Mermelstein, Fred H. (8 Skyview Terrace Clifton, NJ, 07013, US)
|1.||A method of treating solid tumors in a mammal which comprises administering a therapeutically effective amount of one or more arsenic compounds to said mammal.|
|2.||A method of treating metastatic neoplastic disease in a mammal which comprises administering a therapeutically effective amount of one or more arsenic compounds to a mammal in need of such therapy.|
|3.||A method of treating melanoma, breast, colon, ovarian, renal, central nervous system, bladder, prostate or lung cancer in a human which comprises administering a therapeutically effective amount of one or more arsenic compounds to said human.|
|4.||The method of claim 1 wherein said tumor is a tumor of epithelial tissue, lymphoid tissue, connective tissue, bone or central nervous system.|
|5.||The method of claim 2 wherein said metastatic neoplastic disease is a disease of epithelial tissue, lymphoid tissue, connective tissue, bone or central nervous system.|
|6.||The method of claim 4 wherein said tumor of the epithelial tissue is a tumor of epithelial glands, epithelial ducts, liver, biliary tract, gastrointestinal tract, respiratory tract, or urogenital tract.|
|7.||The method of claim 4 wherein said tumor is squamous cell carcinoma of the esophagus, adenocarcinoma of the esophagus, colorectal carcinoma, or gastric carcinoma.|
|8.||The method of claim 4 wherein said tumor of the lymphoid tissue is Hodgkins lymphoma, nonHodgkin's lymphoma, follicular lymphoma, diffuse lymphoma, lymphoblastic lymphoma, large cell lymphoma, or small lymphocytic lymphoma.|
|9.||The method of claim 4 wherein said tumor of the central nervous system is selected from the group consisting of neuroblastoma, retinoblastoma, glioblastoma or oligodendroglioma.|
|10.||The method of claim 1,2 or 3 wherein said arsenic compound is an ionic aqueous solution of arsenic.|
|11.||The method of claim 1,2 or 3 wherein said arsenic compound is selected from the group consisting of arsenic trioxide and Fowler's solution.|
|12.||The method of claim 1,2 or 3 wherein the total daily amount administered is from about 10 pg to about 200 mg.|
|13.||A method for treating neoplastic diseases in a human which comprises administering to a human an effective amount of an arsenic compound, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of at least one other therapeutic.|
|14.||The method of claim 12 in which said other therapeutic agent is a chemotherapeutic or radiotherapeutic.|
|15.||The method of claim 1,2,3, or 12 in which said administration is made parenterally, topically, dermally, directly into the tumor or orally.|
|16.||The method of claim 12 in which said other therapeutic agent is selected from the group consisting of etoposide, cisplatin, carboplatin, estramustine phosphate, vinblastine, methotrexate, hydroxyurea, cyclophosphamide, doxorubicin, 5fluorouracil, taxol, diethylstilbestrol, VM 26 (vumon), BCNU, alltrans retinoic acid, procarbazine, cytokines, therapeutic vaccines, and other immunomodulators.|
|17.||The method of claim 1,2,3 or 12 in which said administration is made via an implantation device.|
|18.||A method of treating hematopoietic disorders in a mammal which comprises administering one or more arsenic compounds to said mammal, wherein said hematapoietic disorder is selected from the group consisting of acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, myeloid metaplasia, myeloid dysplastic syndrome, multiple myeloma and plasmacytoma.|
|19.||An oral pharmaceutical composition useful for treating neoplastic diseases in a human which comprises an effective amount of an arsenic compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.|
|20.||A sterile unit dosage form adapted for parenteral administration which comprises a therapeutically effective nonlethal amount of arsenic trioxide in an aqueous carrier, said dosage form being contained in a sealed sterile glass container.|
More specifically, the present invention relates to novel chemotherapeutic methods--novel uses of arsenic compounds for treating primary and metastatic tumors; primary and metastatic tumors of the central nervous system; refractory primary and metastatic tumors of the central nervous system; breast, lung, bladder and prostate cancer; and refractory breast, lung, bladder and prostate cancer to mention a few.
2. BACKGROUND OF THE INVENTION In 1997 more than one million people will develop some type of cancer in the United States. Approximately 500,000 will be cured or in a state of remission. These numbers represent an improving cure rate seen over the past decade which is largely due to earlier detection, better treatment and advances in chemotherapy. In particular, the advances in chemotherapy include targeted or specific drug therapy in which a drug is developed specifically for the treatment of a certain cancer type. This"disease-oriented" approach is designed to identify compounds which exert selective effects in vitro on particular tumor types and to follow-up these leads in vivo utilizing cell lines, (Fiebig et al., Cancer Treatment Reviews 17: 109-117 (1990)).
However, the incidence of cancer is continuing to climb as our population ages and as new cancers develop or occur more
frequently, such as in patients infected with AIDS virus.
Thus, it is clear that there is a tremendous demand for additional regimens to treat patients with cancer.
2.1. Pathobiolocrv of Cancer Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis).
Clinical data and molecular biologic studies indicate that cancer is a multistep process that begins with minor preneoplastic changes, which may under certain conditions progress to neoplasia.
Pre-malignant abnormal cell growth is exemplified by hyperplasia, metaplasia, or most particularly, dysplasia (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
The neoplastic lesion may evolve clonally and develop an increasing capacity for invasion, growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host's immune surveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12).
2.2. AIDS-related Non-Hodakin's Lymphoma Since the discovery of AIDS, the disease has had a close association with an interesting spectrum of cancers.
Further, the types of malignancies and their incidence rates are increasing as the development of effective antiretroviral therapies and prophylaxis against opportunistic infections leads to prolonged survival in the immunodeficient state for AIDS patients, (Karp and Broder, Cancer Res. 51: 4747-4756 (1991)). AIDS-related non-Hodgkin's lymphoma was found to occur in AIDS patients only after 1981. AIDS-related non- Hodgkin's lymphoma is a very aggressive disease with a very high incidence of central nervous system involvement. It is increasing in incidence in the AIDS population. As patients infected with the AIDS virus now live longer because they are not dying of the usual infections, they are developing lymphoma at an increasing rate. The characteristics of AIDS- related non-Hodgkin's lymphoma are detailed in an article by Karp and Broder, (1991), supra.
The problems the medical oncologist has in treating patients with AIDS-related lymphomas is the recently described predilection for occurrence of the lymphoma in the central nervous system (in brain and surrounding meninges) and the fact that the patient with AIDS has a very weak bone marrow which cannot tolerate treatment with standard chemotherapy. This makes the treatment of lymphoma in patients with AIDS very difficult because standard chemotherapeutic agents are usually very marrow suppressive and do not cross blood brain barrier (to treat the central nervous system disease).
2.3. Primary and Metastatic CNS Tumors The incidence of primary and metastatic brain tumors is increasing in the United States. Indeed, the arsenal of chemotherapeutics for these types of cancers is minimal, while the need for such therapeutics is high.
Glioblastoma multiform and other primary and metastatic central nervous system tumors are devastating malignancies. The treatment of these tumors include surgery, radiation therapy and treatment with agents such as the nitrosourea BCNU. Other chemotherapeutic agents utilized include procarbazine, vincristine, hydroxyurea and cisplatin.
Unfortunately, even when all three modalities (surgery, radiation therapy and chemotherapy) are utilized, the average survival of patients with central nervous system malignancies is still about 57 weeks. Clearly, new treatment approaches are needed both for patients with newly diagnosed primary and metastatic central nervous system tumors, as well as for patients with such tumors which are refractory to the above modalities. Finding such new agents has been complicated by the fact that there is no animal model which appears to predict what agent will be clinically effective against primary and metastatic central nervous system tumors.
2.4. Breast, Lung, Bladder and Prostate Cancers Breast cancer has been known to occur in about one in every 8-9 women in the United States. The treatment for early breast cancer is surgery, with or without radiation therapy, or surgery, with or without radiation therapy, plus chemotherapy and/or hormonal therapy. Despite the best efforts of physicians there are still more than 80,000 deaths each year from breast cancer and the incidence is still rising. Current chemotherapy for patients with primary or metastatic breast cancer includes treatment with cyclophosphamide, methotrexate, doxorubicin, 5-fluorouracil, cisplatin, vinblastine, taxol, taxotere, mitomycin C and occasionally other agents. Unfortunately, even with these agents, almost all women who develop metastatic breast cancer
succumb to their disease. One particular place that metastatic breast cancer does metastasize to is the central nervous system. When central nervous system metastases do occur, the usual treatment is surgery (for a solitary metastasis) or radiation, or surgery plus radiation therapy.
At present there is no chemotherapy which is felt to be helpful in this situation.
Lung cancer is responsible for more than 150,000 deaths each year in the United States. Most patients with lung cancer present a tumor that has already metastasized to a variety of organs, including lung, liver, adrenal gland and other organs. The current treatment for metastatic lung cancer is not yet standardized (Ihde, Daniel C., "Chemotherapy of Lung Cancer", The New England Journal of Medicine 327: 1434-1441,1992 November 12th issue). However, chemotherapy regimens which are utilized include treatment with cisplatin plus etoposide, combinations of cyclophosphamide plus doxorubicin plus cisplatin, and single agents alone or in combination, including ifosfamide, teniposide, vindesine, carboplatin, vincristine, taxol, nitrogen mustard, methotrexate, hexamethylmelamine and others. Despite these chemotherapeutic regimens the average patient with metastatic lung cancer still only survives 7-12 months. One particular troublesome place for metastases of lung cancer is the central nervous system. The treatment for central nervous system metastases includes surgery (to remove a solitary lesion), radiation therapy, or a combination of both. Unfortunately, there is not standard chemotherapy which is felt to be helpful in this situation.
Each year about 11,000 patients die of bladder cancer in the U. S. Although at presentation the disease is usually localized, most patients develop distant metastatic disease. The most recent advances have been in the area of chemotherapy for patients with such metastatic disease. One effective regimen is called the MVAC regimen. It consists of treatment with methotrexate plus vinblastine plus adriamycin (doxorubicin) plus cisplatin. Although the response rate is
high to this chemotherapeutic regimen, medical oncologists are noting that one place the patients fail is with metastases to the central nervous system. Unfortunately, there is no standard chemotherapy which is felt to be helpful in this situation.
It is estimated that more than 100,000 men will be diagnosed with prostate cancer this year and more than 30,000 patients will die from the disease. The most common sites of metastases in patients with prostate cancer are the bone and lymph nodes. The bone metastases are particularly bothersome in that they can create intense pain for the patient. The current treatment for metastatic prostate cancer includes treatment with flutamide, leuprolide, diethylstilbestrol, and other hormonal manipulations, as well as chemotherapy (doxorubicin, estramustine phosphate, vinblastine, suramin, cisplatin, and others). Unfortunately, none of these agents are consistently helpful in the disease. In addition, as patients with prostate cancer live longer with their malignancy, they will most likely develop a higher incidence of metastases to the central nervous system (including the spinal cord).
In general, as patients are living longer with the common malignancies such as breast cancer, lung cancer, bladder cancer, prostate cancer and a variety of other malignancies (because of control of their systemic disease with surgery, radiation therapy and chemotherapy), oncologists are noting that they are developing an increasing incidence of metastatic tumors in the central nervous system including the brain. This is probably because most of the currently available chemotherapy does not cross the blood brain barrier. When the patient (who has their tumor controlled in sites outside of the brain) develops brain metastases, it is a very difficult situation. Options for that patient are usually limited to surgery for a solitary metastasis and/or radiation therapy. However, after those modalities fail, the patient usually has no other options.
For each of the above indications (primary brain tumors and metastases to the brain from other common tumors such as breast, lung, bladder and prostate cancers), there is a tremendous need for a more effective treatment and/or methods for improving the quality of patient life.
2.5. Esophaqeal Cancer In the U. S., carcinoma of the esophagus represents about 6% of all cancers of the gastrointestinal tract but causes a disproportionate number of cancer deaths. (Boring, C. C., et al.: Cancer statistics, 1993. CA Cancer J. Clin.
43: 7,1993). These cancers usually arise from the epithelial layer of the esophagus and are either squamous cell carcinomas or adenocarcinomas. Overall the 5 year survival is about 5%.
Squamous cell carcinoma generally occurs after the age of 50 and is more common in males than in females. The incidence varies widely from country to country and between regions within countries. In the U. S. the incidence is between 2 and 8 persons per 100,000 and is more prevalent in blacks than in whites.
Adenocarcinoma represents 25% of all esophageal CA in the U. S. It is usually located in the distal one third of the esophagus and may invade the adjacent gastric cardia. It tends to occur in people over 40 years of age and is more common in males than in females. It is more common in whites than in blacks.
2.6. Arsenic And Its Medical Uses Arsenic has been considered to be both a poison and a drug for a long time in both Western and Chinese medical practices. In the latter part of the nineteenth century, arsenic was used frequently in attempts to treat diseases of the blood in the West. In 1878, it was reported that treatment of a leukemic patient with Fowler's solution (a solution of potassium arsenite) reduced markedly the count of white blood cells (Cutler and Bradford, Am. J. Med. Sci.,
January 1878,81-84). Further interests in the use of Fowler's solution as a palliative agent to treat chronic myelogenous leukemia (CML) was described by Forkner and Scott in 1931 (J. Am. Med. Assoc., 1931, iii, 97), and later <BR> <BR> <BR> confirmed by Stephens and Lawrence in 1936 (Ann. Intern. Med. _ 9,1488-1502). Typically, Fowler's solution was orally administered to leukemic patients as a solution until the level of white blood cells was depressed to an acceptable level or until toxicities (such as skin keratoses and hyperpigmentation) developed, while the patients enjoyed varying periods of remission. In the 1960's, Fowler's solution was still used occasionally in attempts to treat CML, however, most patients with CML were treated with other chemotherapeutic agents, such as busulfan, and/or radiation therapy (Monfardini et al., Cancer, 1973,31: 492-501).
Paradoxically, one of the long recognized effects of exposure to arsenic, whether the source is environmental or medicinal, is skin cancer (Hutchinson, 1888, Trans. Path.
Soc. Lond., 39: 352; Neubauer, 1947, Br. J. Cancer, 1: 192).
There were even epidemiological data to suggest that the use of Fowler's solution over long periods could lead to an increased incidence of cancer at internal sites (Cuzick et al., Br. J. Cancer, 1982,45: 904-911; Kaspar et al., J. Am.
Med. Assoc., 1984,252: 3407-3408). The carcinogenicity of arsenic has since been demonstrated by the fact that it can induce chromosomal aberration, gene amplification, sister chromatid exchanges and cellular transformation (See e. g., Lee et al., 1988, Science, 241: 79-81; and Germolec et al., Toxicol. Applied Pharmacol., 1996,141: 308-318). Because of the known carcinogenic effect of arsenic, its only therapeutic use in human in Western medicine today is in the treatment of tropical diseases, such as African trypanosomiasis, (melarsoprol, or Arsobal by Rhône Poulenc Rorer, Collegeville, PA; See Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th edition, chapter 66,1659-1662,1997).
In traditional chinese medicine, arsenous acid or arsenic trioxide paste has been used to treat tooth marrow diseases, psoriasis, syphilis and rheumatosis (Chen et al., 1995, in Manual of Clinical Drugs, Shanghai, China, Shanghai Institute of Science and Technology, p. 830). In 1970s, arsenic trioxide had been applied experimentally to treat acute promyelocytic leukemia (APL) in China (commented by Mervis, 1996, Science, 273: 578). The clinical efficacy of arsenic trioxide has recently been re-investigated in 14 of 15 patients with refractory APL, where the use of an intravenous dose at 10 mg/day for 4-9 weeks was reported to result in complete morphologic remission without associated bone marrow suppression (Shen et al., 1997, Blood, 89: 3354- 3360). It was also reported that arsenic trioxide induced apoptosis (programmed cell death) in vitro in NB4 cells, an APL cell line, and that apoptosis was apparently associated with down-regulation of the oncogene bcl-2, and intracellular redistribution of the chimeric PML/RARa protein that are unique to APL cells (Chen et al., 1996, Blood, 88: 1052-1061; Andre et al., 1996, Exp. Cell Res. 229: 253-260). Similarly, melarsoprol has been reported to induce apoptosis in cell lines representative of chronic B-cell leukemia (Konig et al., 1997, Blood 90: 562-570). Whether apoptosis is induced in APL patients is presently unclear, but it is believed by some to be one of the possible mechanisms of the therapeutic effects of certain arsenic compounds.
Although arsenic is well known to be both a poison and a carcinogenic agent, there have been many reports concerning the use of arsenic in medical treatment.
Identification or discussion of the art above must not be construed as an admission that such is prior art.
Further, from the above discussion, it should be clear that there are a plethora of different types of cancers, each of which requires a unique treatment protocol.
Thus, the development of a broad spectrum anti-cancer agent is extremely desirable. At a minimum, additional effective
anti-cancer agents are needed to be added to the arsenal against cancer.
3. SUMMARY OF THE INVENTION Notwithstanding the conflicting reports in the art concerning benefits and risks of the administration of arsenic to patients, applicants have discovered that arsenic compound has broad applicability in the treatment of various cancers, including solid tumors and blood disorders. For example, the present invention encompasses the use of arsenic in the form of a salt, complex, organic compound or ionic solution to treat tumors of epithelial tissue, connective tissue, central nervous system, lymphoid tissue, hematopoietic cells and tumors associated with oncogenic viruses.
Further, the present invention encompasses the use of arsenic compounds to treat mammals suffering from primary and metastatic neoplastic disease as well as infectious diseases related thereto.
In addition, this invention also encompasses the use of arsenic compounds to treat primary and metastatic breast, lung, bladder and prostate cancers in humans.
This invention also encompasses the treatment of hematopoietic disorders in mammals by the administration of one or more arsenic compounds to said mammal. The hematopoietic disorders to be treated include but are not limited to polycythemia vera, Hodgkin's Disease, non- Hodgkin's Disease including Follicular Lymphoma, Diffuse Lymphoma, lymphoblastic lymphoma, small lymphocytic lymphoma, acute lymphocytic leukemia, hairy cell leukemia, myeloid metaplasia, myeloid dysplastic syndrome, multiple myeloma and plasmacytoma.
In accordance with the present invention, arsenic compounds can be used alone or in combination with other known therapeutic agents (including chemotherapeutics, radioprotectants and radiotherapeutics) or techniques to either improve the quality of life of the patient, or to
treat the primary neoplastic disease. For example, the arsenic compounds can be used before, during or after the administration of one or more known antitumor agents including but not limited to mustard compounds, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, floxuridine, methotrexate, vincristine, vinblastine, taxol, etoposide, temiposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, mitomycin, cisplatin, carboplatin, estramustine phosphate, hydroxyurea, BCNU, procarbazine, VM- 26 (vumon), interferons and all-trans retinoic acid (ATRA), (See for example, the Physician Desk References 1997). In addition, the arsenic compounds can be used before, during or after irradiation treatment. For the treatment of HIV- infected individuals, the arsenic compounds can be used alone or in combination with AZT, ddI, ddA, ddC, d4T, 3TC and other known antiviral agents.
The invention described herein encompasses a method of treating primary and metastatic neoplastic diseases, a method of treating solid tumors, a method of treating leukemias, a method of treating cancers related to bcl-2 (oncogene), each of which comprises the administration of a therapeutically effective and non-lethal amount of one or more arsenic compounds to a mammal in need of such therapy.
The invention, as mentioned above also encompasses the use of combination therapy to treat the aforementioned diseases.
In a particular embodiment, the arsenic compounds are used within a method to treat breast, lung, colon, ovarian, renal, non-small cell lung, central nervous system, bladder, prostate and head and neck cancer by administering an effective amount of one or more arsenic compounds alone or in combination with other antineoplastic agents or therapeutic techniques including radiotherapy and surgery.
Without being limited by any theory, the inventors believe that the arsenic compounds of the invention may have one or more mechanisms of action in connection with the methods described herein. For example, the arsenic compounds
may act as a phosphorous analogue which interferes with the phosphorylation events that occur in signal transduction involved in apoptosis. Arsenic may also act as an inhibitor of angiogenesis, i. e., the formation of new blood vessels, thereby limiting blood flow to proliferating preneoplastic cell masses, tumors and metastases. It is well known that if a tumor is not invaded by blood capillaries, it would have to depend on the diffusion of nutrients from its surroundings and cannot enlarge beyond a certain size. Arsenic may also function as a differentiating agent which causes dividing preneoplastic and/or cancer cells that display an undifferentiated or underdifferentiated phenotype to develop into terminally differentiated cells, and die after a finite number of cell divisions. Finally, arsenic may also act to sensitize the cancer cells to radiation and/or chemotherapy.
Thus, the arsenic compounds of the invention are described as being useful against a variety of cancers.
Specific therapeutic regimens, pharmaceutical compositions, and kits are also provided by the invention.
Thus, the invention also encompasses pharmaceutical compositions which comprise one or more arsenic compounds and a pharmaceutically acceptable carrier. The compositions are sterile solutions suitable for intravenous injection or infusion. In another embodiment the invention encompasses a composition suitable for oral delivery; comprising one or more arsenic compounds and a pharmaceutically acceptable excipient or carrier. In another embodiment, the invention also includes compositions suitable for topical or dermal delivery.
Particular compositions of the invention and their properties are described in the sections and subsections which follow.
4. BRIEF DESCRIPTION OF THE FIGURES Figure 1A-1I. Dose response curves showing percentage growth of various cancer cell lines after continuous exposure to 10-5 to 10-9ßg/ml of arsenic trioxide
for 2 days. Figure 1A. Leukemic cell lines CCRF-CEM, HL- 60 (TB), K-562, MOLT-4, RPMI-8226, SR. Figure 1B. Non-Small Cell Lung Cancer cell lines A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-8322M, NCI-H460, NCI-H522. Figure 1C.
Colon Cancer cell lines COLO 205, HCT-116, HCT-15, HT29, KM12, SW620. Figure 1D. CNS Cancer cell lines SF-268, SF- 295, SF-539, SNB-19, SNB-75, U251. Figure 1E. Melanoma cell lines LOX 1MV1, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62. Figure 1F. Ovarian Cancer cell lines IGROV1, OVCAR-3, OVCAR-5, OVCAR-8, SK-OV-3. Figure 1G.
Renal Cancer cell lines A498, CAKI-1, RXE 393, SN12C, TX-10, UO-31. Figure 1H. Prostate Cancer cell lines PC-3, DU-145.
Figure 1I. Breast Cancer cell lines MCF7, NCI/ADR-RES, MDA- MB-435, MDA-N, BT-549, T-47D.
Figure 2. Mean graphs showing selectivity patterns at each of the principal response parameters for all the cell <BR> <BR> lines tested after continous exposure to 10-5 to 10-9 gg/Ml Of arsenic trioxide for 2 days.
Figure 3A-3I. Dose response curves showing percentage growth of various cancer cell lines after continuous exposure <BR> <BR> to 10-5 to 10-9ßg/ml of arsenic trioxide for 6 days. Figure 3A. Leukemic cell lines CCRF-CEM, K-562, MOLT-4, RPMI-8226.
Figure 3B. Non-small Cell Lung Cancer cell lines EKVX, HOP- 62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522.
Figure 3C. Colon Cancer cell lines COLO 205, HCT-116, HCT- 15, HT29, KM12, SW-620. Figure 3D. CNS Cancer cell lines SF-268, SF-295, SF-539, SNB-75, U251. Figure 3E. Melanoma cell lines LOX IMVI, MALMI-3M, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62. Figure 3F. Ovarian Cancer cell lines IGROVI, OVCAR-3, OVCAR-5, OVCAR-8, SK-OV-3. Figure 3G.
Renal Cancer cell lines 786-O, A498, CAKI-1, RXF 393, S12C, TK-10. Figure 3H. Prostate Cancer cell lines DU-145.
Figure 3I. Breast Cancer cell lines MCF7, NCI/ADR-RIS, MDA- MB-231/ATCC, HS 578T, MDA-MB-435, MDA-N, BR-549, T-47D.
Figure 4. Mean graphs showing selectivity patterns at each of the principal response parameters for all the cell
lines tested after continous exposure to 10-5 to 10-9Mg/ml of arsenic trioxide for 6 days.
5. DETAILED DESCRIPTION OF THE INVENTION Methods and compositions for the treatment of primary and metastatic neoplastic diseases are described herein. The invention is based, in part, on a dosage regimen for administration of compositions comprising arsenic. The invention is also based in part, on the potency of the arsenic compounds of the invention against certain cancers.
This invention includes a method of treating primary solid tumors in a mammal which comprises administering to a mammal in need of such therapy a therapeutically effective and non-lethal amount of one or more arsenic compound.
The invention also includes a method of treating metastatic tumors in a mammal which comprises administering to a mammal a therapeutically effective and non-lethal dose of one or more arsenic compound.
The invention includes a method for treating disorders of blood in mammal which comprises administering one or more arsenic compound in a therapeutically effective and non-lethal amount.
The arsenic compound of the invention may be utilized in in a variety of known forms; for example, arsenic can be administered as a salt, an organic or inorganic complex, an organic chelate, an organic compound or an organic or inorganic solution. It is preferred that the form be chosen to reduce toxicity and improve efficacy. Further, the form chosen may also depend on the type and location of the tumor in question. The inorganic salt forms of arsenic are preferred. For example, inorganic salts such as arsenic triiodide, arsenic (III) bromide, arsenic (III) chloride, arsenic pentoxide, arsenic trioxide, Fowler's solution (potassium arsenite), sodium arsenite, and calcium arsenite may be used.
Arsenic trioxide is most preferred. Both arsenous acids and arsenites as well as arsenic acids and arsenates may be used
within the present methods. Aqueous solutions containing arsenite ions are preferred. Further, arsenic sulfides may be used such as arsenous sulfide, arsenic sulfide, arsenic pentasulfide, tetraarsenic trisulfide and tetraarsenic pentasulfide. Without being limited by any theory, certain of these arsenic compounds may be prodrugs to an active species.
Generally, the skilled artisan will recognize that the form of arsenic to be used should be therapeutically effective without unreasonable toxicity. The toxicity is dependent upon the dose, the dosage form, the mode of administration and frequency of dosing. Generally, the skilled artisan can chose from the following known forms of arsenic: arsenic halides, arsenic oxides, arsenic acids, arsenic sulfides, and the like.
Arsenic can also be readily combined with carbon to form a wide variety of organic compounds. These include but are not limited to primary and secondary arsines, tertiary arsines, halo arsines, dihalo arsines, cyclic and polymeric substances containing arsenic; specific examples of organic arsenic compounds include but are not limited to 3-Nitro-4- hydroxyphenylarsonic acid, arsanilic acid, sodium hydrogen 4-aminophenylarsenate, melarsoprol, melarsonyl potassium, carbarsone, arsenamide arsphenamine and sodium arsanilate.
As used herein,"arsenic compound"refers to a pharmaceutically acceptable form of arsenic including salts, solutions, complexes, chelates and organic and inorganic compounds incorporating arsenic. It should be recognized that the invention includes arsenic prodrugs or compounds that are converted in vivo to biologically active forms of arsenic. Such prodrugs may be used to reduce or avoid the well known potential for arsenic toxicity. The arsenic compounds of the present invention can be synthesized or commercially purchased. For example, the compounds can be prepared from well-known chemical techniques. (See for example, Kirk-Othmer, Encyclopedia of Chemical Technology 4 ed. volume 3 pps. 633-655 John Wiley & Sons).
In one embodiment, the arsenic compound of the invention is arsenic trioxide which is dissolved in an aqueous solution of sodium hydroxide, with the pH adjusted to a physiologically acceptable range, e. g. about pH 6-8.
Any suitable mode of administration may be used in accordance with the present invention including but not limited to parenteral administration such as intravenous, subcutaneous, intramuscular and intrathecal administration; oral, intranasal, rectal or vaginal administration may also be used; directly into the tumor; transdermal patches; implant devices (particularly for slow release); finally, topical administration may be used. The mode of administration will vary according to the type of arsenic compound being used and the disease to be treated.
The pharmaceutical compositions to be used may be in the form of sterile physiologically acceptable (aqueous or organic) solutions, colloidal suspensions, creams, ointments, pastes, capsules, caplets, tablets and cachets. The pharmaceutical compositions comprising arsenic compounds of the invention can be contained in sealed sterile glass containers and/or ampoules. Further, the active ingredient may be micro-encapsulated, encapsulated in a liposome, noisome or lipofoam alone or in conjunction with targeting antibodies. It should be recognized that delayed slow or sustained release forms of administration are also included.
The arsenic compounds of the present invention may be used against a variety of primary and metastatic neoplastic diseases including but not limited to primary and metastatic tumors of the central nervous system, breast, colon, ovaries, kidneys, lung, bladder, prostate and head and neck.
More specifically, the arsenic compounds of the present invention can be used to treat tumors of epithelial origin including but not limited to: squamous cell carcinoma basal cell carcinoma melanoma
tumors of epithelial lining of glands or ducts: adenocarcinoma papillary carcinoma papillary adenocarcinoma tumors of the liver and biliary tract: Hepatocellular carcinoma tumors of the gastrointestinal tract: squamous cell carcinoma of the esophagus adenocarcinoma of the esophagus colorectal carcinoma (colon cancer) gastric carcinoma (stomach cancer) tumors of respiratory tract: bronchogenic carcinoma small cell carcinoma large cell carcinoma tumors of the urogenital tract: transitional cell carcinomas of bladder squamous cell carcinoma of bladder carcinoma of prostate carcinoma of cervix tumors of breast tumors of blood cells and related cells (leukemias): acute and chronic lymphocytic leukemia polycythemia vera Cancers of Lymphoid tissue Malignant Lymphomas-Hodgkins Lymphoma Non-Hodgkin's Lymphoma-Follicular lymphoma Diffuse lymphoma Small lymphocytic lymphoma Large cell lymphoma Lymphoblastic lymphoma Multiple myeloma Tumors of Connective Tissue Cancers of Bone Osteosarcoma Tumors of the Nervous System Neuroblastoma
Retinoblastoma Glioblastoma Oligodendroglioma Tumors associated with oncogenic viruses Human Papillomavirus-squamous cell carcinoma of cervix Ebstein-Barr Virus-Burkitts Lymphoma B cell lymphoma's in immuno-comprised individuals Nasopharyngeal carcinoma Hepatitis B Virus-Hepatocellular carcinoma Herpes Virus 8 or Kaposi Sarcoma Herpes Virus (KSHV)-Kaposi's Sarcoma, and the like. Other neoplastic diseases known to the skilled artisan are also encompassed by the present invention including cancer of the oral cavity, larynx, kidney, testis and ovary. The skilled artisan will recognize that other cancers may be treated in accordance with the present invention.
The term"a method for treating primary and metastatic tumors of the central nervous system"as used herein means that the disease and the symptoms associated with the disease are alleviated, reduced, cured, or otherwise placed in a state of remission.
As used herein, the terms"a method for treating primary or metastatic breast, lung, bladder or prostate cancer"and"a method for treating metastases from breast, lung, bladder or prostate cancer"means that the disease and the symptoms associated with the disease are alleviated, reduced, cured, or placed in a state of remission. In addition, the term"a method for treating metastases from breast, lung, bladder or prostate cancer"means that the metastatic tumors and the symptoms associated with the disease are alleviated, reduced, cured, placed in a state of remission.
The term"refractory"when used herein means that malignancies are generally resistant to treatment or cure.
The term"refractory"when used in the above terms, means that the malignancies which are generally resistant to
treatment or cure are alleviated, reduced, cured, or placed in a state of remission.
As used herein the terms"a therapeutic agent", "therapeutic regimen","radioprotectant","chemotherapeutic" mean conventional drugs and drug therapies, including vaccines, for treating cancer, viral infections, and other malignancies, which are known to those skilled in the art.
"Radiotherapeutic"agents are well known in the art.
As used herein,"a method of treating cancer"or"a method of treating solid tumors"or"a method of treating neoplastic diseases"means that the disease and the symptoms associated with the disease are alleviated, reduced, cured, or placed in a state of remission. Further, tumor growth is inhibited and/or tumor size is reduced.
As used herein,"preneoplastic"cell refers to a cell which is in transition from a normal to a neoplastic form; and morphological evidence, increasingly supported by molecular biologic studies, indicates that preneoplasia progresses through multiple steps. Non-neoplastic cell growth commonly consists of hyperplasia, metaplasia, or most particularly, dysplasia (for review of such abnormal growth conditions (See Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79).
Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. As but one example, endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. Atypical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally
large, deeply stained nuclei, and exhibit pleomorphism.
Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
Although preneoplastic lesions may progress to neoplasia, they may also remain stable for long periods and may even regress, particularly if the inciting agent is removed or if the lesion succumbs to an immunological attack by its host.
The therapeutic regimens and pharmaceutical compositions of the invention may be used with additional immune response enhancers or biological response modifiers including, but not limited to, the cytokines IFN-a, IFN-, IL-2, IL-4, IL-6, TNF, or other immunostimulatants/immunomodulators. In accordance with this aspect of the invention, the arsenic compounds are administered in combination therapy with one or more of these agents.
5.1. Formulation The arsenic compounds of the invention may be formulated into pharmaceutical preparations for administration to mammals for treatment of cancer.
Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, packaged, labelled for treatment of and used for the treatment of the indicated tumor, such as human sarcomas and carcinomas, e. g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e. g., acute lymphocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia and chronic lymphocytic leukemia; and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrbmls macroglobulinemia, and heavy chain disease. Alternatively, it can be labeled for treatment of the appropriate infectious disease.
Alternatively, pharmaceutical compositions may be formulated for treatment of appropriate infectious diseases.
If the complex is water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if the resulting complex has poor solubility in aqueous solvents, then it may be formulated with a non-ionic surfactant such as Tween, polyethylene glycol or glycerine. Thus, the compounds and their physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, topical, dermal, vaginal, drug delivery device, e. g., porous or viscous material such as lipofoam, rectal administration or, in the case of tumors, directly injected into a solid tumor.
For oral administration, the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e. g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents
(e. g., lecithin or acacia); non-aqueous vehicles (e. g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e. g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e. g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e. g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e. g., magnesium stearate, talc or silica); disintegrants (e. g., potato starch or sodium starch glycolate); or wetting agents (e. g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e. g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e. g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e. g., by bolus injection or continuous infusion. Such formulations are sterile.
Formulations for injection may be presented in unit dosage form, e. g., in ampules or in multi-dose containers, with an
added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e. g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e. g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
The invention also provides kits for carrying out the therapeutic regimens of the invention. Such kits comprise in one or more containers therapeutically effective amounts of the arsenic compounds in pharmaceutically acceptable form. The arsenic compound in a vial of a kit of the invention may be in the form of a pharmaceutically acceptable solution, e. g., in combination with sterile saline, dextrose solution, or buffered solution, or other
pharmaceutically acceptable sterile fluid. Alternatively, the complex may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e. g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for injection purposes.
In another embodiment, a kit of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of arsenic compounds by a clinician or by the patient.
The magnitude of a therapeutic dose of an arsenic compound in the acute or chronic management of cancer will vary with the severity of the condition to be treated and the route of administration. The dose, and perhaps dose frequency, will also vary according to the age, body weight, condition and response of the individual patient. In general, the total daily dose ranges for the conditions described herein are generally from about 10 Mg to about 200 mg administered in divided doses administered parenterally or orally or topically. A preferred total daily dose is from about 0.5 mg to about 70 mg of the active ingredient.
Desirable blood levels may be maintained by a continuous infusion of an arsenic compound as ascertained by plasma levels. It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions. Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).
Again, any suitable route of administration may be employed for providing the patient with an effective dosage of an arsenic compound. For example, oral, rectal, vaginal, transdermal, parenteral (subcutaneous, intramuscular,
intrathecal and the like) may be employed. Dosage forms include tablets, troches, cachet, dispersions, suspensions, solutions, capsules, patches, and the like. (See, Remington's Pharmaceutical Sciences.) The pharmaceutical compositions of the present invention comprise an arsenic compound as the active ingredient, or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients, for example antivirals. The term"pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases.
The pharmaceutical compositions include compositions suitable for oral, rectal, mucosal routes, transdermal, parenteral (including subcutaneous, intramuscular, intrathecal and intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated.
In the case where an intravenous injection or infusion composition is employed, a suitable dosage range for use is, e. g., from about 0.5 mg to about 150 mg total daily dose.
In addition, the arsenic carrier could be delivered via charged and uncharged matrices used as drug delivery devices such as cellulose acetate membranes, also through targeted delivery systems such as fusogenic liposomes attached to antibodies or specific antigens.
In practical use, an arsenic compound can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e. a., oral or parenteral (including tablets, capsules, powders, intravenous injections or infusions). In preparing the compositions for oral dosage form any of the usual pharmaceutical media may be employed,
e. g., water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like; in the case of oral liquid preparations, e. g., suspensions, solutions, elixirs, liposomes and aerosols; starches, sugars, micro- crystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like in the case of oral solid preparations e. a., powders, capsules, and tablets. In preparing the compositions for parenteral dosage form, such as intravenous injection or infusion, similar pharmaceutical media may be employed, e. g., water, glycols, oils, buffers, sugar, preservatives and the like know to those skilled in the art. Examples of such parenteral compositions include, but are not limited to Dextrose 5% w/v, normal saline or other solutions. The total dose of the arsenic compound may be administered in a vial of intravenous fluid, e. g., ranging from about 2 ml to about 2000 ml. The volume of dilution fluid will vary according the total dose administered.
5.2. Taraet Cancers Cancers that can be treated by the methods of the present invention include, but not limited to human sarcomas and carcinomas, e. g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e. g., acute lymphocytic leukemia (myeloblastic, myelomonocytic, monocytic and erythroleukemia); and chronic lymphocytic leukemia; and polycythemia vera, lymphoma (Hodgkin's disease and non- Hodgkin's disease), multiple myeloma, Waldenström's macroglobulinemia, and heavy chain disease. Specific examples of such cancers are described in the sections below.
In a specific embodiment the cancer is metastatic.
In another specific embodiment, the patient having a cancer is immunosuppressed by reason of having undergone anti-cancer therapy (e. g., chemotherapy radiation) prior to administration of the arsenic compounds of the invention.
In a specific embodiment, the present invention provides compositions and methods for enhancing tumor specific immunity in individuals suffering from colorectal cancer metastasized to the liver, in order to inhibit the progression of the neoplastic disease. Preferred methods of treating these neoplastic diseases comprise administering a composition of arsenic which elicits an immune response against tumor cells.
In another specific embodiment, the present invention provides compositions and methods for enhancing specific immunity in individuals suffering from hepatocellular carcinoma in order to inhibit the progression of the neoplastic disease and ultimately irradiate all preneoplastic and neoplastic cells.
In a specific embodiment, the present invention provides hsp compositions and methods for enhancing specific immunity to preneoplastic and neoplastic mammary cells in women. The present invention also provides compositions and methods for inhibiting cancer cell proliferation and metastasis. These compositions can be applied alone or in combination with each other or with biological response modifiers.
6. WORKING EXAMPLES The following subsections describe the testing of a pharmaceutical composition comprising arsenic trioxide in vitro using a panel of cancer cell lines employed by the National Cancer Institute (NCI). The results demonstrate that arsenic trioxide is effective in inhibiting the growth of a broad range of leukemic cells and cancer cells in vitro.
6.1. METHODS AND MATERIALS Arsenic trioxide (1 mg/ml, 10 mg/ampoule, manufactured by Taylor Pharmaceuticals, Decatur, Illinois) was tested at five concentrations each at 10-fold dilutions, i. e., 10-5,10-6,10-7, lu-', and 10-9 ßg/ml- The in vitro tests were performed by incubating the test cells in the presence of the indicated concentration of arsenic trioxide under standard culture conditions for a designated period of time, which is followed by a sulforhodamine B (SRB) protein assay to estimate cell viability or growth. The cell lines are organized into subpanels according to the origin of the cell lines, e. g., leukemia, breast cancer, etc. A description of the cell lines and method of testing is described in Monk et al.
(1997, Anticancer Drus Des. 12: 533-41) and Weinstein et al., (1997, Science 275: 343-9), which are incorporated herein in their entirety.
Described below are the data analysis procedures and displays.
The measurement of an effect is expressed in Percentage Growth (PG). The measured effect of the compound on a cell line is calculated according to one or the other of the following two expressions: If (Mean ODtest-Mean ODzero) > 0. then PG = 100 X (Mean ODtest-Mean ODzerO)/(Mean ODctrl-Mean OD, If (Mean ODtest-Mean ODzero) < 0. then PG = 100 X (Mean ODtest - Mean ODzero)/Mean ODzero
where: Mean ODzero = the average of optical density measurements of SRB-derived color just before exposure of cells to the test compounds; Mean ODtest = the average of optical density measurements of SRB-derived color after exposure of cells to the test compound for a designated period of time; and Mean ODctrl = the average of optical density measurements of SRB-derived color after with no exposure of cells to the test compound for a designated period of time Table 1 and 2 present the experimental data collected against each cell line. The first two columns describe the subpanel (e. g., leukemia) and cell line (e. g., CCRF-CEM) involved. The next two columns list the Mean ODzero and Mean ODCtrl; the next five columns list the Mean ODtest for each of five different concentrations. Each concentration is expressed as the log,, (molar or ßg/ml). The next five columns list the calculated PGs for each concentration. The response parameters GI50, TGI and LC50 are interpolated values representing the concentrations at which the PG is +50,0, and-50, respectively. Sometimes these response parameters cannot be obtained by interpolation. If, for instance, all of the PGs in a given row exceed +50, then none of the three parameters can be obtained by interpolation. In such a case, the value given for each response parameter is the highest concentration tested and is preceded by a">" sign. This practice is extended similarly to the other possible situations where a response parameter cannot be obtained by interpolation.
A dose-response curve (see Figures 1A-lI and 3A-3I) for the set of data is created by plotting the PGs against the loglo of the corresponding concentration for every cell line. The cell line curves are grouped by subpanel.
Horizontal lines are provided at the PG value of +50,0, and
-50. The concentrations corresponding to points where the curves cross these lines are the GI50, TGI, and LC50, respectively.
A mean graph (Figures 2 and 4) facilitates visual scanning of data for potential patterns of selectively for particular cell lines or for particular subpanels with respect to a selected response parameter. Differences in apparent selectivity patterns may occur for the same compound against the same cell lines when different parameters are compared. The mean graphs page of the data package shows mean graphs at each of the principal response parameters: <BR> <BR> <BR> <BR> GI50, TGI, and LC50. Bars extending to the right represent sensitivity of the cell line to the test agent in excess of the average sensitivity of all tested cell lines. Since the bar scale is logarithmic, a bar 2 units to the right implies <BR> <BR> <BR> <BR> the compound achieved the response parameter (e. g., GI50) for the cell line at a concentration one-hundredth the mean concentration required over all cell lines, and thus the cell line is unusually sensitive to that compound. Bars extending to the left correspondingly imply sensitivity less than the mean. If for a particular drug and cell line, it was not possible to determine the desired response parameter by interpolation, the bar length shown is either the highest concentration tested (and the listed loglo of the response parameter will be preceded by a">") or the lowest concentration tested (and the listed log,, will be preceded by a"<") The values at either limit (> or <) are also calculated in the mean used for the mean graph. Therefore, the mean used in the mean graph may not be the actual mean of the GI50 for instance. For this reason, this value is referred to as the MGMID (for mean graph midpoint).
6.2. RESULTS The results of two sets of tests is presented below. In the first set, the cells from 56 different cancer cell lines were exposed to five concentrations of arsenic trioxide continuously for two days prior to performing the SRB assay. In the second set, the cells from 50 different cell lines (a subset of the first 56 cell lines, plus the renal cancer cell line 786-0) were exposed continuously for six days prior to the SRB assay.
TABLE 1<BR> Log10 Concentration<BR> Mean Optical Densities Percent Growth<BR> Time<BR> Zero Ctrl -8.9 -7.9 -6.9 -5.9 -4.9 -8.9 -7.9 -6.9 -5.9 -4.9 G150 TGI LC50<BR> Leukemia<BR> CCRF-CEM 0.300 1.155 1.203 1.195 1.134 0.704 0.285 106 105 98 47 -5 1.11E-06 1.00E-05 >1.26E-05<BR> HL-60(TB) 0.233 0.530 0.533 0.507 0.535 0.499 0.213 101 92 102 90 -9 3.19E-06 1.03E-05 >1.26E-05<BR> K-562 0.209 1.416 1.387 1.431 1.418 1.124 0.199 98 101 100 76 -5 2.63E-06 1.10E-05 >1.26E-05<BR> MOLT-4 0.134 0.438 0.465 0.454 0.454 0.368 0.146 109 105 105 77 4 2.96E-06 >1.26E-05 >1.26E-05<BR> RPMI-8226 0.257 0.893 0.868 0.848 0.813 0.670 0.204 96 93 87 65 -21 1.88E-06 7.23E-06 >1.26E-05<BR> SR 0.158 0.457 0.454 0.425 0.457 0.338 0.111 99 89 100 60 -30 1.63E-06 5.85E-06 >1.26E-05<BR> Non-Small Cell Lung Cancer<BR> A549/ATCC 0.015 0.477 0.479 0.486 0.476 0.516 0.336 100 102 100 108 69 >1.26E-05 >1.26E-05 >1.26E-05<BR> EKVX 0.342 0.736 0.809 0.849 0.841 0.853 0.385 119 129 127 130 11 5.91E-06 >1.26E-05 >1.26E-05<BR> HOP-62 0.335 1.109 1.088 1.099 1.113 1.086 0.605 97 99 100 97 35 7.18E-06 >1.26E-05 >1.26E-05<BR> HOP-92 0.505 1.694 1.554 1.603 1.477 1.381 0.873 88 92 82 74 31 4.50E-06 >1.26E-05 >1.26E-05<BR> NCI-H226 0.560 0.932 0.967 0.918 0.967 0.967 0.904 109 96 109 109 92 >1.26E-05 >1.26E-05 >1.26E-05<BR> NCI-H23 0.648 1.622 1.769 1.822 1.880 1.635 1.215 115 121 127 101 58 >1.26E-05 >1.26E-05 >1.26E-05<BR> NCI-8322M 0.382 0.997 1.103 1.036 0.976 0.992 0.755 117 106 97 99 61 >1.26E-05 >1.26E-05 >1.26E-05<BR> NCI-H460 0.296 1.235 1.132 1.186 1.234 1.157 0.757 89 95 100 92 49 1.20E-05 >1.26E-05 >1.26E-05<BR> NCI-H522 0.478 1.138 1.332 1.135 1.189 0.892 0.378 129 100 108 63 -21 1.79E-06 7.08E-06 >1.26E-05<BR> Colon Cancer<BR> COLO 05 0.328 1.394 1.425 1.414 1.576 1.434 0.935 103 102 117 104 57 >1.26E-05 >1.26E-05 >1.26E-05<BR> HCT-116 0.301 1.574 1.508 1.480 1.488 1.391 0.685 95 93 93 86 30 5.53E-06 >1.26E-05 >1.26E-05<BR> HCT-15 0.219 1.623 1.800 1.627 1.673 1.522 0.504 113 100 104 93 20 4.90E-06 >1.26E-05 >1.26E-05<BR> HT29 0.095 0.578 0.637 0.599 0.580 0.479 0.169 112 104 100 80 15 3.63E-06 >1.26E-05 >1.26E-05<BR> KM12 0.189 0.741 0.744 0.728 0.777 0.737 0.567 101 98 106 99 68 >1.26E-06 >1.26E-05 >1.26E-05<BR> SW-620 0.153 0.886 0.898 0.868 0.857 0.779 0.267 102 98 96 85 16 4.05E-06 >1.26E-05 >1.26E-05<BR> CNS Cancer<BR> SF-268 0.203 0.767 0.821 0.736 0.815 0.767 0.334 110 94 108 100 23 5.63E-06 >1.26E-05 >1.26E-05<BR> SF-295 0.249 1.007 0.951 0.978 0.938 0.938 0.639 93 96 91 91 51 >1.26E-05 >1.26E-05 >1.26E-05<BR> SF-539 0.132 0.462 0.491 0.516 0.506 0.435 0.110 109 117 113 92 -17 3.06E-06 8.85E-06 >1.26E-05<BR> SNB-19 0.176 0.905 0.857 0.880 0.887 0.883 0.437 93 97 98 97 36 7.38E-06 >1.26E-05 >1.26E-05 SNB-75 0.501 1.051 0.925 1.049 1.134 0.703 0.438 77 100 115 37 -13 8.54E-07 6.98E-06 >1.26E-05<BR> U251 0.192 0.799 0.789 0.792 0.803 0.680 0.061 98 99 101 80 -68 2.02E-06 4.37E-06 9.47E-06<BR> Melanoma<BR> LOX MVI 0.173 1.304 1.175 1.170 0.974 0.997 0.319 89 88 71 73 13 3.03E-06 >1.26E-05 >1.26E-05<BR> MALME-3M 0.476 0.859 1.006 0.949 0.868 0.819 0.275 138 123 102 90 -42 2.51E-06 6.02E-06 >1.26E-05<BR> M14 0.123 0.613 0.602 0.635 0.592 0.549 0.136 98 105 96 87 3 3.45E-06 >1.26E-05 >1.26E-05<BR> SK-MEL-2 0.388 0.704 0.746 0.714 0.735 0.676 0.323 113 103 110 91 -17 3.03E-06 8.81E-06 >1.26E-05<BR> SK-MEL-28 0.513 1.090 1.088 1.107 1.093 1.057 0.546 100 103 101 94 6 3.98E-06 >1.26E-05 >1.26E-05<BR> SK-MEL-5 0.454 2.107 2.049 2.066 2.025 1.748 0.460 96 97 95 78 0 2.90E-06 >1.26E-05 >1.26E-05<BR> UACC-257 0.596 1.142 1.149 1.128 1.165 1.078 0.814 101 97 104 88 40 7.78E-06 >1.26E-05 >1.26E-05<BR> UACC-62 0.306 1.082 1.111 1.105 1.108 1.051 0.355 104 103 103 96 6 4.10E-06 >1.26E-05 >1.26E-05<BR> Ovarian Cancer<BR> IGROV1 0.291 1.679 1.828 1.932 1.743 1.710 0.536 111 118 105 102 18 5.22E-06 >1.26E-05 >1.26E-05<BR> OVCAR-3 0.240 1.066 1.073 1.055 1.045 0.980 0.343 101 99 97 90 12 4.10E-06 >1.26E-05 >1.26E-05<BR> OVCAR-5 0.457 1.206 1.243 1.230 1.157 1.181 0.715 105 103 93 97 34 7.07E-06 >1.26E-05 >1.26E-05<BR> OVCAR-8 0.173 1.340 1.250 1.245 1.159 1.107 0.495 92 92 84 80 28 4.71E-06 >1.26E-05 >1.26E-05<BR> SK-OV-3 0.351 0.865 0.836 0.852 0.853 0.867 0.557 94 97 98 100 40 8.59E-06 >1.26E-05 >1.26E-05<BR> Renal Cancer<BR> A498 0.400 0.678 0.691 0.657 0.649 0.635 0.357 105 93 89 84 -11 2.90E-06 9.71E-06 >1.26E-05<BR> CAKI-1 0.372 0.942 1.058 0.960 1.103 0.819 0.325 120 103 128 78 -13 2.59E-06 9.13E-06 >1.26E-05<BR> RXE 393 0.743 1.243 0.860 1.114 1.290 1.077 0.776 23 74 109 67 7 >1.26E-05 >1.26E-05<BR> SN12C 0.157 0.546 0.533 0.529 0.500 0.474 0.113 97 96 88 81 -28 2.44E-06 6.96E-06 >1.26E-05<BR> TX-10 0.355 1.027 1.014 1.002 1.042 0.999 0.727 98 96 102 96 55 >1.26E-05 >1.26E-05 >1.26E-05<BR> UO-31 0.124 0.765 0.799 0.816 0.830 0.785 0.391 105 108 110 103 42 9.20E-06 >1.26E-05 >1.26E-05<BR> Prostate Cancer<BR> PC-3 0.187 0.419 0.426 0.399 0.390 0.342 0.179 103 91 88 67 -4 2.17E-06 1.10E-05 >1.26E-05<BR> DU-145 0.384 1.151 1.081 1.389 1.194 1.119 0.873 91 131 106 96 64 >1.26E-05 >1.26E-05 >1.26E-05<BR> Breast Cancer<BR> MCF7 0.200 0.928 0.924 0.912 0.985 0.893 0.277 99 98 108 95 11 4.31E-06 >1.26E-05 >1.26E-05<BR> NIC-ADR-RES 0.328 1.386 1.576 1.376 1.425 1.298 0.801 118 99 104 92 45 9.69E-06 >1.26E-05 >1.26E-05<BR> MDA-MB-<BR> 231/ATCC 0.304 0.644 0.635 0.651 0.674 0.667 0.314 97 102 109 107 3 4.43E-06 >1.26E-05 >1.26E-05<BR> HS 578T 0.369 1.255 1.338 1.296 1.219 1.300 0.742 109 105 96 105 42 9.44E-06 >1.26E-05 >1.26E-05<BR> MDA-MB-435 0.465 1.425 1.370 1.354 1.476 1.369 0.666 94 93 105 94 21 5.05E-06 >1.26E-05 >1.26E-05<BR> MDA-N 0.348 1.471 1.499 1.481 1.374 1.316 0.385 102 101 91 86 3 3.44E-06 >1.26E-05 >1.26E-05 BT-549 0.735 1.762 1.910 1.865 1.847 1.599 0.998 114 110 108 84 26 4.82E-06 >1.26E-05 >1.26E-05<BR> T-47D 0.464 1.007 0.941 1.054 1.184 0.905 0.419 88 109 133 81 -10 2.78E-06 9.86E-06 >1.26E-05
In the first set of tests, according to Table 1 and the dose response curves as shown in Figure 1A to 1I, arsenic trioxide was effective in reducing cell growth against almost all the cell lines tested. In particular, leukemic cell lines, melanoma cell lines, and ovarian cancer cell lines responded consistently by showing a reduction of more than 50% growth. According to the mean graph as shown in Figure 2, most of the leukemic and melanoma cell lines, central nervous system cancer cell lines SNB-75 and U251, prostate cancer cell line PC-3, renal cancer cell lines A498, CAKI-1, SN12C, and lung cancer cell line NCI-H522 were especially sensitive (relative to all the cell lines tested) to treatment with arsenic trioxide.
TABLE 2<BR> Log 10 Concentration<BR> Mean Optical Densities Percent Growth<BR> Time<BR> Panel/Cell Line Zero Ctrl -8.9 -7.9 -6.9 -5.9 -4.9 -8.9 -7.9 -6.9 -5.9 -4.9 GI50 TGI LC50<BR> Leukemia<BR> CCRF-CEM 0.047 3.511 3.601 3.592 3.051 0.320 0.048 103 102 87 8 0 3.68E-07 >1.26E-05 >1.261-05<BR> K-562 0.041 3.011 3.042 2.992 3.360 2.931 0.096 101 99 112 97 2 3.941-06 >1.26E-05 >1.26E-05<BR> MOLT-4 0.018 0.503 0.554 0.228 0.432 0.245 0.113 111 43 85 47 20 >1.26E-05 >1.26E-05<BR> RPMI-8226 0.066 1.432 1.607 1.263 1.423 0.368 0.065 113 88 99 22 -2 5.481-07 1.091-05 >1.26E-05<BR> Non-small Cell Lung Cancer<BR> EKVX 0.030 0.682 0.573 0.601 0.628 0.414 0.014 83 88 92 59 -53 1.51E-06 4.22E-06 1.18E-05<BR> HOP-62 0.061 1.026 1.076 0.997 1.019 0.451 0.014 105 97 99 40 -77 8.66E-07 2.781-06 7.41E-06<BR> HOP-92 0.115 1.292 1.455 1.834 1.319 1.229 0.079 114 146 102 95 -31 2.85E-06 7.111-06 >1.26E-05<BR> NCI-H226 0.094 0.498 0.504 0.458 0.489 0.348 0.164 101 90 98 63 17 2.40E-08 >1.261-05 >1.26E-05<BR> NCI-H23 0.075 1.624 1.818 1.814 1.872 1.303 0.086 113 112 116 79 1 2.97E-06 >1.261-05 >1.26E-05<BR> NCI-H322M 0.035 0.702 0.680 0.620 0.674 0.552 0.054 97 88 96 77 3 2.94E-06 >1.261-05 >1.26E-05<BR> NCI-H460 0.034 2.839 2.797 2.806 2.792 2.535 0.062 99 99 98 89 1 3.50E-06 >1.261-05 >1.26E-05<BR> NCI-H522 0.175 1.224 1.269 0.535 1.191 0.128 0.028 104 34 97 -27 -84 7.641-07 3.19E-06<BR> Colon Cancer<BR> COLO 205 0.022 2.351 2.308 2.026 2.283 1.404 0.024 98 86 97 59 0 1.81E-06 >1.26E-05 >1.26E-05<BR> >1.26E-<BR> HCT-116 0.046 3.420 3.395 3.289 3.381 3.284 1.931 99 96 99 96 56 05 >1.26E-05 >1.26E-05<BR> HCT-15 0.033 3.838 3.965 3.921 4.227 3.432 0.404 103 102 110 89 10 3.93E-06 >1.26E-05 >1.26E-05<BR> HT29 0.017 3.060 3.189 2.988 2.706 1.476 0.027 104 98 88 48 0 1.12E-06 >1.26E-05 >1.26E-05<BR> KM12 0.012 0.808 0.487 0.533 0.507 0.036 0.017 80 87 83 4 1 3.29E-07 >1.26E-05 >1.26E-05<BR> SW-620 0.023 2.652 2.613 2.308 2.337 1.475 0.007 99 87 88 55 -72 1.39E-06 3.43E-06 8.50E-06<BR> CNS Cancer<BR> SF-268 0.054 0.798 0.990 0.381 0.356 0.387 -0.004 126 44 41 45 -100 1.06E-08 2.57E-06 5.69E-06<BR> SF-295 0.031 1.324 1.284 1.279 1.244 0.659 0.010 97 97 94 49 -69 1.17E-06 3.25E-06 8.64E-06<BR> SF-539 0.041 1.598 1.727 1.403 1.545 0.691 0.017 108 87 97 42 -59 8.91E-07 3.29E-06 1.04E-05<BR> SNB-75 0.134 1.065 1.143 1.033 1.024 0.815 0.028 108 97 96 73 -79 1.79E-06 3.80E-06 8.08E-06<BR> U251 0.105 2.324 2.129 2.018 1.908 0.088 -0.012 91 86 81 -17 -100 2.63E-07 8.51E-07 3.16E-06<BR> Melanoma LOX IMVI 0.013 2.153 2.000 1.666 0.053 0.002 -0.001 93 77 2 -88 -100 2.90E-08 1.32E-07 4.73E-07<BR> MALMI-3M 0.115 0.650 0.702 0.595 0.823 0.383 0.005 110 90 132 50 -96 1.26E-06 2.78E-06 6.12E-06<BR> SK-MEL-2 0.213 0.500 0.491 0.462 0.426 0.128 . 97 87 74 -40 -100 2.06E-07 5.63E-07 1.85E-06<BR> SK-MEL-28 0.093 2.030 2.012 1.906 1.797 0.488 . 99 94 88 20 -100 4.60E-07 1.86E-06 4.84E-06<BR> SK-MEL-5 0.181 1.907 1.935 1.864 1.929 1.846 0.013 102 97 101 96 -93 2.22E-06 4.07E-06 7.46E-06<BR> UACC-257 0.053 0.883 0.872 0.857 0.832 0.504 0.039 99 97 94 54 -27 1.42E-06 5.83E-06 >1.26E-05<BR> UACC-62 0.030 1.537 1.546 1.460 1.409 0.539 0.001 101 95 92 34 -98 6.60E-07 2.27E-06 5.43E-06<BR> Ovarian Cancer<BR> IGROV1 0.335 3.115 3.541 3.056 3.204 2.877 0.216 115 98 103 91 -36 2.67E-06 6.62E-06 >1.26E-05<BR> OVCAR-3 0.081 1.153 1.047 1.128 1.124 0.482 0.001 90 98 97 37 -99 7.76E-07 2.36E-06 5.49E-06<BR> OVCAR-5 0.038 1.404 1.433 1.460 1.478 1.287 0.028 102 104 105 91 -26 2.83E-06 7.53E-06 >1.26E-05<BR> OVCAR-8 0.016 2.090 2.272 1.848 2.242 1.437 -0.001 109 88 107 68 -100 1.62E-06 3.21E-06 6.36E-06<BR> SK-OV-3 0.041 1.474 1.445 1.410 1.461 1.247 0.020 98 96 99 84 -51 2.25E-06 5.27E-06 1.23E-05<BR> Renal Cancer<BR> 786-0 0.021 1.578 1.582 1.821 1.786 0.832 -0.003 100 116 113 52 -100 1.30E-06 2.77E-06 5.91E-06<BR> A498 0.051 0.741 0.826 0.764 0.837 0.558 0.025 112 103 114 73 -52 1.94E-06 4.85E-06 1.22E-05<BR> CAKI-1 0.032 1.352 1.254 1.725 1.573 0.491 0.004 93 128 117 35 -88 8.22E-07 2.43E-06 6.22E-06<BR> RXF 393 0.198 1.515 1.327 1.301 1.727 0.909 0.017 86 84 116 54 -91 1.34E-06 2.96E-06 6.54E-06<BR> S12C 0.024 0.956 0.882 1.141 1.113 0.829 0.013 92 120 117 86 -46 2.37E-06 5.67E-06 >1.26E-05<BR> TK-10 0.072 1.333 1.356 1.268 1.334 1.103 0.239 102 95 100 82 13 3.66E-06 >1.26E-05 >1.26E-05<BR> Prostate Cancer<BR> DU-145 0.095 2.158 2.147 1.954 1.935 2.153 0.055 99 90 89 100 -43 2.82E-06 6.32E-06 >1.26E-05<BR> Breast Cancer<BR> MCF7 0.028 2.650 2.647 2.611 2.603 1.683 0.008 100 99 98 63 -73 1.57E-06 3.66E-06 8.51E-06<BR> NCI/ADR-RIS 0.050 2.304 2.075 2.253 1.951 1.319 0.072 90 98 84 56 1 1.64E-06 >1.26E-05 >1.26E-05<BR> MDA-MB-231/A<BR> TCC 0.158 0.761 0.759 0.764 0.771 0.563 -0.001 100 101 102 67 -100 1.59E-06 3.18E-06 6.33E-06<BR> HS 578T 0.109 0.728 0.777 0.587 0.890 0.706 0.010 108 77 126 96 -91 2.23E-06 4.12E-06 7.62E-06<BR> MDA-MB-435 0.054 1.500 1.276 1.470 0.865 0.631 0.021 85 98 56 40 -62 3.00E-07 3.10E-06 9.60E-06<BR> MDA-N 0.028 2.652 2.468 2.616 2.606 1.738 0.026 93 99 98 65 -9 2.02E-06 9.55E-06 >1.26E-05<BR> BT-549 0.112 1.798 1.876 1.940 1.963 1.697 0.018 105 108 110 94 -84 2.22E-06 4.24E-06 8.08E-06<BR> T-47D 0.098 0.824 0.883 0.336 0.539 0.398 0.027 108 33 61 41 -73 2.90E-06 7.93E-06
In the second set of tests, according to Table 2, the dose response curves, and the mean graph as shown in Figure 3A to 3I, and Figure 4, arsenic trioxide was effective in reducing cell growth against all the cell lines tested.
The results were consistent with those obtained from the first set of tests. In particular, several melanoma cell lines appeared to be especially sensitive at the various principal response parameters.
In conclusion, these results demonstrate that arsenic trioxide is effective in inhibiting the growth of leukemic cells and cancer cells in vitro, and that arsenic trioxide can be used in human subjects to treat a broad range of leukemia, and cancers, including but not limited to non- small cell lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer.
The present 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 herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
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