CN101020920A - Cytologic process of measuring bioactivity of bone absorption inhibiting medicine - Google Patents
Cytologic process of measuring bioactivity of bone absorption inhibiting medicine Download PDFInfo
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- CN101020920A CN101020920A CNA2007100382523A CN200710038252A CN101020920A CN 101020920 A CN101020920 A CN 101020920A CN A2007100382523 A CNA2007100382523 A CN A2007100382523A CN 200710038252 A CN200710038252 A CN 200710038252A CN 101020920 A CN101020920 A CN 101020920A
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Abstract
The present invention relates to measurement of activity of bone absorption inhibiting medicine, and is especially process of measuring the activity of osteoclast inhibiting medicine quantitatively. The measurement process includes the following steps: 1. establishing method of in vitro inducing and culturing osteoclast and inducing and culturing mature osteoclast; 2. adding medicines of different concentrations into the inducing and culturing system; 3. measuring cell growth state or cell active function state; and 4. calculating the bioactivity of the medicine. The process is precise, reliable, convenient, economic and simple, and may be used in screening new compound, determining the osteoclast inhibiting activity and judging the possibility of being used as candidate medicine for treating osteoporosis.
Description
Technical field
The present invention relates to a kind of method that suppresses the bone resorption pharmaceutical activity of measuring, this method is specifically related to the activity of osteoclast suppressive drug is carried out quantitative mensuration.
Background technology
Osteoporosis is common disease and frequently-occurring disease, owing to this sick sickness rate height, complication are comparatively serious, the shortage ideal prevents and the treatment measure, becomes the focus that people pay close attention to day by day, and along with aged crowd's increase, sickness rate is also in rising trend.
The keeping of bone normal morphology and function depends on the running balance between the synthetic and bone absorption of sclerotin, the osteoporotic pathogenic factor of all kinds is different, it but is identical that but basic pathology changes, thereby promptly because bone absorption surpasses the synthetic bone loss that causes general of sclerotin.Treat osteoporotic main means, the one, promote the medicine of bone mineralising, as the calcium preparation of vitamins D and various kinds etc.; The 2nd, the medicine of anti-bone resorption is as estrogens medicine alternative medicine at present commonly used, diphosphonate, thyrocalcitonin etc.Along with the osteoporosis sickness rate rise and people to the concern of its hazardness, develop safer and more effective osteoporosis control medicine, become one of present ten minutes active research field, the medicine that especially can effectively suppress osteoclast effect minimizing destruction of bone has become current main goal in research.As the thyrocalcitonin of recent listing and just the bone protected protein in clinical trial (Osteoprotegerin OPG), anti-RANKL monoclonal antibody denosumab etc., all is to treat osteoporotic new drug by suppressing the osteoclast effect.
Normal human's osseous tissue is in the bone reconstruction that does not stop, bone is rebuild and is meant that removing the local old bone of bone replaces the process that forms new bone, be a kind of important replacement mechanism of ripe osseous tissue, to be osteoclast paired with one of scleroblast, link coupled cellular activity process mutually, the main pathogenesis of osteoporosis is that the bone reconstruction of body is unbalance, and vital role is wherein played in acting on of osteoclast.Osteoclast derives from blood mononuclear cell, enters behind the osseous tissue that differentiation and maturation is an osteoclast under the effect of marrow stromal cell excretory DIF.The function of osteoclast is a bone resorption, it can as excavator bone surface to around dissolve osseous tissue.The osteoclast bone resorption mainly leans on two kinds of effects, and the one, it is the ruffled border release acidic substance that the brush shape contacts with ground substance of bone, with inorganic salt such as the calcium phosphorus dissolving of bone surface; The 2nd, the bone that the clear zone that links to each other with ground substance of bone will be to be absorbed is surrounded, and discharges at the fold edge under the cooperation of lysosomal enzyme, and the bone organic substrate is degraded.The important proteolytic enzyme in a organized way of the enzyme that the osteoclast lysosome comprises, matrix metalloproteinase enzyme, L-Cysteine HCL Anhydrous, serine protease and acid phosphatase.These enzymes are the necessary enzymes of osteoclast function, also are the features of osteoclast simultaneously, can be used for measuring osteoclast activity.
Anti-bone resorption medicine can be the combining of osteoclast and precursor cell film thereof surface blocking-up activation inducible factor and acceptor, or by suppressing the normal physiological metabolism of osteoclast, suppresses that the formation of osteoclast or inhibition osteoclast activity play a role.This class medicine both may suppress the growth of osteoclast precursor cell or osteoclast, also may be the activity that suppresses the required enzyme of osteoclast performance biological function.
Summary of the invention
The purpose of this invention is to provide a kind of with the bioactive method of cytology quantitative assay inhibition bone resorption medicine.
Purpose of the present invention can reach by following measure:
1. set up external evoked cultivation osteoclast method or and thus the method inducing culture go out sophisticated osteoclast;
2. the medicine that in inducing culture system or culture system, adds different concns;
3. cell growth state or cytoactive functional status are measured;
4. the calculating of medicine to be measured biological activity unit.
The measuring method of cell growth state and active function state, can be MTS[3-(4,5-dimethylthiazol-2-yl)-5 (3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium, inner salt] method, MTT (diMethylThiazol-diphenyl Tetrazolium bromide) method, CCK8 (Cell Counting Kit-8) method, XTT (2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) method, platform Finland staining and kethepsin, matrix metalloproteinase enzyme, L-Cysteine HCL Anhydrous, serine protease, anti-tartaic acid phosphatase substrate reaction solution method.And with IC50 (50% inhibitary concentration) value calculated activity unit.Medicine is that osteoclast produces and the medicine of mature osteoclast active function to suppress, as estrogens medicine, bisphosphonates, thyrocalcitonin medicine and osteoprotegerin or the like.
This class medicine both may suppress the growth of osteoclast precursor cell or osteoclast, also may be the activity that suppresses the required enzyme of osteoclast performance biological function.We have done " medicine is divided into the inhibiting mensuration of osteoclast to broken bone precursor cell " and " medicine is to the mensuration of mature osteoclast activity inhibition " test respectively.Concrete experimental technique is as follows;
One medicine is divided into the inhibiting mensuration of osteoclast to broken bone precursor cell
1 sets up the method for external evoked cultivation osteoclast
At the precursor cell RAW264.7 of external evoked cultivation osteoclast, in the inducing culture system, add inductor 1,25 (OH)
2VitD3 (1,25-dihydroxyvitamin D3), 1,25 (OH)
2The final concentration of VitD3 is 10E6-10E9M, forms by tartaric-resistant and periosteum sheet bone lacuna and identifies the success of osteoclast inducing culture.
2 add the medicine of different concns in the inducing culture system
Pair cell divides into groups.Control group does not add medicine, and the medicine group adds the medicine of different concns when inducing mixed culture, is used to estimate the restraining effect of medicine to broken bone precursor cell differentiation.Specifically be grouped as follows:
1) inductor control group: the Raw264.7 inducing culture of simple inductor effect
2) medicine group: the medicine (medicine and inductor add simultaneously) of the dilution of the Raw264.7 inducing culture of inductor effect+by a certain percentage
3 cell states are measured
Utilize cell growth state and existing state measuring method such as mtt assay, MTS method, the dyeing of platform Finland, CCK-8, methods such as XTT, detection of drugs is to the influence of broken bone precursor cell differentiation with growth.Also can with the active method of osteoclast certain enzyme as select for use the acid phosphatase enzyme substrates right-nitrophenol phosphoric acid ester (NPP), measure the activity of the anti-tartaic acid Phosphoric acid esterase respectively organize cell.
The calculating of 4 activity units
The measured value of the inhibiting rate of medicine (%)=(measured value of the measured value of inductor control group-medicine group) * 100/ inductor control group.With the inhibiting rate that is obtained is ordinate zou, is the X-coordinate mapping with the logarithm of corresponding concentration, with the linear regression treatment of experimental data, calculates the IC50 of medicine, with IC50 calculated activity unit.
Two medicines are to the mensuration of mature osteoclast activity inhibition
1 inducing culture goes out sophisticated osteoclast
With 1,25 (OH)
2VitD3 induces mature osteoclast at the broken bone precursor cell RAW264.7 of external evoked cultivation, forms by tartaric-resistant and periosteum sheet bone lacuna and identifies the success of osteoclast inducing culture.
2 add the medicine of different concns in culture system
Pair cell divides into groups.Control group does not add medicine, and the medicine group adds the medicine of different concns after inducing culture goes out mature osteoclast, is used to estimate the restraining effect of medicine to osteoclast activity.Specifically be grouped as follows:
1) inductor control group: the Raw264.7 inducing culture of simple inductor effect
2) medicine group: the Raw264.7 inducing culture of inductor effect goes out the medicine of the dilution of mature osteoclast+by a certain percentage
The calculating of 3 cell states mensuration and activity unit
Method is the same.
The inventive method can be carried out quantitative assay to the activity of osteoclast suppressive drug, has accurately, characteristics such as reliable, convenient, economy and simple possible.Can be used for screening new compound, determine whether it has the osteoclast activity of inhibition and and then infer whether can treat the osteoporosis drug candidate.
Description of drawings
Fig. 1 suppresses the experimental result of the bioactive quantitative assay of osteoclast precursor cell RAW264-7 (MTS method) for embodiment 1OPG
Fig. 2 is the experimental result that embodiment 2 rhOPG-Fc suppress the quantitative assay (anti-tartaic acid phosphatase activity assay method) of osteoclast precursor activity of cell biology
Fig. 3 suppresses the experimental result of the quantitative assay (anti-tartaic acid phosphatase activity assay method) of mature osteoclast biologic activity for embodiment 3rhOPG-Fc
Embodiment
Embodiment 1:
OPG suppresses the bioactive quantitative assay of osteoclast precursor cell RAW264-7 (MTS method)
The osseous tissue blood mononuclear cell is divided into mature osteoclast under the effect of marrow stromal cell excretory DIF.Induce brokenly in the factor of bone precursor cell the part, the most key with the RANKL-RANK system.Marrow stromal cell secretory cell nuclear Factor-Kappa B receptor activation factor aglucon (receptor activator ofNFKB ligand, RANKL), RANKL and osteoclast are the nucleus factor-kappa B receptor activation factor (receptor activator of NF-κ B, the RANK) combination of cell surface.In a single day RANKL combines with RANK will stimulate the differentiation of precursor osteoclast, and the sophisticated osteoclast of activation, causes bone resorption.OPG is a kind of RANK bait acceptor, its effect be in conjunction with or in and solubility RANKL, thereby the combining of blocking-up RANKL and functional receptor RANK.Therefore OPG is equivalent to the stopper of whole RANKL system, has suppressed the effect of RANKL-RANK system, thereby has suppressed osteoclast differentiation and activation.RANKL-RANK-OPG forms complete bone metabolism regulator control system, regulates the metabolic balance of osseous tissue.
OPG is found by two study group of the U.S. and Japan respectively in 1997 the same period, mature protein is to contain 380 amino acid whose a kind of secretor type glycoprotein, its bioactive functions fragment is the 1-4 structural domain of N end, the i.e. part of 22-201 amino acids.Clear and definite based on to the understanding of OPG/RANKL/RANK system and OPG effect, is the feature disease as a kind of treatment with excessive bone resorption, osteoporosis that causes as multiple myeloma, neoplastic bone transfer, post-menopausal osteoporosis, hormone etc., the pharmaceutical use of OPG causes people's attention day by day.We utilize pichia yeast expression system, expression has prepared 22-201 amino acids functional fragment and the segmental OPG-Fc fusion rotein of human IgG lFc of OPG, and in cell and animal model experiment, observe and suppress osteoclast activity and function of resisting osteoporosis significantly, the biological activity of quick, quantitative mensuration OPG-Fc fusion rotein for convenience,, we adopt osteoclast described above to suppress experiment and the enzymatic substrate decomposes the colorimetric estimation that develops the color, and have set up OPG-Fc fusion rotein biological activity quantivative approach.
Utilize the MTS method to detect the influence of OPG to the RAW264-7 differentiation, compare the difference of various dose and time point, concrete grammar is as follows:
1.RAW264-7 the inducing culture of cell
Get 96 orifice plates, 5000 in 1000 in every hole combined inoculation osteoclast precursor cell RAW264-7 cell and myeloma cell Sp2/0 cell, every hole adds osteoclast and induces differentiation liquid, promptly adds dexamethasone and 1,25-(OH) in the DMEM perfect medium
2VitD
3, final concentration is respectively 1 * 10
-7Mol/L and 2 * 10
-8Mol/L.Plant three blocks of plates altogether, the A1 hole on every block of plate only adds the DMEM perfect medium, surveys periodic blank hole as MTS.
Other one 96 orifice plate of parallel inoculation simultaneously is as identifying whether the osteoclast inducing culture is successful.The method of identifying adopts tartaric-resistant and periosteum sheet bone lacuna to form experiment.
2, experiment grouping
96 orifice plate osteoclast mixed culture systems are divided into 9 groups, three every group multiple holes.The rhOPG-Fc that adds different concns respectively, concentration is 200ug/L, and 20ug/l, 2ug/l, 200ng/l, 20ng/l, 2ng/l, 0.2ng/l, 0.02ng/l, Ong/l (control group), cell are in 37 ℃, and 5%CO2 continues to cultivate.
3, the MTS method is measured
Experimental group is got a culture plate after adding rhOPG-Fc albumen, adds the MTS working fluid of 20 μ l, and 37 ℃, 5%CO
2After cultivating 4h, put on the enzyme connection detector and measure optical density(OD) (OD) value, detect wavelength 490nm.Carried out twice MTS respectively again measured at 96 hours and 120 hours.
4. result
Osteoclast inducing culture qualification result:
Add in the mixed culture system of inductor, do not add the inductor control group with the simple mixing cultivation and compare, TRAP stained positive after 96 hours, periosteum sheet bone lacuna forms.The success of confirmation inducing culture.
The MTS experimental result is seen Fig. 1.
The result shows, the concentration of rhOPG-Fc is lower than 0.02ug/L does not have any restraining effect substantially to the osteoclast precursor cell, be higher than 0.02ug/L restraining effect is appearred in the osteoclast precursor cell, the IC50 that can calculate rhOPG-Fc inhibition RAW264-7 differentiation with graphing method is: 20ug/L.
With rhOPG-Fc IC50 is that 50ug/L calculates, and the rhOPG-Fc biological activity is 5 * 10E4 U/g (being commonly designated as 50U/mg).
RhOPG-Fc suppresses the quantitative assay (anti-tartaic acid phosphatase activity assay method) of osteoclast precursor activity of cell biology
1. the inducing culture of osteoclast
Get 24 orifice plates, 20000 in 4000 in every hole combined inoculation osteoclast precursor cell RAW264-7 cell and myeloma cell Sp2/0 cell, it is to add dexamethasone and 1,25-(OH) in the DMEM perfect medium that every hole adding osteoclast is induced differentiation liquid
2VitD
3, ultimate density is respectively 1 * 10
-7Mol/L and 2 * 10
-8Mol/L.Plant a plate altogether.
Other one 24 orifice plate of parallel inoculation simultaneously is as identifying whether the osteoclast inducing culture is successful.The method of identifying adopts tartaric-resistant and periosteum sheet bone lacuna to form experiment
Cultivate after 96 hours, tartaric-resistant shows that the osteoclast endochylema presents rose, has red positive particle in the kytoplasm, and check figure is numerous.Observe periosteum sheet bone lacuna simultaneously under the Electronic Speculum and form, confirm the success of osteoclast inducing culture.
2. experiment grouping
Divide six groups with the osteoclast on 24 orifice plates, answer holes for three every group, add the rhOPG-Fc of different concns in the time of mixed culture respectively, concentration is respectively 0.0001mg/L, 0.001mg/L, 0.01mg/L, 0.1mg/L, 1mg/L, 10mg/L.
3. anti-tartaic acid phosphatase activity detects:
After 120 hours, detect the activity of anti-tartaic acid Phosphoric acid esterase in the osteoclast.Culture supernatant is discarded, with PBS flushing twice, add cell pyrolysis liquid, every hole 150ul was hatched 25 minutes on ice, with the cell shovel cell was scraped, transfer in the Eppendorf tube, and 10000g, 10min is centrifugal.Collect supernatant.Get 100ul, the substrate that adds anti-tartaic acid Phosphoric acid esterase is right-nitrophenol phosphoric acid ester (NPP), incubated at room 15-30 minute, place and detect optical density value (OD value) on the microplate reader, measure wavelength 405nm.
4. the results are shown in Figure 2.
The result shows that rhOPG-Fc has the obvious suppression effect to the differentiation of osteoclast, and can calculate its IC50 by graphing method is 0.02mg/L, and its activity unit is 5 * 10E4 U/g (being commonly designated as 50U/mg)
Embodiment 3:
RhOPG-Fc suppresses the quantitative assay of mature osteoclast biologic activity.
1. the inducing culture of osteoclast
Get 24 orifice plates, 20000 in 4000 in every hole combined inoculation osteoclast precursor cell RAW264-7 cell and myeloma cell Sp2/0 cell, it is to add dexamethasone and 1,25-(OH) in the DMEM perfect medium that every hole adding osteoclast is induced differentiation liquid
2VitD
3, ultimate density is respectively 1 * 10
-7Mol/L and 2 * 10
-8Mol/L.Plant a plate altogether.
Other one 24 orifice plate of parallel inoculation simultaneously is as identifying whether the osteoclast inducing culture is successful.The method of identifying adopts tartaric-resistant and periosteum sheet bone lacuna to form experiment
Cultivate after 96 hours, tartaric-resistant shows that the osteoclast endochylema presents rose, has red positive particle in the kytoplasm, and check figure is numerous.Observe periosteum sheet bone lacuna simultaneously under the Electronic Speculum and form, confirm the success of osteoclast inducing culture.
2. experiment grouping
After the success of osteoclast inducing culture, divide eight groups with the osteoclast on 24 orifice plates, three every group multiple every group of rhOPG-Fc that add different concns respectively in hole, concentration is respectively 0.0125mg/L, 0.025mg/L, 0.05mg/L, 0.1mg/L, 0.2mg/L, 0.4mg/L, 0.8mg/L.
3. anti-tartaic acid phosphatase activity detects:
Add after rhOPG-Fc24 hour, detect the activity of anti-tartaic acid Phosphoric acid esterase in the osteoclast.Culture supernatant is discarded,, add cell pyrolysis liquid, hatched on ice 25 minutes, cell is scraped, transfer in the Eppendorf tube with the cell shovel with PBS flushing twice, 10000g, 10min is centrifugal.Collect supernatant.Get 100ul, the substrate that adds anti-tartaic acid Phosphoric acid esterase is right-nitrophenol phosphoric acid ester (NPP), incubated at room 15-30 minute, place and detect optical density value (OD value) on the microplate reader, measure wavelength 405nm.
4. the results are shown in Figure 3.
The result shows that rhOPG-Fc has the obvious suppression effect to the function of mature osteoclast, and can calculate its IC50 by graphing method is 0.18mg/L, and its activity unit is 5.5 * 10E3 U/g (being commonly designated as 5.5U/mg).
OPG acts on osteoclast and generates and a plurality of links ripe and the broken bone effect of maturation back performance, rhOPG-Fc suppresses osteoclast treatment osteoporosis and osteonecrosis is the comprehensive embodiment of its multiple effect, in measure its active level that suppresses osteoclast function test with above-mentioned several different methods, find, OPG is discrepant to the activity unit of different link effects, we infer that OPG is different to the ability to function of a plurality of links of broken bone effect thus, for example rhOPG-Fc induces the active 50U/mg of being of inhibition of differentiation to osteoclast, the activity that the mature osteoclast function is suppressed is 5.5U/mg, and the main mechanism of action that can judge OPG thus is that osteoclast induces the inhibition of differentiation, and the mechanism of action that the mature osteoclast function is suppressed is positioned at next.
Claims (5)
1. measure the bioactive method of medicine inhibition bone resorption for one kind, it is characterized in that described method comprises:
A. set up the method for external evoked cultivation osteoclast or go out sophisticated osteoclast with this method inducing culture;
B. the medicine that in inducing culture system or culture system, adds different concns;
C. osteoclast growth conditions or cytoactive functional status are measured;
The calculating of medicine D. to be measured biological activity unit.
2. mensuration medicine as claimed in claim 1 suppresses the bioactive method of bone resorption, it is characterized in that the cell state measuring method is MTS method, mtt assay, the dyeing of platform Finland, CCK-8 method, XTT method and anti-tartaic acid phosphatase substrate reaction solution method, and with IC50 value calculated activity unit.
3. mensuration medicine as claimed in claim 1 suppresses the bioactive method of bone resorption, and osteoclast is induced differentiation and maturation or/and suppress the medicine of mature osteoclast functionally active in order to suppress to it is characterized in that described medicine.
4. mensuration medicine as claimed in claim 1 suppresses the bioactive method of bone resorption, it is characterized in that it is as an activity unit with IC50 that described biological activity unit is calculated.
5. mensuration medicine as claimed in claim 1 suppresses the bioactive method of bone resorption, it is characterized in that this method is used for the mensuration of pair cell different steps, in inducing culture system or culture system, add medicine, by medicine the activity unit that the different steps cell suppresses ability is compared, can study the effect link and the mechanism of medicine.
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