METHOD OF TREATING PATHOLOGIC HETEROTOPIC OSSIFICATION
What is described is a method of preventing or treating heterotopic ossification, vascular calcification, and pathologic mineralization, comprising administering an drug, wherein the drug is an antagonist of the Hedgehog (Hh) pathway. For example the antagonist consists of arsenic trioxide, sodium arsenite, phenylarsine, GANT-58, GANT-61, zerumbone vismodegib (GDC-0449, Genentech), bevacizumab, gemcitabine, nab-paclitaxel, FOLFIR, FOLFOX, RO4929097, cixutumumab, cisplatin, etoposide, LDAC, decitabine, daunorubicin, cytarabin, rosiglitazone, goserelin, leuprolide, capecitabine, fluorouracil, leucovorin, oxaliplatin, irinotecan, diclofenac, BMS-833923 (XL139), IPI-926—Infinity Pharmaceuticals, Inc., LDE225, LEQ506—Novartis Pharmaceuticals, TAK-441 Millennium Pharmaceuticals, Inc., and PF-04449913—Pfizer, alone or in combination therapy. The method targets pluripotent mesenchymal cells, wherein the antagonist inhibits expression of a gene encoding a Hh pathway component, for example by decreasing levels of mRNA encoded Ptch1, Gli1 or HIP.
|Compositions and methods for preventing and treating heterotopic ossification and pathologic calcification|
Patent #US 10,456,409 B2
Current AssigneeNostopharma LLC
Sponsoring EntityNostopharma LLC
|Compositions and methods for preventing and treating heterotopic ossification and pathologic calcification|
Patent #US 10,548,908 B2
Current AssigneeNostopharma LLC
Sponsoring EntityNostopharma LLC
- 1. A method of preventing or treating heterotopic ossification comprising administering a drug, wherein the drug is an antagonist of the Hedgehog pathway.
- 3-5. -5. (canceled)
- 7-8. -8. (canceled)
- 13-17. -17. (canceled)
- 18. A method of inhibiting formation of heterotopic ossification comprising administering an antagonist of the Hedgehog pathway.
- 20-21. -21. (canceled)
- 25-28. -28. (canceled)
- 29. A method of treating heterotopic ossification (HO), vascular calcification, or pathologic mineralization using an antagonist of the Hedgehog pathway, comprising administering said antagonist to a subject in need thereof.
- 32. (canceled)
- 33. A method of using an antagonist of the Hedgehog pathway, comprising exposing mesenchymal cells to said antagonist to prevent activation of said cells.
- 35-40. -40. (canceled)
This applications claims the benefit of U.S. Provisional Application No. 61/504,041 filed Jul. 1, 2011, the contents of which are hereby incorporated by reference in its entirety.
What is described are methods of using antagonists of the Hedgehog pathway to treat heterotopic ossification, vascular calcification, or pathologic mineralization, and to prepare medicaments for treating these diseases.
Heterotopic ossification (HO) can result from osteoid formation of mature lamellar bone in soft tissue sites outside the skeletal periosteum (skeletal system). HO most commonly occurs around proximal limb joints. This osteoid formation often is associated with an inflammatory phase characterized by local swelling, pain, erythema and sometimes fever. This pathological process may occur in sites such as the skin, subcutaneous tissue, skeletal muscle, and fibrous tissue adjacent to joints. Bone may also form in walls of blood vessels as well as in ligaments. Lesions range from small clinically insignificant foci to massive deposits throughout the body.
HO presents rarely as a hereditary disorder, and is sometimes associated with lower motor neuron disorders. More commonly it is associated with spinal cord injury, trauma and brain injuries, burns, fractures, muscle contusion, and joint arthroplasty. HO is a severe complication of hip surgery, acetabular and elbow fracture surgery. It may occur in patients who are on neuromuscular blockade to manage adult respiratory distress syndrome, and in patients with nontraumatic myelopathies. Following combat-related trauma, for example amputation, HO is a frequent occurrence and a common problem. HO may result in joint contracture and ankylosis, pain, spasticity, swelling fever, neurovascular compression, pressure ulcers, and significant disability.
HO can also be caused by genetic diseases such as progressive osseous heteroplasia (POH; MIM #166350) and Fibrodysplasia Ossificans Progressiva (FOP; MIM #135100). POH is associated with inactivating mutation in the GNAS gene, which encodes Gαs, the alpha subunit of the stimulatory guanine nucleotide binding protein that acts downstream of many G protein-coupled receptors in activating adenylyl cyclase (Kaplan, et al. 1994, J Bone Joint Surg Am 76, 425-436; Shore, et al., 2002, N Engl J Med 346, 99-106; and Eddy, et al., 2000, J Bone Miner Res 15, 2074-2083). Patients with inactivating mutations in GNAS can also suffer from Albright'"'"'s hereditary osteodystrophy (AHO) when the genetic mutations are inherited from the mother. Clinically, POH presents during infancy with dermal and subcutaneous ossifications that progress during childhood into skeletal muscle and deep connective tissues (e.g. tendon, ligaments, fascia). Over time these ossifications lead to joint stiffness, bone and joint fusions and growth retardation of the affected limbs. Currently, patients with POH undergo aggressive surgical resection of ectopic bone to abrogate spreading of the lesion. This often results in partial or full amputation of limbs and lesions frequently return (Kaplan, et al., 2000, J Bone Miner Res 15, 2084-94; and Shehab, et al., 2003, J Nucl Med 43, 346-353), which underscores the importance of developing improved therapeutic interventions. Observations made in patients with POH suggest that mesenchymal stem cells present in soft tissues inappropriately differentiate into osteoblasts and begin to deposit bone. Due to a lack of both in vitro and in vivo animal models, the pathogenesis of POH remains unknown and, like all forms of HO, lacks adequate treatments.
During HO there must be an inciting event, usually an episode of trauma which may result in hematoma. There is usually a signal from the site of injury, suggested to be bone morphogenetic protein(s). For HO progression, there must be a supply of pluripotent (multipotent) mesenchymal cells, which can differentiate into osteoblasts or chondroblasts, and an environment conducive to the continued production of heterotopic bone. A mouse model of FOP expressing a strong constitutively active ALK2 R206H mutant, was found to be useful in identifying a selective agonist to nuclear retinoic acid receptor-α (RAR-α) in mesenchymal cells. RAR-α agonists were found to partly inhibit HO, while an agonist to RAR-γ was found to be a potent inhibitor of intramuscular and subcutaneous HOin FOP models (Shimono et al., 2011, Nature Medicine 17:454-60).
HO in FOP occurs through endochondral ossification mechanism where cartilage formation precedes osteoblast differentiation whereas HO in POH occurs through intramembranous ossification where osteoblasts differentiate directly from mechenchymal progenitor cells. Non-genetic forms of HO occur through mechanisms of both endochondral and intramembranous ossification. Therefore, understanding the molecular and cellular mechanisms underlying POH will contribute significantly to our understanding of HO, which is essential in finding methods for treating HO, a pressing need that can only be met by a therapeutic that targets potential signalling pathways associated with this disease.
One aspect of the invention is a method of preventing or treating HO comprising administering a drug, wherein the drug is an antagonist of the Hedgehog pathway. The antagonist is selected from the group consisting of zerumbone epoxide, staurosporinone, 6-hydroxystaurosporinone, arcyriaflavin C, 5,6-dihyroxyarcyriaflavin A, physalin F, physalin B, cyclopamine, HPI-1, HPI-2, HPI-3, and HPI-4, arsenic trioxide (ATO), sodium arsenite, phenylarsine, GANT-58, GANT-61, and zerumbone. Preferably the antagonist is an arsenic compound, most preferably ATO. The antagonist may be administered by injection, preferably by an infusion. If the antagonist is ATO, the preferred dosage ranges between 0.05 to 0.20 mg/kg/day. The antagonist may be administered orally. An embodiment of the invention is a method whereby the antagonist targets pluripotent mesenchymal cells, preferably to prevent proliferation or differentiation of the mesenchymal cells.
Another embodiment of the invention is the method of treating HO in which the antagonist alters expression of a gene expressed in the mesenchymal cells, preferably a gene that encodes a component of the Hedgehog pathway. These genes may be selected from a gene family consisting of Hh, PTCH, GLI, and SMO, preferably a gene is selected from the group consisting of Shh, Dhh, Ihh, Ptch1, Ptch2, Gli1, Gli2, Gli3, and Smo. Preferably, the antagonist inhibits expression of the gene, including decreasing levels of mRNA encoded by the gene, particularly Ptch1, Gli1 or HIP.
Another aspect of the invention is a method of inhibiting formation of heterotopic ossification comprising administering an antagonist of the Hedgehog pathway, particularly to a subject that is susceptible to HO. The subject is preferably a mammal, most preferably a human patient. Such patients include those who experienced trauma, including spinal cord injury, trauma and brain injuries, burns, fractures, muscle contusion, joint arthroplasty, lower motor neurone disorders, hereditary disorders, or combat-related trauma. HO amenable to treatment may be diagnosed by computed tomography, bone scintigraphy, ultrasonography, or X-radiography.
Another aspect of the invention is use of an antagonist of the hedgehog pathway for preparing a medicament for treating HO, vascular calcification, or pathologic mineralization. The antagonist is selected from the group consisting of zerumbone epoxide, staurosporinone, 6-hydroxystaurosporinone, arcyriaflavin C, 5,6-dihyroxyarcyriaflavin A, physalin F, physalin B, cyclopamine, HPI-1, HPI-2, HPI-3, and HPI-4, ATO, sodium arsenite, phenylarsine, GANT-58, GANT-61, and zerumbone.
Another aspect of the invention is a method of using an antagonist of the Hedgehog pathway, comprising administering said antagonist to a subject in need thereof to prevent or treat HO, vascular calcification, or pathologic mineralization. The amount of said antagonist administered to the subject are sufficient to reduce levels of HO in the subject.
Another aspect of the invention is a method of using an antagonist of the Hedgehog pathway, comprising exposing mesenchymal cells to said antagonist to prevent activation of said cells. In an embodiment of this method the antagonist prevents proliferation or differentiation of mesenchymal cells. In another embodiment, mesenchymal cell activation results in increased expression of a gene encoding a component of the Hedgehog pathway. The gene is selected from a gene family consisting of Hh, PTCH, GLI, and SMO, specifically, a gene is selected from the group consisting of Shh, Dhh, Ihh, Ptch1, Ptch2, Gli1, Gli2, Gli3, and Smo. In another embodiment, the antagonist inhibits expression of the gene, preferably by decreasing levels of mRNA encoded by the gene, most preferably wherein the gene is Ptch1, Gli1 or HIP.
What is described in a method of using antagonists of the Hedgehog pathway to treat HO, vascular calcification, and/or pathologic mineralization.
The term “heterotopic ossification” refers to the frequent sequela of central nervous system injury. It is encountered in certain embodiments, in cases of spinal cord injury, head injury, cerebrovascular accident and burns. In one embodiment, neurogenic heterotopic ossification is not associated with local trauma. Osseous trauma is associated with an increased incidence of heterotopic ossification distal to the trauma site, or due to the extent of the original cerebral injury in other embodiment. In one embodiment, the onset of heterotopic ossification may be as early as two weeks postinjury and patients remain susceptible to its onset through the first nine months after injury. In one embodiment, alkaline phosphatase level is raised in the presence of calcium deposition, with the development of heterotopic ossification preceding the elevation of serum alkaline phosphatase. In one embodiment, the hip appears to be the most common site of heterotopic ossification formation, occurring with almost equal frequency in the upper extremities and at both the elbow and the shoulder from craniocerebral injury.
Genetic diseases fibrodysplasia ossificans progressive (FOP) and progressive osseous heterplasia (POH) the most severe manifestations of heterotopic bone formation. FOP occurs rarely and is a result of a mutation in ACVR1, which encodes a bone morphogenetic protein type I receptor. Patients with POH have inactivating mutations of the GNAS gene, which also can give rise to Albright'"'"'s hereditary osteodystrophy (AHO) when the mutations are inherited from the mother.
Myositis ossifican circumscripta is characterized by the intramuscular proliferation of fibroblasts, new bone, and/or cartilage.
The earliest opportunity for acute treatment, HO can be reliably diagnosed by computed tomography, bone scintigraphy and ultrasonography. Two to six weeks later plain radiography can detect it. Bony maturation occurs within six months.
Conventional treatment usually involves non-steroidal anti-inflammatory drugs (indomethecin, rofecoxib), or bisphosphonate (etidronate, pamidronate), Coumadin/warfarin, salicylates, and/or local radiation are administered. Often, surgery is the only option for treatment.
Outcome of treatment can be measured by standard radiological grading system for HO, changes in range of motion in the affected joint measured by goniometry, mean length of time to objective improvement of HO-related clinical symptoms or signs, changes in standardized functional or joint-specific measures
What is meant by “vascular calcification,” or equivalently vascular ossification calcification (VOC), and “pathologic mineralization” is the result of deposition of calcium salts in the neointima of atheromatous plaques or in the media of vascular beds. Vascular smooth muscle cells (VSMCs) become multipotent mesenchymal cells that can transform into osteocytic- or chondrocytic-like cells. VOC and pathological mineralization are major risk factors for cardiovascular morbidity and mortality. Clinically there is a need to develop therapies to prevent calcification in situations of atherosclerosis, chronic kidney disease, type II diabetes, especially in hemodialysis patients. A chronic inflammatory state is commonly associated with VOC in these situations. VOC can usually be detected by X-ray or CT.
Apoptosis and vesicle release from VSMCs are crucial initiating events in VOC. Inflammatory cytokines may play an initiating role. Following the initiating even, a number of secondary responses can give rise to VOC, including hypercalcemia, hyperphosphatemia, oxidative stress, and aging. BMP, osteoprotegerin (OPG) and other osteogenic signalling pathways are also implicated in development of VOC, just as these pathways are central in HO. Evidence of OPG-mediated receptor activation of RANK and RANKL have been implicated in VOC. VOC is treated in a variety of ways. Antihypertensive agents have been implicated in control of VOC, nifedipine for example.
What is meant by “Hedgehog pathway” refers to Hedgehog (Hh) signal transduction. This pathway is initiated by the induction of the Hh precursor protein (45 kDa) in Hh-secreting cells, after which the precursor undergoes autocatalytic processing and modification. The precursor is cleaved to a 20 kDa N-terminal signal domain and a 25 kDa C-terminal catalytic domain. Subsequently, a cholesterol molecule is bound covalently to the carboxy terminus of the N-terminal domain, which is then secreted from the cytosol as a Hh ligand. On the surface of Hh-receiving cells there are two proteins of the pathway. One is Patched (Ptch), a twelve-pass transmembrane protein, interacts with the Hh ligand and the other is Smoothened (Smo), a seven pass transmembrane protein that is a signal transducer. In the absence of Hh ligands, Ptch interacts with Smo to inhibit its function and prevent activation of the downstream signaling cascade. Once the Hh ligand binds to Ptch along with Hh-interacting protein, Smo inhibition is released; this results in the activation of a downstream signaling cascade. This activation results in the release of a transcriptional factor GLI from a macromolecular complex on microtubules that includes the suppressor of fused, fused protein kinas A, GLI and possibly other components. GLI enters the nucleus and alters transcription of several genes, including those of the Hedgehog pathway. In vertebrates, Hh signaling activation requires cilium, a microtube based cell organelle.
“Antagonists of the Hedgehog pathway” refers to one or more molecules known to inhibit the Hedgehog family, including zerumbone epoxide, staurosporinone, 6-hydroxystaurosporinone, arcyriaflavin C, 5,6-dihyroxyarcyriaflavin A, physalin F, physalin B, cyclopamine, HPI-1, HPI-2, HPI-3, and HPI-4, arsenic trioxide (ATO), sodium arsenite, phenylarsine, GANT-58, GANT-61, and zerumbone (Kim et al., 2010, PNAS, 107:13432-37; Beauchamp et al. 2011, JCI 121:148-60; Lauth et al 2007, PNAS 104:8455-60; Hosoya et al., 2008, ChemBioChem 9:1082-92; Hyman et al, 2009, 106:14132-37; and Mas et al, 2010, Biochem. Pharm. 80:712-23, all hereby incorporated by reference in their entirety). Antagonists of the Hedgehog pathway also refers to drugs that are in clinical trials, including vismodegib (GDC-0449, Genentech), bevacizumab, gemcitabine, nab-paclitaxel, FOLFIR, FOLFOX, RO4929097, cixutumumab, cisplatin, etoposide, LDAC, decitabine, daunorubicin, cytarabin, rosiglitazone, goserelin, leuprolide, capecitabine, fluorouracil, leucovorin, oxaliplatin, irinotecan, diclofenac, BMS-833923 (XL139), IPI-926—Infinity Pharmaceuticals, Inc., LDE225, LEQ506—Novartis Pharmaceuticals, TAK-441 Millennium Pharmaceuticals, Inc., and PF-04449913—Pfizer, alone or in combination therapy. Inhibitors of cilium formation can also be used as Hh inhibitors.
As used herein, “arsenic compound” refers to a pharmaceutically acceptable form of arsenic trioxide (As203) or melarsoprol. Melarsoprol is an organic arsenic compound which can be synthesized by complexing melarsen oxide with dimercaprol or commercially purchased (Arsobal® by Rhone Poulenc Rorer, Collegeville, Pa.). Since the non-pharmaceutically formulated raw materials of the invention are well known, they can be prepared from well-known chemical techniques in the art. (See for example, Kirk-Othmer, Encyclopedia of Chemical Technology 4th ed. volume 3 pps. 633-655 John Wiley & Sons).
The arsenic compounds of the invention may be formulated into sterile pharmaceutical preparations for administration to humans for treatment of HO or VOC. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, packaged, labeled for treatment of and used for the treatment of the indicated leukemia, lymphoma, or solid tumor.
In one aspect, the invention provides a method for the manufacture of a pharmaceutical composition comprising a therapeutic effective and non-lethal amount of arsenic trioxide (As203). Arsenic trioxide (raw material) is a solid inorganic compound that is commercially available in a very pure form. However, it is difficult to dissolve As203 in aqueous solution. Arsenic is present in solution in the +5 valence state (pentavalent) or the +3 valence state (trivalent). For example, potassium arsenite (KAs02; which is present in Fowler'"'"'s solution) and salts of arsenious acid contain pentavalent arsenic. It is known that one form of arsenic is more toxic than the other. (Goodman & Oilman'"'"'s The Pharmacological Basis of Therapeutics, 9th edition, chapter 66, 1660, 1997). A fresh solution of arsenic trioxide containing arsenic in the trivalent state will be gradually oxidized to pentavalent state if exposed to air for a prolonged period, and as a result of the accumulation of pentavalent arsenic, the relative toxicity of a solution of As203 will change over time. (Id.) Furthermore, it is observed that the total amount of arsenic in solution decreases over time. This loss of material is caused by the progressive conversion of arsenic in the solution into arsine (AsH3) which is a gaseous compound at room temperature. This is particularly problematic in pharmaceutical applications if the concentration of an active ingredient in the injected material cannot be controlled. It is also undesirable to allow arsine to escape from the solution into the atmosphere because arsine is also toxic.
An arsenic solution may be obtained using methods know in the art, for example by solubilizing solid high purity As203 in an aqueous solution at high pH using mechanical stirring and/or gentle heating, or by dissolving the solid compound overnight. Typically, a solution of 1 M As203 is obtained. To adjust the pH of the As203 solution, the solution may be diluted in water and the resulting solution neutralized with an acid such as hydrochloric acid, with constant stirring until the pH is about 8.0 to 8.5. The partially neutralized As203 solution may then be sterilized for example, by filtration (e.g., through a 0.22 μm filter), and stored in sterile vials.
To make a pharmaceutical composition that can be directly injected into a subject, the composition must be sterile, standard techniques known to the skilled artisan for sterilization can be used. See, e.g., Remington'"'"'s Pharmaceutical Science. The pH of the partially neutralized As203 solution may be further adjusted to near physiological pH by dilution (10-100 fold) with a pharmaceutical carrier, such as a 5% dextrose solution. For example, 10 mL of a partially neutralized As203 solution can be added to 500 mL of a 5% dextrose solution to yield a final pH of about 6.5 to 7.5. The method of the invention reduces the oxidation of arsenic in solution. Pharmaceutical compositions containing arsenic trioxide manufactured by the method of the invention show improved stability and long shelf life.
The active ingredients of the invention are formulated into pharmaceutical preparations (e.g., together in a composition or separately to be used in a combination therapy) for administration to mammals for treatment of HO and/or VOC.
The following table provides a list of Hedgehog pathway antagonists, with dosage appropriate for current use of the drug in clinical trials for treating cancer. The skilled worker could adjust the dosage regime and mode of administration according to the status of the patient being treated for HO.
For oral administration of arsenic compounds, the pharmaceutical preparation can be in liquid form, for example, solutions, syrups or suspensions, or can be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well-known in the art.
Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For oral administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The therapeutic agents consisting of arsenic compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Such formulations are sterile. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as emulsion in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
The pharmaceutical preparations can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.
Any suitable mode of administration may be used in accordance with the present invention including but not limited to parenteral administration such as intravenous, subcutaneous, intramuscular and intrathecal administration; oral, and intranasal administration, and inhalation. The mode of administration will vary according to the degree of HO or VOC, and the condition of the human.
The pharmaceutical compositions to be used may be in the form of sterile aqueous or organic solutions; colloidal suspensions, caplets, tablets and cachets.
In accordance with the present invention, arsenic trioxide or melarsoprol compounds can be used alone or in combination with other known therapeutic agents or techniques to either improve the quality of life of the patient, or to treat HO or VOC. For example, the arsenic compounds can be used before, during or after the administration of one or more known anti-inflammatory agents
It will be recognized by one of skill in the art that the content of the active ingredients in the pharmaceutical preparations of this invention can vary quite widely depending upon numerous factors such as, the desired dosage and the pharmaceutically acceptable carrier being employed. The table above lists Hedgehog pathway antagonists and modes of administration for each.
For administration of an arsenic compound, the dosage amount will usually be in the range of from about 0.1 mg/kg to about 100 mg/kg, in certain embodiments from about 1.0 to about 50 mg/kg, in other embodiments from about 2.5 to about 25 mg/kg, and in other embodiments from about 3 to about 15 mg/kg.
The invention also provides kits for carrying out the therapeutic regimens of the invention. Such kits comprise in one or more containers therapeutically effective amounts of the arsenic compounds in pharmaceutically acceptable form. The arsenic compound in a vial of a kit of the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid. Alternatively, the complex may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for injection purposes.
In another embodiment, a kit of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of arsenic compounds by a clinician or by the patient.
Desirable blood levels may be maintained by a continuous infusion of an arsenic compound as ascertained by plasma levels. It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust therapy to lower dosage due to toxicity, or bone marrow, liver or kidney dysfunctions. Conversely, the attending physician would also know how to and when to adjust treatment to higher levels if the clinical response is not adequate (precluding toxic side effects).
Again, any suitable route of administration may be employed for providing the patient with an effective dosage of an arsenic compound. For example, oral, transdermal, iontophoretic, parenteral (subcutaneous, intramuscular, intrathecal and the like) may be employed. Dosage forms include tablets, troches, cachet, dispersions, suspensions, solutions, capsules, patches, and the like. (See, Remington'"'"'s Pharmaceutical Sciences.)
The pharmaceutical compositions of the present invention comprise an arsenic compound as the active ingredient, pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients, for example all trans retinoic acid. The term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases.
The pharmaceutical compositions include compositions suitable for oral, mucosal routes, transdermal, iontophoretic, parenteral (including subcutaneous, intramuscular, intrathecal and intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated.
In the case where an intravenous injection or infusion composition is employed, a suitable dosage range for use is, e.g., from about one to about 40 mg arsenic trioxide total daily; about 0.001 to about 10 mg arsenic trioxide per kg body weight total daily, or about 0.1 to about 10 mg melarsoprol per kg body weight total daily.
In addition, the arsenic carrier could be delivered via charged and uncharged matrices used as drug delivery devices such as cellulose acetate membranes, also through targeted delivery systems such as fusogenic liposomes attached to antibodies or specific antigens.
In practical use, an arsenic compound can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including tablets, capsules, powders, intravenous injections or infusions). In preparing the compositions for oral dosage form any of the usual pharmaceutical media may be employed, e.g., water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like; in the case of oral liquid preparations, e.g., suspensions, solutions, elixirs, liposomes and aerosols; starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like in the case of oral solid preparations e.g., powders, capsules, and tablets. In preparing the compositions for parenteral dosage form, such as intravenous injection or infusion, similar pharmaceutical media may be employed, e.g., water, glycols, oils, buffers, sugar, preservatives and the like know to those skilled in the art. Examples of such parenteral compositions include, but are not limited to Dextrose 5% w/v, normal saline or other solutions. The total dose of the arsenic compound may be administered in a vial of intravenous fluid, e.g., ranging from about 2 ml to about 2000 ml. The volume of dilution fluid will vary according to the total dose administered. For example, arsenic trioxide supplied as a 10 ml aqueous solution at 1 mg/ml concentration is diluted in 10 to 500 ml of 5% dextrose solution, and used for intravenous infusion over a period of time ranging from about ten minutes to about four hours.
All mice used in these examples have been previously described in the literature, including: Gαsflox (Chen, M., et al., 2005, J Clin Invest 115, 3217-27), Prx1-cre (Logan, M., et al., 2002, Genesis 33, 77-80), Dermo1-cre Yu, K., et al., 2003, Development 130, 3063-74), Ap2-cre (Nelson, et al., 2004, Dev Biol 267, 72-92), and Ptch1flox (Mak, K.K., et al., 2006, Development 133, 3695-3707).
Alizarin Red-Alcian Blue Staining:
Embryos were skinned and placed in 100% EtOH overnight to fix. Embryos were then placed in staining solution for 2 days (50 mL staining solution=2.5 mL 0.3% Alcian Blue, 2.5 mL 0.1% Alizarin Red, 2.5 mL 100% glacial acetic acid, 42.5 mL 70% EtOH). Embryos were rinsed with water, then placed in 1% KOH until destained, and finally in 80% glycerol for storage.
Von Kossa Staining:
Tissue sections were deparaffinized and hydrated in distilled water. silver hydrate, 5%, was added to slides and place under a 60-watt lamp for 1 hour. Slides were rinsed three times in distilled water before adding 5% sodium thiosulfate for 5 minutes. Slides were rinsed three times in distilled water, and counterstained with nuclear fast red for 5 minutes. Slides were again rinsed three times in distilled water, dehydrated, cleared and put under a coverslip.
Tissue sections were deparaffinized and hydrated in distilled water. Slides were placed into boiling 10 mM citrate pH6 for 15 minutes, and then placed at room temperature for 15 minutes. Slides were placed in 3% water/MeOH for 15 minutes. Slides were equilibrated in phosphate buffered saline with 0.1% Tween-20 (PBS-T) then block for 1 hour with 5% normal goal serum in PBS-T. Rabbit anti-osterix antibody (Abcam; ab22552) was added at 1:1000 and incubated overnight at 4° C. Slides were washed and detected using the anti-rabbit ABC elite kit (Vector labs; PK-6101) and DAB tablets (Sigma-Aldrich; D4293). Slides were counterstained with nuclear fast red and Alcian Blue, then dehydrated, cleared, and put under a coverslip.
Using the Prx1-cre, Gαs was removed in undifferentiated limb bud mesenchyme. While the limb pattern was largely normal, Prx1-cre+; Gαsflox/− mice were born with a soft tissue syndactyly (webbing between the digits) and a progressive form of HO. In the skeletal preparations examined, bone was stained red and cartilage was stained blue. HO first appeared by Alizarin Red staining at embryonic day 16.5 and was clearly visible by postnatal day 4 in Prx1-cre+; Gαsflox/− mice (arrows,
Loss of Gαs was also associated with the formation of vascular calcification/ossification. In Prx1-cre+; Gαsflox/− mice ossification is frequently seen surrounding blood vessels between the digits (arrows,
These results demonstrate that loss of Gαs promotes HO formation both in soft tissues and around blood vessels.
The formation of HO indicated inappropriate upregulation of a pro-osteogenic pathway (e.g. BMP, Wnt, and/or hedgehog) outside the skeleton. As Gαs is a physiologically important activator of protein kinase A (PKA) and PKA is a potent inhibitor of Hedghehog (Hh) signaling (Jiang, et al., 1995, Cell 80, 563-572), loss of Gαs is likely associated with an upregulation of Hh that was driving HO formation. Accordingly, Hh signaling would be elevated prior to HO formation and overlap in its tissue expression. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on RNA isolated from control and Prx1-cre+; Gαsflox/− mutant limb tissue at E14.5, prior to HO formation. The following primers were used for qRT-PCR:
Relative expression was quantified using the 2−AAct method (Livak, et al., 2001, Methods 25, 402-08).
Prx1-cre+; Gαsflox/− mice showed a reduction in Gαs mRNA (
Elevation in the protein level of the Gli3 full-length form was also seen during Hh pathway activation (Wang, et al. 2000, Cell 100, 423-34). Western blot analysis was performed on limb tissue isolated from E18.5 control and mutant mice. The presence of the Gli3 full-length form was assayed. This product has a molecular weight of ˜190 kDa versus the repressor form which has an apparent molecular weight of ˜83 kDa. Western blotting was performed using standard techniques using a rabbit anti-Gli3 antibody (Dr. Susan Mackem (NIH/NCI)), a rabbit anti-Gαs (Dr. Lee Weinstein (NIH/NIDDK)) and a rat anti-α-tubulin (Sigma).
Western blotting performed with an antibody raised against Gli3 demonstrated that Gli3 full-length form (Gli3-FL) was present at higher levels and Gli3 repressor form (Gli3-R) at lower levels in the mutant relative to littermate controls (
All of these data are consistent with the Hh pathway being upregulated in the mutant mice that form HO, which suggests inhibiting the Hh pathway may be an important treatment for HO.
Ptch1 is an inhibitor of the Hh pathway and loss of both copies of the Ptch1 gene leads to elevated Hh signaling and embryonic lethality (Goodrich, et al., 1997, Science 277, 1109-13). Mice lacking one copy of Ptch1 are viable and highly sensitized to further increases in Hh signaling. To show Gαs is a biologically important regulator of the Hh pathway, the genetic interaction of Gαs and Ptch1 was measured. male Dermo1-cre+; Gαsflox/−;Ptch1flox/− mice were mated to Gαsflox/flox female mice. If Gαs and Ptch1 do not interact genetically then Dermo1-cre+; Gαsflox/−;Ptch1flox/+ and Dermo1-cre+; Gαsflox/−;Ptch11+/+ would look similar. If Gαs and Ptch1 do interact genetically then Dermo1-cre+; Gαsflox/−;Ptch1flox/− mice would look more severe than either Dermo1-cre+; Gαsflox/−;Ptch11+/+ or Dermo1-cre+; Gαsflox/−;Ptch1flox/+ mice. At E18.5, no Dermo1-cre+; Gαsflox/−;Ptch1flox/+ mice were observed, likely because these mice died prior to this stage. Dermo1-cre+; Gαsflox/−;Ptch1flox/+ mice were present in the correct Mendellian ratio at E13.5, and presented with severe skeletal defects including polydactyly, exencephaly and craniofacial defects (
These data further demonstrate that Gαs and Ptch1 interact which confirms that elevated Hh signaling is associated with multiple pathologic phenotypes, including HO.
HO requires an inappropriate increase in bone forming cells (osteoblasts) The role of Gαs in promoting osteoblastic differentiation was tested. Bone marrow mesenchymal cells (BMMC) (also called mesenchymal stem cells) were isolated from Gαsflox/flox mice. BMMC were isolated by flushing the bone marrow cavity of 6 week old mice and plating cells in Alpha-MEM, 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine. Prior to reaching confluence cells were treated with a Cre- or GFP-containing adenovirus. Upon reaching confluence cells were switched to osteogenic media (DMEM, 10% lot-selected FBS, 100 U/mL penicillin, 100 ng/mL streptomycin, 2 mM glutamine, 10-4M L-ascorbic acid 2-phosphate and 10 mM α-glycerol phosphate). These cells were infected with a Cre-containing adenovirus to remove Gαs and cultured under conditions that favor osteogenic differentiation.
Upon reaching confluence in vitro, a significant rise in Hh target genes in these cells was found (
This result confirms the role of Gαs in promoting osteoblastic differentiation.
The role of inhibitors of Hh signaling in decreasing the elevated Hh signaling observed in Prx1-cre+; Gαsflox/− mutant mice. Limb culture experiments were performed in which the limbs from living embryos at E14.5 were removed and cultured in vitro for 4 days. Limb culture was performed by using BGJB culture media supplemented with 0.2% bovine serum albumin (Sigma Aldrich) and forskolin (Sigma Aldrich), cyclopamine (BIOMOL), or ATO (Sigma Aldrich). ATO was prepared by placing 50 mg of ATO in the bottom of a 50 mL conical tube and dissolving with 1 mL of 1N NaOH. 48 mL of PBS was then added to the tube and 0.82 mL of 1.2N HCl to adjust pH to 7.2. Media was changed daily following addition of culture media. In culture, the limbs were cultured with vehicle or ATO and assayed for reduced Hh pathway activation by qRT-PCR. Mutant mice demonstrated elevated Hh signaling, by increased expression of Ptch1, Gli1 and HIP mRNA (
Treatment of Prx1-cre+; Gαsflox/− mutant limbs with the Hh pathway antagonist ATO led to a dramatic reduction in the Hh pathway activation. This result is consistent with a role of ATO in blocking HO formation.
HO development in adult mice was studied by using adenovirus cre recombinase (Ad-Cre)-driven loss of Gαs (
Results showed ectopic bone forms over the right forelimb and hind limb following Ad-Cre-mediated loss of Gαs in Gαsflox/flox mice and not on the control side left limbs with Ad-GFP injection (
HO in adult loss of Gαs in Gαsflox/flox mice is shown in
Removal of Gαs from bone marrow mesenchymal cells (BMMC) promotes osteoblastic differentiation, which is inhibited by Hh antagonist, GANT-58 treatment in a dose-dependent manner (
HO in with adult gain of function of hedgehog effector protein Smoothed, in Gt(ROSA)26Sortm1(Smo/YFP)Amc/J mice following Ad-Cre subcutaneous injection. Ectopic bone forms over the right forelimb and hind limb following Ad-Cre-mediated loss of Gαs in Gαsflox/flox mice and not on the control side left limb with Ad-GFP injection. Alizarin Red stains bone and Alcian Blue stains cartilage (
Inhibitors of Hh signaling were tested for an ability to inhibit the formation of HO in vivo. Matings were established between Prx1-cre+; Gαsflox/− male and Gαsflox/flox female mice. Pregnant females at gestational days E13.5, E15.5 and E17.5 were injected intraperitoneally with 5 mg/kg ATO. Pregnant mice are first weighed and then injected with care so as to avoid injection into uteri. Mice were sacrificed at E18.5, pups were collected, and stained for bone formation by Alizarin Red-Alcian Blue staining. Prx1-cre+; Gαsflox/− pups from mice injected with vehicle control produced HO as evidenced by enhanced red staining in soft tissues. Prx1-cre+; Gαsflox/− pups from mice injected with 5 mg/kg ATO showed a significant reduction in HO in both forelimb and hind limb (
The ability of hedgehog pathway antagonists to block the formation of vascular calcification/ossification will be tested in established models.
Mating between either Prx1-cre+/−; Gαs+/− or Dermo1-cre+/−; Gαs+/− males and Gαsflox/flox females will be made. The mutant mice from these matings with the genotypes of either Prx1-cre+/−; Gαsflox/− or Dermo1-cre+/−; Gαsflox/− show clear signs of vascular calcification and pathologic ossification at embryonic day 18.5 (E18.5) (
Mutant embryos with the genotypes Prx1-cre+/−; Gαsflox/− or Dermo1-cre+/−; Gαsflox/− who have come from females treated with Hh antagonists will be compared with mutant mice from female mice that have receive control injections. Using this approach, positive results demonstrated that ATO prevents the formation of pathologic ossification (
Results showing GANT58 prevents to formation of pathologic ossification have also been obtained. The effects of Hh antagonists will be quantified in several ways:
- 1) Alizarin red-Alcian blue staining will be performed (as in
FIG. 10) and morphometric analysis will permit quantification of the amount of red staining (bone) in the affected tissues;
- 2) RNA will be isolated from limbs and analyzed by qRT-PCR for markers of Hh signaling and bone formation [assumption is Hh antagonists will decrease expression of Hh signaling markers (Ptch1, Gli1, HIP) and decreased markers of bone formation (osterix, alkaline phosphatase, bone sialoprotein)];
- 3) affected tissue will be removed, fixed, and sectioned for histologic analysis [bone formation (e.g. subcutaneous or perivascular Von Kossa staining as in
FIGS. 2A and 3A, respectively)) will be quantified in sections and compare mutants isolated from treated and untreated females]. For vascular calcification Alizarin red-Alcian blue staining will also be performed on the heart and great vessels of Dermo1-cre+/−; Gαsflox/− mutant mice. FIG. 3Bshows that these mutant mice develop vascular ossification around the great vessels at E18.5. The heart and great vessels of Dermo1-cre+/−; Gαsflox/− mutant mice from females treated with Hh antagonists will be compared to Dermo1-cre+/−; Gαsflox/− mutant mice from females treated with vehicle controls. Morphometric analysis will allow quantification of ossification (Alizarin red staining) between treatment groups.
- 1) Alizarin red-Alcian blue staining will be performed (as in
A second model of heterotopic ossification is described in O'"'"'Connor, 1998, Clin Orthopaedics Related Res 345:71-80, and in Shimono et al., 2011, Nature Medicine 17:454-60, hereby incorporated in their entirety. Briefly, 1 μg of human recombinant bone morphogenic protein 2 (BMP2) will be absorbed onto a small collagen disc and that disc will be inserted intramuscularly into an adult mouse. Within 5-6 days the released BMP2 will recruit bone-forming cells and within 2-3 weeks these cells will begin to ossify the tissue around the disc. The ability of treatment with Hh antagonists to block the formation of pathologic ossification will be measured. Mice will be treated, starting at day 1, with intraperitoneal injections given daily to every-other-day for 3 weeks. The doses will range from 0.01-100 mg/kg. At 3 weeks post disc insertion, the mice will be sacrificed and bone formation will be quantified using several strategies. Computed tomography (CT) and microcomputed tomography (μCT) will visualization and quantification of the size of the ossicle. Also, removing the ossicle and histologic sectioning will allow morphometric analysis and quantify bone formation (e.g. surface area, osteoblast number). Also, qRT-PCR for markers of bone formation (e.g. osterix, alkaline phosphatase, bone sialoprotein) will allow expression of genes associated with bone formation to be quantified. Controls will be mice who received injections of vehicle.
The ability of Hh pathway inhibitors to block the progression of pathologic ossification will be measured using this same BMP2 collagen disc implantation model. For this experiment, the BMP2/collagen disc will be implanted and the mice will be allowed to recover prior to injection of Hh pathway antagonists. Mice will be treated, starting at day 14, with intraperitoneal injections given daily to every-other-day for 2 weeks. The doses will range from 0.01-100 mg/kg. At 4 weeks post disc insertion, the mice will be sacrificed and bone formation will be quantified using several strategies. CT and μCT will be used to visualize and quantify the size of the ossicle. Also, removing the ossicle and histologic sectioning will allow morphometric analysis and quantification of bone formation (e.g. surface area, osteoblast number). Also, qRT-PCR for markers of bone formation (e.g. osterix, alkaline phosphatase, bone sialoprotein) will allow quantification of the expression of genes associated with bone formation. Controls will be mice who received injections of vehicle.
Dosage will be optimized for all experiments by identifying the highest concentration of the Hh pathway antagonist that is tolerated by the recipient (i.e. does not lead to death, does not induce abortion) and pathologic bone formation will be quantified. This dose will be established for both daily and every-other-day injections. Once this dose is identified the dose will be reduced by halves to identify lower doses of the compounds which are still effective at inhibiting pathologic bone formation.
For in vitro cellular models, bone marrow mesenchymal cells (BMMCs) will be isolated from adult Gαsflox/flox mice and embryonic limb mesenchymal cells from E12.5 Gαsflox/flox mice. These mice will be cultured and treated with either a cre recombinase-expressing or green fluorescent protein (GFP)-expressing adenovirus. The cre recombinase-expressing adenovirus will remove Gαs from the cells and these will function as the mutant ossifying cells. The GFP-expressing adenovirus will function as the control cells. Upon reaching confluence cells will be switched to osteogenic media [DMEM, 10% lot-selected FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 10-4M L-ascorbic acid 2-phosphate and 10 mM β-glycerol phosphate]. At all stages of culture cells will be treated either with media or media plus Hh inhibitor. The efficiency of Hh pathway inhibition by qRT-PCR for Ptch1, Gli1 and HIP will be quantified. Oosteoblast differentiation by qRT-PCR for specific markers [osterix (Osx), oollagen 1a1 (Col1a1), alkaline phosphatase (Alk Phos), bone sialoprotein (BSP), osteocalcin (OC)] will also be quantified. Von Kossa and Alizarin red staining will be used to measure in vitro bone formation. The time points to assay are: preconfluence and day 0, 2, 4, 7, 14, 21 in osteogenic media. As shown above, mutant BMMC express Hh signaling and show osteoblast differentiation under osteogenic conditions (
To optimize dosage for these in vitro experiments will identify the highest concentration of the Hh pathway antagonist that allows normal cells growth (i.e. non-toxic) and block in vitro bone formation. Once this dose is identified, doses will be reduced by halves to identify lower doses of the compounds still effective at inhibiting Hh pathway inhibition and in vitro bone formation.
The purpose of this study is to determine if hedgehog antagonists are an effective treatment for patients with non-genetic forms of heterotopic ossification (HO, e.g., neurogenic injury, surgery, trauma or severe burns). The hedgehog antagonists may include one or more of the following drugs: vismodegib (GDC-0449, Genentech), bevacizumab, gemcitabine, nab-paclitaxel, FOLFIR, FOLFOX, RO4929097, cixutumumab, cisplatin, etoposide, LDAC, decitabine, daunorubicin, cytarabin, rosiglitazone, goserelin, leuprolide, capecitabine, fluorouracil, leucovorin, oxaliplatin, irinotecan, diclofenac, BMS-833923 (XL139), IPI-926—Infinity Pharmaceuticals, Inc., LDE225, LEQ506—Novartis Pharmaceuticals, TAK-441 Millennium Pharmaceuticals, Inc., or PF-04449913—Pfizer, alone or in combination therapy.
Condition: non-genetic forms of heterotopic ossification (HO)
Intervention: Drug: hedgehog antagonists through intraperitoneal injection (IP injection)
The clinical trial will be carried out, in accordance with applicable rules and regulations, e.g., 21 CFR 312 and 45 CFR 46.
Study Type: Interventional
Study Design: Allocation: Randomized
Endpoint Classification: Safety/Efficacy Study
Intervention Model: Single Group Assignment
Masking: Single Blind (Caregiver)
Primary Purpose: Treatment
Title: Hedgehog inhibitors for treatment of non-genetic forms of heterotopic ossification
Primary Outcome Measures:
Determine dose-response, tolerability, and adverse effects of Hh inhibitors in 100 patients with non-genetic forms of HO.
Estimated Enrollment: 100
In Phase I, a small group of healthy volunteers (20-100) will be used to evaluate the safety of the drug, determine a safe dosage range, and identify side effects of these compounds.
In Phase II, a larger group of patients (100-300) will be tested to determine the drug'"'"'s effectiveness and further investigate its safety in a randomized manner relative to a placebo. This trial will last from two months to two years. Likely trial outcomes may include the reduced HO as measured by X-ray or computed tomography, quantification of blood markers of bone formation/turnover, analysis of joint stiffness/mobility, and quality of life assessment.
During Phase III, the drug will be given to large groups of patients (1,000-3,000) to confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the experimental drug or treatment to be used safely. Patients will be randomized and blinded. This trial will likely last between one and four years. Likely trial outcomes may include the reduced sizes of HO as measured by X-ray or computed tomography, quantification of blood markers of bone formation/turnover, analysis of joint stiffness/mobility, and quality of life assessment.
In Phase IV, post marketing studies will gather additional information including the drug'"'"'s risks, benefits, and optimal use.
Sleep difficulties in children with autism spectrum disorders (ASD) are common reasons why parents seek medical intervention for their children. Identifying a safe and effective pharmacologic agent that promotes sleep in ASD would favorably impact the lives of these children and their families. In this study we plan to determine the dose-response, tolerability and any adverse effects of supplemental melatonin in 30 children with ASD. The melatonin dose levels are 1 mg, 3 mg, 6 mg, and 9 mg. After a 3 week baseline period, the child will begin melatonin at 1 mg and will dose escalate every three weeks until he/she is falling asleep within 30 minutes of bedtime at least 5/7 nights per week. No child will take more than 9 mg of supplemental melatonin.
Ages Eligible for Study: 10 Years to 60 Years
Genders Eligible for Study: Both
Accepts Healthy Volunteers: Yes
- Patients with HO ages 10-60 years.
- Diagnosis of HO based on X-ray or computed tomography, quantification of blood markers of bone formation/turnover, analysis of joint stiffness/mobility, and quality of life assessment.
- Patients may take seasonal allergy medications.
- Patients taking medications other than those in the inclusion criteria.
- Patients with other skeletal disorders.
- Patients with liver disease or high fat diets, as Hh inhibitors may be affected in these children.
- Patients with known genetic syndromes of skeletal defects.
- Patients who have outside normal limits on blood work for complete blood count, liver and renal function and hormone levels of ACTH, cortisol, LH, FSH, prolactin, testosterone and estradiol.