CA2120944A1 - Stabilization of human interferon - Google Patents
Stabilization of human interferonInfo
- Publication number
- CA2120944A1 CA2120944A1 CA002120944A CA2120944A CA2120944A1 CA 2120944 A1 CA2120944 A1 CA 2120944A1 CA 002120944 A CA002120944 A CA 002120944A CA 2120944 A CA2120944 A CA 2120944A CA 2120944 A1 CA2120944 A1 CA 2120944A1
- Authority
- CA
- Canada
- Prior art keywords
- interferon
- composition
- lithium
- stabilizing agent
- amine
- 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.)
- Abandoned
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
Abstract
ABSTRACT OF THE DISCLOSURE
Described herein are compositions for stabilizing human interferon, including pharmaceutical preparations useful in topical applications. Amine stabilizing agents such as primary aliphatic amines and anionic stabilizing agents such as lithium organo sulfates protect human interferons from degradation and provide enhanced storage stability. These stabilizing agents containing interferon can be added to appropriate pharmaceutical carriers for topical applications.
The topical products also exhibit enhanced storage stability.
Described herein are compositions for stabilizing human interferon, including pharmaceutical preparations useful in topical applications. Amine stabilizing agents such as primary aliphatic amines and anionic stabilizing agents such as lithium organo sulfates protect human interferons from degradation and provide enhanced storage stability. These stabilizing agents containing interferon can be added to appropriate pharmaceutical carriers for topical applications.
The topical products also exhibit enhanced storage stability.
Description
~,WO 93/0883~ 2 ~ 2 ~ PC~ S~2/~)X~()' STABILIZATION OF HUMAN INTER~EROI~
TECHNICAL FIELD OF THE INVENTION
This invention relates to the stabilization of human interferon, and more particularly, to alpha interferon and omega interferon or mixlures thereof.
S BACKGROUND_OF THE INVENTION
Interferons are proteins having a variety of biological actions including antiviral, immunomodulating and antiproliferative effects. Cellular production of interferons may be stimulated by numerous agents, including viruses. Interferons protect anirnal tissues and cells against viral attack and are 10 an important host defense mechanism. Interferon may be produced endogenously by numerous cell types such as leukocytes, fibroblasts and lymphocytes, and may also be produced in cell culture or recombinantly. In most cases, interferons provide better protection to tissues and cells of the kind from which they have been produced than to other types of tissues and cells, 15 indicating that human-derived interferon should be more eMcacious in treatinghuman diseases than interferons from other species. There are several distinct types of interferons, generally classified as alpha, beta, gamma and omega interferons, and a large number of variants thereof.
The alpha and omega interferons are derived primarily from 20 leukocytes. Cell-derived interferon such as leukocyte interferon is difficult to purify to homogeneity and, as a consequence, is most often used as a crude or partially purified preparation. Leukocyte interferon preparations contain two molecular populations (alpha and omega interferons) that are distinguishable physically, chemically and biologically. Alpha interferon comprises 25 approximately 80% of leukocyte interferon activity, and omega interferon comprises about 20% of leukocyte interferon activity. The leukocyte interferon forms can be separated by sodium dodecyl sul~ate - polyacry]amide gel electrophoresis (SDS-PAGE) into slow- and fast-migrating components as described by Stewart II, W. E. and Desmyter, J. Virolo~ 67:68-73 (1975).
~0 93/0~832 ~ s~2/ox~9~
212~9~
The larger leukocyte interferon forrns have apparent molecular weights of about 21,000-25,000 Daltons, while the smaller leukocyte inlerferon forms have ~¦ apparent molecular weights of about 15,000-18,000 Daltons. The larger (omega) interferon are glycosylated, whereas the smaller (alpha) interferon are S not. General discussions of these and other interferons can be found in various texts and monographs including: The Interferon Svs~em by W.E. Stewart, n, Springer-Verlag, New York (1979); and Xnter~eron Therapy, World Health Organization Technical Reports Series 676, World Health Organization, Geneva (1982).
10Ihe method of administering interferon is an irnportant factor in the clinical application of this important therapeutic agent. Systemic administration of interferon by either intravenous, intramuscular or subcutaneous injection has been most frequently used with some success in treating disorders such as haily cell leukemia and Acquired Immune Deficiency 15 Syndrome (AIDS) - related Kaposi's sarcoma. Arnong the problems inherent in intravenous, intrarnuscular or subcutaneous administration is that interferoncan come into contact with uninfected or nonmalignant cells, and may thereby cause unwanted side effects such as fever, malaise and myalgia.
In some cases it would be preferable to administer interferon 20 directly to the affected tissues or organs. This may be accomplished by direct injection of interferon into the diseased site such as done in selected cases ofcondylomata acuminata (genital warts), involving the external surfaces of genital or perianal areas. Interferon can also be administered by local topical application directly to the diseased site, such as a vi~l-induced skin lesion.
25 Topical interferon may be useful for treatment of conditions such as condylomata acuminata, ce~vical dysplasia, rectal cancer, basal cell carcinoma, penile cancer or any other disorder responsive to interferon, particularly thoselinked to papilloma virus in~ection. Topical application in these cases could reduce the unwanted side effects associated with intramuscular, intravenous or . ~.
~ WO 93/08832 PC~/US92/082')~
TECHNICAL FIELD OF THE INVENTION
This invention relates to the stabilization of human interferon, and more particularly, to alpha interferon and omega interferon or mixlures thereof.
S BACKGROUND_OF THE INVENTION
Interferons are proteins having a variety of biological actions including antiviral, immunomodulating and antiproliferative effects. Cellular production of interferons may be stimulated by numerous agents, including viruses. Interferons protect anirnal tissues and cells against viral attack and are 10 an important host defense mechanism. Interferon may be produced endogenously by numerous cell types such as leukocytes, fibroblasts and lymphocytes, and may also be produced in cell culture or recombinantly. In most cases, interferons provide better protection to tissues and cells of the kind from which they have been produced than to other types of tissues and cells, 15 indicating that human-derived interferon should be more eMcacious in treatinghuman diseases than interferons from other species. There are several distinct types of interferons, generally classified as alpha, beta, gamma and omega interferons, and a large number of variants thereof.
The alpha and omega interferons are derived primarily from 20 leukocytes. Cell-derived interferon such as leukocyte interferon is difficult to purify to homogeneity and, as a consequence, is most often used as a crude or partially purified preparation. Leukocyte interferon preparations contain two molecular populations (alpha and omega interferons) that are distinguishable physically, chemically and biologically. Alpha interferon comprises 25 approximately 80% of leukocyte interferon activity, and omega interferon comprises about 20% of leukocyte interferon activity. The leukocyte interferon forms can be separated by sodium dodecyl sul~ate - polyacry]amide gel electrophoresis (SDS-PAGE) into slow- and fast-migrating components as described by Stewart II, W. E. and Desmyter, J. Virolo~ 67:68-73 (1975).
~0 93/0~832 ~ s~2/ox~9~
212~9~
The larger leukocyte interferon forrns have apparent molecular weights of about 21,000-25,000 Daltons, while the smaller leukocyte inlerferon forms have ~¦ apparent molecular weights of about 15,000-18,000 Daltons. The larger (omega) interferon are glycosylated, whereas the smaller (alpha) interferon are S not. General discussions of these and other interferons can be found in various texts and monographs including: The Interferon Svs~em by W.E. Stewart, n, Springer-Verlag, New York (1979); and Xnter~eron Therapy, World Health Organization Technical Reports Series 676, World Health Organization, Geneva (1982).
10Ihe method of administering interferon is an irnportant factor in the clinical application of this important therapeutic agent. Systemic administration of interferon by either intravenous, intramuscular or subcutaneous injection has been most frequently used with some success in treating disorders such as haily cell leukemia and Acquired Immune Deficiency 15 Syndrome (AIDS) - related Kaposi's sarcoma. Arnong the problems inherent in intravenous, intrarnuscular or subcutaneous administration is that interferoncan come into contact with uninfected or nonmalignant cells, and may thereby cause unwanted side effects such as fever, malaise and myalgia.
In some cases it would be preferable to administer interferon 20 directly to the affected tissues or organs. This may be accomplished by direct injection of interferon into the diseased site such as done in selected cases ofcondylomata acuminata (genital warts), involving the external surfaces of genital or perianal areas. Interferon can also be administered by local topical application directly to the diseased site, such as a vi~l-induced skin lesion.
25 Topical interferon may be useful for treatment of conditions such as condylomata acuminata, ce~vical dysplasia, rectal cancer, basal cell carcinoma, penile cancer or any other disorder responsive to interferon, particularly thoselinked to papilloma virus in~ection. Topical application in these cases could reduce the unwanted side effects associated with intramuscular, intravenous or . ~.
~ WO 93/08832 PC~/US92/082')~
2 ~ 2 ~
subcutaneous administration. Also, the patient could self-administer topical interferon without the direct assistance of a qualifled health-care worker. A
topical interferon product could also promote patient compliance by providing a more pleasant therapeutic experience for the patient than interferon treatmentS via injection.
The lack of ~nterferon stability in solutions and other products has heretofore lirnited its utility. Interferon products having enhanced storagestability should faci3itate more wide-spread use of this import~nt therapeutic agent. Several ~actors are important in designing a useful topical interferon 10 preparation. First, conventional topical interferon preparations are gene~llyconsidered unstable, and therefore are considered to have a limited shelf-life.
A topical interferon pre~aration should be stabilized to prevent degradation over time and upon shipping and handling. Second, because interferon generally has a higher molecu1ar weight than the molecular weights of the 15 therapeutic agents usually administered in topical preparations, interferon should be incolporated into a substance which sufficiently holds the high molecular weight interferon in suspension during packaging, shipping and application, and yet also be able to release the substance from the preparation in a reasonable length of time once it has been applied to the diseased site.
20 Third, the preparation must not adversely affect the antivilal activity of interferon.
The present invention describes ne v, improved stabilizers and methods for stabilizing interferon, rneeting the requirements for topical interferon products defined above and for stabilized interferon solutions.
25Other interferon-stabilizing agents have been described and used to stabili7e interferon.
Estis et al. in U.S. Patent No. 4,680,175 describe the use of protease inhibitors such as alpha-l-antitrypsin inhibitor, alpha-2 macroglobulin, soybean inhibitor, N-alpha-tosyl-L-lysine chloromethyl ketone, , ~vo93/08832 ~ s92/08~
~¦ 2 1 2 o 9 4 L~
:¦ phenylmethylsulfonyl fluoride, and N-alpha-iosylphenylalanine chloromethyl :I ketone to stabilize topical interferon preparations. Miller, in U.S. Patent No.
~ 4,957,734, teaches the use of nonionic surface active agents in combination ¦ with interferon and discloses the use of anionic, cationic and nonionic surface S active agents, but does not clainn stabilization of inter~eron. Miller discloses that quarterna~y ammonium detergents such as cetyl pyridin~um chloride and benzalkonium chloride are preferable cationic agents. Hasegawa et al. in U.S.
Patent No. 4,675,184 teach that anionic surface active agents such as sodium cyl sulfates stabilize beta-interferon (fibroblast-derived).
Stewart et al. in U.S. Patent No. 3,981,991 teach that interferon can be stabilized by treating the antiviral agent with a combination of: 1) guanidine-hydrochloride or urea; 2) mercaptoethanol or ethanethiol; and 3) an agent selected from the group consisting of sodium dodecylsulfate, sodium decylsulfate, sodium dodecylsulfonate, dodecylamine and decylamine. Stewart 15 et al. caution that interferon cannot be stabilized with only one or two of the aforesaid three agents. None of these patents teach that human alpha and omega interferons or mixtures thereof can be stabilized with only the stabilizing agents described herein.
SUMMARY OF THE INVEN~ION
~i 20 This invention relates to preparations for stabilizing human interferon. The present invention shows that human alpha and omega interferon, or mLl~tures thereof, can be stabili~ed when placed in solutions containing amines, and more particularly, primary aliphatic amines having from between 8 to 14 car~on atoms. Lithium salts of anionic stabili7ing agents such 25 as lithium organo sulfates also stabilize interferon. Stabilized interferon may 3, b~ placed in a plhannaceutically acceptable ca~ier for topical administration.
Further stability is achieved by adding additional a~nine-stabilizin~ agents to the stabilized interferon product.
r~ WO93/û8832~ /US92/0~29' 2 ~ 2 ~
These and other advan~ges of the present invention will become apparent from the following detailed description.
DETAILED DESCRIPTION OF THE Il~PIENTION
The presenl invention relates to stabilizing interferon to enhance 5 its storage stabili~y. This invention is applicable to all types of interferon including natural interferon, interferon produced by recombinant DNA
technology, and interferon produced by chemical synthesis or modification.
Also, the invention can be used with crude, semi-purified and purified interferon from leukocytes, fibroblasts, Iymphocytes or any other interferon-10 containing or producing tissues from humans or any other appropriate species.A method for producing interferon from leukocytes (comprisingalpha interferon and omega interferon) is described by Cantell, et ah in:
Methods ~ in En~;ymolo~, 78: 29-38 (1981). In general, the interferon produced by this method and other methods is supplied as a liquid. Alpha 15 isolates and omega isolates of interferon are prepared by subjecting leukocyte interferon to non-reducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) as described in Stewart, W.E. II, et al., Proc.
Natl. Acad. Sci. 74:4200-4204 (1977). Briefly, leukocyte interferon samples in 0.01 M sodium phosphate buffer were constituted to contain 5 M urea and 20 1 % sodium dodecyl sulfate. Bromphenol blue in sucrose was added to make the samples 6% sucrose, and the samples were heated to lOO~C for one minute. Aliquots of 0.1 ml of sample were electrophoresed on parallel SDS-PAGE gels. When the dye front had migrated about 18 centimeters into gels, gels were sliced into 2.2 millimeter segments, and two adjacent slices were 25 pooled into e~ch fraction containing the alpha isolates and omega isolates, and were eluted ovemight at 4C into 1 ml of Eagle's minimal essential medium containing 10% fetal calf serum.
Those skilled in the alt will recognize that the amount of interferon which is administered in any particular case, as well as the frequency at which ,~0 93/08g32 ~ JS~)2/()82()' 2~3~
the interferon is administered, will depend upon such factors as the type of interferon used, the disease being treated, the patient's response to interferontreatment, and whether the preparation used is a semi-solid or a liquid. The National Institutes of Health ~United States ~Department of Health and Human S Services, Bethesda, Maryland) have established unit strengths ~or interferon activity. In terms of these unit strengths, dosage levels for topical pharmaceutical preparations using crude or partially purifled natural int~rferons can range from about 10,000 lU/gram of topical preparation to 1,000,000 IU/gram of topical preparation.
The terrn "stabilized interferon composition" is employed herein to describe an agent having an interferon and an amine stabilizing agent present in an effective amount to stabilize interferon. The interferon can be human interferon or interferon from any other appropriate species. Preferably the interferon is human leukocyte-derived interferon comprising alpha interferon, 15 omega interferon or mixtures thereof. The interferon can also be recombinant interferon or interferon produced or modified chemically. The present invention reveals enhanced storage stability and antivilal activity of stabilizing agents for interferon. The term "amine stabilizing agent" is employed herein to includeprimary, secondary, ortertiary amines. Preferably, 20 the amine stabilizing agent is a primary arnine having the general formula RNH2 where "R" is an aliphatic group. Preferably, amine stabilizing agents have aliphatic groups of from about 8 to about 14 carbon atoms, although aliphatic groups having less than 8 or more than 14 carbon atoms are encompassed within the scope of this invention. More preferably, aliphatic 25 primary arnines having from between about 8 to about 14 carbon atoms may be used as amine stabili~ing agents. Most preferably, the arnine stabilizing agent is a primary aliphatic amine having an aliphatic group consisting of 12 carbon atoms. I`he aliphatic groups may be linear or branched, but are preferably linear. Representative examples of amines useful as arnine ~WO 93/08832 Pc~r/v~2/()82~
: 2 ~ 2 ~
stabili~ing agents include octyl amine, decyl amine, dodecyl amine, and tetradecyl amine. Amine stabilizing agents having more than 14 carbon atoms are generally insoluble. Arnine stabilizing agents having less than 8 carbon atoms are generally inefficient stabilizers. Most preferable of the amine 5 stabilizing agents is dodecyl amine~ The ef~ective arnount of amine stabilizing agents to stabilize interferon ranges from about 0.1% to about 10.0% by :¦ weight, although lower or higher concentrations of amine stabilizing agents may be used, as would be ascertainable by a person having ordinary skill in the art.
10The term "lithium stabilized interferon composition" is employed 31 herein to describe an agent having an interferon and a lithium anionic stabilizing agent present in an effective amount to stabilize interferon. The interferon can be human interferon or interferon from any other appropriate species. Preferably the interferon is human leukocyte-derived interferon 15 containing alpha iDterferon or omega interferon or mixtures thereof. The interferon can also be recombinant interferon or interferon produced or modified chemically.
The term "lithium anionic stabilizing agent" is employed herein ~' to include the lithium salts of compounds having a net negative charge.20 Lithium anionic stabilizing agents useful in this invention are lithium organo sulfates, and may be represented by the general formula:
Li RX
where "R" is an aliphatic group, "X" is an anion and "n" is chosen to electrochemically balance the anion. "X" is preferably sul~ate. Lithium 25 anionic stabilizing agents containing aliphatic groups having from about 8 toabout 22 carbon atoms may be used, although aliphatic groups of more than 22 carbons or less than 8 carbons are encompassed within the scope of the present invention. Preferably, lithium anionic stabilizing agents contain 8 to 14 carbonatoms, and most preferably 12 carbon atoms. Representative examples of 0 93/U8832 i~C~/US92/~1829 lithium anionic stabilizing agents include lithium octyl sulfate, lithium decyl sulfate, lithium dodecyl sulfate and lithium tetradecyl sulfate. The concentrations of lithium anionic stabilizing agents may range from about 0.1%
to about 10.0% by weight, although lower or higher concentrations may be 5 used as would be ascertainable by a person having ordinary skill in the art.
The patent of Hasegawa et al., described above, does not disclose the use Or lithium organo sulfate interferon stabili7ers. As will be noted below, lithium-containing stabilizing agents have a clinical utility not possessed by sodium-containing stabilizing agents.
Interferon compositions containing from about I x 104 to 1 x 106 IU/ml are most preferably used in the present invention, although interferon compositions having lower or higher activity may also be used as would be readily ascertainable by a person having ordinary skill in the art.
The precise mechanism of action of the amine stabilizing agents and 15 lithium anionic stabili~ing agents in stabilizing interferon is not known, but is thought to be related to "defensive reversible denaturation" as described by W.E. Stewart II, et al. Na~ure, Vol. 249, pp. 460-61 ~1974) .and W.E.
Stewart I[, et al., Preparative Biochemisty Vol. 4, pp. 383-93 (1974).
The clinical utility of sodium-containing interferon stabilizing agents 20 such as sodium organo sulfates is limited by the side effects associated withtopical administration of sodium-salt-containing products. A not uncommon unwanted side effect associated with use of sodium-salt-containing products is chronic irritant dermatitis. Tupker, R.A. et ak, Acta De_. Venereo!. 70:1-5 ~1990) . Unlike sodium-salt-containingtopical products, lithium-saltp~parations 25 are not associated with such unwanted side effects. A lithium-salt-containinginterferon product would therefore represent an advance over sodium-salt-contair~ing topical interferon preparations.
The term "interferon antiviral preparation" as used herein describes a p~oduct having a stabilized interferon composition and a ~WO 93/0~832 ~'C~/US92/08'97 2~2~9~
g pha~naceutically acceptable carrier, said pharrnacelllically acceptable carrie}
itself containing substantially little or no amine stabilizing agent in the absence of the stabilized interferon composition. E,ffective amounts of amine stabili~ing agent for stabili2ing interferon in an interferon antiviral preparation are S achieved when the weight of amine stabilizing agent exceeds the weight of total protein. Total protein may be determined by conventional techniques generally known to those skilled in the art. For example, total protein may be determined by the Lowry method or modifications thereof. Effective amounts of amine stabilizing agent (gram amine stabilizing agent per g~am total protein)10 range fronn about 1.0 to about 10.0, and are most preferably about 1.44.
The term "lithium stabilized interferon antiviral preparation" as used herein describes a product having a lithium stabili~ed interferon composition and a pharmaceutically acceptable carrier, said pharmaceutically acceptable canier itself containing substantially little or no lithium anionic i5 stabilizing agent in the absence of the lithium stabilized interferon composition.
Effective amounts of lithium stabilizing agent for stabilizing interferon in a lithium stabilized interferon antiviral preparation are achieved when the weightof lithium stabilizing a~ent exceeds the weight of total protein. Total protein may be determined by conventional techniques generally known to those skilled 20 in the art. For example, total protein may be determined by the Lowry method or modifications thereof. ~ffective amounts of lithium stabilizing agent (gram lithium stabilizing agent per gram total protein) range from about 1.0 to about 10.0, and are most preferably about 1.44.
Phannaceutically acceptable carriers should be inert. The 25 canier should not react with or otherwise reduce the effectiveness of the active ingredients, and more particularly, should not significantly diminish the effectiveness or stability of the interferon. Pharmaceutically acceptable carriers include water, ethanol, polyethylene glycol, mincral oil, petrolatum, propylene glycol, lanolin, and similar agents. Appropriate preparations for topical o 93/08832 PCI/lIS92/~X29' 2~ 209 .1 applications are preferably constituted in lotion, cream~ oil or emulsion formulations.
The tenn "interferon ointment" as used herein describes an .~ interferon antiviral preparation containing an amine stabilizing agent which has S been added in an effective amount to the pharmaceutically acceptable carrier to stabilize interferon. The concentration of arnine stabilizing agent added to the pharmaceutically acceptable carrier may range from about 0.1 % to about 10.0% by weight, although lower or higher concentrations may be used depending on application.
10The terrn "lithium interferon ointment" as used herein describes a lithium interferon antivi~l preparation containing a lithium anionic stabilizing agent which has been added in an effective amount to the pharmaceutically acceptable carrier to stabilize interferon. The concentration of lithium anionicstabilizing agent added to the pharmaceutically acceptable carrier may range 15 from about 0.1% to about 10.0% by weight, although lower or higher concentrations may be used as would be ascertainable by a person having ordinary skill in the art.
The term "mixed interferon ointment" as used herein describes an interferon antivi~al preparation containing a lithium anionic stabilizing agent 20 which has been added in an effective amount to the pharmaceutically acceptable carrier to stabilize interferon, or a lithium interferon antiviral preparation containing an amine stabilizing agent which has been added in an effective amount to the pharmaceutically acceptable carrier to stabili2e interferon, or mixtures thereof. The concentration of lithium anionic stabilizing agent or 25 amine stabilizing agent added to the phannaceutically acceptable carrier may range from about 0.1% to about 10.0% by weight, although lower or higher concentrations may be used as would be ascertainable by a person having ordinary skill in the art.
WO 93t08832 2 ~ 2 0 9 4 f~ PCr/l!S92/0829~
The antiviral activity of interferon may be determined by a number of methods such as those described more fillly in: W.E. Ste~art 11, The Intefferon S~stem, Springer-Verlag (2d Fd. 1981). The Cytopathic Effect Inhibition Assay (CPE) is particularly usefill for determining interferon antiviral 5 activity. The method is described more fully in Id. at pp. 17-18 and is incolporated herein by reference. As used herein, the CPE method entails introducing 50 microliters of growth medium (Eagle's minimal essential medium supplemented with 5% to 10% fetal calf serum) per well into microtiter plates. Twenty-five microliters of interferon is introduced into the 10 first well of the microtiter row and the solution mixed with a fresh micro pipette tip. A 25 microliter aliquot is transferred to the next well and mixed with a fresh micro pipette tip. Virus controls, cell controls and standards are treated similarly. The samples are exposed to sterilizing ultraviolet radiation (approximately 6 minutes at 15 centimeters with a General Electric germicidal 15 larnp). Freshly trypsinized cell suspensions of human foreskin fibroblast cells are introduced into each well (0.01 milliliters containing about 2 x 10~ cells) and the microtiter trays are incubated over night at 37C. A suspension of vesicular stomatitis virus ~American Type Culture Collection, Camden, N.J., U.S.A.) containing about I x 104 plaque-forming units in 50 microliters of 20 serum-free Eagle's minirnal essential medium is introduced into each well, except cell controls, and the trays are incubated for approximately 24 hours at 37C, at which tirne virus controls show 100% CPE. End-points are read microscopically as 50 % protection. Residual activity thus measured is compared to the International Reference standard 6, 69/19 for human interferon 25 (leukocyte-derived, Medical Research Council, National Institute for Biological Standards and Control, London, U.K.) tested si nultaneously.
Because of the recognized inaccuracies of this biological assay (differences of less than 0.3 loglO cannot be objectively distinguished), all results are presented as approximate percentages of starting interferon (O 93/08832 ~cr/llss2/(J~2s~
2~209~
activities . Thus, a readin~ cf 100 % activity indicates rlo detectible or significant loss of interferon activity. A reading of between 70 and 80%
activity indicates a slight loss of interferon activity; a reading of between 30 to 60% activity indicates a significant detectible loss; a reading of between I to 5 20% activity indicates approximately a I to 2 loglO decrease in interferon activity; and a reading of 0% activity indicates no detectible activity remaining.
The following examples serve to illustrate embodiments of this invention without lirniting the same, but should not be considered as a limitation on the scope of the invention.
10 Example I
Storage stability of interferon in stabilized interferon compositions and lithium stabilized interferon compositions was assessed.
Human leukocyte interferon containing approximately 80 % alpha inter~eron and approximately 20% omega interferon, prepared as described by Cantell, supra, 15 was dialy~ed in physiological saline. Stabilized interferon compositions wereprepared by mixing aliquants of 0.1 ml human leukocyte interferon (at I x 104, I X 105 or 1 x lo6 IU/ml) with 0.9 ml physiological saline (pH 7.2) containing amine stabilizing agents at the concentrations indicated in Table 1. Lithium stabilized interferon compositions were prepared by mixing aliquants of 0.1 ml 20 human leukocyte interferon (at 1 x 104, I X 105 or I x 10~ IU/ml) with 0.9 mlphysiological saline (pH 7.2) containing the lithium anionic stabilizing agents at the concentrations indicated in Table 1.
The stabili~ed interferon compositions and lithium stabilized interferon compositions were incubated at room temperature (20 to 24C) for 25 I hour, 24 hours, or I month, and then tested for residual antiviral activityusing vesicular stornat;tis virus and human skin fibroblast cell lines, according to the CPE inhibition method described above. Results, expressed as % of initial antivi~l activity, are shown in Table 1.
~WO 93/0883~ s92/08~
2~9~4 Table I
Stabilization of huma~ interferon (20-24C) StabilizerConceotr. % Initial Activity stabilizer storage time (wt. %~_lhr 24hr I mo.
Lithium octyl sulfate 0.1 90 10 0 1.0 100 70 10 10.0 100 100 60 Lithium decyl sulfate 0.1 100 30 10 1.0 100 80 40 10.0 100 100 80 Lithium dodecyl sulfate0.1 100 100 100 1.0 100 100 100 10.0 100 100 100 Lithium tetradecyl sulfate 0.1 100 100 100 1.0 100 100 100 25 Octyl amine 0.1 70 20 0 1.0 ~0 50 10 10.0 100 70 10 Decyl amine 0.1 80 30 10 1.0 80 50 10 10.0 100 70 30 Dodecyl amine 0.1 100 100 100 1.0 100 100 100 10.0 100 100 100 Tetradecyl amine 0.1 100 100 100 1.0 100 100 100 40 Control - 100 30 0 ~0 93/0883~ 92/~)~29~
2 1 2 ~
~4 As shown, the lithium organo sulfates are effective in stabilizing the aqueous human interferons. Most effective was lithium dodecyl sulfate and lithium tetradecyl sulfate, at concentratiorls ranging from 0. I to 10 % by weight. The amine stabilizing agents likewise stabilized human interferon.
Most effective was dodecyl amine at concentrations Or o. I % to 10 % by weight, and tet~decyl a;nine in concentrations of 0.1% to 1.0% by weight.
Exarnple 2 The stabilized interferon compositions (containing dodecyl arnine at 0.1% and 1.0% by weight), prepared as described in Example 1, were stored at 37C for I hour, 24 hours, or 1 month, and tested for residual antivi~l activity using the CPE inhibition assay. Unstabilized interferon in saline served as a control. Results, expressed as % of initial antivi~l activity, are shown in ~able 2.
Table 2 Stability of Stabilized lnterferon Compositions (37C) Stabilizer Stabilizer Corlcentr. % Initial Activity storage tirne (% by wt.! lhr 24hr 1 month Dodecyl amine 0.1 100 100 100 1.0 100 100 100 Control -- 100 30 0 ~
As shown, dodecyl amine at concentrations of 0.1% and 1.0% by weight stabilized the interferon stored at 37C.
Example 3 The stabilized interferon compositions prepared as descnbed in 35 Example 1 were stored at 56C for 1 hour or 24 bours, and tested for residual ,_~WO ~3/08832 PC r/US92/0t~29' i 2~ 2~9l~ ~
-` 15 antivilal activity as shown in Table 3. Unstabilized interferon in saline selvedas a control. Results are expressed as % of initial antiviral activity.
Table 3 Stability of Stabilized Interferon Compositions (56C) Stabilizer Stabili~er Concelltr. % Initia3 Activity storage time (% by wt.~ Ihr 24hr Dodecyl amine 0.1 100 100 Control -- 3 0 As shown, dodecyl amine at concentrations of 0.1% to 1.0% by weight stabilized human interferon stored for up to 24 hours.
ample 4 Storage stability of interferon antiviral preparation was assessed.
25 Stabilized interferon composition, prepared as in Example 2, and a control interferon in sa}ine were each dialyzed against 0.03 M ammonium bicarbonate buffer (pH 7.6) and Iyophilized. The stabilized interferon composition and control interferon in powder forrn were each added to a pharmaceutical carrier comprising (weight %) 10% lanolin and 90% petrolatum, and mixed at 37C
30 for 1 hour. Aliquants of the interferon antiviral preparation thus obtained were stored at 4C for 1 month, 6 months, 12 months, and 36 months.
Following each selected storage period, aliquants of interferon antiviral prepalation were mixed with equal volumes of S ~o fetal calf serum-containing tissue culture medillm (Eagle's mir~imal essential medium) and incubated for 35 4 hours at 37C, and the culture medium tested for eluted residual antiviral /0 93/08837 PC~ !S92/~)~29~
2 1 ~
activity by the CPE inhibition assay. The results, expressed as % of initial antiviral activity, are shown in Table 4.
Table 4 Stability of interferon antiviral preparation (4 C) Interferon% Initial Activity storage time I mo. 6 mo. 12 mo. _36 mo. ~ -Dodecyl amine-stabilized 100 100 100 100 -~
Control 70 10 0 n.t.*
- - - -* Signifies "not tested", because prior activity was already zero.
As shown, dodecyl amine treatment significantly stabilized the interferon antivi-al preparation for periods of up to 36 months in samples stored at 4C. ~ ~-Example 5 The interferon antiviral preparation in Example 4 was stored at room temperature (20-24C). Results, expressed as % of initial antiviral activity, are shown in Table 5.
,~ WO ~3/~)8832 PCr/US9~/082~2 !
2~2~9~
Table 5 Stability of interferon antiviral preparation ~20-24C) Interferon % lnitial Activity storage time I mo. 6 mo12 mo. 3S mo.
Dodecyl amine-stabilized 90 40 20 0 Control 0 n.t.*n.t. n.t.
1 5 - -~--~
* Signifies "no~ tested", because prior activity was already zero.
As shown, dodecyl amine treatment significantly stabilized the interferon antiviral prepa~tion for periods of at least a year in samples stored20 at 20-24~C.
~' ..
,~0~3/0~83~ ~C~ S92/(~X2()7 21209~
Example 6 The interferon antiviral preparation in Exarnple 4 was stor~l at 37C. Results, expressed as % of initial antiviral activity, are shown in Table 6.
Table 6 Stability of interferon antiviral preparation (37 C) Inter~eron% Initial Activity storage time I mo. 6 mo. 12 mo. 36 mo.
Dodecyl amine~
stabilized ~0 20 n.t. *
Control 0 n.t. n.t. n.t.
20 * Signifies "not tested", because prior activity was already zero.
As shown, dodecyl amine-treatment signi~lcantly stabili7ed the interferon antiviral preparation for periods of up to six months in samples stored at 37C.
25 Example 7 The storage stability of interferon ointment was assessed. An interferon antiviIal preparation was prepared as described in E~sample 4, with the exception that the pharmaceutical carrier contained 0.1% tweight %) dodecyl amine. Aliquants of the interferon ointments thus obtained were stored 30 at 37~C, for 1 month, 6 months, 12 months, and 36 months. Aliquants were then mixed with equal volurnes of 5 % fetal calf sen~m-containing tissue culturemedium (Eagle's minimal essential medium) and incubated for 4 hours at 37C, and the culture medium tested for eluted residual antiviral activity by the CPE inhibition assay. The results are shown in Table 7.
. ~
WO 93/OX83 PC~/US92/082~ ~
2~209~
Table 7 Stability of interferon ointment (4 C) Interferon % Initial A~tivi!ty storage time I mo. 6 mo. 12 mo. 36 mo.
Dodecyl amine- ;
ointment 100 100 100 100 Control 70 10 0 n.t.* -* Signifies "not tested", because prior activity was already zero.
Example 8 20The interferon ointment as in Example 7 was stored at room temperature (20-24C) for I month, 6 months, 12 months and 36 months. Results, expressed as % of initial antiviIal activity, are shown in Table 8.
Table 8 Stability of interferon ointment 25(2Q-24C) Interferon % Initial A~tivity storage time I mo.6 mo.12 mo. 36 mo.
Dodecyl arnine-ointment 100 100 100 100 Control 0 n.t. *n.t. n.t.
* Signifies "not tested", because prior activity was already zero.
O 93/OK832 IJCI/l!S92/1)829' 2 ~ 2 ~
Example 9 Interferon ointments as in Example 7 were stored at 37C for I
month, 6 months, 12 months and 36 months. Resul~s, expressed as % of initial antiviral activity, are shown in Table 9.
s Table 9 Stability of interferon ointment (37C) Interferon % Initial Activity storage time 1 mo. 6 mo. 12 mo. 36 mo.
Dodecyl amine-ointment 100 100 100 100 Control 0 n.t.* n.t. n.t.
20 * Signifies "not tested", because prior activity was already zero.
Example 10 Leukocyte interferon extraceed as in Example I was subjected to 25 non-reducing SDS-PAGE and the alpha interferon and omega interferon fractions eluted into serum-containing medium (Eagle's minimal essential medium containing 5% fetal calf serum). Stabili2ed interferon compositions ~1.0% by weight dodecylamine) as in Example 2 were prepared for alpha-isolate interferon and for omega-isolate interferon. Control interferon 30 (containing both alpha and omega interferon) was prepared in saline. The stabilized interferon compositions were stored for up to one month at room tempelature (20-24C). Results are expressed as % of irutial antiviral activity.
".-':' :.:
~WO 93/~883~ PCr/US()2/08. 9' 2~2~
:
Table 10 Stabilization of stabilized interferon compositions: : -Alpha and omega interferon isolates :
Inter~eron % Init;al Astivity .
storage time ~ I hr 24 hrs I month Alpha-isolates 100 100 100 Omega-isolates 100 100 100 Control 100 30 0 Stabilized ~nterferon compositions comprising alpha isolate or omega isolate, as in Example 10, were stored ~or up to one month at 37C.
Results are expressed as % of initial antivi~l activity.
Table 11 Stabilization of stabilized interferon compositions:
Alpha and omega isolates ~37C) Interferon % Initial Activity s~orage tirne I hr 24 hrs I month Alpha-isolates 100 100 100 :~
Omega-isolates 100 100 100 --Control 100 30 0 ~o 93/0883~ ~'CI/IIS9~/~JX29' 2~ 209~q F,xample 12 Interferon ointments prepared as in Pxample 7 comprising alpha isolate or omega isolate (isolates prepared .IS in Example 10) were stored for up to 6 months at room temperature (20-24C). Results are expressed as %
S of initial antiviral activity.
, Table 12 Stability of interferon ointment:
Alpha and omega isolates (20-24C) Interferon% Initial A~ivity storage time I month 6 months Alpha-isolates100 100 Omega-isolates100 100 Control 0 n.t.
2() _ :
* Signifies "not tested", because prior activity ~as aLready zero.
'. ~ ~
wo 93/1)8~32 1 ~U' ~
2~ ~9~
Example 13 Interferon ointments prepared as in Exarnple 7 comprising alpha isolate or omega isolate (isolates prepared as in P,xample 10) were stored for up to 6 months at 37C. Results are expressed as æ of initial antiviral activity.
s Table 13 Stability of interferon ointment:
Alpha and omega isolates (37C) - Inter~eron % Initial Activity storage time l month 6 months Alpha-isolates 100 100 Omega-isolates l 00 100 Control 0 n.t. ~
_ __ _ _ * Signif1es "not tested", because prior activity was already ~ero.
25 Many other variations and modifications may be made in the invention hereinbefore described, by those having experience in this technology, without departing from the concept of the present invention. Accordingly, it should be clearly understood that the methods and compositions described in the foregoing description are illustrative only and not intended as a limitation on 30 the scope of the invention.
subcutaneous administration. Also, the patient could self-administer topical interferon without the direct assistance of a qualifled health-care worker. A
topical interferon product could also promote patient compliance by providing a more pleasant therapeutic experience for the patient than interferon treatmentS via injection.
The lack of ~nterferon stability in solutions and other products has heretofore lirnited its utility. Interferon products having enhanced storagestability should faci3itate more wide-spread use of this import~nt therapeutic agent. Several ~actors are important in designing a useful topical interferon 10 preparation. First, conventional topical interferon preparations are gene~llyconsidered unstable, and therefore are considered to have a limited shelf-life.
A topical interferon pre~aration should be stabilized to prevent degradation over time and upon shipping and handling. Second, because interferon generally has a higher molecu1ar weight than the molecular weights of the 15 therapeutic agents usually administered in topical preparations, interferon should be incolporated into a substance which sufficiently holds the high molecular weight interferon in suspension during packaging, shipping and application, and yet also be able to release the substance from the preparation in a reasonable length of time once it has been applied to the diseased site.
20 Third, the preparation must not adversely affect the antivilal activity of interferon.
The present invention describes ne v, improved stabilizers and methods for stabilizing interferon, rneeting the requirements for topical interferon products defined above and for stabilized interferon solutions.
25Other interferon-stabilizing agents have been described and used to stabili7e interferon.
Estis et al. in U.S. Patent No. 4,680,175 describe the use of protease inhibitors such as alpha-l-antitrypsin inhibitor, alpha-2 macroglobulin, soybean inhibitor, N-alpha-tosyl-L-lysine chloromethyl ketone, , ~vo93/08832 ~ s92/08~
~¦ 2 1 2 o 9 4 L~
:¦ phenylmethylsulfonyl fluoride, and N-alpha-iosylphenylalanine chloromethyl :I ketone to stabilize topical interferon preparations. Miller, in U.S. Patent No.
~ 4,957,734, teaches the use of nonionic surface active agents in combination ¦ with interferon and discloses the use of anionic, cationic and nonionic surface S active agents, but does not clainn stabilization of inter~eron. Miller discloses that quarterna~y ammonium detergents such as cetyl pyridin~um chloride and benzalkonium chloride are preferable cationic agents. Hasegawa et al. in U.S.
Patent No. 4,675,184 teach that anionic surface active agents such as sodium cyl sulfates stabilize beta-interferon (fibroblast-derived).
Stewart et al. in U.S. Patent No. 3,981,991 teach that interferon can be stabilized by treating the antiviral agent with a combination of: 1) guanidine-hydrochloride or urea; 2) mercaptoethanol or ethanethiol; and 3) an agent selected from the group consisting of sodium dodecylsulfate, sodium decylsulfate, sodium dodecylsulfonate, dodecylamine and decylamine. Stewart 15 et al. caution that interferon cannot be stabilized with only one or two of the aforesaid three agents. None of these patents teach that human alpha and omega interferons or mixtures thereof can be stabilized with only the stabilizing agents described herein.
SUMMARY OF THE INVEN~ION
~i 20 This invention relates to preparations for stabilizing human interferon. The present invention shows that human alpha and omega interferon, or mLl~tures thereof, can be stabili~ed when placed in solutions containing amines, and more particularly, primary aliphatic amines having from between 8 to 14 car~on atoms. Lithium salts of anionic stabili7ing agents such 25 as lithium organo sulfates also stabilize interferon. Stabilized interferon may 3, b~ placed in a plhannaceutically acceptable ca~ier for topical administration.
Further stability is achieved by adding additional a~nine-stabilizin~ agents to the stabilized interferon product.
r~ WO93/û8832~ /US92/0~29' 2 ~ 2 ~
These and other advan~ges of the present invention will become apparent from the following detailed description.
DETAILED DESCRIPTION OF THE Il~PIENTION
The presenl invention relates to stabilizing interferon to enhance 5 its storage stabili~y. This invention is applicable to all types of interferon including natural interferon, interferon produced by recombinant DNA
technology, and interferon produced by chemical synthesis or modification.
Also, the invention can be used with crude, semi-purified and purified interferon from leukocytes, fibroblasts, Iymphocytes or any other interferon-10 containing or producing tissues from humans or any other appropriate species.A method for producing interferon from leukocytes (comprisingalpha interferon and omega interferon) is described by Cantell, et ah in:
Methods ~ in En~;ymolo~, 78: 29-38 (1981). In general, the interferon produced by this method and other methods is supplied as a liquid. Alpha 15 isolates and omega isolates of interferon are prepared by subjecting leukocyte interferon to non-reducing sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) as described in Stewart, W.E. II, et al., Proc.
Natl. Acad. Sci. 74:4200-4204 (1977). Briefly, leukocyte interferon samples in 0.01 M sodium phosphate buffer were constituted to contain 5 M urea and 20 1 % sodium dodecyl sulfate. Bromphenol blue in sucrose was added to make the samples 6% sucrose, and the samples were heated to lOO~C for one minute. Aliquots of 0.1 ml of sample were electrophoresed on parallel SDS-PAGE gels. When the dye front had migrated about 18 centimeters into gels, gels were sliced into 2.2 millimeter segments, and two adjacent slices were 25 pooled into e~ch fraction containing the alpha isolates and omega isolates, and were eluted ovemight at 4C into 1 ml of Eagle's minimal essential medium containing 10% fetal calf serum.
Those skilled in the alt will recognize that the amount of interferon which is administered in any particular case, as well as the frequency at which ,~0 93/08g32 ~ JS~)2/()82()' 2~3~
the interferon is administered, will depend upon such factors as the type of interferon used, the disease being treated, the patient's response to interferontreatment, and whether the preparation used is a semi-solid or a liquid. The National Institutes of Health ~United States ~Department of Health and Human S Services, Bethesda, Maryland) have established unit strengths ~or interferon activity. In terms of these unit strengths, dosage levels for topical pharmaceutical preparations using crude or partially purifled natural int~rferons can range from about 10,000 lU/gram of topical preparation to 1,000,000 IU/gram of topical preparation.
The terrn "stabilized interferon composition" is employed herein to describe an agent having an interferon and an amine stabilizing agent present in an effective amount to stabilize interferon. The interferon can be human interferon or interferon from any other appropriate species. Preferably the interferon is human leukocyte-derived interferon comprising alpha interferon, 15 omega interferon or mixtures thereof. The interferon can also be recombinant interferon or interferon produced or modified chemically. The present invention reveals enhanced storage stability and antivilal activity of stabilizing agents for interferon. The term "amine stabilizing agent" is employed herein to includeprimary, secondary, ortertiary amines. Preferably, 20 the amine stabilizing agent is a primary arnine having the general formula RNH2 where "R" is an aliphatic group. Preferably, amine stabilizing agents have aliphatic groups of from about 8 to about 14 carbon atoms, although aliphatic groups having less than 8 or more than 14 carbon atoms are encompassed within the scope of this invention. More preferably, aliphatic 25 primary arnines having from between about 8 to about 14 carbon atoms may be used as amine stabili~ing agents. Most preferably, the arnine stabilizing agent is a primary aliphatic amine having an aliphatic group consisting of 12 carbon atoms. I`he aliphatic groups may be linear or branched, but are preferably linear. Representative examples of amines useful as arnine ~WO 93/08832 Pc~r/v~2/()82~
: 2 ~ 2 ~
stabili~ing agents include octyl amine, decyl amine, dodecyl amine, and tetradecyl amine. Amine stabilizing agents having more than 14 carbon atoms are generally insoluble. Arnine stabilizing agents having less than 8 carbon atoms are generally inefficient stabilizers. Most preferable of the amine 5 stabilizing agents is dodecyl amine~ The ef~ective arnount of amine stabilizing agents to stabilize interferon ranges from about 0.1% to about 10.0% by :¦ weight, although lower or higher concentrations of amine stabilizing agents may be used, as would be ascertainable by a person having ordinary skill in the art.
10The term "lithium stabilized interferon composition" is employed 31 herein to describe an agent having an interferon and a lithium anionic stabilizing agent present in an effective amount to stabilize interferon. The interferon can be human interferon or interferon from any other appropriate species. Preferably the interferon is human leukocyte-derived interferon 15 containing alpha iDterferon or omega interferon or mixtures thereof. The interferon can also be recombinant interferon or interferon produced or modified chemically.
The term "lithium anionic stabilizing agent" is employed herein ~' to include the lithium salts of compounds having a net negative charge.20 Lithium anionic stabilizing agents useful in this invention are lithium organo sulfates, and may be represented by the general formula:
Li RX
where "R" is an aliphatic group, "X" is an anion and "n" is chosen to electrochemically balance the anion. "X" is preferably sul~ate. Lithium 25 anionic stabilizing agents containing aliphatic groups having from about 8 toabout 22 carbon atoms may be used, although aliphatic groups of more than 22 carbons or less than 8 carbons are encompassed within the scope of the present invention. Preferably, lithium anionic stabilizing agents contain 8 to 14 carbonatoms, and most preferably 12 carbon atoms. Representative examples of 0 93/U8832 i~C~/US92/~1829 lithium anionic stabilizing agents include lithium octyl sulfate, lithium decyl sulfate, lithium dodecyl sulfate and lithium tetradecyl sulfate. The concentrations of lithium anionic stabilizing agents may range from about 0.1%
to about 10.0% by weight, although lower or higher concentrations may be 5 used as would be ascertainable by a person having ordinary skill in the art.
The patent of Hasegawa et al., described above, does not disclose the use Or lithium organo sulfate interferon stabili7ers. As will be noted below, lithium-containing stabilizing agents have a clinical utility not possessed by sodium-containing stabilizing agents.
Interferon compositions containing from about I x 104 to 1 x 106 IU/ml are most preferably used in the present invention, although interferon compositions having lower or higher activity may also be used as would be readily ascertainable by a person having ordinary skill in the art.
The precise mechanism of action of the amine stabilizing agents and 15 lithium anionic stabili~ing agents in stabilizing interferon is not known, but is thought to be related to "defensive reversible denaturation" as described by W.E. Stewart II, et al. Na~ure, Vol. 249, pp. 460-61 ~1974) .and W.E.
Stewart I[, et al., Preparative Biochemisty Vol. 4, pp. 383-93 (1974).
The clinical utility of sodium-containing interferon stabilizing agents 20 such as sodium organo sulfates is limited by the side effects associated withtopical administration of sodium-salt-containing products. A not uncommon unwanted side effect associated with use of sodium-salt-containing products is chronic irritant dermatitis. Tupker, R.A. et ak, Acta De_. Venereo!. 70:1-5 ~1990) . Unlike sodium-salt-containingtopical products, lithium-saltp~parations 25 are not associated with such unwanted side effects. A lithium-salt-containinginterferon product would therefore represent an advance over sodium-salt-contair~ing topical interferon preparations.
The term "interferon antiviral preparation" as used herein describes a p~oduct having a stabilized interferon composition and a ~WO 93/0~832 ~'C~/US92/08'97 2~2~9~
g pha~naceutically acceptable carrier, said pharrnacelllically acceptable carrie}
itself containing substantially little or no amine stabilizing agent in the absence of the stabilized interferon composition. E,ffective amounts of amine stabili~ing agent for stabili2ing interferon in an interferon antiviral preparation are S achieved when the weight of amine stabilizing agent exceeds the weight of total protein. Total protein may be determined by conventional techniques generally known to those skilled in the art. For example, total protein may be determined by the Lowry method or modifications thereof. Effective amounts of amine stabilizing agent (gram amine stabilizing agent per g~am total protein)10 range fronn about 1.0 to about 10.0, and are most preferably about 1.44.
The term "lithium stabilized interferon antiviral preparation" as used herein describes a product having a lithium stabili~ed interferon composition and a pharmaceutically acceptable carrier, said pharmaceutically acceptable canier itself containing substantially little or no lithium anionic i5 stabilizing agent in the absence of the lithium stabilized interferon composition.
Effective amounts of lithium stabilizing agent for stabilizing interferon in a lithium stabilized interferon antiviral preparation are achieved when the weightof lithium stabilizing a~ent exceeds the weight of total protein. Total protein may be determined by conventional techniques generally known to those skilled 20 in the art. For example, total protein may be determined by the Lowry method or modifications thereof. ~ffective amounts of lithium stabilizing agent (gram lithium stabilizing agent per gram total protein) range from about 1.0 to about 10.0, and are most preferably about 1.44.
Phannaceutically acceptable carriers should be inert. The 25 canier should not react with or otherwise reduce the effectiveness of the active ingredients, and more particularly, should not significantly diminish the effectiveness or stability of the interferon. Pharmaceutically acceptable carriers include water, ethanol, polyethylene glycol, mincral oil, petrolatum, propylene glycol, lanolin, and similar agents. Appropriate preparations for topical o 93/08832 PCI/lIS92/~X29' 2~ 209 .1 applications are preferably constituted in lotion, cream~ oil or emulsion formulations.
The tenn "interferon ointment" as used herein describes an .~ interferon antiviral preparation containing an amine stabilizing agent which has S been added in an effective amount to the pharmaceutically acceptable carrier to stabilize interferon. The concentration of arnine stabilizing agent added to the pharmaceutically acceptable carrier may range from about 0.1 % to about 10.0% by weight, although lower or higher concentrations may be used depending on application.
10The terrn "lithium interferon ointment" as used herein describes a lithium interferon antivi~l preparation containing a lithium anionic stabilizing agent which has been added in an effective amount to the pharmaceutically acceptable carrier to stabilize interferon. The concentration of lithium anionicstabilizing agent added to the pharmaceutically acceptable carrier may range 15 from about 0.1% to about 10.0% by weight, although lower or higher concentrations may be used as would be ascertainable by a person having ordinary skill in the art.
The term "mixed interferon ointment" as used herein describes an interferon antivi~al preparation containing a lithium anionic stabilizing agent 20 which has been added in an effective amount to the pharmaceutically acceptable carrier to stabilize interferon, or a lithium interferon antiviral preparation containing an amine stabilizing agent which has been added in an effective amount to the pharmaceutically acceptable carrier to stabili2e interferon, or mixtures thereof. The concentration of lithium anionic stabilizing agent or 25 amine stabilizing agent added to the phannaceutically acceptable carrier may range from about 0.1% to about 10.0% by weight, although lower or higher concentrations may be used as would be ascertainable by a person having ordinary skill in the art.
WO 93t08832 2 ~ 2 0 9 4 f~ PCr/l!S92/0829~
The antiviral activity of interferon may be determined by a number of methods such as those described more fillly in: W.E. Ste~art 11, The Intefferon S~stem, Springer-Verlag (2d Fd. 1981). The Cytopathic Effect Inhibition Assay (CPE) is particularly usefill for determining interferon antiviral 5 activity. The method is described more fully in Id. at pp. 17-18 and is incolporated herein by reference. As used herein, the CPE method entails introducing 50 microliters of growth medium (Eagle's minimal essential medium supplemented with 5% to 10% fetal calf serum) per well into microtiter plates. Twenty-five microliters of interferon is introduced into the 10 first well of the microtiter row and the solution mixed with a fresh micro pipette tip. A 25 microliter aliquot is transferred to the next well and mixed with a fresh micro pipette tip. Virus controls, cell controls and standards are treated similarly. The samples are exposed to sterilizing ultraviolet radiation (approximately 6 minutes at 15 centimeters with a General Electric germicidal 15 larnp). Freshly trypsinized cell suspensions of human foreskin fibroblast cells are introduced into each well (0.01 milliliters containing about 2 x 10~ cells) and the microtiter trays are incubated over night at 37C. A suspension of vesicular stomatitis virus ~American Type Culture Collection, Camden, N.J., U.S.A.) containing about I x 104 plaque-forming units in 50 microliters of 20 serum-free Eagle's minirnal essential medium is introduced into each well, except cell controls, and the trays are incubated for approximately 24 hours at 37C, at which tirne virus controls show 100% CPE. End-points are read microscopically as 50 % protection. Residual activity thus measured is compared to the International Reference standard 6, 69/19 for human interferon 25 (leukocyte-derived, Medical Research Council, National Institute for Biological Standards and Control, London, U.K.) tested si nultaneously.
Because of the recognized inaccuracies of this biological assay (differences of less than 0.3 loglO cannot be objectively distinguished), all results are presented as approximate percentages of starting interferon (O 93/08832 ~cr/llss2/(J~2s~
2~209~
activities . Thus, a readin~ cf 100 % activity indicates rlo detectible or significant loss of interferon activity. A reading of between 70 and 80%
activity indicates a slight loss of interferon activity; a reading of between 30 to 60% activity indicates a significant detectible loss; a reading of between I to 5 20% activity indicates approximately a I to 2 loglO decrease in interferon activity; and a reading of 0% activity indicates no detectible activity remaining.
The following examples serve to illustrate embodiments of this invention without lirniting the same, but should not be considered as a limitation on the scope of the invention.
10 Example I
Storage stability of interferon in stabilized interferon compositions and lithium stabilized interferon compositions was assessed.
Human leukocyte interferon containing approximately 80 % alpha inter~eron and approximately 20% omega interferon, prepared as described by Cantell, supra, 15 was dialy~ed in physiological saline. Stabilized interferon compositions wereprepared by mixing aliquants of 0.1 ml human leukocyte interferon (at I x 104, I X 105 or 1 x lo6 IU/ml) with 0.9 ml physiological saline (pH 7.2) containing amine stabilizing agents at the concentrations indicated in Table 1. Lithium stabilized interferon compositions were prepared by mixing aliquants of 0.1 ml 20 human leukocyte interferon (at 1 x 104, I X 105 or I x 10~ IU/ml) with 0.9 mlphysiological saline (pH 7.2) containing the lithium anionic stabilizing agents at the concentrations indicated in Table 1.
The stabili~ed interferon compositions and lithium stabilized interferon compositions were incubated at room temperature (20 to 24C) for 25 I hour, 24 hours, or I month, and then tested for residual antiviral activityusing vesicular stornat;tis virus and human skin fibroblast cell lines, according to the CPE inhibition method described above. Results, expressed as % of initial antivi~l activity, are shown in Table 1.
~WO 93/0883~ s92/08~
2~9~4 Table I
Stabilization of huma~ interferon (20-24C) StabilizerConceotr. % Initial Activity stabilizer storage time (wt. %~_lhr 24hr I mo.
Lithium octyl sulfate 0.1 90 10 0 1.0 100 70 10 10.0 100 100 60 Lithium decyl sulfate 0.1 100 30 10 1.0 100 80 40 10.0 100 100 80 Lithium dodecyl sulfate0.1 100 100 100 1.0 100 100 100 10.0 100 100 100 Lithium tetradecyl sulfate 0.1 100 100 100 1.0 100 100 100 25 Octyl amine 0.1 70 20 0 1.0 ~0 50 10 10.0 100 70 10 Decyl amine 0.1 80 30 10 1.0 80 50 10 10.0 100 70 30 Dodecyl amine 0.1 100 100 100 1.0 100 100 100 10.0 100 100 100 Tetradecyl amine 0.1 100 100 100 1.0 100 100 100 40 Control - 100 30 0 ~0 93/0883~ 92/~)~29~
2 1 2 ~
~4 As shown, the lithium organo sulfates are effective in stabilizing the aqueous human interferons. Most effective was lithium dodecyl sulfate and lithium tetradecyl sulfate, at concentratiorls ranging from 0. I to 10 % by weight. The amine stabilizing agents likewise stabilized human interferon.
Most effective was dodecyl amine at concentrations Or o. I % to 10 % by weight, and tet~decyl a;nine in concentrations of 0.1% to 1.0% by weight.
Exarnple 2 The stabilized interferon compositions (containing dodecyl arnine at 0.1% and 1.0% by weight), prepared as described in Example 1, were stored at 37C for I hour, 24 hours, or 1 month, and tested for residual antivi~l activity using the CPE inhibition assay. Unstabilized interferon in saline served as a control. Results, expressed as % of initial antivi~l activity, are shown in ~able 2.
Table 2 Stability of Stabilized lnterferon Compositions (37C) Stabilizer Stabilizer Corlcentr. % Initial Activity storage tirne (% by wt.! lhr 24hr 1 month Dodecyl amine 0.1 100 100 100 1.0 100 100 100 Control -- 100 30 0 ~
As shown, dodecyl amine at concentrations of 0.1% and 1.0% by weight stabilized the interferon stored at 37C.
Example 3 The stabilized interferon compositions prepared as descnbed in 35 Example 1 were stored at 56C for 1 hour or 24 bours, and tested for residual ,_~WO ~3/08832 PC r/US92/0t~29' i 2~ 2~9l~ ~
-` 15 antivilal activity as shown in Table 3. Unstabilized interferon in saline selvedas a control. Results are expressed as % of initial antiviral activity.
Table 3 Stability of Stabilized Interferon Compositions (56C) Stabilizer Stabili~er Concelltr. % Initia3 Activity storage time (% by wt.~ Ihr 24hr Dodecyl amine 0.1 100 100 Control -- 3 0 As shown, dodecyl amine at concentrations of 0.1% to 1.0% by weight stabilized human interferon stored for up to 24 hours.
ample 4 Storage stability of interferon antiviral preparation was assessed.
25 Stabilized interferon composition, prepared as in Example 2, and a control interferon in sa}ine were each dialyzed against 0.03 M ammonium bicarbonate buffer (pH 7.6) and Iyophilized. The stabilized interferon composition and control interferon in powder forrn were each added to a pharmaceutical carrier comprising (weight %) 10% lanolin and 90% petrolatum, and mixed at 37C
30 for 1 hour. Aliquants of the interferon antiviral preparation thus obtained were stored at 4C for 1 month, 6 months, 12 months, and 36 months.
Following each selected storage period, aliquants of interferon antiviral prepalation were mixed with equal volumes of S ~o fetal calf serum-containing tissue culture medillm (Eagle's mir~imal essential medium) and incubated for 35 4 hours at 37C, and the culture medium tested for eluted residual antiviral /0 93/08837 PC~ !S92/~)~29~
2 1 ~
activity by the CPE inhibition assay. The results, expressed as % of initial antiviral activity, are shown in Table 4.
Table 4 Stability of interferon antiviral preparation (4 C) Interferon% Initial Activity storage time I mo. 6 mo. 12 mo. _36 mo. ~ -Dodecyl amine-stabilized 100 100 100 100 -~
Control 70 10 0 n.t.*
- - - -* Signifies "not tested", because prior activity was already zero.
As shown, dodecyl amine treatment significantly stabilized the interferon antivi-al preparation for periods of up to 36 months in samples stored at 4C. ~ ~-Example 5 The interferon antiviral preparation in Example 4 was stored at room temperature (20-24C). Results, expressed as % of initial antiviral activity, are shown in Table 5.
,~ WO ~3/~)8832 PCr/US9~/082~2 !
2~2~9~
Table 5 Stability of interferon antiviral preparation ~20-24C) Interferon % lnitial Activity storage time I mo. 6 mo12 mo. 3S mo.
Dodecyl amine-stabilized 90 40 20 0 Control 0 n.t.*n.t. n.t.
1 5 - -~--~
* Signifies "no~ tested", because prior activity was already zero.
As shown, dodecyl amine treatment significantly stabilized the interferon antiviral prepa~tion for periods of at least a year in samples stored20 at 20-24~C.
~' ..
,~0~3/0~83~ ~C~ S92/(~X2()7 21209~
Example 6 The interferon antiviral preparation in Exarnple 4 was stor~l at 37C. Results, expressed as % of initial antiviral activity, are shown in Table 6.
Table 6 Stability of interferon antiviral preparation (37 C) Inter~eron% Initial Activity storage time I mo. 6 mo. 12 mo. 36 mo.
Dodecyl amine~
stabilized ~0 20 n.t. *
Control 0 n.t. n.t. n.t.
20 * Signifies "not tested", because prior activity was already zero.
As shown, dodecyl amine-treatment signi~lcantly stabili7ed the interferon antiviral preparation for periods of up to six months in samples stored at 37C.
25 Example 7 The storage stability of interferon ointment was assessed. An interferon antiviIal preparation was prepared as described in E~sample 4, with the exception that the pharmaceutical carrier contained 0.1% tweight %) dodecyl amine. Aliquants of the interferon ointments thus obtained were stored 30 at 37~C, for 1 month, 6 months, 12 months, and 36 months. Aliquants were then mixed with equal volurnes of 5 % fetal calf sen~m-containing tissue culturemedium (Eagle's minimal essential medium) and incubated for 4 hours at 37C, and the culture medium tested for eluted residual antiviral activity by the CPE inhibition assay. The results are shown in Table 7.
. ~
WO 93/OX83 PC~/US92/082~ ~
2~209~
Table 7 Stability of interferon ointment (4 C) Interferon % Initial A~tivi!ty storage time I mo. 6 mo. 12 mo. 36 mo.
Dodecyl amine- ;
ointment 100 100 100 100 Control 70 10 0 n.t.* -* Signifies "not tested", because prior activity was already zero.
Example 8 20The interferon ointment as in Example 7 was stored at room temperature (20-24C) for I month, 6 months, 12 months and 36 months. Results, expressed as % of initial antiviIal activity, are shown in Table 8.
Table 8 Stability of interferon ointment 25(2Q-24C) Interferon % Initial A~tivity storage time I mo.6 mo.12 mo. 36 mo.
Dodecyl arnine-ointment 100 100 100 100 Control 0 n.t. *n.t. n.t.
* Signifies "not tested", because prior activity was already zero.
O 93/OK832 IJCI/l!S92/1)829' 2 ~ 2 ~
Example 9 Interferon ointments as in Example 7 were stored at 37C for I
month, 6 months, 12 months and 36 months. Resul~s, expressed as % of initial antiviral activity, are shown in Table 9.
s Table 9 Stability of interferon ointment (37C) Interferon % Initial Activity storage time 1 mo. 6 mo. 12 mo. 36 mo.
Dodecyl amine-ointment 100 100 100 100 Control 0 n.t.* n.t. n.t.
20 * Signifies "not tested", because prior activity was already zero.
Example 10 Leukocyte interferon extraceed as in Example I was subjected to 25 non-reducing SDS-PAGE and the alpha interferon and omega interferon fractions eluted into serum-containing medium (Eagle's minimal essential medium containing 5% fetal calf serum). Stabili2ed interferon compositions ~1.0% by weight dodecylamine) as in Example 2 were prepared for alpha-isolate interferon and for omega-isolate interferon. Control interferon 30 (containing both alpha and omega interferon) was prepared in saline. The stabilized interferon compositions were stored for up to one month at room tempelature (20-24C). Results are expressed as % of irutial antiviral activity.
".-':' :.:
~WO 93/~883~ PCr/US()2/08. 9' 2~2~
:
Table 10 Stabilization of stabilized interferon compositions: : -Alpha and omega interferon isolates :
Inter~eron % Init;al Astivity .
storage time ~ I hr 24 hrs I month Alpha-isolates 100 100 100 Omega-isolates 100 100 100 Control 100 30 0 Stabilized ~nterferon compositions comprising alpha isolate or omega isolate, as in Example 10, were stored ~or up to one month at 37C.
Results are expressed as % of initial antivi~l activity.
Table 11 Stabilization of stabilized interferon compositions:
Alpha and omega isolates ~37C) Interferon % Initial Activity s~orage tirne I hr 24 hrs I month Alpha-isolates 100 100 100 :~
Omega-isolates 100 100 100 --Control 100 30 0 ~o 93/0883~ ~'CI/IIS9~/~JX29' 2~ 209~q F,xample 12 Interferon ointments prepared as in Pxample 7 comprising alpha isolate or omega isolate (isolates prepared .IS in Example 10) were stored for up to 6 months at room temperature (20-24C). Results are expressed as %
S of initial antiviral activity.
, Table 12 Stability of interferon ointment:
Alpha and omega isolates (20-24C) Interferon% Initial A~ivity storage time I month 6 months Alpha-isolates100 100 Omega-isolates100 100 Control 0 n.t.
2() _ :
* Signifies "not tested", because prior activity ~as aLready zero.
'. ~ ~
wo 93/1)8~32 1 ~U' ~
2~ ~9~
Example 13 Interferon ointments prepared as in Exarnple 7 comprising alpha isolate or omega isolate (isolates prepared as in P,xample 10) were stored for up to 6 months at 37C. Results are expressed as æ of initial antiviral activity.
s Table 13 Stability of interferon ointment:
Alpha and omega isolates (37C) - Inter~eron % Initial Activity storage time l month 6 months Alpha-isolates 100 100 Omega-isolates l 00 100 Control 0 n.t. ~
_ __ _ _ * Signif1es "not tested", because prior activity was already ~ero.
25 Many other variations and modifications may be made in the invention hereinbefore described, by those having experience in this technology, without departing from the concept of the present invention. Accordingly, it should be clearly understood that the methods and compositions described in the foregoing description are illustrative only and not intended as a limitation on 30 the scope of the invention.
Claims (34)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:-
1. A composition comprising a therapeutically effective amount of an interferon which is an alpha interferon, an omega interferon, or a recombinantly produced interferon, and a stabilizing agent which is a primary alkyl amine.
2. The composition of claim 1 which is a powder.
3. The composition of claim 1 wherein the powder is mixed with a pharmaceutically acceptable carrier.
4. The composition of claim 1 wherein the pharmaceutically acceptable carrier is petrolatum or lanolin.
5. The composition of claim 1 with an interferon activity of between about 104 to about 106 international units per milliliter.
6. The composition of claim 1 wherein the stabilizing agent comprises between about 0.1% to about 10% by weight of the composition.
7. The composition of claim 1 wherein the weight ratio of the stabilizing agent to the interferon is between about 100 to about 1.44.
8. The composition of claim 1 wherein the primary alkyl amine is an octyl amine, a decyl amine, a dodecyl amine, or a tetradecyl amine.
9. A composition comprising a therapeutically effective amount of an interferon which is an alpha interferon, an omega interferon, or a recombinantly produced interferon, and a stabilizing agent which has the formula LinRX, wherein Li is lithium, R is an aliphatic, X is an anion, and n isa number chosen to electrochemically balance lithium with the anion.
10. The composition of claim 9 which is a powder.
11. The composition of claim 9 wherein the powder is mixed with a pharmaceutically acceptable carrier.
12. The composition of claim 9 wherein the pharmaceutically acceptable carrier is petrolatum or lanolin.
13. The composition of claim 9 with an interferon activity of between about 104 to about 106 international units per milliliter.
14. The composition of claim 9 wherein the stabilizing agent comprises between about 0.1% to about 10% by weight of the composition.
15. The composition of claim 9 wherein the weight ratio of the stabilizing agent to the interferon is between about 1.00 to about 1.44.
16. The composition of claim 9 wherein the stabilizing agent is a lithium octyl sulfate, a lithium decyl sulfate, a lithium dodecyl sulfate, or a lithium tetradecyl sulfate.
17. A method for making a composition comprising the steps of:
a. providing an amount of interferon which is an alpha interferon, an omega interferon, or a recombinantly produced interferon, b. mixing the provided interferon with an effective amount of a stabilizing agent which a primary alkyl amine to form the composition; and c. dialyzing the composition.
a. providing an amount of interferon which is an alpha interferon, an omega interferon, or a recombinantly produced interferon, b. mixing the provided interferon with an effective amount of a stabilizing agent which a primary alkyl amine to form the composition; and c. dialyzing the composition.
18. The method of claim 17 further comprising the step of lyophilizing the dialyzed composition to a powder.
19. The method of claim 18 wherein the powder is mixed with a pharmaceutically acceptable carrier.
20. The method of claim 19 wherein the pharmaceutically acceptable carrier is petrolatum or lanolin.
21. The method of claim 17 wherein the composition has an interferon activity of between about 104 to about 106 international units per milliliter.
22. The method of claim 17 wherein the stabilizing agent comprises between about 0.1% to about 10% by weight of the composition.
23. The method of claim 17 wherein the weight ratio of the stabilizing agent to the interferon is between about 1.00 to about 1.44.
24. The method of claim 17 wherein the primary alkyl amine is an octyl amine, a decyl amine, a dodecyl amine, or a tetradecyl amine.
-27- PC?/US 92/08292
-27- PC?/US 92/08292
25. A composition made by the method of claim 17.
26. A method for making a composition comprising the steps of:
a. providing an amount of interferon which is an alpha interferon, an omega interferon, or a recombinantly produced interferon, b. mixing the provided interferon with an effective amount of a stabilizing agent which the formula LinRX, wherein Li is lithium, R is an aliphatic, X is an anion, and n is a number chosen to electrochemically balance lithium with the anion to form the composition, and c. dialyzing the composition.
a. providing an amount of interferon which is an alpha interferon, an omega interferon, or a recombinantly produced interferon, b. mixing the provided interferon with an effective amount of a stabilizing agent which the formula LinRX, wherein Li is lithium, R is an aliphatic, X is an anion, and n is a number chosen to electrochemically balance lithium with the anion to form the composition, and c. dialyzing the composition.
27. The method of claim 26 further comprising the step of lyophilizing the dialyzed composition to a powder.
28. The method of claim 27 wherein the powder is mixed with a pharmaceutically acceptable carrier.
29. The method of claim 28 wherein the pharmaceutically acceptable carrier is petrolatum or lanolin.
30. The method of claim 26 wherein the composition has an interferon activity of between about 104 to about 106 international units per milliliter.
31. The method of claim 26 wherein the stabilizing agent comprises between about 0.1% to about 10% by weight of the composition.
32. The method of claim 26 wherein the weight ratio of the stabilizing agent to the interferon is between about 1.00 to about 1.44.
-28- PC?/US92/08292
-28- PC?/US92/08292
33. The method of claim 26 wherein the stabilizing agent is a lithium octyl sulfate, a lithium decyl sulfate, a lithium dodecyl sulfate, or a lithium tetradecyl sulfate.
34. A composition made by the method of claim 26.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/790,670 US5236707A (en) | 1991-11-08 | 1991-11-08 | Stabilization of human interferon |
| US07/790,670 | 1991-11-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2120944A1 true CA2120944A1 (en) | 1993-05-13 |
Family
ID=25151414
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002120944A Abandoned CA2120944A1 (en) | 1991-11-08 | 1992-09-30 | Stabilization of human interferon |
Country Status (10)
| Country | Link |
|---|---|
| US (2) | US5236707A (en) |
| EP (1) | EP0611305A1 (en) |
| JP (1) | JPH07500834A (en) |
| AU (1) | AU664717B2 (en) |
| CA (1) | CA2120944A1 (en) |
| FI (1) | FI942017A (en) |
| MX (1) | MX9206399A (en) |
| NO (1) | NO941383L (en) |
| TW (1) | TW215902B (en) |
| WO (1) | WO1993008832A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL7404590A (en) * | 1974-04-03 | 1975-10-07 | Stichting Rega V Z W | METHOD OF REACTIVATING INTERFERON. |
| NL7404589A (en) * | 1974-04-03 | 1975-10-07 | Stichting Rega V Z W | PROCEDURE FOR STABILIZING INTERFERON. |
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| US4462985A (en) * | 1980-08-22 | 1984-07-31 | University Of Illinois Foundation | Delivery of biologically active components of heterologous species interferon isolates |
| EP0077063B1 (en) * | 1981-10-13 | 1988-03-16 | Exovir, Inc. | Interferon-containing compositions and the use of these compositions in the treatment of herpetic infections, pre-malignant skin lesions, skin malignancies and psoriasis |
| EP0080879B1 (en) * | 1981-11-28 | 1986-10-01 | Sunstar Kabushiki Kaisha | Pharmaceutical composition containing interferon in stable state |
| DE3262575D1 (en) * | 1981-12-23 | 1985-04-18 | Schering Corp | Stabilised interferon formulations and their preparation |
| US4957734A (en) * | 1982-06-14 | 1990-09-18 | Exovir, Inc. | Treatment of certain skin malignancies and pre-malignant skin lesions, herpes zoster and psoriasis |
| US4462986A (en) * | 1982-11-04 | 1984-07-31 | Ens Bio Logicals, Inc. | Synergistic anti-herpes compositions |
| US4680175A (en) * | 1984-02-07 | 1987-07-14 | Interferon Sciences, Inc. | Interferon administration vehicles |
| US5165921A (en) * | 1989-01-23 | 1992-11-24 | National Geno Sciences, Inc. | Method for treating condyloma acuminatum with interferon |
| US5326892A (en) * | 1992-11-24 | 1994-07-05 | Advanced Technology Materials, Inc. | Bimetallic alkoxide reagents and method of making the same |
-
1991
- 1991-11-08 US US07/790,670 patent/US5236707A/en not_active Expired - Fee Related
-
1992
- 1992-09-30 CA CA002120944A patent/CA2120944A1/en not_active Abandoned
- 1992-09-30 EP EP92921622A patent/EP0611305A1/en not_active Withdrawn
- 1992-09-30 AU AU27888/92A patent/AU664717B2/en not_active Ceased
- 1992-09-30 JP JP5508407A patent/JPH07500834A/en active Pending
- 1992-09-30 WO PCT/US1992/008292 patent/WO1993008832A1/en not_active Application Discontinuation
- 1992-10-28 TW TW081108591A patent/TW215902B/zh active
- 1992-11-06 MX MX9206399A patent/MX9206399A/en not_active IP Right Cessation
-
1993
- 1993-08-13 US US08/105,923 patent/US5431909A/en not_active Expired - Fee Related
-
1994
- 1994-04-15 NO NO941383A patent/NO941383L/en unknown
- 1994-05-02 FI FI942017A patent/FI942017A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| FI942017A0 (en) | 1994-05-02 |
| US5236707A (en) | 1993-08-17 |
| AU664717B2 (en) | 1995-11-30 |
| MX9206399A (en) | 1993-08-01 |
| AU2788892A (en) | 1993-06-07 |
| US5431909A (en) | 1995-07-11 |
| NO941383L (en) | 1994-06-29 |
| WO1993008832A1 (en) | 1993-05-13 |
| TW215902B (en) | 1993-11-11 |
| FI942017A (en) | 1994-05-02 |
| NO941383D0 (en) | 1994-04-15 |
| EP0611305A1 (en) | 1994-08-24 |
| JPH07500834A (en) | 1995-01-26 |
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