CN107496935A - A kind of inclusion compound of Sorafenib and beta cyclodextrin and preparation method thereof - Google Patents
A kind of inclusion compound of Sorafenib and beta cyclodextrin and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of inclusion compound of Sorafenib and beta cyclodextrin and preparation method thereof inclusion compound to contain Sorafenib molecule and beta cyclodextrin molecule, and Inclusion ratio, i.e. mol ratio are 1：0.8~1.5；The defects of Sorafenib beta cyclodextrin clathrate provided by the present invention and preparation method thereof overcomes current Sorafenib preparation production technology to a certain extent, obtained inclusion compound is the solid forms dramatically different with known Sorafenib Tosylate crystal formation, can effectively avoid Sorafenib Tosylate polycrystalline present in preparation process or mixed crystal phenomenon.Preparation method is easy to implement, and material loss is small, mild condition, suitable for industrial production.
Description
Technical field
The present invention relates to a kind of preparation method, is a kind of antineoplastic 4- { 4- [({ [chloro- 3- (trifluoros of 4- specifically
Methyl) phenyl] amino carbonyl) amino] phenoxy group-N- picoline -2- formamides (Sorafenib, Sorafenib) β -
Cyclodextrin (β-Cyclodextrin) inclusion compound and preparation method thereof.
Background technology
Sorafenib chemical name is：4- { 4- [({ [4- chloro- 3- (trifluoromethyl) phenyl] amino } carbonyl) amino] benzene oxygen
Base }-N- picoline -2- formamides, molecular formula C21H16ClF3N4O3, molecular weight 464.82, molecular structure is as shown in formula I：
Sorafenib is the antitumor oral multi-kinase inhibitor researched and developed by German Bayer and Onxy companies, for treating
Liver cancer, clear-cell carcinoma and the thyroid cancer of operation can not be implemented.Sorafenib, which has, suppresses growth of tumour cell and angiogenesis
Dual antitumor action, by suppressing tumour cell target site A-RAF, B-RAF, V600EBRAF, C-RAF, FLT3, c-
The steric configuration of KIT, VEGFR-2, VEGFR-3 and PDGFR- beta kinase and change its phosphorylation mechanisms, suppress kinase activity, enter
And RAF/RAF/MEK/ERK the and VEGF signal paths of tumour cell are disturbed, reach and suppress tumor cell proliferation and containment blood vessel
The effect of generation.Sorafenib is stronger to the selectivity of tumour cell as a kind of Mutiple Targets antineoplastic, in treatment of cancer
In it is with the obvious advantage, current widely used medicine type is Sorafenib Tosylate (trade name：Nexavar), in 2005
Granted U.S. FDA, it is granted in 2006 to enter China.
Sorafenib solubility in water minimum (almost insoluble) and stomach permeability is higher, is listed in BCS
(Biopharmaceutics classification system) II class medicine.Sorafenib preparation is oral relatively raw at present
Thing availability is relatively low (about 38%~49%), and oral dose is larger (400mg/ times, 2 times/day), and long-term use has certain
Side effect.Therefore, improve the dissolution rate of Sorafenib preparation, improve oral administration biaavailability, reduction toxic side effect has important
Meaning.
Beta-schardinger dextrin (β-cyclodextrin), molecular formula C42H70O35, molecular structure is as shown in formula II.
Beta-schardinger dextrin is a kind of cyclic oligomer sugar compounds, and containing 7 D- glucopyranose units, its molecular configuration is in micro-
Cone-shaped ring, there is the characteristics of outer rim is hydrophilic, and inner chamber is hydrophobic.The cavity of beta-schardinger dextrin can include numerous guest molecules, form master
Guest inclusion thing (Host-Guest Complex), causes the change of the properties such as guest molecule physics, chemistry, biology, is FDA
The medical auxiliary materials and food additives of approval.Some insoluble drug molecules as object after inclusion, solubility and dispersiveness
Significantly improve, simultaneously because the polyhydroxy hydrophily outside beta-schardinger dextrin improves the biocompatibility of medicine, β-ring is pasted in addition
The natural enzymolysis property of essence is also so that being included the slow releasing function of medicine is improved.
In view of the ultralow dissolubility of Sorafenib, Yuan Yan companies are prepared into salt form (Sorafenib Tosylate, business
The name of an article " Nexavar "), to improve its dissolubility.But the dissolubility of Sorafenib Tosylate still extreme difference, in water almost
Fairly insoluble, bioavilability still has much room for improvement (38-49%, relative to oral dose 100-400mg), and toluenesulfonic acid rope
La Feini is in highly acid (pH<2), for oral drugs, acidity can produce by force obvious stomach and stimulate.Drawn currently for rope
The research of non-Buddhist nun focus primarily upon Sorafenib tosilate preparation (WO0041698, WO2006034796,
CN101052619) and different crystal forms (I, II, III) preparation (WO0042012, WO2006034797, CN101065360).
Chinese patent CN102145175 disclose a kind of Sorafenib Tosylate-hydroxypropyl-beta-cyclodextrin inclusion and
Its preparation method, included, but formed as host molecule Sorafenib Tosylate using hydroxypropyl-β-cyclodextrin
Complex structure characterize insufficient, only disclose the differential scanning calorimetric curve of inclusion compound, therefore to whether really foring
Inclusion compound not can determine that.In addition, CN102145175 is few to inclusion compound Quality Research, its balance in water is only disclosed
Solubility, and solubility study shows, is 1 in rate of charge：When 1, the inclusion compound is compared to Sorafenib Tosylate
Dissolubility almost without lifting (1.1 times).Therefore, it is more preferable to prepare dissolubility, the higher Sorafenib preparation of bioavilability
Form, and clearly structure and property representation are carried out to it there is important practical value.
The content of the invention
For the present situation that Sorafenib dissolubility is small, bioavilability is relatively low, the present invention provides a kind of Sorafenib-β-ring
Cyclodextrin inclusion compound and preparation method thereof, and structural characterization and property research are carried out to it.The inclusion compound obtained improves medicine
Solubility and relative to light, heat, oxidation stability, improve the dissolution rate of medicine, enhance the slow releasing function of medicine, can grow
The blood concentration that it is fixed that time dimension keeps steady, and then improve the bioavilability of bulk drug.Preparation side provided by the present invention simultaneously
Method also provides an effective approach for the research and development of Sorafenib pharmaceutical preparation, and the present invention is to come by the following technical programs in fact
Existing：
A kind of Sorafenib-Benexate Hydrochloride, inclusion compound contain Sorafenib molecule and beta-schardinger dextrin molecule, inclusion
Than that is, mol ratio is 1：0.8~1.5.
As a further improvement, inclusion compound of the present invention contains rubbing for Sorafenib molecule and beta-schardinger dextrin molecule
That ratio preferably 1：1.
As a further improvement, inclusion compound of the present invention carries out diffraction analysis by the use of CuK α as characteristic X-ray, with
The angle of diffraction 2 θ, ± 0.2 ° represents that X-ray diffraction includes 4.0 °, 6.0 °, 9.8 °, 11.8 °, 12.0 °, 13.3 °, 15.0 °,
Characteristic peak at 16.0 °, 17.5 °, 18.4 °, 19.6 °.
The invention also discloses a kind of preparation method of Sorafenib-Benexate Hydrochloride, based on beta-schardinger dextrin,
Using Sorafenib as object, in the presence of the solvent by inclusion reaction, by Sorafenib with comprising, shelve or be embedded in β-ring
Dextrin intramolecular, form inclusion compound.
As a further improvement, the present invention specifically includes following steps：
1), by Sorafenib and beta-schardinger dextrin using mol ratio as 1：0.5~1.5 feeds intake, and is added to the four of certain volume
The in the mixed solvent of hydrogen furans and water；
2), by the mixed solution heating stirring that step 1) obtains to complete dissolved clarification；
3), the settled solution for obtaining step 2) carries out inclusion reaction, heating stirring method, mixing time 4~30 hours；
4), stop heating and stirring, Temperature fall crystallization, until crystallization is complete, filter, drying, produce Sorafenib-β-
Cyclodextrin inclusion compound.
As a further improvement, the volume ml ratios of the volume ml of the tetrahydrofuran described in step 1) and water are 1~4：1.
As a further improvement, the amount mmol of Sorafenib and the body of solution in the mixed solution system described in step 2)
Product ml ratios are 1：20~100.
As a further improvement, the heating stirring method described in step 3), reaction time are 4~30 hours, heating-up temperature
For 50~80 DEG C.
As a further improvement, the recrystallization temperature described in step 4) is 20~30 DEG C；Drying mode is vacuum drying, is dried
Dry temperature is 30~40 DEG C.
The invention also discloses a kind of Sorafenib-Benexate Hydrochloride to prepare the medicine for suppressing tumour growth
Application in preparation.
Having the beneficial effect that relative to prior art of the invention：
(1) Sorafenib-Benexate Hydrochloride provided by the present invention and preparation method thereof overcomes to a certain extent
At present the defects of Sorafenib preparation production technology, obtained inclusion compound is notable not with known Sorafenib Tosylate crystal formation
Same solid forms, can effectively avoid Sorafenib Tosylate polycrystalline present in preparation process or mixed crystal phenomenon.Prepare
Method is easy to implement, and material loss is small, mild condition, suitable for industrial production.
(2) solubility, dissolution rate greatly improve：The present invention is directed to the situation of Sorafenib poorly water-soluble, using inclusion skill
Art, it is water-soluble to improve it.After Sorafenib is via beta-cyclodextrin inclusion compound, dissolubility significantly improves.Solubility experiment shows (table
1), compared to Sorafenib active compound and Sorafenib Tosylate, Sorafenib-Benexate Hydrochloride is in neutral aqueous solution
(pH7.0) greatly increased with the solubility in simulate the gastric juice (pH1.2 acidic aqueous solutions).In addition, in water and acidic aqueous solution,
The dissolution rate of Sorafenib, Sorafenib Tosylate and Sorafenib-Benexate Hydrochloride is as shown in Figure 7.Pass through and compare
Understand, Sorafenib composition is significantly higher than Sorafenib and toluene from the dissolution rate in inclusion compound of the present invention and stripping quantity
Sulfonic acid Sorafenib, show that Sorafenib-Benexate Hydrochloride can effectively improve the dissolution rate of drug ingedient.
1 Sorafenib of table-Benexate Hydrochloride is compared with Sorafenib active compound, the solubility of Sorafenib Tosylate
(mg/mL, 37 DEG C)
(3) slow releasing function of Sorafenib-Benexate Hydrochloride of the present invention and bioavilability are compared to rope
La Feini and Sorafenib Tosylate significantly improve.Pharmacokinetic studies are shown (Fig. 8)：Sorafenib-beta-schardinger dextrin bag
The concentration of compound in blood is Sorafenib and 5.4 times of Sorafenib Tosylate and 3.2 times respectively, shows Suo Lafei
Buddhist nun-Benexate Hydrochloride improves the blood concentration of drug ingedient within a certain period of time, and the sustained release for improving drug ingedient is made
With.Internal distribution experiments show (Fig. 9)：In liver, the concentration of Sorafenib and Sorafenib Tosylate be Sorafenib-
2.6 times of Benexate Hydrochloride and 4.2 times, illustrate that Sorafenib-Benexate Hydrochloride is reduced inside Sorafenib
First pass effect, Sorafenib is less in liver metabolism, is improved into the concentration in blood.It so can both reduce the liver damage of medicine
Hinder effect, can also improve the bioavilability of medicine.
(4) Sorafenib-Benexate Hydrochloride of the present invention can improve the suppression tumour growth of drug ingedient, promote
The effect of apoptosis of tumor cells and antitumor position angiogenesis, improve the bioavilability of drug ingedient and tumour is controlled
Therapeutic effect, while the side effect of drug ingedient is reduced to a certain extent.
Sorafenib-Benexate Hydrochloride structure is detected in the present invention and the instrument of performance is as follows：
X-ray powder diffraction instrument：Rigaku company, model D/Max-2550PC, Cu-K αSpoke
Penetrate, tube voltage 40KV, tube current 250mA, 5 °/min of sweep speed, 0.02 ° of step width, θ -2 θ of 3-40 ° of scanning range (2 θ) connect
Continuous scanning.
Synthesis thermal analyzer：TA companies of the U.S., model SDTQ600, purge gass：Nitrogen 120mL/min, programming rate 10
DEG C/min, temperature range：Room temperature~400 DEG C.
Differential scanning calorimeter：TA companies of the U.S., model DSCQ100, purge gass：Nitrogen 120mL/min, programming rate
10/min, temperature range：Room temperature~300 DEG C.
Fourier infrared spectrograph：German Bruker companies, model Vector 22, scan wave-number range：4000-
400cm-1。
NMR spectrum：INSTRUMENT MODEL：Bruker DMX-500 NMRs, condition determination：DMSO-d6
Ultra-violet absorption spectrum (UV)：Using Dynamica HALO DB-20 ultraviolet specrophotometers, by sample preparation into one
Determine strength solution, and, as blank control, determined by the use of with batch solvent using 1cm absorption cells in the range of 200~400nm.Suo La
The maximum absorption band of Fei Ni-Benexate Hydrochloride is located at 266nm, and the maximum absorption band of Sorafenib is located at 244nm.
High-efficient liquid phase determining method (HPLC)：Chromatographic column is filler with octadecylsilane chemically bonded silica, and mobile phase is
Acetonitrile-water (4：6), flow velocity 0.6mL/min, Detection wavelength 266nm.It is appropriate that precision weighs Sorafenib active compound reference substance,
Add methanol that the solution that concentration is 0.05mg/mL is made, as reference substance solution；It is appropriate that precision weighs sample, is made of methanol dense
The solution for 0.05mg/mL is spent, takes 20 μ L to inject liquid chromatograph respectively, chromatogram is recorded, according to external standard method in terms of peak area
Calculate concentration.
Brief description of the drawings
Fig. 1 be Sorafenib-Benexate Hydrochloride, beta-schardinger dextrin, Sorafenib XRD spectra stacking chart；
In figure, (a) Sorafenib-Benexate Hydrochloride, (b) beta-schardinger dextrin, (c) Sorafenib；Sorafenib in figure-
The spectrogram of Benexate Hydrochloride has unique diffraction maximum, both different from bulk drug Sorafenib also different from beta-schardinger dextrin,
Proof has new thing mutually to generate, and is Sorafenib-Benexate Hydrochloride of the present invention.
Fig. 2 be Sorafenib-Benexate Hydrochloride, beta-schardinger dextrin, Sorafenib TG figures stacking chart；In figure, (a)
Sorafenib-Benexate Hydrochloride, (b) Sorafenib, (c) beta-schardinger dextrin；The decomposition temperature of Sorafenib active compound is 211.9
DEG C, and the decomposition temperature of Sorafenib-Benexate Hydrochloride of the present invention is 319.1 DEG C, improves the heat-resisting of medicine
Capacity of decomposition.
Fig. 3 is Sorafenib-Benexate Hydrochloride, Sorafenib, the DSC stacking charts of beta-schardinger dextrin；
In figure, (a) Sorafenib-Benexate Hydrochloride, (b) Sorafenib, (c) beta-schardinger dextrin；As seen from the figure, this hair
Bright described Sorafenib-Benexate Hydrochloride has two endothermic peaks at 69.9 DEG C, 97.5 DEG C, and the 232.7 of Sorafenib
282.0 DEG C of melting peaks of DEG C fusion and decomposition peak and beta-schardinger dextrin disappear, and show that Sorafenib passes through clathration and beta-schardinger dextrin
New material is formd, while drug ingedient heat endurance improves.
Fig. 4 is Sorafenib-Benexate Hydrochloride, Sorafenib, infrared spectrum (IR) stacking chart of beta-schardinger dextrin；Figure
In, (a) Sorafenib-Benexate Hydrochloride, (b) Sorafenib, (c) beta-schardinger dextrin；As seen from the figure, compared to beta-schardinger dextrin
Infrared spectrum, Sorafenib-Benexate Hydrochloride is in 1505cm-1And 1548cm-1Absworption peak be present in place.The two absorb
Peak corresponds to C=C stretching vibration and C=N stretching vibration respectively, for caused by pyridine ring in Sorafenib molecular structure.
Fig. 5 be beta-schardinger dextrin, Sorafenib, Sorafenib-Benexate Hydrochloride H-NMR spectrum stacking chart；By scheming
Understand, in Sorafenib-Benexate Hydrochloride, the NMR signal of Sorafenib part hydrogen atom weakens, and shows β-ring
Dextrin has included the moieties of Sorafenib, and shielding action is generated to the hydrogen atom on group.
Fig. 6 is the NOESY spectrograms of Sorafenib-Benexate Hydrochloride.
As seen from the figure, the phenoxy group of Sorafenib and pyridine structure are related to the methine and hydroxyl structure of beta-schardinger dextrin,
Prove interaction be present between Subjective and Objective.
Fig. 7 is Sorafenib-Benexate Hydrochloride, and Sorafenib, Sorafenib Tosylate is in water and pH1.2 acidity
Dissolution rate figure in the aqueous solution；
In figure, dissolution rate figure of (a) Sorafenib-Benexate Hydrochloride in water, (b) Sorafenib-beta-schardinger dextrin
Dissolution rate figure of the inclusion compound in acidic aqueous solution, dissolution rate figure of (c) Sorafenib in water, (d) Sorafenib is in acidity
Dissolution rate figure in the aqueous solution, dissolution rate figure of (e) Sorafenib Tosylate in water, (f) Sorafenib Tosylate exist
The dissolution rate figure of acidic aqueous solution.As seen from the figure, dissolution rate of the Sorafenib-Benexate Hydrochloride in two kinds of dissolution mediums
All it is optimal.
Fig. 8 is Sorafenib-Benexate Hydrochloride, and Sorafenib, the medicine of Sorafenib Tosylate is for dynamic curve
Stacking chart.
In figure, (a) Sorafenib-Benexate Hydrochloride, (b) Sorafenib, (c) Sorafenib Tosylate.By scheming
Understand, Sorafenib maintains higher concentration in blood after beta-cyclodextrin inclusion compound in certain time, be Sorafenib active compound
With 5.4 times of Sorafenib Tosylate blood concentration and 3.2 times, show Sorafenib-Benexate Hydrochloride in a timing
The interior blood concentration for improving drug ingedient, improve the slow releasing function of drug ingedient.
Fig. 9 is Sorafenib-Benexate Hydrochloride, Sorafenib, after Sorafenib Tosylate oral 2hr and 4hr,
The distribution of major organs in vivo.
Compared to Sorafenib and Sorafenib Tosylate, Sorafenib-Benexate Hydrochloride is in main metabolic device
The distribution (sum of liver and nephrosis treating medicine concentration) of official does not show significant difference.On the whole, Sorafenib-beta-cyclodextrin inclusion compound
Thing is higher in the distributed density of each organ, illustrates that Sorafenib-Benexate Hydrochloride oral administration biaavailability is better than Suo Lafei
Buddhist nun and Sorafenib Tosylate.
Figure 10 is Sorafenib-Benexate Hydrochloride, and Sorafenib, Sorafenib Tosylate treatment group and feminine gender are right
According to tumor suppression curve inside group；
In figure, (a) is negative control group tumor suppression curve, and (b) is Sorafenib treatment group tumors suppression curve, (c)
For Sorafenib Tosylate treatment group tumors suppression curve, (d) is Sorafenib-Benexate Hydrochloride treatment group tumors suppression
Koji-making line.It was found from figure, relative to negative control group, each treatment group's curve shows the repressed feature of tumour growth.This
Show that active component Sorafenib has played effect in oncotherapy.Between each treatment group, toluenesulfonic acid rope can detect
La Feini tumor inhibitory effect is slightly better than Sorafenib, relative to the above two, the tumour of Sorafenib-Benexate Hydrochloride
Inhibition significantly improves.
Figure 11 is Sorafenib-Benexate Hydrochloride, and Sorafenib, Sorafenib Tosylate treatment group and feminine gender are right
According to the apoptosis of tumor cells rate of group；
In figure, (a) is negative control group apoptosis of tumor cells rate, and (b) is Sorafenib treatment group tumors apoptosis rate,
(c) it is Sorafenib Tosylate treatment group tumors apoptosis rate, (d) is Sorafenib-Benexate Hydrochloride treatment group
Apoptosis of tumor cells rate.It was found from figure, relative to negative control group, each treatment group shows the rush apoptosis work to tumour cell
With.Between each treatment group, the statistical value of the apoptosis of tumor cells rate of Sorafenib-Benexate Hydrochloride is about 24.6%,
Higher than other treatment group.
Figure 12 is Sorafenib-Benexate Hydrochloride, and Sorafenib, Sorafenib Tosylate treatment group and feminine gender are right
According to the tumor locus new vessels rate of group；
In figure, (a) is the new vessels situation of negative control group tumor locus, and (b) is Sorafenib treatment group tumors portion
The new vessels situation of position, (c) are the new vessels situation at Sorafenib Tosylate treatment group tumors position, and (d) draws for rope
The new vessels situation at Fei Ni-Benexate Hydrochloride treatment group tumors position.As seen from the figure, in Sorafenib-beta-schardinger dextrin
Under the therapeutic action of inclusion compound, tumor locus new blood vessel productivity ratio is only the 26~44% of negative control group, better than other treatment
Group.
Embodiment
The present invention is according to the principle of interaction of molecules, and using Sorafenib as object (such as formula I), beta-schardinger dextrin is main body point
Sub (such as formula II), in the presence of the solvent by clathration, Sorafenib molecule is included or is embedded in the molecule of beta-schardinger dextrin
In cavity, inclusion compound is formed.The mol ratio of Sorafenib molecule and beta-schardinger dextrin molecule is 1 in gained inclusion compound：1, inclusion compound
Purity is more than 99%.
Further, inclusion compound of the present invention carries out diffraction analysis by the use of CuK α as characteristic X-ray, with the θ of the angle of diffraction 2, ±
0.2 ° of expression
X-ray diffraction include 4.0 °, 6.0 °, 9.8 °, 11.8 °, 12.0 °, 13.3 °, 15.0 °, 16.0 °, 17.5 °,
18.4 °, 19.6 °, the characteristic diffraction peak at place.Fig. 1 is the XRD spectra of Sorafenib-Benexate Hydrochloride.Can by comparing
Know, inclusion compound has the diffractive features peak dramatically different with Sorafenib and beta-schardinger dextrin, illustrates via after clathration, host and guest
Body is intermolecular to generate interaction, so as to form new thing phase.
Further, the thermogravimetric analysis (TG) of Sorafenib-Benexate Hydrochloride of the present invention is as shown in Figure 2：Suo La
The decomposition temperature of non-Buddhist nun's active compound is 211.9 DEG C, and the decomposition temperature of Sorafenib-Benexate Hydrochloride of the present invention is 319.1
DEG C, show that inclusion compound significantly improves the heat endurance of drugs sorafenib.
Further, the differential thermal analysis (DSC) of Sorafenib-Benexate Hydrochloride of the present invention is as shown in Figure 3：This hair
Bright described Sorafenib-Benexate Hydrochloride has two endothermic peaks at 69.9 DEG C, 97.5 DEG C, causes to slough the crystallization water
Decalescence, and (282.0 DEG C) disappearances of the melting peak of the fusion and decomposition peak (232.7 DEG C) of Sorafenib and beta-schardinger dextrin enter
One step shows that Sorafenib forms new material by clathration and beta-schardinger dextrin, while the heat for improving drug ingedient is steady
It is qualitative.
Yet further, infrared spectrum analysis, inclusion compound collection of illustrative plates are carried out to Sorafenib of the present invention-Benexate Hydrochloride
(Fig. 4) has the characteristics that：(1) compared to Sorafenib and beta-schardinger dextrin monomer, do not occur in the infared spectrum of inclusion compound new
Absworption peak；(2)
Compared to the infrared spectrum of beta-schardinger dextrin, Sorafenib-Benexate Hydrochloride is in 1505cm-1And 1548cm-1Place
There is absworption peak.The two absworption peaks are respectively C=C stretching vibration and C=N stretching vibration, are by Sorafenib molecule
In structure caused by pyridine ring.
Integration analysis shows that Subjective and Objective molecule produces inclusion, the portion of Sorafenib by intermolecular weak effect or hydrogen bond action
Group is divided to be shielded in the cavity of beta-schardinger dextrin.
Nuclear magnetic resonance spectroscopy further demonstrate the architectural feature of inclusion compound, and nuclear magnetic resonance 1H-NMR spectrograms are shown (Fig. 5)：
In Sorafenib-Benexate Hydrochloride of the present invention, the NMR signal of Sorafenib part hydrogen atom weakens, table
The bright beta-cyclodextrin inclusion compound moieties of Sorafenib, to the hydrogen atom on group generate shielding action.Nuclear-magnetism is total to simultaneously
The NOESYspectrum that shakes spectrums show (Fig. 6)：The phenoxy group and pyridine structure of Sorafenib and the methine and hydroxyl of beta-schardinger dextrin
Structure is related, it was demonstrated that interaction between Subjective and Objective be present.
The present invention provides a kind of preparation method of above-mentioned Sorafenib-Benexate Hydrochloride, and this method has operation letter
Just the characteristics of, reproducible, material loss lacks, there is higher practical value.Specific implementation step is as follows：
By Sorafenib and beta-schardinger dextrin using mol ratio as 1：0.5~1.5 feeds intake, and is added to the tetrahydrofuran of certain volume
With the in the mixed solvent of water, heating stirring, up to complete dissolved clarification, it is further continued for stirring some hours progress host-guest inclusions, stops
Heating and stirring, Temperature fall crystallization, until crystallization is complete, filter, drying, produce Sorafenib-Benexate Hydrochloride.Step
It is rapid 1) in, Sorafenib and beta-schardinger dextrin rate of charge, the proportion of composing of mixed solvent, the volume ratio of tetrahydrofuran and water for 1~
4：1；In step 2), the amount (mM) of Sorafenib material and the volume (milliliter) of solution are than being 1 in reaction system：20~
100, in step 3), heating-up temperature is
50 DEG C~80 DEG C, mixing time is 4~30hr,；In step 4), recrystallization temperature is 20~30 DEG C, and drying course is
30~40 DEG C of vacuum drying.
The invention also discloses application of the Sorafenib-Benexate Hydrochloride in tumour growth research is suppressed.
Take 20 nude mices (male；6 week old；Body weight：18~22g, △ m<5g) raise under aseptic condition.Tumor model is adopted
Employment breast tumor cell (MDA-MB-231 cell lines) is established, and in vitro culture MDA-MB-231 cells, pancreatin digests and collected
In
It is made suspension in RPMI-1640 culture mediums, 106Cells/200 μ L are used for tumor inoculation.By 200 μ LMDA-MB-
231 cell infusions are in epidermis stomach wall.Tumor inoculation success, 4 groups are randomly divided into by nude mice after 20 days.Group I is negative control group
(Control), group II-group IV is treatment group.Group II-group IV receives Sorafenib, Sorafenib Tosylate and rope respectively
The treatment of La Feini-Benexate Hydrochloride.Administering mode：Orally；Dosage：10mg (active component)/kg, 1 times/day；
Treat the duration：21 days.During treatment, the body weight, health status and gross tumor volume of each group nude mice are observed and recorded.
After treatment end, nude mice is put to death, takes out tumor tissues.For the mechanism of action of active component Sorafenib, to tumor killing effect, promote
Apoptosis effect and anti-angiogenesis effect are studied.
Specifically, gross tumor volume measures：Gross tumor volume is measured once every three days, and the length of tumour is measured with slide measure
(a) it is and wide
(b) gross tumor volume, is estimated according to formula III.
a×b2/ 2 formulas III
Apoptosis and angiogenesis case study：It is right to observe the apoptosis of tumor cells and angiogenesis situation of tumor tissues
Tumor tissue section carries out TUNEL and CD31 and dyed simultaneously to be observed using Laser Scanning Confocal Microscope, according to being observed under respective channel
The statistical value analysis apoptosis and angiogenesis situation of the fluorescence area arrived.Tumor tissue section through processing is marked, broken
Broken or fracture DNA and nucleus are marked by TUNEL and Hoechst 332589 respectively, and apoptotic cell is in Laser Scanning Confocal Microscope
Blue-green fluorescent is sent under respective channel.Statistical analysis is carried out to study rush apoptosis effect to the fluorescence area of each group section sample
Fruit.Tumor tissue section through processing is marked, blood vessel and nucleus are marked by CD31 antibody and DAPI respectively, are being copolymerized
Under focusing microscope respective channel, labeled blood vessel sends green fluorescence.The fluorescence area of each group section sample is counted
Analyze to study anti-angiogenesis effect.
Result of study is as shown in Figure 10-Figure 12.By it was found that：(1) relative to negative control group, each equal table for the treatment of group
It is slow to reveal tumour growth, there is apoptosis phenomenon in tumour cell, and tumor locus angiogenesis by containment the characteristics of.This
Show that active component Sorafenib has played effect in oncotherapy, by disturb or block RAF/RAF/MEK/ERK and
VEGF signal paths, realize and suppress tumour growth, promote apoptosis of tumor cells and resist the effect of tumor locus angiogenesis.(2)
Between each treatment group, the therapeutic effect of group III is can detect slightly better than group II, but without significant difference；Relative to group II and group
III, organize IV therapeutic effect and significantly improve, show
Go out：Tumour growth is slowly or stagnation, tumor locus apoptosis rate reach the production of 18~25%, tumor locus blood vessel
Rate is only the 26~44% of negative control group.This shows in the case of active compound dosage identical, Sorafenib-beta-cyclodextrin inclusion compound
The therapeutic effect of thing is more excellent, and first cause is：Sorafenib-Benexate Hydrochloride of the present invention improves drug ingedient
Slow release effect, drug ingedient is maintained of a relatively high blood concentration within a certain period of time, improve the biology of drug ingedient
Availability.(3) found by observing and recording, in treatment group, the body weight of each group nude mice has declined, wherein organizing IV nude mice
Changes of weight is minimum, and in group II and group III, nude mice Body weight loss, diarrhoea phenomenon are more obvious.This draws mainly due to rope
Caused by the side effect of non-Buddhist nun in itself, and the spy that chemotherapeutics generally has
Point, after beta-cyclodextrin inclusion compound, the side effect of drug molecule substantially reduces, and reduces drug molecule to biology
The harm of body.
Further heretofore described method is described below by drawings and examples, but the scope of the present invention
It is not limited by the example, following examples are descriptive, are not limited, it is impossible to limit the protection model of the present invention
Enclose.
The preparation of 1 Sorafenib of embodiment-Benexate Hydrochloride
Precision weighs beta-schardinger dextrin 1.134g (1mmol) and Sorafenib 0.464g (1mmol) into eggplant type bottle, adds four
(volume ratio of tetrahydrofuran and water is 4 to the mixed solvent of hydrogen furans and water：1) 40mL, it is put into water-bath and heats while stirring
To 80 DEG C, continue to stir after dissolved clarification, continue stirring 4 hours, then take out and place room temperature (20~30 DEG C), stand crystallization, crystallization
About 8~10 hours time, until crystallization is complete, filter, drying (25 DEG C of vacuum drying), produce.
The preparation of 2 Sorafenibs of embodiment-Benexate Hydrochloride
Precision weighs beta-schardinger dextrin 1.134g (1mmol) and Sorafenib 0.232g (0.5mmol) into eggplant type bottle, adds
(volume ratio of tetrahydrofuran and water is 3 to the mixed solvent of tetrahydrofuran and water：1) 15mL, it is put into water-bath and adds while stirring
Heat continues to stir to 65 DEG C after dissolved clarification, continues stirring 30 hours, then stops stirring and heating, Temperature fall crystallization, is cooled to
At about 20~25 DEG C, that is, there is crystal precipitation, continuation crystallization is complete up to crystallization, filters, and drying (40 DEG C of vacuum drying), produces.
The preparation of 3 Sorafenibs of embodiment-Benexate Hydrochloride
Precision weighs beta-schardinger dextrin 1.134g (1mmol) and Sorafenib 0.696g (1.5mmol) into eggplant type bottle, adds
(volume ratio of tetrahydrofuran and water is 2 to the mixed solvent of tetrahydrofuran and water：1) 90mL, it is put into water-bath and adds while stirring
Heat continues to stir to 70 DEG C after dissolved clarification, continues stirring 25 hours, then stops stirring and heating, Temperature fall crystallization, is cooled to
At about 20~25 DEG C, that is, there is crystal precipitation, continuation crystallization is complete up to crystallization, filters, and drying (40 DEG C of vacuum drying), produces.
The preparation of 4 Sorafenibs of embodiment-Benexate Hydrochloride
Precision weighs beta-schardinger dextrin 1.134g (1mmol) and Sorafenib 0.372g (0.8mmol) into eggplant type bottle, plus
Entering the mixed solvent of tetrahydrofuran and water, (volume ratio of tetrahydrofuran and water is 1：1) 70mL is put into water-bath adds while stirring
Heat continues stirring 10 hours to 60 DEG C after dissolved clarification, then stop stirring and heating, Temperature fall crystallization, be cooled to about 20~25
DEG C when, that is, have a crystal precipitation, continue crystallization until crystallization is complete, filter, drying (30 DEG C of vacuum drying), produce.
The preparation of 5 Sorafenibs of embodiment-Benexate Hydrochloride
Precision weighs beta-schardinger dextrin 1.134g (1mmol) and Sorafenib 0.556g (1.2mmol) into eggplant type bottle, adds four
(volume ratio of tetrahydrofuran and water is 3 to the mixed solvent of hydrogen furans and water：1) 80mL, it is put into water-bath and heats while stirring
To 50 DEG C, continue stirring 8 hours after dissolved clarification, then take out and place room temperature (20~30 DEG C), standing crystallization, the crystallization time about 8~
10 hours, until crystallization is complete, filter, drying (35 DEG C of vacuum drying), produce.
The preparation of 6 Sorafenibs of embodiment-Benexate Hydrochloride
Precision weighs beta-schardinger dextrin 1.134g (1mmol) and Sorafenib 0.650g (1.4mmol) into eggplant type bottle, adds four
(volume ratio of tetrahydrofuran and water is 2 to the mixed solvent of hydrogen furans and water：1) 100mL is put into water-bath and heated while stirring
To 80 DEG C, continue stirring 12 hours after dissolved clarification, then stop stirring and heating, Temperature fall crystallization, be cooled to about 20~25 DEG C
When, that is, there is crystal precipitation, continuation crystallization is complete up to crystallization, filters, and drying (30 DEG C of vacuum drying), produces.
Embodiment 7 surveys equilbrium solubility of the Sorafenib-Benexate Hydrochloride in water and pH1.2 acid solutions, and
Contrasted with Sorafenib and Sorafenib Tosylate
Sorafenib, Sorafenib Tosylate and the Sorafenib-Benexate Hydrochloride of excess are separately added into
10mL pH are 7.0, and in the water that pH is 1.2.60~120min of closing stirring makes solution reach balance under water-bath (37 DEG C).
Solution prepared by saturated solution fast filtering (0.24 μm of miillpore filter) centrifuge tube, being determined in maximum absorption wave strong point
Absorbance.Each sample parallel determination 3 times.Its corresponding saturated concentration is calculated with calibration curve method.
Embodiment 8 determines Sorafenib-Benexate Hydrochloride in water and the dissolution rate of pH1.2 acidic aqueous solutions, and
Contrasted with Sorafenib and Sorafenib Tosylate
Sorafenib (0.5g), Sorafenib Tosylate (0.6g) and Sorafenib-Benexate Hydrochloride are taken respectively
(2g) powder is separately added into 500mL water (adding 0.2%SDS (lauryl sodium sulfate)) and pH1.2 acidic aqueous solutions (add 0.2%
SDS (lauryl sodium sulfate)) in, put in water-bath, with 37 DEG C of temperature, rotating speed 100rpm is slowly stirred, 1,2,3,4,5,
10th, 15,20,25,30,40,50,60,90, the miillpore filter that sampling 5mL crosses 0.24 μm during 120min takes every time into centrifuge tube
Add the dissolution blank medium of isothermal equivalent after the completion of sample.To make absorbance that measure obtains, sampling obtains in prescribed limit
Settled solution dilute certain multiple with corresponding blank medium, in the extinction of solution prepared by maximum absorption wave strong point measure
Degree.Each sample parallel determination 3 times.Its corresponding concentration is calculated with calibration curve method, draws out corresponding stripping curve.
Medicine generation inside the Sorafenib of embodiment 9, Sorafenib Tosylate and Sorafenib-Benexate Hydrochloride
Thank dynamics and in vivo distribution
Similar (18~22g, the △ m of 18 body weight<Male BALB/c nude mices 5g) are randomly divided into three groups of (6/group) progress
Vivo pharmacokinetic and internal distribution experiments.I oral Sorafenib active compound of group, organizes II oral toluenesulfonic acid Suo Lafei
Buddhist nun, organize III oral Sorafenib-Benexate Hydrochloride.Administering mode：Orally；Dosage：10mg (active component)/kg；
Blood concentration detection time：(after oral) 0~24 hour；Take blood mode：Eye socket takes blood, blood sample Quick spin processing (rotating speed：
6000rpm, time：5min), -20 DEG C of preservations are to be measured；Sample of tissue mode：Solution takes nude mouse tissue liver, kidney, pancreas, lung and the heart,
Tissue is washed with 0.1mol/L PBS, blots and weighs, is ground with 2 milliliters of tissue homogenizers, is shaken and extracted with mobile phase, with three
Fluorine acetic acid protein precipitation, is neutralized with NaOH, and deproteinized is centrifuged after constant volume, is accurately pipetted a certain amount of supernatant and is settled to 2 milliliters
Volumetric flask, -20 DEG C of preservations are to be measured.Concentration detection method：High performance liquid chromatography detection.
Finally, it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, it is of the invention
Above example is not limited to, there can also be many deformations.One of ordinary skill in the art can be straight from present disclosure
Export or all deformations associated are connect, are considered as protection scope of the present invention.
Claims (10)
- A kind of 1. Sorafenib-Benexate Hydrochloride, it is characterised in that described inclusion compound contain Sorafenib molecule and β- Cyclodextrin molecular, Inclusion ratio, i.e. mol ratio are 1：0.8~1.5.
- 2. Sorafenib-Benexate Hydrochloride as claimed in claim 1, it is characterised in that described inclusion compound contains rope The mol ratio of La Feini molecules and beta-schardinger dextrin molecule is preferably 1：1.
- 3. Sorafenib-Benexate Hydrochloride as claimed in claim 1, it is characterised in that described inclusion compound CuK α Diffraction analysis is carried out as characteristic X-ray, with the θ of the angle of diffraction 2, ± 0.2 ° represents that X-ray diffraction includes 4.0 °, 6.0 °, 9.8 °, Characteristic peak at 11.8 °, 12.0 °, 13.3 °, 15.0 °, 16.0 °, 17.5 °, 18.4 °, 19.6 °.
- A kind of 4. preparation method of Sorafenib-Benexate Hydrochloride as claimed in claim 1, it is characterised in that with β- Cyclodextrin is main body, using Sorafenib as object, in the presence of the solvent by inclusion reaction, by Sorafenib with comprising, put Beta-schardinger dextrin intramolecular is put or be embedded in, forms inclusion compound.
- 5. the preparation method of Sorafenib-Benexate Hydrochloride as claimed in claim 4, it is characterised in that including following Step：1), by Sorafenib and beta-schardinger dextrin using mol ratio as 1：0.5~1.5 feeds intake, and is added to the tetrahydrochysene furan of certain volume The in the mixed solvent muttered with water；2), by the mixed solution heating stirring that step 1) obtains to complete dissolved clarification；3), the settled solution for obtaining step 2) carries out inclusion reaction, heating stirring method, mixing time 4~30 hours；4), stop heating and stirring, Temperature fall crystallization, until crystallization is complete, filter, drying, produce Sorafenib-β-ring paste Inclusion compounds.
- 6. preparation method as claimed in claim 5, it is characterised in that the volume ml of the tetrahydrofuran described in step 1) and water Volume ml ratios are 1~4：1.
- 7. preparation method as claimed in claim 5, it is characterised in that Sorafenib in the mixed solution system described in step 2) Amount mmol and solution volume ml ratios be 1：20~100.
- 8. preparation method as claimed in claim 5, it is characterised in that the heating stirring method described in step 3), reaction time 4 ~30 hours, heating-up temperature was 50~80 DEG C.
- 9. preparation method as claimed in claim 5, it is characterised in that the recrystallization temperature described in step 4) is 20~30 DEG C；Institute The drying mode stated is vacuum drying, and drying temperature is 30~40 DEG C.
- 10. a kind of Sorafenib-Benexate Hydrochloride as described in claim 1 or 2 or 3 is being prepared for suppressing tumour life Application in long pharmaceutical preparation.
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| CN112891557A (en) * | 2021-02-03 | 2021-06-04 | 柯阳 | ICG-beta-cyclodextrin drug delivery system and preparation method and application thereof |
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