Influence of Alternative Tubulin Inhibitors on the Potency of a Epirubicin-Immunochemotherapeutic Synthesized with an Ultra Violet Light-Activated Intermediate

Immunochemotherapeutics, epirubicin-(C3-amide)-SS-[anti-HER2/neu] with an internal disulfide bond, and epirubicin-(C3-amide)-[anti-HER2/neu] were synthesized utilizing succinimidyl 2-[(4,4 ́-azipentanamido) ethyl]-1,3 ́-dithioproprionate or succinimidyl 4,4-azipentanoate respectively. Western blot analysis was used to determine the presence of any immunoglobulin fragmentation or IgG-IgG polymerization. Retained HER2/neu binding characteristics of epirubicin-(C3-amide)-[anti-HER2/neu] and epirubicin-(C3-amide)-SS-[anti-HER2/neu] were validated by cell-ELISA using a mammary adenocarcinoma (SKBr-3) population that highly over-expresses trophic HER2/neu receptor complexes. Cytotoxic anti-neoplastic potency of epirubicin(C3-amide)-[anti-HER2/neu] and epirubicin-(C3-amide)-SS-[anti-HER2/neu] between epirubicin-equivalent concentrations of 10 M and 10 M was determined by measuring the vitality/proliferation of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3 cell type). Cytotoxic anti-neoplastic potency of benzimidazoles (albendazole, flubendazole, membendazole) and griseofulvin were assessed between 0-to-2 g/ml and 0-to-100 g/ml respectively while mebendazole and griseofulvin were analyzed at fixed concentrations of 0.35 g/ml and 35 g/ml respectively in dual combination with gradient concentrations of epirubicin-(C3-amide)-[anti-HER2/neu] and epirubicin-(C3-amide)-SS-[anti-HER2/neu]. Cytotoxic anti-neoplastic potency for epirubicin-(C3-amide)-[anti-HER2/neu] and epirubicin-(C3-amide)-SS[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) was nearly identical at epirubicin-equivalent concentrations of 10 M and 10 M. The benzimadazoles also possessed cytotoxic anti-neoplastic activity with flubendazole and albendazole being the most and least potent respectively. Similarly, griseofulvin had cytotoxic anti-neoplastic activity and was more potent than methylselenocysteine. Both mebendazole and griseofulvin when applied in dual combination with either epirubicin-(C3-amide)[anti-HER2/neu] or epirubicin-(C3-amide)-SS-[anti-HER2/neu] produced enhanced levels of cytotoxic anti-neoplatic potency.

The anthracycline class of chemotherapeutics is commonly administered for the treatment of breast cancer and many other neoplastic conditions due to their superior level of potency.One of the most common dose-limiting side effects of anthracycline administration is cardiotoxicity (doxorubicin >> epirubicin).Even with the anthracyclines a complete clinical resolution of breast cancer, (particularly resistant forms), is rarely attainable especially when utilized as a monotherapy.Combination chemotherapy regimens are almost invariably more potent in suppressing the growth and metastasis of neoplastic cell types, significantly prolonging quality-of-life, delaying the onset of disease relapse, combating chemotherapeutic resistance, extending the duration of disease remission, and facilitating complete neoplastic disease elimination.Chemotherapeutic resistance is a particularly important development that hinders successful treatment of breast cancer because approximately 20-30% of all affected cases develop metastatic brain lesions which characteristically display moderate-to-high levels refractoriness to chemotherapeutic intervention (Honig et al., 2005).Despite the advantages of combination chemotherapy regimens, they still suffer from a high frequency of toxic sequelae that can limit the extent and duration of administration (Azad, Posadas et al., 2008;Balayssac et al., 2011;Ceresa & Cavaletti, 2011;Chang et al., 2001;Iarussi, Indolfi, Galderisi, & Bossone, 2000;Raschi et al., 2010;Scully & Lipshultz, 2007;Stavridi & Palmieri, 2008;Vantelon et al., 2001;Wachters, Van Der Graaf, & Groen, 2004).
Covalent immunochemotherapeutics designed to selectively bind to external surface membrane receptors whereby the entire complex is internalized by mechanisms of receptor-mediated-endocytosis ultimately liberates the chemotherapeutic moiety through various processes within the acidic endolysosome environment (pH 5.0-5.5).A previously synthesized covalent anthracycline immunochemotherapeutic, epirubicin-[anti-HER2/neu] designed to contain a synthetically introduced acid-labile hydrazone (C 13 -imino) bond structure did not have any detectably greater cytotoxic anti-neoplastic potency against mammary adenocarcinoma (SKBr-3) populations (Coyne, Jones, & Pharr, 2011).Similar covalent anthracycline immunochemotherapeutics with acid-labile/acid-sensitive properties reportedly afford increased or accelerated liberation (cytosol bioavailability) of only 40% of their total chemotherapeutic content within the low pH of endolysosomal environments found in many cancer cell types.In parallel with the concept of acid-sensitive anthracycline immunochemotherapeutics both epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] and epirubicin-(C 3 -amide)-[anti-HER2/neu] were synthesized in order to determined if the synthetic introduction of a disulfide bond structure created an enzyme or acid-sensitive covalent immunochemotherapeutic.Similar covalent maytansinoid immunochemotherapeutics with an integral disulfide bond structure have been synthesized that variably provide increases in intracellular chemotherapeutic bioavailability (Erickson et al., 2010;Kellogg et al., 2011).
Due to the effectiveness of conventional tubulin/microtubule inhibitors in combination with the anthracyclines, and because of their different spectrums of dose-limiting toxic sequelae, there is a distinct need to identify and evaluate alternative tubulin/microtubule inhibitors that have potent cytotoxic anti-neoplastic potency.To date, very little is known about the cytotoxic anti-neoplastic potency of the benzimidazoles (anthelmintic) or griseofulvin (anti-fungal agent) tubulin/microtubule inhibitors against aggressive and resistant breast cancer or their capacity to produce additive or synergistic levels of cytotoxic anti-neoplastic potency when applied in dual combination with covalent anthracycline immunochemotherapeutics.

Non-Reducing SDS-PAGE Size Separation, Western-Blot Immunodetection, and Chemiluminescent Autoradiography
Standardized amounts and concentrations (60 g/ml) of covalent epirubicin immunochemotherapeutics and reference control immunoglobulin fractions were combined 50/50 with an equal volume of conventional PAGE sample preparation buffer (Tris/glycerol/bromphenyl blue/sodium dodecyl sulfate) formulated without 2-mercaptoethanol.Each immunoglobulin sample (0.9 μg/well) was processed without boiling and then developed in parallel with a mixture of pre-stained reference control molecular weight markers by non-reducing SDS-PAGE (11% acrylamide, 100 V constant voltage at 3°C for 2.5 hours).Developed non-reducing SDS-PAGE acrylamide gels were then equilibrated in electrophoresis "tank" buffer devoid of methanol.Lateral transfer of SDS-PAGE separated proteins onto sheets of nitrocellulose membrane for Western (immunodetection) blots was performed at 20 volts constant voltage for 16 hours at 2°C to 3°C with the transfer manifold packed in crushed ice.
Nitrocellulose membranes with laterally transferred immunoglobulin fractions for immunodetection and chemiluminescent autoradiographic analyses were equilibrated in Tris buffered saline (TBS: Tris HCl 0.1 M, NaCl 150 mM, pH 7.5, 40 ml) at 4°C for 15 minutes followed by incubation in TBS blocking buffer solution (Tris 0.1 M, pH 7.4, 40 ml) containing bovine serum albumin (BSA 5%) applied at 2 O to 3 O C for 16 hours in combination with gentle horizontal agitation.Prior to further processing nitrocellulose membranes were vigorously rinsed in Tris buffered saline (Tris 0.1 M, pH 7.4, 40 ml, n = 3 rinses).

Mammary Carcinoma Tissue Culture Cell Culture
Human chemotherapeutic-resistant human mammary adenocarcinoma (SKBr-3) cell line was utilized as an ex-vivo neoplasia model and was acquired directly from American Tissue Cell Culture (ATCC) within 24 months of investigation.Mammary adenocarcinoma (SKBr-3) has been the only cell line or cell type utilized, cultivated or preserved/stored frozen in the laboratory during a period of the past 6 years and during the conduction of research investigations currently described.Characteristically, mammary adenocarcinoma (SKBr-3) uniquely over-expresses epidermal growth factor receptor 1 (EGFR, ErbB-1, HER1) and highly over-expresses epidermal growth factor receptor 2 (EGFR2, HER2/neu, ErbB-2, CD340, p185) at 2.2 x 10 5 /cell and 1 x 10 6 /cell respectively.
Populations of the mammary adenocarcinoma (SKBr-3) cell line were propagated in 150-cc 2 tissue culture flasks containing McCoy's 5a Medium Modified supplemented with fetal bovine serum (10% v/v) and penicillin-streptomycin at a temperature of 37 O C under a gas atmosphere of air (95%) and carbon dioxide (5% CO 2 ).Tissue culture media was not supplemented with growth factors, growth hormones or other growth stimulants of any type.Investigations were all performed using mammary adenocarcinoma (SKBr-3) propagated to a >85% level of confluency.
Contents within individual well of 96-well microtiter plates were removed manually by pipette at 72-hours and then the mammary adenocarcinoma (SKBr-3) monolayers were serially rinsed (n = 3) with PBS followed by incubation with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT 5 mg/ml in RPMI-1640 growth media devoid of pH indicator or bovine fetal calf serum).During a 3-to-4 hour incubation period at 37 O C under a gas atmosphere of air (95%) and carbon dioxide (5% CO 2 ) chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations were allowed to biochemically convert intracellular MTT to navy-blue formazone crystals by the endogenous enzyme, mitochondrial succinate dehydrogenase.Contents of 96-well microtiter plates were then removed, and serially rinsed with PBS (n = 3) followed by dissolving the resulting blue intracellular formazone crystals with DMSO (300 μl/well).Spectrophotometric absorbance of the blue-colored supernatant was then measured at 570 nm using a computer-integrated microtiter plate reader.

Molecular Properties
The percent of non-covalently bound anthracycline contained in covalent epirubicin immunochemotherapeutics following separation by micro-scale desalting/buffer exchange column chromatography was consistently < 4.0% of the total epirubicin content (Coyne, Jones, & Pharr 2011;Coyne et al., 2011;2009).Residual non-covalently bound anthracycline is generally considered to not be available for further removal by serial/repeated column chromatography (Beyer et al., 2001).The anthracycline molar-incorporation-indexes for epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] were 40% (39.65%) and 47% (47.15%) respectively.Related preliminary analyses revealed that the epirubicin UV-photoactivated intermediates when synthesized in DMSO retained reactivity after freezing for at least 48 hours at -20 O C based on an epirubicin molar-incorporation-index of 51% when the reaction mixture was combined with bovine serum albumin at a succinimidyl 4,4-azipentanoate-to-BSA molar-ratio of 7-9:1 in concert with subsequent UV-photoactivation (354 nm, 25°C, 15 minutes).Higher epirubicin molar-incorporation-indexes are possible to achieve with modifications in methodology but the harsher synthesis conditions required for such purposes are accompanied by substantial reductions in final covalent immunochemotherapeutic yield, (Greenfield et al., 1990) and declines in antigen-immunoglobulin binding-avidity (e.g.cell-ELISA parameters).Evaluation of covalent epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] immunochemotherapeutics size-separated by SDS-PAGE and developed by Western blot analysis utilizing anti-murine IgG-strepavidin as a 1° immunoglobulin produced chemiluminescent autoradiography images that detected single (major) 150-kD band profiles for both the anti-HER2/neu reference control and each individual covalent epirubicin immunochemotherapeutic similar to results previously reported for other methodologies (Figure 2
In addition to protein (immunoglobulin) pre-thiolation that can under certain conditions result in intra-molecular and inter-molecular disulfide bond formation, the binding-avidity of epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] for membrane HER2/neu was also likely influenced by the covalent bonding of the anthracycline moiety to different amino acid residues within the Fab' antigen binding region of anti-HER2/neu immunoglobulin.Seemingly modest alterations in synthetic chemistry reactions and elevations in chemotherapeutic molar-incorporation-index can profoundly influence immunoglobulin binding properties (Yang & Reisfeld 1988a).The relatively mild conditions employed during organic chemistry reaction schemes and the relatively low molar-incorporation-index of 40.0%collectively contribute to the high biological integrity of epirubicin (C 3 -amide)-[anti-HER2/neu] and epirubicin (C 3 -amide)-SS-[anti-HER2/neu] based on the collective interpretation of results from SDS-PAGE chemiluminescent autoradiography, cell-ELISA analyses and cytotoxic anti-neoplastic potency against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3).

Cytotoxic Potency of Covalent Epirubicin Immunochemotherapeutics
Creation of a synthetic covalent bond between epirubicin and anti-HER2/neu monoclonal immunoglobulin for the production of epirubicin-(C 3 -amide)-[anti-HER2/neu] or epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] did not decrease the cytotoxic potency of the anthracycline against chemotherapeutic-resistant mammary adenocarcinoma  when assessed between the epirubicin-equivalent concentration range of 10 -10 M to 10 -6 M (Figures 3 & 4).Similar properties have been recognized previously for epirubicin-(C 13 -imino) -[anti-HER2/neu] (Coyne et al., 2011), epirubicin-(C 3 -amide)-[anti-HER2/neu] (Coyne et al., 2009), epirubicin-(C 3 -amide)-[anti-EGFR] (Coyne et al., 2009) and analogous covalent immunochemotherapeutics designed to selectively "target" anthracycline delivery (Dillman et al., 1989;Herbert, Norris, & Sauk 2003;Johnson, Briggs, Gutowski, & Barton 1995;King et al., 1999;Sivam et al., 1995;Stan, Radu, Casares, Bona, & Brumeanu 1999;Yang et al., 1988a).Synthetic incorporation of an internal (integral) disulfide bond into the molecular structure of covalent immunochemotherapeutics has potential merit as a strategy for enhancing the intracellular bioavailability of chemotherapeutic moieties following internalization by mechanisms of receptor-mediated endocytosis.The presumption is largely based on the concept that glutathione (GSH) tri-peptide is found intracellularly at levels that are 100x to 1000x (2-to-10 mM) higher than concentrations found within the extracellular fluid or plasma compartments (2-to-20 μM).Given this difference, disulfide bond structures have been synthetically introduced into maytansinoid-immunochemotherapeutics with the intent of improving their intracellular bioavailability following internalization by "targeted" neoplastic cell populations (Erickson et al., 2010;Kellogg et al., 2011).Due to these considerations epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] was synthesized with an internal (integral) disulfide bond structure in order to determine if such a chemical modification influences cytotoxic anti-neoplastic potency against mammary adenocarcinoma (SKBr-3) presumably by promoting elevations in the intracellular bioavailability of the anthracycline moiety (Figure 1).In contrast to the cytotoxic anti-neoplastic potency of covalent maytansinoid-immunochemotherapeutics against various cancer cell types like human intestinal carcinoma (xenografts) (Erickson et al., 2010;Kellogg et al., 2011), the cytotoxic anti-neoplastic potency of epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] was not significantly greater compared to epirubicin-(C 3 -amide)-[anti-HER2/neu] (Figure 3). Smilar to results detected in comparisons between epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] and epirubicin-(C 3 -amide)-[anti-HER2/neu] several other covalent immunochemotherapeutics with internal disulfide bonds incorporated into their molecular structure have also been found to not possess increased levels of cytotoxic anti-neoplastic potency (Erickson et al., 2006;Lewis Phillips et al., 2008;Sun et al., 2011).Certain maytansinoid-SS-[anti-HER2/neu] immunochemotherapeutics in this regard that contain a synthetically introduced disulfide bond do not exert higher planes of cytotoxic anti-neoplastic potency against HER2/neu positive breast cancer (Lewis Phillips et al., 2008).Potency of epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] against mammary adenocarcinoma (SKBr-3) might have been improved if a modified analog of succinimidyl 2-[(4,4´-azipentanamido)ethyl]-1,3´-dithioproprionate had been utilized to incorporate an internal disulfide bond structure at a physically different location or in a different molecular configuration (Kellogg et al., 2011).A supportive analogy is the observation that mytansinoid-immunochemotherapeutics that contain two methyl groups in close proximity to chemotherapeutic moieties and are devoid of methyl groups on the "linker side" exert only intermediate levels of plasma stability, but superior levels of cytotoxic anti-neoplastic potency against xenografts of human intestinal carcinoma (Kellogg et al., 2011).Synthetic introduction of disulfide bond structures into covalent mytansinoidimmunochemotherapeutics can therefore increase in-vivo susceptibility to premature enzymatic degradation but if they are located in a sterically hindered position they are less susceptible to enzyme-mediated liberation within the intravascular compartment (Kellogg et al., 2011).Unfortunately it was not possible to evaluate the influence of disulfide bond position on the cytotoxic potency of epirubicin-(C 3 -amide) -SS-[anti-HER2/neu] against mammary carcinoma due to a lack of available reagents.
Other biochemical and cell biology associated variables may also account for the lack of improved cytotoxic anti-neoplastic potency of epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] against chemotherapeutic-resistant human mammary adenocarcinoma (SKBr-3) compared to epirubicin-(C 3 -amide)-[anti-HER2/neu] (Figure 3).Covalent epirubicin-(C 13 -imino)-[anti-HER2/neu] immunochemotherapeutic synthesized with an internal bond structure that reportedly has acid-labile properties does not exert significantly higher levels of cytotoxic anti-neoplastic potency against mammary adenocarcinoma (SKBr-3) than a non-acid-labile epirubicin-(C 3 -amide)-[anti-HER2/neu] immunochemotherapeutic (Coyne et al., 2011b).The fact that epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] also did not exert an enhanced level of cytotoxic anti-neoplastic activity against chemotherapeutic -resistant mammary adenocarcinoma (SKBr-3) may therefore reflect a cell biology related variable that explains a lack of enhanced efficacy.Interestingly, minimal or no correlation frequently exists between the in-vitro and in-vivo potency of covalent immunochemotherapeutics with synthetically introduced disulfide bond structures (Kellogg et al., 2011) which is in marked contrast to covalent immunochemotherapeutics devoid of this same internal chemical structure.Differences in cytotoxic anti-neoplastic potency to this degree in-vivo have been attributed to the influence of hepatic metabolization and variations in the creation of lipophilic and hydrophilic metabolites that determine the extent of distribution within fluid compartments and penetration across intact cancer cell membranes (Erickson et al., 2010) .
The cytotoxic anti-neoplastic potency of the prototypic epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin (C 3 -amide)-SS-[anti-HER2/neu] immunochemotherapeutics can potentially be enhanced in-vivo through several molecular strategies that involve exploiting the over-expression of membrane HER2/neu and the molecular properties of anti-HER2/neu as a selective "targeted" delivery platform.Given this perspective, endogenous trophic receptor over-expression is a critical variable that influences the cytotoxic anti-neoplastic potency of anthracycline-[anti-HER2/neu], anthracycline-[anti-EGFR] and related covalent immunochemotherapeutics because it provides opportunities to [i] significantly suppress neoplastic cell growth for populations with proliferation rates heavily dependent on tropic receptor over-expression; [ii] promote continual and selective chemotherapeutic deposition on the external surface membrane of neoplastic cells; and [iii] induce progressive active chemotherapeutic internalization by mechanisms of receptor-mediated endocytosis in a manner that promotes escalating increases in cytosol chemotherapeutic accumulation (Pimm, Paul, Ogumuyiwa, & Baldwin 1988;Shih et al., 1994;Stan et al., 1999;Yang et al., 1988a).The latter consideration is important since receptor-mediated-endocytosis of epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] can lead to increases in cytosol anthracycline concentrations that are 8.5x (Stan et al., 1999) to >100x (Pimm et al., 1988) greater than those that are attainable by simple passive anthracycline diffusion from the plasma or extracellular fluid compartments (e.g.following intravenous injection).Although specific data for HER2/neu and EGFR receptor complexes in chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) is limited, metastatic multiple myeloma internalizes approximately 8 x 10 6 anti-CD74 monoclonal antibody molecules per day following binding to membrane CD74 sites (Hansen, Ong & Diril 1996).Following selective "targeted" delivery more than 50% of an anthracycline at 24-hours is retained intracellularly (Stan et al., 1999) where it is primarily associated with either internal membrane structures or it becomes distributed throughout the cytosol environment (Liu et al., 2010;Shih et al., 1994).Conversely, "free" non-conjugated anthracycline upon passive diffusion across intact cellular lipid bilayer membranes is detected predominately in complex with nuclear DNA less than 30 minutes following initial exposure (Shih et al., 1994) whereas anthracycline liberated from covalent immunochemotherapeutics ultimately distributes into, and accumulates within the nucleus, mitochondria and golgi apparatus (Beyer, Rothen-Rutishauser, Unger, Wunderli-Allenspach & Kratz 2001).
Third, cytotoxic anti-neoplastic potency of covalent immunochemotherapeutics can be assessed by measuring cellular proliferation with either [H 3 ]-thymidine, or an ATP-based assay method because of their reportedly >10-fold greater sensitivity in detecting early cell injury compared to MTT vitality stain based assay methods (Mueller, Kassack, & Wiese 2004;Ulukaya, Ozdikicioglu, Oral, & Dermirci 2008).Despite this consideration, MTT vitality stain continues to be extensively applied for the routine assessment of cytotoxic anti-neoplastic potency of chemotherapeutic agents in part because the detection of true cancer cell death is generally considered superior to the detection of reversible cellular injury (Dery, Van Themsche, Provencher, Mes-Masson, & Asselin 2007;Huang, Pierstorff, Osawa, & Ho 2007;Kars, Iseri, Gunduz, & Molnar 2008;Spee et al., 2006;Varache-Lembège, Larrouture, Montaudon, Robert, & Nuhrich 2008) Fourth, cytotoxic anti-neoplastic potency can be delineated in-vivo utilizing human neoplastic xenographs in animal hosts as a neoplastic disease model where the efficacy of covalent immunochemotherapeutics frequently tends to be higher than in ex-vivo tissue culture models utilizing the same identical cancer cell type (Aboud-Pirak, Hurwitz, Bellot, Schlessinger, & Sela 1989;Johnson et al., 1995;Zhang, Wang, Li, Liu, & Dong 1992).Enhanced levels of covalent immunochemotherapeutic potency measured in-vivo that can not effectively be assessed in ex-vivo tissue culture is presumed to at least in part be dependent upon responses by the endogenous immune system through processes that include antibody-dependent cell cytotoxicity (ADCC) phenomenon in concert with complemented-mediated cytolysis activated by the formation of HER2/neu-immunoglobulin complexes on the exterior surface membrane of "targeted" neoplastic cells.Endogenous immune cell types involved in ADCC responses release cytotoxic mediators known to additively and synergistically enhance the cytotoxic anti-neoplastic activity of conventional chemotherapeutics (Coyne, Fenwick & Ainsworth 1997).The contributions of ADCC and complement-mediated cytolysis to the in-vivo cytotoxic anti-neoplastic potency of covalent anthracycline immunochemotherapeutics would be further complemented by the additive and synergistic properties attained with monoclonal immunoglobulin inhibitors of trophic receptors applied in dual combination with conventional chemotherapeutics (Ciardiello et al., 1999;Fry, Schilke, McGuire, & Bird 2010;García-Sáenz et al., 2008;Jin et al., 2010;Kim et al., 2006;Landriscina, Maddalena et al., 2010;Lynn et al., 2010;Pegram, Lopez, Konecny, & Slamon 2000;Slamon et al., 2001;Slamon & Pegram 2001;Winer & Burstein 2001;Zhang et al., 2002).Additive or synergistic interactions of this type have been detected with anti-HER2/neu in concert with cyclophosphamide, docetaxel, doxorubicin, etoposide, methotrexate, paclitaxel, or vinblastine (Pegram, Lopez, Konecny, & Slamon 2000;Slamon et al., 2001).
Fifth, the synthesis strategy for epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] could have been modified to increase the anthracycline molar-incorporation-index. Unfortunately, such modifications usually entail harsher synthesis conditions that impose a higher risk for declines in retained biological function and substantial reductions in final/total yield (Greenfield et al., 1990;Zhang et al., 1992).In addition to harsher synthesis conditions, excessively high molar incorporation indexes for anthracycline can (as previously discussed) also reduce the biological integrity of immunoglobulin fractions when the number of chemotherapeutic moieties covalently introduced into the Fab' antigen-binding region becomes excessive.Such modifications can result in only modest declines in immunoreactivity (e.g.86% for a 73:1 ratio) but disproportionate declines in anti-neoplastic potency with reductions in activity to levels that are substantially lower than those associated with non-conjugated "free" anthracycline (Zhang et al., 1992).
The benzimidazole anthelmintics and griseofulvin anti-fungal agent have a mechanism-of-action that is highly analogous to that of many conventional tubulin/microtubule inhibitor chemotherapeutics.Even though very little is known about the cytotoxic anti-neoplastic properties of benzimidazole anthelmintic and griseofulvin anti-fungal agents, their mechanism-of-action suggests that they can potentially suppress the growth and vitality of cancer cell populations both alone, and in multi-chemotherapeutic regimens similar to the vinca alkaloids, taxanes (e.g.paclitaxel), podophyllotoxins (e.g.etoposide) and monomethyl auristatin E (MMAE).The cytotoxic anti-neoplastic potency of epirubicin-(C 3 -amide)-[anti-HER2/neu] and epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] formulated between the final epirubin-equivalent concentrations of 10 -10 M to 10 -6 M was markedly increased when applied in concert with mebendazole and griseofulvin (Figures 7 & 8).Mebendazole and griseofulvin evoked slightly higher levels of cytotoxic anti-neoplastic activity in dual combination with epirubicin-(C 3 -amide)-[anti-HER2/neu] than was detected with epirubicin-(C 3 -amide)-SS-[anti-HER2/neu] but the variable responsible for this trend remains unknown although it may possibly be related to variations in intracellular epirubicin bioavailability or differences in the lipophilic characteristics of different epirubicin metabolite analogs (Figures 7,8,9 & 10) (Erickson et al., 2010).
Capacity of the benzimidazole class of tubulin/microtubule inhibitors to evoke additive or synergistic levels of cytotoxic anti-neoplastic potency when applied in dual combination with conventional (e.g.vinblastine) (Spagnuolo et al., 2010) or selectively "targeted"/delivered chemotherapeutic agents has previously been very rarely delineated.Similarly, the potential for griseofulvin in combination conventional chemotherapeutics in multi-combination regimens to create additive or synergistic levels of cytotoxic anti-neoplastic potency has to date been investigated on a very limited scale.In preliminary investigatins, however, griseofulvin has been found to complement the anti-neoplastic properties of nocodazole (Ghadimi et al., 2000;Ho et al., 2001) and vinblastine (Rathinasamy et al., 2010).Despite these voids in knowledge, multiple implications arise upon the acknowledgement that benzimidazole and griseofulvin tubulin/microtubule inhibitors can produce additive or synergistic cytotoxic anti-neoplastic potency when applied in dual combination with conventional or selectively "targeted" chemotherapeutics.Results observed with the benzimidazoles and griseofulvin tubulin/microtubule inhibitors indicate that they potentially have realistic utility as an alternative class of chemotherapeutic capable of providing "new" opportunities for achieving more potent long-term resolution of even the most resistant forms of breast cancer and other neoplastic disease states while simultaneously posing a lower risk of undesirable sequelae.In either a mono-therapy format or as a component of a combination multi-chemotherapeutic regimen these attributes can potentially be attained because benzimidazole and griseofulvin tubulin/microtubule inhibitors are apparently poor P-glycoprotein substrates (Khalilzadeh et al., 2007;Spagnuolo et al., 2010) and they have a relatively wider margin-of-safety than many if not most conventional chemotherapeutics (de Silva, Guyatt & Bundy 1997;Morris et al., 2001b;Pourgholami et al., 2005;2010).The benzimidazole anthelmintics and griseofulvin when applied in additive and synergistic combinations with other chemotherapeutics or anti-cancer modalities can potentially add another level of safety because they are able to ultimately afford lower total dosage requirements.Presumably the highest levels of additive and synergistic cytotoxic anti-neoplastic potency and widest margin-of-safety can be attained when benzimidazole anthelmintics or griseofulvin are substituted for conventional tubulin/microtubule inhibitor chemotherapeutics in combination regimens that also apply covalent anthracyline-immunochemotherapeutics with properties of selective "targeted" delivery.Such considerations are critically important to the development of safer and more effective treatment regimens in order to reduce collateral cardiotoxicity (Danesi et al., 2006;Last et al., 2003;Nakano, Takeshige, Toshima, Tokunaga, & Minakami 1989) and nephroticity (Bulucu et al., 2008) that commonly limit systemic anthracycline administration.

Conclusion
Cardiotoxicity (doxorubicin >> epirubicin) (Danesi et al., 2006;Last et al., 2003;Nakano et al., 1989), nephroticity (Bulucu et al., 2008) and chemotherapeutic resistance represent complications that can commonly limit anthracycline administration in modern clinical oncology.The molecular design and methodology delineated for the synthetic production of covalent epirubicin-immunochemotherapeutics utilizing a UV-photoactivated anthracycline intermediate addresses a need to discover and optimize laboratory methods for the expedient production of anti-cancer therapies at higher end-product yields that possess properties of selective "targeted" delivery that complement the efficacy and potency of conventional and unconventional chemotherapeutics.In this context, selective "targeted" epirubicin delivery affords the opportunity to achieve cytosol concentrations that are greater than can be attained by simple passive diffusion, serve as a molecular mechanism for minimizing the impact of chemotherapeutic-resistance in many forms of neoplastic disease, and it serves as a way of minimizing chemotherapeutic diffusion into innocent tissues and organ systems (potential for a relatively wider margin-of-safety).Covalent anthracycline immunochemotherapeutics are relevant to the treatment of breast cancer and many other neoplastic conditions complicated by aggressive localized growth characteristics or have a high probability for metastasis and therapeutic resistance (Alexander, Greene, Torti & Kucera 2005).Preliminary laboratory analyses that detects and measures over-expression of trophic membrane receptors (e.g.HER2/neu, EGFR, VEGFR), proteins associated with chemotherapeutic resistance (e.g.P-glycorprotein, breast cancer type susceptibility protein/BRCA1 (Chekhun et al., 2009), and endocrine receptor profiles (e.g.estrogen, progesterone, testosterone) could be applied to account for biological variations and identify neoplastic conditions most effectively resolved with covalent immunochemotherapeutics like epirubicin-(C 3 -amide)-[anti-HER2/neu].Base on these considerations, future investigations devoted to delineating the benefit of implementing epirubicin-(C 3 -amide)-[anti-HER2/neu] in the formulation of individualized treatment protocols is warranted.
Discovery of the cytotoxic anti-neoplastic potency of the benzimidazole anthelmintics and griseofulvin against mammary adenocarcinoma (SKBr-3) illustrates their potential applicability as an alternative class of tubulin/microtubule inhibitor chemotherapeutics and encourages support for future investigations devoted to determining their role in the development of new treatment regimens.Relevant attributes include a greater level of efficacy against certain chemotherapeutic-resistant neoplasias that over-express P-glycoprotein and the opportunity they provide to additively or synergistically complement the efficacy of conventional and selectively "targeted"/delivered chemotherapeutics.Such considerations are directly relevant to current trends and objectives in modern clinical oncology directed at identifying multi-treatment protocols with a wider margin-of-safety that more potently resolve locally aggressive, highly metastatic and resistant forms of cancer.