US007 179493B2

(12) United States Patent (10) Patent No.: US 7,179,493 B2 Ellison et al. (45) Date of Patent: Feb. 20, 2007

(54) COMPOSITIONS AND METHODS FOR THE 6,770,304 B2 8/2004 Warrel, Jr. et al. TREATMENT OF PRIMARY AND 2005/0196464 A1* 9, 2005 Hu et al...... 424,623 METASTATC NEOPLASTIC DISEASES USING ARSENC COMPOUNDS FOREIGN PATENT DOCUMENTS (75) Inventors: Ralph M. Ellison, Palm Beach, FL (US); Fred H. Marmelstein, Clifton, CN 1061908 A 6, 1992 NJ (US) CN 1079.391 A 12/1993 CN 1081104. A 1, 1994 (73) Assignee: Polarx Biopharmaceuticals, Inc., CN 1119113 A 3, 1996 Wilmington, DE (US) CN 1121807 A 5, 1996 CN 1122700 A 5, 1996 (*) Notice: Subject to any disclaimer, the term of this CN 1131037 A 9, 1996 patent is extended or adjusted under 35 CN 1133725 A 10, 1996 U.S.C. 154(b) by 438 days. JP 51-88620 3, 1976 WO WO 94,02108 2, 1994 (21) Appl. No.: 10/649,776 WO WO95/O1789 1, 1995 WO WO95/22336 8, 1995 (22) Filed: Aug. 28, 2003 (65) Prior Publication Data OTHER PUBLICATIONS US 2004/O115283 A1 Jun. 17, 2004 “Arsen (III)—Sulfid AS.S.", Gmelin's Arsenic 8". Edition, 17:422 433, 1952. Related U.S. Application Data “Diarsendisulfid AS.S.", Gmelin's Arsenic, 8". Edition, 17:412 (62) Division of application No. 09/173,531, filed on Oct. 422, 1952. 15, 1998, now Pat. No. 6.875451. "Goodman & Gilman's The pharmacological basis of therapeutics'. s s s s 9" Edition, McGraw-Hill, Health Professions Division, pp. 1659 (60) Provisional application No. 60/062.375, filed on Oct. 1662, date unknown. 15, 1997. “Inorganic Arsenic Compounds. Other Than Arsine Health and Safety Guide, Health and Safety Guide No. 70', WHO, Geneva, (51) Int. Cl. 1992. A6 IK33/36 (2006.01) 'Xionghuang Realgar'. Chinese Pharmacopia (1), Guangdong Sci A6 IK 33/24 (2006.01) ence and Technology Publishing House, China, pp. 298-299, 1995. A6 IK 9/00 (2006.01) Akao et al. "Arsenic Induces Apoptosis in B-cell Leukemic Cell A6 IK3I/28 (2006.01) Lines in Vitro. Activation of Caspases and Down-regulation of A6 IK3I/282 (2006.01) Bcl-2 Protein', British J of Hematology, 102. 1055-1060, 1998. A6 IK3I/33 (2006.01) Andre et al., “The PML and PML/RARI Domains: from autoim munity to molecular oncology and from retinoic acid to arsenic'. A 6LX 3/555 (2006.01) Experimental Cell Research, 229:253-260, 1996. St. t%% 3. Arsenic, Environmental Health Criteria 18, Geneva: WHO, 1981. / ( .01) Chemical Abstract, 63-Pharmaceuticals 11:317, #111:219272 and A61 P 35/02 (2006.01) 111:219276p, 1987-1991. (52) U.S. Cl...... 424/623; 424/85.1; 424/184.1; 424/277.1; 424/278.1; 424/620: 424/629; (Continued) 424/649; 514/2: 514/8: 514/12: 514/21: 514/23; Pri E John Pak 514/25; 514/27: 514/28: 514/29:514/34: 514/31;514/45 (74)AST Attorney, 'Can Agent, orO FiFirm—Cephalon, a Cephalon. Inc.I (58) Field of Classification Search ...... 424/422, (57) ABSTRACT 424/620, 623,629, 649, 184.1, 277.1, 85.1 424/278.1: 514/2, 8, 12, 21, 23, 25, 27–29, 514/31, 34, 45, 49,90, 110, 171, 183-185, The invention relates to the use of arsenic compounds to 514/249, 274, 283,444, 449, 492, 504, 588, treat a variety of neoplastic diseases. The present invention 514/615, 733, 50, 109, 251, 559, 575; 600/1 encompasses the administration to a mammal of arsenic in See application file for complete search history. the form of a salt, complex, organic compound or ionic (56) References Cited Solution to treat tumors of epithelial tissue, connective tissue, central nervous system, lymphoid tissue, hematopoi U.S. PATENT DOCUMENTS etic cells and tumors associated with oncogenic viruses. This invention also encompasses the treatment of hematopoietic 132,275 A 10/1872 Gettings 232,807 A 10, 1880 Dennett disorders in mammals by the administration of one or more 3,700.498 A 10, 1972 Kanazawa et al. arsenic compounds to said mammal. Further, the arsenic 4497,780 A 2f1985 Barin et al. compounds may be used to treat metastatic neoplastic dis 5,759,837 A 6/1998 Kuhajda et al. CaSCS. 6,720,011 B1 4/2004 Zhang 6,733,792 B1 5, 2004 Lu 9 Claims, 12 Drawing Sheets US 7,179,493 B2 Page 2

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"Tumorigenicity of inorganic arsenic compounds Kerkvliet et al., “Immunotoxicology Studies of Sodium Arsenate following intratracheal instillations to the lungs of hamsters'. Int. J. effects of Exposure on Tumor Growth and Cell-mediated Tumor Cancer, 40:220-223, 1987. Immunity”. J. Environment Pathology and Toxicology 4:65-79. Yuan et al., “Research on traditional methods for Purifying 1980. Realgar'. Chung Yao Tung Pao, 13(8): 23-26, 1988. Konig et al., "Comparative Activity of Melarsoprol and Arsenic Yuan et al., “Exploring methods for purifying Realgar'. Chung Yao Trioxide in Chronic B-Cell Leukemia Lines”. Blood 90(2):562-570, Tung Pao, 13(8): 17-21, 1988. 1997. Zhang et al., “Traditional Chinese and Western Medicine in the Kwong et al., “Delicious Poison: Arsenic Trioxide for the treatment Treatment of 27 patients with Malignant Lymphoma' Chinese J. of Leukemia, Blood 89:3487-8, 1997. Oncology 10:61-62, 1988. Lee et al., “Induction of Gene Amplification by Arsenic'. Science, 241: 79-81, 1988. Zhang et al. “Treatment of Acute Promyelocytic Leukemia with Li et al. “Traditional Chinese and Western Medicine in the Treat “713”; Clinical Observations and Study of Action Mode on 117 ment of 27 Patients with Malignant Lymphoma'. Chinese J. Oncol Patients”. J. Harbin Medical Univ. 29(3): 243, 1995. ogy, 10:61-62, 1988. Zhang et al., “Clinical Study on the Treatment of Acute Lu et al., “Effective treatment of AML-M3 (APL) and their remis Promyelocytic Leukemia with Ai Ling #1'. J of Traditional Chinese sion maintenance with Realgar: A pilot clinical and laboratory study and Western Medicine 4(1):19, 1984. on 38 patients”. Blood 90(10 Suppl. 1 part 1):416A, #1849, 1997. Zhang et al., “Treatment of Acute Promyelocytic Leukemia with Lu et al., “Study of Realgar in the treatment of acute promyelocytic Intravenous Arsenic Trioxide'. Chinese J. of Hematology 17(2): Leukemia (APL)- a pilot clinical and laboratory study on 38 1996. patients', China-Korea Medical Conference ’97 1997. Zhang et al. “Discussion on methods for removing AS involving use Mervis, “Ancient remedy performs new tricks'. Science 273: 578, of yogurt'. Zhongguo Zhongyao Zaahi, 20(9): 537, 1995. 1996. Zhu et al., “Arsenic-induced PML targeting onto nuclear bodies: Monfardini et al., “Survival in chronic myelogenous leukemia: implications for the treatment of acute promyelocytic leukemia', influence of treatment and extent of disease at diagnosis'. Cancer, Proc. Natl. Acad. Sci. 94:3978-3983, 1997. 31:492-501, 1973. “Letter on Historical Facts Regarding the Development of Ai Ling. Neubauer, "Arsenic Cancer: a review', Arsenical Cancer, Arsenic No. 1 and the Clinical Use of Arsenic Trioxide in the Treatment of Committee of the Medical Research Council, pp. 192-251, 1947. Acute Promyelocytic Leukemia and a Study of its Mechanism” Pershagen et al., “On the pulmonary tumorigenicity of arsenic Heilongjiang Branch of the Chinese Medical Association, Mar. 27. trisulfide and calcium arsenate in hamsters', Cancer Letter, 27:99 1998. 104, 1985. Wang et al., “Arsenic Trioxide and Melarsprol Induced Pro Pories et al., “Trace Elements that Act to Inhibit Neoplastic grammed cell death in myeloid leukemia cell lines and function in Growth'. Annals New York Academy of Sciences, 199: pp. 265 PML and PNL-RARI Independent Manner.” Blood, 92(5):1497 273, 1972. 1504, 1998. US 7,179,493 B2 Page 3

Flamigni et al., “Effect of Sodium Arsenite on the Induction and W. Zhang et al., “The induction of apoptosis and arrest by Turnover of Ornithine Decarboxylase Activity in Erythroleukemia arsenic trioxide in lymphoid neoplasms”. Leukemia (1998), vol. 12, Cells”. Cell Biochemistry and Function vol. 7:213-217 (1989) (C) No. 9, Sep. 1998, pp. 1383-1391. John Wiley & Sons Ltd. 1989. John G. Kidd, “Effects of Arsenic Azoproteins on Mouse Reichi et al., “Effect of Arsenic on Cellular Metabolism. After Single Lymphoma Cells in Vivo? With Observations on the Effects of or Repeated Injection in Guinea Pigs' Arch. Toxicol. Suppl. 13, Other "Anti-Lymphoma' Agents and On the Susceptibility to these 363-365 (1989) (C) Springer Veriag 1989. Effects of Lymphoma Cells of Various Types”, Journal of Experi Remington's Pharmaceutical Sciences, Mack Publishing Company, mental Medicine, vol. 108, 1958, pp. 665-684. Easton PA, Chapter 85, pp. 1570-1580, 1990. Jacques Debray, “Treatment of Hodgkin's Disease'. Therapie, vol. Inorganic Arsenic Compounds other than Arsine Health and Safety 9, 1954, pp. 106-118. Guide, Health and Safety Guide No. 70, WHO, Geneva 1992. G. Lotti, 'Abandonment of arsenic in medical therapy of malignant Sun et al., Ai Ling #1 and Traditional Chinese Medicine in the granuloma (Hodgkin's disease): is it justified?” Revue Medicale Treatment of 32 Patients with Acute Promyelocytic Leukemia, Du Moyen-Orient, vol. 24, No. 2, Mar. 1967 pp. 110-111. Chinese J. of Traditional Chinese and Western Medicine, pp. International Search Report of EP 03029713, dated Mar. 31, 2004. 170-171, vol. 12, No. 3, 1992. S. Rosner, “Immunological Enhancement of in Treleaven et al., “Arsenic and Ayurveda'. Leukemia and Advanced Brain Cancer', ACTA. Neurol. Latinoamer, vol. 21, No. Lymphoma, vol. 10, pp. 343-345 (C) Harword Academic Publishers 4, 1975, pp. 126-132. GmbH 1993. S. Shimotsuura, “Studies on the Antineoplasmic Actions of ASO.” USP Dictionary of USAN and International Drug Names, United Shika Gakuho—Journal of Tokyo Dental College Society, vol. 86. States Pharmacopeial Conventions, Inc., Rockville Md., p. 59, Nov. No. 8, 1986, pp. 1237-1253. 1995. P. Zhang et al., “Treatment of Acute Promyelocytic Leukemia with Fluka 1995/96 Catalog, pp. 152-153 Jul 1995. Intravenous Arsenic Trioxide Zhonghua XueyeXue Wang et al., “Arsenic and the Treatment of Leukemia' J. Harbin Zazhi Chinese Journal of Hematology, vol. 17, No. 2, Feb. 1996, Medical University, vol. 31, No. 5, Oct. 1997. pp. 58-60. International Search Report of EP 03019594, dated Nov. 4, 2003. Matthew Block et al., “Biological Studies with Arsenic IV. The D.A. Sears, “History of the Treatment of Chronic Myelocytic Histopathiologic Effect of Arsenic Upon the Hematopoietic Tissues Leukemia', American Journal of Medical Sciences, vol. 296, No. 2, of Patients with Leukemia', The Journal of Laboratory and Clinical Aug. 1988, pp. 85-86. Medicine, vol. 41, No. 4, Apr. 1953, pp. 499-515. Y.L. Kwong et al., “Delicious Poison: Arsenic Trioxide for the P. Rousellot et al., “Arsenic derivatives: Old drugs for new indica Treatment of Leukemia', Blood, vol. 89, No. 9, May 1997, pp. tions, Les Derives Arsenicaux: De Vieux Medicaments Pour Des 3487-3488. Indications Renouvellees'. Hematologie, vol. 4, No. 3, May 1998, Edward B. Silberstein, "Radio nuclide Therapy of Hematologic pp. 95-97. Disorders'. Seminars in Nuclear Medicine, vol. 9, No. 2, Apr. 1979, P. Rousellot et al., “Arsenic trioxide (AS203) and Melarsoprol pp. 100-107. induce myeloma cell apoptosis in vitro with a preferential effect on International Search Report of EP 04007847, dated Jun. 16, 2004. tumoral cells in patients'bone marrow”. BLOOD, vol. 90, No. 10, Kunio Kitamura et al., “New and arsenic compounds for Nov. 15, 1997, p. 325A. the treatment of refractory acute promyelocytic leukemia: clinical Z. Xie et al., “Melasoprol and arsenic trioxide increase cell death on and basic studies for the next generation'. Cancer Chemother -resistant human leukemia and myeloma cells by regu Pharmacol, (1977), 40 (Suppl) pp. S36-S41. lating expression of BCL-2 apoptosis regulatory family”. BLOOD, Zhu Chen et al., “Acute Promyelocytic Leukemia: Cellular and vol. 90, No. 10, Nov. 15, 1997, pp. 495 A. Molecular Basis of Differentiation and Apoptosis'. Pharmacol. Database Epodoc Online European Patent Office, the Hauge, NL. Ther. vol. 76, No. 1-3, pp. 141-149, 1997. CN1180563, May 6, 1998, Zhao Qingtuan: XP002260136 Database Medline Online US National Library of Medicine “abstract. (NLM), Bethesday, MD US; Aug. 25, 1997, Melino G. et al., International Search Report of EP 03019595, dated Oct. 24, 2003. “Retinoic acid receptors alpha and gamma mediate the induction of Edward B. Silberstein, "Radionuclide Therapy of Hematologic “tissue' transglutaminase activity and apoptosis in human Disorders'. Seminars in Nuclear Medicine, vol. 9, No. 2, Apr. 1979, neuroblastoma cells', XP002284763, Database accession No. pp. 100-107. NLM9281352 A. ABSTRACT. David A. Sears MD, “History of the Treatment of Chronic Database Meline Online US National Library of Medicine (NLM) Myelocytic Leukemia', American Journal of the Medical Sciences, Bethesda, MD, US; Jul. 1997, Cooper M. P. et al., “All-trans retinoic vol. 296, No. 2, Aug. 1988, pp. 85-86. acid induced gene expression and growth inhibition in head and Zhi-Xiang Shen et al., “Use of Arsenic Trioxide (ASO) in the neck cancer cell lines' XP002284764 Database accession No. Treatment of Acute Promyelocytic Leukemia (APL); II. Clinical NLM9307717 At ABSTRACT & Oral Oncology, Jul. 1997, vol. 33, Efficiacy and Pharmacokinetics in Relapsed Patients”. Blood, vol. No. 4, pp. 270-274, ISSN: 1368-8375. 89, No. 9, May 1997, pp. 3354-3360. International Search Report of EP 03019628, dated Oct. 27, 2003. * cited by examiner U.S. Patent Feb. 20, 2007 Sheet 1 of 12 US 7,179,493 B2 100 Irist-a-SS LEUKEMIA so. s 50 s g O -- CCRF-CEM --- HL-60(TB) is -50 --O--- RPMI-8226 --- SR - 100 -9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (ug/ml) FIG. 1A

NON-SMALL CELL 100 free ... ------i.S. NON-SMALL CELL LUNG CANCER is 50 25 -- A549/ATCC ---- EKVX g O --0--- NC-H226 --- NC-H23 --O-- NCI-H522 "0." HOP-92 is -50 - --d- HOP-62 -- NC-H460

"st NCI-H322M -9 -8 7 6 5 4 LOG OF SAMPLE CONCENTRATION (ug/ml) FIG. 1B

COLON CANCER

-- COLO 205 ---- HCT-116 ---0--- KM12 --O-- SW-620 --arre HCT-15 - 0. HT29 -9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (ug/ml) FIG.1C U.S. Patent Feb. 20, 2007 Sheet 2 of 12 US 7,179,493 B2

100 SSS CNS CANCER '', Vyvs. 5 50 d g O -o- SF-268 ---- SF-295 E -50 V ---O-- SNB-75 -- U251

-- SF-539 - O - SNB-19 -9 -8 -7 -6 -5 - 4 LOGOF SAMPLE CONCENTRATION (ug/ml) FIG. 1D

100 E as 50 s O -O- LOX IMV. ---- MALME-5M. s ---0--- SK-MEL-5 --- SK-ME-28 E -50 -- a-- M4 "O" SK-MEL-2 -ra-i- UACC-257 ---- UACC-52 -9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (ug/ml) FIG.1E

OVARAN CANCER

-0- GROV1 ---- OVCAR-3 --O--- SK-OV-3 "0" OWCAR-8

-9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (ug/ml) FIG.1F U.S. Patent Feb. 20, 2007 Sheet 3 of 12 US 7,179,493 B2

is N RENAL CANCER

-O- A-498 --O-- CAK-1 -50 ---0--- TX-10 -o- U0-31 -- a-- RXF393 - O - SN12C -9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (ug/ml) FIG.1G

OO PROSTATE CANCER

O

-9 -8 -7 -6 -5 - 4 LOG 10 OF SAMPLE CONCENTRATION (ug/ml) FIG.1H

BREAST CANCER s se d g ---- NC/ADR-RES --O--- MDA-MB-435 --- MDA-N -- MDA-M8-231/ or HS 578T --dri- BT-549 -- T-47D -9 -8 -7 -6 -5 -4 LOG OF SAMPLE CONCENTRATION (ug/ml) FIG. I U.S. Patent Feb. . 20, 2007 Sheet 4 of 12 US 79 179,493 B2

OÇO]01001 MN M M M M M M M M Yn M. M. M. M. M.

AZOHNOCENNIN00

M M M M A M M M

ENTITEO/ENWd U.S. Patent Feb. . 20, 2007 Sheet 5 of 12 US 7, 179 493 B2

090]

09 0101901 K K K K K K K K A M M M M.

1010100]

O?I901001

830NWOSNO WWONWIEW U.S. Patent Feb. 20, 2007 Sheet 6 of 12 US 7,179,493 B2

M M M M M M M M

OGJI0,01010010||101010013NIT01001O?I?TIBO/TBNWdO?10 U.S. Patent Feb. 20, 2007 Sheet 7 of 12 US 7,179,493 B2

00 LEUKEMA E as 50 35 3 O -0- CCRF-CEM as as as K-562 -50 - MOLT-4 "O." RPM-8226 - 100 -9 -8 -7 -6 -5 -4 LOG OF SAMPLE CONCENTRATION (ug/ml) FIG.5A

NON-SMALL CELL LUNG CANCER E as 95 g --0-- HCP-62 ---0--- NC-H23 --- NC-H522M -- a-- HCP-92 "O". NC-H226 -ar-NC-H460 --- NC-H522 -9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (ug/ml)

COLON CANCER s Se d g -0- COLO 205 --O-- HCT-6 -- KM12 -- SW-620 ----- HC-15 - O - HT29. 9 -8 -7 -6 -5 -4 LOG OF SAMPLE CONCENTRATION (ug/ml) FIG.3C U.S. Patent Feb. 20, 2007 Sheet 8 of 12 US 7,179,493 B2

00 st- S. CNS CANCER E s 50 s 6 O -o- SF-268 --0-- SF-295 s -50 & ---O--- U25 "O" SNB-75 Ol -9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (ag/ml) FIG. 3D

100 a 50 s g O -- LOX IMV. --- MALME-3M a A. -o- SK-MEL-5 - UACC-257 -50 - \\,. --SK-MEL-2 or SK-MEL-28 'S -si- UACC-62 - 100 -9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (pg/ml) FIG.3E

00 OWARIAN CANCER is 50 s g O y - GROV -- OVCAR-3 is -50 w --0--- SK-OV-3 "0" OWCAR-8 -100 -9 -8 - 7 -6 -5 - 4 LOG 10 OF SAMPLE CONCENTRATION (ug/ml) FG.3F U.S. Patent Feb. 20, 2007 Sheet 9 of 12 US 7,179,493 B2

100 RENAL CANCER E as 50 5 g O -- 786-0 ---- A498 -50 --O--- SN12C -- TK-10 -- a-- CAK-1 - O - RXF393 -100 -9 -8 -7 -6 -5 - 4 LOGOF SAMPLE CONCENTRATION (ug/ml)

00 PROSTATE CANCER

O

5 0.

-100 -9 -8 -7 -6 -5 - 4 LOGOF SAMPLE CONCENTRATION (ug/ml)

100ter *SS

0 ---- NC/ADR-RES --0--- MDA-MB-435 --- MDA-N 5 O "a". MDA-MB-231/ "O" HS 578T --d- BT-549 -- T-47D -1 O O 9 -8 -7 -6 -5 -4 LOGOF SAMPLE CONCENTRATION (ug/ml) FIG.3

U.S. Patent Feb. 20, 2007 Sheet 12 of 12 US 7,179,493 B2

01001}NITTT30/TÈNWA01001191019010910OG10090]OÇO] US 7,179,493 B2 1. 2 COMPOSITIONS AND METHODS FOR THE controlled cell proliferation involving an increase in cell TREATMENT OF PRIMARY AND number in a tissue or organ, without significant alteration in METASTATC NEOPLASTIC DISEASES structure or function. As but one example, endometrial USING ARSENC COMPOUNDS hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult CROSS-REFERENCE TO RELATED or fully differentiated cell substitutes for another type of APPLICATION adult cell. Metaplasia can occur in epithelial or connective tissue cells. A typical metaplasia involves a somewhat This application is a divisional of U.S. application Ser. disorderly metaplastic epithelium. Dysplasia is frequently a No. 09/173,531, filed Oct. 15, 1998, which is now U.S. Pat. 10 forerunner of cancer, and is found mainly in the epithelia; it No. 6,875,451, which claims the benefit of Provisional is the most disorderly form of non-neoplastic cell growth, Application Ser. No. 60/062.375, filed Oct. 15, 1997. involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have 1. FIELD OF INVENTION abnormally large, deeply stained nuclei, and exhibit pleo 15 morphism. Dysplasia characteristically occurs where there The present invention relates to methods and composi exists chronic irritation or inflammation, and is often found tions for the treatment of primary and metastatic neoplastic in the cervix, respiratory passages, oral cavity, and gall diseases, including, but not limited to human sarcomas, bladder. carcinomas and hematopoietic disorders. In the practice of The neoplastic lesion may evolve clonally and develop an the treatment of cancer, compositions containing arsenic increasing capacity for invasion, growth, metastasis, and compounds are used to arrest and reverse neoplastic growth. heterogeneity, especially under conditions in which the More specifically, the present invention relates to novel neoplastic cells escape the host’s immune Surveillance chemotherapeutic methods—novel uses of arsenic com (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rd pounds for treating primary and metastatic tumors; primary ed., Mosby, St. Louis, pps. 17.1-17.12). and metastatic tumors of the central nervous system; refrac 25 tory primary and metastatic tumors of the central nervous 2.2. AIDS-related Non-Hodgkin’s Lymphoma system; breast, lung, bladder and prostate cancer; and refrac tory breast, lung, bladder and prostate cancer to mention a Since the discovery of AIDS, the disease has had a close few. association with an interesting spectrum of cancers. Further, 30 the types of malignancies and their incidence rates are 2. BACKGROUND OF THE INVENTION increasing as the development of effective antiretroviral therapies and prophylaxis against opportunistic infections In 1997 more than one million people will develop some leads to prolonged Survival in the immunodeficient state for type of cancer in the United States. Approximately 500,000 AIDS patients, (Karp and Broder, Cancer Res. will be cured or in a state of remission. These numbers 35 51:4747–4756 (1991)). AIDS-related non-Hodgkin’s lym represent an improving cure rate seen over the past decade phoma was found to occur in AIDS patients only after 1981. which is largely due to earlier detection, better treatment and AIDS-related non-Hodgkin’s lymphoma is a very aggres advances in chemotherapy. In particular, the advances in sive disease with a very high incidence of central nervous chemotherapy include targeted or specific drug therapy in system involvement. It is increasing in incidence in the which a drug is developed specifically for the treatment of 40 AIDS population. As patients infected with the AIDS virus a certain cancer type. This “disease-oriented' approach is now live longer because they are not dying of the usual designed to identify compounds which exert selective effects infections, they are developing lymphoma at an increasing in vitro on particular tumor types and to follow-up these rate. The characteristics of AIDS-related non-Hodgkin’s leads in vivo utilizing cell lines, (Fiebig et al., Cancer lymphoma are detailed in an article by Karp and Broder, Treatment Reviews 17:109–117 (1990)). However, the inci 45 (1991), supra. dence of cancer is continuing to climb as our population ages The problems the medical oncologist has in treating and as new cancers develop or occur more frequently, Such patients with AIDS-related lymphomas is the recently as in patients infected with AIDS virus. Thus, it is clear that described predilection for occurrence of the lymphoma in there is a tremendous demand for additional regimens to the central nervous system (in brain and Surrounding treat patients with cancer. 50 meninges) and the fact that the patient with AIDS has a very weak bone marrow which cannot tolerate treatment with 2.1. Pathobiology of Cancer standard chemotherapy. This makes the treatment of lym phoma in patients with AIDS very difficult because standard Cancer is characterized primarily by an increase in the chemotherapeutic agents are usually very marrow Suppres number of abnormal cells derived from a given normal 55 sive and do not cross blood brain barrier (to treat the central tissue, invasion of adjacent tissues by these abnormal cells, nervous system disease). and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites (metastasis). 2.3. Primary and Metastatic CNS Tumors Clinical data and molecular biologic Studies indicate that cancer is a multistep process that begins with minor pre 60 The incidence of primary and metastatic brain tumors is neoplastic changes, which may under certain conditions increasing in the United States. Indeed, the arsenal of progress to neoplasia. chemotherapeutics for these types of cancers is minimal, Pre-malignant abnormal cell growth is exemplified by while the need for such therapeutics is high. hyperplasia, metaplasia, or most particularly, dysplasia (for Glioblastoma multiform and other primary and metastatic review of such abnormal growth conditions, see Robbins 65 central nervous system tumors are devastating malignancies. and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders The treatment of these tumors include Surgery, radiation Co., Philadelphia, pp. 68–79). Hyperplasia is a form of therapy and treatment with agents such as the US 7,179,493 B2 3 4 BCNU. Other chemotherapeutic agents utilized include pro metastases to the central nervous system. Unfortunately, carbazine, , hydroxyurea and . Unfortu there is no standard chemotherapy which is felt to be helpful nately, even when all three modalities (Surgery, radiation in this situation. therapy and chemotherapy) are utilized, the average Survival It is estimated that more than 100,000 men will be of patients with central nervous system malignancies is still 5 diagnosed with prostate cancer this year and more than about 57 weeks. Clearly, new treatment approaches are 30,000 patients will die from the disease. The most common needed both for patients with newly diagnosed primary and sites of metastases in patients with prostate cancer are the metastatic central nervous system tumors, as well as for bone and lymph nodes. The bone metastases are particularly patients with such tumors which are refractory to the above bothersome in that they can create intense pain for the modalities. Finding Such new agents has been complicated 10 patient. The current treatment for metastatic prostate cancer by the fact that there is no animal model which appears to includes treatment with flutamide, leuprolide, diethylstil predict what agent will be clinically effective against pri bestrol, and other hormonal manipulations, as well as che mary and metastatic central nervous system tumors. motherapy (doxorubicin, , vinblas tine, Suramin, cisplatin, and others). Unfortunately, none of 2.4. Breast, Lung, Bladder and Prostate Cancers 15 these agents are consistently helpful in the disease. In addition, as patients with prostate cancer live longer with Breast cancer has been known to occur in about one in their malignancy, they will most likely develop a higher every 8–9 women in the United States. The treatment for incidence of metastases to the central nervous system (in early breast cancer is Surgery, with or without radiation cluding the spinal cord). therapy, or Surgery, with or without radiation therapy, plus 20 In general, as patients are living longer with the common chemotherapy and/or hormonal therapy. Despite the best malignancies such as breast cancer, lung cancer, bladder efforts of physicians there are still more than 80,000 deaths cancer, prostate cancer and a variety of other malignancies each year from breast cancer and the incidence is still rising. (because of control of their systemic disease with Surgery, Current chemotherapy for patients with primary or meta radiation therapy and chemotherapy), oncologists are noting static breast cancer includes treatment with cyclophospha- 25 that they are developing an increasing incidence of meta mide, , doxorubicin, 5-, cisplatin, static tumors in the central nervous system including the , taxol, taxotere, and occasionally brain. This is probably because most of the currently avail other agents. Unfortunately, even with these agents, almost able chemotherapy does not cross the blood brain barrier. all women who develop metastatic breast cancer Succumb to When the patient (who has their tumor controlled in sites their disease. One particular place that metastatic breast 30 outside of the brain) develops brain metastases, it is a very cancer does metastasize to is the central nervous system. difficult situation options for that patient are usually limited When central nervous system metastases do occur, the usual to surgery for a solitary metastasis and/or radiation therapy. treatment is Surgery (for a solitary metastasis) or radiation, However, after those modalities fail, the patient usually has or Surgery plus radiation therapy. At present there is no no other options. chemotherapy which is felt to be helpful in this situation. 35 For each of the above indications (primary brain tumors Lung cancer is responsible for more than 150,000 deaths and metastases to the brain from other common tumors such each year in the United States. Most patients with lung as breast, lung, bladder and prostate cancers), there is a cancer present a tumor that has already metastasized to a tremendous need for a more effective treatment and/or variety of organs, including lung, liver, adrenal gland and methods for improving the quality of patient life. other organs. The current treatment for metastatic lung 40 cancer is not yet standardized (Ihde, Daniel C. “Chemo 2.5. Esophageal Cancer therapy of Lung Cancer. The New England Journal of Medicine 327:1434–1441, 1992 Nov. 12th issue). However, In the U.S., carcinoma of the esophagus represents about chemotherapy regimens which are utilized include treatment 6% of all cancers of the gastrointestinal tract but causes a with cisplatin plus , combinations of cyclophos- 45 disproportionate number of cancer deaths. (Boring, C. C., et phamide plus doxorubicin plus cisplatin, and single agents al.: Cancer statistics, 1993. CA Cancer J. Clin. 43:7, 1993). alone or in combination, including , , These cancers usually arise from the epithelial layer of the vindesine, , Vincristine, taxol, , esophagus and are either squamous cell carcinomas or methotrexate, hexamethylmelamine and others. Despite adenocarcinomas. Overall the 5 year survival is about 5%. these chemotherapeutic regimens the average patient with 50 Squamous cell carcinoma generally occurs after the age of metastatic lung cancer still only Survives 7-12 months. One 50 and is more common in males than in females. The particular troublesome place for metastases of lung cancer is incidence varies widely from country to country and the central nervous system. The treatment for central ner between regions within countries. In the U.S. the incidence Vous system metastases includes Surgery (to remove a Soli is between 2 and 8 persons per 100,000 and is more tary lesion), radiation therapy, or a combination of both. 55 prevalent in blacks than in whites. Unfortunately, there is not standard chemotherapy which is Adenocarcinoma represents 25% of all esophageal CA in felt to be helpful in this situation. the U.S. It is usually located in the distal one third of the Each year about 11,000 patients die of bladder cancer in esophagus and may invade the adjacent gastric cardia. It the U.S. Although at presentation the disease is usually tends to occur in people over 40 years of age and is more localized, most patients develop distant metastatic disease. 60 common in males than in females. It is more common in The most recent advances have been in the area of chemo whites than in blacks. therapy for patients with such metastatic disease. One effec tive regimen is called the MVAC regimen. It consists of 2.6. Arsenic and its Medical Uses treatment with methotrexate plus vinblastine plus adriamy cin (doxorubicin) plus cisplatin. Although the response rate 65 Arsenic has been considered to be both a poison and a is high to this chemotherapeutic regimen, medical oncolo drug for a long time in both Western and Chinese medical gists are noting that one place the patients fail is with practices. In the latter part of the nineteenth century, arsenic US 7,179,493 B2 5 6 was used frequently in attempts to treat diseases of the blood unclear, but it is believed by some to be one of the possible in the West. In 1878, it was reported that treatment of a mechanisms of the therapeutic effects of certain arsenic leukemic patient with Fowler's solution (a solution of potas compounds. sium arsenite) reduced markedly the count of white blood Although arsenic is well known to be both a poison and cells (Cutler and Bradford, Am. J. Med. Sci., January 1878, 5 a carcinogenic agent, there have been many reports con 81–84). Further interests in the use of Fowler's solution as cerning the use of arsenic in medical treatment. Identifica a palliative agent to treat chronic myelogenous leukemia tion or discussion of the art above must not be construed as (CML) was described by Forkner and Scott in 1931 (J. Am. an admission that Such is prior art. Med. Assoc., 1931, iii. 97), and later confirmed by Stephens Further, from the above discussion, it should be clear that and Lawrence in 1936 (Ann. Intern. Med. 9, 1488–1502). there are a plethora of different types of cancers, each of Typically, Fowler's solution was orally administered to which requires a unique treatment protocol. Thus, the devel leukemic patients as a solution until the level of white blood opment of a broad spectrum anti-cancer agent is extremely cells was depressed to an acceptable level or until toxicities desirable. At a minimum, additional effective anti-cancer (such as skin keratoses and hyperpigmentation) developed, agents are needed to be added to the arsenal against cancer. while the patients enjoyed varying periods of remission. In 15 the 1960s, Fowler's solution was still used occasionally in 3. SUMMARY OF THE INVENTION attempts to treat CML, however, most patients with CML were treated with other chemotherapeutic agents, such as Notwithstanding the conflicting reports in the art concern ing benefits and risks of the administration of arsenic to , and/or radiation therapy (Monfardini et al., Can patients, applicants have discovered that arsenic compound cer, 1973, 31:492-501). has broad applicability in the treatment of various cancers, Paradoxically, one of the long recognized effects of expo including Solid tumors and blood disorders. For example, the Sure to arsenic, whether the source is environmental or present invention encompasses the use of arsenic in the form medicinal, is skin cancer (Hutchinson, 1888, Trans. Path. of a salt, complex, organic compound or ionic solution to Soc. Lond, 39:352; Neubauer, 1947, Br. J. Cancer, 1:192). 25 treat tumors of epithelial tissue, connective tissue, central There were even epidemiological data to Suggest that the use nervous system, lymphoid tissue, hematopoietic cells and of Fowler's solution over long periods could lead to an tumors associated with oncogenic viruses. increased incidence of cancer at internal sites (CuZicket al., Further, the present invention encompasses the use of Br. J. Cancer, 1982, 45:904–911; Kaspar et al., J. Am. Med. arsenic compounds to treat mammals Suffering from primary Assoc., 1984, 252:3407 3408). The carcinogenicity of 30 and metastatic neoplastic disease as well as infectious dis arsenic has since been demonstrated by the fact that it can eases related thereto. induce chromosomal aberration, gene amplification, sister In addition, this invention also encompasses the use of chromatid exchanges and cellular transformation (See e.g., arsenic compounds to treat primary and metastatic breast, Lee et al., 1988, Science, 241:79–81; and Germolec et al., 35 lung, bladder and prostate cancers in humans. Toxicol. Applied Pharmacol., 1996, 141:308-318). Because This invention also encompasses the treatment of hemato of the known carcinogenic effect of arsenic, its only thera poietic disorders in mammals by the administration of one or peutic use in human in Western medicine today is in the more arsenic compounds to said mammal. The hematopoi treatment of tropical diseases, such as African trypanoso etic disorders to be treated include but are not limited to miasis, (melarsoprol, or Arsobal(R) by Rhone Poulenc Rorer, 40 polycythemia Vera, Hodgkin’s Disease, non-Hodgkin’s Dis Collegeville, Pa.; See Goodman & Gilman's The Pharma ease including Follicular Lymphoma, Diffuse Lymphoma, cological Basis of Therapeutics, 9th edition, chapter 66, lymphoblastic lymphoma, Small lymphocytic lymphoma, 1659–1662, 1997). acute lymphocytic leukemia, hairy cell leukemia, myeloid In traditional chinese medicine, arsenous acid or arsenic metaplasia, myeloid dysplastic syndrome, multiple trioxide paste has been used to treat tooth marrow diseases, 45 myeloma and plasmacytoma. psoriasis, syphilis and rheumatosis (Chen et al., 1995, in In accordance with the present invention, arsenic com Manual of Clinical Drugs, Shanghai, China, Shanghai Insti pounds can be used alone or in combination with other tute of Science and Technology, p. 830). In 1970s, arsenic known therapeutic agents (including chemotherapeutics, trioxide had been applied experimentally to treat acute radioprotectants and radiotherapeutics) or techniques to promyelocytic leukemia (APL) in China (commented by 50 either improve the quality of life of the patient, or to treat the Mervis, 1996, Science, 273:578). The clinical efficacy of primary neoplastic disease. For example, the arsenic com arsenic trioxide has recently been re-investigated in 14 of 15 pounds can be used before, during or after the administration patients with refractory APL, where the use of an intrave of one or more known antitumor agents including but not nous dose at 10 mg/day for 4–9 weeks was reported to result limited to mustard compounds, nitrogen mustard, chloram in complete morphologic remission without associated bone 55 bucil, , , 6-, marrow suppression (Shen et al., 1997, Blood, 6-thioguanine, , 5-fluorouracil, , meth 89:3354-3360). It was also reported that arsenic trioxide otrexate, Vincristine, vinblastine, taxol, etoposide, temipo induced apoptosis (programmed cell death) in vitro in NB4 side, , , doxorubicin, , cells, an APL cell line, and that apoptosis was apparently mitomycin, cisplatin, carboplatin, estramustine phosphate, associated with down-regulation of the oncogene bcl-2, and 60 hydroxyurea, BCNU, , VM-26 (vumon), inter intracellular redistribution of the chimeric PML/RARC. pro ferons and all-trans retinoic acid (ATRA), (See for example, tein that are unique to APL cells (Chen et al., 1996, Blood, the Physician Desk References 1997). In addition, the 88: 1052–1061; Andre et al., 1996, Exp. Cell Res. 229: arsenic compounds can be used before, during or after 253–260). Similarly, melarsoprol has been reported to irradiation treatment. For the treatment of HIV-infected induce apoptosis in cell lines representative of chronic 65 individuals, the arsenic compounds can be used alone or in B-cell leukemia. (Konig et al., 1997, Blood 90:562–570). combination with AZT, dd, ddA, ddC, d4T, 3TC and other Whether apoptosis is induced in APL patients is presently known antiviral agents. US 7,179,493 B2 7 8 The invention described herein encompasses a method of M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, treating primary and metastatic neoplastic diseases, a UACC-62. FIG. 1F. Ovarian Cancer cell lines IGROV1, method of treating Solid tumors, a method of treating leu OVCAR-3, OVCAR-5, OVCAR-8, SK-OV-3. FIG. 1G. kemias, a method of treating cancers related to bcl-2 (onco Renal Cancer cell lines A498, CAKI-1, RXE 393, SN12C, gene), each of which comprises the administration of a 5 TX-10, UO-31. FIG. 1H. Prostate Cancer cell lines PC-3, therapeutically effective and non-lethal amount of one or DU-145. FIG. 1I. Breast Cancer cell lines MCF7, NCI/ more arsenic compounds to a mammal in need of Such ADR-RES, MDA-MB-435, MDA-N, BT-549, T-47D. therapy. The invention, as mentioned above also encom FIG. 2. Mean graphs showing selectivity patterns at each passes the use of combination therapy to treat the aforemen of the principal response parameters for all the cell lines tioned diseases. 10 tested after continous exposure to 10 to 10 ug/ml of In a particular embodiment, the arsenic compounds are arsenic trioxide for 2 days. used within a method to treat breast, lung, colon, ovarian, FIGS. 3A-3I. Dose response curves showing percentage renal, non-Small cell lung, central nervous system, bladder, growth of various cancer cell lines after continuous exposure prostate and head and neck cancer by administering an to 10 to 10 g/ml cf arsenic trioxide for 6 days. FIG.3A. effective amount of one or more arsenic compounds alone or 15 Leukemic cell lines CCRF-CEM, K-562, MOLT-4, RPMI in combination with other antineoplastic agents or therapeu 8226. FIG. 3B. Non-small Cell Lung Cancer cell lines tic techniques including radiotherapy and Surgery. EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI Without being limited by any theory, the inventors believe H322M, NCI-H460, NCI-H522. FIG.3C. Colon Cancer cell that the arsenic compounds of the invention may have one lines COLO 205, HCT-116, HCT-15, HT29, KM12, or more mechanisms of action in connection with the SW-620. FIG. 3D. CNS Cancer cell lines SF-268, SF-295, methods described herein. For example, the arsenic com SF-539, SNB-75, U251. FIG. 3E. Melanoma cell lines LOX pounds may act as a phosphorous analogue which interferes IMVI, MALMI-3M, SK-MEL-2, SK-MEL-28, SK-MEL-5, with the phosphorylation events that occur in signal trans UACC-257, UACC-62. FIG. 3F. Ovarian Cancer cell lines duction involved in apoptosis. Arsenic may also act as an IGROVI, OVCAR-3, OVCAR-5, OVCAR-8, SK-OV-3. inhibitor of angiogenesis, i.e., the formation of new blood 25 FIG. 3G. Renal Cancer cell lines 786-O, A498, CAKI-1, vessels, thereby limiting blood flow to proliferating preneo RXF 393, S12C, TK-10. FIG. 3H. Prostate Cancer cell lines plastic cell masses, tumors and metastases. It is well known DU-145. FIG. 3I. Breast Cancer cell lines MCF7, NCI/ that if a tumor is not invaded by blood capillaries, it would ADR-RIS, MDA-MB-231/ATCC, HS 578T, MDA-MB have to depend on the diffusion of nutrients from its sur 435, MDA-N, BR-549, T-47D. roundings and cannot enlarge beyond a certain size. Arsenic 30 FIG. 4. Mean graphs showing selectivity patterns at each may also function as a differentiating agent which causes of the principal response parameters for all the cell lines dividing preneoplastic and/or cancer cells that display an tested after continous exposure to 10 to 10 g/ml of undifferentiated or underdifferentiated phenotype to develop arsenic trioxide for 6 days. into terminally differentiated cells, and die after a finite number of cell divisions. Finally, arsenic may also act to 35 5. DETAILED DESCRIPTION OF THE sensitize the cancer cells to radiation and/or chemotherapy. INVENTION Thus, the arsenic compounds of the invention are described as being useful against a variety of cancers. Methods and compositions for the treatment of primary Specific therapeutic regimens, pharmaceutical composi and metastatic neoplastic diseases are described herein. The tions, and kits are also provided by the invention. Thus, the 40 invention is based, in part, on a dosage regimen for admin invention also encompasses pharmaceutical compositions istration of compositions comprising arsenic. The invention which comprise one or more arsenic compounds and a is also based in part, on the potency of the arsenic com pharmaceutically acceptable carrier. The compositions are pounds of the invention against certain cancers. sterile solutions suitable for intravenous injection or infu This invention includes a method of treating primary Solid Sion. In another embodiment the invention encompasses a 45 tumors in a mammal which comprises administering to a composition Suitable for oral delivery; comprising one or mammal in need of Such therapy a therapeutically effective more arsenic compounds and a pharmaceutically acceptable and non-lethal amount of one or more arsenic compound. excipient or carrier. In another embodiment, the invention The invention also includes a method of treating meta also includes compositions Suitable for topical or dermal static tumors in a mammal which comprises administering to delivery. 50 a mammal a therapeutically effective and non-lethal dose of Particular compositions of the invention and their prop one or more arsenic compound. erties are described in the sections and subsections which The invention includes a method for treating disorders of follow. blood in mammal which comprises administering one or more arsenic compound in a therapeutically effective and 4. BRIEF DESCRIPTION OF THE FIGURES 55 non-lethal amount. The arsenic compound of the invention may be utilized in FIGS. 1A-1I. Dose response curves showing percentage in a variety of known forms; for example, arsenic can be growth of various cancer cell lines after continuous exposure administered as a salt, an organic or inorganic complex, an to 10 to 10 ug/ml of arsenic trioxide for 2 days. FIG. 1A. organic chelate, an organic compound or an organic or Leukemic cell lines CCRF-CEM, HL-60(TB), K-562, 60 inorganic solution. It is preferred that the form be chosen to MOLT-4, RPMI-8226, SR. FIG. 1B. Non-Small Cell Lung reduce toxicity and improve efficacy. Further, the form Cancer cell lines A549/ATCC, EKVX, HOP-62, HOP-92, chosen may also depend on the type and location of the NCI-H226, NCI-H23, NCI-8322M, NCI-H460, NCI-H522. tumor in question. The inorganic salt forms of arsenic are FIG. 1C. Colon Cancer cell lines COLO 205, HCT-116, preferred. For example, inorganic salts such as arsenic HCT-15, HT29, KM12, SW620. FIG. 1D. CNS Cancer cell 65 triiodide, arsenic(III)bromide, arsenic(III)chloride, arsenic lines SF-268, SF-295, SF-539, SNB-19, SNB-75, U251. pentoxide, arsenic trioxide, Fowler's solution (potassium FIG. 1E. Melanoma cell lines LOX 1 MV1, MALME-3M, arsenite), sodium arsenite, and calcium arsenite may be US 7,179,493 B2 10 used. Arsenic trioxide is most preferred. Both arsenous acids tumors of the central nervous system, breast, colon, ovaries, and arsenites as well as arsenic acids and arsenates may be kidneys, lung, bladder, prostate and head and neck. used within the present methods. Aqueous solutions con More specifically, the arsenic compounds of the present taining arsenite ions are preferred. Further, arsenic sulfides invention can be used to treat tumors of epithelial origin may be used such as arsenous Sulfide, arsenic Sulfide, arsenic including but not limited to: pentasulfide, tetraarsenic trisulfide and tetraarsenic penta squamous cell carcinoma sulfide. Without being limited by any theory, certain of these basal cell carcinoma arsenic compounds may be prodrugs to an active species. melanoma Generally, the skilled artisan will recognize that the form Tumors of Epithelial Lining of Glands or Ducts: of arsenic to be used should be therapeutically effective 10 without unreasonable toxicity. The toxicity is dependent adenocarcinoma upon the dose, the dosage form, the mode of administration papillary carcinoma and frequency of dosing. Generally, the skilled artisan can papillary adenocarcinoma chose from the following known forms of arsenic: arsenic Tumors of the Liver and Biliary Tract: halides, arsenic oxides, arsenic acids, arsenic sulfides, and 15 Hepatocellular carcinoma the like. Arsenic can also be readily combined with carbon to form Tumors of the Gastrointestinal Tract: a wide variety of organic compounds. These include but are squamous cell carcinoma of the esophagus not limited to primary and secondary arsines, tertiary ars adenocarcinoma of the esophagus ines, halo arsines, dihalo arsines, cyclic and polymeric colorectal carcinoma (colon cancer) Substances containing arsenic; specific examples of organic gastric carcinoma (stomach cancer) arsenic compounds include but are not limited to 3-Nitro Tumors of Respiratory Tract: 4-hydroxyphenylarsonic acid, arsanilic acid, Sodium hydro bronchogenic carcinoma gen 4-aminophenylarsenate, melarsoprol, melarsonyl potas Small cell carcinoma sium, carbarSone, arsenamide arsphenamine and sodium 25 large cell carcinoma arsanilate. Tumors of the Urogenital Tract: As used herein, “arsenic compound” refers to a pharma transitional cell carcinomas of bladder ceutically acceptable form of arsenic including salts, Solu squamous cell carcinoma of bladder tions, complexes, chelates and organic and inorganic com carcinoma of prostate pounds incorporating arsenic. It should be recognized that 30 the invention includes arsenic prodrugs or compounds that carcinoma of cervix are converted in vivo to biologically active forms of arsenic. Tumors of Breast Such prodrugs may be used to reduce or avoid the well known potential for arsenic toxicity. The arsenic compounds Tumors of Blood Cells and Related Cells (): of the present invention can be synthesized or commercially 35 acute and chronic lymphocytic leukemia purchased. For example, the compounds can be prepared polycythemia Vera from well-known chemical techniques. (See for example, Cancers of Lymphoid Tissue Kirk-Othmer, Encyclopedia of Chemical Technology 4 ed. Malignant Lymphomas—Hodgkins Lymphoma volume 3 pps. 633-655 John Wiley & Sons). Non-Hodgkin’s Lymphoma—Follicular lymphoma In one embodiment, the arsenic compound of the inven 40 Diffuse lymphoma tion is arsenic trioxide which is dissolved in an aqueous Small lymphocytic lymphoma solution of sodium hydroxide, with the pH adjusted to a Large cell lymphoma physiologically acceptable range, e.g. about pH 6-8. Lymphoblastic lymphoma Any Suitable mode of administration may be used in Multiple myeloma accordance with the present invention including but not 45 limited to parenteral administration Such as intravenous, Tumors of Connective Tissue Subcutaneous, intramuscular and intrathecal administration; Cancers of Bone oral, intranasal, rectal or vaginal administration may also be Osteosarcoma used; directly into the tumor; transdermal patches; implant Tumors of the Nervous System devices (particularly for slow release); finally, topical 50 Neuroblastoma administration may be used. The mode of administration Retinoblastoma will vary according to the type of arsenic compound being Glioblastoma used and the disease to be treated. The pharmaceutical compositions to be used may be in Oligodendroglioma the form of sterile physiologically acceptable (aqueous or 55 Tumors Associated With Oncogenic Viruses organic) solutions, colloidal Suspensions, creams, ointments, Human Papillomavirus—squamous cell carcinoma of cervix pastes, capsules, caplets, tablets and cachets. The pharma Ebstein-Barr Virus—Burkitts Lymphoma B cell lympho ceutical compositions comprising arsenic compounds of the ma’s in immuno-comprised individuals Nasopharyngeal invention can be contained in sealed sterile glass containers carcinoma and/or ampoules. Further, the active ingredient may be 60 Hepatitis B Virus—Hepatocellular carcinoma micro-encapsulated, encapsulated in a liposome, noisome or Herpes Virus 8 or Kaposi Sarcoma Herpes Virus lipofoam alone or in conjunction with targeting antibodies. (KSHV)—Kaposi's Sarcoma, and the like. Other neoplastic It should be recognized that delayed slow or sustained diseases known to the skilled artisan are also encompassed release forms of administration are also included. by the present invention including cancer of the oral cavity, The arsenic compounds of the present invention may be 65 larynx, kidney, testis and ovary. The skilled artisan will used against a variety of primary and metastatic neoplastic recognize that other cancers may be treated in accordance diseases including but not limited to primary and metastatic with the present invention. US 7,179,493 B2 11 12 The term “a method for treating primary and metastatic ing, but not limited to, the cytokines IFN-O, IFN-Y, IL-2, tumors of the central nervous system' as used herein means IL-4, IL-6, TNF, or other immunostimulatants/immuno that the disease and the symptoms associated with the modulators. In accordance with this aspect of the invention, disease are alleviated, reduced, cured, or otherwise placed in the arsenic compounds are administered in combination a state of remission. therapy with one or more of these agents. As used herein, the terms “a method for treating primary or metastatic breast, lung, bladder or prostate cancer and “a 5.1. Formulation method for treating metastases from breast, lung, bladder or prostate cancer” means that the disease and the symptoms The arsenic compounds of the invention may be formu associated with the disease are alleviated, reduced, cured, or 10 lated into pharmaceutical preparations for administration to placed in a state of remission. In addition, the term "a mammals for treatment of cancer. Compositions comprising method for treating metastases from breast, lung, bladder or a compound of the invention formulated in a compatible prostate cancer means that the metastatic tumors and the pharmaceutical carrier may be prepared, packaged, labelled symptoms associated with the disease are alleviated, for treatment of and used for the treatment of the indicated reduced, cured, placed in a state of remission. 15 tumor, such as human sarcomas and carcinomas, e.g., fib The term “refractory” when used herein means that malig rosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, nancies are generally resistant to treatment or cure. The term osteogenic sarcoma, chordoma, angiosarcoma, endothe “refractory' when used in the above terms, means that the liosarcoma, lymphangiosarcoma, lymphangioendotheliosar malignancies which are generally resistant to treatment or coma, Synovioma, mesothelioma, Ewing's tumor, leiomyo cure are alleviated, reduced, cured, or placed in a state of sarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic remission. cancer, breast cancer, ovarian cancer, prostate cancer, squa As used herein the terms “a therapeutic agent”, “thera mous cell carcinoma, basal cell carcinoma, adenocarcinoma, peutic regimen”, “radioprotectant”, “chemotherapeutic Sweat gland carcinoma, sebaceous gland carcinoma, papil mean conventional drugs and drug therapies, including lary carcinoma, papillary adenocarcinomas, cystadenocarci vaccines, for treating cancer, viral infections, and other 25 noma, medullary carcinoma, bronchogenic carcinoma, renal malignancies, which are known to those skilled in the art. cell carcinoma, hepatoma, bile duct carcinoma, choriocar "Radiotherapeutic' agents are well known in the art. cinoma, seminoma, embryonal carcinoma, Wilms tumor, As used herein, “a method of treating cancer or “a cervical cancer, testicular tumor, lung carcinoma, Small cell method of treating solid tumors” or “a method of treating lung carcinoma, bladder carcinoma, epithelial carcinoma, neoplastic diseases' means that the disease and the Symp 30 glioma, astrocytoma, medulloblastoma, craniopharyngioma, toms associated with the disease are alleviated, reduced, ependymoma, pinealoma, hemangioblastoma, acoustic neu cured, or placed in a state of remission. Further, tumor roma, oligodendroglioma, meningioma, melanoma, neuro growth is inhibited and/or tumor size is reduced. blastoma, retinoblastoma; leukemias, e.g., acute lympho As used herein, “preneoplastic' cell refers to a cell which cytic leukemia (myeloblastic, promyelocytic, is in transition from a normal to a neoplastic form; and 35 myelomonocytic, monocytic and erythroleukemia); chronic morphological evidence, increasingly supported by molecu leukemia and chronic lymphocytic leukemia; and poly lar biologic studies, indicates that preneoplasia progresses cythemia Vera, lymphoma (Hodgkin’s disease and non through multiple steps. Non-neoplastic cell growth com Hodgkin’s disease), multiple myeloma, Waldenström’s monly consists of hyperplasia, metaplasia, or most particu macroglobulinemia, and heavy chain disease. Alternatively, larly, dysplasia (for review of Such abnormal growth con 40 it can be labeled for treatment of the appropriate infectious ditions (See Robbins and Angell, 1976, Basic Pathology, 2d disease. Alternatively, pharmaceutical compositions may be Ed., W. B. Saunders Co., Philadelphia, pp. 68–79). Hyper formulated for treatment of appropriate infectious diseases. plasia is a form of controlled cell proliferation involving an If the complex is water-soluble, then it may be formulated increase in cell number in a tissue or organ, without signifi in an appropriate buffer, for example, phosphate buffered cant alteration in structure or function. As but one example, 45 saline or other physiologically compatible solutions. Alter endometrial hyperplasia often precedes endometrial cancer. natively, if the resulting complex has poor solubility in Metaplasia is a form of controlled cell growth in which one aqueous solvents, then it may be formulated with a non-ionic type of adult or fully differentiated cell substitutes for Surfactant such as Tween, polyethylene glycol or glycerine. another type of adult cell. Metaplasia can occur in epithelial Thus, the compounds and their physiologically acceptable or connective tissue cells. A typical metaplasia involves a 50 solvates may be formulated for administration by inhalation Somewhat disorderly metaplastic epithelium. Dysplasia is or insufflation (either through the mouth or the nose) or oral, frequently a forerunner of cancer, and is found mainly in the buccal, parenteral, topical, dermal, vaginal, drug delivery epithelia; it is the most disorderly form of non-neoplastic device, e.g., porous or viscous material Such as lipofoam, cell growth, involving a loss in individual cell uniformity rectal administration or, in the case of tumors, directly and in the architectural orientation of cells. Dysplastic cells 55 injected into a solid tumor. often have abnormally large, deeply stained nuclei, and For oral administration, the pharmaceutical preparation exhibit pleomorphism. Dysplasia characteristically occurs may be in liquid form, for example, solutions, syrups or where there exists chronic irritation or inflammation, and is Suspensions, or may be presented as a drug product for often found in the cervix, respiratory passages, oral cavity, reconstitution with water or other suitable vehicle before and gall bladder. Although preneoplastic lesions may 60 use. Such liquid preparations may be prepared by conven progress to neoplasia, they may also remain stable for long tional means with pharmaceutically acceptable additives periods and may even regress, particularly if the inciting Such as Suspending agents (e.g., Sorbitol syrup, cellulose agent is removed or if the lesion Succumbs to an immuno derivatives or hydrogenated edible fats); emulsifying agents logical attack by its host. (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond The therapeutic regimens and pharmaceutical composi 65 oil, oily esters, or fractionated vegetable oils); and preser tions of the invention may be used with additional immune Vatives (e.g., methyl or propyl-p-hydroxybenzoates or Sorbic response enhancers or biological response modifiers includ acid). The pharmaceutical compositions may take the form US 7,179,493 B2 13 14 of for example, tablets or capsules prepared by conventional desiccated; in this instance, the kit optionally further com means with pharmaceutically acceptable excipients such as prises in a container a pharmaceutically acceptable Solution binding agents (e.g., pregelatinized maize starch, polyvinyl (e.g., Saline, dextrose solution, etc.), preferably sterile, to pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., reconstitute the complex to form a solution for injection lactose, microcrystalline cellulose or calcium hydrogen purposes. phosphate); lubricants (e.g., magnesium Stearate, talc or In another embodiment, a kit of the invention further silica); disintegrants (e.g., potato starch or sodium starch comprises a needle or syringe, preferably packaged in sterile glycolate); or wetting agents (e.g., Sodium lauryl Sulphate). form, for injecting the complex, and/or a packaged alcohol The tablets may be coated by methods well-known in the art. pad. Instructions are optionally included for administration Preparations for oral administration may be suitably for 10 of arsenic compounds by a clinician or by the patient. mulated to give controlled release of the active compound. The magnitude of a therapeutic dose of an arsenic com For buccal administration, the compositions may take the pound in the acute or chronic management of cancer will form of tablets or lozenges formulated in conventional vary with the severity of the condition to be treated and the a. route of administration. The dose, and perhaps dose fre For administration by inhalation, the compounds for use 15 quency, will also vary according to the age, body weight, according to the present invention are conveniently deliv condition and response of the individual patient. In general, ered in the form of an aerosol spray presentation from the total daily dose ranges for the conditions described pressurized packs or a nebulizer, with the use of a suitable herein are generally from about 10 ug to about 200 mg propellant, e.g., dichlorodifluoromethane, trichlorofluo administered in divided doses administered parenterally or romethane, dichlorotetrafluoroethane, carbon dioxide or orally or topically. A preferred total daily dose is from about other suitable gas. In the case of a pressurized aerosol the 0.5 mg to about 70 mg of the active ingredient. dosage unit may be determined by providing a valve to Desirable blood levels may be maintained by a continuous deliver a metered amount. Capsules and cartridges of, e.g., infusion of an arsenic compound as ascertained by plasma gelatin for use in an inhaler or insufflator may be formulated levels. It should be noted that the attending physician would containing a powder mix of the compound and a suitable 25 know how to and when to terminate, interrupt or adjust powder base Such as lactose or starch. therapy to lower dosage due to toxicity, or bone marrow, The compounds may be formulated for parenteral admin liver or kidney dysfunctions. Conversely, the attending istration by injection, e.g., by bolus injection or continuous physician would also know how to and when to adjust infusion. Such formulations are sterile. Formulations for treatment to higher levels if the clinical response is not injection may be presented in unit dosage form, e.g., in 30 adequate (precluding toxic side effects). ampules or in multi-dose containers, with an added preser Again, any suitable route of administration may be vative. The compositions may take such forms as suspen employed for providing the patient with an effective dosage sions, solutions or emulsions in oily or aqueous vehicles, of an arsenic compound. For example, oral, rectal, vaginal, and may contain formulatory agents such as Suspending, transdermal, parenteral (Subcutaneous, intramuscular, stabilizing and/or dispersing agents. Alternatively, the active 35 intrathecal and the like) may be employed. Dosage forms ingredient may be in powder form for constitution with a include tablets, troches, cachet, dispersions, Suspensions, Suitable vehicle, e.g., sterile pyrogen-free water, before use. Solutions, capsules, patches, and the like. (See, Remington's The compounds may also be formulated in rectal com Pharmaceutical Sciences.) positions such as Suppositories or retention enemas, e.g., The pharmaceutical compositions of the present invention containing conventional Suppository bases Such as cocoa 40 comprise an arsenic compound as the active ingredient, or a butter or other glycerides. pharmaceutically acceptable salt thereof, and may also con In addition to the formulations described previously, the tain a pharmaceutically acceptable carrier, and optionally, compounds may also be formulated as a depot preparation. other therapeutic ingredients, for example antivirals. The Such long acting formulations may be administered by term “pharmaceutically acceptable salts' refers to salts implantation (for example, Subcutaneously or intramuscu 45 prepared from pharmaceutically acceptable non-toxic acids larly) or by intramuscular injection. Thus, for example, the and bases, including inorganic and organic acids and bases. compounds may be formulated with Suitable polymeric or The pharmaceutical compositions include compositions hydrophobic materials (for example, as an emulsion in an Suitable for oral, rectal, mucosal routes, transdermal, acceptable oil) or ion exchange resins, or as sparingly parenteral (including Subcutaneous, intramuscular, intrathe soluble derivatives, for example, as a sparingly soluble salt. 50 cal and intravenous), although the most Suitable route in any Liposomes and emulsions are well known examples of given case will depend on the nature and severity of the delivery vehicles or carriers for hydrophilic drugs. condition being treated. The compositions may, if desired, be presented in a pack In the case where an intravenous injection or infusion or dispenser device which may contain one or more unit composition is employed, a Suitable dosage range for use is, dosage forms containing the active ingredient. The pack may 55 e.g., from about 0.5 mg to about 150 mg total daily dose. for example comprise metal or plastic foil. Such as a blister In addition, the arsenic carrier could be delivered via pack. The pack or dispenser device may be accompanied by charged and uncharged matrices used as drug delivery instructions for administration. devices such as cellulose acetate membranes, also through The invention also provides kits for carrying out the targeted delivery systems such as fusogenic liposomes therapeutic regimens of the invention. Such kits comprise in 60 attached to antibodies or specific antigens. one or more containers therapeutically effective amounts of In practical use, an arsenic compound can be combined as the arsenic compounds in pharmaceutically acceptable form. the active ingredient in intimate admixture with a pharma The arsenic compound in a vial of a kit of the invention may ceutical carrier according to conventional pharmaceutical be in the form of a pharmaceutically acceptable solution, compounding techniques. The carrier may take a wide e.g., in combination with Sterile saline, dextrose solution, or 65 variety of forms depending on the form of preparation buffered solution, or other pharmaceutically acceptable ster desired for administration, e.g., oral or parenteral (including ile fluid. Alternatively, the complex may be lyophilized or tablets, capsules, powders, intravenous injections or infu US 7,179,493 B2 15 16 sions). In preparing the compositions for oral dosage form cinoma in order to inhibit the progression of the neoplastic any of the usual pharmaceutical media may be employed, disease and ultimately irradiate all preneoplastic and neo e.g., water, glycols, oils, alcohols, flavoring agents, preser plastic cells. Vatives, coloring agents, and the like; in the case of oral In a specific embodiment, the present invention provides liquid preparations, e.g., Suspensions, Solutions, elixirs, lipo hsp compositions and methods for enhancing specific immu Somes and aerosols; starches, Sugars, micro-crystalline cel nity to preneoplastic and neoplastic mammary cells in lulose, diluents, granulating agents, lubricants, binders, dis women. The present invention also provides compositions integrating agents, and the like in the case of oral Solid and methods for inhibiting cancer cell proliferation and preparations e.g., powders, capsules, and tablets. In prepar metastasis. These compositions can be applied alone or in ing the compositions for parenteral dosage form, Such as 10 combination with each other or with biological response intravenous injection or infusion, similar pharmaceutical modifiers. media may be employed, e.g., water, glycols, oils, buffers, 6. WORKING EXAMPLES Sugar, preservatives and the like know to those skilled in the art. Examples of Such parenteral compositions include, but 15 The following subsections describe the testing of a phar are not limited to Dextrose 5% w/v, normal saline or other maceutical composition comprising arsenic trioxide in vitro Solutions. The total dose of the arsenic compound may be using a panel of cancer cell lines employed by the National administered in a vial of intravenous fluid, e.g., ranging from Cancer Institute (NCI). The results demonstrate that arsenic about 2 ml to about 2000 ml. The volume of dilution fluid trioxide is effective in inhibiting the growth of a broad range will vary according the total dose administered. of leukemic cells and cancer cells in vitro. 5.2. Target Cancers 6.1. Methods and Materials Cancers that can be treated by the methods of the present Arsenic trioxide (1 mg/ml, 10 mg/ampoule, manufactured invention include, but not limited to human sarcomas and 25 by Taylor Pharmaceuticals, Decatur, Ill.) was tested at five carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, concentrations each at 10-fold dilutions, i.e., 10, 10. chondrosarcoma, osteogenic sarcoma, chordoma, angiosar 107, 10, and 10 g/ml. coma, endotheliosarcoma, lymphangiosarcoma, lymphan The in vitro tests were performed by incubating the test gioendotheliosarcoma, synovioma, mesothelioma, Ewings cells in the presence of the indicated concentration of arsenic 30 trioxide under standard culture conditions for a designated tumor, leiomyosarcoma, rhabdomyosarcoma, colon carci period of time, which is followed by a sulforhodamine B noma, pancreatic cancer, breast cancer, ovarian cancer, (SRB) protein assay to estimate cell viability or growth. The prostate cancer, squamous cell carcinoma, basal cell carci cell lines are organized into Subpanels according to the noma, adenocarcinoma, Sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocar origin of the cell lines, e.g., leukemia, breast cancer, etc. A 35 description of the cell lines and method of testing is cinomas, cystadenocarcinoma, medullary carcinoma, bron described in Monk et al. (1997, Anticancer Drug Des. chogenic carcinoma, renal cell carcinoma, hepatoma, bile 12:533–41) and Weinstein et al., (1997, Science 275:343–9), duct carcinoma, choriocarcinoma, seminoma, embryonal which are incorporated herein in their entirety. carcinoma, Wilms tumor, cervical cancer, testicular tumor, Described below are the data analysis procedures and lung carcinoma, Small cell lung carcinoma, bladder carci 40 displays. noma, epithelial carcinoma, glioma, astrocytoma, medullo The measurement of an effect is expressed in Percentage blastoma, craniopharyngioma, ependymoma, pinealoma, Growth (PG). The measured effect of the compound on a cell hemangioblastoma, acoustic neuroma, oligodendroglioma, line is calculated according to one or the other of the meningioma, melanoma, neuroblastoma, retinoblastoma; following two expressions: leukemias, e.g., acute lymphocytic leukemia (myeloblastic, 45 myelomonocytic, monocytic and erythroleukemia); and If (Mean OD-Meangest OD)20. then chronic lymphocytic leukemia; and polycythemia Vera, lym PG=100x(Mean OD-Mean OD)/(Mean OD phoma (Hodgkin’s disease and non-Hodgkin’s disease), tri-Mean OD'zero) multiple myeloma, Waldenström's macroglobulinemia, and heavy chain disease. Specific examples of Such cancers are If (Mean OD-Meangest OD)<0. then described in the sections below. 50 PG=100x(Mean OD OD). Mean OD In a specific embodiment the cancer is metastatic. In another specific embodiment, the patient having a cancer is where: immunosuppressed by reason of having undergone anti Mean OD, the average of optical density measurements cancer therapy (e.g., chemotherapy-radiation) prior to 55 of SRB-derived color just before exposure of cells to the administration of the arsenic compounds of the invention. test compounds; In a specific embodiment, the present invention provides Mean OD, the average of optical density measurements compositions and methods for enhancing tumor specific of SRB-derived color after exposure of cells to the test immunity in individuals suffering from colorectal cancer compound for a designated period of time; and metastasized to the liver, in order to inhibit the progression 60 Mean OD, the average of optical density measurements of the neoplastic disease. Preferred methods of treating these of SRB-derived color after with no exposure of cells to the neoplastic diseases comprise administering a composition of test compound for a designated period of time arsenic which elicits an immune response against tumor Table 1 and 2 present the experimental data collected cells. against each cell line. The first two columns describe the In another specific embodiment, the present invention 65 subpanel (e.g., leukemia) and cell line (e.g., CCRF-CEM) provides compositions and methods for enhancing specific involved. The next two columns list the Mean OD. and immunity in individuals suffering from hepatocellular car Mean OD; the next five columns list the Mean OD,ies for US 7,179,493 B2 17 18 each of five different concentrations. Each concentration is line to the test agent in excess of the average sensitivity of expressed as the logo (molar or g/ml). The next five all tested cell lines. Since the bar scale is logarithmic, a bar columns list the calculated PGs for each concentration. The 2 units to the right implies the compound achieved the response parameters GI50, TGI and LC50 are interpolated response parameter (e.g., GI50) for the cell line at a con values representing the concentrations at which the PG is 5 centration one-hundredth the mean concentration required +50, 0, and -50, respectively. Sometimes these response over all cell lines, and thus the cell line is unusually sensitive parameters cannot be obtained by interpolation. If, for to that compound. Bars extending to the left correspondingly instance, all of the PGs in a given row exceed +50, then none imply sensitivity less than the mean. If for a particular drug of the three parameters can be obtained by interpolation. In and cell line, it was not possible to determine the desired Such a case, the value given for each response parameter is 10 response parameter by interpolation, the bar length shown is the highest concentration tested and is preceded by a "> either the highest concentration tested (and the listed logo sign. This practice is extended similarly to the other possible of the response parameter will be preceded by a ">') or the situations where a response parameter cannot be obtained by lowest concentration tested (and the listed logo will be interpolation. preceded by a “C”). A dose-response curve (see FIGS. 1A-1I and 3A-3I) for 15 The values at either limit (>or <) are also calculated in the the set of data is created by plotting the PGs against the logo mean used for the mean graph. Therefore, the mean used in of the corresponding concentration for every cell line. The the mean graph may not be the actual mean of the GI50 for cell line curves are grouped by Subpanel. Horizontal lines instance. For this reason, this value is referred to as the are provided at the PG value of +50, 0, and -50. The MG MID (for mean graph midpoint). concentrations corresponding to points where the curves 20 cross these lines are the GI50, TGI, and LC50, respectively. 6.2. RESULTS A mean graph (FIGS. 2 and 4) facilitates visual scanning of data for potential patterns of selectively for particular cell The results of two sets of tests is presented below. In the lines or for particular subpanels with respect to a selected first set, the cells from 56 different cancer cell lines were response parameter. Differences in apparent selectivity pat- 25 exposed to five concentrations of arsenic trioxide continu terns may occur for the same compound against the same ously for two days prior to performing the SRB assay. In the cell lines when different parameters are compared. The mean second set, the cells from 50 different cell lines (a subset of graphs page of the data package shows mean graphs at each the first 56 cell lines, plus the renal cancer cell line 786-0) of the principal response parameters: GI50, TGI, and LC50. were exposed continuously for six days prior to the SRB Bars extending to the right represent sensitivity of the cell assay.

TABLE 1. Log 10 Concentration Time Mean Optical Densities Percent Growth

Zero Ctrl -8.9 -7.9 -6.9 -5.9 -4.9 -8.9 -79 -6.9 -5.9 -4.9 GISO TGI LCSO Leukemia

CCRF-CEM O3OO 115S 1.2O3 1.195 1.134 0.704 O.285 106 105 98 47 -5 111E-06 1.OOE-OS is 1.26E-05 HL-60(TB) O.233 O.S3O O.S33 O.SO7 O.S35 0.499 0.213 101 92 102 90 -9 3.19E-06 1.03E-OS >1.26E-05 K-562 O.209 1416 1387 1431 1418 1.124 O.199 98 101 100 76 -5 2.63E-O6 1.1OE-OS is 1.26E-05 MOLT 4 O.134 0.438 0.465 0.454 (0.454 O.368. O.146 109 10S 105 77 4 2.96E-06 s.1.26E-OS >1.26E-OS RPMI-8226 O.257 0.893 O.868 0.848 0.813 O.67O O.204 96 93 87 65 -21 188E-06 7.23E-06 s.1.26E-05 SR O.158 0.457 0.454 0.425 0.457 0.338 0.111 99 89 100 60 -3O 1.63E-06 5.85E-06 s.1.26E-05 Non Small Cell Lung Cancer

AS49; ATCC OO15 O.477 O.479 0.486 0.476 O.S16 O.336 OO 102 OO 108 69 -1.26E-OS >1.26E-OS is 1.26E-OS EKVX O.342 0.736 O.809 O.849 0.841 O.853 O.385 19 129 27 130 11 5.91E-06 s.1.26E-OS >1.26E-OS HOP-62 O.33S 1.109 1.088 1.099 1.113 1.086 O.6OS 97 99 OO 97 35 7.18E-06 s.1.26E-05 s1.26E-05 HOP-92 OSOS 1.694 1.SS4 1.603 1.477 1.381 0.873 88 92 82 74 31 4.5OE-06 s.1.26E-05 s1.26E-05 NCI-EH226 O.S60 0.932 O.967 O.918 0.967 O.967 O.904 O9 96 09 109 92 >1.26E-OS >1.26E-05 s1.26E-05 NCI-EH23 O648 1622 1769 1822, 1880 1635 1215 1S 121 27 101 58 -1.26E-OS >1.26E-05 s1.26E-05 NCI-8322M O.382 0.997 1.103 1.036 0.976 O.992 O.7SS 17 106 97 99 61 >1.26E-OS >1.26E-OS is 1.26E-OS NCI-EH460 O.296 123S 1.132 1.186 1234 1.157 0.757 89 95 OO 92 49 2OE-05 s1.26E-05 s1.26E-05 NCI-EHS22 O.478 1.138 1.332 1.13S 1.189 O.892 O.378 29 100 O8 63 -21 .79E-06 7.O8E-06 s.1.26E-05 Colon Cancer

COLO 2.05 O.328 1394 1425 1414 1576. 1434 O.935 O3 102 17 104 57 -1.26E-OS >1.26E-05 s1.26E-05 HCT-116 O.301 1.574 1.508 1.480 1488 1.391 O.68S 95 93 93 86 3O 5.53E-06 s.1.26E-05 s1.26E-05 HCT-15 O.219 1623 1.8OO 1627 1.673 1.522 OSO4 13 100 O4 93 2O 4.90E-06 s.1.26E-05 s1.26E-05 HT29 O.095 O.S78 O.637 O.S99 O.S80 O.479 0.169 12 104 OO 8O 15 3.63E-06 s.1.26E-05 s1.26E-05 KM12 O.189 0.741. O.744 O.728 0.777 O.737 O.S67 O1 98 O6 99 68 -1.26E-OS >1.26E-OS is 1.26E-OS SW-62O O.153 0.886 O.898 0.868 0.857 0.779 0.267 O2 98 96 85 16 4.05E-06 s.1.26E-05 s1.26E-05 CNS Cancer

SF-268 O.203 0.767 O.821 O.736 0.815 O.767 0.334 10 94 O8 100 23 5.63E-06 s.1.26E-05 s1.26E-05 SF-295 O.249 1.007 O.951 O.978 O.938 0.938 0.639 93 96 91 91 51 >1.26E-OS >1.26E-05 s1.26E-05 SF-539 O-132 0.462 0.491 O-516 0.506 0.435 0.110 O9 117 13 92 -17 3.06E-O6 8.85E-06 s.1.26E-05

US 7,179,493 B2 23 24 used in human Subjects to treat a broad range of leukemia, 5. The method of claim 1, wherein the total daily amount and cancers, including but not limited to non-Small cell lung administered is from about 0.5 mg to about 70 mg. cancer, colon cancer, central nervous system cancer, mela noma, ovarian cancer, renal cancer, prostate cancer, and 6. The method of claim 1, wherein the arsenic trioxide is breast cancer. administered intravenously. The present invention is not to be limited in scope by the 7. The method of claim 1, wherein the arsenic trioxide is specific embodiments described herein. Indeed, various administered in combination with an effective amount of at modifications of the invention in addition to those described least one other therapeutic agent. herein will become apparent to those skilled in the art from 8. The method of claim 7, wherein theater therapeutic the foregoing description. Such modifications are intended 10 agent is a chemotherapeutic or radiotherapeutic. to fall within the scope of the appended claims. 9. The method of claim 7, wherein the other therapeutic The invention claimed is: agent is selected from the group consisting of etoposide, 1. A method of treating melanoma in a human, which comprises administering parenterally a therapeutically cisplatin, carboplatin, estramustine phosphate, vinblastine, effective amount of arsenic trioxide to said human. 15 methotrexate, hydroxyurea, cyclophosphamide, doxorubi 2. The method of claim 1, wherein said arsenic trioxide is cin, 5-fluorouracil, taxol, diethylstilbestrol, VM-26(vumon), formulated as an ionic aqueous solution. BCNU, all-tans retinoic acid, procarbazine, cytokines, thera 3. The method of claim 1, wherein the total daily amount peutic vaccines, and immunomodulators. administered is from about 10 ug to about 200 mg. 4. The method of claim 1, wherein the total daily amount 20 administered is from about 0.5 mg to about 150 mg.