HK1262168A1 - Compositions and methods of use of phorbol esters - Google Patents

Description

Compositions and methods of use of phorbol esters

The application is a divisional application of a patent application with the application date of 2013, month 1 and day 18, and the application number of 201380014940.X, and the name of phorbol ester composition and using method.

RELATED APPLICATIONS

This application is a U.S. partially-filed application, united states patent application serial No. 12/023,753 filed on 31/2008, which claims priority benefits of U.S. provisional patent application serial No. 60/898,810 filed on 31/2007, U.S. provisional patent application serial No. 61/588,162 filed on 18/2012, U.S. provisional patent application serial No. 61/588,165 filed on 18/2012, and U.S. provisional patent application serial No. 61/588,167 filed on 18/2012, 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.

Background

Plants have long provided many medical uses. The World Health Organization (WHO) estimates that 40 million people (80% of the world's population) are currently using herbal medicines for some aspect of primary health care. (WHO Condition Specification N ° 134 Condition Specification 2008/12). However, specific compounds with medicinal effects may be difficult to isolate and reproduce on a commercial scale. In addition, while the active compounds can be isolated from plants, other parts of the plant such as minerals, vitamins, essential oils, glycosides, alkaloids, bioflavonoids and other substances can also be involved in the action of the active ingredients or known medicinal effects of the plant, thereby presenting challenges to the use, purification and commercialization of plant-based medicaments.

Phorbol is a natural plant-derived organic compound belonging to the family of diterpenes of the crotonaldehyde family. It was first isolated in 1934 as a hydrolysate of Croton oil produced from seeds of Croton (Croton tiglium), a multi-leaved shrub of the family Euphorbiaceae in southeast Asia. Various esters of phorbol have important biological properties, including the reported ability to mimic diacylglycerol and activate protein kinase c (pkc) and to modulate downstream cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathway. In addition, phorbol esters are believed to bind to the chimeric protein (chimaeren), Ras activator RasGRP and vesicle activator Munc-13(Brose N, Rosenmund C., JCelSci; 115: 4399-411 (2002)). Some phorbol esters also induce nuclear factor- κ B (NF- κ B). The most significant physiological property of phorbol esters is their reported ability to act as tumor promoters. (Blumberg, 1988; Goel, G et al, Int, Journal of biology 26,279-288 (2007)).

12-O-tetradecanoyl phorbol-13-acetate (TPA), also known as phorbol-12-myristate-13-acetate (PMA), is a phorbol ester used as an inducer of differentiation and/or apoptosis in various cell lines and primary cells in a model of carcinogenesis. TPA has also been reported to increase circulating white blood cells and neutrophils in patients with chemotherapy-inhibited bone marrow function (Han Z.T. et al Proc. Natl.Acad.Sci.95,5363-5365(1998)) and to inhibit HIV-cytopathic effects on MT-4 cells. (Mekkawy S. et al, Phytochemistry 53,47-464 (2000)). However, TPA has not been shown to be an effective tool for the treatment, management, or prevention of HIV or AIDS or as a chemotherapy adjuvant due to a variety of factors, including the problem of corrosive reactions upon contact with the skin and its potential toxicity. In fact, phorbol esters are generally excluded from potential therapeutic candidates for cancer or inflammatory diseases such as rheumatoid arthritis or conditions involving an inflammatory response such as stroke, autoimmune disorders or prostatic hypertrophy, since phorbol esters play a key role in the activation of protein kinase C (pkc), such that protein kinase C triggers various cellular responses leading to inflammatory responses and neoplasias (Goel et al, Int, Journal of biology 26,279-288 (2007)).

With the development of modern medicine, the survival rates of both chronic and acute diseases have increased, which presents new challenges in managing chronic conditions and sequelae of acute diseases as well as treatment side effects. There is a continuing need to identify agents, including plant-based agents, that can be used to treat disease, prevent acute-onset injury, manage disease sequelae, and manage side effects of disease treatment. While molecular targeting has produced a variety of successful agents, multiple signaling pathways are often involved, and blocking one pathway is easily at an additional cost. There is a clear need for new and more effective treatments and side effect management for individuals suffering from a variety of conditions, particularly chronic or potentially relapsing conditions such as cancer, immune disorders, autoimmune disorders, stroke, rheumatoid arthritis, inflammation, uterine fibroids, prostatic hypertrophy, urinary incontinence, Parkinson's disease and nephropathy.

Summary of The Invention

The present invention relates to compositions containing phorbol esters and methods of use thereof. These compositions and methods are effective in treating chronic or potentially relapsing conditions, or in repairing damage left by an acute episode of a particular disease.

In one embodiment, phorbol esters and derivatives of phorbol esters are used to treat diseases such as HIV and related conditions such as AIDS. The compositions and methods of the present invention may effect treatment of HIV and related conditions such as AIDS by any possible means. In some embodiments, the compositions and methods can improve HIV receptor activity in a mammalian subject. In another embodiment, the compositions and methods as described herein can reduce the number of latent HIV reservoirs in HIV-infected subjects. In yet another embodiment, the compositions and methods as described herein can enhance HIV activation in latent proviral cells. In other embodiments, they may inhibit the cytopathic effects caused by HIV.

In another embodiment, compositions containing phorbol esters and phorbol ester derivatives are useful for treating and managing the symptoms of HIV and AIDS in a mammalian subject. Target conditions for treatment and management using the compositions and methods described herein include, but are not limited to, oral lesions, fatigue, cutaneous thrush, fever, loss of appetite, diarrhea, aphthous ulcers, malabsorption, thrombocytopenia, weight loss, anemia, enlarged lymph nodes, susceptibility to serious secondary conditions such as mycobacterium avium complex, salmonellosis (salmonellosis), syphilis, neurosyphotosis, Tuberculosis (TB), bacillary angiomatosis, aspergillosis, candidiasis, coccidioidomycosis, listeriosis (listeriosis), pelvic inflammatory disease, Burkitt's lymphoma, cryptococcal meningitis, histoplasmosis, Kaposi's sarcoma, lymphomas, systemic non-hodgkins's lymphoma, primary central nervous system lymphoma, lymphomas, Cryptosporidiosis (cryptosporidiosis), isosporidiosis (isosporidiosis), microsporidiosis (microsporidiosis), pneumocystis (pneumocystis cariniponia, PCP), toxoplasmosis (toxoplasmosis), Cytomegalovirus (CMV), hepatitis, herpes simplex, herpes zoster, human papilloma virus (HPV, genital warts, cervical carcinoma), contagious molluscum, oral leukoplakia (OHL), and Progressive Multifocal Leukoencephalopathy (PML).

In yet another embodiment, compounds containing phorbol esters and phorbol ester derivatives may be used to treat neoplastic diseases. Such neoplasms may be malignant or benign. In some embodiments, a neoplasm can be a solid or non-solid cancer. In other embodiments, the neoplasm can be recurrent. In another embodiment, a neoplasm can be refractory. Exemplary neoplasms include, but are not limited to, hematological malignancies/bone marrow disorders, including, but not limited to, leukemias, including Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), chronic myelogenous leukemia blast crisis, myelodysplasia, and myeloproliferative syndrome; lymphomas, including Hodgkin's and non-Hodgkin's lymphomas; subcutaneous adenocarcinoma; ovarian teratocarcinoma; liver cancer; breast cancer; bone cancer; lung cancer; non-small cell lung cancer and prostate cancer. Other neoplasia conditions suitable for treatment using the methods and compositions as described herein include other cancer disorders and conditions, including various types of solid tumors. Successful treatment and/or remission will be judged according to conventional methods, such as determining size reduction of solid tumors, and/or performing histopathological studies to assess growth, stage, metastatic status or potential, presence or expression levels of histological cancer markers, and the like.

The compositions and methods herein may also be used to treat symptoms of neoplastic diseases, including but not limited to anemia; chronic fatigue; excessive or bleeding tendency, such as nasal, gingival and sub-dermal bleeding; easy contusion, especially contusion without obvious reasons; tachypnea; ecchymosis; repeatedly fever occurs; swelling of the gums; the cut heals slowly; bone and joint discomfort; repeated infection; weight loss; scratching; night sweat; lymph node swelling; fever is caused; abdominal pain and discomfort; visual impairment; cough; lack of appetite; chest pain; dysphagia; swelling of the face, neck and upper limbs; frequent urination is required, especially at night; difficulty in starting urination or holding back urine; weak or disrupted urine flow; painful urination or burning sensation; difficulty in erection; ejaculation pain; blood in urine or semen; recurrent pain or stiffness in the waist, hip or thigh; and weakness.

The compositions and methods herein can further be used to treat the side effects of chemotherapy and radiation therapy, which are commonly used as a treatment for neoplastic diseases. Such side effects include, but are not limited to, hair loss, nausea, vomiting, loss of appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, mouth ulcers, itchy skin, scaling, nerve and muscle damage, changes in hearing, weight loss, diarrhea, immunosuppression, bruising, heart damage, bleeding, liver damage, kidney damage, edema, aphtha and sore throats, infertility, fibrosis, hair loss, wet desquamation, dry mucosa, dizziness and encephalopathy.

In yet another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used to modulate a cell signaling pathway. This modulation may have a variety of consequences, for example, in some embodiments, the use of a composition containing a phorbol ester and a phorbol ester derivative may increase the white blood cell count in a mammalian subject. In another embodiment, a composition comprising a phorbol ester and/or a phorbol ester derivative may alter the release of Th1 cytokines in a mammalian subject. In yet another embodiment, a composition comprising a phorbol ester and/or a phorbol ester derivative may alter the release of interleukin 2(IL-2) in a mammalian subject. In another embodiment, a composition comprising a phorbol ester and/or a phorbol ester derivative may alter the release of interferon in a mammalian subject. In yet another embodiment, a composition comprising a phorbol ester and/or a phorbol ester derivative may alter the rate of phosphorylation of ERK.

In yet another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the prevention and treatment of stroke and stroke-induced damage. The effects of stroke, including but not limited to paralysis, spatial disorders, impaired judgment, left-sided ignorance, memory loss, aphasia, coordination and balance problems, nausea, vomiting, cognitive disorders, perceptual disorders, disorientation, ipsilateral hemianopia, and impulsiveness, can be prevented or treated by using phorbol esters and phorbol ester derivatives as described herein.

In yet another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the treatment of rheumatoid arthritis. Rheumatoid arthritis symptoms that may be prevented or treated by phorbol esters and phorbol ester derivatives as described herein include, but are not limited to, joint soreness, morning stiffness, hard bumps in the tissue beneath the arm skin, fatigue, lack of energy, loss of appetite, low fever, or muscle and joint pain.

In other embodiments, phorbol esters and phorbol ester derivatives as described herein may be used to treat prostatic hypertrophy. The compositions and methods as described herein may be used to prevent or treat symptoms of prostate hypertrophy including, but not limited to, end-of-urination dribbling, urinary retention, incomplete bladder emptying, urinary incontinence, excessive urinary frequency, painful urination, hematuria, delayed urination, wasted urination, weak urine flow or a strong and sudden desire to urinate.

In yet another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the treatment of renal disease.

In another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the treatment of urinary incontinence.

In another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the treatment of uterine fibroids.

In another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the treatment of dementia.

In another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the treatment of diabetes.

In one embodiment, phorbol esters and phorbol ester derivatives as described herein may be used to reduce visible signs of aging in an individual.

In another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used to reduce periocular swelling.

In yet another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used to treat autoimmune disorders, including but not limited to myasthenia gravis. Myasthenia gravis symptoms that may be prevented or treated by the use of the compositions and methods described herein include, but are not limited to, ptosis, diplopia, speech impairment, fatiguability, myasthenia, dysphagia, or dysarthria.

In another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the treatment and prevention of central nervous system disorders, such as parkinson's disease. Parkinson's disease symptoms that can be treated or prevented by using the compositions and methods described herein include, but are not limited to, resting tremor, stiffness, bradykinesia, rigidity, speech disorders, cognitive disorders, dementia, mood disorders, drowsiness, insomnia, and postural instability.

In yet another embodiment, phorbol esters and phorbol ester derivatives as described herein may be used for the treatment and prevention of carpal tunnel syndrome.

The present invention achieves the above aspects and meets additional objects and advantages by providing novel and very effective methods and compositions for modulating cell signaling pathways and/or treating diseases and symptoms of diseases or conditions using compositions containing phorbol esters of formula I below or derivative compositions:

wherein R is₁And R₂Can be hydrogen;an alkyl group, wherein the alkyl group contains 1 to 15 carbon atoms;lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof and R₃Can be hydrogen orLower alkenyl and substituted derivatives thereof.

In some embodiments, R₁And R₂At least one of which is different from hydrogen and R₃Is hydrogen orLower alkenyl and substituted derivatives thereof. In another embodiment, R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

The alkyl, alkenyl, phenyl and benzyl groups of the formulae herein may be unsubstituted or substituted with halogen, preferably, chloro, fluoro or bromo; a nitro group; an amino group; and/or similar types of radical substitution.

In yet another embodiment, the present invention achieves these objects and meets additional objects and advantages by providing novel and very effective methods and compositions for modulating cell signaling pathways and/or treating diseases or disease-related conditions using exemplary phorbol ester compositions, such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) of formula II below:

phorbol esters and related compounds and derivatives useful in the formulations and methods of the present invention include, but are not limited to, other pharmaceutically acceptable active salts of the compounds as well as active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates, and/or prodrugs of the compounds. Exemplary forms of phorbol esters useful in the compositions and methods of the present 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-dicaprate; phorbol 12, 13-dihexanoate; phorbol 12, 13-dipropionate; phorbol 12-myristate; phorbol 13-myristate; phorbol 12-myristate-13-acetate (also known as TPA or PMA); 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-cisianoate 13-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; and phorbol 13-acetate.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, subjects with HIV and AIDS, as well as subjects with HIV-and AIDS-related symptoms or secondary or opportunistic diseases (such as oral ulceration, fatigue, cutaneous thrush, fever, loss of appetite, diarrhea, aphthous ulcers, malabsorption, thrombocytopenia, weight loss, anemia, lymphadenectasis, Mycobacterium avium complex, salmonellosis, syphilis, neurosyphosis, Tuberculosis (TB), bacillary angiomatosis, aspergillosis, candidiasis, coccidioidomycosis, Listeria disease, pelvic inflammatory disease, Burkitt's lymphoma, cryptococcal meningitis, histoplasmosis, Kaposi's sarcoma, lymphoma, systemic non-Hodgkin's lymphoma (NHL), primary central nervous system lymphoma, cryptosporidiosis, meningitis, histoplasmosis, Kaposi's sarcoma, lymphoma, systemic non-Hodgkin's lymphoma, or opportunistic diseases, Isosporadic disease, microsporidiosis, pneumocystis disease (PCP), toxoplasmosis, Cytomegalovirus (CMV), hepatitis, herpes simplex, herpes zoster, human papilloma virus (HPV, genital warts, cervical cancer), molluscum contagiosum, Oral Hairy Leukoplakia (OHL), and Progressive Multifocal Leukoencephalopathy (PML)).

Other mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, subjects having neoplastic diseases, including malignant neoplastic diseases, such as solid and non-solid cancers. Non-solid cancers may include hematological malignancies/myeloid disorders, including but not limited to leukemia (including Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), chronic myeloid leukemia blast crisis), myelodysplasia, myeloproliferative syndromes. Solid cancers may include, but are not limited to, lymphoma (including hodgkin's and non-hodgkin's lymphomas), subcutaneous adenocarcinoma, ovarian teratocarcinoma, lung cancer; bone cancer; breast cancer; liver cancer; cancer; oral cancer; non-small cell lung cancer and prostate cancer.

Subjects suitable for treatment with phorbol esters of formula I, particularly TPA, further include subjects with symptoms of such neoplastic disease, such as, but not limited to, anemia; chronic fatigue; excessive or bleeding tendency, such as nasal, gingival and sub-dermal bleeding; easy contusion, especially contusion without obvious reasons; tachypnea; ecchymosis; repeatedly fever occurs; swelling of the gums; the cut heals slowly; bone and joint discomfort; repeated infection; weight loss; scratching; night sweat; lymph node swelling; fever is caused; abdominal pain and discomfort; visual impairment; cough; lack of appetite; chest pain; dysphagia; swelling of the face, neck and upper limbs; frequent urination is required, especially at night; difficulty in starting urination or holding back urine; weak or disrupted urine flow; painful urination or burning sensation; difficulty in erection; ejaculation pain; blood in urine or semen; recurrent pain or stiffness in the waist, hip or thigh; and weakness. In some embodiments, such cancers may be relapsed or refractory.

Other mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, subjects that experience side effects from chemotherapy or radiotherapy to treat neoplastic diseases, including malignant neoplastic diseases, such as solid and non-solid cancers. Such side effects include, but are not limited to, hair loss, nausea, vomiting, loss of appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, mouth ulcers, itchy skin, scaling, nerve and muscle damage, changes in hearing, weight loss, diarrhea, immunosuppression, bruising, heart damage, bleeding, liver damage, kidney damage, edema, aphtha and sore throats, infertility, fibrosis, hair loss and wet desquamation, dry mucosa, dizziness and encephalopathy.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods and compositions of the invention include, but are not limited to, individuals who have suffered a stroke. Subjects suitable for treatment with phorbol esters of formula I, particularly TPA, additionally include subjects who experience the effects of stroke (including, but not limited to, paralysis, steric hindrance, impaired judgment, left-sided ignorance, memory loss, aphasia, coordination and balance problems, nausea, vomiting, cognitive disorders, perceptual disorders, disorientation, ipsilateral hemianopsia, and impulsivity).

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals with rheumatoid arthritis. Rheumatoid arthritis symptoms that can be prevented or treated by phorbol esters of formula I, particularly TPA, include, but are not limited to, joint soreness, morning stiffness, hard bumps in the tissue beneath the arm's skin, fatigue, lack of energy, loss of appetite, low fever, or muscle and joint pain.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals with prostate hypertrophy. The compositions and methods as described herein may be used to prevent or treat symptoms of prostate hypertrophy including, but not limited to, end-of-urination dribbling, urinary retention, incomplete bladder emptying, urinary incontinence, excessive urinary frequency, painful urination, hematuria, delayed urination, wasted urination, weak urine flow or a strong and sudden desire to urinate.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals with renal disease.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals suffering from urinary incontinence.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals with visible signs of aging.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals with swelling around the eye.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals having uterine fibroids.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals suffering from dementia.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals with diabetes.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include those suffering from autoimmune disorders, including but not limited to myasthenia gravis. Myasthenia gravis symptoms that may be prevented or treated by the use of the compositions and methods described herein include, but are not limited to, ptosis, diplopia, speech impairment, fatiguability, myasthenia, dysphagia, or dysarthria.

Other mammalian subjects (including humans) suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include individuals with parkinson's disease. Parkinson's disease symptoms that can be treated or prevented by using the compositions and methods described herein include, but are not limited to, resting tremor, stiffness, bradykinesia, rigidity, speech disorders, cognitive disorders, dementia, mood disorders, drowsiness, insomnia, and postural instability.

In yet another embodiment, a mammalian subject having carpal tunnel syndrome can be treated with a phorbol ester of formula I, in particular TPA, according to the methods of the invention.

Such subjects are prophylactically and/or therapeutically effectively treated by administering to these and other subjects an effective amount of a phorbol ester of formula I sufficient to prevent or reduce viral load, reduce latent HIV reservoirs, increase immune reactivity, increase release of Th1 cytokine, prevent or reduce HIV and AIDS-related symptoms and conditions, reduce and/or eliminate neoplastic cells, increase white blood cell count, induce remission, maintain remission, prevent or reduce malignancy-related symptoms and conditions, increase ERK phosphorylation, reduce or eliminate radiation damage, boost the immune system, reduce nausea, reduce or prevent hair loss, increase appetite, reduce soreness, increase energy levels, relieve gastrointestinal pain, reduce bruise, eliminate oral ulceration, reduce or eliminate skin damage caused by radiation, increase or maintain neutrophil levels, Increasing or maintaining platelet levels, reducing edema, reducing or eliminating wet desquamation, preventing or treating paralysis, increasing spatial awareness, reducing memory loss, reducing aphasia, increasing coordination and balance, improving cognition, reducing or eliminating tremors, reducing or eliminating stiffness and rigidity, improving sleep quality, increasing stability, improving mobility, improving urinary control, improving appetite, reducing muscle or joint pain, improving vision, and/or improving muscle control.

The therapeutically useful methods and formulations of the present invention will effectively employ phorbol esters of formula I in a variety of forms, as described above, including any active pharmaceutically acceptable salts of the 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 other aspects of the invention, combination formulations and methods are provided that use an effective amount of a phorbol ester of formula I in combination with one or more secondary or auxiliary active agents formulated in combination or co-administered with the phorbol ester compound of formula I to produce an effective response in a subject.

Exemplary combination formulations and co-therapies use the phorbol ester compounds of formula I in combination with one or more other retroviruses for the treatment of HIV or AIDS, or secondary or adjunctive therapeutic agents as otherwise noted, in the treatment of viral cytopathic diseases such as HIV and AIDS. Such combination formulations and co-therapies may, for example, follow or be derived from various highly active antiretroviral treatment regimens (HAART regimens) and include regimens such as, but not limited to, two nucleoside analog reverse transcriptase inhibitors plus one or more protease inhibitors or non-nucleoside analog reverse transcriptase inhibitors, and other combinations. Other combination formulations and co-therapeutic methods may, for example, include therapeutic agents for opportunistic infections and compounds for HAART regimens. In these embodiments, the secondary or adjunctive therapeutic agents used in combination with a phorbol ester (e.g., TPA), alone or in combination with, for example, TPA, can have a direct or indirect antiviral effect; other useful adjunctive therapeutic activities may be exhibited in combination with a phorbol ester (e.g., TPA) (such as prevention of HIV, treatment of HIV, activation of HIV reservoirs, increase Th1 cytokine activity); or alone or in combination with a phorbol ester (e.g., TPA), can exhibit adjunctive therapeutic activity useful for the treatment of HIV-associated opportunistic infections.

Auxiliary therapeutic agents useful in these combination formulations and synergistic methods include, for example, protease inhibitors including but not limited to saquinavir (saquinavir), indinavir (indinavir), ritonavir (ritonavir), nefennavir (nelfinavir), azanavir (atazanavir), desrenavir (daunaravir), furamevir (fosamprenavir), tipranavir (tipranavir), and amprenavir (amprenavir), nucleoside reverse transcriptase inhibitors including but not limited to zidovudine (zidovudine), didanosine (didanosine), stavudine (stavudine), lamivudine (lamivudine), latrine (lamivudine), zacitabine (lamivudine), tenofovir fumarate (tenofovir disoproxil), adefovir (felbinavir), adefovir (fel), adefovir (adefovir), adefovir (I), adefovir (I), adefovir (adefovir), adefovir (I), adefovir (I), adefovir (I/or adefovir (I), adefovir (I/or (I), adefovir (I/or adefovir (I), adefovir (I), adefovir (I/or adefovir (I), adefovir (I/or (I), adefovir (I).

In the treatment of neoplastic diseases, exemplary combination preparations and co-therapies use the phorbol ester compounds of formula I in combination with one or more other secondary or adjunctive therapeutic agents that treat neoplastic diseases or are otherwise indicated. The secondary or adjunctive therapeutic agents used in combination with phorbol esters (e.g., TPA) in these embodiments, alone or in combination with, for example, TPA, can have direct or indirect chemotherapeutic effects; other useful adjunctive therapeutic activities may be exhibited in combination with phorbol esters (e.g., TPA) (such as cytotoxicity, anti-inflammation, inhibition of NF- κ B, induction of apoptosis, increased activity of Th1 cytokine); or may exhibit adjunctive therapeutic activity useful for treating neoplasms or associated symptoms, either alone or in combination with, for example, TPA.

Adjunctive or secondary therapeutic agents useful in the treatment of neoplastic diseases in these combined and synergistic formulations and methods of treatment include doxorubicin, vitamin D3, cytarabine (cytarabine), daunorubicin (daunorubicin), cyclophosphamide (cyclophosphamide), gemtuzumab ozogamicin (gemtuzumab ozogamicin), idarubicin, mercaptopurine (mercaptoprine), mitoxantrone (mitoxantrone), thioguanine (thioguanine), aclleukin, asparaginase, carboplatin (carboplatin), etoposide phosphate (etoposide phospate), fludarabine (fludarabine), methotrexate (methotrexate), etoposide, dexamethasone (dexamethasone) and choline magnesium trisalicylate. In addition, adjuvant or secondary therapies such as, but not limited to, radiation therapy, hormonal therapy, and surgery may be used.

Exemplary combination formulations and co-therapies use phorbol ester compounds of formula I in combination with one or more other chemoprotective or otherwise indicated secondary or adjunctive therapeutic agents in the prevention or treatment of side effects of chemotherapy. The secondary or adjunctive therapeutic agents used in combination with phorbol esters (e.g., TPA) in these embodiments may have direct or indirect chemoprotective effects, alone or in combination with phorbol esters (e.g., TPA); other useful adjunctive therapeutic activities may be exhibited in combination with phorbol esters (e.g., TPA) (such as anti-inflammatory, neutrophil stimulation, erythropoiesis stimulation, bone resorption inhibition, bone strengthening, antiemetic, pain relief); or may exhibit adjunctive therapeutic activity useful for the treatment or prevention of chemotherapy side effects or associated symptoms, either alone or in combination with a phorbol ester (e.g., TPA).

Adjunctive or secondary therapeutic agents useful in these combination formulations and co-therapies for preventing or treating the side effects of chemotherapy in a mammalian subject include, but are not limited to, pefilgrastim (pegfilgrastim), exemestane α (epoeitnafa), doypetin α, sodium alendronate (alendronate sodium), risedronate (risedronate), ibandronate (ibandronate), G-CSF, 5-HT₃Receptor antagonists, NK₁Antagonists, olanzapine (olanzapine), corticosteroids, dopamine (dopamin) antagonists, serotonin antagonists, benzodiazepines, aprepitant and cannabinoids (cannabinoids).

Exemplary combination formulations and co-therapies use phorbol ester compounds of formula I in combination with one or more other radioprotective or otherwise indicated secondary or adjunctive therapeutic agents in the prevention or treatment of side effects of radiation therapy as encompassed herein. In these embodiments, the secondary or adjunctive therapeutic agents used in combination with a phorbol ester (e.g., TPA), alone or in combination with a phorbol ester (e.g., TPA), may have direct or indirect radioprotective effects; other useful adjunctive therapeutic activities may be exhibited in combination with phorbol esters (e.g., TPA) (such as anti-swelling, cytoprotection, anti-mucositis, epithelial cell stimulation, anti-fibrosis, platelet stimulation); or may exhibit adjunctive therapeutic activity useful for the treatment or prevention of side effects of radiotherapy or associated symptoms, either alone or in combination with, for example, TPA.

Adjunctive or secondary therapeutic agents useful in these combination formulations and co-therapies for the prevention or treatment of side effects of radiation therapy in mammalian subjects include, but are not limited to, steroids, amifostine (amifostine), chlorhexidine (chlorexidine), benzydamine (benzydamine), sucralfate (sucralfate), Keratinocyte Growth Factor (KGF), palifermin (palifermin), Cu/Zn superoxide dismutase, interleukin 11, or prostaglandins.

In the treatment of stroke, exemplary combination formulations and co-therapies use a phorbol ester compound of formula I in combination with one or more other prophylactic, therapeutic or otherwise indicated secondary or adjunctive therapeutic agents. The secondary or adjunctive therapeutic agents used in combination with a phorbol ester (e.g., TPA) in these embodiments, alone or in combination with the phorbol ester (e.g., TPA), may have a direct or indirect effect on the prevention or recovery of stroke; may exhibit other useful adjunctive therapeutic activities in combination with, for example, TPA (such as anticoagulant, anticholesterolaemia, vasodilation, antihypertensive); or may exhibit adjunctive therapeutic activity useful for the treatment or prevention of stroke or related symptoms, either alone or in combination with, for example, TPA.

In addition, adjunctive or secondary therapies such as, but not limited to, carotid endarterectomy, angioplasty, stenting, craniotomy, endovascular coil embolization, or patent foramen ovale occlusion may be used.

In the treatment of Parkinson's disease, exemplary combination formulations and co-therapeutic methods employ the phorbol ester compounds of formula I in combination with one or more other secondary or adjunctive therapeutic agents that treat Parkinson's disease or are otherwise indicated. The secondary or adjunctive therapeutic agents used in combination with phorbol esters (e.g., TPA) in these embodiments, alone or in combination with, for example, TPA, can have direct or indirect anti-parkinson's disease effects; other useful adjunctive therapeutic activities may be exhibited in combination with, for example, TPA (increase in dopamine, inhibition of catechol-O-methyltransferase, inhibition of aromatic L-amino acid decarboxylase, dopamine agonists, neuroprotection, anticholinergic); or may exhibit adjunctive therapeutic activity useful for treating or preventing chemotherapy side effects or associated symptoms, either alone or in combination with, for example, TPA.

Adjunctive or secondary therapeutic agents useful for preventing or treating the symptoms of parkinson's disease in a mammalian subject in these combination formulations and co-therapies include, but are not limited to, levodopa (levodopa), tolcapone (tolcapone), carbidopa, dopamine agonists, MAO-B inhibitors, pyridoxine (pyridoxine), amantadine (amantidine), pyridoxine, selegiline (seleyiline), rasagiline (rasagiline), or anticholinergics. In addition, adjuvant or secondary therapies may be used, such as, but not limited to, deep brain stimulation or surgery for lesion formation.

In these embodiments, the secondary or adjunctive therapeutic agents used in combination with a phorbol ester (e.g., TPA) may have direct or indirect effects, may exhibit other useful adjunctive therapeutic activities in combination with, for example, TPA (type II 5- α reductase inhibitors, muscle relaxants), or may exhibit adjunctive therapeutic activities useful for treating or preventing prostatic hypertrophy or associated symptoms, either alone or in combination with, for example, TPA.

Adjunctive or secondary therapeutic agents useful for preventing or treating prostatic hypertrophy in mammalian subjects in these combination formulations and synergistic treatment methods include, but are not limited to, finasteride, dutasteride, terazosin, doxazosin, tamsulosin, or α blockers in addition, adjunctive or secondary therapies such as, but not limited to, transurethral prostatectomy, laser surgery, or prostatectomy may be used.

In the treatment of rheumatoid arthritis, exemplary combination formulations and co-therapies use a phorbol ester compound of formula I in combination with one or more other therapeutic agents for the treatment of rheumatoid arthritis or secondary or adjunctive therapeutic agents as otherwise noted. The secondary or adjunctive therapeutic agents used in combination with phorbol esters (e.g., TPA) in these embodiments may have direct or indirect effects, alone or in combination with phorbol esters (e.g., TPA); other useful adjunctive therapeutic activities may be exhibited in combination with a phorbol ester (e.g., TPA) (such as anti-inflammatory, immunosuppressive, TNF-suppressing, antibiotic, calcineurin inhibitor, pyrimidine synthesis inhibitor, 5-LO inhibitor, antifolate, IL-1 receptor antagonist, T cell costimulation inhibitor); or may exhibit adjunctive therapeutic activity useful for the treatment or prevention of rheumatoid arthritis or associated symptoms, either alone or in combination with, for example, TPA.

Adjunctive or secondary therapeutic agents useful in these combination formulations and co-therapies for the prevention or treatment of rheumatoid arthritis in a mammalian subject include, but are not limited to, non-steroidal anti-inflammatory agents, steroids, disease modifying antirheumatics, immunosuppressants, TNF- α inhibitors, anakinra (anakinra), abacatet (abetacept), adalimumab (adalimumab), azathioprine (azathioprine), chloroquine (chloroquine), hydroxychloroquine (hydroxychloroquine), cyclosporine (ciclosporin), D-penicillamine (D-penicillamine), etanercept (etanercept), golimumab (golimumab), aurantium salts, infliximab, leflunomide (leflunomide), methotrexate, minocycline (minocycline), sulfadiazine (sulfasalazine), rituximab (rituximab) or salituzumab.

In the treatment of autoimmune disorders, exemplary combination formulations and co-therapies use phorbol ester compounds of formula I in combination with one or more other secondary or adjunctive therapeutic agents that treat autoimmune diseases or are otherwise indicated. The secondary or adjunctive therapeutic agents used in combination with a phorbol ester (e.g., TPA) in these embodiments, alone or in combination with the phorbol ester (e.g., TPA), may have direct or indirect effects; other useful adjunctive therapeutic activities may be exhibited in combination with the phorbol ester (e.g., TPA) (such as immunosuppression, inhibitory antibodies, anticholinesterase); or alone or in combination with the phorbol ester (e.g., TPA), can exhibit adjunctive therapeutic activity useful for the treatment or prevention of autoimmune disorders, including myasthenia gravis, or related symptoms.

Adjunctive or secondary therapeutic agents useful for preventing or treating an autoimmune disorder in a mammalian subject in these combined preparations and co-therapeutic methods include, but are not limited to, anticholinesterases, corticosteroids, or immunosuppressants.

In the treatment of renal disease, exemplary combination formulations and co-therapies use the phorbol ester compounds of formula I in combination with one or more other secondary or adjunctive therapeutic agents that treat or otherwise indicate renal disease. Secondary or adjunctive therapeutic agents used in combination with, for example, TPA in these embodiments may have direct or indirect effects alone or in combination with, for example, TPA, and may exhibit other useful adjunctive therapeutic activities in combination with, for example, TPA (such as anticholinergic, antidepressant); or may exhibit adjunctive therapeutic activity useful for the treatment or prevention of renal disease or associated symptoms, either alone or in combination with, for example, TPA.

Adjunctive or secondary therapeutic agents useful for preventing or treating kidney disease in a mammalian subject in such combination formulations and co-therapeutic methods include, but are not limited to, anticholinergics, topical estrogens, imipramine (imipramine) or duloxetine (duloxetine).

In the treatment of urinary incontinence, exemplary combination formulations and co-therapies use the phorbol ester compounds of formula I in combination with one or more other secondary or adjunctive therapeutic agents that treat urinary incontinence or are otherwise indicated. Secondary or adjunctive therapeutic agents used in combination with, for example, TPA in these embodiments may have direct or indirect effects alone or in combination with, for example, TPA, and may exhibit other useful adjunctive therapeutic activities in combination with, for example, TPA (such as anticholinergic, antidepressant); or may exhibit adjunctive therapeutic activity useful for the treatment or prevention of urinary incontinence or related symptoms, either alone or in combination with, for example, TPA.

Adjunctive or secondary therapeutic agents useful for the prevention or treatment of urinary incontinence in mammalian subjects in these combined preparations and synergistic methods of treatment include, but are not limited to, anticholinergics, topical estrogens, imipramine, or duloxetine.

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

The present invention also relates to the following items.

1. A method of treating or preventing neoplasms in a mammalian subject comprising administering to the subject an effective amount of a phorbol ester or derivative of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Selected from hydrogen,Lower alkenyl and substituted derivatives thereof; and a combination preparation or a synergistic therapeutic regimen with said phorbol ester or derivative compound of formula IAt least one secondary or adjunctive therapeutic agent effective to treat or prevent a neoplasm in the subject.

2. The method of clause 1, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

3. The method of clause 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

4. The method of clause 1, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

5. The method of clause 1, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

6. The method of clause 1, wherein the at least one secondary or adjunctive therapeutic agent is selected from the group consisting of: doxorubicin, vitamin D3, cytarabine, daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide, dexamethasone, and choline magnesium trisalicylate.

7. The method of clause 1, wherein two secondary or adjunctive therapeutic agents are administered to the subject.

8. The method of clause 7, wherein the two secondary or adjunctive therapeutic agents are dexamethasone and choline magnesium trisalicylate.

9. The method of clause 1, wherein the effective amount comprises between about 10 μ g and 1500 μ g of the phorbol ester or derivative compound of formula I per day.

10. The method of clause 1, wherein the effective amount comprises between about 150 to 500 μ g of the phorbol ester or derivative compound of formula I per day.

11. The method of item 1, wherein the neoplasm is caused by a hematological malignancy/bone marrow disorder.

12. The method of clause 11, wherein the hematological malignancy/bone marrow disorder is leukemia.

13. The method of item 12, wherein the leukemia is acute myeloid leukemia.

14. The method of clause 1, wherein the neoplasm is a solid tumor.

15. The method of clause 1, wherein the neoplasm is a relapsed neoplasm.

16. The method of clause 1, wherein the neoplasm is refractory.

17. A method of preventing or treating one or more neoplastic disease symptoms or conditions in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof; and at least one secondary or adjunctive therapeutic agent effective to treat or prevent symptoms of the neoplastic disease in the subject in a combined formulation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound.

18. The method of clause 17, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

19. The method of clause 17, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

20. The method of clause 17, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

21. The method of clause 20, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

22. The method of clause 20, wherein the at least one secondary or adjunctive therapeutic agent is selected from the group consisting of: doxorubicin, vitamin D3, cytarabine, daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide, dexamethasone, and choline magnesium trisalicylate.

23. The method of item 20, wherein the one or more neoplastic disease symptom or condition is anemia, chronic fatigue, excessive or easy bleeding, contusion, shortness of breath, ecchymosis, repeated fever, swollen gums, slow healing of cuts, bone and joint discomfort, repeated infection, weight loss, itching, night sweats, swollen lymph nodes, fever, abdominal pain and discomfort, visual impairment, cough, lack of appetite, chest pain, difficulty swallowing, swelling, need for frequent urination, difficulty in starting to urinate, difficulty holding urine, weak or interrupted urinary flow, pain or burning in urination, difficulty in erection, pain in ejaculation, blood in urine or semen, recurrent pain or stiffness, or weakness.

24. A method of inducing remission in a mammalian subject having a neoplastic disease, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof; r₃Is hydrogen,Lower alkenyl and substituted derivatives thereof; and at least one secondary or adjunctive therapeutic agent effective to induce remission in the subject in combination formulation or co-therapy regimen with the phorbol ester of formula I or derivative compound.

25. The method of clause 24, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃At least one secondary or adjunctive therapeutic agent that is hydrogen and a combination formulation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound effective to induce remission in the subject.

26. The method of clause 24, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

27. The method of clause 24, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

28. The method of clause 24, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, before, or after the phorbol ester is administered to the subject.

29. The method of clause 24, wherein the at least one secondary or adjunctive therapeutic agent is selected from the group consisting of: doxorubicin, vitamin D3, cytarabine, daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide, dexamethasone, and choline magnesium trisalicylate.

30. A method of inducing neoplastic apoptosis in a mammalian subject having a neoplastic disease, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof; and at least one secondary or adjunctive therapeutic agent effective to induce apoptosis of a neoplasm in the subject in combination formulation or co-treatment regimen with the phorbol ester of formula I or derivative compound.

31. The method of clause 30, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃At least one secondary or adjunctive therapeutic agent that is hydrogen and a combination formulation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound effective to treat or prevent a malignancy in the subject.

32. The method of clause 30, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

33. The method of clause 30, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

34. The method of clause 30, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

35. The method of clause 34, wherein the at least one secondary or adjunctive therapeutic agent is selected from the group consisting of: doxorubicin, vitamin D3, cytarabine, daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide, dexamethasone, and choline magnesium trisalicylate.

36. A composition for preventing or treating a neoplastic disease in a mammalian subject comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof; and at least one secondary or adjunctive therapeutic agent effective to treat or prevent a neoplasm in the subject.

37. The composition of clause 36, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

38. The composition of clause 36, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

39. The composition of clause 36, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

40. The composition of clause 36, wherein the at least one secondary or adjunctive therapeutic agent is selected from the group consisting of: doxorubicin, vitamin D3, cytarabine, daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide, dexamethasone, and choline magnesium trisalicylate.

41. The composition of clause 36, wherein the composition contains at least two secondary or adjunctive therapeutic agents.

42. The composition of clause 41, wherein the at least two secondary or adjunctive therapeutic agents are dexamethasone and choline magnesium trisalicylate.

43. A method of preventing or treating one or more side effects of chemotherapy in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

44. The method of clause 43, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

45. The method of clause 43, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

46. The method of clause 43, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

47. The method of clause 43, further comprising administering to the subject at least one secondary or adjunctive therapeutic agent effective to treat or prevent a chemotherapy treatment side effect in a combination formulation or synergistic therapeutic regimen with the phorbol ester of formula I or a derivative compound.

48. The method of clause 47, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

49. The method of clause 48, wherein the at least one secondary or adjunctive therapeutic agent is selected from the group consisting of pefilgrastim, eltamitan α, dyliprotene α, alendronate sodium, risedronate, ibandronate, G-CSF, 5-HT3 receptor antagonists, NK1 antagonists, olanzapine, corticosteroids, dopamine antagonists, serotonin antagonists, benzodiazepines, aprepitant, and cannabinoids.

50. The method of clause 43, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

51. The method of clause 50, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

52. The method of item 43, wherein the one or more chemotherapy treatment side effects is alopecia, nausea, vomiting, loss of appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, mouth ulcers, itchy skin, scaling, nerve and muscle leprosy, changes in hearing, weight loss, diarrhea, immunosuppression, bruises, a tendency to bleed easily, heart damage, liver damage, kidney damage, dizziness, or encephalopathy.

53. A composition for preventing or treating one or more side effects of chemotherapy in a mammalian subject, comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenesBase ofPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

54. The composition of clause 53, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

55. The composition of clause 53, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-butyrate, phorbol 12-didecanoate, phorbol 12-dipropionate, and phorbol 12-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

56. The composition of clause 53, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

57. The composition of clause 53, further comprising at least one secondary or adjunctive therapeutic agent effective to treat or prevent a chemotherapy side effect in the subject in combination with the phorbol ester of formula I or derivative compound.

58. The composition of clause 57, wherein the at least one secondary or adjunctive therapeutic agent is pefilgrastim, eletriptan α, darbepoetin α, alendronate, risedronate, ibandronate, G-CSF, a 5-HT3 receptor antagonist, an NK1 antagonist, olanzapine, a corticosteroid, a dopamine antagonist, a serotonin antagonist, a benzodiazepine, aprepitant, or a cannabinoid.

59. The composition of item 53, wherein the one or more chemotherapy treatment side effects is alopecia, nausea, vomiting, loss of appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, mouth ulcers, itchy skin, scaling, nerve and muscle leprosy, changes in hearing, weight loss, diarrhea, immunosuppression, bruises, a tendency to bleed easily, heart damage, liver damage, kidney damage, dizziness, or encephalopathy.

60. A method of preventing or treating one or more side effects of radiation therapy in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

61. The method of clause 60, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

62. The method of clause 60, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

63. The method of clause 60, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

64. The method of clause 60, further comprising administering to the subject at least one secondary or adjunctive therapeutic agent effective to treat or prevent a side effect of radiation therapy in a combined preparation or synergistic therapeutic regimen with the phorbol ester of formula I or a derivative compound.

65. The method of clause 64, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

66. The method of clause 65, wherein the at least one secondary or adjunctive therapeutic agent is selected from the group consisting of steroids, amifostine, chlorhexidine, benzydamine, sucralfate, KGF, palifermin, Cu/Zn superoxide dismutase, interleukin 11, or prostaglandins.

67. The method of clause 60, wherein the one or more side effects of radiation therapy are wet desquamation, soreness, diarrhea, nausea, vomiting, loss of appetite, constipation, itchy skin, scaling, canker sores and laryngitis, edema, infertility, fibrosis, hair loss, or dry mucous membranes.

68. The method of clause 60, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

69. The method of clause 60, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

70. A composition for preventing or treating one or more side effects of radiation therapy in a mammalian subject comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

71. The composition of clause 70, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

72. The composition of clause 70, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-butyrate, phorbol 12-didecanoate, phorbol 12-dipropionate, and phorbol 12-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

73. The composition of clause 70, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

74. The composition of clause 70, further comprising at least one secondary or adjunctive therapeutic agent effective to treat or prevent a radiation therapy side effect in the subject in combination with the phorbol ester of formula I or a derivative compound.

75. The composition of clause 70, wherein the one or more secondary or adjunctive therapeutic agents is a steroid, amifostine, chlorhexidine, benzydamine, sucralfate, KGF, palivumin, Cu/Zn superoxide dismutase, interleukin 11, or a prostaglandin.

76. The composition of item 70, wherein the one or more radiation therapy side effects is wet desquamation, soreness, diarrhea, nausea, vomiting, loss of appetite, constipation, itchy skin, scaling, canker sores and laryngitis, edema, infertility, fibrosis, hair loss, or dry mucous membranes.

77. A method of preventing or treating the effects of one or more strokes in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

78. The method of clause 77, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

79. The method of clause 77, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

80. The method of clause 77, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

81. The method of clause 77, further comprising administering at least one secondary or adjunctive therapeutic agent effective to treat or prevent the effects of stroke in the subject in a combination formulation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound.

82. The method of clause 81, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or after the phorbol ester is administered to the subject.

83. The method of item 81, wherein the at least one secondary or adjunctive therapeutic agent is a tissue plasminogen activator, anticoagulant, statin, angiotensin II receptor blocker, angiotensin converting enzyme inhibitor, β -blocker, calcium channel blocker, or diuretic.

84. The method of clause 77, further comprising surgical intervention in combination with a phorbol ester of formula I or a derivative compound to treat or prevent the effects of stroke in the subject.

85. The method of item 84, wherein the surgical intervention is carotid endarterectomy, angioplasty, stenting, craniotomy, endovascular coil embolization, or patent foramen ovale occlusion.

86. The method of item 77, wherein the one or more stroke effects is paralysis, spatial impairment, impaired judgment, left-sided ignorance, memory loss, aphasia, problems with coordination and balance, nausea, vomiting, cognitive impairment, perceptual impairment, disorientation, ipsilateral hemianopia, or impulsivity.

87. The method of clause 77, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

88. The method of clause 87, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

89. A composition for preventing or treating the effects of one or more strokes in a mammalian subject comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

90. The composition of clause 89, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

91. The composition of clause 90, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-butyrate, phorbol 12-didecanoate, phorbol 12-dipropionate, and phorbol 12-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

92. The composition of clause 90, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

93. The composition of clause 90, further comprising at least one secondary or adjunctive therapeutic agent effective to treat or prevent the effects of stroke in the subject in combination with the phorbol ester of formula I or derivative compound.

94. The composition of clause 90, wherein the at least one secondary or adjunctive therapeutic agent is a tissue plasminogen activator, anticoagulant, statin, angiotensin II receptor blocker, angiotensin converting enzyme inhibitor, β -blocker, calcium channel blocker, or diuretic.

95. The composition of clause 90, wherein the one or more stroke side effects is paralysis, a spatial disorder, impaired judgment, left-sided ignorance, memory loss, aphasia, problems with coordination and balance, nausea, vomiting, cognitive disorders, perceptual disorders, disorientation, ipsilateral hemianopsia, or impulsivity.

96. A method of treating or preventing one or more symptoms of Parkinson's disease in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R1 and R2 are selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R3 is hydrogen,Lower alkenyl and substituted derivatives thereof.

97. The method of clause 96, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

98. The method of clause 96, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

99. The method of clause 96, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

100. The method of clause 96, further comprising administering at least one secondary or adjunctive therapeutic agent effective to treat or prevent one or more symptoms of parkinson's disease in a combined preparation or synergistic therapeutic regimen with the phorbol ester of formula I or derivative compound.

101. The method of clause 100, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

102. The method of clause 100, wherein the at least one secondary or adjunctive therapeutic agent is levodopa, carbidopa, pyridoxine, selegiline, rasagiline, tolcapone, a dopamine agonist, a MAO-B inhibitor, amantadine, or an anticholinergic.

103. The method of clause 96, further comprising surgical intervention in combination with a phorbol ester of formula I or a derivative compound to treat or prevent symptoms of parkinson's disease in the subject.

104. The method of item 103, wherein the surgical intervention is deep brain stimulation or surgery for lesion formation.

105. The method of clause 96, wherein the one or more symptoms of parkinson's disease are resting tremor, stiffness, bradykinesia, rigidity, speech disorders, cognitive disorders, dementia, mood disorders, drowsiness, insomnia, and postural instability.

106. The method of clause 96, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

107. The method of clause 96, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

108. A composition for preventing or treating one or more symptoms of Parkinson's disease in a mammalian subject, comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkyl and substituted derivatives thereof.

109. The composition of clause 108, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

110. The composition of clause 108, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-butyrate, phorbol 12-didecanoate, phorbol 12-dipropionate, and phorbol 12-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

111. The composition of clause 108, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

112. The composition of clause 108, further comprising at least one secondary or adjunctive therapeutic agent effective to treat or prevent the effects of stroke in the subject in combination with the phorbol ester of formula I or derivative compound.

113. The composition of clause 112, wherein the at least one secondary or adjunctive therapeutic agent is levodopa, carbidopa, pyridoxine, selegiline, rasagiline, tolcapone, a dopamine agonist, a MAO-B inhibitor, amantadine, or an anticholinergic.

114. The composition of item 108, wherein the one or more symptoms of parkinson's disease are resting tremor, stiffness, bradykinesia, rigidity, speech disorders, cognitive disorders, dementia, mood disorders, drowsiness, insomnia, or postural instability.

115. A method of treating or preventing one or more symptoms of prostate hypertrophy in a mammalian subject comprising administering to said subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and its derivativesGroup of substituted derivatives, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

116. The method of clause 115, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

117. The method of clause 115, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

118. The method of clause 115, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

119. The method of clause 115, further comprising administering at least one secondary or adjunctive therapeutic agent effective to treat or prevent one or more symptoms of prostate hypertrophy in a combined formulation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound.

120. The method of clause 119, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or after the phorbol ester is administered to the subject.

121. The method of clause 119, wherein the at least one secondary or adjunctive therapeutic agent is finasteride, dutasteride, terazosin, doxazosin, tamsulosin, or an α blocking agent.

122. The method of clause 115, further comprising surgical intervention in combination with a phorbol ester of formula I or a derivative compound to treat or prevent symptoms of prostate hypertrophy disease in said subject.

123. The method of clause 122, wherein the surgical intervention is a transurethral prostatectomy, laser surgery, or prostatectomy.

124. The method of clause 115, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

125. The method of clause 115, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

126. The method of item 115, wherein the one or more symptoms of prostate hypertrophy is end of urination dribble, urinary retention, incomplete bladder emptying, urinary incontinence, urinary frequency, painful urination, hematuria, slow or delayed urination, intermittent urine flow, wasted effort to urinate, weak urine flow, or an intense and sudden desire to urinate.

127. A composition for preventing or treating one or more symptoms of prostate hypertrophy in a mammalian subject comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

128. The composition of clause 127, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

129. The composition of clause 127, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

130. The composition of clause 127, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

131. The composition of clause 127, further comprising at least one secondary or adjunctive therapeutic agent effective for the treatment or prevention of symptoms of prostate hypertrophy as a combined preparation with the phorbol ester of formula I or a derivative compound.

132. The composition of clause 131, wherein the at least one secondary or adjunctive therapeutic agent is finasteride, dutasteride, terazosin, doxazosin, tamsulosin, or an α blocking agent.

133. The composition of item 131, wherein the one or more symptoms of prostate hypertrophy is dribbling at the end of urination, urinary retention, incomplete emptying of the bladder, urinary incontinence, urinary frequency, painful urination, hematuria, slow or delayed urination, intermittent urine flow, wasted effort to urinate, weak urine flow, or a strong and sudden desire to urinate.

134. A method of treating or preventing one or more rheumatoid arthritis symptoms in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkyl and substituted derivatives thereof.

135. The method of clause 134, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

136. The method of clause 134, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

137. The method of clause 134, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

138. The method of clause 134, further comprising administering at least one secondary or adjunctive therapeutic agent effective to treat or prevent one or more rheumatoid arthritis symptoms in a combination formulation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound.

139. The method of clause 138, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

140. The method of item 139, wherein the at least one secondary or adjunctive therapeutic agent is a non-steroidal anti-inflammatory agent, a steroid, a disease-modifying antirheumatic, an immunosuppressive agent, a TNF- α inhibitor, adalimumab, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, D-penicillamine, etanercept, golimumab, gold salts, infliximab, leflunomide, methotrexate, minocycline, sulfasalazine

Anakinra, abamectin, rituximab or tositumomab.

141. The method of item 134, wherein the one or more rheumatoid arthritis symptoms is joint soreness, morning stiffness, hard bumps in the tissue under the arm skin, fatigue, lack of energy, loss of appetite, low fever, or muscle and joint pain.

142. The method of clause 134, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

143. The method of clause 134, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

144. A composition for preventing or treating one or more symptoms of rheumatoid arthritis in a mammalian subject comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

145. The composition of clause 144, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

146. The composition of clause 144, 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-didecanate, phorbol 12, 13-dihexanate, phorbol 12, 13-dipropionate, phorbol 12-myristate, phorbol 13-myristate, phorbol 12,13, 20-triacetate, 12-deoxyphorbol 13-angelate 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-butyrate, phorbol 12-didecanoate, phorbol 12-dipropionate, and phorbol 12-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

147. The composition of clause 144, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

148. The composition of clause 144, further comprising at least one secondary or adjunctive therapeutic agent effective for the treatment or prevention of rheumatoid arthritis symptoms in a combined preparation with the phorbol ester of formula I or a derivative compound.

149. The composition of item 148, wherein the at least one secondary or adjunctive therapeutic agent is a non-steroidal anti-inflammatory agent, a steroid, a disease-modifying antirheumatic, an immunosuppressive agent, a TNF- α inhibitor, anakinra, abamectin, adalimumab, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, D-penicillamine, etanercept, golimumab, gold salts, infliximab, leflunomide, methotrexate, minocycline, sulfasalazine, rituximab, or tositumomab.

150. The composition of item 144, wherein the one or more rheumatoid arthritis symptoms are joint soreness, morning stiffness, hard bumps in the tissue under the arm skin, fatigue, lack of energy, lack of appetite, low fever, or muscle and joint pain.

151. A method of treating or preventing one or more myasthenia gravis symptoms in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

152. The method of item 151, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

153. The method of clause 151, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

154. The method of clause 151, further comprising administering at least one secondary or adjunctive therapeutic agent effective to treat or prevent one or more severe myasthenia gravis symptoms in a combined formulation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound.

155. The method of clause 154, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

156. The method of clause 154, wherein the at least one secondary or adjunctive therapeutic agent is an anticholinesterase, corticosteroid, or immunosuppressant.

157. The method of item 151, wherein the one or more myasthenia gravis symptoms is ptosis, diplopia, speech impairment, fatigability, myasthenia, dysphagia, or dysarthria.

158. The method of clause 151, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

159. The method of clause 151, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

160. A composition for preventing or treating one or more myasthenia gravis symptoms in a mammalian subject, comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

161. The composition of clause 160, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

162. The composition of clause 160, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

163. The composition of clause 160, further comprising at least one secondary or adjunctive therapeutic agent effective to treat or prevent the myasthenia gravis symptom in combination with a formulation of the phorbol ester of formula I or a derivative compound.

164. The composition of item 163, wherein the at least one secondary or adjunctive therapeutic agent is an anticholinesterase, corticosteroid, or immunosuppressant.

165. The composition of item 160, wherein the one or more myasthenia gravis symptoms is ptosis, diplopia, speech impairment, fatigability, myasthenia, dysphagia, or dysarthria.

166. A method of treating or preventing symptoms of a kidney disease in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

167. The method of clause 166, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

168. The method of clause 166, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

169. The method of clause 166, further comprising administering at least one secondary or adjunctive therapeutic agent effective to treat or prevent a symptom of renal disease in a combined preparation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound.

170. The method of clause 169, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or after the phorbol ester is administered to the subject.

171. The method of clause 169, wherein the at least one secondary or adjunctive therapeutic agent is an anticholinergic, topical estrogen, imipramine, or duloxetine.

172. The method of item 166, wherein the nephrotic symptom is urinary incontinence, increased urinary frequency, uremia, or oliguria.

173. The method of clause 166, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

174. The method of clause 166, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

175. A composition for preventing or treating kidney disease in a mammalian subject, comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkyl and substituted derivatives thereof.

176. The composition of clause 175, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

177. The composition of clause 175, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

178. The composition of clause 175, further comprising at least one secondary or adjunctive therapeutic agent effective for the treatment or prevention of a symptom of renal disease formulated in combination with the phorbol ester of formula I or a derivative compound.

179. The composition of clause 178, wherein the at least one secondary or adjunctive therapeutic agent is an anticholinergic, topical estrogen, imipramine, or duloxetine.

180. The composition of item 175, wherein the nephrotic symptom is urinary incontinence, increased frequency of urination, uremia, or oliguria.

181. A method of treating or preventing urinary incontinence in a mammalian subject, comprising administering to the subject an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

182. The method of clause 181, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

183. The method of clause 181, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

184. The method of clause 181, further comprising administering at least one secondary or adjunctive therapeutic agent effective to treat or prevent symptoms of urinary incontinence in a combined formulation or synergistic therapeutic regimen with the phorbol ester of formula I or derivative compound.

185. The method of clause 184, wherein in a co-administration regimen, the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or after the phorbol ester is administered to the subject.

186. The method of clause 184, wherein the at least one secondary or adjunctive therapeutic agent is an anticholinergic, topical estrogen, imipramine, or duloxetine.

187. The method of clause 181, wherein the effective amount comprises between about 10 μ g and about 1500 μ g of the phorbol ester or derivative compound of formula I per day.

188. The method of clause 181, wherein the effective amount comprises between about 125 μ g to about 500 μ g of the phorbol ester or derivative compound of formula I per day.

189. A composition for preventing or treating urinary incontinence in a mammalian subject, comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, R₃Is hydrogen,Lower alkenyl and substituted derivatives thereof.

190. The composition of clause 189, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

191. The composition of clause 189, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

192. The composition of clause 189, further comprising at least one secondary or adjunctive therapeutic agent effective for the treatment or prevention of urinary incontinence formulated in combination with the phorbol ester of formula I or a derivative compound.

193. The composition of clause 192, wherein the at least one secondary or adjunctive therapeutic agent is an anticholinergic, topical estrogen, imipramine, or duloxetine.

Detailed Description

Novel methods and compositions have been identified for use in the treatment of chronic or recurrent conditions, or for the repair of lesions left by disease onset or disease treatment in mammalian subjects, including humans.

In various embodiments, the methods and compositions are effective in preventing or treating HIV and AIDS and related conditions, diseases caused by HIV and AIDS, symptoms of HIV and AIDS, and/or diseases resulting from infection by HIV or AIDS. In other embodiments, the methods and compositions are effective in preventing or treating neoplastic diseases and the symptoms of such diseases. Such neoplastic diseases may or may not be malignant. In some embodiments, the neoplastic disease can be a solid or non-solid cancer. In other embodiments, the cancer may be refractory or relapsed. In other embodiments, the methods and compositions are effective to prevent or ameliorate damage or side effects of chemotherapeutic agents. In additional embodiments, the methods and compositions as described herein are effective in preventing or ameliorating the damage or side effects of radiation therapy. In other embodiments, the methods and compositions as described herein are effective in preventing or treating stroke damage. In other embodiments, the methods and compositions as described herein are effective in treating rheumatoid arthritis. In other embodiments, the methods and compositions as described herein are effective in reducing signs of aging. In another embodiment, the methods and compositions as described herein are effective in treating prostatic hypertrophy. In other embodiments, the methods and compositions as described herein are effective in treating autoimmune disorders. In additional embodiments, the methods and compositions as described herein are effective in treating urinary incontinence. In other embodiments, the methods and compositions as described herein are effective in treating kidney disease. In other embodiments, the methods and compositions as described herein are effective in treating parkinson's disease.

The compositions and methods as described herein can prevent or reduce viral load, reduce latent HIV reservoirs, increase immune reactivity, increase release of Th1 cytokine, prevent or reduce HIV and AIDS-related symptoms and conditions, reduce and/or eliminate neoplastic cells, increase white blood cell count, induce remission, maintain remission, prevent or reduce malignancy-related symptoms and conditions, increase ERK phosphorylation, reduce or eliminate radiation damage, boost the immune system, reduce nausea, reduce or prevent hair loss, increase appetite, reduce soreness, increase energy levels, relieve gastrointestinal pain, reduce bruising, eliminate canker sores, reduce or eliminate radiation-induced skin damage, increase or maintain neutrophil levels, increase or maintain platelet levels, reduce edema, reduce or eliminate desquamation, prevent or treat paralysis, increase spatial awareness, increase cancer immunity, decrease cancer immunity, increase cancer immunity, decrease cancer immunity, increase cancer immunity, decrease cancer, Reducing memory loss, reducing aphasia, increasing coordination and balance, improving cognition, reducing or eliminating tremors, reducing or eliminating stiffness and rigidity, improving sleep quality, increasing stability, improving mobility, improving urinary control, increasing urinary continence, improving appetite, reducing muscle or joint pain, improving vision and/or improving muscle control, and enhancing the immune system.

The formulations and methods provided herein use phorbol ester or derivative compounds of formula I, as described in more detail in U.S. patent application No. 12/023,753, filed on 31/1/2008, which claims priority to U.S. provisional patent application serial No. 60/898,810, filed on 31/1/2007, each of which is incorporated herein by reference in its entirety,

wherein R is₁And R₂Can be hydrogen;an alkyl group, wherein the alkyl group contains 1 to 15 carbon atoms;lower alkenylPhenyl radicalBenzyl and substituted derivatives thereof, and R₃Can be hydrogen orLower alkenyl, including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs and prodrugs of these compounds and combinations thereof as novel compounds for the treatment of HIV and AIDS.

The viral load reducing formulations and methods provided herein use phorbol ester or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily providable complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds and combinations thereof) as novel viral load reducing agents.

The formulations and methods of increasing immunoreactivity provided herein use phorbol ester or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds and combinations thereof) as immunostimulatory compounds.

The formulations and methods of increasing Th1 cytokines provided herein use phorbol esters or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily providable complexes, salts, solvates, isomers, enantiomers, polymorphs and prodrugs of these compounds and combinations thereof) as novel agents that increase Th1 cytokines.

The formulations and methods provided herein also use phorbol esters or derivative compounds of formula I above, including all active pharmaceutically acceptable compounds of the present specification, as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof, in the treatment of neoplastic diseases.

The formulations and methods of inducing apoptosis provided herein use phorbol esters or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds and combinations thereof) as chemotherapeutic agents for inducing apoptosis in neoplasms.

The relief-inducing formulations and methods provided herein use phorbol esters or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds and combinations thereof) as antineoplastic agents.

The formulations and methods provided herein also use phorbol ester or derivative compounds of formula I above, including all active pharmaceutically acceptable compounds of the present specification, as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof, in the prevention or treatment of chemotherapy side effects.

The formulations and methods provided herein also use phorbol ester or derivative compounds of formula I above, including all active pharmaceutically acceptable compounds of the present specification, as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof, in the prevention or treatment of side effects of radiation therapy.

The formulations and methods for treating stroke provided herein use phorbol ester or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds and combinations thereof) as anti-stroke agents.

The formulations and methods for treating rheumatoid arthritis provided herein use phorbol ester or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds and combinations thereof) as anti-rheumatoid agents.

The anti-parkinson's disease formulations and methods provided herein use phorbol esters or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification, as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof) as anti-parkinson's disease agents.

The formulations and methods provided herein also use phorbol esters or derivative compounds of formula I above, including all active pharmaceutically acceptable compounds of the present specification, as well as various foreseeable and readily providable complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof, in the treatment of prostatic hypertrophy.

The formulations and methods provided herein also use phorbol esters or derivative compounds of formula I above, including all active pharmaceutically acceptable compounds of the present specification, as well as various foreseeable and readily providable complexes, salts, solvates, isomers, enantiomers, polymorphs and prodrugs of these compounds, and combinations thereof, in the treatment of autoimmune disorders.

The formulations and methods provided herein also use phorbol esters or derivative compounds of formula I above, including all active pharmaceutically acceptable compounds of the present specification, as well as various foreseeable and readily providable complexes, salts, solvates, isomers, enantiomers, polymorphs and prodrugs of these compounds, and combinations thereof, in the treatment of carpal tunnel syndrome.

The formulations and methods provided herein also use phorbol esters or derivative compounds of formula I above, including all active pharmaceutically acceptable compounds of the present specification, as well as various foreseeable and readily available complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof, in the treatment of renal disease.

The formulations and methods for producing continence provided herein use phorbol esters or derivative compounds of formula I above (including all active pharmaceutically acceptable compounds of the present specification as well as various foreseeable and readily providable complexes, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds and combinations thereof) as agents that increase continence.

A variety of mammalian subjects, including human subjects, are amenable to treatment using the formulations and methods of the invention. These subjects include, but are not limited to, individuals with diseases or conditions including neoplastic diseases and viral diseases such as HIV and AIDS, as well as individuals with parkinson's disease, stroke, rheumatoid arthritis, chemotherapy side effects, radiation therapy side effects, prostate hypertrophy, urinary incontinence, myasthenia gravis, and kidney disease.

Subjects suitable for treatment include HIV + humans and other mammalian subjects exhibiting: oral ulceration, fatigue, cutaneous thrush, fever, loss of appetite, diarrhea, aphthous ulcers, malabsorption, thrombocytopenia, weight loss, anemia, lymphadenectasis, susceptible to severe secondary conditions such as mycobacterium avium complex, salmonellosis, syphilis, neurosyphotosis, Tuberculosis (TB), bacillary angiomatosis, aspergillosis, candidiasis, coccidioidomycosis, listeriosis, pelvic inflammatory disease, burkitt's lymphoma, cryptococcal meningitis, histoplasmosis, kaposi's sarcoma, lymphoma, systemic non-hodgkin's lymphoma (NHL), primary central nervous system lymphoma, cryptosporidiosis, isosporosis, microsporidiosis, pneumocystosis (PCP), toxoplasmosis, Cytomegalovirus (CMV), hepatitis, herpes simplex, herpes zoster, human papilloma virus (HPV, anorexia, diarrhea, aphthous ulcers, malabsorption, thrombocytopenia, Tuberculosis (TB), bacillary angiomatosis, aspergillosis, candidiasis, coccidioidomycosis, coccidi, Genital warts, cervical cancer), molluscum contagiosum, Oral Hairy Leukoplakia (OHL), and Progressive Multifocal Leukoencephalopathy (PML).

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as agents effective in the treatment of HIV/AIDS and/or related conditions. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as therapeutic agents in the methods and compositions of the present invention for the treatment of HIV/AIDS and related conditions.

Acquired immune deficiency syndrome or acquired immune deficiency syndrome (AIDS or AIDS) is a collection of symptoms and infections caused by damage to the immune system caused by infection with Human Immunodeficiency Virus (HIV). Impairment of the immune system predisposes individuals to opportunistic infections and tumors. Despite treatments directed to AIDS and HIV to slow viral progression and severity of symptoms, there is no known cure.

HIV is a retrovirus that primarily infects components of the human immune system, such as CD4+ T cells, macrophages, and dendritic cells. When CD4+ T cells are destroyed and their total count is reduced to less than 200 CD4+ T cells/μ L blood or the percentage of CD4+ T-cells to total lymphocytes drops to less than 14%, the cellular immune function is lost, resulting in AIDS.

It is currently believed that T_h1 and T_h2 changes in cytokine balance may contribute to HIV infection-related immune disorders. T is_h1 cells produce cytokines that stimulate the proliferation of cytotoxic T cells. T is_h2 cells produce cytokines responsible for activating the humoral immune response in healthy humans. Progression from HIV infection to AIDS is characterized by T_h1 cytokines IL-2, IL-12 and IFN-gamma levels are reduced with T_h2 cytokines IL-4, IL-5 and IL-10 levels increase. (Clerci, Immunology Today, Vol.14, No. 3, p.107-110, 1993; Becker, Virus Genes 28:1,5-18 (2004)). Resistance to HIV infection and/or resistance to progression to AIDS may therefore depend on T_h1>T_h2.

A portion of the CD4+ memory T cells contain transcriptionally integrated inactive HIV provirus. These latent reservoirs can be activated to produce active infectious virus upon activation with specific antigens or cytokines. These CD4 memory T cells have a half-life of at least 44 months, making HIV eradication extremely difficult and requiring extended duration of antiretroviral therapy, even when HIV levels in the peripheral blood are undetectable.

It has been reported that the vinculin (prostratin) (12-deoxyphorbol 13-acetate, a non-tumor-promoting phorbol ester) has shown some effect on inhibiting cell death and viral replication by HIV. It has been reported that vinculin activates viral expression in latently infected cell lines, but has little or no effect on chronically infected cell lines. (Gulakowski et al, anti Research, Vol.33, 87-97 (1997); Williams et al, JBC, Vol.279, Vol.40, p.42008-42017 (2004)). Vinorelbine represents a distinct subclass of protein kinase C activators that possess unique biological activities other than tumor-promoting phorbol esters such as TPA.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the present invention further include, but are not limited to, mammalian subjects having neoplastic diseases, including solid and non-solid cancers, including hematological malignancies/myeloid disorders, such as leukemias, including Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), chronic myelogenous leukemia blast crisis, myelodysplasia, myeloproliferative syndrome; lymphomas, including hodgkin's and non-hodgkin's lymphomas; subcutaneous adenocarcinoma; ovarian teratocarcinoma; and prostate cancer. In some embodiments, such cancers may be relapsed or refractory.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent that is effective in treating neoplastic diseases. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, that are effective as therapeutic agents in the methods and compositions of the present invention for the treatment of neoplastic diseases and the symptoms of such diseases.

Neoplastic disease is any mass or tumor caused by abnormal and uncontrolled cell division; it can spread to other parts of the body via the lymphatic system or the blood stream. Such growth may be malignant or benign, solid or non-solid.

In some embodiments, the neoplastic disease can be a hematological neoplasm/bone marrow disorder, such as Acute Myeloid Leukemia (AML). AML (also known as acute leukemia, acute myeloid leukemia, acute myelogenous leukemia, and acute non-lymphocytic leukemia) is the most common acute leukemia type in adults. In AML, stem cells produced from the bone marrow usually develop into a class of immature white blood cells called myeloblasts (or medulloblasts). In individuals with AML, these myeloblasts cannot develop to mature into healthy white blood cells. In addition, stem cells of individuals with AML may develop into abnormal red blood cells or platelets. The lack of normal blood cells increases the incidence of infection, anemia, and bleeding. In addition, leukemia cells can spread out of the blood to other parts of the body, including the central nervous system (brain and spinal cord), skin, and gums.

The average age of AML patients is over 64 years. Patients over the age of 60 years had less than 20% remission of AML treated with standard chemotherapy. In addition, patients who progress to AML after a previous hematologic disorder or after leukemia-causing chemotherapy/radiotherapy have similar adverse outcomes.

Chemotherapy is the treatment of cancer with anti-neoplastic drugs or combinations of such drugs. Chemotherapy works by impairing the proliferation of rapidly dividing cells, a property common in cancer cells. However, it cannot automatically distinguish healthy cells from cancer cells that are also dividing rapidly and it has a variety of side effects such as, but not limited to, hair loss, nausea, vomiting, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, mouth ulcers, itchy skin, desquamation, nerve and muscle leprosy, changes in hearing, blood problems, weight loss, diarrhea, immunosuppression, bruising, a tendency to bleed easily, heart damage, liver damage, kidney damage, dizziness and encephalopathy.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention additionally include, but are not limited to, mammalian subjects undergoing chemotherapy.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating the side effects resulting from chemotherapy. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, that are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating side effects resulting from chemotherapy.

Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. It may be administered in vitro, in vivo or systemically. It can produce acute or chronic side effects. Acute side effects occur during treatment and chronic side effects occur months or even years after the end of treatment. The side effects that occur depend on the area of the body being treated, the dose administered daily, the total dose administered, the general medical condition of the patient, and other treatments administered concurrently. (National Cancer Institute, 2011). Common side effects of radiotherapy are wet desquamation, soreness, diarrhea, nausea, vomiting, loss of appetite, constipation, skin itching, desquamation, aphtha and sore throat, edema, infertility, fibrosis, hair loss and dry mucous membranes.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention additionally include, but are not limited to, mammalian subjects undergoing radiotherapy.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating the side effects resulting from radiation therapy. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, that are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating the side effects of radiation therapy.

About 1% of the us population is affected by rheumatoid arthritis. Although the reason is not clear, it is currently considered to be caused by both genetic factors and environmental factors. It is a chronic form of arthritis, usually occurring in joints on both sides of the body and also considered as an autoimmune disease. In rheumatoid arthritis, the immune system acts on the synovium, producing a fluid accumulation in the joints, causing pain and often systemic inflammation. Although the symptoms exhibited by different people vary, they often cause joint pain, stiffness (especially during the morning or after a long sitting), joint swelling, fever, myalgia, joint inflammation, and rheumatoid nodules.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention additionally include, but are not limited to, mammalian subjects with rheumatoid arthritis.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating rheumatoid arthritis symptoms. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating rheumatoid arthritis and its symptoms.

It is estimated that 400 to 600 million people worldwide have parkinson's disease, a chronic progressive neurodegenerative brain disorder. It is thought to have both genetic and environmental triggers, but the exact reason is not known. Many symptoms of parkinson's disease are due to a lack of dopamine and low norepinephrine levels. It is also characterized by the presence of a Lewy body(s), but the exact function of these is not known. Parkinson's disease is characterized by tremor, bradykinesia, rigidity, speech disturbances, postural instability and dementia.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, mammalian subjects with parkinson's disease.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating Parkinson's disease. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating parkinson's disease and its symptoms.

Worldwide, stroke is the second leading cause of death, from which 440 million (9%) of 5050 million total deaths per year result. (http:// www.theuniversityhospital.com/stroke/stats. htm university Hospital, Newark New Jersey,2011) 90% of stroke survivors suffer from a certain type of impairment and it is the leading cause of adult disability in the United states. Stroke occurs when a cerebral vessel is occluded or ruptured. Under hypoxic conditions, brain cells begin to die, resulting in sudden numbness, tingling, weakness, or loss of facial, arm, or leg motor abilities. It also causes sudden vision changes, difficulty speaking, confusion, walking or balance problems, and sudden severe headaches. Following a stroke, the individual may experience paralysis, spatial disturbances, impaired judgment, left-sided ignorance, memory loss, aphasia, coordination and balance problems, nausea, vomiting, cognitive disturbances, perceptual disturbances, disorientation, ipsilateral hemianopsia, and impulsivity.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, mammalian subjects that have suffered from or are at risk of stroke.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent that is effective in preventing or treating the effects of stroke. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating stroke and its symptoms.

More than half of the sexually old men have symptoms of prostate hypertrophy and up to 90% of the sexes in seventies and eighties have such symptoms. As the prostate enlarges, its surrounding tissue layers resist enlargement, causing the glands to compress the urethra. The bladder wall becomes thicker and more irritated and begins to contract, even when it contains a small amount of urine, resulting in more frequent urination. Eventually, the bladder weakens and loses its ability to empty itself. (NIH application No. 07-3012, 2006) the most common symptom of prostate hypertrophy is hesitant intermittent weak urinary stream; urgency and leakage or dribbling; and more frequent urination, particularly at night. Additional symptoms include dribbling at the end of urination, urinary retention, incomplete bladder emptying, incontinence, frequent urination, painful urination, hematuria, slow or delayed urination, or exertion.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, mammalian subjects that have suffered from, or are at risk of, prostate hypertrophy.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating prostatic hypertrophy. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating prostatic hypertrophy and its symptoms.

Autoimmune disorders are conditions that occur when the immune system mistakenly attacks and damages healthy body tissue. In individuals with autoimmune disorders, the immune system is unable to distinguish healthy body tissue from antigens. The result is an immune response that damages normal body tissue. In myasthenia gravis, antibodies target proteins to themselves. Autoantibodies most often act at nicotinic acetylcholine receptors (nachrs), receptors in the motor end plate for the neurotransmitter acetylcholine that stimulates muscle contraction. (Patrick J, Lindstrom J. Autoimmum stress to acetyl Choline receptor science (1973)180: 871-2.) the symptoms of myasthenia gravis include ptosis, diplopia, speech impairment, fatigability, myasthenia, dysphagia, or dysarthria.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, mammalian subjects having or at risk of an autoimmune disorder.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating autoimmune disorders, including myasthenia gravis. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, that are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating autoimmune disorders (including myasthenia gravis) and symptoms thereof.

Carpal tunnel syndrome occurs when the median nerve at the wrist (which runs from the forearm into the palm) undergoes compression or squeezing. Sometimes, thickening or other swelling of the stimulated tendons narrows the carpal tunnel and causes compression of the median nerve. The result may be pain, weakness or numbness in the hands and wrists and spreading up the arms. Although the sensation of pain may be indicative of other conditions, carpal tunnel syndrome is the most common and most well known nerve entrapment in which the peripheral nerves of the body are compressed or traumatized. (NIH application No. 03-4898, 2002)

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, mammalian subjects having or at risk of carpal tunnel syndrome.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating carpal tunnel syndrome. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, that are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating carpal tunnel syndrome.

Chronic kidney disease is a condition in which the kidney is damaged and its ability to regulate water and electrolyte balance, discharge metabolic waste products, and secrete essential hormones of the human body is reduced. Symptoms of renal disease include urinary incontinence, increased frequency of urination, uremia, and oliguria.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, mammalian subjects having or at risk of kidney disease.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating kidney disease. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating renal disease and symptoms thereof.

Urinary incontinence is a common and often embarrassing problem. The severity can range from occasional urine leakage at coughing or sneezing to complete loss of control. Urinary incontinence can be caused by a variety of conditions, including infection, pregnancy, aging, cystolithiasis, prostate cancer, bladder cancer, occlusion, prostatitis, hysterectomy, and drug therapy. It may be transient or persistent.

Mammalian subjects suitable for treatment with phorbol esters of formula I, particularly TPA, according to the methods of the invention include, but are not limited to, mammalian subjects having or at risk of urinary incontinence.

In the methods and compositions of the present invention, one or more phorbol ester compounds of formula I as disclosed herein are effectively formulated or administered as an agent effective in preventing or treating urinary incontinence. In exemplary embodiments, TPA has been demonstrated for illustrative purposes to be an effective agent in pharmaceutical formulations and methods of treatment, either alone or in combination with one or more adjunctive therapeutic agents. The present disclosure further provides additional pharmaceutically acceptable phorbol ester compounds in the form of natural or synthetic compounds, including complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of the compounds disclosed herein, and combinations thereof, which are effective as therapeutic agents in the methods and compositions of the present invention for preventing or treating urinary incontinence and its symptoms.

Phorbol alcohol is a polycyclic alcohol derived from natural plants, and belongs to diterpenes in the crotonaldehyde family. It was first isolated in 1934 as a hydrolysate of croton oil produced from croton seeds. It is very soluble in most polar organic solvents and water. Esters of phorbol have the general structure of formula I below:

wherein R is₁And R₂Selected from the group consisting of hydrogen,Alkyl (wherein the alkyl contains 1 to 15 carbon atoms),Lower alkenyl, lower alkenyl,Phenyl, phenyl,Benzyl and substituted derivatives thereof, and R₃Can be hydrogenLower alkenyl or substituted derivatives thereof.

The term "lower alkyl" or "lower alkenyl" as used herein means a moiety containing from 1 to 7 carbon atoms. In the compounds of formula I, the alkyl or alkenyl group may be straight or branched. In some embodiments, R₁Or R₂Either or both are long chain carbon moieties (i.e., formula I is decanoate or myristate).

The alkyl, alkenyl, phenyl and benzyl groups of the formulae herein may be unsubstituted or substituted with halogen, preferably, chloro, fluoro or bromo; a nitro group; amino groups and groups of similar type.

For use in embodiments herein, phorbol esters (including preparations or extracts of any herbal source such as croton) in both organic and synthetic forms are contemplated as useful compositions comprising phorbol esters (or phorbol ester analogs, related compounds and/or derivatives). Phorbol esters and/or related compounds useful for use in embodiments herein will generally have the structure as illustrated in formula I, but those skilled in the art will also appreciate that functionally equivalent analogs, complexes, conjugates, and derivatives of such compounds are also within the scope of the invention.

In a more detailed embodiment, illustrative structural modifications conforming to formula I above will be selected to provide candidate compounds useful in the treatment and/or prevention of HIV and AIDS and/or neoplastic diseases, wherein: r₁And R₂At least one of which is different from hydrogen and R₃Selected from hydrogenLower alkenyl and substituted derivatives thereof. In another embodiment, R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

Exemplary embodiments of phorbol ester compounds of formula I useful for the treatment of cytopathic diseases such as HIV and AIDS and/or neoplastic diseases, particularly AML, are seen in phorbol 12-myristate-13-acetate (also known as PMA or 12-O-tetradecanoyl-phorbol-13-acetate (TPA)) shown in formula II below.

Other useful phorbol esters and related compounds and derivatives in the formulations and methods of the present invention include, but are not limited to, other pharmaceutically acceptable active salts of the compounds and active isomers, enantiomers, polymorphs, glycosylated derivatives, solvates, hydrates and/or prodrugs of the compounds. Other exemplary forms of phorbol esters for use in the compositions and methods of the present 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-dicaprate; phorbol 12, 13-dicaprate; 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-cisianoate 13-decanoate; 12-deoxyphorbol 13-acetate; phorbol 12-acetate; and phorbol 13-acetate.

The phorbol ester compositions herein comprise compositions for the treatment of HIV and AIDS comprising an anti-AIDS effective amount of a phorbol ester compound of formula I, which is effective in the prevention and/or treatment of HIV, AIDS, and/or HIV-associated symptoms, including opportunistic infections, in a mammalian subject. An "anti-HIV," "anti-AIDS," or "treating AIDS" effective amount of the active compound is therapeutically effective, significantly reducing one or more symptoms of AIDS in a subject and/or reducing one or more symptoms or conditions associated with HIV infection in a subject, in single or multiple unit dosage forms, within a specified therapeutic intervention time. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to alleviate AIDS symptoms or other HIV-related conditions in humans and other mammalian subjects susceptible to HIV infection.

The phorbol ester compositions herein may also comprise a chemotherapeutic composition comprising an anti-neoplastic effective amount of a phorbol ester or derivative compound of formula I effective to maintain and treat a malignancy or symptom resulting from cancer in a mammalian subject. An effective amount of the active compound that is "chemotherapeutic," "anti-tumor," "treating cancer," "inducing apoptosis," "inducing remission," "maintaining remission," is therapeutically effective to significantly reduce one or more symptoms of malignancy in a subject and/or to reduce one or more symptoms or conditions associated with malignancy in a subject, in single or multiple unit dosage forms, over a specified therapeutic intervention time. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to alleviate symptoms of conditions associated with neoplastic disease in humans and other mammalian subjects susceptible to malignancy.

The compositions as described herein comprise chemoprotective compositions comprising an effective amount of a phorbol ester compound of formula I to prevent or mitigate chemotherapy side effects. An effective amount of the active compound that is "chemoprotective", "anti-inflammatory", "neutrophil stimulation", "erythropoiesis stimulation", "bone resorption inhibition", "skeleton strengthening", antiemetic "," pain relief "is therapeutically effective to significantly reduce one or more of the side effects of chemotherapy in a subject in single or multiple unit dosage forms over the specified therapeutic intervention period. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to reduce the side effects of chemotherapy in humans and other mammalian subjects undergoing chemotherapy.

The compositions as described herein comprise a radiation therapy protective composition comprising an effective amount of a phorbol ester compound of formula I to prevent or reduce side effects of radiation therapy. A "radiation protective", "anti-tumescence", "cytoprotection", "anti-mucositis", "epithelial cell stimulation", "anti-fibrosis", "platelet stimulation" effective amount of the active compound is therapeutically effective to significantly reduce one or more chemotherapy side effects in a subject over a given therapeutic intervention time in single or multiple unit dosage forms. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to reduce side effects in radiation-treated humans and other mammalian subjects undergoing radiation therapy.

The compositions as described herein comprise stroke treating compositions comprising an amount of a phorbol ester compound of formula I effective to reduce or prevent stroke damage, and thereby effective to prevent and/or treat stroke or stroke-related symptoms or sequelae in a mammalian subject. An "stroke treating", "anticoagulant", "anticholesterolemia", "vasodilation", "antihypertensive" or "neuroprotective" effective amount of the active compound is therapeutically effective to significantly reduce one or more symptoms or sequelae of stroke in a subject, in single or multiple unit dosage forms, over a specified period of therapeutic intervention. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to prevent or reduce symptoms of stroke or the sequelae of stroke in human and other mammalian subjects susceptible to or suffering from stroke.

The compositions as described herein further comprise a composition for treating parkinson's disease comprising an effective amount of a phorbol ester compound of formula I, which composition is effective for preventing and/or treating parkinson's disease or related symptoms in a mammalian subject. An "treating parkinson's disease," "increasing dopamine," "inhibiting catechol-O-methyltransferase," "inhibiting aromatic L-amino acid decarboxylase," "dopamine agonist," "neuroprotection," or "anticholinergic" effective amount of the active compound is therapeutically effective to significantly reduce or prevent one or more symptoms of parkinson's disease in a subject in single or multiple unit dosage forms over a specified period of therapeutic intervention. In exemplary embodiments, the compositions of the present invention are effective in a method of treatment to prevent or reduce the symptoms of Parkinson's disease in human and other mammalian subjects having or at risk of Parkinson's disease.

The compositions as described herein further comprise a composition for treating prostatic hypertrophy comprising an effective amount of a phorbol ester of a compound of formula I effective for preventing and/or treating prostatic hypertrophy or associated symptoms or sequelae in a mammalian subject "treating prostatic hypertrophy", a type II 5- α reductase inhibitor "or a" muscle relaxant "effective amount of the active compound to be therapeutically effective to significantly reduce one or more symptoms or sequelae of prostatic hypertrophy in a single or multiple unit dosage form in a given treatment intervention time.

The compositions as described herein further comprise a rheumatoid arthritis treatment composition comprising an anti-rheumatoid effective amount of a phorbol ester of a compound of formula I, which is effective in preventing and/or treating rheumatoid arthritis or related symptoms in a mammalian subject. An "anti-rheumatoid", "anti-inflammatory", "immunosuppression", "TNF", "antibiotic", "calpain inhibitor", "pyrimidine synthesis inhibitor", "5-LO inhibitor", "antifolate", "IL-1 receptor antagonist" or "T-cell costimulatory inhibitor" effective amount of the active compound is therapeutically effective to significantly reduce one or more symptoms of rheumatoid arthritis in single or multiple unit dosage form over the indicated therapeutic intervention period. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to prevent or alleviate rheumatoid arthritis symptoms in human and other mammalian subjects having or at risk for rheumatoid arthritis.

The compositions as described herein further comprise a composition for treating an autoimmune disease comprising a phorbol ester of a compound of formula I in an amount effective to treat an autoimmune disease, which is effective to prevent and/or treat an autoimmune disease (such as myasthenia gravis) or associated symptoms or sequelae in a mammalian subject. An "autoimmune disorder treating", "myasthenia gravis treating", "immunosuppression", "antibody inhibiting" or "anticholinesterase" effective amount of the active compound is therapeutically effective to significantly reduce one or more symptoms or sequelae of the autoimmune disorder (particularly myasthenia gravis) in a subject in a single or multiple unit dosage form over a specified period of therapeutic intervention. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to prevent or reduce myasthenia gravis in human and other mammalian subjects having or at risk of myasthenia gravis.

The compositions as described herein further comprise a composition for treating kidney disease comprising an effective amount of a phorbol ester of a compound of formula I, which is effective in preventing and/or treating kidney disease or associated symptoms or sequelae in a mammalian subject. An "nephropathic," "anticholinergic," or "antidepressant" effective amount of the active compound is therapeutically effective to significantly reduce one or more symptoms or sequelae of renal disease (including urinary incontinence) in a subject in single or multiple unit dosage forms over a specified therapeutic intervention period. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to prevent or reduce the symptoms of renal disorders in human and other mammalian subjects having or at risk of renal disease.

The compositions as described herein further comprise a urinary incontinence treatment composition comprising an effective amount of a phorbol ester of a compound of formula I, which is effective in preventing and/or treating urinary incontinence in a mammalian subject. An "increasing urinary continence", "anticholinergic", "antibiotic" or "antidepressant" effective amount of the active compound is therapeutically effective to significantly reduce one or more symptoms of urinary incontinence in a subject in single or multiple unit dosage forms over a specified therapeutic intervention period. In exemplary embodiments, the compositions of the present invention are effective in methods of treatment to prevent or reduce the symptoms of urinary incontinence in human and other mammalian subjects having or at risk of renal disease.

Phorbol ester compositions for therapeutic use (including chemotherapy, chemoprotection, radiation protection, stroke alleviation, treatment of parkinson's disease, treatment of prostatic hypertrophy, treatment of rheumatoid arthritis, anti-aging, treatment of renal disease, increased urinary continence, treatment of autoimmune diseases, and treatment of HIV) of the present invention typically comprise an effective or unit dose of a phorbol ester compound of formula I, which may be formulated with one or more pharmaceutically acceptable carriers, excipients, vehicles, emulsifiers, stabilizers, preservatives, buffers, and/or other additives that may enhance stability, delivery, absorption, half-life, efficacy, pharmacokinetics, and/or pharmacodynamics, reduce adverse side effects, or provide other advantages for pharmaceutical use. An effective amount of a phorbol ester compound of formula I or a related or derivative compound thereof (e.g., a unit dose comprising an effective concentration/amount of TPA or a salt, isomer, enantiomer, solvate, polymorph, and/or prodrug of a selected pharmaceutically acceptable TPA) will be readily determined by one of ordinary skill in the art based on clinical and patient-specific factors. Suitable effective unit dosage ranges for an active compound for administration to a mammalian subject, including a human, can be from about 10 μ g to about 1500 μ g, from about 20 μ g to about 1000 μ g, from about 25 μ g to about 750 μ g, from about 50 μ g to about 500 μ g, from about 150 μ g to about 500 μ g, from about 125 μ g to about 500 μ g, from about 180 μ g to about 500 μ g, from about 190 μ g to about 500 μ g, from about 220 μ g to about 500 μ g, from about 240 μ g to about 5 μ g00 μ g, about 260 μ g to about 500 μ g, about 290 μ g to about 500 μ g. In certain embodiments, the effective dose of phorbol ester compound of formula I or related or derivative compounds for treating disease can be selected within a narrow range, for example, from 10 μ g to 25 μ g, from 30 μ g to 50 μ g, from 75 μ g to 100 μ g, from 100 μ g to 300 μ g, or from 150 μ g to 500 μ g. These and other effective unit doses may be administered in a single dose or in multiple doses per day, week or month, for example in a dosing regimen comprising 1 to 5 or 2 to 3 doses per day, week or month. In an exemplary embodiment, a dose of 10 μ g to 30 μ g, 30 μ g to 50 μ g, 50 μ g to 100 μ g, 100 μ g to 300 μ g, or 300 μ g to 500 μ g is administered once, twice, three times, four times, or five times daily. In more detailed embodiments, doses of 50-100 μ g, 100-. In yet another embodiment, a dose of 50-100. mu.g, 100-300. mu.g, 300-400. mu.g, or 400-600. mu.g is administered every other day. In alternative embodiments, the dose is calculated based on body weight and may be administered, for example, in an amount of about 0.5 μ g/m per day²To about 300. mu.g/m²About 1. mu.g/m²To about 200. mu.g/m²About 1. mu.g/m per day²To about 187.5. mu.g/m²About 1. mu.g/m per day²To about 175. mu.g/m per day²About 1. mu.g/m per day²To about 157 μ g/m per day²About 1. mu.g/m per day²To about 125. mu.g/m²About 1. mu.g/m per day²To about 75 μ g/m²1. mu.g/m per day²To about 50/μ g/m²2. mu.g/m per day²To about 50. mu.g/m²2. mu.g/m per day²To about 30. mu.g/m²Or 3 mug/m per day²To about 30. mu.g/m²。

In other embodiments, the dose may be administered less frequently, e.g., 0.5 μ g/m every other day²To about 300. mu.g/m²About 1. mu.g/m²To about 200. mu.g/m²About 1. mu.g/m every other day²To about 187.5. mu.g/m²About 1. mu.g/m every other day²To about 175. mu.g/m²About 1. mu.g/m per day²To about 157 μ g/m every other day²Every other, ofAbout 1. mu.g/m per day²To about 125. mu.g/m²About 1. mu.g/m every other day²To about 75 μ g/m²1. mu.g/m every other day²To about 50. mu.g/m²2. mu.g/m every other day²To about 50. mu.g/m²2. mu.g/m per day²To about 30. mu.g/m²Or 3 mug/m per day²To about 30. mu.g/m². In other embodiments, the dose may be administered 3 times per week, 4 times per week, 5 times per week, on a weekday only, consistent with other treatment regimens only, for consecutive days, or in any suitable dosage regimen, depending on clinical and patient-specific factors.

The present invention encompasses the delivery amount, time course and mode of delivery of compositions of phorbol ester compounds of formula I (an amount effective to treat AIDS, prevent HIV, treat HIV, activate HIV reservoirs, increase Th1 cytokine, induce ERK phosphorylation, chemotherapy, anti-tumor, treat cancer, induce remission, maintain remission, induce apoptosis) in an amount effective to treat a cytopathic disease, will be routinely adjusted according to the underlying condition of the individual (depending on factors such as the weight, age, sex and personal condition of the individual), the severity of the disease and/or associated symptoms, prophylactic or therapeutic administration, and according to other factors known to affect drug delivery, absorption, pharmacokinetics (including half-life) and efficacy.

The present invention relates to a method for treating an acute disease (or "treating AIDS", "treating HIV", "preventing HIV", "activating HIV reservoirs", or "increasing Th1 cytokine", "inducing ERK phosphorylation", "chemotherapy", "anti-tumor", "treating cancer", "inducing apoptosis", "inducing remission", "maintaining remission", "chemoprotection", "anti-inflammation", "stimulating neutrophils", "stimulating erythropoiesis", "inhibiting bone resorption", "strengthening bone", "arresting", "relieving pain", "radioprotection", "anti-swelling", "cytoprotection", "anti-mucositis", "stimulating epithelial cells", "anti-fibrosis", "stimulating platelets", "treating stroke", "anticoagulant", "anti-cholesterolemia", "vasodilation", "anti-hypertension", "treating parkinson's disease", "increasing dopamine", "inhibiting catechol-O-methyltransferase", "inhibiting aromatic L-amino acid decarboxylase", "dopamine agonist", "neuroprotection", "anti-cholinergic", "treating prostate", "type II 5- α reductase inhibitor", "muscle relaxant", "anti-rheumatoid", "anti-inflammatory", "inhibiting TNF", "antibiotic", "calcium neuroleptic inhibitor", "5-LO synthesis inhibitor", "anti-hypertrophy", "treating prostate disease", "anti-hypertrophy", "treating autoimmune disease", "anti-obesity", "anti-hypertensive", "anti-inflammatory", "inhibiting TNF", "anti-inflammatory", "anti.

The therapeutic effect on the treatment of a cytopathic disease can be easily determined using various assay and model systems. For example, in the treatment of HIV or AIDS, the effect can be confirmed by a reduction in viral load, an increase in CD4 count, an increase in CD3 count, an increase in IL-2 and IFN production, a reduction in IL-4 and IL-10 production, and a reduction or elimination of the symptoms of AIDS, in addition to methods for determining the effect known to those skilled in the art.

The effects of the compositions and methods of the invention can be demonstrated, for example, by blood tests for HIV antibodies, viral load, CD4 levels, CD8 counts, and CD3 counts. Normal levels of CD4 are typically between 600 and 1200 per microliter, or 32-68% of lymphocytes. Individuals with a CD4 count of less than 350 have weakened immune systems. Individuals with a CD4 count of less than 200 are considered to have AIDS. CD8 levels in healthy individuals are typically between 150 and 1000 per microliter. CD3 levels in healthy individuals are typically between about 885 to 2270 per microliter. Levels of CD3, CD4, and CD8 cells can be measured, for example, using flow cytometry. An effective amount of a composition of the invention increases the level of CD3, CD4, and CD8 positive cells by at least a 10%, 20%, 30%, 50% or more decrease, up to 75-90%, or 95% or more. An effective amount will also shift the degree of the amount of individual CD3, CD4, and CD8 towards the optimal category for each type of glycoprotein.

β can also be used₂-microglobulin (β)₂-M) test to evaluate individuals β₂β -microglobulin is a protein released into the blood upon cell death₂An increase in blood levels of M can be used as a measure of the progression of AIDS an effective amount of a composition of the invention will reduce or stop β₂-an increase in the amount of M.

Complete Blood Count (CBC) can be used to further demonstrate the effect. The metrics used in CBC include white blood cell count (WBC), Red Blood Cells (RBC), red blood cell distribution width, hematocrit, and amount of hemoglobin. Among CBCs, specific AIDS-related signs include lower hematocrit, sharp decrease in platelet count, and lower neutrophil levels. An effective amount of a composition of the invention will result in a 10%, 20%, 30%, 50% or more increase in the level measured in a complete blood count, up to 75-90%, or 95% or more. An effective amount will also move the individual's blood proteins to the optimal category for each type of protein.

The effectiveness of the compositions and methods of the present invention may also be evidenced by a reduction in HIV or AIDS symptoms including, but not limited to, oral ulceration, fatigue, skin thrush, fever, loss of appetite, diarrhea, aphthous ulcers, malabsorption, thrombocytopenia, weight loss, anemia, and lymphadenectasis.

The effectiveness of the compositions and methods of the invention may also be evidenced by a reduction in the susceptibility and severity of secondary or opportunistic conditions, such as avian complex mycobacteria, salmonellosis, syphilis, neurosyphilis, Tuberculosis (TB), bacillary angiomatosis, aspergillosis, candidiasis, coccidioidomycosis, listeriosis, pelvic inflammatory disease, burkitt's lymphoma, cryptococcal meningitis, histoplasmosis, kaposi's sarcoma, lymphoma, systemic non-hodgkin's lymphoma (NHL), primary central nervous system lymphoma, cryptosporidiosis, isosporosis, microsporidiosis, pneumocystosis (PCP), toxoplasmosis, Cytomegalovirus (CMV), hepatitis, herpes simplex, herpes zoster, human papilloma viruses (HPV, genital warts, cervical cancer), contagious molluscum, oral leukoplakia (OHL), and Progressive Multifocal Leukoencephalopathy (PML).

The effect can be further demonstrated by detectable HIV reduction in HIV infected subjects, maintenance of normal T cell counts, or maintenance of normal p24 antigen levels.

The efficacy of treatment of neoplastic disease can also be determined by a variety of methods, such as, but not limited to, an ECOG performance scale, a Karnofsky performance scale, a blood cell microscopy, a bone marrow puncture and biopsy, cytogenetic analysis, biopsy, immunophenotyping, blood chemistry studies, complete blood cell counts, lymph node biopsy, peripheral blood smears, visual analysis of tumors or lesions, or any other method known to those of skill in the art for assessing and/or diagnosing malignancy and tumor progression.

For example, Absolute Neutrophil Counts (ANC) can be used to evaluate the effectiveness of the compositions and methods herein in the treatment of hematological malignancies/bone marrow disorders. Normal ANC between 1,500 and 8,000/mm³In the meantime. Individuals with hematological malignancies/bone marrow disordersOften times, the body has less than 1500 pieces/mm³And even up to less than 500/mm³The level of (c). An effective amount of the compositions and methods herein will increase the ANC of an individual by 10%, 20%, 30%, 50% or more, up to 75-90%, or 95% or more. The effective amount may increase the level of ANC above 1500/mm³。

The compositions and methods herein can be used, for example, to further evaluate the efficacy of platelet counts in the treatment of hematological malignancies/bone marrow disorders. Platelet counts are typically between 150,000 and 450,000 platelets per microliter (x 10-6/liter). Individuals with hematological malignancies/bone marrow disorders may have platelet counts below 100,000 per microliter. An effective amount of the compositions and methods herein will increase the platelet count of an individual by 10%, 20%, 30%, 50% or more, up to 75-90%, or 95% or more. The effective amount may increase platelet levels above 100,000 per microliter.

The effectiveness of the compositions and methods herein in the treatment of hematological malignancies/bone marrow disorders can also be evaluated, for example, by measuring the number of myeloblasts. Myeloblasts typically account for less than 5% of the cells in the bone marrow, but should not be present in circulating blood. An effective amount of the compositions and methods herein will result in a 10%, 20%, 30%, 50% or more, up to a 75-90%, 96% or more reduction in the number of myeloblasts. The effective amount may result in a myeloblast reduction of less than 5%.

The effectiveness of the compositions and methods herein in the treatment of hematological malignancies/bone marrow disorders can be further evaluated by examining myeloblasts for the presence of an Auer rod (Auer rod). An effective amount of the composition of the invention will reduce the number of visible orlon rods by 10%, 20%, 30%, 50% or more, up to a reduction of 75-90%, 96% or more, until the complete disappearance of the orlon rods.

The effectiveness of the compositions and methods of the invention may also be evidenced by a reduction in symptoms in a subject having a neoplastic disease, including, but not limited to, anemia; chronic fatigue; excessive or bleeding tendency, such as nasal, gingival and sub-dermal bleeding; easy contusion, especially contusion without obvious reasons; tachypnea; ecchymosis; repeatedly fever occurs; swelling of the gums; the cut heals slowly; bone and joint discomfort; repeated infection; weight loss; scratching; night sweat; lymph node swelling; fever is caused; abdominal pain and discomfort; visual impairment; cough; lack of appetite; chest pain; dysphagia; swelling of the face, neck and upper limbs; frequent urination is required, especially at night; difficulty in starting urination or holding back urine; weak or disrupted urine flow; painful urination or burning sensation; difficulty in erection; ejaculation pain; blood in urine or semen; recurrent pain or stiffness in the waist, hip or thigh; and weakness.

The effects of the compositions and methods of the invention may also be evidenced by a reduction in chemotherapy-treated symptoms including, but not limited to, hair loss, nausea, vomiting, loss of appetite, soreness, neutropenia, anemia, thrombocytopenia, dizziness, fatigue, constipation, mouth ulcers, itchy skin, scaling, nerve and muscle leprosy, changes in hearing, blood problems, weight loss, diarrhea, immunosuppression, bruising, predisposition to bleeding, heart damage, liver damage, kidney damage, dizziness and encephalopathy.

The effectiveness of the compositions and methods of the invention may also be evidenced by a reduction in symptoms associated with radiotherapy (including but not limited to, wet desquamation, soreness, diarrhea, nausea, vomiting, loss of appetite, constipation, itchy skin, scaling, aphthae and sore throats, edema, infertility, fibrosis, hair loss, and dry mucous membranes) as compared to other subjects receiving similar radiotherapy treatments.

The effect in the treatment of rheumatoid arthritis can be confirmed, for example, by using various animal models including a collagen-induced arthritis model (as described in example 30 below), a pristane-induced arthritis model, an adjuvant-induced arthritis model, a streptococcus cell wall-induced arthritis model, an ovalbumin-induced arthritis model, an antigen-induced arthritis model, or a balloon model.

The effectiveness of the compositions and methods of the invention in the treatment of rheumatoid arthritis may also be evidenced by a reduction in the symptoms of rheumatoid arthritis, including but not limited to joint pain, stiffness (especially in the morning or after a sedentary season), joint swelling, fever, myalgia, joint inflammation, and rheumatoid nodules.

The effectiveness of the compositions and methods of the invention in the treatment of rheumatoid arthritis is also evidenced by changes in the erythrocyte sedimentation rate. Individuals with rheumatoid arthritis often have elevated levels of erythrocyte sedimentation. An effective amount of a composition of the invention will reduce the level of erythrocyte sedimentation by 10%, 20%, 30%, 50% or more, up to a reduction of 75-90%, 96% or more, compared to the level of erythrocyte sedimentation initially diagnosed. The effect was also demonstrated by the changes in the levels of rheumatoid factor and anti-cyclic citrulline antibody.

The effectiveness of the compositions and methods of the invention in the treatment of Parkinson's disease is evidenced by a reduction in symptoms of Parkinson's disease, including but not limited to tremor, bradykinesia, rigidity, speech impairment, postural instability and dementia. The effectiveness of phorbol ester compounds of the invention in the treatment of parkinson's disease may be further demonstrated by increased levels of dopamine and/or norepinephrine. Such levels may increase by 10%, 20%, 30%, 50% or more of the increase of normal levels, up to 75-90%, or 95% or more.

The effectiveness of the compositions and methods of the invention in the treatment of Parkinson's disease can be further demonstrated by a reduction in the number of Lewy bodies present, the effectiveness can also be demonstrated by using animal models, such as an MPTP-induced model of Parkinson's disease, a rotenone-induced model of Parkinson's disease, a surgery-induced model of Parkinson's disease, a paraquat-induced model of Parkinson's disease, a 6-OHDA-induced model of Parkinson's disease, or α -synuclein overexpressing mice, the use of the compositions and methods of the invention will reduce the symptoms of Parkinson's disease exhibited in these models by 0%, 20%, 30%, 50% or more, up to 75-90%, 96% or more, as compared to control animals.

Various model systems can be used to demonstrate the efficacy of the compositions and methods of the invention in the treatment of stroke, including temporary middle cerebral artery occlusion as shown in example 22, permanent middle cerebral artery occlusion as shown in example 21, intravascular filament middle cerebral artery occlusion, embolic middle cerebral artery occlusion as shown in example 20, endothelin-1-induced arterial and venous constriction, or cerebral cortical plug therapy. Use of the phorbol ester compositions of the invention will reduce the symptoms exhibited by the model system by 0%, 20%, 30%, 50% or more, up to a reduction of 75-90%, 96% or more, compared to control animals.

The effectiveness of the compositions and methods of the invention in the treatment of stroke may be further demonstrated by the reduction in symptoms exhibited in individuals who have suffered a stroke. Such symptoms include, but are not limited to, paralysis, spatial disorders, impaired judgment, left-sided ignorance, memory loss, aphasia, coordination and balance problems, nausea, vomiting, cognitive disorders, perceptual disorders, disorientation, ipsilateral hemianopsia, and impulsivity. Use of the phorbol ester compositions of the invention will reduce the symptoms exhibited by an individual by 0%, 20%, 30%, 50% or more, up to a reduction of 75-90%, 96% or more, compared to the initial state.

The effectiveness of the compositions and methods of the invention in the treatment of prostatic hypertrophy may be evidenced by a reduction in symptoms associated with prostatic hypertrophy, including, but not limited to, hesitant intermittent weak urinary flow; urgency and leakage or dribbling; more frequent urination; dribbling after urination is finished; urinary retention; incomplete emptying of the bladder; urinary incontinence; the frequency of urination; pain in urination; hematuria; slow or delayed urination; or effort-consuming urination. Use of the phorbol ester compositions of the invention will reduce the symptoms exhibited by an individual by 0%, 20%, 30%, 50% or more, up to a reduction of 75-90%, 96% or more, compared to the initial state.

The effectiveness of the compositions and methods of the invention in the treatment of prostatic hypertrophy can also be demonstrated using various tests, such as a residual urine volume test after urination, a pressure flow rate study, or a cytometric examination. Use of the phorbol ester compositions of the present invention will reduce the amount of residual urine or increase the pressure flow rate phase by 0%, 20%, 30%, 50% or more, up to 75-90%, 96% or more, all as compared to the results prior to treatment with the phorbol ester compound.

The effectiveness of the compositions and methods of the invention in the treatment of myasthenia gravis may be evidenced by a reduction in symptoms associated with myasthenia gravis, including, but not limited to, ptosis, diplopia, speech impairment, fatigability, myasthenia, dysphagia, or dysarthria. Use of the phorbol ester compositions of the invention will reduce the symptoms exhibited by an individual by 0%, 20%, 30%, 50% or more, up to a reduction of 75-90%, 96% or more, compared to the initial state.

The efficacy of the compositions and methods of the invention in the treatment of myasthenia gravis can also be determined using the Tencilron test (Tensilon test) or ice cube test, neurotransmission studies, single fiber EMG or serum antibody detection of acetylcholine receptors. An animal model of myasthenia gravis can also be used to determine effects, such as by immunizing an animal with raja torpedo acetylcholine receptor (AChR) in freund's complete adjuvant. Use of the phorbol ester compositions of the invention will reduce the symptoms exhibited by an individual by 0%, 20%, 30%, 50% or more, up to 75-90%, 96% or more reduction compared to the initial state and/or control.

The effectiveness of the compositions and methods of the invention in the treatment of carpal tunnel syndrome is evidenced by a reduction in the symptoms associated with carpal tunnel syndrome, including but not limited to pain, weakness or numbness in the hands and wrists and spreading up the arms. Use of the phorbol ester compositions of the invention will reduce the symptoms exhibited by an individual by 0%, 20%, 30%, 50% or more, up to a reduction of 75-90%, 96% or more, compared to the initial state.

The effectiveness of the compositions and methods of the present invention in the treatment of renal disease can be evidenced by a reduction in symptoms associated with renal disease, including but not limited to urinary incontinence, increased frequency of urination, uremia, or oliguria. Use of the phorbol ester compositions of the invention will reduce the symptoms exhibited by an individual by 0%, 20%, 30%, 50% or more, up to 75-90%, 96% or more reduction compared to the initial state.

The effectiveness of the compositions and methods of the invention in the treatment of urinary incontinence can be demonstrated by a reduction in symptoms associated with urinary incontinence. Use of the phorbol ester compositions of the invention will reduce the symptoms exhibited by an individual by 0%, 20%, 30%, 50% or more, up to 75-90%, 96% or more reduction compared to the initial state.

For each of the indicated conditions described herein, the test subject will exhibit a 10%, 20%, 30%, 50% or more reduction, up to a 75-90%, or 96% or more reduction in one or more symptoms of (caused by or associated with) the disease or associated disease or condition in the subject as compared to a placebo-treated subject or other suitable control subject.

In other aspects of the invention, there is provided a synergistic therapeutic method for the treatment of a disease ("treatment of AIDS", "prevention of HIV", "treatment of HIV", "activation of HIV reservoirs", "increasing Th1 cytokine", "induction of ERK phosphorylation", "induction of apoptosis", "chemotherapy", "anti-tumor", "treatment of cancer", "induction of remission", "maintenance of remission", "chemoprotection", "anti-inflammation", "stimulation of neutrophils", "stimulation of erythropoiesis", "inhibition of bone resorption", "strengthening bone", "anti-vomiting", "relief of pain", "radioprotection", "anti-swelling", "cytoprotection", "anti-mucositis", "stimulation of epithelial cells", "anti-fibrosis", "platelet stimulation", "treatment", "anti-coagulation", "anti-cholesterolemia", "vasodilation", "anti-hypertension", "increased urinary incontinence", "parkinson therapy", "increased dopamine", "inhibition of catechol-O-methyl transferase", "inhibition of aromatic L-amino acid decarboxylase", "dopamine agonist", "neuroprotection", "anticholinergic", "anti-cholinergic", "treatment of prostate", "type 5- α reductase inhibitor", "muscle relaxant", "anti-rheumatic", "anti-inflammatory", "inhibition of immunosuppressive TNF", "antibiotic", "calcineurin inhibitor", "inhibitory 5-LO-pyrimidine synthesis inhibitor", "anti-depressant inhibitor", "depressor" synergistic therapeutic methods for the administration of a compound anti-cholesterol esterase-proliferative disorder or a synergistic therapeutic agent, or a compound, for the treatment of a disease, or for the treatment of a proliferative disorder, or for the treatment of a synergistic therapeutic method for the production of a synergistic effect of a secondary inhibitory effect of a compound inhibitory activity of a T-nocholinergic agent, for the treatment of a compound, for the T receptor, for the treatment of a disease, or for the treatment of a compound, for the treatment of.

In this context, exemplary combination formulations and synergistic treatment methods use a phorbol ester of formula I in combination with one or more secondary anti-AIDS agents or one or more adjunctive therapeutic agents useful for treating or preventing the target (or associated) disease, condition and/or symptom in selected combination formulations or synergistic treatment regimens formulated or synergistically administered to produce a combination formulation or synergistic treatment method effective or synergistically in treating HIV/AIDS and/or one or more opportunistic or secondary disease or condition symptoms in a subject, in combination with one or more secondary or adjunctive therapeutic agents selected from, for example, protease inhibitors including but not limited to quinavirine, denavirenz, valdecovativiridae, valdecofovir, valdecovativiridae, valfovir, valdecofovir, valdecovativiridavir-fumarate, valdecovativiridae, valdecofovir, valdecovativiridae, valdecovativiridavir-fumarate, valdecovir, valdecovativiridae, valdecovir, valdecovativiridavir-fumarate, valdecovir, valdecovativiridae, valdecovir, valdecovativiridae, valdecovir, valdecovatividine, valdecovir.

Additional exemplary combination formulations and co-therapy methods phorbol esters of formula I may also be used in combination with one or more secondary anti-neoplastic agents or one or more adjunctive therapeutic agents useful for treating or preventing the targeted (or associated) disease, condition, and/or symptom in a selected combination formulation or co-therapy regimen. For most combination formulations and co-therapies of the invention, the phorbol ester compound of formula I or a related or derived compound is formulated in combination or co-administered with one or more secondary or adjunctive therapeutic agents to produce a combination formulation or co-therapy method that is effective or co-synergistic in combination for treating neoplastic disease and one or more symptoms of a secondary disease or condition in a subject. In this context, exemplary combination formulations and co-therapeutic methods use a phorbol ester compound of formula I in combination with one or more secondary or adjunctive therapeutic agents selected from, for example, chemotherapeutic agents, anti-inflammatory agents, doxorubicin, vitamin D3, cytarabine, daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide, dexamethasone, and choline magnesium trisalicylate. In addition, adjuvant or secondary therapies such as, but not limited to, radiation therapy, hormonal therapy, and surgery may be used.

For most combination formulations and co-therapies of the invention, the phorbol ester compound of formula I or a related or derivative compound is formulated or co-administered in combination with one or more secondary or co-therapeutic agents effective in combination or co-synergistically effective in preventing or treating chemotherapy side effects in a subject₃Receptor antagonists, NK₁Antagonists, olanzapine, corticosteroids, dopamine antagonists, serotonin antagonists, benzodiazepines, aprepitant and cannabinoids.

In the prevention or treatment of side effects of radiation therapy, exemplary combination formulations and co-therapies use the phorbol ester compounds of formula I in combination with one or more other radioprotective or otherwise indicated secondary or adjunctive therapeutic agents that may be useful in the treatment or prevention of the condition and/or symptoms of interest. For most combination formulations and co-therapies of the present invention, the phorbol ester compound of formula I or a related or derived compound is formulated in combination or co-administered with one or more secondary or adjunctive therapeutic agents to produce a combination formulation or co-therapy method that is effective in combination or co-therapy for preventing or treating side effects of radiation therapy in a subject. In this context, exemplary combination formulations and co-therapeutic methods use a phorbol ester compound of formula I in combination with one or more secondary or adjunctive therapeutic agents selected from steroids, amifostine, chlorhexidine, benzydamine, sucralfate, Keratinocyte Growth Factor (KGF), palifermin, Cu/Zn superoxide dismutase, interleukin 11, or prostaglandins.

For most of the combination preparations and co-therapies of the present invention, the phorbol ester compound of formula I or a related or derived compound is formulated or co-administered in combination with one or more secondary or co-therapeutic agents that may be effective or co-administered in combination for the prevention or treatment of stroke or the effects of stroke.

In the prevention or treatment of parkinson's disease, exemplary combination formulations and co-therapies use the phorbol ester compounds of formula I in combination with one or more other neuroprotective or otherwise indicated secondary or adjunctive therapeutic agents useful in the treatment or prevention of the targeted disease, condition, and/or symptom. For most of the combination preparations and co-therapies of the present invention, the phorbol ester compound of formula I or a related or derived compound is formulated or co-administered in combination with one or more secondary or adjunctive therapeutic agents to produce a combination preparation or co-therapy which is effective or co-ordinated in the prevention or treatment of parkinson's disease. In this context, exemplary combination formulations and co-therapeutic methods use a phorbol ester compound of formula I in combination with one or more secondary or adjunctive therapeutic agents selected from MAO-B inhibitors, pyridoxine, amantadine, pyridoxine, selegiline, rasagiline or anticholinergics.

For most of the combination formulations and co-therapies of the present invention, the phorbol ester compound of formula I or a related or derived compound is formulated or co-administered in combination with one or more secondary or co-therapeutic agents that are effective or co-effective in combination for the prevention or treatment of prostatic hypertrophy.

For most of the combination formulations and co-therapies of the present invention, the phorbol ester compound of formula I or a related or derivative compound is formulated or co-administered in combination with one or more secondary or co-therapeutic agents that are effective or co-administered in combination for the prevention or treatment of rheumatoid arthritis.

In the prevention or treatment of myasthenia gravis, exemplary combination formulations and co-therapies use the phorbol ester compounds of formula I in combination with one or more indicated secondary or adjunctive therapeutic agents that may be used to treat or prevent the target disease, condition, and/or symptom. For most of the combination preparations and co-therapies of the present invention, the phorbol ester compound of formula I or a related or derived compound is formulated or co-administered in combination with one or more secondary or adjunctive therapeutic agents to produce a combination preparation or co-therapy which is effective or co-ordinately useful in the prevention or treatment of myasthenia gravis. In this context, exemplary combination formulations and co-therapies use a phorbol ester compound of formula I in combination with one or more secondary or adjunctive therapeutic agents selected from anticholinesterase, corticosteroids or immunosuppressants.

In the prevention or treatment of renal disease, exemplary combination formulations and co-therapies use the phorbol ester compounds of formula I in combination with one or more anti-incontinence or otherwise indicated secondary or adjunctive therapeutic agents that may be useful in the treatment or prevention of the target disease, condition, and/or symptom. For most of the combination preparations and co-therapies of the present invention, the phorbol ester compound of formula I or a related or derived compound is formulated or co-administered in combination with one or more secondary or adjunctive therapeutic agents to produce a combination preparation or co-therapy which is effective or co-ordinately useful in the prevention or treatment of renal disease. In this context, exemplary combination formulations and synergistic methods of treatment employ anticholinergics, topical estrogens, imipramine, or duloxetine.

In certain embodiments, the present invention provides a method of treating a disease ("treating AIDS", "preventing HIV", "treating HIV", "activating HIV reservoir", "increasing Th1 cytokine", "inducing ERK phosphorylation", "inducing apoptosis", "chemotherapy", "anti-tumor", "treating cancer", "inducing remission", "maintaining remission", "chemoprotection", "anti-inflammatory", "stimulating neutrophil", "stimulating erythropoiesis", "inhibiting bone resorption", "strengthening bone", "arresting", "relieving pain", "radiation protection", "anti-swelling", "cytoprotection", "anti-mucositis", "stimulating epithelial cells", "anti-fibrosis", "stimulating platelets", "treating stroke", "anti-coagulation", "anti-cholesterolemia", "vasodilation", "anti-hypertension", "treating parkinson's disease", "increasing dopamine", "inhibiting catechol-O-methyltransferase", "inhibiting aromatic L-amino acid decarboxylase", "dopamine agonist", "neuroprotection", "anti-cholinergic", "treating prostate", "type 5- α reductase inhibitor", "muscle relaxation", "anti-rheumatoid", "anti-inflammatory", "inhibiting", TNF "," anti-inflammatory ", or" anti-inflammatory cell accumulation, or "in a combined with one or more of the anti-nociceptive inhibitor, or anti-inflammatory agent, or" anti-nociceptive ", and" anti-nociceptive ", or" anti-nociceptive ", and/nociceptive", or "anti-nociceptive", or "anti-therapeutic co-or anti-therapeutic co-nociceptive or anti-nociceptive", or anti-nociceptive "anti-therapeutic co-nociceptive or anti-therapeutic co-therapeutic.

To practice the co-administration method of the present invention, the phorbol ester compound of formula I may be administered simultaneously or sequentially with one or more secondary or adjunctive therapeutic agents contemplated herein in a co-therapeutic regimen. Thus, in certain embodiments, a compound is administered in conjunction with a non-phorbol ester agent or any other secondary or adjunctive therapeutic agent contemplated herein, using either a single formulation or a combined formulation as described above (i.e., comprising a phorbol ester compound of formula I or both a related or derivative compound and a non-phorbol ester therapeutic agent). The co-administration may be simultaneous or sequential in any order, and may be present for a period of time in which only one or two (or all) of the active therapeutic agents exert their biological activities individually and/or together.

In one embodiment, such a synergistic therapeutic approach may follow or be derived from, for example, various highly active antiretroviral treatment regimens (HAART regimens) and include regimens such as, but not limited to, two nucleoside analog reverse transcriptase inhibitors plus one or more protease inhibitors or non-nucleoside analog reverse transcriptase inhibitors, and phorbol esters of formula I, as well as other combinations. Other co-therapeutic approaches may include, for example, phorbol esters and/or compounds for the treatment of opportunistic infections and from HAART protocols. A salient aspect of all such synergistic therapeutic approaches is that the phorbol ester compounds of formula I exert at least some activity to produce a favorable clinical response, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent, with a supplemental agent that reduces the symptoms of AIDS. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In exemplary embodiments, the phorbol ester compound of formula I is administered in conjunction (simultaneously or sequentially, in combination or as separate formulations) with one or more agents that are secondary to HIV treatment, such as agents selected from, for example, protease inhibitors, including but not limited to saquinavir, Addisoprovir, ritonavir, Nefennavir, atazanavir, Darunavir, furazanavir, Telavir and aminoprenavir, nucleoside reverse transcriptase inhibitors, including but not limited to, zidovudine, didanosine, stavudine, lamivudine, Zaxitadine, Endotetrabine, Tenofovir disoproxil fumarate, AVX754 and Abacavir, non-nucleoside reverse transcriptase inhibitors, including but not limited to, Navirenzapine, delavirdine, valactone A, TMC and Efavirenza combination drug, including but not limited to Enfavirenzam/valtabine/fumarate/tenofovir disoproxil, valdecovatamidine, valdecovatamilide, valdecovatamidine, valdecovatamitriptorexin 91140, valdecovatine, valdecovatamivir, valdecovir, valvudine, valdecovir hydrochloride, valdecovir hydrochloride, valdecovir hydrochloride, valdecovir hydrochloride, val.

In another embodiment, such a synergistic therapeutic approach may, for example, follow or be derived from various chemotherapeutic regimens. Other synergistic treatment methods may, for example, include treatment of phorbol esters and/or additional symptoms for neoplastic disease. A salient aspect of all such synergistic therapeutic approaches is that the phorbol ester compound of formula I exerts at least some activity, thereby producing a favorable clinical response with a supplemental agent that reduces symptoms of neoplastic disease, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered in conjunction (simultaneously or sequentially, in combination or separate formulations) with one or more agents that are secondary to, or otherwise indicated for, treatment of cancer or an adjunctive therapeutic agent (e.g., doxorubicin, vitamin D3, cytarabine, cytosine arabinoside, daunorubicin, cyclophosphamide, gemtuzumab ozogamicin, idarubicin, mercaptopurine, mitoxantrone, thioguanine, aldesleukin, asparaginase, carboplatin, etoposide phosphate, fludarabine, methotrexate, etoposide, dexamethasone, and choline magnesium trisalicylate).

In another embodiment, such a synergistic treatment method may, for example, follow or result from various palliative regimens for chemotherapy patients. The synergistic treatment method may for example comprise treatment of phorbol esters and/or additional side effects against chemotherapy. A salient aspect of all such synergistic therapeutic approaches is that the phorbol ester compound of formula I exerts at least some activity to produce a favorable clinical response with a supplemental agent that mitigates a side effect of chemotherapy, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In exemplary embodiments, a phorbol ester compound of formula I is combined with one or more compounds that reduce secondary chemotherapeutic side effects or other indicated or adjunctive therapeutic agents (e.g., Pegfastin, Estan α, Doripoten α, alendronate, risedronate, ibandronate, G-CSF, 5-HT, etc.)₃Receptor antagonists, NK₁The antagonist, olanzapine, corticosteroids, dopamine antagonists, serotonin antagonists, benzodiazepines, aprepitant and cannabinoid) are administered synergistically (simultaneously or sequentially, in combined or separate formulations).

In another embodiment, such a synergistic treatment method may, for example, follow or result from various palliative regimens for radiation therapy patients. The co-treatment method may for example comprise treatment of phorbol esters and/or additional side effects against radiotherapy. A salient aspect of all such synergistic therapeutic approaches is that the phorbol ester compound of formula I exerts at least some activity, thereby producing a favorable clinical response with a supplemental agent that mitigates a side effect of radiotherapy, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered in conjunction (simultaneously or sequentially, in combination or separate formulations) with one or more compounds that secondarily reduce the side effects of radiation therapy or with additional indicated or adjunctive therapeutic agents, such as steroids, amifostine, chlorhexidine, benzydamine, sucralfate, Keratinocyte Growth Factor (KGF), palifermin, Cu/Zn superoxide dismutase, interleukin 11, or prostaglandins.

In another embodiment, such a synergistic therapeutic approach may, for example, follow or result from various protocols used to treat stroke. The co-therapeutic method may, for example, comprise phorbol esters and/or treatment for preventing or treating damage caused by stroke. A salient aspect of all such synergistic therapeutic methods is that the phorbol ester compound of formula I exerts at least some activity to produce a favorable clinical response with a supplemental agent that prevents or treats stroke, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered in conjunction (simultaneously or sequentially, in combination or as separate formulations) with one or more secondary stroke treating compounds or additional indicated or adjunctive therapeutic agents (e.g., tissue plasminogen activators, anticoagulants, statins, angiotensin II receptor blockers, angiotensin converting enzyme inhibitors, β -blockers, calcium channel blockers, or diuretics).

In another embodiment, such a synergistic therapeutic approach may, for example, follow or be derived from various protocols for treating parkinson's disease. The synergistic therapeutic method may, for example, comprise phorbol esters and/or a treatment for the prevention or treatment of parkinson's disease. A salient aspect of all such synergistic therapeutic methods is that the phorbol ester compounds of formula I exert at least some activity to produce a favorable clinical response with a supplemental agent for the prevention or treatment of Parkinson's disease, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered in conjunction (simultaneously or sequentially, in combination or separate formulations) with one or more secondary therapeutic parkinson's disease compounds or additional indicated or adjunctive therapeutic agents, such as levodopa, tolcapone, carbidopa, dopamine agonists, MAO-B inhibitors, pyridoxine, amantadine, pyridoxine, selegiline, rasagiline or anticholinergic agents. In addition, adjuvant or secondary therapies may be used to treat parkinson's disease, such as, but not limited to, deep brain stimulation or surgery for lesion formation.

In another embodiment, such a synergistic therapeutic approach may, for example, follow or be derived from various protocols for treating prostatic hypertrophy. Synergistic therapeutic methods may, for example, include phorbol esters and/or treatments for preventing or treating prostatic hypertrophy a salient aspect of all such synergistic therapeutic methods is that the phorbol ester compounds of formula I exert at least some activity to produce a favorable clinical response with a supplemental agent for preventing or treating prostatic hypertrophy, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered in conjunction (simultaneously or sequentially, in combination or separate formulations) with one or more compounds that are secondary to treatment of prostatic hypertrophy or with additional indicated or adjunctive therapeutic agents (e.g., finasteride, dutasteride, terazosin, doxazosin, tamsulosin, or α blockers).

In another embodiment, such a synergistic therapeutic approach may, for example, follow or be derived from various protocols for the treatment of rheumatoid arthritis. The co-therapeutic method may for example comprise phorbol esters and/or a treatment for the prevention or treatment of rheumatoid arthritis. A salient aspect of all such synergistic therapeutic methods is that the phorbol ester compound of formula I exerts at least some activity to produce a favorable clinical response with a supplemental agent that prevents or treats rheumatoid arthritis, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered (simultaneously or sequentially, in combination or separate formulations) in conjunction (simultaneously or sequentially) with one or more compounds that are secondary to the treatment of rheumatoid arthritis or otherwise indicated or adjunctive therapeutic agents (e.g., non-steroidal anti-inflammatory agents, steroids, disease modifying antirheumatics, immunosuppressants, TNF- α inhibitors, anakinra, abacatel, adalimumab, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, D-penicillamine, etanercept, golimumab, gold salts, infliximab, leflunomide, methotrexate, minocycline, sulfasalazine, rituximab, or tocilizumab).

In another embodiment, such a synergistic therapeutic approach may, for example, follow or be derived from various protocols for treating autoimmune diseases. The synergistic treatment may, for example, comprise phorbol esters and/or a treatment for preventing or treating myasthenia gravis. A salient aspect of all such synergistic therapeutic methods is that the phorbol ester compound of formula I exerts at least some activity to produce a favorable clinical response with a supplemental agent that prevents or treats myasthenia gravis, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered (simultaneously or sequentially, in combination or in separate formulations) in conjunction with one or more compounds that are secondary to the treatment of myasthenia gravis or with additional indicated or adjunctive therapeutic agents, such as anticholinesterase, corticosteroids or immunosuppressants.

In another embodiment, such a synergistic therapeutic approach may, for example, follow or be derived from various protocols for treating kidney disease. The synergistic treatment may, for example, comprise phorbol esters and/or treatments for preventing or treating renal disease and symptoms of renal disease. A salient aspect of all such synergistic therapeutic approaches is that the phorbol ester compound of formula I exerts at least some activity, thereby producing a favorable clinical response with a supplemental agent that prevents or treats kidney disease, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered in conjunction (simultaneously or sequentially, in combined or separate formulations) with one or more compounds that are secondary to the treatment of renal disease or with an additional indicated or adjunctive therapeutic agent, such as anticholinergics, topical estrogens, imipramine, or duloxetine.

In another embodiment, such a synergistic treatment method may, for example, follow or be derived from various protocols for treating urinary incontinence. The synergistic treatment method may, for example, comprise phorbol esters and/or a treatment for preventing or treating urinary incontinence. A salient aspect of all such synergistic therapeutic methods is that the phorbol ester compound of formula I exerts at least some activity to produce a favorable clinical response with a supplemental agent that prevents or treats urinary incontinence, or a clinical response different from that provided by the secondary or adjunctive therapeutic agent. Generally, the synergistic administration of a phorbol ester compound of formula I with a secondary or adjunctive therapeutic agent will produce improved therapeutic or prophylactic results in a subject, beyond the therapeutic efficacy obtained by administration of the phorbol ester compound of formula I or the secondary or adjunctive therapeutic agent alone. This assessment covers both direct effects as well as indirect effects.

In an exemplary embodiment, the phorbol ester compound of formula I is administered in conjunction (simultaneously or sequentially, in combined or separate formulations) with one or more compounds that are secondary to urinary incontinence or with an additional indicated or adjunctive therapeutic agent, such as anticholinergics, topical estrogens, imipramine, or duloxetine.

As noted above, in all of the various embodiments of the invention encompassed herein, the methods and formulations for treating disease may employ phorbol ester compounds of formula I in any of a variety of forms, including any one or combination of pharmaceutically acceptable salts, solvates, isomers, enantiomers, polymorphs, solvates, hydrates and/or prodrugs of the subject compounds. In an exemplary embodiment of the invention, TPA is used in the therapeutic formulations and methods for illustration purposes.

The pharmaceutical compositions of the present invention may be administered by any means that achieves their intended therapeutic or prophylactic purpose. Suitable routes of administration for the compositions of the present invention include, but are not limited 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.

The compositions of the present invention may further comprise a pharmaceutically acceptable carrier appropriate to the particular mode of administration employed. The dosage form of the composition of the invention comprises excipients recognized in the pharmaceutical compounding art as being suitable for the preparation of dosage units as described above. Such excipients include, but are not intended to be limited to, binders, fillers, lubricants, emulsifiers, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other conventional excipients and additives.

If desired, the compositions of the present invention can be administered in a controlled release form through the use of a sustained release carrier, such as a hydrophilic sustained release polymer. Exemplary controlled release agents in this context include, but are not limited to, hydroxypropylmethylcellulose or other biocompatible matrix (such as cholesterol) having a viscosity in the range of about 100cps to about 100,000 cps.

Some phorbol ester compositions of formula I of the present invention are designed for parenteral administration, e.g., intravenous, intramuscular, subcutaneous, or intraperitoneal administration, including aqueous and non-aqueous sterile injection solutions, which, like many other contemplated compositions of the present invention, may optionally contain antioxidants, buffers, bacteriostats, and/or solutes that render the formulation isotonic with the blood of the mammalian subject; and aqueous and non-aqueous sterile suspensions which may include suspending agents and/or thickening agents. The formulations may be presented in unit-dose or multi-dose containers. Other compositions and formulations of the invention may include polymers for extended release following parenteral administration. The parenteral formulation may be a solution, dispersion or emulsion suitable for such administration. The subject agents may also be formulated into polymers for extended release following parenteral administration. Pharmaceutically acceptable formulations and ingredients will generally be sterile or readily sterilizable, biologically inert, and easy to administer. Such polymeric materials are well known to those of ordinary skill in the art of pharmaceutical compounding. Parenteral formulations typically contain buffers and preservatives, and pharmaceutically and physiologically acceptable injectable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol and the like. 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 above, or an appropriate fraction thereof, of the active ingredient.

In a more detailed embodiment, the composition of the invention may comprise a phorbol ester compound of formula I encapsulated for delivery, which is encapsulated in microcapsules, microparticles or microspheres (such as hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in a macroemulsion.

As noted above, in certain embodiments, the methods and compositions of the present invention may employ pharmaceutically acceptable salts, for example, acid addition or base salts of the phorbol ester compounds of formula I and/or related or derivative compounds described above. Examples of pharmaceutically acceptable addition salts include inorganic and organic acid addition salts. Suitable acid addition salts are formed from acids that form non-toxic salts (e.g., hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate and biphosphate). Other pharmaceutically acceptable salts include, but are not limited to, metal salts such as sodium, potassium, cesium salts, and the like; alkaline earth metals such as calcium salts, magnesium salts, and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N' -dibenzylethylenediamine salt, 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 arginine salts, asparagine salts, glutamic acid salts, tartaric acid salts, and gluconic acid salts. Suitable basic salts are formed from bases which form non-toxic salts such as aluminium, calcium, lithium, magnesium, potassium, sodium, zinc and diethanolamine salts.

Other detailed embodiments, methods and compositions of the present invention employ prodrugs of phorbol esters of formula I. Prodrugs are considered to be any covalently bonded carriers that release the active parent drug in vivo. Examples of prodrugs useful in the present invention include esters or amides having hydroxyalkyl or aminoalkyl groups as substituents, and they may be prepared by reacting such compounds as described above with an anhydride (such as succinic anhydride).

The invention disclosed herein is also to be understood to encompass methods of using in vivo metabolites of the compounds (either produced in vivo after administration of a subject precursor compound or administered directly as the metabolite itself) and compositions comprising phorbol esters of formula I. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, primarily as a result of enzymatic processes. Thus, the present invention includes methods and compositions of the invention using compounds prepared by a process comprising contacting a phorbol ester compound of formula I with a mammalian subject for a time sufficient to produce a metabolite thereof. Such products are typically identified by: the radiolabeled compounds of the invention are prepared, and administered parenterally in detectable doses to animals (such as rats, mice, guinea pigs, monkeys or humans), allowing sufficient time for metabolism to occur and isolation of their transformation products from urine, blood or other biological samples.

It is also understood that the invention disclosed herein encompasses diagnostic compositions useful for diagnosing the level of risk, presence, severity or therapeutic index of a disease (including, but not limited to, neoplastic disease (including malignant neoplastic disease, such as leukemia), stroke, parkinson's disease, myasthenia gravis, rheumatoid arthritis, kidney disease, prostatic hypertrophy and AIDS or related diseases or conditions) in a mammalian subject, or managing the disease, comprising contacting a labeled (e.g., isotopically labeled, fluorescently labeled or otherwise labeled to allow detection of the labeled compound using conventional methods) phorbol ester compound of formula I with a mammalian subject at risk for or exhibiting one or more symptoms of cancer, stroke, parkinson's disease, myasthenia gravis, rheumatoid arthritis, kidney disease, prostatic hypertrophy and/or AIDS (e.g., to a cell, tissue, organ, or individual) and thereafter detecting the presence, location, metabolism, and/or binding status of the labeled compound using any of a number of known assays and labeling/detection methods (e.g., detecting binding to an unlabeled binding partner involved in HIV receptor physiology/metabolism or malignant cell receptor physiology/metabolism). In exemplary embodiments, the phorbol ester compounds of formula I are isotopically labeled by replacing one or more atoms with atoms of different atomic masses or mass numbers. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as²H、³H、¹³C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³¹P、³²P、³⁵S、¹⁸F and³⁶and (4) Cl. The isotopically labeled compounds are then administered to an individual or other subject and subsequently detected as described above according to conventional techniques, yielding useful diagnostic and/or therapeutic management data.

Examples

The experiments described herein demonstrate the novel and potent use of phorbol esters and derivative compounds as HIV therapeutic agents, which are effective in reducing the symptoms of AIDS. In an exemplary clinical trial, individuals who do not respond to traditional treatment for HIV and AIDS respond to TPA treatment. The attending physician "sympathy" with TPA treatment and believing that restoring some patients is life saving. The experiments described below additionally demonstrate the efficacy of phorbol esters and derivative compounds in the treatment of neoplastic diseases, as chemoprotectants, as radioprotectants, in the treatment of stroke, parkinson's disease, prostatic hypertrophy, rheumatoid arthritis, renal disease, urinary incontinence and myasthenia gravis. Phorbol esters additionally provide unexpected cosmetic results in the form of reduced appearance of dark circles and increased skin youth. These and other findings are further detailed and illustrated in the following examples.

Example I

Effect of TPA on peripheral White Blood Cell (WBC) and hemoglobin (Hb) counts in S180 cell injected mice:

sarcoma 180(S180) cells were injected into Kwen-Ming mice. On day three, mice were given TPA intraperitoneally (i.p.) at 50, 100 or 200 μ g/kg/day for 7 days. The next day after completion of treatment, blood samples were taken from the tails of treated mice for WBC and Hb analysis. The WBC counts of the treatment groups (50, 100 or 200 ug/kg/day for 7 days) were 16.1. + -. 7.4, 18.7. + -. 3.0 and 20.7. + -. 3.4X 10, respectively⁹L; WBC count of control group 13.6. + -. 1.8X 10⁹And L. Hb was 136 + -11, 149 + -12 and 149 + -10 g/L for the treatment group and 134+ -15g/L for the control group. The results indicate that intraperitoneal injection of TPA can increase the peripheral WBC count in mice in a dose-dependent manner, while Hb levels in TPA-treated mice are not much affected compared to control mice.

Example II

Dose range studies.

TPA is administered to patients by intravenous (i.v.) infusion because of the intense local irritation that occurs when TPA is administered. The TPA solution in the sterile syringe was injected into 200ml of sterile saline and mixed well for intravenous infusion.

Toxicity and side effects of clinically administered different TPA doses:

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

1mg of TPA in solution was mixed well with 200ml of sterile saline for intravenous infusion, which was completed within 1h at a rate of 16. mu.g/min. One hour after TPA administration, the patient began to have chills lasting about 30 minutes, followed by a fever (patient body temperature reached 37.5-39.5 ℃, continued for 3-5 hours, then returned to normal) with unequal sweating. The above symptoms can be alleviated by administration of a glucocorticoid to the patient. This dose of TPA causes bleeding in a small number of patients, some suffer from short term dyspnea, and Hb is detected in the urine. However, these side effects are transient and recoverable. The heart, liver, kidney and lung were found to function normally.

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

0.5mg TPA in solution was mixed well with 200ml saline for intravenous infusion, which was completed in 1h at a rate of 8. mu.g/min. The response after administration was similar to that of the 1mg TPA dose, but to a lesser extent than the 1mg dose. The patient is more tolerant of lower doses. Occasionally Hb was detected in the urine of the patients. No dyspnea was observed. The heart, liver, kidney and lung function are all normal.

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

0.25mg TPA in solution was mixed well with 200ml saline for intravenous infusion, which was completed in 1h at a rate of 4. mu.g/min. After administration, symptoms such as chills and fever were also observed, but to a much lesser extent than with higher doses. No Hb was detected in the urine and the patient did not experience dyspnea. The heart, liver, kidney and lung function are all normal.

Example III

First clinical study with TPA for HIV + patients

Twelve symptomatic patients (five males and seven females) aged between 35 and 52 years, all infected with HIV by transfusion in 1995 and refractory to standard treatment for HIV, were treated with TPA. Each patient was treated by intravenous administration of a weight-adjusted dose of TPA (75. mu.g/sqm) in 200ml of sterile saline over 1 hour. This dose was administered once daily for the first three days of treatment. This dose was then administered to each patient every other day from day 4 to day 18, followed by a 6 month rest period, followed by a second course of treatment following the same protocol.

Blood samples were collected prior to administration of the first dose of TPA and on days 4 and 40 of the treatment cycle. Levels of CD3, CD4, and CD8 in peripheral blood were measured using monoclonal antibodies (Becton Dickson Scientific co., Franklin Lakes, NJ) and a flow cytometer (b.d. bioscience, San Diego, CA).

As can be seen in table 1, no consistent changes and associations in CD3, CD4, or CD8 levels were observed.

As can be seen in table 2 below, the changes in viral load had similar inconsistent results, with five patients having increased HIV and seven others having no or decreased.

Despite the lack of correlation with viral levels and levels of CD3, CD4, and CD9, eleven patients showed significant improvement following treatment. Eight patients had no symptoms and five of them had remissions for 6 to 12 months. The other three patients had reduced symptoms.

Example IV

Second clinical study with TPA for HIV + patients

Nine patients in example III were given a second TPA treatment. Of these nine, seven were asymptomatic at the start of the second trial. The tenth patient (patient No. 2a) was enrolled in the study and was symptomatic and not previously treated with TPA. Each patient was administered a modulated dose of TPA (75. mu.g/sq m) intravenously in 200ml of sterile saline over one hour. This dose was given once daily to each patient for ten consecutive days, followed by a ten day rest period, for three cycles and a total of 30 doses of TPA. Patients 5a, 6a and 8a stopped taking the anti-AIDS medication one month before starting TPA treatment and started again one month after the third cycle. Patients 1-4a, 7a, and 9a-10a continued to take anti-AIDS medication throughout the treatment.

Blood samples were taken and measured for CD3, CD4, CD8, WBC, RBC, HGB, and platelets three days before the start of treatment, after completion of the first 10 day circulating TPA infusion, and again after the last TPA infusion.

As shown in table 3, CD3 increased in all patients after the first and third infusions of TPA, with the highest values occurring after the third cycle, except for two patients (5a and 10 a). There is a trend toward an increase in CD 8and CD 4. These results indicate that treatment with TPA can boost the immune system. Different results of HIV counts were obtained (table 4). HIV measurements in some patients were below the detection limit of the method (less than 200), while in others they were slightly increased. There were normal changes in measurements of WBCs, RBCs, HGBs and platelets (table 5).

Of the nine patients previously treated with TPA in the first clinical study, only one (No. 9a) exhibited some symptoms of AIDS prior to the start of the second clinical study. After treatment with three cycles of TPA in the second study, this patient and another patient (No. 2a), who had not been treated with TPA, experienced a disappearance of AIDS symptoms and both completely restored normal activity. Eight additional patients had no symptoms of AIDS at the beginning of the study and no symptoms at the end of the study. All patients were still under observation. Treatment with anti-AIDS drugs continues uninterrupted.

As can be seen in table 4, CD3, CD4, and CD8 levels were increased in all patients, and the increase in CD3 levels was the most significant and consistent. HIV varies in viral load. Undetectable in three patients (< 200); there was a slight increase in the other six patients and a decrease in one patient.

Example V

Third clinical study of HIV + patients treated with TPA

Six patients (two males and four females, between the ages of 37 and 52 years) (patient numbers 13-18) were treated with TPA. Four of these patients previously received a combination of TPA treatment with anti-HIV drugs in the first two clinical studies. The other two patients were not treated with TPA but had previously received an anti-HIV drug regimen. All treatments were discontinued three days prior to the start of the third clinical study and were not resumed until 60 days after completion of TPA treatment. Restoration of standard HIV therapy is required by local health authorities.

In the study, each patient received 150 μ g of TPA by intravenous infusion in 200ml of sterile saline over a period of 1.5 to 2 hours daily for 60 days at a total administration dose of 9.0 mg. After completion of 60 days of TPA therapy, these patients were still observed for an additional 60 days, but received no further treatment.

Prior to the start of treatment and again on days 30 and 60, levels of CD3, CD4, and CD8 in peripheral blood were quantified using flow cytometry and appropriate antibodies (obtained from b.d. bioscience, San Diego, CA). Viral loads were determined using conventional methods at Guangan Hospital, Beijing, China (Kuang Ann men Hospital, Beijing, China). RBC, WBC, platelets, and hemoglobin levels of the patient were also measured.

As can be seen in table 6, the viral load was low or undetectable in six patients at the start of the trial and remained low throughout the clinical trial period (despite discontinuation of traditional antiretroviral therapy). In addition, there was no return in viral levels from day 6 to day 15 after cessation of antiretroviral therapy, as was previously reported in patients with plasma viral loads below 50HIV copies/ml. (Harrigan et al, AIDS 13, F59-F62 (1999)). CD3, CD4, and CD8 levels are variable and uncertain.

Test time:

before TPA

Thirty days after TPA

Sixty days after TPA

All values are in the million

White Blood Cells (WBC), Red Blood Cells (RBC), hemoglobin (Rb), and platelets (PLt) were measured before TPA treatment was started, 15 days, 30 days, 45 days, and 60 days after TPA treatment was started, and 30 days after TPA treatment was stopped. As can be seen in table 7, most of the values are within the normal range.

Patients participating in the third clinical study did not experience a viral load rebound as is commonly seen when discontinuing antiretroviral therapy. In addition, during the 120 day observation and treatment period, these patients had no recurrence of AIDS symptoms, were feeling normal and were able to perform general life activities.

Test time:

before TPA

Fifteen days after TPA

Thirty days after TPA

Forty days after TPA

Sixty days after TPA

6. Thirty days after TPA discontinuation

Example VI

Case study

Treatment of initially symptomatic AIDS patients with TPA following the protocol of examples III, IV and V. Patients participating in multiple studies are identified in some cases by more than one patient number. All patient identification numbers correspond to the patient numbers in tables 1-7.

Patient numbers 1 and 15: h.l.y., female, age 35, three clinical trials were involved, diagnosed with AIDS in 2003 and with overt symptoms of this disease. At the beginning of the first study, she had frequent fever, diarrhea, oral ulceration, loss of appetite, weight loss, loss of left eye vision (syncytial formation) and cough (tuberculosis). Patients began receiving antiviral drug therapy, stavudine (D) in 2004₄T), lamivudine (3TC), Nevirapine (NVP) and zidovudine (AZT). Despite the use of anti-AIDS drugs, her CD4 counts as 3 and is unable to perform any physical activity.

During the first study, which was conducted according to the protocol of example III above, she experienced an increase in body temperature of 38-39 ℃ for 2 to 4 hours in four different incidental cases. After treatment with TPA, the symptoms gradually improved. Her appetite improved and diarrhea, oral ulceration and fatigue disappeared, but her vision remained impaired. She gained little weight and returned to be able to resume doing something. She continued to receive antiviral therapy. Improvement of symptoms did not show a correlation with changes in her CD3, CD4, CD8 levels and virus counts.

H.l.y. participated in the second study described in example IV above. At the beginning of the second study, she had no AIDS symptoms. During this subsequent TPA treatment she experienced no side effects. Her levels of CD3, CD4, and CD8 increased after the first and third cycles of TPA treatment, as did her white blood cell count. Her HIV count was high, but her functions were normal and continued to be free of AIDS symptoms.

H.l.y. participated in the third study described in example V above. At the beginning of the third study, her eyes still had problems. During the third study period, she experienced a fever of 38-38.5 ℃ during the third and fourth days of TPA infusion. AIDS symptoms did not recur during the study or the 60 day observation period. She was still asymptomatic, felt normal and was able to perform normal activities, except her vision. She resumed the antiviral therapy after completing the 60 day observation period and continued to be under the physician's care.

Patient number 2: x. female, age 49 years, enrolled in the first clinical study, diagnosed with AIDS in 2004 and had overt symptoms of this disease. She had mild oral ulceration, fatigue, thrush on the skin, fever and loss of appetite. Some of these symptoms are due to herpes virus. She was treated with AZT, DDI and NVP, but drug treatment was discontinued due to side effects. She had not received medication three months prior to TPA treatment. She was frequently hospitalized and out of work. She counted 26 CD4 before treatment.

During TPA treatment according to the protocol of example III, she experienced a 37.5 to 38 degree celsius temperature rise for 1 to 2 hours at three different incidental instances. After treatment with TPA, her oral ulceration, thrush and fever disappeared. Her appetite improved sufficiently that she gained weight and had sufficient energy to return to doing everything. She remained asymptomatic for five months and was not given any anti-AIDS drug during this period. No correlation was shown between the improvement in symptoms and her CD3, CD4, CD8 levels and virus counts.

Patient No. 2a m.s., male, age 48, who was only participating in the second clinical study, had frequent fever, diarrhea, weight loss, a weak immune system, severe depression, and failed to work.

During treatment with TPA according to the protocol of example IV, his body temperature was raised to 38.5 to 39 degrees celsius for 2 to 4 hours in five occasional instances.

After the third cycle of TPA treatment, there were no further fever and diarrhea problems. His CD3, CD4, and CD8 counts tended to rise, as did WBC and HIV counts. His physical and mental condition returns to normal and he is able to work.

Patient number 3: y.p., male, age 51, who was only involved in the first clinical study, was diagnosed with AIDS in 2004 and had overt symptoms of this disease. His major symptoms were diarrhea, fatigue, weight loss, anemia, and bruised skin in both legs; and he can only do light-weight work. He was always treated with AZT, DDI and NVP, but four months prior to TPA administration, severe anemia led to the cessation of drug therapy. His initial CD4 count was 32.

During TPA treatment according to the protocol described in example III, he experienced an increase in body temperature of 38 ℃ to 39 ℃ for 1 to 2 hours in three occasional cases. After treatment with TPA, his symptoms improved significantly, and he was able to recover heavy labor and was living normally. He was asymptomatic for five months following TPA therapy and was not treated with antiviral drugs during this period. There was no correlation shown between CD3, CD4, and CD8 levels and improvement in symptoms, but there was a slight increase in virus counts.

Patient number 4: l.w., male, 34 years old, who was only involved in the first clinical study, tested positive for HIV in 2004 and had overt symptoms of the disease. His main symptoms were diarrhea, fever, weight loss, cough (tuberculosis), enlargement of the right cervical lymph nodes and he failed to work. He had poor initial response to treatment. The schedule of 3TC, DDI and NVP antiviral drug therapy was irregular and stopped during TPA therapy. His initial CD4 count was 173.

During treatment with TPA according to the protocol of example III, he experienced an increase in body temperature of 38 ℃ to 39 ℃ in five occasional cases, lasting 0.5 to 1 hour. After treatment, occasional diarrhea was successfully treated with anti-diarrhea drugs. The improvement in appetite has led to an increase in weight and energy, returning him to a regular work schedule. Lymph nodes returned to normal size. He continued the treatment with anti-viral drugs. No correlation was shown between improvement in symptoms and CD3, CD4, CD8 levels and viral counts.

Patient numbers 5 and 3 a: h.s., female, 37 years old, enrolled in the first two clinical studies, tested positive for HIV in 2004 and had overt symptoms of this disease. At the beginning of the first study, her primary symptoms were skin thrush, hair loss, oral infection, weight loss, and fatigue. She has been using D₄T, DDI and NVP, but stopped due to impaired renal function. Her initial CD4 was counted at 106, but could handle general labor work.

During treatment with TPA according to the protocol of example III, she experienced a 37.5 ℃ to 38 ℃ temperature rise for 0.5 to 1.0 hours in five occasional cases. After treatment with TPA, no improvement in symptoms occurred. After recovery from treatment with the antiviral drug, the previous side effects did not recur and she had reduced intensity of symptoms after one month. This treatment is continuing and she has returned to work. No correlation was shown between improvement in symptoms and changes in CD3, CD4, and CD8 levels or HIV counts.

At the time of the second study, she had no symptoms of AIDS and had not experienced side effects of the course of treatment described in example IV. After the second study, her CD3, CD4, and CD8 levels tended to rise, as did her white blood cell count and platelet levels. Her HIV count was initially undetectable but increased after the third cycle of treatment. She can now work.

Patient number 6, number 4a and number 17: h.s.c., male, 36 years old, three clinical studies were involved, tested HIV positive in 2004 with obvious but mild symptoms. At the beginning of the first study, he experienced dizziness, headache, loss of appetite and was susceptible to upper respiratory infections, but was able to work as normal as the worker. He was always treated with the antiviral drugs AZT, DDI and NVP, but was discontinued due to adverse reactions. His initial CD4 level was 232.

During treatment with TPA according to the protocol of example III, he did not experience an increase in body temperature or any other side effects. After treatment, his symptoms remained unchanged and showed thrombocytopenia not associated with TPA treatment. He continues the treatment with the antiviral drug and can work as before. No correlation was shown between improvement in symptoms and CD3, CD4 and CD8 levels and viral load.

At the time of the second study, he was asymptomatic and his immune system showed normal function. During the second study according to example IV, he also did not experience the side effects of TPA treatment. His CD3, CD4, and CD8 counts increased slightly, as did his white blood cell counts. Viral load was initially undetectable but increased after the third cycle of treatment. However, he did not have any AIDS symptoms and had returned to work.

At the beginning of the third clinical study he had no symptoms. During treatment with TPA according to the protocol of example V, he experienced a local irritation event due to missed needles on day 32, but successfully treated within three days. He was still asymptomatic, felt normal and was able to do gross physical work. He started antiviral therapy after completing the 60 day observation period and continued to be under physician care.

Patient number 7, number 5a and number 16: h.c.l., male, age 49, three clinical studies were involved, tested HIV positive in 2004 and had overt symptoms of the disease. At the time of the first study, his primary symptoms were weight loss, skin thrush, fatigue, loss of appetite, and cough (tuberculosis), but he was able to do light-weight work. He carries out D simultaneously₄T, DDI, NVP, and anti-tuberculosis medication. His firstThe starting CD4 was counted as 10.

During treatment with TPA according to the protocol outlined in example III, he experienced an increase in body temperature to 38 ℃ in two occasional cases, with mild dizziness and headache. After treatment, his symptoms remained unchanged and antiviral therapy was resumed after one month. Over time, his cough, appetite and energy levels improved and he was able to work. He continued to have concurrent antiviral and anti-tuberculosis medications. No correlation was shown between improvement of symptoms and his CD3, CD4, and CD8 levels or viral load.

At the time of the second clinical study, he had no symptoms of AIDS and his immune system showed normal function. During the second clinical study he did not experience the side effects of TPA treatment. After treatment, his CD4 levels were unchanged, but his CD3 and CD8 levels tended to rise, as did his white blood cell count. His viral load was undetectable. He had no AIDS symptoms and had returned to work.

At the beginning of the third clinical study, he did not experience symptoms of AIDS. During treatment according to the protocol outlined in example V he experienced fever in an occasional case. He was still asymptomatic, felt normal and was able to do gross physical work. After completing the 60 day observation period, he resumed the antiviral drug and continued to receive physician attention.

Patient number 8, number 6a and 18: y.x.o., female, 36 years old, three clinical studies were involved, testing positive for HIV in 2004. At the time of the first study, her primary symptom was a greater susceptibility to upper respiratory tract infections. She was treated with AZT, DDI and NVP. At the start of the study, her CD4 level was 524 and she was able to handle general labor work.

During treatment with TPA according to the protocol of example III, she experienced an increase in body temperature to 38.5 ℃ for 4 hours in an occasional case. After treatment, she had a reduced frequency of colds and she had no other symptoms. She continued to be treated with antiviral drugs and was able to work. No correlation was shown between improvement of symptoms and her CD3, CD4, or CD8 levels or viral load.

At the time of the second clinical study, she had no symptoms of AIDS and her immune system was shown to be functioning normally. During the second study, her body temperature was again raised to 38.5 degrees celsius (for two hours) in a single occasional case, following the protocol of example IV. After treatment, her CD3 and CD8 levels increased slightly, while her CD4 and white blood cell counts remained unchanged. Her viral load was undetectable. She showed normal and was able to do physically demanding work.

At the time of the third clinical study, she had no symptoms. The only side effects of treatment according to the protocol of example V were a fever of 38-39℃ for two hours on the second day of treatment and skin irritation (disappearance within two days) by missed needles on day 36. She was still asymptomatic, felt normal and was able to do gross physical work. She resumed the antiviral therapy after completing the 60 day observation period and continued to be under the physician's care.

Patient number 9 and number 7 a: c.t.f., male, age 44, enrolled in the first two clinical studies, tested HIV positive in 2004 and had overt symptoms of the disease. At the beginning of the first study, his symptoms included persistent diarrhea, dizziness, headache, loss of appetite, weight loss, and fatigue. He had a positive response to AZT, DDI and NVP treatment and the blood HIV count was close to the lowest limit. Despite a positive response, his symptoms persisted and he was admitted to the hospital for a diarrhea lasting 20 days. He is very frustrating and unable to do anything.

During treatment with TPA according to the protocol of example III, he experienced a body temperature increase of 37.5 ℃ to 38 ℃ for 2 to 4 hours in six occasional cases. During one TPA administration, missed needles caused severe skin irritation, but were successfully treated. After eight TPA treatments, mild dizziness and headache persisted, but the incidence of diarrhea began to decrease and his appetite improved. After one week, his diarrhea completely disappeared and he had normal appetite. He was able to resume work and was receiving antiviral drug therapy. CD3, CD4, CD8 levels showed an upward trend and the HIV count was undetectable.

At the time of the second clinical study, he had no symptoms of AIDS and his immune system showed normal function. He did not experience side effects during TPA treatment according to the protocol of example IV. After treatment, his CD3, CD4, and CD8 levels increased slightly, while his white blood cell count remained unchanged. His HIV count was still undetectable. He can do heavy work.

Patient number 10 and number 8 a: w.f.w., female, age 47, enrolled in the first two studies, tested positive for HIV in 2003 and had overt symptoms of this disease. At the beginning of the first study, her symptoms included low body temperature, diarrhea, low platelet count, hemoptysis, bloody stools, dizziness, headache, loss of appetite, weight loss, fatigue with mild skin thrush, and deep depression. She was hospitalized for two months with blood at the time of defecation in an occasional case. She was very depressed and unable to work. She had no positive response to AZT, DDI and NVP treatment and her symptoms were not controlled.

During the first TPA treatment she was following the protocol of example III, she experienced an increase in body temperature to 38.5 ℃ for 4 hours in an occasional case. Her dizziness, headache and diarrhea were gradually relieved after TPA treatment. Ultimately, her appetite results in weight gain and an improvement in her energy level. Her platelet counts ranged from 30,000 to 110,000 per microliter and skin thrush and diarrhea were eliminated. She can work again and be treated with antiviral drugs. She occasionally had fever and diarrhea, which could be controlled with medication.

After six months, she experienced mild headache and dizziness and underwent a second TPA treatment. During her second TPA treatment, she experienced a rise in body temperature to 37.5 ℃ to 38 ℃ for 2 to 4 hours in five occasional cases. Twenty hours after the 13 th injection of TPA, her body temperature reached 40.5 degrees celsius and lasted for several hours. It was concluded that the increase in body temperature was not associated with TPA therapy.

After her second TPA treatment, her symptoms disappeared, appetite improved and her weight increased, enabling her to regain energy, return to work and live a normal life. She had no symptoms for one year and had several colds within the first six months after the second TPA treatment. CD3, CD4 and CD8 levels and HIV counts showed an upward trend.

At the time of the second clinical trial according to the protocol of example IV, this patient still showed no symptoms of AIDS and her immune system showed normal function. She did not experience side effects during the treatment period. After treatment, her CD3, CD4, and CD8 counts increased slightly, as did her WBCs. Her HIV count increased slightly. She was healthy and engaged in work since the study was conducted.

Patient numbers 11 and 9 a: female, age 40, enrolled in the first two studies, was diagnosed with AIDS in 2003 and had overt symptoms of this disease. At the beginning of the first study, she had persistent diarrhea, low body temperature, oral ulceration, severe thrush, itching, purpura on her face and lips, dizziness, headache, loss of appetite and fatigue, and depression. She responded poorly to AZT, 3TC and NVP treatment. Her symptoms are uncontrolled and she is unable to work. Her initial CD4 count was 40.

During her first treatment with TPA, she experienced a rise in body temperature to 38 ℃ to 39 ℃ for 2 to 4 hours in four occasional cases. She breathed swiftly in two occasional cases, each time lasting 20 to 30 minutes.

After the sixth dose of TPA, her cutaneous thrush began to disappear, and after completion of TPA treatment, dizziness, headache, fever, and cutaneous thrush were all improving and gradually fading. Her appetite, physical condition and depression improved sufficiently to return her to work.

After 18 months, the patient had relapsed with symptoms (including mild cutaneous thrush, diarrhea and dizziness) and was on a second TPA treatment. During this second treatment, she experienced three temperature increases to 37.5 ℃ to 38 ℃ for 2 to 4 hours. Has no other adverse reactions. After treatment with TPA, her symptoms completely disappeared and her physical condition improved sufficiently to return her to work. She had no symptoms and she had little cold for one year. CD3, CD4, and CD8 levels showed an upward trend, but her HIV count was unchanged.

In a second clinical study conducted according to the protocol of example IV, this patient exhibited AIDS symptoms including headache, dizziness, loss of appetite, and impaired immune function. She did not experience side effects during the treatment period. After treatment, her CD3 and CD8 levels increased, while her CD4 counts remained unchanged. Her HIV count increased slightly, but no other changes were observed. Her mental and physical condition improved significantly and she was doing heavy physical work.

Patient number 12 and number 10 a: c.c.l., female, age 39, enrolled in the first two studies, was diagnosed with AIDS in 2003 and had overt symptoms of this disease. At the beginning of the first study, she had persistent hypothermia, thrush, dizziness, headache, loss of appetite, oral ulceration, fatigue, and deep depression. She was treated with AZT, 3TC and NVP, but the results were poor and she was unable to work. Her initial CD4 count was 84.

This patient was treated twice with TPA during months 3 to 3 of 2006. During the first treatment with TPA, she experienced an increase in body temperature to 38 ℃ to 38.5 ℃ for 2 to 4 hours in eight occasional cases. She experienced shortness of breath for 15 minutes in an occasional case and experienced skin irritation due to missed needles.

After the seventh injection, her oral ulceration disappeared. After all injections were completed, all symptoms disappeared and her physical condition improved sufficiently to return her to work.

Six months later, this patient was retreated with TPA due to recurrent mild diarrhea and dizziness. She experienced an increase in body temperature to 37.5 ℃ to 38 ℃ for 2 to 6 hours in six occasional cases associated with TPA administration. Starting from the eighth injection, the dose was increased from about 150 μ g TPA to 250 μ g TPA. No side effects occurred. After completion of TPA therapy, all her symptoms disappeared. Her physical condition returned to normal and she returned to work and had gone through normal life. She had no symptoms and had few colds a year. CD3, CD4, or CD8 levels did not change, but her HIV count increased.

At the time of the second clinical study, this patient had no symptoms of AIDS, but her immune system was weak. She was treated according to the protocol of example IV and did not experience side effects. After treatment, her CD3, CD4, and CD8 increased slightly, and WBC, RBC, and HGB increased moderately, while platelets showed a decrease. HIV counts were slightly reduced. She was healthy and engaged in heavy physical work since she was treated.

Patient number 13: l.f.l., female, age 53, diagnosed with AIDS in 2004, and only participated in the third clinical study. She presented with mild symptoms of loss of appetite and weight. The long-term antiviral drug was effective and reduced her viral count below detectable levels and increased CD3, CD4, and CD8 counts to higher levels. She had no symptoms and no side effects from her administration prior to TPA treatment. She was still asymptomatic, felt normal and was able to perform normal activities. She resumed antiviral drug therapy after completing the 60 day observation period.

Patient number 14: k.s.m., female, age 45, diagnosed with AIDS in 2004, only participated in the third clinical study. Her symptoms were mild and included loss of appetite and frequent colds. She was treated with antiviral drugs but discontinued due to severe hepatotoxicity. She was asymptomatic prior to TPA treatment and the only TPA side effect was irritation at day 43 due to missed needles, which was easy to treat. AIDS symptoms did not appear during the entire treatment and observation period. She feels normal and is able to perform her daily activities. After the 60-day observation period, she was not enrolled in the study and did not restart antiviral therapy.

Example VII

Treatment of relapsed/refractory malignancies with TPA

Patients with histologically confirmed relapsed/refractory hematological malignancies/bone marrow disorders were treated with a combination of TPA (xchuan Pharmaceuticals, Nan Yang, Henan, China), dexamethasone, and choline magnesium trisalicylate. Similar methods as described 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 conditions and malignancies. Other neoplastic conditions and malignant disorders suitable for treatment using the methods and compositions of the present invention include various forms of cancer, including hematological and bone malignancies, as well as various types of solid tumors. In addition to the protocols specified herein, any of a number of well-known cancer detection and assessment methods will be used to determine successful treatment and/or remission of various target neoplasia and malignant conditions, e.g., by determining size reduction of solid tumors, performing histopathological studies to assess tumor growth, stage, metastatic potential, presence/expression levels of histological cancer markers, and the like.

AML is an invasive disease that usually requires urgent and intensive therapy. The average age of patients diagnosed with AML is 64-68 years, and the probability of a current disease cure for patients treated with standard chemotherapy above the age of 60 is < 20%. Patients who develop AML after a previous hematological disorder or prior to chemotherapy/radiotherapy that causes leukemia have similar adverse outcomes, as do patients with disease associated with specific adverse cytogenetic and clinical characteristics. Thus, most patients diagnosed with AML have patient and/or disease-associated characteristics that correlate with a very poor prognosis. For patients with recurrent disease, there is no standard non-transplant therapy that has proven to be curative. For these patients, AML is often a fatal disease. New methods for treating AML are needed.

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

To date, clinical evaluation of TPA has demonstrated that TPA produces a direct therapeutic cytotoxic effect in at least a subset of AML cases, as measured by cell viability and apoptosis assays. In all primary cultures analyzed by Western analysis, TPA strongly induced ERK phosphorylation after 1 hour of culture. The cytotoxic effect of TPA on primary AML cells was associated with a subsequent loss of phosphorylation-ERK survival signal after 24 hours ex vivo exposure. This observation is in good agreement with other studies that have reported a reduction in survival of primary AML following pharmacological interference with ERK signaling by MEK inhibitors (such as PD98059, U0126 and PD 184352). In our studies, loss of ERK signaling was associated with induction of ERK phosphatase.

In addition to activating protein kinases C and ERK, TPA is also a known inducer of NF-. kappa.B, a pro-survival transcription factor that is constantly activated when present in AML blasts and leukemic stem cells. Recent work in our laboratory has demonstrated that treatment with dexamethasone plus Choline Magnesium Trisalicylate (CMT) for 48h can inhibit the NF- κ B in AML cells in vivo. In addition, we have demonstrated that dexamethasone can induce MKP-1ERK phosphatase expression and enhance the cytotoxicity of TPA on primary AML specimens. In this context, we have selected the use of dexamethasone and CMT as adjunctive drugs for use 24h before and 24h after TPA treatment in the exemplary embodiments below. These drugs are well tolerated and are expected to reduce the inflammatory side effects of treatment and enhance TPA cytotoxicity by increasing ERK phosphatase expression and inhibiting NF- κ B. Dexamethasone and CMT are also used as adjuvant drugs because they are anti-inflammatory, improve side effects, and enhance anti-leukemic activity by inhibiting the anti-apoptotic effects of constitutive NF- κ B expression and inducing phosphatase to decrease signaling pathway activity.

A preliminary TPA phase 1 study recruited 35 patients [23 with relapsed/refractory AML, 2 with other myeloid malignancies (CML-blast crisis, myelodysplasia with blast), 3 with hodgkin's disease, 3 with non-hodgkin's lymphoma and 4 with solid tumors ]. Most patients have relapsed/refractory AML. Our clinical results included an AML patient receiving 8 TPA infusions for >5 months of stable disease. In another AML patient, a significant (5-fold) decrease in circulating blast numbers was seen following TPA administration. This decline in leukemic blast cells lasted for 4 weeks, and the patient eventually died of a fungal infection. Finally, patients with relapsed and refractory hodgkin's disease, despite high dose chemotherapy and assisted by autologous stem cell rescue, have only partial remission of chest wall tumors following TPA administration. TPA dose escalation has been completed and 2 of 3 patients in the last panel were treated at a dose of 0.188mg/m2 on days 1-5 and experienced grade III non-hematologic dose-limiting toxicity (DLT) on days 8-12, establishing a maximum tolerated TPA dose of 0.125mg/m 2/day on days 1-5 and 8-12 as a single agent.

In the case of AML and other hematologic malignancies, patients were given an initial dose of 1 mg/week x 3 weeks (day 1, day 8, day 15) of TPA with administration of continuous/intermittent pulse oximetry for 6 hours. 24 hours prior to the initiation of TPA therapy, patients were given 10mg dexamethasone every 6 hours and 1500mg Choline Magnesium Trisalicylate (CMT) every 8 hours until 24 hours after TPA administration. After administration of the initial dose of TPA, the patients had a 2 week rest period after which they would receive reassessment. Patients with disease response or stable disease due to the initial dose of TPA were treated according to the following protocol for up to 6 28 day periods.

After a 2 week rest period, patients were prodromally dosed 30 minutes prior to TPA administration with 650mg Tylenol and 25-50mg benandryl (depending on patient size and age). They then received an intravenous infusion of TPA daily, 5 days per week, for 2 weeks, followed by a 2 week rest period, via the central venous catheter. TPA was administered in a dose of 1mg in 200ml of physiological saline within 1 hour. 24 hours prior to the initiation of TPA therapy, patients were given 10mg dexamethasone every 6 hours and 1500mg choline magnesium trisalicylate every 8 hours until 24 hours after TPA administration.

Blood levels of TPA were measured before and after TPA infusion using a bioassay that measures organic solvent extractable differentiation activity. 1ml of blood was extracted twice with 5ml of ethyl acetate, the extraction residue was redissolved in 50. mu.L of ethanol and aliquots of HL60 cells were added. After 48 hours, adherent cells were measured.

Blood samples before and after TPA infusion were also tested to determine the levels of white blood cells, platelets, and neutrophils. The samples were also analyzed for the presence of medulloblasts and Oldham's rods. These and subsequent experiments will further elucidate the therapeutic cytotoxic effects and other effects of TPA on neoplastic cell elicitation in AML and other neoplastic and malignant conditions.

Example VIII

Measurement of modulation of ERK activation

Levels of phospho-ERK are measured in circulating malignant cells of patients with leukemia and in peripheral blood mononuclear cells of patients with lymphoma/solid tumors. Blood samples were taken from patients treated according to the protocol of example VII before and after administration of TPA.

In patients with white blood having WBC ≧ 1000/. mu.L, flow cytometry was performed on blood samples using cell surface antigen-specific antibodies and phospho-ERK-specific antibodies (BD Biosciences, San Jose, Calif.). Samples were taken before administration of TPA, after infusion of TPA1 hours on days 1, 2, and 11 of the initial treatment according to the protocol of example VII, and on days 1 and 11 of the subsequent cycle. In patients with leukemia with an absolute leukemic blast number of 2500/μ L or more and other non-leukemic patients, peripheral blood samples were taken before infusion and 1 and 4 hours after infusion on days 1, 8and 15 of the first cycle according to example VII. Samples were also analyzed for phospho-ERK and total ERK1/2 levels using Western blot analysis to confirm the results obtained from flow cytometry and the results associated with clinical response.

The above assays will further elucidate the role of TPA in the treatment of neoplasia and malignant conditions, including the cytotoxic effects of TPA on malignant cells (exemplified by primary AML cells), and the associated decrease in phosphorylation-ERK survival signaling by TPA.

Example IX

Measurement of NF- κ B modulation

In previous studies, we have demonstrated that NF- κ B activity in patients can be modulated following administration of TPA and dexamethasone. In addition, dexamethasone has been shown to induce MKP-1ERK phosphatase expression and enhance TPA cytotoxicity. The following study was designed to further elucidate how NF- κ B activity is therapeutically modulated in patients treated with TPA + dexamethasone.

NF- κ B binding was measured at baseline and in patient peripheral blood samples from patients treated with TPA as in example VII, as well as before and after infusion using an ELISA-based assay (BD Bioscience, San Jose, USA). The level of NF- κ B was quantified using chemiluminescence intensity in a defined amount of cell extract using a 96-well format to detect binding. In addition, electrophoretic mobility shift assays were performed to measure NF- κ B binding in peripheral blood samples of leukemia patients with absolute leukemic blast numbers ≧ 2500/μ L and other non-leukemia patients with normal white blood cell counts.

The above study will further show that PA is an NF-. kappa.B inducer; however, these experiments demonstrated that treatment with semethasone and choline magnesium trisalicylate inhibited AML cells NF- κ B.

Example X

Determination of alterations in leukemia Gene expression

TPA induces RNA levels of several bispecific phosphatases that are capable of terminating pro-survival ERK pathway signaling. RNA expression of AML signaling components (such as MAPK-specific DUSP) was studied using quantitative real-time RT-PCR and oligonucleotide microarray analysis, using blood samples taken before and after infusion from AML patients treated with TPA according to example VII.

Although the foregoing invention has been described in some detail by way of example for purposes of clarity of understanding, it will be obvious to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims and are presented by way of illustration rather than limitation. Various publications and other references are cited herein for economy of description. Each of these references is incorporated by reference herein in its entirety for all purposes. It should be noted, however, that the various publications mentioned herein are incorporated by reference only for their disclosure prior to the filing date of the present application and the inventors reserve the right to antedate such disclosure by virtue of prior invention.

Example XI

Treatment of lymphoma

Patient m.j., 60 years old, male, diagnosed with recurrence of lymphoma and a tumor diameter of 3.5 cm. The patient was dosed with 0.19mg TPA (0.125 mg/m) every other day²) Injections, counted 15 times, were performed for 30 days and the tumor disappeared. Since 2011 he has been in remission for three years.

Example XII

Treatment of breast cancer

Patient m.l., female, 50 years old, was diagnosed with advanced breast cancer. She did not respond to radiation or chemotherapy and the cancer had metastasized to the bone, rendering her need to sit in a wheelchair. She was administered three to four times a week with 0.18mg TPA (1X 0.125 mg/m)²) To 0.26mg TPA (1.5X 0.125 mg/m)²) In ascending dose35 TPA injections were received and are now relieved and able to walk normally.

Example XIII

Treatment of lung cancer

Patient j.l., male, 56 years old, was diagnosed with advanced lung cancer that was refractory to chemotherapy. The cancer has metastasized to his bone, rendering him unable to walk. Three to four times a week with 0.19mg TPA (1X 0.125 mg/m)²) To 0.26mg TPA (1.5X 0.125 mg/m)²) After 35 TPA injections, he had remitted and was able to walk normally.

Example XIV

Treatment of liver cancer

Patient X, male, of unknown age, was diagnosed with metastatic liver cancer. His initial alpha-fetoprotein level was 48,813. He was given chemotherapy and radiation therapy, but his alpha-fetoprotein levels were high at 50,000 +. He then received three times 0.19mg TPA (0.125 mg/m)²) Injected, and his alpha-fetoprotein levels began to decline and returned to normal levels within four months.

Example XV

TPA as an adjuvant for traditional neoplasm treatment

Patient n.k., female, age 54, was diagnosed with advanced metastatic cancer. In addition to chemotherapy, she received five times a week 0.18mg TPA (0.125 mg/m)²) Injections were performed for 12 weeks. The tumor in her pancreas decreased from 6.3cm to 2.4cm as treated. This patient maintained appetite, did not lose hair and vomit and nausea were significantly less frequent than previous chemotherapy treatments without TPA.

The patients were p.t., male,age 42, diagnosed with non-small cell lung cancer. The cancer has metastasized and used(erlotinib) and Iressa^TM(gefitinib) is difficult to treat. Patients were treated with gemcitabine (gemcitabine) in combination with cisplatin (cissplatin) following standard protocol per working day, with injection of 0.19mg TPA (0.125 mg/m)²) And the treatment is carried out for eight weeks. During the combined chemotherapy and TPA treatment he did not have any alopecia and the number of nausea was significantly less than experienced during the previous chemotherapy treatment. He has remitted since 30 days 6 months 2010.

Patient b.l., male, age 59, diagnosed with advanced nasopharyngeal carcinoma and treated with chemotherapy and radiotherapy concurrently. He received 0.19mg TPA (0.125 mg/m) per day before starting radiotherapy²TPA), for five days, and then every other day receives 0.19mg of TPA (0.125 mg/m)²) Total of 20 injections. He had been relieved for two and a half years and did not experience any significant skin damage from radiation treatment.

Example XVI

Chemical protection of TPA

Colony formation assays (including semi-solid media in DMEM and 0.5% agar) were used. For these cultures, monocytes were plated at approximately 2.5X 10⁵Individual cells/mL were plated and GM-CSF and G-CSF were added at a concentration of about 100U/mL. Cells were incubated at 5% CO at 100% humidity₂The culture was carried out in an incubator at 37 ℃ for 14 days. At the end of the incubation period, colonies of 50 or more cells were counted by two independent observers using an inverted microscope. (Hamburger,1977)

Will be at 5 × 10⁵Peripheral stem cells at individual cell/mL concentration in DMEM supplemented with 10% fetal bovine serum were randomly divided into 4 groups. Groups 1 and 4 are noneControl treated, and groups 2and 3 were incubated with 0.05 μ g/mL TPA for 24 hours. After 24 hours, cells were washed with DMEM 10% fetal bovine serum. Groups 3 and 4 were then incubated with 25 μ g/mL 5-fluorodeoxyuridine monophosphate (a metabolite of fluorouracil) for 20 hours. Subsequently, all groups were washed twice and the cells plated in semi-solid agar medium. Colonies were counted on day 14.

Example XVII

Use of TPA to protect against radiation damage

Three cell lines were used to determine the effect of TPA against radiation damage: interleukin-3 dependent murine hematopoietic stem cell line, human bone marrow stromal cell line KM101 and bronchial epithelial (IB3) cells. The 32D cl 3 interleukin-3 (IL-3) -dependent murine committed hematopoietic stem cell line was derived from a long-term bone marrow culture of C3H/HeJ mice, as described in Epperly, 2008. Cells were passaged in 15% WEHI-3 cell conditioned medium (as a source of IL-3), 10% Fetal Bovine Serum (FBS) (Hyclone Laboratories, Logan, UT), and McCoy's supplemented medium. At 24cm³Human bone marrow stromal cell line KM101 cells were passaged weekly in Mackoy's 5A modified medium (GIBCO BRL, Gaithersburg, Md.) supplemented with 10% FBS (Hyclee laboratories, Logan, UT) in Falcon plastic flasks. At 5% CO₂IB3 cells were passaged in a standard Dulbecco's Modified Eagle's Medium (DMEM) (Lonza, Allendale, NJ) supplemented with 10% FBS (Hycleela laboratories, Logan, UT), 1% L-glutamine (GIBCO BRL, Gaithersburg, MD) and 1% penicillin-streptomycin (GIBCO BRL, Gaithersburg, MD) at 37 ℃ for 48-72 hours to uncoated 75cm³Falcon tissue culture flasks to reach 80% confluence twice a week as described in Rwigema, 2011.

Cells from each cell line were plated at 1X 10⁵Individual cells/mL were suspended and irradiated at 0 to 8 Gy. After the irradiation was carried out for 10 minutes,TPA was added to the irradiated cells. The cells were then plated in quadruplicate and incubated in a high humidity incubator with 95% air/5% CO2 at 37 ℃ for 7 days, at which time the cells were stained with crystal violet and colonies of more than 50 cells were counted. Each experiment was performed 3 times separately on three different days. Data were analyzed using a linear quadratic model and a one-click multi-target model (see Epperley, 2001). The Dose Reduction Factor (DRF) of TPA was calculated as the ratio of the dose that produced 50% cell viability in the treated group divided by the dose at 50% viability in the control cell group.

Example XVIII

Protection of TPA against radiation injury in mice

Adult female C57BL/6NHsd mice (20 to 22g, Harlan Sprague Dawley, Chicago, IL) were irradiated with 9.5Gy TBI using gamma β irradiation dose rate (74cGy/min) to achieve the (LD 50/30) dose and received an intraperitoneal injection of 0.125mg/m 10 minutes later²TPA. Survival of mice was monitored (Rigwema, 2011).

Example XIX

Treatment of individuals who have suffered a stroke.

Patient n.c., male, 68 years old, suffered a stroke for 18 months prior to treatment with TPA. When starting TPA treatment he had to walk on a walking stick, with both the left hand and the left leg having difficulty moving and feeling tired and weak. He received 1 ampoule of an injection containing: 0.19mg TPA (0.125 mg/m)²) Every other day for 4 weeks; then 0.24mg TPA (1.25X 0.125 mg/m)²) Every other day for 2 weeks; and then 0.26mg TPA (1.5X 0.125 mg/m)²) Every other day for another 3 weeks. The patient is fully recovered.

Patient m.c., male, age 65 years, suffered a stroke for 7 years before starting treatment with TPA. He weeklyReceive 3 to 4 times 0.19mg TPA (0.125 mg/m)²) Injections were performed for 10 weeks for a total of 35 injections. His face had regained flexibility and the right side improved by 80%.

Example XX

Model for treating embolic stroke by TPA

Male Sprague-Dawley rats (Charles River Japan) were used, each weighing 280-350 g. Embolic stroke was induced according to the modified Kudo et al (1982). Rats for blood collection were treated with 1.0% halothane (Fluorothane) under spontaneous respiration^TM(ii) a Takeda, Osaka, Japan) anesthesia. Mixing Surflo No. 24^TM(Terumo medical Products, Elkton, Md.) was fixed in the femoral artery and 0.1mL of arterial blood was collected with a 1-mL syringe for injection (Terumo medical Products, Elkton, Md.). The arterial blood in the syringe was incubated at 30 ℃ for 2 days to form a blood clot. Thereafter, 0.1mL of physiological saline was added to the syringe for injection and passed through a 26-gauge injection needle (Terumo Medical Products, Elkton, MD) (twice) to break up blood clots.

Rats with induced embolic stroke were anesthetized with 1.0% halothane under spontaneous breathing. The rat neck was made a median incision and the external carotid artery, the superior thyroid artery, the occipital artery and the pterygopalatine artery were electrocauterized with a bipolar electrocoagulator (T-45; Keisei Medical Industrial Co. Ltd, Tokyo, Japan). Cerebral embolism was induced by injecting 0.1mL of the crushed blood clot into the neck.

Cerebral embolism formation was evaluated using a laser Doppler flowmeter (FloC 1; omega wave, Tokyo, Japan). A drop in cerebral blood flow to a level of 30% or less is considered conclusive evidence of the formation of an embolism. After the infusion of the clot, cerebral blood flow was monitored for 30 minutes, and blood flow was monitored to remain at 50% or less of the flow prior to the infusion of the clot. Thereafter, a cannula (PE50) for administration of the drug was secured in the jugular vein and the animal was awakened.

Will have becomeRats that successfully formed cerebral embolism were divided into four groups. The first group of rats was given saline injections every other day. Groups 2-4 were administered at 0.125mg/m every other day²Injection was performed for four weeks. Group 2 was then sacrificed. Groups 3-4 were given more every other day, i.e., 0.156mg/m²Two weeks were run and then group 3 was sacrificed. Group 4 was administered 0.18775mg/m every other day²Three weeks were performed and then sacrificed.

After sacrifice the brains were cut and 10 sections were cut at 1mm intervals using a McIwain tissue chopper (Mickle laboratory engineering, U.K.) and stained by immersion in 2% TTC (2,3, 5-triphenyltetrazolium chloride; Tokyo Kasei) for 20 minutes at 37 ℃. Images of the TTC-stained sections were uploaded to a computer using a digital camera (HC-2500; Fuji Photo Film) and Phatogab-2500 (Fuji Photo Film), and infarct volumes were calculated using Mac Scope (Mitani, Japan). Infarct volume is given as mean ± standard error. Statistical tests on infarct volume results were evaluated by: dunnett's test was performed on the control group as well as each TPA-administered group, compared to the control group, and then t-test was performed on the TPA-administered group.

Neurological symptoms were observed daily until sacrifice and rats were evaluated according to three tests: (1) gently lifting the tail of the rat, suspending the rat at a position one meter away from the ground, and observing the bending of forelimbs; (2) rats were placed on a large sheet of soft plastic-coated paper so that the claws could firmly grip the paper. Holding the rat tail and applying slight lateral pressure behind the rat shoulder until the forelimb glides several inches; (3) the rats were allowed free movement and observed for circling behaviour. The neurological symptoms were scored according to the scale developed by Bederson et al (1986) as follows: 0 minute: no defects were observed; bending forelimbs in a ratio of 1 min; and 2, dividing: the resistance to lateral thrusting is reduced without turning; and 3, dividing: the behavior is the same as 2-level, round.

The neurological symptoms of the control group and each TPA-administered group were evaluated using a Steel test, compared with the control group, and then a Wilcoxon test was performed on the TPA-administered group. In any of the tests, a value of p <0.05 was defined as statistically significant.

Example XXI

Effect of TPA in Stroke treatment Using permanent middle cerebral artery occlusion model

Male Wistar rats (250-320g) were used in this study. Animals were anesthetized with isoflurane (3% induction, 1-2% maintenance). Anesthesia was monitored by squeezing the toes. Sterile techniques were used for all procedures during the study. The surgical site was clamped and cleaned with alcohol and surgical scrub. Animals were placed on warm water heating pads to maintain body temperature. A medial-lateral incision was made in the neck above the carotid artery. Blunt resection of tissue exposes the carotid artery and the bifurcation. Sutures are placed around the proximal portions of the common and external carotid arteries. The sutures are tied. An incision is made in the common carotid artery distal to the ligation site. A pre-prepared filament (4-0 monofilament suture or similar material) is placed in the carotid artery and advanced into the internal carotid artery. The filament is advanced approximately 20mm through the carotid bifurcation until it feels a slight resistance when wedged in the middle cerebral artery. Care must be taken not to damage the artery when inserting the filament. The filaments were knotted in situ and the skin incision was closed. Successful occlusion was assessed while the animals were awake using the Bederson scale as previously described. (see Bederson et al, (1986) Stroke,17: 1304) -1308.) body temperature was taken every 15 minutes to maintain normothermia. Animals that have undergone a middle cerebral artery occlusion procedure may be difficult to thermoregulate hours after surgery, so they are placed in a cooling or heating cabinet depending on the animal's body temperature. Body temperature was maintained at 37.5 ℃. Animals were monitored for 6 hours post-cerebral arteriotomy and then placed in cages overnight.

Rats were divided into four groups. The first group of rats was given saline injections every other day. Groups 2-4 were administered at 0.125mg/m every other day²Injection was performed for four weeks. Group 2 was then sacrificed. Groups 3-4 were given more every other day, i.e., 0.156mg/m²Two weeks were run and then group 3 was sacrificed. Group 4 was administered 0.18775mg/m every other day²Three weeks were performed and then sacrificed.

After sacrifice the brains were cut and 10 sections were cut at 1mm intervals using a McIwain tissue chopper (Mickle laboratory engineering, U.K.) and stained by immersion in 2% TTC (2,3, 5-triphenyltetrazolium chloride; Tokyo Kasei) for 20 minutes at 37 ℃. Images of the TTC stained sections were uploaded to a computer using a digital camera (HC-2500; Fuji Photofilm) and Phatogab-2500 (Fuji Photofilm). Brain sections were photographed and analyzed for infarct size, infarct volume, penumbra, and edema.

Neurological symptoms were observed daily until sacrifice. Neurological symptoms were observed daily until sacrifice and rats were evaluated according to three tests. (1) The tail of the rat was lifted gently, suspended one meter from the ground, and the forelimb was observed to curve. (2) Rats were placed on a large sheet of soft plastic-coated paper so that the claws could firmly grip the paper. The rat tail was pulled and slight lateral pressure was applied behind the rat shoulder until the forelimbs slipped several inches. (3) The rats were allowed free movement and observed for circling behaviour. The neurological symptoms were scored according to the scale developed by Bederson et al (1986) as follows: 0 minute: no defects were observed; bending forelimbs in a ratio of 1 min; and 2, dividing: the resistance to lateral thrusting is reduced without turning; and 3, dividing: the behavior is the same as 2-level, round.

The neurological symptoms of the control group and each TPA-administered group were evaluated using a Steel test, compared with the control group, and then a Wilcoxon test was performed on the TPA-administered group. In any of the tests, a value of p <0.05 was defined as statistically significant.

Example XXII

Effect of TPA in Stroke treatment Using temporary middle cerebral artery occlusion model

Male C57B16 mice (25-30g) were used in this study. Mice were anesthetized with isoflurane (3% induction, 1-2% maintenance). The surgical site was clamped and cleaned with alcohol and surgical scrub. A median cervical incision was made above the carotid artery and the artery was dissected to its bifurcation. The monofilament suture is threaded into the internal carotid artery and advanced until it enters the middle cerebral artery. The suture is tied in place and the incision is closed. Two hours after occlusion, mice were re-anesthetized and the suture was removed from the MCA. Body temperature is maintained with the heating pad both during and after surgery. Animals were monitored for 4 hours after occlusion of the middle cerebral artery.

Rats were divided into four groups. The first group of rats was given saline injections every other day. Groups 2-4 were administered at 0.125mg/m every other day²Injection was performed for four weeks. Group 2 was then sacrificed. Groups 3-4 were given more every other day, i.e., 0.156mg/m²Two weeks were run and then group 3 was sacrificed. Group 4 was administered 0.18775mg/m every other day²Three weeks were performed and then sacrificed.

After sacrifice the brains were cut and 10 sections were cut at 1mm intervals using a McIwain tissue chopper (Mickle laboratory engineering, U.K.) and stained by immersion in 2% TTC (2,3, 5-triphenyltetrazolium chloride; Tokyo Kasei) for 20 minutes at 37 ℃. Images of the TTC stained sections were uploaded to a computer using a digital camera (HC-2500; Fuji Photofilm) and Phatogab-2500 (Fuji Photofilm). Brain sections were photographed and analyzed for infarct size, infarct volume, penumbra, and edema.

Neurological symptoms were observed daily until sacrifice and rats were evaluated according to three tests. (1) The tail of the rat was lifted gently, suspended one meter from the ground, and the forelimb was observed to curve. (2) Rats were placed on a large sheet of soft plastic-coated paper so that the claws could firmly grip the paper. The rat tail was pulled and slight lateral pressure was applied behind the rat shoulder until the forelimbs slipped several inches. (3) The rats were allowed free movement and observed for circling behaviour. The neurological symptoms were scored according to the scale developed by Bederson et al (1986) as follows: 0 minute: no defects were observed; bending forelimbs in a ratio of 1 min; and 2, dividing: the resistance to lateral thrusting is reduced without turning; and 3, dividing: the behavior is the same as 2-level, round.

The neurological symptoms of the control group and each TPA-administered group were evaluated using a Steel test, compared with the control group, and then a Wilcoxon test was performed on the TPA-administered group. In any of the tests, a value of p <0.05 was defined as statistically significant.

Example XXIII

Clinical efficacy of TPA for treatment of stroke

Men and women aged between 30-72 years who had suffered less than a month of previous stroke were enrolled in a 10 week TPA trial.

Enrolled individuals signed informed consent and evaluated using: computed Tomography (CT), physical and neurological tests, neurological examinations, sedation levels, national institute of Health, Stroke Survey (NIHSS), 12-lead electrocardiography, electrocardiographic telemetry, pulse oxygen measurements, vital signs, body weight, patient background, pregnancy tests, measurements of drugs in urine, hematological tests, coagulation panels, general clinical tests, urinalysis. Clinical laboratory tests included a Complete set of Metabolic function tests (Complete Metabolic Panel) (Na, K, Cl, CO2, Glu, BUN, Cr, Ca, TP, Alb, TBili, AP, AST, ALT), hematologic CBC (Hgb, Hct, RBC, WBC, Plt, Diff) and serum hCG for all women.

The individual is administered 0.125mg/m every other day²TPA or placebo for 4 weeks followed by administration of 1.25X 0.125mg/m every other day for week 5 and week 6²Or placebo and administered every other day for weeks 7 to 9 at 1.5X 0.125mg/m²Or a placebo. Subjects were monitored during TPA or placebo administration and 2 hours post administration.

Subjects were evaluated on weeks 5 and 10 using NIHSS (NIH Stroke Scale), Barthel ADL index (Granger,1979) and a modified Rankin Scale (Farrell,1991),

efficacy was determined by measuring the change in NIHSS from baseline in subjects treated with TPA compared to placebo. Secondary efficacy variables are Barthel ADL index and modified Rankin scale. Safety measures were collected and evaluated by the test, specifically measuring the change from baseline visit to week 5. These metrics include adverse event reports, physical examinations, vital signs, weight measurements, ECGs, clinical laboratory test results and vital signs, and the score of suicidal behavior and/or consciousness. An adverse event is any adverse medical event that occurs in a subject administered a study drug, whether or not it is causally related to the study drug. An adverse event may thus be any adverse or undesirable sign (including, for example, abnormal laboratory results), symptom, or condition that is temporarily associated with the study drug, whether or not such condition is considered to be associated with the test drug.

A subject is considered to have completed the study if it has completed all visits. If it does not meet the inclusion/exclusion criteria; suffering from an adverse event, inadequate response to treatment, withdrawal of consent, violation of protocol, self-withdrawal, or death, the study may be terminated.

Example XXIV

Reducing periorbital hyperpigmentation

6 women and 1 man with excessive periorbital hyperpigmentation were treated with TPA. The treatment reduces inflammation and hyperpigmentation in the periorbital region.

Example XXV

Treatment of carpal tunnel syndrome

A female patient, 50 years old, had burning, stinging, pain and numbness in her fingers. She was treated with TPA and returned to full use of her hands.

Example XXVI

Anti-aging Properties of TPA

Patient W.L, male, 82 years old, every other day in 1.25 ampules (0.125 mg/m)²) Treating prostatic hyperplasia for two months. After treatment with TPA, his skin became more tender and less wrinkled.

Example XXVII

Treatment of parkinson's disease

Patient s.k., 55 years old, diagnosed with parkinson's disease. He injected 1 ampoule every other day (containing 0.19mg TPA (0.125 mg/m)²) TPA, for four weeks, then every other day 0.24mg TPA (1.25X 0.125 mg/m)²) For 2 weeks, and every other day 0.26mg TPA (1.5X 0.125 mg/m)²) And then, the treatment is carried out for 3 weeks. The patient no longer showed tremors.

Example XXVIII

Treatment of prostatic hypertrophy

Patient w.l, male, 82 years old, was diagnosed with prostate hypertrophy. He injected 1 ampoule every other day (containing 0.19mg TPA (0.125 mg/m)²) Four weeks apart, followed by injection of 0.24mg TPA (1.25X 0.125 mg/m) every other day²) For 2 weeks, and every other day 0.26mg TPA (1.5X 0.125 mg/m)²) And then, the treatment is carried out for 3 weeks. His PSA index is now below 3.0. The patient also exhibits a younger appearance.

Example XXIX

Treatment of rheumatoid arthritis

Patients received multiple TPA injections and seen a reduction in joint stiffness and an increase in functionality of their hands.

Example XXX

Treatment of collagen-induced arthritis with TPA

Collagen-induced arthritis (CIA) was induced as described previously (Rosloniec 2001) with minor modifications. Briefly, the tail roots of male DBA/1J mice were injected intradermally with 100 μ g of chicken type II collagen (Chondrex, Redmond, WA) in 0.05M acetic acid (emulsified in freund's complete adjuvant (Difco, Detroit, MI)). Twenty-one days after the primary immunization, mice were given booster injections of type II collagen at the same dose level.

Mice were carefully examined daily for the visual appearance of arthritis in their peripheral joints from day 18 after the first collagen injection. The clinical severity of arthritis was scored as follows: each of swollen toes except the thumb was divided into 1 point (highest 4 points), tarsal bones or wrist joints and metatarsal bones or metacarpal joints, wherein the highest point of the hind paw was divided into 6 points and the highest point of the anterior paw was divided into 5 points. Each paw was scored individually, with a cumulative clinical arthritis score of up to 22 per mouse.

After receiving the booster injection, the mice were divided into two groups and received vehicle or 0.125mg/m intraperitoneally on day 0, day 2, day 4, day 6and day 8²TPA. On day 30 after the initial injection, mice were sacrificed and knees were excised and fixed in 10% buffered formalin for 7 days. The fixed tissue was decalcified in 15% EDTA for 3 weeks, dehydrated and embedded in paraffin. Sagittal sections (8 μm) of the total knee were stained with safranin-O and counterstained with fast green/iron hematoxylin. Tissue sections were scored independently by two observers unaware of animal treatment using an established scoring system for synovial hyperplasia (from 0 (no hyperplasia) to 3 (hyperplasia is most severe)) and inflammatory cells in the synovium (0 (no inflammation) to 3 (joint severe inflammation)). Cartilage damage was judged by safranin-O staining as described in Marty,2001, from 0 (no change compared to normal non-arthritic knee joints, complete staining of cartilage) to 3 (total loss of safranin-O staining).

Example XXXI

Treatment of myasthenia gravis

Patient c.l., male, age 63, with myasthenia gravis for more than 40 years. He took dexamethasone and injected 1 ampoule every other day (containing 0.19mg TPA (0.125 mg/m)²) Four weeks apart, followed by injection of 0.24mg TPA (1.25X 0.125 mg/m) every other day²) For 2 weeks, and every other day 0.26mg TPA (1.5X 0.125 mg/m)²) And further 3 weeks for a total of 35 TPA injections. He no longer needs dexamethasone and is today asymptomatic.

Example XXXII

Treatment of urinary incontinence

Patient w.c., female, age 61, had urinary incontinence for more than 30 years. She received six times 0.18mg TPA (0.125 mg/m)²) And has restored normal micturition frequency.

Patient l.j., female, age 48, had urinary incontinence changed for 15 years. She received nine times 0.18mg TPA (0.125 mg/m)²) And has restored normal micturition frequency.

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Claims (16)

1. Use of an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof, for the manufacture of a medicament for preventing or treating one or more radiation side effects in a mammalian subject

Wherein R is₁And R₂Selected from hydrogen and hydroxylA base,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenyl, lower alkenyl,Phenyl, phenyl,Benzyl and substituted derivatives thereof, R₃Is hydrogen,And substituted derivatives thereof, and to the use of such compounds,

wherein the one or more radiation side effects preclude radiation-induced skin damage.

2. The use according to claim 1, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

3. The use 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 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

4. The use according to claim 1, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

5. The use of claim 1, further comprising administering to the subject at least one secondary or adjunctive therapeutic agent effective to treat or prevent a radiation side effect in a combined preparation or co-therapeutic regimen with the phorbol ester of formula I or derivative compound.

6. The use according to claim 5, wherein in a co-administration regimen the at least one secondary or adjunctive therapeutic agent is administered to the subject simultaneously with, prior to, or subsequent to the administration of the phorbol ester to the subject.

7. The use of claim 6, wherein the at least one secondary or adjunctive therapeutic agent is selected from the group consisting of steroids, amifostine, chlorhexidine, benzydamine, sucralfate, KGF, palivumin, Cu/Zn superoxide dismutase, interleukin 11, or prostaglandins.

8. The use of claim 1, wherein the one or more radiation side effects is diarrhea, nausea, vomiting, loss of appetite, constipation, aphtha and sore throats, edema, infertility, fibrosis, hair loss, or mucosal dryness, or a combination thereof.

9. The use of claim 1, wherein the effective amount comprises between about 10 μ g and about 1500 μ g per day of the phorbol ester or derivative compound of formula I.

10. The use of claim 1, wherein the effective amount comprises between about 125 μ g to about 500 μ g per day of the phorbol ester or derivative compound of formula I.

11. A composition for preventing or treating one or more radiation side effects in a mammalian subject comprising an effective amount of a phorbol ester or derivative compound of formula I, or a pharmaceutically acceptable salt, isomer, enantiomer, solvate, hydrate, polymorph or prodrug thereof

Wherein R is₁And R₂Selected from hydrogen, hydroxy,Alkyl, aryl, heteroaryl, and heteroaryl,Lower alkenyl, lower alkenyl,Phenyl, phenyl,Benzyl and substituted derivatives thereof, R₃Is hydrogen,And substituted derivatives thereof, and to the use of such compounds,

wherein the one or more radiation side effects preclude radiation-induced skin damage.

12. The composition of claim 11, wherein R₁Or R₂Is composed ofAlkyl radical, the remainder of R₁Or R₂Is composed ofLower alkyl and R₃Is hydrogen.

13. The composition according to claim 11, 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 20-acetate, 12-deoxyphorbol 13-isobutyrate, phorbol 12-deoxyphorbol 13-isobutyrate, 12-deoxyphorbol 13-isobutyrate-20-acetate, 12-deoxyphorbol 13-phenylacetate 20-acetate, 12-deoxyphorbol 13-tetradecanoate, phorbol 12-tiglate 13-decanoate, 12-deoxyphorbol 13-acetate, phorbol 12-acetate or phorbol 13-acetate.

14. The composition according to claim 11, wherein the phorbol ester is 12-O-tetradecanoyl phorbol-13-acetate.

15. The composition of claim 11, further comprising at least one secondary or adjunctive therapeutic agent effective to treat or prevent radiation side effects in the subject as a combined preparation with the phorbol ester of formula I or derivative compound.

16. The composition of claim 11, wherein the one or more secondary or adjunctive therapeutic agents is a steroid, amifostine, chlorhexidine, benzydamine, sucralfate, KGF, palivumin, Cu/Zn superoxide dismutase, interleukin 11, or a prostaglandin.