Synthesis and evaluation of 90Y-DOTA-colchicine conjugate in murine fibrosarcoma model

Article abstract of DOI:10.1002/jlcr.1115

Colchicine is a cytotoxic bioactive alkaloid that exhibits its action by microtubular binding. With an aim to develop a tumor targeted radio-therapeutic agent, colchicine has been functionalized to trimethylcolchicinic acid and conjugated to the isothiocyanato derivative of DOTA (1,4,7,10-tetraaza cyclododecane tetracetic acid). DOTA coupled colchicine was radiolabeled with ⁹⁰Y, one of the most commonly used therapeutic radioisotope. Complexation of 200 μg of the conjugate with ⁹⁰Y was carried out at pH 4.5 with an incubation time of 45 min at 70°C. Complexation yield of ⁹⁰Y-DOTA-NCS-colchicine was confirmed to be >98% using C-18 reverse phase HPLC system. ⁹⁰Y-colchicine complex could be differentiated from ⁹⁰Y-p-NCS-benzyl-DOTA on the basis of difference in their retention times 8 and 4 min, respectively in a standardized HPLC system. Biodistribution studies in Swiss mice fibrosarcoma tumor model showed an uptake of ～0.8% ID/g tumor at 3 h.p.i. that was retained till 24 h.p.i. ⁹⁰Y-DOTA-NCS-colchicine complex showed excellent pharmacokinetics with major portion of the radioactivity being excreted out within 3 h.p.i. and no accumulation of radioactivity in vital organs. Copyright

Full text of DOI:10.1002/jlcr.1115

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd. Radiopharm 2006; 49: 951–958
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1115
Research Article
90
Synthesis and evaluation of Y-DOTA-Colchicine
conjugate in murine fibrosarcoma model
Drishty Satpati1, Aruna Korde1, Usha Pandey1, Prem Dhami2,
Sharmila Banerjee1 and Meera Venkatesh1,*
1 Radiopharmaceuticals Division, Bhabha Atomic Research Centre,
Mumbai 400 085, India
2 Fuel Reprocessing Division, Bhabha Atomic Research Centre, Mumbai 400 085,
India
Summary
Colchicine is a cytotoxic bioactive alkaloid that exhibits its action by microtubular
binding. With an aim to develop a tumor targeted radio-therapeutic agent, colchicine
has been functionalized to trimethylcolchicinic acid and conjugated to the
isothiocyanato derivative of DOTA (1,4,7,10-tetraaza cyclododecane tetracetic acid).
DOTA coupled colchicine was radiolabeled with 90Y, one of the most commonly used
therapeutic radioisotope. Complexation of 200 mg of the conjugate with 90Y was
carried out at pH 4.5 with an incubation time of 45 min at 708C. Complexation yield
of 90Y-DOTA-NCS-colchicine was confirmed to be >98% using C-18 reverse phase
HPLC system. 90Y-colchicine complex could be differentiated from 90Y-p-NCS-
benzyl-DOTA on the basis of difference in their retention times 8 and 4 min,
respectively in a standardized HPLC system. Biodistribution studies in Swiss mice
fibrosarcoma tumor model showed an uptake of ꢀ0.8% ID/g tumor at 3 h.p.i. that
was retained till 24 h.p.i. 90Y-DOTA-NCS-colchicine complex showed excellent
pharmacokinetics with major portion of the radioactivity being excreted out within
3 h.p.i. and no accumulation of radioactivity in vital organs. Copyright # 2006 John
Wiley & Sons, Ltd.
Received 28 June 2006; Revised 12 July 2006; Accepted 12 July 2006
Key Words: Colchicine; 90Y; tumor therapy; DOTA
*Correspondence to: M. Venkatesh, Radiopharmaceuticals Division, Bhabha Atomic Research Centre,
Mumbai 400 085, India. E-mail: meerav@barc.gov.in
Copyright # 2006 John Wiley & Sons, Ltd.

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D. SATPATI ET AL.
Introduction
Colchicine is an alkaloid occurring in the seeds of the plant Colchicum
autumnale. It is one of the natural products used for the treatment of acute
gout. Colchicine binds to tubulin and exhibits antimitotic action in dividing
cells. The toxic nature of colchicine precludes its use for cancer treatment.
Research is being carried out to develop various analogues of colchicine to
reduce its toxicity. In this respect, various novel structurally modified
colchicine analogues as well as different aspects of mechanism of its biological
activity have been reported in recent literature.1–5 The potential of colchicine
for tumor imaging and assessing antiangiogenic effect has been very well
documented through 99mTc-ethylenedicysteine colchicine (99mTc-EC colchi-
cine).6 Radiolabeling and evaluation of different colchicine derivatives with
[
99mTc(CO)3(H2O)3]+ and [99mTcN]2+ cores for use as tumor imaging agents
has already been reported by us.7 We have tried to explore the potential of
colchicine for tumor therapy by radiolabeling it with 90Y, a therapeutic
radioisotope.
90Y is one of the radioisotopes used for therapy due to its ideal decay
characteristics (T1=2 ¼ 64 h, EbꢁðmaxÞ ¼ 2:28 MeV), availability in no-carrier
added form from a 90Sr/90Y generator system and amenable chemistry for
labeling biomolecules. 90Y, due to its high specific activity has been the
preferred choice for radiolabeling of antibodies, peptides, etc. for development
of receptor specific therapeutic radiopharmaceuticals.8,9 The clinical success of
the FDA approved radiopharmaceutical Zevalin (90Y-ibritumomab) for
treatment of non-Hodgkin’s lymphoma has paved the way for 90Y to be a
preferred radionuclide for targeted radiotherapy. 90Y is also effective for
treatment of solid tumors due to its high range in tissues (11 mm).10
As direct labeling of colchicine or its analogues is not easy to achieve with
lanthanides, an indirect way of using suitable bifunctional chelating agent
(BFCA) is the choice. For labeling with 90Y, colchicine has been modified
suitably and then conjugated to a bifunctional chelating agent DOTA-NCS
[paraisothiocyanato benzyl 1,4,7,10-tetraazacyclododecane 1,4,7,10 tetra
acetic acid (DOTA)] which is known to form kinetically stable complexes
with 90Y.11,12 In this direction, trimethyl colchicinic acid was prepared by acid
hydrolysis of colchicine to introduce an amino group which was then further
used for conjugation with functionalized DOTA derivative viz. p-NCS-benzyl-
DOTA. In the present paper, we report studies on preparation of 90Y labeled
p-NCS-benzyl-DOTA-colchicine and its bio-evaluation in murine fibrosarco-
ma model.
Results and discussion
Modification of colchicine to a suitable precursor was necessary for
subsequent complexation with 90Y. Therefore, acid hydrolysis of colchicine
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

90Y-COLCHICINE FOR TUMOR THERAPY
953
CH O
3
CH O
3
NHCOCH
3
NH
O
2
o
100 C, 5h
CH O
3
CH O
3
30% H2SO4
CH O
3
O
CH O
3
OCH
3
OH
Trimethylcolchicinic acid
Colchicine
HOOCH C
2
CH O
3
CH COOH
2
NH
N
2
N
CH O
3
NCS
+
N
N
CH O
3
O
HOOCH C
2
CH COOH
2
OH
DMF, NaOH
RT, 24h
HOOCCH C
3
CH COOH
2
CH O
3
N
N
N
N
NH
O
HN
CH O
3
S
CH COOH
2
HOOCCH C
2
CH O
3
OH
Scheme 1. Scheme for the synthesis of p-NCS-Benzyl-DOTA-colchicine
using 30% concentrated sulphuric acid was carried out to yield trimethyl
colchicinic acid.13 The hydrolysis of the ꢁNHCOCH3 group as well as the
concomitant demethylation of ꢁOCH3 group of colchicine could be
1
ascertained by the H-NMR. The two 3 H singlets corresponding to ꢁOCH3
and ꢁNHCOCH3 observed in 1H-NMR of colchicine were found to be absent
in trimethylcolchicinic acid. Amino group of trimethylcolchicinic acid was
then conjugated with BFCA under alkaline conditions to yield the DOTA
conjugate of colchicine. (Scheme 1) The formation of the p-NCS-benzyl
DOTA-colchicine was confirmed by NMR.
The radiolabeling of p-NCS-benzyl DOTA-colchicine conjugate with
Yttrium-90 was confirmed by HPLC. In the standardized HPLC system
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

954
D. SATPATI ET AL.
90Y-p-NCS-benzyl-DOTA-Colchicine
300
250
200
150
100
90Y-DOTA-NCS
90Y-acetate
50
0
0
200
400
600
800
1000
1200
RT [sec]
Figure 1. HPLC pattern of 90Y-p-NCS-benzyl-DOTA-colchicine
under the conditions of gradient elution, retention time of 90Y-p-NCS-benzyl-
DOTA was 4 min while that of 90Y-p-NCS-benzyl-DOTA-colchicine was
9 min. (Figure 1) The radiolabeling yield was found to vary with the amount of
conjugate used. With 100 mg of conjugate ꢀ70% radiolabeling yield was
obtained, whereas with 200 mg of the colchicine-DOTA conjugate, >98%
radiolabeling could be achieved as determined by HPLC analysis. The
radiolabeled product retained >98% radiochemical purity even after 72 h at
room temperature as shown by the HPLC analysis. On analysis of the
radiolabeled complex by HPLC after 3 h serum incubation, it was found that
ꢀ80% activity eluted out at retention time same as that of original complex,
indicating ꢀ80% radiochemical purity.
Percentage of injected dose of 90Y-p-NCS-benzyl-DOTA colchicine complex
per gram of all major organ tissues as well as fibrosarcoma tumor in mice is
shown in Table 1. At 3 h.p.i, 0.75 ꢂ 0.15% ID/g tumor uptake was observed
which was retained till 24 h.p.i. (0.85 ꢂ 0.35). For in vivo application of
radiolabeled colchicine, lipophilicity is the major problem as reported in our
Copyright # 2006 John Wiley & Sons, Ltd.
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90Y-COLCHICINE FOR TUMOR THERAPY
955
Table 1. Biodistribution studies of 90Y DOTA-NCS-colchicine in murine fibrosarcoma
model
% ID/g of tissue
Organ/Tissue
3 h.p.i.
24 h.p.i.
Blood
Liver
0.4 ꢂ 0.07
1.2 ꢂ 0.25
3.8 ꢂ 0.68
8.4 ꢂ 2.0
2.1 ꢂ 1.03
0.9 ꢂ 0.25
0.08 ꢂ 0.01
Nil
0.68 ꢂ 0.2
0.3 ꢂ 0.15
0.5 ꢂ 0.3
2.6 ꢂ 0.8
0.6 ꢂ 0.3
Nil
Int þ GB
Kidney
Stomach
Lungs
Muscle
Femur
Spleen
Tumor
T/B
Nil
2.4 ꢂ 0.1
1.5 ꢂ 0.7
0.85 ꢂ 0.35
1.25
0.2 ꢂ 0.1
0.75 ꢂ 0.15
1.9
T/M
9.4
85
earlier studies of colchicine labeled with different technetium cores.7
Conjugation with hydrophilic DOTA derivative has resulted in reduced
lipophilicity as evident from negligible retention of the complex in the liver.
Faster clearance from blood, nearly 90% excretion within 24 h.p.i. and
negligible radioactivity in bone led to ꢀ80% increase in target/nontarget
ratios with time (T/B, T/M). Even though T/B ratio remained almost same (1.5
at 3 h.p.i. and 1.25 at 24 h.p.i.), T/M ratio was found to increase significantly
from 9.3 at 3 h.p.i. to 85 at 24 h.p.i.
Retention of radioactivity in the target organ for longer time is required of a
radiopharmaceutical for use as a clinical therapeutic agent. However, most of
the radiolabeled small molecules developed and reported for tumor therapy
show fast clearance from tumors with negligible retention.9 90Y-DOTA-NCS-
Colchicine has shown better tumor retention than earlier reported 177Lu
labeled sanazole by our group.14 Recent studies report preferred use of 177Lu
along with 90Y for combined therapy of tumors of varied size and stages.15
Therefore to explore its full potential for tumor therapy, the synthesized
colchicine derivative can also be radiolabeled with 177Lu for combined therapy
along with 90Y-DOTA-NCS-Colchicine.
Experimental
90Y was obtained from a 90Sr/90Y generator system developed in-house based
on a supported liquid membrane technique. p-NCS-benzyl DOTA (DOTA-
NCS.6HCl) was obtained from M/s. Macrocyclics, USA. All reagents used
were AR grade and of high purity. HPLC analyses were performed on a Jasco
PU 1580 system with a Jasco 1575 tunable absorption detector and a
radiometric detector system. A C-18 reversed phase HiQ Sil (5 mM,
Copyright # 2006 John Wiley & Sons, Ltd.
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D. SATPATI ET AL.
250 ꢃ 4 mm) column has been used. Proton NMR spectra were recorded on
300 MHz Varian VXR 300S spectrophotometer. Fibrosarcoma cell line was
procured from ‘National Centre of Cell Sciences’, Pune, India. Fibrosarcoma
tumors were raised in Swiss mice by subcutaneous injection of ꢀ106 cells/
animal. 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.
90Sr=90Y generator
90Y-chloride for our studies was obtained from a novel 90Sr-90Y generator.16
In brief, this generator is a cell partitioned into two compartments, namely the
feed and the receiver compartment, with a PTFE membrane impregnated with
2-ethyl hexyl-20-ethyl hexyl phosphonic acid. 90Sr-chloride solution containing
90Sr-90Y at equilibrium (pH 1) in HNO3 medium is placed in the feed
compartment from which 90Y3+ alone is preferentially transported across the
membrane into the receiving chamber filled with 1 M HCl leaving behind the
parent radionuclide and other impurities in the feed compartment.
Chemical synthesis
Trimethylcolchicinic acid. Colchicine (150 mg, 0.37 mmol) was dissolved in
4.5 ml of 30% conc. sulphuric acid and heated for 5 h at 1008C in an oil bath.
The reaction mixture was neutralized while hot with solid sodium carbonate to
pH 7–7.5. The frothy precipitate was filtered and washed with cold water.13
The filtrate was extracted in chloroform (3 ꢃ 10 ml), the combined extracts
were washed with water and dried over anhydrous sodium sulphate. TLC
(silica): 5% glacial acetic acid/acetonitrile. Rf (colchicine)=0.4, Rf (product)
= 0. 1H-NMR (CD3OD, d ppm) 7.9 (s, 1H, 8-H); 7.42 (d, 11H); 7.16 (d, 12H);
6.7 (s, 1H, 4H) 3.9 (s, 3H, -OCH3); 3.86 (s, 3H, -OCH3); 3.6 (s, 3H, -OCH3);
2.58 (m, 1H, 7H); 2.34 (m, 4H, 5,6H).
Colchicine-DOTA-NCS. Trimethylcolchicinic acid (17 mg, 0.05 mmol) was
dissolved in 2 ml DMF and p-NCS-benzyl DOTA (18 mg, 25 mmol) was added
to it along with NaOH (1 mg, 0.025 mmol) to maintain alkaline conditions.
The reaction mixture was stirred at room temperature for 24 h. The solvent
was removed under vacuum and ꢀ20 mg of light yellow product was
1
obtained. H-NMR (D2O, d ppm) 8.3 (s, 1H, 8H); 7.16 (m, 4H, PhDOTA);
6.97 (d,1H, 11H); 6.93 (d, 1H, 12H); 6.7 (s, 1H, 4H); 3.8 (s, 8H, -CH2COOH);
3.78 (s, 3H, -OCH3); 3.76 (s, 3H, -OCH3); 3.74 (s, 3H, -OCH3); 3.43 (t, 1H,
7H); 3.22 (s, 2H, -CH2Ph); 2.68 (m, 1H, -NCHCH2N-); 2.64 (m, 4H, 5,6H); 2.2
(m, 14H, -NCH2CH2N-).
Copyright # 2006 John Wiley & Sons, Ltd.
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90Y-COLCHICINE FOR TUMOR THERAPY
957
Radiolabeling
90YCl3 was converted to 90Y acetate by mixing 90YCl3 (74 MBq) and 0.5 M
ammonium acetate (1:1 v/v). The pH of 90Y acetate was adjusted to 4–5 with
3 N NaOH. Radiolabeling was carried out with varying amounts of conjugate.
In a typical protocol, DOTA-NCS-Colchicine conjugate (200 mg, 0.2 mmol)
was added to 90Y acetate (37 MBq) and the reaction mixture was incubated at
708C for 45 min.
Under similar conditions, 90Y-DOTA-NCS was prepared using 50 mg of
DOTA-NCS and 37 MBq of 90Y acetate.
Characterization
90Y-DOTA-NCS-Colchicine as well as 90Y-DOTA-NCS were characterized by
HPLC. About 25 ml of the radiolabeled complex was injected into the column
(C18 reverse phase) where H2O (solvent A) and acetonitrile (solvent B) with
0.1% trifluoroacetic acid were used for elution in the gradient system: 0–
28 min, 90%A–10%A; 28–30 min, 10%A; 30–32 min, 10%A–90%A. The flow
rate was maintained at 1 ml/min and the elution was monitored by the
radioactivity profile.
Stability studies
In vitro serum stability was studied by incubating the radiolabeled complex in
serum at 378C for 3 h. To determine the radiochemical purity after serum
incubation, the product was extracted in acetonitrile and characterized by
HPLC.
Biodistribution studies
Biodistribution studies were carried out in fibrosarcoma bearing Swiss mice.
0.1 ml of the product containing 3.7 MBq of activity was injected via tail vein.
The studies were carried out in triplicate at 3 h and 24 h post-injection. All
major organs as well as tumor were excised, weighed and counted in a NaI (Tl)
flat geometry detector in order to estimate the percent of injected dose per gm
of the tissue. Blood, muscle and bone were taken as 7, 40 and 10% of total
body weight. All the animal studies were carried out in compliance with
national laws as approved by local animal ethics committee.
Conclusion
Trimethyl colchicinic acid was conjugated to the bifunctional chelating agent,
namely DOTA and this conjugate could be successfully radiolabeled with 90
Y
in high yields. The most promising feature of the product was retention of
radioactivity in fibrosarcoma tumor even after 24 h with fast clearance from
other vital organs. The results lead to a conclusion that colchicine conjugate
Copyright # 2006 John Wiley & Sons, Ltd.
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labeled with Yttrium-90 may have a potential for treatment of large size solid
tumors due to high beta energy of the radioisotope.
Acknowledgements
Authors are grateful to Dr H. D. Sarma for his support to carry out the
biodistribution studies. We also express gratitude for Dr V. Venugopal,
Director Radiochemistry and Isotope Group, Bhabha Atomic Research
Centre, for his constant support and encouragement.
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J Label Compd Radiopharm 2006; 49: 951–958
DOI: 10.1002/jlcr