Simultaneous Dual Selective Targeted Delivery of Two Covalent Gemcitabine Immunochemotherapeutics and Complementary Anti-Neoplastic Potency of [Se]-Methylselenocysteine

The anti-metabolite chemotherapeutic, gemcitabine is relatively effective for a spectrum of neoplastic conditions that include various forms of leukemia and adenocarcinoma/carcinoma. Rapid systemic deamination of gemcitabine accounts for a brief plasma half-life but its sustained administration is often curtailed by sequelae and chemotherapeutic-resistance. A molecular strategy that diminishes these limitations is the molecular design and synthetic production of covalent gemcitabine immunochemotherapeutics that possess properties of selective “targeted” delivery. The simultaneous dual selective “targeted” delivery of gemcitabine at two separate sites on the external surface membrane of a single cancer cell types represents a therapeutic approach that can increase cytosol chemotherapeutic deposition; prolong chemotherapeutic plasma half-life (reduces administration frequency); minimize innocent exposure of normal tissues and healthy organ systems; and ultimately enhance more rapid and thorough resolution of neoplastic cell populations. Materials and Methods: A light-reactive gemcitabine intermediate synthesized utilizing succinimidyl 4,4-azipentanoate was covalently bound to anti-EGFR or anti-HER2/neu IgG by exposure to UV light (354-nm) resulting in the synthesis of covalent immunochemotherapeutics, gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu]. Cytotoxic anti-neoplastic potency of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] between gemcitabine-equivalent concentrations of 10−12 M and 10−6 M was determined utilizing chemotherapeutic-resistant mammary adenocarcinoma (SKRr-3). The organoselenium compound, [Se]-methylselenocysteine was evaluated to determine if it complemented the anti-neoplastic potency of the covalent gemcitabine immunochemotherapeutics. Results: Gemcitabine-(C4-amide)-[anti-EGFR], gemcitabine-(C4-amide)-[anti-HER2/neu] and the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] all had anti-neoplastic cytotoxic potency against mammary adenocarcinoma. Gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] produced progressive increases in anti-neoplastic cytotoxicity that were greatest between gemcitabine-equivalent concentrations of 10−9 M and 10−6 M. Dual simultaneous combinations of gemcitabine-(C4-amide)-[anti-EGFR] with gemcitabine-(C4-amide)-[anti-HER2/neu] produced levels of anti-neoplastic cytotoxicity intermediate between each of the individual covalent gemcitabine immunochemotherapeutics. Total anti-neoplastic cytotoxicity of the dual simultaneous combination of gemcitabine-(C4-amide)-[anti-EGFR] and gemcitabine-(C4-amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) was substantially higher when formulated with [Se]-methylsele-nocysteine.


Introduction
Monoclonal immunoglobulin preparations or pharmaceuticals with binding-avidity for HER2/neu (e.g. anti-HER2/neu: trastuzumab, pertuzumab), EGFR (e.g. anti-EGFR: cetuximab, gefitinib) [1]- [4], HER2/neu and EGFR (e.g. anti-HER2/neu and anti-EGFR: panitumumab) [3]- [6] IGF-1R, VEGFR and inhibitors of trophic membrane receptors can all potentially be effective treatment options for certain neoplastic conditions including cancer affecting the breast, intestinal tract, lung or prostate. The significant advantage of these preparations is their ability to function as a selective anti-cancer treatment modality that also avoids many of the sequelae associated with conventional chemotherapy. Unfortunately, most monoclonal immunoglobulin-based therapies that inhibit the function of trophic membrane receptors are usually only capable of exerting cytostatic properties and as a monotherapy are almost invariably plagued by an inability to evoke cytotoxic activity that is potent enough to effectively resolve most aggressive and advanced forms of neoplastic disease [7]- [12]. Alternatively, enhanced levels of anti-neoplastic cytotoxicity can be attained when monoclonal immunoglobulin-based biotherapies are applied in concert with conventional chemotherapeutics or other anti-cancer treatment modalities [13]- [15].
The potential for selective and simultaneous "targeted" delivery of a single or multiple chemotherapeutic agents or pharmaceuticals at two or more uniquely or over-expressed trophic receptor complexes for the purpose of evoking an enhanced level of anti-neoplastic cytotoxicity or other types of a biological effect against specific cancer cell types remains a facet of oncology and pharmacology that has not been extensively delineated. Based on the increased level of anti-neoplastic cytotoxicity that can potentially be gained through dual simultaneous selectively targeted" epirubicin delivery at trophic receptors over-expressed (EGFR) and highly over-expressed (HER2/neu) by chemotherapeutic resistant mammary adenocarcinoma (SKBr-3) [16] the concept of this molecular strategy does have therapeutic merit. Reported in this research investigation is the anti-neoplastic cytotoxicity of gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) applied simultaneously as a dual selectively "targeted" chemotherapeutic regimen. The strategy has clinical relevance in part due to the effectiveness of gemcitabine, especially in combination with paclitaxel, carboplatin and cisplatin following anthracycline failure in the treatment of metastatic breast cancer [17]. The objective of the research investigations was to determine if simultaneous selective "targeted" delivery of two covalent gemcitabine immunochemotherapeutics is possible at two different endogenous trophic receptor sites over-expressed on the surface membrane of a neoplastic cell type and establish the potential for [Se]-methylselenocysteine to complement the anti-cancer cytotoxic potency attained with this molecular strategy.

Mammary Adenocarcinoma: Neoplastic Disease ex-Vivo Model
Mammary Adenocarcinoma Tissue Culture Cell Culture-The human mammary adenocarcinoma (SKBr-3) was utilized as an ex-vivo model for neoplastic disease. Populations of the mammary adenocarcinoma (SKBr-3) were propagated at ≥85% level of confluency in 150-cc 2 tissue culture flasks containing McCoy's 5a Modified Medium supplemented with fetal bovine serum (10% v/v) and penicillin-streptomycin at a temperature of 37°C under a gas atmosphere of air (95%) and carbon dioxide (5% CO 2 ). Trypsin or any other biochemically active enzyme fraction were not used to facilitate harvest of mammary adenocarcinoma SKBr-3 cell suspensions for seeding of tissue culture flasks or multi-well tissue culture plates. Growth media was not supplemented with growth factors, growth hormones or any other type of growth stimulant.
Anti-neoplastic cytotoxicity for gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 4 -amide)-[anti-EGFR] were measured by removing all contents within the 96-well microtiter plates manually by pipette followed by serial rinsing of monolayers (n = 3) with PBS followed by incubation with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide vitality stain reagent formulated in RPMI-1640 growth media devoid of pH indicator or bovine fetal calf serum (MTT: 5 mg/ml). During an incubation period of 3 -4 hours at 37°C under a gas atmosphere of air (95%) and carbon dioxide (5% CO 2 ) the enzyme mitochondrial succinate dehydrogenase was allowed to convert the MTT vitality stain reagent to navy-blue formazone crystals within the cytosol of mammary adenocarcinoma (SKBr-3) populations (some reports suggest that NADH/NADPH dependent cellular oxidoreductase enzymes may also be involved in the biochemical conversion process). Contents were then removed from each of the 96-wells in the microtiter plate, followed by serial rinsing with PBS (n = 3). The resulting blue intracellular formazone crystals were dissolved with DMSO (300 μl/well) and then spectrophotometric absorbance of the resulting blue-colored supernatant measured at 570 nm using a computer-integrated microtiter plate reader.

Discussion
Despite their common application in modern clinical oncology, conventional chemotherapeutics when given as a monotherapy at dosages that produce safe plasma concentrations almost invariably lack sufficient potency or efficacy to completely resolve most neoplastic disease states. Even when applied as prescribed chemotherapeutic administration is frequently accompanied by some degree of risk for inducing serious sequelae especially during periods of long-term utilization. Newer treatment modalities such as monoclonal immunoglobulin that inhibit function of trophic membrane receptors frequently over-expressed by many neoplastic cell types offer an opportunity to avoid many of the common side effects associated with conventional chemotherapeutics. Unfortunately, most monoclonal immunoglobulin-based therapies that inhibit function of HER2/neu, EGFR, VEGF, IGFR and other uniquely or highly over-expressed trophic receptors are usually only capable of promoting declines in proliferation rate and are largely incapable of evoking cytotoxic activity sufficient to effectively resolve most aggressive or advanced forms of neoplastic disease [7]- [12] [29]- [39]. Inability of most anti-trophic immunoglobulins to exert significant cytotoxic efficacy in-vivo is in part associated with the detection of increases in cell-cycle G 1 -arrest, cellular transformation to states of apoptosisresistance [30], and selection for resistant sub-populations [31] [35] that can be further complicated by frequent reversal of tumor growth inhibition [31] and resumed trophic receptor over-expression [29] following discontinuation of immunoglobulin therapy. Greater levels of anti-neoplastic cytotoxicity are alternatively attainable when anti-trophic receptor immunoglobulin are utilized in dual combination with conventional chemotherapeutics or other cancer treatment modalities [13]- [15].
A small collection of semi-synthetic heterobifunctional organic chemistry reactions can be used to covalently bond gemcitabine to monoclonal immunoglobulin, receptor ligands (e.g. EGFR) or other biologically active protein fractions. One potential method involves creation of a covalent bond structure at the cytosine 2 monoamine group of gemcitabine [40]- [44] either as a direct covalent bond to a ligand or for the purpose of creating a chemically reactive gemcitabine intermediate. Similar molecular strategies have been employed to synthesize covalent anthracycline immunochemotherapeutics through the creation of a covalent bond structure at the α-monoamine (C 3 -amino) group of the carbohydrate-like moiety of doxorubicin, daunorubicin, epirubicin and other related agents in this class of chemotherapeutics [16] [45]- [56]. Generation of a covalent bond at the C 5 -methylhydroxy group of gemcitabine represents an alternative molecular strategy for the synthesis covalent gemcitabine-ligand biopharmaceuticals [41] [44] [57]- [61].
Covalent immunochemotherapeutics can be synthesized that promote selective "targeted" chemotherapeutic delivery in a manner that evoke greater levels of anti-neoplastic cytotoxic potency than the corresponding non-covalent "free" or "parent" form of a chemotherapeutic moiety [16] [18] [25] [63]- [68]. Several molecular mechanisms and cellular processes can be modulated for the purpose of optimizing and enhancing properties that ultimately influence anti-neoplastic cytotoxic potency. Biological activity of the immunoglobulin component of gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] directly facilitates their binding-avidity for trophic membrane receptor sites (e.g. anti-EGFR, anti-HER2/neu) that in turn affords several properties which significantly contribute to the total anti-neoplastic cytotoxic potency of these covalent immunochemotherapeutics. Monoclonal immunoglobulin selected for the synthesis of covalent immunochemotherapeutics should ideally possess several distinct properties that include selective binding-avidity for specific antigenic "sites" on the external surface membrane of cancer cells that are themselves uniquely or highly over-expressed compared to normal, healthy tissues and organ systems. Utilizing immunoglobulin fractions that possess these characteristics allows them to effectively function as a molecular platform that can facilitate selective "targeted" chemotherapeutic delivery in addition to the potential capacity to promote progressive and continual membrane deposition of the chemotherapeutic moiety. The chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) cell type over-expresses EGFR (2.2 × 10 5 /cell) and highly over-expresses HER2/neu (1 × 10 6 /cell) on its exterior surface membrane which promotes selectively "targeted" delivery and progressive membrane deposition of gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] at two different endogenous trophic membrane receptor sites. Progressive membrane deposition of gemcitabine-(C 4 -amide)-[anti-EGFR], gemcitabine-(C 4 -amide)-[anti-HER2/neu] or any other analogous covalent immunochemotherapeutic continues as long as sufficient covalent immunochemotherapeutic is present and EGFR and HER2/neu are expressed and re-expressed on the exterior surface membrane. Given this perspective, one of the most critically important mathematical variables related to cancer cell biology that can significantly determine the anti-neoplastic cytotoxicity of covalent immunochemotherapeutics like gemcitabine-[anti-HER2/neu], [19] [24] gemcitabine-[anti-EGFR], epirubicin-[anti-HER2/neu] [16] [18] [25] or epirubicin-[anti-EGFR], [16] is the expression density of "sites" on the external surface membrane of neoplastic cells utilized to facilitate the selective "targeted" delivery of chemotherapeutic moieties.
In direct accord with the inter-dependent relationship between the immunoglobulin component of covalent immunochemotherapeutics and the biological characteristics of neoplastic cell types, there are other variables in addition to the expression density of membrane-associated "target" sites that significantly determine the anti-neoplastic cytotoxicity of gemcitabine-[anti-HER2/neu], [ [16] and similar covalent immunochemotherapeutics. When uniquely or over-expressed endogenous receptors that are actively internalized by processes of receptor-mediated endocytosis [69] have been selected as sites to facilitate the selective "targeted" delivery and membrane deposition of a chemotherapeutic moieties, it then becomes possible to minimize or avoid simple "coating" of the exterior surface membrane with covalent immunochemotherapeutics like gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu]. Importance of this consideration is based on the realization that in general, it is a prerequisite for most classical chemotherapeutic agents like gemcitabine that possess mechanisms-of-action that is dependent upon enter into the cytosol or nuclear environments in order to create a biological effect. Such processes are assumed to not be a requirement for anti-cancer therapeutics that are membrane-active agents or radioimmunopharmaceuticals that have mechanisms-of-action that do not require entry into cytosol or nuclear environments (e.g. [ 213 Bi or 211 At or 224 Ra]-anti-TAG-72 for colon carcinoma).
Uniquely or over-expressed endogenous receptor types known to be actively internalized by mechanisms of receptor-mediated endocytosis in response to physical binding of immunoglobulin or receptor ligands represents one of the more preferred type of sites on exterior surface membrane of neoplastic populations that can be utilized to selectively "target" chemotherapeutic delivery while also potentially facilitating profound cytosol chemotherapeutic moiety accumulation [69] in addition to preventing or minimizing distribution into and deposition within populations of non-neoplastic cell types (e.g. normal tissues and healthy organ systems). Between different endogenous receptor types and different neoplastic cell populations, variations undoubtedly exist in the rate and extent to which covalent immunochemotherapeutics are deposited on the external surface membrane and are subsequently internalized following the initiation of receptor-mediated-endocytosis [69]. Although specific data for EGFR and HER2/neu receptor-mediated endocytosis in populations of mammary adenocarcinoma (SKBr-3) is somewhat limited, other neoplastic cell types like metastatic multiple myeloma are known to internalize and metabolize approximately 8 × 10 6 molecules of anti-CD74 monoclonal antibody per day [70]. In this context, the collective implications of; [i] selective "targeted" delivery and physical binding at over-expressed and highly over-expressed endogenous receptor sites (e.g. EGFR, HER2/ neu); [ii] continual and progressive membrane deposition; [iii] initiation of receptormediated endocytosis; and [iv] re-expression/replenishment of uniquely or highly over-expressed endogenous receptors is the potential for gemcitabine-(C 4 -amide)-[anti-EGFR] and gem-citabine-(C 4 -amide)-[anti-HER2/neu] and analogous covalent immunochemotherapeutics to promote chemothe-rapeutic moiety accumulation within the cytosol. The degree of cytosol accumulation can approach concentrations that are 8.5× [67] to 100× [71] fold greater than levels attainable by simple passive diffusion of most conventional small molecular weight chemotherapeutics from the extracellular fluid compartment following intravenous administration at clinically-relevant (safe) dosages. Intracellular accumulation of chemotherapeutic moieties of covalent immunochemotherapeutics can therefore continue to occur in neoplastic populations that have been sub-lethally injured as long as they retain the capacity to be uniquely or highly over-express which can be directly influence by the rate at which endogenous membrane receptors are replenished following initial phases of active internalization by mechanisms of receptor-mediated endocytosis [69]. The degree to which such phenomenon occur therefore directly influences and contributes to the potency of gemcitabine-(C 4 -amide)-[anti-EGFR], gemcitabine-(C 4 -amide)-[anti-HER2/neu], gemcitabine-EGF and analogous covalent biochemotherapeutics. Conservative speculation suggests that dual-combinations of covalent immunochemotherapeutics like gemcitabine-(C 4 -amide)-[anti-EGFR] with gemcitabine-(C 4amide)-[anti-HER2/neu] promote greater levels of simultaneous selective "targeted" gemcitabine delivery/membrane deposition and intracellular gemcitabine internalization at both EGFR and HER2/neu endogenous receptors than can be achieved through selective "targeted" gemcitabine delivery at only a single endogenous membrane receptor overexpressed on the exterior surface membrane of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). The promotion of relatively high cytosol chemotherapeutic concentrations within a short confined time period at least in theory decreases the opportunity and frequency that neoplastic cell sub-populations can develop certain forms of (acquired) chemotherapeutic-resistance.
Enhanced levels of anti-neoplastic cytotoxicity that are potentially attainable with a dualcombination of gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] chemotherapeutic-resistant neoplastic cell types can be attributed to physical properties associated with the relatively large molecular weight of selective delivery platforms that chemotherapeutic moieties are often covalently bound to (e.g. IgG MW = 150,000 vs gemcitabine MW = 263.198). Covalent bonding of chemotherapeutics to molecular delivery platforms of relatively large molecular weight effectively prolongs the intravascular pharmacokinetic profile of chemotherapeutic moieties in part because they are no longer removed as rapidly or as extensively from the plasma compartment by renal glomerular filtration (MWCO = 50 -60 kDa) and excreted into the urine. Furthermore, the chemotherapeutic moiety of covalent immunochemotherapeutic agents do not distribute as extensively into cell populations residing within normal tissues and healthy organ systems because of the large molecular weight of the selective delivery platform (e.g. IgG = 150-kDa) which prevents simple passive diffusion across intact lipid bilayer membranes. The latter consideration is significant because a significantly large percentage of the total dose for a conventional small molecular weight chemotherapeutics within the intravascular compartment ultimately does passively diffuse across and enter the cytosol environment of cell populations in normal tissues and healthy organ systems.
The large molecular weight of the immunoglobulin component of gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] or analogous covalent immunochemotherapeutics also inhibits through mechanisms of steric hinderance, the function of biological entities that can utilize chemotherapeutics moieties as a molecular substrate. Enzymes like cytosine deaminase are not as efficient in biochemically degrading or inactivate gemcitabine when it is a moiety within a covalent immunochemotherapeutic. Presumably, at least some degree steric hinderance phenomenon are also responsible for the observation that the non-selective transmembrane efflux "pump", P-glycoprotein (MDR-1: multi-drug resistance protein) [59] commonly responsible for imparting chemotherapeuticresistance in many neoplastic cell types [72]- [77] is less effective in promoting resistance when chemotherapeutic moieties are formulated as covalent immunochemotherapeutics [51] [77]- [80]. Such attributes may in part correlate with the detection of a relatively large proportion of anthracyclines (>50%) retained intracellularly 24-hours post selective "targeted" delivery [67] where they are found primarily associated with membrane structures or it becomes distributed throughout the cytosol environment [69] [81]. Alternatively, non-covalently bound or "free" anthracycline following passive diffusion across an intact lipid bi-layer membrane is detected primarily within complexes associated with nuclear DNA less than 30 minutes after initial exposure [69]. The anthracycline moiety liberated from covalent immunochemotherapeutics reportedly distributes preferentially into, and accumulates within the nucleus, mitochondria and golgi apparatus [26]. The covalent bonding of gemcitabine to monoclonal immunoglobulin similar to gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] could therefore function as a molecular strategy for combating patterns of chemotherapeutic resistance in neoplastic cell types. Fortunately, EGFR and HER2/neu trophic membrane receptors are both overexpressed in several resistant forms of breast cancer [82]- [84] where their refractory response to chemotherapy is associated with an over-expression of transmembrane Pglycoprotein [85]- [90]. Recognition of these inter-relationships between cancer cell biology and selective "targeted" chemotherapeutic delivery directly correlates with the frequent association between chemotherapeutic-resistance, elevated cancer cell survival parameters, and increased proliferation rates (e.g. relevant to local invasiveness and metastatic dissemination) [91] [92].
Utilization of endogenous trophic membrane receptors that regulate neoplastic cell proliferation and viability as "sites" to facilitate selective "targeted" chemotherapeutic delivery on the exterior surface membrane provides an opportunity to potentially exert cytotoxic properties that are independent of those associated with the chemotherapeutic moiety. Most therapeutic immunoglobulins with binding-avidity for uniquely or highly overexpressed endogenous trophic membrane receptors competitively "block" binding of receptor ligands (e.g. EGF ⇉| IgG::EGFR). Suppression of neoplastic cell growth and vitality is therefore achieved by preventing activation, or inhibit the biological function of EGFR, HER2/neu, IGFR, VEGFR and similar trophic membrane receptors that directly or indirectly regulate proliferation kinetics, metastatic behavior and chemotherapeuticresistance. Similarly, internalization of EGFR, HER2/neu, IGFR, VEGFR or analogous endogenous trophic membrane receptors by mechanisms of receptor-mediated-endocytosis promotes transient down-regulation, or partial to complete depletion of their expression resulting in declines in membrane density that lead to suppression of neoplastic cell viability and proliferation rate. In order for this phenomenon to occur trophic membrane receptor "sites" must to a variable degree become physically depleted in a manner that is partially due to a deficient rate of re-expression and replenishment to original baseline levels. The rate and extent at which trophic receptor complexes are internalized by immunoglobulininduced receptor-mediated endocytosis is directly determined by the; [i] availability (quantity and concentration) of covalent immunochemotherapeutics; [ii] expression density of membrane "sites" utilized to facilitate selective "targeted" chemotherapeutic delivery and progressive membrane deposition; and the [iii] corresponding rate and extent that uniquely or highly over-expressed trophic membrane receptors or similar "sites" are re-expressed and replenished on the exterior surface of neoplastic cell populations. [ii] opsonization secondary to immunoglobulin binding at endogenous trophic membrane receptor sites and subsequent formation of IgG/receptor/complement complexes (e.g. induced macrophage phagocytosis); and [iii] antibody-dependent cell-mediated cytotoxicity (ADCC: classically requiring recruitment of NK/natural killer lymphocytes or to a lesser degree participation of macrophages, neutrophils and eosinophils. Collectively these three host immune responses represent the major mechanism of selective anti-neoplastic cytotoxicity evoked by anti-CD20, anti-CD52 and similar monoclonal immunoglobulins utilized for the therapeutic management of haemopoietic neoplasias (e.g. chronic lymphocytic leukemia). Despite the potential for gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] to collectively stimulate complement C9 mediated lysis, ADCC responses and promote IgG/complement facilitated opsonization of neoplastic cells in a manner that attains enhanced levels of selective anti-neoplastic cytotoxicity, it continues to be technically difficult to simultaneously simulate and accurately measure each of these three immune-dependent responses utilizing ex-vivo models for neoplastic disease states.
In clinical scenarios were immunoglobulin fractions are utilized to selectively "target" delivery of therapeutic pharmaceuticals or diagnostic imaging agents in nuclear medicine the antibody component can be biochemically modified with enzyme preparations like papain in order to cleave and remove the Fc segment of the IgG molecule. Biochemical modifications of this type minimize non-selective binding of immunochemotherapeutics to Fc receptors expressed by cell types that comprise the RE system (mononuclear phagocytic system) that anatomically reside within the spleen and liver. Unfortunately, such biochemical modifications create a covalent immunochemotherapeutic composed predominantly of only F(ab′) 2 or Fab′ that have less of a capacity to activate the complement cascade (e.g. C9 cytolysis, C3b/C4b opsonization), increase neoplastic cell opsonization (e.g. macrophage Fc receptor dependent binding), or promote stimulation of ADCC (e.g. NK lymphocyte Fc receptor dependent binding).

Level-1
Greater concentrations of selectively "targeted" chemotherapeutic concentrations within the cytosol of neoplastic cell populations that presents a potential opportunity for resolving neoplastic cell types that are partially resistant when the "parent" conventional chemotherapeutic is administered intravenously at clinically relevant and safe dosages. Dual selective "targeted" chemotherapeutic delivery also represents a strategy for combating chemotherapeutic resistance as can occur with alterations in P-glycoprotein expression [82]- [84].

Level-2
Dual simultaneous inhibition of the biological functions and properties of multiple both endogenous trophic membrane receptors or other sites over-expressed on the exterior surface chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Synergistic levels of antineoplastic cytotoxicity achieved solely through inhibition of multiple endogenous trophic membrane receptors or analogous biological "targets" can only theoretically be achieved if each different site has distinctly different biological functions/properties within a given neoplastic cell type (e.g. EGFR-vs-HER2/neu or CD20-vs-CD74) [93] [94].

Level-4
Simultaneous binding of covalent immunochemotherapeutic combinations like gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] at two different over-expressed trophic receptor types on the exterior surface membrane of a single cancer cell population in-vivo offers the potential to attain a third plane of additive and synergistic anti-neoplastic cytotoxicity from innate immune response mechanisms. Selectively "targeted" additive or synergistic anti-neoplastic cytotoxicity can potentially occur in-vivo through the different combined properties of; [i] complement C9 mediated cytolysis; [ii] IgG/receptor/complement-facilitated opsonization; and [iii] IgG-dependent cell-mediated cytotoxicity (ADCC). Conceptually, at least, the simultaneous binding of gemcitabine-(C 4amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER 2/neu] at two different endogenous trophic receptors on the same cancer cell type offers the probability of evoking a greater degree of selectively "targeted" anti-neoplastic cytotoxicity compared to the selective binding of just a single covalent gemcitabine immunochemotherapeutic.

Dual simultaneous combinations of gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] in-vivo presents an opportunity to potentially attain still another plane of additive and synergistic anti-neoplastic cytotoxicity that involves; [i] gemcitabine in dual-combination with innate immune responses; [ii] trophic receptor inhibition in dual-combination with innate immune responses; and/or [iii] gemcitabine, trophic receptor inhibition and innate immune responses.
In support of this concept, immune cell populations that are involved in ADCC phenomenon release cytotoxic components known to additively and synergistically enhance the cytotoxic anti-neoplastic activity of conventional chemotherapeutic agents [105]. Undoubtedly, other immune responses also contribute to the anti-neoplastic properties of many conventional chemotherapeutic agents. Recognition of the phenomenon where different immune-dependent responses become a significant component of additive and synergistic anti-neoplastic cytotoxicity phenomenon in active partnership with chemotherapeutic moieties and trophic receptor inhibition at least in part delineates how covalent immunochemotherapeutics frequently evoke greater efficacy when implemented in-vivo compared to levels of anti-neoplastic cytotoxicity observed utilizing ex-vivo based models for neoplastic disease even when the same identical cancer cell types (xenographs) are utilized [106]- [108]. Each of the qualities and properties discussed for the selective "targeted" chemotherapeutic delivery and additive or synergistic interactions that can be evoked by gemcitabine-(C 4 -amide)-[anti-EGFR] and gemcitabine-(C 4 -amide)-[anti-HER2/neu] collectively serve to explain how the dual-combination of these two covalent immunochemotherapeutics produced additive levels of anti-neoplastic cytotoxicity measured in chemotherapeutic-resistant mammary-adenocarcinoma (SKBr-3) populations functioning as an ex-vivo model for neoplastic disease (Figure 8). In part, the basis for this perception originates from the observation that when gemcitabine-(C 4 -amide)-  [61] and mammary adenocarcinoma (BG-1) [61] are both known to be relatively resistant to gemcitabine and covalent gemcitabine-(oxyether phopholipid). The two covalent gemcitabine immunochemotherapeutics likely would have evoked greater levels of antineoplastic cytotoxicity if it had been measured utilizing populations of pancreatic carcinoma, [109] small-cell lung carcinoma, [110] neuroblastoma, [111] [61] oral squamous cell carcinoma, [61] and prostatic carcinoma [40] have been found to be sensitive to gemcitabine and gemcitabine-(oxyether phopholipid) covalent chemotherapeutic conjugates.
iv. Assessment of neoplastic cellular proliferation with either [ 3 H]-thymidine, or an ATP-based assay method would likely have resulted in recognizing lower degrees of early anti-neoplastic cytotoxicity because these analytical modalities reportedly are ≥10-fold more sensitive in detecting lower degrees of early sub-lethal antineoplastic cytotoxicity compared to MTT vitality stain based assay methods [115] [116]. In spite of this perception, MTT vitality stain based assays continue to be extensively applied for the routine assessment of true anti-neoplastic cytotoxicity of chemotherapeutics covalently incorporated synthetically into molecular platforms that provide properties of selective "targeted" delivery [16] [59]- [61] [117]- [122]. One notable and significant advantage of MTT vitality stain based assay and other methods applying similar reagents is their ability to indirectly detect and measure lethal anti-neoplastic cytotoxic potency. Potency measured ex-vivo In this manner is generally considered to be superior to merely detecting early-stage sub-lethal cellular injury that could potentially be reversible and be more difficult to correlated with in-vivo levels of efficacy and potency. Levels of anti-neoplastic cytotoxicity vary between different organoselenium compounds when assessed independently as a single agent or in combination with conventional chemotherapeutics [123]- [127]. Various forms of selenium have been reported to additively or synergistically complement the anti-neoplastic cytotoxicity of anthracyclines [123]- [126] [128], irinotecan [127] [129]- [131], docetaxel/paclitaxel [124] [132], and tamoxifen [133].
In the presence of selenium the vulnerability of B-cell lymphoma to the anti-neoplastic cytotoxicity of doxorubicin, etoposide, 4-hydroxyperoxycyclophosphamide, melphalan, and 1-β-D-arabinofuranosyl-cytosine increases approximately 2.5-fold (e.g. methylseleninate 10 -100 μM) [134]. Synergism achieved with selenium in dual-combination with conventional chemotherapeutics can ultimately become additive during prolonged periods of challenge (incubation) or when the duration of clinical administration and treatment is extended [128]. Interestingly, selenium exerts greater cytotoxic anti-neoplastic activity compared to celecoxib [135]- [139] when analyzed at micromolar equivalent concentrations.
Selenium can potentially bestow therapeutically beneficial properties through induction of a number of biological effects or responses in neoplastic cell populations such as its capacity to; [i] induce apoptosis in doxorubicin-resistant lung small-cell carcinoma (selenite 10 μM) [140]; [ii] promote severe ER stress (leukemia cell types) [141]; and [iii] reduce vitality of multidrug-resistant leukemia (selenite-triglycerides 10 μg to 40 μg/ml) [142]. Several specific molecular mechanisms explain the anti-neoplastic cytotoxicity induced by selenium.
Selenium (selenite) causes cell death through activation of the pro-apoptotic transcription factor GADD153 and high concentrations in leukemia cells promote p53 activation [141]. Selenium (selenite) independently mediates anti-neoplastic activity through p53 activation and increased oxidative stress which collectively precipitate mitochondrial dysfunction and caspase activation (leukemia cell types) [141]. Elevated levels of oxidative stress occur at relatively high selenium concentrations [141] which is accompanied by, or a direct result of reductions in catalase enzyme activity (H 2 O 2 → H 2 O + O 2 ) [123]. In addition to the influence of selenium on caspase activation, it also promotes apoptosis by increasing Fasassociated death domain (FADD) expression and enhancement of caspase-8 recruitment for Fas and FADD (MCF7 breast cancer) [126]. Selenium is believed to trigger apoptosis by additionally increasing FOXO3a transcriptional factor activity [125] that occurs in concert with Bim [125] and PUMA up-regulation, or alternatively the down-regulation of FLIP antiapoptotic protein. Gradient increases in selenium concentrations (1 μM to 10 μM) induce dose-dependent elevations in the amount and activity of thioredoxin reductase in nonresistant neoplastic cells while precipitating declines in thioredoxin reductase (e.g. doxorubicin-resistant small cell carcinoma) [140]. Thioredoxin reductase biochemically reduces thioredoxin which mediates the final step of the electron-transfer pathway for nucleoside diphosphate reduction where in cancer cells is essential for cell growth and survival.
In populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3), methylseleninate [125] [126] had greater anti-neoplastic cytotoxic potency than [Se]methylselenocysteine [129] [133] at selenium-equivalent concentrations of 10 μM and 20 μM but they were similar at the selenium-equivalent concentrations of 30 μM, 40 μM and 50 μM (Figure 9). Selenium also can potentially contribute to the efficacy of conventional small molecular weight chemotherapeutic agents. In chemotherapeutic-resistant mammary adenocarcinoma (MCF-7) selenium increases sensitivity to anthracyclines [125] and in combination with doxorubicin it influences Fas signaling [126] at methylseleninate concentrations of 2.5 μM and 5 μM respectively. Selenium also increases mitochondrial caspase-9 activation which promotes apoptosis and produces synergistic levels of antineoplastic cytotoxicity in combination with anthracyclines (e.g. mammary adenocarcinoma MCF7 cell type). [126] Analogous investigations determined that selenium in the form of methylseleninate also complements the anti-neoplastic cytotoxic efficacy of selectively "targeted" covalent immunochemotherapeutics including epirubicin-(C 13 -imino)-[anti-HER2/neu] [25]. The organoselenium agent, [Se]-methylselenocysteine in preference to methylseleninate was evaluated to determine if it could complement the anti-neoplastic cytotoxicity of the two covalent gemcitabine immunochemotherapeutics applied in dual simultaneous combination because it was considered more suitable for in-vivo administration (Figure 9 and Figure  property of selenium that may be particular beneficial for improving the efficacy and potency of covalent immunochemotherapeutics or covalent [receptor ligand]chemotherapeutics with binding-avidity for over-expressed endogenous membrane receptor sites is an ability to potentially improve their internalization by mechanisms of receptormediated-endocytosis [143]. Such claims are however somewhat speculative since they are based on the observation that selenium and α-tocopherol deficiencies reduce receptormediated processes possibly associated with greater levels of membrane oxidation and alterations in membrane fluidity. Interpretation of the anti-neoplastic cytotoxicity analysis of organoselenium analogs in the form of [Se]-methylselenocysteine and methylseleninate suggests that they could be used to achieve specific levels of anti-neoplastic cytotoxicity at lower total gemcitabine-equivalent concentrations of gemcitabine-(C 4 -amide)-[anti-EGFR], gemcitabine-(C 4 -amide)-[anti-HER2/neu] or gemcitabine (Figures 9-11). Conservative extrapolation from this observed result implies that organoselenium compounds when applied in dual simultaneous combination with gemcitabine or covalent gemcitabine immunochemotherapeutics could provide an opportunity for achieving more complete and more rapid resolution of neoplastic conditions while also lowering total chemotherapeutic dosage requirements in a manner that would produce fewer serious side-effects or sequelae.

Conclusions
The covalent immunochemotherapeutics, gemcitabine-(C 4      Relative gemcitabine anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma over challenge (incubation) periods of different duration. Legends: (■) gemcitabine following a 96-hour incubation period; and (◆) gemcitabine following a 182-hour incubation period. Gemcitabine formulated at gradient gemcitabineequivalent concentrations was incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) for a period of either 96-hours or 182-hours. Anti-neoplastic cytotoxicity was measured using a MTT cell vitality assay relative to matched negative reference controls.     for a period of 182-hours. Anti-neoplastic cytotoxicity was measured using a MTT cell vitality assay relative to matched negative reference controls. Relative anti-neoplastic cytotoxicity of organoselenium compounds against chemotherapeutic-resistant human mammary adenocarcinoma. Legends: (◆) [Se]methylselenocysteine; and (■) methylseleninate. Individual organoselenium compounds formulated at gradient selenium-equivalent concentrations was incubated in direct contact with triplicate populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Anti-neoplastic cytotoxicity was measured using a MTT cell vitality assay relative to matched negative reference controls.  Relative gemcitabine anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma over challenge (incubation) periods of different duration. Legends: (◆) gemcitabine following a 96-hour incubation period; and (■) gemcitabine following a 182-hour incubation period. Gemcitabine formulated in triplicate at gradient gemcitabine-equivalent concentrations was incubated in direct contact with triplicate populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) during incubation periods of 96-hours or 182-hours. Anti-neoplastic cytotoxicity was measured using a MTT cell vitality assay relative to matched negative reference controls.