Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Rapamycin (sirolimus) ( Figure 1 ) is a lipophilic macrolide produced by Streptomyces hygroscopicus IsIRRL 5491 (Sehgal ef al., 1975; Vezina ef al., 1975; U.S. 3,929,992; U.S.
• rapamycin is described by the numbering convention of McAlpine ef a/. (1991) in preference to the numbering conventions of Findlay ef al. (1980) or Chemical Abstracts (11 th Cumulative Index, 1982-1986 p60719CS). Rapamycin has significant therapeutic value due to its wide spectrum of biological activities (Huang ef a/, 2003).
• the compound is a potent inhibitor of the mammalian target of rapamycin (mTOR), a serine-threonine kinase downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signalling pathway that mediates cell survival and proliferation.
• mTOR mammalian target of rapamycin
• PI3K phosphatidylinositol 3-kinase
• Akt protein kinase B
• Rapamycin is marketed as an immunosuppressant for the treatment of organ transplant patients to prevent graft rejection (Huang ef a/, 2003).
• rapamycin has potential therapeutic use in the treatment of a number of diseases, for example, cancer, cardiovascular diseases such as restenosis, autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, fungal infection and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease.
• rapamycin has a number of major drawbacks. Firstly it is a substrate of cell membrane efflux pump P-glycoprotein (P-gp, LaPlante ef a/, 2002, Crowe et al, 1999) which pumps the compound out of the cell making the penetration of cell membranes by rapamycin poor. This causes poor absorption of the compound after dosing. In addition, since a major mechanism of multi-drug resistance of cancer cells is via cell membrane efflux pump, rapamycin is less effective against multi-drug resistance (MDR) cancer cells. Secondly rapamycin is extensively metabolised by cytochrome P450 enzymes (Lampen et al, 1998).
• novel rapamycin-like compounds that are not substrates of P-gp, that may be metabolically more stable and therefore may have improved bioavailability.
• these compounds When used as anticancer agents, these compounds may have better efficacy against MDR cancer cells, in particular against P-gp-expressing cancer cells.
• CCI-779 Wyeth-Ayerst an ester of rapamycin which inhibits cell growth in vitro and inhibits tumour growth in vivo (Yu ef al., 2001 ).
• CCI-779 is currently in various clinical trials as a potential anticancer drug.
• a recent publication of CCI-779 phase Il study in patients with recurrent glioblastoma multiforme (Chang, et al., 2005) suggests the low efficacy of this drug in these patients may be due to its poor penetration of blood-brain barrier.
• Studies investigating the pharmacokinetics of RAD001 have shown that, similarly to rapamycin, it is a substrate for P- gp (Crowe ef al, 1999, LaPlante ef al, 2002).
• the compounds of the invention displays a surprisingly different pharmacological profile. In particular they show significantly increased cell membrane permeability and decreased efflux in comparison with rapamycin, and they are not a substrate for P-gp. Additionally, 39-desmethoxyrapamycin shows more potent activity against multi-drug resistant and P-gp-expressing cancer cell lines than rapamycin. When compared with rapamycin 39-desmethoxyrapamycin shows a significantly different inhibitory profile against the NCI 60 cell line panels. Additionally, 39-desmethoxyrapamycin analogues show a significantly different pharmacokinetic profile compared to rapamycin and the leading derivatives in clinical trials.
• 39-desmethoxyrapamycin analogues show an increased ability to cross the blood brain barrier and therefore demonstrate improved availability in the brain. Therefore, the present invention provides for the medical use of 39- desmethoxyrapamycin analogues, these rapamycin analogues have significantly altered pharmacokinetics, improved ability to cross the blood brain barrier, improved metabolic stability, improved cell membrane permeability, a decreased rate of efflux and a different tumour cell inhibitory profile to rapamycin.
• These compounds are useful in medicine, in particular for the treatment of cancer and/or B-ceil malignancies, in the induction or maintenance of immunosuppression, the stimulation of neuronal regeneration or the treatment of fungal infections, transplantation rejection, graft vs.
• the present invention particularly provides for the use of 39-desmethoxyrapamycin in the treatment of cancer and / or B-cell malignancies. Rapamycin has been demonstrated to stimulate autophagy (Raught et al., 2001 ).
• Hyperphosphorylation of the microtubule-associated protein tau and its subsequent aggregation into insoluble paired helical filaments which form intracellular "tangles" is one of the characteristic hallmarks of Alzheimer's disease and the accumulation of this neurofibrillary pathology and the associated neuronal cell death is closely related to the cognitive decline.
• rapamycin increases neuritic outgrowth and neuronal survival in several in vitro and in vivo models (Avramut and Achim, 2002) indicating that rapamycin and analogues thereof may be of use in treating disorders where neuronal regeneration may be of significant therapeutic benefit.
• this utility is dependent on it being able to reach the site of action and therefore rapamycin analogues with an improved ability to cross the blood brain barrier would be particularly preferred.
• the present invention provides the novel and surprising use of 39- desmethoxyrapamycin analogues in medicine, in particular the use of 39- desmethoxyrapamycin, particularly in the treatment of cancer or B-cell malignancies, in the induction or maintenance of immunosuppression, the stimulation of neuronal regeneration or the treatment of fungal infections, transplantation rejection, graft vs. host disease, autoimmune disorders, neurodegenerative conditions, diseases of inflammation vascular disease and fibrotic diseases.
• the present invention provides for the use of 39-desmethoxyrapamycin analogues in the treatment of cancer and B-cell malignancies.
• the present invention provides for the use of 39-desmethoxyrapamycin analogues in the treatment of neurological or neurodegenerative disorders.
• the present invention provides for the use of 39-desmethoxyrapamycin analogues in the treatment of brain tumours, in particular glioblastoma multiforme.
• the 39-desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the present invention relates to the medical use of 39-desmethoxyrapamycin analogues, in particular 39-desmethoxyrapamycin, particularly in the treatment of cancer and / or B-cell malignancies, the induction or maintenance of immunosuppression, the treatment of transplantation rejection, graft vs. host disease, autoimmune disorders, neurodegenerative conditions, diseases of inflammation, vascular disease and fibrotic diseases, the stimulation of neuronal regeneration or the treatment of fungal infections.
• this invention relates to the use of 39-desmethoxyrapamycin analogues for the treatment of cancer and B-cell malignancies.
• the present invention relates to the use of 39- desmethoxyrapamycin in the treatment of cancer and B-cell malignancies.
• the present invention also specifically provides for the use of 39-desmeth ' oxyrapamycin analogues in the treatment of brain tumour(s) or neurodegenerative conditions.
• the present invention provides for the use of 39-desmethoxyrapamycin in the treatment of brain tumour(s) or neurodegenerative conditions.
• the present invention also specifically provides for the use of 39-desmethoxyrapamycin analogues in the treatment of neurodegenerative conditions.
• the present invention provides for the use of 39-desmethoxyrapamycin in the treatment in neurodegenerative conditions.
• analogue means one analogue or more than one analogue.
• autoimmune disorder(s) includes, without limitation: systemic lupus erythrematosis (SLE), rheumatoid arthritis, myasthenia gravis and multiple sclerosis.
• diseases of inflammation includes, without limitation: psoriasis, dermatitis, eczema, seborrhoea, inflammatory bowel disease (including but not limited to ulcerative colitis and Crohn's disease), pulmonary inflammation (including asthma, chronic obstructive pulmonary disease, emphysema, acute respiratory distress syndrome and bronchitis), rheumatoid arthritis and eye uveitis.
• cancer refers to a malignant or benign growth of cells in skin or in body organs, for example but without limitation, breast, prostate, lung, kidney, pancreas, brain, stomach or bowel. A cancer tends to infiltrate into adjacent tissue and spread
• cancer includes both metastatic tumour cell types, such as but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, gliobastoma, primary liver cancer and ovarian cancer.
• the term also specifically encompasses brain tumour(s) as described more fully below.
• brain tumour(s) refers to a malignant or benign growth of cells in the brain, it includes primary and secondary (metastatic) tumours.
• Primary brain tumours include, without limitation, gliomas (e.g. glioblastoma multiforme, astrocytoma, brain stem glioma, ependymoma and oligodendroglioma), medulloblastoma, meningioma, schwannoma (or acoustic neuroma), craniopharyngioma, germ cell tumor of the brain (e.g. germinoma), or pineal region tumor.
• gliomas e.g. glioblastoma multiforme, astrocytoma, brain stem glioma, ependymoma and oligodendroglioma
• medulloblastoma meningioma
• schwannoma or acoustic neuroma
• B-cell malignancies includes a group of disorders that include chronic lymphocytic leukaemia (CLL), multiple myeloma, and non-Hodgkin's lymphoma (NHL). They are neoplastic diseases of the blood and blood forming organs. They cause bone marrow and immune system dysfunction, which renders the host highly susceptible to infection and bleeding.
• CLL chronic lymphocytic leukaemia
• NDL non-Hodgkin's lymphoma
• vascular disease includes, without limitation: hyperproliferative vascular disorders (e.g. restenosis and vascular occlusion), graft vascular atherosclerosis, cardiovascular disease, cerebral vascular disease and peripheral vascular disease (e.g. coronary artery disease, arteriosclerosis, atherosclerosis, nonatheromatous arteriosclerosis or vascular wall damage). It is also used to refer to diseases involving the neogenesis or proliferation of blood vessels in the eye, in particular choroidal neovascularization.
• neurovascular regeneration refers to the stimulation of neuronal cell growth and includes neurite outgrowth and functional recovery of neuronal cells.
• Neuronal regeneration may be of significant therapeutic benefit
• diseases and disorders where neuronal regeneration may be of significant therapeutic benefit include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's chorea (disease), amyotrophic lateral sclerosis, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy, muscular dystrophy, stroke, progressive muscular atrophy, progressive bulbar inherited muscular atrophy, cervical spondylosis, Gullain-Barre syndrome, dementia, peripheral neuropathies and peripheral nerve damage, whether caused by physical injury (e.g. spinal cord injury or trauma, sciatic or facial nerve lesion or injury) or a disease state (e.g. diabetes).
• the terms "medical condition resulting from neural injury or disease” includes without limitation, neurodegenerative condition(s), brain tumour(s), infection or inflammation of the brain and other conditions which may lead to death or dysfunction of nervous or glial cells or tissues.
• neurodegenerative condition(s) includes, without limitation, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), (oculopharyngeal) muscular dystrophy, . (including oculopharyngeal muscular dystrophy), multiple sclerosis, prion diseases (e.g. Creutzfeldt-Jacob disease (CJD)), Pick's disease, Lewy body dementia (or Lewy body disease) and/or motor neurone disease.
• ALS amyotrophic lateral sclerosis
• CJD Creutzfeldt-Jacob disease
• Pick's disease e.g. Creutzfeldt-Jacob disease (CJD)
• Lewy body dementia or Lewy body disease
• motor neurone disease e.g. Creutzfeldt-Jacob disease
• the term "medical condition affecting the central nervous which requires the medicament to cross the blood-brain barrier” includes without limitation medical conditions resulting from neural injury or diseases, and any other condition for which the access of the medicament to the neuronal cells is required for effective therapy.
• fibrotic diseases refers to diseases associated with the excess production of the extracellular matrix and includes (without limitation) sarcoidosis, keloids, glomerulonephritis, end stage renal disease, liver fibrosis (including but not limited to cirrhosis, alcohol liver disease and steato-heptatitis), chronic graft nephropathy, surgical adhesions, vasculopathy, cardiac fibrosis, pulmonary fibrosis (including but not limited to idiopathic pulmonary fibrosis and cryptogenic fibrosing alveolitis), macular degeneration, retinal and vitreal retinopathy and chemotherapy or radiation-induced fibrosis.
• graft vs. host disease refers to a complication that is observed after allogeneic stem cell / bone marrow transplant. It occurs when infection-fighting cells from the donor recognize the patient's body as being different or foreign. These infection- fighting cells then attack tissues in the patient's body just as if they were attacking an infection. GvHD is categorized as acute when it occurs within the first 100 days after transplantation and chronic if it occurs more than 100 days after transplantation. Tissues typically involved include the liver, gastrointestinal tract and skin. Chronic graft vs. host disease occurs approximately in 10-40 percent of patients after stem cell / bone marrow transplant.
• bioavailability refers to the degree to which or rate at which a drug or other substance is absorbed or becomes available at the site of biological activity after administration. This property is dependent upon a number of factors including the solubility of the compound, rate of absorption in the gut, the extent of protein binding and metabolism etc. Various tests for bioavailability that would be familiar to a person of skill in the art are described herein (see also Trepanier et a/, 1998, Gallant-Haidner et al, 2000).
• cancer or B-cell malignancy resistant to one or more existing anticancer agent(s) refers to cancers or B-cell malignancies for which at least one typically used therapy is ineffective. These cancers are characterised by being able to survive after the administration of at least one neoplastic agent where the normal cell counterpart (i.e., a growth regulated cell of the same origin) would either show signs of cell toxicity, cell death or cell quiescence (i.e., would not divide). In particular, this includes MDR cancers or B-cell malignancies, particular examples are cancers and B-cell malignancies which express high levels of P-gp. The identification of such resistant cancers or B-cell malignancies is within the ability and usual activities of a physician or other similarly skilled person.
• 39-desmethoxyrapamycin analogues refers to a compound according to formula (I) below, or a pharmaceutically acceptable salt thereof.
• R 2 and R 3 each independently represents H, OH or OCH 3 .
• 39-desmethoxyrapamycin analogue includes 39- desmethoxyrapamycin itself.
• the pharmaceutically acceptable salts of 39-desmethoxyrapamycin analogues include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like.
• acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts.
• suitable basic salts include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts.
• References hereinafter to a compound according to the invention include both 39-desmethoxyrapamycin and its pharmaceutically acceptable salts.
• the present invention relates to the use of a 39-desmethoxyrapamycin analogue in medicine, in particular in the treatment of cancer, B-cell malignancies, the induction or maintenance of immunosuppression, the treatment of transplantation rejection, graft vs. host disease, autoimmune disorders, neurodegenerative conditions, diseases of inflammation, vascular disease and flbrotic diseases, the stimulation of neuronal regeneration, the treatment of neurological diseases or neurodegenerative conditions or the treatment of fungal infections. Therefore, the present invention provides for the use of a 39-desmethoxyrapamycin analogue, or a pharmaceutically acceptable salt thereof, in the treatment of a medical condition resulting from neural injury or disease.
• the present invention provides for the use of 39-desmethoxyrapamycin, or a pharmaceutically acceptable salt thereof, in the treatment of a medical condition resulting from neural injury or disease.
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27 in the treatment of a medical condition resulting from neural injury or disease.
• the present invention also provides for the use of a 39-desmethoxyrapamycin analogue, i.e. a rapamycin analogue with increased blood-brain barrier permeability, or a pharmaceutically acceptable salt thereof, in the treatment of medical conditions affecting the central nervous which require the medicament to cross the blood-brain barrier i.e. medical conditions where the blood-brain barrier impedes the delivery of the compound.
• a 39-desmethoxyrapamycin analogue i.e. a rapamycin analogue with increased blood-brain barrier permeability
• a pharmaceutically acceptable salt thereof in the treatment of medical conditions affecting the central nervous which require the medicament to cross the blood-brain barrier i.e. medical conditions where the blood-brain barrier impedes the delivery of the compound.
• the present invention provides for the use of 39-desmethoxyrapamycin, or a pharmaceutically acceptable salt thereof, in the treatment of medical conditions affecting the central nervous system where the blood-brain barrier impedes the delivery of the
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27, in the treatment of medical conditions affecting the central nervous system where the blood brain barrier impedes the delivery of the compound.
• this invention relates to the use of a 39- desmethoxyrapamycin analogue for the treatment of cancer and B-cell malignancies. In a further embodiment this invention relates to the use of 39-desmethoxyrapamycin for the treatment of cancer and B-cell malignancies. In a further embodiment, the present invention relates to the use of a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27 for the treatment of cancer and B-cell malignancies. The present invention also specifically provides for the use of a 39- desmethoxyrapamycin analogue in the treatment of brain tumour(s).
• the present invention further provides for the use of 39-de ⁇ methoxyrapamycin the treatment of brain tumour(s).
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27 in the treatment of brain tumour( ⁇ ).
• the present invention provides for the use of a 39- desmethoxyrapamycin analogue in the treatment of glioblastoma multiforme
• the present invention provides for the use of 39-desmethoxyrapamycin in the treatment of glioblastoma multiforme.
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27 in the treatment of glioblastoma multiforme.
• the present invention also provides for the use of a 39-desmethoxyrapamycin analogue in the treatment of neurodegenerative conditions.
• the present invention provides for the use of 39-desmethoxyrapamycin in the treatment of neurodegenerative conditions.
• the present invention provides for the use of a 39- desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27in the treatment of neurodegenerative conditions.
• the neurodegenerative condition may be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington's disease. Therefore, in one embodiment the present invention provides for the use of a 39-desmethoxyrapamycin analogue in the treatment of Alzheimer's disease.
• the present invention provides for the use of 39- desmethoxyrapamycin in the treatment of Alzheimer's disease. In a further embodiment the present invention provides for the use of a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27 in the treatment of Alzheimer's disease. In a further embodiment the present invention provides for the use of a 39- desmethoxyrapamycin analogue in the treatment of multiple sclerosis. In a further embodiment the present invention provides for the use of 39-desmethoxyrapamycin in the treatment of multiple sclerosis.
• the present invention provides for the use of a 39- desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27 in the treatment of multiple sclerosis.
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue in the treatment of Huntington's disease.
• the present invention provides for the use of 39-de ⁇ methoxyrapamycin in the treatment of Huntington's disease.
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27 in the treatment of Huntington's disease.
• the present invention provides a method for the treatment of cancer or B-cell malignancies, a method of induction or maintenance of immunosuppression, the stimulation of neuronal regeneration, a method for the treatment of fungal infections, transplantation rejection, graft vs. host disease, autoimmune disorders, neurodegenerative conditions, diseases of inflammation vascular disease or fibrotic diseases which comprises administering to a patient an effective amount of a 39-desmethoxyrapamycin analogue, in particular 39-desmethoxyrapamycin or a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention provides a method of treatment of a medical condition resulting from neural injury or disease, comprising administering a 39-desmethoxyrapamycin analogue, or a pharmaceutically acceptable salt thereof.
• the present invention provides a method of treatment of a medical condition resulting from neural injury or disease, comprising administering 39-desmethoxyrapamycin.
• the present invention provides a method of treatment of a medical condition resulting from neural injury or disease, comprising administering a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention also provides a method of treatment of medical conditions affecting the central nervous system wherein the blood-brain barrier impedes the delivery of the compound, by administering an effective amount of a 39-desmethoxyrapamycin analogue, i.e. a rapamycin analogue with increased blood-brain barrier permeability, or a pharmaceutically acceptable salt thereof.
• a 39-desmethoxyrapamycin analogue i.e. a rapamycin analogue with increased blood-brain barrier permeability, or a pharmaceutically acceptable salt thereof.
• the 39- desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39- desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention provides a method of treatment of cancer or B-cell malignancies which comprises administering to a patient an effective amount of a 39- desmethoxyrapamycin analogue.
• the present invention provides a method of treatment of brain tumour(s) which comprises administering to a patient an effective amount of a 39-desmethoxyrapamycin analogue.
• the 39- desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39- desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention provides a method of treatment of glioblastoma multiforme which comprises administering to a patient an effective amount of a 39-desmethoxyrapamycin analogue.
• the 39-desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39-desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention provides a method of treatment of a neurodegenerative condition which comprises administering to a patient an effective amount of a 39-desmethoxyrapamycin analogue.
• the 39- desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39- desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the neurodegenerative condition may be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington's disease. Therefore, in one embodiment the present invention provides a method of treatment of Alzheimer's disease which comprises administering to a patient an effective amount of a 39-desmethoxyrapamycin analogue.
• the 39-desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39-desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• a method of treatment of multiple sclerosis which comprises administering to a patient an effective amount of a 39-desmethoxyrapamycin analogue.
• the 39-desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39- desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention provides a method of treatment of Huntington's disease which comprises administering to a patient an effective amount of a 39-desmethoxyrapamycin analogue.
• the 39-desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39-desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention also provides the use of a 39-desmethoxyrapamycin analogue in the manufacture of a medicament for treatment of cancer or B-cell malignancies, for induction or maintenance of immunosuppression, for stimulation of neuronal regeneration, for the treatment of fungal infections, transplantation rejection, graft vs. host disease, autoimmune disorders, neurodegenerative conditions, diseases of inflammation vascular disease or fibrotic diseases.
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a medical condition resulting from neural injury or disease.
• the 39-desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39- desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention also provides for the use of a 39-desmethoxyrapamycin analogue, i.e. a rapamycin analogue with increased blood-brain barrier permeability, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of medical conditions affecting the central nervous system where the blood-brain barrier impedes the delivery of the compound.
• the 39-desmethoxyrapamycin analogue is 39- desmethoxyrapamycin.
• the 39-desmethoxyrapamycin analogue is a 39- desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention also specifically provides for the use of a 39- desmethoxyrapamycin analogue in the manufacture of a medicament for the treatment of brain tumour(s).
• the 39-desmethoxyrapamycin analogue is 39- desmethoxyrapamycin.
• the 39-desmethoxyrapamycin analogue is a 39- desmeth ⁇ xyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention specifically provides for the use of a 39-desmethoxyrapamycin analogue in the manufacture of a medicament for the treatment of glioblastoma multiforme.
• the 39-desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39-desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention also specifically provides for the use of a 39- desmethoxyrapamycin analogue in the manufacture of a medicament for the treatment of neurodegenerative conditions.
• the 39-desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39-desmethoxyrapamycin analogue is a 39- desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the neurodegenerative condition may be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington' ⁇ disease.
• the present invention provides for the use of a 39- desmethoxyrapamycin analogue in the manufacture of a medicament for the treatment of Alzheimer's disease.
• the 39-desmethoxyrapamycin analogue is 39- desmethoxyrapamycin.
• the 39-desmethoxyrapamycin analogue is a 39- desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue in the manufacture of a medicament for the treatment of multiple sclerosis.
• the 39-desmethoxyrapamycin analogue is 39- desmethoxyrapamycin. In a further aspect the 39-desmethoxyrapamycin analogue is a 39- desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27. In an alternative embodiment, the present invention provides for the use of a 39-desmethoxyrapamycin analogue in the manufacture of a medicament for the treatment of Huntington's disease. In a specific aspect the 39-desmethoxyrapamycin analogue is 39- desmethoxyrapamycin. In a further aspect the 39-desmethoxyrapamycin analogue is a 39- desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• 39-Desmethoxyrapamycin analogues are close structural analogues of rapamycin that are made using the methods described in WO 04/007709. However they show a different spectrum of activity to rapamycin, for example as shown by the COMPARE analysis of the NCl 60 cell line panel for 39-desmethoxyrapamycin and related analogues (see table 1 below).
• the COMPARE analysis uses a Pearson analysis to compare the activity of two compounds on the NCI 60-cell line panel and produces a correlation coefficient which indicates how similar the two compounds spectra of activity are and this may indicate how related their mechanism's of action are.
• the Pearson coefficient for rapamycin and 39- desmethoxyrapamycin is 0.614, this should be compared to the Pearson coefficient between rapamycin and CCI-779 (a 40-hydroxy ester derivative of rapamycin) which is 0.86 (Dancey, 2002). Therefore, it can be seen that 39-desmethoxyrapamycin analogues have a different spectrum of activity compared to rapamycin.
• Multi-Drug Resistance is a significant problem in the treatment of cancer and B- cell malignancies. It is the principle reason behind the development of drug resistance in many cancers (Persidis A, 1999). Drugs which worked initially become totally ineffective after a period of time. MDR is associated with increased level of adenosine triphosphate binding cassette transporters (ABC transporters), in particular an increase in the expression of the MDR1 gene which encodes for P-glycoprotein (P-gp) or the MRP1 gene which encodes MRP1.
• ABS transporters adenosine triphosphate binding cassette transporters
• the level of MDR1 gene expression varies widely across different cancer-derived cell lines, in some cell lines it is undetectable, whereas in others may show up to a 10 or 100-fold increased expression relative to standard controls.
• 39-desmethoxyrapamycin analogues are not a substrate for P-gp.
• 39-Desmethoxyrapamycin analogues have a decreased efflux from Caco- 2 cells compared to rapamycin and 39-desmethoxyrapamycin was shown not to be a substrate for P-gp in an in vitro P-gp substrate assay (see examples herein).
• a further aspect of the invention provides for the use of a 39- desmethoxyrapamycin analogue in the treatment of a cancer or B-cell malignancy resistant to 5 one or more existing anticancer agent(s) i.e. MDR cancers or B-cell malignancies.
• the present invention provides for the use of 39-desmethoxyrapamycin in the treatment of P-gp-expressing cancers or B-cell malignancies.
• the present invention provides for the use of 39-desmethoxyrapamycin in the treatment of high P-gp expressing cancers or B-cell malignancies.
• high P-gp expressing cancers or B-cell malignancies particularly, high P-gp expressing cancers or B-cell
• 10 malignancies may have 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold or 100-fold increased expression relative to control levels.
• 39- desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• 39-desmethoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27. Suitable controls are
• suitable cell lines include: 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,
• desmethoxyrapamycin analogue in the preparation of a medicament for use in the treatment of MDR cancers or B-cell malignancies.
• the present invention provides for the use of a 39-de ⁇ methoxyrapamycin analogue in the preparation of a medicament for use in the treatment of P-gp-expressing cancers or B-cell malignancies.
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue
• high P-gp expressing cancers or B-cell malignancies may have 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold or 100-fold increased expression relative to control levels.
• 39-desmethoxyrapamycin analogue is 39- desmethoxyrapamycin.
• 39-desmethoxyrapamycin analogue is a 39-
• 35 desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27. Suitable controls are described above.
• the present invention provides a method for the treatment of P-gp-expressing-cancers or B-cell malignancies comprising administering a therapeutically effective amount of a 39-desmethoxyrapamycin analogue.
• the 39- desmethoxyrapamycin analogue is 39-desmethoxyrapamycin.
• the 39- de ⁇ methoxyrapamycin analogue is a 39-desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• P- glycoprotein (P-gp) in a particular cancer type may be determined by a person of skill in the art using techniques including but not limited to real time RT-PCR (Szakacs et a/, 2004; Stein et al, 2002; Langmann et al; 2003), by immunohistochemistry (Stein ef a/, 2002) or using microarrays (Lee et al, 2003), these methods are provided as examples only, other suitable methods will occur to a person of skill in the art.
• 39-Desmethoxyrapamycin shows increased metabolic stability compared to rapamycin as shown herein in the examples.
• a number of papers have previously identified the 39- methoxy group on rapamycin as being a major site of metabolic attack to convert rapamycin to 39-O-desmethylrapamycin (Trepanier et al, 1998).
• the major metabolites of rapamycin have significantly decreased activity when compared to the parent compound (Gallant-Haidner et al, 2000, Trepanier ef al, 1998).
• 39-desmethoxyrapamycin no longer has available the most significant sites of metabolic attack, which results in an increased stability of the compounds (see examples).
• 39-desmethoxyrapamycin described above (that it is not a substrate for P-gp, has increased metabolic stability and decreased efflux from cells via P-gp) indicate that 39-desmethoxyrapamycin has improved bioavailability compared to its parent compound rapamycin. Therefore, the present invention provides for the use of 39-de ⁇ methoxyrapamycin, a rapamycin analogue with improved metabolic stability, improved cell membrane permeability and a distinct cancer cell inhibitory profile, in medicine, particularly in the treatment of cancer or B-cell malignancies.
• the present invention also provides a pharmaceutical composition comprising a 39- desmethoxyrapamycin analogue, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
• a pharmaceutical composition comprising 39-desmethoxyrapamycin.
• the present invention provides a pharmaceutical composition comprising a 39- desmethoxyrapamycin analogue that additionally differs from rapamycin at one or more of positions 9, 16 or 27.
• the present invention provides a pharmaceutical composition as described above that is specifically formulated for intravenous administration.
• Rapamycin and related compounds that are or have been in clinical trials have poor pharmacological profiles, including poor metabolic stability, poor permeability, high levels of efflux via P-gp and poor bioavailability.
• the present invention provides for the use of a 39-desmethoxyrapamycin analogue or a pharmaceutically acceptable salt thereof which has improved pharmaceutical properties compared to rapamycin.
• a further surprising aspect of the present invention is that 39-desmethoxyrapamycin analogues display a strikingly different pharmacokinetic profile when compared to the existing rapamycin analogues.
• 39-desmethoxyrapamycin analogues show increased blood brain barrier permeability and thus higher exposure of these compounds is seen in the brain compared to related analogues for a given blood level.
• Preferred 39-desmethoxyrapamycin analogues for use in any of the aspects of the invention described above include those which additionally differ from rapamycin at any one of positions 9, 16 or 27, i.e. it is preferred that the 39-desmethoxyrapamycin analogue is not 39- desmethoxyrapamycin itself.
• Further preferred 39-desmethoxyrapamycin analogues include those wherein: the 39-desmethoxyrapamycin analogue has a hydroxy! group at position 27, i.e. R 3 represents OH; the 39-desmethoxyrapamycin analogue has a hydrogen at position 27, i.e. R 3 represents OH; or the 39-desmethoxyrapamycin analogue has a hydroxyl group at position 16, i.e. R 2 represents OH.
• a person of skill in the art will be able to determine the pharmacokinetics and bioavailability of a compound of the invention using in vivo and in vitro methods known to a person of skill in the art, including but not limited to those described below and in Gallant-Haidner et a/, 2000 and Trepanier et a/, 1998 and references therein.
• the bioavailability of a compound is determined by a number of factors, (e.g.
• bioavailability of 39-desmeth ⁇ xyrapamycin or a pharmaceutically acceptable salt thereof may be measured using in vivo methods as described in more detail below, or in the examples herein.
• In vivo assays may also be used to measure the bioavailability of a compound such as
• 39-desmethoxyrapamcyin is administered to a test animal (e.g. mouse or rat) both intraperitoneally (i.p.) or intravenously (i.v.) and orally (p.o.) and blood samples are taken at regular intervals to examine how the plasma concentration of the drug varies over time.
• a test animal e.g. mouse or rat
• intraperitoneally i.p.
• intravenously i.v.
• p.o. orally
• blood samples are taken at regular intervals to examine how the plasma concentration of the drug varies over time.
• the time course of plasma concentration over time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models.
• An example of a typical protocol is described below.
• mice are dosed with 3 mg/kg of 39-desmethoxyrapamycin i.v. or 10 mg/kg of 39- desmethoxyrapamycin p.o.. Blood samples are taken at 5 min, 15 min, 1 h, 4 h and 24 h intervals, and the concentration of 39-desmethoxyrapamycin in the sample is determined via HPLC.
• the time-course of plasma or whole blood concentrations can then be used to derive key parameters such as the area under the plasma or blood concentration-time curve (AUC - which is directly proportional to the total amount of unchanged drug that reaches the systemic circulation), the maximum (peak) plasma or blood drug concentration, the time at which maximum plasma or blood drug concentration occurs (peak time), additional factors which are used in the accurate determination of bioavailability include: the compound's terminal half life, total body clearance, steady-state volume of distribution and F%. These parameters are then analysed by non-compartmental or compartmental methods to give a calculated percentage bioavailability, for an example of this type of method see Gallant-Haidner et a/, 2000 and Trepanier et a/, 1998, and references therein.
• AUC - area under the plasma or blood concentration-time curve
• additional factors which are used in the accurate determination of bioavailability include: the compound's terminal half life, total body clearance, steady-state volume of distribution and F%.
• the efficacy of 39-desmethoxyrapamycin may be tested in in vivo models for neurodegenerative diseases which are described herein and which are known to a person of skill in the art.
• models include, but are not limited to, for Alzheimer's disease - animals that express human familial Alzheimer's disease (FAD) p-amyloid precursor (APP), animals that overexpress human wild-type APP, animals that overexpress p-amyloid 1-42(pA), animals that express FAD presenillin-1 (PS-1) (e. g. German and Eisch, 2004).
• FAD familial Alzheimer's disease
• APP p-amyloid precursor
• pA p-amyloid 1-42
• PS-1 FAD presenillin-1
• For multiple sclerosis - the experimental autoimmune encephalomyelitis (EAE) model see Bradl, 2003 and Example 7).
• Parkinson's disease the 1-methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine (MPTP) model or the 6-hydroxydopamine (6-OHDA) model (see e.g. Emborg, 2004; Schober A. 2004).
• MPTP 1-methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine
• 6-OHDA 6-hydroxydopamine
• Huntington' ⁇ disease there are several models including the R6 lines model generated by the introduction of exon 1 of the human Huntington's disease (HD) gene carrying highly expanded CAG repeats into the mouse germ line (Satha ⁇ ivam et a/, 1999) and others (see Hersch and Ferrante, 2004).
• the aforementioned compound of the invention or a formulation thereof may be administered by any conventional method for example but without limitation they may be administered parenterally, orally, topically (including buccal, sublingual or transdermal), via a medical device (e.g. a stent), by inhalation or via injection (subcutaneous or intramuscular).
• a medical device e.g. a stent
• the treatment may consist of a single dose or a plurality of doses over a period of time.
• a 39-desmethoxyrapamycin analogue Whilst it is possible for a 39-desmethoxyrapamycin analogue to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
• the carrier(s) must be "acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Examples of suitable carriers are described in more detail below.
• a 39-desmethoxyrapamycin analogue may be administered alone or in combination with other therapeutic agents, co-administration of two (or more) agents allows for significantly lower doses of each to be used, thereby reducing the side effects seen.
• the increased metabolic stability of 39-desmethoxyrapamyin has an extra advantage over rapamycin in that it is less likely to cause drug-drug interactions when used in combination with drugs that are substrates of P450 enzymes as occurs with rapamycin (Lampen et al, 1998).
• a 39-desmethoxyrapamycin analogue is co-administered with another therapeutic agent for the induction or maintenance of immunosuppression, for the treatment of transplantation rejection, graft vs. host disease, autoimmune disorders or diseases of inflammation preferred agents include, but are not limited to, immunoregulatory agents e.g. azathioprine, corticosteroids, cyclophosphamide, cyclosporin A, FK506, Mycophenolate Mofetil, OKT-3 and ATG.
• immunoregulatory agents e.g. azathioprine, corticosteroids, cyclophosphamide, cyclosporin A, FK506, Mycophenolate Mofetil, OKT-3 and ATG.
• a 39-desmethoxyrapamycin analogue is co-administered with another therapeutic agent for the treatment of cancer or B-cell malignancies
• preferred agents include, but are not limited to, methotrexate, leukovorin, adriamycin, prenisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti- HER2 monoclonal antibody (e.g.
• HerceptinTM capecitabine, raloxifene hydrochloride, EGFR inhibitors (e.g. lressa ®, TarcevaTM, ErbituxTM), VEGF inhibitors (e.g. AvastinTM), proteasome inhibitors (e.g. VelcadeTM), Glivec ® or hsp90 inhibitors (e.g. 17-AAG). Additionally, 39- desmethoxyrapamyin may be administered in combination with other therapies including, but not limited to, radiotherapy or surgery.
• EGFR inhibitors e.g. lressa ®, TarcevaTM, ErbituxTM
• VEGF inhibitors e.g. AvastinTM
• proteasome inhibitors e.g. VelcadeTM
• Glivec ® or hsp90 inhibitors e.g. 17-AAG.
• 39- desmethoxyrapamyin may be administered in combination with other therapies including, but not limited to, radiotherapy or surgery.
• a 39-de ⁇ methoxyrapamycin analogue is co-administered with another therapeutic agent for the treatment of vascular disease
• preferred agents include, but are not limited to, ACE inhibitors, angiotensin Il receptor antagonists, fibric acid derivatives, HMG-CoA reductase inhibitors, beta adrenergic blocking agents, calcium channel blockers, antioxidants, anticoagulants and platelet inhibitors (e.g. PlavixTM).
• a 39-desmethoxyrapamycin analogue is co-administered with another therapeutic agent for the stimulation of neuronal regeneration, preferred agents include, but are not limited to, neurotrophic factors e.g.
• a 39-desmethoxyrapamycin analogue is co-administered with another therapeutic agent for the treatment of fungal infections; preferred agents include, but are not limited to, amphotericin B, flucytosine, echinocandins (e.g. caspofungin, anidulafungin or micafungin), griseofulvin, an imidazole or a triazole antifungal agent (e.g. clotrimazole, miconazole, ketoconazole, econazole, butoconazole, oxiconazole, terconazole, itraconazole, fluconazole or voriconazole).
• preferred agents include, but are not limited to, amphotericin B, flucytosine, echinocandins (e.g. caspofungin, anidulafungin or micafungin), griseofulvin, an imidazole or a triazole antifungal agent (e.g. clotrimazole, mic
• a 39-desmethoxyrapamycin analogue is co-administered with another therapeutic agent for the treatment of Alzheimer's disease; preferred agents include, but are not limited to, cholinesterase inhibitors e.g. donepezil, rivastigmine, and galantamine; N-methyl-D- aspartate (NMDA) receptor antagonists, e.g. Memantine.
• cholinesterase inhibitors e.g. donepezil, rivastigmine, and galantamine
• NMDA N-methyl-D- aspartate
• a 39-desmethoxyrapamycin analogue is co-administered with another therapeutic agent for the treatment of multiple sclerosis; preferred agents include, but are not limited to, Interferon beta-1 b, Interferon beta-l a, glatiramer, mitoxantrone, cyclophosphamide, corticosteroids (e.g. methylprednisolone, prednisone, dexamethasone).
• preferred agents include, but are not limited to, Interferon beta-1 b, Interferon beta-l a, glatiramer, mitoxantrone, cyclophosphamide, corticosteroids (e.g. methylprednisolone, prednisone, dexamethasone).
• any means of delivering two or more therapeutic agents to the patient as part of the same treatment regime is included any means of delivering two or more therapeutic agents to the patient as part of the same treatment regime, as will be apparent to the skilled person. Whilst the two or more agents may be administered simultaneously in a single formulation this is not essential. The agents may administered in different formulations and at different times.
• the formulations may conveniently be presented in unit dosage form and may 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 invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
• a 39-desmethoxyrapamycin analogue will normally be administered intravenously, orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
• the compositions may be administered at varying doses.
• compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions.
• the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions.
• the fnal injectable form must be sterile and must be effectively fluid for easy syringability.
• the pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
• a 39-desmethoxyrapamycin analogue can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
• Solutions or suspensions of a 39-desmethoxyrapamycin analogue suitable for oral administration may also contain excipient ⁇ e.g. N,N-dimethylacetamide, dispersants e.g. polysorbate 80, , surfactants, and solubilisers, e.g. polyethylene glycol, Phosal 50 PG (which consists of phosphatidylcholine, soya-fatty acids, ethanol, mono/diglycerides, propylene glycol and ascorbyl palmitate),
• excipient e.g. N,N-dimethylacetamide
• dispersants e.g. polysorbate 80
• surfactants e.g. polyethylene glycol
• solubilisers e.g. polyethylene glycol, Phosal 50 PG (which consists of phosphatidylcholine, soya-fatty acids, ethanol, mono/diglycerides, propylene glycol and ascorbyl palmitate)
• Such tablets may contain excipients such as microcrystalline cellulose, lactose (e.g. lactose monohydrate or lactose anyhydrous), sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, butylated hydroxytoluene (E321 ), crospovidone, hypromellose, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium, and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propyicelluiose (HPC), macrogol 8000, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
• excipients such as microcrystalline cellulose, lactose (e.g. lactos
• Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
• Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
• the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
• a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
• Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
• the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
• Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
• the active ingredient may also be presented as a bolus, electuary or paste.
• Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
• compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, transdermal devices, dusting powders, and the like. These compositions may be prepared via conventional methods containing the active agent. Thus, they may also comprise compatible conventional carriers and additives, such as preservatives, solvents to assist drug penetration, emollient in creams or ointments and ethanol or oleyl alcohol for lotions.
• Such carriers may be present as from about 1 % up to about 98% of the composition. More usually they will form up to about 80% of the composition.
• a cream or ointment is prepared by mixing sufficient quantities of hydrophilic material and water, containing from about 5- 10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the desired consistency.
• compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
• the active agent may be delivered from the patch by iontophoresis.
• compositions are preferably applied as a topical ointment or cream.
• the active agent may be employed with either a paraffinic or a water-miscible ointment base.
• the active agent may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
• fluid unit dosage forms are prepared utilizing the active ingredient and a sterile vehicle, for example but without limitation water, alcohols, polyols, glycerine and vegetable oils, water being preferred.
• a sterile vehicle for example but without limitation water, alcohols, polyols, glycerine and vegetable oils, water being preferred.
• the active ingredient depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle.
• the active ingredient can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.
• agents such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle.
• the composition can be frozen after filling into the vial and the water removed under vacuum.
• the dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.
• Parenteral suspensions are prepared in substantially the same manner as solutions, except that the active ingredient is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration.
• the active ingredient can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle.
• a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active ingredient.
• a 39-desmethoxyrapamycin analogue may also be administered using medical devices known in the art.
• a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. 5,399,163; U.S. 5,383,851 ; U.S. 5,312,335; U.S.
• Examples of well-known implants and modules useful in the present invention include : US 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system.
• a 39- desmethoxyrapamycin analogue may be administered using a drug-eluting stent, for example one corresponding to those described in WO 01/87263 and related publications or those described by Perin (Perin, EC, 2005). Many other such implants, delivery systems, and modules are known to those skilled in the art.
• the dosage to be administered of a compound of the invention will vary according to the particular compound, the disease involved, the subject, and the nature and severity of the disease and the physical condition of the subject, and the selected route of administration.
• the appropriate dosage can be readily determined by a person skilled in the art.
• compositions may contain from 0.1 % by weight, preferably from 5-60%, more preferably from 10-30% by weight, of a compound of invention, depending on the method of administration.
• the optimal quantity and spacing of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice.
• Figure 1 shows the structure of rapamycin
• Figure 2 shows the fragmentation pathway for 39-desmethoxyrapamycin
• Figure 3 shows western blots summarisng the mTOR inhibitory activity of 39- desmethoxyrapamycin and rapamycin.
• Figure 4 the %T/C values at all test concentrations for paclitaxel (A and C) and 39- desmethoxyrapamycin (B and D) in normal (A and B) or high P-gp expressing (C and D) cell lines.
• Figure 5 shows the total Area under the Curve (AUC) from 0-24h for brain tissue or blood samples after a single i.v. or p.o. administration of rapamycin and 39- desmethoxyrapamycin.
• AUC Area under the Curve
• B - shows the level of 39-desmethoxyrapamycin and rapamycin detected in the brain tissue over time after a single i.v. administration.
• Figure 6 A - shows disease progression in the EAE model under the prophylactic regime. Values given are the median from the vehicle or treated group. B - shows disease progression in the EAE model under the therapeutic regime. Values given are the median from the vehicle or treated group.
• Figure 7 the graph indicates the relative % survival of mice after induction of glioma by stereotaxic injection of U87-MG cells. Filled diamonds represent the untreated group, filled squares represent the vehicle treated group and open circles represent the 39-desmethoxyrapamycin treated group.
• S. hygroscopicus MG2-10 [IJMNOQLhis] (WO 04/007709; Gregory et al., 2004) was maintained on medium 1 agar plates (see below) at 28 0 C.
• Spore stocks were prepared after growth on medium 1 , preserved in 20% w/v glycerol:10% w/v lactose in distilled water and stored at -80 0 C.
• Vegetative cultures were prepared by inoculating 0.1 ml_ of frozen stock into 50 ml. medium 2 (see below) in 250 mL flask. The culture was incubated for 36 to 48 hours at 28 0 C, 300 rpm.
• Vegetative cultures were inoculated at 2.5 - 5% v/v into medium 3. Cultivation was carried out for 6-7 days, 26 0 C, 300 rpm.
• Avedex W80 dextrin (Deymer Ingredients Ltd) 35 g
• the media was then sterilised by autoclaving 121 0 C, 20 min.
• Avedex W80 dextrin (Deymer Ingredients Ltd) 19 g
• Avedex W80 dextrin (Deymer Ingredients Ltd) 35 g
• the media was then sterilised by autoclaving 121 0 C, 20 min.
• Detection was by L)V absorbance at 254 nm and/or by mass spectrometry electrospray ionisation (positive or negative) using a Micromasss Quattro- Micro instrument.
• Mobile phase A Acetonitrile (100 mL), trifluoracetic acid (1 mL), 1 M ammonium acetate (10 mL) made up to 1 L with deionised water.
• Mobile phase B Deionised water (100 mL), trifluoracetic acid (1 mL), 1 M ammonium acetate (10 mL) made up to 1 L with acetonitrile. Flow rate: 1 mL/minute.
• a linear gradient from 55% B - 95% B was used over 10 minutes, followed by 2 minutes at 95% B, 0.5 minutes to 55% B and a further 2.5 minutes at 55% B.
• Compound detection was by UV absorbance at 280 nm.
• the HPLC system comprised an Agilent HP1100 and was performed on 3 micron BDS C18 Hypersil (ThermoHypersil-Keystone Ltd) column, 15O x 4.6 mm, maintained at 40 0 C, running a mobile phase of:
• Mobile phase A deionised water.
• This system was coupled to a Bruker Daltonic ⁇ Esquire3000 electrospray mass spectrometer. Positive negative switching was used over a scan range of 500 to 1000 Daiton.
• a linear gradient from 55% B - 95% B was used over 10 minutes, followed by 2 minutes at 95% B, 0.5 minutes to 55% B and a further 2.5 minutes at 55% B.
• Oncotest cell lines were established from human tumor xenografts as described by Roth et al. 1999. The origin of the donor xenografts was described by Fiebig et al. 1992. Other cell lines were either obtained from the NCI (H460, SF-268, OVCAR-3, DU145, MDA-MB-231 , MDA-MB-468) or purchased from DSMZ, Braunschweig, Germany (LNCAP). All cell lines, unless otherwise specified, are grown at 37°C in a humidified atmosphere
• Monolayer assay - Protocol 1 A modified propidium iodide assay was used to assess the effects of the test compound(s) on the growth of twelve human tumor cell lines (Dengler et al, 1995).
• cells were harvested from exponential phase cultures by trypsinization, counted and plated in 96 well flat-bottomed microtitre plates at a cell density dependent on the cell line (5 - 10.000 viable cells/well). After 24 h recovery to allow the cells to resume exponential growth, 0.01 mL of culture medium (6 control wells per plate) or culture medium containing 39- desmethoxyrapamycin were added to the wells. Each concentration was plated in triplicate. 39- Desmethoxyrapamycin was applied in two concentrations (0.001 mM and 0.01 mM).
• cell culture medium with or without 39-desmethoxyrapamycin was replaced by 0.2 mL of an aqueous propidium iodide (Pl) solution (7 mg/L).
• Pl propidium iodide
• cells were permeabilized by freezing the plates. After thawing the plates, fluorescence was measured using the Cytofluor 4000 microplate reader (excitation 530 nm, emission 620 nm), giving a direct relationship to the total number of viable cells.
• IC 50 & IC 70 values were estimated by plotting compound concentration versus cell viability.
• the human tumor cell lines of the National Cancer Institute (NCI) cancer screening panel were grown in RPM1 1640 medium containing 5% fetal bovine serum and 2 mM L- glutamine (Boyd and Paull, 1995). Cells were inoculated into 96 well microtiter plates in 0.1 mL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 0 C, 5 % CO 2 , 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs.
• TCA trichloroacetic acid
• the plates were incubated for an additional 48 h at 37 0 C, 5 % CO 2 , 95 % air, and 100 % relative humidity.
• the assay was terminated by the addition of cold TCA.
• Cells were fixed in situ by the gentle addition of 0.05 mL of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4 0 C. The supernatant was discarded, and the plates were washed five times with tap water and air dried.
• SRB Sulforhodamine B
• the LC50 concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning
• Multi-drug resistant cell lines within the 60 cell line panel were identified by the NCI as high P-gp containing cell lines as identified by rhodamine B efflux (Lee et a/., 1994) and by PCR detection of mRNA of mdr-1 (Alvarez et a/., 1995).
• test compounds were prepared in a vehicle consisting of 4% Ethanol, 5% Tween- 20, 5% polyethyleneglycol 400 in 0.15M NaCI.
• a single dose of 10 mg/kg p.o. or 3 mg/kg i.v. was administered to groups of female CD1 mice (3 mice for each compound per time point). At Omin, 4min, 15 min, 1 h, 4h, and 24 h groups were sacrificed and the blood and the brain were collected from each mouse for further analysis.
• the brain samples were snap frozen in liquid nitrogen and stored at -20°C.
• a minimum of 0.2 ml_ of whole blood from each animal was collected in tubes containing ethylene diamine tetra-acetic acid (EDTA) as anticoagulant, thoroughly mixed, and stored at -20 0 C.
• EDTA ethylene diamine tetra-acetic acid
• test compound was dissolved in 100 ⁇ l_ ethanol resulting in a compound solution of 50 mg/mL
• the solution was then diluted to 2 mg/mL by adding approximately 2.4 ml_ 0.15 M NaCI (0.9% w/v saline), 5% v/v Tween 20 and 5% v/v PEG 400 (final ethanol cone. 4% v/v).
• a single dose of 10 mg/kg p.o. or 2 mg/kg i.v. of test compound at a concentration of 10mg/kg p.o or 2mg/kg i.v. was administered to groups of 3 female BaIb C mice. At 5min, 15min, 60min, 4h, 8h and 24h, groups were sacrificed and whole blood samples of approximately 0.2 ml_ were retrieved in EDTA to give a final concentration of 0.5mM, additionally the brains were removed. Both whole blood and brains were snap frozen in liquid nitrogen and stored at -20 0 C until shipment on solid carbon dioxide for analysis
• mice brain or blood 0.05 mL
• internal standard solution 0.1 mL
• Zinc sulphate solution 0.5 mL
• acetone 0.5 mL
• the tubes were then centrifuged in a microfuge for a minimum of 2 minutes.
• the solvent layer was decanted into a 4.5 mL polypropylene tube containing sodium hydroxide (0.1 M, 0.1 mL) and methyl-tert-butyl ether (MTBE, 2 mL). The tube was then mixed for a minimum of 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 a SpeedVac® and evaporated to dryness.
• sodium hydroxide 0.1 M, 0.1 mL
• MTBE methyl-tert-butyl ether
• the dried extracts were 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 auto sampler tubes (NLG Analytical, Adelphi Mill, Bollington, Cheshire, UK) and placed into the auto-sampler tray which was set at ambient temperature.
• the auto-sampler was programmed to inject a 0.03 mL aliquot of each extract onto the analytical column.
• 39-Desmethoxyrapamycin was produced by growing cultures of S. hygroscopicus MG2- 10 [IJMNOQLhis] and feeding with cyclohexanecarboxylic acid (CHCA) as described below.
• CHCA cyclohexanecarboxylic acid
• S. hygroscopicus MG2-10 [IJMNOQLhis] was produced by introducing into the MG2-10 strain described in WO 2004/007709 a plasmid containing the genes rapl, rapj, rapM, rapN, rapO, rapQ and rapL
• the gene cassette was constructed with the rapL gene containing a 5' in- frame histidine tag.
• the plasmid also contained an origin of transfer and an apramycin resistance marker for transformation of MG2-10 by conjugation and selection of exconjugants and a phiBTI attachment site for site-specific integration into the chromosome. Isolation of each of these genes and the method used for construction of gene cassettes containing combinations of post-PKS genes was performed as described in WO 2004/007709.
• rapamycin analogue was proposed to be the desired 39- desmethoxyrapamycin on the basis of the analytical data described under characterisation below.
• a primary vegetative culture in Medium 4 of S. hygroscopicus MG2-10 [IJMNOQLhis] was cultivated essentially as described in Materials & Methods.
• a secondary vegetative culture in Medium 4 was inoculated at 10% v/v, 28 0 C, 250 rpm, for 24h.
• Vegetative cultures were inoculated at 5% v/v into medium 5 (see Materials & Methods) in a 20 L fermenter.
• Cultivation was carried out for 6 days at 26 0 C, 0.5 vvm. > 30% dissolved oxygen was maintained by altering the impeller tip speed, minimum tip speed of 1.18 ms "1 maximum tip speed of 2.75 ms "1 .
• the feeding of cyclohexanecarboxylic acid was carried out at 24 and 48 hours after inoculation to give a final concentration of 2 mM.
• the fermentation broth (30 L) was stirred with an equal volume of methanol for 2 hours and then centrifuged to pellet the cells (10 min, 3500 rpm). 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 strip off the rapamycin analogue and the solvent was removed in vacuo. The aqueous concentrate was then diluted to 2 L with water and extracted with EtOAc (3 * 2 L). The solvent was removed in vacuo to give a brown oil (20.5 g).
• the extract was dissolved in acetone, dried onto silica, applied to a silica column (6 * 6,5 cm diameter) and eluted with a stepwise gradient of acetone/hexane (20% - 40%).
• the rapamycin analogue-containing fractions were pooled and the solvent removed in vacuo.
• the residue (2.6 g) was further chromatographed (in three batches) over Sephadex LH20, eluting with 10:10:1 chloroform/heptane/ethanol.
• the semipurified rapamycin analogue (1.7 g) was purified by reverse phase (C18) preparative HPLC using a Gilson HPLC, eluting a Phenomenex 21.2 * 250 mm Luna 5 ⁇ m C18 BDS column with 21 mL/min of 65% acetonitrile/water.
• the most pure fractions (identified by analytical HPLC, Method B) were combined and the solvent removed in vacuo to give 39-desmethoxyrapamycin (563 mg).
• 27-O-Desmethyl-39-desrnethoxyrapamycin was produced by growing cultures of S. hygroscopicus MG2-10 [JMNOLhis] and feeding with cyclohexanecarboxylic acid (CHCA) as described below.
• CHCA cyclohexanecarboxylic acid
• S. hygroscopicus MG2-10 [JMNOLhis] was produced by introducing into the MG2-10 strain described in WO 2004/007709 a plasmid containing the genes, rapJ, rapM, rapN, rapO, and rapL
• the gene cassette was constructed with the rapL gene containing a 5' in-frame histidine tag.
• the plasmid also contained an origin of transfer and an apramycin resistance marker for transformation of MG2-10 by conjugation and selection of exconjugants and a phiBTI attachment site for site-specific integration into the chromosome. Isolation of each of these genes and the method used for construction of gene cassettes containing combinations of post-PKS genes was performed as described in WO 2004/007709.
• Liquid culture A vegetative culture of S. hygroscopicus MG2-10 [JMNOLhis] was cultivated as described in Materials & Methods. Production cultures were inoculated with vegetative culture at 0.5 ml_ into 7 mL medium 3 in 50 ml_ tubes. Cultivation was carried out for 7 days, 26 0 C, 300 rpm. One millilitre samples were extracted 1 :1 acetonitrile with shaking for 30 min, centrifuged
• rapamycin analogue was proposed to be the desired 27-O-desmethy!-39 ⁇ desmethoxyrapamycin on the basis of the analytical data described under characterisation below.
• Medium 2 was inoculated at 10% v/v, 28 0 C, 250 rpm, for 24h. Vegetative cultures were inoculated at 10% v/v into medium 5 (see Materials & Methods) in a 20 L fermenter. Cultivation was carried out for 6 days at 26 °C, 0.75 wm. > 30% dissolved oxygen was maintained by altering the impeller tip speed, minimum tip speed of 1.18 ms ⁇ 1 maximum tip speed of 2.75 ms "1 .
• the fermentation broth (15 L) was stirred with an equal volume of methanol for 2 hours and then centrifuged to pellet the cells (10 min, 3500 rpm). 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 strip off the rapamycin analogue and the solvent was removed in vacuo. The aqueous concentrate was then diluted to 2 L with water and extracted with EtOAc (3 * 2 L). The solvent was removed in vacuo to give a brown oil (12 g).
• the extract was dissolved in acetone, dried onto silica, applied to a silica column (4 *
• This mass spectrometry fragmentation data narrows the region of the rapamycin analogue where the loss of a methoxy has occurred to the fragment C28-C42 that contains the cyclohexyl moiety and narrows the region of the rapamycin analogue where the loss of an O-methyl has occurred to the fragment C15-C27.
• 16-O-Desmethyl-27-O-desmethyl-39-desmethoxyrapamycin was produced by growing cultures of S. hygroscopicus MG2-10 [IJNOLhis] and feeding with cyclohexanecarboxylic acid (CHCA) as described below.
• CHCA cyclohexanecarboxylic acid
• S. hygroscopicus MG2-10 [IJNOLhis] was produced by introducing into the MG2-10 strain described in WO 2004/00709 a plasmid containing the genes rapl, rapJ, rapN, rapO, and rapL.
• the gene cassette was constructed with the rapL gene containing a 5' in-frame histidine tag.
• the plasmid also contained an origin of transfer and an apramycin resistance marker for transformation of MG2-10 by conjugation and selection of exconjugants and a phiBTI attachment site for site-specific integration into the chromosome. Isolation of each of these genes and the method used for construction of gene cassettes containing combinations of post-PKS genes was performed as described in WO 2004/007709.
• Liquid culture A vegetative culture of S. hygroscopicus MG2-10 [IJNOLhis] was cultivated as described in Materials & Methods. Production cultures were inoculated with vegetative culture at 0.5 mL into 7 mL medium 3 in 50 rnl_ tubes. Cultivation was carried out for 7 days, 26 0 C, 300 rpm. One millilitre samples were extracted 1 :1 acetonitrile with shaking for 30 min, centrifuged 10 min, 13,000 rpm and analysed and quantified according to analysis Method B (see Materials & Methods). Confirmation of product was determined by mass spectrometry using analysis Method C (see Materials & Methods).
• rapamycin analogue was proposed to be the desired 16-O-desmethyl-27- O-desmethyl-39-desrnethoxyraparnycin on the basis of the analytical data described under characterisation below.
• a primary vegetative culture in Medium 2 of S. hygroscopicus MG2-10 [IJNOLhis] was cultivated for 3 days essentially as described in Materials & Methods.
• a secondary vegetative culture in Medium 2 was inoculated at 10% v/v, 28 0 C, 250 rpm, for 48h and a tertiary culture was inoculated at 10% v/v, 28 0 C, 250 rpm, for 24h.
• Vegetative cultures were inoculated at 10% v/v into medium 5 (see Materials & Methods) in 3 x 7 L fermenters. Cultivation was carried out for 6 days at 26 0 C, 0.75 vvm.
• the fermentation broth (12 L) was stirred with an equal volume of methanol for 2 hours and then centrifuged to pellet the cells (10 min, 3500 rpm). 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 strip off the rapamycin analogue and the solvent was removed in vacuo. The aqueous concentrate was then diluted to 2 L with water and extracted with EtOAc (3 * 2 L). The solvent was removed in vacuo to give a brown oil (8.75 g).
• the extract was dissolved in acetone, dried onto silica, applied to a silica column (4 * 6.5 cm diameter) and eluted with a stepwise gradient of acetone/hexane (20% - 40%).
• the rapamycin analogue-containing fractions were pooled and the solvent removed in vacuo.
• the residue (0.488 g) was further chromatographed (in three batches) over Sephadex LH20, eluting with 10:10:1 chloroform/heptane/ethanol.
• the rapamycin analogue-containing fractions were pooled and the solvent removed in vacuo.
• the semipurified rapamycin analogue (162 mg) was purified by reverse phase (C18) preparative HPLC using a Gilson HPLC, eluting a Phenomenex 21.2 x 250 mm Luna 5 ⁇ m C18 BDS column with 21 mL/min of 65% acetonitrile/water. The most pure fractions (identified by analytical HPLC, Method B) were combined and the solvent removed in vacuo to give 16-O-desmethyl-27-O-desmethy!-39-desmethoxyrapamycin (44.7 mg).
• Table 4 shows the results.
• Table 5 shows the IC50 and IC70 for the compounds and rapamycin across the cell lines tested.
• MDR multi-drug resistant
• 39-desmethoxyrapamycin has equivalent or improved efficacy against high MDR-expressing cell lines when compared to rapamycin.
• Confluent Caco-2 cells (Li, A.P., 1992; Grass, G.M., ef a/., 1992, Volpe, D.A., et a/., 2001) in a 24 well Corning Costar Transwell format were provided by In Vitro Technologies Inc. (IVT Inc., Baltimore, Maryland, USA).
• the apical chamber contained 0.15 mL Hank's balanced buffer solution (HBBS) pH 7.4, 1 % DMSO, 0.1 mM Lucifer Yellow.
• the basal chamber contained 0.6 mL HBBS pH 7.4, 1 % DMSO. Controls and tests were incubated at 37 0 C in a humidified incubator, shaken at 130 rpm for 1 h.
• Lucifer Yellow permeates via the paracellular (between the tight junctions) route only, a high Apparent Permeability (P apP ) for Lucifer Yellow indicates cellular damage during assay and all such wells were rejected.
• P apP Apparent Permeability
• Propranolol (good passive permeation with no known transporter effects) & acebutalol (poor passive permeation attenuated by active efflux by P-glycoprotein) were used as reference compounds.
• Compounds were tested in a uni- and bi-directional format by applying compound to the apical or basal chamber (at 0.01 mM). Compounds in the apical or basal chambers were analysed by HPLC- MS (Method A, see Materials & Methods). Results were expressed as Apparent Permeability, P ap p, (nm/s) and as the Flux Ratio (A to B versus B to A).
• a positive value for the Flux Ratio indicates active efflux from the apical surface of the cells.
• HBM Human Liver Microsomal
• Liver homogenates provide a measure of a compounds inherent vulnerability to Phase I (oxidative) enzymes, including CYP450s (e.g. CYP2C8, CYP2D6, CYP1A, CYP3A4, CYP2E1 ), esterases, amidases and flavin monooxygenases (FMOs).
• CYP450s e.g. CYP2C8, CYP2D6, CYP1A, CYP3A4, CYP2E1
• esterases e.g. CYP2C8
• CYP1A CYP1A
• CYP3A4A4 CYP2E1
• esterases e.g. CYP1A, CYP3A4, CYP2E1
• FMOs flavin monooxygenases
• FKBP12 reversibly unfolds in the chemical denaturant guandinium hydrochloride (GdnHCI) and the unfolding can be monitored by the change in the intrinsic fluorescence of the protein (Main et al, 1998).
• Ligands which specifically bind and stabilise the native state of FKBP12 shift the denaturation curve such that the protein unfolds at higher concentrations of chemical denaturant (Main et al, 1999). From the difference in stability, the ligand-binding constant can be determined using equation 1.
• AAG p _ N the difference in the stability of FKBP12 with rapamycin and unknown ligand (at the same ligand concentration), as:
• Kf' is the dissociation constant for rapamycin and K x ⁇ is the dissociation constant for unknown ligand X. Therefore,
• the denaturation curve was fitted to generates values for m D _ N and [-D] 50 o /o , which were used to calculate an average /n-value,
• Inhibition of mTOR can be established indirectly via the measurement of the level of phosphorylation of the surrogate markers of the mTOR pathway and p70S6 kinase and S6 (Brunn et al., 1997; Mothe-Satney et a/., 2000; Tee and Proud, 2002; Huang and Houghton, 2002).
• HEK293 cells were co-transfected with FLAG-tagged mTOR and myc-tagged Raptor, cultured for 24 h then serum starved overnight. Cells were stimulated with 100 nM insulin then harvested and lysed by 3 freeze/thaw cycles.
• Lysates were pooled and equal amounts were immunoprecipitated with FLAG antibody for the mTOR/Raptor complex, lmmunoprecipitates were then processed: samples treated with compound (0.00001 to 0.003 mM) were pre- incubated for 30min at 3O 0 C with FKBP12/rapamycin, FKBP12/39-desmethoxyrapamycin or vehicle (DMSO), non-treated samples were incubated in kinase buffer, lmmunoprecipitates were then subject to in vitro kinase assay in the presence of 3 mM ATP, 10 mM Mn2+ and GST- 4E-BP1 as substrate.
• compound 0.00001 to 0.003 mM
• HEK293 cells were seeded into 6 well plates and pre-incubated for 24h and then serum starved overnight. Cells were then pre-treated with vehicle or compound for 30 min at 30°C, then stimulated with 100 nM insulin for 30 min at 30°C and lysed by 3 freeze/thaw cycles and assayed for protein concentration. Equal amounts of protein were loaded and separated on SDS-PAGE gels. The protein was then wet transferred to PVDF membrane and probed for phospho-S6 (S235/36) or phospho-p70 S6K (T389).
• the cell lines used in the present study were both provided by the National Cancer Institute, USA.
• Assay Protocol A modified propidium iodide assay based on protocol 1 described above was used to assess the effects of 39-desmethoxyrapamycin (Dengler et al, 1995). Briefly, cells were harvested from exponential phase cultures by trypsination, counted and plated in 96 well flat- bottomed microtiter plates at a cell density of 5.000 cells/well. After a 24 h recovery to allow the cells to resume exponential growth, 0.01 ml_ of Verapamil at a concentration of 0.18 mg/mL or 0.01 mL culture medium were added to the cells in order to yield a final concentration of Verapamil in the wells of 0.01 mg/mL. This concentration was found in previous experiments to be non-toxic to the cells.
• Culture medium containing 39-desmethoxyrapamycin, taxol or culture medium alone (for the control wells) was added at 0.01 mL per well.
• the compounds were applied in triplicates in 8 concentrations in half log steps ranging from 0.03 mM down to 10 nM.
• medium or medium with compound was replaced by 0.2 mL of an aqueous propidium iodide (Pl) solution (7 mg/L). Since Pl only passes leaky or lysed membranes, DNA of dead cells will be stained and measured, while living cells will not be stained. To measure the proportion of living cells, cells were permeabilized by freezing the plates, resulting in death of all cells.
• Pl propidium iodide
• Figure 4 shows four graphs demonstrating the %T/C values at all test concentrations for paclitaxel (A and C) and 39-desmethoxyrapamycin (B and D) in normal (A and B) or high P-gp expressing (C and D) cell lines.
• the filled diamonds represent the values after the administration of paclitaxel or 39-desmethoxyrapamycin alone, the open squares represent the values after the administration of paclitaxel or 39-desmethoxyrapamycin in the presence of 0.01 mg/mL Verapamil (a P-gp inhibitor).
• Paclitaxel a known P-gp substrate showed reduced potency in inhibiting P-gp expressing cancer cell line MCF7 ADR and this reduced potency was restored by the coadministration of verapamil, a P-gp inhibitor ( Figures 4A and 4C). 39-desmethoxyrapamycin did not show a significant shift in the growth proliferation curves in the P-gp expressing cell line MCF7 ADR either with or without verapamil ( Figures 4B and 4D) demonstrating that 39-desmethoxyrapamycin is not a substrate for P-gp.
• Example 6 Pharmacokinetic analysis
• the ALJC for each compound in blood or in brain tissue was calculated using Kinetica
• R 1 AUC ' 1 BRAIN
• the AUC for each compound in blood or in brain tissue was calculated using Kinetica 4.4 (InnaPhase Corporation), using a non-compartmental model and the trapezoidal method for AUC calculation.
• EAE Experimental allergic encephalomyelitis
• CNS central nervous system
• MS multiple sclerosis
• MBP myelin basic protein
• CFA complete Freund's adjuvant
• MHO major histocompatibility complex
• T cells Upon activation by antigen, T cells produce several lymphokines which in the case of EAE, may be directly or indirectly responsible for the CNS damage.
• the lymphokines likely to be involved in the pathogenesis of EAE are IL-2, IFN- ⁇ and TNF- ⁇ .
• IL-2 has an important role in T cell activation and proliferation, while IFN- ⁇ is a potent mediator of macrophage activation.
• IFN- ⁇ induces the production of inflammatory cytokines such as IL-I .
• TNF and also the expression of class Il MHC molecules, among others, on the endothelial cells of blood vessels in the CNS, and on astrocytes, which are thought to play an important role in antigen presentation to encephalitogenic T cells.
• Test compounds were be given at different doses (5 or 15 mg/kg bd wt) under both a prophylactic and therapeutic regime.
• the treatment was started one day prior to immunization, and for the therapeutic part of the study it was initiated on day 7 post immunization (p. L).
• Vehicle-treated rats treated under the same experimental conditions, either prophylactically or therapeutically, as were used for controls.
• Treatment was given p.o. daily six times a week until day 30 p.i. Cyclophosphamide was used as a positive control.
• Figure 6A shows the effect of the prophylactic regime of 39-desmethoxyrapamycin at 5 and 15 mg/kg
• figure 6B shows the effect of the therapeutic regime of 39-desmethoxyrapamycin at 5 and 15 mg/kg.
• the effects of 40 mg/kg cyclophosphamide are shown as a positive control.
• the median score of each group is shown. It can be seen that 39-desmethoxyrapamycin has equivalent efficacy in this model to cyclophosphamide and that it reduces not only the severity of symptoms but also reduces the duration of the episode. It should be noted that due to the death during the study of 5 out of 7 vehicle-treated rats, the median value for this group remained at 5, however, the two surviving rats did both eventually return to baseline values by day 28.
• Example 8 Antitumor activity study of 39-desmethoxyrapamycin in a model of glioma orthotopically xenografted in nude mice
• test compound was dissolved in ethanol (0.027 mL/mg compound) and vortexed for 20 rnin until the solution was clear. Ethanolic solutions were aliquoted as appropriate and stored at -20 0 C. The ethanolic solution was then made up to the correct concentration with vehicle (4% Ethanol, 5% Tween-20, 5% polyethyleneglycol 400 in 0.15 M NaCI, prepared with sterile endotoxin free components where possible).
• test substance and control vehicle were administered intravenously (IV, bolus) by injection into the caudal vein of the test mice.
• IV intravenously
• An injection volume of 10 mL/kg was used, based on the most recent body weight of mice.
• the cell line used for the study was U87-MG, a glioblastoma cell line initiated by J.
• Tumor cells were grown as a monolayer at 37°C in a humidified atmosphere (5% CO 2 , 95% air).
• the culture medium was RPM1 1640 (Ref. BE12-702F, Cambrex) containing 2mM L- glutamine supplemented with 10% fetal bovine serum (Ref. DE 14-801 E, Cambrex).
• the cells were adherent to plastic flasks.
• tumor cells were detached from the culture flask by 5 minutes treatment with trypsin-versene (Ref. BE17-161E, Cambrex), in Hanks 1 medium without calcium or magnesium (Ref. BE10-543F, Cambrex). The cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.
• mice were stereotaxically injected with U87-MG cells at DO, 24 to 48 hours after a whole body irradiation with a ⁇ -source (2.5 Gy, Co 60 , INRA BRETENIERE, Dijon).
• a ⁇ -source 2.5 Gy, Co 60 , INRA BRETENIERE, Dijon.
• mice were anesthetised by an intraperitoneal injection of Ketamine 100 mg/kg (Ketamine ⁇ OO ® , Ref 043KET204, Centravet, France) and Xylazine 5 mg/kg (Rompun ® , Ref 002ROM001, Centravet, France) in 0.9% NaCI solution at 10 mL/kg/inj.
• mice were weighed and randomized according to their individual body weight into 3 groups of mice. Four (4) additional mice were added to each treatment group for MRI imaging. The groups were selected such that the mean body weight of each group was not statistically different from the others (analysis of variance). Test substances were administered as defined below.
• mice from group 1 received 5 cycles of daily IV injections of test substances vehicle for 3 consecutive days (at D7 to D9, D14 to D16, D21 to D23, D28 to D30 and D35 to D37: (Q1 Dx3)x5W). Each cycle was separated by a 4-day period of wash out 8.3.2 Mice from group 2 received 5 cycles of daily IV injections of 39-desmethoxyrapamycin at 3 mg/kg/inj for 3 consecutive days at D7 to D9, D14 to D16, D21 to D23, D28 to D30 and D35 to D37: (Q1 Dx3)x5W). Each cycle was separated by a 4-day period of wash out
• MRI analysis of the brain was performed at D23 and D37. All the MRI analyses were performed at 4.7T in the Pharmascan magnet (Bruker, Wissembourg). Mice were positioned within the dedicated mouse cradle and the 38 mm diameter cylindrical coil under continuous anesthesia with i ⁇ oflurane. After tripilot acquisitions, a turboRare T2 weighted sequence was performed. Acquisitions covered the entire brain including the tumour. The tumour volume was determined by manually drawing a region of interest (ROl) around the tumour in each slice and by summation of all the surfaces.
• ROl region of interest
• FIG. 7 shows the survival graph for each treatment group until day 43. Additionally, the results were expressed as a percent (T/C%) where T represents the median survival times of animals treated with 39-desmethoxyrapamycin and C represents the median survival times of control animals treated with vehicle. T/C% was calculated as follows:
• T/C% [T/C] x 100
• Each data point represents the mean of 4 values.

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