WO2009076490A1 - Utilisation d'antagonistes de la cathepsine l dans le traitement de maladie des os - Google Patents

Utilisation d'antagonistes de la cathepsine l dans le traitement de maladie des os Download PDF

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WO2009076490A1
WO2009076490A1 PCT/US2008/086326 US2008086326W WO2009076490A1 WO 2009076490 A1 WO2009076490 A1 WO 2009076490A1 US 2008086326 W US2008086326 W US 2008086326W WO 2009076490 A1 WO2009076490 A1 WO 2009076490A1
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bone
inhibitor
cathepsin
subject
cell
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PCT/US2008/086326
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Michael Long
Daniel Chagnovich
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Velcura Therapeutics, Inc.
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes

Definitions

  • the present invention relates to the fields of molecular biology and medicine. More particularly, it relates to the fields of bone disease & injury, bone repair, bone implants, bone grafts, periodontal disease and cancer. Specifically, it deals with the use of Cathepsin L antagonists to promote bone formation and to treat bone diseases such as osteoporosis, as well as bone trauma and cancers with bone involvement, including multiple myeloma and myeloma bone disease.
  • Osteoporosis represents a large and rapidly growing health care problem with an unmet medical need for therapies that stimulate bone formation.
  • Osteoporosis is known to affect approximately 100 million people worldwide - 35 million of whom live in the
  • bone reconstruction and, specifically, the ability to reconstruct defects in bone tissue that result from traumatic injury, as a consequence of cancer or cancer surgery, as a result of a birth defect, or as a result of aging.
  • traumatic injury as a consequence of cancer or cancer surgery
  • cranial and facial bone are particular targets for this type of reconstructive need.
  • new implant materials e.g., titanium
  • Titanium implants provide excellent temporary stability across bony defects.
  • experience has shown that a lack of viable bone bridging the defect can result in exposure of the appliance, infection, structural instability and, ultimately, failure to repair the defect.
  • Autologous bone grafts are another possibility to deal with bone injury, but they have several demonstrated disadvantages in that they must be harvested from a donor site such as iliac crest or rib, they usually provide insufficient bone to completely fill the defect, and the bone that does form is sometimes prone to infection and resorption.
  • Partially purified xenogeneic preparations are not practical for clinical use because microgram quantities are purified from kilograms of bovine bone, making large scale commercial production both costly and impractical. Allografts and demineralized bone preparations are therefore often employed. Microsurgical transfers of free bone grafts with attached soft tissue and blood vessels can close bony defects and allow an immediate source of blood supply to the graft.
  • these techniques are time consuming, have been shown to produce a great deal of morbidity, and can only be used by specially trained individuals.
  • Another form of bone disease is that resulting from cancer.
  • a number of cancers metastasize to bone and can result in bone weakening, and some are even associated with bone destruction and bone loss, such as breast, lung, thyroid, kidney and prostate cancer.
  • Multiple Myeloma and its associated myeloma bone disease (MBD) is not a metastatic cancer. Rather, myeloma cells are derived from the B-cells of the immune system that normally reside in the bone marrow and are therefore intimately associated with bone.
  • the bone marrow microenvironment plays an important role in the growth, survival and resistance to chemotherapy of the myeloma cells, which, in turn, regulate the increased bone loss associated with this disorder (world-wide-web at multiplemyeloma.org).
  • myeloma patients Over 90% of myeloma patients have bone involvement, versus 40-60% of cancer patients who have bone metastasis, and over 80% of these MBD patients have intractable bone pain. Additionally, approximately 30% of myeloma patients have hypercalcemia that is a result of the increased osteolytic activity associated with this disease (Cavo et al., 2006).
  • the MBD lesions are unique in that they do not heal or repair, despite the patients' having many years of complete remission (world- wide-web at multiplemyeloma.org; Terpos et al, 2005). Mechanistically, this seems to be related to the inhibition and/or loss of the bone- forming osteoblast during disease progression. Indeed, bone marker studies and histomorphometry indicate that both the bone-resorbing osteoclast and osteoblast activity are increased, but balanced early in the disease, whereas overt MBD shows high osteoclast activity and low osteoblast activity (world-wide-web at multiplemyeloma.org).
  • MBD is a disorder in which bone formation and bone loss are uncoupled and would benefit from therapies that both stimulate bone formation and retard its loss. To date, no such therapies exist. Therefore, there continues to be a need for improved methods of stimulating bone formation in vivo to treat bone disease and injury, including cancer, and for producing bone ex vivo in the production and use of implants.
  • a method of promoting bone formation comprising (i) selecting a first Cathepsin L inhibitor; and (ii) administering to a subject the first inhibitor of Cathepsin L.
  • the inhibitor may be an intracellular or extracellular inhibitor of Cathepsin L, or both. It may be biological or an organopharmaceutical small molecule.
  • the biological may be a peptide, an siRNA, an antisense molecule or a single-chain antibody.
  • the organopharmaceutical small molecule may be a structural variant of a known Cathepsin L inhibitor based on hydrophilic and lipophilic characteristics, spatial properties, or other information available from medicinal chemistry and chemical enzymology studies on these compounds.
  • the first inhibitor may also prevent bone resorption.
  • the subject may suffer from a bone -related pathology or injury, such as osteoporosis, vitamin D deficiency, primary bone cancer, metastatic bone cancer, multiple myeloma or myeloma bone disease, bone fracture, periodontal disease, Paget 's disease, osteoporosis secondary to other diseases, or in need of bone grafts or implants.
  • the subject is a human or a non-human animal.
  • the compound may possess or lack bone resorption inhibition.
  • the method may further comprise contacting the subject with a second agent.
  • the second agent may be a compound that inhibits bone loss such, as bisphosphonate, a SERM or a second inhibitor of Cathepsin L that is distinct from the first inhibitor, and may be distinct in its mode (extracellular versus intracellular) of action.
  • the second agent may be an inhibitor of Cathepsin K.
  • the second agent may be a promoter of bone formation such as PTH or an analogue thereof.
  • the first and/or second agent may be formulated to increase transmembrane delivery.
  • the first and/or second agent may be formulated with a cell permeability factor.
  • the first and/or second agent may be formulated in a lipid delivery vehicle.
  • the first and/or second agent may be formulated to enhance its bioavailability.
  • the first and/or second agent may be administered to the subject more than once.
  • the first and/or second agent is administered to the subject by oral or intravenous routes.
  • a method of producing bone from an isolated bone cell comprising (i) selecting a Cathepsin L inhibitor; (ii) contacting the bone cell with the inhibitor of Cathepsin L; and (iii) culturing the cell under conditions promoting bone formation.
  • the method may further comprise implanting the bone cell or bone derived therefrom into a subject.
  • the bone cell may have been obtained, prior to step (ii) from the subject or an allogeneic donor.
  • the obtained cell may have been derived from a stem cell prior to step (ii).
  • the stem cell may have been derived or cultured under conditions promoting the formation of an osteogenic cell prior to step (ii).
  • the compound may possess or lack bone resorption inhibition.
  • a method of identifying an agent that promotes bone formation comprising (i) providing a candidate compound; (ii) admixing the candidate compound with an isolated Cathepsin L enzyme, or a cell expressing Cathepsin L, or an experimental animal, cells of which express Cathepsin L; (iii) measuring Cathepsin L activity or expression or bone formation; and (iv) comparing the characteristic measured in step (iii) with that observed in the absence of the candidate modulator, wherein a difference between the measured characteristic indicates that the candidate compound is, indeed, a modulator of Cathepsin L.
  • the compound may possess or lack bone resorption inhibition.
  • a method of treating a subject with multiple myeloma comprising (i) selecting a first Cathepsin L inhibitor; and (ii) administering to a subject the first inhibitor of Cathepsin L.
  • the inhibitor may be a biological or an organopharmaceutical small molecule.
  • the biological may be a peptide, an siRNA, an antisense molecule or a single-chain antibody.
  • the organopharmaceutical small molecule may be a structural variant of a known Cathepsin L inhibitor based on hydrophilic and lipophilic characteristics, spatial properties, or other information available from medicinal chemistry and chemical enzymology studies on these compounds.
  • the first inhibitor may also prevent bone resorption or stimulate bone formation.
  • the method may further comprise contacting the subject with a second agent.
  • the second agent may be a compound that inhibits bone loss such, as bisphosphonate, a SERM or a second inhibitor of Cathepsin L that is distinct from the first inhibitor.
  • the second inhibitor of Cathepsin L may be distinct in its mode (extracellular versus intracellular) action as the first inhibitor of Cathepsin L.
  • the second agent may be an inhibitor of Cathepsin K.
  • the second agent may be a promoter of bone formation such as PTH or an analogue thereof, or an inhibitor of Cathepsin K such as VEL-0230 or an analogue thereof.
  • the second agent may also be an anti-cancer agent, such as a chemotherapy, a radiotherapy, an immunotherapy, a cytokine therapy, a toxin therapy or a gene therapy.
  • the multiple myeloma may be recurrent, metastatic or drug resistant.
  • the first and/or second agent may be formulated to increase transmembrane delivery.
  • the first and/or second agent may be formulated with a cell permeability factor.
  • the first and/or second agent may be formulated in a lipid delivery vehicle.
  • the first and/or second agent may be formulated to enhance its bioavailability.
  • the first and/or second agent may be administered to the subject more than once.
  • the first and/or second agent may be administered to the subject by oral or intravenous routes, injection into the tumor or tumor vasculature, or administration local or regional to a tumor.
  • compositions and kits of the invention can be used to achieve methods of the invention.
  • FIG 1 Inhibition of Cathepsin L Stimulates Bone Mineralization
  • FIG. 2 Quantitative Analysis of Cathepsin L Inhibitor Stimulation of Human Bone Cell Mineralization.
  • FIG. 3 Exemplary Cathepsin L Inhibitors.
  • Cathepsin Inhibitor 4
  • Cathepsin K a proteolytic enzyme previously thought to be involved only in bone degradation. Its role in modulating bone resorption is that of a classic proteolytic enzyme, functioning to degrade components of the extra-cellular compartment, particularly collagen (Bossard et al., 1996). Moreover, the prevalent viewpoint is that this enzyme functions solely and/or selectively in osteoclasts to degrade extracellular proteins and, as such, is targeted by anti-catabolic therapies that function to inhibit the bone-resorbing osteoclast (Yasuda et al., 2005; Grabowskal et al., 2005; Troen, 2004; Dodds, 2003; Zaidi et al., 2003).
  • Cathepsin K was shown to be expressed by osteoblasts; however, this report still subscribed to the concept that Cathepsin K has an extracellular role and, further, did not test enzymatic function of this enzyme within osteoblasts (Mandelin et al., 2006).
  • Cathepsin K plays a role in bone formation as inhibition of the enzyme unexpectedly results in an increase in bone formation, as described below.
  • the role of Cathepsin K in bone formation intracellular signaling networks is supported by three observations: (1) the inventor's Cathepsin K inhibitors stimulate bone formation both in vivo and in vitro; (2) RNA interference studies of Cathepsin K (RNAi; performed via small-interfering RNA (siRNA) treatment) demonstrate that inhibition of this message results in a bone-formation phenotype in treated human osteoblasts; and (3) stimulation of bone formation with multiple osteogenic growth factors decreases Cathepsin K message and activity within the osteoblast.
  • RNAi RNA interference studies of Cathepsin K
  • siRNA small-interfering RNA
  • Cathepsin K inhibitors Numerous patents exist on the structure of Cathepsin K inhibitors and their use in treating the extracellular activity of the enzyme.
  • the inventors' prior work proposes the use of intracellular inhibition of Cathepsin K to various forms of bone disease and to treat multiple myeloma, including myeloma bone disease.
  • they extend their work on cathepsins to Cathepsin L, and show that similar benefits may accrue in the areas of promoting bone growth, inhibition bone resorption, promoting bone repair and grafting, and treating multiple myeloma and myeloma bone disease through the administration of Cathepsin L preferential or selective inhibitors.
  • Cathepsin L preferential or selective inhibitors.
  • Cathepsins are proteases that function in the normal physiological as well as pathological degradation of connective tissue. Cathepsins play a major role in intracellular protein degradation and turnover, bone remodeling, and prohormone activation (Marx, 1987). Cathepsin B, H, L and S are ubiquitously expressed lysosomal cysteine proteinases that belong to the papain superfamily. They are found at constitutive levels in many tissues in the human including kidney, liver, lung and spleen. Some pathological roles of cathepsins include an involvement in glomerulonephritis, arthritis, and cancer metastasis (Sloan and Honn, 1984). Greatly elevated levels of cathepsin L and B mRNA and protein are seen in tumor cells. Cathepsin L mRNA is also induced in fibroblasts treated with tumor promoting agents and growth factors (Kane and Gottesman, 1990).
  • Cathepsins a cysteine protease family
  • Cathepsin K a member of this protease family
  • Cathepsin K was discovered in 1994, is synthesized as a 37 kDa pre-pro enzyme, and is presumably autoactivated to the mature 27 kDa enzyme at low pH (McQueney et al., 1997; Littlewood-Evans et al., 1997; U.S. Patent 5,861,298).
  • Cathepsin K plays an important role in bone and cartilage break down.
  • Cathepsin L The cysteine protease, Cathepsin L, is produced and secreted by osteoclasts where its primary activity is extracellular (Goto, 1993; Hill, 1994; Kakegawa, 1995). However, the degree expression is variable (Goto, 1993; Hill, 1994; Kakegawa, 1993; Drake, 1996). Cathepsin L appears to function in the degradation of the bone matrix by augmenting that of Cathepsin K during inflammation or other cytokine-mediated events. Thus, collagen degradation by osteoclasts stimulated with inflammatory cytokines is inhibited in response to Cathepsin L inhibition, whereas untreated cells are not (Furuyama, 2000) and may mediate acute versus chronic bone effects (Millest, 1997). Cathepsin L activity is also seen in primary bone cancers and metastatic bone disease (Park, 1996). It also functions to a degree to maintain bon volume in vivo (Potts, 2004).
  • Fracture The first example is the otherwise healthy individual who suffers a fracture. Often, clinical bone fracture is treated by casting to alleviate pain and allow natural repair mechanisms to repair the wound. There has been progress in the treatment of fracture in recent times, however, even without considering the various complications that may arise in treating fractured bones, any new procedures to increase bone healing in normal circumstances would represent a great advance.
  • Periodontal Disease Progressive periodontal disease leads to tooth loss through destruction of the tooth's attachment to the surrounding bone. Approximately 5 - 20% of the U.S. population (15-60 million individuals) suffers from severe generalized periodontal disease, and there are 2 million related surgical procedures. Moreover, if the disease is defined as the identification of at least one site of clinical attachment loss, then approximately 80% of all adults are affected, and 90% of those aged 55 to 64 years. If untreated, approximately 88% of affected individuals show moderate to rapid progression of the disease' which shows a strong correlation with age. The major current treatment for periodontal disease is regenerative therapy consisting of replacement of lost periodontal tissues. The lost bone is usually treated with an individual's own bone and bone marrow, due to their high osteogenic potential.
  • Bone allografts (between individuals) can also be performed using stored human bone. Although current periodontal cost analyses are hard to obtain, the size of the affected population and the current use of bone grafts as a first-order therapy strongly suggest that this area represents an attractive target for bone-building therapies. Osteopenia/osteoporosis.
  • the terms osteopenia and osteoporosis refers to a heterogeneous group of disorders characterized by decreased bone mass and fractures. Osteopenia is a bone mass that is one or more standard deviations below the mean bone mass for a population; osteoporosis is defined as 2.5 SD or lower. An estimated 20-25 million people are at increased risk for fracture because of site-specific bone loss.
  • Risk factors for osteoporosis include increasing age, gender (more females), low bone mass, early menopause, race (Caucasians in general; asian and hispanic females), low calcium intake, reduced physical activity, genetic factors, environmental factors (including cigarette smoking and abuse of alcohol or caffeine), and deficiencies in neuromuscular control that create a propensity to fall.
  • osteoporosis is a significant health care problem.
  • Osteoporosis once thought to be a natural part of aging among women, is no longer considered age or gender-dependent. Osteoporosis is defined as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture. Bone strength reflects the integration of two main features: bone density and bone quality. Bone density is expressed as grams of mineral per area or volume and in any given individual is determined by peak bone mass and amount of bone loss. Bone quality refers to architecture, turnover, damage accumulation (e.g., microfractures) and mineralization. A fracture occurs when a failure-inducing force (e.g., trauma) is applied to osteoporotic bone. Current therapies for osteoporosis patients focus on fracture prevention, not for promoting bone formation or fracture repair.
  • a failure-inducing force e.g., trauma
  • Bone Reconstruction/Grafting A fourth example is related to bone reconstruction and, specifically, the ability to reconstruct defects in bone tissue that result from traumatic injury; as a consequence of cancer or cancer surgery; as a result of a birth defect; or as a result of aging.
  • Titanium implants provide excellent temporary stability across bony defects and are an excellent material for bone implants or artificial joints such as hip, knee and joint replacements.
  • a lack of viable bone binding to implants the defect can result in exposure of the appliance to infection, structural instability and, ultimately, failure to repair the defect.
  • a therapeutic agent that stimulates bone formation on or around the implant will facilitate more rapid recovery.
  • Autologous bone grafts are another possibility, but they have several demonstrated disadvantages in that they must be harvested from a donor site such as iliac crest or rib, they usually provide insufficient bone to completely fill the defect, and the bone that does form is sometimes prone to infection and resorption.
  • Partially purified xenogeneic preparations are not practical for clinical use because microgram quantities are purified from kilograms of bovine bone, making large scale commercial production both costly and impractical. Allografts and demineralized bone preparations are therefore often employed, but suffer from their devitalized nature in that they only function as scaffolds for endogenous bone cell growth.
  • Microsurgical transfers of free bone grafts with attached soft tissue and blood vessels can close bony defects with an immediate source of blood supply to the graft.
  • these techniques are time consuming, have been shown to produce a great deal of morbidity, and can only be used by specially trained individuals.
  • the bone implant is often limited in quantity and is not readily contoured. In the mandible, for example, the majority of patients cannot wear dental appliances using presently accepted techniques (even after continuity is established), and thus gain little improvement in the ability to masticate.
  • Bone cancer occurs infrequently while bone metastases are present in a wide range of cancers, including thyroid, kidney, and lung.
  • Metastatic bone cancer is a chronic condition; survival from the time of diagnosis is variable depending on tumor type. In prostate and breast cancer and in multiple myeloma, survival time is measurable in years. For advanced lung cancer, it is measured in months. Cancer symptoms include pain, hypercalcemia, pathologic fracture, and spinal cord or nerve compression.
  • Prognosis of metastatic bone cancer is influenced by primary tumor site, presence of extra-osseous disease, and the extent and tempo of the bone disease. Bone cancer/metastasis progression is determined by imaging tests and measurement of bone specific markers. Recent investigations show a strong correlation between the rate of bone resorption and clinical outcome, both in terms of disease progression or death. .
  • Multiple myeloma is a B-lymphocyte malignancy characterized by the accumulation of malignant clonal plasma cells in the bone marrow.
  • the clinical manifestations of the disease are due to the replacement of normal bone marrow components by abnormal plasma cells, with subsequent overproduction of a monoclonal immunoglobulin (M protein or M component), bone destruction, bone pain, anemia, hypercalcemia and renal dysfunction.
  • M protein or M component monoclonal immunoglobulin
  • myeloma bone disease is not a metastatic disease. Rather, myeloma cells are derived from the B-cells of the immune system that normally reside in the bone marrow and are therefore intimately associated with bone. Indeed, the bone marrow microenvironment plays an important role in the growth, survival and resistance to chemotherapy of the myeloma cells, which, in turn, regulate the increased bone loss associated with this disorder (world- wide-web at multiplemyeloma.org).
  • myeloma Common problems in myeloma are weakness, confusion and fatigue due to hypercalcemia. Headache, visual changes and retinopathy may be the result of hyperviscosity of the blood depending on the properties of the paraprotein. Finally, there may be radicular pain, loss of bowel or bladder control (due to involvement of spinal cord leading to cord compression) or carpal tunnel syndrome and other neuropathies (due to infiltration of peripheral nerves by amyloid). It may give rise to paraplegia in late presenting cases.
  • Myeloma Bone Disease As discussed above, unlike the osteolysis associated with other bone tumors, the MBD lesions are unique in that they do not heal or repair, despite the patients' having many years of complete remission.
  • MBD bone marker studies and histomorphometry indicate that both the bone-resorbing osteoclast and osteoblast activity are increased, but balanced early in the disease, whereas overt MBD shows high osteoclast activity and low osteoblast activity.
  • MBD is a disorder in which bone formation and bone loss are uncoupled and would benefit from therapies that both stimulate bone formation and retard its loss.
  • SRE skeletal related events
  • a Cathepsin L antagonist of the present invention is one that is capable of inhibiting either or both of the intracellular and extracellular function of Cathepsin L and also promoting the formation of bone in vitro and/or in vivo by the inhibition of intracellular Cathepsin L.
  • the inhibitor of Cathepsin L will have preferential inhibitor activity against Cathepsin L of at least 3-fold over that of other cathepsins in enzymatic assays, in particular over Cathepsin K.
  • the only known activity of Cathepsin L antagonists is to modulate the extracellular activity of Cathepsin L in certain physiological states of osteoclasts.
  • An agent of the present invention is one that is capable of inhibiting the function of Cathepsin L and in so doing promoting the formation of bone in vitro and/or in vivo.
  • the agent may also inhibit bone resorption.
  • the effect of these molecular and cellular actions will be to treat bone disease and injury and to improve the function in the clinical situation (reduction in pain, increase in mobility, etc.) of a subject suffering from bone -related pathologies, such as osteoporosis, bone cancer and metastasis, multiple myeloma, malignant hypercalcemia, Paget's Disease, and arthritis.
  • Cathepsin L inhibitors include those disclosed in U.S. Patents 5,973,110, 5,424,325, 5,422,359, 6,110,967, 5,883,121, 5,843,992, 5,843,508, 5,716,980, 5,698,519, 5,639,783, 5,639,781, 5,556,853, 5,506,243, 5,416,117, 5,395,958, 5,328,909, and 4,507,232, as well as U.S Patent Publications 2006/0258598, 2006/0216297, 2004/0192674 and 2004/0009891.
  • Ravanko et al. (2004), Woo et al. (1996), Yasuma et al. (1998) and Chowdhury et al. also disclose useful inhibitors of Cathepsin L, including those shown in FIG. 3.
  • the present invention provides for the treatment of bones disease and bone trauma by stimulating the production of new bone tissue, both in vivo and ex vzVo for transplant purposes.
  • An agent of the present invention is one that is capable of inhibiting the function of Cathepsin L and also promoting the formation of bone in vitro and/or in vivo by the inhibition of intracellular Cathepsin L within the osteoblast.
  • the agent may also inhibit bone resorption.
  • the effect of these molecular and cellular actions will be to treat bone disease and injury and to improve the patient's ability to function (reduction in pain, increase in mobility, etc.) of a subject suffering from bone-related pathologies.
  • agents may be used in combination with intracellular Cathepsin L inhibitors. More generally, these agents would be provided in a combined amount (along with the intracellular Cathepsin L inhibitor) to produce any of the effects discussed above.
  • This process may involve contacting the cell or subject with both agents at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell or subject with two distinct compositions or formulations, at the same time, wherein one composition includes the intracellular inhibitor and the other includes the second agent.
  • one agent may precede or follow the other by intervals ranging from minutes to weeks.
  • the agents are applied separately to the cell or subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell or subject.
  • the intracellular inhibitor of Cathepsin L is "A” and the other agent is "B":
  • Combination agents include bisphosphonates (DidronelTM, FosamaxTM and ActonelTM), SERMs (Evista) or other hormone derivatives, and Parathyroid Hormone (PTH) analogs.
  • other combination agents may be Cathepsin K inhibitors.
  • treatment of Bone Disease by compounds of the present invention addresses bone loss by stimulating the production of new bone tissue.
  • Cathepsin L inhibitors inhibit the function of osteoclasts with out killing them. This is distinct from the effects of bisphosphonates that stimulate apoptosis, or from those resulting from DensoamabTM, an antibody against RANK ligand that inhibits osteoclast formation. Both of those agents reduce osteoclast numbers, which can also have adverse effects.
  • agents of the present invention will stimulate bone formation to restore lost bone in multiple myeloma, and inhibit osteoclasts in Bone Disease via their anabolic action.
  • the agent may also inhibit bone resorption and in so doing may inhibit further Bone Disease progression.
  • the compounds of the present invention do not reduce osteoclast number, so these important cells can still contribute to bone homeostasis by signaling osteoblasts. Additionally, compounds of the present invention prevent osteoclasts from forming "bone-resorption pits" where the myeloma and/or cancer cells locate themselves, thus reducing the area in which tumor cells can live.
  • agents may be used in combination with Cathepsin L inhibitors to effect greater inhibition of Cathepsin L, and thus provide a more effective therapy for Bone Diseasesa. More generally, these agents would be provided in a combined amount (along with other agents) to produce or increase any of the effects discussed herein.
  • This process may involve contacting the cell or subject with both agents at the same time. This may be achieved by contacting the cell or subject with a single composition or pharmacological formulation that includes both agents, or by contacting the cell or subject with two distinct compositions or formulations, at the same time, wherein one composition includes the intracellular Cathepsin L inhibitor and the other includes the second agent.
  • one agent may precede or follow the other by intervals ranging from minutes to weeks.
  • the agents are applied separately to the cell or subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell or subject.
  • Cathepsin L inhibitors Another type of combination therapy involves the use of Cathepsin L inhibitors with tradition cancer therapies, including chemotherapy, radiotherapy, immunotherapy, hormone therapy, cytokine therapy, gene therapy, or toxin therapy.
  • the intracellular inhibitor of Cathepsin L is "A” and the other agent is "B":
  • Combination agents include bisphosphonates (Didronel , Fosamax and Actonel ), SERMs (E vista) or other hormone derivatives, and Parathyroid Hormone (PTH) analogs.
  • PTH Parathyroid Hormone
  • U.S. Patents 6,642,239, 6,531,612, 6,462,076 and 6,274,336, as well as U.S. Publication Nos. 2006/0074092, 2006/0020001, 2005/0245596, 2005/0107616, 2005/0054819, and 2004/0249153 disclose other Cathepsin K inhibitors that work in an extracellular fashion.
  • agents include, thalidomide, cyclophosphamide, VAD (vincristine, doxorubicin, and dexamethasone), low-dose therapy with melphalan combined with prednisone, plasmapheresis, dialysis, high-dose chemotherapy with melphalan combined with bone marrow transplant, thalidomide combined with reduced-intensity chemotherapy (melphalan and prednisone), cyclophosphamide, dexamethasone, bortezomib (or Velcade®) and lenalidomide (or Revlimid®).
  • compositions of the present invention comprise an effective amount of one or more Cathepsin L antagonists dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one Cathepsin L antagonist, and optionally an additional active ingredient, will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives ⁇ e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
  • the Cathepsin L antagonist may be admixed with different types of carriers depending on whether it is to be administered orally or by injection.
  • the present invention can be administered buccally, intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, intratumorally, into tumor vasculature, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., nanoparticles, liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
  • the Cathepsin L antagonist may be formulated into a composition in a free base, neutral or salt form or ester. It may also be synthesized/formulated in a prodrug form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, fumaric, or mandelic acid.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the composition is combined or mixed thoroughly with a semi-solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include Cathepsin L antagonist, one or more lipids, and an aqueous solvent.
  • lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long- chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally- occurring or synthetic (i.e., designed or produced by man).
  • Lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • the Cathepsin L antagonist may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • Cathepsin L antagonist pharmaceutical compositions may comprise, for example, at least about 0.1% of the antagonist, about 0.5% of the antagonist, or about 1.0% of the antagonist.
  • the antagonist may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of the antagonist in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • a dose of a Cathepsin L antagonist may also comprise from about 0.1 microgram/kg/body weight, about 0.2 microgram/kg/body weight, about 0.5 microgram/kg/body weight, about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the Cathepsin L antagonists are formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et ah, 1997; Hwang et al, 1998; U.S. Patents 5,641,515, 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety).
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as, for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Patent 5,629,001.
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, gel or sublingual orally-administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • a composition may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet, gel or solution form that may be placed under the tongue, along the gum line, brushed on to teeth surfaces, or otherwise dissolved in the mouth.
  • U.S. Patents 6,074,674 and 6,270,750 both incorporated by reference, describe topical, sustained release compositions for periodontal procedures.
  • Cathepsin L antagonist may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Patents 6,537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must 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 (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Methods and injectable compositions for bone repair are described in U.S. Patents 4,863,732, 5,531,791, 5,840,290, 6,281,195, 6,288,043, 6,485,754, 6,662,805 and 7,008,433, all of which are incorporated by reference.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • Cathepsin L inhibitors and pharmaceuticals for administration by routes discussed above, such inhibitors, alone or in combination, maybe used in the context of devices, such as implants.
  • a variety of bone related implants are contemplated, including dental implants, joint implants such as hips, knees, and elbows, vertebral/spinal implants, and others.
  • the Cathepsin L inhibitor may be impregnated in a surface of the implant, including in a bioactive matrix or coating.
  • the inhibitor may further formulated to sustained, delayed, prolonged or time release.
  • the coating may comprise polymers, for example, such as those listed below. The following is a list of U.S. patents relating to bone implants and devices which may be utilized in accordance with this embodiment of the invention:
  • Bone implant in particular, an inter-vertebral implant
  • Bone stabilization implant having a bone plate portion with integral cable clamping means
  • Bone Precursor Cells Human bone precursor cells are characterized as small-sized cells that express low amounts of bone proteins (osteocalcin, osteonectin, and alkaline phosphatase) and have a low degree of internal complexity (Long et ah, 1995). When stimulated to differentiate, these preosteoblast cells become osteoblast in their appearance, size, antigenic expression, and internal structure. Although these cells are normally present at very low frequencies in bone marrow, a process for isolating these cells has been described (Long et al, 1995).
  • U.S. Patent 5,972,703 further describes methods of isolating and using bone precursor cells, and is specifically incorporated herein by reference.
  • MSC mesenchymal stem cells
  • Preosteoblasts are intermediate between osteoprogenitor cells and osteoblasts.
  • The show increasing expression of bone phenotypic markers such as alkaline phosphatase (Kale et ah, 2000). They have a more limited proliferative capacity, but nonetheless continue to divide and produce more preosteoblasts or osteoblasts.
  • Osteoblasts are differentiated cells of the bone-forming cell lineage. They are large cells, possessing a eccentric nucleus, and produce of the extracellular proteins required for bone formation. They can be obtained from bone as populations of both preosteoblasts and osteoblasts as described in U.S. Serial No. 09/753,043, which is specifically incorporated herein by reference.
  • Serum-Free Media Cell culture may be employed using serum-free media for growth of bone- producing cells, and the subsequent conversion of stem cells into bone-forming osteogenic or osteoprogenitor cells.
  • serum-free media for growth of bone- producing cells, and the subsequent conversion of stem cells into bone-forming osteogenic or osteoprogenitor cells.
  • the following section describes attributes and conditions for using serum-free media. See also U.S. Patent 6,811,776.
  • serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells has been thoroughly reviewed (Barnes, 1987; Barnes & Sam, 1980; Broad et al, 1991; Jayme, 1991).
  • the list of the main additives which are used as supplements for serum-free media is summarized by Barnes (1987) and Barnes & Sam (1980).
  • Most commercially available serum-free media contain a carrier protein such as albumin. The presence of carrier protein might be required for protection of the cell viability.
  • serum free culture medium can be found in U.S. Patent 5,063,157, herein incorporated by reference.
  • the media comprises, in addition to the base medium, transferrin, insulin, a peptone, a ⁇ -D-xylopyranose derivative, selenite and a biological polyamine.
  • Another serum free cell growth medium for mammalian cells is disclosed in U.S. Patent 4,443,546.
  • This growth medium in addition to the basic medium, contains seven ingredients.
  • EPA 481 791 discloses a culture medium for CHO cells comprising water, an osmolality regulator, a buffer, an energy source, amino acids, an iron source, a growth factor and other optional components.
  • the two media exemplified contain 19 and 17 components, respectively. Examples of potential additives to serum free media follow below.
  • Albumin is preferably supplied in the form of bovine (BSA) or human serum albumin (HSA) in an effective amount for the growth of cells.
  • BSA bovine
  • HSA human serum albumin
  • Albumin provides a source of protein in the media.
  • Albumin is thought to act as a carrier for trace elements and essential fatty acids.
  • the albumin used in the present formulations is free of pyrogens and viruses, and when necessary, is approved regulatory agencies for infusion into human patients.
  • the HSA may be deionized using resin beads prior to use.
  • the concentration of human serum albumin is 1-8 mg/ml, preferably 3-5 mg/ml, most preferably 4 mg/ml.
  • Soluble Carrier/Fatty Acid Complex The albumin mentioned above could be substituted by a soluble carrier/essential fatty acid complex and a soluble carrier cholesterol complex which can effectively deliver the fatty acid and cholesterol to the cells.
  • An example of such a complex is a cyclodextrin/linoleic acid, cholesterol and oleic acid complex. This is advantageous as it would allow for the replacement of the poorly characterized albumin with a well defined molecule.
  • the use of cyclodextrin removes the need for the addition of human/animal serum albumin, thereby eliminating any trace undesired materials which the albumin would introduce into the media.
  • the use of cyclodextrin simplifies the addition of specific lipophilic nutrients to a serum-free culture.
  • the lipophilic substances which can be complexed with cyclodextrin include unsaturated fatty acids such as linoleic acid, cholesterol and oleic acid.
  • unsaturated fatty acids such as linoleic acid, cholesterol and oleic acid.
  • the linoleic acid, cholesterol and oleic acid are present in effective amounts and can be present in equal proportions such that the total amount is 0.001 to 100 ⁇ g/ml, preferably 0.1 to 10 ⁇ g/ml.
  • the preparation of such complexes is known in the art and is described, for example, in U.S. Patent 4,533,637, the entire contents of which is hereby incorporated by reference.
  • a source of iron in an effective amount and in a form that can be utilized by the cells can be added to the media.
  • the iron can be supplied by saturating transferrin, its carrier molecule, in an effective amount.
  • the transferrin may be derived from animal sera or recombinantly synthesized. It is understood that when transferrin is derived from an animal source, it is purified to remove other animal proteins, and thus is usually at least 99% pure.
  • the transferrin concentration is usually between 80 and 500 ⁇ g/ml, preferably between 120 and 500 ⁇ g/ml, more preferably between 130 and 500 ⁇ g/ml, even more preferably between 275 and 400 ⁇ g/ml and most preferably 300 ⁇ g/ml.
  • An iron salt preferably a water soluble iron salt, such as iron chloride (e.g., FeCIs-OH 2 O) dissolved in an aqueous solution such as an organic acid solution (e.g., citric acid) is used to supply the iron to transferrin.
  • iron chloride e.g., FeCIs-OH 2 O
  • organic acid solution e.g., citric acid
  • concentration of iron chloride is 0.0008 to 8 ⁇ g/ml, preferably 0.08 to 0.8 ⁇ g/ml, most preferably 0.08 ⁇ g/ml.
  • Insulin Growth Factor Insulin also may be added to the media of the present invention in an effective amount.
  • insulin like growth factor such as insulin like growth factor 1 and insulin like growth factor 2 may be used in place of or in addition to insulin in an amount which provides substantially the same result as a corresponding amount of insulin.
  • insulin growth factor includes both insulin and insulin like growth factors.
  • lipids lipids that are preferably not added unless they are necessary for a particular experiment or to grow a particular type of cell.
  • triglycerides and/or phospholipids may be included as additional sources of lipid.
  • a preferable source of lipid contains a mixture of neutral triglycerides of predominantly unsaturated fatty acids such as linoleic, oleic, palmitic, linolenic, and stearic acid. Such a preparation may also contain phosphatidylethanolamine and phosphatidylcholine.
  • Another source of lipid is a human plasma fraction precipitated by ethanol and preferably rendered virus free by pasteurization.
  • the media When the media is to be used to grow cells for introduction into a human patient, the media preferably does not contain ingredients such as bovine serum albumin, mammalian serum, and/or any natural proteins of human or mammalian origin (as explained above). It is preferable that recombinant or synthetic proteins, if they are available and of high quality, are used. Most preferably, the amino acid sequences of the recombinant or synthetic proteins are identical to or highly homologous with those of humans.
  • the most preferable serum-free media formulations herein contain no animal-derived proteins and do not have even a non-detectable presence of animal protein.
  • optional components which are not necessary are preferably not added to the medium.
  • Such optional components are described in the prior art cited above and may be selected from the group consisting of meat extract, peptone, phosphatidylcholine, ethanolamine, anti-oxidants, deoxyribonucleosides, ribonucleosides, soy bean lecithin, corticosteroids, myoinositol, monothioglycerol, and bovine or other animal serum albumin.
  • bone cells stimulated with Cathepsin L antagonists and/or second agents as discussed above of the present invention may be used for autologous or heterologous transplant into subjects in need thereof.
  • supports will be used to provide a scaffold or matrix over with bone cells and grown and deposit bone.
  • polymeric materials including in vivo medical applications. These materials are well suited to implantation as they can serve as a temporary scaffold to be replaced by host tissues, degrade by hydrolysis to non-toxic products, and be excreted, as described by Kulkarni et al. (1971) and Hollinger and Battistone (1986).
  • Either natural or synthetic polymers can be used to form the matrix, although synthetic polymers are preferred for reproducibility and controlled release kinetics.
  • Synthetic polymers that can be used include bioerodible polymers such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and other poly(alpha-hydroxy acids), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes, and non-erodible polymers such as polyacrylates, ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly( vinyl imidazole), chlorosulphonated polyolifms, polyethylene oxide, polyviny
  • non-degradable materials can be used to form the matrix or a portion of the matrix, they are not preferred.
  • natural polymers include proteins such as albumin, fibrin or fibrinogen, collagen, synthetic polyamino acids, and prolamines, and polysaccharides such as alginate, heparin, and other naturally occurring biodegradable polymers of sugar units.
  • PDS poly(paradioxanone)
  • PLA poly(lactic acid)
  • PGA poly(glycolic acid)
  • PLAGA copolymers. Copolymerization enables modulation of the degradation time of the material. By changing the ratios of crystalline to amorphous polymers during polymerization, properties of the resulting material can be altered to suit the needs of the application.
  • These polymers, including poly(lactide-co-glycolic) acid (PLGA) have been used as polymer composites for bone replacement as reported by Elgendy et al. (1993). Substituted polyphosphazenes have been shown to support osteogenic cell growth, as reported by Laurencin et al. (1993).
  • Poly(organophosphazenes) are high molecular weight polymers containing a backbone of alternating phosphorus and nitrogen atoms.
  • polyphosphazenes each derived from the same precursor polymer, poly(dichlorophosphazene).
  • the chlorine-substituted species can be modified by replacement of the chlorine atoms by different organic nucleophiles such as o-methylphenoxide along with amino acids.
  • the physical and chemical properties of the polymer can be altered by adding various ratios of hydrolytic sensitive side chains such as ethyl glycinate, as described by Wade et al. (1978) and Allcock and Fuller (1981). This will affect the degradation of the polymer as an implantable and biodegradable material as well as vary the support of osteogenic cells for bone and tissue implants, as shown by Laruencin et al. (1993).
  • PLA, PGA and PLA/PGA copolymers are particularly useful for forming the biodegradable matrices.
  • PLA polymers are usually prepared from the cyclic esters of lactic acids. Both L(+) and D(-) forms of lactic acid can be used to prepare the PLA polymers, as well as the optically inactive DL-lactic acid mixture of D(-) and L(+) lactic acids.
  • Methods of preparing polylactides are well documented in the patent literature. The following U.S. Patents, the teachings of which are hereby incorporated by reference, describe in detail suitable polylactides, their properties and their preparation: U.S.
  • PGA is the homopolymer of glycolic acid (hydroxyacetic acid).
  • glycolic acid hydroxyacetic acid
  • glycolic acid is initially reacted with itself to form the cyclic ester glycolide, which in the presence of heat and a catalyst is converted to a high molecular weight linear-chain polymer.
  • PGA polymers and their properties are described in more detail in "Cyanamid Research Develops World's First Synthetic Absorbable Suture,” Chemistry and Industry, 905 (1970).
  • the erosion of the matrix is related to the molecular weights of PLA, PGA or PLA/PGA.
  • Poly(lactide-co-glycolide) (50:50) degrades in about six weeks following implantation.
  • All polymers for use in the matrix must meet the mechanical and biochemical parameters necessary to provide adequate support for the cells with subsequent growth and proliferation.
  • the polymers can be characterized with respect to mechanical properties such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC) and bond structure by infrared (IR) spectroscopy, with respect to toxicology by initial screening tests involving Ames assays and in vitro teratogenicity assays, and implantation studies in animals for immunogenicity, inflammation, release and degradation studies. These polymers are particularly useful in forming fibrous or sponge type matrices for implantation. Polymers can also be used to form hydrogels in which the cells are suspended and then implanted.
  • Matrix Materials Another class of materials for making the matrix is hydroxyapatite, or a similar ceramic formed of tricalcium phosphate (TCP) or calcium phosphate (CaPO 4 ). Calcium hydroxyapatites occur naturally as geological deposits and in normal biological tissues, principally bone, cartilage, enamel, dentin, and cementum of vertebrates and in many sites of pathological calcifications such as blood vessels and skin.
  • TCP tricalcium phosphate
  • CaPO 4 calcium phosphate
  • Synthetic calcium hydroxyapatite is formed in the laboratory either as pure Ca 1O (PO 4 ) 6 (OH) 2 or hydroxyapatite that is impure, containing other ions such as carbonate, fluoride, chloride for example, or crystals deficient in calcium or crystals in which calcium is partly or completely replaced by other ions such as barium, strontium and lead.
  • other ions such as carbonate, fluoride, chloride for example
  • the crystals of pure synthetic apatites, geological apatites and many impure synthetically produced apatites are larger and more crystalline than the biological crystals of bone, dentin, cementum and cartilage.
  • the crystals of bone, dentin and cementum are very small, irregularly shaped, very thin plates whose rough average dimensions are approximately 10 to 50 angstroms in thickness, 30 to 150 angstroms in width, and 200 to 600 angstroms in length.
  • the synthetic materials are highly diverse, as reported in the literature. For example, the characterization of four commercial apatites was reported by Pinholt et al (1992); Marden et al (1990) reports on a protein, biodegradable material; Pinholt et al.
  • hydroxyapatite As used herein, all of these materials are generally referred to as "hydroxyapatite.”
  • the hydroxyapatite is particles having a diameter between approximately ten and 100 ⁇ m in diameter, most preferably about 50 ⁇ m in diameter.
  • Another form of hydroxyapatite scaffold is devitalized coral as a scaffold. Le Guehennec et al (2004); Chen et al (2004); Devecioglu et al (2004); Kujala et al (2004).
  • Calcium phosphate ceramics can be used as implants in the repair of bone defects because these materials are non-toxic, non-immunogenic, and are composed of calcium and phosphate ions, the main constituents of bone (Frame, 1987; Parsons et al, 1988).
  • TCP tricalcium phosphate
  • HA hydroxyapatite
  • Ca 1O (PO 4 ) 6 OH 2
  • Calcium phosphate ceramics have a degree of bioresorb ability which is governed by their chemistry and material structure. High density HA and TCP implants exhibit little resorption, while porous ones are more easily broken down by dissolution in body fluids and resorbed by phagocytosis. However, TCP degrades more quickly than HA structures of the same porosity in vitro. HA is relatively insoluble in aqueous environments. However, the mechanical properties of calcium phosphate ceramics make them ill-suited to serve as a structural element under load bearing circumstances. Ceramics are not preferred since they are brittle and have low resistance to impact loading.
  • Polymers that can form ionic hydrogels which are malleable can also be used to support the cells. Injecting a suspension of cells in a polymer solution may be performed to improve the reproducibility of cell seeding throughout a device, to protect the cells from shear forces or pressure induced necrosis, or to aid in defining the spatial location of cell delivery.
  • the injectable polymer may also be utilized to deliver ells and promote the formation of new tissue without the use of any other matrix.
  • the hydrogel is produced by cross-linking the ionic salt of a polymer with ions, whose strength increases with either increasing concentrations of ions or polymer.
  • the polymer solution is mixed with the cells to be implanted to form a suspension, which is then injected directly into a patient prior to polymerization of the suspension.
  • the suspension subsequently polymerizes over a short period of time due to the presence in vivo of physiological concentrations of ions such as calcium in the case where the polymer is a polysaccharide such as alginate.
  • a hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three- dimensional open-lattice structure which entraps water molecules to form a gel.
  • materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazenes, and polyacrylates such as hydroxyethyl methacrylate (HEMA), which are cross-linked ionically, or block copolymers such as PluronicsTM or TetronicsTM, polyethylene oxide-polypropylene glycol block copolymers which are cross-linked by temperature or pH, respectively.
  • HEMA hydroxyethyl methacrylate
  • PluronicsTM or TetronicsTM polyethylene oxide-polypropylene glycol block copolymers which are cross-linked by temperature or pH, respectively.
  • Other materials include proteins such as fibrinogen, collagen, polymers such as polyvinylpyrrolidon
  • these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof.
  • aqueous solutions such as water, buffered salt solutions, or aqueous alcohol solutions
  • polymers with acidic side groups that can be reacted with cations are poly(phosphazenes), poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate), and sulfonated polymers, such as sulfonated polystyrene.
  • Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used.
  • Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups, and acidic OH groups.
  • Examples of polymers with basic side groups that can be reacted with anions are poly(vinyl amines), poly(vinyl pyridine), poly(vinyl imidazole), and some imino substituted polyphosphazenes.
  • the ammonium or quaternary salt of the polymers can also be formed from the backbone nitrogens or pendant imino groups.
  • Examples of basic side groups are amino and imino groups.
  • Alginate can be ionically cross-linked with divalent cations, in water, at room temperature, to form a hydrogel matrix. Due to these mild conditions, alginate has been the most commonly used polymer for hybridoma cell encapsulation, as described, for example, in U.S. Patent 4,352,883. Described therein is an aqueous solution containing the biological materials to be encapsulated is suspended in a solution of a water soluble polymer, the suspension is formed into droplets which are configured into discrete microcapsules by contact with multivalent cations, then the surface of the microcapsules is cross-linked with polyamino acids to form a semipermeable membrane around the encapsulated materials.
  • the polyphosphazenes suitable for cross-linking have a majority of side chain groups which are acidic and capable of forming salt bridges with di- or trivalent cations.
  • preferred acidic side groups are carboxylic acid groups and sulfonic acid groups.
  • Hydro lyrically stable polyphosphazenes are formed of monomers having carboxylic acid side groups that are cross-linked by divalent or trivalent cations such as Ca + or Al + . Polymers can be synthesized that degrade by hydrolysis by incorporating monomers having imidazole, amino acid ester, or glycerol side groups.
  • Bioerodible polyphosphazenes have at least two differing types of side chains, acidic side groups capable of forming salt bridges with multivalent cations, and side groups that hydrolyze under in vivo conditions, e.g., imidazole groups, amino acid esters, glycerol and glucosyl.
  • the water soluble polymer with charged side groups is cross-linked by reacting the polymer with an aqueous solution containing multivalent ions of the opposite charge, either multivalent cations if the polymer has acidic side groups or multivalent anions if the polymer has basic side groups.
  • the preferred cations for cross-linking of the polymers with acidic side groups to form a hydrogel are divalent and trivalent cations such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, although di-, tri- or tetra-functional organic cations such as alkylammonium salts can also be used.
  • Aqueous solutions of the salts of these cations are added to the polymers to form soft, highly swollen hydrogels and membranes.
  • the preferred anions for cross-linking of the polymers to form a hydrogel are divalent and trivalent anions such as low molecular weight dicarboxylic acids, for example, terepthalic acid, sulfate ions and carbonate ions. Aqueous solutions of the salts of these anions are added to the polymers to form soft, highly swollen hydrogels and membranes, as described with respect to cations.
  • polycations can be used to complex and thereby stabilize the polymer hydrogel into a semi-permeable surface membrane.
  • materials that can be used include polymers having basic reactive groups such as amine or imine groups, having a preferred molecular weight between 3,000 and 100,000, such as polyethylenimine and polylysine. These are commercially available.
  • One polycation is poly(L-lysine)
  • examples of synthetic polyamines are polyethyleneimine, poly(vinylamine), and poly(allyl amine).
  • polysaccharide, chitosan There are also natural polycations such as the polysaccharide, chitosan.
  • Polyanions that can be used to form a semi-permeable membrane by reaction with basic surface groups on the polymer hydrogel include polymers and copolymers of acrylic acid, methacrylic acid, and other derivatives of acrylic acid, polymers with pendant SO 3 H groups such as sulfonated polystyrene, and polystyrene with carboxylic acid groups.
  • the present invention provides methods identifying new and useful inhibitors of Cathepsin L for use in stimulating bone production.
  • These assays may comprise random screening of large libraries of candidate substances; alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to modulate the function of Cathepsin L.
  • a method generally comprises:
  • step (d) comparing the characteristic measured in step (c) with that observed in the absence of the candidate modulator, wherein a difference between the measured characteristic indicates that said candidate modulator is, indeed, a modulator of Cathepsin L.
  • Assays may be conducted in cell free systems, in isolated cells, or in organisms including transgenic animals. Bone formation can be identified by the von Koassa or Alzarin Red stains, FTIR or Raman spectrometric analysis, or by fluorochromes linked to compounds that bind bone.
  • U.S. Patent 6,346,373 describes a whole cell based assay for Cathepsin K activity. It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them. VII. Examples
  • Cathepsin K this cysteine protease plays a role in bone formation.
  • Cathepsin L antagonists stimulate osteoblast development and the formation of bone.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Bossard et al J. Biol. Chem., 271(21): 12517-12524, 1996. Broad et al, Cytotechnology 5:47-55, 1991.

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Abstract

La présente invention concerne la modulation d'une enzyme protéolytique, la cathepsine L qui entraîne une production osseuse accrue par la production de cellules formatrices d'os. Des médicaments qui de préférence ciblent cette enzyme dans des cellules formatrices d'os et inhibent son activité représentent une nouvelle classe de thérapies anaboliques, ou de construction osseuse. De tels médicaments sont utiles dans le traitement de maladies ou troubles tels que l'ostéoporose, la maladie de Paget, le cancer métastatique de l'os, la maladie osseuse de type myélome, les fractures de l'os et analogues.
PCT/US2008/086326 2007-12-12 2008-12-11 Utilisation d'antagonistes de la cathepsine l dans le traitement de maladie des os WO2009076490A1 (fr)

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US5607691A (en) * 1992-06-12 1997-03-04 Affymax Technologies N.V. Compositions and methods for enhanced drug delivery
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US5607691A (en) * 1992-06-12 1997-03-04 Affymax Technologies N.V. Compositions and methods for enhanced drug delivery
US5955491A (en) * 1993-02-19 1999-09-21 Takeda Chemical Industries, Ltd. Method for treating osteoporosis
US5830850A (en) * 1996-08-28 1998-11-03 Mount Sinai School Of Medicine Of The City Of New York Methods for the treatment of bone resorption disorders, including osteoporosis
US6060310A (en) * 1997-11-24 2000-05-09 The United States Of America As Represented By The Department Of Health And Human Services Transcription factor decoy and tumor growth inhibitor
US6287857B1 (en) * 1998-02-09 2001-09-11 Genzyme Corporation Nucleic acid delivery vehicles
US20030203919A1 (en) * 2000-02-10 2003-10-30 Martin Missbach Dipeptide nitrile cathepsin K inhibitors
US20070123483A1 (en) * 2001-02-14 2007-05-31 Shi Guo-Ping Method and composition for treating obesity by targeting cathespin
US20070014788A1 (en) * 2004-12-13 2007-01-18 Novo Nordisk A/S Laminin-5 modulators and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023141237A1 (fr) * 2022-01-20 2023-07-27 Cell and Molecular Tissue Engineering, LLC Méthodes et produits pour détecter, minimiser et traiter des réactions tissulaires associées à des trap et une lésion tissulaire associée à des dispositifs médicaux

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