Anti-Neoplastic Cytotoxicity of Gemcitabine-(C4-amide)-[anti-HER2/neu] in Combination with Griseofulvin against Chemotherapeutic-Resistant Mammary Adenocarcinoma (SKBr-3)

Introduction Gemcitabine is a pyrimidine nucleoside analog that becomes triphosphorylated and in this form it competitively inhibits cytidine incorporation into DNA strands. Diphosphorylated gemcitabine irreversibly inhibits ribonucleotide reductase thereby preventing deoxyribonucleotide synthesis. Functioning as a potent chemotherapeutic, gemcitabine decreases neoplastic cell proliferation and induces apoptosis which accounts for its effectiveness in the clinical treatment of several leukemia and carcinoma cell types. A brief plasma half-life due to rapid deamination, chemotherapeuticresistance and sequelae restricts gemcitabine utility in clinical oncology. Selective “targeted” gemcitabine delivery represents a molecular strategy for prolonging its plasma half-life and minimizing innocent tissue/organ exposure. Methods A previously described organic chemistry scheme was applied to synthesize a UV-photoactivated gemcitabine intermediate for production of gemcitabine-(C4-amide)-[anti-HER2/neu]. Immunodetection analysis (Western-blot) was applied to detect the presence of any degradative fragmentation or polymerization. Detection of retained binding-avidity for gemcitabine-(C4-amide)-[anti-HER2/neu] was determined by cell-ELISA using populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) that highly over-express the HER2/neu trophic membrane receptor. Anti-neoplastic cytotoxicity of gemcitabine-(C4-amide)-[anti-HER2/neu] and the tubulin/microtubule inhibitor, griseofulvin was established against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Related investigations evaluated the potential for gemcitabine-(C4-amide)-[anti-HER2/neu] in dual combination with griseofulvin to evoke increased levels of anti-neoplastic cytotoxicity compared to gemcitabine-(C4-amide)-[anti-HER2/neu]. Results Covalent gemcitabine-(C4-amide)-[anti-HER2/neu] immunochemotherapeutic and griseofulvin exerted anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Covalent gemcitabine-(C4-amide)-[anti-HER2/neu] immunochemotherapeutic or gemcitabine in dual combination with griseofulvin created increased levels of anti-neoplastic cytotoxicity that were greater than was attainable with gemcitabine-(C4-amide)-[anti-HER2/neu] or gemcitabine alone. Conclusion Gemcitabine-(C4-amide)-[anti-HER2/neu] in dual combination with griseofulvin can produce enhanced levels of anti-neoplastic cytotoxicity and potentially provide a basis for treatment regimens with a wider margin-of-safety. Such benefits would be possible through the collective properties of; [i] selective “targeted” gemcitabine delivery; [ii] relatively lower toxicity of griseofulvin compared to many if not most conventional chemotherapeutics; [iii] reduced total dosage requirements faciliated by additive or synergistic anti-cancer properties; and [iv] differences in sequelae for gemcitabine-(C4-amide)-[anti-HER2/neu] compared to griseofulvin functioning as a tubulin/microtubule inhibitor.

The anthracyclines have traditionally been the class of chemotherapeutics most commonly bound covalently to (large) molecular platforms that can facilitate "selective" targeted delivery. Gemcitabine, in contrast to the anthracyclines, is a chemotherapeutic that has less frequently been covalently bound to large molecular weight platforms that can provide various biological properties [20,21] including selective "targeted" delivery [22,23]. Gemcitabine is a deoxycytidine nucleotide analog with a mechanism-of-action that is dependent upon intracellular triphosphoralation which allows it to substitute for cytidine during DNA transcription. In this capacity triphosphoralated gemcitabine both inhibits DNA polymerase biochemical activity and it becomes incorporated into DNA strands. A second mechanism-of-action involves gemcitabine inhibiting and inactivating ribonucleotide reductase in concert with suppression of deoxyribonucleotide synthesis, diminished DNA repair, and declines in DNA transcription. Each of these mechanisms-of-action contributes to initiating the onset of apoptosis. In clinical oncology, gemcitabine is administered for the treatment of certain leukemias and potentially different types of lymphoma in addition to a spectrum of adenocarcinomas and carcinomas affecting the lung (e.g. non-small cell), pancrease, bladder and esophogus. The plasma half-life for gemcitabine is brief because it is rapidly deaminated to an inactive metabolite that is then redily eliminated through renal excretion into the urine [24][25][26].
Despite general familiarity with the influence of anti-HER2/neu immunoglobulin on the viability and vitality of cancer cell populations and it's application in clinical oncology, there is surprisingly little known about covalent gemcitabine-(anti-HER2/neu) immunochemotherapeutics and their potential to exert selectively "targeted" anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma [22,23]. Several distinct attributes can be realized through the molecular design and organic chemistry synthesis of a covalent gemcitabine immunochemotherapeutic that in part include the properties of selective "targeted" chemotherapeutic delivery, continual chemotherapeutic deposition, progressive intracellular chemotherapeutic accumulation, and extended plasma chemotherapeutic pharmacokinetic profiles. Presumably due steric hinderance phenomenon, gemcitabine covalently bound to large molecular weight platforms like immunoglobulin is also less vulnerable to MDR-1 (multi-drug resistance efflux pump) [27,28], or biochemical deamination by cytidine deaminase and deoxycytidylate deaminase (following gemcitabine phosphorylation). Covalently bonding gemcitabine to immunoglobulin or molecular ligands also provides opportunities for attaining additive or synergistic levels of anti-neoplastic cytotoxicity. One approach to attaining additive or synergistic anti-neoplastic cytotoxicity properties includes the utilization of large molecular weight platforms like anti-HER2/neu, anti-EGFR and similar monoclonal immunoglobulin fractions that provide a mechanism for simultaneously achieving selective "targeted" chemotherapeutic delivery and suppress biological vitality in neoplastic cell populations that are heavily dependent on trophic receptor over-expression.

Synthesis of Gemcitabine-(C 4 -amide)-[anti-HER2/neu] Immunochemotherapeutic
Phase-I synthesis scheme for UV-photoactivated gemcitabine-(C 4 -amide) intermediates-The cytosine-like C 4 -amine of gemcitabine (0.738 mg, 2.80×10 -3 mmoles) was reacted at a 2.5:1 molar-ratio with the amine-reactive N-hydroxysuccinimide ester "leaving" complex of succinimidyl 4,4-azipentanoate (0.252 mg, 1.12×10 -3 mmoles) in the presence of triethylamine (TEA 50 mM final concentration) utilizing dimethylsulfoxide as an anhydrous organic solvent system ( Figure 1). The reaction mixture formulated from stock solutions of gemcitabine and succinimidyl 4,4-azipentanoate was continually stirred gently at 25°C over a 4-hour incubation period in the dark and protected from exposure to light. The relatively long incubation period of 4 hours was utilized to maximize ester group degradation associated with any residual succinimidyl 4,4-azipentanoate that may not of reacted in the first 30 to 60 minutes with the C 4 cytosine-like mono-amine group of gemcitabine.
Molecular mass/Size-dependent separation by non-reducing SDS-PAGE-The covalent gemcitabine-(C 4 -amide)-[anti-HER2/neu] immunochemotherapeutic and anti-HER2/neu immunoglobulin fraction reference control were adjusted to a standardized protein concentration of 60 μg/ml and then combined 50/50 v/v with conventional SDS-PAGE sample preparation buffer (Tris/glycerol/bromphenyl blue/SDS) formulated without 2-mercaptoethanol or boiling. Each covalent immunochemotherapeutic, the reference control immunoglobulin fraction (0.9 μg/well) and a mixture of pre-stained reference control molecular weight markers were then developed by non-reducing SDS-PAGE (11% acrylamide) performed at 100 V constant voltage at 3°C for 2.5 hours.
Western-blot immunodetection analyses-Covalent gemcitabine-(C 4 -amide)-[anti-HER2/neu] immunochemotherapeutic following mass/size-dependent separation by nonreducing SDS-PAGE were equilibrated in tank buffer devoid of methanol. Mass/sizeseparated gemcitabine and anthracycline anti-HER2/neu immunochemotherapeutics contained in acrylamide SDS-PAGE gels were then transferred laterally onto sheets of nitrocellulose membrane at 20 volts (constant voltage) for 16 hours at 2° to 3°C with the transfer manifold packed in crushed ice.
Populations of the mammary adenocarcinoma (SKBr-3) cell line were propagated 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 ). Tissue culture media was not supplemented with growth factors, growth hormones or other growth stimulants of any type. Investigations were performed using mammary adenocarcinoma (SKBr-3) monolayer populations at a >85% level of confluent.
Cytotoxic potency of gemcitabine-(C 4 -amide)-[anti-HER2/neu] or griseofulvin was measured by removing all contents within the 96-well microtiter plates manually by pipette followed by serial rinsing of monolayers with PBS (n=3) and incubation with 3-[4,5dimethylthiazol-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 a 3-to-4 hour incubation period 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 conversion process). Contents of the 96-well microtiter plate were then removed followed by serial rinsing with PBS (n=3). The resulting blue intracellular formazone crystals were dissolved with DMSO (300 μl/well) and then the spectrophotometric absorbance of the blue-colored supernantant measured at 570 nm using a computer integrated microtiter plate reader.

General
The inhibition of neoplastic cell vitality by anti-trophic immunoglobulin fractions including anti-HER2/neu, anti-EGFR, anti-VEGF or anti-IGF-1 is almost invariably compromised by their inability to evoke effective levels of anti-neoplastic cytotoxicity due to their tendency to promote elevated levels of cell-cycle G 1 -arrest, increased states of apoptosis-resistance [55], and selection for resistant sub-populations [1,2]. Transformations of this type can be further complicated by frequent reversal of tumor growth inhibition [1] and relapse trophic receptor over-expression [56] following discontinuation of administration. The antineoplastic properties of monoclonal immunoglobulin preparations that inhibit the function of trophic receptor complexes can, however, be complemented in scenarios where they are administered in combination with conventional chemotherapeutics or other cancer treatment modalities [18,19,57].
The molecular design and implementation of succinimidyl 4,4-azipentanoate in organic chemistry reactions schemes to create the UV-photoactivated gemcitabine-(C 4 -amide) intermediate for the synthesis of gemcitabine-(C 4 -amide)-[anti-HER2/neu] [23] or other covalent gemcitabine immunochemotherapeutics has not been extensively delineated to date. Somewhat analogous organic chemistry reaction schemes have however been described in a limited number of investigations for the synthetic production of a covalent gemcitabine-(C 5 -methylhydroxy)-[anti-HER2/neu] immunochemotherapeutic [22]. Gemcitabine-(C 4 -amide)-[anti-HER2/neu] and the organic chemistry reactions utilized in the corresponding synthesis scheme offer several distinct advantages including gentler reaction conditions, greater retained biological activity (IgG binding avidity), greater endproduct yield (due to less IgG degradation or polymerization), flexibility of prolonged storage of the UV-photoactivated chemotherapeutic intermediate, and implementation of a covalent bond forming moiety that lacks any aeromatic ring structure which is known to decrease the the probability of inducting humoral immune responses.
Conceptually there are at least five analystical variables that could have alternatively been modified to achieve substantially higher total levels of anti-neoplastic cytotoxicity for gemcitabine-(C 4 -amide)-[anti-HER2/neu]. First, incubation times with chemotherapeuticresistant mammary adenocarcinoma (SKBr-3) could have been lengthened since longer periods of direct contact (>182-hours) appear to be indicated for covalent gemcitabine immunochemotherapeutics [22,23,28,58,59]. Longer direct contact incubation periods allow a greater opportunity for larger amounts of gemcitabine to be internalized by receptormediated endocytosis and subsequently liberated intracellularly from gemcitabine-(C 4amide)-[anti-HER2/neu] within the phagolysosome following internalization (Figure 4). Second, anti-neoplastic cytotoxicity of gemcibatine-(C 4 -amide)-[anti-HER2/neu] could alternatively have been assessed against a non-chemotherapeutic-resistant human neoplastic cell type similar to those utilized to evaluate majority of the covalent biochemotherapeutics reported in the literature to date. Similarly, the cytotoxic anti-neoplatic potency of gemcibatine-(C 4 -amide)-[anti-HER2/neu] could have alternatively been measured against an entirely different neoplastic cell type that has a relatively higher sensitivity to gemcitabine such as pancreatic carcinoma [60], small-cell lung carcinoma [61], neuroblastoma [62], or leukemia/lymphoid [63,64]. In addition, human promyelocytic leukemia [28,64], T-4 lymphoblastoid clones [64], glioblastoma [28,64] cervical epitheliod carcinoma [64], colon adenocarcinoma [64], pancreatic adenocarcinoma [64], pulmonary adenocarcinoma [64], oral squamous cell carcinoma [64], and prostatic carcinoma [58] have been found to be sensitive to gemcitabine and covalent gemcitabine-(oxyether phopholipid). Within this array of neoplastic cell types both human mammary carcinoma (MCF-7/WT-2') [64] and mammary adenocarcinoma (BG-1) [64] are known to be relatively more resistant to gemcitabine and gemcitabine-(oxyether phopholipid). Presumably this pattern of gemcitabine sensitivity is directly relevant to the cytotoxic anti-neoplatic potency detected for gemcibatine-(C 4 -amide)-[anti-HER2/neu] in chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations ( Figure 4). Third, [ 3 H]-thymidine, or an ATP-based assay could have alternatively been applied to measure anti-neoplastic cytotoxicity of gemcitabine-(C 4 -amide)-[anti-HER2/neu] since they are reportedly >10-fold more sensitive in detecting early sub-lethal cell injury compared to MTT vitality stain assay methods [65,66]. Despite this consideration, MTT vitality stain continues to be extensively applied for routine assessment of true anti-neoplastic cytotoxicity in contrast to transient or sub-lethal injury for chemotherapeutics covalently incorporated synthetically into molecular platforms that provide properties of selective "targeted" delivery [28,44,64,[67][68][69][70][71][72][73]. In this context, one distinctly important attribute of MTT vitality stain based assays is that they provide a way of measuring the extent of cell death induced by an anti-cancer agent within a population of neoplastic cells in a manner that tends to have greater relevance to clinical oncology in contrast to assays for biomarkers that simply reflect transient (non-lethal) cell injury.
Forth, anti-neoplastic cytotoxicity of gemcibatine-(C 4 -amide)-[anti-HER2/neu] immunochemotherapeutic could have been delineated in-vivo against human neoplastic xenographs in animal hosts as a model for human cancer. Many if not most covalent immunochemotherapeutics with properties of selective "targeted" delivery frequently have a higher degree of effectiveness and potency when evaluated in-vivo in contrast to levels acquired ex-vivo in tissue culture models utilizing the same cancer cell type [74][75][76].
Enhanced efficacy and potency is in part attributable to endogenous immune responses including antibody-dependent cell cytotoxicity (ADCC) phenomenon [77] in concert with complemented-mediated cytolysis induced by formation of antigen-immunoglobulin complexes on the exterior surface membrane of "targeted" neoplastic cell populations. During ADCC events cytotoxic components are liberated that additively and synergistically enhance the anti-neoplastic cytotoxicity activity of conventional chemotherapeutic agents [78]. Contributions of ADCC and complement-mediated cytolysis to the in-vivo antineoplastic cytotoxicity of covalent immunochemotherapeutics is further complemented by the additive and synergistic anti-neoplastic properties attained wiith anti-trophic receptor monoclonal immunoglobulin when applied in dual combination with conventional chemotherapeutic agents [18,19,[79][80][81][82][83][84][85][86][87][88]. Additive or synergistic interactions of this type have been delineated between anti-HER2/neu when applied in dual combination with cyclophosphamide, docetaxel, doxorubicin, etoposide, methotrexate, paclitaxel, or vinblastine [19,79].
Fifth, strategies for the synthesis of gemcitabine-(C 4 -amide)-[anti-HER2/neu] could have been modified to increase the gemcitabine molar-incorporation-index. Unfortunately, such modifications usually require the implementation of harsher reaction conditions that in turn impose a higher risk of reduced biological activity (e.g. IgG antigen binding avidity) and substantial declines in final/total product yield [75,89]. Aside from overly harsh synthesis conditions, excessively high molar incorporation indexes for any chemotherapeutic agent can also reduce biological integrity of immunoglobulin fractions when the number of pharmaceutical groups introduced into the Fab' antigen-binding region becomes excessive. Such alterations can result in only modest declines in immunoreactivity (e.g. 86% for a 73:1 ratio) but disproportionately large declines in anti-neoplastic activity in addition to substantial reductions in potency [75].
Biological integrity of the immunoglobulin component of covalent immunochemotherapeutics is critically important because it facilitates selective "targeted" delivery of the chemotherapeutic moiety and it's subsequent internalization by mechanisms of receptor-mediated endocytosis when an appropriate "target" site on the external membrane has been selected [90,91]. Immunoglobulin-induced receptor-mediated endocytosis at membrane HER2/neu complexes ultimately can result in increases in the intracellular concentration of selectively "targeted"/delivered chemotherapeutic that are 8.5 [91] to >100 × fold greater [92] than those attainable by simple passive diffusion. Although specific data for HER2/neu and EGFR expression by mammary adenocarcinoma (SKBr-3) is limited [44], other neoplastic cell types like metastatic multiple myeloma are known to internalize approximately 8×10 6 molecules of anti-CD74 monoclonal antibody per day [93].
Griseofulvin while functioning as a tubulin/microtubulin inhibitor exerted detectable levels of anti-neoplastic cytotoxicity against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) particularly between the final concentrations of 1 μM to 20 μM and it was more potent than methylselenocysteine formulated at equivalent concentration levels ( Figure 5). Increasing the incubation period from 72-hours to 182-hours measurably increased the anti-neoplastic cytotoxicity of griseofulvin ( Figure 6).

Anti-neoplastic cytotoxicity of dual combinations-
The griseofulvin mechanismof-action is similar to the vinca alkaloids, taxanes (e.g. paclitaxel), podophyllotoxins (e.g. etoposide) and monomethyl auristatin E (MMAE). Based on these properties it can be speculated that griseofulvin has a potential capacity to additively or synergistically enhance the anti-neoplastic cytotoxicity of conventional and selectively "targeted" chemotherapeutics. Such properties have to date largely remained unknown except for limited preliminary descriptions for the dual combinations of nocodazole/griseofulvin [95,99] and vinblastine/griseofulvin [94].
The anti-neoplastic cytotoxicity profiles for griseofulvin applied in dual combination with the covalent gemcitabine immunochemotherapeutic or gemcitabine collectively validated speculation that this alternative tublin/microtubule inhibitor can exert complementary levels of efficacy against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) and potentially other neoplastic cell types (Figure 7 and 8). The implications of these findings are in accord with results from previous reports that recognized detectable increases in antineoplastic cytotoxicity activity for covalent epirubicin immunochemotherapeutics and epirubicin against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) when applied in dual combination with griseofulvin [47]. Undoubtedly, levels of anti-neoplastic cytotoxicity for gemcitabine-(C 4 -amide)-[anti-HER2/neu] immunochemotherapeutic in dual combination with griseofulvin (15 μM fixed-concentration) would in all probability have been greater during incubation periods longer than 182-hours.
Discovery that griseofulvin (tubulin/microtubule inhibitor) can independently exert antineoplastic cytotoxicity activity against chemotherapeutic-resistant human adenocarcinoma, and enhance (additively or synergistically) the anti-neoplastic cytotoxicity of conventional gemcitabine and selectively "targeted" gemcitabine immunochemotherapeutics is important and has multiple implications. The combination of griseofulvin and gemcitabine-(C 4amide)-[anti-HER2/neu] presents a potential opportunity to attain additive and/or synergistic levels of anti-neoplastic cytotoxicity through three different molecular mechanisms (e.g. griseofulvin/gemcitabine, griseofulvin/[anti-HER2/neu], and gemcitabine/[anti-HER2/neu] dual combination effects). Attributes of this nature are at least in part complemented by both griseofulvin [101] and the chemotherapeutic moiety of covalent immunochemotherapeutics [27,28] like gemcitabine-to-[anti-HER2/neu] functioning as poor P-glycoprotein substrates. Griseofulvin in dual (additive or synergistic) combination with either a covalent gemcitabine-(C 4 -amide)-[anti-HER2/neu] or gemcitabine therefore offer the option for developing treatment schemes that potentially evoke more rapid and long-term (durable) resolution of even chemotherapeutic-resistant neoplastic cell populations. Complementary qualities that griseofulin in dual (additive or synergistic) combination with gemcitabine-(C 4amide)-[anti-HER2/neu] or gemcitabine ultimately can afford are; [i] lower total chemotherapeutic dosage requirements; [ii] reduced frequency and severity of sequelae, and a [iii] decreased probabilty of complete therapeutic resistance. Fewer and less severe sequelae are at least conceptually probable because of the relatively wider marginof-safety of griseofulvin compared to many if not most conventional chemotherapeutics [102][103][104][105] which is further complemented by the selective "targeted" delivery properties of gemcitabine-(C 4 -amide)-[anti-HER2/neu] and related covalent immunochemotherapeutics. Lastly, application of griseofulvin as an alternative tubulin/microtubule inhibitor in dual combination with either a covalent gemcitabine immunochemotherapeutic or gemcitabine is in direct accord with the general recommendation for in-vivo treatment regimens. Current clinical oncology guidelines advocate that different anti-cancer agents administered during the course of multi-chemotherapeutic schedules ideally should exert distinctly different mechanisms-of-action (avoids competitive inhibition) and individually evoke different sets of undesirable sequellae.

Conclusion
Organic The molecular design of gemcitabine-(C 4 -amide)-[anti-HER2/neu] and related covalent gemcitabine immunochemotherapeutics [22,23] result in the synthesis of an anti-cancer preparation that affords a more prolonged plasma pharmacokinetic profiles for the gemcitabine moiety. In the form of a covalent immunochemotherapeutic, gemcitabine has a substantially longer plasma half-life (T 1/2 ) that is at least in part attributable to; [i] a reduced gemcitabine deamination within gemcibatine-(C 4 -amide)-[anti-HER2/neu] due to substrate steric hinderance phenomenon; and [ii] decreased gemcitabine renal clearance rate [24][25][26] due to the substantially larger molecular weight for gemcibatine-(C 4 -amide)-[anti-HER2/ neu] (MW ~ 150 kDa) compared to gemcitabine (MW=263.2) which far exceeds the molecular weight cutoff for excretion by glomerular filtration. Prolongation of the pharmacokinetic profiles for gemcitabine in the form of gemcitabine-(C 4 -amide)-[anti-HER2/neu] ultimately complements, enhances and facilitates the properties of selective "targeted" chemotherapeutic delivery, continual cancer cell membrane deposition, and progressive intracellular chemotherapeutic accumulation.
Anti-neoplastic cytotoxicity of gemcitabine or gemcitabine-(C 4 -amide)-[anti-HER2/neu] was increased when they were applied in dual combination with the tubulin/microtubule inhibitor griseofulvin against chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) populations. The implications of this discovery are important and wide-ranging in scope because they they offer the potential option for developing treatment schemes that more rapidly evoke durable (longterm) resolution of neoplastic disease states while simultaneously providing certain properties that impose a lower frequency and severity of sequelae and susceptibility to resistance. More rapid resolution at least in theory could be achieved with gemcitabine-(C 4 -amide)-[anti-HER2/neu] or gemcitabine in dual combination with with the tubulin/microtubule inhibitor griseofulvin because of the additive or synergistic levels of anti-neoplastic cytotoxicity activity that could be attained that in turn will also lower total dosage requirements and total dose administered. Lower frequency of resistance is attained with gemcitabine-(C 4 -amide)-[anti-HER2/neu] and other chemotherapeutic analogs that are covalently bound to large molecular weight platforms are apparently poor substrates for P-glycoprotein/MDR-1 (multi-drug resistance efflux pump) [27,28]. Similar in concept to the benzimidazole tubulin/microtubule inhibitors [106][107][108], griseofulvin may potentially be a poor P-glycoprotein/MDR-1 substrate but this property remains to be more concisely delineated. Given this perspective, resistant forms of breast cancer that over-expresses EGFR and HER2/neu are often less vulnerable to the cytotoxic potency of chemotherapeutics due to a simultaneous over-expression of trans-membrane Pglycoprotein which functions as a somewhat non-selective membrane "pump" complex for many pharmaceutical agents [109][110][111][112][113][114].
Monomethyl auristatin E (MMAE) is far too toxic for direct systemic administration so instead it must be covalently bound to immunoglobulin (e.g. anti-GPNMB/anti-CRO11/ glembatumumab and anti-CD30/brentuximab) or other similar large molecular weight "carrier" platform.  Characterization of the major molecular weight profile for covalent gemcitabine-(C 4amide)-[anti-HER2/neu] immunochemotherapeutics compared to anti-HER2/neu monoclonal immunoglobulin. Legends: (Lane-1) murine anti-human HER2/neu monoclonal immunoglobulin reference control; and (Lane-2) covalent gemcitabine-(C 4 -amide)-[anti-HER2/neu] immunochemotherapeutic. Covalent gemcitabine immunochemotherapeutic and anti-HER2/neu monoclonal immunoglobulin were size-separated by non-reducing SDS-PAGE followed by lateral transfer onto sheets of nitrocellulose membrane to facilitate detection with biotinylated goat anti-mouse IgG immunoglobulin. Subsequent analysis entailed incubation of nitrocellulose membranes with strepavidin-HRPO in combination with the use of a HRPO chemiluminescent substrate for the acquisition of autoradiography images.   Relative anti-neoplastic cytotoxicity of the tubulin/microtubule inhibitor, griseofulvin compared to methylselencysteine against chemotherapeutic-resistant mammary adenocarcinoma. Legend: (◆) griseofulvin; and (■) methyselenocysteine. Mammary adenocarcinoma (SKBr-3) monolayer populations were incubated 96-hours with griseofulvin or methylselenocysteine formulated at gradient concentrations and antineoplastic cytotoxicity measured as a function of MTT cell vitality stain intensity relative to matched negative reference controls. Relative anti-neoplastic cytotoxicity of griseofulvin functioning as a tubulin/microtubule inhibitor against chemotherapeutic-resistant mammary adenocarcinoma as a function of challenge duration. Legend: (■) griseofulvin following an incubation period of 182-hours, and (◆) griseofulvin following an incubation period of 96-hours. Mammary adenocarcinoma SKBr-3 monolayer populations were incubated with covalent gemcitabine immunochemotherapeutics. Cytotoxicity was measured applying the MTT cell vitality assay relative to matched negative reference controls.  Relative anti-neoplastic cytotoxicity of gemcitabine in dual combination griseofulvin compared to gemcitabine alone against chemotherapeutic-resistant mammary adenocarcinoma. Legend: (■) gemcitabine with griseofulvin; (◆) gemcitabine alone; and (□) gemcitabine-(C 4 -amide)-[anti-HER2/neu] with griseofulvin. Chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) monolayer populations were incubated for 182-hours with gemcitabine (+/-grisofulvin 15 μM fixed-concentration) formulated in triplicate at gradient concentrations. Anti-neoplastic cytotoxicity was measured using a MTT cell vitality assay relative to matched negative reference controls.