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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Bioorg Med Chem Lett. 2014 Dec 13;25(3):571–574. doi: 10.1016/j.bmcl.2014.12.016

A comparative study of trans- and cis-isomers of a bone-seeking agent, DO2A2P

Guiyang Hao a, Wei Liu a,b, Gedaa Hassan a, Orhan K Öz a, Zoltan Kovacs c, Xiankai Sun a,c,*
PMCID: PMC4386885  NIHMSID: NIHMS649283  PMID: 25541387

Abstract

The macrocyclic bone-seeking agent, DO2A2P, bears a cyclen core and two pairs of peripheral phosphonate and carboxylate groups. The geometric disposition of the peripheral functionalities gives arise to a pair of geometric isomers: cis-DO2A2P and trans-DO2A2P. In order to compare the biological behavior of the isomer pair, cis-DO2A2P was synthesized. Both isomers were successfully radiolabeled with 177Lu, which might potentiate their applications in both radiotherapy and imaging of bone diseases. Through a set of biological assays including the hydroxyapatite binding, in vitro stability, and in vivo distribution, we demonstrated that the geometric pair of DO2A2P had virtually identical biological properties.

Keywords: Bone-seeking agent, Bisphosphonate, Geometric isomer, Macrocyclics, Lutetium-177

Bisphosphonate analogs have been long used directly (e.g. Pamidronate) or when labeled with therapeutic radioisotopes (e.g. Quadramet®, 153Sm-EDTMP) for palliative treatment of bone metastases, which are common in many types of cancers at advanced stages.1 In addition, clinical bone scans are mainly performed with 99mTc-labeled bisphosphonates, such as 99mTc methylenebisphosphonate (Medronate). Despite the well-established clinical use, directly radiolabeled bisphosphonate complexes suffer several obvious limitations, which include low kinetic stability, suboptimal specific radioactivity, nonspecific accumulation in non-target tissues, and unknown coordination structure.2 Therefore, significant efforts have been seen to develop bone seeking agents derivatized from bisphosphonates, in which the radiometal is coordinated by a “bifunctional chelator” that oftentimes carries a spacer to optimize the biological behavior.2 In this regard, macrocyclic chelators are preferred over their acyclic counterparts because more kinetically stable complexes can result from their rigid and pre-organized structures.3–6 Furthermore, macrocyclic ligands bearing a mixed set of phosphonate and carboxylate groups, such as TRITP, DOTP, DOA3P, and trans-DO2A2P (Figure 1), have been reported as novel bone targeting agents when labeled with radioisotopes (e.g. 68Ga-DOTP, 153Sm/166Ho-TRITP, and 177Lu-DOTP).2 In this class of complexes, both the macrocyclic core and the peripheral phosphonate or carboxylate moieties contribute to the metal binding, while the phosphonate still maintains the desired bone targeting capability. It has been reported that a positive correlation exists between the bone uptake and the number of methylphosphonate pendent arms.2, 7 While more phosphonate pendent arms lead to higher bone accumulation, agents with mixed phosphonate and carboxylate groups (e.g. trans-DO2A2P) present quicker radiolabeling, higher in vivo stability, and relatively lower osmolarity than those bearing a high number of phosphonates (e.g. TRITP, DOTP, and DOA3P).7–9

Figure 1.

Figure 1

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Structures of relevant bisphosphonate (BP) analogs. DO3A-BP: 2,2′,2″-(10-(2-((2-(3,3-diphosphonopropanamido)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; NOTA-HBP: 2,2′-(7-(1-carboxy-4-((4-hydroxy-4,4-diphosphonobutyl) amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid; DOTP: ((1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(methylene)) tetrakis(phosphonic acid); TRITP: ((1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetrayl)tetrakis(methylene)) tetrakis(phosphonic acid); DOA3P: 2-(4,7,10-tris(phosphonomethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid; trans-DO2A2P: 2,2′-(4,10-bis(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid; and cis-DO2A2P: 2,2′-(7,10-bis(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,4-diyl)diacetic acid).

Unlike TRITP, DOTP, and DOA3P, DO2A2P can present two geometric (cis/trans) isomers, which might incur different biological behavior after labeling with radioisotopes.7 While the synthesis and biological properties of trans-DO2A2P has been well-documented, its cis-counterpart, cis-DO2A2P (2,2'-(7,10-bis(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,4-diyl)diacetic acid), has not been reported. It would be of interest to know whether or not the two geometric isomers behave similarly in vivo.

The synthesis of cis-DO2A2P was accomplished by a similar approach reported for trans-DO2A2P (Scheme 1).10 First, the 1,4-bis-substituted cyclen, 1, was synthesized according to a previously reported procedure.11 It was then converted to 2 using triethyl phosphate and paraformaldehyde in a Mannich-type reaction. The intermediate 2 was directly deprotected by refluxing it in hydrochloric acid to afford cis-DO2A2P, 3. The product was further purified by recrystallization from ethanol/water.12

Scheme 1.

Scheme 1

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Synthesis of cis-DO2A2P. i) triethyl phosphate, paraformaldehyde, 4 days at r.t.; ii) 20% HCl, reflux for 2 days.

Lutetium-177 was chosen for the radiolabeling and biological evaluation of DO2A2P ligands given the fact that 177Lu (Eβ-max: 497 KeV; t1/2: 6.73 d) has been increasingly used for targeted therapy and it is a commercially available. In addition to its low-energy β− emission that provides a tissue penetration range effective for the treatment of small tumors ( 3.0 mm),13 177Lu’s γ energies (113 KeV, 208 KeV) are also suitable for single-photon emission computed tomography (SPECT) imaging, which enables the desirable non-invasive monitoring and radiation dosimetry calculation. The labeling of both cis- and trans- DO2A2P ligands with 177Lu was carried out in virtually quantitative yield at 50 °C and 90 °C within 30 min (Table 1).14 However, when the radiolabeling reactions were conducted at room temperature (r.t.), the isomer pair indeed behaved differently. The cis-isomer needed a much longer time (up to 3.5 h) to be completely labeled than the trans-isomer (within 1 h).

Table 1.

Radiolabeling efficiency of the cis- and trans-DO2A2P isomer pair with 177Lu at room temperature (r.t.), 50 °C, and 90 °C.

Time 177Lu-cis-DO2A2P 177Lu-trans-DO2A2P
r.t. 50 °C 90 °C r.t. 50 °C 90 °C
0.5h 53 >99 >99 96 >99 >99
1h 74 >99 >99 >99 >99 >99
2h 91 / / >99 / /
3.5h >99 / / >99 / /
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/

not tested

The hydrophilicity of the radiolabeled complex is one important factor to determine its in vivo profile, which can be measured by their partition coefficients (logP) in a biphasic solvent system of n-octanol and water.15 The results indicate both 177Lu-labeled cis- and trans- DO2A2P complexes are highly hydrophilic. The in vitro stability of both complexes was evaluated in rat serum by radio-TLC. Both complexes remained virtually intact (> 98%) out to 7 days.16

The comparative binding of 177Lu-labeled cis- and trans-DO2A2P onto hydroxyapatite (HA), the main mineral component of bone, is shown in Figure 2.17 The adsorption profiles of both 177Lu-complexes to HA are nearly identical.

Figure 2.

Figure 2

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Comparative hydroxyapatite (HA) adsorption profiles of 177Lu-cis-DO2A2P and 177Lu-trans-DO2A2P versus the HA amount.

In order to compare their in vivo bone-seeking profiles, a SPECT/CT imaging study was performed in normal BALB/c mice using both 177Lu-cis-DO2A2P and 177Lu-trans-DO2A2P.18 As shown in Figure 3, both agents presented almost identical in vivo distribution patterns in that they preferentially accumulated in the spine and the bone joints at 1 h post-injection. The quantitative analysis of the SPECT/CT images revealed the bone uptake levels of 177Lu-cis-DO2A2P and 177Lu-trans-DO2A2P were 1.2 ± 0.2 %ID/g and 1.1 ± 0.1 %ID/g at 1 h post-injection, respectively. No significant difference was observed between the two radiotracers. At 24 h post-injection, most of the radioactivity had cleared out of the mouse body.

Figure 3.

Figure 3

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Representative SPECT/CT images of 177Lu-cis-DO2A2P and 177Lu-trans-DO2A2P at 1 h post-injection presented as maximum intensity projection (MIP). CT images: gray color scale; SPECT images: red color scale (MIP); sagittal, and coronal: nih fire color scale. Arrows indicate the spine and bone joints.

Taken together, no significant difference was observed for the 177Lu-labeled geometric isomer pair of DO2A2P in their biological properties assayed in this work.

Acknowledgments

This work was partially supported by an NIH R21 grant (CA119219), an NIH NCRR grant (1S10RR029674-01), and the Dr. Jack Krohmer Professorship Funds.

Footnotes

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References and notes

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  • 15.Determination of the partition coefficient (logP): 5 μL of 177Lu-labeled DO2A2P was added to a solution containing 500 μL of Milli-Q water and 500 μL of n-octanol (obtained from a saturated octanol/water solution) (n = 3). The sample vials were shaken for 1 h at room temperature. From each vial, aliquots of 100 μL each were removed from the n-octanol phase and the water phase, respectively, and counted separately by a Perkin-Elmer γ-counter. The partition coefficient was calculated as the ratio of counts in the octanol fraction to the counts in the water fraction. An average of logP value was tried from the 3 samples.
  • 16.Serum stability: The in vitro serum stability experiment was conducted by adding 10 μL of 177Lu-labeled DO2A2P (3.7 MBq/0.1 mCi) to 100 μL of rat serum. The solution was incubated at 37 °C, and sampled for radio-TLC analysis up to 7 days post-addition to rat serum.
  • 17.Adsorption study: The 177Lu-labeled cis- and trans- DO2A2P (370 KBq) was incubated by continuous mixing for 1 h at room temperature with 5, 10, 25, 50, 100, and 150 mg of hydroxyapatite (HA) in 1.5 mL of 0.1 M tris-HCl buffer (pH 7.4), respectively. Immediately after incubation, the mixture was centrifuged at 10,000 rpm to achieve complete sedimentation, and the liquid phase was separated. The solid phase was washed twice with 1.5 mL of 0.1 M tris-HCl buffer (pH 7.4). Washing buffer was pooled with liquid phase. The radioactivity in the liquid and solid phases was measured in a dose calibrator.
  • 18.SPECT/CT imaging procedures: SPECT/CT imaging was performed in a dedicated, multiplexed multi-pinhole small animal SPECT/CT imaging system, NanoSPECT/CT Plus (BioScan). The detection energy window for 177Lu was set at and keV with a 10% difference. The CT projections were recorded at 45 kV and 170 μA of tube voltage and current, respectively. The solution of 177Lu-cis-DO2A2P and 177Lu-trans-DO2A2P was diluted with saline solution to prepare the injection doses, respectively. Each male BALB/c mouse (18–22 g) was injected intraveneously with 20–22 MBq of radioactivity in 0.15 mL. Two imaging sessions started 1 h and 24 h post-injection, respectively (n = 3).

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