Patent Application
20180369273
The invention relates to pharmaceutical field, specifically to a pharmaceutical composition and use thereof.
Colorectal cancer is one of the most common malignant tumors, with incidence listed in the second in developed countries including European and USA, and the third worldwide. Along with continuous improvement of living standard in China and the westernization of diet, the incidence of colorectal cancer, increasing year by year in recent years, has risen to 3-5th among various cancers, especially in big cities. At present, the treatment of colorectal cancer applied drugs with only general effect on tumor but lacked direct targeting colorectal cancer. This situation needs to be improved as soon as possible.
miR-150 Inhibits Colorectal Cancer Cell Proliferation
The key molecule miR-150 was validated through screening from colorectal cancer progresses at different stages (normal, adenomas, adenocarcinomas) by using miRNA microarrays. It was found in large-scale clinical samples analysis that colorectal cancer patients with low miR-150 expression always had shorter survival and with poorer post-operative chemotherapy sensitivity; however, patients with high miR-150 expression survived longer and with post-operative chemotherapy sensitivity. According to the sequences of miRNAs, four oligos: miR-150 mimic, inhibitor and the controls (mimics control, inhibitor control) were acquired (Table 1), which can be obtained commercially. We then developed a new formulation by applying histidine-lysine Polymer (HKP) as carriers to form nanoparticle formulation. This formulation could be applied as a new generation of targeted therapeutic drug, which inhibited the growth of colon cancer by inducing programmed death of cancer cell in a tumor xerographic mouse models. In conclusion, the chemical synthesis and modification of microRNA drug provides a brand-new therapeutic method for colon cancer, which is different from traditional small molecule or monoclonal antibody drugs. This drug modality shows clear mechanism of action, accurate targets, as well as unique advantages in safety, since it is derived from human body.
Regulation of the Target Gene Expression by Synthesized miRNAs
MicroRNAs (miRNAs) are endogenous non-coding RNA in eukaryotes which can control protein expression by regulating the degradation of the target messenger RNA. The miRNA mimics or antagomirs could be obtained by chemical synthesis; Synthesized mimics can simulate endogenous maturity miRNAs with high expression, whereas synthesized antagomirs can specifically binding to target miRNAs and weaken the gene silencing effects of endogenous miRNAs, resulted with regulation of the expression of target proteins.
A Branched Histidine-Lysine Peptide (HKP) for siRNA Delivery In Vivo
Branchedhistidine-lysine peptide (HKP, Histidine-Lysine Co-polymer), a branched polymer with positive charge (
In accordance with the present invention, a pharmaceutical composition is provided, which is a targeted drug for the treatment of colorectal cancer.
A pharmaceutical composition is described, which comprises microRNAs with or without chemical modification, and a carrier that is suitable for delivery in vivo. The carrier is a branched histidine-lysine polypeptide (HKP) or modified compound thereof.
Specifically, the microRNAs include a miR-150, sequence of the miR-150 is 5′-UCUCCCAACCCUUGUACCAGUG-3′. The expression of miR-150 is closely related with prognosis of colorectal cancer by large-scale clinical samples analysis and cellular studies.
Specifically, the miR-150 is a miR-150 analogue constructed according to known sequence of micro RNA in vitro.
Specifically, the microRNA is single strand, which is easily degraded, so the chemical modification is used to improve its stability.
Specifically, the chemical modification is formed on a pentose of a single nucleotide or multi-nucleotides of the microRNAs.
Specifically, a chemical group of the chemical modification is fluoro or methoxy.
More Specifically, the chemical modification is formed on 2′-OH in all bases of a single-stranded of the microRNAs.
Specifically, the microRNAs include a miR-143, sequence of the miR-143 is 5′-UGAGAUGAAGCACUGUAGCUC-3′.
More Specifically, the miR-143 is a miR-143 analogue constructed according to known sequence of micro RNA in vitro.
Specifically, the microRNAs include a miR-195, sequence of the miR-195 is 5′-UAGCAGCACAGAAAUAUUGGC-3′.
More Specifically, the miR-195 is a miR-195 analogue constructed according to known sequence of micro RNA in vitro.
Specifically, the microRNAs include a miR-150 and a miR-143, and the miR-150 and the miR-143 are mixed to form a double-target microRNAs inhibitor to enhance the anti-tumor effect.
Specifically, the microRNAs include a miR-150 and a miR-195, and the miR-150 and the miR-195 are mixed to form a double-target microRNAs inhibitor to enhance the anti-tumor effect.
Specifically, the microRNAs include a miR-143 and a miR-195, and the miR-143 and the miR-195 are mixed to form a double-target microRNAs inhibitor to enhance the anti-tumor effect.
Specifically, the microRNAs include a miR-150, a miR-143 and a miR-195, and the miR-150, the miR-143 and the miR-195 are mixed to form a triple-target microRNAs inhibitor to enhance the anti-tumor effect.
Specifically, the branched histidine-lysine polypeptide as claimed is a positively charged histidine-lysinepolymer (HKP), which is used for nucleic acid delivery in a variety of tissue types.
Specifically, the modified compound of branched histidine-lysine polypeptide is a branched histidine-lysine polymer with a histidine in every branch (HKP+H), which is used for nucleic acid delivery in a variety of tissues with low immune and inflammatory reaction.
Specifically, the branched histidine-lysine polypeptide is H3K4b, which is constituted by three lysine cores and four branches that contain a large number of repetitive histidine and lysine, whose structure is shown in
Specifically, the modified compound of the branched histidine-lysine polypeptide is H3K(+H)4b, with a histidine in each branch of H3K4b. The structure of H3K(+H)4b is the structure shown in
Specifically, the nitrogen and phosphorus mass ratio (N/P) of the carriers and the microRNAs is between 8:1 and 1:8.
Preferably, the nitrogen and phosphorus mass ratio (N/P) of the carriers and the microRNAs is no less than 4:1.
Specifically, the carrier is a three-component system of RGD-PEG-HKP. RGD and HKP are coupled on both ends of PEG, and the RGD is a polypeptide composed of 7-12 amino acids with specific recognition and adhesion effects on new blood vessels endothelial cells, which can be used as a targeting molecule of micro nucleic acid delivery system.
A HKP-end of the three-component system of RGD-PEG-HKP is capable of combining a micro nucleic acid to form a nanoparticle (approximately 150 nm), and a RGD-end on the surface of nanoparticle is capable of targeting tumor cells.
Specifically, a molar concentration of the microRNAs is no less than 1 nM.
HKP of the present invention is synthesized from an outsourced company, which made the peptide according to the techniques and processes of the inventors.
Use of the pharmaceutical composition for the manufacture of a targeted drug for colorectal cancer treatment is described herein.
Use of the pharmaceutical composition synergized with small molecule chemical drug or monoclonal antibody for the preparation of a targeted drug for colorectal cancer treatment is described herein.
The syntheses of the four single stranded miRNAs in the present invention are synthesized by monomer nucleotides via chemical methods, and chemical modification were introduced to increase their stability. The chemical modification is formed on 2′OH in all the bases of single-stranded miRNAs and the chemical group is methoxy(2′-Ome) or fluoro (2′-F) (
Due to the implementation of the technical scheme above, the invention has the following advantages compared with the current technology:
nanoparticles with different sizes are formed after mixed microRNAs with carrier at different N/P ratios, which are suitable for microRNAs delivery and can inhibit the growth of colon cancer by inducing programmed death of cancer cell, so as to depress the growth of colorectal cancer.
The present invention will be further described in detail with reference to specific embodiments, but the present invention is not limited to the following embodiments.
Embodiment 1. Agarose gel electrophoresis analysis of the optimal nitrogen-phosphorus ratio (N/P) between HKP and miR-150.
Agarose gel electrophoresis is an electrophoresis method with the supporter of agarose, which plays a dual role in “molecular sieves” and “electrophoresis”. The separation of charged particles not only depends on the character and amount of net charge, but also on the molecular size. Generally, electrophoresis with the supporter of 1% agarose always be used to isolated nucleic acid including in this study, to observe the HKP entrapment capacity to miRNA by 1% agarose gel electrophoresis.
HKP is obtained from two different resources, which was synthesized according to the technology and process of the invention, and mixed with M, respectively in different mass ratio of 1:1, 2:1, 4:1, 6:1, 8:1, to conduct the pre-screening test of nitrogen and phosphorus suitable mass ratio.
After agarose gel electrophoresis (
Embodiment 2. Particle size and potential measurements to validate the proper nitrogen to phosphorus mass ratio (N/P) of HKPs and miR-150s.
Dynamic light scattering (DLS) particle size measurement has become a conventional characterization method of nanotechnology because of its accuracy, rapidity and repeatability. Particle size distribution of nanoparticles in aqueous suspension can be measured directly by laser granulometer, which using dynamic light scattering principle.
Zeta potential is also called the electric potential (ζ-potential), it refers to the potential of the shear plane, and is an important indicator of nanoparticle solution dispersion stability. It is the potential difference between continuous phase and the fluid layer with stable stratification that attached to dispersed particles, and can be measured directly by electro dynamics. Electric potential of nanoparticles in aqueous suspension can be measured directly by Zeta Potential Meter, which using electrophoretic light scattering principle.
Mixed HKP (API) with two oligos (mimics, inhibitor,respectively) in the five mass ratios: 2:1, 2.5:1, 3:1, 3.5:1, 4:1, to measure the particle size and potential of the formed nanoparticles. The data of all ratios (Table 2 and Table 3) showed stable potentials and particle sizes.
Embodiment 3. Analysis of suitable nitrogen and phosphorus mass ratio (N/P) between HKP and miR-150 by entrapment efficiency measurement
RiboGreen RNA quantitation reagent is an ultra-sensitive nucleic acid fluorescent dyes, and RNA quantification will become simple and quick by using this reagent.
RiboGreen fluorescent dye almost has no fluorescence activity in solution; But when it combines with RNA, its fluorescence activity will increase 1000-folds. The fluorescence excitation and emission wavelength of RiboGreen-RNA complex is 500 nm and 525 nm respectively.
When the RibGreen fluorescent dye is added to the solution, the free miRNA will bind with it and form a RiboGreen-RNAs complex with fluorescent activity. However, HKP-encapsulated miRNAs do not bind to RibGreen fluorescent dyes and do not produce fluorescent activity. Therefore, a standard curve is drawn by using the miRNA fluorescence intensity and the corresponding concentration, and then the corresponding concentration is calculated on the standard curve (
Entrapment Efficiency=(1-CRNA/Cinitial concentration)×100%
HKP (API) and two oligos (mimics, inhibitor) were respectively mixed in the five mass ratios, namely 2:1, 2.5:1, 3:1, 3.5:1, 4:1, to measure the entrapment efficiency.
With the increment of HKP (
Embodiment 4. In vitro cell biology experiments to analyze the relative activity of miR-150 at the ratio (N/P) of 4:1 (miRNA to HKP)
The complexes of HKP (API) and miRNA (unmodified) (N/P ratio=4:1) were transfected into LOVO cell lines and using real-time fluorescence quantification PCR in vitro to detect miR-150 relative expression.
Real-time fluorescence quantitative PCR method: In the real-time fluorescence quantitative PCR reaction system, a fluorescent chemical substance is introduced. As the PCR reaction proceeds, the PCR reaction products are continuously accumulated, and the fluorescence signal intensity also increases in equal proportion. After each cycle, a fluorescence intensity signal is collected, so that changes in the amount of the product can be monitored by changes in the fluorescence intensity, thereby enabling quantitative and qualitative analysis of the starting template.
LOVO cell line was used in the invention. After the cells are transfected for 20-24 hours, the total RNA is extracted from each group of cells, the corresponding cDNA is obtained by reverse transcription PCR, and finally passed through the real-time quantitative PCR to detect the expression of miR-150 in each group of cells. U6 gene was used as a reference gene. Each sample was run in triplicate.
Four oligo miRNAs were detected at a concentration of 100 pM. A group of 100 nM was set up as control. The experimental results showed that the mimics of the three groups had certain effects, especially in 100 nM group (
Embodiment 5. Analysis of miR-mimics (with chemical modification and without) relative expression in vitro entrapped by lipo
Nanocomplexes with unmodified mimics and mimics control, as well as chemically modified (2′- and 2′-fluoro modified) mimics and mimics control were encapsulated with lipo and transfected into LOVO cell lines. The miR-150 relative expression of each group was detected and compared by real-time fluorescent quantitative PCR in vitro, in order to confirm whether the chemical modification was beneficial or not.
The most effective one is 2′methoxyl (2′-Ome) modified single stranded miRNA, which could cause a substantial increase of miR-150 intracellular relative expression (
Embodiment 6. Pharmacodynamic study of miR-150 targeted polypeptide formulation
LoVo cells were inoculated subcutaneously in nude mice to form tumors. HKP-encapsulated miR-150 and other control groups of micro nucleic acid drug preparations were injected into the tumor tissue for 9 consecutive treatments. Subcutaneous xenografts were injected once every two days (injected into the tumor tissue at a level of 0.5 cm along the dorsal surface of the skin, and the needles continued to enter the center of the tumor, intratumoral injection). Each injection was 1 OD/70 uL formulation/once with a total of 9 times administration. The entire process took 18 days from the start of dosing to the final collection. Tests have shown that miR-150 has a significant inhibitory effect on tumors (
Embodiment 7. Changes of tumor volume in the process of targeted therapy
At the beginning of the treatment, the tumor volumes are all approximately 145 mm3. During the course of treatment, except for the miR-150 treatment group, whose tumor growth was significantly slowed, the other groups showed similar growth trends as the untreated control group. From the seventh treatment, tumor growth was significantly inhibited (
Embodiment 8. Expression of target molecule miR-150 in tumor tissues
After treatment, animals were treated with euthanasia and tumor tissues were harvested to obtain total RNA. Quantitative RT-PCR was used to detect the expression levels of targeted micro nucleic acids in tumor tissues. It was shown that the expression of micro nucleic acid in miR-150/HKP treatment group was significantly higher than that in other groups (
Embodiment 9A. Programmed tumor cell death (PCNA) was induced by miR-150/HKP formulation
The treated tumor tissues were detected and proliferating cell nuclear antigen (PCNA) was evaluated. PCNA in tumor tissues treated with miR-150/HKP drugs was significantly different from other control groups and untreated groups. PCNA was substantially reduced in the tumor cell nucleus after miR-150/HKP formulation treatment, indicated tumor cell death (
Embodiment 9B. Programmed tumor cell death (TUNEL) was induced by miR-150/HKP formulation
After the treatment, it was detected that miR-150/HKP treatment group showed significant difference from other control groups by classic TUNEL method. TUNEL nuclei got a substantial increase in the tumor cell after miR-150/HKP formulation treatment, indicated the treatment induced programmed tumor cell death (
Embodiment 10. RGD-HKP can target to deliver miRNA to tumor tissue
The HKP/siRNA nanoparticles modified by application of cRGD can have affinity for α vβ3 and αvAβ5 integrins on the surface of neovascular endothelial cells. Due to the large number of neovascularizations in the tissues near the tumor, and abundant expression of αvβ3 and αvβ5 integrins, the HKP (cRGD modified)/siRNA nanoparticles we designed could enhance the miRNA enrichment near the tumor tissue and miRNA entered into tumor cells effectively to induce cell death.
Embodiment 11. miR-143 for the treatment of colorectal cancer
miR-143 is significantly down-regulated in colorectal cancer tissues, and its expression level can be regulated through different pathways. Studies have shown that PI3K/Akt, MAPK and HGF/MET signal paths were affected by the down-regulation of miR-143. Meanwhile, miR-143 can decrease the expression of Ras, Erk5, Akt and MCC1. If miR-143 is added to rectal cancer cells, apoptosis of cancer cells can be induced and slower tumor growth can be observed in mouse animal experiments. Therefore, the application of miR-143 alone can achieve the treatment of rectal cancer.
Embodiment 12. miR-195 for colorectal cancer treatment to reduce tumor resistance to cytotoxic drugs
Through cytological tests, it was found that knockdown of miR-195 HT29 and LOVO cells significantly inhibited DOX (Doxorubicin)-induced cytotoxicity. At the same time, the direct involvement of miR-195 inhibits the expression of BCL2L2. Further studies have shown that low-profile miR-195 expression can enhance DOX resistance in tumor precursor cells and reduce the rate of tumor cell apoptosis. When miR-195 is overexpressed, tumor cells are more sensitive to DOX and induce apoptosis. These results suggest that the application of miR-195 as a therapeutic method will gradually reduce the resistance of tumor cells to DOX and enhance the therapeutic effect.
Embodiment 13. miR-150/miR143/miR195 miRNAs for the treatment of colorectal cancer
Based on the embodiment 11 and 12, we could assume and confirm that the miRNAs cocktail of miR-150/miR143, the miR-150/miR195, the miR143/miR195, or the miR-150/miR143/miR195 trinity would further enhance the treatment effect.
Embodiment 14. Synergized miR-150 with small molecule-based drug for anti-tumor treatment
Stivarga (regorafenib, Tablets Riegefini) is the FDA approved new indication in 2013 for the treatment of advanced gastrointestinal stromal tumors. Stivarga is a kinase inhibitor apply to the chemotherapy that was used based on [fluoropyrimidine]-, [oxaliplatin]- and [irinotecan]-in the past. It is a kind of anti-VEGF treatment including KRAS wild-type, as well as a kind of anti-EGFR-treatment for metastatic colorectal cancer (CRC). The use of miR-150 in combination with this small molecule targeted drug to treat predictable good outcomes in rectal cancer.
Embodiment 15. Synergized miR-150 with monoclonal antibody for anti-tumor treatment
Lots of monoclonal antibody drugs are approved by the FDA as first-line or second-line drugs for colorectal cancer treatment, such as Bevacizumab (Avastin), Betuximab (Erbitux), and Panitumumab (Vectibix). The combination of miR-150 and antibody drugs which have been applied in clinical application would be an approach with a great prospect for the treatment.
The foregoing has described the present invention in detail, and its purpose is to enable those skilled in the art to understand the contents of the present invention and implement the same, and does not limit the protection scope of the present invention. The equivalent changes and modifications based on the spiritual essence of the invention should be covered within the scope of protection.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201510685343.0 | Oct 2015 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2016/085605 | 6/13/2016 | WO | 00 |
