PLATINUM (II) COMPLEXES CONTAINING N-HETEROCYCLIC CARBENE LIGAND AND PINCER LIGANDS, SYNTHESIS, AND THEIR APPLICATIONS IN CANCER TREATMENT
Provided herein is a method of synthesis of Pt(II) complexes, a pharmaceutical composition comprises thereof. Also provided herein are the methods for the treatment and prevention of cancer/tumor in patients in need thereof by the administration of the Pt(II) complexes. Also provided is a method of detecting the Pt(II) complex in a biological system. Also provided is a method of making the Pt(II) complex The Pt(II) complexes possess anticancer activity such as the induction of cell death, inhibition of cellular proliferation, and inhibition of tumor growth in vivo.
Described herein are platinum (II) complexes containing N-heterocyclic carbene ligand, a method of synthesis of the platinum (II) complexes containing N-heterocyclic carbene ligand, methods of treating and preventing cancer or tumor using the platinum (II) complexes containing N-heterocyclic carbene ligand. The platinum (II) complexes has a dual action including cytotoxic to tumor growth and anti-angiogenesis. Also provided is a method of detecting the platinum(II) complexes containing N-heterocyclic carbene ligand by fluorescence microscopy. Also described are therapeutic and prophylactic compositions containing a purified platinum(II) complexes containing N-heterocyclic carbene ligand. In certain embodiments, the methods of treating and preventing cancer or tumor are in combination with other cancer or tumor treatment. In certain embodiments, the cancer or tumor treatment is chemotherapy, radiation therapy, gene therapy, surgery or a combination thereof.
2. BACKGROUNDAs stimulated by the clinical success of cis-diamminedichloroplatinum (cisplatin), a platinum(II) complex, for the treatment of cancers, scientists have paid great attention to the development of metal-based anticancer drugs which target DNA including the cisplatin analogues and some ruthenium(II)-arene complexes [Sadler, P. J. et al. Curr. Opin. Chem. Biol. 2008, 12, 197]. However, severe side effects and the induced drug resistance are commonly encountered and thus subsequently have hampered the wider applications of these DNA binding agents.
Cisplatin and its derivatives are widely used as chemotherapeutic agents for treating cancer. Yet, most of them fail in combating with metastatic cancer, which is a big problem found in cancer treatment. In view of this, it is important to develop new cytotoxic agents that can at the same time regulate tumor microenvironment which is important for governing tumor progression, growth, angiogenesis and metastasis.
3. SUMMARYDescribed herein are Pt(II)-NHC—BPI complexes, compositions comprising Pt(II)-NHC—BPI complexes, methods of using the Pt(II)-NHC—BPI complexes in cancer/tumor treatment, a method of synthesis of Pt(II)-NHC—BPI complexes, and a method of detecting the Pt(II)-NHC—BPI complexes. In one embodiment, the method of treatment and prevention is in combination with one or more cancer/tumor therapies.
Described herein is a Pt(II) complex comprising a Pt(II)-NHC ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI) ligand, wherein the Pt(II)-NHC ligand is perpendicular to the BPI ligand.
In one embodiment, the NHC ligand and the BPI ligand have a bond angle of about 90°. In one embodiment, the Pt(II) complex has anti-tumor or anti-angiogenic properties.
Described herein is a method of making a Pt(II) complex, comprising reacting [Pt(BPI)Cl] with corresponding imidazolium salt in the presence of a base to form the Pt(II) complex.
In one embodiment, provided herein is a method for cancer or tumor treatment and prevention resulting in induction of cell death, inhibition of cellular proliferation, inhibition of angiogenesis, or inhibition of in vivo tumor growth. In one embodiment, provided herein is a method comprising administering to a subject in need thereof a composition comprising an effective amount of a Pt(II)-NHC—BPI complex. In one embodiment, the Pt(II)-NHC—BPI complexes is a platinum(II) complex described herein represented by the structural formulae of I, derivatives thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof,
wherein R1 is —CH3, —C4H9, -nC6H13, —CH3, or —CH2Ph, and wherein R2 is —CH3, —C4H9, -nC6H13, —C8H17, —C16H33, or —CH2Ph. In another embodiment, provided herein is a method for detecting an effective amount of the Pt(II)-NHC—BPI complexes, depending on the fluorescence changes at proper wavelength. The Pt(II)-NHC—BPI complex is a platinum(II) complex described herein can be represented by the structural formula of I, or an acceptable salt thereof,
wherein R1 is —CH3, —C4H9, -nC6H13, —CH3, or —CH2Ph, and
wherein R2 is —CH3, —C4H9, -nC6H13, —C8H17, —C16H33, or —CH2Ph.
Described herein is a Pt(II) complex which comprises a Pt(II)-NHC ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI) ligand, wherein the Pt(II)-NHC ligand is perpendicular to the BPI ligand, and having the following formula:
wherein R1 is —CH3, —C4H9, -nC6H13, —CH3, or —CH2Ph, and
wherein R2 is —CH3, —C4H9, -nC6H13, —C8H17, —C16H33, or —CH2Ph. In one embodiment, R1 is C4H9 and R2 is C4H9.
Described herein is a composition comprising a Pt(II)-NHC—BPI complex, which comprises a Pt(II)-NHC ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI) ligand, wherein the Pt(II)-NHC ligand is perpendicular to the BPI ligand.
In one embodiment, the NHC ligand and the BPI ligand have a bond angle of about 90°. In one embodiment, the Pt(II) complex comprises anti-tumor and/or anti-angiogenic properties.
Described herein is a method for treatment of tumor or cancer in a subject comprising administering to a subject in need thereof an effective amount of a composition comprising a Pt(II) complex that regulates uPA/uPAR-mediated angiogenic pathway or VEGF-induced angiogenic pathway. In one embodiment, provided herein is a method wherein the tumor is one or more of hepatocellular carcinoma, cervical epithelioid carcinoma, lung carcinoma, breast cancer, colon cancer, melanoma or nasopharyngeal carcinoma. In one embodiment, the effective amount is about 0.1 mg/kg to 50 mg/kg. In one embodiment, the effective amount is about 2.5-5 mg/kg.
The Pt(II)-NHC—BPI complexes are stable in air and aqueous solutions like phosphate-buffered saline (PBS) conditions. The anti-cancer active Pt(II)-NHC—BPI complexes is also accompanied with the release of highly fluorescent ligand. The Pt(II)-NHC—BPI complexes display similar anti-cancer or anti-tumor activity. They can be detected via the fluorescent ligand which makes them to be excellent bio-probes and for prevalent biological applications.
Described herein is a method to detect the Pt(II) complex in a subject, said method comprises administering an effective amount of Pt(II) complex to the subject and detect the Pt(II) complex using fluorescent detection. In one embodiment, the effective amount of Pt(II) complex is 1 μM-500 μM.
Provided herein is a new series of dual cytotoxic and anti-angiogenic platinum(II) complexes with N-heterocylic carbene (NHC) and 1,3-bis(2-pyridylimino)isoindoline (BPI) ligands. The NHC ligand is found to be perpendicular to the plane of BPI ligand, as revealed by X-ray crystallography, thus allowing these platinum(II) complexes to target other biomolecules rather than DNA only. The introduction of NHC ligand, which is a strong σ-donor, also renders the complexes strong luminescence in aqueous solution and live cells, and hence their subcellular localization in endoplasmic reticulum (ER) can be identified by fluorescence microscopy. With their accumulation in ER, they are found to induce ER stress and subsequent apoptotic cell death, accounting for their potent cytotoxicity toward cancer cells.
5.1 Pt(II) ComplexesPlatinum(II) compounds as exemplified by cisplatin and its derivatives have been widely used in the treatment of cancer.[1,2] Their mechanism of action is mainly through covalent crosslinking onto DNA, leading to cancer cell apoptosis or cell cycle arrest.[3,4] Since DNA is the primary molecular target, cancer cells with changes in repair of DNA lesion, such as enhanced nucleotide excision repair or deficiency in mismatch repair, are found to show resistance to these platinum drugs.[5] Moreover, these platinum drugs generally give rise to severe toxic side effects, probably due to the fact that DNA is not a specific biomolecule in cancer cells.[5] As a result, there are continuing efforts on searching metal complexes with new working mechanisms.
It should be advantageous for developing anticancer drugs to target tumor microenvironment. Tumor microenvironment is complex and dynamic, and is regulated by a number of mediators and signaling transduction pathways that govern tumor progression including tumor initiation, growth, angiogenesis and metastasis.[6,7] For example, tumor cells developing their microenvironment by secretion of vascular endothelial growth factor (VEGF) or cytokines to promote abnormal tumor neovasculature formation, which provides nutrients for further tumor growth and metastasis.[8,9] In addition, binding of urokinase plasminogen activator (uPA) to uPA receptor (uPAR) in tumor microenvironment can trigger activation of metalloproteinases (MMPs) to degrade the components of surrounding extracellular matrix (ECM),[10] and hence contributes to tumor cell metastasis. Together with the fact that over 90% of cancer deaths today are due to metastasis formation,[11] regulations of tumor microenvironment including inhibition of tumor growth, metastasis and VEGF-induced angiogenesis, have been considered as effective means in combating tumor progression.[12,13]
A number of metal complexes have been reported to target tumor microenvironment by acting as angiogenesis inhibitors.[14-16] Notably, a ruthenium(III) complex, NAMI-A, was found to be non-cytotoxic toward solid tumor but show promising antitumor activities by inhibition of tumor metastasis and angiogenesis.[14] In addition, platinum complexes showing dual cytotoxic and anti-angiogenic properties have also been explored,[17-19] and they should show improved anticancer efficacy through decreasing acquired-drug resistance and systemic toxicities, as compared to that of a cytotoxic or an anti-angiogenic agent alone.[20,21] However, none of them exhibited promising in vivo antitumor and anti-angiogenic activities.
Disclosed herein are platinum(II) complexes that exhibit dual cytotoxic and anti-angiogenic properties, and are luminescent in vitro so that real-time monitoring of therapeutic progress would be feasible. Provided is an out-of-plane ancillary ligand to the platinum(II) center for targeting biomolecules other than DNA in order to achieve dual cytotoxic and anti-angiogenic properties. NHC is a strong G-donor and can increase the energy level of non-emissive ligand-field (LF) state, rendering platinum(II) complexes strongly luminescent,[24] and this strong luminescence feature can help to elucidate mechanism of anticancer actions of the complexes by fluorescence microscopy. Provided is a new series of platinum(II) complexes containing NHC ligands and 1,3-bis(2-pyridylimino)isoindoline (BPI) which has two accessible nitrogen atoms ([Pt(BPI)(NHC)](OTf);
Provided herein is a Pt(II)-NHC—BPI complex. In one embodiment, the Pt(II)-NHC—BPI complexes is a platinum(II) complex described herein represented by the structural formulae of I, derivatives thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof,
wherein R1 is —CH3, —C4H9, -nC6H13, —CH3, or —CH2Ph, and wherein R2 is —CH3, —C4H9, -nC6H13, —C8H17, —C16H33, or —CH2Ph. Described herein is a Pt(II) complex which comprises a Pt(II)-NHC ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI) ligand, wherein the Pt(II)-NHC ligand is perpendicular to the BPI ligand, and having the following formula:
wherein R1 is —CH3, —C4H9, -nC6H13, —CH3, or —CH2Ph, and
wherein R2 is —CH3, —C4H9, -nC6H13, —C8H17, —C16H33, or —CH2Ph. In one embodiment, R1 is C4H9 and R2 is C4H9.
Also disclosed are the synthesis of platinum(II) [Pt(II)] complexes containing N-heterocyclic carbene ligand (NHC) and BPI ligand, composition comprising platinum(II) [Pt(II)] complexes containing N-heterocyclic carbene ligand (NHC) and BPI ligand, methods of treating and preventing cancer or tumor in a subject, and a method of detecting the Pt(II) complex. Disclosed herein is a method of treating or preventing cancer/tumor comprising administering a pharmaceutical composition comprising at least one of the Pt(II)-NHC—BPI complexes in an effective amount for anti-cancer or anti-tumor activity. In certain embodiments, anti-cancer or anti-tumor activities includes, but are not limited to, the induction of cell death, inhibition of cellular proliferation, inhibition of angiogenesis, and inhibition of in vivo tumor growth. Provided herein is a method of detecting the Pt(II)-NHC—BPI complexes. In an embodiment, a signal is detected depending on fluorescence changes at proper wavelength. As provided herein, in one embodiment, Pt(II)-NHC—BPI complexes refer to a molecule of a platinum(II) ion connected to a N-heterocyclic carbene ligand and a BPI ligand. In one embodiment, platinum(II) [Pt(II)] complexes containing N-heterocyclic carbene ligand (NHC) is represented by structural formula I, derivatives thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
As used herein, the phrase “acceptable salt,” as used herein, includes salts formed from the charged Pt(II)-NHC—BPI complex and counter-anion(s).
As used herein, the phrase “counter-anion” refers to an ion associated with a positively charged Pt(II)-NHC—BPI complex. Non-limiting examples of counter-ions include halogens such as fluoride (F−), chloride (Cl−), bromide (Br−), iodide (I−); sulfate (SO42−); phosphate (PO43−); trifluoromethanesulfonate (triflate, -OTf or CF3SO3−); acetate (−OAc); nitrate (NO3−); perchlorate (ClO4−); hexafluorophosphate (PF6−) and hexafluoroacetylacetonate ([CF3C(O)CHC(O)CF3]−).
In one embodiment, the invention relates to the synthesis of novel platinum(II) [Pt(II)] bearing N-heterocyclic carbene ligand and BPI ligand.
In another embodiment, the invention relates to a pharmaceutical composition for cancer treatment by inhibition of the proliferation of cancer cells in vitro comprising an effective amount of one or more of the Pt(II)-NHC—BPI complexes.
In another embodiment, the invention relates to a pharmaceutical composition for cancer treatment by the inhibition of tumor growth in vivo comprising an effective amount of one or more of the Pt(II)-NHC—BPI complexes.
In another embodiment, the invention relates to fluorescent detecting compounds, and the application in cellular imaging, comprising an effective amount of a Pt(II)-NHC—BPI complex.
The Pt(II)-NHC—BPI complexes of this invention can be represented by one or more of structural formula I, derivatives thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
In one embodiment, the invention relates to a pharmaceutical composition for treating or preventing cancer/tumor. In certain embodiments, the treatment and prevention comprises induction of cell death, inhibition of cellular proliferation, inhibition of angiogenesis, and the inhibition of tumor growth in vivo. In one embodiment, the method comprises administering an effective amount of the Pt(II)-NHC—BPI complexes to a subject. In one embodiment, the method comprises detecting the Pt(II) complex is a subject comprising administering an effective amount of the Pt(II)-NHC—BPI complexes. In one embodiment, the Pt(II) complex is detected by fluorescence changes at proper wavelength. The Pt(II)-NHC—BPI complex has a formula I, derivatives thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof,
wherein R1 is —CH3, —C4H9, -nC6H13, —CH3, or —CH2Ph, and
wherein R2 is —CH3, —C4H9, -nC6H13, —C8H17, —C16H33, or —CH2Ph.
5.2 Human Treatment 5.2.1 FormulationsThe platinum(II) complexes provided herein can be administered to a patient in the conventional form of preparations, such as injections and suspensions. Suitable formulations can be prepared by methods commonly employed using conventional, organic or inorganic additives, such as an excipient selected from fillers or diluents, binders, disintegrants, lubricants, flavoring agents, preservatives, stabilizers, suspending agents, dispersing agents, surfactants, antioxidants or solubilizers.
Excipients that may be selected are known to those skilled in the art and include, but are not limited to fillers or diluents (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate and the like), a binder (e.g., cellulose, carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethyleneglycol or starch and the like), a disintegrants (e.g., sodium starch glycolate, croscarmellose sodium and the like), a lubricant (e.g., magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl sulfate and the like), a flavoring agent (e.g., citric acid, or menthol and the like), a preservative (e.g., sodium benzoate, sodium bisulfite, methylparaben or propylparaben and the like), a stabilizer (e.g., citric acid, sodium citrate or acetic acid and the like), a suspending agent (e.g., methylcellulose, polyvinyl pyrrolidone or aluminum stearate and the like), a dispersing agent (e.g., hydroxypropylmethylcellulose and the like), surfactants (e.g., sodium lauryl sulfate, polaxamer, polysorbates and the like), antioxidants (e.g., ethylene diamine tetraacetic acid (EDTA), butylated hydroxyl toluene (BHT) and the like) and solubilizers (e.g., polyethylene glycols, SOLUTOL®, GELUCIRE® and the like). The effective amount of the platinum(II) complexes provided herein in the pharmaceutical composition may be at a level that will exercise the desired effect.
In another embodiment, provided herein are compositions comprising an effective amount of platinum(II) complexes provided herein and a pharmaceutically acceptable carrier or vehicle, wherein a pharmaceutically acceptable carrier or vehicle can comprise an excipient, diluent, or a mixture thereof. In one embodiment, the composition is a pharmaceutical composition.
Compositions can be formulated to contain a daily dose, or a convenient fraction of a daily dose, in a dosage unit. In general, the composition is prepared according to known methods in pharmaceutical chemistry. Capsules can be prepared by mixing the platinum(II) complexes provided herein with a suitable carrier or diluent and filling the proper amount of the mixture in capsules.
5.3 Method of UseSolid tumor cancers that can be treated by the methods provided herein include, but are not limited to, sarcomas, carcinomas, and lymphomas. In specific embodiments, cancers that can be treated in accordance with the methods described include, but are not limited to, cancer of the breast, liver, neuroblastoma, head, neck, eye, mouth, throat, esophagus, esophagus, chest, bone, lung, kidney, colon, rectum or other gastrointestinal tract organs, stomach, spleen, skeletal muscle, subcutaneous tissue, prostate, breast, ovaries, testicles or other reproductive organs, skin, thyroid, blood, lymph nodes, kidney, liver, pancreas, and brain or central nervous system.
In particular embodiments, the methods for treating cancer provided herein inhibit, reduce, diminish, arrest, or stabilize a tumor associated with the cancer. In other embodiments, the methods for treating cancer provided herein inhibit, reduce, diminish, arrest, or stabilize the blood flow, metabolism, or edema in a tumor associated with the cancer or one or more symptoms thereof. In specific embodiments, the methods for treating cancer provided herein cause the regression of a tumor, tumor blood flow, tumor metabolism, or peritumor edema, and/or one or more symptoms associated with the cancer. In other embodiments, the methods for treating cancer provided herein maintain the size of the tumor so that it does not increase, or so that it increases by less than the increase of a tumor after administration of a standard therapy as measured by conventional methods available to one of skill in the art, such as digital rectal exam, ultrasound (e.g., transrectal ultrasound), CT Scan, MRI, dynamic contrast-enhanced MRI, or PET Scan. In specific embodiments, the methods for treating cancer provided herein decrease tumor size. In certain embodiments, the methods for treating cancer provided herein reduce the formation of a tumor. In certain embodiments, the methods for treating cancer provided herein eradicate, remove, or control primary, regional and/or metastatic tumors associated with the cancer. In some embodiments, the methods for treating cancer provided herein decrease the number or size of metastases associated with the cancer.
In certain embodiments, the methods for treating cancer provided herein reduce the tumor size (e.g., volume or diameter) in a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, 99%, or 100%, relative to tumor size (e.g., volume or diameter) prior to administration of platinum(II) complexes as assessed by methods well known in the art, e.g., CT Scan, MRI, DCE-MRI, or PET Scan. In particular embodiments, the methods for treating cancer provided herein reduce the tumor volume or tumor size (e.g., diameter) in a subject by an amount in the range of about 5% to 20%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 30% to 100%, or any range in between, relative to tumor size (e.g., diameter) in a subject prior to administration of platinum(II) complexes as assessed by methods well known in the art, e.g., CT Scan, MRI, DCE-MRI, or PET Scan.
In certain embodiments, the methods for treating cancer provided herein reduce the tumor perfusion in a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, 99%, or 100%, relative to tumor perfusion prior to administration of platinum(II) complexes as assessed by methods well known in the art, e.g., MRI, DCE-MRI, or PET Scan. In particular embodiments, the methods for treating cancer provided herein reduce the tumor perfusion in a subject by an amount in the range of about 5% to 20%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 30% to 100%, or any range in between, relative to tumor perfusion prior to administration of platinum(II) complexes, as assessed by methods well known in the art, e.g., MRI, DCE-MRI, or PET Scan.
In particular aspects, the methods for treating cancer provided herein inhibit or decrease tumor metabolism in a subject as assessed by methods well known in the art, e.g., PET scanning. In specific embodiments, the methods for treating cancer provided herein inhibit or decrease tumor metabolism in a subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 80%, 85%, 90%, 95%, or 100%, relative to tumor metabolism prior to administration of platinum(II) complexes, as assessed by methods well known in the art, e.g., PET scanning. In particular embodiments, the methods for treating cancer provided herein inhibit or decrease tumor metabolism in a subject in the range of about 5% to 20%, 10% to 20%, 10% to 30%, 15% to 40%, 15% to 50%, 20% to 30%, 20% to 40%, 20% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 99%, 30% to 100%, or any range in between, relative to tumor metabolism prior to administration of platinum(II) complexes, as assessed by methods well known in the art, e.g., PET scan.
5.4 Patient PopulationIn some embodiments, a subject treated for cancer in accordance with the methods provided herein is a human who has or is diagnosed with cancer. In other embodiments, a subject treated for cancer in accordance with the methods provided herein is a human predisposed or susceptible to cancer. In some embodiments, a subject treated for cancer in accordance with the methods provided herein is a human at risk of developing cancer.
In one embodiment, a subject treated for cancer in accordance with the methods provided herein is a human infant. In another embodiment, a subject treated for cancer in accordance with the methods provided herein is a human toddler. In another embodiment, a subject treated for cancer in accordance with the methods provided herein is a human child. In another embodiment, a subject treated for cancer in accordance with the methods provided herein is a human adult. In another embodiment, a subject treated for cancer in accordance with the methods provided herein is a middle-aged human. In another embodiment, a subject treated for cancer in accordance with the methods provided herein is an elderly human.
In certain embodiments, a subject treated for cancer in accordance with the methods provided herein has a cancer that metastasized to other areas of the body, such as the bones, lung and liver. In certain embodiments, a subject treated for cancer in accordance with the methods provided herein is in remission from the cancer. In some embodiments, a subject treated for cancer in accordance with the methods provided herein that has a recurrence of the cancer. In certain embodiments, a subject treated in accordance with the methods provided herein is experiencing recurrence of one or more tumors associated with cancer.
In certain embodiments, a subject treated for cancer in accordance with the methods provided herein is a human that is about 1 to about 5 years old, about 5 to 10 years old, about 10 to about 18 years old, about 18 to about 30 years old, about 25 to about 35 years old, about 35 to about 45 years old, about 40 to about 55 years old, about 50 to about 65 years old, about 60 to about 75 years old, about 70 to about 85 years old, about 80 to about 90 years old, about 90 to about 95 years old or about 95 to about 100 years old, or any age in between. In a specific embodiment, a subject treated for cancer in accordance with the methods provided herein is a human that is 18 years old or older. In a particular embodiment, a subject treated for cancer in accordance with the methods provided herein is a human child that is between the age of 1 year old to 18 years old. In a certain embodiment, a subject treated for cancer in accordance with the methods provided herein is a human that is between the age of 12 years old and 18 years old. In a certain embodiment, the subject is a male human. In another embodiment, the subject is a female human. In one embodiment, the subject is a female human that is not pregnant or is not breastfeeding. In one embodiment, the subject is a female that is pregnant or will/might become pregnant, or is breast feeding.
In some embodiments, a subject treated for cancer in accordance with the methods provided herein is administered platinum(II) complexes or a pharmaceutical composition thereof, or a combination therapy before any adverse effects or intolerance to therapies other than the platinum(II) complexes develops. In some embodiments, a subject treated for cancer in accordance with the methods provided herein is a refractory patient. In a certain embodiment, a refractory patient is a patient refractory to a standard therapy (e.g., surgery, radiation, anti-androgen therapy and/or drug therapy such as chemotherapy). In certain embodiments, a patient with cancer is refractory to a therapy when the cancer has not significantly been eradicated and/or the one or more symptoms have not been significantly alleviated. The determination of whether a patient is refractory can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of a treatment of cancer, using art-accepted meanings of “refractory” in such a context. In various embodiments, a patient with cancer is refractory when one or more tumors associated with cancer, have not decreased or have increased. In various embodiments, a patient with cancer is refractory when one or more tumors metastasize and/or spread to another organ.
In some embodiments, a subject treated for cancer accordance with the methods provided herein is a human that has proven refractory to therapies other than treatment with platinum(II) complexes, but is no longer on these therapies. In certain embodiments, a subject treated for cancer in accordance with the methods provided herein is a human already receiving one or more conventional anti-cancer therapies, such as surgery, drug therapy such as chemotherapy, anti-androgen therapy or radiation. Among these patients are refractory patients, patients who are too young for conventional therapies, and patients with recurring tumors despite treatment with existing therapies.
5.5 DosageIn one aspect, a method for treating cancer presented herein involves the administration of a unit dosage of platinum(II) complexes or a pharmaceutical composition thereof. The dosage may be administered as often as determined effective (e.g., once, twice or three times per day, every other day, once or twice per week, biweekly or monthly). In certain embodiments, a method for treating cancer presented herein involves the administration to a subject in need thereof of a unit dose of platinum(II) complexes that can be determined by one skilled in the art.
In some embodiments, a unit dose of platinum(II) complexes or a pharmaceutical composition thereof is administered to a subject once per day, twice per day, three times per day; once, twice or three times every other day (i.e., on alternate days); once, twice or three times every two days; once, twice or three times every three days; once, twice or three times every four days; once, twice or three times every five days; once, twice, or three times once a week, biweekly or monthly, and the dosage may be administered orally.
5.6 Combination TherapyPresented herein are combination therapies for the treatment of cancer which involve the administration of platinum(II) complexes in combination with one or more additional therapies to a subject in need thereof. In a specific embodiment, presented herein are combination therapies for the treatment of cancer which involve the administration of an effective amount of platinum(II) complexes in combination with an effective amount of another therapy to a subject in need thereof.
As used herein, the term “in combination,” refers, in the context of the administration of platinum(II) complexes, to the administration of platinum(II) complexes prior to, concurrently with, or subsequent to the administration of one or more additional therapies (e.g., agents, surgery, or radiation) for use in treating cancer. The use of the term “in combination” does not restrict the order in which platinum(II) complexes and one or more additional therapies are administered to a subject. In specific embodiments, the interval of time between the administration of platinum(II) complexes and the administration of one or more additional therapies may be about 1-5 minutes, 1-30 minutes, 30 minutes to 60 minutes, 1 hour, 1-2 hours, 2-6 hours, 2-12 hours, 12-24 hours, 1-2 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 26 weeks, 52 weeks, 11-15 weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks, 40-50 weeks, 1 month, 2 months, 3 months, 4 months 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, or any period of time in between. In certain embodiments, platinum(II) complexes and one or more additional therapies are administered less than 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, 2 months, 3 months, 6 months, 1 year, 2 years, or 5 years apart.
In some embodiments, the combination therapies provided herein involve administering platinum(II) complexes daily, and administering one or more additional therapies once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every month, once every 2 months (e.g., approximately 8 weeks), once every 3 months (e.g., approximately 12 weeks), or once every 4 months (e.g., approximately 16 weeks). In certain embodiments, platinum(II) complexes and one or more additional therapies are cyclically administered to a subject. Cycling therapy involves the administration of platinum(II) complexes for a period of time, followed by the administration of one or more additional therapies for a period of time, and repeating this sequential administration. In certain embodiments, cycling therapy may also include a period of rest where platinum(II) complexes or the additional therapy is not administered for a period of time (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 20 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years). In an embodiment, the number of cycles administered is from 1 to 12 cycles, from 2 to 10 cycles, or from 2 to 8 cycles.
In some embodiments, the methods for treating cancer provided herein comprise administering platinum(II) complexes as a single agent for a period of time prior to administering the platinum(II) complexes in combination with an additional therapy. In certain embodiments, the methods for treating cancer provided herein comprise administering an additional therapy alone for a period of time prior to administering platinum(II) complexes in combination with the additional therapy.
In some embodiments, the administration of platinum(II) complexes and one or more additional therapies in accordance with the methods presented herein have an additive effect relative the administration of platinum(II) complexes or said one or more additional therapies alone. In some embodiments, the administration of platinum(II) complexes and one or more additional therapies in accordance with the methods presented herein have a synergistic effect relative to the administration of the Compound or said one or more additional therapies alone.
As used herein, the term “synergistic,” refers to the effect of the administration of platinum(II) complexes in combination with one or more additional therapies (e.g., agents), which combination is more effective than the additive effects of any two or more single therapies (e.g., agents). In a specific embodiment, a synergistic effect of a combination therapy permits the use of lower dosages (e.g., sub-optimal doses) of platinum(II) complexes or an additional therapy and/or less frequent administration of platinum(II) complexes or an additional therapy to a subject. In certain embodiments, the ability to utilize lower dosages of platinum(II) complexes or of an additional therapy and/or to administer platinum(II) complexes or said additional therapy less frequently reduces the toxicity associated with the administration of platinum(II) complexes or of said additional therapy, respectively, to a subject without reducing the efficacy of platinum(II) complexes or of said additional therapy, respectively, in the treatment of cancer. In some embodiments, a synergistic effect results in improved efficacy of platinum(II) complexes and each of said additional therapies in treating cancer. In some embodiments, a synergistic effect of a combination of platinum(II) complexes and one or more additional therapies avoids or reduces adverse or unwanted side effects associated with the use of any single therapy.
The combination of platinum(II) complexes and one or more additional therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, platinum(II) complexes and one or more additional therapies can be administered concurrently to a subject in separate pharmaceutical compositions. Platinum(II) complexes and one or more additional therapies can be administered sequentially to a subject in separate pharmaceutical compositions. Platinum(II) complexes and one or more additional therapies may also be administered to a subject by the same or different routes of administration.
The combination therapies provided herein involve administering to a subject to in need thereof platinum(II) complexes in combination with conventional, or known, therapies for treating cancer. Other therapies for cancer or a condition associated therewith are aimed at controlling or relieving one or more symptoms. Accordingly, in some embodiments, the combination therapies provided herein involve administering to a subject to in need thereof a pain reliever, or other therapies aimed at alleviating or controlling one or more symptoms associated with or a condition associated therewith.
Specific examples of anti-cancer agents that may be used in combination with platinum(II) complexes include: a hormonal agent (e.g., aromatase inhibitor, selective estrogen receptor modulator (SERM), and estrogen receptor antagonist), chemotherapeutic agent (e.g., microtubule dissembly blocker, antimetabolite, topisomerase inhibitor, and DNA crosslinker or damaging agent), anti-angiogenic agent (e.g., VEGF antagonist, receptor antagonist, integrin antagonist, vascular targeting agent (VTA)/vascular disrupting agent (VDA)), radiation therapy, and conventional surgery.
Non-limiting examples of hormonal agents that may be used in combination with platinum(II) complexes include aromatase inhibitors, SERMs, and estrogen receptor antagonists. Hormonal agents that are aromatase inhibitors may be steroidal or nonsteroidal. Non-limiting examples of nonsteroidal hormonal agents include letrozole, anastrozole, aminoglutethimide, fadrozole, and vorozole. Non-limiting examples of steroidal hormonal agents include aromasin (exemestane), formestane, and testolactone. Non-limiting examples of hormonal agents that are SERMs include tamoxifen (branded/marketed as Nolvadex®), afimoxifene, arzoxifene, bazedoxifene, clomifene, femarelle, lasofoxifene, ormeloxifene, raloxifene, and toremifene. Non-limiting examples of hormonal agents that are estrogen receptor antagonists include fulvestrant. Other hormonal agents include but are not limited to abiraterone and lonaprisan.
Non-limiting examples of chemotherapeutic agents that may be used in combination with platinum(II) complexes include microtubule disasssembly blocker, antimetabolite, topisomerase inhibitor, and DNA crosslinker or damaging agent. Chemotherapeutic agents that are microtubule dissemby blockers include, but are not limited to, taxenes (e.g., paclitaxel (branded/marketed as TAXOL®), docetaxel, abraxane, larotaxel, ortataxel, and tesetaxel); epothilones (e.g., ixabepilone); and vinca alkaloids (e.g., vinorelbine, vinblastine, vindesine, and vincristine (branded/marketed as ONCOVIN®)).
Chemotherapeutic agents that are antimetabolites include, but are not limited to, folate anitmetabolites (e.g., methotrexate, aminopterin, pemetrexed, raltitrexed); purine antimetabolites (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, pentostatin, thioguanine); pyrimidine antimetabolites (e.g., 5-fluorouracil, capcitabine, gemcitabine (GEMZAR®), cytarabine, decitabine, floxuridine, tegafur); and deoxyribonucleotide antimetabolites (e.g., hydroxyurea).
Chemotherapeutic agents that are topoisomerase inhibitors include, but are not limited to, class I (camptotheca) topoisomerase inhibitors (e.g., topotecan (branded/marketed as HYCAMTIN®) irinotecan, rubitecan, and belotecan); class II (podophyllum) topoisomerase inhibitors (e.g., etoposide or VP-16, and teniposide); anthracyclines (e.g., doxorubicin, epirubicin, Doxil, aclarubicin, amrubicin, daunorubicin, idarubicin, pirarubicin, valrubicin, and zorubicin); and anthracenediones (e.g., mitoxantrone, and pixantrone).
Chemotherapeutic agents that are DNA crosslinkers (or DNA damaging agents) include, but are not limited to, alkylating agents (e.g., cyclophosphamide, mechlorethamine, ifosfamide (branded/marketed as IFEX®), trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, carmustine (branded/marketed as BiCNU®), lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, busulfan, mannosulfan, treosulfan, carboquone, N,N′N′-triethylenethiophosphoramide, triaziquone, triethylenemelamine); alkylating-like agents (e.g., carboplatin (branded/marketed as PARAPLATIN®), cisplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, satraplatin, picoplatin); nonclassical DNA crosslinkers (e.g., procarbazine, dacarbazine, temozolomide (branded/marketed as TEMODAR®), altretamine, mitobronitol); and intercalating agents (e.g., actinomycin, bleomycin, mitomycin, and plicamycin).
Non-limiting examples of other therapies that may be administered to a subject in combination with platinum(II) complexes include:
(1) a statin such as lovostatin (e.g., branded/marketed as MEVACOR®);
(2) an mTOR inhibitor such as sirolimus which is also known as Rapamycin (e.g., branded/marketed as RAPAMUNE®), temsirolimus (e.g., branded/marketed as TORISEL®), evorolimus (e.g., branded/marketed as AFINITOR®), and deforolimus;
(3) a farnesyltransferase inhibitor agent such as tipifarnib;
(4) an antifibrotic agent such as pirfenidone;
(5) a pegylated interferon such as PEG-interferon alfa-2b;
(6) a CNS stimulant such as methylphenidate (branded/marketed as RITALIN®);
(7) a HER-2 antagonist such as anti-HER-2 antibody (e.g., trastuzumab) and kinase inhibitor (e.g., lapatinib);
(8) an IGF-1 antagonist such as an anti-IGF-1 antibody (e.g., AVE1642 and IMC-A 1) or an IGF-1 kinase inhibitor;
(9) EGFR/HER-1 antagonist such as an anti-EGFR antibody (e.g., cetuximab, panitumamab) or EGFR kinase inhibitor (e.g., erlotinib; gefitinib);
(10) SRC antagonist such as bosutinib;
(11) cyclin dependent kinase (CDK) inhibitor such as seliciclib;
(12) Janus kinase 2 inhibitor such as lestaurtinib;
(13) proteasome inhibitor such as bortezomib;
(14) phosphodiesterase inhibitor such as anagrelide;
(15) inosine monophosphate dehydrogenase inhibitor such as tiazofurine;
(16) lipoxygenase inhibitor such as masoprocol;
(17) endothelin antagonist;
(18) retinoid receptor antagonist such as tretinoin or alitretinoin;
(19) immune modulator such as lenalidomide, pomalidomide, or thalidomide;
(20) kinase (e.g., tyrosine kinase) inhibitor such as imatinib, dasatinib, erlotinib, nilotinib, gefitinib, sorafenib, sunitinib, lapatinib, or TG100801;
(21) non-steroidal anti-inflammatory agent such as celecoxib (branded/marketed as CELEBREX®);
(22) human granulocyte colony-stimulating factor (G-CSF) such as filgrastim (branded/marketed as NEUPOGEN®);
(23) folinic acid or leucovorin calcium;
(24) integrin antagonist such as an integrin α5β1-antagonist (e.g., JSM6427);
(25) nuclear factor kappa beta (NF-κβ) antagonist such as OT-551, which is also an anti-oxidant;
(26) hedgehog inhibitor such as CUR61414, cyclopamine, GDC-0449, and anti-hedgehog antibody;
(27) histone deacetylase (HDAC) inhibitor such as SAHA (also known as vorinostat (branded/marketed as ZOLINZA)), PCI-24781, SB939, CHR-3996, CRA-024781, ITF2357, JNJ-26481585, or PCI-24781;
(28) retinoid such as isotretinoin (e.g., branded/marketed as ACCUTANE®);
(29) hepatocyte growth factor/scatter factor (HGF/SF) antagonist such as HGF/SF monoclonal antibody (e.g., AMG 102);
(30) synthetic chemical such as antineoplaston;
(31) anti-diabetic such as rosaiglitazone (e.g., branded/marketed as AVANDIA®);
(32) antimalarial and amebicidal drug such as chloroquine (e.g., branded/marketed as ARALEN®);
(33) synthetic bradykinin such as RMP-7;
(34) platelet-derived growth factor receptor inhibitor such as SU-101;
(35) receptor tyrosine kinase inhibitorsof Flk-1/KDR/VEGFR2, FGFR1 and PDGFR beta such as SU5416 and SU6668;
(36) anti-inflammatory agent such as sulfasalazine (e.g., branded/marketed as AZULFIDINE®); and
(37) TGF-beta antisense therapy.
6 EXAMPLES Example 6.1: Preparation and Characterization of the NHC ComplexesThe following examples illustrate the synthesis and characterization of the platinum(II) complexes.
[Pt(BPI)(NHC)](OTf) with different alkyl chains and aromatic groups on the NHC ligands were prepared by refluxing [Pt(BPI)Cl][25] with corresponding imidazolium salt in the presence of base (
The UV-visible absorption data and spectra of 1a-1j were depicted in Table 2 and
As compared with the chloro-precursor complex [Pt(BPI)Cl] (1i), both the absorption and emission spectra of platinum(II) complexes with NHC ligands (1b and 1j) displayed distinct blue-shifts (
Interestingly, in one embodiment, proteomics data and in vitro biochemical assays at sub-cytotoxic concentrations reveal that a representative complex, 1b, can regulate uPA/uPAR-mediated and VEGF-induced angiogenic pathways. Ex vivo anti-angiogenic properties of 1b is further demonstrated by chorioallantoic membrane (CAM) assay. More importantly, treatment of nude mice bearing highly metastatic MDA-MB231 xenograft by 1b show significant reduction in tumor volume. Immunohistochemical analysis of tumor tissues from treated mice supports the promising in vivo antitumor as well as anti-angiogenic activities of 1b, while blood biochemistry reveal minimal systemic toxicity found in the treated mice. All of these results indicate that these dual cytotoxic and anti-angiogenic platinum(II) complexes are useful for treating cancer, including non-curable highly metastatic cancer.
The luminescence properties of [Pt(BPI)(NHC)]+ in live cells were also examined. After treating human cervical epithelial carcinoma (HeLa) cells with 1b (5 μM) for 15 min, a strong green luminescence was observed from cytoplasm of the cells (
In view of the enormous success of platinum(II) compounds for anti-cancer treatment,[28] in vitro cytotoxicity of [Pt(BPI)(NHC)]+ towards various cancer cell lines including HeLa, colon carcinoma (HCT116), lung cancer (NCI-H460) and highly invasive triple-negative breast cancer (MDA-MB-231), as well as non-tumorigenic immortalized human hepatocyte (MIHA) were examined. 1a-1h were cytotoxic against the cancer cells with IC50 (dose required to inhibit 50% of cellular growth) ranging from 0.14±0.01 to 18.21±1.52 μM after 72 h treatment. They were found to be more potent in killing most of the cancer cells investigated than cisplatin (11.75±1.36 to 77.19±7.82 μM). Among these platinum(II) complexes, 1b displayed relatively higher cytotoxicity towards NCI-H460 and HCT116 cells than that towards non-tumorigenic MIHA cells (16- and 19-fold difference in IC50 values respectively; Table 4), suggesting its selectivity on killing cancer cells over non-tumorigenic cells. As a result, 1b was selected as a target compound and its anti-cancer properties were further investigated.
Since 1b was found to accumulate in ER domain as revealed by confocal fluorescence microscopy images (
In addition to ER stress, apoptosis-related protein such as poly(ADP-ribose) polymerase (PARP) and caspases 3 and 9 were cleaved in MDA-MB-231 cells treated with 1b for 48 h (
To obtain a holistic insight into the mechanism of action of 1b, proteomic analysis on 1b-treated MDA-MB-231 cells was performed using HPLC-LTQ-Orbitrap MS. A bioinformatics analysis of the proteomic data showed that CHIP (c-terminal Hsp70-interacting protein), c-Kit and Von Hippel-Lindau (VHL) protein pathway were one of the most predominantly modulated pathways in MDA-MB-231 cells treated with 1b (5 μM) for 5 h with high statistical significance (Table 5). Interestingly, these three pathways were related to angiogenic responses of cancer cells. The expression level of CHIP is negatively correlated with VEGFR2 which is an important receptor for initiation of angiogenesis;[32] Von Hippel-Lindau (VHL) protein is capable of suppressing tumor growth through down-regulation of a number of angiogenic factors;[33] c-kit receptor regulate angiogenesis by PI3K/Akt downstream signaling pathway.[34]
In view of the regulation of angiogenesis-related pathways by 1b as identified by proteomic data, anti-angiogenic and anti-metastatic properties of 1b were evaluated. Wound closure assays showed that 1b effectively inhibited migration of MDA-MB-231 cells at sub-cytotoxic concentrations (0.25-1 μM) after a 24 h treatment in a concentration- and time-dependent manner (
To gain better insight into the anti-angiogenic properties of 1b, effects of 1b on uPA/uPAR system were first investigated, as this system has been found to play crucial roles in growth, metastasis and angiogenesis of many solid malignancies, e.g. by activation of MMPs for ECM degradation and triggering downstream intracellular signaling for metastasis.[10,35] Western blotting experiments showed that the expression level of uPA and MMP-9 (examples of MMPs) decreased significantly when MDA-MB-231 cells were treated with increasing concentration of 1b (
In addition to the regulation of proteolysis, 1b was found to significantly suppress phosphorylation of focal adhesion kinase (FAK) at the site of Tyr397, and moderately inhibited levels of phosphorylation of FAK at Tyr925 and Tyr576/577, while exhibited little effect on total protein level of FAK (
The effects of 1b, LY294002 (PI3K inhibitor)[39] and U0126 (ERK inhibitor)[40] on PI3K and ERK signaling pathways were further investigated. LY294002, U0126 or 1b alone displayed insignificant cell growth inhibitory effects (1b is at sub-cytotoxic concentration;
Vascular endothelial growth factor (VEGF) is another critical mediator of angiogenesis and regulates most of the steps in angiogenic cascade, including proliferation, migration and tube formation of endothelial cells.[41,42] Previous studies demonstrated that MMP-9 and uPA were able to facilitate degradation of ECM, leading to release or activation of VEGF, thus promoting tumor growth and angiogenesis.[43-45] Therefore, effect of 1b on secretion level of VEGF in the culture media of MDA-MB-231 cells was investigated by Quantikine® ELISA kit. It was found that VEGF secretion level was reduced by 50% upon treating MDA-MB-231 cells with 1b for 24 h, as compared to the untreated cells (
To further validate anti-angiogenic properties of 1b, effects of 1b on another aggressive cell line, human umbilical vein endothelial cell (HUVEC) line, were studied. HUVEC is a well-established cell line for studying angiogenesis.[46] It was found that exposure of HUVECs to VEGF (50 ng/mL) lead to promoted cell growth, migration, invasion and tube formation (
With the promising dual cytotoxic and anti-angiogenic properties of 1b, in vivo antitumor activity of 1b was investigated. Treatment of nude mice bearing MDA-MB-231 xenografts with 1b through intravenous injection once per two days resulted in a significant growth inhibition by 81 and 64% at concentration of 5 mg/kg and 2.5 mg/kg respectively, as compared to mice treated with solvent control (both with p<0.01;
As a promising candidate for treating cancer in vivo, we further investigate systemic toxicity from 1b. Blood biochemistry of nude mice after treatment with 1b (5 mg/kg, 2.5 mg/kg) showed low systemic toxicity of 1b (
Discussion
Disclosed herein are dual cytotoxic and anti-angiogenic compounds should be new candidates for treating aggressive and highly metastatic cancers which are almost non-curable in this moment. Although platinum(II) compounds are known for their good potency in killing cancer cells, it is quite surprising that anti-angiogenic platinum(II) compounds are less well known and none of the reported platinum(II) compounds demonstrate dual cytotoxic and anti-angiogenic activities in vivo. This can be due to the strong binding of square-planar platinum(II) compounds onto DNA, thus they show less tendency to interact with other biomolecules in tumor microenvironment and hence they are not anti-angiogenic. In order to target other biomolecules, NHC ligands were introduced onto the complexes in order to weaken the interactions of the complexes with DNA by the out-of-plane NHC ligands. Proteomics data and in vitro biochemical assays indicate significant effect of 1b on uPA/uPAR- and VEGF-mediated signaling pathway, further suggesting that 1b can likely interact with biomolecules in tumor microenvironment.
In addition, since NHC is a strong G-donor and can increase the energy level of non-emissive ligand-field (LF) state, the platinum(II) NHC complexes are strongly luminescent and displaying vibronic structured emission spectra upon photo excitation.[24] Due to their luminescence properties, cellular distribution of the complexes can be examined by confocal fluorescence spectroscopy. 1b is found to preferentially accumulate in ER domain of HeLa cells and this information helped us to unravel the mechanism of action of these anticancer complexes: induction of ER stress and subsequent apoptotic cell death. Also, it is conceivable that such strong luminescence can render the metal complexes being both diagnostic and therapeutic agents, i.e. theranostics, and hence real-time monitoring of treatment by the complexes can be feasible.
The promising in vitro and in vivo anticancer activities of this class of platinum(II) complexes are attributable to their dual cytotoxic and anti-angiogenic properties. Their high cytotoxicity against a panel of cancer cells, including highly metastatic MDA-MB-231, can be explained by induction of ER stress, as supported by up regulation of phosphorylated RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK), phosphorylated eukaryotic initiation factor 2α (eIF2α) and C/EBP homologous protein (CHOP) in western blotting analysis. In addition, DNA fragmentation and mitochondria dysfunction are found in the treated cells, as shown by G0/G1 cell-cycle arrest and increase of cell population in sub-G1 phase, and JC1 staining assay respectively. On the other hand, the anti-angiogenic properties of the complexes are first revealed by proteomic analysis on 1b-treated MDA-MB-231 cells, showing that CHIP, c-Kit and VHL protein pathway are the most predominantly modulated pathways and they are all closely related to angiogenic responses of cancer cells. The involvement of 1b in regulation of angiogenesis is further supported by western blotting and in vitro experiments including wound closure assay, transwell invasive and tube formation assay. More importantly, 1b demonstrates excellent ex vivo and in vivo anti-angiogenic properties in chorioallantoic membrane (CAM) assay and immunohistochemical analysis of tumor tissues from treated mice bearing MDA-MB-231 xenograft, respectively. Taken together, treatment of mice bearing highly metastatic MDA-MB-231 xenograft by 1b results in remarkable inhibition of tumor growth, and anti-proliferation and anticancer effects as found in immunohistochemical analysis. Interestingly, there is no significant loss in body weight or death of mice throughout the treatment and blood biochemistry indicates low systemic toxicity of 1b, further demonstrating the potential of this class of [Pt(BPI)(NHC)]+ complexes for treating highly metastatic cancers. The promising dual cytotoxic and anti-angiogenic effects in vivo should be firstly found in platinum(II) compounds, according to the best of our knowledge, and the unique chemical structure of the complexes with out-of-plane NHC ligands for prohibiting strong interactions with DNA can probably accounted for the dual properties.
Dual cytotoxic and anti-angiogenic [Pt(BPI)(NHC)](OTf) were synthesized and characterized. They were cytotoxic against various cancer cells, and this was ascribed to apoptotic cell death induced by ER-stress, mitochondria dysfunction and cell cycle arrest. Proteomic data indicated regulation of angiogenesis by the platinum(II) complexes. Such unique feature allows these complexes to slow down extracellular matrix proteolysis process by inhibiting uPA and MMP expressions. Also, this could inhibit downstream signaling pathways of uPA/uPAR and protect native VEGF from cleavage, thereby accounting for the promising in vitro and ex vivo anti-angiogenic properties of the complexes (
All of the starting materials for synthesis came from commercially available resources such as Sigma Aldrich, Alfa Aesar and Apollo Scientific companies. The solvents used were at least in analytical grade. Elemental analysis was done by Dr. Zong of the Institute of Chemistry at the Chinese Academy of Science located in Beijing. 1H NMR spectra were recorded on Bruker FT-400M Hz or 300M Hz NMR spectrometers with tetramethylsilane as the reference. Fast atom bombardment (FAB) mass spectra were obtained on a Finnigan Mat 95 mass spectrometer. Perkin-Elmer Lambda 19 UV-vis spectrophotometer was used for UV-vis spectral analysis.
Fluorescence images were taken using Carl Zeiss LSM 510 Meta/Axiocam confocal microscopy. For MTT and protein assays, the absorbance was quantified by using a Perkin Elmer Fusion Reader (Packard BioScience Company).
The BPI (1,3-Bis(2-pyridylimino)isoindoline), benz(f)BPI (3-Bis(2-pyridylimino)benz(f)isoindoline) and Pt(BPI)Cl were synthesized according to reported procedures. [47]
6.3 Compound Characterization Synthesis of 1,3-Bis(2-pyridylimino)isoindoline (BPI)A mixture of phthalonitrile (1 g, 7.81 mmol), 2-aminopyridine (1.47 g, 15.62 mmol), and CaCl2 (78.6 mg, 0.7 mmol) in 1-butanol (50 mL) was refluxed for 1 day. After cooling to room temperature, the resulting pale yellow precipitate was filtered off. Then, the crude product was purified by chromatography on a silica gel column using dichloromethane/methanol (200:1, v/v) as the eluent. Yield: 54%.
Synthesis of 3-Bis(2-Pyridylimino)Benz(f)Isoindoline (Benz(f)BPI)A mixture of naphthalene-2,3-dicarbonitrile (1.39 g, 7.81 mmol), 2-aminopyridine (1.47 g, 15.62 mmol), and CaCl2 (78.6 mg, 0.7 mmol) in 1-butanol (50 mL) was refluxed for 5 days. After cooling to room temperature, the resulting pale yellow precipitate was extracted to dichloromethane layer. Then, the crude product was purified by chromatography on a silica gel column using hexane: ethyl acetate (5:1, v/v) as the eluent. Yield: 43%. 1H NMR (400 MHz, CDCl3): δ=8.65-8.64 (m, 2H), 8.60 (s, 2H), 8.09-8.06 (m, 2H), 7.81-7.77 (m, 2H), 7.64-7.61 (m, 2H), 7.51-7.49 (m, 2H), 7.16-7.13 (m, 2H).
Synthesis of Pt(BPI)Cl (1i)Silver triflate (0.138 g, 0.54 mmol) was added dropwise into Pt(COD)Cl2 (0.1 g, 0.26 mmol) in methanol (15 mL). After stirring for 15 min, the mixture was filtered off and added into extend-BPI (101 mg, 0.29 mmol) in methanol (15 mL). Then, triethylamine (27 mg, 0.26 mmol) was added into reaction mixture and it was heated to 50° C. for 24 h. After cooling to room temperature, the crude product was extracted into dichloromethane layer. Finally, the yellow solid was washed with ether. Yield: 64%. 1H NMR (400 MHz, CDCl3): δ=10.37-10.35 (m, 2H), 8.13-8.10 (m, 2H), 7.94-7.90 (m, 2H), 7.63-7.67 (m, 4H), 7.01-7.04 (m, 2H).
Synthesis of Pt (N{circumflex over ( )}N{circumflex over ( )}N) ClSilver triflate (0.138 g, 0.54 mmol) was added dropwise into Pt(COD)Cl2 (0.1 g, 0.26 mmol) in methanol (15 mL). After stirring for 15 min, the mixture was filtered off and added into BPI (88 mg, 0.29 mmol) in methanol (15 mL). Then, triethylamine (27 mg, 0.26 mmol) was added into reaction mixture and it was heated to 50° C. for 24 h. After cooling to room temperature, the crude product was extracted into dichloromethane layer. Finally, the red solid was washed with ether. Yield: 42%. 1H NMR (400 MHz, CDCl3): δ=10.32-10.29 (m, 2H), 8.59 (s, 2H), 8.09-8.03 (m, 2H), 7.98-7.92 (m, 2H), 7.73-7.65 (m, 4H), 7.07-7.03 (m, 2H).
Synthesis of 1aA mixture of [Pt(BPI)Cl] (50 mg, 0.095 mmol), potassium tert-butoxide (10.6 mg, 0.095 mmol) and 1,3-dimethyl-1H-imidazol-3-ium iodide (23.4 mg, 0.104 mmol) in acetonitrile (15 mL) was heated to reflux for 12 hours. After cooling to room temperature, silver trifluoromethanesulfonate (84 mg, 0.33 mmol) was added into reaction mixture and stirred for 30 min. After extracting the crude product into dichloromethane layer, it was purified by column chromatography on silica gel with CH3CN/CH2Cl2 (3:1, v/v) as eluent, and yellow powder was obtained.
Yield 52%; 1H NMR (400 MHz, CD3CN): 8=8.17-8.15 (m, 2H), 8.02 (t, 2H, J=8.0 Hz), 7.80-7.82 (d, 2H, J=4.0 Hz), 7.70-7.72 (m, 2H), 7.60 (s, 2H), 7.42-7.40 (d, 2H, J=4.0 Hz), 7.02 (t, 2H, J=8.0 Hz), 3.98 (s, 6H); MS (FAB, +ve): m/z 589 [M-OTf]+; Elemental analysis calcd (%) for C24H20F3N7O3PtS: C, 39.03, H, 2.73, N, 13.27; found: C, 38.75, H, 2.80, N, 13.22.
Synthesis of 1bThe procedure is similar to that for 1a.
Yield 61%; 1H NMR (400 MHz, CDCl3): δ=8.17-8.16 (m, 2H), 8.06-8.02 (m, 2H), 7.84-7.82 (m, 2H), 7.74-7.72 (m, 2H), 7.62-7.61 (m, 2H), 7.44-7.42 (m, 2H), 7.04-7.00 (m, 2H), 4.38 (t, 4H, J=8.0 Hz), 1.76-1.68 (m, 4H), 1.31-1.22 (m, 4H), 0.77-0.72 (m, 6H); MS (FAB, +ve): m/z 673 [M-OTf]+; Elemental analysis calcd (%) for C30H32F3N7O3PtS: C, 43.79, H, 3.92, N, 11.92; found: C, 43.64, H, 4.05, N, 11.82.
Synthesis of 1cThe procedure is similar to that for 1a.
Yield 49%; 1H NMR (400 MHz, CDCl3): δ=8.20-8.14 (m, 2H), 8.08-8.02 (m, 2H), 7.88-7.82 (m, 2H), 7.78-7.72 (m, 2H), 7.62-7.60 (m, 2H), 7.42-7.38 (m, 2H), 7.02-6.96 (m, 2H), 4.35 (t, 4H, J=8.0 Hz), 1.78-1.66 (m, 4H), 1.28-1.16 (m, 4H), 1.14-1.04 (m, 8H), 0.78-0.70 (m, 6H); MS (FAB, +ve): m/z 729 [M-OTf]+; Elemental analysis calcd (%) for C35H43F3N7O3PtS.0.5H2O: C, 46.56, H, 4.91, N, 10.86; found: C, 46.38, H, 4.71, N, 11.00.
Synthesis of 1dThe procedure is similar to that for 1a.
Yield 51%; 1H NMR (500 MHz, CDCl3): δ=8.17-8.15 (m, 2H), 8.04-8.00 (m, 2H), 7.86-7.80 (m, 2H), 7.74-7.72 (m, 3H), 7.46-7.43 (m, 3H), 7.04-7.01 (m, 2H), 4.28 (t, 2H, J=4.0 Hz), 4.06 (s, 3H), 1.16-1.07 (m, 12H), 0.77 (t, 3H, J=8.0 Hz); MS (FAB, +ve): m/z 687 [M-OTf]+; Elemental analysis calcd (%) for C30H36F3N7O3PtS: C, 43.58, H, 4.39, N, 11.86; found: C, 43.79, H, 4.15, N, 11.53.
Synthesis of 1eThe procedure is similar to that for 1a.
Yield 44%; 1H NMR (300 MHz, CDCl3): δ=8.20-8.14 (m, 2H), 8.06-7.96 (m, 2H), 7.86-7.78 (m, 2H), 7.76-7.70 (m, 3H), 7.48-7.42 (m, 3H), 7.06-7.00 (m, 2H), 4.28 (t, 2H, J=6.0 Hz), 4.06 (s, 3H), 1.35-1.04 (m, 28H), 0.88 (t, 3H, J=6.0 Hz). MS (FAB, +ve): m/z 799 [M-OTf]+; Elemental analysis calcd (%) for C38H52F3N7O3PtS: C, 48.61, H, 5.58, N, 10.44; found: C, 49.01, H, 5.50, N, 10.23.
Synthesis of 1fThe procedure is similar to that for 1a.
Yield 45%; 1H NMR (400 MHz, CD3CN): 8=8.16-8.14 (m, 2H), 7.93-7.91 (m, 2H), 7.81-7.79 (m, 2H), 7.68-7.66 (m, 4H), 7.26-7.24 (m, 4H), 7.06-6.98 (m, 8H), 6.60-6.54 (m, 2H), 5.42 (s, 4H); MS (FAB, +ve): m/z 741[M-OTf]+; Elemental analysis calcd (%) for C36H28F3N7O3PtS.CHCl3: C, 43.99, H, 2.89, N, 9.71; found: C, 44.07, H, 2.97, N, 9.89.
Synthesis of 1 gThe procedure is similar to that for 1a.
Yield 49%; 1H NMR (400 MHz, CDCl3): δ=8.17-8.12 (m, 2H), 8.08-8.02 (m, 2H), 7.80-7.78 (m, 2H), 7.74-7.73 (m, 2H), 7.65-7.62 (m, 1H), 7.54-7.53 (m, 1H), 7.27-7.25 (m, 2H), 7.17-7.15 (m, 2H), 7.04-6.99 (m, 3H), 6.77-6.74 (m, 2H), 5.44-5.42 (m, 2H), 4.33-4.29 (m, 2H), 1.74-1.62 (m, 2H), 1.24-1.15 (m, 2H), 0.72-0.68 (m, 3H); MS (FAB, +ve): m/z 707 [M-OTf]+; Elemental analysis calcd (%) for C33H30F3N7O3PtS: C, 46.26, H, 3.53, N, 11.44; found: C, 46.17, H, 3.55, N, 11.14.
Synthesis of 1hSilver trifluoromethanesulfonate (24.4 mg, 0.095 mmol) was added into a mixture of [Pt(BPI)Cl] (50 mg, 0.095 mmol) and triphenylphosphine (30 mg, 0.114 mmol) in dichloromethane: acetonitrile (20 mL; 1:1, v/v). The reaction mixture was stirred at room temperature for 5 hours.
After extracting the crude product into dichloromethane layer, it was purified by recrystallization by diffusing diethyl ether into acetonitrile. Reddish yellow crystal was obtained.
Yield 39%; 1H NMR (400 MHz, CDCl3): δ=8.71-8.69 (m, 2H), 8.16-8.15 (m, 2H), 7.86-7.78 (m, 9H), 7.58-7.52 (m, 6H), 7.48-7.46 (m, 6H), 6.62-6.58 (m, 2H). 31P NMR (400 MHz, CDCl3): δ=12.27. Elemental analysis calcd (%) for C37H27F3N5O3PPtS: C, 49.12, H, 3.01, N, 7.74; found: C, 48.98, H, 3.02, N, 7.99.
Synthesis of 1jThe procedure is similar to that for 1a.
Yield 47%; 1H NMR (400 MHz, CDCl3): δ=8.68 (s, 2H), 8.16-8.14 (m, 2H), 7.88-7.86 (m, 2H), 7.74-7.72 (m, 6H), 7.24-7.22 (m, 4H), 7.12-7.04 (m, 8H), 6.67-6.64 (m, 2H), 5.53 (s, 4H); MS (FAB, +ve): m/z 791 [M-OTf]+; Elemental analysis calcd (%) for C40H31F3N7O3PtS: C, 51.01, H, 3.32, N, 10.41; found: C, 50.85, H, 3.02, N, 10.15.
6.4 Experimental Procedure 6.4.1 Cell CultureThe cell lines were maintained in cell culture media (Minimum essential medium (MEM) for HeLa; and Dulbecco's modified eagle medium (DMEM) for MDA-MB-231 and MiHa, Roswell Park Memorial Institute (RPMI) medium for NCI-H460 and HCT116 supplemented with fetal bovine serum (10 vol %), streptomycin (100 μg/ml) and penicillin (100 U/ml) in an incubator (5% CO2) at 37° C.
Human umbilical vein endothelial cells (HUVEC) were cultured in endothelial cell growth medium (ECGM): M199 medium (Life Technologies, Invitrogen) supplemented with 15 vol % fetal bovine serum at 37° C. in a humidified (5% CO2, 95% air) atmosphere.
6.4.2 MTT AssayThe inhibition of cell growth by different metal complexes were determined by MTT assay. Firstly, 4×103 to 8×103 cells were seeded on 96-well culture plates for 24 h. Then, different concentrations of complex was added into different wells by serial dilution and the cells were incubated with complex for 48-72 h. After that, 10 μl of MTT solution (5 mg/ml) was added per well and the plate was incubated for 4 h at 37° C. in a humidified atmosphere of 5% CO2. Viable cells with active metabolism converted MTT into a purple colored formazan product. In order to solubilize the formazan for absorbance readings, 100 μl of SDS (0.1 g/ml, 0.01 M HCl) was added per well and the plate was kept in a dark and humidified chamber overnight. Finally, the absorbance at 580 nm of each well was monitored by microtiter plate reader.
The growth inhibition by a specific complex was represented by IC50 (concentration of a complex causing 50% inhibition of cell growth). Each experiment was repeated three times and the results were expressed as means±standard deviation (SD).
6.4.3 Scratch Assay (Wound-Healing Assay)MDA-MB-231 cells were cultured in 6-well plate and allowed to form a confluent monolayer for 24 h. After serum starved for 4 h, cells were scratched by pipette tips, washed with PBS and photographed by using a fluorescence microscope (20× objective). The fresh medium supplemented with 10 vol % FBS was added into each well with different concentrations of Pt complex. After incubated for 24 h, cells were photographed again at three random areas. Then the migrated cells were quantified by manual counting and inhibition ratio was expressed as % of control.
6.4.4 Transwell Invasion AssayEffects of Pt complex on the invasion of MDA-MB-231 or HUVECs cells were performed on Transwell Boyden chamber (8 μm pore, Corning, Lowel, Mass.) pre-coated with matrigel for 4 h at 37° C. The cell suspension (2.5×105 cells/ml, 100 μL) in serum free medium (SFM) was placed to the upper compartment of chamber. The bottom chambers were supplemented with 500 μl complete medium (10 vol % FBS) or conditioned medium (with VEGF=13.4 ng/ml from MDA-MB-231 cells) containing indicated concentrations of Pt complex. After incubated for 24 h, the non-migrant cells from the upper face were scraped using a cotton swab. The invaded cells on the lower face were fixed with methanol, stained with Giemsa, photographed by a phase-contrast microscope (200×, Nikon TS 100). The invaded cells were quantified by manual counting and inhibition ratio was expressed as % of control.
6.4.5 Tube Formation AssayThe In Vitro Angiogenesis Kit (CaymanChemical) was used in the tube formation assay. Firstly, the ECMatrix solution and 10× Diluent Buffer were mixed in 9:1 (v/v) ratio on ice. Then 50 μL of mixture was transferred into each well of 96-well plate and incubated at 37° C. for 1 h for polymerization. Then, around 4×104 of MS-1 cells in 100 μL DMEM medium was pre-mixed with different concentrations of complex and that cell-complex containing medium was added on the top of the polymerized matrix. After 2 h, the tube formation was observed under an inverted microscopy at a 50× magnification.
At the same time, the cell viability under the same condition was determined by MTT assay. Again, around 4×104 MS-1 cells in 100 μL DMEM medium was pre-mixed with different concentrations of complex and they were seeded into 96-well plate. After 2 h, the medium was removed and fresh medium with 10% MTT was added per well and the plate was incubated for 4 h at 37° C. in a humidified atmosphere of 5% CO2. In order to solubilize the formazan for absorbance readings, 100 μl of SDS (0.1 g/ml, 0.01 M HCl) was added per well and the plate was kept in a dark and humidified chamber overnight. Finally, the absorbance at 580 nm of each well was monitored by microtiter plate reader. Each experiment was repeated three times and the results were expressed as means±standard deviation (SD).
6.4.6 Confocal Fluorescence MicroscopyHeLa cells (2×105 cells) were seeded in a one chamber slide (Nalgene; Nunc) with culture medium (2 mL per well) and incubated at 37° C. in a humidified atmosphere of 5% CO2/95% air for 24 h. After treating with 1b (5 μM) for 1 h, ER-tracker™ red (1 μM), Mitotracker® deep red (50 nM) or Lysotracker® deep red (50 nM) were incubated with cells for 10 min, and then the cells were washed with PBS twice. Confocal fluorescence images were captured using a Carl Zeiss LSM510 Meta confocal microscope with the use of 488 and 543 nm/633 nm lasers for the excitation of complex 1b and red/deep red tracker respectively, under a Plan-Apochromat 63×1.40NA oil-immersion objective.
6.4.7 Determination of Extracellular VEGFThe concentrations of extracellular VEGF was determined by Quantikine® ELISA kit (R&D System). Briefly, cells were cultured in 6-well plate for 24 h, washed with PBS twice and replaced with fresh serum-free medium containing different concentrations of Pt complex. After 24 h treatment, the culture media were collected and centrifuged to eliminate cellular debris. Then, the collected medium was added into the detected microplate and incubates for 2 h at room temperature. After three washes, the VEGF conjugate was added and incubated for another 2 h. After another three washes and the addition of substrate solution and stop solution, the detection of VEGF concentration in the culture medium was performed by monitoring absorption at 450 nm using a microplate spectrophotometer (VERSA max, Molecular Devices).
6.4.8 Chorioallantoic Membrane AssayThe effect of Pt complex on the ex vivo angiogenesis was determined by chorioallantoic membrane (CAM) assay. Briefly, fertilized chicken eggs were incubated at 37° C. in a humidified incubator with forced air circulation. After 5-6 days, eggs were cracked open and methylcellulose discs containing different concentrations of 1d (40 al/egg) and VEGF (50 ng/mL) were gently implanted on top of chicken CAM. After one day incubation, the CAM was observed under a microscope (Olympus BX 40) and photographed. VEGF treatment group was used as a positive control. Three eggs per group were used in each experiment and three independent experiments were performed.
6.4.9 Flow Cytometric AnalysisThe effects of 1d on the cell cycle progression and the induction of apoptotic cell death were quantified by flow cytometric analysis. Briefly, treated or untreated cells were trypsinized, washed with PBS and fixed with 70% ethanol overnight at −20° C. The fixed cells were washed with PBS and incubated with a PI working solution for 4 h in darkness. The stained cells were analyzed by flow cytometer (Beckman Coulter, Fullerton, Calif.). Cell cycle distribution was analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, Calif.). The proportion of cells in G0/G1, S, and G2/M phases was represented as DNA histograms. Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. For each experiment, over 10,000 events per sample were recorded.
6.4.10 Western BlottingMDA-MB-231 cells (5×105 cells) were incubated with Pt compound and washed with phosphate-buffered saline (PBS), lysed with radioimmunoprecipitation assay buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 5 mM sodium fluoride, 1 mM sodium vanadate and protein inhibitor cocktail) for 15 min at 4° C. The cell lysates were centrifuged at 13,000 rpm for 15 min at 4° C. The protein concentrations of the extracts were determined using a BCA protein assay kit (Beyotime, Haimen, China). Specific amount of protein sample (30 μg) was then boiled for 5 min in a 5× sample buffer (50 mM Tris (pH 7.4), 4% sodium dodecyl sulfate (SDS), 10% glycerol and 50 μg/mL bromophenol blue) at a volume ratio of 4:1. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride membranes and immunoblotted with primary anti-bodies. After further incubation with horseradish peroxidase (HRP)-conjugated secondary antibody, the blot was stained with a chemiluminescent detection reagent and subsequently analyzed by enhanced chemiluminescence. Protein expression was visualized on Kodak Biomax X-ray film.
6.4.11 Statistic AnalysisExperiments were conducted at least three times and data was expressed as mean±standard deviation (SD). Statistical analysis was performed on SPSS statistical program version 13 (SPSS Inc., Chicago, Ill.). Difference between two groups was analyzed by two-tailed Student's t test and that between three or more groups was analyzed by one-way ANOVA multiple comparisons. Difference with P<0.05 (*) or P<0.01 (**) was considered to be statistically significant.
6.4.12 Proteomic StudiesSample preparation. MDA-MB-231 cells (8×105 cells) were incubated with 5 μM of 1d or DMSO for 5 h under 5% CO2 environment at 37° C. The cells were then washed with PBS to remove excess compound and lysed with urea lysis buffer (20 mM Tris-HCl, 8 M urea, protein phosphatase inhibitor cocktail, pH 8.0). The cell lysates were centrifuged at 13,000 rpm for 15 min at 4° C. Specific amount of protein sample (50 μg) were then precipitated by adding 4× volume of ice-cold acetone and stored at −20° C. for 4 h. The precipitated proteins were centrifuged at 13,000 rpm for 20 min at 4° C. and the acetone solvent were discarded. After that, the protein pellets were dried by SpeedVac (Thermo Fisher Scientific) and re-suspended in 50 μL of urea buffer (100 mM Tris, 8 M urea, pH 8.5). Then, freshly prepared DTT (final concentration: 5 mM) was added into the sample to reduce to disulfide bond for 30 mins. Then, iodoacetamide (final concentration: 25 mM) was added to alkylate the reduced di-sulfide bond and the samples were kept in the dark for 30 min at 25° C. In order to dilute the urea concentration down to 2 M, around 140 μL of 100 mM Tris (pH 8.5) buffer was added into the sample. Then, 1 μg of trypsin was added into the sample mixture and it was kept at 37° C. overnight. About 10 μL of formic acid was added into sample mixtures to stop the digestion. After centrifugation at 14,000 rpm for 15 min, the supernatants were transferred to new eppendorf (can be frozen at −80° C. for long term storage). The resulting peptides were desalted and enriched by StageTips. For each sample, three biological replicates were prepared. The samples were re-dissolved with H2O (containing 0.1% formic acid, v/v) for subsequent MS analysis.
HPLC-MS/MS analysis. MS analysis was performed with a LTQ Orbitrap Velos Orbitrap mass spectrometer (Thermo Scientific) connected online with a HPLC. The analytical column was a self-packed PicoTip® column (360 μm outer diameter, 75 μm inner diameter, 15 μm tip, New Objective) packed with 10 cm length of C18 material (ODS-A C18 5-μm beads, YMC) with a high-pressure injection pump (Next Advance). The mobile phases of HPLC are A (0.1% formic acid in HPLC grade H2O, volume percentage) and B (0.1% formic acid in HPLC grade acetonitrile, volume percentage). 3 μg of sample was loaded onto the analytical column by the auto-sampler and rinsed with 2% B for 6 min and subsequently eluted with a linear gradient B from 2% to 40% for 120 min. For the MS analysis, LTQ-Orbitrap Velos MS was operated in a data-dependent mode cycling through a high-resolution (6000 at 400 m/z) full scan MS1 (300-2000 m/z) in Orbitrap followed by CID MS2 scans in LTQ on the 20 most abundant ions from the immediate preceding full scan. The selected ions were isolated with a 2-Da mass window and put into an exclusion list for 60 seconds after they were first selected for CID.
Proteins identification and quantification. The raw data were directly used for protein identification and quantification using MaxQuant (Version 1.5.3.30). The data were searched against uniprot human database (27 May 2016, 70625), in which trypsin specificity was used with up to two missed cleavages 17 allowed. Methionine oxidation was set as a variable modification, and iodoacetamide derivative of cysteine was set as a fixed modification. Default settings were used for mass tolerance for MS1 and MS2. The false discovery rate (FDR) was determined by searching against a reverse database and kept FDR at 1%. Peptides were quantified in a label-free manner using the area under the extracted ion chromatograph of peptides, and the protein abundances were the sum of the peptide abundances.
Signaling pathway analysis. Lists of quantified proteins (shown as their Protein IDs) were uploaded to the ExPlain™ tool (version 3.1, BIOBASE) for further signaling pathway analysis. Details of procedure for pathway analysis have been described previously.[48,49]
6.4.13 In Vivo Tumor Growth Inhibition ExperimentsAll experiments were followed to the guidelines of the Laboratory Animal Unit of the University of Hong Kong. Ten mice were randomly divided into two groups (5 mice for each group) for two different treatment conditions.
Around 4×106 of cancer cells in 100 μL of PBS were injected into right back flanks of the mice through subcutaneous injection. After tumor formation (around 4 days), the treatment group was injected with drug at the dosage of 10 mg/kg and the control group was injected with solvent only. The size of the tumors were measured by a ruler every 2-3 days until the mice were sacrificed. The longest diameter (a) and shortest diameter (b) of the tumor would be picked up and the volume of the tumor could be calculated through the following formula:
V=0.52×ab2
It should be noted that the body weight of the mice were also be recorded in order to examine the side effect of the drugs.
To calculate the inhibition effect of the drug, the ratio of enlargement of tumor volume between the drug treatment group and control group would be applied in the following formula:
where Vt and Vt′ are the tumor volumes of drug treatment and control group respectively; V0 and V0′ are the tumor volume at the 0 day of drug treatment and control group respectively.
The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
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Claims
1. A Pt(II) complex consisting of a Pt(II)-N-heterocyclic carbene (“NHC”) ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI) ligand, and wherein the Pt(II) complex has formula I,
- wherein R1 is —CH3, —C4H9, -nC6H13, —CH3, or —CH2Ph, and
- wherein R2 is —CH3, —C4H9, -nC6H13, —C8H17, —C16H33, or —CH2Ph.
2. The Pt(II) complex of claim 1 wherein the NHC ligand is about 90° to the BPI ligand.
3. The Pt(II) complex of claim 1 wherein the Pt(II) complex has anti-tumor or anti-angiogenic properties.
4. (canceled)
5. A Pt(II) complex consisting of a Pt(II)-NHC ligand and 1, 3-bis(2-pyridylimino) isoindoline (BPI) ligand, wherein the Pt(II)-NHC ligand is perpendicular to the BPI ligand, and having the following formula:
- wherein R1 is —CH3, —C4H9, -nC6H13, —CH3, or —CH2Ph, and
- wherein R2 is —CH3, —C4H9, -nC6H13, —C8H17, —C16H33, or —CH2Ph.
6. The Pt(II) complex of claim 5 wherein R1 is C4H9 and R2 is C4H9.
7. A composition comprising the Pt(II) complex of claim 5.
8. The composition of claim 7 wherein the Pt(II)-NHC ligand is about 90° to the BPI ligand.
9. The composition of claim 7 wherein the Pt(II) complex comprises anti-tumor and/or anti-angiogenic properties.
10-18. (canceled)
Type: Application
Filed: Aug 9, 2018
Publication Date: Feb 13, 2020
Inventors: Chi Ming CHE (Mid-Level), Sin Ki FUNG (New Territories), Tian Feng CHEN (Guangzhou)
Application Number: 16/059,679
