CN101011237A - Pharmaceutical in vivo dynamics characteristic-nondestructive in situ monitoring system and monitoring method - Google Patents
Pharmaceutical in vivo dynamics characteristic-nondestructive in situ monitoring system and monitoring method Download PDFInfo
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Abstract
The invention relates to a system used in the internal dynamic nondestructive online checking of protein polypeptide drug, and relative checking method. The inventive system comprises a fluorescence probe and a detecting system, while the detecting system is formed by a near-infrared light source, a transmission optical fiber, a receiving optical fiber, a near-infrared high-pass filter, and a near-infrared detector. The laser of light source and the fluorescence light received by the detector can be transmitted by the optical fiber; the near-infrared band-pass or high-pass filter is at the front of detector. The invention can avoid sampling timely in the test, avoid killing animals in the organism distribution test, to and avoid the following sample treatment.
Description
Technical field
The present invention relates to medicine and survey the field, be specifically related to a kind of system and detection method thereof that is used for the detection on the throne of protein and peptide drugs body internal dynamics characteristic-nondestructive in health check-up.
Background technology
The physiologically active of protein and peptide drugs is strong, therapeutic index is high, plays an important role in keeping the body normal function.And the research of pharmaceutical in vivo dynamics process is requisite important step in any medicament research and development process.At present, the detection method of protein and peptide drugs roughly can be summarized as four kinds of immunological detection method, isotopic labeling tracer method, physico-chemical analysis technology and biologic assays in the body.Except that tracer method, other three kinds of detection methods all can not realize real-time monitoring on the throne, need to detect supervisor through timing sampling, sample treatment, analytical tool, and operating process is extremely loaded down with trivial details, and often causes the generation of sample contamination and extra error.The more important thing is and to reflect medicine real-time process in vivo.
Although at present to mostly using the radiolabel method in the research of biomacromolecule tissue distribution, and it has higher sensitivity, accumulates for a long time and can cause damage to organ-tissue.And near-infrared light waves does not have any injury to biological tissue, and its spectrum and image detecting technique can realize dynamic characteristic to be measured in the harmless real-time monitoring bio body on the throne.Further, the near-infrared window has been avoided the absorption of a lot of endogenous material, and the penetration depth in biological tissue is bigger, and its excited fluorescent is subjected to the influence of biological tissue's background less, so can detect the near infrared signal that is produced in the deep tissues.
Along with people to near-infrared light waves the understanding in depth of transmission characteristic in biological tissue, its harmless peculiar advantage of real time monitoring on the throne is fully utilized at biomedical sector, various near-infrared system occurs in succession, mainly show various spectroscopic systems (US6456862, US6594513, US7072701) that carry out hemodynamic parameter monitoring in the body and the picture system that carries out diagnosing tumor (US6615063, US7011817).Spectroscopic system mainly is to monitor at endogenous material such as the more rich blood of in-vivo content, and the absorption characteristic of utilizing multi-wavelength is different to be measured to distinguish.And the array that picture system is often formed by light source scanning or optical fiber is realized the function of multiple light courcess-multi-detector, obtains image reconstruction through complicated algorithm then.More perfect on these Systems Theorys, measuring range is wider, but its detection sensitivity and spatial resolution are difficult to apply in the drug monitoring that distributes in the body seldom.So develop simple near infrared spectrum and image detecting technique research pharmaceutical in vivo dynamics characteristic has actual application value and DEVELOPMENT PROSPECT admirably.
Because most drug does not all have tangible near infrared light spectral property, so need medicine to be measured is carried out the nir dye labelling.In recent years, using maximum nir dyes is the heterocyclic compound that a class is called as the polymethine cyanine dyes, the big pi-conjugated system that it is made up of two ends or intermediary heterocycle, aromatic ring and intramolecular polymethine chain, absorption and emission spectra all are near infrared region (650～1200nm), molar absorption coefficient is big, fluorescence quantum yield is high, is the good reagent that detects in the body.In actual applications, near-infrared cyanine dye except with (Tang Bo, Huang Hui, Xu Kehua etc., CN 1844119A some micromolecule or high polymer combine; Ntziachristos V, Weissleder R, US 6615063B1; Li C, Tansey W, Ke S, et al.JControl Res, 2004,94:39-51), most of research concentrates on the application in the detection in vivo and in vitro of the labelling of biomacromolecules such as protein polypeptide or nucleic acid and these labeled complexs.Have different effects with different macromole in conjunction with nir dye, comprising the following aspects:
1) with after the protein binding cyanine dyes protein binding can be used for albumen in the fluorescence polarization method test sample (middle mountain is great, the sincere .CN1237242A of Miyazaki's core), the synthetic (Wang Kemin of fluorescent nano particle, Tan Weihong, He Xiaoxiao .CN1654592A) and proteinic non-covalent detection (Zheng Hong, Li Donghui, Mao Yuxia .CN1401988A)
2) combine with specific antibody: in the research of cyanine dyes labelled protein, the Biofunctional material that begins at first to relate to is an antibody.It combines the available fluorescence optical fiber immunosensor (FFOI in back with IgG, fluorescent fiber-optic immunosensor) investigates antigen antibody reaction, connect specific monoclonal antibody fragment now, can carry out immunofluorescence to the animal whole body develops, analyze fragment distribution metabolism situation (Daneshvar M I, Peralta J M, Casay G A.et al.J ImmunolMethods in vivo, 1999,226:119-128; Ramjiawan B, Maiti P, Kaplan H, et al.VibrationalSpectroscopy, 2002,28:177-188.).Wherein the IRDye series dyes further has been applied in the In-Cell Western analysis.(Matheny?S?A，Chen?C?Y，Kortum?R，et?al.Nature，2004，427，256-260)。
3) combine with specific polypeptide: in diagnosing tumor and treatment, mainly contain two kinds with the bonded polypeptide of cyanine dyes derivant: the first is at receptor (the Ichinose J of tumor surface overexpression, Morimatsu M, Yanagida T, et al.Biomaterials, 2006,27,3343-3350), especially utilize the synthetic ring-type fluorescent polypeptide of the self structure complex of polypeptide or dyestuff, in vivo than stable more (the Houston J P of straight-chain polypeptide, Ke S, Wang W, et al.J Biomed Opt.2005,10,054010.); Another kind then at the tumor relevant enzymes (Weisssleder R, Mahmood U, Tung C H, et al.Radiology, 1999,213:866-870.).
4) combine with nucleic acid: 5 ' end by labeling nucleic acid, can read out DNA sequence by four colors or Two Colour Fluorescence dyestuff, improved the sensitivity (Shealy DB, et al.Anal Chem, 1995,67,247) of nucleic acid sequencing greatly.In addition, in the target position research of PNA in cell tissue, the dyestuff of cy series also plays pivotal role (Rigby S, Keefe HP, Stender H.US6905824B2).
In the described in the above application, cyanine dyes and the bonded main mode of protein polypeptide have non-covalent the combination and two kinds of covalent bond.Non-covalent combination generally is used for vitro detection, as the pre-column derivatization of protein quantification or biological sample.Because this combination is affected by environment big, stability is used less not as good as the latter in vivo in the fluorescence monitoring.And the research of covalent bond mode is deep relatively, be primarily aimed at hydroxyl, amino or sulfydryl in protein, polypeptide, DNA or the other biological molecule, reach the purpose of coupling protein by the active group that changes nir dye, combine (Salama G as iodoacetyl with sulfydryl in the albumen, Waggoner A S, Abramson J.Biophysical Journal, 1985,47,456a.); The isothiocyanate that activated carboxylic forms (NCS), N-hydroxy-succinamide active ester groups such as (NHS ester) combine (FlanaganJ H Jr, Khan S H, Menchen S with amino or sulfydryl in the macromole, et al.Bioconjug Chem, 1997,8,751-756; Heavy rattan is practiced Buddhism or Taoism, and Miyazaki's core is sincere, the great .CN1238990 in middle mountain).The product of these covalent coupling modes is relatively stable, further extracts purification but need all before the labelling that the cyanine dyes original structure is modified the back, is not suitable for the labelling of dyestuff in a small amount, and molecular weight is lower than 1000 little peptide, and the method related application seldom.Samuel etc. introduce the nir dye cytate with two carboxyls when solid-phase synthetic peptide, make two free amino group condensations on itself and the peptide chain form cyclic octapeptide (Achilefu S, Ye Y P, Li W P, et al.J Am ChemSoc, 2003,125,7766-7767).Yet the prerequisite of this method is a polypeptide to be fixed on the resin, and excessive dyestuff just can be used the organic reagent eluting, and it is not suitable for albumen or polypeptide is the situation of solution state.Therefore, provide the new method that a kind of labeling process is simple, be fit to micro-dye marker, have more potentiality in actual applications.
Summary of the invention
The object of the present invention is to provide the system and the detection method of DYNAMIC DISTRIBUTION in a kind of harmless real-time monitoring protein and peptide drugs body on the throne.This system comprises fluorescent probe and monitoring system, monitoring system of the present invention is made up of near-infrared light source, Transmission Fibers, near-infrared high-pass filter, near infrared detection device, the fluorescent emission that the exciting light that light source sends and detector are accepted all can pass through fiber-optic transfer, and near-infrared high-pass filters sheet is between detector and determinand.
It is bigger that present fluorescent material and small-molecule drug are compared molecular weight, may influence intravital distribution of small-molecule drug and metabolic process behind the labelling.And the protein and peptide drugs molecular weight is bigger usually, and the group of many suitable mark fluorescent materials is arranged on the molecule.The selected fluorescent material of the present invention preferably has the chemical substance of higher fluorescence quantum efficiency near infrared region, by optimizing each reaction condition, make labeling process simple, and labeling effciency meets the demands, and the pharmaceutically active behind the labelling and stability meet the requirements.
The used near-infrared system of the present invention comprises the laser instrument that is used for fluorescence excitation material generation fluorescence, the exciting light of preferred 700-900nm wavelength; Exciting light transmits by fibre bundle; High-pass filter is to enter detection system by incident illumination; The near infrared detection device comprises the detection system with photomultiplier tube and has the detection imaging system of CCD.Wherein, the detection system with photomultiplier tube is used for the detection real-time on the throne of local organization medicine DYNAMIC DISTRIBUTION; The CCD image-forming detecting system is used to monitor the tissue distribution of medicine.Used components and parts all are fixed on the heavier trestle table, and the position of each element of scalable.The trestle table outside is covered with a lucifuge camera bellows.In the process of the test determinand is put into case by wicket, shut and carry out fluorescence excitation behind the wicket and detect, make whole measuring process keep stable.
Local organization detection by quantitative: be fixed on the clamping plate after the Animal Anesthesia.The laser that light source sends projects certain particular organization position of tested animal health by Transmission Fibers, and the fluorescence that send at this position is transferred to the photomultiplier tube detection system that has the high pass light filter by accepting fibre bundle.Transmission Fibers with accept optical fiber and all contact closely with the epidermis at this position, and its relative position can be regulated arbitrarily and makes system reach optimum detection sensitivity.Curve can be obtained by calibration curve by the fluorescence signal that detects during medicine.See Fig. 2.
Medicine body tissue distributes and monitors: fluorescently-labeled protein and peptide drugs places imaging under the CCD system rapidly after entering in the experimental animals by various route of administration.The laser that light source sends projects the tested animal body surface equably by Transmission Fibers, parts of body sends ground fluorescence and enters the CCD imaging system after by the high pass light filter, regulate CCD position, focal length and light billows aperture, make the image resolution ratio that obtains reach optimum state.Simultaneously in order locate and distinguish the tissue location that produces fluorescence, utilize the wavelength can be through the low-light uniform irradiation tested animal of optical filter, the adjusting luminous flux makes to be seen the tested animal profile substantially clearly and is as the criterion.See Fig. 3.
The invention has the advantages that: the optical monitoring system that dynamic characteristic in a kind of harmless real-time monitoring protein and peptide drugs body on the throne is provided.Compare with the pharmacokinetic method of routine relatively, this method need not at the experimentation timing sampling, does not also need to put to death as conventional method many animals in the tissue distribution assays; Simultaneously also saved sample subsequent processes a large amount of in the conventional method.
On the other hand, the invention provides a kind of protein polypeptide of carboxyl nir dye labelling and the synthetic method in solution thereof.One of purpose of the present invention is the protein polypeptide fluorescent marker of synthetic a kind of "dead" pollution, another purpose is the step of province's decarboxylate dye marker front activating purification and realizes carboxyl dyestuff labeling polypeptide in solution that three of purpose is to optimize the dye marker condition to realize that sxemiquantitative is synthetic.
The feature of the protein polypeptide of nir dye labelling is: by nir dye and albumen or polypeptide be combined into, absorption and fluorescence emission peak are arranged in the near infrared region.The carboxyl of dyestuff nitrogen end and the free amino group in the protein polypeptide form covalent bond under the condition that catalyst exists, thus with the protein polypeptide coupling.
The catalyst of selecting for use in the preparation method of nir dye labelled protein polypeptide can be N, N '-dicyclohexylcarbodiimide (DCC), I-hydroxybenzotriazole (HOBt), 2-(1-hydrogen-1-benzotriazole base)-1,1,3,3-tetramethylurea hexafluorophosphoric acid (HBTU), hexafluorophosphoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus (PyBOP), 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) or their mixture in twos.Select for use different catalysts tense marker method to be divided into two kinds: two step successive reaction methods and a step direct reaction method.
Two step successive reaction methods are applicable to the catalyst system and catalyzing that comprises carbodiimide class (DCC, EDC etc.), and concrete steps are:
A) dyestuff activation: dyestuff and equimolar DCC or EDC are dissolved in the anhydrous dimethyl sulfoxide (DMSO), after the vortex mixed, in this solution, drop into second kind of catalyst (as PyBOP, HoBt etc.), stirring reaction again;
B) protein labeling: the activatory suspension of a step is got supernatant after centrifugal, jolting limit, limit slowly is added dropwise to albumen or polypeptide solution, the mass ratio that makes dyestuff and biomacromolecule is 1: O.3 between 1: 10, and both mixing afterreactions, preferably reaction 4-6 hour under 4 degree conditions;
C) purification of labeled complex: behind the reactant liquor mixing, in the PBS buffer, dialyse the lyophilizing of gained solution.And a step direct reaction method is adopted in the reaction that does not have carbodiimide class catalyst to participate in, concrete steps are: with nir dye and equimolar catalyst dissolution in anhydrous DMSO, after the vortex mixed, jolting limit, limit slowly is added dropwise to protein polypeptide solution, mixing, reaction, preferably stoichiometric number 6 hours under 4 degree conditions.The purification process of labeled complex is with " two step successive reaction methods ".
The protein polypeptide solution pH value that wherein participates in reaction is preferably between 8.0 to 10.0.
The present invention has following characteristics:
1. the protein polypeptide of nir dye labelling is "dead", has solved the infringement problem of isotopic labeling to biological tissue's organ.
2. the absorption of the protein polypeptide of nir dye labelling and fluorescence emission peak have reduced the ambient interferences of monitoring in the body in the near infrared region.
3. the protein medicaments of nir dye labelling can keep most of active, and before stability is better than labelling.
4. carboxylic near-infrared cyanine dye does not further become the form of isothiocyanate (NCS), N-hydroxy-succinamide active ester (NHS ester) isoreactivity ester, directly by the catalyst coupling protein.
5. micro-carboxyl dyestuff is not suitable for the technology of purifying in the activation back, the invention provides a kind of simple and feasible micro-reaction approach.
6. by optimizing catalyst system and catalyzing and reaction condition, realized the sxemiquantitative of dye marker, mark rate and traditional method are suitable.
7. polypeptide combines with nir dye after being formulated as solution, does not need to begin or be fixed on holder to react from solid phase synthesis.
Description of drawings
Fig. 1 is the absorption and the fluorescent emission wavelength of used nir dye among the present invention, and 1 is absorption curve, and 2 is the fluorescent emission curve
Fig. 2 is that detector is the near-infrared monitoring device with photomultiplier tube.
Fig. 3 is the imaging monitoring apparatus with CCD
Fig. 4 is cypate labelling insulin curve chart when the medicine that the mice shank records
Fig. 5 is that (wherein A is back 30 seconds of injection to the interior scattergram of the body of different time behind the cypate labelling insulin tail vein injection mice; B is back 1 minute of injection; C is back 2 minutes of injection; D is back 11 minutes of injection; E is back 90 minutes of injection; F is back 11 hours of injection)
Fig. 6 is the feces imaging between the 11h to 19h behind the injected in mice cypate labelling insulin
Fig. 7 is that (wherein A is back 4 minutes of injection to the interior scattergram of the body of different time behind the cypate labelling lysozyme tail vein injection mice; B is back 6 minutes of injection; C is back 9 minutes of injection; D is back 140 minutes of injection; E is back 270 minutes of injection; F is back 24 hours of injection)
Fig. 8 be in the body of different time behind the cypate labelling reorganization altheine enzyme tail vein injection mice scattergram (A is back 1 minute of injection; B is back 5 minutes of injection; C is back 18 minutes of injection; D is back 35 minutes of injection; E is back 130 minutes of injection; F is back 23 hours of injection)
Fig. 9 is the fluorescence imaging of injection back 24h chorista organ
The specific embodiment
Embodiment 1
The synthetic method of cypate labelling insulin
1mg cypate (spectrum is seen Fig. 1) is dissolved among the anhydrous DMSO, to wherein adding 0.4mgHBTU and 0.3mgHOBT, behind the airtight vortex mixing of lucifuge, this jolting limit, activating solution limit slowly is added dropwise in the insulin solutions (pH8.5) of 4ml 1.25mg/ml, stirring reaction is 6 hours under 4 degree.Behind the reactant liquor mixing, dialysis is two days in PBS buffer (pH7.4), changes one time dialysis solution in per 4 hours.The lyophilizing of gained solution is placed on-20 ℃ of refrigerator cryopreservation.Cypate is 0.57: 1 to the mark rate of insulin as calculated.
The synthetic method of cypate labelling lysozyme
1mg cypate is dissolved among the anhydrous DMSO, and to wherein adding 0.4mgDCC, after the vortex mixed, jolting was reacted 30 minutes.In this solution, drop into 0.3mgHOBT again, stirring reaction 2 hours, the centrifugal supernatant that goes is the dyestuff activating solution.This jolting limit, activating solution limit slowly is added dropwise in the lysozyme soln (pH9.2) of 4ml 1.25mg/ml, stirring reaction is 6 hours under 4 degree.Behind the reactant liquor mixing, dialysis is two days in PBS buffer (pH7.4), changes one time dialysis solution in per 4 hours.The lyophilizing of gained solution is placed on-20 ℃ of refrigerator cryopreservation.Dyestuff cypate is 1.94: 1 to the mark rate of lysozyme after calculating.
Embodiment 3
The synthetic method of cypate labelling reorganization L-Asnase
1mg cypate is dissolved among the anhydrous DMSO, to wherein adding 0.4mgHBTU and 0.3mgHOBT, behind the airtight vortex mixing of lucifuge, this jolting limit, activating solution limit slowly is added dropwise in the L-Asnase solution (pH8.5) of 4ml 1.25mg/ml, stirring reaction is 6 hours under 4 degree.Behind the reactant liquor mixing, dialysis is two days in PBS buffer (pH7.4), changes one time dialysis solution in per 4 hours.The lyophilizing of gained solution is placed on-20 ℃ of refrigerator cryopreservation.Cypate is 33: 1 to the mark rate of asparaginase as calculated.
The synthetic method of cypate labelling bovine serum albumin
1mg cypate is dissolved among the anhydrous DMSO, and to wherein adding 0.4mgDCC, after the vortex mixed, jolting was reacted 30 minutes.In this solution, drop into 0.3mgHOBT again, stirring reaction 2 hours, the centrifugal supernatant that goes is the dyestuff activating solution.This jolting limit, activating solution limit slowly is added dropwise in the bovine serum albumin solution (pH9.2) of 4ml 1.25mg/ml, stirring reaction is 6 hours under 4 degree.Behind the reactant liquor mixing, dialysis is two days in PBS buffer (pH7.4), changes one time dialysis solution in per 4 hours.The lyophilizing of gained solution is placed on-20 ℃ of refrigerator cryopreservation.The mark rate of per molecule bovine serum albumin is 28: 1.
Embodiment 5
(1) the different pharmaceutical grade protein pharmaceutical grade protein of Cypate labelling has been selected insulin, and lysozyme and reorganization altheine enzyme carry out labelling with cypate under certain condition respectively.Quantitatively, concentration of insulin is 355 μ g/mL before the final injection, and the get on concentration of cypate of labelling is 22.25 μ g/mL; The concentration of lysozyme is 23.1mg/mL, and the get on concentration of cypate of labelling is 15 μ g/mL; The concentration of reorganization altheine enzyme is 145 μ g/mL, and the get on concentration of cypate of labelling is 20.77 μ g/mL.
(2) the equipment laboratory of the near-infrared system near infrared detection system of being assembled comprises two in the laser instrument that is used to produce exciting light, and exciting light is in the NL-FC-2.0-763 of 765.9nm type semiconductor laser, Shanghai Nlight Photonics Corp.; Exciting light is at the laser instrument of 808nm HLU32F400-808, LIMO company; Being used to detect the instrument that fluorescent material produces fluorescence is to have the detection system of photomultiplier tube (Japanese guest pine) and have the detection imaging system of CCD (Princeton), they have very high fluorescence quantum efficiency in the near infrared region, thereby have higher detection sensitivity.Before detection system, add an optical filter to enter CCD, improve the specificity and the system signal noise ratio of fluoroscopic examination by incident illumination.Detection system with photomultiplier tube is used for the detection real-time on the throne of local organization medicine; The CCD image-forming detecting system is used to monitor the DYNAMIC DISTRIBUTION of systemic drug.All appts all is to be fixed on the support, and the outside whole device with a cover cover every light that has wicket, to reduce the influence of ambient light.All added a high flux optical filter before the detector of collection fluorescence, the light that it can stop before the 800nm wavelength enters detector, fluorescent emission wavelength maximum is positioned at 810nm because the absorption maximum exciting light of the fluorescent dye Cypate that we adopt is positioned at 788nm, so optical filter can stop exciting light and surround lighting on every side and enter detector, and only allowing the fluorescence that produces enter, this has just increased the sensitivity of fluoroscopic examination greatly.
(3) the fixing back of mouse anesthesia local organization depilation, with the Transmission Fibers bundle and accept the end alignment animal health tissue to be measured of fibre bundle, the relative position of two fibre bundles can be regulated arbitrarily, makes fluorescence signal reach the strongest.Fluorescence is by accepting optical fiber, and through the laggard detection system of going into the photoelectricity multiplier tube of high-pass filter, detection signal is shown by computer real-time.Behind the mouse mainline cypate labelled protein medicine be the opening entry fluorescence intensity over time.
(4) after depilation mouse mainline cypate labelled protein medicine enters in the body, place imaging under the CCD detection system rapidly.With excitation wavelength is the exciting light of the NL-FC-2.0-763 type semiconductor laser of 765.9nm as fluorescence, the Transmission Fibers head is apart from the oblique upper of mice certain distance, adjust illuminating area and intensity of illumination, regulate the planar angle of excitation line and animal simultaneously and make the fluorescence signal of acceptance reach maximum.The parameter that the CCD imaging system can be regulated is the size in position, focal length and light billows aperture.Regulate these parameters, make the image resolution ratio that obtains reach optimum state.In order to locate and distinguish the tissue location that produces fluorescence, utilize exciting light to illuminate light source simultaneously, regulate light intensity magnitude and be as the criterion can see the mouse profile substantially clearly in the laser instrument conduct of 808nm HLU32F400-808.
One, the body internal dynamics process of cypate labelling insulin
1, be fixed on the support behind the monitoring mouse anesthesia of leg tissue blood drug level, the insulin of tail vein injection 0.1mLcypate labelling, wherein concentration of insulin is 0.355mg/mL, cypate concentration is 22.25 μ g/mL.The optical fiber direction of transmission exciting light and fluorescent emission meets at right angles, and regulates when optical fiber makes fluorescence intensity maximum and fixes, and detects fluorescence signal with the detection system with photomultiplier tube, and shows in real time.The fluorescence intensity of continuous monitoring, curve during by standard curve conversion patent medicine is seen Fig. 4.
2, in the monitoring injection cypate labelling insulin of body tissue's DYNAMIC DISTRIBUTION, concentration of insulin is 0.355mg/mL, and cypate concentration is 22.25 μ g/mL.Get 0.1mL and inject in the body, place CCD near-infrared system imaging immediately by mouse tail vein.Preserve a series of images, selected part explanation result of the test is seen Fig. 5.
By top a series of images as can be seen, begin to occur fluorescence in the liver in the time of back 30 seconds, the fluorescence that produces in the time of also seeing labeled drug through abdominal vascular simultaneously in injection.Injection in the time of back 1 minute in the liver fluorescence strengthen, to injection in the time of back 2 minutes in the liver fluorescence seem stronger, at this moment can't see the fluorescence in the blood vessel.Begin to drain to intestinal by bile duct from liver when injecting back 11 minutes, fluorescence at this moment is very strong.Substantially detect less than fluorescence in the liver in the time of back 90 minutes to injection, and be extensive distribution in intestinal.Inject and only observed fluorescence faint in the intestinal in back 11 hours.Inject and do not observe any fluorescence in the body in back 21 hours.
In order to embody the accuracy of harmless detection on the throne more realistically, untie kidney position on one side from the mouse back operation, do not detect fluorescence, illustrate that medicine does not pass through to renal excretion.Feces after the collection injected in mice between 11 hours to 19 hours places imaging under the CCD system, result such as Fig. 6, and as seen some feces has fluorescence to occur.Illustrate cypate labelling insulin be by bile excretion in intestinal, excrete at last.
Two, the body internal dynamics process of cypate labelling lysozyme
Test is with in the cypate labelling lysozyme, and the concentration of lysozyme is 23.1mg/mL, and cypate concentration is 15 μ g/mL.Extract 0.1mL and inject in the body, place CCD near-infrared system imaging immediately by mouse tail vein.Preserve a series of images, selected part explanation result of the test is seen Fig. 7.
After injection cypate labelling lysozyme entered in the mice body, fluorescence was not very strong in a few minutes of beginning, as fluorescence intensity also is not very strong when injecting back 4 minutes.But the fluorescence in the liver just begins to strengthen subsequently, and is very strong with respect to the front in the time of back 6 minutes to injection.Have faint fluorescence to occur when injecting back 9 minutes in the intestinal of side, this explanation cypate labelling bacteriolyze is eliminated the speed of intestinal into by bile duct in liver very fast.To injection back 140 minutes the time in the liver fluorescence intensity reduce but still have certain intensity, the fluorescence intensity in the intestinal seems very high relatively.Do not have big variation in the time of back 270 minutes to injection, just the fluorescence intensity at liver position has reduced a bit again always.Injecting did not have fluorescence in the intestinal in back 24 hours, but the liver position also has faint fluorescence.Illustrate that cypate labelling lysozyme removes slower in liver.
Three, the body internal dynamics process of cypate labelling reorganization altheine enzyme
In the used cypate labelling reorganization altheine enzymatic solution, the concentration of enzyme is 145 μ g/mL, and the get on concentration of cypate of labelling is 20.77 μ g/mL.The 1mL syringe extracts 0.1mL marker enzyme solution, injects by the tail vein in the body of depilation mice.Place imaging under the CCD system of near-infrared system immediately, preserve a series of pictures.Choose the result of some of them caption test, see Fig. 8.
Inject in back 1 minute and fluorescence promptly occurs in the liver, but fluorescence intensity not very high.Fluorescence intensity in a few minutes subsequently in the liver is being strengthened always, as in the liver that records when injecting back 5 minutes fluorescence intensity ratio to inject in back 1 minute liver fluorescence intensity much higher.Begin occur faint fluorescence in the intestinal in injection in the time of back 18 minutes, can obviously see in the time of 35 minutes getting fluorescence in the intestinal.And at this moment must fluorescence intensity obviously weaken in the liver.To detecting fluorescence at a lot of positions of intestinal 23 hours the time, liver and kidney position have a bit faint fluorescence to occur.
In order to get observed result above proving conclusively, put to death mice in back 24 hours in injection, get organ-tissue: heart, lung, liver, spleen, kidney, stomach, duodenum, small intestinal, colon.Take out concentrated the putting well in back and directly place imaging under the CCD system that has only exciting light, result such as Fig. 9.
Owing to there is not the laser instrument of 808nm to illuminate, it is invisible to produce being organized in the CCD system imaging of fluorescence.Have only duodenum, small intestinal, colonic segment, liver, kidney to have fluorescence to occur among the figure, and the fluorescence in the intestinal is stronger relatively.Have only faint fluorescence in the liver, wherein have some fluorescence stronger relatively, this in vivo in the imaging 23 hours liver positions have some fluorescence to occur being consistent.The kidney fluorescence intensity relatively a little less than.Heart, lung, spleen, stomach do not have fluorescence and occur, and illustrate that these tissues do not have the distribution of cypate labelling reorganization altheine enzyme.The result is identical with the distribution results that detection on the throne draws.
Utilize cypate labelling three hatching egg BAIYAO thing insulins, lysozyme and reorganization altheine enzyme, utilize the near-infrared system to detect intravital dynamic process.Be presented at dynamic process in the blood in monitoring to cypate labelling insulin huckle blood vessel.Coming behind these medicine tail vein injections as can be seen in the test of whole body DYNAMIC DISTRIBUTION all is to focus in the liver soon.It is in injection back 2 minutes that cypate labelling insulin fluorescence intensity in liver arrives maximum; Cypate labelling lysozyme and cypate labelling reorganization altheine enzyme are respectively injection back 6 minutes and 5 minutes.Illustrate that these medicines all will pass through the first pass effect of liver, and much begin in intestinal, to eliminate by bile duct.Cypate labelling insulin is eliminated the time that enters intestinal in liver be in injection back 11 minutes.Cypate labelling lysozyme and cypate labelling reorganization altheine enzyme are respectively injection back 9 minutes and 18 minutes.It is also different to enter the time that the material in the intestinal excretes.Cypate labelling reorganization altheine enzyme is injected and can also be detected stronger fluorescence in back 23 hours in intestinal, cypate labelling lysozyme has detected less than some fluorescence at back 24 hours intestinal positions of injection, cypate labelling insulin the fluorescence at back 11 hours intestinal positions of injection just seem very a little less than, when injecting back 20 hours 30 minutes in the body after testing less than any fluorescence.
Claims (10)
1, a kind of system that monitors DYNAMIC DISTRIBUTION in the protein and peptide drugs body, comprise fluorescent probe and monitoring system, it is characterized in that: monitoring system by near-infrared light source, Transmission Fibers, accept optical fiber, near-infrared high-pass filter, near infrared detection device and form, the fluorescent emission that exciting light that light source sends and detector are accepted all can be passed through fiber-optic transfer, and the near-infrared band leads to or high-pass filter is positioned at before the detector.
2, the system of claim 1, wherein the near-infrared light source wave-length coverage is 700～900nm.
3, the system of claim 1, wherein detector comprises photomultiplier detector or CCD detector, their fluorescence quantum efficiencies in 700～900nm scope are not less than 60%.
4, the system of claim 1, wherein fluorescent probe is a near infrared fluorescent probe.
5, the system of claim 4, wherein near infrared fluorescent probe is prepared by following method: carboxyl and the free amino group in protein polypeptide of nir dye by the nitrogen end forms covalent bond under the condition that catalyst exists, thereby with the protein polypeptide coupling.
6, the method for claim 5, wherein catalyst is selected from N, N-dicyclohexylcarbodiimide, I-hydroxybenzotriazole, 2-(1-hydrogen-1-benzotriazole base)-1,1,3, one or more in 3-tetramethylurea hexafluorophosphoric acid, hexafluorophosphoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus, 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride.
7, the method for claim 5, wherein the protein and peptide drugs of nir dye labelling is prepared by following method:
A) dyestuff activation: with dyestuff and equimolar catalyst dissolution in anhydrous dimethyl sulfoxide, after the vortex mixed, stirring reaction;
B) protein labeling: the activatory suspension of a step is got supernatant after centrifugal, and jolting limit, limit slowly is added dropwise to albumen or polypeptide solution, and the mass ratio that makes dyestuff and biomacromolecule is between 1: 0.3 to 1: 10, both mixing afterreactions;
C) purification of labeled complex: behind the reactant liquor mixing, in the PBS buffer, dialyse the lyophilizing of gained solution.
8, the method for claim 7, wherein the pH value of protein polypeptide solution is between 8.0 to 10.0.
9, the method for DYNAMIC DISTRIBUTION in a kind of harmless real-time monitoring protein and peptide drugs body on the throne comprises:
Earlier nir dye labelled protein polypeptide drug is entered in the experimental animals, then:
A, local organization detection by quantitative: fixing after the Animal Anesthesia, the laser that light source sends projects certain particular organization position of tested animal health by Transmission Fibers, the fluorescence that send at this position is transferred to the photomultiplier tube detection system that has the high pass light filter by accepting fibre bundle, Transmission Fibers with accept optical fiber and all contact closely with the epidermis at this position, and its relative position can be regulated arbitrarily and make system reach optimum detection sensitivity, and curve can be obtained by calibration curve by the fluorescence signal that detects during medicine;
B, medicine body tissue distribute and monitor: fluorescently-labeled protein and peptide drugs is by after entering in the experimental animals, place imaging under the CCD imaging system, the laser that light source sends projects the tested animal body surface equably by Transmission Fibers, the fluorescence that parts of body sends enters the CCD imaging system after by the high pass light filter, regulate CCD position, focal length and light billows aperture, make the image resolution ratio that obtains reach better state, utilize wavelength can see through the low-light uniform irradiation tested animal of optical filter simultaneously, the adjusting luminous flux makes sees the tested animal profile substantially clearly.
10, the system of claim 1 is used for the purposes of DYNAMIC DISTRIBUTION in the harmless real-time monitoring protein and peptide drugs body on the throne.
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| CN102663218A (en) * | 2011-10-28 | 2012-09-12 | 天津大学 | Prediction model of area under the curve (AUC) based on physicochemical property for quinolones antibacterial |
| CN103336128A (en) * | 2013-06-08 | 2013-10-02 | 天津药物研究院 | Detection method of tissue distribution of small molecule polypeptide drugs |
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| EP0533333A3 (en) * | 1991-09-19 | 1993-07-28 | Texaco Development Corporation | Optical photometry system |
| CN2490584Y (en) * | 2000-10-13 | 2002-05-08 | 岳英 | Immune magnet microsphere fluorescent fiber-optical flow analyser |
| US20080195062A1 (en) * | 2005-06-08 | 2008-08-14 | Vanderbilt University | Sampling of blood analytes |
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| CN102663218A (en) * | 2011-10-28 | 2012-09-12 | 天津大学 | Prediction model of area under the curve (AUC) based on physicochemical property for quinolones antibacterial |
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| CN109490274B (en) * | 2019-01-04 | 2023-06-09 | 齐鲁工业大学 | Experimental device for researching unidirectional mass transfer of enzyme in leather and application method |
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