Subscriber access provided by ALBANY STATE UNIV

Molecular Pharmaceutics is published by the American Chemical Society. 1155Sixteenth Street N.W., Washington, DC 20036Published by American Chemical Society. Copyright © American Chemical Society.However, no copyright claim is made to original U.S. Government works, or worksproduced by employees of any Commonwealth realm Crown government in the courseof their duties.

Article

Cathepsin B Degradable Star Shaped PeptidicMacromolecules for Delivery of 2-methoxyestradiol

Ravi Shankar, Abhilash Samykutty, Corinne Riggin, Sneha Kannan, Ursula Wenzel, and Rohit KolhatkarMol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp400261h • Publication Date (Web): 23 Aug 2013

Downloaded from http://pubs.acs.org on August 27, 2013

Just Accepted

“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are postedonline prior to technical editing, formatting for publication and author proofing. The American ChemicalSociety provides “Just Accepted” as a free service to the research community to expedite thedissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscriptsappear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have beenfully peer reviewed, but should not be considered the official version of record. They are accessible to allreaders and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offeredto authors. Therefore, the “Just Accepted” Web site may not include all articles that will be publishedin the journal. After a manuscript is technically edited and formatted, it will be removed from the “JustAccepted” Web site and published as an ASAP article. Note that technical editing may introduce minorchanges to the manuscript text and/or graphics which could affect content, and all legal disclaimersand ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errorsor consequences arising from the use of information contained in these “Just Accepted” manuscripts.

1

Cathepsin B Degradable Star Shaped Peptidic Macromolecules for Delivery of 2-

methoxyestradiol

Ravi Shankar1, 2*, Abhilash Samykutty1*, Corinne Riggin2, Sneha Kannan2, Ursula Wenzel1 and Rohit Kolhatkar1,2†

1 Department of Biopharmaceutical Sciences, University of Illinois Chicago, Rockford, IL 61107, USA

2 Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Baltimore, MD, 21201, USA

* Both authors contributed equally.

† Corresponding author:

Rohit Kolhatkar

Department of Biopharmaceutical Sciences

University of Illinois Chicago

1601 Parkview Ave, Rm N302

Rockford, IL 61107

Tel: (815) 395 5922

Email: rohitk@uic.edu

Page 1 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

2

Abstract

2-methoxyestradiol (2ME), a natural metabolite of estradiol has antiproliferative and

antiangiogenic activity. However, its clinical success is limited due to poor water solubility and

poor pharmacokinetic parameters suggesting the need for a delivery vehicle. In this study we

evaluated cathepsin B degradable star shaped peptidic macromolecules (SPMs) that can

potentially be used to create higher generation and high molecular weight peptidic polymer as

delivery vehicle of 2ME. Two peptidic macromolecules having positively charged amine

(ASPM) or negatively charged carboxyl surface groups (CSPM) were synthesized and evaluated

for their degradation in the presence of cathepsin B and stability in the presence of neutral or

acidic buffer and serum. Both ASPM and CSPM degraded rapidly in the presence of cathepsin

B. Both were stable in neutral and acidic buffer whereas only CSPM exhibited substantial

stability in the presence of serum. Both macromolecules were nontoxic towards breast cancer

cells whereas 2ME-containing macromolecules exhibited antiproliferative activity in the

micromolar range. Overall, results from current study indicate that tetrapeptide GFLG can be

used to create star-shaped macromolecules that are degraded in the presence of cathepsin B and

have the potential to be developed as delivery vehicle of 2ME.

KEY WORDS: 2-Methoxyestradiol, Cathepsin B, Peptidic dendrimer, Degradable polymer.

Page 2 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

3

INTRODUCTION

2-methoxyestradiol (2ME, Figure 1, I), has been identified as a potential antitumor agent

due to its ability to inhibit angiogenesis and proliferation of the cancer cells in various human

cell lines. This includes cell lines that are resistant to chemotherapeutic agents [1-4]. It inhibits

the activation of HIF-1α [5], which plays a key role in developing resistance of tumor cells to

chemotherapeutic agents. Several in-vivo studies demonstrate the effectiveness of 2ME in

inhibiting tumor growth [1, 3-5]. The results from the Phase I clinical trials demonstrate the

safety of 2ME [6, 7]. However, low plasma levels detected, despite higher drug administration, is

limiting its clinical success. Lower solubility and extensive metabolism are responsible for its

lower plasma levels [1, 6]. The therapeutic benefit of 2ME can be improved by using a delivery

vehicle that can increase its solubility, decrease metabolism and release the drug at the site of

action.

Delivery vehicles in the nanometer size range such as polymers [8-10], liposomes [11,

12], micelles, and inorganic nanomaterials [13] can overcome several limitations associated with

small molecular weight anticancer therapeutics like 2ME. One of the prerequisites for the

effectiveness of such delivery vehicle is to release the free drug at tumor sites. The site specific

release of the free drug can be achieved by using stimuli-sensitive linker that will respond to the

differences between, normal and tumor tissue, or intracellular and extracellular environment

[14]. Two extensively studied stimuli-sensitive linkers are hydrolysable [15] or reducible linkers

[16]. However, these approaches provide limited control over the site-specific degradation after

in-vivo administration. In contrast, the release of the drug triggered by the presence of specific

enzyme entails higher chemical stability and concurrent specificity associated with the enzyme

Page 3 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

4

selected [17]. Therefore, we designed peptidic macromolecules that can be completely degraded

in the presence of an enzyme cathepsin B.

Cathepsin B is one of the most prominent proteases present in normal cells and tissues.

The expression of cathepsin B is substantially increased in human tumors such as prostate,

breast, colon, esophageal, gastric, lung, ovarian, and thyroid carcinomas as well as in gliomas

and melanomas [18-20]. This has been observed at mRNA and protein level. The higher

expression level can be the result of oncogene contribution, gene amplification, alternative

splicing, or post-translational modification [20, 21]. The differences in cathepsin B expression

levels in normal and cancerous tissues can be exploited to achieve higher drug release at tumor

sites using two types of approaches. An extensively studied approach is the use of cathepsin B

sensitive linker for drug release. This has been reported to reduce toxicities associated with small

molecular weight therapeutic molecules like doxorubicin [10, 22-24]. One disadvantage for the

linker approach is the high reliance on kidneys for the clearance of non-degradable polymeric

backbone. Increasing evidence from preclinical studies suggest the induction of intracellular

vacuolation in animal models raising the concerns about biosafety of nondegradable polymers

after chronic administration at high doses [25, 26]. Completely degradable polymers can be

cleared easily and can potentially release their entire therapeutic payload at tumor sites compared

to lower drug release associated with linker strategy. It has been reported that less than 5% of the

injected polymeric conjugate reaches tumor [27]. Furthermore, most polymeric conjugates have

only 10% wt/wt of the therapeutic component. Therefore, it is important for the small

percentage of the drug-containing polymeric conjugates to release its entire drug load after

reaching its destination. We envision a completely degradable delivery system can be developed

to achieve higher drug release at tumor sites. In this manuscript we report the first generation of a

Page 4 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

5

completely degradable system. We report the synthesis of star shaped peptidic macromolecules

(SPMs) that can be completely degraded within a short time period in the presence of cathepsin

B. Using 2ME we then demonstrate that 2ME-conjugated star shaped macromolecule (MESPM)

is as effective as a 2ME-containing peptidic monomer in inhibiting the growth of three triple

negative breast cancer cell lines.

Page 5 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

6

MATERIALS AND METHODS:

Reagents and chemicals:

Estradiol was purchased from Ochem Inc. (Des Plaines, IL), Amino acids were purchased from

Novabiochem (Darmstadt, Germany). Coupling Reagents dicyclohexylcarbodiimide (DCC), 1-

hydroxybenzotriazole (HOBT), and O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium

hexafluorophosphate (HBTU) were obtained from Oakwood Chemicals (West Columbia, SC).

PAMAM dendrimer (Generation 0), N,N-Diisopropylethylamine (DIPEA), trifluoroacetic acid

(TFA), di-tert-butyl dicarbonate ((Boc)2O), dichloromethane (DCM), dimethylsulfoxide

(DMSO), deutrited DMSO-d6, chloroform (CDCl3), D2O and other chemicals were ACS grade

and purchased from Sigma Chemical Co. (St. Louis, MO). Cathepsin B (CPB) and model

substrate N-benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride were also purchased from Sigma

Chemical Co. (St. Louis, MO). 2ME [28], Gly-2ME [29], and BocGFLGOMe [30] were

synthesized as described before. All commercial reagents and anhydrous solvents were used

without further purification or distillation unless otherwise stated. High-performance liquid

chromatography (HPLC) grade solvents were purchased from Fisher Scientific (Pittsburgh, PA).

All small molecules were analyzed using TLC, NMR, MS and HPLC for the purity. Polymers

were purified using HPLC before analysis. Compounds showing >95 % purity form HPLC

analysis were used for biological characterization. Analytical thin layer chromatography was

performed on Whatman silica gel 60 Å with fluorescent indicator (Partisil K6F). Compounds

were visualized by UV light and/or stained with ninhydrin solution followed by heating. Flash

column chromatography was performed on Whatman silica gel 60 Å (230-400 mesh). NMR (1H,

13C) spectra were recorded on a Varian 300/400 MHz or a Bruker 400 MHz spectrometer and

calibrated using an internal reference. ESI mode mass spectra were recorded on a Shimatzu

Page 6 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

7

LCMS 2020. MALDI analysis were performed using Voyager-DE PRO mass spectrometer

(Applied Biosystems, Foster City, CA, USA) equipped with a 337 nm pulsed nitrogen laser.

Synthesis of positively charged amine-terminated star-shaped peptidic macromolecule

(ASPM):

Positively charged amine-terminated star-shaped peptidic macromolecule (ASPM, II, Figure 1)

was synthesized using a sequential approach where a reactive ester of each amino acid was

added to the PAMAM dendrimer (Generation 0, G0) in a stepwise fashion (Scheme 1). In each

step, one amino acid was conjugated to the four surface amine groups through two reactions. In

the first reaction, p-nitrophenylester (ONp) of boc-protected amino acid was conjugated to the

free terminal amines present in PAMAM dendrimer. In the second step, boc groups on amino

acids were deprotected under acidic conditions to get positively charged amine-terminated star-

shaped macromolecule. Typical procedure for the conjugation of amino acid to four arms is

described below using glycine as an example. Briefly, into the solution of PAMAM dendrimer

(G0) (0.1 g, 0.19 mmol) in DMSO was added Boc-Gly-ONp (0.34 g, 1.1 mmol) and the reaction

mixture was stirred overnight. Solvent was evaporated in vacuo and the crude mixture was

dissolved in acetone and precipitated in cold ether to obtain gummy compound that was

dissolved in the mixture of dichloromethane and TFA (50:50, 6 mL). The solution was stirred

for 6 hours. Solvent was evaporated and the crude mixture was dissolved in methanol and

precipitated in cold ether to obtain G0(Gly)4 (0.23 mg, yield 78%). Repetitions of conjugation

and deprotection reactions using p-nitrophenyl esters of phenylalanine (Step 2, Scheme 1),

leucine (Step 3, Scheme 1) and glycine (Step 4, Scheme 1) yielded ASPM with four positively

charged surface amine groups. The crude ASPM was purified using preparative HPLC using

Waters HPLC system equipped with 600E multisolvent delivery system, 717 plus autosampler

Page 7 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

8

and 996 PDA detector. Varian Dynamex column (250×10 mm) was used for preparative

purification using water (0.1 % TFA) as mobile phase A and mixture of 80 % acetonitrile (0.1 %

TFA) and 20 % water (0.1 % TFA) as mobile phase B at the flow rate of 5 ml/min with gradient

of 0 to 60 % B over the period of 60 min. Fractions containing product were pooled together,

solvent was evaporated under vacuo followed by lyophilization to obtain pure ASPM as white

solid. The final product obtained was > 97 % pure as analyzed by HPLC. Dynamex column (250

x 4.6 mm) was used for analytical HPLC run using water (0.1 % TFA) as mobile phase A and

mixture of 80 % acetonitrile (0.1 % TFA) and 20 % water (0.1 % TFA) as mobile phase B at the

flow rate of 1.5 ml/min with gradient of 0 to 100 % B over the period of 45 min followed by a

100% B for 10 min. The same instrument method was used for analysis of all the polymers using

HPLC and retention time reported in Table 1. MS Anal. MALDI Mol wt. Calc 2014.42;

observed 2038 (Figure S1A) 1H NMR (500MHz, D2O): 0.81-0.87 (m, 24H, 8XCH3, -Leu), 1.43-

1.6 (m, 12H, β-Leu, γ-Leu), 2.40-2.53 (m, 12H, -NCH2CH2N-, CH2CO), 2.84-3.10 (m, 16H, β-

Phe, CH2N(CH2)2), 3.13-3.35 (m, 16H, CH2NHCO), 3.61-3.83 (m, 16H, α-Gly), 4.12-4.32 (m,

4H, α-Leu), 4.58-4.66 (m, 4H, α-Phe ), 7.11-7.36 (m, 20H, ArH).

Synthesis of negatively charged carboxyl-terminated star-shaped peptidic macromolecule

(CSPM) and 2ME containing star-shaped peptidic macromolecule (MESPM):

Carboxyl-terminated star-shaped peptidic macromolecule (CSPM, III, Figure 1) and 2ME

containing star-shaped peptidic macromolecule (MESPM, IV, Figure 1) were synthesized using

convergent approach where a tetrapeptide GFLG or GFLGG2ME was concurrently conjugated

to the all four arms of the central core. The synthesis involved three steps i) synthesis of core ii)

synthesis of monomers and iii) conjugation of monomers to core.

Page 8 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

9

Synthesis of core:

Tetrakis-(p-nitrophenyl ester) (EDTA(ONp)4) was used as central core for CSPM and MESPM.

EDTA(ONp)4 was synthesized from ethylene diamine tetraacetic acid and p-nitrophenol using

DCC as a coupling agent (Scheme 2). Briefly, EDTA (1 g, 3.34 mmol) was dissolved in

anhydrous DMF and the solution was cooled down to -10 °C. Into the solution was then added

solution of p-nitrophenol (2.08 g, 14.9 mmol) and solution of DCC (3.08 g, 14.9 mmol) and the

reaction mixture was stirred for 3 h at -10°C. The reaction mixture was then stirred overnight at

4°C followed by stirring for 12 h at room temp. Diclyclohexylurea (DCU) was precipitated,

solvent evaporated and the product was crystalized using ethanol. After two crystallizations pale

yellow color product was obtained (1.7 g, yield 64 %) which was found to be pure according to

TLC. 1H NMR (500MHz, CDCl3): δ 3.1 (s, 4H, N-CH2CH2-N), 4.9 (s, 8H, COCH2N), 7.4 (d,

8H, ArH), 8.5 (d, 8H, ArH); MS Anal.; Mol wt calculated 776.62; observed 777.62 (M+1).

Synthesis of monomers:

Synthesis of Boc-GFLGOH (3): BocGFLGOMe was synthesized according to previously

reported method [30]. Deprotection of methyl ester under basic conditions yielded BocGFLGOH

(3, Scheme 3). Briefly, Boc-GFLGOMe (1 g, 1.9 mmol) was dissolved in THF (10 mL) and

mixed with a NaOH solution in water (0.15 g in 10 mL). After 1 h of stirring, the reaction

mixture was concentrated in vacuo. The crude product was dissolved in ethyl acetate (50 mL)

and the solution was acidified with dilute acetic acid (10%). The ethyl acetate layer was washed

with water (2 x 10 mL) and brine (10 mL), dried over Na2SO4 and concentrated in vacuo to

obtain solid compound that was crystallized from ethyl acetate / diethyl ether to give 0.65 g

(67% yield) as a white crystalline solid. 1H NMR (DMSO; d6): 0.83 (d, 3H, CH3-Leu), 0.88 (d,

Page 9 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

10

3H, CH3-Leu), 1.3 (s, 9H, Boc), 1.45-1.48 (m, 2H, β-Leu), 1.56-1.63 (m, 1H, γ-Leu), 2.78 (dd,

1H, β-Phe), 3.01(dd, 1H, β-Phe), 3.35-3.45 (m, 2H, α-Gly), 3.71(t, 2H, α-Gly), 4.32-4.34 (m,

1H, α-Leu), 4.53-4.54 (m, 1H, α-Phe), 6.92 (t, 1H, NH-Boc) 7.20-7.22 (m, 5H, ArH Phe), 7.83

(d, 1H, -CONH-), 8.04-8.07(t, 1H, -CONH-), 8.14(d, 1H, -CONH-), 12.5(s, 1H, COOH) ); MS

Anal.; Mol wt calculated 492.57; observed 493.26 (M+1).

Synthesis of GFLGOH (4): Deprotection of Boc group in BocGFLGOH (3) under acidic

conditions yielded GFLGOH (4, Scheme 3). Briefly, Boc-GFLGOH (0.1 g, 0.2 mmol) was

dissolved in 50:50 mixtures of TFA and DCM (1 mL). After 1h solvent was evaporated under

vacuum. The residue was dissolved in minimum volume of methanol and precipitated in diethyl

ether to obtain white solid. (TFA salt of GFLGOH; 0.1g, 98% yield). 1H NMR (500MHz,

DMSO-d6): δ 0.85 (d, 3H, CH3-Leu), 0.9 (d, 3H, CH3-Leu), 1.46-1.50 (m, 2H, β-Leu), 1.61-

1.63 (m, 1H, γ-Leu), 2.70-2.76 (m, 1H, β-Phe), 3.03-3.07 (dd, 1H, β-Phe), 3.48-3.54 (m, merged

with water impurities in DMSO, 2H, α-Gly), 3.76 (t, 2H, α-Gly ), 4.35 (dd, 1H, α-Leu), 4.64-

4.67 (m, 1H, α-Phe), 7.18-7.25 (m, 5H, ArH Phe), 7.95 (bs, 2H, NH2), 8.15 (t,1H, NH-), 8.33 (d,

1H, NH), 8.59 (d, 1H, NH) ); MS Anal.; Mol wt calculated 392.45; observed 393.20 (M+1).

Synthesis of GFLGG2ME (5): 2ME containing monomer was synthesized according to scheme

3. Briefly, Boc-GFLG-OH (3) (0.1 g, 0.20 mmol) and HBTU (0.11 g, 0.30 mmol) were

dissolved in 10 mL dry DMF. The solution was cooled to 0 0C and into it was added 2 mL DMF

containing Gly-2ME (72.96 mg, 0.20 mmol). The temperature was maintained at 0 0C for 30 min

after which reaction mixture was allowed to warm to room temperature followed by stirring for

overnight. The reaction mixture was then quenched by 30 mL distilled water, solid precipitated

was filtered and purified by flash column chromatography over silica gel using ethyl

Page 10 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

11

acetate/hexane (50:50) as eluent to obtain BocGFLGG2ME as a white solid (60% yield, 100

mg). The corresponding Boc-GFLGG2ME (100 mg, 0.12 mmol) was dissolved in 50:50

mixtures of TFA and DCM (1 mL). After 1h solvent was evaporated under vacuum. The residue

was dissolved in minimum volume of methanol and precipitated in diethyl ether to obtain white

solid which was purified by flash column chromatography using methanol/ethyl acetate (2:98) as

eluent to obtain GFLGG2ME (5) (TFA salt, 98% yield, 100 mg). 1H NMR (500MHz, DMSO-

d6): δ = 0.77 (s, 3H, 2ME H-18, CH3), 0.85 (d, 3H, CH3-Leu), 0.90 (d, 3H, CH3-Leu), 1.23-1.32

(m, 6H, 2ME H-14(1H), H-7(2H), H-15(2H), H-8(1H)), 1.47-1.52 (m, 3H, β-Leu, 2ME H-12

(1H), ) 1.57-1.65(m, 2H, γ-Leu(1H) & 2ME H-12(1H), 1.74-1.77(m, 2H, 2ME H-16), 2.05-2.11

(m, 2H, 2ME H-11), 2.21-2.24 (m, 1H, 2ME H-9), 2.62-2.66 (m, 1H, 2ME H-6), 2.73-2.75(m,

1H, β-Phe), 3.02 (dd, 1H, β-Phe), 3.37-3.43(m, 1H, 2ME H-17), 3.57-3.58(m, 1H, 2ME H-6),

3.74 (s, 3H, OCH3), 3.75 (d, 2H, α-Gly), 3.86 (d, 2H, α-Gly), 4.0 (br, 2H Gly), 4.30-4.35 (m,

1H, α-Leu), 4.59-4.67 (m, 1H, α-Phe), 6.44 (s, 1H, 2ME H-1), 6.7(s, 1H, 2ME H-4), 7.17-7.20

(m, 5H, ArH Phe), 8.07 (t, 1H, -CONH-), 8.24 (t, 1H, -CONH-), 8.37(d, 1H, CONH), 8.59(d,

1H, CONH) ); MS Anal.; Mol wt calculated 834.38; observed 835.40 (M+1).

Synthesis of SPMs:

Synthesis of CSPM (III): Conjugation of GFLGOH (4) with EDTA(ONp)4 (2) yielded CSPM

(III), EDTA(GFLGOH)4) (Scheme 3). Briefly, EDTA(ONp)4 (0.1 g, 0.12 mmol) was dissolved

in anhydrous DMSO (1mL). Into the solution was then added solution of GFLGOH (4) (0.4 g,

1.02 mmol) in DMSO (0.5 mL) and triethylamine (150 µL, 1.02 mmol). After 8h, reaction

mixture was concentrated in vacuo to obtain crude product which was purified by preparative

HPLC as described before for purification of ASPM. Fractions containing product were pooled

Page 11 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

12

together, solvent was evaporated under vacuo followed by lyophilization to obtain pure

EDTA(GFLGOH)4 (CSPM) as white solid. 1H NMR (500MHz, DMSO-d6): 0.81-0.87 (m, 24H,

CH3, Leu), 1.23-1.25 (m, 4H, γ-Leu), 1.46-1.47 (m, 8H, β-Leu), 1.57-1.60 (m, 4H, β-Leu), 2.73-

2.76 (m, 4H, -NCH2CH2N-), 3.00-3.07 (m, 8H, β-Phe), 3.67-3.72 (m, 12H, α-Gly & -

COCH2NCH2CH2), 3.74-3.80 (m, 12H, α-Gly & -COCH2NCH2CH2 ), 4.33-4.35 (m, 4H, α-Leu),

4.59-4.60 (m, 4H, α-Phe ), 7.15-7.20 (m, 20H, ArH), 8.11-8.19(m, 12H, CONH-), 8.32-8.34 (m,

4H, CONH). MS Anal. Mol wt 1787.9 [M-2], MALDI Mol wt Calc 1788.87 observed 1791.87

(Figure S1B).

Synthesis of MESPM (IV): MESPM (EDTA(GFLGG2ME)4) (IV) was synthesized using

GFLGG2ME (5) as a monomeric unit and EDTA(ONP)4 (2) as central core (Scheme 3). Briefly,

EDTA(ONp)4 (2) (0.1 g, 0.12 mmol) was dissolved in anhydrous DMSO (1 mL). Then solutions

of GFLGG2ME.TFA (5) (0.85 g, 1.02 mmol) in DMSO (0.5 mL) and triethyl amine (0.15 mL,

1.02 mmol) were added dropwise. The reaction was allowed to proceed for 8h at room

temperature. Then reaction mixture was concentrated by vacuum pump with 550C temperature to

obtain yellowish mixture which was washed with ether (2 X 5 mL) and purified by preparative

HPLC using the same column and conditions as described above for purification of ASPM to

obtain EDTA(GFLGG2ME)4 as a white solid (57% yield, 23 mg). 1H NMR (500MHz, DMSO-

d6): 0.76 (s, 12H, 2ME H-18), 0.81 (d, 12H, CH3-Leu), 0.87 (d, 12H, CH3-Leu), 1.28-1.32 (m,

24H, 2ME H-14(4H), H-7(8H), H-15(8H), H-8(4H)), 1.46-1.50 (m, 8H, β-Leu) 1.53-1.66 (m,

8H, 2ME H-12(4H), γ-Leu (4H)), 1.73-1.77 (m, 8H, 2ME H-16), 2.05-2.11 (m, 8H, 2ME H-11),

2.21-2.24 (m, 4H, 2ME H-9), 2.57-2.66 (m, 4H, 2ME H-6), 2.74-2.78 (m, 4H, β-Phe), 2.99-3.04

(m, 4H, β-Phe), 3.16-3.17 (m, 4H, -NCH2CH2N-), 3.70 (s, 12H, OCH3), 3.72-3.75 (m, 8H, α-

Gly), 3.62-3.84 (d, 8H, α-Gly), 4.21-4.29 (m, 8H, α-Leu (4H), -COCH2N (4H)), 4.50-4.56 (m,

Page 12 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

13

4H, α-Phe), 4.59-4.63 (t, 4H, COCH2N-), 6.4 (s, 4H, 2ME H-1), 6.7 (s, 4H, 2ME H-4), 7.14-7.24

(m, 20H, ArH), 8.16 - 8.21 (m, 20H, CONH), 8.5 (s, 4H, OH). MS Anal. MALDI Mw Calc

3155.67 Mw observed 3178.39 (M+23) (Figure 2).

Stability studies:

0.24 mg of the polymer was dissolved in 150 µL of the PBS or PBS (10 % FBS) or acetate buffer

and the solutions were incubated for the respective incubation period. At the end of the

incubation period solutions were placed in boiling water (10 min) followed by addition of cold

acetone to precipitate proteins. Supernatant was collected, dried, reconstituted in 100 µL of

mobile phase and centrifuged. 70 µL of the supernatant was store at -70 °C until further analysis.

At the time of analysis 10 µL of Gly-Phe (4 mg/mL) was added to the supernatant solution to

serve as internal standard and the sample was analyzed using reverse phase HPLC (Waters

Corporation, Milford, MA). Percentage of intact polymer remaining was calculated based on the

calibration curve generated for the intact polymer.

Cathepsin B degradation studies:

The degradation of peptidic dendrimer in the presence of lysosomal enzyme cathepsin B (CPB)

was evaluated according to previously described procedures [31-33] with minor modifications.

Enzyme incubation mixture consisted of 100 µL of CPB stock solution (0.98 mg/mL or 0.49

mg/mL) in 0.1 M ammonium acetate buffer pH=5, 1 mM EDTA) and 50 µL of reduced

glutathione solution (250 mM in acetate buffer pH=5, 1 mM EDTA). The mixture was incubated

at 37 °C for 5 min before addition of polymer solution. 0.24 mg of polymer to be evaluated was

dissolved in ammonium acetate buffer (50 µL) and was added to the preincubated mixture. After

the incubation period the mixture was placed in boiling water (10 min) followed by addition of

Page 13 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

14

cold acetone to precipitate proteins. Supernatant was collected, dried, reconstituted in 100 µL of

mobile phase and centrifuged. 70 µL of the supernatant was stored at -70 °C until further

analysis. At the time of analysis 10 µL of Gly-Phe solution (4 mg/mL) was added to the

supernatant solution to serve as internal standard and the sample was analyzed using reverse

phase HPLC (Waters Corporation, Milford, MA). Percentage of intact polymer remaining was

calculated based on the calibration curve generated for the intact polymer. The activity of

enzyme was determined spectrophotometrically using N-benzoyl-Phe-Val-Arg-p-nitroanilide

hydrochloride as a substrate. The recovery of the polymer from the experimental procedure was

determined by using acetate buffer without cathepsin B and was found to be more than 90 %.

Samples obtained from degradation studies of amine-terminated polymer were also analyzed by

MALDI to determine degradation profile of the polymer.

Cell lines

Breast cancer cell lines MDA-MB-231 human adenocarcinoma (ATCC, Manassas, VA) and BT-

549 (ATCC, Manassas, VA) were cultured in RPMI 1640 media (Invitrogen, Carlsbad, CA)

supplemented with 4 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum (FBS) and

1% 100x antibiotic-antimycotic (Invitrogen) at 37°C in a humidified atmosphere of 5% CO2

(v/v). Breast cancer cell line Hs 578T (ATCC, Manassas, VA) was cultured in Dulbecco’s

modified eagle medium (DMEM) supplemented with 10 % (v/v) non-heat-inactivated FBS and

0.01 mg/mL of bovine insulin. For all experimental procedures, sub-confluent cells were

harvested with 0.05% trypsin/0.02% EDTA in PBS.

Solubility studies:

Page 14 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

15

Stock solutions of 2ME and MESPM were prepared by dissolving 10 mg of drug in 1 mL

DMSO. Five working solutions (1,5, 10, 30, 50 ug/mL for 2ME and 1, 2, 3.1, 6.25, 12.5 ug/mL

for MESPM) were prepared by dilution in DMSO from the stock solution. Solubility was

determined using LCMS. Chromatography was performed on Shimatzu LCMS 2020 (Shimatzu

Scientific Instrument Inc., Addison, IL) which included a quaternary pump with an online

degasser and an autosampler (SIL-20A UFLC). Analysis was performed at ambient temperature

on Atlantis dC18 column (3 µm; 2.1 x 150 mm) using mobile phase composed of water (0.1 %

acetic acid) and acetonitrile (0.1 % acetic acid). The mobile phase was delivered as an isocratic

run (50 % water and 50 % acetonitrile) for 2ME and as a gradient run for MESPM

(Supplementary data Table T1) at a flow rate of 0.4 mL/min. A single quadrupole detector with

electrospray interface operated in negative mode (for 2ME) and positive ionization mode (for

MESPM) was used for ion detection. Data processing and analysis was performed using

LCMSsolution software. Retention time for 2ME and MESPM was found to be 2.88 and 7.98

respectively. A calibration curve was constructed by least-squares linear regression analysis of

peak area ratio of 2ME or MESPM versus the drug concentration (Supplementary data Figure

S4A and S4B). To determine solubility in water, stock solutions of 2ME or MESPM were

diluted with water, kept for 24h, centrifuged at 3000 rpm for 5 min and the supernatant was

filtered through syringe filter (0.45 µM, Whatman) before analysis using LCMS to obtain peak

area ratio that was used to determine the concentration of 2ME or MESPM in solution. Two

independent studies were performed starting from preparing stock solutions and running the

samples in triplicate.

In vitro growth inhibition assay:

Page 15 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

16

The WST (water soluble tetrazolium salt) assay was used to determine the antiproliferative effect

of 2ME, Gly2ME, GFLGG2ME, CSPM, ASPM and MESPM using three triple negative breast

cancer cell lines MDA-MB-231, BT-549 and Hs 578T. Cells were plated at predetermined

densities (2500 cells/well for all cell line) in 96-well plates and allowed to attach and grow for 24

h. Control and various treatments were added and the cells were incubated for another 48 h, 72 h,

4 days or 5 days (depending on the treatment). Medium was aspirated and cells were incubated

in the presence of WST-1 reagent for 4 h and absorbance was read at 440 nm. Cell growth

inhibition was determined by subtracting the cell viability at day 0 from the cell viability after

treatment and expressed as % cell viability compared to untreated cells. Experiments were

routinely conducted in the exponential growth phase. Seven different concentrations of each

compound were used for the experiment. GI50 values were determined by nonlinear regression

analysis using GraphPad Prism software. Mean GI50 values are reported for at least two

independent experiments with n=3 for each concentration.

RESULTS:

Synthesis and characterization of degradable star shaped peptidic macromolecules

The characteristics of SPMs used for biological studies are summarized in Table 1. A single

molecular peak was observed for all the macromolecules in MALDI (Figure 2 B, Figure S1),

indicating the presence of a single molecular entity as opposed to a polymer with polydispersity.

MALDI and NMR analysis for all three SPMs confirmed the conjugation of monomer to all four

branches. NMR spectra for MESPM is shown in Figure 2A. The characteristic peaks at δ 1.2-

1.3, δ 3.6-3.8, and δ 7.2-7.5 in the NMR spectra were used to confirm the number of leucines,

glycines, and phenylalanines, respectively, whereas peaks at δ 6.4 and δ 6.7 were used to

confirm the number of 2ME molecules present in a macromolecule. Based on NMR and MALDI

Page 16 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

17

analysis, 2ME content was calculated to be 38% wt/wt. The absence of the monomeric

tetrapeptide and the free 2ME in MESPM was confirmed by performing HPLC analysis.

Stability and in-vitro degradation of SPMs:

To evaluate stability, SPMs were incubated with PBS (pH =7.4), 10% FBS in PBS, and

acetate buffer (pH = 5) and percentage of intact SPMs remaining is shown in Figure 3A (ASPM)

and Figure 3B (CSPM). Both ASPM and CSPM were quite stable in PBS. No degradation was

observed for either of SPMs at earlier time points (2 and 4 h) and more than 95% of the intact

SPMs were detected even after longer incubation (24 h). Degradation profiles of SPMs were

different in the presence of serum. ASPM showed signs of degradation at earlier time points (74-

84% intact ASPM detected after 2 h and 4 h) with 50% intact macromolecule remaining after 24

h (Figure 3A). In contrast, CSPM was stable in 10% FBS over 24 h period (97% of intact CSPM

detected after 24 h incubation, Figure 3B). Cathepsin B has its maximum activity at pH 5.0.

Thus, degradation studies in the presence of cathepsin B were conducted at acidic pH. To

determine if the pH has any effect on degradation, SPMs were incubated in an acetate buffer

(without cathepsin B) and evaluated for stability. None of the polymers showed any degradation

in acidic pH (Figure 3A,B).

Degradation studies after incubation with cathepsin B revealed the presence of 10%, 1%

and 0 % intact ASPM after 2 h, 4 h, and 24 h respectively (Figure 4A). The lower concentration

of enzyme (0.12 units/µmol of peptide vs 0.25 units/µmol peptide used in the initial studies) used

in subsequent experiments decreased the rate of degradation as indicated by detection of higher

percentages of the intact ASPM (35% and 5% vs 10% and 0% at 2 and 4 h respectively) (Figure

4B). Figure 5 illustrates the percentage of intact CSPM after incubation with cathepsin B (0.25

Page 17 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

18

units/µmol peptide). None of the intact CSPM was detected after 2 h while 46%, 83%, 91%,

93%, and 94% degradation were seen after 4, 10,15, 30, and 60 min, respectively (Figure 5).

Table 2 illustrates the percentages of intact monomer GFLG after incubation with cathepsin B or

FBS. In contrast to macromolecules, 80% of the intact monomer was detected in the presence of

cathepsin B whereas it degraded completely in the presence of FBS. Degradation behavior of

single entities was retained within a mixture (1:1) of monomer and CSPM. Thus, incubation of a

mixture with cathepsin B degraded only CSPM whereas incubation of a mixture with FBS

degraded only monomer (Table 2).

Solubility and in-vitro cytotoxicity of SPMs and MESPM

Solubility of ASPM and CSPM was found to be more than 5mg/mL. Solubility of 2ME

and MESPM in water was determined using LCMS and was found to be 0.3 ug/mL and 8 ug/mL

respectively. Figure 7 shows % cell viability of MDA-MB-231 after treatment with SPMs for

various time periods. After 48 h exposure, cell viability was more than 90% for both SPMs at the

highest concentrations tested indicating their non-toxic behavior. Our previous reports and

literature suggest that surface charge and density affects cell viability [34-36]. Positively

charged amine groups exhibit higher toxicity compared to their negatively charged or neutral

counterparts. Thus, ASPM was incubated for longer periods (5 days) to evaluate cytotoxicity.

No toxicity was seen even after these longer incubation times. GI50 values could not be

calculated for any of the treatments since cell viability was always more than 50%.

Antiproliferative activity of 2ME, Gly2ME, GFLGG2ME, CSPM and MESPM was further

evaluated in three triple negative breast cancer cell lines viz MDA-MB-231, BT-549 and Hs578

T after 48 and 72 h treatment. Representative cell viability graphs after 48 h treatment are shown

in Figure 8A for MDA-MB-231, Figure 8B for BT-549 and Figure 8C for Hs578 T cell line. GI50

Page 18 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

19

values for all drug-containing treatments are listed in Table 3. The rank order for the

antiproliferative effect of 2ME was found to be BT-549 > MDA-MB-231 > Hs 578T. BT-549

cell line was 1.6- and 2-fold more sensitive to 2ME than MBA-MB-231 and Hs 578T cell line

respectively. Irrespective of the treatment GI50 values remained mostly the same after 48 and 72

h. Cell viability for three cell lines, after incubation with MESPM for 48 h at the highest

concentration tested, is reported in Figure 9. MESPM showed significantly higher toxicity in

Hs578T cell line compared to other two cell lines after 48h treatment. As indicated in Figure 9

no significant toxicity was detected for CSPM in all cell lines used in the study.

DISCUSSION

Our aim is to develop a degradable delivery system and trigger its degradation at tumor sites. We

decided to use enzymatic trigger due to the specificity that can be imparted by using enzyme

substrate as a building block. We chose cathepsin B as a trigger because of its higher expression

at tumor sites. The tetrapeptide GFLG was used as a building block because previously it has

been used to conjugate anticancer drugs to the polymeric backbone [8, 23, 37] and as a linker

between low molecular weight HPMA copolymers to create high molecular weight degradable

system [38-40]. The major reason for selection of this tetrapeptide was the stability it entails to

the polymeric conjugates in the presence of serum, whereas the degradability it imparts in the

presence of cathepsin B. Two star-shaped peptidic macromolecules (SPMs) were synthesized.

One in which tetrapeptide was conjugated to the core thorough its carboxyl-terminus leaving free

amines at the surface to yield amine-terminated star shaped peptidic macromolecule (ASPM,

Figure 1, II) and one in which tetrapeptide was conjugated to the core through its amine terminus

to yield carboxyl-terminated star-shaped peptidic macromolecule (CSPM, Figure 1, III). CSPM

was also used to conjugate model drug 2ME to yield 2ME containing star shaped peptidic

Page 19 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

20

macromolecule (MESPM, Figure 1, III). ASPM was synthesized by building the tetrapeptide

sequence on PAMAM dendrimer (G0, ethylenediamine core) by sequential addition of one

amino acid (G, F, L, G) at a time (Scheme 1). During the synthesis, we discovered that coupling

agents could be used instead of reactive ester to conjugate the first and second amino acid to the

central core. However, they failed to give quantitative yield during the conjugation of the third

and fourth amino acid. Sequential approach could not be used for CSPM and MESPM due to the

requirement of cumbersome synthesis of reactive esters at each step. Instead, a convergent

approach was used, where tetrapeptides (GFLG or GFLGG2ME) were conjugated to the central

core (EDTA) in one step. Our initial attempts to conjugate the tetrapeptides to the central EDTA

core using various coupling reagents yielded a mixture of compounds having one to four

branches of tetrapeptides attached to the central core. In contrast, the use of tetrakis-p-

nitrophenyl ester of EDTA was efficient with better yields and less side products. The use of the

convergent approach for the MESPM also precluded the possibility of the presence of free 2ME

in the conjugate.

Both SPMs showed substantial stability in the presence of serum at earlier time points.

CSPM was stable even after longer incubation period (24 h) but only 50 % of intact ASPM was

detected at that time point. The detection of lower amount of ASPM can in part be due to its

binding to plasma proteins or due to its degradation. The stability of GFLG-containing-HPMA

copolymers in the presence of serum has been reported previously and been attributed to

specificities of the enzymes present in serum, or the presence of the protease inhibitors, or

combination of both [41]. The degradation profile of SPMs in the presence of cathepsin B was

evaluated under in-vitro conditions, which mimic the conditions of lysosomal degradation as

described in previous reports [31-33]. Time-dependent degradation was observed for both ASPM

Page 20 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

21

and CSPM. Degradation was complete for CSPM within 2 h whereas 10% of ASPM was

detected at the same time point indicating differences in their rates of degradations that can be

attributed to their surface charge. Previously, HPMA copolymers having GFLG linker have been

shown to release the model-drug or drug (anthracyclin, campthothecin, and ansamycin analogs)

within few hours [31, 42-46]. Approximately, 60-70 % of doxorubicin release within 10 h time

period has been reported for doxorubicin containing HPMA copolymer [42]. In contrast, rapid

and complete degradation of CSPMs observed incurrent study is expected to facilitate the drug

release in an in-vivo setting after its encounter with cathepsin B.

Cathepsin B is a cysteine protease and can act as an endopeptidase [47, 48] or an

exopeptidase [49, 50] depending on the pH of the environment [47, 51, 52]. As an exopeptidase,

it has been reported to cleave a dipeptide bond at the carboxyl terminus. HPLC chromatogram of

incubation mixture of CSPM indicated the release of tetrapeptide GFLGOH (Fig S2) suggesting

that enzyme is acting as an endopeptidase cleaving the GFLG branch from the EDTA core. The

schematic representation of such type of cleavage and degradation is shown in figure 6A.

However, it is not the only pathway for degradation of CSPM since various peaks between 20-25

min of retention time suggests the presence of other fragments formed due to cleavage at other

sites. In contrast to CSPM, HPLC chromatogram of the incubation mixture of ASPM revealed

the absence of the peak corresponding to the monomeric tetrapeptide and the appearance of

various peaks at higher retention time (20-25 min) indicating the presence of other molecular

fragments. MALDI analysis of these samples indicated the presence of fragments corresponding

to the cleavage of dipeptide (GL) from one, two and three branches (Figure S3). No product

corresponding to the cleavage of all four dipeptides from the dendrimer was detected, even after

24 h of incubation. It is unclear at this point why one GFLG branch remains attached to the core.

Page 21 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

22

Perhaps higher incubation time is required to cleave the last branch from the core. Schematic

representation for the degradation of ASPM is shown in Figure 6B. The differences in the

degradation behavior of CSPM and ASPM can be attributed to the surface charge and the

difference in the sequence. CSPM has GFLG as core-to-surface sequence while ASPM has

GFLG as surface-to-core sequence. Very few studies have been conducted where large protein

substrates have been used to understand the mechanism for endopeptidase activity of cathepsin

B. The peptide segment (Ile105-Pro126) termed the occluding loop in the primed side of the active

site of cathepsin B is known to play a critical role in determining an endo- or exopeptidase

activity of the enzyme [48, 53, 54]. Disruption of two salt-bridge interactions between the loop

and the main body of the enzyme (Asp22-His110 and Arg116-Asp224) has been shown to result in

dramatic increase in the endopeptidase activity of the enzyme [48, 50, 54]. Surface acidic groups

present on CSPM might disrupt these salt-bridge interactions resulting in higher endopeptidase

activity. It is surprising, however, not uncommon to report that irrespective of surface positive

charges, cathepsin B starts cleaving terminal dipeptide GL from the first branch of ASPM

followed by the second and the third branch. Cathepsin B has been shown to cleave amide

terminated peptides [53]. To gain more knowledge about the degradative mechanism, we

conducted degradation studies using monomeric tetrapeptide GFLG. Surprisingly, no

degradation was seen for the monomeric tetrapeptide (Table 2), which suggests that the

tetrapeptide itself does not act as a substrate for cathepsin B supporting the specific sequence

requirements for small peptides that must be met in order to behave as cathepsin B substrates.

However, degradation of SPMs indicates the lack of stringent substrate requirements for

macromolecules. Next, we evaluated the degradation of a 1:1 mixture of monomeric tetrapeptide

and CSPM in the presence of cathepsin B and 10% FBS solutions. Table 2 illustrates the

Page 22 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

23

percentages of intact CSPM or tetrapeptide detected after incubation with cathepsin B or FBS.

As expected, within a mixture, CSPM was preferentially degraded in the presence of cathepsin B

while being stable in FBS. Conversely, monomeric tetrapeptide was completely stable in the

presence of cathepsin B and degraded completely in the presence of serum. These results

emphasize the importance of molecular weight and architecture in serum stability and enzymatic

degradation.

Next, we evaluated the potential of SPMs for delivery of 2ME. CSPM was selected due

to its serum stability and complete degradation in the presence of cathepsin B. A glycine

derivative of 2ME (Gly-2ME) was conjugated to the terminal acidic groups to get MESPM.

Solubility studies indicated modest improvement (26-fold) in the solubility of MESPM

compared to 2ME. Growth inhibition studies were performed using three triple negative breast

cancer cell lines viz MDA-MB-231, BT-549 and Hs 578T. Xing et al has reported higher

expression levels of cathepsin B in Hs 578T compared to MDA-MB-231 and BT-549 cell lines

[55]. We wanted to observe if these expression levels could be translated into differential toxicity

profile of MESPM. Although GI50 values for MESPM were quite similar in Hs 578T and BT-

549, MESPM showed significantly higher toxicity at the highest concentration studied in Hs

578T compared to BT-549 (Figure 9). This is in spite of 2-fold lower sensitivity of Hs 578T to

2ME. Comparison of Hs 578 T and MDA-MB-231 cell line proved difficult due to differential

activity of MESPM in MDA-MB-231 cell lines after 48 and 72 h treatment. Overall

antiproliferative activity of MESPM in all cell lines was almost similar to the monomer

GFLGG2ME indicating that GFLGG2ME is probably getting released. In-vitro release studies

can only confirm that hypothesis. Nevertheless, antiproliferative activity of MESPM indicates

that either 2ME or 2ME containing peptide is getting released from MESPM. Increasing the

Page 23 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

24

incubation time from 48 to 72 hours did not change GI50 values significantly for monomers or for

MESPM (except for MESPM in MDA-MB-231). Decrease in the antiproliferative activity of

polymeric conjugate compared to free drug is expected and well known. MESPM exhibited

higher GI50 compared to free drug. However, the loss of activity is significantly less compared

to the loss of activity observed after conjugation of drugs to the water-soluble polymers using

GFLG tetrapeptide as a linker. More importantly drug content (wt/wt) of the peptidic dendrimer

was much higher (38%) compared to other drug containing polymeric conjugates reported in the

literature.

Low solubility of 2ME along with its rapid metabolism is reported to limit its

bioavailability. In the current study solubility of 2ME in water was found to be 0.3 ug/mL,

which is in agreement with the reported value in the literature [56]. The molecular weight of

MESPM is low and will allow for rapid renal clearance. However, a modest increase in solubility

of MESPM (26-fold more than 2ME) along with the potential of lowering the metabolism (due

to the conjugation of the SPM through 17-OH group on the steroidal backbone of 2ME) is

expected to achieve higher plasma levels compared to 2ME alone. The primary intention of this

work was to evaluate if cathepsin B is able to recognize peptide GFLG in the star-like

architecture and if such a system would exhibit any antiproliferative property when conjugated to

the drug. Confirmatory results from current study set a stage for building a high molecular

weight dendritic systems using GFLG as a building block. Such a system that will respond to the

presence of cathepsin B can be benefitted by its higher molecular weight to achieve better in-

vivo profile such as longer circulation time and tumor accumulation. Structural optimization can

also be achieved at this stage to make the system highly water soluble while maintaining its

antiproliferative property.

Page 24 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

25

CONCLUSION

Here, we report the use of degradable SPMs as a potential platform for building delivery

vehicle for 2ME. SPMs were stable in the presence of serum but degraded completely within

short period of time in the presence of cathepsin B, which indicates their potential as stimuli-

sensitive drug carriers. Surface charge affected both serum stability as well as degradation

behavior of SPMs in the presence of cathepsin B. Higher drug loading (38% wt/wt) was achieved

with good antiproliferative activity in a panel of triple negative breast cancer cell lines.

Page 25 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

26

ABBREVIATIONS

ATCC American type culture collection

BOC tert-Butyloxycarbonyl

CCK cell counting kit

DCM dichloromethane

DCC dicyclohexylcarbodiimide

DCU dicyclohexylurea

DIPEA N,N-diisopropyl ethylamine

DMAP 4-dimethyl amino pyridine

DMSO dimethyl sulfoxide

EDTA Ethylenediaminetetraacetic acid

FBS fetal bovine serum

G0 Generation 0

GFLG Gly-Phe-Leu-Gly

HOBt Hydroxybenzotriazole

HBTU O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate

HPLC high performance liquid chromatography

IC50 half maximal inhibitory concentration

MW molecular weight

ONp p-nitrophenol

PAMAM poly (amido amine)

Page 26 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

27

PBS phosphate buffered saline

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

UV ultraviolet

Wt weight

ACKNOWLEDGEMENTS:

Authors would like to thank Dr. Lee, Director of Protein Research lab, Research Resource

Center at UIC and Dr. Campbell from University of Maryland, Baltimore for their assistance

with MALDI analysis. This work was supported by Department of Defense Multidisciplinary

Postdoctoral fellowship W81XWH-06-1-0698 and Department of Defense Breast Cancer

Research Program Concept Award (W81XWH-09-1-0687) to RK. The work was also supported

by start-up funds to RK from Department of Biopharmaceutical Sciences, University of Illinois

Chicago.

Page 27 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

28

Legends to figures:

Figure 1:

Chemical structure of 2-methoxyestradiol (I), schematic representation of amine-terminated star-

shaped peptidic macromolecule (II, ASPM), carboxyl-terminated star-shaped peptidic

macromolecule (III, CSPM) and 2-methoxyestradiol containing peptidic macromolecule (IV,

MESPM).

Figure 2:

A) NMR spectra of MESPM (IV) showing peaks corresponding to all amino acids and 2ME B)

MALDI analysis of MESPM.

Figure 3:

Stability of A) ASPM and B) CSPM in the presence of PBS (pH = 7.4), acetate buffer (pH = 5.0)

and 10% FBS in PBS over the period of 24 hours. Percentage of the intact polymer remaining

after incubation in respective buffers is reported and was determined using HPLC. Area under

curve (AUC) for polymer was normalized with the AUC obtained for Gly-Phe that was used as

an internal standard. (n = 3, mean ± SD).

Figure 4:

Degradation profile of ASPM (II) in the presence of A) Cathepsin B (0.25 units/µmol of peptide)

and B) Cathepsin B (0.12 units/µmol of peptide). Percentage of the intact polymer remaining

after incubation with cathepsin B is reported and is determined using HPLC. Area under curve

Page 28 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

29

(AUC) for polymer was normalized with the AUC obtained for Gly-Phe that was used as an

internal standard. (n = 3, mean ± SD).

Figure 5:

Degradation profile of CSPM (III) in the presence of Cathepsin B (0.25 units/µmol of peptide).

Percentage of the intact polymer remaining after incubation with cathepsin B is reported and is

determined using HPLC. Area under curve (AUC) for polymer was normalized with the AUC

obtained for Gly-Phe that was used as an internal standard. (n = 3, mean ± SD).

Figure 6:

Schematic representation for the degradation of A) CSPM and B) ASPM in the presence of

cathepsin B. CSPM and ASPM degraded differently. HPLC analysis indicated that one of the

cleavage sites during degradation of CSPM is the amide bond between the acidic core and amine

terminal of tetrapeptide releasing GFLG. ASPM degraded by releasing terminal dipeptide GL. ‘-

------‘ indicates cleavage sites.

Figure 7:

Growth inhibition effect of A) CSPM and B) ASPM on breast cancer cell line (MDA-MB-231)

after incubation for 48, 72 h and 5 days. Cell viability was determined using WST-1. Results are

mean ± SD (n=3).

Figure 8:

Growth inhibition effect of 2ME, Gly2ME, GFLGG2ME and MESPM on breast cancer cell

lines. A) MDA-MB-231 B) BT-549 C) Hs 578T. Cell viability after 48 h treatment was

Page 29 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

30

determined using WST-1. Results are mean ± SD (n=6, two independent experiments in

triplicates). 2ME; Gly2ME; GFLGG2ME; MESPM

Figure 9:

Growth inhibition effect of CSPM and MESPM on MDA-MB-231, BT-549 and Hs 578T cells

after incubation for 48 at 15 µM 2ME equivalent concentration. Cell viability was determined

using WST-1. Results are mean ± SD (n=6, two independent experiments in triplicates).

*Statistical difference between cell viabilities of different cell lines was compared using student t

test where * denotes significant difference between cell viability of Hs578T and MDA-MB-231

(p < 0.05) and cell viability of Hs 578T and BT-549 (p < 0.05).

Page 30 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

31

Figure 1

Page 31 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

32

Figure 2:

Page 32 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

33

Figure 3

A)

B)

Page 33 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

34

Figure 4:

A)

B)

Page 34 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

35

Figure 5

Page 35 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

36

Figure 6:

A)

B)

Page 36 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

37

Figure 7

A)

B)

Page 37 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

38

Figure 8

A)

B)

C)

Page 38 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

39

Figure 9:

Page 39 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

40

Table 1: Physicochemical characteristics of peptidic dendrimers

Polymer Mw

Calc.

Mw

Observed by

MALDI

Retention

Time

(HPLC)

(Min)

G0(GLFGNH2)4

(ASPM) 2014.42 2038

(M+Na) 21.77

EDTA(GFLGOH)4

(CSPM)

1788.87 1791.87 27.08

EDTA(GFLGG2ME)4

MESPM 3155.67 3178.39

(M+Na) 46.42

Page 40 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

41

Table 2: Degradation of CSPM and 1:1 mixture of CSPM and monomer in the presence of

cathepsin B.

% intact GFLGOH /

CSPM remaining

Cathepsin B

(15 min)

FBS

(24 hr)

Monomer (GFLG) 80 ± 1.5 0 %

CSPM (III) 8.9 ± 0.6 97 ± 11

Monomer

+

CSPM (1:1 ratio)

Monomer 100 ± 1.9 0 %

CSPM 13.9 ± 2.0 96 ± 15

Page 41 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

42

Table 3: Growth inhibition (GI50) values of 2ME, Gly2ME, GFLGG2ME and MESPM in breast

cancer cell lines MDA-MB-231, BT-549 and Hs 578T

Sample GI50 (µM)

MDA-MB-231 BT-549 Hs578T

48 h 72 h 48 h 72 h 48 h 72 h

2ME 1.0 ± 0.2 1.1 ± 0.4 0.6 ± 0.08 0.66 ± 0.04 1.22 ± 0.50 1.47 ± 0.42

Gly-2ME 2.7 ± 0.8 - 1.9 ± 0.35 1.77 ± 0.11 3.7 ± 0.6 2.44 ± 0.70

GFLGG2ME 7.8 ± 0.3 7.6 ± 0.6 7.9 ± 0.5 7.70 ± 0.10 7.03 ± 1.12 8.22 ± 0.87

MESPM 8.5 ± 0.3 13.8 ± 4 10.3 ± 2.4 10.81 ± 0.62 10.2 ± 0.7 10.93 ± 1.27

GI50 values are calculated from dose response curves obtained by performing nonlinear

regression analysis using GraphPad Software. Values are calculated for at least two

independent experiments with n=3 for each concentration (mean ± SD).

Page 42 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

43

Scheme 1: Synthesis of ASPM using sequential approach

Step 1: i) Boc-Gly-ONp ii) 50:50 TFA/DCM; Step 2: i) Boc-Phe-ONp (ii) 50:50 TFA/DCM; Step 3) i) Boc-Leu-ONp (ii) 50:50 TFA/DCM; Step 4: i) Boc-Gly-ONp(ii) 50:50 TFA/DCM

Page 43 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

44

Scheme 2: Synthesis of Tetrakis(p-nitrophenyl) ester of EDTA (central reactive core)

Page 44 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

45

Scheme 3: Synthesis of CSPM (III) and MESPM (IV) using convergent approach.

Page 45 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

46

TOC figure

Page 46 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

47

References

[1] Verenich S, Gerk PM. Therapeutic promises of 2-methoxyestradiol and its drug disposition

challenges. Mol Pharm. 2010;7:2030-9.

[2] Sutherland TE, Anderson RL, Hughes RA, Altmann E, Schuliga M, Ziogas J, et al. 2-

Methoxyestradiol--a unique blend of activities generating a new class of anti-tumour/anti-

inflammatory agents. Drug discovery today. 2007;12:577-84.

[3] Klauber N, Parangi S, Flynn E, Hamel E, D'Amato RJ. Inhibition of angiogenesis and breast

cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol. Cancer research.

1997;57:81-6.

[4] Fotsis T, Zhang Y, Pepper MS, Adlercreutz H, Montesano R, Nawroth PP, et al. The

endogenous oestrogen metabolite 2-methoxyoestradiol inhibits angiogenesis and suppresses

tumour growth. Nature. 1994;368:237-9.

[5] Mueck AO, Seeger H. 2-Methoxyestradiol--biology and mechanism of action. Steroids.

2010;75:625-31.

[6] Dahut WL, Lakhani NJ, Gulley JL, Arlen PM, Kohn EC, Kotz H, et al. Phase I clinical trial

of oral 2-methoxyestradiol, an antiangiogenic and apoptotic agent, in patients with solid tumors.

Cancer biology & therapy. 2006;5:22-7.

[7] James J, Murry DJ, Treston AM, Storniolo AM, Sledge GW, Sidor C, et al. Phase I safety,

pharmacokinetic and pharmacodynamic studies of 2-methoxyestradiol alone or in combination

with docetaxel in patients with locally recurrent or metastatic breast cancer. Investigational new

drugs. 2007;25:41-8.

[8] Kopecek J. Polymer-drug conjugates: Origins, progress to date and future directions.

Advanced drug delivery reviews. 2013;65:49-59.

Page 47 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

48

[9] Zhou Y, Kopecek J. Biological rationale for the design of polymeric anti-cancer

nanomedicines. Journal of drug targeting. 2013;21:1-26.

[10] Duncan R, Vicent MJ. Polymer therapeutics-prospects for 21st century: The end of the

beginning. Advanced drug delivery reviews. 2013;65:60-70.

[11] Allen TM, Cullis PR. Liposomal drug delivery systems: From concept to clinical

applications. Advanced drug delivery reviews. 2013;65:36-48.

[12] Koshkaryev A, Sawant R, Deshpande M, Torchilin V. Immunoconjugates and long

circulating systems: Origins, current state of the art and future directions. Advanced drug

delivery reviews. 2013;65:24-35.

[13] Kim CS, Tonga GY, Solfiell D, Rotello VM. Inorganic nanosystems for therapeutic

delivery: Status and prospects. Advanced drug delivery reviews. 2013;65:93-9.

[14] Hoffman AS. Stimuli-responsive polymers: Biomedical applications and challenges for

clinical translation. Advanced drug delivery reviews. 2013;65:10-6.

[15] Felber AE, Dufresne MH, Leroux JC. pH-sensitive vesicles, polymeric micelles, and

nanospheres prepared with polycarboxylates. Advanced drug delivery reviews. 2012;64:979-92.

[16] Graf N, Lippard SJ. Redox activation of metal-based prodrugs as a strategy for drug

delivery. Advanced drug delivery reviews. 2012;64:993-1004.

[17] de la Rica R, Aili D, Stevens MM. Enzyme-responsive nanoparticles for drug release and

diagnostics. Advanced drug delivery reviews. 2012;64:967-78.

[18] Gondi CS, Rao JS. Cathepsin B as a cancer target. Expert opinion on therapeutic targets.

2013;17:281-91.

[19] Roshy S, Sloane BF, Moin K. Pericellular cathepsin B and malignant progression. Cancer

metastasis reviews. 2003;22:271-86.

Page 48 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

49

[20] Mohamed MM, Sloane BF. Cysteine cathepsins: multifunctional enzymes in cancer. Nature

reviews Cancer. 2006;6:764-75.

[21] Reiser J, Adair B, Reinheckel T. Specialized roles for cysteine cathepsins in health and

disease. The Journal of clinical investigation. 2010;120:3421-31.

[22] Duncan R, Vicent MJ. Do HPMA copolymer conjugates have a future as clinically useful

nanomedicines? A critical overview of current status and future opportunities. Advanced drug

delivery reviews. 2010;62:272-82.

[23] Vasey PA, Kaye SB, Morrison R, Twelves C, Wilson P, Duncan R, et al. Phase I clinical

and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer

doxorubicin]: first member of a new class of chemotherapeutic agents-drug-polymer conjugates.

Cancer Research Campaign Phase I/II Committee. Clinical cancer research : an official journal

of the American Association for Cancer Research. 1999;5:83-94.

[24] Seymour LW, Ferry DR, Kerr DJ, Rea D, Whitlock M, Poyner R, et al. Phase II studies of

polymer-doxorubicin (PK1, FCE28068) in the treatment of breast, lung and colorectal cancer.

International journal of oncology. 2009;34:1629-36.

[25] Duncan R. Polymer therapeutics as nanomedicines: new perspectives. Current opinion in

biotechnology. 2011;22:492-501.

[26] Rob Webster VE, B. Kevin Park, Donald Walker, Mark Hankin, Philip Taupin. PEG and

PEG conjugates toxicity: towards an understanding of the toxicity of PEG and its relevance to

PEGylated biologicals. In: Veronese FM, editor. PEGylated Protein Drugs: Basic Science and

Clinical Applications: Birkhäuser Basel; 2009. p. 127-46.

[27] Park K. The optimal formulation variables for tumor targeting. Journal of controlled release

: official journal of the Controlled Release Society. 2012;157:315-6.

Page 49 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

50

[28] Rao PN, Cessac JW. A new, practical synthesis of 2-methoxyestradiols. Steroids.

2002;67:1065-70.

[29] Ho A, Kim YE, Lee H, Cyrus K, Baek SH, Kim KB. SAR studies of 2-methoxyestradiol

and development of its analogs as probes of anti-tumor mechanisms. Bioorganic & medicinal

chemistry letters. 2006;16:3383-7.

[30] Pechar M, Ulbrich K, Subr V, Seymour LW, Schacht EH. Poly(ethylene glycol) multiblock

copolymer as a carrier of anti-cancer drug doxorubicin. Bioconjugate chemistry. 2000;11:131-9.

[31] Borgman MP, Ray A, Kolhatkar RB, Sausville EA, Burger AM, Ghandehari H. Targetable

HPMA Copolymer-Aminohexylgeldanamycin Conjugates for Prostate Cancer Therapy. Pharm

Res-Dord. 2009;26:1407-18.

[32] Kasuya Y, Lu ZR, Kopeckova P, Tabibi SE, Kopecek J. Influence of the structure of drug

moieties on the in vitro efficacy of HPMA copolymer-geldanamycin derivative conjugates.

Pharm Res. 2002;19:115-23.

[33] Wang D, Kopeckova JP, Minko T, Nanayakkara V, Kopecek J. Synthesis of starlike N-(2-

hydroxypropyl)methacrylamide copolymers: potential drug carriers. Biomacromolecules.

2000;1:313-9.

[34] Kolhatkar RB, Kitchens KM, Swaan PW, Ghandehari H. Surface acetylation of

polyamidoamine (PAMAM) dendrimers decreases cytotoxicity while maintaining membrane

permeability. Bioconjugate chemistry. 2007;18:2054-60.

[35] Fischer D, Li Y, Ahlemeyer B, Krieglstein J, Kissel T. In vitro cytotoxicity testing of

polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials.

2003;24:1121-31.

Page 50 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

51

[36] Ryser HJ. A membrane effect of basic polymers dependent on molecular size. Nature.

1967;215:934-6.

[37] Seymour LW, Ferry DR, Anderson D, Hesslewood S, Julyan PJ, Poyner R, et al. Hepatic

drug targeting: phase I evaluation of polymer-bound doxorubicin. Journal of clinical oncology :

official journal of the American Society of Clinical Oncology. 2002;20:1668-76.

[38] Pan H, Sima M, Yang J, Kopecek J. Synthesis of Long-Circulating, Backbone Degradable

HPMA Copolymer-Doxorubicin Conjugates and Evaluation of Molecular-Weight-Dependent

Antitumor Efficacy. Macromolecular bioscience. 2013;13:155-60.

[39] Shiah JG, Dvorak M, Kopeckova P, Sun Y, Peterson CM, Kopecek J. Biodistribution and

antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer-

doxorubicin conjugates in nude mice. Eur J Cancer. 2001;37:131-9.

[40] Dvorak M, Kopeckova P, Kopecek J. High-molecular weight HPMA copolymer-adriamycin

conjugates. Journal of controlled release : official journal of the Controlled Release Society.

1999;60:321-32.

[41] Rejmanova P, Kopecek J, Duncan R, Lloyd JB. Stability in rat plasma and serum of

lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl) methacrylamide

copolymers. Biomaterials. 1985;6:45-8.

[42] Etrych T, Chytil P, Jelinkova M, Rihova B, Ulbrich K. Synthesis of HPMA copolymers

containing doxorubicin bound via a hydrazone linkage. Effect of spacer on drug release and in

vitro cytotoxicity. Macromolecular bioscience. 2002;2:43-52.

[43] Caiolfa VR, Zamai M, Fiorino A, Frigerio E, Pellizzoni C, d'Argy R, et al. Polymer-bound

camptothecin: initial biodistribution and antitumour activity studies. Journal of controlled release

: official journal of the Controlled Release Society. 2000;65:105-19.

Page 51 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

52

[44] Duncan R, Kopeckova-Rejmanova P, Strohalm J, Hume I, Cable HC, Pohl J, et al.

Anticancer agents coupled to N-(2-hydroxypropyl)methacrylamide copolymers. I. Evaluation of

daunomycin and puromycin conjugates in vitro. Br J Cancer. 1987;55:165-74.

[45] Kopecek J, Rejmanova P, Duncan R, Lloyd JB. Controlled release of drug model from N-

(2-hydroxypropyl)-methacrylamide copolymers. Annals of the New York Academy of Sciences.

1985;446:93-104.

[46] Rejmanova P, Kopecek J, Pohl J, Baudys M, Kostka V. Polymers Containing Enzymatically

Degradable Bonds .8. Degradation of Oligopeptide Sequences in N-(2-

Hydroxypropyl)Methacrylamide Co-Polymers by Bovine Spleen Cathepsin-B. Makromol Chem.

1983;184:2009-20.

[47] Khouri HE, Plouffe C, Hasnain S, Hirama T, Storer AC, Menard R. A model to explain the

pH-dependent specificity of cathepsin B-catalysed hydrolyses. The Biochemical journal.

1991;275 ( Pt 3):751-7.

[48] Nagler DK, Storer AC, Portaro FC, Carmona E, Juliano L, Menard R. Major increase in

endopeptidase activity of human cathepsin B upon removal of occluding loop contacts.

Biochemistry. 1997;36:12608-15.

[49] Musil D, Zucic D, Turk D, Engh RA, Mayr I, Huber R, et al. The refined 2.15 A X-ray

crystal structure of human liver cathepsin B: the structural basis for its specificity. The EMBO

journal. 1991;10:2321-30.

[50] Illy C, Quraishi O, Wang J, Purisima E, Vernet T, Mort JS. Role of the occluding loop in

cathepsin B activity. The Journal of biological chemistry. 1997;272:1197-202.

Page 52 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

53

[51] Renko M, Pozgan U, Majera D, Turk D. Stefin A displaces the occluding loop of cathepsin

B only by as much as required to bind to the active site cleft. The FEBS journal. 2010;277:4338-

45.

[52] Ruzza P, Quintieri L, Osler A, Calderan A, Biondi B, Floreani M, et al. Fluorescent,

internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin

B. Journal of peptide science : an official publication of the European Peptide Society.

2006;12:455-61.

[53] Krupa JC, Hasnain S, Nagler DK, Menard R, Mort JS. S2' substrate specificity and the role

of His110 and His111 in the exopeptidase activity of human cathepsin B. The Biochemical

journal. 2002;361:613-9.

[54] Quraishi O, Nagler DK, Fox T, Sivaraman J, Cygler M, Mort JS, et al. The occluding loop

in cathepsin B defines the pH dependence of inhibition by its propeptide. Biochemistry.

1999;38:5017-23.

[55] Xing R, Addington AK, Mason RW. Quantification of cathepsins B and L in cells. The

Biochemical journal. 1998;332 ( Pt 2):499-505.

[56] Cho JK, Hong KY, Park JW, Yang HK, Song SC. Injectable delivery system of 2-

methoxyestradiol for breast cancer therapy using biodegradable thermosensitive

poly(organophosphazene) hydrogel. Journal of drug targeting. 2011;19:270-80.

Page 53 of 53

ACS Paragon Plus Environment

Molecular Pharmaceutics

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960