AU2006202364B2 - Process for producing arsenic trioxide formulations and methods for treating cancer using arsenic trioxide or melarsoprol - Google Patents

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Sloan-Kettering Institute For Cancer Research Actual Inventor(s): Raymond P Warrell, Pier Paolo Pandolfi, Janice L Gabrilove Address for Service and Correspondence: PHILLIPS ORMONDE & FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street evnboume 3000 AUSTRALIA Invention Title: PROCESS FOR PRODUCING ARSENIC TRIOXIDE FORMULATIONS AND METHODS FOR TREATING CANCER USING ARSENIC TRIOXIDE OR MELARSOPROL Our Ref: 774471 POF Code: 1060/112794 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 6006q PROCESS FOR PRODUCING ARSENIC TRIOXIDE FORMULATIONS AND METHODS FOR TREATING CANCER USING ARSENIC TRIOXIDE OR MELARSOPROL 5 1. FIELD OF INVENTION This application is a divisional of Australian patent application number 2002300339, which is in turn a divisional of Australian patent application number 747474 (13973/99), the entire content of which is herein incorporated by reference. 10 The present invention relates to methods and compositions for the treatment of leukemia, lymphoma, and certain other cancers. More specifically, the present invention relates to the novel uses of arsenic trioxide and an organic arsenic compound for treating acute leukemia and chronic leukemia. 15 2. BACKGROUND OF THE INVENTION 2.1. 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-bome spread of malignant cells to regional 20 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 as exemplified by hyperplasia, metaplasia, and dysplasia (for review of such abnormal growth conditions, see 25 Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79) precedes the formation of a neoplastic lesion. A neoplastic lesion may evolve clonally to grow into a solid tumor, and develop an increasing capacity for invasion, growth, metastasis, and heterogeneity, especially under conditions in which the neoplastic cells escape the host's immune 30 surveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps. 17.1-17.12). - la WiArwYMNDE1ETM147474iv.dot Leukemia refers to malignant neoplasms of the blood-forming tissues. Transformation to malignancy typically'occurs in a single cell through two or more steps with subsequent proliferation and clonal expansion. In some 5 leukemias, specific chromosomal translocations have been identified with consistent leukemic cell morphology and special clinical features (e.g., translocations of 9 and 22 in chronic myelocytic leukemia, and of 15 and 17 in acute promyelocytic leukemia). Acute leukemias are predominantly 10 undifferentiated cell populations and chronic leukemias more mature cell forms. Acute leukemias are divided into lymphoblastic (ALL) and non-lymphoblastic (ANLL) types. They may be further subdivided by their morphologic and cytochemical 15 appearance according to the French-American-British (FAB). classification or according to their type and degree of differentiation. The use of specific B- and T-cell and. myeloid-antigen monoclonal antibodies are most helpful for classification. ALL is predominantly a childhood disease 20 which is established by laboratory findings and bone marrow examination. ANLL, also known as acute myeloblastic leukemia (AML), occurs at all ages and is the more common acute leukemia among adults; it is the form usually associated.with irradiation as a causative agent. 25 chronic leukemias are described as being lymphocytic (CLL) or myelocytic (CML). CLL is characterized by the appearance of mature lymphocytes in blood, bone marrow, and lymphoid organs. The hallmark of CLL is sustained, absolute lymphocytosis (> 5,000/yL) and an 30 increase of lymphocytes in the bone marrow. Most CLL patients also have clonal expansion of lymphocytes with B cell characteristics. CLL is a disease of older persons. In CML, the characteristic feature is the predominance of granulocytic cells of all- stages of differentiation in blood, 35 bone marrow, liver, spleen, and other organs. In the symptomatic patient at diagnosis the total WBC count is usually about 2O0,000/yL, but may reach'1,000,000/gL. CML is -2relatively easy to diagnose because of the presence of the Philadelphia chromosome. The very nature of hematopoietic cancer necessitates using systemic chemotherapy as the primary 5 treatment modality. Drugs selected according to sensitivities of specific leukemias are usually given in combination. Radiation therapy may be used as an adjunct to treat local accumulations of leukemic cells.. Surgery is rarely indicated as a primary treatment modality, but may be 10 used in managing some complications. Bone marrow transplantation from an HLA-matched sibling is sometimes indicated. 2.2. ARSENIC AND ITS MEDICAL USES 15 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 20 treatment of a leukemic patient with Fowler's solution (a solution containing potassium arsenite, valence +5) 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 25 chronic myelogenous leukemia (CML) was described by Forkner and Scott in.1931 (J. Am. Med. Assoc., 1931, iii, 97), and later confirmed by Stephens and Lawrence in 1936 (Ann .. Intern. Med. 9, 1488-1502). However, while the active chemical ingredient(s) of Fowler's solution was not 30 determined, its toxicity was well recognized. Fowler's solution was administered strictly as an oral composition,.. and was given 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 35 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 -3chemotherapeutic agents, such. as busulf an, 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 5 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 10 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., 15 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 20 trypanosomiasis, (the organic arsenical, melarsoprol; See Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th edition, chapter 66, 1659-1662, 199.7). In traditional chinese medicine, arsenous acid or arsenic trioxide paste has been used to treat tooth marrow 25 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 1970!s, arsenic trioxide had been applied experimentally to treat acute promyelocytic leukemia (APL) in China (commented by 30 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 35 bone marrow suppression (Shen et al., 1997, Blood, 89:3354 3360). It was also shown 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; 5 Andre et al., 1996, Exp. Cell Res. 229:253-260). It has been reported that the biological activity of arsenic is due to the ability of arsenic to direct the nucleoplasmic fraction of PML to nuclear bodies for degradation (Zhu et al., 1997, Proc. Natl. Acad. Sci., 94:3978-3983). 10 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. Further, from the above discussion, it should be clear that there are many different types of leukemias, each of which requires a 15 unique treatment protocol that is modified according to the presence of factors predicting for a risk of treatment failure. Thus, the development of a broad spectrum anti leukemia agent that can be used alone or in combination with other existing drugs is extremely desirable. 20 3. SUMMARY OF THE INVENTION Despite the conflicting reports in the art concerning benefits and risks. of the administration of arsenic to patients, applicants have discovered that arsenic 25 ,rioxide and the organic arsenical, melarsoprol, have broad applicability in the treatment of various types of leukemias, lymphomas, and solid tumors. 30 35 -5- In accordance with the present invention, arsenic 5 trioxide or melarsoprol 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 leukemia, lymphoma or solid tumor. The 10 arsenic compounds can be used before, during or after the administration of one or more known chemotherapeutic agents, including antitumor agents. In addition, the arsenic compounds can be used before, during or after radiation treatment. 15 The pharmaceutical compositions of the invention are sterile solutions suitable for intravenous injection or infusion. In another embodiment the invention encompasses a composition suitable for oral delivery; comprising arsenic trioxide or melarsoprol and a pharmaceutically acceptable 20 excipient or carrier. In another embodiment, the invention also includes compositions suitable for topical or transdermal delivery, including but not limited to iontophoretic methods. Specific therapeutic regimens, pharmaceutical compositions, and kits are also provided by 25 the invention. Particular compositions of the invention and their uses are described in the sections and subsections which follow. 30 4. DETAILED DESCRIPTION OF THE INVENTION Methods and compositions for the treatment of leukemia, lymphoma or -solid tumors are described herein. This invention provides a method of treating acute or chronic leukemia, lymphoma, or solid tumors in a human which 35 comprises administering to a human in need of such therapy a therapeutically effective and non-lethal amount of one or more arsenic compounds, such as arsenic trioxide or melarsoprol. -6- 7 The invention also includes a method of treating leukemia in a human who has become refractory to other forms of treatment which comprises administering to a human arsenic trioxide or melarsoprol in combination with another chemotherapeutic agent, e.g., all-trans retinoic acid (ATRA). 5 Thus, the present invention provides a method for treating acute promyelocytic leukemia in a human comprising administering a therapeutically effective dosage amount of about 0.15 mg/kg arsenic trioxide once per day. The invention also relates to a method for the manufacture of pharmaceutical compositions comprising arsenic trioxide. It is preferred that 10 pharmaceutical compositions of the present invention exhibit reduced toxicity, improved efficacy, improved stability during storage and use, and that the composition has a physiologically acceptable pH. 4.1. THE ARSENIC COMPOUNDS 15 As used herein, "arsenic compound" refers to a pharmaceutically acceptable form of arsenic trioxide (As 2 0 3 ) or melarsoprol. Melarsopral is an organic arsenic compound which can be synthesized by complexing melarsen oxide with dimercaprol or commercially purchased (Arsobal@ by Rh6ne Poulenc Rorer, Collegeville, PA). Since the non-pharmaceutically formulated raw 20 materials of the invention are well known, they can be prepared from well known chemical techniques in the art. (see for example, Kirk-Othmer, Encyclopedia of Chemical Technology 4 th ed. Volume 3 pps.633-655 John Wiley & Sons). W:Files\674761\674781-new spec p7_310506.doc 7A 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" 5 agents are well known in the art. In accordance with the present invention, arsenic trioxide or melarsoprol 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 10 leukemia, lymphoma or solid tumor. For example, the arsenic compounds can be used before, during or after administration of one or more known antitumor W~t,,s'7478T 874781-spec p7.dc 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, 5 etoposide, temiposide, dactinomycin, daunorubicin, doxorubicin, bleomycin, mitomycin, cisplatin, carboplatin, estramustine phosphate, hydroxyurea, BCNU, procarbazine, VM 26, interferons, and all-trans retinoic acid (ATRA), or other retinoids (See, for example, the Physician Desk References 10 1997). In addition, the arsenic compounds can be used before, during or. after radiation treatment. In a specific embodiment, the arsenic compound of the invention and ATRA can be administered as a mixture. In preferred aspects, the lymphoma, leukemia or solid tumor in 15 the human treated by the combination is refractory to general methods of treatment, or is a relapsed case of leukemia. Any suitable mode of administration may be used in accordance' with the present invention including but not limited to parenteral administration -such asf-intravenous, 20 subcutaneous, intramuscular and intrathecal administration; oral, and intranasal administration, and inhalation.. The mode of administration will vary according to the type of cancer, and the condition of the human. The pharmaceutical compositions to be used may be 25 in the form of sterile aqueous or organic solutions, colloidal suspensions, caplets, tablets and cachets. 4.2. METHOD OF TREATMENT The term "a method for treating leukemia" as used 30 herein means that the disease and the symptoms associated with the disease are alleviated, reduced, cured, or placed in a state of remission. For example, the methods of treatment of the invention can lower the white blood cell count, or reduce lymphocytosis in a human under treatment. 35 The term "a method for treating lymphoma" as used herein means that the disease and the symptoms associated with the disease are alleviated, reduced, cured, or placed in a state of remission. - 8- The term "a method for treating solid tumor" as used herein means that the disease and the symptoms associated with the solid tumor are alleviated, reduced, cured, or placed in a state of remission. 5 In addition, the term "a method for treating leukemic -infiltration" means that the infiltration of leukemic cells out of circulation and into other organs and systems and the symptoms associated with such infiltration are alleviated, reduced, cured, or placed in a state of 10 remission. The term "refractory" when used herein means that the leukemia is generally resistant to treatment or cure. As used herein, "preneoplastic" cell refers to a cell which is in transition from a normal to a neoplastic 15 form; or cells that fail to differentiate normally; and morphological evidence, increasingly supported.by molecular biologic studies, indicates that preneoplasia progresses through multiple steps. In one embodiment, the invention provides a method 20 for treatment of leukemia in a human comprising the administration of a therapeutically effective and non-lethal amount of arsenic trioxide or melarsoprol to the human. The invention also provides a weight-based dosing regimen, not heretofore disclosed, that maximizes the safety. in humans of 25 these otherwise highly toxic compounds. Arsenic trioxide (As:o,) inhibits growth and induce apoptosis in NB4 acute promyelocytic leukemic cells. Acute promyelocytic leukemia (APL) is -associated with the t.(15;17) translocation, which generates a PML/RARa fusion protein 30 between PML, a growth suppressor localized on nuclear matrix associated bodies, and RARa, a nuclear receptor for retinoic acid (RA). PML/RARa was proposed to block yeloid differentiation through inhibition of nuclear receptor response, as does a dominant negative RARa mutant. In 35 addition, in APL cells, PML/RARa displaces -PML and other nuclear body (NB) antigens onto nuclear microspeckles, likely resulting in the loss of PML and/or NB functions. It.has been suggested that high concentrations of arsenic trioxide -9promote apoptosis, whereas-low concentrations induce partial differentiation in NB4 cells as well as cells derived from APL patients. It was postulated that As,Q works through its ability to specifically cause PML-RARa in APL cells to be 5 relocalized to nuclear bodies for degradation (Zhu et al., 1997, Proc. Natl. Acad. Sci. USA, 94:3978-3983). However, these findings tend to limit.the use of arsenic trioxide to a subset of leukemias. See Konig et al., 1997, Blood, 90:562 570. . 10 Unexpectedly, the inventors have discovered that both As 2 0: and melarsoprol are able to inhibit cell growth, and induce apoptosis in various myeloid leukemia cell lines in a PML and PML-RARa independent manner. Thus, the inventors have discovered that, contrary to the earlier i5 findings, arsenic trioxide and melarsoprol are both effective against a broad range of leukemias regardless of the underlying molecular mechanism that causes the neoplasia. Working examples of the effect of arsenic compounds on a number of leukemic cell lines are provided in Sections 5.1 20 and 5.2. Accordingly, the arsenic compounds of the present invention can be used against a variety of leukemias, including but not limited to: Acute lymphoblastic leukemia (ALL) 25 Acute lymphoblastic B-cell leukemia Acute lymphoblastic T-cell leukemia Acute -myeloblastic leukemia (AML) Acute promyelocytic leukemia (APL) Acute monoblastic leukemia 30 Acute erythroleukemic leukemia Acute megakaryoblastic leukemia Acute myelomonocytic leukemia Acute undifferentiated leukemia Chronic myelocytic leukemia (CML) 35 Chronic lymphocytic leukemia (CLL) The skilled artisan will recognize that other leukemias may be treated in accordance with the present invention. - 10 - In another embodiment, the invention provides a method for treatment of lymphoma in a human comprising the administration of a therapeutically effective and non-lethal amount of arsenic trioxide or melarsoprol to the human. 5 Lymphoma that can be treated by the methods of the invention include but are not limited to high grade lymphoma, intermediate grade lymphoma, low grade lymphoma, and the various subclassifications. In yet another embodiment, the invention provides a 10 method for treatment of solid tumors, including metastasises, in humans comprising the administration of a therapeutically effective and non-lethal amount of arsenic trioxide or melarsoprol-to the human. Solid tumors that can be treated by the methods of the invention include but are not limited 15 to: cancer of the digestive tract, oesophagus, liver, stomach, and colon; skin; brain; bone; breast; lung; and soft tissues, including but not limited to various sarcomas, and preferably prostate cancer. In various embodiments, the leukemic or tumor cells 20 are infiltrating other organs and systems in a human, for example, the central nervous system. The methods. of the invention are also applicable to reduce the number of preneoplastic cells in a human in which there is an abnormal increase in the number of preneoplastic cells. 25 In a specific embodiment, the invention-provides a method of treatment of acute promyelolytic leukemia (APL) in a human comprising the administration of a therapeutically effective and non-lethal amount of melarsoprol to the human. The inventors discovered, as described in Section 5.2, that 30 concentrations of melarsoprol that are cytotoxic in vitro can readily be achieved in vivo. In one specific embodiment, the invention provides a method of treatment of chronic myelogenous leukemia (CML) in a human comprising the administration of a therapeutically 35 effective and non-lethal amount of arsenic trioxide to the human. The 'inventors discovered, as described in Section 5.3, that arsenic trioxide can also induce apoptosis in a CML cell line. The therapeutic benefits of the pharmaceutical - 11 compositions of the invention comprising arsenic trioxide is far superior to that of potassium arsenite, commonly formulated as Fowler's solution. In yet another specific embodiment, the invention 5 provides a method of treatment of acute promyelocytic leukemia (APL) in a human, in which the APL is associated with a translocation of the RARa locus on chromosome 17 to chromosome 11, comprising the administration of a therapeutically effective amount of arsenic trioxide or 10 melarsoprol to the human. In the majority of APL cases, RARa on chromosome 17 translocates and fuses with the PML gene located on chromosome 15, i.e., t(15;17). In a few cases RARa translocates to chromosome 11 where it fuses to the PLZF. gene. Patients harboring the t(15;17) are uniquely sensitive 15 to treatment with all-trans retinoic acid (ATRA), yielding complete remission rates of 75% to 95%. APL associated with the t(11;17) (PLZF-RARa) shows a distinctly worse prognosis with poor response to chemotherapy and little or no response to treatment with ATRA, thus defining a new APL syndrome. 20 The present invention provides that arsenic trioxide or melarsoprol can be used to treat such cases of -APL. Transgenic animal models of APL associated with t(15;17) and t(11;17) for testing the therapeutic benefits and dosages of arsenic compounds of the invention are described in Section 25 5.4 hereinbelow. Humans having leukemia are sometimes refractory to conventional methods of treatment by reason of having undergone anti-leukemic therapy (e.g., chemotherapy)._ Thus, the invention provides a method of treatment of leukemia in a 30 human who is not responding to conventional therapy comprising the administration of a therapeutically effective and non-lethal amount of a combination of arsenic compound and another chemotherapeutic agent, such as but not limited to, all-trans retinoic acid (ATRA) or other retinoids, to the 35 human. The arsenic compound can either be arsenic trioxide or.melarsoprol or a pharmaceutically acceptable salt thereof. The invention also encompasses the treatment of retinoid resistant patients with an arsenic compound. - 12 - In specific embodiments, the arsenic compound of the invention and the chemotherapeutic agent can be administered either as a mixture or sequentially. When administered sequentially, the arsenic compound may be 5 administered before or after the chemotherapeutic agent, so long as the first administered agent is still providing antileukemic activity in the human when the second agent is administered. Any of the modes of administration described herein may be used to deliver the. combination. In preferred 1o aspects, the leukemia in the human treated by the combination is refractory to general methods of treatment, or is a relapsed case of leukemia. 4.3. PROCESS FOR THE MANUFACTURE OF STERILE 15 ARSENIC TRIOXIDE SOLUTION The arsenic compounds of the invention may be formulated into sterile pharmaceutical preparations for 20 administration to humans for treatment of leukemias, lymphomas and solid tumors. 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 25 leukemia, lymphoma, or solid tumor. In one aspect, the invention provides a method for the manufacture of a pharmaceutical composition comprising a therapeutic effective and non-lethal amount of arsenic trioxide (As:O). Arsenic trioxide (raw material) is a solid 30 inorganic compound that is commercially available in a very pure form. However, it is difficult to dissolve AsO in aqueous solution. Further, the inventors are unaware of any published teachings on how to formulate AsO, as a pharmaceutical composition suitable for injection directly 35 into a human. Arsenic is present in solution in the +5 valence state (pentavalent) or the +3 valence state (trivalent). For example, potassium arsenite (KAsO,; which is present in Fowler's solution) and salts of arsenious acid contain pentavalent arsenic. It is known that one form of -13arsenic is more toxic than the other. (Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th edition, chapter 66, 1660, 1997). A fresh solution of arsenic trioxide containing arsenic in the trivalent state will be 5 gradually oxidized to pentavalent state if exposed to air for a prolonged period, and as a result of the accumulation of pentavalent arsenic, the relative toxicity of a solution of As:O., will change over time. (Id.) Furthermore, it is observed that the total amount of arsenic in solution 10 decreases over time. This loss of material is caused by the progressive conversion of arsenic in the solution into arsine (AsH.) which is a gaseous compound at room temperature. This is particularly problematic in pharmaceutical applications if the concentration of an active ingredient in the injected 15 material cannot be controlled. It is also undesirable to allow arsine to escape from the solution into the atmosphere because arsine is also toxic. The inventors have experimented and successfully developed a method for formulating arsenic trioxide which 20 overcomes the above-described problems of solubility.and stability. The method comprises solubilizing solid high purity As::. in an aqueous solution at high pH, such as -pH greater than 12. For example, a 5 M solution of sodium hydroxide may be used. To aid solubilization and obtain a 25 clear and homogenous solution, mechanical stirring and/or gentle heating may be applied. A solution of AsO... can also be obtained by dissolving the solid compound overnight. Typically, a solution of 1 M AsO. is obtained by this method. However, this solution is too basic to be useful as a 30 pharmaceutical composition. To adjust the pH of the As70, solution, the solution is first diluted in water, for example, to a concentration of about 1 mg/mL, pH 12. The AsO. solution is then back titrated with acid, such as, hydrochloric acid (1 M to 5 M 35 HCl), with constant stirring until the pH is about 8.0 to 8.5. Highly concentrated HCl is not suitable as it causes precipitation to occur in the AsO. solution. The partially neutralized AsO, solution may then be sterilized for example, - 14 by filtration (e.g., through a 0.22 pm filter), and stored in sterile vials. To make a pharmaceutical composition that can be directly injected into a subject, the composition must be 5 sterile, standard techniques known to the skilled artisan for sterilization can be used. See, e.g., Remington's Pharmaceutical Science. the pH of the partially neutralized As-0: solution may be further adjusted to near physiological pH by dilution (10-100 fold) with a pharmaceutical carrier, 10 such as a 5% dextrose solution. For example, lOmL of a partially neutralized AsO:. solution can be added to 500 mL of a 5% dextrose solution to yield a final pH of about 6.5 to 7.5. The method of the invention reduces the oxidation of arsenic in solution. Pharmaceutical compositions containing 15 arsenic trioxide manufactured by the method of the invention show improved stability and long shelf life. 4.4. PHARMACEUTICAL COMPOSITION AND MODES OF ADMINISTRATION According to the invention, the arsenic compounds 20 and their physiologically acceptable solvates may be formulated for oral or parenteral administration. 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 25 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 30 (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 35 conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl -15methylcellulose); 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 5 (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. 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 10 pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbor, dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a 15 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 20 administration by injection, e.g., by bolus injection or continuous- infusion. 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 25 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. 30 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 35 the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile - 16 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 5 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 10 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 is compound in the acute or chronic management of leukemia 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 20 general, the daily dose ranges of arsenic trioxide for the conditions described herein are generally from about 0.05 to about 5 mg per kg body weight administered in divided doses administered parenterally or orally or topically. A preferred total daily dose is from about 2.5 to about 40 mg 25 of arsenic trioxide. Preferably the arsenic trioxide formulation of the invention is given daily for a maximum of 60 days, or until remission, followed by two to ten additional cycles, each lasting about 25 days in duration. For example, depending on the body weight of a patient with 30 acute promyelocytic leukemia, a daily dose of arsenic trioxide greater than or less than 10 mg can be administered. Alternatively, following the weight-based dosing regimen, a therapeutic effect can be obtained with a daily dose of arsenic trioxide less than 10 mg. 35 For treatment of solid tumor, a preferred dosing regimen involves intravenous infusion of about 0.1 to about 5 mg per kg body weight per day for 5 days. This five-day treatment protocol is repeated once per month until the tumor - 17 growth tumor is inhibited or when the tumor shows signs of regression. As for melarsoprol, the total daily dose ranges for the conditions described herein are generally from about 0.1 5 to about 5 mg/kg body weight administered in divided doses administered parenterally or orally or topically. A preferred total daily dose is from about 0.5 to about 4 mg melarsoprol per kg body weight. The effect of the therapy with arsenic trioxide or 10 melarsoprol on development and progression of cancer can be monitored by any methods known in the art, including but not limited to determining: levels of tumor specific antigens and putative biomarkers, e.g., carcinoembryonic antigens (CEA), alpha-fetoprotein; and changes in morphology and/or size 15 using computed tomographic scan and/or sonogram. 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 20 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). 25 Again, any suitable route of administration may be employed for providing the patient with an effective dosage of an arsenic compound. For example, oral, transdermal~, iontophoretic, parenteral (subcutaneous, intramuscular, intrathecal and the like) may be employed. Dosage forms 30 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 35 ingredient, pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients, for example all trans retinoic acid. The term "pharmaceutically acceptable - 18 salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases. The pharmaceutical compositions include 5 compositions suitable for oral, mucosal routes, transdermal, iontophoretic, 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. 10 In the case where an intravenous injection or infusion composition is employed, a suitable dosage range for use is, esgs, from about one to about 40 mg arsenic trioxide total daily; about 0.001 to about 10 mg arsenic trioxide per kg body weight total daily, or about 0.1 to about 10 mg 15 melarsoprol per kg body weight total daily. 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 20 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 25 a wide variety of forms depending on the form of preparation desired for administration, e g., 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, 30 esg., 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, 35 lubricants, binders, disintegrating agents, and the like in the case of oral solid preparations e.g., powders, capsules, and tablets. In preparing the compositions for parenteral dosage form, such as intravenous injection or infusion, -19similar 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 5 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 to the total dose administered. For example, arsenic trioxide 10 supplied as a 10 ml aqueous solution- at 1 mg/ml concentration is diluted in 10 to 500 ml of 5% dextrose solution, and used for intravenous infusion over a period of time ranging from about ten minutes to about four hours. An exemplary course of treatment of a patient with i5 leukemia, lymphoma, or solid cancer can involve daily administration by intravenous infusion of arsenic trioxide in an aqueous solution at a daily dose of about 0.01 to 1 mg arsenic trioxide per kg of body weight of: the patient. Preferably, about 0.15 mg arsenic trioxide per kg body weight 20 per day is used. The course of treatment may continue until bone marrow remission is observed or when side effects are becoming serious. The.course of treatment may be repeated for up to ten times over approximately 10 months with a break of about 3 to 6 weeks in between courses. The post-remission 25 course of treatment involves infusion of arsenic trioxide at a daily dose of about 0.15 mg per kg of body weight of the patient on a daily or weekdays-only basis for a cumulative total of 25 days. 30 5. EXAMPLES Described below are examples of uses of the arsenic compounds of the invention in treatment of various types of leukemia. Through these and other experiments the arsenic trioxide formulation of the invention were found to be well 35 tolerated in humans. For example, three APL patients were given 10 mg of the arsenic trioxide formulation of the invention once daily (flat dose) intravenous dose. - 20 - 5.1. ARSENIC TRIOXIDE AND MELARSOPROL INDUCE APOPTOSIS IN MYELOID LEUKEMIA CELL LINES The activity of AsO, and melarsoprol against 5 myeloid leukemia cell lines, including the APL cell line NB4 306 (a retinoic acid-resistant cell line derived from NB4 that no longer expresses the intact PML-RARa fusion protein), HL60, KG-1, and the myelomonocytic cell line U937 was investigated. To examine the role of PML in mediating 10 arsenical activity, the inventors also tested these.agents using murine embryonic fibroblasts (MEFs) and bone marrow (Bm) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, it is found that both compounds inhibited cell growth and induced apoptosis in 15 all cell lines tested. Melarsoprol was more potent than As,0 at equimolar concentrations ranging from 10- to 10-1 mol/L. As:C, relocalized PML and PML-RARa onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in 20 inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RARa nuclear localization. Moreover, both As:O, and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML-/-MEF, and inhibited colony forming unit erythroid (CFU-E) and CFU'granulocyte-monocyte 25 formation in BM cultures of PML+/+ and PML-/- progenitors. A detailed description of the methods, materials, and results of these experiments is provided in Wang et al., Blood, 1998, 92:1497-1504. Results from the experiments show that the 30 cytotoxic effect of both arsenicals in these cell lines is not mediated by mechanisms that are dependent on PML or PML RARa expression. In most lines, melarsoprol was somewhat more potent compared with As2, in inhibiting growth and inducing apoptosis, and the effects of both drugs were dose 35 dependent. As previously reported, it is confirmed that AsO, relocalized PML protein onto nuclear bodies and induced PML and PML-RARa degradation in NB4 cells while triggering spoptosis. However, similar effects were also observed in HL60 and U937 cells which do not harbor the PML-RARa fusion -21gene. Moreover, melarsoprol induced apoptosis in all the cell lines tested without altering PML and/or PML-RARa. The differentiating action of As,0, and melarsoprol, appeared negligible in vitro, and did not appear to depend on 5 the expression and/or modulation of PML and/or PML-RARa either. In fact, the small effect observed by the inventors in long-term cultures (up to 2 weeks), was comparable in all the cell lines tested with both compounds. It is also found that bcl-2 downregulation, which 10 has been previously linked to the antileukemic effects of As., in APL, was also not dependent on expression of PML-RARa protein, because it occurred in the NB4 subclone 306 in which the intact protein is not detectable. Finally, to test whether PML expression was essential to the antileukemic is effects of arsenicals, both agents were tested in mouse. embryonic fibroblasts and BM cells from animals wherein wild type PML had been eliminated by homologous recombination. In these cells wholly lacking PML expression, both As:O, and melarsoprol were equally effective in inhibiting growth and 20 inducing apoptosis, and both had similar effects on normal CFU-E and CFU-GM colony. formation. Moreover, no differences between wild-type and PML-/- cells were observed. Without being limited by any theory, together, these data strongly support theory that the antileukemic effects of these 25 arsenicals occurs independently of both PML and PML-RARa expression. These results are in keeping with the medicinal history of arsenicals for diseases tnat are not characterized by alterations in PML protein such as, for instance, chronic myelocytic leukemia. 30 The results indicate that both Asych and melarsoprol are broadly active as antileukemic agents in both myeloid and lymphoid diseases. In conclusion, the data indicate that cytotoxic activity is not mediated by the PML protein and therefore. is not limited to diseases that are associated with 35 alterations in PML expression. Thus, the arsenic compounds of the invention have a potentially broader therapeutic role that is not confined to APL. - 22 - 5.2. CLINICAL STUDY OF HELARSOPROL IN PATIENTS WITH ADVANCED LEUKEMIA Melarsoprol, an organic arsenical synthesized by 5 complexing relarsen oxide with dimercaprol, has primarily been used for the treatment of African trypanosomiasis. The effects of melarsoprol upon induction of apoptosis in cell lines representative of chronic B-cell lymphoproliferative disorders have been investigated, and the results are 10 described below. Melarsoprol (supplied as Arsobal [36 mg/mL) by Rhone Poulenc Rorer, Collegeville, PA) was diluted in propylene glycol at a stock concentration of 104 mol/L and stored at room temperature. AsO. (Sigma, St. Louis, MO) was 15 dissolved in 1.65 mol/L sodium hydroxide (NaOH) at a stock solution of 10'' mol/L Serial dilution (10~* to 10- mol/L) were made in RPMI 1640 media. An Epstein-Barr virus (EBV) transformed B-prolymphocytic cell line (JVM-2), an EBV transformed B-cell chronic lymphocytic leukemia (B-CLL) cell 20 line (I83CLL), and one non-EBV-transformed B-CLL cell line (WSU-CLL) were used as targets. Dose-response experiments with melarsoprol (10~ to 10~' mol/L) were performed over 96 hours. Unexpectedly, the inventors found that melarsoprol. 25 caused a dose- and time-dependent inhibition of survival and growth. in all three cell lines. In contrast, As.0, at similar concentrations had no effect on either viability or growth. After 24 hours, all three cell lines treated with melarsoprol (10- mol/L) exhibited morphologic characteristics of 30 apoptosis. A prominent concentration-dependent downregulation of bcl-2 mRNA after 24 hours of exposure to melarsoprol in WSU-CLL 183CLL, and JVM-2 cells was observed. Decrease of bcl-2 protein expression was also observed in all three cell lines, whereas As.01 had no effect on this 35 parameter. Given that the in vitro data above have shown unexpectedly broad antileukemic activity for melarsoprol against both myeloid and lymphoid cells, and generally at - 23 lower concentrations than As,0:, a study was initiated to evaluate the pharmacokinetics, safety, and potential efficacy of melarsoprol in human patients with relapsed or refractory leukemia. 5 Eligible patients were treated with a brief IV injection daily for 3 days, repeated weekly for 3 weeks, with an additional 3 wk course in responding pts. The initial dose was 1 mg/kg on Day 1, 2 mg/kg on Day 2, and 3.6 mg/kg on Day 3 and all days thereafter. Parallel in vitro studies 10 included culture sensitivity of fresh leukemic cells to both melarsoprol and As -. , along with serial flow cytometric studies of surface antigen expression, apoptosis, and bcl-2 expression. Three patients with AML and one with CKL have entered the study. 15 Using a method based on high performance liquid chromatography that is. sensitive to approximately 10 ing/ml, preliminary pharmacokinetic data show that peak plasma drug concentrations were obtained immediately after injection with a Cmax that ranged from 1.2 ng/ml on day 1 to 2.4 ng/ml on 20 day 3. While the initial distribution phase was rapid, a prolonged Thy has suggested release from a deep compartment. Plasma areas under the- concentration x time curves (AUCs) were proportional to the administered dose, ranging from 0.48 ng-hr/ml on Day 1 to 1.48 ng-hr/ml on Day 3. Detectable 25 concentrations of the drug were found in plasma one week after initial dosing. The drug has been relatively well tolerated. Adverse effects have included transient pain at the injection site and mild nausea. No signs of "reactive encephalopathy" (occasionally observed during treatment of. 30 CNS trypanosomiasis) have been observed. Results from these studies suggest that melarsoprol may have broader activity than inorganic AsOy, and that concentrations which are cytotoxic to leukemic cells in vitro, and thus therapeutic, are readily achieved in vivo. 35 5.3. ARSENIC TRIOXIDE INDUCES APOPTOSIS IN K562 CHRONIC MYELOGENOUS LEUKEMIA (CML) CELLS - 24 - A Philadelphia chromosome positive CML cell line K562 is used to determine if arsenic trioxide (As0 2 ) promotes apoptosis in CML. Suspension cultures of cells in log phase' 5 were exposed to As2,O at concentrations of 1 x 10~ M, 5 x 10~4M, and 1 x 10~' M. Aliquots of cells were analyzed at various time points over the course of 72 hours to assess viability and apoptosis. viability was measured using trypan blue exclusion; at the same time, apoptosis was detected by 10 morphology, flow cytometry, and DNA gel electrophoresis. Arsenic trioxide at a concentration of 1 x 10~* M had no effect on K562 cell growth or viability. The greatest effect'on cell growth -and survival was seen with 1 x 10'4 M As2O.. K562 cell growth and viability data after 72 hours of 15 exposure to AsO. are recorded in Table 1: Table 1: % Cell Growth Impairment - t Viability p value control 0 92.1 * 0.9 5 x 10' M As7o 3 63.0 78.8 0.5 0.0001 20 1 x 10". M As0 3 75.3. .61.9 : 2.9 0.0223 Evidence that this arsenic-induced decrease in viability represented apoptosis was analyzed. Morphologic features of apoptosis including membrane blebbing and.nuclear condensation were evident in stained cytospins of K562 cells 25 incubated with 10~1 M As-O for 72 hours. This correlated with evidence of DNA internucleosomal damage as visualized by gel electrophoresis of DNA extracted from K562:-celis exposed to 101 M AsO. Quantitative assessment of apoptosis, as measured by the TUNEL method demonstrated that 75.6%.± 8.6 (1 30 x 10~5 M As'0 3 ) cells exhibited apoptosis as compared with 6.3% 1 3.0 (control) cells at 72 hours. Treatment of K562 cells with 10-1 M As 2 ,O resulted in an upregulation of p21 mRNA, as detected by Northern analysis, suggesting an arrest of the cells in the Gi phase of the cell cycle. This data indicates 35 arsenic trioxide as a therapeutic agent for CML. - 25 - 5.4. THERAPEUTIC TRIALS WITH RETINOIC ACID AND ARSENIC TRIOXIDE (As 2 0 3 ) IN PML-RARQ AND PLZF-RA~a TRANSGENIC MICE 5 -Acute promyelocytic leukemia (APL) is associated with chromosomal translocations which invariably involve the translocation of the Retinoic Acid Receptor a (RARa) locus on chromosome 17 to other loci in the genome, such as in the majority of APL cases, the PML gene located on chromosome 15, 10 and in a few cases the PLZF gene on chromosome 11. Patients harboring the t(15;17) are sensitive to treatment with All Trans Retinoic Acid (ATRA), yielding complete remission rates of 75% to 95%. APL associated with the t(ll;17) (PLZF-RARa) shows a poor response to ATRA. To test the efficacy of As.O. in the treatment of APL, models of the disease were created in transgenic mice. Transgenic mice were generated by standard techniques in which the expression of the PML-RARa or PLZF-RARa fusion proteins is placed under the control of a myeloid 20 promyelocytic specific human Cathepsin-G (hCG) minigene. Both hCG-PML/RARa and hCG-PLZF-RARa transgenic mice develop myeloid leukemia with features of APL similar to those in humans. Therapeutic trials on these leukemic mice with the 25 following regimens were started: 1) ATRA: 1.5 yg per gram of body weight per day administered orally; and 2) ATRA: 6 pg per gram of body weight per day administered intraperitoneally. Mice were bled once a week to evaluate the response. 30 PML/RARa leukemias responded well to ATRA with.high remission rates (80% with regimen 1). Surprisingly, in vitro, ATRA induced differentiation, and inhibited growth of leukemic cells as well as leukemic colony formation in bone marrow and spleen progenitors assays in both PML-RARa and 35 PLZF-RARa leukemias. Furthermore, in ex vivo experiments, leukemic cells from PLZF-RARa mice lost their tunorigenic capacity when transplanted in recipient nude mice upon pre incubation with ATRA, while untreated cells were tumorigenic. - 26 - However, in vivo, PLZF-RARa leukemias responded poorly to ATRA (28% with regimen 1), while higher doses of ATRA appeared more effective (50% with regimen 2). In conclusion, leukemias in PLZF-RARa transgenic mice are sensitive to ATRA 5 treatment, but might require therapeutic regimens with high doses of~ATRA. These findings have direct implications in the treatment of APL patients with t(11;17). In both PML-RARa and PLZF-RARa leukemias, ATRA prolonged survival, but leukemias relapsed shortly after 10 remission was achieved, and were refractory to further ATRA. treatment. The two transgenic mouse models is also used to test the efficacy and dosage of As0-. , and ATRA+As 2 0: in combination for treatment* of APL patients resistant to ATRA, and in APL associated with the t(11;17). A regimen of As 2 ,O 15 at 6 pg per day or a combination of As,0, at 6 yg and ATRA at 1.5 or 6 pg per gram of body weight per day is administered intraperitoneally. Mice are bled weekly to evaluate the remission of the APL. 20 5.5. MANUFACTURE AND STABILITY OF PHARMACEUTICAL FORMULATION Solid ultrapure arsenic trioxide (As.0:) was solubilized in a solution of 5 M sodium hydroxide (NaOH). The suspension was stirred at ambient temperature for 5 25 minutes which yielded a clear, homogenous solution. The AsO. solution (2 mL, 1.0 M) was added to 393.6 mL of H.O in a 500 ml Erlenmeyer flask,.which yielded an As 0a concentration of 1 mg/mL at pH = 12. A 5.0 M HC1 solution was prepared by dilution of HC1 (49.26 mL, 37% wt/wt, 10/15 M) with H10 (50.74 30 mL) in a 250 mL Erlenmeyer flask. The HCl solution was later transferred via syringe to a 1000 mL empty evacuated container. The AsA0 solution was back titrated with HCl (0.725 mL, 5.0 M) to pH 8.0. Approximately 10 mL of the backtitrated As:0 solution was filtered through a Millex-GS 35 0.22 pm filter unit and was added to each of approximately 30 sterile evacuated sterile vials. To make the pharmaceutical composition which would be injected intravenously into patients, 10 mL of this solution was withdrawn from two of -27the vials and was added to a 500 mL 5%-dextrose solution which yielded a final pH of 6.5. The high purity of the bulk starting material was confirmed (see Table 1) by atomic absorptiometry. Duplicate 5 samples of four intermediate or final-step solutions were assayed for total arsenic content. Assay bulk powder confirmed the extremely high purity of the starting material. Data for arsenic content of the intermediate and finished product solutions are presented in Table 2 below. 10 The data below show that the solutions are stable in that there does not appear to be any indication of weight loss of arsenic over time. Table 2 Arsenic content (ppm) of intermediate formulation. 15 and finished product solution of arsenic trioxide. Sample Code A-01* A-02 A-03 A-04 A-05 Aliquot A 146,600 600 707 629 680 Aliquot B 139,000 564 703 688 687 20 Assay 1.1% 6% 0.57% 8.7% Variance * -Identity of sample codes: A-01: Intermediate product solution after initial 25 solubilization in NaOH. A-02: Intermediate product solution prior to HCl titration. A-03: Intermediate product prior to Millex filtration. A-04: Finished product from.sterile 10 ml fill vial 30 immediately after manufacturing. A-05: Finished product from capped vials two months after manufacturing. 35 6. EXAMPLES: CLINICAL TRIALS IN APL PATIENTS Arsenic trioxide was evaluated in patients with APL 40 to determine whether this agent induced either cytodifferentiation or apoptosis. Twelve patients who had relapsed from extensive prior therapy were treated with arsenic trioxide at doses ranging from 0.06 to 0.2 mg/kg per day until a bone marrow remission was achieved. Bone marrow - 28 mononuclear cells were serially m6nitored by flow cytometry for immunophenotype, fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT PCR) assay for PML/RAR-a expression, and Western blot . 5 expression of the apoptosis-associated proteins, caspases 1, 2 and 3.. The results showed that low-doses of arsenic trioxide are highly effective for inducing complete remission in relapsed patients with APL. Clinical response is associated with incomplete cytodifferentiation and induction 10 of apoptosis with caspase activation in leukemic cells. 6.1. METHODS clinical protocol: Eligibility requirements included a diagnosis of APL confirmed by cytogenetics or fluorescence in 15 situ hybridization (FISH) analysis for a t(15;17) translocation, or by reverse transcriptase polymerase reaction (RT-PCR) assay for PML/RAR-a. Patients must have relapsed from standard therapy that had included all-trans retinoic acid plus a combination of cytotoxic drugs. Signed 20 informed consent was required, and the protocol was reviewed and approved by this center's institutional review board Arsenic trioxide treatment: Arsenic trioxide was supplied as an aqueous solution in 10 ml vials containing 1 mg/ml of 25 drug. The drug was.further diluted in 500 ml of 5%-dextrose solution and infused intravenously over 2 to 4 hours once per day. While the initial cohort of patients received either 10 or 15 mg/day as a flat dose, the referral of two children prompted the invention of a weight-based regimen (0.15 30 mg/kg/day) that was heretofore unknown. The drug was given daily until bone marrow remission was observed. Patients who achieved complete remission were eligible for treatment with additional courses of therapy 3 to 6 weeks after the preceding course. Subsequent courses were generally given at 35 a dose of 0.15 mg/kg/day for a cumulative total of 25 days, administered either daily or on a weekdays-only schedule, for a maximum total of 6 courses over approximately 10 months. 29 - Monitoring during study: Patients with coagulopathy were transfused with platelets and fresh-frozen plasma to maintain the platelet count and fibrinogen at target levels z 50,000 cells/cu mm and 2 100 mg/dL, respectively. Blood counts, 5 coagulation studies, serum chemistry profiles, urinalyses, and electrocardiograms were serially obtained. A bone marrow aspiration and/or biopsy was performed at baseline and periodically thereafter until remission was documented. Conventional response criteria were observed, which included 10 recovery of bone marrow to < 5% blasts, peripheral blood leukocytes > 3,000 cells/cu mm, and platelets > 100,000 cells/cu mm. Cellular immunophenotype studies: Heparinized bone marrow or I5 blood samples were collected and mononuclear cells were isolated.by Ficoll-Hypaque centrifugation. Surface membrane antigens were detected by direct immunofluorescence staining using fluorescein isethiocynate (FITC) or phycoerythrin conjugated monoclonal antibodies: CD16 (Leu lla), CD11b, CD33 20 (Leu M9), HLA-DR, CD45, and CD14, purchased from either Becton-Dickinson (Mountainview, CA) or Immunotech Immunology (Marseille, France). Dual-color staining was performed by incubating cells simultaneously with two monoclonal antibodies, including CD33-PE/CDllb-FITC and CD33-PE/CD16 25 FITC. Negative controls using irrelevant monoclonal immunoglobulins of the same isotype were analyzed concurrently. Flow cytometric analyses were performed on an EPICS Profile II flow cytometer (Coulter Electronics) equipped with a 488 nm argon laser. Forward and side'-scatter 30 cell parameters were measured.and combined with CD45/CD14 staining to identify populations of interest and to exclude monocytes from the analysis gate. The Multiparameter Data Acquisition and Display System (MDADS, Coulter Electronics) was used to acquire and analyze data. 35 Fluorescence in situ hybridization (FISH): Selected specimens that had undergone immunofluorescence staining for CD33 and CDllb were sorted for cells that coexpressed both - 30 antigens using a FACStar Plus cell-sorter (Becton-Dickinson). Separated cells were incubated in culture media at 370 C for one hour, treated with hypotonic solution 0.075M KCl for 5 minutes, fixed in 3:1 methanol:acetic acid fixative, and air 5 dried. Interphase FISH was performed using a specific pHjL/RAR-_ translocation dual-color prcbe (Vysis; Downer's Grove, IL).. Briefly, DNA from interphase cells was denatured by immersing slides in a solution of 50% formamide/2xSSC at 730 C for 5 minutes; the slides were then dehydrated in 10 alcohol and air dried. A mixture of probe in hybridization mixture was applied, covered with a cover slip, and sealed with rubber cement. Hybridization was carried out at 37* C in a moist chamber for approximately 12 to 16 hours. Following hybridization, unbound probe was removed by washing 15 the slides at 450 C in 50% formamide/2xSSC solution three times for 10 minutes each, followed by a wash in 2xSSC/0.1 NP-40 solution at 45* C for 5 minutes. Slides were then air dried and counter-stained with 4',6-diamidino-2-phenylindole and covered with a glass coverslip. Analysis of interphase 20 cells for fluorescent signals was performed with a Photometrics Sensys camera fitted to a Zeiss axioscope. A minimum of 300 cells was studied for each sample. Western blot analysis: Cells were lysed in a buffer 25 containing 50 mM Tris-HCl, 0.5 mM ethylene glycol [bis] [aminioacylJ tetra acetic acid, 170 mM NaCl, imM dithiothreitol, 0.2% NP-40, 0.01 U/mL aprotinin, 10 yg/mL leupeptin, -10pg/mL pepstatin, and 1 yM phenylmethylsulfonyl fluoride (all from Sigma). The lysates were then sonicated 30 using a ultrasonic homogenizer (471C series, Cole Parmer Instruments, Chicago, IL) and centrifuged at. 7,500 g (Sorvall Instruments, Newtown, CT). Protein content of the lysates was determined using a BioRad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) at 595 nm with a BSA standard. A 35 sample buffer containing 10 % glycerol, 0.4 % SDS, 0.3 % bromphenol blue, 0.2 % pyronin Y, in lx stacking buffer (Tris base 0.5 M, 0.8 % SDS), 20 % 2-mercaptoethanol, was added to the cell lysates, which were heat-denatured at 95*C for 3 min. - 31 - Subsequently, 15 pg/lane of protein was loaded on a SDS polyacrylamide gel containing 12.5% polyacrylamide and was size-fractionated by electrophoresis. Proteins were electroblotted onto Tras-Blotz transfer medium (Bio-Rad) and 5 stained with Ponceau-S as an internal loading control. Rabbit anti-human monoclonal antibodies, including caspase 1, caspase 2 (both from Santa Cruz Biotechnology, Santa Cruz, CA), and caspase 3 (PharMingen, San Diego, CA) were added, and bound antibodies were detected using the ECLTM 10 chemiluminescence detection system (Amersham, Arlington Heights, IL). Protein bands were quantified by computer densitometry. RT-PCR analysis for PML/RAR-a: RT-PCR was performed using 15 methods previously described (Miller et al., 1992, Proc. Natl. Acad. Sci. 89:2694-8; Miller et al., 1993, Blood, 82:1689-94). 6.2. RESULTS 20 Patients: Twelve patients with relapsed or refractory APL were treated. All patients had received extensive prior therapy with retinoids and cytotoxic drugs (Table 3). Two patients had relapsed from allogeneic bone marrow transplantation, one of whom had also failed donor T-cell 25 reinfusion. One patient was being maintained on-hemodialysis for chronic renal failure. Clinical Efficacy: Eleven of the 12 patients achieved a complete remission after arsenic trioxide treatment. The patient.who entered on hemodialysis sustained an intracranial 30 hemorrhage on day 1 and died on day 5. The median duration of therapy in responding patients was 33 days (range, 12 to. 39 days), the median daily dose was 0.16 mg/kg (range, 0.06 to 0.2 mg/kg), and the median cumulative dose during induction was 360 mg (range, 160 to 515 mg) (Table 3). 35 complete remission by all criteria was attained at a median time of 47 days (range, 24 to 83 days) after initiation of therapy. Remission by bone marrow criteria - the determining factor for discontinuing therapy - was achieved first, - 32 usually followed in sequence by recovery of peripheral blood leukocytes and platelets. Over the range of doses used in this study, no differences in efficacy or time to response were obvious. After 2 courses of therapy, 8 of 11 patients 5 tested had converted their RT-PCR assays for PML/RAR-a from positive.to negative. All 11 patients in complete remission completed at least 1 post-remission treatment course with arsenic trioxide. Four, two, and one patient each have completed a 10 total of three, four and five treatment courses, respectively. The median duration of remission is 5+ months (range, 1 to 9+ months). However, 3 of the 11 patients relapsed during the second treatment course; none of these patients had converted their RT-PCR assays, and each appeared 15 to have rapidly acquired drug resistance. Two of these individuals have since expired from progressive leukemia. Adverse Events: The clinical condition of patients in this study was highly variable, which reflected their extensive 20 prior therapy. The protocol did not require hospitalization; three patients completed induction therapy entirely as outpatients, and one other individual was hospitalized solely for placement of a venous catheter. 'However, 8 patients were hospitalized for complications of leukemia, 5 of whom 25 required transfer to an intensive care unit, endotracheal intubation, and assisted ventilation for complications that included pulmonary hemorrhage, renal failure, sepsis, graft vs. host disease, non-specific pulmonary infiltrates, or hypotension. one patient required insertion of a permanent 30 pacemaker after second-degree heart block developed in the setting of severe metabolic acidosis, hyperkalemia, hypotension, and renal insufficiency. However, the heart block reversed despite rechallenge with further arsenic trioxide therapy. The drug was temporarily withheld due to 35 serious intercurrent medical complications in 5 patients for a median of 2 days (range, 1 to 5 days). Two patients developed symptoms similar to that of the "retinoic acid syndrome"; both were presumptively treated with dexamethasone - 33 and improved. Only 2 patients required no platelet transfusions whatsoever; the median number of platelet units transfused was 61 (range, 0 to 586 units). The median total peripheral blood leukocyte count 5 at entry was 4,700 cells/cu mm (range, 500 to 144,000 cells/cu mm). Six patients developed leukocytosis (i.e., > 20,000 cells/cu mm) that ranged from 20,800 to 144,200 cells/cu mm. No additional therapy was administered to these patients, and the leukocytosis resolved in all cases without 10 further intervention. Common adverse reactions included lightheadedness during the infusion, fatigue, musculoskeletal pain, and mild hyperglycemia. Three patients developed dysesthias presumably due to peripheral neuropathy. However, 2 of these 15 patients had been immobilized for prolonged periods during assisted ventilation, and the other patient had an antecedent neuropathic history. Immunophenotype studies: APL is characterized by cells that 20 express CD33, an antigen typically associated with primitive myeloid cells. Arsenic trioxide therapy induced a progressive decrease in the proportion of cells that solely expressed CD33, along with an increase in the proportion of cells that expressed CDl1b, an antigen associated with mature 25 myeloid elements. While these changes would be anticipated from any agent that induced remission in APL, arsenic trioxide also induced expression of cells that simultaneously expressed both antigens. In most cases, these dual expressing cells dominated the myeloid cell population, and 30 they persisted for extended periods after complete remission was achieved by clinical criteria. Fluorescence in situ hybridization analysis: Bone marrow mononuclear cells taken from a patient both early and later 35 in complete remission were sorted by flow cytometry for coexpression of CD33 and CDllb. Using fluorescence in situ hybridization (FISH) analysis, three hundred cells were examined early in remission. Similar to control APL cells, - 34 the majority of these cells yielded a hybrid signal, indicating a translocation between PML and RAR-a genes and their origin from the neoplastic clone. However, when cells from the same patient were again sorted using these same S parameters later in remission, only the normal pattern of fluorescence signals was detected, indicating their derivation from normal hematopoietic progenitors. Western blot analysis: Protein extracts from bone marrow 10 mononuclear cells were serially examined by Western blot analysis.. The analysis showed that the precursor forms of caspase 2 and caspase 3 were upregulated in vivo.in response to arsenic trioxide treatment. Moreover, this treatment also induced expression of cleaved fragments of caspase 1, 15 indicating activation of the enzyme. There is also some indication that expression of the cleaved form of caspase 3 is increased. The antibody used in these experiments does not react with the cleaved form of caspase 2. 20 6.3. DISCUSSION In this study, with few exceptions, patients admitted to the trial had sustained multiple relapses and were resistant to conventional chemotherapy, retinoids, or bone marrow transplantation. At entry, patients in this 25 study suffered from numerous leukemia-related complications, including respiratory failure, disseminated Varicella zoster infection, cavitary aspergillosis, chronic renal failure, and graft-vs.-host disease. Moreover, 5 of the 12 patients required admission to an intensive care unit for assisted 30 ventilation and supportive care, but these complications were not directly related to arsenic trioxide therapy. Virtually all patients with a confirmed diagnosis of APL attained remission without the early mortality associated with retinoid therapy. Although less commonly 35 observed compared with all-trans retinoic acid treatment, arsenic trioxide induced striking leukocytosis in several patients. Upon withholding other cytotoxic drugs, the leukocytosis disappeared as patients attained remission. - 35 - Despite 3 early relapses, 8 of 11 patients tested converted RT-PCR assays for PML/RAR-a (a molecular marker of residual disease) to negative, a phenomenon that is unusual after all trans retinoic acid treatment alone. Finally, arsenic 5 trioxide is active in APL over at least a three-fold dose range from 0.06 to 0.20 mg/kg. All-trans retinoic acid induces "terminal" differentiation of APL cells, but the cytodifferentiating effects of arsenic trioxide appear to be incomplete. Arsenic 10 induces a population of cells that simultaneously express surface antigens characteristic of both mature and immature cells (i.e. CDllb and CD33,' respectively). Early during induction, these cells retain the t(15;17) translocation that characterizes APL. Unexpectedly, these cells persisted in 15 the bone marrow despite the achievement of a clinically complete remission; however, later in remission, the coexpressing cells - while still readily detectable - were no longer positive by in situ hybridization. The morphologic appearance of leukemic cells during therapy is also far less 20 distinctive than that observed during therapy with all-trans retinoic acid. In fact, leukemic cells from many patients displayed few morphologic changes for 10 or more days, after which the proportion of leukemic cells progressively decreased. 25 Following "non-terminal" differentiation, arsenic trioxide appeared to induce apoptosis, coincident with increased expression and conversion of cysteine proteases (termed caspases) from inactive precursors to activated enzymes. The caspase pathway has only recently been 30 characterized as an important pathway of programmed cell death. Initially recognized due to homology between the C. elegans protein ced-3 and mammalian interleukin-10 converting enzyme (ICE), the family of caspases now encompasses at least 10 different proteins that cleave a number of polypeptides. 35 In leukemic cell lines, caspase. activation is inducible with a number of cytotoxic agents, including all-trans retinoic acid. Since these enzymes induce widespread proteolysis, it is conceivable that PML/RAR-a is a caspase substrate. - 36 - A final similarity shared by arsenic trioxide and all-trans retinoic acid is the rapid development of clinical resistance in some individuals. Leukemic cells taken from two patients who relapsed retained in vitro sensitivity over 5 concentrations ranging from 10~4M to 10~" M. Relative-arsenic resistance due to decreased intracellular transport has been described in association with down-regulation of membrane transporters encoded by the ars operon in bacterial cells. Resistance in mammalian cells is less well-characterized, but 1o alterations in membrane transport or efflux are probably important factors. In summary, arsenic trioxide induces complete remission in patients with APL who have relapsed from extensive prior therapy. This drug causes partial but is incomplete cytodifferentiation of leukemic cells, followed by caspase activation and induction of apoptosis. -37 - Al0 U DVHUnMV) qL 0) 41) 4 o 0D 0 Ci - 'D 0 i N 0 o) AI A - i C U) N L w i) 4.) to 0 0 0 D 0~ 0~ '0 0 m a n N In 14 0 0 0 0 O m 9 %D Ni W 0 -4 0 %D 0 r- ND Ln HV V-4 4 0 4 N 14 1- V-4 N N V4 o 41 Uto to0 44. >9 40 0 0 i n fl 0 -4 * 0 cl 0 0 E-4 11J 4'3B 00 9 X ~ D ~ 0 4 0 10 N 0 inI All patients had previously received one or more courses of all-trans retinoic acid, plus an anthracycline antibiotic plus cytosine arabinoside. * Denotes individuals with proven retinoid resistance (i.e. lack of response during reinduction 5 or relapse while on retinoid maintenance); t Denotes patient who died early. Other treatment: 1 mitoxantrone/etoposide;^ allogeneic bone marrow transplantation; methotrexate/vincristine/6-mercaptopurine; * 9-cis retinoic acid plus M195 (anti-CD33 monoclonal antibody). 10 7. EXAMPLES: CLINICAL USE IN LYMPHOMA Based upon the initial discovery of the antitumor effects of arsenic trioxide in vitro against B-cell lymphocyte lines, the inventors treated one patient with 15 intermediate-grade large cell lymphoma who had relapsed from multiple forms of conventional therapy, including autologous bone marrow transplantation. Despite rapid progression of his disease prior to starting the arsenic trioxide therapy, treatment with arsenic trioxide effected a major (>50%) 20 shrinkage in the size of his cancerous lymph nodes and spleen, which was also associated with a major improvement of his quality of life. 8. EXAMPLES: CLINICAL USE IN NON-HEMATOPOTETIC CANCEl 25 Arsenic trioxide was also used to treat colon cancer. In a preliminary test, one patient with colon cancer who received one treatment with arsenic trioxide showed a major reduction in his serum CEA (carcinoembryonic antigen) level. The patient received daily intravenous infusion of 30 0.1-5 mg arsenic trioxide per kg body weigh per day for five days. A change in the level of CEA from 19,901 ng/ml to 15,266 ng/ml, a 23% reduction, was observed. It is well known that the a reduced level of serum CEA is associated with antitumor response. 35 Clinical data confirms that arsenic trixoide can also be used to treat other non-hematopoietic cancer, such as colon cancer. - 39 - 9. EXAMPLES: PHARMAcOKINETICS STUDIES Several dose-ranging studies were conducted to examine the pharmacokinetics (PK) and biological effects of Asgh in patients with APL and in patients with other 5 hematologic diseases. In patients with APL, marrow mononuclear cells were serially monitored by flow cytometry for immunophenotype, fluorescence in situ hybridization (FISH), and Western blot expression of the apoptosis associated proteins, caspases 1, 2 and 3. Cells that 10 coexpressed CDllb and CD33, and which by FISH analysis carried the t(15;17) translocation, progressively increased during treatment and persisted early in complete remission. Asx0 also induced in vivo expression of the proenzymes of caspase 2 and caspase 3,' and activation of both caspase 1 and is caspase 3. PK analysis of blood and urine for elemental . arsenic (As) content showed that As was distributed in both plasma and red blood cell fractions of whole blood. Parallel elimination curves suggested that these 2 compartments were freely exchangeable, and decayed from peak values with 20 initial half lives of about 60 mins. The mean AUC on day 1 was about 400 ngrhr/ml. Approximately 20% of the. administered dose was recovered in urine within the first 24 hrs. We then initiated a dose-ranging study in patients with diseases other than APL using a daily intravenous dosing 25 schedule for a cumulative total of 25 days per treatment course every 3-5 weeks at dose levels of 0.1 and 0.15 mg per kg body weight-per day. To date, 10 patients have been accrued, including patients with CLL (2 patients), AML (3 patients), lymphoma (4 patients), and CML (1 patient). Five 30 patients were removed from the study early due to rapid progression, and 5 completed the planned 25-day course. Over this dose range, the drug has proved well-tolerated; adverse effects have included skin rash, lightheadedness during the infusion, fatigue, and QTc prolongation on~ EKG. Results from 35 this ongoing study show that clinical use of As:O, induces partial differentiation and apoptosis in APL, but that the therapeutic effects of this agent are not confined to this disorder. - 40 - 41 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. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps. A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was, in Australia, known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims W:\Files\674781\674781-speci p41.doc

Claims (17)

1. The use of arsenic trioxide for manufacturing a medicament for the treatment of acute 5 promyelocytic leukemia by administering to a human a therapeutically effective amount of about 0.15 mg of arsenic trioxide per kg body weight of the human per day.

2. The use of claim 1, wherein arsenic trioxide is administered by intravenous infusion. 10

3. The use of claim 1 or 2, wherein administration of arsenic trioxide is repeated daily until bone marrow remission is observed in the human.

4. The use of claim 3 further comprising one to ten additional cycles of administration of arsenic trioxide after observation of bone marrow remission, each additional cycle comprising 15 first suspending treatment for three to six weeks and then administering arsenic trioxide five to seven times per week for a period of 25 days.

5. The use according to claim I or 2 wherein a course of administration is repeated for up to ten times over approximately 10 months with a break of 3 to 6 weeks in between courses. 20

6. The use according to claim 1 or 2 wherein a post-remission course of administration involves infusion of the arsenic trioxide on a daily or weekdays only basis for a cumulative total of 25 days. 25

7. The use according to claim 1 or 2 wherein the arsenic trioxide is given daily for a maximum of 60 days, followed by two to ten additional cycles each lasting about 25 days in duration.

8. The use according to any preceding claim, wherein all-trans retinoic acid is also 30 administered to the human.

9. A method for treating acute promyelocytic leukaemia in a human comprising administering to the human a therapeutically effective amount of about 0.15 mg of arsenic trioxide per kg body weight of the human per day. W 'Fil\777i\774471 Cims 2. I09 doc 43

10. The method of claim 9, wherein arsenic trioxide is administered by intravenous infusion. 5

11. The method of claim 9 or 10, wherein administration of arsenic trioxide is repeated daily until bone marrow remission is observed in the human.

12. The method of claim I1 further comprising one to ten additional cycles of administration of arsenic trioxide after observation of bone marrow remission, each additional 10 cycle comprising first suspending treatment for three to six weeks and then administering arsenic trioxide five to seven times per week for a period of 25 days.

13. The method according to claim 9 or 10 wherein a course of administration is repeated for ip to ten times over approximately 10 months with a break of 3 to 6 weeks in between 15 courses.

14. The method according to claim 9 or 10 wherein a post-remission course of administration involves infusion of the arsenic trioxide on a daily or weekdays only basis for a cumulative total of 25 days. 20

15. The method according to claim 9 or 10 wherein the arsenic trioxide is given daily for a maximum of 60 days, followed by two to ten additional cycles each lasting about 25 days in duration. 25

16. The method according to any one of claims 9 to 15, wherein all-trans retinoic acid is also administered to the human.

17. The method according to claim 9, as hereinbefore described and with reference to any of the Examples. 30 %VWFIles\774471\ 774471 Cla ims 2.1.09.doc