Octreotide-Mediated Tumor-Targeted Drug ... - ACS Publications

Subscriber access provided by UNIV LAVAL

Article

Octreotide-mediated tumor-targeted drug delivery via cleavable doxorubicin-peptide conjugate Marco Lelle, Stefka Kaloyanova, Christoph Freidel, Marily Theodoropoulou, Michael Musheev, Christof Niehrs, Günter Stalla, and Kalina Peneva Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00487 • Publication Date (Web): 02 Nov 2015 Downloaded from http://pubs.acs.org on November 3, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Octreotide-mediated tumor-targeted drug delivery via a cleavable doxorubicin-peptide conjugate Marco Lellea, Stefka Kaloyanovaa, Christoph Freidela, Marily Theodoropouloub, Michael Musheevc, Christof Niehrsc,d, Günter Stallab and Kalina Peneva*, a, e

a Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

b Max Planck Institute for Psychiatry, Kraepelinstraße 2 -10, 80804 Munich, Germany

c Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany

d DKFZ-ZMBH Alliance, Division of Molecular Embryology, 69120 Heidelberg, Germany

e Laboratory of Organic and Macromolecular Chemistry, Friedrich Schiller University Jena, Lessingstrasse 8, 07743 Jena

*To whom correspondence should be addressed. Phone: +496131379136; Email: [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

Although recent methods for targeted drug delivery have addressed many of the existing problems of cancer therapy associated with undesirable side effects, significant challenges remain that have to be met before they find significant clinical relevance. One such area is the delicate chemical bond that is applied to connect a cytotoxic drug with targeting moieties like antibodies or peptides.

Here we describe a novel platform that can be utilized for the preparation of drug-carrier conjugates in a site-specific manner, which provides excellent versatility and enables triggered release inside cancer cells. Its key feature is a cleavable doxorubicin-octreotide bioconjugate that targets overexpressed somatostatin receptors on tumor cells, where the coupling between the two components was achieved through the first cleavable disulfide-intercalating linker. The tumor targeting ability and suppression of adrenocorticotropic hormone secretion in AtT-20 cells by both octreotide and the doxorubicin hybrid were determined via a specific radioimmunoassay. Both substances reduced the hormone secretion to a similar extent, which demonstrated that the tumor


Page 2 of 29

Page 3 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


homing peptide is able to interact with the relevant cell surface receptors after the attachment of the drug. Effective drug release was quickly accomplished in the presence of the physiological reducing agent glutathione. We also demonstrate the relevance of this scaffold in biological context in cytotoxicity assays with pituitary, pancreatic as well as breast cancer cell lines.

KEYWORDS doxorubicin, octreotide, tumor-targeting, somatostatin receptor, drug delivery, cross-linker INTRODUCTION The anthracycline doxorubicin is a widely applied anticancer agent with a broad spectrum of activities against childhood solid tumors, breast cancer, soft tissue sarcomas, leukemia as well as aggressive lymphomas.1, 2 However, the therapeutic efficacy of the drug is often restricted by side effects such as poor tumor selectivity.3 In order to overcome this limitation anthracyclines and other anticancer drugs have been modified with highly specific ligands (e.g. gonadotropin-releasing hormone, transferrin, folic acid) for cell surface associated receptors.4-6 This enables an active drug targeting mediated by receptors that are overexpressed on tumor cells.7, 8 The somatostatin analog octreotide is such a potent ligand that has a high affinity for somatostatin receptor subtypes 2 and 5.9 Both subtypes and in particular type 2 are highly expressed on various tumor cells and primary tumor tissues, which makes them an attractive target for somatostatin receptor-mediated anticancer therapy.10-12 Moreover, octreotide is equipped with a slight antiproliferative action.13 Furthermore, ligand-receptor complexes are rapidly internalized by endocytotic processes and can translocate to perinuclear regions.10, 14, 15 This intracellular pathway is favorable for the delivery of cytotoxic substances like doxorubicin that exhibit their anticancer activity predominantly in the cell nucleus through DNA intercalation and subsequent inhibition of DNA topoisomerases.2 However, all previously reported anticancer drug-somatostatin analog hybrids share the same structural feature, namely, they utilize an ester bond as linkage between the drug and the ligand that is prone to hydrolysis.16, 17 Thus, any resulting bioconjugates are quickly degraded in blood serum by 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

carboxylesterases.18, 19 The decreased half-life of these hybrids can lead to an early release of the drug and undesired unspecific toxicity. To overcome this drawback more stable bonds have to be exploited for the preparation of such conjugates. Nevertheless, the linkage between doxorubicin and octreotide must allow efficient release of the cytotoxic molecule at the tumor site. Unfortunately, tumor-homing peptides like octreotide possess very limited number of appropriate functional groups, which can be applied for the conjugation of biologically active substances. Utilization of the hydroxyl groups leads to the formation of unstable ester bonds, as discussed above, and the free amines of the peptide can be only used to create highly stable amides. Moreover, the ε

-amino group of lysine residue has an essential function, since this amino acid belongs to the receptor-binding motif (Phe-Trp-Lys-Thr) and is therefore unsuitable for modification.9, 20 In spite of that, there is one structural component left, which can be further modified, namely, the disulfide bond that can be functionalized by intercalating cross-linking reagents (e.g. dibromomaleimides).21-23 Thereby, the crucial cyclic structure of octreotide, responsible for the additional stability against enzymatic digestion and for the maintenance of the shape of the receptor-binding motif, would be ensured. Nonetheless, all intercalating reagents described in the literature generate two widely stable thioether bonds, which cannot be cleaved under physiological conditions and thereby cannot be applied for the release of a toxic freight. In the present work, we introduce a new approach to overcome all the aforementioned limitations. The cytotoxic drug doxorubicin is coupled to the tumor-targeting vector - octreotide via a disulfide-intercalating cross-linking reagent. On the one hand this reagent creates an oxime bond with the drug, and on the other hand two disulfides with octreotide to keep the cyclic structure of the peptide. The combination of a hydrolytically stable oxime bond and disulfides leads to the formation of a novel bioconjugate superior to any previous anticancer drug-somatostatin analog hybrid as it allows the efficient release of the toxic cargo within the reducing environment of cancer cells. The versatility of the linker molecule described here will enable its future application not only in targeted drug delivery, but also in the chemical modification of therapeutic proteins. 4 ACS Paragon Plus Environment

Page 4 of 29

Page 5 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MATERIALS AND METHODS:

General information Solvents, chemicals and reagents were bought from commercial sources (Acros Organics, Alfa Aesar, AppliChem, Deutero, Fisher Scientific, Fluka, Merck and Sigma Aldrich) and used without further purification. Doxorubicin hydrochloride and octreotide diacetate were purchased from Ontario Chemicals, Inc. (Guelph, Ontario, Canada). CellTiter-Glo® luminescent cell viability assay was ordered from Promega (Mannheim, Germany). WST-1 colorimetric cell viability assay was purchased from Roche Molecular Biochemicals (Mannheim, Germany).

Synthesis of 2-(2-pyridyldithio)ethylamine hydrochloride (2) 2,2′-Dithiodipyridine (25 g, 113.47 mmol) was dissolved in 150 ml methanol and degassed in an ultrasonic bath for 30 minutes. To this solution 2-mercaptoethylamine hydrochloride (2.15 g, 18.91 mmol, 1) was slowly added within one hour. Afterwards, the flask was sealed with a septum and the reaction mixture was stirred overnight at room temperature under argon. The yellow solution was precipitated twice in cold diethyl ether and the product was obtained as a colorless crystalline solid (4.21 g, 18.91 mmol, quantitative yield). m/z (MALDI-TOF) 187.00 [M+H]+; 1H-NMR (300 MHz, DMSO-

d6, 298 K) δ (ppm) = 8.56-8.46 (m, 1H), 8.30 (s, br, 3H), 7.88-7.80 (m, 1H), 7.76 (d, 1H, J = 8.1 Hz), 7.34-7.25 (m, 1H), 3.17-3.01 (m, 4H); 13C-NMR (75 MHz, DMSO-d6, 298 K) δ (ppm) = 158.09, 149.80, 137.89, 121.59, 120.00, 37.65, 34.74

Synthesis of (Boc-aminooxy)acetic acid N-hydroxysuccinimide ester (4) Initially, a slurry of N-hydroxysuccinimide (2.79 g, 24.28 mmol) and (Boc-aminooxy)acetic acid (4.42 g, 23.12 mmol, 3) in 55 ml dry dichloromethane (DCM) was prepared. Under argon N,N´diisopropylcarbodiimide (3.06 g, 3.76 ml, 24.88 mmol) was added and the clear solution was stirred for 2 h. Additional N,N´-diisopropylcarbodiimide (244 mg, 300 µl, 1.99 mmol) was added and the reaction mixture was stirred for further 2 h at room temperature. Afterwards, the precipitated urea was filtered off and washed with a small amount of DCM. The obtained solution was diluted with 200 ml DCM and washed four times with water. The organic layer was dried with magnesium sulfate and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the solvent was removed in vacuo to obtain the product as a colorless solid (6.13 g, 21.27 mmol, 92%). m/z (MALDI-TOF) 357.04 [M+3Na]+; 1H-NMR (300 MHz, DMSO-d6, 298 K) δ (ppm) = 10.36 (s, 1H), 4.82 (s, 2H), 2.84 (s, 4H), 1.42 (s, 9H); 13C-NMR (75 MHz, DMSO-d6, 298 K) δ (ppm) = 169.95, 165.14, 156.57, 80.60, 69.90, 27.93, 25.47

Synthesis of (S)-tert-butyl(1,5-dioxo-1,5-bis((2-(2-pyridinyldithio)ethyl)amino)pentan-2yl)carbamate (6) The protected glutamic acid derivative (500 mg, 2.02 mmol, 5), N,N,N′,N′-tetramethyl-O-(Nsuccinimidyl)uronium tetrafluoroborate (1.22 g, 4.04 mmol) and 2 (901 mg, 4.04 mmol) were dissolved in 20 ml dry N,N-dimethylformamide (DMF). N,N-diisopropylethylamine (DIPEA) (1.72 ml, 10.11 mmol) was added and the solution was stirred in an argon atmosphere for 5 h at room temperature. Subsequently, 350 ml ethyl acetate was added and the solution was washed three times with brine. The organic layer was dried with magnesium sulfate and the solvent was removed

in vacuo. Afterwards, the residue was purified by silica gel column chromatography (ethyl acetate – methanol 15:1) to obtain the product as yellow oil (1.02 g, 1.73 mmol, 86%). m/z (MALDI-TOF) 605.99 [M+Na]+; 1H-NMR (300 MHz, DMSO-d6, 298 K) δ (ppm) = 8.51-8.43 (m, 2H), 8.07 (t, 1H, J = 6.3 Hz), 8.04 (t, 1H, J = 6.3 Hz), 7.87-7.72 (m, 4H), 7.28-7.20 (m, 2H), 6.88 (d, 1H, J = 7.8 Hz), 3.92-3.74 (m, 1H), 3.44-3.24 (m, 4H), 2.88 (t, 4H, J = 6.3 Hz), 2.10 (t, 2H, J = 7.8 Hz), 1.92-1.61 (m, 2H), 1.36 (s, 9H); 13

C-NMR (75 MHz, DMSO-d6, 298 K) δ (ppm) = 171.85, 171.66, 159.09, 159.01, 155.20, 149.63,

149.57, 137.76, 121.16, 119.33, 119.23, 78.06, 54.07, 37.78, 37.67, 37.45, 31.82, 28.15, 27.79

Synthesis of (S)-tert-butyl((1,5-dioxo-1,5-bis((2-(2-pyridinyldithio)ethyl)amino)pentan-2-yl)amino)2-oxoethoxycarbamate (7) The modified glutamic acid (250 mg, 428.2 µmol, 6) was dissolved in 10 ml dry DCM and the identical amount of trifluoroacetic acid (TFA) was added. This solution was stirred for 1 h at room temperature followed by removal of solvent and reagent under reduced pressure. The obtained oil was dissolved in 5 ml dry DMF and consecutively DIPEA (387.4 mg, 509.8 µl, 3 mmol) and 4 (135.8 mg, 471 µmol) were added. Subsequently, the reaction mixture was stirred under argon for 3 h at room


Page 6 of 29

Page 7 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


temperature. Afterwards, the solution was diluted with 250 ml ethyl acetate, washed three times with brine and dried over magnesium sulfate. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (ethyl acetate – methanol 15:1) to obtain the product as yellow oil (187.2 mg, 284.9 µmol, 78%). m/z (MALDI-TOF) 695.22 [M+K]+; 1H-NMR (300 MHz, DMSO-d6, 298 K) δ (ppm) = 10.30 (s, 1H), 8.51-8.40 (m, 2H), 8.24 (t, 1H, J = 5.7 Hz), 8.12 (d, 1H, J = 8.0 Hz), 8.05 (t, 1H, J = 5.7 Hz), 7.88-7.70 (m, 4H), 7.28-7.19 (m, 2H), 4.33-4.09 (m, 1H), 3.43-3.24 (m, 4H), 2.89 (t, 2H, J = 7.0 Hz), 2.87 (t, 2H, J = 7.0 Hz), 2.09 (t, 2H, J = 8.0 Hz), 1.97-1.69 (m, 2H), 1.38 (s, 9H); 13

C-NMR (75 MHz, DMSO-d6, 298 K) δ (ppm) = 171.40, 170.88, 167.91, 159.08, 159.04, 156.86,

149.58, 137.76, 121.17, 119.25, 80.61, 74.64, 51.80, 37.84, 37.78, 37.43, 37.27, 31.59, 28.04, 27.91

Synthesis of the bis(2-pyridyl disulfide) carrying doxorubicin derivative (9) 7 (450 mg, 685.1 µmol) was gradually dissolved in 10 ml dry DCM and the identical amount of TFA was added. The solution was vigorously stirred for 1 h at room temperature and the solvent and reagent were removed under reduced pressure. Doxorubicin hydrochloride (397.3 mg, 685.1 µmol) was dissolved in 80 ml DMF/sodium acetate buffer (1:1) – pH 4.8 and was added to the oily residue. Afterwards, the reaction mixture was stirred for 48 h at room temperature and was subsequently purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The solvent of the isolated fractions was removed under reduced pressure, whereby the product was obtained as a red solid (579.1 mg, 507.0 µmol, 74%). m/z (MALDI-TOF) 1104.09 [M+Na]+; RP-HPLC tR = 28.88 min (480 nm), A: 25 mM triethylammonium acetate (TEAA) buffer (pH 7), B: acetonitrile (ACN), 0 min 100% A – 40 min 30% A; 1H-NMR (850 MHz, DMSO-d6, 298 K) δ (ppm) = 8.42-8.37 (m, 2H), 8.24 (t, 1H, J = 5.7 Hz), 8.03 (d, 1H, J = 8.2 Hz), 8.00 (t, 1H, J = 5.7 Hz), 7.93-7.87 (m, 2H), 7.79-7.47 (m, 2H), 7.70 (d, 1H, J = 8.0 Hz), 7.65 (d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 8.0 Hz), 7.21-7.16 (m, 2H), 5.26-5.38 (m, 1H), 5.235.17 (m, 1H), 5.10-5.02 (m, 1H), 4.95-4.90 (m, 1H), 4.57-4.27 (m, 5H), 4.25-4.18 (m, 1H), 4.07 (q, 1H, J = 6.6 Hz), 3.98 (s, 3H), 3.35-3.30 (m, 2H), 3.30-3.27 (m, 1H), 3.23-3.17 (m, 2H), 3.07-2.99 (m, 2H), 2.87-2.81 (m, 1H), 2.84 (t, 2H, J = 6.7 Hz), 2.76 (t, 2H, J = 6.8 Hz), 2.48-2.43 (m, 1H), 2.12-2.07 (m, 1H),



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.06-1.99 (m, 2H), 1.91-1.82 (m, 1H), 1.84 (s, 3H), 1.75-1.67 (m, 1H), 1.63-1.58 (m, 1H), 1.49-1.44 (m, 1H), 1.15 (d, 3H, J = 6.6 Hz)

Synthesis of the doxorubicin-octreotide hybrid (12) Initially, octreotide diacetate (31.6 mg, 27.7 µmol, 10) was reduced under argon in 5 ml TEAA buffer (pH 7, 25 mM) with tris(2-carboxyethyl)phosphine (TCEP) hydrochloride (79.5 mg, 277.2 µmol) for 90 min. The reduced peptide 11 was purified by RP-HPLC and the isolated fraction was added to 9 (26.4 mg, 23.4 µmol) dissolved in 10 ml DMF/phosphate buffer (1:1) – pH 7. Afterwards the reaction mixture was stirred for 1 h at room temperature in an argon atmosphere. Subsequently, the solution was purified by RP-HPLC and the solvent of the isolated fractions was removed in vacuo, whereby the product was obtained as a red solid (39.5 mg, 19.2 µmol, 83%). m/z (MALDI-TOF) 1882.05 [M+H]+; RP-HPLC tR = 26.96 min (480 nm), A: 25 mM triethylammonium acetate (TEAA) buffer (pH 7), B: acetonitrile (ACN), 0 min 100% A – 40 min 30% A

Nuclear magnetic resonance (NMR) spectroscopy 1

H- and 13C-NMR spectra were recorded on Bruker AMX 300 and Bruker WB 850 (Bruker Avance III)

spectrometer. Chemical shifts are expressed in parts per million (ppm) relative to the residual solvent signal: DMSO-d6 (δH = 2.50, δC = 39.52). Coupling constants (J) are given in Hz.

Mass spectrometry Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was performed on a Bruker Reflex II TOF spectrometer equipped with a 337 nm nitrogen laser. 2,5Dihydroxybenzoic acid or α-cyano-4-hydroxycinnamic acid (peptidic sample) were utilized as matrix.

Reversed-phase high-performance liquid chromatography (RP-HPLC) RP-HPLC was conducted on a Jasco LC-2000Plus system (Groß-Umstadt, Germany), with appropriate diode array detector (MD-2015), solvent delivery pumps (PU-2086) and columns. Analytical RP-HPLC was carried out with a ReproSil 100 C18 (250 x 4.6 mm) column from Jasco with 5 µm particle size as a stationary phase and a flow rate of 1 ml/min. Purification of the products was performed on a Jasco ReproSil 100 C18 (250 x 20 mm) column with a flow rate of 15 ml/min and 5 µm silica as stationary


Page 8 of 29

Page 9 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


phase. The applied eluents were 25 mM triethylammonium acetate buffer (pH 7) [A] and acetonitrile [B] with a linear gradient. All substances were detected at 480 nm (characteristic absorbance of doxorubicin).

Glutathione-mediated degradation A 0.5 mM solution of 12 in Dulbecco´s Phosphate-Buffered Saline (DPBS) was incubated in the presence of glutathione (10 mM) at 37°C in an Eppendorf Thermomixer compact (300 rpm). The disulfide cleavage of 12 was monitored by RP-HPLC on a Jasco LC-2000Plus System for 24 h under the above-mentioned conditions. The degradation products were analyzed and identified on a Agilent 1290 UHPLC system equipped with a Agilent 6490 triple quadrupole mass spectrometer.

Cell cultures Human pancreatic carcinoma cells MIA PaCa-2 (ATCC® CRL-1420) were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and cultured in Dulbecco´s Modified Eagle Medium (DMEM) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 2.5% heat-inactivated horse serum and 1% Penicillin/Streptomycin antibiotics (Life Technologies, Darmstadt, Germany). The human pancreatic adenocarcinoma cell line Capan-2 (ATCC® HTB-80™, LGC Standards GmbH, Wesel, Germany) was grown in McCoy´s Medium containing 10% FBS and 1% Penicillin/Streptomycin antibiotics. MCF-7 cells (ATCC® HTB-22, human breast adenocarcinoma cell line) were grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 1% Penicillin/Streptomycin antibiotics. The human lung carcinoma cells A549 (ATCC® CCL-185™) were grown in F-12K Medium (Kaighn´s Modification of Ham´s F-12 Medium) supplemented with 10% fetal calf serum (FCS) and 100 units/ml Penicillin as well as 100 μg/ml Streptomycin. Mouse corticotrophinoma cells AtT-20/D16vF2 (ATCC® CCL-89™) were obtained from the American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% FCS, 2 nM glutamine and 105 IU/l Penicillin-Streptomycin. The above-mentioned cells were maintained at 37°C and 5% CO2 in a humidified incubator and were subcultured every 3-4 days with 0.25% trypsin. Cell culture materials were from Life Technologies, Nunc and Sigma Aldrich.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cell viability assays The cytotoxic effects of doxorubicin, octreotide and the hybrid on MIA PaCa-2, MCF-7, Capan-2 and A549 cells were investigated with the CellTiter-Glo® cell proliferation assay according to manufacturer´s instructions. Briefly, cells were seeded into 96-well plates with a density of 1x103 cells per well in 100 µl medium and incubated to allow attachment. After 24 h the medium was removed and doxorubicin, octreotide as well as 12 were added at various concentrations from 0.1-50 µM in 100 µl serum free medium. The cells were incubated for 3 h with the drugs and subsequently 100 µl fresh medium was added to each well. Cell viability was determined 72 h after treatment with the compounds based on quantitation of ATP by the CellTiter-Glo® luminescent cell viability assay. The evolved luminescence was measured on a Tecan plate reader (Grödig, Austria) to quantify the viability of the cells in each well. Wells without drug treatment were used to obtain 100% cell viability and blank wells with medium only were subtracted from sample wells and control cells. Drug concentrations were transformed into a logarithmic scale prior to analysis and the obtained data from the survival curves were expressed as IC50 values in μM. Every experiment was performed in independent triplicates. The viability of AtT-20 cells was determined in a similar manner 72 h after treatment with the compounds using the WST-1 colorimetric assay at 450 nm (Roche Molecular Biochemicals).

Determination of adrenocorticotropic hormone (ACTH) secretion The ACTH secretion of AtT-20 cells was determined by a radioimmunoassay as previously described by Stalla et al. and the obtained values were normalized with the cell viability results from the WST-1 assay.24 The ACTH antisera were produced in rabbits using a synthetic βM-23-corticotropin-amidebovine thyroglobulinconjugate.25 For the ACTH RIA we used the antibody AC2-VII with a working dilution of 1:10.000. As tracer we used ACTH 1-39 (Bachem) labeled with 125J (Perkin Elmer, Waltham, MA) with the chloramine-T method. 100µl of the diluted supernatants were incubated with 100µl of the primary antibody plus 100µl tracer (20.000cpm/100µl) for 24 hours at +4°C. The day after 100µl secondary goat anti-rabbit (1:50, Upstate) were added and incubated for 1 hour at 10 ACS Paragon Plus Environment

Page 10 of 29

Page 11 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


room temperature. After washing with 6% polyethyleneglycol 6000 (Merck) twice, radioactivity was measured in the gamma counter (Perkin Elmer, Waltham, MA). Final ACTH values are presented as (pg/ml)/OD450nm.

Microscopy studies The subcellular localization of the doxorubicin-octreotide hybrid 12 was investigated in different cell lines (MIA PaCa-2, MCF-7, Capan-2 and A549) with live cells at 37°C and 5% CO2. Fluorescence widefield microscopy images were obtained with a 100x/1.3 oil-immersion objective on a inverse Olympus IX81 microscope using following filters: excitation BP 531/40, emission BP 593/40. Fluorescence confocal laser scanning microscopy (CLSM) was performed on a TCS SP5 (Leica) equipped with a 63x/1.2 water-immersion objective and an incubation chamber for live cell imaging (37°C, 5% CO2). The anthracycline derivative was excited with an argon laser at λex = 488 nm (power set to 20%) and the emission range was set to λem = 550 - 660 nm. Fluorescence signal was detected by hybrid detectors (HyD) with fixed gain values set to 100. Staining of endosomes was achieved by incubation with CellLight® BacMam 2.0 reagent and the nuclear stain was obtained via DRAQ5™ (both from Thermo Fisher Scientific).

RESULTS AND DICSUSSION

Synthesis of disulfide-intercalating cross-linking reagent The chemically reactive sites present in octreotide and doxorubicin required a novel cross-linker 7 that possesses three different functionalities in order to construct the desired drug conjugate (Scheme 1). Amino acids are small molecules that typically bear already several different reactive sites, which makes them an excellent scaffold for building heterofunctional cross-coupling reagents.26 For that reason the glutamic acid derivative 5 was chosen as a cornerstone in the preparation of the linker. Both carboxylic acids were reacted with 2-(2-pyridyldithio)ethylamine hydrochloride 2 while

N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) was used as a coupling reagent to form the amides. The synthesis of 2 was accomplished with an excess of 2,2′-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dithiodipyridine according to a literature procedure.27 Furthermore, an aminooxy component for the condensation with the aliphatic ketone of doxorubicin had to be introduced to the amino acid scaffold of 6. Thus, the preparation of the active ester 4 was carried out as described before.28 Subsequently, 6 was deprotected under strongly acidic conditions with equal amounts of dichloromethane and trifluoroacetic acid.29 Deprotection of the Boc group occurred in quantitative yield within 1 hour and the N-hydroxysuccinimide ester 4 was coupled to the free amine. Thereby the desired cross-linker containing a protected aminooxy group and two 2-pyridyl disulfide functionalities was obtained. This reagent can be applied for the formation of an oxime bond and two disulfides respectively.

Synthesis of doxorubicin-octreotide hybrid The aliphatic keto group of the anthracyclines daunorubicin and doxorubicin can be easily modified with numerous biomolecules and in particular peptides mediated through aminooxy functionalities, which form oximes.30-32 The necessary hydrolytic stability of the oxime bond is a key feature for the drug delivery approach utilized here,3, 31, 33 so that the selective release of the cytotoxic substance by disulfide cleavage in cancer cells can be ensured. Initially, the Boc-protective group of 7 was cleaved to unmask the desired aminooxy group and to attach the disulfide-intercalating cross-linking reagent to doxorubicin (Scheme 2). The oxime condensation between the two components was carried out under slightly acidic conditions in a sodium acetate buffered solution (pH 4.8).3, 4 Consequently, doxorubicin derivative bearing two 2pyridyl disulfides was obtained. Both pyridyl disulfides are suitable for a quick and irreversible exchange with free sulfhydryl groups, such as the two thiols of reduced octreotide. During this process pyridine-2-thione is released that has a characteristic absorbance at 343 nm, which prevents the occurrence of undesired side reactions. Moreover, this small chromophore allows one to monitor the reaction progress.34 The required reduction of the peptide 10 was accomplished in a short period of time with an excess of TCEP hydrochloride. Subsequently, the excess of reducing agent was removed by RP-HPLC and the isolated product fraction of the reduced peptide 11 was added to the


Page 12 of 29

Page 13 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2-pyridyl disulfide containing doxorubicin derivative 9. Due to the equal reactivity of the two pyridyl disulfides and both thiol groups of 11, two isomers of 12 were created during the reaction (supplementary data). After the disulfide exchange was completed, the peptide-drug conjugate was purified by RP-HPLC, characterized by MALDI-TOF MS and obtained in very high yield (83%).

Receptor binding The obtained bioconjugate 12 has the potential to selectively deliver the cytotoxic drug upon binding to the overexpressed somatostatin receptors on tumor cells. Consequently, it is very important that the ability of the peptide to interact with the receptor remains intact after the attachment of the drug. In order to investigate this property a cell line was chosen, which is endogenously expressing somatostatin receptor subtype 2. The AtT-20 pituitary tumor cells respond to synthetic ligands of this receptor by decreasing the adrenocorticotropic hormone (ACTH) secretion.35 Therefore, this feature can be applied to assess the receptor interactions of the hybrid, which are crucial for binding of the modified peptide as well as the subsequent internalization of the ligand-receptor complexes. The treatment of AtT-20 cells with octreotide for 72 h suppressed the ACTH secretion in a dose dependent manner (Figure 1A). 12 suppressed the hormone secretion in a similar fashion as reflected by the obtained values from the radioimmunoassay (mean % suppression of ACTH by 12 compared to octreotide - 23±9 vs. 28±8 at 10 nM and 37±3 vs. 44±6 at 100 nM, P 150

Hybrid (12)

31.50 ± 1.74

27.14 ± 2.47

48.90 ± 5.40


Page 22 of 29

Page 23 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Intracellular trafficking of the doxorubicin-octreotide hybrid 12 in MIA PaCa-2, Capan-2, MCF-7 and A 549 cells imaged by confocal laser scanning microscopy (B, D) and fluorescence widefield microscopy (F) at various time points. All cell lines were incubated with 10 µM of 12 at 37°C. The corresponding brightfield images after 24, 48 and 72 h are depicted on the panels of A, C and E. Red color illustrates doxorubicin fluorescence. Scale bars represent 15 µm.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. (A) Fluorescence widefield microscopy image and its overlay with the brightfield image, accordingly, obtained after the incubation of MIA PaCa-2 cells with 10 µM of 12 for 24 h at 37°C, to visualize drug-associated fluorescence (red) at the cell membranes. Cells were washed three times with DPBS, prior to microscopy. Scale bars represent 10 µm. (B) Confocal laser scanning microscopy images of MIA PaCa-2 cells incubated for 48 h at 37°C with 10 µM of 12 and 10 µl CellLight® endosomal stain. Anthracycline fluorescence is shown in red, whereas endosomes are depicted in green. Colocalization of both appears yellow (right panel). Scale bars represent 15 µm.

Figure 5. Determination of the nuclear localization of the anthracycline drug in MIA PaCa-2 cells by microscopy, 72 h after the incubation with 10 µM of 12. (A) Brightfield image. (B) Confocal fluorescence microscopy image of the doxorubicin derivative. (C) Nuclei staining via incubation with 5 µM DRAQ5™, carried out 5 minutes prior to microscopy. (D) Overlay of the drug-associated fluorescence (red) and the fluorescence of the nuclear stain (blue). The corresponding colocalization is represented by violet color. Scale bars represent 15 µm. 24 ACS Paragon Plus Environment

Page 24 of 29

Page 25 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SUPPORTING INFORMATION All chemical structures, full NMR spectra, MALDI spectra and HPLC chromatograms are displayed in a supplementary file. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding author *Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. Email: [email protected]. Phone: + 49 6131 379 136. Fax: + 496131 379 100. Notes The authors declare no competing financial interest. ACKNOWLEDGEMETNS The authors are grateful to Jose Luis Monteserin Garcia and Johanna Stalla for technical assistance with the experiments in AtT-20 cells. REFERENCES

(1) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: Molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004, 56, (2), 185-229. (2) Nadas, J.; Sun, D. X. Anthracyclines as effective anticancer drugs. Expert Opin. Drug Discov. 2006, 1, (6), 549-568. (3) Schlage, P.; Mezo, G.; Orban, E.; Bosze, S.; Manea, M. Anthracycline-GnRH derivative bioconjugates with different linkages: Synthesis, in vitro drug release and cytostatic effect. J. Controlled Release 2011, 156, (2), 170-178. (4) Szabo, I.; Manea, M.; Orban, E.; Antal, C.; Bosze, S.; Szabo, R.; Tejeda, M.; Gaal, D.; Kapuvari, B.; Przybylski, M.; Hudecz, F.; Mezo, G. Development of an Oxime Bond Containing Daunorubicin-Gonadotropin-Releasing Hormone-III Conjugate as a Potential Anticancer Drug. Bioconjugate Chem. 2009, 20, (4), 656-665. (5) Kratz, F.; Beyer, U.; Roth, T.; Tarasova, N.; Collery, P.; Lechenault, F.; Cazabat, A.; Schumacher, P.; Unger, C.; Falken, U. Transferrin conjugates of doxorubicin: Synthesis, characterization, cellular uptake, and in vitro efficacy. J. Pharm. Sci. 1998, 87, (3), 338-346. (6) Leamon, C. P.; Reddy, J. A. Folate-targeted chemotherapy. Adv. Drug Del. Rev. 2004, 56, (8), 1127-1141. 25 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(7) Danhier, F.; Feron, O.; Preat, V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Controlled Release 2010, 148, (2), 135-146. (8) Lammers, T.; Hennink, W. E.; Storm, G. Tumour-targeted nanomedicines: principles and practice. Br. J. Cancer 2008, 99, (3), 392-397. (9) De Martino, M. C.; Hofland, L. J.; Lamberts, S. W. J. Somatostatin and somatostatin receptors: from basic concepts to clinical applications. Neuroendocrinology: Pathological Situations and Diseases 2010, 182, 255-280. (10) Sun, L. C.; Coy, D. H. Somatostatin Receptor-Targeted Anti-Cancer Therapy. Curr. Drug Del. 2011, 8, (1), 2-10. (11) Reubi, J. C. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr. Rev. 2003, 24, (4), 389-427. (12) Schaer, J. C.; Waser, B.; Mengod, G.; Reubi, J. C. Somatostatin receptor subtypes sst(1), sst(2), sst(3) and sst(5) expression in human pituitary, gastroentero-pancreatic and mammary tumors: Comparison of mRNA analysis with receptor autoradiography. Int. J. Cancer 1997, 70, (5), 530-537. (13) Theodoropoulou, M.; Stalla, G. K. Somatostatin receptors: from signaling to clinical practice. Front. Neuroendocrinol. 2013, 34, (3), 228-52. (14) Roosterman, D.; Roth, A.; Kreienkamp, H. J.; Richter, D.; Meyerhof, W. Distinct agonistmediated endocytosis of cloned rat somatostatin receptor subtypes expressed in insulinoma cells. J. Neuroendocrinol. 1997, 9, (10), 741-751. (15) Lelle, M.; Frick, S. U.; Steinbrink, K.; Peneva, K. Novel cleavable cell-penetrating peptide–drug conjugates: synthesis and characterization. J. Pept. Sci. 2014, 20, (5), 323333. (16) Nagy, A.; Schally, A. V.; Halmos, G.; Armatis, P.; Cai, R. Z.; Csernus, V.; Kovacs, M.; Koppan, M.; Szepeshazi, K.; Kahan, Z. Synthesis and biological evaluation of cytotoxic analogs of somatostatin containing doxorubicin or its intensely potent derivative, 2pyrrolinodoxorubicin. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, (4), 1794-1799. (17) Huang, C. M.; Wu, Y. T.; Chen, S. T. Targeting delivery of paclitaxel into tumor cells via somatostatin receptor endocytosis. Chem. Biol. 2000, 7, (7), 453-461. (18) Orban, E.; Mezo, G.; Schlage, P.; Csik, G.; Kulic, Z.; Ansorge, P.; Fellinger, E.; Moller, H.; Manea, M. In vitro degradation and antitumor activity of oxime bond-linked daunorubicinGnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites. Amino Acids 2011, 41, (2), 469-483. (19) Nagy, A.; Plonowski, A.; Schally, A. V. Stability of cytotoxic luteinizing hormonereleasing hormone conjugate (AN-152) containing doxorubicin 14-O-hemiglutarate in mouse and human serum in vitro: Implications for the design of preclinical studies. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, (2), 829-834. (20) Moller, L. N.; Stidsen, C. E.; Hartmann, B.; Holst, J. J. Somatostatin receptors. BBABiomembranes 2003, 1616, (1), 1-84. (21) Smith, M. E. B.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L. M.; Papaioannou, D.; Waksman, G.; Caddick, S.; Baker, J. R. Protein Modification, Bioconjugation, and Disulfide Bridging Using Bromomaleimides. J. Am. Chem. Soc. 2010, 132, (6), 1960-1965. (22) Brocchini, S.; Godwin, A.; Balan, S.; Choi, J. W.; Zloh, M.; Shaunak, S. Disulfide bridge based PEGylation of proteins. Adv. Drug Del. Rev. 2008, 60, (1), 3-12. (23) Balan, S.; Choi, J. W.; Godwin, A.; Teo, I.; Laborde, C. M.; Heidelberger, S.; Zloh, M.; Shaunak, S.; Brocchini, S. Site-specific PEGylation of protein disulfide bonds using a threecarbon bridge. Bioconjugate Chem. 2007, 18, (1), 61-76. (24) Stalla, G. K.; Stalla, J.; Huber, M.; Loeffler, J.-P.; Höllt, V.; von Werder, K.; Müller, O. A. Ketoconazole Inhibits Corticotropic Cell Function in Vitro. Endocrinology 1988, 122, (2), 618623. (25) Fink, R.; Muller, O. A.; Scriba, P. C. Specific N-terminal ACTH-Radioimmunoassay after Plasma Extraction. Anal. Chem. 1980, 301, 126-127. (26) Lelle, M.; Peneva, K. An amino acid-based heterofunctional cross-linking reagent. Amino Acids 2014, 46, (5), 1243-1251. 26 ACS Paragon Plus Environment

Page 26 of 29

Page 27 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(27) van der Vlies, A. J.; O'Neil, C. P.; Hasegawa, U.; Hammond, N.; Hubbell, J. A. Synthesis of Pyridyl Disulfide-Functionalized Nanoparticles for Conjugating Thiol-Containing Small Molecules, Peptides, and Proteins. Bioconjugate Chem. 2010, 21, (4), 653-662. (28) Deroo, S.; Defrancq, E.; Moucheron, C.; Kirsch-De Mesmaeker, A.; Dumy, P. Synthesis of an oxyamino-containing phenanthroline derivative for the efficient preparation of phenanthroline oligonucleotide oxime conjugates. Tetrahedron Lett. 2003, 44, (46), 83798382. (29) Wuts, P. G. M.; Greene, T. W., Protective Groups in Organic Synthesis. John Wiley & Sons, Inc.: 2006; p 696-926. (30) Braslawsky, G. R.; Kadow, K.; Knipe, J.; Mcgoff, K.; Edson, M.; Kaneko, T.; Greenfield, R. S. Adriamycin(Hydrazone)-Antibody Conjugates Require Internalization and Intracellular Acid-Hydrolysis for Antitumor-Activity. Cancer Immunol Immun 1991, 33, (6), 367-374. (31) Miklan, Z.; Orban, E.; Csik, G.; Schlosser, G.; Magyar, A.; Hudecz, F. New Daunomycin-Oligoarginine Conjugates: Synthesis, Characterization, and Effect on Human Leukemia and Human Hepatoma Cells. Biopolymers 2009, 92, (6), 489-501. (32) Ingallinella, P.; Di Marco, A.; Taliani, M.; Fattori, D.; Pessi, A. A new method for chemoselective conjugation of unprotected peptides to dauno- and doxorubicin. Bioorg. Med. Chem. Lett. 2001, 11, (10), 1343-1346. (33) Kalia, J.; Raines, R. T. Hydrolytic stability of hydrazones and oximes. Angew. Chem. Int. Ed. 2008, 47, (39), 7523-7526. (34) Carlsson, J.; Drevin, H.; Axen, R. Protein Thiolation and Reversible Protein-Protein Conjugation - N-Succinimidyl 3-(2-Pyridyldithio)Propionate, a New Heterobifunctional Reagent. Biochem. J. 1978, 173, (3), 723-737. (35) Veenstra, M. J.; de Herder, W. W.; Feelders, R. A.; Hofland, L. J. Targeting the somatostatin receptor in pituitary and neuroendocrine tumors. Expert opinion on therapeutic targets 2013, 17, (11), 1329-43. (36) Casi, G.; Neri, D. Antibody-drug conjugates: Basic concepts, examples and future perspectives. J. Controlled Release 2012, 161, (2), 422-428. (37) Deneke, S. M.; Fanburg, B. L. Regulation of Cellular Glutathione. Am. J. Physiol. 1989, 257, (4), L163-L173. (38) Meister, A.; Anderson, M. E. Glutathione. Annu. Rev. Biochem. 1983, 52, 711-760. (39) Balendiran, G. K.; Dabur, R.; Fraser, D. The role of glutathione in cancer. Cell Biochem. Funct. 2004, 22, (6), 343-352. (40) Siegfried, J. M.; Burke, T. G.; Tritton, T. R. Cellular-Transport of Anthracyclines by Passive Diffusion - Implications for Drug-Resistance. Biochem. Pharmacol. 1985, 34, (5), 593-598. (41) Huo, M. R.; Zou, A. F.; Yao, C. L.; Zhang, Y.; Zhou, J. P.; Wang, J.; Zhu, Q. N.; Li, J.; Zhang, Q. Somatostatin receptor-mediated tumor-targeting drug delivery using octreotidePEG-deoxycholic acid conjugate-modified N-deoxycholic acid-O, N-hydroxyethylation chitosan micelles. Biomaterials 2012, 33, (27), 6393-6407. (42) Watt, H.; Kumar, U. Colocalization of somatostatin receptors and epidermal growth factor receptors in breast cancer cells. Cancer Cell International 2006, 6, (1), 5. (43) Fisher, W. E.; Doran, T. A.; Muscarella, P.; Boros, L. G.; Ellison, E. C.; Schirmer, W. J. Expression of somatostatin receptor subtype 1-5 genes in human pancreatic cancer. J. Natl. Cancer Inst. 1998, 90, (4), 322-324. (44) Starkey, J. R.; Pascucci, E. M.; Drobizhev, M. A.; Elliott, A.; Rebane, A. K. Vascular targeting to the SST2 receptor improves the therapeutic response to near-IR two-photon activated PDT for deep-tissue cancer treatment. Biochimica et Biophysica Acta (BBA) General Subjects 2013, 1830, (10), 4594-4603. (45) Nayak, T.; Norenberg, J.; Anderson, T.; Atcher, R. A Comparison of High- Versus LowLinear Energy Transfer Somatostatin Receptor Targeted Radionuclide Therapy In Vitro. Cancer Biother. Radiopharm. 2005, 20, (1), 52-57. (46) Amherdt, M.; Patel, Y. C.; Orci, L. Binding and internalization of somatostatin, insulin, and glucagon by cultured rat islet cells. The Journal of Clinical Investigation 1989, 84, (2), 412-417. 27 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(47) Hornick, C. A.; Anthony, C. T.; Hughey, S.; Gebhardt, B. M.; Espenan, G. D.; Woltering, E. A. Progressive Nuclear Translocation of Somatostatin Analogs. J. Nucl. Med. 2000, 41, (7), 1256-1263.


Page 28 of 29

Page 29 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


119x65mm (300 x 300 DPI)

ACS Paragon Plus Environment

Octreotide-Mediated Tumor-Targeted Drug ... - ACS Publications

Recommend Documents