Structural and Epimeric Isomers of HPPH [3-Devinyl 3-{1-(1-hexyloxy

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Structural and Epimeric Isomers of HPPH [3-Devinyl 3{1-(1-hexyloxy) ethyl}pyropheophorbide-a]: Effects on Uptake and Photodynamic Therapy of Cancer Courtney Saenz, Ravindra R Cheruku, Tymish Y. Ohulchanskyy, Penny Joshi, Walter A Tabaczynski, Joseph R. Missert, Yihui Chen, Paula Pera, Erin Tracy, Aimee Marko, Daniel Rohrbach, Ulas Sunar, Heinz Baumann, and Ravindra K. Pandey ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00023 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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ACS Chemical Biology

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

Courtney Saenz1, Ravindra R. Cheruku1, Tymish Y. Ohulchanskyy2,3, Penny Joshi1, Walter A. Tabaczynski1 Joseph R. Missert1, Yihui Chen1 Paula Pera1, Erin Tracy4, Aimee Marko1, Daniel Rohrbach1, Ulas Sunar1 Heinz Baumann4,* and Ravindra K. Pandey1,* Photodynamic Therapy Center, Cell Stress Biology, 4Molecular Biology

1

Roswell Park Cancer Institute, Buffalo, NY 14263 2College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, China 518060 3

Institute for Lasers, Photonics and Biophotonics, SUNY at Buffalo, Buffalo, NY 14260

-------------------------------------*

Corresponding authors

Ravindra K Pandey, Ph. D. E-mail: [email protected] Heinz Baumann, Ph. D. E-mail:[email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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 mitochondria and endoplasmic

reticulum

by

primary

culture of human lung and head & 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

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C-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 reduction in blood flow and immunohistochemical assessment of damage to the vascular endothelium.

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INTRODUCTION Photodynamic therapy (PDT) is increasingly acceptable as a curative or palliative treatment of cancer and some non-cancerous conditions that are generally characterized by overgrowth of transformed cells.1,2 Interest in this procedure was promoted by world-wide approval of Photofrin® (a porphyrin-based photosensitizer) for the treatment of lung, gastric, esophageal, bladder and cervical tumors, in addition to cervical dysplasia and actinic keratosis.3 PhotofrinTM fits some of the criteria for the so called “ideal” photosensitizer, but it suffers from several drawbacks.4 First, it is a complex mixture of porphyrins with various monomers, dimers and higher oligomers linked with ether, ester and carbon-carbon linkages.5-8 Secondly, its long wavelength absorption falls at 630 nm, which lies well below the wavelength necessary for the maximum tissue penetration.4 Finally, due to slow systemic clearance, it causes prolonged cutaneous photosensitivity, a major adverse effect associated with this sensitizer. Therefore, efforts are underway in ours and other laboratories to develop improved agents with higher tumor selectivity.9-11 A more detailed understanding of the mechanisms involved in the photosensitized damage to cells and tissues and a defined correlation between chemical structure and photodynamic activity for various classes of porphyrin-based compounds led to the development of second-generation photosensitizers with improved phototherapeutic properties.12-16 In our previous SAR and QSAR studies with a series of alkyl and aryl ether analogs of certain chlorins (ring D reduced porphyrins),17-21 we observed that besides overall lipophilicity, the presence of a particular substituent at the various peripheral position(s) of the molecule makes a considerable difference in tumor-uptake, cellular retention and PDT efficacy. For example, in a series of the alkyl ether analogs of pyropheophorbide-a, we observed a parabolic relationship between the log P values (determines the overall lipophilicity of a compound) and the PDT activity.22 Among the compounds investigated, the hexyl ether derivative [1 and 2 as

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methyl ester and carboxylic acid (HPPH)] was found to be most effective.17,23,24 HPPH is currently under Phase I/II human clinical trials for various indications (e. g., skin, lung, Barrett esophagus and head & neck cancer) and, in contrast to Photofrin, it does not cause long-term skin phototoxicity.21-29 For quite some time, a number of laboratories have been investigating the structural requirements of various tetrapyrrolic systems for developing more effective PDT agents.30-33 In our laboratories, the SAR approach has been most useful in developing improved photosensitizers with tumor imaging ability.34-40 So far, most of the chlorin-based photosensitizers were derived from chlorophyll a and contain the ring-D reduced system. We were interested in investigating the impact of other pyrrole ring reduced systems, especially related to HPPH methyl ester 1 and the corresponding carboxylic acid analog 2 in which the ring-D pyrrole is reduced.41 Herein, we present the synthesis and photosensitizing efficacy of both ring-D (1, 2) and ring-B reduced (3, 4) isomers (Fig. 1) for the treatment of cancer by PDT. Cell biological studies have indicated that HPPH interacts with the plasma membrane, diffuses into cells and accumulates in mitochondria and endoplasmic reticulum (ER). Not known is whether any of these cellular properties are altered as function of ring structures of the porphyrin. First evidence that these may be the case stems from our recent studies of HPPHrelated isomers of a 3-iodobenzyl derivative of pyropheophorbide42. The amount of HPPH in cells culture or in tumors (in vivo) correlates with the level of photoreaction, cellular PDT response and cell death.43 Hence, in vitro assays have emerged as diagnostic tools to evaluate optimized photosensitizers and to design their in vivo application. In this study we applied tissue culture models of human cancer types for which PDT is considered as a treatment option. The newly synthesized HPPH isomers were analyzed for cellular uptake, intracellular localization, binding to the outer mitochondrial translocator protein (or peripheral benzodiazepine receptor, PBR), photoreaction and skin phototoxicity. We made the discovery of isoform-specific differences in the cellular retention of HPPH.

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Figure 1: Structure of ring-D and ring-B reduced isomers RESULTS AND DISCUSSION Chemistry: Conversion of bacteriochlorophyll-a to ring-B and ring-D reduced chlorins We have recently shown that oxidation of bacteriopheophorbide-a 7 (obtained from bacteriochlorophyll-a by following the standard methodology) with ferric chloride produced the ring-D oxidized (ring-B reduced) chlorin in excellent yield41. When the oxidant was replaced by 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), the ring-B oxidized (ring-D reduced) chlorin was obtained. We extended this approach for the preparation of HPPH (ring-D reduced chlorin, 1 and 2) and the corresponding ring-B reduced isomer 3 and 4. For the preparation of the ring-B reduced isomer 3, bacteriochlorophyll-a 5 extracted from Rb. sphaeroides was used as a substrate, which on treating with 0.5 M hydrochloric acid at room temperature with subsequent reaction with aqueous trifluoric acid (TFA) and then with diazomethane yielded methyl bacteriopheophorbide-a 6 (Scheme 1). Reaction of 6 with collidine at refluxing temperature gave methyl bacteriopyropheophorbide-a 7 in 60% yield. In an attempt to develop regioselective preparation ring-B and ring-D reduced chlorins, the methyl bacteriopyropheophorbide-a 7 (both rings B and D are reduced) was reacted with a series of oxidizing agents.41 Depending on the oxidizing agent used, ring-B or the ring-D reduced chlorin was isolated in >80% yield. Having the desired new chlorin 8 (ring B reduced) in

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hand, our goal was to develop an efficient synthetic methodology for the preparation of its 3-[1(1-hexyloxyethyl) derivative (Scheme 2) and to compare its efficacy with HPPH. Two synthetic Me Me Rb. sphaeroides Me

PhytylO2C

O

N

Me

Me

N Mg N N D

B Et

O CO2Me 5

Me TFA

Me

Me

MeO2C

O

Me

Me

NH N

B Et Me Collidine

N HN D

Me

O CO2Me 6

Me

O

Me

NH N

B Et

N HN D

Me O

MeO2C 7

Scheme 1: Synthesis of methyl bacteriopyropheophorbide-a from bacteriochlorophyll-a

strategies were used for the preparation of 3 and the corresponding carboxylic acid 4. In the first approach, the acetyl group present at position-3 in chlorin 8 was reduced to the corresponding (1-hydroxyethyl) derivative 9, which on reacting with HBr gas and 1-hexanol afforded B-ring reduced chlorin 3 in > 70% yield. The same chlorin can also be prepared on reacting methyl bacteriopyropheophorbide-a 7 with sodium borohydride and then converting it to the corresponding hexyl ether derivative 11 before ferric chloride oxidation. Both methodologies produced chlorin 3 in almost similar yield, but the 2nd approach has a slight advantage as the same bacteriochlorin 11 on subjecting to two different oxidizing agents (DDQ and FeCl3) could generate both ring-B and ring-D reduced isomers 1 and 3 respectively. The methyl ester functionality present in chlorins 1 and 3 on reacting with aqueous lithium hydroxide (LiOH) was converted into the corresponding carboxylic acid 2 (HPPH) and 4 (iso-HPPH) respectively in 9095% yields. The structures of new chlorins (and the intermediates) were confirmed by NMR and HRMS analyses (see the Supporting Material). Striking differences between the ring D and ring B reduced chlorins were observed in the NMR spectra (Fig. 2). In brief, the 131-CH2 protons

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observed as a pair of doublets centered at δ 5.19 ppm in ring-D reduced chlorin 1, appeared as a tight AB pattern in the B ring reduced chlorin 3, suggesting the regioselective oxidation of ringD, which was further confirmed by the presence of triplets at δ 3.90 and 2.94 ppm. These triplets

Scheme 2: Synthesis of HPPH (ring D reduced chlorin) and the corresponding ring B reduced isomer derived from methyl bacteriopyropheophorbide-a.

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each integrated for two protons were assigned to the 17- and 171-CH2 protons of the propionic ester side chain, whereas in the ring-D reduced chlorin (HPPH), these protons were observed at δ 2.70, 2.54 and 2.30 ppm. The meso- region of both the chlorins showed some interesting features. For example, the 5-H proton, which appeared at δ 9.80 for HPPH methyl ester 1, was observed at 9.03 ppm for the ring-B reduced isomer 3. The 20- meso proton, which appeared at δ 8.54 ppm in HPPH methyl ester 1 was observed at δ 9.18 in isomer 3. Compared to HPPH methyl ester 1, the peaks at δ 9.54 integrating for a single proton and assigned to 10- meso proton, appeared at δ 8.65 ppm in HPPH isomer, possibly due to higher electron density at these positions. HPPH methylHPPH-ME ester 1 (ring-D reduced) 2 17 1 -H 17 -H

13 2-H

Iso HPPH methyl ester 3 IHPPH-ME (ring-B reduced)

13 2 -H

17 2 -H

17 1-H

Figure 2: Partial NMR spectra of HPPH methyl ester 1 (ring-D reduced chlorin) and the corresponding ring-B reduced isomer 3. In compound 3, the peak at 3.75 ppm is narrower than the other methyl peaks, and this is typical of the ester methyls of this type.

Furthermore, no methyl peak is observed at this

chemical shift in compound 4 (the closely related acid analog). The peaks at 3.45/3.44 ppm show “doubling” due to stereoisomerism, and this is typical of the 2-CH3 group, which is nearest to the chiral center. The peak at 3.56 ppm is assigned as the 12-CH3 group because its chemical shift is roughly the same as that observed for 12-CH3 in somewhat similar compounds (HPPH and HPPH methyl ester). The peak at 3.19 ppm is assigned to the 18-CH3 group by elimination. For compound 4, following the same approach, the 4 methyl peaks observed in the

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range of 3-4 ppm were assigned as follows: 3.49 (12-CH3), 3.39/3.38 (2-CH3), 3.18 (18-CH3). Photophysical properties of ring-B vs ring-D reduced isomers The absorption and fluorescence spectra of all the isomers were measured in methanol/dichloromethane at equimolar concentrations. Compared to the HPPH methyl ester 1 or the carboxylic acid 2, which showed its longest wavelength absorption at λmax 660 nm, the related B- ring reduced isomers 3 and 4 show a strong absorption band peaked at ~669 nm. The broad ‘Soret’ bands were observed at ~408-441 nm for 2 and 410-459 nm for 4 (Fig. 3A). Both isomers (2 and 4) were highly fluorescent and exhibited an intense fluorescence in the range of ~650-750 nm with maxima at 666 nm and 678 nm, respectively. The generation of singlet oxygen (1O2), a key cytotoxic agent in PDT, by both the

.

isomers 2 and 4 was quantified via measurements of 1O2 phosphorescence peaked at ~1270 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0

250

2 (HPPH) 4 (HPPH Isomer)

Fluorescence (A.U.)

Absorbance (A.U.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2 (HPPH) 4 (HPPH Isomer)

200 150 100 50 0

300

500

600

700

650

700

750

Wavelength (nm)

Wavelength (nm)

Figure 3: (A): Absorption and (B) fluorescence spectra of HPPH 2 and iso-HPPH 4 in methanol at equimolar concentration (0.5 µmol). nm.42

Singlet oxygen quantum yield for iso-HPPH 4 was determined using decays of

phosphorescence of 1O2 sensitized by 4 and 2 (HPPH). The areas under the decay curves were calculated (using Origin software) and the value of singlet oxygen quantum yield for iso-HPPH 4 was obtained from the ratio of the calculated areas, using known value of 1O2 yield for HPPH (48%).43 As illustrated in Fig 4, a slight increase in the singlet oxygen quantum yield was observed for the iso-HPPH 4 (~55%) than the corresponding ring-D reduced HPPH 2.

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Figure 4: Singlet oxygen production of 2 (HPPH) and 4 in methanol detected by measuring the phosphorescence of singlet oxygen at 1270 nm upon excitation with a nanosecond pulsed laser at 532 nm. Decays of singlet oxygen phosphorescence spectra of 2 and the corresponding ringB reduced isomer 4 in methanol are shown. The instrument response function (IRF) obtained using cuvette with methanol is also shown.

In vitro and in vivo photobleaching of ring-B vs. ring-D reduced isomers The photobleaching (or photodestruction) of the photosensitizer by singlet oxygen during PDT treatment is an important phenomenon, which is currently being explored to monitor the light dosimetry to improve PDT outcome. We investigated the photostability of HPPH and isoHPPH both in vitro (13% BSA in phosphate-buffer) and in vivo (BALB/c mice bearing Colon26 tumors). As can be seen from the data summarized in Fig. 5A both isomers as methyl ester or carboxylic acid functionalities (1-4) showed significant photobleaching in vitro with time, ring-B reduced isomer being slightly more stable under these experimental parameters. For determining in vivo photobleaching characteristics of the isomers, these compounds were individually injected (i. v.) into BALB/c mice bearing Colon26 tumors. After 24h, the tumors were exposed to light (therapeutic treatment parameters) and the rate of photobleaching was determined by measuring the decay in their long wavelength absorption. The results are depicted in Fig. 5B, which illustrates that the rate of photobleaching of the isomers in vivo is quite similar. The comparable bleaching for all compounds suggests that an equivalent

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photosensitizing function for each compound can be expected

1

1.2

0.8

1

Normalized R.F.U.

Normalized Percent Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6 2 (HPPH)

0.4

4 (HPPH Isomer)

0.2

1 (HPPH Methyl Ester) 3 (HPPH Methyl Ester Isomer)

0 0

20

40

60

0.8 0.6

2 (HPPH)

0.4

4 (HPPH Isomer) 1 (HPPH Methyl Ester)

0.2

3 (HPPH Methyl Ester Isomer)

0

80

0

Time (minutes)

1

3 5 10 15 Time (minutes)

20

30

Figure 5: (A) Photobleaching properties of ring-B and ring-D isomers 1-4 in 1% bovine serum albumin (BSA) in PBS, (B) HPPH and the respective B-ring reduced isomer as carboxylic acids and their respective methyl esters 1-4 were injected (drug dose: 0.47 µmol/kg) in BALB/c mice bearing Colon26 tumors. At 24 h post-injection, the tumors were exposed to light (665 nm, light dose, 135J/cm2, 75mW/cm2 and the rate of photobleaching was measured at variable time points by both absorption and fluorescence spectroscopy.

quite similar. The comparable bleaching for all compounds suggests that an equivalent photosensitizing function for each compound can be expected. Uptake and retention of HPPH and HPPH methyl ester by primary cultures of human SCC The cellular accumulation of porphyrin-based photosensitizers involves three processes: (a) binding to or diffusion through the plasma membrane of the target cells, (b) internalization and subcellular deposition, and (c) rate of release (or retention). The concentration of the intracellular photosensitizers, in turn, determines the key feature of PDT, the magnitude of photoreaction. The uptake of porphyrins were noted to be subject to cell type-specific properties, i.e., differences in the retention by tumor cells and by non-transformed stromal cells.44 Moreover, we cannot predict a priori what is the influence of the porphyrin structures on each of the uptake processes. Hence, we establish an assay system that permits a direct comparison of cell types relevant to human cancers considered for PDT. This system consists

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of reconstituted co-cultures of stromal and cancer cells derived from human tumor samples of the lung and head and neck. By monitoring over the course of several days these live cultures, which had been treated with the HPPH isomers, we could establish the kinetics of uptake, define the cell specificity, and cross-compare different tumor cell preparations and porphyrin compounds. The key features established for HPPH and HPPH methyl ester are presented by the results with two separate cultures of head and neck tumor cells with different preference for HPPH (Fig. 6). To readily distinguish cancer epithelial cells and tumor fibroblasts, the latter cells were marked by CSFE staining prior to reconstitution of the co-cultures. The binding capability of HPPH and HPPH methyl ester was assessed by incubating the cell cultures with the compounds for 30 minutes on ice (suppressed subcellular membrane movement, Fig. 6A). Both HPPH forms were found primarily associated with the plasma membrane of both cell types as evident from the uniform fluorescent staining of the cells. Quantification of fluorescence indicated that HPPH bound 5-fold more effectively than HPPH methyl ester. Incubation of the cells for 24 h under standard culture condition in the presence of the photosensitizers yielded a maximal accumulation for both cells types. At this stage, the cellular levels of HPPH and HPPH methyl ester were comparable. Higher magnification of cells indicated that HPPH was localized to mitochondria and ER as noted previously.45 In contrast, the fluorescence for HPPH methyl ester was localized in dense vesicles and to a lesser degree to mitochondria and ER. Subsequent incubation of the cells in photosensitizer free medium (=chase) tested the cellular efflux kinetics of the compounds. As noted previously for lung cancer cells44, the retention of HPPH was highly variable among different cell preparations. In the example of HN-85-1 cells (Fig. 6A) the tumor cells lost about 90% of HPPH within 24 h, a loss rate almost as high as observed for stromal cells. In contrast, HPPH methyl ester showed a prolonged retention that was more prominent for the tumor cells. The low HPPH retention phenotype of HN-85-1 cells iscontrasted by that of HNT-1 cells, another head and neck cancer case (Fig. 6B). These cells

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attainted within the 24-h incubation period with photosensitizer a 3 to 5-fold higher accumulation

Figure 6: Cell type-specific uptake and retention of photosensitizers. Co-cultures of tumor epithelial cells derived from head and neck squamous carcinoma HN-85-1 (A) and HNT-1 (B) were combined with CSFE-stained tumor-associated fibroblasts derived from head and neck squamous carcinoma HN-87 and HN-96, respectively. Cultures were incubated for 30 min in serum-free medium with 1.6 µM HPPH or HPPH methyl ester (HPPH-ME) on ice (Binding) or for 24 h in medium containing 10% fetal bovine serum and 1.6 µM photosensitizer (Uptake). After recording cell-associated fluorescence, the co-cultures were incubated for additional 24 h in medium containing 10% fetal bovine serum but without photosensitizers (Chase). The phase microscopic pictures of the cultures and corresponding fluorescent images representing CSFE (green) and HPPH (red) are reproduced.

of HPPH than fibroblasts. The prominence of HPPH retention was still evident after a 24-h

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chase period. HPPH methyl ester, however, was accumulated and retained by HNT-1 cells roughly equal to that of HN-85-1 cells. From the examples shown in Fig. 6, it is evident that the relative concentrations of cell-bound HPPH derivatives is subject to change over time and, thus, predicts a proportionally altered PDT efficacy. The differential uptake and retention of porphyrins by epithelial cells emerged as sensitive markers for probing the functional consequences of structural modifications to the macrocycle, such as position of reduced rings, in the context of carboxylic acid or methyl ester derivatives. The four isomeric HPPH compounds 1 - 4 were compared by applying these under identical conditions to the lung SCC cells, TEC-1-2 (Fig. 7). These cells, like HN-85-1 cells (Fig. 6A) showed high uptake but also a particularly low long-term retention of HPPH. By recording the fluorescence of cell-associated photosensitizer (Fig. 7A), we identified HPPH and iso-HPPH were taken up by a several-fold higher rate, but are also lost from the cells at a higher rate, than the corresponding methyl ester derivatives. While the uptake rate of the methyl ester derivatives was lower, after a continuous incubation for 24 h with the photosensitizers, HPPH methyl ester was accumulated to roughly equal level of HPPH and iso-HPPH. In contrast, the uptake of IsoHPPH methyl ester remained ~ 4-fold below that of HPPH methyl ester, suggesting the porphyrin structure with reduced B-ring, in the context of neutral charge at the periphery, was less favorable in supporting transmembrane movement. Due to the higher retention of the methyl ester derivatives, these were more prominently detected in cells at a later stage of incubation in the absence of photosensitizers relative to the carboxylic acid derivatives. The stronger retention of the methyl ester derivatives suggests a stronger interaction with membrane components at the subcellular site of deposition. Although the quantitative values for uptake and loss of the individual compounds varied somewhat among cell preparations, essentially the same trends governing overall retention patterns were determined for the four isomeric HPPH derivatives in other epithelial cell cultures from lung and head and neck tumor tissues.46

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Figure 7: Effect of porphyrin ring structure on uptake and retention of isomeric HPPH derivatives. (A) Confluent monotypic cultures of tumor epithelial cells derived from lung SCC TEC-1-2 were incubated with medium and 1.6 µM photosensitizers and chased as listed at the top. At the times indicated, HPPH fluorescence of the cultures at 100X magnification was recorded (500 ms exposures). (B) Equivalent cultures of TEC-1-2 cells in 24-well plates were incubated with medium containing 10% fetal bovine serum and 800 nM photosensitizers and chased in photosensitizer-free medium as listed at the bottom. The cultures were washed in serum-free medium, HPPH fluorescence was measured by microscopic imaging (values show normalized as fluorescent units) and the culture plates were treated with light (665 nm or 674 nm) for 9 min at 37°C to a fluence of 3 J/cm2. Immediately following light treatment, the monolayers were solubilized. Equal aliquots of cell lysates were analyzed by Western blotting for the proteins indicated to the right. The percent conversion of STAT3 into the homodimeric complex I was calculated from the values representing the enhanced chemiluminescent signals of these protein bands. Photosensitizer levels correlate with the photoreaction As evident from the data presented in Figs. 6 and 7A, the relative levels of the

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compounds 1 to 4 detectable in cells drastically changed over the course of days in culture. Hence, the effectiveness of the photoreaction mediated by each compound was expected to be proportional to the photosensitizer level at the time of light treatment. This close relationship was confirmed by quantification of the photosensitizer based on intensity of fluorescence and the cumulative effect of the light-induced photoreaction measured by the oxidative cross-linking of STAT3 (Fig. 7B). The latter determination involved Western blotting of electrophoretically separated proteins in cell extracts prepared immediately following light treatment of the cultures. Additional response markers for the magnitude of photoreaction were the activation of the stress mitogen-activated protein kinase (MAPK) pathway leading to the increased phosphorylation of p38 MAPK and the loss of immune-detectable epidermal growth factor receptor (EGFR). The pattern of cellular changes in response to the photoreaction proved also to be a sensitive indicator for the effect of the modifications of porphyrin structure on the cell biology of the compounds 1-4, emphasizing the lower effectiveness of those compounds with reduced B-ring. Evaluation of the HPPH derivatives to bind to PBR The observation that a fraction of intracellularly localized compounds 1-4 are deposited into mitochondria led to the question whether at this site, the porphyrins interact with the outer mitochondrial translocator protein (or peripheral benzodiazepine receptors, PBR) and, if so, whether this interaction functionally contributes to the outcome of PDT. Verma et. al.47 were the first to demonstrate the binding affinity of various porphyrins to the PBR with prominent physiological consequences. Kessel et. al.48 determined that among three very similar analogs of protoporphyrins (III, IX and XIII) only protoporphyrin IX had a significant affinity for the PBR. This finding suggested that a functional relationship between PBR binding and PDT efficacy may hold only for sensitizers with PP-IX-like configurations. A recent report by Olenick49 on the importance of PBR receptor in phthalocyanine photosensitizer PC-4 concluded that the binding of Pc-4 to PBR is less relevant for the phototoxicity of Pc-4-PDT. Other mitochondrial events, such as photodamage of Bcl-2 may account for the observed increase in Pc-4-PDT induced

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apoptosis and to be independent of mechanism involving PBR. In a series of metallated analogs of pyropheophorbide-a system, we have shown that the type of central metal present makes a significant difference in PBR binding affinity.50 100 Specific Binding (% of Control)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60

PK11195 2 (HPPH)

40

4 (HPPH Isomer)

20

1 (HPPH Methyl Ester) 3 (HPPH Methyl Ester Isomer)

0 -10

-9

-8

-7

-6

-5

-4

Log[ligand], M

Figure 8: Displacement of bound [3H]-PK11195 (PBR probe) with HPPH methyl ester 1, the corresponding carboxylic acid (HPPH) and the ring-B reduced isomers 3 and 4 respectively. To investigate the PBR binding affinity of ring-D vs. ring-B reduced isomers 1-4, Colon26 cells were incubated with tritium labeled [3H-]-PK11195 (PBR probe) and then exposed to increasing concentrations of the photosensitizer. Its binding affinity to PBR was determined by the competitive displacement of the 3H-probe by measuring the radioactivity released into the culture medium. The results summarized in Fig. 8 suggest that both ring-B and ring-D reduced isomers (methyl ester or carboxylic acid derivatives), possess similar low binding affinity to PBR with an estimated IC50 ≥ 10-4 M. This result suggests that an interaction of the compounds 1 - 4 with PBR is minimal at the concentration level of the photosensitizers used for PDT (< 10-6M) In vitro Photosensitizing Efficacy The ultimate goal of our work is to apply the photosensitizers for the treatment of cancer lesion in situ and to define optimal conditions for achieving a cure. While xenografts derived from the patient’s cancer cells can be grown in immune-compromised mice and would allow us to determine the systemic fate of the photosensitizer and ability to mediated a photoreaction in

17



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Page 18 of 44

the transplanted human tumor tissue, the model will not accurately address issues related to post-PDT processes. These processes, in particular those involving the immune system, contribute to the control of tumor growth. Hence, we chose the syngeneic mouse model of colon carcinoma Colon26 grown as subcutaneous tumor in BALB/c mice. Separate in vitro experiments (not shown) have indicated that Colon26 cells have a profile of photosensitizer retention that is similar to that displayed by the human HN-85-1 cells (Fig. 6A). However, unlike the primary cultures of human tumor cells, Colon26 cells lack the distinctive epithelial morphology and the feature of segregated growth in co-cultures with stromal fibroblasts. Hence, the tumor cell-specific retention of photosensitizers could not be defined as was done in Fig. 6. As first step in the evaluation of the compounds 1 - 4 for PDT application, we determined the relative activity in mediating cell killing in vitro as a function of light dose (Fig. 9). The results illustrate the in vitro photosensitizing efficacy of photosensitizers 1-4 and indicate that both isomers, as methyl ester or carboxylic acid analogs 1, 3 or 2, 4, showed similar efficacy. However, the compounds 2 and 4 bearing carboxylic acid functionality showed enhanced PDT efficacy than the corresponding methyl ester derivatives 1 and 3. These results are in close agreement with the relative photoreactions determined at the level of STAT3 crosslinking. Both HPPH (ring-D, trans-reduced) and iso-HPPH (ring-B, trans- reduced) contain a chiral center at position 31- and were isolated as a 1:1 epimeric mixture. To investigate the difference in biological efficacy of each epimer, several attempts were made to separate the isomers by HPLC. We were able to separate the isomers of HPPH, but were unsuccessful is isolating the individual epimer of Iso-HPPH for detailed biological studies. However, both the epimers of HPPH (isolated in >95% purity) showed similar in vitro PDT efficacy, intracellular localization and tumor u(Supplemental Information). These findings are similar to those observed with PSs ptake (BALB/c mice bearing Colon26 tumors). For details, see Figures S18 - S26 related to bacteriopurpurinimides51,52.

18


Page 19 of 44

140 H P P H - 0.125 µ M iso-H P P H - 0 .1 25 µM H P P H -M e - 0.125 µ M iso-H P P H -M e - 0 .1 25 µM

120 100

% Control Growth

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40 20 0 -2 0 0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

J/c m 2

Figure 9: Comparative in vitro photosensitizing efficacy of isomers 1-4 at a fixed concentration (125 nM) and variable light doses in Colon26 tumor cell lines at 24 h post incubation (MTT assay). In vivo biodistribution of HPPH and iso-HPPH Since the cytotoxic response to PDT indicated a much greater activity of the carboxylic acid derivatives 2 and 4 than the corresponding methyl ester analogs, the biodistribution and in vivo PDT efficacy of isomers 2 and 4 was investigated in BALB/c mice bearing Colon26 tumors. To determine in vivo biodistribution, the14C-labeled analog 12 was synthesized by following the methodology51 depicted in Scheme 3.

*

*

O-CH2-(CH2)4CH3 Me Me

Me

Me Et HBr/AcOH 14

N HN

Me LiOH

Me HOOC (14C-HPPH)

12

C-hexanol

O

Et

NH N

NH N 14

Me

Me 14

Et

N HN

N HN

MeO2C

Me

Me

Me

Me

Me NH N

OH

C-hexanol LiOH Me

Me Et NH N N HN

Me

Me

Me MeO2C

O

HBr gas

Me

O-CH2-(CH2)4CH3

O 9

HOOC

* = 14C-labeling

* = 14C-labeling

13

O

(14C Iso-HPPH)

Scheme 3: Synthesis of 14C-HPPH and the corresponding ring-B reduced isomer (iso-HPPH) A similar approach was used for the preparation of the corresponding Iso-HPPH. In brief,

19



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Page 20 of 44

the ring-B reduced chlorin 9 was reacted with HBr gas, the solvent was evaporated in vacuum, and the resulting intermediate was immediately reacted with [14C]1-hexanol, which on subsequent treatment with aqueous LiOH gave the desired compound. The purity of both isomers 12 and 13 was confirmed by HPLC analysis (Fig. 10). The HPLC analysis also revealed a substantial difference in elution profile for HPPH and iso-HPPH, suggesting a higher hydrophilicity for HPPH. However, further detailed study revealed the protonation of the isoHPPH under acidic solvent system used as a mobile phase protonates faster than the HPPH.To confirm these findings, we observed that using pure methanol as the mobile phase, the HPPH and iso-HPPH as carboxylic acid analogs eluted very poorly. In both cases a very large portion of the injected sample was retained in the column. Adding acetic acid to the methanol (0.5% v/v acetic acid in methanol as the mobile phase) significantly improved the elution. Both compounds eluted as a single peak. But compared to HPPH, the iso-HPPH peak was much broader. It was apparent from the photodiode array (PDA) uv-vis spectra of the eluted peaks that there was protonation of the free-base nitrogens of the porphyrin core. The long-wavelength absorption peak of the free base HPPH was observed at 660 nm and the iso-HPPH at 680 nm in methanol. On protonation, both the HPPH and iso-HPPH showed broad absorption at 650 nm. A higher degree of protonation of the iso-HPPH versus HPPH in the acidic mobile phase most likely is possibly caused the difference in their retention times. Both compounds in analytical TLC (silica coated) using 5% methanol/dichloromethane as a mobile phase showed similar Rf values, which further confirmed their similar polarity (Fig. S17, Supplemental Information). The

14

C-analogs 12 (HPPH) and the ring-B-reduced isomer 13 were

intravenously injected into mice bearing Colon26 tumors and the tumor-associated radioactivitymeasured at 2, 4, 8, 12 and 24h post-injection (Fig. 11). Both isomers had significant tumor-avidity, but HPPH showed higher uptake than iso-HPPH at all time-points. The

20


Page 21 of 44

maximum uptake was observed at 8-24h post-injection and both isomers showed a significant clearance from tumor between 12-72h post-injection.

Absorbance (A.U.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0

2

4

6

8

10

12

14

Time (Minutes) Figure 10: HPLC chromatogram of 14C-HPPH 12 and the corresponding ring-D reduced isomer 13, eluted as cationic forms (see the supplemental information Fig. S17). HPLC column: Waters Symmetry C18 (Waters Part # WAT045905), dimensions 4.6 x150m, particle size 5µ, mobile Phase 0.5 % (v/v) acetic acid in methanol, flow rate 1.0 ml / min. Wavelength channel data collected between 350 to 800 nm; processed at 410 nm. Both isomers were mixed together before loading to HPLC column.

Figure 11: In vivo biodistribution of 14C HPPH and iso-HPPH (50 µCi/mouse) in BALB/c mice bearing Colon26 tumors (tumor size: 4-5 mm) at various time points (2 – 72 hours), 3 mice per time point for each isomer. Arrows indicate the tumor-uptake of Iso-HPPH at 12 and 24 h postinjection. In vivo photosensitizing activity of HPPH and iso-HPPH The PDT efficacy of isomers 2 and 4 was evaluated in BALB/c mice bearing Colon26

21



tumors. The PSs were injected intravenously (0.47µmol/kg) and in two separate experiments, either at 12 h or 24h post-injection, the tumors were irradiated with laser light (665 nm for HPPH and 674 nm for its isomer, 128J/cm2, 14mW/cm2), and the tumor growth was monitored daily for 60 days. From the results summarized in Fig. 12 [20 mice/compound (10 mice 2 experiments/compound)] it can be seen that both HPPH and iso-HPPH showed similar longterm tumor-cure, with HPPH being slightly more effective. HPPH at a lower drug dose

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Graphical Abstract ____________________________________________________________________________

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

Courtney Saenz, Penny Joshi, Tymish Ohulchanskyy, Walter A. Tabaczynski, Joseph R. Missert, Yihui Chen, Pera Paula, Erin Tracy, Aimee Marko, Daniel Rohrbach, Ulas Sunar, Heinz * * Baumann and Ravindra K. Pandey

A 3D cell culture system demonstrates high tumor-specificity for both ring ‘B’ and ring ‘D’ reduced isomers (HPPH and Iso-HPPH) in head and neck cancer patients. ____________________________________________________________________________

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Structural and Epimeric Isomers of HPPH [3-Devinyl 3-{1-(1-hexyloxy

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