Patent Description:
Combination chemotherapy has given high cure rates for certain types of metastatic cancer, such as childhood leukemia, lymphoma, and testicular cancer. However, most common types of metastatic cancer are currently incurable. The <NUM>-year survival rates of some metastatic cancers are approximately as follows: cervical <NUM>%, colorectal <NUM>%, uterine <NUM>%, esophageal <NUM>%, kidney <NUM>%, liver/biliary <NUM>%, lung/bronchus <NUM>%, melanoma <NUM>%, ovarian <NUM>%, pancreatic <NUM>%, stomach <NUM>%, bladder <NUM>%, breast <NUM>%. The following references relate to this matter: <NPL>; <NPL>). Despite decades of research and hundreds of billions of dollars, the age-adjusted cancer mortality rates reported by the U. National Cancer Institute for many types of cancers showed no decline over a <NUM>-year period, from <NUM>-<NUM>. During the same period, the National Library of Medicine catalogued <NUM>,<NUM>,<NUM> scientific articles about cancer, of which <NUM>,<NUM> related to the treatment of metastatic cancer, and since the <NUM>'s there have been over <NUM>,<NUM> medical papers and scientific reports published on clinical trials for metastatic cancer and over <NUM>,<NUM> scientific papers published on combination cancer therapy. Despite this truly massive scientific effort, obtaining complete responses (CRs)--that is, the absence of all detectable cancer--in patients with most types of metastatic cancer has not been possible. Generally, a <NUM>% or <NUM>-log reduction in cancer cell burden is needed to obtain a CR. A patient with metastatic cancer can have tens of billions of cancer cells distributed throughout his or her body: decreasing the tumor cell burden by <NUM> logs would still leave millions to billions of viable cancer cells in the patient; with time those cancer cells could multiply and cause progressive disease. For example, the CR rate in pancreatic cancer using FOLFIRINOX, the most effective chemotherapy, is only <NUM>%. In patients with metastatic melanoma treated with Nivolumab plus ipilimumab, the state-of-the-art therapy, the CR rate was <NUM>%. The CR rate in patients with melanoma treated with the BRAF inhibitor Vemurafenib was <NUM>%. Similar low rates of CRs are seen with most types of metastatic cancers. Durable, long-term CRs are even rarer in patients with most types of metastatic disease. The following references relate to this matter: <NPL>; <NPL>; <NPL>. There have been thousands of clinical trials with a large number of different combinations of anticancer drugs, yet few drug regimens give high CR rates in patients with metastatic cancer, and cures for most types of metastatic cancer are very rare. Furthermore, the few types of cancers that are currently curable at a high rate with combination chemotherapy are generally characterized by properties that confer hypersensitivity to a particular chemotherapy drug or drugs. Extraordinary effort, resources and time have been expended without success to develop methods capable of high CR rates, and still over <NUM>,<NUM> people in the U. die of metastatic cancer each year. Presently, there are no methods for the effective treatment for most types of metastatic cancer that can give high CR rates or durable, long-term CRs in patients. Thus, a need exists to develop a cancer therapy that can achieve high rates of CRS, especially long-term durable CRs, in patients with metastatic cancer or refractory caner.

<CIT> describes methods for the effective treatment of metastatic cancers that involve treatment with melphalan, BCNU, and redox cycling agents in conjunction with bone marrow stem cell infusion.

In vitro, it is easy to profoundly decrease cellular GSH levels (and to consequently increase sensitivity to DNA-crosslinking drugs such as melphalan) by incubating cells with redox cycling agents or agents that generate reactive oxygen species. Many studies have demonstrated that ascorbic acid undergoes transition metal catalyzed autoxidation to produce hydrogen peroxide. In vitro, ascorbic acid and hydrogen peroxide can deplete GSH, induce oxidative stress, and kill cells. Riordan, in <CIT> (Therapeutic method for the treatment of cancer), teaches the use of high-dose intravenous ascorbic acid for the treatment of cancer. However, multiple clinical trials have failed to demonstrate anticancer activity of high-dose ascorbic acid in patients, and ascorbic acid has not provided a basis for obtaining high rates of complete responses in patients with metastatic cancer. The biologic activity of hydrogen peroxide is a function of the dose per cell or the dose per liter of intracellular fluid. Exposure of cells to ascorbic acid or hydrogen peroxide in vitro can result in doses per cell that are thousands of times higher than those that can be achieved in vivo. Hydrogen peroxide is rapidly decomposed in cells by glutathione peroxidase; in the process GSH is oxidized to GSSG. However, the GSSG in turn is reduced back to GSH by glutathione reductase with NADPH as the reductant. The reductive capacity of cells for GSSG far exceeds the flux of H2O2 that could be generated in vivo from even very high doses of ascorbic acid. This explains in part the absence of anticancer activity of ascorbic acid observed in multiple clinical trials. The following references relate to this matter: <NPL>);<NPL>; <NPL>; <NPL>; <NPL>.

The delivery of drugs into tumors is compromised by a number of factors including poor vascularization, increased interstitial fluid pressure, and increased flow of interstitial tumor fluid out of the tumor. Accordingly, the intravenous administration of redox cycling drugs to a patient will generally result in higher drug levels and greater biologic effect in normal tissues than in tumors. Therefore, the GSH depletion and resulting sensitization to melphalan by intravenously administered redox cycling agents will generally be greater in normal tissues than tumors.

In the presence of oxygen, hydroxocobalamin catalyzes the autoxidation of ascorbic acid. In vitro, the combination of hydroxocobalamin and ascorbic acid generates hydrogen peroxide, lowers GSH levels, and is cytotoxic. The process involves redox cycling of the cobalt between Co(III) and Co(II) oxidation states with ascorbate serving as the reductant and oxygen as the oxidant. The ascorbic acid is oxidized to the ascorbate free radical and ultimately dehydroascorbic acid (DHA). The following reference relate to this matter: <NPL>; <NPL>;<NPL>;<NPL>.

Each of ascorbic acid and hydroxocobalamin distributes in the extracellular fluid and is not preferentially taken up by tumor cells. Accordingly, one skilled in the art would expect that the combination of intravenous ascorbic acid and hydroxocobalamin would not selectively deplete GSH in tumor cells versus normal tissues. One skilled in the art would expect that the combination would equally sensitize normal tissues and tumor tissues to melphalan, and that any gain in tumor cell killing would be offset by increased toxicity to normal cells, which would limit the dose of melphalan that could be safely administered.

In animal models, the combination of DHA and hydroxocobalamin exerted potent anticancer effects, however the combination of ascorbic acid and hydroxocobalamin was ineffective. Initial reports of anticancer activity with hydroxocobalamin and ascorbic acid were corrected in a follow-on publication and attributed to the use of ascorbic acid that had already decomposed to DHA prior to administration. In mouse models of P388 lymphocytic leukemia, the combination of ascorbic acid and hydroxocobalamin had anticancer activity that was limited in extent and duration; survival was prolonged only by about <NUM> days. The following reference relates to this matter: <NPL>(see Appendix A). DHA is unstable in blood and decomposes intravascularly within seconds to <NUM>,<NUM>-diketogulonic acid (<NUM>,<NUM>-DKG). The following references relate to this matter: <NPL>;<NPL> (see Appendix A); <NPL>; <NPL>.

Ascorbic acid alone has been proposed as a means to induce oxidative stress in tumors. The administration of high-dose ascorbic acid was shown to generate ascorbate free radical and hydrogen peroxide in the microdialysis fluid obtained from tumors and subcutaneous tissues. However, the levels of hydrogen peroxide measured in the microdialysis fluid reflect both hydrogen peroxide from the extracellular fluid and hydrogen peroxide generated in the microdialysis tubing. Hydrogen peroxide production in microdialysis tubing could be significant because the flow rate was slow, the ascorbic acid levels were high, and a <NUM>,<NUM> to <NUM>,<NUM> molecular weight serum factor catalyzes the autoxidation of ascorbic acid. This serum factor could be present in the microdialysis concentration at significant levels as the molecular weight cut-off of the dialysis membrane was <NUM>,<NUM>. In addition, the levels of ascorbate free radicals detected in the microdialysate from subcutaneous extracellular fluid were significantly higher than those from tumor extracellular fluid. Since ascorbate free radicals undergo rapid disproportionation to DHA and ascorbic acid, this strongly suggests that the levels of DHA generated in subcutaneous extracellular fluid were higher than that generated in tumor extracellular fluid. The data indicate that ascorbic acid undergoes autoxidation in microdialysate from tumor extracellular fluid and extracellular fluid from normal tissue. It has been postulated, but not demonstrated, that the extracellular environment of tumors may contain higher levels of transition metals that can catalyze the autoxidation of ascorbic acid compared to normal tissues. Even if this is the case, the rate of autoxidation of ascorbic acid in tumors is slow. Mice given an intravenous infusion of hyperpolarized ascorbic acid demonstrated no detectable DHA in tumors. By contrast, after infusion of hyperpolarized DHA to mice, hyperpolarized ascorbic acid was readily detected in tumors. Furthermore, as already discussed, the rate of hydrogen peroxide production from the autoxidation of ascorbic acid is far less than the capacity of tissues to detoxify the hydrogen peroxide. The following references relate to this matter: <NPL>; <NPL>; <NPL>; <NPL>; <NPL>.

Consider the consequences of adding a redox catalyst such as hydroxocobalamin at equal concentrations to the extracellular fluid of normal tissues and the extracellular fluid of tumors at a concentration that results in rapid ascorbic acid autoxidation (compared to the rate of autoxidation in tissues without the catalyst). The result would be essentially equal rates of ascorbic acid autoxidation in extracellular fluid from normal tissues and tumor tissues since the contribution of endogenous catalysts would be minor compared to the catalytic activity of the hydroxocobalamin. Accordingly, one skilled in the art would expect the administration of a catalyst such as hydroxocobalamin, which is taken up equally by tumors and normal tissues, would provide no basis for the selective depletion of GSH in tumors. One skilled in the art would expect that absent selective depletion of GSH in tumor cells, the toxicity of melphalan would be increased in both normal tissues and tumor cells, and that the increased toxicity to normal tissues would require a dose reduction to the patient, which would offset any gain in cytotoxicity to tumor cells by the GSH depleting agents. For example, L-buthionine-SR-sulfoximine (BSO) depletes GSH in both normal tissues and tumor tissues, and in patients BSO increases the toxicity of melphalan to normal bone marrow. Another example is misonidazole, which upon systemic administration depletes GSH non-selectively in both tumors and normal tissues. The combination of misonidazole and nitrogen mustard results in increased DNA crosslinking and increased toxicity to both normal tissues and tumor tissues, with the greatest toxicity increases seen in normal tissues. The enhancement of toxicity to both normal tissues and tumors by DNA-damaging drugs administered in combination with drugs that non-selectively inhibit DNA repair is a general occurrence. For example, it is seen with DNA-damaging agents in combination with O6-benzylguanine and poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors. The following references relate to this matter: <NPL>; <NPL>; <NPL>; <NPL>; <NPL>.

As described herein, the unexpected result that the administration of ascorbic acid and hydroxocobalamin will result in selective delivery of hydrogen peroxide and DHA to tumors and the selective depletion of GSH in tumor cells has been discovered. Despite the fact that the delivery of ascorbic acid and hydroxocobalamin will be equal in tumor and normal tissues, the dose of DHA to tumor cells will be approximately <NUM> to <NUM> times greater than in most normal tissues, and the dose of hydrogen peroxide to tumor cells will be as much as <NUM> times greater. Surprisingly, the unexpected preferential delivery of DHA and hydrogen peroxide will result from increased interstitial fluid pressure, interstitial fluid, and poor vascularity, which are characteristic of tumors. This is unexpected because increased interstitial fluid pressure in tumors and poor tumor vascularity are well known barriers to tumor uptake of drugs. The following references relate to this matter:<NPL>; <NPL>; <NPL>.

Any subject matter falling outside of the extent of protection conferred by the claims is provided for information purposes, only. References to a method of treatment in this description are to be interpreted as references to the compounds, (kits of) pharmaceutical compositions or medicaments of the present invention for use in a method of treatment of the human (or animal) body by therapy (or for diagnosis).

The present invention relates to effective methods of treating metastatic cancers to achieve high rates of complete responses (CRs) and especially long-term, durable CRs. The present invention includes a method for the treatment and effective treatment (as defined below) of metastatic cancers, where the method comprises the administration of a set of drugs comprising melphalan, BNCU, hydroxocobalamin, ascorbic acid, and optionally ethanol and bone marrow stem cell infusion.

In a first aspect, the invention pertains to a method for the treatment of metastatic cancer or refractory metastatic cancer in a subject and comprises administering a combination of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid, or pharmaceutically acceptable salts of any of the foregoing, and optionally administering ethanol and/or bone marrow stem cells, wherein the melphalan, <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, hydroxocobalamin, and ascorbic acid, or pharmaceutically acceptable salts of any of the foregoing, are all administered simultaneously or within a six-hour time period; wherein the melphalan dose is in the range of <NUM> to <NUM>/m2. In one embodiment, <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea is administered at a dose range of <NUM> to <NUM>/m2; the melphalan is administered at a dose of <NUM> to <NUM>/m2; the hydroxocobalamin is administered at a dose of <NUM> to <NUM>,<NUM>/m2, and the ascorbic acid is administered at a dose of <NUM> gram to <NUM> grams. In another embodiment, the <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea is administered at a dose range of <NUM> to <NUM>/m2; the melphalan is administered at a dose of <NUM> to <NUM>/m2; the hydroxocobalamin is administered at a dose of <NUM> to mg to <NUM>/m2, and the ascorbic acid is administered a dose of <NUM> grams to <NUM> grams. In a further embodiment, the <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea is administered at a dose of <NUM>/m2; the melphalan is administered at a dose of <NUM>-<NUM>/m2; the hydroxocobalamin is administered at a dose of <NUM>/m2, and the ascorbic acid is administered a dose of <NUM> grams to <NUM> grams. In one embodiment, the invention further comprises the systemic administration of ethanol at a dose of <NUM> to <NUM> grams. In another embodiment, the invention further comprises bone marrow stem cell transplantation therapy. In a further embodiment, the metastatic cancer is in a subject with an inherited germline mutation in a gene involved in DNA repair, and/or homologous recombination, and/or DNA crosslink repair. In one embodiment, the metastatic cancer is in a patient with an inherited germline mutation in one or more of the following genes: ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1 (FANCJ, BACH1), EME1, ERCC1, ERCC4, FAN1, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCO, FANCP, FANCQ, FANCQ, FANCR, FANCS, FANCT, HELQ, MEN1, MUS81, NBN (NBS1), PALB2, RAD50, RAD51 (FANCR), RAD51C (FANCO), RAD51D, REV1, SLX4 (FANCP), UBE2T (FANCT), USP1, WDR48, XPF, XRCC2, XRCC3, or other genes involved in DNA-crosslink repair, homologous recombination, or DNA repair. In another embodiment, the metastatic cancer is in a subject with an inherited germline mutation in BRCA1 and/or BRCA2. In a further embodiment, the metastatic cancer is selected from pancreatic cancer, ovarian cancer, breast cancer, and prostate cancer.

In a third aspect, the invention pertains to a method of treating cancer comprising the administration of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, hydroxocobalamin, and ascorbic acid, or pharmaceutically acceptable salts of any of the foregoing.

In a fifth aspect, the invention pertains to a set of pharmaceutical compositions for use in effectively treating metastatic cancer or refractory metastatic cancer in a subject, comprising a therapeutically effective dose of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid, or pharmaceutically acceptable salts of any of the foregoing.

In a sixth aspect, the invention pertains to the use of a pharmaceutical compositions for the treatment of metastatic cancer or refractory metastatic cancer in a subject, comprising a therapeutically effective dose of a combination of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin and ascorbic acid, wherein the melphalan dose is in the range of <NUM> to <NUM>/m2.

In a seventh aspect, the invention pertains to a method for the prevention of hemolysis and/or methemoglobin formation in a subjected treated with agents that generate hydrogen peroxide and comprises the systemic administration of a therapeutically effective dose of ethanol.

The present invention relates to methods for the treatment and effective treatment (as defined below) of metastatic cancers, including refractory metastatic cancers.

Acquired Drug Resistance: refers to the ability of populations of cancer cells to escape destruction or inactivation by a drug at levels that are clinically achievable, wherein said lack of sensitivity arises or evolves in an initially drug-sensitive population.

Analog: refers to a compound or moiety possessing significant structural similarity as to possess substantially the same function.

Allogeneic: refers to tissue or cells derived from another individual.

Appropriately selected patients: refers to patients who are good candidates for the treatment and that are likely to benefit. For example, a frail, elderly patient with serious underlying medical conditions (e.g., heart disease, liver disease, renal disease, severe malnutrition) would generally not be a good candidate. A patient with such advanced metastatic disease that he or she would be unlikely to survive the treatment would not be a good candidate. A patient with extensive metastatic disease to the brain would not be a good candidate. Methods for the appropriate selection of patients are well known to one skilled in the art.

Approximately: refers to plus or minus <NUM>% when referring to drug doses and ranges of drug doses.

Area under the curve (AUC): refers to the integral of the drug concentration-time curve for a drug in vitro or in vivo; the AUC is a measure of total drug exposure.

Ascorbate free radical: refers to the radical formed from the one electron oxidation of ascorbic acid. The following reference relates to this matter: <NPL>.

Ascorbic acid: refers to L-ascorbic acid and the molecular species that are in equilibrium with ascorbic acid when ascorbic acid is dissolved in water or aqueous solutions. Ascorbic acid has two ionizable hydroxyl groups with pKa of <NUM> and pKa of <NUM>, respectively. At physiological pH, the ascorbate monoanion is the predominant form, however, small amounts of the ascorbate dianion are also present; both are species are in equilibrium with ascorbic acid. Ascorbic acid also refers to pharmacologically acceptable salts of L-ascorbic acid, such as mono-sodium ascorbate. Ascorbic acid does not refer to DHA or ascorbate free radical. Doses of ascorbic acid are based on the content of L-ascorbic acid, assuming that all the drug were in the form of L-ascorbic acid. The following reference relates to this matter: <NPL>.

AUC-: <NUM> refers to the drug AUC needed to give a <NUM>-log reduction in clonogenic cell survival.

Autologous: refers to tissue or cells derived from the same individual.

BCNU: refers to the drug carmustine, also known as <NUM>,<NUM>-Bis(<NUM>-chloroethyl)-<NUM>-nitrosourea (<NPL>). BCNU inhibits glutathione reductase, which is critical to maintaining cellular GSH levels in the presence of oxidative stress. Glutathione reductase catalyzes the reduction by NADPH of GSSG to GSH. BCNU is also a DNA-crosslinking agent.

Bone marrow stem cell or hematopoietic stem cell: refers to a pluripotent cell that can reconstitute normal bone marrow and give rise to all normal bone marrow cell lineages; these cells are typically CD34+ cells, can be isolated from bone marrow aspirates, peripheral blood, and umbilical cord blood, and can be autologous or allogeneic. Cells that can give rise to bone marrow stem cells for the purposes of this application are also considered to be "bone marrow stem cells.

Bone marrow stem cell infusion, bone marrow stem cell transplantation therapy, and stem cell infusion: refer to the process of intravenously administering bone marrow stem cells to speed recovery of bone marrow function.

BRCA-associated cancer and BRCA-related cancer: refer to cancer that arises in the setting of an inherited BRCA mutation.

Buthionine sulfoximine (BSO): refers to a selective inhibitor of gamma-glutamylcysteine synthetase, the rate limiting enzyme in GSH synthesis.

Cancer: refers to a disease defined by malignant behavior. Only malignant cells (i.e., cells that engage in malignant behavior) can sustain the clinical disease of cancer.

Clonogenic survival: refers to the ability of a cell to multiply and form a colony of cells.

Clonogenic survival fraction: refers to a measure of clonogenic survival, calculated as the fraction of cells that are able to give rise to a colony of cells in a colony-forming assay, also equal to the probability of clonogenic survival.

Combination therapy: refers to the administration of therapeutic compounds (e.g., agents or drugs) in a manner wherein each therapeutic compound is administered at a different time, as well as to the administration of these therapeutic agents, or at least two of the therapeutic agents, concurrently or in a substantially simultaneous manner. Simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or by administering multiple, single capsules for each of the therapeutic agents, or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route, including oral routes, intravenous routes, intramuscular routes, or direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the selected combination may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. Therapeutic agents may also be administered in alternation. The combination therapies featured in the present invention can result in a synergistic effect in the treatment of a disease or cancer. Combination therapy also refers to the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies (e.g., surgery or radiation treatment). Where the combination therapy comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

Complete Response (CR): refers to the absence of all detectable cancer, which is typically determined by CT scan, MRI or other imaging or detection technology; The following reference relates to this matter: <NPL>. It should be noted that the RECIST guidelines equate the presence of tumor mass with the presence of cancer and decreases in tumor mass with anticancer efficacy. While tumor mass is an accurate metric for cytotoxic anticancer drugs and therapies that kill cancer cells, it is not an accurate metric for anticancer drugs that permanently abolish the potential for cell proliferation without necessarily killing cells. For example, bizelesin acts in this manner. By definition, cell populations (i.e., tumor masses) that do not and cannot proliferate, cannot exhibit malignant behavior and are not cancerous, even though said cell populations remain viable.

Current, established therapies: refers to existing regimens used to treat subjects.

Dehydroascorbic acid (DHA) refers to the oxidized form of ascorbic acid; (5R)-<NUM>-[(<NUM>)-<NUM>,<NUM>-dihydroxyethyl]oxolane-<NUM>,<NUM>,<NUM>-trione.

Detoxification: refers to the process of decreasing or abolishing the cellular toxicity of a drug by means of spontaneous or cellular metabolic processes. For example, enzymatic or spontaneous nucleophilic reaction of GSH with an alkylating agent results in detoxification of the alkylating agent.

<NUM>,<NUM>-Diketogulonic acid: refers to (4R,<NUM>)-<NUM>,<NUM>,<NUM>-trihydroxy-<NUM>,<NUM>-dioxohexanoic acid, which is a decomposition product of DHA, (<NPL>).

Distribute into the extracellular fluid: means that the volume of distribution of the drug is essentially the extracellular fluid space in the body.

DNA interstrand crosslinking agent: refers to a drug or chemical agent that binds the DNA strands of the DNA double helix together with sufficient affinity as to preclude strand separation and thereby impairs DNA synthesis. In general, but not always, said binding affinity results from covalent bonds formed between the crosslinking agent and the DNA strands. Examples of DNA-crosslinking agents are provided in the following: <NPL>.

Dose modification factor: refers to the following formula: [drug concentration that gives a <NUM>-log reduction in clonogenic cell survival without the second drug(s) "X"] / [drug concentration that gives a <NUM>-log reduction with drug(s) "X"]. For example, the drug could be melphalan and drug "X" could be BSO.

Durable complete response (also long-term CR): refers to a long-lasting CR; or a CR lasting at least <NUM> year off chemotherapy; or a CR lasting for a period of time greater than <NUM> X, wherein X is the median overall survival of comparable patients with the same type and stage of cancer who are treated with current, established therapies and fail to have a CR. For example, if the median overall survival for a particular type and stage of cancer were <NUM> months with current, established therapies in patients that failed to have a CR, then for a CR to be considered a durable CR in this setting, it would have to exceed <NUM> months in duration.

Effective treatment of metastatic cancer or effectively treating cancer: refers to a treatment or method that in appropriately selected patients gives high rates or high probabilities of one or more of the following: CRs, durable long-term CRs; long-term progression-free survival, long-term overall survival, long-term disease-specific survival, long-term relative survival, long-term disease-free survival, and apparent cures; and which generally preserves or improves the patient's quality of life. A grant of breakthrough therapy designation by the Food and Drug Administration (FDA) would provide supportive evidence of effectiveness; however, a treatment that is statistically superior to placebo, prolonged overall survival or progression free survival by several months and received FDA approval would by our definition not be deemed an effective treatment. Similarly, a treatment that gives high rates (e.g., <NUM>%) of short-term (e.g., several months duration) CRs would not be deemed an effective treatment.

Electrophilic DNA-crosslinking agent: refers to a DNA-crosslinking agent that reacts with nucleophilic sites on DNA; for example, the bifunctional alkylating agent melphalan is an electrophilic DNA-crosslinking agent that reacts with two nucleophilic centers on DNA: N-<NUM> of guanine and N-<NUM> of adenine.

Enzyme: refers to a protein that catalyzes a chemical reaction.

Extracellular fluid: refers to the fluid that resides outside of cells in the body; the corresponding space is referred to the extracellular space. For purposes of this application, extracellular fluid can be viewed as the plasma and interstitial fluid.

Fanconi/BRCA pathways of DNA repair: refers to the cellular machinery, proteins, and processes involved in homologous recombination and the repair of DNA interstrand crosslinks. The following references relates to this matter: <NPL>; <NPL>.

Glutathione (GSH): refers to a tripeptide with a gamma peptide bond between the amine group of cysteine and the carboxyl group of the glutamate side-chain, where the cysteine is attached by peptide bond to glycine. GSH is the major intracellular thiol compound: it is an important antioxidant and an important agent in the intracellular detoxification of reactive electrophiles, such as alkylating agents.

Glutathione peroxidase: refers to an enzyme that catalyzes the conversion of hydrogen peroxide into water and GSH into GSSG.

Glutathione reductase (GR): refers to an enzyme that catalyzes the reduction of GSSG into GSH; NADPH is used as the reducing agent.

Glutathione disulfide (GSSG): refers to the compound formed by linking two GSH molecules by a disulfide bond; also, referred to as "oxidized GSH.

Glutathionylation: refers to the formation of mixed disulfides between glutathione and low-pKa cysteinyl residues of proteins. The following reference relates to this matter: <NPL>.

High rate (or probability) of complete responses: refers to a rate (or probability) of CR that is at least approximately two times the rate (or probability) obtained with current, established treatments for the particular type and stage of cancer, wherein the term "the particular type" of cancer can refer not only to the histological type (i.e., serous ovarian cancer), but also to other clinically relevant qualifying properties such as platinum-resistance; or, alternatively, a rate exceeding approximately <NUM>%. The term "high probability of complete response" is preferred when dealing with a single patient, but otherwise the terms "high rate" and "high probability" are essentially interchangeable.

Homologous recombination: refers to a DNA repair process that results in the removal and repair of DNA interstrand crosslinks and the repair of DNA double stranded breaks. The following references relates to this matter:<NPL>; <NPL>.

Includes (as well as including and other forms of the word): shall be construed as includes (or including, etc.) without limitation or "includes but is not limited to.

Inhibitor of glutathione reductase: refers to a drug or agent that inhibits GR activity or that spontaneously or after metabolic activation generates a chemical species that inhibits GR activity.

Interstitial fluid or interstitial water: refers to extravascular fluid that is located outside of cells.

Interstitial space: refers to the space occupied by interstitial fluid.

Intracellular water or fluid: refers to water or fluid that is located inside cells.

Intracellular GSSG/2GSH reduction potential: refers to a metric that provides a measure of the reducing activity of GSH under the intracellular conditions; it is given by ΔE in the Nernst equation: ΔE = Eph - RT/2F ln [GSH]^<NUM>/ [GSSG], wherein Eph is E0 (the reduction potential under standard state conditions) adjusted to the intracellular pH; R is the gas constant, F is the Faraday constant, T is the temperature, [GSH] is the glutathione concentration, and [GSSG] is the glutathione disulfide concentration at the intracellular location. At pH <NUM> Eph = ~ -<NUM> mV, and at <NUM>, ΔE = ~ -<NUM> -<NUM> log [GSH]^<NUM>/ [GSSG] in mV. The following reference which relates to this matter: <NPL>.

Interstitial fluid pressure: refers to the pressure exerted by interstitial fluid. The following reference relates to this matter:<NPL>.

Intrinsic drug resistance: refers to the ability of populations of cancer cells to escape destruction or inactivation by a drug at levels that are clinically achievable, wherein said lack of sensitivity is manifest prior to drug exposure.

Irreversible inhibitor: refers to an agent that permanently inactivates an enzyme; generally, this occurs by covalent modification of the enzyme at site(s) that are essential for enzyme activity.

Liquid cancer: refers to a cancer derived from the bone marrow or lymphatic tissues; examples include leukemia, lymphoma, and myeloma.

Log reduction in cell survival: is the negative logarithm of the fraction of clonogenic cancer cells that survive the treatment; that is, each log reduction reduces the number of surviving clonogenic cancer cells by nine tenths. For example, a <NUM>-log reduction means that the treatment results in a <NUM>% decrease in clonogenic cell survival, a <NUM>-log reduction corresponds to a <NUM>% decrease in clonogenic cell survival, a <NUM>-log reduction corresponds to a <NUM>% decrease in clonogenic cell survival, etc..

Malignant behavior: refers to proliferation and invasiveness in an abnormal context or setting in the body, wherein invasiveness is the expansion of cells into new space, which can be local or distant (i.e., metastatic), with the remodeling or destruction of existing tissue architecture and the creation of infrastructure to support the metabolic needs of the cells; the mechanisms of invasiveness can be carried out by malignant cells and/or non-malignant cells in the microenvironment. Malignant behavior is the defining property of cancer.

Malignant cell: refers to a cancer cell that expresses or can express malignant behavior; not all tumor cells in a patient with cancer are malignant; many tumor cells in patients with cancer are dead-end, cannot proliferate, cannot engage in malignant behavior, and are not malignant cells.

Melphalan (<NPL>): is a bifunctional alkylating agent that crosslinks DNA and thereby inhibits cancer cell clonogenic survival.

Metastatic cancer: refers to cancer that has spread beyond the local tissue site of origin to distant locations in the body; that is, non-localized cancer. Micro-metastatic cancer is metastatic cancer that is not detectable with conventional imaging technology because of the small size of the metastatic lesions.

mg/m2 and gram/m2: refer to the dose per square meter of body surface area. Methods for calculating body surface area are well known to one skilled in the art. Doses expressed in mg/m2 or grams/m2 can be converted into approximately equivalent or similar doses based on body weight or other metrics well known to one skilled in the art; embodiments of the present invention in which doses are expressed in mg/kg or other such metrics are within the scope of the present invention.

NADPH: refers to the reduced form of nicotinamide adenine dinucleotide phosphate (NADP).

Nitrogen mustard analog: refers to a compound containing two or more chloroethylamine groups or an analog thereof; a compound that can transform in vivo or vitro into one with chloroethylamine groups; or a compound that can form aziridinyl groups. Chloroethylamine undergoes intramolecular nucleophilic reactions with elimination of Cl- and forms aziridinyl groups.

Neoadjuvant setting: refers to the administration of a chemotherapeutic drug or therapy before surgical resection of the primary tumor.

Non-homologous end joining (NHEJ): refers to a process for the repair of double stranded DNA breaks that results in error-prone repair. The following reference which relates to this matter: <NPL>.

Non-refractory metastatic cancer: refers to metastatic cancer of a type that can be effectively treated with current, established therapies; examples include most but not all testicular cancers, childhood acute lymphocytic leukemia, Hodgkins lymphoma, follicular thyroid cancer, and other cancers that are well known to one skilled in the art.

Nucleotide excision repair (NER): refers to a DNA repair process that removes nucleotides with bulky modifications and repairs the damage. The following reference which relates to this matter: <NPL>.

Oxidative stress: refers to the condition that exists when the levels of reactive oxygen species exceed the ability of cells to maintain those reactive chemical species within normal, physiological or acceptable levels; oxidative stress is generally associated with an increase the intracellular GSSG/2GSH reduction potential and oxidative damage to biomolecules. The following reference relates to this matter: <NPL>. Some methods for measuring oxidative stress are reviewed in: <NPL>.

Pharmacologic effect: refers to an action imparted by a drug on a subject or on cells in the subject; for example, a decrease in cellular GSH, or cytotoxicity are pharmacologic effects.

Potential for cell proliferation: refers to the ability of cells to proliferate; or clonogenic survival as measured by the ability to form colonies of cells. The potential for cell proliferation differs from cell proliferation: all malignant cells by definition have the potential for cell proliferation all the time, but most malignant cells are not actively engaged in proliferation most of the time, as cell proliferation is episodic.

Probability of clonogenic survival: refers to clonogenic survival fraction.

Prodrug refers: to a derivative of a drug that can be transformed in vivo or in vitro either spontaneously or as a result of metabolism or enzyme activity into the parent drug.

Reactive oxygen species (ROS): refer to reactive oxygen-related species such as superoxide (O2-), hydrogen peroxide (H2O2), hydroxy radical (OH•), peroxy radicals (ROO•), nitric oxide (NO•), and peroxynitrite anion (ONOO-). The following reference relates to this matter: <NPL>.

Redox cycling: refers to a series of chemical reactions in which a compound is reduced and the product is then oxidized by reaction with molecular oxygen; the catalytic cycle can repeat many times and consume large quantities of the reducing agent and large quantities of oxygen. For example, quinones can be reduced by a variety of cellular enzymes by one electron transfer from NADH or NADPH to give semi-quinone radicals, which can react with oxygen to regenerate the quinone and give superoxide. Redox cycling causes oxidative stress in cells by generating large amounts of superoxide and other reactive oxygen species. Redox cycling can be represented as repetitive cycles of equations <NUM> and <NUM>: Equation <NUM>: E + R → R-* + E+ <MAT> where E is an electron donor, E+ is the oxidized form of E, and R-* is a free radical.

Redox cycling agent (or drug): refers to a compound that engages in redox cycling; the term can refer to the reduced and/or oxidized form of the cycling chemical species that repetitively undergoes changes in oxidation/reduction status; it is also used to refer to compounds that can generate by spontaneous or metabolic processes a redox cycling agent.

Refractory metastatic cancer: refers to metastatic cancer that has failed to adequately respond to therapy; or metastatic cancer of a type that is known to be generally unresponsive to existing therapies and that cannot be effectively treated with current, established therapies. For example, metastatic testicular cancer is highly curable and is generally not a refractory metastatic cancer; by contrast, pancreatic cancer, melanoma, and platinum-resistant ovarian cancers are refractory metastatic cancers. A patient need not have failed on treatment to be considered to have refractory metastatic cancer. A cancer is considered refractory to a particular drug if the type of cancer is known not to respond well to the particular drug. For example, pancreatic cancer is refractory to BCNU, melphalan, and high-dose ascorbic acid. The following references relate to this matter: <NPL>; <NPL>; <NPL>).

Sensitize cancer cells to a DNA-crosslinking agent or DNA-damaging agent: means to increase the sensitivity of cancer cells to the agent, which results in said agent causing a much greater inhibition of cancer clonogenic survival with a decrease in the AUC or dose of the agent needed to give a <NUM>-log reduction in cancer cell clonogenic survival by a factor of at least <NUM>; the degree of sensitization is measured by the dose modification factor (DMF).

Set of drugs (e.g., agents or compositions) for use in a regimen to treat (a specified condition): refers to one or more drugs; if the set comprises drug #<NUM>, drug #<NUM>, drug #<NUM> and drug #<NUM>, then the term "a set of drugs for use in a regimen to treat (a specified disease) means:
drug #<NUM> for use in a regimen to treat (a specified disease), drug #<NUM> for use in a regimen to treat (a specified disease), drug #<NUM> for use in a regimen to treat (a specified disease), and drug #<NUM> for use in a regimen to treat (a specified disease), wherein the regimen involves the combined use of drug #<NUM>, drug #<NUM>, drug #<NUM>, and drug #<NUM>. Set of drugs also refers to a kit comprising said drugs.

Solid cancers or solid tumors: refers to a cancer derived from a solid tissue; examples include pancreatic cancer, colon cancer, lung cancer, and ovarian cancer.

Subject: refers to a mammal in need of treatment or prevention, e.g., humans, companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats, and the like), and laboratory animals (e.g., rats, mice, guinea pigs, and the like). Typically, the subject is a human in need of the specified treatment.

Selective delivery of drugs to a tumor: refers to the systemic administration of one or more agents to a subject and achieving drug levels in tumors and/or the intracellular fluid of tumor cells that are greater than the levels in normal tissue, wherein the magnitude of the increased delivery of the drug to tumors is sufficient to preferentially elicit a desired pharmacologic effect of said drug in tumors.

Selective (effect) in tumors (or tumor cells): refers to achieving a magnitude of an effect in tumors (or tumor cells) that is greater than the magnitude in normal tissue, wherein the magnitude of the increased effect in tumors (or tumor cells) is sufficient to preferentially elicit a desired pharmacologic effect in the tumors (or tumor cells).

Synergy or synergistic effect: refers to a detectable effect that is greater (i.e., in a statistically significant manner relative to an appropriate control condition) in magnitude than the sum of the effects that can be detected when the compounds are used alone: that is, the effect of the combination is greater than the expected additive effect of each component. A synergistic effect may be an effect that cannot be achieved by administration of any of the compounds or other therapeutic agents as single agents. A synergistic effect may include an effect of treating cancer by reducing tumor size, inhibiting tumor growth, or increasing survival of the subject. A synergistic effect may also include reducing cancer cell viability, inducing cancer cell death, or inhibiting or delaying cancer cell growth.

Systemic administration: refers to the administration of a drug the results in drug distribution in the body by means of the blood circulation; it includes intravenous (IV), intraarterial, intraperitoneal, and oral routes of drug administration. A preferred route is IV.

Therapeutically effective dose: refers to a dose that gives the beneficial treatment effect.

Thiolate: the negatively charged conjugate base of a thiol; a deprotonated thiol ion. In cells, the protein thiolate content is largely determined by the content of cysteine thiol groups that have a pKa of approximately <NUM> or less.

Treatment: refers to a therapy that provides a beneficial effect to a patient with a respect to a disease or condition.

USP: refers to The U. Pharmacopeial Convention (USP) drug standards.

The Co(III) atom of hydroxocobalamin cycles between Co(III) and Co(II) states in the presence of a reducing agent and oxygen. A large number of hydroxocobalamin analogs in which the cobalt atom or another transition metal atom can undergo redox cycling are known to one skilled in the art. Examples of analogs, derivatives, prodrugs, and pharmacologically acceptable salts of hydroxocobalamin include hydroxocobalamin acetate (<NPL>), hydroxocobalamin hydrochloride, vitamin B 12r, diaquacob(III)inamide (<NPL>); methylaquacobinamide (<NPL>), adenosylaquacob(III)inamide (<NPL>), and cyanoaquacob(III)inamide (<NPL>). The following references relate to this matter: <NPL>; <NPL>;<NPL>.

A reducing agent that can reduce hydroxocobalamin is D-ascorbic acid (<NPL>), or a racemic mixture of D and L ascorbic acid; or a thiol such as cysteine, n-acetyl cysteine, glutathione, sodium <NUM>-sulfanylethanesulfonate (Mesna), or <NUM>,<NUM>-dimercaptooctanoic acid (dihydrolipoic acid), or pharmacologically acceptable salts or prodrugs thereof. A wide range of other compounds can undergo autoxidation in the presence of hydroxocobalamin with the production of hydrogen peroxide. One skilled in the art will recognize other compounds that would behave in a similar fashion. Methods for the systemic administration of thiols are well known to one skilled in the art.

Hydroxocobalamin and ascorbic acid can react to generate hydrogen peroxide, ascorbate free radical and/or dehydroascorbic acid (DHA) and <NUM>,<NUM>-diketogulonic acid (<NUM>,<NUM>-DKG). Ascorbate free radicals undergo rapid dismutation to ascorbic acid and DHA. The hydrogen peroxide may be generated directly or indirectly, for example by dismutation of superoxide. Hydrogen peroxide, DHA and <NUM>,<NUM>-DKG can mediate useful selective pharmacologic effects in cancer cells including: depletion of GSH, increase in the intracellular GSSG/2GSH reduction potential, inhibition of tumor cell ATP production, inhibition of glycolysis, the sensitization of tumor cells to DNA-damaging agents, sensitization of tumor cells to DNA-crosslinking agents, inhibition of mitosis, and cytotoxicity.

Hydroxocobalamin acts a catalyst for the oxidation of ascorbic acid. Hydroxocobalamin undergoes redox cycling in the presence of ascorbic acid and oxygen. In this cyclic process, hydroxocobalamin is reduced by ascorbic acid, and the reduced form of hydroxocobalamin is then oxidized by electron transfer to oxygen. The net result is that hydroxocobalamin serves as a catalyst for the oxidation of ascorbic acid, and hydrogen peroxide and DHA are produced. In vitro, the combination of hydroxocobalamin and ascorbic acid generates hydrogen peroxide, lowers GSH levels, and is cytotoxic. The following references relate to this matter:<NPL>; <NPL>; <NPL>; <NPL>.

Methods for the intravenous administration of hydroxocobalamin are well known to one skilled in the art. Hydroxocobalamin and ascorbic acid can be given simultaneously or essentially at the same time. Alternatively, hydroxocobalamin can be given hours prior to ascorbic acid because hydroxocobalamin has a plasma half-life of approximately <NUM> to <NUM> hours. Hydroxocobalamin may be given over approximately <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> minutes, such as over approximately <NUM>-<NUM> minutes immediately prior to the administration of ascorbic acid, which may be given over a time period of approximately <NUM>-<NUM> minutes. Hydroxocobalamin is currently used intravenously for the treatment of cyanide poisoning. The following reference relates to this matter: Prescribing information for Cyanokit.

Methods for the intravenous administration of ascorbic acid are well known to one skilled in the art. Ascorbic acid may be given intravenously over approximately <NUM>-<NUM> minutes, such as over approximately <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> minutes. Intravenous ascorbic acid has been used in multiple clinical trials. The following references relate to this matter: <NPL>); <NPL>; <NPL>;<NPL>; <NPL>.

Both hydroxocobalamin and ascorbic acid rapidly distribute into the extracellular fluid compartment following systemic administration. In the presence of oxygen, hydroxocobalamin and ascorbic acid react to generate hydrogen peroxide and DHA. Hydrogen peroxide and DHA are both rapidly cleared from the intravascular compartment and rapidly efflux the interstitial fluid and enter the intracellular fluid. Pharmacologic effects of DHA and hydrogen peroxide include the selective induction of oxidative stress in tumors, the selective depletion of glutathione in tumors, the selective increase in the intracellular GSSG/2GSH reduction potential in tumors, the selective inhibition of tumor cell ATP production, the selective inhibition of glycolysis in tumor cells, the selective sensitization of tumor cells to DNA-damaging agents, the selective sensitization of tumor cells to DNA-crosslinking agents, and selective cytotoxicity to tumor cells. These effects can be increased by inhibition of GR, an enzyme that reduces GSSG to GSH.

Ethanol may be administered orally or intravenously to prevent the inactivation of catalase by hydrogen peroxide generated from the reaction of hydroxocobalamin and ascorbic acid. If the activity of red blood cell catalase is compromised, then hydrogen peroxide can cause hemolysis and methemoglobinemia. Methods for the intravenous administration of ethanol are well known to one skilled in the art. The following reference relates to this matter: FDA prescribing information for <NUM>% Alcohol in <NUM>% Dextrose Injection, USP. The ethanol can also be used as a co-solvent for one or more of the drugs.

Methods for the intravenous administration of melphalan are well known to one skilled in the art. Melphalan may be administered over a period of approximately <NUM> to <NUM> minutes.

Bone marrow stem cells may be infused to reverse bone marrow toxicity from a DNA-damaging drug. Stem cell infusions are generally given if the melphalan dose exceeds approximately <NUM>/m2 or the BCNU dose exceeds approximately <NUM>/m2 or if the patient has, or is expected to have, prolonged bone marrow suppression following the drug treatment. The stem cells are collected prior to the administration of the chemotherapy drugs (i.e., the DNA-damaging drugs) and are purified and stored. The bone marrow stem cells are preferably infused <NUM>-<NUM> days after the chemotherapy drugs; however, the stem cells can be administered at later times. Purified autologous bone marrow stem cells are strongly preferred; however, allogeneic bone marrow stem cells can also be employed. Technology for hematopoietic stem cell collection, purification, storage, and transplantation or infusion are well known to one skilled in the art. The use of purified stem cell preparations enriched for CD34+ hematopoietic cells and depleted of circulating tumor cells is preferred. The following references relate to this matter: <NPL>; <NPL>; <NPL>.

The mechanism by which hydroxocobalamin and ascorbic acid <NUM> will selectively deliver hydrogen peroxide to tumors is unexpected, as there would appear to be no basis for tumor selectivity: both agents will distribute essentially uniformly throughout the extravascular fluid after systemic administration, and furthermore the rate of the reaction to form hydrogen peroxide will be essentially equal throughout the extracellular space both in tumor and in normal tissues. It would therefore seem that there is no basis for tumor selectivity in drug delivery. However, the system of hydroxocobalamin, ascorbic acid, and hydrogen peroxide has unexpected pharmacokinetic properties that can give up to a <NUM>-fold increased drug delivery to tumors. This is especially unexpected because tumors have decreased blood flow and increased interstitial fluid pressure compared to normal tissues, which generally serve as barriers to drug delivery into tumors. The present invention exploits these well-known "barriers" to drug delivery to tumors to paradoxically enhance drug delivery to tumors.

One mechanism of action may be as follows: Normally interstitial fluid pressure is about -<NUM> to -<NUM> mmHg relative to atmospheric pressure. In tumors, the interstitial fluid pressure is significantly greater. The increased interstitial fluid pressure of tumors is due to leaky capillaries that allow extravasation of albumin into the intestinal space (which increases the oncotic pressure in the extracellular fluid), decreased or absent lymph flow, dysregulation of the tumor blood flow (which can lead to higher capillary blood pressure in tumors), and increased production of osmotically active substances such as hyaluronic acid within the tumor microenvironment. When the interstitial fluid pressure increases above <NUM>, there is a large increase in interstitial fluid volume. Accordingly, tumors are characterized by a large increase in interstitial fluid volume compared to normal tissues. The ratio of interstitial fluid volume to intracellular fluid volume is much greater in tumors than in normal tissues: typically, approximately <NUM> to <NUM> times greater in tumors than in most normal tissues. The following references relate to this matter: <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>.

After the intravenous or systemic administration of hydroxocobalamin and ascorbic acid, there will be a rapid equilibration of the concentrations of the agents between the plasma and interstitial fluid. After the distributive phase is complete, there will be essentially no net flux of hydroxocobalamin or ascorbic acid between the plasma and interstitial fluid (except for that resulting from gradients generated by renal elimination of hydroxocobalamin and ascorbic acid from plasma). If the rate of renal clearance of hydroxocobalamin and ascorbic acid is small compared to the production rate of hydrogen peroxide, then its effect will be small. Since the production rate of hydrogen peroxide will be essentially equal in both the plasma and interstitial fluid any net flux of hydrogen peroxide between the plasma and interstitial fluid would result only from concentration gradients that result from differences in the elimination rates in the respective compartments. Since hydrogen peroxide is both rapidly degraded in the intravascular compartment and rapidly taken up from interstitial fluid into intracellular water, the absolute concentration of hydrogen peroxide in both the plasma and interstitial fluid will be low, and the absolute magnitude of any concentration gradients between plasma and interstitial fluid will also be low. Absent a significant concentration gradient between plasma and interstitial fluid, hydrogen peroxide in the interstitial fluid will largely be taken up into the intracellular space in the microenvironment where it is formed. In this case, the dose of hydrogen peroxide received by cells at a particular site will depend upon the ratio of interstitial fluid to intracellular fluid at the site. Since this ratio is much higher in tumors, tumors cells will receive a correspondingly greater dose of hydrogen peroxide than cells in normal tissues. (The same will apply for DHA.

Hydrogen peroxide will degrade much faster in the intravascular space than in the interstitial fluid. The diffusion or uptake of hydrogen peroxide from interstitial fluid into the intravascular space can be much faster than the uptake of hydrogen peroxide into the intracellular space, as hydrogen peroxide is rapidly decomposed in the intravascular space. The rate-limiting step is the diffusion of hydrogen peroxide into red blood cells, where it is decomposed by catalase. Hydrogen peroxide in the intravascular space is rapidly destroyed by catalase: the half-life is ~ <NUM> milliseconds. By contrast, the half-life of hydrogen peroxide decomposition by pancreatic cancer cells in a tumor will be about <NUM> seconds. This estimate is based on extrapolation from the known rates of hydrogen peroxide consumption by pancreatic cancer cells in vitro to in vivo cell densities. The rate of efflux of hydrogen peroxide out of the interstitial fluid into the intravascular compartment will be a function of the surface area of the capillaries per ml of interstitial fluid and the volume of red blood cells (in the capillaries) per ml of interstitial fluid, both of which are much higher in normal tissues than in tumors. For example, the ratio of RBC volume to interstitial fluid volume in rat fibrosarcomas is ~. <NUM>; the ratio is ~ <NUM> times higher in rat lung, <NUM> times higher in rat kidney, and ~ <NUM> times higher in the heart. The capillary surface area/ml of interstitial fluid is also much smaller in tumors than in most normal tissues. In many normal tissues, the ratios of red blood cell volume/interstitial fluid volume and capillary surface area/interstitial fluid volume are so high that nearly all hydrogen peroxide will be consumed in the intravascular space and the dose of hydrogen peroxide delivered to the intracellular space of the normal tissue will be very small compared to that delivered to the intracellular space of tumors.

The following references relate to this matter:<NPL>; <NPL>; <NPL>; <NPL>.

The oxidation of ascorbic acid, which is catalyzed by hydroxocobalamin, generates DHA. DHA is rapidly taken up by cells and reduced to ascorbic acid; in the process <NUM> GSH molecules are oxidized to GSSG. DHA is transported into cells by GLUT transporters, which are highly overexpressed on cancer cells. DHA and its decomposition product <NUM>,<NUM>-DKG are highly electrophilic and can mediate useful pharmacologic effects in cancer cells such as inhibiting mitosis, depleting intracellular GSH, increasing the intracellular GSSG/2GSH reduction potential in tumor cells, inhibiting glycolysis, depleting ATP, and killing tumor cells. The following references relate to this matter:<NPL>;<NPL>; <NPL>; <NPL>; <NPL>.

Hydrogen peroxide in the presence of transition metals generates reactive oxygen species that can damage DNA and cellular components. Hydrogen peroxide can also induce oxidative stress, deplete intracellular GSH, increase the intracellular GSSG/2GSH reduction potential in tumor cells, inhibit glycolysis, deplete ATP, sensitize cells to DNA-damaging agents, and cause cytotoxicity. The effects are increased by high intracellular levels of ascorbic acid, which elevate free iron levels in cells. Inhibition of GR also increases the effects of hydrogen peroxide. The following references relate to this matter: <NPL>;<NPL>; <NPL>; <NPL>;<NPL>; <NPL>; <NPL>; <NPL>; <NPL>.

The concentration of GSH in cells is typically in the range of <NUM> to <NUM>. When GSH is oxidized, the GSSG formed is rapidly reduced back into GSH by glutathione reductase. Cells have a tremendous capacity to reduce GSSG. For example, the reductive capacity of hepatocyte cells for GSSG in vitro is ~ <NUM> mmoles/hour per liter of intracellular fluid. In order to depress intracellular GSH levels, it is necessary to oxidize GSH at a rate that exceeds the cellular reductive capacity for GSSG. This would generally require the delivery of an enormous and impractical quantity of oxidizing agent. The addition of a glutathione reductase inhibitor prevents the reduction of GSSG and thereby allows intracellular GSH levels to be decreased by low levels of oxidizing agents such as hydrogen peroxide. The following reference relates to this matter: <NPL>.

Metastatic cancers that can be treated with the methods and treatment regimens and embodiments of the present invention include: Metastatic cancer, Refractory metastatic cancer, BRCA1-related metastatic cancer (inherited mutation), BRCA2-related metastatic cancer (inherited mutation), PALB2-related metastatic cancer (inherited mutation), Metastatic cancer in the setting of an inherited BRCA/Fanconi pathway mutation(s), Metastatic cancer in the setting of an acquired BRCA/Fanconi pathway mutation(s), BRCA2-related pancreatic cancer (inherited mutation), BRCA2-related prostate cancer (inherited mutation), BRCA2-related ovarian cancer (inherited mutation), BRCA2-related breast cancer (inherited mutation), BRCA2-related fallopian tube cancer (inherited mutation), BRCA1-related pancreatic cancer (inherited mutation), BRCA1-related prostate cancer (inherited mutation), BRCA1-related ovarian cancer (inherited mutation), BRCA1-related fallopian tube cancer (inherited mutation), BRCA1-related breast cancer (inherited mutation), PALB2-related pancreatic cancer (inherited mutation), PALB2-related prostate cancer (inherited mutation), PALB2-related ovarian cancer (inherited mutation), PALB2-related fallopian tube cancer (inherited mutation), PALB2-related breast cancer (inherited mutation), Breast cancer (ductal adenocarcinoma), RAD50-related breast cancer (inherited mutation), Cancers that arise in patients with an inherited germline mutation(s) or an acquired somatic mutation(s) in a gene or genes involved in DNA crosslink repair, homologous recombination, and/or DNA repair, Breast cancer (lobular adenocarcinoma), Breast cancer (sarcoma), Breast cancer (triple negative), Breast cancer (inflammatory), Breast cancer (Paget's), Prostate cancer (adenocarcinoma), Pancreatic cancer (adenocarcinoma, Stage I-IV), Ovarian cancer (serous), Ovarian cancer (endometroid), Ovarian cancer (clear cell), Ovarian cancer (mucinous), Adenocarcinoma, Basal Cell Carcinoma, Bile Duct cancer, Bladder cancer, Bronchial cancer, Carcinoid Tumor, Cervical cancer (squamous), Cervical cancer (adenocarcinoma), Colorectal cancer, Colon cancer, Duodenal cancer, Endometrial cancer, Endometroid endometrial cancer, Esophageal cancer, Esophageal cancer (squamous cell), Esophageal cancer (adenocarcinoma), Ewing sarcoma, Fallopian tube cancer, Ocular melanoma, Malignant fibrous histiocytoma of bone, Osteosarcoma, Gallbladder cancer, Gastric cancer, Gastrointestinal carcinoid tumor, Gastrointestinal stromal tumors (GIST), Germ cell tumors, Head and neck cancer, Hepatocellular cancer, Hypopharyngeal cancer, Malignant islet cell tumors, Renal cell carcinoma, Laryngeal cancer, Lip and oral cavity cancer, Leiomyosarcomas, Lymphoma, Leukemia, T cell leukemia, B-cell lymphoma, B-cell leukemia, Acute myelogenous leukemia, Myeloma, Non-Hodgkins lymphoma, Lung cancer, Non-small cell lung cancer, Small cell lung cancer, Lung cancer (adenocarcinoma), Lung cancer (large cell), Lung cancer (squamous cell), Melanoma, Merkel cell carcinoma, Mesothelioma, Nasal cavity and paranasal sinus cancer, Nasopharyngeal cancer, Neuroendocrine cancer, Oral cancer, Oropharyngeal cancer, Pancreatic neuroendocrine tumors, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Rectal cancer, Renal cell cancer, Renal clear cell cancer, Renal chromophobe cancer, Renal papillary cancer, Renal pelvis and ureter, Transitional cell cancer, Salivary gland cancer, Sarcoma, Squamous cell carcinoma, Rhabdomyosarcoma, Small intestine cancer, Soft tissue sarcoma, Squamous neck cancer with occult primary, Testicular cancer, Thyroid cancer (papillary, follicular, medullary, and anaplastic), Transitional cell cancer of the renal pelvis and ureter, Urethral cancer, Uterine cancer, Undifferentiated cancer, Endometrial uterine Sarcoma, Vaginal cancer, and Vulvar cancer. The cancer treatment methods of the present invention can be used to treat, but are not limited to treating, cancers that arise in patients with an inherited germline mutation(s) or an acquired somatic mutation(s) in ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1 (FANCJ, BACH1), EME1, ERCC1, ERCC4, FAN1, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCO, FANCP, FANCQ, FANCQ, FANCR, FANCS, FANCT, HELQ, MEN1, MUS81, NBN (NBS1), PALB2, RAD50, RAD51 (FANCR), RAD51C (FANCO), RAD51D, REV1, SLX4 (FANCP), UBE2T (FANCT), USP1, WDR48, XPF, XRCC2, XRCC3, or other genes involved in DNA-crosslink repair, homologous recombination, or DNA repair.

The above gene names are based on the HUGO Human Genome Nomenclature System, which is well known to one skilled in the art. Cancer cells with an inherited or acquired mutation(s) in the above genes have an increased sensitivity to DNA-damaging and especially DNA-crosslinking agents. Methods for identifying such inherited and somatic tumor mutations are well known to one skilled in the art. More detailed descriptions of metastatic cancers, all of which are within the scope of the present invention, are provided in the following reference: <NPL>.

The applicability of the present invention to sensitizing cells to DNA-crosslinking agents is due to the common mechanisms of GSH-mediated detoxification of electrophiles and the common mechanisms involved in the repair of DNA interstrand crosslinks, regardless of the particular crosslinking agent. The following reference relates to this matter: <NPL>. The applicability of the present invention to sensitizing tumor cells to DNA-damaging agents in general is the result of the multiple mechanisms of DNA repair that are inhibited by an increase in the intracellular GSH reduction potential. This will result in a profound synergy: the antitumor activity of the drug combinations of the present invention is greater than the additive antitumor activity of the individual drugs.

Multiple steps required for the repair of DNA-drug monoadducts and DNA interstrand crosslinks are redox sensitive and are inhibited by an increase in the intracellular GSSG/2GSH reduction potential. This explains the profound hypersensitivity to melphalan induced by oxidative stress seen with BCNU and adriamycin. The following references relate to this matter: Jevtović-Todorović V, et al. , J Cancer Res Clin Oncol. , <NUM>, <NUM>(<NUM>):<NUM>-<NUM>; Jevtović-Todorović V, et al. , Biochem Pharmacol. , <NUM> Oct <NUM>, <NUM>(<NUM>):<NUM>-<NUM>.

Oxidative stress and an increase in the intracellular GSSG/2GSH reduction potential can inhibit proteins involved in DNA repair by a variety of mechanisms, including S-glutathionylation of the proteins, intermolecular disulfide formation, intramolecular disulfide formation, and by impairing the detoxification of ROS, which causes an increase in levels of ROS that oxidize critical protein thiols. In addition, it can compromise cellular energy production. An increase the intracellular GSSG/2GSH reduction potential leads to global changes in cellular metabolism that affect thousands of redox-sensitive proteins. Redox-sensitive proteins are required for all major pathways of DNA repair.

The enzyme MGMT detoxifies BCNU by catalyzing removal of the drug adducts from guanine bases in DNA. MGMT is a redox-sensitive enzyme, which is dependent upon an active-site cysteine that is glutathionylated and inhibited under oxidative conditions. The following reference relates to this matter: <NPL>.

Ubiquitylation, SUMOylation, and neddylation are critical to multiple steps in multiple pathways of DNA repair, including nucleotide excision repair (NER), homologous recombination (HR), single strand annealing (SSA), non-homologous end joining (NHEJ), alternate NHEJ, and translesion DNA synthesis. Multiple steps in the enzymatic pathways of ubiquitylation, SUMOylation, and neddylation are dependent upon active-site cysteines that are redox sensitive and inhibited by glutathionylation and oxidation of the active sites. The following references relate to this matter:<NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>;<NPL>; <NPL>; <NPL>; <NPL>.

Ku protein is required for the NHEJ repair of DNA double stranded breaks and is redox sensitive. Oxidative stress also inhibits DNA-dependent protein kinase (DNA-PKcs) and inhibits the localization of DNA-PKcspThr2609 at double stranded breaks and impairs repair. The following references relate to this matter: <NPL>;<NPL>; <NPL>; <NPL>.

Topoisomerase II is involved in DNA unwinding, is involved in multiple steps of DNA repair, and is redox sensitive. The following references relate to this matter: <NPL>; <NPL>, <NPL>; <NPL>.

XPA is required for NER, and XPA deficiency sensitizes cells to melphalan. XPA is redox sensitive. The following references relate to this matter:<NPL>; <NPL>.

RPA is required for all major DNA repair pathways and is redox sensitive. The following references relate to this matter: <NPL>; <NPL>; <NPL>; <NPL>.

Deubiquitinases (DUB) are critical to multiple pathways of DNA repair and are redox sensitive. The following references relate to this matter: <NPL>; <NPL>; <NPL>.

XRCC3 is essential to multiple steps of HR; XRCC3 deficiency is characterized by extreme hypersensitivity to DNA-crosslinking agents. The protein has multiple cysteine groups that are redox sensitive, susceptible to modification by electrophilic thiol reactive agents and glutathionylation. The following references relate to this matter: <NPL>; <NPL>.

Ribonucleotide Reductase (RNR) has critical cysteine groups and is a redox-sensitive enzyme involved in DNA damage repair. The following reference relates to this matter: <NPL>.

Human apurinic/apyrimidinic (AP) endonuclease <NUM> (APE1) is a redox-sensitive enzyme that plays a key role in DNA base excision repair pathways. The following reference relates to this matter:<NPL>.

ATP is required for multiple steps in DNA repair. Multiple critical enzymes involved in ATP production are redox-sensitive and are inhibited by oxidative stress. Aconitase is a redox-sensitive enzyme involved in energy production in the Krebs cycle. Glyceraldehyde <NUM>-phosphate dehydrogenase is a redox-sensitive enzyme that is essential for ATP production by glycolysis. The mitochondrial carnitine/acylcarnitine carrier (CAC) is redox sensitive: it is required for the transport of acylcarnitines into mitochondria and the β-oxidation of fatty acids, which is an important source of ATP for prostate cancer cells. α-Ketoglutarate dehydrogenase (KGDH) is a redox-sensitive enzyme critical to energy generation in the Krebs cycle. Isocitrate dehydrogenase is a redox-sensitive enzyme in the Krebs cycle. The following reference relates to this matter: <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>.

Protein tyrosine phosphatases (PTPs) are involved in multiple pathways of DNA repair and are redox-sensitive enzymes. The following reference relates to this matter: <NPL>.

There are many other redox-sensitive enzymes and proteins in addition to those listed above that are critical to the repair of DNA damage and that are inhibited by oxidative stress and that will be inhibited in tumors by the present invention, including by the combination of BCNU, hydroxocobalamin, and ascorbic acid.

The role of the ethanol is to prevent the inactivation of red blood cell catalase. The intravascular decomposition of hydrogen peroxide in the setting of glutathione reductase inhibition is dependent upon the enzymatic activity of red blood cell catalase. Catalase can exist in a number of forms. Hydrogen peroxide oxidizes the heme iron of the resting form of catalase (i.e., ferricatalase) to an oxyferryl group with a porphyrin radical called Compound I, in the process creating one molecule of water. Compound I then oxidizes another molecule of hydrogen peroxide, regenerating the ferricatalase form of catalase, and in the process, creates one molecule of oxygen and another molecule of water. The net result is that the <NUM> molecules of hydrogen peroxide are converted by the catalase into one molecule of molecular oxygen and two molecules of water. Compound I, however, can also be reduced by a single electron to Compound II, which is an inactive form of catalase. NADPH binds to catalase and inhibits the formation of Compound II. Glucose-<NUM> phosphate dehydrogenase (G6PD) deficiency, which is a common inherited genetic disorder, impairs NADPH production and can result in accumulation of Compound II and catalase inhibition. This can lead to hemolysis or methemoglobinemia under conditions of oxidative stress. Acquired G6PD deficiency or impaired NADPH production would lead to the same result. Low concentrations of ethanol are able to prevent the inactivation of catalase by converting Compound I into ferricatalase; in the process the ethanol is oxidized to acetaldehyde. The following references relate to this matter: <NPL>; <NPL>; <NPL>;.

The scope of the present invention includes methods to prevent the loss of catalase function and to prevent oxidant-induced hemolysis and/or methemoglobin formation in subjects treated with oxidant drugs or agents that generate hydrogen peroxide; said methods comprise the systemic administration of ethanol. The ethanol is administered prior to or during exposure to the oxidant. The dose of ethanol is in the approximate range of <NUM> to <NUM> grams. The ethanol can be given orally or intravenously. In a preferred embodiment, the dose of ethanol is approximately <NUM> to <NUM> grams/m2, given over approximately <NUM> hour intravenously. The drug can also be given as a constant intravenous infusion for longer periods of time.

The present invention also relates to a method for the treatment and effective treatment of metastatic cancer and refractory metastatic cancer. The method, referred to as embodiment E2, comprises the following:.

Embodiment Ee2 of the present invention is a set of drugs for use in a regimen for the treatment and effective treatment of metastatic cancer and refractory metastatic cancer. The set of drugs comprises:.

The cancers that can be treated with embodiments E2 and Ee2 are as described in LIST A.

In preferred embodiments, bone marrow stem cells are infused to reverse bone marrow toxicity. Stem cell infusions are generally given if the melphalan dose exceeds approximately <NUM>/m2 or the BCNU dose exceeds approximately <NUM>/m2 or if the patient has, or is expected to have, prolonged bone marrow suppression following the drug treatment. (This applies to all embodiments of the present invention in which BCNU and/or melphalan are used. ) The stem cells are collected prior to the administration of the chemotherapy drugs (e.g., melphalan), and are purified and stored. The bone marrow stem cells are preferably infused <NUM>-<NUM> days after the chemotherapy drugs. However, the stem cells can be administered at later times. Purified autologous bone marrow stem cells are strongly preferred. However, allogeneic bone marrow stem cells can also be employed. The use of purified stem cell preparations enriched for CD34+ hematopoietic cells and depleted of circulating tumor cells is preferred. Non-purified bone marrow stem cells can also be used.

In preferred embodiments of E2 and Ee2, the melphalan is in dose ranges of approximately <NUM> to <NUM>/m2, <NUM> to <NUM>/m2, <NUM> to <NUM>/m2, <NUM> to <NUM>/m2, and <NUM> to <NUM>/m2. In preferred embodiments, the melphalan dose is approximately <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>/m2. The melphalan is administered IV over a period of approximately <NUM> to <NUM> minutes, although longer times can be employed if steps are taken so that the melphalan is not degraded in the IV solution prior to administration. The melphalan is administered either immediately before, concomitantly with, or immediately after the BCNU, hydroxocobalamin, and ascorbic acid. In preferred embodiments, the melphalan, BCNU, hydroxocobalamin, and ascorbic acid are all administered within a <NUM> hour, <NUM> hour, <NUM> hour, <NUM> hour, <NUM> hour, and <NUM> hour time period.

In preferred embodiments of E2 and Ee2, the BCNU dose is approximately <NUM> to <NUM>/m2, <NUM> to <NUM>/m2, <NUM> to <NUM>/m2, <NUM> to <NUM>/m2, and <NUM> to <NUM>/m2. In preferred embodiments, the BCNU dose is approximately <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>/m2. The BCNU is administered IV at a rate of approximately <NUM>/m2/min. The BCNU can be administered before, concurrently, or immediately after the melphalan. The BCNU is preferably administered before the ascorbic acid.

In embodiments of E2 and Ee2, the ethanol dose is in the ranges of approximately <NUM> to <NUM> grams, <NUM> to <NUM> grams/m2, <NUM> to <NUM> grams/m2, and <NUM> to <NUM> grams/m2. In preferred embodiments, the ethanol dose is approximately, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> grams. The ethanol can be given orally or IV. When given intravenously the ethanol is given over approximately <NUM> minutes to <NUM> hours, depending upon the dose. The timing of the ethanol administration is before or concomitant with the administration of the ascorbic acid such that ethanol is present in the blood during the time of ascorbic acid exposure and hydrogen peroxide formation. In a preferred embodiment, the ethanol is given at the time of BCNU administration within a <NUM> hour period of ascorbic acid administration.

In preferred embodiments of E2 and Ee2 the hydroxocobalamin dose is in the ranges of approximately <NUM> to <NUM>,<NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM> grams and <NUM> to <NUM> grams. In other preferred embodiments, the hydroxocobalamin dose is approximately <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> grams. In other preferred embodiments, the hydroxocobalamin dose is in the ranges of approximately <NUM> to <NUM>,<NUM>/m2, <NUM> to <NUM>/m2, <NUM> to <NUM>/m2, <NUM> to <NUM> grams/m2, and <NUM> to <NUM> grams/m2. In other preferred embodiments, the hydroxocobalamin dose is approximately <NUM>/m2, <NUM>/m2, <NUM>/m2, <NUM>/mg, and <NUM>,<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> grams/m2. The hydroxocobalamin is administered IV over approximately <NUM> to <NUM> minutes. Both the hydroxocobalamin and ascorbic acid can be given simultaneously or essentially at the same time. Alternatively, the hydroxocobalamin can be given hours prior to the ascorbic acid, because hydroxocobalamin has a plasma half-life of approximately <NUM> to <NUM> hours. In a preferred embodiment, the hydroxocobalamin is given over approximately <NUM>-<NUM> minutes, immediately prior to the administration of the ascorbic acid, which is given over a time period of approximately <NUM>-<NUM> minutes.

In preferred embodiments of E2 and Ee2, the IV ascorbic acid dose is in the range of approximately, <NUM> to <NUM> grams/m2, <NUM> to <NUM> grams/m2, <NUM> to <NUM> grams/m2, <NUM> to <NUM> grams/m2, and <NUM> to <NUM> grams/m2. In preferred embodiments, the dose of ascorbic acid is approximately, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> grams. The ascorbic acid is given intravenously over approximately <NUM> to <NUM> minutes. In preferred embodiments, the ascorbic acid is given over approximately <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> minutes.

In preferred embodiments of E2 and Ee2, the melphalan, BCNU, ethanol, hydroxocobalamin, and ascorbic acid are all administered within a time period of approximately <NUM> hours. The drugs can also be given as split doses within the time period. In preferred embodiments, the time period is approximately <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> minutes, <NUM> days before the bone marrow stem cell infusion (i.e., on day minus <NUM>, where day <NUM> is the day of stem cell infusion.

For the sake of simplicity and economy of space, embodiments referring to specific doses of multiple drugs and specific types of metastatic cancers are uniquely specified with the nomenclature rules described below:.

"En" refers to the method of treating cancer described in embodiment number n. For example, E2 refers to the methods of embodiment E2. "Een" refers to the set of drugs described in embodiment Een. For example, Ee2 refers to the set of drugs described in embodiment Ee2. "EnS" and "EenS" refer to embodiments En and Een in which stem cells are infused. Note that a lack of an "S" suffix does not imply that stem cells are not infused. "En(ABCDFTUM)" and "Een(ABCDFTUM)" refer respectively to embodiments En and Een, wherein:.

"A", "B", "C", "D", "F", "T", "U" and "M" are numbers equal to <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. "ABCDFTUM" is a number in base <NUM>. Base <NUM> is the mathematical system in which the individual numbers or digits are limited to <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> in the positional numbering system. By contrast, base <NUM> is the standard numbering system with individual numbers <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>. Counting in base <NUM> is well known to one skilled in the art. The following reference relates to this matter: <NPL>. "A" refers to the approximate dose of melphalan, wherein A=<NUM> means the melphalan dose is <NUM> to <NUM>/m2, A=<NUM> means <NUM> to <NUM>/m2, A=<NUM> means <NUM> to <NUM>/m2, A=<NUM> means <NUM> to <NUM>/m2, and A=<NUM> means <NUM> to <NUM>/m2. "B" refers to the approximate dose of BCNU; wherein B=<NUM> means the dose is <NUM> to <NUM>/m2, B=<NUM> means <NUM> to <NUM>/m2, B=<NUM> means <NUM> to <NUM>/m2, B=<NUM> means <NUM> to <NUM>/m2, and B=<NUM> means <NUM>-<NUM>/m2. "C" refers to the approximate dose of ethanol; wherein C=<NUM> means no ethanol, C=<NUM> means the dose of ethanol is <NUM> to <NUM> grams/m2, C=<NUM> means <NUM> to <NUM> grams/m2, C=<NUM> means <NUM> to <NUM> grams/m2, C=<NUM> means <NUM> to <NUM> grams/m2
X. "D" refers to the approximate dose of hydroxocobalamin; wherein D=<NUM> means the dose is <NUM> to <NUM>/m2; D=<NUM> means <NUM> to <NUM>/m2, D=<NUM> means <NUM> to <NUM> grams/m2; D=<NUM> means <NUM> to <NUM> grams/m2, and D=<NUM> means <NUM> to <NUM>,<NUM>/m2 of hydroxocobalamin. "F" refers to the approximate dose of ascorbic acid; wherein F=<NUM> means the dose is <NUM> to <NUM> grams/m2, F=<NUM> means <NUM> to <NUM> grams/m2, F=<NUM> means <NUM> to <NUM> grams/m2, F=<NUM> means <NUM> to <NUM> grams/m2, and F=<NUM> means <NUM> to <NUM> grams/m2. "TUM" refers to the type of metastatic cancer or tumor, wherein when TUM has the values listed below, the metastatic cancer types that can be treated with the embodiment are as indicate below.

Using the above nomenclature, some additional embodiments of E2 and E2S are E2(ABCDFTUM) and E2S(ABCDFTUM) where ABCDFTUM = <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,. To save space, the ellipsis is used to represent all the intervening numbers in the sequence. In other words, ABCDFTUM = <NUM> to <NUM> sequentially in base <NUM>. Therefore, a list of some embodiments of E2 is: E2(<NUM>), E2(<NUM>), E2(<NUM>), E2(<NUM>), E2(<NUM>), E2(<NUM>), E2(<NUM>), E2(<NUM>), E2(<NUM>), E2(<NUM>), E2(<NUM>),. , E2(<NUM>). Similarly, a list of some embodiments of E2S is E2S(<NUM>), E2S(<NUM>), E2S(<NUM>), E2S(<NUM>), E2S(<NUM>), E2S(<NUM>), E2S(<NUM>), E2S(<NUM>), E2S(<NUM>), E2S(<NUM>), E2S(<NUM>),. , E2S(<NUM>).

A list of some embodiments of Ee2 is: Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>), Ee2(<NUM>),. , Ee2(<NUM>). A list of some embodiments of Ee2S is: Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>), Ee2S(<NUM>),. , Ee2S(<NUM>).

E3 is a method for the treatment of metastatic cancer in a subject, comprising administering a combination of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin and ascorbic acid, simultaneously or within a six-hour time period, and optionally administering ethanol and optionally administering stem cells.

Ee3 is a set of drugs or kit comprising <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid, for use in a regimen for the treatment of metastatic cancer; wherein the regimen comprises administering the drugs simultaneously or within a six-hour time period, and optionally administering ethanol and optionally administering stem cells.

A list of some embodiments of E3 is: E3(<NUM>), E3(<NUM>), E3(<NUM>), E3(<NUM>), E3(<NUM>), E3(<NUM>), E3(<NUM>), E3(<NUM>), E3(<NUM>), E3(<NUM>), E3(<NUM>),. , E3(<NUM>). A list of some embodiments of E3S is: E3S(<NUM>), E3S(<NUM>), E3S(<NUM>), E3S(<NUM>), E3S(<NUM>), E3S(<NUM>), E3S(<NUM>), E3S(<NUM>), E3S(<NUM>), E3S(<NUM>), E3S(<NUM>),. , E3S(<NUM>). A list of some embodiments of Ee3 is: Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>), Ee3(<NUM>),. , Ee3(<NUM>). A list of some embodiments of Ee3S is: Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>), Ee3S(<NUM>),. , Ee3S(<NUM>).

E4 is a method for the effective treatment of metastatic cancer in a subject, comprising administering a combination of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid, simultaneously or within a six-hour time period, and optionally administering ethanol and optionally administering stem cells.

Ee4 is a set of drugs or kit comprised of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid, for use in a regimen for the effective treatment of metastatic cancer; wherein the regimen comprises administering the drugs simultaneously or within a six-hour time period, and optionally administering ethanol and optionally administering stem cells.

A list of some embodiments of E4 is: E4(<NUM>), E4(<NUM>), E4(<NUM>), E4(<NUM>), E4(<NUM>), E4(<NUM>), E4(<NUM>), E4(<NUM>), E4(<NUM>), E4(<NUM>), E4(<NUM>),. , E4(<NUM>). A list of some embodiments of E4S is: E4S(<NUM>), E4S(<NUM>), E4S(<NUM>), E4S(<NUM>), E4S(<NUM>), E4S(<NUM>), E4S(<NUM>), E4S(<NUM>), E4S(<NUM>), E4S(<NUM>), E4S(<NUM>),. , E4S(<NUM>). A list of some embodiments of Ee4 is: Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>), Ee4(<NUM>),. , Ee4(<NUM>). A list of some embodiments of Ee4S is: Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>), Ee4S(<NUM>),. , Ee4S(<NUM>).

E5 is a method for the effective treatment of refractory metastatic cancer in a subject, comprising administering a combination of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid, simultaneously or within a six-hour time period, and optionally administering ethanol and optionally administering stem cells.

Ee5 is a set of drugs or kit comprising <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid, for use in a regimen for the effective treatment of refractory metastatic cancer; wherein the regimen comprises administering the drugs simultaneously or within a six-hour time period, and optionally administering ethanol and optionally administering stem cells.

A list of some embodiments of E5 is: E5(<NUM>), E5(<NUM>), E5(<NUM>), E5(<NUM>), E5(<NUM>), E5(<NUM>), E5(<NUM>), E5(<NUM>), E5(<NUM>), E5(<NUM>), E5(<NUM>),. , E5(<NUM>). A list of some embodiments of E5S is: E5S(<NUM>), E5S(<NUM>), E5S(<NUM>), E5S(<NUM>), E5S(<NUM>), E5S(<NUM>), E5S(<NUM>), E5S(<NUM>), E5S(<NUM>), E5S(<NUM>), E5S(<NUM>),. , E5S(<NUM>). A list of some embodiments of Ee5 is: Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>), Ee5(<NUM>),. , Ee5(<NUM>). A list of some embodiments of Ee5S is: Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>), Ee5S(<NUM>),. , Ee5S(<NUM>).

In some preferred embodiments of E2S and Ee2S, and E3S and Ee3S, and E4S and Ee4S, and E5S and Ee5S, the drug doses are as given below:.

In some preferred embodiments of the above embodiments, the cancer is pancreatic, breast, ovarian, or prostate. In some preferred embodiments of the above embodiments, the cancer is in a subject with an inherited BRCA1 and/or BRCA2 mutation.

E6 is a method for the treatment of metastatic cancer or refractory metastatic cancer in a subject, comprising administering a combination of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid, simultaneously or within a six-hour time period; wherein the melphalan dose is in the range of <NUM> to <NUM>/m2.

In a preferred embodiment of E6, <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea is administered at a dose range of <NUM> to <NUM>/m2; the melphalan is administered at a dose of <NUM> to <NUM>/m2; the hydroxocobalamin is administered at a dose of <NUM> to <NUM>,<NUM>/m2; and the ascorbic acid is administered a dose of <NUM> gram to <NUM> grams.

In a preferred embodiment of E6, the <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea is administered at a dose range of <NUM> to <NUM>/m2; the melphalan is administered at a dose of <NUM> to <NUM>/m2; the hydroxocobalamin is administered at a dose of <NUM> to mg to <NUM>/m2; and the ascorbic acid is administered a dose of <NUM> grams to <NUM> grams.

In a preferred embodiment of E6, the <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea is administered at a dose of <NUM>/m2; the melphalan is administered at a dose of <NUM>-<NUM>/m2; the hydroxocobalamin is administered at a dose of <NUM>/m2; and the ascorbic acid is administered a dose of <NUM> grams to <NUM> grams.

In preferred embodiments of the above E6 embodiments, the methods are further comprising systemically administering ethanol at a dose of <NUM> to <NUM> grams.

In preferred embodiments of the above E6 embodiments, the methods are further comprising bone marrow stem cell transplantation therapy.

In preferred embodiments of the above E6 embodiments, the methods are for the treatment for metastatic cancer in a subject with an inherited germline mutation in a gene involved in DNA repair, and/or homologous recombination, and or DNA crosslink repair.

In preferred embodiments of the above E6 embodiments, the methods are for the treatment for metastatic cancer in a subject with an inherited germline mutation in one or more of the following genes: ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1 (FANCJ, BACH1), EME1, ERCC1, ERCC4, FAN1, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCO, FANCP, FANCQ, FANCQ, FANCR, FANCS, FANCT, HELQ, MEN1, MUS81, NBN (NBS1), PALB2, RAD50, RAD51 (FANCR), RAD51C (FANCO), RAD51D, REV1, SLX4 (FANCP), UBE2T (FANCT), USP1, WDR48, XPF, XRCC2, and XRCC3.

In preferred embodiments of the above E6 embodiments, the methods are for the treatment for metastatic cancer in a subject with an inherited germline mutation in BRCA1 and/or BRCA2.

In preferred embodiments of the above E6 embodiments, the methods are for the treatment for metastatic cancer in a subject with one or more of the following types of cancer: pancreatic cancer, ovarian cancer, breast cancer, and prostate cancer.

E8 is a method of treating cancer comprising the administration of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, hydroxocobalamin, and ascorbic acid.

Embodiment E10 is a set or kit of pharmaceutical compositions for use in effectively treating metastatic cancer or refractory metastatic cancer in a subject, comprising a therapeutically effective dose of a combination of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, and ascorbic acid.

Embodiment E11 comprises the use of a pharmaceutical composition for the treatment of metastatic cancer or refractory metastatic cancer in a subject, comprising a therapeutically effective dose of a combination of <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin and ascorbic acid, wherein the melphalan dose is in the range of <NUM> to <NUM>/m2.

It will be appreciated that in the methods and compositions described herein, any suitable form of the active principles (e.g., drugs) may be used, e.g., a salt form, or a prodrug or active metabolite; these forms are within the scope of the present invention.

A preferred melphalan formulation comprises melphalan hydrochloride equivalent to <NUM>/ml of melphalan, <NUM>/ml of povidone, <NUM>/ml of sodium citrate, <NUM> of propylene glycol, <NUM> <NUM>% ethanol, and water to give a volume of <NUM>, which is then diluted with <NUM>% Sodium Chloride for intravenous Injection, USP, to give a melphalan concentration not greater than <NUM>/mL.

A preferred BCNU formulation comprises of <NUM> of BCNU dissolved in <NUM> of <NUM>% ethanol and <NUM> of Water for Intravenous Injection, USP, which is further diluted with <NUM>% Sodium Chloride Injection, USP to a BCNU concentration of approximately <NUM>/ml.

A preferred formulation comprises hydroxocobalamin dissolved in <NUM>% Sodium Chloride for Intravenous Injection, USP at a concentration of not more than <NUM>/ml.

A preferred formulation of ascorbic acid comprises ascorbic acid and an equimolar amount of sodium hydroxide with the pH adjusted to approximately <NUM> to <NUM> (with sodium hydroxide or sodium bicarbonate), which is diluted in Water for Intravenous Injection, USP, to give a final concentration of <NUM>/ml of ascorbic acid, which is isotonic with an osmolarity of ~ <NUM> mOsm/L. In other preferred formulations, the solution can be more concentrated with the ascorbic acid concentration ranging up to <NUM>/ml. Hypertonic solutions need to be given by a central IV line.

In certain embodiments, the pharmaceutical compositions described herein are formulated as a form suitable for oral administration, as a tablet, as a capsule, as a cachet, as a pill, as a lozenge, as a powder, or as a granule. In some embodiments of the present invention, the pharmaceutical compositions are formulated as sustained release formulations, solutions, liquids, or suspensions; for parenteral injection as a sterile solution, suspension or emulsion; for topical administration as an ointment, cream, lotion, spray, foam, gel, or paste; or for rectal or vaginal administration as a suppository or pessary. In certain embodiments, the pharmaceutical compositions are formulated in unit dosage forms suitable for single administration of precise dosages. In certain aspects, the pharmaceutical composition includes a conventional pharmaceutical carrier or excipient and an agent as described herein as an active ingredient. In addition, other medicinal or pharmaceutical agents, carriers, adjuvants, etc. are included. Exemplary parenteral administration forms include solutions or suspensions of active agents in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms are optionally buffered.

Suitable pharmaceutical carriers include inert diluents or fillers, water, and various organic solvents. The pharmaceutical compositions optionally contain additional ingredients such as flavorings, binders, excipients, and the like. For example, in a specific embodiment, tablets containing various excipients, such as citric acid, are employed together with various disintegrants. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate, and talc are optionally used. Other reagents such as an inhibitor, surfactant or solubilizer, plasticizer, stabilizer, viscosity increasing agent, or film-forming agent are also optionally added. In certain embodiments, solid compositions of a similar type are employed in soft or hard filled gelatin capsules. In certain embodiments, the pharmaceutical compositions and/or formulations described herein include lactose or milk sugar or high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active ingredient or ingredients are optionally combined with various sweetening or flavoring agents, coloring agents or dyes or, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof. Those of ordinary skill in the art are familiar with formulation and administration techniques that can be employed with the agents and methods of the invention, e.g., as discussed in <NPL>; and <NPL>. Formulations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active agents, which may contain antioxidants, buffers, biocide, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which optionally include suspending agents or thickening agents. Examples of suitable isotonic vehicles for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes or other microparticulate systems may be used to target the agent to blood components or one or more organs. The concentration of the active ingredient or ingredients in the solution varies depending on intended usage. Non-limiting examples of excipients that are used in conjunction with the present invention include water, saline, dextrose, glycerol, or ethanol. The injectable compositions optionally comprise minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or other such agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins. Drugs that have acidic or basic groups may be administered in formulations as pharmacologically acceptable salts; for example, melphalan may administered as melphalan hydrochloride, and ascorbic acid may be administered as sodium ascorbate. Examples of pharmaceutically acceptable carriers that are optionally used include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances.

The methods and compositions described herein can also further include additional therapeutic agents and drugs for the treatment of the cancer or for alleviating symptoms.

The drugs combinations employed in the present methods have high potential to cause nausea and emesis. Effective methods to control these side effects are known to one skilled in the art and would be employed in conjunction with the current methods. Generally, patients would be pre-treated with dexamethasone and a serotonin antagonist. Suitable protocols are known to one skilled in the art. The following references relate to this matter:<NPL>.

A patient with metastatic pancreatic cancer with an inherited BRCA2 mutation would be treated with the following protocol:.

Stem cell mobilization, collection, purification, and storage:.

Day Before Stem Cell Infusion: (day minus <NUM>).

Next treatment cycle:
<NUM>. Repeat steps <NUM>-<NUM> in approx. <NUM>-<NUM> weeks for a total of <NUM>-<NUM> courses of melphalan, BCNU, ethanol, hydroxocobalamin, ascorbic acid, and stem cell infusions.

In example <NUM>, the treatment is as described in Example <NUM>, however the melphalan is administered at a dose of <NUM>/m2 and the ascorbic acid dose is <NUM>,<NUM>/m2 over <NUM> minutes.

In example <NUM>, the treatment is as described in Example <NUM>, the patient has metastatic prostate cancer in the setting of an inherited BRCA2 mutation. However, the melphalan is administered at a dose of <NUM>/m2.

In example <NUM>, the treatment is as described in Example <NUM>, However, the BCNU dose is administered at a dose of <NUM>/m2.

In example <NUM>, the treatment is as described in Example <NUM>, However, the patient has pancreatic cancer and does not have a BRCA mutation.

Claim 1:
<NUM>,<NUM>-Bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, melphalan, hydroxocobalamin, ascorbic acid, or a pharmaceutically acceptable salt of any of the foregoing, for use in a method for the treatment of metastatic cancer or refractory metastatic cancer in a subject, comprising administering a combination of
(a) <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea,
(b) melphalan,
(c) hydroxocobalamin, and
(d) ascorbic acid,
or pharmaceutically acceptable salts of any of the foregoing,
and optionally administering
(e) ethanol and/or
(f) bone marrow stem cells,
wherein the melphalan, <NUM>,<NUM>-bis(<NUM>-chloroethyl)-<NUM>-nitrosourea, hydroxocobalamin, and ascorbic acid, or pharmaceutically acceptable salts of any of the foregoing, are all administered simultaneously or within a six-hour time period; wherein the melphalan dose is in the range of <NUM>/m<NUM> to <NUM>/m<NUM>.