Stable supersaturated aqueous solutions of silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin via chemical conversion in the presence of a chemically modified β-cyclodextrin

Article abstract of DOI:10.1023/A:1019862629357

Purpose. A method for obtaining clear supersaturated aqueous solutions for parenteral administration of the poorly soluble experimental anti-cancer drug silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin (DB-67) has been developed. Methods. Equilibrium solubilities of DB-67 were determined in various solvents and pH values, and in the presence of chemically modified water-soluble β-cyclodextrins. The stoichiometry and binding constants for complexes of the lactone form of DB-67 and its ring-opened carboxylate with sulfobutyl ether and 2-hydroxypropyl substituted β-cyclodextrins (SBE-CD and HP-CD) were obtained by solubility and circular dichroism spectroscopy, respectively. Kinetics for the reversible ring-opening of DB-67 in aqueous solution and for lactone precipitation were determined by HPLC with UV detection. Results. Solubilities of DB-67 lactone in various injectable solvent systems were found to be at least one order of magnitude below the target concentration (2 mg/ml). DB-67 forms inclusion complexes with SBE-CD and HP-CD but the solubilization attainable is substantially less than the target concentration. Slow addition of DB-67/DMSO into 22.2% (w/v) SBE-CD failed to yield stable supersaturated solutions due to precipitation. Stable supersatured solutions were obtained, however, by mixing a concentrated alkaline aqueous solution of DB-67 carboxylate with an acidified 22.2% (w/v) SBE-CD solution. Ring-closure yielded supersaturated solutions that could be lyophilized and reconstituted to clear, stable, supersaturated solutions. Conclusions. The method developed provides an alternative to colloidal dispersions (e.g., liposomal suspensions, emulsions, etc.) for parenteral administration of lipophilic camptothecin analogs.

Full text of DOI:10.1023/A:1019862629357

Pharmaceutical Research, Vol. 19, No. 8, August 2002 (© 2002)
Research Paper
provide the desired concentration for some very poorly wa-
ter-soluble drugs. In such instances, supersaturated solutions
may be one of the few alternatives, providing that drug pre-
cipitation can be prevented (3).
Stable Supersaturated Aqueous
Solutions of Silatecan
7
-t-Butyldimethylsilyl-10-
Silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin
Hydroxycamptothecin via Chemical
Conversion in the Presence of a
Chemically Modified ␤-Cyclodextrin
(DB-67) is an experimental drug in the early stages of devel-
opment as a cancer chemotherapeutic by the National Cancer
Institute. DB-67, one of a class of A and B ring modified
camptothecin analogs, displays superior binding to cellular
and liposomal membranes and enhanced drug stability in the
presence of human serum albumin when compared with clini-
cally relevant more hydrophilic camptothecin analogs be-
cause of its increased lipophilicity and dual 7-alkylsilyl and
1
1,2
Tian-xiang Xiang and Bradley D. Anderson
10-hydroxy substitution (4). DB-67 has comparable potency
in in vitro cytotoxicity assays to other FDA approved camp-
tothecin analogs (e.g., Camptosar and Hycamtin). As shown
Received April 22, 2002; accepted May 1, 2002
Purpose. A method for obtaining clear supersaturated aqueous solu- in Fig. 1, DB-67 can exist in an E-ring closed lactone (I) and
tions for parenteral administration of the poorly soluble experimental an E-ring opened form (II) depending on solution pH and
anti-cancer drug silatecan 7-t-butyldimethylsilyl-10-hydroxycampto- solvent properties. Structure-activity studies have shown that
thecin (DB-67) has been developed.
Methods. Equilibrium solubilities of DB-67 were determined in vari-
ous solvents and pH values, and in the presence of chemically modi-
nately, as reported in this study, the lactone form of DB-67
fied water-soluble ␤-cyclodextrins. The stoichiometry and binding
has very poor solubility in water. In the past, poor solubility
constants for complexes of the lactone form of DB-67 and its ring-
opened carboxylate with sulfobutyl ether and 2-hydroxypropyl sub-
stituted ␤-cyclodextrins (SBE-CD and HP-CD) were obtained by
solubility and circular dichroism spectroscopy, respectively. Kinetics
successful inhibition of DNA topoisomerase I by camptothe-
cin analogs requires an intact lactone E-ring (5–8). Unfortu-
has prevented the extensive use of highly lipophilic campto-
thecin analogs in the clinical treatment of cancer. Indeed,
while a number of water soluble camptothecin derivatives
for the reversible ring-opening of DB-67 in aqueous solution and for including Camptosar, topotecan, 9-amino-camptothecin, 7-(4-
lactone precipitation were determined by HPLC with UV detection. methylpiperazino-methylene)-10,11-methylenedioxy campto-
Results. Solubilities of DB-67 lactone in various injectable solvent thecin, 10,11-methylenedioxy-camptothecin and 10,11-ethyl-
systems were found to be at least one order of magnitude below the
enedioxy-camptothecin have either been on the market, stud-
target concentration (2 mg/ml). DB-67 forms inclusion complexes
ied preclinically, or used in clinical trials to treat certain types
with SBE-CD and HP-CD but the solubilization attainable is sub-
of human cancer, few clinical studies have been conducted in
stantially less than the target concentration. Slow addition of DB-67/
human patients involving poorly water soluble, highly lipo-
DMSO into 22.2% (w/v) SBE-CD failed to yield stable supersatu-
philic camptothecin analogs (e.g., camptothecin, 10-hydroxy-
rated solutions due to precipitation. Stable supersatured solutions
were obtained, however, by mixing a concentrated alkaline aqueous
solution of DB-67 carboxylate with an acidified 22.2% (w/v) SBE-CD
7-ethyl camptothecin, DB-67, etc.) (9–13). Thus, a viable for-
mulation of DB-67 for intravenous delivery should exhibit
solution. Ring-closure yielded supersaturated solutions that could be sufficient physical and chemical stability to maintain the de-
lyophilized and reconstituted to clear, stable, supersaturated solu- sired concentration (2 mg/mL) exclusively in the lactone
tions.
form.
This paper describes the development of a simple strat-
egy for preparing a stable supersaturated parenteral formu-
lation of DB-67. The solubility of DB-67 in various solvent
Conclusions. The method developed provides an alternative to col-
loidal dispersions (e.g., liposomal suspensions, emulsions, etc.) for
parenteral administration of lipophilic camptothecin analogs.
KEY WORDS: cyclodextrin complexes; camptothecin analogs; par- systems (e.g., aqueous solutions varying in pH, cosolvents,
enteral formulation; lactone hydrolysis; solubilization; parenteral for-
mulation.
emulsions, and liposomes), complexation with cyclodextrin
derivatives (i.e., SBE-CD and HP-CD), hydrolysis kinetics at
different pH values and in the presence and absence of cy-
clodextrins, and physical stability with respect to drug pre-
INTRODUCTION
cipitation have been explored. Based on these experiments, a
The low aqueous solubility and physical and chemical
novel formulation involving chemical conversion in the pres-
instability of many new drug candidates limits the options
ence of the chemically modified ␤-cyclodextrin, SBE-CD, has
available for their formulation in parenteral dosage forms.
While various solubilization approaches are available includ-
been developed.
ing salt formation, cosolvent solubilization, complexation,
and the formation of mixed micelles, liposomes, emulsions,
and micro/nanoparticles (1,2), these approaches often fail to
MATERIALS AND METHODS
Chemicals
1
DB-67 (NSC 708298) and Diluent-12 (NSC 614387; com-
posed of 50% Cremophor and 50% ethanol) were supplied by
the National Cancer Institute (Bethesda, MD, USA). ␤-cy-
Division of Pharmaceutical Sciences, College of Pharmacy, Univer-
sity of Kentucky, 327G Pharmacy Building, Lexington, Kentucky
40536-0082.
2
To whom correspondence should be addressed. (e-mail: bande2@ clodextrin sulfobutyl ether, sodium salt, having an average
uky.edu) degree of substitution of 7 sulfobutyl ether residues per cy-
1215
0724-8741/02/0800-1215/0 © 2002 Plenum Publishing Corporation

1
216
Xiang and Anderson
terol, and DMPG (70:25:5 mole) in ethanol, evaporating un-
der a stream of N2 gas, vacuum-drying at 45°C overnight,
rehydrating with a 10% (w/v) solution of sucrose in 30 mM
acetate buffer at pH 5.1, and extruding the rehydrated sus-
pensions through a 0.1 ␮m polycarbonate filter 15 times by a
LiposoFast-Pneumatic Extruder (Avestin, Inc., Ottawa). The
emulsions were prepared by weighing out egg PC, glycerin,
and Tween 80, dispersing the weighed compounds with water,
adding slowly a weighed amount of soybean oil into the aque-
ous solution with vigorous stirring, sonicating the mixture for
about half an hour, and homogenizing (EmulsiFlex-C5, Aves-
tin, Inc., Ottawa) the mixture at 10,000 psi for 15 cycles.
Circular Dichroism Measurements
Circular dichroism was used to examine inclusion com-
plex formation between the DB-67 carboxylate and cyclodex-
trins (i.e., SBE-CD and HP-CD). The DB-67 concentration
−
4
was set at 2.2 × 10 M and solution pH was maintained at
0.6 with 0.1 M Na CO . Circular dichroism measurements
Fig. 1. The structures of the lactone ring intact DB-67 (species I) and
its E-ring opened counterpart (species II) and the proposed reaction
sequences for the E-ring opening hydrolysis and acid-base dissocia-
tions of DB-67.
1
2
3
were conducted at ambient temperature (24°C) on a Jasco
model J-710 Spectropolarimeter (Jasco Spectroscopic, Ha-
chioji City, Japan) with computer-controlled data acquisition
and analysis. The light source was a Xenon-arc lamp. The
samples were transferred to a 0.5-cm path-length quartz cu-
vette aligned in a holder within a chamber under a constant
flow of nitrogen gas. The scan speed, step resolution, re-
sponse time, and accumulation were set at 50 nm/min, 1 nm,
clodextrin molecule, (SBE-CD, Captisol®) was a gift from
CyDex, Inc. (Overland Park, KS, USA). 2-hydroxypropyl-␤-
cyclodextrin (HP-CD) with an average degree of substitution
of 7.5 (ave. MW ס 1540) was a gift from Pharmatec, Inc.
PEG-400 and propylene glycol (99.5%) were obtained from
Aldrich (Milwaukee, WI, USA). Camptothecin (CPT), di-
methylsulfoxide (DMSO, ACS reagent), sucrose and choles-
terol (+99%) were obtained from Sigma Chemical Inc. (St.
Louis, MO, USA). 1,2-dimyristoyl-sn-glycerol-3-phospho-
3
seconds and 50, respectively.
Analytical Methods
Concentrations of DB-67 or camptothecin in various
choline (DMPC) and 1,2-dimyristoyl-sn-glycerol-3-[phospho- samples were measured by HPLC in an isocratic mode. The
rac-(1-glycerol)] (DMPG), sodium salt, were purchased from HPLC system consisted of a Discovery C18 (5 ␮m) column (15
Avanti Polar Lipids (Pelham, AL, USA) and stored in a cm × 4.6 mm) and guard column (2 cm × 4.6 mm) (Supelco,
freezer upon arrival. All other compounds were reagent or Belleforte, PA, USA), a Beckman 110B solvent delivery
HPLC grade from commercial sources and were used without module (Beckman Instruments, San Ramon, CA, USA), a
further purification. Aqueous solutions were prepared using Rheodyne M7125 injector with a 20 ␮l injection loop (Rainin
deionized water.
Instrument, Woburn, MA, USA), a Waters M2487 Dual ␭
Absorbance detector (Water Associates, Milford, MA, USA)
set at 254 nm, an HP 3392A integrator (Hewlett-Packard,
Avondale, PA, USA), and mobile phases containing 25% and
Solubility Determinations
Solubilities of DB-67 or camptothecin in a given solvent
system (e.g., cosolvents, emulsions, and liposomes) were de-
termined by adding an amount of drug well in excess of its
estimated solubility to 1–2 mL of the solvent of interest in a
41% (v/v) acetonitrile in 2% (w/v) triethylamine acetate for
camptothecin and DB-67, respectively. The retention vol-
umes for the lactone and ring-opened forms of DB-67 were
approximately 4 and 10 mL, respectively. External standards
prepared with 41% (v/v) acetonitrile/59% 2 mM HCl in water
and 2 mM NaOH in water were used for the lactone and
ring-opened species, respectively. The carry-over was less
than 0.02% and the response factors (peak area/
concentration) for both species were constant below 0.03 mg/
mL. If necessary, the samples to be assayed were diluted with
methanol before the HPLC run.
4
-mL glass vial. Buffers employed for pH-solubility studies
were 30 mM acetate (pH 5.2), 30 mM phosphate (pH 7.4), 23
mM carbonate (pH 9.5), and 500 mM carbonate (pH 9.8 &
1
0.2). Inclusion complex formation between the lactone of
DB-67 and SBE-CD or HP-CD was determined by measuring
DB-67 solubility at varying concentrations of cyclodextrin at
a pH Յ4.6 maintained with a 30 mM citric acid buffer. The
capped vials were rotated in a VWR2010 incubator (VWR
Scientific Inc., San Francisco, CA, USA) set at 25°C for a
period of 3 to 14 days. The samples were then filtered (0.45
Forward and Reverse Hydrolysis Reactions
␮
m, 4 mm Gelman Acrodisc, PN 4473 or 1.2 ␮m, 25 mm
The kinetics for hydrolysis of the lactone and for the ring
Gelman Acrodisc, PN 4190 for liposomal or emulsion closure reaction were investigated in aqueous solutions at pH
samples) and the first 6 to 8 drops were discarded while the 7.4 and 4.0, respectively, maintained at 25 ± 0.1°C in a circu-
remaining 2 to 4 drops were collected in a 4-mL glass vial, lating water bath (Haake A81, Berlin, Germany). The reac-
weighed, diluted with methanol, and analyzed by HPLC. Li- tions were initiated by adding 0.1 mL of reactant (DB-67
posome samples were prepared by dissolving DMPC, choles- lactone in DMSO or DB-67 carboxylate in 2 mM NaOH) to

1
217
5
mL of a 30 mM phosphate (pH 7.41, 0.1 ionic strength) or 12, was determined in triplicate after the samples were ro-
acetate (pH 4.04, 0.1 ionic strength) buffer to obtain an initial tated for 3 and 14 days in an incubator controlled at 25°C. A
−
6
reactant concentration of 2–4 × 10 M. After the addition t test analysis indicated no significant difference (p < 0.025)
and at different time intervals, aliquots of the reactant solu- between apparent solubilities at 3 and 14 days. Thus, unless
tions were withdrawn and immediately assayed by HPLC. otherwise specified, the solubility experiments were con-
The concentrations of both the E-ring opened (II; cf., Fig. 1) ducted with an equilibration time of 3 days. The solubility of
and lactone forms of DB-67 (I) were monitored.
DB-67 in various solvent systems is reported in Table I. The
intrinsic solubility of DB-67 in an aqueous solution at pH 5.2,
wherein DB-67 exists predominantly as a lactone, is 0.11 ␮g/
mL, well below the target concentration of 2 mg/mL needed
to afford an appropriate administration time and volume.
Various solubilization approaches such as cosolvents (e.g.,
mixtures of Diluent-12, PEG-400 or PG with water), com-
plexation with water-soluble chemically modified cyclodextrins
Precipitation Studies
Stability of supersaturated DB-67 solutions with respect
to drug precipitation was monitored by measuring changes of
DB-67 concentration in the supernatant after filtration. Ini-
tially, concentrated stock solutions of DB-67 lactone (20–25
mg/ml) in DMSO were added slowly (0.1 mL/min) to a con-
tinuously stirred solution of 22.2% (w/v) SBE-CD/water to a
final concentration of either 1 or 2 mg/mL. Subsequently, a
concentrated solution of DB-67 carboxylate (20 mg/mL) in an
aqueous solution containing a molar excess of NaOH was
filtered through a 0.2 ␮m filter (13 mm Gelman Acrodic, PN
(
e.g., SBE-CD and HP-CD), emulsions (95% Intralipid, 4%
Tween 80, 1% eggPC), and liposomes (DMPC:Chol:DMPG,
0:25:5 (mole)) were explored to find an optimal solubiliza-
7
tion method for the formulation. These vehicles are com-
monly considered for solubilizing highly lipophilic drug can-
didates and, in some cases (e.g., cyclodextrins, emulsions, and
liposomes), it was anticipated that they might stabilize the
lactone form of DB-67. As noted in Table I, however, these
solvent systems were ineffective in achieving the desired con-
centration.
4
423T) and injected slowly (0.11 mL/min) into 22.2% (w/v)
SBE-CD/water containing 2 mM acetic acid and a molar ex-
cess of HCl to neutralize the DB-67 carboxylate, induce ring
closure, and achieve a final solution containing 2 mg/mL DB-
6
7 lactone at a pH of approximately 4. After the dilutions, the
samples were stored at 25°C. At certain time intervals, ali-
quots were filtered through a 0.45 ␮m (4 mm Gelman Acro-
disc, PN4473) filter and after discarding the first 6 to 8 drops,
pH-Solubility Profiles for DB-67
Shown in Fig. 2 is the apparent solubility of DB-67 in
the next 2 to 3 drops were collected, weighed, and diluted aqueous solutions at different pH values. Based on rate con-
with methanol for subsequent HPLC assay.
stants reported previously (14), equilibration times of 3 days
were more than sufficient to achieve equilibrium with respect
to the ring opening-closing reactions of camptothecin ana-
logues. In accordance with the fact that the E-ring opened
form of DB-67 (II) has two ionizable groups (10-OH and the
Lyophilization
Solution formulations (3 mL) in glass vials were frozen in
liquid nitrogen for 10 min, transferred to a freeze-dryer
–
COOH) in the pH range of 4 to 10, the reaction sequences
(FDC206, Savant Instruments Inc.) and lyophilized at <−50°C
described in Fig. 1 must be considered in interpreting the
pH-solubility profile. The overall solubility (S) for DB-67 can
then be written as:
for one day. Secondary drying was performed for 4 h at room
temperature.
−
−
2−
−
RESULTS AND DISCUSSION
S = I + II + II + ͓I + II + IIЈ
(1)
͓
͔
͓
͔
͓
͔
͔
͓
͔
͓
͔
or
Solubilization of DB-67 in Various Solvent Systems
pH−pKa1
pH−pKЈa2
2pH−pKa1−pKa2
S = S ͑1 + K + K 10
+ 10
+ K 10
1
The effect of equilibration time on the solubility of DB-
7 in a 50% Cremophor/50% EtOH (v/v) cosolvent, Diluent-
o
1
1
6
+ KЈ 10pH−pKЈa2͒
(2)
1
a
Table I. Solubilities of DB-67 in Various Solvent Systems at 25°C
Solvent
S (mg/mL)
−
5
Water/30 mM acetate buffer at pH 5.23
Water/0.5 M carbonate buffer at pH 10.20
(1.11 ± 0.00) × 10
1
1.78 × 10
(4.9 ± 0.2) × 10
−
1
4
4
4
1
5
0% (w/v) HPCD/water
−
1
1
0% (w/v) SBE-CD/water/1mM HCl
0% PG, 10% (v/v) EtOH/water
0% (v/v) PEG-400/water
(2.09 ± 0.04) × 10
(1.73 ± 0.04) × 10
−
−
−
2
1
−
(3.3 ± 0.5) × 10
(2.0 ± 0.3) × 10
0% (v/v) PEG-400/water
1
Emulsion (20% soybean oil, 2% glycerin, 73% water,
% Tween 80, 1% eggPC)
Liposome (70:25:5 (mole) DMPC:Chol:DMPG)
(2.06 ± 0.05) × 10
4
−
3
(7.4 ± 0.3) × 10
(7.5 ± 0.2) × 10
2.00 × 10
0
5
5
0% Cremophor, 50% (v/v) EtOH (Diluent-12)
% Cremophor, 5% (v/v) in 5% (w/v) Dextrose/Water
−
1
a
Mean ± standard deviation from duplicate samples except in 10% (v/v) diluent-12 in 5% (w/v)
dextrose/water and water/0.5 M carbonate buffer at pH 10.20 (single).

1
218
Xiang and Anderson
a slope of approximately 2 near pH 10, due to ionization of
the carboxylic group in the E-ring opened species and the
hydroxyl group at position 10 in DB-67. At pH 10.20, the
solubility reaches approximately 18 mg/mL, well above the
target DB-67 concentration of 2 mg/mL, but the E-ring
opened species of a camptothecin analog is therapeutically
inactive, has a significantly shorter plasma half-life, and ex-
hibits greater toxicity than the lactone. This is supported by
pharmacologic evidence from clinical studies in humans and
other mammalian species receiving sodium camptothecin,
9-amino camptothecin and Topotecan (15,16). Thus, an alter-
native strategy to solubilize DB-67 is needed.
Inclusion Complexation between DB-67 and Cyclodextrins
The results in Table I indicate that DB-67 can be solu-
bilized by the water-soluble chemically modified ␤-cyclodex-
trins (e.g., SBE-CD and HP-CD). To understand the com-
plexation mechanisms behind this solubilization effect, two
methods were employed to determine the complex formation
constants for the neutral lactone and ionized carboxylate
forms of DB-67 with two water-soluble ␤-cyclodextrins, SBE-
CD and HP-CD, since no single method was suitable for both
species of DB-67. A solubility method was employed for DB-
67 lactone as the intrinsic solubility for the neutral species in
aqueous solution is very low and sensitive to cyclodextrin
concentration, [CD], as shown in Fig. 3(A). The linear solu-
bility profile for DB-67 with SBE-CD in Fig. 3 indicates that
Fig. 2. Dependence of solubility, S (mg/ml), for DB-67 in aqueous
solution at 25°C on solution pH values. The curve is a least-squares fit
using Eq. (3).
where I and II denote the neutral lactone and E-ring opened
species, respectively, and S is the intrinsic aqueous solubility
o
of the lactone. The equilibrium constants corresponding to
−
+
Fig. 1 and Eq. (2) are: K ס [II]/[I], K ס [II ][H ]/[II], KЈ
1
a1
a2
−
+
−
+
-2
+
−
ס [I ][H ]/[I], KЉ ס [IIЈ ][H ]/[II], K ס [II ][H ]/[II ],
KЈ ס [IIЈ ]/[II ], and KЈ ס [I ][H ]/[I], and KЈ ס [II
H ]/[IIЈ ]. Among these seven equilibrium equations, only
a2
a2
−
−
−
+
−2
]
1
a2
a1
+
−
[
five are independent. Because various A-ring substituents
have only a minor effect on the kinetics and equilibrium of
1:1 complexes predominate. In contrast, a quadratic depen-
dence of DB-67 solubility on [CD] was observed for com-
plexation of the lactone with HP-CD, suggesting the forma-
tion of 1:2 complexes at a higher HP-CD concentration.
In the limit of [CD] >> So, the relevant formation con-
stants can be evaluated as,
E-ring opening/closing and K for unsubstituted camptothe-
1
−
3
cin is small (2 × 10 ) (14), several approximations can be
made in Eq. (2) (K ס KЈ , K ס KЈa2 ס KЉ , K ס KЈa1),
1
1
a2
a2
a1
resulting in the following simplified solubility equation:
pH
pH
2pH
S = S ͑1 + K K 10 + K 10 + K K K 10
͒
(3)
o
1
a1
a2
1
a1 a2
1:1
1:2
2
S = S ͑1 + K ͓CD͔ + Kf ͓CD͔ ͒
(4)
o
f
where S , K K and Ka2 are adjustable parameters. A least-
o
1
a1
squares fit of the solubility data in Fig. 2 yielded K K and where S is the solubility in the absence of cyclodextrin. A
1
a1
o
−
7
pKa2 of (3.4 ± 0.3) × 10 and 8.67 ± 0.20, respectively. For the linear least-squares fit of the data for SBE-CD in Fig. 3(A)
ring-opened DB-67 (II), the carboxylic acid group may have yielded Kf ס 8.5 ± 0.2 × 10 M (r ס 0.999). The inability
1
:1
3
−1
2
a pK value around 3.8 since the pK values for structurally for SBE-CD to form 1:2 complexes with DB-67 apparently
a
a
similar compounds, D-gluconic acid and ␣-hydroxybutyric arises from the presence of an average of seven negatively
acid, are 3.77 and 3.65, respectively, and a pK of 4.00 was charged sulfobutyl ether groups in the molecule preventing
a
assumed for 10-hydroxyl substituted camptothecin by Fass- the approach of two SBE-CDs to each other to form 1:2
berg et al. (14). Assuming that pK ס 3.80, a value of K ס complexes. A least-squares fit of the data for HP-CD in Fig.
a1
1
−
3
1:1
3
2
2
.2 × 10− can be estimated for K . This value is close to K ס 3(A) according to Eq. (4) yielded of K
ס 5.8 ± 0.2 × 10
and Kf ס 3.8 ± 0.2 × 10 M (r ס 1.000).
The ability of HP-CD to form 1:2 complexes with the
1
1
f
3
−1
1:2
4
-2
2
× 10 estimated for unsubstituted camptothecin by Fass-
M
berg et al. (14). The pKa2 value (8.67) obtained from the
present regression analysis is close to the UV spectroscopic lactone form of DB-67 suggests that there are at least two
measurement of pKa2 (8.56) for the phenol group at position binding sites in the lactone. The influence of the 7-t-
1
0 in 10-hydroxyl-camptothecin (14), though the extremely butyldimethylsilyl substituent on the complexation strength
low solubility for DB-67 would preclude the use of this tech- can be evaluated by comparing the complexation of DB-67
nique for the determination of pKa2 for DB-67.
and unsubstituted camptothecin (CPT) using the same solu-
Referring to Fig. 2, at a pH below 7.4 but above the bility method. The results for CPT are presented in Fig. 3 (B)
estimated pKa1 (3.8) for the carboxylic acid group in the ring- and fitted with a linear model. The 1:1 complex formation
opened species, the solubility S increases by less than one constants for CPT with SBE-CD and HP-CD are presented in
order of magnitude over 3 pH units. This result suggests that Table II along with those for DB-67. Interestingly, the 1:1
the ring-opening reaction (I -> II) is unfavorable (i.e., K is complex formation constants for CPT with SBE-CD and HP-
1
small). This is consistent with previous studies of the hydro- CD are about 30 times smaller than those for the lactone form
lysis kinetics of camptothecin analogs (14) in which the K
of DB-67 with the corresponding cyclodextrin. Since the 10-
OH is hydrophilic and thereby less inclined to enter into the
1
−
3
constant was on the order of 2 × 10
.
Above pH 7.4, the solubility increases dramatically with CD cavity, the present results suggest that the 7-t-butyldi-

1
219
ture arises from the fact that cyclodextrins stabilize DB-67
toward hydrolysis as discussed in the next section.
Inclusion complex formation between DB-67 carboxyl-
ate and SBE-CD or HP-CD was examined by circular dichro-
ism. The inverse of the ellipticity difference, ⌬␪, between the
2
-
complexed and free DB-67 (II ) at 280 nm (the wavelength at
which the differences were maximized) was plotted vs. 1/[CD]
as shown in Fig. 4. The complex formation constant K was
f
obtained from a linear least-squares fit according to the Ben-
esi-Hildebrand equation (17),
1
1
1
= ⌬␪
+ ⌬␪
(5)
⌬␪
K ͓CD͔
AB
f
AB
where ⌬␪AB is the difference in molar extinction coefficients
for right and left circularly polarized light between the free
and bound DB-67. The regression analyses yielded K ס 2.5
f
2
−1
2
2
±
1.2 × 10 M (r ס 0.994) and ⌬␪ ס −15.5 ± 6.6 mdeg (r
AB
ס 0.994) for complexes involving the ionized E-ring opened
3
-1
form of DB-67 with SBE-CD and K ס 1.7 ± 0.3 × 10 M and
⌬
f
2
␪AB ס −11.7 ± 1.3 mdeg (r ס 0.996) for complexes involv-
ing the ionized E-ring opened form of DB-67 with HP-CD,
respectively. As shown in Table II, E-ring opening and ion-
ization of DB-67 carboxylate decrease its binding constant
with SBE-CD by about one order of magnitude, though these
effects are less dramatic for complexation with HP-CD. This
difference may be due to charge repulsion between the DB-67
carboxylate and the negatively charged SBE-CD.
Kinetics of DB-67 E-Ring Opening/Closing
Figure 5(A) shows the kinetic profiles for the appearance
of DB-67 lactone (I) and the disappearance of the E-ring
opened DB-67 (II) along with the overall DB-67 concentra-
tion vs. time at pH 4. At this pH, the reaction involves pri-
marily the neutral lactone (I) and unionized ring-opened form
Fig. 3. Solubility for the neutral lactone form of DB-67 (A) and
camptothecin (B) at 25°C as a function of cyclodextrin concentrations
of DB-67 (II) (Fig. 1). The equilibrium constant, K , for this
1
reversible hydrolysis reaction determined from the pH-
(SBE-CD and HP-CD) in aqueous solution. The curves are least-
−
3
solubility profile is 2 × 10 . Thus, the reverse reaction, lac-
tone hydrolysis, can be neglected. Assuming ring closure to be
first-order, the concentration profiles should obey the follow-
squares fits using Eq. (4) or its linear version.
methylsilyl substituent enhances complex formation. The fail- ing relations:
ure of CPT to form 1:2 complexes with HP-CD also implies
that the absence of the 7-t-butyldimethylsilyl in CPT depletes
one binding site to HP-CD. Based on these results, it is con-
cluded that the predominant 1:1 complex for DB-67 involves
inclusion of the 7-t-butyldimethylsilyl residue. The DB-67 E-
ring may be involved in 1:2 complex formation with HP-CD
as steric hindrance may prevent the binding of two cyclodex-
trins near the A and B rings. Further evidence for this struc-
1:1
−1
1:2
−2
Table II. Formation Constants, Kf (M ) and Kf (M ), for 1:1 and
1
:2 Inclusion Complexes of DB-67 and CPT with SBE-CD and
HP-CD
Solute
SBE-CD
HP-CD
3
3
Neutral lactone (DB-67) 8.5 ± 0.2 × 10 (1:1) 5.8 ± 0.2 × 10 (1:1)
4
3
.8 ± 0.1 × 10 (1:2)
2
3
Fig. 4. 1/⌬␪ vs. 1/[CD], where ⌬␪ is the ellipticity difference between
Carboxylate (DB-67)
Neutral lactone (CPT)
Carboxylate (CPT)
2.5 ± 1.2 × 10 (1:1) 1.7 ± 0.3 × 10 (1:1)
2
-
2
2
the bound and free species of the ring opened form of DB-67 (II )
and [CD] is the cyclodextrin concentration. The lines are least-
squares fits using Eq. (5).
2.5 ± 0.1 × 10 (1:1) 1.8 ± 0.0 × 10 (1:1)
—
—

1
220
Xiang and Anderson
Fig. 5(B) yielded the first-order rate constant, k ס 1.1 ±
open
−
4 −1
0
.1 × 10
s
, which is close to the interpolated value of 1.3 ×
−
4 −1
10
s
at pH 7.40 for unsubstituted camptothecin (14), sug-
gesting that substitution at the A and B rings has no signifi-
cant effect on the E-ring closure kinetics. The mass balance
results in Fig. 5(B) indicate that no other reactions contribute
significantly under these conditions.
The hydrolysis of the lactone was also investigated at pH
7.4 in the presence of 20% (w/v) SBE-CD and HP-CD, re-
spectively. The temporal profiles for the disappearance of the
lactone and the appearance of the E-ring opened form of
DB-67 were found to follow first-order kinetic models similar
to those in Eqs. (6) and (7). Values for k
were (7.1 ± 0.1)
open
−
5 −1
−5 −1
×
10
s
and (6.2 ± 0.1) × 10
s
in 20% (w/v) SBE-CD and
HP-CD, respectively. The presence of 20% (w/v) SBE-CD
or HP-CD reduces the rate of E-ring opening by 1.6- and
1.8-fold, respectively. The mechanisms responsible for this
improved stability of the therapeutically viable DB-67 may
differ in the cases of SBE-CD and HP-CD. In 20% (w/v)
HP-CD, >99.9% of DB-67 is included in the CD cavity, and
roughly 46% of the complexes are in the form of 1:2 com-
plexes. If the hydrolysis site on the E-ring is included in the
CD cavity in the 1:2 complexes with HP-CD but not in the
1
:1 complexes, about 46% of the E-rings should be protected
−
from attack by OH ions. This may explain the ca. 44% re-
duction in the observed rate constant. On the other hand, in
2
0% (w/v) SBE-CD, >99.8% of DB-67 is bound to SBE-CD
in 1:1 complexes but perhaps only a small fraction of the
:1 complexes involve those in which the E-ring is included in
1
the CD cavity. However, the negative charged sulfobuty−l
ether groups in SBE-CD bound to the lactone may repel OH
−
ions in the vicinity and reduce the rate of the OH catalyzed
hydrolysis reaction.
Fig. 5. (A) Kinetic profiles for the appearance of the lactone (I) and
the disappearance of the E-ring opened DB-67 carboxylate (II) along
with the overall DB-67 concentration vs. time in pH 4.04 buffer (30
mM acetate, I ס 0.1). (B) Kinetic profiles for the appearance of
species II and the disappearance of species I along with the overall
DB-67 concentration vs. time in pH 7.41 buffer (30 mM phosphate, I
ס 0.1). The curves are least-squares fits using Eqs. (6–7).
Stability with Respect to Drug Precipitation
A successful supersaturated parenteral formulation re-
quires physical stability with respect to drug precipitation.
While supersaturated solutions prepared by dilution of con-
centration stock solutions of DB-67 lactone in DMSO pro-
vided clear solutions at 1 mg/mL with no signs of precipitation
within 3 days, precipitation was evident even during the di-
lution process at 2 mg/mL and continued after the dilution
phase. Since the poorly water-soluble lactone was employed
close
−k t
͓
I͔ = ͓I͔ ͑1 − e
͒ + ͓I͔o
(6)
(7)
t
eq
close
−k t
͓
II͔ = ͓͑II͔ − ͓II͔ ͒e
+ ͓II͔eq
t
o
eq
where the subscripts t, o, and eq stand for time t, initial time, in the DMSO additions, the local drug concentration near the
and at equilibrium, respectively. Simultaneous non-linear injection tip may have been sufficient to induce rapid nucle-
least-squares fits of the kinetic profiles in Fig. 5 (A) according ation and precipitation. Proper control of the release pattern
to Eqs. (6) and (7) yielded the first-order rate constant, k
(local vs. uniform) and rate of DB-67 incorporation into the
, which is close to the interpolated value dilution medium appeared to be critical to the success of this
at pH 4.04 for unsubstituted camptothecin type of formulation approach.
14), suggesting that substitution on the A and B rings has no
The desired slow and uniform release of DB-67 into the
close
−
4 −1
ס 3.3 ± 0.3 × 10
s
−
4 −1
of 3.0 × 10
s
(
significant effect on the E-ring closure kinetics. The mass dilution medium could be accomplished by employing a
balance results, also shown in Fig. 5(A), indicate that no other chemical approach, namely, by converting the highly water-
reactions occur under these conditions.
soluble carboxylate (II) to the lactone (I) in the presence of a
Fig. 5(B) displays profiles for the appearance of the E- chemically modified cyclodextrin. Studies by Fassberg and
ring opened form of DB-67 (II) and the disappearance of the Stella (14) for other camptothecin analogs suggest that the
lactone (I) along with the overall DB-67 concentration vs. rate of this chemical reaction can be regulated by solution pH.
−
time at pH 7.4. At this pH, the ionized carboxylate, II , domi- Consequently, a concentrated alkaline solution of DB-67 car-
nates. Thus, the reverse reaction from II to I can be neglected boxylate was diluted with an acidic buffer to facilitate a timely
−
and Eqs. (6) and (7) are applicable for species I and II + II . conversion from the E-ring opened DB-67 to its lactone. The
Simultaneous non-linear least-squares fits of the profiles in target final drug concentration was 2 mg/mL. Fig. 6 (A) shows

1
221
was examined following the same procedure as described
above. As shown in Fig. 6 (B), the reconstituted solution
remained clear for more than seven days, suggesting that no
significant nucleation/precipitation occurred during or after
lyophilization.
SUMMARY
The present solubility studies indicate that traditional
solubilization approaches failed to achieve an equilibrium tar-
get concentration of 2 mg/mL for the lactone form of DB-67.
To this end, a clear, supersaturated solution was prepared via
pH induced chemical conversion of the ring-opened form of
DB-67 to the lactone in the presence of a chemically modified
water soluble ␤-cyclodextrin, SBE-CD. Solutions both before
and after lyophilization and reconstitution remained clear and
stable for at least three days.
This formulation method can potentially be utilized for
the development of clear injectable solutions for other lipo-
philic and poorly water-soluble camptothecin analogs.
ACKNOWLEDGMENTS
This work was performed under National Cancer Insti-
tute (NCI) contract NO1-CM-77108. The authors thank Dr.
Thomas Burke and Dr. Paul Bummer for the use of their
freeze-dryer and Jasco model J-710 Spectropolarimeter, re-
spectively, in their laboratories and for their assistance in
using these instruments. The authors also thank Mr. Nick
Nash for performing some of the experiments presented in
this paper.
Fig. 6. (A) Concentration of the neutral DB-67 lactone (open circles)
and overall DB-67 concentration (solid circles) in filtered solutions REFERENCES
after 1 + 9 dilution from a high-pH aqueous solution into 20% (w/v)
1
. S. Sweetana and M. J. Akers. Solubility principles and practices
for parenteral drug dosage form development. PDA J. Pharm.
Sci. Technol. 50:330–342 (1996).
. D. J. W. Grant and T. Higuchi. Solubility Behavior of Organic
Compounds, John Wiley & Sons, Inc., New York, 1990.
. J. J. Torres-Labandeira, P. Davignon, and J. Pitha. Oversaturated
solutions of drug in hydroxypropylcyclodextrins: Parenteral
preparation of pancratistatin. J. Pharm. Sci. 80:384–386 (1990).
SBE-CD as a function of time after the completion of mixing. (B)
DB-67 concentration in the filtered solutions of duplicate samples
after reconstitution of the lyophilized formulation. The horizontal
line indicates the averaged overall DB-67 concentrations in the un-
filtered solutions.
2
3
the overall DB-67 concentration and the concentration of
converted neutral DB-67 in the solution containing 20% (w/v)
SBE-CD at different time intervals. The lactone slowly built
up in the final solution in a uniform manner, attaining
completion within 100 m to yield a clear solution at pH 4.39.
The overall DB-67 concentration remained constant over an
extended period of three days, well beyond the target stability
of one day. This slow and uniform formation of the lactone
may explain the superior physical stability of this formulation
approach in comparison to supersaturated solutions prepared
from a concentrated stock solution of the lactone in DMSO.
In the absence of cyclodextrins, however, the precipitation
was found to occur in less than one minute after dilution from
a concentrated DB-67 solution at a high pH (10.50). Thus, it
appears that only a combined approach of chemical conver-
sion via pH adjustment and complexation with cyclodextrins
would provide a stable formulation with the desired drug
concentration.
4. D. Bom, D. P. Curran, S. Kruszewski, S. G. Zimmer, S. J. Thomp-
son, G. Kohlhagen, W. Du, A. J. Chavan, K. A. Fraley, A. L.
Bingcang, J. J. Latus, Y. Pommier, and T. G. Burke. The novel
silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin dis-
plays high lipophilicity, improved human blood stability, and po-
tent anticancer activity. J. Med. Chem. 43:3970–3980 (2000).
. C. Jaxel, K. W. Kohn, M. C. Wani, M. E. Wall, and Y. Pommier.
Structure-activity study of the actions of camptothecin deriva-
tives on mammalian topoisomerase I: Evidence for a specific re-
ceptor site and a relation to antitumor activity. Cancer Res. 49:
1465–1469 (1989).
5
6. Y. H. Hsiang, M. G. Lihou, and L. F. Liu. Arrest of replication
forks by drug-stabilized topoispomerase I-DNA cleavable com-
plexes as a mechanism of cell killing by camptothecin. Cancer
Res. 49:5077–5082 (1989).
7
. Y. H. Hsiang, L. F. Liu, M. E. Wall, M. C. Wani, A. W. Nicholas,
G. Manikumar, S. Kirschenbaum, R. Silber, and M. Potmesil.
DNA topoisomerase I-mediated DNA cleavage and cytotoxicity
of camptothecin analogues. Cancer Res. 49:4385–4389 (1989).
. R. P. Hertzberg, M. J. Caranfa, K. G. Holden, D. R. Jakas, G.
Gallagher, M. R. Mattern, S. M. Mong, J. O. Bartus, R. K.
Johnson, and W. D. Kingsbury. Modification of the hydroxy lac-
tone ring of camptothecin: Inhibition of mammalian topoisomer-
ase I and biologic activity. J. Med. Chem. 32:715–720 (1989).
. R. Kapoor, D. L. Slade, A. Fujimori, Y. Pommier, and W. G.
Harker. Altered topoisomerase I expression in two subclones of
human CEM leukemia selected for resistance to camptothecin.
Oncol. Res. 7:83–95 (1995).
8
For long-term storage, the 2 mg/mL solution of the lac-
tone in 20% (w/v) SBE-CD prepared according to the pro-
cedure described above was lyophilized after the chemical
conversion process was complete. Lyophilized samples were
reconstituted and the stability of the reconstituted solution
9

1
222
Xiang and Anderson
1
1
1
0. L. S. Rosen. Irinotecan in lymphoma, leukemia, and breast, pan- 14. J. Fassberg and V. J. Stella. A kinetic and mechanistic study of
creatic, ovarian, and small-cell lung cancers. Oncology (Hun-
tingt). 12:103–109 (1998).
the hydrolysis of camptothecin and some analogues. J. Pharm.
Sci. 81:676–684 (1992).
1. C. Kollmannsberger, K. Mross, A. Jakob, L. Kanz, and C. Boke- 15. J. G. Supko and L. Malspeis. Pharmacokinetics of the 9-amino
meyer. Topotecan - A novel topoisomerase I inhibitor: pharma-
cology and clinical experience. Oncology. 56:1–12 (1999).
2. D. J. Adams, M. W. Dewhirst, J. L. Flowers, M. P. Gamcsik, O. 16. N. B. Haas, F. P. LaCreta, J. Walczak, G. R. Hudes, J. M. Bren-
and 10,11-methylenedioxy derivatives of camptothecin in mice.
Cancer Res. 53:3062–3069 (1993).
M. Colvin, G. Manikumar, M. C. Wani, and M. E. Wall. Camp-
tothecin analogues with enhanced antitumor activity at acidic pH.
Cancer Chemother. Pharmacol. 46:263–271 (2000).
nan, R. F. Ozols, and P. J. O’Dwyer. Phase I/pharmacokinetic
study of topotecan by 24-hour continuous infusion weekly. Can-
cer Res. 54:1220–1226 (1994).
13. A. Inoue and N. Saijo. Recent advances in the chemotherapy 17. H. A. Benesi and J. H. Hildebrand. A spectrophotometric inves-
of non-small cell lung cancer. Jpn. J. Clin. Oncol. 31:299–304
2001).
tigation of the interaction of iodine with aromatic hydrocarbons.
J. Am. Chem. Soc. 71:2703–2707 (1949).
(