TRIPTONIDE OR A COMPOSITION COMPRISING TRIPTONIDE FOR USE IN TREATING DISORDERS
20230125429 · 2023-04-27
Assignee
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/585
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present application provides Triptonide or a functional equivalent or pharmaceutically acceptable salt thereof, or a composition comprising the same for use in treating or preventing hyperproliferative disorders. Also provided is a method for treating or preventing hyperproliferative disorders, preferably cancer in a subject using the above substances.
Claims
1. A method for treating hyperproliferative disorders caused from protease activated receptor 2 (PAR2)-expressing proliferating cells, immune response related disorders and/or pain in a subject, comprising administering to the subject a therapeutically effective amount of the PAR2 biased agonist that can cause activation of protein kinase A (PKA) and cell cycle arrest at the G2/M transition.
2. The method according to claim 1, wherein the hyperproliferative disorder is cancer.
3. The method according to claim 2, 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 nerve system cancer and melanoma.
4. A method for identifying a PAR2 agonist that can cause activation of PKA, comprising assessing the cellular mitotic catastrophe-inducing effect of a candidate agent.
5. The method according to claim 4, wherein the candidate agent is administered at a cellular interphase.
6. The method according to claim 4, wherein the candidate agent is administered as a short treatment of several minutes to several hours.
7. The method according to claim 4, wherein Triptonide or a pharmaceutically acceptable salt thereof is identified to be a PAR2 agonist.
8. A pharmaceutical composition comprising a PAR2 agonist that can cause activation of PKA and a pharmaceutically acceptable carrier.
9. The pharmaceutical composition according to claim 8, wherein the composition further comprises one or more of other agents that cause activation of PKA.
10. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is formulated into pharmaceutically acceptable dosage forms.
11. The pharmaceutical composition according to claim 10, wherein the pharmaceutically acceptable dosage forms are selected from oral liquids, capsules, powders, tablets, granules, pills, syrups, injections and the like.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF INVENTIONS
[0039] The present application provides an agent that can cause activation of protein kinase A, or a pharmaceutical composition comprising the same for use in treating or preventing hyperproliferative disorders (especially cancer), immune response related disorders and/or pain control in a subject, or for use in a method for treating or preventing these disorders or diseases.
[0040] In certain embodiments, the agent that can cause activation of protein kinase A disclosed herein is an agonist for GPCR receptors. In preferable embodiments, the agent is Triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof.
[0041] In certain embodiments, the agent that can cause activation of protein kinase A can selectively killing cancer cells in a subject. In specific embodiments, the cells are PAR2-expressing proliferating cells.
[0042] In some embodiments, the cancer can be a primary cancer or a metastatic cancer. In specific embodiments, the cancer can be hepatocellular carcinoma, breast cancer, colon cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, renal cancer, prostate cancer, central nerve system cancer, melanoma and the like.
[0043] The availability of the desirable novel targets is a limiting factor for developing new targeted anticancer therapies. This disclosure describes the identification of PAR2 as a novel target that can be exploited for instigating selective killing of PAR2-expressing cancer cells through a unique mode of PAR2 activation by using Triptonide or its functional equivalents. Significantly, our studies found that Triptonide could be used to cause mitotic catastrophe by the non-canonical activation of PAR2 that is associated with a prolonged elevation of PKA, enabling the targeted killing of PAR2-expressing, proliferating cells. Unexpectedly, our data have revealed that although PAR2 expression is primarily restricted to quiescent and/or terminally differentiated non-dividing cells, it is expressed in two transformed human cell lines (LO-2 and GES-1) as well as many human cancer cell lines. This finding provided evidence that abnormal activation of PAR2 could represent an early “driver” alteration (i.e. a driver change) that has occurred before or during oncogenesis and hence a desirable target for instigating the specific killing of cancer cells for therapeutic benefits without causing unacceptable harmful side effects. Importantly, we have shown that orthotopical HepG2 xenograft tumors could be rapidly and completely eliminated from the tumor-bearing mice by a treatment with Triptonide that is many folds below the maximum tolerable dose, providing the proof-of-principle for this new paradigm of targeted anticancer therapy. In some embodiments, given the wide-spread expression of PAR2 in human cancers and the early onset of PAR2 activation during oncogenesis, such a paradigm could be applicable for the treatment and/or prevention of many types of human cancers. In some embodiments, given that PAR2 plays a significant role in both inflammatory response and pain control and the great safety profile of Triptonide, it is possible that Triptonide could be exploited for the management of disorders associated with inflammation and excessive pain by modulating the PAR2-mediated inflammatory and/or pain responses.
[0044] Cancers is a disease of abnormal cell proliferation and therefore killing and/or suppressing the growth of the abnormally proliferating cancer cells constitute a main strategy for treating this disease. Proliferation of human cells, both normal and malignant, is a highly regulated and complex process. In humans, a given cell, once born, could either stay in a quiescent (also known as G0) state, or go on to a new round of proliferation, giving rise to two new daughter cells. A cell proliferation cycle is divided into four sequential phases: the gap 1 (G1), the synthesis (S), the gap 2 (G2) and the mitosis (M) phase. More recently, the G1 phase has been further subdivide into an early G1, or G1-postmitosis (G1-ps) and a late G1 or G1-pre-S (G1-ps). The G1-pm defines a relatively constant period (3-4 hours) that represents the minimum time for the newly born cells to become competent to pass the so-called restriction (or R) point to commit to proliferation; while the G1-ps is much more variable among different cell types or even among individual cells of the same type. Before passing the “R” point, individual cells retain the capacity of exiting the cell cycle to enter a quiescent or G0 state. Activation of the MAPK-ERK pathway by mitogenic stimulations constitutes an important force that drives individual cells to pass the “R” point and to commit to proliferation. Once committed into the cell cycle, the progression of the cell cycle is subjected to additional regulations including those at the G1/S, S/G2, G2/M boundaries and during mitosis. Once a cell passes the G2/M transition, it will be either arrested by the antephase checkpoint or progress into prometaphase. Cells that are arrested at the antephase checkpoint can withdraw back into an interphase state and then could resume forward progression once the conditions become appropriate. In contrast, those that have entered the prometaphase have passed the so-called “point-of-no-return” and can no longer withdraw back to an interphase state. Rather, they could either progress normally to complete a productive mitosis with the production of two diploid daughter cells; or end with a failed mitosis. Some failed mitoses could lead to the formation of tetraploid cells, while the rest will ultimately succumb to death, i.e. result in mitotic catastrophe. Thus, in order to prevent failed mitoses, G2/M transition is highly regulated.
[0045] In mammalian cells, mitosis promoting factor (MPF) plays a critical role in regulating G2/M transition. The core component of this MPF is the cyclin B1-cyclin dependent kinase 1 (CDK1) kinase complex. The activation of the CDK1 kinase is both necessary and sufficient to facilitate the G2/M transition to initiate mitosis. During the interphase of the cell cycle, this kinase complex is inactive as the result of phosphorylation by the Wee1/Tyt1 kinases. At late G2, CDK1 is activated by the action of CDC25 phosphatase, which removes the inhibitory CDK 1 phosphorylation. More recent studies have shown that G2/M transition is initiated first by activating the cyclin B1-CDK1 complex in the cytoplasmic compartment. This is then immediately followed by the import of the activated cyclin B1-CDK1 complex into the nucleus, leading to the highly coordinated events that are associated with mitosis. Intriguingly, in the mammalian female oocytes, the G2/M transition at the prophase I of meiosis is suppressed by the inactivation of initial activation of CDK1 in the cytoplasm through the elevation of cytoplasmic PKA activity. The elevation of the cytoplasmic PKA activity suppresses CDK1 activation by phosphorylating a number of proteins, including Wee1 and CDC25, which results in Wee1 activation and CDC25 inactivation, respectively. Since Wee1 activation and CDC25 inactivation both suppress CDK1 activation, this elevation of PKA activity provides a very effective mechanism for suppressing G2/M transition. Elevation of PKA activity is also an effective means for suppressing G2/M transition in mammalian somatic cells.
[0046] PAR2 is a member of a self-ligand GPCR sub-family of receptors for which the cognate ligand and its corresponding receptor is encoded as a single polypeptide and deployed together onto the cytoplasmic membrane. The canonical ligand of PAR2 is located in the N-terminal of the polypeptide and is positioned outside the cytoplasmic membrane in an unavailable state. It becomes available when the protein is cleaved by a specific protease, such as Trypsin. The two major biological roles of PAR2/Par2 are: 1) A sensory role in pain and itch perception in the nervous system; 2) A role in the regulation of barrier integrity and the inflammatory response in the epithelial linings of the various organs/tissues. Accordingly, human PAR2 and its mouse homologue Par2 are expressed primarily at high levels in the terminally differentiated epithelial cells of the epidermis, on the top of the crypts of the gastrointestinal tract, and in a subset of neurons. Par2 deficient mice are viable and developmentally normal, but have defects in pain perception and in inflammatory response. Thus, the pattern of PAR2/Par2 expression correlates remarkably well with its defined roles in vivo. The important roles of PAR2 in immune response and pain control have led to a great interest in the development of PAR2 modulators as potential drug candidates for managing conditions associated with pain and/or inflammation. Such modulators have proven very useful both as research tools as well as potential leads for developing drugs for treatments related to immune response and/or pain control. Of note, these modulators include small molecule modulators that target the canonical PAR2-mediated signaling pathway, i.e. the Gαq-PLC pathway.
[0047] Alternatively, PAR2 activation can be induced by some proteases such as elastin and cathepsin, which can lead to the activation of the Gαs-cAMP-PKA signaling cascade, resulting in the activation of the PKA kinase. The Inventors first discovers Triptonide as the very first small modulator for the PAR2-mediated Gαs-AC-cAMP-PKA pathway.
[0048] In some embodiments, the agent disclosed herein such as Triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof can activate PAR2, and in turn cause sustained activation of PKA. In preferable embodiments, the agent disclosed herein such as Triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof produces a mitotic catastrophe-inducing effect, thereby leading to the ultimate demise of proliferating cancer cells.
[0049] In some embodiments, the agent disclosed herein such as Triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof can be used to facilitate the selective killing PAR2-expresing proliferating cells without causing unacceptable adverse effects.
[0050] The inventors have demonstrated that Triptonide had the desirable selective lethal effect on proliferating cells including cancer cells, which is in part due to Triptonide’s unique mitotic catastrophe-inducing effect on mitogenically activated cells while sparing the quiescent, non-dividing cells.
[0051] In specific embodiments, Triptonide is identified as a unique agent that can be used to induce PAR2/Par2-mediated mitotic catastrophe in Par2/PAR2-expressing cells, leading to the selective killing of proliferating Par2/PAR2-expressing cells. The inventors found that the mitotic catastrophe-inducing effect of Triptonide is due to its non-canonical agonist effect on the Par2/PAR2-Gαs-AC-cAMP-PKA signaling cascade.
[0052] In some embodiments, Triptonide functions as an unusual PAR2 agonist, leading to abnormal activation of the AC-cAMP-PKA pathway. The inventors first identify Triptonide as a small molecule agonist for the PAR2-Gαs-AC-cAMP-PKA signaling pathway.
[0053] In some embodiments, the duration of exposure of cancer cells to Triptonide or a functional equivalent or pharmaceutically acceptable salt thereof allows sustained activation of PKA kinase and in turn cancer cell-specific growth inhibition without causing PAR2/Par2-independent harmful effects.
[0054] In specific embodiments, the cancer cells are exposed to Triptonide for multiple times. It is preferable that a time span between successive exposures that is sufficient for the significant clearance of the administered Triptonide is implemented so that a possible PAR2/Par2-independent harmful effect would not occur or would not reach an unacceptable level.
[0055] In specific embodiments, the duration of exposure or the time span between successive exposures can be determined by a skilled person in the art as needed in view of the disclosure herein. For example, the desired duration of exposure for HepG2 cells can be 20 minutes to 2 hours, e.g. one hour. For another example, the time span between two successive dosages in tumor-bearing mice by gavage can be one day, two days, three days, etc..
[0056] In one aspect, a pharmaceutical composition comprising Triptonide or a functional equivalent or pharmaceutically acceptable salt thereof is provided. In some embodiments, the pharmaceutical composition can further comprise one or more of other agents through the so-called combinatorial strategy to enhance the desired therapeutic efficacy, to reduce the undesirable effects, or both.
[0057] The pharmaceutical composition disclosed herein can be presented in the form of a unit dose, and can be prepared by any methods well known in the pharmaceutical field. All of the methods comprise the step of combining the active ingredients disclosed herein with one or more pharmaceutically acceptable carriers or other agents or other forms of interventions. Generally, a composition is prepared by combining active ingredients with a liquid carrier or a solid carrier or both, followed by shaping the resultant product as required.
[0058] In certain embodiments, Triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof disclosed herein, or the composition comprising the same can be formulated together with a pharmaceutically acceptable carrier into pharmaceutically acceptable dosage forms, for example, oral liquid, capsule, powder, tablet, granule, pill, syrup, injection, suppository and the like, either by itself, or in combination with other agents or other forms of interventions.
[0059] The “pharmaceutically acceptable carrier” disclosed herein refers to a carrier which does not interfere with the bioactivity of active ingredients, including those commonly used in the pharmaceutical field. The pharmaceutically acceptable carrier disclosed herein can be solid or liquid, including pharmaceutically acceptable excipients, buffers, emulsifiers, stabilizers, preservatives, diluents, encapsulants, fillers and the like. For example, the pharmaceutically acceptable buffer further comprises phosphates, acetates, citrates, borates, carbonates and the like.
[0060] In certain embodiments, Triptonide or a functional equivalent or pharmaceutically acceptable salt thereof, or the composition comprising the same is administered via any appropriate routes, such as orally, subcutaneously, intramuscularly or intraperitoneally. In preferred embodiments, Triptonide or a functional equivalent or pharmaceutically acceptable salt thereof, or the composition comprising the same is administered orally, either by itself, or in combination with other agents or other forms of interventions.
[0061] In another aspect, a method for treating or preventing hyperproliferative disorders such as cancer, immune response related disorders and/or pain control in a subject is provided, which comprises administering a therapeutically or prophylactically effective amount of Triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same to the subject, either by itself, or in combination with other agents or other forms of interventions.
[0062] In a further aspect, use of a therapeutically or prophylactically effective amount of Triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same in treating or preventing hyperproliferative disorders such as cancer, immune response related disorders and/or pain control in a subject is provided, either by itself, or in combination with other agents or other forms of interventions.
[0063] In certain embodiments, the treatment comprises selectively killing cancer cells, preferably PAR2-expressing proliferating cells. In some embodiments, the prevention comprises selectively killing PAR2-expressing cells in premalignant and/or malignant sites.
[0064] The disclosure described herein provides an effective targeted anticancer therapy. The recent advent of targeted anticancer therapies has provided great hope for cancer patients. Traditionally, the development of a targeted anticancer therapy begins with the identification of a cancer specific attribute (for example, a cancer specific mutation or gene expression signatures), followed by the development of the appropriate modulating agents. However, such a strategy has been proven less fruitful for developing targeted therapies for hepatocellular carcinomas (HCCs), and thus the development of effective targeted anticancer drugs had remained an unmet urgent need.
[0065] In some embodiments, the disclosure described herein provides an effective targeted therapy for hepatocellular carcinomas. In other embodiments, the disclosure described herein provides effective targeted therapies for breast cancer, colon cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, renal cancer, prostate cancer, central nerve system cancer, melanoma, etc..
[0066] The “therapeutically effective amount” or “prophylactically effective amount” used herein can be determined as the case may be, and can be easily operated by a person having ordinary skills in the art according to the dosage actually required, for example, according to the body weight, age and conditions of a patient, and/or with the available arts in the field of personalized medicine. Where the composition comprises a pharmaceutically acceptable carrier, the active ingredients and the carrier can be mixed by conventional methods in the pharmaceutical field to manufacture the required medicament.
[0067] In certain embodiments, when Triptonide, or a functional equivalent or pharmaceutically acceptable salt thereof is administered in combination with one or more of other agents or form(s) of interventions in order to enhance the beneficial effect, to reduce the undesirable effects, or both.
[0068] The term “subject” used herein refers to a mammal, including but not limited to primates, bovine, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. The cells used herein can be from a subject, an organ, a tissue, a cell or any other suitable sources.
[0069] In the instant specification and claims, the terms “comprise/comprises/compri sing”, “include/includes/including” and “contain/contains/containing” mean “including but not limited to”, and are not intended to exclude other parts, additives, components or steps.
[0070] It should be understood that the features, characteristics, components or steps described in the specific aspects, embodiments or examples in the present application can be applied to any other aspects, embodiments or examples described herein, unless indicated to the contrary.
[0071] The above disclosures describe the inventions in general, and the following examples further illustrate the inventions. These embodiments, examples and figures described are only intended to illustrate the inventions, but not considered as any limitation to the inventions. The sole and exclusive indicator of the scope of the invention, and what is intended by the applicant to be the scope of the invention, is the literal and equivalent scope of the set of claims that issue from this application, in the specific form in which such claims are issued, including any subsequent correction or corrections. Although specific terms and values are used herein, these terms and values should also be interpreted to be illustrative, and do not limit the scope of the inventions. Unless indicated specifically, the experimental methods and techniques in the specification are methods and techniques well known to a person skilled in the art.
EXAMPLES
Experimental Methods
1. Cell Culture Experiments
1.1 Preparation and Culture of Primary Mouse Hepatocytes and Keratinocytes
[0072] The initiation and culture of primary mouse hepatocytes and keratinocytes were carried out as previously described. Briefly, to initiate hepatocyte culture, animals were first anesthetized with pentobarbital sodium (400 mg/kg, ip), then the peritoneal cavity was opened, and the liver was perfused in situ via the portal vein for 4 min at 37° C. with calcium-free HEPES buffer and for 8 to 10 minutes with HEPES buffer containing 0.5 mg/ml collagenase D (Life Technologies, USA) and 3 mM CaCl2. The perfusion rate was set at 5 ml/min. The cells were seeded onto individual well of 12-well plates (Corning, USA) at a density of 400,000 cells/well in Williams’ medium E (Life Technologies, USA) supplemented with 10% fetal bovine serum (Life Technologies, USA) and allowed 2 hours to attach. Unattached cells were discarded, while the attached cells (the hepatocytes) were kept in fresh culture medium.
[0073] For the neonatal mouse keratinocyte culture, the dorsal skins of neonatal wild type or Par2 knockout mice were harvested from wild type or Par2 knockout mice, respectively. The skins were incubated overnight at 4° C. in 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 adjoining dermis, and the dispersed epidermal cells were collected in suspension Eagle’s minimal essential medium (SMEM) (Life Technologies, USA) supplemented with glutamine and 8% calcium free fetal calf serum (FCS) (Life Technologies, USA). The cells were plated onto individual wells of 48 well plates (Corning, USA), which were pre-coated with collagen (Life Technologies, USA), at a density of 70,000 cells/well. The cell cultures were kept for 12 hours at 34° C. in a humidified incubator containing 8% CO.sub.2. Low calcium (0.05 mM) S-MEM, containing 8% FCS was then added to initiate the culture. The culture medium was changed every 2 days.
1.2 Culture of Established Cell Lines
[0074] Cancer cell lines and immortalized human cell lines were cultured in 1640 tissue culture medium (Corning, USA) with 10% fetal bovine serum in a standard tissue culture condition of 37° C. and 5% CO.sub.2.
1.3 HepG2 Cell Synchronization
[0075] HepG2 cells that are approximately 60% confluent were maintained in a serum-free 1640 medium (Corning, USA) for 48 hours in the absence or presence of 200 .Math.M mimosine for 28 hours.
2. IncuCyte Zoom-Based Proliferation Experiments
[0076] For primary hepatocytes, the seeded cells were monitored for up to 48 hours either without any treatment (as a control) or with various types of treatments. For primary keratinocytes, cells in 48-well plates were monitored either without any treatment or with various types of treatment for up to 96 hours. For HepG2, cells 96-well plates were monitored either without any treatment or with various types of treatments for up to 48 hours. The IncuCyte Zoom was set to take a set of images at fixed locations (4 per well for 96-well plates and 16 per well for 48-well plates and 12-well plates) every three hours.
3. Western Blot
[0077] After treatments, cells were harvested and protein extracts were prepared by lysis in RIPA buffer (Solarbio). Western blots were performed using specific antibodies and SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Antibodies were purchased from commercial venders as described below: polyclonal rabbit anti-PAR2 antibodies (ABGENT), polyclonal rabbit anti-Beta-Actin antibodies (Cell Signaling Technology), anti-ERK (Cell Signaling Technology), anti-phosphorylated ERK (Cell Signaling Technology), anti-phosphorylated histone H3 (Abchem), anti-CDK1 (Abcam).
[0078] To assess the effect of Triptonide on ERK phosphorylation, HepG2 cells were seeded in 6 well plates at 2 x 10.sup.5 cells per well, cultured for 12 hours and then synchronized by serum starvation in serum-free 1640 basal medium for 48 hours. The cells were then immediately treated with either the vehicle solution or with 1 .Math.M Triptonide. Samples were taken at various time points after the treatments for Western blot analysis.
[0079] To assess the effect of Triptonide on histone H3 phosphorylation, HepG2 cells were seeded in 6 well plates at 2 x 10.sup.5 cells per well, cultured for 12 hours and then synchronized in the culture medium containing 200 .Math.M mimosine for 28 hours. Following the mimosine treatment, cells were recovered in regular culture medium for 2 hours and then treated with either the vehicle solution or 1 .Math.M Triptonide for one hour. After the Triptonide treatments, cells were returned to regular culture condition and samples were harvested at various time points for Western blot analysis.
[0080] To assess the levels of PAR2 expression in cultured cells, various cell lines or primary cells were cultured to approximately 90% confluent before being harvested for Western blot analysis.
4. Edu-Incorporation and Detection Experiments
[0081] The experiments were carried out using the “Click-iT Plus EdU Imaging Kits” (Life Technologies, Carlsbad, California, USA) according to the instruction provided by the manufacturer with minor modifications. Briefly, HepG2 cells were seeded onto coverslip inside individual wells of 24-well plates (one coverslip per well) at a density of 30,000 cells/well. The HepG2 cells were either untreated, or at various duration of recovery in regular medium after subjecting to either 28 hours of treatment with 200 .Math.M, or incubation in serum free medium for 48 hours. The cells were then released back into a drug-free medium for various times before being fed with Edu (at 10 .Math.M) for 30 minutes. The cells were then fixed with 3.7% formaldehyde in PBS for 15 minutes at room temperature, followed by permeabilization using 0.5% Triton X-100 for 20 minutes at room temperature. Click-iT Plus reaction cocktail was then added and incubated for 30 minutes. The coverslips were washed with 3% BSA in PBS, and then stained with Hoechst 33342 (5 .Math.g/ml). The Click reaction is designed to attach the Alexa Fluor florescent dye onto Edu, enabling the visualization of Edu-containing DNA and the cells that were undergoing DNA synthesis when Edu was added. The Hoechst 33342 fluorescent dye binds specifically to DNA, allowing the visualization of all nuclei. Following the Click reaction and Hoechst 33342 staining, fluorescent microscopy was performed to assess the Edu-incorporating characteristics of cells that had been subjected to various types of treatments.
5. Preclinical Antitumor Experiments
[0082] The preclinical experiments were carried out by AntiCancer Biotech (Beijing) Co., Ltd.
5.1 Animals
[0083] Female athymic nude mice (6 weeks of age) were used in this study. The 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 irradiation or autoclaving. A total of 30 nude mice were used for the study.
5.2 Reagents Used for the Study
[0084] Triptonide-containing suspensions were prepared as ready-to-administer oral formulations in carboxy methyl cellulose suspension. The concentrations of Triptonide were selected so that the desired amount of the drug for each animal could be obtained in a volume of approximately 0.2 ml. During the course of the animal study, the vehicle suspension and the Triptonide-containing suspension were designated as Reagent A and Reagent B. The information regarding the nature of Reagent A and Reagent B were withheld from AntiCancer Biotech (Beijing) Co., Ltd., which performed the animal experiments.
5.3 Tumor Cells
[0085] HepG2-GFP human hepatocellular carcinoma cells (AntiCancer, Inc., San Diego, CA) were incubated with RPMI-1640 (Gibco-BRL, Life Technologies, Inc.) containing 10% FBS. Cells were grown in a CO.sub.2 Water Jacketed Incubator (Forma Scientific) maintained at 37° C. and a 5% CO.sub.2/95% air atmosphere. Cell viability was determined by trypan blue exclusion analysis.
5.4 Subcutaneous Human Hepatocellular Carcinoma Model
[0086] Female athymic nude mice were each injected subcutaneously with a single dose of 5x10.sup.6 HepG2-GFP cells. Tumors were harvested when their sizes reached approximately 1 cm.sup.3.
5.5 Orthotopic Human Hepatocellular Carcinoma Model
[0087] The method for establishing the Orthotopic Human Hepatocellular Carcinoma Model has been described previously (62). HepG2-GFP cells derived subcutaneous tumors were divided into fragments of approximately 1 mm.sup.3 and implanted orthotopically 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, an upper abdominal incision of was made under anesthesia. Right lobe of the liver was exposed and a part of the liver surface was injured mechanically by scissors. Then a piece of tumor fragment was fixed within the liver tissue, and the liver was returned to the peritoneal cavity followed by the abdominal wall closing. Mice were kept in laminar-flow cabinets under specific pathogen-free-conditions.
5.6 Study Design
[0088] At three days after the implantation, animals with an implanted tumor of approximately 2 mm.sup.2 were selected based on the results of fluorescent imaging. For this experiment, animals with the desired tumors were divided randomly into groups, 10 animals per group. Also, individual mice were each given an earmark for identification.
5.7 Treatments
[0089] First, a preliminary experiment was carried out to determine the maximal non-toxic dose by administering an oral dose of Triptonide at 0, 5, 10, 25, 50, 100, 200 mg/kg body weight once every other day. This experiment revealed that dose levels at up to 100 mg/kg body weight did not cause any significant adverse effects. Thus, a second preliminary experiment was carried out in tumor-bearing nude mice to determine whether an antitumor effect could be achieved within the non-toxic range. In the first set of the experiments, tumor-bearing mice were treated with 0, 1, 5, 10, 25 mg/kg Triptonide by gavage with the regimen of one dosage every other day. The results showed that the treatment with the 25 mg/kg dosing was very effective in reducing the tumor mass within a week. Thus, the 25 mg/kg once every other day regimen was selected for the preclinical experiment. The volumes of individual tumors were assessed by bioluminescent imaging.
5.8 Animal Monitoring
[0090] During the period of the study, all of the experimental mice were checked daily for mortality or signs of distress. The animals were observed until day 28 after tumor implantation.
a. Body Weights
[0091] The body weights of the mice were measured every three days during the study period.
b. Whole-Body Imaging
[0092] Images of tumor growth and progression were acquired every three days during the period of the study with the FluorVivo imaging system, Model 300/Mag (INDEC, CA, USA).
c. Termination
[0093] Animals were euthanized by injection of over-dose pentobarbital sodium.
d. Autopsy
[0094] As soon as each animal was euthanized, its liver was exposed by removing the skin and immediately subjected to a final GFP fluorescent imaging. After imaging, the site for the implantation of each liver was examined for tumor or its remnant. When a tumor was clearly identifiable, it was excised and its weight was determined using an electronic balance (Sartorius BS 124 S, Germany). Tumor tissues or tissues around the implanted sites when tumors were not visible were harvested and kept in formalin for further analysis.
6. Cell Cycle Analysis and Flow Cytometry
[0095] A standard propidium iodide (PI) staining approach was used to analyze the cell cycle profile. Briefly, HepG2 cells were seeded and then treated either with or without 200 .Math.M mimosine for 28 hours. The untreated cells were used as an asynchronized control. Some of the mimosine treated cells were then cultured in the absence of additional drug treatments and were harvested at 0, 11, 24 and 37 hours for flow cytometry analysis. Alternatively, the same mimosine treated cells were cultured in the presence of 1 .Math.M Triptonide, 2 .Math.M Triptolide, or 10 .Math.M Triptonide and were harvested at the same times points as those for the untreated cells. The cells were fixed with 70% ethanol in -20° C., washed with PBS and re-suspended in staining solution (50 .Math.g/ml PI (Sigma), 200 .Math.g/ml RNase A (Roche) for flow analysis. All flow cytometry data were collected using a Coulter EPICS XL-MCL Cytometer (Beckman Coulter) or a BD LSR I Cytometer (Becton Dickinson). Data were analyzed using FACScan (Becton Dickinson) and the WinMDI (J. Trotter, Scripps Institute) software packages.
7. Measurement of Camp Levels
[0096] Cultured HepG2 cells that were approximately 70% confluent were treated with 200 .Math.M mimosine for 28 hours. The mimosine-treated cells were then recovered in the regular culture medium for 2 hours before the treatment with the vehicle solution, 1 .Math.M Triptonide, or 50 nM Trypsin in serum-free 1640 basal medium. Cells were harvested at various time points after the treatments. For one set of samples, the cells were treated with 1.Math.MTriptonide after they had recovered in the regular culture medium for 4 hours. The concentrations of cAMP in individual samples were then determined using a cAMP ELLISA Kit (CELL BIOLABS, Cat. No. STA- 501) according to the protocol provided by the manufacturer.
8. Measurement of PKA Activity
[0097] Cultured HepG2 cells that were approximately 70% confluent were treated with 200 .Math.M mimosine for 28 hours. The mimosine-treated cells were recovered in regular culture medium for 2 hours before the treatment with the vehicle solution or 1 .Math.M Triptonide. To measure the acute effects of the treatments on PKA activities, the treatments were carried out in serum-free 1640 basal medium. Cells were harvested at various time points after the treatments. To measure the long-term effects of the treatments, the treatments were applied in regular culture medium for one hour. After the treatments, cells were returned to the regular culture condition and then harvested at various time points after the treatments. The levels of PKA activity of individual samples were determined by using the PepTag® Non-Radioactive cAMP-Dependent Protein Kinase Assay System (Promega, Cat. No. V5340) according to the protocol provided by the manufacturer.
9. Other Reagents
[0098] PKI22 amide (myristoylated) was purchased from Tocris; and Vidarabine was purchased from Selleck. All other chemicals were purchased from Sigma-Aldrich, unless specified otherwise.
Example 1. Triptonide Can Exhibit a Cancer Cell-Specific Growth Inhibitory Effect
[0099] We devised a screening scheme in which both primary mouse hepatocytes (PMHs, representing non-cancer cells) and HepG2 cells (HCC cancer cells) are exposed to individual testing agents for one hour only and search for the agents that exhibit a significant growth inhibitory effect for the HepG2 cells but not for the PMHs. The one-hour exposure is intended to simulate the transient high concentrations within the liver as the results of either the first pass effects of some orally administered drugs, or of locally delivered drugs, anticipating that such a short term exposure is nevertheless sufficient to affect certain cell surface receptors without causing any significant off-target effects due to intracellular absorption. Such a strategy would also reduce false hits due to reversible cell cycle arrests. The IncuCyte Zoom system (Essen BioScience, Ann Arbor, Michigan) was used to determine the IC50 values for the short-term (one hour) exposure (IC50) for each tested compound, and to record the dynamic change of individual cells by taking photograph of the cultured cells every 2 minutes over the course of the experiment.
[0100] Exposure of HepG2 cells to Triptonide (
[0101] Under the one-hour drug exposure condition, the growth inhibition could be a direct reflection of the effect on irreversible growth inhibition (senescence), cell death, or both. Initially, the assay was carried out with mixed populations of cells at various stages of the cell cycle. Given that the cell cycle status of individual cells could affect their responses toward growth inhibitory and/or cell killing agents and that only a portion of cells were killed with the treatment of 16 .Math.M Triptonide, we then asked whether mitogenic activation and/or the stage(s) of cell cycle progression could affect HepG2′s response toward the treatments. In other words, does Triptonide exert its growth inhibitory effect by targeting the proliferating subpopulation of the cancer cell? To address this question, we carried out additional studies using cell populations that were enriched with cells in specific cell cycle stages. We used two different methods to obtain HepG2 cell populations that were enriched at various stages of the cell cycle. Specifically, serum starvation was used to obtain cell populations that are enriched in G0 and G1 phase of the cell cycle, and mimosine treatment was used to block cell progression at the late G1. Moreover, populations that were enriched for various stages of G1, S, G2 and M phase were then derived from these cell populations by allowing them to recover for certain time periods in the culture medium. The stages of the individual populations were determined by the 5-ethynyl-2′-deoxyuridine (Edu) incorporation experiment. In the Edu incorporation experiment, the cells are fed with Edu, an artificial building block for DNA, for a short period of time (about 30 minutes). The cells that are undergoing DNA synthesis (i.e. in the S phase) are the only cells that could incorporate Edu into their DNA. Thus, the ability of incorporating Edu serves as a marker for S phase cells.
[0102] Edu incorporation experiment revealed that seventy-eight percent of the mimosine treated HepG2 cells incorporated Edu within 30 minutes. The majority of them (>90%) completed the S phase to reach the G2 phase within the next two hours (1.5 hours plus 0.5 hours for pulse labeling,
[0103] Subsequently, an experiment was carried out to assess whether the effects of Triptonide on the serum starved cells were concentration- and/or time-dependent. Specifically, the serum starved cells were subjected to a one-hour treatment of Triptonide at various concentrations (from 0.1 to 10 .Math.M), starting from their release from the serum starvation treatment (GO/early G1 or late G1) to up to 10 hour later (when the second wave of Edu incorporation occurred). We found that the one-hour treatments with Triptonide at concentrations below 1 .Math.M had no significant impacts on the growth of these serum starved HepG2 cells, regardless when the Triptonide treatment was administered. All treatments with 10 .Math.M Triptonide were growth inhibitory. Interestingly, at 1 .Math.M, the treatment was growth inhibitory only when it was administered at the 0 and 2 hours after the starvation (data not shown).
[0104] A more detailed study was carried out to examine the responses when the 1 .Math.M Triptonide treatment was applied at 0, 1, 2, 4 hours after the serum starvation. The data showed that after being released into regular culture medium, some of the serum starved HepG2 cells (presumably the G1 subpopulation) began entering mitosis at hour 13 and reached a peak at hour 16 (
[0105] The 1 .Math.M Triptonide treatment caused a significant accumulation of cells with a spherical morphology and condensed chromatin only when the treatment was administered at 0 and 4 hours after the serum starved cells were cultured in regular medium. The time of on-set for the accumulation of the cells with the unique spherical morphology and condensed chromatin became quite evident by hour 13 (
[0106] A similar series of experiments were then carried out with the mimosine-treated cells in order to rule out potential effects that were specific to serum starvation and to examine whether any other points during the cell cycle when the cells were sensitive to the Triptonide treatment. The mimosine-treated HepG2 cells could also resume cell cycle progression. Of note, a significant fraction of them have entered mitosis at hour 11 and the percentage of mitotic figure peaked at about hour 14 after the release from the mimosine treatment (
[0107] Thus, the combined data from experiments using HepG2 cells that were treated using two different methods show that HepG2 are sensitive to a unique mitotic catastrophe-inducing effect of Triptonide in a cell cycle stage-specific fashion, namely around S/G2 transition points. A sensitive point around the G1/S transition was detected when serum starved HepG2 cells (at (G0/early G1 plus late G1) were used, but not when mimosine-treated cells (at the G1/S boundary) were used. Rather, the mimosine-treated G1/S cells became static following the Triptonide treatment, which is expected to prevent the cells to progress into mitosis or mitotic catastrophe. Thus, the mimosine-treated cells are not suitable for assessing whether Triptonide exerts a mitotic catastrophe-inducing effect around the G1/S transition point. Accordingly, it appears likely that Triptonide can exert a mitotic catastrophe-inducing effect around G1/S transition point. Importantly, quiescent (G0) cells were refractory to the mitotic catastrophe-inducing effect of Triptonide.
Example 2. Triptonide and Triptolide Exhibit Distinctive Effects on HepG2 Cells
[0108] In this example, the effects of Triptonide and Triptolide on HepG2 cells were compared.
[0109] A one-hour treatment of the mimosine-treated HepG2 cells with 1 .Math.M Triptolide was growth inhibitory, but did not cause mitotic catastrophe, regardless when it was administered (
[0110] Cell cycle progression analyses showed that the mimosine treated HepG2 cells were able to resume productive cell cycle progression. The majority of them reached the G2/M phase with a near 4N DNA content at 11 hours after the mimosine release (Mim R11). Meanwhile, those mimosine treated cells that were additionally treated with 1 .Math.M Triptonide exhibited a significant accumulation of G2/M cells (82%, 80%, and 78% at 11, 24, and 37 hours, respectively). These data further validate the conclusion that the spherical cells that occurred in the mimosine-treated cells 2 hours after the mimosine treatment were indeed the result of mitotic catastrophe. In contrast, cells that were treated with either 2 .Math.M Triptolide or 10 .Math.M Triptonide were first arrested at the G1, S, and G2 phases at 11 hours (Mim R11). By 24 hours (Mim R24), the percentages of S and G2 diminished, while significant peaks of the sub-G1 (<2N, apoptotic) population became evident for both. Importantly, there were no accumulations of cells with a 4N DNA content, revealing the lack of a significant mitotic catastrophe effect (
Example 3. Triptonide Targets PAR2 to Induce Mitotic Catastrophe
[0111] In order to identify the target(s) of Triptonide, we examined whether the growth inhibitory effect of Triptonide was dependent on PAR2.
[0112] To address this question, we took a genetic approach. Specifically, since human PAR2 and its mouse homologue Par2 are highly conserved and primary keratinocytes express Par2 (intriguingly, only the differentiated postmitotic keratinocytes express high levels of Par2 in vivo), we decided to examine whether Triptonide could cause cell death in a Par2-dependent manner by comparing the Triptonide sensitivities between primary keratinocytes derived from wild-type and Par2 knockout mice, respectively. We found that 50 nM of Triptonide (the lowest dose tested) was effective in inhibiting the growth of wild-type keratinocytes, whereas 1.6 .Math.M was sufficient to cause a complete growth inhibition (IC50: 1.293 .Math.M) (
[0113] Treatment of wild-type keratinocytes with Trypsin, a cognate activator of Par2/PAR2, did not cause any significant cell death (
Example 4. Triptolide Was Not as Effective as Triptonide in Causing Par2-Dependent Death of PMKs
[0114] Interestingly, while Triptonide exhibited a significant growth suppressive effect on the wild-type keratinocytes at as low as 50 nM, its close relative Triptolide did not have any significant growth inhibitory effect on either the wild-type or the Par2 knockout keratinocytes even at concentration as high as 800 nM (
[0115] Also, it became apparent that the mitotic catastrophe-inducing effect of the Triptonide treatment manifested itself in a delayed fashion. Specifically, based on the data from our Edu incorporation experiment, it took at least 11 hours for mimosine-treated cells to traverse from the G1/S boundary (one of the sensitive point) to the G2/M transition point, when the mitotic catastrophe began to occur (
[0116] Given the Par2/PAR2-dependent nature of the mitotic catastrophe-inducing effect of Triptonide, activation of Par2/PAR2-mediated signaling then became a candidate for causing such an effect. Canonical activation of Par2/PAR2 by trypsin leads to the activation of the Gq-coupled pathway, and indirectly the activation of the MAPK-ERK pathway. Alternatively, Par2/PAR2 activation has also been reported to result in the activation of the Gs-coupled pathway, namely the Gs-Adenylyl cyclase (AC)-cAMP-PKA pathway. Because the MAPK-ERK pathway had been implicated to have important roles during the cell cycle, including G2 and mitosis, and Triptolide had been identified as a potent Par2/PAR2 antagonist, we asked whether Triptonide could cause the unique mitotic catastrophe-inducing effect by perturbing the proper action of the MAPK-ERK pathway. To test this possibility, we examined whether Triptonide could function like Triptolide as a potent Par2/PAR2 antagonist. As a hallmark of Par2/PAR2 activation is the unique two-stage ERK phosphorylation (an acute phase of increase followed by a delayed beta arrestin-dependent increase), we examined the effect of Trypsin (a cognate PAR2 agonist), Triptonide, and Triptolide on ERK phosphorylation in HepG2 cells. We found that, as expected, Trypsin caused a two-stage increase in the levels of phosphorylated ERK in serum-starved G0/G1 HepG2 cells. Triptolide, a Par2/PAR2 antagonist, had no significant effects on the levels of phosphorylated ERK. To our surprise, however, Triptonide caused only an acute transient increase at the levels of phosphorylated ERK (
Example 5. Triptonide Represents the Very First Small Molecule Biased Agonist for the Gαs-Coupled PAR2 Signaling Pathway
[0117] Elevation of the PKA activity in the cytoplasm have proven an effective mechanism for suppressing G2/M transition in both mammalian oocytes and somatic cells, while prolonged or permanent inhibition of G2/M transition could lead to mitotic catastrophe. Since PAR2 activation can be coupled to the activation of the Gαs -AC-cAMP-PKA pathway, i.e. the elevation of PKA activity, we then considered the hypothesis that Triptonide causes the mitotic catastrophe-inducing effect by activating the Gαs-coupled pathway through PAR2, i.e., through the elevation of cytoplasmic activity of PKA by activating the AC-cAMP-PKA pathway. This hypothesis led to several predictions: 1) CDK1 activation would not occur prior to the time of the G2/M transition; 2) the AC-cAMP-PKA pathway would be activated; 3) the level of cytoplasmic PKA would remain elevated around the time of the G2/M transition; 4) the growth inhibitory and mitotic catastrophe-inducing effect of Triptonide would be dependent on the AC-cAMP-PKA pathway. We then carried out the following experiments to examine the validity of these predictions.
[0118] First, we compared the kinetics of CDK1 activation between untreated and Triptonide-treated HepG2 cells, which were mimosine-synchronized, as they progressed through the S, G2 phases into mitosis (hour 0 to hour 12). Specifically, we assessed the activity of CDK1 by monitoring the phosphorylation of histone H3 by Western blot. The results of this experiment showed that in the mimosine-synchronized HepG2 cells, the level of phosphorylated histone H3 (p-H3) increased dramatically at hour 10 and then decreased rapidly with the next hour (
[0119] Next, we examined whether the Triptonide treatment could indeed result in the activation of the AC-cAMP-PKA pathway. Specifically, we examined the effects of the Triptonide treatment on the levels of cAMP and PKA activity. In this case, the experiments were carried out in a serum-free medium in order to minimize the influence of the levels of cAMP by the serum. Mimosine-synchronized cells were subjected to the Triptonide treatment at both a sensitive and a non-sensitive time (hour 2 and 4 after the mimosine treatment in
[0120] Importantly, however, in order to maintain the suppression of the G2/M transition, the effect of the Triptonide on CDK1 activity would need to be maintained until or possibly even beyond the point of the G2/M transition, which is supported by the data shown in
[0121] We next addressed the question regarding whether the growth inhibitory effect or mitotic catastrophe-inducing effect of Triptonide was in fact caused by the abnormal activation of the Gαs-AC-cAMP-PKA pathway. To address this question, we first treated the mimosine-synchronized HepG2 cells with Vidarabine (a small molecule inhibitor for adenylyl cyclase), Triptonide either alone or in combination and then examined their effects on the growth rate and/or mitotic catastrophe-inducing effect. We found that the treatment with Triptonide, but not those with Vidarabine, exhibited a significant growth inhibitory effect on the HepG2 cells. Meanwhile, the treatment with both Vidarabine and Triptonide also had no significant effects on the growth of the HepG2 cells (
[0122] We then carried out a similar set of experiments with myristoylated PKA inhibitor 14-22 amide, a cell permeable PKA inhibitor. We found that the treatment with the PKI inhibitor alone had no significant effect on the growth of the HepG2 cells, while the Triptonide treatment exhibited a significant growth inhibitory effect on these cells. Yet, co-treatment with the PKI inhibitor and the Triptonide did not cause any significant growth inhibitory effect on the HepG2 cells (
[0123] Also, given that the growth inhibitory effect of Triptonide on moues primary keratinocytes was also dependent on Par2 (
Example 6. The Majority of Cell Lines of Multiple Types of Human Cancers Are Sensitive to Triptonide
[0124] Previously, it has been reported that PAR2 is expressed in many cancer cell lines and in a significant fraction of many tumor types examined. Thus, we then examined whether this novel anticancer paradigm could be exploited for the specific killing of cancer cells in many cases of HCCs and/or of other types of cancers.
[0125] To address this possibility, we investigated whether other cell lines, including other HCC cell lines, cell lines from other type of cancers, as well as non-cancer immortalized/transformed cell lines, were also sensitive to Triptonide. Specifically, a total of 51 human cancer cell lines, representing 10 types of human cancers (6 non-small cell lung cancer cell lines, 5 colon cancer cell lines, 5 central nerve system cancer cell lines, 8 Melanomas cell lines, 4 ovarian cancer cell lines, 4 renal cancer cell lines, 2 prostate cancer cell lines, 3 breast cancer cell lines, 7 hepatocellular carcinoma cell lines, and 4 gastric cancer cell lines) were analyzed. The IC50s for the 51 cancer cell lines range from 0.41 .Math.M (NCI-H23, a non-small cell lung cancer line) to 51.039 .Math.M (SNB-19, a central never system cancer line). The overall mean value was 7.308 .Math.M, which was slightly lower than that of HepG2 (7.5 .Math.M) (Table 1). Among them, 34 lines (66.7%), consisting of at least one representative from all of the 10 cancer types, have ID50 values lower than that of HepG2 (Table 1). Thus, the vast majority of these cell lines of 10 different cancer types are more sensitive to Triptonide than HepG2.
[0126] Taken together, these data demonstrate that the majority of cancer cell lines derived from 10 types of human cancers are very sensitive to the one-hour treatment with Triptonide. In particular, 66.7% of the cancer cell lines, with at least one representative from each of the 10 types of cancers examined, has IC50 values lower than that of HepG2, the model cell line that was used in the initial screen as well as many of the subsequent experiments, including the antitumor experiment to be described below.
TABLE-US-00001 Growth Inhibitory Effects of Triptonide (One flour Exposure) Cell Line Tumor Type of Origin IC.sub.50 (.Math.M) EKVX Non-Small Cell Lung 2.383 HOP-62 Non-Small Cell Lung 9.235 NCI-H226 Non-Small Cell Lung 11.218 NCI-H23 Non-Small Cell Lung 0.415 NCI-H460 Non-Small Cell Lung 8.437 NCI-H522 Non-Small Cell Lung 23.142 HCC-2998 Colon 2.774 HCT-116 Colon 2.572 HCT-15 Colon 1.182 HT29 Colon 1.696 SW-620 Colon 11.642 SF-268 CNS 11.797 SF-295 CNS 14.559 SF-539 CNS 1.143 SNB-19 CNS 51.039 U251 CNS 43.176 LOX IMV1 Melanoma 1.774 MALME-3M Melanoma 3.437 M14 Melanoma 4.934 MDA-MB-435 Melanoma 1.913 SK-MEL-2 Melanoma 0.608 SK-MEL-28 Melanoma 17.834 UACC-257 Melanoma 16.002 UACC-62 Melanoma 9.350 IGR-OV1 Ovarian 4.656 OVCAR-3 Ovarian 4.903 OVCAR-4 Ovarian 1.539 OVCAR-5 Ovarian 8.229 OVCAR-8 Ovarian 7.295 NCI/ADR-RES Ovarian 8.335 SK-OV-3 Ovarian 3.887 A498 Renal 4.337 ACHN Renal 14.997 CAKI-1 Renal 6.146 SN12C Renal 1.066 PC-3 Prostate 6.707 DU-145 Prostate 2.376 MCP7 Breast 0.592 HS 578T Breast 1.858 T-47D Breast 1.809 HepG.sub.2 Hepatocellular carcinoma 7.509 Hep3B Hepatocellular carcinoma 1.281 SNU-449 Hepatocellular carcinoma 4.917 SNU-387 Hepatocellular carcinoma 6.817 Huh7 Hepatocelular carcinoma 1.745 LM9 Hepatocellular carcinoma 4.239 SMMC7721 Hepatocellular carcinoma 3,910 SGC7901 Gastric cancer 11.977 MGC803 Gastric cancer 2.580 AGS Gastric cancer 4.065 BGC823 Gastric cancer 10.366 Mean value 7.655 GES-1 16.719 LO2 0.642 *, **: Immortalized cell lines derived from gastric epithelium and hepatocytes respectively.
Example 7. Transformed Non-Cancer Cells Were PAR2 Positive and Sensitive to Triptonide
[0127] In the experiment described above, two immortalized cell lines (LO2, immortalized hepatocytes; and GES-1, immortalized gastric epithelium) were also included as the non-cancer control cell lines. Remarkably, we noticed that both of these two immortalized cell lines examined were sensitive to Triptonide (Table 1). This finding prompted us to hypothesize that PAR2 was expressed in these non-cancer cells, or in other words, PAR2 activation could constitute a common early event (or the so-called “driver event”) of oncogenesis (and hence a desirable target for anticancer drug development). Indeed, we found that PAR2 was clearly expressed not only in HCC cell lines (
Example 8. Triptonide Exhibited a Potent Antitumor Activity in Vivo
[0128] As discussed earlier, the unique Triptonide-mediated, PAR2-dependent killing of proliferating cells in the context of the restricted expression of PAR2 in non-dividing cells in vivo prompted us to further evaluate the feasibility of using Triptonide and/or similar PAR2 ligands to instigate a targeted anticancer therapy for PAR2-expressing cancers. However, given that long-term exposure to Triptonide were quite toxic to the Par2-expressing primary mouse keratinocytes at concentrations as low as 50 nM (
[0129] The essence of targeted anticancer therapy is the selectively enhanced killing of cancer cells for a desirable therapeutic gain with minimal or no adverse effects. Thus, we first determined the lethal dose and the maximum tolerable dose (MTD) of Triptonide for mice based on a once every other day regimen. We found that 200 mg/kg resulted in 100% death within four days, while 100 mg/kg did not result in any significant adverse effects on the mice, establishing the lethal dose and the MTD within the 100 to 200 mg/kg range for this specific one every other day regimen. Meanwhile, we also found that when Triptonide was given at 100 mg/kg but for three times a day with a four-hour interval between each treatment, all animals were dead within four days. Together, these data showed that excessive exposure either at too high a dose or at a too short interval could increase the risk of general toxicity, while repeated dosing of up to 100 mg/kg at the appropriate intervals such as once every other day were well tolerated. It is noted that once every other day might not be the optimal scheme. Nonetheless, we decided to begin with this scheme and the 25% value of the MTD, i.e. 25 mg/kg.
[0130] A preliminary experiment was carried out to test the potential effective antitumor range of dosing by treating the tumor-bearing mice with 0, 1, 5, 10, 25 mg/kg by gavage with the regimen of one dosage every other day. The results of this preliminary experiment showed that compared to the 0 mg/kg control, treatments with 1 or 5 mg/kg had no significant effects on the changes in the intensities of the tumor specific signal; and those with 10 mg/kg reduced the tumor specific signals. In contrast, the treatments with 25 mg/kg Triptonide could not only rapidly reduce the intensity of the tumor specific signals, but eventually reduced the tumor specific signal to the background level (data not shown). Based on the result from this preliminary experiment, we concluded that the treatment with a single gavage dose of 25 mg/kg once every other day was sufficient to provide a potent anti-tumor effect.
[0131] A full fledge preclinical experiment with a Green fluorescent protein (GFP)-expressing HepG2 (HepG2-GFP) orthotopic xenograft tumor model was then carried out. In this case, GFP was used as a tumor specific signal to assess the relative volumes of individual tumors. Thirty mice were randomly divided into three cohorts, 10 mice per cohort; and were treated with the vehicle only, Triptonide at the level of 25 mg/kg body weight, or Sorafenib, respectively. Sorafenib is the only anticancer drug approved by the USFDA for HCCs. It was therefore included as a positive control. The result showed that compared with the vehicle control group, the Sorafenib-treated group exhibited lower intensities of the tumor specific signals at all time points, indicative of a tumor suppressive effect. However, the tumor specific signals remained detectable and exhibited a trend of increased intensities over time for all treated mice throughout the treatment period. In contrast, the Triptonide-treated group was characterized by an initial rapid reduction in the intensities of the tumor specific signals. Remarkably, the tumor specific signals could no longer be detected in any of the ten mice by two weeks after the initiation of the treatment (
[0132] Together, these data have indicated that the treatment with Triptonide effectively eliminated the tumors without causing any adverse effects to the tumor-baring animals.
Discussion and Conclusions
[0133] In an attempt to identify candidates to be used for targeted anticancer therapy for human HCCs, we discovered that Triptonide had the desirable cancer cell-specific growth inhibitory effect (
[0134] We next showed that this unique mitotic catastrophe-inducing effect of Tritonide was distinct from the cell killing effect of Triptolide (
[0135] Mechanistically, Triptonide could cause the activation of ERK (
[0136] Taken together, these data have led to the discovery of a new paradigm for instigating the selective killing of proliferating PAR2-expressing by targeting PAR2 with Triptonide and/or other PAR2 agonist(s) that could be used to cause the unusual activation of the AC-cAMP-PAK signaling pathway, while sparing PAR2 negative cells as well as those PAR2 expressing cells that are not proliferating. In addition, since individual cells are only vulnerable to this killing effect of Triptonide, the ability to activate ERK could increase the efficacy of the killing by promoting the entry of quiescent cells into the cell cycle.
[0137] The inventors next found that at least one cell line from a collection of human cancer cell lines that represent the 12 major types of human cancers was more sensitive than HepG2 (Table 1). Moreover, PAR2 expression was detected in all gastric cancer cell lines examined (
[0138] Intriguingly, the inventors also revealed that PAR2 is expressed in both of the two immortalized cell line examined, suggesting that acquisition of PAR2 expression is an early event for many cancers (
[0139] Significantly, the results from a preclinical experiment showed that a Triptonide single agent therapy had a curative effect on an orthotopical xenograft liver tumor model. Remarkably, the curative effect was achieved with a drug dose that is four folds lower than the maximum non-lethal dose. Together, these data demonstrate that Triptonide can be used to instigate the selective and effective killing of cancer cells for therapeutic gain with little or no adverse side effects. It should be noted that HepG2 has a median IC50 for Triptonide (IC50: 7.509 .Math.M) that is higher than 68% of the cancer cell lines examined (Table 1), suggesting that Triptonide, or its functional equivalents, could potentially be used to treat many types of cancers.
[0140] Intriguingly, the one-hour exposure of 1 .Math.M Triptonide to HepG2 cells around the S/G2 (or perhaps also G1/S) transition caused mitotic catastrophe at a time frame that was coincided with the timing for mitosis. Significantly, this unique mechanism of cell death restricts its damage to only the cycling portion of the PAR2-expressing cells. As PAR2 is primarily expressed in non-cycling postmitotic cells in humans, this has raised the possibility that Triptonide, or other agents with the same unique effect on PAR2, could be used to implement the specific killing of cycling PAR2-expressing cancer cells without inflicting any significant adverse effects to any of the non-cancer cells including the non-cycling PAR2-expressing cells. Thus, taken together, our data have established that the non-canonical activation of PAR2, in a manner that is mediated by Triptonide, constitutes a new candidate paradigm for developing targeted anticancer therapy.
[0141] The concept of targeting PAR2 with specific, biased or abnormal agonists to achieve cancer-cell-specific killing for targeted anticancer therapy has not been reported to date. It also represents the first example for the effectiveness of targeting a GPCR for anticancer benefit. And to the best of our knowledge, the instant application also constitutes the first disclosure for the identification of small molecular agonists for the PAR2-AC-cAMP-PKA signaling pathway.
[0142] The finding that Triptonide has such a unique anticancer property was a major surprise for us. Remarkably, our data clearly showed that Triptolide and Triptonide exhibited contrasting different effects both on ERK phosphorylation in HepG2 cells and on the growth and survival of Par2 knockout keratinocytes. Accordingly, while the antitumor activity of Triptolide that is due to its general, non-cancer cell-specific cytotoxicity, has been well known, the unique mode of PAR2-dependent cytotoxicity and the targeted antitumor activity of Triptonide described here have not been reported previously.
[0143] Furthermore, the use of the cultured primary mouse hepatocytes rather than an immortalized cell line, such as LO2, has proven critical in this discovery, since PAR2 is apparently expressed in the immortalized LO2 cell line (interestingly also the immortalized cell line GES-1). Of note, when applied in the usual continuous exposure fashion, Triptonide did not exhibit any significant cancer cell-enhanced killing effect. Serendipitously, by employing a unique screening strategy that combines the use of primary cultured mouse hepatocytes as a non-cancer control and the one-hour drug treatment scheme, we were very fortunate to have discovered the differential effects of Triptonide on primary cultured mouse hepatocytes and its presumed cancer counterpart HepG2 cells in our initial experiment.
[0144] The subsequent finding that a Triptonide-based treatment at a dose that was four fold below the maximal tolerable dose resulted in a full curative outcome was indeed a surprise given the potential off-target cytotoxic effect(s) of the drug. A drug that targets a cell surface receptor could exhibit such a target-only detrimental effect if it is highly specific for the desirable target of interest; and/or if it has a pharmacokinetic attribute that would limit its access to this desirable target. In this case, it is worth noting that in rodents, Triptonide, when administered via intraperitoneal injection, has a very short T1/2 alpha (0.17-0.195 hours) and T1/2 beta (about 4.95-6.49 hours). It is also worth noting that there has been great interest in the anti-cancer activity of Triptolide, despite its inherent general cytotoxicity. However, it has become increasingly evident that the very narrow range between the effective doses of anti-tumor activities and the minimal dose of general toxicity could represent a major hurdle for its clinical application as an anticancer drug. In contrast, Triptonide is not only less potent in terms of its general cytotoxicity (i.e. PAR2-independent toxicity), but also it has very unique pharmacokinetic characteristics such as rapid redistribution/metabolism and fast clearance. These unique features might prove to be yet another desirable attribute as a drug for targeting PAR2 (a cell surface receptor), for the selective killing of PAR2-expressing cancer cells through targeted anticancer therapies.
[0145] It is likely that other compounds or biologic agents could be used to cause similar PAR2-dependent killing of proliferating cells. Interestingly, a previous study has implicated Triptolide as a potent inhibitor for PAR2-dependent functions, while our data indicate that Triptonide causes the PAR2-dependent killing by acting as an abnormal activator for PAR2.
[0146] In retrospect, given the widespread expression of Par2 in several cell types across many types of organs/tissues, the finding that a treatment with a PAR2 agonist for the Gαs -AC-cAMP-PKA would be expected to cause the elevation of PKA in all these PAR2-expresing cells. Accordingly, our data have also led to the realization that the up-regulation of the PKA activity in these mouse cells is apparently well tolerated. It is likely that such a feature could be common to many other cells types and could be conserved between mouse and humans. In this regards, it is reasonable to predict that other GPCR receptors, which are expressed in human cancer cells, could be exploited for instigating the effective killing of the cancer cells for therapeutic gains with manageable or no major adverse side effects. Likewise, other components that can be used to up-regulate PKA activities in cancer cells, in a manner similar to that of Triptonide, could potentially be exploited for anticancer benefits.
[0147] In addition, it is highly likely that Triptonide and/or its functional analogues could be combined with other drugs or modalities including immunotherapies to develop new anti-cancer treatment strategies.
[0148] In summary, our studies supported that the sustained elevation of PKA activities through the activation of PAR2 GPCR receptor by Triptonide could be used as a novel strategy for targeted anticancer therapy. We have succeeded in the development and implementation of this strategy, providing the proof of concept for developing personalized anticancer therapies through the use of Triptonide and possibly also other compounds or biologics that have a similar effect on PAR2, either by themselves or in the setting of combinatorial therapies with other agents and/or modalities.
[0149] As the sustained elevation of PKA activity could potentially be achieved in many different ways, elevation of PKA could be exploited as a general anticancer paradigm. In particular, many GPCR receptors are coupled to the Gαs-AC-cAMP-PKA signaling cascade and some are expressed in some human cancers. Although under normal physiological conditions, the activation of individual GPCR receptors by their cognate ligands usually results in the up-regulation of PKA activity in a highly controlled manner, it remains possible that some biased ligands for such GPCR receptors could cause a sustained activation of PKA activity just as the activation of PAR2 by Triptonide does. Accordingly, such biased GPCR ligands could be used to instigate the selective killing of cancer cells for therapeutic gain as well. In this regard, the methods described herein for the detection of the effect of Triptonide at the cell biology level could prove useful in identifying new agents, including functional analogs of Triptonide, that can be used to cause the mitotic catastrophe-inducing effect. Such a functional assay as well as the related methods will have important applications in developing companion diagnostic tests that can be used to guide patient selection in the clinical settings.
[0150] It is understood that, although the inventions have been described in some detail by way of illustration and examples, these inventions are not limited to specific details described in these specific forms. Apparently to those skilled in this field, various equivalent changes and modifications to the technical features involved in the inventions may be practiced without deviating from the spirit of the inventions described herein, and such changes and modifications are within the scope of the inventions.
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