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Research Articles: Therapeutics, Targets, and Development
Tumor localization and antitumor efficacy of novel sapphyrin compounds
1 Pharmacyclics, Inc., Sunnyvale, California and 2 Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas
Requests for reprints: Louie Naumovski, Cancer Biology, Pharmacyclics, Inc., 995 East Arques Avenue, Sunnyvale, CA 94085. Phone: 650-400-0015; Fax: 408-328-3689. E-mail: lnaumovski{at}pcyc.com
Abstract
Sapphyrins are pentapyrrolic metal-free expanded porphyrins with potential medical use as anticancer agents. The novel sapphyrin derivative, PCI-2050, functionalized with 2-[2-(2-methoxyethoxy)ethoxy]ethoxy groups to enhance solubility and a modified bipyrrole moiety was found to be more potent in inducing apoptosis than the previously described sapphyrin PCI-2000. Because some sapphyrins may localize to tumors, we took advantage of the intrinsic fluorescence of these compounds to develop a flow cytometry–based assay to track sapphyrin biodistribution in tumor-bearing mice. Ex vivo analysis of sapphyrin-injected animals revealed that PCI-2050 preferentially localized to tumor, whereas PCI-2000 distributed into normal tissues rather than tumor. PCI-2050 uptake in xenograft tumor cells and resultant tumor cell cytotoxicity was dose dependent. To investigate structure–activity relationships, we focused on PCI-2050 and three derivatives that differ by their alkyl substituents on the bipyrrole moiety: PCI-2051, PCI-2052, and PCI-2053. Treatment of Ramos cells in culture or treatment of Ramos xenograft-bearing animals with each of the sapphyrins followed by ex vivo growth of tumor cells revealed the same pattern of cytotoxicity: PCI-2050 > PCI-2052 > PCI-2051 > PCI-2053. Thus, subtle changes in the alkyl substituents on the bipyrrole moiety result in significant changes in antitumor activity. PCI-2050 displayed significant antitumor efficacy in both Ramos and RKO xenograft models without hematologic, hepatic, or renal abnormalities as assessed by complete blood counts and serum chemistries. On the basis of these findings, it is concluded that the sapphyrin PCI-2050 warrants further evaluation as a potential anticancer agent due to its intrinsic proapoptotic activity and tumor localization ability. [Mol Cancer Ther 2006;5(11):2798–805]
Introduction
Sapphyrins are pentapyrrolic metal-free expanded porphyrins with potential medical use in a variety of areas, including cancer therapy (1). Sapphyrins bind to anions, such as phosphate, chloride, and fluoride, and have been postulated to serve as potential anion transporters (1–3). Because sapphyrins absorb light in the red portion of the visible spectrum and can generate singlet oxygen in good quantum yield (4–7), they have been explored as potential photodynamic therapy agents to inactivate viruses in vitro and also to treat superficial squamous cell carcinoma in mice (5, 6). Like porphyrins, some sapphyrins show preferential accumulation in tumors, suggesting that if they had intrinsic cytotoxic or growth inhibitory activity, they could act as anticancer agents (4, 8).
We previously showed that water-soluble sapphyrins, such as the bis-hydroxypropyl derivative PCI-2000, can act as potent inducers of apoptosis in a wide variety of hematopoietic tumor-derived cell lines, including lymphoma, leukemia, and myeloma in the absence of light (9). PCI-2000 was found to trigger an apoptotic pathway in the tumor cells as shown by release of cytochrome c from mitochondria; activation of caspase-9, caspase-8, and caspase-3; cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase; and Annexin V binding (9). Treatment of Ramos lymphoma cells with PCI-2000 or PCI-2010, a congeneric tetrahydroxy bis-carbamate derivative, resulted in enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK), a stress-responsive protein kinase that participates in the regulation of apoptosis. Both of these sapphyrins were found to have potent cytotoxic activity in a wide variety of hematopoietic-derived tumor cells (9).
In the present study, we have synthesized and characterized novel water-soluble sapphyrins that have increased tumor selectivity and cytotoxicity compared with PCI-2000. We developed methods based on assessment of drug fluorescence in normal organs and tumor tissue and in vivo/ex vivo analysis to quickly screen drug candidates for potential antitumor activity. Specifically, we show dose-dependent localization of the sapphyrin PCI-2050 in tumor cells relative to normal tissues in vivo, which correlates with tumor cell killing. In mouse xenograft models, PCI-2050 is active as an anticancer agent at doses that are not toxic to bone marrow, liver, or kidney.
Materials and Methods
Sapphyrin Synthesis
PCI-2000 was prepared using the "3+1+1" procedure as described in the literature (10). The synthesis of PCI-2050 and its derivatives (PCI-2051, PCI-2052, and PCI-2053; see Fig. 1
for chemical structures) will be described elsewhere.3 The purity of each compound was >94% as measured by reversed-phase high-performance liquid chromatography. The identity was ascertained by proton and carbon-13 nuclear magnetic resonance spectra, as well as electrospray ionization mass spectrometry. For animal studies, PCI-2000 was formulated by dissolving in 5% mannitol containing 5% absolute ethanol followed by sterile filtration, whereas other sapphyrins were formulated by dissolving in 5% mannitol followed by sterile filtration.
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Apoptosis Assays
Annexin V–FITC binding was assayed with a FACSCalibur instrument (Becton Dickinson, San Jose, CA) using reagents from Biosource (Camarillo, CA) per the protocol of the manufacturer.
Caspase-3 activity was assayed using the EnzChek Caspase-3 Assay kit 2 (Molecular Probes, Eugene, OR). Cells were harvested, rinsed in cold PBS, and analyzed according to the protocol of the manufacturer. Cell extracts were incubated in a reaction mixture containing Z-DEVD-R110 (0.5 mmol/L) at room temperature for 30 minutes, and fluorescence levels were determined at an excitation of 485 nm and emission of 510 nm using a fluorescence plate reader. For each cell line, measured fluorescence levels were normalized to fluorescence levels of nontreated cell lysates.
In vivo/Ex vivo Analysis of Sapphyrin Fluorescence Uptake and Antitumor Activity
Ten million Ramos cells were implanted s.c. into the right hind flank of 6-week-old crl:CD-1-nuBR nude mice that had been whole body irradiated with 3 Gy at 24 hours before tumor implantation. About 7 days later, when tumor size reached 
100 to 150 mm3, mice (n = 2) were injected i.v. via tail vein with various sapphyrins at doses indicated in text and figure legends. Twenty-four hours after the last dose, mice were sacrificed by CO2 inhalation and tissues were harvested for analysis. Tumor and tissues from sapphyrin-injected mice were disaggregated in PBS and filtered through a 50 µm syringe (BD Biosciences, San Diego, CA) to generate a single-cell suspension. Cells were centrifuged for 5 minutes at 600 x g and treated with RBC lysing buffer (Sigma, St. Louis, MO) for 2 minutes with gentle mixing. Samples were diluted with PBS, mixed, and centrifuged for 5 minutes at 600 x g. The supernatant was aspirated and cells were diluted with PBS before use. To assess the cytotoxic and growth inhibitory activity of sapphyrins injected into tumor-bearing animals, disaggregated tumor cells (50,000/mL) were placed in culture. Twenty-four hours later, Annexin V staining was used to assess cytotoxicity. Four days after plating, cell numbers were determined by Coulter counter.
Sapphyrins are green and absorb light in the 400 to 500 nm range with resultant fluorescence of >650 nm. The relative uptake of the different sapphyrins in cell culture, and tumor and normal tissues, was determined by flow cytometry using an excitation wavelength of 488 nm and an emission wavelength of >650 nm with a FACSCalibur instrument (Becton Dickinson).
Animal Xenograft Models for Antitumor Efficacy
CD1 female mice were irradiated with 3 Gy from a cesium source 24 hours before implantation with either 10 million Ramos or RKO cells injected s.c. into the right hind flank. For the Ramos "minimal disease" xenograft model, 3 days after Ramos cell implantation, mice (n = 6) were treated with PCI-2050 given i.v. in the tail vein at 1, 5, or 10 µmol/kg for two daily doses. For the "palpable disease" xenograft model, mice (n = 6) were treated with drug when tumor size reached 150 mm3 for Ramos and 100 mm3 for RKO. Vehicle control–treated animals received 5% mannitol on the same schedule. Tumor measurements were done every 4 days and the tumor volume was calculated as (
/6 x length x width x height). Error bars represent SEs. Student's t test was used to determine significance. All animal studies were approved by the Pharmacyclics Institutional Animal Care and Use Committee.
Results
PCI-2050 Is More Potent than the Previously Studied Sapphyrin Compound PCI-2000 in Inducing Apoptosis and Growth Inhibition
We had previously identified the sapphyrins PCI-2000 and PCI-2010 as potential anticancer agents based on in vitro and in vivo studies. In an effort to improve the solubility and potentially the efficacy of these sapphyrins, we synthesized PCI-2050, a sapphyrin containing a bipyrole subunit that is modified compared with PCI-2000 and that has four 2-[2-(2-methoxyethoxy)ethoxy]ethoxy groups attached to the ß-pyrrolic hydroxypropyl substituents for water solubility (Fig. 1A). Ramos lymphoma cells were treated with PCI-2000 and PCI-2050 in cell culture and analyzed for markers of apoptosis. We found that PCI-2050 was more active than PCI-2000 in inducing death of Ramos cells as shown by Annexin V–positive cells, caspase-3 activity, and poly(ADP-ribose) polymerase cleavage (Fig. 1B–D). Both PCI-2000 and PCI-2050 were readily detectable by flow cytometry analysis with PCI-2050 displaying greater median fluorescence in sapphyrin-treated Ramos cells (Fig. 1E). However, because the spectral properties of different sapphyrins may vary depending on intrinsic fluorescence, solubility, degradation, and cellular milieu, fluorescence measurements by flow cytometry may not provide a quantitative means of comparing the cellular localization of different sapphyrin analogues to each other. PCI-2050 is also active in inducing apoptosis in solid tumor–derived cell lines, including A549 (lung cancer) and RKO (colon cancer; data not shown).
PCI-2050 Localizes Preferentially to Tumor Cells In vivo with Less Uptake in Normal Tissues than PCI-2000
We developed a Ramos tumor xenograft model in nude mice to assess drug biodistribution in tumor cells versus normal tissues. Mice carrying Ramos tumor in their flanks were injected via tail vein with PCI-2000 or PCI-2050 for 2 consecutive days and then sacrificed for analysis (schematic; Fig. 2A
). Tumor, spleen, and liver tissues were isolated and analyzed by flow cytometry, exploiting the fact that sapphyrin compounds are fluorescent. We found that tumor cells harvested from animals treated with PCI-2050 had substantially more fluorescence than spleen and liver cells (Fig. 2B–D). On the other hand, organs harvested from animals treated with PCI-2000 showed the reverse pattern with greater fluorescence in normal tissues than tumor cells (Fig. 2B–D). Although qualitative, fluorescence served as a useful "surrogate" marker to quickly and conveniently track drug localization. Furthermore, by comparing the pattern of fluorescence of a particular sapphyrin with that of another sapphyrin among tumor and normal body tissues, we were able to determine relative in vivo biodistribution. These patterns of drug fluorescence lead us to conclude that PCI-2050 has more favorable tumor localization properties than PCI-2000.
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Analysis of PCI-2050 Congeners
To dissect the relative contribution of polyethylene glycol groups versus changes in the bipyrole moiety, several congeners of PCI-2050 were synthesized that differed at the bipyrole moiety (see Fig. 1A for structures of PCI-2051, PCI-2052, and PCI-2053). Ramos cells were treated with PCI-2050, PCI-2051, PCI-2052, and PCI-2053 in cell culture and Annexin V–positive cells and caspase-3 activity were determined (Fig. 4A, B
). PCI-2050 and PCI-2052 were the most active derivatives in terms of inducing apoptosis (Fig. 4A, B). PCI-2051 was not as active in inducing apoptosis and PCI-2053 was essentially inactive (Fig. 4A, B). Although we did not specifically quantitate drug uptake by biochemical assays, drug fluorescence in sapphyrin-treated cells showed a pattern consistent with the cytotoxic activity of the drugs (Fig. 4C).
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PCI-2050 Has Minimal Affects on Blood Counts or Serum Chemistries
Because PCI-2050 and PCI-2052 were the most active sapphyrins, as judged in terms of inducing apoptosis of tumor cells in vitro and in vivo, we determined the safety of these compounds in mice by evaluating single dose toxicity of the compounds given i.v. These studies showed that mice could tolerate PCI-2050 up to 25 µmol/kg without any deaths (0 of 3 mice) observed during a 14-day follow-up period. By contrast, mortality was seen in the case of PCI-2052 even at 20 µmol/kg (two of three mice). There were no notable treatment-related histopathology findings when PCI-2050 was administered at nonlethal doses (
20 µmol/kg; n = 3, data not shown).
Treatment of animals (n = 3) with 10 µmol/kg PCI-2050 each day for 2 days revealed no significant changes in peripheral blood counts or serum chemistry values measured at 7, 14, and 21 days after injection (Table 1 ). Specifically, there was no significant decrease in peripheral WBC count (no myelosuppression or lymphosuppression) and platelet counts remained adequate at all time points tested (Table 1). There were no elevations in serum blood urea nitrogen or creatinine to indicate kidney toxicity (Table 1). There were also no increases in various markers of liver function, indicating the absence of overt liver problems (Table 1). PCI-2050–treated animals had minimal weight loss (<10%) during the course of these experiments (data not shown).
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50% (Fig. 5C). Animals treated with 10 µmol/kg PCI-2050 x 2 had minimal weight loss and those treated with 12.5 µmol/kg x 2 had <10% weight loss during the course of these experiments (data not shown). Doses of PCI-2050 > 12.5 µmol/kg x 2 days (although showing better tumor control) were not well tolerated as manifested by >15% weight loss (data not shown). Administration of PCI-2050 via tail vein for 2 days in a row resulted in some injection site irritation that precluded further consistent dosing of the drug among animal cohorts (data not shown).
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Porphyrins and expanded porphyrins, such as texaphyrins and sapphyrins, have potential medical use, including in the area of cancer imaging and therapy (1, 11, 12). Sapphyrins are expanded metal-free porphyrins with interesting chemical properties; however, only limited information is available regarding their biological activity (2). We previously described cytotoxic and selective antitumor activity of sapphyrins PCI-2000 and PCI-2010 that provided the rationale for synthetic modifications of this class of compounds aimed at optimizing their therapeutic potential (9). We showed that these sapphyrins induce apoptosis in numerous cell lines, including those derived from lymphoma, leukemia, and multiple myeloma, in the absence of sufficient light to induce photodynamic effects (9). The biochemical pathway through which sapphyrins induce apoptosis has not yet been fully elucidated; however, similar to other stress-inducing stimuli, sapphyrin treatment was found to result in the phosphorylation of p38MAPK, which may then transduce either survival or death signals (13, 14). The combination of PCI-2000 with a specific inhibitor of p38MAPK phosphorylation resulted in synergistic cytotoxicity, leading to the conclusion that p38MAPK phosphorylation was a protective response in PCI-2000–treated Ramos cells as opposed to a death-transducing signal.
Hematoporphyrin derivative, a mixture of porphyrins, shows enhanced uptake in tumors in vivo, which can be recapitulated in vitro under conditions of low pH and low serum (15). Like porphyrins, sapphyrins are known to localize to tumor tissues (8). Tumor localization is a desirable property for an anticancer compound because it might result in increased antitumor activity while reducing toxicity to normal tissues. In an effort to identify other useful sapphyrin compounds, especially those with potential tumor-localizing ability, we synthesized PCI-2050 that contains amphoteric polyethylene glycol groups to enhance solubility and a modified bipyrrole moiety compared with PCI-2000.
To determine biodistribution in tumor relative to normal tissues, we took advantage of the intrinsic fluorescence of sapphyrins to develop a flow cytometry method based on patterns of drug fluorescence. We could not use whole-body imaging techniques because small amounts of sapphyrin deposition in skin prevented adequate and quantitative imaging of internal organs and s.c. tumors. Using flow cytometry of disaggregated organs and tumors, we found that comparing the relative levels of fluorescence among tumor and normal tissues gave important information regarding biodistribution patterns in vivo. For example, animals injected with PCI-2050 had higher levels of drug fluorescence in tumor than in normal tissues. In contrast, animals injected with PCI-2000 displayed higher levels of drug fluorescence in normal tissues than in tumor cells. This biodistribution pattern suggests that PCI-2000 might have greater toxicity to normal tissues than would PCI-2050. Indeed, this seems to be the case because PCI-2000 results in toxic deaths at doses of 9.2 µmol/kg and above, whereas PCI-2050 results in toxic deaths at doses of 
30 µmol/kg (ref. 9 and this study). Perhaps, in part, PCI-2050 has improved tumor distribution because it has reduced uptake in normal tissues and thus more is available for uptake into tumor cells. The favorable biodistribution profile of PCI-2050 compared with PCI-2000 may be due to the polyethylene glycol moieties, because addition of polyethylene glycol groups to other drugs and proteins has resulted in improved biodistribution and activity (16). The ex vivo fluorescence measurements of sapphyrin biodistribution are believed to be reflective of the unchanged parent compound based on the absence of significant fluorescent metabolites in chromatograms obtained from gradient high performance liquid chromatography analysis of plasma, kidney, and tumors harvested from mice at 0.5, 4, and 24 hours after dosing with PCI-2050.4
We further characterized PCI-2050 fluorescence uptake into tumor cells in vivo. There was a linear correlation between dose injected into Ramos xenograft animals and PCI-2050 fluorescence in tumor cells. Ex vivo flow cytometry of in vivo treated tumor cells revealed uniform fluorescence without evidence of a nonfluorescent subpopulation, suggesting that the drug uptake was relatively homogenous in tumor cells. Furthermore, tumor cells derived from PCI-2050–treated animals showed dose-dependent growth inhibition and induction of apoptosis. Although we cannot completely rule out PCI-2050 metabolism to nonfluorescent species, we found >98% drug recovery when PCI-2050 was spiked into homogenates of kidney, liver, or tumor and incubated for 4 hours at room temperature.4 Additionally, we have observed that nonfluorescent precursors of the final compound have considerably less cytotoxic activity than PCI-2050.4 Therefore, even if PCI-2050 were metabolized, we would expect loss of cytotoxic activity.
Another difference between PCI-2000 and PCI-2050 is a modification on the bipyrrole moiety. To further investigate structure–activity relationships, we focused on PCI-2050 and three derivatives that differ only in their pattern of substitution on the bipyrrole moiety, namely PCI-2051, PCI-2052 and PCI-2053. Although seemingly modest in chemical terms (only permutations of ethyl, methyl, and hydrogen are involved), it was found that these changes had a significant effect on biological activity. Both in cell culture and in tumor cells derived from drug-injected mice (as determined by ex vivo analysis), PCI-2050 showed the highest cytotoxicity followed by PCI-2052. PCI-2051 and PCI-2053 were much less active. Interestingly, a similar pattern of cytotoxicity and drug fluorescence was noted in cells treated in culture and those collected following in vivo treatment. Because spectral properties of various sapphyrins may be intrinsically different and may also depend on other variables, such as cellular context, drug fluorescence measured by flow cytometry cannot be used to precisely quantify the uptake of different sapphyrins. However, measurements of drug fluorescence in tumor cells and tissues, coupled with determination of drug cytotoxicity, allowed us to conclude that subtle changes in the alkyl substituents on the bipyrrole moiety resulted in significant changes in cytotoxic activity and tissue distribution.
We selected PCI-2050 as the most promising candidate to evaluate for antitumor efficacy in xenograft models because it (a) gave rise to superior tumor cell uptake in vivo and subsequent death of explanted tumor cells, and (b) displayed lower toxicity than PCI-2052. In particular, no decrease in the peripheral WBC count, RBC count, or platelet count was observed in PCI-2050–treated mice, leading us to conclude that bone marrow suppression is not a prominent side effect of this sapphyrin. Further, there were no alterations in serum markers to suggest renal or hepatic toxicity. PCI-2050 displayed dose-dependent antitumor efficacy in both minimal tumor (animals treated before tumor was palpable) and established tumor (palpable tumor) models, reducing tumor growth by up to 60% to 75%. Scheduling and optimization of drug dosage has not been adequately investigated to determine if that would enhance antitumor efficacy.
Because the mechanism(s) of action of sapphyrins is currently unknown, we could not chemically manipulate sapphyrins based on a specific molecular target or pathway. We were able to bypass the need for a defined target by improving on the tumor-localizing potential of sapphyrins to enhance antitumor activity. Sapphyrins are particularly attractive because they are readily amenable to chemical manipulation. Accordingly, efforts are currently under way to more thoroughly evaluate the efficacy and toxicity profiles of these and other sapphyrins and to optimize their potency through synthesis of further derivatives and preclinical analysis of biodistribution and activity.
Footnotes
Grant support: NIH grant GM0588907 (J.L. Sessler) and funds from the Texas TD & T program.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5/ 4/06; revised 8/16/06; accepted 9/25/06.
References
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