review phenothiazine

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Running head: Perspective on cancer selective tissue staining by phenothiazines and related compounds Summary: Methylene blue, toluidine blue O, thionin, (phenothiazines) as well as Neutral Red (phenazine) and Nile Blue (phenoxazine) are tricyclic heteroaromatic cationic lipophilic dyes selectively taken up by cancer cells in living (unfixed) tissue; they have been used both for the demarcation of tumor cells within tissue and evaluated as agents for potentially treating cancer by photodynamic therapy. A review of the experimental and clinical literature of phenothiazine dyes and the related phenoxazine and phenazine dyes suggests that the selectivity for staining tumors maybe attributed in some part to their being facultative substrates for the multidrug transporter (MDR) system. The plasma membrane transporter system that is responsible for multidrug resistance plays a role in normal cell physiology and is over expressed in tumor cells. As an influx substrate for the multidrug transporter, the selective and preferential uptake of these planar tricyclic heteroaromatic molecules may reflect the enhanced MDR transporter system as a physiological tumor marker. Selective uptake by tumor cell transporters may also be modulated by the cellular redox status which plays a key role in carcinogenesis and may also be correlated to the MDR system. Further studies and understanding of this correlation will be useful for future photodiagnostic and phototherapeutic applications of this generic class of compounds. Introduction The phenothiazinium chromophore Toluidine blue O was first notably employed as an in vivo cancer imaging stain for the delineation of dysplasia and carcinoma in situ of the uterine cervix by Richart (1963). 1 This staining method served as a practical solution for the difficulty of selecting punch biopsy sites from the exposed portion of the cervix that may contain neoplastic epithelium. The choice of toluidine blue was based on selecting a common biological stain that would provide good contrast of the tissue for colposcopy. Previously, the only clinical test available was the Schiller test which consists of the application of iodine and iodide to the cervix. The Schiller test as an adjunct to cervical biopsy has two limitations; the benign Schiller-positive area adjacent to an area of neoplasia, and the well-differentiated glycogen-containing Schiller-negative area of dysplasia. In a study of 200 patients comparing the toluidine blue staining test to colposcopy more than 95 percent correlated to the distribution of intraepithelial neoplasia. This reported correlation was attributed to toluidine blue generally being recognized as a nuclear stain; the in vivo staining of the cervical epithelium appeared “clinically, to be closely related to the number of nuclei per unit area.” 1

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Page 1: Review Phenothiazine

Running head:

Perspective on cancer selective tissue staining by phenothiazines and related compounds

Summary:

Methylene blue, toluidine blue O, thionin, (phenothiazines) as well as Neutral Red (phenazine) and Nile Blue (phenoxazine) are tricyclic heteroaromatic cationic lipophilic dyes selectively taken up by cancer cells in living (unfixed) tissue; they have been used both for the demarcation of tumor cells within tissue and evaluated as agents for potentially treating cancer by photodynamic therapy.

A review of the experimental and clinical literature of phenothiazine dyes and the related phenoxazine and phenazine dyes suggests that the selectivity for staining tumors maybe attributed in some part to their being facultative substrates for the multidrug transporter (MDR) system. The plasma membrane transporter system that is responsible for multidrug resistance plays a role in normal cell physiology and is over expressed in tumor cells. As an influx substrate for the multidrug transporter, the selective and preferential uptake of these planar tricyclic heteroaromatic molecules may reflect the enhanced MDR transporter system as a physiological tumor marker. Selective uptake by tumor cell transporters may also be modulated by the cellular redox status which plays a key role in carcinogenesis and may also be correlated to the MDR system. Further studies and understanding of this correlation will be useful for future photodiagnostic and phototherapeutic applications of this generic class of compounds.

Introduction

The phenothiazinium chromophore Toluidine blue O was first notably employed as an in vivo cancer imaging stain for the delineation of dysplasia and carcinoma in situ of the uterine cervix by Richart (1963).1 This staining method served as a practical solution for the difficulty of selecting punch biopsy sites from the exposed portion of the cervix that may contain neoplastic epithelium. The choice of toluidine blue was based on selecting a common biological stain that would provide good contrast of the tissue for colposcopy. Previously, the only clinical test available was the Schiller test which consists of the application of iodine and iodide to the cervix. The Schiller test as an adjunct to cervical biopsy has two limitations; the benign Schiller-positive area adjacent to an area of neoplasia, and the well-differentiated glycogen-containing Schiller-negative area of dysplasia. In a study of 200 patients comparing the toluidine blue staining test to colposcopy more than 95 percent correlated to the distribution of intraepithelial neoplasia. This reported correlation was attributed to toluidine blue generally being recognized as a nuclear stain; the in vivo staining of the cervical epithelium appeared “clinically, to be closely related to the number of nuclei per unit area.”

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Based on this apparent correlation, the toluidine blue technique was also applied to the in vivo delineation of oral intraepithelial neoplasia (Niebel and Chomet 1964).2 Using a 1% acetic acid pre-rinse followed by 1% aqueous toluidine blue and subsequent 1% acetic acid rinsing, 20 patients with intraoral lesions were evaluated. Eleven were histologically diagnosed while the remaining nine, with benign traumatic or hyperplastic lesions, served as controls. The results for oral epithelial neoplasia were consistent with Richart’s results for neoplastic cervical epithelium. Early malignant lesions were sharply delineated from the non-neoplastic epithelium and stained more intensely. Non-malignant traumatic ulcers frequently confused the picture; “the stain is retained at the site of inflammatory infiltrate that is associated with eroded mucosa.”

This apparent selectivity for neoplastic epithelial tissue by toluidine blue when stained in vivo was attributed to the dye’s property as a nuclear stain: “the intensity of the stain is correlated with the nuclear density, and the severity of the neoplastic process can be gauged roughly from the shade of blue, which ranges from a pale royal blue in minimal dysplasia, to a very intense royal blue in carcinoma in situ, or early invasive carcinoma”.

The use of toluidine blue for staining oral cancer has been reported in numerous subsequent studies. (3 4 5 6 7 8 9 10 11) In evaluating the use of toluidine blue specifically as a screen for oral cancer, 16 studies conducted between 1964 and 1984 were compiled from the literature.12 The results from 12 were subjected to a “meta-analysis” comprising 2937 subjects. The statistical paradigms employed in the meta-analysis of these sundry reports yielded a sensitivity of toluidine blue in detecting oral cancer of 97.7 to 93.5%. The specificity ranged from 92.9% to 73.3%. The analysts attribute the selectivity of the toluidine blue test to “the staining of acid tissue components such as DNA and RNA”. It was determined that if (toluidine blue) is used to screen high-risk populations, the likelihood of a false negative finding is extremely low, whereas false positive results will be relatively numerous. However, given the high sensitivity of the test, the absolute number of false positive tests will be small. Further analysis is needed to evaluate the economic costs of false positives and false negatives, versus the value of identifying true positives at an early stage.

Toluidine blue has also evaluated as a topical stain for diagnosing skin cancer (Sugerman, et al. (1970)13. Applying 1% toluidine blue to a variety of skin lesions – before biopsy or excision and using a pre-rinse of 1% acetic acid, the time of staining was not controlled. A post-rinse comprised of acetic acid, saline, or water was used. 17/18 squamous tumors and 6/7 premaligant squamous lesions stained positively. No positive stains were recorded for 18 basal cell carcinomas, although “several large” basal cell carcinomas with central ulceration took up the dye in the ulcerated area only but was not interpreted as positive staining. The study noted that extension of toluidine blue staining beyond grossly visible margins sometimes occurred and that “complete excision of the tumor required removal of all the stained tissue”.

Intraoperative staining of fresh mucosal specimens from head and neck surgery for margin assessment was reported using a toluidine blue procedure developed from its extensive use in oral cancer screening (Durante, et al. (1986)14. Noting that intraoperative frozen sections do not always encompass the entire perimeter of the excised specimen, the correlation of intraoperative toluidine blue staining with permanent sections was excellent.

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Compound R1 R2 R3 R7 R8 R9

Methylene Blue H H NMe2 NMe2 H HAzure B H H NMe2 NHMe2 H HAzure A H H NMe2 NH2 H HAzure C H H NHMe2 NH2 H HThionin H H NH2 NH2 H HToluidine Blue H Me NMe2 NH2 H HDimethyl Methylene Blue Me H NMe2 NMe2 H MeNew Methylene Blue H Me HNEt HNEt Me H

Methylene blue is a phenothiazine dye closely related to toluidine blue and differs by not having a methyl group on the ring at position 2, and both phenylamine groups are tertiary, i.e. dimethylated (diagram). Because toluidine blue has a primary ring amino group, it is able to undergo quinoneimine formation which may account for its being “metachromatic”. (diagram) Metachromasia is sometimes attributed to dimer and polymer formation, which broadens the absorption peak toward the blue end of the visible spectrum and is especially notable for methylene blue. However, for toluidine blue as well as thionin and the azures, the ability to undergo neutral quinoneimine formation may confer a shift in the absorption spectra that accounts for the noticeable green staining of RNA by azure B (see Wainwright and Amaral, (200553); a pH effect rather than resulting from dimerization15.

Methylene blue has long been used clinically to reverse methemoglobinemia caused by genetic deficiencies and metabolic poisoning.16 It has been used as an antidote to paraquat poisoning17, to maintain blood pressure in septic shock18 and during orthotopic liver transplantation19 and to treat ifosamide encephalopathy20. In fact, methylene blue is the oldest known synthetic antimalarial drug, its clinical efficacy having been reported in 189121 but because the dye stains tissue of the subject receiving it, it was not seriously considered for clinical use at that time. However, the general staining properties are similar to toluidine blue, and its clinical history and relative safety suggested its use for in vivo staining applications

Methylene blue at 0.2% was used as an in vivo staining test for bladder cancer (Fukui et al. 1983)22. Applied for 1 hour in the bladder of 129 patients grade 1 tumors were poorly stained or unstained in 86% while grade 2 and grade 3 tumors picked up the stain in 74 and 96 % of the cases. While normal mucosa did not pick up the stain, nonpapillary in situ and microinvasive carcinomas did so frequently. Moderate dysplasia stained in half the cases. The intensity of the stain could be histologically correlated with the degree of anaplasia. Chronic cystitis with an

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inflammatory infiltrate took up the stain as well. In a similar study published shortly after these results, the method of staining with methylene blue was modified by reducing the concentration from 0.2 to 0.1%, incorporating saline in the staining solution, and reducing the contact time from 1 hour to five minutes (Gill et al. 1984).23 The bladder was also irrigated with saline whereas distilled water was used in the Fukui study. Thereafter, either endoscopic or open surgery was performed. The transitional cell carcinomas in 45 of 48 patients bound methylene blue to the surfaces of the tumors but not to normal urothelium. Higher grade tumors usually bound the dye more extensively than lower grades. The three patients, whose tumors did not bind methylene blue, had received previous chemotherapy, which might account for their being falsely negative. Carcinoma in situ and dysplasia did stain blue. Areas of hyperplasia and cystitis, however, did not bind methylene blue. In vivo intravesical staining with methylene blue has been a simple and safe procedure which has enhanced the endoscopic localization for biopsy and fulguration/resection of transitional cell carcinomas.

Methylene blue has been used to selectively stain intestinal metaplasia in Barrett’s esophagus (Canto et al. 1996).24 Reporting an overall accuracy of 95% for MB detecting specialized columnar epithelium, the technique for upper endoscopy involved first spraying a 10% solution of N-acetyl-cysteine (to remove superficial mucus) followed immediately by a 0.5% solution of MB followed by tap water rinsing. A subsequent report (Canto (2001)25) examined 551 biopsies from 47 patients with biopsy-proven Barrett’s esophagus and 48 sections from five surgical specimens with Barrett’s esophagus and dysplasia and early adenocarcinoma. The accuracy of ex vivo and in vivo methylene blue staining for specialized columnar epithelium was 87% and 90% respectively.

The results in these studies using methylene blue parallel those of toluidine blue for oral cancer detection and cervical neoplasia. The phenothiazine dyes toluidine blue and methylene blue have been successfully used as selective stains in vivo for demarcating tumor from normal cells in tissue. The underlying premise for this use, as suggested in some of the cited reports, is that these basic dyes are selective for nucleic acids and that nuclear material is over expressed in cancer cells. This follows from their cytochemical staining properties where they preferentially bind to nucleic acids and mucopolysaccharides in fixed thin sections. Their use as a nucleic acid stain for fresh tissue or in vivo staining for cancerous cell localization however overlooks the need for these dyes to first traverse the cell membrane before associating with DNA or RNA.

Mechanism of Staining

An early study on the mechanism of staining with toluidine blue, as used in a screening test for squamous cell carcinoma, used sodium silicotungstate as a stabilizer for the dye for subsequent conventional fixation (Herlin, et al. (1983)26). While the exact procedure used is unclear, the results suggested that selectivity of toluidine blue for cancerous esophageal mucosa is due to “structural disorganization allowing the penetration of toluidine blue into the cancerous tissue and the affinity of the dye for nuclear material”. It was further observed that inflammatory cells accumulated the dye as well as cancer cells and thus cellular specificity is not the determinative mechanism of the staining. “The prime step factor seems to be the tissue and membrane permeability”.

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Using molybdate to precipitate methylene blue after supravital staining, Muller et al. (1996)27 evaluated electron microscopy data on staining of dendritic cells in the epithelia of the soft palate and skin of the mouse after dye injection. The ultra-structural details were compared with corresponding light microscopy findings. Methylene blue stained tissue was fixed by immersion in a paraformaldehyde-glutaraldehyde solution containing phosphomolybdic acid and the dye precipitate was stabilized by ammonium heptamolybdate. The light microscopy investigation revealed that selective staining of dendritic cells depended on the presence of ambient oxygen. Nerve fibers were stained within the epithelium as well as the subepithelial connective tissue. At the electron microscopic level, the dye was clearly identified as an electron dense precipitate that accumulated primarily within the cytoplasm near the plasma membrane. Furthermore, it was bound to the chromatin of the nuclei. No significant staining of mitochondria or other organelles was seen. Within the cytoplasm, the oxygen-dependent binding sites were hypothesized to be associated with heme proteins that attract both the dye in its reduced lipophilic leuco form and oxygen, followed by generation of oxygen radicals and a reoxidation of the leuco form to the cationic blue dye.

Lampidis et al. (1985)28 noted that lipophilic positively-charged compounds are facilitated across biological membranes by the transmembrane potential of intact cells. As one example they showed that one such compound, rhodamine 123, was selectively toxic toward a variety of transformed (carcinoma), epithelial cells in vitro. They suggested one mechanism that could account for the selectivity of this agent would be a difference in the plasma membrane potential between normal and carcinoma cells, reporting that a significantly higher transmembrane potential has been found in a pair of carcinoma (83 mV for human breast and -99 mV for human cervix) as compared to normal (-56 mV for marsupial kidney and -48 mV for monkey kidney) epithelial cell lines. They also identified 3 other positively-charged lipophilic compounds, safranin 0, rhodamine 6G and tetraphenylphosphonium chloride (TPP+), which show selective toxicity toward carcinoma cells in vitro, while an uncharged lipophilic analog, rhodamine 116, does not. Their data suggest that the higher plasma membrane potential of carcinoma cells may in part contribute to the preferential accumulation and selective toxicity of the lipophilic cationic compounds they examined. An extension of this concept to an in vivo environment could lead to a class of cationic compounds which selectively exploit differences between normal and carcinoma cells.

Selectivity

Modica-Napolitano and Aprille (2001)29 proposed that the selectivity displayed by the general class of delocalized lipophilic cations (DLCs) offer a distinct mechanistic approach to cancer chemotherapeutic drug development strategies. Traditional chemotherapies, aimed at DNA replication in rapidly dividing cells, have achieved only limited success in the treatment of carcinomas due largely to their lack of specificity for cells of tumorgenic origin. Therefore they suggest DLC’s to investigate treatment strategies aimed at novel cellular targets that are sufficiently different between normal cells and cancer cells so as to provide a basis for selective tumor cell killing. Delocalized lipophilic cations (DLCs) are concentrated by cells and into mitochondria in response to negative inside transmembrane potentials. The higher plasma and/or mitochondrial membrane potentials of carcinoma cells compared to normal epithelial cells account for the selective accumulation of DLCs

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in carcinoma mitochondria. Since most DLCs are toxic to mitochondria at high concentrations, their selective accumulation in carcinoma mitochondria and consequent mitochondrial toxicity provide a basis for selective carcinoma cell killing. Several of these compounds have already displayed some degree of efficacy as chemotherapeutic agents in vitro and in vivo (for example the work of Lampidis et al. (1985)28. The effectiveness of DLCs can also be enhanced by their use in photochemotherapy or combination drug therapy. Discovery of the biochemical differences that account for the higher membrane potentials in carcinoma cells is expected to lead to the design of new DLCs targeted specifically to those differences for even greater selectivity and efficacy for tumor cell killing. The phenothiazines, phenoxazines, and phenazines are of this class.

The empirically observed selectivity of the thiazine dyes for cancerous cells, putatively based on their specific interaction with nucleic acids and coupled with their strong absorbance in the red portion of the visible spectrum have made them attractive potential agents for photochemical studies. The photodynamic reaction of methylene blue with DNA was systematically reported as early as 196230 A number of subsequent studies have been published on the photosentistizing effects of tricyclic heteroaromatic cationic dyes such as methylene blue and toluidine blue and their use as potential phototherapeutic agents. (31 32 33 34 35 36 37 38 39 40)

Photosensitization combined with cancer selective uptake suggests other DLC compounds to be similarly been evaluated.

Neutral Red

Neutral Red (diagram), 3-amino-7-dimethyleamino-2-methylphenazine, is a weakly cationic, nontoxic vital dye closely related to methylene blue which is widely used as a histology stain for proliferating cells. While Neutral Red stains both normal and cancer mitotic cells, its uptake was found to be distinctly higher by living mitotic cancer cells (Sit et al. 1992).41 The uptake of Neutral Red was also found to be greatly enhanced by a brief exposure to epidermal growth factor (EGF) the receptors of which have been cited as a causal factor in neoplastic cells. Suggesting that the uptake of neutral red by live cells could provide additional criteria for the detection of carcinogenicity, it was also explored as a photodynamic agent to selectively kill cancer cells (VanderWerf, et al. (1997)42. The rapidity of cell uptake of neutral red reported by Sit, et al. is particularly striking; the extraordinarily rapid uptake, usually against a concentration gradient, is a characteristic observed in a number of reports.

Derivatives of Nile Blue (diagram) – tricyclic heteroaromatic cationic dyes of the phenoxazine class - also localize selectively in animal tumors. In evaluating various phenoxazine derivatives for singlet oxygen yield and photokilling potency as

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photosensitizers in a human bladder carcinoma cell line, Lin et al. (1991)43 noted that the uptake of Nile blue derivatives by cells in culture exhibited a pattern of rapid initial uptake followed by a gradual increase in cellular dye contents. The uptake reportedly did not correlate directly with the individual pKa values or hydrophobicities of the derivatives, indicating that the structural modifications that increased singlet oxygen yields did not significantly alter the uptake and retention of Nile blue derivatives. They suggested the highly concentrative uptake by and slow efflux from dye-loaded cells was consistent with an active mechanism for the cellular accumulation of these dyes. On the other hand, the retention of the compounds was directly proportional to dye concentration in the medium over a 1000-fold range of concentrations, and the uptake could proceed at temperatures below 2 degrees C; these observations excluded endocytosis or a carrier-mediated mechanism for the uptake. They hypothesize that Nile blue derivatives transport across the cell membrane possibly as deprotonated forms and, upon entering the cell, either partition into lipophilic areas of the cell membranes and/or become sequestered in certain intracellular organelles. Suggesting here that the high accumulation of the Nile blue derivatives may be the result of dye aggregation, partitioning in membrane lipids, and or sequestration in subcellular organelles, they followed up with a study presenting an investigation of the subcellular localization and mechanism of accumulation of these dyes. 44 Notably they found that treatments with agents affecting the membrane pH gradient reduced the uptake and enhanced the efflux of dyes, while agents that alter cellular membrane potentials had no effect on dye accumulation. The uptake of the dyes was partially inhibited by inhibitors of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results showing that the cellular uptake of these dyes is highly concentrative and proportional to the extracellular dye concentration over a wide range, are consistent with their hypothesis that the dyes are mainly localized in the lysosomes via an ion-trapping mechanism.

Using EMT-6 murine sarcoma cells, benzophenoxazine derivatives were also characterized by a similar uptake/efflux pattern in vitro consisting of a rapid and extensive cellular accumulation followed by a slow efflux rate (Cincotta, et al. 1993) 45. Contrary to their rapid uptake, 50% of the accumulated derivative is retained intracellularly after a 6-h period in dye-free medium. Video-enhanced fluorescence microscopy corroborated the rapid uptake measurements as well as indicating the intracellular localization of the dyes in both living and thermally inactivated cells. Low extracellular dye concentrations (0.05 µM) resulted in a punctate fluorescence pattern in the perinuclear region, while higher dye concentrations (> 0.1 µM) lead to additional fluorescence in the cytoplasm, cytomembranes, and other organelles but apparently not the nucleus. Absorption spectrometry revealed that living cells rapidly reduce the dyes to their colorless leuco form (photoinactive) if oxygen is not readily available in the environment. It is shown that the cellular reduction is an enzymatic process and that an oxygen-free and cell-free medium containing both the coenzyme NADH and the hydride transfer enzyme diaphorase is capable of reducing the dyes to the colorless leuco form.

The Cincotta (1994)46 team again used their EMT-6 murine sarcoma cell line to explore the photodynamic effects of a benzophenothiazine, 5-ethylamino-9-diethylamino benzo[a] phenothiazinium chloride (diagram), in vitro as well as in vivo. The selectivity of the phenothiazine derivative for tumor was shown by spectrophotometrically measuring dye tissue distribution after s.c. injection. The

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ratios of dye in the tumor to the dye in surrounding skin and gastrocnemius muscle 8 h following dye injection were 4:1 and 8:1, respectively. This distribution of a phenothiazine (methylene blue) derivative was almost simultaneously reported (Peng, et al. 1993)47 in tumor and normal tissue of rats. Using Wistar rats bearing fibrosarcoma (Leeds ovarian tumor) after I.V. injection similar kinetics of accumulation and elimination were noted as determined by fluorescence. The tumor:skin and tumor:muscle ratios of the methylene blue derivative fluorescence intensity were found to be 9 and 4, respectively, 4 h after intravenous injection, indicating selective uptake of MBD by the tumor tissue. Cincotta et al. further suggest that is it the redox properties of the dye coupled with the differing metabolic states of the tumor and (surrounding) skin which increase the amount of photoactive oxidized dye present in the tumor and decrease it in the skin.

Two interesting observations resulting from the published studies of the Cincotta group are made: first that the phenoxazine derivatives cross the (cell) membrane as deprotonated (leuco) forms and are subsequently reoxidized; further, that the uptake is at least partially energy dependent being partially inhibited by inhibitors of oxidative phosphorylation: and secondly, that the photodynamic potency (and thus the photodiagnostic “potency”) of a phenothiazine photosensitizer may be dependent on the ratio of the oxidized (chromophore) to reduced (leucophore) species determined by metabolic differences between normal and tumor cells.

The oxidation reduction (redox) properties of methylene blue are generally known if simply from its use in the “methylene blue reduction test” for milk. The cellular reduction of methylene blue and toluidine blue has been reported using bovine endothelial cells in culture (Bongard et al. 1995).48 The uptake of methylene blue (MB), and toluidine blue O (TBO) by bovine pulmonary arterial endothelial cells grown on microcarrier beads was detected as a decrease in the concentration of dye after these thiazine dyes were added to the medium surrounding the cells. Because the reduced forms of these dyes are much more lipophilic than the oxidized forms, the possibility that reduction of the dyes at the cell surface might have preceded the uptake by the cells was studied. The ability of the cells to reduce a toluidine blue O-polyacrylamide polymer (TBOP), which was too large to enter the cells in either the oxidized or reduced form, was evaluated. The TBO moieties of the polymer were reduced by the cells, indicating that the dye did not have to enter the cells to be reduced and that reduction can occur at, or near, the cell surface. The rate of TBOP reduction was found to be about the same as the rate of uptake of the monomeric dyes, indicating that the cell surface reduction mechanism had a sufficient capacity to account for the monomer uptake by the cells. It was also found that ferricyanide ion, which also did not permeate the cells, was reduced by the cells and that external ferricyanide inhibited the monomeric MB uptake. The results with ferricyanide were also consistent with the concept that the monomeric thiazine dyes are reduced at the cell surface before the more lipophilic reduced forms are taken up by the endothelial cells.

Merker et al. (1997)49 further evaluated this hypothesis that the accumulation of thiazine dyes, such as methylene blue, by cultured bovine pulmonary arterial endothelial cells involves reduction on the cell surface, followed by diffusion of the lipophilic reduced form of the dye into the cells and intracellular reoxidation to the relatively membrane-impermeant hydrophilic form. The specific question they addressed was whether inhibition of methylene blue uptake by cyanide and azide is

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via inhibition of extracellular reduction or inhibition of intracellular reoxidation. They used the cell membrane-impermeant ferricyanide ion as a secondary electron acceptor to measure the extracellular reduction of methylene blue independently from its uptake by the cells. In addition, toluidine blue O, incorporated into an acrylamide polymer so that it could not permeate the cells in either the reduced or oxidized forms, was used to examine the effects of cyanide and azide on the extracellular reduction. Microscopic observations of the effect of the inhibitors on the intracellular accumulation of methylene blue were also made. The results showed that the reduction and intracellular sequestration are separate processes and that, in doses that inhibited intracellular reoxidation, and therefore uptake and sequestration, neither cyanide nor azide had an inhibitory effect on extracellular reduction. The intracellular distribution of the observable oxidized form of the dye was consistent with oxidation of the reduced dye within subcellular organelles. The demonstration that extracellular reduction and intracellular sequestration are separate is consistent with the hypothesized sequence of events.

In their discussion of these results, they note that trans-plasma membrane electron transport is apparently a normal function of most, if not all, cells. In mammalian cells, the physiological roles for the electron transport are not all fully understood. Plasma membrane NADPH oxidase activity plays an important role in the respiratory burst in neutrophils, and various functions have been attributed to the reductases on the surfaces of hepatocytes and various tumor cell lines. In general, the processes of plasma membrane electron transport from intracellular electron donors, such as NADPH or NADH, to external acceptors have been found to be cyanide and azide insensitive. Thus, in this regard, the endothelial cell plasma membrane thiazine reductase appears to be similar to other trans-plasma membrane electron transporters that have been described previously50.

In a study of the interaction of methylene blue and blood cells, Sass (1967)51

showed that methylene blue was taken up by erythrocytes in suspension with a 150 to 200 fold increase over extracellular concentrations. It was proposed that the oxidized form of methylene blue (MB+) freely diffuses across the erythrocyte membrane and is then rapidly reduced to the leuco form (MBH) which is then trapped in the cell.

Based on the results of Merker and colleagues48 49 in pulmonary arterial endothelial cells, May, Qu, and Cobb (2004)52 reported that in erythrocytes there is extracellular reduction of MB+ (contrary to the Sass theory), that MB+ accumulates as MB+ and MBH in erythrocytes as a function of extracellular concentration. The transmembrane reduction of the extracellular MB+ seems to be mediated by a protein because it is sensitive to inhibition by thiol reagents and enzyme digestion in erythrocyte ghosts devoid of cytoplasm. Further, they showed that when reduction of MB+ to MBH was prevented by extracellular ferricyanide there was little or no uptake of the dye as MB+ by the cells. In the absence of ferricyanide, the cells took up 71% of the dye (at 20µM) after 30 minutes; approximate half of that occurring within the first five minutes. The cells concentrated free MB+ 14-fold, free MBH 4.3 fold and bound dye (presumed ionic) 18-fold confirming accumulation of both free and reduced methylene blue against a concentration gradient. May and colleagues further confirm the involvement of a membrane protein in MB+

reduction, a “thiazine reductase”. May, Qu, and Cobb make the point that “entry of the positively charged MB+

across the cell membrane will likely be slower than that of the more lipophilic MBH”; their results suggest the positively charged MB+ is not taken up by erythrocyte cells

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to any appreciable extent - if at all. This observation together with that of Merker and colleagues for the pulmonary endothelial cell, and the similar observations of the reduction of a Nile Blue derivative before cell uptake by a bladder carcinoma line, and for a benzophenoxazine derivative in a murine sarcoma cell line - strongly suggests that a mediated reduction of the cationic tricyclic heteroaromatic compounds is requisite for any significant selective uptake, staining, and localization within tumor cells.

In a comprehensive and very informative review of “phenothiazines in antimalarial research”, Wainwright & Amaral (2005)53 note that methylene blue is not an efficient stain for normal red blood cells. The reason given for this is the “hydrophilic nature of the methylene blue molecule”. This appears in part to be at odds with the results of May et al. Never-the-less it is noted the activity of methylene blue against intra-erythrocytic forms of Plasmodium spp. is reportedly the highest of the series. The putative reason given for this, citing Cranston et al. (1984)54, is that the parasite installs transporters in the red cell membrane increasing permeability. The presence of such transporters allows ingress of hitherto excluded molecules, including hydrophilic dyes such as methylene blue. Again, this would appear to be at odds with the findings of May et al. where, at least for erythrocytes, there is a “built-in” transporter system that effectively concentrates the dye against a gradient; however, it is transported across the membrane after mediated reduction as the less hydrophilic LMB form.

There has been considerable recent interest in the red blood cell uptake of phenothiazinium dyes for the photodynamic decontamination of donated blood and blood products due to the AIDS pandemic and the transmission of HIV through the blood supply. Methylene blue is currently being employed for the decontamination of plasma by various European blood collection/treatment agencies55, and thionin has recently been proposed for the similar treatment of platelets56. The American Red Cross is sponsoring similar research57 58 59 in the United States. Nevertheless, recent reports show that exposure of red cells to photodeactivation with methylene blue causes increased lysis when refrigerated over time60.

The results of May et al. showing that erythrocytes have a built-in redox transporter system for which methylene blue is an apparent substrate requires an “approach” re-evaluation for using phenothiazinium (or related tricyclic heteroaromatic) compounds for whole blood photodynamic decontamination. Wagner, et al. (2003) 61 employ quinacrine (structure diagram) together with dimethymethylene blue (DMMB) for enhanced photodynamic decontamination of vesiclular stomatitis virus inactivation with diminished hemolysis of red cells. Quinacrine acts as a substrate analog (for transmembrane transport) which cannot easily be reduced. It may block the binding, reduction, and internalization of dimethymethylene blue by erythrocytes – thus minimizing membrane damage during photolysis. Here, Wagner, et al., are acknowledging that the uptake of DMMB may first require reduction. The use of quinacrine resulted in an increased extracellular concentration of DMMB, increased extracellular viral inactivation kinetics, and decreased hemolysis during 1-6 degrees C storage without alteration of quinacrine absorption properties. These results collectively suggest that despite its recognized affinity for viral nucleic acid, DMMB also binds to RBC membranes and that the bound dye is, in part, responsible for photoinduced hemolysis. Given the broad range of pharmacological effects displayed by quinacrine (an acridine derivative), other non-reducible non-photosensitive membrane transporter competitors may be preferred to accomplish a similar result.

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In analyzing the mode of action of the various antimalarial drugs, Wainwright and Amaral point out that much of the perceived wisdom is “inherited”. For example after showing that some of the simpler aminoacridines intercalate into nucleic acids, this was taken to be the “mode of action” and that similar structurally related compounds, such as methylene blue and toluidine blue also effect their mode of action at the nucleic acid level. There may be some merit to this certainly, especially with respect to the photodynamic effects of methylene blue (Floyd et al.62, Tuite & Kelly63). But with regards to antimalarial properties, it may be that the oxidation-reduction properties of the phenothiazaine/ phenothiazinium nucleus may hold the key – inducing an oxidative stress on the system. This is far more likely given that the antimalarial effects are not photoinduced – at least clinically in erythrocytes. The greatly increased hexose monophosphate shunt (HMPS) activity in parasitized red blood cells – some 78 times greater than in normal red blood cells- may facilitate significantly higher levels of reductive uptake of methylene blue64. Reduced methylene blue is readily oxidized back to the strongly absorbing MB chromophore, and as noted by May et al.52, both MB+ and MBH are concentrated in erythrocytes. The presence of MB+ in erythrocytes in this and a previous study65, as well as in endothelial cells49, shows that intracellular MBH undergoes oxidation with trapping of MB+ in the cells. Oxidation of MBH by Fe3+ in hemoglobin66 and in Fe3+-containing enzymes is responsible for the beneficial effects of MB+, such as reduction of methemoglobin and activation of guanylate or nitric oxide synthases. If these pathways are saturated, however, MBH will react with molecular oxygen to generate superoxide and other reactive oxygen species (ROS) that can be damaging to the cells67 68. This mechanism is present in erythrocytes, because MB+ concentrations increase oxygen uptake69 70 and oxidize ascorbate and GSH. May et al. (2003)71 also recently reported that a similar range of MB+ concentrations also oxidized ascorbate and GSH in cultured endothelial cells, associated with oxidation of dihydrofluorescein. The latter provides strong evidence for the generation of excess ROS72. This study shows in addition that excess ROS are due not as much to the initial reduction of MB+ at the cell surface as to its redox cycling. Thus antimalarial activity may be due to ROS on reduction.

Wainwright and Amaral (2005)53 also explore the issue of resistance to the antimalarials chloroquine and related quinolines in P. falciparium which is thought to occur by inhibition of drug accumulation inside the parasite food vacuole – possibly due to an upregulated P-glycoprotein type analog. The authors note that chloroquine resistance can be reversed by treatment with verapamil, an inhibitor of calcium binding by calmodulin like proteins. Verapamil also inhibits ABC transporters by denying calcium to calcium dependent ATPase. Chlorpromazine, a derivative of methylene blue, also interferes with calcium binding and they postulate it would follow that both methylene blue and toluidine blue might reverse drug pump resistance mediated by antibiotic efflux pump mechanisms that depend on calcium dependent ATPase.73

Reviewing the biochemical, cellular, and pharmacological aspects of the multidrug transporter system, Ambudkar et al. (1999)74, citing Ford et al. (1989)75, note that none of the known modulators (of the MDR transporter system) inhibit ATP binding. Modulators such as verapamil are substrates of the transporter and hence inhibit the transport function in a competitive manner by interrupting the catalytic cycle of P-gp (p-glycoprotein or the “multidrug transporter). In fact, Ford75 reported a study of the structural features that determine the activity of phenothiazines and

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related drugs for inhibition of cell growth and reversal of multidrug resistance, identifying the class – broadly – as competitive MDR substrates.

Mechanistically, P-gp modulators are either high-affinity substrates of the pump or are efficient inhibitors of ATP-dependent transport by P-gp. The ability of a modulator to stimulate or inhibit the ATPase activity in isolated membrane preparations indicates direct interaction of the compound with P-gp modulators. Although enough structural diversity exists among the P-gp modulators so that no consensus structure can be defined, within each class of drugs structure-activity relationships have made it possible to define certain chemical features of these compounds that seem to be essential for functional interaction with P-gp. Several different classes of compounds with tricyclic ring nuclei have been studied to identify important structural features responsible for anti-MDR activity. Among these compounds, which include phenoxazine, phenothiazine, phenoxazone, resurfin acetate, xanthene, xanthene carboxylic acid, acridine carboxylic acid, and 1,10-phenanthroline, phenoxazine proved to be the most active agent for sensitizing MDR cells to vincristine and vinblastine. Hydrophobicity does not correlate with the ability of this series of compounds to modulate the accumulation of vincristine and vinblastine. Knowledge of the anti-MDR features of tricyclic ring– containing compounds has been further extended in studies by Ford et al. 75 with derivatives of phenothiazine and thioxanthene. Results from their studies indicate that increasing hydrophobicity of the phenothiazine and thioxanthene nucleus increases their potency against MDR. A more recent study by Hendrich et al. (2003)78 suggests that there is little correlation with the membrane perturbing properties (hydrophobicity, lipophilicity) and P-gp modulation.

In modulator binding to P-gp, a clear lack of conserved elements of molecular recognition is apparent, which complicates the structural definition of the MDR pharmacophore. Furthermore, much evidence suggests that the drug binding sites on P-gp are multiple and complex. Nevertheless, information compiled from various structure-activity studies can be used to outline a minimum requirement for anti-MDR activity.

Because P-gp is able to recognize drug molecules directly from the membrane bilayer, the overall hydrophobicity of the modulators seems to be an important, but not the sole, requirement for chemosensitizing activity. It is reasonable to state that hydrophobicity of a molecule aids interaction with P-gp by improving its chance of interaction. Because aromatic groups largely contribute to the hydrophobicity of a compound, planar ring structures seem to be a hallmark of anti-MDR compounds. However, one should not underestimate the potential of these ring structures to be involved directly in interaction with P-gp. Apart from the planar aromatic domain, presence of a basic nitrogen atom located within an extended side chain of the aromatic ring structure also seems to play a determining role in the interaction of modulators with P-gp. These structures would fit the definition of DLC’s. Tertiary amino groups increase considerably the anti-MDR potency of a compound compared with primary and secondary amines. The chemosensitizing activity increases even more if the nitrogen atom is incorporated into a nonaromatic ring structure (as in phenothiazines and thioxanthenes).

The partitioning model cited in Ambudkar et al. (1999)74 proposes that overexpression of the P-gp leads to alteration of electrical membrane potential and intracellular pH and to other biophysical characteristics of the cell. Alterations in the

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biophysical parameters of the cell perturb the intracellular level of anticancer drugs. In this model, P-gp indirectly promotes decreased intracellular drug accumulation. In the pump model, the energy of ATP hydrolysis by P-gp is utilized for the removal of drugs from cell membranes and cytoplasm analogous to the ion-translocating pumps. The pump recognizes substrates through a complex substrate recognition region or regions and directly pumps drugs out of the cell by mechanisms that are not yet well understood.

Much of the experimental data supports the drug pump model because evidence for a direct interaction of many of the substrates or reversing agents with the transporter has been obtained, including drug binding studies, photoaffinity labeling experiments, the demonstration of drug-stimulated ATPase activity in direct proportion to the ability of P-gp to transport these drugs, and a variety of amino acid substitutions in P-gp that alter its substrate specificity.

MDR substrate “agency” of phenothiazines and in particular methylene blue was more recently reported in a study (Trindade (2000) 76) where MB was found to revert MDR in resistant cells while further potentiating its photodynamic action. Using five cell lines, some of which did not express an “MDR phenotype” the authors reported no difference in sensitivity to the photodynamic action of methylene blue. To test the MDR reversion in MDR resistant cells, the cells were grown with and without MB and 60 nM of vincristine. All concentrations of MB used in cell growth showed inhibition, and all cells had significantly reduced viability after photoillumination. They concluded that whether or not cells displayed an MDR, MB photodynamic potency was a function of cell type not multidrug resistance. Further, they suggest that methylene blue rather than being a substrate for P-gp, as with hematoporphyrin derivatives, was an MDR reverser.

That methylene blue “preferentially accumulates” in tumor cells is a premise often made without actually addressing a mechanism for such preferential accumulation. Non cancerous cells are shown to accumulate methylene blue against a concentration gradient; in fact, animal studies have shown that there is accumulation of methylene blue by many cell types when it is administered either intravenously or subcutaneously (the concentration of the oxidized and reduced forms will no doubt vary by cell type and redox status as well; see 46 and 47). In the studies of Merker et al. 49 and May et al.52, the uptake of methylene blue by non-cancerous pulmonary endothelial cells and erythrocytes is mediated by some energy dependent mechanism and may involve a specific transmembrane transporter. There is no reason to believe that without extracellular reduction of methylene blue there is any intracellular uptake (by the otherwise membrane impermeable more hydrophilic form) as shown in these studies by the cell lines used. Does methylene blue act as a competitive substrate for drug uptake and influx in these cells? And is this in part the mechanism for MDR reversal by MB? And once internalized, the proposed redox cycling of LMB and MB+ – particularly at intracellular concentrations that may saturate such cycling - also compete with the energy needed for effecting drug efflux with the MDR phenotypes – or does it alter ROS signaling that may regulate the drug efflux systems. To what degree does elevated ROS reduce cell viability without regard to the effects of PDT?

Redox cycling of MB may be an energy “trap”. LMB is easily oxidized and readily serves up its electron to almost any other acceptor, especially oxygen. However, to traverse the plasma membrane, it needs to be in the reduced lipophilic form. For a hypoxic tumor cell, keeping methylene blue reduced requires a

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cytosolic redox adjustment that may preclude the efficient efflux of other MDR substrates – the competitive inhibition of drug efflux. Or, alternatively, its influx (as LMB) is so readily accomplished that accumulation of high concentrations of either the oxidized or reduced species is simply that is competes as a substrate with other MDR substrates; i.e. it may be cycled back and forth across – or within -the plasma membrane because its redox potency matches up with that on the cytosol and extracellular sides of the membrane – a redox energy trap. This is similar to Biaglow and Miller’s (2005)87 (cited infra) “futile redox cycling” mechanism for motexafin gadolinium, a new cancer chemotherapeutic compound.

Lin, et al. 44notes that a mechanism for the selective localization of Nile blue dyes in tumors, observed in earlier studies, is unknown. Lewis et al. (1949)77

indicated that there is a correlation of dye structure and selectivity of tumor staining; all compounds that stained tumor had amino or substituted amino groups both in the 3- and 7- positions. Varying the nature of the substituted amino groups in these positions resulted in compounds with marked differences in tumor staining ability. Noted as an example, the substitution in the 3-amino group with the benzyl radical yielding Nile blue 2B, which stained tumor more intensely than NBA (no 3-amino substitution). Further structural features for selective tumor staining by benzophenothiazines such as dialkylation of the 7-amino group and at least one hydrogen in the 3-amino group were also suggested. Cincotta et al. suggested that for tumor staining ability, pKa, hydrophobicity, and the ability to undergo protonation-deprotonation reactions were important to membrane transport. In the Lin study, there was no apparent correlation with these properties and dye accumulation in tumor cells.

Similar structure-activity relationship analyses have been reviewed for phenothiazines as multidrug resistance (MDR) reversers previously citing Ford et al. who studied 30 phenothiazine derivatives on MDR reversal activity and noted various modifications that modulated MDR reverser effects. Substituent effects that provided the best results were unrelated to hydrophobicity and further suggested that modulation was a result of target specific interactions.

Hendrich et al. (2003)78 studied the anti-MDR activity of six phenothiazine maleates to see if a relationship existed between the effects of the phenothiazine derivatives on lipid bilayers and their ability to modulate the multidrug resistance of cancer cells. Using differential fluorescent probe DiOC3 accumulation with flow cytometry, all of the phenothiazine maleates were effective MDR reversers; no general correlation between measured lipophilicity and anti-MDR activity was observed. This is inconsistent with the conclusions of Ford et al. and Ramu and Ramu (1992)79 suggesting that phenothiazine substitutions increasing lipophilicity also increase anti-MDR activity. The Hendrich et al. observation is supported by Klein et al. (2001)80 This study was performed to elucidate the relationship between steric factors, lipophilicity, and the potency of cationic-amphiphilic compounds to displace calcium ions from phosphatidylserine monolayers. The latter property is considered to be a substance/phospholipid affinity measure. Although the affinity of the model cationic-amphiphilic substances to the phospholipid monolayer tended to increase with lipophilicity, no general interrelation between the two properties was found. Surprisingly, the assay system (a phospholipid monolayer) was quite sensitive towards small steric changes at the 'ligand' molecules. Stereochemical factors have a considerable influence on the interaction of solutes with phospholipid membranes;

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thus questioning whether lipophilicity measures alone, without taking other molecular features into account, can meaningfully be used to explain or predict the influence of solutes on membrane-related processes and properties.

Wesolowska, et al. (2005)81 used a functional fluorescence assay to show that MRP1-mediated efflux of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) out of human erythrocytes is stimulated by the phenothiazine maleates they identified as P-glycoprotein inhibitors (Hendrich et al. (2003) ).

MRP1 is a multidrug resistance associated protein that is a member, with P-glyocprotein, of the ABC transporter family (ATP-binding cassette). Glavinas et al. (2004)82 have provided an excellent overview of the ABC proteins. Many of the ABC transporters function as active transporters, they mediate the transport of substrates against a concentration gradient. This transport activity requires the energy of ATP hydrolysis, controlled by drug interaction, and coupled to the actual substrate translocation. Most probably the substrate recognition and binding occurs in some parts of the transmembrane domains. According to several reports, the hydrophobic substrates of MDR1 are recognized within the membrane bilayer or in its vicinity (Cited in Glavinas et al.). Similarly, in the case of MRP1 it has also been suggested that the transmembrane domains are involved in the drug interactions. The multidrug resistance phenotype in tumors is associated with the overexpression of certain ABC transporters, termed MDR proteins. The P-glycoprotein (Pgp, MDR1, ABCB1) mediated multidrug resistance was the first discovered and probably still is the most widely observed mechanism in clinical multidrug resistance. There are two other ABC transporters, which have been definitely demonstrated to participate in the multidrug resistance of tumors: one is the multidrug resistance protein 1.

Phenothiazine maleates-induced stimulation of ATP-dependent uptake of 2',7'-bis-(3-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) into inside-out membrane vesicles prepared from erythrocyte membranes was also demonstrated by Wesolowska, et al. (2005). Moreover, it was shown that phenothiazine maleates exerted stimulating effect on ATPase activity measured in erythrocyte membranes. This report is the first one demonstrating that compounds able to inhibit transport activity of P-glycoprotein can stimulate MRP1 transporter. They conclude that phenothiazine maleates probably exert their stimulatory effect on MRP1 by direct interaction with the protein at the site different from the substrate binding site.

The role of phenothiazine compounds as a possible MDR modulator has recently been explored by Tegos and Hamblins (2006)83 in their potential use in phototherapeutic antimicrobials. Multidrug resistance pumps (MDRs) have been identified for a wide range of organisms. They asked whether phenothiazinium salts (methylene blue and toluidine blue) with structures that are amphipathic cations could potentially be substrates of MDRs. Using MDR-deficient mutants of Staphylococcus aureus (NorA), Escherichia coli (TolC), and Pseudomonas aeruginosa (MexAB) they found 2 to 4 logs more killing than seen with wild-type strains by use of three different phenothiazinium photosensitizers and red light. Mutants that overexpress MDRs were protected from killing compared to the wild type. Effective antimicrobial photosensitizers of different chemical structures showed no difference in light-mediated killing depending on MDR phenotype. Differences in uptake of phenothiazinium photosensitizers by the cells depending on level of MDR expression were found. Tegos and Hamblins suggest that specific MDR inhibitors could be used in combination with phenothiazinium salts to enhance their photodestructive efficiency.

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Cincotta, et al.46 observed that levels of their benzophenothiazine in non-cancerous tissue PDT produces little cytotoxicity compared to the PDT effects observed at much lower (one third) levels of benzophenothiazine localized in tumor tissue; the difference being attributed to the dye being in the non photosensitizing reduced form in normal tissue versus the photoactive cationic species that predominates in tumor. The redox properties of the dye are important. “The reduction of the benzophenothiazine necessitates that it come in contact with intracellular reductases/dehydrogenase and their coenzymes NAD(P)H which are principally located in the mitochondria, endoplasmic reticulum, and the cytosol. Therefore if a quantitative or qualitative difference exists between the enzymatic redox profiles of malignant and normal tissue it would not be surprising if a simultaneous change occurred in the ratio of oxidized to reduced dye between these two tissues. It has been well established that the metabolic changes which accompany the neoplastic transformation are mediated by altered enzyme profiles at these three intracellular sites (cited in Cincotta et al.; Weber (1977)84, Apffel (1978)85).

In addition it has been proposed that the autofluorescence differences often seen between malignant and normal tissues are the consequence of decreased levels of NADH in malignant tissue (refs + autofluor imaging refs with comment)”

Enhanced tumor glycolysis resulting from an activated hexose monophostate shunt may directly enhance the uptake and influx of planar tricyclic heteroaromatic compounds such as the phenothiazines, phenoxiazines, etc. The energetics required for a transporter mediated influx – or a pharmacophore mediated influx – may be more favorable with enhanced glycolysis. The relationship between expression of the ATP cassette family of transporters, such as P-gp or mrp and tumor hypoxia is well recognized.

Pervaiz and Clement (2004)86 suggest a causal connection between drug resistance and redox status in tumor cells. They note various factors have been implicated in the acquisition of the resistant phenotype, such as activation of drug efflux pumps, overexpression of proteins that inhibit cell death, absence of critical members of the death circuitry, and selective loss of cell cycle checkpoints. Carcinogenesis is in part a product of increased proliferation and defective or diminished cell death signaling. The cellular redox status is a determinate of responsiveness to exogenous stimuli (uptake of delocalized cationic lipophiles for example). Intracellular generation of reactive oxygen species (ROS) is tightly regulated by the intrinsic anti-oxidant defense systems; experimental evidence suggests that ROS also play an important role as signaling molecules in diverse physiological processes. Low levels of intracellular ROS have been linked to cellular proliferation and cell cycle progression in part explaining the pro-oxidant state invariably associated with the transformed phenotype.

Exploiting tumor specific redox status is a current cancer chemotherapeutic strategy; for example the thioredoxin reductase/ thioredoxin system (TRX/TX) and motexafin gadolinium (Biaglow and Miller (2005)).87 Thioredoxin expression is increased in a variety of human malignancies, including lung, colorectal, cervical, hepatic, and pancreatic cancer, which is likely related to changes in protein structure and function. Thioredoxin plays a role in stimulating hypoxia-inducible

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factor. Cancer cells are able to adapt to the hypoxic conditions found in nearly all solid tumors. Hypoxia leads to activation of hypoxia-inducible factor 1 (HIF-1), which is a transcription factor involved in development of the cancer phenotype. Biaglow and Miller note several lines of evidence that suggest TX may be necessary but not sufficient for conferring resistance to anti-cancer drugs. For example, the resistance of adult T-cell leukemia cell lines to doxorubicin and ovarian cancer cell lines to cisplatin has been associated with increased intracellular TX-1 levels. The biological activities of TRX/TX and their apparent relevance to aggressive tumor growth suggest that this system may be an attractive target for cancer therapy. Either individual enzymes or substrates can be altered. For cells that do not contain glutaredoxin, depletion of HMPS-generated NADPH or direct interaction with TX or TRX may be viable approaches to blocking the HMPS/TRX/TX-coupled reactions. When glutaredoxin is present, its reducing activity also may need to be targeted through depletion of glutathione. For example Motexafin gadolinium: Motexafin gadolinium is an expanded porphyrin that contains Gd+3 in the central cavity of the macrocycle. Motexafin gadolinium has a strong affinity for electrons; that is, it is easily reduced. Motexafin gadolinium displays catalytic activity by accepting electrons from various intracellular metabolites, such as ascorbate, NADPH, glutathione, TR and others. In the presence of oxygen, motexafin gadolinium produces superoxide and other reactive oxygen species, and regeneration of the motexafin gadolinium molecule by a process referred to as “futile redox cycling”. An interesting characteristic of motexafin gadolinium is its selective localization into cancer cells, which has been confirmed in vivo using MRI scanning to detect the biodistribution of the paramagnetic, Gd+3 containing complex. In addition to their high rates of anaerobic glycolysis, tumors possess an altered redox status, low pH, nutrient supply limitations, and are often hypoxic. It is possible that the tumor selectivity demonstrated by motexafin gadolinium is a consequence of the altered redox state of these tissues or other factors present in the tumor microenvironment.

Interestingly, methylene blue and toluidine blue are also easily reduced intracellularly accepting electrons from various metabolites such as ascorbate, NADPH, glutathione, and others. They are readily reoxidized by oxygen and as May notes52, will generate superoxide and other reactive oxygen species; and both are shown to selectively localize in tumor cells, properties shared with motexafin gadolinium. Also note among pyridine nucleotide-dependent disulphide reductases, MB selectively inhibits glutathione reductase in P. falciparum (cited in Wainwright and Amaral; Becker et al. 199988).

While methylene blue is an inhibitor of guanlyl cyclase, Lee and Wurster (1995)89 found that it induced cytotoxicity in SK-N-MC human neuroblastoma and U-373 MG human astrocytoma cells in a dose-dependent manner. MB did not significantly alter cellular levels of cGMP in both cells. cGMP may not be a major target of the cytotoxic action of MB. However, hydroxyl radical scavengers or intracellular Ca2+ modulators effectively blocked the MB-induced cytotoxicity. These results suggest that hydroxyl radical and intracellular Ca2+ may have an important involvement in the cytotoxic action of MB. Interestingly these results further suggest that the treatment with MB may be useful for the therapeutic applications of human brain tumors.

Tumor redox status is a basis for cellular imaging strategies: Kuppusamy et al. (2002)90 note the redox environment within the tumor cell is an important parameter that may determine the response of a tumor to certain chemotherapeutic agents, radiation, and bioreductive hypoxic cell cytotoxins. A

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variety of intracellular molecules may contribute to the overall redox status in tissues including GSH, thioredoxins, NADPH, flavins, ascorbate, and others. Using electron paramagnetic resonance spectroscopy and nitroxide redox probe, they have spatially mapped a radiation induced fibrosarcoma in mice and explored the role intracellular glutathione on the tissue redox status. The development of therapeutic regimens to exploit the physiological differences between normal and malignant tissue may also provide options for improved treatment. Some of the physiological differences between normal tissue and tumors include oxygen status, redox status, and intracellular pH. These differences, at least in part, can result from the physical architecture of the tumors with compromised blood supply. Methods that can detect subtle differences in the above physiological parameters would greatly aid in devising appropriate treatment strategies.

Echoing the idea that one of the biochemical "hallmarks" of malignancy is enhanced tumor glycolysis, in part due to the overexpression of glucose transporters (GLUTs) and the increased activity of mitochondria-bound hexokinase in tumors, Zhang, et al. (2004)91 suggest that easy methods for assessing glucose utilization in vitro and in vivo should find widespread application in biological and biomedical studies, as illustrated by the adoption of FDG PET imaging in medicine. They synthesized a NIR fluorescent pyropheophorbide conjugate of 2-deoxyglucose (2DG), Pyro-2DG, as a GLUT-targeted photosensitizer and evaluated the in vivo uptake of Pyro-2DG; Pyro-2DG selectively accumulated in two tumor models compared to surrounding normal muscle tissues at a ratio of about 10:1. By simultaneously performing redox ratio and fluorescence imaging, a high degree of correlation between the PN/(Fp+PN) redox ratio, where PN denotes reduced pyridine nucleotides (NADH) and Fp denotes oxidized flavoproteins, and the Pyro-2DG uptake was found in both murine tumor models, indicating that Pyro-2DG could serve as an extrinsic NIR fluorescent metabolic index for the tumors. The fact that only a low level of correlation was observed between the redox ratio and the uptake of Pyro-acid (the free fluorophore without the 2-deoxyglucose moiety) supports the hypothesis that Pyro-2DG is an index of the mitochondrial status (extent of PN reduction) of a tumor.Summary

Using the phenothiazine toluidine blue O as a cervical cancer diagnostic in vivo, based on its affinity for nucleic acids when used as a histochemical stain, began an interesting history of in vivo cancer imaging with this class of dyes. While the actual mechanism still remains unclear, the demonstrated correlation of dye uptake with cancerous cells, not only for toluidine blue but methylene blue and similar cationic tricyclic heteroaromatics parallels two other features characteristic of neoplastic cells for which “interaction” of phenothiazines have been shown: hypoxia and enhanced tumor glycolysis and as substrates for the MDR system either by direct interaction as a substrate, modulating P-gp directly or by altering the cellular redox physiology, such as creating a “futile redox cycling” as Biaglow and Miller suggested for motexafin gadolinium. Together with tumor specific changes in membrane pH gradient and acidification, some mechanism other than simple nucleic acid affinity is responsible for the tumor selective localization of the phenothiazines and related compounds. However, the “selective” aspect of these observations must be emphasized. The phenothiazines will be taken up to some

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degree by all cell types; the extent dependent up the underlying influx mechanism, the rate of efflux, and the redox state of the cell.

The utility of the tumor selective uptake and staining of methylene blue and toluidine blue has not diminished. Methylene blue has been found to be a useful adjunct for determining biopsy sites in Barrett’s esophagus.92 While most studies report very good correlation with histopathology, other studies have not been as demonstrative. The basic technique involves using 10% N-acetyl cysteine as a mucolytic agent for two minutes prior to spraying with 0.5% methylene blue.

N-acetylcysteine (NAC) has been tested as a medication which can suppress various pathogenic processes in disease93. Besides its well-known and efficient mucolytic action, NAC meets these needs by virtue of its antioxidant and anti-inflammatory modes of action. NAC is a thiol compound which by providing sulfhydryl groups, can act both as a precursor of reduced glutathione and as a direct ROS scavenger, hence regulating the redox status in the cells. In this way it can interfere with several signaling pathways that play a role in regulating apoptosis, angiogenesis, cell growth and arrest and inflammatory response. This may include modulation of the ATP dependent multidrug transporter system and alter drug uptake and efflux. Overall, the antioxidant effects of NAC are well documented in in vivo and in vitro studies.

If the selective uptake and influx of methylene blue by dysplastic cells is a function of the tumor redox status, the use of NAC by endoscopists with methylene blue may play a much more important role than simply acting as a mucolytic agent. Suppressing uptake of methylene blue by inflammatory cells by alleviating oxidative stress may significantly improve the diagnostic accuracy94 of this method and cannot be overlooked as part of the procedure.

Cationic dye influx may be facilitated by upregulation of membrane transporters in response to altered cellular redox status that is characteristic of carcinogenesis such as the activation of the hexose monophosphate shunt. Dye expressed intracellularly as the oxidized chromophore correlates to enhanced cell ROS. More significantly, these redox dyes can modify the redox status and may enhance their own uptake in a permissive fashion. The extremely concentrative and rapid uptake of the dyes as the neutral more lipophilic species followed by intracellular reoxidation may promote and catalyze its mediated influx transport via the ABC system. The significantly enhanced rate of influx via the transporter system in response to the hypoxic redox status of tumor cells may underlie the selective staining for cancerous cells and tissue that have been reported for phenothiazines and related DLC’s.

Vital staining with the cationic planar tricyclic heteroaromatics such as methylene blue, toluidine blue or Nile blue is clearly much more than simple ionic interactions with nucleic acids; their selectivity is based on differences in cellular redox metabolism and an energy dependent interaction with plasma membrane transport mechanisms. Understanding these interactions and mechanisms may improve the utility of these well known selective stains for practical and cost effective clinical applications such as optical imaging and cancer diagnostics.

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