99mTc-labeled NGR-chlorambucil conjugate, 99mTc-HYNIC-CLB-c(NGR) for targeted chemotherapy and molecular imaging

Article abstract of DOI:10.1002/jlcr.3522

Targeted delivery of chemotherapeutic drug at the tumor site enhances the efficacy with minimum systemic exposure. Towards this, drugs conjugated with peptides having affinity towards a particular molecular target are recognized as affective agents for targeted chemotherapy. Thus, in the present study, tumor-homing asparagine-glycine-arginine (NGR) peptide ligand was conjugated to DNA alkylating nitrogen mustard, chlorambucil (CLB). The peptide-drug conjugate (PDC), CLB-c(NGR), was radiolabeled with ^99mTc-HYNIC core to trace its pharmacokinetics and biodistribution pattern. In vitro cell-binding studies of ^99mTc-HYNIC-CLB-c(NGR) were conducted in murine melanoma B16F10 cells. The cytotoxicity studies conducted by incubation of the peptide/drug/PDC with B16F10 cells demonstrated enhanced cytotoxic effect of PDC in comparison to either the peptide or the drug alone. In vivo biodistribution studies in C57BL6 mice bearing melanoma tumor showed maximum tumor uptake at 30?minutes pi (2.45?±?0.28% ID/g), which reduced to 0.77?±?0.1% ID /g at 3?hours pi. The radiotracer being hydrophilic cleared rapidly from the heart, lungs, liver, and muscle. The tumor-to-blood and tumor-to-muscle ratios improved with time. This study opens avenues for conjugation of other targeting peptides with the drug CLB for enhanced toxicity at the diseased site.

Full text of DOI:10.1002/jlcr.3522

99mTc-labeled NGR-Chlorambucil Conjugate, 99mTc-HYNIC-CLB-c(NGR)
for Targeted Chemotherapy and Molecular Imaging
Short title: 99mTc-labeled peptide drug conjugate, 99mTc-HYNIC-CLB-c(NGR)
Kusum Vats1, Drishty Satpati*1, Rohit Sharma1, Chandan Kumar1, Haladhar D Sarma2,
Ashutosh Dash1
1Radiopharmaceuticals Division,
2Radiation Biology and Health Science Division,
Bhabha Atomic Research Centre, Mumbai-400085
Author for correspondence: Drishty Satpati, PhD
Email: drishtys@barc.gov.in
Ph: 91-22-25590748
Fax: 91-22-25505151
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3522
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Abstract
Targeted delivery of chemotherapeutic drug at the tumor site enhances the efficacy
with minimum systemic exposure. Towards this drugs conjugated with peptides having
affinity towards a particular molecular target are recognized as affective agents for targeted
chemotherapy. Thus in the present study tumor homing NGR peptide ligand was conjugated
to DNA alkylating nitrogen mustard, chlorambucil (CLB). The peptide drug conjugate
(PDC), CLB-c(NGR) was radiolabeled with 99mTc-HYNIC core to trace its pharmacokinetics
and biodistribution pattern. In vitro cell binding studies of 99mTc-HYNIC-CLB-c(NGR) were
carried out in murine melanoma B16F10 cells. The cytotoxicity studies carried out by
incubation of the peptide/ drug/ peptide-drug conjugate with B16F10 cells demonstrated
enhanced cytotoxic effect of PDC in comparison to either the peptide or the drug alone. In
vivo biodistribution studies in C57BL6 mice bearing melanoma tumor showed maximum
tumor uptake at 30 min p.i. (2.45  0.28% ID/g) which reduced to 0.77  0.1% ID /g at 3 h
p.i. The radiotracer being hydrophilic cleared rapidly from heart, lungs, liver and muscle. The
tumor-to-blood ratio and tumor-to-muscle ratios improved with time. This study opens
avenues for conjugation of other targeting peptides with the drug chlorambucil for enhanced
toxicity at the diseased site.
Keywords: Chlorambucil, NGR peptide, peptide-drug conjugate, 99mTc-HYNIC, cytotoxicity
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1. Introduction
Chemotherapy is an important clinical tool for treatment of different types of cancers.
However chemotherapeutic drugs do not spare normal cells during their attack at malignant
sites posing limitation on the prolonged drug therapy needed in case of metastatic patients.
The high dose administration of drugs during conventional chemotherapy is responsible for
systemic toxicity and various other side-effects. Drugs conjugated with the targeting vectors
like antibodies and peptides can serve as conduits for site-specific delivery and thus
overcome the problem of non-specific accumulation of drugs in normal tissues.1-3 The tumor
vasculature (tumor homing) targeting arginine-glycine-aspartic acid (RGD) and asparagine-
glycine-arginine (NGR) peptides have been explored and identified as effective drug
carriers.4-6 The RGD and NGR peptides conjugated with human tumor necrosis factor
(hTNF) are reported to exhibit enhanced antitumor action at smaller doses.7-9 The peptide–
drug conjugates (PDCs) can thus precisely deliver the drug to the cancer cells and provide
more efficacious results. The peptide-drug conjugate (PDC), NGR-hTNF is under clinical
investigation for malignant pleural mesothelioma, colorectal, lung (small-cell and non-small-
cell), liver and ovarian cancer, and soft tissue sarcomas.10-12
Chlorambucil (CLB) is a widely used chemotherapeutic drug in the treatment of
chronic lymphocytic leukemia, lymphomas, breast and ovarian carcinoma and a variety of
other solid tumors.13,14 Chlorambucil, 4-{4-[Bis(2-chloroethyl)amino]phenyl}butyric acid
with two reactive chloroethyl side chains belongs to the group of alkylating agents referred to
as nitrogen mustards.14,15 Chlorambucil exerts its cytotoxic action by forming inter-strand
cross-links in DNA through alkylation at N7 of guanine base.15,16 The goal of this study was
to prepare NGR-chlorambucil conjugate for targeted chemotherapy enhancing clinical benefit
of the drug. Radiolabeled PDCs can provide insight into the pharmacokinetics of PDC and
the drug therapy response through non-invasive molecular imaging of tumor sites. 99mTc is an
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ideal radioisotope for diagnostic purposes due to its favourable decay characteristics (t1/2 = 6
h, 140 keV).17,18 Amongst the various approaches available for 99mTc-labeling we focused on
99mTc-HYNIC core (HYNIC = 6-hydrazinonicotinic acid) as it offers high labeling efficiency
and allows for preparation of molecular imaging probes with high specific activity and
promising pharmacokinetic profile.19,20 HYNIC also has an advantage of simple and easier
conjugation with peptides either on-resin during solid phase peptide synthesis or in solution
phase.21-23
In the present study cyclic NGR peptide, cKCNGRC was synthesized manually using
standard Fmoc solid phase peptide synthesis. The peptide was then conjugated with the drug,
chlorambucil and subsequently functionalized with HYNIC chelator. The peptide-drug
conjugate, HYNIC-chlorambucil-cKCNGRC [HYNIC-CLB-c(NGR)] was radiolabeled with
99mTc. The radiotracer, 99mTc-HYNIC-chlorambucil-cKCNGRC [99mTc-HYNIC-CLB-
c(NGR)] was investigated for its efficacy as tumor targeted molecular imaging probe. The in
vitro cell binding study and cytotoxicity studies were carried out in human melanoma
B16F10 cells. In vivo biodistribution studies were performed in C57BL6 mice bearing
melanoma tumor.
2. Materials and Methods
2.1 General
The amino acid derivatives Nα-Fmoc-Nγ-trityl-L-asparagine, Nα-Fmoc-NΩ-
(2,2,4,6,7-pentamethyl-2,3-dihydrobenzo[b]furan-5ylsufonyl)-L-arginine, Nε-Boc-Nα-Fmoc-
L-lysine were purchased from Alfa Aesar, USA. Fmoc-Cys(Acm)-OH and Fmoc-Gly-OH
were purchased from Sigma Aldrich. NovaSyn TGR resin was procured from Novabiochem,
Germany. 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
hexafluorophosphate (HATU) was procured from Alfa Aesar, USA. Chlorambucil, N,N-
diisopropylethylamine (DIPEA), Piperidine, 6-chloronicotinic acid, di-tert-butyl dicarbonate,
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sodium dihydrogen phosphate, disodium hydrogen phosphate, ethylenediamine diacetic acid
(EDDA), N-[Tris(hydroxymethyl)methyl]glycine (Tricine) and hydrogen chloride solution
4.0 M in dioxane were procured from Sigma Aldrich, USA. Stannous chloride dihydrate was
procured from Fluka. The peptide drug conjugate HYNIC- CLB-c(NGR) was purified using a
semi-preparative HPLC system (JASCO, Japan) connected with JASCO-PU-2086 Plus,
intelligent prep pump, JASCO UV-2075 Plus absorption detector and having a Megapak Sil
C18-10 column (7.5 × 250 mm). The analytical HPLC measurements were performed on a
JASCO PU 2080 Plus dual pump HPLC system, Japan, with a JASCO 2075 Plus tunable
absorption detector and a Gina Star radiometric detector system, using a C18 reversed phase
HiQ Sil column (5 μm, 4 x 250 mm). The eluting solvents (1 mL/min) used in HPLC were;
H2O (solvent A) and acetonitrile (solvent B) with 0.1% trifluoroacetic acid following the
gradient: 0-3 min: 0% B; 3-5 min: 0-20% B; 5-10 min: 20% B; 10-20 min: 20-90% B; 20-22
min: 90-0% B; 22-30 min: 0% B. Matrix assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry analysis was carried out at the Mass spectrometry facility
of Tata Memorial Centre, Advanced Centre for Treatment, Research and Education in Cancer
(ACTREC).
2.2 Synthesis
2.2.1 Synthesis of succinimidyl-6-hydrazinopyridine-3-carboxylate hydrochloride (NHS-
HYNIC)
Synthesis of NHS-HYNIC was carried out following a reported procedure, using 6-
chloronicotinic acid as the starting material.23 Briefly, 6-chloronicotinic acid (1 g, 6.35 mmol)
was added to 80% hydrazine hydrate (8 mL) and stirred at 100˚C for 4 h. After cooling to
room temperature the reaction mixture was concentrated under reduced pressure to give white
solid. The solid was dissolved in water and adjustment of pH of the solution to 5.5 resulted in
precipitate formation. The precipitate was filtered, washed with ethanol and dried in vacuum
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to give 6-hydrazinopyridine-3-carboxylic acid (845 mg). Subsequently protection of
hydrazine group was carried out by addition of triethylamine (365 µL, 2.62 mmol) and di-t-
butyl-dicarbonate (285 mg, 1.31 mmol) to a solution of 6-hydrazinopyridine-3-carboxylic
acid (200 mg, 1.31 mmol) in DMF and the reaction was stirred at room temperature for 16 h.
The reaction mixture was then concentrated under reduced pressure to give a brown solid.
The crude product was purified by silica gel column chromatography eluting with ethyl
acetate to obtain 6-BOC-hydrazinopyridine-3-carboxylic acid. In the next step, a solution of
6-BOC-hydrazinopyridine-3-carboxylic acid (200 mg, 0.79 mmol) and N-
hydroxysuccinimide (92 mg, 0.80 mmol) in DMF was added to a solution of
dicyclohexylcarbodiimide (165 mg, 0.80 mmol) in DMF and stirred overnight at room
temperature. Upon completion of the reaction, the reaction mixture was filtered and the
filtrate was concentrated to dryness to obtain a brown residue. The crude product obtained
was purified by silica gel column chromatography using ethyl acetate as the eluting solvent to
obtain succinimidyl-6-BOC-hydrazinopyridine-3-carboxylic acid (105 mg).
To succinimidyl-6-BOC-hydrazinopyridine-3-carboxylic acid (100 mg) in a sealed
flask under nitrogen environment, 4.0 M HCL in dioxane (2 mL) was added. The reaction
mixture was stirred at room temperature for 4 h. The solvent was removed under reduced
pressure and the residue was washed with THF and dried. A yellowish white solid (42 mg)
was obtained. ESI-MS: m/z 251.1 observed (calcd. for C10H10N4O4: 250.1)
2.2.2 Synthesis of Chlorambucil-c(KCNGRC)-NH2 [CLB-c(NGR)]
The fully protected peptide chain Lys(Boc)-Cys(Acm)-Asn(Trt)-Gly-Arg(Pbf)-Cys(Acm)-
NH2 was manually synthesized by standard Fmoc solid phase synthesis on NovaSyn TGR
resin. Coupling of each amino acid was carried out using standard Fmoc strategy where 3-
fold excess of amino acid as well as O-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU) was used along with 6-fold excess of N,N-
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diisopropylethylamine (DIPEA) in dimethylformamide (DMF) for 90 min. The reaction was
monitored by picrylsulphonic acid test and the Fmoc groups were removed by treatment with
20% piperidine in DMF for 30 min. The peptide at N-terminus was conjugated with
chlorambucil, an anticancer DNA-alkylating drug, in presence of HATU and DIPEA. The
peptide-drug conjugate was then cyclized by simultaneous deprotection and oxidation of
acetamidomethyl thiol protected cysteines on solid support with thallium (III) trifluoroacetate
and cleaved from the resin using a cocktail mixture of trifluoroacetic acid
(TFA)/ethanedithiol/triisopropylsilane/water (94:2.5:2.5:1 v/v). The crude peptide-drug
conjugate was precipitated, washed three times with diethyl ether, purified by semi-
preparative HPLC and subsequently lyophilized to obtain white fluffy powder. MALDI-TOF
MS: m/z 962.1 observed (calcd for C38H61Cl2N13O8S2: 963).
2.2.3 Synthesis of HYNIC-Chlorambucil-c(KCNGRC)- [HYNIC-CLB-c(NGR)]
The peptide-drug conjugate, CLB-c(NGR) was functionalized with the HYNIC chelator at ε-
amino group of lysine and the reaction was carried out in solution phase. Briefly, NHS-
HYNIC (2.2 mg, 8 µmol) was added to the purified CLB-c(NGR) (6 mg, 6 µmol) conjugate
dissolved in DMF (500 µL) and stirred at room temperature overnight. The HYNIC-CLB-
c(NGR) conjugate thus formed was purified by semi-preparative HPLC and characterized by
mass spectroscopy. MALDI-TOF MS: m/z 1211.9 [M+CF3COOH] observed (calcd for
C44H66Cl2N16O9S2: 1098.1).
2.3 Radiolabeling
The purified HYNIC functionalized peptide-drug conjugate HYNIC-CLB-c(NGR) was
radiolabeled with 99mTc using stannous chloride (SnCl2) as reducing agent and EDDA and
tricine as co-ligands. The conjugate HYNIC-CLB-c(NGR) (40 µg) was mixed with EDDA
(200 μL, 50 mg/mL in 0.1N NaOH), tricine (100 μL, 200 mg/mL in distilled water),
disodium hydrogen phosphate (6.4 mg), sodium dihydrogen phosphate (21 mg), Na99mTcO4
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(500 μL, 370 MBq) and SnCl2 (20 μg). The reaction mixture was subsequently incubated at
90˚C for 15 min. The radiochemical yield and purity (RCP) of the radiotracer was determined
by TLC and HPLC respectively.
2.4 Determination of octanol-water partition coefficient (Log Po/w)
Radiotracer (50 µL, ~3.7 MBq) was mixed with distilled water (950 µL) and n-octanol (1
mL) on a vortex mixer for about 1 min and then centrifuged (3500 g) for 5 min to get clear
separation of the two layers. The radioactivity associated with 100 μL samples of both the
aqueous and the organic phases was measured in well type NaI (Tl) counter. This was
repeated thrice and the measurements were used to calculate the Log Po/w of the radiotracer.
2.5 Stability studies
The stability of the radiotracer, 99mTc-HYNIC-CLB-c(NGR) was determined in saline and
human serum. The radiotracer was incubated in saline at 37˚C and the stability was assayed 6
h post-preparation by HPLC analyses. To determine serum stability, the radiotracer (50 μL)
was incubated in human serum (250 µL) at 37˚C for 6 h. About 100 μL aliquot of this
solution was withdrawn; the serum proteins were precipitated by the addition of 100 μL of
acetonitrile. The mixture was centrifuged at 3500 g for 10 minutes and the supernatant was
analyzed by TLC and HPLC to check the stability of the radiotracer in serum.
2.6 In vitro cell binding studies
The murine melanoma cells B16F10 were obtained from National Center for Cell Sciences
(NCCS) Pune, India. The cells were cultured in Dulbecco’s modified Minimum Essential
Medium (DMEM) supplemented with 10% fetal calf serum (Invitrogen Carlsbad, CA) and
1% antibiotic/antimycotic formulation. Cells were incubated at 37˚C in a humidified
atmosphere containing 5% CO2. For cell binding studies, B16F10 cells (1 × 105) were seeded
in 24-well tissue culture plates and incubated at 37˚C overnight. The cells were subsequently
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incubated with 99mTc-HYNIC-CLB-c(NGR) (37 kBq/well) at 37˚C for 1 h. After incubation
cells were washed twice with ice-cold phosphate buffer saline (PBS) and the cells were
harvested by trypsinization. At the end of trypsinization, wells were examined under a light
microscope to ensure complete detachment of cells. Cell suspensions were collected and
radioactivity associated with cells was measured in a NaI (Tl) gamma counter. The activity in
these cell suspensions as percentage of total input radioactivity was calculated to determine
the cell uptake data. The experiment was carried out in triplicate. Specific uptake was
determined by pre-incubation of cells with 100-fold excess of peptide [c(KCNGRC)].
Percentage inhibition values were calculated as [{difference between cell binding of
radiotracer in absence and presence of cold peptide)/cell binding in absence of
peptide}×100].
2.7 Cytotoxicity studies
To determine cytotoxicity of the peptide-drug conjugate the colorimetric MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was carried out. All the
samples were prepared with maximal final DMSO concentration <0.05%. The cells
(3×103/well) were cultured in a 96-well plate overnight at 37˚C and subsequently exposed to
varying concentrations of peptide (cNGR), drug (CLB) and PDC (CLB-cNGR) (1-50 µM) for
48 h. Cells treated with medium only served as control group. At the end of incubation, 20
µL of MTT solution (5 mg/mL in PBS) was added in each well and incubated for 3 h. After
removing the supernatant, 150 µL of solubilizing solution (10% Triton X-100 in acidic iso-
propanol) was added. The absorbance was measured at 570 nm with reference to 630 nm in
BioTek Universal Microplate Reader (BioTek USA, Winooski, VT). The difference between
these measurements was used for calculating the % of growth inhibition (% GI) in test wells
compared to the control. The control cells were treated with medium containing only 0.05%
DMSO solvent. All the tests were done in quadruplicates.
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2.8 Biodistribution studies
Biodistribution studies were carried out in C57BL6 mice bearing murine melanoma
(B16F10) tumor. The radiotracer (~3.7 MBq/animal, 100 µL) was injected intravenously into
the tail vein of each mice (n = 4). The animals were sacrificed at different time intervals (30,
60, and 180 min), and the relevant organs excised for measurement of retained activity. For
blocking studies the peptide, c(NGR) (15 mg/kg) was co-administered along with the
radiotracer and mice were sacrificed at 60 min p.i. The excised organs were weighed and the
activity associated with them was measured in a flat-bed type NaI (Tl) counter with suitable
energy window for 99mTc. The activity retained in each organ/tissue was expressed as a
percent value of the injected dose per gram (% ID/g ± S.D). All the animal experiments were
carried out in compliance with the relevant national laws, as approved by the local committee
on the conduct and ethics of animal experimentation.
2.9 Data analysis
Statistical data are reported as mean ± standard deviation (S.D.). Paired two-tailed Student's t
tests were done to evaluate statistical significance, where p<0.05 was considered to be
statistically significant.
3. Results and Discussion
3.1 Synthesis and radiolabeling
The peptide-drug conjugate, HYNIC-CLB-c(NGR) (Scheme 1) could be prepared
with >99% purity [as ascertained by UV-HPLC chromatogram (Figure 2)] by standard Fmoc
solid phase synthesis method and was characterized by mass spectroscopy. Since HYNIC
occupies a single co-ordination site EDDA and tricine were used as co-ligands to complete
the coordination sphere and form a stable complex with 99mTc. The peptide-drug conjugate
HYNIC-CLB-c(NGR) was radiolabeled with 99mTc with specific activity of 9.8 GBq/µmol.
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Schematics of radiosynthesis of 99mTc-HYNIC-CLB-c(NGR) is presented in Figure 3. The
radiochemical yield (RCY) and purity (RCP) of the radiotracer was determined by TLC and
HPLC respectively. In TLC system using methanol: 1 M ammonium acetate (1:1, v/v) as the
-
mobile phase 99mTc-HYNIC-CLB-c(NGR) and 99mTcO4 had Rf = 1.0 whereas colloid,
99mTcO2 remained at the point of spotting with Rf = 0. In case of 0.1 M sodium citrate mobile
-
phase, Rf = 0 was observed for 99mTc-HYNIC-CLB-c(NGR) and 99mTcO2 whereas 99mTcO4
had Rf = 1.0. The RCY of 99mTc-HYNIC-CLB-c(NGR) was thus determined to be 98 ± 0.5%.
The RCP as determined by HPLC radio-chromatograms for 99mTc-HYNIC-CLB-c(NGR) was
98 ± 1.5%. Retention time of 99mTc-HYNIC-CLB-c(NGR) was 14.1 min (Figure 3). The
possibility of any interaction of chlorambucil moiety with the co-ligands, EDDA and tricine
was investigated by incubation of the co-ligands with the peptide-drug conjugate, HYNIC-
CLB-c(NGR) under similar conditions of radiolabeling. No change in the retention
time/pattern of the peptide-drug conjugate, HYNIC-CLB-c(NGR) in UV-HPLC was
observed thus ruling out the possibility of any adduct formation of chlorambucil with co-
ligands. The radiotracer showed excellent in vitro stability in saline as no change in the
HPLC radio-chromatogram pattern was observed after 6 h incubation period. The radiotracer
was found to be 95% intact after 6 h incubation in human serum at 37˚C. Radio-TLC also
confirmed the HPLC results and no increase in any colloidal species were observed on
incubation of the radiotracer with human serum. These data suggest sufficient stability of the
radiotracer for further carrying out in vivo studies.
The partition coefficient (Log Po/w) was determined to be -2.19 ± 0.1 for 99mTc-
HYNIC-CLB-c(NGR) suggesting that the radiotracer is highly hydrophilic.
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3.2 In vitro cell binding studies
Cell binding studies were performed in murine melanoma B16F10 cells. The uptake
value of 0.92 ± 0.09% was observed for 99mTc-HYNIC-CLB-c(NGR). During inhibition
studies with excess of unlabeled c(NGR) peptide the uptake reduced to 0.59 ± 0.05%.
The cytotoxicity of the PDC was compared with that of the peptide, cNGR as well as
with the drug, CLB alone by carrying out cell cytotoxicity assay in murine melanoma
B16F10 cells. The cells cultured for a period of 24 h were incubated with the different
concentrations of compounds for 48 h and the cytotoxicity of the compounds was determined
by using MTT cell viability assay (Figure 4). The results are expressed as % growth
inhibition compared with cells incubated with only the medium. Figure 4 demonstrates that
the exposure of cells to PDC resulted in significant growth inhibition at all the concentrations
as compared to the drug or the peptide alone. It was observed that nearly 25-fold excess
concentration of the drug is required to achieve similar cytotoxicity as obtained on exposure
to PDC. RGD peptide-chlorambucil conjugate has also been reported to show higher growth
inhibition in B16F10 cells as compared to the drug or peptide alone.24 The enhanced
cytotoxic effect of PDC even at lower levels indicates clear advantage in reducing the
systemic exposure and side effects of the drug. The conjugation of the drug with the targeting
peptide would thus allow targeted delivery and killing of cancer cells increasing the
chemotherapeutic efficacy of the drug.
3.3 Biodistribution studies
Results of biodistribution studies of 99mTc-HYNIC-CLB-c(NGR) in C57BL6 mice
bearing melanoma tumor are summarized in Table 1. The maximum tumor uptake of 2.45 ±
0.28% ID/g was observed at 30 min p.i. The radiotracer cleared rapidly from non-target
organs like heart, lungs, liver and muscle. The clearance of 99mTc-HYNIC-CLB-c(NGR)
from the blood pool was reflected in the improved tumor/blood ratio with time. The tumor-to-
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blood ratio and tumor-to-muscle ratios improved with time attaining a maximum of 2.5 ± 0.4
and 4.23 ± 0.57 at 180 min p.i. (Figure 5).
The blocking experiment carried out by co-injection of 15 mg/kg of cold c(NGR)
peptide, resulted in 35% reduction in the uptake of 99mTc-HYNIC-CLB-c(NGR) in melanoma
(B16F10) tumor.
The chemotherapeutic drug chlorambucil radiolabeled with 99mTc-tricarbonyl core
using two different chelators containing N2O and NNN as donor atoms has been reported.25,26
The two 99mTc(CO)3-labeled chlorambucil complexes had high log P values of 1.2 and 2.34
respectively and thus exhibited high liver and intestinal activities. 99mTc(CO)3-chlorambucil
complex with N2O donors showed increase in the stomach activity indicating decomposition
of the radiotracer. However the currently studied radiotracer tagged with the peptide had
lower log P value -2.19±0.1 and the high hydrophilicity of the radiotracer was visible in its
clearance from all the non-target organs. This suggests that off-target toxicity of drugs can be
reduced by conjugation to the targeting vector, peptide. Radiolabeled peptide-drug conjugates
offer an advantage of tracking the pathway of the drug, monitoring the reduction in tumor
growth and thus ascertain the future application of PDC in targeted chemotherapy.
4. Conclusion:
This study reports the conjugation of chemotherapeutic drug, chlorambucil with the tumor
targeting peptide, cNGR. The PDC showed enhanced inhibitory effect on the cell growth as
compared to the peptide or the drug alone during cytotoxicity assays. The PDC was
functionalized with HYNIC chelator for radiolabeling with 99mTc. The radiolabeled PDC,
99mTc-HYNIC-CLB-c(NGR) could be obtained in high radiochemical yield. The high
hydrophilicity of the radiotracer resulted in rapid clearance from the non-target organs. This
study thus paves the way for designing of different peptide-drug conjugates for targeted
chemotherapy that can reduce the limitations while using the drug alone.
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Acknowledgements
Authors are thankful to Dr. B. S. Tomar, Director, Radiochemistry and Isotope Group,
Bhabha Atomic Research Centre (BARC) for his support.
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Scheme 1
Scheme 1: Synthesis of HYNIC-Chlorambucil-c(KCNGRC) [HYNIC-CLB-c(NGR)]
Reagents and conditions: a) N-Fmoc-Cys(Acm)-OH, HATU, DIPEA, DMF, rt, 2 h; b) 20%
piperidine in DMF, rt, 30 min (x 2); c) N-Fmoc-Arg(Pbf)-OH, HATU, DIPEA, DMF, rt, 2 h;
d) N-Fmoc-Gly-OH, HATU, DIPEA, DMF, rt, 2 h; e) N-Fmoc-Asn(Trt)-OH, HATU,
DIPEA, DMF, rt, 2 h; f) N-Fmoc-Cys(Acm)-OH, HATU, DIPEA, DMF, rt, 2 h; g) N-Fmoc-
Lys(Boc)-OH, HATU, DIPEA, DMF, rt, 2 h; h) 20% piperidine in DMF, rt, 30 min (x 2); i)
Chlorambucil, HATU, DIPEA, DMF, rt, 2 h; j) Tl(III)trifluoroacetate, DMF, rt, 2 h; k)
TFA/ethanedithiol/triisopropylsilane/water (94:2.5:2.5:1 v/v), rt, 2 h; l) Succinimidyl-6-
hydrazinopyridine-3-carboxylate hydrochloride (NHS-HYNIC), DMF, rt, 12 h
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Figure 2
Figure 2: UV-HPLC (220 nm) chromatogram of HYNIC-CLB-c(NGR)
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Figure 3
Figure 3: Radio-synthesis and HPLC radio-chromatogram of 99mTc-HYNIC-CLB-c(NGR)
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Figure 4
Figure 4: Growth inhibition effect of c(NGR), CLB and CLB-c(NGR) on B16F10 cells after
48h of exposure. The error bars represent standard deviation. *p < 0.05 and **p <
0.005.
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Figure 5
Figure 5: Variation in the uptake ratios of tumor/blood and tumor/muscle with time for 99mTc-
HYNIC-c(NGR)-CLB
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Table 1: Biodistribution pattern of 99mTc-HYNIC-CLB-c(NGR) in C57BL6 mice bearing
melanoma (B16F10) tumor
% ID/g (s.d) (n = 4)
Organs
60 min
30 min
60 min
180 min
(Blocking)
0.54 (0.04)
0.94 (0.12)
2.73 (0.15)
0.88 (0.08)
2.73 (1.15)
0.35 (0.02)
1.20 (0.4)
Blood
Liver
2.15 (0.11)
2.49 (0.76)
3.32 (1.00)
1.00 (0.35)
3.82 (1.26)
0.33 (0.05)
1.50 (0.55)
0.47 (0.05)
0.90 (0.25)
2.45 (0.28)
1.15 (0.18)
2.75 (0.42)
0.77 (0.15)
1.75 (0.03)
4.03 (0.22)
0.52 (0.22)
1.78 (0.21)
0.67 (0.11)
0.63 (0.38)
0.68 (0.21)
0.51 (0.10)
1.48 (0.16)
1.92 (0.16)
3.00 (0.34)
0.31 (0.24)
0.79 (0.32)
3.25 (1.29)
1.36 (0.77)
1.44 (0.38)
0.28 (0.10)
0.33 (0.19)
0.50 (0.02)
0.17 (0.06)
0.77 (0.10)
2.50 (0.40)
4.23 (0.57)
Intestine
Stomach
Kidney
Heart
Lungs
Spleen
Muscle
Tumor
T/B
0.41 (0.26)
0.34 (0.12)
0.93 (0.06)
T/M
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99mTc-labeled NGR-Chlorambucil Conjugate, 99mTc-HYNIC-CLB-c(NGR)
for Targeted Chemotherapy and Molecular Imaging
Kusum Vats1, Drishty Satpati*1, Rohit Sharma1, Chandan Kumar1, Haladhar D Sarma2,
Ashutosh Dash1
Peptide (cNGR)
NH2
NH
HN
100
O
c(NGR)
CLB
**
**
O
NH2
CLB-c(NGR)
80
60
40
20
0
NH
**
**
PDC [CLB-cNGR]
O
S
NH
S
*
*
*
**
*
HN
O
H
H
N
N
O
N
H
O
O
O
O
N
NH2
Cl
1
5
10
25
50
NH
C
Concentration(µM)
Cl
Drug
(Chlorambucil)
N
NH
NH2
Chelator (HYNIC)
In this study the drug chlorambucil was conjugated with the peptide c(NGR) to prepare the
peptide-drug conjugate (PDC), CLB-c(NGR) for targeted chemotherapy. The PDC exhibited
enhanced cell cytotoxicity than peptide, cNGR or drug, chlorambucil alone. The radiolabeled
PDC, 99mTc-HYNIC-CLB-c(NGR) being highly hydrophilic cleared rapidly from the non-
target organs during biodistribution studies. The PDCs thus have a promising potential for
future use in targeted chemotherapy.
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