Method of imaging cell death in the body of a mammal subject in vivo |
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IPC classes for russian patent Method of imaging cell death in the body of a mammal subject in vivo (RU 2228765):
       
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The invention relates to medicine, in particular to radiology. The method can improve the accuracy of imaging cell death in the body of a mammal subject. The subject is administered annexin labeled biocompatible radionuclide, and then place the subject within the detection field of the radiation detector to construct images of radioactive emissions after a period of time for which labeled annexin reaches localization in the body of the subject, and the specified image is a representation of cell death in this region of the subject. When the radionuclide is selected from the group consisting of iodine 123, iodine-131, gallium 67, indium 111, fluorine 18 and technetium 99m. 27 C.p. f-crystals, 4 tab., 4 Il.
The area of technology to which the invention relates
The invention relates to research in medicine with the use of radioisotopes and relates to a method of imaging cell death in vivo. In particular, it concerns the use of annexin radioactive label for visualization plots the death of cells in the body of a mammal using rays to form images.
This work was partially supported by the Level of technology
Apoptosis or programmed cell death plays a critical role in the development and the number of homeostatic and disease processes (see Thompson, S. C., Science 267: 1456, 1995). New strategies for therapy of some diseases could therefore be implemented through modulation of apoptotic cell death. The lack of non-invasive way(s) detection (detection and monitoring of apoptotic cell death in vivo prevents the study of new pharmacological agents for the stimulation or inhibition of apoptotic cell death.
The possibility of application of spectroscopy proton nuclear magnetic resonance (1N-AMRC) lipids for detection of specific changes in the composition and/or fluidity of plasma membranes of lymphoblasts and other cell lines exposed to apoptotic cell death (see article F. G. Blankenberg et al., Blood 87:1951, 1996).
Clinical application of the study of lipids using1H-AMRC limited now challenging the local magnetic effects of the surrounding microenvironment found in vivo in many tissues and organs.
The invention
In one aspect, the invention includes a method of surrounding the subject in vivo. The method includes steps (a) introduction to the subject of annexin labeled biocompatible radionuclide, (b) placing the subject within the detection field of the radiation detector after a period of time for which labeled annexin can achieve localization in the body of the subject, and (C) measuring the radioactive emissions of the radionuclide, localized in the body of a subject using a radiation detector to construct images of radioactive emissions, the image is a representation of cell death in a certain area of the body of a mammal subject. In one embodiment, the method further includes a step (d) processing the image to isolate the signal due to nonspecific localization of labeled annexin, such as non-specific localization in the kidney.
The radionuclides used in the method include iodine-123, iodine 131, gallium 67, indium 111, fluorine 18 and technetium 99m (Tc99m). Note that fluorine 18 is a positron emitter and can thus be used in positron emission tomography (PET). Iodine 123, iodine 131, gallium 67, indium 111 and technetium 99m is used with standard detection-radiation. TSM is the preferred management TSM associated with annexin through hydrazinonicotinamide (GNA). Labeled TSM annexin usually injected at a dose of between about 5 and about 20 MCI.
In one main embodiment of the invention the radiation detector is a detectorradiation, and measuring radioactive emissions are emissions-rays. In another main embodiment, the radiation detector is a detector positron emission, and the measured radioactive emission is the emission of positrons.
In yet another General embodiment, the method further includes repeating steps (b) and (C) in selected intervals of time, with repetition is effective in detecting changes in the intensity of the radiation (e.g., emission of gamma rays or positrons) from the area of the body over time, reflecting changes in the number of cells in which cell death occurs.
Another main variant of implementation includes repeating steps (b) and (C) in selected intervals of time, while repeating is effective to track changes in localizationradiation in the body of the subject over time, reflecting changes in the localization of cells in cuoricino camera Angera or a camera for obtaining a three-dimensional image.
The preferred annexin for use in the invention is annexin V. It is usually administered in doses not exceeding approximately 300 μg protein/kg, preferably between about 1 and 10 μg protein/kg, there are various methods of administration, including intravenous (i.v.), intraperitoneal (i.p.), intrathecal and intrapleural introduction.
Measurement-radiation (emission-rays) to construct the image is usually carried out between about 5 minutes and about 2 hours after administration of labeled annexin. In one embodiment, the measurement of the emission-rays to construct images spend approximately one hour after administration of labeled annexin.
When presented in this way can be acquired images of different areas of the subject. For example, the region may include the subject almost completely or part of the subject, such as the head or part thereof, heart, or liver or part, etc.,
The invention also provides a kit for imaging cell death in vivo. The kit includes (i) a sealed vial containing labelled GNA Annex shall actor Sn-Tricine, prepared, for example, as described in Materials and methods (b) and stored under N2, (iii) how to obtain the labeled TSM annexin with components (i) and (ii) together with TSM and (iv) instructions introduction TSM-annexin to visualize areas of cell death in vivo. In one embodiment, the set stored at -70 C and transported on dry ice. In another embodiment, labelled GNA annexin lyophilizer.
Data and other objects and features of the invention will be more apparent after reading the detailed description in conjunction with the attached figures.
List of drawings and other materials
Fig. 1 is a computer image showing Fas-mediated fast-paced hepatic apoptosis as determined by DSM-GNA-annexin V.
Fig. 2 is a digital image showing the signal from TSM-GNA-ovalbumin during Fas-mediated fast developing hepatic apoptosis.
Fig. 3 represents the result of quantitative determination of the absorption TSM-GNA-annexin V transplantirovannam heart.
Fig. 4 shows the dependence of histological degrees of ossobranie
I. Definitions
The term "cell death (cells) in the context of the detection cell death" or "localization of cell death" refers to cells that have lost the integrity of the plasma membrane, as well as to the processes by which die mammalian cells. These processes include apoptosis and processes that are believed to include apoptosis (e.g., senescence), as well as necrosis. The term "cell death" is used in this context to refer to the death or impending death of nuclear cells (such as neurons, myocytes, hepatocytes, and so on), as well as the death or impending death of a nuclear-free cells (e.g. red blood cells, platelets, etc).
"Biocompatible radionuclide" or "biocompatible radioisotope is an isotope, which is deemed applicable for injection to a patient when used in radioisotope studies in medicine. Examples of biocompatible radionuclides include iodine 123, iodine 131, gallium 67, indium 111, fluorine 18 and technetium 99m.
II. Cell death - apoptosis and necrosis
Apoptosis means "programmed cell death", in which the cell performs a program of "cell suicide". Now consider that apoptosis is an evolutionary to the. the Roma also believe that in many cases, apoptosis can be a program "default" (rules), which may be actively inhibited in a healthy viable cells.
The decision of cells to "submit" apoptosis may be taken under the influence of a number of regulatory incentives and environmental factors (see Tompson, S. C., Science 267:1456, 1995). Physiological activators of apoptosis include tumor necrosis factor (TNF), Fas ligand, transforming growth factor the neurotransmitters glutamate, dopamine, N-methyl-D-aspartate, removal of growth factors, loss of binding matrix, calcium and glucocorticoids. Associated with damage inducers of apoptosis include heat shock, viral infection, bacterial toxins, oncogenes myc, rel, IEA, tumor suppressor p53, cytolytic T cells, oxidants, free radicals and deprivation of nutrients (antimetabolites). Associated with therapy inducers of apoptosis include-irradiation, UV-irradiation and a number of chemotherapeutic drugs, including cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Associated with toxin inducers of apoptosis including the hen he is a pathological way in the cells, which normally do not regenerate, such as neurons. Because these cells are not replaced when they die, their loss can lead to weakening and sometimes fatal dysfunction of the affected organ. This dysfunction has been proven for several neurodegenerative disorders that have been associated with an increased level of apoptosis, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinopathy and cerebellar degeneration.
The consequences of unwanted apoptosis can be similarly harmful also in other pathologies, including ischemic injury, such as usually occur in cases of myocardial infarction, reperfusion injury and stroke. In particular, apoptosis, as it plays a main role in a very delayed infarction after mild local ischemia (see S. Du et al., J. Cereb. Blood Flow and Metab. 16: 195-201, 1996).
Additional diseases associated with increased apoptosis include, but are not limited to, the following: AIDS, myelodysplastic syndromes such as aplastic anemia, and induced toxins liver disease, including damage due to excessive alcohol consumption.
Necrosis is the Oia cell internal program suicide). Necrosis can be caused by traumatic injury, bacterial infection, acute hypoxia, etc., There is some overlap between the two types of cell death that some incentives may cause either necrosis or apoptosis or both in some degree depending on the severity of the damage.
III. Asymmetry in biological membranes.
In General, believe that biological membranes are asymmetric in terms of specific membrane phospholipids. In particular, the outer layer of the plasma membranes of eukaryotes is formed mainly by cholinesterase, such as sphingomyelin and phosphatidylcholine (PC), while the inner layer contains mainly aminophospholipid, such as phosphatidylserine (PS) and phosphatidylethanolamine (pea). This asymmetry is believed to be supported by the activity of adenosine triphosphate (ATP)-dependent aminophospholipid-translocases, which selectively transports FS, FAA between bimolecular layers (see Seigneuret, M. and P. F. Devaux, Proc. National Acad. Sci. USA 81: 3751, 1984). Other enzymes that are believed to be involved in the transport of phospholipids between layers include ATP-dependent to flippase (see Connor J. et al., J. Biol. Chem. 267:19412, 1992) and scramblase lipids (see statutes, the loss of this asymmetry is associated with certain physiological and pathological processes. For example, it is shown that the asymmetry of the membrane, defined as the presence of PS on the outer layer of the plasma membrane ("FS-manifestation"), is one of the earliest manifestations of apoptosis that precedes DNA fragmentation, formation of bubbles of plasma membrane and loss of membrane integrity (see Martin S. J. et al., J. Exp. Med. 182: 1545, 1995; V. A. Fadok et al., J. of Immunol. 148:2207, 1992; J. of Immunol. 149: 4029, 1992).
Such a shift was found in sickle-cell disease (see A. Lane et al., Am. J. Hematol. 47: 295, 1994),-thalassemia (see Borenstain-Ben Yashar V. et al., Am. J. Hematol. 44: 63, 1993), activation of platelets and the number of mutant tumor cells with defective transport FS. The gradual emergence of FS in the outer layer, as shown, takes place during the aging of red blood cells (see Tait J. F. and D. Gibson, J. Lab. Clin. Med. 123: 741, 1994). When the manifestation of FS on these cells reaches a critical level, cells are removed from the bloodstream by macrophages (see Pak C. S. and Filder I. J., Sernin Cancer il. 2: 189, 1991). The direct culmination of all these States is the death paramparent as apoptosis, and necrosis. His role in the early stages of apoptosis briefly described above. Upon reaching apoptotic cells in the final stages of apoptosis (i.e., loss of membrane integrity) will be taken into account that FS in both layers of the plasma membrane will be "displayed" in the extracellular environment. A similar situation exists in cell death by necrosis, in which the loss of membrane integrity is either the initiating factor, or there is in the early stages of the process necrotic cell death, respectively data necrotic cells also exhibited" FS up until "exhibited" both layers of the plasma membrane.
IV. Annexin
Proteins of the family of annexins use for the practical implementation of the present invention. High levels of annexin V in the norm found in the cytoplasm of some cells, including the placenta, lymphocytes, monocytes, epithelium of the bile ducts and kidney (cortical) tubules. Although the physiological function of annexins are not completely understood, some properties of annexins determine their use as diagnostic and/or therapeutic agents. In particular, it was shown that annexins have a very high affinity to V. Summary of experimental results
The experiments conducted to support the present invention have demonstrated that the introduction of annexin radioactive label can be used for imaging cell death in vivo. For example, the experiments in Example 1 are image acquisition and quantification of Fas-mediated death of hepatocytes in response to injection of purified antibody Jo2 in mice (see Ogasawara J. et al., Nature 364: 806, 1993). The results of these experiments (see, for example, Fig. 1) showed an increase in two and four times the absorption liver annexin V with a radioactive label after one and two hours, respectively, specifically due to Fas-mediated death of hepatocytes after injection of the antibody Jo2. It was also noted that the temporary doubling of the absorption of the spleen immediately after the introduction, which subsequently fell to control values. This decrease in signal from the spleen can be caused by rapid removal of lymphocytes from blood and spleen in response to the explosion of Fas-mediated apoptosis after treatment.
The binding of annexin it was also noted in the kidneys. However, this binding was present in the absence of any apoptosis-induzione activity of the kidneys over time after the introduction of AB (antibodies) against Fas together with the increase of absorption in the liver in the same period suggests, that is not related to apoptosis affinity kidney to annexin V is lower than the affinity of apoptotic tissue. Linking cortical substance of the kidney injected annexin V may be partly due to cross-reactivity with annexin phospholipid renal tubules.
It should be noted that there was a small selection labeled annexin through the kidneys, suggesting that radioactive label (in this case TSM) remained associated with annexin during the experiments. Further, the injected labeled TSM annexin rapidly eliminated from the bloodstream, with the half-life in serum is about 3-7 minutes. These factors allowed us to visualize the radiopharmaceutical signal 1-2 hours after its introduction.
The above features provide the opportunity for daily (or two day) research on production of images, each of which represents a "snapshot" apoptotic activity of a tissue or organ of interest, during injection of annexin V with a radioactive label.
VI. Visualization of cell death in vivo
The invention includes in one aspect a method of imaging cell death (due to, for example, adopt the label (for example, technetium 99m annexin V) to introduce the subject. After a period of time for which the conjugate can achieve localization in the body of the subject, the subject is placed in detectiona field detector-rays. Subject support in practically stationary during measurementradiation technetium 99m using detector-rays. After phase measurement design image issue-rays. Designed so the image is then used to provide the attending physician with a card or localized areas of cell death in the body of a mammal subject, or in the analyzed region of the body of a mammal subject.
To facilitate interpretation of the images obtained using the above method, the image may be processed digitally (digitized) to filter background noise and/or non-specific localization (e.g., localization in the kidneys) conjugate annexin/TSM, as described in more detail below.
The advantage of the method described above is that by measuringradiation and the formation of the image is hurtrays from the subject over time, reflecting changes in the number of cells undergoing cell death. This approach can also be used to track changes in the localization issuerays from the subject over time, reflecting changes in the distribution of cells in which cell death occurs.
Synthesis of annexin radioactive label
The invention can be practically implemented using purified native, recombinant or prepared synthetically annexin. Annexin V, for example, can be appropriately obtained from human placenta (see Funakoshi T. et al., Biochemistry 26: 5572, 1987). However, recombinant annexin represents a number of advantages, including ease of access and economic efficiency. In humans and other organisms have been cloned from a number of different annexins. Their sequences are available in databases for sequences, including Genbank.
The invention is preferably implemented practically using annexin V, for several reasons. First, annexin V is one of the annexins present in the greatest quantities, and secondly, it is easy to get from the wounds (see article Tait J. F. et al., Biochem. 27: 6268, 1988). Annexin V is mol. weight of 36 KD and high affinity (KD=7 nmol/l) to phosphatidylserine (FS). The sequence of annexin V can be obtained in Genbank under registration numbers U05760-U05770.
The example expression systems, suitable for annexin for use in this invention, see Materials and methods. It uses expression vector rate (Novagen, Madison, Wisconsin) in E. coli.
Other bacterial expression vectors can also be used. They include, for example, plasmid pGEX (see Smith, D. B. et al., Gene 67: 31, 1988) and its derivatives (for example, pGEX series from Pharmacia Biotech, Piscataway, NJ). These vectors Express the polypeptide sequence of the cloned insertions, fused in reading frame with the glutathione S-transferase. Recombinant plasmid pGEX can be transformed suitable strains of E. coli, and the formation of the fused protein can be induced by adding isopropyl-thiogalactopyranoside. The solubilized recombinant protein can then be purified from cell lysates of induced cultures using affinity chromatography on agarose in accordance with standard methods nd Sons, Inc., Media, PA). Other commercially available expression systems include yeast expression systems, such as gene-expression set on the basis of Pichia from Invitorgen (San Diego, CA); baculovirus expression system (see article'reilly P. R. et al., Baculovirus expression vectors: a laboratory manual ("Laboratory manual of expression vectors based on the baculovirus"), 1992; Beames, et al., Biotechniques 11: 378, Clontech, Palo Alto, CA) and expression systems based on mammalian cells (Clontech, Palo Alto CA; Gibco-BRL, Gaithersburg MD).
In the expression vectors can be engineering introduced a number of features, such as leader sequences which promote the secretion of expressed sequences in the culture medium. Received, the recombinant polypeptides are usually isolated from lysed cells or culture medium.
Selected recombinant polypeptides obtained as described above can be purified by standard methods of protein purification, including differential precipitation, chromatography using a molecular sieve, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography. Preparations of proteins can be concentrated, for example, filtration (Amicon, Danvers, Mass.).
Anne is avisit from the particular application of the claimed method.
The invention can be practically implemented using any of the currently available radionuclides. When choosing a suitable radionuclide practitioner will usually imply a specific application of the invention along with factors common to obtain a radiological image. These factors include (i) the minimum emission of particles, (ii) the amount of primary photon energy between about 50 and 500 Kev, (iii) physical half-life of the decay is greater than the time required to prepare material for introduction, (iv) the effective half life longer than the time of the study, a suitable chemical form and reactivity, low toxicity and stability and practical stability of annexin labeled this radionuclide.
An example of a radionuclide is Tc99m, which has a half-life of approximately 6 hours and can be used for labeling of annexin with high specific activity. It satisfies most of the above criteria and is used in more than 80% of visualization techniques using radioisotopes in medicine. Other isotopes that can be used include iodine 123 (the period of the iodine half-life of ~ 2.8 days).
Binding of the isotope with annexin can be carried out using known techniques. For example, Tc99m can be associated with annexin using group hydrazinonicotinamide (GNA), available at AnorMED, Langley, British Columbia, Canada, as described below in the section Materials and methods. Gallium 67, indium 111 can be used for introduction into proteins radioisotope labels, using, for example, a method described in the article Hnatowich D. J. et al., J. Immunol. Meth. 65: 147, 1983, included in this context by reference.
There are other ways of labeling of proteins with radioactive nuclides. For example, in U.S. Patent No. 5552525 issued 03 September 1996 (Dean), describe the receipt of peptides labeled with technetium 99m (Tc99m). Methods of labeling peptides and polypeptides Tc99m also described in U.S. patent No. 5443815 and 5508020. In article Lind et al., (J. Nucl. Med. 31: 417-473, 1990) described Tc99m labeled monoclonal antibodies. In article LaMuraglia et al. (J. Vasc. Surg. 10: 20-28,1989) described111In-labeled nonspecific human immunoglobulin and article Fischman et al., (J. Nucl. Med. 32: 482-491, 1991) described the conjugates of the chemotactic formulate (fMLF) -111In-labeled, DTPA (diethylenetriaminepentaacetic acid).
Introduction annexin radioactive label
Annexin radioactive label may be the introduction the two main reasons: (i) the number and type of injected radionuclide and (ii) the amount of injected protein annexin.
Technetium (Te) 99m can be entered adult in doses up to about 20 MCI. The preferred dose for a single injection TSM (a radioactive isotope of technetium t) is between approximately 5 and 20 MCI.
Annexin V begins to exhibit pharmacological effects (effects of anticoagulant) at doses exceeding 300 mg/kg, Respectively, the diagnostic methods according to this invention (in which try to overcome the pharmacological effects labeled annexin), preferably implement almost at doses lower than 300 mg/kg, usually lower than 50 µg/kg Data trace doses (for example, from 10 μg/kg to 50 mg/kg) have no pronounced pharmacological or toxic side effects in animals or humans.
Annexin radioactive label is usually suspended in a suitable carrier for delivery, such as sterile saline solution. The carrier may also contain stabilizing agents, carriers, fillers, anticoagulants, emulsifiers, etc., that are known from the prior art.
Annexin radioactive label can be entered any number of ways that are known to be effective for the introduction of proteins with radioactive label to visualize.v.) the injection. It is especially useful for visualization of internal organs with good vascularization, such as heart, lung, spleen, etc. Ways intravenous radiopharmaceuticals known. For example, it is known that the pharmaceutical preparation with a radioactive label is usually injected bolus injection using the method of Oldendorf/horizontal Bar (Oldendorf/Tourniquet) or intravenous way of introduction (see, for example, the monograph Mettler F. A. and M. J. Guiberteau, Essentials of nuclear medicine imaging, ("fundamentals of imaging studies using radioisotopes in medicine, 2nd ed., W. C. Saunders Company, Philadelphia, PA, 1985, Appendix D).
To obtain the image (imaging) of the brain annexin radioactive label can be entered podvoloshino. Intrathecal introduction gives a connection directly into the space under the arachnoid membrane containing cerebral spinal fluid (CSF). Shipping in the region of the spine may also be accomplished by epidural injections in the spine area above the arachnoid membrane.
Other routes of administration include intraperitoneal (for example, for patients undergoing kidney dialysis) and intrapleural administration. When a special application of the invention predusmatriva suppletion and oral, intravaginal and/or rectal administration.
The ways of practical implementation of the above options introduction known in the prior art.
Localization of annexin radioactive label
After the introduction of labeled annexin he are able to be localized in the tissue or target organ. Localization in this context refers to the condition in which the body of the subject is achieved either equilibrium or pseudostable the ratio between bound, "localized", and unrelated, "free" labeled annexin. The amount of time required for this localization, typically of the order of several minutes or tens of minutes. It can be measured by the half-life of labeled annexin serum. In the case of labeled TSM annexin V, intravenously injectable, the half-life in serum is between approximately 3 and 7 minutes. Time localization also depends on the availability of tissue for labeled annexin. This, in turn, depends on the method of administration, as known from the prior art.
Visualization is preferable to start the localization of most of the labeled annexin on its target(s). For injected labeled TSM annexin is(approximately 30-70 minutes in the case of conjugates of annexin/TSM) is considered sufficient to achieve almost complete localization. The expert will appreciate, however, that it may be desirable visualization in a period of time that is less than or exceeds the above period of ~ 10 half-life periods. For example, when visualizing cell death due to damage to the blood vessel availability of the target tissue is very high, with strong signal can be obtained from the site of the target after a few minutes, especially if you gradually introduce low dose of labeled annexin to minimize the signal from the label, circulating in the bloodstream.
In all the above cases, a reasonable estimate of the time required for localization can be done by a specialist. Moreover, after determining the localization as a function of time can be followed by visualization of signalrays labeled annexin according to the methods corresponding to the invention.
Device for detectionrays
Device for forming an image using-radiation function by accumulation of the signal received fromrays emitted by the subject over time. One of the most widely used methods of detection rays emitted by the radionuclide, the protons (usually with the help of crystals of NaI(TI)), which then reinforce the photomultiplier (FU), is converted into a voltage signal and is used to construct the image. Components of the scintillation camera of Angera typically include a collimator, a scintillation crystal, the set FU analyzer amplitude pulses, a cathode-ray tube (CRT) and remote control. Typically, the camera system also includes a computer. Processing between the near FU and display (e.g., CRT) can be either analog or digital. A detailed description of theory and operation-scintillation cameras Angera can be found in any of the many reviews and/or materials of the radioisotope research in medicine (see, for example, the monograph Mettler and Guiberteau, "fundamentals of imaging studies using radioisotopes in medicine", 1985, introduced in this context by reference).
A more informative image can be obtained by using the emission-computed tomography (POS) to obtain 3-the SPECT), in which the use of isotopes, such as TSM, and positron emission tomography (PET), which is based on the annihilation photons at high energy (511 Kev) that provides localization with high accuracy. The disadvantage of PET is that it is usually used with short-lived obtained in the cyclotron isotopes, such as11C,13N and18F. SPECT, on the other hand, you can use described in this context radiopharmaceutical agents (for example, TSM).
The SPECT system typically includes one or two heads-scintillation camera Angera, controlled by computer, which can rotate around the patient in a circular or elliptical orbit. These cameras SPECT are commercially available from several manufacturers. For example, Siemens (Des Plains, IL) sells a few of these cameras, including the E-HIMSELF", "ORBITER", "ECAT", "MULTISPECT3", "MULTISPECT 2" and "DIACAM".
The camera, as described above, now usually include a mounted image processors that can manipulate the image as digital files, in order to remove the background, add color, etc., When images are in the form of digital files, they can be manipulated using a number of programs primetime PC or Apple Macintosh (Apple Computer, Cupertino, CA) and printed.
The location of the subject in the field of devices for detectionrays
1. Field detection device. Field detection (detection) device is defined as land that can be held stable and reliable measurement of emission-rays. When using Actds image acquisition field detection device is all the space to whichradiation can be reliably measured or part of this space, which is programmed into the POS system, as included in the scan. This space is usually much larger than the field of detection of one non-POS camera.
It should be understood that the animal or subject does not necessarily need to be in the field detection device for detecting-rays. For example, if there is an interest in the analysis of the signal from a particular body, to obtain the desired information, one can measure only the signal from the body areas containing the body, and sufficient area surrounding dark areas.
2. The location of the subject: immobilization. To collect the signal, which is used to generate the image measure-radiation, which will be used to construct images. If the signal is strong enough, i.e. such that it is possible to construct the image at issuerays measured within less than approximately 20 MS, and/or the subject is not moving relative to the image plane enough to ruin the picture, then special measures for immobilization is usually not required. Requires only that the subject was placed in the field of devices for detection during measurement.
If, on the other hand, the dimensionradiation takes more than 20 msec and the subject moves, there should be measures to ensure immobilization of the subject during measurement issuerays corresponding to the degree of mobility of the subject, with the aim of preserving the spatial information in the constructed image. For example, in the case where the subject is represented by a man and a measurement time of photon emission is of the order of several seconds, the subject can simply ask to not move, to the extent possible during the measurement issuerays (visualize the emer, using anesthesia or mechanical restraint devices.
Can be constructed in a number of restraint devices. For example, the retaining device, effective for immobilization mouse over tens of seconds to minutes, can be constructed by fixing plate transparent torays over the foam cushion. At one end of the cushion has a recess for the head of the animal. The animal is placed under the plate so that his head is on deepening that allows you to breathe freely, although the movement of the body is limited to the foam cushion.
It can be understood that the depicted region may include virtually all of the subject or part of the subject, which is needed in the diagnosis or monitoring of cell death. For example, this area may include only an appendage or part of such appendage, head, Central nervous system or internal cavity such as the chest or abdominal cavity. In some cases, of the invention, the data field can contain only the selected organ or part. For example, the method can be used to analyze cell death only in the Central nervous system, the. In addition, the analyzed area can be limited to a tumor, for example, cancer patients undergoing treatment, designed in such a way that causes cell death in the tumor.
Design image issuerays: processing image
In the most suitable cameras emissions measurementsrays generates a voltage signal that is either displayed on a CRT or stored and/or analyzed by the computer in the form of a sequence of numbers. These numbers are used to create images of standard visualization techniques. For example, the image is usually analyzed by standardizing single pulsesrays (or relative to a fixed pre-selected values or relative to the maximum number defined in any pixel (picture element), and the transformation of the standardized numbers in luminance (black-and-white scale) or color (color), which are displayed on the monitor. In the false-color view of a typical color codes are as follows. Pixels with zero values indicate black, with low values in blue, and increasing �947.gif">-rays. The position of the colors on the monitor represents the distribution of emissionrays and, accordingly, the position of the zones of cell death.
If it is desirable to trace the localization and/or the signal over time, for example, to register the effects of treatment on the distribution and/or localization of cell death, dimensionradiation or imaging can be repeated over selected time intervals to create a series of images. Intervals can be short, such as minutes, or long, such as days, weeks, months, or years.
Image created in a manner consistent with this invention can be analyzed by various methods. Their range extends from simple visual examination, produced in the mind of the assessment and/or print copies on a solid medium to complex analysis of digital images.
VII. Applications
The main applications of annexin V with a radioactive label include inappropriate detection of apoptosis in disease States in which it should not happen, for example in immune disorders such as lupus, transplant rejection or in cells of a subject, increasing tirovannyh virus.
The results presented in this paper indicate that annexin radioactive label can be used in several clinical studies, where necessary monitoring of apoptotic and/or necrotic cell death, such as, but not limited to, rejection of, or damage to an organ transplant or bone marrow, infectious and non-infectious inflammatory diseases, autoimmune disease, myocardial and cerebral ischemia or infarction, cardiomyopathies, atherosclerosis, nerve and neuromuscular degenerative diseases, sickle cell disease,-thalassemia, cancer therapy, AIDS, myelodysplastic syndromes and induced toxin liver disease, etc., Annexin radioactive label can also be used as a tool for clinical research to study the normal immune system, embryonic development and immunological tolerance and allergies.
Annexin V with a radioactive label can be used, for example, for visualization and quantitative analysis of apoptotic cell death in normal and malignant tissues exposed Leche which can be used for rapid testing and development of new medicines and methods of treatment of many diseases. In addition, the methods can be used to monitor the progress of treatment, monitoring disease progression, or both. Further, they can be used for early detection of certain diseases.
The following examples illustrate the invention but are in no way intended to limit the present invention.
Materials and methods
A. Preparation of GNA-labeled annexin V
Human annexin V was obtained by expression in E. coli plasmids RETA-RAR and purified as described previously (see B. L. Wood et al., Blood 88: 1873-1880, 1996, introduced in this context by reference). 30 mm stock solution ("mother solution of ester STA") N-hydroxysuccinimide complex ester hydrazinonicotinamide (GNA; obtained from AnorD, Langley, British Columbia, Canada; see J. W. Babich et al., Journal of Nuclear Medicine 34: 1964, 1993, introduced in this context by reference) were prepared by suspendirovanie 220 mg of the hydrochloride Succinimidyl 6-hydrazinonicotinamide (SGBV) 18.5 μl of N,N-dimethylformamide (DMF). Five mg of annexin V, dissolved in 893 μl of buffer A (20 mm HEPES, pH 7.4, 100 mm NaCl), reacted with the mother solution of ester STA for three hours with gentle stirring, in a closed place at room temperature the links. The reaction was stopped by adding 500 μl 500 mm glycine, pH of 5.3, and then spent dialysis at 4With against 20 mm sodium citrate, pH 5,2, 100 mm NaCl. The precipitate was removed by centrifugation at 1500 g for 10 minutes. Aliquots of 100 μl (100 μg) STA-annexin V was stored at -70C.
C. incorporation of radioactive label in the GNA-annexin V
80 μl of SnCl2(50 mg/ml in 0.1 N HCl, after which, within two hours blew gaseous N2) were added to 50 ml of a 20 mm solution of Tricine (pH of 7.1 through which within one hour blew gaseous N2; Trizin=N-[Tris(hydroxymethyl)methyl]glycine). 200 μl of a solution of Sn-tricin was added to 100 μl of Tc99m (activity 4-8 MCI), mixed with an aliquot of 100 ál of annexin V (prepared as described above) according to the methods described in Larson et al., Bioconjugate Chem. 6: 635-638, 1995.
The specific activity of the annexin radioactive label was 20-200 µci/µg of protein (depending on the desired activity) when radiosistemi 92-97%, a specific instant thin-layer chromatography (MDH) using saline as a solvent. Activity binding membrane GNA-annexin V and labeled TSM GNA-annexin replace the I125-annexin V (see B. L. Wood et al., Blood 88: 1873-1880, 1996). After 15 minutes of incubation at room temperature the sample is centrifuged, FITZ-annexin V, associated with the pellets of cells, release using EDTU (ethylenediaminetetraacetic acid) and define released FITZ-annexin V by fluorometry. In this system analysis unmodified annexin, GNA-annexin and TSM-GNA-annexin V was competitive inhibition (50% concentration of binding FITZ-annexin V) 8 nm and 10.5 nm and 12.3 nm, respectively. Introduction GNA in annexin V, as shown, is 0.9 mol/mol annexin V.
C. Visualization and research bearsdley
Mice injected 50-150 µci TSM-GNA-annexin (0,125-0,25 µg protein) after determining the ratio of free and bound TSM using MTCH with a saline solution as a solvent. Images of mice lying on his stomach, get through one to two hours after injection of radiopharmaceutical. Image get in 15 minutes, using low-energy bending (LEM) scintillation camera with a highly sensitive parallel collimator and matrix for image 128128 (Siemens, Des Plains, IL). The same protocols ispolzuetsa cervical lymph nodes/salivary glands, brain, thymus, heart, lungs, liver, spleen, stomach, gastrointestinal tract, kidney, skeletal muscle, fat, blood, and the remaining skeleton. The samples were calculated using autogamma scintillation counter Packard Cobra II (Packard Instrument, Downers Grove, IL), expressed as the adjusted pulses/minute for isotopic labels and background activity.
D. Immune staining associated human annexin V and apoptotic nuclei
From formalin fixed immersed in paraffin tissue to make the cuts with a thickness of 5 μm for staining with hematoxylin/eosin or other means. Immune staining associated human annexin V was performed using rabbit antisera generated against human placental annexin V and affinity purified recombinant annexin V bound to Affi-gel (Bio-Rad). Then immunohistochemical determination was performed by consecutive stages of incubation with labeled Biotin goat antibody against rabbit and a complex of avidin-horseradish peroxidase (Jackson Measurement Research) followed by reaction with 3,3'-diaminobenzidine as described Bindl and Warnke (see Bindl J. M. and R. A. Warnke, Am. J. Clin. Pathol. 85: 490-493, 1986, introduced in this context by reference).
-exonuclease (Life Technologies, Gaithersburg, MD) at a concentration of 5 units/ml for 30 minutes at 37 
Apoptotic nuclei identified in histological sections using the TUNEL staining of disintegration of nuclear DNA using a commercially available kit peroxidase (APORTAG, Oncor, Gaithersburg, MD). As indicated by the data given in table. 3, apoptosis, obviously, is in myocytes and cells of the zone of inflammation at the time of rejection of the cardiac allograft.
As the results, shown in Table 4 below, and the graph shown in Fig. 4, the absorption TSM-GNA-annexin V correlates with histological grades of acute rejection.
Example 3
Visualization of in vivo treatment of lymphoma in mice
TSM-GNA-annexin V cook almost, as described above. Visualization >/p>Murine b-cell lymphoma S (Maloney D. J. et al., Hybridoma 4: 191-209, 1985) were cultured in C3H mice.HeN (Narean Breeders, Indianapolis) after subcutaneous injection of 400 tumor cells suspended in 200 μl of RPMI medium 1640 (without serum) in the left side. Fourteen days after implantation, mice treated by intraperitoneal injection of 100 mg/kg cyclophosphamide. Mice intravenously injected with 25-50 μg/kg TSM-GNA-annexin V (100-150 µci/animal) in 20 hours after administration of cyclophosphamide. Then get a picture of the animal and kill it in 2 hours after injection of radiopharmaceutical preparation after removal of the tumor for scintillation counting and histological studies.
Untreated lateral tumor implants (n=8) are clearly visible when imaging with a scintillation camera and have 365% excess absorption annexin V relative to the normal activity of healthy soft tissue, as shown by image analysis PIO. Treated side of the tumor (n=6) show legkouswoyaema increased activity TSM-GNA-annexin V to 78% relative to the control, expressed as the activity of the whole body/g tumor (P<0.05 using two-way t - student criterion for determining the dignity of irout 132% increase uptake of annexin V, expressed as percentage of injected dose/g of tumor (P<0.05) in 58% wage decrease in weight (P<0.05) as compared to control. The activity of the whole body/g tumor, as shown by the image analysis PIO has a linear dependence on the percentage of injected dose/g of tumor that is determined at the study bearsdley (r2=0,831). Histological analysis demonstrated virtually complete (>95%) apoptosis of all lymphoblasts in treated tumors with less than 5% of apoptotic cells in controls.
Since the invention has been described with reference to specific methods and embodiments of, take into account the possibility of various modifications and changes without going beyond the scope of the invention.
Claims 1. Method of imaging cell death in the body of a mammal subject in vivo, namely, that the subject is administered annexin labeled biocompatible radionuclide, and then place the subject within the detection field of the radiation detector to construct images of radioactive emissions after a period of time for which labeled annexin reaches localization in the body of the subject, and the specified image is odabrao additionally carry out image processing for clipping the signal, due to non-specific localization of labeled annexin. 3. The method according to p. 2, characterized in that the specified nonspecific localization is localized in the kidneys. 4. The method according to any of paragraphs.1-3, wherein the radionuclide is selected from the group consisting of iodine 123, iodine 131, gallium 67, indium 111, fluorine 18 and technetium 99 m (Tc99m). 5. The method according to any of paragraphs.1-4, wherein the radionuclide is represented by technetium 99m (Tc99m). 6. The method according to p. 5, characterized in that Tc99m associated with annexin through hydrazinonicotinamide. 7. The method according to p. 5 or 6, characterized in that the labeled annexin is administered in an amount to provide a dose of between about 5 and about 20 MCI. 8. The method according to any of paragraphs.1-7, characterized in that the radiation detector is a detectorradiation of the radioactive emission is the emission-rays.
9. The method according to p. 8, characterized in that said detectorrays is a-scintillation camera.
10. The method according to p. 8 or 9, characterized in that for constructing the image measure emissions-Lucera>11. The method according to any of paragraphs.8-10, characterized in that for constructing the image measure emissionsrays approximately 1 hour after administration of labeled annexin.
12. The method according to any of paragraphs.1-11, characterized in that the cell death caused by apoptosis.
13. The method according to any of paragraphs.1-12, characterized in that after the selected time intervals of the subject re-placed in the field detected by the radiation detector and measure radioactive emissions radionuclide, localized in the body of the subject, and the specified repeating is effective to track changes in the intensityradiation from a specified area over time, reflecting changes in the number of cells in which cell death occurs.
14. The method according to any of paragraphs.1-13, characterized in that after the selected time intervals of the subject re-placed in the field detected by the radiation detector and measure radioactive emissions radionuclide, localized in the body of the subject, and the specified repeating is effective to track changes in the localizationradiation in this region over time, reflecting changes in localization glue the doctrine is a camera for obtaining a three-dimensional image.
16. The method according to any of paragraphs.1-15, characterized in that the annexin is annexin V.
17. The method according to any of paragraphs.1-16, characterized in that the number entered labeled annexin does not exceed approximately 300 μg protein/kg
18. The method according to any of paragraphs.1-17, characterized in that the number entered labeled annexin is between approximately 1 and 10 μg protein/kg
19. The method according to any of paragraphs.1-18, characterized in that the labeled annexin administered intravenously.
20. The method according to any of paragraphs.1-18, characterized in that the labeled annexin is administered intraperitoneally.
21. The method according to any of paragraphs.1-18, characterized in that the labeled annexin enter podvoloshino.
22. The method according to any of paragraphs.1-18, characterized in. that labeled annexin enter intrapleural.
23. The method according to any of paragraphs.1-18, characterized in that the labeled annexin enter intralymphatically.
24. The method according to any of paragraphs.1-18, characterized in that the labeled annexin is administered intramuscularly.
25. The method according to any of paragraphs.1-24, wherein the designated area includes almost the whole of the subject.
26. The method according to any of paragraphs.1-24, wherein the designated area includes the head or part of it.
27. The method according to any of paragraphs.1-24, Otley is the, that the specified region includes the liver or part of it.
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