Investigation of 3′-debenzoyl-3′-(3-([124I]-iodobenzoyl))paclitaxel analog as a radio-tracer to study multidrug resistance in vivo

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Abstract

A study was carried out to identify a suitable radioactive paclitaxel analog and to use it to investigate tumor multidrug resistance in vivo. 3′-Debenzoyl-3′-(3-([124I]-iodobenzoyl))paclitaxel was prepared by aromatic iodination of 3′-debenzoyl-3′-(3-trimethylstannylbenzoyl)paclitaxel. Uptake of the labeled paclitaxel analog in nude mice bearing tumor with the paclitaxel sensitive cancer cell lines MCF7 and MDA-435/LCC6(WT), and multidrug resistant cell lines NCI/ADR-RES and MDA-435/LCC6(MDR), was studied. There was no difference in drug level between the sensitive and resistant MDA-435/LCC6 tumors at 6 h post-injection. However, at 6 h, there was a significant increase in drug level for the MCF7 tumor as compared with the NCI/ADR-RES tumor, presumably due to increased drug retention. At 24 h, drug uptake/retention was significantly higher in both sensitive tumor cell lines as compared to their drug resistant counterparts. Pretreatment of mice with MDR transport modulators, Cyclosporine or tRA 96029, did not increase the level of labeled paclitaxel analog in the drug resistant MDA-435/LCC6(MDR) tumor. On the other hand, at 24 h Cyclosporine apparently increased analog level in the drug sensitive MDA-435/LCC6(WT) tumor, aiding drug imaging studies.

Highlights

3′-Debenzoyl-3′-(3-iodobenzoyl)paclitaxel cytotoxicity was comparable to paclitaxel. 3′-Debenzoyl-3′-(3-([124I]-iodobenzoyl)paclitaxel was synthesized. Uptake of the drug was higher in sensitive tumor compared to the resistant tumor. The Pgp-modulators had a positive effect on drug-sensitive tumor. The sensitive tumor was visible in images obtained using micoPET.

Introduction

The taxoids paclitaxel and docetaxel (Fig. 1) are primary tubulin-targeting drugs in the treatment of human cancers, particularly breast, ovarian, and lung carcinomas. Their effectiveness in cancer chemotherapy is often limited because of tumor drug resistance, which causes short duration tumoricidal response. Possible mechanisms underlying tumor drug resistance include over-expression of the mdr1 gene and increased levels of membrane glycoprotein P-170 (Pgp), an ATP-dependent transport pump capable of effluxing these drugs (Gottesman et al., 2002).

Early diagnosis of tumors that are resistant to paclitaxel or docetaxel and identification of the underlying mechanisms of resistance are critical pieces of information that would enable the adjustment of therapeutic strategies if available before initiation of treatment (individualized medicine). The available therapeutic options include (1) the use of drugs that are not substrates for Pgp (e.g., Cisplatin), (2) the use of second generation taxoids with higher therapeutic efficacy against multidrug resistant tumors (e.g. IDN 5109, see Vredenburg et al., 2001), and (3) the use of drug resistance reversal agents (e.g., tRA 98006, see Brooks et al., 2003).

Radiotracer based molecular imaging with positron emission tomography (PET), single photon emission computer tomography (SPECT), and gamma camera are effective technologies for monitoring tumor drug uptake and retention (Phelps, 2000). Li et al. (1997) synthesized and injected indium-111-DTPA-paclitaxel into mice implanted with MCA-4, a paclitaxel responsive mammary tumor. The drug was selectively taken up by the tumor with tumor to muscle drug ratios of 2.64, 3.16 and 6.94 at 30 min, 2 h and 24 h, respectively, although the absolute uptake in the tumor decreased from 1.95% (injected dose/g) at 30 min to 0.21% at 24 h after injection. Gamma scintigraphy and autoradiographic studies showed retention of radiolabeled paclitaxel in the tumor up to 24 h after injection. Two major limitations of an indium-111 labeled agent are (1) its high rate of accumulation in the liver, rendering the evaluation of hepatic metastases quite difficult; and (2) its known high affinity for transferrin receptors, leading to excessive radiation burden to the bone marrow.

Hendrikse and Vaalburg (2002) developed a method that uses [11C]-Verapamil as a positron emitting Pgp substrate to measure the Pgp functionality in vivo with PET. They reported that the [11C]Verapamil accumulation at 1 h post-injection in the lungs, heart and tumor was 43, 1.3, and 0.9% respectively, of the total injected dose. The drawback of this tracer is that the high lung uptake would interfere with any breast tumor imaging protocol.

3′-Debenzoyl-3′-(4-([123I]-iodobenzoyl))paclitaxel as an agent for tumor drug resistance screening has been previously reported by Roh et al. (2000). However, its usefulness to image Pgp was not studied. Kiesewetter et al. (2003) reported the synthesis and preliminary evaluation of paclitaxel derivatives labeled with 18F, 76Br, and 124I. They concluded that the increase of [18F]-paclitaxel accumulation in liver and lungs after administration of the Pgp-inhibitor XR9576 is related to Pgp inhibition and hence it is a useful agent for PET imaging of the dynamic of Pgp expression. Roh et al. (2000) and Kiesewetter et al. (2003) have prepared labeled paclitaxel with 123I and 124I at the para position respectively. These drugs were not studied in tumor bearing mice. Kiesewetter et al. (2003) used Sprague-Dawley rats for biodistribution studies. In addition the effect of preadministration of paclitaxel on the biodistribution was also studied. Preadministration of paclitaxel prior to 124IPAC resulted in significantly increased uptake in lung and liver. It was concluded that there Pgp has a role in lung uptake. The higher liver uptake was attributed to enhanced metabolism upon preadministration of paclitaxel. Because of the existence of multiple iodine isotopes, development of paclitaxel derivatives labeled with iodine allows the use of multiple molecular imaging modalities (PET, SPECT and gamma camera) and selection from a wide range of radioisotope half-life for monitoring the drug uptake. We expected a possible negative pharmacological effect by introducing an iodo group on the 3′-phenyl ring. Therefore, we synthesized three iodo isomers of paclitaxel (i.e., 3′-debenzoyl-3′-(2, 3, and 4-iodobenzoyl)paclitaxel). The isomer that was most similar to paclitaxel in the cytotoxicity assays was iodo at 3-position and therefore was chosen for labeling. Bio-distribution studies were performed in mice bearing tumors, using 124I-labeled drug. These mice were also sequentially imaged with a microPET camera after the injection of 124I-labeled drug. The performance of the iodo isomers of paclitaxel and paclitaxel were assessed with MCF7, NCI/ADR-RES and MDA-435/LCC6(WT)/MDA-435/LC6(MDR) tumor cell lines. The bio-distribution and PET imaging studies were performed with labeled 3′-debenzoyl-3′-(3-([124I]-iodobenzoyl))paclitaxel in nude mice bearing MCF7, NCI/ADR-RES and MDA-435/LCC6(WT)/MDA-435/LC6(MDR) tumors. As a useful application of these tracers, we evaluated the effectiveness of two modulators, Cyclosporine and tRA 96023 on radiotracer uptake in Pgp sensitive and resistant tumors.

Section snippets

Materials and methods

All chemical reactions were performed under an argon atmosphere. All air and moisture sensitive reactions were conducted using Schlenk glassware using standard inert atmosphere techniques. Thin layer chromatography (TLC) was performed on Merck Silica Gel 60F254 silica plates and visualized by UV illumination. Flash column chromatography was performed on Merck silica gel. 1H or 13C NMR spectra were recorded using a Varian VXR-500 and 400 NMR in deuterated solvents as indicated in the

Biodistribution and imaging

Female nude athymic mice, 6–10 weeks of age obtained from National Cancer Institute (Fredrick, MD). Animals were housed at the Medical Research Complex at Roswell Park Cancer Institute. Animal care was within IACUC guidelines and meets all Federal and State regulation. Mice were tumored subcutaneously, bilaterally with resistant variant on the right shoulder area and wild type tumor on the left, either MCF7 paired with its resistant variant NCI/ADR-RES or MDA-435/LCC6(WT) paired with

Data analysis

Data were fit with the Sigmoid-Emax concentration-effect model (Holford and Scheiner, 1981) with nonlinear regression, weighted by the reciprocal of the square of the predicted response. The fitting software was developed at RPCI with Microsoft FORTRAN, and uses the Marquardt (1963) algorithm as adapted by Nash (1979) for the nonlinear regression. The concentration of drug, which resulted in 50% growth inhibition (IC50), was calculated.

3′-Debenzoyl-7-(trichloroethoxycarbonyl)paclitaxel (2)

The baccatin III-coupled product 1 (111.4 mg, 0.107 mmol), prepared using the method by Reddy et al. (2001), was dissolved in formic acid (96%, 4.6 mL) and stirred at room temperature for 2 h. The solvent was evaporated under vacuo and the residual solid was dried under high vacuum. The crude free amine was used for the next step without further purification.

3′-Debenzoyl-3′-(4-iodobenzoyl)-7-(trichloroethoxycarbonyl)paclitaxel (3a)

The crude amine 2 (111 mg, 0.107 mmol) was dissolved in EtOAc (3.0 mL) and then a saturated solution of aq. NaHCO3 (3.0 mL) was added and the

3′-Debenzoyl-3′-(3-[124I]iodobenzoyl)paclitaxel (10)

Na124I in 0.1 M NaOH (10–30 μL) was added to acetic acid (100 μL of 5%) in acetonitrile, mixed and transferred to an IODO-GEN coated tube. The stannylated precursor 9 (40 μg) was dissolved in an acetic acid/ acetonitrile (50 μL of 10%) solution and transferred to the IODO-GEN coated tube. The solution was mixed and left at room temperature for 15 min. The reaction mixture was purified on an HPLC using a Phenomenex Luna C18(2) column (4.6×250 mm). The eluent was 60% acetonitrile, 40% water and the flow

Chemistry

The synthesis of the iodobenzoylpaclitaxel analogs 4a4c is outlined in Scheme 1. The iodinated analogs 4 were prepared from paclitaxel precursor 1, which was synthesized, using the method of Reddy et al. (2001). The N,O-acetonide and the Boc-protecting group in precursor 1 were removed using formic acid to furnish 3′-aminopaclitaxel intermediate 2. Reaction of the free amino group in 2 with 2-, 3-, and 4-iodobenzoyl chloride provided the three regioisomeric iodo analogs 3. Deprotection of the

Summary

3′-Debenzoyl-3′-(3-iodobenzoyl)paclitaxel was found to match most closely paclitaxel when compared to the other isomers in antiproliferative assays. The 3′-debenzoyl-3′-(3-[124I]iodobenzoyl)paclitaxel analogs were prepared by aromatic iodination of 3-debenzoyl-3′-(3-trimethylstannylbenzoyl)paclitaxel. The uptake of the labeled drug was higher in sensitive tumors compared to the resistant tumors. Application of Pgp-modulators Cyclosporine increased drug accumulation in drug-sensitive tumors

Acknowledgment

This work was supported by an award from the Roswell Park Alliance Foundation.

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