Journal of Cancer Therapy, 2012, 3, 689-711 Published Online October 2012 (
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu]
Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against
Chemotherapeutic-Resistant Mammary Adenocarcinoma
Cody P. Coyne1, Toni Jones1, Ryan Bear2
1Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Oktibbeha County, USA; 2Wise Cen-
ter, Mississippi State University, Oktibbeha County, USA.
Received August 10th, 2012; revised September 12th, 2012; accepted September 24th, 2012
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 synthe-
sis. Functioning as a potent chemotherapeutic gemcitabine promote decreases in neoplastic cell proliferation and apop-
tosis 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 gemcit-
abine 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 chemo-
therapy. 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 antici-
pated to exert greater levels of cytotoxic anti-neoplastic potency against other neoplastic cell types like pancreatic car-
cinoma, 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 gemcit-
abine covalent conjugates.
Keywords: Gemcitabine; HER2/neu; UV-Photoactivated Gemcitabine-(C4-amide) Intermediate; Organic Chemistry
Reaction; Gemcitabine-(C4-amide)-[anti-HER2/neu]; Covalent Bond Synthesis; Gemcitabine
(C5-methylcarbamate)-[anti-HER2/neu]; Cytotoxic Anti-Neoplastic Potency; Chemotherapeutic-Resistant;
Mammary Adenocarcinoma; Cell-ELISA; SDS-PAGE; Immunodetection; Chemiluminescent
1. Introduction
The anthracyclines have historically been the most
common class of chemotherapeutic covalently bonded to
(large) molecular platforms that can facilitate “selective”
targeted delivery [1-25]. The spectrum of anthracylines
utilized to synthesize covalent anthracycline-immuno-
chemotherapeutics to date has largely included doxoru-
bicin [26-30] and to a lesser extent daunorubicin [31-33]
or epirubicin [7,34,35].
The chemotherapeutic, gemcitabine has in contrast to
the anthracyclines been less frequently bonded cova-
lently to large molecular weight platforms that can fa-
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
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 mechanism-
of-action for gemcitabine involves inhibition and inacti-
vation of ribonucleotide reductase ultimately resulting in
suppression of deoxyribonucleotide synthesis in concert
with diminished DNA repair and reduced DNA tran-
scription. Each of these mechanisms-of-action collec-
tively promotes cellular apoptosis. In clinical oncology,
gemcitabine is administered for the treatment certain
leukemias and potentially lymphoma conditions in addi-
tion to a spectrum of adenocarcinomas and carcinomas
affecting the lung (e.g. non-small cell), pancrease, blad-
der and esophogus. Gemcitabine has a brief plasma
half-life because it is rapidly deaminated to an inactive
metabolite that is rapidly eliminated through renal excre-
tion into the urine [37-39]. The molecular design and
synthesis of a covalent gemcitabine immunochemo-
therapeutics 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 hin-
derance phenomenon that accounts for gemcitabine being
apparently a much poorer substrate for MDR-1 (multi-
drug resistance efflux pump) [40] in addition to the rapid
deaminating enzyme systems, cytidine deaminase, and
deoxycytidylate deaminase (following gemcitabine pho-
sphorylation) when this chemotherapeutic is covalently
incorporated into an immunochemotherapeutic.
The molecular design, synthetic organic chemistry re-
action 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 num-
ber of organic chemistry reaction schemes that have been
utilized to covalently bond gemcitabine to large molecu-
lar 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 adeno-
carcinomas provides an opportunity to achieve additive
or synergistic levels of cytotoxic anti-neoplastic po-
tency. 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-ex-
pression 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 re-
portedly have an inability to exert levels of cytotoxic
activity sufficient to independently resolve many neo-
plastic disease states [41-47] unless they are applied in
concert with conventional chemotherapy or other anti-
cancer 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 evalu-
ation of covalent gemcitabine immunochemotherapetuics
[36]. Even fewer reports exist to date that describe simi-
lar aspects for covalent gemcitabine-[anti-HER2/neu]
immunochemotherapeutics and their potential to exert
selectively “targeted” cytotoxic anti-neoplastic potency
against chemotherapeutic-resis-tant mammary adenocar-
cinoma [36] or other cancer cell types.
2. Materials and Methods
2.1. Gemcitabine-(C4-amide)-[anti-HER2/neu]
Immunochemotherapeutic Synthesis
Phase-I Synthesis Scheme for UV-Photoactivated Gem-
citabine-(C4-amide) Intermediates-The cytosine-like C4-
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 suc-
cinimidyl 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 con-
tinually 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 C4 cyto-
sine-like amine group of gemcitabine.
Phase-II Synthesis Scheme for Covalent Gemcit-
abine-(C4-amide)-[anti-HER2/neu ] Immunochemothera-
peutic Utilizing a UV-Photoactivated Gemcitabine In-
termediate-Immunoglobulin fractions of anti-HER2/neu
(1.5 mg, 1.0 × 10–5 mmoles) in buffer (PBS: phosphate
0.1, NaCl 0.15 M, EDTA 10 mM, pH 7.3) were com-
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
Copyright © 2012 SciRes. JCT
1 2 3
HP-TLC Analysis
Figure 1. Schematic illustration of the organic chemistry reaction schemes utilized in the 2-phase synthesis regimen for gem-
citabine-(C4-amide)-[anti-HER2/neu]. Legends for Reactions: (Phase-I) creation of a covalent amide bond at the C4 cyto-
sine-like monoamine of gemcitabine and the ester group of succinimidyl 4,4-azipentanoate resulting in the creation of a cova-
lent UV-photoactivated gemcitabine-(C4-amide) intermediate. The reaction results in the liberation of the succinimide “leav-
ing” complex. (Phase-II) creation of a covalent bond between the UV-photoactivated gemcitabine-(C4-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-succ inymide groups of disuc cinimidyl glutarate
with the C4 cytosine like “ring amine” of gemcitabine. (Lane-1) gemcitabine reference control; (Lane-2) gemcitabine reacted
with disuccinmidyl glutarate in DMSO with Tri-e thylamide at 50 mM final concentration; and (Lane-3) gemcitabine reacted
with disuccinmidyl glutarate in DMSO and ddH2O (2:1 v/v). Reaction products were developed by silic a gel HP-TLC using a
mobile phase of propanol/ethanol (80:20 v/v) and images visualized under UV light (254 nm).
Figure 2. Molecular design and chemical composition of two covalent gemcitabine-immunochemotherapeutics. Legends:
(Top-Panel) Gemcitabine-(C4-amide)-[anti-HER2/neu] synthesized using a 2-stage organic chemistry reaction scheme that
initially creates a covalent bond at the C4 cytosine-like amine group of gemcitabine. (Bottom-Panel) Gemcitabine-(C5-me-
thylcarbamate)-[anti-HER2/neu] synthesized using a 3-stage organic chemistry reaction scheme that formed covalent bonds at
the chemotherapeutic C5-methylhydroxy group and at/to thiolated lysine α-amine groups residing within the amino acid se-
quence of anti-HER2/neu monoclonal immunoglobulin fractions.
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
bined at a 1:10 molar-ratio with the UV-photoactivated
gemcitabine-(C4-amide) intermediate (Phase- 1 end prod-
uct) and allowed to gently mix by constant stirring for 5
minutes at 25˚C in the dark. The photoactivated group of
the gemcitabine-(C4-amide) intermediate was then re-
acted with side chains of amino acid residues within the
sequence of anti-HER2/neu monoclonal immunoglobulin
during a 15 minute exposure to UV light at 354 nm (re-
agent activation range 320 - 370 nm) in combination with
constant gentle stirring (Figures 1 and 2). Residual che-
motherapeutic was removed from the covalent gemcit-
abine-(C4-amide)-[anti-HER2/neu] immunochemotherapeu-
tic applying micro-scale column chromatography follow-
ing pre-equilibration of exchange media with PBS (phos-
phate 0.1, NaCl 0.15 M, pH 7.3).
2.2. Gemcitabine-(C5-methylcarbamate)-
[anti-HER2/neu] Immunochemotherapeutic
Phase-I: Immunoglobulin Thiolation at Lysine ε-Amine
Groups-A purified fraction of monoclonal immunoglobu-
lin with binding-avidity specifically for human HER2/
neu (ErbB-2, CD 340) was utilized for the semi-synthe-
sis of gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu]
[36]. Desiccated anti-HER2/neu monoclonal immuno-
globulin (1.5 mg) was combined with 2-imino-thiolane
(2-IT: 6.5 mM final concentration) in PBS (0.1 M, pH
8.0, 250 µl) and incubated at 25˚C for 1.5 hours in com-
bination with simultaneous constant gentle stirring [8,
50-52]. Thiolated anti-HER2/neu monoclonal immuno-
globulin was then buffer exchanged into PBS-EDTA
(phosphate 0.1, NaCl 0.15 M, EDTA 10 mM, pH 7.3)
using micro-scale column chromatography. Moles of
reduced sulfhydryl groups covalently introduced into
anti-HER2/neu monoclonal immunoglobulin was meas-
ured with a 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB
reagent) based assay. The average number of thiolated
lysine ε-amine groups introduced into anti-HER2/neu
fractions (R-SH/IgG) was 3:1 based on results with 2-IT
Phase-II: Synthesis of Gemcitabine-(C5-methylcarba-
mate)-PMPI Sulfhydryl Reactive Intermediate-Gemcit-
abine in DMSO (0.738 mg, 2.80 × 10–3 mmoles) was
combined at a 5:1 molar ratio with N-[p-maleimido-
phenyl]-isocyanate (PMPI: 0.120 mg, 5.60 × 10–4 mmoles)
[36,53-55] and allowed to mix by constant gentle stirring
at 25˚C for 3.5 hours. Under these conditions the PMPI
isocyanate moiety exclusively reacts with hydroxyl (R-
OH) groups and preferentially creates a carbamate cova-
lent bond at the terminal C5-methylhydroxy group of
gemcitabine [36,40,56-61]. The highly selective reaction
is reportedly complete within 2 hours under the condi-
tions applied as described. Gemcitabine was formulated
at a large molar excess to deplete un-reacted PMPI and
maximize synthesis of the sulfhydryl-reactive maleimide
Phase-III: Covalent Reaction of Gemcitabine-(C5-
methylcarbamate)-PMPI Intermediate with Thiolated Im-
munoglobulin-The gemcitabine-(C5-methylcarbamate)-PMPI
intermediate with a maleimide moiety that exclusively
reacts with reduced sulfhydryl (R-SH) groups was com-
bined at a 1.5:1 molar ratio with thiolated terminal lysine
ε-amines in anti-HER2/neu monoclonal immunoglobulin
fractions (PBS-EDTA: phosphate 0.1, NaCl 0.15 M,
EDTA 10 mM, pH 7.3) and the formulation mixture in-
cubated with constant stirring at 25˚C for 2 hours
[2,3,7,9,25,26,28,36,53,62-66]. Similar synthesis strate-
gies in concept have previously been applied to produce
covalent anthracycline immunochemotherapeutic prepa-
rations [7,8,50,51,67,68]. Because of the selective char-
acteristics of the synthesis scheme employed to produce
the sulfhydryl-reactive gemcitabine-(C5-methylcarbamate)-
PMPI intermediate and the limited duration of chemical
stability associated with it’s maleimide moiety in aque-
ous buffers, the preparation was directly mixed with
thiolated anti-HER2/neu fractions [7,36,51]. Residual
gemcitabine was removed from the final covalent gem-
citabine-(C5-methylcarbamate)-[anti-HER2/neu] immuno-
chemotherapeutic end-product applying microscale col-
umn chromatography following pre-equilibration of ex-
change media with PBS (phosphate 0.1, NaCl 0.15 M,
pH 7.3) yielding a homogenous purified preparation
(Figure 2).
2.3. Analysis and Property Characteristics
General Analysis-Quantitation of the amount of non-
covalently bound gemcitabine contained within cova-
lent gemcitabine-(C4-amide)-[anti-HER2/neu] and gem-
citabine-(C5-methylcarbamate)-[anti-HER2/neu] im-mu-
nochemotherapeutics following separation by column
chromatography was determined by measured absorb-
ance at 265 - 268 nm [69,70] for the resulting supernatant
after precipitation of gemcitabine-immuno-chemothera-
peutics with methanol:acetonitrile (1:9 v/v).
In contrast to the anthracyclines, [7,71,72] gemcitabine
can not be measured directly within covalent immuno-
chemotherapeutic preparations by spectrophotometric ab-
sorption [36]. Alternatively it is possible to calculate the
amount of gemcitabine that has been covalent incurpo-
rated into immunochemotherapeutics by measuring re-
sidual unbound gemcitabine before and after the Phase II
reaction or by measuring the difference in non-chemo-
therapeutic-occupied sites associated with either amine or
reduced sulfhydryl groups within anti-HER2/neu mo-
noclonal immunoglobulin compared to gemcitabine-im-
munochemotherapeutics [36,51,52].
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
Determination of the gemcitabine molar-incorpora-
tion-Index and gemcitabine molar-equivalent-concentra-
tions for gemcitabine-(C5-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 thio-
lated anti-HER2/neu fractions relative to the covalent
gemcitabine-immuno-chemotherapeutic following sepa-
ration by column chromatography [36,51,52]. Reduced
sulfhydryl groups were measure by combining anti-HER2/
neu or gemcitabine (C5-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 fol-
lowing incubation at 25˚C for 15 minutes. The amount
and concentration of sulfhydryl groups was then calcu-
lated utilizing a linearized standard curve generated with
reference control solutions of cysteine HCl monohy-
drate formulated at known concentrations (molar extinc-
tion coefficient: 14,150 M–1·cm–1).
Determination of the immunoglobulin concentration
for covalent gemcitabine-(C4-amide)-[anti-HER2/neu] and
gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu] im-
munochemotherapeutics was determined by measuring
spectrophotometric absorbance at 280 nm in combina-
tions with utilizing a 235 nm-vs-280 nm standardized
reference curve in order to accommodate for any poten-
tial absorption profile over-lap at 280 nm between gem-
citabine and immunoglobulin.
Mass/Size-Dependent Separation of Gemcitabine-Im-
munochemotherapeutics by Non-Reducing SDS-PAGE-
Covalent gemcitabine-(C4-amide)-[anti-HER2/neu] and
gemcitabine (C5-methylcarbamate)-[anti-HER2/neu] im-
munochemotherapeutics in addition to a anti-HER2/neu
immunoglobulin reference control fraction were ad-
justed 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-mercap-
toethanol or boiling. Each covalent gemcitabine immu-
nochemotherapeutic, the reference control immuno-
globulin 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% acry-
lamide) performed at 100 V constant voltage at 3˚C for
2.5 hours.
Immunodetection Analyses-Covalent gemcitabine-(C4-
amide)-[anti-HER2/neu] and gemcitabine (C5-methyl-
carbamate)-[anti-HER2/neu] immunochemo-therapeutics
following mass/size-dependent separation by non-re-
ducing SDS-PAGE were equilibrated in tank buffer de-
void 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 mem-
brane 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 Tris-
buffered saline (TBS: Tris HCl 0.1 M, NaCl 150 mM, pH
7.5, 40 ml) at 4˚C for 15 minutes followed by incubation
in TBS blocking buffer solution (Tris 0.1 M, pH 7.4, 40
ml) containing bovine serum albumin (5%) for 16 hours
at 2˚C to 3˚C applied in combination with gentle hori-
zontal agitation. Prior to further processing, nitrocellu-
lose membranes were vigorously rinsed in Tris buffered
saline (Tris 0.1 M, pH 7.4, 40 ml, n = 3x).
Rinsed BSA-blocked nitrocellulose membranes de-
veloped for immunodetection (Western-blot) analyses
were incubated with biotinylated goat anti-murine IgG
(1:10,000 dilution) at 4˚C for 18 hours applied in combi-
nation with gentle horizontal agitation. Nitrocellulose
membranes were then vigorously rinsed in TBS (pH 7.4,
4˚C, 50 ml, n = 3) followed by incubation in blocking
buffer (Tris 0.1 M, pH 7.4, with BSA 5%, 40 ml).
Blocking buffer was decanted from nitrocellulose mem-
brane blots and then rinsed in TBS (pH 7.4, 4˚C, 50 ml, n
= 3) before incubation with strepavidin-HRPO
(1:100,000 dilution) at 4˚C for 2 hours applied in combi-
nation with gentle horizontal agitation. Prior to chemi-
luminescent development nitrocellulose membranes were
vigorously rinsed in Tris buffered saline (Tris 0.1 M, pH
7.4, 40 ml, n = 3). Development of nitrocellulose mem-
branes by chemiluminescent autoradiography following
processing with conjugated HRPO-strepavidin required
incubation in HRPO chemiluminescent substrate (25˚C, 5
to 10 mins.). Autoradiographic images were acquired by
exposing radiographic film (Kodak BioMax XAR) to
nitrocellulose membranes sealed in transparent ultraclear
re-sealable plastic bags.
Mammary Adenocarcinoma Tissue Culture Cell Cul-
ture—The chemotherapeutic-resistant (SKBr-3) human
mammary adenocarcinoma cell line was utilized as an
ex-vivo neoplasia model. Mammary adenocarcinoma
(SKBr-3) characteristically over-expresses epidermal
growth factor receptor 1 (EGFR, ErbB-1, HER1) and
highly over-expresses epidermal growth factor receptor 2
(EGFR2, HER2/neu, ErbB-2, CD340, p185) at 2.2 ×
105/cell and 1 × 106/cell respectively.
Populations of the mammary adenocarcinoma (SKBr-3)
were propagated in 150-cc2 tissue culture flasks con-
taining McCoy’s 5a Modified Medium supplemented
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
with fetal bovine serum (10% v/v) and penicillin-strep-
tomycin at a temperature of 37˚C under a gas atmosphere
of air (95%) and carbon dioxide (5% CO2). Tissue cul-
ture media was not supplemented with growth factors,
growth hormones or other growth stimulants of any type.
Investigations were performed using mammary adeno-
carcinoma (SKBr-3) monolayer populations at a >85%
level of confluency.
Cell-ELISA Total Membrane-Bound Immunoglobulin
Assay-Cell suspensions of mammary adenocarcinoma
(SKBr-3) were seeded into 96-well microtiter plates in
aliquots of 2 × 105 cells/well and allowed to form a con-
fluence adherent monolayer over a period of 48 hours.
The growth media contents of individual wells was then
removed manually by pipette and serially rinsed (n = 3)
with PBS followed by stabilization of adherent cellular
monolayers onto the plastic surface of 96-well plates
with paraformaldehyde (4% in PBS, 15 minutes). Stabi-
lized mammary adenocarcinoma (SKBr-3) monolayers
were then incubated with gemcitabine-(C4-amide)-[anti-
HER2/neu] or gemcitabine (C5-methylcarbamate)-[anti-
HER2/neu] immunoconjugates formulated at gradient
concentrations of 0.1, 0.25, 0.5, 1.0, 5.0 and 10 µg/ml in
tissue culture growth media (200 µl/well). Direct contact
incubation between mammary adenocarcinoma (SKBr-3)
cellular monolayers and gemcitabine-(C4-amide)-[anti-
HER2/neu] or gemcitabine-(C5-methylcarbamate)-[anti-
HER2/neu] at 37˚C was performed over an incubation
period of 3-hours using a gas atmosphere of air (95%)
and carbon dioxide (5% CO2). Following serial rinsings
with PBS (n = 3), development of stabilized mammary
adenocarcinoma (SKBr-3) monolayers entailed incuba-
tion with β-galactosidase conjugated goat anti-mouse
IgG (1:500 dilution) for 2 hours at 25˚C with residual
unbound immunoglobulin removed by serial rinsing with
PBS (n = 3). Final cell ELISA development required
serial rinsing (n = 3) of stabilized cellular monolayers
with PBS followed by incubation with nitrophenyl-β-
D-galactopyranoside substrate (100 µl/well of ONPG
formulated fresh at 0.9 mg/ml in PBS pH 7.2 containing
MgCl2 10 mM, and 2-mercaptoethanol 0.1 M). Absorb-
ance within each individual well was measured at 410
nm (630 nm reference wavelength) after incubation at
37˚C for a period of 15 minutes.
Cell Vitality Stain-Based Assay for Measuring Cyto-
toxic Anti-Neoplastic Potency-Individual preparations
of gemcitabine-(C4-amide)-[anti-HER2 /neu] and gem-
citabine-(C5-methylcarbamate)-[anti-HER2/neu] were for-
mulated in growth media at standardized chemotherapeu-
tic-equivalent concentrations of 10–10, 10–9, 10–8, 10–7,
and 10–6 M (final concentration). Each chemotherapeu-
tic-equivalent concentration of covalent immunochemo-
therapeutic was then transferred in triplicate into 96-well
microtiter platesm containing mammary adenocarcinoma
(SKBr-3) monolayers (growth media 200 µl/well). Cova-
lent gemcitabine immunochemotherapeutics where then
incubated in direct contact with monolayer mammary
adenocarcinoma SKBr-3 populations for a period of 182-
hours at (37˚C under a gas atmosphere of air (95%) and
carbon dioxide/CO2 (5%). Following the initial 72-hour
incubation period, mammary adenocarcinoma (SKBr-3)
populations were replenished with fresh tissue culture
media with or without covalent gemcitabine-immuno-
Cytotoxic potencies for gemcitabine-(C4-amide)-[anti-
HER2/neu] and gemcitabine-(C5-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%
CO2) 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. Con-
tents 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
3. Results
Molar-Incorporation Index-Size-separation of covalent
immunochemotherapeutics like gemcitabine-(C4-amide)-
[anti-HER2/neu] and gemcitabine-(C5-methylcarbamate)-
[anti-HER2/neu] by micro-scale exchange column chro-
matography consistently yields preparations that contain
<4.0% of residual chemotherapeutic that is not cova-
lently bound to the immunoglobulin fraction [7,36,71,72].
Small residual amounts of non-covalently bound chemo-
therapeutic remaining within covalent immunochemo-
therapeutic preparations is generally considered to not be
available for further removal through any additional se-
quential column chromatography separations [73]. The
calculated estimate of the molar-incorporation-index
for the covalent gemcitabine-(C4-amide)-[anti-HER2/neu]
immunochemotherapeutic was 2.78 utilizing the organic
chemistry reaction scheme that forms an amide bond at
the C4 cytosine-like amine of gemcitabine resulting in the
initial synthesis of the UV-photoactivated gemcitabine-
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
Copyright © 2012 SciRes. JCT
(C4-amide) intermediate (Figures 1 and 2). The mo-
lar-incorporation-ration of 2.78-to-1 for gemcitabine-
(C4-amide)-[anti-HER2/neu] was relatively larger than
the 1.1-to-1 gemcitabine molar-incorporation-index at-
tained during the synthesis of gemcitabine-(C5-me-thyl-
carbamate)-[anti-HER2/neu] [36].
noglobulin-equivalent concentrations formulated at 0.010,
0.025, 0.050, 0.250, and 0.500 µg/ml (Figure 4). In order
to detect elevations in total membrane-bound gemcit-
abine-(C5-methylcarbamate)-[anti-HER2/neu] standard-
ized total immunoglobulin-equivalent concentrations had
to alternatively be formulated at 0.5, 1.0, 5.0 and 10.0
µg/ml (Figure 4). Collectively each of these sets of
cell-ELISA findings serve to validate the retained selec-
tive binding-avidity of gemcitabine-(C4-amide)-[anti-
HER2/neu] and gemcitabine-(C5-methylcarbamate)-
[anti-HER2/neu] for external membrane HER2/neu re-
Molecular Weight Profile Analysis-Mass/size sepa-
ration of covalent gemcitabine-(C4-amide)-[anti-HER2/
neu] and gemcitabine-(C5-methylcarbamate)-[anti-HER2/
neu] immunochemotherapeutics by SDS-PAGE in com-
bination with immunodetection analysis (Western blot)
and chemiluminescent autoradiography recognized a sin-
gle primary condensed band of 150-kDa between a mo-
lecular weight range of 5.0-kDa to 450-kDa (Figure 3)
Patterns of low-molecular-weight fragmentation (prote-
olytic/hydrolytic degradation) or large-molecular-weight
immunoglobulin polymerization were not detected (Fig-
ure 3). The observed molecular weight of 150-kDa for
both gemcitabine-(C4-amide)-[anti-HER2/neu] and gem-
citabine-(C5-methylcarbamate)-[anti-HER2/neu] directly
corresponds with the known molecular weight/mass of
reference control anti-HER2/neu monoclonal immuno-
globulin fractions (Figure 3). Analogous results have
been reported for similar covalent immunochemothera-
peutics [2,7,36,71,72,74].
150 kDa
1 2
Figure 3. Characterization of the major molecular weight
profile for covalent gemcitabine-(C4-amide)-[anti-HER2/neu]
and gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu] im-
munochemotherapeutics compared to anti-HER2/neu mono-
clonal immunoglobulin. Legends: (Lane-1) murine anti-
human HER2/neu monoclonal immunoglobulin reference
control; (Lane-2) covalent gemcitabine-(C4-amide)-[anti-
HER2/neu] immunochemotherapeutic; and (Lane-3) cova-
lent gemcitabine-(C5-methylcarbamate)-[anti-H ER2/ ne u] im-
munochemotherapeutic. Covalent gemcitabine immuno-
chemotherapeutics or anti-HER2/ neu monoclonal immu-
nogloublin 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 en-
tailed incubation of nitrocellulose membranes with stre-
pavidin-HRPO in combination with the use of a HRPO
chemiluminescent substrate for acquisition of autoradio-
graphy images.
Cell-Binding Analysis-Total bound immunoglobulin in
the form of gemcitabine-(C4-amide)-[anti-HER2/neu] or
gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu] on
the external surface membrane of adherent mammary
adenocarcinoma (SKBr-3) populations was measured by
cell-ELISA (Figure 4). Greater total membrane-bound
gemcitabine-(C4-amide)-[anti-HER2/neu] was detected
with progressive increases in standardized total immu-
Figure 4. Detection of total anti-HER2/neu immunoglobulin in the form of gemcitabine-(C4-amide)-[anti-HER2/neu] and
gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu] bound to the exterior surface membrane of chemotherapeutic-resistant
mammary adenocarcinoma (SKBr-3). Legends: (Left-Panel) gemcitabine-(C4-amide)-[anti-HER2/neu]; and (Right-Panel)
gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu]. Monolayer populations of mammary adenocarcinoma (SKBr-3) were
incubated with the covalent gemcitabine-(C4-amide)-[anti-HER2/neu] or gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu]
immunochemotherapeutics over a 4-hour pe riod and total immunoglobulin bound to the exterior sur f ace membr a ne w a s then
measured by cell-ELISA.
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
ceptor sites highly over-expressed at 1 × 106/cell on the
exterior surface membrane of mammary adenocarcinoma
(SKBr-3) populations (Figure 4) [36].
Cytotoxic Anti-Neoplastic Potency Analysis-Gemcitabine-
(C4-amide)-[anti-HER2/neu] and gemcitabine-(C5-methylcar-
bamate)-[anti-HER2/neu] exerted 41.1% and 30.8% maxi-
mum selective “targeted” cytotoxic anti-neoplastic potency
(58.9% and 69.2% residual survival) against chemothera-
peutic-resistant mammary adenocarcinoma (SKBr-3) at
the gemcitabine-equivalent concentration of 106 M
respectively (Figures 5-7). Profiles for the cytoto-
xic anti-neoplastic potency of gemcitabine-(C4-amide)-
[anti-HER2/neu] and gemcitabine-(C5-methylcarbamate)-
[anti- HER2/neu] after a 182-hour incubation period were
highly analogous to gemcitabine chemotherapeutic fol-
lowing a 72-hour incubation period at the gemcitabine-
equivalent concentrations of 1010 M, 109 M, 108 M,
107 M and 106 M (Figures 5 and 6). The cytotoxic
anti-neoplastic potency of gemcitabine-(C4-amide)-[anti-
HER2/neu], gemcitabine-(C5-methylcarbamate)-[anti-HER2/
neu] and gemcitabine after a 182-hour incubation period
were essentially equivalent at the gemcitabine-equivalent
concentrations of 1010 M and 109 M but not at 107 M or
106 M (Figures 5 and 7) [36]. Mean maximum cytotoxic
anti-neoplastic potencies for gemcitabine-(C4-amide)-
[anti-HER2/neu] at 182-hours, gemcitabine at 72-hours,
and gemcitabine at 182-hours were 41.1%, 48.0% and
88.3% (58.9%, 52.0% and 11.7% residual survival) at the
gemcitabine-equivalent concentration of 10-M respec-
tively (Figure 5). Gemcitabine-(C4-amide)-[anti-HER2/
neu] and gemcitabin-(C5-methylcarbamate)-[anti-HER2/
neu] immunochemotherapeutic both exerted profiles for
cytotoxic anti-neoplastic potency against chemothera-
peutic mammary adenocarcinoma (SKBr-3) that were
similar to epirubicin-(C3-amide)-[anti-HER2/neu] but
only at the chemotherapeutic-equivalent concentrations
of 1010 M, 109 M and 108 M respectively (Figure 8)
[71]. The level of cytotoxic anti-neoplastic potency for
epirubicin-(C3-amide)-[anti-HER2/neu] was substantially
higher at the chemotherapeutic-equivalent concentrations
of 107 M and 106 M after a 72-hour incubation period
(Figure 8). Mean maximum levels of anti-neoplastic
potency for gemcitabine-(C4-amide)-[anti-HER2/neu], gem-
citabin-(C5-methylcarbamate)-[anti-HER2/neu] and epi-
ribicin (C13-imino )-[anti-HER2/neu] were 41.1% (182-
hours), 30.8% (182-hours) and 88.5% (72-hours) at the
chemotherapeuticequivalent concentration of 106 M re-
spectively (Figures 5-8).
Comparison of the cytotoxic anti-neoplastic potency of
gemcitabine-( C 4-amide)-[anti-HER2/neu] and gemcitabine-
(C5-methylcarbamate)-[anti-HER2/neu] as a function of
immunoglobulin-equivalent concentrations (standardized
anti-HER2/neu content) and gemcitabine molarincorpora-
tion-index detected distinct differences between the two
covalent gemcitabine-immunochemo-therapeutics (Figure
9). Given this perspective, gemcitabine (C4-amide)-[anti-
HER2/neu] and gemcitabine-(C5-methylcarbamate)-[anti-
HER2/neu] each exerted an equivalent level of cytotoxic
anti-neoplastic potency against chemotherapeutic-resistant
mammary adenocarcinoma (SKBr-3) at immunoglobu-
lin-equivalent concentrations of 6.9 × 10–8 M and 9.1 ×
10–9 M respectively (Figure 9). Based on these calcula-
tions, gemcitabine-(C4-amide)-[anti-HER2/ neu] was ap-
proximately 7.6-fold more potent than gemcitabine-(C5-
methylcarbamate)-[anti-HER2/neu] at a cytotoxic anti-
neoplastic potency level of approximately 30% when
standardized as a function of immunoglobulin-equivalent
concentration (Figure 9). Monoclonal anti-HER2/neu
[7,36,71,72] and anti-EGFR [7] immunoglobulin frac-
tions alone between 0-to-182-hours do not exert detect-
able levels of ex-vivo cytotoxic anti-neoplastic potency
against chemotherapeutic-resistant mam- mary adenocarci-
noma (SKBr-3) which is in direct accord with previous
investigations (Figure 10) [7,28,29,32, 74,75].
4. Discussion
The creation of a synthetic covalent bond between gem-
citabine and monoclonal immunoglobulin, immuno-
Figure 5. Differences in cytotoxic anti-neoplastic potency
for gemcitabine-(C4-amide)-[anti-HER2/neu] compared to
gemcitabine alone. Legends: (4) covalent gemcitabine-(C4-
amide)-[anti-HER2/neu ] immunochemotherapeutic (182 hour
incubation period); (•) gemcitabine chemotherapeutic (72-
hour incubation period); and (A) gemcitabine chemothera-
peutic (182-hour incubation period). Chemotherapeutic-
resistant mammary adenocarcinoma (SKBr-3) monolayer
populations were incubated with covalent gemcitabine(C4-
amide)-[anti-HER2/neu] or gemcitabine formulated in trip-
licate at gradient gemcitabine-equivalent concentrations. Cyto-
toxic anti-neoplastic potency was measured using a MTT
cell vitality assay relative to matched negative reference con-
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
globulin fragments (e.g. Fab’), receptor ligands or other
biologically active protein fractions can be achieved util-
izing only a relatively small array of organic chemistry
reaction schemes. Chemical sites within gemcitabine that
are potentially available for synthetic covalent bond re-
actions include the (C4’)-NH2, (C3’)-OH and (C5’)-OH
groups that can be reversibly protected utilizing di-tert-
dibutyl dicarbonate [61] when non-selective organic
chemistry reaction schemes are employed. Generation of
a covalent bond at the C5-methylhydroxy group of gem-
citabine represents one molecular approach to synthesiz-
ing covalent gemcitabine-immunochemotherapeutics
or gemcitabine-ligand preparations [36,40,56-61,76]. A
second and possibly more infrequently utilized organic
chemistry reaction involves the creation of a covalent
bond at the cytosine-like C4-amine group of gemcitabine
either in the form of a direct link to a “targeting” plat-
form for selectivey chemotherapeutic delivery or alterna-
tively for the purpose of creating a gemcitabine reactive
intermediate [21,59,61,77,78]. Similar molecular strate-
gies have been employed to synthesize covalent anthra-
cycline immunochemotherapeutics through the formation
of a covalent bond at the α-monoamine (C3-amine) group
associated with the carbohydrate-like moiety of doxoru-
bicin, daunorubicin, or epirubicin [5,7-9,11-16,18,19,23,
71,72].In addition to the anthracyclines [72] and gem-
citabine analogous organic chemistry reaction schemes
employing succinimidyl 4,4-azipentanoate could poten-
tially be applied to covalently bond cytosine arabinoside
(Ara-C), 5-azacytidine, cladribine (2-chloro2’deoxyadenine),
clofarabine, decitabine (5-aza-2’deoxycytidine), fludara-
bine, lenalidamide, troxacitabine or other chemothera-
peutic (pharmaceutical) agents that contain an available
mono-amine group to large molecular weight platforms
like monoclonal immunoglobulin.
Gemcitabine has been covalently bound to biologi-
cally relevant ligands that inludes poly-L-glutamic
acid (polypeptide configuration), [58] cardiolipin, [56,
57] 1-dodecylthio-2-decyloxypropyl-3-phophatidic acid,
[40,60] lipid-nucleosides, [76] N-(2-hydroxypropyl) me-
thacrylamide polymer (HPMA), [21] benzodiazepine re-
ceptor ligand, [59,61] 4-(N)-valeroyl, 4-(N)-lauroyl, 4-
(N)-stearoyl, [78] 1,1’,2-tris-noraqualenecarboxylic acid,
[79] and the 4-fluoro [18F]-benzaldehyde derivative [77]
for application as a positron-emitting radionuclide. Few
if any published have described the molecular design,
chemical synthesis and evaluation of the cytotoxic anti-
neoplastic potency for gemcitabine immunochemothera-
peutic created by generating a covalent bond at either the
C5-methylhydroxy [36] or cytosine-like C4-amine groups
of gemcitabine. In addition, there has to date been
no previously published descriptions of utilizing suc-
cinimidyl 4,4-azipentanoate to create a UV-photoacti-
vated gemcitabine-(C4-amide) intermediate to facilitate
Figure 6 .Relative cytotoxic anti-neoplastic potency for
gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcitabine-
(C4-amide)-[anti-HER2/neu] compared to gemcitabine alone.
Legends: (4) covalent gemcitabine-(C4-amide)-[anti-HER2/
neu] immunochemotherapeutic (182-hours); (A) covalent
gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu] immuno-
chemotherapeutic (182-hour incubation period) and (•) gem-
citabine chemotherapeutic (72-hour incubation period) Chemo-
therapeutic-resistant mammary adenocarcinoma (SKBr-3)
monolayer populations were incubated individually with
gemcitabine-(C4-amide)-[anti-HER2 /neu ], gemcitabine(C5-me-
thylcarbamate)-[anti-HER2/neu] or gemcitabine formulated
in triplicate at gradient gemcitabine-equivalent concentra-
tions. Cytotoxic anti-neoplastic potency w as measur ed using
a MTT cell vitality assay relative to matched negative ref-
erence controls.
Figure 7. Relative cytotoxic anti-neoplastic potency of cova-
lent gemcitabine-(C4-amide)-[anti-HER2/neu] and gemcit-
abine-(C5-methylcarbamate)-[anti-HER2/ne u] immunoc hemo-
therapeutics as a function of gemcitabine-equivalent con-
centrations. Legends: (4) gemcitabine-(C4-amide)-[anti-HER2/
neu]; (A) gemcitabine-(C5-methyl carbonate)-[anti-HER2/neu];
and (•) gemcitabine alone. Chemot herapeutic-resistant mam-
mary adenocarcinoma (SKBr-3) monolayer populations
were incubated 182-hours with covalent gemcitabine im-
munochemotherapeutics or gemcitabine formulated in trip-
licate at gradient gemcitabine-equivalent concentrations.
Cytotoxic anti-neoplastic potency was measured using a
MTT cell vitality assay relative to matched negative refer-
ence controls.
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
Figure 8. Relative cytotoxic anti-neoplastic potency of the
covalent immunochemotherapeutics gemcitabine-(C4-amide)-
[anti-HER2/neu], gemcita bine-(C5-methyl-carbamat e)-[anti-HER2/
neu] and epirubicin-(C3- amide)-[anti-HER2/neu] formulated
at chemotherapeutic-equivalent concentrations. Legends: (4)
gemcitabine-(C4-amide)-[anti-HER2/neu] at 182-hours; (•) gem-
citabine-(C5-methylcarbonate)- [anti-HER 2/ neu] at 182-hours;
and (A) epirubicin-(C3-amide)-[anti-HER2/neu] at 72-hours.
Chemotherapeutic-resistant mammary adenocarcinoma (SKBr-
3) monolayer populations were incubated with the covalent
immunochemotherapeutics or gemcitabine chemotherapeu-
tic that were each formulated in triplicate at gradient che-
motherapeutic -equivale nt concentratio ns. Cytotoxic anti- neo-
plastic potency w as measured using a MTT cell vitality assay
relative to matched negative reference controls.
synthesis of a covalent gemcitabine immunochemothera-
peutic similar to gemcitabine-(C4-amide)-[anti-HER2/
neu] (Figure 1). Analogous synthetic organic chemistry
reaction schemes have however been published on a very
limited scale for the production of a covalent epirubi-
cin-(C3-amide)-[anti-HER2/neu] immunochemotherapeu-
tic [72].
Speicific attributes related to the variables of 1) che-
motherapeutic chemical composition; 2) organic chemis-
try reaction selectivity; 3) molar ratio formulations
(chemotherapeutic/reagent/IgG); 4) specific sequential
order of individual organic chemistry reaction schemes,
and 5) extension of incubation periods for organic chem-
istry reactions during the synthesis of gemcitabine
(C4-amide)-[anti-HER2/neu], gemcitabine-(C5-methyl-
carbamate)-[anti-HER2/neu] and epirubicin-(C3-amide)-
[anti-HER2/neu] collectively minimized side reactions
resulting in the formation of extraneous side-products
(Figure 1) [36,72]. Reaction condition variables are es-
pecially important during the initial phases of synthesiz-
ing gemcitabine-(C5-methylcarbamate) and UV-photo-acti-
vated gemcitabine-(C4-amide) reactive intermediates
(Figure 2). [36] Generation of the UV-photoactivated
gemcit- abine-(C4-amide) intermediate with succinimidyl
4,4-azipentanoate involves the succinimide ester group
Figure 9. Relative cytotoxic anti-neoplastic potency of gem-
citabine-(C4-amide)-[anti-HER2/neu] an d ge mci tab i ne- (C 5-me-
thylcarbamate)-[anti-HER2/neu] as a function of immu-
noglobulin-equivalent concentration. Legends: (4) gemcit-
abine-(C4-amide)-[anti-HER2/neu] with a gemcitabine mo-
lar-incorporation-index of 2.78:1 (182-hour incubation pe-
riod); and (•) gemcitabine-(C5-methyl carbonate)-[anti-HER2/
neu] with a gemcitabine molar-incorporation-index of 1.1:1
(182-hour incubation period). Arrows indicate the approxi-
mate concentration of gemcitabine-(C4-amide)[anti-HER2/neu]
and gemcitabine-(C5-methyl carbonate)[anti-HER2/neu] ne-
cessary to achieve a 30% level of cytotoxic anti-neoplastic
potentcy. Chemotherapeutic-resistant mammary adenocar-
cinoma (SKBr-3) monolayer populations were incubated
with either covalent gemcitabine immunochemotherapeu-
tics formulated in triplicate at gradient concentrations. Cy-
totoxic anti-neoplastic potency measured using a MTT cell
vitality assay relative to matched negative reference con-
preferentially reacting with and forming a colvaent
bond at the C4 cytosine-like amine group of gemcitabine.
In organic solvent systems like DMSO and DMF suc-
cinimidyl 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 re-
portedly dissociate redily with the addition of small
amounts of ddH2O 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-photo-
activated moiety of succinimidyl 4,4-azipentanoate dur-
ing the extended incubation with gemcitabine. Alterna-
tively, an aqueous buffer formulated between the pH
range of 7 to 9 can effectively promote covalent amide
bond formation when shorter incubation periods are in-
dicated. Utilization of aqueous buffer with a pH of 6.5
and implementation of lower reaction condition tem-
peratures (e.g. 4˚C) have reportedly been found to en-
hance the reaction of succinimide ester group with dif
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
Figure 10. Relative cytotoxic anti-neoplastic potency of co-
valent gemcitabine-(C4-amide)-[anti-HER2/neu] immuno-
chemotherapeutic compared to anti-HER2/neu monoclonal
immunoglobulin. Legends: (4) covalent gemcitabine-(C4-
amide)-[anti-HER2/neu] immunochemotherapeutic; and (A)
anti-HER2/neu monoclonal immunoglobulin. Chemothera-
peutic-resistant mammary adenocarcinoma (SKBr-3) mon-
olayer populations were incubated with gemcitabine-(C4-
amide)-[anti-HER2/neu] and anti-HER2/neu monoclonal
immunoglobulin formulated in triplicate at at gradient
concentrations. Cytotoxic anti-neoplastic potency was meas-
ured using a MTT cell vitality assay relative to matched
negative reference controls .
ferent primary amine subtypes (e.g. lysine ε-amine-
vspeptide N-terminal amine).
Conservative speculation suggests that one of the rea-
sons for the differences in molar incorporation indexes
(2.78-vs-1.1) for gemcitabine-(C4-amide)-[anti-HER2
/neu] compared to gemcitabine-(C5-methylcarbamate)-
[anti-HER2/neu] respectively was probably due to a
combination of two critical reaction condition variables.
Most notable in this regard was the application of the
UV-photoactivated gemcitabine-(C4-amide) intermediate
at a 10-to-1 molar ratio to anti-HER2/neu monoclonal
immunoglobulin in concert with a lack of a require-
ment [72] for 2-iminothiolane (2-IT) [36,71] or N-suc-
cinimidyl-S-acetylthioacetate (SATA) [7] to pre-thiolate
immunoglobulin fractions, IgG fragments, receptor li-
gands or other biologically active peptide proteins (Figure
1). Higher molar incorporation indexes are possible to
achieve with certain methodology modifications but the
harsher synthesis conditions required for such purposes
almost invariably are accompanied by substantial reduc-
tions in final product yield of the covalent immuno-
chemotherapeutic [6]. In addition to harsh reaction con-
ditions, immunoglobulin antigen binding-avidity can be
reduced as a function of excessive covalent chemothera-
peutic incorporation into or within the Fab antigen-
binding domain of immunoglobulin fractions. Despite
this consideration, relatively higher molar incorpora-
tion indexes were attained during the synthesis of
ge m citabine-(C4-amide)-[anti-HER2/neu] (2.78-to-1 or
278%) compared to gemcitabine-(C5-methylcarbamate)-
[anti-HER2/neu] (1.1-to-1 or 110%), [36] epirubicin-
(C13-imino )-[anti-HER2/neu] (0.4-to-1 or 40%), [71]
epirubicin-(C3-amide)-[anti-HER2/neu] (0.275 - to- 1 or
27.5%),[7,72] and epirubicin-(C3-amide)-[anti-EGFR]
(0.407-to-1 or 40.7%) [7]. Conservative speculation sug-
gests that one reason for the higher molar incorporation
index observed for covalent gemcitabine-(C4-amide)-
[anti-HER2/neu] immunochemotherapeutic was due to
the implementation of a synthesis scheme that involved a
distinctly different organic chemistry reactions and that
even higher molar incorporation indexes along with
greater levels of cytotoxic anti-neoplastic potency are
possible (Figure 1).
A somewhat unique property of the UV-photoacti-
vated gemcitabine-(C4-amide) intermediate generated
utilizing succinimidyl 4,4-azipentanoate in Phase-I of the
synthetic organic chemistry reaction scheme is that it
does not contain a sulfhydryl-reactive maleimide group
(Figure 1). The lack of a sulfhydryl-reactive maleimide
moiety within the structure of the UV-photoactivated
gemcitabine-(C4-amide) intermediate therefore allows it
to be applied to synthesize covalent immunochemo-
therapeutics without a requirement to pre-thiolateamine
groups associated with lysine residues in the amino acid
sequence of anti-HER2/neu monoclonal immunoglobulin.
Because of this feature it is possible to initiate Phase-II
of the synthetic organic chemistry reaction scheme for
gemcitabine-(C4-amide)-[anti-HER2/neu] without the
introduction of reduced sulfhydryl groups into the amino
acid sequence of immunoglobulin (IgG) fractions, IgG
fragments [F(ab’)2 or Fab’], receptor ligands, receptor
ligand fragments or other biologically relevant pro-
tein fractions (Figure 1). In contrast, the gemcitabine-
(C5-methylcarbamate) reactive intermediate synthesized
with N-[p-maleimidophenyl]-isocyanate does contain a
sulfhydryl-reactive maleimide group (Figure 2) [36].
Similarly, anthracycline reactive intermediates applied to
synthesize many if not most anthracycline-immuno-
chemotherapeutics also employ a sulfhydryl-reactive malei-
mide group to facilitate the creation of a covalent bond
with immunoglobulin or other biologically active protein
fractions [7,71,72]. Such synthetic organic chemistry
reactions schemes are dependent upon the utilization of
heterobifunctional reactants similar to succinimidyl-4-
(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),
[7,80-82] N-ε-maleimidocaproic acid hydrazide (EMCH),
[9,10,71] or N-[p-maleimidophenyl]- isocyanate (PMPI)
[36,53-55]. In the application of these covalent bond-
forming reagents, disruption of disulfide bond structures
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Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
or prethiolation of immunoglobulin or other biological
protein fractions is almost invariably required due to the
relatively low number of non-sterically hindered sulfhy-
dryl groups available within the amino acid sequence of
most biologically active proteins in the form of reduced
cysteine amino acid residues (e.g. R-SH). Increasing the
number of available reduced sulf- hydryl groups can be
achieved by the application of 1,4dithiothreitol which
reduces intramolecular cystinecystine [26-28] and similar
disulfide structures [83] (DTT: R-CH2-S-S-CH2-R—2
R-CH2-SH). The actual synthetic introduction of “new”
or additional reduced sulfhydryl groups at the ε-amine of
lysine amino acid residues is possible utilizing organic
chemistry reaction schemes that employ 2-iminothio-
lane (2-IT), [2,6, 36,71,84] mercaptosuccinimide, [85] or
N-succinimidylS-acetylthioacetate (SATA) [7,84,86]. Al-
ternatively, carboxyl groups on molecules like heparin
and hyaluronic acid (HA) can be thiolated with 3,3’di-
thiobis (propanoic)hydrazide (DPTH) [83,87] or divinyl-
sulfone (DVS), [88, 89] in addition to the hydroxyl
groups of molecules with a cholesterol-like core [90]. In
the application of DTPH the integral disulfide bond is
subsequently reduced with DTT reagent [83,87].
Covalently bonding gemcitabine or other chemothera-
peutic agents to biological protein fractions like immu-
noglobulin 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 intro-
duction 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 separa-
tion procedure. The brief duration of the synthetic or-
ganic chemistry reaction scheme for gemcitabine-
(C4-amide)-[anti-HER2/neu] utilizing succinimidyl 4,4-
azipentanoate is realized because of the relatively rapid
time course for the Phase-I, but especially the Phase-II
organic chemistry reaction. The synthetic organic chem-
istry reaction scheme has also been designed so that ad-
justment of buffer pH to different levels during the pro-
cedure 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 rela-
tively mild conditions thereby possing a lower risk of
protein fragmentation or polymerization (e.g. IgG-IgG)
through premature intra-molecular disulfide bond forma-
tion [2]. Realized benefits therefore include greater re-
tained biological activity (e.g. antigen binding-avidity)
and increased total final yield of a function immuno
chemotherapeutic end-product. Lastly, lack of a require-
ment to either convert existing cystine-cystine disulfide
bonds to their reduced form or the introduction of re-
duced 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, [7,80-82] EMCH [9,10,71] or PMPI [36,53-55]
is only equivalent to or lower than the extent of pre-
thiolation at ε-amine groups associated with the finite
number of lysine residues within the amino acid se-
quence of protein fractions. In prethiolation dependent
synthesis schemes higher epirubicin molar-incorporation-
indexes 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-(C4-amide)-[anti-HER2/neu] com-
pared to gemcitabine-(C5-methylcarbamate)-[anti-HER2/
neu] at the cytotoxic anti-neoplastic potency level of ap-
proximately 30% can be attributed to a combination of a
greater degree retained biological activity for anti-
HER2/neu (cell-ELISA) and a higher gemcitibin mo-
lar-incorporation-index of 2.78-to-1 for gemcitabine-(C4-
amide)-[anti-HER2/neu] in contrast to 1.1-to-1 for gem-
citabine-(C5-methylcarbamate)-[anti-HER2/neu] (Figure
9). Both of these properties are anticipated to be attribut-
able 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-(C4-amide)-[anti- HER2/neu].
Implementation of succinimidyl 4,4-azipentanoate in
the synthesis scheme for gemcitabine-(C4-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 activ-
ity that affords properties of selective “targeted” delivery.
In contrast to SMCC, [7,80-82] EMCH [9,10,71] or
PMPI [36,53-55] the synthesis of gemcitabine-(C4-am-
ide)-[anti-HER2/neu] utilizing succinimidyl 4,4-azipen-
tanoate has the added benefit of not introducing extrane-
ous five and six carbon or carbon/nitrogen ring structures
into the final covalent immunochemotherapeutic end-
product (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
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
organic solvent systems supplemented with triethylamine
[N(CH2CH3)3] or similar proton acceptor molecules at
low concentrations. In stock solutions of reaction mix-
tures formulated in aqueous buffers a significant amount
of hydrolytic degradation of succinimidyl 4,4-azipen-
tanoate is expected to occur to varying degrees. Alterna-
tively, if stock solutions and reaction mixtures of gem-
citabine 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-(C4-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-azipen-
tanoate allow for the convenient option of “presynthe-
sizing” and preserved storage of the UV-photoactivated
gemcitabine-(C4-amide) intermediate for an extend pe-
riod of time for the future production of covalent gem-
citabine-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” pharma-
ceutical delivery.
Cell-Binding Profiles-Increases in standardized im-
munoglobulin-equivalent concentrations of gemcitabine-
(C4-amide)-[anti-HER2/neu] and gemcitabine-(C5-methyl-
carbamate)-[anti-HER2/neu] correlated with elevations
in total immunoglobulin membrane binding profiles in
populations of human mammary adenocarcinoma de-
tected by cell-ELISA (Figure 4). The lower standardized
immunoglobulin-equivalent concentration range for gem-
citabine-(C4-amide)-[anti-HER2/neu] compared to gem-
citabine-(C5-methylcarbamate)-[anti-HER2/neu] implies
that the former covalent gemcitabine immunochemo-
therapeutic may have a higher level of retained anti-
HER2/neu binding-avidity. The most probably explana-
tion for this difference can be attributed to the imple-
mentation of milder organic chemical reaction conditions
and a lack of a requirement for pre-thiolate of anti-
HER2/neu fractions. Previous investigations have simi-
larly noted that modest alterations in synthetic chemistry
and elevations in the chemotherapeutic molar incorpora-
tion index can profoundly influence immunoglobulin
binding properties [29].
Cytotoxic Anti-Neoplastic Activity/Potency-Covalent
gemcitabine conjugates have been synthesized that exert
greater cytotoxic anti-neoplastic potency than gemcit-
abine chemotherapeutic alone, but these preparations
have been produced in the form of gemcitabine-
(oxyether phopholipid) [40,60] or dual gemcitabine/
doxorubicin-HPMA (N-(2-hydroxypropyl) methacryla-
mide polymer). [21] In a very limited number of investi-
gations, the cytotoxic anti-neoplastic activity for majori-
ties of these covalently bonded gemcitabine preparations
were reported against human mammary carcinoma
(MCF7/WT-2’), [60] human mammary adenocarcinoma
(BG-1), [60] promyelocytic leukemia, [40,60] a T-4 lym-
phoblastoid clone, [60] glioblastoma, [40,60] cervical
epithelioid carcinoma, [60] colon adenocarcinoma, [60]
pancreatic adenocarcinoma, [60] pulmonary adenocarci-
noma, [60] oral squamous cell carcinoma, [60] and
prostatic carcinoma [21].
Increases in the molar chemotherapeutic-equivalent
concentrations of gemcitabine-(C4-amide)-[anti-HER2/neu]
and gemcitabine-(C5-methylcarbamate)-[anti-HER2/ neu]
created corresponding elevations in the cytotoxic anti-
neoplastic potency and declines in the residual survival
of chemotherapeutic-resistant mammary adenocarcinoma
(SKBr-3) populations (Figures 5-7). Neither gemcit-
abine-(C4-amide)-[anti-HER2 /neu ] or gemcitabine-(C5-me-
thylcarbamate)-[anti-HER2/neu] exerted substantially
greater selective “targeted” anti-neoplastic potency
against chemotherapeutic-resistant mammary adenocar-
cinoma (SKBr-3) that was greater than gemcitabine alone
when formulated at molar chemotherapeutic-equivalent
concentrations between 1010 M to 106 M and an incu-
bation period of 182-hours (Figures 5-7). Such findings
are in contrast to covalent epirubicin-[anti-HER2/neu]
immunochemotherapeutics that possess equivalent or
greater cytotoxic anti-neoplastic potency levels than
epirubicin alone [7,71,72]. Despite this difference, the
selectively “targeted” cytotoxic anti-neoplastic potency
of covalent gemcitabine-(C4-amide)-[anti-HER2/neu]
and gemcitabine-(C5-methylcarbama te)-[anti-HER2/neu]
immunochemotherapeutics at 182-hours was almost
identicial to levels exerted by gemcitabine after a
72-hour incubation period [92].
In the interpretation of the cytotoxic anti-neoplastic
potency of gemcitabine-(C4-amide)-[anti-HER2/neu] and
gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu] it should
be emphasized that such comparisons were made at
gemcitabine-equivalent concentrations. Alternatively, if
comparisons of cytotoxic anti-neoplastic potency for the
two covalent gemcitabine-immunochemotherapeutics are
made as a function of immunoglobulin-equivalent con-
centrations (e.g. anti-HER2/neu content) and gemcitabine
molar-incorporation-indexes then it is possible to detect a
relatively greater level of potency for gemcitabine-
(C4-amide)-[anti-HER2/neu] compared to gemcitabine (C5-
methylcarbamate)-[anti-HER2/neu] (Figure 9). Given this
perspective, gemcitabine-(C4-amide)-[anti-HER2/neu] and
gemcitabine-(C5-methylcarbamate)-[anti- HER2/neu] for
example each exerted a 30% level of cytotoxic anti-neo-
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
plastic potency at immunoglobulin-equivalent concentra-
tions of 6.9 × 10–8 M and 9.1 × 10–9 M respectively
(Figure 9). Based on these calculations, gemcitabine-
(C4-amide)-[anti-HER2/neu] was approximately 7.6-fold
more potent than gemcitabine-(C5-methylcarbamate)-
[anti-HER2/neu] when cytotoxic anti-neoplastic activity
was standardized as a function of immunoglobulin-
equivalent concentration. Presumably this difference in
cytotoxic anti-neoplastic potency was due to a combina-
tion of a greater degree of retained biological activity for
anti-HER2/neu (cell-ELISA) and a higher gemcitibin
molar-incorporation-index (2.78-to-1) for gemcitabine-
(C4-amide)-[anti-HER2/neu] compared to gemcitabine-
(C5-methylcarbamate)-[anti-HER2/neu]. Both of these
properties are likely attributable to the application of
gentler reaction conditions again due in part to a lack of
a requirement for anti-HER2/neu prethiolation during
Phase-II of the organic chemistryreaction scheme applied
in the synthesis of gemcitabine-(C4-amide)-[anti-HER2/
In contrast to most covalent anthracycline immuno-
chemotherapeutics described to date, a longer 182-hour
incubation period was applied to access the cytotoxic
anti-neoplastic potency of gemcitabine-(C4-amide)-[anti-
HER2/neu] and gemcitabine-(C5-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 explana-
tions may account for the requirement to use longer in-
cubation periods for the ex-vivo evalution of gemcitabine
compared to anthracycline-immunochemotherapeutics or
anthracycline covalent bound to other molecular plat-
forms with properties that afford selective “targeted”
delivery (e.g. receptor ligands). Since the covalent im-
munochemotherapeutics gemcitabine-(C4-amide)-[anti-
HER2/neu], gemcitabine-(C5-methylcarbamate)-[anti-HER2/
neu] [36] and several epirubicin-[anti-HER2/ neu] im-
munochemotherapeutics [7,71,72] all selectively “target”
chemotherapeutic delivery at the same HER2/ neu re-
ceptor site highly over-expressed on the external surface
membrane of mammary adenocarcinoma (SKBr-3), it is
possible that differences in their cytotoxic anti- neoplas-
tic activity may be attributable to, 1) differences in the
vulnerability of covalent bond structures created during
the synthesis of gemcitabine-(C4-amide)-[anti-HER2/neu]
and gemcitabine-(C5-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 en-
zyme fractions necessary for biochemically liberating
gemcitabine versus epirubicin from covalent immuno-
chemotherapeutics; 3) variation in the acidic characteris-
tics associated with the endosome/lysosome microenvi-
ronment 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 rec-
ognized for this class of chemotherapeutic agent; 5) vul-
nerability of the gemcitabine moiety in covalent gemcit-
abine immunochemotherapeutics to inactivation by
deamination. The fact that cytotoxic anti-neoplastic po-
tency profiles for gemcitabine-(C4-amide)-[anti-HER2/
neu] and gemcitabine-(C5-methylcarbamate)-[anti-HER2/
neu] at the end of a 182-hour incubation period were
very similar to those for gemcitabine after a 72-hour in-
cubation period implies that cytotoxic anti-neoplastic
activity of the gemcitabine immunochemotherapeutics is
possibly delayed due to a slow release of the chemo-
therapeutic moiety that is apparently longer compared to
the rate of anthracycline-release from covalent epirubi-
cin-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-(C4-amide)-[anti-HER2/neu] and
could represent a desirable property that can be em-
ployed as a molecular strategy to evoke “super-loading
that in turn can facilitate extensive and sustained chemo-
therapeutic 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 cyto-
toxic anti-neoplastic potency. The positive findings di-
rectly address one of the major objectives that originally
motivated the molecular design and synthesis of gem-
citabine-(C4-amide)-[anti-HER2/neu]. Additionally, there
was a perceived need for the molecular design of a syn-
thesis scheme that was composed of a sequential series of
organic chemistry reactions that could facilitate relatively
rapid production of gemcitabine-(C4-amide)-[anti-HER2/
neu] using mild conditions that affored minimal degrada-
tive low molecular weight fragmentation or large mo-
lecular weight polymerization (e.g. IgG-IgG). Recent
investigations describing the methodology employed for
the synthesis of epirubicin-(C5-amide)-[anti-HER2/neu]
through the application of a UV-photoactivated epirubi-
cin intermediate revealed that there was a high degree of
probability that a similar organic chemistry regimen
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
could be adapted as a model with minor modifications
for the relatively rapid synthesis of a covalent gemcit-
abine-(C4-amide)-[anti-HER2/neu] immunochemothera-
peutic [72]. In this context, a set of organic chemistry
reactions were implemented to synthesize gemcitabine-
(C4-amide)-[anti-HER2/neu] that had not previously been
described for the production of a gemcitabine-immuno-
chemotherapeutic or covalent gemcitabine-ligand prepa-
ration. The organic chemistry synthesis reactions utilized
for the production of gemcitabine-(C4-amide)-[anti-HER2/
neu] also possesses practical utility because it can serve
as a model or template for the molecular design and pro-
duction of other covalent immunochemotherapeutics.
Conceptually there are at least five analytical vari-
ables that could have alternatively been modified to
achieve substantially higher total levels of cytotoxic
anti-neoplastic potency for gemcitabine-(C4-amide)-[anti-
HER2/neu]. First, incubation times with chemothera-
peutic-resistant mammary adenocarcinoma (SKBr-3) could
have been lengthened to a period > 182-hours [36]
there-by allowing greater opportunity for larger amounts
of gemcitabine to be internalized by receptor-mediated
endocytosis and subsequently liberated intracellularly
from gemcitabine-(C4-amide)-[anti-HER2/neu] or gem-
citabine-(C5-methylcarbamate)-[anti-HER2/neu]. Sup-
port for this consideration in based on the observation
that there was a simple dose effect for gemcitabine-(C4-
amide)-[anti-HER2/neu], and because mammary adeno-
carcinoma (SKBr-3) survivability was very similar when
challenged with gemcibatine-(C5-methylcarbamate)-[anti-
HER2/neu] (182-hours) compared to gemcitabine (72-
hours), but increased dramatically for gemcitabine when
the incubation period was extended to 182-hours (Fig-
ures 5-7).[36] Conservative speculation suggests that
incubation of chemotherapeutic-resistant mammary ade-
nocarcinoma (SKBr-3) with gemcitabine-(C4-amide)-
[anti-HER2/neu] or gemcibatine-(C5-methylcarbamate)-
[anti-HER2/neu] for periods greater than 182-hours
would have resulted in even higher levels of cytotoxic
anti-neoplatic potency since there was no indication that
the level of cytotoxic activity achieved against chemo-
therapeutic-resistant mammary adenocarcinoma (SKBr-3)
had reached a “plateau” or maximum level (Figures 5-7).
Second, cytotoxic anti-neoplastic potency of gemci-
batine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C5-
methylcarbamate)-[anti-HER2/neu] could have alterna-
tively been assessed against a human neoplastic cell type
that was not chemotherapeutic-resistant similar to cancer
cell types utilized to evaluate majority of the covalent
biochemotherapeutics reported in the literature to date.
Rare exceptions to this consideration have been the ap-
plication of chemotherapeutic-resistant metastatic mela-
noma M21 (covalent daunorubicin immunochemothera-
peutics synthesized using anti-chondroitin sulfate pro-
teoglycan 9.2.27 surface marker), [29,32,93] chemo-
therapeutic-resistant mammary carcinoma MCF-7AdrR
(covalent anthracycline-ligand chemotherapeutics syn-
thesized utilizing epidermal growth factor/EGF or an
EGF fragment); [94] and chemotherapeutic-resistant
mammary adenocarcinoma (SKBr-3) populations (epiru-
bicin-anti-HER2/neu, epirubicin-anti-EGFR, gemcitabine-
HER2/neu) [7, 36,71,72].
Somewhat analogous to the concept of non-chemo-
therapeutic resistant cancer cell types, the cytotoxic anti-
neoplatic potency of gemcibatine-(C4-amide)-[anti-HER2/
neu] and gemcitabine-(C5methylcarbamate)-[anti-HER2/
neu] could also have alternatively been measured against
an entirely different neoplastic cell type such as pancre-
atic carcinoma, [95] small-cell lung carcinoma, [96]
neuroblastoma, [97] or leukemia/lymphoid [60,98] popu-
lations due to their relatively higher gemcitabine sensi-
tivity. Similarly, human promyelocytic leukemia, [40,60]
T-4 lymphoblastoid clones, [60] glioblastoma, [40,60]
cervical epitheliod carcinoma, [60] colon adenocarci-
noma, [60] pancreatic adenocarcinoma, [60] pulmonary
adenocarcinoma, [60] oral squamous cell carcinoma, [60]
and prostatic carcinoma [21] have all been found to be
sensitive to gemcitabine and gemcitabine-(oxyether
phopholipid) covalently bonded chemotherapeutics.
Within this array of neoplastic cell types, however, hu-
man mammary carcinoma (MCF-7/WT-2’) [60] and
mammary adenocarcinoma (BG-1) [60] are known to be
relatively more resistant to gemcitabine and gemcit-
abine-(oxyether phopholipid) chemotherapeutic conju-
gate. Presumably this pattern of diminished gemcitabine
sensitivity is directly relevant to the cytotoxic anti-neo-
platic potency detected for gemcibatine-(C4-amide)-
[anti-HER2/neu] and gemcitabine-(C5-methylcarbamate)
-[anti-HER2/neu] compared to gemcitabine in chemo-
therapeutic-resistant mammary adenocarcinoma (SKBr-3)
populations (Figures 5-7).
Third, cytotoxic anti-neoplastic potency of gemci-
batine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C5-
methylcarbamate)-[anti-HER2/neu] could have been
evaluated at higher gemcitabine-equivalent concentra-
tions. Since gemcitabine in contrast to the anthracyclines
has rarely been synthetically incorporated into (cova-
lently bonded to) selective “targeted” delivery platforms,
[21,36,40,57-60] it is uncertain if this chemotherapeutic
can be utilized to consistently create covalent gemcit-
abine immunochemotherapeutics that posses signifi-
cantly 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 gemcit-
abine immunochemotherapeutics is the opportu- nity to
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Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
create a new anti-cancer modality that affords reduced
exposure of healthy tissues and organ systems to the cy-
totoxic anti-neoplastic properties of chemothera- peutics.
By design, such attributes are facilitated by selectively
“targeted” delivery of chemotherapeutic moieties in a
manner that produces cytotoxic anti-neoplastic properties
that are largely restricted to malignant lesions. Given this
perspective and applying basic pharmacology principals,
the variable of potency can simply be addressed through
adjustment of concentration (dose administered) within
the limitations of induced side effects and sequelae.
Fourth, anti-neoplastic potency of gemcitabine-(C4-
amide)-[anti-HER2/neu] and gemcitabine-(C5-methylcar-
bamate)-[anti-HER2/neu] would likely have been sub-
stantially greater if cellular proliferation had been as-
sessed with either [3H]-thymidine, or an ATP-based as-
say method because of their reportedly >10-fold greater
sensitivity in detecting early cell injury compared to
MTT vitality stain based assay methods [99,100]. De-
spite this consideration, MTT vitality stain based assays
continue to be extensively applied for the routine as-
sessment of true cytotoxic anti-neoplastic potency of
chemotherapeutics covalently incorporated synthetically
into molecular platforms that provide properties of selec-
tive “targeted” delivery.[7,40,58,60,101-106] 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 cel-
lular injury that could ultimately be reversible.
Fifth, cytotoxic anti-neoplastic potency of gemci-
batine-(C4-amide)-[anti-HER2/neu] and gemcitabine-(C5-
methylcarbamate)-[anti-HER2/neu] immunochemothera-
peutic 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 neo-
plastic cell populations (that genuinely do possess prop-
erties 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 [107-109]. Enhanced
levels of covalent immunochemotherapeutic potency
measured in-vivo is presumably attributable in part to
induced responses by the innate immune system that in-
cludes antibody-de-pendent cell cytotoxicity (ADCC)
phenomenon in concert with complementedmediated
cytolysis initiated or stimulated by the formation of anti-
gen-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 comple-
ment-mediated cytolysis to the in-vivo cytotoxic
anti-neoplastic potency of covalent immunochemothera-
peutics 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 chemo-
therapeutic agents [48,49,83,89,111-118]. 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], meth-
otrexate [111], paclitaxel [49,111], or vinblastine [111].
Sixth, several modifications could have been made in
the synthesis strategy for gemcitabine-(C4-amide)-[anti-
HER2/neu] and gemcitabine-(C5-methylcarbamate)-[anti-
HER2/neu] in order to increase the gemcitabine mo-
lar-incorporation-index. Examples in this regard include
the application of gemcitabine and the covalent bond
forming reagents at higher molar concentrations, imple-
mentation 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 reagent-
to-immunoglobulin molar ratios in a manner that forces
the organic chemistry reactions in a direction that in-
creases final product yield. Unfortunately, such modifi-
cations usually also require or impose harsher reaction
conditions that necessitate an acceptance for a higher risk
of reduced biological activity (e.g. decreased antigen bind-
ing avidity) and substantial declines in final/total product
yield [6, 108]. Aside from overly harsh synthesis condi-
tions, 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’ anti-
genbinding region becomes excessive. Such modifica-
tions can result in only modest declines in immunoreac-
tivity (e.g. 86% for a 73:1 ratio) but disproportionately
large declines in cytotoxic anti-neoplastic activity in ad-
dition 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 com-
ponent of covalent immunochemotherapeutics is criti-
cally important. It not only serves as a means of facili-
tating selectively “targeting” chemotherapeutic delivery,
but it also initiates or induces internalization of covalent
immunochemotherapeutics by mechanism of receptor-
mediated endocytosis assuming an appropriate mem-
brane-associated antigen has been selected as a “target”
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
(e.g. many carcinoma and adenocarcinoma cell types
highly over-express HER2/neu and/or EGFR) [119]. Al-
though specific data for HER2/neu and EGFR expression
by mammary adenocarcinoma (SKBr-3) is limited, [7]
other neoplastic cell types like metastatic multiple mye-
loma are known to internalize and metabolize approxi-
mately 8 × 106 molecules of anti-CD74 monoclonal an-
tibody per day [120]. Immunoglobulin-induced receptor-
mediated endocytosis at membrane HER2/neu complexes
can ultimately lead to increases in the intracellular con-
centration of selectively “targeted”/delivered chemo-
therapeutic that approach and exceed levels 8.5 [121] to
>100 × fold greater [122] than those that can ever possi-
bly be achieved by simple passive chemotherapeutic dif-
fusion from out of the intravascular compartment.
The application of succinimidyl 4,4-azipentanoate in
contrast to succinimidyl-4-(N-maleimidomethyl)-cyc-
lohexane-1-carboxylate (SMCC), [7,80-82] N-ε-male-
imidocaproic acid hydrazide (EMCH), [8-10,51,52,71] or
N-[p-maleimidophenyl]-isocyanate (PMPI) [36,53-55]
can facilitate greater flexibility in synthesis methods de-
signed to increase the chemotherapeutic molar-incorpo-
ration-index during the creation of covalent immuno-
chemotherapeutics without having to use harsher reaction
conditions. The major risk of compromising the biologi-
cal integrity (antigen binding avidity) of gemcitabine-
(C4-amide)-[anti-HER2/neu] synthesized with a UV-photo-
activated 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 re-
ceptor binding region of ligands). Despite the general
validity of the inverse relationship between chemothera-
peutic molar-incorporation-index and retained biological
activity (e.g. anti-HER2/neu mediated selective “tar-
geted” delivery) and the greater potency of covalent im-
munochemotherapeutics 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 immu-
nochemotherapeutics or ligand-chemotherapeutic prepa-
ration. In this regard, it is important that external mem-
brane-associated sites be chosen that are known to func-
tionally undergo phenomenon analogous to receptor-
mediated-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 re-
quirement 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.
5. Acknowledgements
The authors would like to acknowledge Mr. Tom Thomp-
son in the Office of Agriculture Communications for
assistance with the photographic illustration.
[1] T. Kaneko, D. Willner, J. O. Knipe, G. R. Braslawsky, R.
S. Greenfield and D. M. Vyas, “New Hydrazone Deriva-
tives of Adriamycin and Their Immunoconjugates: A Cor-
relation between Acid-Stability and Cytotoxicity,” Bio-
conjugate Chemistry, Vol. 2, No. 3, 1991, pp. 133-141.
[2] G. Di Stefano, M. Lanza, F. Kratz, L. Merina and L.
Fiume, “A Novel Method for Coupling Doxorubicin to
Lactosaminated Human Albumin by an Acid Sensitive
Hydrazone Bond: Synthesis, Characterization. And Pre-
liminary Biological Properties of the Conjugate,” Euro-
pean Journal of Pharmaceutical Sciences, Vol. 23, No.
4-6, 2004, pp. 393-397. doi:10.1016/j.ejps.2004.09.005
[3] F. Kratz, A. Warnecke, K. Scheuermann, C. Stockmar, J.
Schwab, P. Lazar, P. Drückes, N. Esser, J. Drevs, D.
Rognan, C. Bissantz, C. Hinderling, G. Folkers, I. Ficht-
ner and C. Unger, “Probing the Cysteine-34 Position of
Endogenous Serum Albumin with Thiol-Binding, Dox-
orubicin Derivatives. Improved Efficacy of an Acid-Sen-
sitive Doxorubicin Derivative with Specific Albumin-
Binding Properties Compared to That of the Parent Com-
pound,” Journal of Medicinal Chemistry, Vol. 45, No. 25,
2002, pp. 5523-5533. doi:10.1021/jm020276c
[4] C. Unger, B. Häring, M. Medinger, J. Drevs, S. Steinbild,
F. Kratz and K. Mross, “Phase I and Pharmacokinetic
Study of the (6-Maleimidocaproyl) Hydrazone Derivative
of Doxorubicin,” Clinical Cancer Research, Vol. 13, No.
16, 2007, pp. 4858-4866.
[5] C. Mazuel, J. Grove, G. Gerin and K. P. Keenan, “HPLC-
MS/MS Determination of a Peptide Conjugate Prodrug of
Doxorubicin, and Its Active Metabolites, Leucine-Dox-
orubicin and Doxorubicin, in Dog and Rat Plasma,”
Journal of Pharmaceutical and Biomedical Analysis, Vol.
33, No. 5, 2003, pp. 1093-1102.
[6] R. S. Greenfield, T. Kaneko, A. Daues, M. A. Edson, K.
A. Fitzgerald, L. J. Olech, J. A. Grattan, G. L. Spitalny
and G. R. Braslawsky, “Evaluation In-Vitro of Adriamy-
cin Immunoconjugates Synthesized Using an Acid-Sen-
sitive Hydrazone Linker,” Cancer Research, Vol. 50, No.
20, 1990, pp. 6600-6607.
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
[7] C. P. Coyne, M. Ross, J. Bailey and T. Jones, “Dual Po-
tency Anti-HER2/neu and Anti-EGFR Anthracycline-
Immunoconjugates in Chemotherapeutic-Resistant Mam-
mary Carcinoma Combined with Cyclosporin-A and
Verapamil P-Glycoprotein Inhibition,” Journal of Drug
Targeting, Vol. 17, No. 6, 2009, pp. 474-489.
[8] A. Lau, G. Berube, C. H. J. Ford and M. Gallant, “Novel
Doxorubicin-Monoclonal Anti-Carcinoembryonic Anti-
gen Antibody Immunoconjugate Activity In-VivoBio-
organic and Medicinal Chemistry, Vol. 3, No. 10, 1995,
pp. 1305-1312. doi:10.1016/0968-0896(95)00126-2
[9] M. Kruger, U. Beyer, P. Schumacher, C. Unger, H. Zahn
and F. Kratz, “Synthesis and Stability of Four Maleimide
Derivatives of the Anti-Cancer Drug Doxorubicin for the
Preparation of Chemoimmunoconjugates,” Chemical &
Pharmaceutical Bulletin, Vol. 45, No. 2, 1997 pp. 399-
401. doi:10.1248/cpb.45.399
[10] D. Y. Furgeson, M. R. Dreher and A. Chilkoti, “Struc-
tural Optimization of a ‘Smart’ Doxorubicin-Polypeptide
Conjugate for Thermally Targeted Delivery to Solid Tu-
mors,” Journal of Controlled Release, Vol 110, No. 2,
2006, pp. 362-369. doi:10.1016/j.jconrel.2005.10.006
[11] J. F. Liang and V. C. Yang, “Synthesis of Doxorubi-
cin-Peptide Conjugate with Multidrug Resistant Tumor
Cell Killing Activity,” Bioorganic and Medicinal Chem-
istry Letters, Vol. 15, No. 22, 2005, pp. 5071-5075.
[12] M. Sirova, J. Strohalm, V. Subr, D. Plocova, P. Ros-
smann, T. Mrkvan, K. Ulbrich and B. Rihova, “Treatment
with HPMA Copolymer-Based Doxorubicin Conjugate
Containing Human Immunoglobulin Induces Long-Last-
ing Systemic Anti-Tumor Immunity in Mice,” Cancer
Immunol Immunother, Vol. 56, No. 1, 2007, pp. 35-47.
[13] B. K. Wong, D. Defeo-Jones, R. E. Jones, V. M. Garsky,
D. M. Feng, A. Oliff, M. Chiba, J. D. Ellis and J. H. Lin,
“PSA-Specific and Non-PSA-Specific Conversion of a
PSA-Targeted Peptide Conjugate of Doxorubicin to Its
Active Metabolite,” Drug Metabolism and Disposition,
Vol. 29, No. 3, 2001, pp. 313-318.
[14] G. L. Bidwell, A. N. Davis, I. Fokt, W. Priebe and D.
Raucher, “A Thermally Targeted Elastin-Like Polypep-
tide-Doxorubicin Conjugate Overcomes Drug Resistance,”
Investigational New Drugs, Vol. 25, No. 4, 2007, pp.
313-326. doi:10.1007/s10637-007-9053-8
[15] K. A. Ajaj, R. Graeser, I. Fichtner and F. Kratz, “In-Vitro
and In-Vivo Study of an Albumin-Binding Prodrug of
Doxorubicin That Is Cleaved by Cathepsin B,” Cancer
Chemotherapy and Pharmacology, Vol. 64, No. 2, 2009,
pp. 413-418. doi:10.1007/s00280-009-0942-8
[16] C. Ryppa, H. Mann-Steinberg, I. Fichtner, H. Weber, R.
Satchi-Fainaro, M. L. Biniossek and F. Kratz, “In-Vitro
and In-Vivo Evaluation of Doxorubicin Conjugates with
the Divalent Peptide E-[c(RGDfK)2] That Targets In-
tegrin aVb3,” Bioconjugate Chemistry, Vol. 19, No. 7,
2008 pp. 1414-1422. doi:10.1021/bc800117r
[17] Y. F. Huang, D. Shangguan, H. Liu, J. A. Phillips, X.
Zhang, Y. Chen and W. Tan, “Molecular Assembly of an
Aptamer-Drug Conjugate for Targeted Drug Delivery to
Tumor Cells,” A European Journal of Chemical Biology,
Vol. 10, No. 5, 2009, pp. 862-868.
[18] Y. Ren, D. Wei and X. Zhan, “Inhibition of P-Glycopro-
tein and Increasing of Drug-Sensitivity of a Human Car-
cinoma Cell Line (KB-A-1) by an Anti-Sense Oligode-
oxynucleotide-Doxorubicin Conjugate In-Vitro,” Biotech-
nology and Applied Biochemistry, Vol. 41, No. 2, 2005,
pp. 137-143. doi:10.1042/BA20040058
[19] Y. Ren, X. Zhan, D. Wei and J. Liu, “In-Vitro Reversal
MDR of Human Carcinoma Cell Line by an Antisense
Oligodeoxynucleotide-Doxorubicin Conjugate,” Biomedi-
cine & Pharmacotherapy, Vol. 58. No. 9, 2004, pp. 520-
[20] L. Kovar, T. Etrych, M. Kabesova, V. Subr, D. Vetvicka,
O. Hovorka, J. Strohalm, J. Sklenar, P. Chytil, K. Ulbrich
and B. Rihova, “Doxorubicin Attached to HPMA Co-
polymer via Amide Bond Modifies the Glycosylation
Pattern of EL4 Cells,” Tumor Biology, Vol. 31. No. 4,
2010, pp 233-242. doi:10.1007/s13277-010-0019-7
[21] T. Lammers, V. Subr, K. Ulbrich, P. Peschke, P. E. Huber,
W. E. Hennink and G. Storm, “Simultaneous Delivery of
Doxorubicin and Gemcitabine to Tumors in Vivo Using
Prototypic Polymeric Drug Carriers,” Biomaterials, Vol.
30, No. 20, 2009, pp. 3466-3475.
[22] H. Krakovicova, T. Ethch and K. Ulbrich, “HPMA-Based
Polymerconjugates with Drug Combinations,” European
Journal of Pharmacology, Vol. 37. No. 3-4, 2009, pp.
[23] N. Cao and S. S. Feng, “Doxorubicin Conjugated to
D-Alpha-Tocopheryl Polyethylene Glycol 1000 Succinate
(TPGS): Conjugation Chemistry, Characterization, In-
Vitro and In-Vivo Evaluation,” Biomaterials, Vol. 29, No.
28, 2008, pp. 3856-3865.
[24] P. C. Rodrigues, U. Beyer, P. Schumacher, T. Roth, H. H.
Fiebig, C. Unger, L. Messori, P. Orioli, D. H. Paper, R.
Mülhaupt and F. Kratz, “Acid-Sensitive Polyethylene
Glycol Conjugates of Doxorubicin: Preparation, In-Vitro
Efficacy and Intracellular Distribution,” Bioorganic &
Medicinal Chemistry, Vol. 7, No. 11, 1999, pp. 2517-
2524. doi:10.1016/S0968-0896(99)00209-6
[25] F. Kratz, “Albumin as a Drug Carrier: Design of Prodrugs,
Drug Conjugates and Nanoparticles,” Journal of Con-
trolled Release, Vol. 132, No. 3, 2008, pp. 171-183.
[26] K. Inoh, H. Muramatsu, S. Torii, S. Ikematsu, M. Oda, H.
Kumai, S. Sakuma, T. Inui, T. Kimura and T. Muramatsu,
“Doxorubicin-Conjugated Anti-Midkine Monoclonal An-
tibody as a Potential Anti-Tumor Drug,” Japanese Jour-
nal of Clinical Oncology, Vol. 36, No. 4, 2006, pp.
207-211. doi:10.1093/jjco/hyl004
[27] G. L. Griffiths, M. J. Mattes, R. Stein, S. V. Govindan, I.
D. Horak, H. J. Hansen and D. M. Goldenberg, “Cure of
SCID Mice Bearing Human B-Lymphoma Xenografts by
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
an Anti-CD74 Antibody-Anthracycline Drug Conjugate,”
Clinical Cancer Research, Vol. 9, No. 17, 2003, pp.
[28] P. Sapra, R. Stein, J. Pickett, Z. Qu, S. Govindan, V, T. M.
Cardillo, H. J. Hanson, I. D. Horak, G. L. Griffiths and D.
M. Goldenberg, “Anti-CD74 Antibody-Doxorubicin Con-
jugate, IMMU-110, in a Human Multiple Myeloma Xeno-
graph and in Monkeys,” Clinical Cancer Research, Vol.
11, No. 14, 2005, pp. 5257-5264.
[29] H. M. Yang and R. A. Reisfeld, “Doxorubicin Conjugated
with Monoclonal Antibody Directed to a Human Mela-
noma-Associated Proteoglycan Suppresses Growth of Es-
tablished Tumor Xenografts in Nude Mice,” Proceedings
of the National Academy of Sciences, Vol. 85, 1988, pp.
1189-1193. doi:10.1073/pnas.85.4.1189
[30] P. A. Trail, D. Willner, S. J. Lasch, A. J. Henderson, S.
Hofstead, A. M. Casazza, R. A. Firestone, I. Hellstrom
and K. E. Hellstrom, “Cure of Xenografted Human Car-
cinomas by BR96-Doxorubicin Immunoconjugates,”
Science, Vol. 261, No. 5118, 1993, pp. 212-215.
[31] E. Diener, U. Diner, A. Sinha, S. Xie and R. Vergidis,
“Specific Immunosuppression by Immunotoxins Con-
taining Daunomycin,” Science, Vol. 231, No. 4734, 1986,
pp. 148-150. doi:10.1126/science.3484557
[32] R. O. Dillman, D. E. Johnson, J. Ogden and D. Beidler,
“Significance of Antigen, Drug, and Tumor Cell Targets
in the Preclinical Evaluation of Doxorubicin, Daunorubi-
cin, Methotrexate, and Mitomycin-C Monoclonal Anti-
body Immunoconjugates,” Molecular Biotherapy, Vol. 1,
5, 1989, pp. 250-255.
[33] M. Page, D. Thibeault, C. Noel and L. Dumas, “Coupling
a Preactivated Daunorubicin Derivative to Antibody. A
New Approach,” Anticancer Research, Vol. 10, No. 2A,
1990, pp. 353-357.
[34] J. Reményi, B. Balázs, S. Tóth, A. Falus, G. Tóth and F.
Hudecz, “Isomer-Dependent Daunomycin Release and in
Vitro Antitumour Effect of Cis-Aconityl-Daunomycin,”
Biochemical and Biophysical Research Communications,
Vol. 303, No. 2, 2003, pp. 556-561.
[35] J. R. Ogden, K. Leung, S. A. Kunda, M. W. Telander, B.
P. Avner, S. K. Liao, G. B. Thurman and R. K. Oldham,
“Immunoconjugates of Doxorubicin and Murine Antihu-
man Breast Carcinoma Monoclonal Antibodies Prepared
Via an N-Hydroxysuccinimide Active Ester Intermediate
of Cis-Aconityl-Doxorubicin: Preparation and in Vitro
Cytotoxicity,” Molecular Biotherapy, Vol 1, No. 3, 1989,
pp. 170-174.
[36] C. P. Coyne, T. Jones and T. Pharr, “Synthesis of a Co-
valent Gemcitabine-(Carbamate)-[Anti-HER2/neu] im-
munochemotherapeutic and Cytotoxic Anti-Neoplastic
Activity against Chemotherapeutic-Resistant SKBr-3
Mammary Carcinoma,” Bioorganic and Medicinal Chem-
istry, Vol. 19, No. 1, 2011, pp. 67-76.
[37] A. I. Shamseddine, M. J. Khalifeh, F. H. Mourad, A. A.
Chehal, A. Al-Kutoubi, J. Abbas, M. Z. Habbal, L. A.
Malaeb and A. B. Bikhazi, “Comparative Pharmacoki-
netics and Metabolic Pathway of Gemcitabine during In-
travenous and Intra-Arterial Delivery in Unresectable
Pancreatic Cancer Patients,” Clinical Pharmacokinetics,
Vol. 44, No. 9, 2005, pp. 957-967.
[38] E. Giovannetti, A. C. Laan, E. Vasile, C. Tibaldi, S. Nan-
nizzi, S Ricciardi, A. Falcone, R. Danesi and G. J. Peters,
“Correlation between Cytidine Deaminase Genotype and
Gemcitabine Deamination in Blood Samples,” Nucleo-
sides Nucleotides Nucleic Acids, Vol. 27, No. 6, 2008, pp.
720-725. doi:10.1080/15257770802145447
[39] J. A. Gilbert, O. E. Salavaggione, Y. Ji, L. L. Pelley-
mounter, B. W. Eckloff, E. D. Wieben, M. M. Ames and
R. M. Weinshilboum, “Gemcitabine Pharmacogenomics:
Cytidine Deaminase and Deoxycytidylate Deaminase
Gene Resequencing and Functional Genomics,” Clinical
Cancer Research, Vol. 12, No. 6, 2006, pp. 1794-1803.
[40] R. L. Alexander, B. T. Greene, S. Torti and V. G. L.
Kucera, “A Novel Phospholipid Gemcitabine Conjugate
Is Able to Bypass Three Drug-Resistance Mechanisms,”
Cancer Chemotherapy and Pharmacology, Vol. 56, No. 1,
2005, pp. 15-21. doi:10.1007/s00280-004-0949-0
[41] R. J. Pietras, M. D. Pegram, R. S. Finn, D. A. Maneval
and D. J. Slamon, “Remission of Human Breast Cancer
Xenografts on therapy with Humanized Monoclonal An-
tibody to HER-2 Receptor and DNA-Reactive Drugs,”
Oncogene, Vol. 17, No. 17, 1998, pp. 2235-2249.
[42] R. Marches and J. W. Uhr, “Enhan Cement of the
p27Kip1-Mediated Antiproliferative Effect of Trastuzu-
mab (Herceptin) on HER2-Overexpressing Tumor Cells,”
International Journal of Cancer, Vol. 112, No. 3, 2004,
pp. 492-501. doi:10.1002/ijc.20378
[43] M. Sliwkowski, X, J. A. Lofgren, G. D. Lewis, T. E. Ho-
taling, B. M. Fendly and J. A. Fox, “Nonclinical Studies
Addressing the Mechanism of Action of Trastuzumab
(Herceptin),” Seminars in Oncology, Vol. 26, Suppl. 12,
1999, pp. 60-70.
[44] N. U. Lin, L. A. Carey, M. C. Liu, J. Younger, S. E.
Come, M. Ewend, G. Harris, E. Bullitt, A. D. Van den
Abbeele, J. W. Henson, X. Li, R. Gelman, H. J. Burstein,
E. Kasparian, D. G. Kirsch, A. Crawford, F. Hochberg
and E. P. Winer, “Phase II Trial of Lapatinib for Brain
Metastases in Patients with Human Epidermal Growth
Factor Receptor 2-Positive Breast Cancer,” Journal of
Clinical Oncology, Vol. 26, No. 12, 2008, pp. 1993-1999.
[45] M. A. Cobleigh, C. L. Vogel, D. Tripathy, N. J. Robert, S.
Scholl, L. Fehrenbacher, J. Wolter, V. Paton, S. Shak, G.
Lieberman and D. J. Slamon, “Multinational Study of the
Efficacy and Safety of Humanized Anti-HER2 Mono-
clonal Antibody in Women Who Have HER2-Overex-
pressing Metastatic Breast Cancer That Has Progressed
after Chemotherapy for Metastatic Disease,” Journal of
Clinical Oncology, Vol. 17, No. 9, 1999, pp. 2639-2648.
[46] C. L. Vogel, M. A. Cobleigh, D. Tripathy, J. C. Gutheil,
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
L. N. Harris, L. Fehrenbacher, D. J. Slamon, M. Murphy,
W. F. Novotny, M. Burchmore, S. Shak, S. J. Stewart and
M. Press, “Efficacy and Safety of Trastuzumab as a Sin-
gle Agent in First-Line Treatment of HER2-Overex-
pressing Metastatic Breast Cancer,” Journal of Clinical
Oncology, Vol. 20, No. 3, 2002, pp. 719-726.
[47] G. D. Lewis Phillips, G. Li, D. L. Dugger, L. M. Crocker,
K. L. Parsons, E. Mai, W. A. Blättler, J. M. Lambert, R.
Chari, V, R. J. Lutz, W. L. Wong, F. S. Jacobson, H.
Koeppen, R. H. Schwall, S. R. Kenkare-Mitra, S. D.
Spencer and M. X. Sliwkowski, “Targeting HER2-Posi-
tive Breast Cancer with Trastuzumab-DM1, an Antibody-
Cytotoxic Drug Conjugate,” Cancer Research, Vol. 68,
No. 22, 2008, pp. 9280-9290.
[48] J. A. García-Sáenz, M. Martín, A. Calles, C. Bueno, L.
Rodríguez, J. Bobokova, A. Custodio, A. Casado and E.
Díaz-Rubio, “Bevacizumab in Combination with Metro-
nomic Chemotherapy in Patients with Anthracycline- and
Taxane-Refractory Breast Cancer,” Journal of Chemo-
therapy, Vol. 20, No. 5, 2008, pp. 632-639.
[49] D. J. Slamon, B. Leyland-Jone, S. Shak, H. Fuchs, V.
Paton, A. Bajamonde, T. Fleming, W. Eiermann, J.
Wolter and M. Pegram, “Use of Chemotherapy plus
Monoclonal Antibody against HER2 for Metastatic
Breast Cancer That Overexpress HER2,” The New Eng-
land Journal of Medicine, Vol. 344, No. 11, 2001, pp.
786-792. doi:10.1056/NEJM200103153441101
[50] U. Beyer, T. Roth, P. Schumacher, G. Maier, A. Unold, A.
W. Frahm, H. H. Fiebig, C. Unger and F. Kratz, “Synthe-
sis and In-Vitro Efficacy of Transferring Conjugates of
the Anticancer Drug Chlorambucil,” Journal of Medicinal
Chemistry, Vol. 41, No. 15, 1998, pp. 2701-2708.
[51] L. C. Xu, M. Nakayama, K. Harada, A. Kuniyasu, H.
Nakayama, S. Tomiguchi, A. Kojima, M. Takahashi, M.
Ono, Y. Arano, H. Saji, Z. Yao, H. Sakahara, J. Konishi
and Y. Imagawa, “Bis(Hydroxamamide)-Based Bifunc-
tional Chelating Agent for 99mTc Labeling of Polypep-
tides,” Bioconjugate Chemistry, Vol. 10, No. 1, 1999, pp.
9-17. doi:10.1021/bc980024j
[52] Y. Arano, T. Uezono, H. Akizawa, M. Ono, K. Wakisaka,
M. Nakayama, H. Sakahara, J. Konishi and A. Yokoyama,
“Reassessment of Diethylenetriaminepentaacetic Acid
(DTPA) as a Chelating Agent for Indium-111 Labeling of
Polypeptides Using a Newly Synthesized Monoreactive
DTPA Derivative,” Journal of Medicinal Chemistry, Vol.
39 No. 18, 1996, pp. 3451-3460. doi:10.1021/jm950949+
[53] M. E. Annunziato, U. S. Patel, M. Ranade and P. S.
Palumbo, “p-Maleimidophenyl Isocyanate: A Novel He-
terbifunctional Linker for Hydroxyl to Thiol Coupling,”
Bioconjugate Chemistry, Vol. 4, No. 3, 1993, pp. 212-218.
[54] Q. Liu, J. R. de Wijn and C. A. van Blitterswijk, “Cova-
lent Bonding of PMMA, PBMA, and Poly(HEMA) to
Hydroxyapatite Particles,” Journal of Biomedical Materi-
als Research, Vol. 40, No. 2, 1998, pp. 257-263.
[55] X. L. Wang, Y. Huang, J. Zhu, Y. B. Pan, R. He and Y. Z.
Wang, “Chitosan-Graft Poly(p-Dioxanone) Copolymers:
Preparation, Characterization, and Properties,” Carbohy-
drate Research, Vol. 344, No. 6, 2009, pp. 801-807.
[56] S. M. Ali, A. R. Khan, M. U. Ahmad, P. Chen, S. Sheikh
and I. Ahmad, “Synthesis and Biological Evaluation of
Gemcitabine-Lipid Conjugate (NEO6002),” Bioorganic
& Medicinal Chemistry Letters, Vol. 15, No. 10, 2005, pp.
2571-2574. doi:10.1016/j.bmcl.2005.03.046
[57] P. Chen, P. Y. Chien, A. R. Khan, S. Sheikh, S. M. Ali,
M. U. Ahmad and I. Ahmad, “In-Vitro and in-Vivo
Anti-Cancer Activity of a Novel Gemcitabine-Cardiolipin
Conjugate,” Anticancer Drugs, Vol. 17, No. 1, 2006, pp.
53-61. doi:10.1097/01.cad.0000185182.80227.48
[58] L. V. Kiew, S. K. Cheong, K. Sidik and L. Y. Chung,
“Improved Plasma Stability and Sustained Release Profile
of Gemcitabine via Polypeptide Conjugation,” Interna-
tional Journal of Pharmaceutics, Vol. 391, No. 1-2, 2010,
pp. 212-220. doi:10.1016/j.ijpharm.2010.03.010
[59] P. Guo, J. Ma, S. Li, Z. Guo, A. L. Adams and J. M.
Gallo, “Targeted Delivery of a Peripheral Benzodiazepine
Receptor Ligand-Gemcitabine Conjugate to Brain Tu-
mors in a Xenograft Model,” Cancer Chemotherapy and
Pharmacology, Vol. 48, No. 2 , 2001, pp. 169-176.
[60] R. L. Alexander, S. L. Morris-Natschke, K. S. Ishaq, R. A.
Fleming and G. L. Kucera, “Synthesis of Cytotoxic Ac-
tivity of Two Novel 1-Dodecylthio-2-Decyloxypropyl-
3-Phophatidic Acid Conjugates with Gemcitabine and
Cytosine Arabinoside,” Journal of Medicinal Chemistry,
Vol. 46, 19, 2003, pp. 4205-4208.
[61] Z. Guo and J. M. Gallo, “Selective Protection of 2’,2’-
Difluorodexoycytidine (Gemcitabine),” The Journal of
Organic Chemistry, Vol. 64, No. 22, 1999, pp. 8319-8322.
[62] P. A. Trail, D. Willner, J. Knipe, A. J. Henderson, S. J.
Lasch, M. E. Zoeckler, M. D. TrailSmith, T. W. Doyle, H.
D. King, A. M. Casazza, J. Brown, S. J. Hofstead, R. S.
Greenfield, R. A. Firestone, K. Mosure, K. F. Kadow, M.
B. Yang, K. E. Hellstrom and I. Hellstrom, “Effect of
linker Variation on the Stability, Potency and Efficacy of
Carcinoma-Reactive BR64-Doxorubicin Immunoconju-
gates,” Cancer Research, Vol. 57, No. 1, 1997, pp. 100-
[63] T. Etrych, T. Mrkvan, B. Ríhová and K. Ulbrich, “Star-
Shaped Immunoglobulin-Containing HPMA-Based Con-
jugates with Doxorubicin for Cancer Therapy,” Journal of
Controlled Release, Vol. 122, No. 1, 2007, pp. 31-38.
[64] J. Liu, H. Zhao, K. J. Volk, S. E. Klohr, E. H. Kerns and
M. S. Lee, “Analysis of Monoclonal Antibody and Im-
munoconjugate Digests by Capillary Electrophoresis and
Capillary Liquid Chromatography,” Journal of Chroma-
tography A, Vol. 735, No. 1-2, 1996, pp. 357-366.
[65] F. Kratz, G. Ehling, H.-M. Kauffmann and C. Unger,
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
“Acute and Repeat-Dose Toxicity Studies of the (6-
Maleimidocaproyl)Hydrazone Derivative of Doxorubicin
(DOXO-EMCH), an Albumin-Binding Prodrug of the
Anticancer Agent Doxorubicin,” Human & Experimental
Toxicology, No. 26, No. 1, 2007, pp. 19-35.
[66] D. Lebrecht, A. Geist, U. P. Ketelsen, J. Haberstroh, B.
Setzer, F. Kratz and U. Walker, “The 6-Maleimido-
caproyl Hydrazone Derivative of Doxorubicin (Doxo-
emch) Is Superior to Free Doxorubicin with Respect to
Cardiotoxicity and Mitochondrial Damage,” International
Journal of Cancer, Vol. 120, No. 4, 2006, pp. 927-934.
[67] F. Kratz, U. Beyer, P. Collery, F. Lechenault, A. Cazabat,
P. Schumacher, U. Falken and C. Unger, “Preparation,
Characterization and In-Vitro Efficacy of Albumin Con-
jugates of Doxorubicin,” Biological and Pharmaceutical
Bulletin, Vol. 21, No. 1, 1998, pp. 56-61.
[68] F. Kratz, U. Beyer, T. Roth, N. Tarasova, P. Collery, F.
Lechenault, A. Cazabat, P. Schumacher, C. Unger and U.
Falken, “Transferrrin Conjugates of Doxorubicin: Syn-
thesis, Characterization, Cellular Uptake, and In-Vitro Ef-
ficacy,” Journal of Pharmaceutical Sciences, Vol. 87, No.
3, 1998, pp. 338-346. doi:10.1021/js970246a
[69] M. N. Kirstein, I. Hassan, D. E. Guire, D. R. Weller, J. W.
Dagit, J. E. Fisher and R. P. Remmel, “High-Performance
Liquid Chromatographic Method for the Determination of
Gemcitabine and 2’,2’-Difluorodeoxyuridine in Plasma
and Tissue Culture Media,” Journal of Chromatography
B: Biomedical Sciences and Applications, Vol. 835, No.
1-2 , 2006, pp. 136-142.
[70] V. Reichelova, F. Albertioni and J. Liliemark, “Determi-
nation of 2-Chloro-2’-Deoxyadenosine Nucleotides in
Leukemic Cells by Ion-Pair High-Performance Liquid
Chromatography,” Journal of Chromatography B: Bio-
medical Sciences and Applications, Vol. 682, No. 1, 1996,
pp. 115-123. doi:10.1016/0378-4347(96)00048-5
[71] C. P. Coyne, T. Jones, A. Sygula, J. Bailey and L. Pin-
chuk, “Epirubicin-[Anti-HER2/neu] Synthesized with an
Epirubicin-(C13-Imino)-EMCS Analog: Anti-Neo-plastic
Activity against Chemotherapeutic-Resistant SKBr-3 Mam-
mary Carcinoma in Combination with Organic Sele-
nium,” Journal of Cancer Therapy, Vol. 2, No. 1, 2011,
pp. 22-39. doi:10.4236/jct.2011.21004
[72] C. P. Coyne, T. Jones and R. Bear, “Synthesis of Epirubi-
cin-(C3-amide)-[Anti-HER2/neu] utilizing a UV-Photo-
activated Epirubicin Intermediate,” Cancer Biotherapy
and Radiopharmaceuticals, Vol. 27, No. 1, 2012, pp. 41-
55. doi:10.1089/cbr.2011.1097
[73] U. Beyer, B. Rothen-Rutishauser, C. Unger, H. Wunderli-
Allenspach and F. Kratz, “Difference in the Intracellular
Distribution of Acid-Sensitive Doxorubicin-Protein Con-
jugates in Comparison to Free and Liposomal-Formulated
Doxorubicin as Shown by Confocal Microscopy,” Phar-
maceutical Research, Vol. 18, No. 1, 2001, pp. 29-38.
[74] J. A. Sinkule, S. T. Rosen and J. A. Radosevich, “Mono-
clonal Antibody 44-3A6 Doxorubicin Immunoconjugates:
Comparative in Vitro Anti-Tumor Efficacy of Different
Conjugation Methods,” Tumour Biology, Vol. 12, No. 4,
1991, pp 198-206. doi:10.1159/000217705
[75] G. P. Sivam, P. J. Martin, R. A. Reisfeld and B. M.
Mueller, “Therapeutic Efficacy of a Doxorubicin Im-
munoconjugate in a Preclinical Model of Spontaneous
Metastatic Human Melanoma,” Cancer Research, Vol. 55,
No. 11, 1995, pp. 2352-2356.
[76] R. L. Alexander and G. L. Kucera, “Lipid nucleoside
Conjugates for the Treatment of Cancer,” Current Phar-
maceutical Design, Vol. 11, No. 9, 2005, pp. 1079-1089.
[77] P. Lagisetty, P. Vilekar and V. Awasthi, “Synthesis of
Radiolabeled Cytarabine Conjugates,” Bioorganic & Me-
dicinal Chemistry Letters, Vol. 19, No. 16, 2009, pp. 4764-
4767. doi:10.1016/j.bmcl.2009.06.056
[78] F. Castelli, M. G. Sarpietro, M. Ceruti, F. Rocco and L.
Cattel, “Characterization of Lipophilic Gemcitabine Prod-
rug-Liposomal Membrane Interaction by Differential
Scanning Calorimetry,” Molecular Pharmaceutics, Vol. 3,
No. 6, 2006, pp. 737-744. doi:10.1021/mp060059y
[79] L. H. Reddy, C. Dubernet, S. L. Mouelhi, P. E. Marque,
D. Desmaele and P. Couvreur, “A New Nanomedicine of
Gemcitabine Displays Enhanced Anticancer Activity in
Sensitive and Resistant Leukemia Types,” Journal of
Controlled Release, Vol. 124, No. 1-2, 2007, pp. 20-27.
[80] S. H. Frost, H. Jensen and S. Lindegren, “In-Vitro Evalua-
tion of Avidin Antibody Pretargeting Using [211At]-La-
beled and Biotinylated Poly-L-Lysine as Effector Mole-
cule,” Cancer, Vol. 116, No. S4, 2010, pp. 1101-1110.
[81] H. Karacay, R. M. Sharkey, S. Govindan, V, W. J.
McBride, D. M. Goldenberg, H. J. Hansen and G. L.
Griffiths, “Development of a Streptavidin-Anti-Carci-
noembryonic Antigen Antibody, Radiolabeled Biotin
Pretargeting Method for Radioimmunotherapy of Colo-
rectal Cancer. Reagent Development,” Bioconjugate Che-
mistry, Vol. 8, No. 4, 1997, pp. 585-594.
[82] A. Lau, G. Bérubéand and C. H. ford, “Conjugation of
Doxorubicin to Monoclonal Anti- Carcinoembryonic An-
tigen Antibody via Novel Thiol-Directed Cross-Linking
Reagents,” Bioorganic & Medicinal Chemistry, Vol. 3,
No. 10, 1995, pp. 1299-1304.
[83] A. K. Fry, K. F. Schilke, J. McGuire and K. E. Bird,
“Synthesis and Anticoagulant Activity of Heparin Immo-
bilized ‘End-On’ to Polystyrene Microspheres Coated
with End-Group Activated Polyethylene Oxide,” Journal
of Biomedical Materials Research Part B: Applied Bio-
materials, Vol. 94, No. 1, 2010, pp. 187-195.
[84] M. H. Vingerhoeds, H. J. Haisma, S. O. Belliot, R. H.
Smit, D. J. Crommelin and G. Storm, “Immunoliposomes
as Enzyme-Carriers (Immuno-Enzymosomes) for Anti-
body-Directed Enzyme Prodrug Therapy (ADEPT): Op-
timization of Prodrug Activating Capacity,” Pharmaceu-
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
tical Research, Vol. 13, No. 4, 1996, pp. 604-610.
[85] K. Fujiwara, M. Yasuno and T. Kitagawa, “Novel Prepa-
ration Method of Immunogen for Hydrophobic Hapten,
Enzyme Immunoassay for Daunomycin and Adriamycin,”
Journal of Immunological Methods, Vol. 45, No. 2, 1981,
pp. 195-203. doi:10.1016/0022-1759(81)90213-1
[86] Y. Wang, X. Liu and D. J. Hnatowich, “An improved
Synthesis of NHS-MAG3 for Conjugation and Radiola-
beling of Biomolecules with [99mTc] at Room Tempera-
ture,” Nature Protocols, Vol. 2, No. 4, 2007, pp. 972-978.
[87] P. Joshi, K. F. Schilke, A. Fry, J. McGuire and K. Bird,
“Synthesis and Evaluation of Heparin Immobilized ‘Side-
On’ to Polystyrene Microspheres Coated with End-Group
Activated Polyethylene Oxide,” International Journal of
Biological Macromolecules, Vol. 47, No. 2 , 2010, pp. 98-
103. doi:10.1016/j.ijbiomac.2010.05.015
[88] J. Vega, S. Ke, Z. Fan, S. Wallace, C. Charsangavej and
C. Li, “Targeting Doxorubicin to Epidermal Growth Fac-
tor Receptors by Site-Specific Conjugation of C225 to
Poly(L-Glutamic Acid) through a Polyethylene Glycol
Spacer,” Pharmaceutical Research, Vol. 20, No. 5, 2003
pp. 826-832. doi:10.1023/A:1023454107190
[89] R. Jin, L. S. Moreira Teixeira, A. Krouwels, P. J. Dijkstra,
C. A. van Blitterswijk, M. Karperien and J. Feijen, “Syn-
thesis and Characterization of Hyaluronic Acid-Poly
(Ethylene Glycol) Hydrogels via Michael addition: An In-
jectable Biomaterial for Cartilage Repair,” Acta Biomater,
Vol. 6, No. 6, 2010, pp. 1968-1977.
[90] J. Morales-Sanfrutos, A. Megia-Fernandez, F. Hernan-
dez-Mateo, M. D. Giron-Gonzalez, M. D. R. S. Gonzalez
and F. Santoyo-Gonzalez, “Alkyl Sulfonyl Derivatized
PAMAM-G2 Dendrimers as Nonviral Gene Delivery
Vectors with Improved Transfection Efficiencies,” Or-
ganic & Biomolecular Chemistry, Vol. 9, No. 3, 2011, pp.
851-864. doi:10.1039/c0ob00355g
[91] M. Haas, F. Moolenaar, A. Elsinga, E. A. Van der
Wouden, P. E. De Jong, D. K. F. Meijer and D. D. Zeeuw,
“Targeting of Doxorubicin to the Urinary Bladder of the
Rat Shows Increased Cytotoxicity in the Bladder Urine
Combined with an Absence of Renal Toxicity,” Journal
of Drug Targeting, Vol. 10, No. 1, 2002, pp. 81-89.
[92] American Cancer Society, “Cancer Facts and Figures
2004,” American Cancer Society, 2004, pp. 1-60.
[93] H. M. Yang and R. A. Reisfeld, “Pharmacokinetics and
Mechanism of Action of a Doxorubicin-Monoclonal An-
tibody 9. 2. 27 Conjugate Directed to a Human Melanoma
Proteoglycan,” Journal of the National Cancer Institute,
Vol. 80, No. 14, 1988, pp. 1154-1159.
[94] S. Lutsenko, V, N. B. Feldman and S. E. Severin, “Cyto-
toxic and Antitumor Activities of Doxorubicin Conju-
gates with the Epidermal Growth Factor and Its Recep-
tor-Binding Fragment,” Journal of Drug Targeting, Vol.
10, No. 7, 2002, pp. 567-571.
[95] C. W. Michalski, M. Erkan, D. Sauliunaite, T. Giese, R.
Stratmann, C. Sartori, N. A. Giese, H. Friess and J. Kleeff,
Ex-Vivo Chemosensitivity Testing and Gene Expression
Profiling Predict Response Towards Adjuvant Gemcit-
abine Treatment in Pancreatic Cancer,” British Journal of
Cancer, Vol. 99, No. 5, 2008, pp. 760-767.
[96] T. Hoang, K. Kim, A. Jaslowski, P. Koch, P. Beatty, J.
McGovern, M. Quisumbing, G. Shapiro, R. Witte and J.
H. Schiller, “Phase II Study of Second-Line Gemcitabine
in Sensitive or Refractory Small Cell Lung Cancer,” Lung
Cancer, Vol. 42, No. 1, 2003, pp. 97-102.
[97] J. Bierau, A. H. van Gennip, R. Leen, R. Meinsma, H. N.
Caron and A. B. van Kuilenburg, “Cyclopentenyl Cyto-
sine-Induced Activation of Deoxycytidine Kinase In-
creases Gemcitabine Anabolism and Cytotoxicity in
Neuroblastoma,” Cancer Chemotherapy and Pharma-
cology, Vol. 57, No. 1, 2006, pp. 105-113.
[98] V. Santini, G. D’Ippolito, P. A. Bernabei, A. Zoccolante,
A. Ermini and P. Rossi-Ferrini, “Effects of Fludarabine
and Gemcitabine on Human Acute Myeloid Leukemia
Cell Line HL 60: Direct Comparison of Cytotoxicity and
Cellular Ara-C Uptake Enhancement,” Leukemia Re-
search, Vol. 20, No. 1, 1996, pp. 37-45.
[99] H. Mueller, M. U. Kassack and M. Wiese, “Comparison
of the Usefulness of the MTT, ATP and Calcein Assays
to Predict the Potency of Cytotoxic Agents in Various
Human Cancer Cell Lines,” Biomolecular Screening, Vol.
9, No. 6, 2004, pp. 506-515.
[100] E. Ulukaya, F. Ozdikicioglu, A. Y. Oral and M. Dermirci,
“The MTT Assay Yields a Relatively Lower Result of
Growth Inhibition than the ATP Assay Depending on the
Chemotherapeutic Drug Tested,” Toxicol in Vitro, Vol.
22, No. 1, 2008, pp. 232-239.
[101] M. Varache-Lembège, S. Larrouture, D. Montaudon, J.
Robert and A. Nuhrich, “Synthesis and Antiproliferative
Activity of Aryl- and Heteroaryl-Hydrazones Derived
from Xanthone Carbaldehydes,” European Journal of
Medicinal Chemistry, Vol. 43, No. 6, 2008, pp. 1336-
1343. doi:10.1016/j.ejmech.2007.09.003
[102] M. D. Kars, O. D. Iseri, U. Gunduz and J. Molnar, “Re-
versal of Multidrug Resistance by Synthetic and Natural
Compounds in Drug-Resistant MCF-7 Cell Lines,” Che-
motherapy, Vol. 54, No. 3, 2008, pp. 194-200.
[103] H. Huang, E. Pierstorff, E. Osawa and D. Ho, “Active
Nanodiamond Hydrogels for Chemotherapeutic Deliv-
ery,” Nano Letters, Vol. 7, No. 11, 2007, pp. 3305-3314.
[104] M. C. Dery, C. Van Themsche, D. Provencher, A. M.
Mes-Masson and E. Asselin, “Characterization of EN-
1078D, a poorly differentiated human endometrial carci-
Copyright © 2012 SciRes. JCT
Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:
Cytotoxic Anti-Neoplastic Activity against Chemotherapeutic-Resistant Mammary Adenocarcinoma SKBr-3
Copyright © 2012 SciRes. JCT
noma cell line: A Novel Tool to Study Endometrial Inva-
sion In-Vitro,” Reproductive Biology and Endocrinology,
Vol. 5, 2007, pp. 38-39. doi:10.1186/1477-7827-5-38
[105] B. Spee, M. D. Jonkers, B. Arends, G. R. Rutteman, J.
Rothuizen and L. C. Penning, “Specific Down-Regulation
of XIAP with RNA Interference Enhances the Sensitivity
of Canine Tumor Cell-Lines to Trail and Doxorubicin,”
Molecular Cancer, Vol. 5, 2006, p. 34.
[106] N. Denora, V. Laquintana, A. Trapani, A. Lopedota, A.
Latrofa, J. M. Gallo and G. Trapani, “Translocator Pro-
tein (TSPO) Ligand-Ara-C (cytarabine) Conjugates as a
Strategy to Deliver Antineoplastic Drugs and to Enhance
Drug Clinical Potential,” Molecular Pharmaceutics, Vol.
7, No. 6, 2010, pp. 2255-2269.
[107] W. C. Shen and H. J. Ryser, “Cis-Aconityl Spacer be-
tween Daunomycin and Macromolecular Carriers: A
Model of pH-Sensitive Linkage Releasing Drug from a
Lysosomotrophic Conjugates,” Biochemical and Bio-
physical Research Communications, Vol. 102, No. 3,
1981, pp. 1048-1054.
[108] Y. Zhang, N. Wang, N. Li, T. Liu and Z. Dong, “The
Antitumor Effect of Adriamycin Conjugated with Mono-
clonal Antibody against Gastric Cancer In-Vitro and
In-Vivo,” Acta Pharmaceutica Sinica, Vol. 27, No. 5,
1992, pp. 325-330.
[109] E. Aboud-Pirak, E. Hurwitz, F. Bellot, J. Schlessinger and
M. Sela, “Inhibition of Human Tumor Growth in Nude
Mice by a Conjugate of Doxorubicin with Monoclonal
Antibodies to Epidermal Growth Factor Receptor,” Pro-
ceedings of the National Academy of Sciences, Vol. 86,
No. 10, 1989, pp. 3778-3781.
[110] C. P. Coyne, B. W. Fenwick and J. Ainsworth, “Anti-
Neoplastic Activity of Chemotherapeutic ‘Loaded’ Neu-
trophils against Human Mammary Carcinoma,” Biother-
apy, Vol. 10, No. 2, 1997, pp. 145-159.
[111] M. D. Pegram, A. Lopez, G. Konecny and D. J. Slamon,
“Trastuzumab and Chemotherapeutics: Drug Interactions
and Synergies,” Seminars in Oncology, Vol. 27, Suppl. 11,
2000, pp. 21-25.
[112] D. Slamon and M. Pegram, “Rationale for Trastuzumab
(Herceptin) in Adjuvant Breast Cancer Trials,” Seminars
in Oncology, Vol. 28, Suppl. 3, 2001, pp. 13-19.
[113] E. P. Winer and H. J. Burstein, “New Combinations with
Herceptin in Metastatic Breast Cancer,” Oncology, Vol.
61, Suppl. 2, 2001, pp. 50-57. doi:10.1159/000055402
[114] S. Kim, C. N. Prichard, M. N. Younes, Y. D. Yazici, S. A.
Jasser, B. N. Bekele and J. N. Myers, “Cetuximab and Ir-
inotecan Interact Synergistically to Inhibit the Growth of
Orthotopic Anaplastic Thyroid Carcinoma Xenografts in
Nude Mice,” Clinical Cancer Research, Vol. 12, No. 2,
2006, pp. 600-607. doi:10.1158/1078-0432.CCR-05-1325
[115] M. Landriscina, F. Maddalena, A. Fabiano, A. Piscazzi, O.
La Macchia and M. Cignarelli, “Erlotinib Enhances the
Proapoptotic Activity of Cytotoxic Agents and Syner-
gizes with Paclitaxel in Poorly-Differentiated Thyroid
Carcinoma Cells,” Anticancer Research, Vol. 30, No. 2,
2010, pp. 473-480.
[116] F. Ciardiello, R. Bianco, V. Damiano, S. De Lorenzo, S.
Pepe, S. De Placido, Z. Fan, J. Mendelsohn, A. Bianco
and G. Tortora, “Antitumor Activity of Sequential Treat-
ment with Topotecan and Anti-Epidermal Growth Factor
Receptor Monoclonal Antibody C225,” Clinical Cancer
Research, Vol. 5, No. 4, 1999, pp. 909-916.
[117] K. D. Lynn, D. G. Udugamasooriya, C. L. Roland, D. H.
Castrillon, T. J. Kodadek and R. A. Brekken, “GU81, a
VEGFR2 Antagonist Peptoid, Enhances the Anti-Tumor
Activity of Doxorubicin in the Murine MMTV-PyMT
Transgenic Model of Breast Cancer,” BMC Cancer, Vol.
10, 2010, p. 397. doi:10.1186/1471-2407-10-397
[118] L. Zhang, D. Yu, D. J. Hicklin, J. A. Hannay, L. M. Ellis
and R. E. Pollock, “Combined Anti-Fetal Liver Kinase 1
Monoclonal Antibody (Anti-VEGFR) and Continuous
Low-Dose Doxorubicin Inhibits Angiogenesis and Growth
of Human Soft Tissue Sarcoma Xenografts by Induction
of Endothelial Cell Apoptosis,” Cancer Research, Vol. 62,
No. 7, 2002, pp. 2034-2042.
[119] L. B. Shih, D. M. Goldenberg, H. Xuan, H. W. Lu, M. J.
Mattes and T. C. Hall, “Internalization of an Intact Doxo-
rubicin Immunoconjugate,” Cancer Immunology, Immu-
notherapy, Vol. 38, No. 2, 1994, pp. 92-98.
[120] H. J. Hansen, G. L. Ong and H. Diril, “Internalization and
Catabolism of Radiolabeled Antibodies to the MHC Class-
II Invariant Chain by B-Cell Lymphomas,” Biochemical
Journal, Vol. 320, 1996, pp. 293-300.
[121] A. C. Stan, D. L. Radu, S. Casares, C. A. Bona and T. D.
Brumeanu, “Antineoplastic Efficacy of Doxorubicin En-
zymatically Assembled on Galactose Residues of a Mono-
clonal Antibody Specific for the Carcinoembryonic Anti-
gen,” Cancer Research, Vol. 59, No. 1, 1999, pp. 115-
[122] M. Pimm, V, M. A. Paul, T. Ogumuyiwa and R. W.
Baldwin, “Biodistribution and Tumour Localization of a
Daunomycin-Monoclonal Antibody Conjugate in Nude
Mice and Human Tumour Xenografts,” Cancer Immu-
nology, Immunotherapy, Vol. 27, No. 3, 1988, pp. 267-