WO2010044686A1 - Akr1c3 utilisé comme biomarqueur, procédés de sélection et de traitement de patients sur la base d'un profil d'akr1c3 et composés utiles à cet effet - Google Patents

Akr1c3 utilisé comme biomarqueur, procédés de sélection et de traitement de patients sur la base d'un profil d'akr1c3 et composés utiles à cet effet Download PDF

Info

Publication number
WO2010044686A1
WO2010044686A1 PCT/NZ2009/000227 NZ2009000227W WO2010044686A1 WO 2010044686 A1 WO2010044686 A1 WO 2010044686A1 NZ 2009000227 W NZ2009000227 W NZ 2009000227W WO 2010044686 A1 WO2010044686 A1 WO 2010044686A1
Authority
WO
WIPO (PCT)
Prior art keywords
akrl
patient
cancer
activated
agent
Prior art date
Application number
PCT/NZ2009/000227
Other languages
English (en)
Inventor
Adam Vorn Patterson
Christopher Paul Guise
Maria Rosaria Abbattista
William Robert Wilson
Jeffrey Bruce Smaill
Sophie Syddall
William Alexander Denny
Amir Ashoorzadeh
Original Assignee
Auckland Uniservices Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Auckland Uniservices Limited filed Critical Auckland Uniservices Limited
Publication of WO2010044686A1 publication Critical patent/WO2010044686A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
    • 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
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • 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
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/28—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton
    • C07C237/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having the nitrogen atom of the carboxamide group bound to an acyclic carbon atom of a hydrocarbon radical substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/63—Esters of sulfonic acids
    • C07C309/64—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to acyclic carbon atoms
    • C07C309/65—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to acyclic carbon atoms of a saturated carbon skeleton
    • C07C309/66—Methanesulfonates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90209—Oxidoreductases (1.) acting on NADH or NADPH (1.6), e.g. those with a heme protein as acceptor (1.6.2) (general), Cytochrome-b5 reductase (1.6.2.2) or NADPH-cytochrome P450 reductase (1.6.2.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • AKR1C3 AS A BIOMARKER, METHODS OF SELECTING AND TREATING PATIENTS BASED UPON AN AKRIC3 PROFILE AND COMPOUNDS FOR USE
  • the present invention relates generally to the field of biomarkers, and more specifically to methods and procedures to determine drug sensitivity in patients to allow the identification of individualized patient profiles which will aid in treating diseases and disorders.
  • the invention also provides methods of treatment which are based upon such biomarker-related profiles, as well as compounds suitable for use in such methods. BACKGROUND OF THE INVENTION
  • Cancer is a disease with extensive histoclinical heterogeneity. Although conventional histological and clinical features have been correlated to prognosis, the same apparent prognostic type of tumors varies widely in its responsiveness to therapy and consequent survival of the patient. New prognostic and predictive markers, which would facilitate an individualization of therapy for each patient, are needed to accurately predict patient response to treatments, such as small molecule or biological molecule drugs, in the clinic. The problem may be solved by the identification of new parameters that could better predict the patient's sensitivity to treatment. The classification of patient samples is a crucial aspect of cancer diagnosis and treatment.
  • the association of a patient's response to a treatment with specific biomarkers can open up new opportunities for treatment development in non-responding patients, or distinguish a treatment's indication among other treatment choices because of higher confidence in die efficacy. Further, the pre-selection of patients who are likely to respond well to a medicine, drug, or combination therapy may reduce the number of patients needed in a clinical study or accelerate the time needed to complete a clinical development program (M. Cockett et al., Current Opinion in Biotechnology, 11:602- 609 (2000)).
  • HSDs hydroxysteroid dehydrogenases
  • ARR aldo-keto reductase superfamily
  • HSDs function as prereceptor switches by activating/deactivating steroid hormones via redox chemistry.
  • the occupancy of androgen receptors in the prostate may be regulated by reducing the highly potent androgen 5 ⁇ -dihydrotestosterone to the inactive metabolite 3 ⁇ -androstanediol.
  • reduction of 5 ⁇ -dihydroprogesterone to 3 ⁇ , 5 ⁇ -tetrahydroprogesterone produces an allosteric regulator of the GABA receptor in the brain.
  • AKR1C2 human type 3 3 ⁇ -hydroxysteroid dehydrogenase
  • AKRl C3 contains high 17 ⁇ -HSD activity and is involved in the peripheral formation of androgens and estrogens, reactions that may be important in prostate and breast cancer.
  • AKRl C3 also exhibits prostaglandin synthase activity.
  • AKRl C3 has also been identified as a suppressor of cell differentiation in myeloid cells, and has been suggested as an antineoplastic target (e.g. in HL-60 cells, see Desmond et al. Cancer Res. 63, 505-512, (2003)). Overexpression of AKR1C3 resulted in diminished sensitivity to the differentiation promoter ATRA. Inhibition of the activity of the enzyme, such as by competitive inhibition, has been suggested as a potential cancer therapy.
  • NSAIDs Non-Steroidal Antiinflammatory Drugs
  • AKRl C3 may also have a role in cancer therapy other than through inhibition.
  • the applicants have determined that AKRl C3 is capable of exhibiting nitroreductase activity and activating nitroaromatic prodrugs to release a cytotoxic effector.
  • AKRlC3-activated nitroaromatic prodrugs are the derivativeobenzamide mustards (such as those described in WO 2004/033415 and WO 2005/042471), with 2-((2- bronioemyl)-2- ⁇ [(2-hydroxyemyl)arnino]carbonyl ⁇ -4,6-dinitroanilino)ethyl methanesulfonate (PR- 104A, released from pre-prodrug 2-[(2-brornoe ⁇ yl)-2,4-dinitxo-6-[[[2-(phosphonooxy)ethyl]arnino]- carbonyl]anilino]ethyl methanesulfonate (PR-104)) being a representative example.
  • the invention provides an assay method for predicting the response of a patient with cancer to treatment with a therapeutic agent, said method comprising: (a) determining at least one AKRl C3 profile selected from the group consisting of an
  • AKRl C3 nucleic acid profile an AKRl C3 protein profile and combinations thereof in a sample from said patient
  • the sample is of or from a tumour and the response being predicted is whether the tumour is responsive or non-responsive to treatment with said agent.
  • the tumour is a solid tumour.
  • the tumour sample may be from a treatment naive patient or from a patient pre-treated with, and optionally resistant to, at least one other therapeutic agent.
  • the tumour sample is obtained from the patient shortly or immediately prior to the assay being performed.
  • the pre-treatment is with an antifolate or antimetabolite anti-cancer agent or a platinum or anthracycline anti-cancer agent.
  • the sample is a non-tumour sample. Samples from outside the tumour can be tested, for example, to predict whether the therapeutic agent will have an acceptable toxicity profile in normal tissue.
  • the non-tumour sample can be from the bone marrow, brain, lung, liver, kidney, colon, small intestine, mammary gland, uterus, cervix, bladder, prostate or testis, or can be blood, plasma or serum.
  • the AKRl C3 profile of samples from both a tumour and non- tumour source is determined. The patient can then be identified as suitable or non-suitable for treatment with the therapeutic agent based upon a comparison of the AKRl C3 profiles from the respective tumour and non-tumour sources.
  • the therapeutic agent to which a response is predicted is an AICRl C3-activated prodrug or a pre-prodrug thereof.
  • the prodrug is PR-104A
  • the pre-prodrug is PR-104
  • a positive AKRl C3 profile being predictive that the patient will be responsive to treatment with PR-104A or PR- 104.
  • die prodrug is a compound of formula I as defined below or a pharmaceutically acceptable salt thereof
  • the pre-prodrug is a compound of formula II as defined below or a pharmaceutically acceptable salt thereof, with a positive AKRl C3 profile being predictive that the patient will be responsive to treatment with the prodrug, the pre-prodrug, or the pharmaceutically acceptable salt thereof.
  • the therapeutic agent to which a response is predicted is a non-AKRlC3- activated anti-cancer agent with a negative AKRl C3 profile being predictive that the patient will be responsive to treatment with said anti-cancer agent.
  • die invention provides a method of treating a patient with cancer which comprises die step of treating a patient predicted to be a responder to treatment widi an AKRl C3- activated therapeutic agent widi an amount of an AKRlC3-activated agent sufficient to produce a therapeutic effect.
  • the AKRlC3-activated therapeutic agent is PR-104A or PR- 104.
  • the AKRl C3-activated therapeutic agent is a compound of formula I as defined below, a compound of formula II as defined below, or a pharmaceutically acceptable salt of a compound of formula I or of formula II.
  • the AKRl C3-activated agent can be administered alone or, as is preferred, in combination with at least one other anti-cancer agent.
  • the invention provides a method for predicting the response of a patient with cancer to treatment with a therapeutic agent, said mediod comprising: (a) providing at least one AKRl C3 profile selected from the group consisting of an AKRl C3 nucleic acid profile, an AKRl C3 protein profile and combinations thereof; and
  • the invention provides a method of treating a patient with cancer which comprises the step of treating said patient when predicted to be a non-responder to treatment with an AKRl C3-activated agent with a therapeutic amount of at least one anti-cancer agent other than an AKRlC3-activated agent, or with an amount of a combination of an AKRl C3-activated agent and a Non-Steroidal Anti-Inflammatory Drug (NSAID) sufficient to produce a therapeutic effect.
  • NSAID Non-Steroidal Anti-Inflammatory Drug
  • a particularly preferred combination for treating said patient is a combination of PR-104 and naproxen.
  • the AI ⁇ RlC3-activated agent/NSAID combination can also be further combined with at least one other anti-cancer agent.
  • the invention provides a method of cancer treatment which includes the step of treating a patient with cancer having a positive AKRl C3 profile with an amount of an AKRl C3 -activated agent sufficient to produce a therapeutic effect.
  • the AKRlC3-activated agent can be administered alone or, as is preferred, in combination with at least one other anti-cancer agent.
  • the patient is treated with PR-104A, PR-104, a compound of formula I as defined below, a compound of formula II as defined below, or a pharmaceutically acceptable salt thereof, alone or in combination with at least one other anti-cancer agent.
  • the invention provides a method of cancer treatment which comprises the step of treating a patient with cancer having a negative AICRl C3 profile with a therapeutic amount of an anti-cancer agent other than an AKRlC3-activated agent or with an amount of a combination of an AKRlC3-activated agent and a NSAID sufficient to produce a therapeutic effect.
  • the invention provides a method of treating a patient resistant to treatment with-a non-AKRl C-activated anti-cancer agent which comprises the step of treating said patient with an amount of an AKRlC3-activated agent sufficient to produce a therapeutic effect.
  • said patient is treated with PR-104A or PR-104 or with a compound of formula I or II as defined below, or a pharmaceutically acceptable salt thereof.
  • the invention provides an assay method for optimising dose efficacy in a patient with cancer receiving or who is to receive an AKRlC3-activated therapeutic agent, said method comprising:
  • AKRl C3 profile selected from the group consisting of an AKRl C3 nucleic acid profile, an AKRl C3 protein profile and combinations thereof in a tumour sample from said patient as well as in at least one non-tumour sample from said patient;
  • the recommended dose is zero.
  • the invention provides an assay method for optimising dose efficacy in a patient with cancer receiving or who is to receive an AKRl C3-activated therapeutic agent, said method comprising:
  • AKRl C3 profile selected from the group consisting of an AKRl C3 nucleic acid profile, an AKRl C3 protein profile and combinations thereof in a tumour sample from said patient as well as in at least one non-tumour sample from said patient;
  • the invention provides a method of identifying an AKRl C3 -activated prodrug comprising a bioreductive trigger component and an active drug component, which method comprises the steps of:
  • the invention provides a prodrug comprising a bioreductive trigger and an active drug component, wherein said bioreductive trigger is activated by AKRl C3 enzyme to release the active drug, and wherein said prodrug is identified or identifiable by a method as defined above.
  • the active drug component is a cytotoxic agent capable of producing a therapeutic effect when released in, or delivered to, a tumour.
  • the invention provides a kit for use in an assay method as defined above, which kit comprises:
  • the invention further provides a pharmaceutical composition comprising a compound of formula I as defined above, a compound of formula II as defined above, or a pharmaceutically acceptable salt thereof.
  • the invention further provides a method of treating a patient with cancer which comprises the step of administering to a patient in need thereof a therapeutically-effective amount of a compound of formula I as defined above, a compound of formula II as defined above, or a pharmaceutically acceptable salt thereof.
  • the invention further relates to use of a compound of formula I as defined above, a compound of formula II as defined above, or a pharmaceutically acceptable salt thereof in the manufacture of a composition for treating cancer in a patient in need thereof.
  • the invention also contemplates a compound of formula I as defined above, a compound of formula II as defined above, or a pharmaceutically acceptable salt thereof for treating cancer in a patient in need thereof.
  • Figure 1 Mechanism of activation of PR-104 by one-and two-electron transfer.
  • AKRl C3 is able to reduce PR-104A to its cytotoxic metabolites under aerobic conditions. A-Aerobic metabolism was determined by an LC/MS/MS assay for PR- 104H ad PR-104M. B-Detection of expressed AKR enzymes and NQOl in HCT 116 cells by Western blotting.
  • Figure 3 AKR1C3 protein levels correlate with aerobic metabolism of PR-104A in a panel of
  • a - Cell lines are shown in rank order of aerobic metabolism of PR-104A to PR- 104H/M
  • FIG. 4 Effect of AKRl C3 expression on sensitivity of human tumour cell lines to bioreductive drugs in vitro and in vivo.
  • A- 1C50 assays were carried out on HCTl 16WT and HCTl 16 AKRl C3 cells, expression of AKRl C3 sensitises HCTl 16 cells to PR-104A but not to other bioreductive drugs.
  • B Tumour growth delay experiment showing increased sensitivity to PR- 104, but not to cyclophosphamide (CPA) in AKJIl C3 expressing HCTl 16 tumours.
  • C Expression of AKRl C3 in human tumour xenografts by Western blotting.
  • B Graph showing the intensity of AKRl C3 staining for 2700 tumors across 19 tumour types. A conservative cut-off was used so only diffuse staining (score 6 and 3) or strong moderate staining (score 5) was included as a positive result.
  • C A breakdown of lung cancer subtypes (positive scoring represents a score of 6, 5 or 4).
  • Tissue are; 1, adrenal; 2, bladder; 3, bone marrow; 4, eye; 5, breast; 6, cerebellum; 7, cerebral cortex; 8 fallopian tube; 9, esophagus; 10, stomach; 11, small intestine; 12, colon; 13, rectum; 14, heart; 15, kidney; 16, liver; 17, lung; 18, ovary; 19, pancreas; 20, parathyroid; 21, pituitary; 22, placenta; 23, prostate; 24, skin; 25, spinal cord; 26, spleen; 27, muscle; 28, testis; 29, thymus; 30, thyroid; 31, tonsil; 32, cervix; 33, endometrium.
  • B IC50 of bioreductive prodrugs following 4h aerobic exposure of HCTl 16 WT and HCTl 16 AKR1C3 (clones #1 and #2).
  • Figure 7 Pharmacological inhibition of aerobic reduction of PR-104A in vitro and mice.
  • Anti-cancer effects include, but are not limited to, anti-tumour effects, the response rate, the time to disease progression and the survival rate.
  • Anti-tumour effects include but are not limited to inhibition of tumour growth, tumour growth delay, regression of tumour, shrinkage of tumour, increased time to regrowth of tumour on cessation of treatment and slowing of disease progression.
  • Effective amount means an amount of a compound that, when administered to a subject for treating a cancer, is sufficient to effect such treatment for the cancer.
  • the “effective amount” will vary depending on the cancer to be treated, the compound to be administered, the severity of the cancer treated, the age and relative health of the subject, the route and form of administration, whether the treatment is monotherapy or combination therapy, the judgement of the attending clinician, and other factors.
  • “Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.
  • “Pharmaceutically acceptable salts” of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound.
  • Such salts include: acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, methanesulfonic acid, maleic acid, tartaric acid, citric acid and the like; or salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g.
  • Acceptable organic bases include ethanolamine, diethanolamine, N- methylglucamine, triethanolamine and the like.
  • Acceptable inorganic bases include aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.
  • subject is intended to refer to an animal, preferably a mammal, more preferably a mammalian companion animal or human. Preferred companion animals include cats, dogs and horses.
  • mammalian subjects include an agricultural animal, including a horse, a pig, a sheep, a goat, a cow, a deer, or a fowl, or a laboratory animal, including a monkey, a rat, or a mouse.
  • an agricultural animal including a horse, a pig, a sheep, a goat, a cow, a deer, or a fowl
  • a laboratory animal including a monkey, a rat, or a mouse.
  • treat and its derivatives should be interpreted in their broadest possible context. The term should not be taken to imply that a subject is treated until total recovery. Accordingly, “treat” broadly includes maintaining a subject's disease progression or symptoms at a substantially static level, increasing a subject's rate of recovery, amelioration and/or prevention of the onset of the symptoms or severity of a particular condition, or extending a patient's quality of life. The term “treat” also broadly includes the maintenance of good health for sensitive individuals and building stamina for disease prevention.
  • Warm blooded animal means any member of the mammalia class including, but not limited to humans, non-human primates such as chimpanzees and other apes and monkey species, farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. DESCRIPTION OF THE INVENTION
  • aldo-ketoreductase family 1 member C3 (17- beta-hydroxysteroid dehydrogenase type 5; 3-alpha-hydroxysteroid dehydrogenase type 2; Dihydrodiol dehydrogenase 3; Prostaglandin F synthase; ACC No. NM-003739; Prot ID P42330; Gene ID 8644, also known as AKRl C3; DD3; DDX; HAKRB; HAKJIe; HAl 753; HSDl 7B5; hlu PGFS; KlAAOl 19) and its role as a biomarker.
  • the invention is based upon the surprising finding that AKRl C3 has nitroreductase activity. This latter activity delivers the capability to AKRl C3 to bioreduce and therefore activate nitroaromatic prodrugs which, upon activation, release one or more cytotoxic species in vivo.
  • This finding relating to AKRl C3 will be apparent to diose persons skilled in the art.
  • One such application is in a method of screening candidate nitroaromatic prodrugs to determine which are reduced (activated) by AKRl C3 and therefore capable of releasing one or more cytotoxic species in vivo.
  • the primary and preferred application is in methods of screening patients to assess their predicted responder/non-responder status with respect to AKRlC3-activated prodrug therapy in treating cancer, with "cancer” having its widest possible meaning to include any member of a class of diseases characterised by the uncontrolled growth of abberant cells.
  • cancer having its widest possible meaning to include any member of a class of diseases characterised by the uncontrolled growth of abberant cells.
  • the screening approach of the invention involves determining at least one AKRl C3 profile for said patient. That profile may be an AKRl C3 nucleic acid profile, an AKRl C3 protein profile or a combination of these.
  • a number of approaches are available to determine an AKRl C3 nucleic acid profile for a patient. Such approaches include determining a genotypic profile, a gene expression profile or a combination. Any other art-established approach to determining a nucleic acid profile (including those which determine gene copy numbers or methylation profiles, for example) can also be used. Similarly, an AKRl C3 protein profile can be established using any art- known - technique. Examples include determining an AKRl C3 expression profile or an AKRl C3 activity profile.
  • An activity profile may involve, where the AKRlC3-activated prodrug is PR-104A for example, measuring the reduction of PR-104A to PR-104H or PR-104M.
  • the selected profile is an AKRl C3 nucleic acid profile
  • the AKRl C3 nucleic acid assayed for can be the AKRl C3 gene or any representative part thereof.
  • a "gene” in this context is a segment of DNA involved in producing a polypeptide chain, in this case the AKRl C3 protein.
  • the gene can include, without limitation, regions preceding and following the coding region such as the promoter and 3'-untranslated region, as well as the introns.
  • the nucleic acid assayed for can be ribonucleic acid transcribed from the gene during production of the polynucleotide, such as mRNA.
  • the AKRl C3 gene has a number of polymorphisms (Jakobsson, J et al., The P ⁇ karmacogenonomics journal (2007) 7, 282-289). All polymorphisms are included as assay targets. Individual polymorphisms can be targeted by an assay approach which focuses on the (or a) polymorphic site. Those skilled in the art will recognise that the nature of the polymorphism, such as the allele or genotype present at a polymorphic site can also be determined indirectly, e.g., by an analysis of one or more polymorphisms in linkage disequilibrium with an allele at the polymorphic site. Such approaches are described in more detail below.
  • AKRl C3 gene can co-ordinate with expression of other genes, including those in the AKR superfamily. While less desirable than an assay for nucleic acid which is direcdy part of the AKRl C3 gene, an assay which targets such co-ordinated gene expression is in no way excluded.
  • the AKRl C3 protein assayed for will generally be the 323 amino acid 37 kDa protein having wild-type functionality, or any part thereof including any product of in vivo proteolytic digestion. Polymorphic forms (such as the GIn 5 His variant) are however not excluded and neither are fragments (including digestion fragments) of such polymorphic forms. It will be appreciated that retention of the nitroreductive functionality of the protein or fragment and identification of that capability is critical to assigning either a "predicted responder" or "predicted non-responder” status to a patient.
  • the result of the assay whether via an AKRl C3 nucleic acid profile, an AKRl C3 protein profile or both will be to assign a "predicted responder" or "predicted non-responder” status to a patient.
  • Patients whose samples generally a turnout biopsy, preferably taken shordy or immediately prior to the assay) are AICRl C3 positive will be predicted to be responders whereas patients whose samples are AKRl C3 negative will be predicted to be non-responders.
  • Predicted responders will be considered suitable for therapy involving administration of an AKRl C3-activa ted prodrug.
  • Such prodrugs are any compound which is capable, upon exposure to functional AKRl C3 enzyme, of releasing a cytotoxic species to have an anti-tumour effect.
  • activation by AKRl C3 may be the only mechanism.
  • Activation by AKRl C3 need not, however, be the sole activation mechanism for all such compounds.
  • any compound which is activated by both AKRl C3 and a nitroreductase expressed within, or proximate to, hypoxic regions of tumour remains an "AKRl C3-activated" compound or drug as that term is used in this invention.
  • AKRl C3-activated prodrugs include the nitroaromatic prodrugs described in WO 2004/033415 and their pre-prodrug forms described in WO 2005/042471.
  • PR-104A and PR-104 Specific representative examples are PR-104A and PR- 104.
  • Prodrugs PR- 104 and PR-104A are also able to be activated by 1 -electron nitroreduction under hypoxia.
  • prodrugs of formula I are:
  • Pre-prodrugs of formula II are:
  • X and Y are different with X being selected from Cl, Br, I, and OSO 2 R 1 , and Y being selected from Cl, Br, I and OSO 2 R 1 ;
  • Z represents, at any available ring position, -CO- or -SO 2 -;
  • R is methyl or ethyl;
  • R 1 is C 1 — C 6 alkyl; and n is 1 - 6.
  • Non-responders are candidates for treatment widi other cancer therapeutics (particularly those which are AKRl C-independent) or with AKRl C3-activated agents which are also activated via another mechanism.
  • Predicted non-responders to AKRl C3 can therefore be treated with PR- 104 or PR-104A alone or in combination(s) with other agents, including other cancer therapeutics.
  • a patient who has predicted non-responder status may have some level of AKRl C3 expression, albeit less than enough to be predicted to respond therapeutically to treatment with an AKRl C3-activated prodrug on its own.
  • an AKRlC3-independent (or negated) treatment approach is preferred, either by anti-cancer agents which are not activated or otherwise affected by AKRl C3, or by a combination approach where it remains desirable to administer an AKRl C3-activated prodrug.
  • One preferred combination approach is to administer PR-104 or PR-104A together with a Non-Steroidal Anti-Inflammatory Drug (NSAID).
  • NSAID Non-Steroidal Anti-Inflammatory Drug
  • This combination can be administered alone or itself in combination with other cancer therapeutics.
  • Combinations which include both PR-104/PR- 104A and an NSAID are employed where it is viewed as necessary or desirable to functionally eliminate any residual AKRl C3 activation from PR-104/PR-104A when treating the patient.
  • NSAIDs that inhibit AKRl C3 are well known in the art. While any appropriate NSAID can be employed in this approach, it is preferred that the NSAID be naproxen.
  • AKRl C3 negative Patients whose tumours are considered AKRl C3 negative are also candidates for treatment with therapeutics to which resistance is induced through co-expression of other genes in the AKR family (such as AKRlCl, AKRl C2, AKRlBlO and NQOl).
  • Representative cancer therapeutics to treat AKRl C3 negative patients include antifolates (Selga et al., Biochemical Pharmacology 75 (2008)414-426), platinums (such as cis-platin and carboplatin) and anthracyclines (such as oracin (6- [2-(2-hydroxyethyl)-aminoethyl]-5,l l-dioxo-5,6-dihydro-ll i-/-indeno[l,2-c]isoquinoline), doxorubicin and daunorubicin).
  • antifolates Selga et al., Biochemical Pharmacology 75 (2008)414-426
  • platinums such as cis-platin and carboplatin
  • anthracyclines such as oracin (6- [2-(2-hydroxyethyl)-aminoethyl]-5,l l-dioxo-5,6-dihydro-ll i-/-
  • AKRl C3 RNA transcripts have been detected in the brain, lung, liver, kidney, colon small intestine, mammary gland, uterus, cervix, bladder, prostate and testis, and most of these areas will be tumour-free in the patient concerned.
  • Levels of AKRl C3 in normal tissues not implicated in the cancer or in blood, serum or plasma can therefore also be determined for the purpose of predicting whether or not treatment with a particular AKRl C3-activated prodrug will have an acceptable toxicity outcome for a patient.
  • results of such an assay can be used, usually in conjunction with the results of a parallel tumour- focussed assay, to predict the likely cytotoxicity of the prodrug within the patient generally, and to either eliminate the patient as suitable for treatment (where the non-specific toxicity is unacceptably high) or to assist with optimising the dose of the prodrug to be administered to the patient.
  • the results of assays described herein can be used to calculate a likely maximum tolerated dose for an individual, having regard to the level or activity of AKRl C3 in normal tissue (e.g., from the individual) compared to tumour tissue.
  • a variety of techniques can be used for genotypic analysis of a nucleic acid sample in determining a genotypic profile according to the mediods of the present invention. For example, en2ymatic amplification of nucleic acid from a sample can be conveniently used to obtain nucleic acid for subsequent analysis. However, the presence or absence of a nucleic acid sequence can also be determined directly from a nucleic acid sample without enzymatic amplification (e.g., using hybridization techniques). Geno typing of nucleic acid, whether amplified or not, can be performed using any of various techniques known to one of skill in the art. Useful techniques include, without limitation, polymerase chain reaction (PCR)-based analysis, sequence analysis, and electrophoretic analysis, which can be used alone or in combination.
  • PCR polymerase chain reaction
  • PCR amplification of nucleic acids
  • primers for PCR analysis can be designed based on the sequence flanking the nucleic acid of interest.
  • a PCR primer can contain between about 15 to about 60 nucleotides (e.g., 15-50,15-40, or 15-30 nucleotides) of a sequence upstream or downstream of the nucleic acid of interest.
  • Such primers generally are designed to have sufficient guanine and cytosine content to attain a high melting temperature which allows for a stable annealing step in the amplification reaction.
  • Several computer programs, such as Primer Select are available to aid in the design of PCT primers.
  • Sequence analysis can also be useful for genotyping a gene.
  • sequence analysis includes any manual or automated process by which the order of nucleotides in a nucleic acid is determined.
  • sequence analysis can be used to determine the nucleotide sequence of a sample of DNA.
  • the term encompasses, without limitation, chemical and enzymatic methods such as dideoxy enzymatic methods including, for example, Maxam-Gilbert and Sanger sequencing as well as variations thereof.
  • the term also encompasses, without limitation, capillary array DNA sequencing, which relies on capillary electrophoresis and laser-induced fluorescence detection and can be performed using instruments such as the MegaBACE 1000 or ABI 3700.
  • the term encompasses thermal cycle sequencing (Sears et al., Biote ⁇ niques, 13:626-633 (1992)); solid-phase sequencing (Zimmerman et al., Methods MoI. Cell. Biol., 3:39-42 (1992); and sequencing with mass spectrometry, such as matrix - assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Fu et al, Nature Biotech, 16:381-384 (1998).
  • thermal cycle sequencing Sears et al., Biote ⁇ niques, 13:626-633 (1992)
  • solid-phase sequencing Zimmerman et al., Methods MoI. Cell. Biol., 3:39-42 (1992)
  • sequencing with mass spectrometry such as matrix - assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Fu et al, Nature Biotech, 16:381-384 (1998).
  • the term further includes, without limitation, sequencing by hyridization (SBH), which relies on an array of all possible short obligonucleotides to identify a segment of sequence (Chee et al, Science, 274:610-614 (1996); Drmanac et al., Science, 260:1649-1652 (1993); Drmanac et al., Nature Biotech., 16:54-58 (1998)). See, in general, Ausubel et al., Current Protocols in Molecular Biology, Chapter 7 and Supplement 47, John Wiley & Sons, Inc., New York (1999). A number of sequencing methods and platforms are particularly suited to large-scale implementation, and are amenable to use in the methods of the invention.
  • SBH sequencing by hyridization
  • pyrosequencing methods such as that utilised in the GS FLX pyrosequencing platform available from 454 Life Sciences (Branford, CT) which can generate 100 million nucleotide data in a 7.5 hour run with a single machine
  • solid-state sequencing methods such as that utilised in the SOLiD sequencing platform (Applied Biosystems, Foster City, CA).
  • a number of methods currendy used for detection of polymorphsims involve site-specific and/or allele-specif ⁇ c hybridisation. These methods are largely reliant on the discriminatory binding of oligonucleotides to target sequences containing the SNP of interest.
  • the techniques of Illumina (San Diego, CA), Affymetrix (Santa Clara, CA.) and Nanogen Inc. (San Diego, Calif.) are particularly well-known, and utilize the fact that DNA duplexes containing single base mismatches are much less stable than duplexes that are perfectly base-paired. The presence of a matched duplex is usually detected by fluorescence.
  • a number of whole-genome genotyping products and solutions amenable or adaptable for use in the present invention are now available, including those available from the above companies.
  • the method utilises a single-step hybridization involving two hybridization events: hybridization of a first portion of the target sequence to a capture probe, and hybridization of a second portion of said target sequence to a detection probe. Both hybridization events happen in the same reaction, and the order in which hybridisation occurs is not critical.
  • electrophoretic analysis can be useful for genotyping a gene.
  • electrophoretic analysis includes a process whereby charged molecules are moved through a stationary medium under the influence of an electric field. Electrophoretic migration separates nucleic acids primarily on the basis-of their charge, which is in proportion to their size, with smaller molecules migrating more quickly.
  • the term includes, without limitation, analysis using slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, or capillary electrophoresis.
  • Capillary electrophoretic analysis generally occurs inside a small-diameter quartz capillary in the presence of high (kilovolt- level) separating voltages with separation times of a few minutes.
  • nucleic acids are conveniently detected by UV absorption or fluorescent labeling, and single-base resolution can be obtained on fragments up to several hundred base pairs in length.
  • Such methods of electrophoretic analysis, and variations thereof, are well known-in the art, as described, for example, in Ausubel et al., Current Protocols in Molecular Biology, Chapter 2 and Supplement 45, John Wiley & Sons, Inc., New York (1999).
  • genotyping techniques include, without limitation, automated sequencing and RNAase mismatch techniques (Winter et al., Proc. Natl. Acad. Sd., 82:7575-7579 (1985). See, in general, Birren et al., Genome Anayl sis: A Laboratory Manual, Volume 1 (Analyzing DNA), New York, Cold Spring Harbor Laboratory Press (1997).
  • an AKRl C3 nucleic acid profile that comprises data on one or more polymorphisms, such as one or more polymorphisms associated with increased or decreased expression or activity of AKRl C3, can be generated by direcdy determining the one or more polymorphisms, or by detection of one or more other polymorphisms which are in linkage disequilibrium with one or more of said polymorphisms.
  • Linkage disequilibrium is a phenomenon in genetics whereby two or more mutations or polymorphisms are in such close genetic proximity that they are co-inherited. This means that in genotyping, detection of one polymorphism as present implies the presence of the other.
  • the one or more polymorphisms in linkage disequilibrium with the one or more polymorphisms are in greater than about 60% linkage disequilibrium, are in about 70% linkage disequilibrium, about 75%, about 80%, about 85%, about 90%, about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% linkage disequilibrium with the one or more polymorphisms.
  • a gene expression profile is typically evaluated in vitro on a sample collected from a subject in comparison to a normal or reference sample. Determination of a transcriptional expression profile can be accomplished, e.g., using hyridization techniques well-known to those skilled in the art such as Northern analysis and slot blot hybridization or by performing reverse-transcriptase (RT)- PCR amplification followed by gel electrophoresis.
  • hyridization techniques well-known to those skilled in the art such as Northern analysis and slot blot hybridization or by performing reverse-transcriptase (RT)- PCR amplification followed by gel electrophoresis.
  • PCR amplification techniques are described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1999); Theophilus et at, "PCR Mutation Detection Protocols," Humana Press (2002); and Innis et al., “PCR Applications: Protocols for Functional Genomics," 1 st Edition, Academic Press (1999).
  • General nucleic acid hybridization methods are described in Anderson, “Nucleic Acid Hybridization,” BIOS Scientific Publishers (1999).
  • Amplification or hybridization of a plurality of transcribed nucleic acid sequences can also be performed using mRNA or cDNA sequences arranged in a microarray.
  • Microarray methods are generally described in Hardiman, “Microarrays Methods and Applications: Nuts & Bolts,” DNA Press (2003) and Baldi et al., “DNA Microarrays and Gene Expressions: From Experiments to Data Analysis and Modeling,” Cambridge University Press (2002).
  • 5,777,888 discloses the utility of microarray gene expression profiles to evaluate the target specificity of a candidate drug by comparison of an expression profile obtained from cells treated with the candidate drug to a database of expression profiles obtained from cells treated with known drugs.
  • U.S. Pat. No. 6,218,122 provides methods of monitoring the disease state of a subject and determining the effect of a therapy upon the subject through the use of gene expression profiles (see, also, U.S. Pat. No. 6,266,093).
  • a protein biomarker can be analyzed using an immunoassay.
  • a protein expression profile can also be evaluated using electrophoresis, e.g., Western blotting, as well as any other technique known to those skilled in the art.
  • Immunoassay techniques and protocols are generally described in Price and Newman, "Principles and Practice of Immunoassay,” 2nd Edition, Grove's Dictionaries (1997); and Gosling. "Immunoassays: A Practical Approach," Oxford University Press (2000).
  • the presence or amount of the protein biomarker is typically determined using antibodies specific for the biomarker and detecting specific binding.
  • Any suitable immunoassay can be utilized for determining the presence of level of one or more protein biomarkers in a sample.
  • a variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (see, e.g., Self et al., Curr. Opin. Biote ⁇ noL, 7:60-65 (1996)).
  • immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL).
  • EIA enzyme multiplied immunoassay technique
  • ELISA enzyme-linked immunosorbent assay
  • MAC ELISA IgM antibody capture ELISA
  • MEIA microparticle enzyme immunoassay
  • CEIA capillary electrophoresis immunoassays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • FPIA fluor
  • Immunoassays can also be used in conjunction with laser induced fluorescence (see, e.g. Schmalzing et al., Electmphoresis, 18:2184-93 (1997); BaoJ. Chromatogr. B. Biomed. Sa., 699:463-80 (1997)).
  • Liposome immunoassays such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention (see, e.g., Rongen et al.,/. Immunol. Methods, 204:105-133 (1997)).
  • Nephelometry assays in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concertration, are suitable for use in the methods of the present invention.
  • Nephelometry assays are commercially available from Beckman Coulter (Brea, Calif.;- Kit #449-430) and can be performed using a Behring Nephelometer Analyzer (Fink et al.,/. Clin. Chem. Clin. Bio ⁇ em., 27:261-276 (1989)).
  • Direct labels include fluorescent or luminescent tags, metals dyes, radionuclides, and the like, attached to the antibody.
  • An antibody labelled with iodine-125 ( 125 1.) can be used for determining the level of one or more biomarkers in a sample.
  • a chemiluminescence assay using a chemiluminescent antibody specific for the biomarker is suitable for sensitive, non-radioactive detection of biomarker levels.
  • An antibody labelled with fluorochrome is also suitable for determining the level of one or more biomarkers in a sample.
  • fluorochromes examples include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoerythrin, rhodamine, Texas red, and lissamine.
  • Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, urease, and the like.
  • a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethyl-benzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • TMB tetramethyl-benzidine
  • An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm.
  • a ⁇ -galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl- ⁇ -D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm.
  • An urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St. Louis, Mo.).
  • a signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 125 1; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis of the amount marker levels can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) in accordance with the manufacturer's instructions.
  • the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
  • Antigen capture assays can be useful in the methods of the present invention.
  • an antibody directed to a biomarker of interest is bound to a solid phase and sample is added such that the biomarker is bound by the antibody. After unbound proteins are removed by washing, the amount of bound marker can be quantitated using, for example, a radioimmunoassay (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)).
  • Sandwich enzyme immunoassays can also be useful in the methods of the present invention. For example, in a two-antibody sandwich assay, a first antibody is bound to a solid support, and the biomarker is allowed to bind to the first antibody.
  • the amount of the biomarker is quantitated by measuring the amount of a second antibody that binds the biomarker.
  • the antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microliter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like.
  • An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
  • Quantrtiative Western blotting also can be used to detect or determine the level of one or rr ⁇ ore proteinaceous biomarkers in a sample.
  • Western blots can be quantitated by well-known methods such as scanning densitometry or phosphorimaging. In certain instances, autoradiographs of the blots are analyzed using a scanning densitometer (Molecular Dynamics; Sunnyvale, Calif.) and normalized to a positive control. Values are reported, for example, as a ratio between the actual value to the positive control (densitometric index). Such methods are well known in the art as described, e.g., in Parra et alj. Vase. Surg., 28:669-675 (1998).
  • immunohistochemistry or “IHC” encompasses techniques that utilize the visual detection of fluorescent dyes or enzymes coupled (i.e., conjugated) to antibodies that react with the biomarker using fluorescent microscopy or light microscopy and includes, without limitation, direct fluorescent antibody, indirect fluorescent antibody (IFA), anticomplement immunofluorescence, avidinbiotin immunofluorescence, and immunoperoxidase assays.
  • IHC immunohistochemistry
  • An IFA assay for example, is useful for determining whether a sample is positive for a particular marker of interest, the level of that marker, and/or die staining pattern of that marker.
  • concentration of the marker in a sample can be quantiated, e.g., through endpoint titration or through measuring the visual intensity of fluorescence compared to a known reference standard.
  • IHC can be performed according to the following protocol: (1) slides containing the sample (e.g., tumor tissue are deparaffinized with xylene/70% ethanol into phosphate buffered saline (PBS) at pH 7.4; (2) the slides are then immersed in 10 mM citric acid at pH 6.0 microwaved for about 37 minutes, and cooled down at room temperature (RT) for bout 30-60 minutes; (3) endogenous peroxidases ate quenched for about 10 minutes in 1 part 30% H 2 O 2 and 9 parts methanol and the slides are washed 3 times for 3 minutes in PBS; (4) the slides are blocked with blocking reagent at RT for about 30 minutes; (5) antibodies against the biomarker of interest are added and the slides are incubated at 4° C.
  • PBS phosphate buffered saline
  • the slides are washed in PBS at RT for about 30 minutes, changing the wash buffer every 5 minutes; (7) secondary antibodies such as biotinylated antibodies are added and the slides are incubated at RT for about 60 minutes; (8) the slides are washed in PBS at RT for about 30 minutes, changing the wash buffer every 5 minutes; (9) streptavidin is added and the slides are incubated at RT for about 30 minutes; (10) 3,3 — diaminobenzidine (DAB) is added, the slides are incubated for 5 minutes, the DAB is neutralized with bleach, and the slides are washed for 5 minutes with water; (11) the slides are counterstained with methylgreen for 3 minutes washed 3 times with water; (12) the slides are dipped in 95% ethanol, followed by a 100% ethanol and xylene series; and (13) a coverslip is placed on the slide.
  • DAB diaminobenzidine
  • IHC protocols for determining the presence or level of specific antigens of interest are known in the art. These include the IHC protocols described in, e.g., Ishikawa et al, Cancer Res., 65:9176-9184 (2005) for TGF- ⁇ and amphiregulin; Cappuzzo et al.,/. Clin. Oncol., 23:5007-5018 (2005) for HER2; Cappuzzo et al., /. Natl. Caner Inst., 97:643-655 (2005) for EGFR; Abrams et al., MoI. Cancer. Then, 2:471-478 (2003) for c-KIT and PDG-FRB; and Lee et A., Anal. Quant.
  • Tissue staining can be visualized using peroxidase-based immunostaining kits available from Vector Laboratories (Burlingame, Calif.) and DAKO (Glostrup, Denmark).
  • the presence or level of a biomarker can also be determined by detecting or quantifying the amount of the purified marker. Purification of the marker can be achieved, for example, by high pressure liquid chromatography (HPLC), alone or in combination with mass spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, tandem MS, etc.).
  • Qualitative or quantitative detection of a biomarker can also be determined by well-known methods including, without limitation, Bradford assays, Coomassie blue staining, silver staining, assays for radiolabeled protein, and mass spectrometry.
  • the activity of a protein biomarker can also be determined by art-known techniques, including by one or more of the analytical techniques discussed above.
  • Antibodies against AKRl C3 are commercially available.
  • a suitable example is the anti — AKRl C3 antibody, clone NPG. G6. A6 (Lin et al., Steroids, 2004 69:795-801, Sigma product A6229).
  • the invention is not limited to the use of existing antibodies but contemplates generation of new reagents.
  • the generation and selection of antibodies not already commercially available for detecting or determining the level of protein biomarkers generally may be accomplished several ways.
  • one way is to purify polypeptides of interest using e.g., solid phase peptide synthesis methods well known in the art. See, e.g., Guide to Protein Purification, Murray P. Deutcher, ed., Meth. En ⁇ ymol, Vol. 182, 1990; Solid Phase Peptide Synthesis, Greg B. Fields, ed., Meth. En ⁇ ymol, Vol. 289, 1997; Kiso et al., Chem. Pharm. Bull., 38:1192-99 (1990); Mostafavi et al., Biomed. Pept.
  • polypeptides Nucleic Acids, 1:225-60, (1995); Fujiwara et al., Chem. Pharm. Bull., 44:1326-31 (1996).
  • the selected polypeptides may then be injected, for example, into mice or rabbits, to generate polyclonal or monoclonal antibodies.
  • mice or rabbits One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in ⁇ ntibodies, A Laboratory Manual, Harlow and Lane, Eds., Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. (1998).
  • binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (see, e.g., Antibody Engineering: A Practical Approach, Borrebaeck, Ed., Oxford University Press, Oxford (1995);/. Immunol, 149:3914- 3920 (1992)).
  • Hypoxia-activated prodrug PR- 104 is a water-soluble phosphate ester which is converted in ⁇ vo to the corresponding alcohol, PR-104A.
  • This 3,5-dinitrobenzamide-2-nitrogen mustard is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and 5-amine (PR-104M) (Patterson et al., Clin Can Res 2007, 13:3922-32, see Figure 1).
  • neoplastic cell lines display atypical aerobic cell sensitivity that appears to correlate with NQOl expression in vitro (Guise et al., Biochem Pharmacol, 2007, 74:810- 20). NQOl cDNA expression does not however increase aerobic cytotoxicity or PR-104A metabolism. Thus DT-diaphorase is not the aerobic PR-104A reductase but appears to be co- ordinately regulated with it.
  • Aims ⁇ To identify the reductase(s) responsible for the reduction of PR-104A under aerobic conditions. ⁇ To determine the expression of these reductase(s) in human cancers.
  • RNA expression profiles covering 38,500 probes (Affymetrix HG-Ul 33 Plus 2.0) were obtained for 23 human neoplastic cell lines.
  • Oxidoreductase genes were selected, using Gene Ontology, and analysed by hierarchical clustering .
  • To identify candidate PR-104A reductases the correlation coefficient (Kendall TauJ between gene expression and aerobic PRl 04 metabolism (to PR104H/M) was calculated for each probeset; correlation p-values were adjusted using the false discovery rate (FDR) correction for multiple comparisons.
  • FDR false discovery rate
  • Entry vectors encoding AKR family members/NQOl were purchased from Invitrogen's sequence verified ultimate ORF library.
  • Candidate genes were cloned into a Gateway compatible expression vector behind the EF-I ⁇ promoter.
  • the bicistronic mRNA also encoded a protein conferring resistance to puromycin to enable selection of transfected cells.
  • PR-104A metabolites by LC /MS /MS.
  • Cells were exposed to PR-104A in 24 well plates for 1 hr using a 37°C humidified incubator (20% O2, 5% CO2)
  • PR-104A metabolites were extracted from the media and cell monolayer by addition of two volumes of methanol containing tetradeuterated (d4)-PR-104H internal standard and stored at -80 0 C.
  • Samples (75 ⁇ L) of standards and unknowns were diluted with 25 ⁇ L water and assayed by LC/MS/MS (Agilent 6410).
  • Antibodies Anti-AKR1C3 (Sigma, A6229). Anti-AKRIBIO (Abnova, H00057016-M01). Anti-V5-HRP (Invitrogen, 46-0708). Anti-actin (Chemicon, MAB1501R). Anti-NQOl (kindly provided by Prof David Ross, University of Colorado, Denver). Cytotoxicity assay. Growth inhibition of human HCTl 16 WT and HCTl 16 AKRl C3 carcinoma cells was determined from IC50 values for 4 hr drug exposure under oxic conditions, with staining by sulforhodamine B 5 days later.
  • Tumour growth delay assay Antitumour activity of PR-104 and cyclophosphamide in HCTl 16 WT and HCTl 16 AKRl C3 tumours was evaluated. Tumours were grown in CD-I nude mice (SC inoculation) and activity was assessed by tumour growth inhibition. Mice were IP dosed at the MTD with PR-104 (550mg/kg) or cyclophosphamide (62mg/kg) using a q4dx3 schedule. Tumour volumes were monitored until tumour volume increased 4-fold with respect to day 1 treatment volume. Clonogenic survival assay. Tumours were grown in CD-I nude mice (SC inoculation). Mice were IP dosed with PR-104 (348mg/kg).
  • tumours were excised, weighed, minced with surgical scissors and dissociated enzymatically in magnetically stirred suspensions.
  • Cell suspensions were diluted, plated and incubated at 37°C for 10-14 days before staining with methylene blue. Colonies containing >50 cells were counted.
  • Commercial tumour microarrays TMAs. TMAs were purchased from Imgenex Inc. (Sano).
  • oxidoreductase genes identified a cluster of cell lines (SiHa, HT29, HCT8, H460, A549, Skov3) which also had high sensitivity to PR-104A in aerobic IC50 assays (not shown) and high rates of aerobic PR-104A reduction to PR-104H and PR-104M (see Fig 3A).
  • ARR aldo-keto reductase
  • PR-104A can be reduced by AKR1C3 under aerobic conditions ⁇
  • candidate AKR enzymes were expressed in metabolism-null HCTl 16 cells.
  • Expression of proteins was checked using Invitrogen's TAG on demandTM system and * "-' - through specific antibodies for AKRl C3, AKRlBlO and NQOl (representative actin loading shown) (Fig 2B).
  • AKRl C3 ⁇ Expression of AKRl C3 correlates well with aerobic metabolism of PR-104A to its cytotoxic metabolites in human tumour cell lines ( Figure 3). The correlation observed with AKR1C3 appears to be stronger than that observed for NQOl or AKRlBlO.
  • Tumour xenografts expressing AKRl C3 are more sensitive to PR-104 compared to ' tumours with low AKRl C3 expression (Fig 4B-E).
  • ⁇ AKRl C3 was shown to metabolise PR-104A in ⁇ tro.
  • AKRl C3 expression varies between cancer types. Cancer populations with high expression of AKR1C3 present as indications for PR-104 treatment.
  • Patients with cancer can be screened to determine their individual levels of AKRl C3 expression and identified as likely responders or non-responders to treatment with PR- 104/PR-104A.
  • TMAs human tumor biopsy tissue microarrays
  • Table 2 A total of 3932 individual cores representing 19 cancer types (2490 cases) were analyzed across 38 TMAs with an average of 207 cores per disease (median 174; range 31-452).
  • Methodology optimisation was carried out and cross-validated against paired frozen samples by western blot (Fig. S8).
  • TMAs tissue microarrays
  • AKR1C3 expression in human tumor surgical samples is heterogeneous.
  • a scoring system illustrated in Fig. 7A, gave higher ranking to uniform over focal staining - with scores of 4, 5 and 6 considered 'positive'.
  • Expression of AKRl C3 was present in most tumor types (Fig 7B).
  • HCC showed the highest frequency of positive cores with most (58%) staining strongly in all cells (Score 6).
  • Other disease types with >50% positive cores included bladder, renal and gastric carcinomas. A summary of all scores is shown in Table 3.
  • Table 3 Summary of AKR1C3 immunohistochemical staining score frequencies.
  • Esophagus 133 201 4.5 30.8 3.0 38.3 22.4 8.5 9.0 21.9 61.7
  • Lymphoma 36 45 0 4.4 0.0 4.4 15.6 0.0 22.2 57.8 95.6
  • Adrenal 20 21 23.8 4.8 19.0 47.6 23.8 4.8 4.8 19.0 52.4
  • Thyroid 15 15 0 0.0 6.7 6.7 6.7 6.7 20.0 60.0 93.3
  • AKR1C3 expression in normal tissues A survey of 33 normal tissue cores identified small intestine and kidney as containing moderate numbers of cells with strong AKRl C3
  • HCC cores (Fig. 7D). Independently, 23 normal tissue sections were analyzed and demonstrated strong positive AKRl C3 staining in 7 tissues; stomach, small intestine, colon, pancreas, kidney, uterus and ovary, with weak/diffuse staining in the majority of liver cells (Table 1). Most specimens demonstrated both nuclear and cytoplasmic staining but adrenal and liver demonstrated cytoplasmic staining only. Thus the full section histopathology analysis was broadly consistent with the TMA scoring.
  • AKRl C3 Over expression of AKRl C3 has been documented in carcinomas of the breast (24, 37, 38), prostate (39-41), endometrium (42, 43), and kidney (44). Overall, the IHC analysis demonstrates the intensity of AKRl C3 expression is strikingly elevated in certain neoplasia relative to normal tissues (Figure 7D).
  • AKRl C3 activation of certain anti-cancer agents and their cytotoxicity between AKR1C3 activation and the responsiveness of a subject to a particular anti-cancer agent, or between AKRl C3 activation and the susceptibility (or otherwise) of a subject to undesirable side effects including undesirable cytotoxicity, each have application in the design and/or screening of candidate therapeutics.
  • the candidate compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art.
  • libraries include: small molecule libraries, peptide libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone that is resistant to enzymatic degradation); spatially addressable parallel solid phase or solution phase libraries; synthetic libraries obtained by deconvolution or affinity chromatography selection; and the "one-bead one- compound” libraries.
  • libraries comprising compounds of formulae I and II are specifically contemplated. Examples of methods for synthesis of molecular libraries are well known in the art.
  • Libraries of compounds may be presented in any manner amenable to identification of the compounds comprised therein, and may include libraries presented in solution, on beads, chips, bacteria (e.g., U.S. Patent No. 5,223,409), spores (e.g., U.S. Patent No. 5,223,409), plasmids, or phage (e.g., U.S. Patent No. 5,223,409).
  • a system containing an AKRl C3 gene or an AKRl c3 gene product, such as AKRl C3, is contacted with a candidate compound and the level of activation is evaluated relative to that in the absence of an AKRl C3 gene or an AKRl c3 gene product.
  • Activation may be assessed by determining the presence or level of a candidate compound or of an activated product thereof, directly or indirectly.
  • methods of chemical analysis such as mass spectroscopy, NMR, HPLC, elemental analysis, and the like, including those analytical methods exemplified herein, may be utilized to identify an activated product directly.
  • one or more activities of a candidate compound or its activated product can be used to indirectly identify candidate compounds susceptible to activation by AKRl C3. For example, a comparison is made between the cytotoxicity of a candidate compound in the presence or absence of AKRl C3.
  • AKRl C3 contemplates prior contact as well as contemporaneous contact, such that candidate compounds may be contacted with AKRl C3 prior to contacting the candidate compound with a cell, whether or not the AKRl C3 is subsequently removed.
  • candidate compound may be contacted with the cell and AKRl C3 simultaneously.
  • the cells may naturally express AKRl C3, or may be modified to express recombinant AKRl C3, for example, may have one or more AKRl C3 genes or fragments thereof, including fragments comprising one or more AKRl C3 polymorphisms, and one or more AKRl C3 genes or fragments thereof fused to a marker gene(s).
  • a parent cell line known to exhibit AKRl C3 activity is used to generate a daughter cell line deficient in AKRl C3 activity, or vice versa, by methods well known in the art. Both parent and daughter cells are contacted with a candidate compound, whereupon cytotoxicity of the compound in each cell line is assessed. A greater degree of cytotoxicity in a cell line exhibiting AKRl C3 activity compared to a cell line lacking AKRl C3 activity is indicative of a candidate compound that is susceptible to AKRl C3 activation.
  • a candidate compound may be contacted with AKRl C3 enzyme, whether in vitro or in ⁇ vo, prior to or during contacting the candidate compound with a target cell (whether a normal cell or a tumour cell).
  • a difference in the cytotoxicity exhibited by the candidate compound that has not been contacted with AKRl C3 versus that exhibited by the candidate compound that has been contacted with AKRl C3 is indicative of the susceptibility of the candidate compound to AKRl C3 activation.
  • HCT-116 NXT cell ATCC CCL-247
  • HCT-116 AKR1C3 cells engineered to express human aldo-keto reductase 1C3 (AKRl C3; NM_003739) were passaged as monolayers in minimal essential media ( ⁇ MEM; Gibco, Invitrogen Corporation, Grand Island, NY, USA) supplemented with 5% FBS, (GIBCO NZ Ltd, Auckland, New Zealand) without antibiotics for ⁇ 3 months from frozen stocks confirmed to be mycoplamsa free by PCR-ELISA (Roche Diagnostics
  • the drugs were then diluted along the plate in 3-fold serial dilutions using a 12- channel pipette (Biolab Limited, Albany, Auckland, New Zealand). Plates were returned to the 37°C, 5% CO 2 incubator and left for 4 hrs. After the incubation period cells were washed three times and plates were filled with 200 ⁇ l of ⁇ MEM containing 5% FCS and 1% penicillin/streptomycin ⁇ Gibco, Invitrogen Corporation, Grand Island, NY, USA), and left for a further 5 days in a 37 0 C, 5% CO 2 incubator. After five days cells were fixed by adding 67 ⁇ l of cold 40% trichloroacetic acid
  • the stain was solubilised by the addition of lOO ⁇ l of 1OmM unbuffered Tris (AppliChem GmbH, Darmstadt, Germany), left for lhr in the dark shaking at 150 rpm (Barns tead-Labline; Barnstead International, Dubuque, 10, USA). Plates were then read on an ELx 808 Absorbance Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA). Wavelengths on the plate reader were set at 490nm for measurement filter and 450nm for reference filter (the reference filter was subtracted from the measurement filter to give final absorbance: 490-450).
  • IC 50 value (KC4 microplate data analysis software V3.4, Bio- Tek), where 50% suppression of cell growth has occurred relative to untreated controls.
  • N 2-6 independent experiments IC 50 value (Mean ⁇ 1 SD) were determined for each HCTl 16 population and intra-experimental sensitivity ratio calculated. The results are shown in Table 6 below.
  • SN 29236 is at least as sensitive to AKRl C3 activation as compound 11 (PR-104A).
  • AKR1C3 expression sensitises cells to PR-104A but not to other bioreductive drugs.
  • AKR1C3 expression enhances the cytotoxicity of PR-104A
  • two clones from a pool of HCTl 16 cells transfected with AKRl C3 were isolated. Clonogenic survival curves showed clone #1 to be 10-fold more sensitive to PR-104A than the parental cells under aerobic conditions, and was further sensitised (44-fold) under anoxia (Fig. 6A).
  • aerobic IC 50 assays the sensitivity of clone #1 and #2, relative to the parental line, to PR-104A and 10 other bioreductive agents including 6 other nitro compounds, 3 quinones and a tertiary amine N- oxide (Fig. 6B and Table 7 below) were compared.
  • the present invention enables the suitability of a particular treatment regimen to a particular patient (and vice versa) to be determined.
  • the discussion herein refers to aspects of the invention useful to predict or determine a subject's response to one or more cancer therapeutics, such as one or more AKRlC3-activated agents
  • these aspects of the invention are also useful in determining a subject's suitability for a treatment regime, preferably in determining a subject's suitability to treatment with one or more anti-cancer agents, including one or more AKRl C3- activated agents, or in determining a subject's suitability to treatment widi a combination of dierapeutic agents, such as one or more AKRl C3-activated agents and one or more NSAIDs.
  • the methods of the invention allow an AKRl C3 profile to be determined for a particular patient.
  • the profile will usually be determined in a tumour sample from the patient with detection of a high level of AKRl C3 expression (for example) being predictive that a patient will likely therapeutically respond to an AKRl C3-activated prodrug. That patient will be identified as having "likely responder status” or as having a "responder profile”.
  • detection of a low level of ARKl C3 expression (again, for example) will be predictive that the patient will be unlikely to therapeutically respond to an AKRl C3-activated prodrug based therapy.
  • the latter patient will be identified as having "likely non-responder status" or as having a "non-responder profile”.
  • AKRl C3 profile such as an AKRl C3 profile
  • AKRl C3 profile comprising data relating to AKRl C3 levels or activity in normal tissue and in tumour tissue can be used to select a particular anti-cancer agent, such as a particular AKRl C3- activated prodrug.
  • a particular anti-cancer agent such as a particular AKRl C3- activated prodrug.
  • an AKRl C3 profile can be used to determine a suitable dosage regime for a subject.
  • an AKRl C3 profile comprising data relating to AKRl C3 levels or activity in normal tissue and in tumour tissue may indicate an enhanced response of said subject to an increased dose, or increasing doses, of an anti-cancer agent, such as an AKRl C3- activated therapeutic agent.
  • die subject may exhibit high levels of AKRl C3 in tumour tissue, but no or very low levels of AKRl C3 in non-tumour tissues.
  • the AKRl C3-activated prodrug can be administered as a monotherapy or in combination with at least one other anticancer agent.
  • the other anti-cancer drug(s) will primarily be selected based upon the cancer type. For example, where the cancer is NSCLC and the AKRlC3-activated prodrug is PR- 104, a suitable combination agent is docetaxel. Similarly, where the cancer is hepatocarcinoma and the AKRl C3-activated prodrug is again PR- 104, the combination agent is sorafenib.
  • the treatment selected may vary dependent upon the precise AKRl C3 profile determined.
  • the patient can be treated with any appropriate anticancer agent (selected in accordance with cancer type).
  • That agent can be an agent which is not activated by AKRl C3 or can be an AKRlC3-activated agent with another mechanism of activation (e.g. hypoxic activation).
  • Such agents can be administered alone or in combination as appropriate.
  • the therapeutic selection criteria change.
  • Either one or more anti-cancer agents without significant AKRlC3-activation capability are selected, or a dual-mechanism AKRlC3-activated prodrug such as PR-104 is administered in combination with an agent which effectively ablates or inhibits AKRl C3 activity such as a Non-Steroidal Anti-Inflammatory Drug (NSAID).
  • NSAID Non-Steroidal Anti-Inflammatory Drug
  • the NSAID for example, naproxen
  • the combination will desirably be administered together with at least one further anti-cancer agent selected with reference to the cancer to be treated.
  • the combination of PR-104, naproxen and docetaxel can be employed in treating NSCLC, and the combination of PR- 104, naproxen and sorafenib employed in treating hepatocarcinoma.
  • compositions of the invention suitable for administration to a subject may be formulated as a medicament or pharmaceutical, and appropriate formulations may be prepared by an art skilled worker with regard to that skill and the teaching of this specification.
  • compositions useful herein may be formulated to allow for administration to a subject by any chosen route, including but not limited to oral or parenteral (including topical, or subcutaneous, intramuscular, intradermal, and intravenous injections or infusion techniques) administration.
  • routes of administration to a subject will typically take into account the purpose for which the composition is being administered, and die nature of the composition (such as the active agents within the composition).
  • any mode of administration may be suitable for any composition of the invention, including administration by multiple routes, including different routes for different agents. Therefore, inhalation (nasal or buccal inhalation) and vaginal and rectal administration of any composition of the invention is also contemplated. Intramedullar, epidural, intra-articular, and intra-pleural administration of any composition of the invention is also contemplated. Administration of a composition of the invention, optionally with at least one additional therapeutic factor, by a first administration route accompanied by separate, simultaneous or sequential administration of one or more other agents, including one or more other therapeutic agents, by a second administration route is also contemplated; for example, intravenous administration of a composition of the invention accompanied by oral administration of the at least one additional therapeutic agent.
  • a pharmaceutical composition according to the invention may be formulated with an appropriate pharmaceutically acceptable carrier (including excipients, diluents, auxiliaries, and combinations thereof) selected with regard to the intended route of administration and standard pharmaceutical practice. See for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed., Mack Publishing Co., 1980.
  • compositions of the invention may also be formulated as a dosage form.
  • a dosage form useful herein may be administered orally as a powder, liquid, tablet or capsule.
  • Suitable dosage forms may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents, or have an enteric coating. Suitable enteric coatings are known. Enteric coatings surrounding the active ingredients and prevent the release of the active ingredients in the stomach but allow release after the dosage form has left the stomach.
  • Dosage forms useful herein may be adapted for immediate, delayed, modified, sustained, pulsed or controlled release of the active components.
  • Suitable formulations may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents.
  • Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the active ingredients with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. Active ingredients can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tabletting agent.
  • Liquid pharmaceutical compositions may comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • compositions can also be administered via the parenteral route.
  • parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipient. Solubilising agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic agent.
  • the pharmaceutical composition may conveniently be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has a suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has a suitable pH, isotonicity and stability.
  • Those of skill in the art are able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, and Lactated Ringer's injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included as required.
  • Injectable dosage forms may be formulated as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. 5 The dosage form may also be emulsified. Anti-cancer therapeutics, and when present the at least one additional therapeutic factor, may be mixed with carriers such as, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • Sustained-release preparations may be prepared by methods well-known in the art. Suitable examples of sustained-release preparations include semi-permeable matrices of solid 10. hydrophobic polymers containing the therapeutic agents described herein, such as one or more AKRlC3-activated agent, and when present the at least one additional therapeutic agent.
  • the matrices may be in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices examples include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (see US 3,773,919), copolymers of L-glutamic 15 acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, and degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate).
  • polyesters for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)
  • polylactides see US 3,773,919
  • copolymers of L-glutamic 15 acid and ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid- glycolic acid copolymers such
  • the composition is in the form of a powder, a tablet, a caplet, a pill, a hard or soft capsule or a lozenge, or in the form of a cachet, a dispensable powder, granules, a 20 suspension, an elixir, a liquid, a drink, or any other form that can be added to food or drink, including for example water or fruit juice.
  • the composition is an enteral product, a solid enteral product or a liquid enteral product.
  • composition further comprises one or more constituents (such as antioxidants) which prevent or reduce degradation of the composition during storage or after 5 administration.
  • constituents such as antioxidants
  • compositions of the invention can be provided and administered in forms suitable for once-a-day dosing.
  • an acetate, phosphate, citrate or glutamate buffer may be added allowing a pH of the final composition to be from about 5.0 to about 9.5; optionally a carbohydrate or polyhydric alcohol tonicifier and, a preservative selected from the group 30 consisting of m-cresol, benzyl alcohol, methyl, ethyl, propyl and butyl parabens and phenol may also be added.
  • Water for injection, tonicifying agents such as sodium chloride, as well as other excipients may also be present, if desired.
  • formulations are isotonic or substantially isotonic to avoid irritation and pain at the site of administration.
  • buffer when used with reference to 35 hydrogen-ion concentration or pH, refer to the ability of a system, particularly an aqueous solution, to resist a change of pH on adding acid or alkali, or on dilution with a solvent.
  • Characteristic of buffered solutions which undergo small changes of pH on addition of acid or base, is the presence either of a weak acid and a salt of the weak acid, or a weak base and a salt of the weak base.
  • An example of the former system is acetic acid and sodium acetate. The change of pH is slight as long as the amount of hydroxyl ion added does not exceed the capacity of the buffer system to neutralize it.
  • Maintaining the pH of the formulation in die range of approximately 5.0 to about 9.5 can enhance die stability of the parenteral formulation of the present invention.
  • Other pH ranges include, about 5.5 to about 9.0, or about 6.0 to about 8.5, or about 6.5 to about 8.0, or, preferably, about 7.0 to about 7.5.
  • the buffer used in die certain embodiments of the present invention may be selected from any of the following, for example, an acetate buffer, a phosphate buffer or glutamate buffer, die most preferred buffer being a phosphate buffer.
  • Carriers or excipients can also be used to facilitate administration of the compositions and formulations of die invention.
  • carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, polyethylene glycols and physiologically compatible solvents.
  • a stabilizer may be included in the formulations of the invention, but will generally not be needed. If included, however, a stabilizer useful in die practice of the invention is a carbohydrate or a polyhydric alcohol.
  • the polyhydric alcohols include such compounds as sorbitol, mannitol, glycerol, xylitol, and polypropylene/ethylene glycol copolymer, as well as various polyediylene glycols (PEG) of molecular weight 200, 400, 1450, 3350, 4000, 6000, and 8000).
  • the carbohydrates include, for example, mannose, ribose, trehalose, maltose, inositol, lactose, galactose, arabinose, or lactose.
  • Anti-microbial agents in bacteriostatic or fungistatic concentrations are generally added to preparations contained in multiple dose containers.
  • a preservative is, in the common pharmaceutical sense, a substance diat prevents or inhibits microbial growth and may be added to a pharmaceutical formulation for this purpose to avoid consequent spoilage of the formulation by microorganisms. While the amount of the preservative is not great, it may nevertheless affect the overall stability of the active agent(s).
  • die preservative for use in the practice of the invention can range from 0.005 to 1.0% (w/v), the preferred range for each preservative, alone or in combination with others, is: benzyl alcohol (0.1-1.0%), or m-cresol (0.1-0.6%), or phenol (0.1-0.8%) or combination of methyl (0.05- 0.25%) and ethyl or propyl or butyl (0.005%-0.03%) parabens.
  • the parabens are lower alkyl esters of para-hydroxybenzoic acid.
  • the parenteral formulation may be thickened with a thickening agent such as a methylcellulose.
  • a thickening agent such as a methylcellulose.
  • the formulation may be prepared in an emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder, a non-ionic surfactant or an ionic surfactant.
  • aqueous suspensions such as synthetic and natural gums, e.g., tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
  • Such additional ingredients may include wetting agents, oils (e.g., a vegetable oil such as sesame, peanut or olive), analgesic agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatin or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine).
  • oils e.g., a vegetable oil such as sesame, peanut or olive
  • analgesic agents emulsifiers, antioxidants, bulking agents, tonicity modifiers, metal ions, oleaginous vehicles
  • proteins e.g., human serum albumin, gelatin or proteins
  • a zwitterion e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine.
  • the dose of the composition administered, the period of administration, and the general administration regime may differ between subjects depending on the responder status of the subject, in addition to such variables as the severity of symptoms of a subject, the type of disorder to be treated, the mode of administration chosen, and the age, sex and/or general health of a subject.
  • the dosage required may depend on the choice of the route of administration, the nature of the formulation, the nature of the subject's illness, the subject's size, weight, surface area, age, and sex, other drugs being administered, the judgment of the attending physician, and the nature of the anti-cancer agent. Wide variations in the needed dosage are to be expected in view of the variety of compounds available and the different efficiencies of various routes of administration.
  • oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art.
  • the amount of anti-cancer agent administered may be between about 20% and 100% of the maximum tolerated dose of the subject.
  • administration may include a single dose, such as a single daily dose, or administration of a number of discrete divided doses as may be appropriate. It will also be understood that a person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine an effective dosage regime (including dose and timing of administration) for a given condition.
  • Ketoconazole, cimetidine, isoniazid, metyrapone, quinidine, furafylline, celecoxib, naproxen (NAP), diethyldithiocarbamate (DITC) and 1-aminobenzotriazole (ABT) were from
  • Tissue solubiliser Soluene-350
  • liquid scintillants Emulsifier-SafeTM, Hionic-Fluor
  • Cell lines were passaged in ⁇ MEM supplemented with 5% fetal bovine serum without antibiotics for ⁇ 3 mo from frozen stocks confirmed to be Mycoplasma- free by PCR-ELISA (Roche Diagnostics). Cell lines were obtained from ATCC (Manassas, VA) except for the gift of A549 cells (Dr. Martin Brown, Stanford U.) and SiHa (Dr. David Cowan, Ontario Cancer
  • AKR1C3 in which human AKRl C3 is expressed from an EF-I ⁇ promoter in die Gateway compatible F527-V5 plasmid, is as described above. Plasmids containing cDNAs of the murine genes AKR1C6 (MC203314), AKR1C12 (MC203332), AKR1C14 (MC206345) and AKRl C21 (MC203832) were purchased from Origene (Rockville, MD, USA). Custom primers were used to amplify the gene sequences and attach flanking regions compatible with Gateway ® cloning technology.
  • AKRl C 18, AKRl Cl 9 and AKRl C20 were custom synthesised with Gateway compatible flanking regions by GenScript (Piscataway, NJ, USA) and supplied in the pUC57 plasmid. Sequences generated by GenScript were codon optimised for translation in human cells.
  • Murine AKRs were cloned into the Gateway ® compatible F527-V5 vector as described previously for members of the human AKR family 2 (**). Plasmids were transfected into HCTl 16 cells using Fugene ⁇ transfection reagent and stable populations selected with puromycin as described previously (Guise, Wang, et al. 2007
  • HCT116 AKRi ⁇ cells A549 or HCT116 AKRi ⁇ cells, and HCTl 16 cells transfected with murine AKRs, using a modification of the previous method described above.
  • Cells (5 x 10 5 cells per well in 24-well plate) were pre-incubated at 37°C with inhibitors as required for 2 h, followed by addition of PR- 104A to 100 ⁇ M (with maintenance of the inhibitor concentrations). Plates were incubated aerobically for a further 1 h, samples extracted with methanol containing PR-104H-d 4 internal standard as above, and stored at -80 0 C for analysis by LC-MS/MS. Cytotoxicity of PR-104A in the HCTl 16 cell lines was determined in aerobic monolayers, by clonogenic assay after exposure for 2 hr, as described herein. Results Screening Candidate Inhibitors of PR-104A Aerobic Metabolism
  • Celecoxib 278 >300 5.45 200 2 ⁇ 0.01
  • the invention provides compounds of formulae I and II as described below. These and other nitroaromatic compounds described herein are suitable for use in the practise of the • invention as described herein.
  • Y being selected from Cl, Br, I, and from OSO 2 R 1 ,
  • Z represents at any available ring position -CO- or -SO 2 -, R is methyl or ethyl;
  • Certain exemplary compounds are those of formulae Ia or Ha:
  • Still further exemplary compounds are those of formulae Ic or Hc:
  • Exemplary specific compounds of formula I are depicted in Table 9 below and include the following: 2-((2-Bromoethyl)-2- ⁇ [(2-hydroxyethyl)(methyl)amino]carbonyl ⁇ -4,6- dinitroanilino) ethyl methanesulfonate (12); 2-[bis(2-bromoethyl)amino]-iV-ethyl-iV-(6- hydroxyhexyl)-3,5-dinitrobenzamide (107); 2-((2-chloroethyl)-2- ⁇ [(2- hydroxyemyl)(niemyl)arr]ino]carbonyl ⁇ -4,6-dinitroariilino)ethyl methanesulfonate (108); 2-((2- chloroethyl)-2- ⁇ [(3-hydroxypropyl)(rnemyl)arnino]carbonyl ⁇ -4,6-dinitroanilino)ethyl me
  • the preferred dinitrobenzamide mustard prodrug compounds of formula I and II may be prepared employing methods analogous to those described in the literature (Atwell et al, PCT Int. Appl. WO 2008030112 Al; Yang et al, Tetrahedron, 2007, 63, 5470-5476; Atwell et al, J. Med. Chem. 2007, 50, 1197-1212; Denny et al, PCT Int. Appl. WO 2005042471 Al; Denny et al, PCT Int. Appl. WO 2004033415 Al; Fnedlos et al, J. Med. Chem.
  • the preferred dinitrobenzamide mustard prodrug compounds of formula I may be prepared as shown in scheme 1 from the known. 3-chloro-2,6-dinitrobenzoic acid (Palmer et al, J. Med. Chem. 1996, 39, 2518-2528), or the commercially available 2-chloro- 3,5-dinitrobenzoic acid and 5-chloro-2,4-dinitrobenzoic acid, respectively.
  • the asymmetric haloge ⁇ /alkyl sulfonate mustards (VIII) can be prepared from their symmetrical counterparts by reaction with approximately one equivalent of a silver alkylsulfonate salt in an appropriate solvent, such as acetonitrile.
  • the preferred dinitrobenzamide mustard prodrug compounds of formula I may be prepared as shown in scheme 2 from the known. 3- chloro-2,6-dinitrobenzoic acid (Palmer et al, J. Med. Chem. 1996, 39, 2518-2528), or the commercially available 2-chloro-3,5-dinitrobenzoic acid and 5-chloro-2,4-dinitrobenzoic acid, respectively. Reaction with tert-butyl acetate in the presence of perchloric acid provides the respective tert-butyl esters (IX). Reaction of these with diethanolamine in dioxane then affords die respective diols (X).
  • the preferred asymmetric dinitrobenzamide mustard prodrug compounds of formula I may also be prepared following the methodology of Yang et al (Tetrahedron, 2007, 63, 5470-5476) as shown in scheme 3.
  • the above described tert- butyl esters (IX) can be reacted with aziridine ethanol in the presence of a metal halide (LiCl, LiBr or NaI) to provide the haloethyl half-mustards (XIV), which can be derivatised to the halo/alkylsulfonate mustards (XV) by reaction with the appropriate alkylsulfonic anhydride in the presence of pyridine and catalytic dimethylaminopyridine (DMAP).
  • asymmetric halogen/alkyl sulfonate mustards can be prepared from their symmetrical counterparts by reaction with approximately one equivalent of a silver alkylsulfonate salt in an appropriate solvent, such as acetonitrile.
  • phosphates of formula II may be prepared as shown in scheme 5 from the preferred prodrug compounds of formula I by reaction of these alcohol derivatives with di- tert-butyl diisopropylphosphoramidite utilizing lH-tetrazole as die base, followed by oxidation with eidier r ⁇ -chloroperoxybenzoic acid (r ⁇ -CPBA) or 70% aqueous hydrogen peroxide, to provide the di-/ ⁇ -butylphosphate ester intermediates (XXII).
  • Acid mediated hydrolysis employing trifluoroacetic acid (TFA) in dichloromethane, then provides the phosphates of formula II as their free acids.
  • the asymmetric bromo/methyl sulfonate mustard (12) was prepared from diis by reaction with one equivalent of silver mesylate in acetonitrile. Reaction of alcohol 2 with di-tertbutyldiisopropylphosphoramidite utilizing lH-tetrazole as the base, followed by oxidation widi 70% aqueous hydrogen peroxide, provided the di-tertbutylphosphate ester intermediate (33). Acid mediated hydrolysis, employing trifluoroacetic acid (TFA) in dichloromethane, then provided the phosphate (39) of formula II as the free acid.
  • TFA trifluoroacetic acid
  • the compounds of the Formulae I and II of the present invention can be used in the treatment of cancer of the human or animal body.
  • the treatment may be of any cancer type that includes hypoxic regions, as the mustards of Formula II are reduced by enzymes present in such regions.
  • the cancers treated may be solid tumours, such as ovarian, colon, brain, thyroid, pancreas, bladder, breast, prostate, lung (such as small cell lung tumour cells and large cell lung carcinoma), cervical and skin cancer.
  • the cancer may be leukaemia, multiple myeloma or lymphoma. All of these cancers present with hypoxic regions, particularly where tumours are growing or have become large.
  • the compounds of the invention can be administered in the form of pharmaceutical compositions, containing one or more compounds of the invention in combination with one or more pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier(s) should be non-toxic and not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier will depend on the route of administration, which can be oral, or parenteral, including intravenous, cutaneous, subcutaneous, intramuscular, intravascular or by infusion.
  • the preparation of pharmaceutical compositions comprising one or more compounds of the invention may be performed by methods well known in the art, such as those described above.
  • the exact dose of the compound to be administered will be at the discretion of the physician, taking into account the type of cancer, the therapeutic approach (monotherapy or combination therapy) and the overall condition and needs of the patient. Typical doses and administration schedules will be determined by experience in clinical trials. Total doses are expected to be in the range from about 0.1 to 200 mg/kg per subject, such as about 10 mg/kg per subject. The amount of compound administered may be between about 20% and 100% of the maximum tolerated dose of the subject.
  • the compounds of Formula I and II can be used as single agents or in combination with one or more other cytotoxic or other therapeutic agents or therapies, especially those that are relatively ineffective against hypoxic cells, such as radiation therapy. Where such other agents and/or radiotherapy are administered in combination with a compound of the invention, the radiation and/or other agents may be administered before, during or after administration of the compound of Formula I and II.
  • cytotoxicity of compounds 11 (PR-104A) and 12 was assessed as follows.
  • Parental HCT-116 WT cell (ATCC CCL-247) and HCT-Il 6 ⁇ KR1C3 cells engineered to express human aldo-keto reductase 1C3 (AKRl C3; NM_003739) were passaged as monolayers in minimal essential media ( ⁇ MEM; Gibco, In ⁇ trogen Corporation, Grand Island, NY, USA) supplemented with 5% FBS, (GIBCO NZ Ltd, Auckland, New Zealand) without antibiotics for ⁇ 3 months from frozen stocks confirmed to be mycoplamsa free.by PCR-ELISA (Roche Diagnostics Mannheim, Germany).
  • cells were washed three times and plates were filled with 200 ⁇ l of ⁇ MEM containing 5% FCS and 1 % penicillin/streptomycin ⁇ Gibco, In ⁇ trogen Corporation, Grand Island, NY, USA), and left for a further 5 days in a 37 0 C, 5% CO 2 incubator. After five days cells were fixed by adding 67 ⁇ l of cold 40% trichloroacetic acid (Merck KGaA, Darmstadt, Germany) to each well, to give a final concentration of 10%. Plates were held at 4 0 C for 1 hour. After 1 hr, plates were rinsed in tap water 3-4 times, with excess water being drained prior to staining.
  • ⁇ MEM containing 5% FCS and 1 % penicillin/streptomycin ⁇ Gibco, In ⁇ trogen Corporation, Grand Island, NY, USA
  • representative compound 12 is at least as sensitive to AKRl C3 activation as compound 11 (PR- 104A).
  • Carboxylates Selective for Human Aldo-Keto Reductase Isoforms Potential Antineoplastic Agents That Work Independently of Cyclooxygenase Isozymes" MoI Pharmacol. 2005 Jan;67(l):60-8. Epub 2004 Oct 8.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • Hospice & Palliative Care (AREA)
  • Genetics & Genomics (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention repose sur la découverte que l'enzyme AKR1C3 est capable de présenter une activité nitroréductase et d'activer des promédicaments nitroaromatiques en vue de la libération de l'effecteur cytotoxique. Les promédicaments nitroaromatiques activés AKR1C3 comprennent les moutardes de dinitrobenzamide telles que 2-((2-bromoéthyl)-2-{[(2-hydroxyéthyl)amino]carbonyl}-4,6-dinitroanilino)éthyl méthanesulfonate (PR-104A), et le pré-promédicament correspondant -[(2-(bromoéthyl)-2,4-dinitro-6-[[[2- (phosphono-oxy)éthyl]amino]carbonyl]anilino]éthyl méthanesulfonate (PR-104). L'invention concerne des procédés et des examens permettant de déterminer la sensibilité de patients à des médicaments, en vue d'identifier des profils individualisés de patients facilitant le traitement de maladies et de troubles tels que le cancer. L'invention concerne aussi des méthodes de traitement fondées sur de tels profils liés à des biomarqueurs, ainsi que des composés convenant pour être utilisés dans ces méthodes et des compositions comprenant de tels composés.
PCT/NZ2009/000227 2008-10-17 2009-10-19 Akr1c3 utilisé comme biomarqueur, procédés de sélection et de traitement de patients sur la base d'un profil d'akr1c3 et composés utiles à cet effet WO2010044686A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
NZ57212308 2008-10-17
NZ572123 2008-10-17
NZ57213308 2008-10-17
NZ572133 2008-10-17

Publications (1)

Publication Number Publication Date
WO2010044686A1 true WO2010044686A1 (fr) 2010-04-22

Family

ID=42106697

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NZ2009/000227 WO2010044686A1 (fr) 2008-10-17 2009-10-19 Akr1c3 utilisé comme biomarqueur, procédés de sélection et de traitement de patients sur la base d'un profil d'akr1c3 et composés utiles à cet effet

Country Status (1)

Country Link
WO (1) WO2010044686A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016161342A3 (fr) * 2015-04-02 2016-11-10 Threshold Pharmaceuticals, Inc. Dérivés de nitrobenzyle d'agents anticancéreux
US10131683B2 (en) 2014-07-17 2018-11-20 Molecular Templates, Inc. TH-302 solid forms and methods related thereto
CN110057944A (zh) * 2019-05-24 2019-07-26 广东中烟工业有限责任公司 一种氨基酸衍生化方法及其应用
US10364261B2 (en) 2015-03-10 2019-07-30 Obi Pharma, Inc. DNA alkylating agents
US10409869B2 (en) 2012-10-29 2019-09-10 Obi Pharma, Inc. (R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis (ethylene)phosphoramidate, compositions and methods for their use and preparation
WO2019190331A1 (fr) * 2018-03-29 2019-10-03 Achilles Medical Limited Composés de type promédicaments activés par akr1c3 et leur utilisation pour le traitement de troubles hyperprolifératifs
US10668047B2 (en) 2015-06-24 2020-06-02 Molecular Templates, Inc. Aziridine containing DNA alkylating agents
WO2021110085A1 (fr) * 2019-12-03 2021-06-10 深圳艾欣达伟医药科技有限公司 Procédé d'association avec un niveau d'expression d'une enzyme akr1c3 par l'intermédiaire d'une teneur en prostaglandine, et utilisation de criblage pour l'administration de médicament

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993011099A1 (fr) * 1991-11-28 1993-06-10 Cancer Research Campaign Technology Limited Derives de nitroaniline et leur utilisation comme agents anti-tumeurs
WO2008030112A1 (fr) * 2006-09-04 2008-03-13 Auckland Uniservices Limited Procédés de préparation de composés asymétriques à base de moutarde de dinitrobenzamide, composes intermédiaires utiles dans de tells procédés et produits obtenus à partir desdits procédés
WO2008033041A1 (fr) * 2006-09-11 2008-03-20 Auckland Uniservices Limited Traitement contre le cancer
WO2008124824A1 (fr) * 2007-04-10 2008-10-16 Myriad Genetics, Inc. Doses et méthodes de traitement du cancer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993011099A1 (fr) * 1991-11-28 1993-06-10 Cancer Research Campaign Technology Limited Derives de nitroaniline et leur utilisation comme agents anti-tumeurs
WO2008030112A1 (fr) * 2006-09-04 2008-03-13 Auckland Uniservices Limited Procédés de préparation de composés asymétriques à base de moutarde de dinitrobenzamide, composes intermédiaires utiles dans de tells procédés et produits obtenus à partir desdits procédés
WO2008033041A1 (fr) * 2006-09-11 2008-03-20 Auckland Uniservices Limited Traitement contre le cancer
WO2008124824A1 (fr) * 2007-04-10 2008-10-16 Myriad Genetics, Inc. Doses et méthodes de traitement du cancer

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DESMOND J.C. ET AL.: "The Aldo-Keto Reductase AKR1C3 Is a Novel Suppressor of Cell Differentiation That Provides a Plausible Target for the Non-Cyclooxygenase- dependent Antineoplastic Actions of Nonsteroidal Anti-Inflammatory Drugs", CANCER RESEARCH, vol. 63, no. 2, 2003, pages 505 - 512 *
GUISE C.P. ET AL.: "Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A: A role for NADPH:cytochrome P450 oxidoreductase under hypoxia", BIOCHEMICAL PHARMACOLOGY, vol. 74, no. 6, 2007, pages 810 - 820 *
JAMESON M.B. ET AL.: "A phase I trial of PR-104, a nitrogen mustard prodrug activated by both hypoxia and aldo-keto reductase 1C3, in patients with solid tumors", CANCER CHEMOTHERAPY AND PHARMACOLOGY, vol. 65, no. 4, 2010, pages 791 - 801 *
MCKEOWN S.R. ET AL.: "Bioreductive Drugs: From Concept to Clinic", CLINICAL ONCOLOGY, vol. 19, no. 6, 2007, pages 427 - 442 *
PATTERSON A.V ET AL.: "The bioreductive prodrug PR-104 is activated under aerobic conditions by human aldo-keto reductase 1C3 (prostaglandin F synthase)", JOURNAL OF CANCER SUPPLEMENTS, vol. 6, no. 12, 2008, pages 151 *
PATTERSON A.V. ET AL.: "Mechanism of Action and Preclinical Antitumor Activity of the Novel Hypoxia-Activated DNA Cross-Linking Agent PR-104", CLINICAL CANCER RESEARCH, vol. 13, no. 13, 2007, pages 3922 - 3932 *
SINGLETON R.S. ET AL.: "DNA Cross-Links in Human Tumor Cells Exposed to the Prodrug PR-104A; Relationships to Hypoxia, Bioreductive Metabolism, and Cytotoxicity", CANCER RESEARCH, vol. 69, no. 9, 2009, pages 3884 - 3891 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10409869B2 (en) 2012-10-29 2019-09-10 Obi Pharma, Inc. (R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-N,N'-bis (ethylene)phosphoramidate, compositions and methods for their use and preparation
US10131683B2 (en) 2014-07-17 2018-11-20 Molecular Templates, Inc. TH-302 solid forms and methods related thereto
US10654876B2 (en) 2014-07-17 2020-05-19 Molecular Templates, Inc. TH-302 solid forms and methods related thereto
US10766914B2 (en) 2015-03-10 2020-09-08 Obi Pharma, Inc. DNA alkylating agents
US10364261B2 (en) 2015-03-10 2019-07-30 Obi Pharma, Inc. DNA alkylating agents
CN112142692A (zh) * 2015-04-02 2020-12-29 深圳艾欣达伟医药科技有限公司 硝基苄基衍生物抗癌试剂
JP2018511612A (ja) * 2015-04-02 2018-04-26 アセンタ ファーマシューティカルズ リミテッド 抗がん剤のニトロベンジル誘導体
US10829437B2 (en) 2015-04-02 2020-11-10 Obi Pharma, Inc. Nitrobenzyl derivatives of anti-cancer agents
WO2016161342A3 (fr) * 2015-04-02 2016-11-10 Threshold Pharmaceuticals, Inc. Dérivés de nitrobenzyle d'agents anticancéreux
US11535585B2 (en) 2015-04-02 2022-12-27 Obi Pharma, Inc. Nitrobenzyl derivatives of anti-cancer agents
US10668047B2 (en) 2015-06-24 2020-06-02 Molecular Templates, Inc. Aziridine containing DNA alkylating agents
WO2019190331A1 (fr) * 2018-03-29 2019-10-03 Achilles Medical Limited Composés de type promédicaments activés par akr1c3 et leur utilisation pour le traitement de troubles hyperprolifératifs
CN111918864A (zh) * 2018-03-29 2020-11-10 阿基利斯医疗有限公司 通过akr1c3活化的前药化合物及其治疗过度增殖性失调的用途
EP3774743A4 (fr) * 2018-03-29 2021-11-24 Achilles Medical Limited Composés de type promédicaments activés par akr1c3 et leur utilisation pour le traitement de troubles hyperprolifératifs
US11661404B2 (en) 2018-03-29 2023-05-30 Achilles Medical Limited Prodrug compounds activated by AKR1C3 and their use for treating hyperproliferative disorders
CN111918864B (zh) * 2018-03-29 2024-03-05 阿基利斯医疗有限公司 通过akr1c3活化的前药化合物及其治疗过度增殖性失调的用途
CN110057944A (zh) * 2019-05-24 2019-07-26 广东中烟工业有限责任公司 一种氨基酸衍生化方法及其应用
WO2021110085A1 (fr) * 2019-12-03 2021-06-10 深圳艾欣达伟医药科技有限公司 Procédé d'association avec un niveau d'expression d'une enzyme akr1c3 par l'intermédiaire d'une teneur en prostaglandine, et utilisation de criblage pour l'administration de médicament

Similar Documents

Publication Publication Date Title
WO2010044686A1 (fr) Akr1c3 utilisé comme biomarqueur, procédés de sélection et de traitement de patients sur la base d&#39;un profil d&#39;akr1c3 et composés utiles à cet effet
JP6906554B2 (ja) 治療に反応する患者を同定するバイオマーカーおよびそのような患者の治療
US9284368B2 (en) Substituted pyrazines as cadherin-11 inhibitors
JP2019531699A (ja) Ｎｒｆ２及びその遺伝子の下流標的遺伝子の発現状態及び変異状態によるがんの診断及び治療方法
JP2008533053A (ja) ヒストン脱アセチル化酵素阻害剤は、がん細胞を上皮増殖因子受容体阻害剤に対して感受性にする
KR20150079949A (ko) 암의 합성치사 및 치료
US20100028259A1 (en) Use of organic cation transporters for cancer diagnosis and therapy
KR20220123064A (ko) Cdk12/13 억제제에 의한 암 치료
JP2004533603A (ja) 異常な細胞成長に関するマーカとしてのｐｉｎ１
EP3218517A1 (fr) Mir-214 comme biomarqueur de diagnostic et de pronostic spécifique à la rectocolite hémorragique et inhibiteur de mir-214 pour son traitement
JP2021532158A (ja) 転移性癌および化学療法抵抗性癌の治療のためのテトラヒドロキノリノ誘導体
AU2014266278B2 (en) Therapeutic effect prediction method for colorectal cancer patient in whom expression of TK1 protein has increased
WO2016109471A1 (fr) Promédicaments de type phosphates de bufaline et leurs méthodes d&#39;utilisation
JP2020505392A (ja) アンドロゲン非依存性がんを処置するための組成物および方法
CA2697887A1 (fr) Modulation et/ou detection de la deaminase induite par activation et procedes d&#39;utilisation de celle-ci
US20140227172A1 (en) Diagnosis and treatment of prostate cancer
Pinheiro et al. Targeting metabolic reprogramming as an anti-cancer strategy: aiming at monocarboxylate transporters
US11892454B2 (en) Method for the diagnosis of etoposide prodrug treatable cancer
EP2709730A1 (fr) Traitement et pronostic de cancer
US10316319B2 (en) Composition for diagnosis of liver metastasis of colorectal cancer and the use thereof
US20120264803A1 (en) Method for predicting therapeutic effects of chemotherapy on hepatocellular carcinoma patients
US20190227072A1 (en) Biomarkers for Carcinogenesis and Uses Thereof
CN114214418A (zh) 一种评估肺癌患者对质子放疗敏感性的生物标志物及其用途
HUE026447T2 (en) Pharmacodynamic tumor response biomarkers
Class et al. Patent application title: PROGNOSTIC AND/OR PREDICTIVE BIOMARKERS AND BIOLOGICAL APPLICATIONS THEREOF Inventors: Lorenzo Galluzzi (Paris, FR) Annick Harel-Bellan (Paris, FR) Guido Kroemer (Paris, FR) Ken Olaussen (Paris, FR) Jean-Charles Soria (Igny, FR) Assignees: INSTITUT GUSTAVE ROUSSY

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09820808

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09820808

Country of ref document: EP

Kind code of ref document: A1