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
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
• • 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
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/529—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim forming part of bridged ring systems
• • 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
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

• the present invention is concerned with the finding that the macrocyclic quinazoline derivative 4,6-ethanediylidenepyrimido[4,5-b][6,l,12]benzoxadiazacyclo-pentadecine, 17-bromo-8,9,10,l 1,12,13, 14,19-octahydro-20-methoxy-13-methyl, described as compound 22 in PCT publication WO2004/105765, is useful in the manufacture of a medicament for the treatment or prevention of bone cancers and methods for killing bone cancer cells, including osteosarcomas, chondrosarcomas, myeloma bone disease and osteolytic bone metastases from other primary sites. It accordingly provides methods for treating, preventing, delaying or mitigating bone cancer, or for preventing and treating of bone loss associated with cancer metastases.
• Bone cancer includes primary bone cancer cells such as osteosarcoma cells, cells from Ewing's family of tumors, chondrosarcoma cells, malignant giant cell tumor cells, malignant fibrous histiocytoma cells and adamantinoma cells, as well as secondary bone cancer cells that have metastasized from other tissues, including breast, lung, prostate and kidney.
• primary bone cancer cells such as osteosarcoma cells, cells from Ewing's family of tumors, chondrosarcoma cells, malignant giant cell tumor cells, malignant fibrous histiocytoma cells and adamantinoma cells, as well as secondary bone cancer cells that have metastasized from other tissues, including breast, lung, prostate and kidney.
• Osteosarcoma is a malignant tumor of bone, which is most prevalent in adolescents and young adults. Osteosarcoma accounts for approximately 5% of the tumors in childhood and 80% of these tumors originate around the knee. The prognosis is often poor and within 1 year after commencing definitive therapy, about 30% of patients diagnosed with osteosarcoma will develop lung metastasis. The prognosis appears to be determined by the site of metastases and surgical resectability of the metastatic disease, either at diagnosis or following a variable period of chemotherapy. Patients who have complete surgical ablation of the primary and metastatic tumor (when confined to the lung) following chemotherapy may attain long-term survival, although event-free survival remains about 20% for patients with metastatic disease at diagnosis. Patients developing recurrent disease often have a poor prognosis and die within 1 year of the development of metastatic disease.
• Chemotherapy is often ineffective, resulting in a high mortality rate. Hence, it is important that new therapeutic approaches are evaluated for this malignant disease.
• Myeloma bone disease is a cancer of antibody-producing plasma cells in the bone marrow. Proliferation of the cancerous plasma cells, referred to as myeloma cells, causes a variety of effects, including lytic lesions (holes) in the bone, decreased red blood cell number, production of abnormal proteins (with attendant damage to the kidney, nerves, and other organs), reduced immune system function, and elevated blood calcium levels (hypercalcemia).
• myeloma cells When myeloma cells are present at distinct skeletal locations, the disease is referred to as multiple myeloma.
• myeloma is the second most common blood cancer and may be increasing in prevalence, particularly among individuals under age 55 (International Myeloma Foundation).
• Many different treatment options are available or in development, but there is neither a cure nor agreement on an optimal myeloma management regimen.
• Patients are treated with chemotherapy as well as symptom- specific treatments for one or more of hypercalcemia, increased infection risk, kidney failure, anemia, hyperviscosity of blood, elevated stroke risk, bone destruction and pain, and muscle weakness.
• dramatic reduction in the number of myeloma cells does not necessarily translate into longer remissions or survival times, and therapies that were effective before a remission may not prove effective upon relapse of the disease.
• myeloma One of the most prevalent and significant characteristics of myeloma is the activation of osteoclasts, multinucleated cells that absorb bone, leading to bone thinning, lytic bone lesions, and bone fracture. Lytic bone lesions occur in 70-80% of multiple myeloma patients and are frequently associated with severe bone pain and pathologic fractures. In normal bone functioning, a balance exists between osteoclasts, which resorb bone, and osteoblasts, cells that produce bone. This balance is upset in myeloma patients, and more bone is resorbed than produced. The increased osteoclastic bone resorption occurs adjacent to the myeloma cells and not in areas of normal bone marrow, indicating that the osteoclast activation occurs by a local mechanism.
• myeloma cells activate osteoclasts
• Myeloma cells in culture, produce or induce production of several osteoclast-activating factors (OAFs) whose specific roles in vivo are yet to be determined.
• OAFs osteoclast-activating factors
• MIP-Ia chemokine macrophage inflammatory protein-la
• bisphosphonates a class of chemicals that inhibits osteoclast activity or osteoclast attachment to bone surface and eventually leads to osteoclast cell death. They may also affect myeloma cells directly. Bisphosphonates are administered by infusion. Third- generation bisphosphonates are currently under development, but even improved versions of the drugs may have potential side effects including hypocalcemia, kidney damage, and increased pain.
• Bisphosphonates do not completely block the bone destruction process, and patients eventually develop new bone lesions.
• An alternative therapy for bone destruction in multiple myeloma that can be administered orally would be highly beneficial.
• Bone metastases are often associated with advanced cancer and are most common with breast, prostate and thyroid carcinomas and multiple myeloma (supra). Bone metastases are present in 65-75% of patients with advanced (metastatic) breast cancer. Metastatic bone lesions may be lytic or sclerotic in nature depending upon whether increased osteoclastic or osteoblastic activity predominates; if both processes are equally active, they are termed mixed lesions. Bone metastases in breast cancer patients usually involve osteolytic disease, where normal bone homeostasis is disrupted and skewed towards excessive resorption of bone (Coleman RE, Cancer Treat Rev. 27(3), 165-76 -A-
• Tumor-induced skeletal damage is mediated by osteoclasts that are stimulated directly or indirectly to dissolve bone by local factors (e.g. prostaglandin E, interleukin- 1, tumor necrosis factor and procathepsin D) released by tumor cells or associated immune cells, or by systemic factors, such as parathyroid hormone-related peptide.
• local factors e.g. prostaglandin E, interleukin- 1, tumor necrosis factor and procathepsin D
• systemic factors such as parathyroid hormone-related peptide.
• the most frequently affected skeletal sites are the vertebrae, pelvis, ribs, femur and skull.
• the invention is directed in part to methods of treating or preventing bone cancer, and to methods of treating or preventing bone loss associated with cancer metastases, utilizing certain compounds described in WO 2004/105765, the disclosure of which is hereby incorporated by reference in its entirety.
• the present invention provides the use of the macrocyclic quinazoline derivative 4,6-ethanediylidenepyrimido[4,5-b][6,l,12]benzoxadiazacyclo- pentadecine, 17-bromo-8,9, 10, 11 , 12, 13 , 14, 19-octahydro-20-methoxy- 13 -methyl, described as compound 22 in PCT publication WO2004/105765, in the manufacture of a medicament for the treatment or prevention of bone cancers and methods for killing bone cancer cells, including osteosarcomas, chondrosarcomas, myeloma bone disease and osteolytic bone metastases. It accordingly provides methods for treating, preventing, delaying or mitigating bone cancer, or for preventing and treating of bone loss associated with cancer metastases.
• the invention provides a method of inhibiting metastatic spread of a cancer to skeletal system, in a mammalian subject comprising administering to a mammalian subject suspected of having metastatic cancer a compound of the invention, in an amount effective to inhibit metastatic spread of the cancer to the skeletal system; and a method for treating bone cancer comprising administering to a mammalian subject diagnosed with a cancer a composition comprising a compound of the invention, in an amount effect to reduce growth or neoplastic spread of the bone cancer.
• any reduction in the rate of cancer growth or spread is indicative of successful treatment.
• cancer growth is halted completely.
• cancers shrink or are eradicated entirely.
• Preferred subjects for treatment are human subjects, but other animals, especially murine, rat, bovine, porcine, primate, and other model systems for cancer treatment, are contemplated.
• Metastatic cancers as used herein are contemplated to include a variety of cancers can metastasize to the bone, but the most common metastasizing cancers are breast, lung, renal, multiple myeloma, thyroid and prostate.
• cancers that have the potential to metastasize to bone include but are not limited to adenocarcinoma, blood cell malignancies, including leukemia and lymphoma; head and neck cancers; gastrointestinal cancers, including stomach cancer, colon cancer, colorectal cancer, pancreatic cancer, liver cancer; malignancies of the female genital tract, including ovarian carcinoma, uterine endometrial cancers and cervical cancer; bladder cancer ; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer, including malignant melanoma and squamous cell cancer.
• the present invention especially contemplates prevention and treatment of tumor-induced osteolytic lesions in bone
• the compounds are administered along with a second cancer therapeutic agent.
• the second agent can be any chemotherapeutic agent, radioactive agent, radiation, nucleic acid encoding a cancer therapeutic agent, antibody, protein, and/or other anti-lymphangiogenic agent or an anti-angiogenic agent.
• the second agent may be administered before, after, or concurrently with the compounds of the invention.
• the subject to be treated has been diagnosed with an operable tumor, and the administering step is performed before, during, or after the tumor is resected from the subject.
• Compound treatment in conjunction with tumor resection is intended to reduce or eliminate regrowth of tumors from cancer cells that fail to be resected.
• the invention provides a method of treating or preventing bone cancer, and to methods of treating or preventing bone loss associated with cancer metastases comprising the step of administering to a mammal (including, but not limited to humans, rats, canines, bovines, porcines, and primates) in need thereof a compound of the invention.
• a mammal including, but not limited to humans, rats, canines, bovines, porcines, and primates
• Figure 1 Effect of treatment on spontaneous lifting behaviour of the left hind paw. The data is presented as the percentage of the time the paw was raised over an observation period of 4 minutes.
• the vehicle group was dosed by oral gavage, daily with a 20% HPCD solution at pH 4.0.
• Compound 1 was dosed once daily at its maximum tolerated dose (MTD) of 200mg per kg, Iressa was also dosed at its maximum tolerated dose, by oral gavage, of 50mg per kg daily for 14 days.
• MTD maximum tolerated dose
• Figure 2 Representative reconstructions from ⁇ Ct s of the ipsilateral left hindlimbs showing osteolytic bone destruction in the tumor inoculated animals.
• WO-2004/105765 describes the preparation, formulation and pharmaceutical properties of macrocyclic quinazoline derivatives of formula (I) as multi targeted kinase inhibitors (MTKIs).
• the present invention also concerns a method of treating tumor-induced osteolytic lesions in bone of a mammal, comprising the step of administering a therapeutically effective amount of a compound according to the invention to said mammal.
• the present invention provides the use of 4,6- ethanediylidenepyrimido[4,5- ⁇ ] [6,1,12]benzoxadiazacyclo-pentadecine, 17-bromo- 8,9,10,11,12,13, 14,19-octahydro-20-methoxy-13-methyl or a pharmaceutically acceptable acid or base addition salt thereof, in the manufacture of a medicament for the treatment or prevention of bone cancer, including primary bone cancers and bone metastases as defined hereinbefore.
• a further aspect of the present invention is directed to a method for the treatment of prevention of bone cancer in a mammalian subject, comprising administering a therapeutically effective amount of 4,6-ethanediylidenepyrimido[4,5- b] [6, 1 , 12]benzoxadiazacyclo-pentadecine, 17-bromo-8,9, 10, 11, 12, 13, 14,19-octahydro- 20-methoxy- 13 -methyl or a pharmaceutically acceptable acid or base addition salt thereof, to a mammalian subject in need of such treatment.
• the pharmaceutically acceptable acid or base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and non-toxic base addition salt forms which MTKI 1 is able to form.
• the basic properties can be converted in their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid.
• Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i. e.
• butanedioic acid maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, />toluenesulfonic, cyclamic, salicylic, ⁇ -aminosalicylic, pamoic and the like acids.
• the acidic properties may be converted in their pharmaceutically acceptable base addition salts by treating said acid form with a suitable organic or inorganic base.
• suitable organic or inorganic base e.g. the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g., the benzathine, JV-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.
• acid or base addition salt also comprise the hydrates and the solvent addition forms which MTKI 1 is able to form.
• examples of such forms are e.g., hydrates, alcoholates and the like.
• the present invention is concerned with a use of the dihydrobromide salt of 4,6-ethanediylidenepyrirnido[4,5-Z>] [6,1,12]benzoxadiazacyclo-pentadecine, 17- bromo-8,9,10,11,12,13, 14,19-octahydro-20-methoxy-13-methyl, i.e., 17-bromo- 8,9, 10, 11 , 12, 13 , 14, 19-octahydro-20-methoxy- 13-methyl-4,6-ethanediylidenepyrimido [4,5-b][6,l,12]benzoxadiazacyclopentadecine dihydrobromide, in any of the aforementioned uses for MTKI 1.
• the present invention provides the use of the aforementioned MTKI 1 for the preparation of a pharmaceutical composition for the prevention and/or treatment of bone cancers.
• the present invention also concerns a method of preventing and/or treating bone cancer in a mammal, comprising the step of administering a therapeutically effective amount of the aforementioned MTKI 1 to said mammal.
• the present invention provides the use of MTKI 1 for the preparation of a pharmaceutical composition for the prevention and/or treatment of bone loss.
• the present invention also concerns a method for preventing and/or treating of bone loss associated with cancer metastases in a mammal, comprising the step of administering a therapeutically effective amount of MTKI 1 to said mammal.
• the most preferred compounds for use in accordance with the present invention are those selected from the group consisting of compounds having the following structure:
• the compounds according to the invention can be prepared and formulated into pharmaceutical compositions by methods known in the art and in particular according to the methods described in the published patent specification WO-2004/105765 mentioned herein and incorporated by reference.
• compositions a therapeutically effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which may take a wide variety of forms depending on the form of preparation desired for administration.
• a pharmaceutically acceptable carrier which may take a wide variety of forms depending on the form of preparation desired for administration.
• These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for systemic administration such as oral, percutaneous, or parenteral administration; or topical administration such as via inhalation, a nose spray, eye drops or via a cream, gel, shampoo or the like.
• any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions (including nanosuspensions), syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed.
• the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included.
• Injectable solutions may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution.
• Injectable solutions containing compounds of formula (I) may be formulated in an oil for prolonged action.
• Appropriate oils for this purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soy bean oil, synthetic glycerol esters of long chain fatty acids and mixtures of these and other oils.
• Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed.
• the carrier optionally comprises a penetration enhancing agent and/or a suitable wettable agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not cause any significant deleterious effects on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
• These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on or as an ointment.
• compositions for topical application there may be cited all compositions usually employed for topically administering drugs e.g. creams, gels, dressings, shampoos, tinctures, pastes, ointments, salves, powders and the like.
• compositions may be by aerosol, e.g. with a propellent such as nitrogen, carbon dioxide, a freon, or without a propellent such as a pump spray, drops, lotions, or a semisolid such as a thickened composition which can be applied by a swab.
• a propellent such as nitrogen, carbon dioxide, a freon
• a propellent such as a pump spray
• drops lotions
• a semisolid such as a thickened composition which can be applied by a swab.
• semisolid compositions such as salves, creams, gels, ointments and the like will conveniently be used.
• Dosage unit form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• dosage unit forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls and the like, and segregated multiples thereof.
• a therapeutically effective amount of the pharmaceutical composition comprising a compound according to the invention is administered orally or parenterally.
• Said therapeutically effective amount is the amount that effectively prevents metastasis and/or growth or reduces the size of a variety of neoplastic disorders or cell proliferative disorders (supra) in patients.
• a pharmaceutical composition comprising a compound of the present invention, and in particular
• 4,6-ethanediylidenepyrimido[4,5-&] [6,1,12]benzoxadiazacyclo-pentadecine, 17-bromo- 8,9,10,11,12,13,14,19-octahydro-20-methoxy- 13 -methyl (MTKIl, or Compound 1) as the active ingredient can be administered orally in an amount of from 10 mg to several (1 to 5) grams daily, either as a single dose or subdivided into more than one dose, including, e.g., two, three or even four times daily.
• a preferred amount ranges from 500 to 4,000 mg daily.
• a particularly, preferred dosage for such a compound is in the range of 750 mg to 3,000 mg daily.
• the amount of a compound according to the present invention, also referred to here as the active ingredient, which is required to achieve a therapeutic effect will, of course, vary with, the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated.
• the optimum dosage amounts and regimen can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.
• This treatment can be given either continuously or intermittently, including, e.g., but not limited to, cycles of 3-4 weeks with treatment given for 1-21 days per cycle or other schedules shown to be efficacious and safe.
• the product MTKIl can be prepared as a 10-mg/mL oral solution, pH 2. It contains an excipient, Captisol® (chemical name: sulfobutyl ether- ⁇ -cyclodextrin, SBE- ⁇ -CD), citric acid, Tween® 20, HCl, and NaOH in purified water.
• Captisol® chemical name: sulfobutyl ether- ⁇ -cyclodextrin, SBE- ⁇ -CD
• citric acid citric acid
• Tween® 20 HCl
• NaOH NaOH
• the product MTKIl can also be prepared as 50-mg, 100-mg and 300-mg oral immediate release capsules, containing the active chemical entity MTKIl, lactose monohydrate (200 mesh), sodium lauryl sulphate and magnesium stearate in hard gelatin capsules, sizes 3, 4 and 00, respectively.
• the capsules may also contain any or all of the following ingredients: gelatin, red iron oxide and titanium oxide.
• the above MTKI 1 may be used in combination with one or more other cancer treatments.
• Such combinations could encompass any established antitumor therapy, such as, but not limited to, chemotherapies, irradiation, and target based therapies such as antibodies and small molecules (such as for example bisphosphonates, taxanes, anthracyclines, capecitabine, Herceptin, docetaxel, satraplatin, cetuximab, avastin, aromatase inhibitors and methothrexate).
• chemotherapies such as antibodies and small molecules (such as for example bisphosphonates, taxanes, anthracyclines, capecitabine, Herceptin, docetaxel, satraplatin, cetuximab, avastin, aromatase inhibitors and methothrexate).
• target based therapies such as antibodies and small molecules (such as for example bisphosphonates, taxanes, anthracyclines, capecitabine, Herceptin
• the MTKI 1 and the further anti-cancer agent may be administered simultaneously (e.g. in separate or unitary compositions) or sequentially in either order. In the latter case, the two compounds will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved.
• the preferred method and order of administration and the respective dosage amounts and regimens for each component of the combination will depend on the particular MTKI and further anti-cancer agents being administered, their route of administration, the particular tumor being treated and the particular host being treated. The optimum method and order of administration and the dosage amounts and regimen can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.
• MTKI 1 also referred to herein as Compound 1
• Compound 1 has resulted in an extremely favourable tissue distribution profile including the ability to cross the intact blood brain barrier whilst still retaining good cellular activity and oral bioavailability.
• this preferential tissue distribution to the bone marrow compartment results in significant anti tumoral activity using experimental models of bone metastases.
• NCTC2472 fibrosarcoma ATCC Rockvile, MD USA
• MDA-MB231 breast cancer cells Dr. Yoneda, Univ.
• mice for the NCTC2472 mouse fibrosarcoma cells (20-25 g, Charles River, Sulzfeld, Germany) or female NMRI Nude mice for the MDA MB 231 human breast cancer cells (Janvier, France) were used.
• Induction of bone cancer was carried out as previously described (Schwei et al., 1999 ').
• Induction of general anaesthesia was performed under 4 % isoflurane in a mixture of 30% O 2 and 70% air (1000 ml/ min). Anaesthesia was then maintained at 2.5 % isoflurane for the duration of the surgical procedure.
• the left hind paw was shaved and disinfected with povidone- iodine followed by 70% ethanol.
• mice were treated once daily (QlD) with either vehicle (20% Hydroxypropyl- ⁇ -cyclodextrine, pH 4.0) or vehicle formulated to give a dose of 200 or 50 mg/kg of Compound 1 respectively by gavage (p.o.) administered in a volume of 10 ml/kg body weight. Mice were treated up to 18 days after bone tumor induction.
• vehicle 20% Hydroxypropyl- ⁇ -cyclodextrine, pH 4.0
• Pain behaviours were evaluated in the group of sham and bone tumour mice and were behaviourally tested during a 2- week period prior to and 1, 9, 12 and 14 days after tumour inoculation.
• the femur of the left hind limb was sampled and used for ⁇ CT scanning as described in Vermeirsh et al., (2004) 2 .
• Spontaneous lifting behavior Animals were habituated to the laboratory room at least 30 minutes in a transparent acrylic cylinder of 20 cm diameter and thereafter observed during 4 minutes for spontaneous lifting behaviour of the left hind paw.
• Bone analysis was carried out on ipsilateral left hind limbs prior to and 7, 12, 15 and 18 days following cell injection. Limbs were fixed in 10% phosphate-buffered formalin and transfered to a plastic cuvette filled with 70% ethanol for scanning using the SkyScan microtomograph (Skyscan 1067®, Skyscan, Aartselaar, Belgium). For medium resolution measurement, the X-ray beam was collimated to a diameter of 18 mm, line spacing and point resolution were set at 0.254 and 0.127 mm, respectively. After standardized reconstruction, the datasets for each bone were re-sampled using computer software (Ant, 3D-creator vs.
• the vehicle group was dosed by oral gavage, daily with a 20% HPCD solution at pH 4.0.
• Compound 1 was dosed once daily at its maximum tolerated dose (MTD) of 200mg per kg, Iressa was also dosed at its maximum tolerated dose, by oral gavage, of 50mg per kg daily for 14 days.
• the vehicle treated group of animals displayed detectable paw lifting behaviours starting seven days post tumor cell inoculation.

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