KR20070116868A - ３ 의 Medicinal use of desmethoxy rapamycin and its analogs - Google Patents

Abstract

The present invention relates to the medical use of the 39-desmethoxyrapamycin analog.

Description

Medical uses of 39-desmethoxyrapamycin and analogues

Rapamycin (Syrrolimus) (FIG. 1) is known as Streptomyces Streptomyces. hygroscopicus ) is generated by NRRL 5491 (Sehgal et. al . , 1975; Vezina et al . , 1975; US Patent No. 3,929,992; U.S. Patent No. 3,993,749), a lipophilic macrolide with 1,2,3-tricarbonyl moieties linked to pipecolic acid lactones (Paiva et. al . , 1991). For the purposes of the present invention, rapamycin is described in Findlay et. al . (1980) or McAlpine et al. Rather than the numbering arrangement of Chemical Abstracts (11th edition, Cumulative Index, 1982-1986 p60719CS). al . It is described by the numbering arrangement of (1991).

Rapamycin has effective therapeutic value due to its broad biological activity spectrum (Huang et. al . , 2003). The compound is a mammalian target of rapamycin, a serine-threonine kinase downstream of the phosphatidylinositol 3-kinase (PI3K) / Akt (protein kinase B) signaling pathway that mediates cell survival and proliferation. , mTOR) is a potent inhibitor. This inhibitory activity is obtained after rapamycin binds to immunophiliin FK506 binding protein 12 (FKBP12) (Dumont, FJ and QX Su, 1995). In T cells, rapamycin inhibits signaling by IL-2 receptors followed by autoproliferation of T cells, resulting in immunosuppression. Rapamycin is marketed as an immunosuppressive agent for the treatment of organ transplant patients to prevent graft rejection (Huang et. al . , 2003). In addition to immunosuppression, rapamycin can be used in numerous diseases, such as cancer, cardiovascular diseases such as restenosis, autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, fungal infections and Alzheimer's disease, It has strong therapeutic use in the treatment of neurodegenerative diseases such as Parkinson's disease and Huntington's chorea.

Despite the usefulness of rapamycin in various disease states, rapamycin has a number of major problems. First, it is the substrate of the membrane-leave pump, P-glycoprotein (P-gp), which pumps the compound out of the cell, making cell membrane permeation poorly by rapamycin (LaPlante et. al . , 2002, Crowe et al. , 1999). This leads to poor absorption of the compound after administration. In addition, rapamycin is less effective against multi-drug resistance (MDR) cancer cells because the main mechanism of multi-drug resistance of cancer cells is via a cell membrane drain pump. Second, rapamycin is extensively metabolized by cytochrome P450 enzymes (Lampen et al . , 1998). The first pass in the liver is lossy, which also contributes to the low oral bioavailability of rapamycin. The role of CYP3A4 and P-gp in the low bioavailability of rapamycin has been shown that administration of CYP3A4 and / or P-gp inhibitors reduced the release of rapamycin from CYP3A4-transfected Caco-2 cells (Cummins et. al . , 2004). Administration of a CYP3A4 inhibitor decreased the intestinal metabolism of rapamycin (Lampen et. al . , 1998). Low oral bioavailability of rapamycin leads to significant variability between individual individuals, resulting in inconsistent treatment results and difficulties in clinical application (Kuhn et al . , 2001, Crowe et. al . , 1999).

Thus, there has been a need for the development of novel rapamycin-like compounds, which are not substrates of P-gp and can be metabolically more stable and have improved bioavailability. Such compounds, when used as anticancer agents, may have better efficacy against MDR cancer cells, particularly P-gp-expressing cancer cells.

A significant range of synthesized rapamycin analogs have been reported using chemically available sites of the molecule. The following compound description was adapted to the numbering system of rapamycin molecules described in FIG. 1. Chemically available sites in the molecule for derivatization or substitution include C40 and C28 hydroxy groups (eg, US Pat. No. 5,665,772; US Pat. No. 5,362,718), C39 and C16 methoxy groups (eg WO Pat. 96/41807; US Pat. No. 5,728,710), C32, C26, and C9 keto groups (eg, US Pat. No. 5,378,836; US Pat. No. 5,138,051; US Pat. No. 5,665,772). For the purpose of trienes, hydrogenation at C17, C19 and / or C21 retained antimicrobial activity but yielded relatively low immune suppression (eg, US Pat. No. 5,391,730; US Pat. No. 5,023,262). Significant improvements in molecular stability (eg, formation of oximes at C32, C40 and / or C28, US Pat. No. 5,563,145, US Pat. No. 5,446,048), and significant improvements in resistance to metabolic attack ( For example, US Pat. No. 5,912,253), significant improvements in bioavailability (eg US Pat. No. 5,221,670; US Pat. No. 5,955,457; WO 98/04279) and generation of prodrugs (eg For example, US Pat. No. 6,015,815; US Pat. No. 5,432,183) has been achieved through derivatization.

Two of the most advanced rapamycin analogues in clinical development are 40-O- (2-hydroxy) ethyl-rapamycin (RAD001, Certican, everolimus), a semi-synthetic analogue of rapamycin that shows immunosuppressive pharmacological effects. (Sedrani, R. et al . , 1998; Kirchner et al . , 2000; US Pat. No. 5,665,772) and in vitro ( in inhibiting cell growth in vitro) and in vivo (in CCI-779 (Wyeth-Ayerst) (Yu et ), 40-O- [2,2-bis (hydroxymethyl) propionyloxy] rapamycin, an ester of rapamycin that inhibits tumor growth in vivo ) al . , 2001). CCI-779 is currently a potential anticancer drug in various clinical trials. A recent publication (Chang, et al. , 2005) on the CCI-779 Phase II study in patients with recurrent glioblastoma multiforme suggests that the low efficacy of this drug in these patients is associated with the blood brain barrier. It is suggested that this may be due to poor permeation of CCI-779 in the blood-brain barrier. A study examining the pharmacokinetics of RAD001 shows that RAD001, like rapamycin, is a substrate of P-gp (Crowe et. al . , 1999, LaPlante et al . , 2002).

Despite the close structural similarity with rapamycin, the compounds of the present invention show surprisingly different pharmacological profiles. Specifically, they showed significantly increased cell membrane permeability and reduced excretion compared to rapamycin, which were not substrates of P-gp. In addition, 39-desmethoxyrapamycin showed more potent activity against multi-drug resistant and P-gp-expressing cancer cell lines than rapamycin. When compared to rapamycin, 39-desmethoxyrapamycin shows a significantly different inhibition profile for the NCI 60 cell line panel.

In addition, the 39-desmethoxyrapamycin analogue showed a significantly different pharmacokinetic profile compared to rapamycin and the leading derivative in clinic. Unexpectedly, the 39-desmethoxyrapamycin analogue showed increased ability to cross the blood brain barrier and thus demonstrated improved availability in the brain.

Accordingly, the present invention provides the medical use of 39-desmethoxyrapamycin analogs, which have significantly altered pharmacokinetics, improved ability to cross the blood brain barrier, improved metabolic stability, improved Cell membrane permeability, reduced excretion rate and tumor cell inhibition profile different from rapamycin. Such compounds may be used in medicine, specifically for treating cancer and / or B-cell malignancies, inducing or maintaining immune suppression, promoting neuronal regeneration or fungal infections, tissue transplant rejection, graft versus host disease ( graft vs. host disease, autoimmune disorders, inflammatory diseases, vascular diseases and fibrotic diseases. The present invention particularly provides the use of 39-desmethoxyrapamycin in the treatment of cancer and / or B-cell malignancies.

Rapamycin has been shown to promote autophagy (Raught et al. , 2001). Attenuated self depletion or dysregulation of self depletion is associated with a number of diseases including Alzheimer's disease, Parkinson's disease, Huntington's chorea and mad cow disease (including Creutzfeldt-Jakob disease). I suggest you can help. Recent in vitro studies have demonstrated that administration of rapamycin can reduce the appearance and aggregation of aggregates associated with poly (Q) and poly (A) expansion in transfected COS-7 cells (Ravikumar et. al . , 2002). Thus, if rapamycin can cross the blood brain barrier, these results indicate that it can make suitable candidates for the treatment of Huntington chorea and other related diseases. This suggests a need for the development of rapamycin analogs that can cross the blood brain barrier.

Hyperphosphorylation of microtubule-associated protein tau and subsequent aggregation into insoluble paired spiral filaments that produce intracellular "tangles" is one of the typical features of Alzheimer's disease. Neuronal death associated with the accumulation of neurofibrillary pathology is closely related to cognitive decline. An et al . A recent study by (2003) demonstrated that activated p70 S6 kinase was distributed simultaneously with neurofibrotic etiology in the brain of Alzheimer's patients, and especially activated p70 S6 kinase was markedly increased in nerves before the lesions grew ( An et al . , 2003). In in vitro assays where the administration of zinc sulfide resulted in activation of p70 S6 kinase, and overall increased levels of normal and hyperphosphorylated tau, cell pretreatment of rapamycin resulted in a 3-fold decrease in the activity of p70 S6 kinase and was normal and excessive. It was shown that the overall increased level of phosphorylated tau was significantly reduced. Thus, these results indicate that if rapamycin or rapamycin analogs can reach the site of action, administration of such compounds may help to reduce the neurofibrotic etiology of Alzheimer's disease.

In addition, rapamycin has been reported to increase neuritic outgrowth and neuronal survival in many in vitro and in vivo models (Avramut and Achim, 2002), suggesting that rapamycin and its analogs have significant therapeutic regeneration. It may be useful when treating a disease that may be helpful. However, since such utility depends on the compound being able to reach the site of action, rapamycin analogs with improved ability to cross the blood brain barrier would be particularly desirable.

The present invention specifically relates to induction or maintenance of immune suppression, promotion of nerve regeneration or fungal infection, tissue transplant rejection response, graft versus host disease, autoimmune disorders, neurodegenerative conditions, in the treatment of cancer or B-cell malignancies, A novel and surprising use of 39-desmethoxyrapamycin analogs in medicine, specifically 39-desmethoxine, for the treatment of inflammatory diseases, vascular diseases and fibrotic diseases. In particular, the present invention provides the use of 39-desmethoxyrapamycin analogs in the treatment of cancer and B-cell malignancies. In a preferred embodiment, the present invention provides the use of 39-desmethoxyrapamycin analogs in the treatment of neurological or neurodegenerative diseases. In a more preferred embodiment, the present invention provides the use of 39-desmethoxyrapamycin analogs in the treatment of brain tumors, specifically in the treatment of glioblastoma multiforme. In certain embodiments of the invention, the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin.

Summary of the Invention

The present invention specifically relates to the treatment of cancer and / or B-cell malignancies, induction or maintenance of immune suppression, tissue transplant rejection, graft versus host disease, autoimmune disorders, neurodegenerative conditions, inflammatory diseases, vascular diseases and fibrotic It relates to the medical use of 39-desmethoxyrapamycin analogs, in particular the use of 39-desmethoxyrapamycin, in the treatment of diseases, the promotion of nerve regeneration or the treatment of fungal infections. In particular, the present invention relates to the use of 39-desmethoxyrapamycin analogs for the treatment of cancer and B-cell malignancies. In certain embodiments, the present invention relates to the use of 39-desmethoxyrapamycin in the treatment of cancer and B-cell malignancies. The present invention also provides the use of 39-desmethoxyrapamycin analogs in the treatment of brain tumor (s) or neurodegenerative conditions. In certain embodiments, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of brain tumor (s) or neurodegenerative conditions. The present invention also specifically provides the use of 39-desmethoxyrapamycin analogs in the treatment of neurodegenerative conditions. In particular, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of neurodegenerative conditions.

Justice

The article was used in the present specification to refer to "a (a)" and "a (an)" is one or more than one of the grammatical object (that is, at least one) of the article. For example, "an analog" means one analog or more than one analog.

When the term “ autoimmune disorder (s)” is used, this includes, without limitation, systemic lupus erythrematosis (SLE), rheumatoid arthritis, myasthenia gravis and multiple sclerosis.

When the term “ inflammatory disease ” is used herein, it includes, without limitation, psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel disease (including but not limited to ulcerative colitis and Crohn's disease), pulmonary inflammation ( Asthma, chronic obstructive pulmonary disease, emphysema, acute respiratory distress, and bronchitis), rheumatoid arthritis and eye uveitis.

When the term " cancer " is used herein, it is malignant or benign of cells in skin or body organs, such as, without limitation, breast, prostate, lung, kidney, pancreas, brain, stomach or bowel. Refers to growth. Cancer tends to seep into adjacent tissues and spread (transfer) to distant organs, such as bone, liver, lungs, or brain. When the term cancer is used herein, it is not limited to metastatic tumor cell types such as, but not limited to, melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma, rectal cancer, prostate cancer, small Tissues such as small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, kidney cancer, gastric cancer, glioblastoma, primary liver cancer, and ovarian cancer All forms of tumor are included. The term also specifically includes brain tumor (s) as described more fully below.

When the term “brain tumor (s)” is used herein, it refers to malignant or benign growth of cells in the brain, which includes primary tumors and secondary (metastatic) tumors. Primary brain tumors include, but are not limited to, glioma (e.g., polymorphic glioblastoma, astrocytoma, brain stem glioma, ventricular hematoma and oligodendroglioma), stromal blastoma ( medulloblastoma, meningioma, schwannoma (or auditory nerve tumor), craniphaphagiogioma, germ cell tumor of the brain (e.g. germinoma) or pineal area tumor region tumors). The term “ brain cancer ” is also used to describe the set of diseases, which terms are used interchangeably herein.

When the term “B-cell malignancy” is used herein, it refers to a herd of diseases including chronic lymphocytic leukaemia (CLL), multiple myeloma, and non-Hodgkin's lymphoma (NHL). Include. These are tumor diseases of the blood and blood forming organs. They cause dysfunction of the bone marrow and immune system, making the host very sensitive to infection and bleeding.

When the term “vascular disease” is used herein, it is without limitation hyperproliferative vascular disease (eg restenosis and vascular occlusion), graft vascular atherosclerosis, heart Vascular diseases, cerebrovascular diseases and peripheral vascular diseases (eg, coronary artery disease, atherosclerosis, atherosclerosis, nonatheromatous atherosclerosis or vascular wall damage). It is also used to refer to diseases involving the neoplasia or proliferation of blood vessels in the eye, in particular those involving choroidal neovascularization.

When the term " nerve regeneration " is used herein, it refers to the promotion of nerve cell growth and includes neurite proliferation and functional recovery of nerve cells. Physical injury (eg, spinal cord injury or traumatic neuropathy, sciatic or facial nerve lesion or injury), or disease state (eg, diabetes) may include, without limitation, diseases and disorders in which nerve regeneration may be an effective therapeutic benefit. Alzheimer's disease, Parkinson's disease, Huntington's chorea, trigeminal neuralgia, Yeti neuralgia, facial nerve palsy, muscular dystrophy, seizures, progressive muscular atrophy, progressive spherical hereditary muscular atrophy progressive bulbar inherited muscular atrophy, cervical spondylosis, Gullain-Barre syndrome, dementia, peripheral neuropathies and peripheral nerve damage.

When the term “medical condition due to nerve injury or disease” is used herein, it is a neurodegenerative condition (s), brain tumor (s), infection or inflammation of the brain, and death or dysfunction of nerve or glial cells or tissues. Other conditions that can cause are but are not limited to these.

When the term “neurodegenerative state (s)” is used herein, it refers to Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis (ALS), and muscular dystrophy (of optic pharynx). ) (Including muscular dystrophy of the pharynx), multiple sclerosis, mad cow disease (including, for example, Creutzfeldt-Jacob disease (CJD)), PEEK disease, Lewy body dementia (or Lewy body disease), and And / or motor neurological disorders.

As used herein, the term "medical condition affecting the central nerve in need of a medicament that crosses the blood brain barrier," refers to a medical condition resulting from nerve damage or disease, and for the treatment of which the medical use of nerve cells is effective. It includes but is not limited to any other state required.

When the term “fibrotic disease” is used herein, it refers to a disease associated with overproduction of extracellular matrix, and (without limitation) sarcoidosis, keloid, glomerulonephritis , End-stage renal disease, liver fibroids (including but not limited to sclerosis, alcoholic liver disease and steato-hepatitis), chronic graft nephropathy, surgical stenosis, angiopathy, cardiac fibrosis, pulmonary fibrosis (including but not limited to idiopathic pulmonary fibrosis and cryptogenic fibrosing alveolitis), macular degeneration, retinopathy of the retina and vitreous, and chemotherapy or radiation-induced Fibrosis.

When the term “transplantation versus host disease” is used herein, it refers to the complication observed after allogeneic stem cell / bone marrow transplantation. This occurs when infection-fighting cells from the donor perceive the patient's body as different or heterogeneous. These infection-fighting cells then attack the tissues in the patient's body as they attack the infection. GvHD is classified as acute when it occurs within the first 100 days after transplantation and chronic when it occurs more than 100 days after transplantation. Typically, tissues of interest include the liver, the gastrointestinal tract, and the skin. Chronic graft versus host disease occurs in approximately 10-40 percent of patients after transplanting stem cells / bone marrow.

When the term "bioavailability" is used herein, it refers to the extent or proportion after which the drug or other substance is absorbed or made available at the biologically active site. These characteristics depend on a number of factors including the solubility of the compound, the rate of absorption in the digestive tract, the degree of protein binding and metabolism, and the like. Various tests for bioavailability that are familiar to those skilled in the art are described herein (see also Trepanier et. al . , 1998, Gallant-Haidner et al . , 2000).

When the term "cancer or B-cell malignancy tumor resistant to one or more existing anticancer agent (s)" is used herein, it refers to a cancer or B-cell malignancy tumor in which one or more commonly used treatments are ineffective. . Such cancers may occur after administration of one or more tumor agents where normal cell counterparts (ie, growth regulated cells of the same origin) show signs of cytotoxicity, apoptosis or cell arrest (ie, non-dividing). It is characterized by being able to survive. Specifically, this includes MDR cancer or B-cell malignancies, specific examples being cancer and B-cell malignancies that express high levels of P-gp. Identifying such resistant cancers or B-cell malignancies is within the ability and normal activity of the physician or other similar skilled person.

When the term “39 -desmethoxyrapamycin analog” is used herein, it refers to a compound according to formula (I), or a pharmaceutically acceptable salt thereof:

In the above, R ₁ represents (H, H) or = 0, and R ₂ and R ₃ each independently represent H, OH or OCH ₃ . This compound is also referred to as " compound of the present invention" , which term is used interchangeably in this application.

In the present application, the term “39 -desmethoxyrapamycin analogue” includes 39-desmethoxyrapamycin itself.

When the term “39 -desmethoxyrapamycin ” is used herein, it represents a compound of Formula (I) or a pharmaceutically acceptable thereof, wherein R ₁ represents ═O and R ₂ and R ₃ each represent OCH ₃ . Refers to possible salts.

Pharmaceutically acceptable salts of the 39-desmethoxyrapamycin analogues include quaternary ammonium acid addition salts as well as conventional salts produced from pharmaceutically acceptable inorganic or organic acids or bases. More specific examples of suitable acid salts include hydrochloride, hydrobromide, sulfate, phosphate, nitrate, perchlorate, fumarate, acetate, propionate, succinate, glycolate, formate, lactate, maleate, tartarate, citrate , Palmoic acid salt, malonate, hydroxymaleate, phenylacetate, glutamate, benzoate, salicylate, fumarate, toluenesulfonate, methanesulfonate, naphthalene-2-sulfonate, benzenesulfonate Salts, hydroxynaphthoic acid salts, hydroiodide salts, malic acid salts, steroic acid salts, tannic acid salts and equivalents thereof. Other acids, such as oxalic acid, are by themselves not pharmaceutically acceptable, but may be useful in the preparation of salts useful as intermediates when obtaining compounds of the invention and their pharmaceutically acceptable salts. More specific examples of suitable base salts include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and Procaine salts. The following references to the compounds according to the invention include both 39-desmethoxyrapamycin and its pharmaceutically acceptable salts.

Detailed description of the invention

The present invention relates to the use of 39-desmethoxyrapamycin analogs in medicine, specifically for the treatment of cancer, B-cell malignancies, induction or maintenance of immune suppression, tissue transplant rejection, graft versus host disease, autoimmune disorders, It relates to the use of 39-desmethoxyrapamycin analogs in the treatment of neurodegenerative conditions, inflammatory diseases, vascular diseases and fibrotic diseases, the promotion of nerve regeneration, the treatment of neurological or neurodegenerative conditions or the treatment of fungal infections. Accordingly, the present invention provides the use of a 39-desmethoxyrapamycin analog or a pharmaceutically acceptable salt thereof in the treatment of a medical condition due to nerve injury or disease. In certain embodiments, the present invention provides the use of 39-desmethoxyrapamycin or a pharmaceutically acceptable salt thereof in the treatment of a medical condition due to nerve injury or disease. In another embodiment, the present invention relates to the use of rapamycin and another 39-desmethoxyrapamycin analogue at one or more of positions 9, 16 or 27 in the treatment of a medical condition due to nerve injury or disease. to provide.

In addition, the present invention provides a 39-desmethoxyrapamycin analogue in the treatment of a medical condition that affects the central nerve in need of medicine across the blood brain barrier, ie, a medical condition in which the blood brain barrier interferes with the delivery of the compound. That is, the use of a rapamycin analogue or pharmaceutically acceptable salt thereof having increased blood brain barrier permeability. In certain embodiments, the present invention provides the use of 39-desmethoxyrapamycin or a pharmaceutically acceptable salt thereof in the treatment of a medical condition in which the blood brain barrier affects the central nervous system that interferes with the delivery of the compound. In another embodiment, the present invention provides an additional treatment wherein the blood brain barrier affects the central nervous system, which interferes with the delivery of the compound, in addition to rapamycin at one or more of positions 9, 16 or 27. -The use of desmethoxyrapamycin analogs.

In certain embodiments, the invention relates to the use of a 39-desmethoxyrapamycin analog for the treatment of cancer and B-cell malignancies. In another embodiment, the present invention relates to the use of 39-desmethoxyrapamycin for the treatment of cancer and B-cell malignancies. In another embodiment, the present invention is directed to the use of rapamycin and another 39-desmethoxyrapamycin analogue at one or more of positions 9, 16 or 27 for the treatment of cancer and B-cell malignancies. It is about. The present invention also specifically provides the use of 39-desmethoxyrapamycin analogs in the treatment of brain tumor (s). The present invention also provides the use of 39-desmethoxyrapamycin in the treatment of brain tumor (s). In another embodiment, the present invention provides the use of rapamycin and additionally another 39-desmethoxyrapamycin analog at one or more of positions 9, 16 or 27 in the treatment of brain tumor (s). In particular, the present invention provides the use of 39-desmethoxyrapamycin analogs in the treatment of glioblastoma multiforme. In certain embodiments, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of glioblastoma multiforme. In another embodiment, the present invention provides the use of rapamycin and another 39-desmethoxyrapamycin analogue at one or more of positions 9, 16 or 27 in the treatment of glioblastoma multiforme.

The present invention also provides the use of 39-desmethoxyrapamycin analogs in the treatment of neurodegenerative conditions. In another embodiment, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of neurodegenerative conditions. In another embodiment, the present invention provides the use of rapamycin and another 39-desmethoxyrapamycin analog in one or more of positions 9, 16 or 27 in the treatment of a neurodegenerative condition. Specifically, the neurodegenerative condition can be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington chorea. Thus, in one embodiment, the present invention provides the use of a 39-desmethoxyrapamycin analog in the treatment of Alzheimer's disease. In another embodiment, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of Alzheimer's disease. In another embodiment, the present invention provides the use of rapamycin and another 39-desmethoxyrapamycin analog further at one or more of positions 9, 16 or 27 in the treatment of Alzheimer's disease. In another embodiment, the present invention provides the use of a 39-desmethoxyrapamycin analog in the treatment of multiple sclerosis. In another embodiment, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of multiple sclerosis. In another embodiment, the present invention provides the use of rapamycin and another 39-desmethoxyrapamycin analogue additionally at one or more of positions 9, 16 or 27 in the treatment of multiple sclerosis. In an alternative embodiment, the present invention provides the use of a 39-desmethoxyrapamycin analog in the treatment of Huntington's chorea. In yet another embodiment, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of Huntington's chorea. In another embodiment, the present invention provides the use of rapamycin and another 39-desmethoxyrapamycin analogue at one or more of positions 9, 16 or 27 in the treatment of Huntington's chorea.

In an alternative embodiment, the invention relates to 39-desmethoxyrapamycin analogs, specifically 39-desmethoxyrapamycin, or to rapamycin, which further differs from at least one of positions 9, 16 or 27; A method of treating cancer or B-cell malignancies, a method of inducing or maintaining immune suppression and promoting nerve regeneration, fungal hepatitis, tissue transplant rejection, comprising administering an effective amount of desmethoxyrapamycin analog to a patient Provided are methods for treating transplantation versus host disease, autoimmune disorders, neurodegenerative conditions, inflammatory diseases, vascular diseases or fibrotic diseases. In particular, the present invention provides a method of treating a medical condition due to nerve damage or disease, comprising administering a 39-desmethoxyrapamycin analog or a pharmaceutically acceptable salt thereof. In certain embodiments, the present invention provides a method of treating a medical condition due to nerve injury or disease, comprising administering 39-desmethoxyrapamycin. In another embodiment, the invention provides medical care for a nerve injury or disease, comprising administering rapamycin and another 39-desmethoxyrapamycin analog additionally at one or more of positions 9, 16, or 27. Provides a method of treating the condition. In addition, the present invention provides for the administration of 39-desmethoxyrapamycin analogs, ie, rapamycin analogs with increased blood brain barrier permeability, or pharmaceutically acceptable salts thereof, thereby preventing the blood brain barrier from interfering with the delivery of the compound. A method of treating a medical condition affecting the nervous system is provided. In certain embodiments, the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment, the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27.

Preferably, the present invention provides a method of treating cancer or B-cell malignancies comprising administering to a patient an effective amount of a 39-desmethoxyrapamycin analog. In another preferred embodiment, the present invention provides a method of treating brain tumor (s) comprising administering to a patient an effective amount of a 39-desmethoxyrapamycin analog. In certain embodiments, the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment, the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. In certain embodiments, the invention provides a method of treating multiple sclerosis comprising administering to a patient an effective amount of a 39-desmethoxyrapamycin analog. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27.

In another preferred embodiment, the present invention provides a method of treating a neurodegenerative condition comprising administering to a patient an effective amount of a 39-desmethoxyrapamycin analog. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. Specifically, the neurodegenerative condition can be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington chorea. Thus, in one embodiment, the present invention provides a method of treating Alzheimer's disease comprising administering to a patient an effective amount of a 39-desmethoxyrapamycin analog. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. In another embodiment, the present invention provides a method of treating multiple sclerosis comprising administering to a patient an effective amount of a 39-desmethoxyrapamycin analog. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. In an alternative embodiment, the present invention provides a method of treating Huntington's chorea comprising administering to a patient an effective amount of a 39-desmethoxyrapamycin analog. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27.

The invention also provides for the treatment of cancer or B-cell malignancies, or the induction or maintenance of immune suppression, the promotion of nerve regeneration, fungal infection, tissue transplant rejection, graft versus host disease, autoimmune disorders, neurodegenerative conditions, inflammatory In the manufacture of a medicament for the treatment of a disease, vascular disease or fibrotic disease, the use of the 39-desmethoxyrapamycin analog is provided. In particular, the present invention provides the use of a 39-desmethoxyrapamycin analog or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a medical condition due to nerve injury or disease. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27.

In addition, the present invention provides a 39-desmethoxyrapamycin analog, ie, increased vascular brain barrier permeability, in the manufacture of a medicament for the treatment of a medical condition in which the blood brain barrier affects the central nervous system that interferes with the delivery of the compound. Provided is the use of a rapamycin analog or a pharmaceutically acceptable salt thereof. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27.

The present invention also provides the use of 39-desmethoxyrapamycin analogs in the manufacture of a medicament for the treatment of brain tumor (s). In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. In certain embodiments, the invention provides the use of a 39-desmethoxyrapamycin analog in the manufacture of a medicament for the treatment of glioblastoma multiforme. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27.

Also specifically, the present invention provides the use of 39-desmethoxyrapamycin analogs in the manufacture of a medicament for the treatment of neurodegenerative conditions. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. Specifically, the neurodegenerative condition can be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington chorea. Thus, in one embodiment, the present invention provides the use of a 39-desmethoxyrapamycin analog in the manufacture of a medicament for the treatment of Alzheimer's disease. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. In another embodiment, the present invention provides the use of a 39-desmethoxyrapamycin analog in the manufacture of a medicament for the treatment of multiple sclerosis. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. In an alternative embodiment, the present invention provides the use of a 39-desmethoxyrapamycin analog in the manufacture of a medicament for the treatment of Huntington's chorea. In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27.

The 39-desmethoxyrapamycin analog is a structurally close rapamycin analog made using the method described in WO 04/007709. However, they show different activity spectra than rapamycin, for example, as shown by COMPARE analysis of a panel of NCI 60 cell lines of 39-desmethoxyrapamycin and its analogs (see Table 1 below). . The COMPARE assay uses the Pearson assay to compare the activity of two compounds in a panel of NCI 60-cell lines, yielding correlation coefficients showing how similar the activity spectra of these two compounds are and how these mechanisms of action are related. Can be represented. In a particular embodiment, the Pearson coefficient of rapamycin and 39-desmethoxyrapamycin is 0.614, which is the Pearson correlation coefficient of rapamycin and CCI-779 (40-hydroxy ester derivative of rapamycin). It should be compared with 0.86 (Dancey, 2002). Thus, it can be found that the 39-desmethoxyrapamycin analog has a different activity spectrum when compared to rapamycin.

TABLE 1

Multi-Drug Resistance (MDR) is an important issue in the treatment of cancer and B-cell malignancies. This is the main reason behind the development of drug resistance in many cancers (Persidis A, 1999). The first drug acts as a whole after a period of time becomes ineffective. MDR encodes MRP1 gene or MRP1, which encodes an increased level of adenosine triphosphate-binding cassette transporter (ABC transporter), especially P-glycoprotein (P-gp). It is associated with an increase in MRP1 gene expression. The expression level of the MDR1 gene varies widely in various cancer-derived cell lines and cannot be detected in certain cell lines, while in other cell lines it is increased up to 10 or 100 times compared to the standard control. Expression can be shown.

The indicated certain difference in activity spectra between rapamycin and 39-desmethoxyrapamycin can be explained because the 39-desmethoxyrapamycin analogue is not a substrate of P-gp. The 39-desmethoxyrapamycin analog had reduced excretion from Caco-2 cells when compared to rapamycin and 39-desmethoxyrapamycin was found to be not a substrate of P-gp in vitro P-gp substrate analysis (See Examples herein).

Accordingly, another embodiment of the present invention is directed to the treatment of 39-desmethoxyrapamycin analogs in the treatment of cancer or B-cell malignancies, ie MDR cancer or B-cell malignancies, resistant to one or more existing anticancer agent (s). Serves the purpose. In certain embodiments, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of P-gp-expressing cancer or B-cell malignancies. In another more preferred embodiment, the present invention provides the use of 39-desmethoxyrapamycin in the treatment of highly P-gp expressing cancer or B-cell malignancies. Specifically, cancers or B-cell malignancies that express very P-gp may have a 2, 5, 20, 25, 50, or 100 fold increased expression compared to control levels. In certain embodiments of the above uses, the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment of the above uses, the 39-desmethoxyrapamycin analogue is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. Suitable controls are cells that do not express P-gp, which have low expression levels of P-gp or have low MDR function, and those skilled in the art can recognize or identify such cell lines: Suitable cell lines include, but are not limited to, the following: MDA435 / LCC6, SBC-3 / CDDP, MCF7, NCI-H23, NCI-H522, A549 / ATCC, EKVX, NCI-H226, NCI-H322M, NCI-H460, HOP-18, HOP-92, LXFL 529, DMS 114, DMS 273, HT29, HCC-2998, HCT-116, COLO 205, KM12, KM20L2, MDA-MB-231 / ATCC, MDA-MB-435, MDA-N, BT-549, T-47D, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, K-562, MOLT-4, HL-60 (TB), RPMI-8226, SR, SN12C, RXF-631, 786-0, TK-10, LOX IMVI, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL- 28, M14, UACC-62, UACC-257, PC-3, DU-145, SNB-19, SNB-75, SNB-78, U251, SF-268, SF-539, XF 498.

In an alternative embodiment, the present invention provides the use of a 39-desmethoxyrapamycin analog in the manufacture of a medicament for use in the treatment of MDR cancer or B-cell malignancy. In certain embodiments, the present invention provides the use of 39-desmethoxyrapamycin analogues in the manufacture of a medicament for use in the treatment of P-gp-expressing cancer or B-cell malignancy. In another more preferred embodiment, the present invention provides the use of 39-desmethoxyrapamycin analogues in the manufacture of a medicament for use in the treatment of very P-gp expressing cancer or B-cell malignancies. In particular, cancers or B-cell malignancies that express very P-gp may have an expression that is 2, 5, 10, 20, 25, 50, or 100 times increased relative to the level of the control group. . In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. Suitable controls are described above.

Methods of determining the expression level of P-gp in a sample are further discussed herein.

Thus, in another embodiment, the present invention provides a method of treating P-gp-expressing cancer or B-cell malignancy, comprising administering a therapeutically effective amount of a 39-desmethoxyrapamycin analog. . In certain embodiments the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. In another embodiment the 39-desmethoxyrapamycin analog is an additional 39-desmethoxyrapamycin analog that is additionally different from rapamycin at one or more of positions 9, 16 or 27. Expression levels of P-glycoprotein (P-gp) in certain cancer forms are not limited to these, but real-time RT-PCR (Szakacs et al . , 2004; Stein et. al . , 2002; Langmann et al . , 2003, Alvarez et al ., 1995, Boyd et al . , 1995), by immunohistochemistry (Stein et al . , 2002) or using microarrays (Lee et al . , 2003) can be measured by one of ordinary skill in the art using techniques that include. Other suitable methods are known to those skilled in the art, and such methods will only be provided in the Examples.

39-desmethoxyrapamycin shows increased metabolic stability when compared to rapamycin as shown in the examples herein. Already a number of papers have identified the 39-methoxy group in rapamycin as a major site of metabolic attack to convert rapamycin to 39-O-desmethylrapamycin (Trepanier et al . , 1998). The major metabolite of rapamycin has a significantly reduced activity compared to the parent compound (Gallant-Haidner et. al . , 2000, Trepanier et al . , 1998). In contrast, 39-desmethoxyrapamycin is the most effective site subject to metabolic attack, which is no longer valid, increasing the stability of the compound (see Examples). In connection with the higher or equivalent potency of 39-desmethoxyrapamycin relative to the rapamycin parent compound, this gives the compounds of the present invention a longer half-life. This is also a surprising advantage of 39-desmethoxyrapamycin over rapamycin.

The characteristics of 39-desmethoxyrapamycin described above (39-desmethoxyrapamycin is not a substrate of P-gp, has increased metabolic stability, and has a reduced excretion through P-gp) Desmethoxyrapamycin shows improved bioavailability compared to parent compound rapamycin. Accordingly, the present invention relates to 39-desmethoxyrappa, which is a rapamycin analogue in medicine, specifically in the treatment of cancer or B-cell malignancies, with improved metabolic stability, improved cell membrane permeability and completely different cancer cell inhibition profiles. Provides the use of mycin.

The present invention also provides a pharmaceutical composition comprising a 39-desmethoxyrapamycin analog or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier. In certain embodiments, the present invention provides a pharmaceutical composition comprising 39-desmethoxyrapamycin. In yet another embodiment, the present invention provides a pharmaceutical composition comprising rapamycin and an additional 39-desmethoxyrapamycin analogue at one or more of positions 9, 16 or 27. In certain embodiments, the present invention provides a pharmaceutical composition as described above specifically formulated for intravenous injection.

Rapamycin and related compounds in or on clinical trials such as CCI-779 and RAD001 include poor metabolic stability, poor permeability, high levels of excretion through P-gp and poor bioavailability. It has a poor pharmacological profile. The present invention provides the use of 39-desmethoxyrapamycin or a pharmaceutically acceptable salt thereof having improved pharmaceutical characteristics when compared to rapamycin.

In addition, a surprising embodiment of the present invention is that the 39-desmethoxyrapamycin analogue exhibits a significantly different pharmacokinetic profile when compared to the existing rapamycin analogues. Specifically, the 39-desmethoxyrapamycin analogue showed increased blood brain barrier permeability, and thus higher exposure was found in the brain when these compounds were compared to related analogs at a given blood level.

This difference in pharmacokinetics is entirely unexpected and has not been proposed anywhere in the prior art. Known disadvantages of currently available therapies for diseases including neurodegenerative conditions and brain tumors present a challenging task of reaching the site of action of the drug (see Pardridge, 2005). In addition, this has been reported to be a problem when existing rapamycin analogs are used in therapy, and in particular, studies investigating the efficacy of CCI-779 in the treatment of glioblastoma multiforme, although systemic concentrations were adequate, suggest that the blood brain barrier It was concluded that this drug acted as a barrier in the delivery of the drug to tumors (Chang, 2005). Thus, the present invention first disclosed rapamycin analogues with improved blood brain barrier permeability, and therefore the first disclosed rapamycin analogues with significant utility for treating brain tumors and neurodegenerative conditions.

Preferred 39-desmethoxyrapamycin analogs for use in certain embodiments of the invention described above include analogs additionally different from rapamycin at any of positions 9, 16 or 27, ie 39-des Preferably the methoxyrapamycin analog is not 39-desmethoxyrapamycin itself. In addition, preferred 39-desmethoxyrapamycin analogs include:

From above

The 39-desmethoxyrapamycin analogue has a hydroxyl group at position 27, ie R ₃ represents OH;

The 39-desmethoxyrapamycin analog has hydrogen at position 27, ie R ₃ represents OH; or

The 39-desmethoxyrapamycin analog has hydroxy at position 16, ie R ₂ represents OH.

Those skilled in the art will be able to determine the pharmacokinetics and bioavailability of the compounds of the present invention using in vivo and ex vivo methods known to those skilled in the art, including those described below and Gallant-. Haidner et al. , 2000 and Trepanier et al . , 1998, and those described in the references herein, but are not limited to these. The bioavailability of a compound is determined by a number of factors (e.g., water solubility, cell membrane permeability, degree of protein binding and metabolism and stability), each of which can be determined by in vitro tests described below, It will be appreciated by those skilled in the art that one or more of these improvements can lead to improvements in the bioavailability of the compounds. Alternatively, bioavailability of 39-desmethoxyrapamycin or a pharmaceutically acceptable salt thereof can be determined as described in more detail below or using the in vivo methods described in the Examples herein. .

In vivo analysis

In vivo assays can also be used to determine the bioavailability of compounds such as 39-desmethoxyrapamycin. In general, the compound is administered to test animals (eg, mice or rats) in two ways, either intraperitoneally (ip) or intravenously (iv) and orally (po) and at regular intervals The sample is then examined to see how the plasma concentration of the drug changes over time. A time trend of plasma concentration over time can be used to calculate the absolute bioavailability of the compound in percent using a standard model. Examples of conventional protocols are described below.

Mice are administered 3 mg / kg 39-desmethoxyrapamycin iv or 10 mg / kg 39-desmethoxyrapamycin po. Blood samples are taken at 5, 15, 1, 4 and 24 hour intervals and the concentration of 39-desmethoxyrapamycin in the sample is determined via HPLC. The time-trend of plasma or total blood concentration is then used to determine the area under the plasma concentration-time curve (AUC-this is directly proportional to the total amount of unchanged drug reaching the large circulation), the maximum (peak) plasma drug. Additional factors that can be used to derive key parameters such as concentration, peak plasma or time (peak time) at which blood drug concentrations occur, and additional factors used in the accurate determination of bioavailability are the terminal half life, Total body clearance, steady-state distribution volume and F%. These parameters are then analyzed by non-compartmental or compartmental methods to provide calculated bioavailability, and are examples of forms of such methods as Gallant-Haidner et al. , 2000 and Trepanier et al . , 1998 and references herein.

The efficacy of 39-desmethoxyrapamycin can be tested in an in vivo model for neurodegenerative diseases described herein and known to those skilled in the art. Such models include Alzheimer's disease-animals expressing human familial Alzheimer's disease (FAD) p-amyloid precursor (APP), animals overexpressing human wild-type APP, p Animals that express over amyloid 1-42 (pA), animals expressing FAD presenillin-1 (PS-1) (eg German and Eisch, 2004), It is not limited. For multiple sclerosis—experimental autoimmune encephalomyelitis (EAE) model (see Bradl, 2003 and Example 7). For Parkinson's disease-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model or 6-hydroxydopamine (6-OHDA) model (e.g. Emborg, 2004; Schober A. 2004). For Huntington's chorea, the R6 lines model (Sathasivam et ) is produced by the injection of exon 1 of the human Huntington's disease (HD) gene, which carries a highly expanded CAG repeat mainly of mouse bacteria. al . , 1999) and others (see Hersch and Ferrante, 2004).

The above-mentioned compounds of the present invention or preparations thereof can be administered by any conventional method, for example, but not limited to, parenteral, oral, topical (including buccal, sublingual, or transdermal), Administration may be via a medical device (eg stent), by inhalation or via injection (subcutaneous or intramuscular). Treatment may consist of a single dose or multiple doses over a period of time.

The 39-desmethoxyrapamycin analog can be administered alone, but it is preferred to be present as a pharmaceutical formulation, with one or more acceptable carriers. The carrier (s) should be "acceptable" in the sense of being compatible with the compounds of the present invention and should not be harmful to the doser. Examples of suitable carriers are described in more detail below.

39-desmethoxyrapamycin analogs can be administered alone or in combination with other therapeutic agents, and the co-administration of two (or more) substances allows for significantly lower dosages than when each is used, Thus reducing the observed side effects. The increased metabolic stability of 39-desmethoxyrapamycin is an additional advantage over rapamycin, in that it occurs less likely to result in drug-drug interactions when used in combination with drugs that are substrates of the P450 enzyme when occurring with rapamycin. (Lampen et al . , 1998).

Thus, in one embodiment, the 39-desmethoxyrapamycin analog is administered concurrently with another therapeutic agent for the induction or maintenance of immunosuppression, tissue transplant rejection response, graft versus host disease, autoimmune disorder or inflammatory disease Preferred formulations include, for example, azathioprine, corticosteroids, cyclophosphamide, cyclosporin A, FK506, Mycophenolate Mofetil, OKT-3 and ATG It is not limited to these.

In an alternative embodiment, the 39-desmethoxyrapamycin analog is co-administered with another therapeutic agent for the treatment of cancer or B-cell malignancy, with preferred agents methotrexate, leucovorin, adriamycin, prinison (prenisone), bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, togestif Megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g. Herceptin ^™ ), capecitabine, raloxifene hydrochloride, EGFR inhibitors (eg Iressa ^® , Tarceva ^TM , Erbitux ^TM ), VEGF inhibitors (eg Avastin ^TM ), proteasome inhibitors (eg Velcade ^TM ), Glivec ^® or hsp90 inhibitors (eg , 17-AAG), but is not limited to these. In addition, 39-desmethoxyrapamycin can be administered in combination with other therapies including, but not limited to, radiation therapy or surgical operations.

In one embodiment, the 39-desmethoxyrapamycin analog is co-administered with another therapeutic agent for the treatment of vascular disease, and preferred agents include ACE inhibitors, angiotensin II receptor antagonists, fibric acid derivatives. , HMG-CoA reductase inhibitors, beta adrenergic blocking agents, calcium channel blockers, antioxidants, anticoagulants and platelet inhibitors (e.g., Plavix ^™ ). It is not.

In one embodiment, the 39-desmethoxyrapamycin analog is co-administered with another therapeutic agent to promote nerve regeneration, and preferred agents include neurotrophic factors such as nerve growth factor, glial cell derived growth factor. , Brain derived growth factors, ciliary neurotrophic factors and neurotrophin 3, but are not limited to these.

In one embodiment, the 39-desmethoxyrapamycin analog is co-administered with another therapeutic agent for the treatment of fungal infections; Preferred agents include amphotericin B, flucitosine, echinocandin (e.g. caspofungin, anidulafungin or micafungin), gliofulvin, imidazole Or triazole antifungal agents (e.g., clotrimazole, myconazole, ketoconazole, echonasol, buttoconazole, oxyconazole, terconazole, itraconazole, fluconazole or voriconazole) It is not limited.

In one embodiment, the 39-desmethoxyrapamycin analog is co-administered with another agent for the treatment of Alzheimer's disease; Preferred preparations include cholinesterase inhibitors such as donepezil, rivastigmine, and galantamine; N-methyl-D-aspartate (NMDA) receptor antagonists, such as, but not limited to Memantine.

In one embodiment, the 39-desmethoxyrapamycin analog is co-administered with another agent for the treatment of multiple sclerosis; Preferred formulations include interferon beta-1b, interferon beta-1a, glatiramer, mitoxantrone, cyclophosphamide, corticosteroids (e.g. methylprednisolone, prednisone, dexamethasone) However, the present invention is not limited thereto.

As will be apparent to those skilled in the art, co-administration includes any method of delivering two or more therapeutic agents to a patient as part of a treatment method. Two or more agents may be administered simultaneously in a single formulation, but this is not essential. The formulations may be administered in different formulations and at different times.

The formulations can easily be presented in a single dosage form and can be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the present invention) with the carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and directly causing the association of the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, by shaping into products.

39-Desmethoxyrapamycin analogs are usually in the form of pharmaceutical preparations comprising the active ingredient, optionally in the form of acid or base addition salts which are nontoxic and organic or inorganic, in pharmaceutically acceptable dosage forms, Inner, orally or by any parenteral route. Depending on the disease and the patient to be treated, as well as the route of administration, the composition can be administered with varying dosages.

Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. The compositions may also be in the form of sterile powders for the instant preparation of such sterile injectable solutions or dispersions. In all cases, the final state of the injectable formulation must be sterile and must be efficiently flowable for easy syringability.

The pharmaceutical composition must be stable under the conditions of manufacture and storage; Therefore, it should preferably be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium and includes, for example, water, ethanol, polyols (eg glycerol, propylene glycol and liquid polyethylene glycols), vegetable oils, and suitable mixtures thereof.

For example, the 39-desmethoxyrapamycin analog can be orally, into the cheeks, or under the tongue, in the form of tablets, capsules, ovules, elixirs, solutions or suspensions which may include sweetening or coloring agents. It may be administered for immediate-release, delayed-release or controlled-release application.

Solutions or suspensions of 39-desmethoxyrapamycin analogs suitable for oral administration may also include excipients such as N, N-dimethylacetamide, dispersants such as sorbate 80, surfactants, and solubilizers such as Polyethylene glycol, Phosal 50 PG (comprised of phosphatidylcholine, soybean fatty acid, ethanol, mono / diglycerides, propylene glycol and ascorbyl palmitate).

Such tablets include microcrystalline cellulose, lactose (eg, lactose monohydrate, or anhydrous lactose), sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, butylated hydroxytoluene (E321), Disintegrants such as crospovidone, hypromellose, starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain composite silicates, and polypinylpyrrolidone, Granulation binders such as hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), macrogol 8000, sucrose, gelatin and acacia. Lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may also be included.

In addition, solid compositions of a similar type may be used as filling materials in gelatin capsules. In such cases, preferred excipients include lactose, starch, cellulose, lactose or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the compounds of the present invention may be used in combination with various sweetening or flavoring agents, coloring materials or dyes, with emulsifiers and / or suspending agents, and with diluents such as water, ethanol, propylene glycol and glycerin. Together, and combinations thereof.

Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may optionally contain a binder (e.g. povidone, gelatin, hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g. sodium starch glycolate, a crosslinked polydon, By cross-linking sodium carboxymethyl cellulose), surface-active or dispersing agents, to prepare the active ingredients in free-flowing formulations such as powders or granules in a suitable machine. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered compound and the inert liquid diluent in a wet state. Tablets may optionally be coated or scoured and formulated to provide sustained or controlled release of the active ingredient herein, for example in various proportions of hydroxypropylmethylcellulose Can be used to provide the desired release profile.

Suitable for oral administration, the preparations according to the invention can be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of active ingredient; As a powder or granules; As a liquid or suspension in an aqueous liquid or non-aqueous liquid state; Or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a large bolus, electuary, or paste.

Formulations suitable for topical topical administration include lozenges comprising the active ingredient in a sweetened ingredient, generally sucrose and acacia or tragacanth; Pastilles comprising the active ingredient in an inactive ingredient such as gelatin and glycerin or sucrose and acacia; And mouth-washes comprising the active ingredient in a suitable liquid carrier.

In addition to the components specifically mentioned above, the formulations of the present invention may contain other substances conventional in the art, in view of the form of the formulation to be solved, for example, a sweetening agent suitable for oral administration may be used. It may include.

Pharmaceutical compositions adapted for topical administration include ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, drug-infused dressings, sprays, aerosols or oils, transdermal devices, spray powders, and equivalents thereof. It can be formulated as. Such compositions can be prepared by conventional methods, including the active ingredient. Thus, they may also include compatible conventional carriers and additives, such as preservatives, solvents that aid drug penetration, emollients in creams or ointments, and ethanol or oleyl alcohols for lotions. Such carriers may be present from about 1% to about 98% in the composition. More generally, they will form up to about 80% of the composition. By way of illustration only, creams or ointments are prepared in sufficient amounts to produce a cream or ointment with the desired consistency by mixing with a sufficient amount of hydrophilic material and water, including about 5-10% by weight of the compound.

Pharmaceutical compositions adapted for transdermal administration may be presented in separate patches that allow direct contact with the epidermis of the recipient for an extended period of time. For example, the active ingredient can be delivered from the patch by iontophoresis.

For application to external tissues, for example mouth and skin, the composition is preferably applied as a topical ointment or cream. When formulated into an ointment, the active ingredient can be used with paraffinic or water-miscible ointment ingredients.

Alternatively, the active ingredient may be formulated into a cream with an oil-in-water cream component or a water-in-oil component.

For parenteral administration, fluid unit dosage forms are prepared using active ingredients and sterile vehicles, which include, for example, water, alcohols, polyols, glycerin and vegetable oils. But not limited to, water is preferred. Depending on the vehicle and concentration used, the active ingredient may be suspended or dissolved in the vehicle. When preparing a solution, the active ingredient may be dissolved in water for injection and sterilized by filtration before filling and sealing in a suitable vial or ampoule.

Advantageously, agents such as local anesthetics, preservatives and buffers can be dissolved in the vehicle. To increase stability, the composition can be frozen after filling the vial and water can be removed under vacuum. The lyophilized powder in dry state is then sealed in a vial and a vial with water for injection can be supplied to reconstitute the liquid before use.

Parenteral suspensions are prepared in substantially the same manner as liquid solutions, except that the active ingredient is suspended instead of dissolved in the vehicle and sterilization cannot be performed by filtration. The active ingredient can be sterilized by exposure to ethylene oxide, before suspending in the sterile vehicle. Advantageously, surfactants or wetting agents are included in the composition to facilitate uniform distribution of the active ingredient.

In addition, the 39-desmethoxyrapamycin analog can be administered using medical devices known in the art. For example, in one embodiment, the compositions of the present invention are described in U.S. Patent Nos. 5,399,163; US Patent No. 5,383,851; US Patent No. 5,312,335; US Patent No. 5,064,413; US Patent No. 4,941,880; US Patent No. 4,790,824; Or with a needleless subcutaneous injection device, such as the device disclosed in US Pat. No. 4,596,556. Examples of well-known implants and modules useful in the present invention include: US Pat. No. 4,487,603, which discloses a buried micro-infusion pump for dispensing drugs at a controlled rate; US Patent No. 4,486, 194, which discloses a therapeutic device for administering a drug through the skin; US Patent No. 4,447,233 which discloses a drug infusion pump for delivering a drug at the correct infusion rate; US Patent No. 4,447, 224, which discloses a variable flow buried infusion device for continuous drug delivery; US Patent No. 4,447, 224, which discloses an osmotic drug delivery system having a multi-chamber compartment; US Patent 4,475, 196 discloses an osmotic drug delivery system. In certain embodiments, the 39-desmethoxyrapamycin derivative is a drug release stent, for example corresponding to that disclosed in the published application relating to WO 01/87263 or described by Perin (Perin, EC, 2005). It may be administered using a corresponding one. Many other such implants, delivery systems, and modules are known to those skilled in the art.

The dosage to which a compound of the present invention will be administered will vary depending upon the particular compound, the disease and subject involved, and the nature and severity of the disease, and the physical condition of the subject, and the route of administration chosen. Appropriate dosages can be readily determined by one skilled in the art.

The composition may comprise from 0.1% by weight of the compounds of the invention, preferably 5-60% by weight, more preferably 10-30% by weight, depending on the method of administration.

The optimal content and interval of administration of the individual dosage forms of the compounds of the invention will be determined by the nature and extent of the condition to be treated, the dosage form, the route and site, and the age and condition of the particular subject to be treated, and the physician will ultimately determine the appropriate dosage to be used. It will be appreciated by those skilled in the art that the formulation will be determined. Such dosage forms may be repeated as often as appropriate. If side effects occur, the content and / or frequency of the dosage form may be altered or reduced, in accordance with normal clinical practice.

1 shows the structure of rapamycin.

FIG. 2: shows the fragmentation pathway of 39-desmethoxyrapamycin.

FIG. 3: shows a Western blot showing briefly the mTOR inhibitory activity of 39-desmethoxyrapamycin and rapamycin.

Figure 4:% T at all test concentrations of paclitaxel (A and C) and 39-desmethoxyrapamycin (B and D) in normal cell lines (A and B) or highly P-gp expressing cell lines (C and D). / C value

Figure 5: One i.v. of A-rapamycin and 39-desmethoxyrapamycin. Or p.o. Area under the Curve (AUC) is shown in brain tissue or blood samples after administration from 0 to 24 hours.

B-one i.v. The levels of 39-desmethoxyrapamycin and rapamycin detected in brain tissue over time after administration are shown.

Figure 6: A-Disease progression in the EAE model under the prevention plan. Values provided are mean values from vehicle group or treated group.

B-Disease progression in the EAE model under treatment plan. Values provided are mean values from vehicle group or treated group.

7: Graph shows relative percent survival of mice after glioma induction by stereotaxic injection of U87-MG cells. Filled diamonds represent untreated groups, filled squares represent vehicle treated groups, and open circles represent 39-desmethoxyrapamycin treated groups.

Materials and methods

material

Unless otherwise indicated, all reagents used in the examples below were obtained from commercial sources.

culture

S. hygroscopicus MG2-10 [IJMNOQLhis] (WO 04/007709; Gregory et. al . , 2004) were maintained at 28 ° C. in a medium 1 agar plate (see below). After growing in medium 1, cell stocks were prepared and 20% w / v glycerol in distilled water; It was kept at 10% w / v lactose and stored at -80 ° C. In a 250 ml flask, growth cultures were prepared by inoculating 0.1 ml of frozen stock in 50 ml medium 2 (see below). Cultures were incubated at 28 ° C. and 300 rpm for 36 to 48 hours.

Produce Method:

Growth cultures were inoculated in medium 3 at 2.5-5% v / v. For 6-7 days, incubation was performed at 26 ° C. at 300 rpm.

Injection procedure:

24-48 hours after inoculation, injection / addition of cyclohexane carboxylic acid was performed, unless otherwise stated, at 1-2 mM final concentration.

Badge 1:

The medium was then sterilized by autoclaving at 121 ° C. for 20 minutes.

Medium 2: RapV7 seed medium

The medium was then sterilized by autoclaving at 121 ° C. for 20 minutes.

After sterilization, 0.16 mL of 40% glucose is added to 7 mL of each medium.

Medium 3: MD6 medium (fermentation medium)

Prior to sterilization, 0.4 mL of sigma α-amylase (BAN 250) was added to 1 L of medium.

The medium was sterilized at 121 ° C. for 20 minutes.

After sterilization, 0.35 ml of sterile 40% fructose and 0.10 ml of L-lysine (140 mg / mL in water, sterilized by filtration) were added to 7 ml each.

Medium 4: RapV7a Seed Medium

The medium was then sterilized by autoclaving at 121 ° C. for 20 minutes.

Medium 5: MD6 / 5-1 medium (fermentation medium)

The medium was sterilized at 121 ° C. for 30 minutes.

After sterilization, 15 g of fructose per liter were added.

After 48 hours, 0.5 g / L of L-lysine was added.

Analytical Method

Method A

Injection volume: 0.005-0.1 mL (depending on sensitivity).

Mobile phase A: 0.01% formic acid in pure water

Mobile Phase B: 0.01% Formic Acid in Acetonitrile

Flow rate: 1 ml / min mobile phase and HPLC was performed on Agilent "Spherisorb" "Rapid Resolution" cartridges SB C8, 3 microns, 30 mm x 2.1 mm.

A concentration linear gradient was used to maintain 95% B for 5 minutes, 95% B for 4 minutes, and return to 5% B for the next cycle at 0 minutes, at 0 minutes. Detection was by UV absorbance at 254 nm and / or mass spectroscopy electron spray ionization (positive or negative) using a Micromasss Quattro-Micro instrument.

Method B

Injection volume: 0.02 mL.

Mobile Phase A: Fill with 1 L of deionized water with acetonitrile (100 mL), trifluoroacetic acid (1 mL), 1M ammonium acetate (10 mL).

Mobile phase B: Fill with 1 L of acetonitrile with deionized water (100 mL), trifluoroacetic acid (1 mL), 1M ammonium acetate (10 mL).

Flow rate: 1 mL / min

Was carried out in a mobile phase of and maintained at 50 ° C. and HPLC was performed on a 3 micron BDS C18 Hypersil (ThermoHypersil-Keystone Ltd) column, 150 × 4.6 mm.

A concentration linear gradient of 55% B-95% B was used over 10 minutes, then held at 95% B for 2 minutes, made 55% B over 0.5 minutes, and further 2 minutes at 55% B. For a while. Compounds were detected by UV absorbance at 280 nm.

Method C

The HPLC system included an Agilent HP1100,

Mobile Phase A: Deionized Water.

Mobile phase B: acetonitrile.

Flow Rate: Run in 1 mL / min mobile phase, hold at 40 ° C., and perform on 3 micron BDS C18 Hypersil (ThermoHypersil-Keystone Ltd) column, 150 × 4.6 mm.

The system is connected to a Bruker Daltonics Esquire3000 electron injection mass spectrometer. Over a scan range of 500 to 1000 Daltons, positive negative switching was used. A concentration linear gradient of 55% B-95% B was used over 10 minutes, then held at 95% B for 2 minutes, made 55% B over 0.5 minutes, and further 2 minutes at 55% B. For a while.

In vitro anti-cancer activity ( in in vitro ) Bioassay ( bioassay )

In vitro evaluation of anticancer activity of compounds in 12 human tumor cell lines in a monolayer proliferation assay was performed by the Oncotest Testing Facility, Institute for Experimental Oncology, Oncotest GmbH, Freiburg. The characteristics of the 12 selected cell lines are summarized in Table 2 below.

TABLE 2 Test Cell Lines

Roth et al . Oncotest cell lines were generated from human tumor xenografts, as described by, 1999. The origin of donor xenografts is described in Fiebig et al. , 1999. Other cell lines were obtained from NCI (H460, SF-268, OVCAR-3, DU145, MDA-MB-231, MDA-MB-468) or from DSMZ, Braunschweig, Germany (LNCAP).

Unless otherwise specified, all cell lines contain RPMI 1640 medium, 10% fetal calf serum, and 0.1 mg / mL gentamycin in a humidified atmosphere (95% air, 5% CO ₂ ) at 37 ° C. It is grown on a 'ready-mix' badge (PAA, Coibe, Germany).

Monolayer  Assay-Protocol 1:

Modified propidium iodide assays were used to assess the effect of test compound (s) on the growth of 12 human tumor cell lines (Dengler et. al . , (1995)).

Briefly, cells were harvested from exponentially staged cultures by trypsinization, counted, and 96-well bottom-flat at cell concentration dependent (5-10,000 live cells / well). bottomed) onto a microtiter plate. After 24 hours of recovery to allow the cells to grow exponentially, 0.01 mL of culture medium (6 control wells per plate) or 39-desmethoxyrapamycin was added to the wells. Each concentration was plated in triplicates. 39-desmethoxyrapamycin was applied at two concentrations (0.001 μM and 0.01 μM). After 4 consecutive days of exposure, the cell culture medium with or without 39-desmethoxyrapamycin was replaced with 0.2 mL of aqueous propidium iodine (PI) solution (7 mg / L). To determine the percentage of live cells, cells were permeated by freezing the plate. After thawing, the fluorescence is measured using a Cytofluor 4000 microplate reader (excitation 530 nm, emission 620 nm) to obtain a direct relationship to the total number of living cells.

Growth inhibition was expressed as treated group / control x 100 (% T / C). For active compounds, IC ₅₀ and IC ₇₀ values were assessed by plotting compound concentration versus cell viability.

Monolayer  Assay-Protocol 2:

Human tumor cell lines from the National Cancer Institute (NCI) Cancer Screening Panels were grown in RPM1 1640 medium containing 5% fetal calf serum and 2 mM L-glutamine (Boyd and Paull, 1995). Cells were seeded in 96-well microtiter plates at 0.1 mL at plate densities ranging from 5,000 to 40,000 cells / well depending on the time the individual cell lines were doubled. After seeding the cells, the microtiter plates were incubated for 24 hours at 37 ° C., 5% CO ₂ , 95% air and 100% relative humidity before adding the drug to be tested.

After 24 hours, two plates were fixed in situ with trichloroacetic acid (TCA) for each cell line to show the measured cell population (Tz) of each cell line at the time of drug addition. It was. The drug to be tested was dissolved in dimethyl sulfoxide to a target maximum test concentration of 400 fold and stored frozen before use. At the time of drug addition, aliquots of the frozen concentrates were dissolved and diluted to twice the desired final maximum test concentration with complete medium containing 0.05 mg / mL gentamycin. An additional 4-fold, 10-fold, or 1/2 log serial dilution was made to provide five drug concentrations and controls overall. 0.1 mL aliquots of these various drug dilutions were added to the appropriate microtiter wells already containing 0.1 mL of medium, resulting in the required final drug concentration.

After the drug was added, the plates were incubated for an additional 48 hours under 37 ° C., 5% CO ₂ , 95% air and 100% relative humidity. For adhered cells, the assay was terminated by adding cold TCA. Cells were fixed in situ by mild addition of cold 50% (w / v) TCA (final concentration, 10% TCA) and incubated at 4 ° C. for 60 minutes. The supernatant was discarded and the plate washed with tap water five times and air dried. Sulforhodamine B (SRB) solution (0.1 mL) at 0.4% (w / v) in 1% acetic acid was added to each well and the plate was incubated for 10 minutes at room temperature. After dyeing, the unbound dye was removed by washing five times with 1% acetic acid and the plate was dried in air. The bound dye was then dissolved with 10 mM trizma base and the absorbance read in an automated plate reader at 515 nm wavelength. For suspended cells, the method was the same except that the assay was terminated by immobilizing the settled cells at the bottom of the well by light addition of 0.05 mL of 80% TCA (final concentration, 16% TCA). Growth at each drug concentration level using seven absorbance measurements (time zero (Tz), control growth (C), and test group growth in the presence of five concentration levels of drug (Ti)) Percentage growth was calculated Percentage growth inhibition was calculated as follows.

[(Ti-Tz) / (C-Tz)] x 100 for concentrations with Ti≥Tz

At a concentration of Ti <Tz, [(Ti-Tz) / Tz] x 100

Three dose response parameters were calculated for each experimental substance. 50% growth inhibition (GI50) was calculated from [(Ti-Tz) / (C-Tz)] x 100 = 50, which was a net protein increase in control cells (measured by SRB staining) during incubation with drug. Drug concentration reduced by 50%. Drug concentration (TGI), which produces global growth inhibition, was calculated from Ti = Tz. Treated LC50 (the concentration of drug that produced a 50% reduction in protein measured at the end of drug treatment compared to the time of initiation), indicating a net decrease in cells after treatment. [(Ti- Tz) / Tz] x 100 = − Calculated from 50.

Multidrug resistant cell lines within a panel of 60 cell lines were tested for rhodamine B excretion (Lee et al. , 1994) and PCR detection of mdr-1 mRNA (Alvarez et al. al . , 1995) was identified as a cell line containing high P-gp by NCI.

Pharmacokinetics  Assay-Protocol 1:

Test compounds were prepared in a vehicle comprising 4% ethanol, 5% Tween-20, 5% polyethyleneglycol 400 in 0.15M NaCl. 10 mg / kg p.o. Or a single dose of 3 mg / kg i.v. was administered to a group of female CD1 mice (3 mice for each compound per time point). At 0 minutes, 4 minutes, 15 minutes, 1 hour, 4 hours and 24 hours, groups of mice were killed and blood and brain collected from each mouse for further analysis.

Brain samples were snap frozen in liquid nitrogen and stored at -20 ° C. At least 0.2 ml of the total blood of each animal was collected in test tubes containing the anticoagulant ethylene diamine tetraacetic acid (EDTA), mixed thoroughly and stored at -20 ° C.

Pharmacokinetics  Assay-Protocol 2:

To prepare the solution to be administered, 5 mg sample compound was dissolved in 100 μl ethanol to make a 50 mg / mL compound solution. The solution was then added by adding about 2.4 ml of 0.15 M NaCl (0.9% w / v saline), 5% v / v Tween 20, and 5% v / v PEG 400 (final ethanol concentration 4% v / v). Dilute to 2 mg / mL.

10 mg / kg p.o or 2 mg / kg i.v. Test compound at concentration 10 mg / kg p.o. Or a single dose of 2 mg / kg i.v. was administered to a group of three female BaIb C mice. At 5 minutes, 15 minutes, 60 minutes, 4 hours, 8 hours and 24 hours, the group was killed and the whole blood sample was filled in EDTA with about 0.2 ml to a final concentration of 0.5 mM and the brain was also removed. . Both whole blood and brain were snap frozen in liquid nitrogen and stored at −20 ° C. until transported in solid carbon dioxide for analysis.

Pharmacokinetics  Analysis of the Research Sample

The analysis was performed by ASI Limited (St George's Hospital Medical School, London). The concentrations of sample compounds in the provided blood and brain samples were determined by HPLC capable of MS detection. Control blood samples without sample compounds were obtained from Harlan Sera-Lab Limited (Loughborough, England). At time zero, brain samples were provided as brain samples without sample compounds.

Preparation of Brain Samples:

One hemisphere of each brain was homogenized with 5 mL water.

Extraction of Samples:

Control or test samples of mouse brain or blood (0.05 mL), internal standard solution (0.1 mL), 5% zinc sulfide solution (0.5 mL), and acetone (0.5 mL) were placed in 2 mL polypropylene test tubes (Sarstedt Limited, Beaumont Leys). , Leicester, UK), and then the contents obtained were mixed for at least 5 minutes (IKA-Vibrax-VXR mixer, Merck (BDH) Limited, Poole Dorset, UK). The test tubes were then centrifuged in a microfuge tube for at least 2 minutes. The resulting solvent layer was transferred to a 4.5 mL propylene tube containing sodium hydroxide (0.1 M, 0.1 mL) and methyl-tert-butyl ether (MTBE, 2 mL). The tubes were then mixed for at least 5 minutes (IKA-Vibrax-VXR mixer) and then centrifuged at 3500 rpm for 5 minutes. The solvent layer was transferred to a 4.5 mL conical polypropylene tube, placed in SpeedVac ^® , and the solvent was distilled off to dryness.

The resulting dried extract was reconstituted with 0.15 mL 80% methanol and mixed for a minimum of 5 minutes (IKA-Vibrax-VXR mixer) and centrifuged at 3500 rpm for 5 minutes. The extract was transferred to an auto sampler tube (NLG Analytical, Adelphi Mill, Bollington, Cheshire, UK) and placed in an auto-sampler tray fixed at room temperature. An auto-sampler was programmed to inject 0.03 mL aliquots of each extract into the analytical column.

Example  1: Fermentation and Separation of Sample Compounds

1.1. Fermentation and 39- Of desmethoxyrapamycin  detach

As described below, 39-desmethoxyrapamycin was prepared by growing a culture of S. hygroscopicus MG2-10 [IJMNOQLhis] and injecting cyclohexanecarboxylic acid (CHCA).

Plasmids containing the rapI, rapJ, rapM, rapN, rapO, rapQ, and rapL genes are injected into the MG2-10 dye described in WO 2004/007709, thereby expressing S. hygroscopicus MG2-10 [ IJMNOQLhis]. The resulting gene cassette was constructed with the rapL gene containing the 5 'in-frame histidine tag. As described in WO 2004/007709, the plasmid also contains apramycin for the origin of transfer, and for the transformation of MG2-10 by conjugation and selection of an exconjugant. Resistance markers and phiBTI attachment sites were included for site-specific integration into the chromosome. The method used for the isolation of each of these genes and the construction of the gene cassette comprising the combination of post-PKS genes was performed as described in WO 2004/007709.

Liquid culture

Growth cultures of S. hygroscopicus MG2-10 [IJMNOQLhis] were cultured as described in Materials and Methods. Production cultures were inoculated at 0.5 mL with growth cultures in a 50 mL tube with 7 mL medium 3. For 7 days, the incubation was performed at 26 ° C. and 300 rpm. One milliliter of sample was extracted with 1: 1 acetonitrile with shaking for 30 minutes, centrifuged at 13,000 rpm for 10 minutes, analyzed and quantified according to Analytical Method B (see Materials and Methods). Identification of the product was determined by mass spectrometer using Analytical Method C (see Materials and Methods).

Based on the analytical data discussed below, the observed rapamycin analog was proposed to be the target 39-desmethoxyrapamycin.

Fermentation

Primary growth cultures in medium 4 of S. hygroscopicus MG2-10 [IJMNOQLhis] were cultured essentially as described in Materials and Methods. Secondary growth cultures in Medium 4 were inoculated at 10% v / v at 28 ° C., 250 rpm for 24 hours. In a 20 L fermentor, the growth cultures were inoculated at 5% v / v in medium 5 (see Materials and Methods). For 6 days, at 26 ° C., incubation was performed at 0.5 vvm. ≧ 30% dissolved oxygen was maintained by changing the impeller tip speed to a minimum tip speed of 1.18 ms ^{−1 and} a maximum tip speed of 2.75 ms ⁻¹ . At 24 and 48 hours after inoculation, cyclohexanecarboxylic acid was injected to give a final concentration of 2 mM.

Extraction and purification

Fermentation broth (30 L) was stirred with equal volume of methanol for 2 hours and then centrifuged (10 minutes, 3500 rpm) to pellet the cells. The supernatant was stirred with Diaion ^® HP20 resin (43 g / L) for 1 hour and then filtered. The resin was washed batchwise with acetone to remove the rapamycin analog and the solvent was removed in vacuo. The aqueous concentrate was then diluted with 2 L of water and extracted with ethyl acetate (3 x 2 L). The solvent was removed in vacuo to give a brown oil (20.5 g).

The extract was dissolved in acetone, water was removed from the silica, applied to a silica column (6 × 6.5 cm diameter) and eluted with a stepwise concentration gradient of acetone / hexane (20% -40%). Fractions containing rapamycin analogs were collected and the solvent was removed in vacuo. The residue obtained (2.6 g) was further chromatographed (in three batches) eluting with 10: 10: 1 chloroform / heptane / ethanol in Sephadex LH20. Half purified rapamycin analog (1.7 g) was subjected to reverse phase (C18) prep HPLC using Gilson HPLC and eluting with Phenomenex 21.2 x 250 mm Luna 5 μm C18 BDS column at 21 mL / min with 65% acetonitrile / water. Purification by The purest fractions (identified by analytical HPLC, method B) were combined and the solvent removed under vacuum to give 39-desmethoxyrapamycin (563 mg).

Identification

The ¹ H NMR spectrum of 39-desmethoxyrapamycin was identical to that of standard material (P. Lowden, Ph. D. Dissertation, University of Cambridge, 1997).

LCMS and LCMS ⁿ analysis of the culture extract showed that the m / z ratio of rapamycin analog was 30 atomic mass units lower than that of rapamycin, which is consistent with the absence of methoxy groups. Observed ions: [M ⁻ H] ⁻ 882.3, [M + NH ₄ ] ⁺ 901.4, [M + Na] ⁺ 906.2, [M + K] ⁺ 922.2. Fractionation of the sodium adduct gave the expected ions of 39-desmethoxyrapamycin, consistent with the cleavage pathway already identified (FIG. 2) (JA Reather, Ph.D. Dissertation, University of Cambridge, 2000 ). This mass spectroscopic cleavage data narrows the area of novel rapamycin analogues in which loss of methoxy groups occurs in fragments C28-C42 containing a cyclohexyl moiety.

This mass spectroscopic cleavage data is entirely consistent with the structure of 39-desmethoxyrapamycin.

1.2. Fermentation and 27-O- Deathmethyl -39- Of desmethoxyrapamycin  detach

As described below, 27-O-desmethyl-39-desmethoxyrapamycin was prepared by growing a culture of S. hygroscopicus MG2-10 [IJMNOQLhis] and injecting cyclohexanecarboxylic acid (CHCA).

S. hygroscopicus MG2-10 [IJMNOQLhis] was made by injecting plasmids containing the rapJ, rapM, rapN, rapO, and rapL genes into the MG2-10 variant described in WO 2004/007709. The resulting gene cassette was constructed with the rapL gene containing the 5 'in-frame histidine tag. As described in WO 2004/007709, the plasmid also contains apramycin for the origin of transfer, and for the transformation of MG2-10 by conjugation and selection of an exconjugant. PhiBTI attachment sites were included for resistance markers, and for site-specific integration with chromosomes. The method used for the construction of gene cassettes, including the isolation of each of these genes, and the combination of post-PKS genes, was performed as described in WO 2004/007709.

Liquid culture

Growth cultures of S. hygroscopicus MG2-10 [IJMNOQLhis] were cultured as described in Materials and Methods. The resulting cultures were inoculated at 0.5 mL with growth cultures in a 50 mL tube in 7 mL medium 3. For 7 days, the incubation was performed at 26 ° C. and 300 rpm. One milliliter of the sample was extracted with 1: 1 acetonitrile with shaking for 30 minutes, centrifuged at 13,000 rpm for 10 minutes, analyzed and quantified according to Analytical Method B (see Materials and Methods). Identification of the product was determined by mass spectrometer using Analytical Method C (see Materials and Methods).

Based on the analytical data discussed below, the observed rapamycin analogues were proposed to be the target 27-O-desmethyl-39-desmethoxyrapamycin.

Fermentation

Primary growth cultures in medium 2 of S. hygroscopicus MG2-10 [IJMNOQLhis] were cultured essentially as described in Materials and Methods. Secondary growth cultures in medium 2 were inoculated at 10% v / v at 28 ° C., 250 rpm for 24 hours. In a 20 L fermentor, the growth cultures were inoculated at 10% v / v in medium 5 (see Materials and Methods). For 6 days, at 26 ° C., incubation was performed at 0.75 vvm. ≧ 30% dissolved oxygen was maintained by changing the impeller tip speed to a minimum tip speed of 1.18 ms ^{−1 and} a maximum tip speed of 2.75 ms ⁻¹ . At 24 hours and 48 hours after inoculation, cyclohexanecarboxylic acid was injected to obtain a final concentration of 2 mM.

Extraction and purification

Fermentation broth (15 L) was stirred with equal volume of methanol for 2 hours and then centrifuged (10 minutes, 3500 rpm) to pellet the cells. The supernatant was stirred with Diaion ^® HP20 resin (43 g / L) for 1 hour and then filtered. The resin was washed batchwise with acetone to remove the rapamycin analog and the solvent was removed in vacuo. The aqueous concentrate was then diluted with 2 L of water and extracted with EtOAc (3 x 2 L). The solvent was removed in vacuo to give a brown oil (12 g).

The extract was dissolved in acetone, water was removed from the silica, applied to a silica column (4 × 6.5 cm diameter) and eluted with a stepwise concentration gradient of acetone / hexane (20% -40%). Fractions containing rapamycin analogs were collected and the solvent was removed in vacuo. The residue obtained (0.203 g) was purified by reverse phase (C18) prep HPLC using Gilson HPLC and eluting with a Phenomenex 21.2 × 250 mm Luna 5 μm C18 BDS column at 21 mL / min with 65% acetonitrile / water. The purest fractions (identified by analytical HPLC, method B) were combined and the solvent removed under vacuum to give a residue (25.8 mg). The residue obtained was purified by reverse phase (C18) prep HPLC using Gilson HPLC and eluting with a Hypersil 4.6 × 150 mm 3 μm C18 BDS column at 1 mL / min with 60% acetonitrile / water. The purest fractions (identified by analytical HPLC, method B) were combined and the solvent removed under vacuum to give 27-O-desmethyl-39-desmethoxyrapamycin (19.9 mg).

Identification

As shown in Table 3 below, the ¹ H and ¹³ C NMR spectra were consistent with the structure and designation of 27-O-desmethyl-39-desmethoxyrapamycin.

TABLE 3 NMR results of 27-O-desmethyl-39-desmethoxyrapamycin in CDCl ₃ by 500 MHz ¹ H-NMR and 125 MHz ¹³ C-NMR.

* Value showed integration (integration) twice when compared to the other value in the ¹³ C- ¹³ C-NMR spectrum.

* Since the stereochemistry could not be determined, more NMR experiments were needed (such as 1D and 2D NOESY), so these values of ¹ H in the axial and axial directions of methylene were not specified. .

LCMS and LCMS ⁿ analysis of the culture extract showed that the m / z ratio of rapamycin analog was 44 atomic mass units lower than that of rapamycin, which is consistent with the lack of methyl and methyl groups. Observed ions: [M−H] 868.7, [M + NH ₄ ] ⁺ 887.8, [M + Na] ⁺ 892.8. Fractionation of the sodium adduct gave the expected ions of 27-O-desmethyl-39-desmethoxyrapamycin, consistent with the cleavage pathway already identified (FIG. 2) (JA Reather, Ph.D. Dissertation, University of Cambridge, 2000). This mass spectroscopic cleavage data narrows the region of rapamycin analogues in which loss of methoxy groups occurs in fragments C28-C42 containing a cyclohexyl moiety and the region of rapamycin analogues in which loss of O-methyl occurs in fragments C15-C27 Narrow it down.

This mass spectroscopic cleavage data is entirely consistent with the structure of 27-O-desmethyl-39-desmethoxyrapamycin.

1.3. Fermentation and 16-O- Deathmethyl -27-O- Deathmethyl -39- Of desmethoxyrapamycin  detach

As described below, 16-O-desmethyl-27-O-desmethyl-39-desme is grown by growing a culture of S. hygroscopicus MG2-10 [IJMNOQLhis] and injecting cyclohexanecarboxylic acid (CHCA). Toxyrapamycin was prepared.

S. hygroscopicus MG2-10 [IJMNOQLhis] was made by injecting plasmids containing the rapI, rapJ, rapN, rapO, and rapL genes into the MG2-10 variant described in WO 2004/007709. The resulting gene cassette was constructed with the rapL gene containing the 5 'in-frame histidine tag. As described in WO 2004/007709, the plasmid may also contain apramycin resistance markers for mobile origin, and transformation of MG2-10 by conjugation and selection of external conjugates, and site-specific intercalation with chromosomes. The phiBTI attachment site was included for the sake. The method used for the construction of gene cassettes, including the isolation of each of these genes, and the combination of post-PKS genes, was performed as described in WO 2004/007709.

Liquid culture

Growth cultures of S. hygroscopicus MG2-10 [IJMNOQLhis] were cultured as described in Materials and Methods. The resulting cultures were inoculated at 0.5 mL with growth cultures in a 50 mL tube with 7 mL medium 3. For 7 days, the incubation was performed at 26 ° C. and 300 rpm. One milliliter of the sample was extracted with 1: 1 acetonitrile with shaking for 30 minutes, centrifuged at 13,000 rpm for 10 minutes, analyzed and quantified according to Analytical Method B (see Materials and Methods). Identification of the product was determined by mass spectrometer using Analytical Method C (see Materials and Methods). Identification of the product was determined by mass spectrometer using Analytical Method C (see Materials and Methods).

Based on the analytical data discussed below, the observed rapamycin analogues were proposed to be the target 16-O-desmethyl-27-O-desmethyl-39-desmethoxyrapamycin.

Fermentation

Primary growth cultures in medium 2 of S. hygroscopicus MG2-10 [IJMNOQLhis] were incubated for 3 days essentially as described in Materials and Methods. Secondary growth cultures in medium 2 were inoculated at 10% v / v at 28 ° C., 250 rpm for 48 hours, and tertiary cultures at 10% v / v at 28 ° C., 250 rpm , Inoculated for 24 hours. In a 3 x 7 L fermentor, the growth cultures were inoculated at 10% v / v in medium 5 (see Materials and Methods). For 6 days, at 26 ° C., incubation was performed at 0.75 vvm. ≧ 30% dissolved oxygen was maintained by changing the impeller tip speed to a minimum tip speed of 0.94 ms ^{−1 and} a maximum tip speed of 1.88 ms ⁻¹ . At 24 hours after inoculation, cyclohexanecarboxylic acid was injected to a final concentration of 1 mM. L-lysine was injected at t = 0.

Extraction and purification

Fermentation broth (12 L) was stirred with equal volume of methanol for 2 hours and then centrifuged (10 minutes, 3500 rpm) to pellet the cells. The supernatant was stirred with Diaion ^® HP20 resin (43 g / L) for 1 hour and then filtered. The resin was washed batchwise with acetone to remove the rapamycin analog and the solvent was removed in vacuo. The aqueous concentrate was then diluted with 2 L of water and extracted with EtOAc (3 x 2 L). The solvent was removed in vacuo to give a brown oil (8.75 g).

The extract was dissolved in acetone, water was removed from the silica, applied to a silica column (4 × 6.5 cm diameter) and eluted with a stepwise concentration gradient of acetone / hexane (20% -40%). Fractions containing rapamycin analogs were collected and the solvent was removed in vacuo. The residue obtained (0.488 g) was further chromatographed (in three batches) eluting with 10: 10: 1 chloroform / heptane / ethanol in Sephadex LH20. Fractions containing rapamycin analogs were collected and the solvent was removed under vacuum. Semi-purified rapamycin analog (162 mg) was purified by reverse phase (C18) prep HPLC using Gilson HPLC and eluting with a Phenomenex 21.2 × 250 mm Luna 5 μm C18 BDS column at 21 mL / min with 65% acetonitrile / water. Purified. The purest fractions (identified by analytical HPLC, method B) were combined and the solvent removed under vacuum, 16-O-desmethyl-27-O-desmethyl-39-desmethoxyrapamycin (44.7 mg) Got.

Identification

LCMS and LCMS ⁿ analysis of the culture extract showed that there was a new rapamycin analog that eluted much earlier than all other 39-desmethoxy analogs. The m / z ratios of the various ions of the rapamycin analogs showed that they were 58 atomic mass units lower than rapamycin, which is consistent with the absence of two O-methyl groups and one methoxy group. Observed ions: [M ⁻ H] ⁻ 854.7, [M + NH ₄ ] ⁺ 877.8, [M + Na] ⁺ 892.7, [M + Na] ⁺ 908.8. Fractionation of the sodium adduct gave the expected ions of 16-0-desmethyl-27-O-desmethyl-39-desmethoxyrapamycin, consistent with the cleavage pathway already identified (FIG. 2) ( JA Reather, Ph.D. Dissertation, University of Cambridge, 2000). This mass spectroscopic cleavage data narrows the region of the rapamycin analogue that results in loss of methoxy groups in fragments C28-C42 containing a cyclohexyl moiety and the region of the rapamycin analogue that results in loss of O-methyl in fragments C15-C27. Narrow it down. These NMR and mass spectroscopic cleavage data are entirely consistent with the structure of 16-O-desmethyl-27-O-desmethyl-39-desmethoxyrapamycin.

Example  2: in vitro evaluation of anticancer activity

39- Of desmethoxyrapamycin  In Vitro Evaluation of Anticancer Activity

Anticancer activity of 39-desmethoxyrapamycin against a panel of 12 human tumor cell lines in a monolayer proliferation assay, as described in Protocol 1 in the above general method using modified propidium iodide assay In vitro evaluation of was performed.

The results are shown in Table 4 below, with each result representing the average of two replicates. Table 5 shows the IC ₅₀ and IC ₇₀ of the compounds and rapamycin in the cell lines tested.

TABLE 4

TABLE 5

39- Of desmethoxyrapamycin  The drug resistance ( MDR ) In vitro evaluation of selective anticancer activity

In vitro evaluation of the selective MDR anticancer activity of 39-desmethoxyrapamycin against a panel of NCI 60 cell lines in human tumor cell lines in a monolayer proliferation assay described in Protocol 2 of Materials and Methods using an SRB-based assay It was performed according to the bar.

The results are shown in Table 6 below.

TABLE 6 In vitro activity against highly MDR-representing cell lines

Except for only one cell line, it can be seen that 39-desmethoxyrapamycin has an equivalent or improved potency against a very MDR-representing cell line when compared to rapamycin.

Example  3: in vitro ADME  analysis

Caco -2 permeability analysis

Aggregated Caco-2 cells in a 24-well Corning Costar Transwell format (Li, AP, 1992; Grass, GM, et. al . , 1992, Volpe, DA, et al . , 2001) was provided by In Vitro Technologies Inc. (IVT Inc., Baltimore, Maryland, USA). The apical chamber included 0.15 mL Hank's balanced buffer solution (HBBS) pH 7.4, 1% DMSO, 0.1 mM Lucifer Yellow. The bottom chamber contained 0.6 mL HBBS pH 7.4, 1% DMSO. The control and samples were incubated at 37 ° C. in a humidified incubator and shaken at 130 rpm for 1 hour. Lucifer yellow penetrates only through paracellular (between tight junctions), and Lucifer Yellow's high apparent permeability papp indicates cell damage during analysis, and all such wells are removed. Propanolol (good passive permeation without known transporter effect) and acebutalol (poor passive permeation, weakened by active release by P-glycoprotein) were used as standard compounds. The compound can be tested in one direction and in this direction format by applying the compound to the attachment chamber or the bottom chamber (at 0.01 mM). Compounds in the attached chamber or bottom chamber are analyzed by HPLC-MS (see Method A, Materials and Methods). The results are expressed as apparent transmittance, Papp, (nm / s) and flux ratio (for A to B and B to A).

Volume Acceptor: 0.6 ml (A> B) and 0.15 ml (B> A)

Single layer area: 0.33 cm ²

△ Time: 60 minutes

Positive values of flux ratio indicate active discharge from the apical surface of the cell.

Human Liver Microsomal (HLM) Stability Analysis

Hepatic homogenate comprises a compound I, CYP450 (eg, CYP2C8, CYP2D6, CYP1A, CYP3A4, CYP2E1), esterase, amidase, and flavin monooxygenase (FMO). Provide intrinsic weaknesses of measurement for (oxidative) enzymes.

The half-life (T1 / 2) of the test compound can be determined by monitoring the disappearance over time by LC-MS in exposure to human liver microsomes. Compounds were added at 0.001 mM at 37 ° C. for 40 minutes at 0.1 M Tris-HCl, pH 7.4, with saturation levels of NADPH as subcarrier and sub-cellular portion of human liver microsome cells of 0.25 mg / mL protein. Incubated. At regular intervals, acetonitrile was added to the test sample to precipitate protein and stop metabolism. Samples were centrifuged and analyzed for parent compound using Assay Method A (see Materials and Methods).

TABLE 7 In vitro ADME  Analysis

Example  4: in vitro binding assay

FKBP12

FKBP12 is reversibly unfolded in the chemical denaturant guandinibium hydrochloride (GdnHCl), which can be monitored by changes in the intrinsic fluorescence of the protein (Main et al. al . , 1998). Ligands that specifically bind to stabilize the original state of FKBP12 shift the denaturation curve, causing the protein to unfold at higher concentrations of chemical denaturants (Main et. al . , 1999). Ligand-binding constants from the difference in stability can be determined using Equation 1 below.

ΔG _app is the apparent difference in free energy of unfolding between the free and ligand-binding forms, Is the free energy of unfolding in the water of the free state protein, [L] is the concentration of the ligand, and K _d is the dissociation constant of the protein-ligand complex (Meiering et. al . , 1992). The free energy of unfolding can be related to the median value of the unfolding transition using the equation

Where m _{D -N} is a constant depending on the protein provided and the denaturant provided, which is proportional to the change in the degree of exposure of the residues in the spread (Tanford 1968 and Tanford 1970) and [D] _50% of the denaturant corresponding to the median unfold Concentration. We report the differences in the stability of FKBP12 for rapamycin and unknown ligands (at the same ligand concentration).

으로 정의하고 as

Define as

Where < m _{D -N} > is the mean m -value of the unfolded transition state, and Δ [D] _50% is the difference between the median of the rapamycin-FKBP12 unfolded transition state and the unfolded transition state of the unknown ligand-FKBP12 complex. to be. Under conditions where [L]> K _d , ΔΔ G _{D −N} may be related to the relative K _d of the two compounds via Formula 4,

은 라파마이신의 해리 상수이고,

는 알려지지 않은 리간드 X의 해리 상수이다. 따라서,In the above,

Is the dissociation constant of rapamycin,

Is the dissociation constant of unknown ligand X. therefore,

Becomes

을 계산할 수 있다.

의 문헌적 값인 0.2 nM이 사용된다.For the determination of K _d of 39-desmethoxyrapamycin, the respective denaturation curves were fitted to calculate the values for m _DN and [D] _50% , using which the average m − value, < m _{D −N} >, And [Delta] [D] _50% , thus,

Can be calculated.

The literature value of 0.2 nM is used.

TABLE 8 In vitro FKBP12  Join analysis result

mTOR

Inhibition of mTOR was indirectly demonstrated by measuring surrogate markers of the mTOR pathway and the level of phosphorylation of p70S6 kinase and S6 (Brunn et. al ., 1997; Mothe-Satney et al . , 2000; Tee and Proud, 2002; Huang and Houghton, 2002).

HEK293 cells were simultaneously transfected with FLAG-tagged mTOR and myc-tagged Raptor, incubated for 24 hours, and then not given serum overnight. The cells were promoted with 100 nM insulin and then harvested and lysed by three freeze / thaw cycles. The resulting lysates were collected and immunoprecipitation with FLAG antibody for mTOR / Raptor complex at the same content. Immunoprecipitation was then progressed: samples treated with compounds (0.00001-0.003 mM) were previously pretreated at 30 ° C. for 30 minutes with FKBP12 / rapamycin, FKBP12 / 39-desmethoxyrapamycin derivative or vehicle (DMSO). The untreated samples were incubated in kinase buffer solution. Under Then, the immunoprecipitation ^{3 mM ATP, 10 mM Mn 2} + , and the substrate GST-4E-BP1 exists, and the in vitro kinase assay. The reaction was stopped with 4 times sample buffer, then 15% SDS-PAGE, wetted with PVDF membrane, and then probed for phospho-4E-BP1 (T37 / 46).

Alternatively, HEK293 cells were seeded in 6 well plates, precultured for 24 hours, and then not given serum overnight. Cells were pretreated with vehicle or compound at 30 ° C. for 30 minutes, then promoted with 100 nM insulin at 30 ° C. for 30 minutes, and cells were lysed by three freeze / thaw cycles and protein concentration analyzed. Equal amounts of protein were loaded and separated on SDS-PAGE gels. Proteins were transferred to PVDF membranes and then probed for phospho-S6 (S235 / 36) or phospho-p70 S6K (T389).

The results of this experiment are shown according to FIG. 3.

Example  5: in vitro P- gp  Substrate analysis

Cell line

The cell lines used in the current study (MACL MCF7 and MACL MCF7 ADR) were all provided by the National Cancer Center.

Cells were typically passed once or twice a week. Cells were maintained in culture for up to 20 passages. Humidified atmosphere (95% air, 5% CO ₂ ) in RPMI 1640 medium (PAA, Colbe, Germany) supplemented with 5% calf serum (PAA, Colbe, Germany) and 0.1% zetamycin (PAA, Colbe, Germany) All cells were raised at 37 ° C.

Analysis protocol

Based on Protocol 1 described above, a modified propidium iodine assay was used to assess the effect of 39-desmethoxyrapamycin (Dengler et. al . , 1995). Briefly, cells were harvested from exponentially staged cultures by trypsinization, counted, and plated in 96-well bottom-flat microtiter plates at a cell concentration of 5,000 cells / well. . After 24 hours recovery to allow the cells to grow exponentially, 0.01 ml of Verapamil was added to the cells at a concentration of 0.18 mg / mL or 0.01 mL culture medium, resulting in a final concentration of Verapamil in the wells at 0.01 mg. / mL. This concentration was found to be non-toxic to cells in previous experiments. Culture medium containing 39-desmethoxyrapamycin, taxol or culture medium alone (control wells) was added at 0.01 mL per well. The compound was applied three times at eight concentrations in half log steps ranging from 0.03 mM to 10 nM. After 3 days of continuous exposure to the drug, the medium or medium containing the compound was replaced with 0.2 mL of an aqueous propidium iodine (PI) solution (7 mg / L). Since the PI passes only through the well-soaked or lysed membranes, the DNA of dead cells will be stained and measured, whereas live cells will not be stained. To measure the percentage of live cells, cells were permeated by freezing the plate, confirming the death of all cells. After thawing the plate, fluorescence was measured using a Cytofluor 4000 microplate reader (excitation 530 nm, emission 620 nm) to obtain a direct relationship to the total cell number. Growth inhibition was expressed as test / control x 100 (% T / C). If a positive control (taxol) induces migration in tumor growth inhibition with and without verapamil and the vehicle C treated control cells had> 500 fluorescence intensity, the assay was considered valuable.

Preparation of 39-Desmethoxyrapamycin Test Solution

A 3.3 mM stock solution of 39-desmethoxyrapamycin was prepared in DMSO and stored at -20 ° C. The stock solution was then dissolved on the day of use and stored at room temperature prior to and during administration. The dilution step was performed using RPMI 1640 medium, resulting in a solution 18 times final concentration.

result

Figure 4 shows the percentages at all test concentrations of paclitaxel (A and C) and 39-desmethoxyrapamycin (B and D) in normal cell lines (A and B) or highly P-gp expressing cell lines (C and D). Four graphs demonstrating T / C are shown. Filled diamonds show values after paclitaxel or 39-desmethoxyrapamycin alone, and open squares receive paclitaxel or 39-desmethoxyrapamycin in the presence of 0.01 mg / mL verapamil (P-gp inhibitor). The value is then displayed.

Paclitaxel, a known P-gp substrate, showed reduced titers when inhibiting P-gp expressing cancer cell line MCF7 ADR, which was restored by simultaneous administration of verapamil, a P-gp inhibitor (FIG. 4A and 4C)

39-desmethoxyrapamycin did not show an effective shift in the growth proliferation curve of P-gp expressing cell line MCF7 ADR, with or without verapamil (FIGS. 4B and 4D), which showed 39-desmethoxyrappa Prove that mycin is not a substrate of P-gp.

Example  6- Pharmacokinetics  analysis

6.1. Rapamycin and  39- Of desmethoxyrapamycin PK  analysis

As described above, pharmacokinetic analyzes using standard methods were performed for rapamycin and 39-desmethoxyrapamycin (protocols used for each compound are shown in Table 9).

Kinetica 4.4 (InnaPhase Corporation) was used and the AUC was calculated for each compound in blood or brain tissue using a non-compartmental model and numerical integration method for AUC calculation.

p.o. And i.v. After administration, the partition coefficient (Ri) for the individual compounds was calculated as shown below.

The results of this analysis are summarized in Table 9 below and in FIG. 5.

<Table 9>- Pharmacokinetics Data  summary

6.2. Rapamycin , 39- Desmethoxyrapamicin  And 27-O- Deathmethyl -39- Of desmethoxyrapamycin PK  analysis

Pharmacokinetic analyzes for rapamycin, 39-desmethoxyrapamycin and 27-O-desmethyl-39-desmethoxyrapamycin were performed using standard methods as described above (protocol 1 described above). )).

Kinetica 4.4 (InnaPhase Corporation) was used and the AUC was calculated for each compound in blood or brain tissue using a non-compartmental model and numerical integration method for AUC calculation.

i.v. After administration, the partition coefficient (Ri) of each compound was calculated as shown below.

<Table 10>- Pharmacokinetics Data

Example  7-Laboratory of Multiple Sclerosis Allergic  Encephalomyelitis ( Experimentalal  Allergic Encephalomyelitis , EAE Active in the model

Laboratory allergic encephalomyelitis (EAE) is an autoimmune inflammatory and denyelinating disease of the central nervous system (CNS) and is considered the most optimally available animal counterpart for multiple sclerosis (MS). The disease can be induced by injecting genetically sensitive animals with whole spinal cord or myelin basic protein (MBP) in complete Freund's adjuvant (CFA). Antigen-specific effector cells associated with CNS injury are a class II major histocompatibility complex (MHO) that is limited to CD4 + T lymphocytes. Recently, the role of cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF) or interferon (IFN) in the inflammatory response has received high attention. Upon activation by antigen, T cells produce several lymphokines that, in the case of EAE, can respond directly or indirectly to CNS damage. Related to the development of EAE, the same lymphokines are IL-2, IFN-γ, and TNF-β. IL-2 plays an important role in T cell activation and proliferation, whereas IFN-γ is a powerful mediator of macrophage activation. In addition, IFN- [gamma] induces the production of inflammatory cytokines such as IN-1. Expression of class II MHC molecules in TNF, and also among others, in endothelial cells of the CNS vascular wall, and in astrocytes, is believed to play an important role in antigen delivery of T cells that cause encephalitis.

7.1.- Animal and Immunization Procedures

Male Lewis rats, 8 to 10 weeks old, were given free access to food and water and were raised under standard laboratory conditions (no specific pathogens). 50 mL of incomplete adjuvant (Difco, Detroit, Ml) and 25 mg guinea pig spinal cord and 1 mg Mycobacterium tuberculosis strain H 37 RA (Difco) on the tail donor EAE was induced by one injection.

7.2.- Clinical and histological records

Up to 30 days after immunization, rats were examined by measuring rat body weight and clinical signs of EAE daily. The following clinical grading was performed by observers who did not know the treatment: 0 = no disease, 1 = limp tail, 2 = slightly paraparesis, 3 = severe paraparesis Oh, 4 = dying, 5 = death. For rats with grade 0 for 5 consecutive days, it was defined that the disease disappeared when clinical symptoms completely disappeared and the motility of the pre-immunization period was restored.

7.3.- Experimental Treatment

Sample compounds were given at various doses (5 or 15 mg / kg body weight) in both the prophylactic regime and the therapeutic regime. For the preventive planning part of the study, treatment was started one day prior to immunization, and for the treatment planning part of the study, treatment was initiated 7 days after immunization (p.i.). For prophylactic or therapeutic purposes, vehicle-treated rats treated under the same experimental conditions were used as controls. p.i. was treated daily with p.o. six times a week until 30 days. Cyclophosphamide was used as a positive control.

The experimental results are shown in FIG. 6 and Table 11 below. FIG. 6A shows the effect of the prevention plan of 39-desmethoxyrapamycin at 5 and 15 mg / kg, and FIG. 6B shows the effect of the treatment plan of 39-desmethoxyrapamycin at 5 and 15 mg / kg. . In each scheme, the effect of 40 mg / kg cyclophosphamide is shown as a positive control. In both graphs, the median of each group is shown. 39-desmethoxyrapamycin has the same efficacy as cyclophosphamide in this model, and 39-desmethoxyrapamycin not only reduces the severity of symptoms but also reduces the duration of the episode. It can be seen. Due to death during the study of five of the seven vehicle-treated rats, the mean value of this group remained at 5, but note that both live rats returned to their baseline values by the end of 28 days. Should be.

TABLE 11

* Statistically different from vehicle-treated controls, p <0.05, Mann Whitney Rank Sum test.

Example  8-Nude glioma xenografted in the same spot in nude mice ( glioma  orthotopically xenografted Model 39- Of desmethoxyrapamycin  Antitumor Activity Study

8.1. Study preparation

8.1.1-Sample Preparation:

The test compound was dissolved in ethanol (0.027 mL / mg compound) and vortexed for 20 minutes until the resulting solution became clear. The obtained ethanol solution was appropriately aliquoted and stored at -20 ° C. The obtained ethanol solution was then adjusted to the correct concentration with the vehicle (4% ethanol, 5% Tween-20, 5% polyethylene glycol 400 in 0.15 M NaCl, if possible with sterilized endotoxin free components) box).

8.1.2. -Means of administration

Test substance and control vehicle were administered intravenously (IV, mass) by injecting into the tail vein of the test animal. Based on the most recent mouse body weight, an injection volume of 10 mL / kg was used.

8.1.3. -Cancer cell line

The cell line used in the study was U87-MG, the glioblastoma cell host initiated by J. Ponten from grade III glioblastoma of a 44 year old Caucasian female (Poten et. al . , 1968).

8.1.4. Cell culture conditions for cell line formation

Tumor cells were raised in a single layer at 37 ° C. in a humid atmosphere (5% CO ₂ , 95% air). Culture medium was RPM1 1640 (Reference BE12-702F, Cambrex) with 2 mM L-glutamine supplemented with 10% fetal bovine serum (Reference DE 14-801 E, Cambrex). Cells were attached to plastic flasks. For use in experiments 5 treated with trypsin-versene (Ref. BE17-161E, Cambrex) in Hanks' medium (Ref. BE10-543F, Cambrex) without calcium or magnesium For minutes, tumor cells were removed from the flask in culture. Cells were counted on a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.

8.2.- In the brains of nude mice orientation  By injection of method Glioblastoma  Judo

After γ-source (2.5 Gy, Co ⁶⁰ , INRA BRETENIERE, Dijon) was irradiated throughout the body, mice were injected with U87-MG cells in a stereotactic manner at 24-48 h, DO. For injection by tumor positioning method, 100 mg / kg ketamine (Ketamine5OO ^® , see 043KET204, Centravet, France) and 0.95 NaCl solution and Xylazine (Rompun ^® , Ref 002ROM001, Centravet, France) Mice were anesthetized by injecting 5 mg / kg into the peritoneum at 10 mL / kg / injection. 1 x 10 ⁵ U87-MG tumor cells re-suspended in 0.002 mL of RPMI-1640 medium using three independent positioning methods (Kopf Instrument, Germany and Stoelting Company, USA) on the right ear canal surface of the mouse Cells were injected by the stereotactic method. 0.002 mL of the cell suspension was injected at 500 nL / min.

8.3-Treatment Plan

At D7, mice were weighed and mice were randomly divided into three groups according to their individual weights. For MRI imaging, four additional mice were added to each treatment group. The groups were selected so that the mean weight of each group was not statistically different from the other groups (variance analysis). Test substances were administered as defined below.

8.3.1. Mice in group 1 experienced five cycles of daily IV infusion of test substance vehicle for three consecutive days (D9 at D7, D16 at D14, D23 at D21, D30 at D28, and D37 at D35: (Q1Dx3) x5W). . Each cycle is divided into four day periods of enteral lavage.

8.3.2 Mice in group 2 were 39-desmethoxyrappa at 3 mg / kg / injection for 3 consecutive days with D9 at D7, D16 at D14, D23 at D21, D30 at D28 and D37 (Q1Dx3) x5W at D35). We experienced five cycles of daily IV injection of mycin. Each cycle is divided into four day periods of enteral lavage.

8.3.3. Mice in group 3 were not treated.

The treatment plan is summarized in Table 12 below.

TABLE 12

8.4.- MRI  analysis

MRI analysis of the brain was performed at D23 and D37. All MRI analyzes were performed with 4.7T on Pharmascan magnet (Bruker, Wissembourg). The mouse was placed under anesthesia with isoflurane in a mouse-only cradle and a 38 mm diameter circular coil.

After three acquisitions, the TurboRare T2 weighted sequence was performed. Acquisition covered the entire brain, including tumors. Tumor volume was measured by hand drawing the region of interest (ROI) around the tumor in each slice and summing all surfaces.

8.5.- Results

7 shows a survival graph of each treatment group up to 43 days.

The results are also expressed in percent (T / C%), where T represents the mean survival time among animals treated with 39-desmethoxyrapamycin and C represents the average survival time for control animals treated with vehicle. T / C% was calculated as follows.

                       T / C% = [T / C] x 100

In addition, MRI analysis was used to calculate the average of the calculated tumor volume per treatment group and the results are summarized in Table 13 below. Since all vehicle treated animals died until 37 days, it was not possible to compare tumor size at this stage.

TABLE 13

Each data score represents four mean values.

Reference

Claims (23)

In the manufacture of a medicament for the treatment of a medical condition resulting from nerve injury or disease, R ₁ represents (H, H) or = 0 and R ₂ and R ₃ each independently represent H, OH or OCH ₃ 39-desmethoxyrapamycin analogue or a pharmaceutically acceptable salt thereof.

In the manufacture of a medicament for the treatment of a medical condition affecting the central nervous system, R ₁ represents (H, H) or = O and R ₂ and R ₃ each independently represent H, OH or OCH ₃ Use of the 39-desmethoxyrapamycin analogue according to (1), wherein said medical condition requires a medicament that crosses the blood brain barrier.

Use according to claim 1 or 2, wherein the medical condition is selected from the group consisting of brain tumors and neurodegenerative conditions. Use according to claim 3, wherein the medicament is for the treatment of brain tumors. Use according to claim 4, wherein the brain tumor is glioblastoma multiforme. 4. Use according to claim 3, wherein the medicament is for the treatment of neurodegenerative conditions. Use according to claim 6, wherein said neurodegenerative condition is Alzheimer's disease. Use according to claim 6, wherein said neurodegenerative condition is multiple sclerosis. In the manufacture of a medicament for the treatment of cancer or B-cell malignancies, R ₁ represents (H, H) or = 0 and R ₂ and R ₃ each independently represent H, OH or OCH ₃ Use according to 39-desmethoxyrapamycin analogs or pharmaceutically acceptable salts thereof, wherein the cancer or B-cell malignancy is resistant to one or more existing anticancer agents.

10. The use of claim 9, wherein the cancer or B-cell malignant tumor expresses P-glycoprotein. Use according to claim 10, wherein the cancer or B-cell malignancy has a high expression level of P-glycoprotein. The use according to any one of claims 1 to 11, wherein the medicament is for intravenous administration of a 39-desmethoxyrapamycin analog or a pharmaceutically acceptable salt thereof. Use according to any one of the preceding claims, wherein the medicament further comprises one or more other therapeutic agonists. The method according to any one of claims 3 to 5 or 9 to 13, wherein the medicament is for the treatment of cancer or B-cell malignancy, and the medicament is methotrexate, leucovorin, adriamycin, prini. Prenisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, toremifene Guestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g. Herceptin ^™ ), capecitabine, raloxifene hydrochloride And at least one agent selected from the group consisting of EGFR inhibitors, VEGF inhibitors, proteasome inhibitors and hsp90 inhibitors. The use according to any one of claims 1 to 14, wherein the 39-desmethoxyrapamycin analog is 39-desmethoxyrapamycin. The use according to any one of claims 1 to 14, wherein the 39-desmethoxyrapamycin analog is further different from rapamycin at one or more of positions 9, 16 or 27. The use of claim 16, wherein the 39-desmethoxyrapamycin analog is different from rapamycin at one or more of the 16 or 27 positions. 17. The use of claim 16, wherein the 39-desmethoxyrapamycin analog is different from rapamycin at positions 16 and 27. The use according to any one of claims 16 to 18, wherein the 39-desmethoxyrapamycin analogue has a hydroxyl group at position 27, ie R ₃ represents OH. The use according to any one of claims 16 to 18, wherein the 39-desmethoxyrapamycin analog has hydrogen at position 27, ie R ₃ represents OH. 21. The use of any one of claims 16-20, wherein the 39-desmethoxyrapamycin analogue has a hydroxyl group at position 16, ie R ₂ represents OH. Along with the pharmaceutically acceptable carrier, R ₁ represents (H, H) or = 0 and R ₂ and R ₃ each independently represent H, OH or OCH ₃ , 39-desume according to formula (I) A pharmaceutical composition comprising a thioxapamycin analog or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of claim 23 formulated especially for intravenous administration.

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