CN117551008B - Deferoxamine conjugate and preparation method and application thereof - Google Patents
Deferoxamine conjugate and preparation method and application thereofInfo
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- CN117551008B CN117551008B CN202311494778.8A CN202311494778A CN117551008B CN 117551008 B CN117551008 B CN 117551008B CN 202311494778 A CN202311494778 A CN 202311494778A CN 117551008 B CN117551008 B CN 117551008B
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
- C07C309/01—Sulfonic acids
- C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
- C07C309/41—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton
- C07C309/43—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton having at least one of the sulfo groups bound to a carbon atom of a six-membered aromatic ring being part of a condensed ring system
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P39/04—Chelating agents
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- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
- C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
- C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
A deferoxamine conjugate and a preparation method and application thereof, belongs to the technical field of medicines, and in particular relates to a 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate and a preparation method and application thereof. On one hand, the invention solves the problems of high toxicity, short half-life, high administration frequency and the like of the deferoxamine drug by providing the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate, thereby improving the medication compliance of patients and reducing the related toxic and side effects of the drug. In another aspect, the invention provides a pharmaceutical composition comprising a 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate and its use in the preparation of a medicament for treating iron overload and in the preparation of a medicament for treating tumors.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate and application thereof.
Background
Deferoxamine (DFO) as a metal ion complexing agent can form stable deferoxamine-iron complexes with free iron ions through hydroxamic acid groups, ultimately excreted from urine. Deferoxamine can bind free labile iron pools in the body and treat acute or chronic iron overload by removing excess iron in the body. In recent years, deferoxamine has also been used in many clinical trials, such as subarachnoid hemorrhage, diabetic foot ulcers, triple negative breast cancer, etc., with great potential in future clinical applications.
Deferoxamine is administered parenterally as the only FDA approved iron chelator, in a manner suitable for use in more general clinical situations, as compared to other FDA approved iron chelators. Deferoxamine still suffers from many drawbacks in clinical administration, firstly, the half-life of deferoxamine is extremely short (about 5.5 min) and the dosage is large (30-60 mg/kg/day), and a single administration by a patient requires continuous subcutaneous injection for 8-12h, and at least five days a week of injection, which results in extremely poor patient compliance. Secondly, deferoxamine itself is hepatotoxic, and high doses and high dosing frequency can cause some toxic side effects to the respiratory, audiovisual and cardiovascular systems of the patient. Therefore, the method for reducing the toxicity of the deferoxamine drug, prolonging the half-life period of the deferoxamine and reducing the administration frequency of the deferoxamine is expected to improve the medication compliance of patients, and provides more valuable possibility for clinical application.
Most of the previous studies have utilized high molecular weight water-soluble polymers to synthesize conjugates with deferoxamine, which reduce the rate of excretion of the conjugate by escaping renal clearance, thereby extending the deferoxamine half-life. However, clinically, patients with iron overload often have impaired liver function, which prevents liver and gall excretion of deferoxamine-iron ion complexes, in addition, the macromolecular conjugate is difficult to freeze-dry and fill in industrial production, and the macromolecular conjugate improves the viscosity of deferoxamine clinical injection, prevents the application of medicines in subcutaneous injection, and no clinical test application of the macromolecular deferoxamine conjugate exists at present.
Disclosure of Invention
The invention aims to provide a 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate and application thereof, which are used for solving the problems of high toxicity, short half-life, high administration frequency and the like of deferoxamine medicaments in the background art, thereby improving the pharmacokinetics and pharmacodynamics properties of deferoxamine, and being expected to improve the medication compliance of patients and reduce the related toxic and side effects of the medicaments.
In order to achieve the aim, the invention provides a deferoxamine conjugate, which is prepared by connecting 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate and deferoxamine molecules through an amide bond, wherein the 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate is prepared by sulfonation of 9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate, and the deferoxamine conjugate has the following structural formula:
the structural formula of the 9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate is as follows:
the structural formula of the 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate is as follows:
The structural formula of the deferoxamine is as follows:
The invention provides a method for synthesizing a 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate, which comprises the following steps of dissolving 9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate and chlorosulfonic acid in methylene dichloride for reaction, purifying after the reaction is completed, freeze-drying after the purification to obtain the 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate, dissolving the 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate and deferoxamine in a solvent, reacting under the action of DIPEA, purifying after the reaction is completed, and freeze-drying after the purification to obtain a final product of the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate, wherein the synthetic route is shown as follows:
preferably, the reaction solvent is anhydrous N, N-dimethylformamide.
Preferably, the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate obtained by the reaction is purified by preparation of a liquid phase to obtain a conjugate having a molecular weight of 861.37.
Preferably, the in vitro iron chelating ability and cytotoxicity of the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate and deferoxamine are compared;
Preferably, the half-life of the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate in vivo is compared with deferoxamine;
Preferably, the in vivo toxicity of the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate is compared to deferoxamine;
The invention provides an application of a 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate in preparing a medicament for treating iron overload diseases;
The invention provides an application of a 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate in preparing a tumor treatment drug.
The beneficial effects of the invention are as follows:
Compared with the deferoxamine with the same concentration, the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate provided by the invention has lower toxicity, longer half-life, stronger capability of removing superfluous iron in blood plasma, capability of treating iron overload at low administration frequency and better attenuation effect. The invention provides a medicine combination of a 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate and 5-aminolevulinic acid, which can improve tumor diagnosis and treatment effects.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of deferoxamine, 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate, and 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate of the examples;
FIG. 2 is a mass spectrum of deferoxamine, 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate, and 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate in the examples;
FIG. 3 is a graph of iron ion chelation ability of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the examples;
FIG. 4 shows the results of a 24h cytotoxic MTT assay for 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the examples;
FIG. 5 is the results of a 48h cytotoxic MTT assay for 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the examples;
FIG. 6 is a 72h cytotoxic MTT assay result for 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the examples;
FIG. 7 is the results of pharmacokinetic experiments on 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) in the examples;
FIG. 8 is the results of pharmacokinetic experiments comparing 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the examples;
FIG. 9 is the results of a pharmacodynamic iron scavenging ability experiment for five administrations of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the examples;
FIG. 10 is the results of a serum ferritin concentration pharmacodynamic experiment with three doses of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) in the examples;
FIG. 11 is the results of a serum iron level pharmacodynamic experiment with three administrations of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) in the examples;
FIG. 12 is the results of an antioxidant experiment of the pharmacodynamics of five administrations of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the example;
FIG. 13 shows the results of liver histopathological manifestations of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the examples.
FIG. 14 is the experimental results of enhanced protoporphyrin IX accumulation in tumors for 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO in the examples.
FIG. 15 is an experimental result of photodynamic treatment of cancer with 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (FMS-DFO) and DFO enhanced 5-aminolevulinic acid in example.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 Synthesis of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate
9-Fluorenylmethoxycarbonyl-N-succinimidyl carbonate (337.4 mg,1 mmol) (purchased from Shanghai Aba Ding Gongsi) was weighed, placed in 4mL of methylene chloride (CH 2Cl2) and lowered to 0 ℃, 0.03% solution of chlorosulfonic acid CH 2Cl2 was added dropwise, the reaction was continued at 25℃after 15min, and the precipitate was centrifuged after 1h at room temperature. The precipitate was washed 4 times in a mixed solution of cyclohexane: dichloromethane=1:1, and the precipitate was dissolved in water and freeze-dried to obtain 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate.
DFO (26.2716 mg,0.04 mmol) was weighed into 7mLN, N-Dimethylformamide (DMF), heated and stirred at 55℃until DFO was completely dissolved, then 2-sulfo-9-fluorenylmethoxycarbonyl-N-succinimidyl carbonate (34.4 mg,0.04 mmol) was weighed into 1mLDMF, which was added to the DFO solution, and finally 20. Mu.L of DIPEA was added thereto to adjust the reaction system to be weakly basic, and after reacting at 25℃for 4 hours, the reaction solution was dried under reduced pressure. After the generation of new compounds is determined by a High Performance Liquid Chromatography (HPLC), the mixture after the reaction is separated and purified by a preparative liquid chromatograph, and finally the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate is obtained by freeze drying. The structure of the compound of example 1 was first confirmed by nuclear magnetic resonance hydrogen spectrometry, and the results are shown in FIG. 1, and the spectrum analysis is that H at 3, 12, 17, 19, 5, 9;4, 11, 16 in the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate corresponds to H at bdqr, ip, cjq in the deferoxamine molecule, and H at 23-26 corresponds to H at E-H in the 2-sulfo-9-fluorenylmethoxycarbonyl-succinimide structure. H at a, B was only present in the 2-sulfo-9-fluorenylmethoxycarbonyl-succinimide structure and not in the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate, demonstrating successful synthesis of the succinimide structure and the terminal amino group of deferoxamine in the experiment. Thereafter, as shown in FIG. 2, the synthesis of the conjugate having a molecular weight of 861.37 was confirmed by mass spectrometry to confirm that the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate was successfully synthesized.
EXAMPLE 2 examination of the iron chelating ability of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate
The iron ion chelating ability was examined by taking (6.56 mg 0.01 mmol) deferoxamine and (8.61 mg 0.01 mmol) 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate (conjugate and deferoxamine free drug have equivalent deferoxamine), dissolving in 10ml distilled water, mixing the two solutions with equivalent 10mM ferrous ammonium sulfate solution, incubating for 30min, diluting four times, and measuring absorbance at wavelength 430nm by ultraviolet-visible spectrophotometry. As a result, as shown in FIG. 3, the characteristic absorption peak of the deferoxamine chelate iron ion was 430nm, and the absorption peak after the iron ion was chelate by the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate was also 430 nm. Thus, the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate synthesized in the present invention has comparable iron chelating ability with equimolar concentrations of deferoxamine.
EXAMPLE 3 cytotoxicity assay of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate
Cytotoxicity the cytotoxicity was detected by MTT assay using samples of deferoxamine and 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate, respectively. The samples were dissolved in DMEM medium to prepare solutions with equivalent concentrations of deferoxamine of 1mM, and used in the subsequent cytotoxicity experiments, the complete DMEM medium was used to dilute the samples into solutions with equivalent concentrations of deferoxamine of 750 μm, 500 μm, 250 μm, 200 μm, 125 μm, 100 μm, 50 μm, 10 μm. The control group used an equal volume of DMEM medium. RAW246.7 mouse mononuclear macrophages were inoculated in 96-well plates at a density of 5X 10 3 per well, after 12h of incubation the medium was discarded and replaced with DMEM complete medium containing different concentrations of deferoxamine and 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate as described above. Cytotoxicity was detected by MTT method at 24h, 48h and 72h of incubation, respectively. The results of the cytotoxic MTT assay are shown in FIGS. 4, 5 and 6, from which it can be seen that the toxicity of the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate was significantly lower than that of deferoxamine at the same concentration throughout the time frame. This shows that after formation of the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate, deferoxamine toxicity was significantly reduced.
EXAMPLE 4 pharmacokinetic assay of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate
10 Male healthy rats are taken, weight is 180-200g, the rats are randomly divided into 2 groups, each group is 5, the deferoxamine and the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate are weighed according to the equivalent dosage of deferoxamine of 50mg/kg and dissolved in proper physiological saline, the administration is carried out by tail vein injection, blood is taken from the orbit on time, blood plasma is obtained by centrifugation, and the concentration of the drug in the blood plasma is measured by a high performance liquid chromatograph. The results of the pharmacokinetic experiments are shown in fig. 7 and 8, and the pharmacokinetic study parameters are shown in table 1. It can be seen from fig. 7 and 8 that the free deferoxamine is completely removed by metabolism within 30min after the tail vein injection administration, and the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate can be quickly combined with HSA after entering the body and can be completely removed after circulating in plasma for 8h, and at the same time, an amide bond can be broken, and the free DFO is gradually released for 24h, which indicates that the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate obtained after the structural modification of the DFO can successfully prolong the half life of the DFO in the body.
TABLE 1 pharmacokinetic parameters
Example 5 test of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate for the treatment of iron overload Capacity
Healthy BALB/c female mice were selected, kept in separate cages and weighed the day before molding. A solution of iron Dextran (Dextran/Fe, physiological saline) with an iron content of 35% was prepared, and after the solution was filtered and sterilized, tail vein injection was performed on mice at a dose of 150mg/kg to establish a mouse iron overload model. On the eighth day after iron dextran injection, mice were randomly divided into 3 groups of 5 mice each, control group, DFO group and FMS-DFO group, respectively, and a group of healthy mice was also set up as a Blank group (Blank group). The doses corresponding to the DFO group and the FMS-DFO group were 100mg/kg and 153.63mg/kg, respectively, and the Control group and the Blank group were given with equal volumes of physiological saline. Each group of mice was given once every two days by tail vein injection in doses for five times. After starting the administration, mice were fed with the iron-deficiency feed, seven days after the last administration, all mice were collected from the eyes and placed in heparin-coated blood collection tubes, centrifuged at 13000rpm/min for 5min, and the supernatant was taken for use. As shown in the results of the competitive enzyme-linked immunosorbent assay (ELISA) measurement of ferritin in the plasma of the mice, the results are shown in figure 9, and the comparison results of the Blank group and the Control group show that the FE content in the plasma of the Control group is obviously improved, which indicates that the establishment of an iron overload model of the mice is successful, and the other two groups show that the DFO and the FMS-DFO can reduce the ferritin content in the plasma of the mice, but the ferritin content of the FMS-DFO group is lower, and the comparison with the DFO group shows that the difference is obvious, which indicates that the capability of the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate for removing redundant iron in the plasma is obviously improved compared with deferoxamine.
Example 6 test of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate to reduce dosing frequency to treat iron overload
Healthy BALB/c female mice were selected, kept in separate cages and weighed the day before molding. A solution of iron Dextran (Dextran/Fe, physiological saline) with an iron content of 35% was prepared, and after the solution was filtered and sterilized, tail vein injection was performed on mice at a dose of 150mg/kg to establish a mouse iron overload model. On the eighth day after iron dextran injection, mice were randomly divided into 3 groups of 5 mice each, a control group (Positive group), a Single group and a Three-Times group, respectively, and a group of healthy mice was also set up as a blank group (Negative group). The doses corresponding to the DFO group and the FMS-DFO group were 100mg/kg and 153.63mg/kg, respectively, and the Positive group and the Negative group were given with equal volumes of physiological saline. Each group of mice was dosed once every two days by tail vein injection, once in Single group and Three times in Three-Time group. After starting the administration, mice were fed with the iron-deficiency feed, seven days after the last administration, all mice were collected from the eyes and placed in heparin-coated blood collection tubes, centrifuged at 13000rpm/min for 5min, and the supernatant was taken for use. The results of measuring ferritin in the plasma of the mice by adopting a competitive enzyme-linked immunosorbent assay (ELISA) are shown in fig. 10 and 11, and the results are shown in the comparison results of the Positive group and the Positive group, wherein the significant increase of the Fe content in the plasma of the Positive group indicates that the establishment of an iron overload model of the mice is successful, and the other two groups of results show that the ferritin content in the plasma of the mice can be reduced by single administration and multiple administrations, but the ferritin content of the FMS-DFO group is not significantly different from the health level, and the comparison with the Positive group is significantly different, so that the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate still has the capability of removing the excessive iron in the plasma to the health level after the administration frequency is reduced to 3 administrations.
EXAMPLE 7 toxicity test of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate
The iron overload mice were given either five DFO or FMS-DFO doses, seven days after the last dose, all mice were collected from the eyes and placed in heparin-coated blood collection tubes and centrifuged at 13000rpm/min for 5min to obtain supernatants for use. The Malondialdehyde (MDA) content in serum was detected by 2-thiobarbituric acid chromogenic method, and the experimental results are shown in FIG. 12, wherein both free DFO and FMS-DFO have the capability of relieving oxidative stress caused by iron overload, and the FMS-DFO group has obviously higher capability of down regulating oxidative stress under the condition of the same molar dosage of administration, and shows more antioxidant capability.
Example 8 liver histopathological manifestations of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate in mice
Five doses of DFO or FMS-DFO were administered, liver tissue was dehydrated and embedded in paraffin, the embedded samples were cut into 4 μm slices using a tissue sample microtome, the samples were stained with Hematoxylin (hemaloxylin) and Eosin (Eosin), and finally stained slice specimens were observed under a microscope, as shown in fig. 13, positive control slices were seen to contain a wide range of ferrioxaxanthin (arrow 1) and globulocyst distention (arrow 2), while hepatocytes disappeared and nuclei were centered. After DFO administration, the ferrioxacin was reduced, but a greater extent of balloon swelling still occurred (arrow 2). FMS-DFO conjugates did not cause inflammatory infiltrates, only less globular cyst distention was found (arrow 2), indicating that both deferoxamine and 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugates can reduce hepatotoxicity caused by iron overload, but the attenuation effect of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugates was significantly better.
Example 9-2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate in combination with 5-aminolevulinic acid to diagnose tumors
According to the invention, a BALB/c mouse inoculated with 4T1 cells subcutaneously is used as a tumor model, when the tumor volume reaches to 700mm 3, intravenous injection of D-PBS (-), DFO or FMS-DFO is carried out on the mouse, the doses of DFO and FMS-DFO are 560 mug/861 mug/serving respectively, and then 250mg/kg of 5-aminolevulinic acid is orally taken. Mice were sacrificed 3h later, tumors and livers were collected, and fluorescence imaging was analyzed using an IVIS system. Fluorescence semi-quantitative data demonstrated (fig. 14), that 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate was able to enhance accumulation of 5-aminolevulinic acid-internalized protoporphyrin IX at tumor sites with statistically significant advantages over deferoxamine itself, while 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate had no significant effect on liver accumulation. The result shows that the 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate can enhance the fluorescence positioning capability of protoporphyrin IX at a tumor site and improve the tumor diagnosis effect of 5-aminolevulinic acid.
EXAMPLE 10 combination of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate with 5-aminolevulinic acid for treatment of tumors
The invention randomly divides the BALB/c mice with the subcutaneous tumor of the 4T1 cells into four groups, wherein one group is used as a positive control group, and intravenous injection and oral administration of D-PBS (-) are carried out. The other three groups were administered intravenously with D-PBS (-), DFO, or FMS-DFO at doses of 560 μg/and 861 μg/, respectively, followed by oral administration of 100mg/kg 5-aminolevulinic acid. The group tumors were irradiated with 635nm laser (200 mW/cm 2, 10 min) for 10min 3h post-dose. Tumor sizes were measured every 2 days after dosing for the different treated mice until day 8. As shown in fig. 15, the use of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate in combination with 5-aminolevulinic acid provides significant advantages in treating tumors as compared to the use of 5-aminolevulinic acid alone and 5-aminolevulinic acid in combination with deferoxamine, demonstrating the higher tumor treating effect of the use of 2-sulfo-9-fluorenylmethoxycarbonyl-deferoxamine conjugate in combination with 5-aminolevulinic acid.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
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