CA1059029A - Stabilization of interferon against mechanical stress - Google Patents
Stabilization of interferon against mechanical stressInfo
- Publication number
- CA1059029A CA1059029A CA239,141A CA239141A CA1059029A CA 1059029 A CA1059029 A CA 1059029A CA 239141 A CA239141 A CA 239141A CA 1059029 A CA1059029 A CA 1059029A
- Authority
- CA
- Canada
- Prior art keywords
- interferon
- acid
- mechanical stress
- activity
- against mechanical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/565—IFN-beta
Abstract
Abstract of the Disclosure The present invention encompasses a method for stabilizing interferon during purification comprising treating an interferon preparation with an effective stabilizing amount of reduced glutathione, thiodiglycol, thioethanolamine, thioalkanoic acid having 1-7 carbon atoms, monothioglycerol, dithiotreitol, thiotic acid, N-acetylcysteine, or N-acetylhomocysteine as a sulfur containing reducing agent for selectively retaining interferon sulfhydryl groups in the reduced state. The method of the present invention preserves the activity of interferon by selectively retaining interferon sulfhydryl (-SH) functions in the reduced state without reducing interferon disulfide linkages (-S-S-) which are essential for activity.
Description
1059C~Z9 STABILI~ATION OF INTERFERON
AGAINST MECHANICAL STRESS
The present invention encompasses a method for stabilizing interreron during purification comprising treating an interferon preparation with an effective stabilizing amount of reduced glutathione, thiodiglycol, thioethanolamine, thioalkanoic acid having 1-7 carbon atoms, monothioglycerol, dithiotreitol, thiotic acid, N-acetylcysteine, or N-acetylhomocysteine as a sulfur containing reducing agent for selectively retaining interferon sulfhydryl in the reduced state without reduc-tion of essential disulfide linkages. The metho,d of the present invention preserves the activity of interferon by selectively retaining interferon sulfhydryl (-SH) functions in the reduced state without reducing inter-feron disulfide linkages (-S-S-) which are essential for activity. Methods for producing and isolating interferon are extensively taught, Ciba Foundation Symposium-Inter-feron-edited by G. E. W. Wolstanholme and Maeve O'Connor;
Little, Brown and Co., Boston, 1967~
It is well known that interferon may be derived from cells grown in tissue culture and that cultures of non-transformed cells are preferred as a source of inter-feron. However, the,interferon derived from such,cells is highly susceptible to inactivation by mechanical stress, and other procedures involved in conventional purification of the material, and the yields of purified material obtained by these procedures are low.
"For the purpose of the present invention inactivation by mechanical stress is also considered to encompass the in-activation of interferon which can occur on standing."
~.
~059~Z9 It is known that interferon contains disulfide bonds which must remain intact if activity is to be main-tained and reduction of these disulfide bonds with strong reducing agents destroys the actiYity of the interferon.
However, it has now been shown that interferon also con-tains sulfhydryl groups and that these also are essential to the activity of the material. The process of inacti-vation, for example by mechanical stress described above probably involves either the linking together of the sulfhydryl groups or disulfide interchange reactions, to form inter or intra-molecular disulfide bridges, thereby causing the interferon to adopt a biologically inactive configuration.
Certain water soluble sulfur-containing mild reducing agents retain interferon sulfhydryl groups in a reduced state without affecting disulfide linkages which are essential for interferon activity. The preferred reagents are: thioacetlc acid, reduced glutathione (~-L-glutamyl-L-cysteinylglyc$ne), monothioglycerol (3-mercapto-1,2-propanediol), dithiothreitol (1,4-dithiothreitol), thiotic acid (1,2-dithiolane-3-valerlc acid), N-acetyl-cysteine, N-acetylhomocysteine, thioethanolamine (2-aminoethanethiol) and thiodiglycol (2,2~-thiodiethanol).
These compounds are represented by the following structures.
O ' . ' .
CH3-C-SH thioacetic acid, thioalkanoic acids havlng 1-7 carbon atoms are likewise suitable.
S - S thiotic acid(lj2-/ \ dithiolane-3-valeric (CH2)4- C02H acid) ~os9oz~
CHOH monothloglycerol ¦ (3-mercapto-1,2-propanediol) CH2- SH thioethanolamine ¦ (2-aminoethanethiol) . (CH2) - OH
1 2 thiodiglycol (2,2'-thiodlethanol) (CH2)2--OH
H-CI-SH
H-C-OH dithiothreitol ¦ ~1,4-dithiothreitol) HO-CI-H
H-CI-SH
H
¦ N-acetylcysteine HS-CH -CH -CH-CO2H N-acetylhomocysteine
AGAINST MECHANICAL STRESS
The present invention encompasses a method for stabilizing interreron during purification comprising treating an interferon preparation with an effective stabilizing amount of reduced glutathione, thiodiglycol, thioethanolamine, thioalkanoic acid having 1-7 carbon atoms, monothioglycerol, dithiotreitol, thiotic acid, N-acetylcysteine, or N-acetylhomocysteine as a sulfur containing reducing agent for selectively retaining interferon sulfhydryl in the reduced state without reduc-tion of essential disulfide linkages. The metho,d of the present invention preserves the activity of interferon by selectively retaining interferon sulfhydryl (-SH) functions in the reduced state without reducing inter-feron disulfide linkages (-S-S-) which are essential for activity. Methods for producing and isolating interferon are extensively taught, Ciba Foundation Symposium-Inter-feron-edited by G. E. W. Wolstanholme and Maeve O'Connor;
Little, Brown and Co., Boston, 1967~
It is well known that interferon may be derived from cells grown in tissue culture and that cultures of non-transformed cells are preferred as a source of inter-feron. However, the,interferon derived from such,cells is highly susceptible to inactivation by mechanical stress, and other procedures involved in conventional purification of the material, and the yields of purified material obtained by these procedures are low.
"For the purpose of the present invention inactivation by mechanical stress is also considered to encompass the in-activation of interferon which can occur on standing."
~.
~059~Z9 It is known that interferon contains disulfide bonds which must remain intact if activity is to be main-tained and reduction of these disulfide bonds with strong reducing agents destroys the actiYity of the interferon.
However, it has now been shown that interferon also con-tains sulfhydryl groups and that these also are essential to the activity of the material. The process of inacti-vation, for example by mechanical stress described above probably involves either the linking together of the sulfhydryl groups or disulfide interchange reactions, to form inter or intra-molecular disulfide bridges, thereby causing the interferon to adopt a biologically inactive configuration.
Certain water soluble sulfur-containing mild reducing agents retain interferon sulfhydryl groups in a reduced state without affecting disulfide linkages which are essential for interferon activity. The preferred reagents are: thioacetlc acid, reduced glutathione (~-L-glutamyl-L-cysteinylglyc$ne), monothioglycerol (3-mercapto-1,2-propanediol), dithiothreitol (1,4-dithiothreitol), thiotic acid (1,2-dithiolane-3-valerlc acid), N-acetyl-cysteine, N-acetylhomocysteine, thioethanolamine (2-aminoethanethiol) and thiodiglycol (2,2~-thiodiethanol).
These compounds are represented by the following structures.
O ' . ' .
CH3-C-SH thioacetic acid, thioalkanoic acids havlng 1-7 carbon atoms are likewise suitable.
S - S thiotic acid(lj2-/ \ dithiolane-3-valeric (CH2)4- C02H acid) ~os9oz~
CHOH monothloglycerol ¦ (3-mercapto-1,2-propanediol) CH2- SH thioethanolamine ¦ (2-aminoethanethiol) . (CH2) - OH
1 2 thiodiglycol (2,2'-thiodlethanol) (CH2)2--OH
H-CI-SH
H-C-OH dithiothreitol ¦ ~1,4-dithiothreitol) HO-CI-H
H-CI-SH
H
¦ N-acetylcysteine HS-CH -CH -CH-CO2H N-acetylhomocysteine
2 2 1 (2-acetamido-4-mercapto-NH-CQ-CH3 butyric acid) reduced glutathione (y-L-glutamyl-L-cystein-ylglycine) For purposes of the present lnvention an effec-tive stabilizing amount is 0.1 to 10-g molar concentration of the reducing agent in the ~olutlon containlng interferon.
I Preferred embodiment of the present invention.
are represented by the method of stabilizing fibroblast lOS9OZ9 interferon against mechanical stress by treating an inter-feron preparation with at least l x 10-4 moles per liter of thiotic acid, at least 1 x 10 1.5 moies per liter of N-acetylcysteine, or at least 1 x 10 2 moles per liter of dithiothreitol.
Interferon derived from fibroblast cells is most preferably stabilized by treating the crude inter-feron preparation with at least 1 x 10-4 moles per liter of thiotic acid before sub~ecting the interferon to mechanical stress such as ultra filtration, shaking, or precipitation.
Itis to be particularly noted that related structures not having the requisite sulfur moiety are ineffective. For example, thiotic acid and thioalkanoic acids having 1-7 carbon atoms such as thioacetic acid are ; effective interferon stabilizing agents while octanoic acid is not. N-acetylcysteine is effective and N-acetyl valine is not.
The reagent may be added to the crude prepara-tlon of interferon at the appropriate concentration (aneffective stabilizing amount) and the interferon subse-quently purified and concentrated by conventional tech-nlques such as chromatography, ultrafiltration or centri-fugation. Upon completion of the purification the stabllizing agent may be removed by dialysis against a buffer solution ~hich does not contain the agent.
The following exampl0s are illustrations of the present lnvention and should not be construed as limiting the invention in spirit or scope.
~059C31;~9 The abllity of several:compounds to prevent inactivation by mechanical stress of interferon derived from human fibroblasts is shown in Table 1 below:
_ CONCEN- .
COMPOUND TRATION
. ~mM) TIME OF AGITATION(HOURS) . ._ . . 2 4 6 24 . . INTERFERON ACTIVITY
Control interferon 30 20 10 3 (no additives) i Thiotic acid 1 100 100 100 95 3ctanoic acid 1 30 20 10 3 ; N-acetylcysteine 100 90 85 85 80 N-acetylvaline 100 30 15 10 4 : Dithiothreitol 10 90 90 85 85 2-mercaptoethanol 14 10 10 5 10 _ Samples of interferon ln tissue culture medium were agitated by shaking in a tube 50 times per minute at a temperature of 4C. The percentage of the original interferon activity remaining after increasing time inter-vals is given in Table 1. An indication that the reactlve species involved the sulfhydryl group is given by the fact that octanoic acid and N-acetylvaline, which are closely chemically related to thiotic acid and N-acetylcysteine respectlvely, but lack the sulfhydryl moiety, are both totally inactive.
. The strong reduclng agent 2-mercaptoethanol has .
no protective activity against the inactivation and itself caused inactivation of samples which are not agitated.
2-mercaptoethanol ls shown separately to disrupt disulfide bonds essential to the reactivlty of interferon.
It is, therefore, demonstrated that only those preferred reagents which react with interferon sulfhydryl groups without disrupting disulfide bonds are able to stabilize interferon against mechanical inactivation. In contrast potent reagents which do disrupt the disulflde bonds of interferon cause a loss of activity of interferon even ln the absence of physical stress.
Interferon from two types of fibroblast cells, MRC5 and FSA were sub;ected to increasing physical stress ; by shearing in a rotational viscometer comprising two con-centrlc cylinders rotating in relation to each other.
The interferon had initial activity of 1000 u/ml.
RESULTS
Activity of 1 Hour unsheared control - 1000 u/ml FSA/10-4 molar thiotlc acid - about 850 u/ml MRC5/10-4 molar thiotic acld - about 700 u/ml FSA sheared control - about 500 u/ml MRC5 sheared control - about 50 u/ml Samples of lnterferon 0.1 molar in N-acetylcys-teine were shaken at the rate of 50 times per minute for a total period of 24 hours at 4C. After 24 hours the N-acetylcysteine treated interferon samples had essen-tially unchanged activity and untreated controls had 1/10 the original activity after 24 hours of shaking.
1059(~Z9 Reagents of the present lnventlon functlon by maintaining a reducing atmosphere around the critical sulfhydryl groups. They do not bind to these groups and their reaction is therefore reversible if, for example, they are dialysed out of the interferon preparation.
Furthermore, interferon which is rendered unstable by dialysing away a reducing agent stabilizer may be re-stabilized by the addition of a fresh reducing agent.
The properties of reagents according to the present invention are illustrated by the examples des-cribed in Table 2. An aqueous preparation of interferon was sub;ected to treatment with various reagents and then agitated for 24 hours at 4C. The resulting loss of total interferon activity as a result of this agitation was measured.
TABLE ~
No. Treatment % Loss of Activity (shaking for 24 hours at 4C.) 1 None 90 2 0.1 M N-acetylcysteine 0
I Preferred embodiment of the present invention.
are represented by the method of stabilizing fibroblast lOS9OZ9 interferon against mechanical stress by treating an inter-feron preparation with at least l x 10-4 moles per liter of thiotic acid, at least 1 x 10 1.5 moies per liter of N-acetylcysteine, or at least 1 x 10 2 moles per liter of dithiothreitol.
Interferon derived from fibroblast cells is most preferably stabilized by treating the crude inter-feron preparation with at least 1 x 10-4 moles per liter of thiotic acid before sub~ecting the interferon to mechanical stress such as ultra filtration, shaking, or precipitation.
Itis to be particularly noted that related structures not having the requisite sulfur moiety are ineffective. For example, thiotic acid and thioalkanoic acids having 1-7 carbon atoms such as thioacetic acid are ; effective interferon stabilizing agents while octanoic acid is not. N-acetylcysteine is effective and N-acetyl valine is not.
The reagent may be added to the crude prepara-tlon of interferon at the appropriate concentration (aneffective stabilizing amount) and the interferon subse-quently purified and concentrated by conventional tech-nlques such as chromatography, ultrafiltration or centri-fugation. Upon completion of the purification the stabllizing agent may be removed by dialysis against a buffer solution ~hich does not contain the agent.
The following exampl0s are illustrations of the present lnvention and should not be construed as limiting the invention in spirit or scope.
~059C31;~9 The abllity of several:compounds to prevent inactivation by mechanical stress of interferon derived from human fibroblasts is shown in Table 1 below:
_ CONCEN- .
COMPOUND TRATION
. ~mM) TIME OF AGITATION(HOURS) . ._ . . 2 4 6 24 . . INTERFERON ACTIVITY
Control interferon 30 20 10 3 (no additives) i Thiotic acid 1 100 100 100 95 3ctanoic acid 1 30 20 10 3 ; N-acetylcysteine 100 90 85 85 80 N-acetylvaline 100 30 15 10 4 : Dithiothreitol 10 90 90 85 85 2-mercaptoethanol 14 10 10 5 10 _ Samples of interferon ln tissue culture medium were agitated by shaking in a tube 50 times per minute at a temperature of 4C. The percentage of the original interferon activity remaining after increasing time inter-vals is given in Table 1. An indication that the reactlve species involved the sulfhydryl group is given by the fact that octanoic acid and N-acetylvaline, which are closely chemically related to thiotic acid and N-acetylcysteine respectlvely, but lack the sulfhydryl moiety, are both totally inactive.
. The strong reduclng agent 2-mercaptoethanol has .
no protective activity against the inactivation and itself caused inactivation of samples which are not agitated.
2-mercaptoethanol ls shown separately to disrupt disulfide bonds essential to the reactivlty of interferon.
It is, therefore, demonstrated that only those preferred reagents which react with interferon sulfhydryl groups without disrupting disulfide bonds are able to stabilize interferon against mechanical inactivation. In contrast potent reagents which do disrupt the disulflde bonds of interferon cause a loss of activity of interferon even ln the absence of physical stress.
Interferon from two types of fibroblast cells, MRC5 and FSA were sub;ected to increasing physical stress ; by shearing in a rotational viscometer comprising two con-centrlc cylinders rotating in relation to each other.
The interferon had initial activity of 1000 u/ml.
RESULTS
Activity of 1 Hour unsheared control - 1000 u/ml FSA/10-4 molar thiotlc acid - about 850 u/ml MRC5/10-4 molar thiotic acld - about 700 u/ml FSA sheared control - about 500 u/ml MRC5 sheared control - about 50 u/ml Samples of lnterferon 0.1 molar in N-acetylcys-teine were shaken at the rate of 50 times per minute for a total period of 24 hours at 4C. After 24 hours the N-acetylcysteine treated interferon samples had essen-tially unchanged activity and untreated controls had 1/10 the original activity after 24 hours of shaking.
1059(~Z9 Reagents of the present lnventlon functlon by maintaining a reducing atmosphere around the critical sulfhydryl groups. They do not bind to these groups and their reaction is therefore reversible if, for example, they are dialysed out of the interferon preparation.
Furthermore, interferon which is rendered unstable by dialysing away a reducing agent stabilizer may be re-stabilized by the addition of a fresh reducing agent.
The properties of reagents according to the present invention are illustrated by the examples des-cribed in Table 2. An aqueous preparation of interferon was sub;ected to treatment with various reagents and then agitated for 24 hours at 4C. The resulting loss of total interferon activity as a result of this agitation was measured.
TABLE ~
No. Treatment % Loss of Activity (shaking for 24 hours at 4C.) 1 None 90 2 0.1 M N-acetylcysteine 0
3. As 2 above and dialysed 93
4. As 3 above then 0.1 M 0 N-acetyl cysteine added .
A conventional procedure for purification of interferon is ultra filtration on a membrane. ~lowever, under conventional condltlons much of the activity of the interferon is lost in carrying out this procedure, when the interferon is that derived from fibroblasts, It is shown in Table 3 below that interferon may be stabilized against such inactivation by treatment wlth 0.1 molar N-acetylcysteine.
Ultra- Concen- % Yield filtration tration of StabilizerMembrane Fao~or Activity None UM 10 5 20 None UM 20 5 11 0.1 M N-acetylcysteine UM 10 5 100 0.1 M N-acetylcysteine UM 20 5 100 A further conventional purification procedure for interferon is salt precipitation using for instance increasing concentrations of ammonium sulfate or potassium thiocyanate at ~C. at mildly acidic or neutral pH. Under conventional conditions much of the activity of interferon is lost in carrying out this procedure when the interferon is derived from fibroblasts. In Table 4 it is shown that interferon may be e~fectively stabilized in ammonium sul~ate precipitation systems by addition of 1 mM thiotic acid.
USE OF SULFHYDRYL STABILIZER IN INTERFERON
PRECIPITATION BY AMMONIUM SULFATE AT pH 7.0 AT 4C.
Treatment Percent Yield Unprotected interferon 30%
Interferon plus 1 mM Thiotic acid 100%
Reversible treatment with a reducing agent whose reactivity is such that only those sulfhydryl groups which play a part in interferon inactivation are reduced and not _g_ 1~590~9 the essential disulfide bonds stabilizes fibroblast interferon against the inactivation which typically occurs while undergoing conventional purification procedures.
.
Human fibroblast interferon is treated with 10 4 moles per liter of thiotic acid and stored at 4C for 14 weeks.
The stability of thiotic acid treated interferon is compared to that of the control. The log10 of interferon titer remains substantially unchanged for the thiotic acid treated interferon while the interferon titer for untreated interferon decreases from 103-5 to 102 after 14 weeks at 4C. Thus thiotic acid present in concentrations of 10 5 to 10 3 molar preferably at least 10 4 molar prolong shelflife of interferon.
A conventional procedure for purification of interferon is ultra filtration on a membrane. ~lowever, under conventional condltlons much of the activity of the interferon is lost in carrying out this procedure, when the interferon is that derived from fibroblasts, It is shown in Table 3 below that interferon may be stabilized against such inactivation by treatment wlth 0.1 molar N-acetylcysteine.
Ultra- Concen- % Yield filtration tration of StabilizerMembrane Fao~or Activity None UM 10 5 20 None UM 20 5 11 0.1 M N-acetylcysteine UM 10 5 100 0.1 M N-acetylcysteine UM 20 5 100 A further conventional purification procedure for interferon is salt precipitation using for instance increasing concentrations of ammonium sulfate or potassium thiocyanate at ~C. at mildly acidic or neutral pH. Under conventional conditions much of the activity of interferon is lost in carrying out this procedure when the interferon is derived from fibroblasts. In Table 4 it is shown that interferon may be e~fectively stabilized in ammonium sul~ate precipitation systems by addition of 1 mM thiotic acid.
USE OF SULFHYDRYL STABILIZER IN INTERFERON
PRECIPITATION BY AMMONIUM SULFATE AT pH 7.0 AT 4C.
Treatment Percent Yield Unprotected interferon 30%
Interferon plus 1 mM Thiotic acid 100%
Reversible treatment with a reducing agent whose reactivity is such that only those sulfhydryl groups which play a part in interferon inactivation are reduced and not _g_ 1~590~9 the essential disulfide bonds stabilizes fibroblast interferon against the inactivation which typically occurs while undergoing conventional purification procedures.
.
Human fibroblast interferon is treated with 10 4 moles per liter of thiotic acid and stored at 4C for 14 weeks.
The stability of thiotic acid treated interferon is compared to that of the control. The log10 of interferon titer remains substantially unchanged for the thiotic acid treated interferon while the interferon titer for untreated interferon decreases from 103-5 to 102 after 14 weeks at 4C. Thus thiotic acid present in concentrations of 10 5 to 10 3 molar preferably at least 10 4 molar prolong shelflife of interferon.
Claims (5)
1. A method for stabilizing interferon against mechanical stress comprising treating an interferon preparation with 0.1 to 10-5 molar concentration of reduced glutathione, thiodiglycol, thioethanolamine, thioalkanoic acid having 1-7 carbon atoms, monothioglycerol, dithiotreitol, thiotic acid, N-acetylcysteine, or N-acetylhomocysteine as a sulfur containing reducing agent for selectively retaining interferon sulfhydryl groups in the reduced state without reduction of essential disulfide linkages.
2. A method according to claim 1 for stabilizing interferon against mechanical stress comprising treating an interferon preparation with at least 1 x 10 4 moles per liter of thiotic acid.
3. A method according to claim 1 for stabilizing interferon against mechanical stress comprising treating an interferon preparation with at least 1 x 10 -1.5 moles per liter of N-acetylcysteine.
4. A method according to claim 1 for stabilizing interferon against mechanical stress comprising treating an interferon preparation with at least 1 x 10-2 moles per liter of dithiothreitol.
5. A method according to claim 1 wherein the interferon is derived from fibroblast cells.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB4813574A GB1526205A (en) | 1974-11-07 | 1974-11-07 | Treatment of interferon-like material |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1059029A true CA1059029A (en) | 1979-07-24 |
Family
ID=10447515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA239,141A Expired CA1059029A (en) | 1974-11-07 | 1975-11-06 | Stabilization of interferon against mechanical stress |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPS5191320A (en) |
| BE (1) | BE835355A (en) |
| CA (1) | CA1059029A (en) |
| DE (1) | DE2549768A1 (en) |
| FR (1) | FR2290222A1 (en) |
| GB (1) | GB1526205A (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4100150A (en) | 1975-11-04 | 1978-07-11 | G. D. Searle & Co. | Stabilization of interferon against mechanical stress using thioctic acid |
| US4252791A (en) * | 1979-10-19 | 1981-02-24 | The Medical College Of Wisconsin, Inc. | Interferon stabilization |
| JPS5892622A (en) * | 1981-11-28 | 1983-06-02 | Sunstar Inc | Pharmaceutical preparation containing stably compounded interferon |
| AU1234383A (en) * | 1982-03-17 | 1983-09-22 | Inter-Yeda Ltd. | Interferon stabilised with polyvinyl-pyrrolidone |
| US4432895A (en) * | 1982-11-24 | 1984-02-21 | Hoffmann-La Roche Inc. | Monomeric interferons |
| JP2577744B2 (en) * | 1986-07-18 | 1997-02-05 | 中外製薬株式会社 | Stable granulocyte colony-stimulating factor containing preparation |
| CA1321349C (en) * | 1986-11-21 | 1993-08-17 | Robert Zimmerman | Therapeutic combination of free-radical scavenger or metabolic inhibitor and biologically active protein |
| US5508031A (en) * | 1986-11-21 | 1996-04-16 | Cetus Oncology Corporation | Method for treating biological damage using a free-radial scavenger and interleukin-2 |
| GB9226729D0 (en) * | 1992-12-22 | 1993-02-17 | Wellcome Found | Therapeutic combination |
-
1974
- 1974-11-07 GB GB4813574A patent/GB1526205A/en not_active Expired
-
1975
- 1975-11-06 FR FR7533991A patent/FR2290222A1/en active Granted
- 1975-11-06 JP JP50133553A patent/JPS5191320A/ja active Pending
- 1975-11-06 DE DE19752549768 patent/DE2549768A1/en not_active Withdrawn
- 1975-11-06 CA CA239,141A patent/CA1059029A/en not_active Expired
- 1975-11-07 BE BE161679A patent/BE835355A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5191320A (en) | 1976-08-10 |
| DE2549768A1 (en) | 1976-05-20 |
| BE835355A (en) | 1976-03-01 |
| GB1526205A (en) | 1978-09-27 |
| FR2290222A1 (en) | 1976-06-04 |
| FR2290222B1 (en) | 1981-12-04 |
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