99mTc-labeled vasopressin peptide as a radiopharmaceutical for small-cell lung cancer (SCLC) diagnosis

Article abstract of DOI:10.1021/jm500272r

The ^99mTc-labeled conjugates of the vasopressin (AVP) peptide and of its analogue d(CH₂)₅[d-Tyr(Et²)-Ile ⁴-Eda⁹]AVP (AVP(an)) have been synthesized using the technetium complexes with tetradentate tripodal chelator (the tris(2-mercaptoethyl)amine (NS₃)) and the monodentate isocyanide ligand (CN-peptide). The conjugates exhibit high stability in the presence of 100 times the molar excess of standard amino acids cysteine or histidine and also satisfactory stability in human serum. The ^99mTc(NS ₃)(CN-AVP) and ^99mTc(NS₃)(CN-AVP(an)) ability of binding to small-cell lung cancer (SCLC) cell line H69 was studied in vitro. The results suggest that the novel vasopressin conjugate ^99mTc(NS₃)(CN-AVP(an)) is a desirable compound for imaging oncogene receptors overexpressed in SCLC cells and can be an important basis for further consideration the conjugate as a potential diagnostic radiopharmaceutical for patients suffering from small-cell lung cancer.

Full text of DOI:10.1021/jm500272r

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Article
99m
Tc-labeled Vasopressin Peptide as a Radiopharmaceutical
for Small-Cell Lung Cancer (SCLC) Diagnosis
Ewa Gniazdowska, Przemys#aw Ko#mi#ski, Krzysztof Ba#kowski, and Pawe# Ochman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500272r • Publication Date (Web): 27 Jun 2014
Downloaded from http://pubs.acs.org on July 2, 2014
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99mTc-labeled Vasopressin Peptide as a Radiopharmaceutical for Small-Cell
Lung Cancer (SCLC) Diagnosis
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Ewa Gniazdowskaa,*, Przemysław Koźmińskia, Krzysztof Bańkowskib, Paweł Ochmanc,
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aInstitute of Nuclear Chemistry and Technology, Warsaw, Poland
bPharmaceutical Research Institute, Warsaw, Poland
cThe Maria SkłodowskaꢀCurie Institute of Oncology, Warsaw, Poland
ABSTRACT
The 99mTcꢀlabeled conjugates of the vasopressin (AVP) peptide and of its analogue
d(CH2)5[DꢀTyr(Et2)ꢀIle4ꢀEda9]AVP (AVP(an)) have been synthesized using the technetium
complexes with tetradentate tripodal chelator – the tris(2ꢀmercaptoethyl)ꢀamine (NS3) – and
the monodentate isocyanide ligand (CNꢀpeptide). The conjugates exhibit high stability in the
presence of 100 times the molar excess of standard amino acids cysteine or histidine, and also
satisfactory stability in human serum. The 99mTc(NS3)(CNꢀAVP) and 99mTc(NS3)(CNꢀ
AVP(an)) ability of binding to smallꢀcell lung cancer (SCLC) cell line H69 was studied in
vitro. The results suggest that the novel vasopressin conjugate 99mTc(NS3)(CNꢀAVP(an)) is a
desirable compound for imaging oncogene receptors overexpressed in SCLC cells and can be
an important basis for further consideration the conjugate as a potential diagnostic
radiopharmaceutical for patients suffering from the smallꢀcell lung cancer.
Keywords: technetium, vasopressin, radiopharmaceutical, small cell lung cancer (SCLC),
receptor, cell line H69
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1. INTRODUCTION
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The more and more important role in the medicine of presentꢀdays is played by
radiopharmaceuticals, the compounds labeled with various shortꢀlived radionuclides, which
are introduced into the body for either diagnostic or therapeutic purposes. The introduced
doses of radiopharmaceuticals are at so low level (nanomolar) that they do not influence the
function of the biological organ. The radiopharmaceuticals, which selectively distribute
within given tissues or organs, are usually coordination compounds with a central ion of a
radiometal, firmly attached to a biologically active molecule (peptide). The measurement of
space distribution of the intensity of the gamma rays emitted from the body makes possible to
determine localization of the radiopharmaceuticals within the body and to define
abnormalities in the tissue structures or in the functioning of the studied organs. Such methods
are used to image receptors of the central nervous system, functions of heart, brain, kidneys,
liver, bileꢀduct and skeleton, in order to determine and localize cancers.1 The diagnostic
methods of nuclear medicine make possible detection of diseases at their early stage, much
earlier than the accompanying morphological changes could be detected by methods of
classical medicine. Such early and apposite diagnosis strongly promotes the effectiveness of
the consecutive therapy. Among the most important diagnostic radiopharmaceuticals are those
labeled with technetiumꢀ99m – the radionuclide of nearly ideal nuclear properties and of rich
coordination chemistry.2ꢀ4
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The goal of this work was to label the vasopressin peptide Cys1ꢀTyr2ꢀPhe3ꢀGln4ꢀAsn5ꢀ
Cys6ꢀPro7ꢀArg8ꢀGly9 (AVP) and its analogue d(CH2)5[DꢀTyr(Et2),Ile4,Eda9]AVP (AVP(an))
with technetiumꢀ99m radionuclide.
Vasopressin (arginine vasopressin, (Arg8)ꢀVasopressin, AVP), also known as argipressin
or antidiuretic hormone (ADH), is a cyclic peptide containing residues of nine amino acids.
Two of them are the molecules of cysteine (Cys1 and Cys6). Because of the disulfide bond
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between them, vasopressin forms a cyclic sixꢀmembered amino acid ring with three amino
acid residues hanging out. Vasopressin is a peptide hormone found in most mammals,
including humans. It is derived from a preprohormone precursor synthesized in the
hypothalamus, from which it is liberated during transport to the posterior pituitary. AVP
regulates the body retention of water, being released when the body is dehydrated (antiduretic
action of AVP, mediated via V2 type receptors). It causes the kidneys to conserve water (but
not salt) and to reduce urine volume. AVP also raises arteriar blood pressure by inducing
moderate vasoconstriction (pressor action of AVP, mediated via V1 type receptor). In
addition to its predominantly antidiuretic and pressor activities vasopressin, as a neuropeptide,
generates a variety of neurological effects in the central nervous system (CNS). It is also
involved, via different receptor subtypes, in higher brain functions, including cognitive
abilities and emotionality. In recent years interest increased in the role of vasopressin in social
behavior and in its participation in such diseases as schizophrenia and autism.5ꢀ8 Normal
plasma concentration of AVP is lower than 4 pg/ml and a halfꢀlife of AVP is about 10 ÷ 35
min (due to its biodegradation by vasopressinases which are found in the liver and kidney). 9,10
Decreased AVP level leads to diabetes insipidus, and therefore the increase of this period (to
avoid recognition by degrading enzymes) is important and can be achieved by removing the
Nꢀterminal amine group of Cys1 and using the nonꢀnatural Dꢀarginine, instead of natural Lꢀ
arginine at the position 8.11
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The analogue of AVP, the d(CH2)5[DꢀTyr(Et2)ꢀIle4ꢀEda9]AVP compound (AVP(an)), is
one of the several effective antagonists to the antidiuretic arginine vasopressin V2ꢀreceptor.5,6
Arginine vasopressin receptor 2 (AVPR2, also called V2 receptor) belongs to the sevenꢀ
transmembrane, Gꢀprotein–coupled receptor family, and its intracellular signaling pathway
involves a Gs/adenylyl cyclaseꢀstimulating system that induces cAMP formation.12 The V2
receptors are present in greatest amounts in the kidney, but recently, polymerase chain
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reaction studies revealed that V2 receptor mRNA is also present in other tissues or cells such
as brain, liver and lung.13,14
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The overexpression of vasopressin receptor V2 has been found on smallꢀcells of lung
cancer (SCLC).13,15ꢀ18 The SCLC accounts for about 20% of all lung cancers and usually
involves a tumor of neuroendocrine origin with very low survival rate. The SCLC cells have a
high proliferation rate and spread to lymph nodes and other vital organs, that is, brain, liver
and bone. The 5ꢀyear survival rate is below 20%.19
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The essential features of AVP affinity for V2 receptor are: the ring formed via disulfide
bond between Cys1 and Cys6, the three amino acid “tail”, the presence of tyrosine residue at
position 2, and the Cꢀterminal in glycine blocked by NH2. The use of radiolabeled AVP
targeting vectors to selectively target receptorꢀexpressing tumors offers innovative diagnostic
and treatment strategies for patients suffering from smallꢀcell lung cancer.20
The aim of the experimental work was to synthesize and investigate novel conjugates of
‘4+1’ mixedꢀligand Tc(III)ꢀ99m complexes with AVP and its analogue AVP(an) (Figure 1).
Figure 1. (A) 99mTc labeled AVP. (B) 99mTc labeled AVP(an), AVP(an)=d(CH2)5[DꢀTyr(Et2)ꢀ
Ile4ꢀEda9]AVP.
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These two conjugates are very stable not only in the thermodynamic sense, but also
towards ligand exchange in vivo.21ꢀ23 The conjugates 99mTc(RNS3)(CNꢀpeptide) consist of
central metal ion Tc(III) coordinated by the tetradentate RNS3 tripodal chelator (R=H, tris(2ꢀ
mercaptoethyl)ꢀamine) and a monodentate isocyanide species, previously coupled with the
peptide. Introduction of a hydrophilic or hydrophobic group R at the periphery of the NS3
ligand may change the lipophilicity of the complex molecule in such a way that the
lipophilicity of the whole peptideꢀradiometal conjugate will be close to that of the free
peptide.21,24,25
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To verify the identity of the 99mTcꢀlabeled peptides, the nonꢀradioactive ‘cold’ rhenium
reference compounds Re(RNS3)(CNꢀpeptide) have been synthesized and characterized by
elemental analysis and mass spectrometry.
2. RESULTS AND DISCUSSION
2.1. AVP and AVP(an) labeling, stability, electrophoresis and lipophilicity studies
The HPLC chromatograms (using System 2) of the compounds Re(NS3)(CNꢀAVP),
99mTc(NS3)(CNꢀAVP), Re(NS3)(CNꢀAVP(an)) and 99mTc(NS3)(CNꢀAVP(an)) are shown in
Figure 2. The retention times (RT) determined for the studied conjugates are 11.7 min, 12.0
min, 15.9 and 16.3 min, respectively. Practically the same positions of the peaks of
Re(NS3)(CNꢀAVP) and 99mTc(NS3)(CNꢀAVP) as well as of Re(NS3)(CNꢀAVP(an)) and
99mTc(NS3)(CNꢀAVP(an)) can be a proof of the existence of the species 99mTc(NS3)(CNꢀ
AVP) and 99mTc(NS3)(CNꢀAVP(an)) synthesized in n.c.a. (no carrier added) scale. The small
peaks recorded at RT = 3.4 min and 7.9 min correspond to the intermediate complexes 99mTcꢀ
EDTA/mannitol and 99mTc(NS3), respectively.
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Figure 2. The HPLC chromatograms of the Tc/Re conjugates prepared in this study (system 2).
The 99mTcꢀlabeled vasopressin conjugates 99mTc(NS3)(CNꢀAVP) and 99mTc(NS3)(CNꢀ
AVP(an)) were synthesized with radiochemical yield higher than 95% and specific activity of
about 25 GBq/ꢀmol of the conjugate.
Stability of 99mTcꢀlabeled vasopressin conjugates 99mTc(NS3)(CNꢀAVP) and
99mTc(NS3)(CNꢀAVP(an)) is shown in Figure 3. Vertical axis presents the percentage of intact
conjugates in the challenge experiments with an excess (10 mM) of histidine or cysteine. Both
conjugates are sufficiently stable for up to 24 h. In both cases the HPLC chromatograms
showed presence of only one radioactive species in the solution, with the retention time
characteristic for either the conjugate 99mTc(NS3)(CNꢀAVP) or 99mTc(NS3)(CNꢀAVP(an)),
respectively. Thus, we can consider that the conjugates in vitro will not undergo the ligand
exchange reaction with amino acids or other strongly competing natural ligands containing
SH or NH groups.
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Figure 3. Histidine and cysteine challenge experiments showing the percentage of intact
99mTc(NS3)(CNꢀAVP) and 99mTc(NS3)(CNꢀAVP(an)) conjugates remaining after different
periods of incubation at 37 oC.
Stability of 99mTc(NS3)(CNꢀAVP) and 99mTc(NS3)(CNꢀAVP(an)) conjugates in human
and rat serum is shown in Figures 4 and 5, respectively. Investigations of the behavior of the
isolated 99mTc(NS3)(CNꢀAVP) conjugate in human serum have shown that the studied
complex is stable within about 10ꢀ12 minutes. After this period, corresponding to the halfꢀlive
of vasopressin peptide in vivo, a new peak appeared on the HPLC chromatograms as the result
of the enzymatic vasopressin degradation (Figure 4 left). According to the literature data there
are two major sites of cleavage of vasopressin: the TyrꢀPhe and ArgꢀGly bonds.26 The
vasopressin derivative, which lacks free Nꢀterminal amine group (in our case the Nꢀterminal
amine group of vasopressin is blocked by bonding with the isocyanide linker CNꢀBFCA) is
more stable and in this case the main biodegradation product results from the cleavage of the
ArgꢀGly bond.
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Figure 4. HPLC chromatograms of 99mTc(NS3)(CNꢀAVP) conjugate after incubation at 37 oC
in human (left) and rat serum (right).
The HPLC chromatograms obtained after incubation of 99mTc(NS3)(CNꢀAVP) conjugate
in human or in rat serum (Figure 4) show two peaks: (a) at RT=11.8 min, corresponding to the
99mTc(NS3)(CNꢀAVP) conjugate (AVP = CysꢀTyrꢀPheꢀGlnꢀAsnꢀCysꢀProꢀArgꢀGly), and (b) at
RT=2.8 min, corresponding, most probably, to the 99mTc(NS3)(CNꢀCysꢀTyrꢀPheꢀGlnꢀAsnꢀ
CysꢀProꢀArg) analogue. Comparing the results of incubation obtained in the case of human
(Figure 4 left) and rat (Figure 4 right) serum, one can see that biodegradation of vasopressin
in rat serum is more rapid and the peak b at RT=2.8 min appears already after about fifth
minute of incubation. The removal of the Cꢀterminal glycine from the vasopressin “tail”
results in formation of significantly less lipophilic species and, according to the literature26, is
also responsible for the inactivation of the AVP peptide towards its V2 receptor. To avoid the
cleavage of the ArgꢀGly bond in the vasopressin peptide, the (DꢀArg8)ꢀVasopressin (with the
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nonꢀnatural Dꢀarginine instead of naturally existing Lꢀarginine in the position 8) can be
used.11
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The HPLC chromatograms obtained after incubation of 99mTc(NS3)(CNꢀAVP(an))
conjugate in human or rat serum are shown in Figure 5. The results show that the AVP(an)
biomolecule, being the antagonist to the V2 vasopressin receptor, is stable in both human and
rat serum.
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Figure 5. HPLC chromatograms of 99mTc(NS3)(CNꢀAVP(an)) conjugate after incubation at 37 oC
in human (left) and rat (right) serum.
The electropherograms recorded during paper electrophoresis experiments for 99mTcꢀ
labeled AVP and 99mTcꢀlabeled AVP(an) showed only one peak located at the starting
position (a shift neither towards cathode nor anode was observed). These results confirmed
the expected lack of charge on the studied conjugates.
The log D values obtained for 99mTcꢀlabeled AVP and 99mTcꢀlabeled AVP(an) are close to
each other and are equal to ꢀ0.48±0.02 and ꢀ0.44±0.03, respectively (average values from five
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independent measurements). These values, significantly higher than the lipophilicity of the
free peptide, for which log D is equal to ꢀ2.15,27 may be decreased by introducing a
hydrophilic group, R, at the periphery of the NS3 ligand24,25,28, with no changes in the stability
of the whole conjugate.
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2.2. Binding studies
Affinity of different 99mTcꢀspecies to the V2 receptor of H69 cells is shown in Figure 6.
Vertical axis presents affinity of the studied 99mTcꢀspecies, evaluated as a ratio of activity
bound by cells to that of sum of activity bound by cells and that which remained in the
solution above the cells. As one can see only 99mTcꢀspecies containing vasopressin peptide or
its analogue interact with V2 receptors, and the interaction of 99mTc(NS3)(CNꢀAVP(an)) is
more effective than that of 99mTc(NS3)(CNꢀAVP). Basing on these results the conjugate
99mTc(NS3)(CNꢀAVP(an)) has been selected for the subsequent biological studies.
Figure 6. In vitro cell binding of different 99mTcꢀspecies synthesized in this work. Cell binding
levels are expressed as the percentage of added doses (%ID).
Binding of the conjugate 99mTc(NS3)(CNꢀAVP(an)) to V2 receptor of H69 cells was
found to be saturable and specific in the predominant degree (Figure 7A). The nonꢀspecific
binding determined in the presence of 0.1 ꢀM solution of AVP(an) accounts in general for 6%
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of specific binding. The Bmax value was found to be 0.0058 ± 0.0003 nM (n=3), which
corresponds to an approximate number of 7,000 binding sites per cell. The dissociation
constant Kd, usually used to describe the affinity of the conjugate to its receptor (Figure 7B),
was found to be 0.13 ± 0.04 nM (n=3) and the Ki value, calculated according to the Chengꢀ
Prusoff equation29 was found to be 0.11 ± 0.03 nM. Based on literature data concerning
affinity of different peptidic and nonꢀpeptidic agonists and antagonists to arginine vasopressin
receptors one can say that generally both nonꢀpeptide compounds and agonists are
characterized with lower Kd values (higher binding affinity) than peptide compounds and
antagonists, respectively.13,18,30 Nevertheless the binding reaction is temperature dependent
and tissue is temperature sensitive, as well as the same type of receptor expressed on cell
surface of different cell lines can exhibit different biological behavior, the experimentally
measured values (obtained by scientific teams usually under different conditions) can not be
consider as ‘absolute’ and their comparison need to be qualitative only. Comparing our results
with those presented in literature one can see that 99mTc(NS3)(CNꢀAVP(an)) conjugate is
characterized with significantly low values of Kd and Ki which indicate high binding of the
99mTc(NS3)(CNꢀAVP(an)) conjugate to the V2 receptor of H69 cells.
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Figure 7. (A) Saturation curve for 99mTc(NS3)(CNꢀAVP(an)). The specifically bound
radioligand is plotted as a function of increasing concentration of 99mTc(NS3)(CNꢀAVP(an)).
(B) Scatchard plot.
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In a competitive binding experiment AVP(an) inhibited the binding of 99mTc(NS3)(CNꢀ
AVP(an)) to V2 receptor and the inhibitory concentration of 50% (IC50) was found to be 29 ±
1.3 nM (Figure 8). This value also confirms satisfactorily high binding of 99mTc(NS3)(CNꢀ
AVP(an)) conjugate to V2 receptor in SCLC cells H69.
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Figure 8. Displacement of 99mTc(NS3)(CNꢀAVP(an)) by increasing concentration of unlabeled
AVP(an).
3. EXPERIMENTAL PROCEDURES
3.1. Materials and methods
(Arg8)-Vasopressin peptide was a commercially available product, Bachem, Cat. Number: Hꢀ
1780. The analogue d(CH2)5[D-Tyr(Et2)-Ile4-Eda9]AVP was a gift from Professor Maurice
Manning, University of Toledo, Toledo, Ohio, United States. Human male SCLC (Small Cell
Lung Cancer) cell line NCI-H69 was obtained from Ludwik Hirszfeld Institute of
Immunology and Experimental Therapy, Polish Academy of Sciences (Poland, Wrocław).
ATCC-formulated RPMI-1640 Medium (Catalog No. 30ꢀ2001) was obtained from ATCC
(USA). Cell Dissociation Solution Non-enzymatic 1x (Catalog No. C5914) was obtained
from SigmaꢀAidrich. All solvents and commercially available substances were of reagent
grade and used without further purification. Twice distilled water was used throughout.
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The gradient and HPLC conditions were as follows: solvent A, 0.1% (v/v) TFA in water;
solvent B, 0.1% (v/v) trifluoroacetic acid (TFA) in acetonitrile; System 1: semiꢀpreparative
Phenomenex Jupiter Proteo column, 4 ꢁm, 90 Å, 250×10 mm, UV/Vis detection at 220 nm,
gradient elution: 0ꢀ20 min 20 to 80 % solvent B, 20 min 80 % solvent B; 2 ml/min.; System
2: analytical Phenomenex Jupiter Proteo column, 4 ꢁm, 90 Å, 250 × 4,6 mm, γꢀdetection,
gradient elution: 0ꢀ20 min 20 to 80 % solvent B, 20 min 80 % solvent B; 1 ml/min.
Na99mTcO4 was eluted from a commercial 99Mo/99mTc generator (Radioisotope Centre
POLATOM, Institute of Atomic Energy, Poland).
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Mass Spectrometry (MS): Mass spectra were measured on a Bruker 3000 Esquire mass
spectrometer equipped with ESI.
Infrared (IR): IR spectra in solid KBr pellets (investigated species being about 1% of the
pellet) were recorded in the range 4.000ꢀ600 cmꢀ1 using Bruker Equinox 55 FTꢀIR
spectrophotometer. All spectra were registered independently at least three times with 50
scans each and with spectral resolution of 1 cmꢀ1.
1H NMR and 13C NMR spectra were obtained on a 400 MHz Varian Mercury spectrometer at
room temperature. In the case of 1H NMR chemical shifts were reported as δ values relative to
the internal TMS.
The purity of the synthesized compounds was determined using analytical HPLC and was
found to be more than 95%.
3.2. Syntheses
The tetradentate NS3 ligand (tris(2ꢀmercaptoethyl)ꢀamine; 2,2’,2’’ꢀnitrilotriethanethiol) was
prepared by two steps reaction according to the procedure described in ref.31. In the first step
850 mg (3.52 mmol) of tris(2ꢀchloroethyl)amin hydrochloride and 2.04 g (17.85 mmol) of
potassium thioꢀacetate in 15 ml of ethanol were refluxed (at 75oC) for 5 hours. After the
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solvent removing the residue was dissolved in 4 mL of 1 M aqueous solution of KOH and
extracted three times with diethyl ether. The product of this step – tris(thioacetate) was a
yellow oil. Yield ≅ 68%. In the second step tris(thioacetate), 500 mg (1.54 mmol) dissolved in
5 ml of dry THF, was reduced at 0 °C with LiAlH4, 250 mg (7.15 mmol) dissolved in 10 ml
of dry THF. The final product, yield ≈ 45%, was precipitated as the oxalate salt and applied as
such in further reactions.
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EA: Calcd.: C 36.51; H 7.66; N 7.10; Found: C 36.52; H 7.27; N 7.62
1H NMR (CDCl3) δ (ppm): 1.80 (s, 3H, SH); 2.72 (m, 12H, NCH2 and SCH2)
13C NMR (CDCl3) δ (ppm): 822.81 (H2CS); 57.01 (NCH2)
IR (KBr) (cmꢀ1): 2987 (CH); 2425 (SH)
The aliphatic linker CN-BFCA (BFCA = Bifunctional Coupling Agent, isocyanobutyric
succinimidyl ester) was synthesized in a three step according to procedure described in ref.32
In the first step 4ꢀaminobutanoic acid, 30 g (290.92 mmol), dissolved in 1.5 mL of acetic
anhydride was refluxed with 225 mL of formic acid at 100 ꢀ115°C for 8 h. After the solvent
was removed and the residue was stirred in a mixture of diethyl ether, acetonitrile, and ethyl
acetate (30/5/5, v/v/v), the product was collected and dried. Yield ≈ 30%. In the second step
equal molar quantities of 4ꢀ(formylamino)ꢀbutanoic acid, Nꢀhydroxysuccinimide and
dicyclohexylcarbodiimide were dissolved in 25 mL of dimethylformamide (DMF) and the
reaction was stirred at room temperature overnight. The reaction mixture was filtered, the
solvent was evaporated in vacuo and the residue was dissolved in solvent mixture (diethyl
ether/acetonitrile/ethyl acetate 20/5/5, v/v/v) to crystallize out the product ꢀ 4ꢀ(formylamino)ꢀ
butanoic succinimidyl ester. Yield≈ 50%. In the last step to the suspension of 1 g (4.4 mmol)
of 4ꢀ(formylamino)ꢀbutanoic succinimidyl ester and 1.37
g
(5.24 mmol) of
triphenylphosphine in 6 mL of trichloromethane 214 ꢀL (2.2 mmol) of tetrachloromethane
and 305 ꢀL (2.2 mmol) of triethylamine were added and the mixture was allowed to stirred
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for 3 h at 50 °C. The solvent was evaporated in vacuo and the main product ꢀ isocyanobutyric
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MS, m/z: Calcd. 212.21; Found: 213.1 [M+H+]
1H NMR (CD3CN) δ (ppm): 2.07 (m, 2H, CH2CH2CH2); 2.76 (t, 2H, CH2CO); 3.56 (tt, 2H,
CH2NC)
13C NMR (CD3CN) δ (ppm): 25.6 (CH2CH2CH2); 40.4 (CN); 170.2 (CO)
IR (KBr) (cmꢀ1): 2951, 2926 (CH); 2152 (CN); 1814, 1785, 1729 (CO)
Synthesis of CN-peptide (peptide=AVP, AVP(an)). The coupling reaction of the isocyanide
linker CNꢀBFCA with AVP or AVP(an) peptide is shown in Scheme 1.
Scheme 1. Coupling of CNꢀBFCA linker with peptide.
The solution containing 0.47 mg (2.2 ꢁmol) of CNꢀBFCA dissolved in 100 ꢁL of DMF and
0.5 ꢁL (3.6 ꢁmol) of triethylamine was added to 2 mg (1.8 ꢁmol) of AVP (in the synthesis
with AVP(an) the same molar ratios of all components were used). The mixture was allowed
to stay overnight at room temperature and then the solvent was removed under vacuum. The
residue was dissolved in a mixture of 50 ꢁL of acetonitrile and 100 ꢁL of water. The crude
product was purified by the semiꢀpreparative HPLC (System 1), alkalized and lyophilized.
Yield ≈ 38%.
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MS of CNꢀAVP, (m/z): Calcd: 1179.35, Found: 1179.40 [M+H]+
MS of CNꢀAVP(an), (m/z): Calcd: 1231.61, Found: 616.1 [M+H]+, 1254.3 [M+Na]+
The “cold rhenium precursor” Re(NS3)(PMe2Ph) was synthesized according to the
procedure described in ref.28 The mixture of 20 mL of methanol and 4 mL of EtOH/HCl
containing 80.5 mg (0.41 mmol) of the tetradentate ligand NS3, 98.90 mg (0.71 mmol) of the
monodentate ligand PMe2Ph and 317.78 mg (0.41 mmol) of the rhenium(III) precursor
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Re[SC(NH2)2]6Cl3 H2O was refluxed (at 62 C) under argon atmosphere for 3 hours. The
crude product was purify by column chromatography (Silica Gel, 0.040ꢀ0.063 mm (230ꢀ400
mesh), Merck) using the eluent THF/nꢀHexane (4/3, v/v). Yield ≈ 25
EA: Calcd.: C 32.42; H 4.47; N 2.70; S 18.54; Found: C 32.44; H 4.47; N 2.55; S 18.80
MS, m/z: Calcd.: 519.7; Found: 519.3 [M+H+]
Synthesis of Re(NS3)(CN-AVP) and Re(NS3)(CN-AVP(an)). The nonꢀradioactive rhenium
reference compounds Re(NS3)(CNꢀpeptide) were prepared in two steps (Scheme 2). In the
first step 10.1 mg (19.4 ꢁmol) of ‘cold rhenium precursor’ Re(NS3)(PMe2Ph) and 6.1 mg
(28.7 ꢁmol) of isocyanobutyric succinimidyl ester were dissolved in 1.5 mL of CHCl3 and
stirred at room temperature for 2h. In the second step to the mixture containing 1.02 mg (0.94
ꢁmol) of AVP (in the case of AVP(an) the same molar amount was used) and 0.71 mg (1.2
ꢁmol) of Re(NS3)(CNꢀBFCA) dissolved in 100 ꢁL of DMF we added 0.26 ꢁL (1.85 ꢁmol) of
triethylamine. The mixture was allowed to stand overnight at room temperature. After
completion of the reaction, DMF was removed under vacuum and the residue was dissolved
in 200 ꢁL of acetonitrile/water (1:1). The products of both steps were purified on a
semipreparative HPLC column under the conditions described above (System 1) and
lyophilized. Yield ≈ 45%.
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Re(NS3)(CNꢀAVP): MS (m/z): Calcd: 1559.91, Found: 1560.08 [M+H]+
Re(NS3)(CNꢀ(AVP(an)): MS (m/z): Calcd: 1612.1, Found: 806.8 [M+H]+, 1634.5 [M+Na]+
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Scheme 2. Reaction route to Re(NS3)(CNꢀpeptide).
Preparation of 99mTc(NS3)(CN-AVP) and 99mTc(NS3)(CN-AVP(an)) For labeling AVP and
AVP(an) with 99mTc a twoꢀstep procedure21 was applied (Scheme 3).
Scheme 3. Labeling of AVP and AVP(an) with technetiumꢀ99m complex of the ‘4+1’ type.
99
In the first step 1 mL of eluate from the Mo/99mTc generator (200 – 1000 MBq) was added
to a kit formulation containing 1 mg of Na2EDTA, 5 mg of mannitol and 0.1 mg of SnCl2 in
freezeꢀdried form under nitrogen. The mixture was allowed to stand at room temperature for
20 min. The radiochemical purity was checked by HPLC (System 2) and TLC methods. When
Merck 60 WF254 aluminum sheets are used the intermediate complex 99mTcꢀEDTA/mannitol
migrates with the solvent front in water but remains at the origin in acetone. In the second
step the intermediate 99mTcꢀEDTA/mannitol compound reacted with 300 µg of the NS3 ligand
and with about 50 µg of the isocyanideꢀmodified peptide CNꢀAVP or CNꢀAVP(an). The
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reaction progress and radiochemical purity were controlled by HPLC (System 2). The
radiochemical yields of the 99mTcꢀlabeled conjugates were approximately 95%.
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3.3. In vitro stability studies
Stability studies of 99mTc(NS3)(CN-AVP) and 99mTc(NS3)(CN-AVP(an)). The conjugates
isolated from the reaction mixture (using HPLC System 1), present in the solution in
concentration no higher than 10ꢀ4 mM, were incubated at 37 C with 10 mM solutions of
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histidine or cysteine in the PBS buffer (pH 7.4). HPLC analyses of the incubated solutions
were performed at different time periods from 0.5 h up to 24 h, since staring the incubation.
Stability studies of 99mTc(NS3)(CN-AVP) and 99mTc(NS3)(CN-AVP(an)) in human and rat
serum. For that purpose 0.1 mL of the solution of the isolated 99mTc(NS3)(CNꢀAVP) or
99mTc(NS3)(CNꢀAVP(an)) conjugate in the 0.1 M PB buffer, pH=7.4, was added to 0.9 mL of
human or rat serum (obtained from the Centre of Radiobiology and Biological Dosimetry,
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INCT Warsaw and Sigma Aldrich, respectively) and incubated at 37 C. At specified time
intervals small samples (0.1 ꢀ 0.2 mL) of the mixture were withdrawn, mixed in an Eppendorf
tube with ethanol (0.3 ꢀ 0.5 mL) and vigorously shaken to precipitate proteins. Then, the
samples were centrifuged (14000 rpm, 5 min) and the supernatant was separated. The
radioactivity of both supernatant and precipitate was measured using the wellꢀtype NaI(Tl)
detector. To check if the conjugates did not convert into other waterꢀsoluble radioactive
species, aliquots of the supernatant were analyzed by HPLC for the content of the
99mTc(NS3)(CNꢀpeptide) complex.
3.4. Paper Electrophoresis
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Paper electrophoresis experiments were performed on 20 x 1 cm chromatographic paper
strips, Paper Chromedia GF 83 (Whatman), preꢀtreated with the phosphate buffer (0.1 M, pH
7.40), using a midi horizontal electrophoresis unit (SigmaꢀAldrich) device. The experiments
were carried out at 200 V (10 V cmꢀ1 ) for 60 min for both conjugates. The developed paper
strips were left to dry, and then the distribution of radioactivity on the strips was determined
using an automatic TLC analyzer SCꢀ05 (homeꢀmade, INCT, Warsaw).
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3.5. Lipophilicity studies
Lipophilicity of the 99mTc(NS3)(CNꢀAVP) and 99mTc(NS3)(CNꢀAVP(an)) conjugates,
which is an important factor affecting the distribution of drug molecules in the organism, was
characterized by their distribution coefficients, D, in the system nꢀoctanol/PBS buffer pH=7.4.
The activity of each layer (which shows concentration of the 99mTc species in the layer) was
determined by measuring γꢀradiation, using a wellꢀtype NaI(Tl) detector. Distribution
coefficient D was calculated as the ratio of activity of organic to that of aqueous phase (as an
average value from at least three independent measurements). Immediately after the
distribution experiments, the aqueous phases were analyzed by HPLC to check whether the
studied conjugate had not decomposed during the experiment.
3.6. Cell culture
Human small cell lung cancer NCIꢀH69 cell line was maintained in ATCCꢀformulated
RPMIꢀ1640 Medium enriched in fetal bovine serum to a final concentration of 10% and
supplemented with 0.1 IU/ml penicillin and 0.1 mg/ml streptomycin. Cells were cultured at
37oC in a humidified incubator under an atmosphere containing 5% CO2 and subcultured once
a week. The cells grow in suspension, in the form of multicell aggregates (Figure 9), and
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before cell counting as well as before affinity studies the addition of Cell Dissociation
Solution Nonꢀenzymatic 1x is required in order to disperse the H69 cells (the Cell
Dissociation Solution Nonꢀenzymatic 1x medium was applied according to protocol attached
to the product).
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Figure 9. Human male cell line NCIꢀH69 under microscope (IX81 Motorized Inverted
Microscope, Olympus). Besides single cells the multicell aggregates are visible.
3.7. Affinity studies
Affinity studies were carried out on the human SCLC cell line NCIꢀH69 seeded in 6ꢀwell
plates in RPMIꢀ1640 medium in amount of approximately 5x105 cells/well. Cell count was
determined using a hemocytometer and microscope. Preliminary affinity studies were
performed by incubating H69 cells suspended in 1 mL of RPMIꢀ1640 medium in the presence
of conjugates 99mTc(NS3)(CNꢀAVP) or 99mTc(NS3)(CNꢀAVP(an)), as well as other 99mTcꢀ
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species (99mTcO4 , intermediate compounds 99mTcꢀEDTA/mannitol and 99mTc(NS3)). After 45
min of incubation the binding was stopped and suspended cells were centrifuged. Both
phases, medium from above cells and the cells (after two washing steps in PBS in order to
eliminate unbound activity) were measured in a gamma counter. Affinity of the studied 99mTc
species was calculated as a ratio of activity bound by cells to the sum of activity bound by
cells and remained in the solution aspirated from above the cells (Figure 6).
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In order to determine Kd, NCIꢀH69 cells were grown in 6ꢀwell plates in RPMIꢀ1640 medium
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activity, the cells were centrifuged and counted in a gamma counter. Nonspecific binding was
defined as the amount of activity still bound in the presence of 1 ꢀM solution of AVP(an).
Under these conditions virtually all receptors are occupied by the unlabeled conjugate, so that
the radioconjugate can be bound with cells only via nonspecific interactions. In order to
determine Kd value the data were analysed by the Scatchard plot (Figure 7) using the
relationship between the ratio of Specific bound ligand/Free ligand (B/F) vs. Specific bound
ligand (B). The slope of the straight line indicates the negative reciprocal of Kd (ꢀ1/Kd). The
intercept of the straight line with the B axis represents the maximum concentration of peptide
(Bmax) bound to the V2 receptor of cells (represented by the number of binding sites per cell)
at infinite peptide excess.
3.8. Competitive binding assays
Competitive binding assays were performed for AVP(an) using NCIꢀH69 cells line and
99mTc(NS3)(CNꢀAVP(an)) as the radioligand (Figure 8). Briefly, 20,000 cpm of
99mTc(NS3)(CNꢀAVP(an)) in 0.1 M PBS sterile buffer, pH 7.4, was added to each well in 6ꢀ
well plates in RPMIꢀ1640 medium (approximately 5x105 cells in each well) and incubated at
37 oC for 45 min. Then, the incubation was stopped, the suspended cells were centrifuged and
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washed twice with PBS at 37 C, in order to eliminate unbound activity. Next, the cells were
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incubated again at 37 C for 45 min applying concentrations of AVP(an) in the range 0.005–
1000 nM. After that time the incubation was stopped and the solution from above the cells
was quantitatively separated and measured in a gamma counter. The suspended cells were
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Nonspecific binding was defined, as before, as the amount of activity still bound by cells in
the presence of 1 ꢀM solution of AVP(an). The specific binding was obtained by subtracting
nonspecific binding from total binding. IC50 value was determined from the relationship
between the ratio of the activity of the cells to that of quantitatively separated solution from
above the cells and concentration of AVP(an). Inhibition constant Ki value was calculated
from the IC50 value using the ChengꢀPrusoff equation.29
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4. Conclusions
The 99mTcꢀlabeled vasopressin conjugates show high stability in the presence of an excess
of standard amino acids cysteine or histidine. The proof of their stability is that no
transchelation reactions have been observed in the challenge experiments. In human or rat
serum the 99mTc(NS3)(CNꢀAVP) conjugate is cut into two species as the result of enzymatic
degradation of the peptide, while the 99mTc(NS3)(CNꢀAVP(an)) conjugate, where AVP(an) is
an antagonist to the V2 vasopressin receptor, is quite stable even in rat serum.
The Bmax value obtained for 99mTc(NS3)(CNꢀAVP(an)) conjugate is equal to 0.0058 which
corresponds to an approximate number of 7,000 binding sites per cell. The dissociation
constant Kd equal to 0.13 nM, inhibition constant Ki value equal to 0.09 nM and the IC50
value equal to 29 nM, indicate satisfactorily high binding of the 99mTc(NS3)(CNꢀAVP(an))
conjugate to V2 vasopressin receptor in SCLC cells H69.
In the conclusion one can say that the physicochemical and biological properties of
99mTc(NS3)(CNꢀAVP(an)) conjugate can be an important basis for further consideration the
conjugate as a potential diagnostic radiopharmaceutical for patients suffering from the smallꢀ
cell lung cancer.
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Acknowledgements and notes
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The work has been supported by the statutory activity of the Institute of Nuclear
Chemistry and Technology, Warsaw, Poland.
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The authors thank Prof. Maurice Manning, University of Toledo, Toledo, Ohio, United
States, for the sample of vasopressin analogue d(CH2)5[DꢀTyr(Et2)ꢀIle4ꢀEda9]AVP and Prof.
S. Siekierski (INCT, Warsaw, Poland) for valuable discussion and review of the manuscript.
We would particularly like to thank the team of Dr. HansꢀJürgen Pietzsch (Helmholtzꢀ
Zentrum DresdenꢀRossendorf, Germany) for developing our skills in syntheses of ‘4+1’
technetiumꢀ99m complexes.
Abbreviations used: AVP, arginine vasopressin(1ꢀ9); AVP(an), analogue d(CH2)5[DꢀTyr(Et2)ꢀ
Ile4ꢀEda9]AVP; V2, arginine vasopressin receptor 2; SCLC, smallꢀcell lung cancer; ADH,
antidiuretic hormone; CNS, central nervous system; HPLC, highꢀperformance liquid
chromatography; n.c.a., no carrier added; RCP, radiochemical purity; RCY, radiochemical
yield; NS3, tris(2ꢀmercaptoethyl)ꢀamine; CNꢀBFCA, isocyanobutyric acid succinimidyl ester;
PBS, Phosphate buffered saline; TFA, trifluoroacetic acid
ANCILLIARY INFORMATION
*Corresponding author: Phone +48 22 504 1178. E-mail: e.gniazdowska@ichtj.waw.pl
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