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Glycosylated DOTA-r-Melanocyte-Stimulating Hormone Analogues forMelanoma Targeting: Influence of the Site of Glycosylation on in VivoBiodistribution
Jean-Philippe Bapst, Martine Calame, Heidi Tanner, and Alex N. Eberle*
Laboratory of Endocrinology, Department of Biomedicine, University Hospital and University Children’s Hospital, Basel,Switzerland. Received January 8, 2009; Revised Manuscript Received March 9, 2009
R-Melanocyte-stimulating hormone (R-MSH) is known to bind to the melanocortin receptor 1 (MC1R) which isoverexpressed on melanotic and amelanotic melanoma cells. R-MSH analogues are potential candidates for specifictargeting of melanoma metastases. Several linear and cyclic radiolabeled MSH peptides have been designed andtested in the past, showing both high affinity for the MC1R in vitro and good incorporation in tumor xenograftsin vivo. However, considerable kidney reabsorption of the radiopeptides could not be avoided. With the aim toincrease the tumor-to-kidney ratio, we synthesized six glycosylated derivatives of NAPamide, an R-MSH octapeptideanalogue with high tumor selectivity and coupled them to the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The peptides were evaluated in vitro for MC1R binding and bioactivity and, after labelingwith 111In, for in vitro cellular uptake and in vivo tissue distribution in mice carrying B16F1 melanoma tumors.The glycopeptides showed excellent binding affinities in the low nanomolar to subnanomolar range using bothmurine and human melanoma cell lines. However, five glycopeptides displayed lower selectivity in vivo than theparent DOTA-NAPamide, because of either a lower tumor uptake or a higher kidney uptake. In particularC-terminal extension of the amide group by a galactosyl moiety increased the kidney retention dramatically. Bycontrast, an N-terminally positioned galactose residue in DOTA-Gal-NAPamide improved the tumor-to-kidneyratio (4-48 h AUC of 1.34) by a factor of about 1.2 as compared to the parent DOTA-NAPamide (4-48 hAUC of 1.11), thus serving as new lead compound for MC1R-targeting molecules.
INTRODUCTION
The melanocortin type-1 receptor (MC1R), which is over-expressed on the plasma membrane of melanoma cells, is apotential target for the diagnosis or therapy of metastases ofmalignant melanoma (1-3). Radiolabeled R-melanocyte-stimulating hormone (R-MSH), the natural ligand of MC1R,and various types of synthetic linear or cyclic analogues havebeen studied as candidates for MC1R targeting (4-10). Theseanalogues not only showed excellent binding properties to bothmurine and human melanoma cells in vitro but also good tumoruptake in vivo (4-10). However, because of considerableunspecific retention of radioactivity in the kidneys, clinicalstudies with these compounds remained scarce (11).
Generally, renal toxicity of DOTA-containing radiopeptidesremains one of the main dose-limiting factors for peptide-targeted radiotherapy (12, 13), as demonstrated for somatostatinanalogues (14, 15) which are routinely used in the clinic forthe diagnosis and therapy of various tumors expressing soma-tostatin receptors (16, 17). To improve the in vitro and in vivocharacteristics of peptide hormone analogues in general, andof radiolabeled somatostatin or R-MSH derivatives in particular,different types of modification of the chelator-peptide structureshave been examined, such as structural stabilization of thepeptide (16, 18), reduction of the size of the peptide (8),chemical variation of the radiometal chelator (19-21), the useof different radiometals such as 67/68Ga, 111In, 90Y, 177Lu, 99mTc,and 188Re (6, 9, 22, 23), the adjustment of the net charge of
chelator-peptide complexes (7, 24), the position of the chelatoron the peptide (7), cyclization of the peptides via a rheniumcore (5, 9), and the synthesis of dimerized R-MSH analogues(25). Another approach to reduce nonspecific uptake of, forexample, somatostatin (26) or R-MSH (6), by the kidneys isthe coinjection of cationic amino acids. All these strategiesimproved melanoma targeting with radiolabeled R-MSH ana-logues in experimental models, but none of these lowered kidneyuptake sufficiently to allow a broad clinical evaluation ofdiagnostic or therapeutic MSH radiopharmaceuticals.
Alternative approaches to obtain R-MSH compounds withimproved pharmacokinetic properties are needed, yieldingmolecules with particularly high receptor affinity, low nontargettissue retention, and an almost exclusively urinary excretionroute with minimal kidney reabsorption and retention, in orderto further minimize nephrotoxicity. Coupling of carbohydratemoieties to radiopeptides can strongly influence the pharmaco-kinetics of peptides, owing to the more hydrophilic characterof glycosylated molecules (27). The excretion of glycosylatedradiopeptides into the primary urine may be favored, andreuptake by the tubular system of the kidneys reduced, as shownfor glycosylated peptides and proteins (27, 28). It has beenshown that carbohydrates, if properly positioned, preservestructure-activity relationships (SAR) of the peptides with theirtarget receptors. For example, glycosylated enkephalin deriva-tives maintained their binding properties and exhibited prolongedbioavailability (29, 30). The in vivo stability of renin inhibitorscould also be improved by glycosylation (31), and carbohydratedsomatostatin analogues displayed a more favorable pharmaco-kinetic profile with reduced liver uptake and biliary excretion(32-34), thus switching the excretion route from hepato-biliaryto renal. Sugyama and colleagues (27, 28, 35) investigatednumerous modifications of Arg-vasopressin by coupling a
* For correspondence or reprints contact Alex N. Eberle, Ph.D.,Department of Biomedicine, University Hospital Basel, Klingelberg-strasse 23, CH-4031 Basel, Switzerland. Phone: +41-61-265-2382. Fax:+41-61-265-2706. E-mail: [email protected].
Bioconjugate Chem. 2009, 20, 984–993984
10.1021/bc900007u CCC: $40.75 2009 American Chemical SocietyPublished on Web 04/23/2009
variety of different sugar moieties to the peptide in order toimprove kidney targeting. Their investigations revealed that theaffinity of the peptides for kidney tubular cell membranes couldbe influenced, depending on the type of sugar used (27, 28, 35).
In this study, we synthesized and evaluated, both in vitro withmouse and human melanoma cells and in vivo with biodistri-bution experiments using melanoma tumor-bearing mice, severalradiolabeled carbohydrated R-MSH analogues whose structurewas based on [111In]DOTA-NAPamide previously synthesizedin our laboratory and found to be one of the most efficient MSHtargeting derivatives (6). The novel MSH peptides whichcontained different carbohydrate moieties inserted into theC-terminus and N-terminus as well as side chains of theNAPamide peptide, included DOTA-NAPamide-Gal, NR-DOTA-[Lys(Gluc)8]-NAPamide, DOTA-[Asp(Gal)2]-NAP-amide, DOTA-Mtr-NAPamide, DOTA-Gluc-NAPamide,DOTA-Gal-NAPamide (Figure 1).
EXPERIMENTAL PROCEDURES
Peptides and Analytical Procedures. R-MSH was a giftfrom Novartis (Basel, Switzerland), and [Nle4, D-Phe7]-R-MSH(NDP-MSH) was purchased from Bachem (Bubendorf, Swit-zerland). The reference compound DOTA-NAPamide (6) andall other peptides were synthesized in our laboratory using aPioneer peptide synthesizer (PerSeptive Biosystems Inc., Framing-ham, MA) and the continuous-flow strategy with Fmoc (9-fluorenylmethoxycarbonyl) protection of NR-amino groups (forrecent details, see Bapst et al. (25)). Fmoc-amino acids were
purchased from Novabiochem (Laufelfingen, Switzerland).Fmoc-PAL-PEG-PS polystyrene resin was from AppliedBiosystems (Rotkreuz, Switzerland) and TentaGel S TRT-Lys(Boc)-Fmoc acid-labile resin from Fluka (Buchs, Switzer-land). Fmoc-Asp(1,2:3,4-diisopropylidene-6-amino-R-D-galac-tosyl)-OHwasagift fromSynphaBaseAG(Muttenz,Switzerland).Other carbohydrates and organic reagents were purchased fromFluka or Sigma (Buchs, Switzerland).
NMR spectra were recorded on a Bruker Avance DMX-500(500 MHz) spectrometer (Bruker, Fallanden, Switzerland).Assignment of 1H and 13C spectra was achieved using 2Dmethods (COSY, HSQC). Chemical shifts are expressed in ppmusing residual CDCl3 and CHD2OD as references. Mass spectra(MS) were recorded on a Finnigan LCQ Deca (Thermo FisherScientific Inc., Waltham, MA) electrospray ion trap smallmolecules and proteomics MS system. Analytical reversed-phasehigh-performance liquid chromatography (RP-HPLC) of allpeptides was performed on a Jasco PU-980 system (Jasco Inc.,Easton, MD) usually with a Waters (Waters, Milford, MA)Symmetry C18 analytical column (5 µm, 3.9 × 150 mm), or aVydac (Grace, Deerfield, IL) 218TP54 C18 analytical column(5 µm, 4.6 × 250 mm). Preparative RP-HPLC of peptides wasperformed on the same system but either with a Vydac218TP510 C18 semipreparative column (5 µm, 10 × 250 mm)or a Waters SymmetryPrep C18 preparative column (7 µm, 19× 150 mm). Peptides were eluted by applying a gradient of5-95% solvent B (solvent A: 0.1% trifluoroacetic acid (TFA)in H2O; solvent B: 0.1% TFA in 70:30 acetonitrile/H2O; gradient
Figure 1. Structures of glycopeptides used in this study. W, N-terminal position; Z, C-terminal position; X, side chain of residue 2; Y, side chainof residue 8; Ac, acetyl group.
Glycosylated R-MSH Analogues for Melanoma Targeting Bioconjugate Chem., Vol. 20, No. 5, 2009 985
cycle: 0-27 min, 95%-10% A; 27-30 min, 10%-95% A;30-32 min, 95% A) in 32 min at constant flows of 1 mL/minfor the analytical columns, 3 mL/min for the semipreparativecolumn, and 5 mL/min for the preparative column. UV detectionwas performed at 280 nm on a Jasco UV-1570 UV-vis detector.
Preparation of Glycopeptides. DOTA-Gal-NAPamide. Thepeptide with an N-terminal Gal and DOTA on the Lys side chain(DOTA-Gal-NAPamide) was synthesized by first preparing aGal-octanoic acid (36).
Allyl-2,3,4,6-tetra-O-acetyl-R-D-galactopyranoside. 1,2,3,4,6-Penta-O-acetyl-�-D-galactopyranose (10.24 mmol, 1 equiv) wasdissolved in anhydrous acetonitrile (40 mL) and cooled to 0°C. Allyltrimethylsilane (30.76 mmol, 3 equiv) and borontrifluoride diethyl etherate (58.4 mmol, 5.7 equiv) were carefullyadded under an N2 atmosphere. The mixture was slowly warmedup to room temperature and stirred overnight under N2. It wasthen neutralized by pouring the solution into an Erlenmeyer flaskcontaining NaHCO3 (40 mL). Once the bubbling was over, thereaction mixture was transferred to a separation funnel, extractedwith dichloromethane (2 × 40 mL), and dried over Na2SO4.The crude product was purified by column chromatography(petroleum ether/ethyl acetate 3:1 to 2:1), resulting in a clearsyrup. Yield: 6.96 mmol (68%).
1H NMR (CDCl3, 500.1 MHz) δ: 1.97 (s, 3H, CH3), 1.98 (s,3H, CH3), 2.01 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.21 (m, 1H,CH2CHdCH2), 2.40 (m, 1H, CH2CHdCH2), 4.00-4.04 (m, 2H,H-5, H-6a), 4.14 (m, 3J5,6b ) 9.1 Hz, 2J6a,6b ) 12.9 Hz, 1H,H-6b), 4.24 (m, 1H, H-1), 5.03-5.08 (m, 2H, CH2CHdCH2),5.15 (dd, 3J3,4 )3.3 Hz, 3J2,3 ) 9.3 Hz, 1H, H-3), 5.21 (dd, 3J1,2
) 5.0 Hz, 3J2,3 ) 9.3 Hz, 1H, H-2), 5.35 (m, 1H, H-4), 5.69(m, 1H, CH2CH ) CH2). 13C NMR (CDCl3, 125.8 MHz) δ:20.67, 20.73, 20.74, 20.79 (4C, 4 CH3), 30.92 (1C,CH2CHdCH2), 61.45 (C-6), 67.58 (C-4), 67.90 (C-3), 68.24(2C, C-5, C-2), 71.43 (C-1), 117.67 (1C, CH2CH)CH2), 133.30(1C, CH2CHdCH2), 169.81, 169.94, 170.09, 170.56 (4CdO).
2,3,4,6-Tetra-O-acetyl-R-D-galactopyranosyl Aldehyde. Asolution of allyl-2,3,4,6-tetra-O-acetyl-R-D-galactopyranoside(6.96 mmol) was dissolved in anhydrous CH2Cl2 (100 mL) andcooled to -70 °C. Ozone (O3) was bubbled through the solutionfor approximately 1 h, until the solution turned blue. The mixturewas deozonized by bubbling N2 through the solution until itwas colorless. Acetic acid (10 mL) and zinc dust (7.5 g) wereadded to the solution, and the suspension was slowly warmedup to room temperature overnight under stirring. The suspensionwas filtered through Celite, and the filtrate was concentrated todryness. The crude product was purified by column chroma-
Figure 2. Summary of the synthesis and structures of Gal-NAPamide and DOTA-Gal-NAPamide.
986 Bioconjugate Chem., Vol. 20, No. 5, 2009 Bapst et al.
tography (petroleum ether/ethyl acetate 1.5:1) and dried underhigh vacuum. Yield: 4.74 mmol (68%).
1H NMR (CDCl3, 500.1 MHz) δ: 2.05 (s, 6H, 2 CH3), 2.06(s, 3H, CH3), 2.11 (s, 3H, CH3), 2.65-2.76 (m, 2H, CH2CHO),4.05-4.11 (m, 2H, H-5, H-6a), 4.32 (m, 1H, H-6b), 4.86 (m,1H, H-1), 5.17 (dd, 3J3,4 ) 3.2 Hz, 3J2,3 ) 8.5 Hz, 1H, H-3),5.28 (dd, 3J1,2 ) 4.7 Hz, 3J2,3 ) 8.4 Hz, 1H, H-2), 5.41 (m, 1H,H-4), 9.72 (m, 1H, CHO). 13C NMR (CDCl3, 125.8 MHz) δ:20.63, 20.67, 20.70 (4C, 4 CH3), 41.94 (CH2CHO), 60.82 (C-6), 66.62 (C-1), 66.88 (C-4), 67.72 (C-3), 67.84 (C-2), 69.67(C-5), 169.61, 169.72, 169.88, 170.63 (4 CdO), 198.31 (CHO).
2,3,4,6-Tetra-O-acetyl-R-D-galactopyranosylethanoic Acid. Asolution of 2,3,4,6-tetra-O-acetyl-R-D-galactopyranosyl aldehyde(4.74 mmol) in tert-butyl alcohol (20 mL) and a solution ofKH2PO4 (1.25 M, 8 mL) were added to a vigorously stirredsolution of KMnO4 (1 M, 9 mL). The mixture was stirred for20 min. A solution of saturated Na2SO3 was added dropwiseuntil KMnO4 was neutralized, yielding a brown precipitate ofMnO2 which was filtered through Celite and washed withethanol (2 × 25 mL). Ethanol was then removed from the filtrateunder reduced pressure, and the remaining solution was acidifiedto pH 3 using 1 N HCl and then extracted with dichloromethane(4 × 30 mL). The combined extracts were concentrated anddried under high vacuum to yield a white foam (4.31 mmol,91%).
1H NMR (CDCl3, 500.1 MHz) δ: 2.04 (s, 6H, 2 CH3), 2.07(s, 3H, CH3), 2.12 (s, 3H, CH3), 2.63 (dd, 3J ) 5.6 Hz, 2J )15.7 Hz, 1H, CH2CO2H), 2.72 (dd, 3J ) 8.9 Hz, 2J ) 15.6 Hz,1H, CH2CO2H), 4.10-4.17 (m, 2H, H-5, H-6a), 4.24 (dd, 3J5,6b
) 6.9 Hz, 2J6a,6b ) 10.7 Hz, 1H, H-6b), 4.70 (m, 1H, H-1),5.17 (dd, 3J3,4 ) 3.3 Hz, 3J2,3 ) 8.9 Hz, 1H, H-3), 5.33 (dd,3J1,2 ) 5.0 Hz, 3J2,3 ) 8.9 Hz, 1H, H-2), 5.43 (m, 1H, H-4). 13CNMR (CDCl3, 125.8 MHz) δ: 20.65, 20.65, 20.70 (4C, 4 CH3),33.02 (CH2CO2H), 61.04 (C-6), 67.04 (C-4), 67.56 (C-2), 67.77(C-3), 68.73 (C-1), 69.45 (C-5), 169.57, 169.85, 169.98, 170.65(4 CdO), 174.90 (CO2H).
The Gal-octanoic acid was attached to the N-terminus ofpartially protected NAPamide: 2,3,4,6-tetra-O-acetyl-R-D-ga-lactopyranosylethanoic acid (2 equiv) in DMF was preactivatedwith HATU (2 equiv) for 10 min and added to a suspension ofsolid-phase-bound, partially protected NAPamide (1 equiv)containing DIPEA (4 equiv). After 40 h at room temperature,the resin was washed (DMF, MeOH, DMF) and the couplingcycle repeated. After the resin was washed, the peptide wascleaved using a 90% TFA-mixture (25), precipitated in t-butylmethyl ether, and purified by RP-HPLC (tR ) 12.51 min). TheDOTA moiety was then coupled to the ε-amino group of Lys11
and deprotected in acid-treated (2 N HCl; >1 h) glassware usingthe standard procedure (25). Deprotection of the acetyl groupsof the carbohydrate moiety was carried out in liquid phase usinghydrazine hydrate (diluted 1:9 in DMF, 1 mL/20 mg peptide)for 5 h and purified by RP-HPLC (Vydac semipreparativecolumn, tR ) 14.8 min). Calculated monoisotopic mass: 1647.79g mol-1; measured: 1647.5 g mol-1.
DOTA-Gluc-NAPamide. The glucose moiety was coupledto solid-phase bound NAPamide using the Maillard reactionwhich is a slow nonenzymatic chemical reaction of reducingsugars modifying amino groups of peptides, proteins, lipids, andnucleic acids (37), thus leading to the formation of Amadoriproducts (38-40). The resin containing NAPamide (1 equiv)was stirred in the presence of �-D-glucose (10 equiv) underreflux in a mixture of MeOH/AcOH (95:5) at 60 °C for 72 h.After the mixture was washed with MeOH and 2-propanol,completion of the reaction was checked by the Kaiser test. Thepeptide was then cleaved from the resin, precipitated with t-butylmethyl ether, and purified by RP-HPLC (tR ) 8.94 min).Coupling and deprotection of DOTA followed standard proce-dures (see above), followed by purification using RP-HPLC (tR
) 9.1 min). Calculated monoisotopic mass: 1605.6 g mol-1;measured: 1605.5 g mol-1.
DOTA-Mtr-NAPamide. The maltotriose moiety was coupledto solid-phase-bound NAPamide using the Maillard reaction,followed by the addition and deprotection of DOTA, asdescribed above for DOTA-Gluc-NAPamide. RP-HPLC yieldedthe product peak at tR ) 7.8 min. Calculated monoisotopic mass:1930.03 g mol-1; measured: 1930.1 g mol-1.
DOTA-[Asp(Gal)2]-NAPamide. To the solid-phase-boundpartial NAPamide sequence His-D-Phe-Arg-Trp-Gly-Lys-resin(with side-chain protection), Fmoc-Asp(1,2:3,4-diisopropy-lidene-6-amino-R-D-galactosyl)-OH and, subsequently, Fmoc-Nle-OH were coupled manually using the HOBt-DIPC method.After cleavage of Fmoc, the N-terminal acetyl group wasintroduced to the peptide-resin (1 equiv, suspended in a verysmall amount of DMF) by the addition of p-nitrophenyl acetate(2 equiv) activated by HOBt (1 equiv). After 24 h, the resinwas filtered and extensively washed with DMF and with2-propanol. The peptide was cleaved from the resin using a 90%TFA-mixture, precipitated, and purified by RP-HPLC (Vydacanalytical column, tR ) 13.71 min). The DOTA moiety wasthen coupled to the ε-amino group of Lys11 and deprotected as
Table 1. MC1R Affinity and Biological Activity of r-MSH Analogues on B16F1 and HBL Cells
R-MSH analogues B16F1 IC50 (nmol/L)a HBL IC50 (nmol/L)a rEC50 (R-MSH ) 1)b
R-MSH 1.50 ( 0.14 1.91 ( 0.26 1DOTA-NAPamide 1.38 ( 0.35 3.09 ( 1.11 0.66 ( 0.35DOTA-Gal-NAPamide 1.62 ( 0.20 5.08 ( 0.25 0.63 ( 0.13DOTA-Gluc-NAPamide 1.90 ( 0.07 5.57 ( 0.58 0.34 ( 0.03DOTA-Mtr-NAPamide 2.95 ( 0.17c 7.61 ( 0.66c 0.62 ( 0.37DOTA-[Asp(Gal)2]-NAPamide 0.45 ( 0.01c 0.88 ( 0.08 0.26 ( 0.03NR-DOTA-[Lys(Gluc)8]-NAPamide 4.58 ( 0.42c 5.61 ( 2.17 1.02 ( 0.21DOTA-NAPamide-Gal 1.24 ( 0.22 2.00 ( 0.13 0.48 ( 0.17
a MC1R affinity of R-MSH analogues was assessed by competition binding experiments with B16F1 or HBL cells and 125I-NDP-MSH as radioligand(n ) 3-16). b Biological activity of R-MSH analogues was determined by melanin assay with B16F1 cells, and results are expressed as relativeconcentration inducing half-maximal response (rEC50, n ) 3-10) normalized on R-MSH ) 1. c P < 0.05 vs DOTA-NAPamide.
Figure 3. Internalization of 111In-DOTA-Gal-NAPamide into B16-F1 mouse melanoma cells (exemplary experiment showing the meansof n ) 3 individual determinations).
Glycosylated R-MSH Analogues for Melanoma Targeting Bioconjugate Chem., Vol. 20, No. 5, 2009 987
described above. RP-HPLC yielded the product peak at tR )10.68 min. Calculated monoisotopic mass: 1646.8 g mol-1;measured: 1646.8 g mol-1.
NR-DOTA-[Lys(Gluc)8]-NAPamide. Solid-phase boundNAPamide was first N-terminally extended by DOTA andcleaved from the resin, and then Gluc was attached to theε-amino group of Lys11. DOTA was coupled using the standardprocedure. The peptide was then cleaved from the resin, andthe chelator complex was simultaneously deprotected using thestandard 90% TFA-mixture. After precipitation, the peptide waspurified by RP-HPLC (tR ) 9.5 min) and lyophilized. Thecarbohydrate moiety was coupled using a slightly modifiedMaillard reaction: NR-DOTA-NAPamide (1 equiv) and �-D-glucose (10 equiv) were stirred for only 45 h at 60 °C underreflux and an N2 atmosphere using a mixture of MeOH/AcOH(95:5). After the mixture was washed with MeOH and 2-pro-panol, the completion of the reaction was checked by the Kaisertest. The peptide was directly purified by RP-HPLC (tR ) 8.59min) and lyophilized. Calculated monoisotopic mass: 1605.75g mol-1; measured: 1605.8 g mol-1.
DOTA-NAPamide-Gal. NAP (Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys) was assembled on TentaGel S TRT-Lys(Boc)-Fmocresin containing an acid-labile linker producing a free C-terminalacid under very mild acid cleavage conditions, without depro-tection of the side chains. The free NR group of Nle wasacetylated as described above and the peptide cleaved from theresin using DCM/TFE/AcOH 7:2:1 (1 mL/0.1 g of resin; 1 h).The solvent volume was reduced by careful evaporation, andthe peptide was precipitated by the addition of 10 volumes ofice-cold diethyl ether and purified by RP-HPLC (tR ) 21.32min). Calculated monoisotopic mass: 1851.21 g mol-1; mea-sured: 1850.69 g mol-1. The peptide (1 equiv) was dissolvedin DMF and its free C-terminal -COOH group preactivated withHATU (2 equiv) for 10 min. 1-Amino-1-deoxy-�-D-galactose(2 equiv) and DIPEA (4 equiv) were added, and the mixturewas agitated for 1 h and then left at room temperature overnight.It was purified by RP-HPLC (tR ) 20.95 min) and lyophilized.Calculated monoisotopic mass: 1770.05 g mol-1; measured:1770.10 g mol-1. Side-chain protecting groups were then cleavedusing a 94% TFA-mixture (2 h) containing only 1% thioanisole,which kept the formation of side products minimal. Solventswere removed by careful evaporation with the help of addeddiethyl ether as solvent carrier. The peptide was dissolved byconsecutive addition of 20% AcOH, H2O and CH3CN andpurified by RP-HPLC (tR ) 9.9 min). Finally, the DOTA moietywas coupled to the ε-amino group of Lys11 and deprotected usingstandard procedures as described above. Purification wasachieved by RP-HPLC (tR ) 10.12 min). Calculated monoiso-topic mass: 1647.79 g mol-1; measured: 1648.0 g mol-1.
Radioligands. 111In-Labeled Peptides. Incorporation of 111Ininto DOTA-peptides was performed by the addition of 55.5MBq of 111In-Cl3 (Mallinckrodt, Petten, The Netherlands; stockconcentration: 370 MBq/mL) to the DOTA-peptides (10 nmol)dissolved in 54 µL of acetate buffer (0.4 mol/L, pH 5) containing2 mg of gentisic acid. After 10 min of incubation at 95 °C, theradiolabeled DOTA-peptides were purified on a small reversed-phase cartridge (Sep-Pak C18, Waters), using sodium acetatebuffer (pH 7) for washing out unbound radioisotope, followedby elution of the radiopeptide with ethanol. The purity of theresulting radioligands was assessed by RP-HPLC on a JascoPU-980 chromatography system, connected to a Radiomatic500TR LB506C1 γ-detector (Packard) and a Waters SpherisorbODS2/5-µm column under the following conditions: eluent A) 0.1% TFA in water; eluent B ) 0.1% TFA in acetonitrile;gradient ) 0-2 min, 96% A; 2-22 min, 96%-45% A; 22-30min, 45%-25% A; 30-32 min, 25% A; 32-34 min, 25%-96%A; flow rate, 1.0 mL/min. The specific activity of the radioli-T
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988 Bioconjugate Chem., Vol. 20, No. 5, 2009 Bapst et al.
gands was estimated by HPLC analysis of radioligand peaks incomparison with HPLC profiles of the parent radioligandDOTA-NAPamide whose specific activity had previously beendetermined in a competitive binding assay using an antibodywhich exhibited the same affinity to labeled and unlabeledpeptide (for methodological details, see (41)). From theseprofiles, it was estimated that the specific activity of all 111In-labeled peptides was ∼7.4 GBq/µmol.
125I-Labeled Peptide. NDP-MSH was iodinated using theIODO-GEN method. NDP-MSH (12.14 nmol) was mixed with37 MBq of Na125I (Amersham Bioscience, Otelfingen, Swit-zerland; stock concentration: 3.7 GBq/mL) in 60 µL ofphosphate buffer (0.3 M, pH 7.4) in IODO-GEN-precoated tubes(Pierce, Rockford, IL). After 15 min of incubation at roomtemperature under shaking, the mixture was loaded on a smallreversed-phase cartridge (Sep-Pak C18, Waters) and washed
Figure 4. Tissue distribution of [111In]DOTA-Gal-NAPamide at 4, 24, and 48 h postinjection. Results are expressed as % of injected dose pergram of tissue (%ID/g; means ( SEM; n ) 4). †P < 0.05 vs [111In]DOTA-NAPamide.
Figure 5. Tumor vs kidney ratio of the area under the curve (4-48 h) of the glycopeptides compared to DOTA-NAPamide in B16F1 melanoma-bearing mice.
Glycosylated R-MSH Analogues for Melanoma Targeting Bioconjugate Chem., Vol. 20, No. 5, 2009 989
with water and acetic acid (0.5 M), and finally the peptide waseluted with methanol and collected. The fractions containing125I-NDP-MSH were supplemented with dithiothreitol (1.5 mg/mL) and stored at -20 °C. Before each binding experiment, anadditional purification was performed by RP-HPLC usingγ-detection.
Cell Culture. The mouse B16F1 melanoma cell line (42)was cultured in modified Eagle’s medium (MEM; BiochromAG, Germany) supplemented with 10% heat-inactivated fetalcalf serum, 2 mmol/L L-glutamine, 1% nonessential amino acids,1% vitamin solution, 50 IU/mL penicillin, and 50 µg/mLstreptomycin (all from Gibco/Invitrogen, Carlsbad, CA) at 37°C in an atmosphere of 95% air/5% CO2. For expansion orexperiments, the cells were detached with 0.02% ethylenedi-aminetetraacetic acid (EDTA) in PBS (150 mmol/L, pH7.2-7.4).
The human HBL melanoma cell line was cultured in modifiedRPMI medium (Biochrom AG) supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L L-glutamine, 50 IU/mLpenicillin, and 50 µg/mL streptomycin in the same culture andexpansion conditions as B16F1 cells.
In Vitro Binding Assay with B16F1 or HBL Cells. Com-petition binding experiments were performed by incubatingMC1R-expressing B16F1 or HBL cells in microplates with theradioligand 125I-NDP-MSH and a series of dilutions of competi-tor peptides (from 1 × 10-6 to 1 × 10-12 mol/L) as describedpreviously (43). Triplicates of 100 µL of B16F1 or HBL cellsuspensions adjusted to 4 × 106/mL were incubated in 96-wellU-bottom microplates (Falcon 3077) for 3 h at 15 °C (B16F1)or for 2 h at 37 °C (HBL) with 50 µL of 125I-NDP-MSH (50 000cpm). The binding medium consisted of MEM with Earle’s salts(Biochrom AG), 0.2% bovine serum albumin, and 1,10-phenanthroline (0.3 mmol/L; Merck). The reaction was stoppedby incubation on ice for 10 min, and the cell-bound radioactivitywas collected on filters by use of a cell harvester (Packard,Meriden, CT). The radioactivity was counted by use of aTopCount microplate scintillation counter (Packard), and the50% inhibitory concentration (IC50) was calculated with thePrism 5.0 software (GraphPad Software, Inc.).
In Vitro Melanin Assay. The biological activity of theR-MSH derivatives was assessed with an in situ melanin assay(44). B16F1 cells (2500 per well, 100 µL) were distributed incell culture flat bottom 96-well plates in medium consisting ofMEM without phenol red and supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L L-glutamine, 0.31 mmol/LL-tyrosine, 1% nonessential amino acids, 1% vitamin solution,50 IU/mL penicillin, and 50 µg/mL streptomycin. Afterovernight incubation in a cell incubator at 37 °C, serialconcentrations of R-MSH derivatives, ranging from 1 × 10-8
to 1 × 10-12 in 100 µL volume, were added and incubationwas prolonged for an additional 72 h. Melanin production wasthen monitored through measurement of the absorbance at 310nm in a microplate reader (Spectra MAX 190, MolecularDevices, Menlo Park, CA).
In Vitro Internalization Assay. B16F1 cells were seededin six-well plates and incubated overnight at 37 °C in B16F1cell culture medium. For the experiments, B16F1 medium wasremoved and replaced by 1 mL of Mouse Binding Medium(MBM) internalization buffer, consisting of MEM with Earle’ssalts (Biochrom AG, Germany), 0.2% bovine serum albumin,and 1,10-phenanthroline (0.3 mmol/L; Merck). After 1 h ofincubation at 37 °C, 74 kBq of radioligand (111In-labeledpeptides) was added, and the plates were incubated for differenttimes. Nonspecific internalization was assessed by addition of50 µL of a 1 µM nonradioactive R-MSH solution. Samples weretaken from the supernatant after 0.5, 2, and 3.5 h to determinethe total dose added, immediately followed by extensive
washings of the cells (6×) using prewarmed (37 °C) MBM toremove the excess of radioligand. The cells were then incubatedin 2 mL of ice-cold acid buffer (acetate-buffered HBSS, pH 5)for 10 min to dissociate surface-bound ligand. After collectionof the acidic fraction, cells were rinsed once with MBM andthe washing was pooled with the acid buffer fraction. The cellswere washed once more with MBM (37 °C), lysed in 1% TritonX-100, and finally transferred to tubes for quantification. Theradioactivity of all collected fractions was determined in a CobraII Auto-Gamma γ-counter (Packard). One counting plate wassubjected to the same treatment as the plate incubated for thelongest time. Cells from this plate were not lysed but detached(EDTA) and counted. Results of the experiments are expressedas the percentage of added dose per million cells.
Biodistribution in B16F1 Tumor-Bearing Mice and Sta-bility of Radioligands after Kidney Excretion. All animalexperiments were performed in compliance with Swiss regula-tions for animal welfare. Female B6D2F1 mice (C57BL/6 ×DBA/2 F1 hybrids; breeding pairs obtained from IFFA-CREDO)were implanted subcutaneously with 0.5 million B16F1 cellsin phosphate-buffered saline (PBS) to generate a primary skinmelanoma. One week later, 200 µL containing 185 kBq (25pmol) of radioligand diluted in PBS/BSA (pH 7.4) was injectedintravenously in the lateral tail vein of each mouse. To allowdetermination of nonspecific uptake, 50 µg of R-MSH (30 pmol)was coinjected with the radioligand in some mice. The animalswere killed at the indicated time points; organs and tissues ofinterest were dissected and rinsed of excess blood and weighed,and the radioactivity measured in a γ-counter. The percentageof the injected dose per gram tissue (%ID/g) was calculated foreach tissue. The total counts injected per animal were calculatedby extrapolation from counts of a standard taken from theinjected solution for each animal.
As part of the biodistribution experiments, samples of urinewere collected from melanoma-bearing mice 10, 15, 20 min,and 4 h after injection of 185 kBq of 111In-DOTA peptide andkept frozen at -80 °C until use. Urine (1 vol) was mixed withmethanol (2 vol) to precipitate the proteins and was analyzedby RP-HPLC/γ-detection under the conditions mentioned above.
Analysis of Data. Unless otherwise stated, results areexpressed as mean ( standard error of the mean (SEM).Statistical evaluation of the binding assays was performed byusing Student’s t-test. For the analysis of data collected duringbiodistribution experiments, the mean value obtained for eachorgan was compared individually to each DOTA-NAPamideorgan values by using Student’s t-test, and the results werecorrected with the Bonferroni correction. A P-value of <0.05was considered statistically significant. The area under the curve(AUC) was calculated for a particular time period (4-48 h)using the GraphPad Prism software; mean tissue uptake valuesat each time point were used for this calculation.
RESULTS
Synthetic Glycosylated DOTA-NAPamide Analogues.The preparation of glycosylated DOTA-NAPamide analoguesfollowed four different strategies: (1) synthesis of a galactopy-ranosylethanoic acid for straightforward attachment to the NR-terminal amino group of the peptide, as indicated for DOTA-Gal-NAPamide (Figures 1 and 2), requiring a three-step synthesisof the glyco derivative (allylation of its anomeric center,followed by ozonolysis and oxidation (36)); (2) Maillard reactionof �-D-glucose or maltotriose with the NR-terminal or the Nε-side-chain amino group, as exemplified for DOTA-Gluc-NAPamide, DOTA-Mtr-NAPamide, or NR-DOTA-[Lys(Gluc)8]-NAPamide (Figure 1); (3) use of the Fmoc-Asp(1,2:3,4-diisopropylidene-6-amino-R-D-galactosyl)-OH building block fora direct introduction of the carbohydrate during solid-phase
990 Bioconjugate Chem., Vol. 20, No. 5, 2009 Bapst et al.
assembly into the side-chain position of residue 2 of DOTA-[Asp(Gal)2]-NAPamide; (4) functionalization of the C-terminalamide as glycoamide, by attachment of 1-amino-1-deoxy-�-D-galactose to the free carboxy terminus of a side-chain-protectedpeptide, followed by deprotection and attachment of the DOTAgroup at the Nε-side chain of Lys 8.
The glycopeptides were usually obtained in high purity(>99%) but in low yields. Indeed, if DOTA-Gal-NAPamidewas obtained in a total yield of 7.4%, the total yields of otherglycopeptides were 3% for DOTA-Gluc-NAPamide, 3.4% forDOTA-Mtr-NAPamide, 2% for DOTA-[Asp(Gal)2]-NAPa-mide, 1% for NR-DOTA-[Lys(Gluc)8]-NAPamide, and 3.1%for DOTA-NAPamide-Gal.
The retention times of 111In-labeled peptides on analyticalγ-detection RP-HPLC were found to be 18.6 min for DOTA-Gal-NAPamide, 19.2 min for DOTA-Gluc-NAPamide, 18.0 minfor DOTA-Mtr-NAPamide, 20.6 min for DOTA-[Asp(Gal)2]-NAPamide, 19.1 min for NR-DOTA-[Lys(Gluc)8]-NAPamide,and 19.6 min for DOTA-NAPamide-Gal. Their level ofradiochemical purity always exceeded 97%, and the specificactivity did not appear to differ significantly between thedifferent glycosylated peptides as deduced from HPLC analysis.
Receptor Binding. MC1R affinity of the glycosylated NAP-amide analogues was assessed by competition binding assaysagainst 125I-NDP-MSH on both murine B16F1 and human HBLmelanoma cell lines, using R-MSH and DOTA-NAPamide asreference compounds. All peptides showed very good bindingaffinities to mouse and human MC1R with IC50s ranging from0.45 to 7.61 nmol/L (Table 1). DOTA-[Asp(Gal)2]-NAPamidedisplayed the highest affinity for the MC1R on both B16F1 andHBL cells and exceeded that of DOTA-NAPamide by a factorof 3 to 3.5. DOTA-NAPamide-Gal was also slightly moreactive than DOTA-NAPamide. DOTA-Gal-NAPamide andDOTA-Gluc-NAPamide displayed slightly lower IC50s thanDOTA-NAPamide, while the affinity of DOTA-Mtr-NAPa-mide and NR-DOTA-[Lys(Gluc)8]-NAPamide was about 2- to3.5-fold lower than that of DOTA-NAPamide. The glycopep-tides without the DOTA chelator all showed markedly higheraffinity (data not shown).
Biological Activity. In the melanin assay using B16F1 cells,all DOTA-glycopeptides, except for NR-DOTA-[Lys(Gluc)8]-NAPamide, displayed higher biological (agonist) activity thanDOTA-NAPamide, all in the picomolar range. In order tocompare the EC50 of different experiments, these were normal-ized against the EC50 of the reference compound R-MSH whoserEC50 was defined as 1 (Table 1). DOTA-[Asp(Gal)2]-NAP-amide and DOTA-Gluc-NAPamide displayed the lowest rEC50swith 0.26 and 0.34, respectively. The values of the otherglycopeptides were comparable to that of DOTA-NAPamide.The induction of melanin synthesis by B16F1 cells started atdoses matching their IC50s and lower. Only DOTA-Mtr-NAPamide showed a slightly worse rEC50 but still matchingthat of R-MSH. Again, DOTA-[Asp(Gal)2]-NAPamide exhib-ited the best agonist activity in vitro.
Internalization Assay. Internalization assays were carriedout to determine if and how fast the glycopeptides wereinternalized by B16F1 cells. The results were excellent, allglycopeptides being almost totally internalized in 2 h or less.As an example, Figure 3 shows the internalization kinetics ofDOTA-Gal-NAPamide, which reached a plateau after 2 h wheninternalization of the peptide was almost total. These data showthat internalization of the peptide by tumor cells cannot be anissue regarding the in vivo tumor uptake.
Biodistribution in Melanoma-Bearing Mice. Table 2 showsthe tissue distributions of the different 111In-labeled, glycosylatedDOTA-NAPamide peptides in comparison with [111In]-DOTA-NAPamide at 4, 24, and 48 h after injection of the
radioligand. Coinjection of an excess of R-MSH to block MC1Rreduced the 4-h tumor uptake of DOTA-Gal-NAPamide by>90%, of DOTA-Mtr-NAPamide by 89%, of NR-DOTA-[Lys(Gluc)8]-NAPamide by 86%, of DOTA-Gluc-NAPamideby 80%, of DOTA-NAPamide-Gal by 77%, and of DOTA-[Asp(Gal)2]-NAPamide by 75%, indicating that all these deriva-tives are taken up by melanoma cells through receptor-mediatedinternalization. DOTA-Gal-NAPamide showed the highesttumor uptake among the glycopeptides, comparable to that ofDOTA-NAPamide (Figure 4), followed by DOTA-Mtr-NAPamide, DOTA-NAPamide-Gal, DOTA-[Asp(Gal)2]-NAP-amide, NR-DOTA-[Lys(Gluc)8]-NAPamide, and DOTA-Gluc-NAPamide. The ranking of melanoma uptake of the differentglyco-NAPamide peptides did not correspond with the in vitroMC1R binding affinity.
Clearance of 111In from the kidneys was found to be quite aslow process, as also reported for other peptides. Indeed, 46%of the radioactivity measured in the kidneys 4 h after injectionof DOTA-Gal-NAPamide was still present after 24 h, and 26%after 48 h. For DOTA-[Asp(Gal)2]-NAPamide the values were49% and 33%, for DOTA-Mtr-NAPamide 34% and 27%, forDOTA-Gluc-NAPamide 65% and 43%, and for NR-DOTA-[Lys(Gluc)8]-NAPamide 66% and 35%. The slowestclearance from the kidneys was observed for DOTA-NAPamide-Gal whose values were 99% and 34% and hence was opposedto the expectation that glycosylation of the C-terminal amideof Lys8 may increase kidney clearance and reduce reuptake.
Radioactivity release from the tumor was faster than fromthe kidneys, with 25% of the radioactivity measured after 4 hstill present after 24 h and 11% after 48 h for DOTA-Gal-NAPamide, and slightly higher but comparable values forDOTA-[Asp(Gal)2]-NAPamide, DOTA-Gluc-NAPamide, NR-DOTA-[Lys(Gluc)8]-NAPamide, and DOTA-NAPamide-Gal,in part due to their lower overall tumor uptake. Indeed,DOTA-Mtr-NAPamide, which displayed an almost similartumor uptake as that of DOTA-Gal-NAPamide after 4 h,exhibited values of 22% and 8% at 24 and 48 h.
The glycopeptides with the highest in vitro binding affinitydisplayed a lower in vivo accumulation in melanoma than wouldhave been predicted from their in vitro potency. DOTA-Gal-NAPamide, showing an average receptor binding affinitycomparable to DOTA-NAPamide, delivered a more favorablebiodistribution profile with higher tumor uptake after 4 h (8.30%( 1.22%ID/g) and higher tumor-to-kidney ratio after 4 h (1.87( 0.15) than that for any other glycopeptide. Indeed, DOTA-Gal-NAPamide exhibited a 4-48 h AUC tumor-to-kidney ratio of1.34 (Figure 5), which exceeds that of DOTA-NAPamide(1.11) by 17%. It was unfortunately impossible to comparedirectly the tumor-to-kidney ratio of the AUC of the peptidessynthesized in this work with peptides synthesized by others,because of the lack of 48 h data in their work. Nevertheless,their 4 h tumor-to-kidney-ratio could be compared, and itappeared that DOTA-Gal-NAPamide was among the bestradiolabeled R-MSH analogues synthesized so far. Indeed, asmentioned above, DOTA-Gal-NAPamide displayed a 4 htumor-to-kidney ratio of 1.87 while, for instance, 111In-DOTA-ReCCMSH (1.02) (4), 188Re-(Arg11)CCMSH (0.32)(10), 111In-DOTA-NAPamide (1.63) (7), [111In-DOTA-Nle4,Asp5,D-Phe7,Ac-Lys11-NH2]-R-MSH4-11 (1.06) (7), 90Y- or 177Lu-DOTA-Re(Glu2,Arg11)CCMSH (1.30 and 1.57) (9), 111In-DOTA-Cyc-MSH (0.35) (45), or 111In-DOTA-GlyGlu-Cyc-MSH (0.61) (45) exhibited lower tumor-to-kidney ratios after4 h.
DISCUSSION
This study focused on the influence of introducing a fewdifferent types of carbohydrates at the N- or C-terminal end
Glycosylated R-MSH Analogues for Melanoma Targeting Bioconjugate Chem., Vol. 20, No. 5, 2009 991
and at side-chain positions of the R-MSH derivative DOTA-NAPamide. In view of the reports that glycosylation of peptidesor proteins would reduce reuptake by the tubular system of thekidneys(27,28),ourfirstattemptwastostudyaDOTA-NAPamidederivative whose C-terminal amide was glycosylated. Previousobservations in our laboratory (unpublished data) demonstratedthat an important metabolite of [111In]DOTA-NAPamide wasH-Lys([111In]DOTA)-NH2 which was regularly observed in theurine collected from mice treated with this peptide. However,DOTA-NAPamide-Gal showed a higher and much longerkidney retention than all other glycopeptides which we inves-tigated. Most likely, the C-terminal carbohydration led toincreased reuptake and trapping of the peptide or its metabolitesin endosomes in tubular cells of the kidney.
Glycosylation of the amino acid side chains of Asp2 or Lys8
doesnot seemtobeagoodalternativeeither.DOTA-[Asp(Gal)2]-NAPamide and NR-DOTA-[Lys(Gluc)8]-NAPamide both dis-played slightly lower tumor uptake and higher kidney uptakethan the other glycopeptides. Again, the increased kidney uptakemay be the result of higher rate of internalization and markedretention by tubular cells. A precise molecular explanation wouldrequire a detailed analysis, particularly in view of the very highin vitro potency of DOTA-[Asp(Gal)2]-NAPamide whichwould rule out steric hindrance by the side-chain galactosylgroup for receptor binding.
Finally, three different glycopeptides bearing a glucose,galactose, or maltotriose moiety at their N-terminal end werethen examined in order to assess the influence of the type ofsugar on the pharmacokinetics. The derivative with a glucoseresidue showed poor pharmacokinetic properties, displaying anin vivo tumor-to-kidney ratio (4-48 h AUC) of only 0.60(Figure 5), although it exhibited a similarly low kidney uptakeafter4h(4.48%ID/g)as thestructurallysimilar [111In]DOTA-Gal-NAPamide. The analogue with N-terminal maltotriose showedmuch better properties with a tumor-to-kidney ratio (4-48 hAUC) of 0.99. The tumor uptake was similar to that of[111In]DOTA-NAPamide, but the higher kidney uptake led to alower ratio as compared to DOTA-NAPamide. The most encour-aging data were obtained with [111In]DOTA-Gal-NAPamidewhich exhibited the relatively highest melanoma uptake and lowestkidney uptake in vivo, leading to a tumor-to-kidney ratio whichexceeded that of [111In]DOTA-NAPamide by a factor of about1.2. This result is very valuable, as it shows the direction to followin order to improve tumor-to-nontarget-tissue ratios which arefundamental for future therapeutic applications.
In conclusion, we demonstrated that introduction of differenttypes of sugars at various positions along the sequence of 111In-labeled NAPamide affect the in vitro binding affinity and thein vivo pharmacokinetic behavior. It is noteworthy that carbo-hydration generally did not reduce the binding affinity whencompared to the nonglycosylated reference peptide. Also, thesugar moieties appeared to be without influence on the extentof 111In-labeling of DOTA, owing to a rigidly standardizedlabeling protocol. Kidney reuptake and retention, however, wereincreased with side-chain and C-terminal sugar groups butdecreased with an N-terminal galactosyl group. This compound,DOTA-Gal-NAPamide, exhibited favorable pharmacokineticdata. Its high melanoma uptake and lower kidney retention, andabove all its improved tumor-to-kidney uptake ratio, opens thedoor to the development of novel glycosylated radiolabeledR-MSH derivatives for melanoma targeting with further reducednephrotoxicity.
ACKNOWLEDGMENT
The authors thank the Swiss National Science Foundationand the Swiss Cancer League for financial support of this work.
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