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

doi:10.4236/jct.2012.325089

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Journal of Cancer Therapy, 2012, 3, 689-711

http://dx.doi.org/10.4236/jct.2012.325089 Published Online October 2012 (http://www.SciRP.org/journal/jct)

689

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

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.

Email: coyne@cvm.msstate.edu

Received August 10th, 2012; revised September 12th, 2012; accepted September 24th, 2012

ABSTRACT

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

Autoradiography

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-

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

690

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-

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

691

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

692

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

Synthesis

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

reagent.

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

intermediate.

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].

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

693

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

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

694

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-

chemotherapeutics.

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

reader.

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-

Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:

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695

(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:

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696

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 10−6 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 10−10 M, 10−9 M, 10−8 M,

10−7 M and 10−6 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 10−10 M and 10−9 M but not at 10−7 M or

10−6 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 10−10 M, 10−9 M and 10−8 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 10−7 M and 10−6 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 10−6 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-

trols.

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697

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.

Synthesis of Gemcitabine-(C4-amide)-[anti-HER2/neu] Utilizing a UV-Photoactivated Gemcitabine Intermediate:

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698

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-

trols.

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

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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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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

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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 10−10 M to 10−6 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-

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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/

neu].

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

gemcitabine-(C5-methylcarbamate)-[anti-HER2/neu]

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

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703

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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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”

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(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.

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