The Structures of Gd(III) Chelates Conjugated at the Periphery of 3-(1'-Hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH) Have a Significant Impact on the Imaging and Therapy of Cancer

Abstract

3-(1'-Hexyloxyethyl)-3-devinyl-pyropheophorbide-a (HPPH or Photochlor), a tumor-avid chlorophyll-a derivative currently undergoing human clinical trials, was conjugated at various peripheral positions (position-17 or 20) of HPPH with either Gd(III)-aminobenzyl-DTPA (Gd(III) DTPA) or Gd(III)-aminoethylamido-DOTA (Gd(III) DOTA). The corresponding conjugates were evaluated for in vitro PDT efficacy, T₁ , T₂ relaxivities, in vivo fluorescence, and MR imaging under similar treatment parameters. Among these analogs, the water-soluble Gd(III)-aminoethylamido-DOTA linked at position-17 of HPPH, i. e., HPPH-17-Gd(III) DOTA, demonstrated strong potential for tumor imaging by both MR and fluorescence, while maintaining the PDT efficacy in BALB/c mice bearing Colon-26 tumors (7/10 mice were tumor free on day 60). In contrast to Gd(III) DTPA (Magnevist) and Gd(III) DOTA (Dotarem), the HPPH-Gd(III) DOTA retains in the tumor for a long period of time (24 to 48 h) and provides an option of fluorescence-guided cancer therapy. Thus, a single agent can be used for cancer-imaging and therapy. However, further detailed pharmacokinetic, pharmacodynamic, and toxicological studies of the conjugate are required before initiating Phase I human clinical trials.

Figure 1.

Comparative absorbance (A) and fluorescence (B) spectra of conjugates 4, 6, 8, 9 and HPPH (1) in methanol (conc. 0.5 μM).

Figure 2.

(A) MTT assay of conjugates 4 in PBS (4P) and liposomal (4L) formulations and 6, 8, 9 in liposomal formulation (conc. 0.5 μM) incubated in Colon-26 cells ( 0.5 × 10⁶) for 4 h and 24h followed by a light dose of 0.5 J/cm² at a fluence rate of 3.2 mW/cm². (B) Cellular uptake of conjugates 4P, 4L, 6, 8 and 9 studied in Colon-26 cells. 0.5 μM of each conjugate was incubated with cells, which were harvested and examined for cellular uptake 4 hours and 24 hours after incubation.

Figure 3.

Comparative tumor uptake of the conjugates formulated in PBS and liposome determined by fluorescence. The conjugates were administered (i.v.) in mice (BALB/c mice bearing Colon 26 tumors, 3 mice/group) at an imaging dose of 10 μmol/kg bearing subcutaneous Colon-26 tumors. Fluorescence was acquired at variable time points ranging from 4 to 48 hours post injection using the Maestro imaging system.

Figure 4.

The time course study showing comparative tumor accumulation of conjugates 4P, 4L and 6 as measured by in vivo fluorescence. The conjugates were injected (i.v.) at the imaging dose of 10 μmol/kg to BALB/c mice (n=3) bearing subcutaneous Colon-26 tumors.

Figure 5.

Structure of HPPH conjugated with three molecules of Gd(III)DTPA (ref. [21]).

Figure 6.

(A). Overlay of T₁ weighted scan images with false color representing the change in T₁ rate values (s⁻¹) over baseline within subcutaneous tumors for conjugate 4P (top) and HPPH-17-tri-DTPA-Gd(III) (bottom) 4h (left column) and 24h post injection (right column) (B). Increase in MRI signal intensity produced by conjugate 4P at 4h post injection (right panel) in comparison to baseline (left panel) represented by raw image (top panel) and images overlaid with false color (lower panel). (C). Left panel: improved visibility was shown after the use of 17-DOTA conjugate 4P (right), as the yellow arrow points out the vena cava and kidney, shown with higher clarity in comparison to baseline level (left). (D) Right panel: representative slices of co-registered MR data in the coronal view (a) and axial view (c) as well as a volumetric 3D reconstruction (b). A subcutaneous tumor is highlighted with a long arrow. They two cylinders are NMR tubes used as co-registration fiducial markers. 3D optical data generated by an IVIS Spectrum system co-registered with the MR data. Color overlay indicates source of fluorescence signal, with excellent co-localization of signal to the tumor location determined by MR imaging. The white outline (d & f) is the 3D volume generated by the Spectrum’s laser profilometry system (short arrow). An error in the Spectrum’s profilometry reconstruction depth is noted for the right NMR tube (f); otherwise the multimodality co-registration is of high quality. Please note: Conjugate 4 formulated in PBS is coded as 4P.

Figure 7.

Comparative imaging using optical and MR imaging of vital organs and structures (thigh muscle, liver, kidney, spleen) to subcutaneous Colon-26 tumor implanted in the flank. 3D MRI images were acquired 4, 24, and 48h post injection of conjugate 4P at an imaging dose of 10 μmol/kg. Immediately after MRI scanning, in vivo tumor imaging was conducted before mice (n=3 for each time point) were sacrificed and organs excised for ex vivo imaging.

Figure 8.

In vivo photosensitizing efficacy of water soluble HPPH-17-mono-DOTA-Gd (III) 4P at a dose of 10 μmole/kg (light dose: 40 J/cm² at 40 mW/cm²). Mice (10 mice/group), bearing 4–6 mm diameter Colon-26 tumors were implanted in BALB/c mice. The tumors were exposed to light for 16 min 40 sec from a tunable dye laser tuned to the maximum red absorption peak (λ_max= 665 nm) at 48 h post-administration. Tumor regrowth was monitored daily. Three mice with tumor regrowth (>400 mm³) were euthanized following the guidelines of approved animal protocol. At day 60, 7/10 mice did not show any tumor-regrowth (70% cure)

Scheme 1.

Synthesis of 17- and 20- substituted HPPH-Gd(III) DTPA and DOTA conjugates: a) LiOH in H₂0, MeOH, THF; b) 2-Aminoethyl-DOTA-tris(tert-butyl ester), DMTMM, THF; c) 70% TFA/CH₂Cl₂; d) Pyridine, GdCl₃.6H₂0; e) Pyridinium tribromide, pyridine (cat), CH₂Cl₂; f) 3-(tert-Butoxycarbonyl)phenylboronic acid pinacol ester Pd (PPh₃)₄; K₃PO₄, THF; g) 2-Aminoethyl-DOTA-tris(tert-butyl ester), PyBOP, Et₃N, DMF; h) p-NH₂-Bn-DTPA-penta(tert-butyl ester), PyBOP, Et₃N, DMF.