Pharmaceutical Compositions Comprising POH Derivatives

Abstract

The present invention provides a method for treating inflammation in a mammal. The method comprises delivering a therapeutically effective amount of a composition comprising perillyl alcohol (POH) conjugated with linoleic acid to the mammal. The present invention also provides a method for treating or preventing damage caused by UV exposure in a mammal.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 175,901, filed April 16, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates to perillyl alcohol (POH) derivatives. The present invention further relates to methods of treating inflammation and treating or preventing damage from UV exposure using POH derivatives, such as POH carbamates, where the nitrogen substituent can be a therapeutic agent, and POH esters, where the ester functional group can be a fatty acid. [Background technology]

[0003] Malignant gliomas, the most common form of central nervous system (CNS) cancer, are currently considered essentially incurable. Among the various malignant gliomas, anaplastic astrocytoma (grade III) and glioblastoma multiforme (GBM; grade IV) have a particularly poor prognosis due to their rapid growth and resistance to currently available treatments. The current standard of care for malignant gliomas consists of surgery, ionizing radiation, and chemotherapy. Despite recent advances in medicine, there has been no significant improvement in the prognosis of malignant gliomas over the past 50 years. Wen et al.Malignant gliomas in adults.New England J Med.359:492-507,2008.Stupp et al.Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.New England J Med.352:987-996,2005. The poor response of tumors, including malignant gliomas, to various types of chemotherapeutic agents is often due to intrinsic drug resistance. In addition, acquired resistance and undesirable side effects of initially well-responsive tumors are other problems that frequently impede long-term treatment using chemotherapeutic agents. Therefore, to overcome these problems, various analogues of chemotherapeutic agents have been prepared. The analogues include novel therapeutic agents that are hybrid molecules of at least two existing therapeutic agents. For example, cisplatin has been conjugated with Pt-(II) complexes bearing cytotoxic codrugs or with bioactive shuttle moieties, such as porphyrins, bile acids, hormones, or modulators that promote transmembrane transport or drug accumulation in cells. (6-aminomethylnicotinate)dichlorodiplatinum(II) complexes esterified with terpene alcohols were tested in a panel of human tumor cell lines. The terpenyl moiety in these complexes appeared to perform a transmembrane shuttle function and increase the rate and extent of uptake of these conjugates into various tumor cell lines. Schobert et al. Monoterpenes as Drug Shuttles:Cytotoxic(6-minomethylnicotinate)dichlorodiplatinum(II) Complexes with Potential to Overcome Cisplatin Resistance.J.Med.Chem.2007,50,1288-1293. POH, a naturally occurring monoterpene, has been suggested to be an effective agent against a variety of cancers, including CNS, breast, pancreatic, lung, melanoma, 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. There remains a need to prepare perillyl alcohol derivatives, including perillyl alcohol conjugated with other therapeutic agents, and use the materials in the treatment of cancers, such as malignant gliomas, and other brain disorders, such as Parkinson's disease and Alzheimer's disease. The perillyl alcohol derivatives can be administered alone or in combination with other treatment methods, including radiation, standard chemotherapy, and surgery. Administration can also be via various routes, including intranasal, oral, oral-tracheal, and transdermal for pulmonary delivery. There is a need to use perillyl alcohol derivatives, including perillyl alcohol conjugated with other therapeutic agents, in the treatment of inflammation, particularly skin inflammation, and to treat or prevent damage caused by UV exposure. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Wen et al.Malignant gliomas in adults.New England J Med.359:492-507,2008. [Non-Patent Document 2] Stupp et al.Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.New England J Med.352:987-996,2005. [Non-Patent Document 3] Schobert et al. Monoterpenes as Drug Shuttles:Cytotoxic(6-minomethylnicotinate)dichlorodiplatinum(II) Complexes with Potential to Overcome Cisplatin Resistance.J.Med.Chem.2007,50,1288-1293. [Non-Patent Document 4] Gould, M. Cancer chemoprevention and therapy by monoterpenes.Environ Health Perspect.1997 June;105(Suppl 4):977-979. [Non-Patent Document 5] 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. Summary of the Invention [Problem to be solved by the invention]

[0005] [Means for solving the problem]

[0006] The present invention provides a pharmaceutical composition comprising a POH derivative, wherein the POH derivative is selected from the group consisting of a POH carbamate and a POH ester.

[0007] Perillyl alcohol carbamate can be perillyl alcohol conjugated with a therapeutic agent, such as a chemotherapeutic agent.The chemotherapeutic agent that can be used in the present invention includes DNA alkylating agent, topoisomerase inhibitor, endoplasmic reticulum stress inducer, platinum compound, metabolic antagonist, enzyme inhibitor, receptor antagonist, etc.In certain embodiments, the therapeutic agent is dimethylcelecoxib (DMC), temozolomide (TMZ) or rolipram. The perillyl alcohol carbamate can be 4-(bis-N,N'-4-isopropenylcyclohex-1-enylmethyloxycarbonyl[5-(2,5-dimethylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonamide, 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-oxo-pyrrolidine-1-carboxylic acid 4-isopropenylcyclohex-1-enylmethyl ester, and 3-methyl 4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)carbamic acid-4-isopropenylcyclohex-1-enylmethyl ester.

[0008] The POH ester may be perillyl alcohol conjugated with a hydrocarbon selected from the group consisting of alkyl; alkenyl; alkynyl; cycloalkyl; cycloalkenyl; cycloalkyl; heterocyclylalkyl; aryl; heteroaryl. In certain embodiments, the hydrocarbon may be a fatty acid having one or more dehydration sites. In certain embodiments, the fatty acid may be linoleic acid or palmitic acid. In certain embodiments, the fatty acid may be linoleic acid, and the POH-linoleoyl ester is also known as NEO400. In other words, NEO400 is perillyl alcohol conjugated with linoleic acid ("POH-LA conjugate").

[0009] The pharmaceutical composition of the present invention may be administered before, during or after radiation. The pharmaceutical composition may be administered before, during or after administration of a chemotherapeutic agent. The pharmaceutical composition may be administered before, during or after administration of an anti-inflammatory agent. The routes of administration of the pharmaceutical composition include inhalation, intranasal, oral, topical, transdermal, intravenous, subcutaneous or intramuscular administration.

[0010] The present invention further provides a method for treating a disease in a mammal, comprising delivering a therapeutically effective amount of a perillyl alcohol carbamate or perillyl alcohol ester to the mammal. The method may further comprise treating the mammal with radiation, and / or may further comprise delivering a chemotherapeutic agent to the mammal, and / or may further comprise delivering an anti-inflammatory agent to the mammal. The disease to be treated may be cancer, including tumors of the nervous system, such as glioblastoma and melanoma. Routes of administration of the perillyl alcohol carbamate include inhalation, intranasal, oral, topical, transdermal, intravenous, subcutaneous or intramuscular administration.

[0011] The present invention also provides a method for preparing POH carbamates, comprising reacting a first reactant of perillyl chloroformate with a second reactant, which may be dimethylcelecoxib (DMC), temozolomide (TMZ) or rolipram. When the second reactant is dimethylcelecoxib, the reaction may be carried out in the presence of acetone and potassium carbonate catalysts. When the second reactant is rolipram, the reaction may be carried out in the presence of tetrahydrofuran and n-butyllithium catalysts. Perillyl chloroformate may also be prepared by reacting perillyl alcohol with phosgene. [Brief description of the drawings]

[0012] [Figure 1] 1 shows the results of an MTT cytotoxicity assay demonstrating that dimethylcelecoxib (DMC) is effective in killing U87, A172 and U251 human glioma cells. [Diagram 2]1 shows the results of an MTT cytotoxicity assay demonstrating that POH-DMC conjugates are effective in killing U87, A172 and U251 human glioma cells according to the present invention. [Diagram 3] 1 shows the results of an MTT cytotoxicity assay demonstrating that temozolomide (TMZ) is effective in killing U87, A172 and U251 human glioma cells. [Figure 4] 1 shows the results of an MTT cytotoxicity assay demonstrating that POH-TMZ conjugates are effective in killing U87, A172 and U251 human glioma cells according to the present invention. [Diagram 5] 1 shows the results of an MTT cytotoxicity assay demonstrating that POH-rolipram conjugates and rolipram are effective in killing A172 human glioma cells. [Figure 6] 1 shows the results of an MTT cytotoxicity assay demonstrating that POH-rolipram conjugates and rolipram are effective in killing U87 human glioma cells. [Figure 7] 1 shows the results of an MTT cytotoxicity assay demonstrating that POH-rolipram conjugates and rolipram are effective in killing U251 human glioma cells. [Figure 8] 1 shows the results of an MTT cytotoxicity assay demonstrating that POH-rolipram conjugates and rolipram are effective in killing L229 human glioma cells. [Figure 9] Figure 9A shows the inhibition of tumor growth by butyryl-POH in mouse models. Figure 9A shows images of subcutaneous U-87 gliomas in nude mice treated with butyryl-POH, purified (S)-perillyl alcohol with a purity of over 98.5% ("purified POH"), POH purchased from Sigma chemicals ("Sigma"), or phosphate buffered saline ("PBS"; negative control). Figure 9B shows the average tumor growth over time (for a total period of 60 days). [Figure 10] 1 shows colony formation assay (CFA) results demonstrating the cytotoxic effects of TMZ and TMZ-POH on TMZ-sensitive (U251) and TMZ-resistant (U251TR) U251 cells. [Figure 11] 1 shows colony formation assay (CFA) results demonstrating the cytotoxic effect of POH on TMZ-sensitive (U251) and TMZ-resistant (U251TR) U251 cells. [Figure 12] 1 shows the results of an MTT cytotoxicity assay demonstrating that the POH-TMZ conjugate is effective in killing U251 cells, U251TR cells, and normal astrocytes. [Figure 13] 1 shows the results of an MTT cytotoxicity assay demonstrating that the POH-TMZ conjugate is effective in killing normal astrocytes, brain endothelial cells (BECs; confluent and subconfluent) and tumor brain endothelial cells (TuBECs). [Figure 14] 1 shows the results of an MTT cytotoxicity assay demonstrating that TMZ and POH-TMZ conjugates are effective in killing USC-04 glioma cancer stem cells. [Figure 15] 1 shows the results of an MTT cytotoxicity assay demonstrating that POH is effective in killing USC-04 glioma cancer stem cells. [Figure 16] 1 shows the results of an MTT cytotoxicity assay demonstrating that TMZ and POH-TMZ conjugates are effective in killing USC-02 glioma cancer stem cells. [Figure 17] 1 shows the results of an MTT cytotoxicity assay demonstrating that POH is effective in killing USC-02 glioma cancer stem cells. [Figure 18] 1 shows a Western blot demonstrating that TMZ-POH induces ER stress (ERS) in TMZ-sensitive ("U251-TMZs") and TMZ-resistant ("U251-TMZr") U251 glioma cells. [Figure 19A] Photographs of mice receiving localized application of asTPA to the skin for 7 days and further treated with a glycerol / ethanol vehicle. [Figure 19B] Photograph of another mouse that received asTPA application to a portion of the skin for 7 days and was further treated with a glycerol / ethanol vehicle. [Figure 20A]Photograph of mice receiving patchy dermal application of asTPA for 7 days and further treatment with POH-linoleic acid conjugate in a glycerol / ethanol vehicle. [Figure 20B] Photograph of mice receiving patchy dermal application of asTPA for 7 days and further treatment with POH-linoleic acid conjugate in a glycerol / ethanol vehicle. [Figure 21A] Photographs of SKH-1 hairless mice treated with TPA. [Figure 21B] Photographs of SKH-1 hairless mice treated with TPA and POH-linoleic acid conjugate. [Figure 21C] Photographs of SKH-1 hairless mice treated with TPA. [Figure 21D] Photographs of SKH-1 hairless mice treated with TPA and POH-linoleic acid conjugate. [Figure 22A] Photograph of SKH-1 hairless mice treated with sunscreen and irradiated with a UV lamp. [Figure 22B] Photographs of SKH-1 hairless mice treated with POH and irradiated with a UV lamp. [Figure 22C] Photographs of SKH-1 hairless mice treated with linoleic acid and irradiated with a UV lamp. [Figure 22D] Photographs of SKH-1 hairless mice treated with POH-LA conjugates and irradiated with a UV lamp. [Figure 22E] Photographs of SKH-1 hairless mice that were not treated and were irradiated with a UV lamp. [Figure 23A] 1 is a photograph of an SKH-1 hairless mouse before irradiation with a UV lamp. [Figure 23B] Photographs of SKH-1 hairless mice that were not treated and were irradiated with a UV lamp. [Figure 23C] Photographs of SKH-1 hairless mice treated with POH and irradiated with a UV lamp. [Figure 23D] Photographs of SKH-1 hairless mice treated with linoleic acid and irradiated with a UV lamp. [Figure 23E]Photographs of SKH-1 hairless mice treated with a mixture of linoleic acid and POH and irradiated with a UV lamp. [Figure 23F] Photographs of SKH-1 hairless mice treated with POH-LA conjugates and irradiated with a UV lamp. [Figure 24A] Photographs of SKH-1 hairless mice treated with POH-LA conjugates and irradiated with a UV lamp. [Figure 24B] Photographs of SKH-1 hairless mice treated with linoleic acid and irradiated with a UV lamp. [Figure 24C] Photographs of SKH-1 hairless mice treated with POH and irradiated with a UV lamp. [Figure 24D] Photographs of SKH-1 hairless mice treated with a mixture of linoleic acid and POH and irradiated with a UV lamp. [Figure 25A] 1 is a graph showing the change in IL-1β levels after UV exposure with and without treatment of POH-LA conjugates. [Figure 25B] 1 is a graph showing the change in IL-6 levels after UV exposure with and without treatment of POH-LA conjugates. [Figure 25C] 1 is a graph showing the change in TNF-α levels after UV exposure with and without treatment of POH-LA conjugates. [Figure 26A] 1 is a graph showing DNA damage produced by 6-4PP measurements after UV exposure with and without POH-LA conjugate treatment. [Figure 26B] 1 is a graph showing DNA damage produced after UV exposure with and without POH-LA conjugate treatment by CPD measurement. [Figure 27A] 1 is a photomicrograph showing skin staining upon activation of follistatin with a POH-LA conjugate. [Figure 27B] 1 is a photomicrograph showing staining of skin upon activation of follistatin with POH. [Figure 27C]1 is a photomicrograph showing skin staining upon activation of follistatin with linoleic acid. [Figure 27D] 1 is a photomicrograph showing the staining of skin by activation of α6β4 integrin with POH-LA conjugate. [Figure 27E] 1 is a photomicrograph showing skin staining due to activation of α6β4 integrin with POH. [Figure 27F] 1 is a photomicrograph showing skin staining due to activation of α6β4 integrin with linoleic acid. [Figure 27G] 13 shows photomicrographs showing skin staining due to activation of Yap1 with a POH-LA conjugate. [Fig. 27H] 13 shows micrographs showing skin staining due to activation of Yap1 with POH. [Figure 27I] 13 shows micrographs showing skin staining due to activation of Yap1 with linoleic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention provides a monoterpene or sesquiterpene derivative, such as a perillyl alcohol derivative. The present invention also provides a pharmaceutical composition comprising a monoterpene or sesquiterpene derivative, such as a perillyl alcohol derivative. For example, the perillyl alcohol derivative may be a perillyl alcohol carbamate. The perillyl alcohol derivative may be perillyl alcohol conjugated to a therapeutic agent, such as a chemotherapeutic agent. The monoterpene (or sesquiterpene) derivative may be formulated into a pharmaceutical composition, wherein the monoterpene (or sesquiterpene) derivative is present in an amount ranging from about 0.01% (w / w) to about 100% (w / w), about 0.1% (w / w) to about 80% (w / w), about 1% (w / w) to about 70% (w / w), about 10% (w / w) to about 60% (w / w), or about 0.1% (w / w) to about 20% (w / w). The composition can be administered alone or co-administered with radiation or another agent (e.g., a chemotherapeutic agent) to treat a disease such as cancer. The treatment can be sequential, with the monoterpene (or sesquiterpene) derivative administered before or after the administration of the other agent. For example, perillyl alcohol carbamate can be used to sensitize a cancer patient to radiation or chemotherapy. Alternatively, the agents can be administered simultaneously. The route of administration can vary and can include inhalation, intranasal, oral, transdermal, intravenous, subcutaneous or intramuscular injection. The present invention also provides a method of treating a disease such as cancer, comprising delivering a therapeutically effective amount of a monoterpene (or sesquiterpene) derivative to a patient.

[0014] The composition of the present invention may contain one or more types of derivatives of monoterpenes (or sesquiterpenes). Monoterpenes include terpenes that consist of two isoprene units. Monoterpenes may be linear (non-cyclic) or may contain rings. Derivatives of monoterpenoids are also included in the present invention. Monoterpenoids may be produced by biochemical modification, such as oxidation or rearrangement of monoterpenes. Examples of monoterpenes and monoterpenoids include perillyl alcohol (S(-)) and (R(+)), ocimene, myrcene, geraniol, citral, citronellol, citronellal, linalool, pinene, terpineol, terpinene, limonene, terpinene, phellandrene, terpinolene, terpinen-4-ol (or tea tree oil), pinene, terpineol, terpinene; terpenoids, such as p-cymene, which are derived from monocyclic terpenes such as menthol, thymol, and carvacrol; bicyclic monoterpenoids, such as camphor, borneol, and eucalyptol.

[0015] Monoterpenes can be differentiated according to the structure of the carbon skeleton and classified 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, phellandrene, terpinolene, menthol, carveol, etc.), bicyclic monoterpenes (e.g., pinene, myrtenol, myrtenal, verbanol, verbanone, pinocarveol, carene, sabinene, camphene, tsene, etc.) and tricyclic monoterpenes (e.g., tricyclene). See Encyclopedia of Chemical Technology, Fourth Edition, Volume 23, pages 834-835.

[0016] The sesquiterpenes of the present invention include terpenes that are composed of three isoprene units. Sesquiterpenes may be linear (non-cyclic) or may contain rings. Derivatives of sesquiterpenoids are also included in the present invention. Sesquiterpenoids can be produced by biochemical modification, such as oxidation or rearrangement of sesquiterpenes. Examples of sesquiterpenes include farnesol, farnesal, farnesyl acid and nerolidol.

[0017] Derivatives of monoterpenes (or sesquiterpenes) include, but are not limited to, carbamates, esters, ethers, alcohols and aldehydes of monoterpenes (or sesquiterpenes). Monoterpene (or sesquiterpene) alcohols can be derivatized to carbamates, esters, ethers, aldehydes or acids.

[0018] Carbamates are functional groups based on a carbonyl group adjacent to an oxygen and a nitrogen. [ka] R refers to a class of chemical compounds that share the same 1 , R 2 and R 3 may be an optionally substituted alkyl, aryl, etc. group. The R groups on the nitrogen and oxygen may form a ring. R 1 -OH may be a monoterpene, such as POH. 2 -NR 3 The moiety can be a therapeutic agent.

[0019] Carbamates can be synthesized by reacting an isocyanate with an alcohol or by reacting a chloroformate with an amine. Carbamates can be synthesized by reactions utilizing phosgene or a phosgene equivalent. For example, carbamates can 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 produced from the reaction of a urea intermediate with an alcohol. Dimethyl carbonate and diphenyl carbonate are also used to produce carbamates. Alternatively, carbamates may be synthesized by the reaction of an alcohol and / or amine precursor with an ester-substituted diaryl carbonate, such as bismethylsalicylcarbonate (BMSC). US Patent Publication No. 20100113819.

[0020] The carbamates can be synthesized by the following approach. [ka] Suitable reaction solvents include, but are not limited to, tetrahydrofuran, dichloromethane, dichloroethane, acetone, and diisopropyl ether. The reaction may be carried out 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 substrate R-NH2 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, N,N'-diisopropylethylamine, butyllithium, and potassium-t-butoxide.

[0021] Alternatively, the carbamates can be synthesized by the following approach. [ka] Suitable reaction solvents include, but are not limited to, dichloromethane, dichloroethane, toluene, diisopropyl ether, and tetrahydrofuran. The reaction may be carried out at a temperature ranging from about 25° C. to about 110° C., or from about 30° C. to about 80° C., or at about 50° C. The molar ratio of perillyl alcohol to the substrate RN=C=O 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.

[0022] The esters of monoterpene (or sesquiterpene) alcohols of the present invention can be derived from inorganic or organic acids. Inorganic acids include, but are not limited to, phosphoric acid, sulfuric acid, and nitric acid. Organic acids include, but are not limited to, carboxylic acids, such as benzoic acid, fatty acids, acetic acid, and propionic acid, and any therapeutic agent having at least one carboxylic acid functional group. Examples of esters of monoterpene (or sesquiterpene) alcohols include, but are not limited to, carboxylic acid esters (e.g., benzoate esters, fatty acid esters (e.g., palmitate esters, linoleate esters, stearate esters, butyryl esters, and oleate esters), acetates, propionates (or propanoates), and formates), phosphates, sulfates, and carbamates (e.g., N,N-dimethylaminocarbonyl). Retrieved from Wikipedia - Ester URL: http: / / en.wikipedia.org / wiki / Ester.

[0023] A specific example of a monoterpene that can be used in the present invention is perillyl alcohol (commonly abbreviated as POH). Derivatives of perillyl alcohol include perillyl alcohol carbamate, perillyl alcohol ester, perillic aldehyde, dihydroperillic acid, perillic acid, perillic aldehyde derivatives, dihydroperillic acid esters and perillic acid esters. Derivatives of perillyl alcohol can also include its oxidative and nucleophilic / electrophilic addition derivatives. US Patent Publication No. 20090031455. US Patent Nos. 6,133,324 and 3,957,856. Many examples of derivatives of perillyl alcohol have been reported in the chemical literature (see Appendix A: CAS Scifinder search output file, retrieved January 25, 2010).

[0024] In certain embodiments, POH carbamates are synthesized by a process that includes reacting a first reactant of perillyl chloroformate with a second reactant such as dimethylcelecoxib (DMC), temozolomide (TMZ) and rolipram. The reaction can be carried out in the presence of tetrahydrofuran and a base such as n-butyl lithium. Perillyl chloroformate can be made by reacting POH with phosgene. For example, POH conjugated with temozolomide via a carbamate bond can be synthesized by reacting temozolomide with oxalyl chloride, followed by reaction with perillyl alcohol. The reaction can be carried out in the presence of 1,2-dichloroethane.

[0025] POH carbamates encompassed by the present invention include, but are not limited to, 4-(bis-N,N'-4-isopropenylcyclohex-1-enylmethyloxycarbonyl[5-(2,5-dimethylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonamide, 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-oxo-pyrrolidine-1-carboxylic acid 4-isopropenylcyclohex-1-enylmethyl ester, and (3-methyl 4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)carbamic acid-4-isopropenylcyclohex-1-enylmethyl ester. Details of the chemical reactions to produce these compounds are described in the Examples below.

[0026] In certain embodiments, the perillyl alcohol derivative may be a perillyl alcohol fatty acid ester, such as palmitoyl ester of POH and linoleoyl ester of POH, the chemical structures of which are shown below. [ka] [ka] The monoterpene (or sesquiterpene) derivative may be a monoterpene (or sesquiterpene) conjugated with a therapeutic agent. The monoterpene (or sesquiterpene) conjugate encompassed by the present invention is a molecule having a monoterpene (or sesquiterpene) covalently bonded to a therapeutic agent via a chemical linking group. The molar ratio of monoterpene (or sesquiterpene) to 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 ratio. The monoterpene (or sesquiterpene) and therapeutic agent may be covalently bonded via a carbamate, ester, ether bond, or any other suitable chemical functional group. When the monoterpene (or sesquiterpene) and therapeutic agent are conjugated via a carbamate bond, the therapeutic agent may be any agent having at least one carboxylic acid functional group or any agent having at least one amine functional group. In a particular example, the perillyl alcohol conjugate is perillyl alcohol covalently attached to a chemotherapeutic agent via a chemical linking group.

[0027] According to the present invention, therapeutic agents that can be conjugated with monoterpenes (or sesquiterpenes) include, but are not limited to, chemotherapeutic agents, therapeutic agents for the treatment of CNS disorders (including, but not limited to, primary degenerative neurological disorders such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, attention deficit hyperactivity disorder or ADHD, psychiatric disorders, psychological disorders, psychosis, and depression), immunotherapeutic agents, angiogenesis inhibitors, and antihypertensive agents. Anticancer agents that can be conjugated with monoterpenes or sesquiterpenes can have one or more of the following effects on cancer cells or subjects: cell death; reduction in cell proliferation; reduction in cell number; inhibition of cell proliferation; apoptosis; necrosis; mitotic catastrophe; cell cycle arrest; reduction in cell size; reduction in cell division; reduction in cell viability; reduction in cell metabolism; markers of cell damage or cytotoxicity; indirect indicators of cell damage or cytotoxicity, such as tumor shrinkage; improvement in subject survival; or disappearance of markers associated with undesirable, unwanted, or abnormal cell proliferation. US Patent Publication No. 20080275057. Mixtures and / or co-formulations of a monoterpene (or a sesquiterpene) with at least one therapeutic agent are also encompassed by the present invention.

[0028] Chemotherapeutic agents include, but are not limited to, DNA alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducers, platinum compounds, antimetabolites, vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor antagonists, tyrosine kinase inhibitors, boron radiosensitizers (i.e., Velcade), and combination chemotherapy therapies.

[0029] 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; alkylsulfonates, such as busulfan (mannosulfan, treosulfan); aziridines, such as carboquone, triaziquone, triethylenemelamine; hydrazine (procarbazine); triazenes, such as dacarbazine and temozolomide (TMZ); altretamine and mitobronitol.

[0030] Non-limiting examples of topoisomerase I inhibitors include campothecin derivatives including SN-38, APC, NPC, campothecin, topotecan, exatecan mesylate, 9-nitrocamptothecin, 9-aminocamptothecin, lurtotecan, rubitecan, ciratecan, gimatecan, diflomotecan, exatecan, BN-80927, DX-8951f, and MAG-CPT, as described in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and U.S. Patent Publication No. 200510250854; Li et al. (2000) Biochemistry 39(24):7107-7116 and Gatto et al. (1996) Cancer Protoberberine alkaloids and their derivatives, including berberrubine and coraline, as described in Makhey et al. (2003) Bioorg. Med. Chem. 11(8):1809-1820; phenanthroline derivatives, including benzo[i]phenanthridine, nitidine, and fagaronine, as described in Xu (1998) Biochemistry 37(10):3558-3566; and terbenzimidazole and its derivatives, as described in Foglesong et al. (1992) Cancer Chemother. Pharmacol. 30(2):123-25, Crow et al. (1994) J. Med. Chem. 37(19):31913194, and Crespi et al. and anthracycline derivatives including doxorubicin, daunorubicin, and mitoxantrone, as described in U. et al. (1986) Biochem. Biophys. Res. Commun. 136(2):521-8. Topoisomerase II inhibitors include, but are not limited to, etoposide and teniposide.Dual topoisomerase I and II inhibitors include, but are not limited to, Saintopin and other naphthecenediones, DACA and other acridine-4-carboxamides, Intoplicine and other benzopyridoindoles, TAS-I03 and other 7H-indeno[2,1-c]quinolin-7-ones, pyrazoloacridines, 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 drugs inhibit topoisomerase II and have DNA intercalation activity, such as, but not limited to, the anthracyclines (aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin) and the anthracenediones (mitoxantrone and pixantrone).

[0031] Examples of endoplasmic reticulum stress inducers include, but are not limited to, dimethyl-celecoxib (DMC), nelfinavir, celecoxib, and boron radiosensitizers (ie, Velcade (bortezomib)).

[0032] 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 generally CHEMOTHERAPY FOR GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, Angioli et al. Eds., 2004, Series Basic and Clinical Oncology).

[0033] "FOLFOX" is an abbreviation for a type of combination therapy used to treat colorectal cancer. It includes 5-FU, oxaliplatin, and leucovorin. Information about this treatment is available on the National Cancer Institute's website (cancer.gov, last accessed January 16, 2008).

[0034] "FOLFOX / BV" is an abbreviation for a type of combination therapy used to treat colorectal cancer. This therapy includes 5-FU, oxaliplatin, leucovorin, and bevacizumab. Additionally, "XELOX / BV" is another combination therapy used to treat colorectal cancer, which includes a prodrug to 5-FU known as capecitabine (Xeloda) combined with oxaliplatin and bevacizumab. Information about these therapies is available on the National Cancer Institute's website at cancer.gov, or from the National Comprehensive Cancer Network's website at nccn.org (last accessed May 27, 2008).

[0035] Non-limiting examples of antimetabolites include folate-based, i.e., dihydrofolate reductase inhibitors, such as aminopterin, methotrexate, and pemetrexed; thymidylate synthase inhibitors, such as raltitrexed, pemetrexed; purine-based, i.e., adenosine deaminase inhibitors, such as pentostatin, thiopurines, such as thioguanine and mercaptopurine, halogenated / ribonucleotide reductase inhibitors, such as cladribine, clofarabine, fludarabine, or guanine / guanosine:thiopurines, such as thioguanine; or pyrimidine-based, i.e., cytosine / cytidine:hypomethylating agents, such as azacytidine and decitabine, DNA polymerase inhibitors, such as cytarabine, ribonucleotide reductase inhibitors, such as gemcitabine, or thymine / thymidylate:thymidylate synthase inhibitors, such as fluorouracil (5-FU). Equivalents of 5-FU include its prodrugs, analogs and derivatives, such as 5'-deoxy-5-fluorouridine (doxiflurodine), 1-tetrahydrofuranyl-5-fluorouracil (florafur), capecitabine (Xeloda), SI (MBMS-247616, consisting of tegafur and two modulators, 5-chloro-2,4-dihydroxypyridine and potassium oxonate), ralititrexed (Tomudex), nolatrexed (Thymitaq, AG337), LY231514 and ZD9331, as described in Papamicheal (1999) The Oncologist 4:478-487.

[0036] Examples of vincalkaloids include, but are not limited to, vinblastine, vincristine, vinflunine, vindesine, and vinorelbine.

[0037] Examples of taxanes include, but are not limited to, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel. An example of an epothilone is iabepilone.

[0038] Examples of enzyme inhibitors include, but are not limited to, farnesyltransferase inhibitors (tipifarnib); CDK inhibitors (alvocidib, celecilib); proteasome inhibitors (bortezomib); phosphodiesterase inhibitors (anagrelide; rolipram); IMP dehydrogenase inhibitors (tiazofurin); lipoxygenase inhibitors (masoprocol). Examples of receptor antagonists include, but are not limited to, ERAs (atrasentan); retinoid X receptors (bexarotene); and sex steroids (testolactone).

[0039] Examples of tyrosine kinase inhibitors include, but are not limited to, inhibitors against ErbB: HER1 / EGFR (erlotinib, gefitinib, lapatinib, vandetanib, sunitinib, neratinib); HER2 / neu (lapatinib, neratinib); RTK class III: C-kit (axitinib, sunitinib, sorafenib), FLT3 (lestaurtinib), PDGFR (axitinib, sunitinib, sorafenib); and VEGFR (vandetanib, semaxanib, cediranib, axitinib, sorafenib); bcr-abl (imatinib, nilotinib, dasatinib); Src (bosutinib) and Janus kinase 2 (lestaurtinib).

[0040] "Lapatinib" (Tykerb®) is a dual inhibitor of EGFR and erbB-2. Lapatinib is being investigated in several clinical trials as an anticancer monotherapy, in addition to in combination with trastuzumab, capecitabine, letrozole, paclitaxel and FOLFIRI (irinotecan, 5-fluorouracil and leucovorin). It is currently in Phase III trials for the oral treatment of metastatic breast, head and neck, lung, gastric, renal and bladder cancer.

[0041] The chemical equivalent of lapatinib is a small molecule or compound that is a tyrosine kinase inhibitor (TKI) or a HER-1 or HER-2 inhibitor. Several TKIs have been found to have effective antitumor activity and are approved or in clinical trials. Such examples include, but are not limited to, Zactima (ZD6474), Iressa (gefitinib), imatinib mesylate (STI571; Gleevec), erlotinib (OSI-1774; Tarceva), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY43-9006), sutent (SUI 1248) and leflutomide (SU10l).

[0042] PTK / ZK is a broad specificity tyrosine kinase inhibitor that targets all VEGF receptors (VEGFR), platelet derived growth factor (PDGF) receptors, 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-1 (Flt-1), VEGFR-2 (KDR / Flk-1) and VEGFR-3 (Flt-4). The chemical name of PTK / ZK is 1-[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 inhibitors, ZK 222584 and ZK.

[0043] Chemotherapeutic agents that can be conjugated to a monoterpene or sesquiterpene can also include amsacrine, trabectin, retinoids (alitretinoin, tretinoin), arsenic trioxide, asparagine depleters (asparaginase / pegaspargase), celecoxib, demecolcine, elesclomol, elsamitrucin, etoglucide, lonidamine, lucantone, mitoguazone, mitotane, oblimersen, temsirolimus, and vorinostat.

[0044] The monoterpene or sesquiterpene derivatives can be conjugated with an angiogenesis inhibitor. Examples of 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-O-(chloroacetyl-carbonyl)-fumagillol, COL-3, combretastatin, combretastatin A4 phosphate, dalteparin, EMD 121974 (cilengitide), endostatin, erlotinib, gefitinib (Iressa), genistein, halofuginone hydrobromide, Id1, Id3, IM862, imatinib mesylate, IMC-IC11-inducible protein 10, interferon-α, interleukin-12, lavendustin A, LY317615 or AE-941, marimastat, muspin, medroxypreprogesterone acetate, Meth-1, Meth-2, 2-methoxyestradiol (2-ME), neovastat, oteopontin cleaved product, PEX, pigment epithelial growth factor (PEGF), platelet factor 4, prolactin fragment, growth-related protein (PRP), PTK787 / ZK 222584, ZD6474, recombinant human platelet factor 4 (rPF4), restin, squalamine, SU5416, SU6668, SU11248 suramin, taxol, tecogalan, thalidomide, thrombospondin, TNP-470, troponin-1, vasostatin, VEG1, VEGF-Trap and ZD6474.

[0045] Non-limiting examples of angiogenesis inhibitors also include tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1 / KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet-derived growth factors, MMP (matrix metalloproteinase) inhibitors, integrin blockers, pentosan polysulfate, angiotensin II antagonists, cyclooxygenase inhibitors (including nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen, and selective cyclooxygenase-2 inhibitors such as celecoxib and rofecoxib), and steroidal anti-inflammatory drugs (e.g., corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone).

[0046] Other therapeutic agents that modulate or inhibit angiogenesis and that may be conjugated to monoterpenes or sesquiterpenes 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]). U.S. Patent Publication No. 20090328239. U.S. Patent No. 7,638,549.

[0047] Non-limiting examples of antihypertensive 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), telmisartan, irbesartan, tasosartan, olmesartan, olmesartan medoxomil, etc.), calcium antagonists (e.g., manidipine, nifedipine, amlodipine, (or amlodipine), efonidipine, nicardipine, etc.), diuretics, renin inhibitors (e.g., aliskiren, etc.), aldosterone antagonists (e.g., spironolactone, eplerenone, etc.), beta-blockers (e.g., metoprolol (or topolol), atenolol, propranolol, carvedilol, pindolol, etc.), vasodilators (e.g., nitrates, soluble guanylate cyclase stimulators or activators, prostacyclin, etc.), angiotensin vaccines, clonidine, etc. U.S. Patent Publication No. 20100113780.

[0048] Other therapeutic agents that may be conjugated with a monoterpene (or sesquiterpene) include sertraline (Zoloft), topiramate (Topamax), duloxetine (Cymbalta), sumatriptan (Imitrex), pregabalin (Lyrica), lamotrigine (Lamictal), valacyclovir (Valtrex), tansulfocine (Flomax), zidovudine (Combivir), lamivudine (Combivir), efavirenz (Sustiva), abacavir (Epzicom), lopinavir (Kaletra), and pioglitazone (Pioglitazone). These include ritazon (Actos), desloratidine (Clarinet), cetirizine (Zyrtec), pentoprazole (Protonix), lansoprazole (Prevacid), rebeprazole (Aciphex), moxifloxacin (Avelox), meloxicam (Mobic), dorzolamide (Traspot), diclofenac (Voltaren), enlapril (Vasotec), montelukast (Singulair), sildenafil (Viagra), carvedilol (Colleg), and ramipril (Derix).

[0049] Table 1 lists pharmaceutical agents that can be conjugated to monoterpenes (or sesquiterpenes), including the structures of the pharmaceutical agents and preferred derivatives for conjugation. [Table 1] TIFF2024513969000008.tif231153TIFF2024513969000009.tif214152TIFF20245139690 00010.tif233152TIFF2024513969000011.tif217152TIFF2024513969000012.tif139153 The purity of monoterpene (or sesquiterpene) derivatives can be assayed by gas chromatography (GC) or high performance liquid chromatography (HPLC). Other techniques for assaying the purity of monoterpene (or sesquiterpene) derivatives and 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 by measuring optical rotation.

[0050] The monoterpene (or sesquiterpene) derivatives can be purified by methods such as crystallization or by separating the monoterpene (or sesquiterpene) derivatives from impurities according to the intrinsic physicochemical properties of the derivatives (e.g. solubility or polarity). Thus, the monoterpene (or sesquiterpene) derivatives can be separated from the monoterpene (or sesquiterpene) by suitable separation techniques known in the art, such as preparative chromatography, (fractional) distillation, or (fractional) crystallization.

[0051] The present invention also provides a method of using monoterpene (or sesquiterpene) derivatives to treat diseases such as cancer or other nervous system disorders. The monoterpene (or sesquiterpene) derivatives can be administered alone or in combination with radiation, surgery or chemotherapy. The monoterpene or sesquiterpene derivatives can also be co-administered with antiviral, anti-inflammatory or antibiotic agents. The agents can be administered simultaneously or sequentially. The monoterpene (or sesquiterpene) derivatives can be administered before, during or after the administration of other active agents.

[0052] Monoterpene or sesquiterpene derivatives can be used in combination with radiation therapy. In one embodiment, the present invention provides a method of treating tumor cells, such as malignant glioma cells, with radiation, by treating the cells with an effective amount of monoterpene derivatives, such as perillyl alcohol carbamate, and then exposing them to radiation. Monoterpene derivative treatment can be before, during and / or after radiation. For example, monoterpene or sesquiterpene derivatives can be administered continuously starting one week before the start of radiation therapy and continuing for two weeks after the completion of radiation therapy. U.S. Patent Nos. 5,587,402 and 5,602,184.

[0053] In one embodiment, the present invention provides a method of treating tumor cells, such as malignant glioma cells, with chemotherapy, by treating the cells with an effective amount of a monoterpene derivative, such as perillyl alcohol carbamate, and then exposing the cells to chemotherapy. The monoterpene derivative treatment can be before, during and / or after chemotherapy.

[0054] Monoterpene (or sesquiterpene) derivatives can be used to treat nervous system cancers, such as malignant gliomas (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma multiforme), retinoblastoma, pilocytic astrocytoma (grade I), meningioma, metastatic brain tumors, neuroblastoma, pituitary adenoma, skull base meningioma, and skull base cancer. As used herein, the term "nervous system tumor" refers to a condition in which a subject has a malignant proliferation of nervous system cells.

[0055] Cancers that may be treated with the monoterpene (or sesquiterpene) derivatives of the present invention include lung cancer, ear, nose and throat cancer, leukemia, colon cancer, melanoma, pancreatic cancer, breast cancer, prostate cancer, breast cancer, hematopoietic tumors, ovarian cancer, basal cell carcinoma, biliary tract cancer, bladder cancer; bone cancer; breast cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; intraepithelial neoplasia; kidney cancer; laryngeal cancer; acute myeloid leukemia, acute lymphocytic leukemia. leukemias, including chronic myeloid leukemia, chronic lymphocytic leukemia; liver cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; myeloma; fibroma, neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; kidney cancer; respiratory system cancer; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; urinary system cancer, and other carcinomas and sarcomas. U.S. Patent No. 7,601,355.

[0056] The compositions of the present invention have been demonstrated to exhibit their activity in the prevention and treatment of skin diseases and disorders. In some embodiments, these compositions have been demonstrated to prevent and treat skin damage caused by UV radiation. The skin damage caused by UV radiation can be selected from the group consisting of atrophy, pigmentation changes, wrinkles, and malignant tumors. The malignant tumor can be skin cancer. The three most common types of skin cancer are basal cell carcinoma, squamous cell carcinoma, and malignant melanoma. D'Orazio, J., et al., Int. J. Mol. Sci., 2013 Jun 7; 14(6): 12222-48. A portion of the solar spectrum includes wavelengths of electromagnetic energy ranging from approximately 290 to 3,000 nanometers (nm). This range can be divided into various regions: (1) the ultraviolet region (290-400 nm), (2) the visible region (400-760 nm), and (3) the near-infrared region (>760 nm). The ultraviolet region is further divided into three bands, arbitrarily referred to as the UVA, UVB, and UVC bands.

[0057] The UVB band extends from 290 nm to 320 nm. It is the primary cause of sunburn and is also the most effective in stimulating pigmentation reactions in the skin. Although UVC radiation from the sun (200-280 nm) does not reach the surface of the Earth, radiation in this range may be encountered from artificial sources such as germicidal lamps and high and low pressure mercury arc lamps. However, for the purposes of this invention, protection against UVC radiation is generally not of great concern, i.e., in contrast to the dangers posed by UVA and UVB radiation. The UVA band, which extends from 320 to 400 nm, can also cause pigmentation reactions. UVA radiation can also cause sunburn, but to a lesser extent than UVB radiation.

[0058] However, the amount of UVA radiation exposure is increasing. This is due to the fact that most sunscreens only effectively block UVB radiation. As mentioned above, UVB radiation has a greater ability to cause pigmentation and burn reactions than UVA radiation. Therefore, if a person is using a sunscreen that blocks UVB radiation, they tend to stay under the sun for longer periods of time as the immediate effects of sunburn / burn do not appear. The problem is that UVA penetrates further into the skin and although it does not cause immediately obvious effects, it does cause long-term damage. In recent years, it has been well documented that UVA radiation is harmful to the skin, as is UVB radiation. In fact, the latest data reveals that solar radiation containing these wavelengths (A and B) is currently a contributing cause of skin cancer, accounting for 30-40% of all new cancers each year. In the United States alone, 500,000 new cases of skin cancer will be reported this year, and the number is expected to continue to rise. UVA radiation has been shown to promote skin cancer by inhibiting enzymes that repair cells damaged by UVB radiation. UVA radiation also enhances the damage caused by UVB rays, as it penetrates the skin more deeply than UVB radiation, causing vascular changes and premature aging of the skin (see, e.g., Hurwitz, Sidney, "The Sun and Sunscreen Protection: Recommendations for Children," Dermatol Surg. Oncol; 14:6 (June 1988) p. 657). Thus, the goal of a sunscreen should be to protect the user from both UVA and UVB radiation with minimal side effects. This objective is not adequately achieved with the use of currently available sunscreen products.

[0059] Topical sunscreen products can be divided into two broad categories: organic and inorganic (physical) sunscreens.

[0060] Commercially available sunscreen products contain from about 3 to about 26% of one or more UV-absorbing chemicals. When applied to the surface of the skin as a thin film, i.e., about 10 to 15 μm thick, these chemicals act as filters, reducing the penetration of UV radiation to the cells of the epidermis. These sunscreens are typically applied in cream, oil, lotion, alcohol or gel vehicles and are usually colorless because they do not contain any visible light-absorbing chemicals.

[0061] Figures 21A-D show the induction of skin inflammation by daily dermal application of a solution of 12-0-tetradecanoylphorbol-13-acetate in acetone (asTPA) (asTPA) to the dorsum of all SKH-1 hairless mice, except for the control group, for 7 consecutive days. For this purpose, animals were anesthetized by inhalation of isoflurane and 100 μL of asTPA (corresponding to 20 μg of TPA) was applied. As soon as the TPA evaporated, the mice were returned to their cages. Mice were then treated with 10 mg / kg of freshly prepared POH-LA conjugates every day. POH-LA conjugates were formulated in glycerol:ethanol (90:10), and control animals received only the glycerol:ethanol vehicle. 50 μL of the POH-LA conjugate formulation or vehicle was applied to the rump flank of the mice over a 3-day pretreatment period. After these 3 days, asTPA was applied for 7 days along with continued application of POH-LA conjugate formulation and vehicle. Mice were photographed at the end of the 10 days. In Figures 21A and 21B, TPA was used in the glycerol:ethanol vehicle alone, whereas in Figures 21C and 21D, TPA was used with POH-LA conjugate formulation.

[0062] Figures 22A-E show prevention of UV-induced skin inflammation and damage. Subject mice were irradiated with a short-wavelength UV lamp (VWR, Radnor, PA). The peak spectral output of this lamp was approximately 310 nm, with no detectable energy below 260 nm, approximately 0.6% at 260-280 nm (UV-C), 72.7% at 280-320 nm (UV-B), and 26.7% at 320-400 nm (UV-A). The UV-B radiation dose per exposure was 54 mJ / cm in the first week, as confirmed by a UV monitor. 2 , and 72 mJ / cm in the second week. 2 , and 90 mJ / cm in the third week. 2 , and 108 mJ / cm for the rest of the week. 2 was set to.

[0063] SKH-1 hairless mice were treated with 10 mg / kg of POH-LA conjugates, freshly prepared daily. POH-LA conjugates were formulated in glycerol:ethanol (90:10), and control animals received either the glycerol:ethanol vehicle alone or a commercial sunscreen (Well&Good Sun Protection Wipes SPF 15). Additional test groups were treated with POH formulated in a glycerol:ethanol vehicle, or linoleic acid formulated in a glycerol:ethanol vehicle. 50 μL of each formulation was applied to an area of ​​1 cm exposed to UV light. 2The mice were treated with the respective formulations after exposure to UV light. In FIG. 22A, the mice were treated with sunscreen. In FIG. 22B, the mice were treated with POH. In FIG. 22C, the mice were treated with linoleic acid. In FIG. 22D, the mice were treated with POH-LA conjugates. In FIG. 22D, the mice were treated with glycerol:ethanol vehicle only. POH-LA conjugates, POH or linoleic acid solutions were gently spread onto the skin surface and allowed to air dry. Sunscreened mice had the exposed area gently wiped with a sunscreen towelette. This exposure and treatment continued for up to 10 weeks. The animals were photographed and weighed throughout the experimental period. At the end of the experimental period, the mice were photographed and euthanized before skin and blood collection. Blood was analyzed for markers of inflammation and DNA was collected from the exposed areas of the skin and analyzed for markers of DNA damage.

[0064] Figure 23 shows a simulation of sunlight with irradiation by a UVB lamp (310 nm). Irradiation was performed in a phototoxicity test chamber fitted with a UV-B lamp providing a spectral output of approximately 310 nm. The source of UVR was a short-wave UV lamp (VWR, Radnor, PA). The peak spectral output of this lamp was approximately 310 nm, with no detectable energy below 260 nm, approximately 0.6% at 260-280 nm (UV-C), 72.7% at 280-320 nm (UV-B), and 26.7% at 320-400 nm (UV-A). The UV-B radiation dose per exposure was 54 mJ / cm in the first week, as confirmed by a UV monitor. 2 , and 72 mJ / cm in the second week. 2 , and 90 mJ / cm in the third week. 2 , and 108 mJ / cm for the rest of the week. 2 Each irradiation event was 10 min long.

[0065] POH-LA conjugates, linoleic acid, POH, and additive combinations of linoleic acid and POH were each suspended in glycerol:ethanol (90:10 vol / vol) in 50 μL to a final concentration of 10 nM and applied to each SKH-1 hairless mouse after UV-B exposure for treatment. Sunscreen mice were gently wiped on the exposed area with a sunscreen towelette. Figure 23A shows a mouse before irradiation. In Figure 23B, the mouse was treated with glycerol:ethanol vehicle only. In Figure 23C, the mouse was treated with POH. In Figure 23D, the mouse was treated with linoleic acid. In Figure 23E, the mouse was treated with a mixture of linoleic acid and POH. In Figure 23F, the mouse was treated with POH-LA conjugates. In Figure 23G, the mouse was treated with sunscreen. Application of POH-LA conjugates to UV-B exposed tissues limited the amount of observable skin damage compared to untreated controls (B) or application of the individual components of the POH-LA conjugates alone (POH-C and LA-D), mixed together (E), or with sunscreen (G). In all exposed and treated groups except for the POH-LA conjugate (F), the skin exhibited dry reddened skin, often with damage, 5 weeks after exposure and treatment. Linoleic acid, POH, and the linoleic acid-POH mixture all showed damage. Meanwhile, the skin of POH-LA conjugate exposed and treated mice looked similar to the skin of unexposed control mice.

[0066] The mice on the linoleic acid-POH mixture were tested consecutively compared to the rest, and therefore to the different backgrounds of the photographs. However, dry and reddened skin is clearly observed even in this group.

[0067] Figures 24A-D show a simulation of sunlight with irradiation by a UVB lamp (310 nm). Irradiation was performed in a phototoxicity test chamber fitted with a UV-B lamp providing a spectral output of approximately 310 nm. The source of UVR was a short wavelength UV lamp (VWR, Radnor, PA). The peak spectral output of this lamp was approximately 310 nm with no detectable energy below 260 nm, approximately 0.6% at 260-280 nm (UV-C), 72.7% at 280-320 nm (UV-B), and 26.7% at 320-400 nm (UV-A). The UV-B radiation dose per exposure was 54 mJ / cm in the first week as confirmed by a UV monitor. 2 , and 72 mJ / cm in the second week. 2 , and 90 mJ / cm in the third week. 2 was set to.

[0068] POH-LA conjugates (Figure 24A), linoleic acid (Figure 24B), POH (Figure 24C), and additive combinations of linoleic acid and POH (Figure 24D) were each suspended in glycerol:ethanol (90:10 vol / vol) in 50 μL to a final concentration of 10 nM and applied to each mouse after UV-B exposure for treatment. Application of POH-LA conjugates to UV-B exposed tissues limits the amount of observable skin damage compared to the individual components of POH-LA conjugates alone or in mixtures. In all exposed and treated groups except for POH-LA conjugates, the skin exhibited dry reddened skin three weeks after exposure and treatment, often with damage. Linoleic acid, POH, and the mixture of linoleic acid-POH all showed damage. Meanwhile, the skin of POH-LA conjugates in exposed and treated mice showed no damage.

[0069] The present invention also provides a method for treating CNS disorders, including primary degenerative neurological disorders, such as, but not limited to, Alzheimer's disease, Parkinson's disease, psychological disorders, psychosis, and depression. Treatment can consist of using monoterpene or sesquiterpene derivatives alone or in combination with current drugs used to treat Parkinson's disease, Alzheimer's disease, or psychological disorders.

[0070] The present invention also provides a method of improving an immunomodulatory therapy response, comprising exposing cells to an effective amount of a monoterpene or sesquiterpene derivative, such as perillyl alcohol carbamate, before or during immunomodulatory therapy. Preferred immunomodulatory agents are cytokines, such as interleukins, lymphokines, monokines, interferons and chemokines.

[0071] The compositions may be administered by any method known in the art, including, but not limited to, 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, sublingual, mucosal, ocular, spinal, intrathecal, intraarticular, intraarterial, intrathecal, bronchial and lymphatic administration. Topical formulations may be in the form of gels, ointments, creams, aerosols, foams, and the like. Intranasal formulations may be delivered as a spray or in drops. Transdermal formulations may be administered via transdermal patch or iontophoresis. Inhalation formulations may be delivered using a nebulizer or similar device. The compositions may also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other suitable compositions.

[0072] Figures 25A-C show changes in cytokine levels after UV exposure with and without POH-LA conjugate treatment. Compared to unexposed control mice (not shown), serum levels of IL-1β (Figure 25A), IL-6 (Figure 25B) and TNF-α (Figure 25C) were significantly increased in all light-exposed groups, both treated and untreated. For IL-1β, POH-LA conjugates produced the lowest increase in serum levels (6.4±2.5 pg / mL), while untreated animals had the highest increase (10.4±3.1 pg / mL), with both linoleic acid and POH-treated animals showing intermediate levels (8.3±2.1 pg / mL and 8.5±3.0 pg / mL). None of the differences between these three groups were significant. IL-6 levels remained significantly lower in POH-LA conjugate treated animals (iv group, 15.2±4.1pg / mL) compared to the untreated group (24.6±3.2pg / mL), while intermediate levels were observed in both the linoleic acid group (18.3±3.4pg / mL) and the POH treated group (17.9±2.8pg / mL). Similarly, TNF-α levels were significantly lower in the POH-LA conjugate (72.6±22.8pg / mL) compared to the linoleic acid group (126.9±14.6pg / mL) and the untreated group (157.7±22.8pg / mL), while the POH group animals had intermediate levels (103.2±18.0pg / mL).

[0073] Figures 26A and 26B show DNA damage resulting after UV exposure with and without POH-LA conjugate treatment. After the animals were euthanized, skin tissue was collected from the back of the animals, fixed in formalin, and processed for analysis. Absorption of ultraviolet (UV) light results in two major types of DNA damage, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidine photoproducts (6-4PPs). A UV DNA damage standard was used for comparison with UV-exposed mouse skin-extracted DNA. Both standard and sample DNAs were first heat denatured and then adsorbed to a 96-well DNA high-binding plate. 6-4PP (Figure 26A) and CPD (Figure 26B) present in the samples or standards are probed with anti-6-4PP or CPD antibodies followed by HRP-conjugated secondary antibodies. The 6-4PP or CPD content in unknown samples is determined by comparison with a standard curve prepared from predetermined CPD-DNA standards ranging from 1,000 to 1.56 ng / ml. As is evident, there was less DNA damage in POH-LA conjugate-treated mice. The skin has important barrier, sensory and immune functions and contributes to the health and integrity of the organism. Extensive skin damage that threatens the entire organism requires immediate and effective treatment. Wound healing is a natural response, but in severe conditions such as burns and diabetes, this process is insufficient and effective treatment cannot be achieved. Epidermal stem cells (EPSCs) are a multipotent cell type that are dedicated to the formation and differentiation of a functional epidermis.

[0074] POH-LA conjugates have been shown to enhance the activation of cellular proteins. In addition, POH-LA conjugates have been shown to stimulate skin regeneration via stem cell activation.

[0075] Activins are members of the transforming growth factor-β family of growth and differentiation factors. The biological activity of activins is regulated by the secreted glycoprotein follistatin, which captures activins and thus inhibits their biological activity. Follistatin controls keratinocyte proliferation in skin wounds. Most importantly, it has been shown that limiting the activation of activins in keratinocytes is beneficial to the wound healing process. Figures 27A-C show the staining of skin with POH-LA conjugates (Figure 27A), POH, (Figure 27B) and linoleic acid (Figure 27C).

[0076] α6β4 integrin is a component of hemidesmosomes and binds laminin 322 (previously called laminin 5) in the basement membrane. α6β4 integrin in epithelial cells plays an essential role in strengthening and stabilizing skin tissue through the formation of hemidesmosomes. Figures 27D-F show the staining of skin with POH-LA conjugates (Figure 27D), POH (Figure 27E) and linoleic acid (Figure 27F) by activation of α6β4 integrin.

[0077] Yap1 is a key modulator of epidermal stem cell proliferation and tissue expansion. α-catenin controls Yap1 activity and phosphorylation by regulating its interaction with 14-3-3 and PP2A phosphatases. This helps identify Yap1 as a determinant of epidermal stem cell proliferation potential and as a key effector of the "crowd control" molecular circuitry in mammalian skin. Figure 27G-I show the staining of skin with POH-LA conjugates (Figure 27G), POH (Figure 27H) and linoleic acid (Figure 27I) for Yap1 activation.

[0078] POH-LA conjugates have been shown to enhance the activation of cellular proteins. In addition, POH-LA conjugates have been shown to stimulate skin regeneration via stem cell activation.

[0079] To prepare such pharmaceutical compositions, one or more monoterpene (or sesquiterpene) derivatives can be mixed with pharma- ceutically acceptable carriers, adjuvants and / or excipients according to conventional pharmaceutical compounding techniques. Pharmaceutically acceptable carriers that can be used in the compositions of the present invention include standard pharmaceutical carriers, such as phosphate buffered saline, water, and emulsions, such as oil / water emulsions or water / oil emulsions, and any of a variety of wetting agents. The compositions can further 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 semi-solid excipients can be selected from glycerol, propylene glycol, water, ethanol, and a variety of oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Liquid carriers, particularly for injections, include water, saline, aqueous dextrose, and glycols.For examples of carriers, stabilizers, and adjuvants, see Remington's Pharmaceutical Sciences, edited by EW Martin (Mack Publishing Company, 18th Edition, 1990).The composition can also include stabilizers and preservatives.

[0080] As used herein, the term "therapeutically effective amount" is an amount sufficient to treat a particular disorder or disease or to obtain a pharmacological response that treats a disorder or disease. Methods for determining the most effective means of administration and dosages may vary depending on the composition used in the therapy, the purpose of the therapy, the target cells being treated, and the subject being treated. Therapeutic dosages may generally be titrated to optimize safety and efficacy. Single or multiple administrations may be administered, with the dosage level and pattern selected by the treating physician. Appropriate dosage formulations and methods of administering the agent can be readily determined by one of skill in the art. For example, the composition is 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. When the compounds described herein are co-administered with another agent or therapy, the effective amount may be less than when the agent is used alone.

[0081] Transdermal formulations can be prepared by incorporating the active agent into a thixotropic or gelatinous carrier, such as a cellulose medium, for example, methylcellulose or hydroxyethylcellulose, and then the resulting formulation is packaged in a transdermal device adapted to be fixed against the skin of the user. When the composition is in the form of a gel, the composition can be rubbed onto the patient's membrane, for example, the skin of the shoulder or upper arm and / or upper torso, preferably intact, clean and dry skin, and maintained thereon for a period of time sufficient to deliver the monoterpene (or sesquiterpene) derivative to the patient's serum. The composition of the present invention in the form of a gel can be included in a tube, sachet, metered pump. Such a tube or sachet can contain one unit dose or two or more unit doses of the composition. The metered pump can be capable of dispensing one metered dose of the composition.

[0082] The present invention also provides the above composition for intranasal administration. Thus, the composition can further include a permeation enhancer. Southall et al.Developments in Nasal Drug Delivery, 2000. The monoterpene (or sesquiterpene) derivative can be administered intranasally in liquid form, such as a solution, emulsion, suspension, drops, or in solid form, such as a powder, gel, or ointment. Devices for delivering intranasal drugs are well known in the art. Nasal drug delivery can be performed 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 (MDIs), pressurized inhalers, insufflators, and bidirectional devices. The nasal delivery device can be metered to administer an accurate effective dose to the nasal cavity. The nasal delivery device can be for single unit delivery or multiple unit delivery. In a particular example, the ViaNase Electronic Atomizer from Kurve Technology (Bethell, Washington) can be used in the present invention (http: / / www.kurvetech.com). The compounds of the present invention can also be delivered by tube, catheter, syringe, packtail, cotton ball, nasal tampon or submucosal injection. US Patent Publication Nos. 20090326275, 20090291894, 20090281522 and 20090317377.

[0083] The monoterpene (or sesquiterpene) derivatives can be formulated as aerosols using standard procedures. The monoterpene (or sesquiterpene) derivatives can be formulated with or without a solvent, and with or without a carrier. The formulation can be a solution or an aqueous emulsion containing one or more surfactants. For example, an aerosol spray can be generated from a pressurized container containing a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrocarbon, compressed air, nitrogen, carbon dioxide, or other suitable gas. The dosage unit can be determined by providing a valve to deliver a metered amount. A pump spray dispenser can dispense a metered dose or a dose having a specific particle or droplet size. As used herein, the term "aerosol" refers to a suspension of fine solid particles or droplets of a liquid solution in a gas. Specifically, an aerosol includes a gas-borne suspension of droplets of monoterpene (or sesquiterpene), such as may be generated in any suitable device, such as an MDI, a nebulizer, or a mist sprayer. Aerosol also includes dry powder compositions of the compositions of the invention suspended in air or other carrier gas. Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313. Raeburn et al., (1992) Pharmacol. Toxicol. Methods 27:143-159. The monoterpene (or sesquiterpene) derivative may be delivered to the nasal cavity as a powder in the form of microspheres delivered by a nasal insufflator. The monoterpene (or sesquiterpene) derivative may be absorbed onto a solid surface, such as a carrier. The powder or microsphere may be administered in a dry, air-free form. The powder or microsphere may be stored in the container of the insufflator. Alternatively, the powder or microsphere may be filled into a capsule, such as a gelatin capsule, or other single-dose unit adapted for nasal administration.

[0084] The pharmaceutical composition can be delivered to the nasal cavity by placing the composition directly in the nasal cavity, for example, in the form of a gel, ointment, nasal emulsion, lotion, cream, nasal tampon, dropper, or bioadhesive strip. In certain embodiments, it may be desirable to extend the residence time of the pharmaceutical composition in the nasal cavity, for example, to promote absorption. Thus, the pharmaceutical composition can be optionally formulated with bioadhesive polymers, gums (e.g., xanthan gum), chitosan (e.g., highly purified cationic polysaccharides), pectin (or any carbohydrate that thickens like a gel or emulsifies when applied to the nasal mucosa), microspheres (e.g., starch, albumin, dextran, cyclodextrin), gelatin, liposomes, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and / or cellulose (e.g., methyl or propyl; hydroxyl or carboxy; carboxymethyl or hydroxypropyl).

[0085] Compositions containing purified monoterpenes (or sesquiterpenes) can be administered by oral inhalation to the airways, ie, the lungs.

[0086] Typical delivery systems for inhalable medications include nebulizer inhalers, dry powder inhalers (DPIs), and metered dose inhalers (MDIs).

[0087] Nebulizer devices generate a high velocity air stream that causes the therapeutic agent in liquid form to spray as a mist. The therapeutic agent is formulated in liquid form, such as a solution or suspension of particles of appropriate size. In one embodiment, the particles are micronized. The term "micronized" is defined as about 90% or more of the particles have a diameter of less than about 10 μm. Suitable nebulizer devices are commercially available, for example, from PARI GmbH (Starnberg, Germany). Other nebulizer devices include Respimat (Boehringer Ingelheim) and those disclosed, for example, in U.S. Patent Nos. 7,568,480 and 6,123,068, and WO 97 / 12687. Monoterpenes (or sesquiterpenes) can be formulated as aqueous solutions or liquid suspensions for use in nebulizer devices.

[0088] DPI devices typically administer therapeutic agents in the form of a free-flowing powder that can be dispersed in the patient's airstream during inspiration. DPI devices that use an external energy source can also be used in the present invention. To achieve a free-flowing powder, the therapeutic agent can be formulated with a suitable excipient (e.g., lactose). A dry powder formulation can be made, for example, by combining dry lactose having a particle size of about 1 μm to 100 μm with micronized particles of a monoterpene (or sesquiterpene) and dry blending. Alternatively, the monoterpene can be formulated without excipients. The formulation is loaded into a dry powder dispenser or into an inhalation cartridge or capsule for use in a dry powder delivery device. Examples of commercially available DPI devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (see, e.g., U.S. Pat. No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378,519); Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. No. 4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat. No. 4,353,365). Further examples of suitable DPI devices are described in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810, and references therein.

[0089] MDI devices typically release a measured amount of therapeutic agent using compressed propellant gas. Formulations for MDI administration include a solution or suspension of the active ingredient in a liquefied propellant. Examples of propellants include hydrofluoroalkanes (HFAs), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane (HFA 227), and chlorofluorocarbons, such as CCl3F. Additional components of HFA formulations for MDI administration include cosolvents, such as ethanol, pentane, water; and surfactants, such as sorbitan trioleate, oleic acid, lecithin, and glycerin. (See, e.g., U.S. Pat. No. 5,225,183, European Patent No. 0717987, and International Publication No. WO 92 / 22286.) The formulation is loaded into an aerosol canister that forms part of the MDI device. Examples of MDI devices specifically developed for use with HFA propellants are provided in U.S. Patent Nos. 6,006,745 and 6,143,227. For examples of processes for preparing suitable formulations and devices suitable for inhalation administration, see U.S. Patent Nos. 6,268,533, 5,983,956, 5,874,063 and 6,221,398, as well as WO 99 / 53901, WO 00 / 61108, WO 99 / 55319 and WO 00 / 30614.

[0090] Monoterpene (or sesquiterpene) derivatives can be encapsulated in liposomes or microcapsules for delivery by inhalation. Liposomes are vesicles composed of a lipid bilayer membrane and an aqueous interior. The lipid membrane can be made of phospholipids, examples of which include phosphatidylcholines, such as lecithin and lysolecithin; acidic phospholipids, such as phosphatidylserine and phosphatidylglycerol; and sphingophospholipids, such as phosphatidylethanolamine and sphingomyelin. Alternatively, cholesterol may be added. Microcapsules are particles coated with a coating material. For example, the coating material may consist of a mixture of film-forming polymers, hydrophobic plasticizers, surface-activating agents, or / and lubricating nitrogen-containing polymers. U.S. Patent Nos. 6,313,176 and 7,563,768.

[0091] The monoterpene (or sesquiterpene) derivatives 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 melanoma. The monoterpene (or sesquiterpene) derivatives may also be used in combination with anesthetics or analgesics for transdermal delivery of analgesics.

[0092] The present invention also provides the above composition for ocular administration.Accordingly, the composition can further comprise a permeation enhancer.For ocular administration, the composition described herein can be formulated as a solution, emulsion, suspension, etc.Various 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; 5,800,807; 5,776,445; 5,698,219; 5,521,222; 5,403,841; 5,077,033; 4,882,150; and 4,738,851.

[0093] The monoterpene (or sesquiterpene) derivatives can be administered alone or in combination with other drugs for short or long term treatment of the above diseases.The composition can be administered to mammals, preferably humans.Mammals include, but are not limited to, mice, rats, rabbits, monkeys, cows, sheep, pigs, dogs, cats, livestock, sport animals, pets, horses and primates.

[0094] The present invention also provides a method of inhibiting cell proliferation in vitro, ex vivo or in vivo, comprising contacting a cell, such as a cancer cell, with an effective amount of a monoterpene (or sesquiterpene) derivative described herein.

[0095] Pathological cells or tissues, such as hyperproliferative cells or tissues, can be treated by contacting the cells or tissues with an effective amount of the composition of the present invention. The cells, such as cancer cells, can be primary cancer cells or cultured cells available from tissue banks, such as American Type Culture Collection (ATCC). The pathological cells can be cells of systemic cancer, glioma, meningioma, pituitary adenoma, or CNS metastases from systemic cancer, lung cancer, prostate cancer, breast cancer, hematopoietic tumors, or ovarian cancer. The cells can be from a vertebrate, preferably a mammal, more preferably a human. U.S. Patent Publication No. 2004 / 0087651. Balassiano et al. (2002) Intern. J. Mol. Med. 10: 785-788. Thorne, et al. (2004) Neuroscience 127: 481-496. Fernandes, et al. (2005) Oncology Reports 13: 943-947. Da Fonseca, et al. (2008) Surgical Neurology 70:259267. Da Fonseca, et al. (2008) Arch. Immunol. Ther. Exp. 56:267-276. Hashizume, et al. (2008) Neuroncology 10:112-120. The in vitro efficacy of the composition can be determined using methods well known in the art. For example, the cytotoxicity of the monoterpene (or sesquiterpene) derivatives of the present invention and / or therapeutic agents can be tested by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cytotoxicity assay. The MTT assay is based on the principle of uptake by metabolically active cells of MTT, a tetrazolium salt, which is metabolized into a blue formazan product that can be read spectrophotometrically. J. of Immunological Methods 65:55 63, 1983. The cytotoxicity of the monoterpene (or sesquiterpene) derivatives of the present invention and / or therapeutic agents can be investigated by colony formation assay. Functional assays for inhibition of VEGF secretion and IL-8 secretion can be performed by ELISA. The cell cycle blockade by the monoterpene (or sesquiterpene) derivatives of the present invention and / or therapeutic agents can be investigated by standard propidium iodide (PI) staining and flow cytometry. Invasion inhibition can be investigated by Boyden chamber. In this assay, a layer of reconstituted basement membrane, Matrigel, is coated onto a chemotactic filter and acts as a barrier to the migration of cells in the Boyden chamber. Only cells with invasive potential can pass through the Matrigel barrier. Other assays include, but are not limited to, cell viability assays, apoptosis assays, and morphological assays.

[0096] The following are examples of the present invention and should not be construed as limiting.

[0097] [Example] [Example 1] Dimethyl celecoxib bis POH carbamate (4-(bis-N,N'-4-isopropenylcyclohex-1-enylmethyloxycarbonyl[5-(2,5-dimethylphenyl)-3-trifluoromethylpyrazol-1-yl]benzenesulfonamide) The reaction scheme is as follows: [ka] Phosgene (20% in toluene, 13 ml, 26.2 mmol) was added over 30 minutes 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) while maintaining the temperature at 10°C to 15°C. The reaction mixture was warmed to room temperature and stirred under N2 for 8.0 hours. The reaction mixture was quenched with water (30 mL) and the organic layer was separated. The aqueous layer was extracted with toluene (20 mL) and the combined organic layers were washed with water (50 mL x 2), brine (15%, 30 mL) and dried over sodium sulfate (20 grams). The filtered organic layer was concentrated under vacuum to give perillyl chloroformate as an oil. Weight: 2.5 grams; Yield: 89%. 1H-NMR (400MHz, CDCl3): δ 1.5(m,1H),1.7(s,3H),1.8(m,1H),2.0(m,1H),2.2(m,4H),4.7(dd,4H);5.87(m,1H). Perillyl chloroformate (0.11 grams, 0.55 mmol) was added slowly over 5 minutes under N2 to a mixture of dimethylcelecoxib (0.2 grams, 0.50 mmol) and potassium carbonate (0.13 grams, 1.0 mmol) in dry acetone (10 mL). The reaction mixture was heated to reflux and maintained for 3 hours. TLC analysis indicated the presence of dimethylcelecoxib (>60%) so an additional 1.0 equivalent of perillyl chloroformate was added and refluxed for an additional 5 hours. The reaction mixture was cooled and the acetone was concentrated under vacuum to give a residue.

[0098] The resulting residue was suspended in water (15 mL) and extracted with ethyl acetate (3×15 mL). The combined organic layers were washed with water (20 mL), followed by brine (15%, 20 mL), and dried over sodium sulfate. The filtered organic layer was concentrated under vacuum to give a residue which was purified by column chromatography [column dimensions: diameter 1.5 cm, height 10 cm, silica 230-400 mesh], eluted with hexane (100 mL) followed by a mixture of hexane / ethyl acetate (95:5, 100 mL). The hexane / ethyl acetate fractions were combined and concentrated under vacuum to give a gummy mass.

[0099] The product POH carbamate had a weight of 120 mg and a yield of 31%. 1H-NMR (400 MHz, CDCl3): δ 0.9(m, 2H), 1.4(m, 2H), 1.7(m, 7H*), 1.95(m, 8H*), 2.1(m, 4H), 2.3(s, 3H), 4.4(d, 2H), 4.7(dd, 2H), 5.6(br d, 2H), 6.6(s, 1H), 7.0(br s, 1H), 7.12(d, 1H), 7.19(d, 1H), 7.4(d, 2H), 7.85(d, 2H); MS, m / e: 751.8(M+3%), 574.3(100%), 530.5(45%), 396(6%). *Additional 2H overlap from putative impurity subtracted in NBNMR integral.

[0100] [Example 2] In vitro cytotoxicity study of dimethylcelecoxib bisPOH carbamate (POH-DMC) The first cytotoxicity assay was performed after treating the cells with dimethyl-celecoxib (DMC) alone. Figure 1 shows the results of the MTT cytotoxicity assay performed on human malignant glioma cells U87, A172 and U251 with DMC alone.

[0101] U87, A172 and U251 cells were then treated with dimethylcelecoxib bisPOH carbamate (POH-DMC) (e.g., synthesized by the method of Example 1) and an MTT cytotoxicity assay was performed (Figure 2). The results suggest that the POH carbamate POH-DMC showed much better cytotoxicity than DMC alone.

[0102] [Example 3] Synthesis of Temozolomide POH Carbamate (3-Methyl 4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-carbamic acid-4-isopropenylcyclohex-1-enylmethyl ester) The reaction scheme is as follows: [ka] Oxalyl chloride (0.13 grams, 1.0 mmol) was added slowly over 2 minutes to a mixture of temozolomide (OChem Incorporation, 0.1 grams, 0.5 mmol) in 1,2-dichloroethane (10 mL) while maintaining the temperature at 10° C. under N2. The reaction mixture was allowed to warm to room temperature and then heated to reflux for 3 hours. Excess oxalyl chloride and 1,2-dichloroethane were removed by concentration under vacuum. The resulting residue was redissolved in 1,2-dichloroethane (15 mL) and the reaction mixture was cooled to 10° C. under N2. A solution of perillyl alcohol (0.086 grams, 0.56 mmol) in 1,2-dichloroethane (3 mL) was added over 5 minutes. The reaction mixture was allowed to warm to room temperature and stirred for 14 hours. The 1,2-dichloroethane was concentrated under vacuum to give a residue which was triturated with hexane. The resulting yellow solid was filtered and washed with hexane. Weight: 170 mg; Yield: 89%. 1H-NMR (400 MHz, CDCl3): δ 1.4-2.2 (m, 10H), 4.06 (s, 3H), 4.6-4.8 (m, 4H), 5.88 (br s, 1H), 8.42 (s, 1H), 9.31 (br s, 1H); MS, no molecular ion peak observed. m / e: 314 (100%), 286.5 (17%), 136 (12%).

[0103] Alternatively, temozolomide POH carbamate was synthesized according to the following procedure. To a mixture of temozolomide (OChem Incorporation, 0.1 grams, 0.5 mmol) in 1,2-dichloroethane (10 mL) was added oxalyl chloride (0.13 grams, 1.0 mmol) slowly over a period of 2 minutes while maintaining the temperature at 10° C. under N2. The reaction mixture was allowed to warm to room temperature and then heated to reflux for 3 hours. The excess oxalyl chloride and 1,2-dichloroethane were removed by concentration under vacuum. The resulting residue was redissolved in 1,2-dichloroethane (15 mL) and the reaction mixture was cooled to 10° C. under N2. A solution of perillyl alcohol (0.086 grams, 0.56 mmol) in 1,2-dichloroethane (3 mL) was added over a period of 5 minutes. The reaction mixture was allowed to warm to room temperature and stirred for 14 hours. 1,2-Dichloroethane was concentrated under vacuum to give a residue, which was purified by a short silica plug column (column dimensions: 2 cm diameter, 3 cm height, 230-400 mesh silica) and eluted with a mixture of hexane / 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 heptane and filtered to give a white solid. Weight: 170 mg; Yield: 89%. 1H-NMR (400 MHz, CDCl3): 1.4-2.2 (m, 10H), 4.06 (s, 3H), 4.6-4.8 (m, 4H), 5.88 (br s, 1H), 8.42 (s, 1H), 9.31 (br s, 1H); MS, no molecular ion peak was observed. m / e: 314 (100%), 286.5 (17%), 136 (12%).

[0104] [Example 4] In vitro cytotoxicity study of temozolomide POH carbamate (POH-TMZ) The first cytotoxicity assay was performed after treating the cells with temozolomide (TMZ) alone, a standard alkylating agent used in the treatment of malignant gliomas. Figure 3 shows the results of an MTT cytotoxicity assay performed on human malignant glioma cells U87, A172 and U251 with TMZ alone. Increasing concentrations of TMZ were minimally cytotoxic to the cell lines tested.

[0105] TMZ-resistant glioma cell lines U87, A172 and U251 cells were then treated with temozolomide POH carbamate (POH-TMZ) (e.g., synthesized by the method of Example 3). The results of the MTT assay (Figure 4) showed that POH carbamate POH-TMZ exhibited a substantial increase in the death rate of various human glioma cells compared to TMZ alone.

[0106] [Example 5] Synthesis of Rolipram POH Carbamate (4-(3-Cyclopentyloxy-4-methoxyphenyl)-2-oxopyrrolidine-1-carboxylic acid 4-isopropenylcyclohex-1-enylmethyl ester) The reaction scheme is as follows: [ka] Phosgene (20% in toluene, 13 ml, 26.2 mmol) was added over 30 minutes 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) while maintaining the temperature at 10°C to 15°C. The reaction mixture was warmed to room temperature and stirred under N2 for 8.0 hours. The reaction mixture was quenched with water (30 mL) and the organic layer was separated. The aqueous layer was extracted with toluene (20 mL) and the combined organic layers were washed with water (50 mL x 2), brine (15%, 30 mL) and dried over sodium sulfate (20 grams). The filtered organic layer was concentrated under vacuum to give perillyl chloroformate as an oil. Weight: 2.5 grams; Yield: 89%. 1H-NMR (400MHz, CDCl3): δ 1.5(m,1H),1.7(s,3H),1.8(m,1H),2.0(m,1H),2.2(m,4H),4.7(dd,4H);5.87(m,1H). Butyllithium (2.5M, 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 under N2 at -72°C over 5 minutes. The reaction mixture was stirred at -72°C for 1.0 hour, after which perillyl chloroformate (dissolved in 4 mL of THF) was added over 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 (2 x 15 mL). The combined organic layers were 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 [column dimensions: diameter 1.5 cm, height 10 cm, silica 230-400 mesh] eluted with a mixture of 8% ethyl acetate / hexane (100 mL) followed by 12% ethyl acetate / hexane (100 mL). The 12% ethyl acetate / hexane fractions were combined and concentrated under vacuum to give a gummy solid. Weight: 142 mg; Yield: 86%. 1H-NMR (400MHz, CDCl3):δ 1.5(m,1H),1.6(m,2H),1.7(s,3H),1.9(m,6H),2.2(m,5H),2.7(m,1H),2.9 (m,1H),3.5(m,1H),3.7(m,1H),3.8(s,3H),4.2(m,1H),4.7(m,6H),5.8(br s,1H),6.8(m,3H);MS,m / e:452.1(M+1 53%),274.1(100%),206.0(55%). [Example 6] In vitro cytotoxicity study of rolipram POH carbamate (POH-rolipram) To compare the cytotoxicity of Rolipram POH carbamate (POH-Rolipram) (e.g., synthesized by the method of Example 5) to Rolipram, a type IV phosphodiesterase that induces differentiation and apoptosis in glioma cells, A172, U87, U251 and LN229 human glioma cells were treated with either POH-Rolipram or Rolipram for 48 hours. The results of the MTT assays are shown in Figures 5-8. POH-Rolipram demonstrated substantially higher mortality rates compared to Rolipram alone for each of several different human glioma cell types. Figure 5 shows an MTT assay of increasing concentrations of Rolipram and POH-Rolipram against A-172 cells. Rolipram alone shows an IC50 of approximately 1000uM (1mM). In the presence of POH-Rolipram, the IC50 is achieved at concentrations as low as 50uM. Figure 6 shows an MTT assay of increasing concentrations of Rolipram using U-87 cells. The IC50 is not achieved at 1000uM. Meanwhile, the IC50 is achieved at 180uM with POH-Rolipram. Figure 7 shows that the IC50 of Rolipram alone against U251 cells is achieved at 170uM. Plateau cytotoxicity reaches 60%. POH-Rolipram achieves IC50 at 50uM, with nearly 100% cytotoxicity at 100uM. Figure 8 shows that the IC50 of Rolipram alone against LN229 cells is not achieved even at 100uM. Meanwhile, the IC50 of POH-Rolipram is achieved at 100uM, with nearly 100% cytotoxicity at 10uM.

[0107] [Example 7] In vivo tumor growth inhibition by POH fatty acid derivatives Inhibition of tumor growth by butyryl-POH was tested in a nude mouse subcutaneous glioma model. Mice were injected with U-87 glioma cells (500,000 cells / injection) and allowed to form palpable nodules over a period of 2 weeks. Once palpable nodules had formed, mice were treated for 8 weeks with topical application by Q-tip (1 cc / application / day) of various compounds as shown in Figure 9A and Figure 9B. Figure 9A shows images of subcutaneous U-87 gliomas in nude mice treated with butyryl-POH, purified (S)-perillyl alcohol with a purity of greater than 98.5% ("purified POH"), POH purchased from Sigma chemicals, or phosphate buffered saline (PBS; negative control). Figure 9B shows the average tumor growth over time (for a total period of 60 days). Butyryl-POH demonstrated the greatest inhibition of tumor growth, followed by purified POH and Sigma POH.

[0108] [Example 8] In vitro cytotoxicity study of temozolomide (TMZ) and temozolomide POH carbamate (POH-TMZ) in TMZ-sensitive and -resistant glioma cells Colony formation assays were performed after cells were treated with TMZ alone, POH alone, and TMZ-POH conjugate. Colony formation assays were performed 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 colony formation assays performed on TMZ-sensitive (U251) and TMZ-resistant (U251TR) U251 cells using TMZ or TMZ-POH. TMZ showed cytotoxicity to TMZ-sensitive U251 cells, but minimal cytotoxicity to TMZ-resistant U251 cells. TMZ-POH showed cytotoxicity to both TMZ-sensitive and TMZ-resistant U251 cells.

[0109] Figure 11 shows the results of colony formation assays performed on TMZ-sensitive (U251) and TMZ-resistant (U251TR) U251 cells using POH. POH showed cytotoxicity to both TMZ-sensitive and TMZ-resistant U251 cells. POH-TMZ (Figure 10) showed substantially greater potency in colony formation assays compared to POH alone (Figure 11).

[0110] [Example 9] In vitro cytotoxicity study of temozolomide POH carbamate (POH-TMZ) on U251 cells, U251TR cells, and normal astrocytes After cells were treated with TMZ-POH conjugate, MTT cytotoxicity assay was performed. MTT cytotoxicity assay was performed 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 MTT cytotoxicity assay performed on TMZ-sensitive cells (U251), TMZ-resistant cells (U251TR) and normal astrocytes. TMZ-POH showed cytotoxicity to both TMZ-sensitive and TMZ-resistant U251 cells, but not to normal astrocytes.

[0111] [Example 10] In vitro cytotoxicity study of temozolomide POH carbamate (POH-TMZ) on BECs, TuBECs, and normal astrocytes After cells were treated with TMZ-POH conjugates, MTT cytotoxicity assays were performed. MTT cytotoxicity assays were performed 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 MTT cytotoxicity assays performed on normal astrocytes, brain endothelial cells (BECs; confluent and subconfluent) and tumor brain endothelial cells (TuBECs). TMZ-POH did not induce significant cytotoxicity on normal astrocytes, confluent BECs or TuBECs. Mild to moderate cytotoxicity was demonstrated at high concentrations of TMZ-POH in subconfluent BECs.

[0112] [Example 11] In vitro cytotoxicity study of temozolomide (TMZ) and temozolomide POH carbamate (POH-TMZ) against USC-04 glioma cancer stem cells MTT cytotoxicity assays were performed after cells were treated with TMZ alone, POH alone, or TMZ-POH conjugate. MTT cytotoxicity assays were performed 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 MTT cytotoxicity assays performed on USC-04 glioma cancer stem cells. TMZ did not induce significant cytotoxicity at increasing concentrations (0-400uM). TMZ-POH showed evidence of cytotoxicity with IC50 at 150uM. Figure 15 shows the results of MTT cytotoxicity assays performed on USC-04 glioma cancer stem cells treated with POH. POH showed cytotoxicity against USC-04 at increasing concentrations (0-2mM).

[0113] [Example 12] In vitro cytotoxicity study of temozolomide (TMZ) and temozolomide POH carbamate (POH-TMZ) against USC-02 glioma cancer stem cells MTT cytotoxicity assays were performed after cells were treated with TMZ alone, POH alone, or TMZ-POH conjugate. MTT cytotoxicity assays were performed 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 MTT cytotoxicity assays performed on USC-02 glioma cancer stem cells. TMZ did not induce significant cytotoxicity at increasing concentrations (0-400uM). TMZ-POH showed evidence of cytotoxicity with IC50 at 60uM. Figure 17 shows the results of MTT cytotoxicity assays performed on USC-02 glioma cancer stem cells treated with POH. POH showed cytotoxicity against USC-02 at increasing concentrations (0-2mM).

[0114] [Example 13] In vitro study of ER stress induced by temozolomide POH carbamate (POH-TMZ) in TMZ-sensitive and -resistant glioma cells TMZ-sensitive and resistant glioma cells were treated with TMZ-POH conjugate for 18 hours, and then Western blot was performed. Figure 18 shows Western blot demonstrating that TMZ-POH induces ER stress (ERS) in TMZ-sensitive and TMZ-resistant U251 glioma cells. Activation of pro-apoptotic protein CHOP was shown at a concentration as low as 60uM of TMZ-POH.

[0115] [Example 14] Anti-inflammatory effect of POH-linoleic acid conjugate in mice A first group of mice was pretreated with 50 microliters (μL) of a solution of a conjugate of POH and linoleic acid (e.g., linoleoyl ester of POH) in a vehicle of a glycerol:ethanol (90:10) mixture. The POH-linoleic acid conjugate solution was applied to the rump flank of the mice for three days. A second control group of mice was treated with the glycerol:ethanol (90:10) vehicle alone.

[0116] After pretreatment, skin inflammation was induced in the first and second groups of mice by daily dermally applying a solution of 12-0-tetradecanoylphorbol-13-acetate (asTPA), a known irritant, to the backs of the mice for 7 days. An additional control group did not receive asTPA. To facilitate application of asTPA, the mice were anesthetized by inhalation of isofluorane. One hundred microliters of asTPA (corresponding to 20 micrograms (μg) of TPA) was then applied to the skin of the mice. Once the asTPA had evaporated, the mice were returned to their cages.

[0117] During the seven day period that the first and second groups of mice were subjected to application of asTPA, the first group continued to receive treatment with the POH-linoleic acid conjugate and vehicle solution, and the second group continued to receive treatment with the vehicle. At the end of the seven day period, the mice were photographed.

[0118] FIG. 19A is a photograph of the first mouse of the second group that received asTPA and vehicle but did not receive POH-linoleic acid conjugate treatment. FIG. 19B is a photograph of the second mouse of the second group that also received asTPA and vehicle but did not receive POH-linoleic acid conjugate treatment. As FIG. 19A and FIG. 19B show, the area of ​​skin on the right buttock of the mouse where asTPA was applied (1902, 1904, respectively) appears inflamed and dry. FIG. 20A is a photograph of the first mouse of the first group that received asTPA in combination with POH-linoleic acid conjugate / vehicle treatment. FIG. 20B is a photograph of the second mouse of the first group that also received asTPA in combination with POH-linoleic acid conjugate / vehicle treatment. As Figures 20A and 20B show, areas of skin on the right buttocks of mice to which asTPA was applied (2002, 2004, respectively) show no inflammation or peeling. The skin is relatively smooth at the application site and surrounding area, providing evidence of the effectiveness of the POH-linoleic acid conjugate / vehicle combination treatment compared to vehicle alone.

[0119] [Example 15] Anti-inflammatory effect of POH-linoleic acid conjugates in mice (alternative protocol) In an alternative protocol to test the efficacy of POH-linoleic acid as an anti-inflammatory agent, 15 mice are divided into five groups (i)-(v), each containing three mice. Groups (iii)-(v) receive pretreatment, while groups (i) and (ii) are negative and positive control groups. Group (iii) receives pretreatment with 10 mg / kg linoleic acid, group (iv) receives pretreatment with 10 mg / kg POH, and group (v) receives pretreatment with 10 mg / kg of a conjugate of linoleic acid with POH (e.g., linoleoyl ester of POH). All pretreatments last for three days.

[0120] Skin inflammation is induced by daily dermal application of an acetone solution of TPA (asTPA) to the dorsum of mice in groups (ii), (iii), (iv) and (v) for 7 consecutive days. Application of asTPA proceeds by restraining mice in groups (ii)-(v) in an adapted device featuring a skin-exposed opening, through which 100 microliters (μL) of asTPA (equivalent to 20 micrograms (μg) of TPA) is applied.

[0121] Treatment given to groups (iii), (iv) and (v) will continue for the 7 day asTPA application period.

[0122] Groups of mice are observed in a double-blind fashion by two separate individuals who do not know which mice are being treated. The extent and severity of dorsal skin inflammation is assessed to calculate an overall macroscopic score of skin inflammation.

[0123] A scoring system is implemented. A macroscopic score from 0 to 12 is assessed as an index of the degree of inflammation observed. A score of 0 indicates the absence of skin inflammation, a score of 1-3 indicates slight inflammation, a score of 4-6 indicates moderate inflammation, a score of 7-9 indicates significant inflammation, and a score of 10-12 indicates severe inflammation. Skin specimens are fixed and, after paraffin embedding, stored in a preservative for histopathological analysis by classical pathological methods.

[0124] Additionally, blood samples were collected from each mouse 4 hours after the last skin treatment. Approximately 0.5 mL of blood was collected by cardiac puncture into tubes containing citrate as an anticoagulant and centrifuged at 1500g for 15 minutes to collect serum. Serum was aliquoted into Eppendorf tubes and stored at -80°C in preparation for cytokine analysis. Serum samples were then thawed, after which three pro-inflammatory cytokines, IL-1, IL-6 and TNF, were assayed simultaneously with the Bio-Rad Mouse 3-Plex-A Panel Kit using Bio-Plex technology.

[0125] Figures 25A-C show changes in cytokine levels after UV exposure with and without POH-LA conjugate treatment. Compared to unexposed control mice (not shown), serum levels of IL-1β (Figure 25A), IL-6 (Figure 25B) and TNF-α (Figure 25C) were significantly increased in all light-exposed groups, both treated and untreated. For IL-1β, POH-LA conjugates produced the lowest increase in serum levels (6.4±2.5 pg / mL), while untreated animals had the highest increase (10.4±3.1 pg / mL), with both linoleic acid and POH-treated animals showing intermediate levels (8.3±2.1 pg / mL and 8.5±3.0 pg / mL). None of the differences between these three groups were significant. IL-6 levels remained significantly lower in POH-LA conjugate treated animals (iv group, 15.2±4.1pg / mL) compared to the untreated group (24.6±3.2pg / mL), while intermediate levels were observed in both the linoleic acid group (18.3±3.4pg / mL) and the POH treated group (17.9±2.8pg / mL). Similarly, TNF-α levels were significantly lower in the POH-LA conjugate (72.6±22.8pg / mL) compared to the linoleic acid group (126.9±14.6pg / mL) and the untreated group (157.7±22.8pg / mL), while the POH group animals had intermediate levels (103.2±18.0pg / mL).

[0126] [Example 16] A murine study to prevent chemically induced skin inflammation and damage by topical application of POH-LA conjugates in immunocompetent SKH-1 Elite hairless mice This example shows the induction of skin inflammation by daily dermal application of a solution of TPA in acetone (asTPA) to the back of all mice, except the control group, for 7 consecutive days. For this purpose, the animals were anesthetized by inhalation of isoflurane and 100 μL of asTPA (equivalent to 20 μg of TPA) was applied. As soon as the TPA evaporated, the mice were returned to their cages. The mice were then treated with 10 mg / kg of freshly prepared POH-LA conjugates every day. The POH-LA conjugates were formulated in glycerol:ethanol (90:10), while control animals received only the glycerol:ethanol vehicle. 50 μL of the POH-LA conjugate formulation or vehicle was applied to the rump flank of the mice for a pretreatment period of 3 days. After these 3 days, asTPA was applied for 7 days, along with continued application of the POH-LA conjugate formulation and vehicle. The mice were photographed at the end of the 10 days.

[0127] [Example 17] A murine study to prevent UV-induced skin inflammation and damage by topical application of POH-LA conjugates in immunocompetent SKH-1 Elite hairless mice Subjects were irradiated with a short-wavelength UV lamp (VWR, Radnor, PA). The peak spectral output of this lamp was approximately 310 nm, with no detectable energy below 260 nm, and approximately 0.6% at 260-280 nm (UV-C), 72.7% at 280-320 nm (UV-B), and 26.7% at 320-400 nm (UV-A). The UV-B radiation dose per exposure was 54 mJ / cm during the first week, as confirmed by a UV monitor. 2 , and 72 mJ / cm in the second week. 2 , and 90 mJ / cm in the third week. 2 , and 108 mJ / cm for the rest of the week. 2 was set to.

[0128] Mice were treated with 10 mg / kg of POH-LA conjugate prepared freshly daily. POH-LA conjugate was formulated in glycerol:ethanol (90:10) and control animals received either the glycerol:ethanol vehicle alone or a commercial sunscreen. Additional test groups were treated with POH formulated in a glycerol:ethanol vehicle or linoleic acid formulated in a glycerol:ethanol vehicle. 50 μL of each formulation was applied to a 1 cm2 area exposed to UV light. 2 The POH-LA conjugate, POH or linoleic acid solution was gently spread on the skin surface and allowed to air dry. Sunscreened mice had the exposed area gently wiped with a sunscreen towelette. This exposure and treatment continued for up to 10 weeks. Animals were photographed and weighed throughout the experimental period. At the end of the experimental period, mice were photographed and euthanized, after which the skin and blood were collected. Blood was analyzed for markers of inflammation, and DNA was collected from the exposed areas of the skin and analyzed for markers of DNA damage.

[0129] [Example 18] Analysis of the prevention of skin damage by the use of POH-LA conjugates versus the use of the components alone and together This example illustrates the simulation of sunlight by irradiation with a UVB lamp (310 nm) in a phototoxicity test chamber fitted with a UV-B lamp providing a spectral output of approximately 310 nm. The source of UVR was a short wavelength UV lamp (VWR, Radnor, PA). The peak spectral output of this lamp was approximately 310 nm, with no detectable energy below 260 nm, approximately 0.6% at 260-280 nm (UV-C), 72.7% at 280-320 nm (UV-B), and 26.7% at 320-400 nm (UV-A). The UV-B radiation dose per exposure was 54 mJ / cm in the first week as confirmed by a UV monitor. 2 , and 72 mJ / cm in the second week. 2 , and 90 mJ / cm in the third week. 2 , and 108 mJ / cm for the rest of the week. 2 was set to.

[0130] POH-LA conjugates, linoleic acid, POH, and additive combinations of linoleic acid and POH were each suspended in glycerol:ethanol (90:10 vol / vol) in 50 μL to a final concentration of 10 nM and applied to each mouse after UV-B exposure for treatment. Application of POH-LA conjugates to UV-B-exposed tissues limited the amount of observable skin damage compared to untreated controls (A), untreated controls (B), or the individual components of POH-LA conjugates alone (POH-C and LA-D), in mixtures (E), or in combination with sunscreen (G). In all exposed and treated groups except for POH-LA conjugates (F), the skin exhibited dry reddened skin, often with damage, 5 weeks after exposure and treatment. Linoleic acid, POH, and the mixture of linoleic acid-POH all showed damage. Meanwhile, the skin of POH-LA conjugate exposed and treated mice looked similar to the skin of unexposed control mice.

[0131] The mice on the linoleic acid-POH mixture were tested consecutively compared to the rest, and therefore to the different backgrounds of the photographs. However, dry and reddened skin is clearly observed even in this group.

[0132] [Example 19] Analysis of the prevention of skin damage by the use of POH-LA conjugates versus the use of the components alone and together This example illustrates the simulation of sunlight by irradiation with a UVB lamp (310 nm) in a phototoxicity test chamber fitted with a UV-B lamp providing a spectral output of approximately 310 nm. The source of UVR was a short wavelength UV lamp (VWR, Radnor, PA). The peak spectral output of this lamp was approximately 310 nm, with no detectable energy below 260 nm, approximately 0.6% at 260-280 nm (UV-C), 72.7% at 280-320 nm (UV-B), and 26.7% at 320-400 nm (UV-A). The UV-B radiation dose per exposure was 54 mJ / cm in the first week as confirmed by a UV monitor. 2 , and 72 mJ / cm in the second week. 2 , and 90 mJ / cm in the third week.2 was set to.

[0133] POH-LA conjugates, linoleic acid, POH, and additive combinations of linoleic acid and POH were each suspended in glycerol:ethanol (90:10 vol / vol) in 50 μL to a final concentration of 10 nM and applied to each mouse after UV-B exposure for treatment. Application of POH-LA conjugates to UV-B exposed tissues limits the amount of observable skin damage compared to the individual components of POH-LA conjugates alone or in mixtures. In all exposed and treated groups except for POH-LA conjugates, the skin exhibited dry reddened skin, often with damage, 3 weeks after exposure and treatment. Linoleic acid, POH, and the mixture of linoleic acid-POH all showed damage. In contrast, the skin of POH-LA conjugates in exposed and treated mice showed no damage.

[0134] [Example 20] Analysis of the prevention of DNA damage by the use of POH-LA conjugates versus the use of its components This example illustrates the simulation of sunlight with irradiation by a UVB lamp (310 nm). Irradiation was performed in a phototoxicity test chamber fitted with a UV-B lamp providing a spectral output of approximately 310 nm. The source of UVR was a short wavelength UV lamp (VWR, Radnor, PA). The peak spectral output of this lamp was approximately 310 nm with no detectable energy below 260 nm, approximately 0.6% at 260-280 nm (UV-C), 72.7% at 280-320 nm (UV-B), and 26.7% at 320-400 nm (UV-A). The UV-B radiation dose per exposure was 54 mJ / cm in the first week as confirmed by a UV monitor. 2 , and 72 mJ / cm in the second week. 2 , and 90 mJ / cm in the third week. 2 was set to.

[0135] Figures 26A and 26B show DNA damage resulting after UV exposure with and without POH-LA conjugate treatment. After the animals were euthanized, skin tissue was collected from the back of the animals, fixed in formalin, and processed for analysis. Absorption of ultraviolet (UV) light results in two major types of DNA damage, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidine photoproducts (6-4PPs). A UV DNA damage standard was used for comparison with UV-exposed mouse skin-extracted DNA. Both standard and sample DNAs were first heat denatured and then adsorbed to a 96-well DNA high-binding plate. 6-4PP (Figure 26A) and CPD (Figure 26B) present in the samples or standards are probed with anti-6-4PP or CPD antibodies followed by HRP-conjugated secondary antibodies. The 6-4PP or CPD content in unknown samples is determined by comparison with a standard curve prepared from predetermined CPD-DNA standards ranging from 1,000 to 1.56 ng / ml. As is evident, there was less DNA damage in the POH-LA conjugate-treated mice.

[0136] The scope of the present invention is not limited by what has been specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the illustrated examples of materials, configurations, structures and dimensions. Numerous references, including patents and various publications, are cited and discussed in the description of the present invention. Citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed herein are incorporated herein by reference in their entirety. Variations, modifications, and other implementations of what is described herein will occur to those skilled in the art without departing from the spirit and scope of the present invention. While specific embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the present invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not by way of limitation.

Claims

1. A composition for treating or preventing skin damage due to UV exposure in a mammal, comprising perillyl alcohol (POH) conjugated with linoleic acid, wherein a therapeutically effective amount of said composition is applied before, during or after UV exposure.

2. The composition of claim 1, wherein the UV exposure is exposure to UV-B radiation.

3. The composition of claim 1 , wherein the composition is applied topically.

4. The composition described in claim 1, wherein the skin damage is selected from the group consisting of atrophy, pigmentation changes, wrinkles, and malignant tumors.

5. The composition described in claim 4, wherein the malignant tumor is skin cancer.

6. The composition described in claim 5, wherein the skin cancer is selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, and malignant melanoma.

7. The composition of claim 1, wherein the perillyl alcohol conjugated to linoleic acid is in the form of a pharmaceutical composition further comprising a glycerol / ethanol mixture.

8. The composition of claim 7, wherein the glycerol / ethanol mixture is in a weight ratio of 90:

10.

9. The composition of claim 1, comprising a linoleoyl ester of POH.

10. The composition of claim 1, wherein the UV exposure is exposure to UV-A radiation.

11. A composition for preventing an increase in cytokines due to UV exposure in a mammal, comprising perillyl alcohol (POH) conjugated with linoleic acid, wherein a therapeutically effective amount of said composition is applied before, during or after UV exposure.

12. The composition of claim 11, wherein the increase in cytokines is assessed by measuring serum levels of IL-1, IL-6, or TNF in the mammal.

13. A composition for reducing DNA damage caused by UV exposure in a mammal, comprising perillyl alcohol (POH) conjugated with linoleic acid, wherein a therapeutically effective amount of said composition is applied before, during or after UV exposure.

14. The composition described in claim 13, wherein the DNA damage is assessed by measuring the levels of pyrimidine (6-4) pyrimidine photoproduct (6-4PP) or cyclobutane pyrimidine dimer (CPD) in the mammal.

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