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WO2008140450A1 - Méthodes de traitement et de prévention de maladies et de troubles osseux métaboliques - Google Patents

Méthodes de traitement et de prévention de maladies et de troubles osseux métaboliques Download PDF

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WO2008140450A1
WO2008140450A1 PCT/US2007/011391 US2007011391W WO2008140450A1 WO 2008140450 A1 WO2008140450 A1 WO 2008140450A1 US 2007011391 W US2007011391 W US 2007011391W WO 2008140450 A1 WO2008140450 A1 WO 2008140450A1
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bone
ethyl
pla
oxo
methyl
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Yi Shi
Theresa Freeman
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Thomas Jefferson University
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Thomas Jefferson University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention relates generally to methods for the treatment and/or prevention of metabolic bone disease or disorders, and more particularly to treatment and/or prevention of metabolic bone diseases or disorders associated with loss of bone mass and density, such as osteoporosis and osteopenic diseases using agents that inhibit the expression and/or activity of Lp-PLA 2 protein.
  • Lipoprotein-Associated Phospholipase A2 (Lp-PLA 2 ), also previously known in the art as Platelet Activating Factor Acetly Hydrolase (PAF acetyl hydrolase) is a member of the super family of phospholipase A2 enzymes that are involved in hydrolysis of lipoprotein lipids or phospholipids. It is secreted by several cells that play a major role in the systemic inflammatory response to injury, including lymphocytes, monocytes, macrophage, T Lymphocytes and mast cells.
  • PAF acetyl hydrolase Platelet Activating Factor Acetly Hydrolase
  • Lp-PLA 2 is responsible for hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso- phosphatidylcholine and an oxidatively modified fatty acid.
  • Lp-PLA 2 hydrolyzes the sn2 position of a truncated phospholipid associated with oxidized LDL.
  • NEFA non-esterfied fatty acids
  • LYSO PC chematractants for circulating monocytes, play a role in the activation of macrophages and increase oxidative stress as well as affecting the functional and the immediate responses of T lymphocytes.
  • Lp-PLA 2 is bound in humans and pigs to the LDL molecule via lipoprotein B, and once in the arterial wall the oxidized LDL is susceptible to hydrolysis by Lp-PLA 2 .
  • Lp-PLA 2 plays a direct role in LDL oxidation.
  • Lp- PLA 2 inhibitors inhibit LDL oxidation.
  • Lp-PLA 2 inhibitors may therefore have a general application in any disorder that involves lipid peroxidation in conjunction with the enzyme activity, for example in addition to conditions such as atherosclerosis and diabetes other conditions such as rheumatoid arthritis, myocardial infarction and reperfusion injury.
  • Lp-PLA 2 is responsible for hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso-phosphatidylcholine (lysoPC) and an oxidatively modified fatty acid. Both of these products Of Lp-PLA 2 action are potent chemoattractants for circulating monocytes. Therefore, Lp-PLA 2 is thought to be responsible for the accumulation of cells loaded with cholesterol ester in the arteries, characteristic of atherosclerosis. [7] Osteopenia and osteoporosis are characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures.
  • Osteoporosis affects 44 million Americans, or 55 percent of the people 50 years of age and older. One in two women and one in four men over age 50 will have an osteoporosis-related fracture in her/his remaining lifetime. Osteopenia and osteoporosis are responsible for more than 1.5 million fractures annually. The estimated national direct expenditures (hospitals and nursing homes) for osteoporotic hip fractures were $18 billion dollars in 2002, and the cost is rapidly rising.
  • Some approaches for the treatment of bone disorders such as osteoporosis include, for example, estrogens, bisphosphonates, calcitonin, flavonoids, and selective estrogen receptor modulators. Other approaches include peptides from the parathyroid hormone family, strontium ranelate, and growth hormone and insulin-like growth response (see, for example, Reginster et al. "Promising New Agents in Osteoporosis," Drugs R & D 1999, 3, 195-201). Ill] The variety of different approaches represented by the therapeutic agents currently available or under study evidence the variety of biological factors influencing the competing processes of bone production and resorption.
  • the present invention relates to methods for treating and preventing metabolic bone diseases and disoders by inhibiting Lp-PLA 2 , including inhibiting the expression and/or activity of Lp-PLA 2 .
  • Metabolic bone diseases and disorders amenable to treatment and/or prevention by the methods of the present invention are diseases and disoders associated with loss of bone mass and density and include but are not limited osteoporosis and osteopenic related diseases. Osteoporosis and osteopenic related diseases include but are not limited to Paget's diseases, hyperparathyroidism and related diseases.
  • the methods as disclosed herein comprise administering to a patient in need thereof for treating or preventing a metabolic bone disease, a pharmaceutical composition comprising an effective amount of an agent which inhibits Lp-PLA 2 , for example an agent which inhibits the expression OfLp-PLA 2 and/or the activity OfLp-PLA 2 protein. It is not intended that the present invention to be limited to any particular stage of the disease (e.g. early or advanced). [14
  • some embodiments provide methods for inhibiting Lp-PLA 2 by blocking enzyme activity and some embodiments provide methods for inhibiting Lp-PLA 2 by reducing and/o ⁇ downregulating the expression Of Lp-PLA 2 RNA.
  • preventing and/or reducing loss of bone mass and/or loss of bone density leads to preventing or reducing symptoms associated with metabolic bone diseases or disorders such as osteoporosis and/or osteopenia diseases.
  • the methods as disclosed herein provide methods of treating and/or preventing a metabolic bone disorder or disease in a patient, such as a human patient, wherein the methods comprise administering to the patient in need thereof a pharmaceutical composition comprising an effective amount of an agent that inhibits the activity and/or expression of the Lp-PLA2 protein.
  • a metabolic bone disease or disorder includes metabolic bone diseases and disorders associated with loss of bone mass and/or loss of bone density.
  • Such metabolic bone diseases include but are not limited to osteoporosis and osteopenic related diseases such as diseases with bone marrow abnormalities.. These include, including dyslipidemia, type II diseases, metabolic syndrome, insulin resistance, Paget's disease, hyperparathyroidism and related diseases.
  • the patient administered an effective amount of an agent that inhibits the activity or expression of the Lp-PLA 2 protein is a human.
  • the method of this invention can be effected by administering an effective amount of a reversible or irreversible Lp-PLA 2 inhibitor.
  • reversible inhibitors are small molecules and compounds such as l-(N-(2-(diethylamino)ethyl)-N-(4-(4- trifluoromethylphenyl)benzyl)-aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6- trimethylenepyrimidin-4-one (which is also known as SB480848).
  • the method of this inventon comprises identifying a patient at risk for developing a clinical degree of a metabolic bone disease such as osteopenia or having a clinical degree of osteopenia and, for those at risk or having such a condition, administering an effective amount of an Lp-PLA 2 inhibitor to those in need thereof, and,optionally, monitoring the effectiveness of the treatment.
  • Bone density and biomarkers can be used to identify patients who could benefit from the therapy as disclosed herein.
  • the methods can further comprise administering additional therapeutic agents used in the treatment of metabolic bone diseases.
  • the metabolic bone disorder is osteoporosis
  • bisphosphates such as alendronate, ibandronate, risedronate, calcitonin, raloxifene, a selective estrogen modulator (SERM), estrogen therapy, hormone replacement therapy (ET/HRT) and teriparatide.
  • SERM selective estrogen modulator
  • E/HRT hormone replacement therapy
  • teriparatide teriparatide
  • FIG. 1 shows the region of medial femoral condyle scanned and analyzed by micro CT.
  • Bold dashed line represents cut plane. Area to the right indicates wedge of medial femoral condyle scanned. Dotted line box indicates approximate area of analysis.
  • Figure 2 shows examples of diabetes mellitus/hypercholesteremia (DM/HC)-induced osteoporotic changes and the effects of the treatment in trabecular bone.
  • Panel 2A shown bone matrix density on non-treated non-DM/HC control animals
  • panel Fig 2B shows an example from a DM/HC non-treated animals as compared to panel Fig 2C showing DM/HC animals treated with an inhibitor of Lp-PLA 2 .
  • Figure 3 shows examples of DM/HC-induced changes in the trabecular bone environment, as shown in Fig 3B as compared to control non-DM/HC animals, shown in Fig
  • Figure 4 shows the Lp-PLA 2 inhibitor restored normal bone marrow and abnormalities in bone marrow such abnormal bone marrow homeostasis.
  • DM/HC-induced changes in the trabecular bone environment are shown in panels Fig4A-D as compared to DM/HC animals administered an Lp-PLA 2 inhibitor, which are shown in panels Fig4 E-G.
  • Figure 5 shows the Lp-PLA 2 inhibitor preserved normal bone marrow and prevented abnormal bone marrow, such as as abnormal bone marrow homeostasis in remote trabacular bone.
  • Examples of low magnification images of DM/HC-induced changes in the trabecular bone environment are shown in panels Fig5A and B as compared to DM/HC animals administered a Lp-PLA 2 inhibitor, which are shown in panels Fig5 C and D.
  • Figure 6 shows the Lp-PLA 2 inhibitor preserved normal bone marrow and prevented abnormal bone marrow, such as as abnormal bone marrow homeostasis in remote trabacular bone.
  • Examples of high magnification images of DM/HC-induced changes in the trabecular bone environment are shown in panels Fig ⁇ A-C as compared to DM/HC animals administered a Lp-PLA 2 inhibitor, which are shown in panels Fig 6 D-E.
  • Figure 7 shows examples of DM/HC-induced TUNEL positive cells within the trabecular bone environment as shown in panels Fig 7A-D, as compared to the effects of animals treated with the Lp-PLA 2 inhibitor SB480848, as shown in Fig 7 E-H.
  • Figure 8 shows the results of MLO- A5 cells treated with 5 ⁇ M LysoPC after 7 days, showing in panel B reduced mineralization in LysoPC treated cells as compared to control treatment in Panel 8A which has increased intensity of Alizarin red staining.
  • Figure 9 shows MLO-A5 cells treated with 5 ⁇ M LysoPC after 7 days show reduced alkaline phosphatase staining.
  • Figure 9A and 9B show examples of alkaline phosphatase staining in control treated cells
  • Figure 9C and 9D show examples of alkaline phosphatase staining in cells treated with 5 ⁇ M LysoPC
  • Figure 9E and 9F show examples of alkaline phosphatase staining in cells treated with 20 ⁇ M LysoPC, showing reduced alkaline phosphatase staining of MLO-A5 cells treated with at least 5 ⁇ M LysoPC at 7 days as compared to control cells at 7 days.
  • Figure 10 shows the results from the proliferation assay, showing MLO-A5 cells treated with 5 ⁇ M LysoPC for 18 hours show reduced proliferation.
  • the inventors have discovered that animals prone to pathological features of metabolic bone diseases exhibit reduced loss of bone density and bone mass when treated with an Lp-PLA 2 inhibitor.
  • Animals treated with an Lp-PLA 2 inhibitor showed increased bone density and reduced death of osteocytes and osteoblasts as compared to animals not treated with the Lp-PLA 2 inhibitor.
  • the animals treated with an Lp-PLA 2 inhibitor also had normal trabecular bone marrow as compared to animals not treated with the Lp-PLA 2 inhibitor. The latter showed trabecular bone marrow abnormalities such as increased extracellular material, reduced cellularity and shrinkage of adipocytes, demonstrating pathological signs of abnormal bone marrow homeostasis.
  • Lp-PLA 2 inhibitors can be used in treating or preventing metabolic bone diseases and disorders, particularly metabolic bone diseases and disorders associated with loss of bone mass and/or loss of bone density.
  • metabolic bone diseases include osteoporosis and osteopenic related diseases caused by or associated with dyslipidemia, type II diseases, metabolic syndrome, insulin resistance, Paget's disease, and hyperparathyroidism and related diseases.
  • Metabolic bone disease refers to a varied assortment of bone diseases and disorders characterized by gradual and progressive loss of bone tissue. Metabolic bone diseases amenable to prevention and/or treatment using the methods as described herein are metabolic bone diseases whereby there is a condition of diffusely decreased bone density and/or diminished bone strength. Such diseases are characterized by histological appearance. Two examples are osteoporosis which is a common metabolic bone disorder characterized by decreased mineral and bone matrix, and osteomalacia which is characterized by decreased mineral but intact bone matrix.
  • osteoopenic diseases or "osteopenia” are used interchangeably herein, and refer to conditions with decreased calcification and/or bone density, and is a descriptive term used to refer to all skeletal systems in which the condition is noted. Osteopenia also refers to a reduced bone mass due to inadequate osteiod synthesis.
  • osteoporosis refers to conditions in which decreased mineral or bone matrix and reduced bone mass occurs.
  • disease or “disorder” is used interchangeably herein, and refers to any alteration in state of the body or of some of the organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with a person.
  • a disease or disorder can also relate to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, inderdisposion or affectation.
  • abnormalities in bone marrow or "abnormal bone marrow” is used to refer to a dysfunctional or abnormal morphological characteristic of bone marrow, for example where the bone marrow comprises loss or death of osteocytes and/or osteoblasts, resulting in, for example fast bone turn over or reduced bone formation.
  • Abnormal bone marrow can also be used to refer to presence of cells or structures not typically present in normal bone marrow, and/or altered morphology of cells present in the bone marrow, for example presence of increased extracellular material, or altered morphology of adipocytes or reduced numbers of cells present in the bone marrow.
  • Bone marrow abnormalities referred to herein are defined as those where general abnormalities of the biological balance in the bone marrow is indicated, for example, including but not limited to viral or bacterial infections in the bone marrow, cellular infiltration of the bone marrow, abnormalities of the bone marrow haematopoiesis, proliferation of malignant neoplasms in the bone marrow and concentration changes in cell growth differentiaion factors.
  • agent refers to any entity which is normally not present or not present at the levels being administered in the cell. Agent can be selected from a group comprising: chemicals; small molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; aptamers; antibodies; or fragments thereof.
  • a nucleic acid sequence can be RNA or DNA, and can be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc.
  • PNA peptide-nucleic acid
  • pc-PNA pseudo-complementary PNA
  • LNA locked nucleic acid
  • nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.
  • a protein and/or peptide or fragment thereof can be any protein of interest, for example, but are not limited to: mutated proteins; therapeutic proteins and truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell.
  • Proteins can also be selected from a group comprising; mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, midibodies, minibodies, triabodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof.
  • the agent can be intracellular within the cell as a result of introduction of a nucleic acid sequence into the cell and its transcription resulting in the production of the nucleic acid and/or protein inhibitor of Lp-PLA 2 within the cell.
  • the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities.
  • the agents of particular interest are small molecules that reversibly or irreversibly inhibit Lp-PLA 2 . These include unsubstituted or substituted alkyl, aromatic, or heterocyclyl compounds as further illustrated below.
  • inhibiting means that the expression or activity of Lp-PLA 2 or variants or homologues thereof is reduced to an extent, and/or for a time, sufficient to produce the desired effect. The reduction in activity can be due to affecting one or more characteristics of Lp-PLA 2 including decreasing its catalytic activity or by inhibiting a co- factor of Lp-P LA 2 or by binding to Lp-PLA 2 with a degree of avidity that is such that the outcome is that of treating or preventing a metabolic bond disorder.
  • Lp-PLA 2 inhibition of Lp-PLA 2 can be determined using an assay for Lp-PLA 2 inhibition by using the bioassay for Lp-PLA 2 protein as disclosed herein.
  • Lp-PLA 2 refers to the protein target to be inhibited by the methods as disclosed herein.
  • Lp-PLA 2 is used interchangeably with lipoprotein associated phospholipase A 2 , also previously known in the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase).
  • PAF acetyl hydrolase Platelet Activating Factor Acetyl Hydrolase
  • Human Lp-PLA 2 is encoded by nucleic acid corresponding to accession No: U20157 (SEQ ID NO:1) or Ref Seq ID: NM_005084 (SEQ ID NO:2) or and the human Lp-PLA 2 corresponds to protein sequence corresponding to accession No: NP_005075 (SEQ ID NO:3), which are disclosed in U.S. Patent 5,981,252, which is specifically incorporated herein in its entirety by reference.
  • siRNA silencing or “gene silenced” in reference to an activity of n RNAi molecule, for example a siRNA or miRNA refers to a decrease in the mRNA level in a cell for a target gene by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of the mRNA level found in the cell without the presence of the miRNA or RNA interference molecule.
  • the mRNA levels are decreased by at least about 70%, about 80%, about 90%, about 95%, about 99%, about 100%.
  • RNAi refers to any type of interfering RNA, including but are not limited to, siRNAi, shRNAi, endogenous microRNA and artificial microRNA. For instance, it includes sequences previously identified as siRNA, regardless of the mechanism of down-stream processing of the RNA (i.e. although siRNAs are believed to have a specific method of in vivo processing resulting in the cleavage of mRNA, such sequences can be incorporated into the vectors in the context of the flanking sequences described herein).
  • RNA refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA is present or expressed in the same cell as the target gene, for example Lp-PLA 2 .
  • the double stranded RNA siRNA can be formed by the complementary strands.
  • a siRNA refers to a nucleic acid that can form a double stranded siRNA.
  • the sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof.
  • the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is about 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 19-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).
  • shRNA small hairpin RNA
  • stem loop is a type of siRNA.
  • shRNAs are composed of a short, e.g. about 19 to about 25 nucleotide, antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand.
  • the sense strand can precede the nucleotide loop structure and the antisense strand can follow.
  • microRNA or "miRNA” are used interchangeably herein are endogenous RNAs, some of which are known to regulate the expression of protein-coding genes at the posttranscriptional level. Endogenous microRNA are small RNAs naturally present in the genome which are capable of modulating the productive utilization of mRNA.
  • artificial microRNA includes any type of RNA sequence, other than endogenous microRNA, which is capable of modulating the productive utilization of mRNA. MicroRNA sequences have been described in publications such as Lim, et ah, Genes & Development, 17, p.
  • miRNA-like stem-loops can be expressed in cells as a vehicle to deliver artificial miRNAs and short interfering RNAs (siRNAs) for the purpose of modulating the expression of endogenous genes through the miRNA and or RNAi pathways.
  • siRNAs short interfering RNAs
  • double stranded RNA or “dsRNA” refers to RNA molecules that are comprised of two strands. Double-stranded molecules include those comprised of a single RNA molecule that doubles back on itself to form a two-stranded structure. For example, the stem loop structure of the progenitor molecules from which the single-stranded miRNA is derived, called the pre-miRNA (Bartel et al. 2004. Cell 1 16:281 -297), comprises a dsRNA molecule.
  • the terms "patient”, “subject” and “individual” are used interchangeably herein and refer to an animal, particularly a human, to whom treatment is provided.
  • the term "gene” used herein can be a genomic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (e.g., introns, 5'- and 3'- untranslated sequences and regulatory sequences).
  • the coding region of a gene can be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA and antisense RNA.
  • a gene can also be an mRNA or cDNA corresponding to the coding regions (e.g. exons and miRNA) optionally comprising 5 f - or 3' untranslated sequences linked thereto.
  • a gene can also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3'- untranslated sequences linked thereto.
  • nucleic acid or "oligonucleotide” or “polynucleotide” used herein can mean at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a nucleic acid also encompasses the complementary strand of a depicted single strand.
  • many variants of a nucleic acid can be used for the same purpose as a given nucleic acid.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a single strand provides a probe for a probe that can hybridize to the target sequence under stringent hybridization conditions.
  • a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence.
  • the nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo- nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and ⁇ soguanine.
  • Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.
  • a nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs can be included that can have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages.
  • Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U-S. Pat. Nos. 5, 235,033 and 5, 034,506, which are incorporated by reference.
  • Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids.
  • the modified nucleotide analog can be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule.
  • Representative examples of nucleotide analogs can be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase- modified ribonucleotides, i.e. ribonucleotides, containing a non naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g.
  • the 2' OH- group can be replaced by a group selected from H. OR, R. halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is C- C6 alkyl, alkenyl or alkynyl and halo is F. Cl, Br or I.
  • Modifications of the ribose- phosphate backbone can be done for a variety of reasons, e.g., to increase the stability and half- life of such molecules in physiological environments or as probes on a b ⁇ ochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs can be made.
  • vector refers to a nucleic acid sequence containing an origin of replication.
  • a vector can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector can be a DNA or RNA vector.
  • a vector can be either a self replicating extrachromosomal vector or a vector which integrate into a host genome.
  • treating includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a metabolic bone disease or disorder such as osteoporosis and osteopenic related disorders.
  • treating refers to the reduction of a symptom and/or a biochemical marker of a metabolic bone disease or disorder by some useful amount, an amount that can be determined by one skilled in the art.
  • Treating with regards to osteoporosis refers to a measurable reduction in a biochemical marker of osteoporosis, or a reduction in the death or loss of osteocytes and/or osteoblasts, i.e. such results would be considered effective treatments by the methods as disclosed herein.
  • treating with regards to osteoporisis refers to a measurable increase in bone or bone matrix mineralization, such as a measurable increase in re-mineralization.
  • a reduction in a symptom such as a measurable slowing of the rate of decline of bone density or a measurable cessation of the rate bone density loss, or a measurable increase in bone density would also be considered as affective treatments by the methods as disclosed herein.
  • the term "effective amount" as used herein refers to the amount of agent that reduces or stops at least one symptom of the metabolic bone disease or disorder.
  • An example of an effective amount would be considered as the amount sufficient to reduce a symptom of the disease or disorder by some measurable amount.
  • One possible measure for treating or preventing osteoporosis or a metabolic bone disorder is a reduction in the measured parameter of at least about 10%.
  • An effective amount as used herein would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease or reverse a symptom of the disease.
  • vectors are used interchangeably with "plasmid” to refer to a nucleic acid molecule capable of transporting another nucleic acid to which, it has been linked.
  • Vectors capable of directing the expression of genes and/or nucleic acid sequence to which they are operatively linked are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids” which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • expression vectors can be used in different embodiments of the invention, for example, but are not limited to, plasmids, episomes, bacteriophages or viral vectors, and such vectors can integrate into the host's genome or replicate autonomously in the particular cell.
  • Other forms of expression vectors known by those skilled in the art which serve the equivalent functions can also be used.
  • Expression vectors comprise expression vectors for stable or transient expression encoding the DNA.
  • Lp-PLA 2 is also referred to in the art as aliases Lp-PLA 2 , LDL-PLA 2 , lipoprotein associated phospholipase A 2 , PLA2G7, phospholipase A 2 (group VII), or Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase or PAFAH).
  • Human Lp-PLA 2 is encoded by nucleic acid corresponding to GenBank Accession No: U20157 (SEQ ID NO:1) or Ref Seq ID: NM_005084 (SEQ IO NO:2) and the human Lp-PLA 2 corresponds to protein sequence corresponding to GenBank Accession No: NP 005075 (SEQ ID NO:3), which are disclosed in U.S. Patent 5,981,252, which is specifically incorporated herein in its entirety by reference.
  • Phospholipase A2 enzyme Lipoprotein Associated Phospholipase A2 (L ⁇ -PLA2), the sequence, isolation and purification thereof, isolated nucleic acids encoding the enzyme, and recombinant host cells transformed with DNA encoding the enzyme are disclosed in WO 95/00649 (SmithKline Beecham pic), which is specifically incorporated herein in its entirety by reference.
  • LDL-PLA2 a subsequent publication from the same group further describes this enzyme (Tew D et al, Arterioscler Thromb Vas Biol 1996:16; 591-9) wherein it is referred to as LDL-PLA2 and later patent application (WO 95/09921, Icos Corporation) and a related publication in Nature (Tjoelker et al, vol 374, 6 April 1995, 549) describe the enzyme PAF- AH which has essentially the same sequence as Lp-PLA2- [57] It has been shown that Lp-PLA2 is responsible for the conversion of phosphatidylcholine to lysophosphatidylcholine, during the conversion of low density lipoprotein (LDL) to its oxidised form.
  • LDL low density lipoprotein
  • the enzyme is known to hydrolyse the sn-2 ester of the oxidised phosphatidylcholine to give lysophosphatidylcholine and an oxidatively modified fatty acid.
  • Both products of Lp-PLA2 action are biologically active with lysophosphatidylcholine, in particular having several pro-atherogenic activities ascribed to it including monocyte chemotaxis and induction of endothelial dysfunction, both of which facilitate monocyte-derived macrophage accumulation within the artery wall.
  • the inventors have discovered that animals prone to disorders characterized by metabolic bone disorders have been found to exhibit normal or close to normal bone matrix normal bone density and normal bone marrow characteristics when treated with an Lp-PLA 2 inhibitor.
  • Lp-PLA 2 inhibitors can be used to treat and/or prevent metabolic bone diseases and disorders such as osteoporosis and osteopenic related diseases bone marrow abnormalities and Paget's disease.
  • Agents that inhibit Lp-PLA? [59J
  • the present invention relates to the inhibition OfLp-PLA 2 .
  • inhibition is inhibition of nucleic acid transcripts encoding Lp-PLA 2 such as by inhibition of messenger RNA (mRNA).
  • inhibition Of Lp-PLA 2 is effected by the inhibition of the expression and/or inhibition of activity of the gene product Of Lp-PLA 2 , including the polypeptide or protein of Lp-PLA 2 Or its isoforms.
  • gene product refers to RNA transcribed from a gene, or a polypeptide encoded by a gene or translated from RNA.
  • inhibition of Lp-PLA 2 is by an agent such as nucleic acids, nucleic acid analogues, peptides, phage, phagemids, polypeptides, peptidomimetics, ribosomes, aptamers, antibodies, small or large organic or inorganic molecules, or any combination thereof.
  • agents include agents that function as inhibitors OfLp-PLA 2 expression, inhibitors of mRNA encoding Lp-PLA 2 being one example.
  • Agents useful in the methods as disclosed herein can also inhibit gene expression (i.e. suppress and/or repress the expression of the gene).
  • Such agents are referred to in the art as “gene silencers” and are commonly known to those of ordinary skill in the art.
  • Examples include a nucleic acid sequence, for an RNA, DNA or nucleic acid analogue, and can be single or double stranded, and can be selected from a group comprising nucleic acid encoding a protein of interest, oligonucleotides, nucleic acids, nucleic acid analogues such as peptide nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acids (LNA) and derivatives thereof etc.
  • PNA peptide nucleic acid
  • pc-PNA pseudo-complementary PNA
  • LNA locked nucleic acids
  • Nucleic acid agents also include, but are not limited to nucleic acid sequences encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (miRNA), antisense oligonucleotides,etc.
  • agents useful in the method as inhibitors Of Lp-PLA 2 expression and/or inhibition of Lp-P LA2 function can be any type of entity. These include small molecules nucleic acid sequences, nucleic acid analogues, proteins, peptides or fragments thereof. Small molecules [63
  • Irreversible inhibitors of Lp-PLA 2 are disclosed in patent applications WO 96/13484, WO96/19451 , WO 97/02242, WO97/217675, WO97/217676, WO 97/41098, and WO97/41099 (SmithKline Beecham pic) which are specifically incorporated in their entirety herein by reference and disclose inter alia various series of 4-thionyl/sulfinyl/sulfonyl azetidinone compounds which are inhibitors of the enzyme Lp-PLA2- These are irreversible, acyJating inhibitors (Tew et al, Biochemistry, 37, 10087, 1998).
  • Synthetic small molecule Lp-PLA 2 inhibitors effective in humans are commonly known by persons of ordinary skill and include those undergoing pre-clinical and clinical. A number of applications have been filed and published by SmithKline Beecham and its successor GlaxoSmithKline.
  • Lp-PLA 2 inhibitors useful in the methods as disclosed herein are described in published patent applications, for example WO2006063791-A1, WO2006063811-Al, WO2006063812-A1 , WO2006063813-A1, all in the name of Bayer Healthcare; and US2006106017-A1 assigned to Korea Res. Inst. Bioscience & Biotechnology, which are specifically incorporated in their entirety herein by reference.
  • Such Lp-PLA 2 agents can also be admininstered with the Lp-PLA 2 inhibitor agents as disclosed herein.
  • any or all of the compounds disclosed in these documents are useful for prophylaxis or treatment of metabolic bone disorders including preventing or treating osteoporosis or osteopenic diseases.
  • the porcine model of decreased bone density as described herein below and exemplified in the Methods can be used by one of ordinary skill in the art to determine which of the disclosed compounds or other inhibitors OfLp-PLA 2 , for example antibodies, or RNAi are effective for the treatment and/or prevention of metabolic bone diseases or disorders as claimed herein.
  • agents inhibiting Lp-PLA 2 can be assessed in animal models for effect on increasing bone density and/or bone mass.
  • the porcine model of hyperglycemia and hypercholesterolemia as disclosed in the Examples herein, where the bone marrow is abnormal and having decreased bone density, for example the decreased bone matrix, in which the bone matrix can be assessed in the present and absence of inhibitors for Lp-PLA 2 by methods commonly known by persons in the art.
  • assessment of bone density and/or markers of metabolic bone diseases such as osteoporosis can be used, as disclosed herein.
  • Lp-PLA 2 inhibitors as disclosed in U.S. patent 6,649,619 and 7,153,861, which are specifically incorporated in their entirety herein by reference (and International Application WO 01/60805) and U.S. patent 7,169,9 2 4 which is incorporated in its entirety herein by reference (and International Patent Application WO 02/3091 1), are useful in the methods disclosed herein for the prophylaxis or for the treatment of metabolic bone diseases or disorder such as osteoporosis or osteopenia.
  • the Lp- PLA 2 inhibitors as disclosed in U.S. publication No.
  • R2 is phenyl, substituted by one to three fluorine atoms; 175) R 3 is methyl or C(i_3)alkyl substituted by NR 8 R 9 ; or
  • R ⁇ and R ⁇ which can be the same or different are selected from the group consisting of hydrogen, or C(i_g)alkyl);
  • R ⁇ is an aryl group, optionally substituted by 1 , 2, 3 or 4 substituents which can be the same or different selected "from Cn .g- j alkyl, C ⁇ _6)alkoxy, Cn _6)alkylthio, hydroxy, halogen, CN, and mono to perfluoro-Cn _4)alkyl;
  • R ⁇ is halogen, C(i_3)alkyl, C(]_3)alkoxy, hydroxyC(i_3)alkyl, C(i_3)alkylthio., C(l-3)alkylsulphinyl, aminoC(i_3)alkyl s mono- or di-C(i_3)alkylaminoC(i_3)alkyl, C(i_3 ⁇ alkylcarbonylaminoC(i_3 ⁇ alkyl, C ⁇ _3)alkoxyC([_3)alkylcarbonylaminoC/'i_3)alkyl, C( i _3)alkylsulphonylaminoC( ⁇ _3)alkyl, C ⁇ ] _3)alkylcarboxy, C(i _3)alkylcarboxyC(i _3)alkyl, and
  • R 3 is hydrogen, halogen, C(i_3)alkyl, or hydroxyC(i_3)alkyl; or [1021 R2 and R ⁇ together with the pyrimidone ring carbon atoms to which they are attached form a fused 5-or 6-membered carbocyclic ring; or
  • R4 is hydrogen, C(i_6)alkyl which can be unsubstituted or substituted by 1, 2 or 3 substituents selected from hydroxy, halogen, OR 7 , COR 7 , carboxy, COOR 7 , CONR 9 R 10 , NR 9 R 1 O, NR 7 COR 8 , mono- or di-(hydroxyC(] _6)alkyl)amino and N-hydroxyC(i_5)alkyl- N-C(I -6) al kylamino; or
  • R- ⁇ is Het-C(0-4) a lkyl in which Het is a 5- to 7- membered heterocyclyl ring comprising N and optionally O or S, and in which N can be substituted by COR 7 , COOR 7 , CONR 9 R 10 , or Cn .gjalkyl optionally substituted by 1, 2 or 3 substituents selected from hydroxy, halogen, OR 7 , COR 7 , carboxy, COOR 7 , CONR 9 R 10 or NR 9 R 10 , for instance, piperidin-4-yl, pyrrolidin-3-yl;
  • R ⁇ is an aryl or a heteroaryl ring optionally substituted by 1 , 2, 3 or 4 substituents which can be the same or different selected from C(i_g)alkyl, Cn .g ⁇ alkoxy, C ⁇ _5)alkylthio, arylC(i_6)alkoxy, hydroxy, halogen, CN, COR 7 , carboxy, COOR 7 , NR 7 CORS, CONR 9 R 10 , S ⁇ 2NR 9 Rl °, NR 7 S ⁇ 2R 8 , NR 9 R 10 , mono to perfluoro-C(]_4)alkyl and mono to perfluoro- C( i _4)alkoxy;
  • RO is an aryl or a heteroaryl ring which is further optionally substituted by 1, 2, 3 or 4 substituents which can be the same or different selected from Cn
  • R 7 is hydrogen or C(i _ 12)alkyl, for instance C( ⁇ _4)alkyl (e.g. methyl or ethyl);
  • R 8 is hydrogen, OC(j_6)alkyl, or C(]_i2)alkyU for instance C( ⁇ _4)alkyl (e.g. methyl or ethyl); 11101 R ⁇ an( i R ⁇ which can be the same or different is each selected from hydrogen, or Cn _i2) a 'kyU or R ⁇ and R ⁇ together with the nitrogen to which they are attached form a 5- to 7 membered ring optionally containing one or more further heteroatoms selected from oxygen, nitrogen and sulphur, and optionally substituted by one or two substituents selected from hydroxy, oxo, Cn _4 ⁇ alkyl, Cn -4)alkylcarboxy, aryl, e.g. phenyl, or aralkyl, e.g benzyl, for instance morpholine or piperazine; and
  • R* can be a phenyl group optionally substituted by 1, 2, 3 or 4 substituents which can be the same or different selected from halo, Ci-C 6 alkyl, • trifluoromethyl or Ci-C 6 alkoxy. More specifically, phenyl is unsubstituted or substituted by 1 , 2, 3 or 4 halogen substituents, particularly, from 1 to 3 fluoro groups, and most particularly, 2,3-difluoro, 2,4-difluoro or 4-fluoro.
  • a further embodiment of formula (II) here is where Y is -CH 2 CH 2 -.
  • R 2 is hydrogen, by default, or is halo, Ci.C ⁇ alkyl, mono to perfluoro- Ci-C 4 alkyl, mono to perfluoro Ci-C4 6 alkoxy, or Ci-Ce alkoxy; particularly mono to perfluoro- Ci-C 4 alkyl, mono to perfluoro- Ci-C 4 alkoxy, or C I- C O alkoxy.
  • R 2 is other than hydrogen
  • n in (R 2 ) n is 1, 2, or 3, and the substitution pattern is meta and/or para, particularly para, i.e. a 4-position substituent.
  • R 2 is 4- trifluorom ethyl or 4-trifluoromethoxy.
  • R 3 and R 4 can be the same or different and are methyl, ethyl, rc-propyl, or n-butyl.
  • R 3 and R 4 are the same and are methyl, or ethyl; methyl is of particular interest.
  • R 5 can be hydrogen, C( I-6) alkyl which is a straight chain, or branched. Of particular interest is methyl, ethyl, propyl, isopropyl, n-butyl, see-butyl, /so-butyl, f-butyl, «-pentyl or n- hexyl.
  • R 1 is phenyl substituted by 2,3 difluoro; [120J R-2 an d R 3 » together with the pyrimidine ring carbon atoms to which they are attached, form a fused 5-membered cyclopentenyl ring;
  • R 4 is 2-(diethylamino)ethyl
  • [1221 R5 is phenyl
  • [123] R ⁇ is phenyl substituted by trifluoromethyl at the 4-position, or thien-2-yl substituted by trifluoromethyl in the 5-position
  • X is (CH 2 ) 2 .
  • R.1 is an aryl group, optionally substituted by 1, 2, 3 or 4 substituents which can be the same or different selected from Cn _6 ) alkyl, hydroxy, halogen, CN, mono to perfluoro-C(i_4)alkyl, mono to perfluoro-C(i_4)alkoxyaryl, and arylC(i_4)alkyl;
  • R2 is halogen, C(i_3)alkyl, C(]_3)alkoxy, hydroxyC(i_3)alkyl, C(i_3)alkylthio, C(i_3)alkylsulphinyl, aminoC(i_3)alkyl, mono- or di-C(i_3)alkylaminoC(i_3)alkyl, C(i _3)alkylcarbonylaminoC(i _3)alkyl, C(i _3)alkoxyC(i _3)alkylcarbonylaminoC(i _3)alkyl, C(i_3)alkylsulphonylaminoC(i_3)alkyl, C(]_3)alkylcarboxy, C(i_3)alkylcarboxyC(i_3)alkyl, and [151] R 3 is hydrogen, halogen, C(j.3)alkyl, or hydroxyC(i_3)alkyl; or
  • R ⁇ and R-* together with the pyridone ring carbon atoms to which they are attached form a fused 5-or 6-membered carbocyclic ring; or 1153] R ⁇ and R ⁇ together with the pyridone ring carbon atoms to which they are attached form a fused benzo or heteroaryl ring optionally substituted by 1 , 2, 3 or 4 substituents which can be the same or different selected from halogen, C(i_4)alkyl, cyano, Cn _3)alkoxyC(i_
  • R4 is hydrogen, C(i_6)alkyl which can be unsubstituted or substituted by 1, 2 or 3 substituents selected from hydroxy, halogen, OR 7 , COR 7 , carboxy, COOR 7 , CONR 9 R 1 °, NR 9 R 10 , NR 7 COR 8 , mono- or di-(hydroxyC(i_6)alkyl)amino and N-hydroxyC(i_6)alkyl- N-Cn _6)alkylamino; or
  • R4 is Het-C(0-4)alkyl in which Het is a 5- to 7- membered heterocyclyl ring comprising N and optionally O or S, and in which N can be substituted by COR 7 , COOR 7 , CONR 9 RlO, or C(i_6)alkyl optionally substituted by 1, 2 or 3 substituents selected from hydroxy, halogen, OR 7 , COR 7 , carboxy, COOR 7 , CONR 9 R 1 O or NR 9 RlO, f or instance, piperidin-4-yl, pyrrolidin-3-yl;
  • R ⁇ is an aryl or a heteroaryl ring optionally substituted by 1 , 2, 3 or 4 substituents which can be the same or different selected from C(i_6)alkyl, C(i_6)alkoxy, C(i_6)alkylthio, arylC(i_ 6 )alkoxy, hydroxy, halogen, CN, COR 7 , carboxy, COOR 7 , NR 7 CORS, CONR 9 R 10 ,
  • R ⁇ 5 is an aryl or a heteroaryl ring which is further optionally substituted by 1 , 2, 3 or 4 substituents which can be the same or different selected from Cn_5 ) alkyl, C ⁇ _g)alkoxy,
  • R 7 and R 8 are independently hydrogen or C/ j . ⁇ 2)alkyl, for instance C/ j __nalkyl (e.g. methyl or ethyl);
  • R 9 and R 10 which can be the same or different is each selected from hydrogen, or C( 1 - 12) a ⁇ y ⁇ or ⁇ an(1 R ⁇ together with the nitrogen to which they are attached form a 5- to 7 membered ring optionally containing one or more further heteroatoms selected from oxygen, nitrogen and sulphur, and optionally substituted by one or two substituents selected from hydroxy, oxo, Cn__nalkyl, C(i_4)alkyl carboxy, aryl, e.g. phenyl, or aralkyl, e.g benzyl, for instance morpholine or piperazine; and
  • Rl is phenyl optionally substituted by halogen, Cn _5)alkyl, trifluoromethyl, C(i_6)alkoxy, preferably, from 1 to 3 fluoro, more preferably,
  • R ⁇ include piperidin-4-yl substituted at the 1- position by methyl, isopropyl, l-(2-methoxyethyl), 1 -(2-hydroxyethyl), t-butoxycarbonyl or ethoxycarbonylmethyl; ethyl substituted at the 2-position by aminoethyl; 1- ethylpiperidinylmethyl; piperidin-4-yl; 3-diethylaminopropyl; 4-pyrrolidin-l-ylbutyl and 1- ethylpyrrolidin-3-yl.
  • R 4 is l-(2-methoxyethyl)piperidin-4-yl, 1-methylpiperidin- 4-yl or l-ethylpyrrolidin-3-yl.
  • Representative examples of R ⁇ include phenyl and pyridyl.
  • R ⁇ is phenyl.
  • Representative examples of R ⁇ include phenyl optionally substituted by halogen, or trifluoromethyl, preferably at the 4-position and hexyl.
  • R ⁇ is phenyl substituted by trifluoromethyl at the 4-position.
  • Further representative examples of R ⁇ include phenyl substituted by 1 or more Cn _3)alkyl.
  • R ⁇ is phenyl substituted by ethyl in the 4-position.
  • R ⁇ and R ⁇ together form a 4- (phenyl)phenyl or a 2-(phenyl)pyridinyl substituent in which the remote phenyl ring can be optionally substituted by halogen or trifluoromethyl, preferably at the 4-position.
  • X is C(2 ⁇ 4)alkylene, more preferably Co-3) a lkylene, most preferably, (CH.2)2 > or CH2S.
  • RI is phenyl substituted by 2,3-difluoro
  • R2 and R- ⁇ 3 together with the pyridone ring carbon atoms to which they are attached, form a fused benzo or pyrido ring;
  • R 4 is l-(2-methoxyethyl)piperidin-4-yl
  • R5 and R ⁇ together form a 4-(phenyl)phenyl substituent in which the remote phenyl ring is substituted by trifluoromethyl, preferably at the 4-position;
  • R 1 is an aryl group, u ⁇ substituted or substituted by 1 , 2, 3 or 4 substituents which can be the same or different selected from the group consisting OfCi-C 6 alkyl, C 1 ..C 6 alkoxy, C 1- C 6 alkylthio, aryl Ci-C 6 alkoxy, hydroxy, halo, CN, COR 6 , COOR 6 , NR 6 COR 7 ,
  • W is CH and X is N, or W is N and X is CH, W and X are both CH, or W and X are N;
  • Y is C 2 -C 4 alkyl
  • R 2 is hydrogen, Ci-C 6 alkyl, C N C 6 alkoxy, Ci-C 6 alkylthio, aryl C]-C 6 alkoxy, hydroxy, halo, CN, COR 6 , carboxy, COOR 6 , NR 6 COR 7 , CONR 8 R 9 , SO 2 NRSR 9 ,
  • NR 6 SO 2 R 7 , NR 8 R 9 mono to perfluoro- C 1- C 6 alkyl, or mono to perfluoro- Ci-C 6 alkoxy;
  • n 0-5;
  • R 3 is C 1 -C 4 alkyl
  • R 4 is C 1 -C 4 alkyl
  • R 5 is hydrogen, C]-C 10 alkyl, C 2- C 10 alkenyl, C 2- C 10 alkynyl, halo Ci-C 4 alkyl, C 3 -C 8 cycloalkyl, C 3 -C 8 cycloalkyl, C3-C8 cycloalkyl Ci-C 4 alkyl, Cs-Cgcycloalkenyl, Cs-Cgcycloalkenyl Ci-C 4 alkyl, 3-8-membered heterocycloalkyl, 3-8-membered heterocycloalkyl Ci-C 4 alkyl, C 6 -Ci 4 aryl, C 6 -Ci 4 aryl Ci-Cm alkyl, heteroaryl, or heteroaryl Ci.Cioalkyl; wherein each group is optionally one or more times by the same and/or a different group which is Ci-C 6 alkoxy, Ci-Ce alkylthio, aryl Ci-C 6 alkoxy,
  • R 6 and R 7 are independently hydrogen or Ci-C 10 alkyl;
  • 2901 R ⁇ and R ⁇ are the same or different and are hydrogen or Ci-Cio alkyl, or R.9 and R ⁇ together with the nitrogen to which they are attached form a 5- to 7 membered ring optionally containing one or more further heteroatoms selected from oxygen, nitrogen and sulphur, and optionally substituted by one or two substituents selected from the group consisting of hydroxy, oxo, Ci-C 4 alkyl, C f- C 4 alkylcarboxy, aryl, and aryl C)-C 4 alkyl; [291 J or a pharmaceutically acceptable salt thereof.
  • R' it can be an phenyl group optionally substituted by 1, 2, 3 or 4 substituents which can be the same or different selected from halo, Ci-C 6 alkyl, trifluoromethyl or Ci-C 6 alkoxy. More specifically, phenyl is unsubstituted or substituted by 1 , 2, 3 or 4 halogen substituents, particularly, from 1 to 3 fluoro groups, and most particularly, 2,3-difluoro, 2,4-difluoro or 4-fluoro.
  • a further embodiment of formula (I) is where Y is -CH 2 CH 2 -.
  • the invention also provides a compound of formula (I) in which R 2 is hydrogen, by default, or is halo, Ci-C 6 alkyl, mono to perfluoro- Cj-C 4 alkyl, mono to perfluoro Ci_C4 6 alkoxy, or Ci-C 6 alkoxy; particularly mono to perfluoro- Ci-C 4 alkyl, mono to perfluoro- Ci-C 4 alkoxy, or CI-C O alkoxy.
  • R 2 is other than hydrogen
  • n in (R 2 ) n is 1 , 2, or 3, and the substitution pattern is meta and/or para, particularly para, i.e. a 4-position substituent.
  • Exemplified compounds include those where R 2 is 4-trifluoromethyl or 4-trifluoromethoxy.
  • R 3 and R 4 can be the same or different and are methyl, ethyl, ⁇ -propyl, or /z-butyl. Of particular interest are those compounds of formula (I) where R 3 and R 4 are the same and are methyl, or ethyl; methyl is of particular interest.
  • R 5 can be hydrogen, C(I -6 ) alkyl which is a straight chain, or branched. Of particular interest is methyl, ethyl, propyl, isopropyl, «-butyl, sec-butyl, /so-butyl, f-butyl, w-pentyl or n- hexyl.
  • any of the compounds described herein above can be prepared in crystalline or non-crystalline form, and, if crystalline, can be solvated, e.g. as the hydrate.
  • This invention includes within its scope stoichiometric solvates (e.g. hydrates).
  • Certain of the compounds described herein can contain one or more chiral atoms, or can otherwise be capable of existing as two enantiomers.
  • the compounds useful in the methods as described herein include mixtures of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures.
  • Also included within the scope of the invention are the individual isomers of the compounds represented by formulas (I) - (IV), as well as any wholly or partially equilibrated mixtures thereof.
  • the present invention also covers the individual isomers of the claimed compounds as mixtures with isomers thereof in which one or more chiral centers are inverted.
  • any tautomers and mixtures of tautomers of the claimed compounds are included within the scope of the compounds of formulas (I) — (IV).
  • the different isomeric forms can be separated or resolved one from the other by conventional methods, or any given isomer can be obtained by conventional synthetic methods or by stereospecific or asymmetric syntheses.
  • L ⁇ is a C(l-6)alkyl group, for instance methyl
  • R 15 is a Cn _6)alkyl group, for instance ethyl or t-butyl and
  • L 1 , L 2 , R a 3 R b , R c , R 2 , R 3 , R 4 , R 5 , n, X, Y and Z are as defined in WO 01/60805.
  • the ester (IV) is usually prepared by N-I alkylation of (V) using (VI), in which R.' 1 is as hereinbefore defined e.g. (VI) is t-butyl bromoacetate or ethyl bromoacetate, in the presence of a base e.g. BuLi in THF or sodium hydride in N-methyl pyrrolidinone (NMP) (step c).
  • R.' 1 is as hereinbefore defined e.g. (VI) is t-butyl bromoacetate or ethyl bromoacetate, in the presence of a base e.g. BuLi in THF or sodium hydride in N-methyl pyrrolidinone (NMP) (step c).
  • the key intermediate (IV) can be synthesised by reacting (XX) with dimethyloxosulfonium methylide, generated via the treatment of tri methyl sulfoxonium iodide with sodium hydride at low temperature, to yield a sulfur ylid (XXII) (step q). Subsequent treatment of (XXII) with carbon disulfide in the presence of diisopropylamine, followed by R.1 CH2-L4, where L ⁇ is a leaving group, yields intermediate (IV) (step r).
  • the R ⁇ X substituent can be introduced by displacement of a leaving group L 2 (e.g. Cl) (step e) either on a pyridine (VIII) or pyridine N-oxide (XIV), to give 2-substituted pyridines (VII) and (XV). Transformation of (VII) or (XV) to the 4-pyridone (V) is accomplished by deprotection of the 4-oxygen (e.g. using (Ph3P) 3 RhCl when in aq.
  • pyridine (VIII) or pyridine N-oxide (XIV) can be prepared by steps (i), (h), (g), (f), and (j), in which: [329J (j) treatment of (VIII) with m-chloroperbenzoic acid in dichloromethane; [330] (f) treatment of (IX) with R 12 OH (X), in which R 12 is allyl, and sodium hydride in DMF;
  • R 1 - CH2SH (XIX) is typically prepared from the thioacetate, which is formed from the corresponding alkyl bromide R ⁇ -CH2Br.
  • Intermediate (XIX) is formed from the 2,6-dioxo-l,3-oxazine (XX) and ester (XXI) by treatment with a base such as NaH in DMF or l,8-diazabicycIo[5.4.0]undec-7-ene in dichloromethane.
  • J387J A mixture of 1 ,1 -dimethylethyl 2-methyl-2-(4-oxo- 1 - ⁇ iperidinyl)propanoate (Int. A6) (370mg, 1.2 equiv), ⁇ [4'-(trifluoromethyl)-4-biphenylyl]methyl ⁇ amine (Int. Al) (397mg, 1 equiv), sodium triacetoxyborohydride (400mg, 1.5 equiv), DCM (10ml) and acetic acid (0.076ml, 1 equiv) was combined and stirred at room temperature until LCMS confirmed disappearance of the amine starting material (approx. 18 hours). Aqueous sodium carbonate was added and then extracted with DCM.
  • Reverse phase ⁇ PLC (Preparative Method B) gave ethyl 2- ⁇ 4-[ ⁇ [2-[2-(2,3-difluorophenyl)ethyl]-4-oxo-l( ⁇ / ⁇ O- quinazolinyl]acetyl ⁇ ( ⁇ 4'-[(trifluoromethyl)oxy]-4-biphe ⁇ ylyl ⁇ methyl)amino]-l-piperidinyl ⁇ - 2-methylpropanoate as a white solid (174mg). [427
  • Reverse phase HPLC (Preparative Method B) gave ethyl 2- ⁇ 4-[ ⁇ [2-[2-(2,3-difluoro ⁇ henyl)ethyl]-4-oxo-l( ⁇ //)- quinazolinyl]acetyl ⁇ ( ⁇ 4'-[(trifluoromethyl)oxy]-4-biphenylyl ⁇ methyl)amino]-l-piperidinyl ⁇ - 2-methylpropanoate as a white solid (166mg).
  • [488J A mixture of [2-[2-(2,4-difluorophenyl)ethyl]-4-oxopyrido[2,4-d]pyrimidin-l( ⁇ /f>- yl]acetic acid (Int. D2) (120mg, 1 equiv), ethyl 2-methyl-2-[4-( ⁇ [4'-(trifluoromethyl)-4- biphenylyl]methyl ⁇ amino)-l -piperidinyl]propanoate (Int. D2) (120mg, 1 equiv), ethyl 2-methyl-2-[4-( ⁇ [4'-(trifluoromethyl)-4- biphenylyl]methyl ⁇ amino)-l -piperidinyl]propanoate (Int. D2) (120mg, 1 equiv), ethyl 2-methyl-2-[4-( ⁇ [4'-(trifluoromethyl)-4- biphenylyl]methyl ⁇
  • Reverse phase HPLC (Preparative Method B) gave ethyl 2- ⁇ 4-[ ⁇ [2-[2-(2,4-difluorophenyl)ethyl]-4- oxopyrido[2,3-c/3pyrimidin-l( ⁇ /jO-yl]acetyl ⁇ ( ⁇ 4'-[(trifluoromethyl)oxy]-4-biphenylyl ⁇ - methyl)amino]-l-piperidinyl ⁇ -2-methylpropanoate as a white solid (149mg).
  • HATU (180mg, 1.5 equiv) was added in 1 portion and stirred an additional 5 min.
  • the crude reaction mixture was concentrated, filtered through a plug of silica eluted with acetone and evaporated to obtain crude 1,1 -dimethylethyl 2-[4-( ⁇ [2-[2-(2,3- difluorophenyl)ethyl]-4-oxopyrido[2,3-d]pyrirnidin-l (4/f)-yl] acetyl ⁇ ⁇ [4'-(trifluoromethyl)-4- biphenylyl]methyl ⁇ amino)-l -piperidinyl]-2-methylpropanoate.
  • compounds useful as inhibitors of Lp-P LA2 useful in the methods as disclosed herein are: [521] l-(N-(2-(diethyIamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)- aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one, also referred to as "SB480848” or the USAN name "darapladib” which is a pyrimidinone-based compound and a reversible inhibitor of Lp-PLA2 and is used in the Examples herein,
  • Nucleic acid inhibitors ofLp-PLA2 f527J are nucleic acids.
  • Nucleic acid inhibitors of Lp-PLA 2 are, for example, but not are limited to, RNA interference-inducing molecules, for example but are not limited to siRNA, dsRNA, stRNA, shRNA and modified versions thereof, where the RNA interference molecule silences the gene expression of Lp- PLA 2 .
  • the nucleic acid inhibitor Of Lp-PLA 2 is an anti-sense oligonucleic acid, or a nucleic acid analogue, for example but are not limited to DNA, RNA, peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), or locked nucleic acid (LNA) and the like.
  • the nucleic acid is DNA or RNA, and nucleic acid analogues, for example PNA, pcPNA and LNA.
  • a nucleic acid can be single or double stranded, and can be selected from a group comprising nucleic acid encoding a protein of interest, oligonucleotides, PNA, etc.
  • nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.
  • RNAi single-stranded RNA
  • shRNAi siRNA
  • mRNAi micro RNAi
  • antisense oligonucleotides etc.
  • single-stranded RNA a form of RNA endogenously found in eukaryotic cells can be used to form an RNAi molecule.
  • Cellular ssRNA molecules include messenger RNAs (and the progenitor pre-messenger RNAs), small nuclear RNAs, small nucleolar RNAs, transfer RNAs and ribosomal RNAs.
  • Double-stranded RNA (dsRNA) induces a size-dependent immune response such that dsRNA larger than 30bp activates the interferon response, while shorter dsRNAs feed into the cell's endogenous RNA interference machinery downstream of the Dicer enzyme.
  • RNA interference provides a powerful approach for inhibiting the expression of selected target polypeptides.
  • RNAi uses small interfering RNA (siRNA) duplexes that target the messenger RNA encoding the target polypeptide for selective degradation.
  • siRNA-dependent post-transcriptional silencing of gene expression involves cutting the target messenger RNA molecule at a site guided by the siRNA.
  • RNA interference is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene ⁇ see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting expression of the target gene.
  • mRNA messenger RNA
  • dsRNA double stranded RNA
  • RNAi is initiated by the dsRNA-specif ⁇ c endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs.
  • siRNAs are incorporated into a protein complex (termed “RNA induced silencing complex,” or “RISC”) that recognizes and cleaves target mRNAs.
  • RISC RNA induced silencing complex
  • RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target genes.
  • inhibiting target gene expression includes any decrease in expression or protein activity or level of the target gene or protein encoded by the target gene as compared to a situation wherein no RNA interference has been induced.
  • the decrease can be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target gene or the activity or level of the protein encoded by a target gene which has not been targeted by an RNA interfering agent.
  • siRNA Short interfering RNA
  • small interfering RNA is defined as an agent which functions to inhibit expression of a target gene, e.g., by RNAi.
  • An siRNA can be chemically synthesized, can be produced by in vitro transcription, or can be produced within a host cell.
  • siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, 22, or 23 nucleotides in length, and can contain a 3' and/or 5' overhang on each strand having a length of about 0. 1, 2, 3, 4, or 5 nucleotides.
  • the length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.
  • the siRNA is capable of promoting RNA interference through degradation or specific post- transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).
  • PTGS post- transcriptional gene silencing
  • shRNAs small hairpin (also called stem loop) RNAs
  • these shRNAs are composed of a short ⁇ e.g., about J 9 to about 25 nucleotide) antisense strand, followed by a nucleotide loop of about 5 to about 9 nucleotides, and the analogous sense strand.
  • the sense strand can precede the nucleotide loop structure and the anti sense strand can follow.
  • shRNAs can be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA Apr;9(4):493-501, incorporated by reference herein in its entirety).
  • the target gene or sequence of the RNA interfering agent can be a cellular gene or genomic sequence, e.g. the Lp-PLA 2 sequence.
  • An siRNA can be substantially homologous to the target gene or genomic sequence, or a fragment thereof.
  • the term "homologous" is defined as being substantially identical, sufficiently complementary, or similar to the target mRNA, or a fragment thereof, to effect RNA interference of the target.
  • RNA suitable for inhibiting or interfering with the expression of a target sequence include RNA derivatives and analogs.
  • the siRNA is identical to its target.
  • the siRNA preferably targets only one sequence.
  • RNA interfering agents such as siRNAs
  • expression profiling Such methods are known to one skilled in the art and are described, for example, in Jackson et al, Nature Biotechnology 6:635-637, 2003.
  • expression profiling one can also screen the potential target sequences for similar sequences in the sequence databases to identify potential sequences which can have off-target effects. For example, according to Jackson et al. (Id.) 15, or perhaps as few as 11 contiguous nucleotides of sequence identity are sufficient to direct silencing of non-targeted transcripts.
  • siRNA molecules need not be limited to those molecules containing only RNA, but, for example, further encompasses chemically modified nucleotides and non-nucleotides, and also include molecules wherein a ribose sugar molecule is substituted for another sugar molecule or a molecule which performs a similar function. Moreover, a non-natural linkage between nucleotide residues can be used, such as a phosphorothioate linkage.
  • siRNA containing D-arabinoft ⁇ ranosyl structures in place of the naturally-occurring D- ribonucleosides found in RNA can be used in RNAi molecules according to the present invention (U.S. Pat. No. 5,177,196).
  • Other examples include RNA molecules containing the o-linkage between the sugar and the heterocyclic base of the nucleoside, which confers nuclease resistance and tight complementary strand binding to the oligonucleotidesmolecules similar to the oligonucleotides containing 2'-O-methyl ribose, arabinose and particularly D- arabinose (U.S. Pat. No. 5,177,196).
  • the RNA strand can be derivatized with a reactive functional group of a reporter group, such as a fluorophore.
  • a reporter group such as a fluorophore.
  • Particularly useful derivatives are modified at a terminus or termini of an RNA strand, typically the 3' terminus of the sense strand.
  • the 2'- hydroxyl at the V terminus can be readily and selectively derivatized with a variety of groups.
  • RNA bases can also be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence can be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated.
  • the bases can also be alkylated, for example, 7-methylguanosine can be incorporated in place of a guanosine residue.
  • Non-natural bases that yield successful inhibition can also be incorporated.
  • siRNA modifications include 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides and RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages.
  • LNA locked nucleic acid
  • RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages.
  • Most of the useful modifications to the siRNA molecules can be introduced using chemistries established for antisense oligonucleotide technology.
  • the modifications involve minimal 2'-O-methyl modification, preferably excluding such modification. Modifications also preferably exclude modifications of the free 5'-hydroxyl groups of the siRNA.
  • siRNA and miRNA molecules having various "tails" covalently attached to either their 3'- or to their 5'-ends, or to both, are also known in the art and can be used to stabilize the siRNA and miRNA molecules delivered using the methods of the present invention.
  • intercalating groups, various kinds of reporter groups and lipophilic groups attached to the 3' or 5' ends of the RNA molecules are well known to one skilled in the art and are useful according to the methods of the present invention.
  • Descriptions of syntheses of 3 '-cholesterol or 3'-acridine modified oligonucleotides applicable to preparation of modified RNA molecules useful according to the present invention can be found, for example, in the articles: Gamper, H. B., Reed, M.
  • siRNAs useful for targeting Lp-PLA 2 expression can be readily designed and tested. Accordingly, siRNAs useful for the methods described herein include siRNA molecules of about 15 to about 40 or about 15 to about 28 nucleotides in length, which are homologous to an Lp-PLA 2 gene.
  • the Lp-PLA 2 targeting siRN A molecules have a length of about 19 to about 25 nucleotides. More preferably, the Lp-PLA 2 targeting siRNA molecules have a length of about 19, 20, 21, or 22 nucleotides.
  • the Lp-PLA 2 targeting siRNA molecules can also comprise a 3' hydroxyl group.
  • the Lp-PLA 2 targeting siRNA molecules can be single-stranded or double stranded; such molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3').
  • the RNA molecule is double stranded and either blunt ended or comprises overhanging ends.
  • At least one strand of the Lp-PLA 2 targeting RNA molecule has a 3' overhang from about 0 to about 6 nucleotides (e.g., pyrimidine nucleotides, purine nucleotides) in length.
  • the 3 ' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length.
  • the Lp-PLA 2 targeting RNA molecule is double stranded - one strand has a 3' overhang and the other strand can be blunt-ended or have an overhang.
  • the length of the overhangs can be the same or different for each strand.
  • the RNA of the present invention comprises about 19, 20, 21 , or 22 nucleotides which are paired and which have overhangs of from about 1 to about 3, particularly about 2, nucleotides on both 3' ends of the RNA.
  • the 3 ' overhangs can be stabilized against degradation.
  • the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • Lp-PLA 2 mRNA has been successfullytargeted usingsiRNAs and such siRNA or vectors forpreparingthem are commercially available, for example from Invitrogen.
  • assesment ofthe expression and/or knock down OfLp-PLA 2 protein using such Lp-PLA 2 siRNAs can be determined using commercially available kits, for example but are not limited to PLAC assay from diaDexus. Others can be readily prepared by those ofskill in the art based onthe known sequence ofthetargetmRNA. To avoid doubt, the sequenceofa human Lp-PLA 2 cDNA is provided at, for example, GenBank Accession Nos. : U20157 (SEQ ID NO:1) orNM_005084 (SEQ ID NO:2).
  • the sequence at U20157 is the following (SEQ ID NO:1): [544] 1 gctggtcgga ggctcgcagt gctgtcggcg agaagcagtc gggtttggag cgcttgggtcc
  • siRNA sequences are chosen to maximize the uptake of the antisense (guide) strand of the siRNA into RISC and thereby maximize the ability of RISC to target human Lp-PLA 2 mRNA for degradation. This can be accomplished by scanning for sequences that have the lowest free energy of binding at the 5 '-terminus of the antisense strand. The lower free energy leads to an enhancement of the unwinding of the 5'- end of the antisense strand of the siRNA duplex, thereby ensuring that the antisense strand will be taken up by RISC and direct the sequence-specific cleavage of the human Lp-PLA 2 mRNA. [546 ⁇ In a preferred embodiment, the siRNA or modified siRNA is delivered in a pharmaceutically acceptable carrier.
  • the siRNA is delivered by delivering a vector encoding small hairpin RNA (shRNA) in a pharmaceutically acceptable carrier to the cells in an organ of an individual.
  • shRNA small hairpin RNA
  • the shRNA is converted by the cells after transcription into siRNA capable of targeting, for example, Lp-PLA 2 .
  • the vector can be a regulatable vector, such as tetracycline inducible vector.
  • the RNA interfering agents used in the methods described herein are taken up actively by cells in vivo following intravenous injection, e.g., hydrodynamic injection, without the use of a vector, illustrating efficient in vivo delivery of the RNA interfering agents, e.g., the siRNAs used in the methods of the invention.
  • Other strategies for delivery of the RNA interfering agents e.g., the siRNAs or shRNAs used in the methods of the invention, can also be employed, such as, for example, delivery by a vector, e.g., a plasmid or viral vector, e.g., a lentiviral vector.
  • RNA interfering agents e.g., the siRNAs or shRNAs of the invention
  • a basic peptide by conjugating or mixing the RNA interfering agent with a basic peptide, e.g., a fragment of a TAT peptide, mixing with cationic lipids or formulating into particles.
  • the dsRNA such as siRNA or shRNA can be delivered using an inducible vector, such as a tetracycline inducible vector.
  • an inducible vector such as a tetracycline inducible vector.
  • a vector can be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion and foreign sequence and for the introduction into eukaryotic cells.
  • the vector can be an expression vector capable of directing the transcription of the DNA sequence of the agonist or antagonist nucleic acid molecules into RNA.
  • Viral expression vectors can be selected from a group comprising, for example, reteroviruses, lentiviruses, Epstein Barr virus-, bovine papilloma virus, adenovirus- and adeno-associated-based vectors or hybrid virus of any of the above.
  • the vector is episomal.
  • the use of a suitable episomal vector provides a means of maintaining the antagonist nucleic acid molecule in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
  • RNA interference molecules and nucleic acid inhibitors useful in the methods as disclosed herein can be produced using any known techniques such as direct chemical synthesis, through processing of longer double stranded RNAs by exposure to recombinant Dicer protein or Drosophila embryo lysates, through an in vitro system derived from S2 cells, using phage RNA polymerase, RNA-dependant RNA polymerase, and DNA based vectors.
  • Use of cell lysates or in vitro processing can further involve the subsequent isolation of the short, for example, about 21-23 nucleotide, siRNAs from the lysate, etc.
  • Chemical synthesis usually proceeds by making two single stranded RNA-oligomers followed by the annealing of the two single stranded oligomers into a double stranded RNA.
  • Other examples include methods disclosed in WO 99/32619 and WO 01/68836 that teach chemical and enzymatic synthesis of siRNA.
  • numerous commercial services are available for designing and manufacturing specific siRNAs (see, e.g., QIAGEN Inc., Valencia, CA and AMBION Inc., Austin, TX)
  • an agent is protein or polypeptide or RNAi agent that inhibits expression of Lp-PLA and/or activity of the Lp-PLA 2 protein.
  • cells can be modified (e.g., by homologous recombination) to provide increased expression of such an agent, for example by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the natural inhibitor agent of Lp- PLA 2 , for example protein or miRNA inhibitor Of Lp-PLA 2 at higher levels.
  • the heterologous promoter is inserted in such a manner that it is operatively linked to the desired nucleic acid encoding the agent. See, for example, PCT International Publication No. WO 94/12650 by Transkaryotic Therapies, Inc., PCT International Publication No. WO 92/20808 by Cell Genesys, Inc., and PCT International Publication No. WO 91/09955 by Applied
  • Cells also can be engineered to express an endogenous gene comprising the agent under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene can be replaced by homologous recombination.
  • Gene activation techniques are described in U.S. Patent No. 5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; PCT/US92/09627 (W093/09222) by Selden et al.; and
  • the agent can be prepared by culturing transformed host cells under culture conditions suitable to express the miRNA.
  • the resulting expressed agent can then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the peptide or nucleic acid agent inhibitor of Lp-PLA 2 can also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA Sepharose; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; immunoaffhity chromatography, or complementary cDNA affinity chromatography.
  • affinity resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA Sepharose
  • hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether
  • immunoaffhity chromatography or complementary cDNA affinity chromatography.
  • the nucleic acid inhibitors OfLp-PLA 2 can be obtained synthetically, for example, by chemically synthesizing a nucleic acid by any method of synthesis known to the skilled artisan.
  • the synthesized nucleic acid inhibitors of Lp-PLA 2 can then be purified by any method known in the art.
  • Methods for chemical synthesis of nucleic acids include, but are not limited to, in vitro chemical synthesis using phosphotriester, phosphate or phosphoramidite chemistry and solid phase techniques, or via deoxynucleoside H-phosphonate intermediates (see U.S. Patent No. 5,705,629 to Bhongle).
  • nucleic acids having nucleic acid analogs and/or modified internucleoside linkages can be preferred.
  • Nucleic acids containing modified internucleoside linkages can also be synthesized using reagents and methods that are well known in the art.
  • siRNA molecules including shRNA molecules, can be obtained using a number of techniques known to those of skill in the art.
  • the siRNA molecule can be chemically synthesized or recombinantly produced using methods known in the art, such as using appropriately protected ribonucleoside phosphorarnidites and a conventional DNA/RNA synthesizer (see, e.g., Elbashir, S.M. et al. (2001) Nature 411:494-498; Elbashir, S.M., W. Lendeckel and T. Tuschl (2001) Genes & Development 15:188-200; Harborth, J. et al. (2001) J. Cell Science 1 14:4557-4565; Masters, J.R.
  • RNA synthesis suppliers including, but are not limited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL , USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).
  • siRNA molecules are not overly difficult to synthesize and are readily provided in a quality suitable for RNAi.
  • dsRNAs can be expressed as stem loop structures encoded by plasmid vectors, retroviruses and lentiviruses (Paddison, P.J. et al. (2002) Genes Dev. 16:948-958; McManus, M.T. et al. (2002) RNA 8:842-850; Paul, CP. et al. (2002) Nat. Biotechnol. 20:505-508; Miyagishi, M. et al. (2002) Nat. Biotechnol. 20:497-500; Sui, G. et al. (2002) Proc. Natl. Acad. ScL, USA 99:5515-5520; Brummelkamp, T. et al.
  • the targeted region of the siRNA molecule of the present invention can be selected from a given target gene sequence, e.g., a Lp-PLA 2 coding sequence, beginning from about 25 to 50 nucleotides, from about 50 to 75 nucleotides, or from about 75 to 100 nucleotides downstream of the start codon. Nucleotide sequences can contain 5' or 3' UTRs and regions nearby the start codon.
  • One method of designing a siRNA molecule of the present invention involves identifying the 23 nucleotide sequence motif AA(Nl 9)TT (where N can be any nucleotide), and selecting hits with at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% G/C content.
  • the "TT" portion of the sequence is optional.
  • the search can be extended using the motif NA(N21), where N can be any nucleotide.
  • the 3' end of the sense siRNA can be converted to TT to allow for the generation of a symmetric duplex with respect to the sequence composition of the sense and antisense 3' overhangs.
  • the antisense siRNA molecule can then be synthesized as the complement to nucleotide positions 1 to 21 of the 23 nucleotide sequence motif.
  • the use of symmetric 3' TT overhangs can be advantageous to ensure that the small interfering ribonucleoprotein particles (siRNPs) are formed with approximately equal ratios of sense and antisense target RNA-cleaving siRNPs (Elbashir et ⁇ l. (2001) supra and Elbashir et al. 2001 supra).
  • RNA interfering agents e.g., an siRNA, or vectors containing an RNA interfering agent
  • target cells e.g., cells of the brain or other desired target cells, for cells in the central and peripheral nervous systems
  • RNA interfering agents e.g., an siRNA
  • Methods of delivering RNA interfering agents, e.g., an siRNA, or vectors containing an RNA interfering agent, to the target cells can include, for example (i) injection of a composition containing the RNA interfering agent, e.g., an siRNA, or (ii) directly contacting the cell, e.g., a cell of the brain, with a composition comprising an RNA interfering agent, e.g., an siRNA.
  • RNA interfering agents e.g., an siRNA can be injected directly into any blood vessel, such as vein, artery, venule or arteriole, via, e.g., hydrodynamic injection or catheterization.
  • the siRNA is delivered to the bone marrow, where Lp- PLA2 is secreted from bone marrow-derived cells such as leukocytes.
  • RNA interfering agent is delivered in a pharmaceutically acceptable carrier.
  • One or more RNA interfering agents can be used simultaneously.
  • the RNA interfering agents e.g., the siRNAs targeting Lp-PLA 2 mRNA, can be delivered singly, or in combination with other RNA interfering agents, e.g., siRNAs, such as, for example siRNAs directed to other cellular genes.
  • Lp-PLA 2 siRNAs can also be administered in combination with other pharmaceutical agents which are used to treat or prevent metabolic bone diseases or disorders.
  • specific cells are targeted with RNA interference, limiting potential side effects of RNA interference caused by non-specific targeting of RNA interference.
  • the method can use, for example, a complex or a fusion molecule comprising a cell targeting moiety and an RNA interference binding moiety that is used to deliver RNA interference effectively into cells.
  • a complex or a fusion molecule comprising a cell targeting moiety and an RNA interference binding moiety that is used to deliver RNA interference effectively into cells.
  • an antibody-protamine fusion protein when mixed with an siRNA, binds siRNA and selectively delivers the siRNA into cells expressing an antigen recognized by the antibody, resulting in silencing of gene expression only in those cells that express the antigen.
  • the siRNA or RNA interference-inducing molecule binding moiety is a protein or a nucleic acid binding domain or fragment of a protein, and the binding moiety is fused to a portion of the targeting moiety.
  • the location of the targeting moiety can be either in the carboxyl-terminal or amino-terminal end of the construct or in the middle of the fusion protein.
  • a viral-mediated delivery mechanism can also be employed to deliver siRNAs to cells in vitro and in vivo as described in Xia, H. et al. (2002) Nat Biotechnol 20(10):1006). Plasmid- or viral-mediated delivery mechanisms of shRNA can also be employed to deliver shRNAs to cells in vitro and in vivo as described in Rubinson, D.A., et al. ((2003) Nat. Genet. 33:401-406) and Stewart, S.A., et al. ((2003) RNA 9:493-501).
  • RNA interfering agents for e.g., an siRNA, can also be introduced into cells via the .
  • RNA interference e.g., post translational gene silencing (PTGS)
  • PTGS post translational gene silencing
  • target gene expression e.g., inhibition of target gene expression or inhibition of activity or level of the protein encoded by the target gene.
  • RNAi molecules do not have to match perfectly to their target sequence. Preferably, however, the 5' and middle part of the antisense (guide) strand of the siRNA is perfectly complementary to the target nucleic acid sequence.
  • RNAi molecules functioning as nucleic acid inhibitors Of Lp-PLA 2 in the present invention are for example, but are not limited to, unmodified and modified double stranded (ds) RNA molecules including short-temporal RNA (stRNA), small interfering RNA (siRNA), short-hairpin RNA (shRNA), microRNA (miRNA), double- stranded RNA (dsRNA), (see, e.g. Baulcombe, Science 297:2002-2003, 2002).
  • the dsRNA molecules e.g. siRNA, also can contain 3' overhangs, preferably 3 1 UU or 3'TT overhangs.
  • the siRNA molecules of the present invention do not include RNA molecules that comprise ssRNA greater than about 30-40 bases, about 40-50 bases, about 50 bases or more. In one embodiment, the siRNA molecules of the present invention are double stranded for more than about 25%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90% of their length.
  • a nucleic acid inhibitor Of Lp-PLA 2 is any agent which binds to and inhibits the expression of Lp-PLA 2 mRNA, where the expression of Lp-PLA 2 mRNA or a product of transcription of nucleic acid encoded by SEQ ID NO:1 or 2 is inhibited.
  • agents inhibiting Lp-PLA 2 are catalytic nucleic acid constructs, such as, for example ribozymes, which are capable of cleaving RNA transcripts and thereby preventing the production of wildtype protein.
  • Ribozymes are targeted to and anneal with a particular sequence by virtue of two regions of sequence complementary to the target flanking the ribozyme catalytic site. After binding, the ribozyme cleaves the target in a site specific manner.
  • ribozymes which specifically recognize and cleave sequences of the gene products described herein, for example for cleavage of Lp-PLA 2 or homologues or variants thereof can be achieved by techniques well known to those skilled in the art (for example Lleber and Strauss, (1995) MoI Cell Biol 15:540.551, the disclosure of which is incorporated herein by reference).
  • agent that inhibit Lp-PLA 2 are proteins and/or peptide inhibitors or fragments of inhibitors of Lp-PLA 2 , for example, but are not limited to mutated proteins; therapeutic proteins and recombinant proteins. Proteins and peptides inhibitors can also include for example mutated proteins, genetically modified proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof.
  • the agents that inhibit Lp-PLA 2 are dominant negative variants of Lp-PLA2, for example a non-functional variant Of Lp-PLA 2 .
  • inhibitors of genes and/or gene products useful in the methods of the present invention include, for example, antibodies, including monoclonal, chimeric humanized, and recombinant antibodies and antigen-binding fragments thereof.
  • neutralizing antibodies can be used as inhibitors of the Lp-PLA 2 enzyme.
  • Antibodies are readily raised in animals such as rabbits or mice by immunization with the antigen. Immunized mice are particularly useful for providing sources of B cells for the manufacture of hybridomas, which in turn are cultured to produce large quantities of monoclonal antibodies.
  • the inhibitor to the gene products identified herein can be an antibody molecule or the epitope-binding moiety of an antibody molecule and the like.
  • Antibodies provide high binding avidity and unique specificity to a wide range of target antigens and haptens.
  • Monoclonal antibodies useful in the practice of the present invention include whole antibody and fragments thereof and are generated in accordance .with conventional techniques, such as hybridoma synthesis, recombinant DNA techniques and protein synthesis.
  • Useful monoclonal antibodies and fragments can be derived from any species
  • murine monoclonal antibody can be "humanized” by genetically recombining the nucleotide sequence encoding the murine Fv region (i.e., containing the antigen binding sites) or the complementarily determining regions thereof with the nucleotide sequence encoding a human constant domain region and an Fc region.
  • Humanized targeting moieties are recognized to decrease the immunoreactivity of the antibody or polypeptide in the host recipient, permitting an increase in the half-life and a reduction the possibly of adverse immune reactions in a manner similar to that disclosed in European Patent Application No.
  • the murine monoclonal antibodies should preferably be employed in humanized form.
  • Antigen binding activity is determined by the sequences and conformation of the amino acids of the six complementarily determining regions (CDRs) that are located (three each) on the light and heavy chains of the variable portion (Fv) of the antibody.
  • CDRs complementarily determining regions
  • the 25-kDa single-chain Fv (scFv) molecule composed of a variable region (VL) of the light chain and a variable region (VH) of the heavy chain joined via a short peptide spacer sequence, is the smallest antibody fragment developed to date. Techniques have been developed to display scFv molecules on the surface of filamentous phage that contain the gene for the scFv.
  • scFv molecules with a broad range of antigenic-specificities can be present in a single large pool of scFv-phage library.
  • Some examples of high affinity monoclonal antibodies and chimeric derivatives thereof, useful in the methods of the present invention, are described in the European Patent Application EP 186,833; PCT Patent Application WO 92/16553; and US Patent No. 6,090,923.
  • Chimeric antibodies are immunoglobin molecules characterized by two or more segments or portions derived from different animal species.
  • the variable region of the chimeric antibody is derived from a non-human mammalian antibody, such as murine monoclonal antibody, and the immunoglobin constant region is derived from a human immunoglobin molecule.
  • both regions and the combination have low immunogenicity as routinely determined.
  • scFv molecules are their monovalent interaction with target antigen.
  • One of the easiest methods of improving the binding of a scFv to its target antigen is to increase its functional affinity through the creation of a multimer.
  • Association of identical scFv molecules to form diabodies, triabodies and tetrabodies can comprise a number of identical Fv modules. These reagents are therefore multivalent, but monospecific.
  • the association of two different scFv molecules, each comprising a VH and VL domain derived from different parent Ig will form a fully functional bispecific diabody.
  • a unique application of bispecific scFvs is to bind two sites simultaneously on the same target molecule via two (adjacent) surface epitopes.
  • scFv-based structures A number of multivalent scFv-based structures has been engineered, including for example, miniantibodies, dimeric miniantibodies, minibodies, (scFv) 2 , diabodies and triabodies. These molecules span a range of valence (two to four binding sites), size (50 to 120 kDa), flexibility and ease of production.
  • Single chain Fv antibody fragments (scFvs) are predominantly monomelic when the VH and VL domains are joined by, polypeptide linkers of at least 12 residues. The monomer scFv is thermodynamically stable with linkers of 12 and 25 amino acids length under all conditions.
  • the noncovalent diabody and triabody molecules are easy to engineer and are produced by shortening the peptide linker that connects the variable heavy and variable light chains of a single scFv molecule.
  • the scFv dimers are joined by amphipathic helices that offer a high degree of flexibility and the miniantibody structure can be modified to create a dimeric bispecific (DiBi) miniantibody that contains two miniantibodies (four scFv molecules) connected via a double helix.
  • DiBi dimeric bispecific
  • Gene- fused or disulfide bonded scFv dimers provide an intermediate degree of flexibility and are generated by straightforward cloning techniques adding a C-terminal Gly4Cys sequence.
  • scFv-CH3 minibodies are comprised of two scFv molecules joined to an IgG CH3 domain either directly (LD minibody) or via a very flexible hinge region (Flex minibody). With a molecular weight of approximately 80 kDa, these divalent constructs are capable of significant binding to antigens.
  • the Flex minibody exhibits impressive tumor localization in mice. Bi- and tri-specif ⁇ c multimers can be formed by association of different scFv molecules. Increase in functional affinity can be reached when Fab or single chain Fv antibody fragments (scFv) fragments are complexed into dimers, trimers or larger aggregates.
  • the most important advantage of multivalent scFvs over monovalent scFv and Fab fragments is the gain in functional binding affinity (avidity) to target antigens.
  • High avidity requires that scFv multimers are capable of binding simultaneously to separate target antigens.
  • the gain in functional affinity for scFv diabodies compared to scFv monomers is significant and is seen primarily in reduced off-rates, which result from multiple binding to two or more target antigens and to rebinding when one Fv dissociates.
  • scFv molecules associate into multimers, they can be designed with either high avidity to a single target antigen or with multiple specificities to different target antigens.
  • Antibodies conjugated with moieties that improve their properties are also contemplated for the instant invention.
  • antibody conjugates with PEG that increases their half-life in vivo can be used for the present invention.
  • Immune libraries are prepared by subjecting the genes encoding variable antibody fragments from the B lymphocytes of naive or immunized animals or patients to PCR amplification.
  • Immunoglobulin germ line genes can be used to prepare semisynthetic antibody repertoires, with the complementarity-determining region of the variable fragments being amplified by PCR using degenerate primers.
  • These single-pot libraries have the advantage that antibody fragments against a large number of antigens can be isolated from one single library.
  • the phage-display technique can be used to increase the affinity of antibody fragments, with new libraries being prepared from already existing antibody fragments by random, codon-based or site-directed mutagenesis, by shuffling the chains of individual domains with those of fragments from naive repertoires or by using bacterial mutator strains.
  • a SCID-hu mouse for example the model developed by Genpharm, can be used to produce antibodies, or fragments thereof.
  • a new type of high avidity binding molecule termed peptabody, created by harnessing the effect of multivalent interaction is contemplated.
  • a short peptide ligand was fused via a semirigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule.
  • ligands and/or chimeric inhibitors can be targeted to tissue- or tumor-specific targets by using bispecific antibodies, for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target.
  • bispecific antibodies for example produced by chemical linkage of an anti-ligand antibody (Ab) and an Ab directed toward a specific target.
  • molecular conjugates of antibodies can be used for production of recombinant bispecific single-chain Abs directing ligands and/or chimeric inhibitors at cell surface molecules.
  • two or more active agents and or inhibitors attached to targeting moieties can be administered, wherein each conjugate includes a targeting moiety, for example, a different antibody. Each antibody is reactive with a different target site epitope (associated with the same or a different target site antigen).
  • Antibody-based or non- antibody-based targeting moieties can be employed to deliver a ligand or the inhibitor to a target site.
  • a natural binding agent for an unregulated or disease associated antigen is used for this purpose.
  • the methods of the present invention relate to use of inhibitors OfLp-PLA 2 for the prevention and/or treatment of metabolic bone diseases or disorders, for example osteoporosis and osteopenic related diseases.
  • agents that inhibit Lp-PLA 2 protein are assessed using abioassay, as disclosed in U.S. Patent 5,981,252 which is incorporated herein in its entirety by reference.
  • One such assay is testing the effect of the agent on the recombinant Lp-PLA 2 protein.
  • recombinant Lp-PLA 2 is purified to homogeneity from baculovirus infected Sf9 cells, using a zinc chelating column, blue sepharose affinity chromatography and an anion exchange column. Following purification and ultrafiltration, the enzyme can be stored at 6mg/ml at 4°C.
  • Assay buffer comprises Tris-HCl (50 mM), NaCl (150 mM) and 1 mM CHAPS, pH 7.4 at room temperature.
  • Activity is measured by an increase in emission at 535 nm on hydrolysis of N- ((6-(2,4-dinitrophenyl) amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-S-pentanoyO-l-hexadecanoyl-sn-glycero-S-phosphoethanolamine, triethylammonium salt (PED6, Molecular Probes catalogue reference D-23739) as substrate, using a fluorometric plate reader with 384 well microtitre plates. Reaction is initiated by the addition of enzyme (approx 400 pM final by weight) and substrate (5 ⁇ M final) to inhibitor in a total volume of 10 microlitres.
  • One aspect of the present invention relates to methods for the treatment and/or prevention of metabolic bone diseases or disorders. These include metabolic bone diseases and/or disorders are characterized by abnormalities in the bone marrow.
  • the method uses agents that inhibit the expression and/or function Of Lp-PLA 2 .
  • the metabolic bone disease or disorder is osteoporosis and osteopenia, and in other embodiments the metabolic bone disease or disorders. can be caused by metabolic diseases such as dyslipidemia, type II diabetes, metabolic syndrome or insulin resistance and the like.
  • Bone marrow homeostasis is critical for tissue repair and regeneration. Disturbed bone marrow homeostasis may lead to aging and tissue degeneration. As disclosed herein, inhibition Of Lp-PLA 2 can also be used for the prevention and/or treatment of metabolic bone marrow diseases or disorders and bone marrow abnormalities.
  • agents inhibiting Lp-PLA 2 as disclosed herein are useful in the treatment and/or prophylaxis of diseases where metabolic bone disease occurs or disorders where loss of bone mass, or bone density has been determined to play a role, such as osteoporosis and related osteopenic diseases, Paget's disease, hyperparathyroidism and related diseases, such as for example dyslipidemia, type II diabetes, metabolic syndrome or insulin resistance and the like.
  • the osteopenic related disease or osteoporosis is associated with the peri and post menopausal conditions. Also encompassed are the treatment and prophylaxis of Paget's disease, hypercalcemia associated with bone neoplasms and all the types of osteoporotic diseases as classified below according to their etiology: Primary osteoporosis, hypercalcemia, involutional osteoporosis, Type I or postmenopausal osteoporosis, Type II or senile osteoporosis, Juvenile osteoporosis, Idiopathic in young adults osteoporosis, Secondary osteoporosis, Endocrine abnormality, Hyperthyroidism, Hypogonadism, Ovarian agenesis, or Turner's syndrome, Hyperadrenocorticism or Cushing's syndrome, Hyperparathyroidism, Bone marrow abnormalities, Multiple myeloma and related disorders, and Systemic mastocytosis, disseminated carcinoma
  • the methods as disclosed herein are especially useful for patients who do have a known genetic risk of metabolic bone diseases and/or disorders, for example osteoporosis.
  • Such patients include those as identified to have risk of having a metabolic bone disease or disorder, as disclosed herein in the section entitled "osteoporosis risk factors" below, which include analysis of risk factors and analysis of genetic or biochemical markers.
  • patients are women, for example post menopausal, or women at least 65 years of age, or patients who have had previous fractures or have relatives who have had a metabolic bone disease, for example osteoporosis.
  • Patients can be identified as having increased risk of developing metabolic bone disease using methods commonly known by person of ordinary skill in the art, and are disclosed herein in the section entitled "methods to identify patients at risk of, or having a metabolic bone disease or osteoporosis” below, which include analysis of genetic and/or biochemical markers for metabolic bone diseases as disclosed herein.
  • Osteoporosis (gr: osteon bone; poros hole) is described in general terms as a reduction in bone density with retention of a normal chemical composition. More specifically, osteoporosis is a generalized, progressive diminution of bone density, i.e. bone mass per unit volume, causing skeletal weakness, although the ratio of mineral to organic elements is unchanged. 30 to 40% of the skeletal mass must be lost in order to reliably diagnose osteoporosis by radiology. Contemporary medicine distinguishes between primary and secondary osteoporosis (The Merck Manual of Diagnosis and Therapy, 17th ed., 1999).
  • Primary osteoporosis includes idiopathic osteoporosis, rare but occurring in children and young adults; postmenopausal osteoporosis, occurring between the ages of 50 and 75; and involutional or senile osteoporosis associated with the normal process of aging.
  • osteoporosis is characterized by increased osteoclast activity and a disruption of the feedback mechanism between the serum calcium level and the parathyroid hormone (PTH) secretion. It occurs mainly uniformly throughout the whole skeleton.
  • Primary osteoporosis accounting for less than 5% of all osteoporosis cases, includes endocrine dysfunctions. It starts mostly at the main skeleton and progresses centrifugally.
  • Osteoporosis is characterized by pain in the respective bones, diffuse back pain, vertebral body collapse, pathological fractures, in particular, fracture of the neck of the femur.
  • the goal of the management of all types of osteoporosis is therefore to decrease pain, to prevent fractures and to maintain the body functions.
  • osteoporosis is a calcium dysfunction and the use of calcium supplements has been widely suggested. However, so far, no reossification of the osteoporotic bone after calcium therapy could be demonstrated.
  • agents inhibiting Lp-PLA 2 using the agents as disclosed herein are useful in preventing and treating metabolic bone diseases and disorders.
  • Additional examples of metabolic bone diseases include osteoporosis. Osteoporosis is a common clinical feature and common complication in patients affected with chronic inflammatory diseases with joint manifestations.
  • rheumatoid arthritis RA
  • Juvenile Rheumatoid Arthritis JRA
  • psoriatic arthritis Reiter's syndrome (reactive arthritis)
  • Crohn's disease ulcerative colitis and sarcoidosis
  • Orcel, et al. Bone demineralization and cytokines; Rev Rhum MaI Osteoartic.1992; 59:16S-22S; Brown, et al., The radiology of rheumatoid arthritis. Am Fam Physician. 1995. 52:1372-80; De Vos, et al., Bone and joint diseases in inflammatory bowel disease. Aliment Pharmacol Ther.
  • Rheumatoid arthritis is associated with a decrease in bone mass (Cortet, et al., Evaluation of bone mineral density in patients with rheumatoid arthritis. Influence of disease activity and glucocorticoid therapy. Rev Rhum Engl Ed. 1997 July-Sep. 30, 1997; 64(7- 9):451-8).
  • Typical changes of an inflammatory arthritis include juxta- articular osteoporosis, cartilage loss, and cortical or marginal bone erosions (Lawson, et al., Lyme arthritis: radiologic findings. Radiology. 1985; 154(1 ):37-43; Grassi, et al., The clinical features of rheumatoid arthritis.
  • Homocysteinemia (the accumulation of homocysteine in plasma and tissue) is the result of deficiencies of certain enzymes and/or substrates involved in the transmethylation pathways. It is caused by the accumulation of homocysteine and its two disulfides in plasma and tissue (Mudd et al., The Metabolic Basis of Inherited Disease, New York, McGraw-Hill, 1978, p. 458). Homocysteinemia is associated with juvenile arteriosclerosis, recurrent arterial and venous thromboembolic manifestations and osteoporosis.
  • agents that inhibit Lp-PLA 2 as disclosed herein are also useful in the treatment of other metabolic bone disorders, for example but not limited to patients with metabolic bone diseases due to chronic inflammatory diseases with joint manifestations, for example but not limited to rheumatoid arthritis (RA), Juvenile Rheumatoid Arthritis (JRA), psoriatic arthritis, Reiter's syndrome (reactive arthritis), Crohn's disease, ulcerative colitis, sarcoidosis.
  • RA rheumatoid arthritis
  • JRA Juvenile Rheumatoid Arthritis
  • psoriatic arthritis psoriatic arthritis
  • Reiter's syndrome reactive arthritis
  • Crohn's disease Crohn's disease
  • ulcerative colitis sarcoidosis.
  • Other patients with metabolic bone diseases include, for example but are not limited to, related osteopenic diseases, Paget's disease, hyperparathyroidism and related diseases, such as for example dyslipidemia, type II diabetes, metabolic syndrome or insulin resistance and the like, primary osteoporosis, involutional osteoporosis, Type I or postmenopausal osteoporosis, Type II or senile osteoporosis, oral bone loss, metabolic peridontilis, Juvenile Idiopathic osteoporosis in young adults, Secondary osteoporosis, Endocrine abnormality, Hyperthyroidism, Hypogonadism, Ovarian agenesis or Turner's syndrome, Hyperadrenocorticism or Cushing's syndrome, Hyperparathyroidism, Bone marrow abnormalities, Multiple myeloma and related disorders, Systemic mastocytosis, Disseminated carcinoma, Gaucher's disease, Connective tissue abnormalities, Osteogenesis imperfecta, Homocystinuria
  • agents that inhibit Lp-PLA 2 as disclosed herein are also useful in the treatment of other metabolic bone disorders, for example but not limited to patients with metabolic bone diseases where the metabolic bone disease or disorder involves bone reabsorbtion, for example Paget's Disease, primary and secondary hyperparathyroidism, humoral hypercalcameia of malignancy and various cancers, where bone resorbtion is increased.
  • metabolic bone diseases for example Paget's Disease, primary and secondary hyperparathyroidism, humoral hypercalcameia of malignancy and various cancers, where bone resorbtion is increased.
  • Bone marrow abnormalities referred to here are defined as those where general abnormality of the biological balance in the bone marrow is indicated, such as viral and bacterial infections in the bone marrow, cellular infiltration of the bone marrow, abnormalities of the bone marrow haematopoiesis, proliferation of malignant neoplasms in the bone marrow and concentration changes in cell growth-differentiation factors.
  • Bone marrow abnormalites include, but are not limited to, anemia, immunodeficiency, bone marrow, depression, bone marrow fibrosis, bone marrow necrosis and degeneration.
  • Metabolic bone diseases or disorders and abnormal bone marrow can be brought about by many pathogenic factors such as pyogenic bacteria, tuberculosis, syphilis, fungi and specified viruses or exogenous matter is termed osteomyelitis and, when there is osteomyelitis, as a result of the impeded blood circulation and infiltration of neutrophils into the bone marrow region, there occur surrounding bone decalcification and tissue breakdown, with resulting pain.
  • osteomyelitis While the occurrence of acute osteomyelitis is declining due to the widespread use of antibiotics, as a result of the appearance of resistant microorganisms, osteomyelitis which from the outset follows a subacute or chronic course remains a problem [Green, et al., J. Bone Joint Serg., 63-A, pl07-l 14 (1981)].
  • the bone marrow is the location of a markedly increased production of leukaemia cells, and normal blood components decline.
  • a principal feature is the proliferation of tumours of plasma cells, which are cells at the end of the B cell lineage, and a multiplicity of these is produced in the bone marrow at sites of active haernatopoiesis.
  • a metabolic bone disease or disorder also encompasses disease and disorders with overactivity of osteocasts.
  • disease or disorders associated with osteocast activity includes for example but are not limited to osteoporosis, Paget's disease, hypercalcemeia, rheumatoid arthritis, cancer, metastatic bone destruction, oral bone loss, metabolic peridontitis and immune disorders.
  • treatment can be directed to a patient who is affected with asymptomatic metabolic bone diseases; it can prevent bone density loss and/or improve bone matrix density.
  • the efficacy of treatment can be determined by monitoring bone mineral density (BMD) or biochemical markers in biological samples.
  • Biochemical markers include, for example but not limited to, measuring the presence of estrogen in the blood of women, and calcium in the urine, hypercalciuria, or increased excretion of calcium.
  • Other markers also include for example, but are not limited to serum markers of bone turnover, for example bone-specific alkaline phosphatase (BSAP), osteocalcin (BGP), tartrate-resistant acid phosphatase (TRAP) and urinary collagen C-terminal extension peptides (CrossLaps) (Hotchkiss et al, 2001; 29;7- 15).
  • diagnostic test for metabolic bone diseases and/or osteoporosis can be used, for example, as disclosed in European Patents EP 1666883, EP 1680513 and EP 1639946, which are incorporated herein in their entirety by reference.
  • Some methods entail determining a baseline value of the bone density of a patient before administering a dosage of agent, and comparing this with a value for bone density of the patient after treatment. The same level of bone density or an increase in bone density indicates a positive treatment outcome (i.e., that administration of the agent has achieved or prevented a decrease in bone density loss).
  • a decreased in the rate of bone density loss or of the rate of bone density loss also indicates a positive outcome, for example that administration has prevented or reduced the rate of the bone density loss. If the value for level of bone density decreases, a negative treatment outcome is indicated.
  • patients undergoing an initial course of treatment with an agent are expected to show the same level or an increase in bone density with successive dosages of an agent as described herein.
  • a control value i.e., a mean and standard deviation
  • the individuals in the control population have not received prior treatment and do not suffer from a metabolic bone disease or disorder, for example osteoporosis.
  • Measured values of bone density in a patient after administering an inhibitor agent Of Lp-PLA 2 as disclosed herein are then compared with the control value.
  • An increase in the bone density in the patient relative to the control value e.g.. an increase of at least 10% of bone density and/or bone mass in a patient approaching control values signals a positive treatment outcome.
  • a decrease in bone density decrease signals a negative treatment outcome.
  • a control value of bone density is determined from a control population of patients who have undergone treatment with a therapeutic agent that is effective at halting the loss of bone density or increasing bone density. Measured values of bone density in the patient are compared with the control value.
  • a patient who is not presently receiving treatment by agents that inhibit Lp-PLA 2 as disclosed herein, but has undergone a previous course of treatment is monitored for bone density to determine whether a resumption of treatment is required.
  • the measured value of bone density in the test patient can be compared with a level of bone density previously achieved in the patient after a previous course of treatment.
  • An increase in the rate of bone density loss and/or bone mass loss, or a decrease in bone density and/or bone mass relative to the previous measurement is an indication that treatment with an agent inhibitor OfLp-PLA 2 can be resumed.
  • the level of bone density or bone mass in the patient can be compared with a control bone densitity level of determined in a population of patients after undergoing a course of treatment.
  • the bone density in a patient can be compared with a control value in populations of prophylactically treated patients who remain free of symptoms of disease, in particular free of symptoms of metabolic bone diseases, for example osteoporosis or osteopenic related diseases, or populations of therapeutically treated patients who show amelioration of a disease symptom.
  • Patients amenable to treatment using the methods as disclosed herein include patients at risk of developing a metabolic bone disease or disorder such as osteoporosis but not showing symptoms, as well as patients showing symptoms of the metabolic bone disease patients with symptoms of osteoporosis.
  • Patients can be screened for their likelihood of having or developing osteoporosis based on a number of bone density tests, imaging tests, biochemical and genetic markers.
  • a number of diagnostic tests are available for identifying patients who have osteoporosis or osteopenic related diseases, or patients at risk of developing ostepoperosis or osteopenic diseases. These include, but are not limited to a bone density test, for example a dual energy x-ray absorption scan (also known as DEXA or DXA scan by persons of ordinary skill in the art).
  • a DEXA scan can identify a patient with a normal bone density, low bone mass or osteoporosis.
  • bone density testing involves a low level of radiation exposure.
  • an non-isotopic detection method for osteoblastic acitivity can be used, as disclosed in U.S. Patent 6,869,593 which is incorporated herein in its entirety by reference.
  • Other diagnostic tests to identify patients at risk of developing osteoporosis or osteopenic diseases include for example without limitation, X ray test to identify the presence of fractures or a history of fractures, bone mineral density (BMD) tests, for example BMD which can be used to determine bone health, bone scans, which identify a patient with metabolic bone disorders, for example cancer, bone lesions, abnormal bone marrow and new fractures.
  • BMD bone mineral density
  • a T-score a number value that results from comparing the bone density to optimal bone density.
  • a T-score appears as a negative number such as -1, -2 or -2.5, the patient is identified as having low bone mass. The more negative the T-score, the greater risk the patient has of developing or having osteoporosis.
  • diagnostic tests to identify patients at risk of developing osteoporosis or osteopenic diseases include for example without limitation, a three componet X-ray bone densitometry scan, as disclosed in U.S. Patent 6,909,771 which is incorporated herein in its entirety by reference.
  • a metabolic bone disorder for example osteoporosis
  • QUS quantitative ultrasound
  • Other diagnostic methods can be used to identify a patient at increased risk of developing osteoporosis or with osteoporosis, for example diagnostic methods as disclosed in European patents 1666883, 1680513 and 1639946, which are incorporated herein in their entirety by reference.
  • Other methods to diagnose a patient at risk of or having a metabolic bone disease such as osteoporosis and/or abnormal bone marrow includes measurement of Lp-PLA 2 activity and/or expression using the methods as disclosed herein, for example using the PLAC test commercially available from diaDexus. 1616]
  • a number of biomarkers can identify a patient at risk of osteoporosis.
  • laboratory test are also useful in the methods of the present invention to identify a patient at risk of developing and/or having osteoporosis, for example measuring the levels of a number of biochemical markers in a biological sample including blood and/or urine.
  • biochemical markers include blood calcium levels, blood vitamin D levels, thyroid function, parathyroid hormone levels, estradiol levels to measure estrogen (useful in assessing the risk in women), follicle stimulating hormone (FSH) to establish menopause status, testosterone levels (in men) and osteocalcin levels to measure bone formation.
  • FSH follicle stimulating hormone
  • Other biochemical markers include for example, but are not limited to serum markers of bone turnover such as bone-specific alkaline phosphatase (BSAP), osteocalcin (BGP), tartrate- resistant acid phosphatase (TRAP) and urinary collagen C-terminal extension peptides (CrossLaps) (Hotchkiss et al, 2001 ; 29;7-15). [6171] Such diagnostic tests are also useful to monitor response of the patient to administration of the Lp-PLA 2 inhibitor agents as disclosed herein.
  • a patient at risk of osteoporosis include women over 65 years of age, post-menoplausal women under age 65 who have multiple risk factors, women at menopause, patients with abnormal spine x-rays, patients with long term oral steroid use and patients with hyperparathyroidism (an over active parathyroid gland).
  • Osteoporosis risk factors 620] One can also diagnose a patient with increased risk of developing a metabolic bone disease or disorder such as osteoporosis on the basis of risk factors.
  • patients identified to be at risk of metabolic bone diseases or disorders are admininstered agents that inhibit Lp-PLA 2 as disclosed herein.
  • Risk factors for osteoporosis are known by persons of ordinary skill in the art, and include advanced age: (i) patients over the age of 65 years; and (ii) gender.
  • secondary osteoporosis can cause further bone loss in postmenopausal women and older men with primary osteoporosis.
  • certain medications to treat endocrine disorders such as hyperthyroidism, marrow disorders, collagen disorders, gastrointestinal problems and seizure disorders.
  • osteopenic diseases and osteoporosis include, for example, patients with: a history of fractures as an adult, history of fractures in the first degree relative, advanced age, female patients, dementia, patients with poor health, fragility or both, current cigarette smoking, low body weight, anorexia nervosa, estrogen deficiency (past menopause, menopause before the age of 45, having both ovaries removed, or absence of menstral periods for a year or more prior to menopause, low testosterone levels in men, use of certain medications such as cortiosteriods and anticonvulsants, lifelong low calcium intake, excessive alcohol intake, inpaired eyesight despite adequate correction, recurrent falls and inadequate physical activity.
  • certain medications such as cortiosteriods and anticonvulsants
  • the cause of osteoporosis are include the hormone deficiency (estrogen or androgen), hormone excess (cushing's syndrome or glucocorticoid administration, thyrotoxicosis, hyperparathyroidism, excessive vitamin D administration), immobilization, tabacco, malignancy, idiopathic or geriatric, and genetic disorders (Type I collagen mutations, Ehlers- danlos syndrome, Marfan's syndrome, Homocystinuria).
  • hormone deficiency estrogen or androgen
  • hormone excess cushing's syndrome or glucocorticoid administration
  • thyrotoxicosis thyrotoxicosis
  • hyperparathyroidism excessive vitamin D administration
  • immobilization tabacco
  • malignancy idiopathic or geriatric
  • genetic disorders Type I collagen mutations, Ehlers- danlos syndrome, Marfan's syndrome, Homocystinuria
  • inhibitors Of Lp-PLA 2 can be administered to patients considered at especially high risk for developing osteoporosis, such patients include for example but without limitation: all women over age 65, women less than age 65 who are postmenopausal and have one or more of the above described risk factors for osteoporosis, postmenopausal women who experience any type of bone fracture, and men who have a testosterone deficiency.
  • patients considered at especially high risk for developing osteoporosis such patients include for example but without limitation: all women over age 65, women less than age 65 who are postmenopausal and have one or more of the above described risk factors for osteoporosis, postmenopausal women who experience any type of bone fracture, and men who have a testosterone deficiency.
  • one can also diagnose a patient with increased risk of developing a metabolic bone disease using genetic markers for the disease. Genetic markers to identify a patient at risk of developing the likes of osteoporosis are known to person of ordinary skill in the art.
  • polymorphisms or single nucleotide polymorphisms (SNPs) present in patients at risk for osteopoerisis are disclosed in U.S. Patent 6,825,336 which is specifically incorporated herein in its entirety by reference.
  • Patients at risk of developing a metabolic bone disease or disorder such as osteoporosis have variances in the PPAR gamma gene, as disclosed in European Patent 1612279 which is incorporated herein in its entirety by reference.
  • osteogenesis imperfecta is caused by a major mutation in the gene encoding for type I collagen, the major collagen constituent of bone. This causes severe osteoporosis.
  • Marfan's syndrome is caused by mutations in fibrillin gene on chromosome 15.
  • Homocytinuria is caused by cystathionine beta-synthase deficiency and exhibits an autosomal recessive pattern of inheritance.
  • Genes associated with osteoporosis include, but not limit to: alcitonin receptor, collagen subunit (alpha-1 (X)) 3, Kuestner et al MoI. Pharmacol. 46 (2), 246-255 (1994); insulin-like growth factor binding protein 1, Brewer et al., Biochem. Biophys. Res. Commun. 152 (3), 1289-1297 (1988), Brinkman et al., EMBO J. 7 (8), 2417-2423 (1988), Cubbage et al., MoI. Endocrinol. 3 (5), 846-851 (1989), Alitalo et al., Hum. Genet.
  • Treatment can begin at any age (e.g., 10, 20, 30). Usually, however, it is not necessary to begin treatment until a patient reaches 40, 50, 60 or 70. Treatment typically entails multiple dosages over a period of time.
  • treatment with an inhibitor agent of Lp-PLA 2 can be monitored by assaying presence of biomarkers or density of bones as disclosed herein over time. If the bone density indicates low bone mass, additional treatment with agents inhibiting Lp-PLA 2 as disclosed herein are recommended, and/or treatment of additional therapies for metabolic bone disease, for example osteoporosis. In the case of potential patients with an inherited risk of developing osteoporosis, treatment can begin antenatally by administering therapeutic agent to the mother or shortly after birth.
  • agents inhibiting Lp-PLA 2 can be assessed in animal models for effect reducing a symptom of a metabolic bone disease.
  • DM/HC hyperglycemia and hypercholesterolemia
  • the animal exhibits symptoms of a metabolic bone disease, for example reduced bone matrix and abnormal bone marrow, in which bone matrix and/or abnormal bone marrow can be assessed in the presence and absence of inhibitors for Lp-PLA 2 by methods commonly known by persons in the art.
  • bone density can be assessed using methods commonly known by persons of ordinary skill in the art, for example BMD tests or CT-micro assessment as disclosed in Example 1.
  • agents inhibiting Lp-PLA 2 can be assessed in animal models for oesteoporosis, permitting analysis of the effects Of Lp-PLA 2 inhibitory agents on bone formation, bone repair and development and treatment, as well as assessment of drug dosages on the development, prognosis and recovery from metabolic bone diseases, for example osteoporosis.
  • Animal models of osteoporosis are well known by person of ordinary skill in the art.
  • One commonly well established model of postmenopausal bone loss are ovariectomized (OVX) rodents.
  • An effective dosage causes at least a measurable, statistically or clinically significant attenuation of at least one marker, symptom, or histological evidence characteristic of metabolic bone disorder and/or abnormal bone marrow.
  • Markers, symptoms and histological evidence characteristic of osteoporosis include bone mass loss, decrease in bone density, and increase in blood calcium levels, and/or vitamin D levels, thyroid function, parathyroid hormone levels, estradiol levels to measure estrogen (useful in assessing the risk in women), follicle stimulating hormone (FSH) to establish menopause status, testosterone levels (in men) and osteocalcin levels to measure bone formation.
  • FSH follicle stimulating hormone
  • biochemical markers also include calcium and phosphorus in blood, hypercalciuria, or increased excretion of calcium in urine, and presence of serum markers of bone turnover, for example bone-specific alkaline phosphatase (BSAP), osteocalcin (BGP), tartrate-resistant acid phosphatase (TRAP) and urinary collagen C-terminal extension peptides (CrossLaps) (Hotchkiss et al, 2001; 29;7-15).
  • BSAP bone-specific alkaline phosphatase
  • BGP osteocalcin
  • TRIP tartrate-resistant acid phosphatase
  • CrossLaps urinary collagen C-terminal extension peptides
  • the suitability of an inhibitor OfLp-PLA 2 for the treatment of a metabolic bone disease can be assessed in any of a number of animal models for metabolic bone diseases.
  • Animal models of osteoporisos are well known by person of ordinary skill in the art.
  • one commonly well established model of postmenopausal bone loss are ovariectomized (OVX) rodents, for example rat and mouse models and non-rodent animals, as disclosed in Thompson et al, 1995; 17:125S-133S, Iwaniec et al, J.
  • OVX ovariectomized
  • OVX non-rodent anomals include for example but are not limited to monkeys (Hotchkiss et al, Bone, 2001 ; 29:7- 15) and sheep
  • DM/HC DM/HC as disclosed herein, where the animal exhibits symptoms of reduced bone matrix and abnormal bone marrow in which bone matrix and/or abnormal bone marrow can be assessed in the presence and absence of inhibitors for Lp-PLA 2 by methods commonly known by persons in the art.
  • Animals administered the compounds are evaluated for symptoms relative to animals not administered the compounds.
  • a measurable change in the severity of a symptom, (for example a decrease in at least one symptom) or a delay in the onset of a symptom in animals treated with an Lp-PLA 2 inhibitor versus untreated animals is indicative of therapeutic efficacy.
  • Compounds for example agents inhibiting Lp-PLA 2 as disclosed herein, can be used as a medicament or used to formulate a pharmaceutical composition with one or more of the utilities disclosed herein. They can be administered in vitro to cells in culture, in vivo to cells in the body, or ex vivo to cells outside of an individual that can later be returned to the body of the same individual or another. Such cells can be disaggregated or provided as solid tissue.
  • Compounds, for example agents inhibiting Lp-PLA 2 as disclosed herein can be used to produce a medicament or other pharmaceutical compositions. Use of agents inhibiting Lp- PLA 2 which further comprise a pharmaceutically acceptable carrier and compositions which further comprise components useful for delivering the composition to an individual are known in the art. Addition of such carriers and other components to the agents as disclosed herein is well within the level of skill in this art.
  • compositions can be administered as a formulation adapted for delivery to the bone or direct contact with the bone marrow.
  • the compostions may be administered as a formulation adapted for systemic delivery.
  • the compostions may be administered as a formulation adapted for delivery to specific organs, for example but not limited to the liver, bone marrow or systemic delivery.
  • pharmaceutical compositions can be added to the culture medium of cells ex vivo.
  • such compositions can contain pharmaceutically-acceptable carriers and other ingredients known to facilitate administration and/or enhance uptake (e.g., saline, dimethyl sulfoxide, lipid, polymer, affinity-based cell specific-targeting systems).
  • compositions can be incorporated in a gel, sponge, or other permeable matrix (e.g., formed as pellets or a disk) and placed in proximity to the endothelium for sustained, local release.
  • the composition can be administered in a single dose or in multiple doses which are administered at different times.
  • Pharmaceutical compositions can be administered by any known route.
  • the composition can be administered by a mucosal, pulmonary, topical, or other localized or systemic route (e.g., enteral and parenteral).
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection, infusion and other injection or infusion techniques, without limitation.
  • systemic administration means the administration of the agents as disclosed herein such that it enters the animal's system and, thus, is patient to metabolism and other like processes, for example, subcutaneous administration.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • phrases "pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, for example the carrier does not decrease the impact of the agent on the treatment.
  • a carrier is pharmaceutically inert.
  • Suitable choices in amounts and timing of doses, formulation, and routes of administration can be made with the goals of achieving a favorable response in the subject with a metabolic bone disease or disorderj such as osteoporosis or osteopenia or a risk thereof (i.e., efficacy), and avoiding undue toxicity or other harm thereto (i.e., safety). Therefore, "effective" refers to such choices that involve routine manipulation of conditions to achieve a desired effect.
  • a bolus of the formulation administered to an individual over a short time once a day is a convenient dosing schedule. Alternatively, the effective daily dose can be divided into multiple doses for purposes of administration, for example, two to twelve doses per day.
  • Dosage levels of active ingredients in a pharmaceutical composition can also be varied so as to achieve a transient or sustained concentration of the compound or derivative thereof in an individual, especially in and around the bone or bone marrow, and to result in the desired therapeutic response or protection. But it is also within the skill of the art to start doses at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the amount of agents inhibiting Lp-PLA ⁇ administered is dependent upon factors known to a person skilled in the art such as bioactivity and bioavailability of the compound (e.g., half-life in the body, stability, and metabolism); chemical properties of the compound (e.g., molecular weight, hydrophobicity, and solubility); route and scheduling of administration, and the like. It will also be understood that the specific dose level to be achieved for any particular individual can depend on a variety of factors, including age, gender, health, medical history, weight, combination with one or more other drugs, and severity of disease.
  • factors known to a person skilled in the art such as bioactivity and bioavailability of the compound (e.g., half-life in the body, stability, and metabolism); chemical properties of the compound (e.g., molecular weight, hydrophobicity, and solubility); route and scheduling of administration, and the like. It will also be understood that the specific dose level to be achieved for any particular individual can depend on a variety of factors, including age, gender, health,
  • treatment can also involve combination with other existing modes of treatment, for example existing agents for treatment for osteoporosis, for example but not limited to, estrogens, bisphosphonates, calcitonin, flavonoids, and selective estrogen receptor modulators.
  • Other approaches include peptides from the parathyroid hormone family, strontium ranelate, and growth hormone and insulin-like growth response.
  • flavonol aglycone glycoside affords an advantageous function through a chelation delivery system.
  • Flavonols possess a benzene ring structure having available bonds to function as a chelate. Therefore, flavonols, due to their particular molecular structure, are capable of holding and delivering certain minerals, including calcium, to mammalian bone tissue. Also bone tissue would naturally absorb flavonol glycosides from the blood stream. It is further disclosed that the combination of the flavonol aglycone glycoside and calcium leads to an increased bone mineral density which would not have been obtainable through the use of simple calcium supplements.
  • quercetin which is a related bioflavonoid and differs from the aforementioned flavonol compounds in that it does not contain the glycoside residue, has been shown to inhibit TNF- ⁇ induced expression of interleukin 8 (IL-8) and monocyte chemoattractant protein- 1 (MCP-I) in cultured human synovial cells. It was therefore suggested that quercetin can be used in the treatment of rheumatoid arthritis which is an autoimmune disorder and involved synovial tissues of joints (Sato et al., The Journal of Rheumatology, 1997; 24:9, p. 1680).
  • IL-8 interleukin 8
  • MCP-I monocyte chemoattractant protein- 1
  • interleukin 1 is the most potent bone resorption agent.
  • interleukin 1, 6, 11 and the tumor necrosis factors appear to have a depressing effect on bone formation.
  • osteoporotic bone i.e. bone mineral with a lower magnesium content, has larger and more perfect crystals and bone mineral with a higher magnesium content has smaller and less perfect crystals than normal bone mineral. It was consequently suggested that the administering of magnesium supplements may be used in osteoporosis therapy.
  • agents that inhibit Lp-PLA 2 as disclosed herein can be combined with other therapeutic agent to prevent and/or treat metabolic bone disease or disorders.
  • Such agents can be any agent currently in use or being developed for the treatment and/or prevention of osteoporosis, where the agent can have a prophylactic and/or a curative effect and/or reduce a symptom of a metabolic bone disorder or disease.
  • the inhibitor agents of Lp-PLA 2 as disclosed herein can be used in combination with medicaments commonly known by person of ordinary skill in the art that are claimed to be useful as symptomatic treatments of osteoporosis.
  • Such medicaments include, but are not limited to, agents known to modify or inhibit osteoclast activity, promote osteoblast activity and/or regulate cellular events necessary for healthy bone marrow and healthy bone metabolism.
  • the inhibitor agents of Lp-PLA 2 as disclosed herein are used for the treatment of osteoporosis
  • the inhibitor agents OfLp-PLA 2 as disclosed herein can be used in combination with those medicaments mentioned above that are claimed to be useful as symptomatic treatments of osteoporosis and/or disease-modifying agents.
  • Disease modifying agents include, for example but are not limited to, estrogens, bisphosphonates, calcitonin, flavonoids, and selective estrogen receptor modulators.
  • Bisphosphates for example bisphosphate acid compounds includes for example, Alendronate, for example Fosmax, Ibandronate, for example Boniva and Risendronate, for example Actonel.
  • Bisphosphosphate acid compounds suppress excessive bone resorption in tumor-induced osteolysis, Paget's disease and osteoporosis, as disclosed in U.S. Patent 6,555,529, and patents EP177433, EP337706, AU8551534, EP27982 and EP94714 which are incorporated herein in their entirety by reference.
  • Bisphosphate acid compounds are alone disclosed in EP100718, U.S. Patent 4,234,645 and 4,067,971 EP84822, WO/9203451,
  • a bisphosphate acid compounds for use with the methods as disclosed herein includes bisphosphoric acid compounds for the treatment of bone marrow abnormalities, as disclose in U.S. Patent 6,555,529 which is incorporated herein in its entirety by reference.
  • a pyridopyrimidine is useful with the methods as disclosed herein, for example as disclosed in International Application WO/0300001 1 which is incorporated herein in its entirety by reference.
  • Calcitonin includes Miacalcin, Calcimar and Fortical.
  • Selective estrogen receptor modulators can also be used for the treatment of osteoporosis, i.e. Roloxifene, sold as Evista®.
  • Parathyroid hormone can also be used in combination with the agents that inhibit Lp-PLA 2 as disclosed herein.
  • Teriparatide is one such agents. It is sold as Forteo®.
  • estrogen replacement and/or hormone replacement therapy can be used in combination with agents that inhibit Lp-PLA 2 formation as disclosed herein.
  • an increase in calcium and/or vitamin D, and/or an increase in physical activity and/or increase in healthy lifestyle (for example stopping smoking, avoidance of excessive alcohol) can be combined with the methods as disclosed herein.
  • 16581 O ne can a l so use indole derivatives with the agents as disclosed herein for the treatment and/or prevention of metabolic bone disorders as disclosed in U.S. Patent 6,903,1 17 which is incorporated in its entirety herein by reference.
  • One can further also use quinolones with the agents as disclosed herein for the treatment and/or prevention of metabolic bone disorders as disclosed in U.S. Patent Application 2003/0114486 which is incorporated in its entirety herein by reference.
  • agent useful for administration to the patient in combination with the agents that inhibit Lp-PLA 2 as disclosed herein are cancer agents admininstered for surgery, chemotherapy, radiotherapy, hormone therapy and immunotherapy and the like.
  • Such agents include chemotherapeutic agents covering three main categories of therapeutic agent: (1) other antiangiogenic agents that work by different mechanisms from those defined hereinbefore (linomide, angiostatin, razoxin, thalidomide), (ii) cytostatic agents such as antioestrogens ( tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene), progestogens (megestrol acetate), aromatase inhibitors (nastrozole, letrazole, vorazole, exemestane), antiprogestogens, antiandrogens (flutamide, nilutamide, bicalutamide, cyproterone acetate), LHRH agonists and antagonists (goserelin acetate, luprolide), inhibitors of testosterone 5.alpha.-dihydroreductase (finasteride), anti- invasion agents (metalloproteina
  • examples are bisphosphonates, parathyroid hormone (PTH), hormone replacement therapy (HRT), selective estrogen receptor modulators (SERM), calcitonin, RANKL antibody, constitutively active androstane receptor (CaR) antagonist and cathepsin K inhibitors.
  • Examples of bisphosphonates include, for example but are not limited to Alendronate (Fosamax® - Merck), Risedronate (Actonel® — Proctor & Gamble), Ibandronate (Boniva®/Bonviva® - Roche/GSK/), Zoledronate (Reclast®/Aclasts® - Novartis).
  • Examples of PTH drugs include teriparatide (Forteo®/Forsteo® — Eli Lilly) parathyroid hormone.
  • Hormone replacement therapy and/or selective estrogen receptor modulators include estrogen, Premarin® being one example, raloxifene sold under the name Evista® by Eli Lilly, and apeledoxifene under development by Wyeth.
  • An example of a calcitonin therapy is miacaclin® sold by Novartis.
  • One or more of these drugs, and drugs of these types can be administered at the same time as the Lp-PLA2 inhibitor, or they may be administered at another time if the affects of the combination therapy is optimized by the latter regime. While it is believed that the dosage of these second therapeutics will most likely be those approved for use as a standalone therapy, it is possible that a particular combination may warrant adjusting the dosage up or down depending on factors such as drug/drug effects and/or the need or response of a particular patient.
  • treatment can also comprise multiple agents to inhibit Lp-PLA 2 expression or activity.
  • diagnosis according to the invention can be practiced with other diagnostic procedures.
  • the bone marrow or blood or urine can be assayed for a change in gene expression profiles using disease-specific molecular diagnostics kits (e.g., custom made arrays, multiplex QPCR, multiplex proteomic arrays).
  • a non-invasive diagnostic procedure e.g., CAT, MRI, SPECT, or PET
  • CAT CAT
  • MRI magnetic resonance
  • SPECT magnetic resonance imaging
  • PET PET
  • Early and reliable diagnosis is especially useful to for prevention and/or treatment for metabolic bone disorders as disclosed herein.
  • the amount which is administered to a patient is preferably an amount that does not induce toxic effects which outweigh the advantages which result from its administration. Further objectives are to reduce in number, diminish in severity, and/or otherwise relieve suffering from the symptoms of the disease in the individual in comparison to recognized standards of care.
  • Dosages, formulations, dosage volumes, regimens, and methods for analyzing results aimed at inhibiting Lp-PLA 2 expression and/or activity can vary. Thus, minimum and maximum effective dosages vary depending on the method of administration. Suppression of the clinical and histological changes associated with a metabolic bone disease and/or disorder can occur within a specific dosage range, which, however, varies depending on the organism receiving the dosage, the route of administration, whether agents that inhibit Lp-PLA 2 are administered in conjunction with other co-stimulatory molecules, and the specific regimen of inhibitor of Lp-P LA2 administration.
  • tablets can be formulated in accordance with conventional procedures employing solid carriers well-known in the art.
  • Capsules employed for oral formulations to be used with the methods of the present invention can be made from any pharmaceutically acceptable material, such as gelatin or cellulose derivatives.
  • Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated, such as those described in U.S. Pat. No. 4,704,295, "Enteric Film-Coating Compositions," issued Nov. 3, 1987; U.S. Pat. No. 4, 556,552, "Enteric Film- Coating Compositions," issued Dec. 3, 1985; U.S. Pat. No. 4,309,404, "Sustained Release Pharmaceutical Compositions," issued Jan. 5, 1982; and U.S. Pat. No. 4,309,406, “Sustained Release Pharmaceutical Compositions,” issued Jan. 5, 1982.
  • solid carriers examples include starch, sugar, bentonite, silica, and other commonly used carriers.
  • carriers and diluents which can be used in the formulations of the present invention include saline, syrup, dextrose, and water.
  • [670J Enteric Coated Formulation [671] As regards formulations for administering the small chemical entities for inhibitors of Lp-PLA 2 of the likes of formulas (I) - (IV) as disclosed herein, one particularly useful embodiment is a tablet formulation comprising the Lp-PLA inhibitor with an enteric polymer casing. An example of such a preparation can be found in WO2005/021002.
  • the active material in the core can be present in a micronised or solubilised form.
  • the core can contain additives conventional to the art of compressed tablets.
  • Appropriate additives in such a tablet can comprise diluents such as anhydrous lactose, lactose monohydrate, calcium carbonate, magnesium carbonate, dicalcium phosphate or mixtures thereof; binders such as microcrystalline cellulose, hydroxypropylmethylcellulose, hydroxypropyl-cellulose, polyvinylpyrrolidone, pre-gelatinised starch or gum acacia or mixtures thereof; disintegrants such as microcrystalline cellulose (fulfilling both binder and disintegrant functions) cross-linked polyvinylpyrrolidone, sodium starch glycollate, croscarmellose sodium or mixtures thereof; lubricants, such as magnesium stearate or stearic acid, glidants or flow aids, such as colloidal silica, talc or starch, and stabilisers such as desic
  • the tablet comprises lactose as diluent.
  • a binder is present, it is preferably hydroxypropylmethyl cellulose.
  • the tablet comprises magnesium stearate as lubricant.
  • the tablet comprises croscarmellose sodium as disintegrant.
  • the tablet comprises microcrystalline cellulose.
  • the diluent can be present in a range of 10 — 80% by weight of the core.
  • the lubricant can be present in a range of 0.25 — 2% by weight of the core.
  • the disintegrant can be present in a range of 1 — 10% by weight of the core.
  • Microcrystalline cellulose if present, can be present in a range of 10 - 80% by weight of the core.
  • the active ingredient preferably comprises between 10 and 50% of the weight of the core, more preferably between 15 and 35% of the weight of the core, (calculated as free base equivalent).
  • the core can contain any therapeutically suitable dosage level of the active ingredient, but preferably contains up to 150mg as free base of the active ingredient.
  • the core contains 20, 30, 40, 50, 60, 80 or lOOmg as free base of the active ingredient.
  • the active ingredient can be present as the free base, or as any pharmaceutically acceptable salt. If the active ingredient is present as a salt, the weight is adjusted such that the tablet contains the desired amount of active ingredient, calculated as free base of the salt.
  • the active ingredient is present as a hydrochloride salt.
  • the core can be made from a compacted mixture of its components. The components can be directly compressed, or can be granulated before compression. Such granules can be formed by a conventional granulating process as known in the art.
  • the granules can be individually coated with an enteric casing, and then enclosed in a standard capsule casing.
  • the core is surrounded by a casing which comprises an enteric polymer.
  • enteric polymers are cellulose acetate phthalate, cellulose acetate succinate, methylcellulose phthalate, ethylhydroxycellulose phthalate, polyvinylacetate pthalate, polyvinylbutyrate acetate, vinyl acetate-maleic anhydride copolymer, styrene-maleic mono-ester copolymer, methyl acrylate-methacrylic acid copolymer or methacrylate-methacrylic acid-octyl acrylate copolymer.
  • the casing can also include insoluble substances which are neither decomposed nor solubilised in living bodies, such as alkyl cellulose derivatives such as ethyl cellulose, crosslinked polymers such as styrene-divinylbenzene copolymer, polysaccharides having hydroxyl groups such as dextran, cellulose derivatives which are treated with bifunctional crosslinking agents such as epichlorohydrin, dichlorohydrin or 1, 2-, 3, 4-diepoxybutane.
  • the casing can also include starch and/or dextrin.
  • enteric coating materials are the commercially available Eudragit® enteric polymers such as Eudragit® L, Eudragit® S and Eudragit® NE used alone or with a plasticiser. Such coatings are normally applied using a liquid medium, and the nature of the plasticiser depends upon whether the medium is aqueous or non-aqueous.
  • Plasticisers for use with aqueous medium include propylene glycol, triethyl citrate, acetyl triethyl citrate or Citroflex® or Citroflex® A2.
  • Non-aqueous plasticisers include these, and also diethyl and dibutyl phthalate and dibutyl sebacate.
  • a preferred plasticiser is Triethyl citrate.
  • the casing can also include an anti-tack agent such as talc, silica or glyceryl monostearate.
  • an anti-tack agent such as talc, silica or glyceryl monostearate.
  • the anti-tack agent is glyceryl monostearate.
  • the casing can include around 5 — 25 wt% Plasticiser and up to around 50 wt % of anti tack agent, preferably 1-10 wt.% of anti-tack agent.
  • a surfactant can be included to aid with forming an aqueous suspension of the polymer.
  • Many examples of possible surfactants are known to the person skilled in the art.
  • Preferred examples of surfactants are polysorbate 80, polysorbate 20, or sodium lauryl sulphate.
  • a surfactant can form 0.1 — 10% of the casing, preferably 0.2 — 5% and particularly preferably 0.5 - 2%
  • seal coat included between the core and the enteric coating.
  • a seal coat is a coating material which can be used to protect the enteric casing from possible chemical attack by any alkaline ingredients in the core.
  • the seal coat can also provide a smoother surface, thereby allowing easier attachment of the enteric casing.
  • suitable coatings Preferably the seal coat is made of an Opadry coating, and particularly preferably it is Opadry White OY-S-28876.
  • the pharmaceutically active ingredient is l-(N-(2- (diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)aminocarbonylmethyl)-2-(4- fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-one, or a salt thereof.
  • enteric-coated formulation as described in WO2005/021002, comprises varying amounts of l-(N-(2-(diethylamino)ethyl)-N-(4-(4- trifluoromethylphenyl)benzyl)aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6- trimethylenepyrimidin-4-one (called “active" in this example) as hydrochloride salt.
  • lactose monohydrate, microcrystalline cellulose, the active ingredient, the hydroxypropyl methyl cellulose and half of the croscarmellose sodium were screened into a 10 Litre Fielder high-shear blender (any suitable high shear blender could be used) and blended for 5 minutes at 300 rpm with the chopper off. The mixture was then granulated by the addition of about 750 ml water whilst continuing to blend. The granules were dried in a Glatt 3/5 fluid bed drier, screened by Comil into a Pharmatec 5 Litre bin blender and then blended with any lactose anhydrous given in the formula plus the remainder of the croscarmellose sodium over 5 minutes at 20 rpm.
  • Magnesium stearate was screened into the blender and the mixing process continued for a further 1 minute at 10 rpm.
  • the lubricated mix was compressed using a Riva Piccolla rotary tablet press fitted with 9.5mm round normal convex punches (any suitable tablet press could be used).
  • the sealcoat, and subsequently the enteric coat are applied by spraying of an aqueous suspension of the coat ingredients in a Manesty 10 coater using parameters for the coating process as recommended by the manufacturers of the coating polymers (again, any suitable coater could be used).
  • Other enteric-coated preparations of this sort can be prepared by one skilled in the art, using these materials or their equivalents.
  • Test animals were housed separately from control animals to avoid inter-animal transfer of drug due to colcophagia. All animals were fed an atherogenic diet twice daily with free access to water. The custom-made diet contained 0.5 and 2% cholesterol and 10 and
  • TUNEL Assay Apoptosis was measured by the TUNEL assay.
  • The*TUNEL assay takes advantage of the fact that during apoptosis nuclear endonucleases digest genomic DNA into fragments of multiples of approximately 200bp.
  • the nucleotide ends were labeled using the FragEL DNA Fragmentation Kit Colorimetric — Klenow Enzyme (Calbiochem, EMD Biosciences, La Jolla, CA, Q1A21-IEA) according to manufacturer's instructions and positive DAB signal was visualized by microscopy as described below.
  • DNase I recombinant, grade I (Roche Diagnostics, Indianapolis, IN; 04 536 282 001) was used to generate TUNEL positive control sections according to manufacturer's instructions. Counterstained with Methyl Green. Analysis was performed on 4 animals from each group, and representative images taken.
  • MLO- A5 osteoblastic cells isolated from 14-day-old osteocalcin promoter-driven T-antigen transgenic mice, J Bone Miner Res 12:2014-2023, 1997) were cultured on Collagenl coated plates at density 15000cells/ well in 12-well TC plates. After 24 hours, the cells were treated w/ 1 OmM ⁇ - GP and LysoPC at 5 ⁇ M concentration for 3 and 7 days. Treatment was repeated every other day. At each time point alizarin red and alkaline phosphatase staining was performed. Alkaline phosphatase Detection Kit (Chemicon, Intl., Temecula, CA, SCR004) was used for detection and performed as per manufactures instructions.
  • MLO-A5 cells were plated in 48-well TC dishes at a density of 5,000 cells/well and allowed to attach (5-6 hrs.). The cells were treated with 5 uM of Lyso PC for 18 hrs followed by analysis with the CyQUANT Cell Proliferation Assay Kit (Molecular Probes) following manufacturers instructions to measure MLO-A5 proliferation.
  • Tissue sampling The right knee joint was removed and dissected. The femoral bone was split in half in parallel to the long axis of the bone. A piece of bone including both the articular surface and growth plate region (-15-20 mm) was sliced off from the front and parallel to the femoral shaft (Figure. 1). The tissues were fixed in 4% paraformaldehyde and scanned within one week in a ⁇ CT scanner (wCT-40, Scanco Corp.) with a voxel resolution of 20 ⁇ m.
  • MicroCT Analysis generates 3 dimensional data including bone density and indices of micro-architecture. From the central region, i.e., between the articular surface (top) and the growth plate (bottom), 250 slices (—5 mm 2 ) were analyzed. To avoid artifact, the measurements were contoured away from the regions directly below the articular surface and the cutting edges. Paraformaldehyde fixed tissues (2x2 cm) were analyzed using microCT 40 (Scanco Medical, Bassersdorf, Switzerland) and a 3D reconstruction of the images slices was generated. The tissues were scanned at a medium resolution with slice thickness and slice increments of 20 ⁇ m. A Sigma filter setting of 0.8, a support value of 1, and a threshold setting of 195.
  • the structural model index is an indicator of trabecular micro-architecture with 0 indicating parallel plate-like formation (a stronger, more structurally mature form) and 3 indicating cylindrical rod-like trabecular (more fragile, degenerating structure).
  • the higher SMI in DM/HC animals shows degeneration of the bone micro -architecture, as shown in Figure 2B as compared with normal controls ( Figure 2A).
  • the DM/HC-induced degenerative changes were minimized, as shown in figure 2C.
  • the BV /TV bone volume over total volume
  • the BS/BV bone surface over bone volume
  • DM/HC animals exhibited reduced BV/TV and increased BS/BV, demononstrating DM/HC animals have osteoporotic pathogenesis. On the contrary, DM/HC animals treated with the Lp-PLA 2 inhibitor SB480848 showed no osteoporotic changes.
  • Tb.Th is a measurement of trabecular thickness. DM/HC animals showed reduced Tb.Th, whereas the reduced Tb.Th thichness was prevented in DM/HC animals treated with the Lp-PLA 2 inhibitor.
  • Tb.Sp Trabecular separation
  • TbN trabecular number
  • ConnD connectivity density
  • Bone marrow homeostasis was also preserved in bone marrow located remotely from traebucular bone, as shown in a low magnification image of the bone marrow remote from the trabecular bone in Figure 5, animals treated with the Lp-PLA 2 inhibitor have normal bone marrow as shown in Figures 5C and D, whereas abnormal homeostasis occurred in non- treated DM/HC animals, as shown in Figure 5A and B.
  • Higher magnification images of bone marrow remote from the trabecular bone are shown in Figure 6D-F, where the Lp-PLA 2 inhibitor prevented DM/HC-induced bone marrow abnormalities that occurred in non-treated DM/HC animals (as shown in Figure 6A-C).
  • EXAMPLE 3 Inhibition of loss of osteocytes and osteblasts by inhibitors of Lp-P LA?- [717] TUNEL staining of regions of trabecular bone were assessed in both DM/HC animals not treated and DM/HC animals treated with an Lp-PLA 2 inhibitor. The animals not treated with the Lp-PLA 2 inhibitor show dramatic increases in TUNEL positive cells within the trabecular bone, for example increased TUNEL positive osteocytes, and increased TUNEL positive osteoblasts on the surface the trabecular bone, as shown in Figure 7A, as compared to animals treated with the Lp-PLA 2 inhibtor, as shown in Figure 4B.
  • Increased apoptosis in DM/HC animals demonstrates increase bone loss, fast bone turn over and reduced bone formation as compared to DM/HC animals treated with a Lp-PLA 2 inhibitor, which demonstrate less bone loss as demonstrated by micro CT analysis as shown in Example 1.
  • MLO-A5 cells treated with 5 ⁇ M LysoPC for 18 hours show reduced proliferation, as compared to non-treated cells, demonstrating that activation of the Lp-PLA 2 pathway by LysoPC prevents proliferation of osteoblasts.

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Abstract

La présente invention concerne des compositions et des méthodes utilisées dans le traitement et la prévention de maladies et de troubles osseux métaboliques par inhibition de Lp-PLA2. Ces compositions et ces méthodes sont utilisées dans le traitement et la prévention de maladies et de troubles osseux métaboliques tels que, par exemple, l'ostéoporose, l'ostéopénie et les maladies associées à l'ostéopénie, et une moelle osseuse anormale.
PCT/US2007/011391 2007-05-11 2007-05-11 Méthodes de traitement et de prévention de maladies et de troubles osseux métaboliques Ceased WO2008140450A1 (fr)

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Cited By (5)

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CN103906744A (zh) * 2011-09-01 2014-07-02 葛兰素集团有限公司 新晶型
US8962633B2 (en) 2007-05-11 2015-02-24 Thomas Jefferson University Methods of treatment and prevention of metabolic bone diseases and disorders
WO2016012916A1 (fr) 2014-07-22 2016-01-28 Glaxosmithkline Intellectual Property Development Limited Dérivés 1,2,3,5-tétrahydro-imidazo [1,2-c]pyrimidine utiles pour le traitement de maladies et de troubles médiés par la lp-pla2
WO2016012917A1 (fr) 2014-07-22 2016-01-28 Glaxosmithkline Intellectual Property Development Limited Dérivés 1,2,3,5-tétrahydro-imidazo [1,2-c]pyrimidine utiles pour le traitement de maladies et de troubles médiés par la lp-pla2
CN120053454A (zh) * 2025-04-29 2025-05-30 浙江大学 Darapladib在制备治疗骨质疏松药物中的应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8962633B2 (en) 2007-05-11 2015-02-24 Thomas Jefferson University Methods of treatment and prevention of metabolic bone diseases and disorders
CN103906744A (zh) * 2011-09-01 2014-07-02 葛兰素集团有限公司 新晶型
WO2016012916A1 (fr) 2014-07-22 2016-01-28 Glaxosmithkline Intellectual Property Development Limited Dérivés 1,2,3,5-tétrahydro-imidazo [1,2-c]pyrimidine utiles pour le traitement de maladies et de troubles médiés par la lp-pla2
WO2016012917A1 (fr) 2014-07-22 2016-01-28 Glaxosmithkline Intellectual Property Development Limited Dérivés 1,2,3,5-tétrahydro-imidazo [1,2-c]pyrimidine utiles pour le traitement de maladies et de troubles médiés par la lp-pla2
CN120053454A (zh) * 2025-04-29 2025-05-30 浙江大学 Darapladib在制备治疗骨质疏松药物中的应用

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