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was ltered through Celite and the solvent removed dissolved in 10 ml of anhydrous methylene chloride.
from the ltrate 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 solution
13
(91% yield) of product. C NMR d 21.39, 22.30, and the mixture reuxed overnight, followed by
24.47, 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) of
13
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.
R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294 285
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) Briey, for IC determination cells were seeded into
50
3
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 humidied
and 150 ml (0.87 mmol) of DIPEA under nitrogen incubator with 5% CO for 3 days. Cell growth was
2
atmosphere and the mixture was reuxed 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 reuxed The IC values for each compound were determined
50
for 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 modied 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 humidied 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 were
2
dissolved 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 ank
6
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
ltered through a 0.45 mm lter 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 2
ml) 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 rst study assessed the efcacy of all the PEG-podo
3
integration of peaks at 6.87 ppm and 6.82 ppm (H-5, derivatives against tumors of approximately 50 mm
1
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 mice
2.2.1. Materials (eight / group). The second study evaluated only
All PEG conjugated podo compounds were dis- those compounds that showed signicant antitumor
solved (|100 mg/ ml) in sterile saline (0.9%) for activity as compared to the control group in the rst
injection prior to in vivo drug treatments and doses study. Compounds were given intravenously at 30
were 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 grew
50
concentration inhibiting 50% of cells) and in vivo over the course of the experiment would display
286 R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294
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 when
the control groups median tumor volume reached
3
approximately 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 signicant differences existed were determined
by least signicant differences techniques. Statistical
TM
analysis was conducted using the StatView soft-
ware program (Abacus Concepts, Inc., Berkeley,
CA).
Fig. 1. Formation of PEG-podophyllotoxin ester (3). DIPC5 3. Results and discussion
diisopropylcarbodiimide, DMAP5N,N-dimethylaminopyridine.
The solubilization of podo was rst 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 of 1 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 efcient route to the
forms has been shown to enhance efcacy in several nal PEG conjugates was similar to that used for
animal models, including the P388/ 0 mouse model camptothecin (a 38 alcohol) and consisted of esteri-
[9,10]. A delivery strategy that utilized amino acid cation of 1 with 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 efcacy (%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 purication
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
R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294 287
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-amino
mate 9 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 acid
1 / 2
differentiation 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
288 R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294
Fig. 3. Preparation of PEG-podophyllotoxin carbonate (7) and PEG-podophyllotoxin carbamate (9). DMAP5N,N-dimethylaminopyridine.
Table 1
a
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
a
All experiments were done in duplicate. Standard deviation of measurements5610%.
R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294 289
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 efcacy 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 active
strated a slightly lower T/ C than native podo at (IC |24 nM) against human small cell lung
50
equivalent 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 reects the fact
a similar T/ C (1.23). However, derivative 5a at this that macromolecules of roughly 50 kDa which
level produced a signicantly 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 signicantly 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
modied form of podo can now be effectively increased tumor vascular permeability and insuf-
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*
c
Test compound Total Mean time %T/ C % ILS P values P values
a
dose 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 P50.0006
45 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 efcacy 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.
290 R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294
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 efcacy observed with
However, none of the analogs was signicantly the longer, circulating PEG-podo conjugates, as
better than the native form when administered on an inferred from the in vitro t data, was in contrast to
1 / 2
equimolar basis. our ndings 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-
50
1 is 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 congura-
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 efcacy and toxicity (Table 4). Within the rst has very little or no biological activity [19,20]. In
week 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 efcacy 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 efcacy 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 3
a
Multiple dose activity of PEG-podophyllotoxin derivatives against solid lung (A549) tumor xenografts, in vivo
d
Test compound % D Basal % D Basal %T/ C
b c
tumor 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 78
PEG-phe-Podo (5f) 394682* 15* 29
PEG-carbonate-Podo (7) 160687* 28 18
PEG-carbamate-Podo (9) 8346125 28 95
a
In vivo efcacy study of the water soluble podophyllotoxin derivatives using a solid A549 tumor xenograft model. Subcutaneous
3
injections 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 .
*
Signicant compared to control (P,0.05).
R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294 291
Fig. 4. Growth curve of A549 tumor xenografts treated with podophyllotoxin derivatives. All compounds were given i.v. () on day 1, 8
3
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 4
a
Single dose activity of PEG-podophyllotoxin derivatives against solid lung (A549) tumor xenografts, in vivo
Test compound % D Basal % D Basal % Survival
b 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 efcacy study of the water soluble podophyllotoxin derivatives using a solid A549 tumor xenograft model. Subcutaneous
3
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).
*
Signicant compared to control (P,0.05).
292 R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294
1
Fig. 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.
R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294 293
conjugates undergoing epimerization in vivo, the simply, perhaps insufcient 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 is
1 / 2
7.4.24 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 to
1 / 2
7.4.24 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. In
1
ences were observed in the H NMR spectrum of 9a addition, PEG may be depositing greater amounts of
1
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 of 9a 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 large
1
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) of 9a 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 reects the in vivo environment, development.
the efcacy differences seen between slow and fast
hydrolyzing PEG esters of podo may not be due to
epimerization 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 resulting
Table 5
in some derivatives demonstrating, at a minimum,
Comparison of epimerization (%) and hydrolysis (%) of mPEG-
equivalency with podophyllotoxin on an equal molar
podophyllotoxin carbamate (9a) in rat plasma at 378C
basis. Some analogs of the polymer conjugate
Time Epimerization Hydrolysis
a b
showed signicantly better activity in a murine
(h) (%) (%)
leukemia model compared to native podophyllotoxin
0 1.8 0
suspended in an intralipid emulsion. Additionally,
1 4.9 8
when tested intravenously against a solid lung tumor
2 7.5 9
4 13.6 21
(A549) model, certain conjugated analogs were
6 18.1 37
equivalent to the podophyllotoxin/ intralipid emul-
c
8 ND 47
sion, while the slow sustained release of others
a
% Epimerization of compound 9a was calculated from the
appeared to cause greater toxicity. There appeared to
ratio of integration of peaks at 6.87 ppm and 6.82 ppm (H-5, 9a
be an overall correlation between the in vivo activity
1
vs. 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 release
HPLC peak area of 9a using a reverse phase C8 column.
c
ND5not determined. possessing greater antitumor activity. Further studies
294 R.B. Greenwald et al. / Journal of Controlled Release 61 (1999) 281294
glycol): a water soluble prodrug, Anti-cancer Drug Des. 13
may 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
ester transport form, Anticancer Res. 117 (1997) 33613368.
[12] W.J. Gensler, C.D. Gastonis, The podophyllotoxin-picro- Acknowledgements
podophyllin equilibrium, J. Org. Chem. 31 (1966) 3224
3227.
We would like to thank Dr. Chyi Lee, Dr. Prakash
[13] T. Corbett, F. Valeriote, L. Polin et al., Discovery of solid
Sai, and Ms. Michelle Boro for their assistance in
tumor active agents using a soft-agar-colony formation disk-
designing and performing analytical measurements,
diffusion-assay, in: F. Valeriote, T. Corbett, L. Baker (Eds.),
and to Ms. Jenny Hsu for assisting in the in vitro and Cytotoxic Anticancer Drugs: Models And Concepts For
Drug Discovery And Development, Kluwer Academic, Bos-
in vivo assays. We would especially like to express
ton, Dordrecht, London, 1992, pp. 5987.
our gratitude to Dr. Jeff McGuire, Vice President of
[14] C.D. Conover, R.B. Greenwald, A. Pendri, C.W. Gilbert,
Research and Development, for his insight and
K.L. Shum, Camptothecin delivery systems: enhanced efca-
guidance on this project.
cy and tumor accumulation of camptothecin following its
conjugation to polyethylene glycol via a glycine linker,
Cancer. Chemother. Pharmacol. 42 (1998) 407414.
[15] R. Pedley, J. Boden, R. Boden, R. Begent, A. Turner, A.
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