Radiolabeled Divalent Peptidomimetic Vitronectin Receptor

Jun 19, 2007 - Judit Bartis,§ Charles Ellars,| Dave Onthank, Padmaja Yalamanchili, Stuart Heminway,. ⊥. Paula Silva,. Simon Robinson, Joel Lazewats...
0 downloads 0 Views 562KB Size
Bioconjugate Chem. 2007, 18, 1266−1279

1266

Radiolabeled Divalent Peptidomimetic Vitronectin Receptor Antagonists as Potential Tumor Radiotherapeutic and Imaging Agents Thomas D. Harris,* Edward Cheesman, Anthony R. Harris, Richard Sachleben,† D. Scott Edwards, Shuang Liu,‡ Judit Bartis,§ Charles Ellars,| Dave Onthank, Padmaja Yalamanchili, Stuart Heminway,⊥ Paula Silva, Simon Robinson, Joel Lazewatsky, Milind Rajopadhye,3 and John Barrett# Discovery Research, Bristol-Myers Squibb Medical Imaging, 331 Treble Cove Road, North, Billerica, Massachusetts 01862 . Received January 2, 2007; Revised Manuscript Received May 1, 2007

The integrin receptor Rvβ3 is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. A radiolabeled Rvβ3 antagonists belonging to the quinolin-4-one class of peptidomimetics (TA138) was previously shown to exhibit high affinity for integrin Rvβ3 and high selectivity versus other integrin receptors. 111In-TA138 exhibited high tumor uptake in the c-neu Oncomouse mammary adenocarcinoma model and produced excellent scintigraphic images. This study describes the synthesis of eight divalent versions of TA138 and their evaluation as potential tumor radiotherapeutic agents. The two main variables in this study were the length of the spacer bridging the biotargeting moieties and the total negative charge of the molecules imparted by the cysteic acid pharmacokinetic modifiers. Receptor affinity was evaluated in a panel of integrin receptor affinity assays, and biodistribution studies using the 111In-labeled derivatives were carried out in the c-neu Oncomouse model. All divalent agents maintained the high receptor affinity and selectivity of TA138, and six of the eight 111In derivatives exhibited blood clearance that was faster than 111In-TA138 at 24 h postinjection (PI). All divalent agents exhibited tumor uptake and retention at 24 h PI that was higher than 111In-TA138. Tumor/organ ratios were improved for most of the divalent agents at 24 h PI in critical nontarget organs marrow, kidney, and liver, with the agents having intermediate-length spacers (29-43 Å) showing the largest improvement. As an example, 111In-15 showed tumor uptake of 14.3% ID/g at 24 h PI and tumor/organ ratios as follows: marrow, 3.24; kidney, 7.29; liver, 8.51. A comparison of therapeutic indices for 90Y-TA138 and 177Lu-15 indicate an improved therapeutic index for the divalent agent. The implications for radiotherapeutic applications and the mechanism of this multivalent effect are discussed.

INTRODUCTION Integrins are a large family of transmembrane heterodimeric glycoprotein cell surface receptors formed by associations between R and β subunits. Integrins are important facilitators of cell-cell and cell-matrix interactions, and are also involved in cell signaling events (1). This has made integrins an important area of study in cancer metastasis and angiogenesis research (2-5). The integrin Rvβ3 (vitronectin receptor or VnR) is generally expressed at low levels on normal tissues and quiescent blood vessels, but is significantly upregulated on the neovasculature found in tumors and in wound repair (6-9) and is also present on certain tumor cells, including melanoma, kidney, breast, and prostate, and squamous cell carcinoma (10-13). This differential expression has made the vitronectin receptor a promising target in anti-angiogenic research (14, 15). For example, antagonists of integrin Rvβ3 have been demonstrated to inhibit tumor angiogenesis and disrupt metastasis in a number of models (16-19). We and others have utilized integrin Rvβ3 antagonists for selective localization of diagnostic and therapeutic radionuclides in the vicinity of malignant tumors. Cyclic RGD peptides labeled * To whom correspondence should be addressed. Phone: 603-8930031. Fax: 603-898-4605. E-mail: [email protected]. † Present address: Momenta Pharmaceuticals, Cambridge, MA. ‡ Present address: Division of Nuclear Pharmacy, Purdue University, West Lafayette, IN. § Present address: ArQule, Woburn, MA. | Present address: AEA Technology QSA, Burlington, MA. ⊥ Present address: Astra Zeneca Pharmaceutical, Wilmington, DE. 3 Present address: VisEn Medical Inc., Woburn, MA. # Present address: Molecular Insight, Cambridge, MA.

with 18F and 64Cu for PET imaging (20-22) and with 99mTc and 111In for SPECT imaging (23-26) have been shown to exhibit selective tumor uptake in a variety of murine tumor models, and a 18F-labeled RGD peptide has recently been evaluated in 19 human patients with solid tumors (27). The use of radiolabeled peptides to image tumor angiogenesis has recently been reviewed (28-30). Our goal in this area was the development of an integrin Rvβ3 antagonist platform that could be utilized to deliver both imaging and radiotherapeutic isotopes to tumors. While many radiolabeled RGD peptides have demonstrated potential as tumor imaging agents, none have been able to combine the high tumor uptake and retention and low uptake in critical nontarget tissues required of a radiotherapeutic agent (29). We reasoned that peptidomimetics having high affinity and selectivity for integrin Rvβ3 offered the best opportunity to deliver diagnostically and therapeutically useful quantities of radionuclides to tumors while minimizing dose to nontarget organs. We recently described the synthesis and evaluation of a series of peptidomimetic antagonists to integrin Rvβ3 based on a substituted quinolin-4-one ring system (31, 32). The agent providing the best combination of tumor uptake and tumor/organ ratios was 111In-TA138 (Figure 1). This agent uses 2-aminoimidazole and a 2,3-diaminopropionate (DAP) arylsulfonamide as mimics of the guanidine and carboxylate groups of the RGD sequence, respectively. Macrocyclic chelator DOTA allows for chelation to both imaging (111In) and therapeutic (90Y, 177Lu) radionuclides, and is conjugated to the aryl sulfonamide group through a short spacer that includes cysteic acid as a pharmacokinetic modifier (PKM). 111In-TA138 was evaluated in the c-neu Oncomouse model, providing tumor uptake of 9.39% ID/g at 2 h postinjection (PI) and selected tumor/organ ratios at 2 h PI as follows: blood, 17.5; muscle, 17.1; liver,

10.1021/bc070002+ CCC: $37.00 © 2007 American Chemical Society Published on Web 06/19/2007

Rvβ3 Antagonists as Tumor Radiotherapeutic Agents

Bioconjugate Chem., Vol. 18, No. 4, 2007 1267 Table 1. Structural Features of Divalent Agents Shown in Scheme 1

Figure 1. Structures of monovalent parent TA138 and

111

In-TA138.

compd

x

y

z

no. spacer atomsa

TA138 13 14 15 16 17 18 19 20

2 0 0 1 1 1 2 2 2

na 0 0 0 0 0 0 1 2

na 2 4 0 1 2 0 0 0

na 23 23 29 29 29 35 73 111

spacer lengthb

no. cysteic acids

∆ chargec

na 29 29 36 36 36 43 89 134

2 2 4 2 3 4 4 4 4

0 -2 0 -1 -2 -2 -2 -2

a Number of atoms between phenyl rings of arylsulfonamide groups. Maximum spacer length calculated using an average interatomic distance of 1.2 Å, which takes into account the different bond angles and hybridization states of the atoms in the spacer. c Relative to TA138.

b

4.83; kidney, 4.04; bone 6.01. 111In-TA138 exhibited slow washout from the tumors, decreasing to 3.46% ID/g at 24 h PI. These are excellent biodistribution properties from the viewpoint of an imaging agent, but they leave room for improvement for a radiotherapeutic agent. We were especially interested in a further slowing of tumor washout and in improving the tumor/organ ratios for critical nontarget organs such as kidneys and bone marrow. One technique that is commonly employed to improve receptor affinity and retention is polyvalency. Our early work with cyclic RGD peptides has shown that in the c-neu Oncomouse model (33), and in the OVCAR-3 xenograft model (23, 24), divalent cyclic RGD peptide Rvβ3 antagonists give superior tumor uptake and retention compared to the cyclic monovalent peptides. This polyvalency effect has also been noted by others for cyclic RGD peptides (34-36), for R-MSH receptor peptides (37), and for Tuftsin antagonist peptides (38). Herein, we report the complete details of the synthesis of eight divalent analogues of TA138, along with receptor affinity and biodistribution data of the 111In-radiolabeled analogues. The general structures of these compounds are shown in Figure 2. The length of the spacer between the two biotargeting groups was varied over a wide range by the number and position of the cysteic acid PKMs and by the number of repeating PEG units, as shown in Table 1. Varying the total number of cysteic acid groups also provided control of the total charge on the molecule.

EXPERIMENTAL SECTION General Experimental. All reactions were carried out under a dry nitrogen atmosphere at ambient temperatures, unless otherwise noted. All chemicals and solvents (reagent grade) were

used as supplied from the vendors cited without further purification, unless otherwise noted. Solutions were concentrated under reduced pressure on a rotary evaporator, with final drying, if required, under high vacuum (95% radiochemical purity according to a previously published procedure (41). This procedure is included in the Supporting Information. 177Lu-15 was prepared in >95%

radiochemical purity according to a previously published procedure (42). This procedure is included in the Supporting Information. In Vivo Biodistribution Study. Biodistribution in the c-neu Oncomouse mammary adenocarcinoma model was carried out according to a previously published procedure (41) as described in the Supporting Information. Scintigraphic Image Study. Scintigraphic images of 111InTA138 and 111In-15 in the c-Neu Oncomouse model were acquired according to a previously published procedure (31). This procedure is reproduced in the Supporting Information.

RESULTS Synthesis of Divalent rvβ3 Antagonists. The syntheses of vitronectin receptor antagonists 1, 2, and 3, having zero, one, and two cysteic acid PKMs, respectively, were described previously (41). The general synthetic route from these three

Rvβ3 Antagonists as Tumor Radiotherapeutic Agents

starting materials to the divalent radiopharmaceuticals reported here is shown in Schemes 1 and 2. Conjugation of 1, 2, and 3, with 0.5 molar equiv of Boc-Glu(OTfp)-OTfp in DMF gave the bis-conjugates in good to excellent yields. Treatment of these conjugates with 1:1 TFA/DCM removed the Boc protecting group from the glutamyl residue, while leaving intact the trityl protecting group on the imidazole rings, to give 4, 5, and 6 in purified yields of 43%, 74%, and 94%, respectively. Compound 4 was used to synthesize DOTA conjugates 13 and 14, having two and four cysteic acid PKMs, respectively. These compounds are related in that the cysteic acids are conjugated to the glutamic acid R-amine, between the glutamic acid branch point and DOTA. Thus, the compounds have identical spacing between the two biotargeting groups but differ in their lipophilicity and charge. The cysteic acids were added in stepwise fashion using Boc-L-cysteic acid and PyBOP in DMF for the conjugation reactions and 1:1 TFA/DCM for Boc removal. Two coupling/ deprotection cycles gave compound 9 in 60% yield over four reactions. Triturations were used to remove impurities in three of the four synthetic steps, with one HPLC purification used for final purification prior to DOTA conjugation. In a similar fashion, 9 was converted to tetra-cysteic acid derivative 10 in 55% overall yield. Conjugation of the bis- and tetra-cysteic acid derivatives to DOTA(Ot-Bu)3-OH was accomplished using HBTU in DMF. Global deprotection using 97:3 TFA/Et3SiH at 70 °C gave final products 13 and 14 in 31% and 12% yield, respectively. The low yields for these final two steps were due in large part to very challenging HPLC purifications that necessitated taking small heart cuts from the product peaks. Compound 5 was used to synthesize a trio of DOTA conjugates, 15, 16, and 17, having a total of two, three, or four cysteic acid PKMs, respectively. All three of these compounds have a single cysteic acid PKM on each arm of the spacer, and either zero, one, or two cysteic acid PKMs conjugated to the glutamic acid R-amine. As a group, they have identical spacing between the biotargeting groups but differ in their lipophilicity and charge. Placing cysteic acids in the chain linking the biotargeting groups adds a total of six atoms to the spacer length. Conjugation of DOTA(Ot-Bu)3-OH directly to 5 followed by global deprotection gave 15 in 42% yield. Reaction of 5 with Boc-cysteic acid and HBTU, followed by removal of Boc using TFA/DCM, gave 11 in 93% yield. Addition of a second cysteic acid PKM to 11 using the same chemistry gave 12 in 53% yield. Compounds 11 and 12 were converted to DOTA conjugates 16 and 17 in 22% and 45% purified yields, respectively. Conjugation of DOTA(Ot-Bu)3-OH directly to 6 followed by global deprotection gave 18 in 28% yield. The final two compounds of this study utilized Suc-PEG spacers to provide a large additional increase in the distance between the biotargeting groups. The spacer groups were added to Boc-glutamic acid in stepwise fashion as shown in Scheme 2. Conjugation of Boc-glutamic acid with 2 equiv of Cbzprotected PEG diamine 21, followed by catalytic hydrogenolysis, and reaction of the resulting amine with succinic anhydride gave dicarboxylic acid 22a in 67% overall yield. A second Suc-PEG spacer was added to each arm of 22a using the same sequence of reactions to give 22b in 12% overall yield. Conjugation of 22a and 22b with bis-cysteic acid Rvβ3 receptor antagonist 3, followed by removal of the Boc protecting groups, gave divalent antagonists 7 and 8 in 50% and 55% yields, respectively. HBTUmediated conjugation of 7 and 8 to DOTA(Ot-Bu)3-OH followed by global deprotection gave 19 and 29 in 30% and 40% yields, respectively. Receptor Binding Assays. Affinity assays toward four integrin receptors were used to measure the potency and selectivity of the divalent Rvβ3 antagonist described here and to compare them to TA138. Our principle receptor Rvβ3 assay

Bioconjugate Chem., Vol. 18, No. 4, 2007 1273 Table 2. In Vitro Receptor Affinity of Quinolin-4-one DOTA Conjugates IC50, nM ((SEM)a compd

Rvβ3 (293β3)b

RIIbβ3 (PRPc)b

Rvβ5 (SKB)d

R5β1 (Jurkat)d

13 14 15 16 17 18 19 20

17 ( 5 40 ( 24 9(6 57 ( 16 42 ( 17 58 ( 18 6(4 15 ( 11

>10 000 >10 000 >10 000 5000 4400 3600 >10 000 >10 000

>10 000 >10 000 >10 000 >10 000 >10 000 >10 000 >10 000 >10 000

4800 NDe 2000 6100 >10 000 >10 000 2900 2100

a Performed in triplicate. b Assay performed according to procedures outlined in ref 42. c Platelet-rich plasma. d Assays performed according to procedures outlined in ref 31. e Not determined.

was the 293β3-fibrinogen (Rvβ3-mediated) adhesion assay (43). Receptor selectivity was measured using the platelet aggregation RIIbβ3 assay (43), the human SKBR3 cell-vitronectin (Rvβ5mediated) adhesion assay (31), and the human lymphocyte (Jurkat)-fibronectin (R5β1-mediated) adhesion assay (31). The affinity data in Table 2 show that all compounds have high affinity toward receptor Rvβ3, with IC50 values of 6-58 nM in the 293β3 assay. They also show good to excellent selectivity versus receptors RIIbβ3, Rvβ5, and R5β1. Radiolabeling. All DOTA conjugates of this study formed stable complexes with 111In. The complexes were prepared according to a previously published procedure and isolated in >95% radiochemical purity (41). In Vivo Biodistribution Study. The biodistribution properties of the compounds in this study were evaluated in the c-neu Oncomouse mammary adenocarcinoma model according to a previously published procedure (41). Compounds were evaluated as the 111In complexes and administered at 2 mCi/Kg of body weight. Mice were euthanized and dissected at 2 and 24 h postinjection (PI), and activity in the organs and tumors was counted on a gamma counter. The biodistribution data are presented in Figures 3 and 4, expressed as % of injected dose/ gram of tissue (%ID/g). All biodistribution data are decaycorrected. Tumor uptake was rapid for all divalent agents, ranging from 4.81% ID/g at 2 h PI for 111In-20 to 9.99% ID/g at 2 h PI for 111In-17. The parent monovalent agent, 111InTA138, displayed slow washout from the tumor, decreasing from 9.39% ID/g at 2 h PI to 3.46% ID/g at 24 h PI. In contrast, tumor uptake for the divalent agents either remained constant or increased between 2 and 24 h, with 111In-15 showing the highest uptake at 14.3% ID/g at 24 h PI. This continuous uptake was also apparent in the colon and uterus, where the majority of divalent agents displayed increased uptake between 2 and 24 h. As shown in Figures 3 and 4, uptake in the kidneys, marrow, and liver decreased over 24 h for all agents. Uptake in organs not shown in the figures was low for all agents as follows at 24 h PI: eyes, muscle, heart, and bile,