Tumor Cell-Specific Retention and Photodynamic Action of Erlotinib-Pyropheophorbide Conjugates.

To enhance uptake of photosensitizers by epithelial tumor cells by targeting these to EGFR, pyropheophorbide derivatives were synthesized that had erlotinib attached to different positions on the macrocycle. Although the addition of erlotinib reduced cellular uptake, several compounds showed prolonged cellular retention and maintained photodynamic efficacy. The aim of this study was to identify whether erlotinib moiety assists in tumor targeting through interaction with EGFR and whether this interaction inhibits EGFR kinase activity. The activity of the conjugates was analyzed in primary cultures of human head and neck tumor cells with high-level expression of EGFR, and in human carcinomas grown as xenografts in mice. Uptake of erlotinib conjugates did not correlate with cellular expression of EGFR and none of the compounds exerted EGFR-inhibitory activity. One derivative with erlotinib at position 3, PS-10, displayed enhanced tumor cell-specific retention in mitochondria/ER and improved PDT efficacy in a subset of tumor cases. Moreover, upon treatment of the conjugates with therapeutic light, EGFR-inhibitory activity was recovered that attenuated EGFR signal-dependent tumor cell proliferation. This finding suggests that tumor cell-specific deposition of erlotinib-pyropheophorbides, followed by light triggered release of EGFR-inhibitory activity, may improve photodynamic therapy by attenuating tumor growth that is dependent on EGFR-derived signals.

Tumor-Avid 3-(1'-Hexyloxy)ethyl-3-devinylpyrpyropheophorbide-a (HPPH)-3Gd(III)tetraxetan (DOTA) Conjugate Defines Primary Tumors and Metastases.

3-(1'-Hexyloxyethyl)-3-devinylpyropheophorbide-a (HPPH or Photochlor), a tumor-avid chlorophyll a derivative currently undergoing human clinical trials, was conjugated with mono-, di-, and tri-Gd(III)tetraxetan (DOTA) moieties. The T1/T2 relaxivity and in vitro PDT efficacy of these conjugates were determined. The tumor specificity of the most promising conjugate was also investigated at various time points in mice and rats bearing colon tumors, as well as rabbits bearing widespread metastases from VX2 systemic arterial disseminated metastases. All the conjugates showed significant T1 and T2 relaxivities. However, the conjugate containing 3-Gd(III)-aminoethylamido-DOTA at position 17 of HPPH demonstrated great potential for tumor imaging by both MR and fluorescence while maintaining its PDT efficacy. At an MR imaging dose (10 µmol/kg), HPPH-3Gd(III)DOTA did not cause any significant organ toxicity in mice, indicating its potential as a cancer imaging (MR and fluorescence) agent with an option to treat cancer by photodynamic therapy (PDT).

Chiral Alkyl Groups at Position 3(1') of Pyropheophorbide-a Specify Uptake and Retention by Tumor Cells and Are Essential for Effective Photodynamic Therapy.

To investigate the importance of the chirality and precise structure at position 3(1') of pyropheophorbide-a for tumor cell specificity and photodynamic therapy (PDT), a series of photosensitizers (PSs) was synthesized: (a) with and without chirality at position 3(1'), (b) alkyl ether chain with a variable number of chiral centers, (c) hexyl ether versus thioether side chain, and (d) methyl ester versus carboxylic acid group at position 172. The cellular uptake and specificity were defined in human lung and head/neck cancer cells. PSs without a chiral center and with an alkyl chain or thioether functionalities showed limited uptake and PDT efficacy. Replacing the methyl group at the chiral center with a propyl group or introducing an additional chiral center improved cellular retention and tumor cell specificity. Replacing the carboxylic acid with methyl ester at position 172 lowered cellular uptake and PDT efficacy. A direct correlation between the PS uptake in vitro and in vivo was identified.

Synthesis, Tumor Specificity, and Photosensitizing Efficacy of Erlotinib-Conjugated Chlorins and Bacteriochlorins: Identification of a Highly Effective Candidate for Photodynamic Therapy of Cancer.

Erlotinib was covalently linked to 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH) and structurally related chlorins and bacteriochlorins at different positions of the tetrapyrrole ring. The functional consequence of each modification was determined by quantifying the uptake and subcellular deposition of the erlotinib conjugates, cellular response to therapeutic light treatment in tissue cultures, and in eliminating of corresponding tumors grown as a xenograft in SCID mice. The experimental human cancer models the established cell lines UMUC3 (bladder), FaDu (hypopharynx), and primary cultures of head and neck tumor cells. The effectiveness of the compounds was compared to that of HPPH. Furthermore, specific functional contribution of the carboxylic acid side group at position 172 and the chiral methyl group at 3(1') to the overall activity of the chimeric compounds was assessed. Among the conjugates investigated, the PS 10 was identified as the most effective candidate for achieving tumor cell-specific accumulation and yielding improved long-term tumor control.

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.

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.

Pluronic F-127: An Efficient Delivery Vehicle for 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH or Photochlor).

To determine the impact of delivery vehicles in photosensitizing efficacy of HPPH, a hydrophobic photosensitizer was dissolved in various formulations: 1% Tween 80/5% dextrose, Pluronic P-123 and Pluronic F-127 in 0.5%, 1% and 2% phosphate buffer solutions (PBS). HPPH was also conjugated to Pluronic F-127, and the resulting conjugate (PL-20) was formulated in PBS. Among the different delivery vehicles, only Pluronic P-123 displayed significant vehicle cytotoxicity, whereas Pluronic F127 was nontoxic. Compared to PL-20, HPPH formulated in Tween80 and Pluronic F-127 showed higher cell-uptake, but lower long-term retention in Colon26 cell compared to PL-20. The higher retention of PL-20 was similarly observed during in vivo uptake with BALB/c mice baring Ct26 tumors. In contrast to the in vitro uptake experiments, PL-20 showed slightly higher uptake compared to HPPH formulated in Tween or Pluronic-F127. A significant difference in pharmacokinetic profile was also observed between the HPPH-Pluronic formulation and PL-20. Under similar in vivo treatment parameters (drug dose 0.47 µmol kg-1 , light dose: 135 J cm-2 at 24 h post-injection of PS), HPPH formulated either in Tween or Pluronic F-127 formulation showed similar in vivo PDT efficacy (20-30% tumor cure on day 60), whereas PL-20 showed 40% tumor cure (day 60).

Epidermal Growth Factor Receptor-Targeted Multifunctional Photosensitizers for Bladder Cancer Imaging and Photodynamic Therapy.

The in vitro and in vivo anticancer activity of iodinated photosensitizers (PSs) with and without an erlotinib moiety was investigated in UMUC3 [epidermal growth factor (EGFR)-positive] and T24 (EGFR-low) cell lines and tumored mice. Both the erlotinib-conjugated PSs 3 and 5 showed EGFR target specificity, but the position-3 erlotinib-PS conjugate 3 demonstrated lower photodynamic therapy efficacy than the corresponding non-erlotinib analogue 1, whereas the conjugate 5 containing an erlotinib moiety at position-17 of the PS showed higher tumor uptake and long-term tumor cure (severe combined immunodeficient mice bearing UMUC3 tumors). PS-erlotinib conjugates in the absence of light were ineffective in vitro and in vivo, but robust apoptotic and necrotic cell death was observed in bladder cancer cells after exposing them to a laser light at 665 nm. In contrast to 18F-fluorodeoxyglucose, a positron emission tomography agent, the position-17 erlotinib conjugate (124I-analogue 6) showed enhanced UMUC3 tumor contrast even at a low imaging dose of 15 µCi/mouse.

Measurement of Cyanine Dye Photobleaching in Photosensitizer Cyanine Dye Conjugates Could Help in Optimizing Light Dosimetry for Improved Photodynamic Therapy of Cancer.

Photodynamic therapy (PDT) of cancer is dependent on three primary components: photosensitizer (PS), light and oxygen. Because these components are interdependent and vary during the dynamic process of PDT, assessing PDT efficacy may not be trivial. Therefore, it has become necessary to develop pre-treatment planning, on-line monitoring and dosimetry strategies during PDT, which become more critical for two or more chromophore systems, for example, PS-CD (Photosensitizer-Cyanine dye) conjugates developed in our laboratory for fluorescence-imaging and PDT of cancer. In this study, we observed a significant impact of variable light dosimetry; (i) high light fluence and fluence rate (light dose: 135 J/cm², fluence rate: 75 mW/cm²) and (ii) low light fluence and fluence rate (128 J/cm² and 14 mW/cm² and 128 J/cm² and 7 mW/cm²) in photobleaching of the individual chromophores of PS-CD conjugates and their long-term tumor response. The fluorescence at the near-infrared (NIR) region of the PS-NIR fluorophore conjugate was assessed intermittently via fluorescence imaging. The loss of fluorescence, photobleaching, caused by singlet oxygen from the PS was mapped continuously during PDT. The tumor responses (BALB/c mice bearing Colon26 tumors) were assessed after PDT by measuring tumor sizes daily. Our results showed distinctive photobleaching kinetics rates between the PS and CD. Interestingly, compared to higher light fluence, the tumors exposed at low light fluence showed reduced photobleaching and enhanced long-term PDT efficacy. The presence of NIR fluorophore in PS-CD conjugates provides an opportunity of fluorescence imaging and monitoring the photobleaching rate of the CD moiety for large and deeply seated tumors and assessing PDT tumor response in real-time.

Whole body and local hyperthermia enhances the photosensitizing efficacy of 3-[(1'-hexyloxy)ethyl]-3-Devinylpyropheophorbide-a (HPPH).

BACKGROUND AND OBJECTIVES: In this study, we evaluated the impact of hyperthermia in photosensitizing efficacy of 3-[(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-a (HPPH or Photochlor) for the treatment of cancer by photodynamic therapy (PDT). STUDY DESIGN/MATERIALS AND METHODS: The outcome of both whole body hyperthermia (WBH) and local hyperthermia (LH) in combination with HPPH-PDT was determined in BALB/c and nude mice bearing Colon26 and U87 tumors, respectively. LH was performed by using an indigenously designed heating device, that was heated to the required temperature using a circulating water bath. The device which has flexible membrane on one side was placed on skin above the tumor. The temperature of the tumor was monitored using a thermocouple sensor placed on the surface of the tumor capable of measuring the temperature within 0.1°C. Uptake of the photosensitizer in tumors was determined by fluorescence using an IVIS or a Nuance Imaging System. The PDT was performed by exposing the tumors to 665 nm laser loght, (135 J/cm2 , 75 mW/cm2 ) at the maximal uptake time of HPPH. Tumor size was measured daily using vernier calipers. RESULTS: The improved PDT efficacy (long-term percentage tumor cure) in combination with hyperthermia is possible due to an increase in tumor-uptake of the photosensitizer (PS), confirmed by in vivo fluorescence imaging and also by increased tumor perfusion and decreased hypoxia as have been reported previously (Sen et al. [2011] Cancer Res. 71:3872-3880 In Vivo. 20:689-695). Interestingly, compared to whole body hyperthermia, the 14 C- HPPH biodistribution data under local hyperthermia showed similar tumor-uptake in BALB/c mice bearing Colon26 tumors, but significantly lower uptake in other organs and in the blood. CONCLUSION: Our study demonstrates that both, fever range whole body and local hyperthermia in combination with HPPH-PDT enhances the long-term tumor cure of BALB/c and nude mice implanted with Colon26 and U87 tumors respectively. Lasers Surg. Med. 50:506-512, 2018. © 2018 Wiley Periodicals, Inc.

Structural and Epimeric Isomers of HPPH [3-Devinyl 3-{1-(1-hexyloxy) ethyl}pyropheophorbide-a]: Effects on Uptake and Photodynamic Therapy of Cancer.

The tetrapyrrole structure of porphyrins used as photosentizing agents is thought to determine uptake and retention by malignant epithelial cancer cells. To assess the contribution of the oxidized state of individual rings to these cellular processes, bacteriochlorophyll a was converted into the ring "D" reduced 3-devinyl-3-[1-(1-hexyloxy)ethyl]pyropheophorbide-a (HPPH) and the corresponding ring "B" reduced isomer (iso-HPPH). The carboxylic acid analogs of both ring "B" and ring "D" reduced isomers showed several-fold higher accumulation into the mitochondria and endoplasmic reticulum by primary culture of human lung and head and neck cancer cells than the corresponding methyl ester analogs that localize primarily to granular vesicles and to a lesser extent to mitochondria. However, long-term cellular retention of these compounds exhibited an inverse relationship with tumor cells generally retaining better the methyl-ester derivatives. In vivo distribution and tumor uptake was evaluated in the isogenic model of BALB/c mice bearing Colon26 tumors using the respective 14C-labeled analogs. Both carboxylic acid derivatives demonstrated similar intracellular localization and long-term tumor cure with no significant skin phototoxicity. PDT-mediated tumor action involved vascular damage, which was confirmed by a reduction in blood flow and immunohistochemical assessment of damage to the vascular endothelium. The HPPH stereoisomers (epimers) showed identical uptake (in vitro & in vivo), intracellular retention and photoreaction.

Highly Effective Dual-Function Near-Infrared (NIR) Photosensitizer for Fluorescence Imaging and Photodynamic Therapy (PDT) of Cancer.

We report herein the synthesis and biological efficacy of near-infrared (NIR), bacteriochlorin analogues: 3-(1'-butyloxy)ethyl-3-deacetyl-bacteriopurpurin-18-N-butylimide methyl ester (3) and the corresponding carboxylic acid 10. In in vitro assays, compared to its methyl ester analogue 3, the corresponding carboxylic acid derivative 10 showed higher photosensitizing efficacy. However, due to drastically different pharmacokinetics in vivo, the PS 3 (HPLC purity >99%) showed higher tumor uptake and long-term tumor cure than 10 (HPLC purity >96.5%) in BALB/c mice bearing Colon 26 tumors. Isomerically pure R- and S- isomers of 3 (3a and 3b, purity by HPLC > 99%) under similar treatment parameters showed identical efficacy in vitro and in vivo. In addition, photosensitizer (PS) 3 showed limited skin phototoxicity and provides an additional advantage over the clinically approved chemically complex hematoporphyrin derivative as well as other porphyrin-based PDT agents, which makes 3 a promising dual-function agent for fluorescence-guided surgery with an option of phototherapy of cancer.