WO2018005994A1 - Methods of treating neurofibromatosis with perillyl alcohol - Google Patents

Abstract

The present methods treat neurofibromatosis by a administering to a subject perillyl alcohol or iso-perillyl alcohol. The present methods also treat neurofibromatosis by administering to a subject a carbamate of perillyl alcohol, or a carbamate of iso-perillyl alcohol. The perillyl alcohol carbamate may comprise perillyl alcohol conjugated with rolipram or temozolomide.

Description

METHODS OF TREATING NEUROFIBRO ATOSIS WIT RILLyL ALCOHOL

Fiekl of the Invention

The present invention relates t niethods of treating neurofibromatosis b th

admirilstraiioti of perilb/l alcohol (POH), iso-perillyl. alcohol, or POH derivatives.

Baekgrowiid of the Invention.

Neurofibromatosis ty e 1 'NF- Γ') is a t nior disorde that is caused by the mutation of a gene on chromosome 17 that is responsible for controlling cell division, NF-t causes tumors along the nervous system ami can grow n her on the body. Common symptoms of NF-t include brownish-red spots in the colored part of the eye called Lisch nodules, benign skin tumors called neurofibromas, and larger benign tumors o.f nerves called piexifonri

neurofibromas, scoliosis (cnrvatnre of the spine), learning disabilities, vision disorders, mental disabilities, multiple cafe an laii (TCL, name of specifics spots) spots and epilepsy . NF-i was formerly known as von Recklinghausen disease. There is no cure for the disorder itself, instead, patients with neurofibromatosis are monitored by a team of specialists to manage symptoms and/or complications.

Malignant _. liomas, th most common form of cen tral, -nefvotis system (CMS) cancers. Is currently considered esseniiali incurable. Among the various malignant gliomas, anaplastic astrocytomas (Grade 111) and glioblastoma multiforme (GBM; Grade IV) have an especially poor prognosis due to their aggressive growth and resistance to currently available therapies. The present standard of care for malignant gliomas con sists of surgery, ionizing radiati on, and clie otberapy, Despite recent advances in medicine, the past 50 years have not seen any significant improvement in prognosis for malignant gliomas. Wen et al. Malignant gliomas in adults. New England J Med. 359; 492-507, 2008, Stupp et al. Radiotherapy plus concomitant and adjuvant temozo!omide for glioblastoma. New England i Med. 3 2 : 987-996, 005.

The poor response of tumors, including malignant gliomas, to various types of cheffiotherapentjc agents are often due to intrinsic drug resistance. Additionally , acquired resistanc of initially well-responding tumors and unwanted side effects are other problems that

I frequentl thwart long-term treatment using: chemo/tierarjeutic agents. Henee, various analogues of cbemo herapeutic agents have frees prepared in m effort to overcome these problems. Tire analogues include neve! therapeutic agents which are hybrid molecules of at least two existing therapeutic agents. For example, cisplatift has been conjugated with Pt-(1X) complexes with cytotoxic codmgs, of conjugated with Moactive shuttle compon nts such, as orph rins, bile acids,■ hormones, or modulators that expedite the transmembrane transport or the drug

accumulation, within the cell. (o^'-AminQmethylnicGtmate) dichioridoplatinumiii) complexes esterified with terpene alcohols were tested on a panel of human tumor cell lines. The terpeny l moieties in: these complexes appeared to fiilfill a transmembrane shuttle innction and increased the rate aod extent of the uptake of these conjugates into various tumor cell lines. Schobert et a! Monoterpenes as Drug Shuttles^* Cytotoxic (6~mmome y1mcotniate) dichloridoplatinimi(II) Complexes with: Potential To Overcome Qsptatin. Resistance. I, .Med, Chent 2007, 50, 1288- 12¾3.

Perillyi alcohol (POH), a natnraily occurring inonoterpene, has been suggested: to he an effective agent against a variety of cancers, including CNS cancer, breast cancer, pancreatic cancer, lung cancer, melanomas and colon cancer. Gould, M. Cance chemoprevention and therapy by monoterpenes. Environ Health Perspeet 1997 ^' June; 105 (Suppl 4): 977 -979. Hybrid molecules containing both perillyi alcohol and retinoids were prepared to increase apoptesis- indoeing activity. Das et at Design and synthesis of potential^' new apoptosis agents: hybrid compounds containing perillyi alcohol and new constrained retinoids. Tetrahedron Letters .2010, 5! , 5.462-1 66,

There is still a need to prepare perillyi alcohol derivatives including perillyi alcohol conjugated with other therapeutic agents, and use this material in the treatment of cancers such as malignant gliomas, as well as other brain disorders such as Parkinson's and Alzheimer's disease. Perillyi alcohol derivatives may be administered alone or in combination^' with other treatment methods including radiation, standard chemotherapy, and surgery. The administration can also be -through various routes including intranasal oral, oral-tracheal for pulmonary delivery, and transdermal Summa y

The present- disclosure provides lor a .^"method of treatin neurofibromatosis in. a subject ^suffering^' fro - neurofibromatosis^ In certain embodiments:, the method comprises a ta mistering to the subject a therapeutically effective m unt of perillyl alcohol (POH) or n iso-periliyl alcohol (iso-POH or a combination thereof. I certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a carbamate of perillyl alcohol (a perillyl alcohol carbamateK a carbamate of an iso-periliyl alcohol fan. iso-periliyl alcohol carbamate), or a combination, thereof

The present disclosure provides for a method of inhibiting or decreasing as level and/or activity in a cell. In certain embodiments, the method comprises administering to the ceil a merapeniically effective amount of perillyl alcohol (PQH) or an iso-periliyl alcohol (isO-FOJI), or a combination thereof, in certain embodiments, the method comprises administering to the cell a therapeutic-ally effective amount of a carbamate of perillyl alcohol fa perillyl alcohol carbamate), a carbamate of an iso-periliyl alcohol (an iso-periliyl alcohol carbamate), or a combination thereof^'

The present disclosure provides for a method of inhibitin o -decreasing Ras level and/or activity in. a subject, lit certain embodiments, the method comprises administering to the subject a therapeutically effective amount of perillyl alcohol fPOH) or an . iso-periliyl alcohol (iso-POH), or a combination thereof i certain mbod ment the method comprises administering to the subject a . therapeutically effective amount of a carbamate of petillyl alcohol (a perillyl alcohol carbamate), a carbamate of an iso-periliyl alcohol (an iso-periliyl alcohol carbamate), or a combination thereof

In certain embodiments, the method further comprises administering iemozoioaaide or rolipram,..

In certain embodiments, die method further comprises administering a statin.

Non-limiting examples of statin include a OTvastatin,..ftuvastaiiii, lovastatm, pitavastatin, pravastatin, simvastatin and r suvastatin.

1» certain embodiments, the method forther comprises administering prostratin.

In certain embodiments,, the administration is by inhalation, intranasal iy, orally, intravenously, subcutaiieously or intramuscularly. Non-limiting examples of isoperillyl alcohol include {4 sopropyis.dene cyciohex-1- enyl)metii8»oI, (4~isopropyI cyclohexa-l,3-^ien.yl)inetbanol, (4-isopropyl cyclohexa-i,4- dieny jmethanol, (4-l§op.fopyipfcettyl)t«ethaapi and (4-isoprope»ylpbenyl)methaiiol.

In certain embodiments, the perillyl alcohol carbamate is perillyi alcohol coajiigated with temosolo side or rolipram. In certain em odiments, the periHy! alcohol, carbamate is 3-methyi.4- oxo-3,4-dihydroim ^ acid-4-isop.topen.yl cycloi es- i-enylffleihyi ester, ϊ«. certaia fflbodimetiis,, the perillyl alcohol carbamate is 4~(3- cyclopeiityloxy~4~me.dx>xy pben:yl}~2Hixo-pyrroiidijie-l-carboxylk acid 4~isopropenyi cyclohex- 1 -enylniethyl ester,

KM carbamates encompassed by the present disclosure include, but are not limited to, 4- bis-M, '-4-isopropeftyl cyclohex-l -enylmemyloxy car^'bonyl [5-(2,5-di methyl pheiiyl)-3- trifleorametbyl pyt¾zol-l-yl] be«zenes¾ifo»ami.de_s 4~{3-cyclopentyloxy^me^.oxyphe«.yl -"2- oxo-pyrrolidine-I-carboxylic acid 4-isopro:peay1 cyclohex«l-enyh«ethyi ester, and (3-methyl 4- oxo-3,4-djhydroimidazo[5._i 1 ~d][ 1 ,2,3,5 jielxaxijie-S-carboiiy carbamic aeid~4-isepFDperiyl cyclohex-f -enylmethy I ester.

Iso-POH carbamates encompassed, by the present invention inc ude, but are not limited to, (3.~Methy1 4-oxo-3_!4-di ydroimidazo[5_J 1 -djf 1 ,2,3,5]lsP'a2iiie-8-cafbori:yI}-carbamic aci -4- isopfOpyBdefte cyclohex.- .1 -eir imeihyl ester, 4-(3 -Cyclopentyloxy-4-me^ xyp enyl 2>oxo- pyrrolidine- 1 -carboxy lie acid 4-i sopropy lldene cyciohex- 1 -enylmethyl ester 4-(Bis~N,N*~4~ isopropy!klene cyclohex-l -eaylmethyloxy carbonyl f.5-(2₅S-drmefhyl phenyl j-3-trifIitoromethyi p ra¾oi~! -ylj henzepesi femmide.

The present diselosare fiirther provides for a phar aceutical^' compositios. comprising a perillyl alcohol carbamate. The perillyl alcohol carbamate may be -perillyl alcohol conjugated with therapeutic agent, such as a chemotlierapeittic agent, The cfeemeiherapeutic agents that may be used in the present invention include DMA alkylating agent, a topoisomerase inhibitor; endoplasmic reticulum stress inducing agent, a platinum compound,, an antimetabolite, an eniiym Inhibitor, and a receptor antagonist. In certain emb di nts, the therapeutic agents are dimethyl eelocoxib (D iQ, temoxolomide (TMZ) or rolipram.. The _.perillyl^' alcohol carbamates may be 4-Bis-N,N'-4-isopropeiiyl cyciohex- 1 -enylnietbyloxy carbonyl [5-(2₅5-dimethyl phenyl)- 3-trifluorometliyi pyrazol-l-ylj benzenesulfonarmde, 4-(3^yclopentyioxy^mediox hehyl)-2---

Brief Description of the Drawl tigs

Figure 1 shows the remit of the MTT cytotoxicity assays demonstrating me efficacy of dimethyl eelecoxib (PMC) in killing A 172 and 0251 huma glioma cells.

Figure 2 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of th FOH-D conjugate In killing U87, A.172 and U25.J■ human glioma cells■ according to the present invention. Figure 3 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of temozoi miide (TMZ) in killing US , A 172 and U251 human glioma cells.

F ure 4 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-TMZ conjugate in killing U87_s A 172, and 11251 human gllofna ceils accordin to the present invention.

Figure 5 s ows the results of the IT cytotoxicity assays demonstrating me efficacy of the PQH- olipram conjugate and Rolipram in. killing A.172 huma gliom ceils. Figure 6 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-Roliprarn conjugate and Rolipram in killing 1JS7 hiuna glioma ceils.

Figure 7 shows the results of the MTT cytotoxicity assay s demonstrating the efficacy of the POH-Rolipram conjugate and Rolipram in killing 0251 ^'human, glioma ceils.

Figure 8 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-RoKpram. conjugate and Rolipram in killing L229 human^' glioma cells. igures 9 A and 98 sho w the inMbttion of tumor growth by biuyryl-POH in mouse models.

Figure 9 A shows the images of subcutaneous U-87 gliomas m nude mice treated with butyryi- Ρ0Η, purified (^perillyi alcohol having a purity greater than 98.5% ("Purified POH"), POH urchase from. Sigma .chemicals ("SigmaTi, or phosphate buffered saline 'PBS* negative control). Figure 9B shows average rumor growth over time (total time period of 60 days).

Figure 10 shows the results of a Colony forming Assay iCFA) demonstrating the cytotoxic eifect of TMZ a«d TMZ-PGB on TMZ sensitive (U251) d TMZ fesfeiant (1125 l.TR) V2S Ϊ cells.

Figure 1.1 shows the results of a Colony forming Assa (CPA } demonstrating the cytotoxic effect ^■of POH on TMZ sensitive (U251) and TMZ resistant (U25ITR) IJ251 cells. Figure 12 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-TMZ conjugate in killing U253 ce¾ U25I cells_* and normal astrocytes..

Figure 13 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of the POH-TMZ conjugate in kiltin normal astrocytes, brain endothelial ceils (BEG; confluent and subcontinent), and tumor brain endothelial cells (T BEC).

Figure 1 shows the results of the MT T cytotoxicity assays demonstrating the efficacy of TMZ and the POH-TMZ conjugate in killing USC-04 glioma cancer stem cells, Figure 15 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of POH in /killing DSC-0 glioma cancer stem cells.

Figure 16 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of TMZ and the POH-TMZ conjugate in killing USC-02 glioma cancer stem cells.

Figure 17 shows the results of the MTT cytotoxicity assays demonstrating the efficacy of POH in killing USC-02 glioma cancer stem cells.

Figure 18 shows a western blot demonstrating that TMZ-POH induces ER stress (ERS) i TM sensitive ("U25J.-TMZs") -and resistant ("U251-T Zr") IJ251 glioma cells. Figure 1 sllows Wes eoi blot results feat Mas Binding Domain (RBD) Pnl!do ii. Assays in lysates of MF! CRL-28S4 cells with or without tre&troeais of POH at. different concentrations_*

Figures 20A-20C show the cytotoxicity of POH and POH conjugated with rolipram (RP-POH) on NF.1. CRL-2884 cells or Panel cells,. Figure 20A: cytotoxicity of POH on NF 1 cells: Figure 208: cytotoxicity of RP-POH ou NFl cells; Figure 20C: cytotoxicity of POH on Pane l cells.

Figures 1 A-2 l C show results from: Mas Binding Domain (&8.D) Pulldown Assays in lysates of Panel cells with or -without rreahtieuts of POH and Iso-POH at different concentrations.. Figure 21 A is Westem blots showing die results from RBD Pulldown Assays in lysates of Panel cells with or without treatments of POH and isorPOH. Figure 2.1 B shows bar graphs f the ~ as levels of the Western Mots, Figure 2!0 shows bax graphs of the Pao-Ras levels of the Western blots. Figures 22A-22C show the cytotoxicity of POH (Figure 22 A), iso-POB. (Figure 22B) and RP- POH (Figure 22C) on FI-G 23312 cells.

Figures 23A-23C show the cytotoxicity of POH (Figure 23A), Iso-POH (Figure 238) aid RP~ POH (Figure 230, R~P) on NF1.~ MPNST 26T ceils,

Figure 24 shows POH effects en total Ras activity,. 1 ; ^'Normal m di Tja (control), 2: 0.5in.M POH treated for 20 lir. 3; 1 ,0 mM POH treated for 20 hr. 4; 20 μΜ Paroesyltransferase inhibitor (FT!) treated for 2C)hr, 5: positive cont ol. Figures 25A-25C show the cytotoxicity of POH, POH in combination with prostratin (Figure 25 A), and prosiratin (Figure 25B) on NPl - MPNST 26Ί cells. Figure 25C are bar graph showing the cytotoxicity of POH, and POH ÷ prostratin on F1 - MPNST 26T cells .

Figures 26 -26C sho the cytotoxicity of POH, POH in combination with lovastatin (Figure 26A), and lovas hi (Figure 261) on NFI~ MPNST 26T cells. Figure 25C are bar graphs showin the cytotoxicity of POH, and POH + prostratin on NF1- MPNST 26T cells. Figure 27 sliows krtraceikilar catciiiBi levels alter POH treatment_*

Detailed Bes ripiittH

The resen invention provides methods ofMututing Eas expression, or of treating neurofibromatosis by administering POH or iso-POH to a mammal in need of such treatment i n some embodiments, the POH ot iso-POH is administered, along with rolipram. ("RP"). In some embodiments, a statin is also administered. Statins inc ude atorvastatin, lluvastatiri, lovastaiin, pravastatin, pravastatin, simvastatin and rosuvastatin.

The POH or iso-POH ma be administered with rolipram or a statin_* or with both. Such administration may be b coadministration of distinct drug products, each containing only one of said POH, iso-PQH, rolipram and/o statin. Alternatively_* -said POH,. iso-POH, rolipram and/or statin may be administered in a fixed dose combination drug product. Alternatively_., the desired combination of drug substances may be dosed as a conjugate- with POH or iso-POH , with or without other drug products_*

The present methods and compounds can be used to treat neurofibromatosis.

Neurofibromatosis may include three, types, nearoibrbmato is type 1 (NFI), neuOTfihromatosts type 2 (Nf 2), and schwannoraatosis. In certain embodiments, HP! and NF2 are inherited disorder and both encompass mutations which predispose individual to multiple tumors of th central or peripheral nervous system, and occasionally to other malignancies. I certai

mbodi ent, major tumor types associated with NFI and NF2 involve glial ce ls (e.g. Schwann cells and astrocytes). In certain embodiments, the tumors of neurofibromatosis are neurofibromas (tumors of the peripheral nerves), and/or tamoxs of Schwann cells.

Nmro romafosis Type 2

In certain embodiments, n urofibromat sis type 2 (NF2) is characterized by .multiple tumors on the cranial and spinal nerves- In certain embodiments, indi viduals with NP2 are at a high risk for developing brai tumors, in particular tumors o both the seventh and eighth crania! nerves, such as bilateral vestibular schwannomas, a type of tumor which oc curs on these nerves. In certain embodiments, hearing loss, ringing in the ears, and problems with balance are symptoms frequently associated with IS1F2. Schwannomas are tumors consisting of nerve sheath cells or Schwann cells (SCs). In -cestain -esilKxiinients-. ¾$ 2. involves.bllateral vestibular schwannomas, also known as acoustic neuromas, as well as spinal chwa omas and

sch wannomas of the peripheral nerves. I addition to. schwannomas, individuals with F2 may develop other types of tumors emanating frdaithe nerves, m ningeal envelopes, brain and spinal card. In certain embodiments, the tnraor is merungioma, ependymomas and/or astrocytomas. In certain embodiments, NF2 patients may have an increased risk for developing mesotheliomas, in certain embodiments, F2 results from a mutation or a deletion of t s NF2 gene (Sainz et at, 1994, Ham, MoL Genet 3; 885-891; Ruttledge et ai, 1994, Nat. Genet. 6: 180-184; Rubio etal, 1994, Cancer Res. 54; 45-47; Hayrth et al, 1997, J. Neitropai o!. Exp. Neurol. 56: 382-390). The MF2 gene is a tumor -sup ressor gene thai encodes a protein, Merlin, Merlin belongs to the emti, rad n, and nioestn (ERM) family of proteins ( Trofatter et L, 1993 , Ce il 75: 826}..

Over-expression of Merlin can block both cell proliferation and oncogene-mduced transformation (Liuchman -&&ύ ^'Rouleau, 1995, Cancer Res. 55(11)† 2270-2274; Tikoo et al .,

1994, J. Biol Chero. 269(38): 23387-23390). Indeed, Merlin can negatively regulate cyclk Dl levels (Xiao et al, 2002, J. Biol C em. 277; $83-886} and loss of Merlin results in

overexpression of cyclin Dl (Lallemand et a!., 2003, Genes Dev. 17: 1090-1 100). However, given its predominant, localization to the membrane and cytoskeieton interface. Merlin is not likel to directly control the cell cycle machinery.

Several lines of evidence suggest that Merlin can regulate receptor tyrosine kinase activity, trafficking, and degradation. Merlin has been shown to. interact directly with the focal adhesion component paxiHin in a complex that contains infegrm-βΙ and ErbB2 (Fernandez- Yalle et al, 2002, Nat Genet. 31(4); 354-362), HGF receptor substrate (H S) (Scoles et al,.2002. Hun¾ MoL Genet 1.1(25): 31.79-3189; Gnimann er aL, 2001 , Hum. Mo!, Gene 10(8); 825-834; Soles et al, 2000, Hum. MoL Genet. 9(1 1 ); 1567- 1574), and platelet derived growth factor receptor (PDGFR) indirectly through interaction with a PDZ-coniaining adaptor protein

EBP50/HHMF (Maudsley et aL, 2000, MoL Ceil Biol 20(22): 8352-8363; Mmthy et al, J 998, J, Biol, Chem_* 273(3): 1273-1276). Neure ulih growth factors (EOF famil of growth factors), VEGF, and OGF are important mitogens for Schwann cells (SCs) I rasnoselsky et al, 1994, J. Neurosci;. 14:7284-7290; DeCiue et aL, 2000, J, Clin, Invest. 105(9): 1233-1241; Caye-Thomasen et al, 2005, ( N t&L 26(l.};98~ l0i). Nettregnl&i/Bf iB pathways are constltu iyely activated in human HF2 vestibular schwannomas a»& inhibitors of these path ways (e,g. antibody against nenregalin and Iressa) block proliferation of MF2~de.ftcient schwannoma cells

(Stoiiecypher et aL, 2006, J. Neuropaths!. Exp. Neurol, 65: 162-175; Hanse et al, 2006, Cilia 53:593-600), Recent evi ence from D sopki indicates that Merlin can regulate

abundance/turnover of many signaling and adhesion receptors such as Notch, th e EGF receptor, Patched, Smootbened, E-cad¾erin, and Fat Loss of merlin results in aectnnidation of these cell surface receptors and activation of the associated signaling pathways (e.g, the EGPR pathway and the Wingless pathway) (Maitra et ai._s 2006, C¾m Biol 1 (7);7O2~?09).

In^■addition to cell surface receptors, Merlin has been shown to interact with downstream components of various signaling pathway s , including Rac-PAK (p21 -activated kinase) path way, Rae is a member of the Rho family of smalt GTPases, which organize the actin cytoskeleton and control mmy cellular processes such -as cell proliferation, trahsfcr atioa, and cell motility (Eiienne-Manneville and Hail, 2002, Nature. 420(6916); 629-635; Sahai and Marshall, 2002, Nat. Rev. Cancer 2(2): 133-142). PAK can phosphorylate 5518 of Merlin (Xiao et al, 2002, J, Biol Chem. 277: 883-SS6; Kissel et al, 2002, J. Biol. Chem. 277(12); 10394-10399) which leads to confonnationaJ chang and loss of growth- suppressing activity (Shaw et ai, 1998, j. Biol. Chem. 273(13); 7757-7764; Shaw et aL 2001 , Dev. Ceil 1.(1): 63-72). Merlin ca also act as a negative regulator of Rac-PA signaling (Shaw et a!., 2001 , Dev. Cell. 1:63*72;· Kisstl et ai, 2003, Mol. Ceil, 12:841-849: Lalleffiand et l, 20O3, Genes Dev. 17: 1090-1100; Hirokawa et ai, 2004, Cancer J . 10: 20-26). Loss of Merlin results in the inappropriate phosphorylation and activation of PAK. Over-expression of Merlin inhibits PAK activation and blocks Rac-induced transformation. (Shaw et ai, 2001, Dev. Cell 1(1): 63-72; Kissil et al., 2003, Mol. Cell.

12:(4):841 -849), Preliminary evidence indicates that loss of Merlin also leads to activation of the Ras/RarJ ek/Erk pathway and PI3 ~Akt pathway (Rang ala t l_^ 2O0S. J, Biol ζ-ht ,

280(12): 11790-11797). A recent study from BrosophUa has proposed that Merlin and a related protein expanded function upsiream of the Hippo signaling pathwa to regulate ceil proliferation and apoptosis (Hamaratoglu et al, 2006, Nat. cell bml 8:27-36; Wiliecke et ah, 2006, Cu Biol 16(21);2Ο9Ο-210Ο), The link between Merlin and growth factor receptor signaling indicates that growth factor receptors may play direct roles in N P2-associated tumor formation and

progression. However, possible iiivolvemeni of Merlin with molti pie signaling pathways presents a challenge In developin drugs for the treat ent of NF2, Neurofibromatosis Type 1 NFI affects the human nervous system (Soransen S A, Mulv I J J, Nielsen A. Ann Ή Y Acad Sei 1986; 86;30~7.),. in certain embodiments, ^'NFI is an autosomal dorainantly inherited, genetic disorder with frequent germiine deletion or ioss-of-ftmction mutations of the NFJ gene, and is caused by mutation in the NFi gene, which encodes Neurofibroma!,, a tumor suppressor. Netif ofbtOinin shares a region of similari ty wi th the p i 20RasGA.P protein, therefore fimctioning as a negative regulator of the Ras pathway.

In certain ernboditnents, the signs of N F I include ea,re~au~lait macules, skitt freckling, skeletal defects, learning disability; Liseh noduies, dermal and plexiforrn neurofibromas (most common), benign tumors of the brain or other organs (e.g, optic pathway astrocytomas, optic neuromas, optic gliomas, cerebral astrocytomas, cerebral gliomas, ganglioneuromas,

epen ymomas, pheochromoeyiomas and gangM neuromas), and malignant neoplasms (e.g.

rhabdomyosarcomas_* neurofibrosareo as or malignant per spheral nerve sheath, tumors P ST) or malignant schwannomas) (KorfB R. J Child Neurol 2002; 17(8}:573*?· discussion 602-4, 46- 51.) Children affected by NF 1 also have Increased risk for developing a rare form: of leakeinia- juvenile myelomonocytic leukemia {JMML (Stiller C A, Chessells J , Fitchett M. Br J Cancer 1994; 70(S):969-72,).. Dermal neurofibromas, subdernial neurofibromas, plexifomi

neurofibromas and MPNSTs are primarily derived from Schwan cells or their progenitors. Optic liomas and astrocytomas are derived from astrocytes. Fheochroraocytomas are derived from neural crest components (as are neurofibromas and MPNSTs).

In certain embodiments, the typical characteristic of NFi is the neurofibroma, of which there ate eliiiically and histologically distinct types. These tumors may cause disfigurement, chronic pain and pruritus. Certain patients may develop some of the same disfiguring symptoms that, are associated witb Elephant Man's disease, a sepaiate disorder originally thought to he NFi . Plexi fonn nettiO fibromas ma be congenital and are present in a subpopuladon of pa tien ts wi th NF I . The affect long portions of nerves and infil trate the nerve and surrounding tissue, resulting in disfiguration and neuralgic complications. In certain embodiments, plexiforin neurofibromas transform to malignant peripheral nerve-sheath tumors, which have significant mortali ty rate.

The Nfl. gene was identified in 1.990 (Wallace etal 19.90 Science 249;i81.~I86; Cawthon et at 1990 Cell 62:193-201) and its gene product, nenroilbromin, has catalytic domain re laied to the GTFase-activaiin protein (GAP) domain of pi 20!lasGAP (March.uk et al., 1991

O tomies 11:931-940; Gutaann et l, 199L Proc. Nail, Acad Set. U A, SB; 965E-&662 DeChie et aL, 1991. P c. Natl. A . Sc U.S.A. 88:9914- 991 S^rtin et aL, 1990. Cell 63:843-849; Xu et at, 1 90. (¾ 63 ; 835-841; Xu et al, 1990. C 62; 599-608), Loss of Nf 1 w human neurofibromas, FHSl^'s, Ieiskemtas, antd tinner-deri ed cell lines results in the elevation of Ras-GTP levels and activation of as- af- ek-Erk2 and other MAP kinase pathways (Guha et ai, 1996^" 0n gme-i2: S07-S13 .BoJlag fc at, 1996, Na Genet 12: 144-148; Basu et gi 1992. Νάί $6; 71.3-71 ; DeClue et al, 1992 Ceii 69:265-273). For example, Ras-GTP levels torn a few Fl MPNST-derived cell liaes STSS-M, 88-3 m 90-8 ate much, higher compared to other cell lines with normal nettrofibroinm. These cell lines also have activaied downstream M AP kinase pathways. In addition, cell proliferation and soft agar growth of STS8- 1 can be inhibited by injection of an antibody against l as and expression of the GAP domain of neurofibroma, respectively.:

The present hweaiion also provides for a derivative of nionoferpene or sesquiterpene, such as a periliyl: alcohol derivative The present invention also provides for a pharmaceutical composition comprising derivative of moiwterpene or sesquiterpene,_, such as a perillyl. alcohol derivative. For example, the perillyl alcohol derivative may be a perillyl alcohol carbamate. The petiUyl alcohol derivative may be perillyl alcohol conjugated with a therapeutic agent such as a cheniot.herapes.nic agent The monoterpene (or sesquiterpene) derivative tnay be formulated, into a pharmaceutical composition, where the -monoterpene (or sesquiterpene) derivative is present in amounts, ranging from about 0,01 % (ww) to about 100%. (w w)_f from about 0.1% (w/w) to about 80% (w/w), f om about 1% (w/w) to about 70 (w/w), from, about 10% (w/w) to about 60%

(w/w), or f om about 0.1% (w/w) to about 20% (w/w). The present compositions can b

administered alone, or may be co-administered togethe with radiation or another agent (e.g., a chemotherapeutic agent), to treat a disease such as cancer. Treatments may he sequential, with the mormterpene (or sesquiterpene) derivative being administered before or after the

administration of other agents. For example, a perillyl alcohol carbamate may be used to sensitize a cancer patient, to radiation or chemotherapy. Alternatively, agents may be

administered concurrently. The route of adminis tra tion may vary, and can inclnde_* .inhalation_* intranasal, oral, transdermal, intravenous, subcutaneous or istxamnseular injection. The present invention, also provides for a method of treating a disease such as cancer , comprising the step of delivering te a patient a therapeutically effective amount of a derivative of nmmiexp& (or sesquiterpene).

The present invention provides for .methods of treating a disease such as cancer using an isoperiliyl alcohol or a derivati ve of an isoperiliyl alcohol Routes of administiaiion include inhalation, intranasal, oral, transdermal, intravenous, sttbcutaaeoiis and intramuscular injection.

In the present methods, a patient is administered a therapeutically effective amount of art isomer or analog of monoierpene or sesquiterpene, such as an isoperiilyi alcohol. The present invention also provides for a method of treating a disease comprising the step of administering to a patient a ierapeuticaliy effective amount of a derivative of an. isomer or analo o

monoterpene or sesquiter ene,, suc as an isoperiliyl alcohol carbamate. The derivative ma be an isoperiliyl alcoiiol con g te with a therapeutic agent such as a chemotberapeutie agent

For example, the isomer or analog of monoterpene or sesquiterpene can be an isoperiliyl alcohol. (iso-POH), Isoperiliyl alcohols include any isomers or analogs of perillyl alcohol. In one embodiment, the isoperiliyl alcohol is ( -isopropylidene cyclofaex-}-enyi)metha¾ol. Other examples of isoperiliyl alcohol include, hut are not limited to, (4-isopropyl eyclohexa-1,3- dienyl).ra.ethanoi, (4-isopropyl cyclohesa-1 ,4-dienyi).raeihanol_? (44sopropylphenyi)methanol and (4-isopropeny!pheny! )rnethanoi

An exemplary isoperiliyl alcohol (4-isopropylidene cyclohex~l-enyl ¾ethanol, s shown below:

The compounds of the present invention may be used for the treatment of nervous system cancers, such as a malignant glioma (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma multiforme), retinoblastoma, ptlocytic astrocytomas (grade i), meningiomas, metastatic brain tumors, neuroblastoma, pituitary adenomas, skull base meningiomas, and skull bas cancer. The

administered together with radiation or another agent (e.g., a ehemo therapeutic agent),, to treat a disease swell as cancer. TreapBents may be sequential, with isoiner or analo o.f monoterpene or sesquiterpene (or the derivative of an isomer or analog of monoterpene or sesquiterpene) being administered before or after the administration of other agents. For example, an isoperillyl alcohol (or an isoperillyl alcohol carbamate, ester., or ether) may be used to sensitize a cancer patient to radiation or chemotherapy. Alternatively, agents may he administered concurrently_* The route of administration may vary, and can include, inhalation, intranasal, oral, transder mal, intravenous, subcutaneous or intramuscular injection.

The compositions of the present, invention may .contain one or more types of isomers or analogs of monoterpene or sesquiterpene (or the derivatives of isomers or analogs of monoterpene or sesquiterpene). The compositions of the present invention may contain one or more types of derivati ves of manoierpene (or sesquiterpene).

Monoterpenes include terpenes that consist of tw isopreae units. Monoterpen.es may be linear (acyclic) Of contain nags. Derivatives of.monotefpeno.ids are also encompassed by the presem invention. Monoterpenoids may be prodaced by -biochemical modifications such as oxidation, or rearrangement of mqnoterpenes. Examples of monoterpenes and monoterpenoids include, perillyl alcohol. (S(-» and R(+)). ocirnens, myrcene, geraniol citral, citroae io!, eitroneilal, linalooi, pinene, terpineol, ier aien, liraooette, terpineaes, pheilaodreftes, erpinolene, terpinen-4 >i (or tea tree oil), ptnene, terpineol, ierpinen; the ierpenoids such, as -cymene which is derived from monocyclic terpenes Such as menthol, thymol and c-arvacrol; bicyclic

monoterpenoids such as -camphor, borneol nd eucalyptoL

Monoterpenes may be distinguished by the siructure of a carbon skeleton and ma be ^•grouped into acyclic nionoierpenes (e.g. , myrcene, (Z)~ and (E)-ociiaeae, linalooi, geraaiol, nerol, eitroaeHo^'i, myrcenoi, getania!, citral a, neral, citral h, citronellal, etc,), nronocyclic monoterpenes (e.g., limoneae, terp iiene, phellandrene, terpinoiene, menthol, carveol, etc.), bieyelie moiiotei enes (e.g., pinene, aiyrtenol, myrteaal, verbanoi, verbaoon, pmocarveoi, carefte, sabinene, eatnpheae, thujette, etc.) and tricyclic monoterpenes (e.g., tricyclene)> See Encyclopedia of Chemical Technology. Fourth Edition, Volume .23, page 834-835.

Sesquiterpenes of t e present invention include terpenes that consist of three isopreue

•units.- Sesquiterpenes may be linear (acyclic) or contain rings. Derivatives of sesqihterpeaoids are also encompassed by the present invention. Ses uiterpenoids may be prodaced b

hiocheaiical modifications such as oxidation or rearrangement of sesquiterpenes. Examples of sesquiterpenes include famesol fsraesal, farnesylic acid and nerolidoL

The derivatives of monoterpene (or sesquiterpene) include, but are not limited to, carbamates, esters, ethers, alcohols and aldehydes of the n oiiot-erpene (or .sesquiterpene).

Monoterpene (or sesquiterpene) alcohols may be deriv-aiized to carbamates, esters, ethers, aldehydes or acids. The derivatives of^* isomers or analogs o moaoterj>e»e or sesquiterpene include, but are not limited to. carbamates, esters, ethers, alcohols and aldehydes of the monoterpene ^"(or sesquiterpetie). Alcohols may be derivatized to carbamates, esters, ethers, aldehydes or acids.

Carbamate refers to a class of chemical compounds sharing the fractional group

based on a earborryl group flanked by an oxygen and a nlrtoge R^} . it* and: R^J can be a groiip sack as alkyl, aryl, etc,,; which can be -substitute - The R groups <m die nitrogen and the oxygen ma form -a- ring. ^'R*»QH may be a monoterpene, e.g., PGH, or iso-POB. The R^-N-R^'' moiety ma be a therapeutic agent.

Carbamates may be synthesized by reacting isocyanate and alcohol, or by reacting ehloroforoiate with amine. Carbamates may be synthesized by reactions making use of phosgeae or phosgene equivalents* For example, carbamates ma be synthesized b reacting phosgene gas, diphosgene or a solid phosgene precursor such as tripbosgen with two amines or an amine and an alcohol. Carbamates (also known as nrethaftes) can also be made iro ? reaction of a urea intermediate with an alcohol Dimethyl carbonate and diplteny! carbonate are also used for making carbamates. Alternatively, carbamates may be synthesized through the reaction of alcohol and/or amine precursors with an ester-stibstituted diary] carbonate, such as

blsmeihyisalicylcarbonate (BMSC). UJS_* Patent Fublicatiou Ho, 20100113819.

Carbamates may be synthesized by the following approach:: · Carbamate

-OH *- - R,— H_> *~ Carbamate o_r i> €

Suitable reaction solvents include^ but are not limited to, tetrabydro&ran,

dicMorometJbaue, dtcjiloroethaoe, acetone* an diisopropyl ether. The reaction may be

performed at a temperature ranging from about -70° C to about 80°C_» or from about -65 to about 50°C. Tire molar ratio of periliyi c oroformate (or isoperiuyl cMoroformate) to the substrate R - - Nl¾ may range from about. 1:1 to about 2? Ϊ , from about 1 :1 to about lJS;!, from about 2: 1 to about 1: 1, or from about 1.01:1 to about 1J ;L Suitable bases include, but are not limited to, organic bases, such, as trietfeylamine,, potassium earbouate,. Ν,Ν'- diisopropyleftylanurse, butyl iii utn, and . otassium-t-butoxide.

Alternatively, carbamates may be synthesized by the following approach;

R3~GH may be, e.g., POM or isp-POk

Suitable reaction solvents include* but are not limited to, diehlorometbaue,

dichioroethane, toluene, diisopropyl ether, aiid tettabydtoferaB, The reaction ma be performed at a temperature ranging from about 2..5. to about I l(f C, or. from about S T to about 8.QT, o about 5:0^V>C. Tie tuola rati of periliyi alcohol (or isoperill.yl afcobol) to the substrata B.- -C^:::: ) may range from about 1 :1 to about 2: 1 , from about 1 : 1 to about ,5 1 , from about 2: 1 to about 1 : 1 , or from about 1 ,05.; 1. to about 1 , 1 : 1.

Esters o die uiouoterpene (or ^'sesquiterpene) alcohols (o esters of the alcohols of the isomers or analogs of monoterpene or sesquiterpene) can be derived from an inorganic acid or an organic acid. Inorganic acids include, but are not limited to, phospiioric acid, sulfuric acid, and nitric acid, Organic acids include, but are not limited to, earboxyilc acid such as enzoic acid, fatty acid, acetic acid and propionic ac , and any therapeutic agent bearing at least one carbox lic acid functional group, Examples of esters of the alcohols include, but. are not limited to, carboxyiic acid esters (such as benzoate esters, fatty acid esters (e.g. , paimitate ester, inoleate ester, stearate ester, butyryl este and oleaie ester), acetates, propionates (or propauoates), and formates), phosphates, sulfates, and carbamates (e.g., N, -dimethyiaminocarbonyl), A specific exam le of a onoterpene that may be used in the present invention is periHyi alcohol. The derivatives of eriily! alcohol tnclnde, perillyl alcohol carbamates, perillyl alcohol esters, perillic aldehydes, dihydroperiliic acid, perillic acid, perillie aldehyde derivatives, dihydroperil ic acid esters ami perillic acid esters. The derivatives of per illy!, alcohol may also include its oxidative and tit!cleophilic/electrophilic addition derivatives. U.S. Patent Publication No, 20090031455. U.S. Patent Nos, ,133,3.24 and 3_s9S7_s856_t Many examples of derivatives of peri i!yl alcohol are reported in the chemistry literature (see Appendix A; CAS Seifinder search output file, retrieved January 25*_.2010)^·.

The derivatives of isoperiliyl alcohol include isoperiliyl alcohol carbamates, isoperiMyl. alcohol esters, isoperillic aldehydes, isoperillie acid, isoperiilie aldehyde derivatives,, ami isoperillie acid esters. The -derivatives of isoperilly! alcoho! may also include its oxidative and nncleophtlic ectrophi^'lic addition derivatives. Pew examples of derivatives of isoperiliyl alcohol are .reported, in the chemistry literature. See U.S . Patent No, 5,994,598 and Japanese Patent No. 07048264A.

In certain embodiments, a POSH carbamate (or an iso-POH^' arbamate) is synthesized by a process comprising the step of reacting a first reactant of perillyl c loroformate (or isoperilM ehiofoiormate) with a second reactant such as dimethyl eefocoxib (D teraoTOlornide (TMZ) and rolipram. The reaction may be carried out in the presence of tetrahydro&ra and a base such. as n-bttiyl lithium Perillyl diloroforoiate (or isoperiilyl c oroforaiate) may be- made by reacting POH (or iso-POH) with, phosgene. For example, PO (o iso-POrl:) c njug ted with

temozolomide through a carbamate bond ma be synthesized by reacting temozolomide. with oxalyl chloride followed by reaction, with perillyl alcohol, (or iso-POH). The reaction .maybe carried oat in the presence of 1 ,2-diehioroethane.

POH carbamates encompassed by the present disclosure include_* but not limited to, 4-bts-

N,M ^,-4-isopropenyl eyciohex- 1 -enylme yioxy carhonyl [5-(2,5-dimethyi phenyl)-3- triftuoromethyl pyrazol-l -yl] benzenesttlfonarnide, 4-(3-c:yclopentyloxy-4-methoxy pherryl)-2- oxo-pyrioiidine-l-carboxy!ifi- acid 4 sofirepenyl.. eyciohex- 1-enyimeihyl ester, and (3-methyl 4- oxo-3,4-dihydroimidaEG[5,I-d]p j2,3,Sltetra ne-S-carbonyl)carbai»ic acid-4-tsoprop:e»yI eyciohex- 1 -en {methyl ester. The details of tjte chemical reactions generating these compounds are described in the Examples below. iso-P5H carbamates encompa sed by the present -Invention include,, but are not limited to, (3- ethyI 4-t»xo~3_;;4~i31ltydroiraj{la¾o[5 ,1 -d][I,2 _jS] sft¾zme-S-carbon l)-eart?aniiC acid -4- isopropylidene cyc!oliex-i-e«yltt¾ethyl ester, 4-(3 lopen^oxy-4-me&oxyp¾eny.i)-2-< x - pyrro idme-3-carboxyhe acid 4 so£rop Hdene cyclobex-l-enyimethyl ester. 4-(Bis-N,N⁵-4- isoprcpyHdene eyclohex-l-eny metitylaxy carbonyl [5-(2,5~dimethyl phenyi)-3H?*fluoroMei¾yl pyrazoi^»l-yi} ¾eazenesiiIfoBatnide. Th details of the chemical reactions generating these compounds are described in the Examples ^'bel w. in certain em odiments, periUy! alcohol derivatives ma be pertityl. alcohol fatty acid esters, such as pafcniioyt ester of PQH and iiiioleoyi ester of PQU, the chemical^' sfractures of which are shown below

Hexadeeanoic acid 4-isopropen l-cyclohe - 1 -enylmethy 1 ester P^'alm¾oyi ester of ΡΌΗ)

Octadeea~9, I2-dienoie acid 4 sopropenYi~cyeIohex-l ~ewlt«eth.Yl ester (linoieoyi. ester of POH)

In certain .embodiments, iso-peri!lyl alcohol derivatives ma he isaperi!ly! alcohol fatt acid esters, suc as palffiit&y!. ester ofiso-POiiaad. lino!soy! ester of iso~PD:l-i.

The moBoterpene (or.^sq¾itsrpene) derivative may be a mottoterpene (or sesquiterpene) conjugated w ith a therapeutic agent. A monoterpene (or sesquiterpene) conjugate encompassed by me present invention is a molecule having a rnonoterpene (or sesmriterpeae} eova!entl bound via a chemical linking group to a therapeutic -agent the molar ratio of the memoterpene (or sesquiterpene) to ^'the therapeutic agent lo the on ter ene (or sesquiterpene) conjugate may be 1 :1 , 1:2, 1:3, 1:4, 2: 1, 3:1. 4:1.. or any other suitable molar ratios. The .monoterpene (or sesquiterpene) and the therapeutic agent may be covalentiy linked through carbamate,, ester, ether bond$, or any other suitable chemical, func ional groups. When the monoterpene (or

sesquiterpene) and the therapeutic agent are conjugated through a carbamate bond, the therapetitic agent may be any agent bearing at least one carboxylic acid .functio al group, or any agent hearing at least one amine functional group, in a specific example, a perillyl alcohol conjugate is perillyl alcohol covalentiy bound via a chemical linking group to a

chemotherapeutie agent.

The derivative- of an isomer or analog of monoterpene or sesquiterpene ma be an isomer or analog of monoterpene or sesquitetpene conjugated with a therapeutic agent, A conjugate- encompassed by the present invention is a molecule- having an isomer or analog of monoterpene or sesquiterpene covalentiy bound via a chemical linking- group to a therapeutic agent The molar ratio of the isomer or analog of nionoterpene or sesquiterpene to the therapeutic agent in the conjugate may -be I T , 1:2,. 1 :3, 1 :4, . 2:1 , 3:1 , 4: 1, or any other suitable molar ratios. The isomer or analog of monoterpene or sesquiterpene and the therapeutic agent may be covalentiy linked through carbamate, ester, ether bonds, or any other suitable chemical, functional, groups_* When the isomer or analog of monoterpene or sesquiterpene and the therapeutic agent are conjugated through a carbamate bond, the therapeutic agent may be any agent bearing at least one earhoxylic acid Junctional group, or any agent bearing at least one amine - unctional group, in a speeiic example, an isoperil^'lyl alcohol conjugate is isoperillyl alcohol covalentiy bound via a Chemical linking group to a chemotherapeutie agent.

Accordin to the present invention, the therapeutic agents that may be conjugated with monoterpene or sesquiterpene (or an isomer or analog of mouoterpene or sesquiterpene) include, but are not limited to, cheinotherapeutic agents, therape otic agents for treatment of CNS disorders (nieluding, without- limitation, primary degenerative neurological disorders such as Atehe!n ers, Parkinson 's, multiple sclerosis, Attention-Deceit Hyperactivity Disorder or A HD, psychological disorders, psychosis and depression), immnnotherapeutic agen¼ angiogenesis inhibitors, and anti-hyperiensive agents, Anti-cance agents that may be conjugated with monoterpene or sesquiterpene can have one or more of the following effects on

77 cancer cells or the subject cell death; decreased cell proliferation; decreased numbers of cells; inhibition of cell growth; apoptosis necrosis; mitotic catastrophe; cell cycle arrest; decreased col! size; decreased cell division; decreased cell sisrvival; decreased cell, metabolism; markers of cell damage -or cytotoxicity; indirect indicators of eel! damage or cytotoxicity such as tumor shrinkage; improved survival of a subject; or disappearance of .markers associated with undesirable, unwanted, or aberrant cell proliferation.. U .S. Patent Publication No. 20080275057,

Also encompassed by the present .invention is co-administration, admixtures, and coi rmnlatioris of FOB, or iso-PQB, and at least one other therapeutic agent.

Cheittotberapeutie agents include* but are not limited to, DNA alkylating agents, opoisomerase inhibitors, endoplasmic reticulu stress inducing agents, a platinum compound, an. antimetabolite, viacalkaloids_? taxanes, epothilones_* enz me inhibitors, receptor antagonists, tyrosine kinase inhibitors, boron radiosensitizers (ie. Velcade), and ehemotherapeutie

combination therapies.

Non-liniiting examples of DN A alkylating agents are nitrogen .mustards, such as

Cyclophosphamide (I fostamtde, Trofosfamide), Chlorambucil (Meiphalan, Prednimustine), Bendaraustiiie. Ora ustme and Estraniustine; nitrosoureas, such as Caortustine (BC U), Lomustme (Semusiine), Foiemustine, Nimustine, ^'Rahiraastine and Streptozocin;_. alky! sulfonates, such as Busulfan (Maanosuifaft, Treosi fan); A¾iridihes₄ such as Carboquone, Triaziquone, Xriethylenemelamine; Hydrazines (Procarbazine); Triazenes such as Daearbazine and Temozolomide (TM2¾ Alttetaraine and MitobronitoL

Non-limiting examples of T poisornerase I inhibitors inckde Campot iecin derivatives including SN-38, APC, PC, campothecin, topotecan, exatecan mesylate, 9-nitrocampfo.thec i₅. 9-annnocam.ptothecitt, lurtoteean, robiiecan, silaiecan, gintatecan, difiomoteean., e latecan, BN- •80927, 0X-895 It and MAG-CPT as deer ibe in Pommier Y, (2006) Nat. Rev. Cancer

6(1 Q);789-802 and U.S. Patent Pirbllcation No. 200510250854; Prbtoberoeriae alkaloids and deri vatives thereof including herberrubine and coralyne as described in Li et a!. (2000)

Biochemistry 39(24}:7107-7116 and. Gatto et al (1996) Cancer Res. 15(i2);2795-2800;

Phenamhroline derivatives including Benzo{i|phenanthridine, Nitidine, and iagaronine as described in Makhey et al (2003) Bioorg. Med. Cheru. 11 (8); 1809-1820; Terbenzimidazole and deri vatives thereof as described hi .Xu (.1998) Biocheimstry 3^:7(| 0)3558-3566; and

Anthracycline derivatives including Doxorubicin, Dannombicin, and Mitoxantrone as described

Dauiiorubidn, Doxorubicin, Epismbicin, i am icirj^ Amriibicsn, Pirarubici«₅ Ya ubieln,

Zorabicin) and Antracenediones (Mitoxanirone and Pixantrone}..

Examples of endoplasmic retieultiiB stress itidocirig agents include, but are not limited to, diinethyl-celeeoxib (DMC), .ndfrnavlr, ce!ecoxih, and boron mdiosensitizers (i.e. ve!cade

(Bortezomih)),

Platinum based compounds are a subclass of DN A -alkylating agents . Non imitlng examples of such, agents include Cispiatin Nedaplaiin, Oxaliplatim Ttiplati tettanitrate, Satfaplatin, Aroplatin, Lobaplatin, and JM~2.16. (see Mc&eage et al (1997) 1. -Clin. Oncol .201 ; 232- 237 and in general, CHEMOTHERAPY FO GYNECOLOGICAL ^'NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, m the Series Basic and Clinicai Oncology, Angioli et al. Eds., 2004).:

"FOLFOX" is an abbreviation for a type of combination therap that is used to treat colorectal cancer. It includes 5-FU, oxaltplatkt and leucovorin. Information

regarding this treatment is available on the National Cancer Institute's web site,

cancer.gov, last accessed on January 16, 2008.

"FOLFOX BV" is an abbreviation for a type of combination therapy that is used to treat colorectal cancer. This therapy includes oxaliplaiin, leucovoria nd

Bevacizumab, Fiathennore, "XELOX/BV" is another combination therapy used to treat colorectal cancer, which Inclu es the prodrug to 5-FU, known as Capedtahine (Xeloda) in combination with oxaliplatin and bevadxaniab. Infoiraation regarding these t eatraeiils are available oft itie National Cancer institute's we s te, caneer.gov or from 23 the National Comprehensive Cancer Network's web site, ncen.org, last accessed ^'on M

27,2008

Mon-lraiiting examples of antimetabolite■■agents, include Folic acid based, i.e.

dihydro ate reductase inhibitors, such as Amiaopteriix, Methotrexate and Peiaetrex d

tbymidylate synthase inhibitors, such as Raititrexed, Penietrexed; Purine based, i.e. an adenosine deaminas inhibitor., such as Pentostatin, a ti ioporiue, such as Th gua nne and fereaptopurIne, a haiogePatedriboiracleoiide reductase inhibitor_* such as Cladribine, Clo&tabine, Fludarabine, or a guanineYguanosine: thiGpurifte,, such as Thiog anine; or Pyrtoidine based, ie.

cyiosnie cytidrae: hypomethylating agent, suc as Azacitidine an Dedtabihe, a D^'NA

l merase inhibitor, such, as Cytarabine, a ribonucleotide reductase inhibitor, such as

Gemeitabiiie, or a ihymine/ibyrnidme: thyniidytate synthase inhibitor, such as a Flcso.rouracil (5- FU), Equivalents to 5-FU include prodrugs, analog and derivative thereof such as 5^f -deoxy-5- f Borouridifte (doxifiuroidine}, l-teitahydroirran^ {ftofafltr), Capecitahine (Xeloda). S-l (MB S-2 7616, consisting of tegafur and two modulators, a 5-chioro-2.4- dihydtoxypyridme and potassium oxonate), ralititrexed (toniudex), nolatrexed (Thyrniiaq, AG337}, LY231514 and ZD9331, as described for example in Paparnisheal (1999) The

Oncologist 4:478-487.

Examples of vincaika ds, include, but are not limited to Vinblastine, Vincristine,

VinS mne, Vindes ne and Vinotelbine.

Examples of taxanes include, but are not limited to doceiaxel, Larotaxel. Oitataxel, Paclitaxel and Tesetaxel. An example of an epothilane is iabepilone.

Examples of en¾yme inhibitors irsc!ude, hut are not limited to famesylttansferase inhibitors (Tipifamib); GDK inhibitor { Alvocidib, Selicic!ib); ptoteasome inhibitor

(8orte¾arnlb);: phosphodiesterase inhibitor (Anagre!ide; rolipram); IMP dehydrogenase inhibitor (Tiazofurrae); and lipoxygenase inhibitor ( asopfocol). Examples of receptor antagonists include, but are not .limited to ER (Atrasefttan); retinoid X receptor (Bexarotene); and a sex steroid fXestolaeione )..

Examples of tyrosine kinase inhibitors include, but are not limited t inhibitors to Erb : HER 1/EGF (Ertotkiib, Gefttinih, Lapattnib, Vandetaaib, Smiitinib, Neratinib); HER2 »eu

(Lapatinib, lN½ratinib}; RTK. class III; C-kit (Axifimb, Sunitinib, Sorafeaib), FLT3 (Lestanrtinib), PDGFR (Axitinib, SuratMb, Soraienih); and VEGFR (Yandetanib, Semaxaoih, Cediranib, Axitinib, Somfenib); bcr~abl (toatinib, Niloiinib, Dasafinib); Sre (Bosutinib) and^' Janus kinase 2 (Lestaurtimb).

"Lapatinib" (Tykerbi) is an dual EGFR and erbB-2 inhibitor. Lapatinib has been investigated as an ant cancer monotherapy, as well as in com ination w ith tmsteumab., capeeitabine, le roxole, paelitaxel and FOLFIRI(iri»otecan, 5-iiiorouracil and leucovorin , in a .number £ clinical trials. It currently in phase 01 testing for the oral tre tment of metastatic breast, head and neck, lung, gastric, renal and bladder cancer.

chemical equivalent of lapaiinib is a smalt molecule or compound that Is a tyrosine kinase inhibitor (TKi) or alternatively a HER-f inhibitor or a HER-2 inhibitor. Several TK s have been found to have effective antitumor activity and hav been approved or are in clinical trials. Examples oFsjjch include, but are n t limited to, Zaciima (ZD6474), Iressa (geiltinib), i atimb mesylate (STI571 Gleevec), erlotirtib (OS! -1774; Tarceva), canertinib (CI 1033), semaxinib (SU54J6), vatalaaib (FT&787/ZK222584), sorafemb (BAY 43- 9006), sute»t(SUi 1248) and lef tniomide (SO 101).

PTK/Z is a tyrosine kinase inhibitor with broad specificity that targets all VEOF receptors (VEGFR), the platelet-derived growth factor (FD6F) receptor, c-KIT and c-F.ras. Dreys (2003) rugs 6(8): 787-794,. FTJ K. is a targeted drug that blocks angiogenesis and lyrophangiogenesis by inhibiting the activity of all known receptors that bind VEOF including VEGFR-I^' (Flt-1), VEGFR-2 (KDR Fik-1) and VBGFR-3 (Fii-4), The chemical names of PTK/ZK re l- 4-Chloroa»iliuoj-4-^ Succinate or ^"!--

Phthalazinamine, N_'(4-ciiloiT'pheny3)-4-(4»pyridiayto (i : 1). Synonyms and analogs of PT /T . are known a Vatatolb, CGP79787D, PTK787/ZK 222584, CGP-79787, DE-00268, ΠΈ1~787, PT 787A, VEGFR-TK inhibitor, ZK 222584 and ZK,

Chemotherapeutic agents that can be used, in admixtures and/or cot OTulaiions and/or conjugated with raonoterpene or sesquiterpene (or an isomer or analog of monoterpene or sesquiterpene) may also include arnsacrine, Trabectedin, retinoids (Alitretmom, Tretinoin), Arsenic irloxlde, aspsragine deplete?^' Asparaginase/ Pegaspargase),, Ceteeoxih, De eco!cine, ElesclomoL Elsamitrnciri, Etoglueid, Lonidatnine, Lueanthone, M oguazone,. Mitotane, GbMniersen, Ternsirolimos, and Vorinostat. The man terpene or sesquiterpene (or a isomer or analog of nionoterpene or sesquiterpene) may be conjugated and/or used m admixtures and/or cofonmdations with angiogenesis inhibitors. Examples of angiogenesis inhibitors include, but are not limited to, angidstatm, angiozyme, antithrorabixt ill. AG3340, VEGF inhibitors ba imastai bevaciztimab (avast-β), BMS-275291, CA1, 2C3, HtiMV833 Cansiatrn, Captopril, carboxyarnidotriaxoie, cartilage derived inhibitor (GDI), CC-5013, 6-0-(c oroacetyl-C boi¾yl)-t¾«agi!lo!, COL-3, eornbretasiatin, cornbretastatin A.4 Phosphate, Daheparin, EMD 121974 (Cilengkide), endostatift, erlotioib, gefitinib (Iressa), genistein, faalorugiaone ydrobromide, Ml, id3, ΪΜ862,. ititiatmife mesylate, IMC-iCU fed ucible protein 10, terferoft-alpha, raterleukin 12, lavenditstin A, LY3 I 7615 or AE-9 1 , marimastat, inspin, niedroxpregestefone acetate, Mefh-i, eth-2, 2* .methoxyestradiol (2- E), neoyastat, oteopontm cleaved product, PEX, . pigment epithelium growth facto (PEGF), platelet factor 4, prolactin fragment proH.ferin-reiatecl protein (PR.P .PTK787/ZK 222584, ZD6474, recombinant human platelet factor 4 (rPF4), restin, sqoalamine, SU5416, SU6668, 801 1248 suramin, Taxol, Teeogaian, thalidomide, thfombosposdifc, TNP- 470, troponin-!, vasostatm, V^'EGi , VEGF-Trap, and ZD6474.

Mon-Hniiting examples o f an giogen esis inhibitors- also include, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Fit- 1 (VEGFRl) and Fik-l/ DR (YEGFR2), inhibitors of epiderma!-der ved, fihroblast-derived, or piateiet. derived growth factors, M P .(matrix meialloprotease) inhibitors, iniegrin blockers, pentosan poiysulfate, angiotensin H antagonists, cydooxygenase inhibitors (including non-steroidal antWnil nmmor drugs

(MSAlDs) such, as aspirin and. ibnprofen, as well as selective cycIooxygenase-2 inhibitors such, as eelecoxib and rofecoxib), and steroidal anti-inflaiiiniatories (such as corticosteroids,

fflinera!ocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone).

Other therapeutic agents that modulate or inhibit angiogenesis and may also be

-conjugated and/or used in admixtures and/or coformulatiohs with monoterpene or sesquiterpene (or an isomer or analog of monoterpene or sesquiterpene) include agents that modulate or inhibit the coagulation and fibrinolysis systems, including, but not limited.- to, heparin, low molecular weight heparins and catboxypeptidase X) inhibitors (also Imown as inhibitors of acti ve thrombin aetivatable fibrinolysis inhibitor [TAffe]). IIS. Patent Publication No. 200P032S 39, U.S. Patent No, 7,638,549. Noit irffitkg examp es of the anti-trypeT£em ¾ agents melude angiotensin converting enxy e inhibitors (e,g._ captoprii, eualapril, delapril etc.), angiotensi 0 antagonists (e.g., candesartars cikxetil, candesati¾tt₅ losartan (or Cogaar k losarian potassium, eprosar an, valsartaii (or Dioyan), ternusaitan, irbesartan, iasosartan, oimesartan, oiniesartaa medoxonril etc.). calcium antagonists (e.g., inatudipine, nifedipine, arntodipine (or AmJodia), efotudipine, nicardipine etc.), diuretics- renin inhibitor (e.g., aliskiren etc.), aldosterone antagonists (e.g., spironolactone, epterenone etc ), beta-blockers (e g., metoprolol (or Toporof), atenolol propranolol, earvedi!ef pindolol etc.), vasodilators (e.g., nitrate, soluble guanylate cyclase stimulator or activator, jprostacycline etc_*), angiotensin vaccine, cloxtidine and the like. U.S. Patent Publication No. 20100113780.

Oilier ^'therapeutic agents that ma be conju ated, (or an isomer or a alog of nionofer ene or sesquiterpene) with nionoterpene (or sesquiterpene) include, but are not limited to, Sertraline (Zoloft), Topiraraafe (Topamax), Didoxetine(€ytnbalta), Sumatriptan (Imitrex), Pregahalin (Lyrica), Laroottigine (Lamictai), Vaiacieiovir Vajtrex), TarMalosi (Flomax), Zidovudine (Combtvir), Lamivudine (Combivk), Etavirertz (Siistiva), Abaeavir (Epzicon ), Lopinavir (Kalefra), Pioglitazone (Actos), Desloratidine (Clarinex), Ceiirizme (Zyrtec), Pentoprazols (Pratonix), Lansoprazole (Prevaeid), Rebeprazole (Aciphex), Mox t¾xaein (Avelox),

Meloxicani (Mobie), Dorzolamide (Truspot), ^'Diclofenac (Voltaren), Eniapril (Vasotec),

Monteluksst (Siftgitlair), Sildenafil (Viagra), Carvedilol (Coreg), Ra aipril (Delix).

Table 1 lists pharmaceutical agents that ca be conjugated with nionoterpene or sesquiterpene (an isomer o analog of monoterpene or sesquiterpene), including structure ofthe pharmaceutical agent and the preferred derivative for conjugation.

Carbamate

Oelix Ramipril Hypertension

or Este

Sineme

Neurological HCX Λ

(Parcopa_? L-DOPA ,

d sorders

Atamet)

By way ofexaorpfe, an L-DOPA iso-POH conjugate is sho wn below:

The parity of the nionoterpene or sesquiterpene or its deri vatives (an isomer or analog of mofiot rpene or sesquiterpene,., or its derivati ves) may be assayed by gas cinematography (G€) or high pressure liquid chromatography (HPLC)> Other techniques for assayin the purity of nionoterpene (or sesquiterpene) derivatives and for determining the presence -of imparities include, but are not limited to, nuclear magnetic resonance ( ) spectroscopy, mass spectrometry (MS), GO-MS, infrared spectroscopy (I ), and thin layer chromatography (TLC). Chiral parity cm be assessed by ehkal GG or measurement of optical rotation. The monoterpene or sesmilterpene or its derivatives (an isoraer or analog of monoterp me or sesquiterpene,, or its derivatives) m be purified by methods suc as crystallization, or by separating the morsoterpene or sesquiterpene or its derivatives (an isomer or analog of nionotetpene or sesquiterpene, or its derivatives) from irapiuities according to the unique- physieochenucal properties (e.g., solubility or polarity) of the derivative. Accordingly, the monoterpene or sesquiterpene or its derivatives (an isomer or analog of nionotefpene or sesqutterpene, or its derivatives) can be separated by suitable separatiori: techni ues know in the art, such as preparative chromatography, (fractional) distillation, or (fractional) crystallization, The invention also provides for methods of using monoterpene or sesquiterpene or its derivatives (aft isomer or analog of monoterpene or sesquiterpene, or its derivatives) to treat a disease, suc as cancer or other nervous system disorders. The present compound may be administered alone, or in combination with radiation, surgery or chemotherapeutic agents. The present compound may also be co-administered with anti iral agents, anti-inflammatory agents or antibi tics. The agents may be administered concurrently or sequentially. The present compound can be administered before, during or after the admini stration of the other acti ve agent(s).

The present compound may be used in. combination with radiation therapy. In one embodiment the present disclosure provides for a method of treatin tumor ceils, such as malignant glioma cells, with radiation, where the ceils ar treated with an effective -amount of the present compound, and then exposed to radiation. TreatMent by the present compound may be before, during and/or afte radiation. For example, the present compound may be administered continuously beginning one week pr ior to the initiation of radiotherapy and continued for two weeks after the completion of radiotherapy. U.S. Patent Nos. 5,587,402 and 5,602,184,

In one embodimen t, the present invention provides for method of treatin tumor ceils, such as malignant glioma ceils, with chemotherapy, where the ceils are treated with an effective amount of the present compound, and then exposed to chemotherapy. Treatment by the present compound ma he before, during and/or after chemotherapy.

The compounds and methods of the present invention may be used to inhibit the Has protein. The Ras family is a protein family of small GTPases that are involved in .cellular signal transduction. Activation of Ras signaling causes ceil growth, differentiation and survival. tatlons in ras genes can permanently acti vate it artel cause Mapprepriate transmission inside the cell evert in the absence of extracellular signals. Because these- signals result in cell growth: and division, dysreaulated Ras sisnalhts can ultimately lead to oncogenesis and cancer. Aetivatiiia mutations in Ras are found in 20-25% of all human tumors and up to 90% in specific tumor types. Ooodseit BS (1999). Downward J., "The molecular perspective; the ras oncogene", Oncologist 4 (3): 263-4, (January 2003), "Targeting RAS signalling pathways in cancer therapy", Nat. Rev, Cancer 3 (1): ί 1 22, Ras family members include, ^'bat are not limited to, BRAS; KRAS; NRAS; DIRASI; DIRAS2 D1RAS3; ER AS ; GEM; MR AS; ^'N. IRAS 1;

NRIRAS2; NBAS; RALA; RALB; RAPiA; RAP IB; R AP2A; RAP2B; RAF2C; RASD1;

RASD2; RASL10A; RASLiOB; RASLllA; RASL-11B RAS L 12; REM! ; REM2; RERG;

RERGL; ERAD; RRAS; and ERAS. Weflftetberg , Rossman L, Der O (March 2005). "Th Ras superfamily at a glance", J. Ceil, Sci. I I 8 (Pi 5): 843-6.

The present compound may be used for the treatment of nervous system cancers, such as a malignant glioma (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma multiforme), re inoblastoma, piiocytie astrocytomas (grade 1), meningiomas, metastatic brain tumors, neuroblastoma, pituitary adenomas, skull base meningiomas; and skull base cancer. As used herein, the term "nervous syste tumors" refers to a condition in which a subject has a malignant proliferation of nervous system ceils.

Cancers that can be treated by the the present compound include, but are not limited to, fattg cancer, ear, nose and throat cancer, leukemia, colon cancer, melanoma, pancreatic cancer, mammary cancer, prosiate cancer, breast cancer, hematopoietic cancer, ovarian cancer, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; iutra*epitheliai neoplasm; kidney cancer; larynx- cancer; leukemia including acute myeloid leukemia, acute lymphoid leukemia, chronic- myeloid leukemia, chronic lymphoid leukemia; liver cancer; lymphoma Including B dgk n's and N on-H dgkin's lymphoma ;

myeloma; fibroma, neuroblastoma; oral cavity cancer (e.g., Bp, tongue, month, and pharynx) ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the respiratory system; sarcoma: skin cancer; stomach cancer; testicular cancer; thyroid cancer- uterine cancer; car er of the m nary system, as well a s other carcinomas and sarcomas. ^' U.S. Patent No. 7,601,355.

The present methods md compounds may be used to treat: CNS disorders, including, without limitation, primary degenerative neurological disorders such as Alzheimer's,

Parkinson's, psychological disorders, psychosis and depression. Treatment may consist of the use of the present compound alone or in comb ination wi th current medi cations used i the treatment of Parkinson's, ALdieimer or psychological disorders.,

The present invention also provides a method of improving imniimomodttlatory therap responses comprising the steps of exposing cells to an effective amount of the present

compound, before or during immunomodulatory treatment. Freten'ed immunomodulatory agents are cytokines, such Interleu ns, Iyrnphokines, monokines, iutefi%reons and ehemokines.

The present composition may be administered, by an method known in the art, including, without limitation, intranasal, oral tran derma , ocular, inttaperitonea!, inhalation, inu- venous, ICV, intraei sternal injection or infusion, subcutaneous, implant, vaginal sublingual, urethral (e.g., urethral suppository), subcutaneous, Intramuscular, intravenous, rectal, sub-lingual, mucosal, ophthalmic, spinal, intrathecal, intra-articular, tra-arterial, siib-arachinoid, bronchial and lymphatic administration. Topical fe mrfation. may be in the form of ge l ointment, cream, aerosol, etc; intranasa formulation can be delivered as a spray or in a drop; transdermal

formulation may he administered via a transdermal patch or iontorphoresis; inhalation

formulation can be delivered using a nebulizer or sirnilar device. Compositions can also take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions.

To prepare such pharmaceutical compositions, one or more of compound of the present disclosure may be mixed with a pharmaceutical acceptable carrier, adjuvant and/or ex!eipteni* according to conventional pharmaceutical compounding techniques. Pharmaceutically acceptable carriers that ca be used In the present compositions encompass any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oi I emulsion, and various types of wetting agents . Th compositions can additionally contain solid pharmaceutical excipierit such as starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt rice, iour, chalk, silica gel, magnesium stearate, sodium siearate. glycerol i O«osiea % sodram chloride, dried sfelrn rnilk arid the like. Liquid and semisolid excipients. ma be selected from glycerol, propylene glycol, water, ethanoi and various oils* including those of petroleum, animal, vegetable or .synthetic origin, e.g., peanut oil, soybean oil, mineral oil. sesame oil, etc, Liquid carriers, particularly for injectable solutions, include water, sa line, aqueous dextrose_* and glycols. For examples of carriers,, siabiikets and adjuvants, se Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishin Company. IStb ed.,, 1.990), The compositions also can include s abilise s and preservatives.

As used herein, the term ^herapeutieaM effective amount" is an amount sufficient t teat a specified disorder or disease or alternatively to obtain a pharmacological response treating a disorder OF disease. Methods of determining the most effective mean s and dosage of amninistration can vary with the composition used for therapy_* the purpose of the therapy, the target cell being treated, and the subject being treated. Treatment dosages generall may be titrated t optimize safet and efficacy. Single or multiple administrations can be carried out. with the dose level and pattern being selected b the treating physician. Suitable dosage formulations and methods of administering the agents can be readily determined by those of skill in the art. In certain embodiments, the composition is administered at about 0,01 mg kg to b ut 200 nig kg, about 0.1 mg kg to about 100 mg/kg, or about 0.5 mg kg to about 50 nig/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.

Transdermal formulations may be prepared by incorporatin the activ agent in a thixotropic or gelatinous carrier such as a eeliulosie medium, e,.g._s methyl, cellulose or hydroxyethyl cellulose, with the resulting formulation then being packed in a transdermal device adapted to be secured in dermal con tact with the skin of a wearer , if die composition is in the form of a gel, the composition may be rubbed onto a membrane of the patient, for example, the skin, preferably intact* clean, and dry skin, of the shoulder or upper arm and or the upper torso, and maintained thereon for a period of time sufficient for delivery of the monoteipene (or sesquiterpene) derivative to the blood serum of the patient. The composition of the present Invention in gel form ma be contained in a tube, a sachet, or a metered p p_* Such a tube or sachet may contain one unit dose, or more than one unit dose, of the composition . A metered pump ay be capable of dispensing one metered dose of the composition_*

This invention also provides the compositions as described above for intranasal adnhnisttetlofl. As such, the compositions can tot er comprise a permeation enhance Sourhali et ah Developments in Nasal rog ^'Delivery, 2000, The monoterpene (or sesquiterpene) derivative amy be administered intfanasat!y in a liquid form such as a solution, an emulsion, a suspension, drops, or in a solid form such as a powder, gel. or ointment Devices to deliver Intranasal medications are well known in. the art Nasal drug deli very can be carried out using devices including, but not limited to, intranasal inhalers, intranasal spray devices, atomizers, nasal spray hotties, unit dose containers, pumps, droppers, squeeze bottles, nebultee.rs, metered dose inhalers (MDi), pressurized dose inhalers. insufflators, .and bi-directional devices. The nasal delivery device can be metered to administer an accurate effectiv 'dosage amount to the nasal cavity. The nasal delivery device can be for single unit deliver}-' or multiple unit delivery. In a specific example, the ViaNase Electronic Atomizer torn urve Technology (Bethel!_*

Washington) can be used in this invention {Mtp"/ ^'www.kurvetecb_:hcorri . The compounds of the present invention may also be delivered through a tube, a catheter, a syringe, a packtail, a pledget, a nasal tampon or by submucosal infusion. ITS_* Patent Publication os, 20690326275, 20090291894, 20090281522 and 20090317377.

The present compound can be formulated, as aerosols using standard procedures. The compound ma be formulated with or without solvents, and formulated with or without carriers. The formulation may be a solution, or may be an aqueous emulsion with one or more surfactants_* For example, an aeroso spra may he generated from pressurized container with, a suitable propellant such as, dichlorodifluoroniethane, trichlorofl oroniethane, dichlortnetraflooroethane, Irydroearbons, compressed air, nitrogen, carbon dioxide, or other suitable gas. The dosage uni can be determined b providing a valve to deiiver a raetered. amount. Pump spray dispensers can dispense a metered dose or a dose having a specific particle or droplet si¾e. As used herein, the ^•term "aerosol" refers to a suspension of fine solid particles or liquid solution droplets in a gas. Specifically, aerosol includes a gas-borne suspension of droplets of a. monoterpene (or sesquiterpene), as may be produced in any suitable device, such as an MDI, a nebulizer, or a mist sprayer. Aerosol als includes a dry powder composition of the composition of the instant Invention suspended in air or other carrier gas, Gonda (.1.990) Critical Reviews in Therapeutic Prog Carrier Systems 6:2?^'3-313, Eaebum et al._} (1992) PharmacoL Toxicol. Methods 27: 143- .159, The present compound may be delivered to the nasal cavity as a powder in a form suc as microspheres delivered b a .nasal Insis ator. Hie present^' compound may be absorbed to a solid surface, for example, a carrier. The ^'powder or microspheres may be administered in a dry. air- dispensable form. The powder or -microspheres may be stored in a container of the insufflator. Alternatively the powder or -microspheres may be filled into a capsule, such as a gelatin ca sule_* or other single dose unit adapted for nasal administration.

The pharmaceutical compositi n, can be deli vered to the nasal cavity by direct placement of the composition in the nasal cavity; for example, in the form, of a gel, an ointment, a nasal emulsion, a lotion* a cream, a rsasat tarnpoji, a dropper, or a bidadhesive strip_* in certain embodiments, it can be desirable to prolon the residence time of the pharmaceutical

composition in the nasal cavity, for example, to enhance absorption-. Thus_s the pharmaceutical composition can optionally be- fotmulated^' with a hioadhesive polymer, a gum (e.g., xanthan gum), ehiiosan (e.g., highly purified cationic polysaccharide), pectin (or any carbohydrate that thickens like a gel or emulsifies when applied to nasal ucosa), microsphere (e.g., starch, albumin, dextran, cyelodextrin), gelatin, liposome, earhamer, polyvinyl alcohol, alginate, acacia_; chitosans and/o cellulose (e.g. , methyl or propyl; hydroxyl or carboxy; carhoxymethyi or hydiOxy!propyl).

The composition containing the present com und can be administered by oral Inhalation into the respiratory tract,, i.e., the lungs.

Typical deli very systems for inhalabie agents include nebulizer inhalers, dry powder inhalers (DPI), aBdmetered-dose inhalers (MDI).

Nebulizer devices produce a stream of high velocity air that causes a therapeutic agent in the form of liquid to spray as a mist. The therapeutic agent is formulated in a liquid form such as a solution or a suspension of particles of suitable -size. In one embodiment, the particles are microriized. The term "micronized" is defined as having about 90% or more of the parti cles with a diameter of less than about 10 μιη. Suitable nebulizer devices are provided commercially, for example, by PAM GmbH (Starnberg, Germany). Other nebulizer devices include Respirnat (Boefarmgef Ingelheim) arid those disclosed in, Ibr example, ITS, Patent NQS. 7,5 S,4$0 and 6,123,668, and WO 97 12683, The monoter enes (or sesquiterpenes) can be formulated for use hi a nebulizer device a an aqueous solution or as liquid suspension. DPI de ices typically administer a therapeutic agent In the form of a §ee t owing powder that^' can be dispersed in a patient's air-stream daring inspiration, DPI devices which use m external energy source may also be used in the present invention, ia order to achieve a free- flowing powder, the therapeutic agent can be formulated with a siiitabie exciptent (e.g., lactose). A dry powder foraiulaiion can be made, for example, by combining dry lactose having a particle size between, about^"! m and 100 μηι with niieroRized particles of the raonoterpeoes (or sesquiterpenes) and dry blending. Alternatively, the moaoterpene- can be formulated without exeipients. The forrnrslstion is loaded into a dry powder dtspenser, or into inhalation cartridges or Capsules for use with a dry powder delivery device. Examples^' of DPI devices provided comajercMiy include Dislcliaf er (GlaxoSraithKIine, Research Triangle Park, C.) see* e.g., OS. Patent ^'No, 5,035,237); Disfcus (GlaxoSmithKiine) (see, e.g., U .S. Patent No. 6,378,519; Turbrfmler (AstraZeneca, . Wilmington, Del.) (see, e.g., U.S. Patent No. 4,524,769); arid

otahaler (GlaxoS ithiKline) (see, e.g., U.S. Patent No. 4,353,365), further examples of suitable DPI devices are described in U.S. Patent Nos. 5,415,162, 5,239,993, and 5,715,810 and references therein,

MDI devices typically discharge a measured . amount of therapeutic agent usin

■compressed propellant gas, Farmutadoas^' for MDI adnunistraiion include a solution or suspension of active ingredient in a liquefied propejlan - Examples of propellants include hydromroroalklanes (HFA), such as 1,1 ,1 ,2-tetraflooroethane (HFA 134a) and 1 ,1 ,1 ,2,3,3,3- heptaf1aoro- i-propa e, (HFA 227), and ehlorotlooroearhons, such as€ ¾F. Additional components of HFA formulations for MDI administration include co-solvents, such ss-e hariol. pentane, water; and surfactants, such as sorbitan trioleate, oleic acid, lecithin, and glycerin,. (See, tor example, L S. Patent No, 5,225,1 S3, EP 0717987, and WO 92/22286). The formulation is loaded into an aerosol canister , whic tonns a portion of a MDI device, Examples of MDI devices developed specifically for use with HFA propellants are provided hi U.S. Patent Nos, 6,006,745 and 6,143,227, For examples of rocesses of preparing suitable formulations and devices suitable for inhalation dosing see U.S. Patent Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, awl WO 9 /$3901, WO 00/61 108, WO 99/5S31f and WO 00/30614.,

The present eompotuid mm he encapsulated ia liposomes or microcapsules lor delivery via inhalation, A liposome is a vesicle composed of a lipid bilayer membrane and a aqueous interior, The lipid membrane may be made of phospholipids* examples of which include phosphatidykhoflne suc as lecithin arid lysolecitiiki; cinic phospholipids such as phosphatklyiserine and phosphatidylglycerol; and. sphingophospholipids such as

phosphaddylethanolainine and sphingomyelin. Alternatively, cholesterol ma e added. A microcapsule is. a panicle coated with a coating material. For example, the coating material may consist of a mixture of a flnn-forming polymer, a hydrophobic plastic i¾e.r, a surface activa n agent or/and a lubricant »itroge«H∞ntamrag polymer. U.S. Patent Nos. 6,313,176 and 7,563,768,

The present compound may also he used alone or i combination, with other

ehe otherapeutte agents via topical application for the treatment of localized cancers such as breast cancer or melanomas. The present compound may also be used in combination with narcotics or analgesics for transdermal delivery of pain .medication.

This invention also pro ides the compositions as described above for ocular

administration. As such, the compositions can further comprise a periaeation eniiaucer. For ocular administration, the compositions described herein can be formulated as a solution, emulsion, suspension, etc, A variety of vehicles suitabl 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 ,

The present compound can be given, alone or in combination with other drugs for die treatment of the above diseases for a short or prolonged period of time:. The present

compositions can be administered to a mammal, preferably a human. Mammals incl de, but are .not limited to, murines, rats, bbit, simians, bovines, ovine, porcine, canines, feline, to animals, sport animals, pets, equine, and primates.

The invention, also provides a method o inhibiting the growth of a cell in vitro, ex viva or m viva, where a cell, such as a cancer ceil, is contacted with an effective amount of the compound as described herein.

Pathological cells or tissue such as hyperproliferative cells or tissue may be treated by contacting the cells or tissue with an effective amount of a composition of this disclosure. The cells, such, as cancer <¾!¼, can h primary cancer ceils or can b■cultured cells available fern tissue banks such as the American Type Culture Collection {ATCC). The■ pathological cells can be cells of a systemic cancer, gliomas, meningiomas, pituitary adenomas, or a CMS .metastasis from a systemic cancer, lung cancer, prostate cancer, breast cancer, hematopoietic cancer or o arian earicer. The cells can be itora a vertebrate, preferably a m mm l, roore preferably a human. U.S. Pate-ni Publication No. 2004/008765 L Batassiano et ai (2002) Mem. J.. Mot Ivied. 10:785-788. l¾or»e, et ai (2004) Keuroscience 127:481 -496. Femandes, etal (2005) Oncology Reports 13:943-947, Pa Fonseca, et al. (2008) Surgical Neurology 70:259267, Da Fonseca, et at (2008) Arch Jn»n^^ 56:267-276. Hais izume, et al, (2008)

Neutoncoiogy 10:1 12-120.

In vitro efficacy of the present composition ca» be determined using methods well known in the art. For example, the eytoxieity of the present monoterpeae (or sesquiterpene)^■and/or the therapeutic agents ma be studied by MTT [3~(4_J5~dinietiiy1¾lazo1-2-yi}-2

tetrazoliiim bromide) cytotoxicity assay. MTT assay is based on the principle of uptake of MIT, a tet azoliuffi salt, by rnetabolicaliy active cells where it is metabolized into a blue colored fbrmazoii product which can be re d' spectrometries iy. J, of immunological M ethods 65; 55 63,. 1983. The cytoxicity of the present nionoterpene (or sesquiterpene) derivative and/or th therapeutic agents ma be studied by colony formation assay . Functional assays fo inhibition of VEOF secretion and IL-S secretion may be performed via ELISA. Cell cycle block by the present monoterpene (o sesquiterpene) derivative and/or the therapeutic agents may be studied by standard propidmni i.odi.de (PI) staining and flow cytometry. Invasion inhibition may be studied by Boydett chambers. In this assay a layer of reconstituted, basement membrane ,

Matriget is coated onto chemoiaxis liters and acts as a barrier to the migration of cells in the Boyden chambers. Onl cells with in vasive capacity can cross the atrigel barrier. Other assays include, but are not limited to ceil viability assays_s apOptosis assays,, and morphological assays,.

The following are examples of the present invention and are not to be construed as limiting.

Examples

Example ?:; Synthesis of Dimethyl Celecoxib bisPOH Carbamate (4~hIs~M,M ~isopr©pe«yl :cyeIolex-l«enyImethyioxy carbony! [5»(¾5«dimethyl pl)e«yl)*3'-triiuororaetfcyi pyrazol-l-yl] berttenesnlfonami de) Tjbe reaction scheme Is the following:

Ffeosgene (20% m toluene, 13 nil 26.2 mmoi) was added, bo a mixture of peril!y! alcohol (2.0 grams, 13 J mmol) and potassium carboriate (5,4 grams, 39, 1. mmol} in dry toluene (30 mL) ove a period of 30 minutes while maintaining the temperature between 1 ° C to 15" C The reaction mixture was allowed to warm to room temperature and stirred tor 8.0 hours under Hz. 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 layer was 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 perlltyl eWoroforraate as an oil Weight; 2.5 grams Yield: 9%, (400 MHz, CDCfe): 4 1.5 im. IB), 1.7 (s_? 3H), 1.8

(in, 1 H), 2.0 (m, 1 H), 2.2 (m, 4H), 4,7 (dd. 4H); 5.87 (m, ί H),

Fetill l chlorof rmate (0.1 1 grains, 0.55 aimol) was added slowly to a mixture of dimethyl ceiecoxib (0.2 grams, 0.50 mmol) arid potassium carbonate (0.13 grams, 1.0 rnol) in dry acetone (1.0 mL) over a period of 5 minutes under Ms. The reaction mixture was heated to reflux and, maintained for 3 hows. Since TLC analysis indicated the presence of dimethyl ceiecoxib (> 60%), another L0 equivalent ofperii!yl eh!ofoformate was added and reffuxed for an additional 5 hours. The reaction mixture was cooled and acetone was concentrated under vacuum; to gi ve a .residue. lie resulting residue was suspended I» water (1.5 aiL) and extracted with ethyl acetate (3x15 wiL). The combined, orgast c layer was washed witfc water (20 r L) followed by brine (15%, 20 mh) and dried oyer 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] and ehited with hexanes (i 00.a¾L) followed by a mixture of exaoes/eihyi acetate (95:5, 100 mL). The hexane/ethy! acetate fractions were combined and concentrated under vacuum to gi ve gummy mass.

The product POM carbamate exhibited a weight of 120 rag and a yield of 1%. ' H-N R (400 MHz, CDC¾); 3 0.9 (m, 2H), 1.4 ^'(m, 2H), 1.7 Cm,, 7H*), 1.95 (m, 8H* >. 2.1 (m, 4H), 2.3 (s_? 3H), 4.4 (d, 2H), 4.7 (dd_s 2B% 5.6 (fer d, 2Mk 6.6 (s, 1 H), 7.0 ( r s, 1 E}„ 7.12 (d, 1 H), 7.1 (d, i H), 7.4 ¾ 2H), 7,8-5 (d, 2H); MS, m/e: 75 ^' (!vf 3%), 574,3 (100%), 530.5 (45%), 396 (.6%), * N.B. further 2!I overlapping iron* presumed impurity discounted in NME intentio .

Example 2: In viim Cytotoxicity Studies of Dimethyl Celecoxi bisPOH Carbamate (POH* DMC)

First cytotoxicity assays were, carried out after cells were treated with dimettryl-celecoxib (DMC) alone. Figure 1 shows the results of the MTT cytotoxicity assays performed oft human mali&nant alioma -cells 087, A172 and^'U25I with DMC alone.

Then US?, Ai 72 and 0251 cells wer treated with dimethyl celecoxib bisPOH carbamate (POH~DMCj (£.¾., synthesized b the method in Example I ), and the MTT cytotoxicity assays performed (Figure 2). The results suggest that POH carbamate PQH-D C exhibited much better cytotoxicit than DMC alone,

Example 3: Synthesis of Temozolomtde FOB Carbamate (3-methyl 4-oxo-3,4- dihydroiinida2o[5J-d|[l ,2_;3,5 tetra ;me a.cid_'4-isopropeByi cyclohex- 1 - euylmethyf ester

The reaction scheme is the following;

Oxalyl chloride (0.13 grams, 1. tnmol) was added slowly to a mixture of temozo!omide (OChem Incorporation, 0.1 grams, 0.5 ni oS) in f;2~dichloroeth&ne (10 mL). over a period of 2 minutes while inaintainin the temperature at 10° C under f¾. The reaction .mixture was allowed to warm, to room temperature and then heated to reflex for 3 'hours.. The excess of oxalyl chloride and ί ,2-dicbloroeihane were removed by concentration under vacuum. The resulting residue was re-dissolved in 1 _;2~dichloreihane (15 mL) and the reaction mixture was cooled to WC under Nj A solution of pertllyl alcohol ( .086 gram .•Q.5^:£tn»K>I) m L,2-dichlo.roethaoe (3 mL) was added over a period of $ minutes. The reaction mixture was allowed to warm to room temperature and stirred for 14 hours. 1 ,2-dic oroethane was concentrated under vacuum to give a residue, which was triturated with hexanes. The resulting yellow solid was filtered and washed with hexanes. Weight: 170 nig; Yield: 89%. ^lH N H (400 MHz, &Χ%): 3 1 ,4-2.2 (ra_; tOH), 4.06 (s, 3H), 4.6-4.8 (ni, 4H), 5.88 (brs, 1H), 8.42 (s, IH), 9.31 (br s, IH); MS, no molecular ion peak was observed, rnfe 314 (.1 0%), 286.5 (17%), 136 (12%).

Alternatively, temoxolomide POH carbamate was synthesized according to the following procedure, Oxalyl chloride (0.13 grains, 1 ,0 mmol) was added slowly to a mixture of

ieinozoloraide (OChera incorporation, 0,1 grams, 0.5 tnmoJ) in 1 ,2-dichloroeihane (10 mL) over a period of 2 miiiutes while maintaining the temperature at 10 °C under !¾. The reaction raixttare was allowed to -warm to room temperature and then, heated to reflux for 3 hours. The excess of oxalyl chloride and 1,2-dkiiioroethaae were removed by concentration under vacuum. The resulting residue was re-dissolved in 1_^2-dlchloreihane (15 mL) and the reaction mixture was cooled to 10 °C under N2. A solution of periilyl alcohol (0.086 grams, 0.56 ramol) in 1,2- dkhloroethane (3.«iL) was added over a period of 5 minutes. The reaction mixture was allowed to warm to room temperature and stirred for .14 hours. 1 _f2~DicIii0roethane was concentrated uftder vacuum to give a residue, whic was purified by a short silica-plug colum (colurnn dimensions: diameter: 2 cm, height: 3 cm, silica: 230-400 mesh) and eluted with a mixture of hexanes/ethyl acetate (1 : 1, 100 mL), The hexMe ethyl acetate fiactkms were combines! and concentrated under vacuum to give a white solid residue which, wa triturated with heptanes and filtered to obtain a white solid. Weight: 170 mg; Yield: 89%. ^f B~NMR (400 MHz, CDC13): 1.4- 2.2 (ni, I^'OH), 4.06 (s, 3H}_? 4.6-4.8 (rn, H 5.88 (hr s_f 1 H}_; 8.42 (s, i B), 931 (hr s, 1 H); MS, no molecular ion peak was observed, tn e; 314 (100%), 286.5 ( 7%), 136 (12%),

Example 4: 1m v Cytotoxicity Studies of Temozoiomide POH Carbamate (POH-TMZ)

First cytotoxicity assays were carried oat alter cells were treated with temozoiomide (TME) alone, the staadartl alkylating agent used in the treatnient of malignant gliomas. Figure 3 shows the results of the MTT cytotoxicity assays performed on human malignant glioma cells IIS^'7, A372 and U251 with TMZ alone. Increasing concentrations of FMZ had niinhualcytotoxicity towards the ceil lines tested.

Then ^'f Z-resistan gliom cell lines 087, AI 72 and 11251 cells were treated wit teniozoloraide POH carbamate (POH-TMZ) (e.g., synthesized by the method in Example 3). The MTT assay results (Figure 4} showed that POH carbamate PQH-TMZ exhibited substantially higher kill rates of the various human glioma cells compared to TMZ alone.

.Example -5: Synthesis of Rolipram POH Carba ate (4~(3-cyclopeEtyioxy~4>-n'ieihoxy phe¾yi}~2>« oxo-pyrrolidine-l-earboxylic acid 4-isopropenyl.<^clohex^l-eny1meth l ester)

The reaction scheme is the following:

Fiiosgeiie (20% & toluene, 13 ml 26.2 ι«ΐ«ο1 was added/to a ixt&re of eriiiyl alcohol (2.0 grams, 13 J mrnol) and potassium carbonate (5,4 grams, 39.1 niniol) in dry toluene (30 nit) Over a period of 30 nunu es while maintaining the temperature between 10°C to 15"C. The reaction /mixture was allowed to warm to room temperature aod stirred for 8.0 hour under Ms. The reaction mixture was quenched with water (30 mL) and the organic layer separated. The aqueoiss layer was extracted with toluene (20 mL) aid the combined organic layer washed with water (50 rnL x 2), brine ( 15%, 30 mL) and dried ove sodium, sulfate (20 grams). The filtered. .organic layer was concentrate under vacuu to give periliyl chloror nnate as m oil. Weight: 2.5 grams; Yield: 89%, 1H-HM (400 MHz, CDCfe): 3 1.5 (m, I U\ 1.7 (s, Hi, 1.8 (m, IB), 2.0 (m, 1 in. 2.2 (m.. 411), 4.7 (del, 411); 5.87 (m„ IB).

Butyl lithium (2 ,5 M, 0 ,1.8 mL 0.45 ntot) was added -to a solution -of rolipram (GL synthesis, inc., 0J grams, 0.36 mrnol) in dry THF at -72° C over a period of 5 minutes under 1%. After the reaction mixture was stirred for 1,0 hours at -72^s C, periliyl chloroibrmaie (dissolved w 4 mL THF) was added over a period of 15 minutes while maintaining the temperature at -72" C. The reaction mixture was stirred fe 2.5 hours and quenc ed with saturated ammonium chloride (5 mL). The reaction mixture was allowed to warm to room temperature and extracted w th ethyl acetate (2x15 mL). The combined organic layer was washed with water ( 15 mL), brine (15%, 15 mL), and then dried over sodium sulfate: The filtered organic layer was concentrated to give an oil which was purified by column chromatography [column dimensions: diameter: 1.5 cm, height: 10 cm, silica: 230-400 mesh] and elated^■with a mixture of 8% ethyl acetate/hexanes (100 mL) followed b 12% ethyl acetiiie/ iexanss (1.00 mt). The 12% ethyl acetate hexanes fractions were conibiiied and concentrated under vacuum to yield a gammy solid. Weight; 142 nig; Yield: 86%. ^!H-NMR (400 MHz, CDC¾): S 1.5 (m_} 1H)₅ 1.6 ( % IB), 1.7 (s, 3H), 1.9 (m, 611 j. 2.2 ( , 5H), 2.7 (m, IH), 2.9 (m. I H h 3.5 (m, I B). 3.7 (m, I H i. 3.8 (s,.3H), 4.2 (m, ! H). 4.7 (m, 6B), 5.8 (for s, 1H)_S 6,8 (m, 3H); MS, m/e; 452.1 (M .53%), 274.1 (100%), 206,0 (55%),.

Exampl : ·6: i?¾ vitro Cytotoxicity Studies of Rolipram POH Carbamate (POH-i olipram)

To compare the cytotoxicity of Rolipram PQif Carbamate (P031-Rolipram} (e.g., synthesized by the method^' n Example 5) with rolipram, a type IV phosphodiesterase inducing differentiation, a i apoptosis in glioma cells, A 172» 087, U251 and LN229 hnman glioma coils were treated with eiiher FQH-Roliprain or rolipram for 48 hours. The MTT assay results are shown in Figures 5 to 8. POH-RoHpram exhibited substantiall higher kill rates compared to rolipram alone for each of the several different human glioma cell types. Figure 5 shows the MTT assay for increasing concentrations of rolipram and FOH-rolipratn for A- 172 cells.

^'Rolipram alone -demonstrates an IC50 of approximately 1000 uM (1 niM). I the presence of POH-rolipraiti, IC50 is achieved at 'concentrations as low as 50 uM. Figure 6 shows the MTT assay for increasing concentrations of rolipram wi th. U-87 cells. IC50 Is not met at 1.000 tiM. On the other hand, ICSO iss achieved at 180 iM with POH-roliprani. Figure 7- shows that 1C50 for rolipram alone for U251 cells is achieved at 170 uM; plateau cytotoxicity is reached at 60%. POH-rolipraro achieves S-C50 at SO .^'u. , with almost 1.00% cy toxicity at 1.00 uM. Figure 8 shows that IC50 for rolipram alone for LN229 ceils is not achieved even at 100 uM On the other hand, C-50 for POH-mlipram is achieved at 100 uM_s with almost 100% cytotoxicity at 10 uM,

Example 7: In vim Tumor Growth inhibition by POH Fatty Acid Derivatives

inhibition, of tumor growth b butyryi-POH was- studied in: a n ude mous subcutaneous glioma model. Mice were injected with U-S7 glioma eels (500,000 cells/injection) and allowed to form a palpable nodule ove two weeks. Once palpable nodule was formed, the mice were treated with, local application of various compounds as indicated in Figures 9A and 9B via a Q- tip (1 cc application/day) over a period of 8 weeks. Figure 9 A shows th images of subcutaneous 0-87 gliomas; in nude mice treated with butyryl-P H, purified impe il! yl alcohol h ving a purity greater than 98.5% ("purified POH"), POH pwchased.irom Sigma chemicals_* or phosphate buffered saline (PBS; negative control). Figure 9B shows average tumor growt over time (total time peri od of 60 days) . Buiyryl-POH demonstrated the greatest inhibition of turner growth, followed by purified POH and Sigma. POH.

Example 8: viiw Cytotoxicity Studies of Temoxoiomide (TMZ) and Temozoiormde POH

Carbamate (PQfrTMZ) o TMZ -sensitive and resistant glioma cells

Colon forming assays were carried out a ter ceils were treated with TMZ alone, POH alone, and the TMZ-POB conjugate. The colony forming assays were canied out as described in

Chen TC, et at Green tea epigatrocatechm galiate enhances therapeutic efficacy of

teroozolonride in orthotopic mouse glioblastoma models. Cancer Lett, 2011 Mar 28;302(2):i00~

8, Figure 10 shows the results of -the colony forming assays performed on TMZ sensitive (U251) and TMZ resistant (U251 TR) 0251 ceils with TMZ or TMZ~POH. TMZ demonst ated cytotoxicity towards TMZ. sensitive U251 cells, but had minimal cytotoxicity towards TMZ resistant U2S 1 cells. ΤΜΖ-ΡΌΗ demonstrated cytotoxicity towards both TM sensitive and

TMZ resistant U251 cells.

Figure 1 1 shows the results of the colony .forming assays performed on TMZ sensitive

(U2S 1) and TMZ resistant (0251 TR) U251 cells with POH. POH demonstrated cytotoxicit towards both TMZ sensitive and TMZ resistant U251 cells. POH -TMZ (Figure 10) exhibited sn stantialfy greater potency compared t FOB alone (Figure 1 1.) in the colony forming assays.

Example 9; In vii Cytotoxicity Studies of Temozoiomide POH Carbamate (POH-TMZ) on 11251 cells, U25ITR cells, and Normal Astrocytes

MTT cytotoxicity assays were carried out after cells were treated with the IMZ-POH conjugate. The MTT cytotoxicity assays were carried out as described in Chen TC, et ai. Green tea epigalloeatechm galiate enhances therapeutic efficacy of teniozoiomide in orthotopic mouse glioblastoma models,. Cancer Lett. 203.1 Mar 23;302(2); 100-8. Figure 12 shows the results of the MTT cytotoxicity assays performed on TMZ sensitive cells (025 ί% TMZ resistant cells

(U25 TR) and normal astrocytes, TMZ-POH demonstrated cytotoxicity towards both TMZ sensitive and TMZ^' resistant U 51 cells, but not towards normal astrocytes. Exampl 10; in vitro ^"Cytotoxicity Studies of Temomkwuide POH Carbamate (ΡΌΗ-ΊΜΖ) n BEC, TuBEC, and Normal Astrocytes

MTT cytotoxicity assays were carried out after cells were treated with the TMZ-POH conjugate.. The MTT cytotoxicity assays were carried out as described in Cfcen. TC_>. et ai. Greeu tea epigallocatechin gallate enhances therapeutic effeaey of temozolomidein orthotopic mous glioblastoma models. Cancer Lett, 201 1 Mar 28;3O2(2}: 10€ML Figure- .1.3 shows the results of the MTT cytotoxicity assays performed: on. normal astrocytes, team endothelial cells (BEC;

confluent and su confluent and wmox hraiu endothelial cells (TuBBC , TMZ-POil did not induce -significant cytotoxicity on norma! astrocytes, confluent BEC, or TuBEC, Mild to moderate cytotoxicity was demonstrated in subcontinent BEC M high concentrations of TMZ- POfi

E.xau¾pk Jj, /« vtiro Cytotoxic iiy Studies of Teuiozoloriiide (TMZ) and Temo^olorriide FQ I Carbamate (POB-TMZ) on USC-04 Glioma Cancer Stem Cells

MTT cytotoxicity assays were carried, out after cells were treated with the TMZ alone, POH alone, or the TMZ-POPI conjugate. The ^'MTT cytotoxicity assays were carried out as described in Chen TC, et a! . Greeu tea epigaJlocatechin gallate enhances therapeutic efficacy of temozolomide in orthotopic mouse glioblastoma models. Cancer Lett. 20.1.1 Mar 28;302(2); 1Q0- 8. Figure 14 sho s the results of the MTT cytotoxicity assays performed, on OS^'C-0 glioma cancer stent, ceils , TMZ did no Induce significant cytotoxicity with increasin eoneeotfations (0-400 -trM). TMZ-PGH demonstrated evidence of cytotoxicity with 1C50 at 150 uM. Figure 15 shows the results of the MTT cytotoxicity assay performed on USC-04 glioma cancer stem cells treated with POH. POH demonstrated cytotoxicity.- n USC-04 with increasing

concentrations (0-2 vaM%

Example 12: In vitro Cytotoxicity Studies of Teraozoloinide (TMZ) and Temozolornide POH Carbamate (POH-TMZ) on USC-02 Glioma Cancer Stem Cells.

MTT cytotoxicity assays were carried^' out after cells were treated with the TMZ alone*

POH lone or the TMZ-POH conjugate. The MTT cytotoxicity assays were carried out as described in Chen TC, et al. Green tea eoiga ocateehin galiate enhances mer^peurtc efficacy of temo oloraide in orthotopic mouse giiohlastorua models. Cancer Lett, 2011 Mar 28 302(2); 100- 8. Figure 16 shows the resalts of the MI cytotoxicity assays perfor med on USC-02 glioma cancer stem cells, TMZ did not induce significant cytotoxicity with increasing concentrations (0-400 uM)_t TMZ-POH demonstrated evidence of cytotoxicity with iCSO at 60 tiM. Figure 17 shows the results of the MTT cytotoxicity assays performed on OSC-02 glioma cancer stem ceils treated with PQH. PQH demonstrated cytotoxicity on OSC-02 with increasing conceritraiions (0~2.mM)> 'Example 3:7?? viiro Studies of ER stress by ^'Temozolomide FOB Carbamate (£OH~T 2) on

TM sensitive and resistant gliom cells

Western blots were performed after TMZ sensitive and resistant .glioma cells were treated with the TMZ-POH conjugate for S hr. Figure 18 shows a western blot demonstrating that

^"T Z-FOH induces ER stress (ERS) in TMZ sensitive and resistant U251 glioma cells,

Activation of the proapoptic protein: CHOP was shown at concentrations as bw as 60 uM of

TMZ-P0iX

Pr par iion^■Ό/4^ ^ψ ^^Ι,4^ϋ α^ψϊ^ό{4.5}ά&^-Μ- (2);

isopropyltriphenylphosphoninni iodide (83.02 g, 192 rnmol) was added to NaH (60%, in mineral oil, 8.38 g, 1 2 mmo!) in dry dimethyl sulfoxide (1.20 mL) at room temperature under nitrogen atmosphere. The reaction mixture was slowly heated to 50 °€ over a period of 15 mtn. and -maintained at SO °C until the reactio mass became a red color (approximately 30 thin). A. solution of 1 ,4-cyclohexaiiedione monoethylene ketal (1 , 30 g, 192 mmol) in dry dimethyl sulfoxide was added over a period of 45 ml» while keeping the temperature below 50 °C and the reaction was maintained at 50 ^ftC for 16 h. he reaction mixture was cooled to room temperature, quenched wit!) cold water (150 ml), and extracted with ethyl acetate (2*1.60 rat^;). The combined organic layer was washed with water (2x200 mL), followed fe brine (1.0%, 250 mL) and dried over sodiyj i sulfate. The filtered organic layer was concentrated to give a solid which was triturated with hexan.es (300 mL) and the precipitated iriphenylphosplrine oxide was filtered .off. The hexane layer was concentrated to give a oil which was pursfied by colum chromatography. [Column dimensions; dia 6 0 cm, height: 12 cm, silica: 200 mesh_* ehtted exanes (1.0 L) followed by hexase: ethyl acetate (97:3₅ 2.0 L)] The hexane: ethyl, acetate fractions were combined and concentrated under vacuum to give an oil. Weight: 23.36 g. Weight, yield: 66_*7%, !H- MR (400 MHz, CDC ): S IM .63 (t, 48), 1.64 <s, 6B), 2.2 (m, 4H), 3.97 (s, AH). MS (APC1 method): No molecular ion peak was observed.

Preparation qf4*iis0pF(> i e -eyel&he am>tie {$):^'

/i-Teluenesidmnie acid ( .16 g, 164 mmol) was added to a solution of fetal (2_:i .23.0 126 mmol) in acetone (2.3 L) and water (138 mL), The reaction mixture was heated to reflux and maintained at reflux for 3.5 h. The mixtu e was cooled to room temperature,. - treated with saturated sodium bicarbonate (60 mL) and concentrated under vacuum. The resulting oily residue was extracted with ethyl acetate (2^X 1.30 mL), washed with water (100 mL), then brine (100 mL), and dried over sodiu sulfate. The filtered organic layer was concentrated under vacuum to .give an oil. Weight; 16 g:. Weight yield: § %. ^JH-NMR (400 MHz, COCh) $ 1-69 ($, 6HL 2.35 (t, 4H), 2.50 (t, 4H)«. MS (APCI method) No moiecittat ion peak was observed (Note: 'E-NMR showed the presence of --2% ofketal 2 but used without purification).

MHz, CD¾): δ 1.42-1.50 (m, 2H), L55~1.61 (rn, 2BT 1.65 (s, 6B), 2.31 (i, 411), 2,61 (s, 2H). MS (APC1 method); Mo molecular ion peak was observed, Pmpamtton-of 3 ~dMtrobe a a id 4~($opropylkfene eyciohex^ enylmethyl ester (6):

Aluminum isopropoxide (5.93 g, 29,0 mmoi) was added to a mixture of epoxide (4, 4.0 g,

26.2 mmolj in toluene SO mL) and the mixture was heated to reflux for 7.0 h. The mixture was cooled to room temperature and quenched with s turated potassiiim sodiu tartrate solution. The organic layer was separated, washed with water (40 mL), followed by brine (40 mL), and dried over sodium sulfate. The filtered organic layer was concentrated under vacuum to give erode JsoperiHyl alcohol (5) as an oil. Weight: 4.0 g. Weight yield: 100%, l*urity: ·~85~90% (by GC area percent, Aetna! yield ca: 85%).

TriethylaftMrse (5.1 mL, 36.6 mmoi) was added o a solution of crude isoperillyi alcohol (5, 4.0 g, 26,2 mmoi) in dichloronietiiaiie (50 mL). After stirring for 15 rain, 3,5-diniirohen2oyl chloride (6.3 g, 27.5 mmoi) was added over a period of 0.25 h. The reaction mixture was stirred for 3.0 h and quenched, with water (30 mL), The organic layer was separated, washed with water (40 mL), and dried over sodium sulfate. The filtered organic layer was concentrated under vacuum to give a pale yellow solid (8.5 g), which was recrystallized from acetone to give pure ester 6 as a pale yellow solid,. Mp: 1 8-140⁹C (acetone). Weight: 5.7 g. Yield; 62% (fro epoxide). 1H-N R (400 MHz, CD{¾) 1.68 (s, 3H), 1.71 (s, 3H), 2.18 (t, 2H), 2.40 (t, 2H), 2.87 (br s_; 2H)_t 4.85 (s, 2B) 5.88 {s, III), 9.17 (i, IB), 9.24 is, tfl). MS (APCI method).: rn/e: 247 J (5%), 1.49.07 (7%), 135.1 (100%), 1.G7J (9%).

Preparation of isoperillyi alcohol (7);

Aqueous sodium hydroxide (1.43 g, 35,7 η ηιοί, dissolved in 12.5 mL of water) was added: to an ice-cold solution of 3, -dinitTOheiizoic acid 4-isopropylidene~eyclohex- ~erryimethy! este (6, 5.63 g, 16,2 mmoi) in methanol (56 mL) over a period of 0.25 h. The reaction mixture was allowed to warm to room temperature and then stirred for 3.0 h. The methanol was concentrated under vacuum to a minimum stirring volume and the mixture was suspended in water (40 mL), The resulting mixture was extracted with ethyl acetate {2 50 mL). The organic layer was washed with water (2*50 ml,), then brine (50 mL), nd dried over sodium sulfate_* The .filtered organic layer was concentrated under vacuum to give pure isoperillyi alcohol as an oil. Weight: 2.35 g. Yield: 95%. -Parit : 97% ( y GC AUC). (400 MHz, <&Κ¾): «5 1.65 (s, 1.69 (S, 3H),

1.77 (bs, 01¾ 2,09 (m, %H\ .2.33 (f,_; 2H), 2,79 (br s, 2H); ^tsC~HMR: <! 20.38,20.80 26.95, 27:60, 29.86, 67,49, 122.88, 123.04, 127,92, 138,37. MS (APCi method): ra/e: 152 ( ^* 3,5%), 135.07 (100 ¾% 107.12 (5%). However, the mass spectrum showed four small peaks (-5%) atM+:

207.06, 269,1, 287.09 & 301 which were not characterised.

Example 15. Alternative S nth sis of l -POM

The reaction scheme is the following:

Preparation ofifffluofomethmewifank: acid 4^o rapyi^d$n$ ydohex~l- fi- l ester (8):

2.5 M solution of n-Butyl lithium in liexaaes (5,6 raL, 14,1 aim®!) was added to a solution- of diisopropytaaiiue (1,98 rrsL., 14.1 rimol) m dr THF (30 mL) at -78 °C over a. period of 0,5 hr. After stirring for 1 ,0 h at -78 °C_S a solation of ketone (3, 1 ,3g, 9,4 mmol) in dry THF (10 mL) was added over a period of 10 r»i» while maintaining the temperature below ~78°C. The reaction mixture was stirred tot 1 ,0 h at -78 °C, A solution of pheuyltrifiirriide (3,53g, 9,86 mmol) in THF (15 mL) was added slowl while maintaining the temperature below -78 °C, The reaction ..mixture was slowl w¾tmed to 0 °€, maintained for 2,0 h at 0 °C and theft quenched with said ammonium chloride solution. The separated organic layer was washed with water (15 mL), brine (IS mL) and dried over sodium sulfate. The filtered organic layer was concentrated under vacuum, and the resulting residue was purified^' by column chromatography. [Column dimensions: dia: 6.0 cm, height: 12 cm, silica: 200 mesh, e!uted with hexanes (200 mL)] The similar fractions were combined and concentrated under vacuum whic gave an oil. Weight: 0, g. Weight yield: 38%. ¼-WMR (400 MHz, CDCh): 3 1 M is, 3¾ 1.71. (s, 3HX 2.37 (m, 2H), .2,46 (m, 2H), 2,91 {m, 2B)_> 5.73 (m, 1H). MS (APC method): No molecular Son peak was observed.

Mote-!: ^lH-NM indicated the presence of aromatic peaks (-5%) between 3 7.42-7.57 which were attributed to the by-product triiluoro- -phe.nyImetha»esulfonanHde. Note~2: The compound 8 was also synthesized in low yield ( 8%) using tfiflie anhydride in the presence t 2.,6^!^^-bu 'l^iij tliyipy8di«e as a base_*

Preparation qf ^'4 propyiicie evck>hex~i~emearboxy!ic aeid meihyt ester (9):

T a solution of compound 8 (G,2g, 0,74 mmoi) in N¾Hftmethylformamide (1.5 mL) was added methanol (1 ,0 mL), triethylamke (0.17 mL, 1.2 mmolk 1 ,3- bis(dlpbeny piH sphffio);propan.e (0,03 g, 0.07 mmoi) and palladium acetate (0.04g_; 0,07 mfflol). The reaction mixture was degassed and then stirred at .room tempefatme under carbon monoxide (balloo pressure) for 5 li. The reaction . mixture was diluted with ethyl acetate (15 mL) and washed with 0.5 M C (15 mL), brine (15 mL) and dried over sodium sulfate. The filtered, organic layer was concentrated under vacuum and the resulting residue wa purified by column chroma tography. [Column dimensions: dia: 6.0 cm, height; 12 em, silica; 200 niesh, e luted with hexanes (100 mL) followed by ethyl acetate; hexanes (2%. ISO mL)] Th similar fractions wore combined and concentrated under vacuum which gave an oil. While TLC analysis showed only a single spot, and GC analysis indicated that the isolated material was a mixture of two primary components thai co-eUited b TLC, Weight: 0, 1 1 g. Weight yield; 82%. ^!H-NMR (400 MHz, CDC ) indicated the presence of peaks corresponding to the methyl ester (9) along with an unknow impurity, GC analysis confirmed that it is mainly a mixture of two compounds with a ratio of 3:1. MS (APCI method); m/ei 180 (U 5%), 180.9 (M*¹ , 100 %). The other peaks (< 5%) at M+: ΙΨΐ, 247. & 274.0 were not characiefized. The etude mixture was taken forward without puiifieatiarr.

Preparation of isoperitiyl alcohol (?}: Methyl ester (9, 0.1 ! g, 0,6 rnmol) in dry THF ( 10 mL) was added to a cold solution of LAH (0.03 g_s 0.78 mmoi) m dry THF (10 mL) over a period of 2 min. The reactio mixture was slowly heated to reflux and maintained for 3.0 k The mixture was cooled to 5 °C and quenched with said sodium sulfate (1.5 mL). The precipitated lithium salts were filtered off and washed with hot ethyl acetate (20 mL). The filtrate was dried over sodium, sulfate. The filtered organic layer was concentrated under vacuum, which gave an oil. Weight: 74 rag. Weight yield; 79%. While TLC analysis showed only a single spot, ^s FL^'N M and OC analysis indi cated that the isolated material was a mixture of two primary coumouents thai eo-eluied by TLC ^J H-NMR (400 MHz, CDC¾) indicated the presence of peaks

corresponding to the isoperillyi alcohol (7) along with a unknown irapnrity. M (APCI method): rn/e: 153 40%), 15.2 (IvF, 131% 135.09 (M-OH). The other peaks atM+: 169JB (10%)..255.20, (13% 285,25 (15%), 287J9 (70%}_; 290 (68%), & 397.24 (15%) were not characterized, GC analysis confirmed the presence of isoperi!lyl alcohol^' (20.5%, (AUC^'))_.S compared with the iso-POH obtained from the epoxide route along with the unknown im arity (67,5%, (AUG)},

Example 16: Synthesis of Iso-POH Conjugated -with Teraoxolaiuide (TMZ)

The reaction scheme is the following:

pale yellow solid will be filtered and washed with hexanes.

Example 1 : Synthesis of!so-POH Conjugated with Roiipfa

The reaction scheme is as follows.

10% ethyl acetate/hexanes (iSO tnL). The 10% ethyl acetate /hexanes fractions will be combined and concentrated trader vacuum to give a gummy solid.

Example IS: Synthesis of Dimethyl Celecoxtb bis iso-POB carbamate conjugate

The reaction scheme is as follows.

Preparation of 4-(Bis~N, N '~4~i$opropylidme cyclohex~l - iy!m ihyloxy earhony! [5- (2, 5- iHmethyi ph nyl)~S~iriflnoromeikyl pyr soi- 1-yl] b ns esttlfona ide:

Phosgene (20% toiueae, 1,9_*5 nil, 39.4 nirnol) will be added to a tmxture of iso erilfyl alcohol (3.0 g, 1 .7 mmol) and potassium carbonate (8.1 g, 58.6.mmol) in dry toluene (45 ml) ove a period of 45 mm while maintaining the temperature between 10-12 ^yC, The reaction mixture will fee allowed, to warm to room temperature and stirred fo 10 h under N3.. The reactio mixture will be quenched with water (40 mV) and the organic layer separated. The aqueous layer will be extracted wit toluene (30 niL) and the combined organic layer washed with water (40 niL x 2), brine (!0%, 40 nit), and dried over sodium sulfate (25 g). The filtered organic layer will be concentrated under vacuum to gi ve isoperiilyl ehlorofbrnrate as a oil,

Isoperiilyl e lorofbrmate (0.22 g₅. l.Q mmol) will be added slowly to a niixtrtre of

^•dimethyl celecoxib (0_*2 g, 0.50 ramol) and potassium, carbonate (0, 14 g, .1.0 rarnol) in dry acetone (25 mL) over a period of 5 mm under ]%. The reaction mixture will be heated to reflux and. maintained fbr 4 h. The re ction m xture will be cooled and the acetone concentrate under vacuum, TJie resaiiing residue ill be suspended in water (25 tnL) and extracted with ethyl acetate (3x20 mL), The com ined orgaiitc taw will e: washed with water (40 niL), followed by brine (1.6%, 30 rnL), and dried over sodium sulfate. The filtered organic layer will be concentrated under vacyum to give a residiie which will be purified by coiumn chromatography .[Column dimensions: dia; 1.5 cm, height: 15 cm, silica: 230-400 mesh] and elated with hexanes (100 ml,) followed by a mixture of hexanes/ethyi acetate (95 5_:, 100 t»I.)₍ The hexane/ethyl acetate fractions will be combined and concentrated under vacuum to give a gumm mass.

Example 19

The Ras Binding Domain (RBD) Pulldown Assay was pertortsed to ana ze die active* GTP bound Ras in cell lysates with or without treatments of FOB at different concentrations.

NF1 (CRL~2884 cells, .human Schwann cell NFl) or Pan! cells were treated with or without PQH for 24 hours, and cells were collected and lysedwith lysis buffer.

Cleared lysate was assayed for protein concentration and protein-eqiiaiized supernatants were incubated with Rai-1 -BBD agarose heads for 1 h at 4 °C_t Beads were washed, four times in the lysis buffer. Bound proteins were dissolved by the addition of 50 p of Laemmlt loading buffer and boiled for 5 minutes. Samples were run on 12.5% SPS-PAGE gets. The amoun - f aetive-GIT-Ras in the bound traction was analyzed by Western blotting by using specific antibody for pan-Ras, H-Ras and K-Ras.

Figure .1.9 shows Western blot results from Ras Binding Domain Pulldown Assays in lysates of F1 CRL-288 cells with or withouttreatmeats of PQH at different coBceafcations, The results show -that POT treatment can Inhibit car decrease (active) ^'H s level aad ot activity.

Example 20; In vitro Cytotoxicity Studies of Perillyl Alcohol and Rolipram-POH on

Neurofibromatosis Cells

Both. POH and FO conjugated to rolipram (BJP-POH) were cytotoxic for

•neurofibromatosis cells (Figures 2QA-20C). Panel cells contain mutated -Ras.

To analyze the viabilit of cells with drug treatments, NFl (CRL-2884 cells) or Panel cells were seeded (5000 cells/well) m 9o~well assay black plates with flat bottoms (Greiner,

Gemiaay), After 24 hours, drug was added to the cells at different concentrations, and incubated for 48 hours. The alarnar blue assa was performed according to the manufacturer's protocol

(Life Technology, Grand Island, NY), Fluorescence was measured rising an excitation wavele¾tb of 540-570 tan (peak excitation Is 570 ωι), emissio at 580-610 nm (peak emission is 585 nm). The average fluorescence values of the eel! culture medium alone (back ground) were subtracted from tie fluorescence values of experimental wells. Percent viability was ^■calculated relative to untreated control cells. All experiments were performed in triplicate.

Exampl 21:

The Ras Binding Domain ( HD) Pul down Assay was performed to analyze the Active- G P boun Ras in eel! lysates with or without treatments of POH arid 1so~P H at diff rent concentrations.

Panel ^'Cells were treated with or without POH or iso-POH for 24hr_s and cells were collected a»d lysed with lysis buffer.

Cleared lysafe was assayed for protein concentration and proiein~e uaiteed supernatanis were incubated with Raf-1 -RBD agarose beads for 1 h at 4^aC. Beads were washed four times in the lysis buffer. Bound proteins were dissolved by the addition of 50 uL ofLae imM loading buffer and boiled for 5 minutes. Samples were run on 12.5% SDS-PAGE gels. The amoun of active-OTP-Ras in the bound fraction was analyzed by Western blotting by using specifi antibodies for -Ras or Pan-Riss. Figure '21 A shows Western bl t results from Ras Binding Domain Pulldown Assays in lysates of Pane I ceils with or without treatments of POH and iso- POH, Figures 21 B and 21€ are bar graphs quantifying the K-Ras level (Figure 2 IB), and Pan- Ras level (Figure 21 C) from the estern blot results of the Ras Binding Domain Pulldown

Assays in lysate of Panel cells with or without treatments of POH and iso-POH. All the blots were normalized to actio control and each protein, expression was calculated as % of control..

The results sho that POH or iso-POH treatment can inhibit or decrease (active) Ras level and/or acti vity .

E am le.22;.

Figures 22A-22C show the cytotoxicity of POH, iso-POH and RP-PQB (FOH conjugated to rolipram) on NF!~G 233 2 cells. GM23312 cells (Coiiell Institute for Medical Research) are malignant peripheral nerve sheath tumor (MP ^'ST) ceils.

To analyze the viability of cells with drag/treatments* NF1-G 23312 cells were seeded

(5000 eelis/welS) in 96- well assay black plates with fla bottoms (Greiner, Germany). After 24 hours, drug was added to the cells at different concentrations, and incubated for S hours. The alaraar blue assay was performed according to the manufacturer's protocol. All experiments were performed in triplicate.

Figures 23A-23C show the cytotoxicity of POH, iso-POH and RP-POH o NFl-MFNST 26T cells.,

H 1-MPNST 26 cells were seeded (5000 cells/well) in 96» eil assay black plates with Hat bottom (Greirser, Germany). After 24 hours, POH, Iso-PQH and RP-POH were added to the cells at differerit concentrations, and incubated for 48 hours. Hie alaraar blue assay was performed according to the ami&turer's protocol All ex eriments were performed in triplicate.

Example 23;

Figure 24 shows POM effects on total Ras activity, using Ras activation EtiSA assay. Specifically,, NF 1 cells (e.g._s C^'KL-2884, hiuuan Se!r a«n. cell T TI ) are cultured to

approximately 8:5-90% conf!uency. NFi cells were treated with perillyl alcohol (PQ^'H) or Farnesyltomsferase inhibitor (FT!) for 20 hrs . Cultured media was -removed and cells were washed twice with ice-cold PBS (Phosphate Buffered Saline). Cells were scraped from the plate aud cell pellets collected. Then FX Mg"^'' Lysis Wash- buffer with protease and. phosphatase inhibitor cocktail was added to the cell pellet. The pellet was resuspended In the^' buffer and and incubated ø» ice for 15 rainntes. The s mple was centriftiged at 14000 tpra for 10 minutes- at 4°€. in i¾Mctocentrimge> The supernatant was- collecte and protein concentration calcrdated. Cell lysates having 100 μ§ or 50 μ$ protein were used and EL1SA was performed according to the manufacturer's protocol (Miilipore Co., Billerica, Massachusetts),

Irs Figure .24, No. 1 : Normal medium (control); No. 2: 5-m POH treated lor 20 hr; No. 3: 1,0 m.M POH treated for 20 hr; No. 4; 20 μ Faoiesyltrartsferase inhibito (FTl) treated, for 20k; No, 5: positive control.

Example 24:

A c mbinati n of FOB and prostratin was more cytotoxic for neurofthromatosis cells than. POH alone or prostratln alone (Figures 25A-25C). Similarly, a combhmtfoti of POH and vastatin was more cyto oxic for

neurofibromatosis, cells than POH aloue or iovasiatin, alone (Figures 2SA~26C).

NFI cells (e,g., CRL-2884, hum n^' Schwann eel! F.1) were used fbr the experiments shown in Figures 25A-25C arid 26A-26C.

Example .25: Intracellular calcium levels after POH treatment

Free Intracellular calcium was determined by using Ff tu 3AM (foyitrogen). U251 cells were seeded on covershps tire day before the experiment and treated wit POH (ImM or 1 ,5mM) fo 20 hours. Cells were - then stained with Fluo-3 dye containing medium for I hour at 37^¾C. he dye was then washed out with dye-free media and fixed with 3,7% formaldehyde for 30.rain. Coversiips were mounted on glass slides; and intracellular calcium levels were determined by monitoring intracellular fluorescence by fluorescence microscop <

Figure 27 shows that POH treatment can increase (free) intracellular calcium levels.

The scope of the present mvention is not limi ted by what lias been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternati ves to the depicted exam l s of materials » configurations, constructions arid dimensions. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of sncn. 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 mvention described herein. All references cited and discussed, in this specification are incorporated herein b reference in their entirety. Variations, .modifications and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the inventi on. While certai embodi ments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the invention. The matter set forth in. the foregoing description and accompanying drawings is offered by way of illustration only a d: not as a limitation.

Claims

What is claimed hi

L A method of treatmg neuroSbroismtosis, compmm administering to a subject a

therapeutically effective amount of periil l alcohol, an iso-perillyl alcohol, or a com iaatian. thereof

2» A method of treating neurofibromatosis, comprising administering to a subject a

therapeutically effective amount of a carbamate of periliyl alcohol, a carbamate of an iso- perillyJ alcohol, or a combination t ereof,

3 , The method of claim , wherein the carbamate of periliyl al cohol is eri!lyi alco hol

conjugated with temozoiomide or rolipram.

4. The met od of claim 2, wherei the carbamate of periliyl alcohol is 4^3-eyelopeftt.yloxy- 4-methoxy phenyi)-2-oxo-p rroHdine- 1 -earhoxylic acid 4-isopropenyi eydohsx- 1 - enytm«*thyl ester.

5. The method of claim 2, w erein, the carbamate of perDly! alcohol is 3-medryI 4-oxo-3₅4- dlhydfoiiritdazo[5₅1 -djfl ,_:2_;3:_sS]tetra2ii>e-S-carbonyl-car¾affiic acid-4 so ^,Qpeayl eyclohex- 1 -enylmethyl ester.

6. The inethod of claims I or 2_S tether comprising administering tem z iomide m rolipram to the subject . The method of claims 1 or 2, farmer compr ising admimstering to tire subject a statin selected from the group consist ng of atorvast tin> flirvastaiiu, lovastaiin, pitavasiatin, pravastatin, simvastatin and rosuvastatk.

8, The method of claims I or 2, tether comprising administering prostraiio to the subject.

9. The method of claims 1 or 2, wherein the administration s by inhalation, mtraiiasally,. orally, intraveaously, subcutaneonsly or intramnsca!arly, I.0, A method, of inhibiting or decreasing ^"Ras level and/or activity in a. cell, comprising administering to the cell perillyi alcohol, an iso-per llyl alcohol., or combination ihereof.

I I . A method of inhibiting or decreasing Ras level and/or activity in a cell, comprising

Bdnwnlsfeiing: to the cell a carbamate of perillyi alcohol, a carbamate of an iso-perillyl alcohol, or a coinbination ihereof

I2._f A method of inhibiting or decreasing Ras level and/or activit in a scrbject, comprisin administering to the subject an effective amount of perillyi alcohol, an iso-perillyl alcohol, or a combination thereof.

13. A method of inhibiting or decreasing Ras level and/or activity In a subject, comprising administering to the stihjeet an effective amount of a carbamate of perillyi alcohol, carbamate of an iso-peri t iy! alcohol, or a combination thereof.

1 , The .method^' of claims 1 1 or 13, wherein the carbamate of perillyi alcohol is perillyi

alcohol conjugated with teaiozoloniide or rolipram.

IS.. The method of claims 1 1 or 13, wherein the earhao¾te of perillyi alcohol Is 4~(3~

cyclop eiit ioxy^»4-meihoKy phettyl)-2:-ox:o-pyrrol idine>- 1 -carhoxyl ic acid 4 sopropenyl eyelohex-1 -enylmeifeyl ester,

16 , The method of claims 1 1. or 13 , wherein the perillyi alcohol caibdmaife-fe 3~roeihyl - xo*

3_i4~dihydrolrn3da¾o|5.,i -d][i,2_J3,5]te^

cyclohex_" 1 -enylmeihyl ester,

17, The m ethod of any of claims 10 ~ 13 , feriher comprising administering em oxoloinide or rolipram,

18. The method^' f any of claims 10— 13, foriher comprising ad irtisierrag a statin selec ted from the roup consisting of atorvastatm, f¾ivastatiii₅ lovastatin, pit va ad , pravastatin, simvastatin and ros«vastatm>

1 . The method of any of claims

20, The method of claims 12 or 13, wherein said administration is by irthalation, intranasaliy, orally, intravenously, subctstaneously or intrarauscaiarly.

21. A method of treating neiux^fioromatosis, coraprising adaimister ing to a subject a

therapeutically effective atnornit of petitlyl alcohol

22, Th method of claim 21 , tether comprising administering rolipram to the subject 23. The method of claim 21 , tether comprising administering a statin selected from die grou consisting of ato astatk, .fluvastat , lovastatin, pravastatin, pra asta n simvastatin and rosuvastaiin,

24. The method of claim 23, wherein the statin is astatiP_*

25, The method of claim 21, further comprising administering prostratiri to the subject,

2 A method of^" treating ueuroflbromatosis, comprising admiiiistering to a snbject a

therapeutically effective amount of Iso-p tiMyi alcohoL

27, The meth of claim.26; further comprising adtplm'sisrmg rolipram to the subject.

28. The method of claim 26, further comprising administering a statin selected from the group consisting of atorvastatm, ilnvastatin, lovastatin, pitavastatin, pravastatin, simvastatin, and rosuvastattn.

29,, The me thod of claim 28 _¥ wherein th statin, is lovastaiin.