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WO2006010073A1 - Prévention du cancer - Google Patents

Prévention du cancer Download PDF

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Publication number
WO2006010073A1
WO2006010073A1 PCT/US2005/024423 US2005024423W WO2006010073A1 WO 2006010073 A1 WO2006010073 A1 WO 2006010073A1 US 2005024423 W US2005024423 W US 2005024423W WO 2006010073 A1 WO2006010073 A1 WO 2006010073A1
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Prior art keywords
lonidamine
prostate
cancer
subject
prostate cancer
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English (en)
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George Tidmarsh
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Molecular Templates Inc
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Threshold Pharmaceuticals Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles

Definitions

  • the invention relates to methods for preventing prostate cancer and has application in the field of medicine and allied fields including but not limited to chemistry, medicinal chemistry, and biology.
  • Prostate cancer is one of the most prevalent malignancies in adult males and an increasingly prevalent health problem in the United States. It is typically a slow-growing malignancy that affects mostly older men. More than 75 percent of prostate cancers are detected in men over 65 years old. It also occurs in younger men, although it is rare in men under 40 years old. There remains a serious unmet need for new methods of preventing prostate cancer as well as other cancers involving epithelial cells (e.g., breast cancer).
  • Lonidamine also known as 1-(2,4-dichIorobenzyl)-1-H-indazole-3- carboxylic acid
  • the mechanism of action of lonidamine may involve interference with the energy metabolism of neoplastic cells by disruption of the mitochondrial membrane and by inhibition of hexokinase.
  • Lonidamine also has anti-spermatogenic activity and has been shown to inhibit germ cell respiration. See Gatto et al., 2002; Hagen et al., 2003; Heywood et al., 1981.
  • Lonidamine has been studied for use in the treatment of advanced breast cancer.
  • Mansi et al., 1991 reports a phase Il study in which lonidamine was administered in a daily divided oral dose of 600 mg. The investigators concluded that lonidamine appeared to be active against advanced breast cancer. Combination studies of lonidamine with other agents in the treatment of advanced breast cancer have also been reported.
  • Lonidamine has also been studied in lung cancer (Joss et al., 1984) in combination with radiation or anti-cancer agents.
  • Privitera et al. 1987 (lonidamine and radiotherapy); Gallo-Curcio et al., 1988 (chemotherapy or radiation therapy plus and minus LND); Giaccone ef a/., 1989 (lonidamine versus polychemotherapy); lanniello et al., 1996 (cisplatin, epirubicin, and vindesine with or without lonidamine); Gridelli et al., 1997 (VM-26 plus lonidamine); Cornelia et al., 1999 (cisplatin, gemcitabine, and vinorelbine with or without lonidamine); DeMarinis et al., 1999 (vindesine and LND); and Portalone et al., 1999 (cisplatin, epidoxorubicin, vindes
  • Lonidamine has been studied as a treatment for other cancers (see Robustelli et al., 1991 ; and Pacilio et al., 1984), including B-cell neoplasms (Robins et al., 1990); advanced colorectal cancer (Passalacqua et al., 1989, and Zaniboni et al., 1995); advanced gastric carcinoma (Barone et al., 1998); malignant glioma (Carapella et al., 1989; Carapella et al., 1990); metastatic cancers (Weinerman, 1990; DeAngelis et al., 1989; U.S. Patent No.
  • lonidamine is still not approved for use in the treatment of cancer in the United States, Asia, and most countries in Europe. Moreover, the use of lonidamine to prevent prostate cancer or other cancers in humans has not been described. There remains a serious unmet need for new methods of preventing cancer.
  • the present invention provides such methods, including methods for prevention of prostate cancer using lonidamine, lonidamine analogs, and other compounds.
  • the invention provides methods for prevention of prostate cancer and other epithelial cell cancers, such as breast cancer.
  • the invention provides a method of treating a human subject to reduce the likelihood of developing prostate cancer by (a) identifying the subject as being in a prostate cancer susceptibility population and (b) administering a prophylactically effective amount of lonidamine (LND) or a lonidamine analog (LNDA) to the subject.
  • LND lonidamine
  • LNDA lonidamine analog
  • the subject is diagnosed with prostatic intraepithelial neoplasia (PIN).
  • the subject has an elevated serum prostate specific antigen (PSA) level; has a rising PSA level; has a family history of prostate cancer; and/or has an elevated level of a prostate-cancer susceptibility marker.
  • PSA serum prostate specific antigen
  • the subject has undergone a prostate biopsy in which no evidence of prostate cancer was detected.
  • the invention provides a method for preventing prostate cancer in a human subject comprising administering a therapeutically effective amount of an energolytic agent (EA) to said human subject in need of such treatment, wherein the energolytic agent is an agent that interferes with energy metabolism in prostate epithelial cells.
  • the energolytic agent is selected from the group consisting of 2-deoxyglucose, 3-bromopyruvate, gossypol, oxamate, iodoacetate, apoptolidin, and a londamine analog.
  • the invention provides a method for preventing an epithelial cancer other than prostate cancer in a human subject comprising administering lonidamine or a lonidamine analog to a subject in need of prophylaxis.
  • Alkyl refers to a linear saturated monovalent hydrocarbon radical or a branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix.
  • (Ci-C 8 ) alkyl or CrC 8 alkyl includes methyl, ethyl, n-propyl, 2-propyl, n-butyl, 2-butyl, tert-butyl, pentyl, and the like.
  • (CrC 6 ) alkyl can be further optionally be substituted with substituents, including for example, hydroxy, amino, mono or di(CrC 6 ) alkyl amino, halo, C 2 -Ce alkenyl ether, cyano, nitro, ethenyl, ethynyl, CrC 6 alkoxy, Ci-C 6 alkylthio, -COOH, -CONH 2 , mono- or di(Ci-C 6 ) alkylcarbox-amido, -SO 2 NH 2 , -OSO 2 -(Ci-C 6 ) alkyl, mono or di(Ci-C 6 ) alkylsulfonamido, aryl and heteroaryl.
  • substituents including for example, hydroxy, amino, mono or di(CrC 6 ) alkyl amino, halo, C 2 -Ce alkenyl ether, cyano, nitro, ethenyl
  • alkenyl refers to a linear monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one double bond, but no more than three double bonds.
  • (C 2 -C 6 ) alkenyl includes, ethenyl, propenyl, 1 ,3-butadienyl and the like.
  • Alkenyl can be further optionally be substituted with substituents, including for example, hydroxy, amino, mono or di(Ci-C 6 ) alkyl amino, halo, C 2 -C 6 alkenyl ether, cyano, nitro, ethenyl, ethynyl, CrC 6 alkoxy, Ci-C 6 alkylthio, -COOH, -CONH 2 , mono- or di(Ci-Ce) alkyl-carboxamido, -SO 2 NH 2 , -OSO 2 -(Ci-C 6 ) alkyl, mono or di(Ci- C 6 ) alkylsulfonamido, aryl and heteroaryl.
  • substituents including for example, hydroxy, amino, mono or di(Ci-C 6 ) alkyl amino, halo, C 2 -C 6 alkenyl ether, cyano, nitro, ethenyl, ethy
  • Alkylene means a linear saturated divalent hydrocarbon radical having from one to twelve carbon atoms or a branched saturated divalent hydrocarbon radical having from one to twelve carbon atoms optionally substituted with substituents including for example, hydroxy, amino, mono or di(Ci-C 6 )alkyl amino, halo, C 2 -C 6 alkenyl ether, cyano, nitro, ethenyl, ethynyl, Ci-C 6 alkoxy, Ci-C 6 alkylthio, -COOH, -CONH 2 , mono- or di-(Ci-C 6 )alkyl-carboxamido, -SO 2 NH 2 , -OSO 2 -(Ci-C 6 ) alkyl, mono or di(Ci-C 6 ) alkylsulfonamido, aryl and heteroaryl.
  • alkylene includes methylene, ethylene, propylene, 2-methyl-propy
  • Heteroalkylene has essentially the meaning given above for alkylene except that one or more heteroatoms (i.e. oxygen, sulfur, nitrogen and/or phosphorous) may be present in the alkylene biradical.
  • heteroalkylene includes, -CH 2 OCH 2 O T CH 2 CH 2 OCH 2 CH 2 -, -CH 2 CH 2 N(CH 3 )CH 2 CH 2 -, - CH 2 CH 2 SCH 2 CH 2 -, and the like.
  • Aryl refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 10 ring atoms which is substituted independently with one to eight substituents, preferably one, two, three, four ot five substituents selected from alkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), -(CR'R") n -COOR (where n is an integer from O to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl,
  • R x and R y together is cycloalkyl or heterocyclyl. More specifically the term aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, and 2-naphthyl, and the substituted forms thereof.
  • Cycloalkyl refers to a monovalent cyclic hydrocarbon radical of three to seven ring carbons.
  • the cycloalkyl group can have one or more double bonds and can also be optionally substituted independently with one, two, three or four substituents selected from alkyl, optionally substituted phenyl, or -C(O)R 2 (where R z is hydrogen, alkyl, haloalkyl, amino, mono-alkylamino, di-alkylamino, hydroxy, alkoxy, or optionally substituted phenyl).
  • cycloalkyl includes, for example, cyclopropyl, cyclohexyl, cyclohexenyl, phenylcyclohexyl, A- carboxycyclohexyl, 2-carboxamidocyclohexenyl, 2-dimethylaminocarbonyl- cyclohexyl, and the like.
  • Heteroalkyl means an alkyl radical as defined herein with one, two or three substituents independently selected from cyano, -OR W , -NR x R y , and -S(O) P R Z (where p is an integer from 0 to 2 ), with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom of the heteroalkyl radical.
  • R w is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, aralkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, or mono- or di-alkylcarbamoyl.
  • R x is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl or araalkyl.
  • R y is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, mono- or di-alkylcarbamoyl or alkylsulfonyl.
  • R z is hydrogen (provided that n is 0), alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, amino, mono-alkylamino, di-alkylamino, or hydroxyalkyl.
  • Representative examples include, for example, 2- hydroxyethyl, 2,3-dihydroxypropyl, 2-methoxyethyl, benzyloxymethyl, 2-cyanoethyl, and 2-methylsulfonyl-ethyl.
  • R w , R x , R y , and R z can be further substituted by amino, halo, fluoro, alkylamino, di-alkylamino, OH or alkoxy.
  • the prefix indicating the number of carbon atoms refers to the total number of carbon atoms in the portion of the heteroalkyl group exclusive of the cyano, -OR W , -NR x R y , or -S(O) P R Z portions.
  • R x and R y together is cycloalkyl or heterocyclyl.
  • Heteroaryl means a monovalent monocyclic, bicyclic or tricyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, or S, the remaining ring atoms being C, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic ring.
  • the heteroaryl ring is optionally substituted independently with one to eight substituents, preferably one, two, three or four substituents, selected from alkyl, cycloalkyl, cycloalkyl-alkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, -COR (where R is hydrogen, alkyl, phenyl or phenylalkyl, -(CR'R") n -COOR (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), or -(CR'R")n-CONR x R y (where n is an integer from 0 to 5, R'
  • R x and R y together is cycloalkyl or heterocyclyl. More specifically the term heteroaryl includes, but is not limited to, pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrrolyl, pyrazolyl, pyridazinyl, pyrimidinyl, benzofuranyl, tetrahydrobenzofuranyl, isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, indolyl, isoindolyl, benzoxazolyl, quinolyl, tetrahydroquinolinyl, isoquinolyl, benzimidazolyl, benzisoxazolyl or benzothienyl, indazolyl, pyrrolopyrymidinyl, indolizinyl,
  • the heterocyclyl ring can be optionally substituted independently with one, two, three or four substituents selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, -COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), -(CR'R") n -COOR (n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or -(CR'R") n -CONR
  • heterocyclyl includes, but is not limited to, pyridyl, tetrahydropyranyl, N-methylpiperidin-3-yl, N-methylpyrrolidin-3-yl, 2-pyrrolidon-1-yl, furyl, quinolyl, thienyl, benzothienyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 1 ,1-dioxo-hexahydro-1 ⁇ 6 -thiopyran-4-yl, tetrahydroimidazo [4,5-c] pyridinyl, imidazolinyl, piperazinyl, and piperidin-2-only and the derivatives thereof.
  • preventing and grammatical equivalents thereof mean to reduce the risk of occurrence of cancer in an individual or in individuals in a population. That is, administration of an agent to a population of individuals "prevents" a condition when, relative to a control population, fewer individuals in the population develop the condition, and/or appearance of the condition is delayed in the administered population relative to a control population.
  • administering or “administration of a drug to a subject (and grammatical equivalents of these phrases) includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug.
  • a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient.
  • a prophylactically effective amount of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing the onset of disease or symptoms, or reducing or delaying the likelihood of the onset of disease or symptoms.
  • a "prophylactically effective amount" of a drug can also be an amount of a drug that, when administered to a subject, reduces the likelihood of recurrence of disease or symptoms, or delays the onset of disease or symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.
  • cancer has its ordinary medical meaning and refers to a malignancy (including head, neck, prostate and breast cancers, leukemias and lymphomas) generally characterized by clonality, autonomy, anaplasia and metastasis (see Mendelsohn, 1991).
  • Epithelial cancer is any malignant neoplasm originating from epithelium, i.e., a carcinoma. Examples of epithelial cancer include prostate; breast; lung; bladder; colon; esophageal; pancreatic; head and neck; skin, including basal cell carcinoma and squamous cell carcinoma; and stomach cancer.
  • biopsy refers to obtaining tissue from a patient and includes needle biopsy, resection and the like.
  • BPH Benign prostatic hyperplasia (also called benign prostatic hypertrophy); LND, Lonidamine; LNDA, Lonidamine analog; LUTS, Lower Urinary Tract Symptoms, usually associated with benign prostatic hypertrophy (BPH); LND/A; the acronym "LND/A” is used for ease of reading and means "lonidamine, or alternatively a lonidamine analog, or alternatively either lonidamine or a lonidamine analog.”
  • Lonidamine is an agent that can disrupt energy metabolism in a cell.
  • lonidamine was approved in Italy and Portugal for the treatment of cancer, including lung, breast, prostate, and brain cancers, and the use of lonidamine and lonidamine analogs (LND/A) for treatment and prevention of benign prostatic hyperplasia has been described (see PCT patent publication Nos. 2004/064735 and 2004/064736, both incorporated by reference herein).
  • the present invention relates to prevention of prostate cancer or other epithelial cell cancers using lonidamine, lonidamine analogs, and other agents (energolytic agents) that disrupt energy metabolism.
  • the invention relates to prevention of prostate cancer in a subject susceptible to developing prostate cancer (i.e., a subject in a "prostate cancer susceptibility population") by administering lonidamine, a lonidamine analog or another energolytic agent.
  • the invention relates to prevention of an epithelial cancer other than prostate cancer (e.g., breast cancer) by administering lonidamine, a lonidamine analog or another energolytic agent to a subject who will benefit from such administration, i.e., a subject in an "epithelial cancer susceptibility population.”
  • Lonidamine is 1-(2,4-dichlorobenzyl)-1-H-indazole-3-carboxylic acid.
  • Lonidamine was first identified as having anti-spermatogenic activity, and subsequently used in a limited number of European countries for treatment of certain cancers. The use of lonidamine and lonidamine analogs for treatment and prevention of benign prostatic hyperplasia has been described. When lonidamine was administered for 28-days to men with BPH, subjects experienced an average decrease in prostate volume of greater than 10% (see Example 1 , below, and Ditonno ef a/., 2005).
  • prostate gland contains secretory epithelial cells in a stroma of connective tissue and smooth muscle (Barry et al., 2003).
  • the secretory epithelial component in the normal prostate is remarkable in that the level of zinc in this tissue is exceedingly high compared to other normal tissues.
  • a consequence of the high zinc level is that, through a mechanism involving zinc inhibition of the enzyme m- aconitase (a key enzyme in the tricarboxylic acid (TCA) cycle), the generation of energy via the TCA cycle and oxidative phosphorylation is substantially reduced in the secretory epithelium of the prostate, making this tissue far more dependent than other tissues and organs upon glycolysis as an energy source.
  • m- aconitase a key enzyme in the tricarboxylic acid (TCA) cycle
  • TCA tricarboxylic acid
  • Another physiological result of the zinc-based inhibition of m-aconitase is the diversion of citrate from the TCA cycle, enabling the prostate to secrete large quantities of citrate, used by the sperm as an energy source and buffer, into the seminal fluid. See, e.g., Costello and Franklin, 1997, 2000; Costello et al., 1999, 2000.
  • Lonidamine induces apoptosis in primary cultures of human prostate epithelial cells (see Example 2 and US patent application publication no. 2004/0167196 published August 26, 2004 and incorporated by reference herein.
  • Lonidamine induces apoptosis in a cell line (LNCaP) derived from human prostate cells (see Example 2 and US 2004/0167196).
  • the methods of the invention provide for the preferential destruction of the citrate- producing cells in the prostate by inhibiting glycolysis or by further impairing mitochondrial function, or both, in those cells, such that enough of the citrate- producing cells are destroyed to reduce substantially the incidence of prostate cancer.
  • an agent that inhibits or impairs energy production in prostate epithelial cells is administered to a human or other mammal susceptible to prostate cancer at a dose that impairs energy production (decreases ATP levels) for a period of time that results in the preferential destruction of at least some of the citrate producing cells by starving them, relative to the normal cells in the body, of energy.
  • the agent is lonidamine.
  • the agent is a lonidamine analog, examples of which are described below in Section 7.
  • the agent is an agent other than lonidamine or a lonidamine analog that inhibits or impairs energy production in epithelial cells (i.e., an energolytic agent).
  • an energolytic agent i.e., an energolytic agent.
  • exemplary energolytic agents are described below in Section 8. Although for the sake of ease of reading the specification generally refers to lonidamine, it will be appreciated that the disclosure herein that relates to the uses of lonidamine apply mutatis mutandis to the uses of lonidamine analogs and energolytic agents.
  • agents useful in the practice of the invention are identified by their ability to mimic one or more other activities of lonidamine, such as induction of apoptosis or inhibition of hypoxic induction of HIF-1 alpha protein expression/accumulation in prostate epithelial cells or cell lines in vitro (e.g., inhibition of hypoxic induction of HIF-1 alpha protein expression/accumulation).
  • lonidamine, a lonidamine analog, or an energolytic agent is administered to a subject in need of prostate cancer prevention.
  • a subject in need of prostate cancer prevention is a man at risk for developing prostate cancer, e.g., a man diagnosed as at risk for developing prostate cancer.
  • Prostate cancer risk can be determined using art-known methods and criteria.
  • the invention relates to prevention of prostate cancer by administering LND/A to a subject in a specified "prostate cancer susceptibility population.”
  • Prostate cancer susceptibility populations can be defined by both inclusionary and exclusionary criteria. All contemplated prostate cancer susceptibility populations exclude females, males less than 21 years old, and males currently or previously diagnosed with or under treatment for prostate cancer (as defined herein).
  • Certain particular a prostate cancer susceptibility populations may also exclude any male:
  • a man with any of the above attributes is excluded from the prostate cancer susceptibility population.
  • each of the above exclusionary criteria can be independently considered, alone or in combination with inclusionary criteria discussed below, in defining a particular (i.e., different) prostate cancer susceptibility population.
  • the invention provides methods for prevention of prostate cancer in a man in a prostate cancer susceptibility population defined as above (e.g., not previously diagnosed with or under treatment for prostate cancer as defined herein and optionally not diagnosed as having a cancer other than prostate cancer and/or diagnosed with or under treatment for BPH of LUTS) and further characterized as having an indicator associated with an increased likelihood of developing prostate cancer. Since the likelihood of developing prostate cancer increases with age, increased age can be used as an indicator. More often, an indicator other than age known to be associated with an increased likelihood of developing prostate cancer is used.
  • Such non-age indicators include, without limitation, abnormal findings from a digital rectal examination, diagnosis of prostatic intraepithelial neoplasia (PIN), the presence of markers such as elevated and/or rising prostate specific antigen (PSA) levels or other prostate cancer-susceptibility markers, and a genetic predisposition to developing prostate cancer and/or familial history of prostate cancer, as described in more detail below.
  • PIN prostatic intraepithelial neoplasia
  • PSA prostate specific antigen
  • the invention provides a method of treating a human subject to reduce the likelihood of developing prostate cancer comprising (a) identifying the subject as being in a prostate cancer susceptibility population, and (b) administering a prophylactically effective amount of lonidamine (LND) or a lonidamine analog (LNDA) or energolytic agent to the subject.
  • LND lonidamine
  • LNDA lonidamine analog
  • prostate cancer refers to a positive clinical diagnosis that a subject has prostate cancer, i.e., a diagnosis confirmed by analysis of prostate tissue (e.g., obtained by biopsy). It will be appreciated that prostate cancer is a slow growing cancer. Cancers are often clonal, indicating that a single prostate cancer cell may appear the prostate years before cancer can be detected or diagnosed in the clinic. However, a man with a single prostate cancer cell is not considered to have prostate cancer as the term is used herein. Indeed, a man with hundreds or even thousands of prostate cancer cells is not considered to have prostate cancer as the term is used herein if the cancer cannot be detected or positively diagnosed using currently used and generally accepted methods for diagnosing prostate cancer.
  • Methods for diagnosis of prostate cancer are known and can include assessment of multiple factors including abnormal findings from a digital rectal examination, hypoechoic lesions detected by transrectal ultrasound (TRUS), serum PSA levels indicative of cancer, and/or histologically detected abnormality.
  • a diagnosis of prostate cancer as used herein requires the presence of indicators sufficient to establish basis for currently approved treatments (e.g., chemotherapy, radiation therapy or prostatectomy).
  • a diagnosis of prostate cancer as used herein requires histological evidence (i.e., analysis of tissue obtained by biopsy) of cancer.
  • a diagnosis of prostate cancer as used herein requires non-histological evidence (e.g., results from an assay for a cancer marker) that is accepted as a surrogate of histological results.
  • a result from a non-histological assay may be considered a surrogate of histological evidence of cancer when a positive result in the non- histological assay has an at least 90% correlation, and alternatively an at least 95% correlation, with a positive histological indication of cancer.
  • the "a subject in need of prostate cancer prevention" is a man who has undergone a prostate biopsy in which no evidence of prostate cancer was detected.
  • men who have been diagnosed as having a cancer (other than prostate cancer) and/or are under treatment for a cancer (other than prostate cancer) are not included in the prostate cancer susceptibility population.
  • BPH can be diagnosed using methods known to physicians (see, The Merck Manual of Diagnosis and Therapy, Section 17. Genitourinary Disorders Chapter 218. Prostate Disease). The most common test is the digital rectal examination in which a physician determines whether the prostate is of a normal size and firmness.
  • diagnostic assays include a urine flow rate test, determination of post void residual urine volume (e.g., by palpitation of the abdomen, drainage of residual urine, x-ray urogramography, or ultrasonography), moderate or severe symptom scores on the American Urologic Association Symptom Index (AUASI; Barry et al., 1992) or International Prostate Symptom Score (IPSS; Barry et a/., 2001).
  • AUASI American Urologic Association Symptom Index
  • IPSS International Prostate Symptom Score
  • lonidamine is administered to a man not under treatment for BPH.
  • BPH is treated using procedures such as surgery (transurethral resection of the prostate; transurethral incision of the prostate; or open prostatectomy), laser therapy, transurethral microwave thermotherapy, balloon dilatation, placement of a prostatic urethral stent, transurethral needle ablation, and transurethral electrovaporization of the prostate or by administration of drugs such as alpha-adrenergic-blockers (e.g., doxazosin, terazosin, tamsulosin, alfuzosin, and prazosin), 5-alpha-reductase (e.g., finasteride), and lonidamine (see US 2004/0167196).
  • alpha-adrenergic-blockers e.g., doxazosin, terazosin, tamsulosin, alfuzosin, and prazosin
  • subjects predicted to benefit significantly from the methods of the invention can be selected in a population of men with BPH by identifying subjects with a serum PSA value greater than 2 ng/ml.
  • the subject has a PSA level greater than about 4 ng/ml. Because higher PSA levels are closely associated with prostate cancer, in one embodiment, the subject selected for therapy with an energolytic agent has a PSA level greater than about 10 ng/ml.
  • LUTS Lower Urinary Tract Symptoms
  • BPH benign prostatic hypertrophy
  • the prostate cancer susceptibility population does not include men diagnosed as having and under treatment for one or more of the following symptoms: (1) urinary urgency; (2) terminal dribbling of urine; (3) frequent urination; (4) nocturia; (5) a weak/slow stream of urine; (6) a sense of incomplete emptying; (7) intermittency; (8) straining; (9) dysuria; (10) hematuria; (11) acute urinary retention; (12) urinary tract infection; and (13) incontinence.
  • men who have had surgical prostatectomy are not included in the prostate cancer susceptibility population.
  • lonidamine, a lonidamine analog, or an energolytic agent is administered to a subject in need of prostate cancer prevention such as a subject in a specified prostate cancer susceptibility population described herein.
  • the subject may be diagnosed as having Prostatic Intraepithelial Neoplasia (PIN) and/or having one or more (i.e., at least one) non-age indicators associated with an increased likelihood of developing prostate cancer and/or being at an increased likelihood of developing prostate cancer due to age.
  • PIN Prostatic Intraepithelial Neoplasia
  • the invention provides a method for preventing prostate cancer in a human patient by administering a therapeutically effective amount of lonidamine, a lonidamine agent or an energolytic agent to the patient.
  • an agent is administered to a subject diagnosed as having Prostatic Intraepithelial Neoplasia (PIN).
  • PIN is characterized by abnormal cellular proliferation within the prostatic ducts, ductules and acini.
  • PIN has previously been referred to as "intraductal hyperplasia,” “hyperplasia with malignant change,” “large acinar atypical hyperplasia,” “marked atypia” and “ductal-acinar dysplasia.”
  • a subject diagnosed as having PIN is, in one embodiment of the invention, at increased risk of developing prostate cancer.
  • PIN is sometimes characterized as high grade PIN (HGPIN) or low grade PIN.
  • HGPIN is associated with the progressive development of abnormalities in the normal prostatic epithelium, leading to a cancerous condition. See, e.g. t Bostwick, 1992.
  • Patients diagnosed as having HGPIN have an increased likelihood of developing prostate cancer within 10 years.
  • the invention provides a method for preventing prostate cancer in a human patient diagnosed with High-Grade Prostatic Intraepithelial Neoplasia (HGPIN) by administering a therapeutically effective amount of lonidamine, a lonidamine agent or an energolytic agent to the patient.
  • HGPIN is characterized by marked cellular proliferation within prostatic ducts, ductules, and acini.
  • HGPIN is associated with proliferative changes within preexisting acini, nuclear and nucleolar abnormalities (e.g., nuclear enlargement and hyperchromasia, nuclear overlapping or pseudo-stratification, prominent nucleoli), and fragmentation of basal cell layer.
  • Typical architectural patterns are associated with HGPIN are tufting, micropapillary, cribiform and flat. See, e.g., Bostwick et a/., 1993.
  • Other, less common patterns of HGPIN include a signet ring-cell pattern, a small cell neuroendocrine pattern, and a mucinous pattern.
  • Proliferation associated with HGPIN can spread through the prostatic ducts in different patterns.
  • neoplastic cells replace the normal luminal secretory epithelium, but the basal layer and basement membrane are preserved.
  • a second pattern is characterized by direct invasion through the ductal or acinar wall with disruption of the basal cell layer.
  • neoplastic cells invaginate between the basal cell layers, sometimes described as pagetoid spread.
  • an agent e.g., lonidamine
  • an agent is administered to a patient having HGPIN characterized by the first spreading pattern.
  • an agent is administered to a patient having HGPIN characterized by the second or third spreading pattern.
  • the patient is diagnosed with Low Grade Prostatic Intraepithelial Neoplasia (LGPIN).
  • LGPIN Low Grade Prostatic Intraepithelial Neoplasia
  • LGPIN Low Grade Prostatic Intraepithelial Neoplasia
  • the epithelium lining ducts and acini are heaped up, crowded and irregular. See, e.g., Bostwick, 1992.
  • Elongated, hyperchromatic nuclei and small nucleoli can be present. Lesions displaying some, but not all features, are considered atypical, but may not be neoplastic. LGPIN is present in many men by the third decade of life. Patients with LGPIN can be monitored for progression to HGPIN.
  • PIN can be diagnosed by any method known to the skilled artisan and accepted in the medical community.
  • PIN can be diagnosed by needle biopsy, such as a transrectal ultrasound-guided biopsy. See, e.g., Borboroglu et al., 2000; Davidson et al., 1995.
  • Prostate biopsies are often performed due to a presence of a palpable lesion in the prostate or other indicia know to physicians.
  • 8-10 cores are prepared for examination by a pathologist for characteristic morphological changes in the prostate. If evidence of PIN is detected, PIN can be monitored, for example, by monitoring PSA levels, as elevated PSA levels may indicate progression to a cancerous state.
  • markers of secretory differentiation include secretory proteins, cytoskeletal proteins, glycoproteins, and neuroendocrine cells.
  • markers include secretory proteins, cytoskeletal proteins, glycoproteins, and neuroendocrine cells.
  • PIN diagnosis also can be performed on biopsy samples by, for example, measuring uteroglobin protein or mRNA levels. See, e.g., U.S. patent publication No.
  • increased likelihood of having PIN can be determined by assaying for Prostate Stem Cell Antigen (PSCA) positive (+) cells within various biological samples, such as serum and prostate biopsy specimens. See, e.g., U.S. patent publication No. 2005/0059099, published March 17, 2005 (the disclosure of which is incorporated by reference herein).
  • morphological analysis can be used to confirm a diagnosis of a grade of PIN.
  • a subject diagosed with PIN and treated with lonidamine, a lonidamine analog or another energolytic agent is evaluated after a period of treatment (e.g., 1 month, six months, one year, etc.) or number of drug administrations (e.g., one administration, daily administration for 1 to 6 months, etc.) and a needle biopsy of the subject's prostate is preformed and evaluated for evidence of PIN.
  • the absence of indicators of PIN or a reduction of the extent of PIN is indicative of the efficacy of the agent, dose, or administration regimen in preventing prostate cancer (or, equivalently, treating PIN) in the individual.
  • a clinical trial is used to assess the efficacy of a particular energolytic agent, dose, or administration regimen in the practice of the present invention.
  • patients diagnosed with HGPIN can be enrolled and receive the agent (e.g., lonidamine) or a placebo, and subjected to one or more follow-on biopsies at a later time point(s) (e.g., six months, one year and five years the initiation of therapy).
  • An efficacy parameter would be a reduction in the rate of detection of prostate cancer in the agent population compared to the placebo population. For example, the rate might be reduced by at least 20%, at least 30% or more.
  • an agent is administered to a subject not diagnosed with prostate cancer, but instead diagnosed by digital rectal examination as having an abnormal prostate size or shape (e.g., a palpable lump).
  • Prostate size and shape can be used to assess prostate cancer risk; the most common test for prostate size and shape is the digital rectal examination in which a physician determines whether the prostate is of a normal size and firmness.
  • Abnormalaties of size, shape or structure e.g., density
  • an agent is administered to a subject not diagnosed with prostate cancer, but having an elevated serum prostate specific antigen (PSA) level.
  • PSA prostate specific antigen
  • Men 40 years old or younger typically have blood PSA levels below 2.0 ng/ml.
  • the range of "normal" PSA increases with age. Normal PSA levels will vary according to the size of the prostate, race, and assay method as well as age, but typical values are shown in Table 1.
  • the subject has a higher than normal PSA level. In one embodiment of the invention, the subject has a PSA level greater than about 2 ng/ml. In one embodiment of the invention, the subject has a PSA level greater than about 2 ng/ml but less than about 8 ng/ml.
  • the subject has a PSA level greater than about 2 ng/ml but less than about 10 ng/ml. In one embodiment of the invention, the subject has a PSA level from 0 to 9 ng/ml, 0 to 8 ng/ml, 0 to 7 ng/ml, 0 to 6 ng/ml, 0 to 5 ng/ml, 0 to 4 ng/ml, 0 to 3 ng/ml or less than 2 ng/ml. In one embodiment of the invention, the subject has a PSA level greater than about 4 ng/ml. In one embodiment of the invention, the subject has a PSA level greater than about 4 ng/ml but less than about 10 ng/ml.
  • an agent is administered to a subject not diagnosed with prostate cancer, but having a rising PSA level. Increases in PSA levels over time are also correlated with prostate cancer. Subjects predicted to benefit significantly from the methods of the invention can be selected in a population of men with rising serum PSA levels.
  • the phrase "rising PSA levels” means a velocity of at least 0.5Q ng/ml/yr or at least 0.75 ng/ml/yr. In one embodiment, the phrase "rising PSA levels” means a PSA doubling time of less than 120 months. PSA levels can be determined as discussed above.
  • an agent is administered to a subject not diagnosed with prostate cancer, but is identified as having an elevated level of a prostate- cancer susceptibility marker.
  • markers can include, for example, prostatic acid phosphatase (Fong et al., 2003); AMACR (Evans, 2003); prostate-specific membrane antigen (PSMA); Pro109 (Freje et al., 1993); Pro112 WO9514772 and WO9845436); Pro111 (Dubbink et al., 1998); Pro115 (Paoloni-Giacobino et al., 1997; WO9837418 and WO987093); Pro110 (U.S. Patent No.
  • an agent is administered to a subject not diagnosed with prostate cancer, but who instead has been diagnosed as having a genetic predisposition to developing prostate cancer.
  • Prostate cancer risk can also be assessed by screening of genetic markers associated with an increased risk of developing prostate cancer. For example, certain polymorphisms of the CAPB, HPC1/RNASEL, HPC2/ELAC2, HPCX, MSR1 , PCAP, HPC20, SRD5A2, androgen receptor (AR) and VDR loci are associated with increased susceptibility to prostate cancer. See Nieder et a/., 2003. See also Verhage et a/., 2003.
  • a subject in a prostate cancer susceptibility population has polymorphism in at least one of the CAPB, HPC1/RNASEL, HPC2/ELAC2, HPCX, MSR1 , PCAP, HPC20, AR, SRD5A2 and VDR loci, which polymorphism is associated with an increased risk of developing prostate cancer.
  • Methods of detecting polymorphisms in susceptibility loci are known to persons of skill in the art. Such methods can include, for example, polymerase chain reaction-based methods, restriction fragment length polymorphism analysis (RFLP) and single strand conformation analysis (SSCP).
  • Methods of differentiating polymorphic (allelic) variants utilize molecular marker techniques well known to those of skill in the art including such techniques as: 1) single stranded conformation analysis (SSCP); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6) allele-specific PCR.
  • SSCP single stranded conformation analysis
  • DGGE denaturing gradient gel electrophoresis
  • ASOs allele-specific oligonucleotides
  • 5) the use of proteins which recognize nucleotide mismatches such as the E. coli mutS protein
  • allele-specific PCR See generally Ausubel et a/., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. (1998) including supplements
  • an agent is administered to a subject not diagnosed with prostate cancer, but having a familial history of developing prostate cancer.
  • a risk of developing prostate cancer can be correlated with a familial history of prostate cancer.
  • the increased risk has been reported to be two- to three-fold greater. See Verhage et al., 2003.
  • the risk is higher having multiple affected relatives or having relatives diagnosed at an early age with prostate cancer. Id.
  • Such a risk can be determined, for example, from a family history. For example, subjects having a cluster of three or more first-degree relatives with prostate cancer can have an increased risk of developing prostate cancer. See Nieder et al., 2003.
  • Subjects having a family history of prostate cancer in each of three generations in the paternal or maternal lineage also can have an increased risk of developing prostate cancer. Id. Also, subjects having two or more first- or second- degree relatives with prostate cancer under age 55 have an increased risk of developing prostate cancer. Id.
  • a subject in a prostate cancer susceptibility population has a cluster of three or more first-degree relatives with prostate cancer can have an increased risk of developing prostate cancer.
  • a subject in a prostate cancer susceptibility population has a family history of prostate cancer in each of three generations in the paternal or maternal lineage.
  • a subject in a prostate cancer susceptibility population has two or more first- or second-degree relatives under age 55 diagnosed with prostate cancer.
  • a subject in a prostate cancer susceptibility population has at least one male relative selected from the group consisting of sons, father, and uncles who has had prostate cancer.
  • Age is a risk factor for developing prostate cancer, with more than 75% percent of prostate cancer diagnosed in men ages 65 or older.
  • the prostate cancer susceptibility population (i) comprises men 40 years or older (i.e., excludes men younger than 40 years old); (ii) comprises men 50 years or older (i.e., excludes men younger than 50 years old) or (iii) comprises men 60 years or older (i.e., excludes men younger than 60 years old).
  • the prostate cancer susceptibility population (i) comprises men younger than 40 years (i.e., excludes men 40 years or older); (ii) comprises men younger than 50 years (i.e., excludes men 50 years or older); (iii) comprises men younger than 60 years (i.e., excludes men 60 years or older).
  • lonidamine is administered to a man BPH identified as having a serum PSA value greater than about 10 ng/ml.
  • the invention provides a method of treating a human subject to reduce the likelihood of developing prostate cancer comprising (a) identifying the subject as being in a prostate cancer susceptibility population, and (b) administering a prophylactically effective amount of lonidamine (LND) or a lonidamine analog (LNDA) or energolytic agent to the subject.
  • LND lonidamine
  • LNDA lonidamine analog
  • energolytic agent energolytic agent
  • some prostate cancer susceptibility populations can be defined by both exclusionary and inclusionary criteria.
  • exemplary prostate cancer susceptibility populations who would benefit from prevention of prostate cancer include any of the following populations
  • EPITHELIAL CANCER SUSCEPTIBILITY POPULATION [0072]
  • methods for preventing epithelial cancer in a subject are provided.
  • agents e.g., lonidamine, a lonidamine analog or an energolytic agent
  • administration of agents e.g., lonidamine, a lonidamine analog or an energolytic agent
  • agents e.g., lonidamine, a lonidamine analog or an energolytic agent
  • the invention provides a method of prophylaxis of an epithelial cell cancer other than prostate cancer comprising administering a prophylactically effective amount of lonidamine or a lonidamine analog or an energolytic agent to the subject in need of such prophylaxis.
  • the invention provides a method of treating a human subject to reduce the likelihood of developing an epithelial cancer, said method comprising (a) identifying the subject as being in an "epithelial cancer susceptibility population," and (b) administering a prophylactically effective amount of lonidamine or a lonidamine analog to the subject.
  • Patients in the epithelial cancer susceptibility population do not have a diagnosed epithelial cancer but may have an indicator associated with an increased likelihood of developing an epithelial cancer.
  • Epithelial cancer susceptibility populations can be defined by both inclusionary and exclusionary criteria.
  • the epithelial cancer susceptibility population excludes patients:
  • epithelial cancer a malignant neoplasm originating from epithelium, i.e., a carcinoma, e.g., prostate; breast; lung; bladder; colon; esophagus; pancreas; head and neck; skin or stomach cancer;
  • Epithelial cancer is typically indicated by abnormal physical examination results (e.g., abnormal breast examination results), by abnormal results from an X- ray, ultrasonographic or other procedure.
  • a positive diagnosis usually involves histologic confirmation, such as by biopsy or other diagnostic measure.
  • diagnosis confirmed by analysis of tissue e.g., obtained by biopsy.
  • Inclusionary criteria for an epithelial cancer susceptibility population are indicators associated with an increased likelihood of developing epithelial cancer such as, without limitation, a genetic predisposition to developing epithelial cancer and/or familial history of epithelial cancer.
  • a subject having increased risk of developing epithelial cancer has one or more (i.e., at least one) indicators associated with an increased likelihood of developing epithelial cancer.
  • Such indicators include those set forth below.
  • an agent is administered to a subject not diagnosed with epithelial cancer, but instead diagnosed as having an elevated level of an epithelial cancer susceptibility marker.
  • markers can include, for example, CA- 125 (epithelial cancer), HER2 (breast cancer), Topoisomerase Il alpha (ovarian epithelial cancer), Werner helicase interacting protein (ovarian epithelial cancer), HEXIM1 (ovarian epithelial cancer), FLJ20267 (ovarian epithelial cancer), Deadbox protein-5 (ovarian epithelial cancer), Kinesin-like 6 (ovarian epithelial cancer), p53 (ovarian epithelial cancer) and NY-ESO-1 (ovarian epithelial cancer). See, e.g., Menard et al., 2004; WO 03/064593.
  • an agent is administered to a subject not diagnosed with epithelial cancer, but who instead has been diagnosed as having a genetic predisposition to developing epithelial cancer.
  • Epithelial cancer risk can also be assessed by screening of genetic markers associated with an increased risk of developing epithelial cancer.
  • the BRCA1 , BRCA2, p53, PTEN, ATM, NBS1 or LKB 1 loci are associated with increased susceptibility to epithelial breast cancer. See, e.g., Dumitrescu et al., 2005.
  • a subject in an epithelial cancer susceptibility population has a polymorphism in at least one of the BRCA1 , BRCA2, p53, PTEN, ATM, NBS1 and LKB1 , which polymorphism is associated with an increased risk of developing epithelial cancer.
  • a subject in an epithelial cancer susceptibility population has a polymorphism at the BRCA1 locus associated with an increased risk of developing epithelial cancer.
  • a subject in an epithelial cancer susceptibility population has a polymorphism at the p53 locus associated with an increased risk of developing epithelial cancer.
  • a subject in an epithelial cancer susceptibility population has a polymorphism at the PTEN locus associated with an increased risk of developing epithelial cancer.
  • a subject in an epithelial cancer susceptibility population has a polymorphism at the ATM locus associated with an increased risk of developing epithelial cancer.
  • a subject in an epithelial cancer susceptibility population has a polymorphism at the NBS1 locus associated with an increased risk of developing epithelial cancer.
  • a subject in an epithelial cancer susceptibility population has a polymorphism at the LKB1 locus associated with an increased risk of developing epithelial cancer.
  • an agent is administered to a subject not diagnosed with epithelial cancer, but diagnosed instead as having a familial history of developing epithelial cancer.
  • a risk of developing epithelial cancer can be correlated with a familial history of epithelial cancer. See, e.g., Dumitrescu et al., 2005 (breast cancer). The risk is higher for subjects having multiple affected relatives or having relatives diagnosed at an early age with epithelial cancer. Such a risk can be determined, for example, from a family history. For example, subjects having one or more first-degree relatives with epithelial cancer can have an increased risk of developing breast cancer.
  • a subject in an epithelial cancer susceptibility population has a cluster of one or more first-degree relatives with epithelial cancer can have an increased risk of developing epithelial cancer.
  • a variety of routes, dosage schedules, and dosage forms are appropriate for administration of agents according to the invention to prevent prostate or other epithelial cancer. Appropriate dosage schedules and modes of administration will be apparent to the ordinarily skilled practitioner upon reading the present disclosure and/or can be determined using routine pharmacological methods and/or methods described herein. [0084] The dose, schedule and duration of administration of the agent will depend on a variety of factors. The primary factor, of course, is the choice of a specific agent.
  • lonidamine administered to treat cancer is administered in 150 mg or 300 mg doses three times a day for a period of about a month
  • goal e.g., prophylaxis or prevention of relapse
  • preferred mode of administration of the drug the formulation used, patient response to the drug, and the like.
  • Guidance concerning administration is provided by prior experience using the agent for a different indication (e.g., lonidamine administered to treat cancer is administered in 150 mg or 300 mg doses three times a day for a period of about a month) and from new studies in humans (e.g., lonidamine administered to treat BPH has been administered in 150 mg doses once a day for a period of about a month) and other mammals.
  • an agent can be administered for the prevention of prostate cancer at a dose in the range of about 0.1 mg to about 100 mg of the agent per kg of body weight of the patient to be treated per day, optionally with more than one dose being administered per day, and typically with the daily dose being administered on multiple consecutive days.
  • an agent is administered in a dose in the range of about 0.2 mg to about 5 mg per kg of body weight of the patient to be treated per day.
  • an agent is administered in a dose in the range of about 0.2 mg to about 1 mg per kg of body weight of the patient to be treated.
  • an agent is administered in a dose of about 25 to 250 mg.
  • a prophylactically effective dose is about 25 to about 150 mg.
  • the prophylactically effective dose of an agent can be administered daily or once every other day or once a week to the patient.
  • Controlled and sustained release formulations of the agents may be used.
  • multiple administrations of the agent are employed.
  • the entire dose may be administered once daily, or the dose may be administered in multiple smaller doses through the course of a day.
  • the dose may be divided into two smaller doses and administered twice daily, or divided into three smaller doses and administered thrice daily.
  • the dose may be combined and given every other day, or even less frequently, but in any event, the dose is repeatedly administered over a period of time.
  • the administration of the prophylactically effective dose may be continued for multiple days, such as for at least five consecutive days, and often for at least a week and often for several weeks or more.
  • the agent is administered once (qday), twice (bid), three times (tid), or four times (qid) a day or once every other day (qod) or once a week (qweek), and treatment is continued for a period ranging from three days to two weeks or longer.
  • the dosage form is the 150 mg unit dosage form marketed under the trade name DORIDAMINATM (e.g., 150 mg po TID for about thirty days), and this dosage form is administered from once to three times daily for full preventive effect.
  • DORIDAMINATM e.g., 150 mg po TID for about thirty days
  • dosing regimens contemplated include, for example and not for limitation, "low dosing” (e.g., dosages in the range of 1-300 mg per day total daily dosage, 5-300 mg/day, 5-70 mg/day, 1-25 mg/day, 20-45 mg/day, 40-65 mg/day, 40-70 mg/day, 50-100 mg/day, 50-200 mg/day, and 50-300 mg/day), "high dosing (e.g., total daily doses greater than 0.5 g, such as doses in the range 0.5 - 5 g/day, 0.5 - 3 g/day, 0.5 - 1 g/day and 1-3 g/day, or higher doses), and “intermediate dosing” (e.g., doses greater than 300 and less than 500 mg/day, such as doses in the range >300-400 or 400 ⁇ 500 , e.g., 450 mg/day).
  • low dosing e.g., dosages in the range of 1-300 mg
  • the present invention also provides a pharmaceutical formulation of an energolytic agent suitable for oral administration (including tablets, capsules, and pills) and contains between 1 and 100 mg of the compound, and in another embodiment between 1 and 10 mg of the compound. In another embodiment, the formulation contains between 200 and 1000 mg of the compound, and in another embodiment between 500 and 1000 mg of the compound.
  • This treatment time period may include continuous dosing TID for two to six months or more or for only one to eight weeks.
  • a dose of 150 mg po TID for 7-30 days of certain agents of the invention, such as lonidamine (and its analogs) can allow for the full therapeutic benefit for an extended period of time (3 months or longer, including up to 6 months to one year) while limiting or eliminating the unwanted side effects.
  • prophylactic treatment of prostate cancer in accordance with the methods of the invention by administering to a patient a much higher dose of an agent for a shorter period of time (that is, fewer administrations; in one embodiment, a single administration of an agent is sufficient to provide protection for a period of 3 to 6 months or longer).
  • low doses are administered for prophylaxis.
  • Exemplary very low dose regimens include (in mg) less than 25 per day, less than 20 per day, less than 15 per day, less than 10 per day, and less than 5 per day.
  • Exemplary very low dose regimens include (in mg) less than 1 mg/month (administered by any treatment schedule, e.g. one 1 mg dose, 30 time 33 mg dose, 4 time 250 mg dose, etc.), less than 500 mg/month, less than 250 mg/month, or less than 100 mg/month.
  • a low dose form is used for a maintenance dose after a higher initial, priming or loading dose.
  • the agent can be administered a single time or many times over periods as long as several months or years.
  • the treatment is continued for one to three months.
  • the treatment is continued for a year.
  • a patient may be administered the agent for a week, a month, two months, three months, six months, one year, two years, five years, ten years, twenty years or longer.
  • treatment may continue indefinitely throughout the life of the patient.
  • treatment may be suspended temporarily if toxicity is observed or for the convenience of the patient without departing from the scope of the invention.
  • preferred dosage forms include pills, tablets, capsules, caplets, and the like, optionally formulated for sustained release.
  • suitable forms for oral administration include troches, elixirs, suspensions, syrups, wafers, lozenges, and the like.
  • Other modes of administration are also contemplated, including parenteral, inhalation spray, transdermal, rectal, intraprostetic injection (e.g., of agent-containing microparticles) and other routes. See, Ansel et al., 1999; and Marshall, 1979.
  • the dosage form is the 150 mg unit dosage form marketed for the treatment of cancer by ACRAF in Italy under the trade name DORIDAMINATM.
  • DORIDAMINATM contains 150 mg lonidamine, 87 mg starch, 55 mg microgranular cellulose, 40 mg lactose, 15 mg sodium carboxymethyl starch, 11 mg hydroxypropyl methylcellulose, 4.5 mg precipitated silica, 3.5 mg magnesium Stearate, 0.4 mg titanium dioxide, and 0.4 mg polyethylene glycol 400.
  • Another formulation is 150 mg lonidamine (intra-granular), 87 mg cornstarch (intra-granular), 40 mg lactose (intra-granular), 3 mg colloidal silicon dioxide (intra-granular), 1.5 mg colloidal silicon dioxide (extra-granular), 55 mg microcrystalline cellulose (extra- granular), 15 mg sodium starch glycolate (extra-granular), 3.5 mg magnesium stearate (extra-granular), 9 mg hydroxypropyl methylcellulose (extra-granular), and 10.9 mg OPADRY white (coating).
  • dosing schedules are for illustration and not limitation, and that a dosing schedule may change during a course of therapy based on, for example, a patient's response to the drug administered. As is well understood in the medicine, treatment may be suspended temporarily if toxicity is observed or for the convenience of the patient without departing from the scope of the invention.
  • results of prevention of prostate cancer in a subject may include a reduction in prostate size, a reduction in serum PSA, and/or results that will be recognized by a treating physician as indicative of a apoptosis of prostate cells (e.g., epithelial cells).
  • prostate size decreases by at least 10%, at least 20% or at least 40% and/or serum PSA levels decreases by at least 10%, at least 20% or at least about 40%, when determined on or after 60 days after the initiation of treatment and compared to a baseline prior to the initiation of drug administration.
  • An assessment of the response to the agent can be made at any time following the first administration of the drug.
  • an assessment is made about 30 days, about 60 days, or about 90 after beginning treatment.
  • assessment can be made about 6, 12, 18, 24 or more months after beginning treatment.
  • an assessment can be made less than about 30 days, about 30 days, about 60 days, or about 90 days after a course of treatment ends.
  • Analogs of lonidamine can be used for prevention of cancer.
  • Examples of lonidamine analogs include tolnidamine; AF-2364; and AF-2785 (see Ansari et al., 1998; and Corsi et al, 1976); and compounds described by Silvestrini, 1981 ; Lobl et al., 1981 ; Cheng et al., 2001 ; Andreani, F., 1984; Besner ef a/., 1997; Caputo, A., 1981 ; Hagishita et al., 1996; Kakehi et al., 1978; Milanesio et al., 2000; Palacios et al., 1995; Silvestrini et al., 1984; Tapia et al., 1999; Wright and Collins, 1956: U.S.
  • V 1 , V 2 , V 3 , and V 4 is independently selected from the group consisting of hydrogen, substituted or unsubstituted (Ci-C 4 ) alkyl or heteroalkyl, halogen, hydroxy, (Ci-C 4 ) alkoxy, cyano, nitro, amino, (CrC 4 ) alkylamino and (Cr C 4 ) dialkylamino or V 1 and V 3 together form a cycloakyl, a heterocycloalkyl, a cycloalkenyl, an aryl or a heteroaryl ring; with the proviso that if one of V 1 and V 2 is hydroxyl, amino, (CrC 4 ) alkylamino or (CrC 4 )
  • R 2 is an aryl or heteroaryl group, optionally substituted with from one to three R 6 substituents that are independently selected from the group consisting of halo and a straight or branched chain (CrC ⁇ )alkyl;
  • R 3 is H, (Ci-C 8 )alkyl or heteroalkyl, (C 3 -C 8 )cycloalkyl or heterocyclyl, or aryl or heteroaryl;
  • R 4 is NR 3 R 7 ,NR 3 OR 7 , NR 7 NR 3 R 7 or NR 3 CN;
  • R 5 is H, OH or halogen
  • R 7 is H, (Ci-C 8 )alkyl or heteroalkyl, (C 3 -C 8 )cycloalkyl or heterocyclyl, or aryl or heteroaryl;
  • R 3 and R 7 together are cycloalkyl, heteroalkyl or heteroaryl;
  • Ar is a substituted or unsubstituted aryl or heteroaryl
  • each W 1 W 3 , W or W 5 is independently N or C;
  • W 2 is a member selected from the group consisting of N, CR 5 , CO, O, NR 7 and S;
  • each W 6 , W 7 , W 8 or W 9 is independently N or CV 6 wherein V 6 is selected from the group consisting of hydrogen, substituted or unsubstituted (CrC 4 ) alkyl or heteroalkyl, halogen, hydroxy, (Ci-C 4 ) alkoxy, amino, cyano, nitro, (Ci-C 4 ) 4 alkylamino and (C 1 -C 4 ) dialkylamino;
  • Y is CHR 8 , NH, CR 8 , NR 8 , S or O;
  • R 8 is H, a straight or branched chain (Ci-C 8 )aikyl or heteroalkyl group
  • [0111] represents a single, double or normalized bond
  • lonidamine analogs have the formula
  • R 1 , R 2 , X, Y, and n are: [0116] R 2 is -Cl, -Br, -I, or -CH 3 , monosubstituted phenyl, substituted at the 2, 3, or 4 position; dichloro, dibromo, dimethyl, or chloro and methyl disubstituted phenyl, substituted at the 2 and 3 or 2 and 4 positions; or 2, 4, 5 trichlophenyl; [0117] Y is -(CH 2 )-; and
  • the lonidamine analog used according to the present invention is other than tolnidamine and/or other than AF-2364 and/or other than AF- 2785.
  • Lonidamine analogs suited for use in the invention are those that interfere with cellular energy metabolism of prostate epithelial cells when administered to a human, non-human primate, or other mammal.
  • a compound e.g., lonidamine
  • Active forms can be identified by routine screening of analogs for the activity of the parent compound.
  • a variety of assays and tests can be used to assess pharmacological activity of lonidamine analogs, including in vitro assays, such as those described below and elsewhere herein, in vivo assays of prostate function (including citrate production and ATP production) in humans, non-human primates and other mammals, in vivo assays of prostate size in humans, non-human primates and other mammals, and/or clinical studies.
  • Apoptosis assay in cell lines As shown in Example 3, lonidamine induces apoptosis in cell lines derived from human prostate cells. The induction of apoptosis is significantly greater in LNCaP cells (ATCC NO. CLR-1740), a prostate-derived cell line that is citrate-producing, than in PC3 cells (ATCC NO. CLR-1435), a prostate- derived cell line that is citrate-oxidizing, consistent with the susceptibility of the citrate-producing prostate cells to metabolic inhibitors such as lonidamine.
  • LNCaP cells ATCC NO. CLR-1740
  • PC3 cells ATCC NO. CLR-1435
  • a prostate- derived cell line that is citrate-oxidizing consistent with the susceptibility of the citrate-producing prostate cells to metabolic inhibitors such as lonidamine.
  • an analog with similar apoptosis-inducing activity is selected.
  • a lonidamine analog that induces apoptosis (enhances caspase 3 activity) in citrate-producing prostate cells, such as LNCaP cells is administered to treat BPH.
  • a lonidamine analog that induces apoptosis in LNCaP cells to a significantly greater degree than in PC3 cells is administered to treat BPH.
  • the induction of apoptosis by the lonidamine analog is at least about 2-fold greater in LNCaP cells than in PC3 cells (and sometimes at least about 3-fold greater, at least about 4-fold greater, or at least about 10-fold greater) when assayed at the concentration of analog at which the difference in the level of apoptosis in the two cell lines is greatest (provided that the concentration of analog used in the assay is not greater than 1 mM).
  • Apoptosis assay in primary cell cultures As shown in Example 2, lonidamine induces apoptosis in primary cultures of human prostate epithelial cells.
  • apoptosis is significantly greater in primary cultures of prostate epithelial cells than in primary cultures of human prostate stromal cells, consistent with the susceptibility of citrate-producing prostate cells to metabolic inhibitors such as lonidamine.
  • a lonidamine analog in which a lonidamine analog is administered for prevention of cancer, an analog with apoptosis-inducing activity similar to that of lonidamine is selected.
  • a lonidamine analog that induces apoptosis in prostate epithelial cells is administered to prevent cancer.
  • a lonidamine analog that induces apoptosis in primary cultures of prostate epithelial cells to a significantly greater degree than in primary cultures of human prostate stromal cells is used. In some embodiments of the invention, the lonidamine analog does not significantly induce apoptosis in stromal cells.
  • induction of apoptosis by the lonidamine analog is at least 2-fold greater in epithelial cells than in stromal cells (and sometimes at least 4-fold greater, sometimes at 10- fold greater, and sometimes at least 20-fold greater) when assayed at the concentration of analog at which the difference in the level of apoptosis in the two cell lines is greatest (provided that the concentration of analog used in the assay is not greater than 1 mM).
  • HIF-1 alpha expression assays As described in US 2004/0167196 and in Example 2, below, lonidamine reduced HIF-1 alpha expression/accumulation (measured in the nuclear fraction) in cells cultured under conditions of hypoxia by almost 2-fold at 200 micromolar and by more than 5 fold (Ae., more than 10-fold) at higher lonidamine concentrations.
  • an energolytic agent reduces HIF-1alpha expression (prevents HIF-1alpha accumulation) in LNCaP cells cultured under hypoxic conditions by at least about 2- fold, at least about 5-fold or at least about 10-fold compared to culture in the absence of lonidamine.
  • HIF-1 alpha expression in prostate cells appears more pronounced in LNCaP cells than in PC3 cells cultured under hypoxic conditions (oxygen level ⁇ 0.1%). Some lonidamine analogs useful for prevention of cancer according to the present invention may have a similar effect.
  • Hexokinase activity As discussed above, and without intending to be bound to any specific mechanism, the effects of lonidamine on the prostate may be mediated, at least in part, by its effects on mitochondria and mitochondrial hexokinase activity in secretory epithelial cells.
  • some lonidamine analogs useful in the present invention have hexokinase inhibitory activity as great as or greater than that of lonidamine.
  • Assays for hexokinase activity are known in the art. See Fanciulli et al., 1996, and Floridi et al., 1981.
  • Antispermatogenic activity Likewise, it is believed that the anti- spermatogenic activity of lonidamine results, at least in part, from energolytic effects in germ cells.
  • Some lonidamine analogs useful in the present invention have anti ⁇ spermatogenic activity as great, or greater, than that of lonidamine.
  • Assays for anti ⁇ spermatogenic activity are known in the art. See, e.g., Grima et al., 2001 ; Lohiya et a/., 1991.
  • energolytic agents can be evaluated in vivo for use in the methods of the invention.
  • suitable assays include measurements of prostate function and activity.
  • In vivo measurements of prostate function The effect of a compound on prostate function, and, in particular, on respiration, can be assessed by monitoring prostate tissue metabolism following administration of the compound.
  • Some lonidamine analogs useful in the present invention will detectably reduce ATP, citrate, and/or lactate production by the prostate in animals (including humans, non- human primates and other mammals). ATP, citrate, and/or lactate levels can be monitored directly and/or indirectly in vivo using techniques of magnetic resonance spectroscopy (MRS) or other methods.
  • MRS magnetic resonance spectroscopy
  • the prostate function is assessed by measuring serum PSA levels. Some lonidamine analogs and other agents useful in the present invention will detectably reduce serum PSA levels when administered.
  • In vivo measurements of prostate size The effect of a compound on prostate size can be assessed following administration of the compound using standard methods (for example, ultrasonography or digital rectal examination, for humans, and ultrasonography and/or comparison of organ weight in animals).
  • Assays can be conducted in humans or, more usually, in healthy non-human animals or in monkey, dog, rat, or other animal models of BPH (see, Jeyaraj et ai, 2000; Lee et al., 1998; Mariotti et al., 1982), Some lonidamine analogs useful in the present invention will detectably reduce prostate size in such assays and animal models. [0132]
  • the activity of a lonidamine analog of interest in any of the aforementioned assays can be compared with that of lonidamine to provide guidance concerning dosage schedules for the compound, and other information.
  • lonidamine analogs with greater biological activity per mg than lonidamine are of special interest.
  • the agent is a prodrug form of lonidamine, lonidamine analog, or other agent useful in the methods of the invention.
  • Prodrug forms are known in the art and include of ester, amide and other derivatives of agents listed above.
  • Exemplary prodrugs are described in copending US provisional application numbers 60/586,934 (filed July 8, 2004) and 60/624,505 (filed November 1 , 2004). Also see US Pat. No. 6,146,658.
  • a compound that inhibits or impairs energy production in prostate epithelial cells is referred to herein as an "energolytic agent.”
  • agents in addition to lonidamine and lonidamine analogs, can be used for prevention of cancer.
  • Energolytic agents useful in the practice of the invention include compounds that inhibit glycolysis, compounds that impair mitochondrial function, and compounds that do both, including, in all cases, compounds that act directly or indirectly on glucose metabolism in the prostate.
  • the energolytic agent is a compound that impairs glycolysis in prostate epithelial cells.
  • the energolytic agent is a compound that impairs mitochondrial function in prostate epithelial cells. In one embodiment, the energolytic agent interferes with both glycolysis and mitochondrial function. In one embodiment, a combination of agents is used, including, in one embodiment, the administration of one agent that is an inhibitor of glycolysis and simultaneous or contemporaneous administration of a second agent that is an inhibitor of mitochondrial function.
  • One class of energolytic agents includes compounds that inhibit glycolysis (directly or indirectly). [0135] For example, the agent may inhibit an enzyme that catalyses a step in the conversion of glucose to pyruvate, or the oxidation of pyruvate to acetyl-CoA.
  • the agent may be an inhibitor of hexokinase, glucokinase, phosphofructokinase, aldose, phosphoglycerate kinase, enolase, pyruvate kinase and/or pyruvate dehydrogenase.
  • such compounds include those described in U.S. Patent No.
  • 5,824,665 e.g., 6- amino-6-deoxy-glucose; N-acetyl- ⁇ -D-mannosamine; D-mannosamine; N- ⁇ -(p-tosyl)- L-Iysine chloromethyl ketone
  • phosphoglycerate quinone methides; taxodone; taxodione; ct-methylene lactones; euparotin acetate; eupacunin; vernolepin; argaric acid; quinaldic acid; 5'-p-fluorosuflonylbenzoyl adenosine; 5-keto-D-fructose; 5-keto- D-fructose-1 ,6-bisphosphate; Mg-phosphoglycerate; 2,3-diphosphoglycerate; 3- (trans)-chlorophosphoenolpyruvate; 3-(cis)-cyanophosphoenolpyruvate; D-tartron
  • Another class of agents includes compounds that impair mitochondrial function (e.g., a mitochondrial poison).
  • Mitochondrial poisons include but are not limited to the mitochondrial poisons described in U.S. Patent No. 6,670,330.
  • some agents may interfere with both glycolysis and mitochondrial function. It will also be appreciated that agents that impair glycolysis will generally also at least indirectly reduce energy production by mitochondria, by reducing the amount of pyruvate available for entry into the TCA cycle. Several exemplary agents are discussed below.
  • 2-DG 2- deoxy-D-glucose
  • 2-DG is phosphorylated by hexokinase to produce 2-DG-6- phosphate, which is not further metabolized and which inhibits hexokinase.
  • 2-DG has been shown to inhibit glycolysis in cancer cells.
  • a 2-DG analog is any D-glucose analog other than 2-DG that does not have a hydroxyl group at the 2 position of the glucose ring.
  • L- glucose and its L-analogs are not 2-DG analogs for purposes of the present invention.
  • a glucose analog includes mannose, galactose, glucose, and 5-thio- glucose.
  • An analog of glucose or 2-DG can have a fluorine in place of a hydrogen at any position on the glucose ring; thus, 2-fluoro-2-deoxy-D-glucose (2-FDG) and 2- difluoro-2-deoxy-D-glucose are 2-DG analogs.
  • An analog of glucose or 2-DG can have an amino group in place of a hydroxyl group at any position on the glucose ring other than the 6 position; thus, 2-amino-2-deoxy-D-glucose (2-glucosamine) and 2- amino-2-deoxy-D-galactose (2-galactosamine) are 2-DG analogs.
  • Other illustrative 2-DG analogs include 2-F-mannose, 2-mannosamine, 2-deoxygalactose, 2-F- deoxygalactose, and di, tri, and other oligosaccharades that contain one or more of the preceding or following 2-DG analogs.
  • Other 2-DG analogs useful in the methods of the present invention include the analogs described in U.S. patent application Serial No.
  • 2-DG analogs useful in the present invention also include those analogs described in Reinhold, 2000, Oncol. Rep., 7:1093-97 (e.g., 2- deoxy-D-glucose tetraacetate) and in PCT publication WO 01/82926.
  • 2-DG and 2-DGA can be administered for the prevention of prostate cancer at a dose in the range of about 1 mg to about 2 g of 2-DG or 2-DGA per kg of body weight of the patient to be treated.
  • 2-DG or a 2-DGA is administered in a dose in the range of about 10 mg to about 1 g of 2-DG or a 2-DGA per kg of body weight of the patient to be treated.
  • 2-DG or a 2-DGA is administered in a dose of about 50 to 250 mg of a 2-DG or a 2-DGA per kg of body weight of the patient to be treated.
  • a prophylactically effective dose is about 25 mg/kg to about 150 mg/kg.
  • the prophylactically effective dose of 2-DG or a 2-DGA is administered daily or once every other day or once a week to the patient, and multiple administrations of the drug are employed. Depending on the dose selected by the practitioner and the convenience of the patient, the entire dose may be administered once daily, or the dose may be administered in multiple smaller doses through the course of a day.
  • the dose may be divided into two smaller doses and administered twice daily, or divided into three smaller doses and administered thrice daily.
  • the dose may be combined and given every other day, or even less frequently, but in any event, the dose is repeatedly administered over a period of time.
  • the administration of the prophylactically effective dose is continued for multiple days, typically for at least five consecutive days, and often for at least a week and often for several weeks or more.
  • 2-DG or a 2-DGA is administered once (qday), twice (bid), three times (tid), or four times (qid) a day or once every other day (qod) or once a week (qweek), and treatment is continued for a period ranging from three days to two weeks or longer. In one embodiment, the treatment is continued for one to three months. In another embodiment, the treatment is continued for a year. Thus, a patient may be administered 2-DG or a 2-DGA for a week, a month, two months, three months, six months, or a year or longer.
  • Another class of agents suitable for use in the methods of the present invention is the class of 3-halo-pyruvates, including but not limited to 3- bromopyruvate, an inhibitor of hexokinase.
  • 3-halo-pyruvates including but not limited to 3- bromopyruvate, an inhibitor of hexokinase.
  • Another class of agents suitable for use in the methods of the present invention is the class composed of gossypol and its analogs, including but not limited to gossypol-(+), gossypol-(-), mixtures of gossypol-(+) and -(-), gossypol acetic acid, gossypol aldehyde, gossypol hemiacetal, gossypol quinoid, gossypolone, metabolites thereof, and physiologically acceptable salts thereof.
  • Gossypol, gossypol analogs, formulations, and unit dose forms that can be- employed in the methods of the present invention are described in PCT patent publication Nos. WO 02/097053; WO 02/47673; U.S. Patent Nos. 6,114,397 and 4,381 ,298; and U.S. patent publication No. 2002/137801.
  • agents for use as agents in the present invention include the anti-metabolites described in U.S. patent publication No. 2002/0035071 (Pitha) including 3-O-methylglucose (Jay et al., 1990); anhydrosugars such as 1 ,5-Anhydro- D-Glucitol (Polygalitrol) (Sols et al., 1954), 1 ,5-anhydroglucitol-6-phosphate (Crane et al., 1954) and, 2.5-Anhydro-D-Mannitol and 2,5-Anhydroglucitol).
  • anhydrosugars such as 1 ,5-Anhydro- D-Glucitol (Polygalitrol) (Sols et al., 1954), 1 ,5-anhydroglucitol-6-phosphate (Crane et al., 1954) and, 2.5-Anhydro-D-Mannitol and 2,5-Anhydrog
  • Compounds for use as agents in the present invention also include inhibitors of lactate dehydrogenase, such as oxamate, and inhibitors of glyceraldehyde 3-phosphate dehydrogenase, such as iodoacetate (see, Lampidis, WO 01/82926).
  • Any of a variety of agents may be used for prevention of prostate cancer.
  • the agent is an inhibitor of hexokinase.
  • the agent is an inhibitor of glucokinase.
  • the agent is an inhibitor of phosphofructokinase.
  • the agent is an inhibitor of aldose.
  • the agent is an inhibitor of phosphoglycerate kinase. In one embodiment, the agent is an inhibitor of enolase. In one embodiment, the agent is an inhibitor of pyruvate kinase. In one embodiment, the agent is an inhibitor of pyruvate dehydrogenase. In one embodiment, the agent is an inhibitor of lactate dehydrogenase. In one embodiment, the agent is an inhibitor of glyceraldehyde 3- phosphate dehydrogenase. In one embodiment, the agent is an inhibitor of glucose transport. In one embodiment, the agent reduces glucose transporter levels or prevents those levels from rising. In one embodiment, the agent is 2-deoxy-D- glucose (2-deoxyglucose or 2-DG).
  • the agent is an analog of 2- deoxyglucose. In one embodiment, the agent is 2-deoxy-D-glucose tetraacetate or 5- thio-glucose. In one embodiment, the agent is gossypol. In one embodiment, the agent is a gossypol analog. In one embodiment, the agent is 3-bromopyruvate. In one embodiment, the agent is a 3-bromopyruvate analog. In one embodiment, the agent is an analog of lonidamine. In one embodiment, the agent is lonidamine. In one embodiment, the agent is tolnidamine. In one embodiment, the agent is oxamate. In one embodiment, the agent is iodoacetate.
  • the agent is apoptolidin. In one embodiment, the agent is an analog of apoptolidin. [0147] In one embodiment, the agent has a molecular weight less than 1000, optionally less than 500. In one embodiment, the agent is synthetic and does not occur in nature.
  • the agent is other than an inhibitor of hexokinase. In one embodiment, the agent is other than an inhibitor of glucokinase. In one embodiment, the agent is other than an inhibitor of phosphofructokinase. In one embodiment, the agent is other than an inhibitor of aldose. In one embodiment, the agent is other than an inhibitor of phosphoglycerate kinase. In one embodiment, the agent is other than an inhibitor of enolase. In one embodiment, the agent is other than an inhibitor of pyruvate kinase. In one embodiment, the agent is other than an inhibitor of pyruvate dehydrogenase. In one embodiment, the agent is other than an inhibitor of lactate dehydrogenase.
  • the agent is other than an inhibitor of glyceraldehyde 3-phosphate dehydrogenase. In one embodiment, the agent is other than an inhibitor of glucose transport. In one embodiment, the agent does not reduce glucose transporter levels or prevent those levels from rising. In one embodiment, the agent is not a direct or indirect inhibitor of HIF-1 alpha. [0149] In one embodiment, the agent is other than 2-deoxyglucose. In one embodiment, the agent is other than an analog of 2-deoxyglucose. In one embodiment, the agent is other than 2-deoxy-D-glucose tetraacetate. In one embodiment, the agent is other than 5-thio-glucose. In one embodiment, the agent is other than gossypol.
  • the agent is other than a gossypol analog. In one embodiment, the agent is other than 3-bromopyruvate. In one embodiment, the agent is other than a 3-bromopyruvate analog. In one embodiment, the agent is other than lonidamine. In one embodiment, the agent is other than tolnidamine. In one embodiment, the agent is other than an analog of lonidamine. In one embodiment, the agent is other than an inhibitor of aconitase. [0150] In one embodiment, the agent is other than oxamate. In one embodiment, the agent is other than iodoacetate. In one embodiment, the agent is other than apoptolidin.
  • the agent is other than analog of apoptolidin.
  • the agent is other than zinc or any other than any agent known for use for prevention of prostate cancer on June 30, 2004, without regard to whether or not the agent is known to inhibit glycolysis or impair mitochondrial function.
  • an agent suitable for use according to the invention for prevention of prostate cancer will be pharmaceutically acceptable, i.e., will not be toxic to the subject at the doses and formulation administered, or the detrimental effects of any toxicity (e.g., side effects) associated with the agent will be outweighed by the benefit to the subject.
  • Methods for identification and assessment of pharmaceutically acceptable agents are well known in the medical and pharmaceutical arts.
  • the therapeutic (i.e., prophylactic) index i.e., dose ratio of therapeutic effects to toxic effects, which can be expressed as the ED 50 /LD 50 ratio
  • the therapeutic index i.e., prophylactic index
  • the agent is a pharmaceutically acceptable salt of an agent named above.
  • Pharmaceutically acceptable salts include addition salts with acids, as well as the salts with bases.
  • Salts with bases are, for example, alkali metal or alkaline earth metal salts, such as sodium, potassium, calcium or magnesium salts, or ammonium salts, such as those with ammonia or suitable organic amines, e.g. diethylamine, di-(2-hydroxyethyl)-amine or tri-(2-hydroxyethyl) ⁇ amine.
  • Suitable acids for the formation of acid addition salts are, for example, mineral acids, such as hydrochloric, hydrobromic, sulphuric or phosphoric acid, or organic acids, such as organic sulphonic acids, for example, benzenesulphonic, 4-toluenesulphonic or methanesulphonic acid, and organic carboxylic acids, such as acetic, lactic, palmitic, stearic, malic, maleic, fumaric, tartaric, ascorbic or citric acid.
  • mineral acids such as hydrochloric, hydrobromic, sulphuric or phosphoric acid
  • organic acids such as organic sulphonic acids, for example, benzenesulphonic, 4-toluenesulphonic or methanesulphonic acid
  • organic carboxylic acids such as acetic, lactic, palmitic, stearic, malic, maleic, fumaric, tartaric, ascorbic or citric acid.
  • the invention provides methods for determining the usefulness of a compound for prevention of prostate cancer.
  • the method involves (a) contacting a citrate-producing cell with the compound; (b) contacting a citrate-oxidizing cell with the compound; and (c) detecting a differential effect of said contacting on the citrate-producing cell compared to the citrate- oxidizing cell.
  • a differential effect e.g., as described herein indicates that the agent may be useful for prevention of prostate cancer. Further and confirmatory assays can then be conducted.
  • the method can be conveniently carried out in vitro.
  • citrate-producing and citrate-oxidizing cell types are known and can be identified using art-known assays and criteria. See, e.g., Costello and Franklin, 1997, Urology 50:3-12 and Franklin et al., 1995, Endocrine 3:603-607.
  • Suitable citrate-producing cells include primary cultures of prostate epithelial cells and certain established cell lines derived from prostate epithelial cells (e.g., LNCaP cells).
  • Suitable citrate- oxidizing cells include primary cultures of prostate stromal cells and certain established cell lines derived from prostate cells (many malignant prostate epithelial cells undergo an apparent metabolic transformation from citrate-producing to citrate- oxidizing; see Franklin et a/.; 1995, Endocrine 3:603-607).
  • the citrate-producing cell is an LNCaP cell and the citrate oxidizing cell is a PC-3 cell.
  • stromal cells Primary cultures of human prostate epithelial and stromal cells are commercially available (e.g., cells can be obtained from Cambrex Bio Science Rockland, Inc., 191 Thomaston Street, Rockland, Maine 04841) and can be prepared according to well known tissue culture methods (see, e.g., Peehl, DM, Culture of Epithelial Cells: Prostate Culture, 1992, 159-180).
  • Established cell lines derived from prostate are available from the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108 USA, or can be prepared according to well known methods.
  • the cell is a recombinant cell selected from the group consisting of (i) a cell, optionally a cell other than a prostate cell, that has been modified so as to accumulate zinc to levels that inhibit m-aconitase and (ii) a cell other than a prostate cell that cannot metabolize citrate.
  • the cell is a cell that has been modified to not express m-aconitase or to express only an inactive mutant thereof.
  • the cells are human (although cells from other mammals also can be used).
  • the cells have been immortalized by modification such that telomerase expression occurs constitutively.
  • cells are contacted with the test compound, usually at a range of concentrations (e.g., 10, 50, 1pO, 200, 400, 600 and 800 microM).
  • concentrations e.g., 10, 50, 1pO, 200, 400, 600 and 800 microM.
  • the contacting is conveniently achieved by adding the compound to the medium in which the cells are cultured, or any other method of contacting.
  • the compound is introduced into the cell or cell culture in a carrier (e.g., liposomal carrier) or solvent.
  • suitable controls e.g., negative controls
  • Assays can be carried out on whole cells, cell extracts or, alternatively, nuclear extracts.
  • the cells are grown under hypoxic conditions.
  • cells can be cultured in low oxygen levels (e.g., ⁇ 0.1%).
  • Hypoxia can also be induced by culture at high cell density.
  • An example of a differential effect is induction of apoptosis that is greater in citrate-producing cells compared to citrate-oxidizing cells.
  • the differential effect is induction of apoptosis that is greater in citrate-producing cells compared to citrate-oxidizing cells.
  • the differential effect is at least about 10-fold or at least about 20-fold.
  • a number of assays for apoptosis or its markers or other indicators thereof are known and can be used in the present assay.
  • apoptosis assays include assays for caspase 3, DNA fragmentation assays (e.g., TUNEL assays; BD Biosciences No 556381), and Annexin V assays (e.g. BD Biosciences No 556547).
  • DNA fragmentation assays e.g., TUNEL assays; BD Biosciences No 556381
  • Annexin V assays e.g. BD Biosciences No 556547.
  • another differential effect that can be measured is a reduction in HIF-1 alpha expression that is greater in citrate-producing cells than in citrate-oxidizing cells, especially cells cultured under hypoxic conditions.
  • the difference is at least about 2-fold, and sometimes at least about 4-fold.
  • Example 1 Administration of Lonidamine to Men with Benign Prostate
  • This example describes results of a Phase Il Open-Label Study to evaluate the efficacy and safety of oral lonidamine (LND) treatment in subjects with symptomatic benign prostatic hyperplasia (BPH) by measuring the change from baseline of: total prostate volume by transrectal ultrasound (TRUS), maximum flow rate (Q m ax) on uroflowmetry, international prostatic symptoms score (I-PSS) and prostate-specific antigen (PSA).
  • TRUS transrectal ultrasound
  • Q m ax maximum flow rate
  • I-PSS international prostatic symptoms score
  • PSA prostate-specific antigen
  • Pretreatment evaluation included recording of medications taken 2 weeks prior to study entry; serum chemistry panel including sodium, potassium, chloride, carbon dioxide, blood urea nitrogen (BUN), creatinine, AST and ALT; hormonal profile (FSH, LH, PRL, testosterone, free testosterone); TRUS measurement of prostate volume; PSA; uroflowmetry; and I-PSS.
  • serum chemistry panel including sodium, potassium, chloride, carbon dioxide, blood urea nitrogen (BUN), creatinine, AST and ALT
  • hormonal profile FSH, LH, PRL, testosterone, free testosterone
  • TRUS measurement of prostate volume PSA
  • uroflowmetry uroflowmetry
  • I-PSS contra event and concomitant medication assessments.
  • subjects also underwent a physical examination including digital rectal exam, temperature and vital signs; evaluation of serum chemistry; hormonal profile; and I-PSS.
  • subjects were also evaluated for prostate volume and uroflowmetry.
  • Subjects' I-PSS symptom scores decreased from an average of 19.5 at baseline to 12.2 on Day-28 (p O.001). The decreases were consistent across subjects.
  • Prostate volume and BPH-related symptoms continued to improve throughout the 28 days of LND treatment and into the post-treatment period, with further reduction of prostate volume and improvement of Q max at Day-56. Baseline equals Day 0 value.
  • lonidamine was also examined in primary cultures of prostate epithelial cells (which accumulate citrate) or prostate stromal cells (which do not accumulate citrate). Lonidamine induced apoptosis only in prostate epithelial cells in a dose-dependent manner. In contrast, induction of apoptosis was not observed in prostate stromal cells after treatment with lonidamine.
  • Apoptosis Assay Cells were plated at a density of 2 x 10 4 cells per well in a 96 well plate, and then maintained in a 37 0 C incubator (5% CO 2 ) for 16 h. Lonidamine was added into each well at different concentrations, and then incubated for 6 h at 37 0 C.
  • the homogeneous buffer and caspase 3 substrate Promega No G7791 ; Promega Corporation, 2800 Woods Hollow Road, Madison Wl USA 53711
  • the fluorescence intensity of cleaved substrate was determined using a fluorescence plate reader at excitation 485 nm and emission 530 nm.
  • a GLP study was conducted to evaluate the effect of lonidamine on the incidence of tumors in Sprague Dawley rats.
  • a total of 500 rats were assigned to groups of 50 rats of each sex per dosage level.
  • Two groups of each sex served as controls and the other six groups (three of each sex) received lonidamine (20, 60 or 180 mg/kg per day) in the diet for 2 years.
  • the rats were sacrificed and tissues were obtained at necropsy for histological assessment including determination of tumor incidence. Survival probability functions were estimated by the Kaplan-Meier technique.
  • Prostate Disease Diagnostic Possibilities And Future Developments. Prostate

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Abstract

L'invention concerne des procédés pour la prévention du cancer de la prostate et de cancers épithéliaux en administrant de la lonidamine et des agents apparentés.
PCT/US2005/024423 2004-07-08 2005-07-08 Prévention du cancer Ceased WO2006010073A1 (fr)

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EP2331092A4 (fr) * 2008-08-21 2011-10-12 Univ Johns Hopkins Procédés et compositions pour l'administration de 3-halopyruvate et de composés associés pour le traitement du cancer
US9737487B2 (en) 2014-01-14 2017-08-22 The Johns Hopkins University Cyclodextrin compositions encapsulating a selective ATP inhibitor and uses thereof
US10751306B2 (en) 2015-11-06 2020-08-25 The Johns Hopkins University Methods of treating liver fibrosis by administering 3-bromopyruvate

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2331092A4 (fr) * 2008-08-21 2011-10-12 Univ Johns Hopkins Procédés et compositions pour l'administration de 3-halopyruvate et de composés associés pour le traitement du cancer
US9492417B2 (en) 2008-08-21 2016-11-15 The Johns Hopkins University Methods and compositions for administration of 3-halopyruvate and related compounds for the treatment of cancer
US10130598B2 (en) 2008-08-21 2018-11-20 The Johns Hopkins University Methods and compositions for administration of 3-halopyruvate and related compounds for the treatment of cancer
US9737487B2 (en) 2014-01-14 2017-08-22 The Johns Hopkins University Cyclodextrin compositions encapsulating a selective ATP inhibitor and uses thereof
US10751306B2 (en) 2015-11-06 2020-08-25 The Johns Hopkins University Methods of treating liver fibrosis by administering 3-bromopyruvate

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