CN103861084A - Application of Romidepsin in treatment of Parkinson disease - Google Patents
Application of Romidepsin in treatment of Parkinson disease Download PDFInfo
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- CN103861084A CN103861084A CN201410048808.7A CN201410048808A CN103861084A CN 103861084 A CN103861084 A CN 103861084A CN 201410048808 A CN201410048808 A CN 201410048808A CN 103861084 A CN103861084 A CN 103861084A
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Abstract
The invention provides an application of Romidepsin in treatment of the Parkinson disease. In-vitro and in-vivo pharmacological experiments prove that the Romidepsin can induce gene expression of dopaminergic neuron cell strains and substantia nigra brain tissue oxidative stress resistance factor metallothionein 2, reduce the oxidative stress of the dopaminergic neurons of a Parkinson disease model, reduce the apoptosis of the dopaminergic neurons and reduce the damage of the dopaminergic neurons of substantia nigra of a PD animal model so as to play a role in treating the Parkinson disease.
Description
Technical field
The present invention relates to the application of Romidepsin, be specifically related to the application of Romidepsin in treatment of Parkinson disease.
Background technology
Parkinson disease (Parkinson ' s disease, PD) be second largest common neurodegenerative diseases, prevalence 0.3%, over-65s prevalence 1-3%.Its pathological characters is to form with Lewy corpusculum, and dopamine neuron degeneration damage in black substance dense area is principal character.
At present; the treatment of PD is mainly to alleviate motor symptoms; the method of still not curing; clinically be used for the treatment of Parkinsonian medicine and have Benserazide preparation, anticholinergic agents, amantadine, dopamine-receptor stimulant, monoamine oxidase B inhibitors, catechol-O-methyl transferase, COMT inhibitor, and neuroprotective etc.Levodopa (Levodopa, L-dopa) as dopamine precursor, it is the main medicine of PD treatment, by improving dopamine level, alleviate the dyskinesia, but " honeymooners " of levodopa treatment is generally 3-5, after this, often can there is the new dyskinesia such as motor fluctuation or unusual fluctuation disease in patient.For complication such as dyskinesias, to think at present and mainly caused by two reasons, one is that the levodopa half-life is short, the fluctuation of concentration in blood and cerebral tissue is obvious; It two is with disease progression, and the continuous degeneration of dopaminergic neuron, death cause its buffer capacity to decline, and cause the pulsed of dopamine receptor is stimulated.But in the therapeutic strategy stimulating at lasting dopaminergic, 2007, dopamine-receptor stimulant pergolide was withdrawn from the market owing to being found to have the valvular effect of infringement after 19 years in listing.
The key of carrying out property of PD state of an illness progress is the damage of carrying out property of dopamine neuron.The clinical treatment of PD can not stop or slow down the sexual development that carries out of PD dopamine neuron damage at present, and patient is along with the prolongation state of an illness of sick time increases the weight of gradually.Urgently find at present the method for new effective treatment PD.
Romidepsin is by the antitumor drug of Gloucester Pharmaceuticals company of U.S. exploitation, is approved listing by U.S. FDA on November 9th, 2009, and this medicine is used for the treatment of skin and lymphoma peripheral T cell.The inventor finds under study for action, Romidepsin induced oxidation stress be expressed by resistance factor metallothionein 2, reduce PD model dopamine neuron active oxygen (reactive oxygen species, ROS) produce, reduce PD model dopamine neuron apoptosis and damage, play the Parkinsonian effect for the treatment of.
Summary of the invention
The object of this invention is to provide the purposes of Romidepsin in treatment parkinson disease.
Specifically, the invention discloses Romidepsin and reduce PD model dopamine neuron ROS generation, reduce the application of PD model dopamine neuron apoptosis and damage.
Medicine Romidepsin of the present invention obtains by commercially available.
PD model of the present invention is PD cell model and PD animal model.
Cell in vitro PD model of the present invention is rotenone PD cell model and MPP
+(1-methyl-4-phenylpyridinium ion) PD cell model.Cell is dopamine neuron cell strain.
Animal PD model of the present invention is the three-dimensional locating injection rotenone preparation of unilateral nigra.
In PD cell model, the valid density scope of Romidepsin is 0.25-60 μ M, and optimum concentration range is 2.5-15 μ M.In PD animal model, the effective dosage ranges of Romidepsin is 0.2-4 mg/10 g, and optimal dose scope is 0.5-2 mg/10 g.
The present invention measures dopamine neuron cell strain by real-time RT-PCR and black substance oxidative stress resistance factor metallothionein 2 is expressed.
The present invention produces and the effect of apoptosis research Romidepsin to external PD cell model by measuring PD cell model dopamine neuron active oxygen ROS.
The present invention is the rescue effect to PD animal model nigral dopaminergic neuron by tyrosine hydroxylase (Tyrosine hydroxylase, TH) immunostaining research Romidepsin.
In the present invention, the advantage of Romidepsin treatment PD is: 1. Romidepsin induction dopamine neuron cell strain and black substance brain tissue oxidizing stress 2 gene expressions of resistance factor metallothionein, 2. Romidepsin reduces PD model dopamine neuron ROS generation, 3. reduce PD model dopamine neuron apoptosis, 4. increase PD animal model TH positive cell number, reduce the damage of PD model dopamine neuron.
The invention provides the purposes of Romidepsin, i.e. the therapeutic use of Romidepsin to PD.
Accompanying drawing explanation
Fig. 1 is that Romidepsin reduces rotenone PD cell model dopamine neuron apoptosis.Bright blue-fluorescence dyeing represents apoptotic cell.
Fig. 2 is that Romidepsin reduces MPP
+pD cell model dopamine neuron apoptosis.Bright blue-fluorescence dyeing represents apoptotic cell.
Fig. 3 is that Romidepsin reduces the damage of rotenone PD animal model dopamine neuron.
The specific embodiment
Further describe by the following examples the present invention, it should be understood that these embodiment, only for the object of illustration, do not limit the scope of the invention.
The Romidepsin using in the embodiment of the present invention is purchased from TOKYO CHEMICAL INDUSTRY CO.
In embodiment, experiment in vitro object of study is dopamine neuron cell strain SH-SY5Y, integral experiment, and object of study is Kunming mouse.
Measurement data represents with mean ± standard deviation, applies SPSS16.0 software and carries out variance analysis, and p<0.05 is for there being significant difference.
Oxidative stress resistance factor metallothionein 2 can alleviate oxidative stress damage.Embodiment 1 and 2 investigates Romidepsin to dopamine neuron and 2 gene expressions of black substance cerebral tissue metallothionein.
embodiment 1:2 gene expressions of Romidepsin induction dopamine neuron cell strain metallothionein.
Day1: will be incubated at 25cm
2the SH-SY5Y cell of culture bottle is made cell suspension, is laid in 6 orifice plates every porocyte about 1 × 10
6.
Day2: the about 30-40% of cell fusion, give Romidepsin, final concentration is 2.5,5,15 μ M.Cultivate 48 hours.
Abandon culture medium, extract cell RNA, real-time RT-PCR measures human metal thioalbumen 2 and expresses, and human metal thioalbumen 2 mRNA primers are purchased from Qiagen (cat.no.QT00220199).Result shows 2 gene expressions of Romidepsin induction dopamine neuron cell strain metallothionein, and presents dose dependent in table 1.
The impact (n=6) of table 1 Romidepsin on 2 gene expressions of dopamine neuron cell strain metallothionein
| Group | Metallothionein 2 |
| Solvent control | 1.0±0.16 |
| Romidepsin 2.5 μM | 4.2±2.7 * |
| Romidepsin 5 μM | 9.5±4.4 * |
| Romidepsin 15 μM | 21.4±9.1 * |
Note: * and the comparison of solvent control group,
p<0.05.
embodiment 2:2 gene expressions of Romidepsin induction cerebral tissue metallothionein.
Mice gives Romidepsin 0.5,1,2 mg/10 g b.w., after 2 hours, Trizol extracts brain tissue cell RNA, and real-time RT-PCR measures Mouse Metallothionein 2 and expresses, and Mouse Metallothionein 2 mRNA primers are purchased from Qiagen (cat.no. QT00264278).Result shows 2 gene expressions of Romidepsin inducing mouse black substance metallothionein, in table 2.
The impact (n=6) of table 2 Romidepsin on 2 gene expressions of mice black substance metallothionein
| Group | Metallothionein 2 |
| Solvent control group | 1.0±0.12 |
| Romidepsin 0.5 mg/10 g b.w | 3.6±2.1 * |
| Romidepsin 2 mg/10 g b.w | 4.8±4.3 * |
| Romidepsin 2 mg/10 g b.w | 14.5±8.4 * |
Note: * and the comparison of solvent control group,
p<0.05.
embodiment 3:romidepsin reduces rotenone PD cell model dopamine neuron ROS.
Day1: will be incubated at 25cm
2the SH-SY5Y cell of culture bottle is made cell suspension, is laid in 6 orifice plates every porocyte about 1 × 10
6.
Day2: the about 30-40% of cell fusion, give Romidepsin, final concentration is 2.5,5,15 μ M.
After Day3:24 hour, add rotenone, final concentration is 0.5 μ M.
After Day4:24 hour, sucking-off culture medium, PBS cleans, and adds DCFH-DA fluorescent probe 1 ml of 1:1000 serum-free medium dilution, hatches 30min for 37 ℃, serum-free medium washed cell 3 times, after trypsinization, centrifugal, 1ml PBS is resuspended, flow cytometer detects the fluorescence of DCF, the results are shown in Table 3.
Table 3 Romidepsin produces ROS impact (n=6) to rotenone PD cell model dopamine neuron
| Group | ROS |
| Normal control | 240.24±37.86 |
| Rotenone | 2245.46±259.76 |
| Romidepsin 2.5 μM | 1452.18±154.73 * |
| Romidepsin 5 μM | 858.49±117.54 * |
| Romidepsin 15 μM | 474.29±57.37 * |
Note: * and the comparison of rotenone group,
p<0.05.
embodiment 4:romidepsin reduces MPP
+pD cell model dopamine neuron ROS.
Day1: will be incubated at 25cm
2the SH-SY5Y cell of culture bottle is made cell suspension, is laid in 6 orifice plates every porocyte about 1 × 10
6.
Day2: the about 30-40% of cell fusion, give Romidepsin, final concentration is 2.5,5,15 μ M.
After Day3:24 hour, add MPP
+, final concentration is 1 mM.
After Day4:24 hour, sucking-off culture medium, PBS cleans, and adds DCFH-DA fluorescent probe 1 ml of 1:1000 serum-free medium dilution, hatches 30 min for 37 ℃, serum-free medium washed cell 3 times, after trypsinization, centrifugal, 1 ml PBS is resuspended, flow cytometer detects the fluorescence of DCF, the results are shown in Table 4.
Table 4 Romidepsin is to MPP
+pD cell model dopamine neuron produces ROS impact (n=6)
| Group | ROS |
| Normal control | 264.58±41.34 |
| MPP + | 1956.76±241.18 |
| Romidepsin 2.5 μM | 1264.58±128.39 * |
| Romidepsin 5 μM | 564.86±74.53 * |
| Romidepsin 15 μM | 448.49±72.56 * |
Note: * and MPP
+group compares,
p<0.05.
embodiment 5:the impact of Romidepsin on rotenone PD cell model dopamine neuron apoptosis.
Apoptosis--Hoechst 33342 staining examines
Day 1: dopamine neuron cell strain SH-SY5Y spreads 24 orifice plates.
Day2: the about 30-40% of cell fusion, give Romidepsin, final concentration is 2.5,5,15 μ M.
After Day3:24 hour, add rotenone, final concentration is 0.5 μ M.
After Day4:24 hour, Hoechst 33342 dyes, and fluorescence microscope detects apoptosis.
Result demonstration, rotenone PD cell model apoptosis is obvious, reduces and give in advance Romidepsin the apoptosis (see figure 1) that rotenone is induced.
Apoptosis--PI flow cytometer detection
Day1: will be incubated at 25cm
2the SH-SY5Y cell of culture bottle is made cell suspension, is laid in 6 orifice plates every porocyte about 1 × 10
6.
Day2: the about 30-40% of cell fusion, give Romidepsin, final concentration is 2.5,5,15 μ M.
After Day3:24 hour, add rotenone, final concentration is 0.5 μ M.
After Day4:24 hour, sucking-off culture medium, PBS cleans, after trypsinization, centrifugal, adds the cold ethanol of 1 ml 70% resuspended, and 4 ℃ are fixedly spent the night.
Day5: by the fixing cell centrifugation of 70% cold ethanol, discard ethanol, resuspended 5 min of 1 ml PBS, centrifugal, 1 ml PI dye liquor is resuspended, and 4 ℃ of lucifuges are hatched 30min, 400 object nylon net filters, drain cell instrument detects.The results are shown in Table 5.
The apoptosis of table 5 Romidepsin on rotenone PD cell model dopamine neuron (PI dye stream measuring) impact (n=6)
| Group | Apoptosis rate (%) |
| Normal control | 0.27 ±0.22 |
| Rotenone | 8.45 ±2.19 |
| Romidepsin 2.5 μM | 5.42±2.24 |
| |
2.57±1.74* |
| Romidepsin 15 μM | 2.07±1.52* |
Note: * and the comparison of rotenone group,
p<0.05.
embodiment 6:romidepsin reduces MPP
+the impact of PD cell model dopamine neuron apoptosis.
Apoptosis--Hoechst 33342 staining examines
Day 1: dopamine neuron cell strain SH-SY5Y spreads 24 orifice plates.
Day2: the about 30-40% of cell fusion, give Romidepsin, final concentration is 2.5,5,15 μ M.
After Day3:24 hour, add MPP
+, final concentration is 1 mM.
After Day4:24 hour, Hoechst 33342 dyes, and fluorescence microscope detects apoptosis.
Result shows, MPP
+pD cell model apoptosis is obvious, reduces MPP and give in advance Romidepsin
+the apoptosis (see figure 2) of induction.
Apoptosis--PI flow cytometer detection
Day1: will be incubated at 25cm
2the SH-SY5Y cell of culture bottle is made cell suspension, is laid in 6 orifice plates every porocyte about 1 × 10
6.
Day2: the about 30-40% of cell fusion, give Romidepsin, final concentration is 2.5,5,15 μ M.
After Day3:24 hour, add MPP
+, final concentration is 1 mM.
After Day4:24 hour, sucking-off culture medium, PBS cleans, after trypsinization, centrifugal, adds the cold ethanol of 1 ml 70% resuspended, and 4 ℃ are fixedly spent the night.
Day5: by the fixing cell centrifugation of 70% cold ethanol, discard ethanol, resuspended 5 min of 1 ml PBS, centrifugal, 1 ml PI dye liquor is resuspended, and 4 ℃ of lucifuges are hatched 30 min, 400 object nylon net filters, drain cell instrument detects.The results are shown in Table 6.
Table 6 Romidepsin is to MPP
+the apoptosis of PD cell model dopamine neuron (PI dye stream measuring) impact (n=6)
| Group | Apoptosis rate (%) |
| Normal control | 0.31 ±0.24 |
| MPP + | 10.15 ±3.21 |
| Romidepsin 2.5 μM | 6.71±3.45 |
| |
4.11±2.87* |
| Romidepsin 15 μM | 2.27±2.24* |
Note: * and MPP
+group compares,
p<0.05.
embodiment 7:romidepsin rescue rotenone PD animal model.
Animal grouping: DMSO right substantia nigra locating injection group, rotenone right substantia nigra locating injection PD model group, Romidepsin intervention group.
The three-dimensional locating injection of right substantia nigra brain, coordinate: 3.0 mm after (1) anterior anus, (2) lateral center line 1.3 mm, (3) skull surface veutro 4.7 mm.Give every of rotenone or DMSO 0.5 μ g/ μ l/.Romidepsin intervention group application rotenone, with PD model group, gives Romidepsin 1 mg/10 g, lumbar injection every day simultaneously.After 8 weeks, start the circling behavior (the results are shown in Table 7) being brought out by apomorphine (apomorphine, APO) at the spacious region of 50 × 50 cm build-in test.
The impact of table 7 Romidepsin on rotenone PD animal model apomorphine induction rotation, (n=8)
| Group | Rotation (turning/5 min) |
| Normal control | 2.3±2.2 |
| Rotenone | 52.2±4.7 |
| Romidepsin | 13.2±4.4 * |
Note: * and the comparison of rotenone group,
p<0.05.
8 weeks time, tyrosine oxidase (TH) immunohistochemical staining, result shows, rotenone right substantia nigra locating injection PD model group, right substantia nigra TH immunocompetence reduces, and dopamine neuron cell obviously destroys.Romidepsin demonstrates obvious rescue effect, and TH immunocompetence approaches control sides, (see figure 3).
Claims (3)
1. the application of Romidepsin in treatment parkinson disease.
2. the application of Romidepsin claimed in claim 1 in treatment parkinson disease, it is characterized in that Romidepsin induction dopamine neuron cell strain and black substance brain tissue oxidizing stress 2 gene expressions of resistance factor metallothionein, Romidepsin reduces Parkinson disease model dopamine neuron active oxygen and produces.
3. the application of Romidepsin claimed in claim 1 in treatment parkinson disease, it is characterized in that Romidepsin reduces Parkinson disease model dopamine neuron apoptosis, increase animal model for parkinsonism black substance TH immunostaining, reduce dopamine neuron damage.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002090534A1 (en) * | 2001-05-02 | 2002-11-14 | The Regents Of The University Of California | Method for treating neurodegenerative, psychiatric and other disorders with deacetylase inhibitors |
| US20050227915A1 (en) * | 2001-05-02 | 2005-10-13 | Steffan Joan S | Methods and reagents for treating neurodegenerative diseases and motor deficit disorders |
| WO2007146730A2 (en) * | 2006-06-08 | 2007-12-21 | Gloucester Pharmaceuticals | Deacetylase inhibitor therapy |
| CN103108648A (en) * | 2010-07-12 | 2013-05-15 | 细胞基因公司 | Romidepsin solid forms and uses thereof |
-
2014
- 2014-02-12 CN CN201410048808.7A patent/CN103861084A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002090534A1 (en) * | 2001-05-02 | 2002-11-14 | The Regents Of The University Of California | Method for treating neurodegenerative, psychiatric and other disorders with deacetylase inhibitors |
| US20050227915A1 (en) * | 2001-05-02 | 2005-10-13 | Steffan Joan S | Methods and reagents for treating neurodegenerative diseases and motor deficit disorders |
| WO2007146730A2 (en) * | 2006-06-08 | 2007-12-21 | Gloucester Pharmaceuticals | Deacetylase inhibitor therapy |
| CN103108648A (en) * | 2010-07-12 | 2013-05-15 | 细胞基因公司 | Romidepsin solid forms and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| P-S CHEN,ET AL: "Valproate protects dopaminergic neurons in midbrain neuron/glia cultures by stimulating the release of neurotrophic factors from astrocytes", 《MOLECULAR PSYCHIATRY》, vol. 11, 31 December 2006 (2006-12-31), pages 1116 - 1125 * |
| SARAH K. KIDD AND JAY S. SCHNEIDER: "Protection of Dopaminergic Cells from MPP+-Mediated Toxicity by Histone Deacetylase Inhibition", 《BRAIN RES.》, vol. 1354, 1 October 2010 (2010-10-01), pages 172 - 178 * |
| 邱晶等: "丙戊酸钠对抗鱼藤酮诱导的SH-SY5Y细胞损伤的线粒体机制", 《中国细胞生物学学报》, vol. 34, no. 4, 30 April 2012 (2012-04-30), pages 349 - 354 * |
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Application publication date: 20140618 |