Targeted molecular imaging probe and method for in vivo molecular imaging

Abstract

Disclosed is a targeted molecular imaging probe consisting of a signal component, a component with a targeted affinity to Cx43 and a linker. The biochemical variation characteristic of connexin43 (Cx43) associated with cardiovascular diseases (especially arrhythmia) and neoplastic diseases can be reflected in the form of an image by using the targeted molecular imaging probe capable of being detected by an imaging device, to achieve in vivo molecular imaging.

Claims

1. A process for in vivo molecular imaging, comprising: providing a Cx43 targeted molecular probe consisting of three components: a signaling component, a Cx43 targeted affinity component and a linker, wherein the signaling component is a moiety which is detectable by an imaging device, the Cx43 targeted affinity component is a polypeptide moiety which specifically binds to Cx43, and the linker links the signaling component to the targeted affinity component; carrying out optical imaging, positron emission tomography (PET), single photon emission tomography, magnetic resonance imaging, photoacoustic imaging, or ultrasonic imaging the site to be detected in a patient by using the Cx43 targeted molecular probe; wherein the Cx43 targeted affinity component specifically binds to the carboxyl terminal of Cx43, and the targeted affinity component is Cx43SP1, Gly-Ala-Pro-Gly-4 Hyp-Pro-Tyr.

2. The process according to claim 1, characterized in that the signaling component of the Cx43 targeted probe is one or more selected from the group consisting of radioisotope, fluorescent dye, quantum dot, paramagnetic material, magnetic nanoparticle, super-paramagnetic material, ultrasound microbubble, and photoacoustic nanoparticle.

3. The process according to claim 1, characterized in that the linker is a chelating agent selected from the group consisting of DTPA, DOTA, DOTAGA, NOTA, NODAGA, TETA, CB-TE2A, Sar, and NODA, or a direct chemical reaction is used to directly link the signaling component to the Cx43 affinity component.

4. A Cx43 targeted molecular probe consisting of three components: a signaling component, a Cx43 targeted affinity component and a linker linking the signaling component to the targeted affinity component, wherein the signaling component is a moiety which is detectable by an imaging device, the targeted affinity component is a moiety which specifically binds to Cx43, and the linker links the signaling component to the targeted affinity component, wherein the targeted affinity component specifically binds to the carboxyl terminal of Cx43, and the targeted affinity component is Cx43SP1, Gly-Ala-Pro-Gly-4 Hyp-Pro-Tyr.

5. The targeted molecular probe according to claim 4, characterized in that the signaling component is one or more selected from the group consisting of radioisotope, fluorescent dye, quantum dot, paramagnetic material, magnetic nanoparticle, super-paramagnetic material, ultrasound microbubble, and photoacoustic nanoparticle.

6. The targeted molecular probe of claim 4, characterized in that, the linker is a chelating agent selected from the group consisting of DTPA, DOTA, DOTAGA, NOTA, NODAGA, TETA, CB-TE2A, Sar, and NODA, or a direct chemical reaction is used to directly link the signaling component to the Cx43 affinity component.

7. A method for diagnosing a disease associated with abnormal Cx43 expression in a subject, comprising administering the targeted molecular probe of claim 4 to the subject, wherein the disease is a tumor or a cardiovascular disease.

8. The method according to claim 7, wherein the cardiovascular disease is myocardial ischemia, arrhythmia, cardiac failure, hypertension and atherosclerosis.

9. The method according to claim 7, wherein the disease associated with abnormal Cx43 expression is a tumor.

10. The process of claim 1 wherein PET is selected from PET/CT and PET/MRI.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of the structure of Cx43 targeted molecular imaging probe;

(2) FIG. 2 shows fluorescent micrographs of an experiment showing the binding of the probe with HeLa-Cx43 cells in vitro:

(3) a: HeLa-Cx43 cells uptake a large amount of the probe Cy5.5-Cx43SP (green) after co-incubation of Cy5.5-Cx43SP with HeLa-Cx43 cells;

(4) b: Cy5.5-Cx43SP is not uptaken by HeLa cells in the control group after co-incubation of Cy5.5-Cx43SP with HeLa cells;

(5) c: after previous blocking by introduction of excess unlabeled Cx43SP, and subsequent co-incubation of Cy5.5-Cx43SP with HeLa-Cx43 cells, the amount of Cy5.5-Cx43SP uptaken by HeLa-Cx43 cells decreases significantly, with almost no Cy5.5-Cx43SP being uptaken;

(6) d: after blocking by introduction of excess unlabeled Cx43SP and subsequent co-incubation of Cy5.5-Cx43SP with HeLa cells, the HeLa cells still do not uptake Cy5.5-Cx43SP. The probe is proven to have good target specificity.

(7) FIG. 3 shows fluorescent micrographs of frozen sections of tissues using muscles as the control:

(8) a: 1 h after tail vein injection of Cy5.5-Cx43SP, the fluorescent micrograph of a frozen section of muscle, showing that Cy5.5-Cx43SP was not gathered in the muscular tissue;

(9) b: the bright field picture of the frozen section of muscle, showing the morphology of the muscular tissue;

(10) c: merged picture of the frozen section of muscle and the fluorescent micrograph.

(11) FIG. 4 is a fluorescent micrograph of a frozen section of heart tissue after in vivo injection of the probe:

(12) a: 1 h after tail vein injection of Cy5.5-Cx43SP, the fluorescent micrograph of a frozen section of myocardium, showing that Cy5.5-Cx43SP is gathered in the myocardial tissue in large amount;

(13) b: 1 h after tail vein injection of Cy5.5-Cx43SP, the bright field picture of the frozen section of myocardium, showing the morphology of the myocardial tissue;

(14) c: 1 h after tail vein injection of Cy5.5-Cx43SP, a merged photograph of the frozen section of myocardium and the fluorescent micrograph, showing that Cy5.5-Cx43SP is gathered in the myocardial tissue in large amount, and is mainly distributed at the location of gap junctions between myocardial cells.

(15) FIG. 5 shows the binding and blocking experiments of .sup.64Cu-NODA-Cx43SP1 with HeLa-Cx43 cells.

(16) FIG. 6 are near-infrared fluorescence images of ex vivo main tissues and organs: 1 h after tail vein injection of the probe Cy5.5-Cx43SP into a mouse, the probe is significantly gathered in the heart, stomach, liver, gall bladder and intestinal tract, and mostly excreted via liver and intestinal tract.

(17) FIG. 7 shows PET images of .sup.64Cu-NOTA-Cx43SP1 in a normal rat at different times, showing that the probe is significantly gathered in the heart. The injection amount of .sup.64Cu-NOTA-Cx43SP1 is about 200 microcurie.

(18) FIG. 8 shows PET images of .sup.64Cu-NOTA-Cx43SP1 in a normal mouse at 1 h, showing that the probe is significantly gathered in the heart. The injection amount of .sup.64Cu-NOTA-Cx43SP1 is about 50 microcurie.

(19) FIG. 9: near-infrared imaging experiment of a tumor in a living mouse: 1 h after tail vein injection of the probe Cy5.5-Cx43SP1 into a mouse, the probe is gathered at tumor site of HeLa-Cx43 overexpressing Cx43 in large amount, and is not concentrated at the HeLa tumor site in the control group on the right side.

(20) FIG. 10: PET imaging experiment of tumor in a mouse in vivo: 1 h after tail vein injection of the probe .sup.64Cu-NODA-Cx43SP1 into a mouse, the probe is gathered at tumor site of HeLa-Cx43 overexpressing Cx43 in large amount (on the left side).

DETAILED EMBODIMENTS

(21) The present invention discloses a Cx43 targeted molecular imaging probe and a process for molecular imaging in vivo, which can be achieved by a person skilled in the art in light of the present disclosure through appropriately improving process parameters. It should be particularly noted that, all similar substitutions and modifications would be obvious for a person skilled in the art, and should be construed as being included in the present invention. The process and use of the present invention have been described with reference to preferable examples, and those skilled in the art can make modifications or proper alterations or combinations to the process and use as described herein without departing from the contents, spirit and scope of the present invention, in order to achieve and apply the technology of the present invention.

(22) In order for those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further illustrated in detail in combination with specific examples.

EXAMPLE 1

Cx43 Targeted Affinity Component—Cx43 Targeted Binding Peptides (Cx43SP)

(23) Any of the following 4 types of polypeptides (Cx43 targeted binding peptides) can specifically bind to the carboxyl terminal of Cx43, and therefore can be used to synthesize Cx43 targeted molecular probe.

(24) 1) Structure of Cx43SP1:

(25) Gly-Ala-Pro-Gly-4 Hyp-Pro-Tyr, also called: AAP10, molecular formula: C.sub.26H.sub.37N.sub.7O.sub.8, molecular weight: 575.6, the structure thereof is as shown below:

(26) ##STR00003##

(27) Cx43SP1 is the most important targeted affinity component in the molecular probes of the present invention, and is a polypeptide which can specifically recognize and bind to Cx43 in vivo, and consists of 6 amino acids. Cx43SP1 comprises a series of antiarrhythmic polypeptides extracted by Aonuma S et al. in 1980 from the atrial tissue of a cattle, which functions via action on Cx43, and therefore has been named antiarrhythmic peptide10 (AAP10). However, as the structures of AAP10 are not stable in plasma, they have no further clinical applications. The present inventors carried out chemical modifications to the structures of AAP10, and synthesized a probe for Cx43 in vivo imaging, which has been verified to have ideal in vivo stability. Since in the present invention, the chemical structure is no longer used as an antiarrhythmic agent, but as a targeted affinity component of a Cx43 targeted molecular imaging probe, it is named Cx43 Specific Peptide 1. The hippocrepiform domain of Cx43SP1 can specifically bind to the receptor domain of the carboxyl terminal of Cx43.

(28) 2) The Structure of Cx43SP2:

(29) Gly-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly, the structure thereof is as shown below:

(30) ##STR00004##

(31) Cx43SP2 is an analog of Cx43SP1, in which some L form amino acids in Cx43SP are substituted with D form amino acids, so as to increase stability. Cx43SP2 is now on the stage of clinical trial as an antiarrhythmic agent. The trade name of Cx43SP2 is rotigaptide, also called CHEMBL450656, GAP-486, ZP123. Formula: C.sub.28H.sub.39N.sub.7O.sub.9, molecular weight: 617.65076.

(32) 3) The Structure of Cx43SP3:

(33) Cx43SP3 is a functional analog of Cx43SP2, also called GAP 134, with a molecular weight of 291.3, and the structure thereof is as shown below:

(34) ##STR00005##

(35) 4) As Shown in Table 1, Cx43SP4 Comprises 5 Types of Amino Acid Sequences Containing the Characteristic Structure of RXP-E:

(36) TABLE-US-00002 TABLE 1 Amino acid sequences of Cx43SP4 RXP-X Sequences SEQ ID NO: RXP-A DVPGRDPGYIKGGGSAHARVPFFSHSLN 1 RNRKPSLYQ RXP-B EIQPRSPLMFSGGGSAHARVPFFSHSAK 2 EARWPRAHR RXP-C GIAAREPNSHDGGGSAHARVPFFSHSRD 3 LWRKPAKSL RXP-D WEEPRRPFTMSGGGSAETHARVPFYSHS 4 PMRHRLPGVHL RXP-E SDDLRSPQLHNGGGSAVPFYSHSHMVRR 5 KPRNPR

(37) Cx43SP3 is a type of polypeptides screened by Mario Delmar et al. in 2006 through phage display technique, which can specifically bind to carboxyl terminal (amino acids 255-382) of Cx43 and are comprised of 34 amino acids. Their basic binding motif is RXP-X, and thus also called RXP series of polypeptides. They are proposed to be used in antiarrhythmic therapy. Wherein, RXP-E has more potential applications than the other polypeptides.

EXAMPLE 2

Preparation of Cx43 Targeted Molecular Probes

(38) The general formula of Cx43 targeted molecular probe of the present invention is as shown in FIG. 1.

(39) (1) The imaging target is Connexin 43 (Cx43), the carboxyl terminal (C-terminal) of Cx43 has a gating particulate structure, which is functionally equivalent to specific receptor domain, is the main binding target of Cx43SP, and is the target of Cx43 targeted imaging of the present invention.

(40) (2) Signaling component: a moiety in the probe detectable with an imaging device, and in the present patent it is mainly an radioisotope (PET and SPECT imaging), a fluorescent dye and a quantum dot (optical imaging), a paramagnetic material, a super-paramagnetic material and magnetic nanoparticles (magnetic resonance imaging), ultrasound microbubbles (ultrasonic imaging), various photoacoustic nanoparticles (photoacoustic imaging) and an imaging material formed by a combination of the various components as mentioned above for detection by means of a multi-mode imaging technique.

(41) (3) Targeted affinity component: a moiety in the probe specifically binding to the imaging molecular target, the binding therebetween has high specificity and high affinity, like the relationship between “a key and a lock”. The present invention mainly involves polypeptides and small molecular structures in example 1.

(42) (4) Linker: a moiety linking the signaling component to the targeted affinity component. Alternatively, the linker may not be introduced, and the signaling component is directly linked to the Cx43 affinity component via employing a chemical method.

EXAMPLE 3

Preparation of Radioisotope Labeled Cx43 Targeted Molecule Probe

(43) Each of the 4 types of polypeptides in example 1 can specifically bind to the carboxyl terminal of Cx43, and therefore can be used to prepare the labeled Cx43 targeted molecular probe. In the examples illustrating the synthesizing processes as follows, Cx43SP1 is used as a representative polypeptide.

(44) The signaling component is a positron emission radioisotope or a single photon radionuclide, which can be used in positron emission tomography (PET) or single photon emission tomography (SPECT) clinically or for a small animal. Through selecting a proper linker or employing a radioactive chemical method, the radioisotope can bind to targeted polypeptides. The linker employed is also called a bifunctional chelating agent. The bifunctional chelating agent possesses not only a functional motif group, but also a group binding to Cx43SP, so as to link the above two components to form a Cx43 targeted molecular probe.

(45) Divalent (M.sup.2+) or Trivalent Metal Ion (M.sup.3+) Labeled Cx43SP

(46) Depending on different properties of radionuclides, different linkers can be selected.

(47) For details, please refer to Table 2.

(48) TABLE-US-00003 TABLE 2 Commonly used radioisotopes, bifunctional chelating agents and thus synthesized Cx43 targeted probe Radion uclides Linker Linker modified Cx43SP .sup.111In .sup.111In .sup.86/90Y .sup.177Lu .sup.67/68Ga .sup.212Pb .sup.212Bi/.sup.213 Bi .sup.89Zr .sup.225Ac 0 .sup.67/68Ga .sup.111In .sup.64/67Cu .sup.203Pb .sup.212Pb 0 .sup.64/67Cu Note: R is Cx43SP. DTPA: diethylene triamine pentaacetic acid DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTAGA: 1-(1-carboxy-3-carboxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-triacetic acid NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid NODAGA : NOTA subjected to functional modification by a glutaric acid arm 2-(p-SCN-Bz)-NOTA: NOTA subjected to functional modification by a benzyl isothiocyanate TETA: 1,4,8,11-tetraazacyclotetradecane-1,4,8,11,tetraacetic acid CB-TE2A : Attachment of two carboxymethyl pendant arms to cross-bridged (CB)-cyclam leads to CB-TE2A Sar: sarcophagine (3,6,10,13,16,19-hexa azabicyclo [6.6.6] icosane) SarAr and AmBa Sarare carboxy are respectively carboxylic acid and amino derivatives of Sar NODA: sodium 1,4,7-triazacyclononane-1,4-diacetate

(49) Many radioisotopes and bifunctional chelating agents are mentioned above. Therefore, .sup.64 Cu labeled Cx43SP is taken as an example here to illustrate the process for labeling Cx43SP with divalent (M.sup.2+) or trivalent metal ions (M.sup.3+) of a radioisotope.

(50) Modifying Cx43SP with NODA: Into an excess of NODA were added appropriate amount of N,N-dimetbylformamide (DMF) and 2% of N,N-diisopropyl ethylamine (DIPEA), and the mixture was vibrated overnight at room temperature. After isolation and purification on high performance liquid chromatography (HPLC), the products thus obtained were analyzed and identified via mass spectrometry. The chemical reaction formula of modifying Cx43SP1 with NODA is as follows:

(51) ##STR00023##

The Chemical Reaction Formula of Modifying Cx43SP1 with NODA

(52) .sup.64Cu labeling: 10 μg of Cx43SP-NODA was dissolved in 200 μl of ammonium acetate buffer with pH of between 4 and 5, and then 50 μL of .sup.64CuCl.sub.2 (pH between 5 and 6). The mixture was reacted at 37° C. for 1 hour. The radiolabeled product was isolated and purified with radioactive detector—high performance liquid chromatography (RP-HPLC), and the labeling rate, radiochemical purity, and specific activity were tested. The chemical reaction formula of labeling NODA-Cx43SP1 with .sup.64Cu is as follows:

(53) ##STR00024##

The Chemical Reaction Formula of Labeling NODA-Cx43SP1 with 64Cu

(54) 2) Labeling Cx43SP with .sup.18F

(55) .sup.18F is the most commonly used radionuclide clinically. It is very difficult to label a polypeptide with affinitive .sup.18F-F.sup.− directly. Generally, modified .sup.18F precursor such as 4-nitrophenyl 2-[.sup.18F]-fluoropropionate (.sup.18F-NFP) is used. By means of this technique, .sup.18F labeled Cx43 probe suitable for Cx43 targeted molecular imaging of heart and tumor can be obtained. The probe is characterized by more clinical application potentials.

(56) Synthesis of .sup.18F-FP-Cx43SP:

(57) .sup.18F labeled precursor .sup.18F-NFP (4-nitrophenyl 2-[.sup.18F]-fluoropropionate, Rt=22-23 min) and 500 μg of Cx43 targeted binding peptide were dissolved in 150 μL of dry dimethyl sulfoxide (DMSO), and then .sup.18F-NFP and 20 μL of N,N-diisopropyl ethylamine (DIPEA) were added. After the mixture was reacted at room temperature for 30 min, 800 μL of 5% acetic acid was added to neutralize. The product was purified with semi-preparative HPLC C-18 column. The purified product was diluted by adding 20 μL of water. During preparation, the C-18 column was repeatedly eluted with 5 mL of ethanol and 10 mL of water to elute products. The final product was blow-dried with nitrogen gas at 60° C. Finally, the .sup.18F labeled polypeptide was dissolved in PBS and ultrafiltered (0.22 μm) into a sterile dose vial for in vitro and in vivo experiments. Likewise, the labeling rate, radiochemical purity, specific activity, etc. were tested by HPLC. The chemical reaction formula of labeling Cx43SP1 with .sup.18F is as follows:

(58) ##STR00025##

The Chemical Reaction Formula of Labeling Cx43SP1 with 18F

(59) Synthesis of .sup.18F-AIF-NODA-Cx43SP:

(60) NODA-Cx43SP was synthesized by using NODA-Cx43SP. QMA-SepPak column was used to absorb 30 mCi (1.1 GBq) aluminum .sup.18F-fluoride, 2.5 mL of aqueous solution without metal ions was used to elute aluminum .sup.18F-fluoride, 400 μL of 0.4 M KHCO.sub.3 solution was used to rinse aluminum .sup.18F-fluoride, and 200 μL of aluminum .sup.18F-fluoride solution was taken out for use. The pH of the solution was adjusted to 4.0 with acetic acid without metal ions. To the solution were sequentially added aluminium chloride (AlCl.sub.3, 2mM, 3 μL, dissolved in 0.1 M sodium acetate buffer, pH 4) and 5 μL of NOTA-Cx43SP (60 mg/mL, dissolved in DMSO). The reaction mixture was incubated at 100° C. for 15 min, and diluted with 1 mL water without metal ions. The product was purified with semi-preparative HPLC. .sup.18F-AIF-NOTA-Cx43SP analogs were collected and evaporated to dry, dissolved in PBS and ultrafiltered (0.22 μm) into a sterile dose vial for in vitro and in vivo experiments. Likewise, the labeling rate, radiochemical purity, specific activity, etc. were tested by HPLC. The chemical reaction formula of labeling Cx43SP1 with .sup.18F is as follows.

(61) ##STR00026##

The Chemical Reaction Formula of Labeling Cx43SP1 with 18F

(62) Besides .sup.18F-NFP, similar processes may also be employed to label Cx43SP by means of other .sup.18F precursors (i.e., auxiliary groups). Structural formulae of commonly used .sup.18F labeled precursors [.sup.18F] FBA: [.sup.18F] o-fluorobenzoic acid; [.sup.18F] FSB: [.sup.18F] fluorobenzoate; [.sup.18F] FBEM and [.sup.18F] FBBO are as shown below:

(63) ##STR00027##

(64) Besides .sup.18F, Cx43SP may also be labeled by applying radionuclides such as .sup.11C, .sup.13N and .sup.15O and selecting corresponding precursors. Advantages of positron emission radionuclide labeled probes include high sensibility, accurate quantification ability, clinical transformation ability, low molecular weight of probe, and good pharmacokinetic characteristics.

(65) 3): .sup.99mTc labeled Cx43SP

(66) For Example: .sup.99mTcO.sup.3+ or .sup.99mTcO.sup.2+, with HYNIC as a Bifunctional Chelating Agent.

(67) Specific steps are as follows:

(68) 1. The bifunctional chelating agent HYNIC-NHS was reacted with amino (—NH.sub.2) on Cx43SP, with DMF as solvent, 2% of DIPEA as catalyst. After purification by high pressure liquid chromatography (HPLC), the product was identified by mass spectrometry.

(69) 2. Technetium-99m labeled HYNIC-Cx43SP: by means of a protocol of complexation with “3+1” binary mixed ligands, in which a tridentate ligand tricine was used as a synergistic reagent, and stannous chloride (SnCl.sub.2.H.sub.2O) was used as a reducing agent. After reaction at room temperature for 20 min, radiochemical purity of the labeled compound was tested, and the casted colloid was less than 1%.

(70) 3. Analysis: Thin Layer Chromatography (ITLC) and high pressure liquid chromatography (HPLC) were used to analyze the content of colloid in the labeled compound and content of product.

(71) The chemical reaction formula of labeling Cx43SP1 with .sup.99mTc is as follows:

(72) ##STR00028##

The Chemical Reaction Formula of Labeling Cx43SP1 with 99mTc

(73) Different products can be obtained depending on different .sup.99mTc-nuclei, different linkers, and different reaction processes. .sup.99mTc labeled Cx43SP, common bifunctional chelating agents and synthesized probes are as shown in Table 2, wherein R represents Cx43SP:

(74) TABLE-US-00004 .sup.99mTcO.sup.3+ or .sup.99mTcO.sup.2+ + 0 .sup.99mTc(CO).sub.3 (H.sub.2O).sup.3+

(75) 4) .sup.123/124I labeled Cx43SP

(76) Radioisotope iodine can be labeled on Cx43SP by selecting appropriate oxidants. There are mainly 2 types of labeling methods, i.e., direct labeling and indirect labeling.

(77) (1) Direct labeling: iodination reaction may be directly carried out via “iodine protonation”. Structural formulae of oxidants commonly used in direct labeling of Cx43SP with .sup.123/124I are as follows:

(78) ##STR00037##
Chloramine T Iodine bead Water insoluble iodinating reagent

(79) The chemical reaction formula of direct labeling Cx43SP1 with .sup.123/124I is as follows:

(80) ##STR00038##

(81) (2) Indirect labeling: Structural formulae of oxidants commonly used in indirect labeling of Cx43SP with .sup.123/124I are as follows:

(82) ##STR00039##

(83) The chemical reaction formula of indirect labeling Cx43SP1 with .sup.123/124I is as follows:

(84) ##STR00040##

EXAMPLE 4

Preparation of Optically Labeled Cx43 Targeted Molecular Probe

(85) 1) Near-infrared fluorescent dye labeled Cx43SP1

(86) Near-infrared fluorescent dyes at a wavelength of 700-900 nm can reach deeper tissues due to their stronger penetration, and therefore often are used to carry out in vivo optical imaging. Commonly used near-infrared fluorescent dyes are shown in figures. Different bifunctional chelating agents can be selected to label a near-infrared fluorescent dye onto Cx43SP. The chemical structural general formula of cyanine dye is as follows:

(87) ##STR00041##

(88) The chemical structures of commonly used 4 types of near-infrared fluorescent dyes are as follows:

(89) (1a) cyanine, (1b) ICG, (1c) SIDAG, (1d) PPCy

(90) ##STR00042##

Chemical structural general formulae of the cyanine dye

(91) Cy5.5 labeling: Cx43-specific binding peptide dissolved in 100 μL of DMSO was mixed with Cy5.5-NHS (1 equiv.) under light screening condition, co-dissolved in 2% DIPEA, and incubated under vibration overnight at room temperature. The product was isolated and purified via semi-preparative HPLC C18 column (250×10 mm), then collected and lyophilized. The yield of the product was calculated, and molecular weight thereof was measured by mass spectrometry (MALDI-TOF-MS). The chemical reaction formula of labeling Cx43SP1 with Cy5.5 is as follows:

(92) ##STR00043##

(93) 2) Near-infrared nano material labeled Cx43SP

(94) There are four types of most commonly used nano materials at present, including nano probe, quantum dot, carbon nano tube and gold nanocluster containing a near-infrared fluorescent dye.

(95) Taking near-infrared quantum dot as an example, quantum dot was used to label Cx43SP: 1000 eq of Cx43SP was dissolved in water, mixed with 1 eq of quantum dot. 1000 eq of EDC was then added. The reaction mixture was stirred at room temperature for 1 hour, then isolated on PD10 column, concentrated via a centrifuge tube, and tested for concentration. One quantum dot can be labeled with 500-600 Cx43SP molecules. The synthesized probe was subjected to in vivo test via near-infrared fluorescent imaging.

EXAMPLE 5

Preparation of a Magnetically Labeled Cx43 Targeted Molecular Probe

(96) 1) Superparamagnetic nanoparticles (Fe.sub.3O.sub.4)

(97) Superparamagnetic nanoparticles were used to label Cx43SP to synthesize the probe useful in magnetic resonance imaging. Taking superparamagnetic nanoparticles as an example, using superparamagnetic nanoparticles to label Cx43SP: 1000 eq Cx43SP was dissolved in water and mixed with 1 eq superparamagnetic nanoparticles. 1000 eq of EDC was then added. The reaction mixture was stirred at room temperature for 1 hour, then isolated on PD10 column, concentrated via a centrifuge tube, and tested for concentration. The chemical reaction formula of labeling Cx43SP1 with superparamagnetic ferric oxide is as follows:

(98) ##STR00044##

(99) 2) Paramagnetic metal chelate: through selecting proper bifunctional chelating agent, a paramagnetic metal chelate can also be labeled on Cx43SP to form a probe useful in magnetic resonance imaging. The paramagnetic metal element mainly include gadolinium (Gd.sup.3+), Dy.sup.3+, Tm.sup.3+, Mn.sup.2+, CEST reagent, .sup.3He, and .sup.129Xe. Due to the relatively week ability of relaxation generated by the paramagnetic metal chelate, whereas the sensibility of magnetic resonance molecular imaging is relatively low, effective signal magnification mechanism may also be introduced, i.e., by using macromolecules carrying multiple functional groups on the surface, such as dendrimer, liposome, etc., several Cx43SP molecules and plenty of paramagnetic metal were chelated onto their surfaces to form a probe. The chemical reaction formula of labeling Cx43SP1 with a paramagnetic metal chelate is as follows:

(100) ##STR00045##

EXAMPLE 6

Preparation of Ultrasound Microbubble Labeled Cx43 Targeted molecular probe

(101) 1000 eq Cx43SP was dissolved in water, and mixed with 1 eq ultrasound microbubble. 1000 eq EDC was then added. The reaction mixture was stirred at room temperature for 1 hour. The chemical reaction formula of labeling Cx43SP1 with ultrasound microbubble is as follows:

(102) ##STR00046##

EXAMPLE 7

Preparation of Photoacoustic Material Labeled Cx43 Targeted Molecular Probe

(103) At present, some gold nanoparticles, such as gold nanorod, can also be used to label Cx43SP to perform photoacoustic imaging. Using gold nanorod to labelCx43SP: 1000 eq Cx43SP was dissolved in water and mixed with 1 eq gold nanorod. 1000 eq EDC was then added. The reaction mixture was stirred at room temperature for 1 hour, then isolated on PD10 column, concentrated via a centrifuge tube, and tested for concentration. The chemical reaction formula of labeling Cx43SP1 with gold nanorod is as follows:

(104) ##STR00047##

EXAMPLE 8

Preparation of a Multi-mode Labeled Cx43 Targeted Molecular Probe

(105) Two or more types of imaging labeling processes were simultaneously used to generate a Cx43 targeted probe detectable via two or more of imaging detection means, i.e., a multi-mode molecular imaging probe. For example, different functional groups were attached to the surface of a nano material, in order to attach other modes of imaging agents such as polypeptide, radionuclide, fluorescent dye, etc.

(106) After modification with mercapto and carboxyl groups on the surface of near-infrared quantum dots, Cx43SP1 was firstly attached thereto. Quantum dots were modified with NODA-MAL. By means of the reaction between maleic anhydride and mercapto, NODA was attached to the quantum dots. Then the product was labeled with .sup.64Cu. The radiolabeled product was isolated and purified via PD10. The labeling rate, radiochemical purity, and specific activity were tested. The probe can be used simultaneously for near-infrared fluorescent imaging and PET imaging. The chemical reaction formula of double-mode labeling Cx43SP1 with .sup.64Cu and quantum dots is as follows:

(107) ##STR00048##

EXAMPLE 9

Ex Vivo Experiment Verification

(108) 1) Establishment of a Cx43 highly expressing cell line: Cervical cancer HeLa cells were transfected by Cx43 gene, and cervical cancer tumor cell lines HeLa-Cx43 overexpressing Cx43 was successfully prepared. Non-transgenic HeLa cells (without C43 expression) were used as control.

(109) 2) Cy5.5-Cx43SP1 cells binding and blocking experiment: HeLa-Cx43 cells and the control group HeLa-Control cells were applied onto a 12-well plate in an amount of 0.5×10.sup.6/well one day prior to experiment, and were classified into four groups: Group A: HeLa-Cx43 cells group, Group B: HeLa-Control cells group, Group C: HeLa-Cx43 cells blocking group, Group D: HeLa-Control cells blocking group. Each group comprises 3 wells and the experiment was repeated for 3 times. Into each well was added Cy5.5-Cx43SP1 with a concentration of 500 nmol/L and an amount of 1 ml. Unlabeled Cx43SP1 was added into blocking groups with a concentration of 5 μmol/L.

(110) As can be seen from the results, there was significant specific binding between Cy5.5-Cx43SP1 and HeLa-Cx43 cells, and no binding between Cy5.5-Cx43SP1 and the control group HeLa cells without transfecting Cx43 gene (FIG. 2-a and FIG. 2-b). After blocking by introduction of an excess of unlabeled 5 μmol/L Cx43SP, the binding between Cy5.5-Cx43SP and HeLa-Cx43 was substantially reduced (FIG. 2-c). The above results show that, the near-infrared fluorescent probe Cy5.5-Cx43SP can be specifically uptaken by HeLa cells overexpressing Cx43, and the binding between Cy5.5-Cx43SP and HeLa-Cx43 has target specificity.

(111) Meanwhile, frozen sections of tissue were further prepared and imaged under a fluorescence microscope. By using muscle as a control (FIGS. 3-a, 3-b, and 3-c), the distribution profile of the probe Cy5.5-Cx43SP in heart was observed under a fluorescence microscope. At 1 h after tail vein injection of Cy5.5-Cx43SP, frozen myocardium sections showed under a fluorescence microscope that Cy5.5-Cx43SP was gathered in the myocardial tissue in large amount, and was mainly distributed at the location of gap junctions between myocardial cells (FIGS. 4-a, 4-b, and 4-c). It is proved at tissue level that the probe has target specificity to the myocardial gap junctions.

(112) 3) .sup.64Cu-NODA-Cx43SP1 Cells Binding and Blocking Experiment

(113) HeLa-Cx43 cells and control group HeLa-Control cells were applied onto a 12-well plate in an amount of 0.5×10.sup.6/well one day prior to experiment, and were classified into four groups: Group A: HeLa-Cx43 cells group, Group B: HeLa-Control cells group, Group C: HeLa-Cx43 cells blocking group, Group D: HeLa-Control cells blocking group. Each group comprises 3 wells, and the experiment was repeated for 3 times. Into each well was added .sup.64Cu-NODA-Cx43SP1 with a concentration of 3.2 μCi/well and an amount of 1 ml. Unlabeled NODA-Cx43SP1 was added into the blocking groups with a concentration of 50 μg/well (10 times of .sup.64Cu-NODA-Cx43SP1). The results show that, there was significant specific binding between .sup.64Cu-NODA-Cx43SP1 and HeLa-Cx43 cells, and no binding between .sup.64Cu-NODA-Cx43SP1 and the control group HeLa cells without transfecting Cx43 gene. In the blocking experiment, the binding between .sup.64Cu-NODA-Cx43SP1 and HeLa-Cx43 was substantially reduced (FIG. 5). The results show that, -NODA-Cx43SP1 can be specifically uptaken by HeLa cells overexpressing Cx43, and the binding between -NODA-Cx43SP1 and HeLa-Cx43 has target specificity and high affinity.

EXAMPLE 10

Animal Experiments

(114) 1) In Vivo Biological Distribution Characteristics of the Probe:

(115) Biological Distribution of Cy5.5-Cx43SP in Normal Mice

(116) 1 hour after injection of Cy5.5-Cx43SP, the mice were sacrificed, and their organs were taken out to perform ex vivo near-infrared fluorescent imaging of main organs. The results show that, the probe Cy5.5-Cx43SP was mainly distributed in heart, liver, gastrointestinal tract, and was mainly excreted through liver and intestinal tract, and partly through kidney and urinary system. Especially, the relatively high uptake by heart is highly consistent with the fact that normal heart express relatively high Cx43 (FIG. 6).

(117) 2) Normal Rat PET Heart Imaging:

(118) The previous experiment verified at in vivo level the target gathering ability of .sup.64Cu-NOTA-Cx43SP in the heart of a normal rat and the biological distribution characteristics of the probe. The results show that, at 30 min after intravenous injection of .sup.64Cu-NOTA-Cx43SP, it began to gather in the heart significantly, and some of the probe maintained in the heart until 3 h after the injection (FIG. 7), while the probe seldom gather in liver and lung, as the images of the myocardium were clear. Since Cx43 is the main connexin in the myocardium, the results show that .sup.64Cu-NOTA-Cx43SP was target gathered in the myocardium. At 1 h after the intravenous injection, the probe .sup.64Cu-NOTA-Cx43SP was mainly distributed in heart and intestinal tract of mice. These results are consistent to the in vitro optical imaging results of Cy5.5-Cx43SP in mice. The experimental results demonstrate that Cx43 targeted molecular probe .sup.64Cu-NOTA-Cx43S designed in the preliminary experiment can be used in in vivo imaging of the myocardium.

(119) 3) PET Heart Imaging of Normal Mice:

(120) PET imaging experiment of .sup.64Cu-NOTA-Cx43SP in normal mice was simultaneously carried out. The results are shown in FIG. 8. At 1 h after intravenous injection of .sup.64Cu-NOTA-Cx43SP, it was significantly gathered in heart, and was also absorbed in liver. Most of .sup.64Cu-NOTA-Cx43SP was excreted through kidney and urinary bladder. These results are consistent with the rat experiment, but not so good as the heart imaging results in rats. It shows that biological distributions of the probe in different animals are somewhat different. Meanwhile, the .sup.64Cu-NOTA-Cx43SP probe can be further optimized and need to be further optimized.

(121) 4) Optical Tumor Imaging of Nude Mice Bearing Tumor:

(122) HeLa-Cx43 cells overexpressing Cx43 were used to establish a subcutaneous xenograft model in nude mouse (FIG. 9, left), and the conventional subcutaneous xenograft model of HeLa cells without transfection of Cx43 were used as control group (FIG. 9, right). In vivo Cy5.5-Cx43SP1 near-infrared fluorescent imaging of tumor was carried out. The results show that, at one hour after tail vein injection of the probe Cy5.5-Cx43SP1, it was gathered in the location of HeLa-Cx43 tumor overexpressing Cx43 in high concentration, and no obvious gather in the location of HeLa tumor in the control group was observed. The results once again show that, at in vivo level, Cx43 targeted molecular probe Cy5.5-Cx43S has certain degree of target specificity to Cx43 positive tumor.

(123) 5) PET Tumor Imaging of Nude Mice Bearing Tumor:

(124) HeLa-Cx43 cells overexpressing Cx43 were used to establish a subcutaneous xenograft model in nude mouse (FIG. 10, left). .sup.64Cu-NODA-Cx43SP was intravenously injected, with a concentration of 40 microcurie. The results of Micro PET imaging show that, at one hour after tail vein injection of the probe .sup.64Cu-NODA-Cx43SP, it was gathered in the location of HeLa-Cx43 tumor overexpressing Cx43 in high concentration. The results once again show that, at in vivo level, Cx43 targeted molecular probe .sup.64Cu-NODA-Cx43SP has certain degree of target specificity to Cx43 positive tumor.