Chlorophyll-a Analogues Conjugated with Aminobenzyl-DTPA as

Bioconjugate Chem. 2005, 16, 32−42

32

Chlorophyll-a Analogues Conjugated with Aminobenzyl-DTPA as Potential Bifunctional Agents for Magnetic Resonance Imaging and Photodynamic Therapy† Guolin Li,3,‡ Adam Slansky,3,‡ Mahabeer P. Dobhal,3 Lalit N. Goswami,3 Andrew Graham,3,§ Yihui Chen,3 Peter Kanter,| Ronald A. Alberico,‡ Joseph Spernyak,⊥ Janet Morgan,§ Richard Mazurchuk,⊥ Allan Oseroff,§ Zachary Grossman,‡,* and Ravindra K. Pandey3,‡,* Photodynamic Therapy Center, Department of Nuclear Medicine and Radiology, Department of Dermatology, Department of Toxicology/Medicine, and Preclinical MR Imaging Resource, Roswell Park Cancer Institute, Buffalo, New York 14263. Received August 12, 2004; Revised Manuscript Received October 21, 2004

A clinically relevant photosensitizer, 3-devinyl-3-(1-hexyloxyethyl)pyropheophorbide-a (HPPH, a chlorophyll-a derivative), was conjugated with Gd(III)-aminobenzyl-diethylenetriaminepentaacetic acid (DTPA), an experimental magnetic resonance (MR) imaging agent. In vivo reflectance spectroscopy confirmed tumor uptake of HPPH-aminobenzyl-Gd(III)-DTPA conjugate was higher than free HPPH administered intraveneously (iv) to C3H mice with subcutaneously (sc) implanted radiation-induced fibrosarcoma (RIF) tumor cells. In other experiments, Sprague-Dawley (SD) rats with sc implanted Ward Colon Carcinoma cells yielded markedly increased MR signal intensities from tumor regionsof-interest (ROIs) 24 h post-iv injection of HPPH-aminobenzyl-Gd(III)-DTPA conjugate as compared to unconjugated HPPH. In both in vitro (RIF tumor cells) and in vivo (mice bearing RIF tumors and rats bearing Ward Colon tumors) the conjugate produced significant increases in tumor conspicuity at 1.5 T and retained therapeutic efficacy following PDT. Also synthesized were a series of novel bifunctional agents containing two Gd(III) atoms per HPPH molecule that remained tumor-avid and PDT-active and yielded improved MR tumor conspicuity compared to their corresponding mono-Gd(III) analogues. Administered iv at a MR imaging dose of 10 µmol/kg, these conjugates produced severe skin phototoxicity. However, by replacing the hexyl group of the pyropheophorbide-a with a tri(ethylene glycol) monomethyl ether (PEG-methyl ether), these conjugates produced remarkable MR tumor enhancement at 8 h post-iv injection, significant tumoricidal activity (80% of mice were tumor-free on day 90), and reduced skin phototoxicity compared to their corresponding hexyl ether analogues. The poor water-solubility characteristic of these conjugates was resolved by incorporation into a liposomal formulation. This paper presents the synthesis of tumor-avid contrast enhancing agents for MR imaging and thus represents an important milestone toward improving cancer diagnosis and tumor characterization. More importantly, this paper describes a new family of bifunctional agents that combine two modalities into a single cost-effective “see and treat” approach, namely, a single agent that can be used for contrast agent-enhanced MR imaging followed by targeted photodynamic therapy.

INTRODUCTION

For many years, in vivo imaging of human organs was largely dependent upon intravenous administration of radioactive molecules for nuclear scanning or nonradioactive iodinated chemicals for radiography (1-4). However, over the past decade, magnetic resonance (MR) imaging has become the method-of-choice for many procedures (4). Unlike nuclear scanning, conventional * To whom correspondence should be addressed. R.K.P.: Phone: 716-845-3203. Fax: 716-845-8920. E-mail: [email protected]. Z.G.: Phone: 716-845-8015. E-mail: [email protected]. † Part of the Special Issue collection for Imaging Chemistry that began in Issue 6, 2004. A preliminary description of this work was presented at the Symposium on Chemistry and Biological Applications of Imaging Agents and Molecular Beacons, at the spring 2004 National Meeting of the American Chemical Society. 3 Photodynamic Therapy Center. ‡ Department of Nuclear Medicine and Radiology. § Department of Dermatology. | Department of Toxicology/Medicine. ⊥ Preclinical MR Imaging Resource.

radiography or computed tomography, MR often relies on “contrast-enhancing agents” to improve inherent contrast between normal and diseased tissue. MR signal intensity or signal intensity differences (contrast) is dependent upon longitudinal (1/T1) and transverse relaxation rates (1/T2) of tissue protons. Agents containing paramagnetic ions have been shown to effectively alter 1/T1 and/or 1/T2 by changing the local magnetic field when they come in close proximity to water protons. The paramagnetic ion most widely used in this regard is gadolinium [Gd(III)] because of its seven unpaired electrons and large paramagnetic moment. At present, three similar Gd(III)-derived MR contrast enhancing agents have been approved by the Food and Drug Administration (FDA) for clinical use in the United States, the bisN-methylglucamine salt of Gd(III)-diethylenetriamine pentaacetic acid (DTPA) (Magnavist, Berlex Laboratories, Wayne, NJ), the bis-N-methylamide of Gd(III)-DTPA (Omniscan, Amersham Health, Princeton, NJ), and the Gd(III) chelate of the 20-(2-hydroxypropyl) derivative of 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-1,4,7-tetraacetic acid (Prohance, Bracco Diagnostics, Princeton, NJ). All three of these agents are carboxylate-containing,

10.1021/bc049807x CCC: $30.25 © 2005 American Chemical Society Published on Web 01/04/2005

Bifunctional Agents for MRI and Photodynamic Therapy

water-soluble complexes. While several agents look promising, no macromolecular MR contrast-enhancing agent has been approved for human use. Conventional MR contrast media rely on tumor enhancement secondary to altered tumor capillary permeability. The time course of tumor enhancement with these agents, therefore, is to a great extent dependent upon intravascular contrast agent concentration. As an administered MR contrast-enhancing agent clears from the circulation, tumor conspicuity may appear to increase due to rising tumor-to-background ratio. Thereafter, tumor MR signal intensity declines as nonbound contrast media clears from a given lesion. Typical plasma clearance rates for FDA approved agents are on the order of 15 min or less, thereby limiting the usable time frame for MR data acquisition, which can take between 5 and 15 min per acquisition. Moreover, several additional disadvantages exist for current FDAapproved MR contrast media. Because current FDA approved MR agents do not preferentially bind specific molecular targets, they cannot reliably differentiate neoplasm from inflammation. Finally, because the tumor selectivity of current MR contrast-enhancing agents is singularly dependent upon perturbations in normal vascular permeability, small foci of metastatic tumor cells, particularly those in minimally enlarged or normalsized lymph nodes, may not be detectable by current methods. However, by selective or nonselective binding of a MR contrast-enhancing agent to a tumor target or by simply slowing in vivo tumor clearance rates, several improvements might be realized. For example, additional data sets or repeat MR acquisitions might be obtained, thereby enabling screening of additional body regions or more detailed MR characterization of specific locations. Previously, gadolinium-based porphyrin contrast media, particularly gadoporphyrin-2, have been extensively studied and found to be necrosis-avid (5, 6). Although such compounds may be useful for tumor imaging (530% tumor mass is commonly necrotic), a MR agent that targets living tumor cells, particularly those that are actively undergoing metabolic processes, would be superior. For the last several years, our laboratory has focused our research efforts on developing various long wavelength-absorbing photosensitizers (660-800 nm) derived from chlorophyll-a and bacteriochlorophyll-a for photodynamic therapy (PDT) (7-14). Among these compounds, HPPH is currently at Phase II human clinical trials and others are at various stages of preclinical studies (15, 16). Because HPPH demonstrated both tumor avidity and limited skin phototoxicity (a major drawback with most of the porphyrin-based compounds), we were interested in studying HPPH as a “vehicle” for delivering gadolinium complexes to tumors. Our goal was to develop a single bifunctional agent that could be clinically utilized both as a MR contrast-enhancing agent and a photosensitizer for PDT. EXPERIMENTAL PROCEDURES 1H and 13C NMR spectra were recorded on Bruker AMX-400 Spectrometer at 400.1 and 100.6 MHz, respectively. Unless otherwise stated, chemical shifts are reported in ppm and referenced to residual solvent resonance peaks (CDCl3: for 1H, 7.26 ppm and 13C, 77.2 ppm; methanol-d4: for 1H, 3.31 ppm). UV-vis spectra were recorded on a Varian (Cary-50 Bio) spectrophotometer. Column chromatographic separations were performed over silica gel 60 (70-230 mesh) or neutral alumina (Brockmann grade III, ∼150 mesh). HPPH-DTPA-tert-butyl Ester (3). A mixture of HPPH 2 (91 mg, 0.143 mmol), 7 (167 mg, 0.215 mmol), N,N′-dicyclohexylcarbodiimide (DCC, 35 mg, 0.170 mmol), and (dimethylamino)pyridine (DMAP, 21 mg, 0.172 mmol) in dry dichloromethane

Bioconjugate Chem., Vol. 16, No. 1, 2005 33 (5 mL) was stirred at room temperature under nitrogen for 16 h. Water (1 mL) was then added, and the mixture was stirred for 15 min. The organic layer was separated, dried with Na2SO4, filtered, and concentrated. The residue was dissolved in minimum amount of dichloromethane (∼2 mL) and refrigerated for 1 h. It was then filtered, and the solid (1, 3-dicyclohexylurea) was washed with dichloromethane. The filtrate was concentrated and purified with column chromatography over neutral alumina (Brockmann grade III, ∼150 mesh) eluting with hexanes-EtOAc (v/v 3/1) and then hexanes-EtOAc (v/v 5/2). The fractions containing the product 3 were combined and concentrated. The residue was purified by another column chromatography over neutral alumina (Brockmann grade III) eluting with CH2Cl2-EtOAc (v/v 8/1) and then CH3OH-CH2Cl2 (3%) to provide the title compound 3 (167 mg, 0.120 mmol) as a dark blue oil in 84% yield. UV-vis in CH2Cl2 [λmax ()]: 410 (117540), 505 (10650), 536 (10740), 604 (8950), 660 (54460). 1H NMR (CDCl3) δ 9.81 (1H, splitting s, H-5), 9.00 (1H, splitting s, H-10), 8.53 (1H, s, H-20), 7.24 (1H, br, CONH), 7.17 (2H, m, H-2′ and 6′), 7.05 (2H, m, H-3′ and 5′), 5.93 [1H, m, CH3(Ohexyl)CH-3], 5.22 (2H, dd, AB system, J ) 20.0 Hz, COCH215), 4.60 (1H, m, H-18), 4.42 (1H, m, H-17), 3.78-3.17 (20H, m, H-1′′, CH3-2, CH3-7, CH3CH2-8 and 5 × NCH2CO2C), 3.09-2.90, 2.90-2.52, 2.50-2.28, 2.24-2.03 [total 19H, m, CH3-12, HNCOCH2CH2-17, H-7′, 8′, 9′, 10′, 11′, CH3(O-hexyl)CH-3], 1.82 (3H, d, J ) 7.4 Hz, CH3-18), 1.78 (2H, m, H-2"), 1.59 (3H, m, CH3CH2-8), 1.40, 1.39, 1.37 [45H, each s, 5×-OCOC(CH3)3], 1.40 (2H, m, overlapped with tert-butyls, H-3"), 1.25 (4H, m, H-4′′ and H-5′′), 0.80 (3H, m, H-6′′), 0.46 (1H, br, NH), -1.59 (1H, s, NH). 13C NMR (CDCl ): δ 196.5, 172.2, 171.5, 170.9, 170.7, 160.4, 3 155.6, 151.0, 149.2, 145.3, 141.7, 141.6, 140.0, 137.7, 136.3, 136.0, 135.9, 132.6, 132.5, 130.2, 129.8, 128.0, 119.7, 106.2, 104.1, 98.3, 98.2, 92.8, 80.9, 80.7, 73.1, 69.9, 63.2, 56.3, 56.2, 53.8, 53.1, 52.7, 51.9, 50.3, 48.4, 36.8, 34.0, 32.0, 30.4, 28.4, 28.3, 26.3, 25.0, 24.9, 23.3, 22.8, 19.6, 17.5, 14.2, 14.1, 11.7, 11.5, 11.2. MS (FAB) m/z 1397.9 (MH+, 100). HRMS (FAB): Calcd for C80H117N8O13 (MH+) 1397.8739; Found 1397.8750. HPPH-DTPA (4). 3 (130 mg) was dissolved in trifluoroacetic acid (TFA, 3 mL). The solution was stirred at room temperature for 3 h. TFA was removed with under high vacuum at room temperature. The residue was dissolved in toluene (10 mL), and then the solvent was removed with rotavapor. The process was repeated three times to give 4 as dark blue powder. 1H NMR (50% CDCl3, 50% CD3OD and one drop of C5D5N) δ 9.77 (1H, splitting s, H-5), 9.46 (1H, s, H-10), 8.60 (1H, s, H-20), 7.31 (2H, m, H-2′ and 6′), 7.01 (2H, m, H-3′ and 5′), 5.92 [1H, m, CH3(Ohexyl)CH-3], 5.20 (2H, dd, AB system, J ) 19.6 Hz, COCH215), 4.62 (1H, m, H-18), 4.34 (1H, m, H-17), 3.81-2.23 (36H, m, H-1", CH3-2, CH3-7, CH3-12, CH3CH2-8, HNCOCH2CH2-17, H-7′, 8′, 9′, 10′, 11′ and 5×-NCH2CO2C. Note: CH3-2, CH3-7, and CH312 appeared at 3.37, 3.26, and 3.58, respectively), 2.12 [3H, d, J ) 6.6 Hz, CH3(O-hexyl)CH-3], 1.84 (3H, d, J ) 7.2 Hz, CH318), 1.72 (2H, m, H-2′′), 1.69 (3H, t, J ) 7.4 Hz, CH3CH2-8), 1.38 (2H, m, H-3′′), 1.21 (4H, m, H-4′′ and H-5′′), 0.75 (3H, t, J ) 7.2 Hz, H-6′′). MS (ESI) m/z 1117.7 (MH+, 100). HPPH-DTPA-Gd Complex (5). To a solution of 4 (135 mg, 0.121 mmol) in pyridine (15 mL) was added a solution of GdCl3‚ 6H2O (45 mg, 0.121 mmol) in deionized water (2 mL). The solution was stirred at room temperature for 3 h. Solvent was removed, and deionized water (10 mL) was added to the residue. A dark blue solid formed. The mixture was filtered and washed with water. The dark blue solid was collected and dried with high vacuum in a desiccator to provide 5 (141 mg) in 90% yield. Anal. Calcd for C60H75N8O14Gd: C, 55.84; H, 5.86; N, 8.69. Found: C, 56.71; H, 5.88; N, 8.37. Compound 8. A mixture of 2 (228 mg, 0.358 mmol), di-tertbutyl iminodiacetate (132 mg, 0.538 mmol), DCC (89 mg, 0.431 mmol), and DMAP (53 mg, 0.434 mmol) in dry dichloromethane (6 mL) was stirred at room temperature under nitrogen for 15 h. Water (1 mL) was then added, and the mixture was stirred for 15 min. The organic layer was separated, dried with Na2SO4, filtered, and concentrated. 1,3-Dicyclohexylurea was removed as described in the preparation of 3. The crude product was purified with column chromatography over neutral alumina (Brockmann grade III, ∼150 mesh) eluting with hexanesEtOAc (v/v 1/1) to provide the title compound 8 (306 mg) as a

34 Bioconjugate Chem., Vol. 16, No. 1, 2005 dark blue oil in 99% yield. UV-vis in CH2Cl2 [λmax ()]: 318 (25650), 410 (128140), 505 (11750), 536 (11750), 605 (9720), 661 (60760). 1H NMR (CDCl3) δ 9.79 (1H, s, H-5), 9.53 (1H, s, H-10), 8.53 (1H, s, H-20), 5.92 [1H, q, J ) 6.6 Hz, CH3(O-hexyl)CH-3], 5.22 (2H, dd, AB system, J ) 20.7 Hz, COCH2-15), 4.51 (1H, q, J ) 6.9 Hz, H-18), 4.39 (1H, m, H-17), 4.03 (2H, splitting s, H-1′ or H-2′), 3.79-3.53 (9H, m, CH3CH2-8, 1× ring CH3, H-1′ or 2′, H-1′′), 3.39, 3.28 (each 3H, s, 2 × ring CH3), 2.75, 2.47 (each 1H, m, HNCOCH2CH2-17), 2.42, 2.14 (each 1H, m, HNCOCH2CH2-17), 2.12 [3H, two set of doublets, J ) 6.6, 7.3 Hz, CH3(Ohexyl)CH-3], 1.81 (3H, d, J ) 7.6 Hz, CH3-18), 1.75 (2H, m, H-2′′), 1.72 (3H, t, J ) 7.6 Hz, CH3CH2-8), 1.50-1.40 [11H, m, H-3′′, 1 × OCOC(CH3)3], 1.23 (4H, m, H-4′′ and H-5′′), 1.04, 1.03 [9H, two singlets, 1 × OCOC(CH3)3], 0.79 (3H, m, H-6′′), 0.43 (1H, br, NH), -1.72 (1H, s, NH). MS (FAB) m/z 864.4 (MH+, 100). HRMS (FAB): Calcd. for C51H70N5O7 (MH+) 864.5275; Found 864.5280. HPPH-di-DTPA-tert-butyl Ester (10). Compound 8 (626 mg, 0.725 mmol) was dissolved in TFA (10 mL). The solution was stirred at room temperature for 2 h. TFA was removed, and the residue was dried with high vacuum. DCC (907 mg, 4.39 mmol), DMAP (268 mg, 2.19 mmol), 7 (2.21 g, 2.84 mmol), and dry CH2Cl2 (20 mL) were then added. The resultant mixture was stirred at room temperature under nitrogen for 20 h. Water (10 mL) was then added, and the mixture was stirred for 15 min. The organic layer was separated, dried with Na2SO4, filtered, and concentrated. 1,3-Dicyclohexylurea was removed as described in the preparation of 3. The product was purified with column chromatography over neutral alumina (Brockmann grade III, ∼150 mesh) eluting with hexanes-EtOAc (v/v 1/2) to remove excess 7 and some other impurity and then eluting with CH3OH-CH2Cl2 (3%) to give 10 (1.13 g) in 68.6% yield. UVvis in CH2Cl2 [λmax ()]: 319 (21870), 411 (109150), 506 (9960), 537 (10010), 604 (8280), 660 (50080). 1H NMR (CDCl3) δ 10.59 (1H, br, 1 × CONH-phenyl), 9.76 (1H, splitting s, H-5), 9.49 (1H, s, H-10), 8.46 (1H, s, H-20), 8.14 (1H, br, 1 × CONH-phenyl), 7.65 (2H, m, 2 × phenyl H), 7.48 (2H, m, 2 × phenyl H), 7.23 (4H, m, 4 × phenyl H), 5.87 [1H, m, CH3(O-hexyl)CH-3], 5.16 (2H, dd, AB system, J ) 20.0 Hz, COCH2-15), 4.40 (1H, m, H-18), 4.28 (1H, m, H-17), 4.07, 3.84 (4H, m, H-1′ and H-1′′), 3.76-3.53 (7H, m, H-1′′′, CH3-12, CH3CH2-8), 3.53-3.28 (23H, CH3-2 and 10 × NCH2CO2C), 3.25 (3H, s, CH3-7), 3.10 (2H, m, H-3′ and H-3′′), 2.98-2.15 (20H, m, HNCOCH2CH2-17, H-2′, 2′′, 4′, 4′′, 5′, 5′′, 6′, 6′′), 2.07 [3H, m, CH3(O-hexyl)CH-3], 1.69 (8H, m, H-2′′′, CH3CH2-8, CH3-18), 1.61-1.28 [92H, m, H-3′′′, 10 × OCOC(CH3)3], 1.20 (4H, m, H-4′′′ and H-5′′′), 0.76 (3H, t, J ) 6.8 Hz, H-6′′), 0.43 (1H, br, NH), -1.75 (1H, s, NH). MS (FAB) m/z 2274.0 (MH+, 100). HRMS (FAB): Calcd for C125H189N13O25Na (MNa+) 2295.3814; Found 2295.3820. HPPH-di-DTPA (11). 10 (30 mg) was treated with TFA (2 mL) as described in the preparation of 4 to provide 11 in quantitative yield. 1H NMR (60% CDCl3, 40% CD3OD and one drop of C5D5N, TMS as internal standard) δ 9.83 (1H, splitting s, H-5), 9.57 (1H, s, H-10), 8.58 (1H, s, H-20), 7.68-7.37 (4H, m, 4 × phenyl H), 7.22-6.94 (4H, m, 4 × phenyl H), 5.92 [1H, m, CH3(O-hexyl)CH-3], 5.13 (2H, dd, AB system, J ) 19.8 Hz, COCH2-15), 4.58-3.99 (6H, m, overlapped with water signal, H-18, H-17, H-1′ and H-1′′), 3.84-2.20 (55H, m, H-1′′′, CH3-2, CH3-7, CH3-12, CH3CH2-8, 10 × NCH2CO2C, HNCOCH2CH217, H-2′, 2′′, 3′, 3′′, 4′, 4′′, 5′, 5′′, 6′, 6′′), 2.12 [3H, m, CH3(Ohexyl)CH-3], 1.85-1.63 (8H, m, H-2′′′, CH3CH2-8, CH3-18), 1.35 (2H, m, H-3′′′), 1.22 (4H, m, H-4′′′ and H-5′′′), 0.76 (3H, m, H-6′′). MS (ESI) m/z 1713.9 (MH+, 100). HPPH-di-DTPA-di-Gd Complex (12). A solution of 10 (700 mg, 0.308 mmol) in TFA (10 mL) was stirred at room temperature for 3 h. TFA was then removed with a ratovapor. The resultant residue was dissolved in pyridine (10 mL). A solution of GdCl3‚6H2O (230 mg, 0.619 mmol) in deionized water (2 mL) was added to above solution. The mixture was stirred at room temperature for 3 h. Solvent was removed, and deionized water (20 mL) was added to the residue. Dark blue solid formed. The mixture was filtered and washed with water. The dark blue solid was collected and dried with high vacuum in a desiccator to provide 12 (630 mg) in 99.5% yield. Anal. C85H107N13O27Gd2. Calcd: C, 49.57; H, 5.24; N, 8.85. Found: C, 50.63; H, 5.10; N, 8.21.

Li et al. Compound 14. Methylpyropheophorbide-a 13 (2.13 g, 3.88 mmol) was dissolved in HBr-AcOH (30%, 40 mL). The solution was stirred at room temperature for 2 h. HBr and acetic acid were removed under high vacuum. To the residue was added tri(ethylene glycol) monomethyl ether (5 mL) and potassium carbonate (2.0 g) and dry CH2Cl2 (20 mL). The mixture was stirred at room temperature for 1 h. It was then diluted with CH2Cl2 and washed with water and 5% NaHCO3. The organic layer was dried with Na2SO4, filtered, and concentrated. The resultant residue was purified with column chromatography over alumina (Grade III) using CH2Cl2-acetone (v/v 9/1) as eluant to give D-20, which was crystallized from CH2Cl2hexanes to provide pure 14 (1.50 g) in 54.2% yield. 1H NMR (CDCl3) δ 9.75 (1H, s, H-5), 9.50 (1H, s, H-10), 8.54 (1H, s, H-20), 6.02 [1H, q, J ) 6.7 Hz, CH3(O-PEG)CH-3], 5.18 (2H, dd, AB system, J ) 19.8 Hz, COCH2-15), 4.49 (1H, m, H-18), 4.30 (1H, m, H-17), 3.66, 3.62, 3,40, 3.28 (15H, each s, 3 × ring CH3, COOCH3, H-7′′), 3.95-3.48, 3.40 (14H, m, CH3CH2-8, H-1′′, 2′′, 3′′, 4′′, 5′′, 6′′), 2.69, 2.55, 2.28 (1H, 1H, 2H, m, CH3OOCCH2CH217), 2.15 [3H, two set of doublets, J ) 6.8, 6.6 Hz, CH3(O-hexyl)CH-3], 1.82 (3H, d, J ) 7.8 Hz, CH3-18), 1.72 (3H, t, J ) 7.6 Hz, CH3CH2-8), 0.43 (1H, br, NH), -1.72 (1H, s, NH). MS (ESI) m/z 735.9 (MNa+, 100). Compound 15. To a solution of 14 (1.5 g) in THF (80 mL) were added methanol (40 mL) and a solution of LiOH‚H2O (1.0 g) in water (20 mL). The mixture was stirred at room temperature for 4 h. It was then neutralized with 2% acetic acid (200 mL). The mixture was extracted CH2Cl2 (4 × 200 mL). The organic layers were combined and washed with water (5 × 400 mL), dried over Na2SO4, filtered, and concentrated. The resultant residue was crystallized with CH2Cl2-hexanes to provide 15 (1.46 g) in 99.3% yield. UV-vis in CH2Cl2 [λmax ()]: 320 (21230), 410 (106603), 505 (9822), 536 (9671), 604 (8160), 661 (49864). 1H NMR (CDCl3) δ 9.72 (1H, splitting s, H-5), 9.49 (1H, s, H-10), 8.51 (1H, s, H-20), 5.99 [1H, q, J ) 6.4 Hz, CH3(OPEG)CH-3], 5.18 (2H, dd, AB system, J ) 20.2 Hz, COCH2-15), 4.47 (1H, q, J ) 7.0 Hz, H-18), 4.31 (1H, m, H-17), 3.65, 3.37, 3,26, 3.25 (12H, each s, 3 × ring CH3, H-7′′), 3.89-3.45, 3.39 (14H, m, CH3CH2-8, H-1′′, 2′′, 3′′, 4′′, 5′′, 6′′), 2.69, 2.59, 2.29 (1H, 1H, 2H, m, CH3OOCCH2CH2-17), 2.12 [3H, two set of doublets, J ) 6.6, 6.7 Hz, CH3(O-hexyl)CH-3], 1.80 (3H, d, J ) 6.9 Hz, CH3-18), 1.69 (3H, t, J ) 7.7 Hz, CH3CH2-8), -1.72 (1H, s, NH). MS (FAB) m/z 699.2 (MH+, 100). HRMS (FAB): Calcd for C40H51N4O7 (MH+) 699.3757; Found 699.3730. Compound 16. By following the procedure for the preparation of 8, reaction of 15 (1.45 g, 2.07 mmol) with di-tert-butyl iminodiacetate (770 mg, 3.14 mmol) in the presence of DCC (650 mg, 3.15 mmol) and DMAP (382 mg, 3.13 mmol) provided 16 (1.60 g) in 83.3% yield. UV-vis in CH2Cl2 [λmax ()]: 318 (18008), 411 (92100), 505 (8335), 537 (8438), 605 (6895), 661 (43426). 1H NMR (CDCl ) δ 9.74 (1H, splitting s, H-5), 9.53 (1H, s, H-10), 3 8.53 (1H, s, H-20), 6.01 [1H, m, CH3(O-hexyl)CH-3], 5.21 (2H, dd, AB system, J ) 19.8 Hz, COCH2-15), 4.50 (1H, m, H-18), 4.38 (1H, m, H-17), 4.02 (2H, splitting s, H-1′ or 2′), 3.68, 3.34, 3,29, 3.27 (12H, each s, 3 × ring CH3, H-7′′), 3.91-3.50, 3.42 (14H, m, CH3CH2-8, H-1′′, 2′′, 3′′, 4′′, 5′′, 6′′), 3.39 (2H, s, H-1′ or 2′), 2.74, 2.43, 1.94 (1H, 2H, 1H, m, CH3OOCCH2CH2-17), 2.13 [3H, d, J ) 6.7 Hz, CH3(O-hexyl)CH-3], 1.80 (3H, d, J ) 7.5 Hz, CH3-18), 1.72 (3H, t, J ) 7.5 Hz, CH3CH2-8), 1.47, 1.43 1.07, 1.03 [6H, 6H, 3H, 3H, each s, 2 × OCOC(CH3)3], 0.79 (3H, m, H-6′′), 0.39 (1H, br, NH), -1.75 (1H, s, NH). MS (FAB) m/z 926.3 (MH+, 100). HRMS (FAB) Calcd for C52H72N5O10 (MH+) 926.5279; Found 926.5316. Compound 18. By following the procedure for the preparation of 10, compound 16 (3.70 g, 3.99 mmol) was treated with TFA (40 mL) to afford compound 17. Without purification, reacting 17 with 7 (7.50 g, 9.63 mmol) in the presence of DCC (2.04 g, 9.89 mmol) and DMAP (1.22 g, 9.99 mmol) provided 18 (5.91 g) in 63.3% yield. UV-vis in CH2Cl2 [λmax ()]: 318 (22525), 412 (112301), 505 (10336), 537 (10243), 605 (8528), 661 (51863). 1H NMR (CDCl ): δ 10.79 (1H, br, 1 × CONH-phenyl), 9.72 (1H, 3 splitting s, H-5), 9.45 (1H, splitting s, H-10), 8.76 (1H, br, 1 × CONH-phenyl), 8.47 (1H, s, H-20), 7.71 (2H, m, 2 × phenyl H), 7.54 (2H, d, J ) 7.9 Hz, 2 × phenyl H), 7.25 (2H, m, 2 × phenyl H), 7.20 (2H, d, J ) 8.2 Hz, 2 × phenyl H), 5.96 [1H, m, CH3(O-hexyl)CH-3], 5.15 (2H, dd, AB system, J ) 20.4 Hz, COCH2-


Bioconjugate Chem., Vol. 16, No. 1, 2005 35

Scheme 1

15), 4.41 (1H, m, H-18), 4.26 (1H, m, H-17), 3.94 (2H, m, H-1′ or H-1"), 3.87-3.20 (48H, m, H-1′ or H-1′′, H-1′′′, 2′′′, 3′′′, 4′′′, 5′′′, 6′′′, 7′′′, 10 × NCH2CO2C, CH3-2, CH3-7, CH3-12, CH3CH28), 3.11 (2H, m, H-3′ and H-3′′), 2.98-2.30 (20H, m, HNCOCH2CH2-17, H-2′, 2′′, 4′, 4′′, 5′, 5′′, 6′, 6′′), 2.10 [3H, m, CH3(PEG)CH-3], 1.68 (6H, m, CH3CH2-8, CH3-18), 1.55-1.30 [90H, m, 10 × OCOC(CH3)3], 0.38 (1H, s, NH), -1.77 (1H, s, NH). MS (FAB) m/z 2336.1 (MH+, 68), 2358.1 (MNa+, 100). HRMS (FAB): Calcd for C126H191N13O28Na (MNa+) 2357.3818; Found 2357.3820. Gd(III) Complex 20. A solution of 18 (5.90 g, 2.52 mmol) in TFA (50 mL) was stirred at room temperature for 3 h. TFA was then removed under vacuum. The resultant residue was washed with water, dried under vacuum, and dissolved in pyridine (70 mL). To this solution was added a solution of GdCl3‚6H2O (1.91 g, 5.14 mmol) in deionized water (5 mL). The mixture was stirred at room temperature for 2.5 h. Solvent was removed to dryness, and deionized water (100 mL) was added to the residue. The dark blue solid was washed several times with water (5 × 100 mL) and dried under high vacuum in a desiccator to provide 20 (5.20 g) in 97.1% yield. Analysis for C86H109N13O30Gd2. Calculated: C, 48.69; H, 5.18; N, 8.59. Found: C, 48.91; H, 4.99; N, 8.85. Formulation Procedure. To formulate the compound into the liposome mixture, a ratio of 2:1 60% lecithin to cholesterol (Sigma) by mass is dissolved into a minimal amount of dichlo-

romethane in a large-mouthed test tube. The dichloromethane was then evaporated by purging nitrogen slowly while the test tube was kept warm by inserting the test tube in a warm water bath. The test tube was then placed into desiccator under high vacuum for 1 h in order to ensure total removal of the dichloromethane. The compound was weighed out in a ratio of 2:1 60% lecithin to compound by mass and placed into another large test tube. PBS (6 mL) (pH 7.2) was added to the compound and sonicated for 5-10 min. This mixture was then pipetted into the test tube with the liposome film and sonicated for 2 h. The solution must be surrounded by cold water since sonication produces heat. After 2 h the solution was filtered through a 0.45 µm filter into a plastic culture tube and wrapped in aluminum foil. Whole Body MRI Imaging. Rats (female SD rats, three rats/group) with subcutaneously implanted Ward colon carcinomas (1 cm3) were imaged in a 1.5 T magnetic resonance imaging system using a standard wrist coil (GE Horizon 5.8, GE Medical Systems, Milwaukee, WI). Preinjection imaging was T1 weighted (TR ) 500 ms, TE ) 14 ms) in axial and coronal planes. Image thickness was 3 mm with a 1 mm interscan gap. Matrix size was 256 × 192 with 1.5 excitations and an 8 × 8 cm field-of-view. T1/T2 Relaxivity Experiments: The R1 and R2 relaxivity measurements were acquired on a General Electric 4.7T/33 cm horizontal bore magnet (GE NMR instruments, Fremont, CA)

36 Bioconjugate Chem., Vol. 16, No. 1, 2005

Li et al.

Scheme 2

incorporating AVANCE digital electronics (Bruker BioSpec platform with Paravision version 2.1 acquisition software, Bruker Medical, Billerica, MA). T1 relaxation rates (R1) were acquired for a range of contrast agent concentrations with a saturation recovery spin-echo (SE) sequence with a fixed TE ) 10 ms and TR times ranging from 125 to 8000 ms (FOV ) 32 × 32 mm, slice thickness ) 1 mm, slices ) 3, interslice gap ) 2 mm, matrix ) 128 × 128, NEX ) 2). Signal intensities at each repetition time was obtained by taking the mean intensity within ROIs using Analyze 5.0 (Biomedical Imaging Resource, Mayo Foundation, Rochester, MN), and R1 and SMAX were determined by nonlinear fitting of the equation:

S(TR) ) SMAX(1 - e-(R1‚TR)) + background noise using Matlab’s Curve Fitting Toolbox (Matlab 6.5, MathWorks Inc., Natick, MA). Similarly, T2 relaxation rates (R2) were acquired with multi-echo, CPMG SE sequence with a fixed TR of 4500 ms and TE times ranging from 100 to 2000 ms. R2 and SMAX were determined as described above using the equation:

S(TE) ) SMAX(e-(R2‚TE)) + background noise Animal and Tumor Systems. RIF-1 tumors were propagated in 6-9 week old female C3H/He mice. Tumors were obtained by subcutaneous injection of 2 × 105 RIF cells, prepared from established tumors by enzymatic digestion, into the right flanks of the animals. Prior to tumor-cell injection all hair was removed from the inoculation site by shaving and use of a commercially available depilatory, e.g. Nair. Tumors were used for experimentation when they reach a diameter of 5-6 mm and a width and thickness of ∼4 mm. Light Sources. An 18-W argon laser pumping a dye laser (model 171/375; Spectra-Physics) using DCM dye (Exciton, Inc.) tuned to 665 nm wavelength filter was employed. The output beam was split using a custom-made eight-way beam splitter

device coupled to 400 µm diameter optical fibers. Each fiber was terminated with a microlens of a focal length of 3.5 mm in order to supply an even light distribution over the treatment field. The power density (75 mW cm-2) was measured by a thermopile power meter (Coherent), and treatment field size was typically 1 cm in diameter. Assessment of Tumor Response and Cure. The tumor’s length (the longest dimension) and width (perpendicular to the long axis) was measured with electronic calipers. The volume was calculated as V ) 1/2(L × W2). The hours-to-endpoint (HTE; time to 400 mm3) was calculated by linearly interpolating between the times just before and after this volume is reached, using log(tumor volume) for the calculations; this was done using Microsoft Excel-based software developed by the RPCIC Biomathematics Resource. Mice were routinely sacrificed just after their tumors reached the endpoint. For nonrecurrent tumors, animals were followed for at least 30 days from the time of treatment. Individual experiment included at least five mice. Toxic death was defined as deaths taking place within 48 h of treatment. Toxic deaths only occur after intense PDT treatments. Mice were used to elucidate important questions regarding the PDT efficacy. The primary therapeutic endpoint was based on the time for tumor regrowth to 400 mm3. These procedures fall into Class 1 (little or no pain and distress) or Class 2 (mild stress) categories. The designed toxicity endpoint, normal (foot) tissue damage, is a Class 3 procedure; early toxic death is a nondesigned toxicity endpoint. PDT experiments using these endpoints are covered in an approved Institute Animal Care and Use (IACUC) protocol. Determination of Intracellular Localization by Fluorescence Light Microscopy. In our attempt to investigate the site(s) of localization of the conjugates, these compounds were coincubated in the RIF cells with Mitotracker Green (Molecular Probes, Eugene, OR) which target mitochondria. Briefly, 1 × 105 cells were plated on poly-L-lysine-coated coverslips and grown for 2 days. Cells were incubated with photosensitizer(s) for 4 or 24 h at 37 °C, 5% CO2, in the dark, and then washed



Scheme 3

with fresh growth medium for 1 h to remove loosely bound drug, and gently rinsed with PBS immediately prior to microscopy (Zeiss Axiovert 200, Inverted Fluorescence Microscope, Carl Zeiss Inc., Germany). Localization was similar at both timepoints. For the coincubation studies, Mitotracker Green (200 nM) was added 24 h before washing with PBS and examining by microscopy. Fluorescence was imaged with an AxioCam MRM monochrome digital camera. The images were captured and processed by MRGrab Software (Carl Zeiss, Inc., Germany). The cells were illuminated by a Fluoarc HBO 103 mercury vapor arc lamp filtered through a filter cube containing a 390-430 nm excitation filter, a 505 nm dichroic filter, and a 650-700 nm emission filter for detection of photosensitizers. Mitotracker Green was detected using a 450-490 nm excitation filter, a 495

nm dichroic, and a 500-550 nm emission filter. Images of photosensitizers and Mitotracker Green were taken in rapid succession.

RESULTS AND DISCUSSION

Chemistry. HPPH Conjugated with One Gd(III)Aminobenzyl-DTPA Complex. For the preparation of the conjugate, methyl pheophorbide a, 1, was isolated from Spirulina pacifica (17, 18) and was converted into the corresponding 3-devinyl-3-(1-hexyloxy)ethyl-pyropheophorbide a (HPPH), 2, by following the improved methodology developed in our laboratory. For the preparation of the MR agent, HPPH 2 was reacted with 4-aminobenzyl-


Li et al.

Figure 1. MRI images of tumors (Word Colon) implanted in rats (SD) from conjugate 20 (10 µmol/kg) before and at 8 h postinjection (for details see the text).

DTPA penta-tert-butyl ester 7 [which in turn was obtained in several steps from 6 by slightly modifying the methodology reported by Cummins et al. (19)] via the carbodiimide approach as illustrated in Scheme 1. For the preparation of the Gd(III) complex, the tert-butyl ester functionalities in 3 were converted into the corresponding carboxylic acid 4, by reacting with trifluoroacetic acid (yield 100%). It was then dissolved in pyridine and reacted with gadolinium chloride hexahydrate dissolved in deionized water at room temperature for 2 h. The solvents were removed, and the residue was washed with deionized water several times and dried under high vacuum. The desired compound 5 was isolated (92% yield). HPPH Conjugated with Two Gd(III)-AminobenzylDTPA Complexes. The tumor avidity of any scanning agent, whether receptor-based (monoclonal antibodies, peptides) or porphyrin-based, generally improves only slightly by molecular redesign. An alternate approach for improving MR relaxivity would be to increase the number of Gd(III) atoms per molecule. Our approach was based on the hypothesis that HPPH conjugated with two or

more Gd(III) chelates per molecule would further enhance tumor conspicuity and/or lower the dose required for MR imaging. For the synthesis of the desired conjugate, HPPH 2 was first reacted with di-tert-butyl iminodiacetate by following the carbodiimide approach as described for the preparation 3 (Scheme 1), and the intermediate amide analogue 8 was obtained (quantitative yield). The two tert-butyl functionalities present in the intermediate were cleaved on reacting with TFA, and the corresponding carboxylic acid 9 was isolated in quantitative yield. In a sequence of reactions, amide analogue 9 on reacting first with 4-aminobenzyl-DTPA 7 (Scheme 1) and then with TFA and GdCl3 produced the desired compound 12 (90% overall yield). The structures of the intermediates and the final products were confirmed by 1H NMR and/or mass spectrometry. Di-Gd(III)-DTPA Conjugate of Pyropheophorbide-PEG. Previous SAR and QSAR efficacy studies on a series of alkyl ether analogues of pyropheophorbide-a (8) has shown that lipophilicity is one of the determinants for PDT. Therefore, to investigate the effect of hydrophilic substituent in tumor imaging, PDT efficacy, and skin



Figure 2. Tumor images rats implanted with Word Colon tumors) before (A) and 8 h postinjection (B) of Magnavist (1 mmol/kg).

phototoxicity, the hexyl ether group present at position-3 in compound 12 was replaced by a poly(ethylene glycol) methyl ether group by following the methodology depicted in Scheme 3, and conjugate 20 was isolated in good yield (see Experimental Procedures). Body Tumor MR Imaging. HPPH-Mono-Gd(III)aminobenzyl-DTPA Conjugate 5, HPPH-Di-Gd(III)-aminobenzyl-DTPA Conjugate 12, and the Related PEG Derivative 20. Imaging was repeated at 1 and 24 h postinjection of Gd-HPPH 5 (11.25 µmol/kg) with identical imaging parameters (see Experimental Procedures). Region of interest measurements were obtained over the tumor paraspinal muscles and intraabdominal fat, using a workstation (GE Advantage Windows 3.1, GE Medical Systems, Milwaukee, WI). The tumor area of interest increased markedly, from 623 to 881 intensity units. The effect was striking both visually and quantitatively. Signal enhancement was largely restricted to tumor. The signal obtained from fat was essentially unchanged (1998 to 1939 intensity units), and muscle enhancement was minimal. The Gd-HPPH 5 was insoluble in water and required solubilization in 1% Tween-80/water solution, which represents a major barrier to human use because of its known toxicity at higher doses. Therefore, the formulation was revised to Gd-HPPH liposomal formulation (see Experimental Procedures), which has achieved a 25-fold increase in solubility. To prove that the liposome preparation remains tumoravid, we successfully produced images virtually identical to the Tween 80 formulation using 10 µmol/kg of conjugate 5. We also observed that the “higher payload” tumoravid double-Gd-HPPH compound 12 was equally soluble with liposomal capsulation and enhanced tumor signal. However, the effect was not linear (the signal enhancement was largely restricted to tumor, whose intensity rises markedly from 370 (pre-injection) to 582 (24 h) to 715 at 48 h postinjection. No tumor enhancement with conjugate 20 over baseline was detected, either visually or quantitatively, at doses of 2.5 µmol/kg and 5.0 µmol/kg in rats. At a dose of 10 µmol/kg, the best enhancement of tumor signal relative to baseline developed 8 h after drug injection. A baseline supine transaxial image viewed from the caudal end (Figure 1, A) demonstrates transplanted tumor (1 cm3) growing out of the ventral abdominal wall. Muscle, fat, and bowel were clearly defined. Baseline signal measurements (Figure 1B) of tumor, muscle, fat, and control tubes of saline and contrast medium were 538, 403, 172, 385, and 1684. Eight hours after intravenous injection of 10.0 µmol/kg of the Gd(III) complex of PyroPEG 20 (Figure 1C) the tumor was visually enhanced and the tumor signal measurement (Figure 1D) increased 22% to 654. Fat and muscle signal changed minimally, from 172 and 403 (baseline measurement) to 165 and 411 (after the conjugate injection), respectively. On the 8-h images, saline and contrast medium control tubes mea-

Figure 3. T1 relaxivity of conjugate 20.

Figure 4. T2 relaxivity of conjugate 20.

Figure 5. In vivo photosensitizing efficacy of DTPA conjugates 5 and 12 (liposomal formulation) at a dose of 0.47 µmol/kg in C3H mice (six mice/group) implanted with RIF tumors. The 4-6 mm diameter tumors were exposed to light [130 J/cm2 (75 mW/ cm2)] for 30 min from a tunable dye laser tuned to the maximum red absorption peak (λmax665 nm)] 24 h postinjection. At day 50, 3/6 mice were tumor-free.

sured 359 and 1746, representing a variation of 15% and 5% respectively from baseline measurements, due to normal system “drift”. Comparative Study with Magnavist.. At present, Magnavist [Gd(III) complex of DTPA] is a standard MRI agent that clinicians use on an almost daily basis. It, however, works by penetrating damaged tumor vascular beds and does not bind to tumor cells, so that when it rapidly clears from the circulation, the tumor concentration also falls rapidly. It has a very brief temporal “window” for MR imaging (5 to 10 min), and therefore to use Magnavist as a tumor-enhancing agent one must know where to image with MRI in advance as it clears


Figure 6. Mice (C3H/HeJ, six mice/group) bearing RIF tumors were treated with 20 (10 µmol/kg) with light (75mW, 135 J/cm2) for 30 min at 8 h postinjection. Control: No light treatment. At day 90, 5/6 mice were tumor-free.

from tumors within minutes. In the present study, Magnavist was used to image tumors implanted in Fisher rats. The best images were obtained at 5, 10 min postinjection (data not shown). However, at 1, 4, 8, and 24 h postinjection, no tumor enhancement was observed (Figure 2, images at 8 h postinjection), whereas the lead compound (e.g. 20) remains in tumors for a reasonable time, so that MRI could scan several large body areas and later treat transcutaneously by PDT. T1 and T2 relaxivity of each compound was determined by taking the slope of linear least-squares fit of contrast agent concentration vs relaxation rate (for compound 20 see Figures 3 and 4). For comparison, relaxivity of GdDTPA (Magnevist, Berlex, Wayne, NJ) at 4.7 T was acquired under identical conditions. Compound 20 formulated in liposome demonstrated a relatively high T1 relaxivity of 18.70 mM-1‚s-1 at 4.7 T as compared to GdDTPA (3.25 mM-1‚s-1), as well as a high T2 relaxivity of 58.63 mM-1‚s-1.

Li et al.

In Vivo Photosensitizing Efficacy of Liposome-Encapsulated Mono-Gd-HPPH Conjugate 5 and Di-Gd(III) Conjugate 12: The photosensitizing efficacies of the mono- 5 and di-Gd(III)-DTPA 12 were evaluated in C3H mice (six mice/group) bearing RIF tumors (7, 8) at a dose of 0.47 µmol/kg. The compounds in liposomal formulation produced the same PDT efficacy (Figure 3). As can be seen, the presence of an additional Gd(III)-DTPA molecule did not inhibit PDT activity. By day 35 the effect of each compound was equal to 50% tumor cure (3/6 mice were tumor free and both formulations produced the same PDT efficacy (Figure 3). However, compound 12 at the tumor-imaging dose (10 µmol/kg), after light exposure (135 J/cm2, 24 h postinjection), was found to be toxic and produced 100% mortality (without light exposure, no toxicity was observed). In Vivo Photosensitizing Activity of the PEG Analogue 20 at MR Imaging Dose. Compound 20 at the tumor imaging dose (10.0 µmol/kg) was evaluated for PDT efficacy in C3H mice (10 mice/group) bearing RIF tumors under the light treatment conditions described for compounds 5 and 12 [130 J/cm2 (75 mW/cm2) for 30 min] at 8 h postinjection (tumor-imaging time). As can be seen from Figure 6, in preliminary screening, the PEG analogue 20 was found to be quite effective (80% of mice were tumor-free on day 90) and did not produce any mortality or adverse effects at the imaging dose before and after the light treatment. All treated mice showed normal behavior. Skin Phototoxicity of the Conjugates and Advantages of the Related PEG Analogue. Conjugates 5, 12, and 20 (10 µmol/kg, the imaging dose) were injected (iv) individually to rats (three rats/group), and the foot was exposed to light (18). The feet of each rat (one exposed to light and another used as a control) response was observed daily. Skin response was significantly different for conjugates 5, 12, and 20. Both 5 and 12 were found

Figure 7. Foot response in female SD rats injected with 10 µmol/kg of compound 20 [A: before light exposure, B: light exposure at 24 h and C: at 96 h postinjection]. At 24 h, swelling of toes and at 96 h, light edema (almost normal) was observed (for details see the text).

Figure 8. Comparative localization of Mitotracker Green (Mitochondrial marker) and conjugate 20 (1.0 µM) in RIF cells, determined by fluorescence light microscopy (false colors). Similar patterns were obtained from the mono- and di-Gd(III)-aminophenyl-DTPA conjugates 5 and 12 respectively (for details, see the text).


to be extremely phototoxic whereas compound 20 showed significantly reduced skin phototoxicity. The foot response was scored by following the literature procedure and was recorded as follows: At 24 h: 1.8 (moderate dry desquamation and swelling of toes); 72 h: 1.0 (erythema with edema and slight depilation); 96 h: 0.1 (light edema); 120 h: 0 (normal, no reaction) (Figure 7). Thus, initial mild-to-moderate phototoxicity resolved to some extent on day 4 and 5. Therefore, the introduction of a more hydrophilic group certainly helped in clearance of the drug from the skin, resulting in reduced phototoxicity. Intracellular Localization. It has been shown by us and others that photosensitizers that localize in mitochondria are generally more effective than those that localize in lysosomes (20-24). Therefore, the localization characteristics of HPPH (localize in mitochondria) was compared with the related DTPA-Gd(III) conjugates including the PEG analogue (for details, see Experimental Procedures). The HPPH and the related DTPA-Gd(III) conjugates 5 and 12 and the PEG analogue 20 were found to localize in mitochondria. A representative example of the DTPA conjugate 20 is shown in Figure 8. Localization images by fluorescence light microscopy (see Experimental Procedures) were taken in rapid sessions. The results (Figure 8) indicate that Mitotracker Green and conjugate 20 localize to the same subcellular region, the mitochondria. CONCLUSION

The preliminary data suggest that di-DTPA-Gd(III) conjugate 12 is a more effective MRI imaging agent than the related mono-DTPA analogue 5. Therefore, for designing an improved imaging agent, it is advantageous to have two Gd(DTPA) moieties in the conjugates. However, for both HPPH conjugates (mono-DTPAGd(III) 5 and di-DTPA-Gd(III)) 12, the imaging dose (10.0µmol/kg) produced severe skin phototoxicity. Interestingly, conjugate 20 in which the hexyl substituent (conjugate 12) was replaced with PEG-monomethyl ether was found to be an effective MRI agent (at 8 h postinjection) at a dose of 10 µmol/kg, with enhanced in vivo photosensitizing efficacy and reduced skin phototoxicity. Measured T1 and T2 relaxivities for liposome-formulated Pyro-PEG-di-DTPA-Gd(III) contrast media 20 were significantly increased as compared to a commercially available mono-DTPA-Gd(III) MR contrast-enhancing agent and were consistent with theoretical values expected for a di-DTPA-Gd(III) conjugate (25). Though the PEG conjugate is not entirely free from phototoxicity, the imaging and photosensitizing results obtained from our systematic synthetic design provides an excellent approach for developing improved bifunctional agents. These characteristics raise the concept of a single “see and treat” agent that could be injected intravenously to image the tumors by MRI and then treat them by exposing to light (PDT) introduced either endoscopically or by a thin fiber optic cable, following guided needle puncture. On the basis of these results, we extrapolate that an improved dual agent can be developed by (a) selecting those photosensitizers as substrates that possess higher tumor selectivity than HPPH, (b) the desired overall lipophilicity can be tuned by introducing mono- or di-PEG substituents at the peripheral position of the chromophore, (c) the depth of tissue penetration could further be increased by conjugating Gd(III)-DTPA with those photosensitizers that exhibit long-wavelength absorption near 750-800 nm. Such dual conjugates may


have advantages in treating large and deeply seated tumors. These studies are currently in progress. ACKNOWLEDGMENT

The authors are thankful to the NIH (CA 55791, 1 R21 CA109914-01), Oncologic Foundation of Buffalo, Roswell Park Alliance, and the shared resources of the Roswell Park Cancer Center Support Grant (P30CA16056) for financial support. We are thankful to Adam Sumlin, PDT Center, for his help in evaluating the foot-response assay (skin phototoxicity). The technical assistance provided by Schlossin and Carol Kaminskiin in tumor imaging is highly appreciated. LITERATURE CITED (1) Caravan, P., Ellison, J. J., McMurry, T. J., and Lauffer, R. B. (1999) Gadolinium(III) chelates as MRI contrast agents: Structure, dynamics, and applications. Chem. Rev. 99, 2293352. (2) Wagner, H. N., Szabo, Z., and Buchanan, J. W. (1995) Nuclear Medicine: What it is and what it does? In Principles of Nuclear Medicine, 2nd ed., Chapter 1, W. B. Saunders Co., Philadelphia. (3) McAfee J. G., and Subramanian, G. (1984) Clinical Radionuclide Imaging. In Radioactive Agents for Imaging (Freeman, L. M., Ed.) Chapter 3, Grune & Stratton, Inc., Philadelphia. (4) Grossman, Z. D., and Rosebrough, S. F. (1990) Clinical Radioimmunoimaging, Grune & Stratton, Inc., Philadelphia. (5) Ni, Y., Adzamli, K., Miao, Y., Cresens, E., Yu, J., Periasamy, M. P., Adama, M. D., and Marchal, G. (2001) MRI contrast enhancement of necrosis by MP-2269 and gadoporphyrin-2 in a rat model of liver infarction. Invest. Radiol. 36 (2), 97103. (6) Hoffmann, B., Bogdanov A., Jr., Marecos E., Ebert, W., Semmler, W., and Weissleder, R. (1999) Mechanism of gadoporphyrin-2 accumulation in tumor necrosis. J. Magn. Reson. Imaging 9, 336-41. (7) Pandey, R. K., Sumlin, A. B., Potter, W. R., Bellnier, D. A., Henderson, B. W., Constantine, S.; Aoudia, M.; Rodgers, M. A. J., Smith, K. M., and Dougherty, T. J. (1996) Synthesis, photophysical properties and photodynamic efficacy of the alkyl ether analogues of chlorophyll-a derivatives. Photochem. Photobiol. 63, 194-205. (8) Henderson, B. W., Bellnier, D. A., Greco, W. R., Sharma, A., Pandey, R. K., Vaughan, L., Weishaupt, K. R., and Dougherty, T. J. (1997) A quantitative structure-activity relationship for a congeneric series of pyropheophorbide derivatives as photosensitizers for photodynamic therapy. Cancer Res. 57, 4000-4007. (9) Zheng, G., Potter, W. R., Sumlin, A., Dougherty, T. J., and Pandey, R. K. (2000) Photosensitizers related to purpurin18-N-alkylamides. A comparative in vivo tumoricidal ability of ester vs amide functionalities. Bioorg. Med. Chem. Lett. 10, 123-127. (10) Rungta, A., Zheng, G., Missert, J. R., Potter, W., Dougherty, T. J., and Pandey, R. K. (2000) Purpurinimides as photosensitizers: Effect of the presence and position of the substituents in in vivo photodynamic efficacy. Bioorg. Med. Chem. Lett. 10, 1463-1466. (11) Zheng, G., Camacho, S., Potter, W., Bellnier, D. A., Henderson, B. W., Dougherty, T. J., and Pandey, R. K. (2001) Synthesis, tumor uptake and in vivo photosensitizing efficacy of a homologous series of 3-devinyl-3-(1-hexyloxyethyl)-purpurin-18-N-alkylimides. J. Med. Chem. 44, 1540-1559. (12) Chen, Y., Graham. A., Potter, W., Morgan, J., Vaughan, L., Bellnier, D. A., Henderson, B. W., Oseroff, A., Dougherty, T. J., and Pandey, R. K. (2002) Bacteriopurpurinimides: Highly stable and potent photosensitizers for photodynamic therapy. J. Med. Chem. 45, 255-258. (13) Li, G., Graham. A., Dobhal, M. P., Morgan, J., Oseroff, J., Dougherty, T. J., and Pandey, R. K. (2003) Synthesis, in vitro/ in vivo photosensitizing efficacy, intracellular localization,

42 Bioconjugate Chem., Vol. 16, No. 1, 2005 HAS (site II) binding ability of various bacteriochlorins derived from chlorophyll-a. J. Med. Chem. 46, 5349-5359. (14) Chen, Y., Sumlin, A., Morgan, J., Gryshuk, A., Oseroff, A., Henderson, B. W., Dougherty, T. J., and Pandey, R. K. (2004) Synthesis and photosensitizing efficacy of isomerically pure bacteriopurpurinimides. J. Med. Chem. 47, 4814-4817. (15) Dougherty, T. J., Pandey, R. K., Nava, H. R., Smith, J. A., Douglass H. O., Edge, S. B., Bellnier, D. A., O.Malley, L., and Cooper, M. (2000) Preliminary clinical data on a new photodynamic therapy photosensitizer HPPH for treatment of obstructive esophageal cancer. Proc. SPIE, 3909, 25-27. (16) Bellnier, D. A., Greco, W. R., Loewen, G. M., Nava V., Oseroff, A. R., Pandey, R. K., Tsuchida, T., and Dougherty, T. J. (2003) Population pharmacokinetics of the photodynamic therapy agent HPPH in cancer patients. Cancer Res. 63, 1806-1813. (17) Smith, K. M., Goff, D. A., and Simpson, D. J. (1985) Meso substitution of chlorophyll derivatives: direct route for transformation of bacteriopheophorbide-d into bacteriopheophorbide-c. J. Am. Chem. Soc. 107, 4941-4954. (18) Pandey, R. K., Bellnier, D. A., Smith, K. M., and Dougherty, T. J. (1991) Chlorin and porphyrin derivatives as potential photosensitizers in photodynamic therapy. Photochem. Photobiol. 53, 65-72. (19) Cummins, C. H., Rutter, E. W., and Fordyce, W. A. (1991) A convenient synthesis of bifunctional chelating agents based

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Chlorophyll-a Analogues Conjugated with Aminobenzyl-DTPA as

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