WO2023130205A1 - 一种稳定溶液、制备方法、应用、药物组合物及试剂盒 - Google Patents

一种稳定溶液、制备方法、应用、药物组合物及试剂盒 Download PDF

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Publication number
WO2023130205A1
WO2023130205A1 PCT/CN2022/070058 CN2022070058W WO2023130205A1 WO 2023130205 A1 WO2023130205 A1 WO 2023130205A1 CN 2022070058 W CN2022070058 W CN 2022070058W WO 2023130205 A1 WO2023130205 A1 WO 2023130205A1
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Prior art keywords
solution
temperature
stable solution
arginine
water
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PCT/CN2022/070058
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English (en)
French (fr)
Inventor
邹方霖
邹礼常
王建霞
钟晓丽
Original Assignee
成都夸常奥普医疗科技有限公司
夸常股份有限公司
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Application filed by 成都夸常奥普医疗科技有限公司, 夸常股份有限公司 filed Critical 成都夸常奥普医疗科技有限公司
Priority to PCT/CN2022/070058 priority Critical patent/WO2023130205A1/zh
Priority to PCT/CN2023/070268 priority patent/WO2023131142A1/zh
Publication of WO2023130205A1 publication Critical patent/WO2023130205A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system

Definitions

  • the invention relates to the technical field of pharmaceutical preparations, in particular to a stable solution, a preparation method, an application, a medicine, a pharmaceutical composition and a kit.
  • the aqueous solubility of arginine varies with temperature, as do pharmaceutical compositions comprising arginine. Although it is necessary to prepare a high-concentration solution exceeding its water solubility, its stability and feasibility are often limited by low temperature under storage conditions or normal temperature under use environment. Taking injections as an example, it usually needs to be stored at low temperature (such as 0-5°C) for a long time and/or used at room temperature (such as 20-25°C). High-concentration aqueous solutions often precipitate under these temperature conditions, which greatly limits the emergence of related high-concentration formulations. In order to ensure the stability of the solution, the concentration of arginine in the clinically used arginine or the aqueous solution containing arginine is less than 6%.
  • the present invention aims to propose a stable solution containing arginine that avoids precipitation in a supersaturated state, a pharmaceutical composition or drug that contains or can form the stable solution, and their preparation methods and application to prepare a high-concentration stable solution higher than the solubility of arginine, providing more new options for clinical practice.
  • arginine has been considered to be a compound that is too simple and not worthwhile to study. It is not only necessary to discover its new functions and break the traditional cognition, but also to encourage research in this area. try.
  • the inventors of the present application unexpectedly found that an arginine/5-fluorouracil/water solution was divided into two parts, one of which was heat-treated and the other was not, and the non-heat-treated aqueous solution was precipitated during the cooling process , and the heat-treated aqueous solution did not precipitate.
  • a single arginine aqueous solution is divided into two parts, and the stabilization treatment including the above-mentioned overheating treatment can be performed or not, and different temperature-sensitive structures can also be produced.
  • the simple but regular stabilization treatment can actually make the solute and water with the same dosage ratio produce a synergistic effect on anti-cooling crystallization beyond the expected anti-cooling crystallization of their conventional solutions, which in turn can provide a stable solution containing ultra-high concentrations of arginine, and these Stable solutions (although they may have different forms on source products) actually have different phase transition properties, stability, and even safety and effectiveness than the prior art, and can better meet the special clinical drug needs .
  • concentration refers to the weight percent of a given solute I and water (denoted B) in an aqueous solution at temperature T [(W I /W B ) (T) % ].
  • the aqueous solution is essentially a homogeneous mixed state of a composition comprising solute and water.
  • the molar ratio of solute I to water in the composition for example, the molar ratio of solute I dry powder to water for dissolving in the instructions for powder injection, if In the form of weight percent, it is also the concentration of the solute I in its aqueous solution.
  • water solubility (abbreviated as solubility) (recorded as C I0 or C I(T)0 ) reflects the water solubility of solute I at temperature T by its characteristic concentration, and refers to the composition of solute I and water under normal conditions at The maximum concentration in an aqueous solution formed at temperature T, or the maximum weight percent of solute I/water in a homogeneously mixed state of a composition of solute I and water formed at temperature T.
  • Its assay method can be: slowly add solute I under fixed temperature T under stirring slowly and make it dissolve in the water of fixed weight (for example 100g), until it shows no longer dissolved (for example does not meet the insolubility of injection solution) Granules or no longer clarified), the percentage ratio calculated based on the weight of the completely dissolved solute and the weight of water, that is, the maximum weight of solute I that can be dissolved in 100 grams of water, is the "solubility of solute I at a fixed temperature T ".
  • thermosensitive solute refers to a solute whose water solubility (solubility) can change with temperature (T), especially decrease with temperature, eg arginine.
  • the constant, f I (T) is a concentration-temperature function that characterizes the composition.
  • the (T) in the subscript of the concentration C I( T) or the (T) in the function f I (T) both means a function with the temperature T as a variable.
  • the above-mentioned temperature (T) is displayed on the X-axis
  • the above-mentioned concentration (C I(T) ) is displayed on the Y-axis
  • T a coordinate point
  • C Iactual the concentration of solute I
  • C I0 water solubility
  • saturated concentration ", " unsaturated concentration”, “ supersaturated concentration” are respectively the solute I/water percentage ratio in described saturated solution, unsaturated solution, supersaturated solution when temperature T, denote respectively as C I saturated (T) , C I unsaturated (T) , C I supersaturated (T) .
  • Both saturated solution and unsaturated solution are thermodynamically stable and kinetically stable systems, while supersaturated solution is thermodynamically unstable, and the three can be transformed into each other.
  • the term "supersaturated solution generation temperature” (denoted as T supersaturated generation ), such as the aforementioned T 2 , refers to the necessary temperature (T 1 ⁇ T 2 ) to generate the above supersaturated solution (T 1 , C supersaturated (T1) ) .
  • the term "supersaturated solution disappearance temperature” (denoted as T supersaturated disappearance ), such as the aforementioned T 4 , refers to the temperature at which the above supersaturated solution (T 1 , C supersaturated (T1) ) crystallizes out (T 4 ⁇ T 3 ) .
  • T supersaturation refers to the temperature at which the above-mentioned supersaturated solution can exist (T supersaturation disappears ⁇ T supersaturation ⁇ T supersaturation builds ).
  • aqueous solution refers to a homogeneous composition of water and a solute.
  • conventional supersaturated solution refers to a supersaturated solution in a conventional aqueous solution, such as solution 3 above.
  • composition refers to a composition that is stored or administered in a conventional aqueous solution, such as the above used to form a solution The solute and water of 1, or the solution 1 and solute used to form solution 2 above.
  • aqueous solution containing thermosensitive solutes is a thermosensitive conventional aqueous solution, containing thermosensitive solutes and water
  • the composition is a conventional composition in the form of a conventional aqueous solution for storage or administration.
  • ultra-high concentration solution refers to a solution with a concentration exceeding the maximum concentration allowed by a conventional aqueous solution, preferably a conventional supersaturated solution.
  • compositions comprising water and its solutes can be studied comparatively by the similarities and differences of their mixtures.
  • the following comparative study was performed on compositions I and II having the same chemical composition. 1).
  • the same solute I and water are mixed at the same temperature (such as the above-mentioned temperature T 1 ) with the same feed ratio (for example, the feed ratio equivalent to the above-mentioned C 1 ), and the two mixtures are two aqueous solutions of the same concentration (for example, both For the above-mentioned solution 1), compositions I and II are two identical compositions (for example, both are conventional saturated solutions of the above-mentioned T1 ). 2).
  • One of the solutions (II) is treated, while the other solution (I) is not subjected to stabilization treatment (such as infrared heating treatment), and then their temperatures are appropriately lowered (such as the above T 2 to T 1 ),
  • stabilization treatment such as infrared heating treatment
  • the untreated solution (I) was a conventional supersaturated solution (such as the above-mentioned solution 3) of T1
  • the treated solution (I) was also a supersaturated solution, and the combination of different treatments Objects I and II have no difference in appearance, but they begin to have different histories. 4).
  • compositions I and II History makes them significantly different even in appearance (for example, I is a saturated solution and crystallization of T4 above, II is a supersaturated solution).
  • I is a saturated solution and crystallization of T4 above, II is a supersaturated solution.
  • the present invention provides a kind of stable solution
  • described stable solution comprises described medicine and water
  • described medicine comprises arginine
  • arginine is in dissolved state when temperature T and the percentage ratio with water is C 1
  • the solubility of the arginine at temperature T is C 2
  • T is the storage or administration environment temperature of the stable solution
  • the stable solution storage or administration environment temperature is also referred to as target scene temperature, such as preparation, storage, movement, administration, or/and in vivo scene.
  • the percentage ratio C1 of said arginine to water does not change with the drop of temperature T, for example, said ultra-high concentration solution formed by said composition, for example, during said storage or/and administration Solute precipitation does not occur with the change of temperature T within the required time.
  • said lower temperature is selected from 0-37°C.
  • the stable solution of the present invention at temperature T is a supersaturated solution, but not a conventional supersaturated solution.
  • solute refers to a non-gaseous substance intended to dissolve in water.
  • stable solution refers to a supersaturated solution (for example, a supersaturated injection solution that can exist stably within a desired period of time within the temperature T (especially lower temperature T) range that may be experienced. liquid).
  • the actual concentration of said arginine is greater than the water solubility at temperature T.
  • the stable solution of the present invention is a supersaturated solution of arginine, which is also the storage or administration form of the composition of the present invention comprising water and arginine.
  • the temperature changes concerned are within the most meaningful range: 0°C ⁇ T ⁇ 37°C, which basically includes the freezing step in the preparation (e.g. filtration, subpackaging, lyophilization) ), common temperature in storage and use.
  • the actual concentration of arginine that can be maintained at 0° C. is more than four times its water solubility at the same temperature.
  • the actual concentration of arginine at 0°C is more than twice its water solubility at 20°C.
  • the percentage ratio (C A(T) ) of the arginine (A) to water is greater than the conventional supersaturated concentration at the same temperature.
  • the percentage ratio C1 of the arginine and water at a temperature T of 0-37°C is 30-300%, 40-300%, 50-300%, 60-300%, or 70-300% %.
  • the percentage ratio C 1 of the arginine to water at a temperature T of 0° C. is 30-300%, 40-300%, 50-300%, or 60-300%.
  • the stable solution of the present invention includes A/water type and A/B/water type, the former contains at least arginine and water, and the latter contains at least arginine (A), other active ingredients (referred to as B) and water.
  • a stable solution (A/B/water) can be considered as comprising the other active ingredient (B) and a supersaturated aqueous solution of arginine (A/water) in which the other active ingredient (B) is in a supersaturated aqueous solution of arginine (A /water) can show completely different water solubility, especially temperature-sensitive crystallinity; it can also be regarded as containing supersaturated aqueous solution of arginine (A/water) and supersaturated aqueous solution of other active ingredients (B/water) water), the supersaturated aqueous solution of other active ingredients may show completely different supersaturated properties, especially temperature-sensitive crystallinity, in the supersaturated aqueous solution of arginine.
  • the stable solution can be in any dosage form suitable for administration, such as injection (preferably local injection), external liquid and the like.
  • injection refers to a sterile preparation containing an active ingredient and a liquid carrier for in vivo administration.
  • the injections are divided into local injections, intravenous injections, etc. according to the way of administration, and the intravenous injections can be used as local injections only after the local administration concentration is given.
  • Injections are divided into liquid injections, powder injections, etc. according to the product form.
  • the powder for injection comprises sterile dry powder and solvent, wherein the sterile dry powder contains part or all of the active ingredients, and the solvent contains all liquid carriers.
  • the concentration of the active ingredient in the injection is the concentration of the active ingredient in its mixture with all the liquid carriers, usually the end point of the local drug delivery device (syringe, puncture, injection catheter, etc.) ) of the active ingredient concentration in the liquid medicine.
  • the concentration of the active ingredient is the concentration of the active ingredient in the mixture of the sterile dry powder and the specified vehicle (such as reconstitution solution, or the pharmaceutically acceptable liquid carrier).
  • external liquid preparation refers to an active ingredient and a liquid carrier and is supplied to the surface of the body [such as skin, mucous membrane (such as eye mucosa, nasal mucosa, etc.) or/and cavity (such as oral cavity, rectum, vagina, urethra, nasal cavity, ear, Road, etc.)] administration of liquid medicines, which include, for example, lotions, liniments, drops, gargles, varnishes, and the like. During local administration, the liquid medicine often comes from local administration devices such as lotion bottles, drip bottles, droppers, gargle bottles, and cotton swabs.
  • the concentration of the active ingredient in the liquid for external use is the concentration of the active ingredient in the liquid medicine.
  • the drug further includes solute B, and the solute B does not include strong acid.
  • the solute B is in a dissolved state at the temperature T and the percentage ratio of the solute B to water is C 3 , the solubility of the solute B at the temperature T is C 4 , wherein C 3 >C 4 , and the solute B and the water are The percentage ratio C3 of water does not change with the rise and fall of temperature T.
  • the solute B is selected from at least one of locally acting drugs, cytotoxic drugs, and biologically active drugs.
  • the percentage ratio C3 of the solute B to water is 0.25%-400%, 10-300% or 30%-250% or 50%-250%.
  • the term "locally acting drug” is different from conventional drugs, and refers to a drug that does not produce an effective chemotherapeutic effect when administered systemically, but can provide effective single use or synergistic local effects, especially local chemical effects, when administered locally , such as other amino acid nutrients, conventional ineffective aromatic compounds, alkalizing agents, and polysaccharides; while conventional drugs refer to drugs that can produce effective chemotherapy when administered systemically, such as cytotoxic drugs.
  • the term "local effect” is different from the conventional effect, and refers to the pharmacological effect (such as tissue necrosis or tumor cell microenvironment) produced by the drug in the lesion tissue space (such as tumor cell microenvironment) after local administration.
  • the conventional effect refers to the systemic pharmacological effect (such as tumor cytotoxicity) produced by the drug delivered to the target area in the form of blood drug after conventional drug administration through the digestive tract or blood vessels.
  • the term "topical chemical action” refers to local action including chemical action.
  • the locally acting drugs include drugs selected from easily soluble drugs, that is, drugs with a water solubility of ⁇ 5%.
  • the actual concentration (C I(T) ) of the easily soluble drug therein is ⁇ 2%, or 2-150%.
  • the solute B includes nutrients selected from other amino acids, and the percentage ratio C 1 of the solute (A+B) to water is 40-300%, 50-300%, or 60-300%.
  • the term "nutrient” refers to substances with nutritional and health effects, preferably selected from compounds with nutritional and health effects, more preferably selected from compounds with nutritional and health effects listed in Chinese, American or European official pharmacopoeias or guidelines.
  • the term "nutritional health effect” refers to the in vivo effect produced by one or more of the following biological effects: providing energy, participating in the synthesis of biologically active substances (such as proteins), participating in part of the metabolism, maintaining the microecological balance of the intestinal tract of animals, and Participate in other physiological regulation that is beneficial to the health of the organism (eg, regulation of protein synthesis, regulation of immune responses).
  • amino acid nutrient refers to amino acid compounds with nutritional and health effects, preferably selected from amino acids, amino acid polymers and amino acid derivatives with nutritional and health effects, more preferably selected from Chinese, American or European official pharmacopoeia or guidelines Amino acid nutraceuticals and amino acid excipients with nutritional and health effects.
  • the amino acids, amino acid polymers and amino acid derivatives of the other amino acid nutrients are preferably selected from the amino acids in the following groups, or amino acid polymers containing amino acids in the following groups, or amino acids in the following groups or containing amino acids in the following groups Amino acid derivatives of amino acid polymers of amino acids: proteinogenic amino acids and non-proteinogenic amino acids.
  • the proteinaceous amino acids include amino acids selected from the group consisting of non-polar amino acids (such as alanine, valine, leucine, isoleucine, phenylalanine, proline), Polar neutral amino acids (such as tryptophan, tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine), basic amino acids (such as lysine, histidine, arginine), acidic amino acids (eg aspartic acid, glutamic acid). All of the above are L-type ⁇ -amino acids except glycine.
  • non-polar amino acids such as alanine, valine, leucine, isoleucine, phenylalanine, proline
  • Polar neutral amino acids such as tryptophan, tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine
  • basic amino acids such as lysine, histidine, arginine
  • the non-protein amino acid may include the following amino acids: ⁇ -alanine, taurine, ⁇ -aminobutyric acid (GABA), tea polyphenols (theanine), pumpkin seed amino acid (3-amino-3-carboxypyridine alkanoic acid), glutamine, citrulline, ornithine, etc.
  • the amino acid polymer may be selected from oligopeptides and polypeptides comprising amino acids as described above.
  • oligopeptide refers to an amino acid polymer comprising 2-10 identical or different amino acids linked by peptide bonds; and the term “polypeptide” refers to a polymer comprising 11-100 identical or different amino acids linked by peptide bonds thing.
  • amino acids constituting the oligopeptide or polypeptide all of them may be one or more of the above-mentioned amino acids, and other amino acids may also be additionally included.
  • the oligopeptide may be one or more selected from the following: glycyl-L-tyrosine, glycylalanine, glycylglycine, lysine-glycine Dipeptide, CG dipeptide, carnosine ( ⁇ -alanine histidine copolymer), glutathione, collagen oligopeptide, casein hydrolyzed peptide, soybean oligopeptide, oligoarginine, oligoglycine, oligomeric lysine.
  • the polypeptide may be one or more selected from the following: polyaspartic acid, polyglutamic acid, and polylysine.
  • the other amino acid nutrients include glycine, and the percentage ratio of glycine to water is >30%, or 33-100%.
  • the other amino acid nutrients include lysine, and the percentage ratio of the lysine to water is >30%, or 33-300%.
  • the conventional ineffective aromatic compound includes methylene blue, and the percentage ratio of the methylene blue to water is >1%, or 1-20%.
  • the alkalizing agent includes sodium carbonate, and the percentage ratio of the sodium carbonate to water is >3%, or 3-50%.
  • the alkalinizing agent includes tromethamine, and the percentage ratio of tromethamine to water is >3%, or 3-50%.
  • the polysaccharide includes dextran, and the percentage ratio of the dextran to water is >0.3%, or 0.3-30%.
  • the cytotoxic drug is selected from at least one of the following groups: drugs that destroy the structure and function of DNA, drugs that intercalate in DNA and interfere with transcription of RNA, drugs that interfere with DNA synthesis, and drugs that affect protein synthesis.
  • the cytotoxic drug is selected from platinum chemotherapy drugs, such as cisplatin, carboplatin, nedaplatin, oxaliplatin, and loper.
  • the cytotoxic drug is selected from 5-fluorouracil, and the percentage ratio of the 5-fluorouracil to water is >3.5%, or 4-40%.
  • the bioactive drug includes probiotics or bioactive substances derived from probiotics (such as water-soluble polysaccharides), and the probiotics or bioactive substances derived from probiotics mixed with water
  • the component ratio is >1%, or 1-30%.
  • the present invention also provides a method for preparing a stable solution, comprising mixing the drug and water according to the percentage ratio and performing a stabilization treatment required for super-dissolving the mixture.
  • Said admixture includes the admixtures which appear in the manufacturing documents or/and instructions for use.
  • the stabilization treatment includes stabilization treatment in pharmaceutical preparation.
  • the stabilization includes stabilization in drug storage.
  • the stabilization treatment includes stabilization treatment in drug delivery.
  • the stabilization treatment includes the stabilization treatment in use of the drug.
  • stabilization treatment refers to a treatment for the purpose of preparing a stable solution by using conditions not usually required for the preparation of conventional supersaturated solutions, such as superheat treatment, radiation treatment, ultrasonic treatment.
  • radiation treatment refers to treatment using radiant energy not normally required to prepare conventional supersaturated solutions, and includes infrared treatment and microwave treatment.
  • the microwave treatment has a frequency of 890-940 MHz and a central wavelength of 0.330 m, or a frequency of 2400-2500 MHz and a central wavelength of 0.122 m.
  • the wavelength range of the infrared treatment is 0.76-5 ⁇ m, and the peak wavelength is 4 ⁇ m.
  • the treatment time of the radiation treatment is ⁇ 10 minutes, or 0.25-10 minutes or 0.5-5 minutes.
  • the stabilization treatment includes superheating by 115% or more beyond the temperature or/and time necessary for mere dissolution.
  • the necessary temperature refers to the temperature necessary to obtain the same water solubility as the percentage ratio of the active solute to water according to conventional methods (for example, according to the solubility curve).
  • the necessary time refers to the time necessary to obtain the same water solubility as the percentage ratio of the active solute to water at the necessary temperature according to conventional methods.
  • the term "superthermal treatment” refers to non-radiative heat treatment (warming in the usual sense) at a temperature above the solubilization temperature required to achieve the same water solubility as the concentration of one or both of the solutes.
  • the treatment temperature of the superheating treatment is between 95°C and 125°C, and the duration is 3min-110min.
  • the treatment temperature of the overheating treatment is 95°C-120°C or 95°C-115°C or 95°C-110°C or 95°C-105°C.
  • the present invention also provides a pharmaceutical composition, which comprises a drug and water, and the drug includes arginine, and is characterized in that the storage or administration form includes the above-mentioned stable solution.
  • the pharmaceutical composition comprises the stable solution described above.
  • the pharmaceutical composition comprises the drug in the stable solution described above and water which can provide the quantitative ratio.
  • Said pharmaceutical composition comprises said drug in a stable solution as described above and a document setting forth said method.
  • the pharmaceutical composition is prepared by the above method for preparing a stable solution.
  • said pharmaceutical composition prepared from a freeze-dried product is included.
  • the present invention also provides a preparation method of the above-mentioned pharmaceutical composition, comprising preparing the stable solution by the above-mentioned method before or after loading into the final delivery system.
  • said pharmaceutical composition comprises said pharmaceutical composition prepared from a lyophilized product, followed by the method described above.
  • the stabilization process is performed either before or after loading into the final delivery system.
  • the present invention also provides a medicine prepared by the above stable solution or by the above method.
  • the pharmaceutical composition of the present invention includes, for example, injection forms that can provide injections: powder injection (water+A dry powder), powder injection (A dry powder+Aqueous solution of B), powder injection (B dry powder+A solution in water), and the like.
  • the pharmaceutical composition is administered locally, such as intratumoral injection.
  • the drug composition (local active ingredient, composition ratio and component concentration) required for local administration can be administered to the tissue where the local lesion is located through interventional means, and produce the required curative effect in the tissue.
  • the local tissue is the tumor body where the tumor cells are located; when the lesion is a non-neoplastic mass, the local tissue is an abnormality such as a mass, such as hyperplasia, cyst, nodule, etc.; when the lesion is a local In the case of inflammation, the local tissue is an inflamed area, such as an inflamed swollen body; when the lesion is abnormal secretion, the local tissue is the source of the abnormality or the secretory gland where it is located.
  • the source of the abnormality is the islets
  • the local tissue is the islets or the pancreas where the islets are located
  • the local tissue is the lesioned skin or the appendages of the lesioned skin.
  • the preparation method of the medicine includes preparing a stable solution by the above method, and then freeze-drying the stable solution.
  • the preparation method of the medicine includes the medicine prepared from the freeze-dried product obtained by the preparation method of the medicine, followed by the method of preparing the stable medicine.
  • the stable solution, the combination mode during storage or/and administration is the application of the pharmaceutical composition of the stable solution, or the pharmaceutical product of the stable solution in the preparation of medicines for preventing or/and treating diseases.
  • the stabilizing solution includes arginine as an active ingredient that provides an antithermosensitivity effect.
  • the disease is a locally diseased disease.
  • the concentration of arginine can not only meet the requirements of its anti-thermosensitivity effect, but also meet the concentration required for at least one of the following other effects: systemic activity, local activity, stabilizing effect, solubilizing effect , pH adjustment, buffering effect.
  • anti-thermosensitivity refers to an artificially endowed effect of a thermosensitive solute contrary to its normal nature (thermosensitivity), whereby an unstable solution in its supersaturated state is transformed into one that is resistant to cooling (e.g. stable solution down to room temperature or even 0°C).
  • Said use includes, for example, use in product preparation and use in useful processing.
  • Said medical applications include, for example, use in the preparation of medical products and use in medical treatment.
  • Said medical products include, for example, drugs.
  • the medical treatment includes, for example, disease treatment.
  • the disease treatment includes, for example, treating the disease.
  • use refers to any useful use, protocol or method.
  • medical application refers to any application related to medical treatment or medicine.
  • disease management refers to any beneficial action against a disease, including, for example, disease detection, prevention, treatment, and the like.
  • the application of the arginine as an active ingredient capable of providing anti-thermosensitivity effect in the preparation of a drug for preventing or/and treating diseases is preferred.
  • drug refers to any substance that can be used for disease treatment and can achieve its effective activity under safety restrictions, including, for example, pharmaceutical active ingredients in medical devices, test reagents, preventive preparations, therapeutic drugs, and so on.
  • the application of the composition can also highlight the application of the characteristics provided by its components in some cases, for example: preferably, the application includes the anti-thermosensitivity effect provided by arginine in the composition applications, including medical applications. Preferably, said applications include applications in which said solutes are provided in said composition in excess of conventional supersaturation concentrations, including medical applications.
  • the medical application of the anti-thermosensitivity includes the anti-thermosensitivity application in the disease treatment method and the anti-thermosensitivity application in the medicine preparation.
  • the medical application of the supernormal supersaturated concentration of the solute includes the application of the supernormal supersaturated concentration in the disease treatment method and the supernormal supersaturated concentration application in the preparation of medicines.
  • the application of the solute supersaturation concentration in the disease treatment method includes any application suitable for the disease treatment method, such as the application in the detection, prevention, treatment, etc. of the disease.
  • the application of the supernormal supersaturated concentration of the solute in disease treatment includes any application suitable for disease treatment, such as subcutaneous administration of supernormal supersaturated concentration, interventional therapy of supernormal supersaturated concentration of local lesion disease, supernormal supersaturated concentration Sustained release of crystals, dilution beyond conventional supersaturation concentration, etc.
  • localized disease refers to a disease with symptoms of localized disease
  • localized disease refers to a primary or secondary structural, morphological or functional abnormality of a local part of the body of an animal (preferably a human being), which may be, for example, Including one or more of the following: tumor, non-tumor enlargement, local inflammation, abnormal secretion of secretory glands, etc.
  • the local site can be any suitable one known to those skilled in the art, for example, it can be a part of one or more of the following organs: secretory organs where the secretory system is located, cardiovascular organs where the blood circulation system is located, skin, etc.
  • the local disease includes tumor, non-neoplastic tumor, local inflammation, abnormal function of secretory gland and skin disease.
  • tumor refers to a mass formed due to abnormal proliferation of cells or mutated cells, which includes solid tumors.
  • solid tumor refers to a tumor having a tumor mass, which may be due to any pathology (malignant and non-malignant) and at any stage, including, for example, the following groups classified by tumor cell type: epithelial cell tumors, sarcomas, Lymphoma, germ cell tumor, blastoma; and includes tumors named after the organ or tissue in which the tumor cell concentration is located, including, for example, tumors named after the following organs or tissues: skin, bone, muscle, breast, kidney, Liver, lungs, gallbladder, pancreas, brain, esophagus, bladder, large intestine, small intestine, spleen, stomach, prostate, testicles, ovaries, or uterus.
  • the tumors include malignant tumors and non-malignant tumors
  • the malignant tumors include breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, esophageal cancer, gastric cancer, laryngeal cancer, Testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant lymphoma, malignant brain tumor
  • the non-malignant tumors include breast tumors, pancreatic tumors, thyroid tumors, prostate tumors, liver tumors, lung tumors, intestinal tumors, oral tumors, Esophageal tumors, gastric tumors, nasopharyngeal tumors, laryngeal tumors, testicular tumors, vaginal tumors, uterine tumors, fallopian tube tumors, ovarian tumors, lymphomas, and brain tumors.
  • the locally diseased disease includes non-neoplastic tumors.
  • non-neoplastic mass refers to a mass other than a tumor, including, for example, hyperplasia (eg, hyperplasia of the breast, pancreas, thyroid, parathyroid, prostate, etc.), cysts (eg, cysts of the breast, thyroid, parathyroid, etc.) , nodules (such as breast, thyroid, parathyroid nodules, etc.), abnormal venous mass (such as hemorrhoids, etc.), swelling of local inflammation, swelling of microbial infection, etc.
  • Described hemorrhoid comprises internal hemorrhoid, external hemorrhoid, mixed hemorrhoid.
  • the local disease includes local inflammation, especially refractory inflammation.
  • the term "local inflammation” refers to non-neoplastic inflammation at a local site, including, for example, degenerative inflammation (alterative inflammation), exudative inflammation (exudative inflammation) and proliferative inflammation, which can be known in the art
  • Any suitable person known by the skilled person may include one or more of the following: arthritis, mastitis, pancreatitis, thyroiditis, prostatitis, hepatitis, pneumonia, enteritis, stomatitis, pharyngitis, periodontitis, Esophagitis, gastritis, gastric ulcer, rhinitis, sinusitis, laryngitis, tracheitis, bronchitis, vaginitis, metritis, salpingitis, oophoritis, etc.
  • the localized diseases include skin diseases, especially refractory skin diseases.
  • skin disease refers to primary or secondary lesions on the skin or skin appendages, which can be any suitable one known to those skilled in the art, for example, it can include one or more of the following Species: Skin cancer, non-malignant skin tumors, viral skin diseases (eg herpes, warts, rubella, hand, foot and mouth disease), bacterial skin diseases (eg impetigo, boils, leprosy), fungal skin diseases (eg various Ringworm), sexually transmitted diseases (such as syphilis, gonorrhea and genital warts), allergic and autoimmune skin diseases (such as contact dermatitis, eczema, urticaria), physical skin diseases (such as solar dermatosis, chilblains, corns , chapped hands and feet, pressure sores), connective tissue diseases (such as lupus erythe
  • the local lesion includes abnormal secretory function of secretory glands.
  • secretory gland refers to a structure composed of glandular cells or groups of glandular cells that performs a secretory function (secretion), which includes exocrine glands and endocrine glands.
  • Abnormal secretion of secretory glands includes hypersecretory gland dysfunction (such as hyperthyroidism) and hyposecretory gland dysfunction (such as hypothyroidism, hypopancreatic islet dysfunction (a type of diabetes)).
  • the local disease includes cardiovascular disease.
  • Interventional therapy has become an important treatment for cardiovascular diseases.
  • the cardiovascular diseases include, for example, hemangioma, hypertrophic obstructive cardiomyopathy, atrial fibrillation, arrhythmia, arterial embolism, and the like.
  • the present invention also provides a method for preventing and/or treating diseases, comprising the step of administering the stable solution to an individual in need.
  • the treatment method includes any method suitable for disease treatment, including conventional chemotherapy, immunotherapy, interventional therapy for local disease, comprehensive treatment of multiple methods, and so on.
  • a pharmaceutical kit comprising a container containing the following independently packaged preparations: the preparation of the above-mentioned stable solution, the preparation containing the components of the above-mentioned pharmaceutical composition, or the preparation containing the above-mentioned drug.
  • the pharmaceutical product also includes instructions or labels, and the instructions include content that reflects the following conditions: the above-mentioned stable solution, or the stable solution prepared by the above-mentioned method from the above-mentioned pharmaceutical composition or the above-mentioned medicine can be stored at room temperature or / and can be used at a temperature below room temperature or / and stored for more than one hour.
  • the actual concentration of arginine that can be maintained at a lower temperature is not only greater than its solubility at the same temperature, but also further greater than its supersaturation concentration at the same temperature. This means that the concentration of arginine in the composition of the invention (including the concentration allowed by the lower temperature) is greater than its maximum concentration (supersaturated concentration) allowed by the lower temperature in conventional solutions.
  • the actual concentration of arginine that can be maintained at 0°C is greater than its supersaturated concentration at the same temperature. This means that the concentration of arginine in the composition of the present invention (including the concentration allowed at 0° C.) can be greater than its maximum concentration (supersaturated concentration) allowed at 0° C. in conventional solutions.
  • the actual concentration of arginine that can be maintained at 0°C is greater than its supersaturated concentration at 25°C.
  • concentration of arginine in the stable solution of the present invention can be greater than the maximum concentration (supersaturated concentration) allowed in conventional solutions at 25°C.
  • the percentage ratio (C A(T) ) of arginine (A) to water may be greater than 25%, 25-300%, or greater than 30%, 30-300% at 0°C.
  • the stable solution of the present invention has the following advantages: compared with the conventional aqueous solution containing arginine, the super-conventional concentration of arginine in the stable solution described in the present application is not only more feasible, but also can provide Effects that the conventional concentration of arginine in conventional aqueous solutions cannot provide: topical administration of supernormal concentrations, supernormal local effects of supernormal concentrations, high dilution freedom of supernormal concentrations, etc.; the stable solution of the present invention has good chemical stability.
  • mice (BALB/c) and nude mice (BALB/c) c Nude) are healthy females aged 6-8 weeks, weighing 17.5-20.5g.
  • V Local lesion volume
  • r local lesion inhibition rate
  • the efficacy of drug i is recorded as Ei, which can be represented by ri.
  • the joint pharmacodynamic effect of the combined administration of drugs A and B is judged as follows: when the drug effect of the combination of drugs A and B is meaningless (r ⁇ 15%) , the composition does not show a co-action, or is considered to be negligible co-action.
  • the nucleation and crystallization produced by the combined use of solutes in the solution has a high degree of uncertainty, and can also be judged according to the above pharmacological action method, that is, based on the following ratio of actual nucleation and crystallization effect/expected nucleation and crystallization effect at a meaningful temperature T
  • s 2 (s 2A +s 2B )-(s 2A ⁇ s 2B ).
  • s 2A 1
  • drug B without nucleation crystallization
  • s 2B 0
  • One of the bottles was not treated as a control, while the other bottle was placed in an infrared oven (Malata multifunctional electric oven, model ZL-1202, rated power 650W) for unconventional infrared treatment (setting temperature 120°C, timing 2 minutes) ), and then put them into the environment at the target scene temperature (15°C) at the same time, and observe with the naked eye whether there is crystallization after storage for 1 hour.
  • infrared oven Malata multifunctional electric oven, model ZL-1202, rated power 650W
  • unconventional infrared treatment setting temperature 120°C, timing 2 minutes
  • the initial solutions of groups 1-3 are typical temperature-increasing solubilization models, and the concentration of each solute in a higher temperature aqueous solution is greater than 150% of its solubility at 15°C.
  • the solute with this concentration is easy to precipitate with the temperature drop, and it is an unstable solute at this temperature, and its aqueous solution seems to be an unstable solution for granted.
  • the untreated solution (conventional solution) of each initial solution of groups 1-3 had crystallization when the temperature was lowered to 15°C, which was in line with the expectation of an unstable solution.
  • the super-conventional treatment solutions (super-conventional solutions) of the initial solutions of groups 1-2 also crystallized when the temperature was lowered to 15°C, which met the expectation that the aqueous solution of unstable solutes is an unstable solution, and also strengthened the "extra-conventional solution" in the prior art.
  • An unstable solution can only be transformed into a saturated solution or a saturated solution to eliminate its instability".
  • the super-conventional treatment solution (super-conventional solution) of the initial solution of group 3 did not crystallize out when the temperature was lowered to 15 °C, and maintained the same single-phase state and concentration. Aqueous solutions of unstable solutes become stable solutions beyond expectations for their temperature sensitivity.
  • the unstable aqueous solution of the two temperature-sensitive solutes turned into a stable solution that is not temperature-sensitive, which shows that the nucleation and crystallization of the solutes used in combination under this condition antagonizes the precipitation, or resists the temperature-sensitive synergy.
  • the term "super-conventional treatment” is different from the conventional treatment of heating and solubilization necessary to reach the target concentration, and refers to any unstable solution that exceeds the temperature rising and solubilizing, and can convert the temperature rising and solubilizing solution into Stabilization of stable solutions.
  • the purpose of heating and solubilization is to obtain the desired solubilization with the minimum temperature rise.
  • the temperature of 5-fluorouracil is raised to 80°C to obtain a concentration exceeding the solubility of the target scene temperature; and the purpose of super-conventional treatment is to make temperature rise and solubilization
  • the conversion of the unstable solution into a stable solution often requires treatment that is usually not necessary for heating and solubilization, such as infrared treatment in the above examples.
  • the solution that has been treated supernormally is called supernormal solution, and the solution that has not been supernormally treated is called conventional solution.
  • the super-conventional treatment can also be overheated (the treatment temperature is ⁇ 95°C, or 95-125°C; the treatment time is 15-60 minutes, 30-60 minutes, 45-100 minutes, or 30-160 minutes ), or microwave treatment (treatment temperature > 90°C, 95-130°C, treatment time 0.5-5 minutes, 0.5-3 minutes) instead of the above infrared treatment (treatment temperature > 90°C, 95-130°C, treatment time 0.5-10 minutes, 0.5-5 minutes, or 0.5-3 minutes), similar results can be obtained.
  • the super-conventional treatment can make the temperature-sensitive arginine aqueous solution produce an anti-temperature-sensitive effect that exceeds the expectations of the conventional treatment, so that the unstable solution is transformed into stable solution.
  • the stable arginine can also be shared with other thermosensitive solutes to produce a new anti-thermosensitivity synergistic effect.
  • arginine aqueous solutions as shown in Table 2 at different temperatures according to conventional methods and determine their corresponding solubility.
  • Each aqueous solution [initial solution (T, C (T)0 )] is divided into 4 bottles (5ml/bottle). Among them, 2 bottles were used as controls without any treatment (conventional solution), while the other 2 bottles were placed in a 95° C. water bath for 30 minutes with super-conventional treatment. Then put one bottle of the conventional solution and the super-conventional solution in an environment of 15°C, and put the other bottle in an environment of 0°C. After storing for 1 hour, observe whether there is crystallization.
  • Arginine corresponds to a solubility of 15.6% (see Example 1 above) and 7.8% at 15°C and 0°C, respectively.
  • the concentration C 1(0°C) of the initial solution (T, C 0(T) ) in group 1 at its dissolution temperature ( 0°C) is equal to its 0°C solubility and less than its 15°C solubility, which is the temperature (0°C- 15°C) stable solution, there is no difference whether the solution has undergone super-conventional treatment or not.
  • the concentration C 1 ( 35°C) of the initial solution (T, C 0(T) ) in group 2 at its dissolution temperature (35°C) is higher than its 0°C solubility and 15°C solubility, which is the temperature (0°C-15°C °C) unstable solution. Its normal solution precipitated at this temperature (0°C, 15°C), meeting the expectation of instability; while its supernormal solution did not precipitate, and the supersaturated solution became a stable solution, exceeding the expectation of instability. Similarly, the conventional solutions in groups 3 and 4 are also unstable solutions, and the supernormal solutions are also stable solutions, which are beyond unstable expectations.
  • an aqueous solution of arginine was conventionally prepared at the target scene temperature (0° C.) and its solubility was determined to be 7.5%.
  • the conventional solution and the superconventional solution were put into the 15°C environment at the same time, and no crystallization was observed.
  • the normal solution is a supersaturated solution at this temperature (15°C, 20.0% > 15.7%), and there is no difference in appearance between the supernormal solution and the normal solution.
  • the conventional solution has crystallization and becomes a heterogeneous composition (0°C, saturation concentration 7.5% + crystallization), while the superconventional solution has no crystallization and is still homogeneous Composition (0°C, 20.0% > 7.5%).
  • an aqueous solution of arginine was conventionally prepared at the target scene temperature (5° C.) and its solubility was determined to be 9.5%.
  • the conventional solution and the superconventional solution were put into the 20°C environment at the same time, and no crystallization was observed.
  • the normal solution is a supersaturated solution at this temperature (20°C, 25.0%>19.2%), and there is no difference in appearance between the supernormal solution and the normal solution.
  • these two bottles of solutions are put into the environment at 5°C at the same time, then crystals will precipitate out in the conventional solution and become a heterogeneous composition (5°C, saturation concentration 9.5% + crystallization), while the superconventional solution will remain homogeneous without crystallization Composition (5°C, 25.0% > 9.5%).
  • an aqueous solution of arginine was conventionally prepared at the target scene temperature (20° C.) and its solubility was determined to be 18.7%.
  • the conventional solution and the superconventional solution were put into the 25°C environment at the same time, and no crystallization was observed.
  • the normal solution is a supersaturated solution at this temperature (25°C, 60.1% > 24.8%), and there is no difference in appearance between the supernormal solution and the normal solution.
  • these two bottles of solutions are placed in the environment of 20°C at the same time, then the conventional solution has crystallization and becomes a heterogeneous composition (20°C, saturation concentration 18.7% + crystallization), while the superconventional solution has no crystallization and is still homogeneous Composition (5°C, 60.1% > 18.7%).
  • the conventional solution and the supernormal solution formed by different treatments of the original arginine solution have different concentration-temperature functions (f conventional solution (T) ⁇ f supernormal solution (T) ), and have become two different solutions.
  • Embodiment 3 Comparative research on anti-thermosensitivity effect
  • the results of groups 2-12 show that temperature-sensitive solutes can be solubilized by heating to prepare a high-concentration aqueous solution (initial solution) with a solubility exceeding the target scene temperature.
  • the initial solution (without special treatment) all precipitated crystals with the temperature drop, and it was the same after special treatment, and the unstable solution could not be changed into a stable solution.
  • the initial solution in group 1 (without special treatment) also precipitated crystallization with the decrease of temperature, but the arginine aqueous solution treated by super-conventional treatment did not precipitate crystallization, and the unstable solution changed into a stable solution.
  • stable solute refers to a solute whose percentage (or concentration) with water does not exceed its water solubility, so that its normal aqueous solution is a stable solution.
  • unstable solute refers to a solute whose percentage (or concentration) with water exceeds its water solubility, such that its normal aqueous solution is an unstable solution.
  • 5-Fluorouracil was used as the model solute of the organic solute for the study.
  • Each solution is divided into 4 bottles (5ml/bottle) and sealed, wherein 2 bottles are not treated specially (conventional solution), and the other 2 bottles are put into an infrared oven for processing (conditions are as in Example 1, research solution).
  • the results are shown in the table below.
  • Sodium carbonate is used as a model solute for inorganic solutes (such as certain inorganic salts) for research.
  • Each solution is divided into 2 bottles (5ml/bottle) and sealed, wherein 1 bottle is not specially treated (conventional solution), and the other bottle is put into an infrared oven for processing (conditions are as in Example 1, research solution). Then put each conventional solution and research solution into 0°C environment, store for 12 hours and observe whether there is crystal precipitation. The results are shown in the table below.
  • the results of group 1 show that although highly soluble inorganic solutes can be prepared into high-concentration aqueous solution (initial solution) with a solubility exceeding the target scene temperature by heating and solubilizing.
  • the initial solution without special treatment
  • Methylene blue at a concentration of ⁇ 10 was studied as a model solute that is a stable solute when used alone.
  • Each solution is divided into 2 bottles (5ml/bottle) and sealed, wherein 1 bottle is not subjected to special treatment (conditions are as in Example 1, conventional solution), and the other bottle is put into an infrared oven for 2 minutes (research solution). Then put each conventional solution and research solution into 4°C environment, store for 24 hours and observe whether there is crystal precipitation. The results are shown in the table below.
  • Example 7 The discovery of the chemical stability of ultra-high concentration arginine
  • Embodiment 8 comparative study of drug efficacy
  • series A, B, and C respectively used mouse breast cancer 4T1 cells (1 ⁇ 10 5 cells/mouse), mouse embryonic fibroblast 3T3 cells (0.5 ⁇ 10 4 cells/mouse ), and the mixture of mouse breast cancer 4T1 cells and mouse embryonic fibroblasts 3T3 (1.05 ⁇ 10 5 cells/monkey, the ratio of 3T3 to 4T1 cells was 1/20) was used as the local lesion modeling cells, and the cells were all Inject into the right axilla of the animal subcutaneously to model the transplanted nodule.
  • the average volumes of the cell-transplanted lesion nodules in the successfully modeled series A, B, and C animals were 126.3mm 3 , 245.6mm 3 , and 273.5mm 3 (the interstitial ratio of the tumor in series C was ⁇ 28.3% ), each series of model animals were randomly divided into 10 groups.
  • the preparation method of the unconventional solution is as follows: add the solute and water required to prepare a 5ml solution into a small glass bottle according to the ratio shown, put the lid on and move it into an infrared oven for 2 minutes and then remove it. Then put the glass bottles containing each solution into an environment of 20°C, store for 1 hour, and observe whether there is crystal precipitation.
  • the curative effect (local lesion inhibition rate RA, RB, RC) of each group can also be calculated according to the curative effect (local lesion inhibition rate) to calculate the actual/expected ratio q of the drug effect.
  • the average stroma-to-stroma ratio of animal tumors in series C was measured to be 28.6%.
  • compositions of the present invention are compared with the conventional compositions (groups 5, 2), and the former has higher joint drug efficacy in the treatment of related diseases of local lesions (series A, B, C), especially in the treatment of related diseases containing fibroblasts in local lesions (such as tumors with a stroma ratio greater than 28%), which far exceed the latter's drug effect expectations.
  • Embodiment 9 Safety comparative study
  • mice were used as the experimental object, and a mixture of mouse breast cancer 4T1 cells and mouse embryonic fibroblast 3T3 cells (1.1 ⁇ 10 5 cells/mouse, the ratio of the number of 3T3 cells to 4T1 cells was 1/10) was used as the local lesion Modeling cells were injected subcutaneously into the right axilla of animals to model transplanted nodules. Animals with successful modeling (average volume of cell-transplanted lesion nodules were 532.7 mm 3 ) were randomly divided into 10 groups, 10 animals in each group.
  • compositions of the present invention (groups 2 and 4) have higher sharing safety in treating related diseases with local lesions.
  • composition of the present invention in addition to showing that it can be stored or/and administered at a lower temperature at an ultra-high concentration, has a wider range of synergistic effects than the conventional composition of the same dose, including a greater range of indications

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Abstract

本发明提供了一种稳定溶液、储存或给药方式为稳定溶液的药物组合物、稳定溶液的药物制品、以及它们制备方法和应用、及试剂盒，所述稳定溶液包含药物和水，所述药物包括精氨酸，所述稳定溶液中精氨酸在温度T时处于溶解状态且与水的百分量比为C1，所述精氨酸在温度T时的水溶解度为C2，其中T为所述稳定溶液储存或给药环境温度、例如0-37℃，C1＞C2。本发明所述的稳定溶液能够在较低温度下稳定存在且具有高于该温度溶解度的超高浓度，为临床提供更多新的选择。

Description

一种稳定溶液、制备方法、应用、药物组合物及试剂盒 技术领域
本发明涉及药物制剂技术领域，特别涉及一种稳定溶液、制备方法、应用、药物、药物组合物及试剂盒。
背景技术
精氨酸的水溶解度随温度变化而变化，包含精氨酸的药物组合物也是如此。制备超过其水溶解度的高浓度溶液虽时有必要，但其稳定性、可行性往往受限于存储条件下的低温或使用环境下的常温。以注射剂为例，通常需要在低温(如0-5℃)下进行长时间储放和/或室温(如20-25℃)下使用。高浓度水溶液在这些温度条件下经常发生析出，这大大限制了相关高浓度制剂的出现。为了确保溶液稳定，临床上使用的精氨酸或包含精氨酸的水溶液中的精氨酸浓度均小于6％。
提高包含精氨酸的水溶液中的精氨酸或其它溶质的浓度，该问题似乎并非看上去那么简单，因为它在数以千计的研究者面前已存在多年，却仍未得到解决。而对稳定存在的高浓度制剂的需求却一直存在，尤其是存储和/或使用时仍保持稳定的高浓度。传统意义上，温敏溶质的浓度必然随温度下降而下降；热力学不稳定的溶液除非转化为稳定状态，其必然是不稳定的；通常溶液浓度小于等于其溶解度，对稳定性要求超高的药物组合物更是如此。如果能制备稳定的高浓度制剂，不仅能节省包装、存储成本，更能鼓励打破传统观念，满足某些超高浓度应用尝尽的迫切需求，如瘤内注射给药，甚至是开发出某些改变产生新现象的应用场景。
发明内容
有鉴于此，本发明旨在提出一种虽为过饱和状态却避免了降温析出的包含精氨酸的稳定溶液、包含或可形成该稳定溶液的药物组合物或药物、以及它们的制备方法和应用，以制备一种高于精氨酸溶解度的高浓度稳定溶液，为临床提供更多新的选择。
一直以来，精氨酸被认为是一种熟悉得不能再简单也就不值得花功夫去研究的化合物，而从中去发现其新的功能并打破传统认知不仅必要，且尤其需要鼓励这方面的尝试。本申请的发明人意外的发现，将一个精氨酸/5-氟尿嘧啶/水溶液分作两份，其中一份经过热处理而另一份未经过热处理，则未经过热处理的水溶液在降温过程中有析出，而经过热处理的水溶液并无析出。更进一步，单独将一个精氨酸水溶液分作两份，进行或不进行包括如上所述的 过热处理的稳定化处理亦可产生不同温敏性构造。简单但有规律可循的稳定化处理居然可使得相同投料量比的溶质和水产生超出其常规溶液预期的抗降温结晶协同作用，进而可提供包含超高浓度精氨酸的稳定溶液，而这些稳定溶液(尽管它们在来源产品上可有不同形式)居然具有不同于现有技术的相变性、稳定性、甚至于安全性、有效性，且能够更好地满足临床上的特殊的用药需求。
为达到上述目的，本发明的技术方案是这样实现的：
术语“浓度”(记为C I或C I(T))是指指定溶质I和水(记为B)在温度T的含水溶液中的重量百分比[(W I/W B) (T)％]。含水溶液实质上是一种包含溶质和水的组合物的均相混合状态，所述组合物中溶质I和水的量比，例如粉针剂说明书中的溶质I干粉和溶解用水的量比，若为重量百分比的形式，则其也是该溶质I在其水溶液中的浓度。
术语“水溶解度”(简称为溶解度)(记为C I0或C I(T)0)通过其特征浓度反映溶质I在温度T的水溶解性，指常规条件下溶质I和水的组合物在温度T形成的水溶液中的最大浓度，或溶质I和水的组合物在温度T形成的均相混合状态中的溶质I/水最大重量百分比。其测定方法可以是：在固定温度T下缓慢搅拌下将溶质I慢慢加入并使其溶入固定重量(例如100g)的水中，直到其显示不再溶入(例如出现不满足注射溶液的不溶性颗粒或不再澄清)，根据完全溶入的溶质重和水重计算出的百分量比，即100克水里可以溶解的溶质I的最大重量，便是溶质I在固定温度T下的“溶解度”。
术语“温敏性溶质”是指水溶性(溶解度)可随温度(T)变化、尤其是随温度下降而下降的溶质，例如精氨酸。温敏性溶质I的溶解度于是可以表示为:C I(T)0＝f I(T)0≠常数。通常认为，温敏性溶质的水溶液也是温敏的、或不稳性的，其中温敏性溶质I的浓度自然也是一个以温度为变量的函数:C I(T)＝f I(T)≠常数，f I(T)是反映组合物特征的浓度-温度函数。在本申请中，浓度C I(T)的下标中的 (T)、或函数f I(T)中的(T)均意味着以温度T为变量的函数。
如以X轴显示上述温度(T)，Y轴显示上述浓度(C I(T))，将上述溶解度测定方法测出的若干坐标点(T，C I(T)0)合理地连接，便构成溶解度曲线C I(T)0＝f I(T)0。百年以来，溶解度曲线一直是研究温敏性溶质I的水溶液-温度关系(C I(T)＝f I(T))的基本方法。
基于溶解度曲线，水溶液(或溶质与水的均相组合物)可以通过其坐标系内的一个坐标点(T，C I(T)＝f I(T))来表示。在固定温度T，依据溶质I的实际浓度(C I实际)与其同温度水溶性(C I0)的关系，水溶液可以区分为:(1)饱和溶液，其C I实际/C I0＝1，对应于溶解度曲线上的点(T，C I饱和 (T)＝C I(T)0)；(2)不饱和溶液，其C I实际/C I0＜1，对应于溶解度曲线下方的点(T，C I不饱和(T)＜C I(T)0)；(3)过饱和溶液，其C I实际/C I0＞1，对应于溶解度曲线上方某个点(T，C I过饱和(T)＞C I(T)0)。术语“饱 和浓度”、“不饱和浓度”、“过饱和浓度”分别为温度T时所述饱和溶液、不饱和溶液、过饱和溶液中的溶质I/水百分量比，分别记为C I饱和(T)、C I不饱和(T)、C I过饱和(T)。饱和溶液和不饱和溶液均为热力学稳定和动力学稳定的系统，而过饱和溶液是热力学不稳定的系统，且三者之间是可以互相转化的。
按照溶解度曲线的指导，可以通过升温增溶，例如将T 1的饱和溶液1(T 1，C 1＝C 1(T1)0)中加入拟增溶量的溶质，再将温度从T 1升至刚好可以溶解完该溶质的T 2，可发生以下反应:
溶液1+溶质→溶液2。
该反应使饱和溶液1变化为饱和溶液2(T 2，C 2＝C I(T2)0)。
按照溶解度曲线的指导，通过将溶液2从T 2缓慢回降至T 1时可发生以下反应:
溶液2→溶液3。
该反应使T 2的饱和溶液2变化为T 1的过饱和溶液3(T 3，C 3＝C 2＞C (T1)0)。通常认为，过饱和溶液在该温度时缺乏结晶核，导致溶质粒子很难发生有序排列形成晶体析出。而当结晶核的生成条件存立后，例如适当降温(ΔT℃)，过饱和溶液很容易析出晶体转化为热力学和动力学都稳定的饱和溶液。
按照溶解度曲线的指导，通过将溶液3从T 1缓慢降至一个足够低的T 4(T 1-T 4＝ΔT)时可发生以下反应:
溶液3→溶液4+结晶。
该反应使T 1的过饱和溶液3变化为溶质析出后T 4的饱和溶液4(T 4，C 4＝C (T4)0＜C 3)。
术语“过饱和溶液生成温度”(记为T 过饱和生成)，例如前述T 2，指生成上述过饱和溶液(T 1，C 过饱和(T1))所必需的温度(T 1＜T 2)。术语“过饱和溶液消失温度”(记为T 过饱和消失)，例如前述T 4，指上述过饱和溶液(T 1，C 过饱和(T1))出现晶体析出的温度(T 4＜T 3)。术语“过饱和溶液温度”(记为T 过饱和)，例如前述T 1，指上述过饱和溶液可以存在的温度(T 过饱和消失＜T 过饱和＜T 过饱和生成)。
术语“水溶液”指水和溶质构成的均相组合物。““常规水溶液”指可以如上所述地按照温敏性溶质的溶解度曲线(C I(T)0＝f I(T)0)的指导生成的水溶液，例如以上溶液1、溶液2、溶液3、溶液4。术语“常规过饱和溶液”是指常规水溶液中的过饱和溶液，例如以上溶液3。“常规组合物”指储存或给药形式为常规水溶液的组合物,例如以上用以形成溶液1的溶质和水、或以上用以形成溶液2的溶液1和溶质。本领域专业人士的一致认识是，包含温敏性溶质的水溶液是温敏性的常规水溶液，包含温敏性溶质和水的组合物是储存或给药形式为常规水溶液 的常规组合物。术语“超高浓度溶液”是指浓度超过常规水溶液、优选为常规过饱和溶液所允许的最高浓度的溶液。
包含水及其溶质的药物组合物可以通过它们的混合物的异同来进行比较研究。例如对化学组成相同的组合物I、II进行以下比较研究。1).将相同溶质I和水以相同投料比(例如相当于上述C 1的投料比)在相同温度(例如上述温度T 1)进行混合，二个混合物为相同浓度的二个水溶液(例如均为上述溶液1)，组合物I、II是两个相同的组合物(例如均为上述T 1的常规饱和溶液)。2).分别加入相同量的溶质，并经相同升温增溶处理(例如均为上述从T 1至T 2)，二个混合物为相同浓度的二个水溶液(例如均为上述溶液2)，组合物I、II仍是两个相同的组合物(例如均为上述T 2的常规饱和溶液)。3).将其中一个溶液(II)进行、而另一个溶液(I)不进行稳定化处理(例如红外加热处理)，再将它们的温度适当下降(例如均为上述T 2至T 1)，二个水溶液均未析出结晶时，未经处理的溶液(I)为T 1的常规过饱和溶液(例如上述溶液3)，而经处理的溶液(I)也为过饱和溶液，不同处理的组合物I、II在外观上尚看不出差别,但它们开始有不同历史。4).将它们的温度足够下降时(例如均为上述T 1至T 4)，如果一个溶液(I)出现析出，而另一个溶液(II)未出现析出，则组合物I、II的不同历史使得它们既使在外观上也出现明显差异(例如I为上述T 4的饱和溶液和结晶，II为过饱和溶液)。尽管它们的差异在该条件下才显示出来，组合物I、II经不同处理后已经成为两个具有足够不同历史、从而有不同浓度-温度函数(f I(T)≠f II(T))、会在有意义的温度(例如上述T 4)下显示不同稳定性的不同组合物。
本发明提供了一种稳定溶液，所述稳定溶液包含述药物和水，所述药物包括精氨酸，所述稳定溶液中精氨酸在温度T时处于溶解状态且与水的百分量比为C 1，所述精氨酸在温度T时的溶解度为C 2，其中T为所述稳定溶液储存或给药环境温度，C 1＞C 2。所述稳定溶液储存或给药环境温度也被称作目标场景温度,目标场景例如制备、储存、移动、给药、或/和体内场景。优选的，所述精氨酸与水的百分量比C 1不随温度T的下降而变化，例如由所述组合物形成的所述超高浓度溶液，例如在所述贮存或/和给药的所需时间内不随温度T变化而出现溶质析出。优选地,所述较低温度选自0-37℃。本发明的在温度T(例如上述T 4至T 2)的稳定溶液是一种过饱和溶液，却非常规过饱和溶液。
术语“溶质”(记为A)是指用于在水中溶解的非气体物质。
术语“稳定溶液”是指在可能经历的温度T(尤其是较低温度T)范围内溶质不随溶液温度的改变而析出，且能在所需时间内稳定存在的过饱和溶液(例如过饱和注射液)。在本发明的稳定溶液中，所述精氨酸的实际浓度大于温度T的水溶解度。按照上述分类，本发明所述稳 定溶液是精氨酸的一种过饱和溶液，也是本发明的包含水和精氨酸的组合物的储存或给药形式。
实际上，对精氨酸的过饱和溶液的研究不见报道，至少并不深入。通常认为，精氨酸是温敏性溶质(C A(T)0＝f A(T)0(T)≠常数)，包含精氨酸的水溶液自然是常规水溶液(C A(T)＝f A(T)≠常数)，包含精氨酸的过饱和溶液自然是常规过饱和溶液(C A(T)0＜C A(T))。包含精氨酸的水溶液、更别说过饱和溶液普遍地存在低温不稳定性(析出)，这也正是本发明所要解决的问题之一。而在本发明的稳定溶液中，精氨酸的实际浓度不随温度(T)下降而下降，即:C I实际(T)＝f I(T)＝常数。本发明的稳定溶液(T，C A(T)＝常数)的非温敏性完全超出常规过饱和溶液(T，C A(T)≠常数)的温敏性预期。
在本发明中，除非另有说法，关注的温度变化在最有意义的范围内:0℃≦T≦37℃，其基本上包括了在制备(例如过滤、分装、冻干中的冷冻步骤)、贮存、使用中的常用温度。
作为本发明的一个示例，所述精氨酸可在0℃保持的实际浓度为其同温度水溶解度的4倍以上。作为本发明的一个示例，所述精氨酸可在0℃保持的实际浓度为其20℃水溶解度的2倍以上。优选的，所述精氨酸(A)和水的百分量比(C A(T))为大于其同温度常规过饱和浓度。
优选的，所述精氨酸与水在温度T为0-37℃时的百分量比C 1为30-300％、40-300％、50-300％、60-300％、或70-300％。优选的，所述所述精氨酸与水在温度T为0℃时的百分量比C 1为30-300％、40-300％、50-300％、或60-300％。
本发明的稳定溶液包括A/水型和A/B/水型，前者至少包含精氨酸和水，后者至少包含精氨酸(A)、其它活性成分(记为B)和水。稳定溶液(A/B/水)可被视为包含其它活性成分(B)和精氨酸过饱和水溶液(A/水)，其它活性成分(B)在所述精氨酸过饱和水溶液(A/水)中可以显示出与其在水中完全不同的水溶性、尤其是温敏结晶性；也可被视为包含精氨酸过饱和水溶液(A/水)和其它活性成分过饱和水溶液(B/水)，其它活性成分过饱和水溶液在所述精氨酸过饱和水溶液中可以显示出与其在水中完全不同的过饱和性质、尤其是温敏结晶性。
所述稳定溶液可以是任何适用于给药的剂型，如注射剂(优选为局部注射剂)、外用液剂等。术语“注射剂”是指含活性成分和液体载体并供体内给药的无菌制剂。所述注射剂按给药方式分为局部注射剂、静脉注射剂等，静脉注射剂只有在给定局部给药浓度后方可作为局部注射剂使用。注射剂按商品形式分为液体注射剂、注射用粉针剂等。注射用粉针剂包含无菌干粉和溶媒，无菌干粉中包含部分或全部活性成分，溶媒中包含全部液体载体。注射剂中所述活性成分的浓度均为其与全部所述液体载体的混合物中的活性成分浓度，通常是局部给药器械(注射器、穿刺器、注入导管等)终点(例如针孔、导管出口等)的液体药物中的活性成分浓度。对注射用粉针剂而言，所述活性成分的浓度即为无菌干粉和指定溶媒的混合 物(例如复溶液，或所述药物学可接受的液体载体)中的活性成分浓度。术语“外用液剂”是指含活性成分和液体载体并供体表[例如皮肤、粘膜(例如眼粘膜、鼻粘膜等)或/和腔道(例如口腔、直肠、阴道、尿道、鼻腔、耳道等)]给药的液体药物，其包括例如洗剂、搽剂、滴剂、含漱剂、涂剂等。局部给药时，所述液体药物往往来自洗液瓶、滴液瓶、滴液管、潄洗液瓶、棉签等局部给药器械。所述外用液剂中所述活性成分的浓度即该液体药物中的活性成分浓度。
优选的，所述药物还包括溶质B，所述溶质B不包括强酸。优选的，所述溶质B在温度T时处于溶解状态且与水的百分量比为C 3、所述溶质B在温度T的溶解度为C 4，其中C 3＞C 4，所述溶质B与水的百分量比C 3不随温度T的升降而变化。
其它可溶性药物的加入增加了与水作用的竞争，理应降低所述超高浓度精氨酸与水的量比。超高浓度精氨酸和超高浓度其它可溶性药物的共用,更是理应增加溶液的温敏性或不稳定性。出乎意料的是，所述精氨酸与水的的实际百分量比仍然不随温度T下降而下降。超高浓度精氨酸与其它可溶性药物(即使其终浓度大于其溶解度)的共用于是具有超加和作用预期、即抗温敏协同作用的技术效果。
优选的，所述溶质B选自局部作用药物、细胞毒药物、生物活性药物中的至少一种。优选的，所述溶质B与水的百分量比C 3为0.25％-400％、10-300％或者30％-250％或者50％-250％。
术语“局部作用药物”区别于常规药物，是指全身性给药时并不产生有效化疗作用、而局部给药时可提供单用有效的或共用协同的局部作用、尤其是局部化学作用的药物，如其它氨基酸类营养素、常规无效芳香化合物、碱化剂、聚多糖；而常规药物则是指全身性给药时可产生有效化疗作用的药物,如细胞毒药物。术语“局部作用”区别于常规作用，是指局部给药后药物在病变组织间隙渗透所及的局部(如肿瘤细胞微环境)范围内产生的药理作用(如致组织坏死或致肿瘤细胞微环境破坏的作用)，而常规作用则是指常规用药后药物经消化道或血管给药后以血药形式递送至靶区所产生的全身性药理作用(如肿瘤细胞毒作用)。术语“局部化学作用”是指包括化学作用的局部作用。
优选的，所述局部作用药物包括选自易溶性药物，即水溶解度为≧5％的药物。优选的，所述易溶性药物在其中的实际浓度(C I(T))为≧2％、或2-150％。
优选的，所述溶质B包括选自其它氨基酸类营养素，所述溶质(A+B)与水的百分量比C 1为40-300％、50-300％、或60-300％。
术语“营养素”是指具有营养保健效应的物质，优选为选自具有营养保健效应的化合物，更优选为选自中国、美国或欧洲官方药典或指南所载的具有营养保健作用的化合物。术语“营养保健效应”是指由以下之一种或多种生物作用产生的体内效应：提供能量、参与生物活性 物质(例如蛋白质)的合成、参与部分新陈代谢、维持动物肠道微生态平衡、以及参与有利于机体健康的其它生理调节(例如调节蛋白质合成、调节免疫反应)。
术语“氨基酸类营养素”是指具有营养保健效应的氨基酸类化合物，优选为选自具有营养保健效应的氨基酸、氨基酸聚合物及氨基酸衍生物，更优选为选自中国、美国或欧洲官方药典或指南所载的氨基酸类营养药和具有营养保健作用的氨基酸类辅料。所述其它氨基酸类营养素的氨基酸、氨基酸聚合物及氨基酸衍生物优选为选自以下组中的氨基酸、或者包含以下组中的氨基酸的氨基酸聚合物、或者以下组中的氨基酸或者包含以下组中的氨基酸的氨基酸聚合物的氨基酸衍生物：蛋白氨基酸和非蛋白氨基酸。
具体而言，所述蛋白氨基酸包括选自以下组中的氨基酸：非极性氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸)，极性中性氨基酸(例如色氨酸、酪氨酸、丝氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸)，碱性氨基酸(例如赖氨酸、组氨酸、精氨酸)，酸性氨基酸(例如天冬氨酸、谷氨酸)。以上除甘氨酸以外均为L型α-氨基酸。所述非蛋白氨基酸可包括以下氨基酸：β-丙氨酸、牛磺酸、γ-氨基丁酸(GABA)、茶多酚(茶氨酸)、南瓜子氨基酸(3-氨基-3-羧基吡烷酸)、谷氨酰胺、瓜氨酸、鸟氨酸等。
所述氨基酸聚合物可以为选自包含如上所述之氨基酸的寡肽和多肽。
术语“寡肽”是指包含2-10个相同或不同的以肽键相连的氨基酸的氨基酸聚合物；而术语“多肽”是指包含11-100个相同或不同的以肽键相连的氨基酸聚合物。对于组成所述寡肽或多肽的氨基酸，可以全部是上述一种或多种氨基酸，也可以额外包含其他氨基酸。作为本发明的一个示例，所述寡肽可以为选自以下之一种或多种：甘氨酰-L-酪氨酸、甘氨酰丙氨酸、双甘氨肽、赖氨酸-甘氨酸二肽、丙谷二肽、肌肽(β-丙氨酸组氨酸共聚物)、谷胱甘肽、胶原蛋白寡肽、酪蛋白水解肽、大豆寡肽、寡聚精氨酸、寡聚甘氨酸、寡聚赖氨酸。作为本发明的一个示例，所述多肽可以为选自以下之一种或多种：聚天冬氨酸、聚谷氨酸、聚赖氨酸。
作为本发明的一个示例，所述其它氨基酸类营养素包括甘氨酸，且所述甘氨酸与水的百分量比为＞30％、或33-100％。
作为本发明的一个示例，所述其它氨基酸类营养素包括赖氨酸，且所述赖氨酸与水的百分量比为＞30％、或33-300％。
作为本发明的一个示例，所述常规无效芳香化合物包括亚甲蓝，且所述亚甲蓝与水的百分量比为＞1％、或1-20％。
作为本发明的一个示例，所述碱化剂包括碳酸钠，且所述碳酸钠与水的百分量比为＞3％、 或3-50％。
作为本发明的一个示例，所述碱化剂包括氨丁三醇，且所述氨丁三醇与水的百分量比为＞3％、或3-50％。
作为本发明的一个示例，所述聚多糖包括右旋糖酐，且所述右旋糖酐与水的百分量比为＞0.3％、或0.3-30％。
优选的，所述细胞毒药物选自以下组中至少一种：破坏DNA结构和功能的药物、嵌入DNA中干扰转录RNA的药物、干扰DNA合成的药物、影响蛋白质合成的药物。
作为本发明的一个示例，所述细胞毒药物选自包括铂类化疗药物，例如顺铂、卡铂、奈达铂、奥沙利铂、洛珀。
作为本发明的一个示例，所述细胞毒药物选自包括5-氟尿嘧啶，且所述5-氟尿嘧啶与水的百分量比为＞3.5％、或4-40％。
作为本发明的一个示例，所述生物活性药物包括益生菌或源自益生菌的生物活性物质(例如水溶性聚多糖)，且所述益生菌或源自益生菌的生物活性物质与水的百分量比为＞1％、或1-30％。
本发明还提供了一种制备稳定溶液的方法，包括按所述百分量比对所述药物和水进行混合以及对混合物进行超溶解所需的稳定化处理。所述混合包括出现在生产文件或/和使用说明书中的混合。
所述稳定化处理包括药物制备中的稳定化处理。
所述稳定化处理包括药物储存中的稳定化处理。
所述稳定化处理包括药物运输中的稳定化处理。
所述稳定化处理包括药物使用中的稳定化处理。
术语“稳定化处理”是指以制备稳定溶液为目的，通过使用制备常规过饱和溶液通常并不需要的条件进行的处理，例如过热处理、辐射处理、超声处理。术语“辐射处理”是指使用制备常规过饱和溶液通常并不需要的幅射能进行的处理，其包括红外处理和微波处理。优选的，所述微波处理的频率为890～940MHz、中心波长0.330m，或者频率为2400～2500MHz、中心波长为0.122m。所述红外处理的波长范围0.76-5μm，峰值波长4μm。所述辐射处理的处理时间为≦10分钟，或0.25-10分钟或0.5-5分钟。
所述稳定化处理包括超出单纯溶解所必需的温度或/和时间的115％以上的过热处理。
所述必需温度是指按照常规方法(例如依照溶解度曲线)获得与所述活性溶质与水的百分量比相同的水溶解度所必需的温度。所述必需时间是指按照常规方法在所述必需温度获得与所述活性溶质与水的百分量比相同的水溶解度所必需的时间。
术语“过热处理”是指以高于为获得与所述溶质之一或全部的浓度相同的水溶解度所需增溶温度的温度进行的非幅射热处理(通常意义的加温)。优选的，所述过热处理的处理温度在95℃和125℃之间，持续时间为3min-110min。优选的，所述过热处理的处理温度为95℃-120℃或95℃-115℃或95℃-110℃或95℃-105℃。
本发明还提供了一种药物组合物，所述组合物包含药物和水，所述药物包括精氨酸，其特征在于，储存或给药形式包括上述稳定溶液。
所述药物组合物包含上述稳定溶液。
所述药物组合物包含上述稳定溶液中的所述药物以及可提供所述量比的水。
所述药物组合物包含上述稳定溶液中的所述药物以及载有上述方法的文件。
所述药物组合物通过上述制备稳定溶液的方法制备。
优选的，包括从冻干产品制备的所述药物组合物。
本发明还提供了一种上述药物组合物的制备方法，包括在装入最终的递送系统之前或之后，通过上述方法制备所述稳定溶液。
优选的，包括从冻干产品制备的所述药物组合物，随后进行上述方法。优选的，在装入最终的递送系统之前或之后，进行稳定化处理工序。
本发明还提供了一种药物，通过上述稳定溶液制备而来，或通过上述方法制备。本发明的药物组合物包括例如可提供注射液的注射剂型:粉针剂(水+A干粉)、粉针剂(A干粉+B的水溶液)、粉针剂(B干粉+A的水溶液)、等等。优选的，所述药物组合物通过局部给药，如瘤内注射。
局部给药所要求的药物组成(局部活性成分、组成比及组分浓度)可通过介入手段给药于局部病变所在的组织、并在该组织内产生所需要的疗效。例如，当病变为肿瘤时，局部组织为肿瘤细胞所在的瘤体；当病变为非瘤肿大时，局部组织为诸如肿块的异常，例如增生、囊肿、结节等病变肿块；当病变为局部炎症时，局部组织为发炎区，例如发炎肿大体；当病变为分泌异常时，局部组织为异常源或其所在的分泌腺体。又例如，当疾病为胰岛素分泌异常时，异常源在胰岛，局部组织则为胰岛或胰岛所在的胰腺；当疾病为皮肤病时，局部组织为病变皮肤或病变皮肤的附属器官。
所述药物的制备方法，包括通过上述方法制备稳定溶液，随后将稳定溶液冷冻干燥。所述药物的制备方法，包括药物的制备方法而得到的冻干产品制备的药物，随后进行所述稳定 药物制备的方法。
稳定溶液、存放或/和给药时的组合方式为稳定溶液的药物组合物、或稳定溶液的药物制品在制备预防或/和治疗疾病的药物中的应用。优选的，所述稳定溶液包括精氨酸，所述精氨酸作为可提供抗温敏作用的活性成分。优选的，所述疾病为局部病变疾病。优选的，所述精氨酸的浓度不仅能满足其抗温敏作用所需、且能满足其以下其它作用之至少一种所需的浓度：全身性活性、局部活性、稳定作用、增溶作用、pH调节作用、缓冲作用。
术语“抗温敏作用”是指温敏性溶质被人为赋予的与其常规性质(温敏性)相反的作用,在该作用下其过饱和状态的不稳定溶液被转变为可耐受降温(例如降至室温甚至于0℃)的稳定溶液。
所述应用例如包括在产品制备中的应用和在有用处理中的应用。所述医学应用例如包括在制备医学产品中的应用和在医学处理中的应用。所述医学产品例如包括药物。所述医学处理例如包括疾病处理。所述疾病处理例如包括治疗疾病。
术语“应用”是指任何有效用的用途、方案和方法。术语“医学应用”是指任何与医疗、医药有关的应用。术语“疾病处理”是指任何针对疾病的有益行为，包括例如疾病的检验、预防、治疗、等等。
优选的，所述精氨酸作为可提供抗温敏作用的活性成分在制备预防或/和治疗疾病的药物中的应用。
术语“药物”是指任何可用于疾病处理的、可在安全限制条件下实现其有效活性的物质，包括例如医学器械中的药物活性成分、检验试剂、预防制剂、治疗药物、等等。
所述组合物的应用还可以在某些情况下凸现其组分所提供的特征的应用，例如:优选的，所述应用包括精氨酸在所述组合物中所提供的抗温敏作用的应用、包括医学应用。优选的，所述应用包括所述溶质在所述组合物中所提供的超常规过饱和浓度的应用、包括医学应用。所述抗温敏作用的医学应用包括在疾病处理方法中的抗温敏作用应用和在药物制备中的抗温敏作用应用。
所述溶质超常规过饱和浓度的医学应用包括在疾病处理方法中的超常规过饱和浓度应用和在药物制备中的超常规过饱和浓度应用。所述在疾病处理方法中的所述溶质超常规过饱和浓度应用包括在任何适用于疾病处理方法中的应用，例如在疾病的检验、预防、治疗、等等中的应用。所述溶质超常规过饱和浓度在疾病治疗中的应用包括任何适用于疾病治疗的应用，例如超常规过饱和浓度皮下给药、局部病变疾病的超常规过饱和浓度介入疗法、超常规过饱和浓度的结晶缓释、超常规过饱和浓度的稀释、等等。
术语“局部病变疾病”是指具有局部病变症状的疾病，而术语“局部病变”是指动物(优选人类)身体局部部位原生或继生的结构、形态或功能上的不正常，其例如可以是包括以下一种或多种：瘤体、非瘤肿大、局部炎症、分泌腺分泌功能异常等。所述局部部位可以是本领域技术人员已知的任意合适者，例如可以是包括以下一种或多种器官中的局部：分泌系统所在的分泌器官、血液循环系统所在的心血管器官、皮肤等。优选的，所述局部病变疾病包括肿瘤、非瘤肿大、局部炎症、分泌腺功能异常和皮肤病。
在本发明的范围内，术语“肿瘤”是指由于细胞或变异的细胞异常增殖形成的肿块，其包括实体肿瘤。术语“实体肿瘤”是指具有瘤体的肿瘤，其可以是由于任何病理(恶性和非恶性)和处于任何阶段的肿瘤，包括例如按照肿瘤细胞类型进行分类的以下组：上皮细胞肿瘤、肉瘤、淋巴瘤、生殖细胞肿瘤、胚细胞瘤；以及包括按照肿瘤细胞集中区所在的器官或组织来命名的肿瘤，包括例如按照以下器官或组织来命名的肿瘤：皮肤、骨、肌肉、乳腺、肾、肝、肺、胆囊、胰腺、脑、食道、膀肌、大肠、小肠、脾、胃、前列腺、睾丸、卵巢或子宫。
所述肿瘤包括恶性肿瘤和非恶性肿瘤，所述恶性肿瘤包括乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、前列腺癌、肝癌、肺癌、肠癌、口腔癌、食道癌、胃癌、喉癌、睾丸癌、阴道癌、子宫癌、卵巢癌、恶性淋巴瘤、恶性脑瘤，所述非恶性肿瘤包括乳腺瘤、胰腺瘤、甲状腺瘤、前列腺瘤、肝瘤、肺瘤、肠瘤、口腔瘤、食道瘤、胃瘤、鼻咽瘤、喉瘤、睾丸瘤、阴道瘤、子宫瘤、输卵管瘤、卵巢瘤、淋巴瘤、脑瘤。
作为本发明的一个示例，所述局部病变疾病包括非瘤肿大。术语“非瘤肿大”是指肿瘤以外的肿大，包括例如增生(例如乳腺、胰腺、甲状腺、甲状旁腺、前列腺等的增生)、囊肿(例如乳腺、甲状腺、甲状旁腺等的囊肿)、结节(例如乳腺、甲状腺、甲状旁腺等的结节)、异常静脉团(例如痔疮等)、局部炎症发肿、微生物感染发肿等。所述痔疮包括内痔、外痔、混合痔。
作为本发明的一个示例，所述局部病变疾病包括局部炎症、尤其是难治性炎症。在本发明的范围内，术语“局部炎症”是指局部部位的非瘤性发炎，包括例如变质性炎(alterative inflammation)、渗出性炎(exudative inflammation)和增生性炎，其可以是本领域技术人员已知的任意合适者，例如可以是包括以下一种或多种：关节炎、乳腺炎、胰腺炎、甲状腺炎、前列腺炎、肝炎、肺炎、肠炎、口腔炎、咽炎、牙周炎、食道炎、胃炎、胃溃疡、鼻炎、鼻窦炎、喉炎、气管炎、支气管炎、阴道炎、子宫炎、输卵管炎、卵巢炎等。
作为本发明的一个示例，所述局部病变疾病包括皮肤病、尤其是难治性皮肤病。在本发明的范围内，术语“皮肤病”是指原生或继生在皮肤或皮肤附属器官的病变，其可以是本领 域技术人员已知的任意合适者，例如可以是包括以下一种或多种：皮肤癌、皮肤非恶性肿瘤、病毒性皮肤病(例如疱疹、疣、风疹、手足口病)、细菌性皮肤病(例如脓疱病、疖、麻风)、真菌性皮肤病(例如各种癣)、性传播疾病(例如梅毒、淋病及尖锐湿疣)、过敏性与自身免疫性皮肤病(例如接触性皮炎、湿疹、荨麻疹)、物理性皮肤病(例如日光性皮肤病、冻疮、鸡眼、手足皲裂、压疮)、结缔组织疾病(例如红斑狼疮)、色素障碍性皮肤病(例如雀斑、色素痣、各种斑)、皮肤附属器疾病(例如痤疮、酒渣鼻、脂溢性皮炎、斑秃、秃发、多汗症及臭汗症)。
作为本发明的一个示例，所述局部病变疾病包括分泌腺分泌功能异。在本发明的范围内，术语“分泌腺”是指由腺细胞或腺细胞群组成的、执行分泌功能(secretion)的结构，其包括外分泌腺和内分泌腺。分泌腺分泌功能异常包括分泌腺功能亢进症(例如甲状腺功能亢进症)和分泌腺功能减退症(例如甲状腺功能減退症、胰岛功能減退症(糖尿病之一种))。
作为本发明的一个示例，所述局部病变疾病包括心血管疾病。介入治疗己经成为心血管疾病的一种重要治疗手段。所述心血管疾病包括例如血管瘤、肥厚型梗阻性心肌病、心房颤动、心律失常、动脉栓塞等。
本发明还提供了一种预防或/和治疗疾病的方法，其包含以下步骤，将所述稳定溶液给予有需要的个体。
所述治疗方法包括任何适用于疾病治疗的方法，包括例如常规化疗、免疫治疗、局部病变疾病的介入疗法、多种方法的综合治疗、等等。
一种药物试剂盒，包括包含以下独立包装制剂的容器：上述稳定溶液的制剂、分别含有上述药物组合物的组成成分的制剂、或含有上述药物的制剂。
优选的，所述药物制品还包括说明书或标签，所述说明书中包含反映以下情况的内容:上述稳定溶液、或者由上述药物组合物或上述药物通过上述方法制备的所述稳定溶液可在室温或/和可在低于室温的温度下使用或/和储放一小时以上。
在本发明的稳定溶液中，所述精氨酸可在较低温度保持的实际浓度不仅大于其同温度溶解度，还可进一步大于其同温度过饱和浓度。这就意味着，精氨酸在本发明的组合物中的浓度(包括较低温度所允许的浓度)为大于其在常规溶液中较低温度所允许的最高浓度(过饱和浓度)。
作为本发明的一个示例，所述精氨酸可在0℃保持的实际浓度大于其同温度过饱和浓度。这就意味着，精氨酸在本发明的组合物中的浓度(包括0℃所允许的浓度)可以为大于其在常规溶液中0℃所允许的最高浓度(过饱和浓度)。
作为本发明的一个示例，所述精氨酸可在0℃保持的实际浓度大于其25℃过饱和浓度。 这就意味着，精氨酸在本发明的稳定溶液中的浓度(包括0℃所允许的浓度)可以为大于其在常规溶液中25℃所允许的最高浓度(过饱和浓度)。优选的，所述精氨酸(A)和水的百分量比(C A(T))在0℃可为大于25％、25-300％、或大于30％、30-300％。
相对于现有技术，本发明所述的稳定溶液具有以下优势：与包含精氨酸的常规水溶液相比，本申请所述的稳定溶液中精氨酸的超常规浓度不仅更加可行、而且可以提供常规水溶液中精氨酸的常规浓度所不能提供的效用：超常规浓度局部给药、超常规浓度的超常规局部作用、超常规浓度的高稀释自由度等等；本发明所述稳定溶液具有良好的化学稳定性。
具体实施方式
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。本申请所使用的氨基酸(除甘氨酸以外均为L型α-氨基酸)等均可从商业途径得到。下面将结合实施例来详细说明本发明。
以下实施例中所用的实验动物均由专业实验动物公司购入的SPF(Specific Pathogen Free，无特定病原体)级动物，除非另有说明，其中小鼠(BALB/c)和裸小鼠(BALB/c Nude)均为6-8周龄健康雌性、体重17.5-20.5g。
皮下移植细胞生成局部病变(如瘤体)的动物建模为现有技术，当瘤体长至所需体积(如小鼠荷瘤为50-500mm 3)为建模成功，建模后将动物分成若干组，每组6只，定期观察测定一般状态、体重、摄食量、局部病变体积、生存时间等。
局部病变体积(V)、局部病变抑制率(r)分别按照下述公式计算：
局部病变体积V＝0.5×a×b 2，其中a、b分别代表局部病变的长、宽；
局部病变抑制率(r)＝[(CV-TV)/CV]，其中TV和CV分别为研究组和阴性对照组的局部病变体积。
在下述实施例中，药物i的药效记为Ei，可以用ri表示。
在以下实施例中，实验结果(例如瘤重、瘤体积、病变组织体积)采用均数±标准差(x±s)表示，两个实验动物组与组均数之间的差别采用统计学软件SPSS13.0或SPSS19.0进行显著性检验，检验选用统计量t来进行，检验水准α＝0.05，P＜0.05表示差异有统计学意义，P＞0.05则无统计学意义。
药物联合使用产生的药理作用(或药效)具有高度不确定性，业内往往依据以下实际药效/预期药效比q判断进行：q＝q 1/q 2，其中q 1为实际共用药效，q 2为药物A和B的实际单用药效(E A、E B)得出的预期相加药效。若q＝1时说明实际共用药效符合预期，即显示为药效相加作用；当q＜1时说明实际共用药效不及预期，即显示为药效拮抗作用；当q＞1时说明实际共用药效超预期，即显示为药效协同作用。
其中q 2的计算方法很多，如Burgi法(Burgi Y.Pharmacology；Drug actions and reactions.Cancer res.1978，38(2)，284-285)；金正均对Burgi法进行了改进(金正均，等概率和曲线和“Q50”，上海第二医学院学报；1981，1，75-86)。药物作用的单纯相加被认为是互相独立的作用的相加，于是q 2＝E A+E B-E A×E B为根据药物A和B的单药实际药效(E A和E B)通过单纯相加的理论预期药效。
根据金正均法的实际药效/预期药效比q进行的药物A和B联合给药的共用药效作用判断如下：当药物A和B的组合物的药效无意义(r≦15％)时，则组合物未显示出共用作用，亦或者说视为可忽略不计的共同作用。当组合物的药效有意义(r＞15％)时，若实际药效/预期药效比q＝1.00则说明组合物的共用药效为单独药效的相加，符合理论单纯相加预期；若实际药效/预期药效比q＞1.00则说明组合物的共用药效存在明显协同作用，即实际作用超理论单纯相加预期；若实际药效/预期药效比q＜1.00则说明该组合物的共用药效存在明显拮抗作用，即实际作用不及理论单纯相加预期。
药物联合使用产生的毒理作用(或副作用)具有高度不确定性，可以按照上述药理作用的方法进行判断，即依据以下实际副作用/预期副作用比Q判断进行：Q＝Q 1/Q 2，其中Q 1为实际共用副作用，Q 2为药物A和B的实际单用副作用得出的预期相加副作用。通常认为，药物副作用的单纯相加是互相独立的副作用的相加。若Q＝1时说明实际共用副作用符合预期，即显示为副作用相加作用；当Q＜1时说明实际共用副作用不及预期，即显示为副作用之间产生了拮抗作用，或产生了安全性协同；当Q＞1时说明实际共用副作用超预期，即显示为副作用之间产生了协同作用，或产生了安全性拮抗。例如，预期相加副作用至少为药物A和B的实际单用副作用中最大者(Q 2＞Q 2max(A，B))，于是当药物A和B的实际共用副作用小于该最大者(Q 1＜Q 2max(A，B))时，Q＝Q 1/Q 2为＜1，说明A和B的共用产生了安全性协同。
溶质联合使用在溶液中产生的成核结晶具有高度不确定性，也可以按照上述药理作用的方法进行判断，即依据在有意义的温度T的以下实际成核结晶效果/预期成核结晶效果比s判断进行：s＝s 1/s 2，其中s 1为实际共用成核结晶效果，s 2为药物A和B的根据实际单用成核结晶效果得出的预期相加成核结晶效果。药物成核结晶作用的单纯相加可被视作互相独立的成核结晶作用的相加，即s 2＝(s 2A+s 2B)-(s 2A×s 2B)。例如，在相同溶质/水量比时，单用成核结晶(s 2A＝1)的药物A和不成核结晶(s 2B＝0)的药物B的相加成核结晶效果仍为成核结晶，即s 2＝(s 2A+s 2B)-(s 2A×s 2B)＝1。若s＝1时说明实际共用成核结晶效果符合预期，即显示为成核结晶(或溶液不稳定性)的相加作用；当s＜1时说明实际共用成核结晶效果不及预期，即显示为成核结晶(或溶液不稳定性)的拮抗作用、或抗温敏协同作用。
更进一步，相似的方式可用于溶质和水的联合使用的抗成核结晶作用(抗温敏作用)的判 断，即依据有意义的温度T下以下实际抗温敏效果/预期抗温敏效果比t判断进行：t＝C 1(T)/C 2(T)，其中C 1(T)为显示实际抗温敏效果的实际浓度，C 2(T)为显示预期抗温敏效果的浓度，即依据溶解度曲线预期的溶解度(饱和浓度)。若t＝1时说明实际共用抗温敏效果符合预期；当t＞1时说明实际共用抗温敏效果超预期，即显示为溶质和水的抗温敏协同作用。
实施例1 抗温敏协同作用的发现
在15℃按常规方法配制如表1所示溶质的水溶液并测定其溶解度。在如下表所示温度T按常规方法配制如下表所示溶质/水百分量比的溶液[始溶液(T,C (T))]各2瓶(各3ml)。其中一瓶作为对照不进行处理，而另一瓶放入红外烤箱(万利达多功能电烤箱,型号ZL-1202,额定功率650W)中进行超常规的红外处理(设定温度120℃,定时2分钟)，然后将它们同时放入目标场景温度(15℃)环境，存放1小时后肉眼观察有无结晶析出。
表1 不同组别处理的结果
Figure PCTCN2022070058-appb-000001
在上表中,组别1-3的始溶液均为典型的升温增溶模型,各溶质在较高温度水溶液中的浓度均大于其15℃溶解度的150％以上。该浓度溶质容易随降温析出,其为该温度的不稳定溶质,其水溶液似乎理所当然地为不稳定溶液。组别1-3的各始溶液的无处理溶液(常规溶液)均在降温至15℃时有结晶析出,合乎其不稳定溶液预期。组别1-2的各始溶液的超常规处理溶液(超常规溶液)在降温至15℃时也有结晶析出,合乎不稳定溶质的水溶液为不稳定溶液的预期,也加强了现有技术中“不稳定溶液唯有转化为饱和溶液或饱和溶液才能消除其不稳定性”的教导。出人意料地,组别3的始溶液的超常规处理溶液(超常规溶液)在降温至15℃时无结晶析出,保持相同的单相状态和浓度。不稳定溶质的水溶液变成了稳定溶液,超出其温敏性预期。
上述不稳定溶质和稳定溶质的联合使用,最可能接受的共用预期为它们各自稳定性的简单加和作用。组别4-5的各始溶液的无处理溶液均在降温至15℃时析出结晶,与组别1的结果相同,合乎其不稳定溶液预期。然而,组别4-5的各始溶液的超常规处理溶液(超常规溶液)中的溶质共用改变了溶质的单用温敏性,降温至15℃时均无结晶析出,超出其温敏性预期。两 个温敏性溶质的不稳定水溶液居然成为不温敏的稳定溶液,显示出在此条件下溶质联合使用的成核结晶析出拮杭、或抗温敏协同。
在本申请中，术语“超常规处理”区别于为达到目标浓度所必须的升温增溶的常规处理，是指任何超出升温增溶所必须的、且可使得升温增溶的不稳定溶液转化为稳定溶液的稳定化处理。总之，升温增溶的目的是以最小的升温获得所需增溶，例如以上实施例中5氟尿嘧啶升温至80℃以获得超目标场景温度溶解度的浓度；而超常规处理的目的是使得升温增溶的不稳定溶液转化为稳定溶液，往往要求升温增溶通常所不必需的处理，例如以上实施例中红外处理。经超常规处理的溶液称作超常规溶液，未经超常规处理的溶液称作常规溶液。
在另一个实验中，超常规处理还可以过热处理(处理温度为≧95℃、或95-125℃；处理时间为15-60分钟、30-60分钟、45-100分钟、或30-160分钟)，或微波处理(处理温度为＞90℃、95-130℃，处理时间为0.5-5分钟、0.5-3分钟)代替上述红外处理(处理温度为＞90℃、95-130℃,处理时间为0.5-10分钟、0.5-5分钟、或0.5-3分钟)来进行，均可获得类似结果。
根据以上结果，在本发明的包含在目标场景温度(例如10-37℃)下，超常规处理可以使得温敏精氨酸水溶液产生超过常规处理预期的抗温敏作用，使得不稳定溶液转化为稳定溶液。该稳定精氨酸还可以与其它温敏性溶质共用产生新的抗温敏协同作用。
实施例2 精氨酸抗温敏作用的进一步研究
按常规方法在不同温度下配制如表2所示精氨酸水溶液并测定其相应溶解度。每个水溶液[始溶液(T,C (T)0)]分装为4瓶(5ml/瓶)。其中2瓶作为对照不进行任何处理(常规溶液)，而另2瓶超常规处理放入95℃水浴中30分钟。然后将常规溶液和超常规溶液中的各一瓶放入15℃环境，另各一瓶放入0℃环境，存放1小时后观察有无结晶析出。
表2 不同处理后在不同温度下放置稳定性
Figure PCTCN2022070058-appb-000002
精氨酸在15℃和0℃所对应的溶解度分别为15.6％(见以上实施例1)和7.8％。组别1中始溶液(T，C 0(T))在其溶解温度(0℃)的浓度C 1(0℃)等于其0℃溶解度和小于其15℃溶解度，为 该温度(0℃-15℃)的稳定溶液，是否经过超常规处理的溶液显示无差异。
组别2中始溶液(T，C 0(T))在其溶解温度(35℃)的浓度C 1(35℃)高于其0℃溶解度和15℃溶解度，为该温度(0℃-15℃)的不稳定溶液。其常规溶液在该温度(0℃、15℃)析出，合乎不稳定预期；而其超常规溶液却并无析出，过饱和溶液成了稳定溶液，超出不稳定预期。同样地，组别3、4中的常规溶液也都是不稳定溶液，而其中的超常规溶液也都是稳定溶液，均超出不稳定预期。
这些结果说明，当精氨酸浓度＞30％时，其超常规溶液显示出完全不同于其常规溶液的温敏成核结晶性。尽管具体的机理尚待更进一步的研究，就该性质以及提供该性质的相关物质结构(例如热力学组成)而言，化学组成相同的超常规溶液和常规溶液是两种热力学组成(或温敏结构)不同的溶液。在以下实验中，进一步研究了化学组成相同的组合物的不同温敏性质。
在一个实验中，在目标场景温度(0℃)按常规方法配制精氨酸水溶液并测定其溶解度为7.5％。另在15℃按常规方法配制精氨酸和水的百分量比为15.7％的饱和溶液(15℃，15.7％)20ml，将其升温至35℃并加入适量精氨酸配制为该温度不饱和溶液(35℃，20.0％)，然后将其分为两瓶(10ml/瓶)，一瓶仍放于35℃(常规溶液)，另一瓶放入红外烤箱中处理2分钟(超常规溶液)。然后将该常规溶液和超常规溶液分别同时放入15℃环境，均未见结晶析出。显然，常规溶液为该温度的过饱和溶液(15℃，20.0％＞15.7％)，而超常规溶液与常规溶液之间并无外观上的差别。再将这两瓶溶液同时放入0℃环境，则常规溶液中有结晶析出成为非均相组合物(0℃，饱和浓度7.5％+结晶)，而超常规溶液并无结晶析出仍为均相组合物(0℃，20.0％＞7.5％)。
在一个实验中，在目标场景温度(5℃)按常规方法配制精氨酸水溶液并测定其溶解度为9.5％。另在20℃按常规方法配制精氨酸和水的百分量比为19.2％的饱和溶液(20℃，19.2％)20ml，将其升温至35℃并加入适量精氨酸配制为该温度不饱和溶液(35℃，25.0％)，然后将其分为两瓶(10ml/瓶)，一瓶仍放于35℃(常规溶液)，另一瓶放入红外烤箱中处理2分钟(超常规溶液)。然后将该常规溶液和超常规溶液分别同时放入20℃环境，均未见结晶析出。显然，常规溶液为该温度的过饱和溶液(20℃，25.0％＞19.2％)，而超常规溶液与常规溶液之间并无外观上的差别。再将这两瓶溶液同时放入5℃环境，则常规溶液中有结晶析出成为非均相组合物(5℃，饱和浓度9.5％+结晶)，而超常规溶液并无结晶析出仍为均相组合物(5℃，25.0％＞9.5％)。
在一个实验中，在目标场景温度(20℃)按常规方法配制精氨酸水溶液并测定其溶解度为18.7％。另在25℃按常规方法配制精氨酸和水的百分量比为23.8％的饱和溶液(25℃， 23.8％)20ml，将其升温至50℃并加入适量精氨酸配制为该温度饱和溶液(55℃，60.1％)，然后将其分为两瓶(10ml/瓶)，一瓶仍放于55℃(常规溶液)，另一瓶放入红外烤箱中处理2分钟(超常规溶液)。然后将该常规溶液和超常规溶液分别同时放入25℃环境，均未见结晶析出。显然，常规溶液为该温度的过饱和溶液(25℃，60.1％＞24.8％)，而超常规溶液与常规溶液之间并无外观上的差别。再将这两瓶溶液同时放入20℃环境，则常规溶液中有结晶析出成为非均相组合物(20℃，饱和浓度18.7％+结晶)，而超常规溶液并无结晶析出仍为均相组合物(5℃，60.1％＞18.7％)。
综上,精氨酸始溶液经不同处理后形成的常规溶液和超常规溶液有不同的浓度-温度函数(f 常规溶液(T)≠f 超常规溶液(T)),已经成为两个不同溶液。常规溶液是对温度变化敏感的不稳定溶液(f 常规溶液(T)≠常数),而超常规溶液却是对温度变化不敏感的稳定溶液(f 超常规溶液＝常数),或者说是一种稳定的过饱和溶液。
实施例3 抗温敏作用比较研究
在80℃按常规方法配制如下表所示的12种氨基酸水溶液(浓度均为接近其在该温度的溶解度)20ml，每个水溶液分装为4瓶(5ml/瓶)。其中2瓶不作特殊处理(常规溶液)，而另2瓶放入蒸汽煲中蒸汽处理30分钟(超常规处理)。然后将它们的常规溶液和超常规溶液中的各一瓶放入15℃环境，另各一瓶放入0℃环境，存放1小时后观察有无结晶析出。结果示于下表。
表3 不同氨基酸的抗温敏作用研究
Figure PCTCN2022070058-appb-000003
在上表中,组2-12的结果显示,温敏性溶质可以通过升温增溶,制备为超目标场景温度溶解度的高浓度水溶液(初溶液)。该初溶液(不作特殊处理)均随温度下降而析出结晶,作特殊处理后亦是如此,未能将不稳定溶液改变为稳定溶液。组1中的初溶液(不作特殊处理)也随温度下降而析出结晶,但经超常规处理的精氨酸水溶液不析出结晶,不稳定溶液改变为稳定溶液。
实施例4 不稳定溶质间的抗温敏协同作用研究(1)
术语“稳定溶质”是指与水的百分量比(或浓度)未超过其水溶解度、因而其常规水溶液为稳定溶液的溶质。术语“不稳定溶质”是指与水的百分量比(或浓度)超过其水溶解度、因而其常规水溶液为不稳定溶液的溶质。
以5-氟尿嘧啶为有机溶质的模型溶质进行研究。在80℃按常规方法配制如下表所示的初溶液各20ml。每个溶液分装为4瓶(5ml/瓶)密封，其中2瓶不作特殊处理(常规溶液)，而另2瓶放入红外烤箱中处理(条件如实施例1,研究溶液)。然后将各常规溶液和研究溶液中的一瓶放入15℃环境，另一瓶放入0℃环境，存放1小时后观察有无结晶析出。结果示于下表。
表5 浓度对协同作用的影响
Figure PCTCN2022070058-appb-000004
在上表中，当浓度足够高(例如≧其溶解度、或30-300％)的精氨酸与浓度足够高(例如从大于到15倍于其室温溶解度)的有机溶质联合使用，对它们与水的混合物进行超常规处理可产生抗温敏协同作用，成为稳定溶液。
实施例5 不稳定溶质间的抗温敏协同作用研究(2)
以碳酸钠为无机溶质(例如某些无机盐)的模型溶质进行研究。在45℃按常规方法配制如下表所示始溶液各10ml。每个溶液分装为2瓶(5ml/瓶)密封，其中1瓶不作特殊处理(常规溶液)，而另1瓶放入红外烤箱中处理(条件如实施例1,研究溶液)。然后将各常规溶液和研究溶液放入0℃环境，存放12小时后观察有无结晶析出。结果示于下表。
表5 浓度对协同作用的影响
Figure PCTCN2022070058-appb-000005
Figure PCTCN2022070058-appb-000006
在上表中,组1的结果显示,高溶性无机溶质虽可以通过升温增溶制备为超目标场景温度溶解度的高浓度水溶液(初溶液)。该初溶液(不作特殊处理)极不稳定,均随温度下降而析出结晶,作特殊处理后亦是如此,未能将不稳定溶液改变为稳定溶液。
组2-4的结果显示,浓度小于溶解度的精氨酸未能与高溶性无机溶质产生抗温敏协同作用,它们的水溶液不稳定。组5-6的结果显示,浓度大于溶解度(或大于30％)的精氨酸与高溶性有机溶质经超常规处理后可产生抗温敏协同作用,有利于它们的水溶液稳定。
实施例6 精氨酸与较低浓度溶质间的抗温敏协同作用研究
浓度为≦10的亚甲蓝作为单用时为稳定溶质的模型溶质进行研究。在45℃按常规方法配制如下表所示的初溶液各10ml。每个溶液分装为2瓶(5ml/瓶)密封，其中1瓶不作特殊处理(条件如实施例1,常规溶液)，而另1瓶放入红外烤箱中处理2分钟(研究溶液)。然后将各常规溶液和研究溶液放入4℃环境，存放24小时后观察有无结晶析出。结果示于下表。
表6 亚甲蓝对精氨酸溶液稳定性的影响
Figure PCTCN2022070058-appb-000007
在上表中，组1-4中初溶液无处理存放的结果合乎加和预期，即稳定溶质的加入不会使得超饱和浓度的精氨酸更加稳定；而其初溶液经超常规处理后存放效果超过前述预期，超饱和浓度的精氨酸与较低浓度溶质间显示出抗温敏协同作用。
在另一个试验中,分别使用右旋糖酐(C A2(45℃)＝2.0％)和水溶性益生菌β-葡聚糖(C A2(45℃)＝10.0％)作为精氨酸的共用溶质,这些共用也显示出类似的抗温敏协同作用。
实施例7 超高浓度精氨酸化学稳定性的发现
在60℃按常规方法配制如下表所示的精氨酸水溶液各10ml，每种溶液分做2瓶。其中1瓶(对照物)放入8℃环境存放120分钟(常规处理)，另1瓶(研究物)放入100℃环境存放120 分钟(加速稳定性试验)，然后将各溶液按《中国药典》2020年第二部的方法进行含量分析，其结果如下。
表7 精氨酸稳定溶液的化学稳定性
Figure PCTCN2022070058-appb-000008
通常认为,精氨酸的化学不稳定反应符合化学反应的一般规律,即反应速率与反应物浓度成正比。某些精氨酸类似物(例如精氨酸盐酸盐注射液)出于热力学稳定性的考虑采用低于溶解度的浓度,而出于化学稳定性的考虑更是采用远低于(例如低于1/10)溶解度的浓度。在上表组别1中,研究物的含量略低于标准(99.0％),似乎证实了精氨酸的化学不稳定性。然而,在上表组别2-6中,浓度超过其室温溶解度、或≧30％的精氨酸水溶液均显示出良好的化学稳定性。更出人意料地是,它们的研究物中的精氨酸含量居然还好于组别1中的精氨酸水溶液。
实施例8 药效对比研究
以裸小鼠为实验对象，系列A、B、和C分别采用小鼠乳腺癌4T1细胞(1×10 5个细胞/只)、小鼠胚胎成纤维细胞3T3(0.5×10 4个细胞/只)、和小鼠乳腺癌4T1细胞与小鼠胚胎成纤维细胞3T3的混合物(1.05×10 5个细胞/只，3T3与4T1的细胞数量比为1/20)为局部病变建模细胞，细胞均注射进入动物右侧腋部皮下进行移植结节建模。建模成功的系列A、B、和C动物中的细胞移植病变结节的平均体积为126.3mm 3、245.6mm 3、和273.5mm 3(系列C中的瘤体的间质比为≧28.3％)，将每个系列模型动物分别随机分为10组。
表8的组别配置溶液，其中常规溶液的配制方法为：按所示量比将配制5ml溶液所需的溶质和水加入烧杯，在溶质溶解所需的温度(室温至70℃)下搅拌溶解为水溶液，再移入小玻璃瓶。非常规溶液的配制方法为：按所示量比将配制5ml溶液所需的溶质和水加入小玻璃瓶，上盖后移入红外烤箱中处理2分钟然后移出。再将盛有各溶液的玻璃瓶放入20℃环境，存放1小时后观察有无结晶析出。其中无析出的稳定溶液按下表所示的剂量用于病变内给药，给药次数为一次，给药后第7天测量局部病变体积(V)，并根据阴性对照组分别计算各系列中各组的疗效(局部病变抑制率RA、RB、RC)，还可根据疗效(局部病变抑制率)计算药效实际 /预期比q。
表8 不同组别对应的抑瘤率结果
Figure PCTCN2022070058-appb-000009
使用肿瘤瘤体间质比测定的公知方法,测出系列C中动物瘤体的平均间质比为28.6％。
在上表中，组别1、3、4、5中的常规溶液在室温均未出现析出，可用以给药；组别2、6、7中的常规溶液在室温均出现析出，而未出现析出的非常规溶液被用以给药。相同剂量相同方式的给药，理应有相同的药效。然而，组别6在系列A、B、和C中的抑制率均大于组别5。尤其是在系列B和C中，组别6的抑制率是组别5的150％以上。组别7在系列A、B、和C中的抑制率均大于组别2。
根据以上结果，本发明的组合物(组别6、7)与常规组合物(组别5、2)相比较，前者在治疗局部病变的相关疾病中具有更高的共用药效(系列A、B、C)、尤其是在治疗包含成纤维细胞的局部病变(例如间质比大于28％的瘤体)的相关疾病中更是远远超过后者的药效预期。
实施例9 安全性对比研究
以裸小鼠为实验对象，采用小鼠乳腺癌4T1细胞与小鼠胚胎成纤维细胞3T3的混合物(1.1×10 5个细胞/只，3T3与4T1的细胞数量比为1/10)为局部病变建模细胞，细胞均注射进入动物右侧腋部皮下进行移植结节建模。将建模成功的动物(细胞移植病变结节的平均体积为532.7mm 3)随机分为10组，每组10只。
按表10的组别制备溶液，其中常规溶液的配制方法为：按所示量比将配制5ml溶液所需的溶质和水加入烧杯，在溶质溶解所需的温度(室温至45℃)下搅拌溶解为水溶液，再移入小玻璃瓶。非常规溶液的配制方法为：按所示量比将配制5ml溶液所需的溶质和水加入小玻璃瓶，上盖后移入红外烤箱中处理2分钟然后移出。再将盛有各溶液的玻璃瓶放入20℃环境，存放1小时后观察有无结晶析出。其中无析出的稳定溶液按下表所示的剂量用于病变内给药，给药次数为一次，给药后24h统计各组死亡率，结果示于下表。
表9 不同组别药物的动物死亡率
Figure PCTCN2022070058-appb-000010
在上表中，组别1、3中的常规溶液在室温均未出现析出，可用以给药；组别2、4中的常规溶液在室温均出现析出，而未出现析出的非常规溶液被用以给药。相同剂量相同方式的给药，理应有相同的安全性。然而，组别1、3中的常规溶液的大剂量注射产生了严重的安全性问题，而组别2、4中的非常规溶液却没有观察到相应问题。
根据以上结果，本发明的组合物(组别2、4)与常规组合物(组别1、3)相比较，前者在治疗局部病变的相关疾病中具有更高的共用安全性。
综上，本发明的组合物除显示出可以超高浓度在较低温度储存或/和给药外，较之相同剂量的常规组合物还有更大范围的协同作用，包括更大适应症范围内的更高药效协同作用，和局部给药时更高的全身性安全性协同作用。

Claims (21)

  1. 一种稳定溶液，所述稳定溶液包含药物和水，所述药物包括精氨酸，其特征在于，所述稳定溶液中精氨酸在温度T时处于溶解状态且与水的百分量比为C 1，所述精氨酸在温度T时的水溶解度为C 2，其中T代表所述稳定溶液储存或给药环境温度，T为0-37℃，C 1＞C 2。
  2. 根据权利要求1所述稳定溶液，其特征在于，所述稳定溶液中精氨酸与水的百分量比C 1为40-300％、50-300％、60-300％、或70-300％。
  3. 根据权利要求2所述稳定溶液，其特征在于，所述稳定溶液中精氨酸与水的百分量比C 1不随温度T的升降而变化。
  4. 根据权利要求1或2所述稳定溶液，其特征在于，所述药物还包括溶质B，所述溶质B不是强酸。
  5. 根据权利要求4所述稳定溶液，其特征在于，所述溶质B在温度T时处于溶解状态且与水的百分量比为C 3、所述溶质B在温度T的溶解度为C 4，其中C 3>C 4，以及所述C 3不随温度T的下降而变化。
  6. 根据权利要求4或5所述稳定溶液，其特征在于，所述溶质B为局部作用药物、细胞毒药物、生物活性药物中的至少一种。
  7. 一种制备稳定溶液的方法，用于制备权利要求1-6任一种所述稳定溶液，其特征在于，包括对所述药物和水进行混合以及对混合物进行超溶解所需的稳定化处理。
  8. 根据权利要求7所述制备稳定溶液的方法，其特征在于，所述稳定化处理包括过热处理、微波处理、红外处理、超声处理中的至少一种。
  9. 根据权利要求8所述制备稳定溶液的方法，其特征在于，所述过热处理的处理温度在95℃-125℃或95℃-115℃或95℃-110℃或95℃-105℃之间，持续时间为3min-110min。
  10. 一种药物组合物，所述组合物包含药物和水，所述药物包括精氨酸，其特征在于，储存或给药形式包括权利要求1-6任一项所述稳定溶液。
  11. 根据权利要求10所述药物组合物的制备方法，其特征在于，在装入最终的递送系统之前或之后，通过权利要求7-9任一项所述方法制备所述稳定溶液。
  12. 一种药物，其特征在于，通过权利要求1-6任一项所述稳定溶液制备而来，或通过权利要求7-9任一项所述的方法制备。
  13. 权利要求12所述药物的制备方法，其特征在于，包括通过权利要求7-9任一项所述方法制备稳定溶液，随后将所述稳定溶液冷冻干燥。
  14. 权利要求1-6任一项所述稳定溶液、权利要求10所述药物组合物、或权利要求 12所述药物在制备预防或/和治疗疾病的药物中的应用。
  15. 根据权利要求14所述的应用，其特征在于，所述精氨酸作为可提供抗温敏作用的活性成分。
  16. 根据权利要求14所述的应用，其特征在于，所述疾病为局部病变疾病。
  17. 根据权利要求16所述的应用，其特征在于，所述局部病变疾病包括肿瘤、非瘤肿大、局部炎症、分泌腺功能异常和皮肤病。
  18. 一种预防或/和治疗疾病的方法，其特征在于，其包含以下步骤:将权利要求1-6任一项所述稳定溶液、权利要求10所述药物组合物、或权利要求12所述药物给予有需要的个体。
  19. 根据权利要求18所述的方法，其特征在于，其包含以下步骤:进行权利要求7-8之一所述方法获得所述稳定溶液。
  20. 一种药物试剂盒，其特征在于，或者包括包含以下独立包装制剂的容器：包括权利要求1-6任一项所述稳定溶液、分别含有由权利要求10所述药物组合物中的所述药物和水、或含有由权利要求12所述的药物制成的制剂。
  21. 根据权利要求20所述的药物试剂盒，其特征在于，还包括包含反映以下情况的说明书:权利要求1-6任一项所述的稳定溶液、或者由权利要求10所述药物组合物或权利要求12所述药物通过权利要求7-9任一项所述方法制备的所述稳定溶液可在室温或/和可在低于室温的温度下使用或/和储放一小时以上。
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