Synthesis of Gemcitabine-( C 4-amide )-[ anti-HER 2 / neu ] Utilizing a UV-Photoactivated Gemcitabine Intermediate : Cytotoxic AntiNeoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3

Gemcitabine is a pyrimidine nucleoside analog that becomes triphosphorylated intracellularly where it competitively inhibits cytidine incorporation into DNA strands. Another mechanism-of-action of gemcitabine (diphosphorylated form) involves irreversible inhibition of the enzyme ribonucleotide reductase thereby preventing deoxyribonucleotide synthesis. Functioning as a potent chemotherapeutic gemcitabine promote decreases in neoplastic cell proliferation and apoptosis which is frequently found to be effective for the treatment of several leukemias and a wide spectrum of carcinomas. A brief plasma half-life in part due to rapid deamination and chemotherapeutic-resistance restricts the utility of gemcitabine in clinical oncology. Selective “targeted” delivery of gemcitabine represents a potential molecular strategy for simultaneously prolonging its plasma half-life and minimizing innocient tissues and organ systems exposure to chemotherapy. The molecular design and an organic chemistry based synthesis reaction is described that initially generates a UV-photoactivated gemcitabine intermediate. In a subsequent phase of the synthesis method the UV-photoactivated gemcitabine intermediate is covalently bonded to a monoclonal immunoglobulin yielding an end-product in the form of gemcitabine-(C4-amide)-[anti-HER2/neu]. Analysis by SDS-PAGE/chemiluminescent auto-radiography did not detect evidence of gemcitabine-(C4-amide)-[anti-HER2/neu] polymerization or degradative fragmentation while cell-ELISA demonstrated retained binding-avidity for HER2/neu trophic membrane receptor complexes highly over-expressed by chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Compared to chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3), the covalent immunochemotherapeutic, gemcitabine-(C4-amide)-[anti-HER2/neu] is anticipated to exert greater levels of cytotoxic anti-neoplastic potency against other neoplastic cell types like pancreatic carcinoma, small-cell lung carcinoma, neuroblastoma, glioblastoma, oral squamous cell carcinoma, cervical epitheliod carcinoma, or leukemia/lymphoid neoplastic cell types based on their reported sensitivity to gemcitabine and gemcitabine covalent conjugates.

The chemotherapeutic, gemcitabine has in contrast to the anthracyclines been less frequently bonded covalently to large molecular weight platforms that can fa-cilitate selective "targeted" delivery [36].Gemcitabine is a deoxycytidine nucleotide analog that intracellularly has a chemotherapeutic mechanism-of-action that involveds it being triphosphoralated in a manner that allows it to substitute for cytidine during DNA transcription resulting in incorporation into DNA strands and inhibition of DNA polymerase biochemical activity.A second mechanismof-action for gemcitabine involves inhibition and inactivation of ribonucleotide reductase ultimately resulting in suppression of deoxyribonucleotide synthesis in concert with diminished DNA repair and reduced DNA transcription.Each of these mechanisms-of-action collectively promotes cellular apoptosis.In clinical oncology, gemcitabine is administered for the treatment certain leukemias and potentially lymphoma conditions in addition to a spectrum of adenocarcinomas and carcinomas affecting the lung (e.g.non-small cell), pancrease, bladder and esophogus.Gemcitabine has a brief plasma half-life because it is rapidly deaminated to an inactive metabolite that is rapidly eliminated through renal excretion into the urine [37][38][39].The molecular design and synthesis of a covalent gemcitabine immunochemotherapeutics provides several attributes that complement their ability to facilitate selective "targeted" delivery, progressive intracellular deposition, and more prolonged plasma pharmacokinetics for the gemcitabine moiety.Attributes in this regard presumably include steric hinderance phenomenon that accounts for gemcitabine being apparently a much poorer substrate for MDR-1 (multidrug resistance efflux pump) [40] in addition to the rapid deaminating enzyme systems, cytidine deaminase, and deoxycytidylate deaminase (following gemcitabine phosphorylation) when this chemotherapeutic is covalently incorporated into an immunochemotherapeutic.
The molecular design, synthetic organic chemistry reaction schemes, and cytotoxic anti-neoplastic potency of gemcitabine covalently bonded to large molecular weight delivery platforms has been described on only a limited scale in published reports.Due to the type and relatively low number of chemical groups (sites) available within the structure of gemcitabine there are only a small number of organic chemistry reaction schemes that have been utilized to covalently bond gemcitabine to large molecular weight platforms and very few reports have described the synthesis and cytotoxic anti-neoplastic potency of covalent gemcitabine immunochemotherapeutics [36].The covalent bonding of gemcitabine to immunoglobulin or ligands that have binding-avidity for trophic receptors like HER2/neu and EGFR frequently over-expressed in breast cancer and by many other carcinomas or adenocarcinomas provides an opportunity to achieve additive or synergistic levels of cytotoxic anti-neoplastic potency.Monoclonal anti-HER2/neu and anti-EGFR im-munoglobulin fractions provide a molecular mechanism for achieving both selective "targeted" chemotherapeutic delivery and growth suppression of neoplastic cell types that biologically are heavily dependent on the over-expression of HER2/neu and EGFR when they function as trophic receptor complexes.Unfortunately when applied as a monotherapy, anti-HER2/neu, anti-EGFR and other therapeutic monoclonal immunoglobulin fractions reportedly have an inability to exert levels of cytotoxic activity sufficient to independently resolve many neoplastic disease states [41][42][43][44][45][46][47] unless they are applied in concert with conventional chemotherapy or other anticancer modalities [48,49].Despite general familiarity with how anti-HER2/neu affects the vitality of cancer cell populations and it's application in clinical oncology, there has been surprisingly little research devoted to the molecular design, chemical synthesis and potency evaluation of covalent gemcitabine immunochemotherapetuics [36].Even fewer reports exist to date that describe similar aspects for covalent gemcitabine-[anti-HER2/neu] immunochemotherapeutics and their potential to exert selectively "targeted" cytotoxic anti-neoplastic potency against chemotherapeutic-resis-tant mammary adenocarcinoma [36] or other cancer cell types.

Gemcitabine-(C 4 -amide)-[anti-HER2/neu] Immunochemotherapeutic Synthesis
Phase-I Synthesis Scheme for UV-Photoactivated Gemcitabine-(C 4 -amide) Intermediates-The cytosine-like C 4amine 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 (Figures 1 and 2).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 degradation of the ester group of any residual succinimidyl 4,4-azipentanoate that may not of reacted in the first 30 to 60 minutes with the C 4 cytosine-like amine group of gemcitabine.
In contrast to the anthracyclines, [7,71,72] gemcitabine can not be measured directly within covalent immunochemotherapeutic preparations by spectrophotometric absorption [36].Alternatively it is possible to calculate the amount of gemcitabine that has been covalent incurporated into immunochemotherapeutics by measuring residual unbound gemcitabine before and after the Phase II reaction or by measuring the difference in non-chemotherapeutic-occupied sites associated with either amine or reduced sulfhydryl groups within anti-HER2/neu monoclonal immunoglobulin compared to gemcitabine-immunochemotherapeutics [36,51,52].Determination of the gemcitabine molar-incorporation-Index and gemcitabine molar-equivalent-concentrations for gemcitabine-(C 5 -methylcarbamate)-[antiHER2/ neu] were calculated using measurements for the relative difference in moles of reduced sulfhydryl groups (e.g.R-SH: cystine amino acid residues and sulfhydryl groups introduced with Traut's reagent) contained within thiolated anti-HER2/neu fractions relative to the covalent gemcitabine-immuno-chemotherapeutic following separation by column chromatography [36,51,52].Reduced sulfhydryl groups were measure by combining anti-HER2/ neu or gemcitabine (C 5 -methylcarbamate)-[anti-HER2/ neu] in phosphate buffered saline (0.1 M, pH 7.4) with 5,5'-dithiobis-(2-nitrobenzoic acid) formulated in sodium phosphate-EDTA buffer (DTNB: 78 µg/ml with EDTA 1 mM in 0.1 M sodium phosphate buffer 0.1 M, pH 8.0).Spectrophotometric absorbance of mixtures formulated at 1:1 v/v (e.g.250 µl each) was measured at 412 nm following incubation at 25˚C for 15 minutes.The amount and concentration of sulfhydryl groups was then calculated utilizing a linearized standard curve generated with reference control solutions of cysteine HCl monohydrate formulated at known concentrations (molar extinction coefficient: 14,150 M -1 •cm -1 ).
Determination of the immunoglobulin concentration for covalent gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] immunochemotherapeutics was determined by measuring spectrophotometric absorbance at 280 nm in combinations with utilizing a 235 nm-vs-280 nm standardized reference curve in order to accommodate for any potential absorption profile over-lap at 280 nm between gemcitabine and immunoglobulin.
Mass/Size-Dependent Separation of Gemcitabine-Immunochemotherapeutics by Non-Reducing SDS-PAGE-Covalent gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine (C 5 -methylcarbamate)-[anti-HER2/neu] immunochemotherapeutics in addition to a anti-HER2/neu immunoglobulin reference control fraction 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 gemcitabine immunochemotherapeutic, the reference control immunoglobulin fraction (0.9 µg/well) and a mixture of prestained 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.
Immunodetection Analyses-Covalent gemcitabine-(C 4amide)-[anti-HER2/neu] and gemcitabine (C 5 -methylcarbamate)-[anti-HER2/neu] immunochemo-therapeutics following mass/size-dependent separation by non-reducing SDS-PAGE were equilibrated in tank buffer devoid of methanol.Mass/size-separated 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˚C to 3˚C with the transfer manifold packed in crushed ice.
Nitrocellulose membranes with laterally-transferred immunochemotherapeutics were then equilibrated in Trisbuffered 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, ml) containing bovine serum albumin (5%) for 16 hours at 2˚C to 3˚C applied 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 = 3x).
Populations of the mammary adenocarcinoma (SKBr-3) were propagated in 150-cc2 tissue culture flasks containing McCoy's 5a Modified Medium supplemented Synthesis of Gemcitabine-(C 4 -amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate: Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3 694 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 confluency.
Cytotoxic potencies for gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5 -methylcarbamate)[anti-HER2/neu] 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 and 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.Contents of the 96-well microtiter plate was then removed, followed by serial rinsing with PBS (n = 3).The resulting blue intracellular formzone 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.

. Relative cytotoxic anti-neoplastic potency of gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] as a function of immunoglobulin-equivalent concentration. Legends: (4) gemcitabine-(C 4 -amide)-[anti-HER2/neu] with a gemcitabine molar-incorporation-index of 2.78:1 (182-hour incubation period); and (•) gemcitabine-(C 5 -methyl carbonate)-[anti-HER2/ neu] with a gemcitabine molar-incorporation-index of 1.1:1 (182-hour incubation period). Arrows indicate the approximate concentration of gemcitabine-(C 4 -amide)[anti-HER2/neu] and gemcitabine-(C 5 -methyl carbonate)[anti-HER2/neu] necessary to achieve a 30% level of cytotoxic anti-neoplastic potentcy. Chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) monolayer populations were incubated with either covalent gemcitabine immunochemotherapeutics formulated in triplicate at gradient concentrations. Cytotoxic anti-neoplastic potency measured using a MTT cell vitality assay relative to matched negative reference controls.
preferentially reacting with and forming a colvaent bond at the C 4 cytosine-like amine group of gemcitabine.In organic solvent systems like DMSO and DMF succinimidyl 4,4-azipentanoate may also react to a much lesser degree with nitrogen groups embended within five or six member ring structures but such complexes reportedly dissociate redily with the addition of small amounts of ddH 2 O or aqeous buffer An organic solvent in the form of DMSO was applied in these investigations in order to preserve the integrity of the UV-photoactivated moiety of succinimidyl 4,4-azipentanoate during the extended incubation with gemcitabine.Alternatively, an aqueous buffer formulated between the pH range of 7 to 9 can effectively promote covalent amide bond formation when shorter incubation periods are indicated.Utilization of aqueous buffer with a pH of 6.5 and implementation of lower reaction condition temperatures (e.g.4˚C) have reportedly been found to enhance the reaction of succinimide ester group with dif
Covalently bonding gemcitabine or other chemotherapeutic agents to biological protein fractions like immunoglobulin without a requirement to convert existing cystine-cystine disulfide bonds to their reduced form (R1-S-S-R2-R1-SH and R2-SH) or the synthetic introduction of reduced sulfhydryl groups provides several disctinct advantages.Specifically, such synthetic organic chemistry reaction schemes entail the implementation of fewer synthetic chemistry reactions, require fewer critical reagents, and maximize final "end-product" yield due in part to at least one less column chromatography separation procedure.The brief duration of the synthetic organic chemistry reaction scheme for gemcitabine-(C 4 -amide)-[anti-HER2/neu] utilizing succinimidyl 4,4azipentanoate is realized because of the relatively rapid time course for the Phase-I, but especially the Phase-II organic chemistry reaction.The synthetic organic chemistry reaction scheme has also been designed so that adjustment of buffer pH to different levels during the procedure is not necessary in contrast to other techniques [91].Perhaps one of the most important features of the synthesis methodology is a lack of a requirement for cystinecystine disulfide bond reduction or pre-thiolation that in turn allows by design the application of synthetic chemistry reactions that are highly efficient under relatively mild conditions thereby possing a lower risk of protein fragmentation or polymerization (e.g.IgG-IgG) through premature intra-molecular disulfide bond formation [2].Realized benefits therefore include greater retained biological activity (e.g.antigen binding-avidity) and increased total final yield of a function immuno chemotherapeutic end-product.Lastly, lack of a requirement to either convert existing cystine-cystine disulfide bonds to their reduced form or the introduction of reduced sulfhydryl groups into immunoglobulin fractions reduces restrictions and limitations on the magnitude of the molar-incorporation-index that can be attained.In contrast, the chemotherapeutic incorporation index for covalent immunochemotherapeutics synthesized utilizing SMCC, [7,[80][81][82] EMCH [9,10,71] or PMPI [36,[53][54][55] is only equivalent to or lower than the extent of prethiolation at ε-amine groups associated with the finite number of lysine residues within the amino acid sequence of protein fractions.In prethiolation dependent synthesis schemes higher epirubicin molar-incorporationindexes are possible with modifications in methodology but requires the use of harsher synthesis conditions that are frequently accompanied by substantial reductions in total yield of covalent immunochemotherapeutic, [6] and declines in antigen-immubnoglobulin bindingavidity (e.g.cell-ELISA parameters).Presumably the 7.6 fold higher potency of gemcitabine-(C 4 -amide)-[anti-HER2/neu] compared to gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/ neu] at the cytotoxic anti-neoplastic potency level of approximately 30% can be attributed to a combination of a greater degree retained biological activity for anti-HER2/neu (cell-ELISA) and a higher gemcitibin molar-incorporation-index of 2.78-to-1 for gemcitabine-(C 4amide)-[anti-HER2/neu] in contrast to 1.1-to-1 for gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] (Figure 9).Both of these properties are anticipated to be attributable to the application of gentler reaction conditions in part due to a lack of a requirement for anti-HER2/neu prethiolation during Phase-II synthesis reaction sheme for gemcitabine-(C 4 -amide)-[anti-HER2/neu].
Implementation of succinimidyl 4,4-azipentanoate in the synthesis scheme for gemcitabine-(C 4 -amide)-[anti-HER1/neu] offers desirable attributes other than a lack of a requirement for pre-thiolation of immu-noglobulin or similar molecular platforms that possess biological activity that affords properties of selective "targeted" delivery.In contrast to SMCC, [7,[80][81][82] EMCH [9,10,71] or PMPI [36,[53][54][55] the synthesis of gemcitabine-(C 4 -amide)-[anti-HER2/neu] utilizing succinimidyl 4,4-azipentanoate has the added benefit of not introducing extraneous five and six carbon or carbon/nitrogen ring structures into the final covalent immunochemotherapeutic endproduct (Figures 1 and 2).Elimination of extraneous ring structures decreases the probability of inducing in-vivo humoral immune response when administered by IV injection that can ultimately result in the formation of neutralizing antibody titers and an increased risk of post-treatment immune hypersensitivity reactions.In addition, the Phase-I synthetic organic chemistry reaction scheme can be performed in either aqueous buffer, or organic solvent systems supplemented with triethylamine [N(CH 2 CH 3 ) 3 ] or similar proton acceptor molecules at low concentrations.In stock solutions of reaction mixtures formulated in aqueous buffers a significant amount of hydrolytic degradation of succinimidyl 4,4-azipentanoate is expected to occur to varying degrees.Alternatively, if stock solutions and reaction mixtures of gemcitabine and succinimidyl 4,4-azipentanoate are instead formulated in an anhydrous organic solvent like DMSO in combination with a proton acceptor molecule then the resulting UV-photoactivated gemcitabine-(C 4 -amide) intermediate is stable at 4˚C or −20˚C for a period of time when adequately protected from UV-light exposure.Such chemical properties of succinimidyl 4,4-azipentanoate allow for the convenient option of "presynthesizing" and preserved storage of the UV-photoactivated gemcitabine-(C 4 -amide) intermediate for an extend period of time for the future production of covalent gemcitabine-immunochemotherapeutics [72].The synthetic organic chemistry reaction scheme described also offers another added level of convenience because it represents a template model that can be adapted and modified to facilitate the covalent bonding of an array of different chemotherapeutic agents to a wide range of molecular platforms that can facilitate selective "targeted" pharmaceutical delivery.
Cell-Binding Profiles-Increases in standardized immunoglobulin-equivalent concentrations of gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] correlated with elevations in total immunoglobulin membrane binding profiles in populations of human mammary adenocarcinoma detected by cell-ELISA (Figure 4).The lower standardized immunoglobulin-equivalent concentration range for gemcitabine-(C 4 -amide)-[anti-HER2/neu] compared to gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] implies that the former covalent gemcitabine immunochemotherapeutic may have a higher level of retained anti-HER2/neu binding-avidity.The most probably explanation for this difference can be attributed to the implementation of milder organic chemical reaction conditions and a lack of a requirement for pre-thiolate of anti-HER2/neu fractions.Previous investigations have similarly noted that modest alterations in synthetic chemistry and elevations in the chemotherapeutic molar incorporation index can profoundly influence immunoglobulin binding properties [29].
In contrast to most covalent anthracycline immunochemotherapeutics described to date, a longer 182-hour incubation period was applied to access the cytotoxic anti-neoplastic potency of gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] in order to optimally evaluate their cytotoxic anti-neoplastic potency (Figure 5) [36,71,72].Longer incubation periods have also been applied to evaluate other synthetic gemcitabine-ligand preparations in order to more accurately access their ex-vivo cytotoxic anti-neoplastic potency [21,36,40,59].Several explanations may account for the requirement to use longer incubation periods for the ex-vivo evalution of gemcitabine compared to anthracycline-immunochemotherapeutics or anthracycline covalent bound to other molecular platforms with properties that afford selective "targeted" delivery (e.g.receptor ligands).Since the covalent immunochemotherapeutics gemcitabine-(C 4 -amide)-[anti-HER2/neu], gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/ neu] [36] and several epirubicin-[anti-HER2/ neu] immunochemotherapeutics [7,71,72] all selectively "target" chemotherapeutic delivery at the same HER2/ neu receptor site highly over-expressed on the external surface membrane of mammary adenocarcinoma (SKBr-3), it is possible that differences in their cytotoxic anti-neoplastic activity may be attributable to, 1) differences in the vulnerability of covalent bond structures created during the synthesis of gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] to enzyme-mediated degradation or simple hydrolysis within the acidic endosome/lysosome microenvironment; 2) variations in the expression profile for different enzyme fractions necessary for biochemically liberating gemcitabine versus epirubicin from covalent immunochemotherapeutics; 3) variation in the acidic characteristics associated with the endosome/lysosome microenvironment necessary for liberating gemcitabine versus epirubicin from covalent immunochemotherapeutics; 4) greater capacity of the anthracycline moiety within intact covalent epirubicin immunochemotherapeutics to exert one or more of the multiple mechanisms-of-action recognized for this class of chemotherapeutic agent; 5) vulnerability of the gemcitabine moiety in covalent gemcitabine immunochemotherapeutics to inactivation by deamination.The fact that cytotoxic anti-neoplastic potency profiles for gemcitabine-(C 4 -amide)-[anti-HER2/ neu] and gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/ neu] at the end of a 182-hour incubation period were very similar to those for gemcitabine after a 72-hour incubation period implies that cytotoxic anti-neoplastic activity of the gemcitabine immunochemotherapeutics is possibly delayed due to a slow release of the chemotherapeutic moiety that is apparently longer compared to the rate of anthracycline-release from covalent epirubicin-immunochemotherapeutics [7,71,72].One important implication of this possible explanation is that a delayed and prolonged release or liberation of gemcitabine from covalent gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] could represent a desirable property that can be employed as a molecular strategy to evoke "super-loading" that in turn can facilitate extensive and sustained chemotherapeutic deposition and release within populations of neoplastic cells.
Collective interpretation of results from SDS-PAGE/ immunodection/chemiluminscent autoradiography, cell-ELISA and cytotoxic anti-neoplastic potency analyses illustrates how gemcitabine can be covalently bound to a large molecular weight "carrier" (protein) to facilitate selective "targeted" chemotherapeutic delivery and cytotoxic anti-neoplastic potency.The positive findings directly address one of the major objectives that originally motivated the molecular design and synthesis of gemcitabine-(C 4 -amide)-[anti-HER2/neu].Additionally, there was a perceived need for the molecular design of a synthesis scheme that was composed of a sequential series of organic chemistry reactions that could facilitate relatively rapid production of gemcitabine-(C 4 -amide)-[anti-HER2/ neu] using mild conditions that affored minimal degradative low molecular weight fragmentation or large molecular weight polymerization (e.g.IgG-IgG).Recent investigations describing the methodology employed for the synthesis of epirubicin-(C 5 -amide)-[anti-HER2/neu] through the application of a UV-photoactivated epirubicin intermediate revealed that there was a high degree of probability that a similar organic chemistry regimen could be adapted as a model with minor modifications for the relatively rapid synthesis of a covalent gemcitabine-(C 4 -amide)-[anti-HER2/neu] immunochemotherapeutic [72].In this context, a set of organic chemistry reactions were implemented to synthesize gemcitabine-(C 4 -amide)-[anti-HER2/neu] that had not previously been described for the production of a gemcitabine-immunochemotherapeutic or covalent gemcitabine-ligand preparation.The organic chemistry synthesis reactions utilized for the production of gemcitabine-(C 4 -amide)-[anti-HER2/ neu] also possesses practical utility because it can serve as a model or template for the molecular design and production of other covalent immunochemotherapeutics.
Third, cytotoxic anti-neoplastic potency of gemcibatine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5methylcarbamate)-[anti-HER2/neu] could have been evaluated at higher gemcitabine-equivalent concentrations.Since gemcitabine in contrast to the anthracyclines has rarely been synthetically incorporated into (covalently bonded to) selective "targeted" delivery platforms, [21,36,40,[57][58][59][60] it is uncertain if this chemotherapeutic can be utilized to consistently create covalent gemcitabine immunochemotherapeutics that posses significantly higher levels of cytotoxic anti-neoplastic potency than gemcitabine alone (Figures 5-7) [36].Despite this consideration, the paramount objective that moti-vates the molecular design and synthesis of covalent gemcitabine immunochemotherapeutics is the opportu-nity to  H]-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 [99,100].Despite this consideration, MTT vitality stain based assays continue to be extensively applied for the routine assessment of true cytotoxic anti-neoplastic potency of chemotherapeutics covalently incorporated synthetically into molecular platforms that provide properties of selective "targeted" delivery.[7,40,58,60,[101][102][103][104][105][106] One of the most significant advantages of MTT vitality stain based assays and methods that apply similar reagents is that the ability to measure lethal cytotoxic anti-anti-neoplastic activity is generally considered to be superior to mearly the detection of early-stage and potentially transient cellular injury that could ultimately be reversible.
Fifth, cytotoxic anti-neoplastic potency of gemcibatine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5methylcarbamate)-[anti-HER2/neu] immunochemotherapeutic could have been delineated in-vivo against human neoplastic xenographs in animal hosts as a model for human cancer.Effectiveness and potency of many if not most covalent immunochemotherapeutics against neoplastic cell populations (that genuinely do possess properties of selectively "targeted" chemotherapeutic delivery) is frequently higher when evaluated in-vivo compared to results acquired ex-vivo in tissue culture models utilizing the same identical cancer cell type [107][108][109].Enhanced levels of covalent immunochemotherapeutic potency measured in-vivo is presumably attributable in part to induced responses by the innate immune system that includes antibody-de-pendent cell cytotoxicity (ADCC) phenomenon in concert with complementedmediated cytolysis initiated or stimulated by the formation of antigen-immunoglobulin complexes on the exterior surface membrane of "targeted" neoplastic cell types.During ADCC events immune cell types actively involved in this response release cytotoxic components that are known to additively and synergistically enhance the cytotoxic anti-neoplastic activity of conventional chemotherapeutic agents [110].The contributions of ADCC and complement-mediated cytolysis to the in-vivo cytotoxic anti-neoplastic potency of covalent immunochemotherapeutics would be further enhanced by the additive and synergistic levels of anti-neoplastic potency produced by anti-trophic receptor monoclonal immunoglobulin when applied in dual combination with conventional chemotherapeutic agents [48,49,83,89,[111][112][113][114][115][116][117][118].Additive or synergistic interactions of this type have been detected between anti-HER2/neu when applied simultaneously in combination with cyclophosphamide [49,111], docetaxel [111], doxorubicin [49,111], etoposide [111], methotrexate [111], paclitaxel [49,111], or vinblastine [111].
Sixth, several modifications could have been made in the synthesis strategy for gemcitabine-(C 4 -amide)-[anti-HER2/neu] and gemcitabine-(C 5 -methylcarbamate)-[anti-HER2/neu] in order to increase the gemcitabine molar-incorporation-index. Examples in this regard include the application of gemcitabine and the covalent bond forming reagents at higher molar concentrations, implementation of smaller reaction volumes during synthesis procedures, increasing the duration of Phase I and/or Phase II synthesis schemes, and possibly altering the relative gemcitabine-to-covalent bond forming reagentto-immunoglobulin molar ratios in a manner that forces the organic chemistry reactions in a direction that increases final product yield.Unfortunately, such modifications usually also require or impose harsher reaction conditions that necessitate an acceptance for a higher risk of reduced biological activity (e.g.decreased antigen binding avidity) and substantial declines in final/total product yield [6,108].Aside from overly harsh synthesis conditions, excessively high molar incorporation indexes for any chemotherapeutic agent can reduce the biological integrity of immunoglobulin fractions when the number of pharmaceutical groups introduced into the Fab' antigenbinding region becomes excessive.Such modifications can result in only modest declines in immunoreactivity (e.g.86% for a 73:1 ratio) but disproportionately large declines in cytotoxic anti-neoplastic activity in addition to reductions in potency that can decrease to levels substantially lower than those found with non-conjugated "free" chemotherapeutic (e.g.anthracyclines) [108].
The biological integrity of the immunoglobulin component of covalent immunochemotherapeutics is critically important.It not only serves as a means of facilitating selectively "targeting" chemotherapeutic delivery, but it also initiates or induces internalization of covalent immunochemotherapeutics by mechanism of receptormediated endocytosis assuming an appropriate membrane-associated antigen has been selected as a "target" (e.g.many carcinoma and adenocarcinoma cell types highly over-express HER2/neu and/or EGFR) [119].Although specific data for HER2/neu and EGFR expression by mammary adenocarcinoma (SKBr-3) is limited, [7] 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 [120].Immunoglobulin-induced receptormediated endocytosis at membrane HER2/neu complexes can ultimately lead to increases in the intracellular concentration of selectively "targeted"/delivered chemotherapeutic that approach and exceed levels 8.5 [121] to >100 × fold greater [122] than those that can ever possibly be achieved by simple passive chemotherapeutic diffusion from out of the intravascular compartment.
The application of succinimidyl 4,4-azipentanoate in contrast to succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), [7,[80][81][82] N-ε-maleimidocaproic acid hydrazide (EMCH), [8][9][10]51,52,71] or N-[p-maleimidophenyl]-isocyanate (PMPI) [36,[53][54][55] can facilitate greater flexibility in synthesis methods designed to increase the chemotherapeutic molar-incorporation-index during the creation of covalent immunochemotherapeutics without having to use harsher reaction conditions.The major risk of compromising the biological integrity (antigen binding avidity) of gemcitabine-(C 4 -amide)-[anti-HER2/neu] synthesized with a UV-photoactivated gemcitabine intermediate therefore is almost entirely associated with methods devised to introduce an excessive amount of pharmaceutical (chemotherapeutic) into immunoglobulin fractions including regions of the amino acid sequence that are directly responsible for providing properties of selective "targeted" delivery (e.g.Fab antigen bindings regions of immunoglobulin or receptor binding region of ligands).Despite the general validity of the inverse relationship between chemotherapeutic molar-incorporation-index and retained biological activity (e.g.anti-HER2/neu mediated selective "targeted" delivery) and the greater potency of covalent immunochemotherapeutics with high chemotherapeutic molar incorporation indexes, it should be emphasized that mathematically the expression density for external membrane-associated "targets" appears to be one of, if not the most critically important variable that influences the cytotoxic anti-neoplastic potency of covalent immunochemotherapeutics or ligand-chemotherapeutic preparation.In this regard, it is important that external membrane-associated sites be chosen that are known to functionally undergo phenomenon analogous to receptormediated-endocytosis in order to avoid only "coating" of the external surface membrane of "targeted" cancer cell populations.Such a prerequisite is relevant assuming that the chemotherapeutic agent applied has a mechanism-of-action that is dependent upon their ability to modify the function of molecular entities within the cytosol or nucleus in order to exert a biological effect.Such a requirement would not be a prerequisite for anti-cancer agents that instead alter or disrupt the physical integrity of cancer cell membranes or the function of complexes that are an integral component of membrane structures.

Figure 1 .
Figure 1.Schematic illustration of the organic chemistry reaction schemes utilized in the 2-phase synthesis regimen for gemcitabine-(C 4 -amide)-[anti-HER2/neu].Legends for Reactions: (Phase-I) creation of a covalent amide bond at the C 4 cytosine-like monoamine of gemcitabine and the ester group of succinimidyl 4,4-azipentanoate resulting in the creation of a covalent UV-photoactivated gemcitabine-(C 4 -amide) intermediate.The reaction results in the liberation of the succinimide "leaving" complex.(Phase-II) creation of a covalent bond between the UV-photoactivated gemcitabine-(C 4 -amide) intermediate and chemical groups within the amino acid sequence of anti-HER2/neu monoclonal immunoglobulin initiated by exposure to UV light (354 nm).Legends for HP-TLC Analysis: Reaction of the N-hydroxy-succinymide groups of disuccinimidyl glutarate with the C 4 cytosine like "ring amine" of gemcitabine.(Lane-1) gemcitabine reference control; (Lane-2) gemcitabine reacted with disuccinmidyl glutarate in DMSO with Tri-ethylamide at 50 mM final concentration; and (Lane-3) gemcitabine reacted with disuccinmidyl glutarate in DMSO and ddH 2 O (2:1 v/v).Reaction products were developed by silica gel HP-TLC using a mobile phase of propanol/ethanol (80:20 v/v) and images visualized under UV light (254 nm).