WO2021058014A1 - 奥硝唑类药物组合物及其制备方法和用途 - Google Patents

奥硝唑类药物组合物及其制备方法和用途 Download PDF

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WO2021058014A1
WO2021058014A1 PCT/CN2020/118543 CN2020118543W WO2021058014A1 WO 2021058014 A1 WO2021058014 A1 WO 2021058014A1 CN 2020118543 W CN2020118543 W CN 2020118543W WO 2021058014 A1 WO2021058014 A1 WO 2021058014A1
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Prior art keywords
pharmaceutical composition
ornidazole
less
nitroimidazole
methyl
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PCT/CN2020/118543
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English (en)
French (fr)
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杨丹
陈令武
丁菲
张琪
杨海雪
汤传飞
王嬿钧
梁衡
王旭艳
Original Assignee
扬子江药业集团南京海陵药业有限公司
南京海陵中药制药工艺技术研究有限公司
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Publication of WO2021058014A1 publication Critical patent/WO2021058014A1/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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • the invention belongs to the field of medicine, and specifically relates to an ornidazole pharmaceutical composition and a preparation method and application thereof.
  • Ornidazole is a nitroimidazole derivative. It is a powerful anti-anaerobe and antiprotozoal drug. It is also a newly developed drug after metronidazole with higher efficacy, shorter treatment course, better tolerance, and wider distribution in the body.
  • the antimicrobial effect of ornidazole is the reduction of the nitro group in its molecule to an amino group in an anaerobic environment, or the formation of free radicals interacting with cell components to cause the death of microorganisms.
  • Ornidazole is 1-(3-chloro-2-hydroxypropyl)-2-methyl-5-nitroimidazole (CAS No. 16773-42-5), and has the chemical structure shown in the following formula:
  • Levoornidazole (also known as “Levoornidazole”, CAS No.166734-83-4) is the levorotatory isomer of ornidazole, which is mainly used clinically for the treatment of Bacteroides fragilis, Bacteroides diundi, and egg Bacteroides viridis, Bacteroides polymorpha, Bacteroides vulgaris, Clostridium spp, Eubacteria, Peptococcus and Peptostreptococcus, Helicobacter pylori, Bacteroides melanogaster, Fusobacterium, CO 2 weaving bacteria, Bacteroides gingivalis, etc. A variety of infectious diseases caused by anaerobic bacteria, or used to prevent such infections before surgery.
  • L-Ornidazole has a chemical structure shown in the following formula:
  • L-ornidazole phosphate ester of L-ornidazole also known as L-ornidazole phosphate
  • salts such as disodium ornidazole phosphate
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an ornidazole compound and 1-(2,3-epoxypropane)-2-methyl-5-nitro with a content of less than 1000 mg/kg Imidazole, and 1-(2,3-dihydroxypropyl)-2-methyl-5-nitroimidazole whose content is less than 2100mg/kg.
  • the ornidazole compound may be selected from ornidazole, its stereoisomers or their precursor compounds.
  • the ornidazole compound may be selected from ornidazole, levornidazole (also known as “levornidazole”) or dextroornidazole (also known as "right ornidazole”) or their Precursor compound.
  • the ornidazole has a chemical structure represented by the following formula (I-1):
  • L-ornidazole has the structure shown in the following formula (I-2):
  • D-ornidazole has the structure shown in the following formula (I-3):
  • the precursor compound of ornidazole, levornidazole or dextroornidazole may be selected from its pharmaceutically acceptable precursor compounds, such as esters or esters selected from any one of them.
  • the pharmaceutically acceptable salts of the esters, and their hydrates can be selected from any of their amino acid esters, phosphoric acid esters, amino acid salts of phosphoric acid esters, and salts of phosphoric acid esters and alkali metal or alkaline earth metal ions. Or at least one of their hydrates; for example, at least one of the sodium, potassium, calcium, magnesium, etc. or hydrates of phosphate esters, an exemplary example is phosphate disodium salt or Its hydrate.
  • the precursor compound of L-ornidazole may be selected from L-ornidazole amino acid ester, L-ornidazole phosphate, L-ornidazole phosphate amino acid salt, and L-ornidazole phosphate and alkali At least one of metal or alkaline earth metal ions or at least one of their hydrate salts; for example, at least one of the sodium, potassium, calcium, magnesium, etc. of L-ornidazole phosphate or their hydrates Exemplified by L-ornidazole phosphate disodium salt (CAS No. 909133-95-5) or its hydrate.
  • the precursor compound of ornidazole, levornidazole or dextroornidazole may be present in the pharmaceutical composition in the form of its amorphous or polymorphic form.
  • the precursor compound of ornidazole, levornidazole or dextroornidazole can also be selected from solvates of pharmaceutically acceptable salts of esters of any of them, such as hydrates thereof, for example At least one of 1, 2, 3, 4, 5, 6 or 7 hydrates of the pharmaceutically acceptable salts of the esters, examples of which can be selected from the hydrates of levornidazole phosphate disodium salt, such as At least one of pentahydrate, hexahydrate, and heptahydrate.
  • 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole has a structure represented by the following formula (II):
  • the content of 1-(2,3-propylene oxide)-2-methyl-5-nitroimidazole is below 900 mg/kg, for example below 800 mg/kg, 700 mg/kg or less, 600 mg/kg or less, 500 mg/kg or less, 400 mg/kg or less, 300 mg/kg or less, 200 mg/kg or less, preferably 100 mg/kg or less.
  • the content of 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole is below 90mg/kg, such as below 80mg/kg, below 70mg/kg, more preferably below 60mg/kg , 50mg/kg or less, 40mg/kg or less, 30mg/kg or less, 20mg/kg or less or 10mg/kg or less.
  • the content of 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole is below 9 mg/kg, for example, below 8 mg/kg , 7mg/kg or less, more preferably 6mg/kg or less, examples of which can be 5mg/kg or less, 4mg/kg or less, 3mg/kg or less, 2mg/kg or less, or 1mg/kg or less.
  • the content of 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole is below 0.9 mg/kg, such as 0.8 mg /kg or less, 0.7mg/kg or less, more preferably 0.6mg/kg or less, examples of which can be 0.5mg/kg or less, 0.4mg/kg or less, 0.3mg/kg or less, 0.2mg/kg or less, or 0.1mg/kg the following.
  • the content of 1-(2,3-propylene oxide)-2-methyl-5-nitroimidazole may be 0, which means that the pharmaceutical composition Does not contain 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole.
  • the content of 1-(2,3-propylene oxide)-2-methyl-5-nitroimidazole is greater than 0, which means that the pharmaceutical composition contains 1 -(2,3-Epoxypropane)-2-methyl-5-nitroimidazole.
  • 1-(2,3-dihydroxypropyl)-2-methyl-5-nitroimidazole has the structure shown in the following formula (III):
  • the content of 1-(2,3-dihydroxypropyl)-2-methyl-5-nitroimidazole is below 2100mg/kg, for example below 2000mg/kg , 1900mg/kg or less, 1800mg/kg or less, 1700mg/kg or less, 1600mg/kg or less, 1500mg/kg or less, 1400mg/kg or less, 1300mg/kg or less, 1200mg/kg or less, 1100mg/kg or less.
  • the content of 1-(2,3-dihydroxypropyl)-2-methyl-5-nitroimidazole is less than 1000 mg/kg, for example, less than 900 mg/kg , 800mg/kg or less, 700mg/kg or less, 600mg/kg or less, 500mg/kg or less, 400mg/kg or less, 300mg/kg or less, 200mg/kg or less, or 100mg/kg or less.
  • the content of the compound represented by formula (III) in the pharmaceutical composition is below 90 mg/kg, such as below 80 mg/kg, below 70 mg/kg, more preferably below 60 mg/kg, examples thereof It can be 50 mg/kg or less, 40 mg/kg or less, 30 mg/kg or less, 20 mg/kg or less, or 10 mg/kg or less. More preferably, in the pharmaceutical composition, the content of the compound represented by formula (III) is below 9 mg/kg, such as below 8 mg/kg, below 7 mg/kg, more preferably below 6 mg/kg, an example of which may be 5 mg/kg. kg or less, 4 mg/kg or less, 3 mg/kg or less, 2 mg/kg or less, 1 mg/kg or less, or 0. Wherein, when the content of the compound represented by formula (III) is 0, it means that the pharmaceutical composition does not contain the compound represented by formula (III). Alternatively, the content of the compound represented by formula (III) in the pharmaceutical composition may also be >0.
  • the weight percentage of the ornidazole compound as the active ingredient in the pharmaceutical composition in terms of Levo-ornidazole, Levo-ornidazole Disodium Phosphate, or a hydrate of any of them
  • the content may be in the range of 10% to 99%, preferably 15% to 90%, for example 20% to 85%, examples thereof may be in the range of, for example, 72% to 83%, examples thereof may be 72%, 73% , 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%.
  • the pharmaceutical composition may optionally include or not include a compound represented by the following formula (IV):
  • the content of the compound represented by formula (IV) in the pharmaceutical composition may be 15000 mg/kg or less, for example, 14000 mg/kg or less, 13000 mg/kg or less, 12000 mg/kg or less, 11000 mg/kg or less , 10000mg/kg or less, 8000mg/kg, 7000mg/kg or less, 6000mg/kg or less, 5000mg/kg or less, 4000mg/kg or less, 3000mg/kg or less, 2000mg/kg or less.
  • the content of the compound represented by formula (IV) may be 1000 mg/kg or less, for example, 900 mg/kg or less, 800 mg/kg or less, 700 mg/kg or less, 600 mg/kg or less, 500 mg/kg or less, 400 mg/kg or less, 300mg/kg or less, 200mg/kg or less, or 100mg/kg or less.
  • the content of the compound represented by formula (IV) in the pharmaceutical composition is below 90 mg/kg, such as below 80 mg/kg, below 70 mg/kg, more preferably below 60 mg/kg, examples thereof It can be 50 mg/kg or less, 40 mg/kg or less, 30 mg/kg or less, 20 mg/kg or less, 10 mg/kg or less, or zero.
  • the content of the compound represented by formula (IV) in the pharmaceutical composition is 0, it means that the pharmaceutical composition does not contain the compound represented by formula (IV).
  • the content of the compound represented by formula (IV) in the pharmaceutical composition may also be >0.
  • the pharmaceutical composition may also optionally contain or not contain 2-methyl-5-nitroimidazole.
  • the content of 2-methyl-5-nitroimidazole in the pharmaceutical composition is below 2000 mg/kg, for example, 1900 mg/kg or less, 1800 mg/kg or less, 1700 mg/kg or less, 1600 mg/kg or less.
  • kg or less 1500mg/kg or less, preferably 1400mg/kg or less, 1300mg/kg or less, 1200mg/kg or less, 1100mg/kg or less, 1000mg/kg or less, such as 900mg/kg or less, 800mg/kg or less, 700mg/kg or less, 600mg/kg or less, 500mg/kg or less, 400mg/kg or less, 300mg/kg or less, 200mg/kg or less, or 100mg/kg or less.
  • 1400mg/kg or less 1300mg/kg or less, 1200mg/kg or less, 1100mg/kg or less, 1000mg/kg or less, such as 900mg/kg or less, 800mg/kg or less, 700mg/kg or less, 600mg/kg or less, 500mg/kg or less, 400mg/kg or less, 300mg/kg or less, 200mg/kg or less, or 100mg/kg or less.
  • the content of 2-methyl-5-nitroimidazole in the pharmaceutical composition is below 90mg/kg, for example below 80mg/kg, below 70mg/kg, more preferably below 60mg/kg , Examples thereof may be 50 mg/kg or less, 40 mg/kg or less, 30 mg/kg or less, 20 mg/kg or less, 10 mg/kg or less, or zero.
  • the content of 2-methyl-5-nitroimidazole in the pharmaceutical composition is 0, it means that the pharmaceutical composition does not contain 2-methyl-5-nitroimidazole.
  • the pharmaceutical composition when the pharmaceutical composition includes a precursor compound of levornidazole, it may or may not include levornidazole, preferably, wherein the content of levornidazole is 2000mg/kg or less, for example 80mg/kg or less, 70mg/kg or less, more preferably 60mg/kg or less, examples of which can be 50mg/kg or less, 40mg/kg or less, 30mg/kg or less, 20mg/kg or less, or 10mg/kg or less. Below kg or 0. Wherein, when the content of levornidazole in the pharmaceutical composition is 0, it means that levornidazole is not included in the pharmaceutical composition.
  • the pharmaceutical composition may also include pharmaceutically acceptable excipients, such as carriers or excipients.
  • pharmaceutically acceptable excipients are preferably non-chemically reactive or inert to the active ingredients.
  • the pharmaceutically acceptable excipient is selected from at least one of the following including but not limited to: fillers, disintegrants, binders, lubricants, surfactants, flavors, wetting agents, pH adjustment Agent, solubilizer or co-solvent, osmotic pressure regulator, etc.
  • the filler can be selected from lactose, sucrose, glucose, mannitol, sorbitol, calcium sulfate, calcium gluconate, calcium hydrogen phosphate, calcium phosphate, calcium carbonate, calcium hydrogen carbonate, starch, carboxylate At least one of methyl starch, pregelatinized starch, microcrystalline cellulose, and the like.
  • the disintegrant can be selected from pregelatinized starch, microcrystalline cellulose, alginic acid, lignocellulose, sodium carboxymethyl starch, guar gum, cross-linked polyvinylpyrrolidone and cross-linked At least one of sodium carboxymethyl cellulose and the like.
  • the binder can be selected from gelatin, dextrin, maltodextrin, sucrose, acacia, polyvinylpyrrolidone, methyl cellulose, carboxymethyl cellulose, ethyl cellulose , At least one of polyvinyl alcohol, polyethylene glycol and hypromellose.
  • the lubricant can be selected from magnesium stearate, calcium stearate, zinc stearate, talc, glyceryl monostearate, polyethylene glycol 4000, polyethylene glycol 6000 , Polyethylene glycol 8000, sodium benzoate, adipic acid, fumaric acid, boric acid, sodium chloride, sodium oleate, triacetin, polyoxyethylene monostearate, monolaurin sucrose, chlorinated At least one of sodium, sodium lauryl sulfate, magnesium lauryl sulfate, and the like.
  • the surfactant can be selected from sodium lauryl sulfate, poloxamer, polysorbate 80, cetyltrimethylamine bromide, sodium lauryl sulfate, stearic acid sulfonic acid At least one of sodium, polyoxyethylene higher fatty alcohol, sucrose ester, sorbitol fatty ester, soybean phospholipid, and the like.
  • the flavoring agent is steviol, fructose, glucose, fructose syrup, honey, aspartame, protein sugar, xylitol, mannitol, lactose, sorbitol, flavor and maltitol At least one of the others.
  • the pH adjusting agent may be selected from at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine and the like.
  • the co-solvent may be selected from alcohol solvents, for example, at least selected from ethanol, glycerin, propylene glycol, polyethylene glycol (eg polyethylene glycol 300, polyethylene glycol 400, etc.) One kind.
  • the osmotic pressure regulator can be selected from at least one of sodium chloride, glucose, fructose, phosphate, polyethylene glycol, propylene glycol, mannitol and the like.
  • the pharmaceutical composition may be a preparation, for example, a preparation for gastrointestinal administration or a preparation for parenteral administration.
  • the preparations for gastrointestinal administration may be tablets, dispersible tablets, capsules, sustained release agents, granules, oral liquids, syrups, etc.
  • the preparations for parenteral administration may be infusion preparations, injections (Such as liquid injections), freeze-dried preparations (such as freeze-dried powders), effervescent tablets, suppositories, sublingual tablets, etc., preferably capsules, injections (such as liquid injections) or freeze-dried preparations for injection (such as freeze-dried powders) ).
  • the pharmaceutical composition is an injection or a freeze-dried preparation for injection, which contains an ornidazole compound, preferably ornidazole, levornidazole phosphate disodium or any of them
  • an ornidazole compound preferably ornidazole, levornidazole phosphate disodium or any of them
  • the hydrate of sulphate, and 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole with a content of less than 60mg/kg.
  • the ornidazole or its isomer is calculated as the active ingredient ornidazole compound in a percentage by weight of 70% to 85%. Within the range, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 82%, 83%, 84%, 85%, preferably 72% To 83%.
  • the pharmaceutical composition such as a freeze-dried preparation for injection, may contain citric acid, and the weight percentage content of the citric acid may be 10-35%, preferably 15-32%, such as 18 -28%, 20-25%, exemplified are 16%, 17%, 18%, 19%, 21%, 22%, 23%, 24%, 26%, 27%, 29%.
  • the pH of the injection may be 4.0-6.0, preferably 4.5-5.5, such as 4.6-5.4, 4.7-5.3, exemplarily 4.8, 4.9, 5.0, 5.1, 5.2.
  • the content of citric acid when the pH of the injection is 4.0-6.0, the content of citric acid may be 19-45 mg/mL.
  • the injection may contain water for injection, ethanol and/or propylene glycol.
  • the administration route of the pharmaceutical composition includes but not limited to gastrointestinal administration or parenteral administration; wherein, the parenteral administration may be injection administration (such as Intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intradermal injection, etc.), vaginal or mucosal administration, etc.
  • parenteral administration may be injection administration (such as Intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intradermal injection, etc.), vaginal or mucosal administration, etc.
  • the active ingredient of the pharmaceutical composition is an ornidazole compound, preferably ornidazole, levornidazole or a precursor compound of levornidazole.
  • the pharmaceutical composition may not contain other active ingredients other than ornidazole compounds, or may also contain other active ingredients other than ornidazole, levornidazole or precursor compounds of levornidazole Active ingredient.
  • the pharmaceutical composition comprises or disodium levornidazole phosphate and citric acid, and optionally water for injection in the presence or absence.
  • the pH of the pharmaceutical composition is 4.5-5.5.
  • the pharmaceutical composition comprises ornidazole, ethanol, propylene glycol, and optionally water for injection.
  • the present invention also provides a preparation method of the above-mentioned pharmaceutical composition, which comprises mixing an ornidazole compound with pharmaceutically acceptable excipients to obtain the pharmaceutical composition.
  • ornidazole, ethanol, propylene glycol and water for injection are mixed to obtain the pharmaceutical composition.
  • disodium levornidazole phosphate or its hydrate is formulated with water to form a solution, and the solution is freeze-dried to obtain the pharmaceutical composition.
  • the pH of the solution is 4.0-6.0, preferably 4.5-5.5, such as 4.6-5.4, 4.7-5.3, exemplarily 4.8, 4.9, 5.0, 5.1, 5.2.
  • the freeze-drying temperature is -60°C to 30°C, preferably -50°C to 25°C.
  • the freeze-drying time is 30-70h, such as 40-60h, or 45-55h.
  • the freeze-drying is programmed temperature control.
  • the exemplary operation of freeze-drying includes: sending the filled semi-stoppered semi-finished product into a freeze-drying tank when the temperature of the silicone oil drops to 0°C, keeping it for 0.5 hours, and waiting for the temperature of the silicone oil to drop to -50 At °C, keep for 1.0h; increase to -20°C and keep for 3.0h; decrease to -50°C and keep for 3.0h.
  • Turn on the vacuum pump evacuate to 200 ⁇ bar, raise the temperature to -20°C for 1.0h, and keep it for 16.0h.
  • the present invention also provides the use of the pharmaceutical composition for improving the safety of the medicine.
  • the present invention also provides the use of the pharmaceutical composition for improving the stability of the drug.
  • the invention also provides the use of the pharmaceutical composition for preparing medicines.
  • the medicine is used to prevent or treat diseases related to anaerobic bacteria, such as to prevent or treat infectious diseases caused by anaerobic bacteria.
  • the anaerobic bacteria may be selected from at least one of the following including but not limited to: Bacteroides fragilis, Bacteroides diundi, Bacteroides ovalifolia, Bacteroides polymorpha, Bacteroides vulgaris, Clostridium, Bacteroides Anaerobic bacteria such as Bacillus, Peptococcus and Peptostreptococcus, Helicobacter pylori, Bacteroides melanogaster, Fusobacterium, CO 2 phagocytic bacteria, Bacteroides gingivalis and other anaerobic bacteria.
  • the medicine is used to prevent or treat infectious diseases caused by anaerobic bacteria before and/or after surgery.
  • the present invention also provides a method for preventing or treating diseases related to anaerobic bacteria, such as a method for preventing or treating infectious diseases caused by anaerobic bacteria, comprising administering a therapeutically effective amount of the pharmaceutical composition to a patient in need .
  • the term "effective amount” or “therapeutically effective amount” refers to the amount of the compound of the present invention sufficient to achieve the intended application (including but not limited to the treatment of diseases as defined below).
  • the therapeutically effective amount may vary depending on the following factors: the intended application (in vitro or in vivo), or the subject to be treated and the disease condition such as the weight and age of the subject, the severity of the disease condition and the mode of administration, etc. It can be easily determined by a person of ordinary skill in the art.
  • the specific dosage will vary depending on the following factors: the particular compound selected, the dosing regimen on which it is based, whether it is administered in combination with other compounds, the timing of administration, the tissue to be administered, and the physical delivery system carried.
  • patient refers to a patient who needs to prevent or treat a disease related to anaerobic bacteria, where the patient is a mammal, for example selected from rodents, cows, pigs, dogs, cats and primates, especially humans.
  • expiry date means the period during which the quality of the drug can meet the specified requirements under the specified storage conditions, such as 24 months, such as 18 months or 12 months.
  • the present application surprisingly found that the presence of 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole and 1-(2,3-bis) in pharmaceutical compositions containing ornidazole compounds Hydroxypropyl)-2-methyl-5-nitroimidazole is genotoxic, causing cell DNA damage and mutagenic effects, which greatly affects the safety and/or stability of such drugs during the validity period. challenge. Such medication risks are controlled or even eliminated in the pharmaceutical composition of the present invention.
  • This application finds that when the weight percentage content of 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole is reduced to less than 1000mg/kg, preferably less than 100mg/kg, more preferably 60mg/kg When the content of 1-(2,3-dihydroxypropyl)-2-methyl-5-nitroimidazole is reduced to less than 2100mg/kg, preferably less than 2100mg/kg, the safety of such drugs And/or stability can be significantly improved.
  • the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods.
  • the test product is prepared on the day of administration. Weigh an appropriate amount of the test product and add an appropriate amount of sodium chloride injection to dissolve it to a concentration of 50 mg/mL. The solution was filtered with a 0.2 ⁇ m filter membrane, and diluted with sodium chloride injection into a solution with a concentration of 20, 8, 2, and 0.5 mg/mL, respectively.
  • the prepared solution is stored at room temperature before administration.
  • Strains Salmonella typhimurium, histidine-deficient strains (TA97a, TA98, TA100, TA102, TA1535). Strains are provided by Moltox, and the bacterial solution is stored in liquid nitrogen.
  • This experiment uses the S9 component of SD rat liver, the protein concentration is 21.64mg/mL, and it is stored in liquid nitrogen. Before use, the S9 mixture will be prepared under aseptic conditions. Refer to Table 1 for the preparation of the S9 mixture.
  • Strain enrichment culture After rapid thawing of the bacterial liquid stored in liquid nitrogen in a 37°C water bath, 100 ⁇ l is inoculated into 20ml of nutrient broth, and incubated at 37°C for 10-12 hours with shaking (120rpm) in the dark.
  • Grouped administration 2 parallel dishes are processed at each test point. The dosing information is shown in Table 2.
  • Group 1 is the spontaneous control group; Groups 2-6 are the test product groups.
  • test product has antibacterial toxicity to the strain according to the following criteria:
  • 1-(2,3-dihydroxypropyl)-2-methyl-5-nitroimidazole has no obvious bacterial toxicity at a dose of 50 ⁇ 5000 ⁇ g/dish, but at a dose of 800 ⁇ 5000 ⁇ g It is mutagenic to Salmonella typhimurium at the dose of /dish, so it is genotoxic.
  • the preparation process is completed under aseptic conditions, the reagent bottles used are all sterilized, and the preparation process is not protected from light.
  • test product in terms of content
  • solvent an appropriate amount of solvent and stir until it is clear and transparent, and finally use the solvent to dilute to the desired concentration.
  • preparation is filtered with a 0.22 ⁇ m filter membrane to obtain a preparation for administration of the test product.
  • Body weight and age at the time of grouping Female: about 24-28g, 8-10 weeks old; Male: about 25-29g, 8-10 weeks old.
  • X is a female rat and Y is a male rat.
  • Randomized grouping according to gender and body weight, and the 48 animals selected were assigned to 5 groups.
  • the weight difference of the animals used for grouping is within ⁇ 20% of the average weight of the same sex. After grouping, the average body weight of each group of animals was not statistically different under the inspection level of 5.0%. After grouping, the remaining animals are kept and recorded and raised.
  • Route of administration Choose the route of intraperitoneal injection.
  • Administration mode 5 times for 3 consecutive days, 1 time in the morning on the first day, and 1 time in the morning and afternoon on the second and third days.
  • Dosage Calculate the dose based on the last weight weighed.
  • Dosing period weigh the body weight in the morning before D1, D2, and D3, and use it to calculate the dose
  • mice were sacrificed by cervical dislocation method, femurs were removed, muscles removed, epiphyses were cut, bone marrow cavity was exposed, bone marrow smears were taken, fixed with methanol, and stained with Gimesa.
  • Microscopy method select areas with complete, uniformly dispersed, and appropriately colored cells under a low-power microscope, and observe with a high-power microscope. Each smear counts the micronucleus rate of 1000 polychromatic erythrocytes (PCE) with clear and complete cytoplasm. The result is expressed in per thousand ( ⁇ ).
  • PCE polychromatic erythrocytes
  • the micronucleus rates are all in the order of Indicates that compared with the negative control group, the two-sided t test and the ⁇ 2 test were used for statistical processing.
  • P ⁇ 0.05 indicates that the difference is statistically significant
  • P>0.05 indicates that the difference is not significant
  • 0.01 ⁇ P ⁇ 0.05 indicates that the difference is significant
  • P ⁇ 0.01 means the difference is extremely significant.
  • the micronucleus rate of the 100mg/kg dose group was significantly different (p ⁇ 0.05). Although there was no statistical difference in other dose groups, the micronucleus rate also increased slightly, which can be considered to be related to the test product.
  • Kunming mice were intraperitoneally injected with impurities III at concentrations of 2.88, 48, and 100 mg/kg for 3 consecutive days. During the test, no animals died or were dying, and there were no obvious abnormalities related to the test product in their body weight. An increase in the micronucleus rate of polychromatic erythrocytes can be seen at a dose of ⁇ 100mg/kg, which is considered to be related to the test product.
  • the level of harmful effects not observed in this test is 48 mg/kg.
  • Impurity limit daily acceptable intake of impurities (that is, PDE value) / maximum daily dosage of drugs.
  • the test product is prepared on the day of administration. Weigh an appropriate amount of the test product and add an appropriate amount of sodium chloride injection to dissolve it to a concentration of 50 mg/mL. The solution was filtered with a 0.2 ⁇ m filter membrane, and diluted with sodium chloride injection into a solution with a concentration of 20, 8, 2, and 0.5 mg/mL, respectively.
  • the prepared solution is stored at room temperature before administration.
  • Strains Salmonella typhimurium, histidine-deficient strains (TA97a, TA98, TA100, TA102, TA1535). Strains are provided by Moltox, and the bacterial solution is stored in liquid nitrogen.
  • This experiment uses the S9 component of SD rat liver, the protein concentration is 21.64mg/mL, and it is stored in liquid nitrogen. Before use, the S9 mixture will be prepared under aseptic conditions. Refer to Table 5 for the preparation of the S9 mixture.
  • Strain enrichment culture After rapid thawing of the bacterial liquid stored in liquid nitrogen in a 37°C water bath, 100 ⁇ l is inoculated into 20ml of nutrient broth, and incubated at 37°C for 10-12 hours with shaking (120rpm) in the dark.
  • Grouped administration 2 parallel dishes are processed at each test point. The dosing information is shown in Table 6.
  • Group 1 is the spontaneous control group; Groups 2-6 are the test product groups.
  • test product has antibacterial toxicity to the strain according to the following criteria:
  • 1-(2,3-epoxypropane)-2-methyl-5-nitroimidazole has bacterial toxicity at a dose of 50 ⁇ 5000 ⁇ g/dish, and it is mutagenic to Salmonella typhimurium. For this reason, it is genotoxic.
  • the preparation process is completed under aseptic conditions, the reagent bottles used are all sterilized, and the preparation process is not protected from light.
  • test product in terms of content
  • solvent an appropriate amount of solvent and stir until it is clear and transparent, and finally use the solvent to dilute to the desired concentration.
  • preparation is filtered with a 0.22 ⁇ m filter membrane to obtain a preparation for administration of the test product.
  • Body weight and age at the time of grouping Female: about 24-28g, 8-10 weeks old; Male: about 25-29g, 8-10 weeks old.
  • X is a female rat and Y is a male rat.
  • Randomized grouping according to gender and body weight, and the 48 animals selected were assigned to 5 groups.
  • the weight difference of the animals used for grouping is within ⁇ 20% of the average weight of the same sex. After grouping, the average body weight of each group of animals was not statistically different under the inspection level of 5.0%. After grouping, the remaining animals are kept and recorded and raised.
  • Route of administration Choose the route of intraperitoneal injection.
  • Administration mode 5 times for 3 consecutive days, 1 time in the morning on the first day, and 1 time in the morning and afternoon on the second and third days.
  • Dosage Calculate the dose based on the last weight weighed.
  • Dosing period Weigh the body weight in the morning before D1, D2, and D3, and use it to calculate the dose.
  • mice were sacrificed by cervical dislocation method, femurs were removed, muscles removed, epiphyses were cut, bone marrow cavity was exposed, bone marrow smears were taken, fixed with methanol, and stained with Gimesa.
  • Microscopy method select areas with complete, uniformly dispersed, and properly colored cells under a low-power microscope, observe with a high-power microscope, count the micronucleus rate of 1000 polychromatic erythrocytes (PCE) with clear and complete cytoplasm on each smear. The result is expressed in per thousand ( ⁇ ).
  • PCE polychromatic erythrocytes
  • the micronucleus rates are all in the order of Indicates that compared with the negative control group, the two-sided t test and the ⁇ 2 test were used for statistical processing.
  • P ⁇ 0.05 indicates that the difference is statistically significant
  • P>0.05 indicates that the difference is not significant
  • 0.01 ⁇ P ⁇ 0.05 indicates that the difference is significant
  • P ⁇ 0.01 means the difference is extremely significant.
  • the weight of the animals in the 100mg/kg dose group showed a downward trend at D3 and showed a dose correlation. Although there was no statistical difference in the weight changes of other animals, it can also be seen that the weight gain was small and showed a dose correlation. Sample related.
  • the micronucleus rate of the 48mg/kg dose group and the 100mg/kg dose group is extremely different (p ⁇ 0.01). Although there is no statistical difference in the other dose groups, the micronucleus rate can also be seen to increase slightly.
  • the dose correlation can be considered to be related to the test product.
  • Kunming mice were intraperitoneally injected with the test compound at concentrations of 2.88, 48, and 100 mg/kg for 3 consecutive days. During the test, no animals died or were dying. The animals in the test group were ⁇ 100 mg Weight loss can be seen at the dose of /kg, and the micronucleus rate of polychromatic erythrocytes can be increased at the dose of ⁇ 48mg/kg, which is considered to be related to the test product. The level of harmful effects not observed in this test (no observed adverse effect level, NOAEL) was 2.88 mg/kg.
  • Impurity limit daily acceptable intake of impurities (that is, PDE value) / maximum daily dosage of drugs.
  • Example 2 Using the method of Example 1, the difference is that the test substance is replaced with a compound of formula (IV). The results show that the compound does not exhibit genotoxicity at doses of 50, 200, 800, 2000, and 5000 ⁇ g/dish.
  • the prescription of the freeze-dried preparation for injection is as follows:
  • the preparation method of the prescription includes the following steps:
  • the detection methods of each substance are as follows:
  • Ion source type AJS ESI ion source
  • Sheath gas temperature 350°C
  • MRM multiple reactive ion monitoring
  • Reference substance solution accurately weigh out 10 mg of 2-methyl-5-nitroimidazole, formula (II) compound, and formula (III) reference substance into a 10 mL volumetric flask, and dilute to the mark with a blank solution.
  • a reference stock solution Precisely measure 0.1 mL of the reference substance stock solution in a 10 mL volumetric flask, and dilute to the mark with the blank solution as the intermediate solution of the reference substance (ie 10 ⁇ g/mL).
  • System suitability solution accurately weigh 10 mg of the test product (calculated as L-ornidazole disodium phosphate) into a 10 mL volumetric flask, and dilute the blank solution to the mark. Take 1mL of this solution into a 10mL volumetric flask, and add the limit point concentration (take 0.6mL of the "reference substance working solution” under (4) in the above volumetric flask) of the compound of formula (II) (60mg/kg), formula (III) ) Compound (60mg/kg), 2-methyl-5-nitroimidazole (60mg/kg) reference substance, and dilute the blank solution to the mark (ie 60ng/mL) to obtain.
  • test solution accurately weigh 10 mg of the test substance (calculated as disodium levornidazole phosphate) into a 10 mL volumetric flask, and dilute the volume to the mark with a blank solution to obtain.
  • the detection limit concentration of each substance is 0.1ng/mL, and the relative standard deviation (RSD) of the content meets the acceptable standard.
  • the pH is 4.5 to 5.5.
  • the detection limit concentration of each substance is 0.1ng/mL, and the relative standard deviation (RSD) of the content meets the acceptable standard.
  • Example 4-5 According to the genotoxicity test method in Example 1-2, the samples in Example 4-5 were tested, and the results showed that the lyophilized preparation samples of Example 4-5 did not have genotoxicity.
  • the preparation process of ornidazole injection mixing the above amounts of ornidazole, ethanol, propylene glycol and water for injection to prepare ornidazole injection.
  • the content of the compound of formula (II) is less than 60 mg/kg, and the content of the compound of formula (III) is less than 2100 mg/kg.
  • Example 7 According to the genotoxicity test method in Example 1-2, the samples in Example 7 were tested, and the results showed that the injection of Example 7 did not have genotoxicity.

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Abstract

一种药物组合物，其包含奥硝唑类化合物、含量在1000mg/kg以下的1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑，以及含量在2100mg/kg以下的1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑。药物组合物中存在的1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑和1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑具有基因毒性，导致细胞DNA损伤，以及产生致突变作用，对此类药物在有效期内的用药安全性和/或稳定性产生极大的挑战。这样的用药风险在上述药物组合物中得到控制甚至消除。

Description

奥硝唑类药物组合物及其制备方法和用途
本申请要求以下2件在先申请的优先权：2019年9月29日向中国国家知识产权局提交的专利申请号为201910936095.0，发明名称为“包含奥硝唑类化合物的药物组合物及其制备方法和用途”的在先申请，以及2019年9月29日向中国国家知识产权局提交的专利申请号为201910936104.6，发明名称为“安全性高的奥硝唑类药物组合物及其制备方法和用途”的在先申请。所述在先申请的全文均通过引用的方式结合于本申请中。
技术领域
本发明属于医药领域，具体涉及奥硝唑类药物组合物及其制备方法和用途。
背景技术
奥硝唑为硝基咪唑类衍生物，是一种强力抗厌氧菌及抗原虫感染的药物，也是继甲硝唑后新研制的疗效更高、疗程更短、耐受性更好、体内分布更广的第三代硝基咪唑类衍生物。奥硝唑的抗微生物作用是通过其分子中的硝基在无氧环境中还原成氨基，或通过自由基的形成与细胞成分相互作用从而导致微生物的死亡。奥硝唑为1-(3-氯-2-羟丙基)-2-甲基-5-硝基咪唑(CAS No.16773-42-5)，具有下式所示的化学结构：
Figure PCTCN2020118543-appb-000001
左旋奥硝唑(又称“左奥硝唑”，CAS No.166734-83-4)是奥硝唑的左旋异构体，临床上主要用于治疗由脆弱拟杆菌、狄氏拟杆菌、卵园拟杆菌、多形拟杆菌、普通拟杆菌、梭状芽胞杆菌、真杆菌、消化球菌和消化链球菌、幽门螺杆菌、黑色素拟杆菌、梭杆菌、CO 2噬织维菌、牙龈类杆菌等厌氧菌所引起的多种感染性疾病，或者用于术前预防此类感染。左旋奥硝唑具有下式所示的化学结构：
Figure PCTCN2020118543-appb-000002
已有研究表明，相对于奥硝唑的右旋异构体或其消旋体，左旋奥硝唑具有较低的神经毒性，为此其用药安全性得到显著改善。此外，现已开发出左旋奥硝唑的前体药物，包括左旋奥硝唑的磷酸酯(又称为磷酸左旋奥硝唑酯)或其盐(如磷酸左旋奥硝唑酯二钠)等。此类前体药物给药后，可在体内磷酸脂酶作用下迅速降解为左旋奥硝唑进而发挥药效。
目前，奥硝唑、其立体异构体及其前体药物，尤其是奥硝唑、左旋奥硝唑及其前体药物 在有效期内的用药安全性和/或稳定性等特性，仍受到药物研究人员广泛关注，并且需要继续深入研究的方向。
发明内容
为改善上述技术问题，本发明提供一种药物组合物，包含奥硝唑类化合物、含量在1000mg/kg以下的1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑，以及含量在2100mg/kg以下的1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑。
根据本发明的实施方案，所述奥硝唑类化合物可以选自奥硝唑、其立体异构体或它们的前体化合物。作为实例，所述奥硝唑类化合物可以选自奥硝唑、左旋奥硝唑(又称“左旋奥硝唑”)或右旋奥硝唑(又称“右奥硝唑”)或它们的前体化合物。
根据本发明的实施方案，所述奥硝唑具有下式(I-1)所示的化学结构：
Figure PCTCN2020118543-appb-000003
左旋奥硝唑具有如下式(I-2)所示的结构：
Figure PCTCN2020118543-appb-000004
右旋奥硝唑具有如下式(I-3)所示的结构：
Figure PCTCN2020118543-appb-000005
根据本发明的实施方案，所述奥硝唑、左旋奥硝唑或右旋奥硝唑的前体化合物可以选自其药学上可接受的前体化合物，例如选自它们任一种的酯或所述酯药学上可接受的盐，以及它们的水合物，例如可以选自它们任一种的氨基酸酯、磷酸酯、磷酸酯的氨基酸盐、以及磷酸酯与碱金属或碱土金属离子形成的盐或它们的水合物中的至少一种；例如，磷酸酯的钠盐、钾盐、钙盐、镁盐等或它们的水合物中的至少一种，示例性的实例为磷酸酯二钠盐或其水合物。
根据本发明的实施方案，左旋奥硝唑的前体化合物可以选自左旋奥硝唑氨基酸酯、磷酸左旋奥硝唑酯、磷酸左旋奥硝唑酯氨基酸盐、以及磷酸左旋奥硝唑酯与碱金属或碱土金属离子形成的或它们的水合物盐中的至少一种；例如，磷酸左旋奥硝唑酯的钠盐、钾盐、钙盐、镁盐等或它们的水合物中的至少一种，示例性为磷酸左旋奥硝唑酯二钠盐(CAS No.909133-95-5)或其水合物。
根据本发明的实施方案，所述奥硝唑、左旋奥硝唑或右旋奥硝唑的前体化合物可以其无定型或多晶型物的形式存在于药物组合物中。或者作为选择，奥硝唑、左旋奥硝唑或右旋奥 硝唑的前体化合物还可以选自它们任一种的酯的药学上可接受的盐的溶剂合物，例如其水合物，例如所述酯药学上可接受的盐的1、2、3、4、5、6或7水合物中的至少一种，其实例可以选自磷酸左旋奥硝唑酯二钠盐的水合物，如其5水合物、6水合物、7水合物中的至少一种。
根据本发明的实施方案，1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑具有如下式(II)所示的结构：
Figure PCTCN2020118543-appb-000006
根据本发明的实施方案，所述药物组合物中，1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑的含量在900mg/kg以下，例如800mg/kg以下、700mg/kg以下、600mg/kg以下、500mg/kg以下、400mg/kg以下、300mg/kg以下、200mg/kg以下，优选100mg/kg以下。优选地，1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑的含量在90mg/kg以下，例如80mg/kg以下，70mg/kg以下，更优选60mg/kg以下、50mg/kg以下、40mg/kg以下、30mg/kg以下、20mg/kg以下或10mg/kg以下。
根据本发明优选的实施方案，所述药物组合物中，1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑的含量在9mg/kg以下，例如8mg/kg以下、7mg/kg以下，更优选6mg/kg以下，其实例可以为5mg/kg以下、4mg/kg以下、3mg/kg以下、2mg/kg以下或1mg/kg以下。
根据本发明更优选的实施方案，所述药物组合物中，1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑的含量在0.9mg/kg以下，例如0.8mg/kg以下、0.7mg/kg以下，更优选0.6mg/kg以下，其实例可以为0.5mg/kg以下、0.4mg/kg以下、0.3mg/kg以下、0.2mg/kg以下或0.1mg/kg以下。
根据本发明的实施方案，所述药物组合物中，1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑的含量可以为0，其意指所述药物组合物中不含有1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑。在本发明药物组合物的一些实施方案中，1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑的含量大于0，其意指所述药物组合物中含有1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑。
根据本发明的实施方案，1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑具有如下式(III)所示的结构：
Figure PCTCN2020118543-appb-000007
根据本发明的实施方案，所述药物组合物中，1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑的含量在2100mg/kg以下，例如2000mg/kg以下，1900mg/kg以下，1800mg/kg以下，1700mg/kg以下，1600mg/kg以下，1500mg/kg以下，1400mg/kg以下，1300mg/kg以下，1200mg/kg以下，1100mg/kg以下。
根据本发明的实施方案，所述药物组合物中，1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑的含量在1000mg/kg以下，例如900mg/kg以下、800mg/kg以下、700mg/kg以下、600mg/kg以下、500mg/kg以下、400mg/kg以下、300mg/kg以下、200mg/kg以下或100mg/kg以下。
根据本发明优选的实施方案，所述药物组合物中，式(III)所示化合物的含量在90mg/kg 以下，例如80mg/kg以下、70mg/kg以下，更优选60mg/kg以下，其实例可以为50mg/kg以下、40mg/kg以下、30mg/kg以下、20mg/kg以下或10mg/kg以下。更优选地，所述药物组合物中，式(III)所示化合物的含量在9mg/kg以下，例如8mg/kg以下、7mg/kg以下，更优选6mg/kg以下，其实例可以为5mg/kg以下、4mg/kg以下、3mg/kg以下、2mg/kg以下、1mg/kg以下或0。其中，式(III)所示化合物的含量为0时，意指所述药物组合物中不含有式(III)所示化合物。或者，所述药物组合物中式(III)所示化合物的含量也可以>0。
根据本发明的实施方案，以左旋奥硝唑、磷酸左旋奥硝唑酯二钠或它们任一种的水合物计，所述药物组合物中，作为活性成分的奥硝唑类化合物的重量百分比含量可以在10％至99％的范围内，优选为15％至90％，例如20％至85％，其实例可以为例如72％至83％的范围内，其实例可以为72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％。
根据本发明的实施方案，所述药物组合物中还可以任选地包含或不包含下式(IV)所示的化合物：
Figure PCTCN2020118543-appb-000008
根据本发明的实施方案，所述药物组合物中，式(IV)所示化合物的含量可以为15000mg/kg以下，例如14000mg/kg以下、13000mg/kg以下、12000mg/kg以下、11000mg/kg以下、10000mg/kg以下、8000mg/kg、7000mg/kg以下、6000mg/kg以下、5000mg/kg以下、4000mg/kg以下、3000mg/kg以下、2000mg/kg以下。优选地，式(IV)所示化合物的含量可以为1000mg/kg以下，例如900mg/kg以下、800mg/kg以下、700mg/kg以下、600mg/kg以下、500mg/kg以下、400mg/kg以下、300mg/kg以下、200mg/kg以下或100mg/kg以下。
根据本发明优选的实施方案，所述药物组合物中，式(IV)所示化合物的含量在90mg/kg以下，例如80mg/kg以下、70mg/kg以下，更优选60mg/kg以下，其实例可以为50mg/kg以下、40mg/kg以下、30mg/kg以下、20mg/kg以下、10mg/kg以下或为0。其中，所述药物组合物中式(IV)所示化合物的含量为0时，意指所述药物组合物中不含有式(IV)所示化合物。或者，所述药物组合物中式(IV)所示化合物的含量也可以>0。
根据本发明的实施方案，所述药物组合物中还可以任选地包含或不包含2-甲基-5-硝基咪唑。
根据本发明的实施方案，所述药物组合物中，2-甲基-5-硝基咪唑的含量在2000mg/kg以下，例如1900mg/kg以下、1800mg/kg以下、1700mg/kg以下、1600mg/kg以下、1500mg/kg以下，优选1400mg/kg以下、1300mg/kg以下、1200mg/kg以下、1100mg/kg以下、1000mg/kg以下，例如900mg/kg以下、800mg/kg以下、700mg/kg以下、600mg/kg以下、500mg/kg以下、400mg/kg以下、300mg/kg以下、200mg/kg以下或100mg/kg以下。
根据本发明优选的实施方案，所述药物组合物中，2-甲基-5-硝基咪唑的含量在90mg/kg以下，例如80mg/kg以下、70mg/kg以下，更优选60mg/kg以下，其实例可以为50mg/kg以下、40mg/kg以下、30mg/kg以下、20mg/kg以下、10mg/kg以下或0。其中，所述药物组合 物中2-甲基-5-硝基咪唑的含量为0时，意指所述药物组合物中不含有2-甲基-5-硝基咪唑。
根据本发明示例性的实施方案，当所述药物组合物中包含左旋奥硝唑的前体化合物时，其可以包含或不包含左旋奥硝唑，优选地，其中所述左旋奥硝唑的含量在2000mg/kg以下，例如80mg/kg以下、70mg/kg以下，更优选60mg/kg以下，其实例可以为50mg/kg以下、40mg/kg以下、30mg/kg以下、20mg/kg以下或10mg/kg以下或0。其中，所述药物组合物中左旋奥硝唑的含量为0时，意指所述药物组合物中不包含左旋奥硝唑。
根据本发明的实施方案，所述药物组合物中还可以包含药学上可接受的辅料，如载体或赋形剂。所述药学上可接受的辅料优选为对活性成分无化学反应性或呈惰性的。例如，所述药学上可接受的辅料选自包括但不限于下列中的至少一种：填充剂、崩解剂、粘合剂、润滑剂、表面活性剂、矫味剂、湿润剂、pH调节剂、增溶剂或助溶剂、渗透压调节剂等。
根据本发明的技术方案，所述填充剂可以选自乳糖、蔗糖、葡萄糖、甘露醇、山梨醇、硫酸钙、葡萄糖酸钙、磷酸氢钙、磷酸钙、碳酸钙、碳酸氢钙、淀粉、羧甲基淀粉、预胶化淀粉和微晶纤维素等中的至少一种。
根据本发明的技术方案，所述崩解剂可以选自预胶化淀粉、微晶纤维素、海藻酸、木质纤维素、羧甲基淀粉钠、瓜耳树胶、交联聚乙烯吡咯烷酮和交联羧甲基纤维素钠等中的至少一种。
根据本发明的技术方案，所述粘合剂可以选自明胶、糊精、麦牙糖糊精、蔗糖、阿拉伯胶、聚乙烯吡咯烷酮、甲基纤维素、羧甲基纤维素、乙基纤维素、聚乙烯醇、聚乙二醇和羟丙甲纤维素等中的至少一种。
根据本发明的技术方案，所述润滑剂可以选自硬脂酸镁、硬脂酸钙、硬脂酸锌、滑石粉、单硬脂酸甘油醋、聚乙二醇4000、聚乙二醇6000、聚乙二醇8000、苯甲酸钠、己二酸、富马酸、硼酸、氯化钠、油酸钠、三醋酸甘油醋、聚氧乙烯单硬脂酸醋、单月桂蔗糖酸醋、氯化钠、月桂醇硫酸钠和月桂醇硫酸镁等中的至少一种。
根据本发明的技术方案，所述表面活性剂可以选自十二烷基硫酸钠、泊洛沙姆、聚山梨酯80、溴化十六烷三甲胺、月桂醇硫酸钠、硬脂酸磺酸钠、聚氧乙烯高级脂肪醇、蔗糖酯、山梨醇脂肪酯和大豆磷脂等中的至少一种。
根据本发明的技术方案，所述矫味剂为甜菊素、果糖、葡萄糖、果葡糖浆、蜂蜜、阿斯巴甜、蛋白糖、木糖醇、甘露醇、乳糖、山梨醇、香精和麦芽糖醇等中的至少一种。
根据本发明的技术方案，所述pH调节剂可以选自盐酸、硫酸、磷酸、枸橼酸、氢氧化钠、氢氧化钾、碳酸钠、碳酸钾、三乙胺等中的至少一种。
根据本发明的技术方案，所述助溶剂可以选自醇类溶剂，例如选自乙醇、甘油、丙二醇、聚乙二醇(如聚乙二醇300、聚乙二醇400等)等中的至少一种。
根据本发明的技术方案，所述渗透压调节剂可以选自氯化钠、葡萄糖、果糖、磷酸盐、聚乙二醇、丙二醇、甘露醇等中的至少一种。
根据本发明的技术方案，所述药物组合物可以为制剂，例如为胃肠道给药制剂或非胃肠道给药制剂。其中，所述胃肠道给药制剂可以为片剂、分散片、胶囊剂、缓释剂、颗粒剂、口服液、糖浆剂等；所述非胃肠道给药制剂可以为输液制剂、注射剂(如液体注射剂)、冻干制剂(如冻干粉剂)、泡腾片、栓剂、舌下片剂等，优选为胶囊剂、注射剂(如液体注射 剂)或注射用冻干制剂(如冻干粉剂)。
根据本发明示例性的实施方案，所述药物组合物为注射剂或注射用冻干制剂，其中包含奥硝唑类化合物，优选包含奥硝唑、磷酸左旋奥硝唑酯二钠或它们任一种的水合物，以及含量在60mg/kg以下的1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑。
根据本发明的实施方案，所述注射液或注射用冻干制剂中，以奥硝唑或其异构体计，作为活性成分的奥硝唑类化合物的重量百分比含量在70％至85％的范围内，例如71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、82％、83％、84％、85％，优选在72％至83％范围内。
根据本发明的实施方案，所述药物组合物，例如注射用冻干制剂中可以含有枸橼酸，所述枸橼酸的重量百分比含量可以为10-35％，优选15-32％，例如18-28％、20-25％，示例性为16％、17％、、18％、19％、21％、22％、23％、24％、26％、27％、29％。
根据本发明的实施方案，所述注射剂的pH可以为4.0-6.0，优选4.5-5.5，例如4.6-5.4、4.7-5.3，示例性为4.8、4.9、5.0、5.1、5.2。
根据本发明的实施方案，所述注射液的pH为4.0-6.0时，枸橼酸的含量可以为19-45mg/mL。例如，pH＝4.0时，枸橼酸的含量为45mg/mL；pH＝6.0时，枸橼酸的含量为19mg/mL。
根据本发明的实施方案，所述注射液中可以包含注射用水、乙醇和/或丙二醇。
根据本发明的技术方案，所述药物组合物的给药途径包括但不限于胃肠道给药或非胃肠道给药；其中，所述非胃肠道给药可以为注射给药(如静脉注射给药、动脉注射给药、肌内注射给药、皮下注射给药、皮内注射给药等)、阴道给药或粘膜给药等。
根据本发明的实施方案，所述药物组合物的活性成分为奥硝唑类化合物，优选奥硝唑、左旋奥硝唑或左旋奥硝唑的前体化合物。优选地，所述药物组合物可以不包含除奥硝唑类化合物之外的其他活性成分，或者也可以包含除奥硝唑、左旋奥硝唑或左旋奥硝唑的前体化合物之外的其他活性成分。
根据本发明示例性的实施方案，所述药物组合物包含或磷酸左旋奥硝唑酯二钠和枸橼酸，以及任选存在或不存在的注射用水。优选地，所述药物组合物的pH为4.5～5.5。
根据本发明示例性的实施方案，所述药物组合物包含奥硝唑、乙醇、丙二醇以及任选存在或不存在的注射用水。
本发明还提供上述药物组合物的制备方法，包括将奥硝唑类化合物与药学上可接受的辅料混合，得到所述药物组合物。
根据本发明示例性的实施方案，将奥硝唑、乙醇、丙二醇和注射用水混合，得到所述药物组合物。
根据本发明示例性的实施方案，将磷酸左旋奥硝唑酯二钠或其水合物与水配成溶液，溶液经冷冻干燥，得到所述药物组合物。
根据本发明的技术方案，所述溶液的pH为4.0-6.0，优选4.5-5.5，例如4.6-5.4、4.7-5.3，示例性为4.8、4.9、5.0、5.1、5.2。
根据本发明的实施方案，所述冷冻干燥的温度为-60℃～30℃，优选-50℃～25℃。
根据本发明的实施方案，所述冷冻干燥的时间为30-70h，例如40-60h，又如45-55h。
根据本发明的实施方案，所述冷冻干燥为程序控温。
根据本发明的技术方案，所述冷冻干燥的示例性操作包括：将灌装半加塞好的半成品在硅油温度降至0℃时送入冻干箱，保持0.5小时，待硅油温度降温至-50℃时，保持1.0h；升温至-20℃，保持3.0h；降温至-50℃，保持3.0h。打开真空泵，抽真空至200μbar，1.0h升温至-20℃，保持16.0h。5min升温至-15℃，保持6.0h；5min升温至-10℃，保持1.0h；10min升温至0℃，保持1.0h；10min升温至10℃，保持1.0h；15min升温至25℃，保持7.0h；在25℃下抽极限真空，继续保持13.0h。进行压力升试验(压力升合格标准≤15μbar/min)，合格后冻干结束，往箱体内充入氮气破除真空(真空控制范围：850mbar-900mbar)，全压塞后出箱。
本发明还提供所述药物组合物用于改善药物安全性的用途。
本发明还提供所述药物组合物用于改善药物稳定性的用途。
本发明还提供所述药物组合物用于制备药品的用途。
优选地，所述药品用于预防或治疗与厌氧菌相关的疾病，如用于预防或治疗厌氧菌导致的感染性疾病。例如，所述厌氧菌可以选自包括但不限于下列中的至少一种：脆弱拟杆菌、狄氏拟杆菌、卵园拟杆菌、多形拟杆菌、普通拟杆菌、梭状芽胞杆菌、真杆菌、消化球菌和消化链球菌、幽门螺杆菌、黑色素拟杆菌、梭杆菌、CO 2噬织维菌、牙龈类杆菌等厌氧菌。
优选地，所述药品用于预防或治疗手术前和/或手术后厌氧菌导致的感染性疾病。
本发明还提供一种预防或治疗与厌氧菌相关的疾病的方法，如预防或治疗厌氧菌导致的感染性疾病的方法，包括将治疗有效量的所述药物组合物给予有需要的患者。
术语定义和说明
除非另有说明，本申请说明书和权利要求书中记载的基团和术语定义，包括其作为实例的定义、示例性的定义、优选的定义、表格中记载的定义、实施例中具体化合物的定义等，可以彼此之间任意组合和结合。这样的组合和结合后的基团定义及化合物结构，应当属于本申请说明书记载的范围内。
术语“有效量”或者“治疗有效量”是指足以实现预期应用(包括但不限于如下定义的疾病治疗)的本发明所述化合物的量。治疗有效量可以取决于以下因素而改变：预期应用(体外或者体内)，或者所治疗的受试者和疾病病症如受试者的重量和年龄、疾病病症的严重性和给药方式等，其可以由本领域普通技术人员容易地确定。具体剂量将取决于以下因素而改变：所选择的特定化合物、所依据的给药方案、是否与其它化合物组合给药、给药的时间安排、所给药的组织和所承载的物理递送系统。
术语“患者”是指需要定预防或治疗与厌氧菌相关的疾病的患者，其中患者为哺乳动物，例如选自啮齿类、牛、猪、狗、猫和灵长类动物，特别是人。
术语“有效期”意指是药品在规定的贮藏条件下质量能够符合规定要求的期限，例如24个月，如18个月或12个月。
有益效果
本申请出人意料地发现，包含奥硝唑类化合物的药物组合物中存在的1-(2,3-环氧丙烷)-2- 甲基-5-硝基咪唑和1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑具有基因毒性，导致细胞DNA损伤，以及产生致突变作用，对此类药物在有效期内的用药安全性和/或稳定性产生极大的挑战。这样的用药风险在本发明的药物组合物中得到控制甚至消除。本申请发现，当将1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑的重量百分比含量降低至1000mg/kg以下，优选100mg/kg以下，更优选60mg/kg以下时；以及当将1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑含量降低至2100mg/kg以下，优选小于2100mg/kg时，此类药物的安全性和/或稳定性能够得到显著改善。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1：式(III)化合物基因毒性实验
(一)Ames实验(菌株实验)
1.材料与方法
1.1供试品
名称：1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑；
包装：安瓿瓶；
规格：约50mg/支；
保存条件：2-8℃、避光密封保存。
1.2溶媒信息
名称：氯化钠注射液；
生产单位：辰欣药业股份有限公司；
性状和理化性质：无色澄明液体，味微咸；
规格：500mL/瓶；
保存条件：密闭保存。
1.3供试品/对照品配制
供试品于给药当天进行配制，称取适量供试品，加入适量氯化钠注射液溶解使其浓度为50mg/mL。将该溶液用0.2μm的滤膜过滤，并用氯化钠注射液将其稀释成浓度分别为20、8、2、0.5mg/mL的溶液。
配制后的溶液在给药前于室温保存。
1.4实验方法
菌株：鼠伤寒沙门氏菌，组氨酸缺陷型菌株(TA97a，TA98，TA100，TA102，TA1535)。菌株由Moltox提供，菌液保存于液氮中。
SD大鼠由昭衍(苏州)新药研究中心有限公司提供。
S9混合液的配制：
本试验使用SD大鼠肝脏S9组分，蛋白浓度为21.64mg/mL，保存在液氮中。使用之前， S9混合液将在无菌条件下配制，S9混合液的配制参照表1。
表1
Figure PCTCN2020118543-appb-000009
菌株增菌培养：将液氮冷冻保存的细菌菌液，37℃水浴速融后，取100μl接种于20ml的营养肉汤内，置37℃避光振荡(120rpm)培养10～12小时。
分组给药：每个测试点处理2个平行皿。给药信息如表2所示。
表2
Figure PCTCN2020118543-appb-000010
注：组1为自发对照组；组2～6为供试品组。
取相应数量的玻璃试管，先在试管中分装2ml顶层培养基(45～47℃加热)，然后依次加入0.1ml细菌培养液、0.1ml供试品溶液、0.5mlS9混合物或pH7.4的PBS。迅速在振荡器上混匀，倒入基础培养基表面，轻轻旋转，使顶层培养基混合液均匀的铺在基础培养基表面。
将平皿置水平桌面上，待培养基凝固后，倒置平皿，37℃培养48-72小时。
1.5数据采集
培养约48～72小时后，计数所有平皿中回复突变菌落数，同时在显微镜下观察背景菌苔评估供试品对菌株是否有抑菌作用。
观察加药时的沉淀情况。
1.6结果判定
根据下述准则判断供试品对菌株是否有抑菌毒性：
1)背景菌苔变薄，同时可能会伴有回复突变菌落数减少；
2)背景菌苔消失，即细菌生长完全被抑制；
3)出现针尖状非回复突变细小菌落(通常伴有背景菌苔缺失)。
2.结果
2.1沉淀情况
各供试品剂量组在加样过程中均未见沉淀产生。
2.2细菌毒性
背景菌苔观察个体数据见下表3。
表3：背景菌苔观察个体数据
Figure PCTCN2020118543-appb-000011
注：“√”表示背景菌苔正常。结果表明：非代谢活化状态及代谢活化状态下，50～5000μg/皿剂量下，各菌株背景菌苔均未见异常。
2.3致突变性
回复突变菌落计数结果见下表4。
表4：回复突变菌落数计数个体数据(个/皿)
Figure PCTCN2020118543-appb-000012
结果表明：在代谢活化和非代谢活化条件下，自发对照组各菌株的回复突变菌落数，均在正常参考范围内或略有增减；非代谢活化状态及代谢活化状态下，50～200μg/皿剂量下，各菌株的回复突变菌落数，均在正常参考范围内或略有增减；除5000μg/皿剂量下，TA1535回复突变菌落数减少外，800～5000μg/皿剂量下，各菌株的回复突变菌落数明显升高，超过了自发对照组的2倍。
3.结论
在本试验条件下，1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑在50～5000μg/皿剂量下未见明显的细菌毒性作用，但在800～5000μg/皿剂量下对鼠伤寒沙门氏菌均有致突变性，为此具有基因毒性。
(二)微核实验
1.供试品和对照品
1.1供试品
名称：1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑；
包装：安瓿瓶；
规格：约50mg/支；
保存条件：2-8℃、避光密封保存。
1.2对照品
注射用环磷酰胺；
生产单位：江苏恒瑞医药股份有限公司；
状况和理化性质：白；色结晶粉末；
批号：19061521；
规格：0.2g/瓶；
保存条件：遮光密封保存。
1.3实验动物
小鼠KM
等级：SPF；
出售单位：河南斯科贝思生物科技股份有限公司；
质量检测单位：山东省实验动物中心；
许可证号：SCXK(豫)2020-0005。
2.给药制剂的配制
配制过程在无菌条件下完成，所使用的试剂瓶等均经过灭菌处理，配制过程未避光。
溶剂(0.9％氯化钠注射液)：
生产单位：安徽双鹤药业有限责任公司；
性状和理化性质：无色澄明液体，味微咸；
批号：20011112C
规格：500mL/瓶；
保存条件：密闭保存。
阳性对照溶液(环磷酰胺)的配制：
称量所需量的阳性对照品(以含量计)，加入适量溶剂搅拌至澄清透明，最后使用溶剂稀释至所需浓度。配制完成后采用0.22μm的滤膜过滤，得阳性对照溶液。
供试品给药制剂配制：
称量所需量的供试品(以含量计)，加入适量溶剂搅拌至澄清透明，最后使用溶剂稀释至所需浓度。配制完成后采用0.22μm的滤膜过滤，得供试品给药制剂。
各组剂量和浓度如下表所示：
Figure PCTCN2020118543-appb-000013
3.实验系统
3.1实验动物
用于试验的动物：昆明种小鼠，清洁级；选用雌性24只、雄性24只。
分组时体重和年龄：雌性：约24～28g，8～10周龄；雄性：约25～29g，8～10周龄。
3.2分组
本试验共设5个组，溶剂组和供试品组每组10只，阳性对照组每组8只，48只。
3.3动物标识
动物接收当天采用3％苦味酸乙醇溶液进行标识，分组后采用苦味酸和笼牌标识，笼牌标识如下表：
Figure PCTCN2020118543-appb-000014
X为雌鼠，Y为雄鼠。
3.4动物随机分组
根据性别及体重进行随机分组，把筛选出来的48只动物分配到5组。用于分组的动物体重差异在同性别平均体重的±20％之内。分组后，每组动物的平均体重，在5.0％的检验水准下，无统计学差异。将分组后剩余动物进行存栏记录，并进行饲养。
3.5试剂量设计
本试验剂量设计见下表：
Figure PCTCN2020118543-appb-000015
4.试验方法
4.1给药
给药途径：选择腹腔注射给药途径。
给药方式：连续3天给药5次，分别为第一天上午给药1次，第二天与第三天上下午分别给药1次。
给药期：3天。
给药容量：10mL/kg。
给药量：以最近一次称量体重计算给药量。
4.2观察和检查
4.2.1死亡或濒死
所有存活动物每天观察1次。
4.2.2体重
适应期：所有动物分组前称重1次，即分组体重
给药期：分别于D1、D2、D3给药前上午称量体重，并用于计算给药量
4.3试验终末程序
4.3.1非计划死亡动物
实验期间，所有动物未见死亡或濒死(死亡或濒死汇总结果见5.1节)。
4.3.2解剖制片
计划解剖日期：给药期末(D4)
解剖动物：所有存活动物
制片方法：采用颈椎脱臼法处死小鼠，取股骨，剔去肌肉，剪去骨骺，暴露骨髓腔，取骨髓涂片，甲醇固定、Gimesa染色。
4.3.3镜检
计划镜检日期：给药期末(D5)
镜检对象：所有动物骨髓涂片(详见4.3.2)
镜检方法：在低倍显微镜下选择细胞完整、分散均匀、着色适当的区域，用高倍镜观察， 每张涂片计数1000个胞质清晰完整的嗜多染红细胞(PCE)的微核率，结果以千分率(‰)表示。
4.4统计分析
应用SPSS16.0统计软件进行分析，微核率均以
Figure PCTCN2020118543-appb-000016
表示，与阴性对照组进行比较，采用双侧t检验及χ 2检验进行统计学处理，P<0.05为差异有统计学意义，P>0.05为差异不显著，0.01<P<0.05为差异显著，P<0.01为差异极显著。
死亡或濒死结果不进行统计，以频数形式表示。
5结果
5.1死亡或濒死
供试品对动物死亡或濒死结果的影响见下表。至给药期末，所有动物均未发现死亡或濒死。
Figure PCTCN2020118543-appb-000017
5.2体重
体重检查结果见下表。
Figure PCTCN2020118543-appb-000018
至给药期末，所有动物体重未见明显异常。
体重改变未见剂量相关性，有增长也有下降，可能与动物个体差异有关，无毒理学意义。
5.3嗜多染红细胞微核率
嗜多染红细胞微核率统计结果见下表。
Figure PCTCN2020118543-appb-000019
Figure PCTCN2020118543-appb-000020
至给药期末，供试品给药组动物呈现出微核率上升趋势。
100mg/kg剂量组微核率相较于阴性对照组差异显著(p<0.05)，其他剂量组虽未见统计学差异，但也可见微核率小幅度增长，可认为与供试品相关。
6结论
在本试验条件下，昆明种小鼠连续3天腹腔注射给予2.88、48、100mg/kg浓度的杂质Ⅲ，试验期间未见动物死亡或濒死，体重未见与供试品相关的明显异常，在≥100mg/kg的剂量下可见嗜多染红细胞微核率上升，认为与供试品相关。本试验未观察到的有害作用的水平(no observed adverse effect level,NOAEL)为48mg/kg。
根据ICH M7(国际人用药品注册技术协调会)指导原则，遗传毒性杂质限值计算如下：
杂质限值＝杂质每日可接受摄入量(即为PDE值)/药物每日最大用量。
PDE＝NOEL×调整体重/(F1×F2×F3×F4×F5)
其中，小鼠NOEL为48mg/kg/天；F1＝12，从小鼠剂量推断人用剂量；F2＝10，参考FDA ICH-Q3C指导原则；F3＝10，实验周期不超过10天；F4＝1，考虑未发现严重毒性反应；F5＝1；调整体重定为50kg；计算结果如下：
PDE＝48mg/kg(NOEL)×50/(12×10×10×1×1)＝2mg；
杂质限度＝2mg/2g(根据临床试验等确定人体该类化合物每日最大用量为2g)＝1000ppm＝1000mg/kg。
实施例2：1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑(式(II)化合物)的基因毒性实验
(一)Ames实验(菌株实验)
1.材料与方法
1.1供试品
名称：1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑；
包装：安瓿瓶；
规格：约50mg/支；
保存条件：2-8℃、避光密封保存。
1.2溶媒信息
名称：氯化钠注射液；
生产单位：辰欣药业股份有限公司；
性状和理化性质：无色澄明液体，味微咸；
规格：500mL/瓶；
保存条件：密闭保存。
1.3供试品/对照品配制
供试品于给药当天进行配制，称取适量供试品，加入适量氯化钠注射液溶解使其浓度为50mg/mL。将该溶液用0.2μm的滤膜过滤，并用氯化钠注射液将其稀释成浓度分别为20、8、2、0.5mg/mL的溶液。
配制后的溶液在给药前于室温保存。
1.4实验方法
菌株：鼠伤寒沙门氏菌，组氨酸缺陷型菌株(TA97a，TA98，TA100，TA102，TA1535)。菌株由Moltox提供，菌液保存于液氮中。
SD大鼠由昭衍(苏州)新药研究中心有限公司提供。
S9混合液的配制：
本试验使用SD大鼠肝脏S9组分，蛋白浓度为21.64mg/mL，保存在液氮中。使用之前，S9混合液将在无菌条件下配制，S9混合液的配制参照表5。
表5
Figure PCTCN2020118543-appb-000021
菌株增菌培养：将液氮冷冻保存的细菌菌液，37℃水浴速融后，取100μl接种于20ml的营养肉汤内，置37℃避光振荡(120rpm)培养10～12小时。
分组给药：每个测试点处理2个平行皿。给药信息如表6所示。
表6
Figure PCTCN2020118543-appb-000022
注：组1为自发对照组；组2～6为供试品组。
取相应数量的玻璃试管，先在试管中分装2ml顶层培养基(45～47℃加热)，然后依次加入0.1ml细菌培养液、0.1ml供试品溶液、0.5ml S9混合物或pH7.4的PBS。迅速在振荡器上混匀，倒入基础培养基表面，轻轻旋转，使顶层培养基混合液均匀的铺在基础培养基表面。
将平皿置水平桌面上，待培养基凝固后，倒置平皿，37℃培养48-72小时。
1.5数据采集
培养约48～72小时后，计数所有平皿中回复突变菌落数，同时在显微镜下观察背景菌苔评估供试品对菌株是否有抑菌作用。
观察加药时的沉淀情况。
1.6结果判定
根据下述准则判断供试品对菌株是否有抑菌毒性：
1)背景菌苔变薄，同时可能会伴有回复突变菌落数减少；
2)背景菌苔消失，即细菌生长完全被抑制；
3)出现针尖状非回复突变细小菌落(通常伴有背景菌苔缺失)。
2.结果
2.1沉淀情况
各供试品剂量组在加样过程中均未见沉淀产生。
2.2细菌毒性
背景菌苔观察个体数据见下表7。
表7：背景菌苔观察个体数据
Figure PCTCN2020118543-appb-000023
注：“√”表示背景菌苔正常；“-”表示背景菌苔消失，×表示背景菌苔减少。
结果表明：非代谢活化状态下，800～5000μg/皿剂量下，各菌株背景菌苔消失，50、200μg/皿剂量下，各菌株背景菌苔减少。代谢活化状态下，200～5000μg/皿剂量下，各菌株背景菌苔消失，50μg/皿剂量下，各菌株背景菌苔减少。
2.3致突变性
回复突变菌落计数结果见下表8。
表8：回复突变菌落数计数个体数据(个/皿)
Figure PCTCN2020118543-appb-000024
Figure PCTCN2020118543-appb-000025
结果表明：在代谢活化和非代谢活化条件下，自发对照组各菌株的回复突变菌落数，均在正常参考范围内或略有增减；非代谢活化状态50、200μg/皿剂量及代谢活化状态50μg/皿剂量下，各菌株的回复突变菌落数明显升高，超过了自发对照组的2倍。
3.结论
在本试验条件下，1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑在50～5000μg/皿剂量下具有细菌毒性作用，对鼠伤寒沙门氏菌均有致突变性，为此具有基因毒性。
(二)微核实验
1.供试品和对照品
1.1供试品
名称：1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑；
包装：安瓿瓶；
规格：约50mg/支；
保存条件：2-8℃、避光密封保存。
1.2对照品
注射用环磷酰胺；
生产单位：江苏恒瑞医药股份有限公司；
状况和理化性质：白；色结晶粉末；
批号：19061521；
规格：0.2g/瓶；
保存条件：遮光密封保存。
1.3实验动物
小鼠KM
等级：SPF；
出售单位：河南斯科贝思生物科技股份有限公司；
质量检测单位：山东省实验动物中心；
许可证号：SCXK(豫)2020-0005。
2.给药制剂的配制
配制过程在无菌条件下完成，所使用的试剂瓶等均经过灭菌处理，配制过程未避光。
溶剂(0.9％氯化钠注射液)：
生产单位：安徽双鹤药业有限责任公司；
性状和理化性质：无色澄明液体，味微咸；
批号：20011112C
规格：500mL/瓶；
保存条件：密闭保存。
阳性对照溶液(环磷酰胺)的配制：
称量所需量的阳性对照品(以含量计)，加入适量溶剂搅拌至澄清透明，最后使用溶剂稀释至所需浓度。配制完成后采用0.22μm的滤膜过滤，得阳性对照溶液。
供试品给药制剂配制：
称量所需量的供试品(以含量计)，加入适量溶剂搅拌至澄清透明，最后使用溶剂稀释至所需浓度。配制完成后采用0.22μm的滤膜过滤，得供试品给药制剂。
各组剂量和浓度如下表所示：
Figure PCTCN2020118543-appb-000026
3.实验系统
3.1实验动物
用于试验的动物：昆明种小鼠，清洁级；选用雌性24只、雄性24只。
分组时体重和年龄：雌性：约24～28g，8～10周龄；雄性：约25～29g，8～10周龄。
3.2分组
本试验共设5个组，溶剂组和供试品组每组10只，阳性对照组每组8只，48只。
3.3动物标识
动物接收当天采用3％苦味酸乙醇溶液进行标识，分组后采用苦味酸和笼牌标识，笼牌标识如下表：
Figure PCTCN2020118543-appb-000027
X为雌鼠，Y为雄鼠。
3.4动物随机分组
根据性别及体重进行随机分组，把筛选出来的48只动物分配到5组。用于分组的动物体重差异在同性别平均体重的±20％之内。分组后，每组动物的平均体重，在5.0％的检验水准下，无统计学差异。将分组后剩余动物进行存栏记录，并进行饲养。
3.5试剂量设计
本试验剂量设计见下表：
Figure PCTCN2020118543-appb-000028
4.试验方法
4.1给药
给药途径：选择腹腔注射给药途径。
给药方式：连续3天给药5次，分别为第一天上午给药1次，第二天与第三天上下午分别给药1次。
给药期：3天。
给药容量：10mL/kg。
给药量：以最近一次称量体重计算给药量。
4.2观察和检查
4.2.1死亡或濒死
所有存活动物每天观察1次。
4.2.2体重
适应期：所有动物分组前称重1次，即分组体重
给药期：分别于D1、D2、D3给药前上午称量体重，并用于计算给药量。
4.3试验终末程序
4.3.1非计划死亡动物
实验期间，所有动物未见死亡或濒死(死亡或濒死汇总结果见5.1节)。
4.3.2解剖制片
计划解剖日期：给药期末(D4)
解剖动物：所有存活动物
制片方法：采用颈椎脱臼法处死小鼠，取股骨，剔去肌肉，剪去骨骺，暴露骨髓腔，取骨髓涂片，甲醇固定、Gimesa染色。
4.3.3镜检
计划镜检日期：给药期末(D5)
镜检对象：所有动物骨髓涂片(详见4.3.2)
镜检方法：在低倍显微镜下选择细胞完整、分散均匀、着色适当的区域，用高倍镜观察，每张涂片计数1000个胞质清晰完整的嗜多染红细胞(PCE)的微核率，结果以千分率(‰)表示。
4.4统计分析
应用SPSS16.0统计软件进行分析，微核率均以
Figure PCTCN2020118543-appb-000029
表示，与阴性对照组进行比较，采用双侧t检验及χ 2检验进行统计学处理，P<0.05为差异有统计学意义，P>0.05为差异不显著，0.01<P<0.05为差异显著，P<0.01为差异极显著。
死亡或濒死结果不进行统计，以频数形式表示。
5结果
5.1死亡或濒死
供试品对动物死亡或濒死结果的影响见下表。至给药期末，所有动物均未发现死亡或濒死。
Figure PCTCN2020118543-appb-000030
5.2体重
体重检查结果见下表。
Figure PCTCN2020118543-appb-000031
实验期间，供试品给药组动物呈现出体重下降趋势。
100mg/kg剂量组动物D3时体重出现下降趋势，且呈现出剂量相关性，其他动物体重变化虽未见统计学差异，但也可见体重增长较小，且呈现出剂量相关性，可认为与供试品相关。
5.3嗜多染红细胞微核率
嗜多染红细胞微核率统计结果见下表。
Figure PCTCN2020118543-appb-000032
实验期间，供试品给药组动物呈现出微核率上升趋势。
48mg/kg剂量组与100mg/kg剂量组微核率相较于阴性对照组差异极其显著(p<0.01)，其他剂量组虽未见统计学差异，但也可见微核率小幅度增长，呈现出剂量相关性，可认为与供试品相关。
6结论
在本试验条件下，昆明种小鼠连续3天腹腔注射给予2.88、48、100mg/kg浓度的供试品化合物，试验期间未见动物死亡或濒死，供试品给药组动物在≥100mg/kg的剂量下可见体重下降，≥48mg/kg的剂量下可见嗜多染红细胞微核率上升，认为与供试品相关。本试验未观察到的有害作用的水平(no observed adverse effect level,NOAEL)为2.88mg/kg。
根据ICH M7(国际人用药品注册技术协调会)指导原则，遗传毒性杂质限值计算如下：
杂质限值＝杂质每日可接受摄入量(即为PDE值)/药物每日最大用量。
PDE＝NOEL×调整体重/(F1×F2×F3×F4×F5)
其中，小鼠NOEL为2.88mg/kg/天；F1＝12，从小鼠剂量推断人用剂量；F2＝10，参考FDA ICH-Q3C指导原则；F3＝10，实验周期不超过10天；F4＝1，考虑未发现严重毒性反应；F5＝1；调整体重定为50kg；计算结果如下：
PDE＝2.88mg/kg(NOEL)×50/(12×10×10×1×1)＝0.12mg；
杂质限度＝0.12mg/2g(根据临床试验等确定人体该类化合物每日最大用量为2g)＝60ppm＝60mg/kg。
实施例3式(IV)化合物的基因毒性实验
采用实施例1的方法，不同之处在于将供试品替换为式(IV)化合物，结果表明该化合物在50、200、800、2000、5000μg/皿的剂量下均不呈现基因毒性。
实施例4注射用冻干制剂
注射用冻干制剂的处方如下：
磷酸左旋奥硝唑酯二钠   6000g
枸橼酸         1140-2700g
加注射用水至   60L
配制成30000支。
该处方的制备方法包括下列步骤：
将一部分枸橼酸加入到处方总体积80％的20℃以下注射用水中，搅拌至完全溶解后，再加入处方量的磷酸左旋奥硝唑酯二钠，搅拌至完全溶解，配制液pH为4.0～6.0。而后取剩余的枸橼酸加冷却好的注射用水配成浓度为20g/100mL的溶液来调节配制液pH至4.5～5.5。根据药液密度(1.063g/ml)补加注射用水(20℃以下)至处方总量。继续搅拌约15min，将配制好的药液经型号为A05NF2PH4以及KA3NFP1的过滤器(0.2μm/0.22μm；膜材为尼龙66)进行过滤。
将过滤后的药液在硅油温度降至0℃时送入冻干箱，保持0.5小时，待硅油温度降温至-50℃时，保持1.0h；升温至-20℃，保持3.0h；降温至-50℃，保持3.0h。打开真空泵，抽真空至200μbar，1.0h升温至-20℃，保持16.0h。5min升温至-15℃，保持6.0h；5min升温至-10℃，保持1.0h；10min升温至0℃，保持1.0h；10min升温至10℃，保持1.0h；15min升温至25℃，保持7.0h；在25℃下抽极限真空，继续保持13.0h。进行压力升试验(压力升合格标准≤15μbar/min)，合格后冻干结束，往箱体内充入氮气破除真空(真空控制范围：850mbar～900mbar)，得到冻干制剂。
重复上述制备方法，共生产得到3个批次的成品，记为S1、S2、S3，即时检验结果如表9所示。
表9成品检验结果
Figure PCTCN2020118543-appb-000033
对S1、S2、S3成品的加速稳定性进行检测(试验条件：温度25℃±2℃，湿度60％RH±5％RH)，检验结果如表10-12所示。
表10 S1成品加速稳定性检验结果
Figure PCTCN2020118543-appb-000034
表11 S2成品加速稳定性检验结果
Figure PCTCN2020118543-appb-000035
表12 S3成品加速稳定性检验结果
Figure PCTCN2020118543-appb-000036
Figure PCTCN2020118543-appb-000037
对S1、S2、S3成品的长期稳定性进行检测(长期稳定性测试条件：5℃±3℃)，检验结果如表13-15所示。
表13 S1成品长期稳定性检验结果
Figure PCTCN2020118543-appb-000038
表14 S2成品长期稳定性检验结果
Figure PCTCN2020118543-appb-000039
表15 S3成品长期稳定性检验结果
Figure PCTCN2020118543-appb-000040
实验结果说明，冻干制剂具有良好的用药安全性和稳定性。
各物质的检测方法如下：
仪器：液相色谱串联质谱联用仪，型号1290LC-6470LC/TQ，安捷伦科技有限公司。
(1)色谱条件
色谱柱：Svea Core Shell C18,(100mm×4.6mm×2.6μm)
流动相：A:(10mmol甲酸铵水溶液)B:(甲醇)
流速：0.6mL/min
进样体积：5μL
梯度程序：
时间(min) 流动相A(体积％) 流动相B(体积％)
0.00 90 10
0.20 90 10
1.00 80 20
2.50 80 20
4.00 70 30
5.50 70 30
6.00 10 90
8.00 10 90
8.01 90 10
10.00 90 10
(2)质谱条件
离子源类型：AJS ESI离子源
碰撞气(N 2)流量：8L/min
鞘气(N 2)流量：11L/min
鞘气温度：350℃
喷雾电压：3500V
喷嘴电压：500V
EMV增益：200V
采集方式：多反应离子监测(MRM)。
(3)空白溶液配制
空白溶液：即纯水。
(4)对照品溶液：精密称取2-甲基-5-硝基咪唑、式(II)化合物、式(III)化合物对照品10mg分别于10mL容量瓶中，用空白溶液定容至刻度，作为对照品储备液。从中各精密量取0.1mL对照品储备液于10mL容量瓶中，用空白溶液定容至刻度，作为对照品中间溶液(即10μg/mL)。从中精密量取5mL于50mL容量瓶中，用空白溶液定容至刻度，作为对照品工作溶液(即1μg/mL)。从中精密量取0.6mL于10mL容量瓶中，用空白溶液定容至刻度，即得60ng/mL对照品溶液。
(5)系统适用性溶液：精密称取供试品10mg(以磷酸左旋奥硝唑酯二钠计)于10mL容量瓶中，空白溶液定容至刻度。取该溶液1mL于10mL容量瓶中，加入限度点浓度(取(4)项下“对照品工作溶液”0.6mL于上述容量瓶中)的式(II)化合物(60mg/kg)、式(III)化合物(60mg/kg)、2-甲基-5-硝基咪唑(60mg/kg)对照品，空白溶液定容至刻度(即60ng/mL)，即得。
(6)供试品溶液的配制：精密称取供试品10mg(以磷酸左旋奥硝唑酯二钠计)于10mL容量瓶中，用空白溶液定容至刻度，即得。
(7)加杂供试品溶液的配制：精密称取供试品10mg(以磷酸左旋奥硝唑酯二钠计)于10mL容量瓶中，加入限度点浓度(取“3.4”项下“对照品工作溶液”0.6mL于上述容量瓶中)的式(II)化合物(60mg/kg)、式(III)化合物(60mg/kg)、2-甲基-5-硝基咪唑(60mg/kg)对照品，空白溶液定容至刻度(即60ng/mL)，即得。
(8)限度：本品中式(II)化合物、式(III)化合物、2-甲基-5-硝基咪唑对照品残留量均不超过60mg/kg。
上表实验结果中，各物质检测限浓度为0.1ng/mL，含量相对标准偏差(RSD)符合可接受标准。
实施例5注射用冻干制剂
单支注射用冻干制剂的处方：
磷酸左旋奥硝唑酯二钠   200mg
枸橼酸              38-90mg
加注射用水至        2mL
pH为4.5～5.5。
参照实施例4中的冻干制备工艺和检测方法，制备得到12批次冻干制剂。
(1)对冻干制剂中各物质的含量进行即时检测，检测结果如表16所示。
表16样品检测结果
Figure PCTCN2020118543-appb-000041
Figure PCTCN2020118543-appb-000042
上表实验结果中，各物质检测限浓度为0.1ng/mL，含量相对标准偏差(RSD)符合可接受标准。
上述12批冻干制剂的其它指标均满足表9所示的成品要求。
实施例6基因毒性测试
按照实施例1-2中的基因毒性试验方法，将实施例4-5中的样品进行测试，结果表明：实施例4-5冻干制剂样品不具有基因毒性。
实施例7奥硝唑注射液
奥硝唑注射液的处方如下：
奥硝唑   500mg
乙醇     0.9g
丙二醇   1mL
加注射用水至3mL。
奥硝唑注射液的制备过程：将上述用量的奥硝唑、乙醇、丙二醇和注射用水混合，制备得到奥硝唑注射液。按照实施例4中的物质检测方法，奥硝唑注射液中，式(II)化合物的含量小于60mg/kg，式(III)化合物的含量小于2100mg/kg。
按照实施例1-2中的基因毒性试验方法，将实施例7中的样品进行测试，结果表明：实施例7注射液不具有基因毒性。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种药物组合物，其包含奥硝唑类化合物、含量在1000mg/kg以下的1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑，以及含量在2100mg/kg以下的1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑。
  2. 根据权利要求1所述的药物组合物，其中所述奥硝唑类化合物可以选自奥硝唑、其立体异构体或它们的前体化合物，优选选自奥硝唑、左旋奥硝唑或右旋奥硝唑或它们的前体化合物；
    优选地，所述奥硝唑、左旋奥硝唑或右旋奥硝唑的前体化合物可以选自其药学上可接受的前体化合物，例如选自奥硝唑或左旋奥硝唑的酯或所述酯药学上可接受的盐或它们的水合物，例如磷酸左旋奥硝唑酯二钠盐或其水合物。
  3. 根据权利要求1或2所述的药物组合物，其特征在于，以奥硝唑、左旋奥硝唑、磷酸左旋奥硝唑酯二钠或它们任一种的水合物计，所述药物组合物中，作为活性成分的奥硝唑类化合物的重量百分比含量可以在10％至99％的范围内，优选为15％至90％，例如20％至85％。
  4. 根据权利要求1-3任一项所述的药物组合物，其特征在于，所述的1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑具有如下式(II)所示的结构：
    Figure PCTCN2020118543-appb-100001
    所述1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑的含量优选在100mg/kg以下，更优选60mg/kg以下。
    优选地，1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑具有如式(III)所示的结构：
    Figure PCTCN2020118543-appb-100002
    所述1-(2,3-二羟基丙基)-2-甲基-5-硝基咪唑的含量小于2100mg/kg。
  5. 根据权利要求1-4任一项所述的药物组合物，其特征在于，所述药物组合物中还任选地包含或不包含下式(IV)所示的化合物：
    Figure PCTCN2020118543-appb-100003
    优选地，所述药物组合物中，式(IV)所示化合物的含量为15000mg/kg以下；
    优选地，所述药物组合物中还任选地包含或不包含2-甲基-5-硝基咪唑；
    优选地，所述药物组合物中，2-甲基-5-硝基咪唑的重量百分比含量在2000mg/kg以下。
  6. 根据权利要求1-5任一项所述的药物组合物，其特征在于，所述药物组合物中还包含药学上可接受的辅料。
  7. 根据权利要求1-6任一项所述的药物组合物，其特征在于，所述药物组合物为制剂；
    优选地，所述药物组合物为注射剂或注射用冻干制剂，其中包含奥硝唑类化合物，优选包含奥硝唑、左旋奥硝唑、磷酸左旋奥硝唑酯二钠或它们任一种的水合物，以及含量在60mg/kg以下的1-(2,3-环氧丙烷)-2-甲基-5-硝基咪唑；
    优选地，所述药物组合物中含有枸橼酸，所述枸橼酸的重量百分比含量为10-35％；
    优选地，所述注射剂的pH为4.0-6.0；
    优选地，所述注射液中可以包含注射用水、乙醇和/或丙二醇。
  8. 权利要求1-7任一项所述药物组合物的制备方法，其特征在于，所述制备方法包括将奥硝唑类化合物与药学上可接受的辅料混合，得到所述药物组合物。
    优选地，将磷酸左旋奥硝唑酯二钠或其水合物与水配成溶液，溶液经冷冻干燥，得到所述药物组合物。
    优选地，所述溶液的pH为4.0-6.0，优选4.5-5.5。
    优选地，所述冷冻干燥的温度为-60℃～30℃，优选-50℃～25℃。
    优选地，所述冷冻干燥的时间为30-70h，例如40-60h，又如45-55h。
    优选地，所述冷冻干燥为程序控温。
  9. 权利要求1-7任一项所述的药物组合物用于改善药物安全性和/或改善药物稳定性。
  10. 权利要求1-7任一项所述的药物组合物用于制备药品。
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