EP2185516A2 - Formes polymorphes d'agonistes de prostaglandine et procédés de fabrication de celles-ci - Google Patents

Formes polymorphes d'agonistes de prostaglandine et procédés de fabrication de celles-ci

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Publication number
EP2185516A2
EP2185516A2 EP08806952A EP08806952A EP2185516A2 EP 2185516 A2 EP2185516 A2 EP 2185516A2 EP 08806952 A EP08806952 A EP 08806952A EP 08806952 A EP08806952 A EP 08806952A EP 2185516 A2 EP2185516 A2 EP 2185516A2
Authority
EP
European Patent Office
Prior art keywords
crystalline form
amino
tert
phenoxy
butyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08806952A
Other languages
German (de)
English (en)
Inventor
David Burns Damon
Jinyang Hong
Joseph Francis Krzyzaniak
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Products Inc
Original Assignee
Pfizer Products Inc
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Filing date
Publication date
Application filed by Pfizer Products Inc filed Critical Pfizer Products Inc
Publication of EP2185516A2 publication Critical patent/EP2185516A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/70—Sulfur atoms
    • C07D213/71—Sulfur atoms to which a second hetero atom is attached
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the present invention relates to polymorphs of therapeutically active and selective modulators of prostaglandin, specifically agonists of EP 2 , pharmaceutical compositions comprising these compounds, methods for the preparation of these compounds and the use of such compounds for treating conditions that are associated with the modulation of prostaglandin, such as bone disorders, glaucoma and ocular hypertension.
  • the present invention relates to polymorphs of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)- phenoxy)-acetic acid sodium salt, pharmaceutical compositions comprising polymorphs of this compound, methods for the preparation of these polymorphs and the use of polymorphs of this compound for treating conditions that are associated with the modulation of prostaglandin.
  • a drug formulation for administration by various routes, and the optimum drug form for different formulations is likely to be different.
  • a drug formulation must have sufficient shelf life to allow successful distribution to patients in need of treatment.
  • an oral drug formulation must provide the drug in a form which will dissolve in the patient's gastrointestinal tract when orally dosed.
  • an immediate release dosage form such as an immediate release tablet, capsule, suspension, or sachet
  • noncrystalline drug form which will generally have a higher initial solubility than a crystalline form when administered into the gastrointestinal tract.
  • a noncrystalline form of a drug is frequently less chemically stable than a crystalline form.
  • dosage forms which operate better if the drug form is less soluble.
  • a chewable tablet or a suspension or a sachet dosage form exposes the tongue to the drug directly.
  • the desired drug solubility is a complex function of delivery route, dose, dosage form design and desired duration of release.
  • a drug which has high solubility it may be desirable to utilize a lower solubility crystalline salt or polymorph for a controlled release dosage form, to aid in achievement of slow release through slow dissolution.
  • a drug which has low solubility it may be necessary to utilize a higher solubility crystalline salt or polymorph, or a noncrystalline form, in order to achieve a sufficient dissolution rate to support the desired drug release rate from the controlled release dosage form.
  • soft gelatin capsule dosage forms In soft gelatin capsule dosage forms ("soft-gels”), the drug is dissolved in a small quantity of a solvent or vehicle such as a triglyceride oil or polyethylene glycol, and encapsulated in a gelatin capsule.
  • a solvent or vehicle such as a triglyceride oil or polyethylene glycol
  • An optimal drug form for this dosage form is one which has a high solubility in an appropriate soft-gel vehicle.
  • a drug form which is more soluble in a triglyceride oil will be less soluble in water.
  • Identification of an appropriate drug form for a soft-gel dosage form requires study of various salts, polymorphs, crystalline and noncrystalline forms. Thus, it can be seen that the desired solubility of a drug form depends on the intended use and not all drug forms are equivalent.
  • a drug form For a drug form to be practically useful for human or animal therapy, it is desirable that the drug form exhibit minimal hygroscopicity. Dosage forms containing highly hygroscopic drugs require protective packaging, and may exhibit altered dissolution if stored in a humid environment. Thus, it is desirable to identify nonhygroscopic crystalline salts and polymorphs of a drug. If a drug is noncrystalline, or if a noncrystalline form is desired to improve solubility and dissolution rate, then it is desirable to identify a noncrystalline salt or form which has a low hygroscopicity relative to other noncrystalline salts or forms.
  • a drug, crystalline or noncrystalline may exist in an anhydrous form or as a hydrate or solvate or hydrate/solvate.
  • the hydration state and solvation state of a drug affects its solubility and dissolution behavior.
  • the melting point of a drug may vary for different salts, polymorphs, crystalline and noncrystalline forms. To permit manufacture of tablets on commercial tablet presses, it is desirable that the drug melting point be greater than around 60 0 C, preferably greater than 100 0 C to prevent drug melting during tablet manufacture.
  • a preferred drug form in this instance is one that has the highest melting point.
  • a salt, hydrate or solvate affects the potency per unit weight. For example, a drug salt with a higher molecular weight counterion will have a lower drug potency per gram than will a drug salt with a lower molecular weight counterion. It is desirable to choose a drug form which has the highest potency per unit weight.
  • the method of preparation of different crystalline polymorphs and noncrystalline forms varies widely from drug to drug. It is desirable that minimally toxic solvents be used in these methods, particularly for the last synthetic step, and particularly if the drug has a tendency to exist as a solvate with the solvent utilized in the last step of synthesis. Preferred drug forms are those which utilize less toxic solvents in their synthesis.
  • BFI Brittle Fracture Index
  • indices or measures of mechanical properties, flow properties and tableting performance include compression stress, absolute density, solid fraction, dynamic indentation hardness, ductility, elastic modulus, reduced elastic modulus, quasistatic indentation hardness, shear modulus, tensile strength, compromised tensile strength, best case bonding index, worst case bonding index, brittle/viscoelasticbonding index, strain index, viscoelastic number, effective angle of internal friction (from a shear cell test), cohesivity (from a powder avalanche test) and flow variability.
  • a number of these measures are obtained on drug compacts, preferably prepared using a triaxial hydraulic press.
  • Drug form properties which affect flow are important not just for tablet dosage form manufacture, but also for manufacture of capsules, suspensions, and sachets.
  • the particle size distribution of a drug powder can also have large effects on manufacturing processes, particularly through effects on powder flow. Different drug forms have different characteristic particle size distributions.
  • Patent Number 6,498,172 B1 discloses the compound (3-(((4-tert-butyl- benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid as being useful in the treatment of, for example, bone disorders.
  • the application refers in general terms to pharmaceutically acceptable salts and the preparation of the sodium salt is disclosed.
  • the '172 patent neither describes any crystallization procedure nor does the patent discuss any polymorph forms of this compound.
  • transverse fractures were made surgically in the ribs of adult beagles, and 0.5 ml of 10% ethanol Tris-buffer vehicle or 0.5 ml of PGE 1 (containing 0.2 mg of PGE 1 in 10% ethanol Tris-buffer) was injected directly into the fracture sites twice a day for 10 days. It was concluded that administration of PGE 1 induced bone matrix formation on the periosteal envelope adjacent to the fracture site and its contralateral matching site.
  • a pharmaceutical composition comprising a therapeutically effective amount of a crystalline form of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)- phenoxy)-acetic acid sodium salt or a hydrate thereof, and a pharmaceutically acceptable diluent or carrier.
  • a method for treating a mammal having glaucoma, ocular hypertension or a condition which presents with low bone mass comprising administering to the mammal a therapeutically effective amount of a crystalline form of (3-(((4-tert-butyl-benzyl)- (pyridine-3-sulfonyl)-amino)-methyI)-phenoxy)-acetic acid sodium salt or a hydrate thereof.
  • a method for augmenting and maintaining bone mass in a mammal comprising administering to the mammal a therapeutically effective amount of a crystalline form of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)- phenoxy)-acetic acid sodium salt or a hydrate thereof.
  • a method for preparing crystalline form A of (3-(((4-tert-butyl-benzyl)- (pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt hemi-hydrate comprises contacting (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)- amino)-methyl)-phenoxy)-acetic acid, as the free acid, with sodium hydroxide in an organic solvent to form a reaction mixture; warming the reaction mixture to a first temperature from about 5OC to about 9OC and holding the first temperature for a period of at least one hour; cooling the reaction mixture to a second temperature from about 15C to about 25C; and collecting solids from the reaction mixture.
  • the organic solvent used in the method may be isopropyl acetate being present at about 8 to about 12 ml per mmol of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)- methyl)-phenoxy)-acetic acid.
  • the sodium hydroxide used in the method may be a 50% aqueous solution of sodium hydroxide present at about 1 to about 1.3 mmol per mmol of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid.
  • a pharmaceutical composition comprising a therapeutically effective amount of a non-crystalline form or an amorphous form of (3-(((4-tert-butyl-benzyl)-(pyridine- 3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt or a hydrate thereof, and a pharmaceutically acceptable diluent or carrier.
  • a method for treating a mammal having glaucoma, ocular hypertension or a condition which presents with low bone mass comprising administering to the mammal a therapeutically effective amount of a non-crystalline form or amorphous form of (3- (((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt or a hydrate thereof.
  • a method for augmenting and maintaining bone mass in a mammal comprising administering to the mammal a therapeutically effective amount of a non-crystalline form or amorphous form of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt or a hydrate thereof.
  • mammal means animals including, for example, dogs, cats, cows, sheep, horses, and humans. Preferred mammals include humans.
  • substantially pure refers the relative purity of the desired polymorph and/or salt with respect to the overall amount of compound (that is, the amount of the desired polymorph and/or salt plus any other polymorphs and amorphous forms, salts and free acids).
  • a “substantially pure” polymorphic form should contain at least about 90% of the desired polymorph relative to the overall amount of compound.
  • a “substantially pure” polymorphic form should contain at least about 95% of the desired polymorph.
  • a "substantially pure" polymorphic form may contain at least about 99% of the desired polymorph.
  • condition(s) which presents with low bone mass refers to a condition where the level of bone mass is below the age specific normal as defined in standards by the World Health Organization "Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994), Report of a World Health Organization Study Group, World Health Organization Technical Series 843". Included in “condition(s) which presents with low bone mass” are primary and secondary osteoporosis. Secondary osteoporosis includes glucocorticoid-induced osteoporosis, hyperthyroidism-induced osteoporosis, immobilization-induced osteoporosis, heparin-induced osteoporosis and immunosuppressive-induced osteoporosis.
  • condition(s) which presents with low bone mass also includes long term complications of osteoporosis such as curvature of the spine, loss of height and prosthetic surgery.
  • condition(s) which presents with low bone mass also refers to a mammal, e.g., a mammal, known to have a significantly higher than average chance of developing such diseases as are described above including osteoporosis (e.g., post-menopausal women, and men over the age of 60).
  • osteoporosis e.g., post-menopausal women, and men over the age of 60.
  • bone mass augmenting or enhancing uses include bone restoration, increasing the bone fracture healing rate, replacing bone graft surgery entirely, enhancing the rate of successful bone grafts, bone healing following facial reconstruction, maxillary reconstruction, mandibular reconstruction, craniofacial reconstruction, prosthetic ingrowth, vertebral synostosis, long bone extension and spinal fusion.
  • compositions of the present invention may also be used in conjunction with orthopedic devices known to those skilled in the art such as spinal fusion cages, spinal fusion hardware, internal and external bone fixation devices, screws and pins.
  • bone mass actually refers to bone mass per unit area which is sometimes (although not strictly correctly) referred to as bone mineral density (BMD).
  • BMD bone mineral density
  • treating includes preventative (e.g., prophylactic), palliative and curative treatment.
  • an effective amount means an amount of a compound or combination of compounds that ameliorates, attenuates or eliminates a particular disease or condition or a symptom of a particular disease or condition, or prevents or delays the onset of a particular disease or condition or a symptom of a particular disease or condition.
  • patient means an animal, such as a human, a companion animal, such as a dog, cat and horse, and livestock, such as cattle, swine and sheep. Particularly preferred patients are mammals, including both males and females, with humans being even more preferred.
  • prodrug refers to a compound that is a drug precursor which, following administration, releases the drug in vivo via some chemical or physiological process (e.g., a prodrug on being brought to the physiological pH or through enzyme action is converted to the desired drug form). Exemplary prodrugs upon cleavage release the corresponding drug compounds.
  • pharmaceutically acceptable salt refers to anionic salts such as (but not limited to) chloride, bromide, iodide, sulfate, bisulfate, phosphate, acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate, gluconate, methanesulfonate and 4-toluene-sulfonate.
  • the expression also refers to cationic salts such as (but not limited to) sodium, potassium, calcium, magnesium, ammonium or protonated benzathine (N,N'-dibenzylethylenediamine), choline, ethanolamine, diethanolamine, ethylenediamine, meglamine (N-methyl-glucamine), benethamine (N-benzylphenethylamine), piperazine and tromethamine (2-amino-2- hydroxymethyl-1 ,3-propanediol).
  • cationic salts such as (but not limited to) sodium, potassium, calcium, magnesium, ammonium or protonated benzathine (N,N'-dibenzylethylenediamine), choline, ethanolamine, diethanolamine, ethylenediamine, meglamine (N-methyl-glucamine), benethamine (N-benzylphenethylamine), piperazine and tromethamine (2-amino-2- hydroxymethyl-1 ,
  • Figure 1 is a diffractogram of Form A of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt according to the present invention
  • Figure 2 is a diffractogram of Form B of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt according to the present invention
  • Figure 3 is a diffractogram of Form C of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt according to the present invention
  • Figure 4 is a diffractogram of Form E of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt according to the present invention
  • Figure 5 is a diffractogram of Form F of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt according to the present invention
  • Figure 6 is a diffractogram of Form G of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl) ⁇ phenoxy)-acetic acid sodium salt according to the present invention
  • Figure 6B is a diffractogram of Form H of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt according to the present invention
  • Figure 6C is a diffractogram of amorphous (3-(((4-tert-butyl-benzyl)-(pyridine- 3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt according to the present invention
  • Figure 7 is a solid-state 13 C nuclear magnetic resonance spectrum of (3-(((4- tert-butyl-benzylHpyridine-S-sulfonyO-aminoVmethyO-phenoxyJ-acetic acid sodium salt, Form A; and
  • Figure 8 is a solid-state 13 C nuclear magnetic resonance spectrum of (3-(((4- tert-butyl-benzylHpyridine-S-sulfonyiyaminoVmethyO-phenoxy ⁇ acetic acid sodium salt, Form H.
  • (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)- phenoxy)-acetic acid sodium salt may be prepared by methods disclosed in U.S. Patent Number 6,498,172 B1, the subject matter of which is herein incorporated in its entirety by reference.
  • (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)- amino)-methyl)-phenoxy)-acetic acid sodium salt may be prepared by the novel method described in Scheme I, below. Certain processes for the manufacture of specific polymorphs of these compounds are set forth in the experimental section. All starting compounds may be obtained by literature procedures or from general commercial sources, such as Sigma-Aldrich Corporation, St. Louis, MO.
  • Step 1 Preparation of (3-r(4-tert-Butyl-benzylamino)-methyll-phenoxy)-acetic acid ethyl ester succinate (II)
  • Steps 2 and 3 Preparation of (3-(((4-tert-butyl-benzylHpyridine-3-sulfonyl)-amino)- methyl)-phenoxy)-acetic acid (V)
  • reaction mixture was washed with 1 N hydrochloric acid (336 ml_), then with 1 M aqueous sodium bicarbonate (336 ml_). 2B ethanol (384 mL) was added to the organic layer, and the mixture was atmospherically distilled until the pot volume was about 400 mL. 6N sodium hydroxide (19.9 mL, 119 mmol, 1.2 equiv) was added and the mixture was held at room temperature for 19 hours. Concentrated hydrochloric acid (11.0 mL, 134 mmol, 1.3 equiv) was added followed by water (192 mL). The slurry granulated 5 hours at room temperature and was filtered.
  • Step 3R Recrystallization of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)- methyl)-pheno ⁇ y)-acetic acid (V)
  • Crystalline Forms A, B, C, E, F, and G of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt were characterized by their X-ray powder diffraction patterns.
  • the x-ray diffraction patterns of Forms A, B, C, E, F, and G were carried out on a Bruker D5000 diffractometer using copper radiation (wavelength: 1.54056A).
  • the tube voltage and amperage were set to 40 kV and 5OmA, respectively.
  • the divergence and scattering slits were set at 1 mm, and the receiving slit was set at 0.6 mm.
  • Diffracted radiation was detected by a Kevex PSI detector.
  • a theta-two theta continuous scan at 2.4 7min (1 sec/0.04 o step) from 3.0 to 40 ° 2 ⁇ was used.
  • An alumina standard was analyzed to check the instrument alignment. Data were collected and analyzed using Bruker axis software Version 7.0. Samples were prepared by placing them in a quartz holder.
  • the sample is typically placed into a holder which has a cavity.
  • the sample powder is pressed by a glass slide or equivalent to ensure a random surface and proper sample height.
  • the sample holder is then placed into the instrument.
  • the incident X-ray beam is directed at the sample, initially at a small angle relative to the plane of the holder, and then moved through an arc that continuously increases the angle between the incident beam and the plane of the holder.
  • Measurement differences associated with such X- ray powder analyses result from a variety of factors including: (a) errors in sample preparation (e.g., sample height), (b) instrument errors (e.g.
  • the X-ray powder diffraction pattern for Form H was generated with a Siemens D5000 diffractometer using copper radiation.
  • the instrument was equipped with a line focus X-ray tube.
  • the tube voltage and amperage were set to 38 kV and 38 mA, respectively.
  • the divergence and scattering slits were set at 1 mm, and the receiving slit was set at 0.6 mm.
  • a theta two theta continuous scan at 2.4 °2 ⁇ /min (1 sec/0.04°2 ⁇ step) from 3.0 to 40 °2 ⁇ was used.
  • An alumina standard (NIST standard reference material 1976) was analyzed to check the instrument alignment. Data were collected and analyzed using BRUKER AXS DIFFRAC PLUS software Version 2.0. Samples were prepared for analysis by placing them in a quartz holder. The PXRD peak was manually selected using the maximum peak height
  • FIG. 1 there is shown a diffractogram of Form A of (3-(((4-tert- butyl-benzylHpyridine-S-sulfonyO-aminoJ-methy ⁇ -phenoxyJ-acetic acid sodium salt according to an embodiment of the present invention.
  • Form A of (3-(((4-tert-butyl- benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt is characterized by the following x-ray powder diffraction pattern expressed in terms of the degree 2 ⁇ , d-spacings, and relative intensities with a relative intensity of >7% measured on a Bruker D5000 diffractometer with CuKa radiation: Table 1 - X-ray powder diffraction pattern of sodium salt Form A
  • Form B of (3-(((4-tert-butyl- benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt is characterized by the following x-ray powder diffraction pattern expressed in terms of the degree 2 ⁇ , d-spacings, and relative intensities with a relative intensity of >3% measured on a Bruker D5000 diffractometer with CuKa radiation:
  • the relative intensities may change depending on the crystal size and morphology.
  • Form C of (3-(((4-tert- butyl-benzylHpyridine-S-sulfonyO-aminoJ-methyO-phenoxyJ-acetic acid sodium salt is characterized by the following x-ray powder diffraction pattern expressed in terms of the degree 2 ⁇ , d-spacings, and relative intensities with a relative intensity of >5% measured on a Bruker D5000 diffractometer with CuKa radiation: Table 3 - X-ray powder diffraction pattern of sodium salt Form C
  • Form E of (3-(((4-tert- butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt is characterized by the following x-ray powder diffraction pattern expressed in terms of the degree 2 ⁇ , d-spacings, and relative intensities with a relative intensity of ⁇ 4% measured on a Bruker D5000 diffractometer with CuKa radiation:
  • Form F of (3-(((4-tert- butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt is characterized by the following x-ray powder diffraction pattern expressed in terms of the degree 2 ⁇ , d-spacings, and relative intensities with a relative intensity of >5% measured on a Bruker D5000 diffractometer with CuKa radiation: Table 5 - X-ray powder diffraction pattern of sodium salt Form F
  • Form G of (3- (((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt is characterized by the following x-ray powder diffraction pattern expressed in terms of the degree 2 ⁇ , d-spacings, and relative intensities with a relative intensity of ⁇ 6% measured on a Bruker D5000 diffractometer with CuKa radiation: Table 6 - X-ray powder diffraction pattern of sodium salt Form G
  • Table 7 lists the degree 2 ⁇ and relative intensities of the diffraction lines in the sample for crystalline Forms A, B, C, E, F, and G of (3-(((4-tert- butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt:
  • each form can be identified and distinguished from the other crystalline forms by either a single x-ray powder diffraction line, a combination of lines or a pattern that is different from the x- ray powder diffraction of the other forms.
  • Table 8 lists unique peaks, as well as combinations of 2 ⁇ peaks for Forms A, B, C, E, F, and G of (3-(((4-tert-butyl ⁇ benzyl)-(pyridine-3-sulfonyl)- amino)-methyl)-phenoxy)-acetic acid sodium salt, i.e., a set of x-ray diffraction lines that are unique to each form.
  • Form H of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt is characterized by the following x-ray powder diffraction pattern expressed in terms of the degree 2 ⁇ , d-spacings, and relative intensities with a relative intensity of ⁇ 6% measured on a Bruker D5000 diffractometer with CuKa radiation:
  • FIG. 6C there is shown a diffractogram of amorphous Form I of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-rnethyl)-phenoxy)- acetic acid sodium salt according to an embodiment of the present invention.
  • Form I of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt is characterized by the x-ray powder diffraction pattern as shown in the figure.
  • Form A of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt may also be characterized by its solid-state nuclear magnetic resonance spectra (SSNMR).
  • SSNMR solid-state nuclear magnetic resonance spectra
  • TMS trimethylsilane
  • Form H of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt may also be characterized by its solid-state nuclear magnetic resonance spectra (SSNMR).
  • SSNMR solid-state nuclear magnetic resonance spectra
  • Crystalline forms of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)- methyl)-phenoxy)-acetic acid sodium salt of the present invention or the noncrystalline form or amorphous form may exist in anhydrous forms as well as hydrated and solvated forms. In general, the hydrated forms are equivalent to unhydrated forms and are intended to be encompassed within the scope of the present invention.
  • Crystalline Forms A, B, and C of (3-(((4-tert-butyl-benzyl)- (pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt preferably occur as hydrates while Forms E, F and G of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt preferably occur as anhydrous forms.
  • the mixture was heated to 70 0 C over 30 minutes and stirred at this temperature for five hours.
  • the mixture was cooled to 40 0 C over four hours, then cooled to 20 0 C over one hour.
  • the slurry then granulated at 20 0 C for 12 hours.
  • the slurry was filtered through a Buchner funnel (filter paper).
  • Form B (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)- acetic acid sodium salt
  • EtOAc water seed; granulate; sodium 2-ethylhexanoate charcoal filter; concentrate filtrate EtOAo, water; EtOAc, granulate with water; then hexane sodium 2-ethylhexanoate filter, dry
  • the filter cake was washed once with 5 mL of ethyl acetate/2.2% water.
  • the filtrate was 40 0 C.
  • 50 mg of (3-(((4-tert-butyl-benzyl)- (pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt (VIII) were added as seed, and the mixture was allowed to cool to room temperature and stirred for 18 hours.
  • the slurry was filtered to give 3.1 g of (3-(((4-tert-butyl-benzyl)- (pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt.
  • the second filtrate was concentrated to dryness, granulated in 40 mL water at room temperature, filtered and air dried to give 2.4 g of solids. These were combined with 30 mL ethyl acetate and a solution of sodium 2-ethylhexanoate (97%, 1 g) in 10 mL ethyl acetate. The mixture was stirred at room temperature for 18 hours, filtered and dried to give a non-crystalline solid. This material was dissolved in 40 ml_ ethyl acetate and 1 ml_ water, and stirred at room temperature to 18 hours. Hexane was added until the haze point was reached, then the mixture was allowed to stir 24 hours.
  • Form G (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)- acetic acid sodium salt
  • Form H a non-crystalline form of (3-(((4-tert-butyl-benzyl)- (pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt
  • Form H 1 Method 1 A solution of 20-100 mg/mL of of (3-(((4-tert-butyl-benzyl)- (pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt was made by dissolving the drug substance in Water for Injections. The solution was filtered with a 0.22 ⁇ sterile filter. The solution was filled into vials/syringes. The vials/syringes were frozen at -45 0 C and held at that temperature for 2 hours. The freeze-drying was completed by primary drying at (-20 to 25 0 C) at 150 m ⁇ , followed by secondary drying at (20 to 30 0 C). The resulting solid is stored.
  • Form H Method 2. Dissolve 1 gram of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt in 10 ml. water for injection to make 100 mg/mL solution. The solution is filtered with a 0.22 ⁇ sterile filter. Aliquots of 0.5 mL solution are filled into glass vials. The vials are loaded into freeze- dryer. The shelf temperature is ramped to -45°C at the rate of 0.5°C/minute. Hold the shelf temperature at -45 0 C for 2 hours. Apply vacuum at 150 m ⁇ .
  • Form I Method 1 : Amorphous (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)- amino)-methyl)-phenoxy)-acetic acid sodium salt was prepared by first adding excess crystalline Form A to an organic solvent (acetonitrile or dichloromethane) and stirring at room temperature for 72 hours. The samples were then filtered and the solution was evaporated at room temperature under ambient conditions to yield a white solid, which was determined to be an amorphous solid by PXRD analysis.
  • organic solvent acetonitrile or dichloromethane
  • Form I Method 2: Amorphous (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)- amino)-methyl)-phenoxy)-acetic acid sodium salt was prepared by first adding excess crystalline Form A to one of the following solvent systems (Isoprypyl ether, Methyl t- butyl ether, or 97% ethyl acetate/3% water) and stirring at 40 0 C for 72 hours. The samples were then filtered and the solution was evaporated at 40°C to yield a white solid, which was determined to be an amorphous solid by PXRD analysis.
  • solvent systems Isoprypyl ether, Methyl t- butyl ether, or 97% ethyl acetate/3% water
  • polymorphic forms of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)- amino)-methyl)-phenoxy)-acetic acid sodium salt described above are useful as prostaglandin agonists and therefore are useful in methods using such prostaglandin agonists and pharmaceutical compositions containing such prostaglandin agonists.
  • the polymorphic forms of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)- methyl)-phenoxy)-acetic acid sodium salt are useful in the treatment and/or prevention of conditions mediated by the modulation of prostaglandin, particularly conditions mediated by agonists of the EP 2 receptor.
  • the polymorphic forms of (3-(((4-tert-butyl-benzyl)- (pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt of this invention can exist in radiolabeled form, i.e., said compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number ordinarily found in nature. Radioisotopes of hydrogen, carbon, phosphorous, fluorine and chlorine include 3 H, 14 C, 32 P, 35 S, 18 F and 36 CI, respectively.
  • Radiolabeled polymorphic forms of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid sodium salt of this invention can generally be prepared of methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the above Schemes and/or in the Examples by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent.
  • anti-resorptive agents for example progestins, polyphosphonates, bisphosphonate(s), estrogen agonists/antagonists, estrogen, estrogen/progestin combinations, Premarin ® , estrone, estriol or 17 ⁇ - or 17 ⁇ -ethynyl estradiol
  • anti-resorptive agents for example progestins, polyphosphonates, bisphosphonate(s), estrogen agonists/antagonists, estrogen, estrogen/progestin combinations, Premarin ® , estrone, estriol or 17 ⁇ - or 17 ⁇ -ethynyl estradiol
  • progestins are available from commercial sources and include: algestone acetophenide, altrenogest, amadinone acetate, anagestone acetate, chlormadinone acetate, cingestol, clogestone acetate, clomegestone acetate, delmadinone acetate, desogestrel, dimethisterone, dydrogesterone, ethynerone, ethynodiol diacetate, etonogestrel, flurogestone acetate, gestaclone, gestodene, gestonorone caproate, gestrinone, haloprogesterone, hydroxyprogesterone caproate, levonorgestrel, lynestrenol, medrogestone, medroxyprogesterone acetate, melengestrol acetate, methynodiol diacetate, norethindrone, norethindrone
  • Preferred progestins are medroxyprogestrone, norethindrone and norethynodrel.
  • Exemplary bone resorption inhibiting polyphosphonates include polyphosphonates of the type disclosed in U.S. Patent 3,683,080, the disclosure of which is incorporated herein by reference.
  • Preferred polyphosphonates are geminal diphosphonates (also referred to as bis-phosphonates).
  • Tiludronate disodium is an especially preferred polyphosphonate.
  • lbandronic acid is an especially preferred polyphosphonate.
  • Alendronate is an especially preferred polyphosphonate.
  • Zoledronic acid is an especially preferred polyphosphonate.
  • Other preferred polyphosphonates are ⁇ -amino-i-hydroxy-hexylidene-bisphosphonic acid and 1- hydroxy-3(methylpentylamino)-propylidene-bisphosphonic acid.
  • the polyphosphonates may be administered in the form of the acid, or of a soluble alkali metal salt or alkaline earth metal salt. Hydrolyzable esters of the polyphosphonates are likewise included. Specific examples include ethane-1 -hydroxy 1 ,1-diphosphonic acid, methane diphosphonic acid, pentane-1-hydroxy-1,1-diphosphonic acid, methane dichloro diphosphonic acid, methane hydroxy diphosphonic acid, ethane-1 - amino-1 ,1 -diphosphonic acid, ethane-2-amino-1,1 -diphosphonic acid, propane-3- amino-1-hydroxy-1 ,1 -diphosphonic acid, propane-N,N-dimethyl-3-amino-1-hydroxy- 1 ,1 -diphosphonic acid, propane-3,3-dimethyl-3-amino-1-hydroxy-1 ,1 -diphosphonic acid, phenyl amino methane diphosphonic acid,N,N-d
  • the compounds of this invention may be combined with a mammalian estrogen agonist/antagonist.
  • Any estrogen agonist/antagonist may be used as the second compound of this invention.
  • the term estrogen agonist/antagonist refers to compounds which bind with the estrogen receptor, inhibit bone turnover and/or prevent bone loss.
  • estrogen agonists are herein defined as chemical compounds capable of binding to the estrogen receptor sites in mammalian tissue, and mimicking the actions of estrogen in one or more tissue.
  • Estrogen antagonists are herein defined as chemical compounds capable of binding to the estrogen receptor sites in mammalian tissue, and blocking the actions of estrogen in one or more tissues.
  • a preferred estrogen agonist/antagonist is droloxifene: (phenol, 3-(1-(4-(2- (dimethylamino)ethoxy)phenyl)-2-phenyl-1-butenyl)-, (E)-) and related compounds which are disclosed in U.S. patent 5,047,431 , the disclosure of which is incorporated herein by reference.
  • Another preferred estrogen agonist/antagonist is 3-(4-(1 ,2-diphenyl-but-1- enyl)-phenyl)-acrylic acid, which is disclosed in Willson et al., Endocrinology, 1997, 138, 3901-3911.
  • Another preferred estrogen agonist/antagonist is tamoxifen: (ethanamine,2-(-
  • Another related compound is 4-hydroxy tamoxifen which is disclosed in U.S. patent 4,623,660, the disclosure of which is incorporated herein by reference.
  • a preferred estrogen agonist/antagonist is raloxifene: (methanone, (6- hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl)(4-(2-(1-piperidinyl)ethoxy)phenyl)- hydrochloride) which is disclosed in U.S. patent 4,418,068, the disclosure of which is incorporated herein by reference.
  • Another preferred estrogen agonist/antagonist is toremifene: (ethanamine, 2-
  • centchroman 1-(2-((4- (-methoxy-2,2, dimethyl-3-phenyl-chroman-4-yl)-phenoxy)-ethyl)-pyrrolidine, which is disclosed in U.S. patent 3,822,287, the disclosure of which is incorporated herein by reference. Also preferred is levormeloxifene.
  • Another preferred estrogen agonist/antagonist is idoxifene: (E)-1-(2-(4-(1-(4- iodo-phenyl)-2-phenyl-but-1-enyl)-phenoxy)-ethyl)-pyrrolidinone, which is disclosed in U.S. patent 4,839,155, the disclosure of which is incorporated herein by reference.
  • Another preferred estrogen agonist/antagonist is (4-(2-(2-aza- bicyclo[2.2.1 ]hept-2-yl)-ethoxy)-phenyl)-(6-hydroxy-2-(4-hydroxy-phenyl)- benzo[b]thiophen-3-yl)-methanone which is disclosed, along with methods of preparation, in PCT publication no. WO 95/10513 assigned to Pfizer Inc.
  • estrogen agonist/antagonists include compounds as described in commonly assigned U.S. patent 5,552,412, the disclosure of which is incorporated herein by reference. Especially preferred compounds described therein are: c/s-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-phenyl)-5,6,7,8- tetrahydro-naphthalene-2-ol;
  • a bone mass augmenting agent is a compound that augments bone mass to a level which is above the bone fracture threshold as detailed in the World Health Organization Study World Health Organization, "Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994). Report of a WHO Study Group. World Health Organization Technical Series 843.”
  • prostaglandin or prostaglandin agonist/antagonist may be used as the second compound in certain aspects of this invention.
  • IGF-1 sodium fluoride
  • PTH parathyroid hormone
  • active fragments of parathyroid hormone growth hormone or growth hormone secretagogues may also be used.
  • the following paragraphs describe exemplary second compounds of this invention in greater detail.
  • prostaglandin refers to compounds which are analogs of the natural prostaglandins PGDi, PGD 2 , PGE 2 , PGE 1 and PGF 2 which are useful in the treatment of osteoporosis. These compounds bind to the prostaglandin receptors. Such binding is readily determined by those skilled in the art of standard assays (e.g., An S. et al., Cloning and Expression of the EP 2 Subtype of Human Receptors for Prostaglandin E 2 , Biochemical and Biophysical Research Communications, 1993, 197(1):263-270).
  • Prostaglandins are alicyclic compounds related to the basic compound prostanoic acid.
  • the carbon atoms of the basic prostaglandin are numbered sequentially from the carboxylic carbon atom through the cyclopentyl ring to the terminal carbon atom on the adjacent side chain. Normally the adjacent side chains are in the trans orientation.
  • the presence of an oxo group at C-9 of the cyclopentyl moiety is indicative of a prostaglandin within the E class while PGE 2 contains a trans unsaturated double bond at the Ci 3 -C 14 and a cis double bond at the C 5 -C 6 position.
  • a variety of prostaglandins are described and referenced below. However, other prostaglandins will be known to those skilled in the art. Exemplary prostaglandins are disclosed in U.S. patents 4,171,331 and 3,927,197, the disclosures of each of which are incorporated herein by reference.
  • prostaglandin agonist/antagonist refers to compounds which bind to prostaglandin receptors (e.g., An S. et al., Cloning and Expression of the EP 2 Subtype of Human Receptors for Prostaglandin E 2 , Biochemical and Biophysical Research Communications, 1993, 197(1):263-270) and mimic the action of prostaglandin in vivo (e.g., stimulate bone formation and increase bone mass). Such actions are readily determined by those skilled in the art of standard assays. Eriksen E.F.
  • sodium fluoride may be used as the second compound in certain aspects of this invention.
  • the term sodium fluoride refers to sodium fluoride in all its forms (e.g., slow release sodium fluoride, sustained release sodium fluoride). Sustained release sodium fluoride is disclosed in U.S. patent 4,904,478, the disclosure of which is incorporated herein by reference.
  • the activity of sodium fluoride is readily determined by those skilled in the art of biological protocols (e.g., see Eriksen E.F. et al., Bone Histomorphometry, Raven Press, New York, 1994, pages 1-74; Grier S.J. et. al., The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv.
  • Bone morphogenetic protein may be used as the second compound of this invention (e.g., see Ono, et al., Promotion of the Osteogenetic Activity of
  • parathyroid hormone refers to parathyroid hormone, fragments or metabolites thereof and structural analogs thereof which can stimulate bone formation and increase bone mass. Also included are parathyroid hormone related peptides and active fragments and analogs of parathyroid related peptides (see PCT publication no. WO 94/01460). Such bone anabolic functional activity is readily determined by those skilled in the art of standard assays (e.g., see Eriksen E.F. et al., Bone Histomorphometry, Raven Press, New York, 1994, pages 1- 74; Grier S.J. et.
  • growth hormone secretagogue refers to a compound which stimulates the release of growth hormone or mimics the action of growth hormone (e.g., increases bone formation leading to increased bone mass). Such actions are readily determined by those skilled in the art of standard assays well known to those of skill in the art. A variety of these compounds are disclosed in the following published PCT patent applications: WO 95/14666; WO 95/13069; WO 94/19367; WO 94/13696; and WO 95/34311.
  • growth hormones or growth hormone secretagogues will be known to those skilled in the art.
  • a preferred growth hormone secretagogue is N-[1 (R)-[1 ,2- Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperidin]-1'-yl)carbonyl]-2- (phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-677.
  • Some of the preparation methods useful for the preparation of the compounds described herein may require protection of remote functionality (e.g., primary amine, secondary amine, carboxyl in Formula I precursors).
  • remote functionality e.g., primary amine, secondary amine, carboxyl in Formula I precursors.
  • the need for such protection will vary depending on the nature of the remote functionality and the conditions of the preparation methods. The need for such protection is readily determined by one skilled in the art. The use of such protection/deprotection methods is also within the skill in the art. For a general description of protecting groups and their use, see T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.
  • the compounds of this invention, prodrugs thereof and pharmaceutically acceptable salts of said compounds and prodrugs are all adapted to therapeutic use as agents that stimulate bone formation and increase bone mass in a vertebrates, e.g., mammals, and particularly humans. Since bone formation is closely related to the development of osteoporosis and bone related disorders, these compounds, prodrugs thereof and pharmaceutically acceptable salts of said compounds and said prodrugs, by virtue of their action on bone, prevent, arrest and/or regress osteoporosis.
  • the utility of the compounds of the present invention as medical agents in the treatment of conditions which present with low bone mass (e.g., osteoporosis) in a vertebrates, e.g., mammals (e.g. humans, particularly the female) is demonstrated by the activity of the compounds of this invention in conventional assays, including an in vivo assay, a receptor binding assay, a cyclic AMP assay and a fracture healing assay (all of which are described below).
  • the in vivo assay (with appropriate modifications within the skill in the art) may be used to determine the activity of other anabolic agents as well as the prostaglandin agonists of this invention.
  • the estrogen agonist/antagonist protocol may be used to determine the activity of estrogen agonists/antagonists in particular and also other anti-resorptive agents (with appropriate modifications within the skill in the art).
  • the combination and sequential treatment protocol described below is useful for demonstrating the utility of the combinations of the anabolic agents (e.g., the compounds of this invention) and anti- resorptive agents (e.g., estrogen agonists/antagonists) described herein.
  • Such assays also provide a means whereby the activities of the compounds of this invention (or the other anabolic agents and anti-resorptive agents described herein) can be compared to each other and with the activities of other known compounds. The results of these comparisons are useful for determining dosage levels in a vertebrates, e.g., mammals, including humans, for the treatment of such diseases.
  • Anabolic Agent in Vivo Assay The activity of anabolic bone agents in stimulating bone formation and increasing bone mass can be tested in intact male or female rats, sex hormone deficient male (orchidectomy) or female (ovariectomy) rats.
  • Male or female rats at different ages can be used in the study.
  • the rats are either intact or castrated (ovariectomized or orchidectomized), and subcutaneously injected or gavaged with prostaglandin agonists at different doses (such as 1 , 3, or 10 mg/kg/day) for 30 days.
  • prostaglandin agonists such as 1 , 3, or 10 mg/kg/day
  • treatment is started at the next day after surgery (for the purpose of preventing bone loss) or at the time bone loss has already occured (for the purpose of restoring bone mass).
  • the right femur from each rat is removed at autopsy and scanned using dual energy X-ray absorptiometry (DXA 1 QDR 1000/W, Hologic Inc., Waltham, MA) equipped with "Regional High Resolution Scan” software (Hologic Inc., Waltham, MA).
  • the scan field size is 5.08 x 1.902 cm, resolution is 0.0254 x 0.0127 cm and scan speed is 7.25 mm/second.
  • the femoral scan images are analyzed and bone area, bone mineral content (BMC), and bone mineral density (BMD) of whole femora (WF), distal femoral metaphyses (DFM), femoral shaft (FS), and proximal femora (PF) are determined.
  • BMC bone mineral content
  • BMD bone mineral density
  • the right tibia is removed at autopsy, dissected free of muscle, and cut into three parts.
  • the proximal tibia and the tibial shaft are fixed in 70% ethanol, dehydrated in graded concentrations of ethanol, defatted in acetone, then embedded in methyl methacrylate (Eastman Organic Chemicals, Rochester, NY).
  • Frontal sections of proximal tibial metaphyses at 4 and 10 ⁇ m thickness are cut using a Reichert-Jung Polycut S microtome. The 4 ⁇ m sections are stained with modified Masson's Trichrome stain while the 10 ⁇ m sections remained unstained.
  • One 4 ⁇ m and one 10 ⁇ m sections from each rat are used for cancellous bone histomorphometry.
  • Cross sections of tibial shaft at 10 ⁇ m thickness are cut using a Reichert-Jung
  • Cancellous bone histomorphometry A Bioquant OS/2 histomorphometry system (R&M Biometrics, Inc., Arlington, TN) is used for the static and dynamic histomorphometric measurements of the secondary spongiosa of the proximal tibial metaphyses between 1.2 and 3.6 mm distal to the growth plate-epiphyseal junction. The first 1.2 mm of the tibial metaphyseal region needs to be omitted in order to restrict measurements to the secondary spongiosa. The 4 ⁇ m sections are used to determine indices related to bone volume, bone structure, and bone resorption, while the 10 ⁇ m sections are used to determine indices related to bone formation and bone turnover.
  • TBT Trabecular bone thickness
  • TBS Trabecular bone separation
  • Osteoclast number OCN, BS: total number of osteoclast within total metaphyseal area.
  • Osteoclast perimeter OCP, mm: length of trabecular perimeter covered by osteoclast.
  • Osteoclast number/mm OCN / BS.
  • Percent osteoclast perimeter %OCP, %): OCP / BS x 100.
  • tibial shaft cortical bone Total tissue area, marrow cavity area, periosteal perimeter, endocortical perimeter, single labeled perimeter, double labeled perimeter, and interlabeled width on both periosteal and endocortical surface are measured, and cortical bone area (total tissue area - marrow cavity area), percent cortical bone area (cortical area / total tissue area x 100), percent marrow area (marrow cavity area / total tissue area x 100), periosteal and endocortical percent labeled perimeter [(single labeled perimeter/2+double labeled perimeter) / total perimeter x 100], mineral apposition rate (interlabeled width/intervals), and bone formation rate [mineral apposition rate x [(single labeled perimeter/2+double labeled perimeter) / total perimeter] are calculated.
  • Statistics can be calculated using StatView 4.0 packages (Abacus Concepts, Inc., Berkeley, CA). The analysis of variance (ANOVA) test followed by Fisher's PLSD (Stat View, Abacus Concepts Inc., 1918 Bonita Ave, Berkeley, CA 94704- 1014) are used to compare the differences between groups.
  • ANOVA analysis of variance
  • PLSD Fisher's PLSD
  • cDNAs representing the complete open reading frames of the human EP 2 and EP 4 receptors are generated by reverse transcriptase polymerase chain reaction using oligonucleotide primers based on published sequences (Regan, J.W. Bailey, TJ. Pepperl, D.J. Pierce, K.L. Bogardus.A.M. Donello, J. E. Fairbaim, CE. Kedzie, K.M. Woodward, D.F. and Gil, D.W. 1994 Cloning of a Novel Human Prostaglandin Receptor with Characteristics of the Pharmaclogically Defined EP 2 Subtype.
  • cDNAs are cloned into the multiple cloning site of pcDNA3 (Invitrogen Corporation, 3985B Sorrento Valley Blvd., San Diego, CA 92121) and used to transfect 293-S human embryonic kidney cells via calcium phosphate co- precipitation. G418-resistant colonies are expanded and tested for specific [ 3 H]PGE 2 binding. Transfectants demonstrating high levels of specific [ 3 H]PGE 2 binding are further characterized by Scatchard analysis to determine Bmax and Kds for PGE 2 .
  • Constituitive expression of both receptors in parental 293-S cells is negligible.
  • Cells are maintained in RPMI supplemented with fetal bovine serum (10% final) and G418 (700 ug/ml final).
  • cAMP responses in the 293-S/EP 2 and 293-S/EP 4 lines are determined by detaching cells from culture flasks in 1 ml of Ca++ and Mg++ deficient PBS via vigorous pounding, adding serum-free RPMI to a final concentration of 1 X 10 6 cells/ml, and adding 3-isobutyl-1-methylxanthine (IBMX) to a final concentration of 1 mM.
  • IBMX 3-isobutyl-1-methylxanthine
  • the compound to be tested is then added to cells at 1:100 dilutions such that final DMSO or ethanol concentrations is 1%.
  • the tubes are covered, mixed by inverting two times, and incubated at 37 0 C for 12 minutes. Samples are then lysed by incubation at 100 0 C for 10 minutes and immediately cooled on ice for 5 minutes. Cellular debris is pelleted by centrifugation at 1000 X g for 5 minutes, and cleared lysates are transferred to fresh tubes.
  • cAMP concentrations are determined using a commercially available cAMP radioimmunoassay kit RIA (NEK-033, DuPont/NEN Research Products, 549 Albany St., Boston, MA 02118) after diluting cleared lysates 1:10 in cAMP RIA assay buffer (included in kit). Typically, one treats cells with 6-8 concentrations of the compound to be tested in 1 log increments. EC50 calculations are performed on a calculator using linear regression analysis on the linear portion of the dose response curves.
  • the cells are lysed by sonification with a Branson Sonifier (Model #250, Branson Ultrasonics Corporation, Danbury, CT) in 2 fifteen second bursts. Unlysed cells and debris are removed by centrifugation at 100 x g for 10 min. Membranes are then harvested by centrifugation at 45,000 x g for 30 minutes. Pelleted membranes are resuspended to 3-10 mg protein per ml, protein concentration being determined of the method of Bradford [Bradford, M., Anal. Biochem., 72, 248 (1976)]. Resuspended membranes are then stored frozen at -80 0 C until use.
  • Binding Assay Frozen membranes prepared as above are thawed and diluted to 1 mg protein per ml in Buffer A above. One volume of membrane preparation is combined with 0.05 volume test compound or buffer and one volume of 3 nM 3 H-prostaglandin E 2 ( #TRK 431, Amersham, Arlington Heights, IL) in Buffer A. The mixture (205 ⁇ L total volume) is incubated for 1 hour at 25°C. The membranes are then recovered by filtration through type GF/C glass fiber filters ( #1205-401 , Wallac, Gaithersburg, MD ) using a Tomtec harvester ( Model Mach II/96, Tomtec, Orange, CT).
  • the membranes with bound 3 H-prostaglandin E 2 are trapped by the filter, while the buffer and unbound 3 H-prostaglandin E 2 pass through the filter into waste. Each sample is then washed 3 times with 3 ml of [50 mM Tris-HCI (pH 7.4), 10 mM MgCI 2 , 1 mM EDTA].
  • the filters are then dried by heating in a microwave oven. To determine the amount of 3 H-prostaglandin bound to the membranes, the dried filters are placed into plastic bags with scintillation fluid and counted in a LKB 1205 Betaplate reader (Wallac, Gaithersburg, MD). IC50s are determined from the concentration of test compound required to displace 50% of the specifically bound 3 H- prostaglandin E 2 .
  • the full length EP 1 receptor is made as disclosed in Funk et al., Journal of Biological Chemistry, 1993, 268, 26767-26772.
  • the full length EP 2 receptor is made as disclosed in Regan et al., Molecular Pharmacology, 1994, 46, 213-220.
  • the full length EP 3 receptor is made as disclosed in Regan et al., British Journal of Pharmacology, 1994, 112, 377-385.
  • the full length EP 4 receptor is made as disclosed in Bastien, Journal of Biological Chemistry, 1994, 269, 11873-11877. These full length receptors are used to prepare 293S cells expressing the EP 1 , EP 2 , EP 3 and EP 4 receptors. 293S cells expressing either the human EP 1 , EP 2 , EP 3 or EP 4 prostaglandin
  • E 2 receptors are generated according to methods known to those skilled in the art.
  • PCR polymerase chain reaction
  • primers corresponding to the 5' and 3' ends of the published full length receptor are made according to the well known methods disclosed above and are used in an RT-PCR reaction using the total RNA from human kidney (for EP 1 ), human lung (for EP 2 ), human lung (for EP 3 ) or human lymphocytes (for EP 4 ) as a source.
  • PCR products are cloned by the TA overhang method into pCR2.1 (Invitrogen, Carlsbad, CA) and identity of the cloned receptor is confirmed by DNA sequencing.
  • 293S cells (Mayo, Dept. of Biochemistry, Northwestern Univ.) are transfected with the cloned receptor in pcDNA3 by electroporation. Stable cell lines expressing the receptor are established following selection of transfected cells with G418.
  • Clonal cell lines expressing the maximal number of receptors are chosen following a whole cell 3 H-PGE 2 binding assay using unlabeled PGE 2 as a competitor.
  • Sprage-Dawley rats at 3 months of age are anesthetized with Ketamine.
  • a 1 cm incision is made on the anteromedial aspect of the proximal part of the right tibia or femur.
  • the incision is carried through to the bone, and a 1 mm hole is drilled 4 mm proximal to the distal aspect of the tibial tuberosity 2 mm medial to the anterior ridge.
  • Intramedullary nailing is performed with a 0.8 mm stainless steel tube (maximum load 36.3 N, maximum stiffness 61.8 N/mm, tested under the same conditions as the bones). No reaming of the medullary canal is performed.
  • a standardized closed fracture is produced 2 mm above the tibiofibular junction by three-point bending using specially designed adjustable forceps with blunt jaws. To minimize soft tissue damage, care is taken not to displace the fracture.
  • the skin is closed with monofilament nylon sutures. The operation is performed under sterile conditions. Radiographs of all fractures are taken immediately after nailing, and rats with fractures outside the specified diaphyseal area or with displaced nails are excluded. The remaining animals are divided randomly into the following groups with 10 - 12 animals per each subgroup per time point for testing the fracture healing.
  • 10 - 12 rats from each group are anesthetized with Ketamine and sacrificed by exsanguination. Both tibiofibular bones are removed by dissection and all soft tissue is stripped. Bones from 5 - 6 rats for each group are stored in 70% ethanol for histological analysis, and bones from another 5 - 6 rats for each group are stored in a buffered Ringer's solution (+4°C, pH 7.4) for radiographs and biomechanical testing which is performed.
  • Sirius red stained sections are used to demonstrate the characterisitics of the callus structure and to differentiate between woven bone and lamellar bone at the fracture site. The following measurements are performed: (1 ) fracture gap - measured as the shortest distance between the cortical bone ends in the fracture, (2) callus length and callus diameter, (3) total bone volume area of callus, (4) bony tissue per tissue area inside the callus area, (5) fibrous tissue in the callus, and (6) cartilage area in the callus.
  • Biomechanical Analysis The methods for biomechanical analysis have been previously published by Bak and Andreassen (The Effects of Aging on Fracture Healing in Rats. Calcif Tissue lnt 45:292-297, 1989). Briefly, radiographs of all fractures are taken prior to the biomechanical test. The mechanical properties of the healing fractures are analyzed by a destructive three- or four-point bending procedure. Maximum load, stiffness, energy at maximum load, deflection at maximum load, and maximum stress are determined.
  • the fracture side is sawed 3 cm to each side of the fracture line, embedded undecalcified in methymethacrylate, and cut on a Reichert-Jung Polycut microtome in 8 ⁇ m thick of frontal sections.
  • Masson-Trichrome stained mid-frontal sections (including both tibia and fibula) are used for visualization of the cellullar and tissue response to fracture healing with and without treatment. Sirius red stained sections are used to demonstrate the characterisitics of the callus structure and to differentiate between woven bone and lamellar bone at the fracture site.
  • Biomechanical Analysis The methods for biomechanical analysis have been previously published by Bak and Andreassen (The Effects of Aging on Fracture Healing in Rats. Calcif Tissue lnt 45:292-297, 1989) and Peter et al. (Peter, C.P.; Cook, W.O.; Nunamaker, D.M.; Provost, M. T.; Seedor, J.G.; Rodan, G.A. Effects of Alendronate On Fracture Healing And Bone Remodeling In Dogs. J. Orthop. Res. 14:74-70, 1996). Briefly, radiographs of all fractures are taken prior to the biomechanical test. The mechanical properties of the healing fractures are analyzed by a destructive three- or four-point bending procedures. Maximum load, stiffness, energy at maximum load, deflection at maximum load, and maximum stress are determined.
  • Estrogen agonist/antagonists are a class of compounds which inhibit bone turnover and prevent estrogen-deficiency induced bone loss.
  • the ovariectomized rat bone loss model has been widely used as a model of postmenopausal bone loss. Using this model, one can test the efficacy of the estrogen agonist/antagonist compounds in preventing bone loss and inhibiting bone resorption.
  • Sprague-Dawley female rats (Charles River, Wilmington, MA) at different ages (such as 5 months of age) are used in these studies.
  • the rats are singly housed in 20 cm X 32 cm X 20 cm cages during the experimental period. All rats are allowed free access to water and a pelleted commercial diet (Agway ProLab 3000, Agway County Food, Inc., Syracuse, NY) containing 0.97% calcium, 0.85% phosphorus, and 1.05 IU/g of Vitamin D 3
  • a group of rats (8 to 10) are sham-operated and treated p.o. with vehicle (10% ethanol and 90% saline, 1 ml/day), while the remaining rats are bilaterally ovariectomized (OVX) and treated with either vehicle (p.o.), 17 ⁇ -estradiol (Sigma, E- 8876, E 2 , 30 ⁇ g/kg, daily subcutaneous injection), or estrogen agonist/antagonists (such as droloxifene at 5, 10, or 20 mg/kg, daily p.o.) for a certain period (such as 4 weeks). All rats are given subcutaneous injections of 10 mg/kg calcein (fluorochrome bone marker) 12 and 2 days before being sacrificed in order to examine the dynamic changes in bone tissue. After 4 weeks of treatment, the rats are sacrificed and autopsied. The following endpoints are determined:
  • Body Weight Gain Body weight at autopsy minus body weight at surgery.
  • Uterine Weight and Histology The uterus is removed from each rat during autopsy, and weighed immediately. Thereafter, the uterus is processed for histologic measurements such as uterine cross-sectional tissue area, stromal thickness, and luminal epithelial thickness.
  • Total Serum Cholesterol Blood is obtained by cardiac puncture and allowed to clot at 4°C, and then centrifuged at 2,000 g for 10 min. Serum samples are analyzed for total serum cholesterol using a high performance cholesterol calorimetric assay (Boehringer Mannheim Biochemicals, Indianapolis, IN).
  • Femoral Bone Mineral Measurements The right femur from each rat is removed at autopsy and scanned using dual energy X-ray absorptiometry (DEXA, QDR 1000/W, Hologic Inc., Waltham, MA) equipped with "Regional High Resolution Scan” software (Hologic Inc., Waltham, MA).
  • the scan field size is 5.08 x 1.902 cm, resolution is 0.0254 x 0.0127 cm and scan speed is 7.25 mm/second.
  • the femoral scan images are analyzed and bone area, bone mineral content (BMC), and bone mineral density (BMD) of whole femora (WF), distal femoral metaphyses (DFM), femoral shaft (FS), and proximal femora (PF) are determined.
  • BMC bone mineral content
  • BMD bone mineral density
  • Proximal Tibial Metaphyseal Cancellous Bone Histomorphometric Analyses The right tibia is removed at autopsy, dissected free of muscle, and cut into three parts. The proximal tibia is fixed in 70% ethanol, dehydrated in graded concentrations of ethanol, defatted in acetone, then embedded in methyl methacrylate (Eastman Organic Chemicals, Rochester, NY). Frontal sections of proximal tibial metaphyses at 4 and 10 ⁇ m thickness are cut using a Reichert-Jung Polycut S microtome. One 4 ⁇ m and one 10 ⁇ m sections from each rat are used for cancellous bone histomorphometry. The 4 ⁇ m sections are stained with modified Masson's Trichrome stain while the 10 ⁇ m sections remained unstained.
  • a Bioquant OS/2 histomorphometry system (R&M Biometrics, Inc., Nashville, TN) is used for the static and dynamic histomorphometric measurements of the secondary spongiosa of the proximal tibial metaphyses between 1.2 and 3.6 mm distal to the growth plate-epiphyseal junction.
  • the first 1.2 mm of the tibial metaphyseal region is omitted in order to restrict measurements to the secondary spongiosa.
  • the 4 ⁇ m sections are used to determine indices related to bone volume, bone structure, and bone resorption, while the 10 ⁇ m sections are used to determine indices related to bone formation and bone turnover.
  • Total metaphyseal area (TV, mm 2 ): metaphyseal area between 1.2 and 3.6 mm distal to the growth plate-epiphyseal junction.
  • Trabecular bone area (BV, mm 2 ): total area of trabeculae within TV.
  • Trabecular bone perimeter (BS, mm): the length of total perimeter of trabeculae.
  • Trabecular bone volume (BV/TV, %): BV / TV x 100.
  • Trabecular bone number 1.199 / 2 x BS / TV.
  • TBT Trabecular bone thickness
  • Osteoclast number (OCN, #): total number of osteoclast within total metaphyseal area.
  • Osteoclast perimeter OCP, mm
  • Osteoclast number/mm OCN / BS.
  • SLS, mm Single-calcein labeled perimeter labeled with one calcein label.
  • DLS, mm Double-calcein labeled perimeter: total length of trabecular perimeter labeled with two calcein labels.
  • Inter-labeled width (ILW, ⁇ m): average distance between two calcein labels.
  • Bone formation rate/surface ref. (BFR/BS, ⁇ m 2 /d/ ⁇ m): (SLS/2 + DLS) x MAR / BS.
  • Bone turnover rate (BTR, %/y): (SLS/2 + DLS) x MAR / BV x 100.
  • COMBINATION AND SEQUENTIAL TREATMENT PROTOCOL The following protocols can of course be varied by those skilled in the art. For example, intact male or female rats, sex hormone deficient male (orchidectomy) or female (ovariectomy) rats may be used. In addition, male or female rats at different ages (such as 12 months of age) can be used in the studies.
  • the rats can be either intact or castrated (ovariectomized or orchidectomized), and administered to with anabolic agents such as the compounds of this invention at different doses (such as 1 , 3 or 6 mg/kg/day) for a certain period (such as two weeks to two months), and followed by administration of an anti-resorptive agent such as droloxifene at different doses (such as 1,5,10 mg/kg/day) for a certain period (such as two weeks to two months), or a combination treatment with both anabolic agent and anti-resorptive agent at different doses for a certain period (such as two weeks to two months).
  • treatment can be started on the next day after surgery (for the purpose of preventing bone loss) or at the time bone loss has already occurred (for the purpose of restoring bone mass).
  • the rats are sacrificed under ketamine anesthesia.
  • the following endpoints are determined:
  • Femoral bone mineral measurements are performed as described above in the estrogen agonist/antagonist protocol.
  • Lumbar Vertebral Bone Mineral Measurements Dual energy X-ray absorptiometry (QDR 1000/W, Hologic, Inc., Waltham, MA) equipped with a "Regional High Resolution Scan” software (Hologic, Inc., Waltham, MA) is used to determined the bone area, bone mineral content (BMC), and bone mineral density (BMD) of whole lumbar spine and each of the six lumbar vertebrae (LV1 - 6) in the anesthetized rats.
  • the rats are anesthetized by injection (i.p.) of 1 ml/kg of a mixture of ketamine/rompun (ratio of 4 to 3), and then placed on a rat platform.
  • the scan field sized is 6 x 1.9 cm, resolution is 0.0254 x 0.0127 cm, and scan speed is 7.25 mm/sec.
  • the whole lumbar spine scan image is obtained and analyzed. Bone area (BA), and bone mineral content (BMC) is determined, and bone mineral density is calculated (MBC divided by BA) for the whole lumbar spine and each of the six lumbar vertebrae (LV1 - 6).
  • Proximal tibial metaphyseal cancellous bone histomorphometric analyses are performed as described above for in the estrogen agonist/antagonist protocol. Measurements and calculations related to trabecular bone volume and structure are performed as described above in the estrogen agonist/antagonist protocol. Further, measurements and calculations related to bone resorption are also performed as described above in the estrogen agonist/antagonist protocol. Still further, measurements and calculations related to bone formation and turnover are performed as described above in the estrogen agonist/antagonist protocol. Further still, the data obtained is analyzed using the statistical manipulations described above in the estrogen agonist/antagonist protocol.
  • Kidney Regeneration Assay The role of an prostaglandin agonist in kidney regeneration is investigated by the ability of Prostaglandin E 2 (PGE 2 ) or a prostaglandin agonist to induce the expression of Bone Morphogenetic Protein 7 (BMP-7) in wild type 293S cells and in 293S cells transfected with EP 2 .
  • PGE 2 Prostaglandin E 2
  • BMP-7 Bone Morphogenetic Protein 7
  • 293S and EP2 293S cells are grown in Dulbecco's Modified Egale medium (DMEM, Gibco, BRL; Gaithersburg, MD ) .
  • DMEM Dulbecco's Modified Egale medium
  • PGE 2 or an prostaglandin agonist cells are plated at a density of 1.5 x10 6 cells /10 cm dish.
  • OptiMEM OptiMEM
  • DMSO Dulbecco's Modified Egale medium
  • Administration of the compounds of this invention can be via any method which delivers a compound of this invention systemicaliy and/or locally (e.g., at the site of the bone fracture, osteotomy, or orthopedic surgery). These methods include oral routes, parenteral, intraduodenal routes, etc. Generally, the compounds of this invention are administered orally, but parenteral administration (e.g., intravenous, intramuscular, transdermal, subcutaneous, rectal or intramedullary) may be utilized, for example, where oral administration is inappropriate for the target or where the patient is unable to ingest the drug.
  • parenteral administration e.g., intravenous, intramuscular, transdermal, subcutaneous, rectal or intramedullary
  • parenteral administration may be utilized, for example, where oral administration is inappropriate for the target or where the patient is unable to ingest the drug.
  • the compounds are used for the treatment and promotion of healing of bone fractures and osteotomies by the local application (e.g., to the sites of bone fractures of osteotomies) of the compounds of this invention or compositions thereof.
  • the compounds of this invention are applied to the sites of bone fractures or osteotomies, for example, either by injection of the compound in a suitable solvent (e.g., an oily solvent such as arachis oil) to the cartilage growth plate or, in cases of open surgery, by local application thereto of such compounds in a suitable carrier or diluent such as bone-wax, demineralized bone powder, polymeric bone cements, bone sealants, etc.
  • a suitable solvent e.g., an oily solvent such as arachis oil
  • local application can be achieved by applying a solution or dispersion of the compound in a suitable carrier or diluent onto the surface of, or incorporating it into solid or semi-solid implants conventionally used in orthopedic surgery, such as dacron-mesh, gel-foam and kiel bone, or prostheses.
  • the compounds of this invention may also be applied locally to the site of the fracture or osteotomy in a suitable carrier or diluent in combination with one or more of the anabolic agents or bone anti-resorptive agents described above.
  • Such combinations within the scope of this invention can be co-administered simultaneously or sequentially in any order, or a single pharmaceutical composition comprising a polymorphic form of (3-(((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)- amino)-methyl)-phenoxy)-acetic acid and its sodium salt, a prodrug thereof or a pharmaceutical salt of said compound or said prodrug as described above and a second compound as described above in a pharmaceutically acceptable carrier or diluent can be administered.
  • a bone anabolic agent can be used in this invention alone or in combination with an anti-resorptive agent for three months to three years, followed by an anti-resorptive agent alone for three months to three years, with optional repeat of the full treatment cycle.
  • the bone anabolic agent can be used alone or in combination with an anti-resorptive agent for three months to three years, followed by an anti-resorptive agent alone for the remainder of the patient's life.
  • a polymorphic form of (3- (((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-methyl)-phenoxy)-acetic acid and its sodium salt, or a prodrug thereof or a pharmaceutically acceptable salt of the prodrug as described above may be administered once daily and a second compound as described above (e.g., estrogen agonist/antagonist) may be administered daily in single or multiple doses.
  • a second compound as described above e.g., estrogen agonist/antagonist
  • the two compounds may be administered sequentially wherein the polymorphic form of (3-(((4-tert-butyl-benzyl)-(pyridine-3- sulfonyl)-amino)-methyl)-phenoxy)-acetic acid and its sodium salt compound, prodrug thereof or pharmaceutically acceptable salt of the prodrug as described above may be administered once daily for a period of time sufficient to augment bone mass to a level which is above the bone fracture threshold (World Health Organization Study "Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994). Report of a World Health Organization Study Group.
  • the first compound as described above is administered once daily in a rapid delivery form such as oral delivery.
  • the amount and timing of compounds administered will, of course, be dependent on the subject being treated, on the severity of the affliction, on the manner of administration and on the judgment of the prescribing physician.
  • the dosages given below are a guideline and the physician may titrate doses of the drug to achieve the treatment (e.g., bone mass augmentation) that the physician considers appropriate for the patient.
  • the physician must balance a variety of factors such as bone mass starting level, age of the patient, presence of preexisting disease, as well as presence of other diseases (e.g., cardiovascular disease).
  • an amount of a compound of this invention is used that is sufficient to augment bone mass to a level which is above the bone fracture threshold (as detailed in the World Health Organization Study previously cited herein).
  • an effective dosage for the anabolic agents used in this invention described above is in the range of 0.001 to 100 mg/kg/day, preferably 0.01 to 50 mg/kg/day.
  • the following paragraphs provide preferred dosage ranges for various anti- resorptive agents.
  • the amount of the anti-resorptive agent to be used is determined by its activity as a bone loss inhibiting agent. This activity is determined by means of the pharmacokinetics of an individual compound and its minimal versus maximal effective dose in inhibition of bone loss using a protocol such as described above (e.g., Estrogen Agonist/Antagonist Protocol).
  • an effective dosage for an anti-resorptive agent is about 0.001 mg/kg/day to about 20 mg/kg/day.
  • an effective dosage for progestins is about 0.1 to 10 mg per day; the preferred dose is about 0.25 to 5 mg per day.
  • an effective dosage for polyphosphonates is determined by its potency as a bone resorption inhibiting agent of standard assays.
  • Ranges for the daily administration of some polyphosphonates are about 0.001 mg/kg/day to about 20 mg/kg/day.
  • an effective dosage for the treatment of this invention for example the bone resorption treatment of this invention, for the estrogen agonists/antagonists of this invention is in the range of 0.01 to 200 mg/kg/day, preferably 0.5 to 100 mg/kg/day.
  • an effective dosage for droloxifene is in the range of 0.1 to 40 mg/kg/day, preferably 0.1 to 5 mg/kg/day.
  • an effective dosage for raloxifene is in the range of 0.1 to 100 mg/kg/day, preferably 0.1 to 10 mg/kg/day.
  • an effective dosage for tamoxifen is in the range of 0.1 to 100 mg/kg/day, preferably 0.1 to 5 mg/kg/day.
  • an effective dosage for 2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1- yl-ethoxy)-phenoxy]- benzo[b]thiophen-6-ol is 0.001 to 1 mg/kg/day.
  • 1 -(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1 ,2,3,4- tetrahydroisoquinoline is in the range of 0.0001 to 100 mg/kg/day, preferably 0.001 to 10 mg/kg/day.
  • an effective dosage for 4-hydroxy tamoxifen is in the range of
  • the compounds of the present invention are generally administered in the form of a pharmaceutical composition comprising at least one of the compounds of this invention together with a pharmaceutically acceptable vehicle or diluent.
  • the compounds of this invention can be administered individually or together in any conventional oral, parenteral, rectal or transdermal dosage form.
  • a pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the like.
  • Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate are employed along with various disintegrants such as starch and preferably potato or tapioca starch and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
  • binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
  • lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes.
  • compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols.
  • preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols.
  • lactose or milk sugar as well as high molecular weight polyethylene glycols.
  • the compounds of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
  • solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts.
  • aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • these aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.
  • the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
  • dilute sterile, aqueous or partially aqueous solutions are prepared.
  • compositions of the invention may contain 0.1%-95% of the compound(s) of this invention, preferably 1%-70%.
  • the composition or Formulation to be administered will contain a quantity of a compound(s) of this invention in an amount effective to treat the disease/condition of the subject being treated, e.g., a bone disorder.
  • kits comprises two separate pharmaceutical compositions: a compound of Formula I a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug and a second compound as described above.
  • the kit comprises container means for containing the separate compositions such as a divided bottle or a divided foil packet, however, the separate compositions may also be contained within a single, undivided container.
  • the kit comprises directions for the administration of the separate components.
  • kits form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.
  • An example of such a kit is a so-called blister pack.
  • Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed.
  • the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed.
  • the tablets or capsules are sealed in the recesses between the plastic foil and the sheet.
  • the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.
  • a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the dosage form so specified should be ingested.
  • a memory aid is a calendar printed on the card e.g., as follows "First Week, Monday, Tuesday, ...etc.... Second Week, Monday, Tuesday, etc.
  • a "daily dose” can be a single tablet or capsule or several tablets or capsules to be taken on a given day.
  • a daily dose of a Formula I compound, a prodrug thereof or a pharmaceutically acceptable salt of said compound or said prodrug can consist of one tablet or capsule while a daily dose of the second compound can consist of several tablets or capsules and vice versa.
  • the memory aid should reflect this.
  • a dispenser designed to dispense the daily doses one at a time in the order of their intended use.
  • the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen.
  • a memory-aid is a mechanical counter which indicates the number of daily doses that has been dispensed.
  • a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.
  • Hard gelatin capsules are prepared using the following:
  • a tablet Formulation is prepared using the ingredients below: Formulation 2: Tablets
  • the components are blended and compressed to form tablets.
  • tablets each containing 0.25-100 mg of active ingredients are made up as follows: Formulation 3: Tablets
  • Talc 1 The active ingredients, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly.
  • the solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve.
  • the granules so produced are dried at 50° - 60 0 C and passed through a No. 18 mesh U.S. sieve.
  • the sodium carboxymethyl starch, magnesium stearate, and talc previously passed through a No. 60 U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.
  • the active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form smooth paste.
  • the benzoic acid solution, flavor, and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.
  • Aerosol solution is prepared containing the following ingredients: Formulation 5: Aerosol
  • Propellant 22 (Chlorodifluoromethane) 70.00
  • the active ingredient is mixed with ethanol and the mixture added to a portion of the propellant, cooled to 30 0 C, and transferred to a filling device.
  • the required amount is then fed to a stainless steel container and diluted with the remaining propellant.
  • the valve units are then fitted to the container.
  • Suppositories are prepared as follows: Formulation 6: Suppositories
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimal necessary heat. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.
  • An intravenous Formulation is prepared as follows: Formulation 7: Intravenous Solution
  • the solution of the above ingredients is intravenously administered to a patient at a rate of about 1 mL per minute.

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Abstract

La présente invention concerne des formes cristallines polymorphes ou une forme non cristalline ou amorphe du composé sel de sodium d'acide acétique (3-(((4-tert-butyl-benzyl)-(pyridine- 3-sulfonyl)-amino)-méthyl)-phénoxy) ou un hydrate de celui-ci, ainsi que des procédés de préparation de celles-ci, des procédés d'utilisation de celles-ci et des compositions pharmaceutiques les contenant. L'invention concerne également des formes cristallines polymorphes sensiblement pures ou une forme non cristalline ou amorphe du sel de sodium d'acide acétique (3- (((4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino)-méthyl)-phénoxy) ou un hydrate de celui-ci.
EP08806952A 2007-08-29 2008-08-25 Formes polymorphes d'agonistes de prostaglandine et procédés de fabrication de celles-ci Withdrawn EP2185516A2 (fr)

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