CN106551930A - Application of the tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared - Google Patents
Application of the tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared Download PDFInfo
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- CN106551930A CN106551930A CN201510630642.4A CN201510630642A CN106551930A CN 106551930 A CN106551930 A CN 106551930A CN 201510630642 A CN201510630642 A CN 201510630642A CN 106551930 A CN106551930 A CN 106551930A
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Abstract
The present invention provides application of the tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared.The toxicity includes Toxicity of Kidney, ototoxicity and neurotoxicity.Research shows that tetrahydropalmatine is inhibited with MATE1 to OCT2, the cytotoxicity that energy antagonism cis-platinum causes, and is preferably verified on the renal cells model of original cuiture.And with (-)-THP as representative, research finds that tetrahydropalmatine has good protective effect to cis-platinum induced mice injury of kidney.Therefore, tetrahydropalmatine is shared with cis-platinum, is likely to reduced cis-platinum and is accumulated in kidney, cells,cochlear and DRGs, so as to reaching prevention or reducing the expected results of toxicity of cisplatin.The present invention has expanded the new application of tetrahydropalmatine；Due to the high expression OCT2 of kidney, cochlear hair cell and DRGs, and on tumour cell, often lack the expression of OCT2, cis-platinum has certain passive permeability, therefore, tetrahydropalmatine is shared with cis-platinum, while decrease cis-platinum institute is causing toxicity, can't reduce the antitumous effect of cis-platinum.
Description
Technical field
The invention belongs to medicinal usage, is related to application of the tetrahydropalmatine caused by cis-platinum is reduced in drug toxicity, including Toxicity of Kidney, ototoxicity and neurotoxicity.
Technical background
Cis-platinum is one of current clinically maximally effective broad-spectrum anti-cancer drug, it is widely used in various treatment of solid tumor such as oophoroma, prostate cancer, lung cancer and breast cancer, but its serious toxicity, its clinical practice is limited including renal toxicity, ototoxicity, neurotoxicity etc., it is especially serious with renal toxicity and ototoxicity.Clinic is although with hydration therapy or forces the safeguard procedures such as diuresis, and the renal toxicity incidence of cis-platinum is still up to 25 ~ 35%, and occurs at the treatment initial stage immediately.By mechanism such as oxidative stress, DNA damage and inflammatory reactions, cis-platinum can cause renal tubular cell dead, Renal tissues damage, so that glomerular filtration rate(GFR declines, RE substantially reduces, causes which to be accumulated in kidney in a large number, cause serious kidney failure.Cis-platinum can also damage cochlear hair cell, cause hearing disability, cause deafness or tinnitus.Therefore, it is problem demanding prompt solution during clinical chemotherapy that cis-platinum institute is causing toxicity, and the research of its safeguard procedures is also study hotspot in recent years.The measure such as clinical conventional anti-inflammatory, anti-oxidant, with the kidney injury for protecting cis-platinum to cause, cells,cochlear is damaged and peripheral nerve injury, but effect is unsatisfactory.
Cisplatin-induced nephrotoxicity, ototoxicity and neurovirulent generation are a complicated multiple-factor participation processes, but its height concentration in renal cells, cochlear hair cell and dorsal root ganglion neurons cell is the key of its toxicity.Cis-platinum is distributed histoorgan each to whole body with blood circulation Jing after intravenously administrable, and wherein concentration highest is nephridial tissue, especially renal cells.Various intake types and outer row's type drug transporters, including the Organic cation transporter 2 of basilar memebrane side specificity overexpression are expressed on renal cells film（organic
cation transporter, OCT2）And teleblem side multiple medicines and toxin efflux protein (multidrug and
Toxin extrusion proteins, MATEs), play an important role in the kidney transmembrane transport and kidney of cationic drug are disposed.Cis-platinum Jing OCT2 are absorbed into renal tubular cell by blood, are accumulated in renal tubule；Outer row's type transporter MATEs
Cis-platinum is pumped out renal tubular cell by (MATE1 and MATE2-K), reduces its concentration in nephrocyte.Due to huge uptakes of the OCT2 to cis-platinum, and MATEs weaker to which outer row, cis-platinum is seriously accumulated in kidney, so as to cause Toxicity of Kidney.Equally, OCT2 transporters height is expressed in cochlear hair cell and Dorsal Root Ganglion Neurons, mediates the cellular uptake of cis-platinum, causes cis-platinum to cause serious ototoxicity and neurotoxicity in cells accumulation.It is clinical to attempt absorbing cis-platinum nephrocyte by suppressing OCT2 with the OCT2 such as Imatinib, Cimetidine inhibitor, to reduce the Toxicity of Kidney caused by cis-platinum.However, Cimetidine Reverse transcriptase MATEs effects are better than OCT2, strengthen cisplatin-induced nephrotoxicity on the contrary.For Cisplatin Ototoxicity or neurotoxicity, the method for clinical conventional oxidation and removing free radicals carrys out antagonism, but it is very little to produce effects.Therefore, find to OCT2 high inhibitions, to MATEs unrestraints or the medicine of weak suppression, to reduce accumulation of the cis-platinum in kidney, cells,cochlear and DRGs, be to expand the fine approach of cis-platinum clinical practice so as to reduce its toxicity.
Tetrahydropalmatine (tetrahydropalmatine, THP), including rotundine ((-)-THP), dextrorotation tetrahydropalmatine ((+)-THP), and dltetrahydropalmatine ((±)-THP), it is one of main effects composition of corydalis tuber (corydalis tuber), with various physiologically actives such as analgesia, anti-oxidant.Wherein (-)-THP is usually used in alleviating various pain such as headache, pectoralgia, and has the advantages that non-additive, toxic and side effect is little.This research investigates tetrahydropalmatine on the mouse renal epithelial cell of the transgenic cell and original cuiture of coexpression human OCT2 and MATE1, cochlear hair cell, Dorsal Root Ganglion Neurons first in stable expression people OCT2, MATE1, including
The inhibitory action of (-)-THP, (+)-THP and (±)-THP to OCT2 and MATE1, can reduce intracellular cis-platinum accumulation, and so as to antagonism cis-platinum, institute is cytotoxicity caused；Further with (-)-THP be representative study its to cis-platinum mouse kidney, cochlea and DRGs accumulate and toxicity impact, tetrahydropalmatine is illustrated as the meaning of the renal toxicity, ototoxicity and neurotoxicity toxicity-reducing medicament of cisplatin induced, provide for tumor patient and have attenuation, analgesia concurrently, without addicted adjuvant therapy medicaments of tumor.Tetrahydropalmatine structural formula：
The content of the invention
It is an object of the invention to provide application of the tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared.The toxicity includes Toxicity of Kidney, ototoxicity and neurotoxicity.
Research shows, tetrahydropalmatine, including (-)-THP,
(+)-THP and (±)-THP are inhibited with MATE1 to OCT2, the cytotoxicity that energy antagonism cis-platinum causes, and are preferably verified on the renal cells model of original cuiture.And with (-)-THP as representative, research finds that tetrahydropalmatine has good protective effect to cis-platinum induced mice injury of kidney.Therefore, tetrahydropalmatine is shared with cis-platinum, is likely to reduced cis-platinum and is accumulated in kidney, cells,cochlear and DRGs, so as to reaching prevention or reducing the expected results of toxicity of cisplatin.
Usefulness of the present invention is：(1) tetrahydropalmatine is the principle active component of one of " eight Zhe's " corydalis tuber, and Yuanhu Zhitong Prescription principle active component, and (-)-THP is even more clinical conventional antalgesic, has been used for treating the pain that tumour causes, and without into recessiveness.Therefore the present invention will expand the new application of corydalis tuber, Yuanhu Zhitong Prescription and (-)-THP；(2) although existing researcher is wished by Cisplatin medicine to the suppression of OCT2 to reduce cisplatin-induced nephrotoxicity, but the clinical OCT2 inhibitor (such as Cimetidine) attempted is often due to row outside the cis-platinum of MATE1 mediations with higher suppression, cis-platinum is made to accumulate in kidney on the contrary more, toxicity is higher.The study find that, suppression of the tetrahydropalmatine to OCT2 is noticeably greater than the suppression to MATE1, can significantly reduce renal toxicity, ototoxicity and the neurotoxicity of cis-platinum；(3) due to the high expression OCT2 of kidney, cochlear hair cell and DRGs, and on tumour cell, often lack the expression of OCT2, as cis-platinum has certain passive permeability, therefore, tetrahydropalmatine is shared with cis-platinum, while decrease cis-platinum institute is causing toxicity, the antitumous effect of cis-platinum can't be reduced.
Description of the drawings
Figure 1. Cis-platinum exists
MDCK-hOCT2 , MDCK-hOCT2/hMATE1 With mock Concentration dependent toxicity on cell.With solvent control group cytoactive average as 100%, in figure, data are represented with mean ± SD, n=3；Compare with mock cells, *P<0.05, * *P<0.01 and * * *P<0.001。
Figure 2.(-)-THP,(+)-THP And ( ± )-THP Concentration suppresses according to lazyness MPP + MDCK-hOCT2 Accumulation on cell.With solvent control group MPP+ Uptake values are 100%, and in figure, data are represented with mean ± SD, n=3.
Figure 3.(-)-THP,(+)-THP And ( ± )-THP Concentration suppresses according to lazyness MPP + MDCK- hMATE1 Accumulation on cell.With solvent control group MPP+ Uptake values are 100%, and in figure, data are represented with mean ± SD, n=3.
Figure 4. (-)-THP,(+)-THP And ( ± )-THP Cis-platinum is existed MDCK-hOCT2 On cell, the concentration of toxicity is according to lazyness inhibitory action.With solvent control group numerical value as 100%, in figure, data are represented with mean ± SD, n=3；Compare with control group,### P<0.001；Compare with cis-platinum group, * * *P<0.001。
Figure 5. (-)-THP,(+)-THP And ( ± )-THP Cis-platinum is existed MDCK-hOCT2/hMATE1 On cell, the concentration of toxicity is according to lazyness inhibitory action.With solvent control group numerical value as 100%, in figure, data are represented with mean ± SD, n=3；Compare with control group,### P<0.001；Compare with cis-platinum group, *P<0.05, * *P<0.01 and * * *P<0.001。
Figure 6.(-)-THP,(+)-THP And ( ± )-THP Concentration reduces what cis-platinum caused according to lazyness MDCK-hOCT2 Cell lactic dehydrogenase (LDH) Seepage.With solvent control group LDH vigor as 100%, in figure, data are represented with mean ± SD, n=3；Compare with control group,### P<0.001；Compare with cis-platinum group, * *P<0.01 and * * *P<0.001。
Figure 7. (-)-THP,(+)-THP And ( ± )-THP Concentration reduces cis-platinum according to lazyness and causes MDCK-hOCT2/hMATE1 Cell lactic dehydrogenase (LDH) Seepage.With solvent control group LDH vigor as 100%, in figure, data are represented with mean ± SD, n=3；Compare with control group,### P<0.001；Compare with cis-platinum group, *P<0.05, * *P<0.01 and * * *P<0.001。
Figure 8. (-)-THP,(+)-THP And ( ± )-THP To the concentration of cis-platinum toxicity on mouse primary renal cells according to lazyness inhibitory action.With solvent control group numerical value as 100%, in figure, data are represented with mean ± SD, n=3；Compare with control group,### P<0.001；Compare with cis-platinum group, * * *P<0.001。
Figure 9. (-)-THP In the distribution of mice plasma and renal tissue.In figure, data are represented with mean ± SD, n=5.
Figure 10. (-)-THP (20 mg/kg , tail vein injection, iv ) To cis-platinum (10 mg/kg , iv) In the impact of mouse kidney concentration.In figure, data are represented with mean ± SD, n=6-10；Compare with cis-platinum group, * * *P<0.001。
Figure 11. (-)-THP (20mg/kg , tail vein injection, iv ) To cis-platinum (10 mg/kg , iv) The impact of administration Mouse Weight.In figure, data are represented with mean ± SD, n=6-10；Compare with control group,### P<0.001；Compare with cis-platinum group, *P<0.05 and * *P<0.01。
Figure 12. (-)-THP (20 mg/kg , tail vein injection, iv ) To cis-platinum (10 mg/kg , iv) The impact of mouse renal function.With solvent control group biochemical indicator average as 100%, in figure, data are represented with mean ± SD, n=6-10；Compare with control group,### P<0.001；Compare with cis-platinum group, * * *P<0.001.
Figure 13. The representative collection of illustrative plates of kidney of mouse pathological examination.(A) solvent control group
(control) mouse kidney section；(B) cis-platinum group (10mg/kg,iv) mouse kidney section；(C) merge and give cis-platinum
(10mg/kg,iv) and (-)-THP (20mg/kg,iv) group mouse kidney section.Renal tissues pathology changes：Cast in black arrow indication renal tubule, the necrosis of green arrow indication renal cells. leica
DM2500 microscopes, 10*20 times is taken pictures.
Specific embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment
1
Cis-platinum exists
MDCK-hOCT2
,
MDCK-hOCT2/hMATE1
With
mock
Concentration dependent toxicity on cell
1.1 on 96 orifice plates inoculating cell
The MDCK-hOCT2 in exponential phase is chosen, MDCK-hOCT2/hMATE1 and mock cells (are built by pharmaceutical college of Zhejiang University, concrete construction method is shown in a:Lei Hongmei etc. is stable to express
The structure of hMATE1 and coexpression hMATE1 and hOCT1 or hOCT2 cell models.Acta Pharmaceutica Sinica, 2015,50 (7)：842-847.b:Wang K., et al., Involvement of organic cation
transporter 2 inhibition in potential mechanisms of antidepressant action.Prog
Neuropsychopharmacol Biol Psychiatry, 2014,53:90-98), digestion, collection cell.With fresh culture again suspension cell, it is 4.0 × 10 to adjust cell density4Individual/mL, is inoculated in 96 porocyte plates cultures with 0.2 mL/ holes.
1.2
MTT experiment
After 24 h of cell culture, reject culture medium adds the culture medium of medicine containing variable concentrations (cis-platinum), and the culture medium with solvent containing respective concentration (DMSO) is as blank control group.48 h are cultivated after administration carries out MTT experiments, add 5 mg/mL MTT (thiazole bromide blue tetrazolium) reagent, 20 μ L per hole, gently shake up, after incubation 4h, Incubating Solution being discarded, adding 150 μ L DMSO, micro shaker 10 min to be vibrated after formazan crystallization is completely dissolved in every hole, enzyme-linked immunosorbent assay instrument determines 570
Nm wavelength is (with 630
Nm is reference) absorbance of each sample in place, the survival rate of the sample cell is represented with the average of absorbance/blank control group absorbance.Data are with mean ± SD (n
=3) represent, to be not added with the control group of cis-platinum
(control) cell survival rate is 100%；Compare with control group, * * *P
<0.001, * *P<0.01 and *P< 0.05。
1.3 result
MTT experiment result shows, MDCK-hOCT2, MDCK-hOCT2/hMATE1 and mock cell and cis-platinum
After (0.01-1000 mol/L) is incubated 48 h altogether, MDCK-hOCT2 is significantly larger than mock cell, its LD to the toxic sensitivity of cis-platinum with MDCK-hOCT2/hMATE1 cells50Value is respectively 8.77
± 0.21 mol/L, 5.49 ± 0.18 mol/L and 14.8 ± 3.5 mol/L, referring to Fig. 1（With solvent control group cytoactive average as 100%, in figure, data are represented with mean ± SD, n=3；Compare with mock cells, *P<0.05, * *P<0.01 and * * *P<0.001）.The above results are pointed out
HOCT2 mediation cis-platinum cellular uptakes increase cells accumulation, cause its cytotoxicity to strengthen.
Embodiment
2
Tetrahydropalmatine, including
(-)-THP
,
(+)-THP
And
(
±
)-THP
It is right
MPP
+
MDCK-hOCT2
With
MDCK- hMATE1
The inhibitory action gathered on cell
The MDCK-hOCT2 in exponential phase is chosen, MDCK-hOCT2/hMATE1 and MDCK-hMATE1 cells (are built by pharmaceutical college of Zhejiang University, concrete construction method is shown in a:Lei Hongmei etc. is stable to express
The structure of hMATE1 and coexpression hMATE1 and hOCT1 or hOCT2 cell models.Acta Pharmaceutica Sinica, 2015,50 (7)：842-847.b:Wang K., et al., Involvement of organic cation
transporter 2 inhibition in potential mechanisms of antidepressant action.Prog
Neuropsychopharmacol Biol Psychiatry, 2014,53:90-98), digestion, collection cell.With fresh culture again suspension cell, it is 2.0 × 10 to adjust cell density5Individual/mL, is inoculated in 24 porocyte plates cultures with 0.5 mL/ holes, when cell growth can be used for accumulation experiment up to 90% degrees of fusion.
MDCK-hOCT2 and MDCK-hMATE1 cells are the mdck cell of high expression people OCT2 and MATE1 transporters.Study tour 0.001 ~ 100 μm of ol/L (-)-THP, (+)-THP and (±)-THP are to MPP+The impact that (the classical substrate of OCT2 and MATE1 transporters, 1 μm of ol/L, 3min) is gathered in above-mentioned cell.As a result show：(-)-THP,
(+)-THP and (±)-THP can significantly inhibit MPP+Accumulation on MDCK-hOCT2 and MDCK-hMATE1 cells, to OCT2 inhibition constants IC50Respectively 2.6,9.5,0.24 μm of ol/L, to MATE1 inhibition constants IC50Respectively 7.4,8.6,2.6 μm of ol/L, referring to Fig. 2, Fig. 3, with solvent control group MPP+Uptake values are 100%, and in figure, data are represented with mean ± SD (n=3), to be not added with the cell survival rate of THP as 100%.The above results are pointed out：(-)-THP, (+)-THP and (±)-THP are respectively provided with stronger inhibitory action to OCT2 and MATE1, and (-)-THP and (±)-THP are weaker than the suppression to OCT2 to MATE1 rejection abilities.
Embodiment
3(-)-THP, (+)-THP
And
(
±
)-THP
Cis-platinum is existed
MDCK-hOCT2
And
MDCK-hOCT2/hMATE1
The impact of toxicity on cell
3.1 MTT experiment
Choose MDCK-hOCT2 and MDCK-hOCT2/hMATE1 in exponential phase (to be built by pharmaceutical college of Zhejiang University, concrete construction method is shown in a:Lei Hongmei etc. is stable to express
The structure of hMATE1 and coexpression hMATE1 and hOCT1 or hOCT2 cell models.Acta Pharmaceutica Sinica, 2015,50 (7)：842-847.b:Wang K., et al., Involvement of organic cation transporter
2 inhibition in potential mechanisms of antidepressant action.Prog
Neuropsychopharmacol Biol Psychiatry, 2014,53:90-98), digestion, collection cell.With fresh culture again suspension cell, it is 4.0 × 10 to adjust cell density4mL-1, 96 porocyte plates cultures are inoculated in 0.2mL/ holes.After 24 h of cell culture, reject culture medium is added containing 1 ~ 100 μm of ol/L (-)-THP,
The cis-platinum culture medium solution of (+)-THP and (±)-THP, the culture medium with solvent containing respective concentration (DMSO) is as blank control group.48 h are cultivated after administration carries out MTT experiments, add 5 mg/mL MTT (thiazole bromide blue tetrazolium) reagent, 20 μ L per hole, gently shake up, after incubation 4h, Incubating Solution being discarded, adding 150 μ L DMSO, micro shaker 10 min to be vibrated after formazan crystallization is completely dissolved in every hole, enzyme-linked immunosorbent assay instrument determines 570
Nm wavelength is (with 630
Nm is reference) absorbance of each sample in place, the survival rate of the sample cell is represented with the average of absorbance/blank control group absorbance.
The measure of LDH vigor in 3.2 cell culture fluids
The cell culture based specimen collected after administration 48 h of culture carries out LDH vitality tests, determines kit specification according to lactic dehydrogenase (LDH) and requires to determine LDH vigor in cell culture fluid.Relative quantification is carried out as 100% with the solvent control group enzyme activity of each concentration administration.
3.3 result
(-)-THP, impacts of (+)-THP and (±)-THP to cis-platinum toxicity on MDCK-hOCT2 and MDCK-hOCT2/hMATE1 cells are investigated with MTT experiments and the LDH vigor in cell culture fluid.As a result show, cis-platinum significantly reduces cell survival rate, and (-)-THP,
(+)-THP and (±)-THP concentration resist cis-platinum according to lazyness (1 ~ 100 μm of ol/L)
(cisplatin, 15 μm of ol/L)
The cell survival rate of induction is reduced, referring to Fig. 4, Fig. 5（With solvent control group numerical value as 100% in figure, in figure, data are represented with mean ± SD, n=3；Compare with control group,### P<0.001；Compare with cis-platinum group, *P<0.05, * *P<0.01 and * * *P<0.001）.Meanwhile, (-)-THP, (+)-THP and (±)-THP (1 ~ 100
Mol/L the rising of the cell LDH vigor that cis-platinum causes) is can obviously reduce, referring to Fig. 6, Fig. 7, with solvent control group LDH vigor as 100% in figure, data are represented with mean ± SD in figure, n=3；Compare with control group,### P<0.001；Compare with cis-platinum group, *P<0.05, * *P<0.01 and * * *P<0.001。
Embodiment
4(-)-THP, (+)-THP
And
(
±
)-THP
Impact to cis-platinum toxicity on mouse primary renal cells
The separation of 4.1 mouse primary renal cellses and culture
Healthy ICR mouse (male, ~ 25g), after etherization, 75% alcohol-pickled 1 minute, is fixed on operating table.Cut abdominal cavity open, with without Ca2+HBSS solution from vena portae hepatica perfusion to kidney existing canescence, remove rapidly double kidneys and be placed in the aseptic PBS solution added with dual anti-(+1% streptomysin of 1% penicillin).With cleaning several times added with dual anti-PBS solution in aseptic super-clean bench, kidney coating is removed, is shredded；After cleaning 3 ~ 5 times, shredded with aseptic operation blade, be placed in concussion digestion about 40min in the mixed solution of 0.1% type Ⅳ collagenase and 0.1% pancreatin.Supernatant liquid is in 80 mesh sieve net filtrations, with DMEM/F12 culture mediums (culture medium containing DMEM/F12,10% hyclone, 1% is dual anti-, 1% Insulin-Transferrin-selenium, three agent) eccentric cleaning 2 times, it is resuspended in culture medium, plants in 96 orifice plates after crossing 200 eye mesh screens, and liquid is changed every other day, growth can be used for cytotoxicity experiment after about 5 ~ 6 days.
4.2
MTT experiment
After Primary kidney cells culture about 5 days, reject culture medium is added containing 1 ~ 100 μm of ol/L (-)-THP,
The cis-platinum culture medium solution of (+)-THP and (±)-THP, the culture medium with solvent containing respective concentration (DMSO) is as blank control group.48 h are cultivated after administration carries out MTT experiments, add 5 mg/mL MTT (thiazole bromide blue tetrazolium) reagent, 20 μ L per hole, gently shake up, after incubation 4h, Incubating Solution being discarded, adding 150 μ L DMSO, micro shaker 10 min to be vibrated after formazan crystallization is completely dissolved in every hole, enzyme-linked immunosorbent assay instrument determines 570
Nm wavelength is (with 630
Nm is reference) absorbance of each sample in place, the survival rate of the sample cell is represented with the average of absorbance/blank control group absorbance.
4.3 result
MTT experimental results show, (-)-THP,
(+)-THP and (±)-THP can concentration according to lazyness (1 ~ 100 μm of ol/L) reduce cis-platinum
(cisplatin, 15 μm of ol/L)
Toxicity on mouse primary nephrocyte, increases cell survival rate, and referring to Fig. 8, with solvent control group numerical value as 100% in figure, in figure, data are represented with mean ± SD, n=3；Compare with control group,### P<0.001；Compare with cis-platinum group, * * *P<0.001。
Impact to induced by Cisplatin renal toxicity
5.1 animal used as test
ICR mouse (25 ~ 30g), male provide (two grades of cleaning, animal credit number by Zhejiang Academy of Medical Sciences animal center：SCXK (Zhejiang)
20140001).All animals are raised according to the requirement of Zhejiang University's animal used as test Operating Guideline, and feeding environment controls suitable temperature (20 ~ 23 DEG C) and humidity (50 ~ 60%), test front 12 h fasting, free water.
5.2
(-)-THP renal tissues are distributed
5.2.1 animal is administered and sample Bian collection
ICR mouse 25, are randomly divided into 5 groups, 5 per group.Tail vein injection gives 20 mg/kg (-)-THP solution, and 10 min after administration, 20 min, 30 min, 60 min and 120 min time points take blood plasma and kidney respectively.Tissue is washed away into blood with physiological saline, filter paper is blotted and is precisely weighed, dispersal mechanism is homogenized into 1g/15mL using tissue
Tissue homogenate (acetonitrile：Water=1:1), -80 DEG C of preservations, to be measured.
5.2.2
Biological sample is pre-processed
The blood plasma or tissue sample, thaw at RT of -80 DEG C of preservations, take blood plasma and each 50 μ L of tissue homogenate are put in 1.5 mL centrifuge tubes, respectively plus 200 μ L and 450 μ L acetonitriles (containing the internal standard Loratadines 50
Ng/mL), blood plasma or tissue sample revolve 2 min protein precipitations of vibration, 15 min are centrifuged in 13000rpm, take supernatant, carries out LC-MS/MS analyses.With determinand and internal standard peak area ratio, retinue calibration curve is substituted into, quantitative analysis is carried out.
Impacts of 5.3 (-)-THP to cis-platinum concentration in kidney
5.3.1 animal is administered and sample Bian collection
ICR mouse 16 (male, 25 ~ 30g of body weight), are randomly divided into 10 mg/kg cis-platinums groups and 10
Mg/kg cis-platinums+20
Mg/kg (-)-THP groups.After tail vein single injection gives (-)-THP solution, then intravenous 10
Mg/kg cisplatin solutions.After 72 h are administered, cut open to kill and take renal tissue, blood is washed away with physiological saline, filter paper is blotted, -80 DEG C of preservations are to be measured.
5.3.2
Kidney samples A is pre-processed
The kidney samples A of -80 DEG C of preservations, thaw at RT, 50 mg of accurately weighed renal tissue are put in 2.0 mL centrifuge tubes, plus 200 μ L nitric acid and 400 μ L H2O2, 1.5 h are cleared up in 80 DEG C of water-bath vibrations, taking-up is placed in ice bath and is cooled to room temperature.Add 200 μ L ammoniacal liquor to be neutralized, 0.22 μM of aqueous membrane filtration, and 3.0 are settled to 0.1% nitric acid
ML, sample carry out the concentration that ICP-MS analyzes platinum Jing after ultrasound.
Impacts of 5.4 (-)-THP to cis-platinum induced mice renal toxicity
5.4.1 animal is administered and sample Bian collection
22 ICR mouse (male, 25 ~ 30g of body weight), are randomly divided into control groups, 10 mg/kg cis-platinums groups and 10 mg/kg cis-platinums+20
Mg/kg (-)-THP groups (n=6-10).After tail vein single injection first gives (-)-THP solution, then 10 mg/kg cisplatin solutions of intravenous, control groups give the physiological saline of same volume.The body weight of mouse, close observation record mouse skin, the change of hair and general activity situation are recorded daily.72 h before administration and after administration, the blood sampling of eye socket angular vein clump take Virus monitory renal function biochemical indicator (including urea nitrogen BUN, creatinine CRE, uric acid URA), and cut open and kill mouse, take out kidney, precise weighing, after portion of tissue is fixed with 40% formalin buffer, FFPE does pathological section, carry out HE dyeing, the pathologic change of basis of microscopic observation nephridial tissue structure.
5.5 experimental result 5.5.1 (-)-THP renal tissues are distributed
After 20 mg/kg (-)-THP solution of mouse mainline, (-)-THP is distributed in renal tissue rapidly, reachable 22.6 μM during 10 min, about 5 times of Plasma (4.5 μM), referring to Fig. 9.In prompting nephridial tissue, (-)-THP concentration is higher, may occur to interact and the kidney concentration of impact cis-platinum with transporter OCT2/MATE1 in kidney, thus may affect its Toxicity of Kidney.
5.5.2 (-)-THP reduces the kidney concentration of cis-platinum
In order to illustrate whether (-)-THP can affect cis-platinum in the concentration of kidney by suppressing the cis-platinum renal transport of OCT2/MATE1 mediations, we further compare single and give 10 mg/kg cis-platinums, and merging gives cis-platinum and 20 mg/kg (-)-THP
After 72 h, the difference of cis-platinum concentration in kidney.As a result show, individually give cis-platinum in cis-platinum group mouse kidney at concentrations up to 141.9
Ppb, and it is 81.7 that merging give (-)-THP to be organized mouse kidney concentration
Ppb, referring to Figure 10, illustrates that (-)-THP can significantly inhibit the kidney intake of cis-platinum and reduce its kidney concentration.
5.5.3
Animal general status in toxicity of cisplatin test
With the cis-platinum of 10 mg/kg dosage to 24 h after mouse tail vein single injection after, mouse mouse fur starts to hold up, and occur it is dispirited, to external world IR it is blunt, activity is reduced, and anorexia, body weight start to be decreased obviously, and the behavior and activity reaction of control rats are without exception, and body weight increases steadily, there is significant difference in both, illustrate that cis-platinum has certain toxicity to mouse mouse integral level.And merging the mouse for giving 20 mg/kg (-)-THP, the body weight that cis-platinum causes reduces being relieved, and points out (-)-THP suppress the mouse kidney toxicity caused by cis-platinum, referring to Figure 11.
5.5.4 Biochemical Indices In Serum
The urea nitrogen (BUN) of 72 h mice serums, creatinine (CRE) and uric acid before detection administration and after administration
(URA) value.As seen from Figure 12, cis-platinum group mice serum renal function index BUN values are individually given and is significantly higher than solvent control group（control）, after cis-platinum shares (-)-THP of 20 mg/kg, BUN values are substantially reduced and close to control groups.The above results are pointed out, and 10 mg/kg cis-platinums of intravenous injection can cause serious renal toxicity in Mice Body；20
The renal toxicity that (-)-THP of mg/kg dosage causes to cis-platinum has stronger mitigation.
5.5.5 renal pathology inspection
HE is dyeed, and pathological analysis of cutting into slices, cisplatin administration group kidney of mouse pathologic finding find there are damage situations：Main pathological change is cast in renal tubule, and part renal cells is downright bad, and shares 20
Mg/kg (-)-THP group Renal tissues damages significantly take a turn for the better, and have no that obvious pathology is sexually revised.Solvent control group (control) group renal pathology inspection does not show any abnormalities, referring to Figure 13.
Claims (3)
1. application of a kind of tetrahydropalmatine in anti-toxicity of cisplatin medicine is prepared, the toxicity include Toxicity of Kidney, ototoxicity and neurotoxicity.
2. application according to claim 1, it is characterised in that the medicine is made up in the auxiliary material that preparation is allowed of tetrahydropalmatine.
3. application according to claim 2, it is characterised in that the method for administration of the medicine is for intravenous and orally.
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| PCT/CN2016/098657 WO2017054640A1 (en) | 2015-09-29 | 2016-09-12 | Use of tetrahydropalmatine in preparing anti-cisplatin toxicity medicament |
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| CN111110675A (en) * | 2020-02-25 | 2020-05-08 | 浙江大学 | Application of tetrahydropalmatine in preparation of anti-oxaliplatin peripheral neurotoxicity drugs |
| CN116637113A (en) * | 2023-06-20 | 2023-08-25 | 南京鼓楼医院 | Application of palmitin in preparation of medicines for treating or preventing ototoxicity caused by platinum or aminoglycoside medicines |
| WO2024011253A1 (en) * | 2022-07-08 | 2024-01-11 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Impeding platinum-based chemotherapeutic induced ototoxicity using a colony stimulating factor 1 receptor inhibitor |
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| CN111110675A (en) * | 2020-02-25 | 2020-05-08 | 浙江大学 | Application of tetrahydropalmatine in preparation of anti-oxaliplatin peripheral neurotoxicity drugs |
| WO2021169194A1 (en) * | 2020-02-25 | 2021-09-02 | 浙江大学 | Application of tetrahydropalmatine in preparation of drugs against oxaliplatin-induced peripheral neurotoxicity |
| WO2024011253A1 (en) * | 2022-07-08 | 2024-01-11 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Impeding platinum-based chemotherapeutic induced ototoxicity using a colony stimulating factor 1 receptor inhibitor |
| CN116637113A (en) * | 2023-06-20 | 2023-08-25 | 南京鼓楼医院 | Application of palmitin in preparation of medicines for treating or preventing ototoxicity caused by platinum or aminoglycoside medicines |
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