Scholarly article on N-Desethyl sunitinib 356068-97-8 from Cancer Research p. 1181

Author Manuscript Published OnlineFirst on December 8, 2015; DOI: 10.1158/0008-5472.CAN-15-2138

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Peptide-drug conjugate GnRH-sunitinib targets angiogenesis selectively at the

site of action to inhibit tumor growth

Orestis Argyros¹, Theodoros Karampelas¹, Xenophon Asvos², Aimilia Varela³, Nisar

Sayyad², Athanasios Papakyriakou⁴, Constantinos H. Davos³, Andreas G Tzakos²,

Demosthenes Fokas⁵, Constantin Tamvakopoulos¹

¹Division of Pharmacology-Pharmacotechnology, Clinical, Experimental Surgery &

Translational Research Center, Biomedical Research Foundation Academy of Athens

(BRFAA), Athens, 11527, Greece

²Department of Chemistry, Organic Chemistry and Biochemistry, University of

Ioannina, Ioannina, 45110, Greece

³Cardiovascular Research Laboratory, BRFAA, Athens, 11527, Greece

⁴Laboratory of Chemical Biology of Natural Products and Designed Molecules,

Institute of Physical Chemistry, N.C.S.R “Demokritos”, Athens, 15310, Greece

⁵Laboratory of Medicinal Chemistry, Department of Materials Science and

Engineering, University of Ioannina, Ioannina, 45110, Greece

Running Title: A potent Sunitinib-GnRH conjugate for tumor targeting

Keywords: Angiogenesis, gonadotropin releasing hormone receptor, sunitinib

analogs, tumor targeting

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Author Manuscript Published OnlineFirst on December 8, 2015; DOI: 10.1158/0008-5472.CAN-15-2138

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Financial Support

This work was co-funded by the Greek Ministry of Education, National Strategic

Reference Framework (NSRF) 2007-2013 under grant "PostDoc - DENTAD-

PROCAT" (LS7-1682/17156/6.12.10).

*Address correspondence to:

Dr. Constantin Tamvakopoulos,

Clinical, Experimental Surgery, & Translational Research Center

Division of Pharmacology-Pharmacotechnology

Biomedical Research Foundation, Academy of Athens, Athens, 11527, Greece.

E-mail: ctamvakop@bioacademy.gr, Phone: +30-210-6597475

Disclosure of Potential Conflicts of Interest

The authors declare that they have no conflict of interest to disclose

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Author Manuscript Published OnlineFirst on December 8, 2015; DOI: 10.1158/0008-5472.CAN-15-2138

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Abstract

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

analogs 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 to 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 to equimolar amounts

of non-conjugated 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.

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Introduction

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 evidence that certain cell-surface receptors in malignant cells are

either mutated or overexpressed (e.g., EGFR, HER2/neu, GnRH-R), compared to

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 (CaP) 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 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 vascular endothelial growth factor

receptors (VEGFRs) types 1/2 and platelet-derived growth factor receptors (PDGFR-

α/β) (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 (RTKs) including VEGFRs-1/2, PDGFR-α/β, KIT and

FLT3, with an average IC₅₀below 100 nM (16,17). Clinical trials of sunitinib in

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tumors, such as prostate (18) and breast cancer (19) were initiated based on 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).

Based on 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 analogs (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). Based on

the above structure activity relationship studies, some sunitinib analog 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 analogs (designated as SANs 1-5). The analogs were designed to be linkable

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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 to its unconjugated derivative (SAN1) or sunitinib.

Materials and Methods

Chemicals, synthesis and Characterization of compounds

Sunitinib was purchased from Selleckchem (Selleckchem, USA) and [D-Lys⁶]-GnRH

from Proteogenix (Schiltigheim, France). All other chemicals and biologicals, unless

stated otherwise were purchased from Sigma-Aldrich (Sigma-Aldrich, Munich,

Germany). The synthesis of SAN1-SAN5 (Figure 1A) and of SAN1GSC (Figure 4A)

is presented in the supplementary information section (S.I.1 and S.I.3). All molecules

were analyzed by Mass Spectrometry (MS) in order 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, USA).

Computational methods

Detailed docking and molecular dynamic analysis for all the compounds was

performed as described in the S.I.2.

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 S.I.4. Cellular

inhibition of autophosphorylation of VEGFR-2 and PDGFR-β was performed as

described previously (32). Detailed methods are available in the S.I.5. For cellular

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studies, cells were used within six months of purchase or authentication and were

cultured as instructed by the American Type Culture Collection (LGC Standards,

Germany). The HCC1954 cell line was obtained from ATCC and HUVEC from Life

Technologies, UK. The DU145, PC3 and NIH/3T3 cells were authenticated by short

tandem repeat profiling (Microsynth, Switzerland). WPE1-NB26-3 cells were a kind

gift from Professor Robert Millar (University of Pretoria, SA)

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 (IP) in male NOD/SCID mice (8-10 weeks, n=4, from

Charles River, Italy) 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 x 10⁶DU145 cells, a castration-

resistant CaP (CRPC) cell line. Treatment was initiated when tumors reached 200

mm³by daily IP 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 S.I.6. Experiments were terminated after approximately

20 days, at 1h or 2h after the last administered dose by euthanizing the animals under

isoflurane anesthesia. Tumors were excised, weighed and prepared for histopathology,

immunohistochemistry (IHC), kinase activity and quantification of compounds by

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LC-MS/MS.

Immunohistochemistry

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 40x 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 40x 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, UK) was used per

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 gray scale 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 in S.I.7. Hematotoxicity was assessed as described in the S.I.8.

Statistical analyses

Statistical analyses and calculation of all IC₅₀s were performed by SigmaPlot 12

software. Statistical significance was determined using the Student’s two-tailed, two-

sample unequal variance distribution t test.

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Results

Synthesis of SANs

The synthetic route for SANs 1-5 is shown in Figure 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 to 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 analog (SAN1) can be found in S.I.2.

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 K_i(Figure 2A) were in the low nM range, similar to the

estimated values of sunitinib. A representative molecular model of SAN1 complexed

with VEGFR-2 is shown in Figure 2A and S.I.3.

Biochemical kinase assays

The potency of all SANs was experimentally evaluated in biochemical assays against

purified VEGFR-2, PDGFR-β, KIT, FLT3 and presented in Figure 2B. All SANs

inhibited the specific RTK targets with IC₅₀values ranging from 20 to 150 nM

comparable to sunitinib. The EGFR served as a negative control (concentrations up to

10 μM 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 based cellular ligand-dependent

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phosphorylation assay. The calculated cellular IC₅₀value for sunitinib was 24 12 nM

(for VEGFR-2) and 72 19 nM for PDGFR-β respectively. Similar potencies were

observed for all SANs with cellular IC₅₀values ranging from 52-180 nM (Figure 2C).

Cytotoxicity studies

The antiproliferative effect of the five SANs was assessed in three GnRH-R positive

CaP cell lines (DU145, PC3, WPE1-NB26-3) one GnRH-R positive breast cancer cell

line (HCC1954), in HUVEC and NIH/3T3 cells. Results and comparative IC₅₀values

are presented in Figure 2D. SAN1 proved highly potent in HUVEC and DU145 cell

lines (IC_50s: 8 4 μM and 11 2 μM respectively), SANs 2-4 showed intermediate

efficacies while SAN5 appeared inactive (IC₅₀>100 μM) 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 h

(1.5 μM) with an AUC_0−8hof 2.51 h x μM. Similarly, SANs 1-4 were rapidly

absorbed reaching highest blood concentrations averaging in the range of 0.5 to 2.1

μM at 0.25 to 1 h after dosing (Figure 2E). Following dosing, concentrations of SAN1

peaked at 0.25 h with an AUC_0−8h: 1.11 h x μM. Signs of discomfort were observed in

mice that received SAN2, discouraging further evaluation. According to the

biochemical assays, compound concentrations of approximately 100 nM were needed

for the inhibition of the target kinases (Figure 2A-C). Following IP dosing, such

levels could be sustained for 4 h post-dose for all the molecules tested. A single IP

administration of an efficacious dose of sunitinib (100 μmol/Kg) was the point of

reference for subsequent efficacy studies in mice (Figure 2E). Based on the described

pharmacokinetic and biological readouts combined with its facile synthetic route,

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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 (Figure 3A), with the average tumor size at

d19 at 312±123 mm³and an average tumor weight of 0.20±0.08 g compared to

1181±105 mm³(p<0.0001) and 0.87±0.51 g (p=0.005) for vehicle treated mice

(Figure 3B). Tumor growth delay was also observed in DU145 xenografted mice

treated with equimolar SAN1 (figure 3A), with an average tumor size at d19 of

455±137 mm³(p<0.001 compared to vehicle treated mice). The corresponding

average tumor weight at sacrifice was 0.37±0.19 g for SAN1 (Figure 3B, p=0.005

compared to vehicle treated mice). Concentrations of sunitinib and SAN1 in blood

and tumor tissue were measured at 2 h after the final dose. Sunitinib’s blood

concentrations were 4.7 0.1 μM, whereas levels at tumor tissue were 11.0 5.0 μM

(Figure 3C). The values for SAN1 in the tumor tissue were significantly lower at

0.4 0.2 μM, but exceeded the IC₅₀level threshold (100 nM) determined for inhibition

of phosphorylation of the target kinases. It should be noted that sunitinib is more

lipophilic compared to SAN1 (as evident in their clogP values), a property that

explains the higher drug accumulation in the tumor tissue over the treatment period.

In vivo target modulation studies

Insights into the molecular mechanism responsible for the observed in vivo efficacy of

SAN1 were obtained by histological, immunohistochemical and target modulation

analysis of tumors harvested from treated and untreated animals. Hematoxylin and

eosin stained tissue sections were evaluated by an independent histopathologist who

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confirmed that the features of the tumors were consistent with a Gleason score 10,

prostatic acinar adenocarcinoma (Figure 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. IHC 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 (Figure 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%. Additionally 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 analog (Figure

3E).

Generation of SAN1GSC

Based on 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 Figures 4A and S.I.3).

Molecular docking calculations indicated that SAN1GSC could bind to the target

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receptors with a predicted Ki=28 nM for VEGFR-2, Ki=65 nM for PDGFR-β and

Ki=51 nM for KIT (Figure 4B).

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 IC₅₀values

of 97 41, 91 36, 74 36 and 37 6 nM (Figure 4C). Western blot analysis indicated

that SAN1GSC inhibited the phosphorylation of VEGFR-2 and PDGFR-β, with nM

potency (38±12 nM for VEGFR-2 and 76±17 nM for PDGFR-β, Figure 4D). Finally,

SAN1GSC was equipotent to SAN1 in terms of cellular toxicity in various cell lines

with the calculated IC₅₀ranging from 9 to 18 μM (Figure 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 Figure 4F). Such an effect was absent when cells were pretreated with a GnRH-R

antibody (Figure 4G), suggesting that pERK1/2 activation was a result of direct

binding of SAN1GSC to the GnRH-R.

Pharmacokinetic evaluation of SAN1GSC

IP administration of 17 μmol/Kg of SAN1GSC in mice led to conjugate blood

concentrations averaging 20 nM at 0.25 h (Figure 4H). Circulating concentrations of

SAN1 formed from the catabolism of SAN1GSC peaked at 0.5 h with a Cmax of 181

nM at 0.5 h and an AUC_0−8h: 0.53 h x μM. The described experiment indicated that

following dosing of SAN1GSC in mice, pharmacologically critical concentrations of

SAN1 (exceeding 100 nM) could be achieved in circulation. It should be noted that

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the pharmacokinetics of SAN1GSC generating adequate (but low) concentrations of

SAN1 sustained over time, was in contrast with the high Cmax (1100 nM), 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 (Figures 5A and 5B),

with an average tumor volume at d20 of 689±102 mm³significantly smaller in

comparison to SAN1 (1010±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 d20, the average tumor weight for SAN1GSC treated mice was

0.50 0.11 g compared to 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 to the vehicle group (figure S.I.5). To

investigate if 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 1h post-dose after d20 of

treatment (Figure 5C). Measurements of SAN1 at the tumor tissue revealed

approximately four times higher SAN1 in SAN1GSC treated mice compared to SAN1

treated mice (113±35 versus 31±8 nM), with a tumor/blood (t/b) ratio for SAN1

formed from SAN1GSC of 0.55 versus 0.16 for SAN1 treated mice.

In vivo target modulation studies

Histological examination of H&E sections revealed a poorly differentiated high

Gleason CaP. IHC analysis in tumor sections using antibodies against Ki-67 and

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CD31 showed a marked reduction in cell proliferation and reduced angiogenesis in

SAN1GSC treated compared to SAN1, sunitinib or [D-Lys⁶]-GnRH treated mice

(Figure 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 (Figure 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 to all

other treatments (Figure 5E). We performed IHC 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 (Figure 5F). Direct inhibition of RPS6KB1 from SAN1GSC

was subsequently verified in a cell-free assay, with a calculated IC₅₀of 337 72nM

(Figure 5G).

Toxicity evaluation of SAN1GSC

The cardiotoxicity of sunitinib (29,30) was assessed and compared to 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 one week following treatment. This effect was observed at the

efficacious (100 μmol/Kg) and the non-efficacious dose of sunitinib (9.17 μmol/Kg)

but not in vehicle, as measured by the % FS change from baseline measurements

(Figure 6A-B). One week of daily IP equimolar administrations of SAN1GSC and

SAN1 demonstrated that SAN1GSC had a minimal, non-significant reduction in % FS

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compared to its baseline levels (from 46.79 0.55 to 44.99 0.73) as opposed to a

statistically significant % 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 Figure 6C.

Hematological 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 (figure S.I.6). Thus

SAN1GSC was the least toxic of the molecules evaluated under the described

conditions.

Discussion

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 CaP cells, despite prolonged exposure to GnRH agonists (6). A better

understanding of CaP 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.

Due to the lack of “linkable” free groups of sunitinib, we rationally designed and

synthesized five analogs that maintained their efficacy but could also be coupled to

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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

IC₅₀values similar to those of unconjugated SAN1.

Following 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 CaP aggressiveness

and metastasis (39,40). Sunitinib has a reported low nM 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

17

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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 pharmacological

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. Since 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 signalling, 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 to 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 %FS in mice

treated with the various molecules. The reduction of the %FS in SAN1 and sunitinib

was not detrimental enough to cause a phenotypic cardiac alteration, but it was

statistically significant compared to their corresponding baseline measurements, as

opposed to SAN1GSC. Additionally SAN1GSC did not affect WBC populations or

18

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

other hematological 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 CaP 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 analog to a peptide

moiety generating a minimally cardiotoxic compound, efficacious at a lower dose

than equimolar amounts of sunitinib and able to achieve a four-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.

Acknowledgments

We would like to 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 hematology studies.

19

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Figure Legends

Figure 1

Synthetic route of sunitinib analogues and physicochemical properties. (A) Synthesis

of sunitinib analogs 1-5 from the available acid intermediate 1. Reagents and

conditions: (a) DMF, EDCI, HOBt, NEt₃, rt; (b) CH₂Cl₂, TFA, rt. (B) Key properties

and MS conditions for the sunitinib analogues.

Figure 2

In silico, in vitro and pharmacokinetic evaluation of the synthesized SANs. (A)

Docking for sunitinib and SAN1-SAN5 with estimated inhibition constants (K_iin

nM). Molecular model of the VEGFR-2 complex with SAN1 (B) Summary of in vitro

24

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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 3

Efficacy of SAN1 and sunitinib in NOD/SCID mice xenografted with the CRPC

DU145 cell line. (A) Tumor growth inhibition. Mice were dosed (IP) daily with

SAN1 (100 μmol/Kg), sunitinib (50 and 100 μmol/Kg), or vehicle. (B) Average tumor

weight at day of sacrifice (d19) between treatment groups. (C) Average intratumoral

drug concentrations were measured by LC-MS/MS at 2h post a final dose on d19, ±

SD. (D) Histological sections of xenograft tissue harvested on the day of sacrifice

(d19). Brown indicates DAB reaction product. Representative x40 fields are shown.

Scale bar: 50 μm. (E) Heat map analysis showing the In vivo phosphorylation status

of 39 kinases on drug versus vehicle treated mice.

Figure 4

Generation and evaluation of the conjugate SAN1GSC. (A) Synthesis of SAN1GSC

from SAN1 and [D-Lys⁶]-GnRH. Reagents and conditions: (a) DMF, DMAP, NEt₃,

rt; (b) [D-Lys⁶]-GnRH, DMF, HATU, NiPr₂Et. (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 analog SAN1 and orange for the [D-Lys⁶]-GnRH peptide. The inset

illustrates a closeup 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,

25

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yellow for S and pink for F. The predicted inhibition constants K_ifor three kinase

receptors are given in nM. (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 min 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 area under the curve (AUC) for each treatment was calculated as a measure of

drug exposure over time. N.A: Not active

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 (d20) between treatment groups. (C)

Average intratumoral drug levels were measured by LC-MS/MS at one hour post a

final dose on d20, ± 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) Histological 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 one hour

post a final dose on d20. (F) Immunohistochemical analysis on sections of xenograft

tissue harvested on day of sacrifice (d20) against pS6 and pRPS6KB1. A marked

26

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reduction of phosphorylation for both proteins is seen only in SAN1GSC treated

mice. For each IHC photo brown indicates DAB reaction product, with representative

x40 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 6

Cardiac LV function echocardiography measurements in C57BL/6 mice treated daily

with IP doses of the various compounds. (A) Representative M-mode

echocardiograms at one week following treatment. (B) Percentage FS reduction

compared to baseline, for sunitinib treated mice at 9.17 and 100 μmol/Kg for a total

period of four weeks. (C) Percentage FS reduction compared to baseline levels, at one

week of treatment with equimolar doses of SAN1 and SAN1GSC, as well as 100

μmol/Kg of SAN1. Data are expressed as mean ± SΕ and a p<0.05 value was

considered statistically significant.

27

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Peptide-Drug conjugate gnrh-sunitinib targets angiogenesis selectively at the site of action to inhibit tumor growth

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