Triptolide or compositions comprising triptolide for treating disorders
阅读说明：本技术 用于治疗病症的雷公藤内酯酮或包含雷公藤内酯酮的组合物 (Triptolide or compositions comprising triptolide for treating disorders ) 是由 罗广彬 于 2018-07-10 设计创作，主要内容包括：本申请提供了用于治疗或预防过度增生性病症的雷公藤内酯酮或者其功能等效物或药学上可接受的盐、或者包含雷公藤内酯酮或者其功能等效物或药学上可接受的盐的组合物。还提供了使用上述物质治疗或预防对象中的过度增生性病症,优选癌症的方法。(The present application provides triptolide, or a functional equivalent or a pharmaceutically acceptable salt thereof, or a composition comprising triptolide, or a functional equivalent or a pharmaceutically acceptable salt thereof, for use in treating or preventing a hyperproliferative disorder. Also provided are methods of using the above substances to treat or prevent hyperproliferative disorders, preferably cancer, in a subject.)
1. A method for treating or preventing a hyperproliferative disorder, preferably cancer, in a subject, comprising administering to said subject a therapeutically or prophylactically effective amount of an agent capable of causing activation of protein kinase a, preferably triptolide or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptolide or a functional equivalent or a pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein the treatment comprises selectively killing cancer cells, preferably proliferating cells expressing protease activated receptor 2(PAR 2); and/or the preventing comprises selectively killing cells that express PAR2 prior to and/or at the site of the malignancy.
3. A pharmaceutical composition comprising triptolide or a functional equivalent or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; optionally, the composition further comprises one or more other agents that cause activation of protein kinase a.
4. A pharmaceutical composition according to claim 3 for use in the treatment or prevention of a hyperproliferative disorder, preferably cancer, in a subject.
5. A pharmaceutical composition according to claim 3 for use in selectively killing cancer cells, preferably proliferating cells expressing PAR2, in a subject.
6. The pharmaceutical composition of any one of claims 3-5, wherein the pharmaceutical composition is formulated into a pharmaceutically acceptable dosage form, preferably oral liquid, capsule, powder, tablet, granule, pill, syrup, injection, and the like.
7. Use of an agent capable of causing activation of protein kinase a, preferably triptonide or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptonide or a functional equivalent or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing a hyperproliferative disorder, preferably cancer, in a subject.
8. The use of claim 7, wherein the medicament is administered via any suitable route, such as orally, subcutaneously, intramuscularly or intraperitoneally; preferably, the medicament is administered orally.
9. The method of claim 1 or 2, the pharmaceutical composition of any one of claims 4-6, or the use of claim 7 or 8, wherein the cancer is selected from hepatocellular carcinoma, breast cancer, colon cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, renal cancer, prostate cancer, central nervous system cancer, and melanoma.
10. A method for treating or preventing an immune response related disorder and/or pain control in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of triptolide, or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptolide, or a functional equivalent or a pharmaceutically acceptable salt thereof.
11. Use of triptonide or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptonide or a functional equivalent or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing an immune response-related disorder and/or pain control in a subject.
12. A method of identifying an agent capable of causing persistent activation of PKA comprising assessing the effect of a candidate agent in inducing a mitotic disorder.
13. The method of claim 12, wherein the candidate agent is administered during an inter-division period.
14. The method of claim 12 or 13, wherein the candidate agent is administered as a short-term treatment of minutes to hours.
15. A method for inducing sustained activation of PKA in a proliferating cell expressing PAR2, comprising contacting the cell with triptonide or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptonide or a functional equivalent or a pharmaceutically acceptable salt thereof.
Technical Field
The present application relates to triptolide or compositions comprising triptolide and their uses. In particular, the application relates to triptolide or compositions comprising triptolide for use or methods for treating or preventing a disease or disorder.
Background
Cancer is a disease of uncontrolled cellular proliferation, and thus targeting cellular proliferation constitutes a potentially effective strategy against cancer. Targeted anti-cancer therapies represent a revolutionary breakthrough and new paradigm of anti-cancer chemotherapy. In this new paradigm, individualized anti-cancer drugs have been developed based on unique cancer-specific genotypes (mutations of specific genes) or epigenetic attributes (mis-expression of specific genes). Thus, such therapies may not only promote targeted killing of cancer cells to minimize the risk of serious side effects, but may also enable treatment to be provided to patients who are most likely to benefit from treatment, thereby reducing unnecessary treatment for patients who are not likely to have a beneficial response. For this reason, targeted anti-cancer therapies are also referred to as personalized anti-cancer therapies.
However, despite the broad prospects for personalized anti-cancer therapies, only a limited number of targets have been identified and successfully utilized in therapy to date. Furthermore, these targeted drugs are often effective in only a small fraction of patients, even for patients with the same specific type of tumor that has targeting properties. As a result, personalized therapies currently only benefit a very small fraction of the entire cancer patient population. Therefore, there is an urgent and unmet need to expand the application of this new therapy. The identification of new targets for the development of targeted therapies is one of the most promising approaches to achieve this goal.
Members of the G protein-coupled receptor (GPCR) superfamily comprise targets for a number of drugs. These receptors transmit information from the extracellular microenvironment to the internal machinery of the cell, affecting the activity of specific downstream signal transduction pathways. Protease activated receptor 2(PAR2), encoded by the F2RL1 gene, is a member of the self-ligand GPCR subfamily of receptors whose cognate ligands and their corresponding receptors are encoded as a single polypeptide and deployed together on the plasma membrane of cells.
Recent studies have revealed that PAR2 is expressed in many types of tumor cells under in vitro culture conditions and in several types of human primary tumors examined, in contrast to its highly restricted expression pattern in several types of terminally differentiated non-dividing cells under normal circumstances, in addition, activation of the classical PAR2 signaling pathway (G α q-PLC-IP3/DAG pathway) appears to promote growth.
Triptolide is a natural compound that was purified in 1972 from Tripterygium wilfordii (Tripterygium wilfordii) plants along with triptolide and triptolide (fig. 1A). It was found that although triptolide and triptolide differ only in the functional group at C14, triptolide has C14 keto and triptolide has C14 alcohol, triptolide, but not triptolide, has potent anti-leukemia activity. Early studies defined triptolide as a toxicant with potent anti-leukemic properties. In addition, although triptolide has been reported to have modest anti-tumor activity in preclinical models, some diterpene lactone epoxides (including triptonide) have been shown to have anti-fertility activity.
In this application, the inventors investigated the effect of triptonide on cancer, particularly proliferating
Summary of The Invention
In a first aspect, the present application provides a method for treating or preventing a hyperproliferative disorder, preferably cancer, in a subject comprising administering a therapeutically or prophylactically effective amount of an agent capable of causing activation of protein kinase a (pka) or a pharmaceutical composition comprising the agent.
In some embodiments, the treatment comprises selectively killing cancer cells, preferably proliferating cells that express
In a second aspect, the present application provides a pharmaceutical composition comprising triptolide, or a functional equivalent or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
In a third aspect, the present application provides triptolide or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptolide or a functional equivalent or a pharmaceutically acceptable salt thereof, for use in treating or preventing a hyperproliferative disorder, preferably cancer, in a subject. In particular, disclosed herein are triptonide, or a functional equivalent or a pharmaceutically acceptable salt thereof, or a composition comprising triptonide, or a functional equivalent or a pharmaceutically acceptable salt thereof, for selectively killing cancer cells, preferably proliferating cells expressing PAR2, in a subject.
In a fourth aspect, the application provides the use of an agent capable of causing PKA activation in the manufacture of a medicament for the treatment or prevention of a hyperproliferative disorder in a subject. In some embodiments, the hyperproliferative disorder is cancer.
The application also provides for the use of an agent capable of causing PKA activation in the preparation of a medicament for selectively killing cancer cells in a subject. In certain embodiments, the cell is a proliferating
In some embodiments, the agent capable of causing PKA activation is an agonist of the GPCR receptor. In a specific embodiment, the agent is triptonide, or a functional equivalent or a pharmaceutically acceptable salt thereof.
In a fifth aspect, the present application provides a method for treating or preventing an immune response-related disorder and/or pain control in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of triptonide or a functional equivalent or pharmaceutically acceptable salt thereof, or a pharmaceutical composition disclosed herein.
In other aspects, the present application provides the use of triptolide, or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptolide, or a functional equivalent or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing an immune response-related disorder and/or pain control in a subject.
In other aspects, the present application provides triptolide or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptolide or a functional equivalent or a pharmaceutically acceptable salt thereof, for use in treating or preventing an immune response-related disorder and/or pain control in a subject.
In other aspects, disclosed herein are methods of inducing sustained activation of PKA in a proliferating cell expressing PAR2, comprising contacting the cell with triptonide, or a functional equivalent or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising triptonide, or a functional equivalent or a pharmaceutically acceptable salt thereof.
In other aspects, all of the aforementioned compositions further comprise one or more other agents.
In other aspects, methods of identifying an agent capable of causing sustained activation of PKA are provided, wherein the methods comprise assessing the effect of a candidate agent in inducing mitotic disorders (mitotic catastrophe). In some embodiments, the candidate agent is administered at intervals. In some embodiments, the candidate agent is administered as a short-term treatment of several minutes to several hours.
Brief Description of Drawings
FIG. 1 shows the effect of triptonide on cell growth. Fig. 1A shows the structures of triptonide and triptolide. FIGS. 1B-1C show the effect of one hour treatment with increasing concentrations of triptolide on the growth of HepG2(B) and cultured Primary Mouse Hepatocytes (PMH) (C). Cells were seeded into each well of a 96-well plate and growth rates were assessed by the relative density of the cultured cells, while actual images of the cultured cells were monitored using the incucte Zoom system by taking 4 images per well at four fixed locations every 3 hours.
FIG. 2 shows cells undergoing DNA replication detected using the Edu incorporation assay. HepG2 cells were synchronized by mimosine or serum starvation. They were then released into EdU-containing medium for 30 minutes. Those cells that are undergoing DNA replication incorporate EdU into newly synthesized genomic DNA. The presence of alkynyl groups in EdU-containing DNA in proliferating cells allows Edu-containing DNA and fluorescein-containing azide to be labeled and visualized by a "click" reaction. The reaction between the alkyne and azide functional groups results in conjugation of the two moieties, and covalent labeling of the DNA with a fluorescent probe. Individual nuclei were visualized by staining the DNA with DAPI, a DNA-specific fluorescent dye. Fig. 2A shows a photograph of mimosine treated cells after a Click assay based on Edu (Click assay). The percentage of replicating cells positive for the signal based on Edu is presented at the bottom of the respective photographs. It is noted that from 0 to 1 hour after the release of mimosa treatment, most cells were positive for the Edu-based fluorescence signal. Most were undetectable at 1.5 hours. Figure 2B shows a photograph of serum starved cells after a click assay based on Edu. The percentage of replicating cells positive for the signal based on Edu is presented at the bottom of the respective photographs. Edu positive cells of the first wave were observed when Edu was fed at 0 hours, beginning to decrease 4 hours after release from starvation. Edu positive cells were observed as a second wave at 10 hours, decreasing at 18 hours post-release.
Figure 3 shows representative images of live cell imaging of serum-starved cells at various time points after release to conventional media without additional treatment (control) or treatment with 1 μ M triptonide at various time points (0 min, 120 min and 240 min, TR0, TR120, TR240, respectively), demonstrating the effect of triptonide on serum-starved HepG2 cells. The top number indicates the time after the cells were released from serum starvation. It is noted that for control and TR120, a significant increase in mitotic image was evident at 13 hours, after which it remained relatively constant, and a significant increase in total cell number at 37 hours. In contrast, for TR0 and TR240 treatments, total cell numbers did not change significantly throughout, but cell numbers with abnormally condensed chromatin increased significantly at 37 hours. In addition, the accumulation of cells with abnormally condensed chromatin reached its peak at 13 hours.
Figure 4 shows representative images of mimosine-treated HepG2 cells treated with vehicle (control) alone or with 1 μ M triptonide at different time points (0 min, 60 min, 120 min and 240 min post-mimosine treatment, or TR0, TR60, TR120, TR240, respectively) and then returned to the conventional medium, confirming the effect of triptonide on mimosine-treated HepG2 cells. Images obtained at 0 hour, 14 hours, 23 hours and 37 hours after the start of the experiment are shown. The top number indicates the time (hours) after the cells were released from the mimosine treatment. Note that for control (top row), TR60 and TR240 treatments, total cell number increased from left to right; absence of such changes in TR0 and TR120 samples; for TR120 treatment, cells with abnormally condensed chromatin accumulate in large numbers (right-most picture of TR 120). It is also noted that for the controls, TR60 and TR240, starting from 14 hours, a relatively constant number of smaller entities of dark and round metaphase cells appeared; and for TR120 treatment, the number of cells with abnormally condensed chromatin steadily increased from 14 hours on.
Figure 5 shows an image of mimosine-treated cells that were further treated with vehicle solutions (control), 1 μ M or 2 μ M triptolide for one hour at 0 min or 120 min after the start of the experiment, demonstrating the effect of triptolide on mimosine-treated HepG2 cells. Only images obtained 0 and 37 hours after the start of the experiment are shown. Only those cells treated with 2. mu.M triptolide (TR 0-2. mu.M, TR 120-2. mu.M) at 0 and 120 min were noted to have significant accumulation of condensed chromatin at 37 hours after initiation. Those cells treated with 1. mu.M triptolide (TR 0-1. mu.M, TR 120-1. mu.M) did not show this feature.
FIG. 6 shows the effect of triptonide and triptolide on cell cycle progression of HepG2 cells. Asynchronous (AS) HepG2 cells were treated with 200. mu.M mimosine for 28 hours and then returned to conventional medium alone for 0, 11, 24 and 37 hours (
FIG. 7 shows the effect of triptonide on cultured primary keratinocytes. FIGS. 7A-7C show growth curves of wild-type keratinocytes (A) or Par2 knock-out keratinocytes (B, C) after one hour of treatment with different concentrations of triptonide. Including the corresponding
Fig. 8 shows images of western blots showing the presence or absence of PAR 2-or PAR 2-specific bands and those β -actin (ACTB, as loading control) in Primary Mouse Hepatocytes (PMH), the immortalized human hepatocyte line LO2, and the hepatocellular carcinoma cell lines Hep3B and HepG2, demonstrating that PAR2 is expressed in the immortalized human hepatocyte line LO2 and the human hepatocellular carcinoma cell line, but not in the primary mouse hepatocytes.
Figure 9 shows the effect of triptolide on cultured primary keratinocytes. FIGS. 9A-9B show growth curves of wild-type keratinocytes (A), or Par2 knock-out keratinocytes (B), after one hour exposure to different concentrations of triptonide. FIGS. 9C-9D show growth curves of wild-type keratinocytes (C), or Par2 knock-out keratinocytes (D), after continued exposure to varying concentrations of triptolide. It is noted that one hour treatment with triptolide concentrations up to 800nM lacks any significant growth inhibition, while similar effective growth inhibition is achieved with triptolide concentrations as low as 1.25nM when applied in a continuous manner.
FIG. 10 shows the effect of trypsin, triptolide, and triptolide on ERK phosphorylation in HepG2 cells HepG2 cells were serum starved for 48 hours, then cultured in serum-free basal medium 1640 containing DMSO vehicle, trypsin (50nM), triptolide (1 μ M), or triptolide (1 μ M), samples were taken at different time points (0 min, 5 min, 10 min, 20 min, and 40 min, respectively) for total protein extraction, Western blots were performed with antibodies specific for phosphorylated ERK (p-ERK), unphosphorylated ERK (ERK), and β -actin, respectively.
Figure 11 shows the effect of triptonide on phosphorylated histone H3 levels in HepG2 cells HepG2 cells were synchronized and released to conventional media for 2 hours by treatment with mimosine 28 hours then cells were treated with vehicle solution or 1 μ M triptonide for 1 hour after treatment, cells were incubated in conventional culture conditions and samples were harvested from 2 to 12 hours per hour (shown in the top of the top panel) western blots were performed to determine the relative levels of phosphorylated histones H3(p-H3), CDK1 and β Actin (ATCB) (as control) it is noted that the peak level of p-H3 was detected in control samples harvested 10 hours after treatment with vehicle solution and the lack of a significant increase in the level of p-H3 in samples derived from triptonide treated cells.
FIG. 12 shows the effect of trypsin and triptonide on cAMP levels in HepG2 cells. HepG2 cells were synchronized by mimosine treatment for 28 hours and then released to conventional media for two hours (triptonide-2, when cells were sensitive to triptonide mitosis-inducing effects). Then, before harvesting the samples, cells were treated with vehicle solution, 50nM trypsin or 1. mu.M triptonide for various times in serum-free basal medium. In addition, a panel of cells was allowed to recover for 4 hours in conventional media before treatment with 1 μ M triptonide (triptonide-4, when the cells were insensitive to triptonide mitosis-inducing effects). cAMP levels in each sample were determined. It is noted that, after a modest and transient increase in cAMP levels in trypsin-treated cells, and allowing cells to recover in conventional media, much higher levels of two repeated peaks of cAMP were observed in 2 hour (triptonide-2) but not 4 hour (triptonide-4) treated cells with triptonide.
FIG. 13 shows the effect of triptonide on the level of PKA activity in HepG2 cells. HepG2 cells were synchronized by mimosine treatment for 28 hours and then released into conventional medium for two hours. Cells were then treated with vehicle solution, or 1 μ M triptonide, for various times in serum-free basal medium prior to harvesting the samples. The level of PKA activity in each sample was then determined. It is noted that the two peaks of increased PKA activity were much higher in triptonide-treated cells than in untreated cells.
FIG. 14 shows the effect of trypsin and triptonide on the level of PKA activity in HepG2 cells. HepG2 cells were synchronized by mimosine treatment for 28 hours and then released into conventional medium for two hours. Then, cells were treated with conventional medium containing vehicle solution, 50nM trypsin or 1. mu.M triptonide for one hour before returning to conventional medium. Samples were harvested at different times after each treatment and the PKA activity of each sample was determined. It is noted that for untreated cells or those treated with trypsin, a sharp decrease in the level of PKA activity occurred 9 hours after release of mimosine treatment; however, for those cells treated with triptonide, the level of PKA activity did not decrease significantly at the same time point.
FIG. 15 shows the effect of blocking the AC-cAMP-PKA signaling pathway on the effect of triptonide on HepG2 cells. HepG2 cells were seeded overnight in 96 wells and then treated with vehicle solutions (control, Ctrl), triptonide (Trip,1 μ M), vidarabine (Vid,10 μ M), octadecylated PKI-14-22 amide (PKI,2.5 μ M), triptonide plus vidarabine (Trip + Vid), or triptonide plus PKI (Trip + PKI). Cell growth and morphology was monitored by using the IncuteCyte Zoom system. Figure 15A shows the effect of vidarabine, triptonide, and vidarabine plus triptonide. It is noted that vidarabine has no significant effect on cell growth. Triptolide is growth inhibitory. Vidarabine plus triptonide also had no significant effect on cell growth. Figure 15B shows the effect of PKI, triptonide, and PKI plus triptonide. . Note that PKI alone has no significant effect on cell growth. Triptolide is growth inhibitory. Triptonide plus PKI had no significant effect on cell growth.
FIG. 16 shows images of Western blot analysis showing the presence or absence of PAR2 and those β -actin (ACTB, as loading control) in the immortalized gastric epithelial cell line GES-1 and 5 gastric cancer cell lines, demonstrating expression of PAR2 in the gastric cancer cell line and in the immortalized gastric epithelial cell line.
FIG. 17 shows the effect of triptonide on tumor-bearing mice. Fig. 17A shows photographs of GFP fluorescence real-time imaging of three tumor-bearing mice, representing three different treatment cohorts (n ═ 10 for each cohort) at different time points (days) after treatment with vehicle treated for triptolide (top row), at a weight level of 25mg/kg triptolide (by gavage), or sorafenib (by intraperitoneal injection) was initiated. It is noted that for vehicle-treated mice (top row), the intensity and relative area of GFP signal gradually increased over time, while the intensity and relative area of GFP signal for triptonide-treated mice began to decrease at
Detailed Description
The present application provides agents, or pharmaceutical compositions comprising the agents, capable of causing protein kinase a activation for use in methods of treating or preventing hyperproliferative disorders (especially cancer), immune response related disorders and/or pain control in a subject, or for treating or preventing such disorders or diseases.
In certain embodiments, the agents capable of causing activation of protein kinase a disclosed herein are agonists of GPCR receptors. In a preferred embodiment, the agent is triptonide or a functional equivalent or a pharmaceutically acceptable salt thereof.
In certain embodiments, an agent capable of causing activation of protein kinase a can selectively kill cancer cells in a subject. In a specific embodiment, the cell is a proliferating
In some embodiments, the cancer may be a primary cancer or a metastatic cancer. In particular embodiments, the cancer may be hepatocellular carcinoma, breast cancer, colon cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, renal cancer, prostate cancer, central nervous system cancer, melanoma, and the like.
The availability of desirable novel targets is a limiting factor in the development of new targeted anti-cancer therapies. The present disclosure describes the identification of PAR2 as a novel target that can be used to stimulate selective killing of cancer cells expressing PAR2 by a unique PAR2 activation pattern through the use of triptonide or a functional equivalent thereof. Notably, our studies found that triptolide can be used to induce mitotic disorders through non-classical activation of PAR2 associated with sustained elevation of PKA, enabling targeted killing of proliferating
Cancer is a disease in which cells proliferate abnormally, and thus killing and/or suppressing the growth of cancer cells that proliferate abnormally constitutes a major strategy for treating the disease. Proliferation of normal and malignant human cells is a highly controlled and complex process. In humans, once born, a given cell can remain in a quiescent (also known as G0) state or continue a new round of proliferation, resulting in two new daughter cells. The cell proliferation cycle is divided into four successive phases: gap 1(G1) phase, synthetic (S) phase, gap 2(G2) phase and mitotic (M) phase. More recently, stage G1 has been further subdivided into early G1 or G1 postmitotic (G1-ps) and late G1 or G1-pre-S (G1-ps). G1-pm defines a relatively constant time period (3-4 hours) representing the minimum time during which novacells can proliferate through the so-called restriction (or R) point; whereas G1-ps is susceptible to becoming variable between different cell types or even between individual cells of the same type. Individual cells retain the ability to exit the cell cycle to enter the quiescent or G0 state before passing the "R" point. Activation of the MAPK-ERK pathway by mitotic stimulation constitutes an important force to drive individual cells through the "R" site and into proliferation. Once in the cell cycle, progression through the cell cycle is additionally regulated, including those at the G1/S, S/G2, G2/M boundary, and during mitosis. Once the cell passes the G2/M switch, it will be arrested by the promiscuous checkpoint or will progress to promiscuous. Cells blocked at a prior checkpoint may be withdrawn to an interphase state and then, once conditions are appropriate, may resume forward progression. In contrast, those cells that have entered the metaphase have passed a so-called "point-of-no-return" and can no longer return to the interphase state. Rather, they can normally progress to complete productive mitosis to produce two diploid daughter cells; or end in a failing mitosis. Some failed mitosis may lead to the formation of tetraploid cells, while the remainder will eventually yield to death, i.e. lead to mitotic disturbances. Thus, to prevent failed mitosis, the G2/M switch is highly regulated.
In mammalian cells, mitotic-promoting factor (MPF) plays a key role in regulating G2/M switching. The core component of this MPF is the cyclin B1 cyclin-dependent kinase 1(CDK1) kinase complex. Activation of CDK1 kinase is both necessary and sufficient to promote G2/M switching to initiate mitosis. At cell cycle intervals, the kinase complex is inactive due to phosphorylation by Wee1/Tyt1 kinase. Late in G2, CDK1 is activated by the action of CDC25 phosphatase, which removes inhibitory CDK1 phosphorylation. Recent studies have shown that the G2/M switch is first initiated by activation of the cyclin B1-CDK1 complex in the cytoplasmic compartment. The activated cyclin B1-CDK1 complex is then immediately introduced into the nucleus, resulting in highly coordinated events associated with mitosis. Interestingly, in mammalian female oocytes, the G2/M shift in meiotic prophase I is suppressed by the initial activation of CDK1 in the cytoplasm via inactivation of the increased cytoplasmic PKA activity. Elevation of cytoplasmic PKA activity suppresses CDK1 activation by phosphorylating a number of proteins (including Wee1 and CDC25), which leads to Wee1 activation and CDC25 inactivation, respectively. Since both Wee1 activation and CDC25 inactivation repress CDK1 activation, an increase in PKA activity provides a very efficient mechanism for repressing the G2/M switch. An increase in PKA activity is also an effective means of suppressing the G2/M switch in mammalian somatic cells.
PAR2 is a member of the receptor's self-ligand GPCR subfamily, its cognate ligand and its corresponding receptor are encoded as a single polypeptide and are deployed together on the cytoplasmic membrane the classical ligand of PAR2 is located at the N-terminus of the polypeptide and outside the cytoplasmic membrane in an unavailable state when the protein is cleaved by specific proteases such as trypsin, this ligand becomes available the two major biological effects of PAR2/PAR2 are 1) the sensory role in pain and itch perception of the nervous system, 2) the role in the regulation of the barrier integrity and inflammatory response of the epithelial lining of various organs/tissues, thus, human PAR2 and its mouse homolog Par387 865 5 are primarily expressed at high levels on terminally differentiated epithelial cells of the epidermis, on the top of the crypts of the gastrointestinal tract and on a subset of neurons.
Alternatively, activation of PAR2 can be induced by several proteases (e.g., elastin and cathepsin) which can lead to activation of the G α s-cAMP-PKA signaling cascade leading to activation of PKA kinase the inventors of the present application have for the first time discovered that triptolide is the first small modulator of the PAR2 mediated G α s-AC-cAMP-PKA pathway.
In some embodiments, an agent disclosed herein, such as a triptonide or a functional equivalent or pharmaceutically acceptable salt thereof, can activate PAR2, which in turn causes sustained activation of PKA. In a preferred embodiment, an agent disclosed herein, such as triptonide or a functional equivalent or pharmaceutically acceptable salt thereof, produces a mitotic disorder-inducing effect, resulting in the eventual death of proliferative cancer cells.
In some embodiments, an agent disclosed herein, such as a triptonide or a functional equivalent or pharmaceutically acceptable salt thereof, can be used to promote selective killing of proliferating cells expressing PAR2 without causing unacceptable adverse effects.
The present inventors have demonstrated that triptolide has the desired selective lethal effects on proliferating cells, including cancer cells, in part due to the unique mitotic disorder-inducing effects of triptolide on mitotically activated cells, while not affecting quiescent, non-dividing cells.
In particular embodiments, triptolide is identified as a unique agent that can be used to induce PAR2/PAR2 mediated mitotic disorders in cells expressing PAR2/PAR2, resulting in selective killing of proliferating cells expressing PAR2/PAR2 the inventors of the present application found that the mitotic disorder-inducing effects of triptolide are due to its non-classical agonist effects on the PAR2/PAR2-G α s-AC-cAMP-PKA signaling cascade.
In some embodiments, triptolide acts as an unusual PAR2 agonist, resulting in abnormal activation of the AC-cAMP-PKA pathway the inventors of the present application identified triptolide for the first time as a small molecule agonist of the PAR2-G α s-AC-cAMP-PKA signaling pathway.
In some embodiments, the exposure of the cancer cells to triptonide or a functional equivalent or pharmaceutically acceptable salt thereof for a time period allows for continued activation of the PKA kinase, thereby inhibiting cancer cell-specific growth without causing deleterious effects that are independent of PAR2/
In particular embodiments, the cancer cells are exposed to triptonide multiple times. Preferably, a time span between successive exposures sufficient to significantly clear the administered triptolide is performed such that possible deleterious effects that are independent of PAR2/PAR2 will not occur or reach unacceptable levels.
In particular embodiments, in view of the disclosure herein, one skilled in the art can determine the duration of exposure or the time span between successive exposures as desired. For example, a desired duration of HepG2 cell exposure may be 20 minutes to 2 hours, such as one hour. As another example, the time span between two consecutive administrations to tumor-bearing mice by gavage can be one day, two days, three days, etc.
In one aspect, a pharmaceutical composition comprising triptolide, or a functional equivalent or pharmaceutically acceptable salt thereof, is provided. In some embodiments, the pharmaceutical composition may further comprise one or more other agents to enhance the desired therapeutic efficacy, reduce undesired effects, or both, by a so-called combination strategy.
The pharmaceutical compositions disclosed herein may be presented in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredients disclosed herein with one or more pharmaceutically acceptable carriers or other agents or other forms of intervention. Generally, compositions are prepared by combining the active ingredient with a liquid carrier or a solid carrier, or both, and then shaping the resulting product as desired.
In certain embodiments, triptolide ketones disclosed herein, or functional equivalents or pharmaceutically acceptable salts thereof, or compositions comprising them (alone or in combination with other agents or other forms of intervention) can be formulated with a pharmaceutically acceptable carrier into pharmaceutically acceptable dosage forms, e.g., oral liquids, capsules, powders, tablets, granules, pills, syrups, injections, suppositories, and the like.
As disclosed herein, "pharmaceutically acceptable carrier" refers to a carrier that does not interfere with the biological activity of the active ingredient, including those commonly used in the pharmaceutical art. The pharmaceutically acceptable carriers disclosed herein may be solid or liquid and include pharmaceutically acceptable excipients, buffers, emulsifiers, stabilizers, preservatives, diluents, encapsulating agents, fillers, and the like. For example, pharmaceutically acceptable buffers also include phosphates, acetates, citrates, borates, carbonates, and the like.
In certain embodiments, triptolide or a functional equivalent or pharmaceutically acceptable salt thereof, or a composition comprising the same, is administered via any suitable route, such as orally, subcutaneously, intramuscularly, or intraperitoneally. In a preferred embodiment, triptolide, or a functional equivalent or pharmaceutically acceptable salt thereof, or a composition comprising the same, is administered orally, either alone or in combination with other agents or other forms of intervention.
In another aspect, there is provided a method for treating or preventing a hyperproliferative disorder, such as cancer, an immune response-related disorder and/or pain control in a subject, said method comprising administering to said subject a therapeutically or prophylactically effective amount of triptolide, or a functional equivalent or pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, alone or in combination with other agents or other forms of intervention.
In other aspects, there is provided the use of a therapeutically or prophylactically effective amount of triptolide, or a functional equivalent or pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, alone or in combination with other agents or other forms of intervention, for treating or preventing a hyperproliferative disorder, such as cancer, an immune response related disorder and/or pain management, in a subject.
In certain embodiments, the treatment comprises selectively killing cancer cells, preferably proliferating
The disclosure described herein provides effective targeted anti-cancer therapies. The recent emergence of targeted anti-cancer therapies has provided great promise for cancer patients. Traditionally, the development of targeted anti-cancer therapies began by identifying cancer-specific attributes (e.g., cancer-specific mutations or gene expression profiles) and then developing appropriate modulators. However, such strategies have proven to be of little success in developing targeted therapies for hepatocellular carcinoma (HCC), and the development of effective targeted anti-cancer drugs remains an unmet urgent need.
In some embodiments, the disclosure described herein provides effective targeted therapy for hepatocellular carcinoma. In other embodiments, the disclosure described herein provides effective targeted therapy for breast cancer, colon cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, renal cancer, prostate cancer, central nervous system cancer, melanoma, and the like.
As used herein, a "therapeutically effective amount" or a "prophylactically effective amount" may be determined as appropriate, and can be readily manipulated by one of ordinary skill in the art based on the actual desired dosage, e.g., based on the weight, age, and condition of the patient and/or available technology in the personalized medical arts. Where the composition comprises a pharmaceutically acceptable carrier, the active ingredient and the carrier may be combined by conventional methods in the pharmaceutical art to prepare the desired medicament.
In certain embodiments, triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof, when administered in combination with one or more other agents or other forms of intervention, is administered to enhance the beneficial effect, to reduce the undesirable effect, or both.
The term "subject" as used herein refers to mammals, including, but not limited to, primates, cows, horses, pigs, sheep, goats, dogs, cats, and rodents, such as rats and mice. Cells as used herein may be from a subject, organ, tissue, cell, or any other suitable source.
In the present description and claims, the terms "comprising", "including" and "containing" mean "including but not limited to", and are not intended to exclude other moieties, additives, components or steps.
It is to be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example in the present application may be applied to any other aspect, embodiment or example described herein, unless indicated to the contrary.
The foregoing disclosure generally describes the invention and the following examples further illustrate the invention. The embodiments, examples and figures described are only intended to illustrate the invention and should not be seen as any limitation of the invention. The sole and exclusive indicator of the scope of the invention, and what is intended by the applicants to be the scope of the invention, is the literal and equivalent scope of the set of claims that issue from this application, in which such claims issue in a specific form, including any subsequent correction. Although specific terms and values are employed herein, they are to be interpreted as illustrative and not limiting the scope of the invention. Unless otherwise indicated, the experimental methods and techniques described in this specification are those well known to those skilled in the art.
Examples
Experimental methods
1. Cell culture experiments
1.1 preparation and culture of Primary mouse hepatocytes and keratinocytes.
Initiation and culture of primary mouse hepatocytes and keratinocytes was performed as described previously. Briefly, to initiate hepatocyte cultures, animals were first anesthetized with sodium pentobarbital (400mg/kg, ip), then the peritoneal cavity was opened, the liver was perfused in situ via the portal vein with calcium-free HEPES buffer at 37 ℃ for 4min, and with a solution containing 0.5mg/ml collagenase D (Life technologies, USA) and 3mM CaCl 2Is perfused with HEPES buffer for 8 to 10 minutes. The perfusion rate was set at 5 ml/min. Cells were seeded at a density of 400,000 cells/well in Williams' medium E (Life Technologies, USA) supplemented with 10% fetal bovine serum (Life Technologies, USA) in individual wells of a 12-well plate and allowed to adhere for 2 hours. Nonadherent cells were discarded, while adherent cells (hepatocytes) were stored in fresh medium.
For neonatal mouse keratinocyte cultures, dorsal skin of neonatal wild-type or Par2 knockout mice were harvested from wild-type or Par2 knockout mice, respectively. The skin was incubated overnight at 4 ℃ in the following solutions: a 0.25% solution of trypsin (Life technologies, USA) in Phosphate Buffered Saline (PBS) without calcium and magnesium. The epidermis was then separated from the adjacent dermis and the dispersed epidermal cells were collected in a suspension of Eagle minimal essential medium (SMEM) (Life Technologies, USA) supplemented with glutamine and 8% calcium-free Fetal Calf Serum (FCS) (Life Technologies, USA). Cells were plated at a density of 70,000 cells/well onto individual wells of a 48-well plate (Corning, USA), which were pre-coated with collagen (Life Technologies, USA). Cell cultures were incubated at 34 ℃ in 8% CO 2For 12 hours in a humidified incubator. Low calcium (0.05mM) S-MEM containing 8% FCS was then added to initiate the culture. The medium was changed every 2 days.
1.2 Culture of established cell lines
Cancer cell lines and immortalized human cell lines were cultured in 1640 tissue culture medium (Corning, USA) with 10% fetal bovine serum at 37 ℃ and standard tissue culture conditions of 5
1.3 HepG2 cell synchronization
Approximately 60% confluent HepG2 cells were maintained in serum-free 1640 medium (Corning, USA) for 48 hours in the absence of mimosine or in the presence of 200 μ M mimosine for 28 hours.
2. Proliferation experiment based on IncuCyte Zoom
For primary hepatocytes, seeded cells were monitored for up to 48 hours without any treatment (as a control) or with multiple types of treatment. For primary keratinocytes, cells in 48-well plates were monitored without any treatment or with multiple types of treatment for up to 96 hours. For HepG2, cells in 96-well plates were monitored without any treatment or with multiple types of treatment for up to 48 hours. The IncuCyte Zoom was set to take one set of images at fixed positions every three hours (4 per well for 96-well plates and 16 per well for 48-well and 12-well plates).
3. Western blot
After treatment, cells were harvested and protein extracts were prepared by lysis in RIPA buffer (Solarbio). Use of specific antibodies and
To evaluate the effect of triptonide on ERK phosphorylation, HepG2 cells were plated at
To assess the effect of triptonide on histone H3 phosphorylation, HepG2 cells were plated at
To assess the level of PAR2 expression in cultured cells, various cell lines or primary cells were cultured to about 90% confluence and then harvested for western blot analysis.
4, Edu incorporation and detection experiments
Experiments were performed using the "Click-iT Plus EdU imaging kit" (Life Technologies, Carlsbad, California, USA) according to the manufacturer's instructions with minor modifications. Briefly, HepG2 cells were seeded at a density of 30,000 cells/well onto coverslips (one coverslip/well) inside individual wells of a 24-well plate. HepG2 cells were untreated, or were at different recovery periods in regular medium after 28 hours of treatment with 200 μ M or 48 hours of incubation in serum-free medium, HepG2 cells. Cells were then released back into drug-free medium at various times before being supplied for 30 minutes Edu (10 μ M). Cells were then fixed with 3.7% formaldehyde in PBS for 15 minutes at room temperature and then permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. The Click-iT Plus reaction mixture was then added and incubated for 30 minutes. The coverslip was washed with PBS containing 3% BSA and then stained with Hoechst33342 (5. mu.g/ml). The click reaction was designed to attach the Alexa Fluor fluorescent dye to Edu, enabling visualization of DNA containing Edu and cells undergoing DNA synthesis upon addition of Edu. The Hoechst33342 fluorescent dye specifically binds to DNA, allowing visualization of all nuclei. After click reactions and Hoechst33342 staining, fluorescence microscopy was performed to assess Edu incorporation characteristics of cells subjected to various types of treatments.
5. Preclinical antitumor test
Preclinical experiments were performed by AntiCancer Biotech (Beijing) co.
5.1 Animal(s) production
Female athymic nude mice (6 weeks old) were used in this study. Animals were purchased from Beijing HFK Bioscience, co., Ltd and maintained in a high efficiency particulate air filter (HEPA) filtered environment with cages, food, and bedding sterilized by radiation or autoclaving. A total of 30 nude mice were used for the study.
5.2 Reagents for research
The suspension containing triptolide is prepared as a ready-to-use (ready-to-administer) oral formulation in a carboxymethyl cellulose suspension. The concentration of triptolide is chosen so that the desired amount of drug can be obtained in a volume of about 0.2ml for each animal. During the course of the animal study, the vehicle suspension and the suspension containing triptonide were designated as reagent a and reagent B. Information on the nature of reagents a and B is retained in the antibancebetech (beijing) co., Ltd, where animal experiments were performed.
5.3 Tumor cells
HepG2-GFP human hepatocellular carcinoma cells (Anticancer, Inc., San Diego, Calif.) were incubated with RPMI-1640(Gibco-BRL, Life Technologies, Inc.) containing 10% FBS. Cells were maintained at 37 ℃ and 5% CO 2CO in 95% air atmosphere 2Growth in Water Jacketed incubator (Forma Scientific). Cell viability was determined by trypan blue exclusion assay.
5.4 Model of subcutaneous human hepatocellular carcinoma
Female athymic nude mice were each administered a single dose of 5x10 6A single HepG2-GFP cell was injected subcutaneously. When the tumor size reaches about 1cm 3At that time, the tumors were harvested.
5.5 In situ human hepatocellular carcinoma model
Methods for establishing an in situ model of human hepatocellular carcinoma have been previously described (62). HepG2-GFP cell-derived subcutaneous tumors were cut to about 1mm 3And implanted in situ into the right lobe of the liver of 6-week-old female BALB/cnu nude mice (Beijing hfk bioscience co., Ltd.), one implant per animal. Briefly, a 1cm epigastric incision was made under anesthesia. Exposing the right lobe of the liver and mechanically damaging it by scissorsA portion of the liver surface. A piece of tumor fragment was then fixed in the liver tissue, the liver was returned to the peritoneal cavity, and the abdominal wall was sutured closed. Mice were housed in a laminar flow cabinet under specific pathogen-free conditions.
5.6 Design of research
Three days after implantation, implanted tumors were selected to be about 2mm based on the results of fluorescence imaging 2The animal of (1). For this experiment, animals with the desired tumor were randomly grouped, 10 animals per group. In addition, each mouse was given an ear tag for identification.
5.7 Treatment of
First, preliminary experiments were conducted to determine the maximum non-toxic dose by administering an oral dose of triptolide at 0, 5, 10, 25, 50, 100, 200mg/kg body weight once every other day. This experiment revealed that dose levels up to 100mg/kg body weight did not cause any significant adverse effects. Thus, a second preliminary experiment was conducted in tumor-bearing nude mice to determine whether an anti-tumor effect could be achieved within the non-toxic range. In the first set of experiments, tumor-bearing mice were treated with 0, 1, 5, 10, 25mg/kg triptonide by gavage using a one dose every other day regimen. The results show that treatment with 25mg/kg dosing is very effective in reducing tumor mass within one week. Thus, for preclinical experiments, a regimen of 25mg/kg was chosen once every other day. The volume of individual tumors was assessed by bioluminescence imaging.
5.8 Animal monitoring
All experimental mice were examined daily for mortality or distress symptoms during the study. Animals were observed until 28 days after tumor implantation.
a. Body weight
Body weights of mice were measured every three days during the study.
b. Whole body imaging
Images of tumor growth and progression were taken every three days during the study using a FluorVivo imaging system, model 300/Mag (INDEC, CA, USA).
c. End up
Animals were euthanized by injection of excess sodium pentobarbital.
d. Autopsy
Once each animal was euthanized, its liver was exposed by peeling and a final GFP fluorescence image was immediately taken. After imaging, each liver was examined for tumors or their remnants at the implantation site. When the tumor was clearly identifiable, it was excised and its weight determined using an electronic balance (Sartorius BS 124S, Germany). When the tumor was not visible, tumor tissue surrounding the implantation site was harvested and saved in formalin for further analysis.
6. Cell cycle analysis and flow cytometry
Cell cycle profiles were analyzed using standard Propidium Iodide (PI) staining methods. Briefly, HepG2 cells were seeded and then treated with or without 200 μ M mimosine for 28 hours. Untreated cells were used as synchronization controls. Then, some mimosine-treated cells were cultured in the absence of additional drug treatment and harvested at 0, 11, 24 and 37 hours for flow cytometry analysis. Alternatively, cells treated with the same mimosine are cultured in the presence of 1 μ M triptolide, 2 μ M triptolide, or 10 μ M triptolide, and harvested at the same time point as the untreated cells. Cells were fixed with 70% ethanol at-20 ℃, washed with PBS, and resuspended in staining solution (50 μ g/ml PI (Sigma),200 μ g/ml rnase a (Roche)) for flow analysis. All flow cytometry data were collected using a Coulter EPICS XL-MCL Cytometer (Beckmann Coulter) or a BD LSR I Cytometer (Becton Dickinson). Data were analyzed using the facscan (becton dickinson) and WinMDI (j. trotter, Scripps Institute) software packages.
Measurement of cAMP levels
Cultured HepG2 cells, approximately 70% confluent, were treated with 200 μ M mimosine for 28 hours. The mimosine-treated cells were then recovered in conventional medium for 2 hours, followed by treatment with vehicle solution, 1 μ M triptonide or 50nM trypsin in serum-free 1640 basal medium. Cells were harvested at different time points after treatment. For one set of samples, cells were treated with 1 μ M triptonide after 4 hours of recovery in conventional media. The cAMP concentration in individual samples was then determined using the cAMP ELLISA kit (CELL BIOLABS, Cat. No. STA-501) according to the protocol provided by the manufacturer.
Measurement of PKA Activity
Cultured HepG2 cells, approximately 70% confluent, were treated with 200 μ M mimosine for 28 hours. Mimosin-treated cells were allowed to recover in conventional medium for 2 hours before treatment with vehicle solution or 1. mu.M triptonide. To measure the acute effect of treatment on PKA activity, treatment was performed in serum-free 1640 basal medium. Cells were harvested at different time points after treatment. To measure the long-term effect of the treatment, the treatment was applied in conventional medium for one hour. After treatment, the cells were returned to normal culture conditions and then harvested at various time points after treatment. By using
9. Other reagents
PKI-14-22 amide (octadecylation) was purchased from Tocris; vidarabine was purchased from selelck. All other chemicals were purchased from Sigma-Aldrich unless otherwise noted.
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