CN1329922A - Join of C peptide and insulin for treating diabetes complication and C peptide-specific antibody - Google Patents
Join of C peptide and insulin for treating diabetes complication and C peptide-specific antibody Download PDFInfo
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Abstract
The present invention discloses a composite medicine for treating the complication of diabetes contains proinsulin C-peptide and/or C peptide associated peptide with excessive physiological dosage and its pharmaceutically receptible salt, insulin with pharmaceutical receptible dosage and its pharmaceutical receptible salt, and pharmaceutical receptible carrier. A C peptide-specific antibody and its preparing process and disclosed also. The nephrosis caused by diabetes can be effectively treated. Said antibody has specificity.
Description
The present invention relates to C-Peptide associating insulin treatment, this therapy has the form and function effect of the diabetes of improvement nephropathy, can be applicable to the treatment of chronic complicating diseases of diabetes.The invention still further relates to diabetes diagnosis and beta Cell of islet and estimate used C peptide specificity antibody and preparation method thereof.More particularly, the present invention relates to C-Peptide and preparation method thereof, C peptide specificity antibody and preparation method thereof, and the effect of C peptide associating insulinize in improving SD rat diabetes nephropathy (DN).
The main harm of diabetes is various complication.Wherein diabetic nephropathy accounts for 1/3 among the type i diabetes patient, is the lethal one of the main reasons of these patients.Comprise that insulinize can blood sugar control though treat at present medicine that type i diabetes uses clinically, can not block developing of diabetic complication fully, therefore, need development effectively at the medicine of complication.
The C peptide is the connection peptides (connecting peptide) that connects A, two chains of B in the proinsulin.In the beta Cell of islet secretory granule, proinsulin is through protease cracking, forms the insulin and the C peptide that wait mole to be made up of the AB chain, secretes then and enters blood.The race difference of C peptide molecule is bigger, and people C peptide contains 31 aminoacid.C peptide folding to the proinsulin molecule in the proinsulin molecule, the correct pairing equimolecular structure of disulfide bond forms and plays an important role.
People were unclear to free C peptide physiological function in the blood in the past, recent discovers, in the experiment of type i diabetes patient and animal, short-term gives the C peptide can improve renal dysfunction (Johansson.B.-L.etal.Diabetologia 35:121-128), strengthen the utilization of glucose, increase the blood flow (Forst, T.et al.J.Clin.Invest.101:2036-2041) of muscle and skin.In addition, the C peptide can also improve the erythrocytic morphotropism (Kunt of type i diabetes patient, T.et al.Diabetologia 42:465-471), the C peptide can promote fibroblasts proliferation (Hehenberger K.et al.EuropeanJ Endocrin 136 (supple1): 15) from patient IDDM.
In vitro study confirms, the C Toplink strengthens renal tubules and sciatic nerve Na+, K+-ATP enzymatic activity (OhtomoY.et al.Diabetologia 39:199-205, Ido, Y.et al.Science 277:563-566), the effect of possible C peptide and its stimulation Na+, the activity of K+-ATP enzyme and endogenous NO synzyme relevant (OhtomoY.et al.Diabetologia 41:287-291, Forst T et al.Clin Sci 98 (3): 283-290).The C peptide can strengthen neuropeptide tyrosine for the vasoconstrictive influence of type i diabetes people, and this may be also can work in coordination with C peptide and neuropeptide tyrosine stimulates Na+, K+-ATP enzymatic activity be correlated with (Ohtomo Y.et al.Diabetologia39:199-205).The C peptide stimulates the transhipment of skeletal muscle glucose, but this effect be by the outer approach of Insulin receptor INSR carry out (Johansson BL et al.Diabetologia 39,687-695).Experiment showed, and to be incorporated into cell surface-G protein-coupled receptor specifically in the nanomolar concentration scope by the C peptide (RiglerR.et al.PNAS 96 (23): 13318-13323).Give the C peptide associating insulinize of type i diabetes rat excusing from death reason dosage, can prevent hemostatic tube, dysneuria (Ido, Y.et al.Science 277:563-566), (3 months) give the treatment of insulin combination C peptide for a long time, can make diabetic reduce the urinary albumin excretion rate, improve renal function and dysautonomia (Johansson BL et al.Diabet Med17 (3): 181-9).
Yet, improve streptozotocin (STZ) for excusing from death reason dosage C peptide associating insulin and induce the pathology of renal disease of SD rat formation to change the people's report that do not see before.
For the mensuration of insulin level in the type i diabetes patient body, generally adopt the method for directly measuring insulin at present.At present, when estimating the beta Cell of islet function, need the C peptide antibody, because of the type i diabetes patient and late period type ii diabetes patient plasma C peptide level obviously reduce.There is human BSA coupling C peptide to prepare C peptide antibody (Yang S.et.al, Chinese Science Bulletin, 1982,2:110-114), but because contain the antibody of anti-BSA in the antiserum simultaneously, so in measuring human body during the C peptide content, in the antiserum antibody of anti-BSA may with the albumen effect of similar BSA in the human blood, thereby cause false positive.
Therefore, this area presses for the strong C peptide antibody of exploitation specificity.
The Therapeutic Method and the pharmaceutical composition that the purpose of this invention is to provide a kind of diabetic nephropathy comprise C-Peptide and related peptides thereof, insulin and acceptable salt pharmaceutically.Provide the preparation of this C peptide simultaneously, purification process and its application in the medicine of treatment diabetic complication.A kind of preparation method that can accurately measure the antibody of C peptide content in the blood of human body also is provided in addition.
In a first aspect of the present invention, a kind of pharmaceutical composition for the treatment of diabetic complication is provided, comprise
(a) excusing from death reason dosage C-Peptide and/or C peptide related peptides, and pharmaceutically acceptable salt,
(b) can accept the insulin and the pharmaceutically acceptable salt thereof of medication amount,
(c) pharmaceutically acceptable carrier.
In a preference of the present invention, described C peptide is to derive from insulinogenic C chain, has following amino acid sequences:
EAEDLQVGQV?ELGGGPGAGS?LQPLALEGSL?Q。
In another preference of the present invention, described C peptide related peptides is to derive from the C-Peptide C-terminal, has the peptide of following amino acid sequences or partial sequence:
X1-X2-E-G-S-L-Q-X3-X4
Wherein X1 is amino, acetyl group, acetylated amino acids or the aminoacid that deaminizes;
X2, X3 can not exist or single amino acids, also can be a plurality of aminoacid or peptide,
X4 is amino, carboxyl or hydroxyl.
In another preference of the present invention, described C peptide related peptides comprises the circulus of the C-terminal pentapeptide that derives from C-Peptide:
In a second aspect of the present invention, a kind of C-Peptide specificity preparation method for antibody is provided, comprise step:
(a), form poly-propionyl C peptide or C peptide related peptides with described C peptide or C peptide related peptides and polymerization single polymerization monomer polymerization under appropriate condition;
(b) poly-propionyl C peptide or the C peptide related peptides immunity inoculation animal that obtains with step (a);
(c) from the animal of immunity inoculation, isolate C-Peptide specificity antibody.
In a third aspect of the present invention, a kind of C-Peptide specificity antibody is provided, it is specifically at C-Peptide, and is not incorporated into BSA.
In a fourth aspect of the present invention, a kind of poly-propionyl C peptide or the antigenic preparation method of C peptide related peptides are provided, it comprises step:
(a) with C peptide or C peptide related peptides and acryloyl chloride with 1: 10-1: 50 mol ratio is mixed, and makes the N end propylene acidylate of C peptide or C peptide related peptides;
(b) add C peptide or the C peptide related peptides generation self-polymerization that Ammonium persulfate. makes N end propylene acidylate, thereby form poly-propionyl C peptide or C peptide related peptides;
(c) isolate poly-propionyl C peptide or C peptide related peptides.
Preferably, in step (a), reaction is at room temperature to react about 3-10 hours; And in step (b), also add TEMED with catalytic reaction.
In the accompanying drawings,
The TSK-f40 column chromatographic isolation and purification of Fig. 1 C-Peptide, wherein shaded side is for collecting part.
Fig. 2 high-pressure liquid phase is identified C peptide purity.
Fig. 3 capillary electrophoresis identifies that the C peptide shows homogeneity.
Fig. 4 Electrospray Mass Spectrometry is measured synthetic C peptide molecular weight.C peptide theoretical molecular 3020.29 wherein; Actual measurement molecular weight: 3020.0.
Fig. 5 has shown the mensuration of C peptide hapten molecule amount.
Fig. 5 (A) is a standard molecular weight albumen eluting collection of illustrative plates.1 is bovine serum albumin (67KD); 2 is oralbumin (45KD); 3 is Rhizoma sagittariae sagittifoliae protease inhibitor (20KD).
Fig. 5 (B) is the eluting collection of illustrative plates of poly-propionyl C peptide, by standard molecular weight albumen eluting collection of illustrative plates as can be known its molecular weight be about 25KD.
Fig. 6 has shown when being a course of treatment with eight weeks, has used C peptide or the insulinize influence to diabetes rat urinary albumin excretion rate separately.
Y-axis represents respectively to organize rat twenty-four-hour urine albumin excretion rate (24hUAER) among the figure, and numerical value is with natural logrithm transition form Ln (24hUAER) expression of 24hUAER.
#: normal group and C peptide treatment group, do not treat group and compare p＜0.05:
$: insulinize group and C peptide treatment group, do not treat group and compare p＜0.05.
Fig. 7 has shown when being a course of treatment with 12 weeks, has used C peptide or the insulinize influence to diabetes rat urinary albumin excretion rate separately.
Y-axis represents respectively to organize rat twenty-four-hour urine albumin excretion rate (24hUAER) among the figure, and numerical value is with natural logrithm transition form Ln (24hUAER) expression of 24hUAER.
#: normal group and C peptide treatment group, do not treat group and compare p＜0.05;
Fig. 8 has shown that C peptide associating insulinize is to the influence of diabetes rat urinary albumin excretion rate when being a course of treatment with eight weeks.
Y-axis represents respectively to organize rat twenty-four-hour urine albumin excretion rate (24hUAER) among the figure, and numerical value is with natural logrithm transition form Ln (24hUAER) expression of 24hUAER.
#: the insulinize group is compared with normal group, p＜0.05;
$: insulinize group and C peptide and insulinize group, p＜0.05.
Fig. 9 has shown when being a course of treatment with eight weeks, has used C peptide or the insulinize influence to diabetes rat kidney substrate glomerule sectional area ratio separately.
Y-axis represents respectively to organize rat kidney substrate glomerule sectional area ratio among the figure.
*: do not treat group and compare P＜0.001 with other three groups;
▲: the insulinize group is compared with normal group, P=0.026.
Figure 10 has shown when being a course of treatment with 12 weeks, has used C peptide or the insulinize influence to diabetes rat kidney substrate glomerule sectional area ratio separately.
Y-axis represents respectively to organize rat kidney substrate glomerule sectional area ratio among the figure.
*: do not treat group and compare P＜0.001 with other three groups.
Figure 11 has shown that C peptide associating insulinize is to the influence of diabetes rat kidney substrate glomerule sectional area ratio when being a course of treatment with eight weeks.
Y-axis represents respectively to organize rat kidney substrate glomerule sectional area ratio among the figure.
#: insulinize group, C peptide are compared p＜0.05 with normal group with the insulinize group; $: insulinize group and C peptide and insulinize group, p＜0.05.
The inventor finds that through long-term extensive and deep research excusing from death reason dosage C peptide associating insulin can be bright The aobvious streptozotocin (STZ) that improves induces the pathology of renal disease of SD rat formation to change. In addition, by with the C peptide N end propylene acidylate obtains its haptens after the polymerization, only contain the antibody of anti-C peptide with the antiserum of its preparation, Have selectivity, thereby can be used for diabetes diagnosis and islet beta cell function evaluation. Pdef polypeptide after the propylene acidylate Oligopeptides antigen preparation method have no forefathers report.
Insulinogen C peptide among the present invention not only comprises deriving from the connection that connects A, two chains of B in the proinsulin Peptide and analog thereof comprise that also they are at pharmaceutically acceptable salt.
As used herein, term " excusing from death reason dosage " refers to that serum C peptide concentration surpasses physiological dose. At medicine of the present invention Compositions, the mol ratio of described C peptide and/or C peptide related peptide and insulin is 1: 1-10: 1, preferably be 1.5: 1-8: 1, more preferably be 2: 1-5: 1.
As used herein, term " treatment diabetic complication " refers to that C peptide associating insulin is to the diabetes kidney The improvement effect of the form and function of pathology and to the effect of whole body microangiopathies comprises the reduction urinary albumin Excretion rate prevents the effect such as kidney matrix glomerulus sectional area ratio increase due to the diabetes, and to other one The blood vessel of position and the therapeutic action of dysneuria.
In this article, term " amino acid " refers to that all following 20 kinds of natural amino acids are (with trigram symbol table unless refer in particular to Show): Gly, Ala, Asp, Glu, Asn, Gln, Ser, Thr, Leu, Ile, Lys, Arg, Phe, Tyr, Trp, Pro, Cys, Met, His, Val. This term comprises D type and L-type amino acid. In addition, this term also comprises alpha-non-natural amino acid, first Baseization amino acid, allo amino acid.
In pharmaceutical composition of the present invention, contain
(a) excusing from death reason dosage Insulinogen C peptide and/or C peptide related peptide, and pharmaceutically acceptable salt,
(b) can accept insulin and the pharmaceutically acceptable salt thereof of medication amount,
(c) pharmaceutically acceptable carrier.
Except containing described component, pharmaceutical composition of the present invention also can comprise conventional solvent and anticorrosion Agent. For the pharmaceutical composition of solution form, its pH scope is not particularly limited, and is generally from 4 to 8.5. In addition, pharmaceutical composition also can be made into lyophilized formulations.
Following (the X wherein of the amino acid sequence that can be used for Insulinogen C peptide of the present invention0xThe amino acid of having listed people or mouse changes):
X
00-Glu-Ala-Glu-Asp-X
01-Gln-Val-X
02-Gln-X
03-Glu-Leu-Gly-Gly-Gly-Pro-
Gly-Ala-Gly-X
04-Leu-Gln-X
05-Leu-Ala-Leu-Glu-X
06-X
07-X
08-Gln-Lys-Arg-
In the formula, X00Be acetyl group, acetylated amino acids or the amino acid that deaminizes;
X
01Be Leu or Pro;
X
02Be Gly or Ala;
X
03Be Val or Leu;
X
04Be Ser or Asp;
X
05Be Pro or Thr;
X
06Be Gly or Val;
X
07Be Ser or Ala;
X
08Be Leu or Arg;
A kind of preferred C peptide is C-P, and its amino acid sequence is EAEDLQVGQV ELGGGPGAGS LQPLALEGSL Q.
C peptide related peptide also can be used for the present invention. Described C peptide related peptide is to derive from Insulinogen C PEPC end End, the peptide with following amino acid sequences or partial sequence:
Xl-X2-E-G-S-L-Q-X3-X4
Wherein Xl is amino, acetyl group, acetylated amino acids or the aminoacid that deaminizes;
X2, X3 can not exist or single amino acids, also can be a plurality of aminoacid or peptide,
X4 is amino, carboxyl or hydroxyl.
A kind of special C peptide related peptides is the circulus that derives from the C-terminal pentapeptide of C-Peptide:
As for the method that obtains C peptide and related peptides thereof, without any special restriction.They can make with solid phase or liquid phase chemistry of peptides synthetic technology or with gene engineering method or with the enzyme action processing method.
Chemical synthesis
Can use conventional chemistry of peptides synthetic technology, with solid phase method or synthetic C-Peptide of the present invention of liquid phase method and related peptides, but better be to use solid phase synthesis process (for example referring to Birr, C., Aspect of theMerrifield Peptide Synthesis, Springer-Verlag, Heidelberg, 1978; Stewartet al., Solid Phase Peptide Synthesis, 2nd.ed., Pierce Chem.co., Rockford, IL, 1984; Barany, G.And Merrifield R.B.in The Peptides, Vol.2; Gross, E.﹠amp; Meienhoffer J., eds., Academic Press, New York, pp3-284,1979).Briefly, at first, utilize suitable activator and condensing agent to be connected on the solid phase carrier through the peptide chain C-terminal amino acid residue of due care radical protection according to that designed and given aminoacid sequence.According to the amino acid whose difference that connects, can select to use the various synthetic solid phase carrier of peptide that is used for, for example comprise that (but being not limited to) gather polystyrene, the polyacrylamide resin of ethanol, divinylbenzene crosslink.
Choice of Resin
Can select suitable resin carrier and synthesis system for use according to the sequence signature of purpose peptide.For example, can in the Fmoc system, use the resin of acid labile.For this reason, preferential solid phase carrier comprises the acrylic resin of PEG-PS-resin, divinylbenzene crosslink, this resinoid of difference in functionality group's form comprise chloromethyl resin, hydroxymethyl resin, para-position acetyl aminomethyl resin, 4-methyldiphenyl methylamine (MBHA) resin, 4-(2 ',-4 '-the Dimethoxyphenyl aminomethyl)-phenoxymethyl resin, 2,4-dimethoxy benzene methanamine resin and (2,4-Dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy group acetylaminohydroxyphenylarsonic acid norleucyl--mbha resin (being Rink Amide mbha resin).
Blocking group and protection aminoacid are selected
In the present invention, can select blocking group commonly used in the chemistry of peptides synthetic technology for use.A kind of blocking group of preferred a-amino acid is 9-fluorenes methoxy carbonyl acyl group (Fmoc) blocking group.
When using the Fmoc system to carry out solid phase synthesis, can suitably select following protected amino acid residue: Fmoc-Cys (Trt) for use, Fmoc-Asn (Trt); Fmoc-Ser (But), Fmoc-Leu, Fmoc-Thr (But); Fmoc-Val; Fmoc-Gly, Fmoc-Lys (Boc), Fmoc-Gln (Trt); Fmoc-Glu (Obut); Fmoc-His (Trt), Fmoc-Tyr (But), Fmoc-Arg (Pmc) or Fmoc-Arg (Pbf) and Fmoc-Pro.
When using the Boc system to carry out solid phase synthesis; can suitably select following protected amino acid residue: Boc-Cys (4-Mzl) for use; Boc-Lys (CLZ); Boc-His (Bom), Boc-Asp (OCHex), Boc-Gln (Xan); Boc-Thr (Bzl); Boc-Ser (Bzl), Boc-Pro, Boc-Trp (CHO).Fmoc here or Boc are used for protecting various amino acid whose alpha-amidos, and the group in the bracket is used for protecting amino acid whose side chain.
Coupling and activation
In the present invention, can use known various coupling agents and each amino acid residue of coupling method coupling in the chemistry of peptides field.For example can use dicyclohexylcarbodiimide (DCC) to carry out direct coupling, or pass through for example pentafluorophenyl esters of active ester, or use Fmoc-aminoacid-carboxylic acid anhydrides.Can use hydroxyl benzotriazole (HOBt) or 7-azepine hydroxy benzenes a pair of horses going side by side triazole (HOAt) or with 2-(1H-benzene parallel triazol-1-yl), 1,1,3,3-four urea hexafluorophosphoric acid ester (HBTU) activated amino acids.
Synthesize and purification
In the present invention, method is finished the synthetic of above-mentioned C-Peptide by hand, or utilizes the polypeptide automatic synthesizer, for example ABI 433A type or the PE Pioneer type polypeptide automatic synthesizer of being produced by Applied Biosystems company.
In the Fmoc synthesis system,, removing the Fmoc blocking group, and extend to the N end one by one by the C end according to given aminoacid sequence with 20% hexahydropyridine/dimethyl formamide (DMF) room temperature treatment 20 minutes.After synthetic the finishing, synthetic C-Peptide is cut down and removes protecting group from resin with the trifluoroacetic acid that contains cleaning agent.The ether sedimentation separation obtains thick peptide after can crossing the filtering resin.After the solution lyophilizing with products therefrom, with gel filtration and the required peptide of reverse phase HPLC method purification.
In the Boc synthesis system, in going protection, neutralization, link coupled circulation, remove blocking group Boc and use diisopropylethylamine (DIEA)/dichloromethane neutralization with TFA/ dichloromethane (DCM).After the peptide chain condensation is finished,, handled 1-2 hour down, peptide chain is downcut from resin, remove blocking group simultaneously at O ℃ with the fluohydric acid gas (HF) that contains p-cresol (5-10%).Use 0.05M NH
4HCO
3The extracting peptide, further with molecular sieve TSK-40f one step separation and purification (Fig. 1), both having obtained HPLC purity is 99% (Fig. 2), the C-Peptide of capillary electrophoresis homogeneous (Fig. 3) after the solution lyophilizing.
Pharmaceutically acceptable salt
For with above-mentioned technology or the C-Peptide that makes with recombinant DNA technology, also available known method is made into its pharmaceutically acceptable salt.For example, can obtain suitable salt with suitable acid, these peptides of alkali treatment by method well known to those skilled in the art.
The polymerization of C peptide or C peptide related peptides (or poly-propionylization)
In the present invention, C peptide or the C peptide related peptides that is used to gather propionylization can be any source.They can be pure substance or mixture.A kind of preferred raw material is synthetic C peptide of chemical synthesis or C peptide related peptides.
When poly-propionyl reaction, be 1 with mol ratio: 50-1: 10, preferably 1: 40-1: 15 C peptide or C peptide related peptides and polymerization single polymerization monomer mix, (for example 0-50 ℃) reaction time enough (as 1-48 hour) under suitable temperature.In addition, in reaction, can add catalyst such as Ammonium persulfate. and TEMED (N, N, N ', N '-tetramethylethylenediamine) with catalytic polymerization, and can add according to circumstances buffer with the pH that keeps reaction system in OK range (about 6.0-8.5).
The polymerization single polymerization monomer that is applicable to polymerization C peptide or C peptide related peptides comprises: contain the α of 3-6 carbon atoms, β-unsaturated carboxylic acid halides, or similar monomer.A kind of particularly preferred polymerization single polymerization monomer is an acryloyl chloride.
Antibody Preparation
On the other hand, the present invention comprises that also C peptide or C peptide related peptides are had specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into C peptide or C peptide related peptides.Preferably, refer to that those can combine with C peptide or C peptide related peptides but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the C peptide or the C peptide related peptides of poly-propionylization can be applied to animal to induce the generation of polyclonal antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and TCell Hybridomas.Elsevier, N.Y., 1981).
In an example of the present invention, earlier with the synthetic C peptide hapten of chemical synthesis, gather the propionyl processing after, immune White Rabbit obtains specificity antibody.And the titre and the specificity of the immunity of the C peptide hapten by following laboratory observation chemosynthesis White Rabbit gained antibody: (1) .ELISA measures the anti-people C of rabbit peptide antiserum titre; (2). use the BSA wrapper sheet, survey the antiserum titre of poly-propionyl-C peptide and BSA-C peptide with said method.
In one embodiment of the invention, utilize the inductive diabetes rat model of streptozotocin (STZ), use the therapeutical effect of C-Peptide associating subcutaneous injection of insulin observation to the rat diabetes nephropathy, the result shows that not only improving its pathology of renal disease changes, and improves the variation of diabetes rat renal function.Particularly, the present invention unites the effect of insulin for diabetes rat by following laboratory observation excusing from death reason dosage C peptide:
(1) C peptide associating insulin action can reduce its urinary albumin excretion rate in the inductive SD rat diabetes of streptozotocin (STZ) model.
(2) C peptide associating insulin action reduces the substrate glomerule area ratio increase that diabetes cause in the inductive diabetes rat model of streptozotocin (STZ).
The invention has the advantages that:
(1) utilizes C peptide associating insulin for treating diabetes can reduce the kidney urinary albumin excretion effectively, diabetic nephropathy is had therapeutical effect than traditional single insulinize.It is one of its mechanism of action that C peptide associating insulinize can reduce the diabetic glomeruli extracellular matrix.
(2) with the synthetic C peptide hapten of poly-propionyl method; this hapten immune animal gained antiserum contains the antibody of anti-C peptide; but avoided common with BSA, ' when KLH etc. prepares antibody as carrier coupling oligopeptide, produce the problem of the antibody of anti-carrier simultaneously, have specificity.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1
With Boc system insulin synthesis C peptide and related peptides thereof
With the C peptide is example: initial 0.32mmol BocQ-Pam-resin, and press polypeptide sequence and extend to the N end one by one by the C end.The used protecting group of various Boc aminoacid is respectively Ser (OBzl), Asp (OCHex), Glu (OCHex).Condensing agent is DCCI, adds HOBT to activate each amino acid whose carboxyl.Whenever, take turns circulation beginning, remove Boc, neutralize with 10%DIEA then with 50%TFA.After peptide chain is synthetic, handled 60 minutes at 0 ℃, peptide chain cracking from the resin is got off with the dry fluohydric acid gas that contains 5% p-cresol; remove various protecting groups simultaneously, with the ammonium hydrogencarbonate extracting of 0.05M, lyophilization; obtain thick peptide, use TSK Hw-40F gel filtration chromatography separation and purification (Fig. 1) then.
Identify that with the high-pressure liquid phase reversed phase column chromatography purity reaches 99% (Fig. 2); Capillary electrophoresis shows homogeneous (Fig. 3); Through the Electrospray Mass Spectrometry determining molecular weight is 3020Da, meets (Fig. 4) with theoretical molecular 3020.29Da.
In addition, with PE-ABI 491A instrument N end protein complete sequence is measured, the N terminal sequence that records is: E-A-E-D-L-Q-V-G-Q-V-E-L-G-G-G-P-G-A-G-S-L-Q-P-L-A-L-E-G-S-L-.
With PE-ABI 491A instrument C end protein partial sequence is measured, the C terminal sequence that records is ... G-S-L-Q.Comprehensive sequencing result, the aminoacid sequence that shows synthetic C peptide is correct.
Embodiment 2
The foundation of type i diabetes rat model
To male S-D rat (3 monthly ages, body weight 200-250g) through tail vein injection streptozotocin STZ (50mg/kg body weight), thereby the preparation diabetes model.Same rat through the tail vein injection buffer as normal control.
Embodiment 3
C peptide associating insulin action reduces the urinary albumin excretion rate in the type i diabetes rat model
In using C peptide or insulinize test separately, diabetes rat is divided into 3 groups: insulinize group, C peptide treatment group and non-treatment group.The insulinize group gives protamine zine insulin injection (PZI) subcutaneous injection once a day, and blood glucose is maintained near normal level (7-8mmol/l); C peptide treatment group administration of human C peptide twice subcutaneous injection (130nmol/kg body weight) every day, and in addition low dose of PZI once a day subcutaneous injection keep survival of rats; Non-treatment organize low dose of PZI once a day subcutaneous injection keep survival of rats; The normal control group does not give any treatment.Be 8 week or 12 weeks the course of treatment.
In C peptide associating insulinize test, diabetes rat is divided into 2 groups: insulinize group and C peptide associating insulinize group.The insulinize group gives protamine zine insulin injection (PZI) subcutaneous injection once a day, makes blood glucose maintain medium control level (about 10mmol/l); C peptide associating insulinize group administration of human C peptide twice subcutaneous injection (130nmol/kg body weight) every day, and protamine zine insulin injection (PZI) subcutaneous injection once a day in addition make blood glucose maintain medium control level.The normal control group does not give any treatment.Be 8 weeks the course of treatment.
Leave and take rat twenty-four-hour urine amount when C peptide and/or insulinize finish, (Cayman Chemical Company Australia) measures urinaryalbumin concentration, calculates twenty-four-hour urine albumin excretion rate (μ g/24h) with rat albumin EIA test kit.
The result shows, in using C peptide or insulinize test separately, 8 the course of treatment in week rat ascending normal group, insulinize group, C peptide treatment group and the non-treatment group of all being followed successively by of twenty-four-hour urine albumin excretion rate.Variance analysis shows that each group difference has statistical significance (p＜0.001).The analysis of Least-significant difference method shows the urinary albumin excretion rate close with the insulinize group (p=0.124) of normal rats, but less than C peptide treatment group and non-treatment group (being respectively p=0.001 and p＜0.001).The urinary albumin excretion rate of insulinize group rat is also less than C peptide treatment group and non-treatment group (being respectively p=0.019 and p=0.001).The urinary albumin excretion rate of C peptide treatment group rat is less than non-treatment group, but the significance of difference is in critical range (p=0.059).12 the week course of treatment rat ascending normal group, insulinize group, non-treatment group and the C peptide treatment group of all being followed successively by of twenty-four-hour urine albumin excretion rate.Variance analysis shows that each group difference has statistical significance (p=0.021).Least significant difference (Least-significant difference) analysis shows, the urinary albumin excretion rate of normal rats close with the insulinize group (p=0.385), the urinary albumin excretion rate close with non-treatment group (p=0.895) of C peptide treatment group rat.The urinary albumin excretion rate of normal rats is less than C peptide treatment group and non-treatment group (being p=0.011).The urinary albumin excretion rate of insulinize group rat is less than C peptide treatment group and non-treatment group, but the significance of difference is in critical range (being respectively p=0.050 and p=0.054).This results suggest is used C peptide (8 week) treatment diabetes rat in a short time separately under blood sugar control situation not, have the trend that the urinary albumin excretion rate increases due to the diabetes of preventing; But along with the prolongation (12 week) of the course of treatment, use the treatment of C peptide separately and no longer demonstrate therapeutic effect, may be because the toxic action of long-term hyperglycemia have been covered the protective effect (referring to Fig. 6, Fig. 7) of C peptide to kidney.
In C peptide associating insulinize test, ascending normal group, C peptide associating insulinize group and the insulinize group of all being followed successively by of twenty-four-hour urine albumin excretion rate.Variance analysis shows that each group difference has statistical significance (p＜0.05).Least significant difference (Least-significant difference) analysis shows that urinary albumin excretion rate and the normal group of C peptide associating insulinize group rat is close; The urinary albumin excretion rate of insulinize group rat is greater than normal group and C peptide associating insulinize group (being p＜0.05).This prompting C peptide associating insulinize is used C peptide or insulinize more separately to preventing that the increase of urinary albumin excretion rate has better effect (referring to Fig. 8) due to the diabetes.
Embodiment 4
The C peptide is united insulin action in the type i diabetes rat model, the influence that the substrate glomerule area ratio that diabetes are caused changes
In the present embodiment, basic identical described in experimental technique and the embodiment 3.Difference is, when finishing, C peptide and/or insulinize gets rat kidney, fix with neutral formalin, specimens paraffin embedding slices, row PAS dyeing is measured substrate glomerule sectional area ratio (being the area of PAS positive material in the glomerule sectional area and the ratio of glomerule sectional area) with IMS coloured image analysis-synthesis system (Shanghai Shen Teng Information Technology Co., Ltd) under light microscopic.
The result shows, in using C peptide or insulinize test separately, 8 the course of treatment in week rat kidney substrate glomerule sectional area ratio in that not treat group maximum, secondly be insulinize group, the value minimum of C peptide treatment group and normal group.Variance analysis shows that each group difference has statistical significance (P＜0.001).Least significant difference (Least-significant difference) analysis shows does not treat the substrate glomerule sectional area ratio of group greater than other three groups (P＜0.001); Difference between C peptide treatment group and insulinize group and normal group does not have statistical significance (P＞0.05).
12 the course of treatment in week rat kidney substrate glomerule sectional area ratio also be that not treat group maximum, next coming in order are C peptide treatment group, normal group and insulinize group.Variance analysis shows that the difference between each group has statistical significance (P＜0.001).Least significant difference (Least-significant difference) analysis shows does not treat the substrate glomerule sectional area ratio of group greater than other three groups (P＜0.001); Difference between C peptide treatment group, insulinize group and normal group does not have statistical significance (P＞0.05).This prompting diabetes rat is used C peptide or insulinize separately all the effect (referring to Fig. 9, Figure 10) that kidney substrate glomerule sectional area ratio increases due to the diabetes of preventing.
In C peptide associating insulinize test, rat kidney substrate glomerule sectional area ratio is followed successively by insulinize group, C peptide associating insulinize group and normal group from big to small.Variance analysis shows that the difference between each group has statistical significance (P＜0.05).Least significant difference (Least-significant difference) analysis shows that the substrate glomerule sectional area ratio of insulinize group and C peptide associating insulinize group is all greater than normal group (P＜0.05); And the substrate glomerule sectional area ratio of insulinize group is greater than C peptide associating insulinize group (P＜0.05).This prompting diabetes rat C peptide associating insulinize is to preventing due to the diabetes increase of kidney substrate glomerule sectional area ratio and use the C peptide more separately or insulinize having better effect (referring to Figure 11).
Chemosynthesis gathers propionyl C peptide
The C peptide of preparation among the 4.0mg embodiment 1 is suspended in the ultra-pure water of 100 μ l PH7.5, adds 2 μ l acryloyl chlorides (mol ratio of C peptide and acryloyl chloride is 1: 20), room temperature reaction (was used KHCO in 5.5 hours
3Keep solution PH about 7.5), add 20 μ l, 10% Ammonium persulfate. and 3 μ l TEMED catalytic polymerizations then, after the ambient temperature overnight, add the neutralization of 5 μ l, 10% ammonia, to ultra-pure water dialysis back lyophilizing, obtain poly-propionyl C peptide.
Embodiment 6
Poly-propionyl C peptide molecular weight is measured
With BSA (bovine serum albumin, 67KD), oralbumin (Ovalbumin, 45KD), Rhizoma sagittariae sagittifoliae protease inhibitor (Arrowhead protease inhibitor, 20KD) be standard molecular weight albumen, (Φ 0.75 * 60cm), measures poly-propionyl C peptide molecular weight for gel filtration on TSK SW G-300 post.
The result shows that the molecular weight of poly-propionyl C peptide molecular weight is about 25KD.
Embodiment 7
The sero-fast preparation of the anti-people C of rabbit peptide
The poly-propionyl C peptide of preparation among the 0.8mg embodiment 5 is dissolved in (1 new zealand white rabbit C of per injection peptide consumption) in the 0.5ml normal saline, in syringe, push into Emulsion (1: 1) with freund adjuvant, the immunity new zealand white rabbit, groin injection in first day, the 8th day; In syringe, push into Emulsion (1: 1), the 15 day, the 22 day vola multi-point injection with non-Freund's complete adjuvant; The antiserum titer determination is carried out in blood-letting in the 30 day then.
The result shows that the C peptide antiserum titre that obtains reaches 2.5 * 10
4During with the BSA wrapper sheet, with the negative contrast of normal rabbit serum, the antibody that records BSA-C peptide antigen (this prepared in laboratory) preparation shows positive, and poly-propionyl C peptide does not produce anti-BSA antibody.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a pharmaceutical composition for the treatment of diabetic complication is characterized in that, comprises
(a) excusing from death reason dosage C-Peptide and/or C peptide related peptides, and pharmaceutically acceptable salt,
(b) can accept the insulin and the pharmaceutically acceptable salt thereof of medication amount,
(c) pharmaceutically acceptable carrier.
2. pharmaceutical composition according to claim 1 is characterized in that, described C peptide is to derive from insulinogenic C chain, has following amino acid sequences:
EAEDLQVGQV?ELGGGPGAGS?LQPLALEGSL?Q。
3. pharmaceutical composition according to claim 1 is characterized in that, described C peptide related peptides is to derive from the C-Peptide C-terminal, has the peptide of following amino acid sequences or partial sequence:
X1-X2-E-G-S-L-Q-X3-X4
Wherein X1 is amino, acetyl group, acetylated amino acids or the aminoacid that deaminizes;
X2, X3 can not exist or single amino acids, also can be a plurality of aminoacid or peptide,
X4 is amino, carboxyl or hydroxyl.
4. pharmaceutical composition according to claim 1 is characterized in that, described C peptide related peptides comprises the circulus of the C-terminal pentapeptide that derives from C-Peptide:
5. pharmaceutical composition according to claim 1 is characterized in that, the mol ratio of described C peptide and/or C peptide related peptides and insulin is 1: 1-10: 1.
6. a C-Peptide specificity preparation method for antibody is characterized in that, comprises step:
(a), form poly-propionyl C peptide or C peptide related peptides with described C peptide or C peptide related peptides and polymerization single polymerization monomer polymerization under appropriate condition;
(b) poly-propionyl C peptide or the C peptide related peptides immunity inoculation animal that obtains with step (a);
(c) from the animal of immunity inoculation, isolate C-Peptide specificity antibody.
7. a C-Peptide specificity antibody is characterized in that, it is specifically at C-Peptide, and is not incorporated into BSA.
8. the antibody above-mentioned as claim 7 is characterized in that, it is that antigen prepares with poly-propionyl C peptide or C peptide related peptides.
9. poly-propionyl C peptide or the antigenic preparation method of C peptide related peptides is characterized in that it comprises step:
(a) with C peptide or C peptide related peptides and acryloyl chloride with 1: 10-1: 50 mol ratio is mixed, and makes the N end propylene acidylate of C peptide or C peptide related peptides;
(b) add C peptide or the C peptide related peptides generation self-polymerization that Ammonium persulfate. makes N end propylene acidylate, thereby form poly-propionyl C peptide or C peptide related peptides;
(c) isolate poly-propionyl C peptide or C peptide related peptides.
10. method as claimed in claim 9 is characterized in that, in step (a), reaction is at room temperature to react about 3-10 hours; And in step (b), also add TEMED with catalytic reaction.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01112929 CN1219548C (en) | 2001-05-18 | 2001-05-18 | Join of C peptide and insulin for treating diabetes complication and C peptide-specific antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01112929 CN1219548C (en) | 2001-05-18 | 2001-05-18 | Join of C peptide and insulin for treating diabetes complication and C peptide-specific antibody |
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| CN1219548C CN1219548C (en) | 2005-09-21 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381727B (en) * | 2008-10-08 | 2011-06-08 | 西藏金稞科技有限公司 | Construction of human insulinogen C peptide high-yield strains |
| CN105073129A (en) * | 2013-01-29 | 2015-11-18 | 奥莎迪药品管理有限公司 | Pharmaceutical compositions for oral treatment of diabetes |
| CN105601750A (en) * | 2016-01-22 | 2016-05-25 | 宁波美康生物科技股份有限公司 | Genetic recombinant human C-peptide fused protein and preparation method and application thereof |
| CN109705221A (en) * | 2018-12-27 | 2019-05-03 | 美康生物科技股份有限公司 | C peptide based immunogens and its monoclonal antibody pair and the antibody are to the application in C peptide magnetic microparticle chemiluminescence immunoreagent |
-
2001
- 2001-05-18 CN CN 01112929 patent/CN1219548C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381727B (en) * | 2008-10-08 | 2011-06-08 | 西藏金稞科技有限公司 | Construction of human insulinogen C peptide high-yield strains |
| CN105073129A (en) * | 2013-01-29 | 2015-11-18 | 奥莎迪药品管理有限公司 | Pharmaceutical compositions for oral treatment of diabetes |
| CN105601750A (en) * | 2016-01-22 | 2016-05-25 | 宁波美康生物科技股份有限公司 | Genetic recombinant human C-peptide fused protein and preparation method and application thereof |
| CN105601750B (en) * | 2016-01-22 | 2019-05-31 | 美康生物科技股份有限公司 | A kind of gene recombinant human C peptide fusion protein and the preparation method and application thereof |
| CN109705221A (en) * | 2018-12-27 | 2019-05-03 | 美康生物科技股份有限公司 | C peptide based immunogens and its monoclonal antibody pair and the antibody are to the application in C peptide magnetic microparticle chemiluminescence immunoreagent |
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| Publication number | Publication date |
|---|---|
| CN1219548C (en) | 2005-09-21 |
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