CN110115711A - Injection bis-phenol EDTA calcium complex disodium salt and preparation method thereof - Google Patents
Injection bis-phenol EDTA calcium complex disodium salt and preparation method thereof Download PDFInfo
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- CN110115711A CN110115711A CN201810112313.4A CN201810112313A CN110115711A CN 110115711 A CN110115711 A CN 110115711A CN 201810112313 A CN201810112313 A CN 201810112313A CN 110115711 A CN110115711 A CN 110115711A
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- Prior art keywords
- injection
- tetraacetic acid
- diamine tetraacetic
- uranium
- solution
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- 229930185605 Bisphenol Natural products 0.000 title claims abstract description 125
- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 title claims abstract description 104
- 238000002347 injection Methods 0.000 title claims abstract description 60
- 239000007924 injection Substances 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 53
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 title abstract 6
- JHECKPXUCKQCSH-UHFFFAOYSA-J calcium;disodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate;hydrate Chemical compound O.[Na+].[Na+].[Ca+2].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O JHECKPXUCKQCSH-UHFFFAOYSA-J 0.000 title abstract 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 66
- 238000000034 method Methods 0.000 claims abstract description 46
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 30
- 239000001110 calcium chloride Substances 0.000 claims abstract description 30
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 30
- -1 bis-phenol ethylenediamine tetraacetic acid Chemical compound 0.000 claims abstract description 28
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 claims abstract description 25
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- VYTBPJNGNGMRFH-UHFFFAOYSA-N acetic acid;azane Chemical compound N.N.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O VYTBPJNGNGMRFH-UHFFFAOYSA-N 0.000 claims description 103
- 239000000243 solution Substances 0.000 claims description 86
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 52
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- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 2
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 abstract 1
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- 238000011587 new zealand white rabbit Methods 0.000 description 1
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- 239000012188 paraffin wax Substances 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 150000003112 potassium compounds Chemical class 0.000 description 1
- 230000003405 preventing effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000000306 qrs interval Methods 0.000 description 1
- 125000004151 quinonyl group Chemical group 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 230000002618 waking effect Effects 0.000 description 1
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- 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]
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- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P39/02—Antidotes
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/14—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof
- C07C227/18—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton from compounds containing already amino and carboxyl groups or derivatives thereof by reactions involving amino or carboxyl groups, e.g. hydrolysis of esters or amides, by formation of halides, salts or esters
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- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
- C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
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Abstract
The invention belongs to chemical pharmacy field, it is related to a kind of injection bis-phenol EDTA calcium complex disodium salt freeze drying powder injection and preparation method thereof.Injection bis-phenol EDTA calcium complex disodium salt of the invention is mainly made of main ingredient bis-phenol EDTA calcium complex disodium salt and antioxidant sodium pyrosulfite, by bis-phenol ethylenediamine tetraacetic acid (EDTA), sodium bicarbonate and calcium chloride, 1:4:1 is chemically reacted main ingredient bis-phenol EDTA calcium complex disodium salt online in formulation process in molar ratio, adjusts pH value with sodium hydroxide.Injection of the invention is freeze drying powder injection, and solvent is water for injection, and compared with chemical synthesis prepares bis-phenol EDTA calcium complex disodium salt, the present invention is suitable for industrialized production, the pharmacodynamics no significant difference of the product of preparation.
Description
Technical Field
The invention belongs to the field of chemical pharmacy, relates to bisphenol diamine tetraacetic acid calcium sodium salt for injection, and particularly relates to bisphenol diamine tetraacetic acid calcium sodium salt freeze-dried powder injection for injection and a preparation method thereof.
Background
The prior art discloses a compound [2, 3-dihydroxy-1, 4-phenylene ]]Diamine tetraacetate and a medicinal composition thereof (Chinese patent 201210497250.1) have obvious discharge promoting effect and cell injury preventing effect on rats polluted/poisoned in radionuclide uranium and human renal proximal convoluted tubule epithelial cells infected with uranium. [2, 3-dihydroxy-1, 4-phenylene ]]The diamine tetraacetate includes calcium sodium/calcium potassium salt, zinc sodium/zinc potassium salt, magnesium sodium/magnesium potassium salt, etc., and can be prepared from [2, 3-dihydroxy-1, 4-phenylene]Diamine tetraacetic acid, calcium agent/zinc agent/magnesium agent and alkaline inorganic sodium/potassium compound, wherein the [2, 3-dihydroxy-1, 4-phenylene ] is]Calcium diamine tetraacetic acid sodium salt (CBMIDA-CaNa)2) Has a molecular weight of: 482, prepared by chemical synthesis;
the animal experiment result shows that CBMIDA-CaNa2The intramuscular injection has obvious effect of promoting the excretion of acute uranium contaminated rats, can obviously promote the excretion of uranium in urine, obviously reduce the accumulation of kidney and bone, and is obviously superior to DTPA-CaNa3(ii) a Furthermore, intramuscular injection of CBMIDA-CaNa2Can obviously reduce the renal function injury of rats with acute uranium poisoning, and is characterized by reducing the serum creatinine and urea nitrogen contents of the rats with acute uranium poisoning to the level of a blank control group, which is obviously superior to DTPA-CaNa3The function of (1); intramuscular injection of CBMIDA-CaNa2Can also obviously relieve the pathological injury of the kidney of the rat with acute uranium poisoning, and is characterized in that the degeneration and the necrosis of renal tubular epithelial cells are obviously relieved, the protein cast in the renal tubular is obviously reduced, and DTPA-CaNa3The pathological injury of the kidney tissue of the rat with acute uranium poisoning cannot be obviously relieved;
the cell test result shows that CBMIDA-CaNa2Can prevent the uranium uptake of human kidney proximal tubular epithelial cells (HK-2 cells); the delayed administration of 24h and 48h can also promote the discharge of uranium in HK-2 cells, obviously reduce the accumulation of uranium in the cells, and the effect is obviously superior to that of DTPA-CaNa3。CBMIDA-CaNa2Immediate or delayed 24h administration also protected the HK-2 cells from damage and genotoxicity caused by uranium, the mechanism of action of which is related to its clearance of intracellular oxygen free Radical (ROS) production induced by uranium; and DTPA-CaNa3The effect of obviously relieving the damage of HK-2 cells induced by uranium and the effect of eliminating ROS are not generated;
the result of an intravenous injection mouse acute toxicity test shows that CBMIDA-Na4Toxicity (LD)50＝0.87g/kg)>CBMIDA-ZnNa2(LD50＝1.922g/kg)>CBMIDA-CaNa2(maximum dose of 5g/kg did not die), its calcium sodium salt toxicity is very low;
however, research practice shows that CBMIDA-CaNa is synthesized by chemical synthesis method2There are significant drawbacks, such as: the problem that the synthesis can not be carried out in a large scale (such as hectogram level), and the industrial production of the raw material medicine is difficult to carry out, and the like. Based on the current situation of the prior art, the inventor of the present application intends to provide a [2, 3-dihydroxy-1, 4-phenylene ] for injection suitable for industrial production]A method for preparing diamine tetraacetic acid calcium sodium salt.
Disclosure of Invention
The object of the present invention is to overcome the drawbacks of the prior art, in particular the chemical synthesis of CBMIDA-CaNa2The existing defects provide a bisphenol diamine tetraacetic acid calcium sodium salt for injection and a preparation method thereof, in particular to a [2, 3-dihydroxy-1, 4-phenylene ] for injection which is suitable for industrial production]Diamine tetraacetic acid calcium sodium salt and a preparation method thereof.
In the present invention, [2, 3-dihydroxy-1, 4-phenylene ]]The chemical name of diamine tetraacetic acid can also be: 2,2 ', 2 ", 2'" - ((((2, 3-dihydroxy-1, 4-phenylene) bis (methylene)) bis (aminomethyl)) tetraacetic acid, abbreviated as: bisphenol diamine tetraacetic acid, molecular formula is: c16H20N2O10(ii) a The molecular weight is: 400.34, simple chemical synthesis process, easily available starting material, low cost, and easy industrialized production, and can be used for preparing [2, 3-dihydroxy-1, 4-phenylene ] for injection]The raw material medicine of diamine tetraacetic acid calcium sodium salt (bisphenol diamine tetraacetic acid sodium salt for injection).
In the invention, the bisphenol diamine tetraacetic acid is difficult to dissolve in water, and the bisphenol diamine tetraacetic acid calcium sodium salt for injection is prepared in an on-line chemical reaction mode in the preparation process.
In the present invention, bisphenol diamine tetraacetic acid calcium sodium salt for injection (code: FDFY001) is prepared from bisphenol diamine tetraacetic acid, sodium bicarbonate/sodium carbonate/sodium hydroxide, calcium chloride/calcium gluconate, sodium metabisulfite/sodium bisulfite/sodium sulfite, water for injection, sodium hydroxide and activated carbon, and is preferably prepared into lyophilized powder injection from bisphenol diamine tetraacetic acid, sodium bicarbonate, calcium chloride, sodium metabisulfite, water for injection, sodium hydroxide and activated carbon, wherein the bisphenol diamine tetraacetic acid: sodium bicarbonate: calcium chloride molar ratio of 1:4:1, bisphenol diamine tetraacetic acid: sodium bicarbonate: pharmaceutical grade calcium chloride: the weight ratio of the water for injection is 50.00: 42.00: 18.38: 400-500, wherein the concentration of sodium metabisulfite is 0.2% (w/v); the invention fills nitrogen gas to protect the whole process in the process of on-line chemical reaction; the sodium hydroxide only plays a role in adjusting the pH value of the solution, the dosage of the sodium hydroxide is not particularly limited as long as the addition amount of the sodium hydroxide can enable the pH value of the bisphenol diamine tetraacetic acid calcium sodium salt solution to be in the range of 7.3-7.8, and the dosage of the activated carbon is 0.1% (w/v).
In the invention, sodium metabisulfite is used as an antioxidant, and the concentration of the sodium metabisulfite only needs to meet the requirement of Chinese pharmacopoeia (2015 edition) of 0.1-0.2 percent, and preferably 0.2 percent (w/v).
In the invention, the dosage of the active carbon only needs to meet the common dosage of 0.1-0.5%, preferably 0.1% (w/v) specified in Chinese pharmacopoeia (2015 edition).
The dosage range of the main component of the bisphenol diamine tetraacetic acid in the bisphenol diamine tetraacetic acid calcium salt for injection is as follows: 50mg to 500mg per count, preferably 400mg to 500mg per count.
The invention provides a preparation method of bisphenol diamine tetraacetic acid calcium sodium salt for injection, which comprises the following steps:
adding 80% redissolution volume of water for injection (35 deg.C) into a preparation barrel, introducing nitrogen gas, and stirring with a stirrer; pouring the sodium bicarbonate and the sodium metabisulfite which are precisely weighed into a preparation barrel, and fully stirring for 15min until the solution is clear; precisely weighing bisphenol diamine tetraacetic acid with a prescription amount, pouring the bisphenol diamine tetraacetic acid into the solution cooled to room temperature, rapidly generating a large amount of gas, stirring for about 10min until the solid is completely dissolved, clarifying the solution, completely discharging bubbles, adjusting the pH to 8.3-8.5 by using a 4mol/L NaOH solution prepared in advance, and continuously stirring for 5 min; during the period, the nitrogen is continuously introduced for protection; precisely weighing medicinal calcium chloride in a prescription amount, pouring the medicinal calcium chloride into the solution, stirring the solution for about 15min until solid calcium chloride is completely dissolved, clarifying the solution, and measuring the pH value to be within the range of 7.3-7.8; if the solution does not reach the specified pH value, adjusting the pH value by adopting 4mol/LNaOH solution; then adding water for injection to full dose; during the period, the nitrogen is continuously introduced for protection; precisely weighing the active carbon in the prescribed amount, pouring the active carbon into the solution, stirring for about 5min, and continuously introducing nitrogen for protection; filtering with 0.45 μm filter membrane and 0.22 μm filter core sequentially into sterile sealed liquid storage bag, collecting fine filtrate, detecting intermediate content, packaging, and lyophilizing.
In the preparation method of the invention, the freeze-drying process comprises the following steps: placing a 10ml penicillin bottle which is filled with a semi-stoppered stopper into a freeze dryer, setting the temperature of a shelf at minus 40 ℃ and preserving heat for 1h, -12 ℃ and 3h and minus 40 ℃ and preserving heat for 2h for pre-freezing; then vacuumizing until the vacuum degree in the box reaches 8Pa, raising the temperature of the shelf to-20 ℃, preserving heat for 8h, -15 ℃, preserving heat for 30h, preserving heat for 12h at 0 ℃ and preserving heat for 6h at 10 ℃ for primary sublimation; under the vacuum degree of 8Pa, the temperature of the shelf is raised to 35 ℃, the shelf is dried for 6 hours at constant temperature, the shelf is continuously vacuumized, and the shelf is continuously dried for 6 hours at constant temperature under 35 ℃ for secondary drying; after the freeze drying is finished, filling nitrogen to protect to-0.6 bar, and pressing and plugging; the total lyophilization process run time was about 74 h.
The bisphenol diamine tetraacetic acid calcium sodium salt for injection is characterized in that the bisphenol diamine tetraacetic acid calcium salt for injection is prepared by freeze-drying aqueous solutions of all components, the water content of the bisphenol diamine tetraacetic acid calcium salt for injection only meets the medicinal standard of freeze-dried powder injection, and the stability and the clinical applicability of the preparation are not affected; the water content is in the range of 0-5% (w/w).
The invention provides a bisphenol diamine tetraacetic acid calcium sodium salt for injection and a preparation method thereof. Compared with the chemical synthesis method for preparing the bisphenol diamine tetraacetic acid calcium sodium salt, the invention is suitable for industrial production, and the pharmacodynamics of the prepared product has no obvious difference with the chemical synthesis method.
Drawings
FIG. 1 shows the excretion promoting effect of bisphenol diamine tetraacetic acid calcium sodium salt prepared by different methods on uranium-infected rats,
wherein: p <0.001 compared to the uranium-contaminated control group.
FIG. 2 is a graph showing the effect of a single immediate dose of FDFY001 on 24h uranium excretion from urine (A), uranium accumulation in the kidney (B) and uranium accumulation in the femur (C) in acute intrauranium contaminated rats,
wherein: rat ip 10. mu.g238U (VI)/rat, 21, 42 and 84mg/kg FDFY001 with238The molar ratio of U (VI) is 250:1, 500:1 and 1000:1 respectively, 66mg/kg of Tiron and238the molar ratio of U (VI) is 1000:1, expressed as (mean. + -. SD), to238Comparison of contaminating groups within U (vi), p < 0.01, p < 0.001; the FDFY001 administration group was compared with the positive control drug Tiron group,###p is less than 0.001; compared with the group administered with 21mg/kg FDFY001,&p＜0.05，&&p＜0.01，&&&p is less than 0.001; compared with the group administered with 42mg/kg FDFY001,$p＜0.05，$$p＜0.01，$$$p＜0.001。
FIG. 3 shows the effect of single immediate FDFY001 administration on urinary uranium excretion (A), renal uranium accumulation (B) and femoral uranium accumulation (C) in acute depleted uranium poisoned rats for 72h,
wherein: compared with the control group with the lean uranium poisoning,￥￥￥P<0.001; compared with the 42mg/kg FDFY001 group,###P<0.001。
FIG. 4 is a graph of the effect of a single immediate dose of FDFY001 on serum creatinine (A) and urea nitrogen levels (B) for 72h in acutely depleted uranium poisoned rats,
wherein: p compared to blank control<0.001; compared with the control group with the lean uranium poisoning,￥￥￥P<0.001。
FIG. 5 is a graph of the effect of immediate single administration of low dose FDFY001 on urinary uranium excretion (A), renal uranium accumulation (B) and femoral uranium accumulation (C) from rat contaminated with uranium,
wherein: rat ip 10. mu.g238U (VI)/rat, 1.3125, 2.625, 5.25, 10.5 and 21mg/kg FDFY001 and238the molar ratios of U (VI) are 15.625:1, 31.25:1, 62.5:1, 125:1 and 250:1, respectively; data are expressed as (mean. + -. SD) with238Comparison of contaminating groups within U (vi), p <0.05, p < 0.01, p < 0.001; compared with the groups administered with 2.625 and 5.25mg/kg FDFY001,#p <0.05, compared with 1.3125, 2.625, 5.25 and 10.5mg/kg FDFY001 administration groups,&&&p＜0.001。
FIG. 6 is a graph of the effect of FDFY001 delayed for 3-4h multiple dosing on urinary uranium excretion (A), renal uranium accumulation (B) and femoral uranium accumulation (C) from dogs contaminated with uranium,
wherein: dog iv 0.2mg238U (VI)/kg, 12.5 and 25mg/kg FDFY001 with238The molar ratio of U (VI) was 37:1 and 74:1, respectively. Data are expressed as (mean ± SE), p <0.05, p < 0.01, compared to the contaminated control in uranium.
Detailed Description
Example 1 recipe screening and optimization
The bisphenol diamine tetraacetic acid calcium sodium salt is prepared from bisphenol diamine tetraacetic acid and Na+And Ca2+According to the molar ratio of 1:4: 1; because the bisphenol diamine tetraacetic acid is difficult to dissolve in water, the calcium sodium salt is prepared by firstly dissolving the bisphenol diamine tetraacetic acid by using an alkaline solution containing sodium ions and then adding a calcium compound to provide Ca capable of being chelated2+(ii) a In the prescription screening, the dosage of the main component bisphenol diamine tetraacetic acid is 05g, the final volume of the solution is 5ml, the color change and the content change of the bisphenol diamine tetraacetic acid calcium sodium solution prepared by different sodium salts and different calcium compounds under different temperature conditions are inspected, the influence of different antioxidants and different dosage thereof and nitrogen protection on the stability of the solution is inspected, the influence of pH value on the precipitation and stability of the solution and the influence of the dosage of active carbon on the content are inspected, and an optimal prescription is determined; wherein,
(0) screening of sodium salt
Respectively adopting NaHCO3、Na2CO3And NaOH as sodium salt to dissolve bisphenol diamine tetraacetic acid, Na+The molar ratio of the bisphenol diamine tetraacetic acid to the bisphenol diamine tetraacetic acid is 4: 1;
the method comprises the following steps: the sample preparation information for 1 vial of preparation was as follows:
① bisphenol diamine tetraacetic acid 0.5g, NaHCO3：0.42g
② bisphenol diamine tetraacetic acid 0.5g, Na2CO3：0.265g
③ bisphenol diamine tetraacetic acid 0.5g, NaOH 0.2g
Accurately weighing according to the scheme, and respectively adding 5ml of pure water to dissolve until the solution is clear; dividing the three solutions into 2 parts, placing one part in room temperature, placing the other part in a 60 deg.C water bath kettle, heating for 15min, and observing the color of the solution and the content change of bisphenol diamine tetraacetic acid as the main component; in addition, 0.42g of NaHCO was weighed separately3、0.265g Na2CO3And 0.2g NaOH, adding 5ml pure water to dissolve until the solution is clear, and detecting the pH value of the solution;
the results show a color change in the solution, NaHCO No. ① at room temperature3Light yellow color, ② Na2CO3Dissolving with color slightly darker than ①, dissolving with ③ NaOH with color deepest and close to tan, heating the 3 solutions to deepen color, and measuring the above NaHCO solutions3、Na2CO3And NaOH solution without drugThe pH values are 7.94, 11.86 and 13.28, which shows that the bisphenol diamine tetraacetic acid is sensitive to the pH, and the stronger the alkalinity is, the darker the color change is;
detecting the content of the bisphenol diamine tetraacetic acid by adopting a high performance liquid chromatography: NaHCO 23、Na2CO3And three sodium salts of NaOH dissolved bisphenol diamine tetraacetic acid (room temperature), wherein the main peak contents are respectively (95.21 +/-0.82)%, (94.37 +/-0.38)% and (93.36 +/-0.63)%, wherein NaHCO is used as a main peak3The content of main dissolved peak is slightly higher than that of NaOH and Na2CO3But the three have no significant difference;
therefore, NaHCO was finally selected3The sodium salt with relatively weak alkalinity dissolves the bisphenol diamine tetraacetic acid;
(2) screening for calcium compounds
Respectively adopting calcium chloride, calcium gluconate and calcium citrate as calcium compound, Ca2+The molar ratio of the bisphenol diamine tetraacetic acid to the bisphenol diamine tetraacetic acid is 1:1, Na+The molar ratio of the bisphenol diamine tetraacetic acid to the bisphenol diamine tetraacetic acid is 4: 1;
the method comprises the following steps: the sample preparation information for 10 bottles of preparation was as follows:
① bisphenol diamine tetraacetic acid 5g NaHCO3: 4.2g, calcium chloride: 1.387g, total volume: 50ml of
② bisphenol diamine tetraacetic acid 5g NaHCO3: 4.2g, calcium gluconate: 5.6g, total volume: 50ml of
③ bisphenol diamine tetraacetic acid 5g NaHCO3: 4.2g, calcium citrate: 7.13g, total volume: 50ml of
Dissolving sodium bicarbonate with 40ml of pure water, adding bisphenol diamine tetraacetic acid powder, performing ultrasonic treatment for 5min to dissolve and remove gas in the solution, adding calcium compound, performing ultrasonic treatment at room temperature until the calcium compound is completely dissolved, or stirring and dissolving at 35 ℃, and adding pure water to a constant volume of 50 ml;
the results show that: after the calcium chloride is added, the solution can be completely dissolved after the ultrasonic treatment at room temperature for about 5min, and the solution is clear; when calcium gluconate is adopted, the ultrasonic treatment time at room temperature needs to be prolonged to about 15min, so that the solution can be clarified, but the color is slightly deepened; or can be dissolved completely by stirring at 35 deg.C for about 15min, but with darker color; when calcium citrate is adopted, the calcium citrate can not be completely dissolved even if the ultrasonic treatment is carried out for 1 hour, and the solution is in an opaque state; the dissolution speed difference after adding different calcium compounds is probably because calcium chloride is easily dissolved in water, the solubility of calcium gluconate in water is low, and calcium citrate is slightly soluble in water; the bisphenol diamine tetraacetic acid calcium sodium salt solution prepared by adopting calcium gluconate under the condition of 35 ℃ is dark and dark brown, probably because the bisphenol diamine tetraacetic acid is easy to be oxidized and discolored along with the rise of temperature;
the final formulation preferably uses calcium chloride as the calcium compound and then calcium gluconate;
(3) screening for antioxidants
The benzene ring of the chemical structure of the bisphenol diamine tetraacetic acid contains two phenolic hydroxyl groups which can be easily oxidized into a quinone structure, so that the color of the solution is deepened; therefore, in the design of the prescription, the antioxidant is considered to be added to reduce the oxidation of the bisphenol diamine tetraacetic acid, thereby reducing the deepening of the solution color and improving the stability of the medicine; the common antioxidants in the injection are sodium bisulfite, sodium sulfite and sodium metabisulfite, and the concentration of the antioxidants is generally 0.1-0.2% according to the stipulation of Chinese pharmacopoeia 2015 edition, so the three antioxidants and the antioxidation of the antioxidants with different concentrations are considered;
the method comprises the following steps: by using NaHCO3Dissolving bisphenol diamine tetraacetic acid to prepare a solution with the concentration of 100mg/ml as stock solution, and simultaneously preparing 1%, 1.5% and 2% of sodium bisulfite mother solution, 1%, 1.5% and 2% of sodium sulfite mother solution, 1%, 1.5% and 2% of sodium metabisulfite mother solution which are different antioxidants; adding 100 μ l of different antioxidants with different concentrations into 900 μ l of bisphenol diamine tetraacetic acid sodium salt solution, preparing into antioxidant-containing solution with concentration of 0.1%, 0.15%, and 0.2%, respectively, and placing the solution in 60 deg.C water bathHeating for 45min, and observing color change;
the results show that: three antioxidants, namely sodium bisulfite, sodium sulfite and sodium pyrosulfite, have antioxidant effects and can obviously reduce the color deepening (brown → yellow) of the solution; and the solution color of each antioxidant is lighter and lighter (yellow → light yellow) along with the increase of the concentration of the antioxidant, which shows that the antioxidant effect has dose dependence, wherein the effect of 0.2 percent sodium metabisulfite is better than the effect of other two antioxidants with the same concentration;
comprehensively considering the antioxidant effect and different application ranges of different antioxidants, wherein sodium bisulfite is suitable for a neutral solution, sodium sulfite is suitable for a slightly alkaline solution, and sodium metabisulfite is suitable for an acidic condition; bisphenol diamine tetraacetic acid is an acidic substance, so 0.2% sodium metabisulfite is preferably used as an antioxidant;
(4) protective action of Nitrogen
In order to prevent the oxidation of oxygen to the medicine, except adding antioxidant in the preparation prescription, nitrogen protection is adopted in the preparation process, so as to achieve the purposes of removing oxygen in saturated solution and isolating oxygen;
the method comprises the following steps: the above optimized prescription is adopted: bisphenol diamine tetraacetic acid NaHCO3The molar ratio of calcium chloride is 1:4:1, each injection contains 0.5g of bisphenol diamine tetraacetic acid as a main component, the filling amount of each bottle is 5ml, and the bottle contains 0.2 percent of sodium metabisulfite; the sample preparation information for 10 bottles of preparation was as follows: bisphenol diamine tetraacetic acid: 5g of the total weight of the mixture; NaHCO 23: 4.2 g; calcium chloride: 1.387 g; sodium metabisulfite: 0.1 g; total volume: 50 ml;
40ml of pure water with the prescription amount of 80 percent is taken and added into a beaker, the temperature of the beaker is kept in a water bath at 35 ℃, and NaHCO with the prescription amount is added3And sodium pyrosulfite, after stirring, dissolving and clarifying and the temperature of the solution is reduced to room temperature, sequentially adding bisphenol diamine tetraacetic acid and calcium chloride, stirring, dissolving and clarifying to prepare a bisphenol diamine tetraacetic acid calcium sodium salt solution; then evenly dividing the mixture into 2 parts, filling nitrogen into one part for protection,sealing by tying a cover; the other part is not filled with nitrogen, covered with a rubber plug, and the color changes of the two parts are observed under the same condition;
the results show that: after the nitrogen-filled group is placed for 24 hours, the color is almost unchanged and still brown; after the nitrogen-free group is placed for 24 hours, the color is dark and is dark brown; the nitrogen has good protection effect;
therefore, the whole preparation process needs to adopt nitrogen protection, and pre-charging nitrogen protection is still adopted after the freeze-drying is finished;
(5) influence of pH value on precipitation of bisphenol diamine tetraacetic acid calcium sodium salt solution
The experiment adopts the optimized formula and preparation process, inspects the influence of adjusting different pH values on the precipitation of the final product of the bisphenol diamine tetraacetic acid calcium sodium salt solution after the main component of bisphenol diamine tetraacetic acid is added, and determines the pH value range of the bisphenol diamine tetraacetic acid sodium salt solution and the calcium sodium salt solution;
the method comprises the following steps: the purity of the bisphenol diamine tetraacetic acid adopted in the experiment is 97.92%, and the water content is 2.21%. The sample preparation information for 5 bottles of preparation amounts is as follows: the bisphenol diamine tetraacetic acid has 100 percent of purity and the dosage after water is subtracted is as follows: 2.6108g of NaHCO3：2.1g，CaCl2·2H2O: 0.9187g, sodium metabisulfite: 0.05g, total volume: 25 ml; preparing 3 parts according to the formula;
the preparation process comprises the following steps: 20ml of 80% by volume ultrapure water (35 ℃ C.) was added to the beaker, and the prescribed amount of NaHCO was added thereto3Stirring with a stirrer for about 5min until the sodium pyrosulfite is completely dissolved, and introducing nitrogen into the solution all the time for protection; cooling to room temperature, adding the bisphenol diamine tetraacetic acid with the formula amount respectively, stirring for about 5min, and clarifying the solution after 2-3 min, wherein a large number of bubbles are generated; at the moment, 4mol/L sodium hydroxide solution is used for adjusting the pH values to 8.10, 8.30 and 8.50 respectively; weighing CaCl in the prescribed amount2·2H2Pouring O into a beaker, stirring for 5min, clarifying the solution, sampling, measuring pH value, filtering the solution with 0.45 μm filter membrane, standing for 4h, and observing the solutionA state;
the results show (as shown in table 1): when the pH value of the bisphenol diamine tetraacetic acid sodium salt solution is adjusted to 8.1, the prescribed amount of CaCl is added2·2H2Stirring for 5min, clarifying the solution, and controlling the pH value to be 7.16; after the solution is filtered, a small amount of precipitate is separated out after the solution is placed for 4 hours; after the pH value is adjusted to 8.3 and 8.5, CaCl is added2·2H2After the O is dissolved and clarified, the pH values are respectively 7.32 and 7.39, and no precipitate is generated after the solution is filtered and placed for 4 hours, which shows that the pH value can obviously change the precipitation of the bisphenol diamine tetraacetic acid calcium sodium salt solution, and the pH value of the solution without the precipitate is above 7.3;
TABLE 1 influence of the pH of the bisphenol diamine tetraacetic acid sodium salt and calcium sodium salt solutions on the precipitation of the calcium sodium salt solutions
(6) Effect of pH on the stability of the solution of calcium sodium bisphenoltetraacetate
The experiment adopts the above determined formula and preparation process, and examines the stability of the bisphenol diamine tetraacetic acid calcium sodium salt solution with the pH values of 7.3 and 7.5 so as to further determine the pH value range of the bisphenol diamine tetraacetic acid calcium sodium salt solution;
the method comprises the following steps: the purity of the adopted bisphenol diamine tetraacetic acid is 99.08 percent; the sample preparation information for 5 bottles of preparation amounts is as follows: the consumption of the bisphenol diamine tetraacetic acid after being converted into 100 percent of purity is as follows: 2.5232g of NaHCO3：2.1g，CaCl2·2H2O: 0.9187g, sodium metabisulfite: 0.05g, total volume: 25 ml. 2 parts are prepared as described herein. The formulation procedure was the same as in the above "(5)", except that: in the experiment, 4mol/L NaOH solution is adopted to adjust the pH value of bisphenol diamine tetraacetic acid sodium salt solution to be 8.3, CaCl is added into one part of the solution2·2H2Stirring and dissolving O, dropwise adding 4mol/LNaOH solution to adjust the pH value to 7.3, filtering through a 0.45-micron filter membrane, standing at room temperature for 0, 2, 4, 6 and 8 hours, and determining the change of the purity of the main component by adopting an HPLC method; adding CaCl into the other part2·2H2Stirring and dissolving O, dropwise adding 4mol/L NaOH solution to adjust the pH value to 7.5, filtering the solution through a 0.45-micron filter membrane, standing at room temperature for 0, 4, 8 and 12 hours, and determining the change of the purity of the main component by an HPLC method;
the results show (as shown in table 2): the bisphenol diamine tetraacetic acid calcium sodium salt solution with the pH values of 7.3 and 7.5 is placed at room temperature for 0 to 12 hours, the purity change is only about 0.27 percent, and the change is not obvious, which shows that the calcium sodium salt solution with 2 pH values is stable for at least 12 hours at room temperature;
TABLE 2 results of purity measurements of sodium calcium tetraacetate solutions of bisphenol diamine at pH 7.3 and 7.5 after standing at room temperature for different periods of time
(7) Screening of active carbon dosage
The bisphenol diamine tetraacetic acid calcium sodium salt injection prepared by the invention has strict requirements on heat source and sterility of medicines in a preparation process, so that a certain proportion of activated carbon is considered to be added in the solution preparation process to adsorb heat source, pigment and other organic impurities, the common dosage of the activated carbon is 0.1-0.5% according to the specification of 'Chinese pharmacopoeia' 2015 edition, and the influence of the dosages of different activated carbons on the purity of main components is investigated in the experiment;
the method comprises the following steps: the sample preparation information for 10 bottles of preparation was as follows: main component bisphenol diamine tetraacetic acid: 5g of NaHCO3：4.2g，CaCl2:1.387g, total volume: 50ml of bisphenol prepared according to the above-identified formulationRespectively adding 0.1% and 0.5% of activated carbon into the diamine tetraacetic acid calcium sodium salt solution, stirring for 5min, observing the color change of the solution after the activated carbon is filtered, and measuring the change of the area of the main component;
the results show that: the color of the solution treated by the activated carbon has no obvious influence, and the color of the solution treated by the 0.1 percent activated carbon and the 0.5 percent activated carbon has no obvious difference; HPLC (high performance liquid chromatography) determination shows that the treatment of the activated carbon with two concentrations has no obvious influence on the main peak area;
therefore, the preparation process adopts 0.1 percent of activated carbon as the filter aid.
Example 2 laboratory Freeze drying Process experiments
The above formula and preparation process research show that the bisphenol diamine tetraacetic acid calcium sodium salt solution is easy to oxidize, and the color change of the solution cannot be completely prevented even if an antioxidant is added and nitrogen protection is adopted, so that the invention is based on the advantages of ensuring the stability of the medicine and improving the convenience of use and carrying, and the solution is prepared into a freeze-dried powder injection form; the experiment specifies the freeze-drying curve of the bisphenol diamine tetraacetic acid calcium sodium salt solution according to the determined prescription and preparation process, and provides freeze-drying process data for pilot scale production;
the method comprises the following steps: LYO-0.5m was used in this experiment2And LYO-1.0m2A freeze dryer (Shanghai Dongfulong science and technology Co., Ltd.), wherein the specification of a freeze-dried product is 0.5g/5ml, the preparation amount of the sample is gradually enlarged from 20 bottles to 50 bottles and 100 bottles, and a freeze-drying curve conforming to the product is formulated;
sample preparation information (100 bottles)
The preparation process comprises the following steps: (1) adding purified water with the prescription amount of about 80% into a beaker, controlling the water temperature at 35 ℃, continuously introducing nitrogen, adding sodium bicarbonate and sodium metabisulfite with the prescription amount, and stirring with a stirrer until the sodium bicarbonate and the sodium metabisulfite are completely dissolved, wherein the stirring time is about 15 min. (2) The solution cooled to room temperature was slowly added with the prescribed amount of bisphenol diamine tetraacetic acid (which rapidly generates a large amount of gas) and stirred with a stirrer until completely dissolved, with a stirring time of about 15 min. (3) Adding a prescribed amount of CaCl into the solution2And stirring the mixture by a stirrer until the mixture is completely dissolved, wherein the stirring time is 10-20 min. (4) Adding activated carbon in the amount of the prescription into the solution, and uniformly stirring the solution by using a stirrer for about 5 min. (5) Supplementing purified water to the total amount, stirring with a stirrer, decarbonizing with 0.45 μm filter membrane, coarse filtering, and fine filtering with 0.22 μm filter membrane. (6) Subpackaging according to 5 mL/bottle, half plugging, and freeze-drying in a freeze-drying box;
and (3) establishing a freeze-drying curve: the influence of the retention time (60 min and 120min) of the prefreezing temperature of-40 ℃, the variation of the prefreezing temperature and the retention time (the first stage: 60min of the prefreezing temperature is maintained at-40 ℃, the second stage: 180 min and 240min of the prefreezing temperature are respectively maintained at-10 ℃, 11 ℃, 12 ℃ and-15 ℃, and the third stage: 120min of the prefreezing temperature is maintained at-40 ℃) on the appearance, redissolution speed, pH value, clarity, drying weight loss and content of the freeze-dried product is mainly examined;
the results show that: the freeze-drying yield is more than 99%, the penicillin bottle bottom falling phenomenon rarely occurs, and the determined freeze-drying curve is shown in table 3; the quality inspection result of the freeze-dried product shows that the freeze-dried product is light brown loose block, the redissolution speed is high, the solution is clear and bright brown, the pH value is 7.24-7.34, the drying weight loss is 1.79% -2.87%, and the content is 90% -92%.
TABLE 3 Freeze-drying Curve of bisphenol diamine tetraacetic acid sodium salt for injection
EXAMPLE 3 Pilot scale-up production of bisphenol diamine tetraacetic acid calcium salt lyophilized powder injection (code: FDFY001)
According to the prescription and the laboratory freeze-drying process determined by the research, a GC-11-S-0001 freeze dryer (a product of Shanghai Dongfulong science and technology Co., Ltd.) is adopted for pilot scale production in the experiment;
the method comprises the following steps: the purity of 3 batches of bisphenol diamine tetraacetic acid was 99.08%, 98.55% and 98.84%, respectively, as charged at 0.5g/5ml, and the sample formulation information was as follows:
the preparation method comprises the following steps: in a clean area, according to the formulation proportion, 80% redissolved volume of water for injection (35 ℃) is added into a preparation barrel (weighed according to water density), nitrogen is continuously introduced, and a stirrer is adopted for stirring. And pouring the sodium bicarbonate and the sodium metabisulfite which are precisely weighed into a preparation barrel, and fully stirring for 15min until the solution is clear. Accurately weighing bisphenol diamine tetraacetic acid with the prescription amount, pouring the bisphenol diamine tetraacetic acid into the solution cooled to room temperature, rapidly generating a large amount of gas, stirring for about 10min until the solid is completely dissolved, clarifying the solution, completely discharging bubbles, adjusting the pH to 8.3 by using a 4mol/LNaOH solution prepared in advance, and continuously stirring for 5 min. During this period, nitrogen gas was continuously introduced for protection. Accurately weighing CaCl in prescription2·2H2And O, pouring the generated gas into the solution, stirring for about 15min until the solid calcium chloride is completely dissolved, clarifying the solution, adjusting the pH value to 7.4-7.5 by using a 4mol/L NaOH solution, and then adding the water for injection to full dose. During this period, nitrogen gas was continuously introduced for protection. Precisely weighing the active carbon in the prescribed amount, pouring into the solution, stirring for about 5min, and continuously introducing nitrogen for protection. A0.45 μm filter was used followed by 0.Filtering with 22 μm filter core, collecting the fine filtrate, detecting content, endotoxin and clarity, packaging, and lyophilizing. The freeze-drying process comprises the following steps: placing a 10ml penicillin bottle which is filled with a semi-stoppered stopper into a freeze dryer, setting the temperature of a shelf at minus 40 ℃ and preserving heat for 1h, -12 ℃ and 3h and minus 40 ℃ and preserving heat for 2h for pre-freezing; rapidly vacuumizing until the vacuum degree in the box reaches 8Pa, heating the shelf to-20 deg.C, keeping the temperature for 8h, -15 deg.C for 30h, keeping the temperature for 0 deg.C for 12h, and keeping the temperature for 10 deg.C for 6h for first sublimation; under the vacuum degree of 8Pa, the temperature of the shelf is raised to 35 ℃ for constant-temperature drying for 6h, and the shelf is continuously vacuumized and continuously dried at 35 ℃ for 6h for secondary drying. After the freeze-drying is finished, re-aerating to-0.6 bar by using nitrogen, and pressing and plugging. The total lyophilization process run time was about 74 h. Randomly sampling from 3 batches of freeze-dried products, and inspecting indexes such as properties, pH value, drying weight loss, loading difference, solution clarity, solution color, endotoxin, sterility, content and the like;
the results show that: the average yield of 3 batches of freeze-dried preparations is about 85 percent, and the phenomenon of penicillin bottle bottom falling rarely occurs; the quality inspection result shows that the bisphenol diamine tetraacetic acid calcium sodium salt for injection meets the pharmaceutical standard of the freeze-dried powder injection (shown in table 4).
TABLE 4 examination results of the finished product of bisphenol diamine tetraacetic acid sodium salt for injection
Example 4 accelerated test and test results thereof
According to the 9000 guiding principle of the general rules of the four ministry of the national pharmacopoeia 2015 edition, the bisphenol diamine tetraacetic acid calcium sodium salt preparation for injection is subjected to accelerated test research, and the content of the main component is mainly determined;
the method comprises the following steps: placing 3 batches of freeze-dried products produced in the pilot scale production in a closed container with the relative humidity of 75% + -5%, placing for 6 months at the temperature of 40 +/-2 ℃, sampling for 1 month, 2 months, 3 months and 6 months respectively, and detecting the content of the main component by adopting an HPLC method;
as a result, as shown in Table 5, no significant change was observed in the main component content when FDFY001 was left for 6 months.
TABLE 5 accelerated test results for FDFY001 (40 ℃. + -. 2 ℃ C., relative humidity 75%. + -. 5%)
Example 5 comparison of the excretion promoting effects of sodium calcium bisphenoldiaminetetraacetate salts prepared by different methods on uranium-infected rats
The research compares the uranium discharge effect of bisphenol diamine tetraacetic acid calcium sodium salt prepared by different methods on rats polluted in uranium, and comprises ① bisphenol diamine tetraacetic acid calcium sodium salt prepared in a small amount by adopting a chemical synthesis method, ② bisphenol diamine tetraacetic acid calcium sodium salt prepared by adopting calcium chloride, ③ bisphenol diamine tetraacetic acid calcium sodium salt prepared by adopting calcium gluconate, and the uranium discharge effect of the bisphenol diamine tetraacetic acid calcium salt is compared with that of the bisphenol diamine tetraacetic acid sodium salt;
the experimental method comprises the steps of uniformly dividing 25 SPF male SD rats with the weight of 180-grade SD into 5 groups according to the weight balance, wherein each group comprises 5 experimental components, namely a ① uranium internal pollution control group, an ② uranium internal pollution + bisphenol diamine tetraacetic acid calcium sodium salt (prepared by a chemical synthesis method), a ③ uranium internal pollution + bisphenol diamine tetraacetic acid calcium sodium salt (prepared by calcium chloride), a ④ uranium internal pollution + bisphenol diamine tetraacetic acid calcium sodium salt (prepared by calcium gluconate), an ⑤ uranium internal pollution + bisphenol diamine tetraacetic acid sodium salt group, and injecting 10 mu g (ip) into the abdominal cavity of the rat238U (VI)/rat was immediately followed by intramuscular injection (im) of 22.5mg/kg of bisphenol diamine tetraacetic acid calcium or sodium salt (about the equivalent of 1/2 commonly used in a single dose of 0.4 g/human) into the uranium contaminating control im saline. Placing the rat inIn the metabolism cage, one mouse and one cage are used for freely taking food and drinking water. Collecting urine for 0-24h, digesting into white solid residue with mixed acid (nitric acid + perchloric acid), dissolving and diluting with 2% nitric acid, and detecting urine uranium content with inductively coupled plasma mass spectrometer (ICP-MS) (Nexion 300, PerkinElmer company, USA);
the results show that: the chemical synthesis method and the bisphenol diamine tetraacetic acid calcium sodium salt prepared by calcium chloride and calcium gluconate can obviously increase the urine uranium discharge amount of the rat infected with acute uranium for 24 hours by about 73 percent, 85 percent and 89 percent compared with a uranium-infected control group, and the three have no obvious difference and the uranium discharge effect of the bisphenol diamine tetraacetic acid sodium salt (as shown in figure 1);
the result shows that the bisphenol diamine tetraacetic acid calcium sodium salt prepared by the calcium chloride and the calcium gluconate has the same high-efficiency uranium discharging effect as the bisphenol diamine tetraacetic acid calcium salt prepared by the chemical synthesis method.
Example 6 the shedding efficacy of an immediate single dose of FDFY001 on acute intrauranium-contaminated rats
According to the conversion from the single dose of 0.4 g/human commonly used by human to the equivalent dose of 42mg/kg in rats, the dose and 1/2 times and 2 times of the dose are adopted in the experiment, and the acute side effect is observed238Excretion promoting effect of contaminated rat in U (VI);
the experimental method comprises the steps that 50 SPF-grade SD rats with the weight of 180-class are evenly divided into 5 groups according to the weight of 10 rats each with the male and female halves, and the experimental groups comprise ① uranium internal pollution control group, ② uranium internal pollution +21mg/kg FDFY001 administration group, ③ uranium internal pollution +42mg/kg FDFY001 administration group, ④ uranium internal pollution +84mg/kg FDFY001 administration group, ⑤ uranium internal pollution +66mg/kg Tiron administration group, and the rats ip 10 mu g238U (VI)/mice were immediately followed by im 21, 42 and 84mg/kg FDFY001 and 66mg/kg tiron, uranium internal contamination control group im formulation adjuvants. Rats were placed in metabolic cages, one mouse per cage, with free access to food and water. Urine was collected 0-24h after uranium infection, rats were sacrificed at the same time, and bilateral kidneys and femurs were taken. Digesting a sample by using mixed acid (nitric acid and perchloric acid)The resulting white solid residue was dissolved and diluted with 2% nitric acid, and urine was measured by ICP-MS (Nexion 300, PerkinElmer, USA)238U (VI) output and kidney and bone238U (VI) accumulation;
as shown in FIG. 2, the administration of 21, 42 and 84mg/kg FDFY001 to im immediately after uranium contamination significantly increased the urine and uranium output of rats 0-24h, which were 1.93 times, 1.96 times and 2.3 times, respectively, higher than those of the control group contaminated with uranium; the uranium accumulation amount of the kidney is obviously reduced by about 88 percent, 86 percent and 93 percent compared with the uranium contaminated control group, and the uranium accumulation amount of the femur is obviously reduced by about 54 percent, 51 percent and 68 percent compared with the uranium contaminated control group; the positive control drug Tiron can obviously increase the urine and uranium output of rats for 0-24h, which is about 1.7 times of that of the uranium internal pollution control group, and the accumulation of the kidney and the femur uranium is obviously reduced by about 73 percent and 11 percent compared with the uranium internal pollution control group; it is noted that the effect of reducing renal and femoral uranium accumulation in the group administered with 3 doses of FDFY001 is significantly better than that of the positive control drug Tiron, wherein the low dose of FDFY001(21mg/kg) is compared with that of FDFY001238The molar ratio of U (VI) is 250:1, and the effect of reducing the accumulation of uranium in kidney and femur is also obviously better than that of Tiron and238an effect of a molar ratio of U (VI) of 1000: 1; when FDFY001 is administered at a dose (84mg/kg) of238When the molar ratio of U (VI) is 1000:1, the effect of improving the urine uranium discharge for 24 hours is obviously better than that of Tiron. Compared with the excretion promoting effect of 3 FDFY001 administration doses, the effect of improving the uranium excretion and reducing the uranium accumulation of the kidney and the femur of the 84mg/kg FDFY001 administration group is obviously higher than that of the 21mg/kg and 42mg/kg 2 administration dose groups, and the uranium excretion effect of the last 2 administration doses is not obviously different;
experiments show that: FDFY001 can obviously promote the excretion of uranium from the acute uranium contaminated rat within 24h by immediate administration, obviously reduce the accumulation of uranium and kidney within 24h, and is obviously superior to a positive control drug Tiron; the uranium expelling effect of 2 times rat equivalent to the administration of single dose of FDFY001 commonly used by human is obviously better than that of 1/2 times and 1 time rat equivalent to the administration of single dose of FDFY001 commonly used by human, which indicates that the first administration dose after uranium infection is doubled, and the effect of promoting uranium excretion can be obviously improved.
Example 7 Effect of immediate single administration of FDFY001 on promoting excretion and reducing uranium-induced renal injury in acutely depleted uranium-poisoned rats
The experiment adopts a rat equivalent dose (42mg/kg) of a single dose commonly used by people and a rat equivalent dose (84mg/kg) of a single dose commonly used by 2 times of people to further observe the effects of the rat equivalent dose on promoting excretion and relieving renal injury caused by depleted uranium in acute depleted uranium poisoning;
the experimental method comprises the steps of dividing 20 SPF-grade male SD rats into 4 groups according to weight balance, and dividing the SPF-grade male SD rats into 5 groups according to weight balance, wherein the experimental groups comprise an ① blank control group, a ② lean uranium poisoning control group, a ③ lean uranium poisoning +42mg/kg FDFY001 administration group, a ④ lean uranium poisoning +84mg/kg FDFY001 administration group, and the rats ip 500 mu g238U (VI)/rat, im 42 and 84mg/kg FDFY001, uranium poisoning control group im preparation auxiliary material and blank control group im normal saline are respectively and immediately carried out. Rats were placed in metabolic cages, one mouse per cage, with free access to food and water. Collecting urine of 0-24h, 24-48h, 48-72h for use238Detecting the content of U (VI); anesthetizing rats 72h after the infection, performing heart blood sampling, centrifuging, collecting serum, and detecting the contents of serum Creatinine (CREA) and urea nitrogen (BUN) by using a full-automatic biochemical analyzer (Hitachi 7180 type); one kidney and two femurs were dissected and harvested, digested and dissolved in 2% nitric acid as described in example 6, and diluted, and examined by ICP-MS (Nexion 300, PerkinElmer, USA)238U (VI) content; fixing the kidney on the other side with 10% neutral formalin, embedding paraffin, slicing, HE staining, and observing the pathological morphology change of the kidney tissue under a microscope;
the results show that: 42mg/kg FDFY001 is administrated by im immediately after the lean uranium poisoning, so that the urine and uranium output of 0-72h is obviously higher than that of a pure lean uranium poisoning group and is respectively 3.5 times and 4 times of that of a lean uranium poisoning control group, wherein the urine and uranium output of 0-24h of rats is obviously higher than that of the pure lean uranium poisoning group and is respectively 4.6 times and 5.1 times of that of the lean uranium poisoning control group, and the urine and uranium output of different dosage administration groups of 24-48h and 48-72h after the lean uranium poisoning is not obviously different from that of the lean uranium poisoning control group; the urine and uranium output of the group with 84mg/kg FDFY001 for 0-72h is increased compared with that of the group with 42mg/kg, but the difference is not obvious (shown in figure 3A); after 42 and 84mg/kg FDFY001 administration, the uranium accumulation amount of the kidney is remarkably reduced by about 85 percent and 82 percent compared with the uranium-depleted group of rats 72h after the uranium-depleted rats are subjected to administration, the uranium accumulation amount of the femur is remarkably reduced by about 58 percent and 88 percent compared with the uranium-depleted group, the effect of remarkably reducing the uranium accumulation amount of the femur of the 84mg/kg administration group is higher than that of the 42mg/kg administration group, and no remarkable difference exists between the effects of remarkably reducing the uranium accumulation amount of the kidney of the two administration groups (as shown in figures 3B and 3C);
rat ip 500. mu.g238Serum CRE and BUN levels of U (VI)/mice were both significantly higher than those of the blank control group after 72h, and immediate administration of 42mg/kg FDFY001 significantly reduced serum CRE and BUN of the acutely depleted uranium poisoned rats to the blank control group (as shown in FIG. 4A); pathological observation of kidney tissue shows that rats ip 500 mug238U (VI) 72h later, a large number of protein casts are arranged in the renal tubules, part of renal proximal tubular epithelial cells are necrotized and shed, a large number of inflammatory cells are scattered in the interstitium and infiltrated, but the morphological structure of the glomerulus is approximately normal; the kidney injury caused by acute depleted uranium poisoning can be obviously relieved by immediately administering 42mg/kg of FDFY001, and only a few renal tubular epithelial cell abscissions and protein cast formation are seen, the bleeding is obviously relieved, and the like;
the results show that: FDFY001 has remarkable effect of promoting excretion of rats suffering from acute uranium deficiency poisoning, and can remarkably relieve pathological injury of renal tissues of rats suffering from acute uranium deficiency poisoning and protect renal functions.
Example 8 minimal effective dose exploration of immediate single dose FDFY001 in acute intrauranium contaminated rat voiding
On the basis of the dose which is proved to have obvious discharge promoting effect on acute uranium internally contaminated rats by 21mg/kg of FDFY001 in example 6, the doses are sequentially reduced by times of 1/2 to 10.5mg/kg, 5.25mg/kg, 2.625mg/kg and 1.3125mg/kg, and the minimum effective dose is searched;
the method comprises the steps of dividing 30 SPF male SD rats into 6 groups according to weight balance, and dividing each group into 5 male SD rats, wherein the experimental groups comprise ① uranium internal pollutionControl group, ② uranium internal contamination +1.3125mg/kg FDFY001 group, ③ uranium internal contamination +2.625mg/kg FDFY001 group, ④ uranium internal contamination +5.25mg/kg FDFY001 group, ⑤ uranium internal contamination +10.5mg/kg FDFY001 group, ⑥ uranium internal contamination +21mg/kg FDFY001 group, rat ip 10 mug238U (VI)/rat, im 1.3125, 2.625, 5.25, 10.5 and 21mg/kg FDFY001, uranium internal contamination control group im preparation auxiliary material. Rats were placed in metabolic cages, one mouse per cage, with free access to food and water. Collecting 0-24h urine, anesthetizing rat 24h after infection, dissecting to obtain bilateral kidney and bilateral femur, digesting by the method of example 6, dissolving in 2% nitric acid, diluting, and detecting urine by ICP-MS (Nexion 300, PerkinElmer product, USA)238U (VI) output and kidney and bone238U (VI) accumulation;
the results show that: 1.3125 and 2.625mg/kg FDFY001 immediately after uranium contamination, im administration can not obviously improve the urine and uranium discharge of uranium-contaminated rats in uranium, and the 2 doses can obviously reduce the uranium accumulation amount of the femur, which is respectively reduced by about 14 percent and 17 percent compared with a uranium-contaminated control group, but obviously lower than the effect of a 21mg/kg FDFY001 administration group; wherein 2.625mg/kg FDFY001 can also obviously reduce the accumulation of renal uranium, which is obviously reduced by about 46 percent compared with a uranium internal pollution control group, but is obviously lower than the effect of a 21mg/kg FDFY001 administration group (as shown in figure 5); the urine uranium output of the rats polluted in uranium can be obviously improved by FDFY001 administration from 5.25mg/kg to 21mg/kg, and is respectively 1.91 times, 2.24 times and 1.96 times of the control group polluted in uranium; the accumulation of uranium in the kidney is obviously reduced, and is respectively reduced by about 59 percent, 73 percent and 90 percent compared with the uranium internal pollution control group; the accumulation of the uranium in the femur is obviously reduced by about 11 percent, 20 percent and 54 percent respectively compared with the uranium internal pollution control group; the 3 administration doses of 5.25, 10.5 and 21mg/kg FDFY001 have no obvious difference in improving urinary uranium discharge, while the effect of obviously reducing the uranium accumulation amount by 21mg/kg FDFY001 is obviously higher than that of a 5.25mg/kg FDFY001 administration group, the effect of obviously reducing the uranium accumulation of femur is obviously higher than that of 1.3125-10.5 mg/kg FDFY001 administration group, and the effect of obviously reducing the uranium accumulation of kidney and femur is not obviously different between 5.25 and 10.25mg/kg FDFY001 administration doses;
the results show that: 5.25mg/kg was the minimum effective dose for immediate single administration of FDFY001 to improve urinary uranium excretion and reduce renal and femoral uranium accumulation, 2.625mg/kg was the minimum effective dose to reduce renal uranium accumulation, and 1.3125mg/kg was the minimum effective dose to reduce femoral uranium accumulation.
Example 9 dog uranium discharge experiment
The experiment was carried out to stain dogs at a single dose of 0.4 g/human, based on the common human single dose, converted to an equivalent dose of 12.5mg/kg for human and dog body surface area to a dog equivalent dose238U (VI) was followed by a 3-4h delay, the first dose (25mg/kg) was equivalent to 2-fold the single dose commonly used in humans, after which a single, canine equivalent dose (12.5mg/kg) commonly used in humans was given daily, and delayed multiple administration of FDFY001 was observed for the pair238Promoting excretion of contaminated dogs in U (VI) and promoting renal injury caused by uranium;
the experimental method comprises the steps of randomly dividing 11 common-grade male Beagle dogs with the weight of 8-10kg into 2 groups according to the weight of ① uranium internal pollution control groups and ② uranium internal pollution + FDFY001 administration groups, one dog and one cage, freely drinking water, feeding 300-350g feed once every day, collecting 24h urine before the experiment, taking venous blood as self blank control, and taking 0.2mg of dog iv238After U (VI)/kg, the delay was 3-4him 25mg/kg FDFY001, then im12.5mg/kg FDFY 0011 times daily for 3 consecutive days, for a total of 4 administrations. Urine was collected every 24h and volume was measured; taking venous blood and centrifugally separating serum 96 hours after uranium contamination, detecting serum creatinine by using a full-automatic biochemical analyzer (Hitachi 7180 type), and detecting urine protein by using a plurality of urine detection test strips (dry chemical method); after dog iv45mg/kg sodium pentobarbital anesthesia, carotid bleeding is performed to kill the dog, bilateral kidneys and bilateral femurs are taken and respectively weighed, and 2.0g samples of the middle sections of the left kidney and the right femur are precisely weighed and used for sampling238Measuring the content of U (VI);
the results show that: the administration of FDFY001 is delayed for 3-4h after uranium infection, the administration is carried out for 1 time and 4 times every day, and the urine and uranium discharge amount is obviously increased by 0-96h compared with that of a uranium internal pollution control group and is 1.89 times of that of the uranium internal pollution control group; the renal and femoral uranium accumulation was significantly reduced by about 77% and 41% compared to the uranium contaminated control group (as shown in figure 6); table 6 shows that 96h after uranium contamination of the uranium internal contamination control group, serum creatinine was significantly increased compared with the blank control group, and urine protein detection was weak positive to strong positive; FDFY001 can remarkably reduce serum creatinine to a normal value, and urine protein is detected to be negative or weakly positive;
experiments show that: the FDFY001 is delayed for 3-4h for multiple times, has the effects of remarkably increasing the uranium excretion from dog urine, reducing the accumulation of the kidney and the femur uranium and protecting the kidney function.
TABLE 6 delay of Effect of FDFY001 on urinary protein and serum creatinine in uranium contaminated dogs by multiple 3-4h dosing
Note: dog iv 0.2mg238U (VI)/kg, 12.5 and 25mg/kg FDFY001 with238The molar ratio of U (VI) was 37:1 and 74:1, respectively. Data are expressed as (mean ± SE). P <0.05 compared to the blank control group.
Example 10 Beagle dog safety pharmacological test
The test adopts 3 dosage points of low, medium and high dosage (25, 50 and 100mg/kg), respectively about 0.4 g/human single dose commonly used by people is converted into 2, 4 and 8 times of Beagle dog equivalent dose (12.5mg/kg), adopts im administration consistent with clinical administration route, adopts an emkaPACK4G remote measuring system to detect the influence of single im FDFY001 on the electrocardiogram, blood pressure and respiration of the conscious Beagle dog, and evaluates the safety of the test sample;
the method comprises the following steps: 8 Beagle dogs, each male and female dog adopts a cross administration method, after each administration, the next administration is carried out after a cleaning period of at least 7 days, and in 4 administration periods, imFDFY001(25, 50 and 100mg/kg) and an auxiliary material reference substance are respectively used for one time;
analyzing and evaluating electrocardio (heart rate, P-R interval, Q-T interval, QRS interval, P wave, R wave, T wave, ST segment and QTcF), blood pressure (systolic pressure, diastolic pressure and average pressure) and respiration (respiratory frequency and tidal volume) data at each time point of 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 24h before and after administration;
the results show that: electrocardiogram: compared with the animal electrocardiogram of each dose group of the test sample and the animal electrocardiogram of the negative control group before self administration, no obvious abnormal change is seen;
blood pressure: compared with a negative control group, the 2h rise of systolic blood pressure after the 50mg/kg group is statistically different (p is less than 0.05); after the 100mg/kg group is administrated, the mean pressure and the systolic pressure are increased for 1h, 2h and 4h, and the diastolic pressure is increased for 4h after the administration, which has statistical difference (p <0.05) (shown in a table 7);
compared with the group before self administration, the average pressure rise of the 50mg/kg group after administration is 0.25h, and the systolic pressure rise of the group after administration is statistically different from the systolic pressure rise of the group after administration for 0.25h and 0.5h (p is less than 0.05); after the group of 100mg/kg is administrated, the average pressure is increased at 0.25h, 0.5h and 1h, the systolic pressure is increased at 0.25h and 0.5h, and the diastolic pressure is increased at 0.25h after the group is administrated, so that the statistical difference exists (p is less than 0.05);
breathing: compared with the self-administration pre-administration and negative control groups, the respiratory frequency and tidal volume of animals of each dose group of the test sample have no obvious abnormal change;
the change of the blood pressure index is considered to have relevance to the stress response generated by animal pain caused by strong stimulation of the test sample to the injection part, and is unrelated to the toxicological action of the test sample;
the results show that: a single intramuscular injection of FDFY001 had no significant effect on waking Beagle dog electrocardiogram, blood pressure and respiration.
TABLE 7 Effect of single intramuscular administration of FDFY001 on blood pressure in Beagle dogs
Note: n is 8; p <0.05 compared to negative control; compared with the administration before the drug administration, the # p is less than 0.05.
Example 11 toxicity test of FDFY001 rat Single intramuscular administration
In this test, according to the preliminary results, the dose of FDFY001 administered is 1000mg/kg, which is about 24 times the equivalent dose (42mg/kg) of the rat after conversion of the single dose of 0.4 g/human commonly used in humans. Evaluating the safety of the test sample by observing the toxic reaction condition of the SD rat after single im FDFY 001;
the method comprises the following steps: 40 SD rats with half male and female, the weight of 177.6-233.4 g at the beginning of administration, the male and female animals are divided into 2 groups according to the weight, 10 male and female animals in each group are respectively an auxiliary material control group (giving an auxiliary material control) and a test sample group (giving FDFY 0011000 mg/kg), the administration is carried out by injecting the muscle of the left and right hind limbs, the administration is carried out 1 time in the morning and afternoon, and the administration volume is 2 mL/kg/time;
the detection indexes are as follows: general observations, body weight, food intake, pathology examination;
the results show that: animals in each group are not moribund or dead, brown urine can be seen when the animals in 1000mg/kg group are administrated in the 1 st day, bilateral hind limb swelling can be seen from the 1 st day to the 3 rd day, and other animals are not obviously abnormal;
weighing the weights of 2d, 7d and 14d before and after administration, wherein the results show that the weights of the male animals 2d and 7d in the 1000mg/kg group are slowly increased and have statistical difference (p is less than 0.05) compared with the auxiliary material control group, and the female animals have no obvious abnormality;
the food intake of 1d, 6d and 13d after administration is detected, the food intake of the male animals of 1000mg/kg group of 1d is slightly reduced compared with that of the auxiliary material control group, and no obvious abnormality is found in the rest of the groups;
the animals are roughly dissected at 15d after the administration, and no obvious morphological abnormality is found in the gross examination of the body surfaces and all main organs of the animals;
experiments show that: the Maximum Tolerated Dose (MTD) of FDFY001 in rats given a single intramuscular injection is 1000mg/kg, which is about 150 times the single dose commonly used in humans.
Example 12 toxicity test of FDFY001 rat Single intravenous administration
The clinical administration route is intramuscular injection, referring to the technical guidance principle of single drug administration toxicity research, 2 acute toxicity tests of the administration routes are usually required, except for the clinical administration route, the route (such as intravenous injection) which can enable the original drug to enter the circulation completely is usually investigated, and the intravenous injection route is selected and investigated in the test;
according to the experimental result, the safety of the test sample is evaluated by observing the toxic reaction condition of SD rats after single intravenous injection of FDFY001, wherein the dosage of FDFY001 is 750mg/kg, which is about 18 times of the equivalent dosage (42mg/kg) of rats after the conversion of single dosage of common use of human, 0.4 g/human;
the method comprises the following steps: 40 SD rats with half male and female bodies respectively, wherein the weight of the SD rats is 176.0-237.5 g at the beginning of administration, the male and female animals are respectively divided into 2 groups according to the weight, each group of male and female bodies is 10 respectively, and the SD rats are respectively an auxiliary material control group (giving an auxiliary material control product) and a test sample group (giving FDFY001750mg/kg), and are administered by single tail vein injection, and the administration volume is 5 mL/kg;
the detection indexes are as follows: general observations, body weight, food intake, pathology examination;
the results show that: animals in each group are not endangered or killed, brown urine can be seen in the 1d after the animals in the 750mg/kg group are administrated, and other animals are not obviously abnormal;
weighing the weights of 2d, 7d and 14d before and after administration, wherein the results show that the 2d weight of female animals in the 750mg/kg group is reduced, the statistical difference (p is less than 0.05) is obtained compared with the auxiliary material control group, and no obvious abnormality is found in other groups;
the food intake of 1d, 6d and 13d after administration is detected, the 1d food intake of 750mg/kg animals is slightly reduced compared with that of the auxiliary material control group, and other animals have no obvious abnormality;
the animals are roughly dissected at 15d after the administration, and no obvious morphological abnormality is found in the gross examination of the body surfaces and all main organs of the animals;
experiments show that: the Maximum Tolerated Dose (MTD) of FDFY001 in rats is 750mg/kg in a single intravenous injection, which is about 112 times the usual single dose in humans for intramuscular injection.
Example 13 Rabbit subcutaneous stimulation test
According to FDFY001 clinical planned maximum concentration not exceeding 80mg/mL, the test adopts the administration concentration of 80mg/mL, the administration volume of 1 mL/mouse and the administration dosage of 80 mg/mouse, and the administration dosage is 32mg/kg calculated according to 2.5kg of rabbit body weight and is about 4.8 times of single dosage (0.4 g/human) commonly used by human; through FDFY001 rabbit subcutaneous stimulation test, the local stimulation of the test sample to a rabbit injection part is observed, and reference is provided for the clinical safety application of the preparation:
the method comprises the following steps: 18 rabbits, all male, were randomly stratified in body weight into a negative control group (0.9% sodium chloride injection), an adjuvant control group (adjuvant control), and a test article group (FDFY001, 80mg/mL), each of 6 rabbits. The administration is performed by subcutaneous injection on the back 1 time every afternoon for 5 days continuously, and the administration volume is 1 mL/tube. The animals were observed daily for general status, behavior, signs during the experiment. Local skin irritation was observed before, within 1h after and 24h, 48h after the last administration on the day of administration for all animals. 3 rabbits in each group are taken 48 hours after the last administration for general autopsy and histopathology examination, and the undivided rabbits are subjected to general autopsy and histopathology examination at 16d after the last administration;
the results show that: the general state, behavior and physical signs of the animals are not abnormal;
visual observation of injection site: mild erythema was seen before and after the last dose day and 24h after the last dose in the negative control group 1 animals, mild to moderate erythema was seen in the test 3d to 24h after the last dose in the adjuvant control group 2 animals, and mild to moderate erythema was seen in the test 3d to 5d in the test group 3 animals. According to the evaluation standard of skin irritation intensity, the negative control group is non-irritant, and the auxiliary material control group and the test sample group are mildly irritant;
general pathological examination: when dissection is carried out 48 hours after the last administration, the injection parts of 2 animals in the negative control group and 1 animal in the test sample group have red spot block-shaped color changes with different sizes; when dissecting 16d after the last administration, no obvious abnormal change is observed under the skin of all the injection parts of the animals,
histopathological examination: after the last administration is finished, the animals are dissected for 48 hours, and microscopic examination shows that the subcutaneous tissues of the injection part of 1 animal in the negative control group bleed slightly, the subcutaneous tissues of the injection part of 1 animal bleed slightly and the muscle layer focus infiltrates slightly with mononuclear cells; the test group 2 animals had slight mononuclear cell infiltration of the subcutaneous tissue foci at the injection site. Microscopic examination of the dissected animals at 16d after the last administration shows that the local focus of the subcutaneous tissue of the injection part of 1 animal in the auxiliary material control group is slightly infiltrated by mononuclear cells,
experiments show that: new Zealand white rabbits were injected subcutaneously in the back with FDFY 0011 mL at a concentration of 80mg/mL, at a dose about 4.8 times that of the single dose commonly used in humans, and no significant irritation was observed at the site of administration.
Example 14 FDFY001 allergy test
(1) Active systemic anaphylaxis test in guinea pigs
In the experiment, the adopted FDFY001 low-dose and high-dose 2 sensitization doses are respectively 31mg/kg and 62mg/kg (about 0.4 g/human equivalent dose and 2 times of equivalent dose of single dose commonly used by human), and the sensitization dose of a low-dose group is 12.5 mg/mouse, the sensitization dose of a high-dose group is 25 mg/mouse and the excitation dose is 2 times of the sensitization dose calculated according to 400g of the weight of a guinea pig. The safety of the test sample is judged through an FDFY001 guinea pig active systemic anaphylaxis test, and a reference is provided for the clinical safety application of the preparation;
the method comprises the following steps: the method comprises the following steps of selecting 40 animals, namely male and female guinea pigs, wherein the animals are divided into a negative control group (0.9% sodium chloride injection), an auxiliary material control group (auxiliary material control product), a low and high dose test sample group (FDFY001 sensitizing doses are 12.5 mg/guinea pig and 25 mg/guinea pig respectively) and a positive control group (ovalbumin and 2 mg/guinea pig respectively) according to weight stratification, each group comprises 8 animals and the rest 4 animals are used for standby. 0.5mL of low and high dose test sample is injected subcutaneously for sensitization, and sensitization is carried out for 3 times in total. At 14d after the last sensitization, 4 animals per group were rapidly injected intravenously for the 1 st challenge, and another 4 animals per group were injected for the 2 nd challenge at 21d, with the challenge dose being 2 times the sensitization dose. Observing animal anaphylaxis immediately within 30min after intravenous injection excitation, measuring the body weight of the administered animal on the first and last sensitization days and the excitation administration day, and observing clinical symptoms of the animal every day;
the results show that: general observations were: no obvious abnormal reaction is seen in all groups of animals during the test period;
excitation observation: at 14d and 21d after the last sensitization, the guinea pigs are excited by intravenous injection, the animal reaction is immediately observed to 30min, no anaphylactic reaction symptom appears in the negative control group, the auxiliary material control group and the guinea pigs of the low and high dose test sample groups, the anaphylactic reaction incidence rate is 0%, and the results are negative; allergic reaction symptoms of nasal pruritus, cough, urination, defecation, gait instability, jumping, spasm, rotation and tidal breathing can be seen after the guinea pigs of the positive control group are excited, the guinea pigs of 8/8 die within 5min, the allergic reaction incidence rate is 100%, and the result is extremely positive;
weight: no obvious abnormality is found in the body weight of the animals during the test period;
experiments show that when the FDFY001 administration dose is about a single dose commonly used by human and an equivalent dose 2 times of the single dose commonly used by human, no sensitization is shown on guinea pigs, and the result of an active systemic anaphylaxis test of the guinea pigs is negative; (2) guinea pig passive skin allergy test
The sensitization dose in the FDFY001 low dose and high dose groups used in this test was the same as in the "(1) guinea pig active systemic anaphylaxis test". Through the FDFY001 guinea pig passive skin allergy test, the condition of the test article causing anaphylactic reaction is judged, and reference is provided for the clinical safety application of the preparation;
the method comprises the following steps: 10 Hartley guinea pigs, male and female animals were randomly divided into a negative control group (0.9% sodium chloride injection), an adjuvant control group (adjuvant control), a low dose group (FDFY00112.5 mg/mouse), a high dose group (FDFY00125 mg/mouse) and a positive control group (ovalbumin 3 mg/mouse) by weight, 2 animals per group, and male and female animals were half each. Sensitizing (0.5 mL/mouse) by subcutaneous injection 1 time every other day for 3 times, and collecting blood 14d after the last sensitization to prepare sensitized serum;
another 30 Hartley guinea pigs were taken and the males and females were randomly stratified by body weight into 5 groups (corresponding to the groups prepared with serum), each group consisting of 6 animals each half male and female. Diluting sensitizing serum with 0.9% sodium chloride injection at a ratio of 1:2, 1:4, 1:8, and 1:16, intradermally injecting serum with different dilution times into depilatory regions at two sides of spinal column of guinea pig, passively sensitizing at 0.1 mL/injection site, exciting by intravenous injection 48h after passive sensitization, exciting administration volume of 1 mL/body (mixed solution containing exciting antigen with sensitizing dose and isovolume of 0.5% Evans blue dye solution), and exciting CO for 30min2Anaesthetizing and euthanizing, cutting back skin, measuring the size of a blue spot of an intradermal injection site in the inner layer of the skin, judging that the blue spot is positive if the diameter of the blue spot is more than 5mm, and observing clinical symptoms of the animal every day during the test period;
the results show that: under each dilution multiple of the sensitizing serum, after guinea pigs are excited by intravenous injection for 30min, no blue spots appear in a negative control group, and the result is negative; the auxiliary material control group has no blue spots and the result is negative; the blue spots do not appear in the low and high dose groups of the test article, and the result is negative. After the positive control group is excited, 6/6 animals show blue spots under the dilution times of 1:2, 1:4, 1:8 and 1:16 of sensitizing serum, and the diameter of the blue spots is more than 5mm, and the result is positive;
no obvious abnormality is observed in clinical symptom observation of animals during the test period;
experiments show that: the results of the guinea pig passive cutaneous anaphylaxis were negative when FDFY001 was administered at a dose of approximately one single dose commonly used in humans and an equivalent dose of 2 times the single dose commonly used in humans.
Claims (7)
1. The bisphenol diamine tetraacetic acid calcium sodium salt for injection is characterized in that bisphenol diamine tetraacetic acid, sodium bicarbonate, calcium chloride, sodium metabisulfite and water for injection are prepared into a freeze-dried powder injection, wherein the bisphenol diamine tetraacetic acid: sodium bicarbonate: calcium chloride molar ratio of 1:4:1, bisphenol diamine tetraacetic acid: sodium bicarbonate: pharmaceutical grade calcium chloride: the weight ratio of the water for injection is 50.00: 42.00: 18.38: 400-500, and the concentration of sodium metabisulfite is 0.2% (w/v).
2. The calcium sodium bisphenoldiaminetetraacetic acid for injection according to claim 1, wherein the amount of the calcium disodium diaminetetraacetic acid component in the lyophilized powder for injection is 50mg to 500mg per unit.
3. The injection of bisphenol diamine tetraacetic acid calcium salt of claim 1 or 2, wherein the amount of bisphenol diamine tetraacetic acid component in the lyophilized powder for injection is 400 mg-500 mg/injection.
4. The bisphenol diamine tetraacetic acid calcium salt for injection as claimed in claim 1, wherein the lyophilized powder for injection further contains sodium hydroxide as a pH regulator, and the amount of the sodium hydroxide is such that the pH value of the injection is within the range of 7.3-7.8.
5. The bisphenol diamine tetraacetic acid calcium salt for injection of claim 1, wherein activated carbon is further used to adsorb heat source, pigment and other organic impurities in the freeze-dried powder injection, and the dosage is preferably 0.1% (w/v).
6. The method for preparing a sodium calcium salt of bisphenol diamine tetraacetic acid for injection according to claim 1, comprising the steps of:
A. adding 80% redissolution volume of water for injection into a preparation barrel according to the formula ratio of the preparation, continuously introducing nitrogen, stirring by a stirrer, sequentially pouring precisely weighed sodium bicarbonate and sodium metabisulfite into the preparation barrel, and stirring for 15min until the solution is clear;
B. weighing bisphenol diamine tetraacetic acid with the formula amount, pouring the bisphenol diamine tetraacetic acid into the solution cooled to room temperature to generate gas, stirring for 10min until the solid is completely dissolved, clarifying the solution, discharging bubbles, adjusting the pH to 8.3-8.5 by using the prepared 4mol/L NaOH solution, then continuously stirring for 5min, and continuously introducing nitrogen for protection;
C. weighing calcium chloride according to the formula amount, pouring the calcium chloride into the solution to generate gas, stirring for 15min until solid calcium chloride is dissolved, clarifying the solution, and measuring the pH value to be within the range of 7.3-7.8; if the solution does not reach the specified pH value, adjusting the pH value by using 4mol/LNaOH solution; then adding water for injection to full dose; during the period, nitrogen is continuously introduced for protection;
D. weighing the active carbon in the formula amount, pouring the active carbon into the solution, stirring for about 5min, and continuously introducing nitrogen for protection;
E. filtering with 0.45 μm filter membrane and 0.22 μm filter core sequentially into sterile sealed liquid storage bag, collecting fine filtrate, detecting intermediate content, packaging, and lyophilizing.
7. The method of claim 6, wherein the lyophilization process comprises: placing a 10ml penicillin bottle which is filled with a semi-stoppered stopper into a freeze dryer, setting the temperature of a shelf at minus 40 ℃ and preserving heat for 1h, -12 ℃ and 3h and minus 40 ℃ and preserving heat for 2h for pre-freezing; then vacuumizing until the vacuum degree in the box reaches 8Pa, raising the temperature of the shelf to-20 ℃, preserving heat for 8h, -15 ℃, preserving heat for 30h, preserving heat for 12h at 0 ℃ and preserving heat for 6h at 10 ℃ for primary sublimation; under the vacuum degree of 8Pa, the temperature of the shelf is raised to 35 ℃ and the shelf is dried for 6 hours at constant temperature; continuously vacuumizing, and continuously drying at the constant temperature of 35 ℃ for 6 hours on a shelf for secondary drying; and after the freeze drying is finished, filling nitrogen to protect to-0.6 bar, and pressing and plugging.
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| 关志宇: "《药物制剂辅料与包装材料》", 31 January 2017, 中国医药科技出版社 * |
| 张华麟等: "邻苯二酚-3,6-二甲撑亚氨基二乙酸的酸离解平衡及其与碱土金属、锰(II)、镉(II)、锌(II)、钴(II)、镍(II)、铜(II)、铁(III)、钍(IV)及铀酰离子配合物稳定性的研究", 《化学学报》 * |
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