Patent Application
20180125923
The present invention relates to the treatment of cerebrovascular diseases. In particular, it relates to a method for treating or ameliorating cerebrovascular diseases in a subject by using peptides isolated from extracts from rabbit skin inflamed by vaccinia virus.
Cerebrovascular diseases are neural function injuries caused by abnormal blood supply of regional brain. In most countries, cerebrovascular diseases, the top three causes of all deaths, can result in a brain injury in adult. Cerebrovascular disease is a major cause for endangering the health of the middle-aged and the aged people, and a major cause of death or disability of the middle-aged and the aged people in most countries.
Stroke, one of the acute cerebrovascular diseases, is the third leading cause of death in worldwide population and induces a highest disabling rate among various diseases. The cerebrovascular diseases could severely affect the life quality of the elderly, bring an enormous burden to patients' family and the society. It also tends to increase in young population.
The cerebrovascular diseases are primarily classified into two types, hemorrhagic and ischemic, of which the latter is 60-70%, and is the most common type of cerebrovascular diseases. It is important to study the pathophysiological mechanism of ischemic cerebrovascular diseases and search for drugs which function as neuroprotection.
The research of the pathophysiologic mechanism of cerebral ischemia has been one of the most focuses of the neuroscience field since 1980s, and so far theories of cerebral ischemia concerning such as energy metabolism, acid intoxication, peroxidation injury, excitatory amino acid induced toxicity injury and calcium overload have been proposed, in which the last two play an important role in ischemic neuronal injury and death. According to pathophysiologic basis of ischemic cerebrovascular diseases, drugs currently used to clinically treat cerebral ischemia mainly comprise calcium ion antagonists (nimodipine), oxygen radical scavengers (VitE, SOD), neurotrophic factors (nerve growth factor, neurotrophic factor), excitatory amino acid antagonists, antioxidants and drugs which improve late-onset neuronal injury. These drugs function via various mechanisms of action, with uncertain therapeutical effects or less specificity or with the concomitancy of severe side-effects, and thus can not meet the clinical requirements yet. There are many commercially available drugs which can be used to improve cerebral circulation, metabolism and functions, such as piracetam, flunarizine, calan, ginkgo extracts. Although they all have certain characteristics, the treatment effects thereof on cerebrovascular diseases are uncertain. Research and development of novel drugs for treating ischemic cerebrovascular diseases becomes increasingly important in the field of pharmaceutics and pharmacology.
The extracts from rabbit skin inflamed by vaccinia virus are commercially available, with the trade name of analgecine, which is manufactured by Vanworld Pharmaceutical (Rugao) Co. Ltd. Previous experiment evidence shows that crude extract of the inflammatory rabbit skins induced by inoculation of vaccinia virus can exert its pharmacological effect on analgesia. However, the active ingredients of analgecine contributing to this effect have not been reported yet. Therefore, there is a need to gain an insight into which components are involved in this effect.
In one aspect, the present invention provides a method for treating or ameliorating cerebrovascular diseases in a subject, wherein the method comprises administering to the subject an effective amount of one or more peptide comprising an amino acid sequence having at least 70% identity to DEAQETAVSSH (SEQ ID NO: 1) or variants, mutants, derivatives, or fragments thereof.
In another aspect, the present invention provides the use of one or more peptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO: 1 or variants, mutants, derivatives, or fragments thereof in the manufacture of a medicament for treating cerebrovascular diseases in a subject.
In one embodiment, the cerebrovascular diseases as described herein are those which lead to damages of the neurological systems with functional deficit. In a particular embodiment, the cerebrovascular diseases as described herein are selected from the group consisting of: ischemic brain injury or hemorrhage brain damage. In a more particular embodiment, the cerebrovascular disease is stroke.
The expression “having at least 70% identity to SEQ ID NO: 1” is intended to mean an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% or more identity to SEQ ID NO: 1.
In a further embodiment, wherein the peptide comprises SEQ ID NO: 1 and/or DEAQETAVSSHEQD (SEQ ID NO: 2). In a more particular embodiment, the peptide consists of SEQ ID NO: 1 and/or SEQ ID NO: 2.
In various embodiments, the peptide comprises at least one amino acid addition, deletion, and/or substitution, preferably 1-5, preferably 1-3 amino acid additions, deletions, and/or substitutions. In another embodiment, the amino acid addition, deletion and/or substitution are located at the C-terminal and/or N-terminal.
In yet another embodiment, the disease is treated or ameliorated by protecting nerve cells.
In a particular embodiment, the subject is human.
In another aspect, the present invention relates to a polynucleotide which encodes the peptide according to the invention; a vector which comprises such polynucleotide; as well as a host cell which comprises such polynucleotide or such vector.
In another aspect, a pharmaceutical composition is provided comprising an effective amount of the peptide according to the invention, the polynucleotide, the vector, or the host cell as described herein, and a pharmaceutically acceptable carrier.
The present invention also relates to the peptide, the polynucleotide, the vector, the host cell, or the pharmaceutical composition as described herein, for use as a medicament. In particular, the present invention relates to a peptide composition comprising or consisting of SEQ ID NO: 1 and SEQ ID NO: 2; a drug combination comprising such a composition; as well as a method of using the same.
The present inventors surprisingly found that 2 peptides isolated from extracts from rabbit skin inflamed by vaccinia virus significantly preserve the neural cell viability under hypoxic, hypoglycemia conditions or a combination of both, upon exposure to H2O2 or L-glutamic acid in vitro and/or in vivo. More surprisingly, the peptide 1 and peptide 5 exhibit synergistic effects to inhibit cytotoxicity of neuronal cell (PC12) induced by hypoxic, hypoglycemia conditions or a combination of both, H2O2 and L-glutamic acid, with a similar potency as seen in the use of AGC (eg, 1 U/ml). Our data therefore successfully identify two short peptides in the AGC as active ingredients that could account, at least in part, for the neuro-protective role of AGC in the ischemic stroke.
H2O2 is an important reactive oxygen component which is involved in the onset of nervous system diseases such as cerebral ischemia, trauma, brain aging, Alzheimer's disease etc. It will peroxidize the membrane lipid, decrease cell membrane fluidity, change components and activities of intracellular proteins, make chromatin concentrated and DNA broken, and finally result in cell death.
Excitatory amino acids, such as glutamic acids, play an important role in the course of a variety of chronic or acute neuropathy which will be accompanied by neuronal death. Glutamic acid can damage nerve cell line and primary nerve cell in a dose dependent manner. It is responsible for the increased intracellular calcium ion and the blocked cystine uptake, and it induces the loss of intracellular reduced glutathione (GSH), the increased oxygen radical and nerve cell death. Therefore, H2O2 or glutamic acid-induced nerve cell injury model can be used as a screening model of neuroprotective agents.
Therefore, in one embodiment, the peptide according to the invention is used to preserve the neural cell viability upon exposure to H2O2 in vitro and/or in vivo, with an unexpected synergistic effect seen in the combinatorial use of peptide 1 and 5. In another embodiment, the peptide according to the invention demonstrates protective effect on the L-glutamic-acid-treated PC12 cells, with an unexpected synergistic effect seen in the combinatorial use of peptide 1 and 5
Neuronal activity requires a continuous supply of oxygen and glucose. The impairment of oxidative metabolism causes a cascade of ionic and metabolic events leading to neuronal death. Therefore, in one embodiment, the peptide according to the invention is used to preserve the neural cell viability under hypoglycemic and hypoxic conditions in vitro and/or in vivo, with an unexpected synergistic effect seen in the combinatorial use of peptide 1 and 5.
In one embodiment, the present invention relates to a method for preserving the neural cell viability upon exposure to H2O2 in vitro or in vivo, comprising treating the neural cell with peptide 1 and/or 5 or administering peptide 1 and/or 5 to the subject in need thereof.
In another embodiment, the present invention relates to a method for preserving the neural cell viability under hypoglycemic and/or hypoxic conditions in vitro or in vivo, comprising treating the neural cell with peptide 1 and/or 5 or administering peptide 1 and/or 5 to the subject in need thereof.
In yet another embodiment, the present invention relates to a method for protecting the neural cell from glutamic acid-induced injury in vitro or in vivo, comprising treating the neural cell with peptide 1 and/or 5 or administering peptide 1 and/or 5 to the subject in need thereof.
Unless defined otherwise, all scientific terms used herein have the same meaning as that commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although the equivalents thereof can be used. All publications and other references mentioned herein are incorporated by reference in their entirety.
The present invention will now be described further with reference to the following examples which, however, are not intended to limit the scope of the present invention.
1) Sample Preparation
About 200 μL of the mixture of biological agents generated from the rabbit skin suffering from inflammation induced by inoculation of vaccinia virus was dried in a vacuum centrifuge.
The lyophilized material was reconstituted with 100 μL of 0.5 M ammonia bicarbonate buffer (pH 8.5) containing 8 M urea and 0.5 M dithiothreitol (DTT) for 1 hr at 37° C., and for another 2 hr at 4° C. under dark condition when 10 μL of 0.5 M iodoacetamide (IAM) was added for alkylation prior to dilution with 400 μL ammonium bicarbonate. Subsequently, the resulting solutions were then digested with 0.2 μg of trypsin for 18 h at 37° C., and then the trypsin-digested solutions were acidified by 10% trifluoroacetic acid (TFA)/H2O to pH 3.0 value. After the reaction, the totally acidified solutions were applied onto the reverse phase C18 column pre-equilibrated with 200 L of 0.1% TFA/H2O (pH 3.0). The column was also washed with 200 μL of 0.1% TFA/H2O (pH 3.0) and then eluted with a stepwise acetonitrile gradient from 50% to 100% in 0.1% TFA at room temperature. As a control in nano-LC-MS/MS, the reconstituted liquid from lyophilized products was also treated by reduction, alkylation, desalting and acidified as mentioned above, except for trypsin digestion.
2) Nano-LC-MS/MS Analysis
The eluted fractions were collected, dried in a vacuum centrifuge, and then reconstituted in 25 μL of 80% ACN in H2O and analyzed by LTQ Orbitrap XL (Thermo Fisher Scientific, San Jose, Calif.). Reverse phase nano-LC separation was performed on an Agilent 1200 series nanoflow system (Agilent Technologies, Santa Clara, Calif.). A total of 10 μL sample from fraction was loaded onto an Agilent Zorbax XDB C18 precolumn (0.35 mm, 5 μm), followed by separation using a C18 column (i.d. 75 μm×25-cm, 3 μm, Micro Tech, Fontana, Calif.). The mobile phases used were (A) 0.1% FA and (B) 0.1% FA in 100% ACN. A linear gradient from 5% to 35% (B) over a 90-min period at a flow rate of 300 nL/min was applied. The peptides were analyzed in the positive ion mode by applying a voltage of 1.8 Kv to the injection needle. The MS was operated in a data-dependent mode, in which one full scan with m/z 300-2000 in the Orbitrap (R=60 000 at m/z 400) using a rate of 30 ms/scan. The six most intense peaks for fragmentation with a normalized collision energy value of 35% in the LTQ were selected. A repeat duration of 30 s was applied to exclude the same m/z ions from the reselection for fragmentation.
3) Database Search and Identification
Peptides were identified by peak lists converted from the nanoLC-MS/MS spectra by searching against animal taxonomy in the Swiss-Prot databases for exact matches using the MASCOT search program (http://www.matrixscience.com; Hirosawa et al., 1993). The mass tolerance of both precursor ion and fragment ions was set to 10 ppm and 0.8 Da, respectively. Searches were performed to allow for the carbamidomethylation (C) as fixed modification, and no trypsin as variable modification.
Peptides were considered identified if their Mascot individual ion score was higher than 20 (p<0.05).
1.1.2. Results
Previous experiment evidence shows that crude extract of the inflammatory rabbit skins induced by inoculation of vaccinia virus can exert its pharmacological effect on analgesia. However, the protein concentrations in these extracts were extremely low and below the detection limit, which demonstrated that the protein-level agent hardly played a crucial role in analgesia. To gain an insight into which kind of components were involved in this effect, we employed a proteomic approach to determine the differences in the mass-to-charge ratios (m/z) by using nano LC-MS/MS. The mass spectral patterns of protein fragments generated with or without trypsin digestion were then used for comparison with those of previously known proteins deposited in databanks to confirm peptides, which can be used for identification of intact proteins (protein ID). Therefore, we can achieve an extensive coverage of peptides by shotgun analysis, elucidate the expression profiles of polypeptides or small peptides and identify sequences as well as the biochemical characterization. A flow chart of the methods used in this work is displayed in
1) Peptide Synthesis
Peptides were synthesized by using first-amino-acid-preloaded Wang resin, N-Fmoc amino acids, and PyBOP through solid phase synthesizer (PS3, Protein Technologies, Inc.). The crudes peptides were purified with RP-18 column by medium pressure liquid chromatography (MPLC) and their purities were confirmed as >95% by high performance liquid chromatography (HPLC) under UV detection at 220 nm wavelength. The characterizations of these peptides were proceeded by MALDI-TOF mass spectrometer (Autoflex III system, Bruker Daltonics) and Nuclear Magnetic Resonance spectroscopy (Varian 400 MHz) prior to further studies. Peptide-5 was also received from Mission Biotech Co. (M. B. Taipei, Taiwan) and presented same analgesic activity with our synthesized one.
2) Stability Test
Peptide-5 was dissolved in series of solution with different pH value and stood for 3 days. Adequate amount of each resulting solution was analyzed by HPLC (Agilent 1100 series) using a Nucleodur Pyramid C18 column (250 mm×4.6 mm, 5 μm) and gradient elution (0 min: 0.1% TFA in H2O; 10 min: 0.1% TFA in MeOH; 15 min: 0.1% TFA in MeOH) with 0.2 mL/min flow. The signals were monitored by UV detector at 220 nm wavelength. The sample collected in the stability test was also analyzed by MALDI-TOF Mass (Bruker Daltonics, Autoflex III) with 2,5-dihydroxybenzoic acid (DHB) as matrix. Furthermore, the portion respond to the signal at 7.8 min were collected and subject to MALDI-MS analysis. Peptide-5 was also prepared 50 μM in PBS and recorded from circular dichroism (Jasco, J-810).
1.2.2. Results
As shown in
As shown in
As shown in
The stability of peptide 1 and its CD pattern was similar to those of peptide 5 (data not shown).
1.2.3. Summary
From the experiments above, we confirmed that the peptides isolated from AGC indeed were very stable, at least from the change of pH values in the solvent and the CD patterns. Hence, these peptides could be present in vivo with no significant changes in their structures and sequences.
2.1 Protective Effect on Hydrogen Peroxide Treated PC12 Cells
2.1.1 Materials and Methods
1) Isolated Peptides or AGC in PC12 Cell Survival Upon Exposure to H2O2
PC12 cells, derived from a pheochromocytoma of the rat adrenal medulla that exhibit certain characteristics of neuro-ganglion cells, were obtained from the Chinese Academy of Medical Sciences, Beijing, China. To examine cell survival upon exposure to H2O2, PC12 cells were grown to confluence in 96 well plate and the medium was changed to serum-free medium containing 200 μM H2O2 (final volume 100 μl) for 24 h. In cells receiving treatment, the isolated peptide or AGC was given into wells for 1 h before exposure to H2O2. After 24 h incubation, 100 μL MTT (final concentration 0.5 mg/ml) was added for additional 4 h incubation, and the cell survival was subsequently assessed by absorbance at 540 nm under a Fluostar microplate reader (BMG, Germany).
2) Statistical Analysis
Data were expressed as mean±standard error (SE) and multiple comparisons were evaluated by the analysis of variance and Neuman-Keuls test. Differences were considered significant at P<0.05. Neurological deficit data were evaluated by the nonparametric Mann-Whitney test.
2.1.2 Results
In the experiment, we showed that AGC at 0.25 u/ml, 0.5 u/ml and 1 u/ml have protective effect on the H2O2-treated neural cells (
2.1.3. Summary
From the screening of the effects of protecting cells from the H2O2-induced cytotoxicity, we provide evidence that (1) AGC indeed possess protective effects against the cytotoxicity derived from exposure of PC12 cells to H2O2, at concentrations of 0.25-1 U/ml; (2) the peptide 1 and 5 could effectively prevent, at least in part, the H2O2-induced cytotoxicity of PC12 cells neuronal cells. And, (3) the combinatorial use of peptide 1 and 5 has synergistic effects in protecting PC12 cells from H2O2-induced cytotoxicity, and the protective effect was as potent as the use of 1 U/ml of AGC.
2.2 Protective Effect on L-Glutamic Acid Treated PC12 Cells
2.2.1 Materials and Methods
1) Effects of Identified Peptides or AGC in PC12 Cell Survival Upon Exposure to L-Glutamic Acid
PC12 cells were obtained from the Chinese Academy of Medical Sciences, Beijing, China. To examine cell survival upon exposure to L-glutamic acid, PC12 cells were grown to confluence in 96 well plate in serum-free RPMI-1640. In cells receiving treatment, the identified peptide or AGC at a given dose was put into wells for 1 h before exposure to exposure to L-glutamic acid in the medium of Mg2+-free Eagle's medium containing 1 mM L-glutamic acid. After 15-min incubation, medium was changed to serum-free RPMI-1640. After 24 h incubation, 100 μL MTT (final concentration 0.5 mg/ml) was added for additional 4 h incubation, and the cell survival was subsequently assessed by absorbance at 540 nm under a Fluostar microplate reader (BMG Germany).
2) Statistical Analysis
Data were expressed as mean±standard error (SE) and multiple comparisons were evaluated by the analysis of variance and Neuman-Keuls test. Differences were considered significant at P<0.05. Neurological deficit data were evaluated by the nonparametric Mann-Whitney test.
2.2.2. Results
In this experiment, we showed that different concentrations of AGC (0.25 u/ml, 0.5 u/ml, and 1 u/ml) had positive effect on the cell survival rate of PC12 cells (
2.2.3 Summary
Similar to the observation made from exposure of LC12 cells to L-glutamic acid, from the screening of the effects of protecting cells from the L-glutamic acid-induced cytotoxicity, we provide evidence that (1) AGC indeed possess protective effects against the cytotoxicity derived from exposure of PC12 cells to L-glutamic acid, at concentrations of 0.25-1 U/ml; (2) the peptide 1 and 5 could effectively prevent, at least in part, the L-glutamic acid-induced cytotoxicity of PC12 cells neuronal cells. And, (3) the combinatorial use of peptide 1 and 5 has synergistic effects in protecting PC12 cells from L-glutamic acid-induced cytotoxicity, and the protective effect was as potent as the use of 1 U/ml of AGC.
2.3 Protective Effect on Cell Survival of PC12 Cells Under Hypoglycemic, Hypoxic or Hypoglycemic-Hypoxic Conditions
2.3.1 Materials and Methods
1) Effects of AGC and Peptide 1 or 5 in PC12 Cell Survival Cultured in Hypoglycemic, Hypoxic or Hypoglycemic-Hypoxic Conditions
PC12 cells were propagated and cultured as described above. For hypoglycemic condition, 125 μM Cobaltous Chloride (CoCl2, Sigma, USA) was added into the culture media when PC12 cells were in monolayer for further 24 h incubation as described. For induction of hypoglycemia, culture media was changed to glucose-free Eagle's medium when PC12 grew to monolayer for additional 24 h incubation. For hypoglycemic and hypoxic conditions, PC12 cells were cultured in glucose-free Eagle's media with addition of 125 μM CoCl2. After 24 h, cells were harvested and subject to MTT assay.
2) Statistical Analysis
Data were expressed as mean±standard error (SE) and multiple comparisons were evaluated by the analysis of variance and Neuman-Keuls test. Differences were considered significant at P<0.05. Neurological deficit data were evaluated by the nonparametric Mann-Whitney test.
2.3.2 Results
The experiment showed that AGC (0.5 u/ml, 1 u/ml) had protective effect on PC12 from cell death under hypoglycemic and hypoxic conditions (
2.3.3 Summary
In this experiment, we provide data showing that (1) AGC indeed possess protective effects against the cytotoxicity derived from exposure of PC12 cells to hypoxia, hypoglycemia, or (hypoxia+hypoglycemia), at concentrations of 0.25-1 U/ml; (2) when used individually, peptide 1 or peptide 5 had some protective effect against cell death induced by hypoxia, but no protective effects against the cytotoxicity induced by hypoxia or by (hypoglycemia+hypoglycemia). (3) Nevertheless, when used in combination, peptide 1+5 was protective against the death of PC12 cells exposed to hypoxia, hypoglycemia, or the combination of hypoxia plus hypoglycemia, and the protective effect was with a similar extent when compared to the use of 1 U/ml of AGC.
2.4 In Vivo Distribution of Peptide 1 and 5 after i.v. Injection
2.4.1 Materials and Methods
The C57B/L6 mice (6˜8 week-old, female) were purchased from National Laboratory Animal Center, Taiwan. For optical imaging, animals were anesthetized by 2% isoflurane gas mixed with oxygen and intravenous injection (i.v.) of Cy5.5-conjugated peptide 1 or Cy5.5-conjugated peptide 5 (35 nmole/per mouse). After administration the mice were placed prone or supine in a dark box and a photographic image was first acquired at low light (exposure time: 5 seconds). At indicated time points after administration of probes, fluorescent signals were obtained using a CCD camera thermoelectrically cooled to −70° C. (Xenogen—IVIS® Spectrum Imaging System, Xenogen Technology). The photon integration period was 1-2 sec. Fluorescent signals were recorded as maximum photos/second/centimeter2/steradian (photon/sec/cm2/sr) and displayed in pseudocolors and superimposed on the photographic image using Xenogen's Living Image software.
2.4.2 Results
Using the in vivo imaging system (IVIS; Perkin Elmer, USA), we can observed that distribution of the 2 peptides injected to the mice through i.v. (
2.4.3 Summary
We thus confirm that both peptide 1 and 5 could migrate from the peripheral circulation through the blood-brain barrier to the brain and spinal cord, as quickly at 15 to 30 minutes after intraperitoneal administration.
This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2015/076747 | 4/16/2015 | WO | 00 |
