TW534815B - Stable protein and nucleic acid formulations using non-aqueous, anhydrous, aprotic, hydrophobic, non-polar vehicles with low reactivity - Google Patents

Abstract

This invention relates to stable non-aqueous formulations which are suspensions of proteinaceous substances or nucleic acids in non-aqueous, anhydrous, aprotic, hydrophobic, non-polar vehicles with low reactivity. More specifically, the present invention relates to stable protein or nucleic acid formulations wherein the compound remains in stable, dry powder form, yet the formulation is flowable and, therefore amenable to delivery to an animal via injection, transdermal administration, oral delivery or using an implantable device for sustained delivery. These stable formulations may be stored at elevated temperatures (e.g., 37 DEG C) for long periods of time and are especially useful as flowable formulations which can be shipped and/or stored at high temperatures or in implantable delivery devices for long term delivery (e.g., 1-12 months or longer) of drug.

Description

534815 A7 _B7_________ V. Description of the Invention (1) Field of the Invention The present invention relates to stable non-aqueous protein and nucleic acid formulations. The stable formulation of the present invention is a suspension of particles containing proteins or nucleic acids in a non-aqueous, anhydrous, aprotic, hydrophobic, non-polar carrier having low reactivity. BACKGROUND OF THE INVENTION References: The following references are indicated by the numbers in brackets ([]) at the relevant parts of this specification. 1. Ahern and Manning, Eds., Stability of Protein Pharmaceuticals, A: Chemical and Physical Pathways of Protein Degradation, Plenum Press, New York, 1992. 2. Wang et al., 1 988, J. Parenteral Science and Technology 42: S4-S26 3. Deetz et al., 1988, Trends in Biotechnol. 6: 15-19 4. Chin et al., 1994, Biotechnol. Bioeng. 44: 140-145 5. Klibanov, 1989, TIBS 14: 141- 144 6. Zaks et al.f 1984, Science 224: 1249-1251 7. Affleck et aL, 1 992, Proc. Natl. Acad. Sci. USA 89: 1100-1104 8. Zaks et al., 1988, 丄 Biol · Chem. 263: 8017-8021 '9. Volkin et al., 1991, Biotechnol. Bioeng. 37: 843-853 Printed by the Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs 10. Guagliardi et al., 1 989, Chimicaoggi 31; 36 11. Paulaitis et al., 1992, Annals New York Acad. Sci. 672: 278-282 1 2. Matsuura et aL, 1 993, J. Amer. Chem. Soc. 1 1 5: 1 261 -1 264 13 Zaks et al., 1 988, 丄 Biol. Chem. 263: 31 94-3201. 14. Prestrelski et al., 1993, Biophys. J. 65: 661-671 15. Zhang et al., 1995, Pharm. Res. 12f 1447-1452 1 6. Singer et al., 1962, Adv. Prot. Chem. 1-68 -4- This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) 534815 A7 B7 V. Description of the invention (2) 17. Volkin et a \ .t 1991, Biotechnol. Bioeng. 37: 843-853 1 8. Aldercreutz et al., 1 987, Biocatalysis 1: 99-1 08 1 9. Guinn et al., 1991, Biotechnol. Bioeng. 37: 303- 308 20. Desai et al., 1995, J. Am. Chem. Soc. 117: 3940-3945 21. Yu et al., 1996, J. Pharm. Sci. 85: 396-401 22. Burke et al. ,, 1 989, J. Am. Chem. Soc. 111: 8290-8291 23. Kanerva et al., 1 989 # J. Am. Chem. Soc. 111: 6865-6866 24. Desai et al., 1994, J. Am. Chem. Soc. 116: 9420-9422 25. Chang et al., January 1 996, Pharm. Tech. 80-84 26. Manning et al., 1989, Pharm. Res. 6: 903-918 27. Hageman , 1988, Drug Dev. Ind. Pharm. 14: 2047-2070 28. Bell et al., 1995, Biopolymers 35: 201-209 29. Meadows, 1 996, US Patent No. 5,480,914 30. Meadows, 1996, US Patent No. 5,518,731 31. Hageman, 1994, International Publication No. W094 / 06452 32. Hofland et al., 1996, Proc. Natl. A cad. Sci. 93: 7305-7309 33. Sullivan, 1996, BioPharm September: 50-51 and 65-66. 34. Huang et al., 1996, International Publication No. W096 / 27393. 35. Debs et al. ,, 1993, International Publication No. W093 / 25673. 36. Lemoine and Cooper, Ed ·, Gene Therapy, Bios Scientific Publishers, Oxford, UK, 1996. 37. Debs et al., 1993, International Publication No. W093 / 24640. Economics Printed by Gibco technical report. 39. Boehringer Mannheim technical report. ^ 40 · Avanti polar lipid technical report. 41. Szoka et al., 1996, International Publication No. W096 / 41873. 42 · Huang et aL, 1990, Nucl. Acids Res. 18 (4): 937-947. This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) 534815

'Explanation of the invention (too long and long ago, the disclosure of a patent or patent application is in its entirety and is used as a reference as if the terms requested in the individual articles, patents and patents are specifically and individually incorporated as reference) The same degree. Love it and peptides, peptides, proteins and other protein-containing substances (such as viruses, antibodies), 'herein referred to as protein f, has great utility in medicine used as a disease, treatment and diagnosis. Protein Essentially those who are active in an aqueous environment. Therefore, the preferred formulation of protein is in an aqueous solution. However, tritium protein has only marginal stability in an aqueous solution. Therefore, protein medicine often needs to be frozen or it has only short-term properties under ambient conditions. Shelf life. In addition, many proteins have only limited solubility in aqueous solutions. Even if they are soluble: concentration, they are easy to agglutinate and precipitate. Q Proteins can be degraded through a variety of chemical mechanisms, including asparagine Deamination of glutamine and glutamine; methionine and, to a lesser extent, oxidation of tryptophan, tyrosine, and histamine Hydrolysis of peptide bonds; cross-linking of disulfide bonds and racemization of palmitic amino acid residues [1,2, and 2 4-2 8]. Water is a reactant in almost all of these degradation pathways. Furthermore, water Can be used as a plasticizer, which helps protein unfolding and irreversible agglutination. Since water is almost a participant in the protein degradation pathway, concentrating an aqueous protein solution into a dry powder also provides a white matter improvement medicine. Stable and other — ”technology. Proteins can be dried using a variety of techniques, including cold; dry, spray-dried, and desiccant-dried. The aqueous protein solution is therefore dried and stored as a dry powder until needed. Protein-dried A serious flaw is that people often prefer to use some kind of liquid. # This paper size applies to China National Standards (CNS) A4 specifications (210X297 mm). R Page order. Printed by the employee consumer cooperative 534815 V. Description of the invention (4 = quality. The use of parenteral injection and drug delivery devices is wrong. Application example. When used in dramas, the "dried protein" must be contaminated ... * White shoulders, which add time-consuming and probable problems, and expose the protein to potentially unraveling conditions :: : Parenteral drug delivery ', especially for proteins and nucleic acids, can be mentioned:; ::. The use of implantable devices for continuous delivery of a wide range of drugs or their ΓΓ style art towels is well known. Typical packaging «for example, 1 Γ ^ 5, 〇34,229E: 5,057,318 ^: ^ 5,1 10,596¾ ^. The disclosures of the h patent are incorporated herein by reference. Proteins can only be marginally soluble in non-aqueous solvents and can disintegrate proteins in a moderate manner. And denaturation [4, 16]. The dissolution of protozoan eggs in sexual solvents generally requires derivatization or reconstitution of proteins. When looking for senile catalysis for aging, Cai and others have already met These catalytic enzymes can be suspended in a non-aqueous carrier in the form of powder, typically in hydrophilic organic solvents including alcohols, ketones, and vinegar [3 =, 13 and U-23]. These enzymes may have sufficient conformational motility to exhibit significant enzyme activity under the enzyme hydration level of ⑶% and the addition of low molecular protonic compounds. The optimum level of activity can be reached significantly at approximately 30% of the enzyme hydration level. At a minimum, their enzyme activity requires a level of necessary water "to hydrate protein t. No, hydration is at a chemical level (usually 10-40% W / W water / protein) and / or protic solvents, such as Used in these studies. Typically results in protein that is unacceptable for pharmaceutical purposes. Another requirement for catalytic action in non-aqueous solvents is the application of the Chinese National Standard (CNS) on this paper standard (please read the Note: Please fill in this page again): Packing ------ 丨 3 = tv 534815 A7 B7 V. Description of the invention (^ The enzyme should be dried from a solution of pH 値 with the optimum pH close to the enzyme activity: Species pH: Gen It is harmful to the storage system of protein medicines, because the degradation mechanisms of protein and protein are all related, and usually the protein is the most stable when it is dry away from its biologically active pH. Furthermore, & Other catalytic enzyme systems are not allowed to add stabilizers, especially carbohydrates that achieve stabilization functions through lice bonds, mouth to protein, and reduction of enzyme hydration) [14]. In addition, there are 7F Fluorocarbons as a Ingredients of drug delivery vehicles used in ophthalmic compositions [29, called. Similarly, suspensions of growth hormone in sweet, diacetate or polyethylene glycol have also been published [31]. Genetic, or &; Because the field of metastasis lies in experimental and clinical progress. Already, there are also transfers into cells by disease mother media such as adenovirus, retrovirus 4, adeno-associated virus, vaccine virus, and Sindbis virus M-W (). Those who use non-viral methods include squatting acid, adding dextran, naked DNA injection, electrophysiology, cochieates, cationic lipid complexes, liposomes, polymers (such as dendritic bodies) And pLGA), disease protein, etc. The printed DNA printed with cationic lipids and / or liposomes by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs has been marked as an effective tool for transfecting a variety of mammalian cells. Their preparation is simple And can be used with a wide variety of DNA's and R-s with few restrictions on the size of nucleic acids. They have the ability to efficiently transfect many different cell types and are not siblings [3 2, 33, 35, 36 ]. Nucleic acid formulations, including DNA / liposomes and RNA / liposomal complexes, must be mixed immediately before administration, resulting in inconveniences in manufacturing, shipping, storage, and administration [35, 37_ This paper is applicable to China National Standard (CNS) A4 specification (210X297 male 534815 printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs)

A7 B7 V. Description of the invention (6) 4j〇]. Often, their two-part formulations are not very highly concentrated and require a large volume of solution. Dry powder formulations containing freeze-dried nucleic acid / liposome complexes have also been used [34, 4 丨], but they all need to be reconditioned with an appropriate aqueous solution just before administration. As for the aqueous complexes, they are internally unstable, and lose most of them within a few hours or days', if not all, the transfection activity [4 丨]. -Therefore, there is a need for pharmaceutical compositions that can overcome these limitations of prior art. Their composition must maintain the stability of the active compound, preferably below room temperature and body temperature (25 and 37 ° F), and present in at least a flowable state for injection, incorporated into the design for immediate , Delay, or long-term administration within the delivery system, or other means of administration. Summary of the Invention The present invention proposes stable, non-aqueous formulations, which include money, skin, proteins, and other protein-containing substances ("proteins" or "protein-containing substances") or compositions containing ⑽A and RNA (" Nucleic acid ") in a non-reactive, anhydrous: suspension in negative, hydrophobic, non-polar carriers. More specifically, the present invention relates to stable formulations which contain proteinaceous substances or: acids It is kept in the form of a dry powder, but the formulation is flowable, so it is used to deliver to the thing through a device such as an 'injectable' ambulatory infusion or continuous delivery. The preparation of these money It can be stored at high temperature (such as 2370 ° C) for a long time and is particularly useful as an implantable delivery device capable of being transported and stored at high temperature, or for long-term delivery of drugs (such as 12 months or longer) ≫ In the formula, the present invention proposes a stable protein composition, which contains -9- Zhang Jiajia standard (CNS) A view (2 similar) 97 public celebration) ~~ ---- 534815 V. Description of the invention (7 a protein powder, In the preferred embodiment, at least one anhydrous, non-protonic, reactive carrier is used. In the preferred embodiment, the η ': polarity'low N γ' can be used in the foot variability group. Up to about 30% (one) of the protein-containing powder, the other-part of the invention, the present invention proposes a method for the preparation of Aman protein composition, these methods include the protein temporarily > About 10% of the protein-containing shellfish powder is suspended in at least one beneficial water g ..., water, aprotic, hydrophobic, non-polar, low-reactivity carrier. In the other part, the present invention proposes a treatment Method for a patient suffering from or susceptible to a condition that can be relieved or prevented by administering a protein-containing compound 'Herman Method includes administering to the patient an effective amount of a stable proteome' which includes a protein-containing powder, wherein Protein in the powder: less than about 10% combined, and at least one anhydrous, non-polar, aprotic, hydrophobic, low-reactivity carrier. In yet another part, the present invention proposes stable nucleic acids A composition comprising a nucleic acid-containing powder, wherein The nucleic acid hydration system in the powder is less than about 10%; and at least one anhydrous, non-polar, non-protonic, hydrophobic, and low-reactivity carrier. Printed in another part by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs The present invention proposes a method for preparing a stable nucleic acid composition, which comprises suspending a nucleic acid-containing powder having a core hydration of less than about 10% in at least one kind of anhydrous, non-polar, aprotic, hydrophobic, In a low-reactivity carrier. In another aspect, the present invention proposes a method for treating patients who are susceptible to or susceptible to a condition that can be relieved or prevented by administration of a nucleic acid-containing compound, the paper size of which is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 534815 A7 ___ _ B7 V. Description of the invention (8) includes taking an effective amount of a stable nucleic acid composition for the patient, wherein the nucleic acid composition includes a nucleic acid-containing powder Wherein the hydration system of the nucleic acid in the powder is less than about 10%; and at least one anhydrous, non-polar, aprotic, hydrophobic, and low-reactivity carrier. Detailed description of the invention The present invention relates to an unexpected discovery, that is, the suspension of dried protein-containing or nucleic acid-containing particles in anhydrous, aprotic, hydrophobic, non-polar, and low-reactivity carriers can lead to stability. Flowable non-aqueous formulation. The previously known protein-containing compounds are all formulated with excipients such as EDTA or polysorbate 80, which must be stored in a dilute buffered aqueous solution at low temperature (4-25 ° c), or often must be stored in Freeze-dried powders or granules that are low temperature and often must be reconditioned prior to use, all use degradation pathways such as acid / base catalyzed hydrolysis, deamination, racemization and oxidation to form degradation products. Similarly, previously known nucleic acid formulations, even those made with freeze-dried powder, are given in dilute aqueous solutions that do not have long-term stability and must be stored at low temperatures. In contrast to this, the formulations disclosed in the present invention can stabilize proteins and nucleic acid compounds at high temperatures (such as 37 ° C) and high concentrations (such as up to about 30 ° / 〇). Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economy Standard peptide and protein formulations include dilute aqueous solutions. Drug stability is usually achieved by varying one or more of the following parameters: pH, buffer type, ionic strength, excipients (EDTA, Tween 80, etc.). For these formulations, degradation pathways (hydrolysis, deamination, racemization) that require water cannot be stabilized. Conversely, in the present invention, it is formulated in a non-aqueous, anhydrous, aprotic, hydrophobic, non-polar, and low-reactive carrier, such as -11- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 (Mm) 534815 Μ ---_____ B7 V. Description of the invention (9) Mineral oil (MO), perfluoronaphthyl (PFD), methoxyflurane (mf), perfluorotributylamine (PTA) and fourteen Proteins in alkanes (TD) have proven to be more chemically and physically stable than those formulated in aqueous solutions. MO, PFD, MF, PTA and TD are all considered as anhydrous, aprotic, hydrophobic, non-polar, low-reactivity carriers. These carriers can reduce the rate of degradation because they can leave the protein-containing compounds away from water and they lack the protons or other reactive moieties that can be provided for the degradation reaction. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economics This invention includes the use of anhydrous, non-protonic, non-polar, hydrophobic, low-reactivity carriers such as MO, PFD, MF, PTA or TD to stabilize protein formulations to combat chemistry And physical degradation. This discovery includes the use of MO, PFD, MF, PTA, or TD to improve the overall stability of proteins in a wide range of conditions, including radon concentration and high temperature, thereby enabling protein formulations to be transported at ambient temperature and / Or storage and other methods of protein delivery in long-term implantation devices become feasible. The present invention proposes a flowable proteinaceous substance pharmaceutical formulation that exhibits desired protein stability. This type of non-aqueous formulation includes two ingredients: 丨) the protein stabilized in a powder formulation with low protein hydration; and 2) anhydrous, hydrophobic, = protonic, non-polar, low reactivity and oriented towards the protein Compound / Solubility Foot Loader. If desired, the dry protein form may contain stabilizers and other excipients. Both stabilizers and excipients are those that can reduce protein water degradation or protect them from interfacial tension or other dehydration that is known to those skilled in the art. Among other factors, it was surprisingly found that when dispersed in certain carriers, the protein powder may show a better contrast to what was observed with the dry powder alone. ⑽) A4 size (210 ^ 97 ^^ 7 534815 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

Α7 Β7 V. Invention description (i0) Significantly more stable. These carriers include long chain alkanes, the most preferred being alkanes in perfluorinated form. The present invention is particularly advantageous in that it can provide the ability to store protein for a long period of time at ambient temperature conditions or the ability to deliver protein for a long period of time with an implanted pump. Lipid / DNA and lipid / RNA complexes facilitate the uptake of nucleic acids into cells in vivo and in vitro. However, their complexes are inherently unstable in solution, losing most of them within a few days at ambient temperature, and if not all, their activity. This property severely limits their applicability in devices such as implantable pumps, storage injections, or other sustained release delivery systems that require long-term stay at 37 ° C. Freeze-drying these complexes results in more stable compositions, but their powders need to be reconditioned to make them flowable before administration, and the reconditioned solution is not stable. The present invention proposes a flowable pharmaceutical formulation of nucleic acid, which exhibits the required stability. This type of aqueous formulation includes two components: 1) a nucleic acid, which is contained in a stabilized powder formulation with low hydration; and 2) anhydrous, hydrophobic, aprotic, low reactivity and has Carrier for the solubility of nucleic acids. Optionally, the nucleic acid in the form of dry powder may contain nucleic acids in the form of lipid / DNA complexes, liposome Y ribosomes, viral media, viral lipoprotein bodies, dendritic bodies, cationic compounds, PLGA particles, and / or the like. Stabilizers and other excipients may be included as appropriate. Their stabilizers and excipients are those that can further reduce or protect them from interface distractions or other processes known to those skilled in the art. The formulation of the invention can be used in a variety of delivery systems, including, but not limited to, various pumping devices (syringes, infusion sets, syringe pumps, implanted skin reservoir systems, liquid-filled capsules, injectable storage compositions, etc. -13- This paper has a size of Chaoxian County (c is called Gongqing) 534815 A7 B7 V. Description of the invention (11 = Another advantage over the previous technology is that the preparation of the present invention can prevent water-based = reverse contact (and subsequent Hydrolysable degradation), due to the hydrophobic nature of the formulation: it can be a barrier to water and moisture. This is because the formulation is used for implanted devices that must be kept in a warm and water environment for a long time. Internal time is particularly important. Another advantage of the invention is that it can formulate proteins or nucleic acids into a flowable state at extremely high concentrations (: up to about 30% W / W). Because the protein or nucleic acid system 2 is in a dry state, so its Not subject to the degradation procedures (eg, agglutination, precipitation, or fragmentation) observed with high-concentration aqueous solutions. Δ · _ · Definitions As used herein, the following terms have the following meanings: &quot; Chemical stability &quot; means Means formed via chemical pathways such as The percentage of degradation products produced by oxidative hydrolysis or enzyme action is acceptable and / or retains acceptable biological activity. In particular, if the formulation forms no more than about 40% decomposition products after one week of capture and / Or retain at least 40% biological activity, it can be regarded as chemically stable. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs • The term 'physical stability' means forming an acceptable percentage of agglutinates ( Such as 'dibody, ginseng body and larger forms) and / or cleaved products. In particular, if a formulation forms agglomerates and / or cleaved products of not more than 10% after one week at 37' It can be regarded as having physical stability. "The term" stability formulation "means that a protein or nucleic acid compound with chemical and physical stability of at least about 50/0 is retained after the next week at 37 ° C. Particularly preferred Formulations are those that retain at least about 65%, and most particularly, at least about 80% of chemically and physically stable compounds under these conditions. Particularly preferred stability formulations include those in high protein or nucleic acid loadings Amount (such as Appropriate finances at 1% or μ's scale_ -14. 534815 A7 B7 V. Description of the invention (12) ~ '-more) Those who maintain fluidity. A M protein &quot; and / or "protein-containing compounds "And / or &quot; Protein-containing substances, thinkers include peptides, polypeptides, proteins, viruses, antibodies, etc. of polymers in which amino acid residues are bound together via a guanamine (CONH) chain. The term includes both natural derivatives or purified and recombinantly produced parts. These terms also include lipoproteins and post-translationally modified forms, such as glycated protein. Analogs of any of these are derived Substances, agonists, antagonists and pharmaceutically acceptable salts are also included in these terms. These nouns also include amino acids with D-amino acids, modified, derivatized or non-naturally occurring in 0 or L-configuration, and / or have peptinimetic units as Structured portions of proteins and / or protein compounds and / or protein substances. The term "π excipient" means a more or less inert ingredient added to the finished formulation in addition to the therapeutic ingredient. The term "non-polar carrier" means a dielectric constant of less than or equal to about 15 Vehicle. η Aprotic Carrier π — The word means a carrier that does not contain acidic hydrogen (that is, hydrogen attached to oxygen or nitrogen). ”Anhydrous carrier η — The word means a carrier that does not contain water, including those adsorbed on its surface or combined into crystal water. Printed by the Consumer Cooperatives of the Central Standards Bureau, Ministry of Economic Affairs, with low reactivity carrier π and / or" low Reactive carrier π — The word means a carrier that is generally free of the protein-containing compound k / or nucleic acid solubilized or reacted with. Low-reactivity carriers are all non-polar and have Hildebrandt <(R)> below about 8.0. Examples of carriers having low reactivity include: a) saturated hydrocarbons, b) halogenated saturated or unsaturated hydrocarbons, and c), a) or b) vinegars and acids. -15- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (2 × TX297 mm) 534815 A7 B7 V. Description of the invention (13 Printed with “protein particles” and / or • by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, including The term "protein powder" means particles containing eggs' protein-containing compounds or protein-containing substances. The present invention = particles may optionally contain a _form as defined above. These agents can be used for hearing, non-ionic interface activity Agents, buffers, tinctures, carrier eggs, preservatives, etc. However, the protein-containing powder of the present invention contains no polymer and is not encapsulated by the polymer substance (that is, they are not as described in US Patent No. 5,518,731. Definition of microparticles or microcapsules.); Body; "combination"-the word means water molecules and proteins or nucleic acids, excipients or carriers "hydrophobic ...-word means that it cannot be dissolved in water to any appreciable degree. The term "nucleic acid" means an unbranched (linear or circular) nucleotide chain in which-the nucleoside of the acid # 5, the phosphate group is adjacent to the acid 3, ie the term includes ribonucleic acid (RNA) and Money RNA ) Constituents. The term nucleic acid includes single-stranded and double-stranded molecules, oligonucleotides, gene expression constructs, mRNA molecules, ribosomes, etc. Naturally derived or purified, synthetically produced, and recombinantly produced Parts are also included in this word. The word also includes analogs 'derivatives, and constructs which include promoter genes, leader genes' signals 'polyadenoic acid' or intervening subsequences, position control regions, targets, Gene |. Contains modified, derivatized, or non-naturally occurring nuclear acid units as part of the structure of zhinucleic acid is also included in this term. '· Lipid / DNA complex &quot; and lipid / RNA complex &quot; The two terms mean that the complex formed by the electrostatic interaction between the nucleic acid and the small cationic monolayer carrier is maintained by the electrostatic interaction instead of being enclosed by the nucleic acid in the liposome. A variety of topological arrangements, such as DN a condensation, liposome-16- Zhang scale applicable to Chinese National Standards (CNS) ^ specifications (2 like 297 public (please read the precautions on the back before filling in this page)

-, Τ '.I- 1 HI I--I · Agglutination and fusion. It is a multi-layered or single-layered vesicle formed between lipids and macromolecules, especially nucleic acids, which are used as drug carriers. Nucleic acid particles and / or 'nucleic acid powder', the terms mean DNA or RNA-containing particles. Nucleic acids may be complexed with lipids or in liposomes, ribosomes, viruses = somatic 'viral lipoprotein bodies, dendritic bodies, cationic polymers, PLG A filial son = ^. The nucleic acid particles of the present invention may optionally contain excipients, as described above. Such excipients may include sugars, non-ionic surfactants, buffers, salts, carrier proteins, preservatives, and the like. Highlights of weekly preparations ▲ The present invention relates to protein-containing particles and acid particles with less than 1G% hydration. The sweat is anhydrous, aprotic, hydrophobic, non-polar, and has a low-allergenic carrier. Medium non-aqueous formulation that is stable even at high temperatures: long periods. Standard dilute peptide and protein aqueous formulations need to deal with retarder type, ionic strength, Qiu and excipients (such as EDTA and ascorbic acid) and other parameters to achieve the desired L nucleic acid formulations need to be prepared or reconstituted before use. Tune. In contrast, the formulations disclosed in the present invention use dry and hydrophobic &quot; anhydrous &quot; non-polar &quot; gardenic low reactivity carriers to linearize protein or nucleic acid compounds. In particular, the stability and flowability of compounds at high concentrations (up to about 3 G%, w / w) can be provided via the formulations of the present invention. Examples of Θ W Τ depleted compounds include proteins that are biologically active or can be used to treat diseases or other conditions and include, but are not limited to, growth hormone, factor νπι, factor U and two-17-green polyfactors, pancreas Chymotrypsin, trypsinogen, ^ interferon n lactase, lactate dehydrogenase, growth factor, coagulation factor, enzyme, immune response stimulating hormone, cytokinin, lymphokine, interferon, immunoglobulin, Interleukins, peptides, growth hormone release inhibitors, growth hormone analogues, growth factor-C, gonadotropin release hormones, deletion hormones, luteinizing hormones, LHRH, LHRH analogues such as leuprude, nafarehn and goserelin, LHRH Agonists and antagonists, printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Health and Economics, printed on the factors' Mortonin, Colchicine, gonadotropins such as M hair gonadotropin, oxytocin, octreotide, growth hormone + amino acid, posterior leaf Vasopressin, adrenocorticotropic hormone, epithelial cell growth factor, prolactin, growth hormone + protein, COSyntropin, lypressin, polypeptides such as Thyroid: releasing factor, thyroid stimulating hormone, hormone secretion live intestinal pancreozymin, brain, peptide, glucagon, endocrine and blood flow distribution through the secretion of an agent. Other agents that can be delivered include antitrypsin, insulin and other peptide hormones, adrenocortical stimulating hormone, thyroid stimulating hormone, and other pituitary hormones, interferon alpha, lu and r, sympathetic interferon, erythropoietin, growth Factors such as GCSF, GM-CSF, insulin-like growth factor 1 'tissue plasminogen activator, C f 4, d DAVP, tumor necrosis factor receptor, pancreatin, lactase, melanin- 丨 receptor Antagonist 'Metaleukin-2' tumor suppressor: protein, cytotoxic protein, main, long-lasting green disease mother and other viruses, viral proteins, antibodies, recombinant antibodies, anti-contact and so on. ^ ^ Examples of nucleic acid compounds that can be formulated according to the present invention include proteins that are biologically active or can be used to treat diseases or other conditions, such as the above -18- This paper applies Chinese National Standard (CNS) A4 specifications (210X297) Nucleic acid for protein compounds. Nucleic acids that can block or reduce the production of bad proteins, including intentional or anti-sense oligonucleotides, can also be used in the present invention. In addition: Nucleic acids that can be used in the present invention also include those that directly or indirectly stimulate the animal to produce a disease (e.g., cancer) or pathogenic organisms such as bacteria, viruses, or protozoa that cause infection. Drugs can be used to treat or prevent various conditions including but not limited to hemophilia and other blood diseases, growth diseases, diabetes, leukemia, hepatitis, renal failure, HIV infection, genetic diseases such as cerebral lipase deficiency and adenosine degeneration. Aminase deficiency, hypertension, septic shock, autoimmune diseases such as multiple sclerosis, Graves disease, systemic lupus erythematosus and wind-temperature arthritis, shock and dementia, cystic fibrosis, lactose intolerance, Kroger Disease, inflammatory bowel disease, renal bowel cancer and other cancers. The aforementioned analogs, derivatives, antagonists, agonists and pharmaceutically acceptable salts can also be used. The proteins and nucleic acids useful in the formulations and methods of the present invention can be used in the form of salts, preferably in the form of pharmaceutically acceptable salts. Useful salts are known to those skilled in the art. JL includes salts formed with inorganic, organic, inorganic or organic bases. Nucleic acids can also be compounded with lipids to form liposomes, ribosomes, printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, viral media, viral lipoproteins, dendritic bodies, cationic polymers, PLGA particles, etc. The ratio of protein or nucleic acid may vary depending on the condition to be treated or prevented by the compound &apos;, the expected dosage, and the route and duration of administration. (See, for example, Ih ^ harmacological ^ i ^ LThera ^ e ^, Gilman et al. 7th ed. (1985) — Post ^^^ 11, Ying, Remington, 18th ed. (1990), which is disclosed in This article serves as a reference.) Applicable ways -19- This paper size applies Chinese National Standards (CNS)) 534815 V. Description of the invention (including oral, intestinal, and intimate contact are those who know what this person knows) : 2 The concentration of all non-intestinal, transmucosal, etc. can be from at least about two eggs v / W) to about 30% and still maintain. The preferred protein range is about 10% to about 3 〇The carrier used in the present invention is a non-aqueous, anhydrous, aprotic, non-polar, hydrophobic, low-reactivity carrier. Their carriers have a low ^ or equal to about 15: the number of electric suspensions; does not contain Acidic hydrogen, that is, plutonium that receives oxygen or nitrogen; and can be used to decompose protein-containing compounds or other ways of reacting. Preferred carriers include: a) saturated hydrocarbons, b) autochemical saturation Or unsaturated hydrocarbons, and c) ”) or b) _ purpose and chain. Particularly preferred carriers are domain hydrocarbons and unsubstituted saturated hydrocarbons. The best carriers are those that are biocompatible, such as perfluorodistillation, perfluorobutylamine ', perfluorotripropylamine, perfluoro · methyldecachloroline bite, full gas _ octahydropyridinium, full Fluoro-N-cyclohexylpyridine, perfluoro_N, N_dimethylcyclohexylmethylamine'ifluorodimethyl_adamantine, completely defeated trimethylbicyclo (3,3 behenane, bis (perfluorohexyl ) Ethylene, bis (perfluorobutyl) ethylene, perfluorobutyl-2-hexylethylene, tetradecane, methoxyflurane or mineral oil. The protein-containing or nucleic acid-containing powders used in the present invention are all solid particles, of which The hydration system of the particles is less than about 10% (W / W7J &lt; / compound). Unlike the previous protein formulations that require hydration and stretchability to maintain enzyme activity, the proteins contained in the particles used in the present invention are due to proteins Hydration is minimized with the lowest flexibility and the lowest exposure to moisture-degrading effects. Unlike previous nucleic acid formulations that required hydration to take the formulation, the formulations of the present invention can reduce the hydration of nucleic acid compounds And degradation at the same time to provide a fluid formulation suitable for use. Milled and spray-dried 20- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X297 mm) Please read the ins and outs _ Xiangzaipai printed by the Central Standards Bureau of the Ministry of Economic Affairs and Consumer Cooperatives 534815 A7 B7 Economy Printed by the Consumer Standards Cooperative of the Ministry of Standards of the People's Republic of China. 5. Description of the invention (is' spray freeze-dried, freeze-dried, precipitated, etc .. These protected powder particles are preferably prepared by solid processing. They may be subject to availability. Including protecting agents such as vinegar, sucrose, trehalose, sorbitol, raffinose dextran or cyclodextrin, which can, for example, bind to proteins via hydrogen bonding to reduce their effective hydration. They It may also contain buiking agents such as glycine or mannitol, which can change the morphology and / or processing characteristics of proteins or nucleic acids; buffers that change pH 値; and non-ionic surfactants, which can Protected from surface absorption and solubilizes the protein or nucleic acid. The formulation of dry protein or nucleic acid powder is known to those skilled in the art ... Here, the protein Or nucleic acid hydration rate refers to the total moisture fraction bound to proteins or nucleic acids in powder formulations. Certain excipients (eg, vinegars) reduce the amount of protein [14] or nucleic acid binding moisture. For For this application, T, the protein or nucleic acid hydration rate is equal to the powder's moisture content (as measured by, for example, KadFischer analysis). It is expressed as a percentage, multiplied by the weight fraction of protein or nucleic acid in the powder. -In general, '纟 invention' qualitative formulations can be prepared by simply suspending the desired amount 'which can be a therapeutically effective amount of a desired protein-containing or nucleic acid powder suspended in a selected carrier. Preferred carriers include Mo, pFD, H, PTA and D. D. Q. Methods We have found that stable non-aqueous formulations of proteins or nucleic acid compounds can be prepared by drying (less than about 10% hydration) Those with chemical conversion rate) The particles are suspended in anhydrous, aprotic, hydrophobic, low -21-: Paper size applicable standard (CNS) A4 size (2l0x29 ^^ "packed 1T — (Please read the precautions on the back before filling in this Page) • m HH · 534815 A7 B7 five, the description of the invention (L9) and the reactive carrier prepared. -We also tested the stability of these formulations by exposing them to high temperatures (37.0 ° C and measuring the chemical and / or physical stability of their formulations. The results of these studies (eg, Examples 1, 2 and 3) The one shown) proves that these formulations are stable for at least one month at 37 ° C. A major part of the invention is that the fluid non-aqueous formulations of the present invention have long-term &amp; scientific and physical stability at high temperatures. Ten years. Even when using high concentrations, their formulations are stable. So 'the advantage of these formulations is that they can be transported and stored at room temperature or above for a long time, they are also applicable. The following methods are used to implement the researchers in the following examples. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page) )

Karl Fischer Moisture Analysis: Place the vial and stopper in a 8 (Γ (:) air oven to dry overnight. Weigh approximately 6 mg of the sample in a dry vial and stopper the vial. Control vial system Prepared by just plugging an empty dry vial (ie, a vial without a sample). Subsequently, a 250 microliter Hamilton syringe (Hamiltocanc, ⑽) was washed three times with anhydrous methanol. , Nv) Add 150 microliters of anhydrous methanol to the sample vial and the control vial. Then, treat each vial with ultrasonic waves to μ until all solids are dispersed and stopped, centrifuge, and Take 100 microliters of Shangcheng methanol and inject it into A called Mat Coulometry Moisture Analyzer (SeraDyn Inc.?

Indianapolis, IN). Record the readings and calculate the water content of the samples by subtracting the control readings from the sample readings. The following agents were used to carry out the studies in the following examples. -22- This paper size applies to Chinese National Standard (CNS) A4 specifications (21〇 &gt; &lt; 297 male thin) 534815 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Α7 Β7 V. Description of the invention (20 perfluoro Perfluorotributylamine and tetradecane were purchased from Aldrich Chemical Company (MilWaukee, WI). Methoflurane was purchased from Hachiman Laboratories (North Chicag0, IL). Light mineral oil usp was purchased from

Spectrum Chemical Corp. (Gardena, CA) The following examples are presented to illustrate the invention and are not intended to be construed as limiting the scope of the invention in any way. Example 1 Stability of α-Interferon Formulation α-Interferon (α-1F Ν) Suspension A human being is heavy and interferon a-2 a (α -1FN) (Scitech Genetics Ltd

Lot # O3 6R2 801) was formulated as a 5 mg / ml solution containing 5 mM citric acid salt, 05% sucrose, and 0.005% Tween 80, pH 4.5.

Then 200 microliters of this solution was dispensed into a 丨 ml glass freeze-drying vial, partially plugged with a freeze-drying stopper, and freeze-dried using a F ds system freeze dryer according to the following cycle: V will be frozen The cabinet is pre-cooled to 5 ° C; put on a vial; freeze to 2.5 ° C at 2.5 ° C / min; when the product has reached -30 ° C, adjust to empty to 丨 2 5 m τ; keep at- 50 ° C for 30 minutes; jump to 0.5 ° C / min to 0 ° c; keep at 0 ° C for 120 minutes; jump to 1 ° C / min to 20 ° C; keep at 20 ° C 120 minutes; ____ -23- ^ 's Zhang Zhishi Shicai Guanjia g ^ TcNS) Α4 ^^ Τ ^ ΐ〇Χ297 ^^ &quot; &quot; ^-(Please read the precautions on the back before filling in this page}

534815 A7 ____ B7 V. Description of the invention (21) Jump to 1 ° C at 1 ° C / min; keep at 20 ° C for 1000 minutes; and stopper the vial. The obtained powder had a moisture content of about 5% and a protein hydration rate of about 2.5% as determined by Karl Fischer analysis. In a vial containing π-IFN powder, add 100 μl of perfluorofluorene (PFD), trifluoromethane (MF), or mineral oil (ΜΟ) to prepare a suspension, and place the vial at 37 ° Warm dipping at C. Samples were taken at 2 and 4 weeks, 700 μl of buffer (containing 5 mM citrate, 0.5% sucrose, and 0.05% Tween 8 0, p Η 4 · 5) and The vial was inverted gently to extract a-IFN from the non-aqueous phase. After 15 minutes, one aliquot of the aqueous phase was removed and analyzed for stability by reverse-phase HPLC and reduced and non-reduced SDS-PAGE electrophoresis. The chemical stability of these formulations was determined by the inverse phase Plc (Table 1). In addition, no agglomerated or cleaved products were observed on reduced or non-reduced SDS-PAGE gels. Table 1 Measurement of the stability time of a -1F N suspension at 37 ° C by reverse phase chromatography% recovery% recovery% recovery (week) PFD suspension 37 ° C MF suspension 37 ° C MO suspension Liquid 37 ° C 0 98 ± 3 92 ± 6 101 ± 1 2 103 soil 2 81 soil 3 94 soil 3 4 98 soil 1 81 soil 1 84 ± 2 printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

* Number table 2-Average sample standard deviation of 3 samples RP-HPLC -24- This paper size is applicable to Chinese National Standard (CNS) M specification (2 丨 0X297 mm) 534815 A7 s___BT___ 5. Description of the invention (22) Instrument : Hewlett Packard HP-1090 Flow rate: 0.3 ml / min Detection: 210 nm

Column: Waters Dalta-Pak, cl8, 150X2 mm, 300A

Mobile phase: A = 30/70 / 0.2 acetonitrile / water / TFA Β = 80/20/0 · 2 acetonitrile / water / TFA gradient:

% B 0 23 45 3 5 55 52 60 90 65 90 68 23

SDS-P AGE Printed by the Consumer Cooperatives of the Central Standards Bureau, Ministry of Economic Affairs: Life Technologies Vertical Gel Electrophoresis system Gel: 15% discontinuous, 15 x 17 cm, 0.8 mm thick Operating conditions: 2 0 0 V, 50 m A, staining for about 3 hours. · Coomassie R-250 gel analysis: Bio-Rad GS-700 image analyzer, using Molecular Analyst software. Example 2 Stability preparation of chymotrypsin formulation Contains 2% chymotrypsin (Worthington Biochemical Corp., -25- This paper is in accordance with Chinese National Standard (CNS) 8-4 specifications (210 × π7 mm) 534815 A7 B7 V. Description of the invention (23 and printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs of the People's Republic of China, 8 and 2 L0t # H5B7), the analysis of κ &amp; η analysis: two ... Water content and hydration rate of protein, which are soluble = salt buffer, pH 8.0, or suspended (as dry powder) in all Naman or light mineral oil 'USP. Store the samples in 37 In the next 10 weeks, casein was used as a substrate to analyze the activity of chymotrypsin. The results are listed in Table 2 and prove the stability of the formulation. The chymotrypsin bioactivity test dilutes the sample to ^ 丨"Borate buffer, pH 80, so that the final chymotrypsin concentration used for the assay is about 2-50 micrograms / ml. Ig of casein is suspended in 95 ml of borate buffer, pH 80 and in a boiling water bath Heat until the casein dissolves (about 10 minutes, as Then, U ml of 5 / 〇CaCl2 was added, and the solution was diluted to 100 ml with 0.1 μ borate buffer solution pjj 8.0 to prepare a complex protein solution. The enzyme solution (10 ml) was placed After preheating in the 37 ° C electric heating group, 10 ml of the sample was added thereto. The solution was mixed and warm immersed in 37 ° C for exactly 20 minutes. Then, 3.0 ml of 5 ° /. Was added. Acetic acid, and the resulting mixture was allowed to stand at room temperature for 30 minutes, and then centrifuged at 3,000 g for 20 minutes. Read the absorbance of the supernatant at 28 nm in the UV spectrum photometric information and calculate its activity using the following equation ( The unit is unit / mg. Activity = ^ one (C) (t) where: At = absorbance (at 280 nm) of the supernatant solution at reaction time t (20 minutes in this example); C = in sample Chymotrypsin concentration; and t = reaction time (20 minutes). -26- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) Please read the note on the back * first

t binding B7 V. Description of the invention (24) Time of chymotrypsin (weeks) π Table 2 Activity of enzyme preparations stored at 37 t% Residual rate% Residual rate% Residual rate PFD * Μ 0 * Buffer solution * U 1 103 soil 5 100 ± 5 100 ± 4 1 97 soil 2 86 soil 1 23 ± 2 3 102 ± 3 96 ± 3 19 soil 2 6 10 102 ± 2 89 soil 1 22 ± 4 102 soil 3 92 ± 2 Mean standard deviation of 6 samples. Stability of plasma protein suspension. Preparation of a post-translationally modified plasma protein formulation with a molecular weight of 5 5 Daltons, which contains 1 mg / ml protein and about 30 mg. / Ml of excipient, buffered to neutral pH. A milliliter of the above solution was pipetted into a 3 milliliter glass vial, covered with a freeze-drying stopper, installed in a freeze-drying chamber (FTS Systems Inc.) and freeze-dried. The powder printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs has a final moisture content of about 0.025% (~ sentence water) and a protein hydration rate of about 0.008%, as measured by Karl Fischer analysis. The vial was filled with 1 ml of perfluoronaphthalene (PFE)) or metho (MF), and the vial was then placed at 37 ° C to prepare a suspension. A control cold-rolled dry powder sample was kept at -80 ° C. Samples were taken at 0, 4 5, 6.5, 8.5, and 12 · 5 weeks, respectively, and their activity was analyzed by bioactivity assay, and their chemical stability was analyzed by particle size exclusion chromatography. -27- This paper size applies Chinese National Standard (CNS) Ad specifications (210X297 mm) 534815 A7 B7 V. Description of the invention (25) The results are summarized in Tables 3 and 4 and show that these formulations maintain chemical stability Properties (measured by biological activity) and physical stability (measured by S e C). Particle size exclusion chromatography official column: TSD G3000 sw X 1 column, 7 8 X 300 mm, 5 microns (Toso-Haas T08541 or equivalent) mobile phase-· 50 mM Na2HP04, 150 nM NaCl, pH 7.0 flow rate : 1.0 ml / min Detector: 214 nm Injection volume: 50 microliters Table 3 Printing time of employees' cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs% LS +% LS +% LS + (week) Freeze-dried powder and PFD suspension In the presence of MF suspension -80 C 37 ° C 3 7 ° C 0 92 ± 14 84 ± 14 92 soil 4 1.5 98 ± 12 109 ± 9 107 ± 16 4.5 89 ± 2 86 ± 4 61 soil 20 6.5 94 ± 7 101 soil 0 68 soil 15 8.5 110 ± 2 97 ± 2 62 soil 5 12.5 111 soil 7 105 ± 11 [protein μ * h% LS =% labeled strength =-[protein] control sample * number represents Average standard deviation of 3 soil samples -28- This paper size applies to China 534815 A7 _______B7 V. Description of the invention (26) Table 4 Stability time of plasma protein suspension measured by particle size exclusion chromatography at 37 ° C % LS +% LS +% LS + (week) Freeze-dried powder PFD suspension MF suspension is stored at -8 0 ° C * 3 7〇C * 3 7〇C * 0 92 ± 096 ± 1 84 ± 7 1.5 107 soil 3 106 ± 2 104 ± 4 4.5 108 ± 2 96 ± 1 67 soil 35 6.5 1 13 soil 2 1 0 1 soil 2 79 ± 12 8.5 105 ± 1 9 5 ± 4 57 ± 5 12.5 100 soil 3 98 ± 1 4- Λ y _ [Protein μ • Sample% LS = = ° / 〇 Indication Intensity = —-— [Protein] Control sample * The number represents the average soil standard deviation of 3 samples. Example 4 Preparation of high-concentration fluidity Printed and prepared by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Material Economy containing albumin (Sigma, Lot 129FO1431), lysozyme (Sigma Lot 65H7025) or trypsinogen (Worthingt〇n L〇t # 38 £ 273) l (w / w) ratio solution. This solution was placed on a Yamat〇adl 31 spray dryer (Yamat (3 cOrp ·, NY) and spray dried with the following parameters: inlet temperature 120 ° C, The outlet temperature is 65 ° C, the atomizer is 1.2 kg / cm2. The powder was then transferred to an air-drying oven and allowed to dry at 30 ° C and then dried overnight. The powder under investigation was Karl Fischer tiller and its moisture content was about 4.5% (w / w). Its protein was hydrated. The conversion rate is about -29- This paper size applies the Chinese National Standard (CNS) M specification (210 &gt; &lt; 297 mm) 534815 Α7 B7 V. Description of the invention (27) 2.25% 〇 700mg of each powder and 1.0 ml of perfluoroamidine is fully mixed to prepare a paste (about 28% w / w). This paste is loaded into a 30 cm syringe (Becton Dickinson) and squeezed. All pastes are squeezed out uniformly and completely with very little force. FExample 5 The stability of a factor IX suspension

Novobiochem, La Jolla, CA) was formulated into a 0.5 mg / ml solution containing 60 ¾ g / ¾ liter of sesame sugar, 60 mg / ml mannitol, 1 mg / ml Tween 80 and 1.6 mg / ml histamine An acid buffer, buffered to a pH of about 7. Take 1 ml of this solution and freeze-dry it according to the above cycle. The obtained powder had 1 obtained by Karl Fischer analysis. /. Moisture content. To a vial containing dry FIX powder was added i ml of perfluoronaphthylmanganese (] ??)), perfluorotributylamine (PTA) or tetradecane (TD) to prepare suspensions. Each vial was warmly immersed at 37 ° C, and a control sample of the freeze-dried powder was stored at -8 (rc.). Samples were taken at 0 and 2 weeks and analyzed by coagulation biological activity assay. Chromatographic analysis of chemical stability. The results printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (Tables 5 and 6) show that these formulations maintain chemical stability (as measured by biological activity) and physical stability (according to Sec tester). __- 30- The scale of the scale is applicable 534815 A7 B7 V. Description of the invention (28) Table 5 The stability time of the factor IX suspension measured at 37 ° C according to the biological activity test% LS +% LS +% LS + (week) PFD suspension 37 ° C PTA suspension 37 ° C TD suspension 37 ° C 0 9 7 ± 2 89 ± 3 95 soil 3 2 9 8 ± 2 96 ± 1 96 soil 1 +% LS = [Protein] Check pine sample = 〇 / «Set up the old sore one _____ [Protein] Control sample * Number 値 represents the average standard deviation of the three samples Table 6 Factor IX suspension at 37 ° C in particle size Stability time for tomography% LS +% LS +% LS + (week) PFD suspension 37 ° C PTA suspension 37 ° C TD suspension 37 ° C 0 94 1 93 soil 1 97 soil 1 2 94 ± 2 9 5 ± 2 96 ± 1 +% LS: [protein μ =% labeled strength = ___ r test sample [protein] control sample * number 値 represents the average soil of 3 samples Standard deviation Example 6 The printed nucleic acid suspension of the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs was poorly stabilized by colonizing the coding sequence of the bacterial amycin acetyltransferase (CAT) into the plastid pClneo (Promega). Prepare pCIN.CAT plastids. Isolate the CAT coding region from plastids psiS-CAT [42] by PCR amplification using standard techniques (PCR Technology 1989, ΑΑ · Erlich, ed -31- This paper is applicable to China National Standard (CNS) A4 specification (210X 297 mm) '534815 Α7 Β7 V. Description of the invention (29)

Stockton Press, and is incorporated herein by reference). These promoter genes produce a unique Xhol restriction site at the 5 ′ end and a unique 1 ^ 〇1ϊ restriction site at the 3 ′ end. These fragments are cloned into xh〇l and pCleno using standard techniques.

Notl Shaw (Molecular Cloning, 2nd edition, 1 89, Sambrook, J., Fritsch, E.F., and Maniatis, T., and incorporated herein by reference). Plastid DNA is grown and isolated in bacterial culture. Preparation of a formulation containing 100 mg / ml sucrose, 100 mg / ml mannitol, 10 μg / ml pCIN-CAT DNA, 50 μg / ml DOTMA (Gased Dioleyloxy) Propane Group] -n, n, n-trimethylammonium) and DOPE (Lipofection, GIBCOBRL) in 10 mM Tris buffer (pH 7.1) Thing. Pipette 200 microliters of the above formulation into a 1 ml glass vial and freeze dry it using the following procedure: Pre-chill the cabinet temperature to 5 ° C; install the vial; freeze at 0.4 ° C / min To -400 ° C and hold at · 40aC for 120 minutes; jump to _ 丨 ° at a rate of 0.4 ° C / minute and hold for 240 minutes; jump at a rate of 0.4 ° C / minute To _ 4 5 ec and hold for 120 minutes; the economy, which is printed by the Consumer Standards Cooperative of the Central Bureau of Standardization, set the air conditioner to 1000mT; keep at -45 ° C, 36m under 100mT vacuum; At 0.04 ° C / min, the temperature was raised to -25 ° C under 100mT airspace; it was maintained at 25C '100mT airspace for 15,000 minutes. The dried powder had a moisture content of about 2% as measured by Kari Fischer analysis. In a glove box under anhydrous nitrogen, add 3,000 microliters of -32- using China's national ΙΤΤ ^ ΰ ^ 7ΐΗ) X 297 mm)-534815 V. Description of the invention (30 ^ gas expired) (PFD) to prepare a suspension. The suspension, dry powder, and solution samples were warmly immersed in the coffee for 1 day, 4 days, and 7 days, and then the gene transfer efficiency was monitored by measuring CAT performance in HEK 293 cells to Recovery of biological activity. Transfection of HEK 293 cell lines with lipid / DNA complexes is described as an example by the manufacturer (GIBCO BRL). Σ The results are shown in Table 7, and it demonstrates that when the lipid / DNA complexes are in aqueous solution After the solution was stored in the coffee for the next week, several K had lost its activity. In contrast, both the freeze-dried nucleic acid powder and the nucleic acid powder suspended in PFD remained almost all after one week of storage at 3rc. Biological activity (within the range of the experimental variability of the test). Table 7 Lipid / DNA constituents at 37. (: Transfection activity after warm soaking. (The number is the average standard deviation of the soil in 12 replicates) Performance CAT protein per milligram of total cells Egg-closing time solution dry powder PFD suspension formulation formulation formulation 47 8 ± 254 219 ± 114 n .d. 1 1 5 soil 46 62 8 ± 192 27 3 ± 122 13 soil 12 2 5 5 soil 137 2 84 soil 267 6 +3 3 77 ± 202 3 3 9 ± 151 (days) 0 1 4 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 7 The above modifications to the methods of implementing the embodiments of the present invention are made by the skilled person following this article It is obvious that the content of the present invention can be completed. The foregoing embodiments are not restrictive. They are merely examples of the present invention, and the scope of the present invention is defined by the scope of the following patent applications. Applicable to China National Standard (CNS) A4 specification (2 丨 〇 &gt; &lt; 297 mm

Claims (1)

534815 〇 &gt; Month '^ Day No. 086115162 patent application in Chinese, please replace # (March 1992) Patent application range 1. A stable non-aqueous composition comprising: a) an active agent-containing powder, wherein the activity in the powder is active The hydration ratio of the agent is less than about 10%; and b) at least one anhydrous, aprotic, hydrophobic, non-polar, and low-reactivity carrier selected from the group consisting of perfluoronaphthyl, methoxyflurane, and all A group consisting of fluorotributylamine, wherein the active agent is selected from the group consisting of proteins and protein compounds. 2. A composition according to item 1 of the scope of patent application, wherein at least about 80% of the active agent is at 37. (: Remain stable for at least one month. 3. The composition according to item 丨 of the scope of patent application, wherein the hydration rate of the active agent is less than about 5%. 4. The composition according to item 1 of the scope of patent application, where The powder includes up to about 30% (w / w) of the composition. 5. The composition according to item 1 of the scope of patent application, wherein the active agent is protein shellfish, which is selected from the following combinations: : Factor IX, Factor VJ] J, α-interferon, sympathetic interferon, no-galactosidase, lactate dehydrogenase 'chymotrypsin, trypsinogen, antibodies, and their analogs 〇6. A composition according to item 1 of the patent application scope, wherein the active agent is medicinal useful. 7. A method for preparing a composition according to item 1 of the patent application scope, comprising suspending the active agent-containing powder in at least one It is selected from anhydrous, aprotic, hydrophobic, non-polar, and low-reactivity carriers in the group consisting of perfluoronaphthyl, methoxyfluorine and perfluorotributylamine. The paper size is suitable for the national standards of fiscal countries (CNS) Α4 specification (21G X 297 public envy) 534815 8 8 8 8 AB c D, patent application scope 8. The composition according to the application scope of the patent application item 丨 or the patent application scope item 7.1 method, wherein the powder accounts for about 10 to about 30% of the composition (w / w) ° 9. According to the composition of the scope of patent application or the method of scope of patent application, the active agent-containing powder accounts for about 10% (~ / ~) of the composition to About 30% (Boundary / \ ¥). 10. According to the composition of the scope of application for patent No. 1, wherein the composition of the composition is long-term continuous administration. -2- ____-__ A paper size applies Chinese national standards (CNS) A4 size (210 X 297 mm)