CN101798588A - Method for testing bioactivities of GLP-1 receptor agonist - Google Patents
Method for testing bioactivities of GLP-1 receptor agonist Download PDFInfo
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- CN101798588A CN101798588A CN200910265928A CN200910265928A CN101798588A CN 101798588 A CN101798588 A CN 101798588A CN 200910265928 A CN200910265928 A CN 200910265928A CN 200910265928 A CN200910265928 A CN 200910265928A CN 101798588 A CN101798588 A CN 101798588A
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Abstract
The invention relates to a method for testing bioactivities, in particular to a method for testing the bioactivities of a GLP-1 receptor agonist. Based on the function of the GLP-1 receptor agonist which can promote mouse insulinoma cells (Min-6) to secrete the insulin, the method comprises the steps of: using insulin quantitatively secreted by a mouse/rat insulin kit to improve the precision of determination through a series of condition optimization processes, and testing the absorbency at the wavelength of 450 nm and the reference wavelength of 590 nm to obtain the effect curve that the GLP-1 receptor agonist induces the Min-6 cells to secrete the insulin; and finally testing the bioactivities of the GLP-1 receptor agonist on the basis of the effect curve.
Description
Technical field
The present invention relates to biological activity determination method, relate to the biological activity determination method of GLP-1 receptor stimulant in particular.
Background technology
Diabetes have become the major disease that threatens human life's health, and [medicine is estimated .2009,24 (1): 32-33 to the GLP-1 receptor stimulant in occupation of more and more important position in the diabetes medicament research; Medicine is estimated .2008,5 (11): 504-505]; The GLP-1 receptor stimulant mainly contains glucagon-like-peptide-1 (GLP-1), insulin secretion accelerating peptide (Exendin-4), people GLP-1 analogue Li Lalu peptide (Liraglutide), the mensuration of its biologic activity is owing to there not being the determination of activity international standard substance to become a big difficult point [the Chinese biological goods are learned magazine .2004,17 (1): 29-31].
The biological function of GLP-1 receptor stimulant mainly be specifically with the GLP-1 acceptor interaction of islet cells, cause that corresponding signal path changes, and induces secretion of insulin.According to these biological functions, measure its biologic activity and mainly contain three major types at present: experimentation on animals [Chin J Chin Pharmacol Ther2004,9 (5): 527-531], amylase discharge analyzes experiment and raji cell assay Raji experiment.
Experimentation on animals needs animal pattern usually, is difficult for obtaining, the cost height, and can only whether activity be arranged statistical study, can only carry out qualitative analysis; It is relatively poor that amylase discharges the susceptibility of analyzing experiment, and linear relationship is bad, only can test as sxemiquantitative.
The raji cell assay Raji experiment comprises second messenger (cAMP) enzyme chain immune response analysis experiment [pharmaceutical analysis magazine.2006,26 (12): 1691-1693] and chemiluminescent substance determination experiment [the journal .2007 of Nankai University, 40 (1): 92-98].The former is the mensuration to cAMP in the signal path change process, but because cAMP unstable existence in cell easily is degraded, and cAMP can produce by number of ways, so specificity is not high; Moreover the RIN-5F cell that uses not is quick to this excitomotor Turin of GLP-1 acceptor, does not have general suitability.The chemiluminescent substance determination experiment need make up specific cell, makes it to secrete specific substrate for enzymatic activity chemoluminescence, needs a large amount of cost of cost and higher technical force [biological chemistry and biophysics progress .2004,31 (4): 36-40; Biotechnology circular .2007,3:118-121].And the two all is the mensuration to middle product, is not this principle design of biological function (secretion inducing Regular Insulin) at the GLP-1 receptor stimulant.
Comparatively speaking, design of the present invention this pharmacology principle of just combining closely designs, and single-minded measures end product Regular Insulin, more can embody the actual effect of medicine, makes more reasonable, the science, more practical more of method.Its advantage is in particular in:
At first, present method can obtain the effect curve that the GLP-1 receptor stimulant promotes excreting insulin, measures the biologic activity of GLP-1 receptor stimulant with this, can draw relatively accurately and tire.And existing method by animal pattern can only determine qualitatively whether the GLP-1 receptor stimulant has the effect that promotes excreting insulin.
Secondly, measuring crucial thing in present method is Regular Insulin, after it is GLP-1 receptor stimulant and GLP-1 acceptor interaction, causes that corresponding signal path changes, the inductive final product, and stability is high, is not subjected to the influence of other material in the cell substantially.And the combine closely pharmacology principle of GLP-1 receptor stimulant of design of the present invention, single-minded at end product Regular Insulin, more can embody the actual effect of medicine, make more reasonable, the science, more practical more of method.And the current experiments method no matter be with cAMP, is the test cell line method of target detect thing with the chemiluminescent substance still, and the two all is the mensuration to middle product, easily degraded, and instability, and intermediate product has multiple generation approach.
Moreover the required cell of chemiluminescent substance assay method needs particular build, just can make it to secrete can be detected chemiluminescent substance, need a large amount of cost of cost, higher technical force and long time.And the Min-6 cell culture condition that the present invention uses is simple, and growth is convenient to operation easily.
Summary of the invention
This genealogy of law can promote the effect of mouse islets oncocyte (Min-6) excreting insulin according to the GLP-1 receptor stimulant, with the quantitative excretory Regular Insulin of mouse/rat insulin test kit, in wavelength 450nm, reference wavelength 590nm place measures its absorbancy, can obtain the GLP-1 receptor stimulant and induce the effect curve of Min-6 emiocytosis Regular Insulin, measure GLP-1 receptor stimulant biologic activity with this.
Because the GLP-1 receptor stimulant acts on the Min-6 cell certain glucose concn requirement is arranged, the present invention is by regulating the concentration of substratum glucose, before adding the standard substance trial-product, use the culture medium culturing target cell of sugar-free, and with high sugar determination culture medium solution dilution standard product trial-product, improve the susceptibility of Min-6 cell.The glucose concn of the mensuration substratum of dilution standard product and trial-product is preferably 25mM to 75mM among the present invention, more preferably 50mM.
This law has certain requirement for the concentration of the cell suspension made from Digestive system; In the present invention, the density of cell suspension is preferably 5 * 10
5Individual/ml.
This law has certain requirement for the incubation time that cell adds behind the sugar-free DMEM substratum; Incubation time is preferably 1-6 hour in the present invention, more preferably 2-4 hour.
This law has certain requirement for the incubation time that test sample adds behind the cell culture medium; Incubation time is preferably 4-6 hour in the present invention.
Description of drawings
When accompanying drawing 3 is 16.7mM for glucose concn, GLP-1 standard substance and placebo biological activity comparison diagram.
When accompanying drawing 4 is 25mM for glucose concn, GLP-1 standard substance and placebo biological activity comparison diagram.
When accompanying drawing 5 is 50mM for glucose concn, GLP-1 standard substance and placebo biological activity comparison diagram.
When accompanying drawing 6 is 75mM for glucose concn, GLP-1 standard substance and placebo biological activity comparison diagram.
When accompanying drawing 7 is 16.7mM for glucose concn, Exendin-4 standard substance and placebo biological activity comparison diagram.
When accompanying drawing 8 is 25mM for glucose concn, Exendin-4 standard substance and placebo biological activity comparison diagram.
When accompanying drawing 9 is 50mM for glucose concn, Exendin-4 standard substance and placebo biological activity comparison diagram.
When accompanying drawing 10 is 75mM for glucose concn, Exendin-4 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 11 is under the constant situation of other experiment conditions, changes the incubation time of sugar-free culture-medium, measures the comparison diagram of emiocytosis Regular Insulin.
Accompanying drawing 12 is under the constant condition of other experiment conditions, changes cell kind plate density, measures the comparison diagram of emiocytosis Regular Insulin.
Accompanying drawing 13 is under the constant situation of other conditions, and change adds the incubation time behind the standard substance, measures the comparison diagram of emiocytosis Regular Insulin.
Accompanying drawing 14 is under the constant situation of other conditions, adds incubation time behind the standard substance and be the comparison diagram of 4 hours and 8 hours.
Embodiment:
Measure the biologic activity of recombinant human GLUCAGON LIKE PEPTIDE-1 (7-36) (GLP-1 (7-36)) with this law
Reagent and material (1) DMEM high glucose medium is got 1 bag in DMEM high glucose medium powder (specification is 1L), is dissolved in water and is diluted to 1000ml, and wherein the Sodium.alpha.-ketopropionate final concentration is that 1mmol/L, HEPES final concentration are 0.5%, adds penicillin 10
5IU and Streptomycin sulphate 10
5IU, sodium bicarbonate 3.7g, after the dissolving, mixing, Sterile Filtration, 4 ℃ of preservations.
(2) perfect medium is measured foetal calf serum 10ml, adds DMEM high glucose medium 90ml, 4 ℃ of preservations.
(3) measure substratum (50mM glucose) DMEM high glucose medium 88ml, add 10%BSA 2ml, 250mM glucose solution 10ml.
(4) 10%BSA gets BSA10g, adds the 100ml water dissolution and is diluted to 100ml, Sterile Filtration ,-20 ℃ of preservations.
(5) PBS gets sodium-chlor 8.0, Repone K 0.20g, and Sodium phosphate dibasic 1.44g, dipotassium hydrogen phosphate 0.24g is dissolved in water and is diluted to 1000ml, through 121 ℃ of sterilizations in 15 minutes.
(6) Digestive system is got 2.5g pancreatin, disodium ethylene diamine tetraacetate 0.2g, sodium-chlor 8.0, Repone K 0.20g, and Sodium phosphate dibasic 1.152g, dipotassium hydrogen phosphate 0.2g is dissolved in water and is diluted to 1000ml, Sterile Filtration ,-20 ℃ of preservations.
(7) the 250mM glucose solution is got glucose (H
2O) 1g, add 20ml sugar-free DMEM substratum dissolving after, mixing, Sterile Filtration, 4 ℃ of preservations.
(8) target cell strain mouse islets oncocyte (Min-6) is given by the Shanghai City diabetes study, goes down to posterity and preserve in this chamber.
(9) RAT/MOUSE INSULIN KIT test kit LINCO company produces, article No.: EZRMI-13K
(10) standard substance, trial-product intestinal bacteria are recombinant human GLUCAGON LIKE PEPTIDE-1 (7-36) freeze-dried preparation of host cell expression
Recombinant human GLUCAGON LIKE PEPTIDE-1 (7-36) standard substance are got in the preparation of standard solution, are diluted to 500ug/ml with the mensuration substratum.In the 1.5ml centrifuge tube, do 2 times of serial dilutions, totally 8 extent of dilution, each extent of dilution is done 2 holes.Under aseptic condition, operate.
Recombinant human GLUCAGON LIKE PEPTIDE-1 (7-36) trial-product is got in the preparation of need testing solution, is diluted to 500ug/ml with the mensuration substratum.In the 1.5ml centrifuge tube, do 2 times of serial dilutions, totally 8 extent of dilution, each extent of dilution is done 2 holes.Under aseptic condition, operate.
Experimental technique makes the Min-6 cell in the perfect medium substratum, cultivates under 37 ℃, 5% carbon dioxide conditions.The cell of taking the logarithm vegetative period, after washing with PBS, cell suspension is made in Digestive system digestion, adjusts cell density to 5.0 * 10
5Individual/ml.Add cell suspension 100ul/ hole in 96 well culture plates, CO
2Incubator is cultivated 40h.Remove cell conditioned medium, add PBS 200ul/ hole, supernatant discarded adds PBS 200ul/ hole again, supernatant discarded, so clean 2 times after, add sugar-free DMEM substratum 100ul/ hole, continue to cultivate 4h.Remove cell conditioned medium, adding PBS cleans 2 times, the standard substance and the need testing solution 100ul/ hole that add the good different concns (500ug/ml-250ug/ml-125ug/ml-62.5ug/ml-31.25ug/ml-15.6ug/m l-7.8ug/ml-3.9ug/ml) of dilution, each concentration do 2 parallel, set up simultaneously and measure the substratum contrast, under 37 ℃, 5% carbon dioxide conditions, continue to cultivate 2h.Collect supernatant, dilute 5 times after, with the detection of Regular Insulin test kit.The detection wavelength is 450nm, and reference wavelength is 590nm.Carry out data analysis with four parametric regression computing methods at last, calculate, draw the extension rate of inducing Regular Insulin 50% maximal stimulation reaction.Unit definition: with the extension rate that can induce Regular Insulin 50% maximal stimulation reaction is 8000 units (being AU).
Trial-product (the AU/ml)=standard substance of tiring are tired * (A
Trial-product/ A
Standard substance) * (ED
50 trial-products/ ED
50 standard substance)
A
Trial-product, A
Standard substance: trial-product, the pre-extension rate of standard substance
ED
50Trial-product, ED
50Standard substance: trial-product, standard substance median effective dose extension rate
Result such as Fig. 1, data are as follows
Standard substance: ED
50 standard substance=25.384, A
Standard substance=0.8, standard substance are tired=8000AU/ml
Trial-product (lot number 20080601): ED
50Trial-product=20.965, A
Trial-product=0.2
Trial-product (lot number 20080701): ED
50Trial-product=24.041, A
Trial-product=0.2
Trial-product (lot number 20080706): ED
50Trial-product=25.149, A
Trial-product=0.2
Placebo:
Trial-product (the AU/ml)=standard substance of tiring are tired * (A
Trial-product/ A
Standard substance) * (ED
50 trial-products/ ED
50 standard substance)
Measure the Exendin-4 biologic activity with this law.
Experiment material, method and embodiment 1 operation are basic identical.
The preparation of need testing solution: get the Exendin-4 trial-product, be diluted to 50ug/ml with the mensuration substratum.
In the 1.5ml centrifuge tube, do 2 times of serial dilutions.
Experimental result is seen Fig. 2, and data are as follows:
Standard substance:
Trial-product:
Placebo:
As shown in Figure 2, sample is parallel with the standard substance curve, and linearity is arranged, according to ED
50, after tiring, the product of settling the standard can tire by working sample, proved the feasibility of method.
Dilution contrasts with measuring substratum bioactive result of bioassay standard product GLP-1 (7-36) under different glucose concn conditions.
Experiment material, method and embodiment 1 operation basic identical (no trial-product).The glucose concn of measuring substratum is configured to 17.5mM respectively, 25mM, 50mM and 75mM.Experimental result is seen Fig. 3,4,5 and 6.By Fig. 3,4,5 and 6 contrast as can be known, the concentration that improves glucose helps emiocytosis Regular Insulin, and the scale of the standard substance excreting insulin of different concns reveals tangible dose-effect relationship, more is tending towards obvious with the contrast of placebo.
Dilution contrasts with the biological activity determination result who measures substratum standard substance Exendin-4 under different glucose concn conditions.
Experiment material, method and embodiment 2 operations basic identical (no trial-product).The glucose concn of measuring substratum is configured to 17.5mM respectively, 25mM, 50mM and 75mM.Experimental result is seen Fig. 7,8,9 and 10, and its comparing result is similar with embodiment 3, all demonstrate the raising glucose concn and helped to improve the bioactive accuracy of mensuration, and its measurement effect the best when 50mM.
Change the incubation time after cell adds sugar-free DMEM substratum, the measurement result contrast of emiocytosis Regular Insulin.
Experiment material method (no trial-product does not have 8 extent of dilution) substantially the same manner as Example 1 is got recombinant human GLP-1 (7-36) standard substance, is diluted to 500ug/ml with the mensuration substratum, measures.Change the incubation time after cell adds sugar-free DMEM substratum, after cultivating 2,4,5,6,12,16,20,24,30 hours, measure cell secretion of insulin situation respectively, its experiment the results are shown in Figure 11.Can find out obviously that from figure to cultivate after 2 hours, 4 hours the insulin-producing amount the highest, help detection by quantitative most.
Change the density of cell, the measurement result contrast of emiocytosis Regular Insulin.
Experiment material method (no trial-product does not have 8 extent of dilution) substantially the same manner as Example 1 is got recombinant human GLP-1 (7-36) standard substance, is diluted to 500ug/ml with the mensuration substratum, measures.After cell is washed with PBS, adjust the density that cell suspension is made in Digestive system digestion, its cell density is adjusted into 5 * 10
5Individual/ml, 5.5 * 10
5Individual/ml, 6.0 * 10
5Individual/ml, 6.5 * 10
5Individual/ml, 7.0 * 10
5Individual/ml, 7.5 * 10
5Individual/ml, 8.0 * 10
5Individual/ml and 9.0 * 10
5Individual/ml, it the results are shown in Figure 12.Can find out obviously that from figure working as cell concn is 5 * 10
5The time, the insulin-producing amount is the highest, helps most detecting.
Change adds the incubation time behind the standard substance, the measurement result contrast of emiocytosis Regular Insulin.
Experiment material method (no trial-product does not have 8 extent of dilution) substantially the same manner as Example 1 is got recombinant human GLP-1 (7-36) standard substance, is diluted to 500ug/ml with the mensuration substratum, measures.After adding the 500ug/ml standard substance, under 37 ℃, 5% carbon dioxide conditions, continue to cultivate after 0.5,1,2,3,4,6 and 16 hour respectively, detect, experimental result is as shown in figure 13.Can obviously find out from figure has tangible jump 4 hours to 6 hours, select such time point proper,
The continuation that adds behind the standard substance was cultivated 8 hours, with incubation time among the embodiment 1 be that 4 hours cell insulin secretion situation compares.
Experiment material method (no trial-product) substantially the same manner as Example 1 is got recombinant human GLP-1 (7-36) standard substance, is diluted to 500ug/ml with the mensuration substratum, measures.After adding the 500ug/ml standard substance, under 37 ℃, 5% carbon dioxide conditions, continue to cultivate after 8 hours, detect.Its result as shown in figure 14.As we know from the figure, continue to cultivate after 8 hours (greater than 6 hours), the point of different standards product concentration drops on being tending towards on the oblique line and reduces fewly, and a large amount of points drops on two ends (upper and lower platform), is unfavorable for detection, thus 4-6 little be good.
Claims (13)
1. GLP-1 receptor stimulant biological activity assay method, its step comprises:
(a) cultivate suitable detection target cell strain;
(b) prepare the GLP-1 receptor stimulant standard solution and the need testing solution of several weaker concns
(c) the cell bacterial strain in vegetative period of taking the logarithm through treating processes, adds the standard substance and the need testing solution of preparation in the step (b), and continues to cultivate;
(d) after the dilution of collection supernatant, obtain the GLP-1 receptor stimulant with the detection of Regular Insulin test kit and induce the effect curve that detects emiocytosis Regular Insulin;
(e) activity of calculating GLP-1 receptor stimulant.
2. the method described in claim 1 is characterized in that described GLP-1 receptor stimulant is GLP-1, Exendin-4, or their analogue.
3. the method described in claim 1 is characterized in that the detection described in the step (a) is mouse islets tumor cell strain Min-6 with the target cell strain.
4. the method described in claim 1 is characterized in that some dilution GLP-1 receptor stimulant standard substance described in the step (b) and need testing solution are diluted by high glucose concn solution allocation.
5. the method described in claim 1 is characterized in that the treating processes described in the step (c) comprises:
(a) wash with PBS after, add Digestive system digestion and make cell suspension, and adjust cell density, add the cell suspension in culture plate, continue to cultivate;
(b) remove cell conditioned medium, add PBS and clean, add sugar-free DMEM substratum, continue to cultivate;
(c) remove cell conditioned medium, add PBS and clean, add standard solution and need testing solution after diluting, continue to cultivate;
6. the method described in claim 1 is characterized in that detection wavelength used in the step (d) is 450nm, and reference wavelength is 590nm.
7. the method described in claim 1 is characterized in that in the step (e), and the method for described calculating GLP-1 receptor stimulant is four parametric regression computing methods.
8. the method described in claim 4 is characterized in that described high glucose solution glucose concn scope is 25mM to 75mM.
9. the method described in claim 8 is characterized in that described high glucose solution glucose concn is 50mM.
10. the method described in claim 5 step (a) is characterized in that described cell density is 5 * 105/ml.
11. the method described in claim 5 step (b) is characterized in that described continuation incubation time is 1 to 6 hour.
12. the method described in claim 11 is characterized in that described continuation incubation time is 2 to 4 hours.
13. the method described in claim 5 step (c) is characterized in that described continuation incubation time is 4 to 6 hours.
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| CN103267752A (en) * | 2013-05-31 | 2013-08-28 | 北京大学 | Method for determining proportion of number of A cells to number of B cells in pancreatic islets |
| CN104870009A (en) * | 2012-12-21 | 2015-08-26 | 赛诺菲 | Functionalized exendin-4 derivatives |
| US9694053B2 (en) | 2013-12-13 | 2017-07-04 | Sanofi | Dual GLP-1/glucagon receptor agonists |
| US9750788B2 (en) | 2013-12-13 | 2017-09-05 | Sanofi | Non-acylated exendin-4 peptide analogues |
| US9751926B2 (en) | 2013-12-13 | 2017-09-05 | Sanofi | Dual GLP-1/GIP receptor agonists |
| US9758561B2 (en) | 2014-04-07 | 2017-09-12 | Sanofi | Dual GLP-1/glucagon receptor agonists derived from exendin-4 |
| US9771406B2 (en) | 2014-04-07 | 2017-09-26 | Sanofi | Peptidic dual GLP-1/glucagon receptor agonists derived from exendin-4 |
| US9775904B2 (en) | 2014-04-07 | 2017-10-03 | Sanofi | Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists |
| US9789165B2 (en) | 2013-12-13 | 2017-10-17 | Sanofi | Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists |
| US9982029B2 (en) | 2015-07-10 | 2018-05-29 | Sanofi | Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists |
| US10758592B2 (en) | 2012-10-09 | 2020-09-01 | Sanofi | Exendin-4 derivatives as dual GLP1/glucagon agonists |
| US10806797B2 (en) | 2015-06-05 | 2020-10-20 | Sanofi | Prodrugs comprising an GLP-1/glucagon dual agonist linker hyaluronic acid conjugate |
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| US9932381B2 (en) | 2014-06-18 | 2018-04-03 | Sanofi | Exendin-4 derivatives as selective glucagon receptor agonists |
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| US10758592B2 (en) | 2012-10-09 | 2020-09-01 | Sanofi | Exendin-4 derivatives as dual GLP1/glucagon agonists |
| CN104870009A (en) * | 2012-12-21 | 2015-08-26 | 赛诺菲 | Functionalized exendin-4 derivatives |
| US9745360B2 (en) | 2012-12-21 | 2017-08-29 | Sanofi | Dual GLP1/GIP or trigonal GLP1/GIP/glucagon agonists |
| US10253079B2 (en) | 2012-12-21 | 2019-04-09 | Sanofi | Functionalized Exendin-4 derivatives |
| CN103267752A (en) * | 2013-05-31 | 2013-08-28 | 北京大学 | Method for determining proportion of number of A cells to number of B cells in pancreatic islets |
| CN103267752B (en) * | 2013-05-31 | 2015-04-08 | 北京大学 | Method for determining proportion of number of A cells to number of B cells in pancreatic islets |
| US9751926B2 (en) | 2013-12-13 | 2017-09-05 | Sanofi | Dual GLP-1/GIP receptor agonists |
| US9789165B2 (en) | 2013-12-13 | 2017-10-17 | Sanofi | Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists |
| US9750788B2 (en) | 2013-12-13 | 2017-09-05 | Sanofi | Non-acylated exendin-4 peptide analogues |
| US9694053B2 (en) | 2013-12-13 | 2017-07-04 | Sanofi | Dual GLP-1/glucagon receptor agonists |
| US9758561B2 (en) | 2014-04-07 | 2017-09-12 | Sanofi | Dual GLP-1/glucagon receptor agonists derived from exendin-4 |
| US9771406B2 (en) | 2014-04-07 | 2017-09-26 | Sanofi | Peptidic dual GLP-1/glucagon receptor agonists derived from exendin-4 |
| US9775904B2 (en) | 2014-04-07 | 2017-10-03 | Sanofi | Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists |
| US10806797B2 (en) | 2015-06-05 | 2020-10-20 | Sanofi | Prodrugs comprising an GLP-1/glucagon dual agonist linker hyaluronic acid conjugate |
| US9982029B2 (en) | 2015-07-10 | 2018-05-29 | Sanofi | Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists |
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