Compositions and Methods of Use of Phorbol Esters

In the Treatment of Parkinson's Disease

Technical Field

[001] The present invention relates to compositions and methods for the treatment of Parkinson's disease and the symptoms of Parkinson's disease. Specifically, the present invention relates to the use of phorbol esters in the treatment and prevention of Parkinson's disease and related symptoms.

Additional Disclosures

[002] Additional disclosures relating to the instant application may be found in "Compositions And Methods Of Use Of Phorbol Esters' ^' United States Patent Application Serial No. 12/023,753, tiled January 31, 2008, PCT Patent Application Serial No. PCT/US08/012 9, filed January 31, 2008, to Richard L. Chang, et al, which claims priority benefit of United States Provisional Patent Application Serial No.60/898,810, filed January 31, 2007, United States Continuation Patent

Application Serial No. 13/595,072, tiled August 27, 2012, United States Continuation Patent Application Serial No.13/794,467, filed March 11, 2013, United States Continuation Patent Application Serial No.13/999,347, tiled February 13.2014; ^• 'Compositions And Methods Of Use Of Phorbol Esters In TheTreatment Of

Neoplasms ^" United States Patent Application Serial No. 13/745,745, filed January 18, 2013, to Richard L. Chang, et al, which claims priority benefit of United States Provisional Patent Application Serial No.61/588.165, tiled January 18.2012, United States Continuation Patent Application Serial No. 14/025.206. filed September 12. 2013. United States Continuation Patent Application Serial No.14/026.473. filed September 13, 2013, United States Continuation Patent Application Serial No.

13/999,339, filed February 13, 2014; "Compositions And Methods Of U se Of Phorbol Esters In The Treatment Of Stroke" United States Patent Application Serial No.13/745,742, filed January 18, 2013, PCT Patent Application Serial No.

PCT/US2013/022325, filed January 18, 2013, to Richard E. Chang, etal, which claims priority benefit of United States Provisional Patent Application Serial No. 61/588,167, filed January 18.2012, United States Continuation Patent Application Serial No.14/025,176, filed September 12, 2013, United States Continuation Patent Application Serial No.14/026,534, filed September 13, 2013, United States Continuation Patent Application Serial No. 13/999,331 , filed February 13, 2014; "Compositions A nd Methods Of Use Of Phorbol Esters" Un ited States Continuation- In-Part Patent Application Serial No. 13/745,740, filed January 1 8, 2013, PCT Patent Application Serial No. PCT/US201 3/022324, filed January 18, 2013, to Richard L. Chang, et al., wh ich claims priority benefit of United States Provisional Patent Application Serial No. 61 /588, 1 62, filed January 1 8, 2012, United States Continuation Patent Application Serial No. 14/025, 1 63, filed September 1 2, 20 1 3, United States Continuation-In-Part Patent Application Serial No. 1 4/027,320, filed September 1 6, 2013, and United States Continuation Patent Application Serial No. 1 3/999,332, filed February 1 3, 201 4, each of which is incorporated herein by reference in its entirety for all purposes.

Background

[003] Parkinson's disease is a progressive, degenerative neurological disorder affecting 1 in 500 people. It is believed that between 7 and 10 million people worldwide are afflicted with Parkinson's disease. Approximately 60,000 new cases per year are clinical ly diagnosed in the United States alone. Parkinson's disease and its symptoms are a result of neuronal death in an area of the brain known as the substantia nigra which controls voluntary movement, plays a role in the production of dopamine and also regulates mood. The substantia nigra is located in a part of the m id brain (mesencephalon) cal led the basal ganglia. The speci fic etiology of Parkinson ^' s disease is not wel l understood although much i s known about the biological functions that arc affected. It is bel ieved that Parkinson ^' s disease occurs as a result of both environmental and genetic factors including but not limited to oxidative stress, mitochondrial dysfunction, amino acid imbalances, protein m isfolding, immune system reactions, vitam in and m ineral deficiencies, lipid peroxidation, the presence of lewy bodies and other types of undiagnosed medical disease.

[004] The main pathologic feature of Parkinson ^' s disease is the loss of

dopaminergic cel ls in the basal gangl ia, especially in the substantia nigra which leads to progressive loss of control over voluntary movement, speech and other autonomic dysfunction. Whi le various therapies have been attempted since the late 1 800' s, there is no cure for Parkinson ^' s disease, only symptomatic treatmen t and the condition is considered chroni c as well as being progressive. Drugs currently used in the treatment of Park inson's disease can help control some of the symptoms but may lose their effectiveness over time as well as having deleterious side effects.

[005] Plants have historically served many medicinal purposes. The World Heal th Organization (W HO) estimates that 4 billion people, 80% of the world population, presently use herbal medicine for some aspect of primary health care. (WHO Fact sheet Fact sheet N° 1 34 December 2008) However, it can be d ifficult to isolate the specific compound that has the curative effect and reproduce it on a commercial scale. Additionally, wh i le active compounds may be isolated from a plant, the other parts of a plant such as m inerals, vitamins, volatile oils, glycosides, alkaloids, bioflavonoids and other inert or unidentified compounds may not be clearly identified or listed in their therapeutic use. Other substances may also be involved in the functioning of the active ingredient or creating the medicinal effect for which the plant is known making the use, purification and commercialization of plant based pharmaceutical agents a challenge.

[006] Phorbol is a naturally derived plant based organ ic com pound of the tigl iane family of diterpenes. It was first isolated in 1 934 as a hydrolysis product of croton oil derived from the seeds of Croton tiglhan, a leafy shrub of the Fuphorbiaceae fam ily that is native to Southeastern Asia. Various esters of phorbol have demonstrated important biological properties inc luding the reported abi l ity to m im ic diacylglycerols by activating protein kinase C (PKC) and modulating downstream cell signal ing pathways (includ ing the mitogen-activated protein kinase (MAPK) pathways).

Phorbol esters are additional ly thought to bind to chimaerins, the Ras activator RasGRP, and the vesicle-prim ing protein Munc- 1 3 (Brose N, Rosenmund C, JCell Sci; 1 1 5 :4399-41 I (2002)). Some phorbol esters also induce nuclear factor-kappa B (N F- B). The most notable physiological property of phorbol esters is their reported capacity to act as tumor promoters. (Blumberg, 1988; Goel, G et al , Int., Journal of Toxicology 26, 279-288 (2007)).

[007] 1 2-0-tetradecanoylphorbol- 1 3-acetate (TPA), also cal led phorbol- 12- myristate- 1 3-acetate (PMA), is a phorbol ester used in models of carcinogenesis as an inducer for differentiation and/or apoptosis in multiple cel l lines and primary cel ls. TPA has also bee n reported to cause an increase in c irculating white blood cel ls and neutrophils in patients whose bone marrow function has been depressed by chemotherapy (Han Z. T. et al. Proc. Natl. Acad. Sci. 95, 5363 -5365 ( 1 998)) and inhibit the HIV-cytopathic effects on MT-4 cells. (Mekkawy S. et al , Phytochernistry 53, 47-464 (2000)). However, due to a variety of factors, including caustic react ions when contacted with the skin and concerns for its potential toxicity, TPA has not been shown to be an effective tool in the treatment of diseases. Indeed, as phorbol esters play a key role in activation of protein kinase C (PKC), which triggers various cellular responses resulting in inflammatory responses and tumor development (Goel et al, Int, Journal of Toxicology 26, 279-288 (2007)), phorbol esters would generally be excluded from possible treatment candidates for i nflammatory diseases or conditions that involve inflammatory reactions such as stroke or Parkinson's Disease. However, treatment with retinoic acid followed by phorbol ester 12-0-tetradecanoylphorbol- 1 3- acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents.

(Xie, H., Hu, L. & Li, G . SH-SY5Y human neuroblastoma cell line: in vitro cel l model of dopami nergic neurons in Parkinson's disease. Chin. Med. J. 123, 1086- 1 092, 2010).

[008] Treatment for Parkinson's disease is currently symptomatic; there is no one treatment that is effective for al l sym ptoms of Parkinson's disease. There is an essential need for new and more e ffective treatments for individuals suffering from Parkinson's disease and other related disorders.

Summary

[009] The present invention relates to a compositions and methods for treating Parkinson's disea se using phorbol esters. These compositions and methods are effective in treati ng chronic or potentially recurring conditions, or to repair the damage caused by acute cases of chronic diseases such as Parkinson' s Disease.

Symptoms of Parkinson's disease that may be treated or prevented by the use of the compositions and methods described herein include, but are not l imited to, tremor at rest, stiffness, bra dykinesia, rigidity, speech impairment, a feeling of weakness in the limbs, lack of dexterity, poor coordination, imbalance, cogniti ve impairment, dementia, mood impairment, drowsiness, sleep disturbances, autonomic dysfunction and postural insta bi l ity.

[010] There is currently is no specific test for Parkinson ^' s di sease, although recent MR1 findings have indicated the appearance of a ' swallow Tai l ' in a weighted 3T image have concluded that: The healthy nigrosome- 1 can be readily depicted on high- resolution 3T - SWI giving rise to a ' swallow tail' appearance of the dorsolateral substantia nigra, and this feature is lost in PD. Visual radiological assessment yielded a high diagnostic accuracy for PD vs. an unselected clinical control population. Assessing the substantia nigra on SWI for the typical 'swallow tail' appearance has potential to become a new and easy applicable 3T MRI diagnostic tool for nigra degeneration in F'D. (Schwarz ST, Afzal M, Morgan PS, Bajaj N, Gowland F*A, et al. (2014) The 'Swallow Tail' Appearance of the Healthy Nigrosome - A New Accurate Test of Parkinson's Disease: A Case-Control and F trospective Cross-Sectional MRl Study at 3T. PLoS ONE 9(4): e93814). If this is adopted and accepted, it will offer a clinical biomarker to accurately assess the efficacy of new treatments in Parkinson's disease. Currently successful treatment will be determined according to the accepted conventional method based on the Unified Parkinson's Disease Rating Scale (UPDRS) including decreases in tremor at rest, stiffness, bradykinesia, rigidity, speech impairment, a feeling of weakness in the limbs, lack of dexterity, poor coordination, imbalance, cognitive impairment, dementia, mood impairment, drowsiness, sleep disturbances, autonomic dysfunction and postural instability.. The compositions and methods described herein achieve the foregoing and satisfy additional objects and advantages by providing novel and surprisingly effective methods and compositions for modulating cell signaling pathways and/or treating diseases and symptoms of diseases or conditions using compositions contain ing a phorbol ester or derivative composition of the Formula 1. below :

Formula wherein Ri and R2 may be hydrogen; hydroxyl; ^{0 c il1i} ' ^yl , wherein the alkyl o

II

group contains 1 :o 15 carbon atoms; ; , wherein a lower al

— -( v— ( I"I :„. _Η ·ι ilkoi _j i.

substituted derivatives thereof. R3 may be hydrogen or

[Oi l] In some embodiments, at least one of Ri and R2 are other than hydrogen and o

II

R3 is hydrogen or and substituted derivat ives thereof. In another

o

embodiment, either R i or R ₂ is __ _n ⁰ __!L _r _ _r ³ > · the remaining R] or R2 isa u -~c k, i _ vvherein a lower alkyl is between I and 7 carbons, and R ₃ is hydrogen.

[012] The alkyl, alkenyi, phenyl and benzyl groups of the formulas herein may be unsubstituted or substituted with halogens, preferab ly, chlorine, fluorine or bro ine, nitro, am ino, and/or similar type radicals.

[013] In a further embodiment, the invention achieves these objects and satisfies additional objects and advantages by providing novel and surprisingly effective methods and compositions for modu lating cel l signal ing pathways and/or treating diseases or conditions associated with d iseases such as Parkinson ' s disease using an exemplary phorbol ester composition such as 1 2-0-tetradecan oylphorbol- 1 3-acetate (TPA) of Formula I I. below:

Formula II.

[014] Useful phorboi esters and related compounds and derivatives within the formulations and methods of the invention include, but are not limited to, other pharmaceutically acceptable active salts of said compounds, as well as active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and/or prodrugs of said compounds. Exemplary forms of phorboi esters for use within the compositions and methods of the invention include, but are not l im ited to, phorboi 1 3- butyrate; phorboi 1 2-decanoate; phorboi 1 3-decanoate; phorboi 1 2. 1 3-diacetate; phorboi 1 3.20-diacetate; phorboi 1 2, 1 3-d ibenzoate; phorboi 1 2, 1 3-dibutyrate; phorboi 1 2, 13-didecanoat ; phorboi 1 2, 1 3-dihexanoate; phorboi 1 2, 1 3-dipropionate; pho rboi 12-myristate; phorboi 1 3-myristate; phorboi 12-myristate- l 3-acetate (also known as TPA or PMA); phorboi 12, 1 3,20-triacetate; 1 2-deoxyphorbol 1 3-angelate; 1 2- deoxyphorbol 1 3-angelate 20-acetate; 1 2-deoxyphorbol 1 3-isobutyrate; 1 2- deoxyphorbol 1 3- isobutyrate-20-acetate; 12-deoxyphorbol 1 3-phenylacetate; 12- deoxyphorbol 1 3-phenylacetate 20-acetate; 12-deoxyphorbol 1 3-tetradecanoate; phorboi 12-tigliats 1 3-decanoate; 1 2-deoxyphorbol 1 3-acetate; phorboi I 2-acetate; and phorboi 1 3-acetate.

[ 015] Mammalian subjects amenable to treatment w ith phorboi esters of Formu la I, particularly TPA, accord ing to the methods of the invention inc lude, but are not limited to individuals with Parkinson ^' s disease. Symptoms of Parkinson ^' s disease that may be treated or prevented by the use of the compositions and methods described herein include, but are not limited to, tremor at rest, stiffness, bradykinesia, rigidity, speech impairment, a feeling of weakness in the limbs, lack of dexterity, poor coordination, imbalance, cognitive impairment, dementia, mood impairment, drowsiness, sleep disturbances, autonomic dysfunction and postural instability.

Subjects amenable to treatment with phorboi esters of Formula I, particularly TPA, or derivatives of the phorbol esters of Formula I including pharmaceutically acceptable salts, enantiomers, isomers, polymorphs, prodrugs, solvates and hydrates include those suffering from symptoms of Parkinson's disease or sym ptoms related to Parkinson's disease such as tremor at rest, stiffness, bradyki nesia, rigidity, speech impairment, a feeling of weakness in the l imbs, lack of dexterity, poor coordinat ion, imbalance, cogni tive impairment, dementia, mood impairment, drowsiness, sleep disturbances, autonomic dysfunction and postural instability.

[016] These and other subjects are effectively treated, prophylactically and/or therapeutically, by administering to the subject an effective amount of a phorbol ester of Formula I sufficient to prevent or treat paralysis, increase spatial awareness, decrease memor loss, decrease aphasia, increase coordination and balance, improve cognition, decrease or el iminate tremors, decrease or el iminate stiffness and rigidity, improve sleep qu ality, increase stability, improve mobility, improve bladder control, ease muscle or joint aches, improve vision, and/or im prove muscle control.

[017] Therapeutical ly useful methods and formulations of the invention wi l l effectively use a phorbol ester of Formula I in a variety of forms, as noted above , including any act ive, pharmaceutical ly acceptable salts of said compounds, as well as active isomers, enantiomers, polymorphs, solvates, hydrates, prodrugs, and/or combinations thereof. TPA of formula I I is employed as an i llustrative embodiment of the invention within the examples herein below.

[018] Within additional aspects of the invention, combinator ial formulations and methods are prov ided wh ich employ an effective amount of a phorbol ester of Formula I in com bination with one or more secondary or adj u nctive active agent s) that is/are combinatorial ly formulated or coordinately adm inistered with the phorbol ester compound of Formula I to yield an effecti ve response in the subject.

[019] Exemplary combinatorial formulations and coordinate treatment methods in the treatment of Parkinson' s disease employ the phorbol ester compound of Formula I in combination w ith one or more additional Parkinson's disease treating or other indicated, secondary, or adj unctive therapeutic agents. The secondary or adj unctive therapeutic agents used in combination with a phorbol ester, e.g., TPA, in these embodiments may possess direct or indirect anti-Parkinsonian effects, alone or in combination with. e.g. TPA; may exhibit other useful adjunctive therapeutic activity in combination with, e.g. TPA (dopamine increasing, catechol-O-methyl transferase inhibiting, aromatic L-amino acid decarboxylase inhibiting, dopamine agonist, neuroprotective, anticholinergic); or may exhibit adjunctive therapeutic activity useful for treating or preventing side effects of Parkinson's disease or associated symptoms alone or in combination with, e.g. TPA.

[020] Useful adjunctive or secondary therapeutic agents in these combinatorial formulations and coordinate treatment methods for the prevention or treatment of symptoms of Parkinson's disease in a mammalian subject include, but are not limited to, carbidopa/levodopa, tolcapone, MAO-B inhibitors, pyridoxine, amantadine, selegiline, rasagil ine, antichol inergics, cholinesterase inhibitors, COMT Inhibitors, and dopamine agonists including, but not limited to, apomorphine, atropine, benztropine, biperiden, bromocriptine, cabergoline, ciladopa, dihydrexidine,

dinapsol ine, doxE.nthrine, entacapone, epicriptine, l isuride, pergolide, piribedil, pramipexole, pro sylnorapomorphine, quinagol ide, rivastigminc, ropinirole, rotigotine, roxindole, suman irole, trihexphenidyl, . In addition, adj unctive or secondary therapies may be used such as, but not l imited to, antioxidants, am ino acid therapy, deep brain stimulation, diet modification, exercise, herbal supplementation, hormone therapy, mineral supplementation, physical therapy, and/or lesion form ation.

[021] The forgoing and additional objects, features, aspects and advantages of the present i nvention wil l become apparent from the followi ng detai led description. Detailed Description

|022] Novel methods and compositions have been identified for use in treating chronic or recurri ng conditions, or to repair the damage left by episodes of i l lness or treatment of illness in mammalian subjects, including humans. In various

embodiments, the methods and compositions as described herein are effective in treating Parkinson ^' s disease and sym ptoms caused by or resembl ing to Parkinson ^' s disease.

[023] In additiond embodiments, the methods and compositions described herein are effective to prevent or treat paralysis, increase spatial awareness, decrease memory loss, decrease aphasia, improve coordination and balance, improve cognition, decrease or eliminate tremors, decrease or eliminate stiffness and rigidity, improve sleep quality, increase stability, improve mobil ity, improve bladder control, increase continence, improve appetite, ease muscle or joint aches, improve vision, and/or improve muscle c ontrol,. [024] Formulations and methods provided herein employ a phorbol ester or derivative compound of Formula I, below,

Formula I [025) wherein R I and R2 may be hydrogen; hydroxy I ; ^~ ° ^C;,1ky1 , wherein the

alkyl group contains 1 to 1 5 carbon atoms; ; w herein a — <. -nhenv]

lower alkenyl group contains between 1 to 7 carbon atoms;

-O— C benzyl and

substituted derivatives thereof. R;, may be hydrogen or

including al l active pharmaceutical ly acceptable com pounds of this description as well as various tc reseen and readi ly provided complexes, salts, solv ates, isomers, enantiorners, polymorphs and prodrugs of these compounds and combinations thereof as novel Parkinson' s Disease treating compounds.

[026] I n some embodiments, at least one of Ri and R2 are other than hydrogen and

i l

R3 is hydrogen or and substituted derivatives thereof. In another o

embodiment, either Ri or R2 is ^{C ( C} " ^aik>i' the remaining R i or R ₂ is a o

I!

o— '. . ^iik . ⁱ ^ wherein a lower alkyl is between 1 and 7 carbons, and R3 is hydrogen. The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein may be unsubstituted or substituted with halogens, preferably, chlorine, fluorine or bromine; nitro; amino; and/or similar type radicals. The compounds of Formula I are more fully described in U.S. Patent Application No. 12/023,753, filed January 3 I , 2008, which claims priority benefit of United States Provisional patent appl ication Serial No. 60/898,8 10, filed January 31 , 2007, each of which is incorporated herein in its entirety by reference.

[027] Anti-Parkinsonian formulations and methods provided herein employ a phorbol ester or derivative compound of Formula I, above, including all active pharmaceutical ly acceptable compounds of this description as well as various foreseen and readily provided complexes, salts, solvates, isomers, enantiomers, polymorphs and prodrugs of these compounds and combinations thereof as antiparkinsonian age nts.

(028] It is estimated that 7 to 10 mi llion people worldwide suffer from Parkinson' s disease, a chron ic and progressive neurodegenerative disorder . It is believed to have both genetic and environmental triggers but the exact etiology is not ful ly understood. Although multipl e gene mutations have been identified in Parkinson ^' s disease, only about 1 0% of all cases are thought to be inherited. Most movement related and autonomic symptoms of Parkinson ^' s disease result from an im balance betw een dopam ine and acetylcholine. Add itionally, low levels of seroton in and norepinephrine contribute to mocd changes and depression in the disease. It is also characterized by the presence of Lew y bodies in post mortem observations. The relationship between Parkinson ^' s disease and lew s bodies is not entire ly c lear but it is believed that lewy bodies may be a product of oxidative stress or perhaps even an immune response to aberrant protein synthesis. Parkinson ^' s disease is characterized by bradykinesia, resting tremor, rigidity, speech impairment, postural instability and dementia.

[029] A broad range of mammalian subjects, including human subjects, are amenable to treatment using the formulations and methods of the invention. These subjects include, but are not lim ited to, individuals suffering from Parkinson's disease or related conditions or individuals at risk for developing Parkinson ^' s disease or related conditions. [030] Mammalian subjects amenable to treatment with phorbol esters of Formula I, particularly TPA , according to the methods of the present invention include, but are not limited to, mammalian subjects with Parkinson's disease and/or symptoms related to Parkinson's di sease.

[031] Within the methods and compositions of the invention, one or more phor bol ester compound(s) of Formula I as disclosed herein is/are effectively formulated or administered as an agent effective for preventing or treating F'arkinson's disease, In exemplary embodiments, TPA is demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and therapeutic methods, alone or in combination with one or more adj unctive therapeutic agent(s). The present disclosure further provides additional, pharmaceutical ly acceptable phorbol ester compounds in the form of a native or synthetic compound, including comple xes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of Ihe compounds disclosed herein, and combinations thereof, which are effective as therapeutic agents within the methods and compositions of the invention in the prevention or treatment of Parkinson's disease and symptoms thereof.

[032] Phorbol is a natural, plant-derived polycyclic alcohol of the tigliane fam ily of diterpenes. It was first isolated in 1 934 as the hydrolysi s product of croton oi l derived from the seeds ol ^' Croton tiglium. It is well soluble in most polar organic solvents and in water. Esters of phorbol have the general structure of Form ula I, below:

Formula I

wherein Ri and R.2 are selected from the group consisting of hydrogen; hydroxyl;

o

O C II iUs l

, wherein the alky] group contains 1 to 1 5 carbon atoms, ;

, wherein a lower alkenyl group contains between 1 to

; and substituted derivatives < )— ί I!

thereof and R3 may be hydrogen, or subst ituted derivatives thereof as wel l as pharmaceutical ly acceptable salts, enantiomers, polymorphs, prodrugs solvates and hydrates of compounds of Formula I an d substituted derivatives thereof.

[033] The term "lower alkyl" or "lower alkenyl" as used herein means moieties containing 1 -7 carbon atoms. In the compounds of the Formu la I, the alkyl or alkenyl groups may be straight or branched chain. In some embodiments, either or both R i or R2, are a long cha in carbon moiety (i.e., Formula I is decanoate or myristate).

[034] The alkyl, alkenyl, phenyl and benzyl groups of the formulas herein may be unsubstituted or substituted with halogens, preferably, ch lorine, fluorine or brom ine; n itro; am ino and sim i lar type radicals.

[035] Organic and synthetic forms of phorbol esters, includ ing any preparations or extracts from herbal sources such as croton tiglium. are contemplated as useful compositions comprising phorbol esters (or phorbol ester analogs, related compounds and/or derivatives) for use within the embodiments herein. Useful phorbol esters and/or related compounds for use within the embodiments herein will typically have a structure as illustrated in Formula I, although functionally equivalent analogs, complexes, conj ugates, and derivatives of such compounds wi ll also be appreciated by those skilled in the art as within the scope of the invent ion.

[036] In more detailed embodiments, illustrative structural modifications according to Formula I above w i ll be selected to provide useful candidate compounds for treating Parkinson's disease, wherein : at least one of Ri and R ₂ are other than hydrogen and R3 is selected from the group consisti ng of hydrogen,

and substituted derivatives thereof. In another embodiment,

either Ri or R ₂ is ' ^" the remaining Ri or R ₂ is

⁽ )

1

— —c i d, .uk i _{and R3 is} hydrogen.

[037] An exemplary embodiment of a phorbol ester compound of Formula I useful in the treatment of Parkinson's disease, is found in phorbol 12-myristate- 13-acetate (also known as PVIA or 12-O-tetradecanoyl-phorbol-l 3-acetate (TPA)) shown in Formula II, below.

Formula II

[038] Additional useful phorbol esters and related compounds and derivatives within the formulations and methods of the invention include, but are not limited to, other pharmaceutically acceptable active salts of said compounds, as well as active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and/or prodrugs of said compounds. Further exemplary forms of phorbol esters for use within the compositions and methods of the invention include, but are not limited to, phorbol 13-butyrate; phorbol 12-decanoate; phorbol 13-decanoate; phorbol 12,13- diacetate; phorbol 13,20-diacetate; phorbol 12,13-dibenzoate; phorbol 12,13- dibutyrate; phorbol 12,13-didecanoate; phorbol 12, 13-dihexanoate; phorbol 12,13- dipropionate; phorbol 12-myristate; phorbol 13-tnyristate; phorbol 12,13,20-triacetate; 12-deoxyphorbol 13-angelate; 12-deoxyphorbol 13-angelate 20-acetate; 12- deoxyphorbol 13-isobutyrate: 12-deoxyphorbol 13-isobutyrate-20-acetate; 12- deoxyphorbol 13-phenylacetate: 1 -deoxyphorbol 13-phenylacetate 20-acetate: 12- deoxypliorbol 13-tetradecanoate; phorbol 12-tigliate 1 3-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; and phorbol 13-acetate as shown in Table 1.

Table 1 Exemplary

Phorbol Esters

Phorbol 13- Butyrate

Phorbol 12- Decanoate

Phorbol 1 3- Decanoate

Phorbol 12, 13- 0

o

Diacetate \

OH _.

O HO

^■ OH

Phorbol 13,20- o

Diacetate

HO^

S

H Phorbol 12,13- Dibenzoate

Phorbol 12,13-

Dibutyra.tc

Phorbol 12,13- Didecanoate

H ^{' '} . H

OH.

.r V

;-)HO

^■ -OH

Phorbol 12,13- Dihexanoate

Phorbol 12,13- Dipropionate

Phorbol 12- y

Myri state A

OH

W

12-Deox phorbol o

13-Acetate o

^' OH\

ΉΟ --OH Phorbol 12

Acetate

Phorbol 1 3

HO " ^'

Acetate

OH

o---<

[039] Compositions as described herein further comprise Parkinson's disease treating composit ions comprising an effective amount of a phorbol ester compound of Formula 1 which is effective for prophylaxis and/or treatment of Parkinson's disease or related symptoms in a mammalian subject. A "'Parkinson ^' s disease treating," "dopam ine enhancing," "catechol-O-methyl transferase inhibi ting," "aromatic L - am ino acid decarboxylase inhibiting, ^" "dopam ine agonist," "neuroprotective," or "anticholinergic" effective amount of the active compound is therapeutical ly effective i n a single or multiple unit dosage form, over a spec ified period of therapeutic intervention, to measurably al leviate or prevent one or more o f the symptoms of Parkinson' s disease in the subject. Within exemplary embodiments, the compositions of the invention are effective in treatment methods to prevent or al leviate symptoms of Parki nson ^' s disease in human and other mammal ian subjects suffering from o r at risk for Parkinson ^' s disease.

[040] Phorbol ester treating, including Parkinson ^' s disease treating, compositions of the invention typically comprise an effective amount or unit dosage of a phorbol ester compound of Formula 1, which may be formulated with one or more pharmaceutical ly acceptable carriers, excipients, vehicles, emulsifiers, stabi l izers, preservatives, buffers, and/or other additives that may enhance stabi lity, delivery, absorption, half- l ife, efficacy, pharmacokinetics, and/or pharmacodynam ics, reduce adverse side effects, or provide other advantages for pharmaceutical use. E ffective amounts o f a phorbol ester compound or related or derivative compound of Formula I (e.g., a unit dose comprising an effective concentration/amount of TPA. or of a selected pharmac eutically acceptable salt, isomer, enantiomer, solvate, polymorph and/or prodrug of TPA) will be readily determined by those of ordinary skill in the art, depending on clinical and patient-specific factors. Suitable effective unit dosage amounts of the active compounds for administration to mammalian subjects, including human:?, may range from about 10 to about 1 500 about 20 to about 1 000 μg, about 25 to a aout 750 μg, about 50 to about 500 μg, about 1 50 to about 500 μg, about 1 25 μg to cibout 500 μg, about 1 80 to about 500 μg, about 1 90 to about 500 μg, about 220 to about 500 μg, about 240 to about 500 g, about 260 to about 500 g, about 290 to about 500 μg. In certain embodiments, the disease treating effective dosage of a phorbol ester compound or related or derivative compound of Formula I may be selected within narrower ranges of, for example, 1 0 to 25 μg, 30-50 μ , 75 to 100 μg, 100 to 300 μg, or 1 50 to 500 μg. These and other effective unit dosage amounts may be administered in a single dose, or in the form of multiple daily, weekly or monthly doses, for example in a dosing regimen comprising from 1 to 5, or 2 to 3, doses administered per day, per week, or per month. In one exemplary embodiment, dosages of 1 0 to 30 μg, 30 to 50 μg, 50 to 1 00 fig, 1 00 to 300 μ , or 300 to 500 μg, are administered one, two, three, four, or five times per day. In more detailed embodiments, dosages of 50- 1 00 μ«, 1 00-300 μ«, 300-400 μβ, or 400-600 μg are adm inistered once or twice dai ly. In a further embod iment, dosages of 50- 1 00 μg, 1 00-300 μg, 300-400 μ , or 400-600 μg are admin istered every other day. In alternate embodiments, dosages are calculated based on body weight, and may be administered, for example, in amounts from about 0^g/m ² to about 300μg/m ² per day. about 1 μg/ul ² to about 200 g m ² . about 1 μg/m ² to about 1 87.5 μg ^' m ² per day. about I μg/m per day to about 1 75 μg/m ² per day, about 1 μ Ίη ² per day to abo ut 1 57 μg/m per day about 1 μg/m ² to about 1 25 μ§/ηι ² per day, about 1 μ »/ιτΓ to about 75 g/m ² per day. 1 μg/m ² to about 50/ μg/tn ² per day, 2 μ£/ιη ^" to about 50 μ /m ² per day, 2 μ /m ² to about 30 μg/m per day or 3 μg/m ² to about 30 μg/m ² per day .

[041 ] In other em odiments, dosages may be administered less frequently, for example. 0^ /m ² to about 300μg/m every other day. about I μg/m to about 200 μg/m , about 1 μg/m ² to about 1 87.5 g/m every other day, about 1 μg/m to about 1 75 μ /m ² every other day, about 1 μg/m ² per day to about 1 57 μg/m ² every other day about 1 μg/m ² to about 125 μ /ηι ² every other day, about ^ig/rn to about 75 |ig/m ² every other day, 1 μg/m ² to about 50μ§/ιη ² every other day, 2 μg/m ² to about 50 μg/m every other day, 2 μg/m ² to about 30 μg/m ² per day or 3 μg/m ² to about 30 μg/m ² per day. In additional embodiments, dosages may be administered 3 times/week, 4 times/week, 5 times/week, only on weekdays, only in concert with other treatment regimens, on consecutive days, or in any appropriate dosage regimen depending on clinical and patient-specific factors

[042] The amount, timing and mode of delivery of compositions of the invention comprising a Parkinson's disease treating effective amount of a phorbol ester compound of Formula I wi ll be routinely adjusted on an individual basis, depending on such factors as weight, age, gender, and condition of the individual, the acuteness of the cytopathic disease and/or related symptoms, whether the administration is prophylactic or therapeutic, and on the basis of other factors known to effect drug delivery, absorpti on, pharmacokinetics, including half-life, and efficacy.

[043] An effective dose or multi-dose treatment regimen for the instant disease treating "dopamine enhancing," "catechol-O-methyl transferase inhibiting," "aromatic L-amino acid decarboxylase inhibiting," "dopam ine agonist," "neuroprotective," "anticholinergic." "muscle relaxant" formulations of the invention will ordinarily be selected to approximate a m inimal dosing regimen that is necessary and sufficient to substant ially prevent or al leviate the symptoms of the disease including Park inson's disease, and/or to substantial ly prevent or al leviate one or more symptoms assoc iated with Parkinson ^' s disease, in the subject. A dosage and administration protocol wi l l often include repeated dosing therapy over a course of several days or even one or more weeks or years. An effective treatment regime may also involve prophylactic dosage adm inistered on a day or multi-dose per day basis lasting over the course of days, weeks, months or even years.

[044] Effectiveness of the compositions and methods of the i nvention in the treatment of Parkinson ^' s disease may be demonstrated by a decrease in the symptoms of Parkinson ^' s including, but not l im ited to tremor., bradykinesia. rigidity, speech impairment, postural instabi l ity and dementia. Effectiveness of the phorbol ester compounds of the present invention in the treatment of Parkin son ^' s disease may further be demonstrated by an increase in dopam ine and/or norepinephrine levels. Such levels may ncrease 1 0%. 20%. 30%, 50% or greater increase, up to a 75-90%, or 95% or greater of normal levels.

[045] Effectiveness of the compositions and methods of the invention in the treatment of Parkinson ^' s disease may be indicated by a decrease in the presence of Lewy bodies and a-synuclein in cases of Parkinson ^' s disease with dementia.

Effectiveness may also be demonstrated through the use of animal models, such as MPTP induced Parkinson's, rotenone induced Parkinson's, surgically induced Parkinson's, paraquat induced Parkinson's, 6-OHDA induced Parkinson's, or a- synuclein overexpression in mice. The use of the compositions and methods of the invention will decrease the symptoms of Parkinson's disease expressed in these models by 0%, 20%, 30%, 50% or more, up to a 75-90%, 96% or greater decrease over control animals.

[046] Within additional aspects of the invention, combinator ial disease treating "Parkinson's disease treating," "dopamine enhancing," "catechol-O-methyl transferase inhibiting," "aromatic L-am ino acid decarboxylase inhibiting," "dopamine agonist," "neuroprotective," "anticholinergic," "muscle relaxant" formulations and coordinate admin istration methods are provided which employ an effective amount of a phorbol ester compound of Formula 1 and one or more secondary or adjunctive agent(s) that is/are combinatorially formulated or coordinatelv administered with the phorbol ester compound of Formula I to yield a combined, multi-active disease treating composition or coordinate treatment method. Exemplary combinatorial formulations and coordinate treatment methods in this context employ the phorbol ester of Formula I in combination with the one or more secondary Parkinson ^' s treatment agent(s), or with one or more adjunctive therapeutic agent(s) that is/are useful for treatment or prophylaxis of the targeted (or associated) d isease, condition and/or symptom( ) in the selected combinatorial formulation or coordinate treatment regimen. For most combinatorial formulations and coordinate treatment methods of the invention, a phorbol ester compound of Formula I or related or derivative compou nd is formu lated, or coordinate!}' adm inistered, in com bination with one or more secondary cr adj unctive therapeutic agent(s). to yield a combined formulat ion or coordinate treatment method that is combinatorially effective or coordinatelv useful to treat in the prevention or treatment of Parkinson ^' s disease employ the phorbol ester compound of Formula 1 in combination w ith one or more add itional, neuroprotective or other indicated, secondary or adj unctive therapeutic agents that is/are useful for treatment or prophylaxis of the targeted disease, condition and/or symptom(s). For most combinatorial fonnulations and coordinate treatment methods of the invention, a phorbol ester compound of Formula I or related or derivative compound is formulated, or coordinatelv adm inistered, in combination with one or more secondary or adjunctive therapeutic agent(s), to yield a combined formulation or coordinate treatment method that is combinatorially effective or coordinately useful to prevent or treat Parkinson's disease. Exemplary combinatorial formulations and coordinate treatment methods in this context employ a phorbol ester compound of Formula I in combination with one or more secondary or adjunctive therapeutic agents selected from carbidopa/levodopa, tolcapone, MAO-B inhibitors, pyridoxine, amantadine, selegiline, rasagil ine, anticholinergics, cholinesterase inhibitors, COMT Inhibitors, and dopamine agonists including, but not lim ited to, apomorphine, atropine, benztropine, biperiden, bromocriptine, cabergoline, ciladopa, dihydrexidine, dinapsoline, doxanthrine, entacapone, epicriptine, lisuride, pergolide, piribedil, pramipexole, propylnorapomorphine, quinagolide, rivastigmime, ropinirole, rotigotine, roxindole, sumanirole, trihexphenidyl, . In addition, adjunctive or secondary therapies may be used such as, but not limited to, antioxidants, amino acid therapy, deep brain stimulation, diet modification, exercise, herbal supplementation, hormone therapy and/or mineral supplementation.

[047] In certain embodiments the invention provides combinatorial disease treating "Parkinson's disease treating," "dopamine enhancing," "catechol-O-methyl transferase inh ibi ting," "aromatic L-am ino acid decarboxylase inhibiting," "dopam ine agonist," "neurop rotective," "anticholinergic, ^" "muscle relaxant' ^* formulations comprising a phorbol ester and one or more adjunctive agent(s) having disease treating activity. Within such combinatorial formu lations, a phorbol ester of Formula 1 and the adjunctive agent(s) having disease treating activity wil l be present in a combined formul ation i n disease treating ("Parkinson ^' s disease treating. ^" "dopam ine enhancing," "catechol-O-methyl transferase inhibiting," "aromatic L-amino acid decarboxylase inhibiting." "dopamine agonist," "neuroprotective," "anticholinergic," "muscle relaxant,") effective amounts, alone or in combination. In exemplary embodiments, a phorbol ester compound of Formula I and a non-phorbol ester agent(s) wil l each be present in a d isease treating/preventing amount (i.e., in singular dosage which will alone el icit a detectable alleviation of s\ mptoms in the subject). Alternatively, the combinatorial formulation may comprise one or both the phorbol ester compound of Formula I and the non-phorbol ester agents in sub-therapeutic singular dosage amounts), wherein the combinatorial formulation comprising both agents features a combined dosage of both agents that is col lectively effective in eliciting a cytopathic disease or condition symptom alleviating response. Thus, one or both of the phorbol ester of Formula I and non-phorbol ester agents may be present in the formulation, or administered in a coordinate administration protocol, at a sub- therapeutic dose, but collectively in the formulation or method they elicit a detectable decrease in symptoms of cytopathic disease in the subject.

[048] To practice coordinate administration methods of the invention, a phorbol ester compound of Formula I may be administered, simultaneously or sequential ly, in a coordinate treatment protocol with one or more of the secondary or adjunctive therapeutic agents contemplated herein. Thus, in certain embodiments a compound is administered coordinately with a non-phorbol esteir agent, or any other secondary or adjunctive therapeutic agent contemplated herein, using separate formulations or a combinatorial formulation as described above (i.e., comprising both a phorbol ester compound of Formula I or related or derivative compound, and a non-phorbol ester therapeutic agent). This coordinate administration may be done simultaneously or sequentially in ei her order, and there may be a time period while only one or both (or al l) active therapeutic agents individually and/or col lectively exert their biological activities.

[049] Within ex3tnplary embodiments, a phorbol ester compound of Formula I wil l be coordinately adm inistered (simu ltaneously or sequentially, in combined or separate formulation(s)), with one or more Parkinson ^' s disease treating agents. Such coordinate treatment methods ma . for example, fol low or be derived from various protocols for the Treatment of Parkinson's disease. Coordinate treatment methods may, for example, include a phorbol ester and/or treatments for prevention or treatment of Parkinson's disease. A distinguishing aspect of a l l such coordinate treatment methods is that the phorbol ester compound of Form ula I exerts at least some activity, wh ich yields a favorable clinical response in co njunction w ith a complementary Parkinson ^' s disease preventing or treating agent, or dist inct, clin ical response provided by the secondary or adj unctive therapeutic agent. Often, the coordinate adm inistration of the phorbol ester com pound of Formula I with the secondary or adjunctive therapeutic agent w i ll y ield improved therapeutic or prophylactic resu lts in the subject beyond a therapeutic effect el icited by the phorbol ester compound o f Formula 1, or the secondary' or adjunctive therapeutic agent administered alone. This qual ification contemplates both direct effects as well as indirect effects. V/ithin exemplary embodiments, a phorbol ester compound of Formula I will be coordinately administered (simultaneously or sequentially, in combined or separate formulation(s)), with one or more secondary Parkinson' s disease treating compounds or other indicated or adjunctive therapeutic agents, e.g. carbidopa/levodopa, tolcapone, MAO-B inhibitors, pyridoxine, amantadine, selegiline, rasagiline, anticholinergics, cholinesterase inhibitors, COMT Inhibitors, and dopamine agonists including, but not lim ited to, apomorphine, atropine, benztropine, biperiden, bromocriptine, cabergoline, ciladopa, dihydrexidine, dinapsoline, doxanthrine, entacapone, epicriptine, lisuride, pergolide, piribedil, pramipexole, prc pylnorapomorphine, quinagolide, rivastigmine, ropinirole, rotigotine, roxindole, sumariirole, trihexphenidyl, . In addition, adjunctive or secondary therapies may be used such as, but not limited to, antioxidants, amino acid therapy, deep brain stimulation, diet modification, exercise, herbal supplementation, horimone therapy, mineral supplementation, physical therapy, and/or lesion formation.

[050] As noted above, in al l of the various embodiments of the invention

contemplated herein; the disease treating methods and formulations may employ a phorbol ester compound of Formula 1 in any of a variety of forms, including any one or combination of the subject compound's pharmaceutical ly acceptable salts, solvates, isomers, enantiorners, polymorphs, solvates, hydrates, and/or prodrugs. In exemplary embodiments of t he invention, TPA is employed within the therapeutic formulations and methods for i ll ustrative purposes.

[051] The pharmaceutical compositions of the present invention may be

administered by .ny means that achieve their intended therapeutic or prophylactic purpose. Suitable routes of administration for the compositions of the invention include, but are n ot l imited to, conventional delivery routes, devices and! methods including injectable methods such as. but not limited to. intravenous, intramuscular, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intraarterial, subcutaneous and intranasal routes.

[052 ] The compositions of the present invention may further inc l ude a pharmaceutically acceptable carrier appropriate for the particular mode of administration be ng employed. Dosage forms of the compositions of the present invention include excipients recognized in the art of pharmaceutical compounding as being suitable for the preparation of dosage units as discussed above. Such excipients include, without i ntended limitation, binders, fil lers, lubricants, emulsifiers, suspending agents, sweeteners, flavorings, preservatives, buffers, wetting agents, disintegrants, effervescent agents and other conventional excipients and additives.

[053] If desired, the compositions of the invention can be administered i n a controlled release form by use of a slow release carrier, such as a hydrophilic, slow release polymer. Exemplary controlled release agents in this context include, but are not limited to, hydroxypropyl methyl cellulose, having a viscosity in the range of about 100 cps to about 1 00,000 cps or other biocompatible matrices such as cholesterol.

[054] Some phorbol ester compositions of Formula I of the invention are designed for parenteral administration, e.g. to be administered intravenously, intramuscularly, subcutaneously or intraperitoneal ly, including aqueous and nonaqueous sterile injectable solutions which, like many other contemplated

compositions of the invention, may optionally contain anti-oxidants, buffers, bacteriostats and'or solutes which render the formulation isotonic with the blood of the mammal ian subject; and aqueous and non-aqueous steri le suspensions which may i nclude suspendi ng agents and/or thicken ing agents. The formulations may be presented in unit-dose or multi-dose containers. Additional compositions and formulations of tie invention may include polymers for extended release fol lowing parenteral admin istration. The parenteral preparations may be solutions, dispersions or emulsions suitable for such administration. The subject agents may also be formulated into polymers for extended release fol lowing parenteral admin istration. Pharmaceutically acceptable formulations and ingredients wi ll typical ly be steri le or readi ly steri lizable, biological ly inert, and easi ly adm in istered. Such po lymeric materials are wel known to those of ordinary ski ll in the pharmaceutical

compounding arts. Parenteral preparations typical ly contain buffering agents and preservatives, and injectable fluids that are pharmaceutically and physiological ly acceptable such as water, physiological sal ine, balanced salt sol utions, aqueous dextrose, glycerol or the l ike, extemporaneous injection solutions, emulsions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as described herein above, or an appropriate fraction thereof, of the active ingredient(s).

[055] In more detailed embodiments, compositions of the invention may comprise a phorbol ester compound of Formula 1 encapsulated for delivery in microcapsules, microparticles, or microspheres, prepared, for example, by coacervation techniques or by interfacial poly merization, for example, hydroxymethylcellulose or gelatin- m icrocapsules and poly(methylmethacylate) microcapsules, respectively; in col loidal drug delivery systems (for example, liposomes, albumin microspheres,

microemulsions, nano-particles and nanocapsules); or within macroemulsions.

[056] As noted above, in certain embodiments the methods and compositions of the invention may employ pharmaceutical ly acceptable salts, e.g., acid addition or base salts of the above-described phorbol ester compounds of Formula I and/or related or derivative compounds. Examples of pharmaceutically acceptable addition salts include inorganic and organic acid addition salts. Suitable acid addition salts are formed from acids which form non-toxic salts, for example, hydrochloride, hydrobrom ide, hydroiodide, sulphate, hydrogen sulphate, nitrate, phosphate, and hydrogen phosphate salts. Additional pharmaceutically acceptable salts include, but are not limited to, metal salts such as sodium salts, potassium salts, cesium salts and the like; alkaline earth metals such as calcium salts, magnesium salts and the like; organic amine salts such as triethylam ine salts, pyridine salts, picoline salts, ethanolamine sal ts, triethanolamine salts, dicyclohexylamine salts, Ν,Ν '- dibenzylethylenediamine salts and the l ike; organic ac id salts such as acetate, citrate, lactate, succinate, tartrate, maleate, fumaratc, mandelate, acetate, dich loroacetate, trifluoroacetate, oxalate, and formate salts; su l fonates such as mcthanesul fonate, benzenesulfonate, and p-toluenesulfonate salts; and amino acid salts such as arginate, asparginate, glutamate, tartrate, and gluconate salts. Suitable base salts are formed from bases that form non-toxic salts, for example aluminum, calcium , l ithium, magnesium, potassium, sodium, zinc and diethanolam ine salts.

[057] Other detailed embodiments, the methods and compositions of the invention for employ prodrjgs of phorbol esters of Formula I . Prodrugs are considered to be any covalently bonded carriers which release the active parent drug in vivo. Examples of prodrugs usefu l within the invention include esters or am ides with hydroxyalkyl or aminoalkyl as a substituent, and these may be prepared by reacting such compounds as described above with anhydrides such as succinic anhydride.

[058] The invention disclosed herein wil l also be understood to encompass methods and compositions comprising phorbol esters of Formula 1 using in vivo metabol ic products of the sa id compounds (either generated in vivo after admini stration of the subject precursor compound, or directly administered in the form of the metabol ic product itself). Such products may result for example from the oxidat ion, reduction, hydrolysis, amidation, esterification and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the invention includes methods and compositions of the invention employing compounds produced by a process comprising contacting a phorbol ester compound of Formula I with a mammalian subject for a peri od of time sufficient to yield a metabolic product thereof. Such products typically are identified by preparing a radiolabeled compound of the invention, administering it parenterally in a detectable dose to an animal such as rat, mouse, guinea p ig, monkey, or to man, al lowing sufficient time for metabolism to occur and isolating its conversion products from the urine, blood or other biological samples.

[059] The invention disclosed herein wil l also be understood to encompass diagnostic compositions for diagnosing the risk level, presence, severity, or treatment indicia of, or otherwise managing diseases including, but not lim ited to, Parkinson's disease or a related disease or condition in a mammal ian subject, comprising contacting a labeled (e.g., isotopical ly labeled, fluorescent labeled or otherwise labeled to permit detection of the labeled compound using conventional methods) phorbol ester compound of Formula I to a mammal ian subject (e.g., to a cell, tissue, organ, or individ ual) at risk or presenting with one or more symptom(s) of

Parkinson's disease, and thereafter detecting the presence, location, metaboli sm, and/or binding state of the labeled compound using any of a broad array of known assays and labeli ng/detection methods. In exemplary embodiments, a phorbol ester compound of Formula 1 is isotopically-labeled by havi ng one or more atoms replaced by an atom havin g a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ² H, ³ H , ¹³ C. 1 ⁴ C, ^{, S} N, ^{, 8} 0. ^{, 7} 0. ^I P. ³² P, ³⁵ S, ^{, S} F. and ³⁶ C1, respectively. The isotopically-labeled compound is then adm inistered to an ind ividual or other subject and subsequently detected as described above, yield ing usefu l diagnostic and/or therapeut ic management data, according to conventional techniques.

Examples

[060] The experiments described below demonstrate novel and powerful uses for phorbol esters and related derivative compounds in the treatment of Parkinson' s disease and symptoms of Parkinson' s disease. These and additional Findings are further expanded and elucidated within the following examples. Example ] Dose

Ranging Study

[061] Due to the strong local irritation caused by TPA application, TPA was given to patients by intravenous (i.v.) infusion. TPA solution in a sterile syringe was injected into 200 ml of sterile saline and mixed well for i.v. infusion.

The Toxicity and Side Effects of Different TPA Doses Adm inistered Clinically:

[062] ( 1 ) TPA given at 1 mg/patient/week:

[063] One mg TPA in solution was mixed well with 200 ml of steri le saline for intravenous infusion which was completed in 1 h at the rate of 16 μg/min. One hour after TPA administration, patients started to have chills which lasted for about 30 min, followed by fever, (the patients' temperature reached 37.5-39.5°C. which lasted for 3- 5 h, then returned to normal) with light to heavy perspiration. The above symptoms could be alleviated by giving the patients glucocorticoids. TPA at this dose caused a m inority of patients to bleed, several patients suffered for a short period of time difficulty in breathing, and Hb was detected in the urine. However, these side effects were short lived and reversible. The cardiac, hepatic, renal and pulmonary functions were al l found to be normal.

[064] (2) TPA given at 0.5 mg/patient x 2/week: (two doses a week)

[065] 0.5 mg of TPA in solution was mixed well with 200 ml of sal ine for intravenous infus ion which was completed in 1 h at the rate o f 8 μg/rnin. The reactions after adm inistration were sim ilar to that of the 1 mg TPA dosage, but to a lesser extent than the 1 mg dose. The patients tolerated the lower dose more easily. Occasional ly, Hb was detected in patients ^' urine. Di fficulty in breathing was not observed. The cardiac, hepatic, renal and pulmonary functions were al l normal.

[066] (3 ) TPA giv en at 0.25 mg/patient x 4/week:

0.25 mg of TPA i n solution was mixed wel l w ith 200 m l of sal ine for intravenous infusion which was completed in 1 h at the rate of 4 μg/m in. After administration, symptoms such as chills and fever were also observed, but to a much lesser extent than with the higher dosages. No Hb was detected in the urine, and no patient suffered difficulty in breathing. The cardiac, hepatic, renal and pulmonary functions were all normal. Example II

Effectiveness of ^' TPA in the treatment of Parkinson's disease using a 6-OHDA model

[067] The action of TPA is investigated using the 6-hydroxydopamiine (6-OHDA)- lesioned rat model of Parkinson's disease where the animals have a partial, uni lateral loss of dopaminergic neurons induced by a single injection of 6-OHDA in the median forebrain bundle. Upon amphetamine stimulation, the lesioned animals wil l display ipsiversive rotational behavior due to the imbalance in dopamine release resulting from the uni lateral loss of dopaminergic cells. This al lows the functional effects of a compound tested in this model to be quantified by counting the number of amphetamine-induced rotations (Ungerstedt, U., 1970). Animals with complete lesion will exhibit contraversive rotational behavior after apomorphine stimulation, making it possible to exclude any fully lesioned animals from the study.

[068] Male Sprague-Dawley rats weighing 280-320 g are housed in a temperature- controlled room jnder a 1 2 h l ight/dark cycle with access to food and water ad l ibitum. Thi rty m i nutes prior to surgery, animals are injected intraperitoneally with pargyline (5 mg/kg) and desipramine (25 mg/kg). Rats are then placed in a stereotactic frame under general anesthesia and a small bur-hole is made in the right side of the skul l. Each animal is given a uni lateral injection of 4 [ig, 6-OHDA (in 2 μΙ sterile water with 0. 1 % ascorbic acid) over 5 m inutes into the right medial forebrain bundle at co-ordinates -2.8mm from bregma, 2 mm lateral to the mid l ine, and 8.6 mm below skull and the rats are then given 5 weeks to recover. The rats are each given 0.05 mg/kg s.c. apomorhine and 5 mg/kg i .p. amphetam ine and assessed for partial or complete lesions. The animals with partial les ions as described above are then randomly divided into two groups and injected with either placebo or 0. 1 25mg/m ² of TPA once per day for six days. After six days they are then inj ected again with 0.05 mg/kg s.c. apomorphine and 5 mg/kg i .p. amphetamine and rotational activity is measured by counting the total number of turns in the 60 minutes following apomorphine or a mphetamine administration. The rats are then left alone for two weeks and sacrificed. The brains of the rats are then fixed and immunostained for the tyrosine hydroxyl ase, and the dopaminergic neurons and their fibers quantified stereologically. Example I I I

Therapeutic effect of TPA in the MPTP model of Parkinson ^' s Disease.

[069] The neurot oxin l -methyl-4-phenyl- l ,2,3,6-tetrahydropyridine (MPTP) is a complex 1 (NAD H dehydrogenase) mitochondrial respiratory chain inhibitor that is used to induce dopaminergic cell loss (Varastet et al., 1994). This toxin is currently widely used as an animal model for PD (Bezard et al., 1997).

[070] Male C57/BL6 mice of 6-9 months old weighing 30-40g are div ided into two groups. All mice are given MPTP (30 mg/kg i.p.) b.i.d. for 6 days. Mice are injected with 0. 125mg/m ^'! TPA or vehicle 2 hours prior to toxin administration until the day before sacrifice. Eight days after stopping injection of M PTP, the mice are sacrificed by CCh and the s :riata from both sides are dissected out on cold surface and frozen on dry ice. The dopaminergic neuronal survival is assessed by striatal dopam ine (DA) content. The dopam ine content is assayed by a radioenzymatic method under GLP conditions, but DA can also be measured using high pressure l iquid chromatography with electrochem ical detection as previously described (Friedemann & Gerhardt, 1 992).

Example I V

Therapeut ic Effect of TPA in the Paraquat model of Parkinson ^' s Disease

[071 ] Experimental evidence demonstrates that the herbicide Paraquat is invol ved in the pathogenesis of Parkinson's Disease. Both rnicroinfusion of Paraquat into the Substantia Nigra of the an imal and system ic or intraperitoneal treatment lead to selective degeneration of dopaminergic neurons accompanied by behavioral and neuropathological signs of severe non-selective neurotoxicity (Brooks A . I . et al ., 1 999; Thiruchelvam M. et al.. 2002 : McCornack A . L. et a l ., 2002).

[072] In this experiment, male Sprague Dawley rats (21 0-220 g weight) are kept on a standard laboratory diet for three weeks (relative hum idity 50%± 1 0%, temperature 22° C.± l and in a light/darkness cycle of 1 2 hours/ 1 2 hours) prior to the tests ith food and water avai lable ad libitum.

[073] Prior to treatment, the rats are anaesthetized by intraperitoneal injection of 380 mg/kg weight of chloral hydrate; a stainless steel guide cannula (size 25 ) is implanted unilaterally in the Substantia Nigra with stereotaxic guiding and is secured to the cranium with deni al acrvlic material. [074] The animals (20 per test group) are allowed a recovery period of seven days prior to treatment; during this period no change in motor activity or posture is observed. Micro infusions are performed by means of a 10 μΐ Hamilton syringe connected to an i njection cannula by means of a Teflon tube. The Paraquat, TP A (0.125mg/m ² ) + Paraquat or the vehicle (0.8% NaCl) are injected at a total volume of 1 μΐ/minute. The animals are then observed for motor activity and general well-being

Example V

Therapeutic Effect of TPA in the MPTP model of Parkinson's disease

[075] MPTP ( 1 - triethyl-4-phenyl- 1 .2,3,6-tetrahydropyridine) is

a neurotoxin precursor to MPP+, which causes permanent symptoms of Parkinson's disease by destroying dopam inergic neurons in the substantia nigra of the brain .

[076] C57BL/6J mice are pretreated with cither vehicle ( 1 % methyl cell ulose) or TPA 30 min prior to administration of MPTP. M PTP is dissolved in isotonic saline (0.9%) and given subcutaneously as a single dose of 1 5 mg free base/kg body weight to produce a reduction in striatal dopamine to about 0.5 nanomoles/mg protei n. Groups of mice (n==8- 1 0 per group) receive either vehicle plus sal ine, vehicle plus MPTP, or TPA p lus M PTP. Seventy two hours after receiv ing MPTP, mice are sacrificed using cervical dislocation and the striata are excised. The tissue is homogenized in 0.4 N perchloric acid, centrifuged, and the supernatant analyzed by high performance li quid chromatography/electro-chem ical detection (H PLC/ED) for dopamine levels. Supernatants are stored in a -90° C. freezer between the time of collection and analysis. TPA is combined with methyl cel lulose and homogenized in water for dosing.

[077] Additional studies are carried out to determ ine the long-term effect on dopamine depletion of TPA. Using the general test method described above, C57BL/6J mice are pretreated with either vehicle or TPA and MPTP and then sacrificed either 3 or 14 days after dosing.

Example V I

Effect of TPA on Reserpine and Haloperidol model of Parkinson's disease

[078] Reserpine (usual dose 4-5 mg kg ^~ ' s.c. ) works by inhibiting the vesicular monoamine transporter, VMAT2. This leads to loss of storage capacity and hence depletion of brain (and peripheral) monoamines including noradrenaline and 5-HT as wel l as dopamine. Behaviorally, reserpine induces features of akinesia and hind limb rigidity in rats that are representative of symptoms associated with PD. [079] Haloperidol works by antagonizing dopamine D2 and, to a lesser extent, Di receptors in medium spiny neurons that comprise the indirect and direct pathways of the motor circuit respectively. The resultant block of striatal dopamine transmission results in abnormal downstream firing within the basal ganglia circuits that is manifest as symptoms of muscle rigidity and catalepsy within 60 min of haloperidol (0.5-5 mg kg ^"1 , i.p.) injection (see, e.g., Sanberg PR. Haloperidol-induced catalepsy is mediated by postsynaptic dopamine receptors. Nature.1980;284:472-473).

[080] Animals are administered injections of dextrose (vehicle), reserpine, or haloperidol. Eighteen hours after reserpine is administered, 0. 125mg/m ² TPA is administered. Fifteen minutes after haloperidol is administered, 0. 1 25mg/m ^'? TPA is administered.

[081 ] Animals are mon itored over a 60 min period in al l instances in transparent shoebox cages that measure 45 x25 x20 cm, with a 1 -cm depth of wood chips on the cage floor and a plastic grid on top of the cage. Rectangular photocel l monitors with a bank of 3 photocell beams surround each test cage. Ambulations (locomotor activity: total number of beam breaks in 1 hour) are recorded by computer and stored for each test session. All data are expressed as total counts over the entire 1 h test period.

Example VI I

Therapeutic effectiveness of TPA on Rotenone model of Parkinson ^' s disease

[082] The insecti cide rotenone is highly l i pophi lic, readi ly crossing the blood-brain barrier and d i ffus ing into neurons w here, it accumu lates with in mitochondria and inhibits complex I. The production of ROS, subsequent to glutathione depletion, is thought to induce oxidative stress (Sherer TB. Betarbet R, Testa CM, Seo BE!.

Richardson JR. Kirn JH. et al. Mechanism of toxicity in rotenone models of

Parkinson's disease. J Neurosci. 2003 ; 23 : 1 0756-1 0764.). Oxidative damage, in the form of protein carbonyl formation, has certainly been found in the midbrain , olfactory bulb, striatum and cortex of rats treated with rotenone (Sherer TB, Betarbet R, Testa CM, Seo BB, Richardson JR, Kim J H, et al. Mechanism of toxicity in rotenone models of Parkinson's disease. J Neurosci . 2003 :23: 10756- 10764.), just as is reported in the PD brain at post-mortem (Alam ZI, Daniel SE, Lees AJ, Marsden DC, Jenner P, Halliwe ll B. A generalised increase in protein carbonyls in the brain in Parkinson's but not incidental Lewy body disease. J Neurochem. 1 997;69: 1326- 1 329). [083] Forty four male Sprague-Dawley rats weighing 239± 5g (range: 232-249 g) at the start of testing are evaluated for baseline performance in the Corridor, Stepping and Whisker Tests was established over 1 week pre-operatively.

[084] For the Corridor test, hungry rats (deprived of food overnight) are placed into a long, narrow al leyway ( 150 cm) and are free to retrieve treats from pots on the left and right sides. Trials are completed when rats made a total of 20 retrievals or after a maximum trial time of 5 min has elapsed. Data are expressed as the number of retrievals made from the ipsilateral side as a percentage of the total number of retrievals made. (Dowd E. Monville C, Torres EM, Dunnett SB (2005) The corridor task: a simple test of lateralised response selection sensitive to

unilateral dopamine deafferentation and graft-derived dopamine replacement in the striatum. Brain Res Bull 68( l -2):24-30)

[085] For the Stepping Test both hindlimbs and one forelimb are gently restrained by the experimenter, and the number of forehand and backhand ad justing steps made by the unrestrained forelimb is counted when the rat is moved sideways along a table surface for 90cm over5s.( Olsson M, Nikkhah G, Bentlage C, Bjorklund A ( 1995) Forelimb akinesia in the rat Parkinson model: differential effects of dopamineagonists and nigral transplants as assessed by a new stepping test. .1 Neurosci 1 5(5 Pt 2):3863— 3875.)

[086] For the Whisker Test of sensorimotor integration both hind limbs and one forelimb are genl ly restrained by the experimenter, and the number of vibrissae- evoked forelimb-placings made by the unrestrained forelimb is counted when the rats ^' whiskers are brushed against the side of a table. This is repeated 1 0 times for each rat on both sides. (Schallert T, Fleming SM. Leasure JL, Tillerson JL, Bland ST

( 2000)CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.

Neuropharmacology 39(5):777-787

[087] After evaluation, rats are divided into performance-matched groups and receive rotenone, control infusions, TPA + rotenone, or TPA + control into the striatum at four points along its rostrocaudal axis. Unilateral lesion surgery is conducted under isofluorane anesthesia (5% in O2 for induction and 2% in 0 ₂ for maintenance) in a stereotaxic frame w ith the nose bar set at 2.3 mm. Rotenone (Sigma, Ireland) is dissolved in a solution of DM SO (Sigma reland), Cremophor ® (Sigma, Ireland) and saline ( 1 : 1 : 1 8). The striatum is lesioned by infusion (Ι μ/min for W

3 min with 2 min for diffusion) at four points along its rostrocaudal axis at stereotaxic coordinates AP+ 1 .3, ML±2.7; AP+0.4, ML±3.1 ; AP -0.4 ML,±4.3; AP - 1.3, ML, ± 4.7 (from bregma) and DV -5.0 below dura. All rats are lesioned on the side opposite their preferred si de as indicated by pre-operative testing in the Corridor Test.

[088] Post-operative behavioral testing is carried out over a 5 week period after which the rats are sacrificed by transcardial fixation and used for quantitative tyrosine hydroxylase and a-synuclein immunohistochemistry. A separate cohort of rats are administered the same rotenone or control infusions and are sacrificed by decapitation 5 weeks after surgery for HPLC analyses.

|089] Rats are sacrificed by term inal anesthesia (50 mg/kg pentobarbital i .p.) and trans-card ially perfused with 1 00 ml heparinized (5000 U/L, Sigma, Ire land) saline followed by 1 50 ml 4% paraformaldehyde. Brains are rapidly removed, post- fixed in 4% para-formaldehyde for 4 h and stored in a 25% sucrose plus 0.1 %sodium azide solution. Serial coronal sections (40 μηι) are cut using a freezing sledge microtome and a 1 :6 series of sections is used for all quantitative imm unohistochem istry. For immunoh istochern ical analyses, following quenching of endogenous peroxid ase activity (using a solution of 3% hydrogen peroxide/ 1 0% methanol in distilled water) and blocking of non-specific secondary antibody binding (using 3% normal horse serum in Tris-buffered sal ine (TBS) w ith 0.2% Triton X- 1 00 at room temperature for 1 h),sections are incubated overn ight at room temperature with the appropriate primary antibody di luted in TBS w ith 0.2% TritonX- 1 00 ( mouse anti -tyrosine hydroxylase (MAB 3 1 8. 1 : 1 000. M ill ipore. U K ) and mouse anti-a-synuclein

(AB 1903, 1 : 1 000. Abcam. U K) ). Sections are then incubated in a biotiny lated secondary antibody for 3 h (horse anti-mouse ( 1 :200, Vector, U K))

followedby 2 h in tubation in streptavidin-biotin-horseradish peroxidase

solution(Vector, U K). Sections are developed in 0.5% solution of diaminobenzidine tetrahydrochloride (Sigma, Ireland) in Tris buffer containing 0.3 μ1/ιη Ι of hydrogen peroxide. Sections are mounted on gelatin coated microscope slides, dehydrated in ascending concentrations of alcohols, cleared in xylene and cover slipped using DPX mountant (BDH chemicals, U K). Photomicrographs of immunostained sections are captured under bright field i llumination and all image analysis is completed using Image J software. For quantification of any rotenone-induced nigro striatal degeneration, tyrosine hydroxylase immunopositive cell bodies in the ipsilateral and contralateral substantia nigra are counted in three coronal sections, and the optical W

density of tyrosine hydroxylase immunoreactivity in the ipsilateral and contralateral striata are quantified in three coronal sections. For quantification of any rotenone- induced a-synuc leiin expression, a-synuclein immunopositive cell bodies in the ipsilateral and contralateral substantia nigra are counted in a

5 single coronal section, and the optical density of a-synuclein immunoreactivity in the ipsilateral and contralateral striata are quantified in a single coronal section. To quantify any rotenone-induced striatal damage, ipsilateral and contralateral striatal volume is determ ined from three coronal sections through the

striatum. A l l quantitative immunohistochcm ical data on the lesioned side is

10 expressed as a percentage of the intact side.

HPLC Analyses

[090] Striatal concentrations of dopamine, noradrenal ine and 5-HT in the intact and lesioned striata are determined using HPLC with electrochemical detection. Striatal tissue samples are briefly sonicated in I ml of mobile phase (0.1 M citric acid, 0. 1 M

15 sodium dihydrogenphosphate, 0. 1 mM EDTA, 1 .4 niM octane- 1 -sulfonic acid,

10%(v/v) methanol in distilled water, pH 2.8) containing the N -methyl5- hydroxytryptamirie (2ng/20μ I) as the internal standard. Homogenates are centrifuged at 1 5,000 x g for 1 5 min and 20μ1 of the resultant supernatant is injected onto a reverse-phase col umn (1.1 Chrosorb RP- 1 8, 25 cm x 4 mm internal diameter, particle 0 size 5 μηι) for separation of catecholamines (flow rate 1 ml/min). Neurotransm itter concentrations are quantified by electrochemical detect ion (Shimadzu )

and chromatograrns are generated using a Merck-H itachi D-2000 integrator. Striatal concentrations of GABA are determ ined using reverse-phase HPLC with fluorometric detection as described previously described (Rea , Cremers TLWesterink

5 BH(2005)HPL Conditions are critical for the detection of GABA by m icrodialysis. J Neurochem 94(3) :672-679). In brief, tissue is sonicated and centrifuged as described above and GABA concentrations in the supernatant are determined off-l ine by pre- column derivatization with o-phthaldialdehyde/mercaptoethanol reagent (50 rng o- phthaldialdehyde (Sigma) dissolved in 1 ml methanol and added to 99 ml0.5 rnol/L0 NaHCC (pH 9.5) containing 10 μ Ι 2-mercaptoethanol), and separation by reverse- phase HPLC with fl orometric detection. Samples (50μ Ι) are derivatized with 30 μ1 o- phthaldialdehyde/mercaptoethanol reagent, m ixed and a llowed to react for 2min. Then 50μ 1 of the reaction m ixture is inj ected by a Gi lson401 C auto sampler (Gilson, Villiers le Bel, France) onto the HPLC apparatus. The derivatization mixture is separated using anisocratic mobile phase and measured by fluorometric detection. The mobile phase consisted of 70 mm di-sodium hydrogen phosphate, 400 μιη EDTA, 0.1 5% (v/v) tetrahydrofurane and methanol(30% (v/v)). The pH of the mobile phase is adjusted to 5.26 with phosphoric acid. The HPLC system

consisted of a SupercosilLC- 18-DB column ( 1 50x4.6 mm, particle size 3μπι; Supelco Inc., Bella Fonte, PA, USA), a Gynkotec 300C high precision pump(flow rate 0.95, or 1 .00 ml/min; Gynkotec, Germering, Germany)and a JASCO FP- 1 20 fluorometric detector (excitati on λ-350ηιη, emission λ-450 nm; Jasco Corporation, Tokyo, Japan ). (Connor TJ, Kelly J P, Leonard BE ( 1 997) Forced swim test-induced neurochemical endocrine, and immune changes in the rat. Pharmacol Biochem Behav 58(4): 961™ 967.)

Statistical Analyses

[091 J Data are analyzed using a Student's t -test or ANOVA (one-way or two-way with repeated measures fol lowed by post-hoc Newman-Keuls) where appropriate. All tests used are ind icated at the relevant point in the text. Differences between groups are considered statistically significant when P <0.05. All data are expressed as mean ± SE .

Example V I I 1

Effectiveness of Phorbol Esters as catechol O-methyltransferase inhibitors

[092] The activity of the phorbol esters as catechol O-methyltransferase (COMT) inhibitors may be readi ly determ ined without undue experimentation using a fluorescence or fl uorescence polarization (FP) methodology that is well known in the art (Kurkela M et al ., Anal Biochem (33 1 ) 2004, 198-200 and Graves, TL et al . Anal Biochem (373) 2008, 296-306). Any compound exhibiting an IC50 below 1 μΜ would be considered a COMT inhibitor as defined here in.

Assay 1

[093] Recombinant human S-COMT ( 12 nM) is preincubated with the phorbol ester and 400 μΜ S-adenosyl-L-meth ionine in 1 00 mM Na2FlP04 buffer, pH 7.4, containing 5 mM MgCb for 60 min at 37 °C . The reaction is started with the addition of the substrate esculetir for a final concentration of 2 μΜ and the production of O- methylated esculetin is followed with the FlexStation fluorescence plate reader (Molecular Probes, USA) using excitation at 355 nm and emission at 460 nm. The inhibitor dissociat ion constant, K, of the studied compounds is calculated using the Morrison equation, which takes the tight binding inhibition into account (Copeland, R. A. Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists, John Wiley & Sons, Inc., Hoboken, NJ, 2005, pp. 185-187): v, _ _t (E + I + Kj)-V(E + I + ,) ² - 4E - 1 wherein v ₀ and v; are the reaction velocities in the absence and presence, respectively, of the inhibitor, E is the active enzyme concentration, and I is the inhibitor concentration. The data are analyzed with GraphPad Prism version 4.00 software (GraphPad Software, San Diego, CA, USA). Assay 2

[094] Recombinant human S-COMT (0.8 n ) is incubated with the phorbol ester and 200 μΜ S-aclenosyl-L-methionine in 1 00 m Na ₂ HP04 buffer, pH 7.4, containing 5 mM MgCb for 30 min at 37 °C. The reaction is started with the addition of esculetin for a final concentration of 0.5 μΜ and the reaction mixture with total volume of 200 μΙ is incubated for 30 min at 37 °C. The reaction is stopped with 20 μΐ of 4 M HC 104 and the precipitated protein is removed by Sirocco protein precipitation plate (centrifuged at 4 °C for 10 min at 3000g). O-Methylated esculetin is detected by Waters HT All iance HPLC setup with Waters 474 fluorescence detector (Ex 460 nm, Em 460 nm, Gain 1 00). The anahtes are separated isocratically using 0. 1 M Na2HP04, 20 mM citric acid, 0. 1 5 mM EDTA, pH 3.2, in 40% methanol as mobile phase and Waters Spherisorb ODS2 ( 3 μηι, 4.6 mm x 100 mm) column. O-Methylated esculetin concentrations arc calculated based on the standard curve and the ¾ values are calculated using the Morrison equation as in Assay 1 .

Example IX

Effectiveness of Phorbol esters as decarboxylase inhibitors

[095] Phorbol esters are assayed for the inhibition of mammalian decarboxylase based on the formation of serotonin. Serotonin decreases generation of dopaminergic neurons from mesencephalic precursors via serotonin type 7 and type 4 receptors (Parga, J., Rodriguez-Pallares, J., Mufioz, A., Guerra, M. J. and Labandeira-Garcia, J.L. (2007), Serotonin decreases generation of dopaminergic neurons from

mesencephalic precursors via serotonin type 7 and type 4 receptors. Devel Neurobio, 67: 10-22. doi: 10. 1002/dneu.20306). Groups of mice are pretreated 16 hours in advance with an MAO-B inhibitor are given a combined dose of the phorbol ester being tested and 100 mg kg i.p. of 5-hydroxytryptophane. Oral doses are

administered separately 30 m inutes prior to the 5-hydroxytryptophane. Forty-five minutes after administration of the 5-hydroxytryptophane, the mice are sacrificed. The kidneys of each group are pooled, homogenized in water assayed for serotonin. The results are expressed in terms of percent inhibition of serotonin in the kidneys as compared to a control group similarly treated but not given a protease ester.

[096] Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the artisan that certain changes and modifications may be practiced within the scope ofthe appended claims which are presented by way of il lustration not l imitation. In this context, various publications and other references have been cited with the foregoing disclosure for economy of description. Each of these references is incorporated herein by reference in its entirety for al l purposes. It is noted, however, that the various publications discussed herein are incorporated solely for their d isclosure prior to the filing date of the present application, and the inventors reserve the righ t to antedate such disclosure by virtue of prior invention.

References

Abrahm J. L., Gerson S. L., Hoxie J. A., Tannenbaum s. h., Cassileth p. A., Cooper R. A. Differential effects of phorbol esters on normal myeloid precursors and leukemic cells. Cancer Res.46, 3711-3716(1986).

Ando I., Crawfor D. H. et al. Phorbol ester-induced expression and function of the interleukin 2 receptor in human B lymphocytes. Eur J Immunol.15(4), 341-4 (1985). Aye M. T., Dunne J.V. Opposing effects of 12-O-tetradecanoylphorboI 13-acetate on human myeloid and lymphoid cell proliferation. J Cell Physiol. 114(2), 209-14 (1983).

Bauer I., Al Sarraj J. et al. Interleukin-I beta and tetradecanoylphorbol acetate- induced biosynthesis of tumor necrosis factor alpha in human hepatoma cells involved the transcription factors ATF2 and c-Jun and stress-activated protein kinases. J Cell Biochem.100(1), 242-255 (Epub ahead of print), (2006).

Beaupre D M and urzrock R. RAS and leukemia: from basic mechanisms to gene- directed therapy. J Clin Oncol 1999; 17: 1071-9.

Bederson JB, Pins LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke. 1986; 17:472-476.

Berenblum I. A re-evaluation of the concept of co-carcinogenesis. Prog. Lxp. Tumor Res.11, 21-30 (1969).

Bezard et al., Effects of different schedules of MPTP administration on dopaminergic neurodegeneration in mice. Exp Neurol.148: 288-92, 1997.

Boutwell R. . Biochemical mechanism of tumor promotion, in mechanisms of tumor promotion and co-carcinogenesis. Eds. Slaga. T.J., Sivak. A.J. and Boutwell, R.K. Raven, New York.49-58 (1978).

Boutwell R.K. The function and mechanism of promoters of carcinogensis. CRC Crit. Rev. Toxicol 2, 419-443 (1974).

Brooks A. I. et al. Paraquat elicited neurobehavioral syndrome caused by

dopaminergic neuron loss Brain Res.823. 1-10, 1999.

Brose N, Rosenmund C. Move over protein kinase C, you ^' ve got company:

alternative effectors of diacylglycerol and phorbol esters. JCell Sci; 115:4399- 11 (2002).

Cancer Chemother Pharmacol. Jun;57(6):789-95 (2006). Cheson B D, Cassileth P A, Head D R, Schiffer C A, Bennett J M, Bloomfield C D, Brunning R, Gale R P, Grever M R, Keating M J, and et al. Report of the National Cancer Institute-sponsored workshop on definitions of diagnosis and response in acute myeloid leukemia. J Clin Oncol 1990; 8: 813-9.

Cui X X, Chang R L, Zheng X, Woodward D, Strair R, and Conney A H. A sensitive bioassay for measuring blood levels of 12-0-tetradecanoylphorbol-13-acetate (TPA) in patients: preliminary pharmacokinetic studies. Oncol Res 2002; 13: 169-74.

Deegan M. J„ Maeda k. Differentiation of chronic lymphocytic leukemia cells after in vitro treatment w ith Epstein-Barr vims or phorbol ester. Immunologic and morphologic studies. Am J Hermatol.17(4), 335-47 (1984).

Falcioni F., Rautmann A. et al. Influence of TPA (12-0-tetradodecanoyl-phorbol-13- acetate) on human B lymphocte function. Clin Exp Immunol.62(3), 163-2 (1985). Farrell B, Godwin J, Richards S, Warlow C, et al. (1991). "The United Kingdom transient ischaemic attack (UK-TIA) aspirin trial: final results." J Neurol Neurosurg Psychiatry 54 (12): 1044-1054.

Forbes I. J., Zalewski P. D., Letarte M. Human B-lymphocyte maturation sequence revealed by TPA-induced differentiation of leukaemi cells. Immunobiology 163(1), 1- 6 (1982).

Friedemann & Gerhardt, Neurobiol Aging, Regional effects of aging on dopaminergic function in the Fischer-344 rat. 13: 325-32, 1992.

Fujisawa K., Nasj K. et al. Production of interleukin (I L)-6 and 1L-8 by a chorio- carcinama cell line, BeWo. Placenta 21(4), 354-60 (2000).

Gunjan Goel, Harinder P. S. Makkar. George Francis, and Klaus Becker. Phorbol

Esters: Structure, Biological Activity, and Toxicity in Animals. International Journal of Toxicology, 26:279-288, 2007.Gogusev J., Barbey S., Nezelof C. Regulation of

TNF-alpha and IL.-l gene expression during TPA-induced differentiation of

"Malignant histiocyosis" DEL cell line t(5:6) (q35:P21). Anticancer Res.16(1), 455-

60 (1996).

Granger CV, Devis LS, Peters MC, Sherwood CC, Barrett JE. Stroke rehabilitation: analysis of repeated Baithel Index measures. Arch Phys Med Rehabil. 1979:60:14-17. Hamburger, A. W., and Salmon, S. E. Primary bioassay of human tumor stem cells. Science (Wash. DC), 797: 461-463,1977., Han Z T, Zhu X X, Yang R Y, Sun J Z, Tian G F, Liu X J, Cao G S, Newmark H L, Conney A H, and Chang R L. Effect of intravenous infusions of 12-0- tetradecanoylphorbol-13-acetate (TPA) in patients with myelocytic leukemia:

preliminary studies on therapeutic efficacy and toxicity. Proc Natl Acad Sci U S A 1998; 95: 5357-61.

Han Z. T., Tong Y. K., He L. M., Zhang Y., Sun J. Z., Wang T. Y., Zhang H., Cui Y. L., Newmark H. L., Conney A. H., Chang R. L. 12-0-Tetradecanoyl-phorbol-13- acetate (TPA) -induced increase in depressed white blood cell counts in patients treated with cytotoxic cancer chemotherapeutic drugs. Proc. Natl. Acad. Sci.95, 5363-5365 (1998).

Han Z.T., Zhu X. X., Yang R. Y., Sun J. Z., Tian G. F., Liu X. J., Cao G. S., NewMark H. L., Conney A. H., and Chang R. L. Effect of intravenous infusion of 12· O-tetradecanoyl-phorbol-13-acetate (TPA) in patients with myelocytic leukemia: preliminary studies on therapeutic efficacy and toxicity. Pro. Natl. Acad. Sci.95, 5357-5361 (1998).

Harada S. el al: Tumor Promoter, TPA, Enhances Replication of HTLV-II1/LAV. Virology 154, 249-258 (1986).

Hecker E. In handbuch der allgemeinen patholgie, ed. Grundniann, E. (Springer- Verlag, Berlin-Heideiberg. Vol. IV 16, 651-676 (1975).

Hecker E. Structure -activity relationships in deterpene esters irritant and co- carcinogenic to mouse skin, in mechanisms of tumor promotion and co- carcinogenesis. Eds. Slaga. T. J.. Sevak, A. j. and Boutwell, R.K. Raven, New York. 11-49 (1978).

Hofmann J. The potential for isoenzyme-selective modulation of protein kinase C. FASEB J.11, 649-669 (1997).

Huberman E., Callaham M. F. Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents. Proc. Natl. Acad. Sci.76, 1293-1297 (1979).

Hunter T. Signaling 2000 and beyond. Cell 100.113-117 (2000).

Jordan C T. Unique molecular and cellular features of acute myelogenous leukemia stem cells. Leukemia 2002: 16: 559-62.

Kassel O, Sancono A, Kratzschmar J. reft B. Stassen M. and Cato A. C.

Glucocorticoids inhibit MAP kinase via increased expression and decreased degradation of M P-1. Embo J 2001; 20: 7108-16. Kazanietz M.G. Eyes Wide Shut: protein kinase C isoenzymes are not the only receptors for the phorbol ester tumor promoters. Mol. Carcinog.28, 5-12 (2000). Keoffler H. P., E!ar-Eli M., Territo M. C. Phorbol ester effect on differentiation of human myeloid leukemia cells lines blocked at different stages of maturation. Cancer Res.41,919-926(1981).

Kim S C, Hahn J S. Min Y H, Yoo N C, Ko Y W, and Lee W J. Constitutive activation of extracellular signal-regulated kinase in human acute leukemias:

combined role of activation of MEK, hyperexpression of extracellular signal- regulated kinase, and downregulation of a phosphatase, PAC1. Blood 1999; 93: 3893- 9.

Kiyoi H, Naoe T, Nakano Y, Yokota S, inami S, Miya aki S, Asou N, Kuriyama K, Jinnai I, Shimazaki C, Akiyama H, Saito K, Oh H, Motoji T, Omoto E, Saito H, Ohno R, and Ueda R. Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia. Blood 1999; 93: 3074-80.

Kobayashi M., Okada N. et al. Intracellular interleukin-1 alpha production in human gingival fibroblasts is differentially regulated by various cytokines. J Dent Res.78(4), 840-9 (1999).

Koefflcr H. P. Phorbol dicster-induced macrophge differentiation of leukemic blasts from patients with human myelogenous leukemia. J. Clin. Invest.66, 1101-1108 (1980).

Kudo ., Aoyama A., Ichimori S. and Fukunaga N. An animal model of cerebral infarction: homologous blood clot emboli in rats. Stroke 13: 505-508 (1982)

Lebien T. W., Bollum F. J. et al. Phorbol ester-induced differentiation of a non-T. non-B leudemic cell line: model for human lymphoid progenitor cell development. J Immunol.128(3), 1316-20 (1982).

McCornack A. L. et al. Environmental risk factors and Parkinson's disease: selective degeneration of nigral dopaminergic neurons caused by the herbicide paraquat.

Neurobiol Dis. 10, 119-127, 2002.

Meinhardt G., Roi:h J., Hass R. Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis. Cell Death Differ.7, 795-803 (2000).

Milella M, Kornblau S M, Estrov Z, Carter B Z, Lapillonne H, Harris D, Konopleva M, Zhao S, Estey E, and Andreeff M. Therapeutic targeting of the EK/ ^' MAPK signal transduction module in acute myeloid leukemia. J Clin Invest 2001; 108: 851-9. 01

Mochty-Rosen D., Kauvar L. M. Modulating protein kinase C signal transduction. Adv. Pharmacol.44, 91-145 (1998).

Morgan M A, Dolp O, and Reuter C W. Cell-cycle-dependent activation of mitogen- activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and induction of growth inhibition and apoptosis by inhibitors of RAS signaling. Blood 2001; 97: 1823-34.

Nagasawa ., Chechgik B. E. et al. Modulation of human T-cell differentiation markers by 12-0-tetradecanoyIphorbal-13-acetate. Thymus.3(4-5), 307-18, (1981).

Nakao Y., Matsuda S. et al. Paradoxical anti-leukemic effects of plant-derived tumor promoters on a human thymic lymphoblast cell line. Int J Cancer 30(6), 687-95 (1982).

Nakao Y., Matsuda S. et al. Phorbol ester-induced differentiation of human T- lymphoblastic cell line HPB-ALL. Cancer Res.42(9), 33843-50 (1982).

Newton A.C. Protein kinase C: structure, function and regulation. J. Biol. Chem.270, 28495-28499 (1995).

Niederman T. M. J., Ratner L. et al. Human Immunodeficiency Virus Type I Nef Protein Inhibits NF- B Induction in Human T Cells. J. Virology 66 (10), 6313-6219 (1992).

Norwell P., Shan key T. V. et al. Proliferation, differentiation and cytogenetics of chronic leukemic B lymphocytes cultured with mitomycin-treated normal cells. Blood 57(3), 444-51 (1981).

O'Banion M. K., Miller J. C. el al. Interleukin-1 beta induces prostaglandin G/H synthase-2 (cyclooxygenase-2) in primary murine astrocyte cultures. J Neurochem

66(6), 2532-40 (1996).

Okamura J., Geffand E. W., Letarte M. Heterogenneity of the response of chronic lymphocytic leukemia cells to phorbol ester. Blood 60(5).1082-8 (1982).

Pa!ombella V J, Rando O J. Goldberg A L, and Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-kappa B I precursor protein and the activation of NF-kappa B. Cell 1994; 78: 773-85.

Platanias L C. Map kinase signaling pathways and hematologic malignancies, Blood 2003; 101 : 4667-79.

Polliack A., Leizerowitz R., Korkesh A.. Gurfel D., Gamliel H., Galili L . Exposure to TPA in vitro as an aid in the classification of blasts in human myelogenous and lymphoid leukemias. Am. J. Hematol.13, 199-211 (1982). Redondo P., Garci-Foncillas J. et al. Differential modulation of IL-8 and TNF-alpha expression in human keratinocytes by buffomedil chlorhydrate and pentoxifylline. Exp. Dermatol. 6(4), 1 86-94 ( 1997).

Rovera G., Santoli D., Damsky C. Human promyelocytic cells in culture differentiate into macrophage-like cells treated with a phorbol diester. Pro. Natl. Acad. Sci. 7, 2779-2783 ( 1 979).

Rul las J., Alcami J. et .al. Receptors in peripheral blood lymphocytes. Antivir. Ther. 9 (4). 545-554 (2004).

Rwigema JC, Beck B, Wang W, Doemling A, Epperly MW, Shields D, Goff

JP, Franicola D, Dixon T, Frantz MC, Wipf P, Tyurina Y, Kagan VE,

Schaar D, GoodcII L, Aisner J, Cui XX, Han ZT, Chang R, Martin J, Grospe S, Dudek

L, Riley J, Manago J, Lin Y, Rubin EH, Conney A, Strair R . A phase I clinical trial of 12- O-tetradecanoylphorbol- 13-acetate for patients with relapsed/refractory malignancies.

Scheinman R 1, Cogswel l P C. Lofquist A , and Baldwin A S, Jr. Role of

Transcriptional Activation of lkappaBalpha in Mediation of I mm unosuppression by Glucocorticoi ds. Science 1995 ; 270: 283-286.

Shkolnick T., Schlossman S. F., Griffin J. D. Acute undifferentiated leukem ia:

induction of partial differentiation by phorbol ester. Leuk. Res. 9, 1 1 - 17 ( 1985). Shwarz M. et .al. H igh-level l L- 1 0 production by monoclonal antibody-stimulated human T cel ls. Immunology 86, 364-371 ( 1 995 ).

Staber P B, Linkesch W, Zauner D, Beham-Schmid C, Guelly C, Schauer S, Si ll H. and Hoefler G. Common alterations in gene expression and increased prol iferation in recurrent acute myeloid leukemia. Oncogene 2004; 23 : 894-904.

Steube . G., Meyer C. Drexler Fl. G. Constitutive excretion of hematopoiet ic cytokines by human carcinoma cel l lines and its up-regulation by interleukin- 1 and phorbol ester. Oncol. Rep. 6(20), 427-32 ( 1 999).

Strair R K, Schaa - D, Goodel l L, Aisner J, Chin V. E id J. Senzon R. Cui X X, Han Z T, Knox B. Rabson A B, Chang R, and Conney A. Adm i nistration of a phorbol ester to patients w ith hematological mal ignancies: prelim inary results from a phase I clinical trial of 12-0-tetradecanoylphorbol- 1 3-acetate. Clin Cancer Res 2002; 8: 25 12-8

Tanner, C. M ., Kamel, F., Ross, G. W. Hoppin, J. A., Goldman, S. M ., Korell, M ., Marras, C, Bhudhikanok, G. S., Kasten, M., Chade, A. R. Comyns, K., Richards. M. W

B., Meng, C, Priestley, B., Fernandez, H. H., Cambi, F., Umbach, D. M, Blair, A., Sandier, D. P., and Langston, J. W., Rotenone, Paraquat and Parkinson's Disease, 201 1, Environmental Health Perspectives, 1 19: 866-872.

Thiruchelvam M. et al. Developmental exposure to the pesticides paraquat and maneb and the Parkinson's disease phenotype. Neurotoxicology 23, 621 -633, 2002.

Totterman T. H.. Nilsson K., Sundstrom C. Phorbol ester-induced differentiation of chronic lymphoctic leukaemia cells. Nature 288(5787), 176-8 ( 1980)

Towatari M, lid i H, Tanimoto M, Iwata H, Hamaguchi M, and Saito H. Constitutive activation of mit3gen-activated protein kinase pathway in acute leukem ia cells.

Leukemia 1997; 1 1 : 479-84.

Ungerstedt, U. and G.W. Arbuthnott, Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res, 1 70. 24(3): p. 485-93.

Van Duuren, B.L. Tumor-promoting agents in two-stage carcinogenesis. Prog. Exp. Tumor Res. 1 1 , 1 -68 ( 1969).

Varastet et al., Chronic MPTP treatment reproduces in baboons the differential vulnerability of mesencephalic dopaminergic neurons observed in Parki nson's disease. Neuroscience, 63 : 47-56. 1 994.

Yamamoto Y, iyoi H, Nakano Y, Suzuki R, odera Y, Miyawaki S, Asou N, Kuriyama K, Yagasaki F, Shimazaki C, Akiyama H, Saito , Nishimura M, Motoji T, Shinagawa , Takeshita A, Saito H, Ueda R, Ohno R, and Naoe T. Activating mutation of D835 w ithin the activation loop of FLT3 in human hematologic malignancies. Blood 2001 : 97: 2434-9.

YIP, Y.K.. et al. Stimulation of human gamma interferon production by diterpene esters. Infection and Immunity 34( 1 ) 1 3 1 - 139 ( 1 981 ).

Zhao J., Sharma Y., Agarwal R. Significant inhibition by the flavonoid antioxidant silymarin against 12-O-tetradecanoylphorbol 1 3-acetate-caused modulation of antioxidant and inflammatory enzymes and cyclooxygenase2 and interlukin-1 alpha expression in SENCAR mouse epidermis: implications in the prevention of stage 1 tumor promotion. Mol Carcinog. 26(4), 321 -33 ( 1999).