EP1221977A2 - Detection de tumeurs par imagerie et leur traitement - Google Patents

Detection de tumeurs par imagerie et leur traitement

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Publication number
EP1221977A2
EP1221977A2 EP00969788A EP00969788A EP1221977A2 EP 1221977 A2 EP1221977 A2 EP 1221977A2 EP 00969788 A EP00969788 A EP 00969788A EP 00969788 A EP00969788 A EP 00969788A EP 1221977 A2 EP1221977 A2 EP 1221977A2
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EP
European Patent Office
Prior art keywords
level
cells
blood flow
oxygenation
tumor cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00969788A
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German (de)
English (en)
Inventor
Ilan Tsarfaty
Miriam Shaharabany
Rinat Abramovitch
Tammar Kushnir
Galia Tsarfaty
Yacov Itzchak
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FBIT Ltd
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FBIT Ltd
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Publication of EP1221977A2 publication Critical patent/EP1221977A2/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention concerns methods for the detection of tumor foci by imaging and methods for enhancing the efficacy of anti-tumorigenic treatments as well as treatments of other diseases.
  • the invention also concerns compositions for use in such methods and kits for carrying them out.
  • Imaging of tumors is based on structural analysis of mammography, ultrasound and magnetic resonance (MR) images.
  • contrast agents are often added which make the blood vessels more clear and the tumor site more visible.
  • An endogenous contrast agent, the blood is also used in magnetic resonance imaging (MRI) techniques and is known as blood oxygen level dependent (BOLD) contrast. Changes in oxygenation of the blood can be observed as signal changes in BOLD MRI measurements.
  • deoxyhaemoglobin which is a paramagnetic molecule
  • oxyhaemoglobin which is diamagnetic
  • MRI functional magnetic resonance imaging
  • angiogenesis During the development of the tumor, through a mechanism termed angiogenesis, the tumor becomes substantively vasculated. As the tumor grows in size, a large part of the blood flow reaches the outer part of the tumor, while its inner part receives much less blood flow.
  • Various agents have been used for increasing tumor blood flow and vascular permeability of a tumor in the attempt to enhance the effectivity of various anti-tumorigenic treatments.
  • Angiotensine (APII) and tumor necrosis factor (TNT) were, for example, used to try and enhance the activity of monoclonal anti-tumor antibody immunoconjugates (Takeda, A. et al, 1999). All of the known agents used for this purpose caused non-specific vasodilatation of blood vessels.
  • Angiotensine was also shown to enhance general tumor microvascular pressure leading to an increase in uptake of specific antibody in the tumor (Netti P.A. et /., 1999).
  • Non-specific haemodynamic changes were also shown to be induced by other agents such as, for example, by the calcium-channel blocker, diltiazem (Muruganandham M. et al, 1999).
  • BOLD contrast MR imaging measurements show the intensity enhancement after treatment with diltiazem, which was used as a radiosensititator for tumor radiotherapy. The increase in tumor blood flow caused by diltiazem was non-specific.
  • Various growth factors and cytokines such as epidermal growth factor
  • EGF EGF
  • TNF- ⁇ TNF- ⁇
  • HGF/SF Hepatocyte Growth Factor/Scattering Factor
  • Met tyrosine kinase
  • Met-HGF/SF signaling and Met amplification, over expression or activation have been associated with a variety of additional human cancers including sarcomas, brain tumors, renal carcinomas, ovary carcinoma, prostate cancers and thyroid cancers.
  • the effect of the Met receptor and its ligand on tumor growth was shown to be by increasing division rate of the tumorgenic cells and/or by increasing angiogenesis of the tumor (Rosen, E.M., et al, 1995 and Tolnay E., et al, 1998).
  • HGF/SF was also shown to decrease mean arterial pressure and to increase the heart rate in conscious instrumented rats (Yang et al, 1997).
  • injection of an agent which activates specific receptors expressed by tumor cells in a tumor to an individual having such a tumor enables to detect the presence of such tumor cells in the individual using imaging methods.
  • the activation of the tumor cells results in a substantive in vivo increase of oxygen consumption by active tumor cells.
  • the increase in oxygen consumption leads to an increase in blood flow to the tumor which, in itself is detectable by imaging methods such as Doppler ultrasound.
  • the increase in blood flow results in increased oxygenation in the area of the tumor cells which is detectable by imaging methods such as functional MRI(fMRI).
  • the blood flow and blood oxygenation will be, at times, referred to as "blood hemodynamics ".
  • the detection of tumor cells in an individual according to the invention is thus based on the specific physiological reaction of the tumor to the administered agent and, for the first time, detection of the changes in the tumor by imaging techniques.
  • HGF/SF Hepatocyte Growth Factor/Scattering Factor
  • injection of HGF/SF to mice bearing the DA3 mammary carcinoma tumor resulted in in vivo enhancement of BOLD images and changes in Doppler ultrasound flow measurements in regions comprising cells which express high levels of the HGF/SF receptor.
  • the changes in blood hemodynamics is specific to those areas in which there are cells expressing high levels of the receptor which the injected agent activates. Other areas in which the specific receptor was expressed at a low level or not expressed at all did not show such changes in blood hemodynamics.
  • the method of the present invention enables to change the blood hemodynamics specifically in regions in which there are cells which physiologically react to the activating agent in a manner which is detectable by imaging techniques.
  • the differences in blood hemodynamics following administration of the agent may vary in different regions of the tumor.
  • BOLD measurements blood oxygenation
  • a decrease in oxygenation levels in these peripheral regions is also typically found.
  • a relatively simple and short term method for detecting or monitoring tumor cells is provided.
  • routinely used imaging techniques such as MRI and ultrasound techniques
  • the detection of tumor cells is carried out non-invasively by externally monitoring the level of oxygenation (measured as BOLD images) and blood flow (seen by Doppler ultrasound measurements) to the cells in a tested tissue of the individual.
  • the method is used to identify such regions in an individual that does not have a pre-known tumorigenic lesion. Such identification is based on comparing measured oxygenation and blood flow in a tested tissue before and after administration of an agent to the tested individual.
  • the level of oxygenation relates to the amount or concentration of oxygen in the blood reaching the cells in the tested tissue. Changes in the level of oxygenation result from changes in the level of oxygen consumption of the cells.
  • the oxygenation level is detected by any of the known imaging methods such as functional magnetic resonance imaging (fMRI) using any of the devices routinely used in these methods and is measured as alterations in BOLD imaging signals.
  • fMRI functional magnetic resonance imaging
  • ROI analysis by ROI analysis.
  • ROI region of interest
  • time lapse analysis is carried out by using appropriate computer programs such as an
  • the term "blood flow" relates to the amount of blood flowing into the close vicinity of the tumor cells and to the tumor cells themselves in the blood vessels surrounding them.
  • the change following administration of the agent in accordance with the invention may be elevation of 5 the blood flow to the tumor, while in other areas of the tumor or its surrounding the effect of the agent administered may be a reduction in the blood flow.
  • the blood flow may be measured by Doppler ultrasound imaging which measures volume and velocity of the blood flowing to the cells. This technique is especially suitable for screening of a large number of regions of an individual, and in wide-scale l o screening of a population.
  • an average value is calculated for detected regions based on measurements over several minutes following a period of time after administration of the activating agent as compared 15 to measurements over the same period of time following administration of a control agent.
  • a method for identifying regions having a high probability of containing tumor cells in a tested tissue of an individual, said method comprising the steps of: 20 (i) measuring the level of oxygenation and/or blood flow in a tested tissue of a tested individual; (ii) administering to said individual an agent which enhances oxygen consumption and/or blood flow of said tumor cells; (iii) measuring the level of oxygenation and/or blood flow of said tested tissue; 25 and
  • the level of oxygenation and/or blood flow which is measured before administration of the agent may be measured following administration of a control agent.
  • control agent may be any physiologically tolerant agent which has no influence on blood oxygenation or blood flow in the treated individual such as, for example, saline.
  • the "agent" used in accordance with the invention may be any substance which, when coming in contact with tumor cells, results eventually in enhancement of the tumor cells oxygen consumption that will result in higher oxygenation and in a change in the blood flow to the tumor cells.
  • the increase in oxygen consumption of cells following their contact with the tested agent in vitro may be measured by any of the known in vitro methods.
  • oxygen consumption can be measured in vitro by perfusion experiments, in which the oxygen content in the perfusion medium is compared to the oxygen content in the effluent medium of the cells (see Examples).
  • Such an agent may, for example, be a ligand of a receptor expressed by the cells, an antibody directed against such receptors or other components of the cells, as well as derivatives and analogues of such substances which maintain their ability to cause enhanced oxygen consumption in tumor cells.
  • the amount of the activating agent necessary for obtaining the desired enhancement in oxygenation and/or blood flow level to the tumor cells which may be required to enable detection by any of the imaging techniques mentioned above, is a very low amount which is below the amount of activating agent which may cause a long-term activation effect in the tumor.
  • the amount of the agent is typically close to the physiological level of said agent, which, for example, in the case of HGF/SF is an amount similar to that which is freed into the bloodstream in an individual having a typical cut or injury.
  • the administered agent may be administered to the individual by any of the known administration modes, such as intravenously (i.v.), per os, subcutaneously (s.c), interperitoneally (i.p.) locally, etc.
  • the agent is adminstered i.v. and the oxygenation level of the tested tissue is then measured at various time intervals following its injection.
  • substantially higher should be understood to mean a level of oxygenation of the tested tissue following administration of the agent which is statistically significantly higher than the level of the oxygenation of the same tested tissue before administration of the agent to the individual.
  • a cut-off value is predetermined on the basis of a large number of measurements of these parameters prior to and following administration of the activating agent in non-malignant tissue. These measurements may be accumulated at a database which may be updated from time to time on the basis of accumulation of additional measurements.
  • the cut-off value is expressed as a percentage of enhancement or reduction of the signal following administration of the activating agent.
  • the measured value of the tested tissue is calculated as described above, and its value is then analyzed in comparison to the predetermined cut-off value.
  • the cut-off value is determined for a specific set of parameters (e.g.
  • the type of activating agent and/or kind of imaging technique used can be used as a basis of comparison to the calculated value of the tested tissue under the same parameters.
  • the value of the calculated index of the tested tissue differs from the predetermined cut-off value, there is a high probability that the tested tissue contains tumor cells.
  • the predetermined cut-off value may be a range of values having a low cut-off value and a high cut-off value, to which the calculated value of the tested tissue is compared.
  • the enhanced oxygen consumption of tumor cells and/or enhanced blood flow to the tumor cells following administration of said agent to the tested individual is a result of activation of said tumor cells.
  • activation may be a result of binding of a specific ligand to its receptor which initiates a signal pathway in the cells resulting in their activation.
  • activation of the cells may be as a result of their contact with any other substance which activates the cells by any cellular pathway.
  • activation renders any change that occurs within the cells as compared to their inactivated condition including metabolic changes, over-expression of a DNA sequence, or a protein activation of signal transduction pathways, effecting ion channels, etc.
  • Such activation is manifested in enhanced oxygen consumption resulting in enhanced oxygenation, alteration of the BOLD signal and/or a change in the blood flow level of the tumor cells which is measured by any of the methods mentioned above.
  • the activation of the tumor cells is obtained by activation of a specific receptor expressed by the cells.
  • a typical example of such receptors is various growth factor receptors expressed on the membrane of tumor cells.
  • the activation of the receptor may be direct or indirect activation. Direct activation is a result of contact of a specific ligand or its derivatives or any other substance which is capable of specific binding to the receptor (e.g. a specific ligand or antibody) resulting in activation of the receptor.
  • Indirect activation of a receptor may be caused by any substance which does not specifically bind to the receptor, but its contact with the tumor cells results, eventually, in activation of said receptor.
  • Activating agents may also be substances which activate such receptors indirectly by elevating the level of receptor ligands in the blood which, in turn, activate one or more receptors.
  • the tumor cells may express more than one receptor in which case several receptors may be activated one at a time or several at a time.
  • the activation of more than one receptors may be obtained by administration of several different agents, each activating (directly or indirectly) one or more receptors or, alternatively, by administering an agent which activates more than one of the receptors. Combined or sequential activation of more than one receptor on the tumor cells may enhance the signal detected by the various imaging techniques.
  • tyrosine kinase receptors such as the Met, erbll and others are expressed on breast cancer tumors as well as other kinds of tumors at a higher level than their expression on normal cells.
  • the tumor cells present in a tested tissue may be activated by activation of such tyrosine kinase receptors by an agent which directly or indirectly activates the receptors in a manner which is manifested in enhanced oxyge consumption of the activated cells, which may be detected by an imaging technique.
  • more than one kind of tyrosine kinase receptor may be activated on the tumor cells either by a single administered agent or by a combination of several agents and the combined activation of several tyrosine kinase receptors may further enhance the oxygen consumption of the tumor cells and as a result increase the signal detected by the imaging technique.
  • Met tyrosine kinase growth factor receptor and its ligand HGF/SF have been shown to be over-expressed in breast tumors.
  • Met expression has been shown to be upregulated in metastatic breast tumors, as compared to the primary breast tumor.
  • Met-HGF/signalling and Met amplification, over expression or activation by point mutation have been shown to be associated with a variety of human cancers including sarcomas, brain tumors, renal carcinoma, ovary carcinoma, prostate cancer and thyroid cancer. Being over expressed in primary metastatic tumors, the Met receptor is a good candidate for being activated by an agent in a manner which can be selectively detected by imaging techniques in accordance with the invention.
  • the method of the invention is used for detecting regions having a high probability of comprising tumor cells in a pre-suspected lesion of an individual.
  • a suspected lesion may be a region in which there is a high probability of existence of primary tumor cells based on analysis by routinely used methods such as histological methods, radioimmunological methods, etc.
  • Such a suspected region may also be in an individual having a high risk of developing a primary tumor due, for example, to hereditary risk factors, exposure to carcinogenic agents, etc.
  • such a lesion may be a region in which there is high probability of existence of a secondary metastatic tumor such as in an individual who had a preliminary tumor which was treated or irradicated by routine methods such as irradiation, chemotherapy or surgery.
  • areas which are considered "non-malignant" areas are determined and the suspected lesion is compared to these areas.
  • the level of blood oxygenation and blood flow is compared between the suspected lesion and the non-malignant lesion before administration of the agent in accordance with the invention, and the two areas are compared once more following administration of the agent to the tested individual.
  • the enhanced blood oxygenation and difference in blood flow following administration of the agent in the suspected lesion is compared to the increase in blood oxygenation and/or blood flow following administration of the agent in the non-malignant areas of the same individual.
  • the method measures the increase in blood oxygenation levels this will typically be carried out by fMRI.
  • the blood flow to the tumor cells this will typically be carried out by Doppler ultrasound imaging.
  • a method for detection of a region with a high probability of comprising tumor cells in a suspected tissue of an individual comprising:
  • a method for detection of a region with a high probability of comprising tumor cells in a suspected tissue of an individual comprising: (i) measuring the level of blood flow in said suspected tested tissue; (ii) measuring the level of blood flow in a non-malignant tested tissue of said individual;
  • the values measured in the malignant and non-malignant tissues are analyzed with respect to a cut-off value or range of cut-off values based on measurements of the non-malignant tissue as described above.
  • a large number of measurements of the enhancement of blood oxygenation or the changes in blood flow in the non-malignant tissues may serve as a basis for calculating such a cut-off value.
  • a ratio is calculated on the basis of the measured values of each of these parameters before and after administration of the agent in accordance with the invention in the suspected area are than compared to the predetermined cut-off value or values.
  • a calculated ratio of oxygenation which is higher than the cut-off value calculated for this parameter or a calculated ration of blood flow which differs from the cut-off value calculated for this parameter indicates a high probability that tumor cells exist in the suspected lesion.
  • the enhancement in oxygen consumption of the tumor cells and thus the enhancement in blood oxygenation of the tumor in the suspected tissue may be as a result of activation of the tumor cells by the administered agent.
  • Such activation may be as a result of activation of a receptor expressed by the tumor cells activated by an agent which activates a receptor directly or indirectly.
  • such a receptor is a tyrosine kinase receptor and the agent in the composition of the invention will be an agent which is capable of activating tyrosine kinase receptors directly or indirectly.
  • an agent may, for example, be HGF/SF, Her2/ neu ligand, EGF etc.
  • the tumor cells may at times be actived by more than one agent. Thus for example, two different ligands may be used, each activating a different receptor on the cells.
  • the tyrosine kinase receptor is a Met receptor.
  • the agent in the composition will be any agent which directly or indirectly activates the Met receptor.
  • Direct activators may be the ligand HGF/SF and derivatives and analogues of HGF/SF which specifically bind to the receptor and activate it.
  • Indirect activators may, for example, be substances such as Heparin which administration results in an increase in the level of HGF/SF and eventually in the activation of the Met receptor and the cells expressing it.
  • agents which activate the Met receptor and may be used in accordance with the invention are specific ligands and antibodies which bind and activate the Met receptor.
  • the tumor cells are metastatic breast cancer tumor cells.
  • the patient undergoes an additional operation during which lymph nodes are removed and checked for the existence of metastatic tumor cells.
  • This procedure which is very important for determination of disease staging, is very difficult for the patient to recover from and also only provides sample information from the several lymph nodes which were removed.
  • the existence of areas having a high probability of comprising metastatic breast tumor cells in all the regional lymph nodes of such a patient may easily be detected without the need for further operating or hospitalization. Additional distal metastasis may also be discovered using this method. Since in the lymph node the non-malignant cells express very low levels of the Met protein, it is especially advantageous to use the method in accordance with the invention, wherein an agent which activates the Met receptor in the tumor cells is used.
  • the method is used in an individual following removal of a primary tumor and during chemotherapy or radiation treatment.
  • secondary metastic tumors are usually not removed, but are expected to shrink and disappear following such treatment.
  • the method of the invention may be used at various times during the administration of such treatment to monitor the existence of secondary metastatic cells in such an individual and/or the response of such cells to the treatment.
  • the method of the invention may be used as a confirmation procedure of currently used diagnostic methods such as mammography.
  • the detection of areas in a lesion having high probability of comprising mammary tumor cells may also be based on activation of additional tyrosine kinase receptors other than the Met receptors such as the erb II receptor which is found to be over-expressed in about 20%-30% of breast cancer patients.
  • compositions are provided for use in any of the above methods of the invention.
  • Such compositions comprise as an active ingredient an effective amount of at least one agent which enhances the oxygen consumption of tumor cells present in the tested tissue.
  • the agent administered to the individual is such which can activate said tumor cells
  • the agent s in the composition will be agents capable of activating tumor cells.
  • the agents in the composition will be such which can activate said receptor directly or indirectly on the tumor cell.
  • compositions will also comprise a pharmaceutically acceptable carrier which may be any of the carriers known in the art.
  • a pharmaceutically acceptable carrier may be, for example, soluble physiologically acceptable carriers such as saline, PBS, etc. or solid state carriers such as, for example, latex beads.
  • the composition may also comprise additional non-active substances such as diluents.
  • kits are provided for carrying out any of the above methods.
  • a kit will typically comprise at least one agent which enhances the oxygen consumption of the tumor cells to be detected, at least one predetermined cut-off value, optionally an algorithm for calculating a diagnostic index and instructions for use.
  • said kit may contain means for being connected to a computerized database comprising a collection of measured values and cut-off values which are periodically updated.
  • the present invention provides also the following therapeutic aspects.
  • the present invention thus provides a method for testing the sensitivity of tumor cells to a specific therapeutic drug or treatment comprising:
  • the change in blood oxygenation or blood flow of tumor cells which is detectable by imaging techniques may enable testing susceptibility of the tumor in an individual to an intended chemotherapeutic drug or to a potential anti-tumorigenic treatment.
  • the individual may be treated with a low dose of the intended drug, and the change in blood flow to the tumor cells and/or the enhancement in blood oxygenation levels of the tumor in an individual may be analyzed by appropriate imaging techniques.
  • This pre-treatment testing will enable to prevent non-efficient chemotherapeutic treatments and the side effects associated with such treatments and will enable to treat the individual with a chemotherapeutic drug to which the specific tumor in the individual is most sensitive.
  • the invention further provides a method for testing the sensitivity of a tumor in an individual to a tested anti-tumorigenic treatment or drug comprising: (i) measuring the level of blood oxygenation of said tumor and/or of blood flow to said tumor in said individual; (ii) administering a low dose of said tested anti-tumorigenic treatment or drug to said individual; (iii) measuring the level of blood oxygenation of the tumor and/or of blood flow to the tumor in said individual; wherein a higher level of measured blood oxygenation and or blood flow in (iii) as compared to the level of measured oxygen in (i) indicating susceptibility of said tumor to the tested tumorigenic treatment or drug.
  • low dose is to be understood as a dose of anti-tumorigenic treatment or drug which is sufficient to enhance the hemodynamics of the tumor and which is not higher than the dose intended for treatment of the tested individual.
  • the dose to be used may be determined by a person versed in the art relatively easily, for example, by carrying out several preliminary experiments in vitro, in which the target tumor cells are contacted with various doses of the intended antitumorgenic treatment or drug.
  • a cut-off value or range of cut-off values may be determined on the basis of preliminary experiments in which the intended anti-tumorigenic treatment or drug is contacted with tumor cells which are not susceptible to said drug or treatment.
  • the measured levels of oxygen consumption of the tumor cells in vitro, or of the hemodynamics of the tumor cells in vivo before and after administration of the tested anti-tumorigenic treatment or drug may then be compared to the cut-off values, wherein measured levels substantially differing from the cut-off values indicate sensitivity of the tumor cells to the tested anti-tumorigenic treatment or drug.
  • the method of the invention is especially suitable for screening for susceptibility to inhibitors of receptors which are intended to be used as therapeutic drugs.
  • receptor inhibitors may, for example, be small molecules, peptides or antibodies which inhibit the expression or activity of such receptors, antibodies directed against such receptors, etc.
  • the common method today to screen patients for susceptibility to such inhibitors is by measuring expression levels of the receptors themselves in biopsies obtained from the individual to be treated containing target cells expressing the receptors against which the inhibitors are intended.
  • this method is not applicable in many cases such as, for example, wherein the target cells are metastatic tumor cells, and the results of this method are very often not sufficiently indicative. In such cases, patients are treated for a long period of time with a drug which has no effect on the target cells before it is realized that the treatment is non-effective and that alternative treatment should be searched for.
  • a ligand of the receptor is administered to the individual and the effect of the ligand on the oxygen consumption of the cells expressing the receptor is tested by fMRI and Doppler ultrasound imaging as described above.
  • the administered ligand does not change the oxygen consumption of the cells, it is then most likely that it will not be effective to use an inhibitor of the receptor to which the ligand binds as a therapeutic treatment in the tested individual.
  • the intended inhibitor drug is then administered to the individual in conjunction with the activating ligand and the effect of the inhibitor on the level of oxygen consumption of the target cells by the ligand is measured again using the above-mentioned methods.
  • the ligand itself resulted in reduction of the oxygen consumption of the cells, an inhibitor which will elevate the oxygen consumption of the cells to the normal level will be regarded as a potentially effective drug.
  • the ligand itself enhanced the oxygen consumption of the target cells, an inhibitor which will reduce the oxygen consumption of the target cells to the normal level will be regarded as a potentially effective drug.
  • a receptor inhibitor which has a good chance of being effective in the treatment of the tested individual is one which has a contrasting effect on the oxygen consumption of the target cells as compared to the effect of the ligand on the same target cells.
  • Any receptor inhibitors may be evaluated by this method such as. for example, tyrosine kinase receptor inhibitors such as inhibitors of EGF receptor. Her2 receptor (the target of Herceptine), inhibitors or hormone receptors such as estrogen receptors, etc.
  • the tested inhibitor ⁇ vill be a Met receptor inhibitor.
  • the above method is suitable for testing receptor inhibitors of receptors which are involved in a wide scope of disorders and diseases.
  • the receptor inhibitors are such which are intended to be used as anti- cancer drugs.
  • a method for screening for inhibitors of receptors having potential in the treatment of disorders and diseases involving target cells expressing receptors inhibited by said tested inhibitor comprising: (i) measuring the level of blood oxygenation of the cells expressing said receptor and/or blood flow to said cells in said individual; ( ⁇ ) administering a ligand capable of binding to said receptor to said individual; (iii) measuring the level of blood oxygenation of said cells and/or of blood flow to said cells in said individual; (iv) wherein there is no substantial difference in the measurements of (i) and (iii) above, said tested receptor inhibitor is determined to be non-suitable for treatment of said individual and wherein the measurements in (iii) is substantially different than those in (i), then: (v) repeating steps (i) - (iii) above and administrating said tested inhibitor together with the ligand and measuring the level of blood oxygenation and/or blood flow to said cells; and (vi) wherein the measurement in (v) is substantially different
  • a method for direct screening of a receptor inhibitor as a potentially effective drug is provided.
  • the effect of the receptor inhibitor on the oxygen consumption of the target cells is measured and i o compared to the level of the blood consumption of the cells before their contact with the inhibitor.
  • the level of blood consumption of these cells contacted with the receptor inhibitor is substantially different than the level of the oxygen consumption of the cells before their contact with the inhibitor, the tested inhibitor is most likely to be potentially effective as a drug.
  • the invention thus further provides a method for screening for inhibitors of receptors having a potential in the treatment of disorders and diseases involving target cells expressing receptors inhibited by said tested inhibitor comprising: (i) measuring level of blood oxygenation of the cells expressing said receptor and/or of blood flow to said cells in said individual; 2 0 ( ⁇ ) administering at the receptor inhibitor to said individual;
  • said tested receptor inhibitor may have a potential as a drug for the 25 treatment of said disorders and diseases.
  • the present invention further provides a kit for evaluating the efficacy of inhibitors of receptors in the treatment of disorders or diseases involving target cells expressing receptors inhibited by said tested inhibitor comprising at least one ligand capable of binding to said receptor; at least one inhibitor of said receptor to be tested, optionally predetermined cut-off values of the level of blood oxygenation and blood flow of said target cells and instructions for use.
  • the enhanced oxygen consumption of tumor cells and blood flow to tumor cells following their activation in accordance with the methods of the invention is useful for increasing tumor permeability and thus susceptibility to various anti-tumorigenic treatments and drugs, such as chemotherapeutical drugs, antibodies and radiation.
  • drugs such as chemotherapeutical drugs, antibodies and radiation.
  • the activating agent is administered to an individual as an auxiliary treatment in conjunction with the anti-tumorigenic treatment or drug.
  • An activating agent may be administered to the individual prior to, simultaneously with, or following the administration of the anti-tumorigenic treatment to achieve the maximal enhancement of the therapeutic effect of the administered drug or therapy.
  • the present invention provides an auxiliary composition for administration in conjunction with an anti-tumorigenic treatment administered to an individual for the treatment of a tumor, said composition comprising as an active ingredient an agent which enhances the oxygenation of said tumor cells and/or changes the blood flow to said tumor cells and a pharmaceutically acceptable carrier.
  • the invention further provides a method for enhancing the efficacy of an anti-tumorigenic treatment administered to an individual having a tumor comprising administering to said individual an effective amount of an agent which enhances the oxygenation of said tumor and/or changes the blood flow to said tumor cells in conjunction with said anti-tumorigenic treatment.
  • the efficacy of irradiation treatment in an individual suffering from a tumor may be enhanced by administrating to the individual an activating agent which enhances the oxygenation of said tumor cells in conjunction with the irradiation treatment.
  • the invention further provides a method for enhancing the efficacy of an anti-tumorigenic irradiation treatment administered to an individual having a tumor comprising administering to said individual an effective amount of an agent which enhances the oxygenation of said tumor in conjunction with said anti- tumorigenic irradiation treatment.
  • the term "effective amount” should be understood to mean an amount which when administered to the individual enhances the anti-tumorigenic effect of the drug, antibody or irradiation.
  • the effective amount may readily be determined by a person versed in the art by preliminary measurements on tumor cells in vitro in conjunction with the intended anti-tumorigenic treatment as well as such measurements in vivo.
  • a chemical contrast medium is a substance, which is introduced into the body to change the contrast between the tissues.
  • MRI contrast agents also called paramagnetic agents
  • Such agents are administered by injection into the vein before or during MRI to help diagnose problems or diseases of the brain or the spine, and to help diagnose problems in other parts of the body, such as the bones and joints, breast, liver, soft tissues, and uterus.
  • a typical chemical contrast media is a complex of a paramagnetic metal ion such as gadolinium (Gd).
  • Gd gadolinium
  • many paramagnetic metal ions are toxic. To decrease their toxicity, these metal ions are typically complexed with other molecules or ions to prevent them from complexing with molecules in the body. Other molecules are used as contrast agents in intrasound.
  • a contrast enhancement is obtained by one tissue having a higher affinity or vascularity than another. Most tumors for example have a greater Gd uptake than the surrounding tissues, causing a larger Ti signal.
  • the present invention thus provides a method for enhancing the efficacy of chemical contrast substances used during magnetic resonance imaging of cells (MRI) or ultrasound comprising administering said chemical contrasts medium in conjunction with an activating agent capable of enhancing the level of blood oxygenation of said cells and/or blood flow to the cells.
  • MRI magnetic resonance imaging of cells
  • ultrasound comprising administering said chemical contrasts medium in conjunction with an activating agent capable of enhancing the level of blood oxygenation of said cells and/or blood flow to the cells.
  • the above method is used for enhancing the efficacy of chemical contrast substances used during MRI or ultrasound of an individual having a tumor and said activating agent is an agent capable of enhancing the level of blood oxygenation of the tumor cells or blood flow to the tumor cells.
  • the activating agent is an agent which activates the tumor cells by activation of at least one cellular receptor expressed by the tumor cells such as tyrosine kinase receptors, an example being the Met tyrosine kinase receptor.
  • the present invention also provides an auxiliary composition for administration in conjunction with chemical contrast substances used during MRI or ultrasound of cells, said composition comprising as an active ingredient an agent which enhances the oxygenation of said cells and/or changes the blood flow to said cells and a pharmaceutically acceptable carrier.
  • the term "in conjunction” should be understood to mean administration of the agent either prior to, simultaneously to or following the said treatment being either the anti-tumorigenic chemotherapeutical drugs, the irradiation treatment or contrast agents.
  • FIG. 1 is a photograph showing a series of BOLD measurements obtained from BALB/c mice which were injected with DA3 into their mammary glands and later treated first with saline and then with HGS/SF. The measurements were taken in one minute intervals for 55 mins. The percentage of BOLD alteration was calculated comparing base line (average of 7 measurements after saline injection) to peak reaction (average of 7 measurements 30 mins after HGF/SF injection).
  • Fig. 1A shows a transverse BOLD image through the mouse.
  • Fig. IB shows negative alteration of signal intensity level superimposed on the first BOLD image.
  • Fig. 1C shows positive alteration of signal intensity level superimposed on the first BOLD image.
  • a substantive increase in blood oxygenation level is seen in the liver where Met is highly expressed.
  • the kidneys and tumor show similar levels of reaction to HGF/SF while lungs show no reaction to HGF/SF.
  • Fig. 2 is a photograph showing differential alteration of blood oxygenation in the tumor as measured by BOLD images of mice treated as explained in Fig. 1 above. The percentage of BOLD alteration was calculated as described in Fig. 1 above.
  • Fig. 2 A shows an axial BOLD image of the tumor.
  • Fig. 2B shows positive (+4% - +250% - green) and negative (-80% - -4% - red) alteration of signal intensity levels superimposed on the BOLD image.
  • Fig. 2C shows time lapse analysis of the elevation of the BOLD signal.
  • X axis is time in seconds and Y axis is average values of signal intensity in ROI.
  • Fig. 2D shows a reduction of the BOLD signal intensity level superimposed on the BOLD image.
  • the X and Y axes are like in Fig. C.
  • Fig. 3 is a photograph showing a series of ultrasound/Doppler measurements in 2-5 min. intervals for 20-30 mins. which were obtained from mice treated as explained in Fig. 1 above.
  • Dl-DMBA-3 is a cell line derived from a poorly differentiated mammary adenocarcinoma induced in BALB/C mice by dimethylbenzanthracene. Limiting dilution cloning produced the cell line designated DA3 (Fu, Y., et al, Cancer Res., 50:227-234, 1990).
  • DA3 cells were grown in DMEM (GibcoBRL Gaithersburg, MD) supplemented with 10% heat - inactivated fetal calf serum (FCS) (GibcoBRL, Gaithersburg, MD), penicillin-streptomycin-nystatine and L-glutamine, under 5% CO2 environment.
  • FCS heat - inactivated fetal calf serum
  • the growth medium contained 11 mM glucose.
  • the cells were grown to approximately 90% confluency, harvested with 0.25% trypsin-0.05% EDTA, centrifuged at 4°C at 1000 x g for 5
  • Assay medium DMEM with 5% calf serum, GIBCO
  • HGF/SF Assay medium
  • 150 ⁇ l was added in 150 ⁇ l to 96 well plates (Costar). Cells were added in 150 ⁇ l assay medium and incubated overnight. Cells were fixed, air dried, stained with Giemsa and examined for scattering (spreading and dispersion of epithelial colonies).
  • Average procedure length was 15-20 min., and the time in CaCl2 was kept below 5 min.
  • the perfusion was performed through an insert with inlet and outlet tubing, and the volume of the perfusion chamber was 2 ml.
  • the perfusion solution flowed from the opening of the inlet near the bottom of the tube through the packed alginate capsules, and the outflow was directed through opening in the insert to the outlet tubing.
  • a constant flow of 0.9 ml/min. in a single pass mode was maintained by a peristaltic pump throughout all experiments, and the temperature was maintained at 37°C.
  • the perfusion solution contained 5.5 mM of glucose, similar to glucose physiological concentration, unless otherwise indicated. In each experiment control perfusion with 31p NMR recording was carried out for about 90 min., to ensure metabolic stability of the cells, before adding the HGF/SF to the perfusion solution.
  • Magnetic Resonance Imaging MRI
  • +2 assay which is based on the reaction of protein with Cu , and spectrophotometric quantitation at 562nm. Absorbance measurements were performed with an ELISA reader (Elx808 BioTEC Instruments, Inc.)
  • Glucose consumption assays 25x1 ⁇ 4 cells were plated in 25-cm flasks in 10 ml growth medium. When the cells reached 50, 5% confluency the medium was replaced by 11 mM glucose DMEM with 80u/ml of HGF/SF. 24 hours later samples of medium were taken for glucose measurements, and a cell count was performed in one flask. After 48 hours of HGF/SF stimulation glucose levels were measured again, and cell counts were performed in the remaining flasks.
  • Glucose concentrations were determined by the hexokinase enzymatic assay, utilizing the coupled enzyme reaction catalysed by hexokinase and glucose-6-phosphate dehydrogenase, and measuring the product, NADH, at 340 nm. Cell counts were performed by trypsinization and suspension of the cells in trypan blue.
  • the cells were embedded in alginate capsules and were perfused as described above. Two three-way valves were inserted in the inflow and the effluent tubes, in close proximity to the perfusion chamber. After 90 min of perfusion for stabilization, baseline control samples were collected at 10 min intervals for an hour. Then, 400 u/ml HGF/SF were added to 50 ml of medium, which were perfused for 1 hour, followed by perfusion with medium for additional 120 min. At each time point samples were withdrawn from the inflow medium and the effluent.
  • the NAD(P)H fluorescence was excited at 325 nm using the CLSM UV laser.
  • the flavoprotein fluorescence- was excited at 488 nm using a 75-mW argon ion laser (model OMI-532 AP; Omnichrome).
  • the emission wavelengths 450 (NAD(12)H fluorescence) or 520 nm (flavoprotein fluorescence) were collected by the CLSM PMTs using a 410 Zeiss (Oberkochen, Germany) confocal laser scanning microscope (CLSM) with the following configuration: 25 mW HeNe lasers, Krypton Argon UV laser lines.
  • NAD(P)H elevation the cells were treated with lmM octanoylcarnitine and 5 mM malate.
  • PPA percentage positive area
  • Images were printed using Codonics dye sublimation color printer. When comparing fluorescence intensity, we used identical parameters for each image (e.g., scanning line, laser light, contrast brightness).
  • mice were injected with solution that contained 1 mg/ml Xylazine and lOmg/ml Imalgene 1000. 100 ⁇ l per lOgr body weigh were injected, if needed another 50 ⁇ l were injected after 1 hour. Catheter was inserted intra-peritoneal to the mice, so that additional injection was done without moving the mouse out of the MR devise. Heparin injection
  • Heparin was injected i.v. to mice in doses of 6, 3, 0.3 or 0.003 units per mice (Intramed Heparin Sodium Injection - Pharmacare Limited).
  • the doses of Heparin used in accordance with the invention are non-hemolytic doses.
  • mice were held on a surface with tape to prevent any movement during MRI measurements. Catheter was inserted IV in the mice tail, so that injection is done without moving the mouse out of the MR machine. 0.1 ml Saline was injected and 15 minutes later HGF/SF was injected. A series of BOLD measurements in intervals of about 1 minute started after the saline injection and proceed for about 55 min.
  • ROI analysis This analysis was done on "Omnipro” computer or “Matlab” programming. We defined an region of interest (ROI), and a time lapse analysis was done. The resulting graph described the average signal intensity in the ROI (Y axis)at the different time points (X axis).
  • mice injected with the DA3 cells were taken to ultra-sound experiments, there was no need for anesthesia.
  • a series of Doppler measurements were taken before (for 10 minutes) and after (for 30 minutes) HGF/SF injection. The measurements were performed in ACUSON 128XP Computer
  • Met expression in DA3 cells was determined by Western blot (WB) analysis with SP260 rabbit anti-peptide antibody. High levels of pl40 met were detected (Fig. 1A lane 1). This band was not evident in the presence of SP260 immunizing peptide (Fig. 1 A lane 2), confirming the specificity of the antibody.
  • the influence of HGF/SF on Met phosphorylation was determined by immunoprecipitation (IP) using SP260 followed by WB analysis using anti-phosphotyrosine antibody (anti-pTyr). Low levels of phosphorylated Met were detected in the untreated DA3 cells (Fig. 1A lane 3). A 5-minute exposure to HGF/SF increased Met phosphorylation (Fig 1 A lane 4).
  • DA3 cells which were treated with HGF/SF displayed fibroblast-like "scattered" morphology (Fig IB ?) as compared to the epithelial appearance of untreated cells (Fig IB ?).
  • This change in morphology is similar to that of HGF/SF-treated MDCK cells (the classical model for HGF/SF-induced cell motility exhibiting disruption and scattering of epithelial cell colonies.
  • DA3 cells were plated in tissue culture flasks. When confluency was approximately 50% the growth medium was replaced with medium containing 2g/l glucose, with or without HGF/SF. 24 and 48 hours later samples from the medium were tested for glucose concentration using hexokinase enzymatic assay kit (Sigma) according to manufacturer's instructions, and cell number in one flask was counted using trypan blue. Glucose consumption levels after 48 hours are displayed as mg/dL/million cells.
  • HGF/SF significantly increased glucose consumption in the cultured DA3 cells, from 79.85+12.2 to 126.0+5.2 mg/dL/million cells (PO.0001).
  • HGF/SF hematomase-derived neurotrophic factor
  • Oxygen consumption was measured by comparing oxygen content between perfusion medium and effluent. Measurements were done in the present and absent of HGF/SF. We found that HGF/SF treatment increased oxygen consumption by 20.8% compared to control untreated cells. Based on the solubility of oxygen in aqueous solutions at 37°C, perfusion rate and protein content, oxygen consumption was determined to be 0.58 0.02 mole/hour/mg protein (control cells) and 0.71 0.03 mole/hour/mg protein (HGF/SF- stimulated cells (p ⁇ 0.05).
  • 2-Deoxyglucose is a metabolic inhibitor that competes with glucose on transport into the cells, and once entering cells is phosphorylated by hexokinase to 2-DG-6P which undergoes no further metabolism (10, 16).
  • 2-DG as a "probe" to measure this initial step of glycolysis.
  • the cells were perfused with DMEM (containing 5.5 mM glucose) into which 5 mM 2-DG were added, and the accumulation of 2-DG-6P was followed serially (Fig. 3A). Because 2-DG treatment causes energy deprivation, and the cells are no longer in homeostasis, it was necessary to perform the control and the HGF/SF stimulation experiments with different samples.
  • 2-DG and HGF/SF had some effects on all the 31p signals and a reference was mandatory.
  • HGF/SF significantly increased 2-DG phosphorylation rate, from 2.2+0.4 x 10"3 normalized integral/mg protein/min in the control perfusions to 3.5+0.3 x 10 " 3 normalized integral/mg protein/min after HGF/SF stimulation (p ⁇ 0.05).
  • 1 C NMR The effects of HGF/SF on cellular glucose uptake and lactate production were studied by continuous 13 C NMR measurements. The following protocol was used: cells were perfused with [l- ⁇ Ci ]-glucose enriched medium and control ⁇ 3c spectra were continuously recorded (Fig.
  • DA3 cells were injected into the mammary glands of Balb/c mice as described above.
  • the tumor bearing mice were injected IV with saline and 15 minutes later received an injection of HGF/SF.
  • a series of BOLD measurements in 1 minute intervals for 55 minutes were obtained.
  • Functional MRI scans, before and after HGF/SF injection of coronal sections of a mouse that included longitudinal section of the tumor as well as liver, lungs and kidneys were obtained and can be seen in Fig. la.
  • Analysis of the pixel activation was calculated as the percentage of BOLD alteration comparing base line (average of 7 measurements after saline injection), to peak reaction (average of 7 measurements 30 min. after HGF/SF injection).
  • Analysis of BOLD signal revealed certain areas exhibiting decreased signal such as seen in Fig lb.
  • BOLD signal also revealed areas exhibiting increased signal such as those shown in Fig. lc.
  • the most significant elevation in the BOLD signal was shown in organs expressing high levels of Met such as the liver. Organs that express low levels of Met, such as lungs and muscles, did not exhibit significant elevation of BOLD signal following HGF/SF injection. In the kidneys, which express moderate levels of Met, BOLD activation levels resembled those of the tumor.
  • Fig 2 shows the BOLD image (Fig. 2a), pixel activation (Fig.2b) and ROI analysis (Fig. 2c-d) of the tumor. In areas primarily bordering the tumor, the signal dramatically decreases (red). On the other hand, areas within the tumor predominantly exhibit increased BOLD signal (green) (Fig.2b).
  • the ROI graphs display time course of BOLD signal in different regions of the tumor before and after HGF/SF injection.
  • Fig 2c describes time course derived from ROI that included most of the internal region of the tumor (green area). This ROI clearly demonstrates the increase in the BOLD signal typical of Met-expressing tumors, following injection of HGF/SF.
  • Fig 2d shows ROI in the tumor margin (red region) in which the BOLD signal decreases.

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Abstract

L'invention porte sur un procédé de détection et de suivi de cellules tumorales chez un patient à base de techniques d'imagerie usuelles telles que l'IRM ou l'échographie s'exécutant non invasivement par observation externe du niveau d'oxygénation des cellules (mesurées en imagerie BOLD) et du flux sanguin les alimentant (mesuré par Doppler aux ultrasons) dans un tissu du patient soumis à examen. Ledit procédé peut servir à identifier des zones du patient ne présentant pas de lésions tumorogènes préconnues et à détecter des zones présentant une probabilité élevée de contenir des cellules dans une lésion présuspectée du patient (par exemple une zone à forte probabilité de présence de cellules tumorales primaires se basant sur une analyse au moyen de méthodes de routine). Le procédé consiste à utiliser un agent dont l'administration au patient ou le contact avec ses cellules produit un accroissement de la consommation d'oxygène des cellules, détectable par différentes techniques d'imagerie. L'agent activateur peut par exemple agir sur les cellules par fixation à un récepteur spécifiquement exprimé par les cellules. L'invention porte également sur des procédés d'évaluation de l'efficacité de traitements anti-tumorogènes basés sur la mesure du niveau d'oxygénation des cellules et du flux sanguin les alimentant par différentes techniques d'imagerie. Elle porte en outre sur des procédés accroissant l'efficacité des traitements anti-tumorogènes, notamment par chimiothérapie, par des anticorps et par irradiation, consistant à administrer au patient une quantité d'un agent accroissant l'oxygénation des cellules cibles et le flux sanguin les alimentant. Ledit procédé permet par ailleurs d'accroître l'efficacité des agents chimiques de contraste utilisés en association avec l'IRM ou l'échographie.
EP00969788A 1999-10-22 2000-10-20 Detection de tumeurs par imagerie et leur traitement Withdrawn EP1221977A2 (fr)

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US6979999B2 (en) * 2004-02-26 2005-12-27 General Electric Company Method and system of mapping oxygen concentration across a region-of-interest
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JPWO2011155168A1 (ja) 2010-06-07 2013-08-01 パナソニック株式会社 組織悪性腫瘍検出方法、組織悪性腫瘍検出装置

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