New somatostatin-drug conjugates for effective targeting pancreatic cancer

Article abstract of DOI:10.1016/j.bmc.2018.06.032

Pancreatic cancer poorly responds to available drugs, and finding novel approaches to target this cancer type is of high significance. Here, based on a common property of pancreatic cancer cells to express somatostatin receptors (SSTR), we designed drug conjugates with novel somatostatin-derived cyclic peptides (SSTp) with broad selectivity towards SSTR types to facilitate drug targeting of the pancreatic cancer cells specifically. Uptake of our newly designed SSTps was facilitated by SSTRs expressed in the pancreatic cancers, including SSTR2, SSTR3, SSTR4 and SSTR5. Three major drugs were conjugated to our best SSTps that served as delivery vehicles, including Camptothecin (CPT), Combretastatin-4A (COMB) and Azatoxin (AZA). All designed drug conjugates demonstrated penetration to pancreatic cancer cell lines, and significant toxicity towards them. Furthermore, the drug conjugates specifically accumulated in tumors in the animal xenograft model, though some accumulation was also seen in kidney. Overall these findings lay the basis for development of novel drug series that could target the fatal pancreatic cancer.

Full text of DOI:10.1016/j.bmc.2018.06.032

Accepted Manuscript
New somatostatin-drug conjugates for effective targeting pancreatic cancer
E. Ragozin, A. Hesin, A. Bazylevich, H. Tuchinsky, A. Bovina, T. Shekhter
Zahavi, M. Oron-Herman, G. Kostenich, M.A. Firer, T. Rubinek, I. Wolf, G.
Luboshits, M.Y. Sherman, G. Gellerman
PII:
S0968-0896(18)30710-7
https://doi.org/10.1016/j.bmc.2018.06.032
BMC 14424
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 April 2018
14 June 2018
24 June 2018
Please cite this article as: Ragozin, E., Hesin, A., Bazylevich, A., Tuchinsky, H., Bovina, A., Zahavi, T.S., Oron-
Herman, M., Kostenich, G., Firer, M.A., Rubinek, T., Wolf, I., Luboshits, G., Sherman, M.Y., Gellerman, G., New
somatostatin-drug conjugates for effective targeting pancreatic cancer, Bioorganic & Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.bmc.2018.06.032
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New somatostatin-drug conjugates for effective targeting pancreatic cancer.
Ragozin E.,1# Hesin A.,2,3# Bazylevich A.,1 Tuchinsky H.,4 Bovina A.1, Shekhter Zahavi T.,5 Oron-Herman
*
M.,6 Kostenich G.,6 Firer M.A,7 Rubinek T., 2,3 Wolf I., 2,3 Luboshits G.7 Sherman M.Y.4 and Gellerman G.1
1 Department of Chemical Sciences, Ariel University, Ariel, 40700, Israel
2 The Oncology Institute, Tel Aviv Souraski Medical Center, Tel-Aviv, 62431, Israel
3 Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, 69978, Israel
4 Department of Molecular Biology, Ariel University, Ariel, 40700, Israel
5 The Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences,
Tel-Aviv University, Tel-Aviv, 69978, Israel
6 The Advanced Technologies Center, Sheba Medical Center, Tel Hashomer, 52621, Israel
7 Department of Chemical Engineering, Ariel University, Ariel, 40700, Israel
# Equal contribution
Address for correspondence
Prof. Gary Gellerman,
Dept. Chemical Sciences,
Ariel University, Ariel 40700, Israel
Email: garyg@ariel.ac.il
1

Graphical Abstract
Targeting
Pancreatic
Cancer
Abstract
Pancreatic cancer poorly responds to available drugs, and finding novel approaches to target
this cancer type is of high significance. Here, based on a common property of pancreatic cancer
cells to express somatostatin receptors (SSTR), we designed drug conjugates with novel
somatostatin-derived cyclic peptides (SSTp) with broad selectivity towards SSTR types to
facilitate drug targeting of the pancreatic cancer cells specifically. Uptake of our newly
designed SSTps was facilitated by SSTRs expressed in the pancreatic cancers, including
SSTR2, SSTR3, SSTR4 and SSTR5. Three major drugs were conjugated to our best SSTps that
served as delivery vehicles, including Camptothecin (CPT), Combretastatin-4A (COMB) and
Azatoxin (AZA). All designed drug conjugates demonstrated penetration to pancreatic cancer
cell lines, and significant toxicity towards them. Furthermore, the drug conjugates specifically
accumulated in tumors in the animal xenograft model, though some accumulation was also seen
in kidney. Overall these findings lay the basis for development of novel drug series that could
target the fatal pancreatic cancer.
Keywords: SSTR; peptide conjugate; targeted drug delivery; pancreatic cancer; SPPS; stability profiles.
Abbreviations
ACN – Acetonitrille; cAMP – 3',5'-cyclic adenosine monophosphate; DCM – Dichloromethane;
DIPEA-Diisopropylethylamine; DMAP - 4-N,N-(dimethylamino)pyridine; DMBA – Dimethyl
barbituric acid; DMF - N,Ndimethylformamide; DMSO - Dimethyl sulfoxide; Fmoc - 9-
fluorenylmethoxycarbonyl; HPLC - High-pressure liquid chromatography; LC-MS - liquid
chromatography-mass spectroscopy; MALDI - Matrix-assisted laser desorption/ionization; PyBOP -
Benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate; rpm - rounds per minute; SST
– somatostatin; TFA - Trifluoroacetic acid; TIPS – Triisopropylsilane; XTT - 2,3-bis(2-methoxy-4-
nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide
2

1. Introduction
Pancreatic cancer is the most lethal gastrointestinal cancer. The disease is usually diagnosed
at advanced metastatic stages, it responds poorly to chemotherapeutics and has an overall 5-
year survival rate between 6-37% [1]. Because of such medical urgency, selective drug delivery
to pancreatic cancer using small molecules [2] proteins [3], nanoparticles [4-7], liposomes [8,
9], quantum dot [10] and micelles [11, 12] has been developed. However, the progress in the
field has not led so far to dramatic breakthrough in patient treatments.
During the last 20 years there has been a substantial effort to target receptors overexpressed in
cancer cells [13], in order to achieve specific receptor-mediated endocytotic delivery of drugs
[14-17]. Since somatostatin is a natural peptide hormone, a somatostatin receptor family
(SSTRs) constituting of 5 subtypes (SSTR 1-5), has drawn attention of medicinal chemists as
promising targets for peptide-based targeted drug delivery [18]. SSTRs are heterogeneously
expressed in the human gastrointenstinal tumors, including pancreatic and neuroendocrine
pancreatic cancer, and intratumoral vessels, and often multiple SSTR subtypes can co-express
in the same cell [19-24]. So far, the most useful peptide developed as a drug carrier for SSTR
overexpressed malignancies has been the modified (linker armed) S-S-bridged octapeptide
octreotide (sandostatin) that preferentially targets SSTR2 [25], though it also has a significant
affinity towards SSTR3 and SSTR5. Several drug conjugates based on octreotide have been
reported for pancreatic cancer, including camptothecin (CPT) [26] and doxorubicin [27]
conjugates. These conjugates displayed significant antitumor activities against pancreatic
cancers and reduced toxicity to benign tissues. However, existing peptides show significant
specificity towards different SSTR types, which may narrow their use to cancers that express
only these receptor types. Our goal was to develop peptides with broad specificity as vehicles
for targeting drugs to pancreatic cancers.
Here we report synthesis of novel conjugates comprising cyclic peptides SSTp-58 and SSTp-
86 that are based on the sandostatin motive and incorporating the S-S-bridged backbone GlyS2
building unit introduced at the beginning of the assembly [28]. Previous in vivo studies of
tumor targeting by SSTp-86 revealed its selective accumulation in HT-29 colon (expressing
SSTR3 and SSTR5) and H69 SCLC xenografts (expressing SSTR2) [29-32].
3

Scheme 1. Structures of SSTp-58, SSTp-86 and octreotide
Here, we constructed drug conjugates with SSTp-58 and SSTp-86 peptides, and investigated
their antitumor activities with pancreatic cancer models. We found that these drug conjugates
effectively and specifically penetrated pancreatic cancer cells, depending of expression of a
broad range of SSTRs, and accumulated in the pancreatic tumors in a xenograft model. Tumor
growth inhibition, maintenance of body weight and overall survival of pancreatic xenograft
models are currently under investigation in our lab.
2. Results and Discussion
2.1 Synthesis of SSTp - mono drug or fluorescein conjugates 1-4, SSTp-dual drug and
fluorescein conjugates 7
Several peptide–drug conjugates 1-4 employing analogs of the SSTp 58 and -86 peptides as
targeting moiety (Scheme 2), were synthesized using solid phase organic synthesis (SPOS).
Mono conjugates of STTp-86 bearing fluorescein (1b), CPT (2b) and COMB (4b) have been
synthesized and characterized previously [35]. The STTp-58-fluoresceine (1a) was synthesized
in the same manner as for 1b. In general, the fully protected cyclic peptide SSTp-58 (5a) and
SSTp-86 (5b) with GABA linker (Scheme 2) were synthesized on an acid-labile Rink amide
MBHA resin (substitution level, 0.56 mmol/g, 1g) using standard Fmoc solid-phase peptide
synthesis (SPPS) by following a published procedure [28].
4

Scheme 2. Synthesis of peptide conjugates 1a – 4b.
As stated above, we decided to develop peptide-drug conjugates with broad specificity than
octreotide as peptide vehicle for targeting drugs to pancreatic cancers. To synthesize
conjugates 2 - 4, first the N-terminal Fmoc group of the GABA linker of preloaded SSTp-58
and -86 respectively was removed (20% piperidine in NMP, 10 mL) releasing the primary
amine toward 5a or 5b correspondingly. Consequently, activated CPT, AZA, and COMB
(CPT, AZA, or COMB [3 eq.], 4-nitrophenyl chloroformate [3 eq.], pyridine, 90oC, 1 h; then
DMAP [6 eq.] DCM, 0oC) were reacted with the primary amine, providing conjugates 2a,b,
3a,b and 4a,b with an aliphatic and aromatic carbamate conjugation site, respectively. Finally,
the on-resin-synthesized conjugates were cleaved from the solid support (cold, 95:2.5:2.5
TFA/TIS/H2O, room temperature, 1h), precipitated by diethyl ether, purified by preparative
HPLC, and characterized by MS (supporting information material). Hetero-nuclear peptide
drug conjugates 7a,b were assembled on Rink amide MBHA resin similarly to mono-
conjugates 2-4 accept the introduction of split functional orthogonally protected tail Fmoc-
5

Lys(Dde)-OH [11] (Scheme 3). First, on-resin intermediates 6a-b with deprotected Lys split
functional tail were obtained after Fmoc removal of their precursors (20% piperidine in NMP,
10 mL). At this stage, the cyclic SSTp targeting carrier peptides were adapted for double drug
conjugation of CPT and FITC. Notably, in its free form, CPT in particular is very potent but it
has poor solubility and produces off-target cytotoxicity, factors that have precluded its clinical
development. We hypothesized that in general, conjugation of CPT to a targeting peptide
would improve its pharmacological properties.
Initially, the free N-terminus amine of the resulting 6a and 6b were loaded with activated CPT
(CPT [3 eq.], 4-nitrophenyl chloroformate [3 eq.], pyridine, 90oC, 1 h; then DMAP [6 eq.]
DCM, 0oC) leading to creation of biodegradable carbamate linkage. Then the second protecting
group Dde was also removed (2% hydrazine in DMF, 3 min (x 3)) and the primary ω−amine of
Lys was allowed to react with FITC (FITC [1.5 eq.], DIEA [3 eq.] in DMF). Finally, all the
on-resin synthesized conjugates were cleaved from the solid support and precipitated by the
addition of cold diethyl ether, isolated, lyophilized and identified by LC-MS and HRMS as dual
conjugates 7a-b.
Scheme 3. Synthesis of hetero-nuclear conjugates 7a, 7b with FITC and CPT
6

2.2 Receptor type specificity of SSTp-drug conjugates.
In order to explore the targeting properties of STTps we established in vitro model using
subclones of HEK-293 cells each overexpressing a single SSTR subtype. Though pancreatic
cancer cell lines mostly express SSTR2 and SSTR5 receptors, human pancreatic tumors can
also express other SSTRs [22, 23]. Here we assess the role of all five SST receptor subtypes in
binding and transport of our novel peptides to cells. First, we tested the ability of peptides 1a
and 1b to inhibit the cAMP signaling pathway activity in a receptor-mediated manner via G-
protein coupled mechanism [37, 39, 40]. cAMP signaling pathway activity was assessed using
a reporter gene system of cAMP response element (CRE) fused to firefly luciferase, where
CRE promoter activity reflects intracellular cAMP accumulation level [36, 41, 42]. First, we
calibrated the system using native somatostatin SST-14 and the SSTR 2, 3, 5-selective agonist
octreotide [25]. As expected, SST-14 inhibited the CRE promoter activity in SSTR 1-5
transfected cells, whereas octreotide inhibited the CRE promoter activity in SSTR 2, 3 and 5-
transfectants selectively (Fig. S-1, also see supplementary info). In this assay, peptide 1a was
less potent than 1b in cells transfected with SSTR5 or SSTR4, while in cells expressing SSTR2
or SSTR3, they demonstrated similar suppression of CRE promoter. Neither of the tested
peptides was effective in inhibition of the cAMP signaling pathway activity in SSTR1-
transfectants (Fig. 1).
SSTR3
SSTR1
SSTR2
100
50
0
100
50
0
100
50
0
86
58
86
58
86
58
10
100
1000
10
100
1000
10
100
1000
Log Concentration (nM)
Log Concentration (nM)
Log Concentration (nM)
SSTR4
SSTR5
control
100
100
100
50
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86
58
86
58
86
58
50
50
0
10
0
10
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100
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10
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Log Concentration (nM)
Log Concentration (nM)
Log Concentration (nM)
Figure 1: Inhibition of the CRE promoter activity by 1a and 1b. HEK-293 cells were transfected with a
7

plasmid encoding reporter luciferase gene under the control of the cAMP response element (CRE), and
co-transfected with SSTR 1-5 expression vectors or with empty vector. After 1 day cells were treated
with FSK or FSK plus SST analog 1a or 1b, and CRE promoter activity was evaluated by monitoring
luminescence. Results are given as mean SD of at least three independent experiments.
To further study the receptor specificity, we examined the build-up of 1b in HEK-293 cells
over-expressing SSTR 1- 5 and found remarkable accumulation in all-transfectants, except for
SSTR1 (Fig. 2). These data indicate strong receptor-specific accumulation of the peptides in
cells, and a broad specificity towards the SSTR types, which justifies using these moieties as
vehicles for specific drug delivery to pancreatic cancers that express various SSTRs.
Figure 2: Accumulation of 1b in SSTR-transfected HEK-293: HEK-293 cells were transfected with
SSTR 1-5 expression vectors or control empty vector and co-transfected with RFP expression vector to
identify transfected cells. 48 hours later 400 nM 1b was added for 30 minutes, and both FITC (1b
contains FITC moiety) (a) and RFP (b) fluorescence were monitored. (c) Merged RFP and FITC
images. Phase-contrast imaging (d) shows total cell number in the samples. (е) Histogram showing
quantitative image analysis of the fluorescence intensity of FITC+ cells (mean SD; n = 10). The
8

numbers denote mean fluorescence intensity (MFI). Data are representative of three independent
experiments (P < 0.001).
In further confirm binding specificity in our receptor transfection protocol, we chose 1b to
conduct a competition assay against octreotide, which effectively binds SSTR2, but not SSTR4
(Fig. 3) [25]. The concentrations of the peptides in this experiment were 400 nM for 1b and
800 nM for octreotide. As shown in Figure 3, octreotide competed with 1b in SSTR2
transfected cells, but not in the SSTR4-transfectants, indicating strong receptor specificity of
peptides in our assay. Together, data on the effects of 1a and 1b on activity of receptors and
receptor-specific penetration into cells suggest that these peptides could provide receptor-
specific targeting of drugs into the pancreatic cancer cells. The prediction from the receptor-
mediated accumulation of the peptides and their effects on cAMP levels was that 1a peptide
should be effective with cells that express SSTR2 (and to lesser extent SSTR4 and SSTR5),
100 µm
100 µm
while 1b with cells expressing any of these receptors.
Figure 3: Competition assay between 1b and octreotide: HEK-293 cells transfected with SSTR2 or
SSTR4 were pre-incubated with 800nM octreotide for 20 min, and then incubated with 400nM 1b for
15 min, and both FITC (1b contains FITC moiety) (a) and RFP (b) fluorescence were monitored. (c)
Merged RFP and FITC images. Phase-contrast imaging (d) was used in order to show total cell number
in the samples. (е) Histogram showing quantitative image analysis of the fluorescence intensity of
FITC+ cells (mean SD; n = 10). The numbers denote mean fluorescence intensity (MFI). Data are
representative of three independent experiments (P < 0.001).
9

2.3 Flow cytometry analysis on Panc-1 and Capan-1 cell lines.
The ability of the peptides 1a and 1b to promote drug delivery was further tested using human
pancreatic cancer lines Capan-1 and Panc-1. We used flow cytometry analysis to confirm
robust expression of SSTR2 and SSTR5 in these cell lines (Fig. 4). Both cell lines possess
similar pattern of somatostatin receptors expression: SSTR2 was detected in 27% and 32% of
cells in Panc-1 and Capan-1 consequently while SSTR5 is expressed by all cells in the
population in both cell lines. In addition, 30% of Panc-1 and Capan-1 cells express both types’
receptors, SSTR2 and SSTR5.
A
I
ii
iii
B
I
ii
iii
Figure 4: SSTR-2 and SSTR-5 expression in human pancreatic cancer cell lines Panc-1(A) and Capan-
1(B). The cells were stained with both anti-SSTR2-PE(I) and SSTR5-AF670(II) antibodies and analysed
by flow cytometry. The percentage of cells expressing both receptors is presented in III. Red: unstained
cells; Aqua: cells stained with anti-SSTR2-PE(I) or SSTR5-AF670(II); Blue: Overlap between red and
aqua.
Though expression of the SSTR2 and SSTR5 receptors on these cell lines was similar, Panc-1
has an inactivating mutation in SSTR5 [43], unlike Capan-1.
2.4 Peptide internalization into Panc-1 and Capan-1 cells
In spite of similar expression of the receptors in Panc-1 and Capan-1 cells, accumulation of the
peptides could be very different, since it also dependent of the rate of penetration. Therefore,
10

we directly measure internalization of 1a and 1b peptides in these pancreatic lines. Cells were
incubated with the fluorescent peptides, and their accumulation was monitored microscopically
after the washout of the external peptides. In Panc-1 cells peptide 1a started to show
accumulation already after 5 h incubation, and the accumulation progressed during the
following 19 hours. In contrast, peptide 1b demonstrated poor internalization (Figure 5).
a.
b.
c.
d
5 μm
5 μm
5 μm
5 μm
e.
f.
g.
h
5 μm
5 μm
5 μm
5 μm
Figure 5: Confocal images of internalization of 1a and 1b into Panc-1 cells. Panc-1 cells were
incubated with peptide FITC conjugates (green) for different time intervals Cell nuclei were stained
with Dapi(blue). . For 1a: (a). 1h, (b). 5h, (c). 12h, (d). 24h (scale bar: 5 μm each), For 1b: (e). 1h; (f).
5h; (g). 12h; (h). 24h (scale bar: 5 μm each).
By comparison, Capan-1 cells accumulated 1a and 1b at similar rates (Figure 6). However, the
effectiveness of accumulation was lower compared with peptide 1a in Panc-1 cells.
a.
b.
c.
d.
5 μm
5 μm
5 μm
5 μm
e.
f.
g.
h.
11

5 μm
5 μm
5 μm
5 μm
Figure 6: Confocal images of internalization of 1a and 1b into Capan-1 cells. Capan-1 cells
were incubated with peptide FITC conjugates (green) for different time intervals. Cell nuclei were
stained with Dapi(blue). For 1a: (a). 1h, (b). 5h, (c). 12h, (d). 24h (scale bar: 5 μm each), For 1b: (e).
1h; (f). 5h; (g). 12h; (h). 24h (scale bar: 5 μm each).
After making sure that peptides 1a and 1b can potentially provide specific targeting of anti-
cancer drugs to pancreatic cancer cells, we evaluated conjugates of these peptides with three
anti-cancer agents, including CPT, COMB and AZA, synthesized as described above.
2.5 Stability of conjugates in IMDM medium
Beside accumulation, an important factor that may affect anti-cancer activity of the drug
conjugates is their stability. The stabilities of the peptide drug conjugates were measured in cell
culture IMDM medium. The stability of conjugates and drug release rates varied depending on
the peptide and the linked drug. Conjugates based on SSTp58 2a (with CPT), 3a (with AZA)
and 4a (with COMB) (t1/2 =3 h, t1/2 =3 h, t1/2 =15 h, correspondingly) decompose faster in
comparison to counterpart SSTp86 2b (with CPT), 3b (with AZA) and 4b (with COMB) (t1/2
=20 h, t1/2 =5 h, t1/2 =18 h, correspondingly) (Fig. 7). Nevertheless, the stabilities of both series
of peptide-drug conjugates were potentially sufficient to provide potent anticancer activities.
Therefore, we next tested the ability of these conjugates to kill pancreatic cancer cells in
culture.
BS 2a
BS 3a
BS 4a
100
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3a
2a
4a
AZA
COMB
CAMP
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10 20 30 40 50 60 70 80
Time(h)
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Time(h)
0
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10 20 30 40 50 60 70 80 90100110120130140
Time(h)
Time(h)
Figure 7: Biostability of peptide drug conjugates 2a-4b in medium IMDM. Compounds were added to
DME containing 20% bovine serum, amino acids and vitamins, sodium pyruvate, HEPES buffer and
potassium nitrate, and incubated at a 37°C incubator for indicated time periods. Aliquots were taken and
12

immediately quenched with 2.5 vol of ethanol and analyzed by LC-MS. Results are given as mean
SD of at least three measurements.
2.6. Cell cytotoxicity of peptide conjugates
Panc-1 and Capan-1 cells were exposed to increasing doses of drugs or conjugates, and at the
end of 48h or 72h incubation, metabolic activities of cells were measured using the XTT assay.
Table 1 shows that peptides SSTp86 and SSTp58 alone are not cytotoxic to either cell type,
while drugs alone or their conjugates with the peptides demonstrated significant toxicities.
Table 1. IC50 values (µM) for SSTp58, SSTp86, 2a-b, 3a-b, 4a-b, CPT, Aza and Comb in Capan-1 and
Panc-1 cell lines. IC50 values were calculated using three-parameter non-linear regression. IC50 values
marked with * were calculated using four-parameter non-linear regression. NA** mean some fits were
ambiguous and statistical comparisons of IC50 curve fits (sum-of-squares F-test), IC50 values and shifts
could not be reliably calculated.
To our surprise, Capan-1 cells were more sensitive to 2a and 2b conjugates than Panc-1, though
both cells have similar levels of SSTR2 and SSTR5 receptors. However, cytotoxic effects of
peptide-drug conjugates could depend on a number of factors beside binding to receptors,
including the rate of penetration, efficiency of the drug release, interaction of drugs with their
targets, etc. Therefore, it is possible that for one of these reasons CAMP is more effective with
Capan-1 than with Panc-1.
Notably, instability of the conjugates can lead to the release of the anti-cancer agent, which may
start killing cells in culture after penetrating cells bypassing a receptor, and therefore may lead to a
receptor-non-specific toxicity. Therefore, it is very difficult to make any conclusion about specific
receptor-mediated anti-cancer effects of the drug conjugates based on the in vitro toxicity data.
13

Accordingly, in planning further tests we used two criteria: (a) a peptide must most effectively
penetrate into cells in a receptor-specific manner, (b) the drug conjugate should show the most
delayed drug accumulation due to the conjugate instability. Upon considering these criteria, we
further focused on the CPT conjugates of the a-series of peptides, which demonstrated strong
accumulation and most delayed drug release (conjugate 2a). Furthermore, as a model for testing
drug conjugate activity in vivo, we chose xenografts of Panc-1 cells that demonstrated the most
efficient accumulation of 1a peptide, compared to 1b peptide and compared to 1a peptide in
Capan-1 cells. This study is currently under investigation in our lab.
2.7. In vivo targeting of Panc-1 tumour tissues.
To evaluate accumulation of the CPT conjugate 2a in xenograft tumor and tissues in vivo, we
prepared a fluorescent versions of the conjugate 1a and 7a (see above). Nude mice bearing 100
mm3 tumors of subcutaneously injected Panc-1 cells were intravenously administered with
either the peptide 1a (SSTp-58-FITC) or the FITC-labeled 1a-CPT conjugate 7a compounds.
After 24 hours the compounds were found to accumulate in the tumor, though some
accumulation was also seen in kidney (Fig. 8). Of note, no significant accumulation of the
conjugate was seen in pancreas. Furthermore, previous studies demonstrated that fluorescein
alone does not concentrate either in pancreas [44] or in tumor [45]. Therefore, accumulation in
the pancreatic tumor is a specific property of the peptide-drug conjugate, which is likely to be
mediated by the SSTRs
Figure 8: Tumor-specific retention of 1a and 7a. Panc-1 cells were injected subcutaneously into nude
mice to induce a tumor. Mice bearing a 100 mm3 tumor were intravenously injected (single IP
injection, 120 mg/Kg) with the fluorescent conjugates. 24 hours post injection the tumor, spleen,
pancreas, kidney and the liver were excised and evaluated for accumulation of fluorescence
microscopically.
14

These data suggested that the peptide 1a-CPT (namely 2a) drug conjugate could show specific
inhibitory effects against pancreatic cancer.
3. Conclusions
Here we designed cyclic peptide that interact with wide range of somatostatin receptors present
in pancreatic cancer cells and in human pancreatic tumors. These peptides penetrate cells via
receptor-mediated mechanisms and selectively deliver anti-cancer drugs to pancreatic tumors in
a xenograft model. The peptide-drug conjugates demonstrate strong activity against pancreatic
cancer. Therefore, this approach lay the basis for development of novel drugs against pancreatic
cancer.
4. Materials and Methods
4.1 Reagents and cell lines
Camptothecin (CPT), all protected amino acids, resin and coupling reagents were purchased
from Tzamal D-Chem Laboratories Ltd. Petah-Tikva, Israel. Combretatstatin 4A (COMB)
[33] and Azatoxin (AZA) [34] were synthesized according to the literature procedures. All the
solvents were purchased from Bio-Lab Ltd. Jerusalem, Israel or Gas Technologies Ltd. Kefar
Saba, Israel. All other chemicals including IMDM medium were purchased from Holland
Moran or Sigma-Aldrich. All the cell lines were cultured in an RPMI medium supplemented
with 2 mM glutamine, 10% fetal bovine serum and with penicillin streptomycin (100 IU/ml of
each). (The cell culture growth medium and all of its additives were purchased from Biological
Industries, Bet-Ha'emek, Israel). All cell cultures were grown at a 37°C incubator in an
environment containing 6% CO2. The cytotoxicity of the materials was determined by
measuring the mitochondrial enzyme activity, using a commercial XTT assay kit (Biological
Industries, Bet-Ha'emek, Israel). All samples contained DMSO at final concentration <0.05%.
All the cancer cell lines were kindly provided by Prof. Arie Ornstein (Sheba Medical Center,
Israel). Noncancerous HEK-293 were obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA).
4.2 High performance liquid chromatography (HPLC)
All HPLC purifications were done via reverse phase on ECOM semi-preparative system with
dual UV detection at 254 and 214 nm. Phenomenex Gemini® 10 µm C18 110 Å, LC 250 x
21.2 mm prep column was utilized. The column was kept at room temperature. The eluent
solvents were 0.1% TFA in H2O (A) and 0.1% TFA in ACN (B). A typical elution was a
15

gradient of 100% A to 50% B over 45 min at a flow rate of 25 mL/min. Analytical RP-HPLC
was performed on an UltiMate 3000 system (Dionex) using a Vydac C18 column (250 x 4.6
mm) with 5 µm silica (300 Å pore size). Linear gradient elution (0 min 0% B; 5 min 0% B; 50
min 90% B) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile: H2O
(80:20, v/v)) was used at a flow rate of 1 mL/min.
4.3 Liquid Chromatography Mass Spectrometry (LCMS)
Electron spray mass spectra (ESI-MS) were obtained using an Autoflex III smart-beam
(MALDI, Bruker), Q-TOF micro (Waters) or an LCQ FleetTM ion trap mass spectrometer
(Finnigan/Thermo). HPLC/LC-MS analyses were made using Agilent infinity 1260 connected
to Agilent quadruple LC-MS 6120 series equipped with ZORBAX SB-C18, 2.1 x 50mm,
1.8µm column. In all cases the eluent solvents were A (0.1% FA in H2O) and B (0.1% FA in
ACN) and the elution gradient profile was: 100% A for first 3 min, followed by 5 min (from
min 3 to min 8) during which it reached 100% B, followed by 5 min (from min 8 to min 13) of
100% B, followed by two min (from min 13 to min 15) during which it returned back to A,
followed by 2 min (from min 15 to min 17) of 100% A. The UV detection was at 254 nm. The
column temperature was kept at 500C. The flow rate was of 0.3 ml /min. The MS fragmentor
was tuned on 30V or 70V on positive or negative mode.
4.4 Synthesis of Peptide- mono-Drug conjugates 1-4.
Coupling step: The synthesis of all the cyclic conjugates 1-4 were provided using previously
described procedure [15, 35]. For more detailed procedures see supplementary material.
Briefly, in a reaction vessel equipped with a sintered glass bottom, rink amide MBHA resin,
(substitution level 0.56mmol/g, 1g) was swelled in NMP by agitation overnight. The Fmoc
group was removed from the resin upon treatment with 20% piperidine in NMP (10ml) for 15
min. This action was repeated twice. After washing the resin with NMP (7 times, 10 ml, 2 min
each time), Fmoc-GlyS2(Acm)-OH [28] building unit (3 eq, 10.5 mmol, 0.64 g) dissolved in
NMP (7 ml) was activated with PyBoP (3 eq, 10.5 mmol, 0.7 g) and DIEA (6 eq, 21 mmol,
0.521 ml) for 4 min at room temperature, transferred to the reaction vessel and allowed to react
for 1h at rt. Following coupling, the peptidyl resin was washed with NMP (5 times, 7 ml) for 2
min each time, completion of reaction was monitored by ninhydrin test (Kaiser test, yellow),
The deblock mixture was a mixture of 80:20 DMF/piperidine (v/v).
Cyclization step: After coupling of Fmoc-Cys(Acm)-OH and NMP wash, the resin was washed
with 4:1 DMF/water (3 times, 6.5 ml, 2min each time). A solution of I2 (10eq, 35 mmol, 1.29
g) in 4:1 DMF/water (10 ml) was added to the peptidyl – resin followed by agitation at rt for 1h
16

to afford the disulfide bridge cyclization [37]. The peptidyl – resin was filtered and washed
extensively with 4:1 DMF/water (7 times, 10 ml, 2 min each time), DMF ( 6 times, 10 ml, 2
min each time), DCM (6 times, 10 ml, 2 min each time), CHCl3 (4 times, 10 ml, 2 min each
time), 2% ascorbic acid in DMF (6 times, 10 ml, 2 min each time) and last wash with DMF (6
times, 10 ml, 2 min each time). Finally, coupling of last amino acid Fmoc-D-Phe-OH after
cyclization, gave cyclic peptides.
Coupling of Fmoc-g-aminobutyric acid (linker): Fmoc-g-aminobutyric acid (3eq, 10.5mmol,
0.49 g) dissolved in NMP (7 ml) was activated with PyBroP (3 eq, 10.5 mmol, 0.7 g) and
DIEA (6 eq, 21mmol, 0.521 ml) for 4 min at room temperature, transferred to the reaction
vessel and allowed to react for 1h at rt. After post coupling wash and Fmoc-deprotection the
peptidyl resin is ready for drug conjugation.
Coupling of anticancer agents to 5a and 5b: The peptidyl resin was washed and a DMF/DCM
(1:1) and solution of premade 4-nitrophenylcarbonate derivative of CPT, AZA and COMB
(anticancer agent (1.2 eq.), 4-nitrophenyl chloroformate (1.2 eq.), DMAP (1.2 eq.), DIPEA (3
eq.) in DCM, 3h, rt) of was added to the exposed primary amine for overnight at rt, leading to
the corresponding carbamate conjugation site. The resin was thoroughly washed (DCM, NMP,
2 x DCM) and the peptide conjugate was cleaved from the polymeric support with the cold
TFA/TIS/H2O (95:2.5:2.5) cocktail. The solvents were removed under a gentle flow of N2 and
then the crude was precipitated from diethyl ether. The supernatant centrifuged and dried by
lyophilization. The purification was conducted on semi-preparative HPLC (AcCN, 0.1% TFA
in H2O) led to final conjugates with carbamate linkage 2a-b, 3a-b and 4a-b correspondingly.
For 2a: (42% yield) LC-MS: RT = 8.25 min; ESI-MS m/z calcd: 854.3 found: 854.8 (M/2+H),
calcd: 569.9 found: 570.2 (M/3+H); For 2b: (41% yield) LC-MS: RT = 9.04 min; HRMS (ESI-
MS): m/z calcd: 1759.691 found: 1759.679 (M+Na) For 3a: (38% yield) LC-MS: RT = 8.57
min; ESI-MS m/z calcd: 871.37 found: 871.0 (M/2+2H), calcd: 580.5 found: 580.9 (M/3+H).
For 3b: (36% yield) LC-MS: RT = 9.17 min; ESI-MS m/z calcd: 885.36 found: 885.0 (M/2+H).
For 4a: (32% yield) LC-MS: RT = 8.18 min; HRMS: ESI-MS m/z calcd: 839.37 found: 839.5
(M/2+2H), calcd: 560.2, found: 560.1 (M/3+2H). For 4b: (43% yield) LC-MS: RT = 9.76 min;
HRMS (ESI-MS): m/z calcd: 1705.718 found: 1705.739 (M+H), calcd: 1727.713, found:
1727.716 (M+Na).
4.5 Synthesis of hetero peptide conjugates 7a,b.
To the intermediates 5a and 5b, Fmoc-Lys(Dde)-OH was coupled to the primary amine using
standard coupling procedure (PyBoP, DIEA in NMP) with 2-fold excess of the AA, leading to
17

resin bound 6a and 6b (Scheme 3). Initially, the Fmoc group was removed (7 ml of 20%
piperidine in DMF, 10 min, 2 cycles) and the deblocked N-terminus amine was reacted with the
preactivated form of CPT by the procedure mentioned above for 2a,b. Then, Dde was removed
by three cycles of 2% hydrazine in 7 ml DMF, 3 min each and after washing the primary
ω−amine of Lys was allowed to react with FITC (FITC, 1.5 eq., DIEA, 3 eq. in 7 ml DMF).
Finally, the on-resin synthesized conjugates were cleaved from the solid support in the same
manner as for 2-4 and precipitated by the addition of cold diethyl ether, isolated
(centrifugation), lyophilized, purified (HPLC, AcCN, 0.1% TFA in H2O) and identified by LC-
MS and HRMS as dual conjugates 7a-b. 7a 40% yield. LC-MS: RT = 9.86 min; MS: ESI-MS
m/z calcd: 2226.9, found: 1113.8 (M/2+2H), calcd: 742.28, found: 742.8 (M/3+1H). 7b 38%
yield. LC-MS: RT 10.08 min; MS: ESI-MS m/z calcd: 1127.92 (M/2+2H), found: 1128.87
(M/2+2H).
4.6 cAMP signaling pathway activity assessment
cAMP signaling pathway activity was assessed using reporter gene system of the cAMP
response element (CRE) fused to firefly luciferase [36]. 70,000 HEK-293 cells were seeded in
24 well plates and a day later the cells were co-transfected with CRE-luciferase reporter
plasmid, β-galactosidase expression construct and with SSTR 1-5 expression vectors or with
control empty vector (pcDNA3.1). Transfections were carried out with JetPei transfection
reagent (Polyplus-transfection, NY, USA) according to the manufacturer’s recommendation.
After 1 day cells were treated with forskolin (FSK) 10 µM or FSK plus SST analog. Forskolin-
induced CRE promoter activity inhibition by SSTRs agonists was evaluated by monitoring
luminescence, assessed relative to FSK alone and normalized to β-galactosidase activity
calculated by ONPG/ beta GAL assay [37, 38]. Experiments were conducted in triplicates. The
data shown are representative of three experiments.
4.7 Bio-stability in IMDM Medium
This medium is a modification of Dulbecco’s Modified Eagle’s Medium (DME) containing
20% bovine serum, amino acids and vitamins, sodium pyruvate, HEPES buffer and potassium
nitrate. Incubation procedure: Incubations were conducted at a 37°C incubator with 1 ml
IMDM medium per incubation tube. The samples were prepared by adding 10 µl of stock
solution (see below) to the medium at the beginning of the incubation period. Aliquots of the
incubation mixture were removed after 1, 3, 6, 24, 48, 72 and 96 h and then immediately
quenched with 2.5 vol of ethanol. The samples were centrifuged at 14000 rpm for 15 min.
Supernatants were collected, filtered and analyzed by LC-MS.
18

4.8 Cytotoxicity test
The cytotoxicity of the peptide-drug conjugates was determined by measuring the
mitochondrial enzyme activity, using a commercial XTT assay kit. All samples contained
DMSO at final concentration <0.05%. Cells were cultured in micro wells at 5-10 x 104 cells/mL
and incubated for 24 h, 48h, and 72h. After the first incubation period the cultures were washed
and then given a fresh medium containing different concentrations of the tested substances. At
the end of the second incubation, XTT reagent was added and the cells were re-incubated for
additional 2-4 h. During that time the absorbencies in the wells were measured with a TECAN
Infinite M200 ELISA reader at both 480 and 680nm. The difference between these
measurements was used for calculating the % Growth Inhibition (GI) in test wells compared to
two controls: cells that were exposed to the medium and solvent, and those which were exposed
to a solvent-free medium. All the tests were done in tetra-plicate. Each experiment was
conducted twice.
4.9 Direct immunofluorescence assay and flow cytometry
For evaluating SSTR2 and SSTR5 expression the Panc-1 and Capanc-1 cells were washed with
PBS and scrapped from the culture flask. 106 of cells were incubated with 10 µL of ready for
use Anti-Human Somatostatin R2/SSTR2-PE and Anti-Human Somatostatin R5/SSTR5-Alexa
Fluor 647 that specifically binds SSTR2 and SSTR5 correspondingly (FAB4224P, IC4448RBD
Bioscience, San Jose, CA, USA) at 4°C for 45 min for the antibody. Following two washings
with PBS the cells were re-suspended in 400 µL of PBS and analyzed with Becton Dickson
FACS Calibur cell analyzer equipped with an argon-ion laser (15W) at 488 nm with a 637 DF
filter. For each sample ~ 104 cell were analyzed. FlowJo software was used to analyze the
collected data. For background measurement, the cells were treated at the same conditions but
without adding the antibody.
4.10 Fluorescent microscopy on non-cancerous HEK-293: Binding affinity assay
70,000 HEK-293 cells were seeded in 24 well plates and a day later the cells were co-
transfected with SSTR 1-5 expression vectors or with control empty vector (pcDNA3.1) and
with an RFP expression vector, in order to confirm successful transfection and correct imaging.
Transfections were carried out as described above. After 1 day the cells were passaged at ≈
30% confluence and a day later the cells were incubated with FITC-labeled peptide 1b in
serum-free DMEM for 30 min. Peptide accumulation was examined by fluorescence
microscopy (cell^R-Imaging Station, Olympus IX2-ZDC, Hamburg, Germany).
4.11 Confocal microscopy on cancer cell lines
19

For confocal microscopy analysis, the cells were treated with two different peptides 1a and 1b
and subjected to accumulation in different period of time, the images were collected with a
Zeiss LSM700 confocal microscope.
4.12 Ex vivo fluorescent imaging on cancer cell lines
Five-week-old athymic nude mice (Harlan Labs, Nes Ziona, Israel) were subcutaneously
inoculated in the dorsal left side with Panc-1 cells, and tumors allowed to establish over time.
When tumor volume reached 100 mm3, 1a and 7a (120 mg/kg) was intravenously administered.
After 24 hours, fluorescent images of 1a and 7a were acquired by Photometrics CoolSNAP
HQ2 camera mounted on an Olympus iX81 fluorescent microscope.
Acknowledgements
The authors thank Prof. Dan Meyerstein from the Department of Chemical Sciences, Ariel
University for technical support and fruitful advices in performing mass spectroscopy. The
authors also thank Mrs. Cherna Moskowitz for generous stipend to E.R. Financial support was
provided by Ariel University Internal Research and Development Program (ASI-GG-2017-
004).
Appendix A. Supplementary data
Supplementary data related to this article can be found at:
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24

Highlights
•
•
•
New cyclic peptide-drug conjugates for effective targeting pancreatic cancer were discovered.
These conjugates comprising cyclic peptides SSTp-58 and SSTp-86 that are based on the sandostatin motive.
The drug conjugates specifically accumulated in tumors in the animal xenograft model.
25