WO2007039318A2 - Neuropeptide y analogs - Google Patents

Abstract

The neuropeptideY (NPY) -receptor-subtype Yl is expressed differentially from breast tumor cells and is therefore an advantageous target molecule for the molecular imaging of breast cancer. Peptide analogs were synthesized, whose sequence is reduced to the receptor-binding sections of the natural ligand NPY. These Yl receptor-selective peptide analogs contain unnatural amino acids that increase the receptor affinity and are to ensure the stability of the greatly shortened peptide. New NPY analogs, which are to be used as radioligands, were tested for their binding affinity and selectivity for the Yl receptor.

Description

NEUROPEPTIDE Y ANALOGS

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.

In certain aspects of the invention, the neuropeptide Y polypeptide analogs are specific or selective for the neuropeptide Y| receptor ("Yi"). By the terms "selective" or "specific," it is meant that the analog has a higher affinity for the Yl receptor, than other neuropeptide receptor subtypes, e.g., Y₂, Y₄, and Y₅. 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. In certain embodiments, 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| selectivity, using the assays described below, or selectivity for any neuropeptide Y receptor.

In a futher embodiment 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. In the disclosure below, many examples of detectable labels are provided which it is attached. In the disclosure below, many examples of 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 ¹⁷⁷Lu, ¹⁸F, ⁶⁸Ga, ^99mTc 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. Other carriers are described in Remington: The Science and Practice of Pharmacy (Gennaro and Gennaro, eds, 20th edition, Lippincott Williams & Wilkins, 2000); Theory and Practice of Industrial Pharmacy (Lachman et al., eds., 3rd edition, Lippincott Williams & Wilkins, 1986); Encyclopedia of Pharmaceutical Technology (Swarbrick and Boylan, eds., 2nd edition, Marcel Dekker, 2002).

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. By the phrase "effective amount," it is meant the quantity of the polypeptide, which when contacted with the cell, allows the cells to be detected. This amount can be determined routinely, and depends, e.g., on the polypeptide, whether it comprises a detectable label, and the route through which it is administered (e.g., intravenously, directly into the tissue or suspected tumor). 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. In a further embodiment 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 Yι, Y₂ or Y₅ 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. The term "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). When coupled to a chemotherapeutic agent, the analogs can be used to target the agent to the cell type of interest.

In a further embodiment the present invention provides the use of neuropeptide Y analog as defined above for the manufacture of a medicament.

More preferably the treatment concerns the treatment of cancer expressing the receptor Y receptor and more preferably the Yi, Y₂ or Y₅ 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:

1. An isolated polypeptide comprising an amino acid sequence listed in Table 8, and derivatives thereof.

2. An isolated polypeptide consisting of an amino acid sequence listed in Table 8, and derivatives thereof.

3. An isolated polypeptide comprising or consisting of P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(DOTA), or derivatives thereof.

4. An isolated polypeptide of any of embodiments 1-3, wherein said polypeptide consists of 12 or less naturally-occurring or non-naturally-occurring amino acids.

5. An isolated polypeptide of any of embodiments 1-4, wherein said polypeptide is selective for the Yi receptor.

6. An isolated polypeptide of any of embodiments 1-5, further comprising a detectable label.

7. 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.

8. 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.

9. A method of embodiment 7, further comprising contacting said cell with a polypeptide which is selective for a Y₂ or Y₅ receptor, and detecting said binding.

10. A method of embodiment 8, further comprising contacting said cell with a polypeptide which is selective for a Y₂ or Y₅ receptor, and detecting said binding.1 11. A method of embodiments 7 or 8, wherein the detecting is performed using positron emission tomography.

12. 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.

13. 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.

14. A method of any of embodiments 7, 8, 12, or 13, wherein said method is performed in situ, on a tissue section comprising a biopsy tissue.

15. A method of any of embodiments 7, 8, 12, or 13, wherein cells in a primary tumor or a metastatic site are detected.

16. 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.

17. A method of embodiment 16, wherein said agent is attached to a chemotherapeutic agent.

18. An antibody which is specific for P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(D0TA), an analog listed in Table 8, or derivatives thereof.

Abbreviations

AchC Aminocyclohexanecarboxylic Acid β-ACC β-Aminocyclopropanecarboxylic Acid

Bmax Maximum Number of Binding Sites

B_s Specific Binding

B_t Total Binding

B₁₁ Non-Specific Binding cpm Enumerated Radioactive Decompositions Per Minute DOTA 1 ,4,7, 10-Tetraazacyclododecane- 1 ,4,7, 10-tetraacetic Acid dpm Radioactive Decompositions Per Minute

FDG ¹⁸F-Deoxyglucose

FDG-6P ' ⁸F-Deoxyglucose-6-Phosphate

FLT ^{I 8}F-L-Thymidine

IC₅₀ Inhibitory Concentration at a 50% Specific Ligand Binding

K_d Equilibrium Dissociation Constant of the Ligand

Kj Equilibrium Dissociation Constant of the Inhibitor

NPY Neuropeptide Y

MRT Magnetic Resonance Tomography

PET Positron Emission Tomography

PP Pancreas Polypeptide

PYY Peptide YY

%ID/g Percentage Distribution of the Injected Dose per Gram (Organ) Weight

SPECT Single Photon Emission Computer Tomography

1 Introduction

1.1 Breast Cancer

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].

A number of factors are now known that increase the risk of developing breast cancer (Table 1). These include, i.a., elements of lifestyle (e.g., smoking, eating foods with a high fat content, alcohol consumption), hormonal status and genetic dispositions. In recent years, the tumor suppressor genes BRCA-I and BRCA-2 that are responsible for the development of breast cancer in about 5-10% of all cases [3] were identified. In addition, reproductive factors, such as early onset of menstruation (first menstruation), a late first pregnancy, a small number of pregnancies, brief or no breastfeeding, and a late menopause increase the risk of breast cancer. With age, the risk of developing breast cancer increases.

Breast cancer can also occur in males. Here, however, the disease is significantly more rare (only about 1% of all cases of breast cancer) and is often only detected late [3]. As causes of breast cancer in males, disorders in hormonal balance and a genetic disposition are assumed. Table 1 : Risk Factors for Breast Cancer in Women [4]

Relative risk High-risk group

Age >10 Etderr/ individuals

Geographical location 5 Developed countries

Breast density >5 Extensive dense breast tissue visible on mammogram

Ageatmenarchc- 3 Before age 11 years

Age at menopause 2 After age 54 years

Age at first full pregnancy 3 First child after age 40years

Famify history 3»2 Breast cancer in first-degree relative

Previous benign breast disease 4-5 Atypical hyperplasia

Cancer in other breast >4 Previous breast cancer

Socioeconomic group 2 Groups I and II*

Body-mass index

Pre menopause 0-7 High body-mass index

Postmenopause 2 High body-mass index

Alcohol consumption 107 7% increase with every dairy drink

Exposure to ionising radiation 3 Abnormal exposure to young girls after age 10 years

Breastfeeding and parity Relative risk falls by 4-3% for Women who do not breastfeed every 12 months of breastfeeding in addition to a J% reduction for every birth

Use of exogenous hormones

Oral contraceptives 1-2 Current users

Hormone-replacement therapy 1'66 Current users

Diethylstilbestrol 2 Use during pregnancy

^*l and Il represent high and low socioeconomic status, respective^.

1.1.1 Clinical Picture

Breast cancer (Carcinoma mammae) is invasive, i.e., malignant tumors that penetrate into the tissue. In general, in comparison to normal 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].

Most 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. When breast cancer propagates in the body, often the axillary lymph nodes are first affected by tumor cells. Then, individual cancer cells can propagate in the body and form additional metastases there.

Krebs

Fig. 1: Various Forms of Breast Cancer (Source: Rϋdiger Anatomy, Anatomical Plates)

[Key to Fig. 1:] duktales carcinoma in situ (Milchgang aufgeschnitten) = Ductal Carcinoma In Situ

(Milk Duct Cut Open) lobulares carcinoma in situ (Drusenlappchen aufgeschnitten) = Lobular Carcinoma In Situ

(Gland Lobules Cut Open) invasives duktales Karzinom (Milchgang aufgeschnitten) = Invasive Ductal Carcinoma (Milk

Duct Cut Open) invasives lobulares Karcinom (Drusenlappchen aufgeschnitten) = Invasive Lobular

Carcinoma (Gland Lobules Cut Open) 1.1.2 Diagnosis

The earlier a breast cancer is detected, the greater the chance for complete recovery. As long as the propagation of the tumor cells is limited to the breast, the recovery rates are over 95%. About 80% of all tumors are found by the affected women in self-examinations.

Primarily mammography and ultrasonography are part of the standard techniques for breast cancer diagnosis. These imaging processes do not make it possible, however, to make reliable statements on whether a significant finding is benign or malignant. For this purpose, an inspection under the microscope of the tissue from the significant area is necessary. The sampling of this tissue is carried out by a biopsy.

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. In this connection, 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. During the imaging, 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.

In most cases, magnetic resonance tomography (MRT) is used to solve problems according to conventional diagnostic processes. With an MRT (also nuclear spin tomography), cross-sectional images of the human body, which often make possible an excellent evaluation of the organs and many changes in the organs, can be produced. The MRT uses magnetic fields, no x rays. The method is very sensitive and is also used in high- risk patients (e.g., #Z?C4-positive patients). The rate of false-positive diagnoses is relatively high, however, such that the MRT is never used as the sole diagnostic process [4]. In addition to the standard imaging processes, diagnostic processes in 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.

1.2 Positron Emission Tomography

Positron Emission Technology (PET) 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

Table 2: Imaging Processes and Their Properties [7]

MRS +++ >2 - Mmol +++

SPECT +++ >4 ++ μmol ++ ++

PET +++ >5 ++ Nmol +++ ++

Ultrasound ++ >0.4 - μmol

Optical + >1 - nmol ++ ++

(e.g., with

Fluorescence

Labels)

MRT: Magnetic Resonance Tomography; MRS: Magnetic Resonance Spectroscopy; SPECT: "Single Photon Emission Computer Tomography"; PET: Positron Emission Tomography

For PET, primarily the radionuclides ¹⁸F, ^{1 1}C, ¹³N or ¹⁵O 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 γ-quanta with

511 keV each, which move away from one another at an angle of 180°, are produced. This "destructive radiation" simultaneously strikes two opposite positions of the detector ring of the positron camera and makes it possible to detect and locate positron emission [8].

Fig. 2: ^ISF-Deoxyglucose (FDG).

It has been known for a long time that malignant tumors have a high glycolysis rate (Warburg, 1930). Cells in which a malignant transformation has occurred usually have an elevated glucose intake and an elevated glucose metabolism [9]. The most frequently used radiopharmaceutical agent for cancer diagnosis with PET is the glucose analog ¹⁸F- deoxyglucose (FDG) (Fig. 2). FDG is also taken up into the cells, just like glucose, via an active transport mechanism, and there, just like glucose, found from hexokinase and phosphorylated to FDG-6-phosphate (FDG-6P). FGD-6P cannot be used as a substrate, however, for additional enzymatic reactions of glycolysis. In addition, it is not completely dephosphorylated in most tissues, including tumors. 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]. P — ♦ Glycolysis

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].

A number of new PET tracers are found in the development. Many of them are concentrated in the visualization of cellular processes that are more specific than the glucose metabolism. Relative to breast cancer, e.g., 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. In addition, 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]. Via the graphic visualization of these target molecules (targets), therapy batches 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. At this time, 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]. Here, 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 (NPY) 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].

C -teππi nus

pNPY YPSKPDNPGBDAPAEDLARYYSALRHYINLITRQRY pPYY YPAKPEAPGEDASPEELSRYYASLRHYLWLVTRQRY hPP APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY

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 is common both in the peripheral and in the central nervous system and is one of the most frequently occurring 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, Y₂, Y₄, Y₅ und Y₆ were identified and were partially characterized extensively [13].

Breast cancer cells express various types of peptide receptors, such as 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(Y₂) 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.

Neuropeptide Y Analogs

As natural ligands, NPY and PYY have a high affinity for Yi- and Y₂-receptors. The latter are not selective for a receptor subtype, however. To date, several selectively-binding NPY analogs have been developed. These include the Yi-selective [Leu³¹, Pro³⁴]-NPY [15], which has substitution in the C-terminal end of the peptide, and the Y₂-selective NPY fragment (13-36) [16].

By substitution experiments with individual amino acids, it was possible to show that the C-terminus of NPY with two arginine radicals at positions 33 and 35 and tyrosine at position 36 constitutes the specific binding to the receptor subtypes [17].

Table 3: Comparison of Yi- and Y₂-Receptors Relative to Affinities of NPY Hormones and NPY Analogs. PYY shows the highest affinity for the two receptors. [Leu³¹, Pro³⁴]-NPY binds better to Yi receptors, while NPY (13-36) has the higher affinity for Y₂ receptors. PP shows the lowest binding affinity for the two subtypes. [1 1] Receptors Affinities

PYY > NPY > [Leu³¹, Pro³⁴]NPY » NPY(13-

Yi Receptors 36) » PP

PYY > NPY > NPY(13-36) > [Leu³¹, Pro³⁴]NPY

Y₂ Receptors > PP

To be able to use NPY analogs in the molecular imaging of breast cancer, 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. In addition, 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].

The 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. These include, i.a., β-amino acids (Fig. 5), which can stabilize defined secondary structures and support the rigidity of a peptide.

α -Aminosaure β² -Aminosaure β³ -Aminosaure

[α-Amino Acid β²-Amino Acid β³-Amino Acid] Fig. 5: General Structure of α- and β-Amino Acids. With β-amino acids, radical (R) can be either on the 2^nd atom (β²) or on the 3 ^rd C atom (β³).

1.5 Radioactive Labeling of 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., ¹⁸F, a positron radiator with a physical half-life of 110 minutes. The labeling of the peptide with ¹⁸F is generally carried out indirectly via the conjugation of a previously F-labeled synthon.

For the organ distribution experiments on tumor-bearing mice, which were performed within the scope of this work, and the gamma-scintigraphy for imaging, iodine isotopes are also used in addition to metallic radionuclides such as ¹⁷⁷Lu, ^99mTc 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. For labeling with ^{1 1 1}In and ¹⁷⁷Lu, for example, DOTA (1,4,7,10- tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid) is coupled covalently to the peptide (Fig. 6).

Fig. 6: DOTA-Chelating Agent Coupled to a Peptide.

For in-vitro characterization of peptides, the latter are labeled in most cases with the iodine isotope ¹²⁵I. For this purpose, the peptides have to contain tyrosine, histidine or primary amino groups. The iodine isotope is eiiner oυund directly to the aromatic radical of these amino acids or reacted indirectly via ¹²⁵I-labeled N-succinimidyl compounds, such as the Bolton-Hunter reagent (Fig. 7), with the primary amino group [19].

Fig. 7: ^l2SI-Labeled Bolton-Hunter Reagent.

1.6 Ligand-Receptor Binding Studies

To determine the binding strength of a peptide (ligand) to its receptor, binding experiments are performed with the radiolabeled ligands. To characterize the ligand-receptor relationship, three types of tests can be carried out:

Saturation experiments, competitive binding tests, and test for binding kinetics. The latter are performed, however, only with complete characterization of ligand-receptor interactions.

These tests can be performed both with whole cells, which express the receptor, and with isolated membrane fractions of cells. For the studies of the NPY-receptor subtypes, cell lines are used that primarily or exclusively express a receptor subtype. Known cell lines that strongly over-express the Yi receptor are the human neuroblastoma cells SK-N-MC [13] and the human breast cancer cells MCF-7 [20]. The human neuroblastoma cells MHH-NB-11 or SMS-KAN, however, express the Y₂ receptor [17, 21].

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 (association), determines constant Ic_0n. The rate of the dissociation of the complex is determined, however, by constant

koff.

LR images per unit of time = [L] * [R] * Ic_0n

LR dissociation per unit of time = [LR] * k_off

Both partial reactions are found in equilibrium, thus:

holds true.

The dissociation constant K_d quantitatively detects the affinity between ligand and receptor and has the dimension of a concentration. If the ligand concentration is equal to K_d, then [R] : [LR] = 1. This means that half of the receptors have ligands. If the ligand has a high affinity for the receptor, the K_d is correspondingly low, since only a small concentration of ligand is necessary to cover half the receptors. A highly affine binding typically has a Kd that is smaller than 10 nmol [19].

1.6.1 Saturation Experiments

In a saturation experiment, both the affinity in the form of K_d and the maximum number of binding sites for a ligand can be determined. To this end, 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. In a saturation diagram, 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. To determine this non-specific binding, inhibitor molecules are used that competitively displace the specifically bound ligands or block all receptor sites. As inhibitors, usually 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).

Specific Binding (B_s) = Total Binding (B_t) - Non-Specific Binding (B₁₁)

The curve of the specific binding approaches a maximum value (asymptote), which corresponds to the maximum number of binding sites (B_maχ 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]

A straight line in the Scatchard Plot is obtained, however, only in the simplest case, if specifically one binding site exists for a ligand on the receptor. If several binding sites exist with different affinities, a curve that results from the various binding sites is thus produced in

the Scatchard Plot (Fig. 9).

[Radioligand] Specific Binding

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].

1.6.2 Competitive Binding Test

Since not all potential binding partners can be radiolabeled to determine their receptor affinity, the substances to be studied in competitive binding tests are used as inhibitors in a radioligand. In this case, 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:

logβJnlabeled Drug]

Fig. 10: Competitive Binding Curve. The IC₅₀ 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 IC₅₀ (inhibitory concentration) or else EC₅₀ (effective concentration).

The value of the IC₅₀ is determined by three factors. Most important is the affinity of the receptor for a competitive substance. If the affinity is high, the IC₅₀ 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, IC₅₀ 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).

Because of this, IC₅₀ values can then only be compared if the determination is made under the same conditions. This is not always achievable. Therefore, equilibrium dissociation constant K₁ is introduced to quantify the affinity of the inhibitor. Index i stands for the inhibition of the radioligand binding by the competitor. K, is calculated by the Cheng- Prusoff equation, which takes into consideration the above-mentioned factors [24].

IC₅₀

V ¹SC —- [Ligand]

1 + κ_d

K, values can be interpreted just as K_d values. The lower a K,-value, the higher the affinity of the substance. Material and Methods

2.1 Material

2.1.1 Devices and Materials

Milli-Q Plus Ultra Pure Water System Millipore

Milli-Q Filter Millipore

Gamma Counter "1480 Automatic Gammacounter Wizard 3" Perkin Elmer

Shaker, Unimax 1010 Heidolph

Incubator 1000 Heidolph

Mixer IKA Labortechnik

Axiovert S 100 Microscope Zeiss

C0₂-Auto-Zero Gassing Incubator Heraeus

Cytoperm 2 Gassing Incubator Heraeus

Sterile Cell, HERA Safe Heraeus

Freezing Container Heraeus

Water Bath GFL

Centrifuge 5810R Eppendorf

Photometer SPECTRAmax plus 384 Molecular Devices

HPLC Agilent 1 100; System 1 Agilent Technologies

RP-HPLC Column Luna, 5 μm, Cl 8 (2) Phenomenex

Neubauer Counting Chamber Labor Optik Centrifuging Tubes, 50 ml, 15 ml TPP Cell Culture Flasks T25, T75, T225 Corning Inc. 1.7 ml Microcentrifuging Tubes Corning Inc.

48-Hole Cell Culture Plates Becton Dickinson

150mm Pasteur Pipette WU Mainz

Cryotubes Nunc

One-way Cannulae Braun

One-way Syringes Braun

Ether Bell Aesculap

Preparation Instruments Aesculap

Metabolic Cages Becker

Metabolic Glasses Becker

Scintillation Vessels Roth

2.1.2 Reagents, Buffers and Solutions

D-PBS without Mg²⁺/Ca²⁺ Invitrogen

HEPES Buffer Invitrogen

Non-essential Amino Acids Invitrogen

0.4% Trypan Blue Solution Invitrogen

Fetal Bovine Serum (FBS) Invitrogen

Sodium Pyruvate Invitrogen

Dulbecco's Modified Eagle Medium with Glutamax I Invitrogen

RPMI 1640 with Glutamax I Invitrogen

MEM with Earle's Salts and Glutamax I Invitrogen

Trypsin/EDTA Solution Biochrom AG

Insulin (Cow) Biochrom AG Sodium Hydroxide Merck

Ethanol, Absolute Merck

Dimethyl Sulfoxide (DMSO) Merck

Bovine Serum Albumin (BSA) Sigma-Aldrich

Sodium Acetate Sigma-Aldrich

Bis-Cinchoninic Acid Solution (BCA) Sigma-Aldrich

Copper(II) Sulfate Solution Sigma-Aldrich

N, N-Dimethylformamide Roth

BD Matrigel Matrix BD Biosciences

Isoflurane Cura MED Pharma

177τ Lutetium Chloride ( rl'7"7LuCl₃) (19.27 GBq/200 μl) IDB Holland BV

2.1.3 Cell Lines

Cell Line Description Special Properties Source

SK-N-MC Human Neuroblastoma, NPY 1 -Receptor Expression ATCC

From Supraorbital Metastasis, Adherently Growing

MCF-7 Human Breast Cancer, NPY 1 -Receptor Expression ATCC

Adherently Growing

MHH-NB- 11 Human Neuroblastoma, NPY2-Receptor Expression DSMZ Adherently Growing 2.1.4 Peptides

Peptides YY (PYY), Human Sigma-Aldrich

Peptide YY Fragment (3-36) (PYYQ-36)), Human Sigma-Aldrich

Neuropeptide Y Fragment (13-36) (NPY(13-36)) Sigma-Aldrich

Leu³ ' -Pro³⁴-Peptide YY (Leu³ ' -Pro³⁴-PY Y) Sigma-Aldrich

¹²⁵I-Peptides YY (¹²⁵I-PYY), Human (81.4 TBq/mmol) PerkinElmer

The NPY analogs, which were studied with regard to their receptor binding within the framework of this thesis, were synthesized by AG Prof. Beck-Sickinger of Universitat Leipzig [Leipzig University], as well as a Schering intern of Dr. Srinivasan, AG Peptidchemie [Peptide Chemistry], and made available.

2.2 Methods

2.2.1 Cell Cultivation

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 μg/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% CO₂ and 95% atmospheric humidity in a gassing incubator.

2.2.1.1 Thawing of Cells

Before the cell line was thawed, the corresponding culture medium was preheated to 37°C. The cells were removed from the nitrogen container and heated as quickly as possible

in a water bath at 37⁰C. After complete thawing, the cryo vessels were disinfected with 70%

ethanol and set under the sterilizing bench. 9 ml of heated medium was introduced into a centrifuging tube and mixed with 1 ml of the cells. This suspension was centrifuged for 5 minutes at 200xg. Then, the supernatant was suctioned off, the pellet was resuspended in 10 ml of freshly preheated medium, and an aliquot for cell counting and a vitality test was removed. The cells were disseminated confluently in T25 culture flasks and cultivated in an incubator for 24 hours without a change in medium. Then, the cells optionally were further subcultivated in larger culture vessels.

2.2.1.2 Regular Cultivation

Maintenance cultures were cultured in T225 culture flasks, and the latter were passaged one to two times per week after achieving confluence. To this end, first the culture

medium and PBS in a water bath were heated to 37°C, and the trypsin/EDTA solution

(0.2%/0.08% (w/v)) was preheated to room temperature. The consumed medium was drawn off from the cells, the cells were washed once with 10 ml of PBS, and the culture flask was mixed with 3 ml of trypsin/EDTA solution, such that the bottom of the flask is completely covered. The cells were incubated for 1 to 8 minutes (depending on the cell line) at 37°C and then dissolved by light tapping on the flask. Then, the cells were rinsed with 10 ml of fresh medium from the bottom of the flask, the cell suspension was moved into a centrifuging tube and then centrifuged for 4 minutes at 200xg and at room temperature. The supernatant was suctioned off, the cell pellet was resuspended in 10 ml of fresh medium, and an aliquot was removed for the cell counting. 2x10⁶ to 1x10⁷ cells were disseminated and cultivated in new T225 culture flasks with 50 ml of medium.

2.2.1.3 Freezing of Cells

Healthy, subconfluent cultures were used for cryopreservation. To this end, the cells were first trypsinized and the cell count determined. The residual cell suspension was

centrifuged for 5 minutes at 200xg and at 4°C and then put on ice for at least 20 minutes.

Then, the supernatant was discarded, and the pellet was resuspended in precooled freezing medium (FBS with 10% (v/v) DMSO), such that a cell concentration of 5x10⁶ cells/ml is achieved. In each case, 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.

2.2.2 Growth Kinetics of the Cell Lines Used

To determine the growth kinetics of the cell lines used, various cell counts were seeded in 48-hole culture plates and incubated. After various points in time (about every two days), the cell counts were determined in each case by three holes at each point in time. To this end, the medium was suctioned off, and 100 μl of trypsin/EDTA solution was pipetted into the holes. Depending on the estimated cell count, at least 100 μl of 37°C culture medium was added; the cells thus were flushed from the bottom of the hole and resuspended in the medium. The cell suspension was moved into a reaction vessel and again very well suspended. The cell counting was carried out with the Neubauer counting chamber. To this end, an aliquot of the cell suspension was removed, and was diluted 1 :2 with 0.2% trypan

blue solution. About 10 μl thereof was pipetted into the counting chamber, and the living

cells (unstained) were counted in at least 4 large quadrants and then averaged. The cell concentration in cells/ml is then produced from the average cell count x 2 x 10⁴. To determine the overall cell count, the cell concentration must be multiplied with the suspension volumes. The total cell counts of the three counted holes were averaged at the respective times and plotted in the diagram against time in semilogarithmic scale.

2.2.3 Binding Studies with Cells

2.2.3.1 Determination of the NPY Receptor Subtypes of the Various Cell Lines

For in-vitro determination of the NPY receptor status, cells in cell culture plates with 48 cavities were cultivated until confluence was completed (about 3x10⁵ 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

medium was suctioned off, the cells were dissolved by incubation with 100 μl of

trypsin/EDTA solution and diluted in a respective cultivating medium. The cells were stained with 0.2% trypan blue solution and counted in the Neubauer counting chamber. For protein determination, the untreated cells were lysed for about 10 minutes in 0.5 ml of MiIH-Q water (lysed). The determination of protein of the extracts was carried out according to the BCA method (see 2.2.5).

To perform the competitive peptide binding studies, the cultivating medium was

suctioned off from the cavities, and replaced by 150 μl of 37°C medium (respective

cultivating medium plus 1% (m/v) BSA). To determine which NPY receptor subtype is

expressed on the respective cell line, 50 μl of a control peptide (PYY, Leu³¹-Pro³⁴-PYY,

PYY(3-36) or NPY(13 -36)) was added as an inhibitor for the binding of radiolabeled universal ligands ¹²⁵I-PYY to three cavities in each case, so that this 100 nmol was present in the batch. To determine total binding B_t (batch without inhibitor), the corresponding amount of medium was added in several cavities. Finally, 50 μl of the ligands ^l25I-peptide YY (1 nmol) was pipetted into all cavities. A total volume of 250 μl 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 μl aliquots on the gamma counter. Within one passage, the samples were measured for 30 seconds in each case. A 3x determination was made.

Table 4: Pipetting Scheme for Receptor Subtype Determination.

The plates were set at a temperature of 37⁰C for 2 hours in the incubator. After the

incubation, 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). By two

washing cycles with 500 μl of ice-cold sodium acetate solution (20 mmol in PBS) for 3 minutes, 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. To determine the amount of the internalized ligand, the cells were lysed with 0.5 ml of a 1 M sodium hydroxide solution. To

this end, the cell culture plates were incubated at a temperature of 37°C in the shaker at 200

rpm. After about 10 minutes, the cell lysates were moved into the reaction vessels. The cavities were washed once more with 0.5 ml of sodium hydroxide solution, and the solutions were combined with extracts. The measurement of the cell extracts was also carried out in a gamma counter.

From the measured activities, the amount of protein, and the specific activity of the radioligands, the amount of bound ligand was calculated in fmol/mg (see 2.2.4).

2.2.3.2 Determination of Dissociation Constants IQ of the Ligand ^U5I-Peptide YY

On whole cells, a saturation experiment was performed to determine the dissociation constants K_d and the number of maximum binding sites B_max of the ligand ¹²⁵I-PYY at the various cell lines. The binding studies are carried out essentially as in the above-described subtype determination. In 48-hole plates, cells of the respective cell line are disseminated and cultivated until confluence is completed. MCF-7 Cells were cultivated up to a cell count of about 5x10⁴ cells per cavity (cells isolated). Before the study, both the cell count and the protein content were determined in two cavities per culture plate in each case.

For the binding studies, the old culture medium was removed and replaced by 150 μl of medium heated to 37⁰C (respective cultivation medium plus 1% (m/v) BSA). The ligand 125I-PYY was used in 12 different concentrations of 10 to 0 nmol. To this end, a dilution sequence in medium was produced, and 50 μl of a dilution was pipetted into six cavities in each case. Another 50 μl 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). To determine the non-specific ligand binding (B₁₁), excess cold peptide YY (50 μl, 5000 nmol) was added in each case to three batches per ligand dilution to displace ligands from the specific binding sites. In the case of batches for determining the overall or total binding (B_t), inhibitor PYY was replaced by a corresponding volume of medium, such that the total volume remained constant (Table 5).

Table 5: Pipetting Scheme for K_d Determination

The plates were set at a temperature of 37°C for 2 hours in the incubator. After the

incubation, 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

culture plates was carried out at a temperature of 37°C in the shaker at 200 rpm. After about

10 minutes, 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.

From the difference of activities for B_t and B₁₁, the activity of the specific binding B_s was determined for ligand concentrations [L] that were used. The concentration of specifically bound ligand [L_b0Und] thus could be calculated (see 2.2.4). In addition, the necessary concentration of free ligand [L_free] for the determination of K_d was calculated from the difference of total ligand concentration [L] and bound ligand [Lboun_d]- The generation of the saturation curve and the Scatchard Plot, as well as the calculation of the K_d was carried out with the aid of the software GraFit 4 (Version 5.0) of Robin J. Leatherbarrow. The program also yielded the value of maximally-bound ligand

in mol/1. The latter was converted into fmol/mg of protein (see 2.2.4).

2.2.3.3 Determination of the Dissociation Constants of Inhibitor Kj

To determine the K, value of the NPY analogs, the latter were used as inhibitors in binding studies in different concentrations. The ligand ¹²⁵I-PYY was used in the concentration range of the K_d determined for the cell line (see information in the Results portion).

In 48-hole plates, cells of one cell line were disseminated and cultivated until confluence was completed. MCF-7 Cells were disseminated with a cell count of 5 x 10⁴ cells per hole, and the binding experiments were performed on the next day. Before each experiment, both the cell count and the protein content in two cavities per culture plate in each case were determined.

For the binding studies, the old culture medium was removed and replaced by 150 μl 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 μmol to 15 pmol. To this end, a dilution series in medium was produced, and 50 μl of a dilution was pipetted into three cavities in each case. To determine the non-specific ligand binding (B₁₁), excess cold peptide YY (50 μl, 5000 nmol) was added to three batches without an NPY analog. In the batches for determining the complete or total binding (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 μl of a ligand concentration was used (Table 6). The exact determination of the ligand concentration used was carried out with 50 μl of aliquots in a gamma counter.

All additional steps, including the determination of the activity of cell lysates, were carried out as in the determination of K_d. Table 6: Pipetting Scheme for Kj-Determination.

To determine the K₁, first the IC₅₀ 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 IC₅₀ 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 IC₅₀ and the ligand concentration that is used.

Since the number of analogs to be studied was very high, the exact IC₅₀ was not determined by any peptide. Instead, first the peptides were used in only two concentrations,

in each case of 50 μmol and 5 μmol (final concentration in the test of 10 and 1 μmol). The

binding studies are carried out as described above. From the cpm values for these two concentrations, the inhibition of the ligand by the analog was then calculated (see 2.2.4).

If the percentage of inhibition for a concentration of the analog was 50%, it was assumed that the IC₅₀ lies in this concentration range. If the inhibition of the ligand binding was > 50%, the IC_5O in one area was smaller than the tested concentration. Thus, the approximate IC₅₀ could be estimated. With IC₅₀ values of less than 1 μmol, the exact IC₅₀ or K₁ was then determined from this analog. 2.2.4 Calculations with Regard to the Binding Assay

Ligand Concentration

First, the specific activity must be calculated on test day.

A = Specific Activity at Test Time t

Ao = Specific Activity with Regard to Calibration Time t₀ t,_/2 = Half-life of ¹²⁵I - 59.4 d

A and A₀ can be indicated in TBq/mmol or dpm/fmol.

The ligand concentration is then calculated:

Bound Ligand LhrmnH in fmol/mg of Protein

The amount of bound ligand is calculated from the cpm values:

Measured Activity [cpm]

T-bound [tmol/mg ot ProteinJ = Specific Activity A [dpm/tmolj * 0.98 * Protein

Content/Cavity [mg] Maximum Binding Sites B_mav

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:

Bmax [M] * Batch Volumes [μl] * 10⁹ Bmax [fmol/mg of Proteinl =

Protein Content/Cavity [mg]

Inhibition of Ligands

The percentage of the inhibition of ligands by an NPY analog is calculated from the measured values of the specific binding with and without inhibitors (analog):

Specific Binding with Inhibitor (NP Y- Analog) |cpm) * 100% % Inhibition =

Specific Binding without Inhibitor [cpm]

The values for the specific binding (with or without NPY analogs) were obtained by subtraction of the cpm values of the batches for the non-specific binding of the values that are measured in each case.

2.2.5 Protein Determination with the BCA Method

To determine the protein content of an unknown solution, first a calibration curve was generated. 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 μg/ml, was used as a standard protein. In each case, 50 μl of the dilutions was removed and pipetted into 1.5 ml reaction vessels. In the control batch, the dilution stage was replaced by the same volume of milli-Q water. The samples were diluted with 950 μl 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.

For protein solutions of unknown concentration (cells lysed by milli-Q water from the binding studies), the test was performed as described above for the BSA standards. The protein solutions were used in undiluted form. Based on the straight-line equation for the calibration curve, the exact protein concentration could be calculated from the extinction values.

2.2.6 In-Vivo Studies

For in-vivo tests, immunodeficient hairless mice (NMRI nude/nude, female, about 20 g) were inoculated subcutaneously by the Taconic M&B Company (Denmark) with 5x10⁶ SK-N-MC cells in 100 μl of Matrigel.

2.2.6.1 Inoculation

First, 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 μl (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

cannula and in each case 100 μl was injected subcutaneously into the thigh area of the right

leg. 2.2.6.2 Growth Kinetics

To determine the in-vivo growth kinetics of the SK-N-MC-tumor xenograft, respectively 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.

2.2.6.3 Radiolabeling of a Peptide with ¹⁷⁷Lu

25 μg of the peptide (1 mg/100 μl of saline) was dissolved together with about 15 mCi (= 525 MBq) of ¹⁷⁷Lu (¹⁷⁷LuCl₃ in 0.05 M HCl supra pure) in ascorbic acid/NaOAc buffer (15 mmol of ascorbic acid plus 30 mmol of NaOAc, pH 5.5) in a total volume of 100 μl. The batch was heated for 30 minutes at 80°C in an oil bath and then cooled for 10 minutes. After the reaction, the batch was made up to 1 ml with ascorbic acid//NaOAc buffer. The radiochemical purity of the product was determined by chromatography with the aid of Reversed Phase-HPLC (RP-HPLC).

For RP-HPLC, 10 μl of the sample (4.0 MBq/100 μl) was injected into the unit. The eluants were A: water plus 0.1% trifluoroacetate (TFA) and B: acetonitrile/water at a 1 :9 ratio plus 0.1% TFA. The flow rate was 1.0 ml/minute. The elution was carried out with the following gradients:

0 minute 22% B

20 minutes 42% B

21 minutes 100% B

26 minutes 100% B

27 minutes 22% B 35 minutes 22% B The radioactivity was detected with a gamma-radiation detector. The sample was frozen at -30°C and used on the following day for the in-vivo study.

2.2.6.4 Organ Distribution of a Radiolabeled Peptide

100 μl of the ^l77Lu-labeled peptide with the indicated activity (about 70 kBq) was administered i.v. into a caudal vein. To determine the full activity, 4-6 scintillation vessels with the same volume and activity, which also was administered in the individual animals, were filled. The animals were put into metabolic cages or into metabolic glasses (in the case of short incubation time) directly after i.v. administration for the indicated incubation period. At least three animals were studied per incubation time (1, 3, 5 and 24 hours). At the respective end of the test, 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. First, a weight determination was made on all of the organs removed, blood, urine, feces and the rest of the body. Then, the radioactivity in the gamma counter, as also by the activity standard (= 100%ID), was determined with the corresponding isotope program. The percentage distribution of the administered dose per gram of weight (%ID/g) was determined from the organ weights as well as the gamma counter measured values. Results

3.1 Establishment and Optimization of the Binding Test

3.1.1 Growth Kinetics of the Cell Lines

The growth behavior of cell lines SK-N-MC, MCF-7 and MHH-NB-11 in 48-hole cell culture plates was studied to determine the incubation time until the binding experiments are performed in the late growth phase (90% confluence).

The SK-N-MC cells showed a growth that was dependent on the disseminated cell count. If a few cells were seeded (1x10⁴), 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 10⁵ (Fig. 1 1). The more thickly seeded cells (5x10⁴) 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 1.5x10⁵ and died after about 10 days. The maximum possible cell count per hole (100% confluence) was 1.5x10⁵ cells.

6 8 io 12

Zeit [d] Fig. 11: Growth Curve for Human SK-N-MC Cells. In 48-hole cell culture plates, 1x10 and 5x10⁴ cells per hole were seeded, and after various points in time, the cell count was determined. Mean values from n = 3.

[Key to Fig. 11:]

Gesamtzellzahl = Total Cell Count

Zeit [d] = Time [Days]

Growth curves of the MCF-7 cells show that 1x10⁴ disseminated cells reached the log phase after 48 hours and divided until a density of 2x10⁵ cells per hole was reached (Fig. 12). Then, the cells passed into the stationary phase. The more thickly disseminated cells (5x10⁴ and 1x10⁵ cells per cavity) showed a less steeply increasing proliferation phase and passed into the stationary phase also starting from a cell count of about 2x10⁵. The maximum cell count was approximately 2.5x10⁵. After reaching this cell count, the cells died.

4 6 10 12

Zeit [d]

Fig. 12: Growth Curve for Human MCF-7 Cells. In 48-hole cell culture plates, IxIO⁴, 5x10⁴ and 1x10⁵ cells were seeded per hole, and the cell count was determined after various points in time. Mean values from n = 3. [Key to Fig. 12:]

Gesam ze_itzellzahl = Total Cell Count

Zeit [d] = Time [Days]

For the MHH-NB-1 1 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 5x10⁵ 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.

6 10

Zeit [d]

Fig. 13: Growth Curve for Human MHH-NB-Il Cells. In 48-hole cell culture plates,

1x10⁴ and 2x10⁴ cells per hole were seeded, and after various points in time, the cell count was determined. Mean values from n = 3.

[Key to Fig. 13:]

Gesamtzellzahl = Total Cell Count

Zeit [d] = Time [Days] 3.1.2 Subtype Determination and Internalization Behavior

To determine which NPY receptor subtypes express the cell lines SK-N-MC, MCF-7 and MHH-NB-11, competitive binding tests were performed with the control peptides PYY, Leu^{3 l}-Pro³⁴-PYY, PYY(3-36) and NPY(13-36) as inhibitors of the ligand ¹²⁵I-PYY. PYY is the only one of the control peptides that is not selective and binds to all receptor subtypes. Leu³¹-Pro³⁴-PYY binds to the Yi - and Y₅-receptors, but not to the Y₂ receptor. PYY(3-36) binds selectively to the Y₂- and Y₅ receptors. NPY(13-36) binds exclusively to the Y₂ receptor.

w/o PYY LP-PYY PY^Λ 1^(3-36) NPN '(13-36) lnhibitoren

D Oberflachenbindung a lnternalisation ■ Summe

Fig. 14: Binding of ¹²⁵I-PYY to SK-N-MC Cells in the Presence of Receptor-Subtype- Specific Control Peptides. 2x10⁵ Cells were incubated for 2 hours with 0.124 nmol of ¹²⁵I- PYY and 100 nmol of PYY(not selective), Leu³¹-Pro³⁴-PYY (Y, -selective), PYY(3-36) or NPY(13-36) (in each case Y₂-selective). The control (w/o) shows the pure binding of the ligand without an inhibitor. Mean values from n = 3 ± SD. [Key to Fig. 14:] gebundenes = Bound lnhibitoren = Inhibitors Oberflachenbindung = Surface Binding Internalisation = Internalization Summe = Sum

The total amount of the bound radiolabeled ligands ¹²⁵I-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}-Pro³⁴-PYY that binds to Yi and Y₅ 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 Y₂ and Y₅ 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.

w/o PYY LP-PYY PYY(3-36) NPY(13-36) lnhibitoren

□ Oberflachenbindung 0 Internalisation ■ Summe

Fig. 15: Binding of • 1 '2"51-PYY to MCF-7 Cells in the Presence of Subtype-Specific Control

Peptides. 2xl(T Cells were incubated for 2 hours with 0.125 nmol of ²⁵I-PYY and 100 nmol of PYY, Leu³¹-Pro³⁴-PYY, PYY(3-36) or NPY(13-36). The control (w/o) shows the pure

binding of the ligand without an inhibitor. Mean values from n = 3 ± SD.

[Key to Fig. 15:] gebundenes = Bound

Inhibitoren = Inhibitors

Oberflachenbindung = Surface Binding

Internalisation = Internalization

Summe = Sum

The amount of bound ¹²⁵I-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 ¹²⁵I-PYY was measured (Fig. 15). After inhibitor PYY was added, 1.3 fmol/mg of ¹²⁵I-PYY was bound, which is comparable to the inhibition by the Yi -receptor-specific Leu³¹-Pro³⁴-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 ¹²⁵I-PYY. The proportion of internalization in the MCF-7 cells was not as high as in the SK-N-MC cells. Surface-bound peptide and receptor-internalized peptide were equally involved in the total binding. Also in these cells, the strongest inhibition with PYY and Leu³¹-Pro³⁴-PYY was achieved, which confirmed the presence of Yi receptors. Based on the inhibition by the Y₂-selective control peptides, the expression of Y₂ receptors, although in a lower number, cannot be excluded.

w/o PYY LP-PYY PYY(3-36) NPY(13-36) lnhibitoren

D Oberflachenbindung a lntemalisation ■ Summe

Fig. 16: Binding of ¹²⁵I-PYY to MHH-NB-Il Cells in the Presence of Subtype-Specific

Peptides. 7xlO⁴ Cells were incubated for 2 hours with 0.121 nmol of ¹²⁵I-PYY and 100 nmol of PYY, Leu³¹-Pro³⁴-PYY (LP-PYY), PYY(3-36) or NPY(13-36). The control (w/o) shows

the pure binding of the ligand without an inhibitor. Mean values from n = 3 ± SD.

[Key to Fig. 16:] gebundenes = Bound lnhibitoren = Inhibitors

Oberflachenbindung = Surface Binding lntemalisation = Internalization

Summe = Sum

For the MHH-NB-1 1 cells, the Y₂ 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. Leu³¹-Pro³⁴-PYY showed no binding, since the amount of bound ¹²⁵I-PYY with about 16.5 fmol/mg was just as high as without adding inhibitor, thus accordingly no inhibition occurred (Fig. 16). By adding PYY (3-36) or NPY(13-36), the ligand binding could be reduced by 7 or 8 fmol/mg. Thus, it can be concluded that the Y₂-receptor subtype, to which these two pepides have bonded, is expressed. After 2 hours of incubation time, 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.

3.1.3 Determination of the Dissociation Constant K₄ of ¹²⁵I-Peptid YY

After the expression of an NPY-receptor subtype was identified with the cell lines, the next step was that dissociation constant K_d was determined for the universal ligand ' ⁵I-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.

1-125 Peptide YY [M]

Fig. 17: Saturation Curve and Scatchard Plot (Right) of the Specific Binding of

to SK-N-MC Cells. 2x10⁵ Cells with an amount of protein of 57 μg were incubated for 2 hours at various concentrations (0.001 to 2 nmol) of ¹²⁵I-PYY. The non-specific binding was

determined by cold PYY in excess (1000 nmol). Mean values from n = 3 ± SD. The K_d of

0.251 nmol and B_max of 46.0 fmol/mg were determined from the Scatchard Plot.

First, a saturation curve and the related Schatchard Plot were generated in a saturation experiment with 2x10⁵ SK-N-MC cells (57 μg of protein) with increasing concentrations of ¹²⁵I-PYY (Fig. 17). The evaluation of the Scatchard Plot yielded a dissociation constant K_d of 0.251 nmol and a maximum binding B_max of 46.03 fmol/mg of protein for ¹²⁵I-PYY. In the following competitive tests with the NPY analogs, the ligand was used in this concentration range. The cell count used was retained for the competitive tests.

o o ώ ύ so

1-125 Peptide YY [M]

Fig. 18: Saturation Curve and Scatchard Plot (Right) of the Specific Binding of 1 '2"51,-PYY to MCF-7 Cells. 5x10⁴ Cells with an amount of protein of 10.5 μg were incubated for 2 hours at various concentrations (0.001 to 2 nmol) of ¹²⁵I-PYY. The non-specific binding was

determined by cold PYY in excess (1000 nmol). Mean values from n = 3 ± SD. The Kj of

0.339 nmol and B_max of 37.6 fmol/mg were determined from the Scatchard Plot.

For the Kj determination on MCF-7 cells, 5xlO⁴ cells (10.5 μg of protein) and a maximum ligand concentration of 2 nmol were used (Fig. 18). For the ligands ¹²⁵I-PYY, the Scatchard analysis yielded a K_d of 0.339 nmol and a B_max of 37.6 fmol/mg of protein. For the competitive tests, 4-5x10⁴ cells and a ligand concentration of about 0.3 to 0.4 nmol were then used.

ω

1-125 Peptide YY [M]

Fig. 19: Saturation Curve and Scatchard Plot (Right) of the Specific Binding of 1 '25³iI-PYY

to MHH-NB-Il cells. 2x10⁵ Cells with an amount of protein of 27.8 μg were incubated for 2

hours at various concentrations (0.001 to 2 nmol) of ¹²⁵I-PYY. The non-specific binding was

determined by cold PYY in excess (1000 nmol). Mean values from n = 3 ± SD. The K^ of

0.034 nmol and B_maχ of 35.3 fmol/mg were determined from the Scatchard Plot.

In MHH-NB-11 cells, a very much lower K_d was determined for ¹²⁵I-PYY than in the case of the other two cell lines, which are also shown in the steeper increase of the saturation curve in comparison to the other cell lines (Fig. 19). The K_d was 0.034 nmol with 2x10⁵ used cells and a protein content of 27.8 μg. The B_max was estimated at 35.3 fmol/mg of protein. The ligand was used in the competitive experiments in the range of this K_d and with 2x10⁵ cells. 3.1.4 ^-Determination of the Standard Peptides PYY and PYY(3-36)

To evaluate the parameters optimized in the saturation experiments, competitive experiments were performed with the non-selective control peptide PYY. The K, values determined for PYY should be comparable to the Kj values of the radiolabeled ligands.

The determination of the K₁ 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). In contrast to this, 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). In comparison to the greatly varying K, values of PYY in the competitive binding experiment, 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 Y₂-receptor-expressing MHH-NB-11 cells. There, 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.

It was clear that the cold PYY as well as the hot PYY on MHH-NB-1 1 cells shows the highest affinity and on MCF-7 cells the lowest affinitiy.

The binding affinity of the Y₂-selective PYY(3-36) showed very different binding affinities on the tested cell lines SK-N-MC and MHH-NB-11. The K₁ on the MHH-NB-11 cells was lower by the factor 763 (Table 7). This cell line thus could be used as a Y₂-receptor selective control.

Table 7: Binding Parameters for the Different Cell Lines.

SK-N-MC MCF-7 MHH-NB-Il

K_d (¹²⁵I-PYY) [nmol] 0.251 0.339 0.034 B_max (¹²⁵I-PYY) [fmol/mg 46.0 37.6 35.3 SK-N-MC MCF-7 MHH-NB-Il

Protein]

Kj (PYY) [nmol] 0.78 5.69 0.1 1

Kj (PYY(3-36)) [nmol] 305.4 Not Determined 0.40

10^"12 10^"" 10-¹⁰ 10-» 10-* 10^"7 10^"8

PYY [M]

K (PYY) 0.11 nmol [¹²⁵I-PYY] ^; 0.039 nmol

10 ¹² 10 ' 10 ' 10* 10-" 10⁷ 10-⁶

PYY [M]

Fig. 20: Competition Curves and K₁ of PYY at Various Cell Lines. A) SK-N-MC, B) MCF-7, C) MHH-NB-11. The cells were incubated for 2 hours in a competitive binding test

with the ligand ¹²⁵I-PYY and cold PYY as an inhibitor. Mean values from n = 3 ± SD.

[Key to Fig. 20:] Peptid = Peptide

3.2 In- Vitro Studies of NPY Analogs

3.2.1 Binding of NPY Analogs to the Yi Receptor

For this work, peptides developed by the Schering Company and the Prof. Beck- Sickinger of Universitat Leipzig (Leipzig University) that should be tested with regard to the Yl binding and selectivity were available.

About 40 peptides were tested first in two relatively high concentrations of 10 and 1 μmol in competitive binding tests with ¹²⁵I-PYY as a ligand with regard to their binding affinity for the Yi receptor on SK-N-MC cells. Based on the inhibition of the ligand by the NPY analog, first the IC₅₀ of the NPY analog was estimated. The latter was under a value of 1 μmol, the exact IC₅₀ was determined in a renewed competitive test, and the K, of the analog was calculated therefrom. Of the approximately 40 tested analogs, eight peptides had a K₁ of less than 1 μmol (Table 8). Peptide P2489 with a K₁ of 49.2 nmol showed the highest binding affinity.

Table 8: Binding Affinities of Selected NPY Analogs and Control Peptides on SK-N-MC Cells. The K₁ was determined with competitive binding tests with the ligand ¹²⁵I-PYY. The amino acids, in which the analogs are distinguished (positions 32 and 34 of the original peptide NPY), are underscored.

^κ) Use for Organ Distribution Test

3.2.2 Binding Affinity and Selectivity of Peptide P2489

Of the peptides studied on SK-N-MC cells, only the peptide P2489 showed a binding affinity of less than 100 nmol (K, = 49.2 nmol). This result was confirmed on the MCF-7 cells also expressing the Yi receptor. Here, a K₁ of 176.1 nmol was determined. In addition, the Yi selectivity was determined by a binding test of Y₂-receptor-expressing MHH-NB-11 cells. No binding could be detected on the MHH-NB-11 cells. The determined competition curve dropped only starting at a P2489 concentration of about 10 μmol (Fig.21). P2489 accordingly shows a Yi -receptor-selective binding.

nM ng SK-N-MC Cells

_1oi2 io» ₁₀ιo i_O ⁹ 10-⁸ 10⁷ 10*

P2489

[Ml

ng MCF-7 Cells

10 10' 10 10 ⁹ 10-⁰ 10 10^"6 10 P2489 [M]

K,(P2489)>10μM Y₂-Receptor-expressing MHH-NB-11 Cells

10 '² 10 " 10 '° 10-⁹ 10^"8 10⁷ 10-⁶ 10-⁵

P2489

[M]

Fig. 21: Competition Curves and Kj of P2489 with Different Cell Lines. A) SK-N-MC, B) MCF-7, C) MHH-NB-1 1. The cells were incubated for 2 hours in a competitive binding test

with the ligand ¹²⁵I-PYY and P2489 as inhibitors. Mean values from n = 3 ± SD.

[Key to Fig. 21 :] Peptid = Peptide

3.3 In- Vivo Studies of NPY Analogs

For a first characterization of peptides in the animal, organ distribution tests were performed with radiolabeled peptides. A simple and already established labeling method is the ¹⁷⁷Lu labeling, for which, however, the DOTA chelating agent is necessary. Therefore, for the in-vivo studies, the DOTA-coupled peptides P2471 and fW7 were used, which showed relatively high affinities for the Yi receptor in the in-vitro tests. The latter were radiolabeled with ¹⁷⁷Lu, and their biodistribution in SK-N-MC-tumor-bearing mice was studied. First, however, the growth behavior of the tumors was studied to determine the growth time until the tumor reached a size of 100-150 mm² for the organ distribution tests.

3.3.1 Growth Kinetics of the 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 5x10⁶ 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 mm² of tumor surface area was shown. Then, the tumors grew only slowly, and after about 45 days, achieved an average tumor size of 135 mm². The growth within the 5 tested animals was very different. In the observed period, a maximum tumor size of 160 mm² was reached.

Zeit [d]

Fig. 22: In-Vivo Growth Curve of SK-N-MC Tumor Xenografts. 5xlO⁶ SK-N-MC cells in

100 μl of Matrigel were injected subcutaneously into the right flank of female hairless mice

(NMRI nude/nude). Mean values from n = 5 ± SD.

[Key to Fig. 22:]

Tumorflache = Tumor Surface Area

Zeit [d] - Time [Days]

3.3.2 Organ Distributions of Radiolabled NPY Analogs The DOTA-coupled peptides P2471 and fW7 were injected into the tumor-bearing mice after labeling with ¹⁷⁷Lu, and their distributions into the tumor and into the organs were measured after various points in time.

3.3.2.1 ¹⁷⁷Lu-P2471

Peptide P2471 was labeled with 177 Lu, and the purity was examined by means of RP- HPLC. The purity of ¹⁷⁷Lu-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"7τLu-P2471

For the organ distribution test, 100 μl of ¹⁷⁷Lu-P2471 with an activity of 70.2 kBq was injected into the caudal vein of the tumor-bearing mice, and the distribution in the organs and in the tumor was determined after 1 , 4 and 24 hours.

The concentration of this pepide in the tumor was very low after 1 hour with a maximum of 0.25%ID/g (Table 9). The percentage distribution in the tumor is almost identical to that in the other organs. Also, in the blood, no more could be detected after a very short time. Only the concentration in the kidneys already after 1 hour with 6.85% ID/g was remarkable, and after 4 hours, a value of 9.36%ID/g was reached, and then slowly dropped to 4.96%ID/g after 24 hours. The majority of the activity (about 84%) was already discovered in the urine after 1 hour. Also in the brain, no concentration of ¹⁷⁷Lu-P2471 took place.

Table 9: Organ Distribution of ¹⁷⁷Lu-P2471 in SK-N-MC Tumor-Bearing Mice. 100 μl of the labeled peptide with an activity of 70.2 kBq was injected into the caudal vein and after various points in time, the activity in the individual organs was determined (n = 3).

I h 4 h 24 h

Percentage Distribution[% ID/g]

SD SD SD

Tumor 0.25 0.04 0.14 0.02 0.16 0.03

Spleen 0.17 0.02 0.14 0.03 0.15 0.03

Liver 0.49 0.14 0.59 0.09 0.44 0.16

Kidneγ 6.85 3.05 9.36 0.33 4.96 1.02

Lung 0.23 0.04 0.09 0.02 0.06 0.00

Bones 0.32 0.05 0.31 0.06 0.24 0.04

Heart 0.13 0.00 0.09 0.01 0.07 0.00

Brain 0.03 0.01 0.02 0.00 0.02 0.00

Thyroid Gland 0.46 0.17 0.42 0.09 0.38 0.02

Muscle 0.10 0.02 0.05 0.01 0.06 0.03

Skin 0.21 0.04 0.09 0.02 0.06 0.00

Blood 0.14 0.02 0.04 0.01 0.01 0.00

Stomach 0.14 0.01 0.14 0.01 0.08 0.01

Ovary 0.45 0.06 0.38 0.06 0.33 0.09

Uterus 0.23 0.06 0.18 0.03 0.13 0.01

Intestine 0.16 0.01 0.34 0.16 0.05 0.02

Pancreas 0.09 0.01 0.07 0.03 0.05 0.01

Adrenal Glands 0.61 0.1 1 0.53 0.10 0.85 0.10

Tumor/Blood 1.79 0.42 3.35 0.59 14.69 4.17

Ratio

Retrieval [%] Organs 6.24 1.69 5.67 0.51 3.26 0.66

Rest of the Body 2.62 0.73 1.35 0.20 1.06 0.06

Urine 83.40 6.34 86.65 3.06 85.13 9.96

Feces 0.07 0.07 3.14 1.94

3.3.2.2 ¹⁷⁷Lu-fW7

The peptide fW7 was labeled with ¹⁷⁷Lu, 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 ^l77Lu-fW7.

In the animal test, 100 μl of ¹⁷⁷Lu-fW7 with an activity of 64.5 kBq was injected into the caudal vein of the SK-N-MC-tumor-bearing mouse, and the organ distribution was determined after 1, 3, 5 and 24 hours.

The organ distribution of ' ⁷⁷Lu- fW7 showed very similar values to those for ¹⁷⁷Lu- P2471 (Table 10). Almost the entire peptide (83%) was already excreted with the urine after 1 hour. As in the case of ¹⁷⁷Lu-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 ¹⁷⁷Lu-P2471. The tumor/blood ratio was at 15.25 after 5 hours and increased to 18.89 after 24 hours. Table 10: Organ Distribution of ¹⁷⁷Lu-fW7 in SK-N-MC-Tumor-Bearing Mice. 100 μl of the labeled peptide with an activity of 64.5 kBq was injected into the caudal vein, and after various points in time, the activity in the individual organs was determined (n = 3).

1 h 3 h 5 h 24 h

Percentage Distribution [% ID/g]

SD SD SD SD

Tumor 0.71 0.09 0.19 0.06 0.21 0.05 0.19 0.05

Spleen 0.19 0.02 0.21 0.04 0.27 0.05 0.16 0.01

Liver 0.92 0.15 1.17 0.16 1.36 0.06 0.85 0.15

Kidney 5.61 1.65 9.03 0.62 8.69 1.70 5.16 1.24

Lung 0.22 0.04 0.09 0.01 0.09 0.01 0.04 0.01

Bones 0.32 0.04 0.22 0.02 0.19 0.03 0.20 0.02

Heart 0.11 0.01 0.07 0.004 0.06 0.01 0.05 0.003

Brain 0.03 0.002 0.04 0.01 0.02 0.01 0.02 0.004

Thyroid Gland 0.55 0.13 0.42 0.23 0.50 0.12 0.25 0.06

Muscle 0.09 0.06 0.04 0.003 0.04 0.01 0.05 0.01

Skin 0.20 0.05 0.09 0.01 0.09 0.02 0.05 0.002

Blood 0.11 0.02 0.02 0.005 0.02 0.01 0.01 0.01

Stomach 0.12 0.04 0.08 0.02 0.08 0.04 0.07 0.02

Ovary 0.55 0.33 0.23 0.10 0.37 0.27 0.20 0.05

Uterus 0.20 0.01 0.17 0.01 0.16 0.08 0.18 0.05

Intestine 0.15 0.03 0.17 0.07 0.24 0.19 0.05 0.03

Pancreas 0.08 0.04 0.04 0.01 0.08 0.03 0.04 0.01

Adrenal Gland 0.46 0.13 0.42 0.04 0.60 0.37 0.24 0.07

Tumor/Blood 6.46 0.96 10.12 1.75 15.25 7.42 18.89 8.29

Ratio

Retrieval [%]

Organ 6.09 0.64 7.29 1.73 6.90 0.97 3.69 0.90

Rest of the Body 1.77 0.22 0.92 0.10 1.23 0.51 0.39 0.01

Urine 83.07 1.95 92.03 8.85 96.37 8.09 92.12 6.13

Feces - - 2.43 4.15 0.22 0.18 1.10 0.30 4 Discussion

4.1 Characterization of the Cell Lines

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 Y₂ receptors.

In the literature, only a few cell lines are described that were studied with regard to the NPY receptor subtype expression. Most frequently described is the human neuroblastoma cell line SK-N-MC, whose Y| receptor expression was detected by Fuhlendorff et al. [15]. In various publications in which NPY and NPY analogs were studied, this cell line was used in the test with regard to Yi receptor binding. Thus, Fabry et al. performed binding and internalization studies with regard to the Yi receptor with SK-N-MC cells and [³H]propionyl- NPY as a ligand [25]. Matthews et al. also studied the binding affinity of various peptides, such as NPY, PYY, Leu³¹-Pro³⁴-PYY and NPY(13-36), to the Yi receptor with competitive binding tests on SK-N-MC cells [26]. With the determination of the NPY receptor subtype performed in this work, the exclusive expression of the Yi subtype could be confirmed with regard to SK-N-MC cells.

To control the peptides for selectivity for the Yi receptor, the human neuroblastoma cell line MHH-NB-11 was used. By binding experiments with receptor-subtype-specific peptides, such as the Y₂ receptor-selective NPY(3-36) and the Yi -selective Leu^{3 l}-Pro³⁴-NPY, and the ligand [³H]propionyl-NPY, Hδfliger 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 Leu³¹-Pro³⁴-PYY has not bonded to the MHH-NB-1 1 cells and thus receptor subtypes Yi and Y₅ could be ruled out. The Y₂-selective control peptides, however, have bonded very well.

Since the NPY analogs are to be used later in the molecular imaging of breast cancer, the Yi receptor binding also should be studied on a breast cancer cell line. In this respect, the human breast cancer cell line MCF-7 was used. In 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. Based on the inhibition of the binding of ¹²⁵I-PYY by PYY(3-36) (Y₂- and Y₅- selective) and NPY(13-36) (Y₂-selective), several Y₂ receptors and optionally Y₅ receptors could be found on the MCF-7 cells. The histological studies by Reubi et al. on breast cancers showed, in 24% of the patient samples studied, a mixture of Yi- and Y₂ receptors, in which, however, the Yi receptor occurred in greater numbers and densities [13]. Since the MCF-7 cell line came from a breast cancer, this clarifies the Co expression of Yi- and Y₂ receptors in these cells. Since the levels of the reduction of the ¹²⁵I-PYY binding by PYY(3-36) and NPY(13-36) were almost the same and NPY(13-36) binds exclusively to Y₂ receptors, it can be assumed that PYY(3-36) also has bonded to the Y₂ receptors, and the expression of Y₅ receptors can be ruled out to a very large extent.

4.2 Determination of the Dissociation Constant Kj of the Ligand ¹²⁵I-Peptide YY

Before newly-synthesized NPY-peptide analogs could be studied with regard to their binding to the Yi receptor in m-vitro binding experiments, the affinity of the ligand ¹²⁵I-PYY to the corresponding cell lines first had to be studied. The K_d determination is of great importance, since the binding of a peptide to the receptor via the displacement of ¹²⁵I-PYY was determined. Based on these constants, the K, of the respective inhibitor peptide is calculated via the IC₅₀ (Formula, see 1.6.2).

The K_d value of 0.251 nmol for ¹²⁵I-PYY that is determined on SK-N-MC cells is comparable to values from the literature. Thus, Poindexter et al. determined a Ka value for ¹²⁵I-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 [³H]-propionyl-NPY could be found in the literature (Fabry et al.), which with 0.37 nmol is also near the K_d of ¹²⁵I-PYY that is determined here [25]. In contrast to binding studies on whole cells, the ligand binding to isolated membranes determines the pure receptor binding. The internalization of the ligand receptor complex or other possible influences of the cell on the binding of the ligand are not considered in this system. If, in whole cells, it is desired to determine the pure receptor binding without the influence of the uptake of ligands into the cell, the peptide binding experiment can be performed on whole cells at 4°C. At this temperature, the active uptake of the ligand in the cell is inhibited. At the same time, however, the association and dissociation rates of the ligand are also slowed, which would not change the K_d (since K_d = k_off/k_on), but the time until equilibrium sets in would be greatly extended.

On the breast cancer cells MCF-7, 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. 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. As the experiments for subtype determination showed, the MCF-7 cells also express several Y₂ receptors, but the latter are only present in a small number compared to the Yi receptors. The Scatchard Plot for the MCF-7 cells, however, does not allow any conclusions on two different binding sites with different affinities. As an alternative to this, 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. Thus, the accessibility of the receptors for the ligands on various cell lines can vary. Also, 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. In the case of the SK-N-MC cells, 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 ^l25I-peptide YY. Saturation experiments with the Y₂ receptor-expressing MHH-NB-11 cells yielded a high binding affinity of ¹²⁵I-PYY (K_d = 0.034 nmol). Also, Gehlert et al. were to describe a higher affinity of this ligand to the Y₂ receptor than to the Yi receptor [29].

The K_d values for the ligands ¹²³I-PYY determined in the saturation experiments were reproducible and were thus used for the determination of IC₅₀ values and K, values of the NPY peptide analogs to be tested with regard to binding affinity.

4.3 Binding Affinities of Standard Peptides and NPY Analogs P2489

The binding behavior of the unlabeled PYY to the cell lines under study was comparable to that of the ¹²⁵I-labeled PYY. PYY had the lowest binding affinity for the MCF-7 cells (K₁ = 5.69 nmol). A K₁ of 0.78 nmol was determined on SK-N-MC cells. The highest affinity of PYY was determined on the MHH-NB-11 cells (K₁ = 0.11 nmol). The differences in the dissociation constants between the three cell lines were clearer for the unlabeled PYY, however, than for ¹²⁵I-PYY. A possible clarification of this can be different growth phases of cells in which the regulation of the receptor expression was different. Thus, with a high regulation of the Yi receptors in the MCF-7 cells, more inhibitors would be required to displace the ligands, which then is reflected in a higher K₁ of PYY (in comparison to the K_d of ¹²⁵I-PYY).

A similar difference of the binding affinities of PYY between Yi- and Y₂ receptor- expressing cells was noted by Matthews et al. [26]. There, an IC₅₀ of 0.59 nmol on SK-N-MC cells was determined for PYY, whereby the Y₂ receptor-expressing cell line KAN-TS showed an IC₅₀ of 0.19 nmol. This difference in the IC₅₀ values between the Yi- and Y₂- expressing cell lines is comparable to the difference of the K₁ values for the SK-N-MC- and MHH-NB-11 cells that we determined in the competitive test.

The competition curve for PYY on MHH-NB-11 cells showed an untypical, almost linear plot (Fig. 20C). Even at the lowest inhibitor concentrations, a displacement of the ligand, which is present in excess, already seems to take place. A similar curve plot was also observed for the Y₂-selective PYY(3-36), and a K, of 0.40 nmol was determined (curve not shown). For this Y₂ receptor-selective peptide, an IC₅₀ of 1.3 nmol [27] was determined by Hofliger et al. on MHH-NB-1 1 cells. The related K, was unfortunately not indicated in the publication, but presumably is under 1 nmol, which is comparable to the K, that we determined in the competition experiment.

The K₁ for PYY(3-36) determined on SK-N-MC cells was 750 x higher (K₁ = 305.4 nmol) than on MHH-NB-11 cells, which excludes binding of Y₂-selective peptides on SK-N- MC cells. The selection of peptides, which bind to the Y₂ receptor, is thus made possible by competitive binding tests with MHH-NB-1 1 cells. This is important, since the binding to the Y₂ receptor for the selective binding of an NPY analog to breast tumor cells is expressly not desired.

Of the synthesized NPY analogs, 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. Also, here, the K₁ 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 Y₂ receptor-expressing MHH-NB-11 cells.

In competitive binding studies, the equilibrium dissociation constant K₁ could be determined in a reproducible manner both for standard peptides and for a Yi receptor- selective NPY analog, and thus the binding tests could be established with the human cell lines SK-N-MC, MCF-7 and MHH-NB-11. 4.4 Influence of Various Amino Acids on the Binding Affinity of the NPY Analogs

Substitution experiments by Soil et al., in which individual amino acids of NPY were replaced by L-AIa, have shown that the C-terminus and especially the positively charged side chains of the two arginines at positions 33 and 35, as well as the tyrosinamide at position 36, are essential for the receptor recognition [17]. To date, however, no defined secondary structures in the C-terminus could be identified, to which the important role of the C-terminus can be attributed. For some time, it has been known that the presence of proline at position 34 leads to the loss of binding affinity of an NPY analog to the Y₂ receptor, without influencing the affinity for the Yi receptor (Fuhlendorff et al. [15]). This finding is used as a starting point for the development of new NPY analogs, which are to have a preference for the Yi receptor. Bases for this are β-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 β-amino acids leads to improved resistance to chemical or enzymatic degradation by peptidases and to increased lipophilia. It is suggested by Koglin et al. that the most effective positions for these β-amino acids in the case of NPY analogs are the positions 32 and 34, since the latter are in direct proximity with the arginines of positions 33 and 35, and thus optional secondary structures, which are produced therefrom, can be stabilized [30].

The synthesized 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-AS³²-Arg-AS³⁴-Arg-Tyr-NH₂

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 (AS³²) and 34 (AS³⁴) are either the Thr (position 32) and GIn (position 34), naturally occurring in NPY, or various unnatural β-amino acids.

In the in-vitro binding test, several peptides, whose β-amino acids positive influence the binding, could be identified. The affinities shown in Table 11 and structural characteristics of the NPY analogs give indications regarding structural binding relationships.

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.

* determined on Yi receptor-expressing SK-N-MC cells

Fig. 25: Structures of β-Amino Acids That Are Contained in the NPY Analogs.+

The NPY analogs, which contain aminocyclohexanecarboxylic acid (AchC) (Fig. 25) as β-amino acid, had greater affinities for the Yi receptor than the other NPY analogs. The latter contained, e.g., D-Cys, Cys or the β-amino acid derivatives of Pro (β-hPro) or Leu (β- 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. If the amino group and carboxyl group are in transposition, this leads to the loss of the binding, while the cis-position improves the receptor binding. In the case of these two peptides, AchC is found in position 32. If cis-AchC is shifted to position 34, and position 32 is occupied by Thr that originally occurs in NPY, this leads to a slight improvement of the affinity. The difference of the K₁ values of P2467 (K, >665 nmol) and P2466 with cis-AchC in position 34 and a K, of 668 nmol is not significant, however, since it is not known how far the K, of P2467 is beyond 665 nmol. The removal of Thr in position 32 in peptide P2487 also had the result of a slight, but not significant increase of the binding affinity for a K₁ of 594 nmol.

The importance of a monocyclic amino acid, such as Pro, or the β-amino acids visualized in Fig. 25, in position 34 for the binding and selectivity was also evident with standard peptide Leu^{3 l}-Pro³⁴-PYY. It shows a high binding strength (K₁ = 1.00 nmol) and selectivity for the Yi receptor. PYY with GIn in position 34 has a higher affinity (K, = 0.78 nmol), but is not receptor-subtype-selective. Since adequately high binding affinity still could not be achieved with the previously synthesized NPY analogs, it has to be assumed that not only the C-terminus but also the other components of the NPY, such as the PP loops, are responsible for a tight binding. McCrea et al. thus showed that NPY sequences 1-7 and 19-23 play an important role in the binding to the Yi receptor [31].

Several of the NPY analogs studied are based on the acetyl-[β-ACC³²'³⁴](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 β-aminocyclopropanecarboxylic acid (β-ACC), consisting of a cyclopropane ring with a methylated carboxyl group (Fig. 25), as components in the binding-relevant positions 32 and 34. The substitution with β-ACC leads to a stabilization of the peptide secondary structure in that the binding to the Yi receptor is influenced in a positive manner.

The amino acid sequence and affinities for the Yi receptor of these peptides are combined in Table 12. If the N-terminal acetylation of Ac-fQ6 is removed (fQ6), the binding affinity of the peptide is lost. The natural NPY does not have any such acetylation. The acetylation of the N-terminus, however, seems to be essential for the binding of NPY fragments or C-terminal sequences.

Table 12: Amino Acid Sequence and Binding Affinity (Yi Receptor) of NPY Analogs That Contain β-ACC-Amino Acids.

4.5 Influence of the Chelating Agent DOTA on the Binding Affinity of the NPY Analogs

The coupling of peptide P2466 with the chelating agent DOTA (P2471), which is required for radiometal labeling with ¹⁷⁷Lu, had the result of the total loss of the receptor binding. The chelating agent is comparatively large in comparison to peptide, so that it can be assumed that because of a steric inhibition, no binding of the peptide to the receptor can take place.

In the case of fQ6(DOTA), 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 β-Ala chain on Lys in the N-terminal end of the peptide. The affinity of the original peptide Ac-fQ6 of 29 nmol decreased, despite introduction of the relatively large DOTA chelating agent, to only 62.8 nmol. Whether this affinity determined in vitro also remained in vivo was studied with biodistribution experiments on tumor-bearing mice.

4.6 In-Vivo Behavior of NPY Analogs

To study the in-vivo behavior of the NPY analogs, organ distribution tests were performed with the ¹⁷⁷Lu-labeled peptides P2471 and fW7(D0TA) on SK-N-MC-tumor- bearing mice.

The labeling of P2471 with ¹⁷⁷Lu was successful with 95% purity. With this peptide, the DOTA complex is coupled via a methylated glycine to the N-terminal end, from which two stereoisomers exist. Moreover, the β-amino acid cis-AchC occurs as a racemate. Thus, four labeled isomers should be seen in the chromatogram. Two of the isomers have an almost identical retention time, such that they have superimposed only three peaks that were to be seen in the chromatogram. A concentration of the ¹⁷⁷Lu-labeled P2471 in the SK-N-MC tumor had not taken place, which was not to be expected otherwise, however, because of the relatively poor binding affinity in the in-vitro tests. ^17?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. In addition, it is advantageous that 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. ¹⁷⁷Lu-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 ¹⁷⁷Lu 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. Under physiological conditions, 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.

In the in-vitro studies, peptide fW7(DOTA) showed a relatively high affinity of 62.8 nmol to the Yi receptor despite DOTA coupling. The labeling with ¹⁷⁷Lu was also successful. Also, several peaks were shown here in the chromatogram based on the racemates of β-ACC. In the case of fW7(DOTA) in the tumor, a weak concentration could be detected, and the tumor/blood ratio was relatively high in all measuring times. Presumably, 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. As also in the case of ¹⁷⁷Lu-P2471, the concentration of ¹⁷⁷Lu- fW7(D0TA) in the kidney was quite high. The values for kidney and urine were very similar in the two peptides P2471 and fW7(D0TA). The peptide fW7(DOTA) had a slight concentration in the liver, since it is presumably more lipophilic than P2471 and also is partially degraded there. 5 Literature

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Claims

Claims

What we claim: 1. A neuropeptide Y analog selected from

Ac-Arg-His-Tvr-Ile-Asn-Leu-Ile-trans(AchCVArg-Gln-Arg-Tvr-NH2 (P2468); Ac-Arg-His-Tvr-Ile-Asn-Leu-Ile-cis(AchCVArg-Gln-Arg-Tvr-NH2 (P2467); Ac-Arg-His-Tw-Ile-Asn-Leu-Ile-Thr-Arg-cis(AchCVArg-Tvr-NH2 (P2466) ; DOTA-Glv(MeVArg-His-Tvr-Ile-Asn-Leu-Ile-Thr-Arg-cis(AchCVArg-Tvr-NH2 (P2471);

Ac-Arg-His-Tyr-Ile-Asn-Leu-Ile Arg-cis(AchCVArg-Tvr-NH2 TP2487):

Ac-Arg-His-Tyr-Ile-Asn-Leu-Ile-Arg-(1 S,2R)AchC-Arg-Tyr-NH2 (P2489); Arg-His-Tyr-Ile-Asn-Leu-Ile-βACC-Arg-βACC-Arg-Tyr-NH2 (fQ6); DOTA-Arg-His-Tyr-Ile-Asn-Leu-Ile-BACC-Arg-J3ACC-Arg-Tyr-NH2 (fQ6(DOTA)); Ac-Lys-βAla -Arg-His-Tyr-Ile-Asn-Leu-Ile-βACC-Arg-βACC-Arg-Tyr-NH2 (fW7(DOTA)*) and derivatives thereof.

2. The neuropeptide Y analog according to claim 1 further comprising a detectable label.

3. The neuropeptide Y analog according to claim 2 wherein the detectable label is a radioactive atom, a fluorescent molecule, a magnetic material, or an energy-emitting material.

4. The neuropeptide Y analog according to claim 3 wherein the energy-emitting material is radionuclide.

5. The neuropeptide Y analog according to claim 4 wherein the radionuclide is Lu, F, ⁶⁸Ga, ^99mTc or ¹¹¹In.

6. The neuropeptide Y analog according to any preceding claims wherein said neuropeptide Y analog is selective for the Y receptor.

7. The neuropeptide Y analog according to claim 6 wherein the Y receptor is a Yi receptor.

8. A composition comprising a neuropeptide analog Y according to any preceding claims and a pharmaceutically acceptable carrier or diluent.

9. A method for detecting a cell expressing a Y receptor, comprising: contacting a cell with an effective amount of a neuropeptide Y analog according to claims 1 to 4 then detecting binding of said polypeptide to said cell.

10. The method according to claim 9 wherein the cell is a breast cancer cell.

11. The method according to claims 9 and 10 wherein the neuropeptide Y analog is selective for Y receptor.

12. The method according to claim 11 wherein the Y receptor is a Y] receptor.

13. The method according to claims 9 to 12 wherein the detecting is performed using positron emission tomography.

14. The method according to claims 9 to 13 wherein the detectable label of the neuropeptide Y analog according to claims 1 to 4 is selected from ¹⁷⁷Lu, ¹⁸F, ⁶⁸Ga, ^99mTc or ¹¹¹In .

15. A method for diagnosing breast cancer comprising the followed steps: contacting a breast cancer with an effective amount of a neuropeptide Y analog according to claims 1 to 4; detecting binding of said neuropeptide Y analog to said cell, wherein binding indicates the presence of breast cancer cells.

16. The method according to claims 9 or 15, wherein said method is performed in situ, on a tissue section comprising a biopsy tissue.

17. The method according to claims 9 or 15, wherein cells in a primary tumor or a metastatic site are detected.

18. A method of treating a breast cancer, comprising: administering an effective amount of a neuropeptide Y analog according to claims 1 which is P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(DOTA) or derivatives thereof.

19. The method according to claim 18, wherein neuropeptide Y analog is attached to a chemotherapeutic agent.

20. An antibody which is specific for P2468, P2467, P2466, P2471 (DOTA), P2487, P2489, fQ6, fQ6(DOTA), fW7(D0TA), or derivatives thereof.

21. Use of neuropeptide Y analog according to claim 1 for the manufacture of a diagnosis agent.

22. Use of neuropeptide Y analog according to claim 1 for the manufacture of a medicament.

23. Use according to claim 22 for the treatment of cancer expressing the receptor Y receptor.

24. Use according to claim 23 wherein neuropeptide Y analog according to claim 1 is attached to a chemotherapeutic agent.

25. A neuropeptide Y analog according to claim 5 selected from ¹⁷⁷Lu-P2471.