CN101798588B - GLP-1 receptor stimulant Determination of biological activity method - Google Patents
GLP-1 receptor stimulant Determination of biological activity method Download PDFInfo
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- CN101798588B CN101798588B CN200910265928.1A CN200910265928A CN101798588B CN 101798588 B CN101798588 B CN 101798588B CN 200910265928 A CN200910265928 A CN 200910265928A CN 101798588 B CN101798588 B CN 101798588B
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Abstract
The present invention relates to biological activity determination method, relate to the biological activity determination method of GLP-1 receptor stimulant in particular.This genealogy of law can promote the effect of murine insulinoma cell (Min-6) excreting insulin according to GLP-1 receptor stimulant, with the Regular Insulin of mouse/rat insulin kit secretion, and the tolerance range of mensuration is improved by a series of condition optimizing process, in wavelength 450nm, reference wavelength 590nm place measures its absorbancy, obtain the effect curve of GLP-1 receptor stimulant induction Min-6 cells secrete insulin, measure GLP-1 receptor stimulant biologic activity with this.
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 threatening human life health, and in diabetes medicament research, GLP-1 receptor stimulant is in occupation of more and more consequence [drug assessment .2009,24 (1): 32-33; Drug assessment .2008,5 (11): 504-505]; GLP-1 receptor stimulant mainly contains glucagon-like-peptide-1 (GLP-1), insulin secretion accelerating peptide (Exendin-4), people GLP-1 analogue Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] (Liraglutide), the mensuration of its biologic activity becomes a large difficult point [Products in China magazine .2004,17 (1): 29-31] owing to not having determination of activity international standard substance.
The biological function of GLP-1 receptor stimulant mainly specifically with the GLP-1 acceptor interaction of islet cells, cause corresponding signal path to change, the secretion of induced 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 release analysis design mothod and raji cell assay Raji experiment.
Experimentation on animals needs animal pattern usually, not easily obtains, cost is high, and statistical study whether can only have activity, can only carry out qualitative analysis; The susceptibility of amylase release analysis design mothod is poor, and linear relationship is bad, only can test as sxemiquantitative.
Raji cell assay Raji experiment comprises second messenger (cAMP) enzyme chain immune response analysis design mothod [pharmaceutical analysis magazine.2006,26 (12): 1691-1693] and chemiluminescent substance determination experiment [Nankai University journal .2007,40 (1): 92-98].The former is the mensuration to cAMP in signal path change process, but exists because cAMP is unstable in cell, and be easily degraded, and cAMP can be produced by number of ways, therefore specificity is not high; Moreover the RIN-5F cell used not is quick to this excitomotor Turin of GLP-1 acceptor, does not have general suitability.Chemiluminescent substance determination experiment needs to build specific cell, makes it to secrete specific substrate for enzymatic activity chemoluminescence, needs a large amount of cost of cost and higher technical force [Progress in Biochemistry and Biophysics .2004,31 (4): 36-40; Biotechnology circular .2007,3:118-121].And both is all the mensuration to intermediate product, it is not biological function (secretion inducing Regular Insulin) this principle design for GLP-1 receptor stimulant.
Comparatively speaking, design of the present invention this pharmacological principle of just combining closely designs, and single-minded measures end product Regular Insulin, more can embody the actual effect of medicine, makes method more reasonable, more scientific, more practical.Its advantage is in particular in:
First, present method can obtain the effect curve that GLP-1 receptor stimulant promotes excreting insulin, measures the biologic activity of GLP-1 receptor stimulant, can draw and tire relatively accurately with this.And the existing method by animal pattern can only determine whether GLP-1 receptor stimulant has the effect promoting excreting insulin qualitatively.
Secondly, measuring crucial thing in present method is Regular Insulin, it be GLP-1 receptor stimulant with GLP-1 acceptor interaction after, cause corresponding signal path to change, the final product of induction, stability is high, is substantially subject to the impact of cell other material interior.The pharmacological principle of GLP-1 receptor stimulant and design of the present invention is combined closely, single-minded for end product Regular Insulin, more can embody the actual effect of medicine, make method more reasonable, more scientific, more practical.And existing experimental technique, no matter be with cAMP, or take chemiluminescent substance as the Cellular Assay Experimental Method of target detect thing, both is all the mensuration to intermediate product, easily degrades, unstable, and intermediate product has multiple the way of production.
Moreover the cell needed for chemiluminescent substance assay method needs particular build, just can make it to secrete the chemiluminescent substance that can be detected, a large amount of costs, higher technical force and longer time need be spent.And the Min-6 cell culture condition that the present invention uses is simple, growth is easy, convenient operation.
Summary of the invention
This genealogy of law can promote the effect of murine insulinoma cell (Min-6) excreting insulin according to GLP-1 receptor stimulant, with the Regular Insulin of mouse/rat insulin kit secretion, in wavelength 450nm, reference wavelength 590nm place measures its absorbancy, the effect curve of GLP-1 receptor stimulant induction Min-6 cells secrete insulin can be obtained, measure GLP-1 receptor stimulant biologic activity with this.
Certain glucose concn requirement is had because GLP-1 receptor stimulant acts on Min-6 cell, the present invention is by regulating the concentration of substratum glucose, the culture medium culturing target cell of sugar-free is used before adding standard substance trial-product, and with high sugar determination culture medium solution dilution standard product trial-product, improve the susceptibility of Min-6 cell.In the present invention, the glucose concn of the mensuration substratum of dilution standard product and trial-product is preferably 25mM to 75mM, is more preferably 50mM.
This law has certain requirement for the concentration of the cell suspension made with 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 after cell adds sugar-free DMEM substratum; Incubation time is preferably 1-6 hour in the present invention, is more preferably 2-4 hour.
This law adds the incubation time after cell culture medium for detection sample certain requirement; Incubation time is preferably 4-6 hour in the present invention.
Accompanying drawing explanation
Accompanying drawing 1 is GLP-1 (7-36) Determination of biological activity experimental result.
Accompanying drawing 2 is Exendin-4 Determination of biological activity experimental result.
Accompanying drawing 3 for glucose concn be 16.7mM time, GLP-1 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 4 for glucose concn be 25mM time, GLP-1 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 5 for glucose concn be 50mM time, GLP-1 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 6 for glucose concn be 75mM time, GLP-1 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 7 for glucose concn be 16.7mM time, Exendin-4 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 8 for glucose concn be 25mM time, Exendin-4 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 9 for glucose concn be 50mM time, Exendin-4 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 10 for glucose concn be 75mM time, Exendin-4 standard substance and placebo biological activity comparison diagram.
Accompanying drawing 11 be other experiment conditions constant when, change the incubation time of sugar-free culture-medium, measure the comparison diagram of cells secrete insulin.
Accompanying drawing 12 is under the condition that other experiment conditions are constant, changes cell kind plate density, measures the comparison diagram of cells secrete insulin.
Accompanying drawing 13 be other conditions constant when, change the incubation time added after standard substance, measure the comparison diagram of cells secrete insulin.
Accompanying drawing 14 be other conditions constant when, add the comparison diagram that the incubation time after standard substance is 4 hours and 8 hours.
Embodiment:
Embodiment 1
The biologic activity of glucagon-like peptide-1 (7-36) (GLP-1 (7-36)) is measured by this law
Reagent and material (1) DMEM high glucose medium get 1 bag, DMEM high glucose medium powder (specification is 1L), be dissolved in water and be diluted to 1000ml, wherein Sodium.alpha.-ketopropionate final concentration is 1mmol/L, HEPES final concentration is 0.5%, adds penicillin 10
5iU and Streptomycin sulphate 10
5iU, sodium bicarbonate 3.7g, after dissolving, mixing, Sterile Filtration, 4 DEG C of preservations.
(2) perfect medium measures foetal calf serum 10ml, adds DMEM high glucose medium 90ml, 4 DEG C 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 100ml water dissolution and is diluted to 100ml, Sterile Filtration ,-20 DEG C of preservations.
(5) PBS gets sodium-chlor 8.0, Repone K 0.20g, Sodium phosphate dibasic 1.44g, and dipotassium hydrogen phosphate 0.24g is dissolved in water and is diluted to 1000ml, through 121 DEG C of sterilizings in 15 minutes.
(6) Digestive system gets 2.5g pancreatin, disodium ethylene diamine tetraacetate 0.2g, sodium-chlor 8.0, Repone K 0.20g, Sodium phosphate dibasic 1.152g, and dipotassium hydrogen phosphate 0.2g is dissolved in water and is diluted to 1000ml, Sterile Filtration ,-20 DEG C of preservations.
(7) 250mM glucose solution gets glucose (H
2o) 1g, after adding the dissolving of 20ml sugar-free DMEM substratum, mixing, Sterile Filtration, 4 DEG C of preservations.
(8) target cell strain murine insulinoma cell (Min-6) given by Shanghai City diabetes study, goes down to posterity and preserve in this room.
(9) RAT/MOUSE INSULIN KIT test kit LINCO company produces, article No.: EZRMI-13K
(10) standard substance, trial-product intestinal bacteria are glucagon-like peptide-1 (7-36) freeze-dried preparation of host cell expression
Glucagon-like peptide-1 (7-36) standard substance are got in the preparation of standard solution, are diluted to 500ug/ml with mensuration substratum.In 1.5ml centrifuge tube, do 2 times of serial dilutions, totally 8 extent of dilution, each extent of dilution does 2 holes.Aseptically operate.
Glucagon-like peptide-1 (7-36) trial-product is got in the preparation of need testing solution, is diluted to 500ug/ml with mensuration substratum.In 1.5ml centrifuge tube, do 2 times of serial dilutions, totally 8 extent of dilution, each extent of dilution does 2 holes.Aseptically operate.
Experimental technique makes Min-6 cell in perfect medium substratum, 37 DEG C, cultivate under 5% carbon dioxide conditions.The cell of taking the logarithm vegetative period, after washing with PBS, cell suspension is made in Digestive system digestion, adjustment cell density to 5.0 × 10
5individual/ml.Add cell suspension 100ul/ hole in 96 well culture plates, CO
240h cultivated by incubator.Removing cell conditioned medium, adds PBS 200ul/ hole, supernatant discarded, then adds PBS 200ul/ hole, supernatant discarded, after so cleaning 2 times, adds sugar-free DMEM substratum 100ul/ hole, continues to cultivate 4h.Removing cell conditioned medium, add PBS and clean 2 times, add standard substance and the need testing solution 100ul/ hole of the different concns (500ug/ml-250ug/ml-125ug/ml-62.5ug/ml-31.25ug/ml-15.6ug/m l-7.8ug/ml-3.9ug/ml) diluted, each concentration do 2 parallel, set up simultaneously and measure substratum contrast, 37 DEG C, continue under 5% carbon dioxide conditions to cultivate 2h.Collect supernatant, after diluting 5 times, detect with Insulin Kit.Determined wavelength is 450nm, and reference wavelength is 590nm.Finally carry out data analysis by four parametric regression computing methods, calculate, draw the extension rate that induced insulin 50% maximal stimulation is reacted.Unit definition: can the extension rate of induced insulin 50% maximal stimulation reaction be 8000 units (i.e. AU).
Trial-product is tired (AU/ml)=standard substance tire × (A
trial-product/ A
standard substance) × (ED
50 trial-products/ ED
50 standard substance)
A
trial-product, A
standard substance: trial-product, standard substance pre-dilution multiple
ED
50trial-product, ED
50standard substance: trial-product, standard substance median effective dose extension rate
Result is as Fig. 1, and 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 is tired (AU/ml)=standard substance tire × (A
trial-product/ A
standard substance) × (ED
50 trial-products/ ED
50 standard substance)
Embodiment 2
Exendin-4 biologic activity is measured by this law.
Experiment material, method and embodiment 1 operate substantially identical.
The preparation of need testing solution: get Exendin-4 trial-product, is diluted to 50ug/ml with mensuration substratum.
In 1.5ml centrifuge tube, do 2 times of serial dilutions.
Experimental result is shown in Fig. 2, and data are as follows:
Standard substance:
Trial-product:
Placebo:
As shown in Figure 2, sample and standard substance oriented parallel, linear, according to ED
50, can tire by working sample after confirmed standard product are tired, demonstrate the feasibility of method.
Embodiment 3
Dilution measures the bioactive Comparative result of substratum bioassay standard product GLP-1 (7-36) under different glucose concn condition.
Experiment material, method and embodiment 1 operate substantially identical (without trial-product).The glucose concn measuring substratum is configured to 17.5mM, 25mM, 50mM and 75mM respectively.Experimental result is shown in Fig. 3,4,5 and 6.From Fig. 3,4, the contrast of 5 and 6, the concentration improving glucose is conducive to cells secrete insulin, and the scale of the standard substance excreting insulin of different concns reveals obvious dose-effect relationship, is more tending towards obvious with the contrast of placebo.
Embodiment 4
Dilution measures the biological activity determination Comparative result of substratum standard substance Exendin-4 under different glucose concn condition.
Experiment material, method and embodiment 2 operate substantially identical (without trial-product).The glucose concn measuring substratum is configured to 17.5mM, 25mM, 50mM and 75mM respectively.Experimental result is shown in Fig. 7,8,9 and 10, and its comparing result is similar with embodiment 3, all show raising glucose concn and contribute to improving the bioactive accuracy of mensuration, and its measurement effect is best when 50mM.
Embodiment 5
Change cell adds the incubation time after sugar-free DMEM substratum, the measurement result contrast of cells secrete insulin.
Experiment material method (without trial-product, without 8 extent of dilution) substantially the same manner as Example 1, gets recombinant human GLP-1 (7-36) standard substance, is diluted to 500ug/ml, measures with mensuration substratum.Change cell and add the incubation time after sugar-free DMEM substratum, respectively in cultivation after 2,4,5,6,12,16,20,24,30 hours, measure the secretion situation of cell Regular Insulin, its experiment the results are shown in Figure 11.Can obviously find out from figure cultivation after 2 hours, 4 hours insulin-producing amount the highest, be conducive to detection by quantitative most.
Embodiment 6
Change the density of cell, the measurement result contrast of cells secrete insulin.
Experiment material method (without trial-product, without 8 extent of dilution) substantially the same manner as Example 1, gets recombinant human GLP-1 (7-36) standard substance, is diluted to 500ug/ml, measures with mensuration substratum.After cell PBS washes, the density of cell suspension is made in the digestion of adjustment Digestive system, and its cell density is adjusted to 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 obviously find out from figure when cell concn is 5 × 10
5time, insulin-producing amount is the highest, is conducive to most detecting.
Embodiment 7
Change the incubation time after adding standard substance, the measurement result contrast of cells secrete insulin.
Experiment material method (without trial-product, without 8 extent of dilution) substantially the same manner as Example 1, gets recombinant human GLP-1 (7-36) standard substance, is diluted to 500ug/ml, measures with mensuration substratum.After adding 500ug/ml standard substance, respectively 37 DEG C, under 5% carbon dioxide conditions, continue cultivation after 0.5,1,2,3,4,6 and 16 hour, detect, experimental result as shown in figure 13.Obviously can find out there is obvious jump 4 hours to 6 hours from figure, select such time point proper,
Embodiment 8
Add the continuation after standard substance and cultivate 8 hours, the cell insulin secretion situation being 4 hours with incubation time in embodiment 1 contrasts.
Experiment material method (without trial-product) substantially the same manner as Example 1, gets recombinant human GLP-1 (7-36) standard substance, is diluted to 500ug/ml, measures with mensuration substratum.After adding 500ug/ml standard substance, 37 DEG C, under 5% carbon dioxide conditions, continue cultivation after 8 hours, detect.Its result as shown in figure 14.As we know from the figure, continue cultivation and (be greater than 6 hours) after 8 hours, the point of different standards product concentration drops on being tending towards on oblique line and reduces few, and a large amount of points drops on two ends (upper and lower platform), is unfavorable for detecting, thus 4-6 little be good.
Claims (10)
1. a GLP-1 receptor stimulant biological activity assay method, its step comprises:
A () cultivates suitable detection target cell strain; Wherein, described detection is murine insulinoma cell strain Min-6 with target cell strain;
B () prepares GLP-1 receptor stimulant standard solution and the need testing solution of several weaker concns; Wherein, described some dilution GLP-1 receptor stimulant standard substance and need testing solution configure dilution by the glucose solution of 25mM to 75mM;
C () is taken the logarithm cell strain in vegetative period, through treating processes, add standard substance and the need testing solution of preparation in step (b), and continue to cultivate;
D () collects supernatant dilution after, detect with Insulin Kit and obtain the effect curve that the induction of GLP-1 receptor stimulant detects cells secrete insulin;
E () calculates the activity of GLP-1 receptor stimulant.
2. the method for claim 1, wherein described GLP-1 receptor stimulant is GLP-1, Exendin-4, or their analogue.
3. the treating processes the method for claim 1, wherein described in step (c) comprises:
(1), after washing with PBS, add Digestive system digestion and make cell suspension, and adjust cell density, add cell suspension in culture plate, continue to cultivate;
(2) remove cell conditioned medium, add PBS cleaning, add sugar-free DMEM substratum, continue to cultivate;
(3) remove cell conditioned medium, add PBS cleaning, add the standard solution after dilution and need testing solution, continue to cultivate.
4. the method for claim 1, wherein determined wavelength used is 450nm in step (d), and reference wavelength is 590nm.
5. the method for claim 1, wherein the middle method calculating GLP-1 receptor stimulant of step (e) is four parametric regression computing methods.
6. the method for claim 1, wherein described glucose solution glucose concn is 50mM.
7. method as claimed in claim 3, wherein, described cell density is 5 × 10
5individual/ml.
8. method as claimed in claim 3, wherein, the continuation incubation time in step (2) is 1 to 6 hour.
9. method as claimed in claim 8, wherein, described continuation incubation time is 2 to 4 hours.
10. method as claimed in claim 3, wherein, the continuation incubation time in step (3) is 4 to 6 hours.
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| UA116217C2 (en) | 2012-10-09 | 2018-02-26 | Санофі | Exendin-4 derivatives as dual glp1/glucagon agonists |
| WO2014096149A1 (en) | 2012-12-21 | 2014-06-26 | Sanofi | Exendin-4 Derivatives |
| 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 |
| EP3080154B1 (en) | 2013-12-13 | 2018-02-07 | Sanofi | Dual glp-1/gip receptor agonists |
| WO2015086733A1 (en) | 2013-12-13 | 2015-06-18 | Sanofi | Dual glp-1/glucagon receptor agonists |
| TW201609795A (en) | 2013-12-13 | 2016-03-16 | 賽諾菲公司 | EXENDIN-4 peptide analogues as dual GLP-1/GIP receptor agonists |
| WO2015086730A1 (en) | 2013-12-13 | 2015-06-18 | Sanofi | Non-acylated exendin-4 peptide analogues |
| TW201625670A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Dual GLP-1/glucagon receptor agonists derived from EXENDIN-4 |
| TW201625668A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists |
| TW201625669A (en) | 2014-04-07 | 2016-07-16 | 賽諾菲公司 | Peptidic dual GLP-1/glucagon receptor agonists derived from Exendin-4 |
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