Patent Application
20180185303
The present invention relates generally to inhibition of monoamine oxidases (MAOs) and their inhibitors (MAOIs) as strategies to treat cancer, particularly prostate cancer. This invention also relates to imaging, screening, diagnostics, and therapeutic methods of cancer. In addition, this invention further relates to cancer biomarkers and methods for differentiating indolent from virulent prostate cancer.
Prostate cancer is the third most common cause of death from cancer in men of all ages and is the most common cause of death from cancer in men over age of 75. Current treatments for prostate cancer include (1) hormonal therapy, (2) chemotherapy, (3) radiation therapy, and (4) surgery. However, they are only effective for patients during the early stages of the disease. There are also undesired side effects associated with each of these treatment modalities. Moreover, for patients with advanced stages of castration-resistant and metastatic prostate cancers, these treatments are only partially effective.
Supplemental or combination therapies may improve the outcome in advanced patients. For instance, patients subjected to androgen ablation therapy with either chemical castration with a LH-RH agonist or surgical castration have benefited by the combination with an antiandrogen like bicalutamide. Patients who failed these hormonal therapies are often benefited by selective chemotherapy such as docetaxel and denosumab and additional hormonal therapy to deplete residual endogenous androgen synthesis (e.g. a CYP17 inhibitor, abiraterone). Despite the improvement, these additional therapies, in general, are only capable of prolonged survival by a few months.
Prognosis and staging of prostate cancer are typically evaluated using the Gleason grading system. A Gleason score is given to prostate cancer based on its microscopic appearance. Cancers with a higher Gleason score are more aggressive and have a worse prognosis. A Gleason score is determined by a pathologist who visually inspects a biopsy sample and then assigning a score to the observed tumor pattern. However, the Gleason system is entirely reliant upon human visual examination, which is prone to error with significant limitations on early detection.
In view of the above, there is an urgent, unmet need for more effective mechanism-based therapies and noninvasive early-stage diagnostic techniques to differentiate indolent from virulent forms of prostate cancer so that overtreatment of this disease can be avoided.
Briefly, the present invention is based, in part, on the surprising discovery that monoamine oxidases exhibit differential expressions/activities in cancerous cells and that inhibitors of monoamine oxidases (MAOs) are capable of repressing the growth of cancer cells in vitro and tumor xenografts in vivo.
MAOs are a family of enzymes that catalyze the oxidation of monamines. They are bound to the outer membrane of mitochondria in most cell types in the body. In humans, there are two isoforms of MAO, MAO-A and MAO-B. The two forms of MAOs are a crucial pair of oxidative enzymes that deaminate biogenic and dietary amines, including monoamine neurotransmitters, resulting in the production of hydrogen peroxide (H2O2). Both isoforms of MAO play key roles and have diverse functions in normal physiology and disease states, such as modulating emotions and behaviors. Because of the vital role that MAOs play in the inactivation of neurotransmitters, MAO dysfunction (too much or too little MAO activity) is thought to be responsible for a number of psychiatric and neurological disorders. For example, unusually high or low levels of MAOs in the body have been associated with depression, schizophrenia, substance abuse, attention deficit disorder, migraines, and irregular sexual maturation. Therefore, MAO was previously known as a target for psychiatric and neurological disorders.
In the present invention, it was unexpectedly discovered that increase of MAO-A activity or expression is correlated with the progression of human prostate cancer. For example, it has been demonstrated that clorgyline, a potent MAO-A inhibitor, is capable of repressing the growth of human prostate cancer cells in vitro and tumor xenografts in vivo. This finding establishes MAOs as a target for cancer.
Accordingly, a first aspect of the present invention is directed to a novel MAO inhibitor selected from the group consisting of compounds 11-14 as shown below:
and a salt thereof. These compounds are commercially available compounds with newly discovered MAO inhibitory activities. They may be purchased from commercial sources, including but not limited to, Aurora Screening Library, Enamine HTS Collection and/or Interchim Screening Library. Thus, this aspect of the invention provides compositions comprising useful for inhibiting MAO activity, comprising one or more compounds selected from the group consisting of compounds 11-14. This aspect of the invention also provides a method for inhibiting MAO activity by contacting a cell with one or more MAO inhibitors selected from the group consisting of compounds 11-14.
In addition to the above disclosed inhibitors, the present invention has also unexpectedly discovered that nanoparticles that are preferential uptaken by cancer cells (e.g. near infrared dies) may be used as a delivery vehicle to deliver a pharmaceutically active agent (e.g. a cytotoxic compound) to cancer cells. For example, the present invention has succeeded in conjugating near-infrared dye nanoparticles such as IR-783 to an active agent such as a MAO inhibitor described above and demonstrated that the resulting nano-conjugates remain preferentially uptaken by cancer cells.
Hence, a second aspect of the present invention is directed to a nano-conjugate capable of preferentially or selectively targeting cancer cells. Nano-conjugates in accordance with this aspect of the invention will generally have an NIR dye nanoparticle conjugated to a cytotoxic compound. Exemplary NIR dyes may include conjugated polyene functional groups, such as one found in IR-783, IR-780, IR-786, and MHI-148 but are not limited thereto. Exemplary cytotoxic compound may include MAO inhibitors, docetaxel, cisplatin, carboplatin, oxaliplatin, doxorubicin, temozolomide, gemcitabine, anthramycin, camptothecin, topotecan, lonidamine, mitomycin, imexon, dacarbazide, PK-11195, but are not limited thereto. Conjugation of the NIR dye nanoparticle to the cytotoxic compound may be achieved by any suitable chemical means known in the art.
In one preferred embodiment, exemplary nano-conjugates of the present invention will generally have at least two functional groups with a cytotoxic element (e.g. an MAO inhibitor) attached to a light emissive element (e.g. NIR dye nanoparticle) via a linker containing at least one C and two H atoms. Preferably, at least two unsaturated structures containing one unsaturated double or triple bond are linked via a backbone chain of 1-3, 1-5, or 1-15 atoms to one heterocycle.
An exemplary linker is one having the following general formula:
wherein M1 is O or S; and wherein at least two of X, Y, and Z participate in bonds to unsaturated and/or aromatic groups A and B (not shown) which proceed through additional carbon, oxygen or nitrogen atoms. Any of X, Y, and Z not participating in a bond to group A or B is substituted with hydrogen or lower aliphatic group, such as C1-C6 alkyl.
As used herein, the term “backbone chain” refers to the chain of atoms linking the two unsaturated structures together, not taking into account said chain.
For example, a conjugate or nanoparticle-encapsulated conjugate (herein referred to as nano-conjugate) in accordance with embodiments of the invention may be one having the following formula:
In another embodiment, X and Y are as above and Z is selected from the group consisting of
wherein the covalent link is attached to the aromatic ring. This compound is herein referred to as MHI-moclobemide, a MAO-A specific reversible inhibitor.
In another embodiment, X and Y are same as above, and Z is
wherein the covalent bond is also attached to the aromatic ring. This compound is herein referred to as MHI-phenelzine, a MAO-A and -B inhibitor.
In still another embodiment, X and Y are same as above, and Z is (±)-trans-2-phenylcyclopropan-1-amine having the formula:
wherein covalent attachment is through the aromatic ring. This compound is herein referred to as MHI-tranylcypromine, which is a MAO-A and -B inhibitor.
In still another embodiment, X and Y are same as above, and Z is N-Benzyl-N-methylprop-2-yn-1-amine, having the following formula:
wherein covalent linkage is attached to the nitrogen as indicated by the curly line. This compound is herein referred to as MHI-pargyline, a MAO-A and -B inhibitor with a preference for MAO-B.
In a preferred embodiment, Y is S; X is a group having the following formula:
and Z is a group having the following formula:
This compound is referred to herein as
MHI-clorgyline, which is a MAO-A specific irreversible inhibitor.
In yet another embodiment, X and Y are same as above, Z is one selected from the following:
wherein covalent linkage is attached to the aromatic rings. This group of compounds is collectively referred to herein as MHI-MAOIs.
The MHI-MAOIs can be conveniently prepared in two steps from MHI-148 and inhibitor through reduction of MHI-148 with lithium aluminum hydride or diborane and subsequent conjugarion of the resulting diol with the MAOI by, for example, but without being limited to, Mitsunobu reaction, to give conjugate 11D.
In still another embodiment, Y and Z are same as above, X is one having the following formula:
wherein the covalent linkage is attached to the cyclohexene ring of the molecule. This group of compounds is collectively referred to herein as NIR-MAOIs.
A third aspect of the present invention is directed to a method for forming an NIR dye-based nano-conjugate capable of preferentially targeting cancerous cells. Methods in accordance with this aspect of the invention will generally include the steps of chemically conjugating an NIR dye nanoparticle to a cytotoxic compound. Suitable NIR dyes and cytotoxic compounds are as described above.
A forth aspect of the present invention is directed to a pharmaceutical composition useful for treating cancer, and methods of treating cancer using the compositions. Compositions in accordance with this aspect of the invention will generally include an active agent capable of inhibiting MAO activity; and a physiologically suitable carrier. In some preferred embodiments, the active agent is a MAO inhibitor known in the art. Exemplary MAO inhibitor may include, but not limited to moclobemide, phenelzine, tranylcypromine, pargyline, and clorgyline. Nucleic acids capable of inhibiting, down-regulating or silencing the expression of MAO may also be advantageously used. Exemplary nucleic acid MAO inhibitors may include siRNA, shRNA, antisense, or any other type of nucleic acid-based gene silencing agents commonly known in the art, such as decoys, ribozymes, and aptamers. Such preferred embodiments can be used, either alone or in combination with the described herein pharmaceutical compositions as cancer therapeutics.
In one exemplary embodiment, gene silencing or knock-down of MAO-A in human prostate cancer cells with shRNA can be exemplified as follows: in a 48-well tissue culture plate, 6×104 human prostate cancer cells per well in 250 ul normal culture medium were seeded 24 hrs prior to viral infection, and the cells should be approximately 50% confluent on the day of infection. A mixture of 40 ul of human shMAOA lentiviral transduction particles (5×106 titer/ml) with polybrene (at a final concentration of 5 ug/ml) in 100 ul medium (without FBS and anti-biotics) was prepared and added into cells for a subsequent incubation for 4 hrs to overnight. The culture medium was replenished after 4 hrs to overnight. Cells were then treated with 2-10 ng/ml puromycin 48 hrs after infection for selection consecutively for 2 weeks, and the medium supplemented with puromycin was replenished every 3-4 days. Stable MAOA-KD cells were validated by Western blot and real-time RT-PCR examination of MAOA gene expression, and were maintained in the culture medium supplemented with puromycin at the same concentrations for selection. The shRNA sequence against human MAOA cDNA is: CCGGCGGATATTCTCTGTCACCAATCTCGAGATTGGTGACAGAGAATATCCG TTTTTG (SEQ ID NO:1), as adapted from a Sigma-Aldrich product (catalog # NM_000240_TRCN0000046009).
In other preferred embodiments, the active agent is an NIR dye-based conjugate as described above. In still other preferred embodiments, the active agent is one selected from compounds 11-14 that are available from commercial sources, including but not limited to, Aurora Screening Library, Enamine HTS Collection and/or Interchim Screening Library.
A fifth aspect of the present invention is directed to a method of delivering a pharmaceutical agent to a cancer cell. Methods in accordance with this aspect of the invention will generally include the steps of conjugating the pharmaceutical agent to an NIR dye; and contacting the conjugate with the cancer cell. In some preferred embodiments, the pharmaceutical agent is a cytotoxic agent. Exemplary cytotoxic agent may include an alkylating agent, an inhibitor of microtubule formation, and an aromatase inhibitor, but are not limited thereto.
A sixth aspect of the present invention is directed to a method of inhibiting MAO activity in a cancer cell. Methods in accordance with this aspect of the invention will generally include the steps of contacting a cell with an inhibitory agent, wherein said inhibitory agent is selected from the group consisting of a MAO inhibitor, a nano-conjugate with a NIR dye conjugated to a MAO inhibitor, and a combination thereof. Any MAO inhibitor known in the art or herein disclosed, both pharmacological and nucleic-acid based, may be advantageously used.
Where prostate cancer is concerned, it is a further discovery of the present invention that MAO-A is associated with chemo and radiation resistance in human prostate cancer whereas MAO-B has a unique expression pattern in human prostate cancer-associated stromal cells. As mentioned above, MAOs are mitochondrial-bound enzymes that catalyze the degradation of monoamine neurotransmitters and dietary amines via oxidative deamination. They are encoded by their genes located in the X chromosome [1, 2]. The by-product of MAO catalysis is hydrogen peroxide, a major source of reactive oxygen species (ROS), which can predispose cancer cells to DNA damage and promote tumor initiation and progression [3]. Modulation of intracellular ROS levels in prostate cancer cells could affect the sensitivity of prostate cancer cells toward hormonal, chemo- and radiation therapy [4]. Moreover, MAOs are responsible for the generation of ROS, in the presence of their biogenic amine substrates from the diet or physiological sources in an epithelial versus stromal cellular compartments. In addition, since prostate stroma is known to drive the progression of prostate cancer, by differentiating the forms, the amount, and the physical location of MAOs in prostate cancer tissue specimens, indolent forms of human prostate cancer may be differentiated from virulent forms. In short, it is an unexpected discovery of the present invention that MAOs are capable of serving as biomarkers for screening, diagnosing, and differentiating prostate cancer forms in patients. Based on the observation that MAO-A and MAO-B differs in their localization, a treatment strategy targeting both MAO-A in prostate cancer epithelium and MAO-B in prostate cancer-associated stroma is also devised.
Accordingly, a seventh aspect of the present invention is directed to a method of differentiating different forms of prostate cancer, comprising assaying MAO activity and location patterns in prostate tissues; and determining a cancer form characterization according to said MAO activity and location patterns. The said MAO activity can be determined, for example, by real-time PCR that measures the MAO-A expression in prostate biopsy as described below:
The biopsy samples should be homogenized in Trizol, and RNA isolated. Next, 1 ug of total RNA will be reverse transcribed in 25 ul volume, then 2 ul of the sample (cDNA) is diluted 1/10 into 20 ul, 5 ul of this sample will be used as template for MAO A measurement. Another 2 ul will be diluted 1/50 into 100 ul, 5 ul of this sample is used for ribosomal RNA control template.
The primer sequence for human MAO A specific primer can be as follows:
113 bp PCR product
The primer sequence for 18s ribosomal RNA are as follows:
253 bp PCR product
PCR condition: 95 degrees C.×4 min 1 cycle
95 degree×30 sec
60 degree×30 sec
72 degree×30 sec 40 cycles.
Those skilled in the art will recognize that the above example is for illustration only and other currently known or future invented methods of measurement may also be used to determine MAO activity.
An eighth aspect of the present invention is directed to a method of screening a patient for risk of cancer, comprising assaying MAO activity in the patient; comparing said activity to a reference; and determining a risk level based on the comparison. The said MAO activity can be determined, for example, by real-time PCR that measures the MAO-A expression in prostate biopsy as described below:
The biopsy samples should be homogenized in Trizol, and RNA isolated. Next, 1 ug of total RNA will be reverse transcribed in 25 ul volume, then 2 ul of the sample (cDNA) is diluted 1/10 into 20 ul, 5 ul of this sample will be used as template for MAO A measurement. Another 2 ul will be diluted 1/50 into 100 ul, 5 ul of this sample is used for ribosomal RNA control template.
The primer sequence for human MAO-A specific primer can be as follows:
113 bp PCR product
The primer sequence for 18s ribosomal RNA are as follows:
253 bp PCR product
PCR condition: 95 degrees C. x 4 min 1 cycle
95 degree×30 sec
60 degree×30 sec
72 degree×30 sec 40 cycles.
Those skilled in the art will also recognize that the above example is for illustration only and other currently known or future invented methods of measurement may also be used to determine MAO activity.
A ninth aspect of the present invention is directed to a method of treating cancer. Methods in accordance with this aspect of the invention will generally include the steps of administering to a subject a pharmaceutical agent capable of inhibiting MAOs in cancer cells. The type of cancers that may be treated by methods in accordance with this aspect of the invention may include prostate, brain, colon, aggressive fibromatosis, but not limited thereto. The pharmaceutical agent may be any of the above described compositions, nano-conjugates, or inhibitors. In a preferred embodiment, the cancer is prostate cancer. In a further preferred embodiment, treatment of prostate cancer may include administering a first pharmaceutically active agent targeting MAO-A in epithelium with clorgyline and a second pharmaceutically active agent targeting MAO-B in stroma with deprenyl. Said first and second pharmaceutically active agent may be different agent or the same agent, so long as they are effective in inhibiting the respective MAO isoform in the respective tissue type.
An tenth aspect of the present invention is directed to a method of monitoring treatment progress in a cancer patient being treated with a pharmaceutical composition comprising a NIR dye-based nano-conjugate. Methods in accordance with this aspect of the invention will generally include the steps of obtaining successive NIR image of the patient; and comparing said successive NIR images to determine progression of said treatment. The effect of conjugate on prostate tumor growth and metastasis can be determined by imaging and IHC analysis. The said imaging can be done, for example, with Xenogen IVIS 200 instrument. This system allows researchers to use real-time, non-invasive imaging to monitor and record cellullar and genetic activity in vivo. Integrated into the system are both a bioluminescence system and a fluorescence system and the capability to easily switch between modalities. A laser scanner also provides 3D surface topography for single-view diffuse tomographic reconstructions of internal sources. Background noise is minimized while sensitivity is maximized using a 26 mm square CCD which is cryogenically cooled. Scans generally take 1-10 minutes to complete with five field of view options ranging from 4 cms to 25 cms.
An eleventh aspect of the present invention is directed to a method of modulating ROS levels in cells. Methods in accordance with this aspect of the invention will generally include the steps of contacting a cell with a MAO inhibitory agent. Suitable MAO inhibitory agent may be any of the MAO inhibitors, nano-conjugates, or pharmaceutical compositions described above. These agents can be used either alone or in combination with mitochondria-directed antioxidants, such as lipoic acid, N-acetyl-L-carnitine and N-Acetyl-L-cysteine.
Other aspects and advantages of the present invention will become apparent from the following detailed description and the appended claims.
Compound 10 was pre-incubated with 1×106 prostate C4-2B cells at 37° C. for 20 min. Then, MAO A substrate C-14 serotonin was added to the incubation solution for 37° C. 20 min. At the end of the incubation, the reaction product was extracted and the radioactivity was counted. The MAO-A activity was expressed as 69.6 nM product formed/20 min/mg protein. The activity without the presence of inhibitor, compound 10, was taken as 100%.
Unless otherwise indicated herein, all terms used herein have the meanings that the terms would have to those skilled in the art of the present invention. Practitioners are particularly directed to current textbooks for definitions and terms of the art. It is to be understood, however, that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.
“Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
A “therapeutically effective amount” of a monoamine inhibitor is an amount sufficient to carry out a specifically stated purpose. An “effective amount” may be determined empirically and in a routine manner in relation to the stated purpose.
A “Carrier” or “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. The physiologically acceptable carrier may be a sterile aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants.
Recent studies indicate that increased MAO-A is associated with prostate cancer progression [5]; conversely, pharmacological MAO-A inhibition or lentiviral shRNA-mediated silencing of MAO-A significantly reduced the growth of prostate cancer cells in vitro and tumor xenograft in vivo [6-8]. Furthermore, our data showed that MAO-A induced epithelial-to-mesenchymal transition (EMT) in human prostate cancer cells, by promoting the loss of E-cadherin (an epithelial marker) expression, up-regulation of vimentin (a mesenchymal marker) levels as well as increased invasion and migration of prostate cancer cells. These results suggest that MAO-A expression may be correlated with the metastatic potential of prostate cancer cells. Taken together, this evidence strongly supports the role of MAO-A as a potential novel target for the treatment of human prostate cancer.
We have found that monoamine oxidase A (MAO-A) knock-down (KD) prostate cancer cells did not grow when injected into mice. This result was dramatically different from the WT prostate cancer cells (See
One aspect of the present invention is a method in which a MAO-A inhibitor, clorgyline, can be delivered to cancer cells and tissues by chemically conjugating clorgyline to a NIR dye. The NIR-dye-clorgyline conjugate is expected to be uptaken by cancer but not normal cells thus avoiding systemic toxicity of this MAOI. Laser-scanning confocal microscopy can be used to determine cellular uptake and localization of the nano-clorgyline within cells (LNCaP, C4-2, and ARCaPM prostate cancer cell lines). This class of NIR dyes can be readily uptake into cancer cells via organic anion transporting peptides.
To determine the ability of nano-clorgyline to inhibit activity of MAO-A. MAO-A inhibition curve is obtained for prostate cancer cells LNCaP, C4-2, and ARCaPM cell lines and compared with that clorgyline itself. These cell lines have moderate to high MAO-A activity. IC50 of nano-clorgyline is determined and compared to that of clorgyline.
To study the effect of nano-clorgyline and NIR dyes (IR-783, IR-780 and MHI-148)-clorgyline on MAO-A and MAO-B inhibition. MAO-A and MAO-B inhibition curves is performed in mice. ICs50 are determined.
To study the localization of nano-clorgyline, and NIR dyes-clorgyline conjugates and its effect on tumor growth, prostate cells are injected into mice. In one exemplary experiment, mice are divided into 3 groups separately injected with (a) nano-clorgyline of NIR dyes-clorgyline, (b) clorgyline itself, and (3) dye only, respectively. The location of the nano-clorgyline was imaged, the tumor's growth (size, number, and weight) was monitored, and the results from the 3 groups were compared.
The result of the experiment demonstrated the effect of nano-clorgyline on tumor growth. Having established the effectiveness of the clorgyline nano-conjugate, it will be appreciated by those skilled in the art that other parameters such as the concentration required for inhibition of MAO-A activity and tumor growth may be readily determined via routine experimentation.
The MAO-A inhibitors, including the NIR dyes-MAOI conjugates thereof, may be used alone or in combination with the existing treatments on tumor growth and metastasis such as (a) surgical castration; (b) radiation (c) docetaxel; (d) abiraterone.
Furthermore, this clorgyline-dye conjugate may be used in connection with methods for treatment of prostate cancer and methods for diagnosis and monitoring of the progression of the prostate cancers by administering to a patient a composition comprising an effective amount of the clorgyline-dye conjugate.
Preparation of Clorgyline-Dye Conjugate.
We have identified a class of near-infrared (NIR) fluorescent heptamethine cyanine dyes, IR-783 (1) (see
Encapsulation of the Clorgyline-Dye Conjugates in Nanoparticles.
In order to achieve water solubility of the clorgyline-dye conjugates and enhance their delivery to tumors, we will encapsulate them in calcium phosphate/silicate nanoparticles of average size of 22 nm that are freely dispersible in water [10]. This encapsulation process results only in minor changes to the photophysical properties of the dyes. Such an encapsulation method has been reported by Adair as effective means of delivery of hydrophobic dyes to cells [11]. Calcium phosphate is an excellent matrix for nanoparticle encapsulation because moderate concentrations of Ca2+ ions are not toxic to cells and in vivo (found in human bone and teeth). It was shown that calcium phosphate dissolves below pH 5.5, liberating the cargo, but it is stable at pH 7.4 [12]. In addition, particles of this matrix disperse freely in aqueous media.
Typically, such nanoparticles are prepared using aqueous co-precipitation of calcium chloride and disodium hydrogen phosphate in the presence of disodium silicate within water-in-oil microemulsions [11, 13]. The encapsulation of the clorgyline-dye conjugate in nanoparticles will be accomplished through its addition into the microemulsion during precipitation. This process will yield nano-clorgyline as a colloidal suspension of nanoparticles of 22-30 nm in size. Its characterization will be done through analysis of the size distribution, morphology, and colloidal state of dispersion of the nanoparticle suspensions using transmission electron microscopy (TEM).
To Determine the Inhibition of MAO-A Activity by Nano-Clorgyline.
We will study the ability of nano-clorgyline to inhibit MAO-A activity by performing the inhibitory curves in human prostate cancel cell lines, LNCaP (non-metastatic cell line), C4-2 and ARCaPm, two metastatic cell lines. IC50 will be determined. All these cell lines have moderate to high MAO-A activity. Shih's lab routinely performs MAO-A inhibition assay in vitro and in vivo.
To Determine if Nano-Clorgyline is Targeted to Mitochondria of Cancer Cells.
We will use laser-scanning confocal microscopy to determine cellular uptake and localization of the nano-clorgyline within cells. The near-infrared dye moiety within the conjugate will serve as a directing group, guiding the conjugate to mitochondria. It has been shown that redox potential of mitochondria is different between cancer and normal cells.
Human cancer cell lines (LNCaP, non-metastastic cell line, C4-2, ARCaPM, two metastastic cell lines), will be tested. As negative controls, normal human prostate epithelial cells (P69 and NPE), normal human prostate fibroblasts (NPF), will be used.
It is anticipated the following studies will show that the growth of prostate cancer will be significantly reduced after the treatment with nano-clorgyline. The outcome will be determined by reduced tumor growth in rate, size and weight. We expect nano-clorgyline will be more effective with fewer side effects than clorgyline itself. In addition, we will establish MAO-A as a biomarker for prostate cancer, with nano-clorgyline to be used for diagnostic tool; further, we will follow the progression of prostate cancer during treatment through imaging of the uptake of the nano-clorgyline.
Synthesis of MHI-Corgyline:
MHI-clorgyline has been prepared through a series of synthetic steps by conjugating dye MHI-148 and clorgyline as outlined in
Preparation of MHI-clorgyline started with commercially available 2,4-dichlorophenol 1. This compound was alkylated under standard conditions (NaOH, H2O) with 1,3-dibromopropane. The product 2 was then reacted with NaN3 in DMF to yield the azide 3, which was subjected to the next step as a solution in MTBE without further purification. The solution of 3 was hydrogenated under low H2 pressure using Pd on activated charcoal as a catalyst in the presence of Boc2O and resulting in the formation of the carbamate 4. This compound was alkylated with propargyl bromide using NaH in dry DMF, producing Boc-protected alkyne 5. The Boc protecting group was removed under acidic conditions using TFA in DCM. The product 6 was alkylated again with 1-bromo-3-thioacetylpropane 7, resulting in the formation of 8, albeit in low yield. Removal of the acetyl protective group was carried out in methanolic HCl and afforded intermediate 9. This intermediate was then coupled with MHI-148 dye using EDC and 4-DMAP to afford the product MHI-clorgyline 10. This product was purified using reverse-phase HPLC and its identity was confirmed by mass spectrometry.
Imagining of MHI-Clorgyline in Live Cells:
Previous study showed that the near-IR hepatamethine cyanine dyes IR-783 and MHI-148 can be retained in cancer cells but not normal cells in tumor xenografts and in spontaneous tumors in transgenic mice. Moreover, the two dyes also have strong emission at 820-860 nm upon excitation at 750-780 nm, which can be easily detected and visualized by NIR imaging equipment and laser-scanning confocal microscopes. Here, we resorted to the use of Zeiss LSM 510 confocal microscope as the imaging equipment. Although cellular uptake of the conjugate was uncertain, due to the similarity in structure, MHI-clorgyline conjugates were expected to have similar fluorescence properties as the NIR dye itself. Therefore, NIR imaging with laser-scanning confocal microscope was used to examine the cellular uptake of MHI-clorgyline in human prostate epithelial cancer cells (C4-2B). This cell line was selected due to its high levels of the expressed MAO-A.
In the preliminary study we found that IR-783 dye co-localized in mitochondria of the live cells. An imaging study with the newly synthesized MHI-clorgyline 10 was carried out (
In order to test an inhibitory activity of the MHI-clorgyline 10, a standard MAO A inhibition assay was carried out in C4-2B cells using radiolabeled substrates. The results clearly indicate that our designed conjugate 10 inhibits MAO A activity with mean IC50 of 2.18×10−5 M (
To Study the Location of Nano-Clorgyline, and its Effect on Tumor Progression in Mice.
We will inject human LNCAP non-metastatic prostate cancer cells (1×106) into immunodeficient nude mice with (1) nano-clorgyline, concentration required to inhibit nearly 100% of MAO-A activity using results from Specific Aim 1b; (2) clorgyline itself (positive control, 10 mg/kg for 100% inhibition of MAO-A activity); (3) dye itself (negative controls, same concentration as nano-clorgyline). A total of 18 mice is required for this part of the study (6 per each group).
Then, we will image the tumor location, monitor the tumor growth (size, number) every other day for one month. The tumor will be located by NIR imaging. The Olenyuk lab has an extensive experience in NIR imaging and the needed equipment is available at the Norris Cancer Center. If our hypothesis is correct, the nano-clorgyline will be located in prostate cancer, and the tumor growth will be reduced. At the end of 30 days, the mice will be sacrificed, the MAO-A activity will be determined in the tumor (if there is still tumor) and the normal prostate tissues. See
To Study the Effect of Nano-Clorgyline on the Metastasis of Prostate Cancer in Mice.
Human prostate cancer cells with metastatic potential will be injected to mice, human C4-2, ARCaPm, (1×106 cells), same three group of mice will be used, the procedure and the experiments will be the same as described in Specific Aim 2a. The tumor growth rate and size and locations will be determined. The presence of absence of the tumor in the bone, will be examined and as an indication of metastasis. A total of 36 mice will be used (18 mice per each cell line with 3 groups of treatment; 2 metastatic cell lines).
The effects of nano-clorgyline alone or in combination with the existing treatments on tumor growth and metastasis in mice.
First line treatment for advanced prostate cancer is androgen ablation therapy (ADT). Unfortunately the duration of response to ADT is limited (about 18 months) and the patients eventually develop castration resistance. The first line treatment for patients with castration resistant PCA is usually chemotherapy with the microtubule inhibitor, docetaxel. Recently, FDA approved the specific CYP17 inhibitor, abiraterone, for the treatment of castration resistant patients who fail docetaxel therapy. This study will evaluate the effects of nano clorgyline alone or in combination with one of these treatment approaches on tumor growth. Since the nano clorgyline can be read non-invasively by INR imaging, the prognosis of each treatment can be easily determined.
Human LNCAP cells (non-metastatic prostate cancer cell line). Alternatively, ARCaPm (with potential for metastasis) will be injected to immunodeficient nude. Next, mice will be divided to three groups as described in A. The tumor location, size, number of lesions will be determined every other day from day 1 to day 30. On day 31 mice will be sacrificed, tumor and host MAO Activity will be determined.
For the castration group, trans-scrotal castration will be performed under isoflurane anesthesia with proper aseptic and antiseptic technique. A total of 108 mice will be used.
Synthesis of Nanoparticle Conjugates with Other Novel MAO-A Inhibitors Obtained from High Throughput Screening.
Optionally, other novel MAO-A inhibitors, disclosed herein may be conjugated for use in the methods and treatments of the present invention. Specifically, below are examples of four high affinity novel MAO-A inhibitors 11-14. They may be conjugated with the near-infrared dye, such as IR-783. The phenol functionality (—OH) presents a viable choice for linker attachment and the subsequent derivatization of these molecules with fluorescent precursor 3 in order to generate novel nanoparticle based MAO-A inhibitors.
High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit elevated MAO-A expression. We have found that a key function of MAO-A is to promote an epithelial-to-mesenchymal transition (EMT). EMT is the process of cellular development characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell mobility. In the context of cancer, promotion of EMT correlates with the increased cell invasion, migration and metastatic potential, hence, the EMT-promoting effect of MAO-A connects MAO-A activity to cancer. More specifically, we have found that overexpression of MAO-A in human prostate cancer cells induces the loss of E-cadherin (an epithelial marker), up-regulates Vimentin/N-cadherin (mesenchymal markers) and increases cell migration and invasion Conversely, knockdown of MAO-A impedes EMT in human prostate cancer cells.
Without being bound to any particular theory, we offer the following experimental observations (
First, we found that MAO-A enhances HIF1α stability by reducing prolyl hydroxylase (PHD) activities and increasing intracellular ROS levels. We then found that by treating prostate cancer cells with a ROS scavenger (N-acetylcysteine), MAO-A-induced HIF1α expression is diminished, which in turn, also decreased MAO-A-enhanced cell proliferation. Moreover, we also found that MAO-A mediated the activation of VEGF and its receptor Neuropilin-1 (NRP1) in response to hypoxia, which in turn stimulated the Akt/FoxO1 signaling pathway and reduced FoxO1 activity by promoting its phosphorylation followed by nuclear export. We further discovered that FoxO1 acts as a transcriptional repressor of Twist1 and binds to a response element in the proximal region of Twist1 promoter. Twist1 is known to be an oncogene in several cancers and is involved in tumor metastasis.
Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF1α, VEGF and Twist1 expression, but less FoxO1 nuclear localization compared to low Gleason grade cancers. Therefore, expression levels of MAO-A, HIF1α, VEGF and Twist1 serve as a biomarker for objectively differentiating high Gleason grade cancers from low Gleason grade cancers.
The following examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. While such examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.
Cell # injected: 1×105 Mice #: WT (N=9) and MAO-A KO (N=9) Tumor injection site #: WT (2×9=18) and MAO-A KO (2×9=18) Tumor incidence rate: WT (11/18=61.1%) and MAO-A KO (3/18=16.7%) Tumor growth: WT>MAO-A KO (p<0.05) Tumor weight: WT>MAO-A KO (p<0.05)
Cell # injected: 1×106 Mice #: WT (N=3) and MAO-A KO (N=3) Tumor injection site #: WT (4×3=12) and MAO-A KO (4×3=12) Tumor incidence rate: WT (11/12=91.7%) and MAO-A KO (10/12=83.3%) Tumor growth: WT>MAO-A KO (p<0.05) Tumor weight: WT>MAO-A KO (p=0.25)
Cell # injected: 1×105 Mice #: WT (N=4) and MAO-A KO (N=5) Tumor injection site #: WT (3×4=12) and MAO-A KO (3×5=15) Tumor incidence rate: WT (10/12=83.3%) and MAO-A KO (0/15=0) Tumor growth: No MCP3 tumor growth in MAO-A KO mice
Cell # injected: 1×106 Mice #: Mice for WT MCP3 cells (N=6) and mice for MAO-A-KD MCP3 cells (N=4) Tumor injection site #: WT MCP3 cells (4×6=24) and MAO-A-KD MCP3 cells (4×4=16) Tumor incidence rate: WT MCP3 cells (21/24=87.5%) and MAO-A-KD MCP3 cells (0/16=0) Tumor growth: With MAO-A KD in tumor, there is no tumor growth.
Cell # injected: 1×106 Mice #: Mice for WT MCP3 cells (N=6) and mice for MAO-A-KD MCP3 cells (N=6) Tumor injection site #: WT MCP3 cells (3×6=18) and MAO-A-KD MCP3 cells (3×6=18) Tumor incidence rate: As of July 16, WT MCP3 cells (15/18=83.33%) and MAO-A-KD MCP3 cells (0/18=0).
General Synthesis:
All reagents and solvents were obtained from commercial sources and were used as received unless otherwise stated. All reactions involving moisture-sensitive reagents were conducted under argon atmosphere with anhydrous solvents and flame-dried glassware. Hygroscopic liquids were transferred via a syringe and were introduced into reaction vessels through rubber septa. Reaction product solutions were concentrated using a rotary evaporator at 30-150 mm Hg. Column chromatography was performed on silica gel (230-400 mesh) using reagent grade solvents. Analytical thin-layer chromatography (TLC) was performed on glass-backed, pre-coated plates (0.25 mm, silica gel 60, F-254, EM Science). Analytical HPLC were performed on Microsorb-MV C8 reverse-phase column (250×4.6 mm, Varian) using Shimadzu LC-10A VP pump and Shimadzu SPD 10A VP UV-vis variable-wavelength detector. Preparative HPLC purifications were carried out with C8 reverse phase preparative column (Grace Davison). The flow rate for preparative reverse-phase HPLC was 4 mL/min. In all cases, 5%-95% gradients of acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) were used as eluents. Water (18 MΩ) was obtained from a Barnstead water purification system, and all buffers were 0.2 μm filtered. Nuclear magnetic resonance (NMR) spectra were collected on Varian 400 MHz instruments in the indicated solvents. The peak positions are reported with chemical shifts (δ) in parts per million (ppm) downfield from the signal for tetramethylsilane (0 ppm) and referenced to the signal resulting from the incomplete deuteration of a solvent used in the experiment (CDCl3: 7.26 ppm, or the center line of the multiplet of DMSO-D6: 2.50 ppm). Carbon-13 chemical shifts are reported as δ values in ppm and referenced to the cabon-13 signal of a solvent used in the experiment (CDCl3: 77.0 ppm, or the center line of the multiplet DMSO-D6: 39.51 ppm). The coupling constants (J) are reported in Hertz (Hz). The following abbreviations are used: singlet (s), doublet (d), triplet (t), doublet of doublets (dd), multiplet (m). Mass spectra were obtained from the Agilent 6520 time-of-flight mass spectrometer.
A mixture of 2,4-dichlorophenol 1 (4.1 g, 25 mmol), 1,3-dibromopropane (10 g, 50 mmol) and a solution of sodium hydroxide (1.0 g) in water (4 mL) was stirred at reflux for 1.5 h. A solution of sodium hydroxide (1.0 g) in water (6 mL) was added and the mixture was refluxed for an additional 1.5 h. After cooling, the reaction mixture was extracted with chloroform (50 mL) and washed with water (30 mL×3). The organic layer was dried over sodium sulfate and evaporated in vacuo. Crude product was obtained (10.49 g) and then purified by silica gel column (79.16 g). Yield 10.7% (0.794 g).
To a solution of 1-(3-bromopropoxy)-2,4-dichlorobenzene (600 mg, 2.11 mmol) in 6.0 mL DMF in a 25 mL round-bottom flask equipped with a stir bar, a thermocouple in a thermowell and a rubber septum stopper with sleeve, 234.0 mg (3.60 mmol) of NaN3 was added at room temperature. Under N2 pressure, the mixture was stirred overnight. The formation of off-white suspension was observed. 20 μL of the reaction mixture was partitioned with 0.5 mL MTBE and 0.5 mL water, and the MTBE layer was used for TLC (silica gel, 100% hexane). The rest of the reaction mixture was partitioned with MTBE and water. The water layer was washed by MTBE. The MTBE layer were washed sequentially with water and NaHCO3, and then used in the next step without further purification.
The MTBE layer obtained from the previous step was transferred into a 500 mL round-bottom flask equipped with a stir bar and a rubber septum stopper with sleeve. In N2 atmosphere, Boc2 (571.0 mg, 2.616 mmol) and Pd/C (518 mg) was added. N2 was carefully replaced by H2. A rubber balloon was used to keep the system under positive gas pressure. TLC (silica gel, 100% hexane to detect starting material and MTBE: hexane=1:1 to detect the product, ninhydrin stain) was used to follow the process of the reaction. Under N2 pressure, the mixture was stirred for 21 h. The reaction mixture was filtered under vacuum through glass microfiber and Celite 545. The filtrate was partition by MTBE and water. The water layer was washed by MTBE. The MTBE layers were washed sequentially with water, saline and NaHCO3, dried by MgSO4, filtered and evaporated. Crude product (991.2 mg) was obtained after work up and purified by silica gel column. Yield 11.4% (77.3 mg).
To a solution of tert-butyl 3-(2,4-dichlorophenoxy)propylcarbamate (77.3 mg, 0.24 mmol) in 0.8 mL DMF in a 20 mL vial equipped with a stir bar and a rubber septum stopper with sleeve and under Ar pressure, NaH (11.9 mg, 0.30 mmol) was added with cooling by ice bath. The reaction mixture was kept under Ar atmosphere at all times. Propargyl bromide in toluene (44.5 mg, 0.30 mmol) was added. The reaction mixture was stirred at room temperature. Next, a small portion of the reaction mixture (10-15 μL) was partitioned with 350 μL MTBE and 350 μL water, and the MTBE layer was used for TLC (silica gel, hexane: MTBE=1:1). The rest of the reaction mixture was partitioned with 20 mL MTBE and 20 mL water. The water layer was washed by 20 mL MTBE. The MTBE layers were washed sequentially with water, saline and NaHCO3, dried by MgSO4, filtered and evaporated. Crude product was purified by silica gel column (1.23 g). Yield 27.4% (23.7 mg).
To a solution of tert-butyl 3-(2,4-dichlorophenoxy)propyl(prop-2-ynyl)carbamate (23.7 mg, 66.1 μmol) in 600 μL DMF in a 20 mL vial equipped with a stir bar, 600 μL TFA was added at room temperature while stirring. In 0.5 h, TLC (MTBE: hexane=1:1, ninhidrin stain) indicated the completion of the reaction. The volatiles were evaporated. The residue was co-evaporated with ACN for 3 times and then used in the next step without further purification.
Structure of the product was proved by NMR and LC-MS.
A 250 mL three-neck round-bottom flask equipped with a thermocouple in a glass sleeve, a magnetic stirrer, a vigreux column with an Argon inlet (middle stem) and a sleeved rubber septum stopper was assembled and dried with a heat gun under flow of Ar. Approximately 110-120 mL of anhydrous DMF was added via cannula under Ar. AcSK (11.68 g, 102.3 mmol) was added by portions into the flask while cooled with ice-MeOH bath. The reaction went on for 7 h at about −10° C. The ice-MeOH bath was removed after quenching the reaction by adding 165 mL water. The reaction mixture was partitioned with 300 mL MTBE and 700 mL water. The water layer was washed by 200 mL MTBE. The MTBE layers were washed sequentially with water, saline and NaHCO3, dried by MgSO4, filtered and evaporated. Yield 98.7% (19.1 g).
To a solution of N-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium 2,2,2-trifluoroacetate (2.14 mg, 0.05 mmol) in 100 μL ACN in a 5 mL vial equipped with a stir bar, 12.1 mg (0.09 mmol) of K2CO3 and 142.4 mg (0.720 mmol) S-3-bromopropyl ethanethioate was added. The mixture was stirred while heated to 50° C. in an oil bath. TLC was performed (silica gel, MTBE: hexane=9:1 to detect starting material and MTBE: hexane=1:9 to detect the consumption of the thioacetate reagent) to follow the process of the reaction. To a solution of N-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium 2,2,2-trifluoroacetate (17.1 mg, 0.05 mmol) in 800 μL ACN in a 20 mL vial equipped with a stir bar, 120.0 mg (0.870 mmol) of K2CO3 and 95.2 mg (0.48 mmol) S-3-bromopropyl ethanethioate was added. The mixture was stirred while heated by 50° C. oil bath for 5 h. The reaction mixtures of the two reactions were combined, filtered and evaporated. Crude product (168.4 mg) was obtained and co-evaporated with hexane for 3 times to remove ACN. Silica gel column (1.25 g) was used to purify the crude product. Yield 14% (2.6 mg).
Structure of the product was proved by NMR and LC-MS
A solution of S-3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propyl ethanethioate (1.17 mg, 3.10 μmol) in 200 μL ACN was added into a 20 mL vial equipped with a stir bar, evaporated and then co-evaporated with MeOH for 3 times to remove ACN. MeOH/HCl (200 μL) was added into the vial and then the vial was heated by 85° C. oil bath for 6 h. The reaction mixture was evaporated, co-evaporated sequentially by MeOH for 3 times and ACN for 3 times, and then used in the next step without further purification.
MHI-148 (4.7 mg, 6.2 mmol) and EDC (1.5-2.4 mg, 7.8-12 mmol), followed by 1.5 mg of DMAP (12 mmol) were added into a 20 mL vial equipped with a stir bar. ACN (400 μL) was added to make solution. The reaction mixture of the previous step was transferred dropwise to the vial with 200 μL ACN at room temperature. The reaction mixture was purified by HPLC (GRACE Davison Apollo C8 5 u column, 250 mm×10 mm).
Our preliminary data suggest that MAO-A is closely related to prostate cancer (PCa) metastasis to bone and for the first time demonstrate that MAO-A protein expression was elevated in PCa bone metastasis relative to normal and low Gleason grade cancerous epithelium (
Manipulation of levels of MAO-A expression in human bone-metastatic PC-3 and ARCaPM PCa cells resulted in altered tumor growth in mice. PC-3 cells overexpressing wild-type MAO-A enhanced its growth whereas ARCaPM cells with specific lentiviral shRNA-mediated silencing completely abrogated the growth of this invasive and bone-metastatic PCa tumor in mice (
Our experiments with murine prostate carcinoma TRAMPC-2 (neuroendocrine phenotype), subcutaneously injected into the MAOA neo mice, showed significantly reduced growth rate of PCa (
Specifically in the bone microenvironment, knockdown of MAO-A in two castration-resistant human PCa cell lines, ARCaPM and C4-2, also significantly reduced cancer-induced local bone destruction by osteolytic lesions (
Mechanistically, MAO-A was found to induce epithelial-to-mesenchymal transition (EMT) in human PCa cells, by promoting the loss of E-cadherin expression (an epithelial marker), up-regulation of Vimentin-N-cadherin (mesenchymal markers) and increased migration and invasion in PC-3 cells (
We also observed that overexpression of MAO-A enhanced hypoxia-inducible factor 1α (HIF1α) expression, and select HIF1α target genes known to promote PCa progression and metastasis, such as VEGF and EMT-promoting genes (Snail2 and Twist1), are also influenced by MAO-A in PCa cells (
A class of fluorescent heptamethine cyanines with near-infrared (NIR) emission maxima, such as MHI-148 dye, which has been identified recently, are non-toxic and have dual functions as tumor-specific targeting and imaging modalities. These dyes, partially mediated by hypoxia, are specifically retained in cancer but not normal cells, and also in tumor xenografts as well as spontaneous tumors in transgenic mice. We have shown enhanced uptake of MHI-148 NIR dye in MAO-A-overexpressing PC-3 tumor xenografts (
Although the present invention has been described in terms of specific exemplary embodiments and examples, it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims.
The following references are incorporated herein by reference:
This application is a continuation of U.S. application Ser. No. 13/559,431 (now U.S. Pat. No. 9,771,625; issued Sep. 26, 2017) filed on Jul. 26, 2012, which claims the benefit of priority of U.S. Provisional Application No. 61/511,920 filed Jul. 26, 2011, all of which are incorporated herein by reference in their entirety.
This invention was made with government support under Contract Nos. P01-CA98912, DAMD-17-03-02-0033, R01-CA122602, R01-MH39085 awarded by the National Institute of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61511920 | Jul 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13559431 | Jul 2012 | US |
| Child | 15688497 | US |
