The potential to heighten the efficacy of antiangiogenic agents was explored in this study based on active targeting of tumor cells overexpressing the gonadotropin-releasing hormone receptor (GnRH-R). The rational design pursued focused on five analogues of a clinically established antiangiogenic compound (sunitinib), from which a lead candidate (SAN1) was conjugated to the targeting peptide [d-Lys6]-GnRH, generating SAN1GSC. Conjugation of SAN1 did not disrupt any of its antiangiogenic or cytotoxic properties in GnRH-R–expressing prostate and breast tumor cells. Daily SAN1GSC treatments in mouse xenograft models of castration-resistant prostate cancer resulted in significant tumor growth delay compared with equimolar SAN1 or sunitinib alone. This efficacy correlated with inhibited phosphorylation of AKT and S6, together with reduced Ki-67 and CD31 expression. The superior efficacy of the peptide–drug conjugate was also attributed to the finding that higher amounts of SAN1 were delivered to the tumor site (∼4-fold) following dosing of SAN1GSC compared with equimolar amounts of nonconjugated SAN1. Importantly, treatment with SAN1GSC was associated with minimal hematotoxicity and cardiotoxicity based on measurements of the left ventricular systolic function in treated mice. Our results offer preclinical proof-of-concept for SAN1GSC as a novel molecule that selectively reaches the tumor site and downregulates angiogenesis with negligible cardiotoxicity, thus encouraging its further clinical development and evaluation. Cancer Res; 76(5); 1181–92. ©2015 AACR.

The concept of generating novel cancer therapeutics that target specific cell surface receptors has been a driving force of drug development over the past few years (1–3). This idea is based on the evidence that certain cell surface receptors in malignant cells are either mutated or overexpressed (e.g., EGFR, HER2/neu, GnRH-R), compared with normal cells (4–6) and that these alterations provide “druggable” opportunities. We recently demonstrated the successful targeting of gonadotropin-releasing hormone receptor (GnRH-R) in GnRH-R–positive prostate cancer using a conjugate of a GnRH peptide linked to the cytotoxic drug gemcitabine (7). These studies were inspired by evidence of a doxorubicin-based conjugate (AN152) that has now advanced to the phase III clinical trials for various solid malignancies (8, 9).

The GnRH-R is primarily located in the pituitary gland (10, 11) but upregulation of its expression has been reported in prostate, breast, and other cancers (10, 12) which persists despite prolonged exposure to GnRH agonists (6). Interestingly, an antiproliferative role has also been reported for GnRH, providing additional opportunities for the treatment of tumors by directly affecting cancer cells apart from the pituitary (13, 14).

Tumor growth depends on the ability of new vessels to sprout in a process called angiogenesis. Numerous antiangiogenic agents have been developed which inhibit the phosphorylation of certain receptors like the VEGFR types 1/2 and platelet-derived growth factor receptors (PDGFR-α/β; ref. 15). A typical example of a clinically successful antiangiogenic agent is sunitinib, an orally available small molecule that inhibits phosphorylation of several receptor tyrosine kinases (RTK) including VEGFRs-1/2, PDGFR-α/β, KIT, and FLT3, with an average IC50 below 100 nmol/L (16, 17). Clinical trials of sunitinib in tumors, such as prostate (18) and breast cancer (19) were initiated on the basis of successful preclinical evidence (16, 20) but were halted in phase III, despite prolongation of progression-free survival, due to the lack of statistically significant overall survival in patients. Experience with sunitinib in the clinic demonstrated that a considerable subset of sunitinib-treated patients develop left ventricular (LV) dysfunction and overt heart failure (21, 22).

On the basis of the findings of the above studies, we surmised that increased specificity of sunitinib delivery at the site of action through conjugation to a targeting agent may lead to improved efficacy while minimizing peripheral toxicities. We decided to pursue this concept by generating novel GnRH-based molecules conjugated to sunitinib, with the prospect of exploiting the additional antiproliferative effects of the GnRH peptide itself.

Conjugation of sunitinib to a targeting group is challenging due to the possibility of altering the potency and pharmacokinetics of the pharmacophore. Few groups have experimented with the generation of sunitinib analogues (23–29), based on cocrystallization studies (30) that revealed indolin-2-one core as the moiety of the drug involved in kinase inhibition. Substitutions at the C4 position might serve as a handle for improving pharmaceutical properties as shown by toceranib (26). On the basis of the above structure–activity relationship studies, some sunitinib analogue conjugates have been synthesized and evaluated either as imaging tools (27) or as lysozyme-based targeting agents (25), but to our knowledge no conjugation to a peptide moiety has been reported.

Herein, we present the results of preclinical studies for a GnRH–sunitinib–based conjugate (called SAN1GSC), generated after the evaluation of various novel sunitinib analogues (designated as SANs 1-5). The analogues were designed to be linkable to a variety of peptides while maintaining their antiangiogenic properties, a desired feature that expands their translational applications. Enhanced efficacy, minimal toxicity, and improved biodistribution properties of SAN1GSC were shown, in comparison with its unconjugated derivative (SAN1) or sunitinib.

Chemicals, synthesis, and characterization of compounds

Sunitinib was purchased from Selleckchem and [d-Lys6]-GnRH from Proteogenix. All other chemicals and biologicals, unless stated otherwise, were purchased from Sigma-Aldrich. The synthesis of SAN1-SAN5 (Fig. 1A) and of SAN1GSC (Fig. 4A) is presented in the Supplementary Information section (Supplementary Information Sections S1 and S3). All molecules were analyzed by mass spectrometry (MS) to obtain the key spectral features necessary for their quantification in blood and tissue as described previously (7, 31). Total polar surface area and clogP values were predicted by Chemdraw Ultra (v10, PerkinElmer Informatics).

Figure 1.

Synthetic route of sunitinib analogues and physicochemical properties. A, synthesis of sunitinib analogues 1–5 from the available acid intermediate 1. Reagents and conditions: DMF, EDCI, HOBt, NEt3, room temperature (a); CH2Cl2, TFA, room temperature (b). B, key properties and MS conditions for the sunitinib analogues.

Figure 1.

Synthetic route of sunitinib analogues and physicochemical properties. A, synthesis of sunitinib analogues 1–5 from the available acid intermediate 1. Reagents and conditions: DMF, EDCI, HOBt, NEt3, room temperature (a); CH2Cl2, TFA, room temperature (b). B, key properties and MS conditions for the sunitinib analogues.

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Computational methods

Detailed docking and molecular dynamic analysis for all the compounds was performed as described in the Supplementary Information Section S2.

In vitro evaluation of compounds

The trans-phosphorylation activity of VEGFR-2, PDGFR-β, KIT, FLT3, ribosomal protein S6 kinase (RPS6KB1), and EGFR was evaluated as described before (17). Detailed methods and a list of the kinases used are available in the Supplementary Information Section S4. Cellular inhibition of autophosphorylation of VEGFR-2 and PDGFR-β was performed as described previously (32). Detailed methods are available in the Supplementary Information Section S5. For cellular studies, cells were used within 6 months of purchase or authentication and were cultured as instructed by the ATCC (LGC Standards). The HCC1954 cell line was obtained from ATCC and human umbilical vein endothelial cell (HUVEC) from Life Technologies. The DU145, PC3, and NIH/3T3 cells were authenticated by short tandem repeat profiling (Microsynth). WPE1-NB26-3 cells were a kind gift from Prof. Robert Millar (University of Pretoria, Pretoria, South Africa).

Pharmacokinetic analysis

All animal procedures were approved by the Bioethical Committee of BRFAA based on the European Directive 86/609. Equimolar (17 μmol/kg) dosing solutions for each compound were prepared in 20% 2-hydroxypropyl β-cyclodextrin in water (HP-b-CD), except for SAN1GSC that was solubilized in saline. All compounds were administered intraperitoneally in male NOD/SCID mice (8–10 weeks, n = 4, from Charles River Laboratory) and blood samples were collected and prepared as described previously (7).

In vivo efficacy of sunitinib, SAN1, and SAN1GSC

NOD/SCID mice were injected in each flank with 3 × 106 DU145 cells, a castration-resistant prostate cancer (CRPC) cell line. Treatment was initiated when tumors reached 200 mm3 by daily i.p. administrations of sunitinib or equimolar doses of SAN1 (100 μmol/kg). Control mice received saline or HP-b-CD. In separate experiments, mice were treated with equimolar amounts (9.17 μmol/kg) of SAN1GSC, SAN1, and [d-Lys6]-GnRH. Mice were weighed regularly and phenotypic signs of discomfort were monitored as described in the Supplementary Information Section S6. Experiments were terminated after approximately 20 days, at 1 hour or 2 hours after the last administered dose by euthanizing the animals under isoflurane anesthesia. Tumors were excised, weighed, and prepared for histopathology, IHC, kinase activity, and quantification of compounds by LC/MS-MS.

IHC

Excised tumors were fixed in neutral buffered formalin, paraffin embedded, sectioned, and stained against CD31, Ki-67, pS6, and pRPS6KB1. Microvessel density was assessed by counting the number of CD31-positive vessels in a 40× microscope field in a blinded fashion and presented as the amount of blood vessels/mm2. Cell proliferation was measured by counting Ki-67–positive cells in a 40× microscope field. Images were acquired by a Leica DFC350-FX camera mounted on a Leica DMLS2 microscope.

In vivo kinase activity

The PathScan RTK signaling array kit (Cell Signaling Technology) was used as per the manufacturer's direction to detect the phosphorylation status in supernatants from tumor extracts (n = 10 for each treatment). Images were analyzed with ImageJ software (v1.28) by loading the image as a grayscale picture. Each kinase array dot was manually selected, normalized, and an average intensity for each kinase was calculated as described previously (33).

Toxicity evaluation

Cardiotoxicity was evaluated through echocardiography as described previously (34) and is mentioned in Supplementary Information Section S7. Hematotoxicity was assessed as described in the Supplementary Information Section S8.

Statistical analyses

Statistical analyses and calculation of all IC50s were performed by SigmaPlot 12 software. Statistical significance was determined using the Student two-tailed, two-sample unequal variance distribution t test.

Synthesis of SANs

The synthetic route for SANs 1-5 is shown in Fig. 1A. These modified versions of sunitinib maintain the RTK inhibition indolin-2-one core while, in addition, they provide a handle for conjugation to a targeting peptide. Figure 1B depicts the MS features as well as some key properties for all SANs. Although the lipophilicity of sunitinib variants (SANs 1-5) was decreased compared with sunitinib, the polar surface area remained unaffected (<100 Å2) for all SANs, a parameter known to influence cell membrane permeability (35). A representative mass spectrum and LC/MS-MS chromatogram for a chosen analogue (SAN1) can be found in Supplementary Information Section S2.

Molecular docking calculations

Molecular docking calculations were performed for the synthesized SANs based on the catalytic domain of three key kinase receptors: VEGFR-2, PDGFR-β, and KIT. Estimated inhibition constants Ki (Fig. 2A) were in the low nanomolar range, similar to the estimated values of sunitinib. A representative molecular model of SAN1 complexed with VEGFR-2 is shown in Fig. 2A and Supplementary Information Section S3.

Figure 2.

In silico, in vitro, and pharmacokinetic evaluation of the synthesized SANs. A, docking for sunitinib and SAN1–5 with estimated inhibition constants (Ki in nmol/L). Molecular model of the VEGFR-2 complex with SAN1. B, summary of in vitro kinase activity in multiple RTKs in the presence of sunitinib or SANs 1–5, (±SD). C, cell-based autophosphorylation assay for PDGFR-β and VEGFR-2 for sunitinib or SANs 1–5, (±SD). D, MTT cytotoxicity assay in a panel of cell lines, (±SD). E, pharmacokinetic evaluation of the most potent SANs versus sunitinib in mice. N.D., not determined; N.A., not active.

Figure 2.

In silico, in vitro, and pharmacokinetic evaluation of the synthesized SANs. A, docking for sunitinib and SAN1–5 with estimated inhibition constants (Ki in nmol/L). Molecular model of the VEGFR-2 complex with SAN1. B, summary of in vitro kinase activity in multiple RTKs in the presence of sunitinib or SANs 1–5, (±SD). C, cell-based autophosphorylation assay for PDGFR-β and VEGFR-2 for sunitinib or SANs 1–5, (±SD). D, MTT cytotoxicity assay in a panel of cell lines, (±SD). E, pharmacokinetic evaluation of the most potent SANs versus sunitinib in mice. N.D., not determined; N.A., not active.

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Biochemical kinase assays

The potency of all SANs was experimentally evaluated in biochemical assays against purified VEGFR-2, PDGFR-β, KIT, and FLT3 and presented in Fig. 2B. All SANs inhibited the specific RTK targets with IC50 values ranging from 20 to 150 nmol/L comparable with sunitinib. The EGFR served as a negative control (concentrations up to 10 μmol/L did not inhibit EGFR phosphorylation).

Cellular ligand–dependent phosphorylation assay

The inhibition of phosphorylation of VEGFR-2 and PDGFR-β, was further confirmed in HUVEC and NIH/3T3 cells, using a Western blot analysis based cellular ligand–dependent phosphorylation assay. The calculated cellular IC50 value for sunitinib was 24 ± 12 nmol/L (for VEGFR-2) and 72 ± 19 nmol/L for PDGFR-β, respectively. Similar potencies were observed for all SANs with cellular IC50 values ranging from 52 to 180 nmol/L (Fig. 2C).

Cytotoxicity studies

The antiproliferative effect of the five SANs was assessed in three GnRH-R–positive prostate cancer cell lines (DU145, PC3, WPE1-NB26-3) and one GnRH-R–positive breast cancer cell line (HCC1954), in HUVEC and NIH/3T3 cells. Results and comparative IC50 values are presented in Fig. 2D. SAN1 proved highly potent in HUVEC and DU145 cell lines (IC50s: 8 ± 4 μmol/L and 11 ± 2 μmol/L, respectively), SANs 2-4 showed intermediate efficacies whereas SAN5 appeared inactive (IC50 > 100 μmol/L) and was discarded from further evaluation.

Pharmacokinetic evaluation

The most promising molecules (SANs 1–4) were evaluated for their pharmacokinetic parameters in mice. Sunitinib maximum blood concentrations were achieved at 0.25 hours (1.5 μmol/L) with an AUC0–8h of 2.51 hours × μmol/L. Similarly, SANs 1–4 were rapidly absorbed reaching highest blood concentrations averaging in the range of 0.5 to 2.1 μmol/L at 0.25 to 1 hour after dosing (Fig. 2E). After dosing, concentrations of SAN1 peaked at 0.25 hour with an AUC0–8h: 1.11 hours × μmol/L. Signs of discomfort were observed in mice that received SAN2, discouraging further evaluation. According to the biochemical assays, compound concentrations of approximately 100 nmol/L were needed for the inhibition of the target kinases (Fig. 2A–C). After intraperitoneal dosing, such levels could be sustained for 4 hours postdose for all the molecules tested. A single intraperitoneal administration of an efficacious dose of sunitinib (100 μmol/kg) was the point of reference for subsequent efficacy studies in mice (Fig. 2E). On the basis of the described pharmacokinetic and biologic readouts combined with its facile synthetic route, SAN1 was selected as the lead candidate compound for follow-up efficacy studies in mice.

In vivo antitumor efficacy of SAN1

Mice bearing established DU145 tumors were treated with each molecule at 100 μmol/kg/day (equivalent to 40 mg/kg). A significant tumor growth delay was observed in all mice treated with sunitinib (Fig. 3A), with the average tumor size at day 19 at 312 ± 123 mm3 and an average tumor weight of 0.20 ± 0.08 g compared with 1,181 ± 105 mm3 (P < 0.0001) and 0.87 ± 0.51 g (P = 0.005) for vehicle-treated mice (Fig. 3B). Tumor growth delay was also observed in DU145 xenografted mice treated with equimolar SAN1 (Fig. 3A), with an average tumor size at day 19 of 455 ± 137 mm3 (P < 0.001 compared with vehicle-treated mice). The corresponding average tumor weight at sacrifice was 0.37 ± 0.19 g for SAN1 (Fig. 3B, P = 0.005 compared with vehicle-treated mice). Concentrations of sunitinib and SAN1 in blood and tumor tissue were measured at 2 hours after the final dose. Blood concentrations of the sunitinib were 4.7 ± 0.1 μmol/L, whereas levels at tumor tissue were 11.0 ± 5.0 μmol/L (Fig. 3C). The values for SAN1 in the tumor tissue were significantly lower at 0.4 ± 0.2 μmol/L, but exceeded the IC50 level threshold (100 nmol/L) determined for the inhibition of phosphorylation of the target kinases. It should be noted that sunitinib is more lipophilic compared with SAN1 (as evident in their clogP values), a property that explains the higher drug accumulation in the tumor tissue over the treatment period.

Figure 3.

Efficacy of SAN1 and sunitinib in NOD/SCID mice xenografted with the CRPC DU145 cell line. A, tumor growth inhibition. Mice were dosed (intraperitoneally) daily with SAN1 (100 μmol/kg), sunitinib (50 and 100 μmol/kg), or vehicle. B, average tumor weight at day of sacrifice (day 19; d19) between treatment groups. C, average intratumoral drug concentrations were measured by LC/MS-MS at 2 hours post a final dose on day 19, ±SD. D, histologic sections of xenograft tissue harvested on the day of sacrifice (day 19). Brown, DAB reaction product. Representative ×40 fields are shown. Scale bar, 50 μm. E, heatmap analysis showing the in vivo phosphorylation status of 39 kinases on drug versus vehicle-treated mice.

Figure 3.

Efficacy of SAN1 and sunitinib in NOD/SCID mice xenografted with the CRPC DU145 cell line. A, tumor growth inhibition. Mice were dosed (intraperitoneally) daily with SAN1 (100 μmol/kg), sunitinib (50 and 100 μmol/kg), or vehicle. B, average tumor weight at day of sacrifice (day 19; d19) between treatment groups. C, average intratumoral drug concentrations were measured by LC/MS-MS at 2 hours post a final dose on day 19, ±SD. D, histologic sections of xenograft tissue harvested on the day of sacrifice (day 19). Brown, DAB reaction product. Representative ×40 fields are shown. Scale bar, 50 μm. E, heatmap analysis showing the in vivo phosphorylation status of 39 kinases on drug versus vehicle-treated mice.

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In vivo target modulation studies

Insights into the molecular mechanism responsible for the observed in vivo efficacy of SAN1 were obtained by histologic, immunohistochemical, and target modulation analysis of tumors harvested from treated and untreated animals. Hematoxylin and eosin (H&E)-stained tissue sections were evaluated by an independent histopathologist who confirmed that the features of the tumors were consistent with a Gleason score 10, prostatic acinar adenocarcinoma (Fig. 3D). Tumors were composed of malignant epithelial cells distributed in solid sheets without glandular formations. The nuclei were pleomorphic, vesicular, and contained a prominent nucleolus. Isolated mitotic figures were noted. Immunohistochemical analysis using antibodies against Ki-67 and CD31 showed a marked reduction in cell proliferation and reduced angiogenesis in sunitinib-treated mice and SAN1-treated mice (Fig. 3D). The Ki-67–assessed proliferation indices (PI) of mice treated with the compounds of interest were as follows: for sunitinib (100 μmol/kg): 9.1 ± 3.3% (P < 0.001, treated/untreated PI ratio = 0.176), for SAN1 (100 μmol/kg): 16.8% ± 3.1% (P < 0.001, treated/untreated PI ratio = 0.325), and for vehicle-treated animals: 51.7% ± 6.5%. In addition, for CD31, the average number of CD31+ cells of mice treated with sunitinib at 100 μmol/kg and mice treated with SAN1 at 100 μmol/kg versus vehicle-treated mice were 5.3 ± 2.2, 6.8 ± 1.6, and 34.5 ± 8.9, respectively.

A marked downregulation of pAKT (Ser473), pS6, and pSTAT1 was noted when the phosphorylation status of 39 kinases was investigated in extracts of tumor tissue in drug-treated versus vehicle-treated mice, consistent with the multikinase phosphorylation inhibition ability of sunitinib and the designed SAN1 analogue (Fig. 3E).

Generation of SAN1GSC

On the basis of the in vivo efficacy profile, SAN1 was selected for conjugation to the [d-Lys6]-GnRH–targeting peptide by utilizing the free hydroxyl group of SAN1. Prior experience (7) allowed us to implement a succinate linker strategy for SAN1 to [d-Lys6]-GnRH, resulting in the SAN1GSC molecule as shown in Fig. 4A and Supplementary Information Section S3. Molecular docking calculations indicated that SAN1GSC could bind to the target receptors with a predicted Ki = 28 nmol/L for VEGFR-2, Ki = 65 nmol/L for PDGFR-β, and Ki = 51 nmol/L for KIT (Fig. 4B).

Figure 4.

Generation and evaluation of the conjugate SAN1GSC. A, synthesis of SAN1GSC from SAN1 and [d-Lys6]-GnRH. Reagents and conditions: DMF, DMAP, NEt3, room temperature (a); [d-Lys6]-GnRH, DMF, HATU, NiPr2Et (b). B, molecular representation of the energy minimized model of VEGFR-2 catalytic domain (blue surface) in complex with the SAN1GSC conjugate (sticks) that is color coded with cyan carbons for the sunitinib analogue SAN1 and orange for the [d-Lys6]-GnRH peptide. The inset illustrates a close-up view of the ATP-binding site with the SAN1 moiety of the conjugate (cyan) and the key interacting residues (orange). Intermolecular hydrogen bonds are shown with dotted lines and the other atom colors are blue for N, red for O, yellow for S, and pink for F. The predicted inhibition constants Ki for three kinase receptors are given in nmol/L. C, summary of in vitro kinase activity (IC50 ± SD) in various RTKs in the presence of SAN1GSC and [d-Lys6]-GnRH. D, cell-based autophosphorylation assay for PDGFR-β and VEGFR-2 in the presence of SAN1GSC and [d-Lys6]-GnRH. E, MTT cytotoxicity assay in a panel of cell lines. F, SAN1GSC activates the ERK1/2 pathway through binding to the GnRH-R. G, phosphorylation of ERK1/2 following treatment with SAN1GSC, [d-Lys6]-GnRH or GSG is absent when cells are pretreated for 30 minutes with an antibody against the human GnRH-R. H, pharmacokinetic evaluation of SAN1GSC. Drug levels of SAN1GSC and SAN1 formed from its breakdown were monitored by LC/MS-MS. The AUC for each treatment was calculated as a measure of drug exposure over time. N.A., not active.

Figure 4.

Generation and evaluation of the conjugate SAN1GSC. A, synthesis of SAN1GSC from SAN1 and [d-Lys6]-GnRH. Reagents and conditions: DMF, DMAP, NEt3, room temperature (a); [d-Lys6]-GnRH, DMF, HATU, NiPr2Et (b). B, molecular representation of the energy minimized model of VEGFR-2 catalytic domain (blue surface) in complex with the SAN1GSC conjugate (sticks) that is color coded with cyan carbons for the sunitinib analogue SAN1 and orange for the [d-Lys6]-GnRH peptide. The inset illustrates a close-up view of the ATP-binding site with the SAN1 moiety of the conjugate (cyan) and the key interacting residues (orange). Intermolecular hydrogen bonds are shown with dotted lines and the other atom colors are blue for N, red for O, yellow for S, and pink for F. The predicted inhibition constants Ki for three kinase receptors are given in nmol/L. C, summary of in vitro kinase activity (IC50 ± SD) in various RTKs in the presence of SAN1GSC and [d-Lys6]-GnRH. D, cell-based autophosphorylation assay for PDGFR-β and VEGFR-2 in the presence of SAN1GSC and [d-Lys6]-GnRH. E, MTT cytotoxicity assay in a panel of cell lines. F, SAN1GSC activates the ERK1/2 pathway through binding to the GnRH-R. G, phosphorylation of ERK1/2 following treatment with SAN1GSC, [d-Lys6]-GnRH or GSG is absent when cells are pretreated for 30 minutes with an antibody against the human GnRH-R. H, pharmacokinetic evaluation of SAN1GSC. Drug levels of SAN1GSC and SAN1 formed from its breakdown were monitored by LC/MS-MS. The AUC for each treatment was calculated as a measure of drug exposure over time. N.A., not active.

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In vitro evaluation of SAN1GSC

Incubation of SAN1GSC with VEGFR-2, PDGFR-β, KIT, and FLT3 resulted in the inhibition of phosphorylation of each of the above kinases with respective IC50 values of 97 ± 41, 91 ± 36, 74 ± 36, and 37 ± 6 nmol/L (Fig. 4C). Western blot analysis indicated that SAN1GSC inhibited the phosphorylation of VEGFR-2 and PDGFR-β, with nanomolar potency (38 ± 12 nmol/L for VEGFR-2 and 76 ± 17 nmol/L for PDGFR-β, Fig. 4D). Finally, SAN1GSC was equipotent to SAN1 in terms of cellular toxicity in various cell lines with the calculated IC50 ranging from 9 to 18 μmol/L (Fig. 4E).

The SAN1GSC-mediated GnRH-R activation of the pERK1/2 signaling pathway was investigated by setting up an indirect method based on the WPE1-NB26-3 cell line. Incubation of WPE1-NB26-3 cells (stably modified to overexpress the GnRH-R) with SAN1GSC resulted in a robust phosphorylation of ERK1/2 similar to a result observed when the same cells were stimulated by [d-Lys6]-GnRH or GSG (lanes 1-3 in Fig. 4F). Such an effect was absent when cells were pretreated with a GnRH-R antibody (Fig. 4G), suggesting that pERK1/2 activation was a result of direct binding of SAN1GSC to the GnRH-R.

Pharmacokinetic evaluation of SAN1GSC

Intraperitoneal administration of 17 μmol/kg of SAN1GSC in mice led to conjugate blood concentrations averaging 20 nmol/L at 0.25 hours (Fig. 4H). Circulating concentrations of SAN1 formed from the catabolism of SAN1GSC peaked at 0.5 hours with a Cmax of 181 nmol/L at 0.5 hour and an AUC0–8h: 0.53 hour × μmol/L. The described experiment indicated that following dosing of SAN1GSC in mice, pharmacologically critical concentrations of SAN1 (exceeding 100 nmol/L) could be achieved in circulation. It should be noted that the pharmacokinetics of SAN1GSC generating adequate (but low) concentrations of SAN1 sustained over time, was in contrast with the high Cmax (1100 nmol/L), resulting from the dosing of unconjugated SAN1, a potential risk factor for peripheral toxicity.

In vivo antitumor efficacy of SAN1GSC

Mice bearing established DU145 tumors were dosed with equimolar amounts of SAN1GSC, SAN1, sunitinib, and [d-Lys6]-GnRH for a period of 20 days. Tumor growth delay was evident in all mice treated with SAN1GSC (Fig. 5A and B), with an average tumor volume at day 20 of 689 ± 102 mm3 significantly smaller in comparison with SAN1 (1,010 ± 114 mm3, P < 0.001) and sunitinib-treated mice (1197 ± 248, P < 0.001) or for mice treated with [d-Lys6]-GnRH (1248 ± 108 mm3, P < 0.001). On day 20, the average tumor weight for SAN1GSC-treated mice was 0.50 ± 0.11 g compared with 0.75 ± 0.33 g for SAN1, (P = 0.038 vs. SAN1GSC), 0.91 ± 0.27 g for sunitinib, (P = 0.003 vs. SAN1GSC), and 0.82 ± 0.22 g for [d-Lys6]-GnRH–treated mice (P = 0.003 vs. SAN1GSC). All treatments were well tolerated with no noticeable body weight loss or overt toxicity compared with the vehicle group (Supplementary Fig. S5). To investigate whether SAN1GSC dosing increased the drug payload to the tumor site, concentrations of SAN1, and of SAN1 derived from SAN1GSC administration, were determined in blood and tumors in samples obtained 1 hour postdose after day 20 of treatment (Fig. 5C). Measurements of SAN1 at the tumor tissue revealed approximately four times higher SAN1 in SAN1GSC-treated mice compared with SAN1-treated mice (113 ± 35 vs. 31 ± 8 nmol/L), with a tumor/blood ratio for SAN1 formed from SAN1GSC of 0.55 versus 0.16 for SAN1-treated mice.

Figure 5.

Efficacy of SAN1GSC in NOD/SCID mice xenografted with the CRPC DU145 cell line. A, efficacy of SAN1GSC (9.17 μmol/kg), versus equimolar amounts of SAN1, sunitinib, and [d-Lys6]-GnRH in NOD/SCID mice xenografted with DU145 cells. B, average tumor weight at day of sacrifice (day 20) between treatment groups. C, average intratumoral drug levels were measured by LC/MS-MS at 1 hour post a final dose on day 20, ± SD. SAN1 formed from the SAN1GSC is four times higher than SAN1-treated mice, despite the fact that blood levels are similar, resulting in a higher blood/tumor ratio on SAN1GSC-treated mice. D, histologic sections of xenograft tissue harvested on day of sacrifice (d20). E, heat map analysis showing the in vivo phosphorylation status of 39 kinases on drug versus vehicle-treated mice at 1 hour post a final dose on day 20. F, immunohistochemical analysis on sections of xenograft tissue harvested on day of sacrifice (day 20) against pS6 and pRPS6KB1. A marked reduction of phosphorylation for both proteins is seen only in SAN1GSC-treated mice. For each IHC photo brown indicates DAB reaction product, with representative ×40 fields shown. Scale bar: 50 μm. G, summary of the in vitro kinase activity in RPS6KB1 in the presence of various molecules (IC50 ± SD).

Figure 5.

Efficacy of SAN1GSC in NOD/SCID mice xenografted with the CRPC DU145 cell line. A, efficacy of SAN1GSC (9.17 μmol/kg), versus equimolar amounts of SAN1, sunitinib, and [d-Lys6]-GnRH in NOD/SCID mice xenografted with DU145 cells. B, average tumor weight at day of sacrifice (day 20) between treatment groups. C, average intratumoral drug levels were measured by LC/MS-MS at 1 hour post a final dose on day 20, ± SD. SAN1 formed from the SAN1GSC is four times higher than SAN1-treated mice, despite the fact that blood levels are similar, resulting in a higher blood/tumor ratio on SAN1GSC-treated mice. D, histologic sections of xenograft tissue harvested on day of sacrifice (d20). E, heat map analysis showing the in vivo phosphorylation status of 39 kinases on drug versus vehicle-treated mice at 1 hour post a final dose on day 20. F, immunohistochemical analysis on sections of xenograft tissue harvested on day of sacrifice (day 20) against pS6 and pRPS6KB1. A marked reduction of phosphorylation for both proteins is seen only in SAN1GSC-treated mice. For each IHC photo brown indicates DAB reaction product, with representative ×40 fields shown. Scale bar: 50 μm. G, summary of the in vitro kinase activity in RPS6KB1 in the presence of various molecules (IC50 ± SD).

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In vivo target modulation studies

Histologic examination of H&E sections revealed a poorly differentiated high Gleason prostate cancer. Immunohistochemical analysis in tumor sections using antibodies against Ki-67 and CD31 showed a marked reduction in cell proliferation and reduced angiogenesis in SAN1GSC-treated compared with SAN1, sunitinib or [d-Lys6]-GnRH–treated mice (Fig. 5D). The Ki-67–assessed PI of mice treated with SAN1GSC were 21.5% ± 2.7%, (P < 0.001, treated/untreated PI ratio = 0.416), for SAN1 51.9% ± 3.7% (P > 0.01, treated/untreated PI ratio = 1.005), and for sunitinib 51.5 ± 3.9% (P > 0.01, treated/untreated PI ratio = 0.998), respectively. The average number of CD31+ cells in tumor sections of mice treated with SAN1GSC were 12.7 ± 3.5, for SAN1 32.14 ± 11, for sunitinib 30.03 ± 7, and for [d-Lys6]-GnRH 28.1 ± 5.4 (Fig. 5D). Finally, results from the panel of 39 kinases revealed a marked downregulation of pAKT (Ser473), pERK1/2, and pS6 only in SAN1GSC-treated mice compared with all other treatments (Fig. 5E). We performed immunohistochemical analysis on tissue sections of treated mice against the pS6 protein, in parallel with its activating kinase (RPS6KB1). Reduced levels of phosphorylation were observed for both proteins only in the SAN1GSC-treated mice (Fig. 5F). Direct inhibition of RPS6KB1 from SAN1GSC was subsequently verified in a cell-free assay, with a calculated IC50 of 337 ± 72 nmol/L (Fig. 5G).

Toxicity evaluation of SAN1GSC

The cardiotoxicity of sunitinib (29, 30) was assessed and compared with treatment with SAN1GSC, SAN1 by measurements of the cardiac LV function in C57BL/6 mice. Initially, we verified that cardiotoxicity related to sunitinib becomes established and reaches a plateau, at 1 week after treatment. This effect was observed at the efficacious (100 μmol/kg) and the nonefficacious dose of sunitinib (9.17 μmol/kg) but not in vehicle, as measured by the percentage of fractional shortening (FS) change from baseline measurements (Fig. 6A and B). One week of daily intraperitoneal equimolar administrations of SAN1GSC and SAN1 demonstrated that SAN1GSC had a minimal, nonsignificant reduction in the percentage of FS compared with its baseline levels (from 46.79 ± 0.55 to 44.99 ± 0.73) as opposed to a statistically significant percentage of FS reduction following equimolar dosing of SAN1 (from 48.22 ± 0.46 to 46.29 ± 0.60, P = 0.01) or equimolar dosing of sunitinib (from 46.35 ± 0.70 to 42.58 ± 0.62, P = 0.0001) as shown in Fig. 6C.

Figure 6.

Cardiac LV function echocardiography measurements in C57BL/6 mice treated daily with intraperitoneal doses of the various compounds. A, representative M-mode echocardiograms at 1 week following treatment. B, percentage FS reduction compared with baseline for sunitinib-treated mice at 9.17 and 100 μmol/kg for a total period of 4 weeks. C, percentage FS reduction compared with baseline levels at 1 week of treatment with equimolar doses of SAN1 and SAN1GSC, as well as 100 μmol/kg of SAN1. Data are expressed as mean ± SE and a P < 0.05 value was considered statistically significant.

Figure 6.

Cardiac LV function echocardiography measurements in C57BL/6 mice treated daily with intraperitoneal doses of the various compounds. A, representative M-mode echocardiograms at 1 week following treatment. B, percentage FS reduction compared with baseline for sunitinib-treated mice at 9.17 and 100 μmol/kg for a total period of 4 weeks. C, percentage FS reduction compared with baseline levels at 1 week of treatment with equimolar doses of SAN1 and SAN1GSC, as well as 100 μmol/kg of SAN1. Data are expressed as mean ± SE and a P < 0.05 value was considered statistically significant.

Close modal

Hematologic analyses of drug-treated C57BL/6 mice demonstrated that SAN1GSC did not affect white blood cell (WBC) populations, with only a mild erythrocytosis observed in [d-Lys6]-GnRH and SAN1GSC-treated mice (Supplementary Fig. S6). Thus, SAN1GSC was the least toxic of the molecules evaluated under the described conditions.

The active targeting of GnRH-R–expressing tumors with GnRH-based therapeutics is a promising field in the area of personalized treatment. Such a strategy may overcome off-target toxicity in tandem with continuous efficacy due to persistent expression of the GnRH-R on prostate cancer cells, despite prolonged exposure to GnRH agonists (6). A better understanding of prostate cancer microenvironment biology (36) and the central role of angiogenic kinases in tumor growth (37) led us to investigate an alternative strategy by combining sunitinib, a small antiangiogenic molecule with the targeting peptide [d-Lys6]-GnRH. Sunitinib has been extensively used in the clinic for the treatment of solid tumors with a known mechanism of action (32) and known off-target cardiotoxicity (38). We hypothesized that conjugation of sunitinib to [d-Lys6]-GnRH would deliver the multikinase inhibitor to the site of action, reducing off-target effects and the dose required for efficacy.

Because of the lack of “linkable” free groups of sunitinib, we rationally designed and synthesized five analogues that maintained their efficacy but could also be coupled to different ligands, expanding their therapeutic potential to other cancer types. Molecular docking analysis revealed that all SANs assumed a bound conformation similar to sunitinib while remaining efficacious at the low nanomolar scale. Evaluation of our data combined with facile chemical synthesis singled-out SAN1 as the lead compound for further development using an in vivo CRPC xenograft animal model.

SAN1 treatment was well tolerated and proved efficacious in inhibiting tumor growth by reduced CD31 and Ki-67 cells in treated animals, suggesting that inhibition of angiogenesis was at least one mechanism by which SAN1 achieved its efficacy.

Building on the efficacy of SAN1, we investigated whether linking of [d-Lys6]-GnRH to SAN1 would lead to an enhanced delivery of the active molecule to the tumor site. SAN1GSC was generated followed by comprehensive in vitro and in silico evaluation. SAN1GSC maintained its antiangiogenic and cytotoxic properties with IC50 values similar to those of unconjugated SAN1.

After dosing of SAN1GSC, the detectable levels of free circulating SAN1 in the blood were low, possibly a desirable feature expected to minimize peripheral toxicities. SAN1GSC was well tolerated and proved efficacious in our CRPC xenograft model. Importantly, measurements of SAN1 in tumor sample specimens showed approximately four times higher levels of intracellular SAN1 released from SAN1GSC than levels of SAN1 delivered following SAN1 dosing. A deeper mechanistic insight of the efficacy observed with SAN1GSC was provided by the direct inhibition of the RPS6KB1 enzyme, a molecule linked to prostate cancer aggressiveness and metastasis (39, 40). Sunitinib has a reported low nmol/L binding affinity for RPS6KB1 (41) a putative target for the development of new drugs (42). Our finding that in vivo inhibition of RPS6KB1 was achieved only in SAN1GSC-treated mice was presumably due to the enhanced targeting of the tumor microenvironment by the conjugate.

SAN1GSC contains the potent [d-Lys6]-GnRH agonist peptide, and it is possible that it exerts an additional central effect through the pituitary in the described dosing scheme, leading to the lowering of gonadal hormone levels (31).

Considering that GnRH agonists are typically used for the CRPC treatment in combination with other drugs (e.g., docetaxel, abiraterone,), a dual pharmacologic approach based on a conjugate monotherapy resulting into intratumoral and circulating testosterone ablation coupled with inhibition of angiogenesis locally, might provide additional benefit in the clinic. As it is not clear how such an approach would alleviate the problem of resistance to treatment that often develops through mechanisms involving ligand-dependent or ligand-independent androgen receptor signaling, future studies may be needed to address this hypothesis (40). Our finding that SAN1GSC inhibited RPS6KB1, a molecule with an important role in CRPC and the upregulated PI3K–mTOR–S6 pathway (40), is further encouraging in terms of its clinical potential.

Importantly, SAN1GSC appeared to have a minimal cardiotoxic and hematotoxic effect in treated mice compared with sunitinib and SAN1 in our dosing scheme. Elevations of blood pressure that might contribute to cardiac dysfunction were not detected following dosing of sunitinib and its analogues, verifying previously published reports (22). However, we did observe a reduction in the percentage of FS in mice treated with the various molecules. The reduction of the percentage of FS in SAN1 and sunitinib was not detrimental enough to cause a phenotypic cardiac alteration, but it was statistically significant compared with their corresponding baseline measurements, as opposed to SAN1GSC. In addition, SAN1GSC did not affect WBC populations or other hematologic parameters measured. Mild erythrocytosis was observed in the [d-Lys6]-GnRH and SAN1GSC-treated mice, possibly linked to the agonist effects of the [d-Lys6]-GnRH moiety and temporary increase of testosterone levels (flare effect), an effect that has been reported with GnRH agonists in the clinic (43).

One limitation of the current study is the lack of SAN1GSC evaluation in other clinically relevant models, where prostate cancer is either orthotopically generated, or by using patient-derived xenografts which reflect the heterogeneity within different cancer histotypes. These studies will assess the efficacy of SAN1GSC in the metastatic setting, where several small molecules often do fail despite initial success in the primary tumor.

In conclusion, we have generated a series of novel linkable antiangiogenic compounds to treat various forms of cancer based on the clinically successful drug sunitinib. We demonstrated for the first time a direct conjugation of a sunitinib analogue to a peptide moiety generating a minimally cardiotoxic compound, efficacious at a lower dose than equimolar amounts of sunitinib and able to achieve a 4-fold enhanced delivery/benefit to the tumor. The concept is certainly applicable to other solid tumor types expressing the GnRH-R or even to other types of receptors by altering the targeting peptide.

No potential conflicts of interest were disclosed.

Conception and design: O. Argyros, A.G. Tzakos, D. Fokas, C. Tamvakopoulos

Development of methodology: O. Argyros, X. Asvos, N. Sayyad, C.H. Davos, C. Tamvakopoulos

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): O. Argyros, T. Karampelas, A. Varela, C.H. Davos, A.G. Tzakos, C. Tamvakopoulos

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): O. Argyros, T. Karampelas, A. Varela, A. Papakyriakou, C.H. Davos, C. Tamvakopoulos

Writing, review, and/or revision of the manuscript: O. Argyros, T. Karampelas, A. Papakyriakou, C.H. Davos, A.G. Tzakos, D. Fokas, C. Tamvakopoulos

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): O. Argyros, A. Varela, C. Tamvakopoulos

Study supervision: O. Argyros, A.G. Tzakos, C. Tamvakopoulos

Other (synthesis and characterization of sunitinib, GnRH, and its conjugation): N. Sayyad

The authors thank Dr. Zainol Harun, pathologist of Island Hospital Penang Malaysia, for characterization of the tumors, Ms. Anna Agapaki for tissue sectioning, and Ms. Efi Panagouli for assistance with the hematology studies.

This work was cofunded by the Greek Ministry of Education, National Strategic Reference Framework (NSRF) 2007–2013 under grant “PostDoc - DENTAD-PROCAT” (LS7-1682/17156/6.12.10).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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