CN104902929A - Uses of protein precursors as prodrugs - Google Patents
Uses of protein precursors as prodrugs Download PDFInfo
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- CN104902929A CN104902929A CN201380038582.6A CN201380038582A CN104902929A CN 104902929 A CN104902929 A CN 104902929A CN 201380038582 A CN201380038582 A CN 201380038582A CN 104902929 A CN104902929 A CN 104902929A
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- fusion rotein
- proinsulin
- protein precursor
- amyloid protein
- insulin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
Abstract
The present invention provides compositions useful as prodrugs and methods for making the same. The compositions include a fusion protein having a first delivery domain and a second protein precursor domain linked together via a linker sequence. The delivery domain is a protein capable of facilitating entry to a target cells via the endocytotic pathway, such as transferrin. The protein precursor is a prohormone or a profactor, such as proinsulin. Methods of this invention include the steps of selecting a protein suitable as the delivery domain, constructing a vector to encode the fusion protein, and expressing the fusion protein in a suitable expression host. Also disclosed is a method for targeted-delivery of prodrugs to livers and a method of reducing hepatic glucose production.
Description
To the cross reference of related application
The application is the U.S. Patent application 13/174 submitted on June 30th, 2011, the part continuation application of 520, it enjoys in the rights and interests of the U.S. Provisional Application 61/361,248 that on July 2nd, 2010 submits, and therefore these all the elements of applying for all is incorporated herein by quoting.
About the statement of the research and development by federal funding
The contract GM 063647 that the present invention authorizes according to NIH and HIGMS completes under governmental support.Government enjoys certain right of the present invention.
Technical field
The invention belongs to drug delivery and protein engineering field.More specifically, the present invention relates to the method and composition for sending the therapeutic agent (such as prohormone or the factor former (profactor)) based on albumen, it just can produce the effective medicine for the treatment of without the need to iii vitro chemical processing or Proteolytic enzyme processing.
Background technology
Many have bioactive peptide or albumen, comprises hormone, cytokine, neuropeptide and somatomedin, is at first to produce with the form of larger inactive precursor peptide.These precursor peptides or propetide (comprise prohormone or the factor is former) the usually specific intracellular proteolysis of needs are processed and are made it be transformed into its activity form to play biological function [1,2].For albumen manufacture, the precursor forms of frequent first synthetic peptide instead of mature form.This is because the mature form of peptide often has the low or structural instability of complicated conformation, yield.For protein drug is sent, propetide (but not peptide of maturation) is merged by chemically conjugated or restructuring and is connected with another protein part, thus realize specifically sending target and the stability in the large [3] strengthening albumen.Therefore, in order to show biologic activity, need process propetide and activate, this is important and challenging step when manufacturing restructuring treatment peptide.
The conventional method of sending prodrug be usually directed to by propetide with send that albumen links together chemically conjugated.But two chemically conjugated major obstacles together of domain be, the composition of end product and size can be allos, and this is unacceptable for therapeutic use.Therefore, a kind of better method is still needed to form the fusion rotein linked together in delivery configuration territory and pro-drugs territory.
Summary of the invention
The present invention is based on following beat all discovery: in the endocytosis of Mediated by Transferrin Receptor, hepatocyte and epithelial intracellular region room have ability proinsulin being converted into insulin.By the inspiration that this finds, the present inventor imagines and has put into practice a kind of method by the propetide of protein factor and transferrins domain being puted together the medicine prepared based on albumen.Such as, can by the propetide (i.e. proinsulin) of insulin and transferrins be puted together the protein drug formed based on insulin.When with hepatocyte incubation, this proinsulin-transferrins conjugate or fusion rotein will be converted into completely active Insulin-Transferrin.Except serving as activation elements, transferrin moiety can also improve the stability of insulin or other treatment peptide and long-lasting activity (compared with its unconjugated homologue).
Those skilled in the art know, the propetide of many protein factors, such as proinsulin and proglucagon, medicine can not be used as, unless cut off specific peptide bond by chemical treatment or proteolytic treatment and they changed into bioactive peptide, such as, remove c-peptide from proinsulin.Or, can with two peptide recombination method insulin synthesis, the method comprises carries out CNBr process and produces A chain and B chain respectively, then carries out redox reaction to form interchain disulfide bond [4].The modification processing that this type of carries out after propetide produces not only has challenge technically for generation therapeutic peptide, and cost benefit is very low.The invention enables and propetide can be used as medicine when not carrying out extra chemical process or enzymatic processing.
Reduce the target spot that Hepatic glucose production has been treating diabetes strategy.The biological activity of insulin (INS) can affect Hepatic glucose production and periphery glucose removes (glucose disposal).Usually, INS is directly delivered to hepatic portal vein by pancreas, under therefore liver is exposed to the INS of high concentration, causes larger impact thus on Hepatic glucose production (HGP).In the INS therapy of routine, subcutaneous administration INS and/or INS analog, thus make liver obtain insulinize (under-insulinized liver).The effect removed periphery glucose afterwards becomes large, causes Developmental and Metabolic Disorder, comprises excessive blood glucose fluctuation, dyslipidemia, blood plasma IGF-1 reduces and growth hormone blood plasma level raises [9].Therefore, the effect of HGP is greater than will has advantage than existing therapeutic scheme to the INS therapy of the effect that periphery glucose removes.Unexpected discovery of the present invention is that fusion rotein of the present invention can be sent to liver target in vivo, and specifically, proinsulin-transferrin fusion protein as herein described can be sent to liver target in vivo.
Therefore, on the one hand, the invention provides a kind of fusion rotein being used as prodrug.The fusion rotein of the embodiment of this aspect of the invention has the first delivery configuration territory be connected with the second amyloid protein precursor domain by catenation sequence usually.This delivery configuration territory is the albumen that can promote to be entered by endocytic pathway target cell.Second amyloid protein precursor domain is preferably prohormone or the factor is former.
On the other hand, the present invention also provides a kind of method be delivered to by amyloid protein precursor domain in the study subject needing this front body structure territory.The method of the embodiment of this aspect of the invention generally includes following steps: form the fusion rotein with the delivery configuration territory be connected with amyloid protein precursor domain, and this fusion rotein is administered to patient.
In another, the present invention also provides a kind of method forming the fusion rotein being used as prodrug.The method of the embodiment of this aspect of the invention generally includes: for amyloid protein precursor selects the albumen being used as delivery configuration territory; Carrier construction, the described delivery configuration territory that described vector encoded is connected with described amyloid protein precursor by the catenation sequence be applicable to; Described fusion rotein is expressed with in the expressive host be applicable to.
In another, the present invention also provides a kind of method extending the plasma half-life of amyloid protein precursor domain.The method of the embodiment of this aspect of the invention generally includes following steps: before being imported by this amyloid protein precursor domain in blood plasma, amyloid protein precursor domain is conjugated to transferrins domain.Herein, transferrins domain serves as Increased Plasma Half-life element, is used for extending the plasma half-life of amyloid protein precursor in blood plasma.
In another, the present invention also provides a kind of method extending the therapeutic effect of amyloid protein precursor in study subject.The method of the embodiment of this aspect of the invention generally includes following steps: amyloid protein precursor is conjugated to transferrins domain, to form the amyloid protein precursor domain be connected with transferrins domain by catenation sequence.Herein, transferrins domain serves as treatment effectiveness stabilisation element, and it extends the treatment effect duration of amyloid protein precursor.
In another, the present invention also provide a kind of by amyloid protein precursor prodrug targeting to the method for the liver of study subject.The method of this aspect of the invention generally includes following steps: use prodrug to study subject, and wherein, described prodrug is fusion rotein, and described fusion rotein comprises the first delivery configuration territory be connected with the second amyloid protein precursor domain by catenation sequence.This hepatic delivery domain be can in vivo targeted delivery to the albumen of liver, it is further preferred that it also promotes to enter hepatocyte by endocytic pathway.Preferably, hepatic delivery domain is transferrins.Second amyloid protein precursor domain is preferably prohormone or the factor is former.In a preferred embodiment, described fusion rotein is proinsulin-transferrin fusion protein.
Other aspects of the present invention and advantage will become obvious from following explanation and claims.
Accompanying drawing explanation
Fig. 1 illustrates the preproinsulin-Tf antigen-4 fusion protein gene construct in pcDNA 3.1 (+) carrier.
Fig. 2 shows and uses anti-Tf antibody and glucagon (former) antibody to carry out the result of Western blotting to proinsulin (PI)-transferrins (Tf) fusion rotein, and this result demonstrates Tf in fusion rotein and proinsulin is all expressed.1st to the 5th road is anti-Tf trace, and the 7th road is glucagon (former) trace.1st road: 5ng deferrization Tf (apo-Tf).2nd road: 20ng deferrization Tf.3rd road: PI-Tf.4th road: 5ng deferrization Tf+PI-Tf.5th road: 20ng deferrization Tf+PI-Tf.6th road: labelling.7th road: PI-Tf.
Fig. 3 shows the glycogenetic figure of Fructus Vitis viniferae of hepatocarcinoma H4IIE cell.Which demonstrate the activity of the suppression Hepatic glucose production of PI-Tf fusion rotein higher than insulin and proinsulin.In addition, Fig. 3 B shows the activity can destroying PI-Tf when there is greatly excessive Tf, shows that this activity is subject to the mediation of TfR combination.(A) the suppression curve of proinsulin, insulin and PI-Tf fusion rotein.(B) Hepatic glucose production increased by 1000 times of Tf altogether incubation block.
Fig. 4 shows the figure of the measurement result of the insulin in solution.Which demonstrate PI-Tf under the existence of hepatocarcinoma H4IIE cell and change into insulin-Tf.
Fig. 5 shows the block diagram illustrated the competition that Insulin receptor INSR combines.The Insulin receptor INSR binding affinity which demonstrating at 37 DEG C the PI-Tf fusion rotein of incubation in advance in H4IIE cell improves.
Fig. 6 shows the block diagram of the glucose uptake of adipose cell, and the absorption of adipose cell to 2-deoxidation-D-[2,6-3H] glucose which demonstrating cultivation receives the stimulation through the pretreated PI-Tf fusion rotein of H4IIE.(A) Dose-dependent curve of insulin and the stimulation of the glucose uptake caused by proinsulin.(B) comparison of the proinsulin of 100nM insulin, proinsulin, equimolar ratio and the glucose uptake caused by Tf and PI-Tf fusion rotein.(C) activity of increase compared with the fusion rotein without cell process has been shown through the pretreated PI-Tf fusion rotein of H4IIE.Asterisk represents p<0.01 in t inspection assessment.
Fig. 7 shows the comparison to Pharmacokinetic Characteristics in exemplary proinsulin-Tf fusion rotein and insulinogenic body.
Fig. 8 shows comparing the effect of lowering blood sugar of exemplary proinsulin-Tf fusion rotein and PBS, insulin and proinsulin (ProINS).
Fig. 9 shows the importance of Hepatic glucose production approach and the suppression to blood glucose levels of prolongation.(A) the blood-glucose source under as fed and fasting state.(B) to the insulin (INS) of STZ mice single subcutaneous injection PBS or 22.5nmol/kg, proinsulin (ProINS) or proinsulin-Tf (ProINS-Tf).Mice fasting is made before the injection in 2 hours and experimentation.OneTouch blood-glucose meter is used to measure blood glucose levels.Data representation meansigma methods ± standard deviation (n=3 ~ 5).
Detailed description of the invention
Term used herein " amyloid protein precursor " refers to inactive protein or the peptide that can be converted into activity form by post translational modification.Exemplary " amyloid protein precursor " can comprise proinsulin, proglucagon and proopiomelanocortin, but is not limited thereto.
Term used herein " prodrug " refers to the pharmacological agents used with inactive form or active significantly lower form, but its in vivo by metabolic activity in cell or extracellular activation.Exemplary prodrug can comprise prohormone or other factors are former.
" albumen " is the macromole comprising one or more polypeptide chain.Albumen can also comprise non-peptide composition, such as glycosyl.Sugar and other non-peptide substituent groups can be added on albumen by the protedogenous cell of product, and different according to cell type.Albumen defines with its amino acid backbone structures in this article; The substituent groups such as such as glycosyl specifically do not indicate usually, but may exist.
Except as otherwise noted, all terms used herein all have the definition hereafter provided, and usually all consistent with the implication of these terms known to those skilled in the art in the invention.For definition and the term of this area, practitioner can be with particular reference to: Sambrook etc. (1989) Molecular Cloning:A Laboratory Manual (second edition), Cold Spring Harbor Press, Plainview, N.Y.; With (1993) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. such as Ausubel F M.These it should be understood that and the invention is not restricted to described ad hoc approach, rule of operation and reagent, because can change.
Term " carrier " refers to the nucleic acid construct that design is used for carrying out shifting between different host cells." expression vector " refers to the carrier that can be incorporated to foreign cell expressing heterologous DNA fragmentation wherein.The all commercially available acquisition of many prokaryotic expression carriers and carrier for expression of eukaryon.Select suitable expression vector in the knowledge of those skilled in the range.Therefore, " expression cassette " or " expression vector " is the nucleic acid construct that restructuring produces or synthesizes, and it has a series of specific nucleic acid elements to allow specific nucleic acid at target cell transcription.Recombinant expression cassettes can be incorporated in plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment.Usually, except other sequences, the recombinant expression cassettes part of expression vector comprises nucleotide sequence to be transcribed and promoter.
Can prepare pharmaceutical preparation of the present invention according to routine techniques known in pharmaceuticals industry, it can exist with unit dosage forms easily.This type of technology comprises the step that active component is combined with one or more pharmaceutical carriers or excipient.Usually, by combining with making the solid carriers uniform close of active component and liquid carrier and/or fine and closely woven segmentation, and making product shaping if desired, preparing preparation.
In order to describe and disclose the compositions and method that may use in the context of the present invention, all publications quoted herein are incorporated herein to quote mode all clearly.
The albumen being suitable as delivery configuration territory will depend on target cell.Preferably, this albumen is the albumen that can promote to be entered by endocytic pathway target cell.
Catenation sequence is preferably shorter and stable.In some embodiments, catenation sequence can resist proteolytic cutting, thus makes fusion rotein keep complete in vivo.In other embodiments, catenation sequence is designed to be cut under suitable environment, the sour environment of such as endocytic vesicles or proteolytic environment.
As demonstration, the present inventor obtains proinsulin-transferrin fusion protein, and demonstrates this fusion rotein and can be converted into Insulin-Transferrin in liver cell culture thing.Different from the proinsulin of non-activity, proinsulin-transferrin fusion protein with hepatocyte incubation after, there is the Glucose transport activity that higher gluconeogenesis is active and equal compared with activated insulin.Therefore, the fusion rotein of embodiments of the present invention can be used as prodrug.
Now further illustrate the present invention with reference to the detailed description of the invention shown in following examples and accompanying drawing.It should be understood that and provide following examples to be to show and further illustrating some embodiment of the present invention and aspect, and should not be construed as and limit the scope of the invention.Although just concrete illustrative embodiments and embodiment describe the present invention, be understood that, only for illustration purposes, those skilled in the art can make various modifications and variations when not deviating from the spirit and scope of the invention illustrated by claims to embodiment disclosed herein.
Embodiment
Embodiment 1
the expression of PI-Tf recombination fusion protein and sign
Utilize molecular cloning, will with preproinsulin sequence (NM_000207) through engineering approaches of Tf sequence (NM_001063) frame endomixis to (Fig. 1) in pcDNA3.1 (+) expression vector (Invitrogen, CA).By the DNA transfection of polyethyleneimine, will the plasmid transient transfection of preproinsulin-Tf fusion gene be comprised in HEK 293 cell.Collect the conditioned medium of serum-free, use laboratory scale tangential flow filtration system (Millipore, MA) to be concentrated, then use Centricon (Millipore, MA) to carry out ultrafiltration.Use anti-Tf antibody (Sigma, MO) and glucagon (former) antibody (Abeam, MA), by Western blotting, sign is carried out with quantitative to PI-Tf fusion rotein.Anti-Tf and glucagon (former) Western blotting demonstrate exists the main band that molecular weight is about 89kD, and this shows that PI-Tf fusion rotein is successfully expressed and is secreted in culture medium.Utilize Xho1 Restriction Enzyme to cut site, Leu-Glu dipeptide sequence is imported between proinsulin and Tf.The Tf shown in 3rd road of Fig. 2 from original serum-free cell culture medium CD 293 (Invitrogen), instead of is produced by the HEK293 cell of transfection, and dipeptides junctional complex keeps stable in production process.
Embodiment 2
pI-Tf fusion rotein in H4IIE hepatoma carcinoma cell strengthens the inhibitory action of Hepatic glucose production
Rat liver cancer H4IIE cell is cultivated in the high glucose DMEM comprising 10% hyclone.After converging, with different drug treating cell 24 hours at 37 DEG C.With phosphate buffered saline (PBS) washed cell 2 times.By by be supplemented with 2mM Sodium Pyruvate and 40mM LD-sodium lactate serum-free, generate culture medium add in cell without glucose, the glucose that form without phenol red DMEM, then incubation 3 hours.Results supernatant, and carry out measure glucose concentration [5] with Amplex Red means of glucose/glucose oxidase test kit (Invitrogen, CA).Cell lysis in 1M NaOH, determines protein content with BCA (Thermo Scientific, IL).
In glucose generation, proinsulin and insulin illustrate suitable inhibition, its IC
50value is respectively 1441.3 ± 641.6pM and 1093.9 ± 105.6pM (Fig. 3 A).Proinsulin is combined with Insulin receptor INSR, but its binding affinity is starkly lower than insulin [6].But insulinogenic higher stability makes its degradation rate lower, this can cause the activity similar to insulin in 24 hours incubations measure.The IC of PI-Tf fusion rotein
50value is 4.60 ± 5.78pM, and itself and proinsulin compare the stronger activity creating about 300 times with insulin.But, proinsulin/insulin and Tf etc. molar mixture do not resemble fusion rotein and significantly improve inhibit activities.Show thus, the inhibitory action of enhancing is owing to this two-part fusion.The common incubation of fusion rotein and excessive Tf (1000 times) can block the inhibitory action strengthened, thus makes activity be down to insulin and insulinogenic level.But, during with excessive albumin incubation, do not observe blocking effect (Fig. 3 B).Therefore, Tf part and proinsulin are merged into a single fusion rotein, significantly can strengthen insulinogenic hepatic glucose rejection ability.
Embodiment 3
pI-Tf is converted into insulin-Tf fusion rotein by hepatoma carcinoma cell
In DMEM culture medium with PI-Tf fusion rotein process rat liver cancer H4IIE cell (ATCC, VA), and at 37 DEG C incubation.Do not collecting culture medium in the same time, and insulin specific radioimmunoassays mensuration and proinsulin specific radioimmunoassays mensuration (Millipore, MA) are being carried out to it.Based on the standard curve from radioimmunoassay, obtain the concentration of proinsulin and insulin.Process at the most after 24 hours in H4IIE cell, continue to create the material containing insulin in the sample through the process of PI-Tf fusion rotein, but in the sample through proinsulin process, be far from it (Fig. 4).The material containing insulin that display produces is insulin-Tf but not the insulin discharged, this is because these two parts are connected by stable peptide bond.Through estimation, the transformation efficiency of PI-Tf to insulin-Tf is 8.8% when PI-Tf dosage is 10nM, is 21.6% when PI-Tf dosage is 1nM.These results prove, prohormone fusion rotein PI-Tf can be converted into insulin-Tf in hepatoma carcinoma cell.Also show, this liver conversion process is subject to the mediation of Tf, appears at recirculation zone in born of the same parents indoor during inferring its endocytosis in TfR mediation and recirculation.
Embodiment 4
in H4IIE hepatoma carcinoma cell to the pretreatment of PI-Tf fusion rotein cause receptors bind affinity increase and to Portugal
the stimulation of grape sugar transport strengthens
Use H4IIE hepatoma carcinoma cell to carry out Insulin receptor INSR and combine mensuration [7].With the unmarked fusion rotein process cell 2 hours of [125I]-TyrA14 insulin (Perkin Elmer, MA) and variable concentrations at 4 DEG C.With phosphate buffered saline (PBS) washed cell 2 times, and with 0.1N NaOH cell lysis under room temperature.The radioactivity of total cell lysate is measured by gamma counter.Measure with BCA and determine protein content.
Compared with insulin, proinsulin show about 100 times low with the binding affinity of the Insulin receptor INSR in H4IIE hepatoma carcinoma cell.In order to confirm that the liver of PI-Tf to insulin-Tf transforms, measure the binding affinity through the pretreated PI-TF fused cell of H4IIE.In brief, first with PI-Tf process cell 1 hour at 37 DEG C or 4 DEG C, at 4 DEG C, balance 15 minutes subsequently, then add [I25I]-TyrA14 insulin.Subsequently at 4 DEG C Incubate cells 2 hours to make in conjunction with abundant.Compared with untreated fusion rotein, at 37 DEG C, pretreated PI-Tf fusion rotein solution has shown the binding ability (Fig. 5) significantly improved.This shows, the combination of this increase may be because carry out the insulin-Tf produced in pretreated process in hepatoma carcinoma cell.In addition, for PI-Tf pretreated at 4 DEG C, significant change is not observed.These data show, liver is converted into insulin-Tf and is undertaken by the protease on cell membrane, but need cellular uptake to carry out the short processing of endocellular enzyme to make PI-Tf fusion rotein.Think that the endocytosis that internalization process is mediated by Tf-TfR and recirculation promote.
Known insulin can promote the picked-up of glucose in muscle and fatty tissue.In order to test PI-Tf and whether have activity through the pretreated PI-Tf of H4IIE in glucose uptake stimulates, the adipose cell of use differentiation as discussed previously establishes glucose uptake and measures [8].In brief, with hormone intermixture induction preadipocyte (the Mus 3T3-L1 fibroblast) differentiation be made up of bovine insulin, dexamethasone and 3-isobutyl-1-methylxanthine.After 10 ~ 14 days, cell reaches and breaks up completely.Make adipose cell serum starvation on pretreatment.In Krebs-Ringer phosphate (KRP) buffer being supplemented with 0.1% bovine serum albumin, by cell and different medicine incubation 30 minutes.Glucose uptake is measured by adding 2-deoxidation-D-[2,6-3H] glucose (Perkin Elmer, MA).By suction, reaction is stopped after 10 minutes, with ice-cold KRP buffer solution cell 4 times.Lysis is made with the KRP containing 0.1M NaOH/0.1%SDS.Quantifying radioactive is carried out by flicker numeration.For measuring with BCA the protein content measured, result is normalized.For the PI-Tf process in H4IIE cell, be that the solutions for administration of the fusion rotein of 10nM is to H4IIE cell by dosage.Incubation is after 24 hours, centrifugal protein solution, collects supernatant to assess its glucose uptake stimulating activity in adipose cell.
In glucose uptake, insulin has shown strong stimulation, and its EC50 value is 2nM, and insulinogenic activity is much lower (Fig. 6 A) then.This is because the binding affinity of proinsulin and Insulin receptor INSR is much lower.Similar to proinsulin, in the glucose uptake of 30 minutes, PI-Tf fusion rotein also shows low stimulating activity (Fig. 6 B).But, when being used in PI-Tf fusion rotein pretreated in H4IIE cell to process adipose cell, show and the glucose uptake (Fig. 6 C) significantly increased compared with pretreated PI-Tf in blank well under same experimental conditions.This result proves, the insulin-Tf that hepatoma carcinoma cell transforms has biologic activity in stimulation glucose uptake.Therefore, liver pretreatment can transform fully and activate PI-Tf fusion rotein.These data show, can apply PI-Tf fusion rotein as the prodrug of being treated diabetes by invasive or non-invasive delivery approach.
Embodiment 5
the prolongation of plasma half-life in the body of proinsulin-Tf fusion rotein
By the 0.5mg/kg proinsulin-Tf of single dose or the intravenous injection of 0.053mg/kg proinsulin in CF-1 mice.Proinsulin-Tf and insulinogenic plasma concentration is measured with proinsulin specificity RIA (Millipore, MA).Data are shown in Figure 7 available from 4 mices.
Pharmacokinetics in body.Before drug administration, make male CF-1 mice (6 ~ 7 week age) fasting 6 hours.Proinsulin-the Tf of intravenous injection single dose or proinsulin.Blood sample is not being taked in the same time by saphena.Whole blood is mixed with heparin, and collected by centrifugation blood plasma.Use proinsulin-Tf and proinsulin as standard curve respectively, determine proinsulin-Tf and insulinogenic plasma concentration by proinsulin specificity RIA.
Embodiment 6
proinsulin-Tf fusion rotein continue, strengthen body in effect of lowering blood sugar
To the PBS of the identical molar dose of diabetic mice single subcutaneous injection of inducing through STZ, insulin, proinsulin or proinsulin-Tf fusion rotein.Mice fasting is made in experimentation.OneTouch blood-glucose meter is used to measure blood glucose levels.All moment all represent the hourage after injection.Data are expressed as relative to 0 hour the blood-glucose percent of (the initial blood glucose level before injection).Fig. 8 and following table summarize experimental result.Data representation meansigma methods ± standard deviation (N=3 ~ 5).
Effect of lowering blood sugar in body.To male C 57 BL/6 J mouse (6 ~ 7 week age) single intraperitoneal injection 175mg/kg streptozotocin.Inject latter 6 days, mice becomes diabetic, and fasting blood glucose levels is about 500mg/dl.Make diabetic mice fasting 2 hours, then single subcutaneous injection albumen.Not in the same time from tail vein blood.OneTouch blood-glucose meter is used to measure blood glucose levels.
Embodiment 7
pressing down to the blood glucose levels under fasted conditions after processing with proinsulin-transferrins (ProINS-Tf)
prolonged action processed
It is the vital approach making glucose homeostasis keep normal blood glucose levels by the Hepatic glucose production (HGP) of glycogenolysis approach and gluconeogenesis approach.Under fasting and starvation conditions, the main source of glucose in blood is by HGP (Fig. 9 A).Therefore, in order to assess the impact of proinsulin-Tf on HGP, the blood glucose levels in long-time after have evaluated injection.Processing male C57Bl/6 mice (6 ~ 7 week age) with single intraperitoneal injection 150mg/kg streptozotocin (STZ), is that the mice of about 500mg/dl is thought diabetogenous by fasting blood glucose levels.To mice single subcutaneous injection buffer control (PBS), insulin (INS), proinsulin (ProINS) or proinsulin-Tf, after application do not measure blood glucose levels in fasted condition in the same time.As shown in Figure 9 B, the blood sugar decreasing effect of proinsulin-Tf raises gradually, maximum efficiency occurs 4 hours after injection, (namely it remain on blood glucose normal level, similar to the mice induced without STZ), and last till tested last 12 hour moment (compared with PBS, have dropped 72% ~ 77%).The blood glucose levels of proinsulin and insulin is respectively from using latter 8 hours with 10 hours compared with PBS group without significant difference.Therefore, the digital proof shown in Fig. 9 B, as indicated in extend the inhibitory action of the blood glucose levels under fasted conditions, proinsulin-Tf suppresses HGP specifically.
Embodiment 8
proinsulin-Tf is on the impact of HGP enzyme level
In order to assess the impact of proinsulin-Tf on HGP, determine the mRNA level in-site of the G-6-Pase (G6Pase) in the STZ mice processed through proinsulin-Tf.G6Pase is the key enzyme of the final step of catalysis HGP approach.With proinsulin-Tf or buffer control treatment S TZ mice 12 hours, and RT-PCR is used to determine G6Pase expression in liver homogenate.Result shows, and the G6Pase in the mice that proinsulin-Tf processes expresses and is only about 10% in control mice, shows to create suppression to HGP.
List of references
Following citing document is incorporated to herein by quoting.
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Claims (12)
1. by prodrug targeted delivery to the method for the liver of study subject, described method comprises:
Use the fusion rotein forming described prodrug, described fusion rotein has the first delivery configuration territory be connected with the second amyloid protein precursor domain by catenation sequence,
Wherein, described first delivery configuration territory be can in vivo targeted delivery to the albumen of liver, and promote enter hepatocyte by endocytic pathway.
2. fusion rotein as claimed in claim 1, wherein, described first delivery configuration territory is transferrins.
3. fusion rotein as claimed in claim 2, wherein, described second amyloid protein precursor domain is proinsulin.
4. reduce a method for the Hepatic glucose production of study subject, described method comprises:
Described study subject is used to the fusion rotein of effective dose,
Wherein, described fusion rotein comprises the proinsulin be connected with transferrins domain.
5. method as claimed in claim 4, wherein, described catenation sequence is Leu-Glu dipeptides.
6. form a method for the fusion rotein being used as prodrug, described method comprises:
For amyloid protein precursor selects the albumen being used as delivery configuration territory;
Carrier construction, the described delivery configuration territory that described vector encoded is connected with described amyloid protein precursor by the catenation sequence be applicable to; With
Described fusion rotein is expressed in suitable expressive host.
7. method as claimed in claim 9, wherein, described delivery configuration territory is transferrins.
8. method as claimed in claim 9, wherein, described amyloid protein precursor is proinsulin.
9. method as claimed in claim 9, wherein, described catenation sequence is Leu-Glu dipeptides.
10. by amyloid protein precursor prodrug delivery to the method for the liver of study subject, described method comprises:
Before being applied in described study subject by described amyloid protein precursor, described amyloid protein precursor is conjugated to transferrins domain,
Wherein, described transferrins domain serves as delivery configuration territory, and described amyloid protein precursor is the amyloid protein precursor be converted into activated protein in liver after with therapeutic activity.
11. methods as claimed in claim 10, wherein, described amyloid protein precursor is that prohormone or the factor are former.
12. methods as claimed in claim 11, wherein, described amyloid protein precursor is proinsulin.
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| US13/476,812 | 2012-05-21 | ||
| US13/476,812 US10513563B2 (en) | 2010-07-02 | 2012-05-21 | Method for uses of proinsulin transferrin fusion proteins as prodrugs |
| PCT/US2013/041867 WO2013177063A1 (en) | 2012-05-21 | 2013-05-20 | Uses of protein precursors as prodrugs |
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|---|---|---|---|---|
| CN109180819A (en) * | 2018-09-14 | 2019-01-11 | 苏州康宇生物科技有限公司 | A kind of preparation method of insulin derivates |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101128587A (en) * | 2003-12-23 | 2008-02-20 | 诺维信达尔塔有限公司 | Gene expression technique |
| WO2012003398A1 (en) * | 2010-07-02 | 2012-01-05 | University Of Southern California | Method for uses of protein precursors as prodrugs |
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|---|---|---|---|---|
| CN101128587A (en) * | 2003-12-23 | 2008-02-20 | 诺维信达尔塔有限公司 | Gene expression technique |
| WO2012003398A1 (en) * | 2010-07-02 | 2012-01-05 | University Of Southern California | Method for uses of protein precursors as prodrugs |
Non-Patent Citations (1)
| Title |
|---|
| YAN WANG等: ""Receptor-mediated activation of a proinsulin-transferrin fusion protein in hepatoma cells"", 《JOURNAL OF CONTROLLED RELEASE》 * |
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|---|---|---|---|---|
| CN109180819A (en) * | 2018-09-14 | 2019-01-11 | 苏州康宇生物科技有限公司 | A kind of preparation method of insulin derivates |
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