Patent Application
20100311823
The present invention relates to pharmaceuticals comprising nucleic acid pharmaceutical agents comprising sequences that bind to arbitrary transcriptional regulatory factors involved in bone formation, and methods of use thereof.
Bones are reconstructed through repeated formation by osteoblasts, and degradation and resorption by osteoclasts. Ordinarily, bone formation and bone resorption are in a state of balance, and a mutual response mechanism exists between osteoblasts and osteoclasts. However, when the balance between the two is disturbed, bone metabolism abnormalities develop, such as osteoporosis or bone destruction due to chronic rheumatoid arthritis. Such bone metabolism abnormalities are becoming one of the large problems of the current aging society.
For example, osteoporosis is a disease frequently found among middle-aged and older people, and particularly among women, and symptoms appearing with advanced age include pain in the back and hip, as well as stooping of the back and bending of the hip when the bones are crushed due to the body's weight.
At present, calcium agents (for example, calcium aspartate, calcium gluconate, and calcium lactate), vitamin D preparations (for example, Alfacalcidol and Calcitriol), female hormone preparations (Estriol and Ipriflavone), vitamin K2 preparations (Menatetrenone), and such are being used for osteoporosis, but each of them have side effects (for calcium agents and vitamin D preparations, these side effects include hypercalcemia, calcium stones, nephropathy, kidney stones, nausea, diarrhea, constipation, headache, insomnia, malaise, and memory decline; for female hormone preparations (estrogen), they include carcinogenesis, thrombophlebitis, and myocardial infarctions; for Ipriflavone, they include peptic ulcers, gastrointestinal bleeding, dizziness, staggering, rashes, and anemia; and for vitamin K2 preparations, they include gastric distress, nausea, diarrhea, rashes, and headaches). Therefore, there is still a desire for very safe and effective pharmaceutical agents.
Further, Paget's disease and the like are examples of diseases caused by a disturbed balance between osteoclasts and osteoblasts. Paget's disease is a chronic disease of the skeleton in which growth of the bone in the diseased parts becomes abnormal, and the bone enlarges and softens. In Paget's disease, both osteoclasts and osteoblasts become excessively active at certain sites in the bone, and the rate of metabolic turnover at these sites increases significantly. The excessively active sites enlarge, but since they are structurally abnormal, they become weaker than normal sites.
Furthermore, modulation of balance between osteoclasts and osteoblasts can be used to prevent, ameliorate, or treat bone tumors, traumatic chondral defects, osteochondritis dissecans, osteoarthritis, rheumatoid arthritis, bone fractures, dislocations, periodontal diseases, and dental caries, and to ameliorate or treat conditions during or after orthodontic and artificial dental implant treatments.
For example, in bone tumors, tumors originating from various tissues eventually cause bone resorption (bone destruction), and induce bone fracture, bone pain, and hypercalcemia. There are two types of bone resorption: local bone resorption in which tumor cells migrate directly to the bone, activate osteoclasts at that site, and lead to bone destruction; and systemic bone resorption in which bone resorption-promoting factors such as parathyroid hormone-related protein (PTH-rP) produced from tumors activate osteoclasts and lead to bone destruction. In either case, the modulation of osteoclasts may prevent, ameliorate, or treat the disease.
In rheumatoid arthritis, inflammation is said to be followed by the induction of inflammatory cytokines such as IL-1, TNF-α, and IL-6, leading to osteoclast formation and bone resorption. Therefore, suppression of bone resorption by modulating osteoclasts may prevent, ameliorate, or treat diseases.
Maintenance of such balance by osteoclasts and osteoblasts is not limited to bones, but also takes place in teeth.
For example, in periodontal diseases, osteoclasts are activated by the toxins of anaerobic eubacteria, and the bone is destroyed; however, in this case as well, inactivation of osteoclasts may lead to prevention, amelioration, or treatment.
Orthodontics utilizes the phenomenon by which a force applied to a tooth from a certain direction causes osteoclasts that appear in the part of the bone where the force was applied to destroy and resorb the bone, the tooth moves to the region resorbed by the osteoclasts, and osteoblasts that appear at the region after displacement then regenerate the bone. In these cases also, it is thought that treatment may be ameliorated by modulating the balance between osteoclasts and osteoblasts.
In this context, recent attempts have been made to develop novel bone formation-modulating agents, or more specifically, to develop pharmaceutical agents with the activity of modulating the differentiation of osteoclasts and osteoblasts.
Patent Document 1 reports that lactacystin, peptidyl aldehyde, pentoxyfilline (PTX), and such can regulate bone formation and hair growth by inhibiting proteasomes or NF-κB.
However, in Example 4 of Patent Document 1, the inventors reveal that in an in vitro calvaria assay, those compounds that inhibit NF-κB but do not inhibit proteasome activity do not enhance bone formation to the pharmacologically active levels demonstrated by proteasome inhibitors.
The invention of Patent Document 2 discloses a method for treating diseases characterized by undesirable bone resorption using an NMDA-receptor antagonist. It also discloses that antisense mRNAs with the ability to inhibit translation of NMDA receptor mRNAs in osteoclasts may also be used for such treatment.
However, although the inventors show Examples relating to known NMDA-receptor antagonist MK-801 (Merck) and phencyclidine (PCP), they do not give specific examples of antisense mRNAs which are nucleic acid pharmaceuticals.
The invention of Patent Document 3 relates to methods for enhancing bone formation by administering an effective dose of one or more oligomer complexes selected from among RANKL (OPGL), RANKL fusion proteins, analogs, derivatives, or mimics, or osteogenic compounds.
The Examples of Patent Document 3 disclose that administration of GST-RANKL stimulates the proliferation of osteoblasts; however, this method calls for administration of a protein, which differs from the nucleic acid pharmaceuticals of the present invention. Furthermore, although RANKL has been identified as a potent inducer of bone resorption and a positive regulatory factor for osteoclast development, the focus in Patent Document 3 is on the stimulation of osteoblasts by RANKL, and thus it differs from the present invention.
[Patent Document 1] WO 00/02548 pamphlet.
[Patent Document 2] Japanese Patent Kohyo Publication No. (JP-A) 2001-513757 (unexamined Japanese national phase publication corresponding to a non-Japanese international publication).
[Non-Patent Document 1] Garrett I. R. et al., J. Clin. Invest., 2003, 111(11); 1771-1782.
A problem to be solved by the present invention is the development of preventive agents, ameliorating agents, and/or therapeutic agents for diseases caused by a disturbed balance between osteoclasts and osteoblasts.
The present inventors developed pharmaceutical agents with the activity of modulating the differentiation of osteoclasts and osteoblasts.
When bone resorption exceeds bone formation, bone mass and bone density become insufficient, but in such cases, the inhibition of bone resorption can prevent, ameliorate, and/or treat diseases.
The differentiation of osteoclasts, which play an important role in bone resorption, is regulated by various hormones and cytokines, such as IL-1 and TNF-α. Recently, it has also been revealed that osteoclast differentiation requires RANK (Receptor Activator of NF-κB)—RANKL (Receptor Activator of NF-κB Ligand) signal transduction through cell-cell contact.
As the result of dedicated research, the present inventors discovered that osteocyte differentiation can be modulated by inhibiting transcriptional regulatory factors using decoys.
More specifically, the present inventors used decoys to inhibit NF-κB, which is heavily involved in osteoclast differentiation, and discovered that these suppress osteoclast differentiation.
Therefore, the present invention provides bone formation modulating agents comprising nucleic acids comprising bone formation-related transcriptional regulatory factor binding sequence as active ingredients. Specifically, the present invention provides nucleic acid pharmaceutical agents (decoys) that inhibit transcriptional regulatory factors involved in osteocyte differentiation to correct a disturbed balance between bone formation and bone resorption.
In summary, the present invention relates to:
(1) a bone formation modulating agent, in which a nucleic acid comprising a bone formation-related transcriptional regulatory factor binding sequence is an active ingredient;
(2) the bone formation modulating agent of (1), wherein the nucleic acid is selected from a group consisting of a decoy, an antisense, a ribozyme, an aptamer, and an siRNA;
(3) the bone formation modulating agent of (1), wherein the nucleic acid is a decoy comprising a DNA or an RNA;
(4) the bone formation modulating agent of (2) or (3), wherein the decoy comprises a DNA oligonucleotide;
(5) the bone formation modulating agent of any one of (2) to (4), wherein the decoy is a decoy of NF-κB, STAT-1, STAT-3, STAT-6, Ets, AP-1, E2F, Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, Smad8, or Runx2/Cbfa1;
(6) the bone formation modulating agent of (5), wherein the decoy is a decoy of NF-κB;
(7) the bone formation modulating agent of any one of (2) to (6), wherein the decoy comprises the NF-κB binding sequence GGGRHTYYHC (wherein R means A or G, Y means C or T, and H means A, C, or T) (SEQ ID NO:1);
(8) the bone formation modulating agent of (7), wherein the decoy comprises the NF-κB binding sequence GGGATTTCCC (SEQ ID NO: 23) or GGGACTTTCC (SEQ ID NO: 24);
(9) the bone formation modulating agent of any one of (2) to (8), wherein the decoy is a decoy represented by SEQ ID NO: 2;
(10) the bone formation modulating agent of any one of (2) to (5), wherein the decoy comprises the STAT-1 or STAT-3 binding sequence TTCNNNGAA (wherein N means A, G, T, or C) (SEQ ID NO:3);
(11) the bone formation modulating agent of (10), wherein the decoy comprises the STAT-1 or STAT-3 binding sequence TTCCGGGAA (SEQ ID NO:4);
(12) the bone formation modulating agent of any one of (2) to (5), wherein the decoy comprises the STAT-6 binding sequence TTCNNNNGAA (wherein N means A, G, T, or C) (SEQ ID NO:5);
(13) the bone formation modulating agent of (12), wherein the decoy comprises the STAT-6 binding sequence TTCCCAAGAA (SEQ ID NO:6);
(14) the bone formation modulating agent of any one of (2) to (5), wherein the decoy comprises the Ets binding sequence MGGAW (wherein M means A or C, and W means A or T) (SEQ ID NO:7);
(15) the bone formation modulating agent of (14), wherein the decoy comprises the Ets binding sequence CGGAA (SEQ ID NO:8);
(16) the bone formation modulating agent of any one of (2) to (5), wherein the decoy comprises the AP-1 binding sequence TGASTMA (wherein S means G or C, and M means A or C) (SEQ ID NO:9);
(17) the bone formation modulating agent of (16), wherein the decoy comprises the AP-1 binding sequence TGAGTCA (SEQ ID NO:10);
(18) the bone formation modulating agent of any one of (2) to (5), wherein the decoy comprises the E2F binding sequence TTTSSCGS (wherein S means G or C) (SEQ ID NO:11);
(19) the bone formation modulating agent of (18), wherein the decoy comprises the E2F binding sequence TTTCCCGC (SEQ ID NO:12);
(20) the bone formation modulating agent of any one of (2) to (5), wherein the decoy comprises the Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, or Smad8 binding sequence GTCT (SEQ ID NO:13), CAGA (SEQ ID NO:15), or GCCG (SEQ ID NO:17);
(21) the bone formation modulating agent of (20), wherein the decoy comprises the Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, or Smad8 binding sequence GTCTAGAC (SEQ ID NO:14) or CAGACA (SEQ ID NO:16);
(22) the bone formation modulating agent of any one of (2) to (5), wherein the decoy comprises the Runx2/Cbfa1 binding sequence ACCACA (SEQ ID NO:18);
(23) the bone formation modulating agent of (22), wherein the decoy comprises the Runx2/Cbfa1 binding sequence AACCACA (SEQ ID NO:19);
(24) the bone formation modulating agent of any one of (2) to (23), wherein the decoy comprises a double-stranded DNA;
(25) a preventive agent, ameliorating agent, or therapeutic agent for a disease caused by a disturbed balance between osteoclasts and osteoblasts, wherein the agent comprises the bone formation modulating agent of any one of (1) to (24);
(26) a preventive agent, ameliorating agent, or therapeutic agent, wherein the disease of (25) is any one selected from osteoporosis, Paget's disease, bone tumor, traumatic chondral defect, osteochondritis dissecans, osteoarthritis, rheumatoid arthritis, bone fracture, dislocation, periodontal disease, and dental caries;
(27) an ameliorating agent for an orthodontic or artificial dental implant treatment, which comprises the bone formation modulating agent of any one of (1) to (24);
(28) a method for modulating bone formation that uses a nucleic acid comprising a bone formation-related transcriptional regulatory factor binding sequence;
(29) the method of (28), wherein the nucleic acid is selected from a group consisting of a decoy, an antisense, a ribozyme, an aptamer, and an siRNA;
(30) the method of (28), wherein the nucleic acid is a decoy comprising a DNA or an RNA;
(31) the method of (29) or (30), wherein the decoy comprises a DNA oligonucleotide;
(32) the method of any one of (29) to (31), wherein the decoy is a decoy of NF-κB, STAT-1, STAT-3, STAT-6, Ets, AP-1, E2F, Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, Smad8, or Runx2/Cbfa1;
(33) the method of (32), wherein the decoy is a decoy of NF-κB;
(34) the method of any one of (29) to (33), wherein the decoy comprises the NF-κB binding sequence GGGRHTYYHC (wherein R means A or G, Y means C or T, and H means A, C, or T) (SEQ ID NO:1);
(35) the method of (34), wherein the decoy comprises the NF-κB binding sequence GGGATTTCCC (SEQ ID NO: 23) or GGGACTTTCC (SEQ ID NO: 24);
(36) the method of any one of (29) to (35), wherein the decoy is a decoy represented by SEQ ID NO: 2;
(37) the method of any one of (29) to (32), wherein the decoy comprises the STAT-1 or STAT-3 binding sequence TTCNNNGAA (wherein N means A, G, T, or C) (SEQ ID NO:3);
(38) the method of (37), wherein the decoy comprises the STAT-1 or STAT-3 binding sequence TTCCGGGAA (SEQ ID NO:4);
(39) the method of any one of (29) to (32), wherein the decoy comprises the STAT-6 binding sequence TTCNNNNGAA (wherein N means A, G, T, or C) (SEQ ID NO:5);
(40) the method of (39), wherein the decoy comprises the STAT-6 binding sequence TTCCCAAGAA (SEQ ID NO:6);
(41) the method of any one of (29) to (32), wherein the decoy comprises the Ets binding sequence MGGAW (wherein M means A or C, and W means A or T) (SEQ ID NO:7);
(42) the method of (41), wherein the decoy comprises the Ets binding sequence CGGAA (SEQ ID NO:8);
(43) the method of any one of (29) to (32), wherein the decoy comprises the AP-1 binding sequence TGASTMA (wherein S means G or C, and M means A or C) (SEQ ID NO:9);
(44) the method of (43), wherein the decoy comprises the AP-1 binding sequence TGAGTCA (SEQ ID NO:10);
(45) the method of any one of (29) to (32), wherein the decoy comprises the E2F binding sequence TTTSSCGS (wherein S means G or C) (SEQ ID NO:11);
(46) the method of (45), wherein the decoy comprises the E2F binding sequence TTTCCCGC (SEQ ID NO:12);
(47) the method of any one of (29) to (32), wherein the decoy comprises the Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, or Smad8 binding sequence GTCT (SEQ ID NO:13), CAGA (SEQ ID NO:15), or GCCG (SEQ ID NO:17);
(48) the method of (47), wherein the decoy comprises the Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, or Smad8 binding sequence GTCTAGAC (SEQ ID NO:14) or CAGACA (SEQ ID NO:16);
(49) the method of any one of (29) to (32), wherein the decoy comprises the Runx2/Cbfa1 binding sequence ACCACA (SEQ ID NO:18);
(50) the method of (49), wherein the decoy comprises the Runx2/Cbfa1 binding sequence AACCACA (SEQ ID NO:19);
(51) the method of any one of (29) to (50), wherein the decoy comprises a double-stranded DNA;
(52) the method of any one of (28) to (51) for preventing, ameliorating, or treating a disease caused by a disturbed balance between osteoclasts and osteoblasts;
(53) the method of (52), wherein the disease caused by the disturbed balance between osteoclasts and osteoblasts is selected from the group consisting of osteoporosis, Paget's disease, bone tumor, traumatic chondral defect, osteochondritis dissecans, osteoarthritis, rheumatoid arthritis, bone fracture, dislocation, periodontal disease, and dental caries;
(54) the method of any one of (28) to (51) for ameliorating an orthodontic or artificial dental implant treatment;
(55) a use of a nucleic acid comprising a bone formation-related transcriptional regulatory factor binding sequence for producing a pharmaceutical agent for modulating bone formation;
(56) the use of (55), wherein the nucleic acid is selected from a group consisting of a decoy, an antisense, a ribozyme, an aptamer, and an siRNA;
(57) the use of (55), wherein the nucleic acid is a decoy comprising a DNA or an RNA;
(58) the use of (56) or (57), wherein the decoy comprises a DNA oligonucleotide;
(59) the use of any one of (56) to (58), wherein the decoy is a decoy of NF-κB, STAT-1, STAT-3, STAT-6, Ets, AP-1, E2F, Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, Smad8, or Runx2/Cbfa1;
(60) the use of (59), wherein the decoy is a decoy of NF-κB;
(61) the use of any one of (56) to (60), wherein the decoy comprises the NF-κB binding sequence GGGRHTYYHC (wherein R means A or G, Y means C or T, and H means A, C, or T) (SEQ ID NO:1);
(62) the use of (61), wherein the decoy comprises the NF-κB binding sequence GGGATTTCCC (SEQ ID NO: 23) or GGGACTTTCC (SEQ ID NO: 24);
(63) the use of any one of (56) to (62), wherein the decoy is a decoy represented by SEQ ID NO: 2;
(64) the use of any one of (56) to (59), wherein the decoy comprises the STAT-1 or STAT-3 binding sequence TTCNNNGAA (wherein N means A, G, T, or C) (SEQ ID NO:3);
(65) the use of (64), wherein the decoy comprises the STAT-1 or STAT-3 binding sequence TTCCGGGAA (SEQ ID NO:4);
(66) the use of any one of (56) to (59), wherein the decoy comprises the STAT-6 binding sequence TTCNNNNGAA (wherein N means A, G, T, or C) (SEQ ID NO:5);
(67) the use of (66), wherein the decoy comprises the STAT-6 binding sequence TTCCCAAGAA (SEQ ID NO:6);
(68) the use of any one of (56) to (59), wherein the decoy comprises the Ets binding sequence MGGAW (wherein M means A or C, and W means A or T) (SEQ ID NO:7);
(69) the use of (68), wherein the decoy comprises the Ets binding sequence CGGAA (SEQ ID NO:8);
(70) the use of any one of (56) to (59), wherein the decoy comprises the AP-1 binding sequence TGASTMA (wherein S means G or C, and M means A or C) (SEQ ID NO:9);
(71) the use of (70), wherein the decoy comprises the AP-1 binding sequence TGAGTCA (SEQ ID NO:10);
(72) the use of any one of (56) to (59), wherein the decoy comprises the E2F binding sequence TTTSSCGS (wherein S means G or C) (SEQ ID NO:11);
(73) the use of (72), wherein the decoy comprises the E2F binding sequence TTTCCCGC (SEQ ID NO:12);
(74) the use of any one of (56) to (59), wherein the decoy comprises the Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, or Smad8 binding sequence GTCT (SEQ ID NO:13), CAGA (SEQ ID NO:15), or GCCG (SEQ ID NO:17);
(75) the use of (74), wherein the decoy comprises the Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, or Smad8 binding sequence GTCTAGAC (SEQ ID NO:14) or CAGACA (SEQ ID NO:16);
(76) the use of any one of (56) to (59), wherein the decoy comprises the Runx2/Cbfa1 binding sequence ACCACA (SEQ ID NO:18);
(77) the use of (76), wherein the decoy comprises the Runx2/Cbfa1 binding sequence AACCACA (SEQ ID NO:19);
(78) the use of any one of (56) to (77), wherein the decoy comprises a double-stranded DNA;
(79) the use of any one of (55) to (78), wherein the bone formation modulating agent prevents, ameliorates, or treats a disease caused by a disturbed balance between osteoclasts and osteoblasts;
(80) the use of (79), wherein the disease caused by a disturbed balance between osteoclasts and osteoblasts is selected from the group consisting of osteoporosis, Paget's disease, bone tumor, traumatic chondral defect, osteochondritis dissecans, osteoarthritis, rheumatoid arthritis, bone fracture, dislocation, periodontal disease, and dental caries; and
(81) the use of any one of (55) to (78), wherein the bone formation modulating agent ameliorates an orthodontic or artificial dental implant treatment.
The present invention provided decoys that are nucleic acid pharmaceuticals that inhibit transcriptional regulatory factors involved in bone formation. Furthermore, such decoys can be used for diseases caused by a disturbed balance between osteoclasts and osteoblasts, such as osteoporosis and Paget's disease. In addition, they can be used as preventive, ameliorating, or therapeutic agents for bone tumors, traumatic chondral defects, osteochondritis dissecans, osteoarthritis, rheumatoid arthritis, bone fractures, dislocations, periodontal diseases, and dental caries, as well as ameliorating agents for orthodontic and artificial dental implant treatments.
a) and (b) show the effects of NF-κB decoy in the ovariectomy-induced osteoporosis model. (a) shows the body weight and serum estradiol levels. (b) The left panel shows a micrograph after hematoxylin staining and the right panel shows a fluorescence micrograph of the cryosections of proximal tibia after FITC-labeled NF-κB decoy treatment (30 μg/kg/h) (×40). Sham=sham operation; OVX=bilateral ovariectomy; NF-κB=bilateral ovariectomy and treatment with NF-κB decoy (30 μg/kg/h); Scb=scramble decoy (30 μg/kg/h)=bilateral ovariectomy and treatment with scramble decoy (30 μg/kg/h). (* P<0.05 compared to Sham; † P<0.05 compared to Scb; N=8)
c) and (d) show the effects of NF-κB decoy in the ovariectomy-induced osteoporosis model. (c) TRAP staining images of the distal femur at 14 days after treatment (×100); and (d) TRAP activities in the proximal tibia and distal femur in four groups at 14 days after treatment. Sham=sham operation; OVX=bilateral ovariectomy; NF-κB=bilateral ovariectomy and treatment with NF-κB decoy (30 μg/kg/h); Scb=scramble decoy (30 μg/kg/h)=bilateral ovariectomy and treatment with scramble decoy (30 μg/kg/h). (* P<0.05 compared to Sham. † P<0.05 compared to Scb; N=8)
a) and (b) show the effects of NF-κB decoy on (a) calcium concentrations in proximal tibia and distal femur and (b) hydroxyproline concentrations in proximal tibia and distal femur in four groups of ovariectomy-induced osteoporosis models at 14 days after treatment. Sham=sham operation; OVX=bilateral ovariectomy; NF-κB=bilateral ovariectomy and treatment with NF-κB decoy (30 μg/kg/h); Scb=scramble decoy (30 μg/kg/h)=bilateral ovariectomy and treatment with scramble decoy (30 μg/kg/h). (* P<0.05 compared to Sham; † P<0.05 compared to Scb; N=8)
c) shows the effects of NF-κB decoy on (c) urinary deoxypyridinoline (which is regulated by the urinary creatine concentration) in four groups of ovariectomy-induced osteoporosis models at 14 days after treatment. Sham=sham operation; OVX=bilateral ovariectomy; NF-κB=bilateral ovariectomy and treatment with NF-κB decoy (30 μg/kg/h); Scb=scramble decoy (30 μg/kg/h)=bilateral ovariectomy and treatment with scramble decoy (30 μg/kg/h). (* P<0.05 compared to Sham; † P<0.05 compared to Scb; N=8)
The bone formation modulating agents of the present invention are nucleic acid pharmaceutical agents that comprise nucleic acids comprising bone formation-related transcriptional regulatory factor binding sequence as active ingredients. The bone formation modulating agents of the present invention can modulate the process of bone formation (for example, the balance between osteoclasts and osteoblasts) by acting on bone formation-related transcriptional regulatory factors.
Various factors relating to bone formation are included in the “bone formation-related transcriptional regulatory factors” described in the present description. Specific examples of bone formation-related transcriptional regulatory factors include NF-κB, STAT-1, STAT-3, STAT-6, Ets, AP-1, E2F, Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, Smad8, and/or Runx2/Cbfa1, but are not limited thereto. In the present description, a “bone formation-related transcriptional regulatory factor binding sequence” means a sequence that can bind to a transcriptional regulatory factor involved in bone formation, such as those transcriptional regulatory factors mentioned above.
The nucleic acid pharmaceutical agents used in the present invention are ordinarily decoys, antisenses, ribozymes, aptamers, siRNAs, or such, but are preferably decoys. In the present description, decoys, decoy oligonucleotides, and decoy ODNs are used synonymously.
Examples of decoys that may be used include decoys of NF-κB, STAT-1, STAT-3, STAT-6, Ets, AP-1, E2F, Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, Smad8, and Runx2/Cbfa1, but NF-κB decoys are preferred. In one embodiment, NF-κB decoys comprise an NF-κB-binding sequence. An NF-κB-binding sequence can be selected from among the consensus sequences of NF-κB binding sequence, GGGRHTYYHC (R(A,G); Y(C, T); and H(A, C, T)) (SEQ ID NO: 1). An example of an NF-κB decoy is a decoy indicated by SEQ ID NO: 2, but is not limited thereto.
In another embodiment, it is possible to use a decoy comprising a sequence selected from among consensus sequences of the STAT-1 and STAT-3 binding sequence, TTCNNNGAA (N(A, G, T, C)) (SEQ ID NO: 3); consensus sequences of the STAT-6 binding sequence, TTCNNNNGAA (N(A, G, T, C)) (SEQ ID NO: 5); consensus sequences of the Ets binding sequence, MGGAW (M(A, C); W(A, T)) (SEQ ID NO: 7); consensus sequences of the AP-1 binding sequence, TGASTMA (S(G, C); M(A, C)) (SEQ ID NO: 9); consensus sequences of the E2F binding sequence, TTTSSCGS (S(G, C)) (SEQ ID NO: 11); consensus sequences of the Smad binding sequence, GTCT (SEQ ID NO: 13), CAGA (SEQ ID NO: 15), or GCCG (SEQ ID NO: 17); and the consensus sequence of the Runx2/Cbfa1 binding sequence, ACCACA (SEQ ID NO: 18).
Specific examples of the decoys that can be used include decoys comprising sequences selected from among the STAT-1 and STAT-3 binding sequence, TTCCGGGAA (SEQ ID NO: 4); the STAT-6 binding sequence, TTCCCAAGAA (SEQ ID NO: 6); the Ets binding sequence, CGGAA (SEQ ID NO: 8); the AP-1 binding sequence, TGAGTCA (SEQ ID NO: 10); the E2F binding sequence, TTTCCCGC (SEQ ID NO: 12); the Smad binding sequence, GTCTAGAC (SEQ ID NO: 14) or CAGACA (SEQ ID NO: 16); and the Runx2/Cbfa1 binding sequence, AACCACA (SEQ ID NO: 19), but are not limited thereto.
The decoys of the present invention may be composed of oligonucleotides. The oligonucleotides may be DNAs or RNAs, and they may comprise modified nucleic acids and/or pseudo nucleic acids. The oligonucleotides may be single stranded or double stranded, but are preferably double stranded. Mutants of these, compounds intramolecularly comprising these, or oligonucleotides comprising complements of these may also be used.
The decoys of the present invention can be produced using standard methods, such as by using DNA synthesizers.
The bone formation modulating agents of the present invention (for example, decoys) can be administered as pharmaceutical compositions locally, systemically, orally, or parenterally, alone or after mixing with common pharmaceutical carriers. For example, when inflammation is localized, as in rheumatoid arthritis, the agents can be injected locally. Furthermore, for periodontal diseases such as alveolar pyorrhea, the agents can be injected into the gingival sulcus using an ointment as the base material.
The present pharmaceutical compositions can be provided in liquid dosage forms such as solutions, suspensions, syrups, liposomes, or lotions, or as solid dosage forms such as tablets, granules, powders, and capsules. If necessary, the pharmaceutical compositions may be supplemented with various vehicles, excipients, stabilizers, lubricants, and/or other conventional pharmaceutical additives.
The pharmaceutical compositions of the present invention can be used to prevent, ameliorate, and/or treat diseases caused by a disturbed balance between osteoclasts and osteoblasts, such as osteoporosis or Paget's disease. Alternatively, they can be used as preventive, ameliorating, and/or therapeutic agents for bone tumors, traumatic chondral defects, osteochondritis dissecans, osteoarthritis, rheumatoid arthritis, bone fractures, dislocations, periodontal diseases, and dental caries, as well as ameliorating agents for orthodontic and artificial dental implant treatments.
The doses of the decoys of the present invention differ depending on the age, body weight, symptoms, the methods of administration, administration routes, and such, but for intraarticular administration, intravascular administration, or intramuscular administration, for example, 10 nmol to 10,000 nmol can be administered to an adult per day at a single site or as divided in multiple sites.
Hereinbelow, the present invention will be specifically described with reference to Examples, but it is not to be construed as being limited thereto.
Bone marrow cells were prepared from three-day-old white rabbits. Bone marrow cells were taken out from rabbit femurs and tibias, collected into tubes, and washed twice with PBS. The fraction rich in mononucleated cells was separated from the bone marrow cells and cultured at 1×105 cells/well using α-MEM medium supplemented with 10% fetal calf serum. Medium exchange was carried out every three days using α-MEM supplemented with vitamin D3 (1×10−8 M). In addition, luciferase gene (Clontech) regulated by an NF-κB binding sequence was transfected into the bone marrow cells using LipofectAMINE 2000 (Invitrogen). 24 hours after transfection, the cells were cultured for six hours in a serum-free medium. Resting state cells were treated with 1×10−8 M vitamin D3 for four hours or 48 hours, washed with PBS, and then lysed at 4° C. for 15 minutes with 200 μL of cell lysis buffer. 20 μL of the cell extracts were mixed with 100 μL of Luciferase Assay Reagent and measured for two seconds each using a luminometer. Cells that were not treated with vitamin D3 but that were otherwise similarly treated were used as the control.
The following were used as the NF-κB decoys and scramble decoys. NF-κB decoy:
Scramble decoy:
The decoy oligonucleotides were prepared using a DNA synthesizer according to standard methods.
Osteoclasts were detected by staining for TRAP, which is considered to be an osteoclast marker. Cells were treated with vitamin D3 (1×10−8 M) and scramble decoy or NF-κB decoy, then fixed on day 7 of culturing using 4.0% paraformaldehyde/PBS at room temperature for ten minutes. The cells were then TRAP stained (Code # OP04, Hokudo).
Vitamin D3 (1×10−8 M) and scramble decoy or NF-κB decoy were added to the cells, which were then stained on day 7 of culturing using Hoechst 33258 (10 μL) for ten minutes. This was observed using a non-confocal microscope (Olympus BX60) at an excitation wavelength of 360 nm. (Since Hoechst 33258 stains all cell nuclei with a disrupted membrane, necrotic cells are observed as intact blue nuclei, while apoptotic cells are observed as fragmented (or condensed) nuclei.)
To store the cells and substrates, samples were fixed with 0.2 M cacodylate buffer supplemented with 5% glutaraldehyde overnight at 4° C. and with 2% osmium for three hours. This was followed by stepwise dehydration of the samples using ethanol (70-100%). To use for scanning electron microscopy (SEM), samples were critical point dried using carbon dioxide. The samples were then coated with gold using a sputtering method, and observed by SEM (JEOL, T-200). To use for transmission electron microscopy (TEM), samples were dehydrated stepwise using ethanol (70-100%), and then embedded in Epok812. Ultra-thin sections were produced using an ultramicrotome (MT-7000, RMC), and these were placed on a collodion-coated copper grid. They were then stained using tannic acid, uranyl acetate, and lead citrate, and then observed by TEM (H-7000, Hitachi).
Results) After treating the cells with vitamin D3, TRAP-positive multinucleated cells were observed (see
Electron microscopic observations also showed that multinucleated cells covering the bone surface with a clear zone (Cz) are absorbed by the bone mineral through the ruffled borders (Rb). In
When luciferase gene regulated by an NF-κB binding sequence was transfected into the bone marrow cells of newborn rabbits to confirm NF-κB activation during osteoclast differentiation, luciferase activity was induced in cells treated with vitamin D3, 48 hours after treatment. This means that NF-κB activity increased (see
To confirm whether vitamin D3-induced NF-κB activity regulates both differentiation and activation of osteoclasts, NF-κB decoy was transfected into osteoclasts, and its inhibitory activity was investigated.
In cells transfected with FITC-labeled decoy (1 μM), activity was observed as a strong fluorescence in the nucleus one, four, and seven days after vitamin D3 treatment (see
When luciferase gene regulated by an NF-κB sequence was transfected together with NF-κB decoy, luciferase activity decreased greatly in undifferentiated osteoclasts four days after vitamin D3 treatment (see
Osteoclasts that differentiated from bone marrow cells due to vitamin D3 are detected as TRAP staining-positive multinucleated cells. TRAP-positive multinucleated osteoclasts significantly increased in the presence of vitamin D3 (see
Since NF-κB is well known as a factor that suppresses cell death, the effect of NF-κB decoy on osteoclast apoptosis was investigated. The cells underwent nuclear condensation, which is recognized as apoptosis (see
Hence, induction of osteoclasts by vitamin D3 was inhibited by NF-κB decoy. This took place partly due to the induction of apoptosis.
TRAP staining was carried out with rat osteoclasts induced by M-CSF and RANKL. NF-κB decoy weakened induction of differentiation to rat osteoclasts by M-CSF and RANKL (see
Osteoclasts were examined using a rat osteoclast formation kit (Hokudo, CAT# CUOC01). Rat osteoclast precursor cells seeded in 24-well plates were incubated in a medium supplemented with M-CSF and RANKL. TRAP staining was performed seven days later.
The effects of NF-κB decoy on resorption pit formation were examined. Three to four mm-thick dentine sections produced from human teeth were placed into 24-well plates. Bone marrow cells on the sixth day after vitamin D3 treatment (1×104 cells/mL) were incubated for six hours on the dentine sections and then washed with PBS, then vitamin D3 (1×10−8 M) and NF-κB decoy or scramble decoy were further added, and this was cultured for three days. After removing the organic matter using sodium hypochlorite, the sections were stained with toluidine blue (SIGMA) and the resorption pits were visualized. Furthermore, the number of circular dots (osteoclast resorption pits) in the sections, which are reported to be related to osteoclast activity, were counted under a light microscope.
Results) TRAP staining was performed seven days after vitamin D3 treatment to measure osteoclast activity. The NF-κB decoy-administered dentine sections showed significant suppression of osteoclast activity compared to scramble decoy-administered dentine sections (see
Female adult Wistar rats (ten weeks old) were purchased from Japan SLC (Shizuoka, Japan). After the rats were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (10 mg/kg), bilateral ovariectomies or sham operations (comparison subjects) were performed and osmotic mini-pumps (Alzet model 2004; Alza Corp) containing either NF-κB decoy or scramble decoy (infusion rate of 30 μg/kg/hour) were implanted. The body weights of these mice were recorded for two weeks. At two weeks after the operations, the mice were deeply anesthetized and their femurs, tibias, blood, and urine were collected for biochemical analyses. The proximal tibia and distal femur were cut out and homogenized in 10 mM triethanolamine buffer (pH 7.5), and supernatants were used to measure TRAP activity using the method of Walter (Walter, K. and Schutt, C., Acid and alkaline phosphatase in serum. Method of Enzymatic Analysis, Academic Press, New York & London: 1974; 856-870). The precipitates were hydrolyzed (hydized) for 24 hours at 105° C. in hydrochloric acid (6 M), hydroxyprolines were measured by the method of Bergman, and the Ca content was measured based on the o-cresolphthalein complexone (OCPC) method by Gitelman (Gitelman, H. J., Kukolj, S., Welt, L. G. Inhibition of parathyroid gland activity by hypermagnesemia. Am. J. Physiol. 1968; 215:483-485). The estradiol level in the serum was measured by EIA (Mitsubishi Kagaku Iatron, Tokyo, Japan), and urinary deoxypyridinoline level was measured on day 14 of the experiments by EIA (Metra Biosystems, Mountain View, Calif.).
Results) The effects of NF-κB decoy on osteoporosis in the ovariectomized rat model
To further clarify the role of NF-κB in the activation of osteoclasts, the authors employed an estrogen-deficient ovariectomized rat model as a model of osteoporosis.
At 14 days after bilateral ovariectomy, serum estradiol levels were significantly decreased in the ovariectomized group, while there was no significant difference in body weight (
In contrast, TRAP activity in the tibia and femur and the increase of TRAP staining-positive area were lowered by continuous treatment with NF-κB decoy using an osmotic mini-pump, but not with scramble decoy (
These results were accompanied with changes in urinary deoxypyridinoline released from the bone by the processing of tissue collagen. Treatment with NF-κB decoy suppressed the ovariectomy-induced increase in urinary deoxypyridinoline (P<0.01,
Osteogenic disorder Shionogi (ODS) rats are a mutant strain of Wistar rats that are deficient in a key enzyme for ascorbate synthesis, in which several hydroxylases such as prolinehydroxylase are decreased (Togari, A., Arai, M., Nakagawa, S., Banno, A., Aoki, M., Matsumoto S., Alteration of bone status with ascorbic acid deficiency in ODS (osteogenic disorder Shionogi) rat, Jpn. J. Pharmacol., 1995 July; 68(3):255-261). When these rats are raised normally on a vitamin C-supplemented diet until they are nine weeks old and then given a deficient diet, collagen synthesis cannot be carried out, causing disintegration of blood vessels and bones and leading to death in approximately five weeks.
Male osteogenic disorder Shionogi rats (eight weeks old) were purchased from CLEA laboratory and fed a diet supplemented with L-ascorbate at 800 mg/kg body weight. One week later, the diet was changed to an L-ascorbate-deficient diet, and osmotic mini-pumps containing either NF-κB or scramble decoy ODN (infusion rate of 30 μg/kg/hour) were implanted in the rats. The body weight of these rats was recorded before and on the fourth week after terminating L-ascorbate administration. The tibias and femurs were analyzed on the fourth week after the operation.
Bone density (BMD) (g/cm2) was measured using a bone density X-ray apparatus, the dual-photon X-ray absorptiometry (DEXA) bone densitometer (GE-Lunar DPX-IQ, Madison, Wis.). High- and low-beam energies for all scans were 80 and 35 kV respectively, at 0.5 mA, as described by Venken, K. et al., Bone 2005, 36:663-670.
Results) Effects of NF-κB decoy ODN on osteoporosis in a vitamin C-deficient rat model
Osteogenic disorder Shionogi rats, which are deficient in a key enzyme for ascorbate synthesis, were used to further verify the therapeutic effect of NF-κB decoy in osteoporosis.
After termination of the L-ascorbate-supplemented diet, the body weight of the osteogenic disorder Shionogi rats dramatically decreased and bone length was shortened. However, NF-κB decoy ODN treatment inhibited the decrease in body weight (
Bone fracture was frequently found in the proximal femur of the groups treated with scramble decoy ODN, but not in the groups treated with NF-κB decoy ODN. As shown in
All values were indicated as a mean±standard error. Variance was determined by the Bonferroni/Dunnett method, and significance was examined for numerous comparisons. P<0.05 was considered as a significant difference.
The present invention provides preventive, ameliorating, and/or therapeutic agents for diseases caused by a disturbed balance between bone formation and bone resorption.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-365148 | Dec 2004 | JP | national |
| 2005-234311 | Aug 2005 | JP | national |
This application is a Continuation of U.S. application Ser. No. 11/721,834, which is the U.S. National Stage application of PCT/JP2005/023078, filed Dec. 15, 2005, which claims priority from Japanese applications JP 2004-365148, filed Dec. 16, 2004, and JP 2005-234311, filed Aug. 12, 2005, the entire contents of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11721834 | Sep 2007 | US |
| Child | 12855242 | US |
