HK1248113B - High surface-area lyophilized compositions comprising arsenic for oral administration in patients - Google Patents

Description

High surface area lyophilized composition containing arsenic for oral administration in patients

Cross-references to related applications

This application claims the benefit of U.S. Provisional Application No. 62/110,574, filed February 1, 2015, and U.S. Provisional Application No. 62/142,709, filed April 3, 2015, the entire contents of which are incorporated herein by reference.

Technical Field

This invention relates to the treatment of malignant tumors, such as tumors or cancers, by administering a lyophilized composition containing arsenic to a subject. Malignant tumors include various hematologic malignancies, such as acute myeloid leukemia (AML) (including acute promyelocytic leukemia (APL)), myelodysplastic syndromes (MDS), multiple myeloma (MM), and lymphoma; and solid tumors, including glioblastoma multiforme and breast cancer.

Conventional arsenic therapy has shown great promise in the treatment of several cancers, but requires daily intravenous (IV) administration. In contrast, the oral formulation of the present invention provides systemic bioavailability comparable to that of intravenous (IV) administration of arsenic trioxide currently in practice. It also exhibits a shelf life of more than three (3) months and provides a far more convenient, less risky, and cheaper method of administering arsenic trioxide compared to intravenous administration. The present invention also relates to methods for preparing lyophilized compositions containing arsenic, methods for preparing the oral formulation of the present invention, methods for orally administering the formulation to a subject, and methods for treating a subject with malignant tumors (e.g., hematologic malignancies) using the oral formulation.

background

Hematologic malignancies

Hematologic malignancies are cancers of the body's hematopoietic and immune systems. Examples include leukemia, lymphoma (Hodgkin's and non-Hodgkin's lymphoma), and myeloma. Abnormal cell growth interferes with the production of healthy blood cells, thus preventing the body from protecting itself from infection.

Hematologic malignancies account for about 9% of new cancer diagnoses in the United States and cause about 59,200 deaths each year. Many of these diseases occur in children.

leukemia

Leukemia is a cancer of the bone marrow and blood. It is characterized by the uncontrolled growth of blood cells. Approximately 30,000 new cases of leukemia are reported annually in the United States. Most cases occur in older adults, although leukemia is the most common type of cancer in childhood.

Leukemia can be acute or chronic. In acute leukemia, the abnormal blood cells are immature cells that remain very immature and cannot perform their normal functions. The number of immature cells increases rapidly, and the disease progresses rapidly. In chronic leukemia, some immature cells are present, but generally these cells are more mature and can perform some of their normal functions. Moreover, the number of immature cells increases more slowly than in acute leukemia. Therefore, chronic leukemia progresses gradually.

Leukemia can occur in either of two main types of white blood cells—lymphocytes (lymphocytic leukemia) or bone marrow cells (myeloid or idiopathic leukemia). Common types of leukemia include acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML) (sometimes called acute non-lymphocytic leukemia (ANLL)), such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemic leukemia, acute neutrophilic leukemia, and myelodysplastic syndromes (MDS); chronic lymphocytic leukemia (CLL); chronic myeloid (granulocytic) leukemia (CML); chronic myelomonocytic leukemia (CMML); hairy cell leukemia; and polycythemia vera and myeloproliferative neoplasms, including myelofibrotic polycythemia vera and essential thrombocythemia.

Lymphoma

There are two main types of lymphoma—Hodgkin's lymphoma and non-Hodgkin's lymphoma. Hodgkin's lymphoma, also known as Hodgkin's disease, is a special form of lymphoma in which specific cells called Reed-Sternberg (R-S) cells are present. These cells are not typically found in other lymphomas.

The cause of Hodgkin's disease is unknown. Like other cancers, Hodgkin's disease is not contagious and cannot be passed on to others. It is not hereditary. The initial symptom of Hodgkin's disease is usually a painless swelling in the neck, armpits, or groin. Other symptoms may include night sweats or unexplained fever, weight loss and fatigue, cough or difficulty breathing, and persistent itching all over the body.

There are approximately 20 different types of non-Hodgkin lymphoma. Non-Hodgkin lymphoma is classified according to its appearance under a microscope and cell type (B cells or T cells). Risk factors include older age, female sex, weakened immune system, infection with human HTLV-1 and EB virus, and exposure to chemicals such as pesticides, solvents, and fertilizers.

multiple myeloma

Myeloma is a malignant tumor composed of plasma cells, a type of cell typically found in the bone marrow. Myeloma cells tend to aggregate in the bone marrow and on the hard outer surface of bone. Sometimes, they aggregate in only one bone and form a single mass or tumor called a plasmacytoma. However, in most cases, myeloma cells aggregate in many bones, often forming multiple tumors and causing other problems. When this occurs, the disease is called multiple myeloma, such as, but not limited to, giant cell myeloma, painless myeloma, focal myeloma, multiple myeloma, plasma cell myeloma, sclerosing myeloma, solitary myeloma, condensing multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma.

Myelodysplastic syndrome

Myelodysplastic syndromes (MDS) are conditions in which the bone marrow produces one or more types of cells (white blood cells, red blood cells, or platelets) that are ineffective and appear abnormal. Most patients are men over sixty years of age. Secondary MDS can occur after chemotherapy and radiation therapy.

Signs and symptoms depend on the type of cells affected. Abnormal white blood cells make a person more susceptible to infection; abnormal platelets make a person more prone to bruising and spontaneous bleeding; and abnormal red blood cells cause anemia and fatigue.

While chemotherapy and radiation therapy are available for treating hematologic malignancies, there remains a need to find more effective and less toxic treatments and methods to manage the disease, especially as clinical oncologists place greater emphasis on the quality of life for cancer patients. This invention provides an alternative approach to the treatment and management of hematologic malignancies by using an oral composition containing arsenic trioxide.

arsenic

Arsenic has been used as a medicine for over 2000 years. In the 18th century, a solution of arsenic trioxide (empirical formula _{As₂O₃ )} in 1% w/v potassium bicarbonate (Fowler's solution) was developed to treat various infections and malignancies. Its leukocyte-suppressing efficacy was first described in 1878 (Kwong Y. L. et al. Delicious poison: arsenic trioxide for the treatment of leukemia, Blood 1997;89:3487-8). Therefore, arsenic trioxide was used to treat chronic myeloid leukemia until it was superseded by more effective cytotoxic drugs in the 1940s. However, interest in this therapy resurfaced when arsenic trioxide was found to induce apoptosis and differentiation in acute promyelocytic leukemia (APL) cells (Chen _G. Q. et al. Use of arsenic trioxide ( _As₂O₃ ) in the treatment of acute promyelocytic leukemia ( _APL ): I. _As₂O₃ exerts dose-dependent dual effect on APL cells in vitro and in vivo, Blood 1997;89:3345-53; Soignet S. L. et al. United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol. 2001;19:3852-60). The clinical significance of these in vitro observations has since been confirmed, as arsenic trioxide induces remission in over 90% of these patients (Shen Z. X. et al. Use of arsenic trioxide ( _{As₂O₃ )} in the treatment of acute promyelocyticle leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients, Blood 1997; 89: 3354-60; Soignet S. L. et al. Complete remission after treatment of acute promyelocyticle leukemia with arsenic trioxide, N Engl J Med 1998; 339: 1341-8; Niu C. et al. Studies on treatment of acute promyelocyticle leukemia with arsenic trioxide: remission induction, follow-up and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocyticle leukemia patients, Blood). 1999;94:3315-24).

A typical course of treatment for arsenic trioxide involves daily intravenous (IV) administration for 4 to 8 weeks, along with the inconvenience, risks, and costs associated with maintaining adequate vascular access and prolonged hospitalization. Currently, there are no FDA-approved oral arsenic trioxide formulations for clinical use. Fowler's solution is no longer detailed in modern pharmacopoeias or listed in formularies (1941, Arsenum. Martindale, The Extra Pharmacopoeia 22: 209-15; British Pharmacopoeia. London: Her Majesty's Stationery Office, 1988; Appendix 1A, page A12). Therefore, orally administered arsenic-containing formulations could offer significant advantages.

The inventors of this invention have obtained a novel formulation comprising a lyophilized composition containing arsenic, which can be administered orally. The inventors have also developed a method for preparing this novel formulation comprising a lyophilized composition containing arsenic. The lyophilized powder containing arsenic is suitable for oral administration to patients, for example, via capsules and tablets.

_{As₂O₃ powder} dissolves slightly and very slowly in cold water; even in boiling water, it dissolves only in a 1:15 ratio (Arsenic Trioxide, in: Budavari S O'Neil M J (Eds), The Merck Index. An encyclopedia of chemicals, drugs and biologicals. NJ: Merck & Co., Inc. 11th ed., Rahway, NJ, USA. 1989. Monograph 832, p. 127). Therefore, and due to other problems described above, it has not yet been formulated into an orally available composition.

Overview

This invention addresses the problems of insolubility and insufficient bioavailability _{of As₂O₃} and provides an arsenic-containing lyophilized composition that makes it bioavailable. Arsenic is introduced as _{As₂O₃ powder} , then dissolved and lyophilized as described below. More specifically, this invention relates to an arsenic-containing lyophilized composition wherein arsenic exists as one or more arsenic salts and/or solvates, and/or _{as As₂O₃} and/or one or more arsenic compounds.

Therefore, as described herein, "arsenic-containing lyophilized composition" (LCCA) means a composition containing arsenic as one or more of its salts, and/or solvates thereof, and/or arsenic trioxide and/or any other arsenic-containing compound, said composition being obtained by applying the method steps of the present invention described below. Alternatively, the arsenic-containing lyophilized composition may be referred herein as lyophilized arsenic trioxide or lyophilized _As₂O₃ ( _LAT ).

Obviously, the present invention envisions preparing this "arsenic-containing freeze-dried composition" by other methods.

In other words, an independently lyophilized composition containing arsenic is one aspect of the present invention. On one hand, the present invention also relates to a method of manufacturing LCCA. On another hand, the present invention also relates to an oral formulation containing LCCA. On yet another hand, the present invention relates to a method of preparing such an oral formulation. The present invention also relates to a pharmaceutical composition in a solid dosage form suitable for oral administration, the composition comprising an arsenic-containing lyophilized composition. The present invention also relates to a pharmaceutical composition in a solid dosage form suitable for oral administration, the composition comprising LCCA, at least one building agent, and at least one lubricant. On one hand, the present invention also relates to a kit comprising a pharmaceutical composition containing LCCA. On yet another hand, the present invention relates to a method of treating malignancies, such as hematological malignancies, in patients in need, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition containing LCCA to the patient.

Implementation Plan—Method for preparing arsenic-containing lyophilized compositions

This invention relates to a method for preparing an arsenic-containing lyophilized composition (LCCA), the method comprising: (A) dissolving _{As₂O₃ powder} in an aqueous medium to form an _As₂O₃ solution; and (B) _lyophilizing the _As₂O₃ solution. In one embodiment, dissolving the _{As₂O₃ powder} in the aqueous medium comprises: (I) adding an alkalizing agent to the _{As₂O₃ powder} in a container with _or without stirring and with or without adding water to adjust the pH to about 12 or higher; (II) adding an acid to the container with or without stirring and with or without adding water to adjust the pH to about 7 to about 8; (III) optionally adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally adding water to the container with or without stirring to produce _{an As₂O₃} solution. In another embodiment, for the purposes of the methods described herein to date, the alkalizing agent comprises sodium hydroxide (NaOH), sodium carbonate ( _Na₂CO₃ ), or a mixture _thereof . In another embodiment, the amount of the alkalizing agent added in the above methods is from about 10% to about 100% of the amount of _{As₂O₃ powder} .

In one embodiment of the invention, for the methods previously described in this section, the acid comprises hydrochloric acid (HCl). In another embodiment, the HCl is about 6M HCl. In another embodiment, the acid is added to the container to adjust the pH to about 7.2. In another embodiment, for the methods described to date in this section, the surfactant comprises at least one of sodium lauryl sulfate; Tween 80®; β-cyclodextrin; poloxamer; and tocopheryl polyethylene glycol succinate (TPGS). In another embodiment, the surfactant is added to about 0.5% v/v to about 4.0% v/v, but not exceeding about 50% _{As₂O₃ concentration} .

In another embodiment of the invention, for the method described above in this section, the freeze-drying step comprises: (A) freezing _{the As₂O₃} solution to produce _a frozen _As₂O₃ product; and (B) drying _{the As₂O₃} product to produce the arsenic-containing freeze-dried composition. In another embodiment, for the method described to date, the freezing step comprises freezing the _As₂O₃ solution at a temperature ranging from about -50°C to about 0° _C . In another embodiment, the _As₂O₃ solution is frozen at about -40° _C for at least about 6 hours. In another embodiment, the drying includes at least one of the following three conditions: (I) drying the _{As₂O₃ product at at least one temperature in the range of about -40°C to about 50°C for about 5 minutes to about 500 minutes; (II) drying the frozen As₂O₃ by} gradually increasing the temperature from at least one first temperature in the range of about -40°C to about 50°C to at least one second temperature in the range of about -40°C to about 50° _C over a period of about 5 minutes to about 500 minutes, wherein the at least one second temperature _is higher than the at least one first temperature; and (III) applying a vacuum to the frozen _As₂O₃ product in the range of about ₃₀₀ mTorr to about 1000 mTorr for about 5 minutes to about 500 minutes.

In one embodiment of the invention, for the methods described herein to date, the drying step includes heating the _As₂O₃ product at about -30˚C and about 800 mTorr for about 60 minutes; heating the _As₂O₃ product from the aforementioned step at -20˚C and 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the aforementioned step at about -5˚C and about 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the aforementioned step at about 10˚C and about 500 _mTorr for about 60 minutes; and heating the _As₂O₃ product from the aforementioned step at about 25˚C and about _{500 mTorr} for a period ranging from about 180 minutes to 300 minutes. In another embodiment, the drying step includes heating the _As₂O₃ product to approximately -30˚C and approximately 800 mTorr for approximately 60 minutes and maintaining it at approximately -30˚C and approximately 800 mTorr for approximately 60 minutes; heating the _As₂O₃ product from the aforementioned step to approximately -20˚C and approximately 500 _mTorr for approximately 60 minutes and maintaining it at approximately _-20˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃ product} from the aforementioned step to approximately -5˚C and approximately 500 mTorr for approximately 300 minutes and maintaining it at approximately -5˚C and approximately 500 mTorr for approximately 120 minutes; heating the _As₂O₃ product from the aforementioned step to approximately 10˚C and approximately 500 mTorr for approximately 120 minutes and maintaining it at approximately 10˚C and approximately 500 mTorr for approximately 60 minutes; and drying the _As₂O₃ product from the aforementioned step for approximately 60 minutes _{. 3.} The product is heated to about 25˚C and about 500 mTorr and held at about 25˚C and about 500 mTorr for a period of about 180 minutes to 300 minutes.

Implementation plan—Arsenic-containing lyophilized composition

This invention relates to a composition comprising an arsenic-containing lyophilized composition (LCCA). In one embodiment, the composition further comprises at least one filler and at least one lubricant. In another embodiment, the composition is prepared by a method comprising the steps of: (A) dissolving _{As₂O₃ powder} in an aqueous medium to form an _As₂O₃ solution _; (B) lyophilizing the _As₂O₃ solution to produce a lyopremix; (C) sieving the lyopremix to produce _{lyophilized As₂O₃ powder} ; (D) optionally, adding at least one filler to the lyophilized _{As₂O₃ powder} ; and (E) optionally, adding one or more lubricants to the lyophilized _{As₂O₃ powder} to produce an oral formulation of _{the As₂O₃} .

In one embodiment of the invention, for the previously described compositions, dissolving the _As₂O₃ powder in an aqueous medium comprises: (I) adding an alkalizing agent to _{the As₂O₃ powder in} a container with or without stirring and with or without adding water, to adjust the pH to about 12 or higher; (II) adding an acid to the container with or without stirring and with or without adding water, to adjust the pH to about 7 to about 8; (III) optionally, adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally, adding water to the container with or without stirring to produce an _As₂O₃ solution. In another embodiment, for the compositions described so far, the alkalizing agent comprises sodium hydroxide ₍ NaOH), sodium carbonate ( _Na₂CO₃ ), or a mixture _thereof . In another embodiment, the amount of the alkalizing agent added to the above compositions is about 10% to about 100 _% of the amount of the _As₂O₃ powder.

In one embodiment of the invention, for the previously described composition, the acid comprises hydrochloric acid (HCl). In another embodiment, the HCl is about 6M HCl. In another embodiment, the acid is added to the container to adjust the pH to about 7.2. In another embodiment, for the composition described to date, the surfactant comprises at least one selected from sodium lauryl sulfate; Tween 80®; β-cyclodextrin; poloxamer; and tocopheryl polyethylene glycol succinate (TPGS). In another embodiment, the surfactant is added to about 0.5% v/v to about 4.0% v/v, but not exceeding about 50% _{As₂O₃ concentration} .

In another embodiment of the invention, for the aforementioned composition, the lyophilization step comprises: (A) freezing _{the As₂O₃} solution to produce _a frozen _As₂O₃ product; and (B) drying the _As₂O₃ product to produce the arsenic-containing lyophilized composition. In another embodiment, for the compositions described so far, the freezing step comprises freezing _{the As₂O₃ solution} at a temperature ranging from about -50°C to about 0° _C . In another embodiment, the _As₂O₃ solution is frozen at about _-40˚C for at least about 6 hours. In another embodiment, the drying includes at least one of the following three conditions: (I) drying the _{As₂O₃ product at at least one temperature in the range of about -40°C to about 50°C for about 5 minutes to about 500 minutes; (II) drying the frozen As₂O₃ by} gradually increasing the temperature from at least one first temperature in the range of about -40°C to about 50°C to at least one second temperature in the range of about -40°C to about 50° _C over a period of about 5 minutes to about 500 minutes, wherein the at least one second temperature _is higher than the at least one first temperature; and (III) applying a vacuum to the frozen _As₂O₃ product in the range of about ₃₀₀ mTorr to about 1000 mTorr for about 5 minutes to about 500 minutes.

In one embodiment of the invention, for the compositions described herein to date, the drying step includes heating the _As₂O₃ product at about -30˚C and about 800 mTorr for about 60 minutes; heating the _As₂O₃ product from the preceding steps at -20˚C and 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the preceding steps at about -5˚C and about 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the preceding steps at about 10˚C and about 500 _mTorr for about 60 minutes; and heating the _As₂O₃ product from the preceding steps at about 25˚C and about _{500 mTorr} for a period ranging from about 180 minutes to 300 minutes. In another embodiment, the drying step includes heating the _As₂O₃ product to approximately -30˚C and approximately 800 mTorr for approximately 60 minutes and maintaining it at approximately -30˚C and approximately 800 mTorr for approximately 60 minutes; heating the _As₂O₃ product from the aforementioned step to approximately -20˚C and approximately 500 _mTorr for approximately 60 minutes and maintaining it at approximately _-20˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃ product} from the aforementioned step to approximately -5˚C and approximately 500 mTorr for approximately 300 minutes and maintaining it at approximately -5˚C and approximately 500 mTorr for approximately 120 minutes; heating the _As₂O₃ product from the aforementioned step to approximately 10˚C and approximately 500 mTorr for approximately 120 minutes and maintaining it at approximately 10˚C and approximately 500 mTorr for approximately 60 minutes; and drying the _As₂O₃ product from the aforementioned step for approximately 60 minutes _{. 3.} The product is heated to about 25˚C and about 500 mTorr and held at about 25˚C and about 500 mTorr for a period of about 180 minutes to 300 minutes.

In another embodiment of the invention, for the compositions previously described in this section, the filler comprises mannitol; and/or said one or more lubricants comprise talc and/or magnesium stearate. In another embodiment, the composition is a controlled-release, orally administered solid composition. In one embodiment, the compositions previously described in this section are encapsulated in capsules.

Implementation Scheme—Method for preparing an oral pharmaceutical formulation containing LCCA

This invention relates to a method for preparing an oral pharmaceutical formulation comprising a lyophilized composition containing arsenic or lyophilized arsenic trioxide ( _As₂O₃ ), the method comprising: ₍ A) dissolving _As₂O₃ powder in an aqueous medium to form _{an As₂O₃ solution} ; (B) lyophilizing the _As₂O₃ solution to produce a lyophilized product; (C) sieving the lyophilized product to produce _{lyophilized As₂O₃} powder; (D) optionally, adding at least _one filler to the _{lyophilized As₂O₃ powder} ; and (E) optionally, adding one or more lubricants to the lyophilized _{As₂O₃ powder} to produce an oral formulation of _{the As₂O₃} .

The present invention also relates to the previously described method of dissolving _{As₂O₃ powder} in an aqueous medium, comprising: (I) adding an alkalizing agent to the _{As₂O₃ powder} in a container with or without stirring and with or without adding water, to adjust the pH to about 12 or higher; (II) adding an acid to the container with or without stirring and with or without adding water, to adjust the pH to about 7 to about 8; (III) optionally adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally adding water to the container with or without stirring to produce an _As₂O₃ solution. In another embodiment, for the purposes of the methods described herein to date, the alkalizing agent comprises sodium hydroxide ₍ NaOH), sodium carbonate ( _Na₂CO₃ ), or a mixture _thereof . In another embodiment, the amount of the alkalizing agent added in the above methods is about 10% to about 100% of the amount of _{the As₂O₃} powder.

In one embodiment of the invention, for the methods previously described in this section, the acid comprises hydrochloric acid (HCl). In another embodiment, the HCl is about 6M HCl. In another embodiment, the acid is added to the container to adjust the pH to about 7.2. In another embodiment, for the methods described to date in this section, the surfactant comprises at least one of sodium lauryl sulfate; Tween 80®; β-cyclodextrin; poloxamer; and tocopheryl polyethylene glycol succinate (TPGS). In another embodiment, the surfactant is added to about 0.5% v/v to about 4.0% v/v, but not exceeding about 50% _{As₂O₃ concentration} .

In another embodiment of the invention, for the method described above in this section, the freeze-drying step includes: (A) freezing _{the As₂O₃} solution to produce _a frozen _As₂O₃ product; and (B) drying _{the As₂O₃} product to produce the arsenic-containing freeze-dried composition. In another embodiment, for the method described to date, the freezing step includes freezing the _As₂O₃ solution at a temperature ranging from about -50°C to about ₀ °C. In another embodiment, the _As₂O₃ solution is frozen at about _-40˚C for at least about 6 hours. In another embodiment, the drying includes at least one of the following three conditions: (I) drying the _{As₂O₃ product at at least one temperature in the range of about -40°C to about 50°C for about 5 minutes to about 500 minutes; (II) drying the frozen As₂O₃ by} gradually increasing the temperature from at least one first temperature in the range of about -40°C to about 50°C to at least one second temperature in the range of about -40°C to about 50° _C over a period of about 5 minutes to about 500 minutes, wherein the at least one second temperature _is higher than the at least one first temperature; and (III) applying a vacuum to the frozen _As₂O₃ product in the range of about ₃₀₀ mTorr to about 1000 mTorr for about 5 minutes to about 500 minutes.

In one embodiment of the invention, for the methods described herein to date, the drying step includes heating the _As₂O₃ product at about -30˚C and about 800 mTorr for about 60 minutes; heating the _As₂O₃ product from the aforementioned step at -20˚C and 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the aforementioned step at about -5˚C and about 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the aforementioned step at about 10˚C and about 500 _mTorr for about 60 minutes; and heating the _As₂O₃ product from the aforementioned step at about 25˚C and about _{500 mTorr} for a period ranging from about 180 minutes to 300 minutes. In another embodiment, the drying step includes heating the _As₂O₃ product to approximately -30˚C and approximately 800 mTorr for approximately 60 minutes and maintaining it at approximately -30˚C and approximately 800 mTorr for approximately 60 minutes; heating the _As₂O₃ product from the aforementioned step to approximately -20˚C and approximately 500 _mTorr for approximately 60 minutes and maintaining it at approximately _-20˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃ product} from the aforementioned step to approximately -5˚C and approximately 500 mTorr for approximately 300 minutes and maintaining it at approximately -5˚C and approximately 500 mTorr for approximately 120 minutes; heating the _As₂O₃ product from the aforementioned step to approximately 10˚C and approximately 500 mTorr for approximately 120 minutes and maintaining it at approximately 10˚C and approximately 500 mTorr for approximately 60 minutes; and drying the _As₂O₃ product from the aforementioned step for approximately 60 minutes _{. 3.} The product is heated to about 25˚C and about 500 mTorr and held at about 25˚C and about 500 mTorr for a period of about 180 minutes to 300 minutes.

In another embodiment of the invention, for the method previously described in this section, the filler comprises mannitol; and/or said one or more lubricants comprise talc and/or magnesium stearate. In another embodiment, the previously described method further includes the step of filling a capsule with the oral formulation.

Implementation plan—oral drug formulations containing LCCA

This invention relates to pharmaceutical compositions in solid dosage forms suitable for oral administration, the compositions comprising a lyophilized composition containing arsenic. In one embodiment, such pharmaceutical composition further comprises at least one filler and at least one lubricant. In another embodiment, the pharmaceutical compositions described to date in this section are prepared by a method comprising the steps of: (A) dissolving _{As₂O₃ powder} in an aqueous medium to form _{an As₂O₃} solution; (B) lyophilizing the _{As₂O₃ solution} to produce a lyophilized product; (C) sieving the _lyophilized product to produce lyophilized _As₂O₃ powder; (D) optionally, adding at least one filler to the lyophilized _{As₂O₃ powder} ; and (E) optionally, adding one or more lubricants to the lyophilized _{As₂O₃ powder} to produce an oral formulation of _{the As₂O₃} .

In one embodiment of the invention, for the pharmaceutical compositions previously described in this section, dissolving the _As₂O₃ powder in an aqueous medium comprises: (I) adding an alkalizing agent to the _As₂O₃ powder in a container with or without stirring and with or without _adding water, to adjust the pH to about 12 or higher; ( _II ) adding an acid to the container with or without stirring and with or without adding water, to adjust the pH to about 7 to about 8; (III) optionally, adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally, adding water to the container with or without stirring to produce _{an As₂O₃} solution. In another embodiment, for the compositions described so far, the alkalizing agent comprises sodium hydroxide (NaOH), sodium carbonate ( _{Na₂CO₃ )} , or a mixture thereof. In another embodiment, the amount of the alkalizing agent added to the above pharmaceutical compositions is about 10% to about 100% of the amount of _{the As₂O₃} powder.

In one embodiment of the invention, for the previously described pharmaceutical composition, the acid comprises hydrochloric acid (HCl). In another embodiment, the HCl is about 6M HCl. In another embodiment, the acid is added to the container to adjust the pH to about 7.2. In another embodiment, for the pharmaceutical composition described to date, the surfactant comprises at least one of sodium lauryl sulfate; Tween 80®; β-cyclodextrin; poloxamer; and tocopheryl polyethylene glycol succinate (TPGS). In another embodiment, the surfactant is added to about 0.5% v/v to about 4.0% v/v, but not exceeding about 50% _{As₂O₃ concentration} .

In another embodiment of the invention, for the pharmaceutical composition described above, the lyophilization step comprises: (A) freezing _{the As₂O₃} solution to produce a frozen _As₂O₃ product; and (B) drying the _As₂O₃ product to produce the arsenic-containing lyophilized composition. In another embodiment, for the composition described so far, the freezing step comprises freezing the _As₂O₃ solution at a temperature ranging from about -50°C to about ₀ °C. In another embodiment, _{the As₂O₃ solution is} frozen at about _-40˚C for at least about 6 hours. In another embodiment, the drying includes at least one of the following three conditions: (I) drying the _{As₂O₃ product at at least one temperature in the range of about -40°C to about 50°C for about 5 minutes to about 500 minutes; (II) drying the frozen As₂O₃ by} gradually increasing the temperature from at least one first temperature in the range of about -40°C to about 50°C to at least one second temperature in the range of about -40°C to about 50° _C over a period of about 5 minutes to about 500 minutes, wherein the at least one second temperature is higher than the at least one first temperature; and (III) applying a vacuum to the frozen _As₂O₃ product in the range of about _{300 mTorr} to about 1000 mTorr for about 5 minutes to about 500 minutes.

In one embodiment of the invention, for the pharmaceutical composition described to date, the drying step includes heating the _As₂O₃ product at about -30˚C and about 800 mTorr for about 60 minutes; heating the _As₂O₃ product from the preceding step at -20˚C and 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the preceding step at about -5˚C and about 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the preceding step at about 10˚C and about 500 _mTorr for about 60 minutes; and heating the _As₂O₃ product from the preceding step at about 25˚C and about _{500 mTorr} for a period ranging from about 180 minutes to 300 minutes. In another embodiment, the drying step includes heating the _As₂O₃ product to approximately -30˚C and approximately 800 mTorr for approximately 60 minutes and maintaining it at approximately -30˚C and approximately 800 mTorr for approximately 60 minutes; heating the _As₂O₃ product from the aforementioned step to approximately -20˚C and approximately 500 _mTorr for approximately 60 minutes and maintaining it at approximately _-20˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃ product} from the aforementioned step to approximately -5˚C and approximately 500 mTorr for approximately 300 minutes and maintaining it at approximately -5˚C and approximately 500 mTorr for approximately 120 minutes; heating the _As₂O₃ product from the aforementioned step to approximately 10˚C and approximately 500 mTorr for approximately 120 minutes and maintaining it at approximately 10˚C and approximately 500 mTorr for approximately 60 minutes; and drying the _As₂O₃ product from the aforementioned step for approximately 60 minutes _{. 3.} The product is heated to about 25˚C and about 500 mTorr and held at about 25˚C and about 500 mTorr for a period of about 180 minutes to 300 minutes.

In another embodiment of the invention, for the pharmaceutical compositions previously described in this section, the filler comprises mannitol; and/or wherein the one or more lubricants comprise talc and/or magnesium stearate. In another embodiment, the pharmaceutical composition is a controlled-release, orally administered solid composition. In one embodiment, the pharmaceutical composition previously described in this section is encapsulated in a capsule.

In another set of embodiments, the invention also relates to capsules comprising about 1 mg, about 5 mg, 10 mg, or about 20 mg of the pharmaceutical compositions previously described in this section. The invention also relates to kits comprising the pharmaceutical compositions described in this section and their instructions for use.

Implementation plan—Administration of the drug composition to the patient

This invention relates to a method for orally administering a pharmaceutical composition comprising a lyophilized composition containing arsenic to a subject, the method comprising the steps of providing the pharmaceutical composition and orally administering the pharmaceutical composition to the subject. The invention also relates to a method for treating malignant tumors such as cancer or tumors in patients in need, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a lyophilized composition containing arsenic to the patient.

In another embodiment, the present invention relates to the methods previously described in this section, wherein the cancer is a hematologic malignancy. In another embodiment, the hematologic malignancy is at least one of the following: acute myeloid leukemia; acute non-lymphocytic leukemia; myeloblastic leukemia, promyelocytic leukemia; chronic myelomonocytic leukemia; monocytic leukemia; erythroleukemia; acute neutrophilic leukemia; myelodysplastic syndrome; acute promyelocytic leukemia; chronic lymphocytic leukemia; chronic myeloid leukemia; hairy cell leukemia; myeloproliferative neoplasm; Hodgkin lymphoma; non-Hodgkin lymphoma; myeloma; giant cell myeloma; painless myeloma; localized myeloma; multiple myeloma; plasma cell myeloma; sclerosing myeloma; solitary myeloma; condensed multiple myeloma; non-secreting myeloma; osteosclerosing myeloma; plasma cell leukemia; solitary plasmacytoma; and extramedullary plasmacytoma.

In another embodiment, for the purposes of the methods described to date in this section, the hematological malignancy is acute promyelocytic leukemia (APL). In one embodiment, the APL is newly diagnosed APL. In another embodiment, the APL is relapsed or refractory APL.

In one embodiment, in the treatment methods previously described in this section, the myeloproliferative tumor is one of myelofibrotic polycythemia vera and essential thrombocythemia.

In one embodiment of the invention, the pharmaceutical composition is administered daily for the method described in this section. In another embodiment, the pharmaceutical composition is administered in a single dose range of about 1 mg to about 50 mg. In yet another embodiment, the pharmaceutical composition is administered in a single dose range of about 0.1 mg/kg body weight to about 0.3 mg/kg body weight.

In another embodiment, for the methods previously described in this section, the patient has previously received or is receiving chemotherapy and/or radiation therapy. In another embodiment, the patient is further administered one or more chemotherapeutic agents. In one embodiment of the method, the chemotherapeutic agent is administered before, after, or concurrently with the pharmaceutical composition.

Implementation Plan—Improvement of the Physical/Chemical Properties of Formulation Powders

This invention relates to a method for increasing the surface area of an initial API powder containing arsenic trioxide to about 2X to about 80X, the method comprising the steps of: (A) dissolving _{As₂O₃ powder} in an aqueous medium to form _{an As₂O₃ solution} , comprising the steps of: (I) adding an alkalizing agent to the _As₂O₃ powder in a container with or without stirring and with or without adding water to adjust the pH to about 12 or higher; (II) adding an acid to the container with or without stirring and with or without adding water to adjust the pH to about 7 to about 8; (III) optionally adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally adding water to the container with or without stirring to produce an _As₂O₃ solution; and (B) lyophilizing _{the As₂O₃} solution, comprising _the steps of: (V) freezing _{the As₂O₃} solution to produce _a frozen _As₂O₃ product; and (VI) drying the As₂O₃ solution. _{The 2O3 product} is used to produce the lyophilized composition containing arsenic.

The present invention also relates to a method for increasing the solubility of arsenic trioxide powder in water or in alcohol to about 2X to about 30X, the method comprising the steps of: (A) dissolving _{As₂O₃ powder} in an aqueous medium to form _{an As₂O₃} solution, comprising the steps of: (I) adding an alkalizing agent to _{the As₂O₃} powder in a container with or without stirring and with or without adding water to adjust the pH to about 12 or higher; (II) adding an acid to the container with or without stirring and with or without adding water to adjust the pH to about 7 to about 8; (III) optionally adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally adding water to the container with or without stirring to produce _{an As₂O₃} solution; and (B) lyophilizing _{the As₂O₃} solution, comprising the steps of: (V) freezing the _As₂O₃ solution to produce _{a frozen As₂O₃ product ;} and (VI) The _As₂O₃ product is dried to produce _the arsenic-containing lyophilized composition.

The present invention also relates to a method for increasing the dissolution rate of an arsenic-containing pharmaceutical composition by at least five times relative to an initial _arsenic trioxide-containing API, the method comprising the steps of: (A) dissolving _As₂O₃ powder in an aqueous medium to form _{an As₂O₃} solution, comprising the steps of: (I) adding an alkalizing agent to the _{As₂O₃ powder} in a container with or without stirring and with or without adding water to adjust the pH to about 12 or higher; (II) adding an acid to the container with or without stirring and with or without adding water to adjust the pH to about 7 to about 8; (III) optionally adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally adding water to the container with or without stirring to produce an _As₂O₃ solution; and (B) lyophilizing _{the As₂O₃} solution, comprising the steps of: ( _V ) freezing the _{As₂O₃ solution} to produce _a frozen _As₂O₃ product; and (VI) The _As₂O₃ product is dried to produce _the arsenic-containing lyophilized composition.

The present invention also relates to a method for providing an oral bioavailability of arsenic to a subject, wherein the oral bioavailability is in the range of about 50% to about 100% of the bioavailability of an intravenously administered pharmaceutical composition containing arsenic, the method comprising the steps of: (A) dissolving _{As₂O₃ powder} in an aqueous medium to form _{an As₂O₃} solution, comprising the steps of: (I) adding an alkalizing agent to the _{As₂O₃ powder} in a container with or without stirring and with or without adding water to adjust the pH to about 12 or higher; (II) adding an acid to the container with or without stirring and with or without adding water to adjust the pH to about 7 to about 8; (III) optionally adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally adding water to the container with or without stirring to produce an _As₂O₃ solution; and _{( B} ) lyophilizing the _As₂O₃ solution, comprising the step of: (V) freezing the _As₂O₃ powder. (VI) Solve _the solution to produce _a frozen _As₂O₃ product; and (VI) dry _{the As₂O₃} product to produce the arsenic-containing lyophilized composition.

The present invention also relates to compositions comprising an arsenic-containing lyophilized composition (LCCA), wherein the arsenic-containing lyophilized composition comprises particles with a D(90) size ranging from about 2 micrometers to 10 micrometers. In another embodiment, the arsenic-containing lyophilized composition comprises particles with a D(90) size of about 1/10 to about 1/50 of the particle size of the initial API powder containing arsenic trioxide. In another embodiment, the arsenic-containing lyophilized composition comprises particles with a BET surface area ranging from about 0.5 ^m² /g to 5 ^m² /g. In another embodiment, the arsenic-containing lyophilized composition comprises particles with a BET surface area of about 5 to about 80 times the surface area of the initial API powder containing arsenic trioxide. In another embodiment, the solubility of the arsenic-containing lyophilized composition in cold water or alcohol is about 2 to about 30 times that of the initial API powder containing arsenic trioxide. In another embodiment, the arsenic-containing lyophilized composition is soluble in cold water or alcohol in the range of about 4 g/100 g to about 60 g/100 g of the lyophilized composition/cold water or alcohol.

In one embodiment, the present invention relates to a pharmaceutical composition described in the preceding section, wherein the dissolution of the pharmaceutical composition, as measured by ICP-OES, is about 5 to about 15 times the dissolution of the initial API powder containing arsenic trioxide.

The present invention also relates to an oral dosage form in capsule form, having a weight of 100 units and comprising: (I) a lyophilized composition containing arsenic, in an amount providing 10 units of arsenic trioxide and 5 units of sodium lauryl sulfate; (II) mannitol, in an amount of 73 units; (III) talc, in an amount of 1; and (IV) magnesium stearate, in an amount of 1 mg; such that the total weight of the composition reaches 100 units. In one set of embodiments of the invention, the capsule weight is 10 mg, 50 mg, 100 mg, or 200 mg.

For example, an oral dosage form in capsule form, weighing 10 mg, contains:

(I) A lyophilized composition containing arsenic, wherein the amount is 1 mg of arsenic trioxide and 0.5 mg of sodium lauryl sulfate;

(II) Mannitol, in an amount of 7.3 mg;

(III) Talc, in an amount of 0.1 mg; and

(IV) Magnesium stearate, in an amount of 0.1 mg.

This results in a total weight of 10 mg for the composition.

Similarly, an oral dosage form in capsule form, weighing 50 mg, contains:

(I) A lyophilized composition containing arsenic, wherein the amount is 5 mg of arsenic trioxide and 2.5 mg of sodium lauryl sulfate;

(II) Mannitol, in an amount of 36.5 mg;

(III) Talc, in an amount of 0.5 mg; and

(IV) Magnesium stearate, in an amount of 0.5 mg.

This results in a total weight of 50 mg for the composition.

In another example, an oral dosage form in capsule form, weighing 100 mg, contains:

(I) A lyophilized composition containing arsenic, wherein the amount is 10 mg of arsenic trioxide and 5 mg of sodium lauryl sulfate;

(II) Mannitol, in an amount of 73 mg;

(III) Talc, in an amount of 1 mg; and

(IV) Magnesium stearate, in an amount of 1 mg

This results in a total weight of 100 mg for the composition.

In another example, an oral dosage form in capsule form, weighing 200 mg, contains:

(I) A lyophilized composition containing arsenic, wherein the amount is 20 mg of arsenic trioxide and 10 mg of sodium lauryl sulfate;

(II) Mannitol, in an amount of 146 mg;

(III) Talc, in an amount of 2 mg; and

(IV) Magnesium stearate, in an amount of 2 mg

This results in a total weight of 200 mg for the composition.

These and other aspects of the invention are described in detail below.

Brief description of the attached figures

Figure 1 shows the pharmacokinetic analysis of the novel lyophilized formulation containing arsenic in dogs, compared with reference arsenic trioxide.

Figure 2 shows a process flow diagram for freeze-drying and capsule manufacturing.

Figure 3 shows the X-ray diffraction pattern of arsenic trioxide (API).

Figure 4 shows the X-ray diffraction pattern of the lyophilized composition containing arsenic.

Figure 5 shows the X-ray diffraction pattern of the final mixture (pharmaceutical formulation) containing the lyophilized composition containing arsenic.

Figure 6 shows the X-ray diffraction pattern of the placebo.

Figure 7 shows SEM images of arsenic trioxide API; the lyophilized product; and the final mixture.

Figure 8. Figure 8 provides a BET surface area plot of arsenic trioxide API, which shows how 1/[Q(Po/P-1)] depends on the relative pressure P/Po.

Figure 9 provides a BET surface area diagram of the lyophilized product (a lyophilized composition containing arsenic), showing how 1/[Q(Po/P-1)] depends on the relative pressure P/Po.

Invention Description

This invention relates to a method for preparing an arsenic-containing lyophilized composition (LCCA), the method comprising: (A) dissolving _{As₂O₃ powder} in an aqueous medium to form an _As₂O₃ solution; and (B) _lyophilizing the _As₂O₃ solution. In one embodiment, dissolving the _{As₂O₃ powder} in the aqueous medium comprises: (I) adding an alkalizing agent to the _{As₂O₃ powder} in a container with _or without stirring and with or without adding water to adjust the pH to about 12 or higher; (II) adding an acid to the container with or without stirring and with or without adding water to adjust the pH to about 7 to about 8; (III) optionally adding a surfactant to the container with or without stirring and with or without adding water; and (IV) optionally adding water to the container with or without stirring to produce _{an As₂O₃} solution.

In another embodiment of the invention, for the method described above in this section, the freeze-drying step includes: (A) freezing _{the As₂O₃} solution to produce _a frozen _As₂O₃ product; and (B) drying _{the As₂O₃} product to produce the arsenic-containing freeze-dried composition. In another embodiment, for the method described to date, the freezing step includes freezing the _As₂O₃ solution at a temperature ranging from about -50°C to about ₀ °C. In another embodiment, the _As₂O₃ solution is frozen at about _-40˚C for at least about 6 hours.

The present invention also relates to a composition comprising an arsenic-containing lyophilized composition (LCCA). In one embodiment, the composition further comprises at least one filler and at least one lubricant. In another embodiment, the composition is prepared by a method comprising the steps of: (A) dissolving _{As₂O₃ powder} in an aqueous medium to form _{an As₂O₃} solution _; (B) lyophilizing the _As₂O₃ solution to produce a lyophilized product; (C) sieving the lyophilized product to produce lyophilized _{As₂O₃ powder} ; (D) optionally, adding at least one filler to the lyophilized _{As₂O₃ powder} ; (E) optionally, adding one or more lubricants to the lyophilized _{As₂O₃ powder} to produce an oral formulation of _{the As₂O₃} .

In another embodiment of the invention, for the compositions previously described in this section, the filler comprises mannitol; and/or said one or more lubricants comprise talc and/or magnesium stearate. In another embodiment, the composition is a controlled-release, orally administered solid composition. In one embodiment, the compositions previously described in this section are encapsulated in capsules.

This invention also relates to a method for preparing an oral pharmaceutical formulation comprising a lyophilized composition containing arsenic or lyophilized _arsenic trioxide ( _As₂O₃ ), the method comprising: (A) dissolving _As₂O₃ powder in an aqueous medium to form _{an As₂O₃ solution} ; (B) lyophilizing _{the As₂O₃} solution to produce a lyophilized product; (C) sieving the lyophilized product to produce lyophilized _As₂O₃ powder; (D) optionally, adding at least _one filler to _the lyophilized _{As₂O₃ powder} ; (E) optionally, adding one or more lubricants to the lyophilized _As₂O₃ powder to produce an oral formulation _{of the As₂O₃ .} This invention relates to a pharmaceutical composition suitable for oral administration in a solid dosage form, the composition comprising a lyophilized composition containing arsenic. In one embodiment, such pharmaceutical composition further comprises at least one filler and at least one lubricant. In another embodiment, the pharmaceutical composition described to date in this section is prepared by the methods described herein. In another embodiment of the invention, for the pharmaceutical compositions previously described in this section, the filler comprises mannitol; and/or wherein the one or more lubricants comprise talc and/or magnesium stearate. In another embodiment, the pharmaceutical composition is a controlled-release, orally administered solid composition. In one embodiment, the pharmaceutical composition previously described in this section is encapsulated in a capsule.

In another set of embodiments, the invention also relates to a capsule comprising about 1 mg, about 5 mg, 10 mg, or about 20 mg of the pharmaceutical composition previously described in this section. The invention also relates to a kit comprising the pharmaceutical composition described in this section and its instructions for use.

This invention relates to a method for orally administering a pharmaceutical composition comprising a lyophilized composition containing arsenic to a subject, the method comprising the steps of providing the pharmaceutical composition and orally administering the pharmaceutical composition to the subject. The invention also relates to a method for treating cancer in a patient in need, the method comprising the step of administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a lyophilized composition containing arsenic.

The present invention relates to a method for increasing the surface area of an initial API powder containing arsenic trioxide to about 5 X to about 80 X; a method for increasing the solubility of arsenic trioxide powder in water or in alcohol to about 2 X to about 30 X; a method for increasing the dissolution of an arsenic-containing pharmaceutical composition relative to the initial API by at least 5 times; a method for providing an oral bioavailability of arsenic to a subject, wherein the oral bioavailability is in the range of about 50% to about 100% of the bioavailability of an intravenously administered arsenic-containing pharmaceutical composition; and a method for reducing the D(90) size range of particles to about 2 micrometers to about 10 micrometers.

The present invention also relates to an oral dosage form in capsule form, having a weight of 100 units and comprising: (I) a lyophilized composition containing arsenic, in an amount providing 10 units of arsenic trioxide and 5 units of sodium lauryl sulfate; (II) mannitol, in an amount of 73 units; (III) talc, in an amount of 1; and (IV) magnesium stearate, in an amount of 1 mg; such that the total weight of the composition reaches 100 units. In one set of embodiments of the invention, the capsule weight is 10 mg, 50 mg, 100 mg, or 200 mg.

How to use

For patients with hematologic malignancies

This invention also provides a method of using an orally administered arsenic-containing lyophilized composition. In one embodiment, the arsenic-containing lyophilized composition is used as a medicament for treating hematologic malignancies such as leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and myeloma. The method includes administering an effective amount of the arsenic-containing lyophilized composition to a subject in need. The arsenic-containing lyophilized composition may be administered orally (in lyophilized form) or enterally via a feeding tube. As used herein, the term "effective amount" refers to an amount sufficient to provide a therapeutic or health benefit in the context of a hematologic malignancy.

In one embodiment, the lyophilized composition containing arsenic can produce health benefits in subjects suffering from hematologic malignancies. Preferably, the subject is a human being. The subject in need is a subject diagnosed with a hematologic malignancy (with or without metastasis) at any stage of the disease. As used herein, the term "hematologic malignancy" includes, but is not limited to, leukemia, lymphoma, and myeloma. As used herein, the term "leukemia" includes, but is not limited to, acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML) (sometimes referred to as acute non-lymphocytic leukemia (ANLL)), such as myeloblastic, premyeloid, myelomonocytic, monocytic, erythroleukemic leukemia, and myelodysplastic syndromes; chronic lymphocytic leukemia (CLL); chronic myeloid (granulocytic) leukemia (CML); chronic myelomonocytic leukemia (CMML); hairy cell leukemia; and polycythemia vera. As used herein, the term "lymphoma" includes, but is not limited to, Hodgkin's disease and non-Hodgkin's lymphoma. As used herein, the term "myeloma" includes, but is not limited to, giant cell myeloma, painless myeloma, focal myeloma, multiple myeloma, plasma cell myeloma, sclerosing myeloma, solitary myeloma, condensing multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma.

The target population may include patients concurrently receiving other treatments for hematologic malignancies. The target population may include patients with hematologic malignancies who have already received treatment (e.g., chemotherapy and/or radiation) and whose cancer is regressing. The target population may include patients with hematologic malignancies who have already received treatment and appear to be clinically free of hematologic malignancies. The arsenic-containing lyophilized composition of the present invention can be administered orally in conjunction with any treatment (such as, but not limited to, chemotherapy and/or radiation). For example, arsenic trioxide compositions can be combined with one or more chemotherapeutic or immunotherapeutic agents, such as acridine (AMSA), busulfan (Myleran®), chlorambucil (Leukeran®), cladribine (2-chlorodeoxyadenosine; "2-CDA"; Leustatin®), cyclophosphamide (Cytoxan®), cytarabine (ara-C; Cytosar-U®), daunorubicin (Cerubidine®), doxorubicin (Adriamycin®), etoposide (VePesid®), fludarabine phosphate (Fludara®), and hydroxyurea (Hydrea®). Combinations of idarubicin (Idamycin®), L-asparaginase (Elspar®), methotrexate sodium plus 6-mercaptopurine (6-MP; Purinethol®), mitoxantrone (Novantrone®), pentostatin (2-deoxymyopicycin; "DCF"; Nipent®), prednisone, retinoic acid (ATRA), vincristine sulfate (Oncovin®), 6-thioguanine (Tabloid®), cyclosporine A, paclitaxel®, cisplatin®, carboplatin®, doxil®, topotecan®, methotrexate®, bleomycin®, and epirubicin® are used. Arsenic trioxide compositions can also be used after other treatment regimens have been completed.

The subjects can be those who have not yet been diagnosed with hematologic malignancies but are susceptible to hematologic malignancies due to genetic and/or environmental factors or have a high risk of developing hematologic malignancies.

Depending on the individual, the range of treatment and health benefits includes inhibiting or delaying the growth of hematologic malignancies and/or their spread to other parts of the body (i.e., metastasis), alleviating cancer symptoms, increasing the survival rate of individuals with cancer, extending life expectancy, improving quality of life, and/or reducing the probability of recurrence after successful treatment (e.g., chemotherapy, radiation). Symptoms associated with hematologic malignancies include, but are not limited to, a weakened immune system, infections, fever, decreased red blood cells and platelets, weakness, fatigue, loss of appetite, weight loss, lymph nodes, enlarged or tender liver or spleen, easy bleeding or bruising, tiny red spots under the skin (called petechiae), swollen or bleeding gums, sweating (especially at night), bone or joint pain, headache, vomiting, confusion, loss of muscle control, and sudden onset.

In particular, the present invention provides a method for achieving complete remission of hematologic malignancies in a subject (e.g., a human), comprising orally administering the arsenic trioxide composition of the present invention to the subject. In other embodiments, the present invention provides remission of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% of hematologic malignancies. The present invention also provides a method for prolonging the survival of a subject suffering from a hematologic malignancy (preferably a human patient), the method comprising orally administering the arsenic-containing lyophilized composition of the present invention to the subject.

Effective doses will vary depending on the patient and route of administration. The effective dose for the patient will also vary depending on the condition being treated and its severity. Dosage, and perhaps frequency, will also vary based on the individual patient's age, weight, and response. Typically, the total daily dose range (in mg) of arsenic-containing lyophilized compositions for patients with hematologic malignancies is as follows: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 mg/day. Depending on the patient's needs, the dosage can be higher, for example, 60, 70, 80, 90, and 100 mg/day or any intermediate dose in between. The lyophilized premixture containing the arsenic composition is administered orally to the subject.

The duration of treatment should be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 7 weeks, at least 10 weeks, at least 13 weeks, at least 15 weeks, at least 20 weeks, at least 6 months, at least 1 year, or at least 2 years. In some cases, dosages outside these ranges may be necessary, as will be apparent to those skilled in the art. In some embodiments, the lyophilized composition containing arsenic may be administered for a period of time until symptoms are controlled or when the disease has partially or completely subsided. Furthermore, it should be noted that, based on the individual patient's response, the clinician or treating physician will know how and when to discontinue, adjust, or terminate the use of the arsenic trioxide composition as a medicine.

The effects of the arsenic-containing lyophilized compositions of the present invention on the development and progression of hematologic malignancies can be monitored by any method known to those skilled in the art, including but not limited to measuring: a) changes in tumor size and morphology (using imaging techniques such as computed tomography (CT) scans or ultrasound images); and b) changes in the levels of biomarkers of hematologic malignancy risk.

In some embodiments, the toxicity and efficacy of the preventive and/or therapeutic regimens of the present invention can be determined by standard pharmaceutical procedures in cell cultures or laboratory animals, such as those used to determine the LD50 (the dose that is 50% lethal to a population) and ED50 (the dose that is therapeutically effective in 50% of a population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, and it can be expressed as the LD50/ED50 ratio. Preventive and/or therapeutic agents exhibiting a large therapeutic index are preferred. While preventive and/or therapeutic agents exhibiting toxic side effects can be used, care should be taken to design delivery systems that target such agents to the site of the affected tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects.

In other embodiments, data obtained from cell culture assays and animal studies can be used to determine dosage ranges for prophylactic and/or therapeutic agents for human use. The dosages of these agents are preferably within a range of circulating concentrations, including the ED50, that have little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration. For any agent used in the methods of the present invention, the therapeutically effective dose can be initially estimated from cell culture assays. Dosages can be developed in animal models to achieve a range of circulating plasma concentrations, including the IC50 (i.e., the concentration of the test compound at which half-maximal inhibition of symptoms is achieved) determined in cell cultures. This information can be used to more precisely determine the useful dose in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

The anticancer activity of the therapy used according to the present invention can also be determined by using various experimental animal models used to study cancer, such as the SCID mouse model or transgenic mice. The following are some assays provided as examples and not as limitations.

The arsenic-containing lyophilized compositions of the present invention are prepared as described below in the specification and experimental sections. Arsenic or one or more chemical forms thereof are the active ingredient, and may optionally contain pharmaceutically acceptable carriers or excipients and/or other ingredients, provided that these ingredients do not impair (e.g., reduce) the potency of the arsenic-containing lyophilized compositions. Other ingredients that may be incorporated into the arsenic-containing lyophilized compositions of the present invention include, but are not limited to, herbs (including traditional Chinese medicine products), herbal extracts, vitamins, amino acids, metal salts, metal chelates, colorants, flavor enhancers, preservatives, etc.

Oral compositions can be used in any dosage form to provide an effective dose to a subject. Dosage forms include tablets, capsules, dispersions, suspensions, solutions, etc. In one embodiment, the compositions of the present invention suitable for oral administration can be presented as individual units, such as capsules, blister packs, blister packs, or tablets, each containing a predetermined amount of activated and conditioned yeast cells, as powders or granules, or as solutions or suspensions, oil-in-water emulsions, or water-in-oil emulsions in aqueous or non-aqueous liquids. In a preferred embodiment, the oral composition is in the form of a lyophilized powder or a fluffy solid. Typically, compositions are prepared by uniformly and tightly mixing the active ingredient with a liquid carrier or a finely dispersed solid carrier, or both, and then shaping the product into the desired form if necessary. These products can be used as pharmaceuticals or dietary supplements, depending on the dosage and circumstances of their use.

The oral compositions of the present invention may further include a binder (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); a binder or filler (e.g., lactose, pentosan, microcrystalline cellulose, or calcium hydrogen phosphate); a lubricant (e.g., magnesium stearate, talc, or silica); a disintegrant (e.g., potato starch or sodium starch hydroxyacetate); or a wetting agent (e.g., sodium lauryl sulfate). Tablets or capsules may be coated using methods well known in the art.

In some embodiments, the lyophilized composition containing arsenic comprises about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 mg equivalents of arsenic trioxide per ml.

Tablets and capsules typically represent the most advantageous form of oral dosage unit due to their ease of administration, in which case a solid drug carrier as described above is used. In a preferred embodiment, the composition is a capsule. The capsule can be formulated by any commercially available method. In some embodiments, the composition contains 0.25 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, etc. Capsules containing arsenic-containing lyophilized compositions in powder form of g, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, or 50 mg.

Other examples of anticancer agents that can be used in various embodiments of the present invention (including the pharmaceutical compositions and dosage forms and kits of the present invention) include, but are not limited to: acivitine; ararubicin; acodazole hydrochloride; acroline; adorine; interleukin; hexamethylmelamine; ambroxol; ametrine acetate; aminoglutethimide; acridine; anastrozole; atrazolyl; asparaginase; triamcinolone; azacitidine; azatiprine; azotocin; palmastat; Benzotiprexate; Bicalutamide; Bismuth subsalicylate; Dinefadroxil dimethsulfate; Bleomycin sulfate; Buquina sodium; Brompirimidine; Busulfan; Actinomycin C; Capprotestone; Carbetamide; Carbetin; Carboplatin; Carmustine; Carrubicin hydrochloride; Carzelaicin; Sildenafil; Chlorisac; Sirloin; Cisplatin; Cladribine; Clinapordine mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Actinomycin D; Daunorubicin hydrochloride Decitabine; Dextromethorphan; Dezaguanine; Dezaguanine Mesylate; Dexaquinone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifen; Droloxifen Citrate; Drotadazole Propionate; Dazomycin; Edatraxa; Ifluornithine Hydrochloride; Exalucin; Enloplatin; Enpromethazine; Epilapiidine; Epirubicin Hydrochloride; Ibuproazole; Exorubicin Hydrochloride; Estrostimine; Estrostimine Sodium Phosphate; Ethidazole; Etoposide; Etoposide Phosphate Glycosides; Chlorpheniramine; Fazodazole Hydrochloride; Fazalabin; Feniveryl Aamine; Azobarbital; Fludarabine Phosphate; Fluuracil; Flucitabine; Phosphorione; Phosphoric Acetonide Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Imofocin; Interleukin II (including recombinant interleukin II or rIL2); Interferon α-2a; Interferon α-2b; Interferon α-n1; Interferon α-n3; Interferon β-Ia Interferon γ-Ib; Isopropylplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprorelin acetate; Riazol hydrochloride; Lometraxo sodium; Lomustine; Loxoanthraquinone hydrochloride; Masrophenone; Metformin; Nitrogen mustard hydrochloride; Medroxyprogesterone acetate; Meropenem; Minoril; Mercaptopurine; Methotrexate; Methotrexate sodium; Chlorpheniramine; Metotep; Mildolipid; Mitocarcin; Mitocarcin Mitox; Mitoxazole; Mitoxicin; Mitosperidone; Mitosperidone; Mitoxanthraquinone hydrochloride; Mycophenolic acid; Nocodazole; Nogamycin; Omaplatin; Oxysulphurine; Paclitaxel; Pegaspargase; Peliberamide; Pendimethicone sulfate; Peprofenamide; Piperabromide; Piperabromide; Porcamycin; Promethene; Porphyrom sodium; Pofibromycin; Prenimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofuranin; Lipoadenosine; Rogulam; Safungo; Safungo in Hydrochloride; Semustine; Cimetrazine; Sodium Phosphate Aspartate; Sparmycin; Spiramustine; Spiramycin; Streptomycin; Strepzotocin; Sulfonamide; Talimycin; Ticogalan Sodium; Tegafur; Teloanthraquinone Hydrochloride; Temopofen; Teniposide; Tirocizolone; Testrolide; Thiomipril; Thioguanine; Thiotepa; Thiazole Carboxyl Nucleoside; Tirazamine; Torrecate Citrate Miphen; Tritolon acetate; Tricerebrolysin phosphate; Trimethotraxa; Trimethotraxa gluconate; Triptorelin; Tobaccochloride hydrochloride; Uramustin; Uretipa; Vapotetide; Vertepofen; Vincristine sulfate; Vincristine sulfate; Vincristine sulfate; Vincristine sulfate; Vinpicidin sulfate; Vincristine sulfate; Vincristyl sulfate; Vinrocin sulfate; Vinorelbine tartrate; Vinrocin sulfate; Vorticillium sulfate; Zorniplatin; Netostatin; Zorrabicin hydrochloride. Other anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; ararubicin; acylfulvin; adenocyclopentol; adoretin; adeleukin; ALL-TK antagonists; hexamethylmelamine; amimastatin; amidox; amifostine; aminolevulinic acid; ararubicin; acridine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antraloxetine; anti-dorsalizing morphogenetic protein-1 (ADMM-1). Phogenetic protein-1; anti-androgen substances, prostate cancer; anti-estrogenic drugs; antitumor ketones; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene regulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atametastine; amustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; berry gibberellin II I derivatives; balanol; balanol; BCR/ABL antagonists; benzochlorins; benzoyl astrocytin; β-lactam derivatives; β-alethine; β-clamycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisaziridinylspermine; bisaziridinylspermine; bistratene A; breflate; bromipridine; bdotimetamine; sulfoxide imine; calcipotriol; carvacrol C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole Carboxyamine triazole; CaRest M3; CARN 700; chondrogenic inhibitor; carboxylic acid; casein kinase inhibitor (ICOS); succinylamine; cephalosporin B; cetroprost; chlorlns; chloroquine sulfonamide; cis-porphyrin; cladribine; clomiphene analogue; clotrimazole; colismmycin A; colismmycin B; cobustatin A4; cobustatin analogue; conagenin; crambescidin 816; clanetouracil; cryptophycin 8; cryptophycin A derivative; cur acin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine phospholipids; cytolysin; hexestrol phosphate; dacizumab; decitabine; dehydrometasine B; dilorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diazoquinone; metasine B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dihydropaclitaxel; dioxamycin; diphenylspiromostin; docetaxel; docosanol; Dolastron; Deoxyfluorouridine; Droloxifen; Drocannabinol; Duocarmycin SA; Ebuselenium; Ecomustine; Edifosine; Ezocurumab; Eflunomide; Elemene; Ethiritidine; Epirubicin; Eprepitant; Estrogen-mustine analogs; Estrogen agonists; Estrogen antagonists; Ethidazole; Etoposide phosphate; Exemestane; Fazodazole; Fazalabin; Feniveryl Aamine; Filgrass; Finasteride; Flavopiridol; Flucallastine; Fluasterone; Fludarabine; Fludaunorunicin hydrochloride; Folican; Formistane; Forstracin; Formustine Gadolinium texaphyrin; Gallium nitrate; Galotetabine; Ganirelix; Gelatinase inhibitor; Gemcitabine; Glutathione inhibitor; Hepsulfam; Heregulin; Hexamethylene bisacetamide; Hypericin; Ibandronic acid; Idarubicin; Edocifene; Icaramone; Imofocin; Ilomasta; Imidazolidine; Imidazolidine; Imiquimod; Immunostimulatory peptides; Insulin-like growth factor-1 receptor inhibitors; Interferon agonists; Interferon; Interleukin; Iodobenzylguanidine; Iodoxorubicin; 4-Sweet potato picrol; Iloprapine; Is obengazole; isohomohalicondrin B; isotasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; levofloxacin; mushroom sulfate polysaccharide; letolstatin; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprorelin + estrogen + progesterone; leuprorelin; levamisole; riazol; linear polyamine analog; lipophilic disaccharide peptide; lipophilic platinum compound; lissoclinamide 7; lobaplatin; earthworm phospholipids; lometrozole; chlordamine; loxoanthraquinone Lovastatin; Loxoribine; Letotecan; Lutetium texaphyrin; Lysofylline; Cleavage peptide; Metformin; Nystatin A; Mamasrophenone; Maspin; Matrix dissolution factor inhibitor; Matrix metalloproteinase inhibitor; Merbarone; Metformin; Methionase; Metoclopramide; MIF inhibitor; Mifepristone; Mitefoxin; Milistatin; Mismatched double-stranded RNA; Mitoguanidine; Dibromoceramide; Mitomycin analogue; Mitonaphthylamine; Mitotoxin fibroblast growth factor-saponin; Mitoantrone; Motrin Farotine; Moraxetine; Monoclonal antibody, Human chorionic gonadotropin; Mycobacterium monophosphate A+ cell wall SK; Mopiperol; Multidrug resistance gene inhibitor; Therapy based on multiple tumor suppressor gene 1; Mustard anticancer drug; Mycaperoxide B; Mycobacterium cell wall extract; Myriaporone; N-acetyl-B-naphthylamine; N-substituted benzamide; Nafarelin; Naretepen; Naloxone + Pentazocine; Napavin; Naphterpin; Natosine; Nedaplatin; Nemorubicin; Neridonic acid; Neutral endopeptidase; Nilumet; Nisamycin; Nitric oxide regulator Nitrogen oxide antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; ondansetron; ondansetron; oracin; oral cytokine inducers; oxaunomycin; oxatatropine; oxaliplatin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronate; ginsenosides; parabactin; parazeptin; pegaspargase; pegaspargase; sodium pentosulfonate; pentostatin; pentrozole; perfluorobromoethane; Pesophosphatamide; Perilla frutescens alcohol; Phenazinomycin; Phenyl acetate; Phosphatase inhibitors; Hemolytic streptococcal preparations (picibanil); Pilocarpine hydrochloride; Pirarubicin; Pyrithione; Placetin A; Placetin B; Platinum activator inhibitors; Platinum complexes; Platinum compounds; Platinum-triamine complexes; Porphyrin sodium; Pofibromycin; Prednisone; Propyldiacrylone; Prostaglandin J2; Proteasome inhibitors; Protein A-based immunomodulators; Protein kinase C inhibitors; Protein kinase C inhibitors; Microalgal; Protein tyrosine phosphatase inhibitors; Purine nucleoside phosphorylase inhibitors; Red pigments; pyrazoline acridine; pyridoxylated hemoglobin polyoxyethylene conjugate; RAF antagonists; raltitrexed; ramosetron; ras farnesyltransferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated retelliptine; rhenium etidronate Re 186; rhizomycin; ribosomalases; RII retinoic acid; roglutamine; roxithromycin; romotide; roquimetac; rubiginone B1; ru boxyl; Safingo; Saintopin; SarCNU; Sarcophytol A; Saxaglastine; Sdi 1 mimic; Semustine; Senescence-derived inhibitor 1; Right-signal oligonucleotide; Signal transduction inhibitor; Signal transduction modulator; Single-chain antigen-binding protein; Cizonan; Sobuzosen; Borcarnazone; Sodium phenylacetate; Solvent; Somatostatin-binding protein; Sonamamine; Phosphatidylcholine; Spiramycin D; Spiromustine; Senna; Spongistatin 1; Squalamine; Stem cell inhibitor; Stem cell division inhibitor; Stipiamide; Matrix lysin inhibitor; Sulfinosine; Potent vasoactive intestinal Peptide antagonists; suradista; suramin; suramin; synthetic glucosamine; tamustine; tamoxifen methyl iodide; taurolimustine; tazarotene; tecogallan sodium; tegafur; tellurapyrylium; telomere-terminal transferase inhibitors; temopofol; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; bacterial embryo extract; thiacloprid; thrombopoietin; thrombopoietin mimics; thymosin; thymopoietin receptor agonists; thymic trehalone; thyroid-stimulating hormone; ethylporphyrin tin; tetrazolomide; dichlorodicyclopentadiene; topsentin Toremifene; pluripotent stem cell factor; translation inhibitors; retinoic acid; triacetyluridine; tricerebroside; trimethotraxate; triptorelin; tropisetron; tolostaniel; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitors; urokinase receptor antagonists; valproic acid; variolin B; vector systems, erythrocyte gene therapy; veraracetasol; verrucine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorticol; zanoterol; zebuline; zilascorb; and netsistatin ester. Other preferred anticancer drugs are 5-fluorouracil and leucovorin. These two agents are particularly useful when used in approaches employing thalidomide and topoisomerase inhibitors.

preparation

In a preferred embodiment, the present invention relates to a method for preparing a lyophilized composition containing arsenic, or lyophilized _arsenic trioxide ( _As₂O₃ ) herein, the method comprising:

a. Dissolving _{As₂O₃ powder} in water in a container, wherein the dissolution is performed in the following order:

i. Add an alkalizing agent to the container to a pH of approximately 12 or higher.

ii. Add acid to the container to adjust the pH to approximately 7 to approximately 8.

iii. Add a surfactant to the container, and

iv. Add water to the container to produce _{an As₂O₃} solution; and

b. Freeze-dry _{the As₂O₃} solution.

In one embodiment of the above method, the alkalizing agent comprises sodium hydroxide (NaOH) or sodium _carbonate ( _Na₂CO₃ ). In another embodiment, the amount of the alkalizing agent added is about 10% to about 100% of the amount of _{As₂O₃ powder} . In other words, the amount of the alkalizing agent added is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, ₂₉ , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, of the amount of _As₂O₃ powder. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100%. In another embodiment, the amount of the alkalizing agent added is any number within the range defined by any two of the above figures and includes both of the above figures.

In another embodiment, the acid comprises hydrochloric acid (HCl). In one embodiment, the HCl is about 6M HCl. In one embodiment, acid is added to the container to adjust the pH to about 7.2. In one embodiment, water is added to the container after steps a)i), a)ii), a)iii), and/or a)iv).

In one embodiment of the invention, the surfactant comprises sodium lauryl sulfate; Tween 80; β-cyclodextrin; poloxamer; and tocopheryl polyethylene glycol succinate (TPGS). In one embodiment of the invention, the surfactant is added to about 0.5% v/v to about 4.0% v/v, but not exceeding about 50% _As₂O₃ concentration _.

In one embodiment of the invention, the freeze-drying step includes freezing _{the As₂O₃} solution to produce a _{frozen As₂O₃} product and drying _the frozen _As₂O₃ product to produce the freeze- _{dried As₂O₃} or a freeze-dried composition containing arsenic. In another embodiment, the freezing step includes freezing _{the As₂O₃} solution at about -40˚C. In another embodiment, the _As₂O₃ solution is frozen at about _-40˚C for at least about 6 hours. In one embodiment, the drying step includes applying heat and vacuum to _{the As₂O₃} solid. For example, the drying steps include heating the _As₂O₃ product at about -30˚C and about 800 mTorr for about 60 minutes; heating the _As₂O₃ product at -20˚C and 500 mTorr for about 120 minutes; heating the _As₂O₃ product at about -5˚C and about 500 _mTorr for about 120 minutes; heating the _As₂O₃ product at about 10˚C and about 500 _mTorr for about 60 minutes; and heating _{the As₂O₃} product at about 25˚C and about 500 _mTorr for about 180 minutes.

In another embodiment, the drying step includes heating the _As₂O₃ product to approximately -30˚C and approximately 800 mTorr for approximately 60 minutes and maintaining this temperature at approximately -30˚C and approximately 800 mTorr for approximately 60 minutes; heating _{the As₂O₃} product to approximately -20˚C and approximately 500 mTorr for approximately 60 minutes and maintaining this temperature at approximately -20˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃ product} to approximately -5˚C and approximately 500 mTorr for approximately 300 minutes and maintaining _this temperature at approximately -5˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃} product to approximately 10˚C and approximately 500 mTorr for approximately 120 minutes and maintaining this temperature at approximately 10˚C and approximately 500 mTorr for approximately 60 minutes; and drying the _As₂O₃ product for approximately 60 minutes... _3. The product was heated to about 25˚C and about 500 mTorr and held at about 25˚C and about 500 mTorr for about 180 minutes.

In one embodiment, the drying step further includes heating the _As₂O₃ product at about 25˚C and about 500 mTorr for about 120 minutes after heating _{the As₂O₃} product at about 25˚C and about 500 mTorr _for about 180 minutes.

The present invention also relates to a method for preparing an oral formulation containing LCCA, the method comprising:

c. Dissolving _{As₂O₃ powder} in water in a container, wherein the dissolution is performed in the following order:

i. Add an alkalizing agent to the container to a pH of approximately 12 or higher.

ii. Add acid to the container to adjust the pH to approximately 7 to approximately 8.

iii. Add a surfactant to the container, and

iv. and adding water to the container to produce _{an As₂O₃} solution; and

d. Freeze-dry _{the As₂O₃} solution to produce a lyophilized product;

e. Sieve the lyophilized material to produce LCCA powder;

f. Add filler to the LCCA powder;

g. Add one or more lubricants to the LCCA powder to produce the oral formulation containing LCCA.

In one embodiment of the above method, the alkalizing agent comprises sodium hydroxide (NaOH) or sodium _carbonate ( _Na₂CO₃ ). In another embodiment, the amount of the alkalizing agent added is from about 10% to about 100% of the amount of _{As₂O₃ powder} . In another embodiment, the acid comprises hydrochloric acid (HCl). In one embodiment, the HCl is about 6M HCl. In one embodiment, acid is added to the container to adjust the pH to about 7.2. In one embodiment, water is added to the container after steps c)i), c)ii), c)iii), and/or c)iv).

In one embodiment of the invention, the surfactant comprises sodium lauryl sulfate; Tween 80; β-cyclodextrin; poloxamer; and tocopheryl polyethylene glycol succinate (TPGS). In one embodiment of the invention, the surfactant is added to about 0.5% v/v to about 4.0% v/v, but not exceeding about 50% _As₂O₃ concentration _.

In one embodiment of the invention, the freeze-drying step includes freezing _{the As₂O₃} solution to produce a frozen _{As₂O₃ product} and drying the frozen _{As₂O₃ product} to produce the freeze-dried _As₂O₃ . In another embodiment, the freezing step includes freezing _{the As₂O₃} solution at about -40˚C. In yet another embodiment, _{the As₂O₃} solution is frozen at about -40˚C for at least about 6 hours. In one embodiment, the drying step includes applying heat and vacuum to _{the As₂O₃ product solid} . For example, the drying steps include heating _{the As₂O₃} product at about -30˚C and about 800 mTorr for about 60 minutes; heating the _As₂O₃ product at -20˚C and 500 mTorr for about 120 minutes; heating _{the As₂O₃} product at about -5˚C and about 500 mTorr for about 120 minutes; heating _{the As₂O₃} product at about 10˚C and about 500 _mTorr for about 60 minutes; and heating the _As₂O₃ product at about 25˚C and about 500 _mTorr for about 180 minutes.

In another embodiment, the drying step includes heating the _As₂O₃ product to approximately -30˚C and approximately 800 mTorr for approximately 60 minutes and maintaining this temperature at approximately -30˚C and approximately 800 mTorr for approximately 60 minutes; heating _{the As₂O₃} product to approximately -20˚C and approximately 500 mTorr for approximately 60 minutes and maintaining this temperature at approximately -20˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃ product} to approximately -5˚C and approximately 500 mTorr for approximately 300 minutes and maintaining _this temperature at approximately -5˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃} product to approximately 10˚C and approximately 500 mTorr for approximately 120 minutes and maintaining this temperature at approximately 10˚C and approximately 500 mTorr for approximately 60 minutes; and drying the _As₂O₃ product for approximately 60 minutes... _3. The product was heated to about 25˚C and about 500 mTorr and held at about 25˚C and about 500 mTorr for about 180 minutes.

In one embodiment, the drying step further includes heating the _As₂O₃ product at about 25˚C and about 500 mTorr for about 120 minutes after heating _{the As₂O₃} product at about 25˚C and about 500 mTorr _for about 180 minutes.

In one embodiment of the invention described above, the filler comprises mannitol. In another embodiment, the one or more lubricants comprise talc and/or magnesium stearate.

In another step, the invention includes filling a capsule with the oral formulation.

The present invention also relates to a pharmaceutical composition suitable for oral administration in a solid dosage form, the composition comprising lyophilized arsenic trioxide (also known as lyophilized arsenic-containing composition), at least one filler, and at least one lubricant.

In one embodiment, the above-mentioned pharmaceutical composition is prepared by a method comprising the following steps:

h. Dissolving _{As₂O₃ powder} in water in a container, wherein the dissolution is performed in the following order:

v. Add an alkalizing agent to the container to a pH of approximately 12 or higher.

vi. Add acid to the container to adjust the pH to approximately 7 to approximately 8.

vii. Add a surfactant to the container, and

viii. and adding water to the container to produce _{an As₂O₃} solution; and

i. Lyophilize _{the As₂O₃} solution to produce a lyophilized product;

j. Sieve the lyophilized material to produce LCCA powder;

k. Add filler to the LCCA powder;

1. Adding one or more lubricants to the LCCA powder to produce an oral formulation of _{the As₂O₃} .

In one embodiment, the average particle size distribution D(90) of the lyophilized material or LCCA is about 2 to 10 micrometers. In other words, the particle size distribution D(90) of the lyophilized material, measured in micrometers, is 2, 3, 4, 5, 6, 7, 8, 9, and 10 micrometers or a number within a range defined by any two of these numbers.

In one embodiment, the average particle size distribution D(10) of the lyophilized material or LCCA is about 0.2 micrometers to 3 micrometers. In other words, the particle size distribution D(10) of the lyophilized material, measured in micrometers, is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1.0, 1.1, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 micrometers or a number within a range defined by any two of these numbers.

In one embodiment, the average particle size distribution D(50) of the lyophilized product or LCCA is about 0.5 micrometers to 4 micrometers. In other words, the particle size distribution D(50) of the lyophilized product, measured in micrometers, is 0.5, 0.6, 0.7, 0.9, 1.0, 1.1, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0 micrometers or a number within a range defined by any two of these numbers.

In one embodiment, the particle size D(90) of the lyophilized product or LCCA is 1/10 and at most 1/50 of the API. In other words, the D(90) of the lyophilized product is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50 of the API's D(90).

In one embodiment, the surface area of the lyophilized product or LCCA is in the range of about 0.5 m²/g to about 5 m²/g. In other words, the surface area can be 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0 m²/g or a number within the range defined by any two figures herein.

In one embodiment, the surface area of the lyophilized material or LCCA, measured by the BET method, is 2 to 80 times the surface area of the API powder. In other words, the BET surface area of the lyophilized product is 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, and 80 times that of the API powder.

In one embodiment, the lyophilized product or LCCA is soluble in cold water. In another embodiment, the solubility of the lyophilized product or LCCA in cold water (room temperature) is about 2 to about 30 times that of the API powder. In other words, the solubility of the lyophilized product or LCCA in cold water (room temperature) is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times that of the API powder. In one embodiment, the lyophilized product or LCCA is soluble in alcohol. In another embodiment, the solubility of the lyophilized product or LCCA in alcohol is about 2 to 30 times that of the API powder. In other words, the solubility of the lyophilized product or LCCA in alcohol is approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 times that of the API powder.

Scanning electron microscopy images revealed that the lyophilized material or LCCA particles exhibited porous properties, which may contribute to the higher solubility, higher surface area, and smaller average particle size of the lyophilized material.

In one embodiment of the above pharmaceutical composition, the alkalizing agent comprises sodium hydroxide (NaOH) or sodium _carbonate ( _Na₂CO₃ ). In another embodiment, the amount of the alkalizing agent added is from about 10% to about 100% of the amount of _{As₂O₃ powder} . In another embodiment, the acid comprises hydrochloric acid (HCl). In one embodiment, the HCl is about 6M HCl. In one embodiment, acid is added to the container to adjust the pH to about 7.2. In one embodiment, water is added to the container after steps h)i), h)ii), h)iii), and/or h)iv).

In one embodiment of the invention, the surfactant comprises sodium lauryl sulfate; Tween 80; β-cyclodextrin; poloxamer; and tocopheryl polyethylene glycol succinate (TPGS). In one embodiment of the invention, the surfactant is added to about 0.5% v/v to about 4.0% v/v, but not exceeding about 50% _As₂O₃ concentration _.

In one embodiment of the invention, the freeze-drying step includes freezing _{the As₂O₃} solution to produce a frozen _{As₂O₃ product} and drying the frozen _{As₂O₃ product} to produce the freeze-dried _As₂O₃ . In another embodiment, the freezing step includes freezing _{the As₂O₃} solution at about -40˚C. In yet another embodiment, the _As₂O₃ solution is frozen at about _-40˚C for at least about 6 hours. In one embodiment, the drying step includes applying heat _and vacuum to _{the As₂O₃} solid. For example, the drying steps include heating _{the As₂O₃} product at about -30˚C and about 800 mTorr for about 60 minutes; heating the _As₂O₃ product at -20˚C and 500 mTorr for about 120 minutes; heating _{the As₂O₃} product at about -5˚C and about 500 mTorr for about 120 minutes; heating _{the As₂O₃} product at about 10˚C and about 500 _mTorr for about 60 minutes; and heating the _As₂O₃ product at about 25˚C and about 500 _mTorr for about 180 minutes. In another embodiment, the drying step includes heating the _As₂O₃ product to approximately -30˚C and approximately 800 mTorr for approximately 60 minutes and maintaining this temperature at approximately -30˚C and approximately 800 mTorr for approximately 60 minutes; heating _{the As₂O₃} product to approximately -20˚C and approximately 500 mTorr for approximately 60 minutes and maintaining this temperature at approximately -20˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃ product} to approximately -5˚C and approximately 500 mTorr for approximately 300 minutes and maintaining _this temperature at approximately -5˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃} product to approximately 10˚C and approximately 500 mTorr for approximately 120 minutes and maintaining this temperature at approximately 10˚C and approximately 500 mTorr for approximately 60 minutes; and drying the _As₂O₃ product for approximately 60 minutes... _3. The product was heated to about 25˚C and about 500 mTorr and held at about 25˚C and about 500 mTorr for about 180 minutes.

In one embodiment, the drying step further includes heating the _As₂O₃ product at about 25˚C and about 500 mTorr for about 120 minutes after heating _{the As₂O₃} product at about 25˚C and about 500 mTorr _for about 180 minutes.

In one embodiment of the invention described above, the filler comprises mannitol. In another embodiment, the one or more lubricants comprise talc and/or magnesium stearate.

In one embodiment, the pharmaceutical composition comprises lyophilized arsenic trioxide, mannitol, talc, and magnesium stearate. In another embodiment, the pharmaceutical composition is a controlled-release, orally administered solid pharmaceutical composition. In yet another embodiment, the pharmaceutical composition is encapsulated in a capsule.

In one embodiment, the capsule contains about 1 mg, 5 mg, 10 mg, and 20 mg of the above-described pharmaceutical composition.

The present invention also relates to a kit comprising the above-described pharmaceutical composition and its instructions for use.

In one embodiment, the present invention relates to a method for treating hematologic malignancies in patients in need, the method comprising the step of administering a therapeutically effective amount of the above-described pharmaceutical composition to the patient.

For example, the hematological malignancies mentioned are acute myeloid leukemia, acute non-lymphocytic leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, myelodysplastic syndrome, acute promyelocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, polycythemia vera, Hodgkin lymphoma, non-Hodgkin lymphoma, myeloma, giant cell myeloma, painless myeloma, localized myeloma, multiple myeloma, plasma cell myeloma, sclerosing myeloma, solitary myeloma, condensed multiple myeloma, non-secreting myeloma, osteosclerosing myeloma, plasma cell leukemia, solitary plasmacytoma, or extramedullary plasmacytoma.

In one embodiment, the hematological malignancy is acute promyelocytic leukemia (APL). In another embodiment of the invention, the APL is newly diagnosed APL, relapsed APL, or refractory APL.

In one embodiment, the pharmaceutical composition is administered daily. In another embodiment, the pharmaceutical composition is administered daily for 52 weeks/year. In another embodiment, the pharmaceutical composition is administered in a single dose range of about 1 mg to about 50 mg. In yet another embodiment, the pharmaceutical composition is administered in a single dose range of about 0.1 mg/kg body weight to about 0.3 mg/kg body weight.

In one embodiment of the invention, the patient has previously received or is receiving chemotherapy and/or radiation therapy. In another embodiment, the treatment method further includes administering one or more chemotherapeutic agents to the patient. The chemotherapeutic agents are administered before, after, or concurrently with the pharmaceutical composition. The invention also relates to a composition comprising LCCA or lyophilized arsenic trioxide, at least one filler, and at least one lubricant. In one embodiment, the lyophilized arsenic trioxide is prepared by a method comprising the following steps:

m. Dissolving _{As₂O₃ powder} in water in a container, wherein the dissolution is performed in the following order:

ix. Add an alkalizing agent to the container to a pH of approximately 12 or higher.

x. Add acid to the container to adjust the pH to approximately 7 to approximately 8.

xi. Add a surfactant to the container, and

xii. and adding water to the container to produce _{an As₂O₃} solution; and

The _As₂O₃ solution is freeze _- dried. In one embodiment, the alkalizing agent comprises sodium hydroxide (NaOH) or sodium _carbonate ( _Na₂CO₃ ). In another embodiment, the amount of the alkalizing agent added is from about 10% to about 100% of the amount _{of As₂O₃} . In one embodiment, the acid comprises hydrochloric acid (HCl), and preferably about 6M HCl. In another embodiment, acid is added to the container to adjust the pH to about 7.2. In another embodiment, water is added to the container after steps m)i), m)ii), m)iii) and/or m)iv).

In one embodiment, the surfactant comprises sodium lauryl sulfate; Tween 80; β-cyclodextrin; poloxamer; and tocopheryl polyethylene glycol succinate (TPGS). The surfactant is added to about 0.5% v/v to about 4.0% v/v, but not exceeding about 50% _{As₂O₃ concentration} .

freeze-dried

Freezing steps

In one embodiment, the freeze-drying step includes freezing _{the As₂O₃} solution to produce a frozen _{As₂O₃ product} and drying the frozen As₂O₃ product to produce freeze- _{dried As₂O₃ .} In one embodiment, the freeze-drying step includes freezing _{the As₂O₃} solution to at least one temperature selected from -50, -49, -48, -47, -46, -45, -44, -43, -42, -41, -40, -39, -38, -37, -36, -35, -34, -33, -32, -31, -30, 29, -28, -27, -26, -25, -24, -23, -22, -21, -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -09, -08, -07, -06, -05, -04, -03, -02, -01, and 0˚C. In another embodiment, the freezing temperature is selected from and includes a range defined by any two of the above numbers. In another embodiment, the freezing step includes freezing _{the As₂O₃} solution at about -40˚C for at least 6 hours. The freezing step may have several freezing sub-steps, with the temperature gradually decreasing. The freezing step and/or freezing sub-steps may each be from about 5 minutes to about 500 minutes, i.e., each number included in that range, for example, 6, 7, 8, 9, ..., 497, 498, 499, and 500 minutes.

Sublimation steps

In one implementation, sublimation (primary drying) or secondary drying is performed at a temperature ranging from -50°C to 35°C. In other words, sublimation can be performed at the following temperatures: -50, -49, -48, -47, -46, -45, -44, -43, -42, -41, -40, -39, -38, -37, -36, -35, -34, -33, -32, -31, -30, -29, -28, -27, -26, -25, -24, -23, -22, -21, -20, -19, -18, -17. The values are: -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35. In one embodiment, the sublimation temperature can be any temperature within a range defined by and including any two of the above numbers. In one embodiment, more than one sublimation temperature is used.

Secondary drying can be carried out at higher temperatures, such as between 35°C and 75°C.

Single or double drying at different temperatures can be carried out for periods ranging from 5 minutes to 500 minutes, i.e., inclusivity, such as 6, 7, 8, 9, ..., 497, 498, 499, and 500 minutes. Double drying can last for longer periods, such as up to 1000 minutes.

Vacuum applications

Vacuum can be applied simultaneously with or independently of drying. Vacuum can be applied during drying, either while it continues from one temperature to a second (higher) temperature or while it is maintained at a temperature. Vacuum can be applied during multiple drying steps or only during some drying steps. Vacuum application can be alternated with drying. The vacuum applied during the drying steps can range from about 300 mTorr to about 1000 mTorr. In one embodiment, the vacuum is 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 50, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 68 0, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, and 1000 millitors, or numbers within a range defined by any two numbers herein, and including a vacuum of said numbers.

The drying step includes applying heat and vacuum to _{the As₂O₃} product. In one embodiment, the drying step includes heating the _As₂O₃ product at about -30˚C and about 800 mTorr for about 60 minutes; heating _{the As₂O₃} product at about -20˚C and about 500 mTorr for about 120 minutes; heating the _As₂O₃ product at about -5˚C and about 500 _mTorr for about 120 minutes; heating the _As₂O₃ product at about 10˚C and about 500 _mTorr for about 60 minutes; and heating _{the As₂O₃ product} at about 25˚C and about 500 _mTorr for about 180 minutes. In another embodiment, the drying step includes heating _{the As₂O₃} product to approximately -30˚C and approximately 800 mTorr for approximately 60 minutes and maintaining this temperature at approximately -30˚C and approximately 800 mTorr for approximately 60 minutes; heating the _As₂O₃ product to approximately -20˚C and approximately 500 mTorr for approximately 60 minutes and maintaining this temperature at approximately -20˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃ product} to approximately -5˚C and approximately 500 mTorr for approximately 300 minutes and maintaining _this temperature at approximately -5˚C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃} product to approximately 10˚C and approximately 500 mTorr for approximately 120 minutes and maintaining this temperature at approximately 10˚C and approximately 500 mTorr for approximately 60 minutes; and drying the _As₂O₃ product for approximately 60 minutes... _3. The product was heated to about 25˚C and about 500 mTorr and held at about 25˚C and about 500 mTorr for about 180 minutes.

In another embodiment, after heating the _As₂O₃ product at about 25˚C and about 500 _mTorr for about 180 minutes, the _As₂O₃ product is then heated at about 25˚C and about 500 _mTorr for about 120 minutes.

In one embodiment of the composition, the filler comprises mannitol, and the one or more lubricants comprise talc and/or magnesium stearate.

The present invention also relates to the above composition comprising freeze-dried arsenic trioxide, mannitol, talc and magnesium stearate.

In one embodiment, LCCA is used in a therapeutically effective amount for the compositions and methods described herein.

experiment

The reference product was _{injectable As₂O₃} available in injectable form with a strength of 10 mg/10 mL. The test product was developed in solid form for oral administration. Test product development began with 1 mg, 5 mg, 10 mg, and 20 mg capsules. Lyophilization techniques were selected to modify the physical and/or chemical properties of arsenic trioxide, including reducing particle size, increasing surface area, and generating other morphological characteristics that may contribute to its solubility, dissolution of pharmaceutical formulations, and/or bioavailability. Suitable excipients were selected and their concentrations optimized to achieve a smooth lyophilization process that improves the solubility of arsenic trioxide. Satisfactory laboratory-scale stability data were found at 40 °C/75% RH for 15 days.

Reference product—injectable form—arsenic trioxide

According to the package insert for injectable arsenic trioxide, arsenic trioxide induces characteristic morphological changes and DNA breaks in NB4 human promyelocytic leukemia cells in vitro, leading to apoptosis. Arsenic trioxide also causes damage or degradation of the fusion protein PML/RAR-α.

Arsenic trioxide in solution hydrolyzes into its pharmacologically active substance, arsenous acid (AsIII). In addition to arsenic acid (Asv) (a product of AsIII oxidation), monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) are the main pentavalent metabolites formed during metabolism.

Following a once-daily dose of 0.15 mg/kg for 5 days/week, the pharmacokinetics of arsenic substances ([AsIII], [Asv], [MMAV], [DMAV]) were determined in 6 patients with acute arsenic pneumonia (APL). Systemic exposure (AUC) appeared linear throughout the total single-dose range of 7 to 32 mg (administered at 0.15 mg/kg). Peak plasma concentrations of arsenite (AsIII), the major active arsenic substance, were reached at the end of the infusion (2 hours). Plasma concentrations of AsIII decreased in a biphasic manner, with a mean elimination half-life of 10 to 14 hours, characterized by an initial rapid distribution phase followed by a slower terminal elimination phase. Daily exposure to Asm (mean AUC 0–24) was 194 ng-hr/mL on day 1 of cycle 1 (n=5) and 332 ng-hr/mL on day 25 of cycle 1 (n=6), representing approximately a 2-fold cumulative effect.

The main pentavalent metabolites MMAV and DMAV appear slowly in plasma (approximately 10–24 hours after the first administration of arsenic trioxide), but accumulate more than AsIII after multiple administrations due to their longer half-lives. The mean estimated terminal elimination half-lives for MMAV and DMAV are 32 hours and 72 hours, respectively. The approximate accumulation range after multiple administrations is 1.4 to 8 times compared to a single dose. Asv is present in plasma only at relatively low levels.

distributed

The distribution volume (Vss) of AsIII was relatively large (average 562 L, N = 10), indicating that ^AsIII is widely distributed throughout the body. Vss also depends on body weight and increases with increasing body weight.

metabolism

Most As ^III is distributed to tissues where it is methylated by methyltransferases, primarily in the liver, into its less cytotoxic metabolites—monomethylarsonic acid (MMA ^V ) and dimethylarsinomethyl acid (DMA ^V ). The metabolism of arsenic trioxide also involves the oxidation of As ^III to As ^V , which can occur in many tissues via enzymatic or non-enzymatic processes. Following administration of arsenic trioxide, only relatively low levels of As ^V are present in plasma.

excretion

Approximately 15% of the injected dose of arsenic trioxide is excreted in the urine as unaltered As ^III . Methylated metabolites of As ^III (MMAV, DMAV) are primarily excreted in the urine. The total clearance of As ^III is 49 L/h, and the renal clearance is 9 L/h. Clearance is independent of body weight or the dose administered in the range of 7–32 mg.

_{As₂O₃ under} development

The active pharmaceutical ingredient used in these experiments, arsenic trioxide, was purchased as a white powder from Sigma-Aldrich with a molecular weight of 197.8 g/mol and the general chemical _{formula As₂O₃} .

For the comparative examples, injectable arsenic trioxide was purchased as Arsenox® (manufactured by Naprod Life Sciences, a privately held limited company in Intaspharma, India). The arsenic trioxide injection was indicated for inducing remission and consolidation in patients with acute promyelocytic leukemia (APL). It was obtained in vials containing 10 mg/10 mL (1 mg/mL) ampoules.

Since the reference product is available in injectable form and the test product is developed in a solid form for oral use, the stability of the reference product is not investigated.

No drug excipient studies were conducted. Solubility studies were performed using different surfactants and solvents as part of the development process.

Formulation process development

Arsenic trioxide available in injectable form and developmental powder for oral administration were tested. _{As₂O₃ powder} was lyophilized to alter the physical and/or chemical properties of arsenic trioxide, including reducing particle size, increasing surface area, and producing other morphological characteristics that may contribute to arsenic dissolution and/or bioavailability.

Freeze-drying, also known as lyophilization, involves removing purified water from liquid, paste, or solid forms using a cycle of freezing and vacuum evaporation without melting the ice. Water (ice) in a solid state exposed to very low pressures, such as a vacuum, sublimates or transitions directly from a solid to a gaseous state. Purified water vapor (or other solvents) is frozen without being trapped by a condenser or cold trap. This technique preserves the volume, appearance, and properties of the processed product.

Generally, a freeze-drying cycle includes the following steps:

1. Freezing: Freezing the product. This provides the necessary conditions for low-temperature drying.

2. Vacuum: After freezing, the product is placed under a vacuum or low pressure. This prevents the frozen solvent in the product from evaporating through the liquid phase; this process is called sublimation.

3. Heating: Applying heat to the frozen product to accelerate sublimation.

4. Condensation: Cryogenic condensation removes the evaporated solvent from the vacuum chamber by converting it back into a solid. This completes the separation process.

The first step in the freeze-drying process is to freeze the product to solidify all its purified water molecules. Once frozen, the product is placed in a vacuum and gradually heated without melting it. This process, called sublimation, converts the ice directly into purified water vapor without first passing through a liquid state. The purified water vapor emitted by the product during the sublimation stage condenses into ice in a collection trap called a condenser within the vacuum chamber of the freeze dryer.

Compared to other drying and preservation technologies, freeze-drying offers numerous advantages. For example, freeze-drying preserves the quality of foods/biochemicals and chemical reagents because they remain at temperatures below their freezing point during sublimation. Freeze-drying is particularly important when processing lactic acid bacteria, as these products are highly susceptible to heat. Freeze-dried foods/biochemicals and chemical reagents can often be stored without refrigeration, significantly reducing storage and transportation costs. Freeze-drying significantly reduces weight, making the product easier to transport. For instance, many foods contain up to 90% purified water. These foods become 1/10 lighter after freeze-drying. Because they are porous, most freeze-dried products can be easily rehydrated. Freeze-drying does not significantly reduce volume; therefore, purified water rapidly regains its position within the molecular structure of the food/biochemical and chemical reagent.

Based on literature and methods, different solubilizers, such as sodium lauryl sulfate, Tween 80®, poloxamer, and pH adjusters such as sodium hydroxide, sodium carbonate, sodium alginate, and hydrochloric acid, were selected to improve solubility for initial development. The dissolution medium used was a 900 ml paddle (mixing) of 0.1 N HCl running at 100 rpm for initial activity development. The reference product was available at a single strength (10 mg/10 mL), and the test product was started at different strengths for oral administration according to dose-weight ratios. Since the reference product was available at a 10 mg strength, formulation development began at a 10 mg strength.

Experiment number L040/1/001

The purpose of this experiment is to study the solubility of arsenic trioxide using sodium hydroxide as a pH adjuster, surfactants such as sodium lauryl sulfate and Tween 80®, and different solvents such as isopropanol and ethanol.

In the first step, under stirring, 4 g of arsenic trioxide was added to 600 mL of purified water in a container. Then, approximately 35 mL of 3M NaOH (dissolved in 12 g of NaOH in 100 mL of purified water) was added dropwise over 15 to 30 minutes with magnetic stirring at 1000 rpm until a clear solution was formed. 1 L of purified water was added to this solution. The pH was measured to be 12.7. Approximately 16.5 mL of 6M HCl (added to 105.6 mL of 200 mL of purified water) was used to adjust the pH to a target of 7 to 8 units. A pH of approximately 7.2 was observed. Purified water was added to bring the solution to a final volume of 2 L. The pH remained constant at 7.3.

Table 1 serial number Element L040/1/001A L040/1/001A mg/unit Quantity/Batch 1. Arsenic trioxide 10 4 g 2. 3M NaOH 10.5 35 mL 3. 6M HCl Qs* 16.5 mL 4. Purified water Qs* Up to 2000 mL

*Qs - Sufficient quantity.

Experiment number L040/1/001B

The purpose of this experiment was to study the solubility of arsenic trioxide using sodium lauryl sulfate. In the first step, 50 g of purified water was added to a glass beaker, and 0.5 g of sodium lauryl sulfate was added while stirring until a clear solution was formed. Next, 5 g _{of As₂O₃} was added to the solution while stirring. Even after stirring for 30 minutes, _As₂O₃ did not dissolve in the solution. In the next step, 30 g of purified water was added to determine _{whether As₂O₃ would} dissolve. No solubility was observed. Even after adding a further 2 g of sodium lauryl sulfate, no solubility was observed after stirring for 30 minutes. Two batches were prepared: L040/1/001B1 and L040/1/001B2. The data are reported in Table 2 below.

Table 2

*Qs - Sufficient quantity.

Experiment number L040/1/001D

The purpose of this experiment was to improve the solubility of arsenic trioxide using cyclodextrin. In the first step, 50 g of purified water was added to a glass beaker, and 0.5 g of cyclodextrin was added while stirring until a clear solution was formed. Then, 5 g _{of As₂O₃} was added to the solution. Even after stirring for 30 minutes, _As₂O₃ remained insoluble. Another 30 g of purified water was added to the solution. _{The As₂O₃} did not dissolve. Even after adding _a further 2 g of cyclodextrin and stirring for another 30 minutes, _{the As₂O₃} still did not dissolve. Table 3 summarizes the data from this experiment.

Table 3 serial number Element L040/1/001D1 L040/1/001D1 L040/1/001D2 L040/1/001D2 mg/unit Quantity/batch (g) mg/unit Quantity/batch (g) 1. Arsenic trioxide 10 5 10 5 2. Cyclodextrin 1 0.5 5 2.5 3. Purified water Qs 80 Qs 80

*Qs - Sufficient quantity.

Experiment number L040/1/001E

The purpose of this experiment was to improve the solubility of arsenic trioxide using Tween 80®. In the first step, 50 g of purified water was added to a glass beaker, and 0.5 g of Tween 80® was added while stirring until a clear solution was formed. Then, 5 g _{of As₂O₃} was added to the solution while stirring. Even after stirring for 30 minutes, _{the As₂O₃} remained undissolved. Another 30 g of purified water was added to the solution, but _{the As₂O₃} still did not dissolve. Even after adding another 2 g of Tween 80®, _{the As₂O₃} failed to dissolve (even after stirring for 30 minutes).

Table 4 serial number Element L040/1/001E1 L040/1/001E1 L040/1/001E2 L040/1/001E2 mg/unit Quantity/batch (g) mg/unit Quantity/batch (g) 1. Arsenic trioxide 10 5 10 5 2. Tween 80® 1 0.5 5 2.5 3. Purified water Qs 80 Qs 80

*Qs - Sufficient quantity.

Experiment number L040/1/001F

The purpose of these experiments is to study the solubility _{of As₂O₃} using various solvents.

Isopropanol

In a beaker, 1.5 g _{of As₂O₃} was added to 50 mL of isopropanol. Even after stirring for 30 minutes, _{the As₂O₃} did not dissolve. Even after adding another 30 mL of isopropanol and stirring for another 30 minutes, the drug still did not dissolve.

ethanol

In a beaker, 1.5 g of the drug _As₂O₃ was added to 50 mL of _ethanol . The drug did not dissolve after stirring for 30 minutes. Even after adding another 30 mL of ethanol and stirring for another 30 minutes, the drug still did not dissolve.

Therefore, the above series of experiments showed that only the L040/1/001A composition formed a clear solution.

Experiment number L040/1/002

The purpose of this experiment is to assess the exact amount of sodium hydroxide required to dissolve arsenic trioxide and to evaluate the effect of pH on the solubility of arsenic trioxide using different excipients.

Experiment number L040/1/002A—Amount of NaOH used to dissolve arsenic trioxide

30 mL of purified water was added to a glass beaker, and the pH was measured to be between 6.7 and 7.0. 200 mg _{of As₂O₃} was added to the solution with stirring. The pH was observed to be approximately 5.6. _{The As₂O₃} particles were observed to float in the solution. 1N NaOH solution (4 g of sodium hydroxide pellets dissolved in 100 mL of purified water) was added dropwise to the solution. Even after adding 0.6 mL of 1N NaOH and stirring for 10 minutes, the drug remained undissolved. The pH was measured to be approximately 9.21. An additional 1.4 mL of 1N NaOH was added with stirring. The drug remained undissolved, and the pH was 12.21. After subsequently adding 1.6 mL of NaOH with stirring, the drug was observed to dissolve at a pH of 12.37. An additional 200 mg _{of As₂O₃} was added to the solution, but it did not dissolve (the pH was found to be 12.27). Adding 2 mL of 1N NaOH with stirring revealed that the drug dissolved at pH 12.33. Adding an additional 600 mg of the drug showed that it was insoluble at pH 12.27. However, when 6.9 mL of 1N NaOH was gradually added, the drug dissolved at pH 12.39. Therefore, 5 mg of sodium hydroxide is required to dissolve 10 mg of arsenic trioxide (by maintaining a constant volume of 30 mL of purified water).

Table 5 Element quantity mg/unit Arsenic trioxide 1000 mg 10 mg NaOH 12.5 mL (500 mg NaOH) 5 mg Purified water 30 mL Qs*

*Qs - Sufficient quantity.

Experiment number L040/1/002B

The purpose of this experiment was to evaluate the pH-dependent solubility of arsenic trioxide using L-arginine (pH approximately 4). In a beaker, 200 mg of L-arginine was dissolved in 100 mL of purified water (2 mg/mL solution) with continuous stirring. From this solution, 50 mL was used to dissolve 50 mg _{of As₂O₃} . However, no dissolution was observed; note that the pH was 4.94.

Table 6 Element quantity mg/unit Arsenic trioxide 50 mg 10 mg L-arginine 100 mg 20 mg Purified water 50 mL Qs*

*Qs - Sufficient quantity.

Experiment number L040/1/002D

The purpose of this experiment was to assess the pH-dependent solubility of arsenic trioxide using sodium bicarbonate (pH approximately 8). 200 mg of sodium bicarbonate was dissolved in 100 mL of purified water (2 mg/mL solution). 50 mL of this solution (pH 8.2) was taken and 50 mg _{of As₂O₃} was added to it. No solubility was observed (at pH 8.0).

Table 7 Element quantity mg/unit Arsenic trioxide 50 mg 10 mg Sodium bicarbonate 100 mg 20 mg Purified water 50 mL Qs*

*Qs - Sufficient quantity.

Experiment number L040/1/002E

The purpose of this experiment was to assess the pH-dependent solubility of arsenic trioxide using L-arginine base (pH approximately 12). 200 mg of L-arginine base was dissolved in 100 mL of purified water (2 mg/mL solution) in a beaker with continuous stirring. From this solution, 50 mL was used to dissolve 50 mg _{of As₂O₃} (pH 10.90). However, no dissolution was observed; note that the pH was 10.53.

Table 7.1 Element quantity mg/unit Arsenic trioxide 50 mg 10 mg L-arginine 100 mg 20 mg Purified water 50 mL Qs*

*Qs - Sufficient quantity.

Experiment number L040/1/002F

The purpose of this experiment was to assess the pH-dependent solubility of arsenic trioxide using sodium carbonate (pH approximately 12). In a beaker, 50 mg of sodium carbonate was added to 50 mL of purified water (1 mg/mL solution). Then, 50 mg of the _{drug As₂O₃} was added to this solution with stirring at pH 11.25. No solubility of the drug was observed. However, as the concentration of sodium carbonate was gradually increased to 50 mg/mL, arsenic trioxide was found to be soluble in this solution at pH 11.46.

Table 8 Element quantity mg/unit Arsenic trioxide 50 mg 10 mg Sodium carbonate 2500 mg 500 mg Purified water 50 mL Qs*

*Qs - Sufficient quantity.

Therefore, in general, the drug is insoluble across the entire pH range of 4–12 when using arginine, arginine base, or sodium bicarbonate, but it has been found to be soluble when using higher concentrations of sodium carbonate (500 mg of sodium carbonate is required to dissolve 10 mg of arsenic trioxide).

Experiment number L040/1/002G

The aim of this experiment was to replicate the experiment of L040/1/002A, but using fillers such as lactose monohydrate and mannitol for subsequent lyophilization. In the first step, 10 g of arsenic trioxide was added to 300 mL of purified water with stirring, and then 125 mL of 1N NaOH was added with stirring. The drug was found to be soluble. The solution was divided into two equal portions:

L040/1/002G1: In Part 1, lactose monohydrate (3.5 g) was added with stirring and the solution was kept aside for physical observation, followed by lyophilization (pH 12.34). After approximately 24 hours at room temperature, the clear solution turned brown, and after 36 hours, the color intensity increased and turned dark brown, therefore lyophilization was not performed.

L040/1/002G2: For Part 2, mannitol (3.5 g) was added with stirring, and the solution was kept aside for physical observation, followed by lyophilization (pH 12.64). After approximately 24 hours, the solution showed no physical change, and the clarified solution was lyophilized for another 24 hours. After lyophilization, the substance appeared very viscous and was difficult to fill into capsules. The viscosity may be due to the corrosive properties of NaOH, the high pH of the solution (12.5), or simply the presence of mannitol.

Table 8.1

*Qs - Sufficient quantity.

Table 8.2 Element L040/1/002G1 L040/1/002G2 Solution (L040/1/002G) 212.5 mL 212.5 mL Lactose monohydrate (Pharmatose 200M) 3.5 g __ Mannitol (Pearlitol SD 200) __ 3.5 g

*Qs - Sufficient quantity.

In the next step, other surfactants were evaluated as solubilizers for arsenic trioxide. Additionally, another experiment was conducted using sodium carbonate and an increased amount of purified water (instead of NaOH). Another experiment was also conducted using NaOH, with the pH adjusted between 7 and 8. A further experiment was performed to evaluate the effect of mannitol on viscosity after lyophilization.

Experiment number L040/1/003

The purpose of this experiment is to study the solubility of arsenic trioxide using poloxamer and sodium carbonate.

L040/1/003A: Use poloxamer to improve solubility

In a beaker, 200 mg of poloxamer was dissolved in 100 mL of purified water (2 mg/mL solution) with stirring. 50 mL of this solution was taken and 50 mg of the drug was added. The drug was found to be insoluble. Even after a 20-minute stirring step, adding an additional 50 mg of poloxamer did not dissolve the drug.

Table 9

*Qs - Sufficient quantity.

L040/1/003B: Increase solubility by adding more purified water.

This experiment is similar to L040/1/002F. 1250 mg of sodium carbonate was added to 50 mL of purified water (25 mg sodium carbonate/mL) in a beaker. 50 mg of arsenic trioxide was added to this solution with stirring, but no solubility of the drug was observed (pH 11.25). Adding 100 mL of purified water also failed to dissolve the drug.

Table 10

*Qs - Sufficient quantity.

Experiment L040/1/004

The purpose of this experiment was to conduct tests using sodium hydroxide and a filler (mannitol) and to evaluate the effect on viscosity by adjusting the pH to approximately 8 (using 6M hydrochloric acid) after lyophilization. Arsenic trioxide was dispersed in purified water, and 12 mL of 3M NaOH was added with stirring until a clear solution was formed. The pH was adjusted to between 7 and 8 using 6M HCl. Purified water was added to the above solution to a final volume of 500 mL, and the solution was divided into two batches.

Batch 1: L040/1/004A1: Load 250 mL and freeze-dry for 24 hours.

Batch 2: L040/1/004A2: 250 mL, add mannitol with stirring and load for freeze drying for 24 hours.

Table 11

*Qs - Sufficient quantity.

Table 11.1

Experiment L040/1/004B

The purpose of this experiment was to prepare lyophilized products for freeze-drying. Sodium hydroxide in solid form was used, along with a reduced volume of purified water, and mannitol was used as a packing agent. In the first step, arsenic trioxide and NaOH were dispersed in purified water and stirred until a clear solution was formed. 6M HCl was added to this solution, and it was freeze-dried for 24 hours. After 24 hours of freeze-drying, a white, fluffy powder was observed for batch L040/1/004A1. For batch L040/1/004A2, a grayish-white, sticky substance was observed, which was difficult to remove from the petri dish. For batch L040/1/004B, a white, fluffy powder was observed. Based on these experiments, it was concluded that the stickiness of the substance (lyophilized product) was due to the mannitol in the solution.

In the first step, lyophilized arsenic trioxide and NaOH were added to pearlitol SD 200, sieved through a #40 mesh (400 microns) sieve, and mixed for 10 min. In the next step, talc and magnesium stearate were sieved through a #60 mesh (250 microns) sieve and added to the mixture from the first step, and mixed for 5 min. The final mixture was then filled into #0 hard gelatin capsule shells using a Laminar Airflow System.

Table 12—Composition of freeze-dried products

Qs* - Sufficient quantity.

Batch L040/1/004B was further processed using mannitol as a filler.

Table 12.1 — Composition of the final mixture

Experiment number L040/1/005

The purpose of this experiment was to evaluate the dissolution of a drug such as arsenic trioxide (equivalent to 10 mg) filled in a hard gelatin capsule shell of size "0". It was found that dissolution was incomplete even after 60 minutes.

Table 13 – Comparison of Dissolution and Measurement Results of Reference and Test Products

The next trial is planned, which will use different surfactants in the formulation to improve dissolution.

In experiment L040/1/006, the experiment was similar to that in L040/1/004B, and based on the precipitation effect of the solution, the solvent (purified water) amount was optimized to 25 mg/mL of arsenic trioxide. In experiment L040/1/007, experiments were conducted with and without different surfactants to evaluate dissolution and differential scanning calorimetry (DSC) studies. For arsenic trioxide capsules, the aim was to prepare drug solutions (25 mg/mL of arsenic trioxide) with different surfactants incorporated.

In the first step, arsenic trioxide was dispersed in 64 mL of purified water. In the next step, 4 g of sodium hydroxide was added to the solution, and the solubility of the drug was observed at pH 12.82. 6M HCl was added to the solution, and the pH was observed to be 8.05. Purified water was then added to the solution, and the solution was divided into several fractions (each containing 40 mL):

L040/1/007: 40 mL drug solution;

L040/1/007A: Add 0.5 g of sodium lauryl sulfate to 40 mL of the drug solution; and

L040/1/007B: Add 0.5 g of Tween 80® to 40 mL of drug solution.

The melting points of solutions L040/1/007 and L040/1/007A were assessed by differential scanning calorimetry (DSC). The DSC study clearly showed no significant change in the melting points.

Table 14

Experiment L040/1/008

The next experiment aimed to perform lyophilization using two different concentrations of sodium lauryl sulfate, similar to experiment L040/1/007. 160 mL of purified water was added to a beaker, along with 10 g of arsenic trioxide and 10 g of NaOH, and stirring was continued until a clear solution (pH 12.38) was formed. To this solution, 40 mL of 6M HCl (pH 8.04) was added with stirring. Additionally, 200 mL of purified water (pH 8.18) was added to the above solution with stirring. The solution was then divided into portions (each containing 80 mL):

Part 1: L040/1/008A: As is a solution.

Part 2: L040/1/008E: Add 1 g of sodium lauryl sulfate to 80 ml of solution with stirring (labeled amount: 25 mg contains 10 mg of arsenic trioxide).

Part 3: L040/1/008F: Add 0.2 g of sodium lauryl sulfate to 80 mL of solution with stirring (labeled amount: 21 mg contains 10 mg of arsenic trioxide).

Subsequently, all solutions were loaded and lyophilized for 24 hours, and samples were collected after lyophilization.

Table 15

Table 15.1

The actual yield of each batch was higher than the theoretical yield, which was attributed to the hygroscopicity of the lyophilized material. Based on the measured values, the lyophilized material was mixed with the extragranular material and filled into capsules.

Table 16 -- Measurement and Moisture Content Results:

Based on the above measurements, the following filling test was conducted.

Table 17--Composition of L040/1/009B

Table 18--Composition of L040/1/012B

Table 19--Composition of L040/1/013B

Add lyophilized arsenic trioxide and NaOH (lyophilized product) to pearlitol SD 200, pass through a #40 mesh sieve, and mix for 10 minutes. Add talc and magnesium stearate, pass through a #60 mesh sieve, to the above mixture, and mix for 5 minutes. Fill the powder into capsules of size "2".

Table 20

As clearly seen from the data above, complete dissolution was observed in all batches. In the next step, larger batch samples similar to L040/1/012B were filled with all strengths of 1 mg, 5 mg, 10 mg, and 20 mg. Based on batch number L040/1/012B, validation batches (batch number L040/4/014) were manufactured with all strengths (1 mg, 5 mg, 10 mg, and 20 mg).

Table 21 -- Solution Preparation and Lyophilization

*Qs – Sufficient quantity.

Table 22—Mixing & Lubrication

In the next step, arsenic trioxide and sodium hydroxide pellets were dispersed in 480 mL of purified water and stirred until a clear solution was formed. 120 mL of 6M HCl was added to this solution with stirring. 15 g of sodium lauryl sulfate was then added to the solution with stirring. Another 20 mL of purified water was added to the above solution. The solution was divided into two portions (L040/4/014A and L040/4/014B) and lyophilized for approximately 30 hours.

Table 23

Table 24

In the next step, lyophilized arsenic trioxide and NaOH (lyophilized product) were added to pearlitol SD200, sieved through a #40 mesh, and mixed for 10 minutes. Talc and magnesium stearate were sieved through a #60 mesh and added to the above mixture, which was then mixed for 5 minutes. The final mixture and suitable capsules and vials were prepared as shown below.

Table 25 strength batch number batch Package 20 mg L040/4/014B1 400 units HDPE 10 mg L040/3/014B1 400 units HDPE 10 mg of lubricating mixture L040/3/014B2 350 units glass bottle 5 mg L040/2/014B1 400 units HDPE 1 mg L040/1/014B1 400 units HDPE

Table 26 batch number L040/4/014B1 L040/3/014B1 L040/3/014B2 L040/2/014B1 L040/1/014B1 strength 20 mg 10 mg 10 mg 5 mg 1 mg Capsule type/vial filling 2 3 glass bottle 5 5 Fill weight (mg) 200 100 100 50 10 Dissolution (0.1 N HCl; 100 rpm; 900 mL; paddle, duration 15 min). 86 92 77 93

Table 27—Stability Study—L040/4/014B1 (20 mg) batch number L040/4/014B1 (20 mg) initial 40℃/75% RH (15 days) Measurement 95.15 101.41 Moisture content 2.24 2.69 Dissolution rate (0.1 N HCl; 100 rpm; 900 mL; paddle, 15 min.) 86 98

Table 28 – Stability Study – L040/3/014B1 (10 mg) batch number L040/3/014B1 (10 mg) initial 40℃/75% RH (15 days) Measurement 102.4 99.15 Moisture content 2.38 2.19 Dissolution rate (0.1 N HCl; 100 rpm; 900 mL; paddle, 15 min.) 92 100

Table 29 – Stability Study – L040/3/014B2 (10 mg lubricating mixture) batch number L040/3/014B2 (10 mg lubricating mixture) initial 40℃/75% RH (15 days) Measurement 98.2 99.78 Moisture content 1.90 2.32

Table 30 – Stability Study – L040/2/014B1 (5 mg) batch number L040/2/014B1 (5 mg) initial 40℃/75% RH (15 days) Measurement 98.8 99.95 Moisture content 2.63 2.82 Dissolution rate (0.1 N HCl; 100 rpm; 900 mL; paddle, 15 min.) 77 84

Table 31 – Stability Study – L040/1/014B1 (1 mg) batch number L040/1/014B1 (1 mg) initial 40℃/75% RH (15 days) Measurement 94 94.16 Moisture content 3.54 3.43 Dissolution rate (0.1 N HCl; 100 rpm; 900 mL; paddle, 15 min.) 93 92

Pharmacokinetic analysis of a novel lyophilized formulation containing arsenic in dogs and its comparison with reference arsenic trioxide. Compare

The aim of these studies was to determine the oral bioavailability and systemic exposure of novel arsenic-containing formulations using lyophilization technology in dogs. Dogs are a well-documented nonrodent species routinely used to estimate the pharmacokinetic properties of novel drugs and formulations in humans.

Research Design

Several groups of male dogs (n=3) were administered arsenic trioxide IV formulation (ARSENOX®) at 0.3 mg/kg, unformulated _As₂O₃ ( _SV100 ) orally at a dose of 2 mg/dog, or lyophilized _As₂O₃ formulation (SV101) orally at a dose of 2 mg/dog. A fourth group of dogs was administered SV101 orally at 2 mg/dog for future validation analysis. Blood samples were obtained from animals in group 1 at 0.083, 0.25, 0.5, 1 _, 2, 4, 6, 8, and 24 h before and after administration, and from animals in groups 2, 3, and 4 at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration. Blood samples were immediately placed on wet ice and processed into plasma within 30 minutes of collection. Arsenic levels in plasma samples were analyzed using ICP-MS at QPS Netherlands BV, with a limit of quantitation (LLOQ) of 2.50 ng/mL. Concentration-time data were used to calculate the pharmacokinetic parameters for arsenic (using WinNonlin™ version 5.2.1).

result

Following an IV bolus administration of ARSENOX®, plasma arsenic levels reached a Tmax of 146 ng/ml at 1.3 h and decreased over a half-life of 10.0 h (t _1/2 ) (Figure 1). The mean systemic clearance was 226 mL/h/kg, and the mean Vss was 2734 mL/kg. Following oral administration, SV100 showed very limited absorption, with oral bioavailability ( ^Fb %) <10%. The oral absorption of SV101 (Group 3) was nearly identical to that of ARSENOX® (IV), with a Cmax of 182 ± 34.5 ng/ml (vs. 146 ± 17.6 ng/ml for ARSENOX®) and an AUC0-last (h.ng/mL) of 1416 ± 345 (vs. 1115 ± 127 for ARSENOX®), indicating complete oral absorption. These data were confirmed by repeated administration of SV101 (Group 4), which produced nearly identical PK exposure parameters. No tolerance issues were observed during oral administration _{of As₂O₃} .

in conclusion

_As₂O₃ was administered to animals via a single intravenous or oral administration _. Results showed that the SV101 treatment group had significantly higher arsenic absorption rates and drug exposure than the SV100 group. Complete oral absorption _{of As₂O₃} in the SV101 treatment group was confirmed by nearly identical systemic arsenic exposure compared to IV-administered ARSENOX®.

Characterization of pharmaceutical capsules containing lyophilized compositions containing arsenic

The purpose of these experiments was to characterize arsenic-containing capsule formulations and summarize their physicochemical properties. Capsules were prepared in 1 mg, 2 mg, 5 mg, 10 mg, and 20 mg lyophilized formulations containing arsenic. Four thousand capsules were prepared. The manufacturing process consisted of (A) lyophilization and (B) mixing and lubrication steps. Figure 2 shows the process flow diagram for capsule manufacturing.

(A) Freeze-dried

The freeze-drying process is carried out in three stages as follows.

1. Freezing

After dissolving, the clarified solution is placed in a freeze dryer set at -40°C to freeze the product.

Table 32—Freezing Steps in Freeze-drying stage Temperature (ºC) Time (min) Vacuum (millitort) freezing -40 60 none freezing -40 300 none Additional freezing (ºC) -40 5 none

2. Single drying

This stage is also known as the "sublimation" stage. In this stage, drying is carried out under vacuum, and the temperature is gradually increased until the substance transforms into a solid form. The process parameters followed in this stage are shown in the table below.

Table 33—Primary Drying

3. Secondary drying

The material, after being dried once, is dried again using optimized vacuum and temperature to remove bound water.

Table 34—Secondary Drying Temperature (ºC) Time (min) Vacuum (millitort) 25 120 500

(B) Mixing and Lubrication

Estimate the amount of lyophilized material to be measured, and calculate the actual amount of lyophilized material required for each capsule as given below.

Actual amount of lyophilized product per capsule (in mg): (A) = [(amount/capsule) x 100] / [as determined]

Amount of mannitol to be dispersed per capsule (in mg): (B) = Theoretical amount of mannitol – [A – Theoretical amount of lyophilized product] Amount of mannitol to be dispersed / batch = B x Batch

As shown above, any extra amount of lyophilized material taken based on actual measurements was made up with mannitol. The lyophilized material and mannitol were passed through an ASTM #40 sieve and mixed by hand in a polyethylene bag for 10 minutes. The mixture was lubricated with magnesium stearate and talc, which were pre-sieved by hand through an ASTM #60 sieve in a polyethylene bag for 5 minutes. The mixture was then filled into capsules according to the details given below.

Table 35—Fill Weight and Capsule Type strength Fill weight (mg) Capsule Model 20 mg 200 1 10 mg 100 3 5 mg 50 4 2 mg 20 4 1 mg 10 4

Physicochemical characterization

Characterize the physicochemical properties of the drug product and conduct a comparative evaluation with the raw materials.

1. Particle size analysis

The particle size distribution of the API and the final formulation was characterized using a Malvern Mastersizer. The data are presented in the table below.

Table 36—Particle Size Distribution Particle size distribution API (micrometer) Lyophilized product (micrometers) Final formulation (micrometers) D(10) 3.9 0.8 1.9 D(50) 21.3 2.0 80.4 D(90) 139 4.7 210.4

The above data indicate a significant reduction in PSD during the lyophilization stage, which may contribute to improved solubility of arsenic trioxide in the formulation. However, the applicant does not wish to be bound by this theory or any scientific theory regarding that point. It should be noted that the final formulation's PSD also includes approximately 75% of other excipients.

2. Polymorphism

The API polymorphisms in the final formulation were analyzed after processing to assess any polymorphic changes from APIs like this. Even the intermediate stage (lyophilized product) was characterized using XRD diffraction patterns. This study concludes that the API does not undergo form changes in the formulation and is found to be stable during the intermediate stages of manufacturing. The diffraction patterns, shown in Figures 3-6, confirm that the same polymorphisms were found to be stable throughout the process and in the final formulation.

3. Images obtained using a scanning electron microscope (SEM):

The particle size and morphological characteristics of the three samples—pure API, lyophilized product, and final mixture—were characterized by SEM. The images are shown in Figure 7.

4. FTIR

The API, lyophilized product, and final formulation were characterized by FTIR. The spectra of the lyophilized product and final formulation were comparable to those of the observed API.

5. Solubility Study

According to the literature, arsenic trioxide is slightly soluble in cold water and insoluble in alcohol. Therefore, the solubility of API and lyophilized products in these media was investigated to evaluate their behavior, and the results are presented below.

Table 37—Solubility Study solvent API (g/100 g) Freeze-dried product (g/100 g) cold water 2.25 40.31 alcohol 1.86 41.90

Solubility studies show that the solubility of API is increased by about 20 times using the freeze-drying method of the present invention.

6. Chemical characterization of pharmaceutical products of all strengths

Table 38—Chemical Characterization serial number test API 1 mg 2 mg 5 mg 10 mg 20 mg 1 describe* N/A conform to conform to conform to conform to conform to 2 Identification test N/A conform to conform to conform to conform to conform to 3 Average weight (mg) of the filled capsules N/A 50.1 60.7 90.2 149.8 275.3 4 Average weight (mg) of capsule filler N/A 10.4 20.6 51.3 101.2 201.7 5 Water content determined by KF, % w/v 2.4 2.5 2.4 2.6 2.3 2.4 Disintegration time (min) N/A 5 min 5 min 6 minutes 6 minutes 5 min 7 Dissolution rate determined by ICP-OES (0.1 N HCl, paddle, 100 rpm, 900 mL, 30 min). 10 88 86 95 98 98 8 Determination by ICP-OES (%) 97.4 96.0 95.6 97.1 98.5 98.6  

*Compliant with their respective specifications

N/A - Not applicable.

7. BET Surface Area Measurement

The surface area of the API and the lyophilized product was measured using the BET method for surface area measurement. The specific surface area of the arsenic trioxide API was determined to be 0.05 ^m² /g. The specific surface area of the lyophilized composition or lyophilized product containing arsenic trioxide was determined to be 2.68 ^m² /g, which is more than 50 times the increase in surface area.

Table 39 below provides the surface area data for the API. Figure 8 provides a BET surface area plot of the API, showing how 1/[Q(Po/P-1)] depends on the relative pressure P/Po.

Table 39—BET Surface Area—API

Table 40 below provides surface area data for the lyophilized material. Figure 9 provides a BET surface area plot of the lyophilized material, showing how 1/[Q(Po/P-1)] depends on the relative pressure P/Po.

Table 40—BET Surface Area—Freeze-dried Products

Polymorphism studies showed that the API remained unchanged during manufacturing and in the final formulation, demonstrating its stability. SEM images in Figure 7 show a sharp decrease in API particle size during lyophilization. The final mixture contained other excipients, resulting in a coarser particle size. Formulations of all strengths (1, 2, 5, 10, and 20 mg) were found to have satisfactory chemical properties and conform to finished product specifications. A significant improvement in formulation dissolution was observed compared to the API, indicating a many-fold increase in the solubility of arsenic trioxide in the formulation.

Claims (47)

1. A method for preparing an arsenic-containing lyophilized composition (LCCA), the method comprising: _{As₂O₃ powder} and an alkalizing agent are dissolved in an aqueous medium to form an _As₂O₃ solution having a pH of 12.33 or _higher , wherein the alkalizing agent is sodium hydroxide and the amount of sodium hydroxide is 50% to 100% of the amount of _{As₂O₃ powder} , wherein the step of dissolving the _As₂O₃ powder further comprises: adding an acid to adjust the pH of _{the As₂O₃ solution} to pH 7 to pH 8. A surfactant is added to _{an As₂O₃ solution} , wherein the surfactant is added in an amount from 0.5% v/v to 4.0% v/v but not exceeding 50% of the _{As₂O₃ powder} concentration; and Lyophilizing _{the As₂O₃ solution} , wherein the lyophilization step includes: Freezing _{the As₂O₃} solution to form a _{frozen As₂O₃} product; and _The frozen _As₂O₃ product is dried to form particles of the LCCA. 2. The method of claim 1, wherein the acid is hydrochloric acid (HCl). 3. The method of claim 2, wherein the HCl is about 6M HCl. 4. The method of claim 1, wherein the acid is added to adjust the pH to approximately 7.2. 5. The method of claim 1, wherein the surfactant is at least one of sodium lauryl sulfate; Tween β-cyclodextrin; poloxamer; or tocopherol polyethylene glycol succinate (TPGS). 6. The method of claim 1, wherein the amount of the surfactant added by weight is in the range of 1:10 to 1:2 compared to the amount of _{As₂O₃ powder} by weight. 7. The method of claim 1, wherein the freezing step comprises freezing _{the As₂O₃} solution at a temperature in the range of -50°C to 0°C. 8. The method of claim 7, wherein the _As₂O₃ solution is frozen at about -40°C _for at least 6 hours. 9. The method of claim 1, wherein the freezing step comprises a freezing sub-step and each sub-step gradually lowers the temperature of the _{As₂O₃ solution} . 10. The method of claim 1, wherein the freezing step lowers the temperature over a period of 5 minutes to 500 minutes. 11. The method of claim 9, wherein each of the freezing sub-steps lowers the temperature over a period of 5 minutes to 500 minutes. 12. The method of claim 1, wherein the drying step comprises a primary drying step and a secondary drying step. 13. The method of claim 12, wherein the primary drying step comprises a heating step. 14. The method of claim 1, wherein the drying step comprises heating the _{frozen As₂O₃} product under vacuum. 15. The method of claim 1, wherein the drying step comprises at least one of the following three conditions: (i) drying the _As₂O₃ product at at least one temperature in the range of -40°C to 50°C for a time ranging from 5 minutes to 500 minutes; (ii) drying the frozen _As₂O₃ product by gradually increasing the temperature from at least one first temperature in the range of -40°C to 50°C to at least one second temperature in the range of -40°C to 50° _C over a time ranging from 5 minutes to 500 minutes, wherein the at least one second temperature is higher than the at least one first temperature; and (iii) applying a vacuum to _{the frozen As₂O₃ product in} the range of 300 mTorr to 1000 mTorr for a time ranging from 5 minutes to 500 minutes. 16. The method of claim 1, wherein the drying step comprises (a) Heating _{the As₂O₃} product at about -30°C and about 800 mTorr for about 60 minutes; heating the _As₂O₃ product from the preceding step at -20°C and 500 mTorr for about 120 minutes; heating the _As₂O₃ product from the preceding step at about -5°C and about 500 _mTorr for about 120 minutes; heating the _As₂O₃ product from the preceding step at about 10°C and about 500 _mTorr for about 60 minutes; and heating the _As₂O₃ product from the preceding step at about 25°C and about _{500 mTorr} for a period ranging from 180 minutes to 300 minutes; or (b) Heating the _As₂O₃ product to approximately -30°C and approximately 800 mTorr for approximately 60 minutes and maintaining it at approximately -30°C and approximately 800 mTorr for approximately 60 minutes; heating _{the As₂O₃} product from the preceding step to approximately -20°C and approximately 500 mTorr for approximately 60 minutes and maintaining it at approximately -20°C and approximately 500 mTorr for approximately 120 minutes; heating _{the As₂O₃} product from the preceding step to approximately -5°C and approximately 500 mTorr for approximately 300 minutes and maintaining it at approximately -5°C and approximately 500 mTorr for approximately 120 minutes; heating the _As₂O₃ product from the preceding step to approximately 10°C and approximately 500 mTorr for approximately 120 minutes and maintaining it at approximately 10°C and approximately 500 mTorr for approximately 60 minutes; and heating _{the As₂O₃ product} from the preceding step to approximately 60 minutes _{... 3.} The product is heated to about 25°C and about 500 mTorr and held at about 25°C and about 500 mTorr for a period of 180 to 300 minutes. 17. The method of claim 1, wherein _{the As₂O₃} powder has an average particle size, and wherein the particles of the LCCA have an average particle size that is 1/10 to 1/50 of the average particle size of the _{As₂O₃ powder} . 18. The method of claim 1, wherein the LCCA particles have a D(90) size range of 2 micrometers to 10 micrometers. 19. The method of claim 1, wherein _{the As₂O₃} powder has an average BET surface area, and wherein the LCCA particles have an average BET surface area that is 2 to 80 times the average BET surface area of the _{As₂O₃ powder} . 20. The method of claim 1, wherein the LCCA particles have a BET surface area in the range of 0.5 ^m² /g to 5 ^m² /g. 21. The method of claim 1, wherein _{the As₂O₃} powder has solubility in cold water or alcohol, and wherein the solubility of the LCCA particles in cold water or alcohol is 2 to 30 times that of the _As₂O₃ powder _. 22. The method of claim 1, wherein the solubility of the LCCA particles in cold water is in the range of 400 mg/ml to 600 mg/ml. 23. The method of claim 1, wherein the _{As₂O₃ powder} has a dissolution rate in water, and wherein the dissolution rate of the LCCA particles in water, as measured by ICP-OES, is 5 to 10 times the dissolution rate of the _{As₂O₃ powder} . 24. The method of claim 1, wherein _{the As₂O₃} powder has a polymorphic form, and wherein the particles of the LCCA have the same polymorphic form as the _{As₂O₃ powder} , as measured by XRD diffraction. 25. The method of any one of claims 1-24, further comprising: sieving the particles of the LCCA to produce lyophilized _{As₂O₃ powder} ; optionally, adding at least one filler to the lyophilized _As₂O₃ powder; and optionally, adding one or more lubricants to the _{lyophilized As₂O₃ powder} to produce an oral formulation of _{As₂O₃ .} 26. The method of claim 25, wherein the method comprises adding mannitol as the at least one filler; and/or wherein the method comprises adding talc and/or magnesium stearate as the one or more lubricants. 27. A pharmaceutical composition prepared according to the method of claim 25. 28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is a solid dosage form suitable for oral administration. 29. The pharmaceutical composition of claim 28, wherein the solid dosage form is a capsule or tablet comprising the lyophilized _{As₂O₃ powder} , the at least one filler, and/or one or more lubricants. 30. The pharmaceutical composition of claim 29, comprising mannitol as said at least one filler and talc and magnesium stearate as said one or more lubricants. 31. A capsule comprising the pharmaceutical composition of claim 30. 32. A capsule comprising 0.25 mg to 50 mg _of the lyophilized _As₂O₃ powder of the pharmaceutical composition of claim 28. 33. A capsule comprising about 1 mg, about 5 mg, about 10 mg, or about 20 mg of the lyophilized _As₂O₃ powder of the pharmaceutical composition of claim ₂₈ , wherein a capsule comprising about 1 mg of the lyophilized _{As₂O₃ powder} optionally has a fill weight of 10 mg; a capsule comprising about 5 mg of the lyophilized _{As₂O₃ powder} optionally has a fill weight of 50 mg; a capsule comprising about 10 mg of the lyophilized _{As₂O₃ powder} optionally has a fill weight of 100 mg; and a capsule comprising about 20 mg of the lyophilized _{As₂O₃ powder} optionally has a fill weight of 200 mg. 34. A kit comprising the pharmaceutical composition of claim 27 and instructions for use thereof. 35. The pharmaceutical composition of claim 27, used for treating cancer in patients in need. 36. A pharmaceutical composition for use according to claim 35, wherein the cancer is a hematologic malignancy. 37. A pharmaceutical composition for use according to claim 36, wherein the hematological malignancy is acute myeloid leukemia; acute non-lymphocytic leukemia; myeloblastic leukemia; promyelocytic leukemia; chronic myelomonocytic leukemia; monocytic leukemia; erythroleukemia; acute neutrophilic leukemia; myelodysplastic syndrome; acute promyelocytic leukemia; chronic lymphocytic leukemia; chronic myeloid leukemia; hairy cell leukemia; myeloproliferative neoplasm; Hodgkin lymphoma; non-Hodgkin lymphoma; myeloma; giant cell myeloma; painless myeloma; localized myeloma; multiple myeloma; plasma cell myeloma; sclerosing myeloma; solitary myeloma; condensing multiple myeloma; non-secreting myeloma; osteosclerosing myeloma; plasma cell leukemia; solitary plasma cell tumor; and At least one of extramedullary plasmacytomas. 38. A pharmaceutical composition for use according to claim 37, wherein the hematologic malignancy is acute promyelocytic leukemia (APL). 39. A pharmaceutical composition for use according to claim 38, wherein the APL is a newly diagnosed APL. 40. A pharmaceutical composition for use according to claim 38, wherein the APL is relapsed or refractory APL. 41. A pharmaceutical composition for use according to claim 37, wherein the myeloproliferative neoplasm is one of myelofibrotic polycythemia vera and essential thrombocythemia. 42. A pharmaceutical composition for use according to claim 35, wherein the pharmaceutical composition is administered daily. 43. A pharmaceutical composition for use according to claim 35, wherein the pharmaceutical composition is administered in a single dose range of 1 mg to 50 mg. 44. A pharmaceutical composition for use according to claim 35, wherein the pharmaceutical composition is administered in a single dose range of 0.1 mg/kg body weight to 0.3 mg/kg body weight. 45. A pharmaceutical composition for use according to claim 35, wherein the patient has previously received or is receiving chemotherapy and/or radiation therapy. 46. A pharmaceutical composition for use according to claim 45, further comprising administering one or more chemotherapeutic agents to the patient. 47. A pharmaceutical composition for use according to claim 45, wherein the chemotherapeutic agent is administered before or simultaneously with the pharmaceutical composition.