc or im 
administration. 
Substances suitable for the purpose according to the invention are, for 
example, lysine, ornithine, arginine, diaminopimelic acid, agmatine, 
creatinine, guanidino-acetic acid, acetylornithine, citrulline, 
arginino-succinic acid, tranexamic acid or epsilon-aminocaproic acid. A 
feature common to them all is the presence of a basic group in the form of 
an amino or guanidino group. 
A particularly suitable combination has proved to be arginine and lysine, 
preferably 0.001 to 1 mol/l, particularly, preferably 0.01 to 0.5 mol/l. 
The bioavailability of polypeptides administered sc or im is distinctly 
increased by such an addition in comparison to a solution which contains 
the polypeptide and, if desired, customary additives such as stabilizers. 
This favorable influence of these substances on the bioavailability and/or 
improvement in the tolerability could be observed particularly for t-PA 
(tissue plasminogen activator), a t-PA mutant, hirudin, urokinase, AT III 
(antithrombin III), factor XIII, EPO (erythropoietin), yon Willebrand 
factor and PP4 (placenta protein 4). The activity of the proteins to 
which, according to the invention, the described substances were added is 
maintained even after lyophilization and dissolving before administration 
in sterilized water. 
In an embodiment according to the invention, a procedure is used in which 
the polypeptide-containing solution is dialyzed against a buffer; for 
example a phosphate, tris-glycine, acetate or citrate buffer, having a 
concentration of 0.001 to 0.1 mol/l, preferably of 0.01 to 0.05 mol/l, 
having a pH of 2 to 10, preferably of 4 to 9, containing at least one 
amino acid which can contain another amino group or a-guanidino group, or 
an amino acid derivative, for example lysine, ornithine, arginine, 
diaminopimelic acid,-agmatine, creatinine, guanidino-acetic acid, 
acetylornithine, citrulline, arginino-succinic acid, tranexamic acid or 
epsilon-aminocaproic acid, preferably lysine, ornithine or arginine in a 
concentration of 0.005 to 0.5 mol/l, preferably of 0.01 to 0.3 mol/l, at a 
temperature of 2.degree. to 30.degree. C., preferably of 4.degree. to 
15.degree. C. 
The conductivity of the dialysis solution is preferably 1 to 50 mSi 
(20.degree. C.), preferably 5 to 30 mSi (20.degree. C.). The osmoeality is 
10 to 2000 mOsmol, preferably 100 to 1000 mOsmol. 
After completion of the dialysis, the polypeptide-containing solution is 
adjusted to the desired final concentration by concentration or dilution. 
The solution is then sterile filtered, bottled and, if desired, 
lyophilized. 
The solutions according to the invention are equally suitable for use in 
human and veterinary medicine. As solely physiologically tolerable 
substances are added, neither inflammation nor skin irritation or vascular 
damage occurs in the administration of the solutions according to the 
invention. 
Using the preparation forms according to the invention, it is now possible 
by means of sc or im administration to achieve plasma concentrations of 
polypeptides which make possible both prophylaxis and therapy. 
The examples illustrate the invention. 
EXAMPLE 1 
Preparation of the protein solutions for sc and im administration 
1. r-t-PA (.sup.R Actilyse) was purchased from Boehringer Ingelheim. 
2. Urokinase (UK) (.sup.R Actosol) was obtained from Behringwerke. P0 3. F 
XIII (.sup.R Fibrogammin) was obtained from Behringwerke. 
4. Antithrombin III (AT III) (.sup.R Kybernin) was obtained from 
Behringwerke. 
5. r-Hirudin was prepared by the process of EP 0,316,650. 
6. r-t-PA mutant (No. 1) was prepared by the process of EP 0,227,462. 
7. von Willebrand (vW) was prepared by the process of DE 3,904,354 (1 mg of 
vW corresponds to 100 U of vW antigen). 
8. PP4 was prepared by the process of EP 0,123,307. 
9. r-erythropoietin (EPO) was prepared by the process of EP 0,123,307 (1 mg 
of EPO corresponds to 80,000 U of EPO antigen). 
The proteins 1 to 4 were dissolved in distilled water. The proteins 1 to 9 
were then dialyzed against the following buffers (Table 1). 
TABLE 1 
__________________________________________________________________________ 
r-t-PA- 
Uro- 
t-t-PA 
mutant 
kinase 
FXIII 
ATIII 
r-Hirudin 
vW PP4 
rEPO 
P** 
Final concentration 
1* 0.5* 
3.1* 
5.5 mg 
8.3* 
1.2* 150 U/ml 
5* 200 U/ml 
__________________________________________________________________________ 
1 Dialysis against 
x x x x x x x x x 
0.05 M tris 
0.1 M NaCl 
2 idem pH 4.5 
x x 
3 0.2 M arginine 
x x x x x x x x x 
0.2 M lysine pH 7.5 
4 0.2 M arginine 
x x 
0.2 M lysine pH 4.5 
5 0.05 M tris (l) 
x x 
0.1 M NaCl pH 7.5 
0.1% Tween 80 
50* hydroxylamine 
6 idem pH 4.5 x 
7 0.05 M tris (1) 
x x 
*0.1 M NaCl pH 7.5 
*0.1% Tween 80 
3% dimethyl sulfoxide 
8 0.2 M arginine pH 7.5 
x 
9 0.2 M lysine pH 7.5 
x 
__________________________________________________________________________ 
(1) according to PNAS 82, 4258-4262 (1985); *mg/ml; **buffer 
After the dialysis, the solutions obtained are sterile filtered and frozen 
at -20.degree. C. until use. 
EXAMPLE 2 
Each ml of the thawed solutions from Example 1 was administered sc and im 
(femoral biceps) in animals (rats or rabbits). In the case of im 
administration, the total volume of 1 ml was divided between four types of 
injection. 
The animals were treated as follows: 
______________________________________ 
Number of 
Animals 
Rats Rabbits 
Test Substance 
Dose per group 
______________________________________ 
r-t-PA 3.3/8.2 mg/kg 2 3 
Urokinase 5 mg/kg 3 
F XIII 10 mg/kg 3 
AT III 10 mg/kg 3 
r-Hirudin 10 mg/kg 3 
r-t-PA-mutant 
1.7 mg/kg 2 
von Willebrand 
300 U/kg 3 
PP4 10 mg/kg 3 
EPO 2000 U/kg 3 
______________________________________ 
After 0, 15, 30, 60, 120, 180, 240 and 340 minutes (r-t-PA mutant) or 0, 
30, 60, 120, 180, 240, 300, 360, 420 and 450 minutes (r-t-PA, hirudin, 
urokinase AT III, F XIII, von Willebrand, EPO, PP4), blood samples (+ 
citrate) were taken. The blood samples were then immediately centrifuged 
and the resulting citrate plasma was frozen at -20.degree. C. until the 
determination. The plasma concentration of the test substance was measured 
as follows: 
______________________________________ 
r-t-PA ELISA (Biopool, Sweden) 
Urokinase ELISA (Biopool, Sweden) 
F XIII: Activity test 
(Behringwerke) 
Antithrombin III: 
ELISA (Behringwerke) 
r-Hirudin: ELISA (Behringwerke) 
r-t-PA mutant ELISA (Biopool Sweden) 
von Willebrand: 
ELISA (Stago, France) 
PP4: ELISA (Behringwerke) 
r-Erythropoietin: 
ELISA (Behringwerke) 
______________________________________ 
The data determined were represented graphically, as shown in FIGS. 1 and 
2. The area under the curve (AUC) was then calculated. This value is an 
expression of the bioavailability and makes possible comparison between 
the different presentation forms. The data show that the administration of 
the solutions according to the invention, with the exception of urokinase 
and hirudin ensures a distinctly better bioavailability, in particular in 
the case of sc administration. 
Surprisingly, it has been found that the hemorrhages occurring on sc and im 
administration of hirudin and of urokinase at the administration site 
using the presentation form according to the invention occur distinctly 
less than when these substances are administered in their customary 
presentation forms. 
______________________________________ 
Number of animals with hemorrhages 
im sc 
______________________________________ 
Hirudin 0.05 M tris 3/3 2/3 
0.1 M NaCl (1 animal died as a 
result of the hemor- 
rhages) 
Hirudin 0.2 M arginine 
0/3 0/3 
0.2 M lysine 
pH 7.5 
Urokinase 0.05 M tris 2/3 2/3 
0.1 M NaCl 
pH 7.5 
Urokinase 0.2 M arginine 
1/3 0/3 
0.2 M lysine 
pH 7.5 
______________________________________ 
______________________________________ 
I. Bioavailability of t-PA in the rat 
Type of 
Buffer Administration 
n AUC + Variation (%) 
______________________________________ 
1 im 2 358 .+-. 10% 
sc 2 36 .+-. 13% 
2 im 2 386 .+-. 2% 
sc 2 53 .+-. 14% 
3 im 2 756 .+-. 2% 
sc 2 423 .+-. 3% 
4 im 2 669 .+-. 17% 
sc 2 382 .+-. 11% 
5 im (all animals died) 
sc (all animals died) 
7 im 2 184 .+-. 18% 
sc 2 87 .+-. 12% 
8 im 2 600 .+-. 4% 
sc 2 320 .+-. 2% 
9 im 2 610 .+-. 10% 
sc 2 290 .+-. 11% 
______________________________________ 
______________________________________ 
II. Bioavailability of t-PA mutant in the rat 
Type of 
Buffer Administration 
n AUC + Variation (%) 
______________________________________ 
1 im 2 113 .+-. 2% 
sc 2 13 .+-. 20% 
2 im 2 295 .+-. 0.5% 
sc 2 161 .+-. 12% 
3 im 2 1524 .+-. 13% 
sc 2 1119 .+-. 14% 
4 im 2 1414 .+-. 2% 
sc 2 1277 .+-. 21% 
5 im 2 32 .+-. 5% 
sc 2 34 .+-. 10% 
6 im (all animals died directly after 
administration) 
sc (all animals died directly after 
administration) 
7 im 2 30 .+-. 12% 
sc 2 39 .+-. 9% 
______________________________________ 
______________________________________ 
III. Bioavailability of antithrombin III (rabbits) 
Type of AUC + Variation 
Buffer Administration n (%) 
______________________________________ 
1 im 3 456,000 .+-. 28 
sc 3 57,000 .+-. 19 
3 im 3 601,000 .+-. 15 
sc 3 116,400 .+-. 18 
______________________________________ 
______________________________________ 
IV. Bioavailability of F XIII (rabbits) 
Type of AUC + Variation 
Buffer Administration n (%) 
______________________________________ 
1 im 3 228,000 .+-. 25 
sc 3 35,000 .+-. 12 
3 im 3 60,600 .+-. 23 
sc 3 62,000 .+-. 20 
______________________________________ 
______________________________________ 
V. Bioavailability of von Willebrand factor (rabbits) 
Type of AUC + Variation 
Buffer Administration n (%) 
______________________________________ 
1 im 3 231 .+-. 14 
sc 3 61 .+-. 20 
3 im 3 715 .+-. 16 
sc 3 12 .+-. 14 
______________________________________