RU2775972C2 - Compositions and methods for using phorbol ethers for treatment of stroke - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: group of inventions relates to the pharmaceutical industry, namely to a composition for the prevention or treatment of one or more consequences of a stroke in a mammal and a method for the prevention or treatment of one or more consequences of a stroke in a mammal. The composition for the prevention or treatment of one or more consequences of a stroke in a mammal includes the effective amount of phorbol ester of the formula I or its pharmaceutically acceptable salt, isomer or enantiomer,

where R₁ and R₂ are selected from a group consisting of hydrogen, hydroxyl,

, where an alkyl group contains from 1 to 15 of carbon atoms;

, where a lower alkenyl group contains from 1 to 7 carbon atoms;

and

; and R₃ is hydrogen and

, where the specified phorbol ether of the formula I excludes 12-O-tetradecanoyl-phorbol-13-acetate (TPA), where one or more of side effects of a stroke is paralysis, spatial perception disorder, blurred consciousness, left ignoring syndrome, aphasia, problems with coordination and balancing, nausea, vomiting, cognitive disorder, perception disorder, orientation disorder, homonymous hemianopia, impaired mobility or impulsivity. The method for the prevention or treatment of one or more consequences of a stroke in a mammal includes the administration of the composition containing the effective amount of phorbol ether of the formula I or its pharmaceutically acceptable salt, isomer or enantiomer to the specified mammal.

EFFECT: above-described composition is effective for the prevention or treatment of one or more consequences of a stroke in a mammal, where one or more of side effects of a stroke is paralysis, spatial perception disorder, blurred consciousness, left ignoring syndrome, aphasia, problems with coordination and balancing, nausea, vomiting, cognitive disorder, perception disorder, orientation disorder, homonymous hemianopia, impaired mobility or impulsivity.

13 cl, 1 tbl, 10 ex

Description

Related Applications

This application is a US patent application claimed priority under US Provisional Patent Application No. 61/588167, filed January 18, 2012, which is incorporated herein by reference in its entirety.

Additional description

Additional disclosures relevant to this application can be found in "Compositions and Methods of Use of Phorbol Esters" US Patent Application No. 12/023753, filed January 31, 2008, by Richard L. Chang et al., the priority of which is claimed in the US provisional patent application No. 60/898810, filed January 31, 2007, each of which is incorporated herein by reference in its entirety.

Technical field

The present invention relates generally to the medical use of phorbol esters in the treatment or prevention of stroke and the consequences of stroke.

State of the art

Plants have historically been used for many medicinal purposes. The World Health Organization (WHO) has estimated that 4 billion people, 80% of the world's population, currently use herbal medicine for some aspect of first aid. (WHO Fact sheet Fact sheet #134, December 2008) However, it can be difficult to isolate a particular compound from a plant that has the desired medicinal effect and replicate it commercially. In addition, while the active compound may be isolated from the plant, other constituents of the plant, such as minerals, vitamins, essential oils, glycosides, alkaloids, bioflavonoids, and other substances, may also be involved in the functioning of the active compound or the medicinal effect that known to the plant, which makes the use, purification and commercialization of herbal pharmaceutical agents problematic.

Phorbol is a natural, organic plant-derived compound from the tigliane family of diterpenes. It was first isolated in 1934 as a hydrolysis product of croton oil obtained from the seeds of Croton tiglium, a green shrub of the Euphorbiaceae family that grows in Southeast Asia. Various phorbol esters have important biological properties, including the known ability to mimic diacylglycerols and activate protein kinase C (PKC); and downstream modulation of cell signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway. Phorbol esters are thought to additionally bind chymaerins, the Ras activator RasGRP, and the vesicle excitatory protein Munc-13 (Brose N, Rosenmund C., JCell Sci;115:4399–411 (2002)). Some phorbol esters also induce nuclear factor-kappa B (NF-κB). The most notable physiological property of phorbol esters is their known ability to act as a tumor growth promoter. (Blumberg, 1988; Goel, G et al., Int, Journal of Toxicology 26, 279-288 (2007)).

12-O-tetradecanoylphorbol-13-acetate (TPA), also called phorbol-12-myristate-13-acetate (PMA), is a phorbol ester used in models of carcinogenesis as an inducer of differentiation and/or apoptosis in several cell lines and primary cells. TPA has also been reported to cause an increase in circulating leukocytes and neutrophils in patients in whom bone marrow function has been suppressed by chemotherapy (Han Z. T. et al. Proc. Natl. Acad. Sci. 95, 5363-5365 (1998)), and inhibits HIV -induced cytopathic effect on MT-4 cells. (Mekkawy S. et al., Phytochemistry 53, 47-464 (2000)). However, due to a variety of factors, including caustic reactions upon skin contact and concerns about its potential toxicity, TPA has not been shown to be an effective treatment, management, or prevention of disease. Indeed, although phorbol esters play a key role in the activation of protein kinase C, which induces various cellular responses leading to inflammatory responses and tumor development (Goel et al., Int, Journal of Toxicology 26, 279-288 (2007)) , phorbol esters are generally excluded as possible candidates for the treatment of conditions that involve inflammatory responses such as stroke.

Heart disease and stroke kill approximately 17 million people a year, accounting for almost a third of all deaths worldwide. They are projected to become the leading cause of death and disability worldwide, with the death toll projected to increase to more than 20 million per year by 2020 and to 24 million per year by 2030. (Atlas of Heart Disease and Stroke, World Health Organization 2004).

While there are more than 300 risk factors associated with coronary heart disease and stroke (Atlas of Heart Disease and Stroke, World Health Organization 2004), in developed countries, at least 1/3 of all cardiovascular diseases are associated with the use of tobacco, alcohol use, high blood pressure, high cholesterol and obesity.

Current therapies for the treatment and prevention of stroke typically combine drugs such as ACE inhibitors, aspirin, beta-blockers, and lipid-lowering drugs; devices such as pacemakers, implantable coronary stent defibrillators, artificial valves, and artificial hearts; and surgeries such as coronary artery bypass grafting, balloon angioplasty, valve repair and replacement, heart transplants, and artificial heart surgeries. “Increasingly, high-tech procedures are being chosen over less expensive but nonetheless effective strategies” (Atlas of Heart Disease and Stroke, World Health Organization 2004), leading to higher healthcare costs and resulting in reported inconsistencies in quality of care between different groups of people.

However, even where advanced technologies and services are available, 60% of stroke sufferers die or become dependent, and each stroke significantly increases the risk of further strokes. "Worldwide, treatment for cardiovascular disease and its risk factors remains inadequate for most patients." (Atlas of Heart Disease and Stroke, World Health Organization 2004). Therefore, there is a clear need for new and more effective interventions for the treatment and prevention of stroke and for the treatment or prevention of long-term effects caused by stroke.

Brief description of the invention

The present invention relates to compositions and methods of using phorbol esters. The compositions and methods described herein are effective in the prevention and treatment of stroke and in the management of stroke complications, including acute ischemic events.

Sequelae of stroke that can be prevented or treated with phorbol esters and phorbol ester derivatives as described herein include paralysis, spatial disturbance, clouding of consciousness, left neglect syndrome, memory loss, aphasia, coordination and balance problems, nausea , vomiting, cognitive impairment, perceptual impairment, disorientation, homonymous hemianopia, and impulsivity, but are not limited to. In some embodiments of the invention, the use of phorbol esters and phorbol ester derivatives can prevent the occurrence and subsequent strokes.

Successful treatment will be determined according to traditional methods, such as reducing the severity or consequences of a stroke, reducing or eliminating the consequences of a stroke, reducing risk factors that predispose an individual to stroke, and/or reducing the number and severity of strokes, including subsequent strokes. .

In another embodiment, phorbol esters and phorbol ester derivatives as described herein can be used to modulate cellular signaling pathways. Such modulation can have various results, for example, in some embodiments of the invention, the use of compositions containing phorbol esters and phorbol ester derivatives can alter the release of Th1 cytokines in mammals. In an additional embodiment, compositions containing phorbol esters and/or phorbol ester derivatives can alter the release of interleukin 2 (IL-2) in mammals. In a further embodiment of the invention, compositions containing phorbol esters and/or phorbol ester derivatives can alter the release of interferon in mammals. In yet another embodiment of the invention, compositions containing phorbol esters and/or phorbol ester derivatives can alter the rate of ERK phosphorylation.

The invention achieves the foregoing and additional objects and benefits by providing a novel and surprisingly effective method and composition for the treatment and prevention of stroke, modulation of cell signaling and/or control pathways, treatment and prevention of complications of stroke, using a formula I phorbol ester containing composition as shown below. :

Formula I

, где алкильная группа содержит от 1 до 15 атомов углерода;

, где низшая алкенильная группа содержит от 1 до 7 атомов углерода;

;

; и их замещенными производными. R₃ может независимо быть водородом или

и их замещенные производные. Способы и композиции настоящего изобретения дополнительно включают любые фармацевтические соли, энантиомеры, изомеры, полиморфы, пролекарства, гидраты и сольваты соединений формулы I.where R ₁ and R ₂ may independently be hydrogen; hydroxyl;

, where the alkyl group contains from 1 to 15 carbon atoms;

, where the lower alkenyl group contains from 1 to 7 carbon atoms;

;

; and their substituted derivatives. R ₃ may independently be hydrogen or

and their substituted derivatives. The methods and compositions of the present invention further include any pharmaceutical salts, enantiomers, isomers, polymorphs, prodrugs, hydrates and solvates of the compounds of formula I.

и их замещенными производными. В другом варианте реализации изобретения или R₁, или R₂ является

алкилом и оставшийся R₁ или R₂ является

алкилом, где низший алкил содержит от 1 до 7 углеродов, а R₃ является водородом.In some embodiments of the invention, at least one of R ₁ and R ₂ are other than hydrogen and R ₃ is hydrogen or

and their substituted derivatives. In another embodiment, either R ₁ or R ₂ is

alkyl and the remaining R ₁ or R ₂ is

alkyl, where the lower alkyl contains from 1 to 7 carbons, and R ₃ is hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups in the formulas of formula I herein may be unsubstituted or substituted with halogens, preferably chlorine, fluorine or bromine; nitro; amino and/or similar types of radicals.

An example of a phorbol ester that is used in the methods and compositions described herein is 12-O-tetradecanoylphorbol-13-acetate (TPA) of formula II below:

Formula II

The formula I phorbol esters and related compounds and derivatives used in the compositions and methods of the present invention include other pharmaceutically acceptable active salts of said compounds, such as active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and/or prodrugs of said compounds, but not limited to them. Exemplary forms of phorbol esters for use in the compositions and methods of the present invention include 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-myristate, 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, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12- but not limited to deoxyphorbol 13-tetradecanoate, phorbol 12-tigliate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate, or phorbol 13-acetate.

Mammalian subjects amenable to treatment with formula I phorbol esters or formula I phorbol ester derivatives, in particular TPA, in accordance with the methods of the present invention include, but are not limited to, individuals who have suffered or are at risk of stroke. Subjects amenable to formula I phorbol ester treatment, in particular TPA, additionally include those suffering from the consequences of a stroke, including paralysis, spatial disturbance, clouding of consciousness, left neglect syndrome, memory loss, aphasia, impaired coordination and balance, nausea, vomiting, cognitive disorders, perceptual disorders, disorientation, homonymous hemianopia and impulsivity, but not limited to.

These and other subjects are effectively treated prophylactically and/or therapeutically by administering to the subject an effective amount of a Formula I phorbol ester or Formula I phorbol ester derivatives in sufficient amounts to modulate NF-κB activity, increase Th1 cytokine activity, prevent or treat paralysis, improve orientation in space, reduce memory loss, reduce aphasia, improve coordination and balance, prevent or reduce the frequency and severity of stroke, and improve cognitive function.

Therapeutically useful methods and compositions of the present invention will effectively use a phorbol ester of formula I or a phorbol ester derivative of formula I in various forms, as noted above, including any active, pharmaceutically acceptable salts of these compounds, as well as active isomers, enantiomers, polymorphs, solvates, hydrates , prodrugs, and/or combinations thereof. TPA of formula II is used as an illustrative embodiment of the invention in the examples below.

In further aspects of the invention, combination compositions and methods that use an effective amount of a Formula I phorbol ester or a Formula I phorbol ester derivative in combination with one or more secondary or co-active agent(s) that are co-formulated or co-administered with a Formula I phorbol ester , to obtain an effective response from the subject.

Combination compositions and coordinated treatments in the treatment of stroke utilize a Formula I phorbol ester or Formula I phorbol ester derivative in combination with one or more additional secondary or conjugate therapeutic agents for the prevention, treatment of stroke, or other indication. Secondary or conjugate therapeutic agents used in combination with a phorbol ester, eg TPA, in these embodiments may have a direct or indirect effect on stroke prevention or recovery, alone or in combination with a phorbol ester, eg TPA; may exhibit other beneficial additional therapeutic activities when combined with, for example, TPA (eg, anticoagulant, anticholesterolemic, vasodilatory, antihypertensive, lowering arteriolar resistance, increasing venous capacity, reducing cardiac oxygen demand, decreasing heart rate, stabilizing heart rate, or neuroprotection ); or may exhibit additional therapeutic activity useful in the treatment or prevention of stroke, either alone or in combination with associated symptoms, such as TPA. Such secondary or conjugate therapeutic agents may be administered before, simultaneously with, or after administration of the Formula I phorbol ester or Formula I phorbol ester derivative.

Useful additional or secondary therapeutic agents in these combination compositions and coordinated treatments for the prevention or treatment of stroke in a mammalian subject include tissue plasminogen activators, anticoagulants, statins, angiotensin II receptor blockers, angiotensin converting enzyme, antiplatelet agents, beta- blockers, aspirin, fibrates, calcium channel blockers or diuretics, but are not limited to. In addition, ancillary or secondary therapies may be used, such as surgery, including carotid endarterectomy, angioplasty, balloon angioplasty, valve repair and replacement, coronary artery bypass grafting, stent placement, craniotomy, endovascular coil embolization, or closure of the foramen ovale, but not limited to this.

The foregoing and additional objects, features, aspects and advantages of the present invention will become apparent from the following detailed description.

Detailed description of the invention

New methods and compositions have been identified for use in the prevention and/or treatment of stroke and stroke complications in mammals, including humans.

In various embodiments, the compositions and methods described herein can increase Th1 cytokine release, increase ERK phosphorylation, modulate NF-κB activity, prevent or treat paralysis, improve spatial orientation, reduce memory loss, reduce aphasia, improve coordination and balance. , improve cognitive function, improve orientation, reduce the incidence of subsequent strokes, and reduce impulsivity.

The compositions and methods provided herein use a formula I phorbol ester or formula I phorbol ester derivatives as described in more detail in US Patent Application No. 12/023753, filed Jan. 60/898810, filed January 31, 2007, which is incorporated herein in its entirety by reference,

Formula I

, где алкильная группа содержит от 1 до 15 атомов углерода;

, где низшая алкенильная группа содержит от 1 до 7 атомов углерода;

;

; и их замещенными производными. R₃ независимо может быть водородом или

.where R ₁ and R ₂ may be hydrogen; hydroxyl;

, where the alkyl group contains from 1 to 15 carbon atoms;

, where the lower alkenyl group contains from 1 to 7 carbon atoms;

;

; and their substituted derivatives. R ₃ independently can be hydrogen or

.

и их замещенные производные, как новые соединения для применения в лечении хронических или рецидивирующих состояний. В другом варианте реализации изобретения, или R₁, или R₂ представляет собой

алкил, а оставшийся R₁ или R₂ представляет собой

, где низший алкил содержит от 1 до 7 углеродов, и R₃ представляет собой водород.In some embodiments, at least one of R ₁ and R ₂ is other than hydrogen and R ₃ is hydrogen or

and their substituted derivatives as new compounds for use in the treatment of chronic or relapsing conditions. In another embodiment of the invention, either R ₁ or R ₂ is

alkyl, and the remaining R ₁ or R ₂ is

, where the lower alkyl contains from 1 to 7 carbons, and R ₃ represents hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups in the formulas herein may be unsubstituted or substituted with halogens, preferably chlorine, fluorine or bromine; nitro; amino and/or similar types of radicals.

Compositions and methods for the treatment of stroke provided herein use a phorbol ester of formula I or a phorbol ester derivative of formula I as set forth above, including all active pharmaceutically acceptable compounds of this disclosure, as well as various contemplated and contemplated complexes, salts, solvates, isomers , enantiomers, polymorphs and prodrugs of said compounds and combinations thereof as anti-stroke agents.

Compositions and methods for elevating Th1 cytokines provided herein use a phorbol ester or formula I derivative as defined above, including all active pharmaceutically acceptable compounds as described herein, as well as various complexes, salts, solvates, isomers, enantiomers provided and readily provided. , polymorphs and prodrugs of these compounds and their combinations as new agents for increasing the level of Th1 cytokines. A wide range of mammals, including humans, as a subject amenable to treatment with the compositions and methods of the invention. These subjects include, but are not limited to individuals who have suffered or are at risk of stroke.

Compositions and methods for increasing ERK phosphorylation provided herein using a phorbol ester or a formula I derivative as defined above include all active pharmaceutically acceptable compounds as described herein, as well as various contemplated and contemplated complexes, salts, solvates, isomers, enantiomers , polymorphs and prodrugs of these compounds and combinations thereof, as new agents for increasing ERK phosphorylation. A wide range of mammals, including a human subject, are amenable to treatment with the compositions and methods of the invention. These subjects include, but are not limited to individuals who have suffered or are at risk of stroke.

In the methods and compositions of the invention, one or more Formula I phorbol esters or Formula I phorbol ester derivatives as described herein are effectively formulated or administered as an agent effective in the treatment and prevention of stroke or stroke complications. In exemplary embodiments of the invention, TPA has demonstrated, for illustrative purposes, effectiveness as an agent in pharmaceutical compositions and therapeutic methods, alone or in combination with one or more auxiliary therapeutic agents. The present invention further provides additional pharmaceutically acceptable phorbol esters in the form of a natural or synthetic compound, including the complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs disclosed herein, and combinations thereof, which are effective as therapeutic agents in the methods and compositions of the invention in the treatment and prevention of stroke and stroke complications.

A stroke is caused by a disruption in the blood supply to the brain. This can be the result of either blockage (ischemic stroke) or rupture of a blood vessel (hemorrhagic stroke). Stroke symptoms include sudden numbness or weakness, especially on one side of the body; sudden confusion or problems speaking or understanding speech; sudden loss of vision in one or both eyes; sudden trouble walking, dizziness, or loss of balance or coordination; or a sudden severe headache with no known cause. Risk factors for stroke include high blood pressure, abnormal blood lipid levels, tobacco use, physical inactivity, obesity, stress, diabetes, alcohol use, excess blood homocysteine, inflammation, and abnormal coagulation. There are also non-modifiable risk factors such as age, genetics, gender, and ethnicity.

Stroke treatment has three distinct phases: prevention, immediate post-stroke treatment, and post-stroke rehabilitation. The compositions and methods described herein may be used at any stage in the treatment of stroke, alone or in combination with one or more adjunctive therapies, including other pharmaceutical agents, devices, or surgical interventions.

Phorbol is a natural plant-derived polycyclic alcohol of the tigliane family of diterpenes. It was first isolated in 1934 as a hydrolysis product of croton oil obtained from the seeds of Croton tiglium. It is highly soluble in most polar organic solvents and in water. Phorbol esters have the general structure of Formula I as shown below:

Formula I

, где алкильная группа содержит от 1 до 15 атомов углерода;

, где низшая алкенильная группа содержит от 1 до 7 атомов углерода;

;

; и их замещенные производные. R₃ может быть водородом или

и их замещенными производными, так же как фармацевтически приемлемые соли, энантиомеры, полиморфы, пролекарства, сольваты и гидраты соединений формулы I.where R ₁ and R ₂ are selected from the group consisting of hydrogen; hydroxyl;

, where the alkyl group contains from 1 to 15 carbon atoms;

, where the lower alkenyl group contains from 1 to 7 carbon atoms;

;

; and their substituted derivatives. R ₃ may be hydrogen or

and their substituted derivatives, as well as pharmaceutically acceptable salts, enantiomers, polymorphs, prodrugs, solvates and hydrates of the compounds of formula I.

The terms "lower alkyl" or "lower alkenyl", as used here, means substituents containing from 1 to 7 carbon atoms. In compounds of formula I, the alkyl or alkenyl group may be straight or branched. In some embodiments, either one or both of R ₁ or R ₂ is a long chain carbon substituent (ie Formula I is decanoate or myristate).

Alkyl, alkenyl, phenyl and benzyl groups in the formulas of this document may be unsubstituted or substituted with halogens, preferably chlorine, fluorine or bromine; nitro; amino and similar types of radicals.

Organic and synthetic forms of phorbol esters, including any preparations or extracts from plant sources such as Croton tiglium, are contemplated as useful compositions comprising phorbol esters (or phorbol ester analogues, related compounds and/or derivatives) for use in embodiments inventions in this document. Useful phorbol esters and/or related compounds for use in the embodiments of the invention herein generally have the structure as shown in formula I, although functionally equivalent analogs, complexes, conjugates and derivatives of these compounds will also be understood by those skilled in the art in within the scope of the present invention.

и их замещенных производных. В другом варианте реализации изобретения, или R₁, или R₂ представляет собой

алкил, а оставшийся R₁ или R₂ представляет собой

и R₃ является водородом.In more detailed embodiments, exemplary structural modifications according to Formula I above will be selected to provide useful candidate compounds for the treatment and/or prevention of strokes, stroke injuries, and/or management of the consequences or complications of strokes in mammals. , including a human, wherein at least one of R ₁ and R ₂ is other than hydrogen and R ₃ is selected from the group consisting of hydrogen, or

and their substituted derivatives. In another embodiment, either R ₁ or R ₂ is

alkyl, and the remaining R ₁ or R ₂ is

and R ₃ is hydrogen.

In an exemplary embodiment, a phorbol ester of Formula I useful in the treatment or prevention of strokes, stroke disorders, and/or management of the effects or consequences of strokes in mammals, including humans, is selected from phorbol 12-myristate-13-acetate (also known as PMA or 12-O-tetradecanoyl-phorbol-13-acetate (TPA)) shown in formula II below.

Formula II

Additional useful phorbol esters and related compounds and derivatives thereof within the compositions and methods of the invention include 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, but not limited to them. The phorbol ester derivatives of formula I may or may not be proper phorbol esters. Further examples of phorbol ester forms for use in the compositions and methods of the present invention include 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-myristate, 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, 12-deoxyphorbol 13-phenylacetate 20- acetate, 12-deoxyphorbol 13-tetradecanoate, but not limited to phorbol 12-tigliate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate, and phorbol 13-acetate; as shown in Table 1.

The compositions described herein comprise compositions for the treatment of stroke comprising an effective amount of a Formula I phorbol ester or a Formula I phorbol ester derivative that ameliorates or prevents post-stroke damage and that is effective in preventing and/or treating stroke or symptoms associated with stroke or stroke. complications in mammals. "Stroke treatment", "anticoagulant", "anticholosteromic", "vasodilating", "antihypertensive", "reducing arteriolar resistance", "increasing venous capacity", "reducing the heart's oxygen demand", "decreasing heart rate", "stabilizing heart rate" or "neuroprotective" effective amount of the active compound is therapeutically effective, in single or multiple unit dosage form, for a specified therapeutic intervention period, to markedly reduce one or more symptoms or complications of stroke in subjects. In exemplary embodiments, the compositions of the present invention are effective in methods of treating, preventing or alleviating the symptoms of stroke or complications of stroke in humans and other mammals susceptible to or who have suffered a stroke.

Phorbol ether compositions for treatment according to the invention generally contain an effective amount or unit dose of a phorbol ester of formula I or a phorbol ester derivative of formula I, which may be formulated with one or more pharmaceutically acceptable carriers, excipients, diluents, emulsifiers, stabilizers, preservatives, buffers and/or other additives that may increase stability, release, absorption, half-life, efficacy, pharmacokinetics and/or pharmacodynamics, reduce adverse side effects, or provide other benefits for pharmaceutical use. Effective amounts of the phorbol ester or the corresponding derivative or compound of Formula I (e.g., unit dose containing an effective concentration/amount of TPA or a selected pharmaceutically acceptable salt, isomer, enantiomer, solvate, polymorph, and/or prodrug of TPA) will be readily determined by one of ordinary skill in the art. , depending on clinical and patient-specific factors. Suitable effective unit dose amounts of active compounds for administration to mammals, including humans, may range from about 10 to about 1500 µg, about 20 to about 1000 µg, about 25 to about 750 µg, about 50 to about 500 µg, about 150 to about 500 µg, about 125 µg to about 500 µg, about 180 to about 500 µg, about 190 to about 500 µg, about 220 to about 500 µg, about 240 to about 500 µg, about 260 to about 500 micrograms; about 290 to about 500 micrograms. In some embodiments of the invention, effective doses of the phorbol ester or the corresponding derivative or compound of formula I for the treatment of a disease can be selected within narrow ranges, for example, from 10 to 25 μg, 30-50 μg, 75 μg to 100, from 100 to 300 mcg, or 150 to 500 mcg. These and other effective unit dosage amounts may be administered as a single dose or as multiple daily, weekly or monthly doses, for example, in a dosing regimen containing 1 to 5, or 2 to 3, administered doses per day, per week , or per month. In one embodiment, doses of 10 to 30 micrograms, 30 to 50 micrograms, 50 to 100 micrograms, 100 to 300 micrograms, or 300 to 500 micrograms are administered one, two, three, four or five times a day. In further embodiments of the invention, doses of 50-100 μg, 100-300 μg, 300-400 μg, or 400-600 μg are administered once or twice a day. In a further embodiment of the invention, doses of 50-100 μg, 100-300 μg, 300-400 μg, or 400-600 μg are administered every other day. In alternative embodiments, doses are based on body weight and may be administered, for example, in amounts from about 0.5 µg/m ² to about 300 µg/m ² per day, from about 1 µg/m ² to about 200 µg /m ² , from about 1 µg/m ² to about 187.5 µg/m ² per day, from about 1 µg/m ² per day to about 175 µg/m ² per day, from about 1 µg/m ² per day to about 157 µg/m ² per day, about 1 µg/m ² to about 125 µg/m ² per day, about 1 µg/m ² to about 75 µg/m ² per day, 1 µg/m ² to about 50/ µg/m ² per day, 2 µg/m ² to about 50 µg/m ² per 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 one embodiment of the invention, doses can be administered less frequently, for example, from 0.5 µg/m ² to about 300 µg/m ² every other day, from about 1 µg/m ² to about 200 µg/m ² , from about 1 µg /m ² to about 187.5 µg/m ² every other day, from about 1 µg/m ² to about 175 µg/m ² every other day, from about 1 µg/m ² per day to about 157 µg/m ² every other day , from about 1 µg/m ² to about 125 µg/m ² every other day, from about 1 µg/m ² to about 75 µg/m ² every other day, from 1 µg/m ² to about 50 µg/m ² every other day, 2 µg/m ² to about 50 µg/m ² 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 a further embodiment of the invention, doses may be administered 3 times/week, 4 times/week, 5 times/week, on weekdays only, only when interacting with other treatment regimens, on subsequent days, or at the appropriate dosing regimen depending on the clinical and patient-specific factors.

The number, timing and mode of release of compositions of the invention containing (alternatively "treating stroke", "anticoagulant", "anticholerosthemic", "vasodilating", "hypotensive", "reducing arteriolar resistance", "increasing venous capacity", "reducing the need of the heart in oxygen", "decreasing heart rate", "stabilizing heart rate", "causing ERK phosphorylation", "modulating IL-2" and/or "neuroprotective") an effective amount of a phorbol ester compound of formula I or a phorbol ester derivative of formula I, will generally be adjusted on an individual basis, depending on factors such as the weight, age, sex and condition of the individual, the severity of the disease and/or associated symptoms, whether the administration is prophylactic or therapeutic, and based on other factors known to effect drug release. means, absorption and pharmacokinetics, including half-life and effect activity.

An effective dose or multi-dose regimen for the treatment of the current disease (alternatively, "treatment of stroke", "anticoagulation", "anticholerosthemic action", "vasodilator", "antihypertensive action", "ERK phosphorylation", "reduction of arteriolar resistance", increase in venous capacity ", "reducing the oxygen demand of the heart", "reducing the heart rate", "stabilizing the heart rate", "reducing blood clotting", "neuroprotective action", "modulating IL-2" or "modulating NFκB") compositions of the invention will generally chosen to approximate the minimum dosage regimen that is necessary and sufficient to substantially prevent or alleviate the symptoms of stroke in a subject. Dosages and administration protocols often include repeated administration over a course of several days or even one or more weeks or years. An effective treatment regimen may also include a prophylactically administered dose per day or multiple doses per day, which generally lasts for days, weeks, months or even years.

The effectiveness of the compositions and methods of the invention for the treatment of stroke can be demonstrated using various model systems, including temporary occlusion of the middle cerebral artery, as shown in example 9, permanent occlusion of the middle cerebral artery, as shown in example 8, occlusion of the endovascular thread of the middle cerebral artery, embolism with blockage of the middle cerebral artery, as shown in example 7, endothelin-1-induced narrowing of arteries and veins or cerebrocortical photothrombosis. The use of phorbol esters in the compositions of the present invention reduces the symptoms or long-term effects shown by the model systems by 0%, 20%, 30%, 50% or more, up to 75-90%, 96% or more reduction in control animals.

The effectiveness of the compositions and methods of the invention in the treatment of stroke can be further demonstrated by the reduction in symptoms in individuals who have suffered a stroke. Such symptoms include, but are not limited to, paralysis, spatial disturbance, clouding of consciousness, left neglect syndrome, memory loss, aphasia, coordination and balance problems, nausea, vomiting, cognitive impairment, perceptual disturbances, disorientation, homonymous hemianopia, and impulsivity. The use of the phorbol ester compositions of the present invention will reduce symptoms in individuals by 0%, 20%, 30%, 50% or more, up to a 75-90%, 96% or more reduction compared to baseline conditions.

In additional aspects of the invention, a combined treatment of the disease is provided ("treatment of stroke", "anticoagulation", "anticholerostomy effect", "vasodilating effect", "antihypertensive effect", "ERK-inducing phosphorylation", "lowering arteriolar resistance", "increasing venous capacity ", "reduction of cardiac oxygen demand", "decrease in heart rate", "stabilization of heart rate", or "NFκB modulation"), compositions and methods of coordinated administration, which use an effective amount of a phorbol ester of formula I or a derivative of formula I and one or more secondary or additional agents that are combined into the composition or co-administered with the formula I phorbol ester to provide a combined, multi-active disease treatment composition or coordinated treatment modality.

Exemplary combinatorial compositions and coordinated treatments for the prevention or treatment of stroke use a Formula I phorbol ester or a Formula I phorbol ester derivative in combination with one or more additional, neuroprotective, or other indicated, secondary, or additional therapeutic agents that are useful in treating or preventing the target disease, condition and/or symptom(s). For most combination compositions and coordinated treatments of the invention, a Formula I phorbol ester or similar or derivative compound is formulated or co-administered in combination with one or more secondary or adjuvant therapeutic agent(s) to provide a combination composition or coordinated treatment that is combinatorially effective. or coordinatively useful in the prevention or treatment of stroke or the consequences of stroke. Exemplary combinatorial compositions and coordinated treatments in this context use a formula I phorbol ester or formula I phorbol ester derivative in combination with one or more secondary or additional therapeutic agents selected from tissue plasminogen activator, anticoagulant, statin, angiotensin II receptor blockers, angiotensin inhibitors converting enzyme, anti-platelet agents, fibrates, beta-blockers, calcium channel blockers, or diuretics. Examples of anticoagulants include, but are not limited to, heparin, warfarin, phenindione, heparinoids, atomentin, acenocoumarol, phenprocoumon inhibitors, idraparinux, fondaparinux, and thrombin. Examples of statins include, but are not limited to, lovastatin, amlodipine, atorvastatin, rosuvastatin, simvastatin, fluvastatin, pitavastatin, and pravastatin. Examples of angiotensin II receptor blockers include, but are not limited to, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan. Angiotensin-converting enzyme inhibitors include, but are not limited to, enazipril, captopril, enalapril, fosinopril, isinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril. Typical beta blockers include but are not limited to alprenolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol propranolol, sotalol, timolol, eucommia, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, and nebivolol. them. Examples of calcium channel blockers include, but are not limited to, amlodipine, clevidipine, diltiazem, felodipine, isradipine, nifedipine, nicardipine, nimodipine, nizoldipine, and verapamil. Examples of diuretics include, but are not limited to, chlorothiazide, hydrochlorothiazide, bumetanide, ethacrynic acid, furosemide, amiloride, spironolactone, eplerenone, and triamterene. Examples of fibrates include, but are not limited to, benzafibrate, ciprofibrate, clofibrate, gemfibrozil, or fenofibrate. Examples of antiplatelet agents include, but are not limited to, clopidogrel and ticlopidine.

Coordinated treatments may additionally use surgery, including the use of pacemakers, implantable defibrillators, coronary stents, prosthetic valves, coronary artery bypass grafting, balloon angioplasty, valve repair and replacement, carotid endarterectomy, angioplasty, stent placement, craniotomy, endovascular coil embolization, open ventricular closure. foramen ovale and heart transplantation, but not limited to.

In some embodiments, the present invention provides a combinatorial treatment of a disease ("treatment of stroke", "anticoagulation", "anticholesterolemic effect", "vasodilating effect", "antihypertensive effect", "induction of ERK phosphorylation", "reduction of arteriolar resistance", "increased venous capacitance", "reduction of cardiac oxygen demand", "decrease in heart rate", "stabilization of heart rate" or "NFκB modulation") composition containing phorbol ester and one or more additional agents active in the treatment of disease. In such combinatorial compositions, the phorbol ester of formula I and the additional agent(s) active in the treatment of the disease will be present in the combined composition for the treatment of the disease ("treatment of stroke", "anticoagulation", "anticholesterolemic action", "vasodilating action", " antihypertensive action", "induction of ERK phosphorylation", "reduction of arteriolar resistance", "increase in venous capacity", "reduction of cardiac oxygen demand", "decrease in heart rate", "stabilization of heart rate" or "NFκB modulation") in effective amounts, singly or in combination. In exemplary embodiments of the invention, the phorbol ester of Formula I and the non-phorbol ester co-agent(s) will be present in a disease-treating/preventive amount (i.e., at a specific dosage that will itself produce a detectable improvement in symptoms in the subject). On the other hand, the combinatorial composition may contain one of or both a phorbol ester of formula I and a non-phorbol ester in subtherapeutic unit dose(s), where the combinatorial composition comprising both agents is characterized by a combined dosage of both agents that are collectively effective in alleviating the disease caused. , condition or symptom. Thus, both the phorbol ester of formula I or the phorbol ester derivative of formula I and the nonphorbol ester may be present in the composition or administered according to the administration protocol at subtherapeutic doses, but together in the composition or method they cause a detectable reduction in the symptoms of the disease, the occurrence or recurrence of stroke or complications. from a stroke in the subject. In yet another embodiment, the combinatorial composition may include one or more neuroprotective agents. In an additional embodiment, the combinatorial composition may include one or more anti-inflammatory agents or other secondary or additional therapeutic agents, as described herein.

In order to practice the coordinated modes of administration of the invention, it is contemplated herein that a Formula I phorbol ester or a Formula I phorbol ester derivative may be administered simultaneously or sequentially, in accordance with a treatment protocol with one or more secondary or additional therapeutic agents. Thus, in some embodiments of the invention, the compound is administered concurrently with a non-phorbol ester or any other secondary or additional therapeutic agent provided herein, using separate compositions or a combination composition as described above (i.e., including and phorbol ester of formula I or a related or derivative compound, and a therapeutic non-phorbol ester). This coordinated administration may be carried out simultaneously or sequentially in any order, and there may be a period of time in which only one or both (or all) active therapeutic agents individually and/or jointly exhibit their biological activity.

In another embodiment of the invention, such coordinated treatments may be, for example, those that follow or are derived from various stroke treatment protocols. Coordinated treatments may, for example, include phorbol ester and/or procedures to prevent or treat damage caused by stroke. The hallmark of all such coordinated therapies is that the formula I phorbol ester or formula I phorbol ester derivative exhibits at least some activity that results in a favorable clinical response when combined with additional stroke prevention or treatment agents, or conversely, a clinical response. caused by secondary or additional therapeutic agents. Often, coordinating the administration of a Formula I phorbol ester or Formula I phorbol ester derivative with a secondary or additional therapeutic agent provides improved therapeutic or prophylactic results in a subject beyond the therapeutic effect induced by the Formula I phorbol ester or Formula I phorbol ester derivative or secondary or additional therapeutic agent. means administered alone. This qualification includes both direct manifestations and indirect ones.

In exemplary embodiments, a Formula I phorbol ester, or a Formula I phorbol ester derivative, is co-administered (simultaneously or sequentially, in combined or separate formulations), with one or more secondary stroke compounds or other indicated or additional therapeutic agents, for example, tissue plasminogen activator, anticoagulant, statin, angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, fibrate, beta-blocker, calcium channel blocker, lipid-lowering drugs, anti-platelet agents, or diuretic. In addition, ancillary or secondary therapy can be used in the treatment of stroke or effects such as, but not limited to stroke, pacemaker, implantable defibrillator, coronary stent, prosthetic valve, prosthetic heart, coronary artery bypass grafting, balloon angioplasty, plasty and valve replacement, heart transplantation, carotid endarterectomy, angioplasty, stenting, craniotomy, endovascular coil embolization, or closure of the foramen ovale.

As noted above, in all of the various embodiments of the present invention discussed herein, the methods of treating a disease and compositions can use the phorbol ester of formula I in any of a variety of forms, including any one or combination of salts, solvates, isomers pharmaceutically acceptable to the subject , enantiomers, polymorphs, solvates, hydrates and/or prodrugs of the compound. In exemplary embodiments of the present invention, TPA is used in therapeutic compositions and methods for the purpose of illustration.

The pharmaceutical compositions of the present invention may be administered by any means that will achieve their intended therapeutic or prophylactic purpose. Suitable routes of administration for the compositions of the invention include, but are not limited to, conventional release routes, devices, and methods, including but not limited to injection routes such as, but not limited to, intravenous, intramuscular, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intraarterial, subcutaneous, and intranasal routes. .

The compositions of the present invention may further include a pharmaceutically acceptable carrier suitable for the particular route of administration employed. Dosage forms of the compositions of the present invention include excipients recognized in the field of pharmaceutical formulation as suitable for the preparation of dosage units, as discussed above. Such excipients include, but are not limited to, binders, fillers, lubricants, emulsifiers, suspending agents, sweeteners, flavors, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other conventional excipients and additives.

If desired, the compositions of the invention may be administered in a controlled release form using a sustained release carrier such as a hydrophilic slow release polymer. Exemplary controlled release agents in this context include, but are not limited to, hydroxypropyl methylcellulose having a viscosity ranging from about 100 centipoise to about 100,000 centipoise, or other biocompatible matrices such as cholesterol.

Some of the formula I phorbol ester compositions of the invention are intended for parenteral administration, for example, for intravenous, intramuscular, subcutaneous or intraperitoneal administration, including aqueous and non-aqueous sterile injectable solutions, which, like many other contemplated compositions of the invention, may additionally contain antioxidants, buffers , bacteriostatic and/or solutes that make the composition isotonic with respect to the blood of a mammal; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and/or thickeners. The compositions may be presented in single-dose or multi-dose containers. Additional compositions and combinations of the invention may include sustained release polymers after parenteral administration. Parenteral preparations may be solutions, dispersions or emulsions suitable for such administration. The agents in question can also be formulated into polymers for sustained release after parenteral administration. Pharmaceutically acceptable compositions and ingredients are generally sterile or readily sterilizable, biologically inert, and readily administered. Such polymeric materials are well known to those skilled in the art of pharmacy. Parenteral preparations usually contain buffering agents and preservatives and injectable liquids that are pharmaceutically and physiologically acceptable, such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, or the like. Raw solutions for injections, emulsions and suspensions can be prepared from sterile powders, granules and tablets of the type previously described. Preferred unit dosage compositions are those containing a daily dose, or a single, daily sub-dose, as described above herein, or the appropriate portion of the active ingredient(s).

In more detailed embodiments, the compositions of the present invention may contain a phorbol ester of formula I or a phorbol ester derivative of formula I encapsulated for release into microcapsules, microparticles or microspheres, prepared, for example, by coacervation methods or by interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsules and poly ( methyl methacrylate) microcapsules, respectively; in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or in macroemulsions.

As noted above, in some embodiments, the methods and compositions of the invention may use pharmaceutically acceptable salts, for example acid addition or base salts of the above Formula I phorbol esters and/or related compounds or derivatives. Examples of pharmaceutically acceptable addition salts include inorganic and organic acid addition salts. Suitable acid addition salts are derived from acids which form non-toxic salts, such as hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, nitrate, phosphate and hydrogen phosphate salts. Additional pharmaceutically acceptable salts include 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, but not limited to, triethylamine salts, pyridine salts, picoline salts, ethanolamine salts, triethanolamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, and the like; organic acid salts such as acetate, citrate, lactate, succinate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, and formate; sulfonates such as methanesulfonate, benzenesulfonate and p-toluenesulfonate; and amino acid salts such as arginate, aspartate, glutamate, tartrate and gluconate. Suitable base salts are derived from bases which form non-toxic salts, such as aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and diethanolamine.

In other detailed embodiments, the methods and compositions of the invention use phorbol ester prodrugs of formula I. Prodrugs are any covalently linked carriers that release the active parent drug in vivo. Examples of prodrugs useful in the invention include esters or amides with hydroxyalkyl or aminoalkyl as a substituent, and they can be prepared by reacting such compounds as described above with anhydrides such as succinic anhydride.

The invention described herein is also to be understood as including methods and compositions containing phorbol esters of formula I using in vivo metabolic products of said compounds (either generated in vivo after administration of a precursor compound to a subject, or directly administered in the form of the metabolic product itself) . Such products can be obtained, for example, as a result of oxidation, reduction, hydrolysis, amidation, esterification, etc. input compounds, mainly due to enzymatic processes. Accordingly, the invention includes methods and compositions of the invention using compounds obtained by a method comprising contacting a phorbol ester of formula I with a mammal for a period of time sufficient to obtain its metabolic product. Such products are typically determined by preparing a radiolabelled compound of the present invention, parenterally administering it at a detectable dose to an animal such as a rat, mouse, guinea pig, monkey, or human, and allowing sufficient time for metabolism and excretion of its converted product from the urine. , blood or other biological samples.

The present invention as disclosed herein should also be understood to include diagnostic compositions for diagnosing the level of risk, presence, severity, or treatment of signs, or otherwise managing a disease, including but not limited to stroke, in a mammal, comprising contacting labeled (eg, isotopically labeled, fluorescently labeled, or otherwise labeled to permit detection of the labeled compound using conventional methods) phorbol ester of formula I with a mammal (eg, cell, tissue, organ, or individual) that is at risk or has one or more symptoms of a stroke and upon detection of the presence, location, metabolism and/or associated state of the labeled compound using any of a wide variety of known labeling/detection assays and methods. In exemplary embodiments of the invention, the phorbol ester of formula I is isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that can be included in the described compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, and ³⁶ Cl, respectively. The isotopically labeled compound is then administered to an individual or other subject and then detected as described above to provide useful diagnostic and/or therapeutic management data, in accordance with conventional methods.

Examples

The experiments described below demonstrate new and potential uses for phorbol esters and derivatives as agents for the treatment and prevention of stroke. These and additional discoveries are also expanded upon and clarified in the following examples.

Example I

Effect of TPA on peripheral leukocyte (WBC) and hemoglobin (Hb) counts in mice injected with S180 cells:

Sarcoma 180 (S180) cells were administered to Kwen-Ming mice. On the third day, mice were injected with TPA intraperitoneally (ip) at a concentration of 50, 100, or 200 μg/kg/day for 7 days. On the second day after the end of treatment, blood samples were taken from the tails of the treated mice for WBC and Hb analyses. The number of WBCs for the treated groups (50, 100, or 200 mcg/kg/day for 7 days) were 16.1±7.4, 18.7±3.0 and 20.7±3.4 x10 ⁹ /l , respectively; the amount of WBC in the control group was 13.6±1.8x10 ⁹ /l. The Hb of the treated groups was 136±11, 149±12 and 149±10 g/L, and the Hb in the control group was 134±15 g/L. The results show that intraperitoneal injection of TPA can increase the peripheral amount of WBC in mice in a dose-dependent manner, while hemoglobin levels are not significantly changed in TPA-treated mice compared to control mice.

Example II

Dose Range Study

Due to the strong local irritation caused by the use of TPA, TPA was administered to patients by intravenous (IV) infusion. The TPA solution in a sterile syringe was injected into 200 ml of sterile saline and mixed well for i.v. infusion.

Toxicity and side effects of various doses of TPA that have been clinically administered:

(1) TPA administered at 1 mg/patient/week:

One mg of TPA in solution was well mixed with 200 ml of sterile saline for intravenous infusion, which was completed over 1 hour at a rate of 16 μg/min. One hour after TPA administration, patients began to feel chills that lasted approximately 30 minutes, followed by fever (patients' temperature reached 37.5-39.5ºC, which lasted for 3-5 hours, then returned to normal) with mild and severe sweating. The above symptoms can be alleviated by the administration of glucocorticoids to patients. TPA at this dosage caused bleeding in a small number of patients, several patients suffered for a short period of time with difficulty in breathing, and hemoglobin was detected in the urine. However, these side effects were short-lived and reversible. Functions of the heart, liver, kidneys and lungs were normal.

(2) TPA administered at 0.5 mg/patient x 2/week: (two doses per week)

0.5 mg of TPA in solution was well mixed with 200 ml of physiological saline for intravenous infusion, which was completed after 1 hour at a rate of 8 μg/min. Post-administration reactions were similar to TPA 1 mg but to a lesser extent than the 1 mg dose. Patients tolerated the lower dose more easily. Occasionally, Hb was found in the patient's urine. Difficulty breathing was not observed. Functions of the heart, liver, kidneys and lungs were normal.

(3) TPA was administered at 0.25 mg/patient x 4/week:

0.25 mg of TPA in solution was well mixed with 200 ml of physiological saline for intravenous infusion, which was completed after 1 hour at a rate of 4 μg/min. After administration, symptoms such as chills and fever were also observed, but to a much lesser extent than with higher doses. Hb was not detected in the urine, and none of the patients suffered from difficulty in breathing. Functions of the heart, liver, kidneys and lungs were normal.

Example III

Treatment of recurrent/refractory malignancies with TPA

Patients with histologically described relapses/refractory malignancies/bone marrow disorders were treated with a combination of TPA (Xichuan Pharmaceuticals, Nan Yang, Henan, China), dexamethasone, and magnesium choline trisalicylate. The comparable methods set forth below for demonstrating the therapeutic use of TPA in the treatment of acute myeloid leukemia (AML) will be used to demonstrate the use of TPA for the treatment of other neoplastic diseases and malignancies. In addition to the specific protocols herein, successful treatment and/or remission will be determined for a variety of target tumors and malignancies under conditions using any of the wide range of known cancer detection and evaluation methods, for example, by determining the size reduction of solid tumors by histopathological studies to evaluate tumor growth. , stage, metastatic potential, levels of presence/expression of histological markers of cancer, etc.

AML is an aggressive disease that usually warrants urgent and intensive care. The median age of patients diagnosed with AML is 64-68 years, and patients over the age of 60 treated with standard chemotherapy are cured of their disease in <20% of cases. Patients who develop AML after chemotherapy/radiotherapy for a prior hematologic disorder or prior leukemia have similar poor outcomes as do patients whose disease is associated with specific adverse cytogenetic and clinical features. Thus, most patients diagnosed with AML have patient and/or disease-related features that are associated with a very poor prognosis. For patients with disease recurrence, standard non-transplant therapy does not show a cure trend. For these patients, AML is often fatal. New approaches to AML therapy are needed.

Using the methods and compositions of the present invention, TPA has been developed as a therapeutic agent for the treatment of AML patients based on the novel role of TPA in modulating intracellular signaling pathways, its ability to induce differentiation and/or apoptosis in cell lines, and clinical data indicating efficacy. TPA in the treatment of neoplastic and malignant diseases, including myeloid malignancies.

So far, clinical evaluation of TPA has shown that TPA has a direct therapeutic cytotoxic effect on at least a subset of AML cases as measured by cell viability and apoptosis assay. In all primary cultures analyzed by Western analysis, TPA strongly induced ERK phosphorylation at 1 hour in culture. The cytotoxic effect of TPA on primary AML cells is associated with the subsequent loss of the survival-promoting phosphor-ERK signal after 24 hours of ex vivo contact. This observation is in good agreement with the results of other studies that have reported reduced survival of primary AML after pharmacological interruption of ERK signaling with MEK inhibitors such as PD98059, U0126 and PD184352. In our studies, the loss of ERK signaling was associated with the induction of ERK phosphatase.

In addition to protein kinase C and ERK activation, TPA is a known inducer of NF-κB, a pro-survival transcription factor that often constitutively acts on AML blasts and leukemic stem cells. Recent work from our laboratory has shown that NF-κB of AML cells can be inhibited in vivo with 48-hour treatment with dexamethasone + magnesium choline trisalicylate (CMT). In addition, we have shown that dexamethasone can induce the expression of ERK MKP-1 phosphatase and increase TPA cytotoxicity in primary AML samples. In this context, we have chosen in exemplary embodiments of the invention to use dexamethasone and CMT as additional drugs to be used 24 hours before and 24 hours after TPA treatment. These drugs are well tolerated and it is expected to reduce inflammatory side effects from treatment and increase TPA cytotoxicity by increasing ERK phosphatase expression and inhibiting NF-κB. In addition, dexamethasone and SMT will be used as complementary drugs because they are anti-inflammatory, can improve side effects, and can enhance antileukemic activity by inhibiting the anti-apoptotic effects of NF-κB constitutive expression and induction of phosphatases that reduce signaling pathway activity.

The original Phase 1 TPA study included 35 patients [23 with relapsed/refractory AML, 2 with other myeloid malignancies (CML blast crisis, myelodysplasia with blast excess), 3 with Hodgkin's disease, 3 with non-Hodgkin's lymphoma, and 4 with solid tumors]. Most patients developed recurrent/refractory AML. Our clinical results include one AML patient with stable disease for >5 months who received 8 TPA infusions. In the second AML patient, a pronounced (5-fold) decrease in the number of circulating blasts was observed after TPA administration. This decrease in leukemic blasts persisted for 4 weeks and the patient eventually died of a fungal infection. In conclusion, a patient with relapsing and refractory Hodgkin's disease, despite high doses of chemotherapy with preserved autologous stem cells, had a partial remission of the chest tumor after TPA administration. The TPA dose escalation was completed, in the last cohort, 2 of 3 patients treated with doses of 0.188 mg/m ² d1-5, 8-12 treated with non-haematological toxicity limiting dose (DLT) class III showed the maximum tolerated dose of TPA as monotherapy at 0.125 mg / m ² /d at d1-5 and 8-12.

In the case of AML and other hematological malignancies, patients are given an initial dose of TPA 1 mg/week x 3 weeks (days 1, 8, 15), administered with continuous/intermittent pulse oximetry for 6 hours. Twenty-four hours before starting TPA therapy, patients are given 10 mg dexamethasone every six hours and 1500 mg magnesium choline trisalicylate (CMT) every eight hours until 24 hours after TPA administration. Following the initial dose of TPA, patients have a two-week rest period after which they may be re-evaluated. Those patients who have disease response or stabilization from the initial dose of TPA are considered for up to six cycles of twenty-eight days according to the protocol below.

After a two-week rest period, patients pre-administer Tylenol 650mg and Benadryl 25-50mg (depending on patient size and age) thirty minutes prior to TPA administration. They are given an intravenous infusion of TPA through a central venous catheter for 5 days a week for two consecutive weeks followed by a 2-week rest period. TPA is administered at a dose of 1 mg in 200 ml of saline over 1 hour. Twenty-four hours before starting TPA therapy, patients are given 10 mg dexamethasone every six hours and 1500 mg magnesium choline trisalicylate, this is continued every eight hours until 24 hours after TPA administration.

The level of TPA in the blood is measured before and after the infusion using a bioassay that measures the differentiation activity of the organic extractable solvent. 1 ml of blood is extracted twice in 5 ml of ethyl acetate, the extraction residue is dissolved in 50 μl of ethanol and an aliquot of HL60 cells is added. After 48 hours, adherent cells are measured.

Tests are also performed on blood samples taken before and after infusion with TPA to determine the level of leukocytes, platelets and neutrophils. Samples are further analyzed for the presence of myeloblasts and Auer bodies. These and subsequent experiments will further reveal the therapeutic cytotoxic and other effects that TPA has against tumor cells in AML and other neoplastic and malignant conditions.

Example IV

ERK activation modulation measurement

Phosphor-ERK levels are measured in circulating malignant cells of leukemia patients and in peripheral blood mononuclear cells of lymphoma/solid tumor patients. A blood sample is taken from patients according to the protocol of Example III both before and after TPA administration.

In leukemia patients with WBC ≥ 1000/µl, flow cytometry is performed on a blood sample using a cell surface-specific antigen-specific antibody and phosphor-ERK directly conjugated to fluorophores (BD Biosciences, San Jose, CA). Samples were taken before TPA administration and one hour after TPA infusion on days 1, 2 and 11 in the initial treatment according to the protocol of Example III and on days 1 and 11 in subsequent cycles. In leukemia patients with an absolute number of leukoblastomas ≥ 2500 per µl and other leukemic patients, peripheral blood samples were taken on days 1, 8 and 15 of the initial cycle according to the protocol of example III before and 1 and 4 hours after the infusion. Samples were also analyzed by Western blot analysis for phosphor-ERK and total ERK1/2 levels to confirm flow cytometric results and to correlate with clinical responses.

The above assays will further elucidate the role of TPA in the treatment of neoplastic and malignant diseases, including the cytotoxic effect of TPA on malignant cells, as evidenced by primary AML cells, and the associated reduction in TPA survival-promoting phosphor-ERK signal.

Example V

Measurement of NF-κB modulation

Previous studies have shown that NF-κB activity may be altered in patients after administration of TPA with dexamethasone. In addition, dexamethasone has been shown to induce the expression of MKP-1 ERK phosphatase and increase TPA cytotoxicity. The following studies are designed to further elucidate how NF-κB activity is therapeutically modulated in patients treated with TPA and dexamethasone.

NF-κB binding is measured in patients' peripheral blood samples at baseline and before and after infusion from patients treated with TPA according to Example III using ELISA-based assays (BD Bioscience, San Jose, US). NF-κB levels are quantified using chemiluminescence severity for signal detection in cell extract limiting using a 96-well format. In addition, an electrophoretic mobility change assay is performed to measure NF-κB binding in the peripheral blood of a leukemia patient with an absolute leukoblast count of ≥2500/µl and other non-leukemic patients with a normal white blood cell count.

The above studies further show that TPA is an inducer of NF-κB; However, these experiments show that NF-κB AML cells can be inhibited by treatment with dexamethasone and magnesium choline trialicylate.

Example VI

Treatment of people who have had a stroke.

Patient NC, male, 68 years of age, suffered a stroke eighteen months prior to treatment with TPA. At the time of starting TPA treatment, he could not walk without a cane, had difficulty with his left arm and left leg, and was tired and weak. He received an injection of 1 ampoule containing 0.19 mg TPA (0.125 mg/m ² ) every other day for four weeks, then 0.24 mg TPA (1.25 x 0.125 mg/m ² ) every other day for 2 weeks, and then 0.26 mg TPA (1.5 x 0.125 mg/m ² ) every other day for an additional 3 weeks. The patient made a full recovery.

Patient MC, male, 65 years of age, suffered a stroke seven years prior to treatment with TPA. He received 3-4 injections of 0.19 mg TPA (0.125 mg/m ² ) per week for ten weeks for a total of 35 injections. He regained facial mobility and had an 80% improvement in the mobility of his right side.

Example VII

Treatment of the embolic stroke model with TPA

Male Sprague-Dawley rats (Charles River, Japan), each weighing 280-350 g, are used. Embolic stroke is induced by a modification of the method of Kudo et al. (1982). Rats to be used for blood collection were anesthetized with 1.0% halothane (Fluorotan™; Takeda, Osaka, Japan) by spontaneous inhalation. Surflo™ with 24 probes (Terumo Medical Products, Elkton, MD) is fixed in the femoral artery and 0.1 ml of arterial blood is withdrawn using a 1 ml injection syringe (Terumo Medical Products, Elkton, MD). The arterial blood in the syringe is incubated at 30° C. ^for 2 days to form a blood clot. Thereafter, 0.1 ml of physiological saline solution is added to an injection syringe and passed through a 26-gauge injection needle (Terumo Medical Products, Elkton, MD) twice so that the blood clot is crushed.

Rats induced with embolic stroke under anesthesia with 1.0% halothane by spontaneous inhalation. The neck of the rats is subjected to a median incision and the external carotid artery, superior thyroid artery, occipital artery and pterygopalatine artery are cauterized with a bipolar coagulator (T-45; Keisei Medical Industrial Company Ltd, Tokyo, Japan). Cerebral embolism is induced by injecting 0.1 ml of crushed thrombus into the internal carotid artery.

The formation of cerebral embolism is assessed using laser Doppler flowmetry (FloC1; Omegawave, Tokyo, Japan). A decrease in cerebral blood flow to a level of 30% or less is taken as positive evidence for the formation of an embolism. Cerebral blood flow is monitored for 30 minutes after clot infusion and blood flow is monitored as the remaining 50% or less of flow prior to thrombus injection. After that, the catheter (PE50) for drug administration is fixed in the jugular vein and the animals wake up.

Rats that have successfully formed a cerebral embolism are divided into four groups. The first group of rats receives saline injections every other day. Groups 2-4 receive 0.125 mg/m ² TPA injection every other day for four weeks. Group 2 is then sacrificed. Groups 3-4 receive an additional 0.156 mg/m ² TPA every other day for two weeks and then Group 3 is euthanized. Group 4 was administered 0.18775 mg/m ² TPA every other day for three weeks and then sacrificed.

The brains are excised after the animals are sacrificed and cut into ten pieces at 1 mm intervals using a McIwain cloth knife (Mickle Laboratory Engineering, UK) and stained for 20 minutes in 2% TTC (2,3,5-triphenyltetrazolium chloride; Tokyo Kasei) at 37° C. Images ^of the TTC-stained slices were loaded into a computer using a digital camera (HC-2500; Fuji PhotoFilm) and Phatograb-2500 (Fuji Photo Film) and infarct volume was calculated using a Mac Scope (Mitani, Japan). Infarct volume is given by mean ± standard error. With regard to the statistical test of the infarct volume result, evaluation is performed by performing a Dunnett test for the control group and for each of the TPA-administered groups compared to the control group, and then by performing a T-test for the TPA-administered group.

Neurological symptoms were observed daily until sacrifice and the rats were evaluated on three tests. (1) Rats were held gently by the tail, held one meter above the floor, and flexion of the forelimbs was observed. (2) The rats were placed on a large sheet of soft, plastic-coated paper that could be firmly gripped by their claws. The tail was held by the hand, gently pressed on the sides behind the shoulders of the rats until the forelimbs slipped a few inches. (3) The rats were allowed to roam freely and the rotational behavior was observed. The calculation of neurological symptoms was carried out in accordance with the scale developed by Bederson et al. (1986) as follows: 0: no observed deficit; 1: flexion of the forelimbs; 2: reduced resistance to lateral pressure without rotation; 3: same behavior as class 2, with rotation.

Neurological symptoms were assessed using the Steele test for the control group and for each of the TPA-administered groups for comparison with the control group, and then by conducting the Wilcoxon test for the TPA-administered group. In any of the tests, the value at p<0.05 was determined to be statistically significant.

Example VIII

Efficacy of TPA in the treatment of stroke using a continuous model of middle cerebral artery occlusion

Male Wistar rats (250-320 g) are used for this study. Animals are anesthetized with isoflurane (3% administration, 1-2% maintenance). Anesthesia is controlled by pinching the toe on the paw. The aseptic method is used for all procedures during this study. The surgical site is trimmed and cleaned with alcohol and a surgical scrub. Animals are placed in warm heated water in a container to maintain body temperature. A paramedian incision is made in the neck over the carotid artery. The tissue is directly dissected to identify the carotid artery and bifurcation. Threads are placed around the proximal or common carotid artery and the outer part of the carotid artery. These threads are tied. An incision is made in the common carotid artery, distal to the vessel ligation. Pre-prepared sutures (4-0 monofilament sutures or similar material) are placed in the carotid artery and advanced into the carotid artery. The thread is advanced approximately 20 mm through the bifurcation of the carotid artery until a slight resistance is felt, similar to wedging into the middle cerebral artery. Care must be taken not to rupture the artery when the thread is inserted. The thread is tied in place and the skin incision is closed. The conscious animal is assessed for successful occlusion using the Bederson scale. (See Bederson et al., (1986) Stroke, 17:1304-1308.) Body temperature is measured every 15 minutes to maintain normothermia. Animals that have undergone a middle cerebral artery occlusion procedure may have difficulty thermoregulating for several hours after surgery. Animals are placed in a cooling or heating field determined by their temperature. Body temperature is maintained at 37.5 ^° C. Animals are monitored for 6 hours after the middle cerebral artery and then placed in cages overnight.

Rats are divided into four groups. The first group of rats is given saline injections every other day. Groups 2-4 receive 0.125 mg/m ² injections of TPA every other day for four weeks. Group 2 is then sacrificed. Group 3-4 receive an additional 0.156 mg/m ² TPA every other day for two weeks and then Group 3 is euthanized. Group 4 was administered 0.18775 mg/m ² TPA every other day for three weeks and then sacrificed.

The brains are excised after the animals are sacrificed and cut into ten pieces at 1 mm intervals using a McIwain cloth knife (Mickle Laboratory Engineering, UK) and stained for 20 minutes in 2% TTC (2,3,5-triphenyltetrazolium chloride; Tokyo Kasei) at 37° C. Images ^of the TTC-stained pieces are loaded onto a computer using a digital camera (HC-2500; Fuji PhotoFilm) and Phatograb-2500 (Fuji Photo Film). Parts of the brain are photographed and analyzed for infarct size, infarct volume, numbra and edema.

Neurological symptoms were observed daily until sacrifice and the rats were evaluated on three tests. (1) Rats were held gently by the tail, held one meter above the floor, and flexion of the forelimbs was observed. (2) The rats were placed on a large sheet of soft, plastic-coated paper that could be firmly gripped by their claws. The tail was held by the hand, gently pressed on the sides behind the shoulders of the rats until the forelimbs slipped a few inches. (3) The rats were allowed to roam freely and the rotational behavior was observed. The calculation of neurological symptoms was carried out in accordance with the scale developed by Bederson et al. (1986) as follows: 0: no observed deficit; 1: flexion of the forelimbs; 2: reduced resistance to lateral pressure without rotation; 3: same behavior as class 2, with rotation.

Neurological symptoms were assessed using the Steele test for the control group and for each of the TPA-administered groups for comparison with the control group, and then by conducting the Wilcoxon test for the TPA-administered group. In any of the tests, the value at p<0.05 was determined to be statistically significant.

Example IX

Efficacy of TPA in the Treatment of Stroke Using a Variable Model of Middle Cerebral Artery Occlusion

Male C57B16 mice (25-30 g) are used in this study. Mice are anesthetized with isoflurane (3% administration, 1-2% maintenance). The surgical site is clipped and cleaned with alcohol and a surgical scrub. An incision in the midline of the neck is made through the carotid artery and the artery is dissected at the bifurcation. The monofilament sutures were inserted into the carotid artery and expanded until they entered the middle cerebral artery. The placed threads were tied and the incision was closed. Two hours after occlusion, mice were reanaesthetized and the suture material was removed from the MCA. Body temperature was maintained by using a heating pad during and after surgery. Animals were monitored for 4 hours after occlusion of the middle cerebral artery.

The rats were divided into four groups. The first group of rats was given saline injections every other day. Groups 2-4 received a 0.125 mg/m ² injection of TPA every other day for four weeks. Group 2 was then sacrificed. Groups 3-4 received an additional 0.156 mg/m ² TPA every other day for two weeks and then Group 3 was euthanized. Group 4 was administered 0.18775 mg/m ² TPA every other day for three weeks and then sacrificed

The brains were excised after the animals were sacrificed, and cut into ten pieces at 1 mm intervals using a McIwain tissue knife (Mickle Laboratory Engineering, UK) and dipped for 20 minutes in 2% TTC (2,3,5-triphenyltetrazolium chloride; Tokyo Kasei) at 37°C. Images ^of the TTC-stained pieces were loaded onto a computer using a digital camera (HC-2500; Fuji PhotoFilm) and Phatograb-2500 (Fuji Photo Film). Parts of the brain are photographed and analyzed for infarct size, infarct volume, numbra and edema.

Neurological symptoms were observed daily until sacrifice and the rats were evaluated on three tests. (1) Rats were held gently by the tail, held one meter above the floor, and flexion of the forelimbs was observed. (2) The rats were placed on a large sheet of soft, plastic-coated paper that could be firmly gripped by their claws. The tail was held by the hand, gently pressed on the sides behind the shoulders of the rats until the forelimbs slipped a few inches. (3) The rats were allowed to roam freely and the rotational behavior was observed. The calculation of neurological symptoms was carried out in accordance with the scale developed by Bederson et al. (1986) as follows: 0: no observed deficit; 1: flexion of the forelimbs; 2: reduced resistance to lateral pressure without rotation; 3: same behavior as class 2, with rotation.

Neurological symptoms were assessed using the Steele test for the control group and for each of the TPA-administered groups for comparison with the control group, and then by conducting the Wilcoxon test for the TPA-administered group. In any of the tests, the value at p<0.05 was determined to be statistically significant.

Example X

Clinical efficacy of using TPA for stroke treatment

Men and women aged 30-72 who have had a previous stroke less than one month ago are recruited to participate in the 10-week TPA study.

Recruited individuals sign an informed consent form and are assessed with computed tomography (CT), physical and neurological tests, neurological examinations, sedation level, National Institutes of Health Stroke Study (NIHSS), 12-lead electrocardiogram, ECG telemetry, oxygen sensor pulse measurements, body measurements, body weight, patient history, pregnancy test, urine drug measurement, hematology test, coagulation kit, general clinical trial, urinalysis. Clinical laboratory testing includes complete blood chemistry (Na, K, Cl, CO2, Glu, BUN, Cr, Ca, TP, Alb, TBili, AP, AST, ALT), CBC hematology (Hgb, Hct, RBC, WBC, Plt , Diff), and serum hCG for all women.

Individuals are administered 0.125 mg / m² TPA or placebo every other day for four weeks, then 1.25 x 0.125 mg/m² or placebo every other day for weeks five and six, and 1.5 x 0.125 mg/m² or placebo every other day during the seventh to the ninth week. Individuals are monitored during and within two hours of TPA or placebo administration.

At weeks five and ten, subjects are scored using the NIHSS (NIH Stroke Scale), Barthel's ADL index (Granger, 1979), and modified Rankine scale (Farrell, 1991).

Efficacy is determined by measuring change from baseline NIHSS data in individuals treated with TPA versus placebo. Secondary efficacy variables are the Barthel ADL index and the modified Rankine scale. Safety measures were taken and honestly evaluated, in particular the measurement of change from baseline in the fifth week. These measures include side-effect reports, physical exams, vital signs, weights, ECGs, clinical laboratory test results, and vital signs as well as scores for suicidal behavior and/or ideas. Side effects are any adverse medical event that occurs in a subject who is administered an investigational drug, whether or not it has a causal relationship with the investigational drug. Therefore, side effects can be any adverse or unintended signs (including abnormal laboratory findings, for example), symptoms, or disorders temporally associated with the study drug or not considered to be related to the study drug.

Subjects are considered to have completed the study if they have completed all visits. They may be excluded from the study if they do not meet the inclusion/exclusion criteria; suffer from side effects, have an inadequate therapeutic response, withdraw their consent, violate protocol, stop attending, or die.

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Claims (21)

1. Composition for the prevention or treatment of one or more consequences of stroke in a mammal, comprising an effective amount of a phorbol ester of formula I or a pharmaceutically acceptable salt, isomer or enantiomer thereof,

where R ₁ and R ₂ are selected from the group consisting of hydrogen, hydroxyl,

, where the alkyl group contains from 1 to 15 carbon atoms;

, where the lower alkenyl group contains from 1 to 7 carbon atoms;

and

; and R ₃ is hydrogen and

, wherein said phorbol ester of formula I excludes 12-O-tetradecanoyl-phorbol-13-acetate (TPA), where one or more of the side effects of a stroke is paralysis, spatial perception disorder, clouding of consciousness, left neglect syndrome, aphasia, problems with coordination and balance, nausea, vomiting, cognitive impairment, perceptual disorder, orientation disorder, homonymous hemianopia, impaired mobility or impulsivity . 2. The composition according to claim 1, where R ₁ or R ₂ is

alkyl, and the remainder of R ₁ or R ₂ is

and R ₃ is hydrogen. 3. Composition according to claim 1, wherein the phorbol ester is 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-myristate, 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, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tigliate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate, or phorbol 13-acetate. 4. The composition of claim 1 further comprising one secondary or additional therapeutic agent that is effective in combination with said formula I phorbol ester to treat or prevent the effects of stroke in said mammal. 5. The composition of claim 4, wherein at least one secondary or additional therapeutic agent is a tissue plasminogen activator, an anticoagulant, a statin, a fibrate, an angiotensin-II receptor blocker, an angiotensin-converting enzyme inhibitor, a beta-blocker, an antiplatelet agent, a calcium channel blocker, or diuretic. 6. A method for preventing or treating one or more consequences of stroke in a mammal, comprising administering a composition according to claim 1, containing an effective amount of a phorbol ester of formula I or a pharmaceutically acceptable salt, isomer or enantiomer thereof, to said mammal,

where R ₁ and R ₂ are selected from the group consisting of hydrogen, hydroxyl,

, where the alkyl group contains from 1 to 15 carbon atoms;

, where the lower alkenyl group contains from 1 to 7 carbon atoms;

and

; and R ₃ is hydrogen and

, wherein said phorbol ester of formula I excludes 12-O-tetradecanoyl-phorbol-13-acetate (TPA), where one or more of the side effects of a stroke is paralysis, spatial perception disorder, clouding of consciousness, left neglect syndrome, aphasia, problems with coordination and balance, nausea, vomiting, cognitive impairment, perceptual disorder, orientation disorder, homonymous hemianopia, impaired mobility or impulsivity . 7. The method according to p. 6, where R ₁ or R ₂ is

alkyl, and the remainder of R ₁ or R ₂ is

and R ₃ is hydrogen. 8. The method of claim 6 wherein the phorbol ester is 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-myristate, 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, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tigliate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate, or phorbol 13-acetate. 9. The method of claim 6, further comprising administering at least one secondary or additional therapeutic agent effective in a combination composition or coordinated treatment regimen with said composition of claim 1 containing a formula I phorbol ester for the treatment or prevention of the consequences of stroke in the specified mammal. 10. The method of claim 9, wherein at least one secondary or additional therapeutic agent is administered to said mammal according to an administration protocol simultaneously with, before, or after said composition of claim 1 containing a formula I phorbol ester to said mammal. 11. The method of claim 9, wherein the at least one secondary or additional therapeutic agent is a tissue plasminogen activator, an anticoagulant, a statin, a fibrate, an angiotensin-II receptor blocker, an angiotensin-converting enzyme inhibitor, a beta-blocker, an antiplatelet agent, a calcium channel blocker, or diuretic. 12. The method of claim 6, further comprising surgery in combination with a composition of claim 1 comprising a phorbol ester of formula I, to treat or prevent the effects of stroke in said mammal. 13. The method of claim 12, wherein the surgical intervention is carotid endarterectomy, angioplasty, stenting, craniotomy, pacemaker implantation, defibrillator implantation, valve implantation, coronary artery bypass grafting, heart transplantation, endovascular coil embolization, or foramen ovale closure.