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WO2007039318A2 - Analogues du neuropeptide y - Google Patents

Analogues du neuropeptide y Download PDF

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Publication number
WO2007039318A2
WO2007039318A2 PCT/EP2006/009812 EP2006009812W WO2007039318A2 WO 2007039318 A2 WO2007039318 A2 WO 2007039318A2 EP 2006009812 W EP2006009812 W EP 2006009812W WO 2007039318 A2 WO2007039318 A2 WO 2007039318A2
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Prior art keywords
receptor
neuropeptide
binding
cells
arg
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WO2007039318A3 (fr
Inventor
Ananth Srinivasan
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Bayer Pharma AG
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Bayer Schering Pharma AG
Schering AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57545—Neuropeptide Y

Definitions

  • the present invention relates to compositions and methods for diagnosing, detecting, imaging, and treating tissues, where the compositions, including pharmaceutical compositions, are provided which comprise neuropeptide Y polypeptide analogs that can be used to selectively target and label cells, especially breast cancer cells.
  • the present invention also relates to antibodies to the neuropeptide Y polypeptide analogs.
  • the neuropeptide Y polypeptide analogs are specific or selective for the neuropeptide Y
  • Yi neuropeptide Y
  • selective it is meant that the analog has a higher affinity for the Yl receptor, than other neuropeptide receptor subtypes, e.g., Y 2 , Y 4 , and Y 5 .
  • the affinity can be 5-fold, 10-fold, 100-fold, 1000- fold, 10,000-fold or more higher.
  • An analog of neuropeptide Y is a polypeptide which has a sequence that is not naturally- occurring. As described in detail below, it can comprise non-naturally occurring amino acids and derivatives thereof; non-peptide bonds (e.g., to enhance stability); amino acid substitutions, deletions, or additions; and various organic and non-organic substitutions for amino acids.
  • the analogs can further be modified by attaching (covalently, non-covalently) moieties to the peptide, e.g., detectable labels, carbohydrates, chemotherapeutic agents, nanoparticles, particles, magnetic materials, lipids, nucleic acids, energy-emitting materials, etc.
  • Polypeptides can comprises about amino acids 25-36 according to the NPY numbering system, and can be optionally substituted as described herein.
  • Table 8 provides examples of several neuropeptide Y analogs. These can be further modified by the substitution, deletion, or addition of non-naturally occurring amino acids; nonpeptide bonds (e.g., to enhance stability); amino acid substitutions, deletions, or additions; and various organic and non-organic substitutions for amino acids.
  • amino acid residue 32 can be deleted (e.g., P2489, between He and Arg) for any of the peptides listed in Table 8, and derivatives thereof.
  • the polypeptide analogs can be routinely made and then selected for a desired activity, e.g., Y
  • the neuropeptide Y analog can further comprise a detectable label.
  • a detectable label is any moiety that facilitates detection or visualization of the polypeptide to which it is attached.
  • detectable labels are provided which it is attached.
  • detectable labels are provided for PET.
  • Other examples of labels include, but are not limited to, e.g., radioactive atoms, fluorescent molecules (including quantum dots), magnetic materials, and energy-emitting materials. Labels can be associated with the analog by any suitable means, e.g., direct conjugation; indirectly using linkers; using binding pairs (e.g., biotin/avidin); chelating agents; etc.
  • a detectable label which is an energy-emitting material is preferred. More preferably the energy-emitting material is a radionuclide, preferably 177 Lu, 18 F, 68 Ga, 99m Tc or 1 11 In.
  • the neuropeptide Y analogs of the present invention can be formulated as pharmaceutical compositions, e.g., comprising a pharmaceutically acceptable carrier.
  • a "pharmaceutically acceptable carriers” is an agent or substance that is combined with a analog of the present invention and which can be administered safely to a subject for clinical purposes. These include, but are not limited to, antioxidants, preservatives, dyes, tablet- coating compositions, plasticizers, inert carriers, excipients, polymers, coating materials, osmotic barriers, devices and agents which slow or retard solubility, etc.
  • the present invention also provides methods of detecting a cell expressing a Yi receptor (or other receptor subtypes), comprising, contacting a cell with an effective amount of a polypeptide which is neuropeptide Y analog selected from P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(DOTA), analogs listed in Table 8, or derivatives thereof, and detecting binding of said polypeptide to said cell.
  • the method can be used to detect any cell type with Yl receptors, including breast cancer cells, and to diagnose the presence of breast cancer cells, especially where the polypeptide is selective for the Yi receptor.
  • the methods can be accomplished in any environment, including in situ (e.g., where the breast of a human is visualized using PET); in vivo, in vitro; on biopsy samples; on slides; on tissue culture dishes; in multi-well plates; etc.
  • Detection can be accomplished by any suitable method without limitation, including by PET; direct visualization; using antibodies to the polypeptide analogs of the present invention (e.g., in an ELISA format); by detecting moieties incorporated into the analogs (e.g., where the analog contains biotin and a strepavidin-fluorescent marker is used to directly detect it.
  • the present invention provides the use of neuropeptide Y analog as defined above for the manufacture of a diagnosis agent. More preferably the diagnosis agent concerns the diagnosis of cancer expressing the receptor Y receptor and more preferably the Y ⁇ , Y 2 or Y 5 receptor. Yi is more preferred.
  • the present invention also provides methods of treating a breast cancer (or other cancer expressing the receptor subtype to which the analog specifically binds), comprising: administering an effective amount of a polypeptide which is neuropeptide Y analog selected from P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(DOTA), analogs listed in Table 8, or derivatives thereof.
  • the polypeptide can be administered alone in therapeutically effective amounts or conjugated to a chemotherapeutic agent.
  • the phrase "effective amount" indicates that the amount of the polypeptide, or polypeptide associated with the chemotherapeutic agent, is effective to treat any symptom or aspect of the cancer. Effective amounts can be determined routinely.
  • treating is used conventionally, e.g., the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving, etc., one or more of the symptoms associated with a breast cancer.
  • Administering effective amounts of the polypeptide alone or with a chemotherapeutic agent can treat one or more aspects of the cancer disease, including, but not limited to, causing or resulting in tumor regression; causing or resulting in cell death; causing apoptosis; causing necrosis; inhibiting cell proliferation; inhibiting tumor growth; inhibiting tumor metastasis; reducing disease progression; stabilizing the disease; reducing or inhibiting angiogenesis; prolonging patient survival; enhancing patient's quality of life; reducing adverse symptoms associated with cancer; and reducing the frequency, severity, intensity, and/or duration of any of the aforementioned aspects.
  • Chemotherapeutic agents are conventional. See, e.g., Cancer: Principles and Practice of Oncology, ed., DeVito et al., 7 th Edition, 2005, Part I, Chapters 15 and 16; Part 4, Chapter 63.
  • the chemotherapeutic agents can be routinely coupled to the polypeptides and administered in effective amounts, e.g., intravenous or intratumoral (i.e., directly into the tumor).
  • the analogs can be used to target the agent to the cell type of interest.
  • the present invention provides the use of neuropeptide Y analog as defined above for the manufacture of a medicament.
  • the treatment concerns the treatment of cancer expressing the receptor Y receptor and more preferably the Yi, Y 2 or Y 5 receptor. Yi is more preferred.
  • the present invention also provides antibodies to the analogs.
  • Antibodies can be routinely prepared.
  • the present invention provides the following aspects:
  • An isolated polypeptide comprising or consisting of P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(DOTA), or derivatives thereof.
  • polypeptide of any of embodiments 1-3, wherein said polypeptide consists of 12 or less naturally-occurring or non-naturally-occurring amino acids.
  • a method of detecting a cell expressing a Yi receptor comprising: contacting a cell with an effective amount of a polypeptide of embodiments 1-6, detecting binding of said polypeptide to said cell.
  • a method of detecting a breast cancer comprising: contacting a breast cancer with an effective amount of a polypeptide of embodiments 1 -6, and detecting binding of said polypeptide to said cell.
  • a method of embodiment 8 further comprising contacting said cell with a polypeptide which is selective for a Y 2 or Y 5 receptor, and detecting said binding.1 11. A method of embodiments 7 or 8, wherein the detecting is performed using positron emission tomography.
  • a method of diagnosing breast cancer comprising: detecting Yi positive cells in breast tissue, wherein said detection is accomplished using a polypeptide of any of embodiments 1-6.
  • a method diagnosing breast cancer comprising: contacting a breast cancer with an effective amount of a polypeptide of embodiments 1-6, detecting binding of said polypeptide to said cell, wherein binding indicates the presence of breast cancer cells.
  • a method of treating a breast cancer comprising: administering an effective amount of a polypeptide which is P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(DOTA), an analog listed in Table 8, or derivatives thereof.
  • a polypeptide which is P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(DOTA), an analog listed in Table 8, or derivatives thereof.
  • breast cancer is the most common cause of cancer deaths in women worldwide [I]. In the year 2002 alone, over 1.1 million new cases of breast cancer were diagnosed worldwide, and approximately over 4.4 million women are now living with this disease [2]. Moreover, breast cancer is the most common cancer in younger women. At ages between 35 and 59 years, about 40% of new cancers and just under 30% of cancer-induced deaths can be attributed to breast cancer. In 2003, over 17,000 women died of breast cancer in Germany. Since 1997, the breast cancer death rate has slightly declined. The proportions of early detection, on the one hand, and improved therapy options, on the other hand, in this development are still unclear, however [3].
  • Cancer is invasive, i.e., malignant tumors that penetrate into the tissue.
  • tumors consist of more or less undifferentiated cells, which usually have lost the specific biochemical function that characterizes the original tissue. Moreover, they have the capacity to proliferate, uncontrolled, with elimination of normal interaction between the cells, and thus to infiltrate adjacent structures [5].
  • breast tumors grow in the upper outside quarter of the breast with the most gland tissue, thus the side that is more likely facing the shoulder.
  • the most common form is the ductal breast cancer (starting from the milk duct), followed by lobular breast cancer (starting from gland lobules) (Fig. 1) [3].
  • Most malignant tumors are not limited to local growth, however, but rather form evacuations (metastases; in other organ systems.
  • evacuations metalastases; in other organ systems.
  • Fig. 1 Various Forms of Breast Cancer (Source: R ⁇ diger Anatomy, Anatomical Plates)
  • the mammography which is performed as a measure of routine early detection, is a special x-ray examination of the breast, which can make visible nodes even below the palpable size of about one centimeter.
  • the tissue density of the breast is imaged with the aid of very low-dosed x-rays.
  • Each breast is recorded from two or more directions.
  • the breast is compressed between two Plexiglas plates to prevent movement and to keep the dose of radiation low [6].
  • Bright spots in this figure show areas with higher tissue density. In this case, this can be both a malignant tumor and a benign tumor or microcalcifications. This differentiation is only slightly possible with mammography in dense breast tissues. In such cases, an ultrasound study, which is the most effective for a diagnosis of small tumors in women with dense breast tissue, is carried out.
  • MRT magnetic resonance tomography
  • nuclear medicine such as SPECT (Single Photon Emission Computer Tomography) and Position Emission Tomography (PET) are used. The latter always obtains increasing importance in breast cancer diagnosis.
  • Positron Emission Technology is an imaging technique in nuclear medicine that uses radiopharmaceutical agents (also named indicators or “tracers”) to detect metabolic changes within cells.
  • Anatomical imaging processes such as, e.g., computer tomography (CT) and magnetic resonance tomography (MRT), yield primarily structural or anatomical information with high spatial resolution. In contrast to this, the PET imaging yields functional or biochemical information. "Molecular Imaging” is therefore also mentioned. Properties of individual imaging processes are referred to in Table 2
  • MRT Magnetic Resonance Tomography
  • MRS Magnetic Resonance Spectroscopy
  • SPECT Single Photon Emission Computer Tomography
  • PET Positron Emission Tomography
  • radionuclides 18 F, 1 1 C, 13 N or 15 O are used for labeling radiopharmaceutical agents. During decomposition, they emit positrons, which then strike electrons in their vicinity. In this case, both particles are destroyed, and two ⁇ -quanta with
  • Fig. 2 IS F-Deoxyglucose (FDG).
  • FDG F- deoxyglucose
  • the phosphorylated FDG cannot pass through the cell wall and thus accumulates in the cell (Fig. 3).
  • the level of the FDG uptake and retention is a quantitative indicator of the glucose metabolism [2].
  • the elevated glycolytic rate and elevated glucose consumption by malignant cells, in comparison to the normal tissue, is the basis for the capacity of FDG-PET imaging for exact differentiation of cancer and benign tissue [10].
  • Fig. 3 FDG-Metabolism in Comparison to Glucose.
  • FDG is phosphorylated from the hexokinase and remains in the cell. This leads to an elevated FDG uptake and retention in metabolically active tissue [2].
  • thymidine analogs are those such as the [F- 18]- fiuoro-L-thymidine (FLT), which targets the DNA replication and is used for the visualization of cell proliferation.
  • FLT fiuoro-L-thymidine
  • estrogen-receptor indicators, annexin V derivatives for apoptosis visualization and specially altered antibody fragments, which directly detect HER- 21 new receptors, are found in development [10].
  • these target molecules can be quantified and, for example, the amount of estrogen receptors can be determined based on which then the use of hormone therapy (e.g., with tamoxifen) can be weighed.
  • hormone therapy e.g., with tamoxifen
  • these targets in the tumor tissue are determined by in-vitro studies of biopsy material. This is employed only in the early stages of the disease, however, since with increasing disease, the target expression is more heterogeneous in the primary tumor, and sometimes metastases are present that cannot be biopsied for tissue studies [2].
  • the results of a needle biopsy are no longer representative of the entirety of the diseases, respectively of all tumors.
  • the PET diagnosis offers the possibility of detecting all foci of disease, as well as the quantification of the corresponding target in the individual tumors. Additional uses for PET imaging are the rating of the stage of the disease, the identification of (distant) metastases and the monitoring of success of the therapy [10]. 1.3 Neuropeptide Y and Its Receptors
  • Neuropeptide Y is a neurotransmitter that consists of 36 amino acids and is amidated in a C-terminal manner. It is included in the NPY-hormone family, to which the peptide YY (PYY) and the pancreas polypeptide (PP) belong. NPY and PYY show 70% homology to one another and are only about 50% homologous to PP. Both NPY and PYY show a structure that is similar to that of Vogel-PP. The latter consists of an N-terminal globular structure and a flexible C-terminus (Fig. 4). The globular, hairpin-like structure is also referred to as a PP-folding [H].
  • Fig. 4 Amino Acid Sequence of pNPY, pPYY and hPP. For each peptide, the constant positions within all previously studied species are underscored. In the upper area of the image, the characteristic PP-folding is shown, and the seven constant positions between NPY, PYY and PP are registered. [12]
  • NPY neuropeptides in the brain. It acts primarily on the central nervous system, where, i.a., it stimulates eating behavior and anxiety. Actions on the peripheral nervous system include vasoconstriction, effects on the gastrointestinal movement and secretion, insulin release and renal secretion. These effects of NPY are mediated by G- protein-coupled NPY receptors, from which previously five different subtypes Yi, Y 2 , Y 4 , Y 5 und Y 6 were identified and were partially characterized extensively [13].
  • NPY(Yi) receptors include those for somatostatin, VIP ("vasoactive intestinal peptide"), GRP ("gastrin-releasing peptide”) and NPY(Yi) receptors. Histological studies of tumor tissue showed an over-expression of the NPY(Y)) receptor both in the case of primary tumors and in (lymph node) metastases [14]. In addition, studies have found that in healthy breast tumor, the NPY(Y 2 ) receptor is expressed almost exclusively. If the Yi receptor occurs in healthy tissue, then it is never the sole subtype and only in a low number and density [13]. An attempt is then made, therefore, to use this differential NPY-receptor expression for breast cancer imaging and to develop corresponding peptide analogs that bind selectively to these receptors.
  • VIP vasoactive intestinal peptide
  • GRP gastrin-releasing peptide
  • NPY and PYY have a high affinity for Yi- and Y 2 -receptors. The latter are not selective for a receptor subtype, however.
  • NPY analogs include the Yi-selective [Leu 31 , Pro 34 ]-NPY [15], which has substitution in the C-terminal end of the peptide, and the Y 2 -selective NPY fragment (13-36) [16].
  • the peptides should be as short as possible, if possible smaller than 12 amino acids. This makes possible a quick tissue penetration, quick excretion and low antigenicity. Their selective binding to the Yi -receptor should be maintained regardless.
  • peptides that are internalized according to the receptor binding are advantageous in that they accumulate in the cell and thus provide a stronger signal for scintigraphy.
  • a major problem of radiolabeled peptides is their stability under physiological conditions. On the one hand, peptides are often fragmented by peptidases and, on the other hand, the stability of the radiolabeling (see Section 1.5) is also not always ensured [18].
  • NPY analogs that are to be studied in this work consist of the modified C- terminus of the NPY, in which, for example, unnatural, more stable amino acids were incorporated, which generally are not found from peptidases or proteases and thus can increase the plasma stability of the peptides.
  • modified C- terminus of the NPY in which, for example, unnatural, more stable amino acids were incorporated, which generally are not found from peptidases or proteases and thus can increase the plasma stability of the peptides.
  • ⁇ -amino acids Fig. 5
  • radical (R) can be either on the 2 nd atom ( ⁇ 2 ) or on the 3 rd C atom ( ⁇ 3 ).
  • the peptides For the use of the NPY analogs in imaging with PET or gamma scintigraphy, the peptides must be labeled with radionuclides, which are selected depending on later use.
  • Peptides which are to be used for PET diagnosis, are labeled with, e.g., 18 F, a positron radiator with a physical half-life of 110 minutes.
  • the labeling of the peptide with 18 F is generally carried out indirectly via the conjugation of a previously F-labeled synthon.
  • iodine isotopes are also used in addition to metallic radionuclides such as 177 Lu, 99m Tc or 1 11 In.
  • Metallic radionuclides are not bound directly to the peptide, but rather via chelating agents, with which the radiometals form a complex.
  • the selection of the chelating agent depends on the metal nuclide.
  • DOTA 1,4,7,10- tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid
  • Fig. 6 DOTA-Chelating Agent Coupled to a Peptide.
  • the latter are labeled in most cases with the iodine isotope 125 I.
  • the peptides have to contain tyrosine, histidine or primary amino groups.
  • the iodine isotope is eiiner o ⁇ und directly to the aromatic radical of these amino acids or reacted indirectly via 125 I-labeled N-succinimidyl compounds, such as the Bolton-Hunter reagent (Fig. 7), with the primary amino group [19].
  • Fig. 7 l2S I-Labeled Bolton-Hunter Reagent.
  • Radioligand-binding experiments are based on the law of mass action. This means that the binding between ligand (L) and receptor (R) is reversible; the ligand-receptor complex (LR) that is produced is thus dissociated again.
  • the rate at which the ligand-receptor complex forms determines constant Ic 0n .
  • the rate of the dissociation of the complex is determined, however, by constant
  • both the affinity in the form of K d and the maximum number of binding sites for a ligand can be determined.
  • the radioligand is used in various concentrations, the bound ligands are separated from the free ligands, and the amount of bound ligands is determined via the measurement of radioactivity.
  • the bound ligand [Lb ound ] is plotted against the ligand concentration [L] that is used (Fig. 8a).
  • the overall binding of the ligand in general contains a non-specific portion that increases linearly with the ligand concentration, and a specific portion that shows a saturation sequence.
  • the non-specific binding is produced by the fact that each ligand binds with very low affinity for a virtually infinite number of binding sites.
  • inhibitor molecules are used that competitively displace the specifically bound ligands or block all receptor sites.
  • the unlabeled ligand is used in excess, such that only the displaced, non-specifically bound radioligand is detected.
  • the curve for the specific binding is obtained when the curve of the non-specific binding is subtracted from the total or overall binding (without inhibitor).
  • the curve of the specific binding approaches a maximum value (asymptote), which corresponds to the maximum number of binding sites (B ma ⁇ or R t ).
  • the K d is then produced from the ligand concentration, in which one-half of the maximum binding sites is occupied (Fig. 8a).
  • the preferred method to determine these values is, however, the linearization of the saturation curve in a Scatchard Plot.
  • the latter is obtained by the concentration of the bound ligands [Lbound] being divided by that of the free ligand [Lf ree ] and the values that are obtained being plotted against the concentration of the bound ligands (Fig. 8b).
  • the point of intersection with the x-axis yields B max (or R t ) and the K d is obtained from the negated reciprocal values of the increase.
  • Fig. 8 Saturation Curve and Scatchard Plot for Determining the Dissociation Constant K d and the Maximum Binding R t .
  • (a) Ligand-saturation curves for the total binding, specific binding and non-specific binding. The specific binding is obtained by subtraction of the lower curve (non-specific) from the upper curve (total). The specific binding approaches a maximum value Rt. K d is produced from the ligand concentration at 50% of the maximum binding, (b) Scatchard Plot of the specific binding. K d is produced from the negation of the reciprocal increase. R t corresponds to the point of intersection with the x-axis. [22]
  • Fig. 9 Saturation Curve (Left) and Scatchard Plot (Right) for a Ligand with Two Different Affine Receptor Binding Sites.
  • the curve in the Scatchard Plot resulted from the sum of the highly affine binding (broken lines) and the lower-affine binding (dotted lines) [23].
  • the substances to be studied in competitive binding tests are used as inhibitors in a radioligand.
  • the binding of a constant radioligand concentration is measured in the presence of varying unlabeled substance concentrations. If the bound radioligand concentration is plotted against the logarithm of the inhibitor concentration in the diagram, the following curve is obtained in an ideal case:
  • Fig. 10 Competitive Binding Curve.
  • the IC 50 is produced from the substance concentration at 50% inhibition of the specific radioligand binding [23]. At very low inhibitor concentrations, the total binding first remains on a plateau and then drops with increasing inhibitor concentration until it again reaches a plateau, which corresponds to the non-specific binding.
  • the concentration of the inhibitor or the substance to be identified (NPY analog), in which 50% of the specific binding of the radioligand (difference of the total binding and non-specific binding) is inhibited is referred to as IC 50 (inhibitory concentration) or else EC 50 (effective concentration).
  • the value of the IC 50 is determined by three factors. Most important is the affinity of the receptor for a competitive substance. If the affinity is high, the IC 50 is low. In addition, the concentration of the radioligand plays a role. The higher the ligand concentration that is used, the more that is required from the competitor to displace the ligand. Therefore, in the experiment, the ligand concentration that is used is in the range of the Kd. Also, IC 50 influences the affinity of the ligand to receptor (K d ). More inhibitors are required to displace a solidly binding ligand (low Ka) than for a poorly binding ligand (high K d ).
  • K, values can be interpreted just as K d values.
  • BSA Bovine Serum Albumin
  • Neuropeptide Y Fragment (13-36) (NPY(13-36)) Sigma-Aldrich
  • the human neuroblastoma cells SK-N-MC were cultivated in Dulbecco's Modified Eagle Medium with Glutamax I and the addition of 0.1 mmol of non-essential amino acids and 10% fetal bovine serum (heat-inactivated).
  • the human breast cancer cells MCF-7 were cultivated in a minimum essential medium with Earle's salts and Glutamax I and the addition of 0.1 mmol of non-essential amino acids, 1 mmol of sodium pyruvate, 10 ⁇ g/ml of insulin (cow) and 10% fetal bovine serum.
  • the human neuroblastoma cell line MHH-NB-1 1 was cultivated in RPMI 1640 medium with Glutamax I and the addition of 0.1 mmol of non-essential amino acids and 10% fetal bovine serum. All cell lines were cultivated at 37°C, 5% CO 2 and 95% atmospheric humidity in a gassing incubator.
  • the corresponding culture medium was preheated to 37°C. The cells were removed from the nitrogen container and heated as quickly as possible
  • cryo vessels were disinfected with 70%
  • precooled freezing medium FBS with 10% (v/v) DMSO
  • a cell concentration of 5x10 6 cells/ml is achieved.
  • 1 ml was pipetted into precooled cryotubes. The tubes were set in the freezing container at -70°C and left there for at least 48 hours. Then, the cryotubes were stored in liquid nitrogen.
  • NPY receptor status For in-vitro determination of the NPY receptor status, cells in cell culture plates with 48 cavities were cultivated until confluence was completed (about 3x10 5 cells per cavity). The number of receptors is indicated per cell as well as per mg of whole protein content of the disseminated cells per cavity. To this end, before each binding study, the cell count and the protein content of in each case two cavities per plate was determined. For cell counting, the
  • PYY(3-36) or NPY(13 -36)) was added as an inhibitor for the binding of radiolabeled universal ligands 125 I-PYY to three cavities in each case, so that this 100 nmol was present in the batch.
  • B t batch without inhibitor
  • 50 ⁇ l of the ligands l25 I-peptide YY (1 nmol) was pipetted into all cavities.
  • a total volume of 250 ⁇ l with the final ligand concentration of 0.2 nmol (Table 4) was produced.
  • the exact determination of the ligand concentration used based on the total activity was carried out by measuring 50 ⁇ l aliquots on the gamma counter. Within one passage, the samples were measured for 30 seconds in each case. A 3x determination was made.
  • the plates were set at a temperature of 37 0 C for 2 hours in the incubator. After the
  • the bound ligand was dissolved from the cell surface and thus the surface binding of the ligand was determined.
  • the supematants from one cavity in each case were combined, and the activity was determined in a gamma counter.
  • the cells were lysed with 0.5 ml of a 1 M sodium hydroxide solution.
  • the cell culture plates were incubated at a temperature of 37°C in the shaker at 200
  • the amount of bound ligand was calculated in fmol/mg (see 2.2.4).
  • the old culture medium was removed and replaced by 150 ⁇ l of medium heated to 37 0 C (respective cultivation medium plus 1% (m/v) BSA).
  • the ligand 125I-PYY was used in 12 different concentrations of 10 to 0 nmol.
  • a dilution sequence in medium was produced, and 50 ⁇ l of a dilution was pipetted into six cavities in each case.
  • Another 50 ⁇ l was pipetted in each case into a reaction vessel, and the exact activity of the individual dilutions on the gamma counter was determined, with which the ligand concentration could be calculated (see 2.2.4).
  • the plates were set at a temperature of 37°C for 2 hours in the incubator. After the
  • the medium was carefully suctioned off with a Pasteur pipette with the aid of a vacuum. Then, the cells were washed twice with 0.5 ml of cold PBS solution (4°C). The cells were lysed with 0.5 ml of a 1 M sodium hydroxide solution, such that ligands both bound on the surface and internalized are found in the cell lysate.
  • the incubation of the cell was carefully suctioned off with a Pasteur pipette with the aid of a vacuum. Then, the cells were washed twice with 0.5 ml of cold PBS solution (4°C). The cells were lysed with 0.5 ml of a 1 M sodium hydroxide solution, such that ligands both bound on the surface and internalized are found in the cell lysate. The incubation of the cell
  • the cell lysates were moved into reaction vessels.
  • the cavities were washed once more with 0.5 ml of sodium hydroxide solution, and the solutions were combined with the extracts.
  • the measurement of the cell extracts was carried out for 30 seconds respectively in the gamma counter.
  • the NPY analogs were used as inhibitors in binding studies in different concentrations.
  • the ligand 125 I-PYY was used in the concentration range of the K d determined for the cell line (see information in the Results portion).
  • the old culture medium was removed and replaced by 150 ⁇ l of medium heated to 37°C (respective cultivation medium plus 1% (m/v) BSA).
  • the NPY- analog was used in 12 different concentrations of 50 ⁇ mol to 15 pmol.
  • a dilution series in medium was produced, and 50 ⁇ l of a dilution was pipetted into three cavities in each case.
  • excess cold peptide YY 50 ⁇ l, 5000 nmol
  • B t the inhibitor (PYY or NPY analog) was replaced by a corresponding volume of medium.
  • the ligand was used in various concentrations, depending on K ⁇ j, which was determined for the individual cell lines. Within one cell assay, in each case 50 ⁇ l of a ligand concentration was used (Table 6). The exact determination of the ligand concentration used was carried out with 50 ⁇ l of aliquots in a gamma counter.
  • the K 1 To determine the K 1 , first the IC 50 from the competition curve is determined. For this purpose, the amount of bound ligands [fmol/mg of protein] was calculated (see 2.2.4) from the measured activities, and then the specific binding was determined from the difference of total and non-specific binding. The specifically bound ligand was plotted in the diagram against the NPY-analog concentration, and the IC 50 was determined. This also took place with the aid of the software GraFit 4. The K, value was then calculated with the Cheng- Prusoff equation (see 1.6.2) via the IC 50 and the ligand concentration that is used.
  • a and A 0 can be indicated in TBq/mmol or dpm/fmol.
  • the amount of bound ligand is calculated from the cpm values:
  • the GraFit program indicates the B max in mol/1.
  • the information is usually in fmol/mg of protein, however.
  • the conversion is as follows:
  • a working solution that consists of 4% copper(II) sulfate solution and biscinchoninic acid solution (BCA) was produced at a 1 :50 ratio.
  • BSA whose 1 mg/ml stock solution in milli-Q water was diluted to concentrations of 100, 200, 300, 400, 500, 600, 700, 800 and 1000 ⁇ g/ml, was used as a standard protein.
  • 50 ⁇ l of the dilutions was removed and pipetted into 1.5 ml reaction vessels.
  • the dilution stage was replaced by the same volume of milli-Q water.
  • the samples were diluted with 950 ⁇ l of working solution (1 :20) and incubated for 30 minutes in a thermomixer at a temperature of 37°C. During the incubation, the batches were shaken at 800 rpm. After the incubation, the samples were cooled to room temperature for about 5 minutes. The determination of extinction by photometer was carried out at a wavelength of 562 nmol. In all samples, a 3x determination was performed. To generate the calibration lines, the extinction values were plotted against the BSA concentration, and a straight-line equation was created.
  • immunodeficient hairless mice (NMRI nude/nude, female, about 20 g) were inoculated subcutaneously by the Taconic M&B Company (Denmark) with 5x10 6 SK-N-MC cells in 100 ⁇ l of Matrigel.
  • the cells were prepared for the inoculation. To this end, the cells were trypsinized as described under 2.2.1.2 and taken up in 10 ml of culture medium. After the cell count was determined, the cell suspension was centrifuged for 5 minutes at 200 x g and the medium supernatant was suctioned off. The resuspension of the cell pellet was carried out in Matrigel corresponding to the counted cells and the necessary cell count per 100 ⁇ l (per mouse). For cell implantation (inoculation), the animals were subjected to short-term anesthesia with isoflurane to be able to place the injection accurately. The injection site was purified with 70% ethanol, the cell suspension was taken up in a 1 ml syringe with a one-way
  • the weight and tumor size (length and width) often tumor-bearing animals were determined 3 x per week over about eight weeks.
  • the tumor surface area was calculated according to the length x width formula and plotted in a diagram against time.
  • the animals were killed by decapitation (beheading) under anesthesia with isoflurane, and the following organs were removed: tumor, spleen, liver, lung, heart, brain, muscles, ovary, uterus, thyroid, kidney, adrenal glands, pancreas, stomach (without contents), intestine (with contents), skin, and blood.
  • Urine and feces were collected separately during the incubation. The rest of the body that remains after the removal of organs was used to balance the substance dose that was still not eliminated at the time of the distribution. The rest of the body was uniformly distributed into 5 scintillation vessels for this purpose.
  • the SK-N-MC cells showed a growth that was dependent on the disseminated cell count. If a few cells were seeded (1x10 4 ), the logarithmic growth phase (log phase) began after a short lag phase (48 hours); until after about 11 days, the cells died at a cell count of Ix 10 5 (Fig. 1 1). The more thickly seeded cells (5x10 4 ) first showed a similar growth. After a very short log phase, the cells entered into the stationary phase without growth starting at a cell count of about 1.5x10 5 and died after about 10 days. The maximum possible cell count per hole (100% confluence) was 1.5x10 5 cells.
  • Fig. 12 Growth Curve for Human MCF-7 Cells.
  • the growth sequence in the cells seeded with different densities was almost the same (Fig. 13). After a short adaptation phase, they showed a steady growth. Starting from a density of 5x10 5 cells per cavity, the cells reached the stationary phase. The microscopy of the cells showed that the adhesion surface area of the culture plate was covered with cells, and it formed additional cell clusters; the cell growth was thus not only two-dimensional but also three-dimensional.
  • Fig. 13 Growth Curve for Human MHH-NB-Il Cells. In 48-hole cell culture plates,
  • the total amount of the bound radiolabeled ligands 125 I-PYY without the addition of inhibitor was about 8.5 fmol/mg of protein with SK-N-MC cells (Fig. 14). With PYY as an inhibitor, a total of about 2 fmol/mg of I-PYY was detected. With this peptide, the binding of the radioactive ligand was almost completely inhibited.
  • the Leu 3 l -Pro 34 -PYYY that binds to Yi and Y 5 receptors showed an almost equally high inhibition, Hike PYY, which points to the expression of these two receptor subtypes.
  • Peptides PYY(3-36) and NPY(13-36) showed no inhibition of the ligand, such that the expression of the receptors Y 2 and Y 5 can be ruled out. Thus, only the Yj receptor was expressed on the SK-N-MC cells. After 2 hours of incubation of the cells with the control peptides, about two thirds of the bound peptide was internalized with the receptor.
  • Fig. 15 Binding of • 1 '2"51-PYY to MCF-7 Cells in the Presence of Subtype-Specific Control
  • the amount of bound 125 I-PYY was lower in MCF-7 cells than in SK-N-MC cells. Without an inhibitor, a total of about 3.7 fmol/mg of bound 125 I-PYY was measured (Fig. 15). After inhibitor PYY was added, 1.3 fmol/mg of 125 I-PYY was bound, which is comparable to the inhibition by the Yi -receptor-specific Leu 31 -Pro 34 -PYY. The addition of PYY(3-36) and NPY(13-36) produced a slight reduction of the ligand binding by 0.5 fmol/mg to a total of 3.2 fmol/mg of bound 125 I-PYY.
  • Fig. 16 Binding of 125 I-PYY to MHH-NB-Il Cells in the Presence of Subtype-Specific
  • the Y 2 receptor could be identified as the single NPY- receptor subtype.
  • the amount of bound radioactive ligands in the presence of PYY was about 6.4 fmol/mg.
  • Leu 31 -Pro 34 -PYY showed no binding, since the amount of bound 125 I-PYY with about 16.5 fmol/mg was just as high as without adding inhibitor, thus accordingly no inhibition occurred (Fig. 16).
  • PYY (3-36) or NPY(13-36) the ligand binding could be reduced by 7 or 8 fmol/mg.
  • the Y 2 -receptor subtype, to which these two pepides have bonded is expressed.
  • the proportion of internalization was about 40% of the bound peptide, if no inhibition had taken place (w/o and LP-PYY). If an inhibition had occurred, the proportion of internalization was higher in comparison to the pure surface binding of the ligand, since the already internalized ligand can no longer be displaced by an inhibitor, in contrast to the ligand that is found on the surface.
  • the cell lines SK-N-MC and MCF-7 thus could be used for testing NPY analogs with regard to the binding to the Yi receptor.
  • the MHH-NB-1 1 cells were used as a negative control, to which Yi-selective peptides should not bind.
  • dissociation constant K d was determined for the universal ligand ' 5 I-PYY on the respective cells. Based on this K d , the concentration range of the ligand in the competition assays can be selected for determining the equilibrium dissociation constant Kj of the NPY analogs.
  • Fig. 17 Saturation Curve and Scatchard Plot (Right) of the Specific Binding of to SK-N-MC Cells. 2x10 5 Cells with an amount of protein of 57 ⁇ g were incubated for 2 hours at various concentrations (0.001 to 2 nmol) of 125 I-PYY. The non-specific binding was
  • Fig. 18 Saturation Curve and Scatchard Plot (Right) of the Specific Binding of 1 '2"51,-PYY to MCF-7 Cells. 5x10 4 Cells with an amount of protein of 10.5 ⁇ g were incubated for 2 hours at various concentrations (0.001 to 2 nmol) of 125 I-PYY. The non-specific binding was
  • Fig. 19 Saturation Curve and Scatchard Plot (Right) of the Specific Binding of 1 '25 3 iI-PYY
  • the determination of the K 1 of PYY in the competitive binding experiment yielded very different values for the various cell lines.
  • the K, on SK-N-MC cells was 0.78 nmol (Fig. 20A).
  • the K, determined on MCF-7 cells with 5.69 nmol was 7.3 x as high; the binding affinity of PYY thus was lower (Fig. 20B).
  • the K d values of the labeled PYY on these two cell lines did not show drastic differences (Table 7).
  • the strongest binding affinity showed PYY on the Y 2 -receptor-expressing MHH-NB-11 cells.
  • the K, was 0.11 nmol.
  • the competition curve on MHH-NB-1 1 cells differs from that of the other cell lines (Fig. 20C): it proved very much flatter and showed an untypical plot with an almost linear drop over a wide concentration range.
  • the binding affinity of the Y 2 -selective PYY(3-36) showed very different binding affinities on the tested cell lines SK-N-MC and MHH-NB-11.
  • the K 1 on the MHH-NB-11 cells was lower by the factor 763 (Table 7). This cell line thus could be used as a Y 2 -receptor selective control.
  • Fig. 20 Competition Curves and K 1 of PYY at Various Cell Lines.
  • Table 8 Binding Affinities of Selected NPY Analogs and Control Peptides on SK-N-MC Cells. The K 1 was determined with competitive binding tests with the ligand 125 I-PYY. The amino acids, in which the analogs are distinguished (positions 32 and 34 of the original peptide NPY), are underscored.
  • Fig. 21 Competition Curves and Kj of P2489 with Different Cell Lines.
  • SK-N-MC Tumor Xenografts For the growth kinetics of the SK-N-MC tumor xenografts, 10 hairless nude/nude NMRI mice were inoculated subcutaneously with 5x10 6 cells each. The growth rate of tumors was 60%. To generate the growth curve, 5 animals were used. The SK-N-MC tumors showed only a slow growth in the hairless mice (Fig. 22). Only 14 days after inoculation, a slight growth jump of 40 to 80 mm 2 of tumor surface area was shown. Then, the tumors grew only slowly, and after about 45 days, achieved an average tumor size of 135 mm 2 . The growth within the 5 tested animals was very different. In the observed period, a maximum tumor size of 160 mm 2 was reached.
  • Fig. 22 In-Vivo Growth Curve of SK-N-MC Tumor Xenografts. 5xlO 6 SK-N-MC cells in
  • Tumorflache Tumor Surface Area
  • Peptide P2471 was labeled with 177 Lu, and the purity was examined by means of RP- HPLC. The purity of 177 Lu-P2471 was approximately 95.33%. The chromatogram showed a mixture that consists of at least three isomers (Fig. 23).
  • Fig. 23 HPLC Chromatogram of 1 '7"7 ⁇ Lu-P2471
  • the peptide fW7 was labeled with 177 Lu, and the purity was examined by means of RP-HPLC. The purity of l 77 Lu-fW7 was 94.83%. The chromatogram showed a double peak, which indicates a mixture of isomers (Fig. 24).
  • Fig. 24 HPLC Chromatogram of l77 Lu-fW7.
  • the organ distribution of ' 77 Lu- fW7 showed very similar values to those for 177 Lu- P2471 (Table 10). Almost the entire peptide (83%) was already excreted with the urine after 1 hour. As in the case of 177 Lu-P2471, a concentration in the kidneys in a similar amount range (about 9%ID/g after 3 hours) also took place here. A smaller portion (0.92%ID/g) accumulated in the liver 1 hour after injection and increased to 1.36%ID/g after 5 hours. The concentration in the tumor was at a maximum value of 0.71%ID/g after 1 hour and then at values around 0.2%ID/g, somewhat higher than with 177 Lu-P2471.
  • the tumor/blood ratio was at 15.25 after 5 hours and increased to 18.89 after 24 hours.
  • NPY analogs For the in-vitro studies of the NPY analogs relative to their binding strength and selectivity with regard to an NPY receptor subtype, cell lines should be used that primarily express Yi- or Y 2 receptors.
  • the human neuroblastoma cell line MHH-NB-11 was used.
  • receptor-subtype-specific peptides such as the Y 2 receptor-selective NPY(3-36) and the Yi -selective Leu 3 l -Pro 34 -NPY, and the ligand [ 3 H]propionyl-NPY, H ⁇ fliger et al. could detect the Y2 receptor expression of MHH-NB-1 1 cells [27]. Also, this was confirmed in the experiments performed here, since Leu 31 -Pro 34 -PYY has not bonded to the MHH-NB-1 1 cells and thus receptor subtypes Yi and Y 5 could be ruled out. The Y 2 -selective control peptides, however, have bonded very well.
  • the Yi receptor binding also should be studied on a breast cancer cell line.
  • the human breast cancer cell line MCF-7 was used.
  • the gene expression analyses by Kuang et al. it was possible to identify the mRNA of the Yi receptor [20].
  • the experiments for subtype characterization showed that the majority of binding sites on these cells were Yi receptors.
  • PYY(3-36) Y 2 - and Y 5 - selective
  • NPY(13-36) Y 2 -selective
  • the K d value of 0.251 nmol for 125 I-PYY that is determined on SK-N-MC cells is comparable to values from the literature.
  • Poindexter et al. determined a Ka value for 125 I-PYY of 0.35 nmol [28]. This value, however, was determined by binding studies on SK- N-MC-membrane fractions and not on whole cells. For whole SK-N-MC cells, only the Kd of [ 3 H]-propionyl-NPY could be found in the literature (Fabry et al.), which with 0.37 nmol is also near the K d of 125 I-PYY that is determined here [25].
  • the ligand binding to isolated membranes determines the pure receptor binding.
  • 125I-PYY has a somewhat higher K d of 0.339 nmol in comparison to the K d (0.251 nmol) that is determined in comparison to the neuroblastoma cells SK-N-MC.
  • K d 0.251 nmol
  • This can be attributed to the binding of the ligand to several NPY receptor subtypes with regard to the MCF-7 cells or with regard to morphological characteristics of these cells.
  • the MCF-7 cells also express several Y 2 receptors, but the latter are only present in a small number compared to the Yi receptors.
  • the Scatchard Plot for the MCF-7 cells does not allow any conclusions on two different binding sites with different affinities.
  • the affinities for both receptor subtypes on MCF-7 can be very similar and therefore cannot be differentiated. Morphological differences between the cell lines can also be the cause of different affinity constants of the ligand to a receptor.
  • the accessibility of the receptors for the ligands on various cell lines can vary.
  • the varying internalization behavior of the cell lines which has been revealed in the experiments for subtype determination, can have an influence on the varying K d values.
  • the proportion of internalization was higher than in the MCF-7 cells and can thus contribute to a higher measured binding affinity of the ligand l25 I-peptide YY.
  • the K d values for the ligands 123 I-PYY determined in the saturation experiments were reproducible and were thus used for the determination of IC 50 values and K, values of the NPY peptide analogs to be tested with regard to binding affinity.
  • the binding behavior of the unlabeled PYY to the cell lines under study was comparable to that of the 125 I-labeled PYY.
  • a K 1 of 0.78 nmol was determined on SK-N-MC cells.
  • the differences in the dissociation constants between the three cell lines were clearer for the unlabeled PYY, however, than for 125 I-PYY. A possible clarification of this can be different growth phases of cells in which the regulation of the receptor expression was different.
  • the selection of peptides, which bind to the Y 2 receptor, is thus made possible by competitive binding tests with MHH-NB-1 1 cells. This is important, since the binding to the Y 2 receptor for the selective binding of an NPY analog to breast tumor cells is expressly not desired.
  • a peptide (P2489) showed a binding affinity of less than 100 mmol in a first screening with regard to binding affinity.
  • This preliminary result was confirmed in competitive binding studies with the Yi -expressing SK-N-MC- and MCF-7 cells.
  • the K 1 on SK-N-MC Cells (49.2 nmol) was lower than on MCF-7 cells (176.1 nmol), which is in conformance with the results of the binding studies with PYY.
  • the selectivity for the Yi receptor was confirmed by the fact that there is no binding to Y 2 receptor-expressing MHH-NB-11 cells.
  • ⁇ -amino acids which have similar properties, like proline. Moreover, they increase the strength of the peptide backbone and stabilize defined secondary structures, primarily in small peptides. In addition, the incorporation of such ⁇ -amino acids leads to improved resistance to chemical or enzymatic degradation by peptidases and to increased lipophilia. It is suggested by Koglin et al.
  • NPY analogs that are studied within the framework of this work consist in their basic structure of the last 12 amino acids of the C-terminus of NPY (amino acids 25-36): Ac-Arg-His-Tyr-Ile-Asn-Leu-Ile-AS 32 -Arg-AS 34 -Arg-Tyr-NH 2
  • the C-terminal end of the shortened NPY analogs is amidated as with NPY, and the N-terminal end is acetylated.
  • the two positively charged arginine radicals at positions 33 and 35, as well as tyrosine at position 36, are contained in each peptide, since they are decisive for the specific binding to the receptor subtypes.
  • the amino acids at positions 32 (AS 32 ) and 34 (AS 34 ) are either the Thr (position 32) and GIn (position 34), naturally occurring in NPY, or various unnatural ⁇ -amino acids.
  • Table 11 Sequence Comparison of the C-Terminus of NPY, PYY and LP-PYY with the NPY Analogs and Binding Affinities of These Peptides to the Yi Receptor.
  • Fig. 25 Structures of ⁇ -Amino Acids That Are Contained in the NPY Analogs.+
  • the NPY analogs which contain aminocyclohexanecarboxylic acid (AchC) (Fig. 25) as ⁇ -amino acid, had greater affinities for the Yi receptor than the other NPY analogs.
  • the latter contained, e.g., D-Cys, Cys or the ⁇ -amino acid derivatives of Pro ( ⁇ -hPro) or Leu ( ⁇ - Leu) at positions 32 and/or 34 as well as at other positions.
  • the binding of these peptides to the Y i receptor was weak, however, so that the binding constants were not determined. If peptides P2467 and P2468 were compared to one another, the influence of the stereochemistry of AchC is clear.
  • NPY analogs studied are based on the acetyl-[ ⁇ -ACC 32 ' 34 ](25-36)NPY (Ac-fQ6) developed by Koglin et al., which has a Kj of 29 nmol on SK-N-MC cells [30].
  • This linear C-terminal NPY-analog contains ⁇ -aminocyclopropanecarboxylic acid ( ⁇ -ACC), consisting of a cyclopropane ring with a methylated carboxyl group (Fig. 25), as components in the binding-relevant positions 32 and 34.
  • ⁇ -ACC ⁇ -aminocyclopropanecarboxylic acid
  • Fig. 25 methylated carboxyl group
  • Table 12 Amino Acid Sequence and Binding Affinity (Yi Receptor) of NPY Analogs That Contain ⁇ -ACC-Amino Acids.
  • a DOTA chelating agent was also coupled to the N- terminal end of the original peptide of Koglin et al. This resulted, as also in the case of P2471, in the reduction of the binding affinity (Ki > 665 nmol). Therefore, an attempt was made to change the coupling position of the DOTA to the peptide to keep the loss of the affinity as low as possible. This could also be achieved with the peptide fW7(D0TA). In this connection, the DOTA was indirectly introduced via a ⁇ -Ala chain on Lys in the N-terminal end of the peptide.
  • Lu-P2471 showed very low activities in the blood, no concentration whatsoever in organs, and a quick excretion, which can end in a low stability of the peptide in the organism. This is presumably caused by a quick enzymatic degradation of the peptide.
  • the blood-brain barrier of l 77 Lu-P2471 could not be overcome, and no concentration took place in the brain. Passing the blood-brain barrier is possible only for lipophilic substances or via active transport systems. Also, the molecule size is decisive for the permeability. 177 Lu-P2471 is presumably too large and, moreover, not lipophilic enough, which also is evident by the low concentration in the liver.
  • the peptide that is labeled with 177 Lu and DOTA remains comparatively long in the kidney, which can be attributed to the fact that peptides are held up in the kidney in conjugated form with radiometal-chelate complexes.
  • peptides are taken up via endocytosis in cells of the proximal tubule in the kidney, where then the intracellular degradation in the lysosomes takes place. The degradation products are then excreted or travel further into the circulation.
  • the peptides that are labeled with radiometals bind to metal-binding proteins after uptake into the cells of the proximal tubule and are thus held up intracellularly. An accumulation of radioactivity in the kidneys followed by an only slow excretion of the metal-chelate complex are carried out. P2471 was thus very quickly degraded in the organism and excreted for the most part via kidney and urine.
  • peptide fW7(DOTA) showed a relatively high affinity of 62.8 nmol to the Yi receptor despite DOTA coupling.
  • the labeling with 177 Lu was also successful.
  • several peaks were shown here in the chromatogram based on the racemates of ⁇ -ACC.
  • the binding affinity that is too low is less the cause for only slight concentration than the metabolic instability and the quick excretion of the peptide.
  • the concentration of 177 Lu- fW7(D0TA) in the kidney was quite high.

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Abstract

Le sous-type Y1 du récepteur du neuropeptide Y (NPY) est exprimé de façon différentielle à partir de cellules tumorales mammaires et constitue ainsi une molécule cible avantageuse pour l'imagerie moléculaire du cancer du sein. Des analogues peptidiques dont la séquence est réduite aux sections de liaison au récepteur du ligand naturel NPY ont été synthétisés. Ces analogues peptidiques sélectifs du récepteur Y1 contiennent des acides aminés non naturels qui augmentent l'affinité au récepteur et sont destinés à assurer la stabilité du peptide fortement raccourci. De nouveaux analogues du NPY, destinés à être utilisés comme ligands radioactifs, ont été testés au niveau de leur affinité de liaison et leur sélectivité pour le récepteur Y1. A cet effet, des essais de liaison in vitro avec des lignées cellulaires exprimant le récepteur Y1 ou Y2 ont été mis au point et optimisés, puis les affinités de liaison des analogues du NPY ont été déterminées. Dans ce cas, on a identifié un peptide (P2489) dont l'affinité de liaison la plus élevée a été déterminée avec un Ki de 42,8 nmol de cellules SK-N-MC exprimant le récepteur Y1 et dont la sélectivité pour le récepteur Y1 a pu être détectée du fait de l'absence de liaison aux cellules MHH-NB-11 exprimant le récepteur Y2. En tant qu'analogue du NPY additionnel, le peptide fW7 contenait l'acide aminé non naturel acide β-aminocyclopropanecarboxylique aux positions 32 et 34, cet acide influençant de façon positive la liaison au récepteur Y1. Un couplage direct de l'agent chélateur DOTA, qui est nécessaire pour le marquage des peptides par métaux radioactifs, à l'extrémité N-terminale des peptides a entraîné la perte de l'affinité de liaison. Par couplage indirect du DOTA au peptide fW7 par l'intermédiaire d'un séparateur, cette perte a pu être réduite, fW7(DOTA) présentant alors une affinité de liaison élevée (Ki = 62,8 nmol) similaire à celle de P2489.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010096188A2 (fr) 2009-02-20 2010-08-26 Ipsen Pharma S.A.S Analogues de neuropeptide y ayant une substitution par une proline en position 34
WO2010096186A1 (fr) 2009-02-20 2010-08-26 Ipsen Pharma S.A.S Analogues de neuropeptide y ayant au moins une substitution par un acide aminé synthétique
JP2012518636A (ja) * 2009-02-20 2012-08-16 イプセン ファルマ ソシエテ パール アクシオン サンプリフィエ 神経ペプチドｙ受容体結合化合物を有する細胞毒性コンジュゲート
CN104755105A (zh) * 2012-09-17 2015-07-01 昂图赫姆有限责任公司 受体配体连接的细胞毒性分子

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4311756A1 (de) * 1993-04-08 1994-10-13 Thomae Gmbh Dr K Modifizierte Peptide, diese Verbindungen enthaltende Arzneimittel und Verfahren zu ihrer Herstellung
EP1208852B1 (fr) * 2000-11-24 2007-08-01 Jean-Claude Reubi Composés fixantes de récepteur de Neuropeptide Y1 dans le traitement et le diagnostic du cancer
AU2002332054B2 (en) * 2001-09-24 2007-11-08 Imperial Innovations Limited Modification of feeding behavior
GB0217015D0 (en) * 2002-07-23 2002-08-28 Bioacta Ltd Peptide 4

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010096188A2 (fr) 2009-02-20 2010-08-26 Ipsen Pharma S.A.S Analogues de neuropeptide y ayant une substitution par une proline en position 34
WO2010096186A1 (fr) 2009-02-20 2010-08-26 Ipsen Pharma S.A.S Analogues de neuropeptide y ayant au moins une substitution par un acide aminé synthétique
JP2012518636A (ja) * 2009-02-20 2012-08-16 イプセン ファルマ ソシエテ パール アクシオン サンプリフィエ 神経ペプチドｙ受容体結合化合物を有する細胞毒性コンジュゲート
EP2398492A4 (fr) * 2009-02-20 2013-07-17 Ipsen Pharma Sas Conjugués cytotoxiques ayant un composé de liaison au récepteur de neuropeptide y
US8877715B2 (en) 2009-02-20 2014-11-04 Ipsen Pharma S.A.S. Cytotoxic conjugates having neuropeptide Y receptor binding compound
CN104755105A (zh) * 2012-09-17 2015-07-01 昂图赫姆有限责任公司 受体配体连接的细胞毒性分子

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