Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0043—Nose
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0085—Brain, e.g. brain implants; Spinal cord

Definitions

• the present invention relates to compositions of perillyl alcohol (POH) derivatives such as POH carbamates, as well as the use thereof for treating cancers.
• POH perillyl alcohol
• Malignant gliomas the most common form of central nervous system (CNS) cancers, is currently considered essentially incurable.
• CNS central nervous system
• anaplastic astrocytomas Grade III
• GBM glioblastoma multiforme
• the present standard of care for malignant gliomas consists of surgery, ionizing radiation, and chemotherapy.
• analogues of chemotherapeutic agents have been prepared in an effort to overcome these problems.
• the analogues include novel therapeutic agents which are hybrid molecules of at least two existing therapeutic agents.
• cisplatin has been conjugated with Pt-(II) complexes with cytotoxic codrugs, or conjugated with bioactive shuttle components such as porphyrins, bile acids, hormones, or modulators that expedite the transmembrane transport or the drug accumulation within the cell.
• Metastasized cancer such as breast cancer
• This challenge once was a late aspect of disease progression, but increasingly is becoming a first site of disease progression after otherwise successful treatment of primary tumor and metastases outside the cranium.
• Traditional breast cancer therapeutics such as paclitaxel or doxorubicin, only reach brain metastases at concentrations that are far lower than needed to be therapeutically active.
• P.R. Lockman, et al. Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer, Clin Cancer Res 16 (2010) 5664-5678.
• BBB blood brain barrier
• Perillyl alcohol a naturally occurring monoterpene, has been suggested to be an effective agent against a variety of cancers, including CNS cancer, breast cancer, pancreatic cancer, lung cancer, melanomas and colon cancer. Gould, M. Cancer chemoprevention and therapy by monoterpenes. Environ Health Perspect. 1997 June; 105 (Suppl 4): 977-979.
• Hybrid molecules containing both perillyl alcohol and retinoids were prepared to increase apoptosis-inducing activity.
• Das et al. Design and synthesis of potential new apoptosis agents hybrid compounds containing perillyl alcohol and new constrained retinoids. Tetrahedron Letters 2010, 51, 1462- 1466.
• TMZ The alkylating agent temozolomide
• GBM glioblastoma multiforme
• TMZ acts as a prodrug. Its mechanism of activation involves hydrolytic opening of its tetrazinone ring, which takes places spontaneously in aqueous solution at 37°C, and does not require the participation of cellular enzymes.
• the resulting product the unstable monomethyl MTIC (5 -(3 -methyltriazen- 1 -yl)-imidazole-4-carboxamide), reacts with water to liberate AIC (4-amino-5-imidazole-carboxamide) and the highly reactive methyldiazonium cation, which methylates DNA purine residues.
• MGMT 0(6)-Methylguanine-DNA methyltransferase
• Knizhnik et al. Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide -induced DNA damage, PLoS One 8 (2013) e55665.
• MGMT expression was investigated in breast cancer metastases to the brain, it was found that over half of the intracranial lesions analyzed were strongly positive for MGMT immunoreactivity.
• B. Ingold et al. Homogeneous MGMT immunoreactivity correlates with an unmethylated MGMT promoter status in brain metastases of various solid tumors, PLoS One 4 (2009) e4775.
• MGMT activity is unusual in that it represents a "suicide” mechanism, whereby acceptance of the alkyl group from DNA irreversibly inactivates the enzyme and leads to its rapid degradation. This feature is exploited by the use of specific MGMT inhibitors, such as 06-benzylguanine (06-BG), which act as
• the invention provides for a method for treating brain metastases of a cancer in a mammal, comprising delivering to the mammal a therapeutically effective amount of a perillyl alcohol derivative, such as a perillyl alcohol carbamate.
• a perillyl alcohol derivative such as a perillyl alcohol carbamate.
• the invention also provides for a method for treating a metastatic cancer of a mammal that has spread to the brain by delivering to the mammal a therapeutically effective amount of a perillyl alcohol derivative, such as a perillyl alcohol carbamate.
• the perillyl alcohol derivative may be perillyl alcohol conjugated with a therapeutic agent, such as a chemotherapeutic agent.
• a therapeutic agent such as a chemotherapeutic agent.
• the chemotherapeutic agents that may be used in the present invention include a DNA alkylating agent, a topoisomerase inhibitor, an endoplasmic reticulum stress inducing agent, a platinum compound, an antimetabolite, an enzyme inhibitor, and a receptor antagonist.
• the therapeutic agent can be temozolomide (TMZ).
• the perillyl alcohol carbamate may be 3 -methyl 4-oxo-3,4-dihydroimidazo[5,l- d][l,2,3,5]tetrazine-8-carbonyl)-carbamic acid -4-isopropenyl cyclohex-l-enylmethyl ester (TMZ-POH).
• the method may further comprise treating the mammal with radiation before, during, or after the administration of the pharmaceutical composition, and/or further comprise delivering to the mammal another chemotherapeutic agent.
• the brain metastasis or metastases to be treated can originate or spread from a cancer such as a systemic cancer, lung cancer, prostate cancer, breast cancer, hematopoietic cancer, ovarian cancer, bladder cancer, germ cell tumors, kidney cancer, leukemia, lymphoma, and melanoma.
• the brain metastases originate or are spread from metastatic breast cancer.
• the routes of administration of the perillyl alcohol derivative include inhalation, intranasal, oral, intravenous, subcutaneous or intramuscular administration.
• the perillyl alcohol derivative can be administered intranasally using a nasal delivery device selected from the group consisting of an intranasal inhaler, an intranasal spray device, an atomizer, a nebulizer, a metered dose inhaler (MDI), a pressurized dose inhaler, an insufflator, a unit dose container, a pump, a dropper, a nasal spray bottle, a squeeze bottle and a bi-directional device.
• a nasal delivery device selected from the group consisting of an intranasal inhaler, an intranasal spray device, an atomizer, a nebulizer, a metered dose inhaler (MDI), a pressurized dose inhaler, an insufflator, a unit dose container, a pump, a dropper, a nasal
• Figure 1 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of dimethyl celecoxib (DMC) in killing U87, A 172 and U251 human glioma cells.
• DMC dimethyl celecoxib
• Figure 2 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-DMC conjugate in killing U87, A172 and U251 human glioma cells according to the present invention.
• FIG. 3 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of temozolomide (TMZ) in killing U87, A172 and U251 human glioma cells.
• TMZ temozolomide
• Figure 4 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the TMZ-POH conjugate in killing U87, A172, and U251 human glioma cells according to the present invention.
• Figure 5 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-Rolipram conjugate and Rolipram in killing A172 human glioma cells.
• Figure 6 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-Rolipram conjugate and Rolipram in killing U87 human glioma cells.
• Figure 7 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-Rolipram conjugate and Rolipram in killing U251 human glioma cells.
• Figure 8 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-Rolipram conjugate and Rolipram in killing L229 human glioma cells.
• Figures 9A and 9B show the inhibition of tumor growth by butyryl- POH in mouse models.
• Figure 9A shows the images of subcutaneous U-87 gliomas in nude mice treated with butyryl-POH, purified (5)-perillyl alcohol having a purity greater than 98.5% (“Purified POH”), POH purchased from Sigma chemicals (“Sigma”), or phosphate buffered saline (“PBS”; negative control).
• Figure 9B shows average tumor growth over time (total time period of 60 days).
• Figure 10 shows the results of a Colony forming Assay (CFA) demonstrating the cytotoxic effect of TMZ and TMZ-POH on TMZ sensitive (U251) and TMZ resistant (U251TR) U251 cells.
• Figure 11 shows the results of a Colony forming Assay (CFA) demonstrating the cytotoxic effect of POH on TMZ sensitive (U251) and TMZ resistant (U251TR) U251 cells.
• CFA Colony forming Assay
• Figure 12 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the TMZ-POH conjugate in killing U251 cells, U251TR cells, and normal astrocytes.
• Figure 13 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the TMZ-POH conjugate in killing normal astrocytes, brain endothelial cells (BEC; confluent and subconfluent), and tumor brain endothelial cells (TuBEC) .
• Figure 14 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of TMZ and the TMZ-POH conjugate in killing USC-04 glioma cancer stem cells.
• Figure 15 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of POH in killing USC-04 glioma cancer stem cells.
• Figure 16 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of TMZ and the TMZ-POH conjugate in killing USC-02 glioma cancer stem cells.
• Figure 17 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of POH in killing USC-02 glioma cancer stem cells.
• Figure 18 shows a western blot demonstrating that TMZ-POH induces ER stress (ERS) in TMZ sensitive (“U251 -TMZs”) and resistant (“U251-TMZr”) U251 glioma cells.
• ERS ER stress
• Figure 19 shows survival of breast cancer cells after drug treatment, where various breast cancer cell lines were exposed to increasing concentrations of TMZ or TMZ-POH for 48 hours, and survival was determined via colony formation assay (CFA). Shown is the fraction of colony-forming cells, where colony formation by control cells (treated with DMSO vehicle only) is set at 1. Graphs with error bars display mean ( ⁇ SD) from >3 independent experiments; graphs without error bars show the average from two independent experiments.
• Figures 20A-20B show cytotoxic potency of TMZ-POH and its individual components, where survival of drug-treated MDA-MB-231 cells was determined by CFA.
• FIG 20A cells were exposed for 48 hours to increasing concentrations of TMZ (diamonds), TMZ-POH (circles), POH (triangles), or equimolar concentrations of TMZ plus POH (squares). Colony formation by control cells (treated with vehicle only) is set at 1 ; graphs display mean ( ⁇ SD) from >3 independent experiments.
• Figure 20B cells were exposed to 10 ⁇ TMZ-POH, TMZ, or POH, or to 10 ⁇ TMZ-POH or TMZ combined with 10 ⁇ POH. Shown is a photo of one representative CFA.
• Figures 21A-21C show MGMT expression levels in various cell lines, where all parts show Western blot analysis of MGMT protein levels with actin as the loading control.
• Figure 21 A shows MGMT basal levels in the six breast cancer cell lines used in this study.
• Figure 2 IB shows MGMT basal levels in three GBM cell lines compared to MCF7 breast cancer cells.
• MDA-MB-468 cells were treated with the indicated concentrations of TMZ-POH, TMZ, or 06-BG for 17 hours before harvest of cellular lysates.
• vh . cells treated with vehicle only.
• Figures 22A-22B show drug sensitivity of MGMT-transfected cells, where MDA-MB-231 cells were stably transfected with MGMT cDNA.
• FIG 22A two individually selected clones, 231 -MGMT- 1 and -2, were analyzed by Western blot for basal level MGMT protein expression in comparison to parental cells.
• Figure 22B 231-MGMT-l and -2 were treated with increasing
• Figures 23A-23C show effect of inclusion of 06-BG, where cells were exposed to TMZ or TMZ-POH for 48 hours in the presence or absence of 06-BG, and cell survival was determined by CFA.
• Figure 23 A shows colony survival of MDA- MB-231 cells;
• Figure 23B shows MGMT-transfected 23 l-MGMT-2 cells, and
• Figure 23C shows MDA-MB-468 cells. Shown is mean number of colonies ( ⁇ SD) from >3 wells treated in parallel.
• Figures 24A-24D show drug effects on DNA damage marker, where cells were treated with different concentrations of TMZ-POH or TMZ and analyzed by Western blot analysis for expression levels of ⁇ - ⁇ 2 ⁇ , a marker for double-strand DNA damage. Actin was used as a loading control.
• MDA-MB-231 cells were treated with 50 ⁇ TMZ-POH for the indicated time periods ( Figure 24A); MDA-MB-231 cells were treated with 50 ⁇ TMZ-POH or 50 ⁇ TMZ for the indicated time periods (Figure 24B); MDA-MB-231 cells were treated with TMZ-POH, TMZ, POH, or TMZ combined with POH (all at 10 ⁇ each) for 24 hours (Figure 24C); MCF7 cells were treated with or without 50 ⁇ TMZ-POH in the presence or absence of 30 ⁇ 06-BG for 48 hours ( Figure 24D).
• Figures 25A-25B show DNA damage and cell death marker analysis, where MDA-MB-231 cells were used for Western blot analysis of expression levels for markers of DNA damage ( ⁇ - ⁇ 2 ⁇ ) and cell death (activated caspase 7 and cleaved PARP).
• FIG 25 A cells were treated with 15 ⁇ TMZ-POH and harvested every 24 hours up to 6 days; control cells remained untreated, or received vehicle (vh.) only.
• Figure 25B cells were treated with 20 ⁇ of either TMZ-POH, TMZ, or POH individually, or with 20 ⁇ TMZ combined with 20 ⁇ POH (TMZ+POH) and harvested after 24 hours or 5 days; control cells remained untreated, or received vehicle (vh.) only.
• Figures 26A-26C depicts determination of drug stability, where MDA- MB-231 cells were analyzed in colony formation assays.
• cells were treated with 15 ⁇ TMZ-POH or 30 ⁇ TMZ for 30 min or 1, 2, 4, and 24 hours. Thereafter, drug-containing medium was removed, fresh medium (without drug) was added, and cells remained undisturbed until colony staining 12 days later.
• cells were exposed to supernatant (i.e., the drug-containing medium removed from cells shown in Figure 26A). The arrows indicate which cells received which supernatant.
• FIG. 26C shows a representative 6-well plate with stained colonies.
• Figures 27A-27B show drug effects on intracranial tumor growth, where luciferase-positive D3H2LN cells were implanted into the brains of 24 nude mice. Ten days later, tumor take was confirmed via bioluminescent imaging, and treatment was initiated with vehicle only (control group), 25 mg/kg TMZ-POH, or 25 mg/kg TMZ, once daily over the course of 10 days. In Figure 27A, all surviving animals were imaged again on days 21, 28, and 36. The top panel shows one representative mouse from the vehicle -only treated group. Note 12-fold increased ROI radiance (representative of tumor growth) from 1.65E7 to 1.92E8 between days 10 and 21. The bottom panel shows a representative mouse from the group of TMZ- POH-treated animals.
• radiance increased only 1.7-fold (from 1.11E7 to 1.92E7) between days 10 and 21, but reached 1.88E8 (similar to control mouse on day 21) by day 43.
• Heat bar to the right shows scale of radiance.
• Figure 27B shows Kaplan-Meier survival plot of all animals carrying intracranial tumors. Arrow labeled Rx indicates the time period of treatment. Statistical difference between groups of TMZ-treated and TMZ-POH-treated animals: /? ⁇ 0.001.
• the present invention provides for a derivative of monoterpene or sesquiterpene, such as a perillyl alcohol derivative.
• the present invention also provides for a pharmaceutical composition comprising a derivative of monoterpene or sesquiterpene, such as a perillyl alcohol derivative.
• the perillyl alcohol derivative may be a perillyl alcohol carbamate.
• the perillyl alcohol derivative may be perillyl alcohol conjugated with a therapeutic agent such as a chemotherapeutic agent.
• a therapeutic agent such as a chemotherapeutic agent.
• sesquiterpene) derivative may be formulated into a pharmaceutical composition, where the monoterpene (or sesquiterpene) derivative is present in amounts ranging from about 0.01% (w/w) to about 100% (w/w), from about 0.1% (w/w) to about 80% (w/w), from about 1%> (w/w) to about 70%> (w/w), from about 10%> (w/w) to about 60%) (w/w), or from about 0.1 %> (w/w) to about 20%> (w/w).
• the monoterpene (or sesquiterpene) derivative is present in amounts ranging from about 0.01% (w/w) to about 100% (w/w), from about 0.1% (w/w) to about 80% (w/w), from about 1%> (w/w) to about 70%> (w/w), from about 10%> (w/w) to about 60%) (w/w), or from about 0.1 %> (w/w) to about 20%> (w/w).
• compositions can be administered alone, or may be co -administered together with radiation or another agent (e.g., a chemotherapeutic agent), to treat a disease such as cancer.
• Treatments may be sequential, with the monoterpene (or sesquiterpene) derivative being administered before or after the administration of other agents.
• a perillyl alcohol carbamate may be used to sensitize a cancer patient to radiation or chemotherapy.
• agents may be administered concurrently.
• the route of administration may vary, and can include, inhalation, intranasal, oral, transdermal, intravenous, subcutaneous or intramuscular injection.
• the present invention also provides for a method of treating a disease such as cancer, comprising the step of delivering to a patient a therapeutically effective amount of a derivative of monoterpene (or sesquiterpene).
• compositions of the present invention may contain one or more types of derivatives of monoterpene (or sesquiterpene).
• Monoterpenes include terpenes that consist of two isoprene units.
• Monoterpenes may be linear (acyclic) or contain rings.
• Derivatives of monoterpenoids are also encompassed by the present invention.
• Monoterpenoids may be produced by biochemical modifications such as oxidation or rearrangement of monoterpenes.
• monoterpenes and monoterpenoids examples include, perillyl alcohol (S(-)) and (R(+)), ocimene, myrcene, geraniol, citral, citronellol, citronellal, linalool, pinene, terpineol, terpinen, limonene, terpinenes, phellandrenes, terpinolene, terpinen-4-ol (or tea tree oil), pinene, terpineol, terpinen; the terpenoids such as /?-cymene which is derived from monocyclic terpenes such as menthol, thymol and carvacrol; bicyclic monoterpenoids such as camphor, borneol and eucalyptol.
• S(-)) and (R(+) perillyl alcohol
• ocimene myrcene, geraniol, citral, cit
• Monoterpenes may be distinguished by the structure of a carbon skeleton and may be grouped into acyclic monoterpenes (e.g., myrcene, (Z)- and (E)- ocimene, linalool, geraniol, nerol, citronellol, myrcenol, geranial, citral a, neral, citral b, citronellal, etc.), monocyclic monoterpenes (e.g., limonene, terpinene,
• acyclic monoterpenes e.g., myrcene, (Z)- and (E)- ocimene, linalool, geraniol, nerol, citronellol, myrcenol, geranial, citral a, neral, citral b, citronellal, etc.
• monocyclic monoterpenes e.
• bicyclic monoterpenes e.g., pinene, myrtenol, myrtenal, verbanol, verbanon, pinocarveol, carene, sabinene, camphene, thujene, etc.
• tricyclic monoterpenes e.g. tricyclene
• Sesquiterpenes of the present invention include terpenes that consist of three isoprene units. Sesquiterpenes may be linear (acyclic) or contain rings.
• Sesquiterpenoids may be produced by biochemical modifications such as oxidation or rearrangement of sesquiterpenes.
• sesquiterpenes include farnesol, farnesal, farnesylic acid and nerolidol.
• the derivatives of monoterpene (or sesquiterpene) include, but are not limited to, carbamates, esters, ethers, alcohols and aldehydes of the monoterpene (or sesquiterpene).
• Monoterpene (or sesquiterpene) alcohols may be derivatized to carbamates, esters, ethers, aldehydes or acids.
• Carbamate refers to a class of chemical compounds sharing the functional group
• R 1 , R 2 and R 3 can be a group such as alkyl, aryl, etc., which can be substituted.
• the R groups on the nitrogen and the oxygen may form a ring.
• R'-OH may be a monoterpene, e.g., POH.
• the R 2 -N-R 3 moiety may be a therapeutic agent.
• Carbamates may be synthesized by reacting isocyanate and alcohol, or by reacting chloroformate with amine. Carbamates may be synthesized by reactions making use of phosgene or phosgene equivalents. For example, carbamates may be synthesized by reacting phosgene gas, diphosgene or a solid phosgene precursor such as triphosgene with two amines or an amine and an alcohol. Carbamates (also known as urethanes) can also be made from reaction of a urea intermediate with an alcohol.
• dimethyl carbonate and diphenyl carbonate are also used for making carbamates.
• carbamates may be synthesized through the reaction of alcohol and/or amine precursors with an ester-substituted diaryl carbonate, such as
• BMSC bismethylsalicylcarbonate
• Carbamates may be synthesized by the following approach:
• Suitable reaction solvents include, but are not limited to, tetrahydrofuran,
• the reaction may be performed at a temperature ranging from about -70°C to about 80°C, or from about -65°C to about 50°C.
• the molar ratio of perillyl chloroformate to the substrate R - NH 2 may range from about 1 : 1 to about 2: 1, from about 1 : 1 to about 1.5 : 1 , from about 2: 1 to about 1 : 1 , or from about 1.05 : 1 to about 1.1 : 1.
• Suitable bases include, but are not limited to, organic bases, such as triethylamine, potassium carbonate, ⁇ , ⁇ '- diisopropylethylamine, butyl lithium, and potassium-t-butoxide.
• carbamates may be synthesized by the following approach:
• Suitable reaction solvents include, but are not limited to, dichloromethane, dichloroethane, toluene, diisopropyl ether, and tetrahydrofuran.
• the reaction may be performed at a temperature ranging from about 25°C to about 110°C, or from about 30°C to about 80°C, or about 50°C.
• Esters of the monoterpene (or sesquiterpene) alcohols of the present invention can be derived from an inorganic acid or an organic acid.
• Inorganic acids include, but are not limited to, phosphoric acid, sulfuric acid, and nitric acid.
• Organic acids include, but are not limited to, carboxylic acid such as benzoic acid, fatty acid, acetic acid and propionic acid, and any therapeutic agent bearing at least one carboxylic acid functional group Examples of esters of monoterpene (or
• sesquiterpene alcohols include, but are not limited to, carboxylic acid esters (such as benzoate esters, fatty acid esters (e.g., palmitate ester, linoleate ester, stearate ester, butyryl ester and oleate ester), acetates, propionates (or propanoates), and formates), phosphates, sulfates, and carbamates (e.g., N,N-dimethylaminocarbonyl).
• carboxylic acid esters such as benzoate esters, fatty acid esters (e.g., palmitate ester, linoleate ester, stearate ester, butyryl ester and oleate ester), acetates, propionates (or propanoates), and formates
• phosphates, sulfates e.g., N,N-dimethylaminocarbonyl
• perillyl alcohol (commonly abbreviated as POH).
• the derivatives of perillyl alcohol include, perillyl alcohol carbamates, perillyl alcohol esters, perillic aldehydes, dihydroperillic acid, perillic acid, perillic aldehyde derivatives,
• the derivatives of perillyl alcohol may also include its oxidative and nucleophilic/electrophilic addition derivatives.
• Many examples of derivatives of perillyl alcohol are reported in the chemistry literature (see Appendix A: CAS Scifmder search output file, retrieved January 25, 2010).
• a POH carbamate is synthesized by a process comprising the step of reacting a first reactant of perillyl chloroformate with a second reactant such as dimethyl celocoxib (DMC), temozolomide (TMZ) and rolipram.
• the reaction may be carried out in the presence of tetrahydrofuran and a base such as n- butyl lithium.
• Perillyl chloroformate may be made by reacting POH with phosgene.
• POH conjugated with temozolomide through a carbamate bond may be synthesized by reacting temozolomide with oxalyl chloride followed by reaction with perillyl alcohol.
• the reaction may be carried out in the presence of 1 ,2- dichloroethane.
• POH carbamates encompassed by the present invention include, but not limited to, 4-(bis-N,N'-4-isopropenyl cyclohex-l-enylmethyloxy carbonyl [5-(2,5- dimethyl phenyl)-3-trifluoromethyl pyrazol-l-yl] benzenesulfonamide, 4-(3- cyclopentyloxy-4-methoxy phenyl)-2-oxo-pyrrolidine-l-carboxylic acid 4- isopropenyl cyclohex-l-enylmethyl ester, and (3 -methyl 4-oxo-3,4- dihydroimidazo[5,l-d][l,2,3,5]tetrazine-8-carbonyl)carbamic acid-4-isopropenyl cyclohex-l-enylmethyl ester.
• the details of the chemical reactions generating these compounds are described in the Examples below.
• perillyl alcohol derivatives may be perillyl alcohol fatty acid esters, such as palmitoyl ester of POH and linoleoyl ester of POH, the chemical structures of which are shown below.
• the monoterpene (or sesquiterpene) derivative may be a monoterpene (or sesquiterpene) conjugated with a therapeutic agent.
• a monoterpene (or sesquiterpene) conjugate encompassed by the present invention is a molecule having a monoterpene (or sesquiterpene) covalently bound via a chemical linking group to a therapeutic agent.
• the molar ratio of the monoterpene (or sesquiterpene) to the therapeutic agent in the monoterpene (or sesquiterpene) conjugate may be 1 : 1, 1 :2, 1 :3, 1 :4, 2: 1 , 3 : 1 , 4: 1 , or any other suitable molar ratios.
• the monoterpene (or sesquiterpene) and the therapeutic agent may be covalently linked through carbamate, ester, ether bonds, or any other suitable chemical functional groups.
• the therapeutic agent may be any agent bearing at least one carboxylic acid functional group, or any agent bearing at least one amine functional group.
• a perillyl alcohol conjugate is perillyl alcohol covalently bound via a chemical linking group to a chemotherapeutic agent.
• the therapeutic agents that may be conjugated with monoterpene (or sesquiterpene) include, but are not limited to, chemotherapeutic agents, therapeutic agents for treatment of CNS disorders
• Anti-cancer agents that may be conjugated with monoterpene or sesquiterpene can have one or more of the following effects on cancer cells or the subject: cell death; decreased cell
• Chemotherapeutic agents include, but are not limited to, DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducing agents, a platinum compound, an antimetabolite, vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, tyrosine kinase inhibitors, boron
• radiosensitizers i.e. velcade
• chemotherapeutic combination therapies i.e. velcade
• Non-limiting examples of DNA alkylating agents are nitrogen mustards, such as Cyclophosphamide (Ifosfamide, Trofosfamide), Chlorambucil (Melphalan, Prednimustine), Bendamustine, Uramustine and Estramustine;
• nitrosoureas such as Carmustine (BCNU), Lomustine (Semustine), Fotemustine, Nimustine, Ranimustine and Streptozocin; alkyl sulfonates, such as Busulfan
• Triethylenemelamine Triethylenemelamine; Hydrazines (Procarbazine); Triazenes such as dacarbazine and Temozolomide (TMZ); Altretamine and Mitobronitol.
• Topoisomerase I inhibitors include
• Campothecin derivatives including SN-38, APC, NPC, campothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan, silatecan, gimatecan, diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT as decribed in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and U.S. Patent Publication No. 200510250854; Protoberberine alkaloids and derivatives thereof including berberrubine and coralyne as described in Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer Res. 15(12):2795-2800;
• Phenanthroline derivatives including Benzo[i]phenanthridine, Nitidine, and fagaronine as described in Makhey et al. (2003) Bioorg. Med. Chem. 11 (8): 1809- 1820; Terbenzimidazole and derivatives thereof as described in Xu (1998)
• Topoisomerase II inhibitors include, but are not
• Dual topoisomerase I and II inhibitors include, but are not limited to, Saintopin and other Naphthecenediones, DACA and other Acridine-4-Carboxamindes, Intoplicine and other Benzopyridoindoles, TAS-I03 and other 7H-indeno[2,l-c]Quinoline-7-ones, Pyrazoloacridine, XR 11576 and other Benzophenazines, XR 5944 and other Dimeric compounds, 7-oxo-7H- dibenz[f,ij]Isoquinolines and 7-oxo-7H-benzo[e]pyrimidines, and Anthracenyl-amino Acid Conjugates as described in Denny and Baguley (2003) Curr.
• Top. Med. Chem. 3(3):339-353 Some agents inhibit Topoisomerase II and have DNA intercalation activity such as, but not limited to, Anthracyclines (Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin) and Antracenediones (Mitoxantrone and Pixantrone).
• Anthracyclines Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin
• Antracenediones Mitoxantrone and Pixantrone
• endoplasmic reticulum stress inducing agents include, but are not limited to, dimethyl-celecoxib (DMC), nelfmavir, celecoxib, and boron radiosensitizers (i.e. velcade (Bortezomib)).
• DMC dimethyl-celecoxib
• nelfmavir nelfmavir
• celecoxib nelfmavir
• boron radiosensitizers i.e. velcade (Bortezomib)
• Platinum based compounds are a subclass of DNA alkylating agents.
• Non-limiting examples of such agents include Cisplatin, Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin, Aroplatin, Lobaplatin, and JM-216. (see McKeage et al. (1997) J. Clin. Oncol. 201 : 1232-1237 and in general, CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL
• FOLFOX is an abbreviation for a type of combination therapy that is used to treat colorectal cancer. It includes 5-FU, oxaliplatin and leucovorin.
• FOLFOX/BV is an abbreviation for a type of combination therapy that is used to treat colorectal cancer.
• This therapy includes 5-FU, oxaliplatin, leucovorin and Bevacizumab.
• Furthennore, "XELOX/BV” is another combination therapy used to treat colorectal cancer, which includes the prodrug to 5-FU, known as Capecitabine (Xeloda) in combination with oxaliplatin and bevacizumab. Infonnation regarding these treatments are available on the National Cancer Institute's web site, cancer.gov or from 23 the National Comprehensive Cancer Network's web site, nccn.org, last accessed on May 27, 2008.
• Non-limiting examples of antimetabolite agents include Folic acid based, i.e. dihydrofolate reductase inhibitors, such as Aminopterin, Methotrexate and Pemetrexed; thymidylate synthase inhibitors, such as Raltitrexed, Pemetrexed; Purine based, i.e.
• an adenosine deaminase inhibitor such as Pentostatin, a thiopurine, such as Thioguanine and Mercaptopurine, a halogenated/ribonucleotide reductase inhibitor, such as Cladribine, Clofarabine, Fludarabine, or a guanine/guanosine: thiopurine, such as Thioguanine; or Pyrimidine based, i.e. cytosine/cytidine: hypomethylating agent, such as Azacitidine and Decitabine, a DNA polymerase inhibitor, such as Cytarabine, a ribonucleotide reductase inhibitor, such as Gemcitabine, or a
• thymine/thymidine thymidylate synthase inhibitor, such as a Fluorouracil (5-FU).
• 5-FU Fluorouracil
• Equivalents to 5-FU include prodrugs, analogs and derivative thereof such as 5' - deoxy-5-fluorouridine (doxifluroidine), l-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda), S-I (MBMS-247616, consisting of tegafur and two
• vincalkaloids examples include, but are not limited to Vinblastine,
• Vincristine, Vinflunine, Vindesine and Vinorelbine are Vincristine, Vinflunine, Vindesine and Vinorelbine.
• taxanes examples include, but are not limited to docetaxel,
• Larotaxel, Ortataxel, Paclitaxel and Tesetaxel Larotaxel, Ortataxel, Paclitaxel and Tesetaxel.
• An example of an epothilone is iabepilone.
• enzyme inhibitors include, but are not limited to farnesyltransferase inhibitors (Tipifarnib); CDK inhibitor (Alvocidib, Seliciclib); proteasome inhibitor (Bortezomib); phosphodiesterase inhibitor (Anagrelide;
• rolipram IMP dehydrogenase inhibitor
• Tiazofurine IMP dehydrogenase inhibitor
• Miazofurine lipoxygenase inhibitor
• receptor antagonists include, but are not limited to ERA (Atrasentan); retinoid X receptor (Bexarotene); and a sex steroid (Testolactone).
• tyrosine kinase inhibitors include, but are not limited to inhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib, Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib, Neratinib); RTK class III: C-kit
• “Lapatinib” (Tykerb®) is an dual EGFR and erbB-2 inhibitor.
• Lapatinib has been investigated as an anticancer monotherapy, as well as in combination with trastuzumab, capecitabine, letrozole, paclitaxel and
• FOLFIRI (irinotecan, 5-fluorouracil and leucovorin), in a number of clinical trials. It is currently in phase III testing for the oral treatment of metastatic breast, head and neck, lung, gastric, renal and bladder cancer.
• a chemical equivalent of lapatinib is a small molecule or compound that is a tyrosine kinase inhibitor (TKI) or alternatively a HER-1 inhibitor or a HER-2 inhibitor.
• Zactima ZD6474
• Iressa gefitinib
• imatinib mesylate STI571; Gleevec
• erlotinib OSI-1774; Tarceva
• canertinib CI 1033
• semaxinib SU5416
• vatalanib PTK787/ZK222584
• sorafenib BAY 43- 9006
• sutent SUI 1248
• lefltmomide SU101
• PTK/ZK is a tyrosine kinase inhibitor with broad specificity that targets all VEGF receptors (VEGFR), the platelet-derived growth factor (PDGF) receptor, c-KIT and c-Fms. Drevs (2003) Idrugs 6(8):787-794. PTK/ZK is a targeted drug that blocks angiogenesis and lymphangiogenesis by inhibiting the activity of all known receptors that bind VEGF including VEGFR-I (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4).
• VEGFR-I Flt-1
• VEGFR-2 KDR/Flk-1
• VEGFR-3 Flt-4
• PTK/ZK The chemical names of PTK/ZK are l-[4-Chloroanilino]-4-[4- pyridylmethyl] phthalazine Succinate or 1 -Phthalazinamine, N-(4-chlorophenyl)-4-(4- pyridinylmethyl)-butanedioate (1 : 1). Synonyms and analogs of PTK/TK are known as Vatalanib, CGP79787D, PTK787/ZK 222584, CGP-79787, DE-00268, PTK-787, PTK787A, VEGFR-TK inhibitor, ZK 222584 and ZK.
• Chemotherapeutic agents that can be conjugated with monoterpene or sesquiterpene may also include amsacrine, Trabectedin, retinoids (Alitretinoin, Tretinoin), Arsenic trioxide, asparagine depleter Asparaginase/ Pegaspargase), Celecoxib, Demecolcine, Elesclomol, Elsamitrucin, Etoglucid, Lonidamine,
• the monoterpene or sesquiterpene derivative may be conjugated with angiogenesis inhibitors.
• angiogenesis inhibitors include, but are not limited to, angiostatin, angiozyme, antithrombin III, AG3340, VEGF inhibitors, batimastat, bevacizumab (avastin), BMS-275291, CAI, 2C3, HuMV833 Canstatin, Captopril, carboxyamidotriazole, cartilage derived inhibitor (CDI), CC-5013, 6-0- (chloroacetyl-carbonyl)-fumagillol, COL-3, combretastatin, combretastatin A4 Phosphate, Dalteparin, EMD 121974 (Cilengitide), endostatin, erlotinib, gefitinib
• Iressa genistein, halofuginone hydrobromide, Idl, Id3, IM862, imatinib mesylate, IMC-IC11 Inducible protein 10, interferon-alpha, interleukin 12, lavendustin A, LY317615 or AE-941, marimastat, mspin, medroxpregesterone acetate, Meth-1, Meth-2, 2-methoxyestradiol (2-ME), neovastat, oteopontin cleaved product, PEX, pigment epithelium growth factor (PEGF), platelet factor 4, prolactin fragment, proliferin-related protein (PRP), PTK787/ZK 222584, ZD6474, recombinant human platelet factor 4 (rPF4), restin, squalamine, SU5416, SU6668, SU11248 suramin, Taxol, Tecogalan, thalidomide, thrombo
• Non-limiting examples of angiogenesis inhibitors also include, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFRl) and Flk-l/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, pentosan polysulfate, angiotensin II antagonists, cyclooxygenase inhibitors (including non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen, as well as selective cyclooxygenase-2 inhibitors such as celecoxib and rofecoxib), and steroidal anti-inflammatories (such as corticosteroids,
• NSAIDs non-steroidal anti-inflammatory drugs
• NSAIDs non-steroidal anti-inflammatory drugs
• NSAIDs non-steroidal
• mineralocorticoids dexamethasone, prednisone, prednisolone, methylpred, betamethasone).
• therapeutic agents that modulate or inhibit angiogenesis and may also be conjugated with monoterpene or sesquiterpene include agents that modulate or inhibit the coagulation and fibrinolysis systems, including, but not limited to, heparin, low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]).
• heparin low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]).
• TAFIa active thrombin activatable fibrinolysis inhibitor
• Non-limiting examples of the anti-hypertensive agents include angiotensin converting enzyme inhibitors (e.g., captopril, enalapril, delapril etc.), angiotensin II antagonists (e.g., candesartan cilexetil, candesartan, losartan (or Cozaar), losartan potassium, eprosartan, valsartan (or Diovan), termisartan, irbesartan, tasosartan, olmesartan, olmesartan medoxomil etc.), calcium antagonists (e.g., manidipine, nifedipine, amlodipine (or Amlodin), efonidipine, nicardipine etc.), diuretics, renin inhibitor (e.g., aliskiren etc.), aldosterone antagonists (e.g., spironolactone, eplerenone etc.
• therapeutic agents that may be conjugated with monoterpene (or sesquiterpene) include, but are not limited to, Sertraline (Zoloft), Topiramate (Topamax), Duloxetine(Cymbalta), Sumatriptan (Imitrex), Pregabalin (Lyrica), Lamotrigine (Lamictal), Valaciclovir (Valtrex), Tamsulosin (Flomax), Zidovudine (Combivir), Lamivudine (Combivir), Efavirenz (Sustiva), Abacavir (Epzicom), Lopinavir (Kaletra), Pioglitazone (Actos), Desloratidine (Clarinex), Cetirizine
• Table 1 lists pharmaceutical agents that can be conjugated with monoterpene 0 (or sesquiterpene), including structure of the pharmaceutical agent and the preferred derivative for conjugation.
• the purity of the monoterpene (or sesquiterpene) derivatives may be assayed by gas chromatography (GC) or high pressure liquid chromatography (HPLC).
• Other techniques for assaying the purity of monoterpene (or sesquiterpene) derivatives and for determining the presence of impurities include, but are not limited to, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), GC- MS, infrared spectroscopy (IR), and thin layer chromatography (TLC). Chiral purity can be assessed by chiral GC or measurement of optical rotation.
• NMR nuclear magnetic resonance
• MS mass spectrometry
• IR infrared spectroscopy
• TLC thin layer chromatography
• the monoterpene (or sesquiterpene) derivatives may be purified by methods such as crystallization, or by separating the monoterpene (or sesquiterpene) derivative from impurities according to the unique physicochemical properties (e.g., solubility or polarity) of the derivative. Accordingly, the monoterpene (or sesquiterpene) derivative can be separated from the monoterpene (or sesquiterpene) by suitable separation techniques known in the art, such as preparative
• the invention also provides for methods of using monoterpenes (or sesquiterpenes) derivatives to treat a disease, such as a cancer or other nervous system disorders.
• a monoterpene (or sesquiterpene) derivative may be administered alone, or in combination with radiation, surgery or chemotherapeutic agents.
• monoterpene or sesquiterpene derivative may also be co-administered with antiviral agents, anti-inflammatory agents or antibiotics.
• the agents may be administered concurrently or sequentially.
• a monoterpene (or sesquiterpene) derivative can be administered before, during or after the administration of the other active agent(s).
• the monoterpene or sesquiterpene derivative may be used in combination with radiation therapy.
• the present invention provides for a method of treating tumor cells, such as malignant glioma cells or brain metastases, with radiation, where the cells are treated with an effective amount of a monoterpene derivative, such as a perillyl alcohol carbamate, and then exposed to radiation.
• Monoterpene derivative treatment may be before, during and/or after radiation.
• the monoterpene or sesquiterpene derivative may be administered continuously beginning one week prior to the initiation of radiotherapy and continued for two weeks after the completion of radiotherapy.
• the present invention provides for a method of treating tumor cells, such as malignant glioma cells or brain metastases, with chemotherapy, where the cells are treated with an effective amount of a monoterpene derivative, such as a perillyl alcohol carbamate, and then exposed to chemotherapy.
• a monoterpene derivative such as a perillyl alcohol carbamate
• Monoterpene derivative treatment may be before, during and/or after chemotherapy.
• Monoterpene (or sesquiterpene) derivatives may be used for the treatment of nervous system cancers, such as a malignant glioma (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma multiforme), retinoblastoma, pilocytic astrocytomas (grade I), meningiomas, metastatic brain tumors, neuroblastoma, pituitary adenomas, skull base meningiomas, and skull base cancer.
• glioma e.g., astrocytoma, anaplastic astrocytoma, glioblastoma multiforme
• retinoblastoma retinoblastoma
• pilocytic astrocytomas grade I
• Cancers that can be treated by the present monoterpene (or sesquiterpene) derivatives include, but are not limited to, lung cancer, ear, nose and throat cancer, leukemia, colon cancer, melanoma, pancreatic cancer, mammary cancer, prostate cancer, breast cancer, hematopoietic cancer, ovarian cancer, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia including acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia; liver cancer; lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; myel
• neuroblastoma e.g., oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the respiratory system; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; cancer of the urinary system, as well as other carcinomas and sarcomas.
• oral cavity cancer e.g., lip, tongue, mouth, and pharynx
• ovarian cancer pancreatic cancer
• prostate cancer retinoblastoma
• rhabdomyosarcoma rectal cancer
• renal cancer cancer of the respiratory system
• sarcoma skin cancer
• stomach cancer testicular cancer
• thyroid cancer thyroid cancer
• uterine cancer cancer of the urinary system, as well as other carcinomas and sarcomas.
• the present monoterpene (or sesquiterpene) derivatives can be used for treating brain metastases that originate or spread from a primary cancer such as a systemic cancer, lung cancer, prostate cancer, breast cancer, hematopoietic cancer, ovarian cancer, bladder cancer, germ cell tumors, kidney cancer, leukemia, lymphoma, and melanoma.
• a primary cancer such as a systemic cancer, lung cancer, prostate cancer, breast cancer, hematopoietic cancer, ovarian cancer, bladder cancer, germ cell tumors, kidney cancer, leukemia, lymphoma, and melanoma.
• the present invention provides for a method for treating a mammal having a metastatic cancer, such as metastatic breast cancer that has spread to the brain, by administering to the mammal a monoterpene (or sesquiterpene) derivative described herein, e.g., a POH carbamate, such as TMZ- POH.
• the present invention also provides methods of treating CNS disorders, including, without limitation, primary degenerative neurological disorders such as Alzheimer's, Parkinson's, psychological disorders, psychosis and depression. Treatment may consist of the use of a monoterpene or sesquiterpene derivative alone or in combination with current medications used in the treatment of Parkinson's, Alzheimer's, or psychological disorders.
• primary degenerative neurological disorders such as Alzheimer's, Parkinson's, psychological disorders, psychosis and depression.
• Treatment may consist of the use of a monoterpene or sesquiterpene derivative alone or in combination with current medications used in the treatment of Parkinson's, Alzheimer's, or psychological disorders.
• the present invention also provides a method of improving
• immunomodulatory therapy responses comprising the steps of exposing cells to an effective amount of a monoterpene or sisquiterpene derivative, such as a perillyl alcohol carbamate, before or during immunomodulatory treatment.
• a monoterpene or sisquiterpene derivative such as a perillyl alcohol carbamate
• immunomodulatory agents are cytokines, such interleukins, lymphokines, monokines, interfereons and chemokines.
• composition may be administered by any method known in the art, including, without limitation, intranasal, oral, transdermal, ocular, intraperitoneal, inhalation, intravenous, ICV, intracisternal injection or infusion, subcutaneous, implant, vaginal, sublingual, urethral (e.g., urethral suppository), subcutaneous, intramuscular, intravenous, rectal, sub-lingual, mucosal, ophthalmic, spinal, intrathecal, intra-articular, intra-arterial, sub-arachinoid, bronchial and lymphatic administration.
• Topical formulation may be in the form of gel, ointment, cream, aerosol, etc; intranasal formulation can be delivered as a spray or in a drop; transdermal formulation may be administered via a transdermal patch or
• compositions can also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions.
• one or more of monoterpene (or sesquiterpene) derivatives may be mixed with a pharmaceutical acceptable carrier, adjuvant and/or excipient, according to conventional
• compositions encompass any of the standard
• compositions can additionally contain solid pharmaceutical excipients such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like.
• Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.
• Liquid carriers particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols.
• stabilizers and adjuvants see Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).
• the compositions also can include stabilizers and preservatives.
• the term "therapeutically effective amount” is an amount sufficient to treat a specified disorder or disease or alternatively to obtain a pharmacological response treating a disorder or disease.
• Methods of determining the most effective means and dosage of administration can vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Treatment dosages generally may be titrated to optimize safety and efficacy. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents can be readily determined by those of skill in the art.
• the composition are administered at about 0.01 mg/kg to about 200 mg/kg, about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kg to about 50 mg/kg.
• the effective amount may be less than when the agent is used alone.
• Transdermal formulations may be prepared by incorporating the active agent in a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose, with the resulting formulation then being packed in a transdermal device adapted to be secured in dermal contact with the skin of a wearer.
• a thixotropic or gelatinous carrier such as a cellulosic medium, e.g., methyl cellulose or hydroxyethyl cellulose
• the composition may be rubbed onto a membrane of the patient, for example, the skin, preferably intact, clean, and dry skin, of the shoulder or upper arm and or the upper torso, and maintained thereon for a period of time sufficient for delivery of the monoterpene (or sesquiterpene) derivative to the blood serum of the patient.
• composition of the present invention in gel form may be contained in a tube, a sachet, or a metered pump.
• a tube or sachet may contain one unit dose, or more than one unit dose, of the composition.
• a metered pump may be capable of dispensing one metered dose of the composition.
• compositions as described above for intranasal administration can further comprise a permeation enhancer.
• the monoterpene (or sesquiterpene) derivative may be administered intranasally in a liquid form such as a solution, an emulsion, a suspension, drops, or in a solid form such as a powder, gel, or ointment.
• Devices to deliver intranasal medications are well known in the art.
• Nasal drug delivery can be carried out using devices including, but not limited to, intranasal inhalers, intranasal spray devices, atomizers, nasal spray bottles, unit dose containers, pumps, droppers, squeeze bottles, nebulizers, metered dose inhalers (MDI), pressurized dose inhalers, insufflators, and bi-directional devices.
• the nasal delivery device can be metered to administer an accurate effective dosage amount to the nasal cavity.
• the nasal delivery device can be for single unit delivery or multiple unit delivery.
• the ViaNase Electronic Atomizer from Kurve Technology (Bethell, Washington) can be used in this invention (http://www.kurvetech.com).
• the compounds of the present invention may also be delivered through a tube, a catheter, a syringe, a packtail, a pledget, a nasal tampon or by submucosal infusion.
• the monoterpene (or sesquiterpene) derivative can be formulated as aerosols using standard procedures.
• the monoterpene (or sesquiterpene) derivative may be formulated with or without solvents, and formulated with or without carriers.
• the formulation may be a solution, or may be an aqueous emulsion with one or more surfactants.
• an aerosol spray may be generated from pressurized container with a suitable propellant such as, dichlorodifluoromethane,
• aerosol refers to a suspension of fine solid particles or liquid solution droplets in a gas.
• aerosol includes a gas-borne suspension of droplets of a monoterpene (or sesquiterpene), as may be produced in any suitable device, such as an MDI, a nebulizer, or a mist sprayer.
• Aerosol also includes a dry powder composition of the composition of the instant invention suspended in air or other carrier gas.
• the monoterpene (or sesquiterpene) derivative may be delivered to the nasal cavity as a powder in a form such as microspheres delivered by a nasal insufflator.
• the monoterpene (or sesquiterpene) derivative may be absorbed to a solid surface, for example, a carrier.
• the powder or microspheres may be administered in a dry, air-dispensable form.
• the powder or microspheres may be stored in a container of the insufflator.
• the powder or microspheres may be filled into a capsule, such as a gelatin capsule, or other single dose unit adapted for nasal administration.
• the pharmaceutical composition can be delivered to the nasal cavity by direct placement of the composition in the nasal cavity, for example, in the form of a gel, an ointment, a nasal emulsion, a lotion, a cream, a nasal tampon, a dropper, or a bioadhesive strip.
• it can be desirable to prolong the residence time of the pharmaceutical composition in the nasal cavity, for example, to enhance absorption.
• the pharmaceutical composition can optionally be formulated with a bioadhesive polymer, a gum (e.g., xanthan gum), chitosan (e.g., highly purified cationic polysaccharide), pectin (or any carbohydrate that thickens like a gel or emulsifies when applied to nasal mucosa), a microsphere (e.g., starch, albumin, dextran, cyclodextrin), gelatin, a liposome, carbamer, polyvinyl alcohol, alginate, acacia, chitosans and/or cellulose (e.g., methyl or propyl; hydroxyl or carboxy; carboxymethyl or hydroxylpropyl).
• a bioadhesive polymer e.g., xanthan gum
• chitosan e.g., highly purified cationic polysaccharide
• pectin or any carbohydrate that thickens like a
• composition containing the purified monoterpene (or sesquiterpene) can be administered by oral inhalation into the respiratory tract, i.e., the lungs.
• Typical delivery systems for inhalable agents include nebulizer inhalers, dry powder inhalers (DPI), and metered-dose inhalers (MDI).
• DPI dry powder inhalers
• MDI metered-dose inhalers
• Nebulizer devices produce a stream of high velocity air that causes a therapeutic agent in the form of liquid to spray as a mist.
• the therapeutic agent is formulated in a liquid form such as a solution or a suspension of particles of suitable size.
• the particles are micronized.
• the term "micronized” is defined as having about 90% or more of the particles with a diameter of less than about 10 ⁇ .
• Suitable nebulizer devices are provided commercially, for example, by PARI GmbH (Starnberg, Germany).
• Other nebulizer devices include Respimat (Boehringer Ingelheim) and those disclosed in, for example, U.S. Patent Nos.
• the monoterpenes can be formulated for use in a nebulizer device as an aqueous solution or as a liquid suspension.
• DPI devices typically administer a therapeutic agent in the form of a free flowing powder that can be dispersed in a patient's air- stream during inspiration. DPI devices which use an external energy source may also be used in the present invention.
• the therapeutic agent can be formulated with a suitable excipient (e.g., lactose).
• a suitable excipient e.g., lactose
• a dry powder formulation can be made, for example, by combining dry lactose having a particle size between about 1 ⁇ and 100 ⁇ with micronized particles of the monoterpenes (or sesquiterpenes) and dry blending.
• the monoterpene can be formulated without excipients.
• the formulation is loaded into a dry powder dispenser, or into inhalation cartridges or capsules for use with a dry powder delivery device.
• DPI devices provided commercially include Diskhaler (GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Patent No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Patent No. 6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Patent No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Patent No. 4,353,365). Further examples of suitable DPI devices are described in U.S. Patent Nos. 5,415,162, 5,239,993, and 5,715,810 and references therein.
• MDI devices typically discharge a measured amount of therapeutic agent using compressed propellant gas.
• Formulations for MDI administration include a solution or suspension of active ingredient in a liquefied propellant.
• propellants include hydro fluoroalklanes (HFA), such as 1,1,1 ,2-tetrafluoroethane (HFA 134a) and 1,1, 1,2,3, 3,3-heptafluoro-n-propane, (HFA 227), and
• HFA formulations for MDI administration include co-solvents, such as ethanol, pentane, water; and surfactants, such as sorbitan trioleate, oleic acid, lecithin, and glycerin. (See, for example, U.S. Patent No. 5,225,183, EP 0717987, and WO 92/22286).
• co-solvents such as ethanol, pentane, water
• surfactants such as sorbitan trioleate, oleic acid, lecithin, and glycerin.
• the formulation is loaded into an aerosol canister, which forms a portion of an MDI device. Examples of MDI devices developed specifically for use with HFA
• propellants are provided in U.S. Patent Nos. 6,006,745 and 6,143,227.
• the monoterpene (or sesquiterpene) derivative may be encapsulated in liposomes or microcapsules for delivery via inhalation.
• a liposome is a vesicle composed of a lipid bilayer membrane and an aqueous interior.
• the lipid membrane may be made of phospholipids, examples of which include phosphatidylcholine such as lecithin and lysolecithin; acidic phospholipids such as phosphatidylserine and phosphatidylglycerol; and sphingophospholipids such as phosphatidylethanolamine and sphingomyelin. Alternatively, cholesterol may be added.
• a microcapsule is a particle coated with a coating material.
• the coating material may consist of a mixture of a film-forming polymer, a hydrophobic plasticizer, a surface activating agent or/and a lubricant nitrogen-containing polymer.
• the monoterpene (or sesquiterpene) derivative may also be used alone or in combination with other chemotherapeutic agents via topical application for the treatment of localized cancers such as breast cancer or melanomas.
• the monoterpene (or sesquiterpene) derivative may also be used in combination with narcotics or analgesics for transdermal delivery of pain medication.
• compositions as described above for ocular administration can further comprise a permeation enhancer.
• the compositions described herein can be formulated as a solution, emulsion, suspension, etc.
• a variety of vehicles suitable for administering compounds to the eye are known in the art. Specific non-limiting examples are described in U.S. Patent Nos. 6,261,547; 6, 197,934; 6,056,950;
• the monoterpene (or sesquiterpene) derivative can be given alone or in combination with other drugs for the treatment of the above diseases for a short or prolonged period of time.
• the present compositions can be administered to a mammal, preferably a human. Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primates.
• the invention also provides a method for inhibiting the growth of a cell in vitro, ex vivo or in vivo, where a cell, such as a cancer cell, is contacted with an effective amount of the monoterpene (or sesquiterpene) derivative as described herein.
• Pathological cells or tissue such as hyperproliferative cells or tissue may be treated by contacting the cells or tissue with an effective amount of a composition of this invention.
• the cells such as cancer cells, can be primary cancer cells or can be cultured cells available from tissue banks such as the American Type Culture Collection (ATCC).
• the pathological cells can be cells of a systemic cancer, gliomas, meningiomas, pituitary adenomas, or a CNS metastasis or brain metastasis from a systemic cancer, lung cancer, prostate cancer, breast cancer, hematopoietic cancer, ovarian cancer, bladder cancer, germ cell tumors, kidney cancer, leukemia, lymphoma, and melanoma.
• the cells can be from a vertebrate, preferably a mammal, more preferably a human.
• MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cytotoxicity assay.
• MTT assay is based on the principle of uptake of MTT, a tetrazolium salt, by metabolically active cells where it is metabolized into a blue colored formazon product, which can be read spectrometrically. J. of Immunological Methods 65: 55 63, 1983.
• the cytoxicity of the present monoterpene (or sesquiterpene) derivative and/or the therapeutic agents may be studied by colony formation assay.
• Functional assays for inhibition of VEGF secretion and IL-8 secretion may be performed via ELISA.
• Cell cycle block by the present monoterpene (or sesquiterpene) derivative and/or the therapeutic agents may be studied by standard propidium iodide (PI) staining and flow cytometry.
• Invasion inhibition may be studied by Boyden chambers. In this assay a layer of reconstituted basement membrane, Matrigel, is coated onto chemotaxis filters and acts as a barrier to the migration of cells in the Boyden chambers. Only cells with invasive capacity can cross the Matrigel barrier.
• Other assays include, but are not limited to cell viability assays, apoptosis assays, and morphological assays.
• Example 1 Synthesis of Dimethyl Celecoxib bisPOH Carbamate (4-(bis-N,N'-4- isopropenyl cyclohex-l-enylmethyloxy carbonyl [5-(2,5-dimethyl phenyl)-3- trifluoromethyl pyrazol-l-yl] benzenesulfonamide) (also referred to as POH-DMC or DMC-POH herein)
• the reaction scheme is the following:
• Phosgene (20% in toluene, 13 ml, 26.2 mmol) was added to a mixture of perillyl alcohol (2.0 grams, 13.1 mmol) and potassium carbonate (5.4 grams, 39.1 mmol) in dry toluene (30 mL) over a period of 30 minutes while maintaining the temperature between 10° C to 15° C.
• the reaction mixture was allowed to warm to room temperature and stirred for 8.0 hours under N 2 .
• the reaction mixture was quenched with water (30 mL) and the organic layer was separated.
• Perillyl chloroformate (0.11 grams, 0.55 mmol) was added slowly to a mixture of dimethyl celecoxib (0.2 grams, 0.50 mmol) and potassium carbonate (0.13 grams, 1.0 mmol) in dry acetone (10 mL) over a period of 5 minutes under N 2 .
• the reaction mixture was heated to reflux and maintained for 3 hours. Since TLC analysis indicated the presence of dimethyl celecoxib (> 60%), another 1.0 equivalent of perillyl chloroformate was added and refluxed for an additional 5 hours.
• the reaction mixture was cooled and acetone was concentrated under vacuum to give a residue.
• Figure 1 shows the results of the MTT cytotoxicity assays performed on human malignant glioma cells U87, A172 and U251 with DMC alone.
• Example 3 Synthesis of Temozolomide POH Carbamate (3 -methyl 4-oxo-3,4- dihydroimidazo[5,l-d][l,2,3,5]tetrazine-8-carbonyl)-carbamic acid-4-isopropenyl cyclohex-l-enylmethyl ester) (also referred to as TMZ-POH or POH-TMZ herein)
• the reaction scheme is the following:
• Oxalyl chloride (0.13 grams, 1.0 mmol) was added slowly to a mixture of temozolomide (OChem Incorporation, 0.1 grams, 0.5 mmol) in 1,2-dichloroethane (10 mL) over a period of 2 minutes while maintaining the temperature at 10° C under N 2 .
• the reaction mixture was allowed to warm to room temperature and then heated to reflux for 3 hours.
• the excess of oxalyl chloride and 1,2-dichloroethane were removed by concentration under vacuum.
• the resulting residue was re-dissolved in 1 ,2-dichlorethane (15 mL) and the reaction mixture was cooled to 10° C under N 2 .
• temozolomide POH carbamate was synthesized according to the following procedure. Oxalyl chloride (0.13 grams, 1.0 mmol) was added slowly to a mixture of temozolomide (OChem Incorporation, 0.1 grams, 0.5 mmol) in 1,2-dichloroethane (10 mL) over a period of 2 minutes while maintaining the temperature at 10 °C under N 2 . The reaction mixture was allowed to warm to room temperature and then heated to reflux for 3 hours. The excess of oxalyl chloride and 1 ,2-dichloroethane were removed by concentration under vacuum.
• 1,2- Dichloroethane was concentrated under vacuum to give a residue, which was purified by a short silica-plug column (column dimensions: diameter: 2 cm, height: 3 cm, silica: 230-400 mesh) and eluted with a mixture of hexanes/ethyl acetate (1 : 1, 100 mL). The hexane/ethyl acetate fractions were combined and concentrated under vacuum to give a white solid residue which was triturated with heptanes and filtered to obtain a white solid. Weight: 170 mg; Yield: 89%.
• TMZ temozolomide
• TMZ-resistant glioma cell lines U87, A172 and U251 cells were treated with TMZ-POH (e.g., synthesized by the method in Example 3).
• the MTT assay results ( Figure 4) showed that TMZ-POH exhibited substantially higher kill rates of the various human glioma cells compared to TMZ alone.
• the reaction scheme is the following:
• Phosgene (20%> in toluene, 13 ml, 26.2 mmol) was added to a mixture of perillyl alcohol (2.0 grams, 13.1 mmol) and potassium carbonate (5.4 grams, 39.1 mmol) in dry toluene (30 mL) over a period of 30 minutes while maintaining the temperature between 10° C to 15° C.
• the reaction mixture was allowed to warm to room temperature and stirred for 8.0 hours under N 2 .
• the reaction mixture was quenched with water (30 mL) and the organic layer separated.
• Butyl lithium (2.5 M, 0.18 mL, 0.45 mmol) was added to a solution of rolipram (GL synthesis, Inc., 0.1 grams, 0.36 mmol) in dry THF at -72° C over a period of 5 minutes under N 2 .
• perillyl chloroformate (dissolved in 4 mL THF) was added over a period of 15 minutes while maintaining the temperature at -72° C.
• the reaction mixture was stirred for 2.5 hours and quenched with saturated ammonium chloride (5 mL).
• the reaction mixture was allowed to warm to room temperature and extracted with ethyl acetate (2x15 mL).
• the combined organic layer was washed with water (15 mL), brine (15%, 15 mL), and then dried over sodium sulfate.
• the filtered organic layer was concentrated to give an oil which was purified by column chromatography
• FIG. 5 shows the MTT assay for increasing concentrations of rolipram and POH-rolipram for A- 172 cells.
• Rolipram alone demonstrates an IC50 of approximately 1000 uM (1 mM). In the presence of POH-rolipram, IC50 is achieved at concentrations as low as 50 uM.
• Figure 6 shows the MTT assay for increasing concentrations of rolipram with U-87 cells. IC50 is not met at 1000 uM. On the other hand, IC50 iss achieved at 180 uM with POH- rolipram.
• Figure 7 shows that IC50 for rolipram alone for U251 cells is achieved at 170 uM; plateau cytotoxicity is reached at 60%.
• POH-rolipram achieves IC50 at 50 uM, with almost 100% cytoxicity at 100 uM.
• Figure 8 shows that IC50 for rolipram alone for LN229 cells is not achieved even at 100 uM.
• IC50 for POH-rolipram is achieved at 100 uM, with almost 100% cytotoxicity at 10 uM.
• Figure 9A shows the images of subcutaneous U-87 gliomas in nude mice treated with butyryl-POH, purified (S)- perillyl alcohol having a purity greater than 98.5% ("purified POH"), POH purchased from Sigma chemicals, or phosphate buffered saline (PBS; negative control).
• Figure 9B shows average tumor growth over time (total time period of 60 days). Butyryl- POH demonstrated the greatest inhibition of tumor growth, followed by purified POH and Sigma POH.
• Example 8 In vitro Cytotoxicity Studies of TMZ and TMZ-POH on TMZ sensitive and resistant glioma cells
• Colony forming assays were carried out after cells were treated with TMZ alone, POH alone, and the TMZ-POH conjugate.
• the colony forming assays were carried out as described in Chen TC, et al. Green tea epigallocatechin gallate enhances therapeutic efficacy of temozolomide in orthotopic mouse glioblastoma models. Cancer Lett. 2011 Mar 28;302(2): 100-8.
• Figure 10 shows the results of the colony forming assays performed on TMZ sensitive (U251) and TMZ resistant (U251TR) U251 cells with TMZ or TMZ-POH.
• TMZ demonstrated cytotoxicity towards TMZ sensitive U251 cells, but had minimal cytotoxicity towards TMZ resistant U251 cells.
• TMZ-POH demonstrated cytotoxicity towards both TMZ sensitive and TMZ resistant U251 cells.
• Figure 11 shows the results of the colony forming assays performed on TMZ sensitive (U251 ) and TMZ resistant (U251 TR) U251 cells with POH.
• POH demonstrated cytotoxicity towards both TMZ sensitive and TMZ resistant U251 cells.
• TMZ-POH Figure 10) exhibited substantially greater potency compared to POH alone ( Figure 11) in the colony forming assays.
• Example 9 In vitro Cytotoxicity Studies of TMZ-POH on U251 cells, U251 TR cells, and Normal Astrocytes.
• MTT cytotoxicity assays were carried out after cells were treated with the TMZ-POH conjugate.
• the MTT cytotoxicity assays were carried out as described in Chen TC, et al. Green tea epigallocatechin gallate enhances therapeutic efficacy of temozolomide in orthotopic mouse glioblastoma models. Cancer Lett. 2011 Mar 28;302(2): 100-8.
• Figure 12 shows the results of the MTT cytotoxicity assays performed on TMZ sensitive cells (U251), TMZ resistant cells (U251TR) and normal astrocytes.
• TMZ-POH demonstrated cytotoxicity towards both TMZ sensitive and TMZ resistant U251 cells, but not towards normal astrocytes.
• Example 10 In vitro Cytotoxicity Studies of TMZ-POH on BEC, TuBEC, and Normal Astrocytes.
• MTT cytotoxicity assays were carried out after cells were treated with the TMZ-POH conjugate.
• the MTT cytotoxicity assays were carried out as described in Chen TC, et al. Green tea epigallocatechin gallate enhances therapeutic efficacy of temozolomide in orthotopic mouse glioblastoma models. Cancer Lett. 2011 Mar 28;302(2): 100-8.
• Figure 13 shows the results of the MTT cytotoxicity assays performed on normal astrocytes, brain endothelial cells (BEC; confluent and subconfluent), and tumor brain endothelial cells (TuBEC).
• BEC brain endothelial cells
• TuBEC tumor brain endothelial cells
• Example I I In vitro Cytotoxicity Studies of TMZ and TMZ-POH on USC-04 Glioma Cancer Stem Cells.
• MTT cytotoxicity assays were carried out after cells were treated with the TMZ alone, POH alone, or the TMZ-POH conjugate.
• the MTT cytotoxicity assays were carried out as described in Chen TC, et al. Green tea epigallocatechin gallate enhances therapeutic efficacy of temozolomide in orthotopic mouse glioblastoma models. Cancer Lett. 2011 Mar 28;302(2): 100-8.
• Figure 14 shows the results of the MTT cytotoxicity assays performed on USC-04 glioma cancer stem cells.
• TMZ did not induce significant cytotoxicity with increasing concentrations (0- 400 uM).
• TMZ-POH demonstrated evidence of cytotoxicity with IC50 at 150 uM.
• Figure 15 shows the results of the MTT cytotoxicity assays performed on USC-04 glioma cancer stem cells treated with POH. POH demonstrated cytotoxicity on USC- 04 with increasing concentrations (0-2 mM).
• Example 12 In vitro Cytotoxicity Studies of TMZ and TMZ-POH on USC-02 Glioma Cancer Stem Cells.
• MTT cytotoxicity assays were carried out after cells were treated with the TMZ alone, POH alone, or the TMZ-POH conjugate.
• the MTT cytotoxicity assays were carried out as described in Chen TC, et al. Green tea epigallocatechin gallate enhances therapeutic efficacy of temozolomide in orthotopic mouse glioblastoma models. Cancer Lett. 2011 Mar 28;302(2): 100-8.
• Figure 16 shows the results of the MTT cytotoxicity assays performed on USC-02 glioma cancer stem cells.
• TMZ did not induce significant cytotoxicity with increasing concentrations (0- 400 uM).
• TMZ-POH demonstrated evidence of cytotoxicity with IC50 at 60 uM.
• Figure 17 shows the results of the MTT cytotoxicity assays performed on USC-02 glioma cancer stem cells treated with POH. POH demonstrated cytotoxicity on USC- 02 with increasing concentrations (0-2 mM).
• Example 13 In vitro Studies of ER stress by TMZ-POH on TMZ sensitive and resistant glioma cells
• Example 14 In vitro and in vivo studies of TMZ-POH on certain breast cancer cells Pharmacological agents
• TMZ was obtained from the pharmacy at the University of Southern California (USC) and dissolved in ethanol to a concentration of 50 mM.
• TMZ-POH which is also referred to as T-P in this example, was provided by NeOnc
• the human cancer cell lines were obtained from the American Tissue Culture Collection (ATCC; Manassas, VA), except for HCC-1937, which was provided by Dr. Michael Press. Cells were propagated in DMEM (provided by the Cell Culture Core Lab of the USC/Norris Comprehensive Cancer Center and prepared with raw materials from Cellgro/MediaTech, Manassas, VA) supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a humidified incubator at 37°C and a 5% C0 2 atmosphere.
• ATCC American Tissue Culture Collection
• VA Manassas, VA
• 200-350 cells were seeded into each well of a 6-well plate. After cells had fully attached to the surface of the culture plate, they were exposed to drug treatment (or DMSO solvent alone) for various times up to 48 hours. Thereafter, the drugs were removed, fresh growth medium was added, and the cells were kept in culture undisturbed for 12-16 days, during which time the surviving cells spawned colonies of descendants. Colonies (defined as groups of >50 cells) were visualized by staining for 4 hours with 1% methylene blue (in methanol), and then were counted.
• drug treatment or DMSO solvent alone
• MDA-MB-231 cells were co-transfected in 6-well plates with the use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer's instructions.
• 2 ⁇ g pSV2MGMT (containing the human MGMT cDNA) was combined with 0.2 ⁇ g pSV2neo (containing the neomycin gene for selection of cells in G418). Both plasmids were provided by Bernd Kaina (Mainz, Germany).
• Individual clones of transfected cells were selected in medium containing 750 ⁇ g/mL G418 and propagated in 250 ⁇ g/mL G418.
• G418 was obtained as G418 disulfate salt from Sigma-Aldrich and dissolved in PBS at 75 mg/mL. Selection medium was removed from cells several days before experimental drug treatment.
• Total cell lysates were analyzed by Western blot analysis as described in P. Pyrko et al. Downregulation of survivin expression and concomitant induction of apoptosis by celecoxib and its non-cyclooxygenase-2-inhibitory analog, dimethyl- celecoxib (DMC), in tumor cells in vitro and in vivo. Mol Cancer 5 (2006) 19.
• the primary antibodies were purchased from Cell Signaling Technology (Beverly, MA) or Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used according to the manufacturers' recommendations. All immunoblots were repeated at least once to confirm the results.
• mice were intravenously injected with 50 mg/kg D-Luciferin (Perkin Elmer, Waltham, MA) and imaged using the Xenogen IVIS-200 Imaging System (Caliper/Perkin Elmer). Images were analyzed by region-of-interest (ROI) analysis using the Living Image software package (Caliper/Perkin Elmer) to quantitate light output (radiance, i.e., photons per second per square centimeter per steradian).
• ROI region-of-interest
• Group 1 was the control group that received vehicle only (45% glycerol, 45% ethanol, 10%> DMSO) via subcutaneous injection.
• Group 2 was the experimental group that received 25 mg/kg TMZ-POH via subcutaneous (s.c.) injection.
• Group 3 was the comparison group and animals received 25 mg/kg TMZ via gavage. Treatment was once per day for a period of 10 days (i.e., 10 treatments total). Thereafter, all surviving animals were imaged again, once per week.
• TMZ-POH The cytotoxic potency of TMZ-POH, was analyzed by colony formation assay (CFA) in a variety of human breast cancer cell lines and compared to the cytotoxicity of TMZ.
• CFA colony formation assay
• low micromolar concentrations of TMZ-POH prevented colony formation in all six cell lines, and in all instances TMZ-POH's potency was substantially stronger than that of TMZ.
• MDA-MB-231 cells were treated with the individual compounds (TMZ-POH, TMZ or POH) alone, or with an equimolar mix of TMZ plus POH, and cell survival was analyzed by CFA. As shown in Figs.
• TMZ-POH was much more potent than a mix of TMZ plus POH, i.e., mixing TMZ with POH was unable to achieve the strong cytotoxic potency of TMZ- POH, and in fact, the addition of equimolar concentrations of POH to TMZ did not increase the potency over TMZ alone.
• 10 ⁇ TMZ reduced colony formation by about 50%
• the combination of 10 ⁇ TMZ with 10 ⁇ POH also caused a 50%> reduction
• 10 ⁇ TMZ-POH caused about 95% fewer colonies (Fig. 20A).
• POH by itself required concentrations well above 100 ⁇ in order to become cytotoxic, and its IC50 in MDA-MB-231 cells was about 700 ⁇ (Fig 20A).
• Fig. 20B shows a representative example of an individual CFA. It illustrates that 10 ⁇ blocks colony formation substantially more potently than TMZ, and that the addition of equimolar concentrations of POH to either TMZ or TMZ- POH is unable to enhance toxicity any further.
• TMZ-POH has with increased potency over TMZ that cannot be matched by merely mixing its individual parts, TMZ and POH.
• MGMT DNA repair protein
• HCC- 1937, MCF7 three cell lines
• T47D, MDA-MB-231, MDA-MB-231 -br had undetectable levels of MGMT protein, as determined by Western blot analysis.
• MGMT expression was aligned with the cytotoxic potency of TMZ- POH in comparison to TMZ.
• IC50 of TMZ-POH i.e., the concentration required to decrease colony formation by 50%
• MGMT -negative cell lines ranged from 1.2 to 4.6 ⁇ , it increased to 31 to 33 ⁇ in the three MGMT-positive lines. Nonetheless, these IC50 values still were
• TMZ-POH The major cytotoxic DNA lesion set by TMZ is methylation of 06- guanine, and it is well known that removal of this methyl group by MGMT leads to rapid degradation of the DNA repair protein.
• the pseudosubstrate 06-BG also activates the suicide mechanism of MGMT, which is confirmed in Fig. 21C, showing that treatment of cells with 06-BG strongly decreases MGMT protein levels.
• Treatment of cells with TMZ also down-regulates MGMT levels, but the effect is fairly weak and high concentrations of the drug are required.
• TMZ- POH affects MGMT levels more potently than TMZ; for instance, while 50 ⁇ TMZ has no effect, 50 ⁇ TMZ-POH causes a significant decrease (Fig. 21C). Together, these results indicate that TMZ-POH's superior potency over TMZ may involve more extensive methylation of 06-guanine targets.
• 06-BG Cells were pre-treated with 06-BG for 60 minutes before addition of TMZ- POH or TMZ. As shown in Fig. 23 A, 06-BG had no effect on the survival of drug- treated MDA-MB-231 cells, consistent with their MGMT-negative status that does not provide a target for 06-BG. In contrast, 06-BG greatly enhanced toxicity of TMZ-POH and TMZ in 231 -MGMT- 1 (Fig. 23B) and 231 -MGMT-2 cells (not shown). Similarly, 06-BG also increased the cytotoxic outcome of TMZ-POH and TMZ treatment in MGMT-positive MDA-MB-468 (Fig. 23C) and MCF7 cells (not shown). Altogether, these results indicate that the key trigger for cell death caused by TMZ-POH is methylation of 06-guanine, which appears to be achieved much more effectively by TMZ-POH as compared to TMZ.
• TMZ-POH As an alkylating agent with cytotoxic mechanism similar to TMZ, but with potency that is substantially greater than the original compound.
• TMZ-POH treatment resulted in pronounced increase in ⁇ - ⁇ 2 ⁇ expression levels, which except for an unexplained dip at 3 days continued to increase over time (Fig. 25 A).
• Both active caspase 7 and cleaved PARP started to increase at day 3 and remained elevated for several more days until day 6 (Fig. 25 A), which is about the time when microscopic examination of treated cells reveals increasing deterioration of the monolayer.
• TMZ-POH-induced cell death similar to what has been reported for physiological concentrations of TMZ, is a slow process and involves apoptotic mechanisms.
• Figs. 20A-20B an equimolar combination of TMZ + POH was unable to achieve the same potency in blocking colony survival as the TMZ-POH conjugate.
• TMZ-POH's impact on DNA damage and its activation of apoptosis we next determined whether TMZ-POH's superior effect would also be reflected at the molecular level of these marker proteins.
• TMZ is a prodrug, and it is well known that its activation takes place spontaneously in aqueous solution at 37°C (i.e., no cellular functions are required for this conversion). As well, the half- lives of both prodrug and active product are fairly short, where all cytotoxic triggers are set within the first few hours of treatment.
• TMZ-POH and TMZ differed in their half-lives, we determined how quickly, and for how long, the drugs exhibited cytotoxic activity in cell culture. First, we exposed cells to variably short periods of drug treatment, washed off the drug, and then continued to keep cells in medium without drug to determine survival and colony- forming ability. For most of these experiments, we used 15 ⁇ TMZ-POH and 30 ⁇ TMZ, because these concentrations are approximately equipotent in the >90% cytotoxicity range (when measured by CFAs and a drug exposure time of 24 hours).
• TMZ-POH TMZ- POH unfolding slightly more potency over the course of 24 hours
• TMZ displayed noticeably greater efficacy when cells were exposed for shorter time periods.
• a one-hour exposure to TMZ reduced colony formation by >50%, whereas during the same time period TMZ-POH reduced it by only 20%; similarly, a two-hour exposure to TMZ had more than double the cytotoxic impact (23% survival) than TMZ-POH (51%).
• TMZ acted more quickly than TMZ-POH; it required only 4 hours to exert maximum toxicity, whereas TMZ-POH had not yet reached its maximum impact at this time point.
• TMZ-POH phosphate-buffered saline
• TMZ-POH and TMZ were added to cells for 24 hours, and survival was determined by CFA.
• both drugs were also added to cells without prior incubation in aqueous solution.
• a representative CFA is shown in Fig. 26C, where the middle panel confirms that both drugs were used at approximately equipotent concentrations; i.e., when added straight to cells, they reduced survival by ⁇ 95%.
• TMZ-POH would be able to exert its anticancer effects in vivo as well, and whether it would be able to do so with a mouse tumor model representing breast cancer spread to the brain.
• D3H2LN cells which are a bioluminescent variant of the MDA-MB-231 cell line with aggressive tumor growth in mice. These cells were implanted into the brains of nude mice, and 10 days later all animals were imaged for luciferase expression in order to confirm efficient tumor take.
• FIG. 27 A Another whole -body imaging after this 10-day treatment period showed (Fig. 27 A) that all vehicle-only treated animals exhibited much increased bioluminescent radiance (indicative of vigorous intracranial tumor growth), some of which had conspicuously spread along the spine. Most of these animals also exhibited behavioral signs of neurological problems and reduced body weight, which necessitated euthanasia. In stark contrast, all animals in the TMZ-POH-treated group seemed to thrive, and their imaging analysis showed only small changes in radiance (Fig. 27 A). In comparison, all animals in the TMZ-treated group showed clearly increasing bioluminescence over time, indicating that tumor growth had continued throughout the 10-day treatment period, and had begun to include the spine in some of the animals. Overall, the TMZ-treated group seemed to have fared somewhat better than the vehicle-treated group, but clearly worse than the animals treated with TMZ-POH.
• Antitumor imidazotetrazines 32. Synthesis of novel imidazotetrazinones and related bicyclic heterocycles to probe the mode of action of the antitumor drug
• TMZ-POH would preserve the release of the reactive methyldiazonium, and therefore that the cytotoxic activity of TMZ-POH would involve DNA methylation, similar to its parental molecule TMZ.
• POH is known to affect several intracellular processes. For instance, it has been shown to inhibit the activity of telomerase and of sodium-potassium pump (Na+/K+-ATPase) [52; 53]. As well, it has been described as a farnesyl-transferase inhibitor that results in the blockage of ras oncoprotein activity (I.R. et al., Inhibition of protein prenylation by metabolites of limonene. Biochem Pharmacol 57 (1999) 801-809; P.L.
• TMZ-POH significantly more potent than TMZ? It has been well established that TMZ (and its active degradation product) exhibits rapid turnover in vitro and in vivo, with a half- life in the range of 1-2 hours. Consistent with these characteristics, we find that after 4 hours of incubation in medium, nearly 100% of TMZ's cytotoxic activity has been spent (Figs. 26A-26C). In contrast, TMZ-POH appears significantly longer-lived, where after 4 hours about 50% activity remains (Figs. 26A-26C). Thus, while not wishing to be bound by any particular theory, we propose that the extended presence of TMZ-POH may provide for greater opportunity to set DNA lesions, resulting in increased cytotoxicity.
• TMZ-POH While the extended half-life of TMZ-POH may suffice to explain its greater potency in vitro, it remains to be established whether it also contributes to its substantially increased in vivo potency in our brain metastasis model (Figs. 27A-27B). Because the lipophilicity of TMZ-POH is increased over TMZ (data not shown), it is also possible that TMZ-POH may cross the BBB more efficiently than TMZ. In the case of TMZ, it is known that drug levels achieved in the cerebrospinal fluid (CSF) are 80% lower than drug levels in the systemic circulation, i.e., in plasma. It is therefore conceivable that TMZ, despite its established therapeutic benefit, would exert even greater activity, if only higher intracranial concentrations could be achieved. In this regard, TMZ-POH might be the vehicle to achieve this.
• CSF cerebrospinal fluid
• TMZ displayed only minor activity in our intracranial in vivo model (Figs. 27A-27B).
• the breast cancer cell line we used a variant of MDA-MB-231, does exhibit extraordinar in vitro sensitivity to TMZ (IC50 ⁇ 10 ⁇ ), and therefore is more sensitive to TMZ than most MGMT-negative GBM cell lines reported in the literature and inclusive of several GBM cell lines we analyzed in parallel (data not shown).
• the TMZ dosage used 25 mg/kg is well within the range of dosages shown to exert potent activity in GBM mouse models, where even 5 mg/kg has significant activity.

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