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

To investigate the impact of mono- and di-β-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 β-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.


Introduction
Since the approval of Photofrin for the treatment of cancer by photodynamic therapy (PDT), a wide variety of porphyrins and non-porphyrin-based photosensitizers have been reported [1][2][3][4]. Efforts have also been made to investigate the tumor targeting ability of photosensitizers by conjugating certain small molecules and peptides to target receptors known for their over-expression in tumors [5,6]. Among these targeting moieties, carbohydrates-especially β-galactose have gained attention [7][8][9][10][11]. The other strategy, which has also made a significant impact in developing improved agent(s) for PDT is the structure activity relationship (SAR) approach, and some of the agents, e. g. HPPH (Photochlor) [12], Foscan [13], Tookad [14], Talaporphin [15], Photobac [16], benzoporphyrin derivative (BPD) [17] and (PC4, a Phthalocyanine) [18] are at various stages of human clinical trials for the use in oncologic and non-oncological applications. Another approach, which has been equally useful in selecting effective agents before evaluating them for in vivo efficacy is to define the cell-specificity of PSs in various tumor cell types, especially PS uptake and retention [19].
Most of the porphyrin-based compounds currently being investigated for the use in PDT show poor water solubility. The common substituents that have been introduced in tetrapyrrolic systems (porphyrins, phthalocyanines or expanded porphyrins) are amine, carboxylic acid, sulfonic acid and ethylene glycol side chains, which do not show much impact in water solubility [20,21]. However, some of the glycosylated porphyrin systems may improve the hydrophilic nature by attaching the sugar moiety at the peripheral positions of the macrocycle. In general, (Glycan-protein) lectin-type receptors are highly expressed in some type of malignant cells, which specifically interact with carbohydrate-conjugated tetrapyrroles [22]. Among certain proteins the galectins are highly conserved carbohydrate recognition domain and exhibit high affinity for β-galactoside [23]. Galectins are known to involve in the modulation of cell adhesion [24], cell growth [25], immune response [26] and angiogenesis [27] and changes in their expression play a critical role in tumor progression.
In our attempt to enhance the efficacy and selectivity of HPPH [3-(1′-hexyloxy)ethyl-3devinylpyropheophorbide-a], currently under human clinical trials [1,28,29], was conjugated with a series of mono-di-and tetrasaccharides, with the goal to target carbohydrate-binding proteins, especially galectin-1 and galectin-3. Among the compounds investigated HPPH-Gal in which a β-galactose moiety was conjugated with HPPH at position 17 2 showed the best efficacy [30]. The increased activity (both in vitro and in vivo) could not be confirmed on the basis of its binding to cellular galectins. However, the overall lipophilicity and the lack of transport by the ABCG2 transporter may contribute to improved efficacy [31].
In a parallel study, to determine the effect of substituents at various peripheral positions of the HPPH moiety, the 20-phenyl substituted analogs were of particular interest due to their higher tumor-cell selectivity. The meso-phenyl substituted analog provides a unique opportunity not only to introduce the targeting moieties at two different positions (17 and 20) of the molecule, but also to investigate the impact of overall charge in tumor cell-uptake and retention. Therefore, in present study, a series of HPPH-based PSs containing monoand di-galactose moiety with and without the presence of hexyl ether side chain at position examined here. Even protected conjugates 29 and 30, which are pre-cursors of 4, fail to exhibit the additional peaks despite the fact that each of these is structurally similar to 4.
These facts lead us to the following hypothesis. Hydrogen bonding -from a hydroxyl group of the unprotected galactose to the E-ring's keto oxygen -may be responsible for the presence of the unexpected carbon resonances observed in 4. The additional carbon peaks could arise due to the presence of two states of each diastereomer: hydrogen bound, and non-hydrogen bound. This would account for our observation of up to four peaks for each of the affected carbons. The lack of extra peaks in 29 and 30 is consistent with this hypothesis because the acetyl-protected galactose moieties in these two precursors lack the hydroxyl groups required for hydrogen bonding to the keto oxygen. The localized nature of this effect (E-ring only) is also consistent with the hydrogen bonding scenario because the influence on carbon chemical shifts is likely to be greatest near the site of the hydrogen bond.
Although plausible, the hydrogen bond explanation fails to account for some of our observations. No other mono-or di-galactose-containing conjugate examined here exhibits the same effect in its E-ring carbons. One might expect 6 and 9 to behave like 4 because each also has an unprotected galactose at position-17, and (relative to 4) each has a similar general structure. Conjugate 6 differs from 4 only in its additional galactose linked through position-20. Conjugate 9 differs from 4 in its lack of the O-hexyl group at position-3 [1]. To be consistent with our hydrogen bonding explanation, we must assume that the O-hexyl group and second galactose moiety somehow prevent hydrogen bond formation, which would account for the behavior of 6 and 9. However, we recognize that the unusual behavior of the E-ring carbons of conjugate 4 may be due to some other source. For example, it might be due to two different ring conformations, or two different stacking geometries. Each of these could manifest itself in two distinct structural states (per diastereomer) resulting in two distinct magnetic environments (per diastereomer) in the vicinity of the E-ring. This could account for the number of carbon peaks observed, up to four per E-ring carbon. Proof of any of these proposed explanations would obviously require further studies.
in both cell types. The efficacy of PS 11 was reduced on replacing the ethyl group at position 3 with 3 (1′-hexyloxy)ethyl substituent. The in vitro efficacy (percentage cell survival) results of PS 2-6 and 9-11 in both FaDu and Colon26 cell lines are shown in Fig. 3.

A difference in mitochondrial and lysosomal localization of HPPH
and the corresponding carbohydrate analogs-The importance of subcellular localization of a photosensitizer and its impact on the destruction of tumor cells by reactive oxygen species (mainly singlet oxygen) produced after exposing the tumors/cells with an appropriate wavelength of light has been a subject of discussion from a long time [35,36]. However, it is now well accepted that most of the porphyrin-based mainly localize in mitochondria and lysosomes [37]. Depending on the type of PS, both diffusion and endocytosis play an important role in cell-uptake. It is now also well accepted that apoptosis [38] and necrosis [39] are the two main mechanisms of cell death. The in vivo studies have suggested that vascular shutdown play crucial role in reducing the survivability of tumor cells as it starves them to nutrients and oxygen [40]. However, direct cell cytotoxicity has not been ruled [41] out in less extent, and the overall mechanism of cell death by PDT is complex. In any event PSs localize either in mitochondria or lysosomes (or both) are reported to be effective, and the tumor specificity seems to depend on over all lipophilicity, charge and the nature of substituents present at peripheral position of the PDT agent.
In our present study, we compared the subcellular localization characteristics of HPPH 1, with its mono-and di-galactose conjugates 2 and 11 respectively. Cells were plated in 6 well plate and allowed to adhere before 1 μM of PS 1 was added. After 24 h, Lysotracker Green DND-26 and Mitotracker Red CmxRos were added to enable fluorescent labeling of the lysosome or mitochondria, respectively. The images were analyzed using Amnis IDEAS v6.2 where the bright detail similarity score was generated by comparing co-localization between the photosensitizer and the organelle Results depicted in Fig. 4 indicate that PS 2 (non-galactose analog) shows the preference to mitochondria over the lysosome, whereas the mono-and di-galactose analogs show preference to lysosome with almost similar localization pattern.

Comparative tumor uptake and PDT efficacy PSs 1, 2 and 11-We
have previously shown that conjugation of β-galactose at position 17 2 -HPPH enhances PDT efficacy of mice bearing BALB/c tumors. In present study, the tumor-uptake and PDT efficacy of HPPH 1 was compared with the corresponding mono-and di-b-galactose conjugates 2 and 11 (at a dose of 0.47 mmol/kg) in SCID mice bearing FaDu tumors. and imaged at various time points using IVIS Spectrum. The fluorescence intensity of tumor, liver and skin was measured at variable time points and the results are shown in Fig. 5. Interestingly, both β-galactose conjugated PSs in SCID mice bearing FaDu tumors showed similar pattern of uptake in tumor, liver and skin from 1 to 8 h post-injection. However, compared to 2, the uptake of PS 11 in tumor was slightly higher at 8 h. A significant clearance from tumor, liver and skin at 24 h post-injection was observed by both the PSs. In contrast to carbohydrate analogs, the uptake and biodistribution characteristics of HPPH was significantly different. For example, the tumor-uptake increased with time, and maximum uptake was observed at 24 h post injection, with a significant clearance from both liver and skin. The high uptake of mono and di-galactose analogs in liver could be due to hepatic uptake and metabolism of galactose moiety (see Fig. 6).

Comparative in vivo PDT efficacy of HPPH (1) with mono-and digalactose conjugates (2 and 11)-
The SCID mice bearing FaDu (Head & Neck tumors) were selected to study the antitumor activity of PSs 1, 2 and 11. When the tumors were of treatment size (4-5 mm in diameter), the mice were injected with PS formulated in 1% Tween 80/5% dextrose solution at a dose of 0.47 μmol/kg. The tumors were exposed to light (665 nm, 135 J/cm 2 , 75 mW/cm 2 ) at 24 h post-injection for PS 1 and 8 h post-injection for the galactose analogs 2 and 11 (due to their optimal tumor uptake at those time points), and tumor growth was measured daily. The PS 2 and 11 injected intravenously in SCID mice bearing FaDu tumors gave complete response (CR) 50% (3/6 mice were tumor free on day 60) and 77.5% (7/9 mice were tumor free on day 60) respectively on day 60, whereas HPPH, at similar drug and light dose was 60% effective (3/5 mice were tumor free on day 60). The statistical evaluation of the survival curves was analyzed by Mantel-Cox test.

Conclusions
In this study, a series of pyropheophorbides containing β-galactose moiety at position-17 [2] and/or position-20(4′) with variable lipophilicity by altering the substituents at position-3 were synthesized. The compounds were characterized by NMR ( 1 H & 13 C), and the purity was ascertained by HPLC. These compounds were insoluble in water and formulated in 1% Tween 80/5% dextrose (D5W) solution [27] The in vitro PDT efficacy of the conjugates was determined in two tumor cell lines: FaDu (head & neck) and Colon16. In both cell lines, PS 2 in which galactose moiety was conjugated at position-17 [2] of HPPH and compound 11 containing β-galactose moieties at positions 17 2 and 20(4′) of meso pyropheophorbidea showed similar efficacy than the non-galactose analog 1 (HPPH). The PS 1 (HPPH) and the corresponding carbohydrate analogs 2 and 11 showed significant tumor uptake in mice bearing FaDu tumors. However, as expected compared to PS 1, the PSs 2 and 11 showed higher uptake in liver, which could be due to higher hepatic uptake/metabolism of β-galactose. Among the compounds tested for in vivo efficacy, the di-β-galactose conjugate 11 gave the best long-term tumor cure in SCID mice bearing FaDu tumors (7/9 mice = 77.7% tumor free on day 30). The higher efficacy of PS 11 over 2 could be due to (a) the difference in biodistribution of the PS across the tumor, (b) the consequences of PDT (immunological impact) after light treatment, and (c) the role of ABCG 2 pump in efflux of the PS (with and without the galactose moiety. These studies using a larger group of mice are currently underway, and hope will help to address these questions. Compared to PS 1 (HPPH), the carbohydrate analogs 4,5,6, and 10 were significantly less effective than the non-carbohydrate analog 1 in vitro, and were not evaluated for in vivo efficacy.

Experimental section
3.1.1. Chemistry-Chemicals used for the synthesis were purchased from Aldrich and Synthose and were reagent grade unless otherwise specified. All reactions were carried out in heat gun-dried glassware under an atmosphere of argon and magnetic stirring. Thin layer chromatography (TLC) was performed on pre-coated silica gel sheets (layer thickness 0.2 mm). Column chromatography was performed using silica gel 60 (70-230 mesh) purchased from Merk. Preparative TLC was also used for the purification of compounds (Analtec, 20 × 20 cm, silica gel GF, 1000 μm). Purity of the compounds was ascertained by TLC, NMR and HPLC. All compounds including the intermediates were >95% pure. UV-visible spectra were recorded on an UV-visible spectrophotometer using DCM and MeOH as solvents. Mass spectrometry analyses were performed at the Mass Spectrometry Facility, University of Buffalo, NY.
NMR data were acquired at 28 °C on a Bruker Avance III HD NMR spectrometer equipped with a 9.4 T narrow-bore magnet, a 5-mm BBO Z-gradient probe, and Topspin 3.2 software.
Frequencies for 1 H and 13 C observations were 400 MHz and 100 MHz, respectively. Chemical shifts were calibrated to the residual solvent resonance and are reported relative to tetramethylsilane (TMS) at 0.00 ppm. 1 H multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad.

3.1.3.
General procedure for deprotection of tert-butyl esters for synthesis of compounds 18, 19, 24, and 30-Starting material (15-25 mg) was stirred in 3 ml of 70% solution of trifluoroacetic acid in DCM for 25 min under Ar atmosphere at room temperature. The reaction mixture was then diluted with 20 ml of DCM, washed with H 2 O (4 × 20 ml, until wash water pH > 6.5), dried over Na 2 SO 4 , and filtered. After removing the solvent, clean product was obtained in quantitative yield.

3.1.4.
General procedure for the synthesis of the compounds 20, 21, 26, and 31-Starting material (1 eq.), 1-β-amino-galactose tetraacetate (2.5 eq.) and benzotriazol-1yloxytris(dimethylamino) phosphonium hexafluorophosphate (2.5 eq.) were dissolved in dry DMF (7 ml) under Ar atmosphere. To this solution, triethylamine (2.5 eq.) was added, and the reaction mixture was stirred overnight (for compounds 26 and 31 for 32 h) at room temperature. After this, 20 ml of DCM was added to the reaction mixture, and it was washed with H 2 O (3 × 20 ml) and dried over Na 2 SO 4 . After filtration, the solvent was removed under reduced pressure. The resulting residue was purified by three separate runs of preparative TLC using silica plates and the following eluents: (1) 4% MeOH in DCM, and (2 & 3) 3% MeOH in DCM.
3.1.5. General procedure for the synthesis of the compounds 5, 10, 22, and 25-Starting material (1 eq.) was dissolved in 10-20 ml of a degassed (3 times, vacuum -Ar) mixture of MeOH and THF (1:1). LiOH monohydrate (15-25 eq.) was dissolved in 5-10 ml of H 2 O and degassed (3 times, vacuum -Ar). Then, the solution of LiOH was added to the solution of starting material and the reaction mixture was degassed (5 times, vacuum -Ar) and stirred overnight at room temperature. The reaction mixture was then neutralized with a cold (0 °C) 5% aqueous solution of CH 3 COOH. The product was extracted from the reaction mixture with DCM (50 ml). DCM solution was washed with H 2 O (5 × 50 ml). The organic layer was then dried over Na 2 SO 4 , filtered, and solvent was removed under reduced pressure. Yield was quantitative for compounds 22 and 25. Compounds 5 and 10, however, required purification by silica column chromatography, using gradient 30-90% MeOH in DCM as eluent.

3.1.7.
General procedure for the deacetylation of galactose moieties for obtaining compounds 4, 6, 9, and 11-Starting material (1 eq.) was dissolved in a mixture of dry DCM (10 ml) and MeOH (1.0 ml). Sodium methoxide in MeOH (0.5 M, 250 μL) was added to this solution, and the reaction mixture was stirred for 25 min under Ar atmosphere at room temperature. It was then neutralized with a solution of 5% acetic acid in DCM and diluted with DCM to 50 ml total volume. The reaction mixture was then washed with H 2 O (3 × 50 ml), dried over Na 2 SO 4 , filtered, and solvents were evaporated under vacuum. The crude product was purified by silica column chromatography, using gradient 35-95% MeOH in DCM as eluent.   Fig. S43, purity >96.0%).   Fig. S43, purity >97.3%).
3.1.9. Method of tumor-implantation-Colon 26 and FaDu tumors were transplanted in 6-8-week-old Female Balb/c (NCI Balb/cAnNCr) and SCID mice (Strain C.B Igh-1b Icr Tac Prkdc Scid) respectively and were maintained in the animal facility. Mice bearing an established tumor (~ 7 days after implantation) were treated with the PDT. Tumor size was measured on two axes (in millimeters; L, longest axis, and W, shortest axis) with the aid of Vernier calipers. Tumor size (mm 3 ) was calculated by using the formula: tumor weight = 1/2 (L × W 2 ). All studies were performed in accordance with protocols approved by the institutional animal care and use committee at Roswell Park Cancer Institute. 3 were injected intravenously (i.v) with photosensitizer PS 2 and PS 11 at dose of 0.47 μmol/kg in Tween formulations. The PS uptake in FaDu tumors was determined by fluorescence imaging using a PerkinElmer IVIS Spectrum at variable timepoints, and the maximum uptake was observed post-injection. At this timepoint the tumors were irradiated with light (fluence:135 J/cm 2 ; fluence rate: 75 mW/cm 2 ) for 30 min at 665 nm using a Lightwave™ laser diode. Mice were restrained without anesthesia in plexiglass holders designed to expose only the tumor and a 2-4 mm annular margin of skin to light. The tumor assessment and measurements were taken daily, then three times a week for 4 weeks, and twice a week thereafter for a total of 30 days post treatment. Tumor volume (mm 2 ) was estimated using a formula: tumor volume = ½ (L x W 2 ). Two axes (mm) of tumor (L, longest axis; W, shortest axis) were measured with the aid of a Vernier caliper. The complete tumor regression (CR) was defined as the inability to detect tumor by palpation at the initial site of tumor appearance for more than two-month post-therapy. The Partial tumor regression (PR) was defined as ≥ 50% reduction in initial tumor size. The edema, erythema, and scar formation in the treatment field was observed and recorded. Tumor response for each treatment was evaluated fot the tumor response.

HSQC
heteronuclear single quantum coherence

ROS
Reactive oxygen species Structures of photosensitizer-carbohydrate conjugates investigated in current study.
Dukh et al.   PS uptake: SCID mice bearing FaDu tumors (3 mice/group) were injected with PS 1 (HPPH), 2 and 11 at a dose of 0.47 μmol/kg. The tumor vs liver and skin (adjacent to tumor) uptake were measured by fluorescence at variable timepoints using IVIS spectrum (excitation wavelength: 665 nm, emission: > 720 nm). Tumor vs liver and skin uptake was determined based on the fluorescence intensity measured by IVIS Spectrum, and was plotted against time. Tumor Images with PS 1, 2 & 3 (2/3 mice/group) are shown at the time of their optimal uptake in tumors. . In case of PS1 injected mice, the tumors were exposed to light at 24 h and for PSs 2 and 11 at 8 h post-injection of the PS (the timepoint of maximal PS uptake). Tumor growth was measured. Among the compounds, PS 11 (di-β-galactose analog) showed the best anticancer activity. Note: None of the mice after the PDT treatment. The mice with tumor regrowth (>400 mm 3 ) were euthanized following the IACUC approved animal protocol.