IS2070B

Stable liquid interferon formulations

Abstract

Liquid interferon compositions having a pH between 4.0 and 7.2 are described. The compositions comprise interferon-beta and a stabilizing agent at between about 0.3 % and 5 % by weight which is an amino acid selected from the group consisting of acidic amino acids, arginine and glycine. If needed, salt is added to provide sufficient ionic strength. The liquid composition has not been previously lyophilized or previously cavitated. The liquid is preferably contained within a vessel having at least one surface in contact with the liquid that is coated with a material inert to adsorption of interferon-beta. A kit for parenteral administration of a liquid interferon formulation and a method for stabilizing liquid interferon compositions are also described.

Term

Term ended

Expired 21 June 2019, 7.3 years ago.

65 claims: 39 independent, 26 dependent

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2ύ70 2ύ70 Demands Kröfur 1. Vökvalyfjablanda sem gerð er af interferóni og varðveisluefhi sem er amínósýra valin úr flokki þeim er samanstendur af sýrumyndandi amínósýrum, arginíni og glýsíni;þar sem amínósýruvarðveisluefnið er í magni milli u.þ.b. 0.3% og 5% (þ/r);þar sem vökvalyfjablandan hefur ekki verið endurgerð úr frostþurrkuðu interferóni;og hefur vökvalyfjablandan ekki verið frostþurrkuð frekar. An interferon-type liquid compound and a preservative selected from the group consisting of acidic amino acids, arginine and glycine;wherein the amino acid detergent is in an amount between about 0.3% and 5% (th / r);since the liquid composition has not been remodeled from lyophilised interferon;and the liquid preparation has not been lyophilized further. 2. The liquid formulation of claim 1 and the amino acid antagonist is valid from arginine and glycine, and there is no plasma albumin in the pharmaceutical formulation. 2. Vökvalyfjablanda sú, sem lýst er í 1. kröfu, og amínósýruvarðveisluefhið er valid úr arginíni og glýsíni, og ekki er blóðvökvaalbúmín í lyfjablöndunni.
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The liquid formulation described in claim 1, and the interferon is interferon-beta or interferon generated by joining. 4. Vökvalyfjablanda sú, sem lýst er ί 1. krdfu, og interferónið er interferón-beta eða interferón myndað með samskeytingu. The liquid composition described in claim 1, with a pH between 4.0 and 7.2. Vökvalyfjablanda sú, sem lýst er ί 1. krdfu og er pH hennar milli 4.0 og 7.2. б. The liquid formulation described in Section 5 and its pH from 4.8 and 5.2. б. Vökvalyfjablanda sú, sem lýst er í 5. krdfu og er pH hennar miUi 4.8 og 5.2.
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7. The aqueous solution as described in claim 6 and having a pH of 5.0. 7. Vökvaly^ablanda sú, sem lýst er í 6. kröfu og er pH hennar 5.0.
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10. The liquid formulation described in claim 1, the concentration of the interferon between 6 MIU and 50MIU. 10. Vökvalyfjablanda sú, sem lýst er í 1. krdfu og er styrkur interferonsins milli 6 MIUog50MIU. 2Ú70 2Ú70
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11. Vökvalyfjablanda sú, sem lýst er í 1. kröfu og er styrkur interferönsms milli 6 MIU og 50 MIU í hverjum skammti. The fluid composition as described in claim 1, is a concentration of interferon between 6 MIU and 50 MIU per dose.
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12. The liquid formulation described in claim 3, and at least one surface of the container is comprised of a material selected from the group consisting of silicone and poly tetrafluoroethylene. 12. Vökvalyfjablanda sú, sem lýst er í 3. kröíu, og er a.m.k. einn flötur ílátsins þakinn efhi völdu úr flokki efha sem samanstendur af silikoni og fjöltetraflúoretýleni.
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13. The liquid formulation described in claim 12, and time is injected. 13. Vökvalyfjablanda sú, sem lýst er ί 12. krdfu, og ílátid er sprauta.
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14. The liquid formulation described in claim 1, additionally comprising a buffer of pH 5.0, wherein the interferon is interferon-beta and the vasodilator is 170 mM L-glutamic acid. 14. Vökvalyfjablanda sú, sem lýst er ί 1. krdfu, og er ad auki ί henni dúi med pH 5.0 þar sem interferónið er interferón-beta og vaiðveisluefoið er 170 mM L-glútamsýra.
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16. The drug preparation described in Clause 14, and at least one additional component selected from the group consisting of 15 mg / ml human plasma albumin and 0.1% Pl / Fonic 68. 16. Vdkvalyfjablanda sú, sem lýst er ί 14. krdfu, og er a.m.k að auki ί henni einn eftiaþáttur valinn úr flokki þeim er samanstendur af 15 mg/ml blóðvökvaalbúmíni úr mönnum og 0.1% (þ/r) Plutonic F-68.
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18. The pharmaceutical composition described in claim 17, comprising a liquid composition in a container of at least one liquid, which comprises a substance valid from the class consisting of silicone and poly tetrafluoroethylene. 18. Vdkvalyfjablanda sú, sem lýst er ί 17. krdfu, og er téð vdkvalyfjablanda í íláti með a.m.k. einn fldt, sem þakinn er efni sem valid er úr þeim flokki efha sem samanstendur af silikoni og fjöltetraflúoretýleni. Vdkvalyfjablanda described in Article 18, and the time is injected. Vdkvalyfjablanda sú sem lýst er ί 18. krdfu, og ílátid er sprauta. 2u70 2u70
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20. The liquid formulation described in claim 1, in addition to which is a dope that maintains pH within the range of 4.0 to 6.0, and the liquid preparation is suitable for extraditorial administration into mammals;and where the interferon is interferon-beta. 20. Vökvaly^ablanda sú, sem lýst er ί 1. kröfu og að auki er ί henni dúi sem heldur pHi innan markanna 4.0 til 6.0, og er vökvalyfjablandan heppileg til inngjafar utan meltingarvegar í spendýrum;og þar sem interferónið er interferón-beta.
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22. The liquid formulation described in claim 21, and no contact areas for liquids containing oxygen are present. 22. Vökvalyfjablanda sú, sem lýst er ί 21. kröfu, og engir snertifletir við vökva, sem innihalda súrefei, eru fyrir hendi.
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23. The liquid formulation described in claim 21, and the mterferon-beta has not been cavity stored or stored during storage. 23. Vökvalyfjablanda sú, sem lýst er ί 21. kröfu, og mterferónið-beta hefur ekki orðið fyrir holrýmingu fyrir geymslu í ílátinu né meðan á henni stóð.
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27. The hydrolysis mixture described in claim 20, which is the hydrolysis mixture sterile. 27. Vðkvalyijablanda sú, sem lýst er í 20. krðfu og er vökvalyfjablandan dauðhreinsuð.
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30. The fluid composition described in claim 29, which is the activity of human interferon beta-beta in the range of 6 to 50 MIU. 30. Vökvalyfjablanda sú, sem lýst er ί 29. krðfu og er virkni samskeytta interferónsins-beta úr mönnum á bilinu 6 til 50 MIU.
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31. The liquid formulation described in claim 1, wherein the amino acid antagonist is present at concentrations between. 0.3% and 3.13% (th / r) and the liquid mixture has been frozen 31. Vökvalyfjablanda sú sem lýst er ί 1. krdfu gerð og er amínósýruvarðveisluefnið til staðar í styrk á milli. 0.3% og 3.13% (þ/r) og hefur vökvalyfjablandan verið fryst
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32. A liquid-liquid mixture made of;(a) interferon, (b) acetate dope and (c) arginine;and the pH of the pharmaceutical composition is between 4.0 and 6.0;and is not plasma albumin in the formulation. 32. Vökvalyljablanda sem gerð er af;(a) interferóni, (b) asetatdúa og (c) arginíni;og er pH lyfjablöndunnar milli 4.0 og 6.0;og er ekki blóðvökvaalbúmín í lyfjablöndunni.
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34. The fluid composition described in 32 or 33, and is the interferon interferon-beta. 34. Vökvalyfjablanda sú sem lýst er í 32. eða 33. kröftim og er interferónið interferón-beta.
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36. The fluid composition as described in 34rd and the concentration of the interferon between 6 MIU and 50 MIU per dose. 36. Vökvalyfjablanda sú, sem lýst er í 34. krdfu og er styrkur interferónsins milli 6 MIU og 50 MIU í hverjum skammti.
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37. The liquid composition described in each of 32.-36. requirements and the concentration of acetate duan is 20 mM. 37. Vökvalyfjablanda sú, sem lýst er í hverri sem er af 32.-36. kröfum og er styrkur asetatdúans 20 mM.
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38. The liquid formulation described in each of the foregoing. 32.-37. krdfum and the concentration of arginine or arginine-HCl is between 0.3% and 5% th / r. 38. Vökvalyfjablanda sú, sem lýst er ί hverri sem er af. 32.-37. krdfum og er styrkur arginínsins eða argininsins-HCl milli 0.3% og 5% þ/r.
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39. The liquid formulation described in each of the foregoing. 32.-38. requirements and in addition to its overboard issue. 39. Vökvalyfjablanda sú, sem lýst er ί hverri sem er af. 32.-38. kröfum og er að auki ί henni yfirbordsvirkt efili.
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41. The liquid formulation described in each of the foregoing. 32.-40. requirements and oxygen concentration of said liquid composition is less than 30% of its equilibrium concentration. 41. Vökvalyfjablanda sú, sem lýst er í hverri sem er af. 32.-40. kröfum og súrefnisstyrkur téðrar vökvalyfjablöndu er minni en 30% af jafhvægisstyrk þess ί andrúmslofti.
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43. Aðferð til að gera interferón stöðugt í vökvalyfjablöndu og felst hún í að blanda saman:a) interferóni;b) dúa;og c) amínósýruvarðveisluefni völdu úr flokki þeim er samanstendur af sýrumyndandi amínósýrum, arginíni og glýsíni;og er styrkur amfnósýruvarðveisluefhisins milli 0.3% og 5% þ/r;þar sem téð vökvalyfjablanda hefur ekki verið endurblönduð úr frostþurrkuðu interferóni, og téð vökvablanda verður ekki frekar fiostþurrkuð. A method of stabilizing interferon continuously in a liquid formulation and comprising mixing: a) interferon;b) dúa;and c) amino acid detergents selected from the group consisting of acidic amino acids, arginine and glycine;and the concentration of the amino acid protective agent is between 0.3% and 5% thereto;since said liquid composition has not been reconstituted from lyophilised interferon, and said fluid mixture is not further freeze dried.
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46. Aðferð eins og lýst er ί 43. krðfu og er téð vökvalyfjablanda í íláti og hefur interferónið ekki orðið fyrir holrýmingu fyrir geymslu í ílátinu né meðan á geymslu stóð. 46. A process as described in claim 43, comprising a liquid detergent composition in a container, and the interferon does not become a cavity for storage in the container nor during storage.
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50. Aðferð sú sem lýst er ί 48. krdfu, og pH téðs dúa er er 5.0. 50. The method described in claim 48, wherein the pH of said buffer is 5.0.
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52. Aðferð sú sem lýst er í 43. krdfu og er styrkur mterferónsins milli 6 MIU og 50 MIU. 52. The method described in 43rd and the concentration of mterferon is between 6 MIU and 50 MIU.
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53. A method as described in claim 45, and at least one surface of the container is comprised of material valid from the class consisting of silicone and poly tetrafluoroethylene. 53. Aðferð eins og lýst er í 45. krdfu og er a.m.k. einn flötur ílátsins þakinn efni sem valid er úr þeim flokki eftia sem samanstendur af silikoni og fjöltetraflúoretýleni.
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54. A method as claimed in 53, and the container is an injection. 54. Aðferð eins og lýst er í 53. kröfu, og ílátið er sprauta.
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57. Adferd sú sem lýst er ί 56. krdfu, og ílátid er sprauta. 57. The behavior described herein is 56, and the time is injected.
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58. Adferd til ad vardveita interferon í vökvalyfjablöndu og felst hún ί ad blanda saman:a) interferóni;b) dúa;og c) arginini;þar sem pH lyfjablöndunnar er milli 4.0 og 6.0;og téd vökvalyijablanda hefur ekki verid endurblöndud úr frostþurrkudu interferóni, og téd vökvalyfjablanda verdur ekki frekar frostþurrkuð. 58. Adherence to interferon interferon in a liquid drug mix, which consists of mixing: a) interferon;b) dúa;and c) arginini;wherein the pH of the pharmaceutical composition is between 4.0 and 6.0;and such liquorice mixtures have not been reconstituted from freeze-dried interferons, and the liquid preparations are not further lyophilized.
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59. Adferd sú sem lýst er ί 58. krdfu og er argininid arginin-HCl.. 59. The behavior of which is described as 58 and is arginine arginine-HCl.
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60. Adferd sú sem lýst er ί hvorri sem er af 58-59. krdfum og er interferónid interferón-beta. 60. The behavior described in each of 58-59. krdfum and is interferonid interferon-beta.
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63. Aðferð sú, sem lýst er í hverri sem er af 58.-62. kröfum og er styrkur asetatdiians 20 mM. 63. The method described in each of 58.-62. requirements and the concentration of acetate is 20 mM.
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64. Aðferð sú, sem lýst er ί hverri sem er af. 58-63. kröftun og er styrkur arginmsins eða argininsins-HCl milli 0.3% og 5% þ/r. 64. The method described in each of the foregoing. 58-63. the concentration of arginamine or arginine-HCl is between 0.3% and 5% th / r.
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65. Aðferð sú, sem lýst er ϊ hverri sem er af. 58-64. kröfum og felst að auki i að blanda yfirborðsvirku eftii út i. 65. The method described in each of the foregoing. 58-64. requirements, and in addition to mixing surface active substances into
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67. Aðferð sú, sem lýst er í hverri sem er af. 58-66. kröftun;og felst téð aðferð ekki ί að blanda blóðvökvaalbúmíni út i. 67. The method described in each of the foregoing. 58-66. forces;and the aforementioned method does not involve mixing plasma albumin out into.
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68. Aðferð sú, sem lýst er í hverri sem er af. 58-67. kröfum og er téð vökvalyfjablanda í íláti og hefur interferónið ekki orðið fyrir holrýmingu fyrir geymslu ί ílátinu né meðan á geymslu stóð. 68. The method described in each of the foregoing. 58-67. requirements and is contained in a liquid detergent composition, and the interferon has not become a cavity for storage of the container or during storage.
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69. Aðferð sú, sem lýst er í hverri sem er af. 58-67. kröfum, téð vökvaly^ablanda er í íláti, og eru engir snertifletir við vökva sem innihalda súrefni fyrir hendi milli vökvalyfjablöndunnar og ílátsins. 69. The method described in each of the foregoing. 58-67. requirements, said liquid reservoir is in a container, and there are no contact areas for liquids containing oxygen between the liquid preparation and the container.

Independent claims39

215 paragraphs in 6 sections, as filed

Description

SUMMARY OF THE INVENTION (0001) The invention provides these methods for continuous human interferon beta and stable interferon-beta preparations.

BACKGROUND OF THE INVENTION (0002) Interferons are proteins with various types of biological activity, some of which are against viruses, some of which are immunosuppressive and, in part, against the multiplication of viruses. Your relatively small monopeptide polypeptide, specific to species, and formed by mammalian cells in response to exposure for all kinds of progenitors such as viruses, polypeptides, mitogenes and other forms. Interferon protects animal tissues and cells against viral attacks and play a major role in the host cell host. Usually, interferons attach better tissue to the tissues and cells that form them from other types of tissues and cells, suggesting that interferon derived from human tissues should be more effective in treating diseases of 1 human than interferon from other species.

For your all sorts of different types of human interferons, they are usually classified into whitening interferon (interferon-alpha), fibromyalgia interferon (interferon beta) and immune interferon (interferon gamma) and a large number of variants. The overall coverage of interferon can be found in various books and scripts, including The Interferon System (WE Stewart Π, Springer-Verlag, NY 1979) and Interferon Therapy (World Health Organization Technical Reports Series 676, World Health Organization, Geneva 1982).

Method of administration of interferon is an important factor in the utilization of this important therapeutic therapeutic agent. Cervical, intravenous, intramuscular or intravenous interferon has most often been used to treat tinnitus, such as leukaemic leukemia, immune deficiency (AIDS) and Karposis sarcoma, which is bound to it. However, it is known that the protein in your purified form is particularly susceptible to nephropathy. The reaction rate (those reactants) that is (most) involved in interferon-degradation of interferon-beta is (u) clustering and parenting. The lack of stability of interferons in solutions and other products has a high level of usefulness.

Intravenous drug interferon formulations are commonly used for the interferon in the form of a solvent (i.e. lyophilised) medium mixed with complex organic adjuvants and preservatives such as non-ionic surfactant, various sugars, organic polyalcohol and / or albumin from human blood. The deficiency of lyophilised drugs is thirty in complex methods of propagation, as packaged sterile water until the year of administration is required. Furthermore, it is necessary to moisten in various ways with lyophilisate prior to use, which may cause the needle to clog and to dissolve a mixture during preparation. Sore tonsils cause particular problems in people with weak muscles and poor muscle coordination such as people with multiple sclerosis (MS). Potential for MS patients to self-administer interferon so that drug doses are easier to administer than the currently available lyophilisates have high levels of value for those patients with whom these medicines are intended. Simple, interferon-based chemotherapy is extremely convenient compared to freeze-dried medicines that need to be reconstituted during use. In non-dermatologically active forms, you can also have complex carriers such as human plasma, polyalcohol, sugars and surfactant anionic viral agents. See, for example, WO 89/10756 (Hara et al., Such as quaternary alcohol and p-hydroxybenzoate era).

WO 88/09674 discloses pharmaceutical derivatives of gamma interferon which have been stable and contain lactic acid (4-OBD-galactopyranosyl-D-gluconic acid) and acetate / glycine dye. The lactic acid prevents the formation of clusters at a higher level with the recovery of freeze-dried elements. EPO163111 demonstrated that amino acids such as arginine or glutamate may increase the solubility of interferon drug compounds in water. EPR284249 describes frost-depressed interferon drugs as in your glycine or albumin from human blood (HSA), duodenum (eg acetate), and equilibrium & i (eg NaCl). US 4496537 discloses lyophilized interferon drugs such as phosphite duct, glycine and HSA. The use of glycine with HSA is considered to improve the recovery of the lyophilisate.

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BACKGROUND OF THE INVENTION With this invention, the above-mentioned problems have been solved by the discovery that may be done by interferon beta from the diet by providing it in a buffer solution with a pH between about 4 and 7.2, and is (the solution of amino acid as a living fluid and, in some cases, salt (if there is no charged side chain of the amino acid). Interferon beta is not lyophilised but is used directly in the pharmaceutical industry of this invention once it has been smithed with behaviors that each skill The industry knows.

Therefore, one aspect of the invention is a fluid composition comprising an interferon and a living medium between about 0.3% and 5% by weight and is related to amino acids selected from the group consisting of acid-forming amino acids, arginine and glycine. This vdkvalyi mix has not been freeze dried. Furthermore, it is desirable that a liquid composition be disposed of as a syringe, and on the inside surface of the skin which is in contact with the skin may be coated with substances such as silicone and polyfluoroethylene which is resistant to interferon. In the medically sensitive drug, interferon-beta and recycled interferon in doves with a pH between about 4.0 and about 7.2. The medal of other pharmaceutical compositions of the invention are:

(1) 20 mM acetate buffer at pH 5.0 which has not previously been lyophilized and epithelial factors of interferon-beta-plus selected from (a) 150 mM argmin-HCl; (b) 100 mM sodium chloride and 70 mM glycine; c 150 mM arginine-HCl and 15 mg / ml albumin human blood; (d) 150 mM arginine HCl and 0.1 Pluronic F-68; (e) 140 mM sodium chloride and 15 mg / ml albumin albumin; and (g) 140 mM sodium chloride and Pluronic F-68.

(2) vdkvi at pH 5.0 which is interferon beta, 170 mM L-glutamic acid and 150 mM sodium hydroxide, and has previously been previously freeze dried;

(3) 20 mM phosphetudium at pH 7.2 which has not previously been lyophilized, and are comprised of interferon-beta-plus components selected from: (a) 140 mM arginine-HCl; and (b) 100 mM sodium disodium and 70 mM glycine.

[0010] Another preferred embodiment of the invention is a medical kit for the administration of the intravenous interferon lymph node. In the hospital there are containers in a liquid mixture with a pH between 4 and 6, and the amount of interferon-beta, which has no evidence of frost-proofing, is sufficient in the fluid for treatment and approximately 5% or less based on the weight of a detergent which is amino acid; and instructions for use.

Yet another aspect of the invention is a liquid preparation suitable for extravagant administration in mammals, and it is quite a sufficient amount of interferon beta that has not previously been lyophilized, to provide an effective effect, 1 buffer holding pH within maize 4 and 6 volumes of suitable ionic strength. The pharmaceutical mixture is stored in a storage container such as a syringe. Preferably, there is no interface between the liquid storage tank and oxygen (ie, the interferon solution never comes into contact with a gas that is oxygen during the mixing and storage). Interferon-beta then maintains all its activity against viruses, and is stored at a temperature between about 2 degrees C and approx. 25 degrees C for a.mk. 3 months.

One of the processes of the invention for power make Inteifeión-beta stable in liquid formulations so that it has some degree of inherent stability when stored between about one and a half. 2 ° C and approx. 25 ° C for 1 at least 3 months, consist of a mixture of: a) a sufficient amount of interferon-beta to produce a powerful effect; b) a dope that maintains a pH in the range of 4.0 to 7.2 inclusive of a suitable ionic species; and c) a reflux fluid which is an amino acid, and has not previously grown to be lyophilized or exposed to a gas in which oxygen is interfered with and stored.

The liquid formulations of the invention had many advantages over lyophilised pharmaceuticals. The median cost is: (i) less volume in each injection dose for a drug preparation which has less discomfort for the patient than the dose volume; (ii) single amino acids replacing complex adjuvants make monitoring of finalized precursors more targeted; (iii) Patching is much simpler as no packet of water for injection (E: WFI, i: VF1) need to have and have a glass and syringe; (iv) higher dosing accuracy can be achieved with fewer fluid transfers; and (v) the safety of the product is improved as a simpler method of administration reduces the likelihood of defects in needles and that the components of the composition fail in the preparation of administration.

It is one of the objects of the present invention to provide a biologically active, stable liquid interferon-beta formulation for use with syringe delivery.

Another object of this invention is to provide a pharmaceutical formulation which does not require the pre-freeze-drying of the interferon-beta mixture.

It is an object of the present invention to prevent the loss of stability of the interferon-beta pharmaceutical composition in a liquid form by: (a) preventing cavity formation and / or formation of lofiboliuria during the preparation of a liquid formulation, or b) keep the liquid preparation so that the air bubble compartment is inert gas such as argon or

---- - - A- *

ΚΟΠΙΙΙΠβΓβΙΠΙ.

It is another object of this invention to provide a liquid formulation which can be stored for a long time in liquid form, thus facilitating storage and transportation prior to ingress. Another object of this invention is to provide a suitable drug preparation which is easy to create and administer, as the freeze-drying and reconstitution steps are derived from the story.

Furthermore, it is one object of the invention to use simple amino acids as a preservative in addition to albumins from human plasma commonly used, thus monitoring the quality of products is much easier than was.

In addition, it is one object of this invention to provide a pharmaceutical formulation having an anti-freeze-free interferon beta, which can be processed less costly than lyophilized.

Other features of the invention will be described in the following description, which are partly apparent with reference to these disclosures or may be discussed with the practice according to the invention. Drawings of pear as a guide are included in this specification and are actually part of it, and show and serve together with the description for the purpose of explaining the fundamental features of the invention.

STATEMENT OF THE INVENTION [0021]

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Figure 1 is a line showing the percentage of interferon-beta monomer remaining in the total molecular weight used for the process, which amounts to the percentage of the oxygen dissolved in the liquid.

Figure 2 is a line showing the percentage of protein, relative to the starting point, versus time for the preparation of BG9589-1 in a liquid form. Samples marked "4 ° C" (filled females) are grown at a temperature range of 2-8 ° C. other samples are grown at 25 ° C (filled rings); 33 ° C (filled with triangles) and 40 ° C (filled with mirrors).

Figure 3 is a line showing the percentage of protein, relative to the starting material starting material, versus time for drug-based BG9589-3. The samples labeled "4 ° C" (filled females) are fast at a temperature range of 2-8 ° C. Oven samples are incubated at 25 ° C (filled rings); 33 ° C (full throttle) and 40 ° C (filled with mirrors).

DETAILED DESCRIPTION OF THE INVENTION The invention described herein addresses the problems and disadvantages inherent in organizational structure and the ability to maintain interferon sustainability and conveys single behavior, which at the same time improves its stability during storage. In part, the invention is based on these findings:

(a) interferon-beta is particularly unstable and forms clusters when it comes into contact with oxygen which is blown through a liquid either or that the liquid is in contact with it in a bubble chamber;

b) interferon-beta drugs on non-carrier fluid such as plasma albumin are particularly susceptible to adsorption (ie, any adverse reaction to the edema) at the top of the glass; and

c) interferon-beta forms colonies at low ionic strength, requiring ionic environments to stay supported in water.

Accordingly, the invention is directed to methods of stabilizing interferon beta human but at the same time as described in the above described gryijur, and to those stable fluidized interferon-beta-pharmaceuticals thus obtained.

A. DEFINITIONS The term "Dutts" refers to the solutions of the acidic acid and salts of which anion is shown, or the solutions of the dead base and its salts. In particular, the term "acetate" in this specification (see also Table I below) has a dummy system which is most preferred to be sodium acetate and acetic acid and the term "fbsfet" has a dummy system which is preferable to being dibasic and monobasic sodium phosphate hepta and monohydrate, as appropriate. Furthermore, the definition of the solutions in Table Π (below) of acidic amino acids with sodium hydroxide, although in the traditional sense of the article, they are not considered to be religions.

The term "adjuvant" has any compound that is added in a pharmaceutical formulation while in process and / or storage, and serves as a function of changing overall properties, enhancing and / or adjusting osmotic concentrations.

The term "preservatives" has an auxiliary means that enhances or improves stubbornness.

The term "stability" a * necessity defined as a function and means the temporal immune function of interferon such as activity against viruses and / or interferon building. [0028] The term "cavity" contains any liquid formulation of interferon drug that is exposed to acne, oxygen in (e.g., breathing air) at least while on its forehead, storage at a time, due to changes in the appearance of self-esteem. The term "cavity" means and at the interface between gas as oxygen and liquid has formed at some stage while on the verge of storing a liquid liquid formulation. The term "cavity" also means that dissolved dissolved oxygen in the liquid interferon drug mixtures exceeds approximately 10% of the equilibrium strength of the air at the temperatures prevailing at least during storage and storage.

The term "non-gastrointestinal", as used herein, includes techniques for administration of subcutaneous, intravenous, intramuscular, intravenous, intravenous, or intravenous infusion.

The term "pharmacologically acceptable salt" is a useful ingredient of any liver or non-essential salt and is relatively undisturbed and harmful to patient patients compatible with therapeutic viruses, thus resulting from the side effects of the salt, do not overestimate the interfering effect.

"Estimated Amount to Behavior Impact" is the amount that results in the outcome, which affects your average life expectancy. "Abundant amount of drug behavior" means the amount that produces a positive result (ie, induces viral effects) in the CPE test against antiviral activity.

"Pharmacologically effective amount" of interferon herein means the percentile fraction of a chemical that is known in the art and pharmaceuticals as being desired and effective in the treatment of certain disorders.

The "pressurized blood pressure" (used alternately in "evenly pressed") is a liquid formulation of mterferon formulation which is sufficiently strong for its various components until its osmotic adhesion is somewhat the same as bdd, þ ie. Cellular contact with the drug mixture will, to a significant extent, maintain its Idgun and not a threat to netted water intake, otherwise due to osmotic pressure.

"Fungicides" (used in conjunction with "polyphosphate species") have a high molecular weight molecular weight, which maximizes the concentration at a particular osmotic concentration. This understanding is the basis of our discovery of interferon-beta is stable at high ionic strength, but the total ionic strength is limited because the solution is supposed to be equilibrated midad at bldd. (see Dsmi 7). Preferred leads to maximum strength by using a multitude of specialists as a helmet.

An article "interfering" refers to a substance that has at least the property of non-interferon activity, not necessarily as essential.

BACKGROUND OF THE INVENTION Generally, this invention may be used for all types of interferons, including natural interferons, interferons processed by DNA junction technology, and interferon semi-purified and purified interferon from fibrous cells, whitewashes, lymphocytes, or any fatty tissue or other suitable species that are interferon or produce interferon. Preferably, however, the invention is used for human fibroblast interferon (interferon beta).

Preferably, the total interferon is a crosslinked form, and DNA hybridization methods for producing proteins contained in the various interferons are known and are not intended to limit the invention in any way. See, for example, U.S. Patents 4,399,216, 5,149,636, 5,179,017 (Axel et al.); 4,470,461 (Kaufman). The combined form of interferon-beta has been formed. See for example. European Patent 0 41313 (Fiber Expression Interferon Beta); U.S. Patent 4,966,843 (McMormick et al., Interferon-expression CHO-cells); U.S. Patent 5,326,859 (Sugano et al., -DNA encoding interferon-beta). Interferon beta may also be altered, either by joint or chemical, and may be formed as blood in the bloodstream. Among the interferon-beta forms may be varied on a table by systine mutants (U.S. Patents 4,588,585 and 4,737, 462; Mark et al.) And methionine-free dentifrices (EP 260 350-Wang et al.). The first-stage amino acid acidification of the protein can be stamped with derivation and used for the other molecules of syrup molecules (glycosylation) of other complementary molecules. Other changes can take place in the process of the most advanced protein formation in the host cell. Individual amino acid residues in the chain can change even further by oxidation, reduction of other forms of derivation, and the protein may break into active fractures. The precise chemical structure of single interferon beta will be subject to many factors and is not strictly limited to the scope of the invention, Oil such interferon-beta proteins as in the pharmaceutical compositions described herein will maintain their viability when present at a suitable environmental site. The first-stage amino acid acidification of the protein can be stamped with derivation and used for the other molecules of syrup molecules (glycosylation) of other complementary molecules. Other changes can take place in the process of the most advanced protein formation in the host cell. Individual amino acid residues in the chain can change even further by oxidation, reduction of other forms of derivation, and the protein may break into active fractures. The precise chemical structure of single interferon beta will be subject to many factors and is not strictly limited to the scope of the invention, Oil such interferon-beta proteins as in the pharmaceutical compositions described herein will maintain their viability when present at a suitable environmental site. The first-stage amino acid acidification of the protein can be stamped with derivation and used for the other molecules of syrup molecules (glycosylation) of other complementary molecules. Other changes can take place in the process of the most advanced protein formation in the host cell. Individual amino acid residues in the chain can change even further by oxidation, reduction of other forms of derivation, and the protein may break into active fractures. The precise chemical structure of single interferon beta will be subject to many factors and is not strictly limited to the scope of the invention, Oil such interferon-beta proteins as in the pharmaceutical compositions described herein will maintain their viability when present at a suitable environmental site. Other changes can take place in the process of the most advanced protein formation in the host cell. Individual amino acid residues in the chain can change even further by oxidation, reduction of other forms of derivation, and the protein may break into active fractures. The precise chemical structure of single interferon beta will be subject to many factors and is not strictly limited to the scope of the invention, Oil such interferon-beta proteins as in the pharmaceutical compositions described herein will maintain their viability when present at a suitable environmental site. Other changes can take place in the process of the most advanced protein formation in the host cell. Individual amino acid residues in the chain can change even further by oxidation, reduction of other forms of derivation, and the protein may break into active fractures. The precise chemical structure of single interferon beta will be subject to many factors and is not strictly limited to the scope of the invention, Oil such interferon-beta proteins as in the pharmaceutical compositions described herein will maintain their viability when present at a suitable environmental site.

One behavior of interacting interferdn-beta is that of the chromosomal chromosomal cells of which human interference has been widely used.

2U70

Combined interferon-beta is secreted from the clusters of clonal hamstring, which has been found in a cystic fibrosis. Cells may be grown in a COj breeding container (5% CO2) at approximately 35 degrees Celcius (hereinafter "C"). Fillable vials can be assembled and immune in a fermentation container in a growing condition if desired to scale up the operation. Vessels in a certain gravel fleet are allowed to take place in approx. six dap, and at that time active interferon-beta myndefhid accumulates in the bacteria. Then you can crop the crop and remove the cells from the food you have collected, for example. by contact flow filtration.

CLEANING INTERFERENCES Interferon cleaning systems have been well described and are approachable to the industry. Medal behavior is a single or multiple process that involves a variety of chromatography. See for example. U.S. Patent 5,015,730 (Friesen et al. -Traction chromatography and HPLC); 4,541,952 (Hosoi et al., Silica gel chromatography).

Good behavioral behavior involves the utilization of the unusual hydrophobic and beneficial alkaline edible interferon-beta molecule, in addition to its immense abrasion in binding metal ions. See, for example, Knight and Fahey, "Human Fibroblast Interferon, an Improved Purification," J. Biol, Chem. 256: 3609-3611 (1981) and Edy et al., "Purification of Human

Fibroblast Interferon by Zinc Chelate Chromatography ", J, Biol, Chem., 232: 5934-5935 (1981), from which the foregoing is filed.

In summary, the absorption and purification procedures include binding of interferon beta to a series of Sepharose® columns (manufactured by Pharmacia Biotech) and rinsing of the columns with polyhydric and polyalcohol. When the Sepharose wash solution is thawed and adjusted to its low pH, its interferon beta pathway will bind SP Sepharose® (Pharmacia Biotech). Most of the pau prdtin remaining on the column are basics in edelium, but hidden in the interferon-beta, and bind the column bound to the interferon. DNA and viruses are distinguished from interferon beta on this column. The column is sideways with a series of fabrics that you are sodium chloride.

The interferon protein now binds to a clustered Sepharose® (Pharmacia Biotech) column previously used in zinc. See Edy et al. above. Unnid σ with a pillar pillar

2d70 oxygen-free air to protect the free sulfonyl group in the molecule, and remains hidden in the cube in all of the following steps. Purified interferonid is acidified and maintained at low pH to disable all the viruses still visible in the skin. After neutralization, the interferon is potentiated using a direct-flow flow filtration and switching the dough into a neutral buffer solution. The exchange rate reduces the concentration of zinc and chemical compounds. After this, the total supply of interferon at -70 ° C can be stored before the stages of preparation of the drug.

D AT A GLOBAL INTERFERENCE IN ORGANIZATION IN THE STRENGTHENING PROCEDURE AS DERIVED AS FOLLOWS AFTER THE FIRST DUBBLE CHARACTERISTICS, A CITY OF ANOTHER DUBBLING PROCESS IS DIFFERENTLY DIFFERENT, whereas in the city of neutral duplex comes a buffer solution with a pH between 4 and 7.2 and ί is a living room liner, which is described in more detail below. The resulting medicinal product and interferon is called "fermenter" and may be frozen for storage. See and Dsmi 7. If it is stored frozen (in a non-gaseous air, for example, argon eda kdfhunarefhi), the side can be thawed through 0.22 micron in a packed container, poured into stainless steel, where the process medium is a unified blend have turned and cleaned until the desired amount of desired product is reached. The diluent is made of the same fabric as was used in a different fabrication process. The vapor detergent is filtered and cleared with cyclic processes, for example, a row of 0.22 microns can be used, and release a stainless steel stopper, which includes non-activated gas, combined vent valve and sia, and inlet / outlet diaphragm . The lid is passed through the diaphragm and into the final container. Lokamyndefhid is transported with pressure, which is formed with gas on a table at the bottom of the door, which can be used to fill daudhremsad syringes with antibacterial additives.

There are many behaviors upon the addition of cleared syringes to the cytotoxic agent and the behaviors that are used in this respect are not strictly limited to the scope of the invention as described herein. As a behavioral behavior, behavior involving the use of HYPAK® GFP (Becton Dickinson Pharmaceutical Systems, Franklin Lakes, NJ). The syringes are steam-cooled with condoms

2υ70 over the odds. Generally, there is such a vacuum compartment that injects those that are filled with an interferon drug mixture. The cell is contained in a germicidal environment. Each syringe is so close that it lies vertically and closes open end of the bulb nose, which is so damaged as it fits the open end of the syringe. The pin is male in such a way that he puts tap on the resemblance to hold the fluid inside. a little bubbles are left in the syringe after the plug has been inserted. The lid is air-tight and backflushed with an inert, oxygen-free gas (eg argon, kiwifruit) several times and when the closure is reached, the pins are mechanically compressed into the open syringe and are prone to leaking each syringe. Then the air is vented into the cell and the pressure within its bed reaches atmospheric pressure. The amount of air gap determines the size of the airbag of the inactive gas.

In a system of use, the syringes rotate vertically and held in a city with a chain on a rotary disk. At first, the syringes are positioned below the point where the syringe is inserted. The needle should be rinsed inside the syringe with the same gas (eg.

köfhunarefhi, argon) The needle is pulled sideways from the syringe. Then the syringe is placed under a needle that is inserted into the syringe. The needle is attached to the syringe for injection into the syringe. The other needle is the side drawn from the syringe. Then the syringe is a city under the third needle that is inserted into the syringe. Bullu (vaporized) is blown into the syringe with an inert, non-oxygen-free gas (eg, cardiovascular, argon), and then draws the needle out of the syringe. Bullan is thus a city of luxury airspace, with inactive gas present between the hydraulic table and the bottom of the bubble.

1. Excipient The excipients are referred to as micronucleus species that maximize ionic strength at determined osmotic levels, such as polyurethane adhesives that may be heparin or polymeric species. As discussed in connection with Dsmi 4, interferon beta is stable at high ionic strength, but healing factor is limited by the need for the solution to be isotonic by means of blood. Preferred lead to maximize ionic strength at certain osmotic concentrations is therefore to use multiple species. Interferon-beta solutions of this invention are hypotensive mediated blood (approximately milliosmol / kilogram).

2u70 Preferred of all preservatives of this invention is amino acid which may be any of the following antibodies: any acidic amino acid (e.g., glutamic acid, aspartic acid) or amino acid selected from arginine and glycine. Preferably, the amino acid residue is arginine in acidic form (arginine-HCl) in solutions at pH 5.0. Preferred acid-forming amino acid is L-glutamic acid. Although no particular theory is used, it is the fact that the multifunctional helper is the oldest, and if so, arginine and lysm (with 3 charged groups) work better to make interferon stable than glycine (with 2 loaded groups), however, it works better to make interferon stable than any of the unspecified specia tested.

If the auxiliary agent arginine-HCl, its concentration ranges from 0.5% (w / v) to 5% but rises 3.13% (equivalent to 150 mM arginine-HCl). If the excipient is glycine, its concentration ranges from 0.50% (w / v) to 2.0% but rises to 0.52% (equivalent to 66.7 mM to 266.4 mM, and 0.70 mM). If the excipient of glutamic acid is in the range of 100 mM to 200 mM, the amount of 170 mM (equivalent to 1.47% to 2.94%, but 2.5%) is increased to 170 mM (equivalent to weight / v / v).

[0050] Different auxiliaries and I will be used as a preservative for interferon beta lipid mixtures of fluid and the pH-buffer system used in 50 mM sodium acetate and glacial acetic acid with 100 mM sodium chloride, pH 5.0. The interferon interferon is either subjected to thermal load by 37 degree cultivation C for approximately 1 to 3 weeks or on a rotary wheel for 1 to 3 days to test mechanical strain. Tests that have been reviewed have been evaluated for the stability of interferon beta using the methods described in Example 1. As described in more detail in Dsmi 2, the greatest stability is observed in pharmaceutical formulations in sodium acetate buffer at pH 5.0 as an amino acid helper component (and may and may be sodium chloride).

2. Interferon Preferred interferon is tachycin interferon beta, all fusible as co-administered interferon-beta human from mammalian cells. In a crosslinked intererone-beta human there may be one free sulfhydryl group at position 17 and one disulfide bond between positions 31 and 141 in each molecule. As is known in natural IFN-beta humans, N13 glycosylation is expected at Asn-80. The concentration range of the pharmaceutical compositions of the invention is from approximately 30 μg / ml to about 250 μg / ml. The desired concentration range is 48 to 78 μg / ml, but the preferred concentration is approximately. 60 μg / ml. Based on international standard values, the Biogen Internal Standard has been standardized according to the WHO International Standard for Interferon, Natural # Gb-23-902-531, so that the IU (International Units) IU (International Units) (for 0.5 ml Input & Volume) is from U.C. b. 6 MIU to 50 MIU, while the desired concentration is 12 MIU.

3.Duinn from the acidic acid and phosphate organisms to be used in the invention described herein to maintain a pH in the range of approximately 4.0 to 7.2 but still from about 4.5 td approx. 5.5 but preferably 5.0, can be conventional buffers of organic acids and their salts such as sítratdúar (eg mónónatríumsítrat-dínatríumsítratblanda, sítónsýru-trínatriumsítratblanda, citric acid-mónonatríumsítratblanda etc.), Súkkinatdúar (eg. Succinic acid-mónónaíríumsúkkinatblanda, succinic acid-natríumhýdroxíðblanda, rafsýrudínatrnunsúkkinatblanda etc. ), tartrate diets (eg tartaric acid-sodium tartrate mixture, tartaric acid-sodium tartrate mixture, tartaric acid-sodium triacetate mixture, tartaric acid-sodium tartrate mixture, tartaric acid-sodium hydroxide mixture), fumarate diets (eg fumaric acid-roononate fumarate mixture, fumaric acid-dinatrium edible mixture, monosodium fumarate-disodium fumarate mixture) gluconic acid-natríumhýdroxíðblanda, gluconic acid-kalíumglúkonatblanda etc.), oxalatdúar (eg oxalic acid-natríumoxalatblanda, oxalic acid-natriumhýdroxíðblanda, oxalic acid-kalíumoxalatblanda etc.), laktatdúar (eg. lactic acid-natríumlaktatblanda, lactic acid-natriumhýdroxíðblanda, lactic acid-kalíumlaktatblanda etc.), phosphate ( monobasic nairium phosphate / dibasic sodium phosphate / dibasic sodium phosphate / acetate) and acetates (such as acetic acid-sodium acetate mixture, acetic acid-nium hydroxide mixture, etc.). In the examples described below, we use different fabric strengths and different pH sodium phosphate, sodium citrate, sodium succinate, sodium carbonate, and sodium acetate to evaluate which one is the most suitable dude. Interferon-beta samples are either at 37 degrees C for 6 days, for example, 2 weeks or rotating for 7 to 9 hours, for example, accelerating breakdown processes. The chemical properties of the samples are then determined. The samples are analyzed for example, light levels, peptide shortages, size exclusion-HPLC, shrinkage

2 μ70 or unadoxidated SDS-PAGE / Westeni stain and equilibrium / Westem stain (ŒF), all of which are described below in Case 1. EYE test of interferon-beta is compared to the starting interferon beta compound or to interferon- beta samples in the range of 2 to 8 degrees C. Our data indicate that pH is the largest factor determining the stability of our interferon-beta display and that samples with a pH between 4.0 and 5.0 are more stable than those with pH 7.0 or higher. See Example 2. However, we could develop a wide range of mterferon-beta formulations at pH-value (pH 7.2), see Example 6.

4. Hollow formation Most residues of free sulfide in interferon-beta oxidize at high pH (pH> 8.0), ie. the pH at which the disulfide bond is replaced. We have experienced some interferon-beta clusters in intermediate storage when using single-cell exclusion chromatography, unreacted SDS-PAGE and dispersion of laser light. Later we found that the formation of clusters-interferon-beta may be dependent on dissolved oxygen levels. Among the process conditions we have developed to ensure that interferon-beta preparations are not cavityed are: (a) if there is any possibility, there should never be any contact between the oxygen and oxygen of the gas during construction and storage; and / or (b) no bubbles may form during construction and storage; and / or (c) dissolved oxygen concentration in the preparation must be kept below 10% of its concentration at ambient temperature at the construction and storage temperature. See Case 3.

5. Adsorption of interferon overboard. In the same way, interferon adsorption on a certain surface and storage in a glass container requires that, for example, one surface of the container in contact with the interferon is coated or otherwise covered with a chemical that prevents adsorption. Such a surface may be chemically or physically inert to adsorption. Examples of substances used for this purpose are known to those of skill in the art, including, for example, sprayed and baked lung, polypropylene, or tetrafluoropropylene (PTFE). We took the desired 60 pg / ml of drug preparations (BG9589-1,2,3 and 4;

given in Table 1 below) and filled with 1 ml of Type I stained syringes injected with Beckon Dickinson and Into 0.75 ml Type I glass. The samples were analyzed by Motifesa HPLC (ipHPLC) for to determine protein strength. The data suggest that less protein has appeared in samples filled with glass containers compared to those filled with silicone-coated syringes. SjáDæmi5.

6. Preferred Drug Blend We performed a rapid diagnosis of protein stability and used for the four drug endpoints shown in Table 1 below, but in each of them 60 μg / ml of interferon-beta. Other types of drug formulations, some of which contain surfactant compounds, such as Pluronic F68 (manufactured by BASF), provide ί tdflu 2.

Table 1: Safe drug breakdown

<td>pHKERFli</td><td>LIST OF SUBSTANCES</td><td>LOKApH</td>

<td>20 mM acetate</td><td>150 mM arginine HCl</td><td>5.0 ("BG9859-1")</td>

<td>20 mM acetate</td><td>70mMglýsín 100 mM sodium chloride</td><td>5.0 ("BG9859-2")</td>

<td>20 mM phosphate</td><td>140 mM arginine HCl</td><td>7.2 ("BG9859-3")</td>

<td>20 mM phosphate</td><td>70mMglýsín 100 mM sodium chloride</td><td>7.2 ("BG9859-4")</td>

The oil in the pharmaceutical formulations is USP-pure. The exact chemical composition is as follows:

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BG9589-1

<td>Component (as raw material)</td><td>amount</td>

<td>Arginfa-HCl, USP</td><td>15.8 mg</td>

<td>glacial acetic acid, USP</td><td>0.167 mg</td>

<td>Sodium acetate trihydrate, USP</td><td>0.972 mg</td>

<td>Interferon-beta</td><td>30pgm</td>

<td>Water for administration, USP</td><td>0.5 ml</td>

BG9589-2

<td>Component (as raw material)</td><td>amount</td>

<td>Glycine, USP</td><td>2.628 mg</td>

<td>glacial acetic acid, USP</td><td>0.185 mg</td>

<td>Sodium acetate trihydrate</td><td>0.932 mg</td>

<td>Interferon-beta-la</td><td>30pgm</td>

<td>Water for administration, USP</td><td>0.5 ml</td>

<td>sodium</td><td>2.922 mg</td>

BG9589-3

<td>Component (as raw material)</td><td>amount</td>

<td>Arginfa-HCl, USP</td><td>154.725 mg</td>

<td>Dibasic sodium phosphate-7H 2 O</td><td>2.332 mg</td>

<td>Monobasic sodium phosphate-1H10</td><td>0.359 mg</td>

<td>Interferon-beta-la</td><td>30 pg</td>

<td>Water for administration, USP</td><td>0.5 ml</td>

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BG9589-4

<td>Component (as raw material)</td><td>amount</td>

<td>Dibasic sodium phosphate-71</td><td>1.984 mg</td>

<td>Monobasic sodium phosphate-1H2O</td><td>0.359 mg</td>

<td>Interferon-beta-la</td><td>30 μg</td>

<td>glycine</td><td>2.628 mg</td>

<td>sodium</td><td>2.922 mg</td>

<td>Water for administration, USP</td><td>0.5 ml</td>

Table 2: Other Possible Drug Disorders

<td>pHKERFIi</td><td>LIST OF SUBSTANCES</td><td>LOKApH</td>

<td>20 mM acetate</td><td>150 mM arginine HCl and 15 mg / ml human hematopoietic albumin</td><td>5.0</td>

<td>20 mM acetate</td><td>150 mM arginine HCl and 0.1% Pluronic F-68</td><td>5.0</td>

<td>20 mM acetate</td><td>140 mM sodium chloride</td><td>5.0</td>

<td>20 mM acetate</td><td>15 mg / ml human hematopoietic albumin 140 mM sodium chloride</td><td>5.0</td>

<td>20 mM acetate</td><td>0.1% Pluronic F-68 and 140 mM sodium chloride</td><td>5.0</td>

<td>170 mM L-glutamic acid 150 mM sodium hydroxide</td><td>15 mg / ml human hematopoietic albumin</td><td>5.0</td>

<td>170 mM L-glutamic acid 150 mM sodium hydroxide</td><td>0.1% Pluronic F-68</td><td>5.0</td>

Other compounds may be added to the pharmaceutical compositions of this invention. Among them may be the following rotavirus, and the desired volatile concentration is represented by weight: phenol approx. 0.2%); methylparaben (0.08%); propylparaben (0.008%); m-cresol (0.1%);

chlorobutanol (0.25%); benzyl alcohol (0.1%); and thymosalom (0.1%). On the basis of

2J70 analysis for determining clot formation and protein deamination (data not shown) are the preferred rotavirin chlorobutanol and benzyl alcohol.

7. Gastrointestinal Disorders With Intravenous Gastrointestinal Disorders Among the preferred illustrative inventions is a medical cassette equipped with the means for administering them to the excipients described below, by way of parenteral administration. The box may contain syringes filled with the liquid composition of the invention, a plurality of cotton wool alcohols, at least one needle, one patch or more, and instructions for use. It is advisable that the formulations described herein may be used with conventional needle-free administration systems.

E. INTERVENTION INTERFERON Interferon formulations of this invention function against viruses. See Example 7. The amount of interferon administered in each individual case and the frequency of administration is dependent on factors such as the type of interferon used, the condition of the patient and the response of the patient to interferon treatment.

Preferred therapeutic agents are useful in co-administration of cerebrospinal sclerosis. Frost-dried (ie reconstituted) liquid preparations of natural mterferon-beta and cross-linked interferon-beta have been administered to patients suffering from cerebrovascular failure and msnusiggi. See Jacobs et al., Armais of Neurology 39: 285-294 (March 1996) and sources cited therein and Jacobs and Munschauer, "Treatment of Multiple Sclerosis with Interferons" (bis 223-250). 1 Treatment of multiple sclerosis; trial design; Results and Future Perspectives, (Editors RA, Rudrick et al.), London: Springer, 1992. The use of the fluid compounds described herein by the brainchild and msnusigg follows the rules and measurements and results in first-class relationships with variables and Jacobs et al. above.

One of the results of the usefulness of the liquid formulations described herein is to provide toxicological assessment and evaluation of the tissue binding of the associated injected liquid preparation.

We have done a toxicological study of rabbits with chemotherapy, as described below. See Example 8.

The following examples are given to describe the inventor's illustrations, but they can not be construed as limiting in any way the invention.

EXAMPLE 1. Testing Methods Many methods previously described in detail are used to determine the physical and chemical properties of the interferon beta contained in our liquid preparations and additionally, note methods may be used to monitor the characteristics of other interferons.

To observe whether insoluble clusters are present or not, light gauge is at 320 nm and emission at 580 nm is measured. The concentration of solvent protein is determined either by measurement of light intensity at 278-280 nm (with a coagulation factor

1.5) or by reverse phase HPLC, using a known different concentration of interferon beta that has been added to the drug formulation and are used as standards. The liquid formulation samples are divorced before the test is performed. The percentage of soluble clusters is determined by understanding interferon-beta monomer coagulation by size exclusion chromatography on the TSK-Gel® G2000SWXL column (Toso Haas, Montgomeryville, PA). The peak area of the peaks observed at 280 nm is used to calculate the percentage of soluble thieves.

The stability of the peptide-frame is confirmed by electrolyte on n-diododecylsulfate polyacrylamide gel (SDS-PAGE). Interferon beta is reduced with mersaptoethanol with sodium dodecyl sulphate present before it is electroplated on 10-20% stigbell gel (MiniPlus Sepragel®, Integrated Separation Systems, Natick, MA). The proteins are then transferred by electrodepositor to nitrous silosin and developed by immunosensitization as an anti-interferon-beta antibody and goat-and-mouse antibodies associated with horseradish peroxidase are used. See, for example., Gel Electrophoresis of Proteins, A Practical Approach, 2nd Edition,

BD Hames and D. Rickwood, IRL Press.

Consideration is made of the change in the net overload charge for induction of deamidation and «unarmed changes with self-loading

2-cell polyacrylamide gel (IEF 3-10 MiniPlus Sepragel®, Integrated Separation Systems). See, Gel Electrophoresis of Proteins, A Practical Approach, 2nd Edition, as mentioned below.

Monitoring of methionine, deamidation of asparagine and other potential changes with peptide mapping is also monitored. Interferon beta is digested with Endoprothinase Lys-C (Wako Pure Chemicals), and dithiothreitol is present, and the peptide fragments are separated by reverse phase HPLC. See generally, Kalgahtgi; K. and Horvath, C. "Rapid Peptide Mapping by High Performance Liquid Chromatography". J. Chromatography 443,343-354 (1988).

Phases of N-linked oligonucleotides are determined using a Fluorophore-AssistedElectrophoresis (FACE®) system from Glyko, Inc. (Novato, CA). Asparagine-linked (N-linked) oligonucleotides are released from the glycoprotein with the aid of the enzyme Peptide-N-glycosidase F, then labeled with fluorophor in the reducing end group with decreasing amine, separated and finally quantitated on the parachylamide gel.

Antiviral activity is determined by methods such as those described in more detail. WE Stewart Π, The Interferon System, Springer Verlag ("2nd Ed. 1981). The Cytopathic Effect Inhibition Assay (CPE) test is particularly useful for determining the effectiveness of interferon against viruses. The method we consider most desirable is described in the WHO Technical Report Series No. 725, Annex 1, (1985), and is hereby referred to as the source. In summary, this CPE approach begins with the creation of a working solution of an interferon-beta standard previously adjusted in compliance with the WHO reference standard. This stock solution is prepared in a D-MEM + path containing 10% bovine fetal thrombus and 4mM of L-glulamine at a concentration of 10,000 units (U) in ml. On the test day are standard, reference solution and sample diluted in D-MEM + (three separate dilution series: a) The presence of 32 U / ml followed by diluted dilutions; b) Start with 12 U / ml followed by 1.5-fold dilutions; and c) Byijun has 6 U / ml followed by 1.2-fold thawing. Fifty microliters of diluted solutions are added to the sugars in the wells of 96-well microtitre plates, and one plate is used for each dilution sequence. Nsst is A549 cells (ATCC, Catalog Number CCL-185, Rockville, MD) in D-MEM + bstt 1, which burns 5x10 and one plate is used for each dilution sequence. Nsst is A549 cells (ATCC, Catalog Number CCL-185, Rockville, MD) in D-MEM + bstt 1, which burns 5x10 and one plate is used for each dilution sequence. Nsst is A549 cells (ATCC, Catalog Number CCL-185, Rockville, MD) in D-MEM + bstt 1, which burns 5x10s cells / ml, 50 micrograms in who burns and causes double dilution of cells and cells

2U70 interferon beta. Cells and interferon-beta are grown at 37 degrees C in 5% carbon dioxide for 15 to 20 hours. Shake the contents of the platelets into a bleach pad and add 100 ml of EMC (cerebrovascular and hepatitis) virus to a well diluted well. The virus and cells are counted at 37 degrees Celsius 5% carbon dioxide for 30 hours. The contents of the plates are shaken on a pinkish pad, and 0.75% of crystal purple coloring is placed on the plates. After 5 to 10 minutes, the plates are washed with distilled water and allowed to dissolve.

Where the test plate is the benchmark for fir growth, such as neither interferon nor EMC ct, reference cells for viruses such as EMC and cells, but no interferon, and also dilution order for interferon standard. Plates are examined by visual inspection to determine which is the lowest well on each column of viable cells (> 25% conjugated purpura). Sensitivity limits are determined as the lowest standard concentration that protects virus virus toxicity to cells. The dilution of the sample into the first positive well is multiplied by the sensitivity limits determined for the standard and dilution factor of the samples to release the activity of the interferon (MU / ml). The results from each album are converted to log units to determine the central ratio and calculate 95% significance.

EXAMPLE 2. Selection of Dual Systems We made three dummy pairs and were 9 to 10 different components in each set. in set I is a series of sodium phosphate and / or 100 mM sodium chloride solutions at a pH between 4.0 and 7.2. In addition, an array of sodium citrate diuretics is present at a pH between 4.0 and 7.0. In series ΠΙ is a series of narium succinate, sodium acetate and sodium carbonate buffer solutions, all of which are mixed with 100 mM sodium chloride, and their pH values are from 4.0 to 7.2. instead of sodium chloride, in two other solutions 50 mM sodium sulphate was at pH 4.0 and

7.2.

Supports of interferon-beta that have been extinguished are celiac disease over 1 different buffer overnight at 2-8 degrees C and carried out at least 2 exchanges, and then filtered under sterile conditions. Protein resistance is determined by measurement of light intensity at 278 nm (with a buffering factor of 1.5 mg '1 ml.cm '1) and in all samples 140 pg / ml or

2u70

150 μg / ml of interferon beta. The samples were siud and divided into four sets by filling in a portion of 2.2 ml Eppendorf glasses. One set is at 2-8 degrees C; one set has 37 degrees C; There is a rotating wheel for 7 to 9 hours; and the final link is used for initialization. Protein loss In percentages due to insoluble thieves, it is calculated from the decrease of protein strength by the various behaviors divided by initial strength.

Results:;

Protein loss percentages of non-destructive clusters are calculated by decreasing protein strength divided by baseline. Metrological analysis of all nostrils suggests that the percentages of protein due to cluster formation have less negative interferon samples at pH 4.0 and 5.0 than shown at this pH. From interferon samples cultured at 37 degrees C, between 10% and 15% were due to cluster formation. At pH, pH 6.0 increased tapide In 40-50%. At meetings and in samples at haerri pH values than 6.0 were more of soluble clusters. Furthermore, peptide mapping, for example, revealed a slight increase in the diminished interferon when the pH rises from 4.0 to

7.2 .; while the test showed that at pH 7.0 and above, more than 85% of the interferon is afamidad. At the msldum equilibrium point (μl) protein sessions in the sample (ie, the protein that does not elute the protein in the electrode and median enhancer protein is mill) with IEF / Westem spots and reveal additional pl-bands in those samples as per sodium citrate and for example, the concentrations of the bands in samples of sodium succinate. Dúageta fbsfats is none at pH 5.0. Sodium acetate sodium acetate at pH 5.0 showed no changes in the pattern of the strength of the band.

Dsmi 3; Impact of cavities While in the summary experiments, the pH, described in the literature, found that the cavity space of the storage vials appeared to be crucial for protein loss in some of the samples. When 1.5 ml of samples were stored in 2.2 ml glds, no protein loss was observed. In contrast, a significant increase in the size of the cluster if

The volume of sample was 1.2 ml. Is this 1 consistent with our findings that the formation of interferon-beta clusters in the stage where the viruses are inactivated depends on the concentration of dissolved oxygen.

In summary, the level of viral inversion involves changing the pH chlorine Sepharose wash solution (See Article C) from 7.85 ± 0.25 between 2.5 and 3.5 with 15% phosphoric acid, keep the elution solution acidic in 120- 135 minutes, then adjust the pH to 6.7 ± 0.7 with 0.5 N sodium hydroxide. All these steps are carried out at 2-8 degrees C. We designed a survey to check whether there were interferon-beta clusters at this stage and the oxygen dissolves.

Methods Coating solution from the clot-Sepharose column was divided into 50 ml or 100 ml doses and placed 100 ml of emulsion. In each bottle was the argon-blown phosphoric acid. Hrsrt is welcome in Flaskunoi in approx. 2 minutes, and hold it without stirring

approximately 2 hours at 2-8 degrees C. After that time, 6.5 ml of argon-blown sodium hydroxide is added and the solution is tested by size exclusion chromatography after a period of time

The dissolved oxygen in the liquid is temporarily mixed with the oxygen barrels (Orion, Model 860) and recorded when the base is in. In samples where the concentration of dissolved oxygen is equal to or less than 10%, argon gas is swept over the air bubble space of the reaction vessel.

Results: Data are shown in Figure 1 and show a clear relationship between the amount of oxygen present at the base and the interferon-beta-monolithic level where viruses are made unevenness. There is a significant difference between the yields obtained at an oxygen concentration of less than or equal to 10% and with all other oxygen levels. We analyzed Uka between thieves (the data are not published here) and found that specific activity decreases approximately. 30-40-fold from the total activity of the millies). We also found that more than at least 90% of the clusters provide resistance to SDS resistance to non-invasive access, indicating the same cross-linking. In diminishing conditions (2% beta-mersaptoethanol) collapses the clusters and become mononuclear, indicating cross-linking of the disulfide bondage.

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Example 4: Selection of Excipients A range of mterferon-beta formulations comprising different excipients are prepared in a pH 5.0 buffer, which is 50 mM sodium acetate and 100 mM sodium chloride. Among the excipients are glycine, arginine-HCl, lysine-HCl, sucrose, glycerin, PEG3350, glutathione and Pluronic F-68. Inventories of interferon-beta bilayer membrane dialysis to 50 mM sodium acetate and 100 mM sodium chloride, pH 5.0 overnight at 2-8 degrees C with at least two exchanges, and then sieved before use. Interferon-beta concentration is determined by measuring light gauge at 278 nm and baseline distribution. all acids are diluted so that the final concentration of the interferon is approximately 60 μg / ml. All of the samples prepared are filtered, 2 milliliters of age are supplied with 4 ml glass capsules (without silicone skin), argon blown air bubbles and capsules closed. The manifold era was at 2-8 degrees C and 37 degrees C for several weeks. Other samples are applied to mechanical shock due to room temperature for 3 days.

In addition, the percentage of dissolved oxygen in the pharmaceutical compositions with Ciba-Corning Model 248 blood gas degradation is shown. The "experimental value" is the partial pressure of oxygen (mm Hg) in the samples minus the partial pressure of oxygen in the blast-screen chromatograph, and the "reference value" is the partial pressure of oxygen in the diabetic sample, which was stored at room temperature minus the partial pressure of oxygen in the blinds that had been cleansed with a nitrogen. The percentage of dissolved oxygen ("experimental value" / "reference value") is always less than 30%.

Results:

IEF / Westem patches and SDS-PAGE / Westem patches for samples at 37 degrees C for twelve weeks indicate the displacement of tear and biliary loss, in addition to the presence of interferon polynomials in samples such as PEG3350 and glutathione era in . After one week still at 37 degrees C, there is an increase in glycerin helper on our spots. The strength of the ribbon decreases in sucrose aid. This original survey showed

2u70 we could investigate more arginine-HCl, glycine, sodium chloride and mannitol in further studies.

Example 5. Adsorption of interferon Thresholds of interferon era membranes over BG9589-1,2,3 and 4 (see Table 1) overnight at 2-8 ° C with at least two exchanges, and then filtered before use. Protein pieces are determined by measuring light intensity at 280 nm (with a buffering factor of 1.5 mg '1 ml.cm '1). OU samples are diluted so that the final concentration of the interferon value is approximately. 60 μg / ml. Dilute your sample and simulate 0.5 ml three-liter, 1.0 ml long silicone-coated BD syringes (Glertegund I) with a cooled air bubble or 0.75 ml filled in a triple, 0.75 ml glass I glass vial with argon-lined air bubbles. Protein resistance is then determined by reverse phase HPLC (Example 1).

Results:

In Table 3 below is a list of the protein strength found in antiphase HPLC decisions. The data indicate that less protein is in those who fill the glass capsule compared to pre-filled silicone-coated syringes. Therefore, silicone-coated syringes are used for the liquid interferon-beta formulation.

Table 3

<td></td><td>Glass capsule (μg / ml) (SJD)</td><td>Silicone-coated syringes (pg / ml) (SD)</td>

Example 6. Pharmaceutical compositions at biochemical pH

2 μ70 Ionic strength / phosphate. We conducted initial observations in phosphate / sodium chloride, pH

7.2 dovetails with different concentrations of duodenal factors where the concentration of phosphorus changed between 10.50 and 75 mM with a concentration (defined as I = EciZi2, where Ci and zi are mdlstyikur and validate ionic species I in that order) is 0.2.0.4 and 0.6 and is adjusted by addition of sodium chloride.

We related, inter alia, to parameters of phosphate strength (10.50 and 75 mM) and concentration (I - 0.2, 0.4 and 0.6). Mixtures of monounsaturated sodium phosphate, dibasic sodium phosphate and sodium chloride (to obtain the desired desired ionic strength) are calculated using a spreadsheet commissioned from Ellis and Morrison, "Buffers of Constant Ionic Strength for Studying pH-dependent Processes", Methods Enzymol. 87: 405-426 (1982). Jöfiiums allowed us to determine the amount of duodenal components required for pH, a certain phosphate concentration, and the determined concentration. Each of the other solutions used in the experimental trial is obtained with dumasponds supplied by Interferon-Beta via Pharmacia PD-10 afsðltunarsúlur. The pH of all of the solutions obtained is 7.20 ± 0.15. Strength is assessed by 280 μm gauge at 280 nm and the solutions are diluted to 150 μg / ml of interferon-beta in the known tissues. The resulting solutions are described in the argon atmosphere by 0.22 micron sieve, and 1.3 ml doses are placed in 5 ml glass vial with argon in the air bubble space. Samples are nektud at 37 degrees C for 6 days in triplicate. Samples are analyzed by the percentage of radiation emitted at 580 nm, the percentage of protein residues and IEF-PAGE / Western spots.

Results:

Analysis of the percentage of radiation radiation at the concentration indicates the relationship between increased radiation (ie decreasing amount of soluble protein clusters)) with increased levels of resistance. Data on recovery of prdtina show a similar tendency to the ŒF-PAGE Western blots, so there is no tendency for demodulation with different levels, so the dll samples are evenly aligned. Thus, there is a tendency to show less cluster formation with decreasing phosphate strength and increased potency after storage for six days at 37 ° C. Initial experiments on the percentage of radiation and a hundred percent of recoveries that fall from different phosphate levels (not shown here) show deaf

2 μ70 tendency toward decreasing% radiation with increased phosphate resistance and diffusion analysis shows no significant difference in mean samples with different phosphate strength Data on percentage recovery show improved protein yield at lower phosphate concentration (mapping difference with 94% confidence). IEF-PAGE Western patches show no appreciable tendency for decomposition based on different phosphate levels.

Ratio of helper / salt. Results of a study (not shown) indicate that some of the excipients need salts (eg sodium chloride) to maintain ionic strength and maintain stability at pH 7.2. Viö designed a particle survey using glycine agents (glycine, lysine, arginine, sucrose and mannitol) and a fraction of sodium chloride that boosted blood pressure (f u = 0.0.0.25.0.75 and 1.0). The fraction is calculated according to: = / Ο · »+ Ohjáiperefci) where Ο η and Ohj ^ m are the osmolarity of sodium chloride and auxiliary material in the solution in mOsm / kg, as appropriate. Salt break gives a fire to a way to compare the salt effect of different auxiliaries. In all samples were additional substances that rendered the solutions at different ratios of helper fitis / salts (cf.

Definition of FSAIT) Ten percent (s) solutions of each adjuvant in 20 mM phosphate, pH 7.2, are degassed and blown with argon. A solution of 250 mM sodium chloride, 20 mM phosphate, at pH 7.2, is prepared, degassed and blown with argon. Interferon-beta intermediate stocks are largely separated from argon-blown 20 mM phosphate-pH 7.2 dowels by dialysis. The concentration of interferon beta in the solution obtained is assessed by measurement of light intensity at 280 nm and then diluted with phosphate buffer and the appropriate auxiliary and saline solutions to produce 60 μg / ml of interferon beta and the conditions of excipient and salt as requested. Show as follows: Filter-sterile 0.22 microns) and filled with 1.0 ml Becton Dickinson powdered icicle I silica gel. 5 ml volumetric volume) and air bubbles filled with cobwebs. Samples are stored at 40 degrees C.

After 6 days, arginine, glycine and sucrose were analyzed by light gauge at 320 and 280 nm both before and after filtration of 0.22 microns. After 2 weeks, arginine, lysine and mannitol were analyzed in the same manner but also with IEF-PAGE, decreasing SDS-PAGE and non-invasive SDS-PAGE. Criterion samples were stored at temperatures between 2 and 8 degrees C and analyzed in the same way.

2ό70

Results:

The repercussion of interferon-beta 1a (as a percentage of the reference material) increases with increasing sucrose and mannitol fan yielding a maximum recovery rate of 130 nm sodium chloride. For arginine and lysine, decrease recovery with rising fat. Hamarks recovered for glycine glycols at pH 7.2 reaches approximately faen = 0.75. The results of the Surveillance Assay on auxiliary materials using phosphatodu at pH 7.2 together with a variety of adjuvants such as glycine, lysine, arginine, mannitol and sucrose added in qualitative amounts to achieve equilibrium resulted in poor recovery. all unhelped helper & i. These additives had no effect on the extent of afamination. For example, reductive and non-invasive SOS / PAGE indicates loss of glycococcal interferon-beta species from all pharmaceutical formulations, and heavier polygonal ribbon for self-pressurized sodium chloride alone and mannitol. In general, there is a strong relationship between ionic brain assistant attachment and its ability to render interferon-beta stable to cluster formation in these physiological pH systems. Non-steroidal additives such as sucrose and mannitol appear to yield no protein or may even cause protein loss at physiological pH. Sodium chloride with one charge on each solvent specimen works better than both mannitol and sucrose. In amino acids there are two bonds on each molecule at an oxygen-rich pH. In the case of glycine, the binary function of the molecule itself appears insufficient to make interferon beta stable. Arginine and lyophilis, which are preloaded on each molecule, make interferon beta even more stable than either the sodium chloride alone or glycine / sodium chloride mixtures.

EXAMPLE 7: STABILITY AND INVESTIGATION OF REACTIVITY QUALITY QUALITY IN QUALITY ARE INJECTED BY PHARMACEUTICAL FORMULATIONS IN DISPOSAL CONDITIONS, INSURANCE OF THE INJECTIONS DISTRIBUTED AT VARIOUS TEMPERATURE FOR ANY HOUR AND THEN EFFICIENTLY. In short, thieves of interferon-beta hemodialysis are present for BG9589-1, -2, -3 and 4 overnight at 2-8 degrees C and at least two exchanges are performed. Protein resistance is determined by measurement of light intensity at 280 nm with a fading factor

1.5 ml / mg / cm. all samples are diluted to the final interferon-beta-la concentration upb 60 μg / ml. Then it's

2 μ70 interferon-beta-1α lipids, shown in tdflu 1, were filtered and 0.5 ml were placed in 1 ml long Becton Dickinson (BD) syringes coated with bubbles of silicone soybean sprays. The samples were identified by "optical density density", "size density", HPLC (SEC), auto-resonance gel (IEF) Western spots, peptide mapping, fluorophoretic carbohydrates (FACE) and CPE bioassay in the airspace The syringe is kd & unarefitis gas. The contents of the syringes are fed at 2-8 degrees C, 25 degrees C, 33 degrees C and 40 degrees C For up to ninety days. The visions are focused on the behavior of peer as lysteria 1.

Results We analyzed our prototype display, with reference to the protein strength of those initiated for up to ninety days at different temperatures. Figure 2 shows that BG98959-1 showed absolute resistance to the protein (no protein loss) after three months Rectification at a temperature in the range of 2-8 degrees C (average 4 degrees C) to 25 degrees C. At a storage temperature (33 degrees C) approaching the liquid temperature, it was corrupted approximately. 18% of the proteins. At temperatures (about 40 degrees C) ofen likamhita was corrupted approximately. thirty hundred percent of protein after 3 months. Somewhat unanimous results were obtained for BG9589-2 (not shown). Figure 3 shows the last two months of storage tests

BG9589-3. Corruption of protein was at a minimum at 4 and up to 25 degrees C, but was reduced at a high temperature. The BG9589-4 launcher is somewhat unanimous to those shown in Figures 2 and 3. These nostrils were confirmed with SDS-PAGE / Westem patches.

No evidence of soluble clusters were found in the "baked" syringes during this survey. No noticeable changes were found on protein strength, amounts found with CPE test, percentage of Oxads APS and carbohydrate patterns. No significant changes were observed in the SDS-PAGE / Westem spots or in IEF / Westera spots. A few increase in the percentage of degeneration of the middue at the start of the session occurs. However, the percentage of deamidation in the stock of intermediates used to fill syringes was 37%, which is higher than 33.8% of the value obtained after the syringe had been filled with it. The reason for this latter & da, low quality can

2u70 have been variables in exams. There were no signs of soluble clusters but the "injected" syringes were found while on the survey. No significant changes were found in protein strength, amount detected by CPE test, hundred percent of deamidation, percent of oxidized APS and carbohydrate patterns. No significant changes were observed in the SDS-PAGE / Westem spots or in IEF / Westem spots. In short, they reveal the precursor already available for the final BG9589-1 final for up to 3 months 2-8 degrees C in the "baked" silicone syringes and 6 months at 2-8 degrees C in "extinguished" silicone -sprautunum.

We conducted a CPE anti-viral test for BG9589-1 and BG9589-2 formulations (see Table 1) after injection of antiseptic drugs. The ratios recorded were found to be 12.0 MU / ml for both BG9589-1 and BG9589-2 . The CPE test against viruses was repeated after storage of samples for up to 3 months at a temperature between 2 - 8grádaC. The recorded activity values for BG9589 are 11.6 MU / ml (n = 8) with 95% confidence in the range of 10.2 -13.3 MU / ml.

At the same time, the stability of the suppliers of intermediates BG9589-1 and BG9589-2 at 2-8 degrees Celsius and -70 degrees C for 6 months. Samples of BG9589-1 that had a history of experience were identified with the behavior of peams described in Example 1. The results obtained by the BG9569-1 synthesis process used for the process are stable at 2-8 degrees Celsius for 5 months and at -70 degrees C for 6 months.

During a period of time, this particular discovery did not show any signs of soluble thawing. No significant changes in the percentage of deamidation and carbohydrate synthesis were observed. (The difference is expressed in the error profile of the test). No noticeable changes were found in the samples when pauced with reductive SDS / Page Western spots and IEF / Westem spots. Orlilil reduction in protein strength is observed. The reduction in protein content obtained at -70 degrees C may be due to the presence of a predisposed fusion and punching in vixl. Protein resistance has not yet decreased by more than 15% of the original protein level.

2ϋ70

Example 8. Medical Test Examinations Bone tolerance in one individual localized intravenous dose (IM) is a method for assessing local toxicity of interferon when administered to new pharmaceutical formulations. Reaction of localized injections by the administration of the liquid diluent described herein or with lyophilized and reconstituted interferons is comparable to the extent of the usual salt dilution.

1. Irritation / Biopsy Surgery (Epidemiologic Quantity) Following a single IM dose of interferon-beta-lymphatic disorder. The twenty-one male kidney-white tumor was given each in a muscle (IM) one 30 μg administered by interferon-beta in the form BG9589-1 (pH 5.0, acetyl acetate, glycine / NaCl preservative, 0.5 ml / dose), BG9589-3 (pH 7.2), BG9589-L (pH 5.0, acetate buffer, arginine retardant fluid, 0.5 ml / dose) , phosphite diii, arginine vaginal fluid, 0.5 ml / dose), BG9589-4 (pH 7.2, phosphate duct, glycine / NaCl preservative, 0.5 ml / dose) and lyophilised interferon beta drug at pH 7.2, which contains 1.5% HSA human hemodialysis), 1.0 ml / dose (see Jacobs et al. above).

Each ingredient was applied to four animals. The animals who received the BG9589-1 oral lyophilisate were also given a standard saline solution on the other side of the carcasses as a negative target. Blood samples are collected 72 hours after insertion for plasma interferon beta activity.

Eye examination on the skin for examination of erythema erythema, erythema and edema is gerd 6,12,24,48 and 72 hours after insertion. When the blood sample taken 72 hours after insertion is the end time, the animals are slaughtered, the injection sites are checked with the eyes to check whether the tissue is confirmed and the side is attached with 10% non-formal formaldehyde solution. The musculoskeletal system is injected under the microscope for examination of inflammation, killing, bleeding and tissue damage.

2u70

Decomposition No of the above compounds was defined as more than a small amount of skins when used as a stage shift according to the Primary Irritation Index scores (EPA Dermal Classification System). Augmentation of the BG9589-4 site of administration indicated a minor irritation (bleeding) at the site of administration in one animal; On the other hand, microscopic examination was not revealed except irritation, and the result of the eye examination was considered to be a defect of a sample. In short, microscopic examination shows that the reaction of the site of test sites was everywhere from being minimized to small scale and no response was greater than the response to the administration of the lyophilised preparation or normal saline.

In addition, it is easy to test rabbit skin rash for repeated IM injections of the combination compositions using a number of rabbit classes, all of which receive intramuscular injection of drug preparations or normal saline solution every other day for eight days (five doses of all). Your dose of injection is predetermined at the end of each animal so that the local contact with the test solution is at its peak. Eye skin inspection takes 4-6 hours after each injection and 24 hours after the last administration in each treatment group. A daily survey is performed at the same time as each skin examination. At the end of 24 hours after ingestion, the animals are slaughtered, the sites are examined for tissue damage and the web is attached to 10% neutral formalin buffer. The remaining tissues are examined for inflammation,

EXAMPLE 9. Medical Tests The therapeutic formulations described herein are in a very heterogeneous range of interferon mixtures. The potential for changes in the behavior of the interferon with respect to drug speed and drug analogue is available in relation to any medical indication shown when administered to humans. Unfortunately, the activity of interferon beta is extremely specific and the most reliable information is obtained from the samples of strains of human origin that have been cultivated and, to a lesser extent, from the surveys of resuscitated cells. A preferable way to investigate pharmacological changes, if any, is to make bioequivalence trial in humans.

The amount of plasma interferon beta required to act on viruses can be estimated using a cytopathic effect, CPE, such as, for example, cytopathic effect. What is described in Example 1. Human fibrosis detection can be done by any number of fluid formulations and lyophilised drug preparations of interferon. By analysis of plasma parameters, area under curve (AUC) and Cmax activity, a qualified professional may determine whether lyophilised drug-based and chemotherapeutic compounds are hepatocellular. As a single example of bioavailability tests, we described a brief survey using a double blind, single dose and transducer to demonstrate the bioavailability of a liquid drug preparation according to the invention and lyophilised drug preparation in healthy volunteers. design: Each volunteer receives the same dose (eg 60 pg / 12 MU) of interferon-beta formulations, and is two blind and two-time transplant (Table 4). The examiners are 18 to 45 years old and their body weight is within 15% average marks based on body height and Ukamsbygging. Blood samples for blood clotting, chemical composition, plasma interferon beta activity, and drug screening are safe immediately after and at different doses every 144 hours after injection. Estimation of the pain of administration and response of the administration status depends on Ukafram. Blood samples for blood clotting, chemical composition, plasma interferon beta activity, and drug screening are safe immediately after and at different doses every 144 hours after injection. Estimation of the pain of administration and response of the administration status depends on Ukafram. Blood samples for blood clotting, chemical composition, plasma interferon beta activity, and drug screening are safe immediately after and at different doses every 144 hours after injection. Estimation of the pain of administration and response of the administration status depends on Ukafram.

Items related to test performance. For the treatment of interferon-related influenza syndrome, all test subjects are given asetaminophene immediately prior to administration and in the present-day interventions.

Pharmacovigilance rate [0102] Determination of plasma interferon beta. The amount of plasma is measured in units of the amount of virus acting on a (CPE) test. in the amount of what it works on the virus is measured

AUC, CnuK and Ton. The AUC value will be calculated from the time of administration

2 July 70 until the last measurable stomach ache (AUC0-τ) and ί 144 hours after dose administration (AUC0.144). The standard analysis of the results of the treatment is performed using SAS (version 6.08, SAS Institute, Cary, North Carolina).

Table 5

<td colspan="5">Estimation of doses in typical nucleotides</td>

<td>close Category</td><td>type administration</td><td>dose (MU)</td><td>Treatment Therapies 1</td><td>Treatment-period 2</td>

<td>1</td><td>IM</td><td>12</td><td>Freeze dried (60 meg)</td><td>Liquid (60 meg)</td>

<td>2</td><td>IM</td><td>12</td><td>Liquid (60 meg)</td><td>Freeze dried (60 meg)</td>

The efficacy of operation. The bioavailability of neopterin, an image of the interferon-induced enzyme GTP cyclohydrolase, indicating stimulation of absorptive cells and T-eirilfrinima (C. Huber et al., J Exp Med 1984; 160: 310-314; 20 September 1996; D. Fuchs, et al., Immunol. Today 9: 150-155, 1988), which have been described in the surveys, both medical and non-medical, on human co-administered interferon beta, is a neoplasm-induced enzyme dependent plasma following a variety of treatments involving human administration of interferon beta.

Neopterin is recommended for laboratory procedures. The pattern of drug trauma is quantitatively described by calculating the variables of the plasma. The first change, EAUC. the area under the curve of neopterin versus time when it has been corrected, for example, the baseline. The second variable is Emax; This change is the difference between the peak point of the neopterin and the baseline. The third parameter is the induction rate, IR; This change is calculated as the peak of neopterin divided by the baseline of neopterin.

Tölfneði. Wilcox-Mann-Whitney single-sided test two are used at AUC to determine equivalence. In order to evaluate the bioavailable available amount of interferon in the composition, based on the bioavailable amount available in the lyophilized region, and their 90% confidence rating is applied to the ANOVA AUC (ANOVA) analysis following lymphatic alteration. Runes and sex are isolated from the variability between "traffickers". Factors that originate in the υ / periods and treatments are isolated by variability are isolated based on the variability "among treatment participants."

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3435396	United States of America	P	1996-12-24
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Stable liquid interferon formulations

Abstract

Term

2U70

BG9589-1

BG9589-2

BG9589-3

2U70

BG9589-4

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