meso- and ß-Pyrrole-Linked Chlorin-Bacteriochlorin Dyads for Promoting Far-Red FRET and Singlet Oxygen Production.

A series of chlorin-bacteriochlorin dyads (derived from naturally occurring chlorophyll-a and bacteriochlorophyll-a), covalently connected either through the meso-aryl or ß-pyrrole position (position-3) via an ester linkage have been synthesized and characterized as a new class of far-red emitting fluorescence resonance energy transfer (FRET) imaging, and heavy atom-lacking singlet oxygen-producing agents. From systematic absorption, fluorescence, electrochemical, and computational studies, the role of chlorin as an energy donor and bacteriochlorin as an energy acceptor in these wide-band-capturing dyads was established. Efficiency of FRET evaluated from spectral overlap was found to be 95 and 98 % for the meso-linked and ß-pyrrole-linked dyads, respectively. Furthermore, evidence for the occurrence of FRET from singlet-excited chlorin to bacteriochlorin was secured from studies involving femtosecond transient absorption studies in toluene. The measured FRET rate constants, kFRET , were in the order of 1011  s-1 , suggesting the occurrence of ultrafast energy transfer in these dyads. Nanosecond transient absorption studies confirmed relaxation of the energy transfer product, 1 BChl*, to its triplet state, 3 Bchl*. The 3 Bchl* thus generated was capable of producing singlet oxygen with quantum yields comparable to their monomeric entities. The occurrence of efficient FRET emitting in the far-red region and the ability to produce singlet oxygen make the present series of dyads useful for photonic, imaging and therapy applications.

Effect of chirality on cellular uptake, imaging and photodynamic therapy of photosensitizers derived from chlorophyll-a.

We have previously shown that the (124)I-analog of methyl 3-(1'-m-iodobenzyloxy) ethyl-3-devinyl-pyropheophorbide-a derived as racemic mixture from chlorophyll-a can be used for PET (positron emission tomography)-imaging in animal tumor models. On the other hand, as a non-radioactive analog, it showed excellent fluorescence and photodynamic therapy (PDT) efficacy. Thus, a single agent in a mixture of radioactive ((124)I-) and non-radioactive ((127)I) material can be used for both dual-imaging and PDT of cancer. Before advancing to Phase I human clinical trials, we evaluated the activity of the individual isomers as well as the impact of a chiral center at position-3(1) in directing in vitro/in vivo cellular uptake, intracellular localization, epithelial tumor cell-specific retention, fluorescence/PET imaging, and photosensitizing ability. The results indicate that both isomers (racemates), either as methyl ester or carboxylic acid, were equally effective. However, the methyl ester analogs, due to subcellular deposition into vesicular structures, were preferentially retained. All derivatives containing carboxylic acid at the position-17(2) were noted to be substrate for the ABCG2 (a member of the ATP binding cassette transporters) protein explaining their low retention in lung tumor cells expressing this transporter. The compounds in which the chirality at position-3 has been substituted by a non-chiral functionality showed reduced cellular uptake, retention and lower PDT efficacy in mice bearing murine Colon26 tumors.

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).

Impact of mono- and di-ß-galactose moieties in in vitro / in vivo anticancer efficacy of pyropheophorbide-carbohydrate conjugates by photodynamic therapy.

To investigate the impact of mono- and di-ß-galactose moieties in tumor uptake and photodynamic therapy (PDT) efficacy, HPPH [3-(1'-hexyloxy)ethyl-3-devinylpyropheophorobide-a], the meso pyropheophorbide-a [3-ethyl-3-devinyl-pyropheophorbide-a], and the corresponding 20-benzoic acid analogs were used as starting materials. Reaction of the intermediates containing one or two carboxylic acid functionalities with 1-aminogalactose afforded the desired 172- or 20(4')- mono- and 172, 20(4')-di galactose conjugated photosensitizers (PSs) with and without a carboxylic acid group. The overall lipophilicity caused by the presence of galactose in combination with either an ethyl or (1'-hexyloxy)ethyl side chain at position-3 of the macrocycle made a significant difference in in vitro uptake by tumor cells and photoreaction upon light exposure. Interestingly, among the PSs investigated, compared to HPPH 1 the carbohydrate conjugates 2 and 11 in which ß-galactose moieties are conjugated at positions 172 and 20(4') of meso-pyro pheophorbide-a showed similar in vitro efficacy in FaDu cell lines, but in SCID mice bearing FaDu tumors (head & neck) Ps 11 gave significantly improved long-term tumor cure.

Meso-Biphenyl-Linked, Near- and Far-Infrared Emitting, Chlorin and Bacteriochlorin Dimers: Synthesis, Excitation Transfer, and Singlet Oxygen Production.

A series of meso-biphenyl linked chlorin and bacteriochlorin dimers, derived from naturally occurring chlorophyll (Chl-a) and bacteriochlorophyll (BChl-a) were synthesized in 32 % to 44 % yields and characterized, as photosynthetic antenna mimics, and a new class of singlet oxygen producing agents. The dimers are characterized by absorption, fluorescence, electrochemical, spectroelectrochemical and computational methods to evaluate their physico-chemical properties, and to identify ground and excited state interactions. Evidence of excited energy exchange among the chromophores in the dimer is derived from femtosecond transient absorption spectral studies. Rate constants for excitation hopping were in the order of 1011  s-1 , indicating occurrence of efficient processes. Nanosecond transient absorption studies confirmed relaxation of the singlet excited chlorin and bacteriochlorin dimers to their corresponding triplet states (3 Chl* and 3 Bchl*). As predicted by the established energy level diagrams, both 3 Chl* and 3 Bchl* are shown to be capable of producing singlet oxygen with appreciable quantum yields (ϕSO ∼0.3).

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.

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).

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.

Standard operating procedure for metabonomics studies of blood serum and plasma samples using a 1H-NMR micro-flow probe.

A standard operating procedure (SOP) is presented for high-throughput metabonomics studies using a 600 MHz NMR spectrometer equipped with a micro-flow probe that is connected to an auto-sampler. The procedure is designed to minimize random and systematic variation of NMR data collection. In addition, a protocol is described to assess the quality of the data acquired by a given NMR spectroscopist to ensure that (i) all researchers involved in the NMR data acquisition of a metabonomics research program perform equally and (ii) operator-associated variation of NMR data collection is statistically not relevant for the interpretation of results obtained from multivariate data analyses.

A Pyropheophorbide Analogue Containing a Fused Methoxy Cyclohexenone Ring System Shows Promising Cancer-Imaging Ability.

Herein we report the synthesis, photophysical properties, positron emission tomography (PET) imaging and photodynamic therapy (PDT) efficacy of methyl 3-(1'-m-iodobenzyloxy)ethyl-3-devinyl-verdin 4 (with or without the 124 I isotope). The PET imaging ability and ex vivo biodistribution of [124 I]4 were compared with the well-studied methyl [3-(124 1'-m-iodobenzyloxy)ethyl]-3-devinyl-pyropheophorbide-a methyl ester (PET-ONCO or [124 I]2) and [18 F]fluorodeoxyglucose ([18 F]FDG) in BALB/c mice bearing colon-26 tumors. Whole-body PET images of [124 I]4 containing a fused methoxy cyclohexenone ring system showed excellent tumor contrast with time (72>48>24 h post-injection). Ex vivo biodistribution results indicate that relative to the current clinical standard [18 F]FDG and [124 I]2 in 2 % ethanol formulation, [124 I]4, at the same radioactive dose (25 µCi per mouse), showed higher tumor uptake at 24 h post-injection and longer tumor retention. In biological environments, compound 4 showed lower fluorescence and lower singlet oxygen yield than 2, which is possibly due to higher aggregation caused by the presence of a fused cyclohexenone ring system, resulting in limited in vitro/in vivo PDT efficacy. Therefore, the chlorophyll-a analogue [124 I]4 provides easy access to a novel PET imaging agent (with no skin phototoxicity) to image cancer types-brain, renal carcinomas, pancreas-in which [18 F]FDG shows limitations.

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.

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.

Photosensitizer (PS)-cyanine dye (CD) conjugates: Impact of the linkers joining the PS and CD moieties and their orientation in tumor-uptake and photodynamic therapy (PDT).

To investigate the impact of linker(s) joining the photosensitizer HPPH [3-(1'-hexyloxy) ethyl-3-devinylpyropheophorbide-a] and the cyanine dye (CD) in tumor-imaging and photodynamic therapy (dual-function agents), a series of HPPH-CD conjugates were synthesized. The modifications were done in an attempt to minimize Forster Resonance Energy Transfer (FRET) between the two chromophores and maximize singlet oxygen production. Among the conjugates containing variable length of linkers, the HPPH-CD conjugate, in which the photosensitizer (PS) and the CD was joined by four Carbon [(CH2)4] units showed higher tumor uptake, improved tumor contrast and limited skin uptake in mice bearing Colon-26 (BALB/c) or U87 tumors in Nude mice. The bi-functional agents in which the HPPH was linked at the meta-position of phenyl-substituted CD 5, 6 and 7 showed longer tumor response (cure) than the corresponding para-substituted analogs 2, 3, and 4, which suggests that the orientation of the PS and CD moieties within the conjugate also makes a substantial difference in tumor-specificity. Compared to HPPH, the singlet oxygen yields of all the HPPH-CD conjugates were significantly low, and required a higher therapeutic dose to achieve the same in vivo response obtained by HPPH-PDT alone. However, conjugate 6 produced a higher singlet oxygen yield with reduced FRET and exhibited enhanced long-term PDT efficacy in mice bearing Colon-26 (BALB/c) and U87 tumors (nude) than its counterparts, including our lead compound (HPPH-CD), making it the most efficacious of the series. Thus, these conjugates bearing cyanine dye moiety (CD) provide an opportunity of imaging deeply seated tumors for fluorescence-guided surgery with an option of PDT.

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.

A novel synthetic route to fused propenochlorin and benzochlorin photodynamic therapy probes.

Reaction of meso-(2-formylvinyl)octaethylporphyrin with (CH3)3SiCN-Cu(OTf)2 produced unexpected 10(3)-trimethylsiloxyl and 10(3)-hydroxyl fused propenochlorins which, in H2SO4, underwent subsequent migration of the 8-ethyl group to the 10(3)-position of the exocyclic benzene ring to form a novel benzochlorin.

Characterization of porphyrins, chlorins, and bacteriochlorins formed via allomerization of bacteriochlorophyll a. Synthesis of highly stable bacteriopurpurinimides and their metal complexes.

Allomerization of bacteriochlorophyll a (Bchl a) was studied under various reaction conditions. Bchl a on stirring with KOH/propanol produced an "unstable bacteriochlorin", which decomposed in acidic conditions to give a complex mixture containing bacteriopurpurin a as a principal component. The yields of other compounds varied and were found to be dependent on reaction condition. The structures of the isolated porphyrins, chlorins, and bacteriochlorins, related to Bchl a, were assigned on the basis of 1D, 2D NMR (ROESY), and mass spectroscopy analyses. The presence of fused anhydride rings in porphyrin, chlorin, and bacterichlorin systems showed a significant influence on their optical properties. Compared to bacteriochlorophyll a and bacteriopheophytin, the related structurally modified analogues, e.g., the bacteriopurpurin a, 13(1)/15(1)-N-alkyl isoimide, and the imide analogues were found to be more stable with a significant difference in spectroscopic properties. Bacteriochlorins containing anhydride, imide, or isoimide cyclic rings demonstrated a significant bathochromic shift of their Q bands in their electronic absorption spectra. Under basic conditions the formation of the 12-hydroxymethyl, 12-formyl, and 12-methylene analogues as byproducts from the 12-methyl-bacteriopurpurin-N-hexylimide could be due to subsequent oxidation of the vinylogous enolate intermediates. To investigate the effect of the central metal in the electronic spectra, the stable bacteriopurpurin-18-N-hexylimide was converted to a series of metal complexes [Zn(II), Cd(II), and Pd(II)] by following the direct or transmetalation approaches. Compared to the free-base analogue, these complexes showed a remarkable shift in their electronic absorption spectra.