MAY/JUNE 2006 Volume 17, Number 3 © Copyright 2006 by the American Chemical Society
COMMUNICATIONS Single-Drug Multiligand Conjugates: Synthesis and Preliminary Cytotoxicity Evaluation of a Paclitaxel-Dipeptide “Scorpion” Molecule Ahmad Safavy,*,§ Kevin P. Raisch,§ Damien Matusiak,⊥ Saloni Bhatnagar,§ and Lawrence Helson‡ Department of Radiation Oncology and the Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, Department of Medicine, University of Illinois at Chicago and Jessie Brown Veterans Administration Medical Center, Chicago, Illinois 60612, and Tapestry Pharmaceuticals, Boulder, Colorado 80301. Received July 26, 2005; Revised Manuscript Received March 7, 2006
To improve the targeting properties of receptor-directed drug-peptide conjugates, a multiligand approach was proposed and a model “scorpion” conjugate (6, Figure 1), consisting of two peptide “claws” and a paclitaxel (PTX) “tail”, was synthesized. The cell surface receptor-directed peptide used in this single-drug multiligand (SDML) model was a segment of the amphibian peptide bombesin (BBN) which had the Y6Q7W8A9V10G11H12L13M14NH2 sequence, designated here as BBN[6-14] (2, Figure 2). Due to the lipophilic nature of both PTX and BBN[6-14], compound 6 had a low water solubility. To enhance the solubility, PEG derivatives of this conjugate were prepared with the polymer inserted either in the claws or in the tail regions. In a preliminary random screening, conjugate 6 showed superior cytotoxic activity in several GRPR-positive human cancer cell lines as compared to free PTX and two single-drug single-ligand (SDSL) conjugates. In a receptor blocking experiment, addition of excess unconjugated BBN[6-14] ligand reduced the cytotoxicity of conjugate 6, indicating the receptor-mediated mechanism of drug delivery. The PEG-derived conjugates showed activities which were intermediate between SDSL and the SDML congeners. Also, an increase in the number of the PEG segments lowered cytotoxicity, possibly due to steric hindrance against ligand-receptor binding. Taken together, these results demonstrate the potential of the multiligand approach in the design of receptor-targeting conjugates for tumor-specific drug delivery.
INTRODUCTION Targeted delivery of antitumor agents is an active and promising area of oncology research (1, 2). Two of the main * Correspondence to Ahmad Safavy, Ph.D., University of Alabama at Birmingham, Department of Radiation Oncology, 1530 3rd Ave. South, 674 Wallace Tumor Institute, Birmingham, Alabama 352946832. E-mail:
[email protected]. § University of Alabama at Birmingham. ⊥ University of Illinois at Chicago and Jessie Brown Veterans Administration Medical Center. ‡ Tapestry Pharmaceuticals.
classes of targeting molecules used for the delivery of cytotoxic agents are MAbs1 (3, 4) and tumor cell surface receptor-directed 1 Abbreviations: BBN, bombesin; Boc, (tert)-butyloxycarbonyl; DCM, dichloromethane; DIEA, N,N-diisopropyl ethylamine; EEDQ, (2-ethoxy-1-ethoxycarbonyl)-1,2-dihydroquinoline; ESMS, electrospray mass spectrometry; Fmoc, fluorenylmethoxycarbonyl; MAbs, monoclonal antibodies; MALDI, matrix-assisted laser desorption/ionization; NHS, N-hydroxysuccinimidyl ester; PEG, poly(ethylene glycol); PTX, paclitaxel; SDML, single-drug multiligand; SDSL, single-drug singleligand; TA, thioanisole; TBST, Tris-buffered saline Tween-20; TFA, trifluoroacetic acid; TIPS, triisopropylsilane.
10.1021/bc050224c CCC: $33.50 © 2006 American Chemical Society Published on Web 04/06/2006
566 Bioconjugate Chem., Vol. 17, No. 3, 2006
Safavy et al.
Figure 1. Structure of the PTX-di-BBN[6-14] “scorpion” conjugate 6.
small-molecule peptides (SMPs) (5, 6). The interest in the use of SMPs for drug targeting is related to their better tumor penetration and faster circulatory clearance when compared to the large MAb molecules. Although the use of SMPs in oncology started mainly with radiolabeled analogues (7-9), research on the development of drug-conjugated SMPs has also attracted interest. To this end, peptides targeting a variety of tumor cell membrane receptors with different drugs have been evaluated (10, 11). An SDSL-type PTX-BBN conjugate (structure 1) was recently reported by our group which showed a higher cytotoxic activity against a human non small cell lung cancer cell line, and as compared to the unconjugated PTX (12). As therapeutic agents, peptide conjugates may suffer from short circulation half-lives, and thereby, low tumor uptake (13, 14). This, in turn, may prevent the tumor tissue from receiving a therapeutically significant dose of the drug. By analogy to the divalent antigen-binding feature of MAbs, which along with their long circulation times account for their characteristic high tumor uptake, we hypothesized that doubling the number of the peptide segments of an SDSL conjugate might improve the tumor targeting ability of the molecule. This hypothesis may be supported by the previously reported investigations on receptor-directed multivalent peptide and protein ligands (15-17). The improvement was expected to be the result of an increase in the frequency of ligand-receptor collisions and/or an augmented stability of the ligand-receptor complex. This communication reports on the synthesis of a PTXderived SDML peptide conjugate as well as on its cytotoxic activity against some human cancer cell lines. To evaluate only the effect of the ligand multiplicity on the overall activity, the drug:conjugate molar ratio was kept at 1 and equal to that in the previously reported SDSL (12). The SDML conjugates are part of a study on the design, synthesis, and evaluation of multidrug multiligand drug conjugates currently underway in this laboratory.
EXPERIMENTAL PROCEDURES Peptide Synthesis. The tyrosylbombesin (BBN[6-14], 2) used in this work was synthesized by standard solid-phase peptide synthesis methods using Rink amide resin and the Fmoc protocol. After TFA cleavage, the peptide was purified by reversed-phase HPLC and identified by ESMS.
For all products, reversed-phase (RP) HPLC analysis/ purification were performed with a Beckman System Gold instrument consisting of a SG 125 solvent module and a SG 166 UV/vis detector, operated by Beckman 32 Karat Version 5.0 software. For analytical HPLC, a 4.6 × 250 mm C18 column (Vydac, Hesperia, CA), and for preparative runs, a Vydac 2.5 × 25 Cm C8 column, were used. Columns were eluted with 0.1% TFA/water (solvent A) and a 10% to 90% gradient of 0.1:60:40, TFA:CH3CN:H2O (v/v) (solvent B). Analytical retention times (tR) in minutes are reported with the corresponding percent purities in parentheses. Paclitaxel-2′-hemisuccinate was prepared as described previously (18). PTXGlu(BBN[6-14])2, 6. To a solution of Boc-Glu (3, Fluka, Milwaukee, WI, 3.4 mg, 13.8 µmol) in dry DMF were added solutions of BBN[6-14] (2, 36.7 mg, 27.6 µmol) and HOBT (5.6 mg, 41.4 µmol) in the same solvent. Under argon, DIEA (20 µL) and then DCC (23 mg, 111.5 µmol) were added and the solution was stirred at ambient temperature for 48 h. A second and a third addition of 2 (19.5 mg, 14.7 µmol), HOBT (1.6 mg, 11.8 µmol), and DCC (7 mg, 34 µmol) were made and stirring continued for 48 h. Product formation was monitored by peak separation and identification by RP-HPLC and ESMS, respectively. On reaction completion, the Boc protection of the intermediate 4 was removed by adding 3 mL of a deblocking cocktail consisting of 5:1:1:0.5, v/v, TFA:DCM:TIPS:TA, and incubation at ambient temperature for 1 h. The intermediate product 5 was purified by preparative RP-HPLC. Yield: 28 mg (87.5%); HPLC, tR: 18.8 min (96%); ESMS: 1159 (M + 2H), 773 (M + 3H). Compound 5 (20 mg, 7.52 µmol) was dissolved in dry DMF, and the vessel was cooled in an ice bath under dry argon. PTXSX (13.3 mg, 15.6 µmol), NHS (5.4 mg, 46.9 µmol), and EEDQ (7.7 mg, 31.2 µmol) were added in sequence and the solution was stirred at 4 °C for 24 h. The final product 6 was purified by RP-HPLC. Yield: 23.5 mg (89.8%); HPLC, tR: 24.4 min (93%); ESMS: 1627 (M + 2H), 1085 (M + 3H). PTXGlu(OH)YBBN[6-14], 9. The same procedure as for 6 was used except that equal molar amounts of Boc-Glu, DCC, and BBN[6-14] were used with no HOBT. The peptide was added 1 h after the addition of DCC. The intermediate conjugate 7 was not isolated and was converted directly to the deprotected
Bioconjugate Chem., Vol. 17, No. 3, 2006 567
Communications Table 1. Analytical Data for PEG-Derived Scorpion Conjugates conjugate
MWcala
MWmalb
tR (min)c
% purityd
PTXPEGGlu(BBN[6-14])2, 13 PTX(PEGGlu)2(BBN[6-14])2, 16 PTXGlu(PEGBBN[6-14])2, 20
5500 10250 10250
5532 10118 10172
24.3 22.9 24.2
>86 99 99
a
Calculated average MW based on the average MW of the starting material PEG 10. b MALDI average MW. c HPLC retention time. d By HPLC.
Figure 2. Structures of different GRPR-directed bombesin-derived peptides. Scheme 2
Scheme 1
derivative 8. The latter conjugate and the final product 9 were purified by RP-HPLC. 8: Yield: 1.85 mg (37.7%); HPLC, tR: 19.3 min (95%); ESMS: 1232 (M + H), 616 (M + 2H) 9: Yield: 1.3 mg (89.8%); HPLC, tR: 24.0 min (85%); ESMS: 2167 (M + H), 1084 (M + 2H). General Procedure for PEG Conjugation. Fmoc-PEGNHS, the N-Fmoc-protected NHS ester of amino-PEG carboxylic acid, with an average MW of ∼3400 (Nektar, Huntsville, AL) was used as the solubilizing linker. The peptide component (1 equiv) was added in dry DMF to the solution of Fmoc-PEGNHS (10, 1.2 equiv) followed by the addition of DIEA (1 equiv) at 0 °C. On reaction completion, piperidine (as 50% DMF solution, v/v) was added and the mixture was incubated for 20 min at ambient temperature. The intermediate conjugates 11, 12, 14, 15, and 17-19 were purified by dialysis against water and were identified by MALDI MS. The final products were purified by RP-HPLC and identified by MALDI MS. The analytical data for these PEG conjugates are shown in Table 1. PTX Conjugation. All PTX couplings (including that for conjugate 21) were carried out according to the procedure of Scheme 1 (last step), described above for the synthesis of conjugate 6. Quantitative Immunohistochemistry. All reagents were supplied as a kit by DAKO (Carpentaria, CA). Cells were plated on two-well permonox chamber slides (Nalge Nunc International, Rochester, NY) at a density of 25 000 cells/well and allowed to grow in defined media. Cells were fixed in 3.7% formaldehyde in PBS for 20 min at 37 °C, followed by rinsing and permeabilizing with TBST. Immunohistochemistry was performed using a two-step indirect immunoperoxidase technique. Cells were incubated in 0.03% H2O2 solution and were rinsed with water and TBST in sequence. The primary (rabbit anti-GRPR) antibody was applied for 1 h at room temperature before rinsing with TBST. The secondary antibody (Labeled Polymer Rabbit HRP) was added with incubation for 30 min. After TBST rinse, the cells were incubated with DAB+Chromogen for 5 min, rinsed with water, and counterstained for 2 min with hematoxylin. Slides were washed with TBST and covered. Control cells were processed identically except that they were not exposed to the primary antibody. Slides were cover-slipped using pristine mount (Invitrogen, Carlsbad, CA). Chromogen abundance was quantified by quantitative immunohistochemistry as previously described (19, 20). Briefly, images were acquired using a Diagnostic Instruments SPOT RT
Digital Scanning Camera (Sterling Heights, MI) attached to a Nikon E600 microscope (Stamford, CT). The amount of chromogen per pixel was determined by subtracting the mathematical energy (EM) of the control slide from that in the homologous region of the experimental slide exposed to primary antibody. Chromogen quantity (EM) is expressed as energy units per pixels (eu/pix). Cytotoxicity Assays. Tumor cells were grown as monolayers in 25-cm2 tissue culture flasks using their respective cell culture medium. All media contained 10% fetal bovine serum. Incubation was at 37 °C under a 5% CO2:air atmosphere (standard conditions) for 5 days. The cells were harvested when in midlog growth, and their concentration was determined using a Coulter particle counter. An aliquot of the cell suspension was diluted in culture medium for delivery to a 96-well plastic plate at a range of 3000 to 10 000 per 100 µL media. Twenty-four hours later, sextuplicate wells were inoculated with either 100 µL per well of medium (controls), or 100 µL of medium containing PTX or a conjugate. Concentrations were adjusted to deliver 0.1 µg/mL of PTX equivalent to all treatment groups. After 1 h incubation, the wells were aspirated and refilled with 200 µL conjugate-free medium. Following an additional 120 h incubation under standard conditions, and removal of the floating nonviable cells by aspiration, the plate-attached viable cells were counted and normalized to the percent of untreated controls. The extent of cytotoxicity in treated wells as compared to the controls, were calculated using a Magellin software program (Dynex Technology, Chantilly, VA). Receptor-Blocking Assay. Tumor cells were cultured, in sextuplicate, in 96-well plates as described above. After a 24 h incubation, 100 µL of medium containing DMSO (control), or 100 µL of medium containing PTX, 6, BBN[6-14], BBN[614]+PTX, or BBN[6-14]+6 was added to sextuplicate wells. Additions were such that a 120 nM of PTX or 6, and 2400 nM of BBN[6-14] were delivered to each well. The BBN[614]+PTX or BBN[6-14]+6 were mixed immediately prior to the addition of the agents to their respective wells. After 1 h incubation at 37 °C, the medium was removed, the cells were washed 1× with PBS, and 200 µL drug-free medium was added to each well. Following an additional 120 h incubation period, the cells were washed 1× with PBS, then detached with trypsinEDTA and counted with a hemacytometer (Hausser Scientific, Horsham, PA). The cell counts were normalized to the percent of untreated control cells. The data were statistically analyzed using Microsoft Excel Software (Microsoft Corporation, Redmond, WA).
568 Bioconjugate Chem., Vol. 17, No. 3, 2006
Safavy et al.
Table 2. Cytotoxicitya of PTX-Di-BBN SDML Conjugates in Different Human Cancer Cell Lines as Compared to Those of PTX and Mono-Ligand Conjugatesb,c conjugate
structure
6
PTXGlu(BBN[6-14])2
9
PTXGlu(OH)BBN[6-14]
13
PTXPEGGlu(BBN[6-14])2
16
PTX(PEG)2Glu(BBN[6-14])2
20
PTXGlu(PEGBBN[6-14])2
21e
PTXBBN[6-14]
SKNASd
FADU
MO59J
JNPRSLT1
HuTu-80
87 ( 4.9 (59 ( 3.0) 43 ( 10.4 (59 ( 3.0) 70 ( 2.8 (55 ( 4.8) 23 ( 4.5 (67 ( 3.4)
89 ( 2.8 (87 ( 2.3)
64 ( 4.2 (58 ( 4.0)
86 ( 1.6 (31 ( 7.8)
93 ( 0.93 (77 ( 11.3)
55 ( 8.1 (26 ( 2.3)
90 ( 0.9 (84 ( 4.1)
16 ( 6.1 (31 ( 7.8)
29 ( 11.1 (77 ( 11.3) 16 ( 1.7 (85 ( 0.02)
22 ( 3.9 (51 ( 2.2)
33 ( 6.0 (51 ( 7.0) 56 ( 6.5 (87 ( 2.3) 26 ( 3.5 (85 ( 2.8)
50 ( 4.2 (58 ( 4.0)
a Defined as %growth inhibition ) 100% - %viable treated cells ( SD; the untreated controls were normalized to 100%. b See footnote 3. c Numbers in parentheses refer to %inhibition induced by unconjugated PTX at equimolar concentrations and in the same experiment. d SKNAS, neuroblastoma; FADU, squamous cell tumor of the pharynx; MO59J, glioblastoma multiform; JNPRSLT1, desmoplastic small cell tumor; HuTu-80, intestinal tumor. All of human origin. e Prepared the same procedure as used for the synthesis of 6 from 5.
Scheme 3
Scheme 4
RESULTS AND DISCUSSION
Scheme 5
The amino acid sequence of the peptide used in this work (BBN[6-14], 2, Figure 2) made possible the solution-phase synthesis of the final conjugates without side-chain protection.2 As the central branching scaffold, NR-(tert)-butyloxycarbonylL-glutamic acid (Boc-Glu, 3, Scheme 1) was used to which the BBN[6-14] segments were connected. A standard DCC/HOBT coupling reaction worked satisfactorily although some condition manipulations seemed to improve both the yield of product recovery and the product purity. Briefly, starting with a 2:1 mole ratio of 2:3, and a larger excess of HOBT/DCC with respect to Boc-Glu, it was noticed that a gradual increase in the molarities of 2 and the reagents resulted in better product yields. Removal of the Boc protection from compound 4, and condensation of the bis-peptide 5 with an equivalent of paclitaxel-2′-hemisuccinate, afforded the target conjugate 6. A mono-BBN conjugate (compound 9) was also prepared for studying the effect of BBN duplicity on cytotoxicity. Synthesis of this product was through the formation of Boc-Glu cyclic anhydride followed by the R-addition of BBN[6-14]. The preferential R-opening of the anhydride by the peptide was presumably due to the presence of the electron-withdrawing Boc group (21). Chromatographic and mass spectrometric analyses indicated formation of only one major product. The low water solubility of paclitaxel has been one of the disadvantages of this drug, which has necessitated the use of solubilizing excipients (e.g., cremophore EL, a poly-ethoxylated castor oil) in the clinically used formulations (22, 23). These additives have been reported to be the cause of allergic reactions and posttreatment toxicities (22, 23). We, and others, have reported the use of PEG as a solubilizing attachment to PTX (24) and its peptide conjugate (12). In the latter case, PEG was inserted between the drug and the peptide segments resulting in high overall aqueous solubility. Here, a PEG amino acid (from Fmoc-PEG-NHS, 10, Schemes 3-5) was used as the solubi-
lizing linker. Furthermore, to study the effect of positional isomerism on both solubility and biological activity, the PEG linker was inserted, separately, in both the claws and the tail regions. Conjugates with one (13, Scheme 3) or two (16, Scheme 4, and 20, Scheme 5) PEG linkers were synthesized. It should be noted that addition of only one PEG to each claw (conjugate 20) results in the formation of a regioisomer of compound 16. Analytical data for these readily water-soluble derivatives are presented in Table 1. Prior to the cytotoxicity experiments, the cell lines were screened for the presence of BBN-related receptors (BBNRs). Immunohistochemistry assays demonstrated the presence of GRP receptors, the mammalian counterpart of BBNR (25, 26) in four of the cell lines (Figure 3). The results of cytotoxicity assays (Table 2) indicated a superior activity for the scorpion conjugate, as compared to both free PTX and the mono-BBN[6-14] control (conjugate 21) in all tested cell lines.3 The MO59J cells, with the lowest GRPR expression (35 000 eu/ pix), showed a weaker response to the di-BBN conjugate than did the other, high-density lines. The human squamous cell tu-
2 Numbering based on the native bombesin sequence. This sequence includes an N-terminal tyrosine residue. Although not relevant to this report, the tyrosine would serve as a radioiodination site for the synthesis of radiolabeled analogues.
3 Selection of the cell lines of Table 2 is for elucidating the general trend of cytotoxicity of the conjugates. The cell lines have different sensitivity towards PTX, and therefore each column of the table has to be viewed individually.
Bioconjugate Chem., Vol. 17, No. 3, 2006 569
Communications
Figure 3. Density of GRPR expression in different human cancer cell lines as determined by immunohistochemistry. Each bar is the mean of values from four independent experiments. Error bars are too small to be visible.
Figure 5. Indiscriminate killing of FaDu cells by PTX and PTXGlu(BBN[6-14])2 (6) in the presence and absence of the blocker peptide BBN[6-14] (2) due to extreme PTX sensitivity to this cell line. Each bar shows the percentage of viable cells after treatment. Treated cell numbers normalized to the untreated control. Table 3. Conjugate:PTX Cytotoxicity Ratios for PTX-Di-BBN SDMLCs in Different Human Cancer Cell Lines as Compared to Those of Free PTX and Mono-Ligand Conjugatea conjugate
structure
6 9 13 16 20 21
PTXGlu(BBN[6-14])2 PTXGlu(OH)BBN[6-14] PTXPEGGlu(BBN[6-14])2 PTX(PEG)2Glu(BBN[6-14])2 PTXGlu(PEGBBN[6-14])2 PTXBBN[6-14]
a
Figure 4. Reduction of PTXGlu(BBN[6-14])2 (6) cytotoxicity due to receptor blockage by excess BBN[6-14] (2) in JNPRSLT1 human desmoplastic small cell tumor. Each bar shows the percentage of viable cells after treatment. The untreated controls were normalized to 100%.
mor of the pharynx, FADU, does not express BBN/GRP receptors (Figure 3) but is highly sensitive to paclitaxel. To verify the receptor-mediated drug delivery of conjugate 6, a blocking experiment was carried out using the JN-PRSLT-1 cell line (GRPR 203 000 eu/pix). Addition of a 20-fold molar excess of the free BBN[6-14] ligand (structure 2) reduced the cytotoxicity of 6 by about 45%whereas it did not have a significant effect on PTX cytotoxicity (Figure 4). On the other hand, when noGRPR FADU cells were used, both PTX and conjugate 6, with or without the blocker 2, killed the cells to the same extent (Figure 5), presumably due to the extreme sensitivity of this cell line to PTX. And for reasons not clear at this time, this sensitivity is reflected in the cytotoxicity of 6 but not with any other conjugate of Table 2. Activity enhancement due to the simultaneous presence of two BBN moieties in the same molecule was also demonstrated by the low relative cytotoxicity of 9. It should be noted that the higher activity of conjugate 9 against the other mono-BBN compound (21) might be due to a higher water solubility of the former under the experimental conditions. Separation of the ligand segment from the bulky PTX by the Glu spacer in 9 also might have reduced the PTX-resulted steric hindrance (SH) and facilitated the peptide-receptor binding. Addition of one PEG spacer to the tail region (compound 13) lowered the activity probably due to a more crowded environment around the molecule, and thereby, jeopardizing conjugate-receptor interactions (e.g., collision), in this case due to the large size of the PEG segments relative to those of the
JNPR- HuTuSKNAS FADU MO59J RLT1 80 1.5 0.73 1.3 0.34 0.43
1.02
0.65 0.64 0.31
1.1
0.86
2.8
1.2
2.1
1.1
0.52
0.38 0.19
Calculated from values of Table 2.
peptide moieties. This SH-caused deactivating effect was more apparent when addition of a second PEG in conjugate 16 led to a considerably lower activity. Incorporation of PEG segments into the claws (compound 20) caused deactivation with respect to both 6 and free PTX. At the same time, conjugate 20 was more cytotoxic than its regioisomer 16, indicating that a regional separation between the PEG segments, even when located in the proximity of the ligands, had less of a deactivating effect than a tandem arrangement. An outline of the cytotoxicity results, in the form of conjugate-to-PTX activity ratios, is shown in Table 3. Advantage of a multiligand approach in the design of receptor-targeted drug delivery molecules was demonstrated in this work by the synthesis of a di-bombesin-paclitaxel conjugate. With the exception of the highly PTX-sensitive FaDu cells, the SDML conjugate showed enhanced cytotoxicity when screened against both the unconjugated drug and mono-ligand conjugates, in a panel of GRPR-positive human cancer cell lines. The cytotoxicity was reduced with receptor blocking by the free ligand and increased with increases in GRPR density. On the basis of these results, further investigation of these conjugates, including antitumor activity studies, is warranted and is underway in this laboratory.
ACKNOWLEDGMENT Valuable assistance and contributions of Dr. Madhavi Chander and Mark Jones (Tapestry Pharmaceuticals), Marion C. Kirk, D. Ray Moore, Landon Wilson (UAB Comprehensive Cancer Center Mass Spectrometry Core Facility), and David Fisher (UAB Medical Education and Design Services) is greatly acknowledged.
570 Bioconjugate Chem., Vol. 17, No. 3, 2006
LITERATURE CITED (1) Guillemard, V., and Saragovi, H. U. (2004) Novel approaches for targeted cancer therapy. Curr. Cancer Drug Targets 4, 31326. (2) Ranson, M., and Jayson, G. (2005) Targeted antitumour therapys future perspectives. Br. J. Cancer 92 Suppl 1, S28-31. (3) Green, M. C., Murray, J. L., and Hortobagyi, G. N. (2000) Monoclonal antibody therapy for solid tumors. Cancer Treat. ReV. 26, 269-286. (4) Lin, M. Z., Teitell, M. A., and Schiller, G. J. (2005) The evolution of antibodies into versatile tumor-targeting agents. Clin. Cancer Res. 11, 129-138. (5) Garrett, M. D., and Workman, P. (1999) Discovering novel chemotherapeutic drugs for the third millennium. Eur. J. Cancer 35, 2010-30. (6) Stefanidakis, M., and Koivunen, E. (2004) Peptide-mediated delivery of therapeutic and imaging agents into mammalian cells. Curr. Pharm. Des. 10, 3033-44. (7) Fischman, A. J., Babich, J. W., and Strauss, H. W. (1993) A ticket to ride: Peptide radiopharmaceuticals. J. Nucl. Med. 34, 22532263. (8) Rogers, B. E., Manna, D. D., and Safavy, A. (2004) In vitro and in vivo evaluation of a 64Cu-labeled polyethylene glycol-bombesin conjugate. Cancer Biother. Radiopharm. 19, 25-34. (9) Safavy, A., Khazaeli, M. B., Qin, H., and Buchsbaum, D. J. (1997) Synthesis of bombesin analogues for radiolabeling with Rhenium188. Cancer (Suppl) 80, 2354-2359. (10) Burkhart, D. J., Kalet, B. T., Coleman, M. P., Post, G. C., and Koch, T. H. (2004) Doxorubicin-formaldehyde conjugates targeting alphavbeta3 integrin. Mol. Cancer Ther. 3, 1593-604. (11) Chau, Y., Tan, F. E., and Langer, R. (2004) Synthesis and characterization of dextran-peptide-methotrexate conjugates for tumor targeting via mediation by matrix metalloproteinase II and matrix metalloproteinase IX. Bioconjugate Chem. 15, 931-41. (12) Safavy, A., Raisch, K. P., Khazaeli, M. B., Buchsbaum, D. J., and Bonner, J. A. (1999) Paclitaxel derivatives for targeted therapy of cancer: Toward the development of smart taxanes. J. Med. Chem. 42, 4919-4924. (13) Weiner, R. E., and Thakur, M. L. (2005) Radiolabeled peptides in oncology: role in diagnosis and treatment. BioDrugs 19, 14563. (14) Zhang, H., Chen, J., Waldherr, C., Hinni, K., Waser, B., Reubi, J. C., and Maecke, H. R. (2004) Synthesis and evaluation of bombesin derivatives on the basis of pan-bombesin peptides labeled with indium-111, lutetium-177, and yttrium-90 for targeting bombesin receptor-expressing tumors. Cancer Res. 64, 6707-15.
Safavy et al. (15) Jones, D. S. (2005) Multivalent compounds for antigen-specific B cell tolerance and treatment of autoimmune diseases. Curr. Med. Chem. 12, 1887-904. (16) Kok, R. J., Schraa, A. J., Bos, E. J., Moorlag, H. E., Asgeirsdottir, S. A., Everts, M., Meijer, D. K., and Molema, G. (2002) Preparation and functional evaluation of RGD-modified proteins as alpha(v)beta(3) integrin directed therapeutics. Bioconjugate Chem. 13, 12835. (17) Terskikh, A. V., Le Doussal, J. M., Crameri, R., Fisch, I., Mach, J. P., and Kajava, A. V. (1997) “Peptabody”: A new type of high avidity binding protein. Proc. Natl. Acad. Sci. U.S.A. 94, 16631668. (18) Deutsch, H., Glinski, J., Hernandez, M., Haugwitz, R., Narayanan, V., Suffness, M., and Zalkow, L. (1989) Synthesis of congeners and prodrugs: Water soluble prodrugs of taxol with potent antitumor activity. J. Med. Chem. 32, 788-792. (19) Matkowskyj, K. A., Cox, R., Jensen, R. T., and Benya, R. V. (2003) Quantitative immunohistochemistry by measuring cumulative signal strength accurately measures receptor number. J. Histochem. Cytochem. 51, 205-14. (20) Matkowskyj, K. A., Schonfeld, D., and Benya, R. V. (2000) Quantitative immunohistochemistry by measuring cumulative signal strength using commercially available software photoshop and matlab. J. Histochem. Cytochem. 48, 303-12. (21) Lloyd-Williams, P., Albericio, F., and Giralt, E. (1997) Chemical approaches to the synthesis of peptides, CRC Press, Boca Raton, FL. (22) Dorr, R. T. (1994) Pharmacology and toxicology of Cremophor EL diluent. Ann. Pharmacother. 28, S11-S14. (23) Sharma, A., Mayhew, E., Bolcsak, L., Cavanaugh, C., Harmon, P., Janoff, A., and Bernacki, R. J. (1997) Activity of paclitaxel liposome formulations against human ovarian tumor xenografts. Int. J. Cancer 71, 103-107. (24) Greenwald, R., Pendri, A., and Bolikal, D. (1995) Highly water soluble-taxol derivatives: 7-poly(ethylene glycol) carbamates and carbonates. J. Org. Chem. 60, 331-336. (25) Cuttitta, F., Carney, D. N., Mulshine, J., Moody, T. W., Fedorko, J., Fischler, A., and Minna, J. D. (1985) Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature 316, 823-6. (26) Kroog, G. S., Jensen, R. T., and Battey, J. F. (1995) Mammalian bombesin receptors. Med. Res. ReV. 15, 389-417. BC050224C
