NZ235153A

NZ235153A - Stabilised alpha-interferon compositions

Info

Publication number: NZ235153A
Application number: NZ235153A
Authority: NZ
Inventors: Alberto Ferro
Original assignee: Hoffmann La Roche
Priority date: 1989-09-28
Filing date: 1990-09-03
Publication date: 1993-03-26
Also published as: HU205555B; CA2024046A1; DD298054A5; AU636653B2; PH27531A; IE903479A1; YU184090A; KR910005886A; HU906021D0; NO904218L; MY106615A; JPH03130232A; FI904756A0; ZA907579B; CS432890A3; MX22522A; CZ277712B6; ATE92334T1; AR244551A1; EP0420049B1

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £35153 235 1 5 3 . .'ority Dats(s): . ■. & Complete Specification Filed: H-c: Publication Date: P.O. Journai, l':o: 2 is; MAR 893; ./it '" A' T ! ' «, — " £ f%i .:*£s & ;NEW ZEALAND PATENTS ACT, 1953 ;NEW ZEALAND PATENT OFFICE ;-3SEP1990 ;W=r ;RECEIVED ;No.: Date: ;COMPLETE SPECIFICATION ;STABILIZED LEUKOCYTE-INTERFERONS ;K/We. F. HOFFMANN-LA ROCHE AG 124 Grenzacherstrasse, CH-4002 Basle, Switzerland, a Swiss Company hereby declare the invention for which X / we pray that a patent may be granted to XHg/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - ;- 1 - ;(followed by page la) ;235 1 5 ;- l<3 " RAN 4100/73 ;10 ;The present invention is concerned with the stabilization of leucocyte interferon (IFN-a), ;especially in the form of lyophilizates, using disaccharides and optionally bile acids or bile acid derivatives. ;Under IFN-a there is to be understood a body--specific protein having antiviral and immunoregulatory activity. The antiviral effect is achieved not by a direct influence on the vicuses themselves, but by an activity on their target cells in the sense of a protection against the viral infection. In addition to the antiviral 1g activity, a-interferon can exert objectifiable effects on cancer tumors, which makes it suitable for use in cancer therapy, and it influences the immune system of the body in that e.g. it activates macrophages and NK cells and increases the expression of various immunologically 2q significant constituents of the cell membrane. ;Thanks to recombinant DNA technology, IFN-a can today be prepared in a microbiological mariner in amounts which hitherto could not be made available by isolation 25 from natural material (leucocytes, lymphocytes) and purification in spite of the greatest efforts. ;This new technology has opened for the first time a way for the intensive clinical testing and possible wide 30 therapeutic use of IFN-a and has made possible an adequate supply of the active substance for persons seeking a treatmeriL with the active substance. ;35 ;Ar/5.7.90 ;o ;10 ;15 ;- 2 - tLm B <fci/ ;It has now emerged that IFN-a in pure form is not completely stable and suffers loss of its biological activity during storage. ;Different adjuvants have therefore already been used for the stabilization of IFN-a. A certain stabilization of IFN-a has been achieved by the addition of glycine (nee e.g. publ. PCT Application 86/00531 and European Patent. Publ. No. 82 481), whereby, however, albumin is preferably simultaneously added. ;In efforts to achieve a good stabilization of IFN-a over the longest possible period while avoiding the use of high-molecular compounds such as :;erum albumin, gelatine, dextran or starch derivatives, it has been found that this is successful using disaccharides and optionally bile acids or bile acid derivatives. ;The present invention is accordingly concerned with compositions, especially in the form of lyophilizates, ;which contain IFN-a, 0.5% to 15% (wt./vol.) of a disac-charide and optionally bile acids or bile acid derivatives, as well as a process for their manufacture which comprises treating an IFN-a solution with a disaccharide and optionally bile acids oc bile acid 25 derivatives and, if desired, lyophilizing the solution obtained. The present invention is also concerned with pharmaceutical preparations based on the compositions in accordance with the invention as well as the use of disaccharides and optionally bile acids or bile acid 30 derivatives for tho stabilization of IFN-a. ;The compositions i ri accordance with the invention can be uned for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of a large 35 number of viral infections and immunoregulatory anomalies, ;> t : ;20 ;especially neoplasms. ;C' ;A ;. <- /> ;- 3 - ;23 5 1 5 ;The stabilization can be applied to natural or recombinant IFN-a (r-IFN-a). Recombinant IFN-aA (r-IFN-aA) is an especially preferred IFN-a in connection with the present invention. The content of g IFN-a in the compositions in accordance with the invention is not critical. The concentration range extends over more than the power of ten and is limited at the upper end otisentially only by the solubility of IFN-a. In the cane of human IFN-a there come into g ;10 consideration, for example, concentrations up to 5*10 international units (I.U.)/ml, with a preferred range , . 6 8 being 0.1 x 10 to 1 x 10 I.U./ml, especially 1 x 106 to 5 x 107 I.U./ml. 15 20 The disaccharide, preferably lactose or saccharose, is added to the IFN-a solution in a concentration of 0.5% to 15% (wt./vol.), preferably 5% (wt./vol.). The bile acids or the bile acid derivatives can be e.g. cholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, glycocholic acid or taurocholic acid, with glycocholic acid being especially preferred. It is added to the 25 IFN-a solution in a concentration, of 0.01 to 3% (wt./vol.), preferably 0.1% to 1% (wt./vol.), more preferably 0.5% (wt./vol.). Further adjuvants for the pH-adjustment, e.g. NaOH, pH-buffering, e.g. phosphate buffer and citrate buffer, 30 isotonization, e.g. sodium chloride, and preservation of the preparation, e.g. methyl p-hydroxybenzoate and propyl p-hydroxybenzoate, as well as for strengthening the structure of the lyophilixate, e.g. glycine, can also be added. 35 Particulars of the invention are described in the following Examples. <••• c 3 20 25 30 35 235 1 5 3 - 4 - The r-IFN-aA used in the Examples can be obtained in pure form according to known procedures described in the 5 literature or according to procedures obvious to a person skilled in the art. In order to determine the antiviral activity of the r-IFN-aA. there was used a cytopathological test with MDBK 20 (Madison Darby Bovine Kidney) cells and VSV (vesicular stomatitis virus) viruses, which has been described by Rubinstein et al. [J. Virol. 37. 755-758 (1981)]. MDBK cells and VSV viruses are generally accessible and can be obtained e.g. from the American Type Culture Collection 15 (ATCC) (MDBK: ATCC Nos. CCL 21 and CRL 6071; VSV: ATCC No. VR-1 59). For the stability testing, solutions with r-IFN-aA and the additives under investigation were lyophilized and stored at different temperatures (5°. 25°, 35°, 45°, 55' and 65°C). After fixed time intervals the antiviral activity of the IFN-aA still present in the lyophilizates was determined. . o The following Examples illustrate the invention without limiting it. Example 1 3 mio. I.U. of r-IFN-aA were taken up in 1 ml of water containing human serum albumin, glycine or lactose and optionally further adjuvants (see Table 1). The solutions obtained were sterile-filtered (membrane filter, 0.2 vim pore size) and loaded in 10 ml glass vials in a steam-sterilized freeze-dryer. After freezing the solutions at -40°C during 4 h the primary drying of the lyophilization process was carried at about -30°C under a pressure of 0.1 mbar during 14 h. Subsequently, the r> 235 1 53 - 5 - secondary drying was carried out under a full vacuum at +20°C during about 4 h. The glass vials were closed with 5 suitable aluminium caps in the freeze-dryer under a N2 atmosphere. They were subsequently subjected to different stability tests, the results of which are compiled in Table 1. 10 15 20 o 25 j 30 35 V,' 235 1 5; - 6 - Table 1 O Composition of the lyophilizates containing r-IFN-aA G 10 15 20 25 30 35 1 2 2 4 5 r-IFN-aA (mio. I.U.) 3.0 3.0 3 .0 3.0 3.0 Sodium chloride (mg) 9.0 - - - - Human serum albumin (mg) 5.0 - - - - Mannitol pyrogen-free (mg) - - - - 20.0 Glycine (mg) - 20.0 - 10.0 - Lactose (mg) - - 50 .0 - - NaH2P04.H20 ad pH 7.4 - q. s . - - q. s NaOH IN ad pH 7.4 - - q . s. q. s. - Water for injection 1.0 ml 1.0 ml 1 .0 ml 1.0 ml 1.0 Antiviral activity (in mio. I.U.) after manufacture of the 3.0 3.0 2 .7 3 . 3 2.3 lyophilizate 2 weeks/ 5°C 3.2 3.0 2 .8 3.3 1.8 " /25°C 3.2 2.7 3 .4 3.1 1.5 " /35°C 3.3 2.8 3 .3 2.7 1.2 " /45°C 2.7 2.0 3 .0 2.9 0.9 " /55°C 2.8 1.5 2 .4 2.1 0.8 " /65°C 2.1 0.6 - 0.4 3 months/ 5°C 2.7 2.5 3 .0 3.6 - " /25°C 2.5 2.3 3 .1 3.2 - " /35°C 2.4 2.1 3 . 1 2.7 - /45°C 1.7 1.2 2 .8 1.3 - 6 months/ 5°C 3.5 3.1 3 .2 3.1 - /25°C 3.3 3 .1 3 .2 2.4 - /35°C 2.6 1.9 3 .0 2.4 - /45°C n. t. 1.7 3 .0 1.1 — 235 1 5 3 - 7 - o 12 months/ 5°C 3.7 n.t. 3.0 CO (M " /25°C 2.9 n.t. 2.9 2.5 5 " /35°C 2.1 n.t. 2.7 1.3 " /45°C n. t. n.t. 2.7 1.6 n.t. = not tested g.s. = quantum satis 10 15 20 o 25 30 35 235 15 3 - 8 - The best results were achieved with the lactose--containing lyophilizate (see column 3). This preparation exhibited an outstanding stability even after long-term storage at the storage temperature of 45°C, which is almost prohibitive for IFN-a. This result could also be confirmed unequivocally in experiments with lyophilizates containing 1 mio. I.U. of r-IFN-aA (see Table 2). r> 235 1 - 9 - Table 2 r~\ Composition of the lyophilizates containing r-IFN-aA 10 15 Lyophilization from: r-IFN-aA (mio. I.U.) Glycine (mg) Lactose (mg) NaOH IN ad pH 7.4 Water for injection Antiviral activity (in mio. I.U.) after 0.5 10.0 q. s. 1.0 ml 0.5 50.0 q. s. 1.0 ml 20 manufacture of the lyophilizate 0.4 0.4 25 2 weeks/ 5°C " /25°C " /35°C " /45°C " /65°C 0.5 0.4 0.4 0.3 0.2 0.4 0.4 0.4 0.3 0.3 30 3 months/ 5°C " /25°C " /35°C " /45°C 0. 5 0.4 0.3 0.2 0.5 0.4 0.4 0.4 35 6 months/ 5°C " /25°C " /35°C ■■ /45°C 0.4 0.4 0.2 0.05 0.5 0.4 0.4 0.3 © 235153 - 10 - Example 2 The stability of lyophilizates containing r-IFN-aA and saccharose was investigated in an analogous manner to Example 1. It was established that the stabilization of r-IFN-aA obtained with lactose can also be achieved with saccharose (see Table 3). 10 15 20 25 30 35 23 5 1 - 11 - Table 3 •w' 5 Composition of the lyophilizates containing r-IFN-aA 10 15 Lvophilizate from: r-IFN-aA (mio. I.U.) Sodium chloride (mg) Human serum albumin Glycine Saccharose (mg) NaH2P04 H20 ad pH 7.4 NaOH IN ad pH 7.4 Water for injection ad 3 9.0 5.0 20. 0 q.s. 10.0 50.0 q. s. 1.0 ml 1.0 ml 1.0 ml 2Q Antiviral activity (in mio. I.U.) after O 25 manufacture of the lyophilizate 3 months/ 5°C /25°C " /35°C /45°C 2.7 2.7 2.5 2.4 1.7 3.0 2.5 2.3 2.1 1.2 3.4 3.9 3 . 6 3.7 30 6 months/ 5°C /25°C /35°C /45°C 3.5 3.3 2.6 3 . 1 3.1 1.9 1.7 4.0 3.7 3.7 35 235 1 5 3 - 12 - Example 3 The stability of lyophilizates containing r-IFN-aA in high concentrations and lactose or saccharose was investigated in an analogous manner to Example 1. It was established that the stabilization of IFN-a produced using disaccharides can also be achieved in the case of high rIFN-aA concentrations (see Table 4). 15 20 25 30 35 235 1 5 3 10 - 13 - Table 4 5 Composition of lyophilizates containing r-IFN-aA Lyophilized from: 1 2 3. 15 r-IFN-aA (mio. I.U.) 9.0 9.0 18.0 18.0 Lactose 50.0 mg - 50.0 mg Saccharose - 50.0 mg - 50.0 mg Glycine 10.0 mg 10.0 mg 10.0 mg 10.0 mg NaOH IN ad pH 7.4 - - - q.s. Water for injection ad 1.0 ml 1.0 ml 1.0 ml 1.0 ml 20 Antiviral activity (in mio. I.U.) after manufacture of the lyophilizate 8.8 8.9 18.3 19.9 25 3 months/ 5°C " /25°C " /35°C " /45°C 9.9 10.0 10.3 10.4 9.6 10.5 10.1 10.9 17.8 19.1 20.5 22 .0 23.4 23.2 n.t, n.t. 30 6 months/ 5°C " /25°C /35°C /45°C 9.7 9.9 8.9 8.4 n.t. n.t. n.t. n.t. 17.8 21.1 16.6 16.1 19.1 21.9 n.t, n.t, 35 235 1 © - 14 - Example 4 The stability of lyophilizates containing r-IFN-aA, lactose and glycocholic acid was investigated in an 5 analogous manner to Example 1. The results obtained are compiled in Table 5. 10 15 20 O 25 30 35 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> n 235 153 15 - Table 5 ComposiLi on of the lyophilizates containing r-IFN-aA lyophilizate from: r-IFN-aA (mio. I.U.) Glycocholic acid (mg) Lactose (mg) >1 g Sodium chloride (mg) NaOH IN ad pH 7.4 Water for injection ad 1 3 9 18 5.0 5.0 5 . 0 5.0 50.0 50.0 50.0 50. 0 LO • CM 2.5 2.5 2.5 q. s. q. s. q. s. q. s. 1.0 ml 1.0 ml 1.0 ml 1.0 15 Antiviral activity (in mio. I.U.) after manufacture of the lyophilizate 0.96 2. 97 8. 56 18. 8 14 days/ 5°C 0.85 - - - " / RT 0.92 - - - " /35°C 0.85 - - - " /45°C 0.83 - - - 6 months/ 5°C 1.02 2. 89 9.2 18.7 » / RT 1.1 2. 82 8.7 18.7 / 3 5 °C 1.06 3.11 8.9 18. 3 /45°C 0.99 2.71 8. 94 n. t 35 23 5 1 5 - 16 - WHAT WE CLAIM IS: o 1. A composition containing a-interferon, 0.5% to 15% (wt./vol.) of a disaccharide and optionally bile acids or g bile acid derivatives. r~^j P.. A compos it. ion according to claim 1, wherein the a-interferon is a natural or a recombinant human leucocyte interferon. 10 3. A composition according to claim 2, which contains r-IFN-aA. 4. A composition according to any one of claims 1 to 3 in the form of a lyophilizate. 5. A composition according to any one of claims 1 to 4, wherein the disaccharide is lactose or saccharose. 6. A composition according to any one of claims 1 to 25 5* whi-ch contains 5% (wt./vol.) of lactose or saccharose. 7. A composition according to any one of claims 1 to 5, which contains 0.01% to 3.0% (wt./vol.) of bile acids or bile acid derivatives, preferably 0.1% to 1.0% (wt. . 30 vol.) of glycocholic acid. 8. A composition according to any one of claims 1 to 5, which contains 0.5% (wt./vol.) of bile acid derivatives, preferably 0.5% (wt./vol.) of glycocholic 35 acid. 15 IS. 23 5 1 5 - 17 - o ■ 5 10 15 20 O 25 30 9. A composition containing r-IFN-aA. 5% (wt./vol.) of lactose and 0.5% (wt./vol.) of glycocholic acid. 10. A composition according to any one of claims 1-9 as a pharmaceutical^ active material. 11. A composition according to any one of claims 1-9 as an antivirally and immunoregulatory active material. 12. A process for the manufacture of a composition according to any one of claims 1 to 9. which process comprises treating a-interferon with a disaccharide and optionally bile acids or bile acid derivatives, preferably glycocholic acid, and. if desired, lyophilizing the solution obtained. 13. Pharmaceutical preparations based on a composition according to any one of claims 1 to 9. 23 5 1 5 - 18 - 14. The use of disaccharides and optionally bile acids or bile acid derivatives. preferably glycocholic acid, for the stabilization of a-interferon. 15. The use of a composition according to any one of claims 1-9 for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of illnesses. 16. The use of a composition according to any one of claims 1-9 foe the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of viral infections. 17. The use of a composition according Lo any one of claims 1-9 for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of immunoregulatory anomalies. 18. The use of a composition according to any one of claims 1- 9 for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of neoplasms. 19. A composition containing a-interferon, a disaccharide and optionally bile acids or bile acid derivatives yrypared according to a process as claimed in claim 12. 20. A composition according to claim 1, substantially as hereinbefore described, especially with reference to the Examples. JAft'D THIS 3 Dty QF/Tt-b 15^3 A. J. PLICAMTS ;/v ,;n1 | £• C- ' «■« • <:■ </div>