Drug Delivery of Anticancer Agents- Water Soluble 4-PolyEG

8/11/2019 Drug Delivery of Anticancer Agents- Water Soluble 4-PolyEG

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Journal of Controlled Release 61 (1999) 281294

www.elsevier.com/locate/jconrel

Drug delivery of anticancer agents: water soluble 4-poly(ethylene glycol) derivatives of the lignan, podophyllotoxin

a b , a a*Richard B. Greenwald , Charles D. Conover , Annapurna Pendri , Yun H. Choe ,a a a b b

Anthony Martinez , Dechun Wu , Shuiyun Guan , Zuliang Yao , Kwok L. ShumaDepartment of Organic and Medicinal Chemistry, Research and Development, Enzon, Inc., 20Kingsbridge Road, Piscataway,

NJ 08854, U SAbDepartment of Pharmacology and Toxicology, Research and Development, Enzon, Inc., 20Kingsbridge Road, Piscataway, NJ 08854,

USA

Received 2 April 1999; received in revised form 2 April 1999; accepted 1 June 1999

Abstract

This paper reports on the synthesis and in vivo oncolytic activity of a series of water-soluble acyl derivatives of

polyethylene glycol (PEG) conjugated podophyllotoxin. Some analogs of the polymer conjugate showed significantly better

activity in a murine leukemia model than native podophyllotoxin suspended in an intralipid emulsion. Additionally, when

tested intravenously against a solid lung tumor (A549) model, some conjugated analogs were equivalent to the

podophyllotoxin/ intralipid emulsion, while those compounds demonstrating slower rates of plasma hydrolysis (in vitro)appeared to cause greater toxicity. There appeared to be an overall correlation between the in vivo antitumor activity of the

conjugate and its rate of hydrolysis in vitro, with those showing faster release possessing greater antitumor activity. In

conclusion, the solubilization and predictable release of podophyllotoxin from a PEG carrier was achieved and resulted in

some derivatives demonstrating, at a minimum, equivalency with podophyllotoxin when administered on an equal molar

basis. Further studies may be warranted to assess the PEG-conjugates pharmacokinetics and therapeutic indices in leukemic

models. 1999 Elsevier Science B.V. All rights reserved.

Keywords: Podophyllotoxin; Conjugates; Polyethylene glycol; Delivery; Cancer

1. Introduction the dried roots of the North American perennial

Podophyllum peltatum (also known as AmericanPodophyllotoxin (podo) is an aryltetralin lactone mandrake). Podo (1) has a wide array of effects on

that belongs to the class of compounds known as biological systems including inhibition of viral repli-

lignans: these are formed in nature by the oxidative cation: 1 has been employed as a topically applied

dimerization of cinnamic acids with cinnamic al- drug for the treatment of the viral condition,

cohols [1]. It is the principal active compound of the Condyloma acuminatum(venereal warts), for over 50

resin mixture known as podophyllin obtained from years [2]. Podo is also known to inhibit nucleoside

transport into mammalian cells. In particular, podo

possesses powerful cytotoxic properties and is*Corresponding author. Tel.: 11-732-980-4958; fax: 11-732-885-2950. known to be a spindle poison similar to colchicine

0168-3659/ 99/ $ see front matter 1999 Elsevier Science B.V. All rights reserved.

P I I : S 0 1 6 8 - 3 65 9 ( 9 9 ) 0 0 1 5 3 - 4


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282 R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281 294

and the vinca alkaloids: by binding to tubulin during This work continues the exploration of drug delivery

mitosis it inhibits microtubule assembly. In one of of insoluble anti-cancer agents using conjugation

the earliest antitumor studies of lignans, a-peltatin with PEG; a strategy which we first initiated with

(the 5-hydroxy isomer of 1) was evaluated in a paclitaxel [8] (a tubulin depolymerization inhibitor)

clinical setting, since it had been shown earlier that and camptothecin [9] (a topoisomerase I inhibitor).against rodent tumors it was the most active com- For these two agents, PEG modification has been

ponent of the podophyllin extract. The conclusion of shown to extend the circulatory exposure, reduce the

the trial was that intravenous administration of a- toxicity and increase therapeutic index of the parent

peltatin (infusion of a finely divided suspension compound [10,11].

obtained by dilution in an acetone solution with 10%

glucose) was not of significant therapeutic value.

Although evidence of acute induced tumor necrosis2. Material and methods

was observed, progressive tumor growth was often

resumed about 5 days after treatment was discon-

tinued; even with maximum tolerated doses [3]. 2.1. Chemistry

Podo shares the property (with the anticancer drugspaclitaxel and camptothecin) of being virtually in-2.1.1. General methods

soluble in water, and early in vivo experiments wereAll reagents and solvents were purchased from

carried out by dissolution of 1 in either alcohol/Sigma-Aldrich and used without further purification

water or propylene glycol solution followed byunless stated otherwise. Podo was obtained from

subcutaneous injection [4]. However, synthetic modi-Spectrum Chemical Mfg. Corp. (Gardena, CA) and

fication of podo has led to the development of thePEG diol (40 kDa) from Crescent Chemical Com-

more soluble antineoplastic agents, etoposide andpany (Hauppauge, NY). t-Boc protected amino acids

teniposide, both 4-glucopyranosyl derivatives ofwere purchased from Advanced ChemTech (Louis-

epipodophyllotoxin [5]. It was soon recognized thatville, KY). All PEG compounds were dried under

both of these drugs had a different mode of actionvacuum or by azeotropic distillation from toluene

than1, and acted by inhibiting the enzymatic activity 1 13

prior to use. H and C NMR spectra were obtainedof DNA-topoisomerase II. with a JEOL FT NMR System JNM GSX-270Clinicians and research scientists have long specu-

instrument using deuterochloroform as the solventlated that podos lackluster therapeutic index could

unless specified. Chemical shifts (d) are reported inbe a result of its insolubility and unpredictable

parts per million (ppm) downfield from tetra-systemic behavior [5,6], which has generated interest 13

methylsilane. C NMR spectra were obtained atin its modification. Early efforts by Lee and co-

67.80 MHz on the JEOL JNM GSX-270.workers to reduce the toxicity of 1, and maintain or

enhance activity were directed along the lines of

simple ester synthesis [7]. While several of the esters 2.1.2. HPLC method

showed significant activity, none were water soluble Analytical HPLCs were performed using a C8

or demonstrated any increase in activity compared to reverse phase column (Beckman, ultrasphere) under

podo when tested at the same dosage level in a P388 isocratic conditions with an 80:20 mixture (v / v) oflymphocytic leukemia mouse model. The objective methanolwater as the mobile phase. Peak elutions

of the work reported within this paper, was the were monitored at 290 nm using UV detector. To

conversion of 1 into a series of water soluble poly detect the presence of any free PEG and also to

(ethylene glycol) [PEG] 40 kDa acyl conjugate confirm the presence of pegylated product, an

derivatives possessing predictable rates of release. In evaporative light scattering detector (ELSD), Model

vitro rates of hydrolysis and cytotoxicity (IC ) were PL-EMD 950 (Polymer Laboratories), was em-50measured and in vivo anti-cancer activity of these ployed. Based on ELSD analysis, all the final

analogs were then tested in both our standard P388/ 0 pegylated products were free of native podo and

leukemia screen and a human lung xenograft model. were$95% pure by HPLC.


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R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281 294 283

2.1.3. Analysis of podo contents in PEG 2.1.5. Chemical synthesis

derivatives The following procedure used for PEG-leu-podo

The UV absorbance of native podo in N,N-di- (5d) illustrates the method followed for the synthesis

methylformamide (DMF) was determined at 300 nm of the other amino acid ester derivatives: PEG-gly-

for six different concentrations ranging from 0.02 podo (5a), PEG-ala-podo (5b), PEG-met-podo (5c),mmol / ml to 0.3 mmol / ml. From the standard plot of PEG-pro-podo (5e) and PEG-phe-podo (5f). All PEG

absorbance vs. concentration, the absorption coeffi- derivatives employed in this study were 40 kDa,

cient, e, for podophyllotoxin was calculated to be 7.4 except for compound 9a and 11, which used 5 kDa

(O.D. at 300 nm for 1 mg/ml with 1.0 cm light PEG to study the epimerization of podo in vitro.

path). Pegylated podo derivatives were dissolved in

DMF at an approximate concentration of 0.06 mmol/ 2.1.6. PEG-podophyllotoxin (3)

ml (based on a MW of 40 kDa) and the UV 11.5 g (0.29 mmol) of PEG dicarboxylic acid (2)

absorbance of these compounds at 300 nm was [8] was dissolved in 50 ml of anhydrous methylene

determined. Using this value and exploying the chloride at room temperature. The solution was

absorption coefficient e obtained from the above, the chilled to 08C and 0.175 ml (1.12 mmol) of DIPC,

concentration of podophyllotoxin in the sample was 18.2 mg (0.15 mmol) ofN

,N

-dimethylaminopyridinedetermined. Dividing this value by the sample con- (DMAP), and 477 mg (1.15 mmol) of podo were

centration provided the percentage of podo in the added, in that order, and the mixture was stirred for 4

sample. h at 08C. The reaction mixture was allowed to warm

to room temperature and stirred for 16 h. The2.1.4. Determination of rates of hydrolysis of reaction solution was washed with 0.1N HCl, dried

PEG-podo derivatives over anhydrous MgSO , and the solvent was re-4

The rates of hydrolysis were obtained by employ- moved by distillation in vacuo. The residue was

ing a C8 reverse phase column (Zorbax SB-C8) recrystallized from 2-propanol to give 9.52 g (82%13

using a gradient mobile phase consisting of (a) 0.1 M yield) of3 as a white solid. C NMRd37.62, 42.68,

triethylammonium acetate buffer and (b) acetonitrile. 44.34, 55.07, 59.52, 67.53, 69.0271.22 (PEG),

A flow rate of 1 ml/ min was used, and chromato- 73.06, 77.47, 100.84, 105.85, 105.95, 107.18,

grams were monitored using a UV detector at 290 108.67, 127.03, 131.41, 133.89, 136.19, 146.70,nm. For hydrolysis in buffer, PEG-podo derivatives 147.27, 151.60, 169.96, 172.36. UV assay showed

were dissolved in 0.1 M pH 7.4 PBS at a con- 2.00 equivalents of podo present in the product.

centration of 5 mg/ml, while for hydrolysis in

plasma, the derivatives were first dissolved in dis- 2.1.7. N-t-Boc-leucine-podophyllotoxin (3d)

tilled water at a concentration of 20 mg/100 ml and A mixture of 1.0 g (4 mmol) of N-t-Boc leucine,

then added to 900 ml of either rat or human plasma. 0.83 g (2 mmol) of podo, 1.6 g (12 mmol) of DIPC,

The mixture was vortexed for 2 min and divided into and 0.8 g (6.6 mmol) of DMAP in 20 ml of dry

2 ml glass vials with 100 ml of the aliquot per each dichloromethane was stirred for 18 h at room

vial. The solutions were incubated at 378C for temperature followed by dilution with 20 ml of

various periods of time. A mixture of methanol dichloromethane. This mixture was washed with 1N

acetonitrile (1:1, v/v, 400 ml) was added to a vial at HCl, saturated aqueous sodium bicarbonate, andthe proper interval and the mixture was vortexed for water. The organic layer was dried over anhydrous

1 min, followed by filtration through 0.45 mm filter sodium sulfate and filtered, followed by removal of

membrane (optionally followed by a second filtration the solvent by distillation in vacuo to yield 1.2 g13

through 0.2 mm filter membrane). An aliquot of 20 (92% yield) of product. C NMR d 21.71, 22.66,ml of the filtrate was injected into the HPLC. On the 24.83, 28.14, 38.48, 41.03, 43.62, 45.33, 52.54,

basis of the peak area, the amounts of native podo 55.95, 60.56, 71.05, 73.98, 80.11, 101.49, 107.05,

and PEG-podo were estimated, and the half-life of 107.93, 109.50, 127.86, 132.11, 134.74, 136.99,

each compound in different media was calculated 147.61, 148.13, 152.52, 155.40, 173.43, 173.88. In

using linear regression analysis. addition, the NMR spectrum also indicates reso-


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nances corresponding to diisopropyl urea as an azeotroped in 200 ml of toluene for 2 h. The solution

impurity. Doubling of peaks corresponding to the was cooled to 308C and 1.0 ml (5.8 mmol) of

t-Boc region of the product was also observed and is diisopropylethyl amine (DIPEA) and 1.0 g (5.8

probably due to a restricted rotation of the t- mmol) of p-nitrophenyl chloroformate were added.

butylcarbamate bond. This material was used without The mixture was stirred at 558C overnight, cooled tofurther purification. room temperature, and concentrated in vacuo. The

residue was recrystallized from methylene chloride/13

2.1.8. Leucine-podophyllotoxin (4d) ethyl ether to give 9.5 g (95% yield) of product. C

A quantity of 0.9 g (1.4 mmol) of t-Boc-leu-podo NMR d67.2570.42 (PEG), 120.98, 124.24, 144.30,

was combined with 10 ml solution of 1N HCl in 151.31, 154.52.

acetic acid and stirred for 30 min at room tempera-

ture. This solution was then diluted with 40 ml of

dichloromethane followed by adjusting the pH to 8.0 2.1.11. PEG podophyllotoxin carbonate (7)

with saturated aqueous sodium bicarbonate. The A mixture of 1.0 g (0.025 mmol) of 6, 100 mg

organic layer was separated, washed with water, (0.24 mmol) of podo, and 50 mg (0.41 mmol) of

dried over anhydrous sodium sulfate and filtered. DMAP in 20 ml of anhydrous dichloromethane wasThe solvent was removed from the filtrate by distilla- refluxed overnight. The solvent was removed in

tion in vacuo to yield 0.6 g (75% yield) of product. vacuo and the residue was recrystallized from 200 ml13

C NMRd 21.41, 22.57, 24.47, 38.29, 40.56, 43.28, of 2-propanol to give 0.87 g (87% yield) of product.13

44.90, 54.77, 55.62, 60.26, 70.85, 73.58, 101.28, C NMR d 37.44, 42.75, 44.29, 55.09, 59.55,

106.66, 107.62, 109.26, 127.71, 131.96, 134.51, 66.5470.53 (PEG), 100.86, 106.09, 107.08, 108.62,

136.60, 147.24, 147.81, 152.22, 173.25, 174.43. In 126.87, 131.33, 133.86, 146.72, 147.34, 151.60,

addition, the NMR spectrum showed resonances 154.39,172.41. UV assay showed 1.91 equivalents of

corresponding to trace amounts of diisopropyl urea, podo present in the product.

acetic acid, and a small amount of unreacted N-t-

Boc-leu-podo. This material was used without fur-

ther purification. 2.1.12. PEG podophyllotoxin carbamate (9)

1.0 g (0.025 mmol) of PEG diamine hydrochloride2.1.9. PEG-leucine-podophyllotoxin (5d) [9] was azeotroped in 30 ml of toluene for 2 h. The

A mixture of 7.5 g (0.19 mmol) of2, 0.4 g (0.75 solution was cooled to room temperature. 9.9 mg

mmol) of leu-podo (4d), 0.18 g (0.95 mmol) of (0.033 mmol) of triphosgene and 43 ml (0.25 mmol)

1 - [3 - (dimethylamino)propyl] - 3 - ethylcarbodiimide of DIPEA were added to the solution and the mixture

hydrochloride (EDC), and 0.24 g (2.0 mmol) of was stirred at 708C for 3 h, followed by cooling to

DMAP in 75 ml of dry dichloromethane was stirred room temperature. Ethyl ether was added to precipi-

for 18 h at room temperature. The reaction mixture tate PEG-isocyanate (8), which was collected and

was filtered through Celite and the solvent removed dissolved in 10 ml of anhydrous methylene chloride.

from the filtrate by distillation in vacuo. The residue 126 mg (0.20 mmol) of dibutyltin dilaurate and 82

was recrystallized from 2-propanol to yield 7.1 g mg (0.20 mmol) of podo were added to the solution13

(91% yield) of product. C NMR d 21.39, 22.30, and the mixture refluxed overnight, followed by24.47, 38.08, 40.22, 43.19, 44.90, 50.21, 55.62, stirring at room temperature for 48 h. The solvent

60.20, 70.0670.61 (PEG), 70.79, 73.82, 101.18, was removed in vacuo and the residue recrystallized

106.82, 107.59, 109.05, 127.42, 131.65, 134.36, from 2-propanol to give 0.65 g (65% yield) of13

136.63, 147.24, 147.77, 152.14, 169.86, 172.76, product. C NMR d 37.62, 39.78, 42.54, 44.00,

172.94. The amount of available 1 as determined by 54.88, 59.21, 67.5972.54 (PEG), 100.55, 105.82,

UV assay was 1.9 equivalents. 107.17, 108.32, 128.31, 130.83, 134.12, 135.98,

146.31, 146.70, 151.32, 155.41, 172.49. An UV

2.1.10. PEG p-nitrophenyl carbonate (6) assay showed 1.84 equiv. of podo present in the

A quantity of 10.0 g (0.25 mmol) of PEG diol was product.


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2.1.13. PEG picropodophyllotoxin carbamate (11) screens were conducted as previously reported [8].

To a solution of 1 g (0.2 mmol) of mPEG (5 kDa) Briefly, for IC determination cells were seeded into503

amine hydrochloride in 10 ml of anhydrous chloro- the plates at a density of 2310 cells per 50 ml per

form were added 23 mg (0.08 mmol) of triphosgene well. Plates were incubated at 378C in a humidified

and 150 ml (0.87 mmol) of DIPEA under nitrogen incubator with 5% CO for 3 days. Cell growth was2atmosphere and the mixture was refluxed for 2 h. To measured by the addition of 10 ml/well of Alamar

this reaction mixture were added 140 mg (0.34 Blue (Alamar Biosciences, Inc., Sacramento, CA)

mmol) of picropodo [12] and 110 mg (0.17 mmol) and the plates were incubated a further 4 h at 378C.

of dibutyltin dilaurate and the mixture was refluxed The IC values for each compound were determined50for 48 h. The solvent was removed in vacuo and the from absorbance vs. dilution factor plots. A549

residue crystallized from 2-propanol to give 0.8 g (human lung carcinoma-ATCC/ CCL185) was grown

(80% yield) of product. Compound 9a was prepared in a 1:1 mixture of Dulbeccos modified eagles

in a similar fashion using podo instead of picropodo. medium and Hams F-12, supplemented with 10%

fetal bovine serum. All cultures were maintained at

2.1.14. Analysis of epimerization 378C in a humidified atmosphere of 5% CO / 95%2

PEG (5 kDa)-podo carbamate (9a

, 1.0 g) was O and subcultured once a week. All cell lines were2dissolved in 16 ml of rat plasma. 4 ml aliquots of periodically tested for Mycoplasma and were Myco-

this solution were transferred to 5 ml polypropylene plasma free. For solid tumor studies, female nu/ nu

tubes and incubated at 378C for various periods of mice (Harlan Sprague Dawley, Madison, WI), 1824

time. At the end of the incubation, the solution was g were inoculated subcutaneously at the left flank6

transferred to a 50 ml polypropylene centrifuge tube with tumor cells (1310 ) in 0.1 ml of medium.

and 20 ml of acetonitrile was added. The mixture

was vortexed and centrifuged and the turbid solution 2.2.3. Anticancer activity

filtered through a 0.45 mm filter membrane and Podo and its conjugated forms were tested intra-

evaporated under reduced pressure. The solid ob- peritoneal in a murine ascites model against the

tained was recrystallized (CH Cl : ether55 ml: 40 leukemia cell line P388/ 0 (mouse, lymphoid neo-2 2ml) to remove small molecule impurities and yielded plasm) as previously described [11]. Two experi-

approximately 160200 mg of recovered PEG carba- ments evaluating PEG-podo conjugates were con-mate derivative. The percent of epimerization of ducted in nude mice bearing A549 solid tumors. The

compound 9a was calculated from the ratio of first study assessed the efficacy of all the PEG-podo3

integration of peaks at 6.87 ppm and 6.82 ppm (H-5, derivatives against tumors of approximately 50 mm1

9a vs. 11) in the H NMR spectrum (Fig. 5). (day 1). In this study, the derivatives were adminis-

tered intravenously (|200 ml/mouse) at 15 mg/kg/2.2. Biological studies dose on day 1, 8 and 19 and their activity compared

to control (untreated) and native podo treated mice2.2.1. Materials (eight / group). The second study evaluated only

All PEG conjugated podo compounds were dis- those compounds that showed significant antitumor

solved (|100 mg/ ml) in sterile saline (0.9%) for activity as compared to the control group in the first

injection prior to in vivo drug treatments and doses study. Compounds were given intravenously at 30were calculated based on their podo equivalents mg/kg on only day 1 (initial tumor volume of 250

3(absolute amount of podo given). For in vivo ad- mm ) and the mice (six/ group) observed for 2

ministration, podo was dispersed in intralipid weeks. In both studies, the overall growth of tumors

(Liposyn III 10%, Abbott Laboratories, North (% change in tumor volume) was calculated as the

Chicago, IL) by sonication. mean tumor volume at the end of the treatment

minus the mean initial (pretreatment) tumor volume,2.2.2. Cell lines and cytotoxicity assays divided by the initial tumor volume. Thus, any tumor

Studies using P388 / 0 cell lines for both IC (drug group which did not respond to treatment and grew50

concentration inhibiting 50% of cells) and in vivo over the course of the experiment would display


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positive percent change and treatment groups in

which tumors regressed would exhibit a negative

percent change. Tumor growth inhibition (%T/ C)

was used to determine antitumor effectiveness [13].

Treatment and control groups were measured whenthe control groups median tumor volume reached

3approximately 600800 mm (exponential growth

phase). All animals received humane care in com-

pliance with the Principles of Laboratory Animal

Care formulated by the National Society of Medical

Research and the Guide for the Care and Use of

Laboratory Animals published by the National

Institute of Health. These experimental protocols

were approved by the Institutional Animal Care and

Use Committee of UMDNJ-Robert Wood Johnson

Medical School.

2.2.4. Statistics

The differences between treatment groups were

assessed by one-way anova. Multiple comparisons,

when significant differences existed were determined

by least significant differences techniques. StatisticalTM

analysis was conducted using the StatView soft-

ware program (Abacus Concepts, Inc., Berkeley,

CA).

Fig. 1. Formation of PEG-podophyllotoxin ester (3). DIPC53. Results and discussiondiisopropylcarbodiimide, DMAP5N,N-dimethylaminopyridine.

The solubilization of podo was first accomplished

by reaction of the 4-OH group with PEG dicarbox- 4-amino acid esters was synthesized and conjugated

ylic acid (2) in the same fashion as reported for to PEG. In the case of paclitaxel (a 28 alcohol),

paclitaxel [8]. The relatively unhindered 28 OH at the simple amino acid ester derivatives which are gener-

4-position of 1 (ring C) reacted rapidly with diiso- ally unstable were avoided by condensing PEG

propylcarbodiimide (DIPC) and 2 to produce the amino acid derivatives directly with the 29-OH group

diester 3 in high yield (Fig. 1). Formation of a [8]. However, in the case of1 conjugation with PEG

tripartate prodrug by introduction of a glycine spacer amino acids led to incomplete reaction and impure

in the paclitaxel and camptothecin PEG transport products. Ultimately the most efficient route to the

forms has been shown to enhance efficacy in several final PEG conjugates was similar to that used foranimal models, including the P388/ 0 mouse model camptothecin (a 38 alcohol) and consisted of esterifi-

[9,10]. A delivery strategy that utilized amino acid cation of1with t-Boc amino acids and carbodiimide

spacer groups also seemed warranted for 1. These reagents. Removal of the t-Boc moiety using mild

was initially accomplished with glycine which acid treatment (1N HCl / HOAc) followed by neutral-

showed greater efficacy (%ILS) in the P388/ 0 ization gave amino acid esters (4af) which were

leukemia screen, however its activity against solid conjugated directly with 2 to give the desired PEG

tumors was not impressive. In order to investigate derivatives (5af) in good yield. Further purification

whether other amino acid spacers would produce was best effected at this stage (Fig. 2). In addition,

better results against solid tumors, a series of podo the PEG carbonate 7 was synthesized from PEG


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Fig. 2. Synthesis of PEG-podophyllotoxin amino acid esters (5af). DIPC5diisopropylcarbodiimide, DMAP5N,N-dimethylaminopyridine.

p-nitrophenylcarbonate (PEG-PNP, 6), and the PEG and in vitro data are presented in Table 1, and

carbamate 9 from PEG isocyanate 8 (Fig. 3) to illustrate the powerful cytotoxic properties possessed

determine their utility as transport forms. The carba- by 1 and its derivatives. For the various a-aminomate9 was, as expected, distinctly different from the acid spacer groups employed, the IC and hydrol-

50

other members of the series as demonstrated by a ysis data appeared to parallel each other. In addition,

longer t . Interestingly, a great deal of in vivo it was observed that the use of different amino acid1 / 2differentiation was later observed which we have spacers resulted in changes to the in vitro liberation

attempted to correlate with physical measurements. of 1. Similar observations have been made with

All of the PEG podophyllotoxin conjugates syn- PEG-paclitaxel and PEG-camptothecin conjugates

thesized exhibited aqueous solubilities similar to [9,10]. However, in contrast to PEG-paclitaxel [10]

unfunctionalized PEG of molecular weight 40 kDa, and PEG-camptothecin [11] transport forms, which

and were approximately 120150 mg/ ml. Kinetic demonstrate a correlation between slower hydrolysis


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Fig. 3. Preparation of PEG-podophyllotoxin carbonate (7) and PEG-podophyllotoxin carbamate (9). DMAP5N,N-dimethylaminopyridine.

Table 1a

In vitro measurements of PEG-podophyllotoxin derivatives

Compound IC t (h)50 1 / 2

(P388, nm) Buffer Rat Human

(pH57.4) plasma plasma

Podophyllotoxin (1) 4 PEG-Podo (3) 8 5 1.5 1

PEG-gly-Podo (5a) 13 7 5 1

PEG-ala-Podo (5b) 21 21 8 2

PEG-met-Podo (5c) 37 19 7 2

PEG-leu-Podo (5d) 28 19 7 3

PEG-pro-Podo (5e) 52 22 8 2

PEG-phe-Podo (5f) 24 24 8 3

PEG-carbonate-Podo (7) 10 3 2 1

PEG-carbamate-Podo (9) 205 65 9 18

aAll experiments were done in duplicate. Standard deviation of measurements5610%.


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and improved therapeutic index against solid tumors, using suspensions or dispersions of native drug

the slow sustained release of podophyllotoxin ap- where nearly comparable doses (18 mg/ kg) results

peared to reduced efficacy and increase toxicity in in a T/ C of 1.24 [7].

tumor bearing mice. From a small in vitro cytotoxicity panel (ie. colon,

The simple a-alkoxy ester derivative 3 demon- breast, lung, ovarian), 1 appeared particularly activestrated a slightly lower T/C than native podo at (IC |24 nM) against human small cell lung50equivalent doses (Table 2). We therefore prepared carcinoma (A549). Thus we chose a human lung

PEG-glycine-podo (5a) which manifested a substan- A549 carcinoma xenografted onto nude mice as a

tially greater T/C than the simple transport form 3. predictive model which, it was hoped, would identify

Our initial in vivo studies employed an ascites P388/ the most effective polymeric transport form for

0 leukemic mouse model and allow the results shown delivery of 1 to solid tumors. We were especially

in Table 2 to be compared directly with previous interested in solid tumors since our lab [14] and

data [7]. Thus, at a total dose of 22 mg/kg a T /C others [15,16] have observed enhanced tumor ac-

value of 1.36 for native 1 was obtained (vs. 1.71 at cumulated of drugs following their conjugation to

27 mg/kg by Levy et al.), while transport form 3 had PEG. This observation presumably reflects the fact

a similar T/C (1.23). However, derivative5a

at this that macromolecules of roughly 50 kDa whichlevel produced a significantly increased T / C of 2.30. circulate for extended periods, show substantial

An increase in total dose to 30 mg/ kg did not change tumor accumulation [17]. Indeed, PEG molecules of

the T/C for 1 (1.40), but clearly produced toxicity 10,000 or greater molecular weight demonstrate a

for 3 (T/C50.93) and 5a (T/C50.70). This data significantly higher accumulation in tumors than

demonstrates that although solubilization of 1 (as within normal tissue, irrespective of the tumor site

transport form 5a) ultimately increases toxicity, this [18]. This is the end result of the combination of

modified form of podo can now be effectively increased tumor vascular permeability and insuffi-

employed at lower levels to achieve an increased life cient tissue drainage that results in what is termed

span (%ILS) as measured against the P388/ 0 murine the enhanced permeability and retention effect,

leukemia model. Such results are not obtainable by which is thought to be an universal solid tumor

Table 2

Activity of PEG-podophyllotoxin derivatives against P388/0 murine leukemia, in vivo*

cTest compound Total Mean time %T / C % ILS P values P values

adose to death vs. control vs. podophyllotox.

b(mg / kg) (days) [cures / group] at equiv. dose

Control 13.161.1 [0 / 20]

Podophyllotoxin (1) 22 17.861.3 [0 / 10] 136 36 P50.0027

30 18.461.8 [0 / 10] 140 40 P50.0008

PEG-Podo (3) 8 20.761.7 [0 / 10] 158 58 P,0.0001 NA

16 22.062.3 [0 / 10] 168 68 P,0.0001 NA

22 16.165.9 [0 / 10] 123 23 P50.0521 P50.3373

30 12.267.2 [0 / 10] 93 27 P50.5570 P50.000645 5.960.9 [0 / 10] 45 255 P,0.0001 NA

PEG-gly-Podo (5a) 16 27.564.4 [0 / 10] 210 110 P,0.0001 NA

22 30.164.3 [0 / 10] 230 130 P,0.0001 P,0.0001

30 9.267.1 [1 / 10] 70 230 P50.0158 P,0.0001

*In vivo efficacy study of the water soluble podophyllotoxin derivatives using the P388/0 murine leukemia model. Derivatives were

given daily [intraperitoneal (i.p.)35], 24 h following an injection of P388/ 0 cells into abdominal cavity with survival monitored for 40 days.

Animals surviving at 40 days were considered cures.a

Equivalent dose of podophyllotoxin (based on podo content).b

Kaplan-Meier estimates with survivors censored.c

Increased life span (%ILS) is (T/C21)3100.


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phenomenon for macromolecular drugs [17]. The tween the in vivo antitumor activity of the conjugate

multiple dose study demonstrated that a number of and its rate of release in rat plasma: the faster the

PEG-podo analogs were as effective as 1 in inhib- hydrolysis, the greater the inhibition on tumor

iting A549 tumor growth (Table 3 and Fig. 4). growth. The lack of increased efficacy observed with

However, none of the analogs was significantly the longer, circulating PEG-podo conjugates, asbetter than the native form when administered on an inferred from the in vitrot data, was in contrast to

1 / 2

equimolar basis. our findings with PEG-paclitaxel and PEG-camp-

To determine the level of toxicity of those PEG- tothecin [10,11]. One possibility was that the ac-

podo conjugates that exhibited antitumor activity cumulated podo conjugate was being converted via

equal to 1, a single high dose of the compounds was C-2 epimerization and released as its inactive form,

administered. The literature reports that the LD for picropodophyllotoxin (1a). It has been well docu-501is 35 mg/ kg in normal mice [2]. Therefore a single mented that under weak alkaline conditions podo

dose of 30 mg/kg (|400 ml / mouse) was chosen in readily converts from its trans lactone ring configura-

this study in an attempt to observe any differences tion to its thermodynamically more stable cis epimer,

between native podo and its analogs in relation to picropodophyllotoxin [19]. Unfortunately, picropodo

both efficacy and toxicity (Table 4). Within the first has very little or no biological activity [19,20]. Inweek after treatment, a majority of animals in the fact, it has been suggested that cells may themselves

PEG-podo (3), PEG-gly-podo (5a) and PEG-ala- utilize this conversion when exposed to podo [19].

podo (5b) groups appeared in poor condition (i.e. So, although the biodistribution of stable macro-

thin, lethargic, hypothermic and/ or hunched stature), molecular carriers has been shown to increase the

which ultimately produced test article related deaths. therapeutic efficacy of their cargo by means of

The PEG-podo carbonate (7) and 1 expressed equiv- favorable passive solid tumor accumulation [17,21],

alent antitumor effects, which resulted in tumor the conversion of podo to an inactive form may

growth inhibition. It still remains to be determined negate this fortuitous physical mechanism and reduce

whether greater efficacy could be achieved with the amount of active podo available when trans-

higher doses of 7. ported via conjugation.

There appeared to be an overall correlation be- In order to investigate the possibility of PEG-podo

Table 3a

Multiple dose activity of PEG-podophyllotoxin derivatives against solid lung (A549) tumor xenografts, in vivo

dTest compound % D Basal % D Basal %T/ C

b ctumor volume body weight

Control 8866150 26

Podophyllotoxin (1) 174688* 19 20

PEG-Podo (3) 249648* 27 22

PEG-gly-Podo (5a) 374668* 23 40

PEG-ala-Podo (5b) 5236154 28 75

PEG-met-Podo (5c) 8126201 23 78

PEG-leu-Podo (5d) 6306162 27 68

PEG-pro-Podo (5e) 6856248 22 78PEG-phe-Podo (5f) 394682* 15* 29

PEG-carbonate-Podo (7) 160687* 28 18

PEG-carbamate-Podo (9) 8346125 28 95

aIn vivo efficacy study of the water soluble podophyllotoxin derivatives using a solid A549 tumor xenograft model. Subcutaneous

3injections of A549 cells were allowed to reach an average tumor volume of 50 mm prior to treatments (day 1). Test compounds were dosed

(15 mg/kg/dose) intravenously on day 1, 8 and 19. Changes in tumor volume and body weight were evaluated on day 43.b

Percent individual tumor volume change (mean6sem) from initial (day 1).c

Percent group mean body weight change (mean) from initial (day 1).d 3

Treatment and control groups were measured when the control groups median tumor volume reached approximately 600800 mm .*

Significant compared to control (P,0.05).


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Fig. 4. Growth curve of A549 tumor xenografts treated with podophyllotoxin derivatives. All compounds were given i.v. () on day 1, 83

and 19 (15 mg/kg/dose) in established (|50 mm ) subcutaneous tumors. PEG-podophyllotoxin dosages were based on podophyllotoxin

equivalents (absolute amount of podophyllotoxin given).

Table 4a

Single dose activity of PEG-podophyllotoxin derivatives against solid lung (A549) tumor xenografts, in vivo

Test compound % D Basal % D Basal % Survivalb c

tumor volume body weight

Control 175650 4 100 (6 / 6)

Podophyllotoxin (1) 57622* 4 100 (6 / 6)

PEG-Podo (3) 62623* 8 83 (5 / 6)

PEG-gly-Podo (5a) 6360 12 17 (1 / 6)*

PEG-ala-Podo (5b) 2460 12 17 (1 / 6)*

PEG-carbonate-Podo (7) 52620* 0 100 (6 / 6)a

In vivo efficacy study of the water soluble podophyllotoxin derivatives using a solid A549 tumor xenograft model. Subcutaneous3

injections of A549 cells were allowed to reach an average tumor volume of 250 mm prior to treatments (day 1). Test compounds were

dosed once (30 mg/kg) intravenously on day 1. Changes in tumor volume, body weight and survival were evaluated on day 23.b

Percent individual tumor volume change (mean6sem) from initial (day 1).c

Percent group mean body weight change (mean) from initial (day 1).*

Significant compared to control (P,0.05).


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1Fig. 5. 270 MHz H NMR spectra of PEG-podophyllotoxin carbamate (9a, A), PEG-picropodophyllotoxin carbamate (11, B), and 9a in rat

plasma after 6 h (C). Chemical shift assignments have been made relative to internal standard tetramethylsilane.


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conjugates undergoing epimerization in vivo, the simply, perhaps insufficient amounts of podo are

relatively stable PEG-podo carbamate (9a, MW55 liberated to attain therapeutic responses. Conceiv-

kDa, t hydrolysis: in rat plasma58.6 h, PBS pH ably, the use of congeners in which epimerization is1 / 27.4.24 h) and PEG-picropodo carbamate (11, MW5 blocked, for example 2-aza-podo (as reviewed by

5 kDa, t hydrolysis in rat plasma54.4 h, PBS pH [23]), is another means which could be employed to1 / 27.4.24 h) were synthesized as model compounds for assess both the role of epimerization and the impact

kinetic studies in rat plasma. Compound 1a clearly of slow release in vivo.

showed different chemical shifts for the aromatic The toxicity of the slower hydrolyzing conjugates

protons in rings B and E and the aliphatic protons in could simply result from the bodys prolonged

rings C and D compared to 1 [22]. Similar differ- exposure to released podo within the circulation. In1

ences were observed in the H NMR spectrum of9a addition, PEG may be depositing greater amounts of1

and 11 (Fig. 5A and B) and therefore H NMR active (non-epimerized) podo in sensitive locations

spectroscopy was suitable for the epimerization that results in increased toxicity compared to the

investigation. Percent epimerization of9a was calcu- non-conjugated drug. While, we are not aware of any

lated from the ratio of integration of singlets at 6.87 thorough quantitative investigation of native podos

ppm (H-5, 9a

) and 6.82 ppm (H-5, 11

): These biodistribution following intravenous administration,signals were found to be easy to differentiate during one would expect that while conjugated to PEG, its

epimerization without any interference from other systemic distribution would be governed by the large1

protons (Fig. 5C). Analysis of the H NMR spectra molecular weight PEG conjugate. Large molecular

of recovered plasma treated samples (Table 5) weight PEG is known to have low urinary clearance

indicated that epimerization occurred only to a small which increases its circulatory retention time, while

extent in rat plasma. The half-life of hydrolysis concurrently allowing it to accumulate in tumor,

(release) of9a was faster (8.6 h) than epimerization muscle, skin, bone and liver [24]. Studies investigat-

(23 h) which implies that the drug released in plasma ing the biodistribution of free podo and its PEG

is mostly in the active podo form. Thus, if the in conjugate would be necessary for this compounds

vitro data accurately reflects the in vivo environment, development.

the efficacy differences seen between slow and fast

hydrolyzing PEG esters of podo may not be due toepimerization of the conjugate, but rather may be 4. Conclusion

related to metabolic and biodistributive factors asso-

ciated both with free and conjugated podo. Quite The solubilization and predictable release of

podophyllotoxin from a PEG carrier was achieved in

this study with ester and carbonate linkages resultingTable 5

in some derivatives demonstrating, at a minimum,Comparison of epimerization (%) and hydrolysis (%) of mPEG-equivalency with podophyllotoxin on an equal molarpodophyllotoxin carbamate (9a) in rat plasma at 378Cbasis. Some analogs of the polymer conjugate

Time Epimerization Hydrolysisa b showed significantly better activity in a murine(h) (%) (%)

leukemia model compared to native podophyllotoxin0 1.8 0

suspended in an intralipid emulsion. Additionally,1 4.9 8when tested intravenously against a solid lung tumor2 7.5 9

4 13.6 21 (A549) model, certain conjugated analogs were6 18.1 37 equivalent to the podophyllotoxin/ intralipid emul-

c8 ND 47

sion, while the slow sustained release of othersa

% Epimerization of compound 9a was calculated from the appeared to cause greater toxicity. There appeared toratio of integration of peaks at 6.87 ppm and 6.82 ppm (H-5, 9a be an overall correlation between the in vivo activity

1vs. 11) in the H NMR spectrum.

b against solid tumors of the conjugate and its rate of% Hydrolysis (rate of disappearance) was calculated from the

hydrolysis in vitro, with those showing faster releaseHPLC peak area of 9a using a reverse phase C8 column.c

ND5not determined. possessing greater antitumor activity. Further studies


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glycol): a water soluble prodrug, Anti-cancer Drug Des. 13may be warranted to assess the PEG-conjugates(1998) 387395.pharmacokinetics and therapeutic indices in treating

[11] C.D. Conover, A. Pendri, C. Lee, C.W. Gilbert, K.L. Shum,leukemia.

R.B. Greenwald, Camptothecin delivery systems: the anti-

tumor activity of a camptothecin-20-polyethylene glycol

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